WO2024075803A1 - 脳神経疾患の検査、治療もしくは予防のための方法 - Google Patents

脳神経疾患の検査、治療もしくは予防のための方法 Download PDF

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WO2024075803A1
WO2024075803A1 PCT/JP2023/036304 JP2023036304W WO2024075803A1 WO 2024075803 A1 WO2024075803 A1 WO 2024075803A1 JP 2023036304 W JP2023036304 W JP 2023036304W WO 2024075803 A1 WO2024075803 A1 WO 2024075803A1
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autoantibody
polypeptide
antibody
autoantibodies
nrxn1α
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裕紀 塩飽
英彦 高橋
均 岡澤
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Tokyo Medical and Dental University NUC
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Definitions

  • the present disclosure relates to a marker useful for testing for cranial nerve diseases or susceptibility to cranial nerve diseases, as well as a testing method and kit using the same. More specifically, the present disclosure relates to a testing method and kit using a specific autoantibody as a marker. The present disclosure also relates to a method for treating or preventing cranial nerve diseases.
  • Schizophrenia is a neurological disease that develops during adolescence and young adulthood and presents with chronic and progressive psychiatric symptoms. Its main symptoms are positive symptoms (hallucinations, delusions, disorganized speech and behavior, etc.), negative symptoms (flattened emotions, poor thinking, decreased motivation, etc.), and cognitive disorders (attention disorders, decreased working memory, impaired executive function, etc.). Schizophrenia patients are symptomatically and genetically heterogeneous, and various pathological mechanisms are thought to be behind them (Non-Patent Document 1). However, there are no biomarkers for these heterozygous subgroups, and the pathological mechanisms underlying treatment resistance in patients and symptoms have not been fully elucidated.
  • Autoantibodies are a major factor in autoimmunity. In fact, autoantibodies specific to molecules in synaptic membranes have been found in patients with encephalitis. Some of these molecule-specific autoantibodies cause symptoms of neurological disorders in patients with encephalitis. The most widely studied form of autoantibody-mediated encephalitis is anti-N-methyl-d-aspartate (NMDA) receptor antibody encephalitis, which is associated with schizophrenia-related symptoms (Non-Patent Document 2). Anti-GABA A R ⁇ 1 receptor antibody encephalitis also causes symptoms of neurological disorders (Non-Patent Document 3). These autoantibodies are also present in patients with schizophrenia, but it is unclear whether they are involved in the symptoms of schizophrenia.
  • NMDA N-methyl-d-aspartate
  • Pettingill P., Kramer, H.B., Coebergh, J.A., Pettingill, R., Maxwell, S., Nibber, A., Malaspina, A., Jacob, A., Irani, S.R., Buckley, C., et al.(2015).
  • Antibodies to GABAA receptor a1 and g2 subunits clinical and serologic characterization. Neurology 84, 1233-1241.
  • the inventors analyzed biological samples isolated from subjects in order to find biomarkers specific to neurological disorders such as schizophrenia.
  • the target antigen there are several prerequisites for the target antigen: (1) a membrane molecule expressed in the nervous system (autoantibodies do not normally enter living cells); and (2) the target antigen must be a molecule involved in schizophrenia and whose function is affected by the autoantibody or at least involved in synaptic function. From this perspective, the inventors used a cell-based assay and ELISA to successfully identify multiple novel autoantibodies that are thought to contribute to the pathophysiology of schizophrenia.
  • the inventors have identified multiple autoantibodies specific to patient-derived samples. More specifically, the inventors have demonstrated that autoantibodies against NCAM1 and other proteins derived from schizophrenia patients induce schizophrenia-related behaviors and changes in spines and synapses in mice. These autoantibodies can induce symptoms of neurological disorders such as schizophrenia, and therefore can be considered as therapeutic targets in patients who are positive for autoantibodies such as anti-NCAM1 autoantibodies.
  • NCAM1 also binds to glial cell line-derived neurotrophic factor (GDNF) and contributes to synapse formation.
  • GDNF glial cell line-derived neurotrophic factor
  • NCAM1 knockout mice and dominant-negative transgenic mice show changes in schizophrenia-related behavior.
  • the inventors have identified anti-NCAM1 autoantibodies in schizophrenia patients. Using a disease model in which immunoglobulin G (IgG) purified from schizophrenia patients was administered to mice, they have demonstrated that anti-NCAM1 autoantibodies inhibit spine and synapse formation in the frontal cortex and induce schizophrenia-related behavior.
  • IgG immunoglobulin G
  • a method for testing a cranial nerve disease or a susceptibility to a cranial nerve disease in a subject comprising: i) providing a sample isolated from a subject; ii) determining the presence or absence of autoantibodies in said sample,
  • the autoantibody is at least one autoantibody selected from the group consisting of an anti-NRXN1 ⁇ antibody, an anti-NRG1 antibody, an anti-NLGN2 antibody, an anti-NLGN3 antibody, an anti-ephrin B1 antibody, and an anti-TRPA1 antibody;
  • the method, wherein the presence of the autoantibody in the sample is indicative of the subject having a neurological disorder or a susceptibility to a neurological disorder.
  • Aspect 2 The method according to aspect 1, wherein the neurological disease is selected from the group consisting of schizophrenia, pain disorder, encephalopathy, epilepsy, psychotic disorder, mood disorder and encephalitis.
  • Aspect 3 The method according to aspect 1, which is carried out in vitro.
  • Embodiment 4 The method according to embodiment 1, wherein the sample is blood, plasma, serum or cerebrospinal fluid.
  • step of determining the presence or absence of an autoantibody comprises performing ELISA, CLEIA (chemiluminescent enzyme immunoassay), CLIA (chemiluminescent immunoassay), latex agglutination, radioimmunoassay, immunoblotting, immunoprecipitation, immunochromatography, or cell-based assay (CBA).
  • ELISA ELISA
  • CLEIA chemiluminescent enzyme immunoassay
  • CLIA chemiluminescent immunoassay
  • latex agglutination e.g., radioimmunoassay, immunoblotting, immunoprecipitation, immunochromatography, or cell-based assay (CBA).
  • kits for use in testing a subject for a cranial nerve disease or susceptibility to a cranial nerve disease comprising at least one of the following 1) to 3): 1) an NRXN1 ⁇ polypeptide, an NRG1 polypeptide, an NLGN2 polypeptide, an NLGN3 polypeptide, an Ephrin B1 polypeptide, and/or a TRPA1 polypeptide; a fragment of an NRXN1 ⁇ polypeptide, an NRG1 polypeptide, an NLGN2 polypeptide, an NLGN3 polypeptide, an Ephrin B1 polypeptide, and/or a TRPA1 polypeptide having binding activity with an anti-NRXN1 ⁇ antibody, an anti-NRG1 antibody, an anti-NLGN2 antibody, an anti-NLGN3 antibody, an anti-ephrin B1 antibody, and/or an anti-TRPA1 antibody; or a polypeptide comprising an epitope recognized by an anti-
  • kits according to aspect 8 wherein the neurological disease is selected from the group consisting of schizophrenia, pain disorder, encephalopathy, epilepsy, psychotic disorder, mood disorder, and encephalitis.
  • the polypeptide is immobilized on a substrate.
  • the substrate is a test strip, a bead, a multiwell plate or a microchip.
  • the kit according to aspect 8 comprising an expression vector containing a nucleic acid encoding the polypeptide.
  • kits according to embodiment 8 further comprising an antibody for detection that binds to anti-NRXN1 ⁇ autoantibody, anti-NRG1 autoantibody, anti-NLGN2 autoantibody, anti-NLGN3 autoantibody, anti-ephrin B1 autoantibody, and/or anti-TRPA1 autoantibody.
  • Aspect 14 Use of anti-NRXN1 ⁇ autoantibodies, anti-NRG1 autoantibodies, anti-NLGN2 autoantibodies, anti-NLGN3 autoantibodies, anti-ephrin B1 autoantibodies, and/or anti-TRPA1 autoantibodies as biomarkers for assessing a cranial nerve disease or susceptibility to a cranial nerve disease in a subject.
  • Aspect 15 The use according to Aspect 14, wherein the brain and nervous system disease is selected from the group consisting of schizophrenia, pain disorder, encephalopathy, epilepsy, psychotic disorder, mood disorder and encephalitis.
  • a method for treating or preventing a neurological disease in a subject comprising a step of removing, reducing, sequestering, inactivating or decomposing anti-NRXN1 ⁇ autoantibodies, anti-NRG1 autoantibodies, anti-NLGN2 autoantibodies, anti-NLGN3 autoantibodies, anti-ephrin B1 autoantibodies, and/or anti-TRPA1 autoantibodies in the subject.
  • the neurological disease is selected from the group consisting of schizophrenia, pain disorder, encephalopathy, epilepsy, psychotic disorder, mood disorder and encephalitis.
  • the method according to aspect 16 comprising a step of performing plasma exchange.
  • a method for treating blood, plasma, serum or cerebrospinal fluid isolated from a subject having or susceptible to a cranial nerve disease comprising treating the blood, plasma, serum or cerebrospinal fluid with a NRXN1 ⁇ polypeptide, an NRG1 polypeptide, an NLGN2 polypeptide, an NLGN3 polypeptide, an Ephrin B1 polypeptide, and/or a TRPA1 polypeptide; a fragment of an NRXN1 ⁇ polypeptide, an NRG1 polypeptide, an NLGN2 polypeptide, an NLGN3 polypeptide, an Ephrin B1 polypeptide, and/or a TRPA1 polypeptide having binding activity with an anti-NRXN1 ⁇ autoantibody, an anti-NRG1 autoantibody, an anti-NLGN2 autoantibody, an anti-NLGN3 autoantibody, an anti-ephrin B1 autoantibody, and/or an anti-TRPA1 autoantibody; or an anti-NRXN1
  • a method for removing or reducing anti-NRXN1 ⁇ autoantibodies, anti-NRG1 autoantibodies, anti-NLGN2 autoantibodies, anti-NLGN3 autoantibodies, anti-ephrin B1 autoantibodies, and/or anti-TRPA1 autoantibodies contained in the blood, plasma, serum, or cerebrospinal fluid by contacting a substrate with at least one selected from the group consisting of a polypeptide containing an epitope recognized by anti-TRPA1 autoantibodies and/or anti-NRXN1 ⁇ autoantibodies, anti-NRG1 autoantibodies, anti-NLGN2 autoantibodies, anti-NLGN3 autoantibodies, anti-ephrin B1 autoantibodies, and/or anti-TRPA1 autoantibodies.
  • a pharmaceutical composition for use in the treatment or prevention of a brain and nervous system disease comprising an NRXN1 ⁇ polypeptide, an NRG1 polypeptide, an NLGN2 polypeptide, an NLGN3 polypeptide, an Ephrin B1 polypeptide, and/or a TRPA1 polypeptide; or a fragment thereof having binding activity with an anti-NRXN1 ⁇ autoantibody, an anti-NRG1 autoantibody, an anti-NLGN2 autoantibody, an anti-NLGN3 autoantibody, an anti-ephrin B1 autoantibody, and/or an anti-TRPA1 autoantibody; or a polypeptide comprising an epitope recognized by an anti-NRXN1 ⁇ autoantibody, an anti-NRG1 autoantibody, an anti-NLGN2 autoantibody, an anti-NLGN3 autoantibody, an anti-ephrin B1 autoantibody, and/or an anti-TRPA1 autoantibody.
  • a method for treating or preventing a cranial nerve disease in a subject comprising: i) providing a sample isolated from a subject; ii) determining the presence or absence of an autoantibody in the sample, wherein the autoantibody is at least one autoantibody selected from the group consisting of an anti-NRXN1 ⁇ antibody, an anti-NRG1 antibody, an anti-NLGN2 antibody, an anti-NLGN3 antibody, an anti-ephrin B1 antibody, and an anti-TRPA1 antibody; iii) When the autoantibody is present in the sample, the subject is subjected to at least one of the following methods selected from the group consisting of 1) to 6): 1) Immunoglobulin therapy, 2) steroid therapy, 3) administration of immunosuppressants
  • the method further comprises a step of determining the presence or absence of an additional autoantibody in the sample, wherein the additional autoantibody is at least one autoantibody selected from the group consisting of an anti-NMDA receptor antibody, an anti-GABA A R ⁇ 1 antibody, and an anti-NCAM1 antibody, and when the autoantibody is present in the sample,
  • the additional autoantibody is at least one autoantibody selected from the group consisting of an anti-NMDA receptor antibody, an anti-GABA A R ⁇ 1 antibody, and an anti-NCAM1 antibody, and when the autoantibody is present in the sample.
  • the subject is administered an antigen polypeptide corresponding to the autoantibody whose presence has been confirmed, an antigen polypeptide fragment having binding activity with the autoantibody, or a polypeptide including an epitope recognized by the autoantibody.
  • B Immunocytochemistry results using a commercial anti-NCAM1 antibody, serum and CSF from schizophrenia patient 1, and serum from a healthy control. NCAM1 and EGFP were expressed from plasmids. Confocal images show antibodies bound to the membrane of EGFP-positive HeLa cells. Similar results were obtained for all anti-NCAM1 antibody-positive schizophrenia patients.
  • Serum antibodies did not react with EGFP, since (1) they did not react with EGFP in the nucleus, and (2) they did not react with cells transfected with an empty plasmid expressing only EGFP. Scale bar: 10 ⁇ m, Ab: antibody, Sz: schizophrenia.
  • A Structure of NCAM1 deletion constructs.
  • B Immunocytochemistry results using serum from schizophrenia patient 1 who was positive for anti-NCAM1 autoantibodies. NCAM1 deletion constructs and EGFP were expressed from plasmids.
  • AAV1-SYN1-EGFP and AAV2-VAMP2-mCherry were injected into the frontal cortex of 6-week-old mice, and purified IgG was injected into the CSF of 8-week-old mice. Molecular, histological, two-photon microscopic, and behavioral analyses were performed in 9-week-old mice.
  • IHC immunohistochemistry
  • IP immunoprecipitation
  • WB western blot.
  • B Immunoprecipitation analysis of mouse frontal cortex tissues revealed that anti-NCAM1 autoantibodies obtained from schizophrenia patients inhibited NCAM1-Fyn interaction.
  • CT computed tomography.
  • A Experimental protocol for IgG injection of anti-NCAM1 autoantibodies from schizophrenia patients. AAV1-SYN1-EGFP and AAV2-VAMP2-mCherry were injected into the frontal cortex of 6-week-old mice, and purified IgG was injected into the CSF of 8-week-old mice. Two-photon microscopy and behavioral analysis were performed in 9-week-old mice.
  • B Immunocytochemical staining using a commercial anti-NRXN1 ⁇ antibody, serum and CSF from schizophrenia patient 1, and serum from a healthy control. Bar: 10 ⁇ m.
  • D Schematic diagram of NRXN1 ⁇ deletion construct.
  • NRXN1-NLGN1 and NRXN1-NLGN2 interactions were inhibited by anti-NRXN1 autoantibodies present in schizophrenia patients.
  • A Representative mEPSC traces recorded from frontal cortical pyramidal neurons.
  • B Cumulative distribution of inter-event intervals for each group. Recording times ranged from 7.5 to 15 min (a total of 34,523 inter-events detected) for healthy controls and patient 1 with schizophrenia, respectively, and 9.5 to 10 min (a total of 27640 inter-events detected).
  • Nine cells from three mice were analyzed per group. **p ⁇ 0.01; Kolmogorov-Smirnov and Mann-Whitney tests.
  • C Cumulative distribution of amplitudes per group.
  • A Experimental protocol for IgG injection of autoantibodies from schizophrenia patients.
  • AAV1-SYN1-EGFP and AAV2-VAMP2-mCherry were injected into the frontal cortex of 6-week-old mice, and purified IgG was injected into the CSF of 8-week-old mice. Two-photon microscopy and behavioral analysis were performed in 9-week-old mice.
  • B Two-photon microscopy of dendritic spines in layer I of the frontal cortex of mice injected with AAV1-SYN1-EGFP and purified IgG from healthy controls, schizophrenia patient 2, and schizophrenia patient 3.
  • Anti-NLGN3 autoantibodies were positive in 2 of 122 patients with schizophrenia and none of 250 healthy controls.
  • Anti-Ephrin B1 autoantibodies were positive in 2 of 122 schizophrenia patients and in none of 250 healthy controls.
  • the presence of autoantibodies against TRPA1 in samples from patients with pain disorders was examined by cell-based assay. The results show that sera from patients with pain disorders react with cells expressing TRPA1. 13 is a photograph showing that anti-NLGN2 autoantibodies were positive in patients who had been misdiagnosed with schizophrenia.
  • FIG. 1 shows electroencephalograms of a patient with anti-NLGN2 autoantibody-positive autoimmune-mediated encephalopathy who had been misdiagnosed as having schizophrenia, before and after plasma exchange.
  • the first aspect of the present disclosure relates to a method for testing cranial nerve disease or cranial nerve disease susceptibility in a subject.
  • the method can include, for example, i) preparing a sample isolated from a subject, and ii) determining the presence or absence of a specific autoantibody in the sample, and the presence of a specific autoantibody in the sample can be used as an indicator that the subject has schizophrenia or a susceptibility to schizophrenia.
  • the testing method according to the present disclosure is useful as a means for determining whether or not a subject suffers from or has developed a cranial nerve disease such as schizophrenia, or as a means for determining the possibility of developing a cranial nerve disease such as schizophrenia in the future, and can provide information useful for diagnosing a cranial nerve disease such as schizophrenia.
  • the cranial nerve disease can be a cranial nerve disease that exhibits psychiatric symptoms.
  • the cranial nerve disease can be a cranial nerve disease caused by an autoantibody.
  • the subject is a human, particularly a patient with a neurological disease or a patient suspected of having a neurological disease, or a subject suspected of having a susceptibility to a neurological disease.
  • the diagnosis can be judged based on the objective indicator of the presence or absence of a specific autoantibody, and can be used as information to assist or support a conventional diagnosis. Such information is useful for determining a more appropriate treatment policy, and can promote improvement of the treatment effect and improvement of the patient's QOL.
  • the method according to the present disclosure can be used to monitor the disease state to prevent the disease from becoming intractable, becoming severe, recurring, etc.
  • Subjects who are estimated to be at high risk of developing a neurological disease such as schizophrenia (high-risk subjects) based on family background, etc. are also suitable subjects. Applying the method according to the present disclosure to such subjects before the symptoms of a neurological disease such as schizophrenia appear makes it possible to prevent or delay the onset of the disease or to intervene in the treatment early.
  • the method according to the present disclosure is also useful for identifying those at high risk of developing a neurological disease such as schizophrenia, that is, those who are susceptible to the disease.
  • Such identification makes it possible to reduce the likelihood of developing (possibility of contracting) the disease, for example, by taking preventive measures or improving lifestyle habits.
  • Those who have no subjective symptoms and for whom it is impossible or difficult to determine whether they have a neurological disease using conventional diagnostic methods are also suitable targets for the method disclosed herein. This testing method may also be carried out as one item in a health check.
  • Neurological disorders include, but are not limited to, schizophrenia, pain disorders, encephalopathy, epilepsy, psychotic disorders, mood disorders, and encephalitis.
  • the presence of a specific autoantibody in a sample obtained from a subject is an indicator that the subject has a neurological disorder, and can be used, for example, as a basis for differential diagnosis from other disorders with similar symptoms.
  • the neurological disorder is schizophrenia.
  • "Schizophrenia" is a mental disorder that causes a disorder in the integration of brain function and presents hallucinations, delusions, negative symptoms, and cognitive decline.
  • the target disease is not limited to schizophrenia, but can also be applied to a wide range of psychiatric disorders, i.e., other neurological disorders such as pain disorders, encephalopathy, epilepsy, psychotic disorders, mood disorders, and encephalitis.
  • Pain disorder is defined as pain at one or more anatomical locations severe enough to cause distress or impairment in social, occupational, or other important areas of functioning.
  • the current diagnosis is based on medical history and exclusion of physical disorders that adequately explain the pain and its severity, duration, and degree of impairment.
  • Encephalopathy is a condition in which the patient exhibits neurological symptoms also seen in encephalitis, such as abnormal electroencephalograms, impaired consciousness, and mental symptoms, but no obvious inflammation is found within the brain tissue.
  • Epilepsy is a group of non-communicable neurological disorders characterized by recurrent seizures. It refers to a neurological disorder or condition that causes epileptic seizures due to abnormal neural activity ("epileptic discharges") occurring in cells in the brain. Epileptic seizures can vary from brief, barely detectable episodes to prolonged, violent, shaking episodes due to abnormal electrical activity in the brain.
  • Symptoms include delusions and hallucinations, among others, but other symptoms include incoherent speech and behaving inappropriately in a given situation. There may also be sleep disorders, social withdrawal, lack of motivation, and difficulty carrying out daily activities.
  • Mood disorders also known as affective disorders, are conditions in which a person experiences distress or experiences significant impairment in daily life due to mood (emotion) changes that last for a certain period of time. Mood disorders are classified according to the World Health Organization's (WHO) International Classification of Diseases, 10th edition, ICD-10, and the American Psychiatric Association's Diagnostic and Statistical Manual of Mental Disorders, 5th edition, DSM-5.
  • WHO World Health Organization's
  • Encephalitis is a general term for inflammatory diseases of the brain, and acute encephalitis refers to a condition in which inflammation in the brain parenchyma causes acute symptoms such as fever, headache, impaired consciousness, and paralysis. Complications may include convulsions, hallucinations, speech disorders, memory disorders, and hearing impairments.
  • susceptibility to a neurological disease refers to a tendency to be susceptible to a neurological disease or a state of being at high risk of developing a neurological disease.
  • the presence of a specific autoantibody in a sample obtained from a subject is an indicator that the subject is susceptible to a neurological disease, and for example, even if the subject does not develop a neurological disease at the time of testing, it can be determined that the subject is at high risk of developing a neurological disease in the future.
  • a testing method refers to an in vitro analysis of components in a sample obtained from a subject to obtain some kind of measurement result.
  • the obtained measurement result i.e., the presence of autoantibodies in the sample
  • the testing method according to the present disclosure can also be considered an in vitro test method for cranial nerve disease or susceptibility to a cranial nerve disease in a subject.
  • the method according to the present disclosure can also be considered a method for obtaining an indicator of the possibility of a cranial nerve disease or a susceptibility to a cranial nerve disease in a subject, or a method for testing the presence or absence of a cranial nerve disease or the likelihood of developing a cranial nerve disease in a subject.
  • the step of preparing a sample isolated from a subject should be understood to mean the step of preparing a sample after it has been taken from a human subject, and should not be understood to include the step of performing an invasive procedure on a human subject, such as surgically harvesting tissue or blood, unless expressly specified.
  • the methods of this disclosure may, in some embodiments, be expressly specified as including the step of harvesting a biological sample from a subject.
  • the sample may be a biological sample derived from a subject, such as, but not limited to, blood, plasma, serum, or cerebrospinal fluid.
  • the sample obtained from the subject may be pre-treated or stored (including frozen, thawed) prior to being analyzed for the presence or absence of a particular autoantibody.
  • the step of determining the presence or absence of a specific autoantibody in a sample can include, but is not limited to, performing, for example, ELISA, CLEIA (chemiluminescent enzyme immunoassay), CLIA (chemiluminescent immunoassay), latex agglutination, radioimmunoassay, immunoblotting, immunoprecipitation, immunochromatography, or cell-based assay (CBA). Note that it is not essential to strictly quantify the amount of autoantibodies present in a sample, and it is sufficient to be able to detect the level of autoantibodies to the extent that the possibility of developing a neurological disease can be determined.
  • the presence of an autoantibody is determined when the measured value of a specific autoantibody in a sample, for example, measured values by ELISA, CLEIA (chemiluminescent enzyme immunoassay), CLIA (chemiluminescent immunoassay), latex agglutination, radioimmunoassay, immunoblot, immunoprecipitation, immunochromatography, or cell-based assay (CBA), exceeds a predetermined value or a control measured value.
  • the predetermined value or control measured value can be determined, for example, based on the measured value of a sample derived from a healthy individual or a sample known not to contain the autoantibody being tested, but is not limited thereto.
  • a positive autoantibody can be defined as a value exceeding two standard deviations from the average value of a measured value such as absorbance. Note that the determination here can be made automatically or mechanically without relying on the judgment of a person with specialized knowledge, such as a physician or laboratory technician, as is clear from the criteria for determination.
  • the ELISA method has many advantages, including high detection sensitivity, high specificity, excellent quantitative performance, simple operation, and suitability for simultaneous processing of multiple samples.
  • An example of a specific operation method when using the ELISA method is shown below.
  • a polypeptide to which a specific autoantibody binds is immobilized on a solid substrate.
  • the surface of a microplate is coated with an autoantibody-binding polypeptide.
  • a sample is then brought into contact with the immobilized polypeptide.
  • an autoantibody that recognizes the immobilized antigen is present in the sample, an immune complex is formed.
  • an enzyme-linked secondary antibody is added to label the immune complex, which is then reacted with the enzyme substrate to produce color, and the amount of color produced is used as an indicator to detect the immune complex.
  • a commercially available kit may be used when performing the ELISA method.
  • the autoantibody is an anti-NCAM1 (Neural Cell Adhesion Molecule 1) antibody.
  • the autoantibody is at least one autoantibody selected from the group consisting of an anti-NRXN1 ⁇ (Neurexin-1-alpha) antibody, an anti-NRG1 (Neureglin-1) antibody, an anti-NLGN2 (Neuroligin-2) antibody, an anti-NLGN3 (Neuroligin-3) antibody, an anti-Ephrin B1 antibody, and an anti-TRPA1 antibody.
  • the autoantibody is at least one autoantibody selected from the group consisting of an anti-NCAM1, an anti-NRXN1 ⁇ antibody, an anti-NRG1 antibody, an anti-NLGN2 antibody, an anti-NLGN3 antibody, an anti-Ephrin B1 antibody, and an anti-TRPA1 antibody.
  • These autoantibodies may recognize naturally occurring subtypes, isoforms, or variants of each of the above proteins.
  • those skilled in the art can easily obtain the gene sequence and amino acid sequence of the appropriate subtype, isoform, or variant of each of the above proteins, and can express them in cells using known techniques.
  • the expressed proteins may be further isolated and purified.
  • NCAM1 also known as CD56, is a cell adhesion molecule belonging to the immunoglobulin superfamily and is expressed primarily in human neurons, glial cells, skeletal muscle cells, NK cells and a subset of T cells. There are three subtypes of NCAM1: a 140/180 kD transmembrane molecule and a 120 kD GPI-anchored molecule. Each isoform is generated by alternative splicing.
  • NRXN1 ⁇ is a single-pass transmembrane protein that is expressed at high levels in the brain and is present at the presynaptic terminal. It binds to the postsynaptic membrane protein Neuroligin in the synaptic cleft and is involved in synapse construction and the release mechanism of neurotransmitters. There are many splice variants, and it is thought to affect the selection of glutamatergic and GABAergic neuronal synapse construction.
  • NRG1 Neuroglin-1
  • NRG2 is a member of the EGF family of proteins and forms a family with the structurally related proteins NRG2, NRG3, and NRG4. Multiple isoforms of NRG1 are generated by alternative splicing and can exert a variety of functions. NRG1 is essential for normal development of the nervous system and heart.
  • NLGN2 Neuron-2
  • NLGN1 NLGN2, NLGN3, NLGN4, and NLGN4Y
  • NLGN2 encodes a member of a family of neuronal cell surface proteins that act as splice-site-specific ligands for ⁇ -neurexins (NRXNs) and are thought to be involved in the formation and remodeling of synapses in the central nervous system.
  • NRXNs ⁇ -neurexins
  • NLGN3 Neuroligin-3 is one of five neuroligin genes (NLGN1, NLGN2, NLGN3, NLGN4, NLGN4Y) in humans.
  • Neuroligins are single-pass transmembrane proteins present in the postsynaptic domain and are endogenous ligands for neurexins (NRXNs) present in presynaptic terminals, regulating synaptic maturation and function.
  • NLGN3 is abundant in the brain, and NLGN3 protein levels increase during postnatal development, coinciding with the peak of synapse formation.
  • Ephrin B1 is one of eight mammalian ephrins (Ephrin A1-A5 and Ephrin B1-B3).
  • B-type ephrins are type I membrane proteins with molecular weights of 30-45 kDa, consisting of an extracellular domain, a transmembrane domain, and an intracellular domain with a PDZ-binding sequence at the C-terminus.
  • Ephrin B1 is encoded by the EFNB1 gene.
  • the encoded protein is a ligand for Eph-related receptor tyrosine kinases. It is involved in cell adhesion and may function in the development and maintenance of the nervous system.
  • TRPA1 is an ion channel present in the plasma membrane of many human and animal cells. This ion channel is known as a pain, cold, and itch sensor in humans and other mammals, as well as a sensor of environmental irritants that elicit other protective responses (tears, airway resistance, coughing).
  • multiple autoantibodies may be measured simultaneously or consecutively.
  • two or more autoantibodies for example, 2, 3, 4, 5, 6, or 7 autoantibodies selected from the group consisting of anti-NCAM1 antibody, anti-NRXN1 ⁇ antibody, anti-NRG1 antibody, anti-NLGN2 antibody, anti-NLGN3 antibody, anti-Ephrin B1 antibody, and anti-TRPA1 antibody, may be measured in any combination.
  • the autoantibody to be analyzed may further include anti-NMDA receptor antibody and anti-GABA A R ⁇ 1 antibody.
  • Kit for use in testing for cranial nerve disease or cranial nerve disease susceptibility A second aspect of the present disclosure relates to a kit for use in testing for cranial nerve disease or cranial nerve disease susceptibility in a subject.
  • the cranial nerve disease may be a cranial nerve disease that presents psychiatric symptoms.
  • the cranial nerve disease may be a cranial nerve disease caused by an autoantibody.
  • the kit may include, for example, at least one of the following 1) to 3): 1) an antigen polypeptide of a specific autoantibody, a fragment of an antigen polypeptide having binding activity with a specific autoantibody, or a polypeptide containing an epitope recognized by a specific autoantibody; 2) a nucleic acid encoding the polypeptide described in 1) above; or 3) a cell expressing the polypeptide described in 1) above.
  • the neurological disorder may be selected from the group consisting of, but is not limited to, schizophrenia, pain disorders, encephalopathy, epilepsy, psychotic disorders, mood disorders, and encephalitis.
  • the kit can include, for example, at least one of the following 1) to 3): 1) an NCAM1 polypeptide, an NCAM1 polypeptide fragment having binding activity with an anti-NCAM1 autoantibody, or a polypeptide containing an epitope recognized by an anti-NCAM1 autoantibody; 2) a nucleic acid encoding the polypeptide described in 1) above; or 3) a cell expressing the polypeptide described in 1) above.
  • the kit may include at least one of the following items 1) to 3): 1) an NRXN1 ⁇ polypeptide, an NRG1 polypeptide, an NLGN2 polypeptide, an NLGN3 polypeptide, an Ephrin B1 polypeptide, and/or a TRPA1 polypeptide; a fragment of an NRXN1 ⁇ polypeptide, an NRG1 polypeptide, an NLGN2 polypeptide, an NLGN3 polypeptide, an Ephrin B1 polypeptide, and/or a TRPA1 polypeptide having binding activity with an anti-NRXN1 ⁇ autoantibody, an anti-NRG1 autoantibody, an anti-NLGN2 autoantibody, an anti-NLGN3 autoantibody, an anti-Ephrin B1 autoantibody, and/or an anti-TRPA1 autoantibody; or a polypeptide comprising an epitope recognized by an anti-NRXN1 ⁇ autoantibody, an anti-NRG1 autoantibody, an
  • the antigen polypeptide of the autoantibody may be a full-length wild-type polypeptide.
  • the antigen polypeptide of the autoantibody may include a mutation or variation compared to the wild-type sequence, such as the addition of a tag sequence to facilitate purification or fixation, or other substitutions, deletions, or additions.
  • the mutation or variation may be naturally occurring or non-naturally occurring.
  • the antigen polypeptide of the autoantibody may be derived from an organism other than a human, provided that the organism is useful for the intended application.
  • the antigen polypeptide may be a fusion protein, such as with GST, beta-galactosidase, maltose binding protein, or a histidine (His) tag.
  • the kit according to the present disclosure may include a fragment of an antigen polypeptide having binding activity with an autoantibody.
  • the fragment of an antigen polypeptide having binding activity with an autoantibody includes an epitope recognized by the autoantibody.
  • the kit according to the present disclosure may include a polypeptide including an epitope recognized by an autoantibody.
  • the inventors have found that the epitope recognized by anti-NCAM1 autoantibody is present in the Ig1 domain of NCAM1.
  • the inventors have also found that the epitope recognized by anti-NRXN1 ⁇ autoantibody is present in the LNS6 domain of NRXN1 ⁇ .
  • the polypeptide may be immobilized on a substrate.
  • the substrate may be, for example, but is not limited to, a test strip, beads, a multi-well plate, or a microchip.
  • a plurality of types of autoantibody-binding polypeptides may be immobilized on the substrate.
  • the substrate may be, for example, a solid-phase substrate made of a resin such as polystyrene resin, polycarbonate resin, silicone resin, or nylon resin, or a water-insoluble substance such as glass.
  • the antigen polypeptide may be supported on the substrate by, for example, physical or chemical adsorption.
  • kits disclosed herein may include a nucleic acid encoding a polypeptide that binds to the autoantibody.
  • the nucleic acid encoding the polypeptide may be used to generate the polypeptide in a cell or in vitro.
  • the kits disclosed herein may include an expression vector that includes a nucleic acid encoding the polypeptide. The expression vector may be used, for example, by transfecting a cell.
  • the kit of the present disclosure may include cells expressing a polypeptide that binds to the autoantibody.
  • the cells may include prokaryotic or eukaryotic cells, such as human cells, non-human mammalian cells, insect cells, fungal cells, etc.
  • the cells may secrete the polypeptide or express it on the cell membrane.
  • cells expressing the polypeptide may be used in cell-based assays (CBAs), etc.
  • kits of the present disclosure may further include an antibody for detection that binds to the autoantibody.
  • an antibody may include, for example, an antibody that recognizes the constant region of a human antibody.
  • the antibody for detection may be labeled with, for example, an enzyme such as peroxidase, microperoxidase, horseradish peroxidase (HRP), alkaline phosphatase, ⁇ -D-galactosidase, glucose oxidase, and glucose-6-phosphate dehydrogenase; a fluorescent substance such as fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC), and europium; a chemiluminescent substance such as luminol, isoluminol, and acridinium derivatives; a coenzyme such as NAD; biotin; and a radioactive substance such as 131I and 125I.
  • an enzyme such as peroxidase, micro
  • Kits according to the present disclosure will typically be accompanied by instructions for use.
  • Other reagents e.g., buffers, blocking reagents, enzyme substrates, color reagents, etc.
  • devices or instruments e.g., containers, reaction devices, spectrophotometers, fluorescence readers, etc.
  • the kit may also include a container containing a known concentration or amount of antibody as a standard sample.
  • Biomarkers for Evaluating Neurological Disease or Neurological Disease Susceptibility The third aspect of the present disclosure relates to the use of autoantibodies as biomarkers for evaluating neurological diseases or neurological disease susceptibility in a subject.
  • the neurological disease may be a neurological disease that exhibits psychiatric symptoms.
  • the neurological disease may also be a neurological disease caused by an autoantibody.
  • a biomarker refers to a substance (biological indicator compound) that is an indicator of the presence or absence of a disease, changes in the condition, or the effect of treatment.
  • the levels of each biomarker can be detected to such an extent that the possibility of developing a neurological disease can be determined.
  • detection can be performed so that it can be determined whether the level of each biomarker in a sample exceeds a predetermined reference value.
  • the detection of the biomarkers can be performed using the methods described herein or any method known to those skilled in the art.
  • the autoantibody is an anti-NCAM1 autoantibody. In some embodiments, the autoantibody is at least one autoantibody selected from the group consisting of anti-NRXN1 ⁇ autoantibody, anti-NRG1 autoantibody, anti-NLGN2 autoantibody, anti-NLGN3 autoantibody, anti-Ephrin B1 autoantibody, and anti-TRPA1 autoantibody.
  • At least one autoantibody selected from the group consisting of anti-NCAM1 autoantibody, anti-NRXN1 ⁇ autoantibody, anti-NRG1 autoantibody, anti-NLGN2 autoantibody, anti-NLGN3 autoantibody, anti-Ephrin B1 autoantibody, and anti-TRPA1 autoantibody may be used as a biomarker for evaluating a cranial nerve disease or a cranial nerve disease susceptibility. A plurality of these biomarkers may be combined and used as a biomarker set.
  • the biomarker or biomarker set according to the present disclosure is useful in testing for the presence or absence of a cranial nerve disease such as schizophrenia or for susceptibility to a cranial nerve disease, and may be used to determine and evaluate a current pathology or a future possibility of developing the disease.
  • the neurological disorders with which the biomarkers are associated include, but are not limited to, schizophrenia, pain disorders, encephalopathy, epilepsy, psychotic disorders, mood disorders, and encephalitis.
  • the biomarkers of the present disclosure may be useful in distinguishing subgroups of neurological disorders such as schizophrenia.
  • the fourth aspect of the present disclosure relates to a method for treating or preventing cranial nerve disease in a subject, which may include, for example, a step of removing, reducing, isolating, inactivating or decomposing a specific autoantibody in a subject.
  • the cranial nerve disease may be a cranial nerve disease that exhibits a psychiatric symptom.
  • the cranial nerve disease may be a cranial nerve disease caused by an autoantibody.
  • the neurological disease for which the treatment or prevention method according to the present disclosure is used may be selected from the group consisting of schizophrenia, pain disorder, encephalopathy, epilepsy, psychotic disorder, mood disorder, and encephalitis, but is not limited thereto.
  • the autoantibody identified by the present inventors damages the central nervous system of the patient by binding to the protein that the antibody recognizes, causing neurological diseases such as schizophrenia, pain disorder, encephalopathy, epilepsy, psychotic disorder, mood disorder, and encephalitis, and that removing, reducing, isolating, inactivating, or decomposing the antibody is believed to lead to the improvement or prevention of the neurological disease.
  • the examples in this specification clearly show that removing the autoantibody identified by the present inventors prevents the induction of symptoms of the neurological disease.
  • the method for treating or preventing a neurological disease according to the present disclosure may include a step of performing plasma exchange.
  • plasma exchange By performing plasma exchange, autoantibodies that cause a neurological disease in a subject can be removed or reduced.
  • Immunological interventions such as plasma exchange and immunoglobulin therapy are used to treat autoantibody encephalitis, and are considered to be effectively used in the method for treating or preventing a neurological disease according to the present disclosure.
  • steroid therapy, administration of immunosuppressants, administration of plasma preparations, and the like, which are used to treat diseases caused by autoantibodies, are also considered to be effectively used in the method for treating or preventing a neurological disease according to the present disclosure.
  • the method for treating or preventing a neurological disease may include a step of removing, reducing, isolating, inactivating, or decomposing at least one autoantibody selected from the group consisting of anti-NCAM1 autoantibody, anti-NRXN1 ⁇ autoantibody, anti-NRG1 autoantibody, anti-NLGN2 autoantibody, anti-NLGN3 autoantibody, anti-Ephrin B1 autoantibody, and anti-TRPA1 autoantibody in a subject.
  • Some embodiments relate to a method for treating blood, plasma, serum, or cerebrospinal fluid isolated from a subject with or susceptible to a cranial nerve disease, comprising contacting the blood, plasma, serum, or cerebrospinal fluid with a substrate having immobilized thereon at least one selected from the group consisting of a specific polypeptide, a fragment of a specific polypeptide having binding activity with a specific autoantibody, or a polypeptide containing an epitope recognized by a specific autoantibody, thereby removing or reducing a specific autoantibody contained in the blood, plasma, serum, or cerebrospinal fluid.
  • the specific polypeptide is selected from the group consisting of an NCAM1 polypeptide, an NRXN1 ⁇ polypeptide, an NRG1 polypeptide, an NLGN2 polypeptide, an NLGN3 polypeptide, an Ephrin B1 polypeptide, and a TRPA1 polypeptide
  • the specific autoantibody may be selected from the group consisting of an anti-NCAM1 autoantibody, an anti-NRXN1 ⁇ autoantibody, an anti-NRG1 autoantibody, an anti-NLGN2 autoantibody, an anti-NLGN3 autoantibody, an anti-Ephrin B1 autoantibody, and an anti-TRPA1 autoantibody.
  • some embodiments relate to a method for treating blood, plasma, serum, or cerebrospinal fluid isolated from a subject with a cranial nerve disease or a susceptibility to a cranial nerve disease, comprising contacting the blood, plasma, serum, or cerebrospinal fluid with a substrate on which at least one selected from the group consisting of an NCAM1 polypeptide, an NCAM1 polypeptide fragment having binding activity with an anti-NCAM1 autoantibody, or a polypeptide containing an epitope recognized by an anti-NCAM1 autoantibody is immobilized, thereby removing or reducing anti-NCAM1 autoantibodies contained in the blood, plasma, serum, or cerebrospinal fluid.
  • Some embodiments also relate to a method for treating blood, plasma, serum or cerebrospinal fluid isolated from a subject having or susceptible to a cranial nerve disease, comprising: treating the blood, plasma, serum or cerebrospinal fluid with an NRXN1 ⁇ polypeptide, an NRG1 polypeptide, an NLGN2 polypeptide, an NLGN3 polypeptide, an Ephrin B1 polypeptide, and/or a TRPA1 polypeptide, an anti-NRXN1 ⁇ autoantibody, an anti-NRG1 autoantibody, an anti-NLGN2 autoantibody, an anti-NLGN3 autoantibody, an anti-Ephrin B1 autoantibody, and/or an anti-TRPA1 autoantibody.
  • the present invention also relates to a method for removing or reducing anti-NRXN1 ⁇ autoantibodies, anti-NRG1 autoantibodies, anti-NLGN2 autoantibodies, anti-NLGN3 autoantibodies, anti-Ephrin B1 autoantibodies, and/or anti-TRPA1 autoantibodies contained in blood, plasma, serum, or cerebrospinal fluid by contacting a substrate with at least one selected from the group consisting of a polypeptide, a fragment of an Ephrin B1 polypeptide, and/or a TRPA1 polypeptide, or a polypeptide containing an epitope recognized by an anti-NRXN1 ⁇ autoantibody, an anti-NRG1 autoantibody, an anti-NLGN2 autoantibody, an anti-NLGN3 autoantibody, an anti-Ephrin B1 autoantibody, and/or an anti-TRPA1 autoantibody, said method comprising the steps of:
  • the sequestration, inactivation, or degradation of a specific autoantibody can be achieved, for example, by allowing a substance that binds to the specific autoantibody to interact with the autoantibody.
  • the sequestration of a specific autoantibody can be achieved, but is not limited to, by targeting a complex of the autoantibody and a substance that binds to the specific autoantibody to a specific organ or tissue.
  • the inactivation of a specific autoantibody can be achieved, but is not limited to, by forming a complex between the autoantibody and a substance that binds to the specific autoantibody, thereby inhibiting the antigen-binding ability of the autoantibody.
  • the destruction of a specific autoantibody can be achieved, but is not limited to, by forming a complex between the autoantibody and a substance that binds to the specific autoantibody, thereby directing the complex to a proteolytic mechanism.
  • Some embodiments relate to a pharmaceutical composition for use in treating or preventing a neurological disease, comprising a specific polypeptide or a fragment thereof having binding activity with a specific autoantibody, or a polypeptide comprising an epitope recognized by a specific autoantibody. Some embodiments also relate to the use of a specific polypeptide or a fragment thereof having binding activity with a specific autoantibody, or a polypeptide comprising an epitope recognized by a specific autoantibody, in the manufacture of a medicament for use in treating or preventing a neurological disease.
  • the specific polypeptide is selected from the group consisting of an NCAM1 polypeptide, an NRXN1 ⁇ polypeptide, an NRG1 polypeptide, an NLGN2 polypeptide, an NLGN3 polypeptide, an Ephrin B1 polypeptide, and a TRPA1 polypeptide
  • the specific autoantibody may be selected from the group consisting of an anti-NCAM1 autoantibody, an anti-NRXN1 ⁇ autoantibody, an anti-NRG1 autoantibody, an anti-NLGN2 autoantibody, an anti-NLGN3 autoantibody, an anti-Ephrin B1 autoantibody, and an anti-TRPA1 autoantibody.
  • some embodiments also relate to a pharmaceutical composition for use in treating or preventing a brain and nerve disease, comprising an NCAM1 polypeptide or a fragment thereof having binding activity with an anti-NCAM1 autoantibody, or a polypeptide comprising an epitope recognized by an anti-NCAM1 autoantibody.
  • Some embodiments also relate to a pharmaceutical composition for use in treating or preventing a neurological disease, comprising an NRXN1 ⁇ polypeptide, an NRG1 polypeptide, an NLGN2 polypeptide, an NLGN3 polypeptide, an Ephrin B1 polypeptide, and/or a TRPA1 polypeptide, or a fragment thereof having binding activity with an anti-NRXN1 ⁇ autoantibody, an anti-NRG1 autoantibody, an anti-NLGN2 autoantibody, an anti-NLGN3 autoantibody, an anti-Ephrin B1 autoantibody, and/or an anti-TRPA1 autoantibody, or a polypeptide comprising an epitope recognized by an anti-NRXN1 ⁇ autoantibody, an anti-NRG1 autoantibody, an anti-NLGN2 autoantibody, an anti-NLGN3 autoantibody, an anti-Ephrin B1 autoantibody, and/or an anti-TRPA1 autoantibody.
  • the neurological disorders for which the pharmaceutical compositions disclosed herein are used include, but are not limited to, schizophrenia, pain disorders, encephalopathy, epilepsy, psychotic disorders, mood disorders, and encephalitis.
  • Some embodiments provide a method for treating or preventing a neurological disease in a subject, comprising: i) providing a sample isolated from a subject; ii) determining the presence or absence of specific autoantibodies in said sample, iii) When the autoantibody is present in the sample, a subject is subjected to at least one of the following procedures selected from the group consisting of 1) to 6): 1) Immunoglobulin therapy, 2) steroid therapy, 3) administration of immunosuppressants; 4) administration of plasma products; 5) Plasma exchange, or 6) Administration of a specific polypeptide, a specific polypeptide fragment having binding activity with a specific autoantibody, or a polypeptide containing an epitope recognized by a specific autoantibody.
  • Immunoglobulin therapy is a treatment in which IgG with Fc activity is administered intravenously, intramuscularly, or subcutaneously, and is used to treat autoimmune diseases (see, for example, Nature Reviews Immunology vol. 13, pp. 176-189 (2013)). Depending on the disease, high-dose immunoglobulin therapy is performed by administering large amounts of the drug.
  • the administered preparation contains polyvalent IgG (immunoglobulin G) extracted from the plasma of more than 1,000 blood donors, and the effect of IVIG lasts for several weeks.
  • Steroid therapy is a treatment used to suppress the body's immune system by administering steroids, which are corticosteroids made by the adrenal glands (located at the top of both kidneys).
  • steroids which are corticosteroids made by the adrenal glands (located at the top of both kidneys).
  • Prednisolone for example, may be used in steroid therapy.
  • Immunosuppressants are drugs used to suppress excessive and abnormal immune responses in the body.
  • Immunosuppressants that can be used include cyclosporine, mizoribine, cyclophosphamide, azathioprine, tacrolimus, and mycophenolate mofetil.
  • Plasma preparations are preparations that use plasma, a liquid that is obtained by centrifuging blood to remove red blood cells, white blood cells, platelets, and other cellular components. Furthermore, products that separate and extract proteins contained in plasma, such as albumin, immunoglobulins, and blood coagulation factors, are also called plasma fraction preparations.
  • Plasma exchange is a treatment in which blood is separated into blood cell and plasma components using a plasma separator, the plasma containing disease-causing substances such as autoantibodies is discarded, and an equal amount of healthy plasma (fresh frozen plasma) is added to replace it.
  • Double filtration plasma exchange (DFPP) is also used, in which after blood cells and plasma are separated using a plasma separation membrane, the plasma is passed through a secondary separation membrane to further remove only substances of a specific size.
  • the specific polypeptide may be selected from the group consisting of an NCAM1 polypeptide, an NRXN1 ⁇ polypeptide, an NRG1 polypeptide, an NLGN2 polypeptide, an NLGN3 polypeptide, an Ephrin B1 polypeptide, and a TRPA1 polypeptide
  • the specific autoantibody may be selected from the group consisting of an anti-NCAM1 autoantibody, an anti-NRXN1 ⁇ autoantibody, an anti-NRG1 autoantibody, an anti-NLGN2 autoantibody, an anti-NLGN3 autoantibody, an anti-Ephrin B1 autoantibody, and an anti-TRPA1 autoantibody.
  • some embodiments provide a method for treating or preventing a neurological disease in a subject, comprising: i) providing a sample isolated from a subject; ii) determining the presence or absence of an autoantibody in the sample, wherein the autoantibody is an anti-NCAM1 antibody; iii) A method for performing at least one of the following steps 1) to 6) on a subject when the autoantibody is present in the sample: 1) Immunoglobulin therapy, 2) steroid therapy, 3) administration of immunosuppressants; 4) administration of plasma products; 5) Plasma exchange, or 6) Administration of an NCAM1 polypeptide, an NCAM1 polypeptide fragment having binding activity with an anti-NCAM1 autoantibody, or a polypeptide containing an epitope recognized by an anti-NCAM1 autoantibody.
  • some embodiments provide a method for treating or preventing a neurological disease in a subject, comprising: i) providing a sample isolated from a subject; ii) determining the presence or absence of autoantibodies in the sample, wherein the autoantibodies are selected from the group consisting of anti-NRXN1 ⁇ autoantibodies, anti-NRG1 autoantibodies, anti-NLGN2 autoantibodies, anti-NLGN3 autoantibodies, anti-Ephrin B1 autoantibodies, and anti-TRPA1 autoantibodies; iii) When the autoantibody is present in the sample, a subject is subjected to at least one of the following procedures selected from the group consisting of 1) to 6): 1) Immunoglobulin therapy, 2) steroid therapy, 3) administration of immunosuppressants; 4) administration of plasma products; 5) Plasma exchange, or 6) Administration of an NRXN1 ⁇ polypeptide, an NRG1 polypeptide, an NLGN2 polypeptide, an NLGN
  • the method for treating or preventing a cranial nerve disease according to the present disclosure further comprises a step of determining the presence or absence of an additional autoantibody in the sample, wherein the additional autoantibody is at least one autoantibody selected from the group consisting of an anti-NMDA receptor antibody, an anti-GABA A R ⁇ 1 antibody, an anti-NCAM1 autoantibody, an anti-NRXN1 ⁇ antibody, an anti-NRG1 antibody, an anti-NLGN2 antibody, an anti-NLGN3 antibody, an anti-Ephrin B1 antibody, and an anti-TRPA1 antibody, and when the autoantibody is present in the sample,
  • the subject may be administered an antigen polypeptide corresponding to the autoantibody whose presence has been confirmed, an antigen polypeptide fragment having binding activity with the autoantibody, or a polypeptide containing an epitope recognized by the autoantibody.
  • the neurological disease to which the above-mentioned method for treating or preventing a neurological disease is applied may be selected from the group consisting of schizophrenia, pain disorder, encephalopathy, epilepsy, psychotic disorder, mood disorder, and encephalitis.
  • Some embodiments provide a pharmaceutical composition for use in treating or preventing a neurological disease in a subject in whom the presence of a specific autoantibody has been confirmed in blood, plasma, serum, or cerebrospinal fluid, comprising: Immunoglobulin preparations, Steroid preparations, Immunosuppressants,
  • the present invention also relates to a plasma preparation, or a pharmaceutical composition comprising a specific polypeptide, a specific polypeptide fragment having binding activity with a specific autoantibody, or a polypeptide containing an epitope recognized by a specific autoantibody.
  • Some embodiments relate to a pharmaceutical for use in treating or preventing a neurological disease in a subject in which the presence of a specific autoantibody has been confirmed in blood, plasma, serum, or cerebrospinal fluid, Immunoglobulin preparations, Steroid preparations, Immunosuppressants,
  • the present invention also relates to the use of plasma preparations, or specific polypeptides, specific polypeptide fragments having binding activity with specific autoantibodies, or polypeptides containing an epitope recognized by a specific autoantibody.
  • the specific polypeptide may be selected from the group consisting of an NCAM1 polypeptide, an NRXN1 ⁇ polypeptide, an NRG1 polypeptide, an NLGN2 polypeptide, an NLGN3 polypeptide, an Ephrin B1 polypeptide, and a TRPA1 polypeptide
  • the specific autoantibody may be selected from the group consisting of an anti-NCAM1 autoantibody, an anti-NRXN1 ⁇ autoantibody, an anti-NRG1 autoantibody, an anti-NLGN2 autoantibody, an anti-NLGN3 autoantibody, an anti-Ephrin B1 autoantibody, and an anti-TRPA1 autoantibody.
  • some embodiments provide a pharmaceutical composition for use in treating or preventing a cranial nerve disease in a subject in whom the presence of an anti-NCAM1 autoantibody has been confirmed in the blood, plasma, serum, or cerebrospinal fluid, comprising: Immunoglobulin preparations, Steroid preparations, Immunosuppressants,
  • the present invention relates to a plasma preparation, or a pharmaceutical composition comprising an NCAM1 polypeptide, an NCAM1 polypeptide fragment having binding activity with an anti-NCAM1 autoantibody, or a polypeptide containing an epitope recognized by an anti-NCAM1 autoantibody.
  • the present invention relates to a pharmaceutical composition for use in treating or preventing a cranial nerve disease in a subject in whom the presence of anti-NRXN1 ⁇ autoantibody, anti-NRG1 autoantibody, anti-NLGN2 autoantibody, anti-NLGN3 autoantibody, anti-Ephrin B1 autoantibody, and/or anti-TRPA1 autoantibody has been confirmed in blood, plasma, serum, or cerebrospinal fluid, comprising: Immunoglobulin preparations, Steroid preparations, Immunosuppressants,
  • the present invention relates to a plasma preparation, or a pharmaceutical composition comprising a fragment of an NRXN1 ⁇ polypeptide, an NRG1 polypeptide, an NLGN2 polypeptide, an NLGN3 polypeptide, an Ephrin B1 polypeptide, and/or a TRPA1 polypeptide having binding activity with an anti-NRXN1 ⁇ autoantibody, an anti-NRG1 autoantibody, an anti-NLGN2 auto
  • the neurological disease to which the pharmaceutical composition is applied may be selected from the group consisting of schizophrenia, pain disorder, encephalopathy, epilepsy, psychotic disorder, mood disorder, and encephalitis, but is not limited thereto.
  • the pharmaceutical composition according to the present disclosure may take any form, such as a tablet, powder, liquid, or semisolid, but is preferably a liquid.
  • the pharmaceutical composition according to the present disclosure may be prepared by blending various main ingredients with a base.
  • the pharmaceutical composition according to the present disclosure may contain pharma- ceutical acceptable excipients, additives, buffers, salts for adjusting isotonicity, antioxidants, preservatives, drug stabilizers, and the like.
  • excipients include, but are not limited to, water, purified water, alcohol, glycerin, lactose, starch, dextrin, sucrose, precipitated silica, honey, rice starch, and tragacanth.
  • the pharmaceutical composition according to the present disclosure may also contain other active ingredients.
  • the amount of each ingredient to be blended may be appropriately determined within a medicament-acceptable range.
  • the dosage of the composition may also be appropriately determined depending on the type of drug used and the subject to which it is administered.
  • the active ingredient may be 0.01 to 15% by weight, for example, 0.1 to 5% by weight.
  • the route of administration can also be appropriately determined depending on the type of drug used and the subject to which it is administered.
  • the method of administration of the pharmaceutical composition according to the present disclosure is not particularly limited, but suitable examples include intravascular administration (preferably intravenous administration), intrathecal administration, intraperitoneal administration, intraintestinal administration, subcutaneous administration, intradermal administration, intramuscular administration, and eye drops, with intravenous administration being a more suitable example.
  • the dosage can be determined appropriately depending on the type of drug used and the recipient.
  • the route of administration can also be determined appropriately depending on the type of drug used and the recipient.
  • Preferred routes of administration include subcutaneous injection of liquid, intravenous injection, intrathecal injection, eye drops, solid preparations, and oral administration of liquid preparations.
  • Example 1 Identification of anti-NCAM1 autoantibodies in patients with schizophrenia Serum samples were obtained from 201 healthy controls (125 men, 76 women, age 22-90 years, median 48 years) and 223 patients with schizophrenia (112 men, 111 women, age 16-84 years, median 52 years). Schizophrenia was diagnosed according to the Diagnostic and Statistical Manual of Mental Disorders-5 (DSM-5). There were no significant differences between groups with regard to age. All samples were tested by ELISA and cell-based assays. ELISA analysis detected anti-NCAM1 autoantibody positivity in 15 patients with schizophrenia. Autoantibody positivity was defined as >2 standard deviations from the mean absorbance (Fig. 1A).
  • ELISA for anti-NCAM1 autoantibodies was performed as follows. First, polystyrene microtiter plates were coated with 100 mL of NCAM1 recombinant protein (2 mg/mL) dissolved in TBS buffer and incubated overnight at 4°C. The plates were washed three times with TBS, and TBS containing 1% BSA (100 mL/well) was added to block non-specific binding, and incubated at 24°C for 1 h. Then, 100 mL of each dilution of serum and CSF samples (1:50 for serum and 1:1 for CSF in TBS containing 1% BSA) was added and incubated at 24°C for 1 h.
  • anti-human IgG alkaline phosphatase (1:50000; purchased from Sigma-Aldrich) was incubated in TBS containing 0.1% Tween 20 for 1 h at room temperature. After washing with TBS, 1 mg/mL p-nitrophenyl phosphate in substrate buffer was added to each well and absorbance was read at 405 nm using a microplate reader.
  • Cell-based assays were performed as follows: HeLa cells and primary cortical neurons were fixed in 2% paraformaldehyde (prepared in phosphate buffer) for 30 min at room temperature, treated with 0.1% Triton X-100 in PBS for 10 min, blocked with 10% FBS or 1% BSA in PBS for 30 min at room temperature, and incubated with serum or primary antibody diluted in blocking buffer.
  • sera with autoantibody titers ⁇ 1:30 were defined as positive for autoantibodies.
  • Previous studies have shown that titers of NMDA receptor and GABA receptor autoantibodies are usually higher than 1:30.
  • diluting serum has the advantage of preventing nonspecific staining.
  • NCAM1 is highly expressed in the nervous system. Western blot analysis was performed to confirm the expression of NCAM1 in mouse brain, and indeed, it was expressed at a much higher level than in peripheral organs.
  • NCAM1 is a synaptic cell adhesion molecule with a transmembrane domain. The extracellular domain of NCAM1 is cleaved by ADAM10 and ADAM17, and a small amount of its soluble form is found in serum. Since changes in soluble NCAM1 have been reported in schizophrenia patients, the inventors examined whether autoantibodies against NCAM1 affect soluble NCAM1 in serum. ELISA was performed to analyze soluble NCAM1 in serum, and it was found that soluble NCAM1 was significantly reduced in schizophrenia patients (Figure 1D).
  • Example 2 The main epitope recognized by anti-NCAM1 antibodies in schizophrenia is located within the Ig1 domain.
  • Fig. 2A The extracellular region of NCAM1 consists of five N-terminal immunoglobulin domains (Ig1-Ig5) and two fibronectin type III domains (FN3).
  • Sera from 12 schizophrenia patients reacted with the truncated forms ⁇ Ig2 and ⁇ Ig2-5, which lack the Ig2 and Ig2-5 domains, respectively, but not with the truncated forms ⁇ Ig1 and ⁇ Ig1-5, which lack the Ig1 and Ig1-5 domains, respectively (Fig. 2B, 2C).
  • Fig. 2D Western blotting
  • Polysialylated NCAM1 (PSA-NCAM) is abundant in the developing nervous system and is associated with cell migration and axonal elongation. Polysialylation occurs on the Ig5 domain of NCAM1. Therefore, it was hypothesized that anti-NCAM1 autoantibodies also detect PSA-NCAM.
  • Western blot analysis using the cerebral cortex of postnatal day 0 mice revealed that anti-NCAM1 autoantibodies detect both NCAM1 and PSA-NCAM. There are approximately 500 molecules that contain Ig domains. Therefore, we analyzed whether anti-NCAM1 autoantibodies cross-react with other molecules that contain Ig domains, such as NCAM2, L1CAM, and TAG1. It was found that the anti-NCAM1 autoantibodies of 12 patients identified by cell-based assay did not react with any of these molecules. These results suggest that anti-NCAM1 autoantibodies react with NCAM1-specific sequences.
  • Example 3 Anti-NCAM1 autoantibodies inhibit NCAM1-NCAM1 and NCAM1-GDNF interactions
  • NCAM1 forms synapses by homophilic binding via immunoglobulin domains including the Ig1 domain. Furthermore, GDNF promotes spine development by binding to NCAM1. Therefore, it was hypothesized that anti-NCAM1 autoantibodies inhibit NCAM1-NCAM1 and NCAM1-GDNF interactions. Pull-down assays showed that IgG purified from schizophrenia patient 1 inhibited NCAM1-NCAM1 and NCAM1-GDNF interactions, whereas IgG purified from a healthy control did not ( Figures 3A and 3B). Pull-down assays also showed that IgG purified from schizophrenia patients 2 and 3 inhibited NCAM1-NCAM1 and NCAM1-GDNF interactions.
  • Example 4 Anti-NCAM1 autoantibodies from schizophrenia patients inhibit the NCAM1-Fyn-FAK-MEK1-ERK1 pathway in miceIf the anti-NCAM1 antibodies found in schizophrenia patients inhibit the NCAM1-GCAM1 and NCAM1-GDNF interactions, it was assumed that they would also cause abnormal molecular signaling, abnormal formation of spines and synapses, and schizophrenia-related behavior in mice.
  • IgG was purified from a schizophrenia patient (patient 1) and an age- and sex-matched healthy individual, and injected into the CSF of mice (8 weeks old). Molecular signaling, spine and synapse formation, and behavior of 9-week-old mice were then analyzed (Figure 4A).
  • Example 5 Anti-NCAM1 autoantibodies from schizophrenia patients reduce the number of spines and synapses in the frontal cortex of mice Anti-NCAM1 autoantibodies inhibited phosphorylation of FAK, ERK1, and MEK1, disrupting pre- and post-synaptic trans-homophilic NCAM1 interactions, a process that maintains synapses, suggesting that they lead to changes in spines and synapses.
  • AAV1-EGFP adeno-associated virus 1
  • AAV1-SYN-EGFP adeno-associated virus 1
  • Example 6 Anti-NCAM1 autoantibodies from schizophrenia patients induce schizophrenia-related behaviors in mice To verify whether anti-NCAM1 autoantibodies induce schizophrenia symptoms, we performed behavioral analysis of autoantibody-treated mice. Administration of purified IgG from schizophrenia patient 1 impaired cognitive function in the Y-maze test (Fig. 4G). Furthermore, mice administered IgG from schizophrenia patients exhibited a deficit in prepulse inhibition, an established endophenotype of schizophrenia (Fig. 4H). Mice administered IgG from healthy controls had normal prepulse inhibition. Mice administered IgG from schizophrenia patient 1 showed no abnormalities in locomotor activity, anxiety behavior, or social interaction, but showed abnormalities in the open field test, elevated plus maze test, and three-chamber test, respectively.
  • Example 7 Adsorption and removal of anti-NCAM1 antibody improved molecular, spinal cord, and behavioral changes
  • anti-NCAM1 antibodies inhibit phosphorylation, reduce the number of spines and synapses, and induce schizophrenia-related behaviors in IgG purified from schizophrenia patient 1
  • Adsorption and removal of anti-NCAM1 antibodies by glutathione S-transferase (GST) pull-down were confirmed by cell-based assays and immunohistochemistry. Phosphorylation, spines and synapses, and behaviors were improved after the adsorption experiment ( Figures 4B–4H).
  • Example 8 Anti-NCAM1 autoantibodies derived from schizophrenia patients were found to induce schizophrenia-related behaviors and synaptic changes in mice. To confirm that the results of schizophrenia patient 1 could be observed in other patients with positive anti-NCAM1 autoantibodies, we performed two-photon and behavioral analyses using purified IgG from schizophrenia patients 2 and 3 (Fig. 5A). IgG from these patients also reduced spine and synapse numbers in the frontal cortex and induced cognitive impairment and deficits in prepulse inhibition (Fig. 5B–5E).
  • Example 9 Identification of anti-NRXN1 ⁇ autoantibodies in patients with schizophrenia. Serum samples were collected from 362 healthy control subjects (181 men, 181 women, age 22-90 years, median 49 years) and 387 patients with schizophrenia (195 men, 192 women, age 16-84 years, median 51 years). Schizophrenia was diagnosed according to the Diagnostic and Statistical Manual of Mental Disorders (DSM5). There were no significant differences between the groups with regard to age. All samples were tested by enzyme-linked immunosorbent assay (ELISA) and cell-based assay.
  • ELISA enzyme-linked immunosorbent assay
  • Anti-NRXN1 ⁇ autoantibodies were also detected in the cerebrospinal fluid (CSF) of anti-NRXN1 ⁇ autoantibody-positive schizophrenic patients (Fig. 6B). The protein concentrations and white blood cell counts in the CSF of these patients were normal.
  • NRXN1 ⁇ truncated forms of NRXN1 ⁇ (Fig. 6D).
  • the extracellular domain of NRXN1 ⁇ is composed of six laminin, neurexin, and sex hormone binding protein (LNS) domains.
  • LNS sex hormone binding protein
  • Example 10 Anti-NRXN1 ⁇ autoantibodies inhibit NRXN1 ⁇ -NLGN1 and NRXN1 ⁇ -NLGN2 interactions NRXN1 ⁇ is highly expressed in the nervous system. Western blot analysis was performed to confirm the expression of NRXN1 ⁇ in mouse brains, and it was found that NRXN1 ⁇ was indeed expressed at a much higher level than in peripheral organs.
  • NRXN1 ⁇ is a presynaptic cell adhesion molecule that interacts with NLGN (postsynaptic cell adhesion molecule) via the LNS6 domain to form synapses.
  • NRXN1 ⁇ functions as a platform and hub for synaptic molecular interactions and signals.
  • Example 11 Decreased mEPSC frequency by anti-NRXN1 ⁇ autoantibodies NRXN1 ⁇ knockout mice had reduced miniature excitatory postsynaptic current (mEPSC) frequency. If anti-NRXN1 ⁇ autoantibodies found in schizophrenia patients inhibit NRXN1 ⁇ -NLGN1 and NRXN1 ⁇ -NLGN2 interactions, we speculated that the electrophysiological properties of synapses would be altered in mice. To test this, we performed electrophysiological analysis of mEPSCs in the frontal cortex. The results showed that the frequency of mEPSCs was significantly reduced in mice treated with anti-NRXN1 ⁇ antibodies ( Figures 8A, 8B). However, the amplitude of mEPSCs was unchanged ( Figures 8A, 8C). These data are consistent with previous findings in knockout mice and confirm that anti-NRXN1 ⁇ autoantibodies alter electrophysiological synaptic properties.
  • mEPSC miniature excitatory postsynaptic current
  • Example 12 Decreased synapses and spines by anti-NRXN1 ⁇ autoantibodies The interaction between NRXN1 ⁇ and NLGN is necessary for synapse formation and maintenance. These previous findings indicate that anti-NRXN1 ⁇ autoantibodies may induce changes in spines and synapses.
  • Example 14 Anti-NRXN1 ⁇ autoantibodies induce schizophrenia-related behaviors and synaptic changes in miceTo confirm that the results from schizophrenia patient 1 could be observed in other patients who tested positive for anti-NRXN1 ⁇ autoantibodies, we performed two-photon and behavioral analyses using purified IgG from schizophrenia patients 2 and 3 (Figure 10A). IgG from these patients also reduced the number of spines and synapses in the frontal cortex of mice, as well as induced cognitive impairment, deficits in prepulse inhibition, and impaired social novelty preference ( Figures 10B-10E).
  • Example 15 Other autoantibodies identified in schizophrenia patients
  • the present inventors also identified autoantibodies against NLGN2, NRG1, NLGN3, and Ephrin B1 as biomarkers for schizophrenia.
  • Figure 11 shows the results of cell-based assays to determine the presence of autoantibodies against NRXN1 ⁇ , NLGN2, NRG1, NLGN3, and Ephrin B1 in samples obtained from schizophrenia patients.
  • Anti-NRXN1 ⁇ autoantibodies were positive in 7 of 380 schizophrenia patients and none of 250 healthy controls.
  • Anti-NLGN2 autoantibodies were positive in 7 of 120 schizophrenia patients and 2 of 201 healthy controls.
  • Anti-NRG1 autoantibodies were positive in 9 of 223 schizophrenia patients and 2 of 201 healthy controls.
  • Anti-NLGN3 autoantibodies were positive in 2 of 122 schizophrenia patients and none of 250 healthy controls.
  • Anti-Ephrin B1 autoantibodies were positive in 2 of 122 schizophrenia patients and none of 250 healthy controls. These autoantibodies can also be used in the testing method, treatment, or prevention method of the present disclosure.
  • Example 16 Other autoantibodies identified in patients with pain disorders
  • the present inventors have also identified autoantibodies against TRPA1 as a biomarker for pain disorders.
  • the present inventors have collected and analyzed the serum of more than 280 patients with pain disorders.
  • Figure 12 shows the results of a cell-based assay to determine the presence of autoantibodies against TRPA1 in samples obtained from patients with pain disorders. The results show that this autoantibody can also be utilized in the diagnostic, testing, therapeutic or preventive methods of the present disclosure.
  • Example 17 Cases where autoantibody-induced encephalopathy was identified from cases misdiagnosed as schizophrenia, and patients positive for novel autoantibodies were treated with plasma exchange. Retrospectively, we verified whether the novel autoantibody of the present invention was the cause of encephalitis that had already been treated for which the cause was unknown. Among nine cases of encephalitis with unknown causes over the past 10 years, one case positive for anti-NRG1 autoantibody and one case positive for anti-NLGN2 autoantibody were found. The patient who tested positive for anti-NLGN2 autoantibodies had been admitted to a psychiatric hospital with schizophrenia after acute psychosis, and was diagnosed as resistant to antipsychotic drugs.
  • the testing method disclosed herein enables highly accurate identification of neurological diseases such as schizophrenia.
  • the testing method disclosed herein is useful as a means for determining whether or not a neurological disease such as schizophrenia has developed. It is also useful as a means for understanding the possibility of developing a neurological disease in the future.
  • This disclosure also provides methods for preventing and treating neurological diseases, as well as pharmaceutical compositions used therein. It is expected that early detection using the testing method disclosed herein and early treatment using the treatment method disclosed herein will prevent neurological diseases from becoming difficult to treat, becoming severe (progression of the disease), or recurring.

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JP2022540913A (ja) * 2019-07-16 2022-09-20 ドイチェス ツェントラム フューア ノイロデジェネラティヴ エアクランクンゲン エー． ファウ．（ディーゼットエヌイー） Ｎｍｄａｒ自己抗体を検出及び単離するためのｎｍｄａ受容体コンストラクト

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JP2022540913A (ja) * 2019-07-16 2022-09-20 ドイチェス ツェントラム フューア ノイロデジェネラティヴ エアクランクンゲン エー． ファウ．（ディーゼットエヌイー） Ｎｍｄａｒ自己抗体を検出及び単離するためのｎｍｄａ受容体コンストラクト

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