Drebrin-derived imadp, method for measuring imadp, and kit for measuring imadp

Abstract

The present invention addresses the problem of providing: a biomarker for determining cognitive impairment or nerve function disorder in an early stage with high accuracy; and others.　In the present invention, a drebrin metabolite peptide (iMADP) selected from the below-mentioned peptides (i) to (iii) is used as a biomarker for determining cognitive impairment or nerve function disorder. (i) A peptide comprising the amino acid sequence represented by SEQ ID NO: 4; (ii) a peptide comprising the amino acid sequence represented by SEQ ID NO: 5; and (iii) a peptide which comprises an amino acid sequence having a structure such that one or more amino acid residues are deleted, substituted, and / or added in the amino acid sequence represented by SEQ ID NO: 4 or SEQ ID NO: 5.

Description

Drebrin-derived iMADP, method for measuring iMADP, and kit for measuring iMADP

[0001] The present invention relates to iMADP derived from drebrin, a method for measuring iMADP, a kit for measuring iMADP, etc. The present invention can be used in a method for determining cognitive dysfunction and / or neurological dysfunction in a subject at an early stage with high accuracy.

[0002] Alzheimer's disease, the primary cause of dementia, is an irreversible, progressive brain disorder believed to result from the dysfunction of synapses in the brain. The increasing number of Alzheimer's disease patients has become a major social concern in recent years, spurring the development of early diagnostic methods and therapeutic drugs. Senile plaques, known to be aggregates of amyloid beta protein (amyloid plaques), are observed in postmortem brains of Alzheimer's disease patients. Amyloid beta protein deposition is the earliest pathologically identifiable lesion, and it has been reported that amyloid beta protein aggregates and directly exhibits neuronal toxicity. Genetic analysis of familial Alzheimer's disease patients has now widely accepted that abnormalities in the production and accumulation of amyloid beta protein are widely associated with the onset of Alzheimer's disease. This is known as the amyloid cascade hypothesis. Numerous studies have established that amyloid beta protein is the primary cause of Alzheimer's disease. Furthermore, it has been shown that amyloid beta protein accumulation in the brain begins more than 20 years before the onset of the disease.

[0003] Synaptic dysfunction observed in cognitive impairments such as Alzheimer's disease can be caused by reduced cerebral blood flow (depression, etc.), cerebral hemorrhage, cerebral infarction, drug addiction, etc. Furthermore, in addition to Alzheimer's disease-type dementia, mitochondrial genetic disorders and dementia caused by diabetes are also known. Alzheimer's disease can only be diagnosed by a postmortem autopsy, but diagnosis during life is performed using questionnaire tests and imaging tests. In this case, a diagnosis is made using questionnaire tests or other methods after the patient experiences subjective symptoms such as severe memory loss, but an objective diagnosis cannot be made. Furthermore, cognitive function assessment methods such as the Mini-Mental State Examination (MMSE) and Clinical Dementia Rating Scale (CDR) have difficulty distinguishing between pre-disease and mild cognitive impairment (MCI), making it difficult to make early diagnoses and to start treatment early.

[0004] Drebrin is known as an actin-binding protein, and its isoforms mainly include drebrin E and drebrin A. Drebrin E is expressed in neurons and some non-neuronal cells during fetal and juvenile stages, while drebrin A is expressed specifically in neurons during maturation. Drebrin E affects process extension and axon growth, while drebrin A accumulates at postsynaptic sites and promotes the formation of dendritic spines. Drebrin A is also known to be involved in synaptic plasticity (Non-Patent Documents 1, 2, 3).

[0005] The present inventors discovered an antibody that specifically binds to drebrin, and further developed a method for detecting drebrin using the antibody as a biomarker (Patent Documents 1 and 2). Furthermore, the present inventors previously developed a method for assessing mild cognitive impairment by measuring the amounts of drebrin A and drebrin A degradation products using an antibody that recognizes Ins2, an amino acid sequence specific to drebrin A (Patent Document 3). However, there is a demand for a method that can assess cognitive impairment in subjects earlier and with higher accuracy.

[0006] International Publication No. 2019 / 216345 Pamphlet Japanese Patent Application Laid-Open No. 2022-1838 International Publication No. 2024 / 122459 Pamphlet

[0007] Sekino, Y., Shirao, T., et. al. The Journal of comparative neurology, 483(4), 383-402, 2005Sekino, Y., Shirao, T., et. al. PloS one, 9(1), e85367, 2014Shirao, T., et. al. Journal of neurochemistry, 109(2), 611-22, 2009

[0008] An object of the present invention is to provide a biomarker or the like for early and highly accurate determination of cognitive dysfunction or neurological dysfunction.

[0009] The present inventors have discovered that among drebrin metabolites present in the cerebrospinal fluid and / or blood of cognitively normal individuals, there is a special drebrin metabolite that is not present or present in small amounts in the cerebrospinal fluid and / or blood of patients with mild cognitive impairment due to Alzheimer's disease (ADMCI) or Alzheimer's disease dementia (ADD), and have identified the amino acid sequence of the isolated drebrin metabolite, thereby arriving at the present invention. This discovery indicates that there are drebrin metabolites containing sites where cleavage and modification are enhanced during cognitive impairment, and the present inventors have named such drebrin metabolites iMADP (intact memory-associated drebrin peptide). While iMADP can be found in biological samples from individuals with normal cognitive function, the amount of iMADP is reduced in the cerebrospinal fluid and blood of patients with mild cognitive impairment due to Alzheimer's disease (ADMCI) and Alzheimer's disease dementia (ADD). On the other hand, the present inventors have found that the amount of iMADP is increased in the blood of patients with neurological disorders such as amyotrophic lateral sclerosis (ALS), immune-mediated peripheral neuropathy (IMN), and multiple sclerosis (MS). This is because in patients with cognitive dysfunction, drebrin metabolism becomes abnormal, causing iMADP to be broken down into smaller fragments and released into the blood, resulting in a decrease in the amount of iMADP. On the other hand, in patients with neurological dysfunction, immune abnormalities cause inflammation, leading to a large amount of iMADP being released into the blood from damaged neurons, which is thought to result in an increase in the amount of iMADP in the blood. The present invention relates to iMADP, a drebrin metabolite that specifically disappears / decreases with the onset of mild cognitive impairment and specifically increases with the onset of neurological dysfunction, as well as a method for measuring iMADP and a kit for measuring iMADP. Measuring iMADP in a biological sample can provide information related to cognitive dysfunction and / or neurological dysfunction, and can be used in a method for determining cognitive dysfunction and / or neurological dysfunction early and with high accuracy.

[0010] Accordingly, the present invention is as follows: (1) A drebrin metabolite peptide (iMADP) selected from the following (i) to (iii): (i) a peptide comprising the amino acid sequence of SEQ ID NO: 4, (ii) a peptide comprising the amino acid sequence of SEQ ID NO: 5, or (iii) a peptide comprising an amino acid sequence in which one or more amino acid residues in the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 5 have been deleted, substituted, and / or added (hereinafter sometimes referred to as "the iMADP of the present invention"). (2) A method for measuring the amount of a drebrin metabolite peptide (iMADP) in a biological sample, wherein the iMADP is (i) a peptide comprising the amino acid sequence of SEQ ID NO: 4, (ii) a peptide comprising the amino acid sequence of SEQ ID NO: 5, or (iii) a peptide comprising an amino acid sequence in which one or more amino acid residues in the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 5 have been deleted, substituted, and / or added (hereinafter sometimes referred to as "the measurement method of the present invention"). (3) The method according to (2) above, wherein the amount of iMADP in a biological sample is measured by an immunological technique using an antibody that binds to the N-terminal region and an antibody that binds to the C-terminal region of iMADP.(4) The antibody that binds to the N-terminal region of iMADP is an antibody that binds to the N-terminal region of the amino acid sequence of SEQ ID NO: 1 from the 365th amino acid residue; or an antibody that binds to the N-terminal region of the amino acid sequence of SEQ ID NO: 1 from the 365th amino acid residue; or an antibody that binds to the N-terminal region of the amino acid sequence of SEQ ID NO: 3 from the 319th amino acid residue; or an antibody that binds to the N-terminal region of the amino acid sequence of SEQ ID NO: 3 from the 319th amino acid residue; or an antibody that binds to the N-terminal region of the amino acid sequence of SEQ ID NO: 3 from the 319th amino acid residue; (5) The method according to (4), wherein the N-antibody is an antibody that binds to the C-terminal region of the amino acid sequence of SEQ ID NO: 1 from the 320th amino acid residue onwards; or an antibody that binds to the C-terminal region of the amino acid sequence of SEQ ID NO: 1 from the 320th amino acid residue onwards, or an antibody that binds to the C-terminal region of the amino acid sequence of SEQ ID NO: 3 from the 320th amino acid residue onwards, or an antibody that binds to the C-terminal region of the amino acid sequence of SEQ ID NO: 3 from the 320th amino acid residue onwards, or an antibody that binds to the C-terminal region of the amino acid sequence of SEQ ID NO: 3 from the 320th amino acid residue onwards, or an antibody that binds to the C-terminal region of the amino acid sequence of SEQ ID NO: 3 from the 320th amino acid residue onwards, or(6) The method according to (5) above, wherein the N-antibody is an antibody that binds to the amino acid sequence of SEQ ID NO: 2, and the C-antibody is an antibody that binds to the amino acid sequence of SEQ ID NO: 7, or the N-antibody is an antibody that binds to the amino acid sequence of SEQ ID NO: 6 or 8, and the C-antibody is an antibody that binds to the amino acid sequence of SEQ ID NO: 2, or the N-antibody is an antibody that binds to the amino acid sequence of SEQ ID NO: 6 or 8, and the C-antibody is an antibody that binds to the amino acid sequence of SEQ ID NO: 9, or the N-antibody is an antibody that binds to the amino acid sequence of SEQ ID NO: 6 or 8, and the C-antibody is an antibody that binds to the amino acid sequence of SEQ ID NO: 10, or the N-antibody is an antibody that binds to the amino acid sequence of SEQ ID NO: 6 or 8, and the C-antibody is an antibody that binds to the amino acid sequence of SEQ ID NO: 7. (7) The method according to any of (2) to (6) above, wherein iMADP is used as a biomarker to determine the presence or absence of cognitive dysfunction or neurological dysfunction, or the risk thereof. (8) The method according to (7) above, wherein the cognitive dysfunction is mild cognitive impairment or Alzheimer's disease dementia, and the neurological dysfunction is amyotrophic lateral sclerosis, immune-mediated peripheral neuropathy, or multiple sclerosis.(9) For use in the method according to any one of (2) to (8) above, an antibody that binds to the N-terminal region of the amino acid sequence of SEQ ID NO: 1 from the 365th amino acid residue; or an antibody that binds to the N-terminal region of the amino acid sequence of SEQ ID NO: 1 from the 365th amino acid residue; or an antibody that binds to the N-terminal region of the amino acid sequence of SEQ ID NO: 3 from the 319th amino acid residue; or an antibody that binds to the N-terminal region of the amino acid sequence of SEQ ID NO: 3 from the 319th amino acid residue; for use in the method according to any one of (2) to (8) above, (10) A combined antibody with (C-antibody) (sometimes referred to herein as the "combined antibody") an antibody that binds to the C-terminal region of the amino acid sequence of SEQ ID NO: 1 from the 320th amino acid residue onwards; or an antibody that binds to the C-terminal region of the amino acid sequence of SEQ ID NO: 1 from the 320th amino acid residue onwards, in which one or more amino acid residues have been deleted, substituted, and / or added; or an antibody that binds to the C-terminal region of the amino acid sequence of SEQ ID NO: 3 from the 320th amino acid residue onwards ..., in which one or more amino acid residues have been deleted, substituted, and / or added (C-antibody). (11) A combined antibody according to (9), wherein the N-antibody is an antibody that binds to the amino acid sequence of SEQ ID NO: 2, 6, or 8, and the C-antibody is an antibody that binds to the amino acid sequence of SEQ ID NO: 2, 7, 9, or 10.(11) The combination antibody according to (10) above, wherein the N-antibody is an antibody that binds to the amino acid sequence of SEQ ID NO: 2 and the C-antibody is an antibody that binds to the amino acid sequence of SEQ ID NO: 7, the N-antibody is an antibody that binds to the amino acid sequence of SEQ ID NO: 6 or 8 and the C-antibody is an antibody that binds to the amino acid sequence of SEQ ID NO: 2, the N-antibody is an antibody that binds to the amino acid sequence of SEQ ID NO: 6 or 8 and the C-antibody is an antibody that binds to the amino acid sequence of SEQ ID NO: 9, the N-antibody is an antibody that binds to the amino acid sequence of SEQ ID NO: 6 or 8 and the C-antibody is an antibody that binds to the amino acid sequence of SEQ ID NO: 10, or the N-antibody is an antibody that binds to the amino acid sequence of SEQ ID NO: 6 or 8 and the C-antibody is an antibody that binds to the amino acid sequence of SEQ ID NO: 7. (12) A kit for measuring drebrin metabolite peptide (iMADP), comprising one or more antibodies that specifically bind to iMADP, wherein the iMADP is: (i) a peptide comprising the amino acid sequence of SEQ ID NO: 4; (ii) a peptide comprising the amino acid sequence of SEQ ID NO: 5; or (iii) a peptide comprising an amino acid sequence in which one or more amino acid residues in the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 5 have been deleted, substituted, and / or added (sometimes referred to as the "kit" in this specification).(13) The antibody is an antibody that binds to the N-terminal region of the amino acid sequence of SEQ ID NO: 1 from the 365th amino acid residue; or an antibody that binds to the N-terminal region of the amino acid sequence of SEQ ID NO: 1 from the 365th amino acid residue; or an antibody that binds to the N-terminal region of the amino acid sequence of SEQ ID NO: 3 from the 319th amino acid residue; or an antibody that binds to the N-terminal region of the amino acid sequence of SEQ ID NO: 3 from the 319th amino acid residue; or an antibody that binds to the N-terminal region of the amino acid sequence of SEQ ID NO: 3 from the 319th amino acid residue; (12) The kit according to (12), comprising: an antibody that binds to the C-terminal region of the amino acid sequence of SEQ ID NO: 1 from the 320th amino acid residue; or an antibody that binds to the C-terminal region of the amino acid sequence of SEQ ID NO: 1 from the 320th amino acid residue; or an antibody that binds to the C-terminal region of the amino acid sequence of SEQ ID NO: 3 from the 320th amino acid residue; or an antibody that binds to the C-terminal region of the amino acid sequence of SEQ ID NO: 3 from the 320th amino acid residue; (14) The kit according to (13), wherein the N-antibody is an antibody that binds to the amino acid sequence of SEQ ID NO: 2, 6, or 8, and the C-antibody is an antibody that binds to the amino acid sequence of SEQ ID NO: 2, 7, 9, or 10. (15) The kit according to (14) above, wherein the N-antibody is an antibody that binds to the amino acid sequence of SEQ ID NO: 2 and the C-antibody is an antibody that binds to the amino acid sequence of SEQ ID NO: 7; the N-antibody is an antibody that binds to the amino acid sequence of SEQ ID NO: 6 or 8 and the C-antibody is an antibody that binds to the amino acid sequence of SEQ ID NO: 2; the N-antibody is an antibody that binds to the amino acid sequence of SEQ ID NO: 6 or 8 and the C-antibody is an antibody that binds to the amino acid sequence of SEQ ID NO: 9; the N-antibody is an antibody that binds to the amino acid sequence of SEQ ID NO: 6 or 8 and the C-antibody is an antibody that binds to the amino acid sequence of SEQ ID NO: 10; or the N-antibody is an antibody that binds to the amino acid sequence of SEQ ID NO: 6 or 8 and the C-antibody is an antibody that binds to the amino acid sequence of SEQ ID NO: 7.(16) The kit according to any one of (12) to (15) above, for determining the use or effectiveness of a therapeutic drug for cognitive dysfunction or neurological dysfunction. (17) The kit according to (16) above, wherein the therapeutic drug for cognitive dysfunction is lecanemab or donanemab. (18) The kit according to (16) or (17) above, wherein the cognitive dysfunction is mild cognitive impairment or Alzheimer's disease dementia, and the neurological dysfunction is amyotrophic lateral sclerosis, immune-mediated peripheral neuropathy, or multiple sclerosis. (19) A control serum, control plasma, or control substance for measurement accuracy control, containing the iMADP according to (1) above (sometimes referred to herein as the "control serum, etc."). (20) A nucleotide encoding the iMADP according to (1) above (sometimes referred to herein as the "iMADP nucleotide"). (21) A vector comprising a promoter and the nucleotide described in (20) above operably linked downstream of the promoter (sometimes referred to as the "vector of the present invention" in this specification).

[0011] Other embodiments of the present invention include: a method for diagnosing cognitive dysfunction or neurological dysfunction, comprising the step (a) of measuring the amount of the present iMADP in a biological sample collected from a subject; and the step (b) of using the amount of the present iMADP measured in the step (a) as an index to determine whether the subject is suffering from cognitive dysfunction or neurological dysfunction or whether the subject is at high risk of suffering from cognitive dysfunction or neurological dysfunction (in other words, a method for assisting a doctor in diagnosing the presence or absence of cognitive dysfunction or neurological dysfunction, but not involving the doctor's diagnostic act); a method for creating data for diagnosing cognitive dysfunction or neurological dysfunction, comprising the step (a) and the step (B) of creating data for determining whether the subject is suffering from cognitive dysfunction or neurological dysfunction or whether the subject is at high risk of suffering from cognitive dysfunction or neurological dysfunction, sequentially comprising the step (a) and the step (B) of using the amount of the present iMADP measured in the step (a) as an index; a method for diagnosing cognitive dysfunction or neurological dysfunction, comprising the steps (a) and (b); Examples of the methods include a method for treating cognitive dysfunction or neurological dysfunction or a method for preventing the worsening of cognitive dysfunction or neurological dysfunction, which comprises the above steps (a) and (b) and includes a step of administering a treatment for treating cognitive dysfunction or neurological dysfunction or a treatment for preventing the worsening of cognitive dysfunction or neurological dysfunction (e.g., drug therapy using a therapeutic agent for cognitive dysfunction or neurological dysfunction, surgical treatment, immunotherapy, etc.) to a subject determined in step (b) to be highly likely to have cognitive dysfunction or neurological dysfunction or to be at high risk of having cognitive dysfunction or neurological dysfunction; and a kit for diagnosing cognitive dysfunction or neurological dysfunction, which comprises one or more antibodies that specifically bind to the iMADP of the present invention.

[0012] By utilizing the present invention, the diagnosis of cognitive dysfunction and / or neurological dysfunction, which has previously relied on imaging tests and the like, can be performed early, simply, and with high accuracy, which can lead to the prevention of the onset of these diseases, early treatment, assessment of treatment efficacy, prediction of recurrence, and prognosis management. That is, the drebrin metabolite peptide (iMADP) of the present invention can be used to produce anti-iMADP antibodies used to measure the iMADP in biological samples. Furthermore, a diagnostic kit can be provided that can be used to determine the use or effectiveness of a therapeutic drug for cognitive dysfunction and / or neurological dysfunction by measuring the amount of the iMADP in a biological sample.

[0013]

[0023] Figure 1 shows the results of comparing the sensitivity and characteristics of 10 antibody combinations to the present iMADP, including nine of the present monoclonal antibodies (#2-1, #7-1, #2-2, #6, #7-2, #8, #9-1, #9-2, and #9-3) and a known drebrin antibody (M2F6). Black plates indicate a background of 0.15 or higher (low antibody specificity). Each plate is numbered 1 to 7, indicating the antibody sensitivity in descending order of sensitivity.

[0024] Figure 1 shows the results of measuring iMADP in human cerebrospinal fluid using an antibody (#7-2) that binds to the amino acid sequence of SEQ ID NO: 7 and an antibody (#2-1) that binds to the amino acid sequence of SEQ ID NO: 2.

[0025] Figure 1 shows the results of measuring iMADP in human cerebrospinal fluid using an antibody (#8) that binds to the amino acid sequence of SEQ ID NO: 8 and an antibody (#2-1) that binds to the amino acid sequence of SEQ ID NO: 2.

[0023] Figure 1 shows the results of measuring iMADP in human cerebrospinal fluid using an antibody (#6) that binds to the amino acid sequences of SEQ ID NOs: 6 and 8, and an antibody (#7-1) that binds to the amino acid sequence of SEQ ID NO: 7. Figure 2 shows the results of measuring iMADP in 1x, 2x, and 4x diluted human plasma using an antibody (#6) that binds to the amino acid sequences of SEQ ID NOs: 6 and 8, and an antibody (#7-1) that binds to the amino acid sequence of SEQ ID NO: 7. Figure 3 shows the results of measuring the amount of iMADP in cerebrospinal fluid collected from cognitively normal individuals ("Normal" in the figure), patients with mild cognitive impairment due to Alzheimer's disease (ADMCI) ("MCI" in the figure), and patients with dementia due to Alzheimer's disease (ADD) ("Dementia" in the figure). "**" and "***" in the figure indicate statistically significant differences (p<0.01 and p<0.001), respectively, relative to Normal by t-test. This figure shows the results of measuring the amount of iMADP in plasma collected from individuals with normal cognitive function ("Normal" in the figure), ADMCI patients ("MCI" in the figure), and ADD patients ("Dementia" in the figure). "**" and "***" in the figure indicate statistically significant differences (p<0.01 and p<0.001), respectively, compared to Normal by t-test. This figure shows the results of measuring the amount of iMADP in plasma collected from individuals with normal neurological function ("Normal" in the figure) and amyotrophic lateral sclerosis (ALS) patients ("ALS" in the figure)."**" in the figure indicates a statistically significant difference (p<0.01) from healthy subjects by t-test. Figure 1 shows the results of measuring the amount of iMADP in plasma collected from subjects with normal neurological function ("healthy subjects" in the figure) and patients with immune-mediated peripheral neuropathy (IMN) ("IMN" in the figure). "**" in the figure indicates a statistically significant difference (p<0.01) from healthy subjects by t-test. Figure 1 shows the results of measuring the amount of iMADP in plasma collected from subjects with normal neurological function ("healthy subjects" in the figure) and patients with multiple sclerosis (MS) ("MS" in the figure). "**" in the figure indicates a statistically significant difference (p<0.01) from healthy subjects by t-test.

[0014] <Definition of Terms and Basic Technical Content> In this specification, the term "cognitive dysfunction" refers to a state in which some kind of impairment has occurred substantially in brain functions related to cognition, such as perceptual function, memory function, attention function, and executive function, and is not limited to the presence or absence of symptoms or their severity. Examples of cognitive dysfunction include mild cognitive impairment (MCI) such as mild cognitive impairment due to Alzheimer's disease (ADMCI); dementia such as Alzheimer's disease dementia (ADD) and Parkinson's disease dementia; and the like, with MCI and ADD being preferred.

[0015] As used herein, "cognitively normal individuals" refers to individuals without cognitive impairment, and "cognitively impaired individuals" refers to patients with mild cognitive impairment or dementia.

[0016] As used herein, "neurologic dysfunction" refers to a state in which some kind of disorder has occurred substantially in the structure of synapses, and the presence or absence of symptoms and the severity of the symptoms are not limited. However, cognitive dysfunction such as mild cognitive impairment and dementia is excluded. Examples of neurologic dysfunction include amyotrophic lateral sclerosis (ALS), immune-mediated peripheral neuropathy (IMN), multiple sclerosis (MS), Parkinson's disease, etc., with ALS, IMN, or MS being preferred.

[0017] As used herein, "individuals with normal neurological function" refers to individuals without neurological dysfunction, and "individuals with neurological dysfunction" includes individuals with neurological dysfunction such as amyotrophic lateral sclerosis (ALS), immune-mediated peripheral neuropathy (IMN), and multiple sclerosis (MS).

[0018] Drebrin is involved in neuronal morphogenesis, particularly process formation, by changing the properties of actin fibers. In developing and migrating neurons, it is present in the cell body and the entire process, but in mature neurons, it is specifically present in the spine structures (dendritic spines). There are two isoforms of drebrin: embryonic type drebrin E and adult type drebrin A. Drebrin A, which is specifically found in the dendritic spines of mature neurons, is characterized by being expressed only in neurons. It is also known that drebrin in dendritic spines is widely lost in dementias such as Alzheimer's disease. Meanwhile, drebrin E is also expressed in the cell bodies of newly formed neurons and migrating neurons in the mature brain.

[0019] As used herein, "drebrin" refers to a protein produced from the human drebrin gene DBN1, and among drebrins, drebrin A (SEQ ID NO: 1) is an isoform expressed in mature nerve cells, and drebrin E (SEQ ID NO: 3) is an isoform produced in nerve cells (immature nerve cells, mature nerve cells) and cells other than nerve cells.

[0020] As used herein, the term "drebrin metabolite" refers to a part or fragment of drebrin A or drebrin E that is produced in the human body and then released into cerebrospinal fluid, blood, etc., or a peptide obtained by any modification of these. Drebrin metabolites do not include drebrin A and drebrin E.

[0021] As used herein, "information related to cognitive impairment" refers to information related to the presence or absence of cognitive impairment, the degree of progression of cognitive impairment, and the possibility of developing cognitive impairment.

[0022] As used herein, "information related to neurological dysfunction" refers to information related to the presence or absence of neurological dysfunction, the degree of progression of neurological dysfunction, and the possibility of onset of neurological dysfunction.

[0023] In this specification, examples of substances used for "labeling" include enzymes such as peroxidase (e.g., horseradish peroxidase [HRP]), alkaline phosphatase, β-D-galactosidase, glucose oxidase, glucose-6-phosphate dehydrogenase, alcohol dehydrogenase, malate dehydrogenase, penicillinase, catalase, apoglucose oxidase, urease, luciferase, and acetylcholinesterase; fluorescent substances such as fluorescein isothiocyanate, phycobiliprotein, rare earth metal chelates, dansyl chloride, and tetramethylrhodamine isothiocyanate; fluorescent proteins such as green fluorescent protein (GFP), cyan fluorescent protein (CFP), blue fluorescent protein (BFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP), and luciferase; ３ H. １４ C. １２５ I or １３１ Examples of the fluorescent substance include radioisotopes such as I, biotin, avidin, or chemiluminescent substances.

[0024] As used herein, the phrase "one or more amino acid residues have been deleted, substituted, and / or added" means that, for example, 1 to 26, preferably 1 to 25, more preferably 1 to 20, even more preferably 1 to 16, even more preferably 1 to 13, even more preferably 1 to 10, and even more preferably 1 to 5 amino acid residues have been deleted, substituted, and / or added. Furthermore, as used herein, "an amino acid sequence in which one or more amino acid residues have been deleted, substituted, and / or added in the amino acid sequence of SEQ ID NO: ..." refers to an amino acid sequence that has, for example, 90% or more sequence identity to the amino acid sequence of SEQ ID NO: .... Here, "90% or more sequence identity" means that the sequence identity is at least 90%, preferably 92% or more, more preferably 93% or more, even more preferably 94% or more, even more preferably 95% or more, particularly preferably 96% or more, especially more preferably 97% or more, especially even more preferably 98% or more, even more especially preferably 99% or more, and most preferably 100% sequence identity. Nucleotide sequence identity can be determined using a program called BLASTN (Altschul SF, et al: J Mol Biol 215: 403, 1990), which is based on the algorithm BLAST (Proc. Natl. Acad. Sci. USA 87: 2264-2268, 1990, Proc Natl Acad Sci USA 90: 5873, 1993). When analyzing a nucleotide sequence using BLASTN, the parameters are, for example, score = 100 and word length = 12.

[0025] <The iMADP of the present invention> The iMADP of the present invention is not particularly limited as long as it is a peptide (drebrin metabolite peptide) (i.e., the iMADP of the present invention) selected from (i) to (iii) of the following: (i) a peptide comprising the amino acid sequence of SEQ ID NO: 4 (specifically, a peptide comprising amino acid residues at positions 221 to 475 of the amino acid sequence of human drebrin A [amino acid sequence of SEQ ID NO: 1]), (ii) a peptide comprising the amino acid sequence of SEQ ID NO: 5 (specifically, a peptide comprising amino acid residues at positions 221 to 429 of the amino acid sequence of human drebrin E [amino acid sequence of SEQ ID NO: 3]), or (iii) a peptide comprising an amino acid sequence in which one or more amino acid residues in the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 5 have been deleted, substituted, and / or added, and an isolated one or a recombinant protein is preferred.

[0026] For the peptide (i) in the present iMADP, the upper limit of the amino acid residue length can be, for example, 300 or less, 290 or less, 280 or less, 270 or less, or 260 or less, etc. For the peptide (ii) in the present iMADP, the upper limit of the amino acid residue length can be, for example, 260 or less, 250 or less, 240 or less, 230 or less, 220 or less, or 215 or less, etc.

[0027] The recombinant iMADP protein can be obtained by introducing the vector described below into host cells, culturing the cells in a culture medium appropriate for the host cells, and isolating and purifying the iMADP produced in the culture medium. Examples of such host cells include Escherichia coli, yeast, mammalian (e.g., human, mouse, rat, dog, cat, etc.) cells, insect (e.g., Spodoptera frugiperda, Trichoplusia ni, etc.) cells, and plant (e.g., tobacco, potato, tomato, carrot, soybean, rapeseed, alfalfa, rice, wheat, barley, etc.) cells.

[0028] As shown in Examples 5 and 6 below, the present iMADP is a drebrin metabolite peptide that is detected in subjects without cognitive impairment when blood samples are measured from subjects with normal cognitive function and patients with mild cognitive impairment and dementia, but is not detected in patients with mild cognitive impairment or dementia, or the amount detected is relatively small compared to subjects without cognitive impairment.

[0029] The present invention is based on the discovery that the amount of iMADP, which is human drebrin A (SEQ ID NO: 1) or human drebrin E (SEQ ID NO: 3), or a fragment or splice variant of either thereof, or a dimer, oligomer, or polymer composed thereof, varies depending on the stage of cognitive and / or neurological dysfunction of a subject.

[0030] <Present Measurement Method> The method of the present invention is not particularly limited as long as it involves measuring the amount (specifically, concentration) of the present iMADP in a biological sample (i.e., the present measurement method). Examples of methods for measuring the amount of the present iMADP in a biological sample include mass spectrometry and immunological techniques. Immunological techniques using an antibody that specifically binds to the present iMADP (anti-present iMADP antibody) are preferred, immunological techniques using two types of anti-present iMADP antibodies are more preferred, and immunological techniques using an antibody that binds to the amino (N)-terminal region of the present iMADP (N-antibody) and an antibody that binds to the carboxyl (C)-terminal region of the present iMADP (C-antibody) are even more preferred. When performing immunological techniques using N-antibodies and C-antibodies, either the N-antibody or the C-antibody may be labeled.

[0031] Examples of the immunological techniques include methods that use labels, such as enzyme immunoassay, chemiluminescent immunoassay, electrochemical immunoassay, fluorescent immunoassay, radioimmunoassay, immunochromatography, and Western blotting, and methods that do not use labels, such as latex agglutination and immunoturbidimetry. More specifically, examples of the immunological techniques include sandwich immunoassay and sandwich ELISA.

[0032] The "antibody that binds to the N-terminal region of the iMADP of the present invention" may be an antibody whose epitope is on the iMADP of the present invention that is closer to the N-terminal side than the epitope of the "antibody that binds to the C-terminal region of the iMADP of the present invention". More specifically, examples include an antibody that binds to the N-terminal region of the amino acid sequence of SEQ ID NO: 1 (the amino acid sequence of human drebrin A) from the 365th amino acid residue; or an antibody that binds to the N-terminal region of the amino acid sequence of SEQ ID NO: 1 from the 365th amino acid residue (sometimes referred to herein as the "amino acid sequence of a human drebrin A variant"), in which one or more amino acid residues have been deleted, substituted, and / or added; or an antibody that binds to the N-terminal region of the amino acid sequence of SEQ ID NO: 3 (the amino acid sequence of human drebrin E) from the 319th amino acid residue; or an antibody that binds to the N-terminal region of the amino acid sequence of SEQ ID NO: 3 from the 319th amino acid residue (sometimes referred to herein as the "amino acid sequence of a human drebrin E variant"), in which one or more amino acid residues have been deleted, substituted, and / or added.

[0033] The above-mentioned "antibody that binds to the C-terminal region of the iMADP of the present invention" may be any antibody that has an epitope on the iMADP of the present invention that is more C-terminal than the epitope of the above-mentioned "antibody that binds to the N-terminal region of the iMADP of the present invention". More specifically, examples include an antibody that binds to the human drebrin A-specific sequence Ins2 (SEQ ID NO: 2); an antibody that binds to the C-terminal region of the amino acid sequence of SEQ ID NO: 1 from the 320th amino acid residue; or an antibody that binds to the C-terminal region of the amino acid sequence of SEQ ID NO: 1 from the 320th amino acid residue; or an antibody that binds to the C-terminal region of the amino acid sequence of SEQ ID NO: 3 from the 320th amino acid residue; or an antibody that binds to the C-terminal region of the amino acid sequence of SEQ ID NO: 3 from the 320th amino acid residue; or an antibody that binds to the C-terminal region of the amino acid sequence of SEQ ID NO: 3 from the 320th amino acid residue. The amino acid residues at positions 320 and 365 in the amino acid sequence of the human drebrin A mutant and the amino acid residues at positions 319 and 320 in the amino acid sequence of the human drebrin E mutant can be identified by performing alignment analysis with the amino acid sequence of SEQ ID NO: 1 or the amino acid sequence of SEQ ID NO: 3.

[0034] According to the present measurement method, by measuring the amount of the present iMADP in a biological sample (cerebrospinal fluid, blood, urine, etc.) collected from a subject, information related to cognitive dysfunction and / or neurological dysfunction can be obtained, and therefore it is extremely useful for preventing the onset of cognitive dysfunction and / or neurological dysfunction, treating it early, assessing the effectiveness of treatment for patients, predicting recurrence, and managing the prognosis.

[0035] As used herein, the term "biological sample" refers to any sample derived from a subject (living body), including, for example, cerebrospinal fluid, blood, urine, saliva, and nasal mucosal fluid. Blood includes whole blood, serum, and plasma. Although the biological sample may be subjected to measurement of the concentration of the present iMADP as is, it is preferable to subject the sample to pretreatment prior to measurement to enhance the detection sensitivity of the present iMADP. Examples of pretreatment methods include immunoprecipitation using an antibody that binds to the present iMADP.

[0036] The present measurement method can be used to determine the presence or absence of cognitive dysfunction or neurological dysfunction, or the risk thereof, using the present iMADP as a biomarker. Specifically, if the amount of the present iMADP is lower than a cutoff value (e.g., the amount of the present iMADP in a cognitively normal individual), the subject can be determined to have or be at high risk of cognitive dysfunction. Conversely, if the amount of the present iMADP is higher than the cutoff value (e.g., the amount of the present iMADP in a cognitively normal individual), the subject can be determined to have or be at high risk of neurological dysfunction.

[0037] When using the present measurement method to determine the presence or risk of cognitive impairment, the cutoff value can be set based on the amount of the present iMADP in biological samples from cognitively normal individuals, patients with mild cognitive impairment, and / or patients with dementia. Furthermore, when using the present measurement method to determine the presence or risk of neurological impairment, the cutoff value can be set based on the amount of the present iMADP in biological samples from neurologically normal individuals and / or patients with neurological impairment. Here, the "cutoff value" refers to the amount of the present iMADP in a biological sample above and below which the presence or risk of cognitive impairment and / or neurological impairment can be determined. The cutoff value can be set appropriately depending on the type of biological sample used for the determination, the type of the present iMADP used for the determination, the characteristics of the subject, etc. For example, when the biological sample is cerebrospinal fluid, the cutoff value for the amount of the present iMADP used to determine cognitive impairment is expected to be set at, for example, about 30 pg / mL or less, about 40 pg / mL or less, or about 50 pg / mL or less. Furthermore, when the biological sample is blood (plasma), the cutoff value for the amount of iMADP used in assessing cognitive impairment is expected to be set at, for example, about 1000 pg / mL or less, about 2000 pg / mL or less, about 3000 pg / mL or less, and the cutoff value for the amount of iMADP used in assessing neurological impairment is expected to be set at, for example, about 7000 pg / mL or more, about 9000 pg / mL or more, about 11000 pg / mL or more, etc. The cutoff value for the amount of iMADP may vary depending on the measurement method and the subject of measurement.

[0038] In the present measurement method, the amount of the present iMADP in a biological sample collected from a subject is measured, and the measured amount of the present iMADP is combined with information obtained from symptoms, interviews, other tests, etc. related to cognitive dysfunction and / or neurological dysfunction in the subject to determine the presence or absence of cognitive dysfunction and / or neurological dysfunction in the subject, or the risk of such dysfunction.

[0039] <Combined antibodies of the present invention> The combined antibodies of the present invention are specified for use "for use in the measurement method of the present invention" and include: an antibody that binds to the N-terminal region of the 365th amino acid residue of the amino acid sequence of SEQ ID NO: 1; or an antibody that binds to the N-terminal region of the 365th amino acid residue of the amino acid sequence of SEQ ID NO: 1 in which one or more amino acid residues have been deleted, substituted, and / or added; or an antibody that binds to the N-terminal region of the 319th amino acid residue of the amino acid sequence of SEQ ID NO: 3; or an antibody that binds to the N-terminal region of the 319th amino acid residue of the amino acid sequence of SEQ ID NO: 3 in which one or more amino acid residues have been deleted, substituted, and / or added (N-antibody); The present invention is not particularly limited as long as it is a combination antibody (i.e., the present combination antibody) with an antibody that binds to the C-terminal region of the amino acid sequence of SEQ ID NO: 1 from the 320th amino acid residue onwards; or an antibody that binds to the C-terminal region of the amino acid sequence of SEQ ID NO: 1 from the 320th amino acid residue onwards, in which one or more amino acid residues have been deleted, substituted, and / or added; or an antibody that binds to the C-terminal region of the amino acid sequence of SEQ ID NO: 3 from the 320th amino acid residue onwards, in which one or more amino acid residues have been deleted, substituted, and / or added (C-antibody), wherein the N-antibody is an antibody whose epitope is on the present iMADP more N-terminally than the epitope of the C-antibody, and the C-antibody is an antibody whose epitope is on the present iMADP more C-terminally than the epitope of the N-antibody. The N-antibody and C-antibody may be labeled.

[0040] The kit of the present invention is not particularly limited as long as it contains one or more antibodies (i.e., anti-iMADP antibodies) that specifically bind to the iMADP of the present invention and is intended for the purpose of measuring the iMADP of the present invention (in a biological sample). Such anti-iMADP antibodies may be labeled. The kit may further include a carrier, a pH buffer, a stabilizer, an instruction manual, a container, etc.

[0041] The anti-iMADP antibodies of the present invention include antibodies that bind to the N-terminal region of the amino acid sequence of SEQ ID NO: 1 from the 365th amino acid residue; or antibodies that bind to the N-terminal region of the amino acid sequence of SEQ ID NO: 1 from the 365th amino acid residue; or antibodies that bind to the N-terminal region of the amino acid sequence of SEQ ID NO: 3 from the 319th amino acid residue; or antibodies that bind to the N-terminal region of the amino acid sequence of SEQ ID NO: 3 from the 319th amino acid residue; or antibodies that bind to the N-terminal region of the amino acid sequence of SEQ ID NO: 3 from the 319th amino acid residue; Preferably, the antibody comprises an antibody that binds to the C-terminal region of the amino acid sequence of SEQ ID NO: 1 from the 320th amino acid residue onwards; or an antibody that binds to the C-terminal region of the amino acid sequence of SEQ ID NO: 1 from the 320th amino acid residue onwards, in which one or more amino acid residues have been deleted, substituted, and / or added; or an antibody that binds to the C-terminal region of the amino acid sequence of SEQ ID NO: 3 from the 320th amino acid residue onwards; or an antibody that binds to the C-terminal region of the amino acid sequence of SEQ ID NO: 3 from the 320th amino acid residue onwards, in which one or more amino acid residues have been deleted, substituted, and / or added (C-antibody). Here, the N-antibody is an antibody whose epitope is on the iMADP of the present invention that is more N-terminal than the epitope of the C-antibody, and the C-antibody is an antibody whose epitope is on the iMADP of the present invention that is more C-terminal than the epitope of the N-antibody.

[0042] The N-antibody in the present measurement method, the present combination antibody, and the present kit is preferably an antibody that binds to the amino acid sequence of SEQ ID NO: 2, 6, or 8, and the C-antibody in the present measurement method, the present combination antibody, and the present kit is preferably an antibody that binds to the amino acid sequence of SEQ ID NO: 2, 7, 9, or 10.

[0043] The combination of the N-antibody and the C-antibody in the present measurement method, the present antibody combination, and the present kit is preferably a combination of an antibody that binds to the amino acid sequence of SEQ ID NO: 2 (N-antibody) with an antibody that binds to the amino acid sequence of SEQ ID NO: 7 (C-antibody); a combination of an antibody that binds to the amino acid sequence of SEQ ID NO: 6 or 8 (N-antibody) with an antibody that binds to the amino acid sequence of SEQ ID NO: 2 (C-antibody); a combination of an antibody that binds to the amino acid sequence of SEQ ID NO: 6 or 8 (N-antibody) with an antibody that binds to the amino acid sequence of SEQ ID NO: 9 (C-antibody); or a combination of an antibody that binds to the amino acid sequence of SEQ ID NO: 6 or 8 (N-antibody) with an antibody that binds to the amino acid sequence of SEQ ID NO: 7 (C-antibody); and a combination of an antibody that binds to the amino acid sequence of SEQ ID NO: 6 or 8 (N-antibody) with an antibody that binds to the amino acid sequence of SEQ ID NO: 7 (C-antibody) is more preferred.

[0044] The kit of the present invention is preferably a kit (diagnostic drug kit) whose use is further specified as "for use in determining (diagnosis) the use or effectiveness (diagnosis) of a therapeutic drug for cognitive dysfunction and / or neurological dysfunction." Examples of therapeutic drugs for cognitive dysfunction include lecanemab and donanemab, which are therapeutic drugs effective in suppressing the progression of mild cognitive impairment and mild dementia due to Alzheimer's disease.

[0045] The present iMADP in a biological sample is bound to the anti-iMADP antibody in the present kit, and the amount of the present iMADP can be measured by detecting the antigen-antibody complex through color development, luminescence, fluorescence, radioactivity, light transmittance, etc. The present kit can be suitably used in the present measurement method.

[0046] The origin, type, class, form, etc. of the antibodies in the antibody combination and kit of the present invention are not particularly limited, and these antibodies include, for example, antibodies derived from mice, rats, cats, etc.; polyclonal antibodies, oligoclonal antibodies (a mixture of several to several dozen types of antibodies); monoclonal antibodies; chimeric antibodies in which a partial region of an antibody (for example, a constant region) is replaced with a region derived from a different biological species; and F(ab') obtained by digesting a monoclonal antibody with pepsin. ２ Antibody fragment, F(ab') ２These include Fab' antibody fragments obtained by reducing antibody fragments, Fab and other antibody fragments obtained by digesting monoclonal antibodies with papain, and recombinant antibodies such as scFv (single-chain antibody) in which an antibody heavy (H) chain variable region and an antibody light (H) chain variable region are linked via an amino acid bridge.

[0047] <The Control Serum, etc. of the Present> The control serum, control plasma, or control substance for measurement quality control of the present invention is not particularly limited as long as it contains a certain amount of the present iMADP and is a control serum, control plasma, or control substance for measurement quality control (i.e., the present control serum, etc.). Examples of such control substances include buffer solutions, physiological saline, etc. The present control serum, etc. can be prepared for quality control when measuring the present iMADP by immunoassay methods, etc. The present control serum, etc. can be used as a standard or control sample in a diagnostic kit for assessing cognitive dysfunction and / or neurological dysfunction.

[0048] <iMADP Nucleotides of the Present Invention> The nucleotide of the present invention may be any nucleotide encoding the iMADP of the present invention (i.e., the iMADP nucleotide of the present invention). The nucleotide constituting the iMADP nucleotide may be DNA or RNA having a natural structure, or may be a nucleotide having a structure in which DNA or RNA having a natural structure has been chemically modified (also referred to as a "modified nucleic acid"). For example, when the iMADP nucleotide of the present invention is in the form of mRNA, a modified nucleic acid is used to confer resistance to degradation by RNase. The modified nucleic acid is preferably a nucleotide in which the base moiety is modified. For example, a pyrimidine nucleotide substituted at the 5th position or a pseudouridine optionally substituted at the 1st position is preferable. Specific examples of the modified nucleic acid include 5-methylcytidine, 5-methoxyuridine, 5-methyluridine, pseudouridine, and 1-alkylpseudouridine. The 1-alkylpseudouridine may be a 1-(C1-C6 alkyl)pseudouridine, preferably 1-methylpseudouridine or 1-ethylpseudouridine.

[0049] The iMADP nucleotides can be readily prepared by conventional methods based on the amino acid sequence of the iMADP. The nucleotide sequences encoding the amino acid sequences can be obtained based on the amino acid sequences listed in the Sequence Listing, and the iMADP nucleotides can be prepared using standard molecular biological and / or chemical procedures.

[0050] The iMADP nucleotides of the present invention may contain nucleotide sequences that have been codon-optimized for expression in a particular host cell. Such optimized nucleotide sequences can be obtained by applying known algorithms and software to the amino acid sequence of interest.

[0051] The iMADP nucleotide may be a single-stranded (sense strand) nucleotide encoding the iMADP, or a double-stranded nucleotide consisting of the sense strand and its complementary antisense strand, and the form is selected to be appropriate for the method of introducing the nucleotide into cells. For example, in the case of mRNA or lentiviral vector, it is single-stranded, and in the case of plasmid DNA, it may be double-stranded.

[0052] <Vector of the Present Invention> The vector of the present invention is not particularly limited as long as it contains a promoter and the present iMADP nucleotides operably linked downstream of the promoter, and is a vector (i.e., the present vector) from which mRNA encoded by the present iMADP nucleotides is transcribed.

[0053] The vector can be appropriately selected depending on the purpose, and examples thereof include non-viral vectors (e.g., episomal vectors, artificial chromosome vectors, and plasmid vectors) and viral vectors. The vector may be circular or linear.

[0054] The promoter in the present vector is not particularly limited as long as it is a region to which RNA polymerase (preferably RNA polymerase and general transcription factor) binds and initiates transcription of mRNA encoded by the present iMADP nucleotide located downstream thereof, and examples thereof include the SRα promoter, SV40 early promoter, viral LTR (long terminal repeat), CMV (cytomegalovirus) promoter, RSV (Rous sarcoma virus) promoter, HSV-TK (herpes simplex virus thymidine kinase) promoter, EF1α promoter, metallothionein promoter, and heat shock promoter.

[0055] In addition to a promoter, the present vector may optionally contain an enhancer, a poly(A) addition signal, a marker gene, a replication origin, a gene encoding a polypeptide that binds to the replication origin and controls replication, and the like. Such a marker gene refers to a gene that enables cell sorting or selection by introducing the marker gene into cells. Specific examples of the marker gene include drug resistance genes, fluorescent protein genes, luciferase genes, and chromogenic enzyme genes. These may be used alone or in combination. Specific examples of the drug resistance gene include neomycin resistance genes, tetracycline resistance genes, kanamycin resistance genes, zeocin resistance genes, and hygromycin resistance genes. Specific examples of the fluorescent protein gene include blue fluorescent protein (BFP), green fluorescent protein (GFP) genes, yellow fluorescent protein (YFP) genes, and red fluorescent protein (RFP) genes. Specific examples of the luciferase enzyme gene include the luciferase gene. Specific examples of the chromogenic enzyme gene include the β-galactosidase gene, the β-glucuronidase gene, and the alkaline phosphatase gene.

[0056] The present invention has been described above by showing preferred embodiments for ease of understanding. The present invention will be described below based on examples. However, the above description and the following examples are provided for illustrative purposes only and are not intended to limit the present invention. Therefore, the scope of the present invention is not limited to the embodiments or examples specifically described herein, but is limited only by the claims.

[0057] (Example 1: Investigation of antibody combinations suitable for sandwich ELISA for measuring iMADP) (1) Two types of monoclonal antibodies (#2-1 and #2-2) of the present invention that bind to drebrin A and / or its metabolites (SEQ ID NO: 2) were produced. (2) A monoclonal antibody (#6) of the present invention that binds to drebrin A, drebrin E and / or their metabolites (SEQ ID NO: 6 and 8) was produced. (3) A monoclonal antibody (#8) of the present invention that binds to drebrin A, drebrin E and / or their metabolites (SEQ ID NO: 8) was produced. (4) Two types of monoclonal antibodies (#7-1 and #7-2) of the present invention that bind to drebrin A, drebrin E and / or their metabolites (SEQ ID NO: 7) were produced. (5) Three types of monoclonal antibodies (#9-1, #9-2 and #9-3) of the present invention that bind to drebrin A, drebrin E and / or their metabolites (SEQ ID NO: 9) were produced. (6) An ELISA was constructed using a combination of two antibodies from each of the nine present monoclonal antibodies prepared in (1) to (5) above and the known drebrin antibody M2F6, a total of 10 antibodies, on a solid-phase plate and labeled antibodies. (7) His-Human Drebrin A recombinant protein expressed in Escherichia coli and purified on a Ni column was diluted with buffer and used as a standard substance. (8) The sensitivity of each antibody combination for iMADP measurement was examined for samples with a background OD450nm of 0.15 or less. (9) Sensitivity was divided into seven levels, with the highest sensitivity ranked from 1 to 7. The results of a comparison of the sensitivity and characteristics of each antibody combination for iMADP are shown in Figure 1.

[0058] (Example 2: Preparation of sandwich ELISA kits for measuring iMADP) Using the nine types of monoclonal antibodies of the present invention prepared in Example 1, multiple ELISA kits (iMADP measurement kits) were prepared, each consisting of an N-antibody that binds to the amino acid sequence of SEQ ID NO: 2, 6, or 8 and a C-antibody that binds to the amino acid sequence of SEQ ID NO: 2, 7, 9, or 10.

[0059] Example 3: Measurement of iMADP using an iMADP measurement kit. Of the iMADP measurement kits prepared in Example 2, three types of kits that showed good sensitivity in Example 1 were used to measure iMADP in cerebrospinal fluid (CSF) (a kit in which the N-antibody is an antibody (#2-1) that binds to the amino acid sequence of SEQ ID NO: 2 and the C-antibody is an antibody (#7-2) that binds to the amino acid sequence of SEQ ID NO: 7; a kit in which the N-antibody is an antibody (#8) that binds to the amino acid sequence of SEQ ID NO: 8 and the C-antibody is an antibody (#2-1) that binds to the amino acid sequence of SEQ ID NO: 2; and a kit in which the N-antibody is an antibody (#6) that binds to the amino acid sequences of SEQ ID NO: 6 and 8 and the C-antibody is an antibody (#7-1) that binds to the amino acid sequence of SEQ ID NO: 7). To prepare a standard curve, His-Human Drebrin A recombinant protein expressed in Escherichia coli and purified through a Ni column was diluted with CSF and measured. iMADP standard solutions of 0.78 to 50.00 ng / mL or 0.31 to 20.00 ng / mL and human cerebrospinal fluid were added to the plate, and after reacting with the labeled antibody, the OD at 450 nm was measured.

[0060] The results of measuring iMADP are shown in Figures 2 to 4. As shown in Figures 2 to 4, a response correlated with the concentration of iMADP added was observed in all kits.

[0061] Example 4: Measurement of iMADP in plasma using an iMADP measurement kit Human plasma containing iMADP was prepared and diluted 1-fold, 2-fold, and 4-fold. The iMADP concentration was measured using a measurement kit in which the N-antibody is an antibody that binds to the amino acid sequence of SEQ ID NO: 8 and the C-antibody is an antibody that binds to the amino acid sequence of SEQ ID NO: 7. As a result, it was confirmed that iMADP in plasma can be measured linearly (i.e., the concentration of iMADP in plasma can be quantified). The results are shown in Figure 5.

[0062] (Example 5: Measurement results of iMADP using an iMADP measurement kit in patients with cognitive impairment) The amount of iMADP in cerebrospinal fluid and plasma collected from a total of 33 subjects, including 20 subjects without cognitive impairment (cognitively normal subjects), 8 patients with ADMCI, and 5 patients with ADD, was measured using an iMADP measurement kit using an antibody that binds to the amino acid sequence of SEQ ID NO: 8 as the N-antibody and an antibody that binds to the amino acid sequence of SEQ ID NO: 7 as the C-antibody (Figures 6 and 7).

[0063] As a result, the iMADP concentration in the cerebrospinal fluid of cognitively normal individuals without cognitive impairment was 50 to 827 pg / mL, and the iMADP concentration in the cerebrospinal fluid of ADMCI patients and ADD patients was below the detection limit of 110 pg / mL. Furthermore, the iMADP concentration in the plasma of cognitively normal individuals without cognitive impairment was 2,737 to 16,365 pg / mL, and the iMADP concentration in the plasma of ADMCI patients and ADD patients was 400 to 2,466 pg / mL. As shown in Figures 6 and 7, significant differences were observed in the iMADP measurements in the cerebrospinal fluid and plasma between the cognitively normal group and the ADMCI patient group or ADD patient group. These results confirmed that the iMADP measurement kit can be used to determine mild cognitive impairment.

[0064] (Example 6: Measurement results of iMADP using an iMADP measurement kit in patients with neurological dysfunction) The amount of iMADP in plasma collected from a total of 75 subjects, including 21 subjects without neurological dysfunction (subjects with normal neurological function), 31 ALS patients, 13 IMN patients, and 10 MS patients, was measured using an iMADP measurement kit using an antibody that binds to the amino acid sequence of SEQ ID NO: 8 as the N-antibody and an antibody that binds to the amino acid sequence of SEQ ID NO: 7 as the C-antibody (Figures 8 to 10).

[0065] As a result, the iMADP concentration in the plasma of subjects with normal neurological function without neurological dysfunction was 400 to 30,800 pg / mL, the iMADP concentration in the plasma of ALS patients was 2,300 to 31,400 pg / mL, the iMADP concentration in the plasma of MN patients was 2,300 to 32,200 pg / mL, and the iMADP concentration in the plasma of MS patients was 3,150 to 39,330 pg / mL. As shown in Figures 8 to 10, significant differences were observed in the iMADP measurement values ​​in plasma between the group of subjects with normal neurological function and the group of amyotrophic lateral sclerosis (ALS) patients, immune-mediated peripheral neuropathy (IMN) patients, and multiple sclerosis (MS) patients. These results confirmed that the iMADP measurement kit can be used to assess neurological dysfunction.

[0066] The amino acid sequences of human drebrin A, Ins2, human drebrin E and iMADP are shown below.

[0067] SEQ ID NO: 1: (amino acid sequence of human drebrin A) 1 MAGVSFSGHR LELLAAYEEV IREESAADWA LYTYEDGSDD LKLAASGEGG LQELSGHFEN 61 QKVMYGFCSV KDSQAALPKY VLINWVGEDV PDARKCACAS HVAKVAEFFQ GVDVIVNASS 121 VEDIDAGAIG QRLSNGLARL SSPVLHRLRL REDENAEPVG TTYQKTDAAV EMKRINREQF 181 WEQAKKEEEL RKEEERKKAL DERLRFEQER MEQERQEQEE RERRYREREQ QIEEHRRKQQ 241 TLEAEEAKRR LKEQSIFGDH RDEEEETHMK KSESEVEEAA AIIAQRPDNP REFFKQQERV 301 ASASAGSCDV PSPFNHRPGR PYCPFIKASD SGPSSSSSSS SSPPRTPFPY ITCHRTPNLS 361 SSLPCSHLDS HRRMAPTPIP TRSPSDSSTA STPVAEQIER ALDEVTSSQP PPLPPPPPPA 421 QETQEPSPIL DSEETRAAAP QAWAGPMEEP PQAQAPPRGP GSPAEDLMFM ESAEQAVLAA 481 PVEPATADAT EIHDAADTIE TDTATADTTV ANNVPPAATS LIDLWPGNGE GASTLQGEPR 541 APTPPSGTEV TLAEVPLLDE VAPEPLLPAG EGCATLLNFD ELPEPPATFC DPEEVEGEPL 601 AAPQTPTLPS ALEELEQEQE PEPHLLTNGE TTQKEGTQAS EGYFSQSQEE EFAQSEELCA 661 KAPPPVFYNK PPEIDITCWD ADPVPEEEEG FEGGD

[0068] SEQ ID NO: 2: (Ins2 sequence) R PYCPFIKASD SGPSSSSSSS SSPPRTPFPY ITCHRTPNLS SSLPC

[0069] SEQ ID NO: 3: (amino acid sequence of human drebrin E) 1 MAGVSFSGHR LELLAAYEEV IREESAADWA LYTYEDGSDD LKLAASGEGG LQELSGHFEN 61 QKVMYGFCSV KDSQAALPKY VLINWVGEDV PDARKCACAS HVAKVAEFFQ GVDVIVNASS 121 VEDIDAGAIG QRLSNGLARL SSPVLHRLRL REDENAEPVG TTYQKTDAAV EMKRINREQF 181 WEQAKKEEEL RKEEERKKAL DERLRFEQER MEQERQEQEE RERRYREREQ QIEEHRRKQQ 241 TLEAEEAKRR LKEQSIFGDH RDEEEETHMK KSESEVEEAA AIIAQRPDNP REFFKQQERV 301 ASASAGSCDV PSPFNHRPGS HLDSHRRMAP TPIPTRSPSD SSTASTPVAE QIERALDEVT 361 SSQPPPLPPP PPPAQETQEP SPILDSEETR AAAPQAWAGP MEEEPPQAQAP PRGPGSPAED 421 LMFMESAEQA VLAAPVEPAT ADATEVHDAA DTIETDTATA DTTVANNVPP AATSLIDLWP 481 GNGEGASTLQ GEPRAPTPPS GTEVTLAEVP LLDEVAPEPL LPAGEGCATL LNFDELPEPP 541 ATFCDPEEVE GEPLAAPQTP TLPSALEELE QEQEPEPHLL TNGETTQKEG TQASEGYFSQ 601 SQEEEFAQSE ELCAKAPPPV FYNKPPEIDI TCWDADPVPE EEEGFEGGD

[0070] SEQ ID NO: 4: (Amino acid sequence of iMADP derived from drebrin A) The numbers indicate the positions (amino acid residue numbers) in the amino acid sequence of drebrin A. 221 RERRYREREQ QIEEHRRKQQ 241 TLEAEEAKRR LKEQSIFGDH RDEEEETHMK KSESEVEEAA AIIAQRPDNP REFFKQQERV 301 ASASAGSCDV PSPFNHRPGR PYCPFIKASD SGPSSSSSSS SSPPRTPFPY ITCHRTPNLS 361 SSLPCSHLDS HRRMAPTPIP TRSPSDSSTA STPVAEQIER ALDEVTSSQP PPLPPPPPPA 421 QETQEPSPIL DSEETRAAAP QAWAGPMEEP PQAQAPPRGP GSPAEDLMFM ESAEQ

[0071] SEQ ID NO: 5 (amino acid sequence of iMADP derived from drebrin E) The numbers indicate the positions in the amino acid sequence of drebrin E. 221 RERRYREREQ QIEEHRRKQQ 241 TLEAEEAKRR LKEQSIFGDH RDEEEETHMK KSESEVEEAA AIIAQRPDNP REFFKQQERV 301 ASASAGSCDV PSPFNHRPGS HLDSHRRMAP TPIPTRSPSD SSTASTPVAE QIERALDEVT 361 SSQPPPLPPP PPPAQETQEP SPILDSEETR AAAPQAWAGP MEEPPQAQAP PRGPGSPAED 421 LMFMESAEQ

[0072] In addition, examples of amino acid sequences to which the N-antibody and C-antibody bind are shown below.

[0073] SEQ ID NO: 6: (amino acid sequence from positions 221 to 232 of the amino acid sequence of drebrin A) RERRYREREQ QI

[0074] SEQ ID NO: 7: (amino acid sequence from positions 421 to 475 of the amino acid sequence of drebrin A) QETQEPSPIL DSEETRAAAP QAWAGPMEEP PQAQAPPRGP GSPAEDLMFM ESAEQ

[0075] SEQ ID NO: 8: (amino acid sequence from positions 221 to 256 of the amino acid sequence of drebrin A) RERRYREREQ QIEEHRRKQQ TLEAEEAKRR LKEQSI

[0076] SEQ ID NO: 9: (amino acid sequence from positions 573 to 620 of the amino acid sequence of drebrin A) CATLLNFD ELPEPPATFC DPEEVEGEPLAAPQTPTLPS ALEELEQEQE

[0077] SEQ ID NO: 10: (amino acid sequence from positions 366 to 420 of the amino acid sequence of drebrin A) SHLDS HRRMAPTPIP TRSPSDSSTA STPVAEQIER ALDEVTSSQP PPLPPPPPPA

[0078] By utilizing the iMADP and the iMADP measurement method and measurement kit of the present invention, risk assessment of cognitive dysfunction and / or neurological dysfunction can be performed early, simply, and with high accuracy, which has previously relied on imaging tests, etc., leading to the prevention of the onset and early treatment of these diseases, and therefore, applications in the medical and pharmaceutical fields are expected.

Claims

1. A drebrin metabolite peptide (iMADP) selected from the following (i) to (iii): (i) a peptide comprising the amino acid sequence of SEQ ID NO: 4, (ii) a peptide comprising the amino acid sequence of SEQ ID NO: 5, or (iii) a peptide comprising an amino acid sequence in which one or more amino acid residues in the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 5 have been deleted, substituted, and / or added.

2. A method for measuring the amount of drebrin metabolite peptide (iMADP) in a biological sample, wherein the iMADP is: (i) a peptide comprising the amino acid sequence of SEQ ID NO: 4; (ii) a peptide comprising the amino acid sequence of SEQ ID NO: 5; or (iii) a peptide comprising an amino acid sequence in which one or more amino acid residues in the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 5 have been deleted, substituted, and / or added.

3. The method according to claim 2, wherein the amount of iMADP in a biological sample is measured by an immunological technique using an antibody that binds to the N-terminal region and an antibody that binds to the C-terminal region of iMADP.

4. The antibody that binds to the N-terminal region of iMADP is an antibody that binds to the N-terminal region of the amino acid sequence of SEQ ID NO: 1 from the 365th amino acid residue; or an antibody that binds to the N-terminal region of the amino acid sequence of SEQ ID NO: 1 from the 365th amino acid residue; or an antibody that binds to the N-terminal region of the amino acid sequence of SEQ ID NO: 3 from the 319th amino acid residue; or an antibody that binds to the N-terminal region of the amino acid sequence of SEQ ID NO: 3 from the 319th amino acid residue; and the antibody that binds to the C-terminal region of iMADP is The method of claim 3, wherein the antibody is an antibody that binds to the C-terminal region of the amino acid sequence of SEQ ID NO: 1 from the 320th amino acid residue onwards; or an antibody that binds to the C-terminal region of the amino acid sequence of SEQ ID NO: 1 from the 320th amino acid residue onwards, in which one or more amino acid residues have been deleted, substituted, and / or added; or an antibody that binds to the C-terminal region of the amino acid sequence of SEQ ID NO: 3 from the 320th amino acid residue onwards ..., in which one or more amino acid residues have been deleted, substituted, and / or added (C-antibody).

5. The method of claim 4, wherein the N-antibody is an antibody that binds to the amino acid sequence of SEQ ID NO: 2, 6, or 8, and the C-antibody is an antibody that binds to the amino acid sequence of SEQ ID NO: 2, 7, 9, or 10.

6. The method of claim 5, wherein the N-antibody is an antibody that binds to the amino acid sequence of SEQ ID NO: 2 and the C-antibody is an antibody that binds to the amino acid sequence of SEQ ID NO: 7; the N-antibody is an antibody that binds to the amino acid sequence of SEQ ID NO: 6 or 8 and the C-antibody is an antibody that binds to the amino acid sequence of SEQ ID NO: 2; the N-antibody is an antibody that binds to the amino acid sequence of SEQ ID NO: 6 or 8 and the C-antibody is an antibody that binds to the amino acid sequence of SEQ ID NO: 9; the N-antibody is an antibody that binds to the amino acid sequence of SEQ ID NO: 6 or 8 and the C-antibody is an antibody that binds to the amino acid sequence of SEQ ID NO: 10; or the N-antibody is an antibody that binds to the amino acid sequence of SEQ ID NO: 6 or 8 and the C-antibody is an antibody that binds to the amino acid sequence of SEQ ID NO:

7.

7. The method according to claim 2 or 3, wherein iMADP is used as a biomarker to determine the presence or absence of cognitive dysfunction or neurological dysfunction or the risk thereof.

8. The method of claim 7, wherein the cognitive dysfunction is mild cognitive impairment or Alzheimer's disease dementia, and the neurological dysfunction is amyotrophic lateral sclerosis, immune-mediated peripheral neuropathy, or multiple sclerosis.

9. An antibody for use in the method of claim 2 or 3, which binds to the N-terminal region of the amino acid sequence of SEQ ID NO: 1 from the 365th amino acid residue; or an antibody which binds to the N-terminal region of the amino acid sequence of SEQ ID NO: 1 from the 365th amino acid residue; or an antibody which binds to the N-terminal region of the amino acid sequence of SEQ ID NO: 3 from the 319th amino acid residue; or an antibody which binds to the N-terminal region of the amino acid sequence of SEQ ID NO: 3 from the 319th amino acid residue; for use in the method of claim 2 or 3, An antibody that binds to the C-terminal region of the amino acid sequence of SEQ ID NO: 1 from the 320th amino acid residue onwards; or an antibody that binds to the C-terminal region of the amino acid sequence of SEQ ID NO: 1 from which one or more amino acid residues have been deleted, substituted, and / or added from the 320th amino acid residue onwards; or an antibody that binds to the C-terminal region of the amino acid sequence of SEQ ID NO: 3 from the 320th amino acid residue onwards; or an antibody that binds to the C-terminal region of the amino acid sequence of SEQ ID NO: 3 from which one or more amino acid residues have been deleted, substituted, and / or added from the 320th amino acid residue onwards; (C-antibody) 10. The combination antibody of claim 9, wherein the N-antibody is an antibody that binds to the amino acid sequence of SEQ ID NO: 2, 6, or 8, and the C-antibody is an antibody that binds to the amino acid sequence of SEQ ID NO: 2, 7, 9, or 10.

11. The combination antibody of claim 10, wherein the N-antibody is an antibody that binds to the amino acid sequence of SEQ ID NO: 2 and the C-antibody is an antibody that binds to the amino acid sequence of SEQ ID NO: 7; the N-antibody is an antibody that binds to the amino acid sequence of SEQ ID NO: 6 or 8 and the C-antibody is an antibody that binds to the amino acid sequence of SEQ ID NO: 2; the N-antibody is an antibody that binds to the amino acid sequence of SEQ ID NO: 6 or 8 and the C-antibody is an antibody that binds to the amino acid sequence of SEQ ID NO: 9; the N-antibody is an antibody that binds to the amino acid sequence of SEQ ID NO: 6 or 8 and the C-antibody is an antibody that binds to the amino acid sequence of SEQ ID NO: 10; or the N-antibody is an antibody that binds to the amino acid sequence of SEQ ID NO: 6 or 8 and the C-antibody is an antibody that binds to the amino acid sequence of SEQ ID NO:

7.

12. A kit for measuring drebrin metabolite peptide (iMADP), comprising one or more antibodies that specifically bind to iMADP, wherein the iMADP is: (i) a peptide comprising the amino acid sequence of SEQ ID NO: 4; (ii) a peptide comprising the amino acid sequence of SEQ ID NO: 5; or (iii) a peptide comprising an amino acid sequence in which one or more amino acid residues in the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 5 have been deleted, substituted, and / or added.

13. An antibody that binds to the N-terminal region of the amino acid sequence of SEQ ID NO: 1 from the 365th amino acid residue; or an antibody that binds to the N-terminal region of the amino acid sequence of SEQ ID NO: 1 from the 365th amino acid residue; or an antibody that binds to the N-terminal region of the amino acid sequence of SEQ ID NO: 3 from the 319th amino acid residue; or an antibody that binds to the N-terminal region of the amino acid sequence of SEQ ID NO: 3 from the 319th amino acid residue; or an antibody that binds to the N-terminal region of the amino acid sequence of SEQ ID NO: 3 from the 319th amino acid residue; The kit according to claim 12, comprising: an antibody that binds to the C-terminal region of the amino acid sequence of SEQ ID NO: 1 from the 320th amino acid residue; or an antibody that binds to the C-terminal region of the amino acid sequence of SEQ ID NO: 1 from the 320th amino acid residue; or an antibody that binds to the C-terminal region of the amino acid sequence of SEQ ID NO: 3 from the 320th amino acid residue; or an antibody that binds to the C-terminal region of the amino acid sequence of SEQ ID NO: 3 from the 320th amino acid residue; or an antibody that binds to the C-terminal region of the amino acid sequence of SEQ ID NO: 3 from the 320th amino acid residue; 14. The kit of claim 13, wherein the N-antibody is an antibody that binds to the amino acid sequence of SEQ ID NO: 2, 6, or 8, and the C-antibody is an antibody that binds to the amino acid sequence of SEQ ID NO: 2, 7, 9, or 10.

15. The kit of claim 14, wherein the N-antibody is an antibody that binds to the amino acid sequence of SEQ ID NO: 2 and the C-antibody is an antibody that binds to the amino acid sequence of SEQ ID NO: 7; the N-antibody is an antibody that binds to the amino acid sequence of SEQ ID NO: 6 or 8 and the C-antibody is an antibody that binds to the amino acid sequence of SEQ ID NO: 2; the N-antibody is an antibody that binds to the amino acid sequence of SEQ ID NO: 6 or 8 and the C-antibody is an antibody that binds to the amino acid sequence of SEQ ID NO: 9; the N-antibody is an antibody that binds to the amino acid sequence of SEQ ID NO: 6 or 8 and the C-antibody is an antibody that binds to the amino acid sequence of SEQ ID NO: 10; or the N-antibody is an antibody that binds to the amino acid sequence of SEQ ID NO: 6 or 8 and the C-antibody is an antibody that binds to the amino acid sequence of SEQ ID NO:

7.

16. A kit according to any one of claims 12 to 15 for determining the use or effectiveness of a therapeutic drug for cognitive dysfunction or neurological dysfunction.

17. The kit of claim 16, wherein the therapeutic agent for cognitive impairment is lecanemab or donanemab.

18. The kit of claim 16, wherein the cognitive dysfunction is mild cognitive impairment or Alzheimer's disease dementia, and the neurological dysfunction is amyotrophic lateral sclerosis, immune-mediated peripheral neuropathy, or multiple sclerosis.

19. A control serum, control plasma or control substance for measurement accuracy control, containing the iMADP described in claim 1.

20. A nucleotide sequence encoding the iMADP of claim 1.

21. A vector comprising a promoter and the nucleotide of claim 20 operably linked downstream of the promoter.

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