DDIT4L Splicing Products as Biomarkers for Senile Dementia Diagnosis

Abstract

This application relates to digested DDIT4L products as diagnostic markers for Alzheimer's disease and their use in the diagnosis of Alzheimer's disease. In particular, this application relates to the use of substances that detect digested intron-retention (DIR) products encoding DDIT4L in a subject's sample for the diagnosis of Alzheimer's disease or mild cognitive impairment, or for creating products for assessing the progression (e.g., evaluation or staging) of cognitive impairment, and to related products and screening methods for pharmaceuticals using DIR products.

Description

Technical Field

[0001] The present application relates to the field of biopharmaceuticals, and specifically to the use of DIR, an intron-retaining splicing product of DNA damage-inducible transcript 4-like transcript (DDIT4L), as a blood marker for young-onset dementia or mild cognitive impairment, as well as corresponding products and methods therefor.

Background Art

[0002] Young-onset dementia, also known as Alzheimer's Disease (AD), is the most prevalent neurodegenerative disease that affects the human brain. It not only causes great suffering to the patients themselves, but also imposes a heavy burden on their families and society.

[0003] According to the "World Alzheimer Report 2017" of the Alzheimer's Disease International (ADI), the global dementia care cost increased from $604 billion in 2010 to $1 trillion in 2017, with a growth rate reaching 65.6%. Among them, there are 5 million AD patients in the United States, and caregivers spend 18 billion hours per year on patient care. In 2017, the global number of AD patients was 50 million, and with the increase in the aging population worldwide, this number is expected to continue to increase in the future. At present, on the premise that there is no drug that can treat, slow down the progression, or prevent this disease, the number of patients is increasing at a pace of doubling every 20 years, and it is predicted to reach 82 million in 2030 and 150 million in 2050.

[0004] According to the "China Development Report 2020: Trends and Policies of China's Population Aging" released by the China Development Research Foundation (a national social organization established with the initiative of the Development Research Center of the State Council), in 2020, the number of elderly people aged 65 and above in China was approximately 180 million, accounting for about 13% of the total population. By 2025 when the "14th Five-Year Plan" is completed, the number of elderly people aged 65 and above will exceed 210 million, accounting for about 15% of the total population. By 2035 and 2050, the number of elderly people aged 65 and above in China will reach 310 million and 380 million respectively, accounting for 22.3% and 27.9% of the total population.

[0005] The incidence of AD is related to age, and the incidence in the overall population is 2% - 4%. In particular, the prevalence rate among middle-aged and elderly people aged 65 and above is 5%, and it increases exponentially with age. According to the analysis, the prevalence rate among elderly people aged 85 and above is 25%, and the prevalence rate among those aged 95 and above is as high as 60%. China has entered an aging society. According to statistics, the number of AD patients is approximately 10 million, and every year, more than 300,000 new cases are increasing.

[0006] The main symptom of AD is cognitive impairment. The fundamental causes of cognitive impairment are synaptic and neuronal damage, and abnormalities in the structure and function of neural circuits. The main reason why the clinical research of new AD drugs has continued to fail is that the mechanisms of synaptic and neural damage are still unclear, and there is a lack of cell / animal models similar to the pathophysiological characteristics of human diseases (such as neurodegenerative diseases). As a result, the research on effective prevention and treatment methods for AD and early diagnostic markers reflecting the disease outcome has been delayed.

[0007] Metabolic abnormalities may be one of the main mechanisms underlying the onset of AD. In AD patients, lipid metabolism abnormalities typified by cholesterol and glucose metabolism abnormalities typified by glucose are the most common metabolic abnormalities, and also include energy metabolism disorders caused by cerebral ischemia and hypoxia due to insufficient blood supply to the brain typified by degenerative changes in cerebral small blood vessels. However, how abnormalities in cerebral lipid metabolism and glucose metabolism affect the occurrence and progression of AD remains unclear. Mitochondria are important organelles involved in the metabolism of major energy substances, and mitochondrial dysfunction causes abnormalities in energy metabolism and damages the functions of nerve cells and cerebral small blood vessel endothelial cells. However, the relationship between mitochondrial dysfunction and lipid metabolism and glucose metabolism abnormalities in the onset and progression of early AD remains unclear.

[0008] As a syndrome accompanied by objective cognitive function decline, mild cognitive impairment (MCI) is a state between mild dementia and healthy aging. Although the prominent symptom is memory impairment, a certain degree of decline can also be observed in other cognitive functions. However, the ability to perform daily activities is normal, and the diagnostic criteria for dementia are not met. Approximately 10% - 15% of MCI cases progress to AD each year, which is 10 times that of normal controls. As a transitional state from normal aging to dementia, MCI has been attracting increasing attention, and the study of MCI is an important stage in the early diagnosis and early intervention of AD.

[0009] Current AD diagnostic markers mainly include detection of Aβ and Tau contents in cerebrospinal fluid, PET and MRI neuroimaging methods, etc. Although these methods have relatively high, and in some cases very high, diagnostic effects, they also have drawbacks such as difficult operation, high cost, invasiveness, and low correlation with disease progression. Therefore, there is an urgent need to develop new markers that are simple, easy to implement, have high cost performance, are suitable for large-scale population screening, and can be effectively used for the early diagnosis of AD.

Summary of the Invention

[0010] The present application provides a blood marker for novel early-onset Alzheimer's disease (AD) or mild cognitive impairment (MCI).

[0011] In one aspect, the present application provides the use of a DIR, which is an intron-retention splicing product of DNA damage-inducible transcript 4-like transcript (DDIT4L), and / or a nucleic acid encoding said DIR, in disease diagnosis and / or disease progression evaluation, wherein the disease includes cognitive impairment.

[0012] In one aspect, the present application provides the use of a DIR, which is an intron-retention splicing product of DNA damage-inducible transcript 4-like transcript (DDIT4L), and / or a nucleic acid encoding said DIR, in disease diagnosis and / or disease progression evaluation, wherein the disease includes neurodegenerative diseases.

[0013] In some embodiments, the DIR includes the amino acid sequence shown in SEQ ID NO: 1.

[0014] In some embodiments, the nucleic acid encoding the DIR includes the nucleotide sequence shown in SEQ ID NO: 8.

[0015] In some embodiments, the nucleic acid encoding the DIR includes the nucleotide sequence shown in SEQ ID NO: 93.

[0016] In some embodiments, the cognitive impairment includes normal age-related cognitive impairment, Lewy body dementia (LBD), frontotemporal dementia, and / or vascular dementia.

[0017] In some embodiments, the inducing diseases of the cognitive impairment include Alzheimer's disease, multiple infarcts, Parkinson's disease, AIDS, and / or Creutzfeldt-Jakob disease (CJD).

[0018] In some embodiments, the cognitive impairment includes mild cognitive impairment (MCI), moderate cognitive impairment, and severe cognitive impairment.

[0019] In some embodiments, the cognitive impairment includes amnestic mild cognitive impairment with multiple cognitive domain impairments (aMCI-m).

[0020] In some embodiments, the neurodegenerative disease includes acute neurodegenerative diseases and chronic neurodegenerative diseases.

[0021] In some embodiments, the neurodegenerative disease includes neurodegenerative diseases caused by neuronal cell death and glial cell homeostasis, neurodegenerative diseases caused by aging, neurodegenerative diseases caused by affected CNS cell function, neurodegenerative diseases caused by abnormal intercellular transmission, and / or neurodegenerative diseases caused by disorders of cell motility.

[0022] In some embodiments, the neurodegenerative disease includes Alzheimer's disease, Parkinson's disease, multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), and / or Huntington's disease (HD).

[0023] In some embodiments, the neurodegenerative disease includes juvenile dementia.

[0024] In some embodiments, the neurodegenerative disease includes mild juvenile dementia, moderate juvenile dementia, and / or severe juvenile dementia.

[0025] In another aspect, the present application provides the use of a DIR, which is an intron-retaining splicing product of DNA damage-inducible transcript 4-like transcript (DDIT4L), and / or a substance that detects a nucleic acid encoding the DIR, in the preparation of a product for disease diagnosis and / or disease progression evaluation, wherein the disease includes cognitive impairment.

[0026] In another aspect, the present application provides the use of a DIR, which is an intron-retaining splicing product of DNA damage-inducible transcript 4-like transcript (DDIT4L), and / or a substance that detects a nucleic acid encoding the DIR, in the preparation of a product for disease diagnosis and / or disease progression evaluation, wherein the disease includes neurodegenerative diseases.

[0027] In another aspect, the present application provides a diagnostic kit containing a DIR, which is an intron-retaining splicing product of DNA damage-inducible transcript 4-like transcript (DDIT4L) described in the present application, and / or a substance that can detect a nucleic acid encoding the DIR, for use in cognitive impairment diagnosis and / or cognitive impairment progression evaluation, and / or for use in neurodegenerative disease diagnosis and / or neurodegenerative disease progression evaluation.

[0028] In some embodiments, the diagnostic kit includes an instruction manual that describes a method for performing cognitive impairment diagnosis and / or cognitive impairment progression evaluation, and / or neurodegenerative disease diagnosis and / or neurodegenerative disease progression evaluation using the diagnostic kit.

[0029] In some embodiments, the DIR includes the amino acid sequence shown in SEQ ID NO:1.

[0030] In some embodiments, the nucleic acid encoding the DIR includes the nucleotide sequence shown in SEQ ID NO:8.

[0031] In some embodiments, the nucleic acid encoding the DIR includes the nucleotide sequence shown in SEQ ID NO:93.

[0032] In some embodiments, the cognitive impairment includes normal age-related cognitive impairment, Lewy body dementia (LBD), frontotemporal dementia and / or vascular dementia.

[0033] In some embodiments, the inducing diseases of the cognitive impairment include Alzheimer's disease, multiple infarcts, Parkinson's disease, AIDS and / or Creutzfeldt-Jakob disease (CJD).

[0034] In some embodiments, the cognitive impairment includes mild cognitive impairment (MCI), moderate cognitive impairment and severe cognitive impairment.

[0035] In some embodiments, the cognitive impairment includes amnestic MCI (aMCI-m) associated with multiple cognitive domain impairments.

[0036] In some embodiments, the neurodegenerative diseases include acute neurodegenerative diseases and chronic neurodegenerative diseases.

[0037] In some embodiments, the neurodegenerative diseases include neurodegenerative diseases caused by nerve cell death and glial cell homeostasis, neurodegenerative diseases caused by aging, neurodegenerative diseases caused by affected CNS cell functions, neurodegenerative diseases caused by abnormal intercellular transmission and / or neurodegenerative diseases caused by disorders of cell motility.

[0038] In some embodiments, the neurodegenerative diseases include Alzheimer's disease, Parkinson's disease, multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS) and / or Huntington's disease (HD).

[0039] In some embodiments, the neurodegenerative diseases include juvenile dementia.

[0040] In some embodiments, the neurodegenerative diseases include mild juvenile dementia, moderate juvenile dementia and / or severe juvenile dementia.

[0041] In some embodiments, the substance includes a reagent and / or device capable of detecting the DIR and / or a nucleic acid encoding the DIR.

[0042] In some embodiments, the substance includes a reagent that specifically detects the DIR and / or the nucleic acid encoding the DIR.

[0043] In some embodiments, the substance includes an antigen-binding protein that can specifically bind to DIR, a probe that can specifically bind to DIR, a primer that can specifically amplify the nucleic acid encoding DIR, a gene chip that can specifically detect the nucleic acid encoding DIR, an aptamer that can specifically bind to the nucleic acid encoding DIR, and / or a guide RNA that can specifically bind to the nucleic acid encoding DIR.

[0044] In some embodiments, the antigen-binding protein includes LCDR2, and the LCDR2 includes the amino acid sequence shown in SEQ ID NO:74.

[0045] In some embodiments, the LCDR2 includes the amino acid sequence shown in SEQ ID NO:26 or 16.

[0046] In some embodiments, the antigen-binding protein includes LCDR1, and the LCDR1 includes the amino acid sequence shown in SEQ ID NO:73.

[0047] In some embodiments, the LCDR1 includes the amino acid sequence shown in any one of SEQ ID NO:25, 52, or 15.

[0048] In some embodiments, the antigen-binding protein includes LCDR3, and the LCDR3 includes the amino acid sequence shown in SEQ ID NO:75.

[0049] In some embodiments, the LCDR3 may include the amino acid sequence shown in any one of SEQ ID NO:27, 53, 67, or 17.

[0050] In some embodiments, the antigen-binding protein comprises LCDR1, LCDR2, and LCDR3, wherein a) the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:25, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:26, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:27, b) the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:52, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:26, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:53, c) the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:52, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:26, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:67, or d) the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:15, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:16, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:17.

[0051] In some embodiments, the antigen-binding protein comprises HCDR1, and the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:70.

[0052] In some embodiments, the HCDR1 may comprise the amino acid sequence shown in any one of SEQ ID NO:20, 35, 56, 10.

[0053] In some embodiments, the antigen-binding protein comprises HCDR2, and the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:71.

[0054] In some embodiments, the HCDR2 may comprise the amino acid sequence shown in any one of SEQ ID NO:21, 36, 42, 48, 11.

[0055] In some embodiments, the antigen-binding protein comprises an HCDR3, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:70.

[0056] In some embodiments, the HCDR3 may comprise the amino acid sequence shown in any of SEQ ID NO:22, 30, 37, 43, 49, 57, 62, 12.

[0057] In some embodiments, the antigen-binding protein comprises HCDR1, HCDR2, and HCDR3, where a) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:20, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:21, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:22, b) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:20, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:21, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:30, c) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:35, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:36, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:37, d) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:20, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:42, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:43, e) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:20, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:48, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:49, f) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:56, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:36, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:57. g) The HCDR1 comprises the amino acid sequence shown in SEQ ID NO:35, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:36, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:62, h) The HCDR1 comprises the amino acid sequence shown in SEQ ID NO:35, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:42, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:37, i) The HCDR1 comprises the amino acid sequence shown in SEQ ID NO:56, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:36, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:57, or j) The HCDR1 comprises the amino acid sequence shown in SEQ ID NO:10, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:11, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:12.

[0058] In some embodiments, the antigen-binding protein comprises a heavy chain variable region VH, and the VH comprises the amino acid sequence shown in any one of SEQ ID NO:13, 23, 31, 38, 44, 50, 58, 63 and 65.

[0059] In some embodiments, the antigen-binding protein comprises a light chain variable region VL, and the VL comprises the amino acid sequence shown in any one of SEQ ID NO:18, 28, 33, 40, 46, 54, 60 and 68.

[0060] In some embodiments, the antigen-binding protein comprises a heavy chain variable region VH and a light chain variable region VL, wherein a) the VH comprises the amino acid sequence shown in SEQ ID NO:23, and the VL comprises the amino acid sequence shown in SEQ ID NO:28, b) The VH comprises the amino acid sequence shown in SEQ ID NO: 31, and the VL comprises the amino acid sequence shown in SEQ ID NO: 33. c) The VH comprises the amino acid sequence shown in SEQ ID NO: 38, and the VL comprises the amino acid sequence shown in SEQ ID NO: 40. d) The VH comprises the amino acid sequence shown in SEQ ID NO: 44, and the VL comprises the amino acid sequence shown in SEQ ID NO: 46. e) The VH comprises the amino acid sequence shown in SEQ ID NO: 50, and the VL comprises the amino acid sequence shown in SEQ ID NO: 54. f) The VH comprises the amino acid sequence shown in SEQ ID NO: 58, and the VL comprises the amino acid sequence shown in SEQ ID NO: 60. g) The VH comprises the amino acid sequence shown in SEQ ID NO: 63, and the VL comprises the amino acid sequence shown in SEQ ID NO: 60. h) The VH comprises the amino acid sequence shown in SEQ ID NO: 65, and the VL comprises the amino acid sequence shown in SEQ ID NO: 54. i) The VH comprises the amino acid sequence shown in SEQ ID NO: 58, and the VL comprises the amino acid sequence shown in SEQ ID NO: 68, or j) The VH comprises the amino acid sequence shown in SEQ ID NO: 13, and the VL comprises the amino acid sequence shown in SEQ ID NO: 18.

[0061] In some embodiments, the antigen-binding protein comprises a heavy chain constant region, and the heavy chain constant region comprises a constant region derived from IgG.

[0062] In some embodiments, the heavy chain constant region comprises a constant region derived from a protein selected from the group consisting of IgG1, IgG2, IgG3, and IgG4.

[0063] In some embodiments, the antigen-binding protein comprises a light chain constant region, and the light chain constant region comprises a constant region derived from Igκ or a constant region derived from Igλ.

[0064] In some embodiments, the light chain constant region comprises a constant region derived from human Igκ.

[0065] In some embodiments, the antigen-binding protein comprises an antibody or an antigen-binding fragment thereof.

[0066] In some embodiments, the antigen-binding fragment is selected from the group consisting of Fab, Fab’, F(ab)2, Fv fragment, F(ab’)2, scFv, di-scFv, VHH and / or dAb.

[0067] In some embodiments, the antibody is selected from the group consisting of a monoclonal antibody, a mouse antibody, and a chimeric antibody.

[0068] In some embodiments, the product comprises a diagnostic kit.

[0069] In another aspect, the present application provides a method for diagnosing cognitive impairment and / or evaluating the progression of cognitive impairment, comprising detecting the content of DIR, which is an intron-retaining splicing product of DNA damage-inducible transcript 4-like transcript (DDIT4L) in a sample derived from a subject in need thereof, and / or the nucleic acid encoding the DIR.

[0070] In another aspect, the present application provides a method for diagnosing neurodegenerative diseases and / or evaluating the progression of neurodegenerative diseases, comprising detecting the content of DIR, which is an intron-retaining splicing product of DNA damage-inducible transcript 4-like transcript (DDIT4L) in a sample derived from a subject in need thereof, and / or the nucleic acid encoding the DIR.

[0071] In some embodiments, the DIR comprises the amino acid sequence shown in SEQ ID NO:1.

[0072] In some embodiments, the nucleic acid encoding the DIR comprises the nucleotide sequence shown in SEQ ID NO:8.

[0073] In some embodiments, the nucleic acid encoding the DIR comprises the nucleotide sequence shown in SEQ ID NO:93.

[0074] In some embodiments, the cognitive impairment includes normal age-related cognitive impairment, Lewy body dementia (LBD), frontotemporal dementia and / or vascular dementia.

[0075] In some embodiments, the inducing diseases of the cognitive impairment include Alzheimer's disease, multiple infarcts, Parkinson's disease, AIDS and / or Creutzfeldt-Jakob disease (CJD).

[0076] In some embodiments, the cognitive impairment includes mild cognitive impairment (MCI), moderate cognitive impairment and severe cognitive impairment.

[0077] In some embodiments, the cognitive impairment includes amnestic MCI (aMCI-m) with multiple cognitive domain impairments.

[0078] In some embodiments, the neurodegenerative diseases include acute neurodegenerative diseases and chronic neurodegenerative diseases.

[0079] In some embodiments, the neurodegenerative diseases include neurodegenerative diseases caused by neuronal death and glial cell homeostasis, neurodegenerative diseases caused by aging, neurodegenerative diseases caused by affected CNS cell functions, neurodegenerative diseases caused by abnormal intercellular transmission and / or neurodegenerative diseases caused by disorders of cell motility.

[0080] In some embodiments, the neurodegenerative disease includes Alzheimer's disease, Parkinson's disease, multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS) and / or Huntington's disease (HD).

[0081] In some embodiments, the neurodegenerative disease includes juvenile dementia.

[0082] In some embodiments, the neurodegenerative disease includes mild juvenile dementia, moderate juvenile dementia and / or severe juvenile dementia.

[0083] In some embodiments, the subject includes a mammal.

[0084] In some embodiments, the subject includes a cancer patient.

[0085] In some embodiments, the subject is in an elderly stage.

[0086] In some embodiments, the sample includes a blood sample and / or a tissue or cell sample.

[0087] In some embodiments, the sample includes whole blood, serum, plasma and / or cerebrospinal fluid.

[0088] In some embodiments, the method includes comparing the content of the DIR and / or the nucleic acid encoding the DIR in the sample of the subject with a healthy control value, and the healthy control value includes the content of the DIR and / or the nucleic acid encoding the DIR in a healthy subject.

[0089] In some embodiments, the healthy subject does not suffer from the cognitive impairment and / or the neurodegenerative disease.

[0090] In some embodiments, if the content of the DIR and / or the nucleic acid encoding DIR in the sample of the subject is significantly higher than the healthy control value, the subject is diagnosed as suffering from the cognitive impairment and / or the neurodegenerative disease.

[0091] In some embodiments, the method includes comparing the content of the DIR and / or the nucleic acid encoding DIR in the sample of the subject with an initial control value, and the initial control value includes the content of the DIR and / or the nucleic acid encoding DIR detected in the past of the same subject.

[0092] In some embodiments, the subject is definitely diagnosed as suffering from the cognitive impairment and / or the neurodegenerative disease.

[0093] In some embodiments, if the content of the DIR and / or the nucleic acid encoding DIR in the sample of the subject is significantly higher than the initial control value, the subject is diagnosed as having the progression of the cognitive impairment and / or the neurodegenerative disease worsened.

[0094] In some embodiments, the method further includes detecting the content of a marker related to the cognitive impairment and / or the neurodegenerative disease, which is derived from the sample of the subject.

[0095] In some embodiments, the marker related to the cognitive impairment and / or the neurodegenerative disease includes AD7C-NTP, pTau-181, pTau-217, and / or Aβ1-42.

[0096] In some embodiments, the method further includes observing a brain image of the subject.

[0097] In another aspect, the present application includes a system for diagnosing cognitive impairment and / or evaluating the progression of cognitive impairment, which includes a substance that detects DIR, which is an intron-retention splicing product of DNA damage-inducible transcript 4-like transcript (DDIT4L), and / or a nucleic acid encoding said DIR.

[0098] In another aspect, the present application includes a system for diagnosing neurodegenerative diseases and / or evaluating the progression of neurodegenerative diseases, which includes a substance that detects DIR, which is an intron-retention splicing product of DNA damage-inducible transcript 4-like transcript (DDIT4L), and / or a nucleic acid encoding said DIR.

[0099] In some embodiments, said DIR includes the amino acid sequence shown in SEQ ID NO:1.

[0100] In some embodiments, the nucleic acid encoding said DIR includes the nucleotide sequence shown in SEQ ID NO:8.

[0101] In some embodiments, the nucleic acid encoding said DIR includes the nucleotide sequence shown in SEQ ID NO:93.

[0102] In some embodiments, said cognitive impairment includes normal age-related cognitive impairment, Lewy body dementia (LBD), frontotemporal dementia and / or vascular dementia.

[0103] In some embodiments, the inducing diseases of said cognitive impairment include Alzheimer's disease, multiple infarction, Parkinson's disease, AIDS and / or Creutzfeldt-Jakob disease (CJD).

[0104] In some embodiments, said cognitive impairment includes mild cognitive impairment (MCI), moderate cognitive impairment and severe cognitive impairment.

[0105] In some embodiments, said cognitive impairment includes amnestic MCI with multiple cognitive domain impairments (aMCI-m).

[0106] In some embodiments, the neurodegenerative disease includes acute neurodegenerative diseases and chronic neurodegenerative diseases.

[0107] In some embodiments, the neurodegenerative disease includes a neurodegenerative disease caused by nerve cell death and glial cell homeostasis, a neurodegenerative disease caused by aging, a neurodegenerative disease caused by affected CNS cell function, a neurodegenerative disease caused by abnormal intercellular transmission, and / or a neurodegenerative disease caused by a disorder of cell motility.

[0108] In some embodiments, the neurodegenerative disease includes Alzheimer's disease, Parkinson's disease, multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), and / or Huntington's disease (HD).

[0109] In some embodiments, the neurodegenerative disease includes juvenile dementia.

[0110] In some embodiments, the neurodegenerative disease includes mild juvenile dementia, moderate juvenile dementia, and / or severe juvenile dementia.

[0111] In some embodiments, the system exists in the form of a kit.

[0112] In some embodiments, the system includes a collection module for collecting a sample from a subject in need thereof.

[0113] In some embodiments, the subject includes a mammal.

[0114] In some embodiments, the subject includes a tumor patient.

[0115] In some embodiments, the subject is in an elderly stage.

[0116] In some embodiments, the sample includes a blood sample and / or a tissue or cell sample.

[0117] In some embodiments, the sample includes whole blood, serum, plasma and / or cerebrospinal fluid.

[0118] In some embodiments, the system includes a detection module for detecting the content value of the DIR and / or the nucleic acid encoding the DIR.

[0119] In some embodiments, the detection module detects the content value of the DIR and / or the nucleic acid encoding the DIR in the sample collected by the collection module.

[0120] In some embodiments, the detection module includes a substance capable of detecting the content of the DIR and / or the nucleic acid encoding the DIR.

[0121] In some embodiments, the substance includes an antigen-binding protein capable of specifically binding to DIR, a probe capable of specifically binding to DIR, a primer capable of specifically amplifying the nucleic acid encoding DIR, a gene chip capable of specifically detecting the nucleic acid encoding DIR, an aptamer capable of specifically binding to the nucleic acid encoding DIR, and / or a guide RNA capable of specifically binding to the nucleic acid encoding DIR.

[0122] In some embodiments, the antigen-binding protein includes LCDR2, and the LCDR2 includes the amino acid sequence shown in SEQ ID NO:74.

[0123] In some embodiments, the LCDR2 includes the amino acid sequence shown in SEQ ID NO:26 or 16.

[0124] In some embodiments, the antigen-binding protein comprises LCDR1, and the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:73.

[0125] In some embodiments, the LCDR1 comprises the amino acid sequence shown in any one of SEQ ID NO:25, 52, 15.

[0126] In some embodiments, the antigen-binding protein comprises LCDR3, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:75.

[0127] In some embodiments, the LCDR3 may comprise the amino acid sequence shown in any one of SEQ ID NO:27, 53, 67, 17.

[0128] In some embodiments, the antigen-binding protein comprises LCDR1, LCDR2 and LCDR3, wherein a) the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:25, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:26, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:27, b) the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:52, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:26, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:53, c) the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:52, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:26, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:67, or d) the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:15, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:16, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:17.

[0129] In some embodiments, the antigen-binding protein comprises HCDR1, and the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:70.

[0130] In some embodiments, the HCDR1 may comprise the amino acid sequence shown in any one of SEQ ID NO:20, 35, 56, 10.

[0131] In some embodiments, the antigen-binding protein comprises HCDR2, and the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:71.

[0132] In some embodiments, the HCDR2 may comprise the amino acid sequence shown in any one of SEQ ID NO:21, 36, 42, 48, 11.

[0133] In some embodiments, the antigen-binding protein comprises HCDR3, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:70.

[0134] In some embodiments, the HCDR3 may comprise the amino acid sequence shown in any one of SEQ ID NO:22, 30, 37, 43, 49, 57, 62 and 12.

[0135] In some embodiments, the antigen-binding protein comprises HCDR1, HCDR2 and HCDR3, wherein a) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:20, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:21, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:22, b) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:20, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:21, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:30, c) The HCDR1 comprises the amino acid sequence shown in SEQ ID NO:35, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:36, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:37, d) The HCDR1 comprises the amino acid sequence shown in SEQ ID NO:20, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:42, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:43, e) The HCDR1 comprises the amino acid sequence shown in SEQ ID NO:20, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:48, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:49, f) The HCDR1 comprises the amino acid sequence shown in SEQ ID NO:56, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:36, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:57, g) The HCDR1 comprises the amino acid sequence shown in SEQ ID NO:35, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:36, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:62, h) The HCDR1 comprises the amino acid sequence shown in SEQ ID NO:35, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:42, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:37, i) The HCDR1 comprises the amino acid sequence shown in SEQ ID NO:56, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:36, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:57, or, j) The HCDR1 comprises the amino acid sequence shown in SEQ ID NO:10, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:11, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:12.

[0136] In some embodiments, the antigen-binding protein comprises a heavy-chain variable region VH, and the VH comprises an amino acid sequence shown in any of SEQ ID NOs: 13, 23, 31, 38, 44, 50, 58, 63, and 65.

[0137] In some embodiments, the antigen-binding protein comprises a light-chain variable region VL, and the VL comprises an amino acid sequence shown in any of SEQ ID NOs: 18, 28, 33, 40, 46, 54, 60, and 68.

[0138] In some embodiments, the antigen-binding protein comprises a heavy-chain variable region VH and a light-chain variable region VL, wherein a) the VH comprises the amino acid sequence shown in SEQ ID NO: 23, and the VL comprises the amino acid sequence shown in SEQ ID NO: 28, b) the VH comprises the amino acid sequence shown in SEQ ID NO: 31, and the VL comprises the amino acid sequence shown in SEQ ID NO: 33, c) the VH comprises the amino acid sequence shown in SEQ ID NO: 38, and the VL comprises the amino acid sequence shown in SEQ ID NO: 40, d) the VH comprises the amino acid sequence shown in SEQ ID NO: 44, and the VL comprises the amino acid sequence shown in SEQ ID NO: 46, e) the VH comprises the amino acid sequence shown in SEQ ID NO: 50, and the VL comprises the amino acid sequence shown in SEQ ID NO: 54, f) the VH comprises the amino acid sequence shown in SEQ ID NO: 58, and the VL comprises the amino acid sequence shown in SEQ ID NO: 60, g) the VH comprises the amino acid sequence shown in SEQ ID NO: 63, and the VL comprises the amino acid sequence shown in SEQ ID NO: 60, h) the VH comprises the amino acid sequence shown in SEQ ID NO: 65, and the VL comprises the amino acid sequence shown in SEQ ID NO: 54, i) The VH contains the amino acid sequence shown in SEQ ID NO:58, and the VL contains the amino acid sequence shown in SEQ ID NO:68, or j) The VH contains the amino acid sequence shown in SEQ ID NO:13, and the VL contains the amino acid sequence shown in SEQ ID NO:18.

[0139] In some embodiments, the antigen-binding protein contains a heavy chain constant region, and the heavy chain constant region contains a constant region derived from IgG.

[0140] In some embodiments, the heavy chain constant region contains a constant region derived from a protein selected from the group consisting of IgG1, IgG2, IgG3, and IgG4.

[0141] In some embodiments, the antigen-binding protein contains a light chain constant region, and the light chain constant region contains a constant region derived from Igκ or a constant region derived from Igλ.

[0142] In some embodiments, the light chain constant region contains a constant region derived from human Igκ.

[0143] In some embodiments, the antigen-binding protein contains an antibody or an antigen-binding fragment thereof.

[0144] In some embodiments, the antigen-binding fragment is selected from the group consisting of Fab, Fab’, F(ab)2, Fv fragment, F(ab’)2, scFv, di-scFv, VHH and / or dAb.

[0145] In some embodiments, the antibody is selected from the group consisting of monoclonal antibodies, mouse antibodies, and chimeric antibodies.

[0146] In some embodiments, the detection module includes an apparatus capable of detecting the content of DIR, which is an intron-retaining splicing product of DNA damage-inducible transcript 4-like transcript (DDIT4L), and / or the nucleic acid encoding the DIR.

[0147] In some embodiments, the detection module includes a substance and / or an apparatus capable of detecting the content of a marker associated with cognitive impairment and / or neurodegenerative disease.

[0148] In some embodiments, the markers associated with cognitive impairment and / or neurodegenerative disease include AD7C-NTP, pTau-181, pTau-217, and / or Aβ1-42.

[0149] In some embodiments, the detection module outputs a content value of the DIR and / or the nucleic acid encoding the DIR.

[0150] In some embodiments, the system includes a judgment module for judging the diagnosis result and / or disease progression result of the subject based on the content of the DIR and / or the nucleic acid encoding the DIR and the healthy control value and / or initial control value. The healthy control value includes the content of the DIR and / or the nucleic acid encoding the DIR in a healthy subject, and the initial control value includes the content of the DIR and / or the nucleic acid encoding the DIR detected in the past of the same subject.

[0151] In some embodiments, the healthy subject does not suffer from the cognitive impairment and / or the neurodegenerative disease.

[0152] In some embodiments, when the content of the DIR and / or the nucleic acid encoding the DIR in the sample of the subject is significantly higher than the healthy control value, the judgment module diagnoses that the subject suffers from the cognitive impairment and / or the neurodegenerative disease.

[0153] In some embodiments, the subject was definitively diagnosed with the cognitive impairment and / or the neurodegenerative disease.

[0154] In some embodiments, when the content of the DIR and / or the nucleic acid encoding DIR in the specimen of the subject is significantly higher than the initial control value, the determination module diagnoses that the progression of the cognitive impairment and / or the neurodegenerative disease in the subject has worsened.

[0155] In some embodiments, the determination module outputs a diagnostic result of the subject.

[0156] In some embodiments, the system includes a display module for displaying the diagnostic result of the determination module.

[0157] In some embodiments, the system includes a storage module for storing the content value output to the detection module and / or the diagnostic result output to the determination module.

[0158] In another aspect, provided is a drug screening method including detecting a change in the content of DIR and / or a nucleic acid encoding DIR in a subject after administration of a candidate drug for use in preventing, treating and / or alleviating cognitive impairment and / or neurodegenerative disease.

[0159] In some embodiments, the DIR includes the amino acid sequence shown in SEQ ID NO: 1.

[0160] In some embodiments, the nucleic acid encoding DIR includes the nucleotide sequence shown in SEQ ID NO: 8.

[0161] In some embodiments, the nucleic acid encoding DIR includes the nucleotide sequence shown in SEQ ID NO: 93.

[0162] In some embodiments, the cognitive impairment includes normal age-related cognitive impairment, Lewy body dementia (LBD), frontotemporal dementia and / or vascular dementia.

[0163] In some embodiments, the inducing diseases of the cognitive impairment include Alzheimer's disease, multiple infarction, Parkinson's disease, AIDS and / or Creutzfeldt-Jakob disease (CJD).

[0164] In some embodiments, the cognitive impairment includes mild cognitive impairment (MCI), moderate cognitive impairment and severe cognitive impairment.

[0165] In some embodiments, the cognitive impairment includes amnestic MCI with multiple cognitive domain impairments (aMCI-m).

[0166] In some embodiments, the neurodegenerative disease includes acute neurodegenerative disease and chronic neurodegenerative disease.

[0167] In some embodiments, the neurodegenerative disease includes neurodegenerative diseases caused by nerve cell death and glial cell homeostasis, neurodegenerative diseases caused by aging, neurodegenerative diseases caused by affected CNS cell functions, neurodegenerative diseases caused by abnormal intercellular transmission and / or neurodegenerative diseases caused by disorders of cell motility.

[0168] In some embodiments, the neurodegenerative disease includes Alzheimer's disease, Parkinson's disease, multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS) and / or Huntington's disease (HD).

[0169] In some embodiments, the neurodegenerative disease includes juvenile dementia.

[0170] In some embodiments, the neurodegenerative disease includes mild juvenile dementia, moderate juvenile dementia and / or severe juvenile dementia.

[0171] In some embodiments, when the candidate drug is administered, if the content of DIR and / or the nucleic acid encoding DIR in the subject decreases, the candidate drug has a therapeutic effect.

[0172] In some embodiments, the administration includes an injection.

[0173] In some embodiments, the drug includes a small molecule drug and / or a biopolymer drug.

[0174] In the first aspect of the present application, provided is the use of a substance for detecting DIR, which is an intron-retaining splicing product encoding DNA damage-inducible transcript 4-like transcript (DDIT4L) derived from a subject sample, in the preparation of a product (such as a kit) for performing the diagnosis of the subject's juvenile dementia, mild cognitive impairment diagnosis and / or the evaluation of the progression of juvenile dementia (such as grading or disease stage).

[0175] In some embodiments, the subject is selected from a human, a non-human primate, a rodent (e.g., a rat, a mouse, a guinea pig), a dog, a cat, a horse, a cow, a sheep.

[0176] In some embodiments, the subject does not suffer from cancer.

[0177] In some embodiments, the subject does not suffer from a malignant tumor.

[0178] In some embodiments, the DIR has a nucleotide sequence shown in SEQ ID NO:2 (mRNA), a coding nucleotide molecule corresponding to the DIR peptide having the nucleotide sequence shown in SEQ ID NO:3, or a DIR peptide having the amino acid sequence shown in SEQ ID NO:4, a variant obtained by one or more nucleotide and / or amino acid substitutions, deletions and / or additions to the sequence, and a sequence selected from the group consisting of sequences having a high degree of homology with the sequence (e.g., sequence homology higher than 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% and 99.5%).

[0179] In some embodiments, the juvenile dementia includes mild (amnesia stage), moderate (neuropathy stage) and severe (severe cognitive impairment stage).

[0180] In some embodiments, the substance is selected from reagents, devices or combinations thereof used for DIR detection.

[0181] In some embodiments, the substance is a substance for detecting the DIR at the gene level and / or protein level. For example, the substance is a substance used in one or more detection techniques or methods selected from the group consisting of immunohistochemistry (immunofluorescence assay, chemiluminescence method, reverse enzyme-linked immunosorbent assay, immunocolloidal gold method), Western blotting, Northern blotting, PCR, biochip method, protein chip method.

[0182] In some embodiments, the substance is selected from substances having specificity for the DIR such as antibodies (such as monoclonal antibodies) of the DIR, splicing product-specific probes, gene chips, PCR primers, gRNAs, etc.

[0183] In some embodiments, the specimen is selected from blood specimens such as whole blood, serum, plasma, tissue or cell specimens, cerebrospinal fluid.

[0184] In some embodiments, the blood sample is fresh blood or cryopreserved blood.

[0185] In some embodiments, an increase in the DIR level in the sample as compared to healthy control values indicates that the subject has early-onset dementia or mild cognitive impairment.

[0186] In some embodiments, an increase in the DIR level in the sample as compared to a mild cognitive impairment control or as compared to an initial control level diagnosed with early-onset dementia indicates that the subject's early-onset dementia is progressing (e.g., the grade classification or disease stage has worsened).

[0187] In some embodiments, an increase in the DIR level in the sample as compared to control values at a low grade or low disease stage indicates that the subject's early-onset dementia is progressing (e.g., the grade classification or disease stage has worsened).

[0188] In another aspect, the present application provides a diagnostic kit comprising a substance capable of detecting DIR, which is an intron-retaining splicing product of DNA damage-inducible transcript 4-like transcript (DDIT4L) as described in the present application, and / or a nucleic acid encoding said DIR.

[0189] For example, the present application provides (i) a substance used for detecting DIR in a control sample, and (ii) a product (such as a kit) used for diagnosing early-onset dementia, diagnosing mild cognitive impairment, and / or evaluating the progression of early-onset dementia (such as grade classification or disease stage), comprising, optionally selected, other substances such as substances used for detecting existing early-onset dementia markers.

[0190] In some embodiments, the substance in (i) is a reagent, device, or combination thereof used for detecting DIR Substances used in one or more detection techniques or methods selected from the group consisting of immunohistochemistry (immunofluorescence assay, chemiluminescence method, reverse enzyme-linked immunosorbent assay, immunocolloidal gold method), Western blotting, Northern blotting, PCR, biochip method, and protein chip method, etc., substances for detecting the DIR at the gene level and / or protein level, or Substances having specificity for the DIR, such as antibodies against the DIR (preferably monoclonal antibodies), splicing product-specific probes, gene chips, PCR primers, gRNAs, etc., are selected from one or more of the group.

[0191] In some embodiments, the other substances in (ii) are substances used for detecting one or more markers of early-onset dementia, such as AD7C-NTP, pTau-181, pTau-217, Aβ1-42, etc., and / or reagents for brain amyloid imaging, and / or reagents used for brain imaging diagnosis of a subject.

[0192] In some embodiments of the present application, after administering the candidate drug, detecting the effect of the candidate drug on the DIR level in a subject, such that the decrease in the level indicates that the candidate drug has a therapeutic effect on early-onset dementia, to provide a method for screening a therapeutic drug for early-onset dementia.

[0193] In some embodiments, the detection is performed using the products described in the present application.

[0194] In some embodiments of the present application, providing a method for diagnosing early-onset dementia, diagnosing mild cognitive impairment, and / or evaluating the progression of cognitive impairment, including detecting the DIR level in a sample of the subject, thereby diagnosing early-onset dementia and / or evaluating the progression of early-onset dementia (such as grading or disease stage, etc.) of the subject.

[0195] In some embodiments, the detection is performed using the product described in the present application.

[0196] In some embodiments, an increase in the DIR level in the specimen, compared to a control value, indicates that the subject has early-onset dementia, or an increase in the DIR level in the specimen, compared to a low-grade or early-stage control, indicates that the subject's early-onset dementia is progressing (e.g., the grade classification or stage has worsened).

[0197] In some embodiments of the present application, (a) an apparatus for collecting and / or receiving DIR level data in a specimen, (b) an apparatus for analyzing data to perform an early-onset dementia diagnosis, mild cognitive impairment diagnosis, and / or assessment of the progression of cognitive impairment (such as grade classification or stage), such that an increase in the DIR level in the specimen, compared to a healthy control value, indicates that the subject has early-onset dementia or mild cognitive impairment, or an increase in the DIR level in the specimen, compared to a mild cognitive impairment control or an initial control level diagnosed with early-onset dementia, indicates that the subject's early-onset dementia is progressing (e.g., the grade classification or stage has worsened). The present application further provides a system corresponding to the method of the present application, including the above.

[0198] In some embodiments, the system further includes (a') an apparatus for collecting and / or receiving other clinical indicators or basic characteristic data related to the early-onset dementia diagnosis and / or progression of early-onset dementia in a specimen.

[0199] In some embodiments, for performing a prognostic evaluation related to the early-onset dementia diagnosis and / or progression of early-onset dementia of the subject, the apparatus described in (b) analyzes the DIR level data together with other clinical indicators or basic characteristic data related to the early-onset dementia diagnosis and / or progression of early-onset dementia.

[0200] Alternatively, in some embodiments, the system further includes an apparatus for analyzing other clinical indicators or basic feature data related to early-onset dementia diagnosis and / or early-onset dementia progression, and / or an apparatus for jointly analyzing DIR level data with other clinical indicators or basic feature data related to early-onset dementia diagnosis and / or early-onset dementia progression in order to evaluate the early-onset dementia diagnosis and / or early-onset dementia progression of the subject.

[0201] In some embodiments, the system further includes one or more apparatuses selected from the group consisting of an apparatus used for data input and / or output and / or storage, an apparatus for uploading and / or storing the analysis results to a cloud database, or an apparatus used for output and / or storage of the analysis results such as its corresponding cloud database apparatus, and an apparatus for intelligently analyzing and recommending treatment methods based on the prognosis evaluation results.

[0202] It should be understood that those skilled in the art can add other general apparatuses or programs to the system as needed.

[0203] Those skilled in the art can easily gain insights into other aspects and advantages of the present application from the following detailed description. The following detailed description only shows and describes exemplary embodiments of the present application. As those skilled in the art will recognize, the content of the present application enables those skilled in the art to make changes to the disclosed specific embodiments without departing from the spirit and scope of the invention according to the present application. Therefore, the descriptions in the accompanying drawings and the specification of the present application are merely exemplary and not restrictive.

Brief Description of the Drawings

[0204] The specific features of the invention according to the present application are as described in the claims. By referring to the exemplary embodiments and the accompanying drawings described in detail in the present application, the features and advantages of the invention according to the present application can be better understood. A schematic description of the accompanying drawings is as follows.

[0205] Figures 1a-1b show the splicing pattern of DDIT4L mRNA precursor and the situation where the DIR protein is secreted into the medium from transfected cells. Here Panel a: The splicing pattern of DDIT4L. The length of the mRNA obtained by removing introns through normal splicing is 582 nt (SEQ ID NO:95). In the mRNA obtained by abnormal intron retention splicing, the length of the RNA involved in the coding of DIR is 255 nt (SEQ ID NO:8). Panel b: DIR is secreted from cells into the medium. It was confirmed that after transfection of HEK293 cells with the pCMV-flag-DIR plasmid and culturing for 48 hours, the DIR protein secreted from the medium could be detected. A DIR protein band was observed in the lysate DIR plasmid lane, and its size is 12 kDa.

[0206] Figures 2a-2b show the changes in the DIR protein content in the blood of AD patients and patients with various other diseases.

[0207] Panel a: Changes in the DIR content in the blood of AD patients and patients with various other diseases detected by Western blotting. The plasma protein in the figure is the light chain of immunoglobulin used as an internal standard.

[0208] Panel b: Changes in the DIR content in the blood of AD patients, MCI patients, and AD / MCI patients detected by ELISA. *** in the figure represents P<0.001, and * represents P<0.05.

[0209] Figure 3 is the calibration curve of the standard DIR protein detected by the ELISA method.

[0210] Figure 4 is the R℃ curve of clinical specimens detected by ELISA.

[0211] Figure 5 shows the changes in DIR content in the blood of different types of MCI patients.

[0212] Figure 6 shows the changes in DIR and other indicator contents in the blood of different types of MCI and AD patients.

[0213] Figure 7 shows the correlation of DIR expression levels in plasma and cerebrospinal fluid.

[0214] Figure 8 shows the detection results of the binding activity of the DIR antibody described in the present application.

Embodiments for Carrying Out the Invention

[0215] Specific Embodiments Hereinafter, embodiments of the present invention will be described using specific specific embodiments. However, those skilled in this technology can easily understand other advantages and effects of the present invention by the disclosure in the present application. [Term Definitions] The term "DDIT4L" usually refers to DNA damage-inducible transcript 4-like, DNA damage-inducible transcript 4-like. It is also called REDD2 / RTP801L. DDIT4L has been shown in research to be associated with cardiac dysfunction. It can also be used in the treatment of gliomas. The GenBank accession number of the human DDIT4L gene is 115265, and the GenBank accession number of the human DDIT4L protein is NP_660287.1.

[0216] ​​The term "QDLIR" generally refers to the DDIT4L intron-retaining splicing product. In the present application, the amino acid sequence of the QDLIR may be SEQ ID NO:1. In the present application, QDLIR can be used interchangeably with the intron-retaining DIR gene generated by abnormal splicing during the expression of the human DDIT4L gene. In the present application, the gene encoding the DIR is also called the DIR gene. DIR may include the amino acid sequence encoded by the first exon (the amino acid sequence is shown in SEQ ID NO.2) and the amino acid sequence encoded by the retained intron (the amino acid sequence is shown in SEQ ID NO.3). Here, the amino acid sequence (IR) encoded by the retained intron can be divided into two parts: IR-I consisting of the first 27 amino acids from the N-terminus (i.e., DIR-I with the amino acid sequence shown in SEQ ID NO.4) and DIR-II consisting of the last 27 amino acids (i.e., DIR-II with the amino acid sequence shown in SEQ ID NO.5). The term "expression level" usually refers to the protein, RNA, or mRNA level of a specific related gene. The expression level of a specific related gene (such as the human DDIT4L gene) can be detected using any method known in the art. In the present application, the term "expression" generally refers to the process of converting the information encoded by a gene into a structure that exists and functions within a cell. For example, it may include reverse transcription and amplification analysis (such as PCR, ligation RT-PCR, or quantitative RT-PCT), hybridization analysis, RNA blotting method (Northern blotting), dot blotting method, in situ hybridization, gel electrophoresis, capillary electrophoresis, column chromatography, Western blotting method, immunohistochemistry, immunostaining, or mass spectrometry. A biological sample or a protein / nucleic acid separated from the sample can be directly analyzed.

[0217] The term "neurodegenerative disease" generally refers to cognitive impairments such as dementia caused by the progressive loss of nerve cell structure and function, including nerve cell death and glial cell homeostasis. In some cases, age (e.g., Alzheimer's disease (AD), Parkinson's disease (PD)) or genetic mutations that affect CNC cell function (Huntington's disease, early-onset AD or PD, amyotrophic lateral sclerosis (ALS)) can cause the neurodegenerative disease. The neurodegenerative disease may have changes and / or disorders resulting from protein misfolding and aggregation, neuroinflammation (CNS inflammation caused by toxic stimuli (protein aggregation), infection, trauma, or stimulation by autoimmune signaling, etc.), changes in cell signaling, acquired aging / cell death (e.g., disruption of apoptosis signaling, mitochondrial dysfunction, impairment of autophagy, activation of necroptosis by stress / inflammation), damage to motor cells, and epigenetic changes.

[0218] The term "Alzheimer's disease" generally refers to early-onset dementia and dementia, which are neurodegenerative diseases that develop slowly and worsen over time. The most common initial symptom is loss of short-term memory (difficulty remembering recent events), and as the disease progresses, at least one symptom such as language impairment, disorientation (e.g., getting lost easily), emotional instability, loss of motivation, inability to take care of oneself, and behavioral problems may gradually appear. The true cause of Alzheimer's disease is still unknown, and its progression may be related to the deposition of fibrillar amyloid plaques and Tau protein in the brain. Currently, there is no treatment that can stop or reverse the progression, and there is only treatment that can temporarily relieve or improve a few symptoms.

[0219] The term "young-onset dementia" can be used interchangeably with the term "Alzheimer's disease" in this application. The young-onset dementia may include mild young-onset dementia, moderate young-onset dementia and / or severe young-onset dementia. For example, the learning and memory impairments of the young-onset dementia patients will become increasingly prominent, and in some cases, language impairment, executive impairment, cognitive impairment (agnosia) and / or skill execution impairment (apraxia) will occur. For example, the moderate young-onset dementia patients may lose the ability to live independently and may be unable to perform most of their daily lives (in some cases, they may suffer from anomia, paraphasia, and / or anosognosia). For example, the severe young-onset dementia patients may rely on caregivers. For example, language skills may be completely lost. For example, they may be unable to eat by themselves.

[0220] The term "cognitive impairment" generally refers to diseases and symptoms that are considered to be, actually involved in, or related to the progressive loss (including neuronal death) of neuronal structure and / or function. For example, the characteristics of the cognitive impairment may include impairments in cognition (memory, attention, perception and / or thinking). These impairments may include pathogen-induced cognitive impairments such as HIV-related cognitive impairment and Lyme disease-related cognitive impairment. Examples of cognitive impairment include Alzheimer's disease, Huntington's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), autism, mild cognitive impairment (MCI), stroke, traumatic brain injury (TBI) and / or age-associated memory impairment (AAMI).

[0221] "Mild Cognitive Impairment (MCI)" generally refers to an intermediate clinical state between normal cognition and cognitive impairment. In some cases, the MCI may include a cognitive impairment that meets the criteria for dementia but progresses more than normal aging. MCI is diverse in clinical symptoms, etiology, prognosis, and prevalence. In some cases, MCI can be a pathological stage of Alzheimer's disease. Some forms of cognitive impairment are considered early symptoms of neurodegenerative diseases that ultimately lead to dementia. In some cases, the MCI may include subtypes selected from the group consisting of aMCI-s: amnestic MCI with a single cognitive domain impairment, aMCI-m: amnestic MCI with multiple cognitive domain impairments, naMCI-s: non-amnestic MCI with a single cognitive domain impairment, and naMCI-m: non-amnestic MCI with multiple cognitive domain impairments.

[0222] The term "normal age-related cognitive impairment" generally refers to a cognitive impairment caused by normal aging. For example, the normal age-related cognitive impairment may appear as confusion about the location of familiar places, taking more time than usual to complete daily tasks, changes in mood or personality, etc.

[0223] The term "Lewy Body Dementia (LBD)" generally refers to Lewy Body Dementia. The characteristic of Lewy Body Dementia is the abnormal accumulation of proteins into masses called Lewy bodies. Lewy Body Dementia causes a gradual decline in mental abilities. Patients with Lewy Body Dementia may experience hallucinations and changes in attention and concentration. Other effects include muscle rigidity, slow movement, difficulty walking, tremors, etc. Patients with Lewy bodies in the brain may also have plaques and tangles related to Alzheimer's disease.

[0224] The term "frontotemporal dementia" generally refers to Pick's disease, a progressive rare disease in which the tau protein affects only the frontal and temporal lobes of the brain. Patients with frontotemporal dementia have difficulty with higher-level reasoning, expressive language, language perception, and memory formation. The frontal and temporal lobes of the brains of patients with frontotemporal dementia atrophy over time.

[0225] The term "vascular dementia" generally refers to problems with reasoning, judgment, and memory caused by impaired blood flow to the brain. For example, the vascular dementia may include dementia caused by risk factors for heart disease and stroke such as high blood pressure and high cholesterol.

[0226] The term "multiple infarcts" generally refers to small non-cortical infarcts caused by occlusion of a single perforating branch of a major cerebral artery. The multiple infarcts may be a specific type of cerebral infarction also called ischemic cerebral infarction. The multiple infarcts may present as hemisensory disturbance, aphasia, dysarthria, bradykinesia, and clumsiness (especially difficulty with fine movements such as writing).

[0227] The term "Parkinson's disease" generally refers to a progressive neurodegenerative disease. The clinical features of the Parkinson's disease (PD) may include motor symptoms (such as tremors, bradykinesia, muscle rigidity, and postural instability), neuropsychiatric symptoms, and other non-motor symptoms. For example, the non-motor symptoms may include cognitive impairment, dementia, mood disorders (such as depression, anxiety, and apathy), and sleep disorders.

[0228] The term "Creutzfeldt-Jakob disease (CJD)" generally refers to a transmissible spongiform encephalopathy that occurs in humans. CJD is a disease caused by prion infection. CJD patients may exhibit delusional behavior, confusion, loss of appetite, weight loss, depression, and in a few cases, visual and auditory abnormalities, and may also exhibit progressive neurological deterioration (such as paresthesia, language disorders, aphasia, etc.) in the advanced stage.

[0229] The term "multiple sclerosis (MS)" generally refers to a demyelinating neuropathy. When the insulating substance (myelin sheath) on the surface of nerve cells in the brain or spinal cord of MS patients is damaged and the signal transmission of the nervous system is damaged, a series of symptoms may be caused, which may affect the patient's activities, psychology, and even mental state. These symptoms may include diplopia, unilateral visual impairment, muscle weakness, sensory dulling, and impaired coordination.

[0230] The term "amyotrophic lateral sclerosis (ALS)" generally refers to amyotrophic lateral sclerosis, a motor neuron disease, which is a progressive and fatal neurodegenerative disease. Among them, a small number of ALS patients may develop frontotemporal dementia. Some ALS patients have reduced hearing, vision, touch, smell, and taste, and a very small number of amyotrophic lateral sclerosis patients develop dementia simultaneously.

[0231] The term "Huntington's disease (HD)", also known as Huntington's chorea, generally refers to a genetic disease that causes brain cell death. As the disease progresses in HD patients, the body's movement disorders become more apparent, their abilities gradually decline, and ultimately they have difficulty moving and cannot speak. Their mental abilities usually decline into dementia.

[0232] The term "advanced age" generally refers to the advanced age of a subject. For example, in the case of humans, the advanced age can be 60 years or older, 70 years or older, or 75 years or older, and in the case of mice, the advanced age can be 10 months or older, such as 13 months or older or 18 months or older. In some cases, the subjects at the advanced age may have one or more symptoms of learning disability, memory impairment, memory deficit, and / or brain dysfunction.

[0233] The term "variant" generally refers to a polypeptide comprising an amino acid sequence that differs from the amino acid sequence of a parent polypeptide or a reference polypeptide (such as a wild-type polypeptide) by at least one amino acid residue. In the present application, the variant may have a high (e.g., at least 80%) homology with the parent polypeptide or the reference polypeptide. The homology may include sequence similarity or identity. In the present application, the homology can be determined using standard techniques known in the art (see, for example, Smith and Waterman, Adv. Appl. Math.). The percentage of identity shared by a polynucleotide or polypeptide sequence is obtained by direct comparison of the sequence information between molecules, and the comparison is performed by sequence alignment and determination of identity by methods known in the art. An example of an algorithm applied to sequence similarity determination is the BLAST algorithm (see Altschul et al., J. Mol. Biol., 215:403-410

[1990] ). The software used for performing BLAST analysis is generally available from the National Center for Biotechnology Information (NCBI).

[0234] In the present application, the term "CDR" is also referred to as "complementary determining region" and generally refers to the region of the variable domain of an antibody that has a highly variable sequence and / or forms a structurally defined loop. For example, an antibody can include six CDRs, three in VH (HCDR1, HCDR2, HCDR3) and three in VL (LCDR1, LCDR2, LCDR3). In some embodiments, naturally occurring camel antibodies composed of only heavy chains function normally and are stable in the absence of light chains. Antibody CDRs are determined by various coding systems such as CCG, Kabat, Chothia, IMGT, Kabat / Chothia. These coding systems are known in the art and specific reference can be made to www.bioinf.org.uk / abs / index.html#kabatnum. For example, the amino acid sequence numbering of the antigen-binding protein can refer to the IMGT numbering scheme (IMGT, the international ImMunoGeneTics information system @imgt.cines.fr; imgt.cines.fr; Lefranc et al., 1999, Nucleic Acids Res. 27: 209-212; Ruiz et al., 2000 Nucleic Acids Res. 28: 219-221; Lefranc et al., 2001, Nucleic Acids Res. 29: 207-209; Lefranc et al., 2003, Nucleic Acids Res. 31: 307-310; Lefranc et al., 2005, DevComp Immunol 29: 185-203).For example, the CDRs of the antigen-binding protein can be determined by the Kabat numbering system (see, for example, Kabat EA & Wu TT (1971) Ann NY Acad Sci 190:382-391 and Kabat EA et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242).

[0235] In the present application, the term "FR" generally refers to the more highly conserved portions of the variable domain of an antibody, which are generally referred to as framework regions. For example, the variable domains of the natural heavy and light chains can each include four FR regions, namely, four in VH (H-FR1, H-FR2, H-FR3, and H-FR4), and four in VL (L-FR1, L-FR2, L-FR3, and L-FR4).

[0236] In the present application, the terms "variable domain" and "variable region" are used interchangeably and generally refer to portions of the antibody heavy and / or light chains. The variable domains of the heavy and light chains are referred to as "VH" and "VL" (or are called "VH" and "VL", respectively). These domains generally have the greatest variability (relative to other antibodies of the same type) and may include the antigen-binding site. In the present application, the term "variable" generally refers to the possibility that specific segments of the variable domain can vary greatly in sequence between antibodies. The variable domain mediates antigen binding and can determine the specificity of a particular antibody for its specific antigen. However, the variability is not uniformly distributed throughout the variable domain. Generally, it can be concentrated in three segments called the hypervariable regions (CDRs or HVRs) of the light and heavy chain variable domains. The more conserved portions of the variable domain are called the framework regions (FRs). The variable domains of the native heavy and light chains each contain four FR regions, mostly adopt a β-folding structure, form a loop connection via three CDR linkages, and in some cases form part of the β-folding structure. The CDRs of each chain are held in close proximity to each other via the FR regions and together with the CDRs of the other chain form the antigen-binding site of the antibody.

[0237] In the present application, the term "antibody" generally refers to an immunoglobulin or a fragment or derivative thereof that contains an antigen-binding site, whether generated in vitro or in vivo. This term can include, but is not limited to, polyclonal, monoclonal, monospecific, multispecific, nonspecific, humanized, single-chain, chimeric, synthetic, recombinant, hybrid, mutant, and xenogeneic antibodies. Unless the term "complete" is specifically modified, the term "antibody" in "complete antibody" includes, for the purposes of the present application, antibody fragments such as Fab, F(ab’)2, Fv, scFv, Fd, VHH, dAb, and other antibody fragments that retain the antigen-binding function (e.g., specifically bind to human DIR). Generally, such fragments can contain an antigen-binding domain. The basic four-chain antibody unit can be a heterotetrameric glycoprotein consisting of two identical light (L) chains and two identical heavy (H) chains. IgM antibodies are composed of five basic heterotetrameric units and another polypeptide called the J chain and can contain ten antigen-binding sites, while IgA antibodies contain two to five basic four-chain units and can bind to the J chain to form a multivalent bond. In the case of IgG, the four-chain unit is usually about 150,000 daltons. Each L chain is linked to the H chain by one covalent disulfide bond, and the two H chains can be linked to each other by disulfide bonds that depend on one or more H-chain isotypes. Each H and L chain can also have regularly spaced intrachain disulfide bridges. Each H chain can have a variable domain (VH) at the N-terminus, followed by three constant domains (CH) in the α and γ chains and four CH domains in the μ and ε isotypes. Each L chain can have a light-chain variable region (VL) at the N-terminus and a constant domain at the other end. VL corresponds to VH, and the light-chain constant region (CL) can correspond to the first constant domain (CH1) of the heavy chain. Certain amino acid residues are thought to form the interface between the light and heavy-chain variable domains. The pair of VH and VL can together form a single antigen-binding site. The L chains of all vertebrates can be classified into one of two distinct types called κ and λ based on the amino acid sequence of their constant domains.The amino acid sequence of the constant domain of the heavy chain constant region (CH) can classify immunoglobulins into different classes or isotypes. Currently, there are five types of immunoglobulins, namely IgA, IgD, IgE, IgG, and IgM, such as IgG1, IgG2, IgG3 and / or IgG4, and IgM, which are named α, δ, ε, γ, and μ, respectively.

[0238] In this application, the term "antigen-binding fragment" generally refers to one or more fragments having the ability to specifically bind to an antigen (such as DIR). In this application, the antigen-binding fragment may include Fab, Fab’, F(ab)2, Fv fragment, F(ab’)2, scFv, di-scFv, VHH and / or dAb.

[0239] In this application, the term "Fab" generally refers to the antigen-binding fragment of an antibody. As described above, a complete antibody can be digested using papain. The antibody is digested by papain to generate two identical antigen-binding fragments, namely, the "Fab" fragment and the remaining "Fc" fragment (i.e., the Fc region). The Fab fragment may be composed of a complete light chain, the variable region of the heavy chain, and the first constant region (CH1) of the H chain (VH).

[0240] In this application, the term "F(ab)2" generally refers to the antigen-binding fragment of an antibody. For example, F(ab)2 may be composed of two linked Fab fragments.

[0241] In this application, the term "Fab′" generally refers to a monovalent antigen-binding fragment of a human monoclonal antibody that is slightly larger than the Fab fragment. For example, the Fab′ fragment may include all light chains, all heavy chain variable regions, and all or part of the first and second constant regions of the heavy chain. For example, the Fab′ fragment may further include all or part of the 220-330 amino acid residues of the heavy chain.

[0242] In the present application, the term "F(ab’)2" generally refers to an antibody fragment produced by digesting a complete antibody with pepsin. The F(ab’)2 fragment contains two Fab fragments linked by disulfide bonds and a portion of the hinge region. The F(ab’)2 fragment has bivalent antigen-binding activity and can crosslink antigens.

[0243] In the present application, the term "Fv fragment" generally refers to a monovalent antigen-binding fragment of a human monoclonal antibody consisting of all or part of the heavy-chain variable region and the light-chain variable region and lacking the heavy-chain constant region and the light-chain constant region. The heavy-chain variable region and the light-chain variable region may contain, for example, CDRs. For example, the Fv fragment contains all or part of the amino-terminal variable regions of approximately 110 amino acids of the heavy and light chains.

[0244] In the present application, the term "scFv" generally refers to a fusion protein containing an antibody fragment having at least one light-chain variable region and an antibody fragment having at least one heavy-chain variable region, wherein the variable regions of the light and heavy chains are contiguous (e.g., via a synthetic linker such as a short flexible polypeptide linker), can be expressed as a single-chain polypeptide, and the scFv retains the specificity of the complete antibody from which it is derived. Unless otherwise specified, as used in the present application, the scFv may have the VL and VH variable regions in any order (e.g., with respect to the N-terminus and C-terminus of the polypeptide), and the scFv may contain VL-linker-VH or VH-linker-VL.

[0245] In the present application, the term "dAb" generally refers to an antigen-binding fragment consisting of an H domain or a VL domain. See, for example, Ward et al. (Nature, 1989 Oct 12; 341(6242): 544-6), Holt et al., Trends Biotechnol., 2003, 21(11): 484-490.

[0246] In the present application, the term "VHH" generally refers to an antibody having a variable antigen-binding domain of a heavy-chain antibody (see Vanlandschoot P. et al., 2011, Antiviral Research 92, 389-407). VHH is also called a nanobody (Nb).

[0247] In the present application, the term "monoclonal antibody" generally refers to an antibody molecule product consisting of a single molecule. Monoclonal antibodies are generally highly specific for a single antigenic site. Furthermore, while conventional polyclonal antibody preparations usually have different antibodies against different determinants, each monoclonal antibody can be directed against a single determinant cluster on the antigen. Monoclonal antibodies, in addition to their specificity, have the advantage that they can be synthesized by hybridoma culture and are not contaminated with other immunoglobulins. The modifier "monoclonal" indicates the property of an antibody obtained from a substantially homogeneous antibody population and is not construed as requiring a specific antibody production method. For example, the monoclonal antibodies used in the present application may be prepared using hybridoma cells or by recombinant DNA methods.

[0248] In the present application, the term "chimeric antibody" generally refers to an antibody in which the variable region is derived from one species and the constant region is derived from another species. Generally, since the variable region is derived from an antibody of an experimental animal such as a rodent ("parent antibody") and the constant region is derived from a human antibody, the resulting chimeric antibody is less likely to cause an immune reaction harmful to a human individual than the parent (e.g., mouse-derived) antibody.

[0249] In the present application, the term "humanized antibody" generally refers to an antibody in which some or all of the amino acids outside the CDR domains of a non-human antibody (e.g., a mouse antibody) are replaced with the corresponding amino acids from a human immunoglobulin. Also, small additions, deletions, insertions, substitutions, or modifications of amino acids in the CDR regions may be tolerated as long as the antibody still retains its ability to bind to a specific antigen. A humanized antibody may optionally include at least a portion of a human immunoglobulin constant region. A "humanized antibody" may retain the same antigen specificity as the original antibody. The "humanized" form of a non-human (e.g., mouse) antibody can be composed of a minimal configuration from a chimeric antibody derived from the sequence of a non-human immunoglobulin. In certain embodiments, the residues of the CDR domains of a human immunoglobulin (acceptor antibody) may be replaced with the residues of the CDR domains of a non-human species (donor antibody) (e.g., mouse, rat, rabbit, or non-human primate) having the desired properties, affinity, and / or ability. In certain embodiments, the residues of the FR domains of a human immunoglobulin may be replaced with the corresponding non-human residues. Additionally, a humanized antibody may include amino acid modifications that are not present in the recipient antibody or the donor antibody. These modifications can be made to further improve the performance of the antibody, such as its binding affinity.

[0250] In the present application, the term "fully human antibody" generally refers to an antibody that contains only human immunoglobulin protein sequences. A fully human antibody may contain mouse glycans if produced in a mouse, mouse cells, or a hybridoma derived from mouse cells. Similarly, a "mouse-derived antibody", "mouse antibody", or "rat antibody" refers to an antibody that contains only the immunoglobulin sequences of a mouse or a rat, respectively. A fully human antibody can be generated in transgenic animals having human and human immunoglobulin germline sequences by phage display or other molecular biology methods. Exemplary techniques for making antibodies are known in the art.

[0251] In the present application, the term "antigen-binding protein" generally refers to a portion that binds to an antigen and, optionally, a scaffold or framework portion that enables the adoption of a conformation that promotes the binding of the antigen-binding protein to the antigen. Examples of antigen-binding proteins include, but are not limited to, antibodies, antigen-binding fragments (Fab, Fab’, F(ab)2, Fv fragment, F(ab’)2, scFv, di-scFv, VHH and / or dAb), immune complexes, multispecific antibodies (such as bispecific antibodies), antibody fragments, antibody derivatives, antibody analogs, or fusion proteins, provided that they exhibit the desired antigen-binding activity. The "antigen-binding protein" of the present application may be composed of a portion that binds to an antigen and, optionally, a scaffold portion or conformational portion that enables the adoption of a conformation that facilitates the binding of the antigen by the antigen-binding portion.

[0252] The term "about" generally refers to a numerical range that is 20% more or less than the specified value. For example, "about X" includes a numerical range of ±20%, ±10%, ±5%, ±2%, ±1%, ±0.5%, ±0.2% or ±0.1% of X, where X is a numerical value.

[0253] Detailed Description of the Invention A prerequisite for the early detection and treatment of early-onset dementia is to find its specific diagnostic markers. In the research of the present inventor, it was found that the DIR protein is present in the blood of Alzheimer's disease patients but not detected in blood samples from healthy individuals. This result suggests that DIR may be a blood marker for the diagnosis (presumably early diagnosis) of Alzheimer's disease. At the same time, as a result of detecting blood samples of other patients with cognitive impairment, the inventor found that the level of DIR protein in blood samples of patients with cognitive impairment such as MCI (mild cognitive impairment) is significantly increased. Therefore, it was found that the increase in DIR in blood becomes a new marker in the diagnosis and treatment of early-onset dementia.

[0254] ​​Specifically, under pathological conditions (such as tumors and neurodegenerative diseases), the diseased tissue lacks blood supply, causing a hypoxic state, leading to a series of changes in gene expression, and the appearance of abnormal spliceosomes. Figuring out how to identify specific genes and their spliceosomes may be the key to elucidating the treatment methods for diseases. The inventor has found in previous studies that DIR is an abnormal protein formed by abnormal splicing in the human body and exists only in human tissues and cells. DIR is an important molecule that can adaptively regulate the functions of cells and tissues through high expression and secretion in the hypoxic environment of cells.

[0255] Therefore, the inventor hypothesized that DIR might enter the blood through the blood supply system of the diseased tissue. Subsequently, the inventor further proved the correctness of this hypothesis through the following tests. After introducing the DIR plasmid into HEK293 cells, the inventor detected the presence of a large amount of DIR protein in the culture medium, proving that DIR can be secreted extracellularly. Furthermore, DIR was detected in the plasma of AD and MCI patients, but not detected or only low levels of DIR were detected in the plasma of healthy individuals and non-AD patients. Since this result suggests that DIR specifically exists in the blood of AD and MCI patients, it can be used as a blood marker for AD diagnosis (such as early diagnosis). At the same time, since the DIR protein level has been found to increase significantly in the blood samples of many other patients with cognitive impairment, the blood level of DIR can be used as a new marker in the diagnosis and treatment of AD (juvenile dementia).

[0256] All numerical ranges provided by this application are intended to explicitly include all numerical values between the endpoints of the range and the numerical ranges therebetween. The features mentioned in this application can be combined with the features mentioned in the examples. All features disclosed in this application can be used in any combination, and each feature disclosed in this specification can be replaced with any alternative feature that serves the same, equivalent, or similar purpose. Therefore, unless otherwise specified, the disclosed features are merely general examples of equivalent or similar features.

[0257] As used in this application, "comprising," "having," or "including" includes "comprising," "consisting essentially of," "consisting of," and "consisting of," and "consisting essentially of" and "consisting of" are subordinate concepts of "comprising," "having," or "including."

[0258] In this application, the neurodegenerative disease may include acute neurodegenerative diseases and chronic neurodegenerative diseases. For example, the neurodegenerative disease may include neurodegenerative diseases caused by neuronal death and glial cell homeostasis, neurodegenerative diseases caused by aging, neurodegenerative diseases caused by affected CNS cell function, neurodegenerative diseases caused by abnormal intercellular transmission, and / or neurodegenerative diseases caused by disorders of cell motility.

[0259] In this application, the neurodegenerative disease may include juvenile dementia. For example, the neurodegenerative disease may include mild juvenile dementia, moderate juvenile dementia, and / or severe juvenile dementia.

[0260] In the present application, the cognitive impairment may include mild cognitive impairment (MCI), moderate cognitive impairment, and severe cognitive impairment. For example, the cognitive impairment may include normal age-related cognitive impairment, Lewy body dementia (LBD), frontotemporal dementia, and / or vascular dementia. For example, the inducing diseases of the cognitive impairment may include Alzheimer's disease, multiple infarcts, Parkinson's disease, AIDS, and / or Creutzfeldt-Jakob disease (CJD).

[0261] In the present application, the cognitive impairment may include mild cognitive impairment (MCI) (for example, loss of short-term memory, difficulty in expressing or understanding abstract things, changes in mood and behavior, difficulty in learning new things or following complex instructions, decline in judgment, and / or decline in basic self-awareness ability, etc.), moderate cognitive impairment (for example, confusion between long-term memory and real-life situation memory, inability to express oneself, changes in behavior and personality, emotional instability, and / or the need for assistance in self-care, etc.) or severe cognitive impairment (memory impairment, decline in physical activity and mental state, inability to effectively express or communicate, inability to take care of oneself, and / or disruption of biological rhythms).

[0262] In the present application, the cognitive impairment may include diseases that can induce and cause the cognitive impairment. For example, the subject may suffer from Alzheimer's disease, multiple infarcts, Parkinson's disease, AIDS, and / or Creutzfeldt-Jakob disease (CJD).

[0263] [Diagnostic target DIR] In one aspect, the present application provides the use of DIR, which is an intron-retaining splicing product of DNA damage-inducible transcript 4-like transcript (DDIT4L), and / or a nucleic acid encoding the DIR, in disease diagnosis and / or disease progression evaluation, wherein the disease may include cognitive impairment.

[0264] In one aspect, the present application provides the use of DIR, an intron retention splicing product of DNA damage-inducible transcript 4-like transcript (DDIT4L), and / or a nucleic acid encoding said DIR, in disease diagnosis and / or disease progression assessment, wherein the disease may include neurodegenerative diseases.

[0265] DDIT4L (DNA Damage Inducible Transcript 4 Like, SEQ ID NO:92) is a protein-coding gene related to the mTOR signaling pathway, and its level is increased by oxidized LDL and hypoxia in macrophages.

[0266] After removing the intron from the DDIT4L gene by normal splicing, a nucleotide sequence (SEQ ID NO:94) encoding a 193aa-long DDIT4L protein (SEQ ID NO:95) can be obtained. However, the applicant unexpectedly discovered that this gene also has another special splicing method, namely intron retention splicing. By this special splicing method, a 2212 nt nucleotide sequence can be obtained, and since it can contain a terminator within the intron, a peptide (SEQ ID NO:1), which is a splicing product of 84 aa, was finally obtained. In the present application, the protein obtained by intron retention splicing may be "QDLIR or DIR" (shown in Figure 1a).

[0267] Unless otherwise specified or distinguishable, the term "DIR" as used in the present application may include the mature mRNA of the DDIT4L gene obtained by intron retention splicing, the splicing peptide coding molecule, or the peptide (or DIR protein) that is the splicing product.

[0268] When the splicing product can be an mRNA, it can be selected from (a) a nucleotide molecule having a sequence shown in SEQ ID NO: 93, or (b) a nucleotide molecule having a high sequence homology with the nucleotide molecule of SEQ ID NO: 93 (for example, the sequence homology can be at least 80%, 85%, 90%, 92%, 95%, 98%, 99%, 99.5% and 99.8%).

[0269] When the splicing product can be a DIR coding molecule, it can be selected from (a) a nucleotide molecule having a sequence shown in SEQ ID NO: 8, or (b) a nucleotide molecule having a high sequence homology with the nucleotide molecule of SEQ ID NO: 8 (for example, the sequence homology can be at least 80%, 85%, 90%, 92%, 95%, 98%, 99%, 99.5% and 99.8%).

[0270] When the splicing product can be a DIR protein, it can be selected from (a') a polypeptide molecule having a sequence shown in SEQ ID NO: 1, (b') a protein molecule having a high sequence homology with the polypeptide molecule of SEQ ID NO: 1 (for example, the sequence homology can be at least 80%, 85%, 90%, 92%, 95%, 98%, 99%, 99.5% and 99.8%), (c') a protein molecule obtained by translating any of the nucleotide molecules of (a) or (b) above, or (d') a conservative variant polypeptide of the protein molecule described in any of (a') to (c') above, or its homologous protein or polypeptide, or an active fragment thereof.

[0271] [Diagnostic substances and diagnostic kits] In another aspect, the present application provides a substance for detecting DIR, which is an intron-retaining splicing product of DNA damage-inducible transcript 4-like transcript (DDIT4L), and / or a nucleic acid encoding said DIR, in the preparation of a product for disease diagnosis and / or evaluation of disease progression, wherein the disease may include cognitive impairment.

[0272] In another aspect, the present application provides a substance for detecting a DNA damage-inducible transcript 4-like transcript (DDIT4L) intron retention splicing product DIR and / or a nucleic acid encoding said DIR in the preparation of a product for disease diagnosis and / or disease progression assessment, wherein the disease may include neurodegenerative diseases.

[0273] As used herein, the "substance for detection", "detection reagent", "DIR detection reagent" or "DIR expression level detection reagent" can be used interchangeably, and all refer to substances that are specific for DIR molecules and can be used for the direct or indirect detection of the presence and / or content of DIR molecules. These detection substances can detect DIR at the gene level or protein level.

[0274] In the present application, the substance may include an antigen-binding protein that can specifically bind to DIR, a probe that can specifically bind to DIR, a primer that can specifically amplify a nucleic acid encoding DIR, a gene chip that can specifically detect a nucleic acid encoding DIR, an aptamer that can specifically bind to a nucleic acid encoding DIR, and / or a guide RNA that can specifically bind to a nucleic acid encoding DIR.

[0275] In the present application, the kit may include at least one type (e.g., one type, two types, three types or more) of the above-mentioned substances for detection. For example, the kit may include at least one type (e.g., one type, two types, three types or more) of antigen-binding proteins (such as DIR antibodies) that can specifically bind to DIR. In the present application, when there are antigen-binding proteins with different amino acid sequences in at least one CDR, they can be regarded as different species of antigen-binding proteins.

[0276] Depending on the sequence of the DIR molecule, those skilled in the art can prepare reagents specific to the DIR molecule based on conventional means or obtain them commercially. For example, the detection reagents used in the present application include, but are not limited to, antigen-binding proteins (which can be monoclonal antibodies, etc.) with detection specificity for the DIR molecule, probes, gene chips, PCR primers, gRNAs, etc.

[0277] Furthermore, to facilitate detection, the detection reagents of the present application can also be provided with detectable labels including, but not limited to, radioisotopes, fluorophores, chemiluminescent moieties, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, dyes, metal ions, ligands (such as biotin and haptens), etc.

[0278] The detection reagents of the present application can exist in solution, be immobilized on a carrier (such as a plate, adsorbent, etc.), or exist by other conventional methods in this field as long as their existence method is suitable for DIR detection in the biological field. For example, when the detection reagent of the present application can be a nucleotide probe, it can exist in the form of a biochip (or called a "microarray").

[0279] In the present application, the said product can exist in the form of a kit.

[0280] In the present application, there is further provided a product for diagnosing and / or evaluating the progression of juvenile dementia (such as grade classification or disease stage), which includes any optionally selected other substances such as substances used for detecting existing AD markers, etc.

[0281] In the present application, the said diagnostic kit may include an instruction manual describing a method for performing a cognitive impairment diagnosis and / or an evaluation of the progression of cognitive impairment, and / or a neurodegenerative disease diagnosis and / or an evaluation of the progression of neurodegenerative diseases using the said diagnostic kit.

[0282] According to the requirements of the detection method to be used, an appropriate DIR detection substance can be selected and prepared into a product such as a kit suitable for the detection method to be used. Those skilled in the art can adjust and change the detection method and the reagents included in the product according to the actual conditions and needs.

[0283] Therefore, in the present application, there is further provided a product (such as a detection kit) that may include (i) one or more reagents used for DIR detection in an effective amount for detection, and (ii) one or more substances arbitrarily selected from the group consisting of a container, an instruction manual, a positive control, a negative control, a buffer, a builder, or a solvent (such as a solution for cell suspension or fixation, a detectable label or mark, a solution facilitating nucleic acid hybridization, a solution for cell lysis, or a solution used for nucleic acid purification).

[0284] In one example, the present application provides a detection kit suitable for detecting the expression of DIR protein in a biological specimen by immunohistochemistry. The detection kit may include a blocking solution such as 10% goat serum, a primary antibody such as a rabbit anti-human or mouse DIR monoclonal antibody, a secondary antibody such as a labeled (such as HRP-labeled) or unlabeled goat anti-rabbit, a substrate buffer such as a DAB substrate buffer, a chromogenic solution, optionally a container filled with the above reagents, and an instruction manual.

[0285] The detection kit of the present application may be attached with an instruction manual of the kit that describes a method for detecting using the kit, a method for diagnosing cognitive impairment and / or evaluating the progression of cognitive impairment based on the detection result, and / or a method for diagnosing neurodegenerative diseases and / or evaluating the progression of neurodegenerative diseases. For example, using the kit described in the present application, AD diagnosis and / or AD progression evaluation (such as grading or disease stage) can be performed.

[0286] The product of the present application may further include other reagents used clinically for AD diagnosis and / or AD progression evaluation (such as grading or disease stage) in a subject to assist or verify the results obtained by DIR detection. Those skilled in the art can make ordinary selections based on specific needs.

[0287] [Diagnostic method] In another aspect, the present application provides a method for diagnosing cognitive impairment and / or evaluating the progression of cognitive impairment, which may include detecting the content of DIR, which is an intron-retaining splicing product of DNA damage-inducible transcript 4-like transcript (DDIT4L) in a sample derived from a subject in need thereof, and / or the nucleic acid encoding said DIR.

[0288] In another aspect, the present application provides a method for diagnosing neurodegenerative diseases and / or evaluating the progression of neurodegenerative diseases, which may include detecting the content of DIR, which is an intron-retaining splicing product of DNA damage-inducible transcript 4-like transcript (DDIT4L) in a sample derived from a subject in need thereof, and / or the nucleic acid encoding said DIR.

[0289] In the present application, the subject may include a mammal.

[0290] In the present application, the subject may include a tumor patient.

[0291] In the present application, the subject is in an elderly stage.

[0292] In the present application, the sample may include a blood sample and / or a tissue or cell sample.

[0293] In the present application, the sample may include whole blood, serum, plasma and / or cerebrospinal fluid.

[0294] In the present application, the method includes comparing the content of the DIR and / or the nucleic acid encoding DIR in the sample of the subject with a healthy control value, and the healthy control value may include the content of the DIR and / or the nucleic acid encoding DIR in a healthy subject.

[0295] In the present application, the method may perform a diagnosis of cognitive impairment and / or an evaluation of the progression of cognitive impairment using the kit described in the present application and / or the detection substance described in the present application.

[0296] In the present application, the healthy subject may not be suffering from the cognitive impairment. The healthy subject may not be suffering from the neurodegenerative disease.

[0297] In the present application, when the content of the DIR in the specimen of the subject and / or the nucleic acid encoding DIR is significantly higher than the healthy control value (for example, at least about 20%, at least about 50%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 200%, at least about 500% or more), the subject can be diagnosed as suffering from the cognitive impairment and / or the neurodegenerative disease.

[0298] In the present application, the method may include comparing the content of the DIR in the specimen of the subject and / or the nucleic acid encoding DIR with an initial control value, and the initial control value may include the content of the DIR detected in the past of the same subject and / or the nucleic acid encoding DIR.

[0299] In the present application, the subject has been definitely diagnosed as suffering from the cognitive impairment and / or the neurodegenerative disease. For example, the subject has been definitely diagnosed as suffering from the cognitive impairment, or the subject has been definitely diagnosed as suffering from the neurodegenerative disease.

[0300] In the present application, when the content of the DIR in the specimen of the subject and / or the nucleic acid encoding DIR is significantly higher than the initial control value (for example, at least about 20%, at least about 50%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 200%, at least about 500% or more), the subject can be diagnosed as having the cognitive impairment and / or the neurodegenerative disease progressing.

[0301] In the present application, the method may further include a step of detecting the content of markers related to cognitive impairment and / or neurodegenerative diseases, which are derived from the specimen of the subject.

[0302] In the present application, the markers related to cognitive impairment and / or neurodegenerative diseases may further include AD7C-NTP, pTau-181, pTau-217, and / or Aβ1-42. The detection results of the content of the markers can be used for assisting in the diagnosis of cognitive impairment, and / or evaluating the progression of cognitive impairment, and / or diagnosing neurodegenerative diseases, and / or evaluating the progression of neurodegenerative diseases.

[0303] In the present application, the method may further include a step of observing the brain image of the subject. The results of the brain image can be used for assisting in the diagnosis of cognitive impairment, and / or evaluating the progression of cognitive impairment, and / or diagnosing neurodegenerative diseases, and / or evaluating the progression of neurodegenerative diseases.

[0304] [Diagnostic system] In another aspect, the present application may include a system for diagnosing cognitive impairment and / or evaluating the progression of cognitive impairment, which includes DIR, which is an intron-retaining splicing product of DNA damage-inducible transcript 4-like transcript (DDIT4L), and / or a substance for detecting a nucleic acid encoding the DIR.

[0305] In another aspect, the present application may include a system for diagnosing neurodegenerative diseases and / or evaluating the progression of neurodegenerative diseases, which includes DIR, which is an intron-retaining splicing product of DNA damage-inducible transcript 4-like transcript (DDIT4L), and / or a substance for detecting a nucleic acid encoding the DIR.

[0306] In the present application, the system may include a detection module for detecting the content value of the DIR and / or the nucleic acid encoding the DIR.

[0307] In the present application, the detection module detects the content value of the DIR and / or the nucleic acid encoding the DIR in the sample collected by the collection module.

[0308] In the present application, the detection module may contain a substance capable of detecting the content of the DIR and / or the nucleic acid encoding the DIR.

[0309] In the present application, the substance may include an antigen-binding protein capable of specifically binding to DIR, a probe capable of specifically binding to DIR, a primer capable of specifically amplifying the nucleic acid encoding DIR, a gene chip capable of specifically detecting the nucleic acid encoding DIR, an aptamer capable of specifically binding to the nucleic acid encoding DIR, and / or a guide RNA capable of specifically binding to the nucleic acid encoding DIR.

[0310] In the present application, the kit may include at least one type (for example, one type, two types, three types or more) of the detection module. For example, the detection module may include at least one type (for example, one type, two types, three types or more) of antigen-binding proteins (such as DIR antibodies) capable of specifically binding to DIR. In the present application, when there are antigen-binding proteins with different amino acid sequences in at least one CDR, they can be regarded as antigen-binding proteins of different species.

[0311] In the present application, the detection module may include a device capable of detecting the content of DIR, which is an intron-retaining splicing product of DNA damage-inducible transcript 4-like transcript (DDIT4L), and / or the nucleic acid encoding the DIR. For example, the device may include devices required for qPCR, ELISA, Western blotting, and / or gene chips.

[0312] In the present application, the detection module may include a substance and / or a device capable of detecting the content of markers related to cognitive impairment and / or neurodegenerative diseases.

[0313] In the present application, the markers related to cognitive impairment and / or neurodegenerative diseases may further include AD7C-NTP, pTau-181, pTau-217, and / or Aβ1-42. For example, the substance may include an antibody capable of specifically binding to the marker, a probe capable of specifically binding to the marker, a primer capable of specifically amplifying the nucleic acid encoding the marker, a gene chip capable of specifically detecting the nucleic acid encoding the marker, an aptamer capable of specifically binding to the nucleic acid encoding the marker, and / or a guide RNA capable of specifically binding to the nucleic acid encoding the marker. For example, the device may include qPCR, ELISA, devices required for Western blotting, and / or a gene chip.

[0314] In the present application, the detection module may output the content value of the DIR and / or the nucleic acid encoding the DIR.

[0315] In the present application, the system may include a judgment module for judging the diagnosis result and / or the disease progression result of the subject based on the content of the DIR and / or the nucleic acid encoding the DIR and the healthy control value and / or the initial control value. The healthy control value may include the content of the DIR and / or the nucleic acid encoding the DIR in a healthy subject, and the initial control value may include the content of the DIR and / or the nucleic acid encoding the DIR detected in the past of the same subject.

[0316] In the present application, the past may include at least 10 days ago, at least 20 days ago, at least 1 month ago, at least 6 months ago, at least 12 months ago, at least 24 months ago or even earlier. In the present application, the subject may be definitely diagnosed as suffering from the cognitive impairment and / or the neurodegenerative disease. By using the system of the present application to follow up the content of the DIR and / or the nucleic acid encoding the DIR in each time period of the subject, it may be helpful for the diagnosis of the progression of the cognitive impairment and / or the neurodegenerative disease of the subject.

[0317] In the present application, the healthy subject may not suffer from the cognitive impairment and / or the neurodegenerative disease.

[0318] For example, when the content of the DIR and / or the nucleic acid encoding the DIR in the sample of the subject is significantly higher than the healthy control value (for example, at least about 20%, at least about 50%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 200%, at least about 500% or more), the determination module may diagnose that the subject is suffering from the cognitive impairment and / or the neurodegenerative disease.

[0319] For example, when the content of the DIR and / or the nucleic acid encoding the DIR in the sample of the subject is significantly higher than the initial control value (for example, at least about 20%, at least about 50%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 200%, at least about 500% or more), the determination module may diagnose that the cognitive impairment and / or the neurodegenerative disease of the subject is progressing.

[0320] In the present application, the determination module may output the diagnosis result of the subject.

[0321] In the present application, the system may include a display module for displaying the diagnosis result of the determination module. For example, the diagnosis result displayed on the display module may include a qualitative result and / or a quantitative result.

[0322] In the present application, the system may include a storage module for storing the content value output to the detection module and / or the diagnosis result output to the determination module.

[0323] In the present application, the system a) a device for collecting and / or receiving DIR level data in a sample b) a device for analyzing the data and, when DIR is higher than the control level, diagnosing AD and / or evaluating the progression of AD in the subject if the subject has AD and / or the AD is progressing.

[0324] In the present application, the system may further include (a’) a device for collecting and / or receiving other clinical indicators or basic feature data related to AD diagnosis and / or AD progression in a sample.

[0325] In the present application, in order to perform a prognostic evaluation related to AD diagnosis and / or AD progression of the subject, the device described in (b) analyzes the DIR level data together with other clinical indicators or basic feature data related to AD diagnosis and / or AD progression.

[0326] Alternatively, in the present application, the system may further include a device for analyzing other clinical indicators or basic feature data related to AD diagnosis and / or AD progression, and / or a device for analyzing the DIR level data together with other clinical indicators or basic feature data related to AD diagnosis and / or AD progression in order to evaluate AD diagnosis and / or AD progression of the subject.

[0327] In the present application, the system may further include one or more devices selected from the group consisting of a device used for data input and / or output and / or storage, a device for uploading and / or storing analysis results in a cloud database, or a device used for output and / or storage of analysis results such as its corresponding cloud database device, and a device for intelligently analyzing and recommending treatment methods based on evaluation results.

[0328] It should be understood that those skilled in the art can add other general devices or programs to the system as needed.

[0329] Generally, AD diagnosis and / or AD progression evaluation (such as grading or disease stage) can be carried out by the following method: measuring the DIR molecular level in a subject or a sample derived from the subject, comparing the level with a control level, and if the DIR molecular level in the subject is higher than the control level according to the comparison result, it is suggested that the subject is suffering from AD or that AD is progressing. In some embodiments, the method of the present application may optionally further include obtaining a sample to be detected from the subject and contacting the sample to be detected with a reagent or kit for detecting the DIR level.

[0330] The term "control" used in the present application may include a control with a healthy person or a control with a specific grade or disease stage of AD. "Control" may refer to a reference DIR molecular level including, but not limited to, the DIR molecular level detected in a non-AD normal biological sample (a sample obtained from a non-AD patient or a healthy person) of the same subject, a statistically confirmed population standard level, or a standardized level. The term "control with a specific grade or disease stage of AD" refers to the DIR molecular level detected in a grade or disease stage confirmed in the past of the same subject, a statistically confirmed population standard level in the grade or disease stage, or a standardized level.

[0331] DIR Antigen-Binding Protein ​​In the present application, the antigen-binding protein may include LCDR2, and the LCDR2 may include the amino acid sequence shown in SEQ ID NO:74. For example, the antigen-binding protein of the present application may have DIR-binding ability. For example, the CDR may be determined by the IMGT numbering scheme.

[0332] X 1 X 2 is S (SEQ ID NO.74), wherein X 1 is A or Y, and X 2 is A or Y.

[0333] In the present application, the LCDR2 may include the amino acid sequence shown in SEQ ID NO:26 or 16.

[0334] In the present application, the antigen-binding protein may include LCDR1, and the LCDR1 may include the amino acid sequence shown in SEQ ID NO:73. For example, the antigen-binding protein of the present application may have DIR-binding ability. For example, the CDR may be determined by the IMGT numbering scheme. For example, the LCDR1 may include the LCDR1 in the light chain variable region of IR-II-1 to IR-II-10.

[0335] Q X 1 X 2 D X 3 X 4 X 5 X 6 X 7 is Y (SEQ ID NO.73), wherein X 1 is a deletion or S, and X 2 is a deletion or V, and X 3 is a deletion or Y, and X 4 is a deletion or D, and X 5 is G or I, and X 6 is D, E or S, and X 7 is N or S.

[0336] In the present application, the LCDR1 includes the amino acid sequence shown in any one of SEQ ID NO:25, 52, and 15.

[0337] In the present application, the antigen-binding protein may include LCDR3, and the LCDR3 may include the amino acid sequence shown in SEQ ID NO:75. For example, the antigen-binding protein of the present application may have DIR-binding ability. For example, the CDR may be determined by the IMGT numbering scheme. For example, the LCDR3 may include the LCDR3 in the light chain variable region of IR-II-1 to IR-II-10.

[0338] X 1 Q X 2 X 3 X 4 X 5 P X 6 X 7 (SEQ ID NO.75), wherein X 1 is L or Q, and X 2 is S or Y, and X 3 is N, S or Y, and X 4 is D, E or K, and X 5 is D or L, and X 6 is F or R, and X 7 is A or T.

[0339] In the present application, the LCDR3 may include the amino acid sequence shown in any of SEQ ID NO:27, 53, 67 and 17.

[0340] In the present application, the antigen-binding protein may include LCDR3, LCDR2 and LCDR1. For example, the LCDR2 of the antigen-binding protein may include the amino acid sequence shown in SEQ ID NO:74, LCDR1 may include the amino acid sequence shown in SEQ ID NO:73, and LCDR3 may include the amino acid sequence shown in SEQ ID NO:75. For example, the antigen-binding protein of the present application may have DIR-binding ability. For example, the CDR may be determined by the IMGT numbering scheme. For example, the LCDR1-3 may include the LCDR1-3 in the light chain variable region of IR-II-1 to IR-II-10.

[0341] In the present application, the antigen-binding protein may include LCDR3, LCDR2, and LCDR1. For example, the LCDR1 of the antigen-binding protein may include the amino acid sequence shown in SEQ ID NO: 25, the LCDR2 may include the amino acid sequence shown in SEQ ID NO: 26, and the LCDR3 may include the amino acid sequence shown in SEQ ID NO: 27.

[0342] For example, the LCDR1 of the antigen-binding protein may include the amino acid sequence shown in SEQ ID NO: 52, the LCDR2 may include the amino acid sequence shown in SEQ ID NO: 26, and the LCDR3 may include the amino acid sequence shown in SEQ ID NO: 53.

[0343] For example, the LCDR1 of the antigen-binding protein may include the amino acid sequence shown in SEQ ID NO: 52, the LCDR2 may include the amino acid sequence shown in SEQ ID NO: 26, and the LCDR3 may include the amino acid sequence shown in SEQ ID NO: 67.

[0344] For example, the LCDR1 of the antigen-binding protein may include the amino acid sequence shown in SEQ ID NO: 15, the LCDR2 may include the amino acid sequence shown in SEQ ID NO: 16, and the LCDR3 may include the amino acid sequence shown in SEQ ID NO: 17.

[0345] For example, the antigen-binding protein of the present application may have DIR-binding ability. For example, the CDR may be determined by the IMGT numbering scheme. For example, the LCDR1-3 may include the LCDR1-3 in the heavy chain variable region of IR-II-1 to IR-II-10.

[0346] In the present application, the antigen-binding protein may include HCDR1, and the HCDR1 may include the amino acid sequence shown in SEQ ID NO:70. For example, the antigen-binding protein of the present application may have DIR-binding ability. For example, the CDR may be determined by the IMGT numbering scheme. For example, the HCDR1 may include the HCDR1 in the heavy chain variable region of IR-II-1 to IR-II-10.

[0347] GYTFX 1 X 2 YX 3 (SEQ ID NO.70), wherein X 1 is S or T, X 2 is E, N, R or S, and X 3 is T or W.

[0348] In the present application, the HCDR1 may include the amino acid sequence shown in any one of SEQ ID NO:20, 35, 56 and 10.

[0349] In the present application, the antigen-binding protein may include HCDR2, and the HCDR2 may include the amino acid sequence shown in SEQ ID NO:71. For example, the antigen-binding protein of the present application may have DIR-binding ability. For example, the CDR may be determined by the IMGT numbering scheme. For example, the HCDR1 may include the HCDR2 in the heavy chain variable region of IR-II-1 to IR-II-10.

[0350] I X 1 P X 2 X 3 X 4 X 5 X 6 T(SEQ ID NO.71), wherein X 1 is L or Y, X 2 is G or H, X 3 is G, N or S, X 4 is a deletion or Y, X 5 is G or Y, X 6 is G, N, S or T.

[0351] In the present application, the HCDR2 may include the amino acid sequence shown in any one of SEQ ID NO: 21, 36, 42, 48, and 11.

[0352] In the present application, the antigen-binding protein may include HCDR3, and the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 72. For example, the antigen-binding protein of the present application may have DIR-binding ability. For example, the CDR may be determined by the IMGT numbering scheme. For example, the HCDR3 may include the HCDR3 in the heavy chain variable region of IR-II-1 to IR-II-10.

[0353] X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 X 11 X 12 X 13 X 14 X 15 DY (SEQ ID NO.72), wherein X 1 is a deletion or A, and X 2 is a deletion or R, and X 3 is a deletion, S or T, and X 4 is a deletion, D, G or Y, and X 5 is a deletion, D, I, M or V, and X 6 is a deletion, G or I, and X 7 is a deletion, T or Y, and X 8 is a deletion, A, L or T, and X 9 is a deletion, R, S or T, and X 10 is a deletion, G or T, and X 11 is a deletion, D or E, and X 12 is D or Y, and X 13 is F, L or Y, and X 14 is A, T or V, and X 15 is F or M.

[0354] In the present application, the HCDR3 may include the amino acid sequence shown in any one of SEQ ID NO: 22, 30, 37, 43, 49, 57, 62, and 12.

[0355] In the present application, the antigen-binding protein may include HCDR3, HCDR2, and HCDR1. For example, the HCDR2 of the antigen-binding protein may include the amino acid sequence shown in SEQ ID NO: 71, the HCDR1 may include the amino acid sequence shown in SEQ ID NO: 70, and the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 72. For example, the antigen-binding protein of the present application may have DIR-binding ability. For example, the CDR may be determined by the IMGT numbering scheme. For example, the HCDR1-3 may include the HCDR1-3 in the heavy chain variable region of IR-II-1 to IR-II-10.

[0356] For example, the HCDR1 of the antigen-binding protein may include the amino acid sequence shown in SEQ ID NO: 20, the HCDR2 may include the amino acid sequence shown in SEQ ID NO: 21, and the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 22.

[0357] For example, the HCDR1 of the antigen-binding protein may include the amino acid sequence shown in SEQ ID NO: 20, the HCDR2 may include the amino acid sequence shown in SEQ ID NO: 21, and the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 30.

[0358] For example, the HCDR1 of the antigen-binding protein may include the amino acid sequence shown in SEQ ID NO: 35, the HCDR2 may include the amino acid sequence shown in SEQ ID NO: 36, and the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 37.

[0359] For example, the HCDR1 of the antigen-binding protein may include the amino acid sequence shown in SEQ ID NO:20, the HCDR2 may include the amino acid sequence shown in SEQ ID NO:42, and the HCDR3 may include the amino acid sequence shown in SEQ ID NO:43.

[0360] For example, the HCDR1 of the antigen-binding protein may include the amino acid sequence shown in SEQ ID NO:20, the HCDR2 may include the amino acid sequence shown in SEQ ID NO:48, and the HCDR3 may include the amino acid sequence shown in SEQ ID NO:49.

[0361] For example, the HCDR1 of the antigen-binding protein may include the amino acid sequence shown in SEQ ID NO:56, the HCDR2 may include the amino acid sequence shown in SEQ ID NO:36, and the HCDR3 may include the amino acid sequence shown in SEQ ID NO:57.

[0362] For example, the HCDR1 of the antigen-binding protein may include the amino acid sequence shown in SEQ ID NO:35, the HCDR2 may include the amino acid sequence shown in SEQ ID NO:36, and the HCDR3 may include the amino acid sequence shown in SEQ ID NO:62.

[0363] For example, the HCDR1 of the antigen-binding protein may include the amino acid sequence shown in SEQ ID NO:35, the HCDR2 may include the amino acid sequence shown in SEQ ID NO:42, and the HCDR3 may include the amino acid sequence shown in SEQ ID NO:37.

[0364] For example, the HCDR1 of the antigen-binding protein may include the amino acid sequence shown in SEQ ID NO:56, the HCDR2 may include the amino acid sequence shown in SEQ ID NO:36, and the HCDR3 may include the amino acid sequence shown in SEQ ID NO:57.

[0365] For example, the HCDR1 of the antigen-binding protein may include the amino acid sequence shown in SEQ ID NO:10, the HCDR2 may include the amino acid sequence shown in SEQ ID NO:11, and the HCDR3 may include the amino acid sequence shown in SEQ ID NO:12.

[0366] For example, the antigen-binding protein of the present application may have DIR-binding ability. For example, the CDR may be determined by the IMGT numbering scheme. For example, the HCDR1-3 may include the HCDR1-3 in the heavy chain variable region of IR-II-1 to IR-II-10.

[0367] In the present application, the antigen-binding protein may include HCDR3, HCDR2, HCDR1, LCDR3, LCDR2, and LCDR1.

[0368] For example, the HCDR1 of the antigen-binding protein described in the present application may include the amino acid sequence shown in SEQ ID NO:20, the HCDR2 may include the amino acid sequence shown in SEQ ID NO:21, the HCDR3 may include the amino acid sequence shown in SEQ ID NO:22, the LCDR1 may include the amino acid sequence shown in SEQ ID NO:25, the LCDR2 may include the amino acid sequence shown in SEQ ID NO:26, and the LCDR3 may include the amino acid sequence shown in SEQ ID NO:27.

[0369] For example, the HCDR1 of the antigen-binding protein described in the present application may include the amino acid sequence shown in SEQ ID NO:20, the HCDR2 may include the amino acid sequence shown in SEQ ID NO:21, the HCDR3 may include the amino acid sequence shown in SEQ ID NO:30, the LCDR1 may include the amino acid sequence shown in SEQ ID NO:25, the LCDR2 may include the amino acid sequence shown in SEQ ID NO:26, and the LCDR3 may include the amino acid sequence shown in SEQ ID NO:27.

[0370] For example, the antigen-binding protein HCDR1 described in the present application may include the amino acid sequence shown in SEQ ID NO:35, HCDR2 may include the amino acid sequence shown in SEQ ID NO:36, HCDR3 may include the amino acid sequence shown in SEQ ID NO:37, LCDR1 may include the amino acid sequence shown in SEQ ID NO:25, LCDR2 may include the amino acid sequence shown in SEQ ID NO:26, and LCDR3 may include the amino acid sequence shown in SEQ ID NO:27.

[0371] For example, the antigen-binding protein HCDR1 described in the present application may include the amino acid sequence shown in SEQ ID NO:20, HCDR2 may include the amino acid sequence shown in SEQ ID NO:42, HCDR3 may include the amino acid sequence shown in SEQ ID NO:43, LCDR1 may include the amino acid sequence shown in SEQ ID NO:25, LCDR2 may include the amino acid sequence shown in SEQ ID NO:26, and LCDR3 may include the amino acid sequence shown in SEQ ID NO:27.

[0372] For example, the antigen-binding protein HCDR1 described in the present application may include the amino acid sequence shown in SEQ ID NO:20, HCDR2 may include the amino acid sequence shown in SEQ ID NO:48, HCDR3 may include the amino acid sequence shown in SEQ ID NO:49, LCDR1 may include the amino acid sequence shown in SEQ ID NO:52, LCDR2 may include the amino acid sequence shown in SEQ ID NO:26, and LCDR3 may include the amino acid sequence shown in SEQ ID NO:53.

[0373] For example, the antigen-binding protein HCDR1 described in the present application may include the amino acid sequence shown in SEQ ID NO:56, HCDR2 may include the amino acid sequence shown in SEQ ID NO:36, HCDR3 may include the amino acid sequence shown in SEQ ID NO:57, LCDR1 may include the amino acid sequence shown in SEQ ID NO:25, LCDR2 may include the amino acid sequence shown in SEQ ID NO:26, and LCDR3 may include the amino acid sequence shown in SEQ ID NO:27.

[0374] For example, the antigen-binding protein HCDR1 described in the present application may include the amino acid sequence shown in SEQ ID NO: 35, HCDR2 may include the amino acid sequence shown in SEQ ID NO: 36, HCDR3 may include the amino acid sequence shown in SEQ ID NO: 62, LCDR1 may include the amino acid sequence shown in SEQ ID NO: 25, LCDR2 may include the amino acid sequence shown in SEQ ID NO: 26, and LCDR3 may include the amino acid sequence shown in SEQ ID NO: 27.

[0375] For example, the antigen-binding protein HCDR1 described in the present application may include the amino acid sequence shown in SEQ ID NO: 35, HCDR2 may include the amino acid sequence shown in SEQ ID NO: 42, HCDR3 may include the amino acid sequence shown in SEQ ID NO: 37, LCDR1 may include the amino acid sequence shown in SEQ ID NO: 52, LCDR2 may include the amino acid sequence shown in SEQ ID NO: 26, and LCDR3 may include the amino acid sequence shown in SEQ ID NO: 53.

[0376] For example, the antigen-binding protein HCDR1 described in the present application may include the amino acid sequence shown in SEQ ID NO: 56, HCDR2 may include the amino acid sequence shown in SEQ ID NO: 36, HCDR3 may include the amino acid sequence shown in SEQ ID NO: 57, LCDR1 may include the amino acid sequence shown in SEQ ID NO: 52, LCDR2 may include the amino acid sequence shown in SEQ ID NO: 26, and LCDR3 may include the amino acid sequence shown in SEQ ID NO: 67.

[0377] For example, the HCDR1 of the antigen-binding protein described in the present application may include the amino acid sequence shown in SEQ ID NO:10, the HCDR2 may include the amino acid sequence shown in SEQ ID NO:11, the HCDR3 may include the amino acid sequence shown in SEQ ID NO:12, the LCDR1 may include the amino acid sequence shown in SEQ ID NO:15, the LCDR2 may include the amino acid sequence shown in SEQ ID NO:16, and the LCDR3 may include the amino acid sequence shown in SEQ ID NO:17.

[0378] For example, the antigen-binding protein of the present application may have DIR-binding ability. For example, the CDRs may be determined according to the IMGT numbering scheme. For example, the HCDR1-3 may be the HCDR1-3 in the heavy-chain variable region of IR-II-1 to IR-II-10, and the LCDR1-3 may be the LCDR1-3 in the light-chain variable region of IR-II-1 to IR-II-10.

[0379] In the present application, the antigen-binding protein may include H-FR1, and the C-terminus of the H-FR1 may be directly or indirectly linked to the N-terminus of the HCDR1. In the present application, the antigen-binding protein may include H-FR2, and the H-FR2 may be located between the HCDR1 and the HCDR2. In the present application, the antigen-binding protein may include H-FR3, and the H-FR3 may be located between the HCDR2 and the HCDR3. In the present application, the antigen-binding protein may include H-FR4, and the N-terminus of the H-FR4 may be linked to the C-terminus of the HCDR3. In the present application, the antigen-binding protein may include H-FR1, H-FR2, H-FR3, and H-FR4.

[0380] In the present application, the antigen-binding protein may include L-FR1, and the C-terminus of the L-FR1 may be directly or indirectly linked to the N-terminus of the LCDR1. In the present application, the antigen-binding protein may include L-FR1, and the C-terminus of the L-FR1 may be directly or indirectly linked to the N-terminus of the LCDR1. In the present application, the antigen-binding protein may include L-FR2, and the L-FR2 may be located between the LCDR1 and the LCDR2. In the present application, the antigen-binding protein may include L-FR3, and the L-FR3 may be located between the LCDR2 and the LCDR3. In the present application, the antigen-binding protein may include L-FR4, and the N-terminus of the L-FR4 may be linked to the C-terminus of the LCDR3. In the present application, the antigen-binding protein may include L-FR1, L-FR2, L-FR3, and L-FR4.

[0381] In the present application, the antigen-binding protein may include the heavy-chain variable region VH, and the VH may include the amino acid sequence shown in any of SEQ ID NO: 13, 23, 31, 38, 44, 50, 58, 63, and 65. For example, the antigen-binding protein of the present application may have DIR-binding ability.

[0382] In the present application, the antigen-binding protein may include the light-chain variable region VL, and the VL may include the amino acid sequence shown in any of SEQ ID NO: 18, 28, 33, 40, 46, 54, 58, 60, and 68. For example, the antigen-binding protein of the present application may have DIR-binding ability.

[0383] In the present application, the antigen-binding protein may include the VH and the VL. In some embodiments, the VH may include the amino acid sequence shown in SEQ ID NO: 23, and the VL may include the amino acid sequence shown in SEQ ID NO: 28.

[0384] In some embodiments, the VH may include the amino acid sequence shown in SEQ ID NO: 31, and the VL may include the amino acid sequence shown in SEQ ID NO: 33.

[0385] In some embodiments, the VH may comprise the amino acid sequence shown in SEQ ID NO:38, and the VL may comprise the amino acid sequence shown in SEQ ID NO:40.

[0386] In some embodiments, the VH may comprise the amino acid sequence shown in SEQ ID NO:44, and the VL may comprise the amino acid sequence shown in SEQ ID NO:46.

[0387] In some embodiments, the VH may comprise the amino acid sequence shown in SEQ ID NO:50, and the VL may comprise the amino acid sequence shown in SEQ ID NO:54.

[0388] In some embodiments, the VH may comprise the amino acid sequence shown in SEQ ID NO:58, and the VL may comprise the amino acid sequence shown in SEQ ID NO:60.

[0389] In some embodiments, the VH may comprise the amino acid sequence shown in SEQ ID NO:63, and the VL may comprise the amino acid sequence shown in SEQ ID NO:60.

[0390] In some embodiments, the VH may comprise the amino acid sequence shown in SEQ ID NO:65, and the VL may comprise the amino acid sequence shown in SEQ ID NO:54.

[0391] In some embodiments, the VH may comprise the amino acid sequence shown in SEQ ID NO:58, and the VL may comprise the amino acid sequence shown in SEQ ID NO:68.

[0392] In some embodiments, the VH may comprise the amino acid sequence shown in SEQ ID NO:13, and the VL may comprise the amino acid sequence shown in SEQ ID NO:18. For example, the antigen-binding protein of the present application may have DIR binding ability.

[0393] In the present application, the antigen-binding protein may include at least one CDR in the VH described in the present application. In the present application, the antigen-binding protein may include at least one CDR in the VL described in the present application. The CDR is obtained by division according to any division method. In the present application, the CDR may cover the CDR sequences obtained by division according to any CDR division method, and may cover variants thereof.

[0394] In the present application, the antigen-binding protein may include HCDR1, HCDR2, and HCDR3 in the VH described in the present application.

[0395] In the present application, the antigen-binding protein may include HCDR1, HCDR2, and HCDR3 in the VH described in the present application. The VH may include the amino acid sequence shown in any of SEQ ID NO: 13, 23, 31, 38, 44, 50, 58, 63, and 65. For example, the antigen-binding protein of the present application may have DIR-binding ability. In the present application, the CDR may cover the CDR sequences obtained by division according to any CDR division method, and may cover variants thereof.

[0396] In the present application, the antigen-binding protein may include LCDR1, LCDR2, and LCDR3 in the VL described in the present application. The VL may include the amino acid sequence shown in any of SEQ ID NO: 18, 28, 33, 40, 46, 54, 60, and 68. For example, the antigen-binding protein of the present application may have DIR-binding ability. In the present application, the CDR may cover the CDR sequences obtained by division according to any CDR division method, and may cover variants thereof.

[0397] In the present application, the antigen-binding protein may include an antibody heavy chain constant region. The antibody heavy chain constant region may be derived from a human IgG, IgA, IgD, IgE, and / or IgM heavy chain constant region. The antibody heavy chain constant region may be derived from a human IgG heavy chain constant region. In some embodiments, the antigen-binding protein may include an antibody heavy chain constant region, and the antibody heavy chain constant region may be derived from a human IgG1, IgG2, IgG3, and / or IgG4 heavy chain constant region. In some embodiments, the antigen-binding protein may include an antibody heavy chain constant region, and the antibody heavy chain constant region may be derived from a human IgG1 heavy chain constant region.

[0398] In the present application, the antigen-binding protein may include an antibody light chain constant region. The antibody light chain constant region may include a constant region derived from Igκ or a constant region derived from Igλ. The antibody light chain constant region may be derived from a human Igκ constant region.

[0399] In the present application, the antigen-binding protein may include an antibody or an antigen-binding fragment thereof.

[0400] In some embodiments, the antigen-binding fragment may include Fab, Fab’, Fv fragment, F(ab’) 2 , F(ab) 2 , scFv, di-scFv, VHH, and / or dAb.

[0401] In some embodiments, the antibody may include a monoclonal antibody. In some embodiments, the antibody may include a mouse-derived antibody and / or a chimeric antibody.

[0402] Furthermore, it should be noted that the antigen-binding protein described in the present application may contain a heavy chain and / or light chain sequence modified by one or more conserved sequences. "Modification by conserved sequences" refers to amino acid modifications that do not significantly affect or change the binding properties of the antibody. Such conservative modifications include amino acid substitutions, additions, and deletions. The modifications can be introduced into the antigen-binding protein described in the present application by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitution is the substitution of an amino acid residue with an amino acid residue having a similar side chain. Groups of amino acid residues having similar side chains are known within the domain. These groups of amino acid residues include amino acids having basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In some embodiments, one or more amino acid residues in the CDR region of the antigen-binding protein described in the present application may be substituted with other amino acid residues of the same side chain group. Those skilled in the art will know that antigen-binding activity will not be lost by some conservative sequence modifications.For details, see, for example, Brummell et al., (1993) Biochem 32:1180-8; de Wildt et al., (1997) Prot. Eng. 10:835-41; Komissarov et al., (1997) J. Biol. Chem. 272:26864-26870; Hall et al., (1992) J. Immunol. 149:1605-12; Kelley and O’Connell (1993) Biochem.32:6862-35; Adib-Conquy et al., (1998) Int. Immunol.10:341-6 and Beers et al., (2000) Clin. Can. Res. 6:2835-43.

[0403] The antigen-binding proteins described in the present application can be identified, screened, or characterized by various assays known in the art.

[0404] For example, known methods such as enzyme-linked immunosorbent assay (ELISA), immunoblotting (such as Western blotting), flow cytometry (FACS), immunohistochemistry, and immunofluorescence can be used to test the antigen-binding activity of the antigen-binding proteins or fusion proteins of the present application.

[0405] In the present application, the antigen-binding protein can specifically bind to DIR or its functionally active fragment. In the present application, the antigen-binding protein can specifically bind to DIR-II.

[0406] In some embodiments, the DIR or its functionally active fragment can be the full-length DIR or its functionally active fragment, or can be a fragment (such as DIR-II) that exhibits the functional activity of DIR. In some embodiments, the DIR or its functionally active fragment can be an isolated DIR or its functionally active fragment, or can be a mixture of various forms of DIR or its functionally active fragments. For example, the DIR or its functionally active fragment can be a human DIR or its functionally active fragment.

[0407] In some embodiments, the antigen-binding protein can measure the binding activity between the antigen-binding protein and an antigen (such as DIR-II) by an ELISA method. For example, the antigen-binding protein can have the ability to bind to the DIR or its functional fragment at a value of about 10 ng / ml or higher (such as at least 15 ng / ml, at least 20 ng / ml, at least 25 ng / ml, at least 30 ng / ml, at least 35 ng / ml, at least 40 ng / ml, at least 45 ng / ml, at least 50 ng / ml, at least 55 ng / ml, at least 60 ng / ml, at least 65 ng / ml, at least 70 ng / ml, at least 75 ng / ml, at least 80 ng / ml, at least 85 ng / ml, at least 90 ng / ml, at least 95 ng / ml, at least 100 ng / ml, at least 500 ng / ml, at least 1000 ng / ml or higher).

[0408] [Drug Screening Method] In another aspect, provided is a drug screening method that may include a step of detecting a change in the content of DIR and / or a nucleic acid encoding DIR in a subject after administration of a candidate drug for use in preventing, treating and / or alleviating cognitive impairment and / or neurodegenerative diseases.

[0409] In the present application, when the content of DIR and / or the nucleic acid encoding DIR in a subject decreases upon administration of the candidate drug, the candidate drug has a therapeutic effect.

[0410] In the present application, the administration may include an injection.

[0411] In the present application, the drug may include a small molecule drug and / or a biopolymer drug.

[0412] According to the disclosure of the present application, the level of DIR is closely related to the presence and / or degree of cognitive impairment, and can be used as an indicator for AD diagnosis and / or AD progression evaluation (such as grading or disease stage).

[0413] Furthermore, the present application provides a method for screening an AD therapeutic drug, including detecting the effect of a candidate drug on the DIR level in a subject or a specimen derived from the subject. After administration of the candidate drug, a decrease in the level indicates that the candidate drug has an AD therapeutic effect. Each feature included in the candidate drug screening method of the present application is as defined or detailed in the present application. In some embodiments, the candidate drug may be a DIR inhibitor.

[0414] In the present application, it has been found that DIR is specifically expressed in the blood specimens and / or cerebrospinal fluid of AD patients. Importantly, DIR can be detected in a small amount of serum (for example, about 100 μl), which becomes a promising test method for early diagnosis of AD for clinical application. According to the current results, the positive detection rates of diseases such as AD and MCI are all above 90%, suggesting that DIR is highly expected to be applied as a specific marker for early screening of AD.

[0415] pTau-181, pTau-217, and Aβ1-42, which are currently used in clinical blood tests, are newly implemented AD early screening test items, with relatively low specificity and relatively high costs. Compared with these conventional AD early screening tests, the AD early screening test based on the DIR level of the present application has a high detection rate, excellent specificity, and / or low cost, and has a price advantage. For example, the cost of detecting the DIR level by ELISA can be controlled below 10 yuan per person. Therefore, the detection indicators and methods of the present application have greater effects and cost advantages compared with the conventional methods.

[0416] Furthermore, in the present application, it has been found that DIR is very specific and sensitive for the diagnosis of cognitive impairment and / or neurodegenerative diseases, and can distinguish potential AD and / or MCI patients in situations where the target differences such as pTau-181, pTau-217, and Aβ1-42 are not significant, and can effectively diagnose patients at an overall earlier stage.

[0417] In the present application, the method can detect the change in the content of DIR and / or nucleic acid encoding DIR in a subject before and / or after administration of a candidate drug using the kit described in the present application and / or the detection substance described in the present application. In the present application, the method can detect the change in the content of DIR and / or nucleic acid encoding DIR in a subject before and / or after administration of a candidate drug using at least one type (for example, one type, two types, three types, or more) of detection substances. For example, the method can use at least one type (for example, one type, two types, three types, or more) of antigen-binding protein (such as a DIR antibody) that can specifically bind to DIR. In the present application, when there are antigen-binding proteins with different amino acid sequences in at least one CDR, they can be regarded as different types of antigen-binding proteins. TIFF2025517103000001.tif60170

[0418] The present application provides the following embodiments.

[0419] [Embodiment 1] Use of a substance for detecting DIR, which is an intron-retaining splicing product encoding DNA damage-inducible transcript 4-like transcript (DDIT4L) derived from a subject sample, in the preparation of a product (such as a kit or a detection system) for performing the diagnosis of the subject early-onset dementia, mild cognitive impairment diagnosis and / or the evaluation of the progression of early-onset dementia (such as grade classification or disease stage).

[0420] [Embodiment 2] The use according to Embodiment 1, wherein the splicing product is selected from the group consisting of mRNA having the nucleotide sequence shown in SEQ ID NO: 93, a nucleic acid encoding a peptide which is a splicing product having the nucleotide sequence shown in SEQ ID NO: 8, or a peptide which is a splicing product having the amino acid sequence shown in SEQ ID NO: 1.

[0421] [Embodiment 3] The use according to Embodiment 1, wherein the early-onset dementia includes early-stage early-onset dementia (amnesia stage), moderate early-onset dementia (neuropathy stage) and severe early-onset dementia (severe cognitive impairment stage).

[0422] [Embodiment 4] The use according to Embodiment 1, wherein the substance is selected from a reagent, a device or a combination thereof used for detecting DIR.

[0423] [Embodiment 5] The use according to Embodiment 1, wherein the substance is selected from a substance having specificity for the DIR, such as an antibody (such as a monoclonal antibody) of the DIR, a splicing product-specific probe, a gene chip, a PCR primer, a gRNA, etc.

[0424] [Embodiment 6] The use according to Embodiment 1, wherein the sample is selected from a blood sample such as whole blood, serum, plasma, a tissue or cell sample, and cerebrospinal fluid.

[0425] [Embodiment 7] An increase in the DIR level in the specimen as compared to the healthy control value indicates that the subject suffers from early-onset dementia or mild cognitive impairment, or an increase in the DIR level in the specimen as compared to the mild cognitive impairment control or as compared to the initial control level diagnosed with early-onset dementia indicates that the early-onset dementia of the subject is progressing (e.g., the grade classification or disease stage has worsened), the use according to Embodiment 1.

[0426] [Embodiment 8] (i) A substance for detecting DIR, which is an intron-retaining splicing product of DNA damage-inducible transcript 4-like transcript (DDIT4L) in a target specimen, (ii) Any other substance optionally selected for use in diagnosing early-onset dementia, diagnosing mild cognitive impairment, and / or evaluating the progression of early-onset dementia (such as grade classification or disease stage), such as substances used for detecting existing early-onset dementia markers, For example, in certain cases, the substance in (i) is a reagent, device, or combination thereof used for detecting DIR, a substance for detecting the DIR at the gene level and / or protein level, such as a substance used in one or more detection techniques or methods selected from the group consisting of immunohistochemistry (immunofluorescence assay, chemiluminescence method, reverse enzyme-linked immunosorbent assay, immunogold method), Western blotting method, Northern blotting method, PCR, biochip method, protein chip method, or a substance having specificity for the DIR, such as an antibody (preferably a monoclonal antibody) against the DIR, a splicing product-specific probe, a gene chip, a PCR primer, a gRNA, and / or The other substances in (ii) include substances used for detecting one or more markers of juvenile dementia such as AD7C-NTP, pTau-181, pTau-217, Aβ1-42, and / or reagents for brain amyloid imaging, and / or reagents used for brain imaging diagnosis of a subject, and are products (such as kits) used for diagnosing juvenile dementia, diagnosing mild cognitive impairment, and / or evaluating the progression of juvenile dementia (such as grading or staging).

[0427] [Embodiment 9] (a) An apparatus for collecting and / or receiving level data of DIR, which is an intron-retaining splicing product of DNA damage-inducible transcript 4-like transcript (DDIT4L) in a sample, and (b) An apparatus for analyzing data for diagnosing juvenile dementia, diagnosing mild cognitive impairment, and / or evaluating the progression of cognitive impairment (such as grading or staging) of a subject, such that an increase in the DIR level in the sample compared to a healthy control value indicates that the subject has juvenile dementia or mild cognitive impairment, or an increase in the DIR level in the sample compared to a mild cognitive impairment control or compared to an initial control level diagnosed with juvenile dementia indicates that the juvenile dementia of the subject is progressing (for example, the grading or staging has worsened). A system for diagnosing juvenile dementia, diagnosing mild cognitive impairment, and / or evaluating the progression of juvenile dementia (such as grading or staging) of a subject, comprising the above.

[0428] [Embodiment 10] A method for screening a therapeutic agent for juvenile dementia, which detects the effect of a candidate drug on the level of DIR, which is an intron-retaining splicing product of DNA damage-inducible transcript 4-like transcript (DDIT4L) of a subject, such that a decrease in the level after administration of the candidate drug indicates that the candidate drug has a therapeutic effect on juvenile dementia.

[0429] Regardless of any theory, the following examples are not intended to limit the scope of the present invention, but only to explain the detection method, applications, etc. of the present application.

Example

[0430] The following further describes the present application in more detail in accordance with specific examples. It should be understood that these examples are used only to explain the present application and are not used to limit the scope of the present application. Those skilled in the art can make appropriate modifications and changes to the present application, and these modifications and changes are within the scope of the present application.

[0431] For experimental methods where specific conditions are not specified in the following examples, general methods in this field can be used, such as those described in "Molecular Cloning: A Laboratory Manual" (Third Edition, New York, Cold Spring Harbor Laboratory Press, New York: Cold Spring Harbor Laboratory Press, 1989) or conditions recommended by the supplier. The DNA sequencing method is a general method in this field and can also be provided by private companies for testing.

[0432] Unless otherwise specified, percentages and parts are based on weight. Unless otherwise defined, all technical and scientific terms used in the present application have the same meaning as those known to those skilled in the art. Also, any methods and materials similar or equivalent to those described can be applied to the methods of the present application. The preferred embodiments and materials described in the present application are for illustrative purposes only.

[0433] [Example 1] The DIR protein is a secreted protein [Test Materials and Methods] Plasmid construction and cell transfection method: To express the flag-DIR protein, the cDNA of human DIR is cloned into the pCMV-flag vector. In HEK293 cells (Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences), the pCMV-flag plasmid and the pCMV-flag-DIR plasmid are transfected with the Lippo3000 reagent. After culturing the cells for 48 hours, the cells and the medium are obtained. An appropriate amount of lysis buffer is added to each, and they are lysed by rotating 30 times at 4°C. After centrifuging at 12,000 g for 30 minutes, the supernatant is collected, and a small amount of the lysis buffer is collected for Western blot analysis.

[0434] Western blot experiment: The Western blot experiment was carried out according to the SDS-PAGE electrophoresis, blotting, immunoreaction, and chemiluminescence procedures (the antibodies used were purchased from Gil Biochemicals (Shanghai) Co., Ltd.).

[0435] [Results and Discussion] After culturing HEK293 cells for 48 hours, a large amount of DIR was expressed in the cells transfected with the pCMV-flag-DIR plasmid, and a large amount of DIR was also detected in the cell culture medium (Figure 1b). This indicates that after DIR is transcribed and translated into protein in the cell, it can be secreted into the extracellular medium through the intracellular secretory pathway. As for the secretion amount, since a part of the DIR synthesized in the cell was secreted into the extracellular medium, it is suggested that DIR is secreted extracellularly in vivo and further enters the blood circulation system, which provides a theoretical basis for DIR to become a blood marker under specific pathological conditions.

[0436] [Example 2] Changes in the content of DIR protein in the blood of AD patients [Test Materials and Methods] Tissue specimens: Venous blood specimens (a total of 58 cases) from AD and MCI patients, non-AD patients (hypertension, coronary artery disease, or diabetes patients), and healthy individuals were collected from Huashan Hospital Affiliated to Fudan University and Xuhui District Central Hospital.

[0437] Method for collecting venous blood and obtaining plasma: Using vacuum blood collection technology, the subject's cubital fossa venous blood was collected into an EDTA vacuum tube, and the test tube was gently inverted 8 times immediately to fully mix the blood and anticoagulant. Next, it was transported to the laboratory on ice and centrifuged at 4°C and 1500 g for 10 minutes. The supernatant was aspirated, dispensed, and stored at -80°C.

[0438] Plasma Western blot method: The dispensed plasma was thawed on ice, appropriately diluted with PBS, and then buffer was added and denatured in a metal bath at 100°C for 5 minutes. The subsequent operations were the same as those for tissue Western blot.

[0439] Plasma ELISA method: Plasma specimens were diluted 1:1 with 1× diluent (purchased from Bio-Rad, OBT1998G). 100 μl of blocking solution was taken with a pipettor and blocked at 37°C for 1 hour. Next, 100 μl of two-fold diluted standard product and diluted plasma were added to a well plate coated with capture antibody (Gil Biochemicals, AB009458) with a pipettor, and the pipettor tip was replaced every time a sample was taken. Incubated at 37°C for 1.5 hours. The liquid in each well was removed and washed 3 times with 100 μl of 1× wash solution (purchased from Bio-Rad, BUF031C). 100 μl of detection antibody (i.e., DIR antibody IR-II-6 in which the amino acid sequence of VH is shown in SEQ ID NO.58 and the amino acid sequence of VL is shown in SEQ ID NO.60) was added to each well and incubated at 37°C for 1 hour. The liquid in each well was removed and washed 3 times with 100 μl of 1× wash solution. 100 μl of 1× avidin-HRP solution was added to each well and incubated at 37°C for 1 hour. The liquid in each well was removed and washed 3 times with 100 μl of 1× wash solution. 100 μl of 1× substrate reaction solution (purchased from Bio-Rad, BUF062C) was added to each well and incubated at room temperature in the dark for 10 - 30 minutes. Detected at a wavelength of 655 nm with a microplate reader and a calibration curve was prepared.

[0440] [Results and Discussion] Clinically, blood samples were collected from AD, MCI patients, non-AD patients, and healthy individuals. As a result of detection for each patient group, the DIR content in the blood samples of AD and MCI patients was the highest and was detected in approximately 95% of the patients. A high concentration of DIR was also detected in the blood samples of MCI patients, and it was detected in approximately 90% of the patients. On the other hand, DIR was not detected or only a trace amount of DIR was detected in the blood samples of non-cognitive impairment patients, and DIR was not detected in the blood samples of healthy individuals (Figure 2a).

[0441] By ELISA method, the DIR content in the blood samples of AD patients was high and reached the level of 10 - 100 μg / ml. However, the DIR content of non-AD patients was at the pg / ml level and was not detected in healthy individuals. Therefore, the DIR level of AD patients was 10^3 - 10^4 times that of other patients. This suggests that the specificity of DIR is very high (Figure 2a and 2b).

[0442] The above results indicate that DIR is present at a high concentration in the blood of AD and MCI patients, and can be used as a blood marker for detecting the presence of AD, especially for patients with mild cognitive impairment such as MCI.

[0443] Furthermore, the DIR content is related to the severity of AD (see Figure 2b).

[0444] [Example 3] Methodological study on DIR protein detection DIR protein (Gill Biochemicals, 752084) was collected and prepared into a stock solution of 1 mg / ml with DPBS solution, and diluted with ELISA detergent (BUF031C, purchased from Bio-Rad) before the experiment to prepare a standard DIR protein solution.

[0445] The concentration of the standard DIR protein was detected by ELISA method and fitted by polynomial regression. The obtained calibration curve is shown in Figure 3.

[0446] The R of the regression model 2The value is 0.99, indicating a high degree of fitness. The hatched area represents the 95% confidence interval of the regression model. A narrow confidence interval can be confirmed from the figure, suggesting that the estimation of the DIR content in the sample based on the model has high reliability.

[0447] [Example 4] ROC Curve of DIR as a Clinical Test Index Blood samples (the same as in Example 2) from AD, MCI patients, non-AD patients, and healthy subjects clinically collected by the ELISA method were detected, and the detection results were analyzed using the "pROC" package of R software (version number 3.6.2). A logistic regression model was constructed with the detected DIR concentration, and an ROC curve was obtained by the analysis of the self-sampling method.

[0448] The results are shown in Figure 4. As a result, ideal estimation results can be obtained only by DIR single-parameter modeling. AUC = 0.807 (95% confidence interval: 0.688 - 0.926)

[0449] [Example 5] Changes in the Content of DIR Protein in the Blood of Different Types of MCI Patients [Test Materials and Methods] Tissue specimens: Venous blood specimens from different types of MCI patients and normal observers were collected from Huashan Hospital Affiliated to Fudan University.

[0450] Method for collecting venous blood and obtaining plasma: The same as in Example 2.

[0451] Plasma ELISA method: The same as in Example 2.

[0452] MCI classification method: aMCI-s: Amnestic MCI with a single cognitive domain impairment, aMCI-m: Amnestic MCI with multiple cognitive domain impairments, naMCI-s: Non-amnestic MCI with a single cognitive domain impairment, naMCI-m: Non-amnestic MCI with multiple cognitive domain impairments.

[0453] In Example 5, there were 122 specimens from aMCI-m patients, 85 specimens from aMCI-s patients, 11 specimens from naMCI-m patients, 88 specimens from naMCI-s patients, and 279 specimens from normal observers.

[0454] [Results and Discussion] As a result of detecting blood specimens of different types of MCI patients and normal observer groups collected clinically, the DIR level in the blood specimens of aMCI-m type patients was elevated, and a significant difference was observed compared with the normal observer group (Figure 5).

[0455] By ELISA method, it was found that the blood DIR content of aMCI-m type patients was high, and aMCI-m type patients were more likely to progress to AD compared with other types of MCI patients, suggesting that the index DIR has the ability to identify the aMCI-m type.

[0456] The above results suggest that DIR can be used as an effective diagnostic marker for AD.

[0457] [Example 6] Changes in the DIR protein content in the blood of different types of MCI patients [Test Materials and Methods] Tissue specimens: Venous blood specimens of different types of MCI patients, AD patients, and normal observers were collected from Huashan Hospital Affiliated to Fudan University.

[0458] Method for collecting venous blood and obtaining plasma: The same as in Example 2.

[0459] Plasma ELISA method: DIR was the same as in Example 2, and Tau, Aβ40, and Aβ42 were detected according to the operation steps using a triple kit (product number: 101995) from Hangzhou Jingbai Biotechnology Co., Ltd.

[0460] MCI classification method: The same as in Example 5.

[0461] [Results and Discussion] Results of detecting blood samples of different types of MCI patients, AD patients and normal control groups collected clinically showed that the DIR level in the blood samples of patients with the aMCI-m type was elevated, and a significant difference was observed compared with the normal control group and other MCI types. On the other hand, for conventional indicators such as Tau, Aβ40 and Aβ42 in the detected blood samples, some indicators (such as Aβ40) tended to increase in the blood samples of patients with the aMCI-m type and AD patients, but no significant difference was observed among the groups (see Figure 6).

[0462] By ELISA method, the blood DIR content of patients with the aMCI-m type and AD patients was high. Compared with the conventional auxiliary diagnostic indicators Tau, Aβ40 and Aβ42, DIR could distinguish patients with the aMCI-m type, AD patients and other types of MCI patients or healthy people earlier and more significantly. Therefore, DIR can be used as an effective diagnostic marker for aMCI-m and AD.

[0463] [Example 7] Comparison of DIR protein content in plasma and cerebrospinal fluid of patients [Test materials and methods] Tissue specimens: Venous blood specimens and cerebrospinal fluid specimens derived from the same patients (1 case each of cerebral edema, meningioma and glioma) were all collected from Huashan Hospital affiliated to Fudan University.

[0464] Method for collecting venous blood and obtaining plasma: The same as in Example 2.

[0465] Method for collecting cerebrospinal fluid: Cerebrospinal fluid collected during the patient's surgery.

[0466] ELISA methods for plasma and cerebrospinal fluid: The same as in Example 2.

[0467] [Results and discussion] The consistency of DIR content in plasma and cerebrospinal fluid was high, and the R 2 value was 0.99 (see Figure 7).

[0468] By ELISA, the DIR content in the patient's blood is consistent with that in the cerebrospinal fluid, and the content detected from the blood can directly reflect the DIR content in the cerebrospinal fluid. Therefore, it is suggested that the DIR described in this application can be applied as an effective diagnostic marker using cerebrospinal fluid as a specimen.

[0469] [Example 8] Detection of the binding activity of DIR antibodies The sequencing results of the DIR antibodies IR-II-1 to IR-II-10 (IR-II-10, that is, A2D10) are shown in Table 1. TIFF2025517103000002.tif166170

[0470] The binding activities of the IR-II-1 to IR-II-10 antibodies to the DIR-II fragment were detected. The specific method is as follows.

[0471] Using coating buffer, the target fragment DIR-II was coated on a 96-well plate and blocked with blocking buffer. Next, monoclonal antibodies (1 mg / mL) were each diluted at the same ratios (1:2k, 1:6k, 1:18k, 1:54k, 1:162k, 1:486k, 1:1458k, and 1:4374k), then added to each well with a pipettor, shaken at 37 °C for 2 hours, and washed 3 times with washing buffer. Next, a secondary antibody (1:5k) added with horseradish peroxidase was added and incubated at 37 °C. After washing 3 times, an appropriate volume of TMB substrate solution was added, incubated at 37 °C for a certain period of time, then set on a microplate reader, and the absorbance value at a wavelength of 450 nm was recorded.

[0472] As shown in Table 2 and Figure 8, from this result, it was found that the IR-II-1 to IR-II-10 antibodies specifically bind to the DIR-II fragment at an action concentration of about 10 ng / ml or more. Therefore, all of the IR-II-1 to IR-II-10 antibodies can be used for DIR protein detection and the diagnosis of aMCI-m and AD. TIFF2025517103000003.tif103170

[0473] All documents related to the present application are cited for reference in this application so that each document is cited independently. After reading the above content of the present application, those skilled in the art can make various changes and modifications to the present invention, but it should be understood that those equivalent forms are included in the scope of the claims of the present application.

Claims

1. Use of DIR, which is an intron-retaining splicing product of DNA damage-inducible transcript 4-like transcript (DDIT4L), and / or a nucleic acid encoding said DIR, in the diagnosis of a disease and / or evaluation of disease progression, wherein the disease includes cognitive impairment.

2. Use of DIR, which is an intron-retaining splicing product of DNA damage-inducible transcript 4 (DDIT4L), and / or a nucleic acid encoding said DIR, in the diagnosis of a disease and / or evaluation of disease progression, wherein the disease includes neurodegenerative diseases.

3. The use according to any one of claims 1 to 2, wherein the DIR comprises the amino acid sequence shown in SEQ ID NO:

1.

4. The use according to any one of claims 1 to 3, wherein the nucleic acid encoding the DIR comprises the nucleotide sequence shown in SEQ ID NO:

8.

5. The use according to any one of claims 1 to 4, wherein the nucleic acid encoding the DIR comprises the nucleotide sequence shown in SEQ ID NO:

93.

6. The use according to any one of claims 1 to 5, wherein the cognitive impairment includes normal age-related cognitive impairment, Lewy body dementia (LBD), frontotemporal dementia and / or vascular dementia.

7. The use according to any one of claims 1 to 6, wherein the inducing diseases of the cognitive impairment include Alzheimer's disease, multi-infarct type, Parkinson's disease, AIDS and / or Creutzfeldt-Jakob disease (CJD).

8. The use according to any one of claims 1 to 7, wherein the cognitive impairment includes mild cognitive impairment (MCI), intermediate cognitive impairment and severe cognitive impairment.

9. The use according to any one of claims 1 to 8, wherein the cognitive impairment includes amnestic MCI with multiple cognitive domain impairments (aMCI-m).

10. The use according to any one of claims 2 to 9, wherein the neurodegenerative diseases include acute neurodegenerative diseases and chronic neurodegenerative diseases.

11. The use according to any one of claims 2 to 10, wherein the neurodegenerative disease includes a neurodegenerative disease caused by neuronal cell death and glial cell homeostasis, a neurodegenerative disease caused by aging, a neurodegenerative disease caused by affected CNS cell function, a neurodegenerative disease caused by abnormal intercellular transmission, and / or a neurodegenerative disease caused by a disorder of cell motility.

12. The use according to any one of claims 2 to 11, wherein the neurodegenerative disease includes Alzheimer's disease, Parkinson's disease, multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), and / or Huntington's disease (HD).

13. The use according to any one of claims 2 to 12, wherein the neurodegenerative disease includes young-onset dementia.

14. The use according to any one of claims 2 to 13, wherein the neurodegenerative disease includes mild young-onset dementia, moderate young-onset dementia, and / or severe young-onset dementia.

15. Use of a DIR, which is an intron-retaining splicing product of DNA damage-inducible transcript 4-like transcript (DDIT4L), and / or a substance for detecting a nucleic acid encoding the DIR, in the preparation of a product for disease diagnosis and / or disease progression evaluation, wherein the disease includes cognitive impairment.

16. Use of a DIR, which is an intron-retaining splicing product of DNA damage-inducible transcript 4-like transcript (DDIT4L), and / or a substance for detecting a nucleic acid encoding the DIR, in the preparation of a product for disease diagnosis and / or disease progression evaluation, wherein the disease includes a neurodegenerative disease.

17. The use according to any one of claims 15 to 16, wherein the DIR includes the amino acid sequence shown in SEQ ID NO:

1.

18. The use according to any one of claims 15 to 17, wherein the nucleic acid encoding the DIR includes the nucleotide sequence shown in SEQ ID NO:

8.

19. The use according to any one of claims 15 to 18, wherein the nucleic acid encoding the DIR includes the nucleotide sequence shown in SEQ ID NO:

93.

20. The use according to any one of claims 15 to 19, wherein the cognitive impairment includes normal age-related cognitive impairment, Lewy body dementia (LBD), frontotemporal dementia, and / or vascular dementia.

21. Use according to any one of claims 15 to 20, wherein the disease inducing cognitive impairment includes Alzheimer's disease, multiple infarction, Parkinson's disease, AIDS and / or Creutzfeldt-Jakob disease (CJD).

22. Use according to any one of claims 15 to 21, wherein the cognitive impairment includes mild cognitive impairment (MCI), moderate cognitive impairment and severe cognitive impairment.

23. Use according to any one of claims 15 to 22, wherein the cognitive impairment includes amnestic MCI (aMCI-m) accompanied by multiple cognitive domain impairments.

24. Use according to any one of claims 16 to 23, wherein the neurodegenerative disease includes acute neurodegenerative disease and chronic neurodegenerative disease.

25. Use according to any one of claims 16 to 24, wherein the neurodegenerative disease includes a neurodegenerative disease caused by nerve cell death and glial cell homeostasis, a neurodegenerative disease caused by aging, a neurodegenerative disease caused by affected CNS cell function, a neurodegenerative disease caused by abnormal intercellular transmission and / or a neurodegenerative disease caused by a disorder of cell movement.

26. Use according to any one of claims 16 to 25, wherein the neurodegenerative disease includes Alzheimer's disease, Parkinson's disease, multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS) and / or Huntington's disease (HD).

27. Use according to any one of claims 16 to 26, wherein the neurodegenerative disease includes juvenile dementia.

28. Use according to any one of claims 16 to 27, wherein the neurodegenerative disease includes mild juvenile dementia, moderate juvenile dementia and / or severe juvenile dementia.

29. Use according to any one of claims 15 to 28, wherein the substance includes a reagent and / or a device capable of detecting the DIR and / or the nucleic acid encoding the DIR.

30. Use according to any one of claims 15 to 29, wherein the substance includes a reagent specifically detecting the DIR and / or the nucleic acid encoding the DIR.

31. The use according to any one of claims 15 to 30, wherein the substance comprises an antigen-binding protein capable of specifically binding to the DIR, a probe capable of specifically binding to the DIR, a primer capable of specifically amplifying a nucleic acid encoding the DIR, a gene chip capable of specifically detecting a nucleic acid encoding the DIR, an aptamer capable of specifically binding to a nucleic acid encoding the DIR, and / or a guide RNA capable of specifically binding to a nucleic acid encoding the DIR.

32. The use according to claim 31, wherein the antigen-binding protein comprises LCDR2, and the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:

74.

33. The use according to claim 32, wherein the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 26 or 16.

34. The use according to any one of claims 31 to 33, wherein the antigen-binding protein comprises LCDR1, and the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:

73.

35. The use according to claim 34, wherein the LCDR1 comprises the amino acid sequence shown in any of SEQ ID NO: 25, 52, and 15.

36. The use according to any one of claims 31 to 35, wherein the antigen-binding protein comprises LCDR3, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:

75.

37. The use according to claim 36, wherein the LCDR3 comprises the amino acid sequence shown in any of SEQ ID NO: 27, 53, 67, and 17.

38. The antigen-binding protein comprises LCDR1, LCDR2, and LCDR3, wherein a) the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 25, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 26, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 27; b) the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 52, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 26, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 53; c) The LCDR1 contains the amino acid sequence shown in SEQ ID NO: 52, the LCDR2 contains the amino acid sequence shown in SEQ ID NO: 26, and the LCDR3 contains the amino acid sequence shown in SEQ ID NO: 67, or d) The LCDR1 contains the amino acid sequence shown in SEQ ID NO: 15, the LCDR2 contains the amino acid sequence shown in SEQ ID NO: 16, and the LCDR3 contains the amino acid sequence shown in SEQ ID NO: 17, the use according to any one of claims 31 to 37. **Claim 39** The use according to any one of claims 31 to 38, wherein the antigen-binding protein contains HCDR1, and the HCDR1 contains the amino acid sequence shown in SEQ ID NO:

70. **Claim 40** The use according to claim 39, wherein the HCDR1 contains the amino acid sequence shown in any one of SEQ ID NO: 20, 35, 56 and 10. **Claim 41** The use according to any one of claims 31 to 40, wherein the antigen-binding protein contains HCDR2, and the HCDR2 contains the amino acid sequence shown in SEQ ID NO:

71. **Claim 42** The use according to claim 41, wherein the HCDR2 contains the amino acid sequence shown in any one of SEQ ID NO: 21, 36, 42, 48 and 11. **Claim 43** The use according to any one of claims 31 to 42, wherein the antigen-binding protein contains HCDR3, and the HCDR3 contains the amino acid sequence shown in SEQ ID NO:

70. **Claim 44** The use according to claim 43, wherein the HCDR3 contains the amino acid sequence shown in any one of SEQ ID NO: 22, 30, 37, 43, 49, 57, 62 and 12. **Claim 45** The antigen-binding protein contains HCDR1, HCDR2 and HCDR3, wherein a) The HCDR1 contains the amino acid sequence shown in SEQ ID NO: 20, the HCDR2 contains the amino acid sequence shown in SEQ ID NO: 21, and the HCDR3 contains the amino acid sequence shown in SEQ ID NO: 22, b) The HCDR1 contains the amino acid sequence shown in SEQ ID NO: 20, the HCDR2 contains the amino acid sequence shown in SEQ ID NO: 21, and the HCDR3 contains the amino acid sequence shown in SEQ ID NO: 30, c) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 35, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 36, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 37; d) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 20, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 42, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 43; e) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 20, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 48, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 49; f) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 56, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 36, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 57; g) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 35, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 36, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 62; h) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 35, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 42, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 37; i) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 56, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 36, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 57, or j) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 10, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 11, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 12, the use according to any one of claims 31 to 44.

46. the antigen-binding protein comprises a heavy chain variable region VH, and the VH comprises the amino acid sequence shown in any one of SEQ ID NOs: 13, 23, 31, 38, 44, 50, 58, 63 and 65, the use according to any one of claims 31 to 45. Claim 47 The use according to any one of claims 31 to 46, wherein the antigen-binding protein comprises a light chain variable region VL, and the VL comprises an amino acid sequence shown in any of SEQ ID NOs: 18, 28, 33, 40, 46, 54, 60 and 68. Claim 48 The antigen-binding protein comprises a heavy chain variable region VH and a light chain variable region VL, wherein a) the VH comprises the amino acid sequence shown in SEQ ID NO: 23, and the VL comprises the amino acid sequence shown in SEQ ID NO: 28; b) the VH comprises the amino acid sequence shown in SEQ ID NO: 31, and the VL comprises the amino acid sequence shown in SEQ ID NO: 33; c) the VH comprises the amino acid sequence shown in SEQ ID NO: 38, and the VL comprises the amino acid sequence shown in SEQ ID NO: 40; d) the VH comprises the amino acid sequence shown in SEQ ID NO: 44, and the VL comprises the amino acid sequence shown in SEQ ID NO: 46; e) the VH comprises the amino acid sequence shown in SEQ ID NO: 50, and the VL comprises the amino acid sequence shown in SEQ ID NO: 54; f) the VH comprises the amino acid sequence shown in SEQ ID NO: 58, and the VL comprises the amino acid sequence shown in SEQ ID NO: 60; g) the VH comprises the amino acid sequence shown in SEQ ID NO: 63, and the VL comprises the amino acid sequence shown in SEQ ID NO: 60; h) the VH comprises the amino acid sequence shown in SEQ ID NO: 65, and the VL comprises the amino acid sequence shown in SEQ ID NO: 54; i) the VH comprises the amino acid sequence shown in SEQ ID NO: 58, and the VL comprises the amino acid sequence shown in SEQ ID NO: 68; or j) the VH comprises the amino acid sequence shown in SEQ ID NO: 13, and the VL comprises the amino acid sequence shown in SEQ ID NO: 18, the use according to any one of claims 31 to 47. Claim 49 The use according to any one of claims 31 to 48, wherein the antigen-binding protein comprises a heavy chain constant region, and the heavy chain constant region comprises a constant region derived from IgG. Claim 50 The use according to claim 49, wherein the heavy chain constant region comprises a constant region derived from a protein selected from the group consisting of IgG1, IgG2, IgG3, and IgG4.

51. The use according to any one of claims 31 to 50, wherein the antigen-binding protein comprises a light chain constant region, and the light chain constant region comprises a constant region derived from Igκ or a constant region derived from Igλ.

52. The use according to claim 51, wherein the light chain constant region comprises a constant region derived from human Igκ.

53. The use according to any one of claims 31 to 52, wherein the antigen-binding protein comprises an antibody or an antigen-binding fragment thereof.

54. The use according to claim 53, wherein the antigen-binding fragment is selected from the group consisting of Fab, Fab’, F(ab)2, Fv fragment, F(ab’)2, scFv, di-scFv, VHH and / or dAb.

55. The use according to any one of claims 53 to 54, wherein the antibody is selected from the group consisting of a monoclonal antibody, a mouse antibody, and a chimeric antibody.

56. The use according to any one of claims 15 to 55, wherein the product comprises a diagnostic kit.

57. A diagnostic kit comprising a substance according to any one of claims 15 to 56 for use in diagnosing cognitive impairment and / or evaluating the progression of cognitive impairment and / or for use in diagnosing neurodegenerative diseases and / or evaluating the progression of neurodegenerative diseases.

58. The diagnostic kit according to claim 57, comprising an instruction manual describing a method of using the diagnostic kit for performing a diagnosis of cognitive impairment and / or an evaluation of the progression of cognitive impairment and / or a diagnosis of neurodegenerative diseases and / or an evaluation of the progression of neurodegenerative diseases.

59. A method for diagnosing cognitive impairment and / or evaluating the progression of cognitive impairment, comprising detecting the content of DIR, which is an intron-retaining splicing product of DNA damage-inducible transcript 4-like transcript (DDIT4L) in a sample derived from a subject in need thereof, and / or a nucleic acid encoding the DIR.

60. A method for diagnosing a neurodegenerative disease and / or evaluating the progression of a neurodegenerative disease, comprising the step of detecting the content of a DIR, which is an intron-retaining splicing product of a DNA damage-inducible transcript 4-like transcript (DDIT4L) in a sample derived from a subject in need, and / or a nucleic acid encoding said DIR.

61. The method according to any one of claims 59 to 60, wherein said DIR comprises the amino acid sequence shown in SEQ ID NO:

1.

62. The method according to any one of claims 59 to 61, wherein the nucleic acid encoding said DIR comprises the nucleotide sequence shown in SEQ ID NO:

8.

63. The method according to any one of claims 59 to 62, wherein the nucleic acid encoding said DIR comprises the nucleotide sequence shown in SEQ ID NO:

93.

64. The method according to any one of claims 59 to 63, wherein said cognitive impairment includes normal age-related cognitive impairment, Lewy body dementia (LBD), frontotemporal dementia and / or vascular dementia.

65. The method according to any one of claims 59 to 64, wherein the inducing diseases of said cognitive impairment include Alzheimer's disease, multiple infarction, Parkinson's disease, AIDS and / or Creutzfeldt-Jakob disease (CJD).

66. The method according to any one of claims 59 to 65, wherein said cognitive impairment includes mild cognitive impairment (MCI), moderate cognitive impairment and severe cognitive impairment.

67. The method according to any one of claims 59 to 66, wherein said cognitive impairment includes amnestic MCI (aMCI-m) with multiple cognitive domain impairments.

68. The method according to any one of claims 60 to 67, wherein said neurodegenerative disease includes acute neurodegenerative diseases and chronic neurodegenerative diseases.

69. The method according to any one of claims 60 to 68, wherein said neurodegenerative disease includes neurodegenerative diseases caused by nerve cell death and glial cell homeostasis, neurodegenerative diseases caused by aging, neurodegenerative diseases caused by affected CNS cell functions, neurodegenerative diseases caused by abnormal intercellular transmission and / or neurodegenerative diseases caused by disorders of cell motility.

70. The method according to any one of claims 60 to 69, wherein the neurodegenerative disease includes Alzheimer's disease, Parkinson's disease, multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS) and / or Huntington's disease (HD).

71. The method according to any one of claims 60 to 70, wherein the neurodegenerative disease includes juvenile dementia.

72. The method according to any one of claims 60 to 71, wherein the neurodegenerative disease includes mild juvenile dementia, moderate juvenile dementia and / or severe juvenile dementia.

73. The method according to any one of claims 59 to 72, wherein the subject includes a mammal.

74. The method according to any one of claims 59 to 73, wherein the subject includes a cancer patient.

75. The method according to any one of claims 59 to 74, wherein the subject is in an elderly stage.

76. The method according to any one of claims 59 to 75, wherein the sample includes a blood sample and / or a tissue or cell sample.

77. The method according to any one of claims 59 to 76, wherein the sample includes whole blood, serum, plasma and / or cerebrospinal fluid.

78. The method according to any one of claims 59 to 77, including comparing the content of the DIR and / or the nucleic acid encoding DIR in the sample of the subject with a healthy control value, wherein the healthy control value includes the content of the DIR and / or the nucleic acid encoding DIR in a healthy subject.

79. The method according to claim 78, wherein the healthy subject does not suffer from the cognitive impairment and / or the neurodegenerative disease.

80. The method according to any one of claims 78 to 79, wherein when the content of the DIR and / or the nucleic acid encoding DIR in the sample of the subject is significantly higher than the healthy control value, the subject is diagnosed as suffering from the cognitive impairment and / or the neurodegenerative disease.

81. The method according to any one of claims 59 to 80, including comparing the content of the DIR and / or the nucleic acid encoding DIR in the sample of the subject with an initial control value, wherein the initial control value includes the content of the DIR and / or the nucleic acid encoding DIR detected in the past of the same subject.

82. The method according to claim 81, wherein the subject has been definitely diagnosed with the cognitive impairment and / or the neurodegenerative disease.

83. The method according to any one of claims 81 to 82, wherein when the content of the DIR and / or the nucleic acid encoding DIR in the sample of the subject is significantly higher than the initial control value, the subject is diagnosed as having an aggravated progression of the cognitive impairment and / or the neurodegenerative disease.

84. The method according to any one of claims 59 to 83, further comprising the step of detecting the content of a marker related to cognitive impairment and / or neurodegenerative disease in a sample derived from the subject.

85. The method according to claim 84, wherein the marker related to cognitive impairment and / or neurodegenerative disease comprises AD7C-NTP, pTau-181, pTau-217, and / or Aβ1-42.

86. The method according to any one of claims 59 to 85, further comprising the step of observing a brain image of the subject.

87. A system for diagnosing cognitive impairment and / or evaluating the progression of cognitive impairment, comprising a substance for detecting DIR, which is an intron-retaining splicing product of DNA damage-inducible transcript 4-like transcript (DDIT4L), and / or a nucleic acid encoding the DIR.

88. A system for diagnosing neurodegenerative disease and / or evaluating the progression of neurodegenerative disease, comprising a substance for detecting DIR, which is an intron-retaining splicing product of DNA damage-inducible transcript 4-like transcript (DDIT4L), and / or a nucleic acid encoding the DIR.

89. The system according to any one of claims 87 to 88, wherein the DIR comprises the amino acid sequence shown in SEQ ID NO:

1.

90. The system according to any one of claims 87 to 89, wherein the nucleic acid encoding the DIR comprises the nucleotide sequence shown in SEQ ID NO:

8.

91. The system according to any one of claims 87 to 90, wherein the nucleic acid encoding the DIR comprises the nucleotide sequence shown in SEQ ID NO:

93.

92. The system according to any one of claims 87 to 91, wherein the cognitive impairment comprises normal age-related cognitive impairment, Lewy body dementia (LBD), frontotemporal dementia and / or vascular cognitive impairment.

93. The system according to any one of claims 87 to 92, wherein the disease inducing the cognitive impairment includes Alzheimer's disease, multiple infarction, Parkinson's disease, AIDS and / or Creutzfeldt-Jakob disease (CJD).

94. The system according to any one of claims 87 to 93, wherein the cognitive impairment includes mild cognitive impairment (MCI), moderate cognitive impairment and severe cognitive impairment.

95. The system according to any one of claims 87 to 94, wherein the cognitive impairment includes amnestic MCI (aMCI-m) accompanied by multiple cognitive domain impairments.

96. The system according to any one of claims 88 to 95, wherein the neurodegenerative disease includes acute neurodegenerative disease and chronic neurodegenerative disease.

97. The system according to any one of claims 88 to 96, wherein the neurodegenerative disease includes a neurodegenerative disease caused by nerve cell death and glial cell homeostasis, a neurodegenerative disease caused by aging, a neurodegenerative disease caused by affected CNS cell function, a neurodegenerative disease caused by abnormal intercellular transmission and / or a neurodegenerative disease caused by a disorder of cell movement.

98. The system according to any one of claims 88 to 97, wherein the neurodegenerative disease includes Alzheimer's disease, Parkinson's disease, multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS) and / or Huntington's disease (HD).

99. The system according to any one of claims 88 to 98, wherein the neurodegenerative disease includes juvenile dementia.

100. The system according to any one of claims 88 to 99, wherein the neurodegenerative disease includes mild juvenile dementia, moderate juvenile dementia and / or severe juvenile dementia.

101. The system according to any one of claims 87 to 100, which exists in the form of a kit.

102. The system according to any one of claims 87 to 101, including a collection module for collecting a specimen derived from a subject in need.

103. The system according to claim 102, wherein the subject includes a mammal.

104. The system according to any one of claims 102 to 103, wherein the subject includes a tumor patient.

105. The system according to any one of claims 102 to 104, wherein the subject is in an elderly stage.

106. The system according to any one of claims 102 to 105, wherein the sample includes a blood sample and / or a tissue or cell sample.

107. The system according to any one of claims 102 to 106, wherein the sample includes whole blood, serum, plasma and / or cerebrospinal fluid.

108. The system according to any one of claims 87 to 107, comprising a detection module for detecting the content value of the DIR and / or the nucleic acid encoding the DIR.

109. The system according to claim 108, wherein the detection module detects the content value of the DIR and / or the nucleic acid encoding the DIR in the sample collected by the collection module.

110. The system according to any one of claims 108 to 109, wherein the detection module includes a substance capable of detecting the content value of the DIR and / or the nucleic acid encoding the DIR.

111. The system according to claim 110, wherein the substance includes an antigen-binding protein capable of specifically binding to the DIR, a probe capable of specifically binding to the DIR, a primer capable of specifically amplifying the nucleic acid encoding the DIR, a gene chip capable of specifically detecting the nucleic acid encoding the DIR, an aptamer capable of specifically binding to the nucleic acid encoding the DIR, and / or a guide RNA capable of specifically binding to the nucleic acid encoding the DIR.

112. The system according to claim 111, wherein the antigen-binding protein includes LCDR2, and the LCDR2 includes the amino acid sequence shown in SEQ ID NO:

74.

113. The system according to claim 112, wherein the LCDR2 includes the amino acid sequence shown in SEQ ID NO: 26 or 16.

114. The system according to any one of claims 112 to 113, wherein the antigen-binding protein includes LCDR1, and the LCDR1 includes the amino acid sequence shown in SEQ ID NO:

73.

115. The system according to claim 114, wherein the LCDR1 includes the amino acid sequence shown in any one of SEQ ID NO: 25, 52 and 15.

116. The system according to any one of claims 111 to 115, wherein the antigen-binding protein comprises an LCDR3, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:

75.

117. The system according to claim 116, wherein the LCDR3 comprises the amino acid sequence shown in any one of SEQ ID NO: 27, 53, 67, and 17.

118. The antigen-binding protein comprises LCDR1, LCDR2, and LCDR3, wherein a) the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 25, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 26, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 27; b) the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 52, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 26, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 53; c) the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 52, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 26, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 67; or d) the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 15, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 16, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:

17. The system according to any one of claims 111 to 117.

119. The system according to any one of claims 111 to 118, wherein the antigen-binding protein comprises an HCDR3, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:

70.

120. The system according to claim 119, wherein the HCDR1 comprises the amino acid sequence shown in any one of SEQ ID NO: 20, 35, 56, and 10.

121. The system according to any one of claims 111 to 120, wherein the antigen-binding protein comprises an HCDR2, and the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:

71.

122. The system according to claim 121, wherein the HCDR2 comprises the amino acid sequence shown in any one of SEQ ID NO: 21, 36, 42, 48, and 11.

123. The system according to any one of claims 111 to 122, wherein the antigen-binding protein comprises an HCDR3, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:

70.

124. The system according to claim 123, wherein the HCDR3 comprises the amino acid sequence shown in any one of SEQ ID NOs: 22, 30, 37, 43, 49, 57, 62 and 12.

125. The antigen-binding protein comprises HCDR1, HCDR2 and HCDR3, wherein a) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 20, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 21, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 22; b) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 20, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 21, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 30; c) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 35, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 36, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 37; d) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 20, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 42, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 43; e) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 20, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 48, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 49; f) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 56, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 36, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 57; g) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 35, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 36, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:

62. h) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 35, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 42, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 37; i) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 56, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 36, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 57, or j) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 10, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 11, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 12, the system according to any one of claims 111 to 124. **Claim 126** The system according to any one of claims 111 to 125, wherein the antigen-binding protein comprises a heavy-chain variable region VH, and the VH comprises the amino acid sequence shown in any one of SEQ ID NOs: 13, 23, 31, 38, 44, 50, 58, 63 and 65. **Claim 127** The system according to any one of claims 111 to 126, wherein the antigen-binding protein comprises a light-chain variable region VL, and the VL comprises the amino acid sequence shown in any one of SEQ ID NOs: 18, 28, 33, 40, 46, 54, 60 and 68. **Claim 128** The antigen-binding protein comprises a heavy-chain variable region VH and a light-chain variable region VL, wherein a) the VH comprises the amino acid sequence shown in SEQ ID NO: 23, and the VL comprises the amino acid sequence shown in SEQ ID NO: 28; b) the VH comprises the amino acid sequence shown in SEQ ID NO: 31, and the VL comprises the amino acid sequence shown in SEQ ID NO: 33; c) the VH comprises the amino acid sequence shown in SEQ ID NO: 38, and the VL comprises the amino acid sequence shown in SEQ ID NO: 40; d) the VH comprises the amino acid sequence shown in SEQ ID NO: 44, and the VL comprises the amino acid sequence shown in SEQ ID NO: 46; e) the VH comprises the amino acid sequence shown in SEQ ID NO: 50, and the VL comprises the amino acid sequence shown in SEQ ID NO: 54; f) the VH comprises the amino acid sequence shown in SEQ ID NO: 58, and the VL comprises the amino acid sequence shown in SEQ ID NO: 60; g) the VH comprises the amino acid sequence shown in SEQ ID NO: 63, and the VL comprises the amino acid sequence shown in SEQ ID NO: 60; h) the VH comprises the amino acid sequence shown in SEQ ID NO: 65, and the VL comprises the amino acid sequence shown in SEQ ID NO: 54; i) the VH comprises the amino acid sequence shown in SEQ ID NO: 58, and the VL comprises the amino acid sequence shown in SEQ ID NO: 68, or j) the VH comprises the amino acid sequence shown in SEQ ID NO: 13, and the VL comprises the amino acid sequence shown in SEQ ID NO: 18, the system according to any one of claims 111 to 127.

129. The system according to any one of claims 111 to 128, wherein the antigen-binding protein comprises a heavy chain constant region, and the heavy chain constant region comprises a constant region derived from IgG.

130. The system according to claim 129, wherein the heavy chain constant region comprises a constant region derived from a protein selected from the group consisting of IgG1, IgG2, IgG3, and IgG4.

131. The system according to any one of claims 111 to 130, wherein the antigen-binding protein comprises a light chain constant region, and the light chain constant region comprises a constant region derived from Igκ or a constant region derived from Igλ.

132. The system according to claim 131, wherein the light chain constant region comprises a constant region derived from human Igκ.

133. The system according to any one of claims 111 to 132, wherein the antigen-binding protein comprises an antibody or an antigen-binding fragment thereof.

134. The system according to claim 133, wherein the antigen-binding fragment is selected from the group consisting of Fab, Fab’, F(ab)2, Fv fragment, F(ab’)2, scFv, di-scFv, VHH and / or dAb.

135. The system according to any one of claims 133 to 134, wherein the antibody is selected from the group consisting of a monoclonal antibody, a mouse antibody, and a chimeric antibody.

136. The system according to any one of claims 108 to 135, comprising an apparatus capable of detecting the content of DIR, which is an intron-retaining splicing product of DNA damage-inducible transcript 4-like transcript (DDIT4L), and / or the nucleic acid encoding said DIR.

137. The system according to any one of claims 108 to 136, comprising a substance and / or an apparatus capable of detecting the content of a marker associated with cognitive impairment and / or neurodegenerative disease in the detection module.

138. The system according to claim 137, wherein the marker associated with cognitive impairment and / or neurodegenerative disease comprises AD7C-NTP, pTau-181, pTau-217, and / or Aβ1-42.

139. The system according to any one of claims 108 to 138, wherein the detection module outputs a content value of the DIR and / or the nucleic acid encoding the DIR.

140. Including a judgment module for judging the diagnosis result and / or disease progression result of the subject based on the content of the DIR and / or the nucleic acid encoding the DIR and the healthy control value and / or initial control value, a) The healthy control value includes the content of the DIR and / or the nucleic acid encoding the DIR in a healthy subject, and the initial control value includes the content of the DIR and / or the nucleic acid encoding the DIR detected in the past of the same subject. The system according to any one of claims 87 to 139.

141. The system according to claim 140, wherein the healthy subject does not suffer from the cognitive impairment and / or the neurodegenerative disease.

142. The system according to any one of claims 140 to 141, wherein when the content of the DIR and / or the nucleic acid encoding the DIR in the sample of the subject is significantly higher than the healthy control value, the judgment module diagnoses that the subject suffers from the cognitive impairment and / or the neurodegenerative disease.

143. The system according to any one of claims 140 to 142, wherein the subject is definitely diagnosed as suffering from the cognitive impairment and / or the neurodegenerative disease.

144. If the content of the DIR and / or the nucleic acid encoding the DIR in the specimen of the subject is significantly higher than the initial control value by the determination module, the system according to any one of claims 140 to 143 diagnoses that the progression of the cognitive impairment and / or the neurodegenerative disease in the subject has worsened.

145. The system according to any one of claims 140 to 144, wherein the determination module outputs a diagnosis result of the subject.

146. The system according to any one of claims 87 to 145, comprising a display module for displaying a diagnosis result of the determination module.

147. The system according to any one of claims 87 to 146, comprising a storage module for storing the content value output to the detection module and / or the diagnosis result output to the determination module.

148. A drug screening method comprising a step of detecting a change in the content of DIR and / or a nucleic acid encoding DIR in a subject after administration of a candidate drug for use in the prevention, treatment and / or alleviation of cognitive impairment and / or neurodegenerative disease.

149. The method according to claim 148, wherein the DIR comprises the amino acid sequence shown in SEQ ID NO:

1.

150. The method according to any one of claims 148 to 149, wherein the nucleic acid encoding the DIR comprises the nucleotide sequence shown in SEQ ID NO:

8.

151. The method according to any one of claims 148 to 150, wherein the nucleic acid encoding the DIR comprises the nucleotide sequence shown in SEQ ID NO:

93.

152. The method according to any one of claims 148 to 151, wherein the cognitive impairment includes normal age-related cognitive impairment, Lewy body dementia (LBD), frontotemporal dementia and / or vascular dementia.

153. The method according to any one of claims 148 to 152, wherein the inducing diseases of the cognitive impairment include Alzheimer's disease, multiple infarction, Parkinson's disease, AIDS and / or Creutzfeldt-Jakob disease (CJD).

154. The method according to any one of claims 148 to 153, wherein the cognitive impairment includes mild cognitive impairment (MCI), moderate cognitive impairment and severe cognitive impairment.

155. The method according to any one of claims 148 to 154, wherein the cognitive impairment includes amnestic MCI (aMCI-m) accompanied by multiple cognitive domain impairments.

156. The method according to any one of claims 148 to 155, wherein the neurodegenerative disease includes an acute neurodegenerative disease and a chronic neurodegenerative disease.

157. The method according to any one of claims 148 to 156, wherein the neurodegenerative disease includes a neurodegenerative disease caused by neuronal death and glial cell homeostasis, a neurodegenerative disease caused by aging, a neurodegenerative disease caused by affected CNS cell function, a neurodegenerative disease caused by abnormal intercellular transmission, and / or a neurodegenerative disease caused by a disorder of cell motility.

158. The method according to any one of claims 148 to 157, wherein the neurodegenerative disease includes Alzheimer's disease, Parkinson's disease, multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), and / or Huntington's disease (HD).

159. The method according to any one of claims 148 to 158, wherein the neurodegenerative disease includes juvenile dementia.

160. The method according to any one of claims 148 to 159, wherein the neurodegenerative disease includes mild juvenile dementia, moderate juvenile dementia, and / or severe juvenile dementia.

161. The method according to any one of claims 148 to 160, wherein when the administration of the candidate drug reduces the content of DIR and / or the nucleic acid encoding DIR in the subject, the candidate drug has a therapeutic effect.

162. The method according to any one of claims 148 to 161, wherein the administration includes an injection.

163. The method according to any one of claims 148 to 162, wherein the drug includes a small molecule drug and / or a biopolymer drug.

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