CN118647405A

CN118647405A - Methods to improve cognitive impairment

Info

Publication number: CN118647405A
Application number: CN202380018625.8A
Authority: CN
Inventors: 李开诚; 史海翔; 攸璞; 李震
Original assignee: Shanghai Quetedi Biotechnology Co ltd
Current assignee: Shanghai Quetedi Biotechnology Co ltd
Priority date: 2022-01-25
Filing date: 2023-01-20
Publication date: 2024-09-13
Also published as: WO2023143425A1

Abstract

The present application relates to a method of improving cognitive impairment. The method comprises administering a modulator that modulates the intron retention cleavage product DIR of the DNA damage inducing transcript 4-like transcript and/or a functional fragment thereof, whereby a disease can be prevented and/or treated, wherein the disease comprises a cognitive disorder and/or a neurodegenerative disease.

Description

Methods of improving cognitive disorders

Technical Field

The application relates to the field of biological medicine, in particular to a method for improving cognitive dysfunction.

Background

The number of the aging population in China is increased year by year, the aged population suffers from various diseases due to the influence of various factors such as immunity decline, arteriosclerosis and the like, wherein the incidence rate of senile dementia in the current aged population is not neglected. Dementia patients not only themselves are afflicted with pain, but also put a great burden on their own families. Therefore, the development of therapeutic drugs for senile dementia is of great significance.

Over the course of many years, more and more senile dementia-related genes have been reported, such as APP, PSEN1, etc. These genetic mutations accelerate neuronal damage and thus cause cognitive impairment. Drugs based on these targets, particularly monoclonal antibody drugs against aβ, have been developed in a number of production lines, and FDA-approved drugs are also marketed in the present year. However, such monoclonal antibodies, while effective in scavenging aβ accumulation in the human brain, are not ideal for improving cognitive ability in patients.

It can be seen that there is a need to seek new methods of improving cognitive disorders.

Disclosure of Invention

The present application provides a novel method of ameliorating cognitive disorders.

In one aspect, the application provides a modulator for modulating the intron retention cleavage product DIR of a DNA damage inducible transcript 4-like transcript and/or a functional fragment thereof for use in the manufacture of a medicament for the prevention and/or treatment of a disease, wherein the disease comprises a cognitive disorder.

In one aspect, the application provides a modulator for modulating the intron retention cleavage product DIR of a DNA damage inducible transcript 4-like transcript and/or a functional fragment thereof for use in the manufacture of a medicament for the prevention and/or treatment of a disease, wherein the disease comprises a neurodegenerative disease.

In certain embodiments, the modulator reduces the expression level and/or biological activity of the DIR and/or functional fragment thereof in the subject.

In certain embodiments, the reducing comprises reducing the expression level and/or biological activity of the DIR and/or functional fragment thereof by at least about 10% as compared to the expression level and/or biological activity of the original DIR and/or functional fragment thereof in the subject.

In certain embodiments, the expression level comprises the expression level of a gene encoding the DIR and/or a functional fragment thereof, the transcription level of a gene encoding the DIR and/or a functional fragment thereof, and/or the expression level of the DIR and/or a functional fragment thereof.

In certain embodiments, the expression level is measured by performing an assay selected from the group consisting of: qPCR, qRT-PCR, hybridization analysis, northern blotting, dot blotting, in situ hybridization, gel electrophoresis, capillary electrophoresis, column chromatography, western blotting, immunohistochemistry, immunostaining, and mass spectrometry.

In certain embodiments, the expression level is measured by using a substance selected from the group consisting of: a primer capable of specifically amplifying a gene encoding the DIR and/or a functional fragment thereof, a nucleic acid molecule specifically binding to the DIR and/or a functional fragment thereof, a small molecule specifically binding to the DIR and/or a functional fragment thereof, a probe specifically binding to the DIR and/or a functional fragment thereof, and a polypeptide specifically binding to the DIR and/or a functional fragment thereof.

In certain embodiments, the functional fragment of the DIR retains at least a portion of the biological activity of the DIR.

In certain embodiments, the biological activity comprises affecting excitability of a neuron and/or inhibiting activity of a neuron.

In certain embodiments, the biological activity comprises being capable of reducing the frequency of excitatory postsynaptic currents (EPSCs), and/or being capable of reducing the amplitude of EPSCs.

In certain embodiments, the reducing comprises administering the DIR and/or functional fragment thereof and/or a nucleic acid encoding the DIR and/or functional fragment thereof, with a reduced frequency of excitatory postsynaptic currents (EPSCs) in the subject and/or with a reduced magnitude of EPSCs in the subject as compared to the biological activity of the original DIR and/or functional fragment thereof in the subject.

In certain embodiments, the biological activity comprises affecting cognitive ability.

In certain embodiments, the biological activity comprises participation in a signaling pathway associated with aβ deposition and/or participation in a signaling pathway associated with Tau entanglement production.

In certain embodiments, the biological activity comprises inducing aβ deposition and/or amyloid plaque formation by gelsolin.

In certain embodiments, the DIR and/or functional fragment thereof induces aβ deposition and/or amyloid plaque formation by binding to gelsolin.

In certain embodiments, the expression level of the DIR and/or functional fragment thereof is positively correlated with the expression level of aβ.

In certain embodiments, the reducing comprises administering the DIR and/or functional fragment thereof and/or a nucleic acid encoding the DIR and/or functional fragment thereof to reduce cognitive ability of the subject as compared to the biological activity of the original DIR and/or functional fragment thereof in the subject.

In certain embodiments, the DIR and/or functional fragment thereof is derived from a mammal.

In certain embodiments, the DIR and/or functional fragment thereof is derived from a primate.

In certain embodiments, the DIR and/or functional fragment thereof is of human origin.

In certain embodiments, the DIR comprises the amino acid sequence shown in SEQ ID No. 1.

In certain embodiments, the functional fragment of the DIR comprises an amino acid sequence encoded by an intron residing in DDIT 4L.

In certain embodiments, the functional fragment of a DIR comprises the amino acid sequence set forth in any one of SEQ ID NOS.4-5.

In certain embodiments, the cognitive disorder comprises a cognitive disorder caused by normal aging, lews Body Dementia (LBD), frontotemporal dementia, and/or vascular dementia.

In certain embodiments, the cognitive disorder-inducing disease comprises alzheimer's disease, multiple infarctions, parkinson's disease, aids, and/or creutzfeldt-jakob disease (CJD).

In certain embodiments, the cognitive disorders include early cognitive disorders (MCI), mid-term cognitive disorders, and late cognitive disorders.

In certain embodiments, the cognitive disorder comprises impaired amnestic MCI multi-cognitive domain (aMCI-m).

In certain embodiments, the neurodegenerative disease comprises acute neurodegenerative disease and chronic neurodegenerative disease.

In certain embodiments, the neurodegenerative disease comprises a neurodegenerative disease caused by neuronal death and glial cell homeostasis, a neurodegenerative disease caused by aging, a neurodegenerative disease caused by an affected CNS cell function, a neurodegenerative disease caused by abnormal intercellular communication, and/or a neurodegenerative disease caused by impaired cell motility.

In certain embodiments, the neurodegenerative disease comprises alzheimer's disease, parkinson's disease, multiple Sclerosis (MS), amyotrophic Lateral Sclerosis (ALS), and/or Huntington's Disease (HD).

In certain embodiments, the neurodegenerative disease comprises Alzheimer's disease.

In certain embodiments, the neurodegenerative disease comprises early, mid and/or late presenile dementia.

In certain embodiments, the subject comprises a mammal.

In certain embodiments, the subject comprises a human.

In certain embodiments, the subject comprises a patient with a neurodegenerative disease and/or a patient with a cognitive disorder.

In certain embodiments, the subject comprises a patient with alzheimer's disease.

In certain embodiments, the subject is in an aging stage.

In certain embodiments, the agent is formulated for oral administration and/or injection administration.

In certain embodiments, the modulator comprises a small molecule compound, a polymer, and/or a biological macromolecule.

In certain embodiments, the modulator comprises an antibody or antigen-binding fragment thereof.

In certain embodiments, the modulator comprises an antibody or antigen-binding fragment thereof that specifically binds to the intron retention cleavage product DIR of the 4-like transcript of the DNA damage inducing transcript and/or a functional fragment thereof.

In certain embodiments, the modulator comprises an antisense oligonucleotide.

In certain embodiments, the modulator comprises an siRNA.

In certain embodiments, the modulator comprises an siRNA that specifically binds to an intron retention cleavage product DIR of a 4-like transcript of a DNA damage inducing transcript and/or a functional fragment thereof.

In certain embodiments, the modulator comprises the nucleotide sequence set forth in any one of SEQ ID NOS.84-87.

In another aspect, the present application provides a method for preventing and/or treating cognitive disorders comprising the steps of: the level of expression and/or biological activity of the intron retention cleavage product DIR and/or functional fragment thereof of the DNA damage inducible transcript 4-like transcript is reduced in a subject in need thereof.

In another aspect, the present application provides a method for preventing and/or treating a neurodegenerative disease, comprising the steps of: the level of expression and/or biological activity of the intron retention cleavage product DIR and/or functional fragment thereof of the DNA damage inducible transcript 4-like transcript is reduced in a subject in need thereof.

In certain embodiments, the subject comprises a mammal.

In certain embodiments, the subject comprises a human.

In certain embodiments, the subject is in an aging stage.

In certain embodiments, the method comprises the steps of: administering to a subject in need thereof a modulator capable of reducing the expression level and/or biological activity of the DIR and/or functional fragment thereof, and/or a nucleic acid encoding the DIR and/or functional fragment thereof.

In certain embodiments, the administration comprises oral administration and/or injection administration.

In certain embodiments, the modulator comprises an antisense oligonucleotide.

In certain embodiments, the modulator comprises an siRNA.

In another aspect, the present application provides a method of screening for a drug capable of preventing and/or treating cognitive disorders and/or treating neurodegenerative diseases, comprising the steps of: detecting the effect of a candidate drug on the expression level and/or biological activity of a DIR and/or functional fragment thereof in a subject, wherein upon administration of the candidate drug, the expression level and/or biological activity of the DIR and/or functional fragment thereof is reduced, the candidate drug is capable of preventing and/or treating cognitive disorders and/or treating neurodegenerative diseases.

In certain embodiments, the subject comprises a mammal.

In certain embodiments, the subject is in an aging stage.

In certain embodiments, the candidate drug comprises a small molecule compound, a polymer, and/or a biological macromolecule.

In another aspect, the application provides an isolated antigen binding protein having the following properties: specifically binds to human DIR and/or functional fragments thereof in ELISA assays at a working concentration of about 10ng/ml or more;

In certain embodiments, the antigen binding protein comprises LCDR2, and the LCDR2 comprises the amino acid sequence shown in SEQ ID NO. 74.

In certain embodiments, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO. 26 or 16.

In certain embodiments, the antigen binding protein comprises LCDR1, wherein LCDR1 comprises the amino acid sequence depicted in SEQ ID NO. 73.

In certain embodiments, the LCDR1 comprises an amino acid sequence set forth in any one of SEQ ID NOs 25, 52, 15.

In certain embodiments, the antigen binding protein comprises LCDR3, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO. 75.

In certain embodiments, the LCDR3 comprises an amino acid sequence set forth in any one of SEQ ID NOs 27, 53, 67, 17.

In certain embodiments, the antigen binding protein comprises LCDR1, LCDR2, and LCDR3, wherein,

A) The LCDR1 comprises an amino acid sequence shown in SEQ ID NO. 25, the LCDR2 comprises an amino acid sequence shown in SEQ ID NO. 26, and the LCDR3 comprises an amino acid sequence shown in SEQ ID NO. 27;

b) The LCDR1 comprises an amino acid sequence shown as SEQ ID NO. 52, the LCDR2 comprises an amino acid sequence shown as SEQ ID NO. 26, and the LCDR3 comprises an amino acid sequence shown as SEQ ID NO. 53;

c) The LCDR1 comprises an amino acid sequence shown as SEQ ID NO. 52, the LCDR2 comprises an amino acid sequence shown as SEQ ID NO. 26, and the LCDR3 comprises an amino acid sequence shown as SEQ ID NO. 67; or alternatively

D) The LCDR1 comprises an amino acid sequence shown in SEQ ID NO. 15, the LCDR2 comprises an amino acid sequence shown in SEQ ID NO. 16, and the LCDR3 comprises an amino acid sequence shown in SEQ ID NO. 17.

In certain embodiments, the antigen binding protein comprises HCDR1, and the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO. 70.

In certain embodiments, the HCDR1 comprises an amino acid sequence set forth in any one of SEQ ID NOs 20, 35, 56, 10.

In certain embodiments, the antigen binding protein comprises HCDR2, and the HCDR2 comprises the amino acid sequence depicted in SEQ ID NO. 71.

In certain embodiments, the HCDR2 comprises an amino acid sequence set forth in any one of SEQ ID NOs 21, 36, 42, 48, 11.

In certain embodiments, the antigen binding protein comprises HCDR3, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO. 70.

In certain embodiments, the HCDR3 comprises an amino acid sequence set forth in any one of SEQ ID NOs 22, 30, 37, 43, 49, 57, 62, 12.

In certain embodiments, the antigen binding protein comprises HCDR1, HCDR2, and HCDR3, wherein,

A) The HCDR1 comprises an amino acid sequence shown as SEQ ID NO. 20, the HCDR2 comprises an amino acid sequence shown as SEQ ID NO. 21, and the HCDR3 comprises an amino acid sequence shown as SEQ ID NO. 22;

b) The HCDR1 comprises an amino acid sequence shown in SEQ ID NO. 20, the HCDR2 comprises an amino acid sequence shown in SEQ ID NO. 21, and the HCDR3 comprises an amino acid sequence shown in SEQ ID NO. 30;

c) The HCDR1 comprises an amino acid sequence shown in SEQ ID NO. 35, the HCDR2 comprises an amino acid sequence shown in SEQ ID NO. 36, and the HCDR3 comprises an amino acid sequence shown in SEQ ID NO. 37;

d) The HCDR1 comprises an amino acid sequence shown in SEQ ID NO. 20, the HCDR2 comprises an amino acid sequence shown in SEQ ID NO. 42, and the HCDR3 comprises an amino acid sequence shown in SEQ ID NO. 43;

e) The HCDR1 comprises an amino acid sequence shown in SEQ ID NO. 20, the HCDR2 comprises an amino acid sequence shown in SEQ ID NO. 48, and the HCDR3 comprises an amino acid sequence shown in SEQ ID NO. 49;

f) The HCDR1 comprises an amino acid sequence shown as SEQ ID NO. 56, the HCDR2 comprises an amino acid sequence shown as SEQ ID NO. 36, and the HCDR3 comprises an amino acid sequence shown as SEQ ID NO. 57;

g) The HCDR1 comprises an amino acid sequence shown in SEQ ID NO. 35, the HCDR2 comprises an amino acid sequence shown in SEQ ID NO. 36, and the HCDR3 comprises an amino acid sequence shown in SEQ ID NO. 62;

h) The HCDR1 comprises an amino acid sequence shown in SEQ ID NO. 35, the HCDR2 comprises an amino acid sequence shown in SEQ ID NO. 42, and the HCDR3 comprises an amino acid sequence shown in SEQ ID NO. 37;

i) The HCDR1 comprises an amino acid sequence shown as SEQ ID NO. 56, the HCDR2 comprises an amino acid sequence shown as SEQ ID NO. 36, and the HCDR3 comprises an amino acid sequence shown as SEQ ID NO. 57; or alternatively

J) The HCDR1 comprises an amino acid sequence shown in SEQ ID NO. 10, the HCDR2 comprises an amino acid sequence shown in SEQ ID NO. 11, and the HCDR3 comprises an amino acid sequence shown in SEQ ID NO. 12.

In certain embodiments, the antigen binding protein comprises a heavy chain variable region VH comprising an amino acid sequence set forth in any one of SEQ ID NOs 13, 23, 31, 38, 44, 50, 58, 63, 65.

In certain embodiments, the antigen binding protein comprises a light chain variable region VL comprising the amino acid sequence set forth in any one of SEQ ID NOs 18, 28, 33, 40, 46, 54, 60, 68.

In certain embodiments, the antigen binding protein comprises a heavy chain variable region VH and a light chain variable region VL, wherein:

a) The VH comprises an amino acid sequence shown in SEQ ID NO. 23, and the VL comprises an amino acid sequence shown in SEQ ID NO. 28;

b) The VH comprises an amino acid sequence shown in SEQ ID NO. 31, and the VL comprises an amino acid sequence shown in SEQ ID NO. 33;

c) The VH comprises an amino acid sequence shown in SEQ ID NO. 38, and the VL comprises an 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 an amino acid sequence shown as SEQ ID NO. 50, and the VL comprises an amino acid sequence shown as 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 an amino acid sequence shown as SEQ ID NO. 63, and the VL comprises an amino acid sequence shown as SEQ ID NO. 60;

h) The VH comprises an amino acid sequence shown as SEQ ID NO. 65, and the VL comprises an amino acid sequence shown as 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 alternatively

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.

In certain embodiments, the antigen binding protein comprises a heavy chain constant region, and the heavy chain constant region comprises a constant region derived from IgG.

In certain 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.

In certain embodiments, the antigen binding protein comprises a light chain constant region, and the light chain constant region comprises an igκ -derived constant region or an igλ -derived constant region.

In certain embodiments, the light chain constant region comprises a constant region derived from human igκ.

In certain embodiments, the antigen binding protein comprises an antibody or antigen binding fragment thereof.

In certain 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.

In certain embodiments, the antibody is selected from the group consisting of: monoclonal antibodies, murine antibodies, and chimeric antibodies.

In another aspect, the application provides a polypeptide comprising an isolated antigen binding protein of the application.

In another aspect, the application provides an immunoconjugate comprising the isolated antigen binding protein of the application or the polypeptide of the application.

In another aspect, the application provides an isolated nucleic acid molecule encoding an isolated antigen binding protein of the application, or a polypeptide of the application.

In another aspect, the application provides a vector comprising an isolated nucleic acid molecule of the application.

In another aspect, the application provides a cell comprising an isolated antigen binding protein of the application, a polypeptide of the application, an immunoconjugate of the application, an isolated nucleic acid molecule of the application and/or a vector of the application.

In another aspect, the application provides a method of making an isolated antigen binding protein of the application or a polypeptide of the application, comprising culturing a cell of the application under conditions such that the isolated antigen binding protein of the application or the polypeptide of the application is expressed.

In another aspect, the application provides a pharmaceutical composition comprising an isolated antigen binding protein of the application, a polypeptide of the application, an immunoconjugate of the application, an isolated nucleic acid molecule of the application, a vector of the application, a cell of the application, and/or a pharmaceutically acceptable adjuvant and/or excipient.

In another aspect, the application provides the use of an isolated antigen binding protein of the application and/or a polypeptide of the application in the manufacture of a medicament for the prevention and/or treatment of a disease or disorder, wherein the disease or disorder comprises a cognitive disorder and/or a neurodegenerative disease.

Other aspects and advantages of the present application will become readily apparent to those skilled in the art from the following detailed description. Only exemplary embodiments of the present application are shown and described in the following detailed description. As those skilled in the art will recognize, the present disclosure enables one skilled in the art to make modifications to the disclosed embodiments without departing from the spirit and scope of the application as claimed. Accordingly, the drawings and descriptions of the present application are to be regarded as illustrative in nature and not as restrictive.

Drawings

The specific features of the application related to the application are shown in the appended claims. A better understanding of the features and advantages of the application in accordance with the present application will be obtained by reference to the exemplary embodiments and the accompanying drawings that are described in detail below. The brief description of the drawings is as follows:

FIGS. 1A-1D show the identification of DIRs according to the application.

FIG. 2 shows a schematic structural diagram of a functional fragment of the DIR of the application.

FIG. 3 shows the effect of functional fragments of DIRs of the application on excitatory postsynaptic currents.

FIG. 4 shows the biological function of the DIR of the application.

FIGS. 5a-5i show that DIR induces A.beta.deposition by binding gelsolin, wherein:

Dir was present in thioflavin s-positive plaques (arrow) in the hippocampus of AD patients (n=3). Scale = 50 μm.

B. Aβ (n=3) was found in proteins precipitated with Flag antibodies in HEK293T cell lysates transfected with Flag-DIR expressing plasmids and added with artificially synthesized aβ42.

Pla experiments showed that DIR was unable to bind aβ in the hippocampus of AD patients. Scale bar = 50 μm.

D. coomassie blue staining showed that in U87MG cell lysates transfected with Flag-DIR plasmid, DIR antibodies could precipitate out a protein with a molecular weight of about 85kDa (n=3).

E. mass spectrometry identified the first 8 molecules between-85 kDa size intervals. gelsolin is the most abundant molecule.

F. Gelsolin (n=3) was found in the protein precipitated with DIR and aβ antibodies in hippocampal lysates of DIR-knock-in mice.

Pla experiments showed gelsolin binding to aβ (arrow) in the hippocampus of AD patients. Scale bar = 50 μm.

H. Different doses of aβ42 were added to the Flag-DIR expressing HEK293T cell lysates, gelsolin was found in the Flag antibody precipitated protein, and the binding level of both was increased in the high dose aβ42 group (10 μg, n=3).

I. In the mixture of gelsolin-his with synthetic aβ42 or DIR (31-84), DIR (31-84) causes insolubility of aβ42 by gelsolin (n=3).

FIGS. 6a-6d show that DIR does not bind directly to Abeta, but rather induces Abeta deposition. Wherein,

A. Artificially synthesized Abeta 42 is added into HEK293T cell lysate transfected with myc-DIR plasmid or DDIT4L-myc, and then myc antibodies are used for immunoprecipitation experiments respectively. Of these, only myc-DIR-expressing lysates were able to precipitate Aβ (n=3).

B. co-immunoprecipitation experiments with synthetic DIR (31-84) and aβ42 showed no direct interaction between the two (n=3).

C. In HEK293T cell lysates co-transfected with Flag-DIR and gelsolin-GFP plasmid, gelsolin-GFP (n=3) was found to be present in the protein precipitated with Flag antibody.

D. Synthetic aβ42 or DIR (31-84) was added to HEK293T cell lysates, DIR (31-84) induced aβ42 insolubility by gelsolin (n=3).

Figures 7a-7c show DIR mediated aβ plaque formation, wherein:

a. Synthetic human aβ40 (100 μm) was added to DIR-KI mice (n=3) hippocampal slices, and thioflavin s-positive plaques (arrows) appeared after 6h incubation and were co-labeled with DIR and gelsolin. Scale bar = 100 μm.

B. In AD patients (n=3), but not in the control group (n=3), thioflavin s-positive dense nuclear plaques co-localized to the hippocampal DG region with DIR and gelsolin (arrows). Scale bar = 50 μm. Quantitative analysis showed that 86.3% of dense nuclear plaques in the cerebral cortex were DIR, gelsolin and thioflavins positive, and this ratio could reach 92.8% in the hippocampus.

Dir was present in aβ -and thioflavine S-positive plaques (arrow) in the hippocampus of AD patients (n=3). The A.beta.positive region is greater than thioflavin S-and DIR-positive signals. Scale bar = 50 μm.

Figure 8 shows that in human hippocampus, aβ -positive and thioflavine s-negative plaques do not overlap with DIR and gelsolin. Wherein,

A. In AD patients (n=3), whereas in control group (n=3), aβ -positive and thioflavine s-negative diffuse plaques (arrows) were not co-localized to the hippocampal DG region with DIR and gelsolin. Scale bar = 50 μm.

Figures 9a-9l show that plasma DIR is a potential biomarker for AD and aMCI, where:

a. the plasma DIR of naMCI (n=44), aMCI (n=42) or AD (n=31) patients was gradually increased compared to the cognitive normal control group (NC, n=33). * P <0.01, p <0.001.

B. Patient plasma aβ42/aβ40 is gradually reduced by naMCI (n=44), aMCI (n=42) or AD (n=31) compared to NC (n=33). * P <0.05, p <0.01.

C. Patient plasma pTau181 is gradually elevated for naMCI (n=44), aMCI (n=42), or AD (n=31) compared to NC (n=33). * P <0.05, p <0.001.

D. there was no significant change in naMCI (n=44), aMCI (n=42), AD (n=31) patient plasma tTau compared to the control group (n=33).

Subject work feature (ROC) analysis of nc (n=33) and AD (n=31) patient plasma DIR.

F. Plasma pta181 ROC analysis for NC (n=33) and AD (n=31).

G. NC (n=33) and AD (n=31) plasma aβ42/aβ40 ROC were analyzed.

H. ROC analysis (red) was performed on NC (n=33) and AD (n=31) in combination with plasma DIR, pTau181 and aβ42/aβ40. ROC analysis (blue) was performed on NC (n=33) and AD (n=31) in combination with plasma pTau181 and aβ42/aβ40.

ROC analysis of nc (n=33) and aMCI (n=42) plasma DIR.

ROC analysis of nc (n=33) and aMCI (n=42) plasma pTau.

K. ROC analysis of NC (n=33) and aMCI (n=42) plasma aβ42/aβ40.

ROC analysis (red) was performed on NC (n=33) and aMCI (n=42) in combination with plasma DIR, pta181 and aβ42/aβ40. ROC analysis (blue) was performed on NC (n=33) and aMCI (n=42) in combination with plasma pta181 and aβ42/aβ40.

FIGS. 10a-10b show ROC analysis of plasma tTau. Wherein,

ROC analysis of nc (n=33) and AD (n=31) plasma tTau.

ROC analysis of nc (n=33) and aMCI (n=42) plasma tTau.

FIGS. 11a-11e show a correlation analysis of plasma DIR with Abeta, wherein:

a. three years ago (2018-2021) normal but currently MCI patients (n=12) had plasma DIR. MCI patients had plasma DIR concentrations higher than blood samples taken 3 years ago.

Mci and AD patients (n=117) and cognitive normal control (n=33) plasma DIR were positively correlated with plasma aβ40.

Three-dimensional visualization of amyloid 18F-AV-45 SUV in AD/MC patient I or NC. Color bars represent SUV indices derived from amyloid 18F-AV-45 images. Plasma DIR values for AD/MCI patients correlated with cortical positive signals, especially temporal lobe positive signals.

D. The plasma DIR was significantly elevated in amyloid PET positive individuals (n=11) compared to amyloid PET negative individuals (n=26). ROC analysis of plasma DIR in amyloid PET positive individuals (n=11) and in amyloid PET negative individuals (n=26). * P <0.01.

DIR-induced amyloid plaque deposition and blood secretion model. In several pathological conditions of local micro-cerebral vascular circulation, hypoxia leads to abnormal retention of introns, leading to translation of DIR proteins. Aβ is produced by the proteolytic process of the Amyloid Precursor Protein (APP), with direct binding to gelsolin. DIR, by binding gelsolin, results in aβ deposition, ultimately forming amyloid plaques in the brain. DIR may also be released into the blood system.

FIGS. 12a-12b show the correlation of plasma DIR with Abeta 42, quantitatively analyzing the SUV values of Abeta-PET. Wherein,

Mci and AD patients (n=117) and cognitive normal control (n=33) plasma DIR were positively correlated with plasma aβ42.

Quantitative analysis of AD/MCI patients or NC SUV values. * P <0.05.

FIG. 13 shows the results of detection of binding activity of DIR antibodies of the application.

FIGS. 14a-14h show that DIR is an aberrant splice of human DDIT4L in a patient, wherein:

a. Two products (-200 bp and (-2000 bp) were found from GBM mRNA of patient 1 (G2913) using the primers for DDIT4L mRNA, but not in Low Grade Glioma (LGG) tissue of LGG patient 1 (L1002).

DIR is a splice subtype of human DDIT4L with introns remaining between exon 2 and exon 3.

Immunoblotting of GBM lysates showed that the antibodies targeting the L N ends of DDIT4 detected two immunoreactive bands (-22 kDa and (-12 kDa, arrow) but not pre-absorbed (pre-absorbed) antibodies.

D. In GBM lysates of two patients (G2913, G1359), a band of 12kDa was detected using DIR antibodies targeting the intron translational region.

E. HEK293T cells transfected with Flag-DIR-expressing plasmids were detected in both cell lysates and culture medium.

F. Brain magnetic resonance imaging of the patient showed that glioblastoma invaded the hippocampal region (hippocampal region, G8098 in G) or that in which glioblastoma did not occur (G8409 in G).

G. DIR was detected only in the plasma of GBM patients (G8098 and G7200) who affected the hippocampus.

DIR is present in the plasma of AD patients and is named A7098, etc.

Figures 15A-15B show the effect on cognitive levels after reduced DIR expression levels.

Figures 16A-16C show the effect of administering DIR antibodies of the application on cognitive levels.

A.Y maze test showed that homozygous DIR-KI mice (n=17) reversed working memory after DIR antibody management as described in the present application compared to mice treated with IgG (n=13). * P <0.05.

B. The DIR antibody treatment described in the present application reversed the ability of new object recognition in homologous DIR-KI mice (n=14) compared to mice treated with IgG (n=11). * P <0.01.

Morris water maze test showed that administration of the DIR antibody of the application (n=11) treatment reduced escape latency after a 5 day training period compared to IgG treatment in homologous DIR-KI mice (n=9). In the exploratory test for evaluating spatial memory, mice treated with IgG showed no preference for the target quadrant, whereas mice treated with DIR antibodies of the application entered a shorter latency and stayed longer in the target quadrant. * P <0.05.

Detailed Description

Further advantages and effects of the present application will become readily apparent to those skilled in the art from the present disclosure, by describing embodiments of the present application with specific examples.

Definition of terms

The term "DDIT4L" generally refers to DNA DAMAGE-inducible transcript 4-like, DNA damage inducing transcripts 4-like. Which may also be referred to as REDD/RTP 801L. It was found that DDIT4L may be associated with cardiac dysfunction. Can also be used for treating glioma. The accession number of the human DDIT4L gene in GenBank is 115265; the human DDIT4L protein has accession number NP-660287.1 at GenBank.

The term "DIR" generally refers to the intron retention cleavage product of DDIT 4L. The scission reaction of DDIT4L can be seen in fig. 1. In the present application, the amino acid sequence of the DIR can be shown as SEQ ID NO. 1.

The term "expression level" generally refers to the protein, RNA or mRNA level of a particular gene of interest. The expression level of a particular gene of interest (e.g., the human DDIT4L gene) may be determined using any method known in the art. In the present application, the "expression" generally refers to a process of converting information encoded by a gene into a structure that exists in a cell and operates in the cell. For example, reverse transcription and amplification assays (e.g., PCR, ligation RT-PCR or quantitative RT-PCT), hybridization assays, northern blotting (Northern blotting), dot blotting, in situ hybridization, gel electrophoresis, capillary electrophoresis, column chromatography, western blotting, immunohistochemistry, immunostaining, or mass spectrometry may be included. The analysis may be performed directly on the biological sample or on proteins/nucleic acids isolated from the sample.

The term "activity" generally refers to any activity associated with a particular protein. In the present application, the activity may include, for example, any activity associated with DIR proteins. The activity may include protease-related enzymatic activity. In some cases, the activity may include biological activity. In some cases, the activity may include binding of the protein to a receptor, e.g., which may produce a measurable downstream effect. In the present application, the activity may include any activity that will be attributed to the protein by those skilled in the art.

The term "cognitive disorder" generally refers to diseases and conditions that are believed or actually involved in progressive loss of neuronal structure and/or function (including neuronal death) or associated with the above. For example, the features of cognitive impairment may include impairment of cognition (e.g., memory, attention, perception, and/or thinking). These disorders may include pathogen-induced cognitive dysfunction, such as HIV-related cognitive dysfunction and lyme disease-related cognitive dysfunction. Examples of cognitive disorders may include alzheimer's disease, huntington's disease, parkinson's disease, amyotrophic Lateral Sclerosis (ALS), autism, early cognitive impairment (MCI), stroke, traumatic Brain Injury (TBI), and/or age-related memory impairment (AAMI).

The term "neurodegenerative disease" generally refers to cognitive disorders such as dementia caused by progressive loss of neuronal structure and function, including neuronal death and glial cell balance. In certain cases, age (e.g., alzheimer's Disease (AD), parkinson's Disease (PD)) or genetic mutations affecting CNS cell function (e.g., huntington's disease, early-onset AD or PD, amyotrophic Lateral Sclerosis (ALS)) may cause the neurodegenerative disease. The neurodegenerative disease may have a change and/or disorder selected from the group consisting of: misfolding and aggregation of proteins; neuroinflammation (e.g., CNS inflammation that occurs upon stimulation of a signal such as a toxic stimulus (e.g., protein aggregation), infection, traumatic injury, or autoimmunity); alterations in cellular signal transduction; acquired senescence/cell death (e.g., interrupted apoptotic signaling, mitochondrial dysfunction, impaired autophagy, activation of necrotic corpuscles by stress/inflammation); motor cell damage and epigenetic changes.

The term "Alzheimer's disease" generally refers to Alzheimer's disease and senile dementia, and is a neurodegenerative disease with slow onset and progressive deterioration with time. The most common early symptom is the loss of short-term memory (it is difficult to remember what happens recently), and as the disease progresses gradually, at least one of the following symptoms may develop: language handicaps, disorientation (e.g., easy getting lost), emotional instability, loss of motivation, inability to self-care, and behavioral problems. The true cause of Alzheimer's disease remains unknown until now, and its course may be related to fibrous amyloid plaque deposition and Tau protein in the brain. There are no treatments that can prevent or reverse the course of the disease, and only a few methods or permissions to temporarily alleviate or ameliorate symptoms.

The term "Alzheimer's disease" is used interchangeably with the term "Alzheimer's disease" in the present application. The Alzheimer's disease may include early Alzheimer's disease, mid Alzheimer's disease and/or late Alzheimer's disease. For example, the early stage Alzheimer's disease patients may experience more pronounced learning and memory disorders, and in some cases, may have language disorders, performance disorders, cognitive disorders (disuse), and/or skill performance disorders (disuse). For example, the mid-Alzheimer's disease patient will lose the ability to live independently and may not be able to perform most of the daily activities (in some cases, may suffer from naming disabilities, confusion, and/or loss of sense of illness). For example, the Alzheimer's disease patients may be advanced dependent caregivers. For example, language capability may be completely lost. For example, it may not be possible to eat by itself.

The term "early cognitive impairment (MCI)" generally refers to an intermediate clinical state between normal cognition and cognitive impairment. In some cases, the MCI may include cognitive impairment that meets dementia criteria but exceeds normal aging to a degree. MCI is diverse in terms of clinical manifestation, etiology, prognosis, and prevalence. In some cases, MCI may be a pathological stage of alzheimer's disease. Certain forms of cognitive impairment may be considered early manifestations of neurodegenerative diseases, ultimately leading to dementia. In some cases, the MCI may comprise a subtype selected from the group consisting of: aMCI-s: amnestic MCI single cognitive domain impairment; aMCI-m: amnestic MCI multi-cognitive domain impairment; naMCI-s: a non-amnestic MCI single cognitive domain is impaired; naMCI-m: the non-amnestic MCI multi-cognitive domain is impaired.

The term "cognitive impairment due to normal aging" generally refers to cognitive impairment due to normal aging. For example, the cognitive impairment caused by normal aging may be manifested as: memory loss, confusion with locations familiar with places, longer than usual for doing daily work, or changes in mood and personality.

The term "Lews Body Dementia (LBD)" generally refers to Lewy Body Detmentia, lewy body dementia. Dementia with lewy bodies is characterized by abnormal accumulation of proteins as bumps called lewy bodies. Dementia with lewy bodies results in a gradual decline in heart intelligence. Patients with dementia with lewy bodies may experience pseudoscopy and changes in alertness and attention. Other effects include muscle stiffness, slow motion, difficulty walking, and tremors. Patients with lewy bodies in the brain may also have plaques and tangles associated with alzheimer's disease.

The term "frontotemporal dementia" generally refers to pick's disease, a progressive rare disease in which tau protein affects only the frontal and temporal lobes of the brain. Frontotemporal dementia patients have difficulties in higher level reasoning, expression language, language perception and memory formation. The frontal and temporal lobes of the brain of frontotemporal dementia patients may atrophy over time.

The term "vascular dementia" generally refers to the problem of reasoning, judgment and memory due to impaired cerebral blood flow. For example, the vascular dementia may include dementia due to factors at risk of heart disease and stroke, such as hypertension and high cholesterol.

The term "multi-infarct" generally refers to a non-cortical small infarct caused by a single through branch occlusion of a large cerebral artery. The multiple infarct type may be a specific type of cerebral infarction, also known as ischemic stroke. The multiple infarctions may be manifested as off-body sensory disturbances, aphasia, dysarthria, slow movements (especially with fine movements such as writing being more difficult).

The term "parkinson's disease" generally refers to a progressive neurodegenerative disease. Clinical features of Parkinson's Disease (PD) may include motor symptoms (e.g., tremor, bradykinesia, myotonia, and postural instability), as well as neuropsychiatric and other non-motor manifestations. For example, the non-motor manifestations may include cognitive dysfunction and dementia, mood disorders (e.g., depression, anxiety, apathy), and sleep disorders.

The term "Creutzfeldt-Jakob disease (CJD)" generally refers to an infectious spongiform encephalopathy that occurs in humans. CJD is a disease caused by prion infection. CJD patients may manifest as paranoid behavior, confusion, loss of appetite and weight, depression, with a few patients with visual or auditory abnormalities; in the progressive phase, progressive neurological exacerbations (e.g., paresthesias, language disorders, and aphasia) are manifested.

The term "Multiple Sclerosis (MS)" generally refers to a demyelinating neuropathy. The destruction of the insulating material (i.e., myelin sheath) on the surface of nerve cells in the brain or spinal cord of the MS patient, impaired signal transduction of the nervous system, can lead to a range of possible symptoms that affect the patient's activity, mental, and even mental state. These symptoms may include double vision, unilateral vision impairment, muscle weakness, dysesthesia, or coordination dysfunction.

The term "Amyotrophic Lateral Sclerosis (ALS)" generally refers to a febrile, motor neuron disease, a progressive and fatal neurodegenerative disease. Among them, a few ALS patients may develop frontotemporal dementia. Some ALS patients experience degeneration in sense, vision, touch, smell and taste, and few amyotrophic lateral sclerosis patients experience dementia at the same time.

The term "Huntington's Disease (HD)", i.e., huntington's disease, generally refers to a genetic disorder that causes brain cell death. The disorder of body movement becomes more pronounced in HD patients as the disease progresses, with progressive deterioration in ability until movement becomes difficult and impossible to speak. Heart mental stress is often reduced to dementia.

The term "aging stage" generally refers to the aging stage of a subject. For example, for a person, the aging period may be over 60 years old, over 70 years old, or over 75 years old; for mice, the aging period may be 10 months or more, for example, 13 months or more or 18 months or more. In some cases, the aged-stage subject may have one or more symptoms of learning deficit, memory deficit, and/or brain dysfunction.

The term "modulator" generally refers to a compound that alters the amount of expression and/or activity of a molecule. For example, a modulator may include a compound that increases or decreases the intensity of a certain activity and/or the amount of expression of a molecule as compared to the intensity of activity and/or the amount of expression in the absence of the modulator. For example, the modulator may comprise an inhibitor that reduces the intensity and/or amount of expression of one or more activities of the molecule.

The term "neuron" generally refers to a nerve cell, which is the primary functional unit of the nervous system. Neurons may be composed of cell bodies and their protruding axons, and one or more dendrites. Neurons can transmit information to other neurons or cells by releasing neurotransmitters at synapses.

The term "excitatory postsynaptic current (EPSC)" generally refers to the flow of ions that cause an excitatory postsynaptic potential (excitatory postsynaptic potential, EPSP). The EPSP is a post-synaptic potential that makes it easier for post-synaptic neurons to trigger an action potential. This temporary depolarization of the postsynaptic membrane potential caused by the influx of positively charged ions into postsynaptic cells is a result of the opening of ligand-gated ion channels. The frequency and/or amplitude of the EPSC may be recorded with a voltage clamp.

The term "cognitive ability" generally refers to the ability to obtain mental behavior of knowledge and understanding through thought, experience, and feel. The cognitive concepts may not be limited to psychological concepts/domains and may include, for example, performing functions, memory, perception, attention, emotion, motor control, and/or intervention processes.

The term "aβ" generally refers to any peptide resulting from cleavage of the β Amyloid Precursor Protein (APP) mediated by β secretase. For example, the aβ may comprise 37, 38, 39, 40, 41, 42 and 43 amino acid peptides and extend from the β secretase cleavage site to amino acids 37, 38, 39, 40, 41, 42 or 43. The Abeta may also be in the N-terminal truncated form of the above peptide, such as pyroglutamic acid forms pE3-40, pE3-42, pE3-43, pE11-42, pE11-43 and the like.

The term "Tau" generally refers to a component of Tau protein and broad aggregates of Tau (e.g., neurofibrillary tangles) that are involved in the stabilization of microtubules in nerve cells. The Tau entanglement can include oligomeric and/or fibrous forms of Tau, which are toxic. The Tau may also include all types and forms (e.g., different alternatively spliced forms) of Tau.

The term "small molecule compound" generally refers to any chemical moiety having a molecular weight of less than about 5000 daltons (Da). In the present application, the small molecule compound may include an organic or inorganic molecule synthesized or found in nature. The small molecule compounds may include non-peptide, non-oligomeric organic compounds. The small molecule compounds may include peptides, peptide analogs (peptidomimetics), amino acids, amino acid analogs, polynucleotides, polynucleotide analogs, aptamers, nucleotides, and/or nucleotide analogs.

The term "polymer" generally refers to a molecule composed of repeating structural units linked by covalent chemical bonds. Typically the polymers are characterized by a large number of repeating units (e.g., 10 repeating units or more, 50 repeating units or more, or typically 100 repeating units or more) and a high molecular weight (50,000 da or more). The polymer may include random copolymers, block copolymers, alternating copolymers, multiblock copolymers, graft copolymers, and/or tapered (tapered) copolymers.

The term "biomacromolecule" generally refers to macromolecules that occur in living materials and organisms, such as nucleic acids, proteins, peptides, carbohydrates, polysaccharides, lipids, and fats. Their respective mono/oligomers may be the corresponding nucleotides, peptides, amino acids, sugars, fatty acids in their respective mono/oligomer form. In the present application, the biomacromolecule may include a protein, and/or a nucleic acid. For example, the biological macromolecule may include an antibody.

In the present application, the term "isolated" generally refers to those obtained from a natural state by artificial means. For example, a polynucleotide or polypeptide that has not been isolated naturally occurs in a living animal, and the same polynucleotide or polypeptide in high purity isolated from such natural state can be referred to as isolated. The term "isolated" may not exclude the presence of artificial or synthetic substances, or the presence of other impure substances that do not affect the activity of the substance.

In the present application, the term "isolated antigen binding protein" generally refers to a protein having antigen binding ability that is free from its naturally occurring state. An "isolated antigen binding protein" of the application may comprise an antigen-binding moiety and optionally, a framework or framework portion that allows the antigen-binding moiety to adopt a conformation that promotes binding of the antigen by the antigen-binding moiety. The antigen binding proteins may comprise, for example, an antibody-derived protein Framework Region (FR) or an alternative protein framework region or artificial framework region with grafted CDRs or CDR derivatives. Such frameworks may include, but are not limited to, framework regions comprising antibody sources that are introduced, for example, to stabilize mutations in the three-dimensional structure of the antigen binding protein, as well as fully synthetic framework regions comprising, for example, biocompatible polymers. Examples of antigen binding proteins may include, but are not limited to: human antibodies, humanized antibodies; a chimeric antibody; a recombinant antibody; a single chain antibody; a bifunctional antibody; a trifunctional antibody; a four-functional antibody; fab, fab ', fv fragments, F (ab') 2,F (ab) 2, scFv, di-scFv, dAb, VHH, igD antibodies; igE antibodies; igM antibodies; an IgG1 antibody; an IgG2 antibody; an IgG3 antibody; and/or IgG4 antibodies and fragments thereof.

In the present application, the term "CDR" also referred to as "complementarity determining region", generally refers to a region in an antibody variable domain, the sequence of which may be highly variable and/or form a structurally defined loop. For example, an antibody may include six CDRs; three in VH (HCDR 1, HCDR2, HCDR 3), and three in VL (LCDR 1, LCDR2, LCDR 3). In certain embodiments, naturally occurring camelid antibodies consisting of only heavy chains are also able to function normally and stably in the absence of light chains. Antibody CDRs can be determined by a variety of coding systems, such as CCG, kabat, chothia, IMGT, a combination of Kabat/Chothia et al. Such coding systems are known in the art and can be found, for example, in www.bioinf.org.uk/abs/index. Html # kabatnum. For example, the amino acid sequence numbering of the antigen binding proteins may be according to the IMGT numbering scheme (IMGT, the international ImMunoGeneTics information system@imgt.cines.fr; IMGT. Circuits. 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 may be determined according to the Kabat numbering system (see, e.g., kabat EA & Wu TT (1971) ANN NY ACADSCI 190:190:382-391 and Kabat EAet al.,(1991)Sequences of Proteins of Immunological Interest,Fifth Edition,U.S.Department of Health and Human Services,NIH Publication No.91-3242).

In the present application, the term "FR" generally refers to the more highly conserved portion of an antibody variable domain, which is referred to as the framework region. For example, the variable domains of the natural heavy and light chains may each comprise four FR regions, namely four in VH (H-FR 1, H-FR2, H-FR3 and H-FR 4), and four in VL (L-FR 1, L-FR2, L-FR3 and L-FR 4).

In the present application, the term "variable domain" is used interchangeably with "variable region" and generally refers to a portion of an antibody heavy and/or light chain. The variable domains of the heavy and light chains may be referred to as "VH" and "VL", respectively (or "VH" and "VL", respectively). These domains may generally be the most variable portions of an antibody (relative to other antibodies of the same type) and may contain antigen binding sites. In the present application, the term "variable" generally means that there may be a large difference in sequence in some segments of the variable domain between antibodies. The variable domain-mediated antigen can bind and determine the specificity of a particular antibody for its particular antigen. However, the variability may not be evenly distributed throughout the variable domains. It may be generally concentrated in three segments called hypervariable regions (CDRs or HVRs) in the light and heavy chain variable domains. The more highly conserved portions of the variable domains may be referred to as Framework Regions (FR). The variable domains of the natural heavy and light chains may each comprise four FR regions, mostly in a β -sheet configuration, connected by three CDRs, which form a loop connection and in some cases form part of a β -sheet structure. The CDRs in each chain can be held together in close proximity by the FR regions, and the CDRs from the other chain together promote the formation of the antigen binding site of the antibody.

In the present application, the term "antibody" generally refers to an immunoglobulin or fragment or derivative thereof, encompassing any polypeptide comprising an antigen binding site, whether produced in vitro or in vivo. The term may include, but is not limited to, polyclonal, monoclonal, monospecific, multispecific, nonspecific, humanized, single chain, chimeric, synthetic, recombinant, hybrid, mutant, and grafted antibodies. Unless otherwise modified by the term "intact", as in "intact antibodies", for the purposes of the present application, the term "antibody" may also include antibody fragments such as Fab, F (ab') 2, fv, scFv, fd, VHH, dAb, and other antibody fragments that retain antigen binding function (e.g., specifically bind to human DIR). Typically, such fragments may include an antigen binding domain. The basic 4-chain antibody unit may be a heterotetrameric glycoprotein consisting of two identical light (L) chains and two identical heavy (H) chains. IgM antibodies may consist of 5 basic heterotetramer units with another polypeptide called the J chain and contain 10 antigen binding sites, while IgA antibodies may include 2-5 basic 4-chain units that can polymerize in conjunction with the J chain to form multivalent combinations. In the case of IgG, the 4-chain unit can generally be about 150,000 daltons. Each L chain may be linked to the H chain by one covalent disulfide bond, while the two H chains may be linked to each other by one or more disulfide bonds depending on the H chain isotype. Each H and L chain may also have regularly spaced intrachain disulfide bridges. Each H chain may have a heavy chain variable region (VH) at the N-terminus, followed by three constant domains (CH) for each of the alpha and gamma chains, followed by four CH domains for the mu and epsilon isoforms. Each L chain may have a light chain variable region (VL) at the N-terminus and a constant domain at its other end. VL may correspond to VH, and light chain constant region (CL) may correspond to the first constant domain of the heavy chain (CH 1). Specific amino acid residues can be considered to form an interface between the light chain and heavy chain variable domains. VH and VL may be paired together to form a single antigen binding site. L chains from any vertebrate species can be divided into one of two distinct types, termed kappa and lambda, based on the amino acid sequence of their constant domains. Immunoglobulins may be assigned to different classes or isotypes based on the amino acid sequence of the heavy chain constant region (CH) constant domain. Currently there are five classes of immunoglobulins: igA, igD, igE, igG, such as IgG1, igG2, igG3, and/or IgG4, and IgM, have heavy chains designated α, δ, ε, γ, and μ, respectively.

In the present application, the term "antigen binding fragment" generally refers to one or more fragments that have the ability to specifically bind an antigen (e.g., DIR). In the present application, the antigen binding fragment may comprise a Fab, fab ', F (ab) 2, fv fragment, F (ab') 2, scFv, di-scFv, VHH and/or dAb.

In the present application, the term "Fab" generally refers to antigen binding fragments of antibodies. As described above, papain can be used to digest intact antibodies. The antibodies, after digestion with papain, produce two identical antigen binding fragments, a "Fab" fragment, and a residual "Fc" fragment (i.e., fc region). Fab fragments can consist of a complete L chain with a heavy chain variable region and the first constant region (CH 1) of the H chain (VH).

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

In the present 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, a Fab' fragment may include all light chains, all heavy chain variable regions, and all or part of the first and second constant regions of a heavy chain. For example, a Fab' fragment can also include part or all of the 220-330 amino acid residues of the heavy chain.

In the present application, the term "F (ab') 2" generally refers to an antibody fragment produced by pepsin digestion of an intact antibody. The F (ab') 2 fragment contains two Fab fragments held together by disulfide bonds and a partial hinge region. F (ab') 2 fragments have divalent antigen binding activity and are capable of cross-linking antigens.

In the present application, the term "Fv fragment" generally refers to a monovalent antigen-binding fragment of a human monoclonal antibody, comprising all or part of the heavy and light chain variable regions, and lacking the heavy and light chain constant regions. The heavy chain variable region and the light chain variable region include, for example, CDRs. For example, fv fragments comprise all or part of the amino terminal variable region of about 110 amino acids of the heavy and light chains.

In the present application, the term "scFv" generally refers to a fusion protein comprising at least one variable region antibody fragment comprising a light chain and at least one antibody fragment comprising a variable region of a heavy chain, wherein the light chain and heavy chain variable regions are contiguous (e.g., via a synthetic linker such as a short flexible polypeptide linker) and are capable of expression as a single chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless specifically stated otherwise, as used herein, an scFv may have the VL and VH variable regions described in any order (e.g., with respect to the N-terminus and C-terminus of the polypeptide), an scFv may comprise a VL-linker-VH or may comprise a VH-linker-VL.

In the present application, the term "dAb" generally refers to an antigen binding fragment having a composition of VH or VL domains, see, e.g., ward et al (Nature, 1989Oct 12;341 (6242): 544-6), see Holt et al, trends Biotechnol.,2003,21 (11): 484-490.

In the present application, the term "VHH" generally refers to antibodies comprising the variable antigen binding domain of a heavy chain antibody (see Vanlandschoot p. Et al, 2011,Antiviral Research 92, 389-407). VHH may also be referred to as Nanobody (Nb).

In the present application, the term "monoclonal antibody" generally refers to a preparation of antibody molecules consisting of single molecules. Monoclonal antibodies are generally highly specific for a single antigenic site. Moreover, unlike conventional polyclonal antibody preparations (which typically have different antibodies directed against different determinants), each monoclonal antibody may be directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies have the advantage that they can be synthesized by hybridoma culture without contamination by other immunoglobulins. The modifier "monoclonal" may refer to the characteristics of the antibody as obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies used in the present application may be prepared in hybridoma cells or may be prepared by recombinant DNA methods.

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. Typically, 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 such that the resulting chimeric antibody has a reduced likelihood of eliciting an adverse immune response in a human individual as compared to the parent (e.g., mouse-derived) antibody.

In the present application, the term "humanized antibody" generally refers to an antibody in which some or all of the amino acids other than the CDR regions of a non-human antibody (e.g., a mouse antibody) are replaced with the corresponding amino acids derived from a human immunoglobulin. Small additions, deletions, insertions, substitutions or modifications of amino acids in the CDR regions may also be permissible, provided that they still retain the ability of the antibody to bind to a particular antigen. The humanized antibody may optionally comprise at least a portion of a human immunoglobulin constant region. A "humanized antibody" may retain antigen specificity similar to that of the original antibody. A "humanized" form of a non-human (e.g., murine) antibody may minimally comprise chimeric antibodies derived from sequences of non-human immunoglobulins. In some cases, CDR region residues in a human immunoglobulin (recipient antibody) may be replaced with CDR region residues of a non-human species (donor antibody) such as mouse, rat, rabbit or non-human primate having the desired properties, affinity and/or capability. In some cases, the FR region residues of the human immunoglobulin may be replaced with corresponding non-human residues. In addition, the humanized antibody may comprise amino acid modifications that are not in the recipient antibody or in the donor antibody. These modifications may be made to further improve the properties of the antibody, such as binding affinity.

In the present application, the term "fully human antibody" generally refers to an antibody comprising only human immunoglobulin protein sequences. If it is produced in a mouse, in a mouse cell or in a hybridoma derived from a mouse cell, the fully human antibody may contain a murine sugar chain. Similarly, "murine antibody", "mouse antibody" or "rat antibody" refer to antibodies comprising only mouse or rat immunoglobulin sequences, respectively. Fully human antibodies can be produced in humans by phage display or other molecular biological methods in transgenic animals with human immunoglobulin germline sequences. Exemplary techniques useful for making antibodies are known in the art.

In the present application, the term "antigen binding protein" generally refers to a protein comprising an antigen binding moiety, and optionally a scaffold or scaffold moiety that allows the antigen binding moiety to adopt a conformation that facilitates binding of the antigen binding protein to an antigen. Examples of antigen binding proteins include, but are not limited to, antibodies, antigen binding fragments (Fab, fab ', F (ab) 2, fv fragments, F (ab') 2, scFv, di-scFv, VHH and/or dAb), immunoconjugates, multispecific antibodies (e.g., bispecific antibodies), antibody fragments, antibody derivatives, antibody analogs, or fusion proteins, and the like, so long as they exhibit the desired antigen binding activity. An "isolated antigen binding protein" of the application may comprise an antigen-binding moiety and optionally, a scaffold or framework moiety that allows the antigen-binding moiety to adopt a conformation that promotes binding of the antigen by the antigen-binding moiety.

In the present application, the terms "polypeptide molecule" and "polypeptide", "peptide" are used interchangeably and generally refer to a polymer of amino acid residues. The term "fusion protein" generally refers to a polypeptide having at least two moieties covalently linked together. Wherein each moiety may be a polypeptide having a different property. The property may be a biological property, such as in vitro or in vivo activity. The property may also be a simple chemical or physical property, such as binding to a target molecule, catalysis of a reaction, etc. The two moieties may be directly linked by a single peptide bond or by a peptide linker.

In the present application, the term "nucleic acid molecule" generally refers to an isolated form of a nucleotide, deoxyribonucleotide or ribonucleotide of any length, or an analogue isolated from the natural environment or synthesized synthetically.

In the present application, the term "vector" generally refers to a nucleic acid vector into which a polynucleotide encoding a protein can be inserted and the protein expressed. The vector may be expressed by transforming, transducing or transfecting a host cell such that the genetic element carried thereby is expressed within the host cell. One vector may contain a variety of elements that control expression. In addition, the vector may also contain a replication origin. The carrier may also include components that assist it in entering the cell.

In the present application, the term "cell" generally refers to a single cell, cell line or cell culture that may or may not be the recipient of a subject plasmid or vector, which comprises a nucleic acid molecule according to the present application or a vector according to the present application. Cells may include progeny of a single cell. The offspring may not necessarily be identical to the original parent cell (either in the form of the total DNA complement or in the genome) due to natural, accidental or deliberate mutation. Cells may include cells transfected in vitro with the vectors of the application.

In the present application, the term "immunoconjugate" generally refers to a conjugate formed by conjugation (e.g., covalent attachment via a linker molecule) of the other agent (e.g., a chemotherapeutic agent, a radioactive element, a cytostatic agent, and a cytotoxic agent) to the antibody or antigen-binding fragment thereof, which conjugate can specifically bind to an antigen on a target cell through the antibody or antigen-binding fragment thereof, delivering the other agent to the target cell (e.g., a tumor cell).

In the present application, the term "pharmaceutical composition" generally refers to a composition for preventing/treating a disease or disorder. The pharmaceutical composition may comprise an isolated antigen binding protein of the application, a nucleic acid molecule of the application, a vector of the application and/or a cell of the application, and optionally a pharmaceutically acceptable adjuvant. In addition, the pharmaceutical compositions may also comprise one or more suitable formulations such as (pharmaceutically effective) carriers. The acceptable ingredients of the composition may be non-toxic to the recipient at the dosages and concentrations employed. Pharmaceutical compositions of the application include, but are not limited to, liquid, frozen and lyophilized compositions.

In the present application, the term "pharmaceutically acceptable carrier" generally refers to a pharmaceutically acceptable carrier, excipient or stabilizer that is non-toxic to the cells or mammals to which it is exposed at the dosages and concentrations employed. The physiologically acceptable carrier may include a suitable substance. Refers to a pharmaceutically acceptable carrier (carrier) that is not typically the same substance as the carrier (vector) used for insertion of nucleic acids in genetic engineering.

In the present application, the term "specific binding" or "specific" generally refers to a measurable and reproducible interaction, such as binding between a target and an antibody, that can determine the presence of a target in the presence of a heterogeneous population of molecules (including biomolecules). For example, an antibody that specifically binds a target (which may be an epitope) may be an antibody that binds the target with greater affinity, avidity, more readily, and/or for a greater duration than it binds other targets. In certain embodiments, the antibodies specifically bind to epitopes on proteins that are conserved among proteins of different species. In certain embodiments, specific binding may include, but is not required to be, exclusively binding.

In the present application, the term "subject" generally refers to a human or non-human animal, including but not limited to, cats, dogs, horses, pigs, cows, sheep, rabbits, mice, rats, or monkeys.

In the present application, the variant may be, for example, a protein or polypeptide having one or more amino acids substituted, deleted or added in the amino acid sequence of the protein and/or the polypeptide (e.g., an antibody or fragment thereof that specifically binds to a DIR). For example, the functional variant may comprise a protein or polypeptide that has been altered in amino acids by at least 1, such as 1-30, 1-20, or 1-10, and yet another such as 1,2, 3, 4, or 5 amino acid substitutions, deletions, and/or insertions. The functional variant may substantially retain the biological properties of the protein or the polypeptide prior to alteration (e.g., substitution, deletion, or addition). For example, the functional variant may retain at least 60%,70%,80%,90%, or 100% of the biological activity (e.g., antigen binding capacity) of the protein or the polypeptide prior to alteration. For example, the substitution may be a conservative substitution. For example, the variant may also be a polypeptide comprising a functionally active fragment thereof, not limited to polypeptides comprising a functionally active fragment of the protein resulting from processing and/or modification occurring in the cell.

In the present application, the homolog may be a protein or polypeptide having at least about 85% (e.g., having at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more) sequence homology to the amino acid sequence of the protein and/or the polypeptide (e.g., an antibody or fragment thereof that specifically binds to DIR).

In the present application, the homology generally refers to similarity, similarity or association between two or more sequences. "percent sequence homology" can be calculated by: the two sequences to be aligned are compared in a comparison window, the number of positions in the two sequences where the same nucleobase (e.g., A, T, C, G, I) or the same amino acid residue (e.g., ala, pro, ser, thr, gly, val, leu, ile, phe, tyr, trp, lys, arg, his, asp, glu, asn, gln, cys and Met) is present is determined to give the number of matched positions, the number of matched positions is divided by the total number of positions in the comparison window (i.e., window size), and the result is multiplied by 100 to produce the percent sequence homology. Alignment to determine percent sequence homology can be accomplished in a variety of ways known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. One skilled in the art can determine suitable parameters for aligning sequences, including any algorithms needed to achieve maximum alignment over the full length sequence being compared or over the region of the target sequence. The homology can also be determined by the following method: FASTA and BLAST. For a description of FASTA algorithm, see w.r.pearson and d.j.lipman, "improved tools for biological sequence comparison", proc.Natl. Acad.Sci., U.S. Proc., 85:2444-2448, 1988; "quick sensitive protein similarity search" by d.j.lipman and w.r.pearson, science,227:1435-1441, 1989. For a description of the BLAST algorithm, see "a basic local contrast (alignment) search tool", journal of molecular biology, 215:403-410, 1990.

In the present application, the term "antisense oligonucleotide" refers to a single stranded oligonucleotide molecule having a nucleobase sequence complementary to a corresponding fragment of a target nucleic acid (e.g., a genomic sequence of interest, a pre-mRNA, or an mRNA molecule). In certain embodiments, the antisense oligonucleotide is 12 to 30 nucleobases in length. In certain embodiments, an antisense oligonucleotide is an unmodified or modified nucleic acid having a nucleotide sequence complementary to a target nucleic acid (e.g., DIR mRNA) sequence.

In the present application, the term "dsRNA" refers to a complex of ribonucleic acid molecules having a duplex structure comprising two antiparallel and substantially complementary nucleic acid strands, referred to as having "sense" and "antisense" orientations relative to a target RNA (e.g., DIR gene). In some embodiments of the application, double-stranded RNA (dsRNA) triggers degradation of target RNA (e.g., mRNA) by a post-transcriptional gene silencing mechanism (referred to herein as RNA interference or RNAi). The duplex structure may be any length that allows for specific degradation of the desired target RNA by RISC pathway, e.g., may be in the range of about 19 to 36 base pairs in length, e.g., about 19-30 base pairs in length, e.g., about 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 base pairs in length. Ranges and lengths intermediate to those described above are also included as part of the application.

In the present application, the term "short interfering RNA (siRNA)" refers to small double stranded RNAs that interfere with gene expression. siRNAs are transmitters of RNA interference (the process of double stranded RNA silencing homologous genes). siRNAs typically comprise two single-stranded RNAs of about 15-25 nucleotides in length, which form a duplex, which may comprise a single-stranded overhang. The cleavage of double-stranded RNA by enzyme complexes, e.g., polymerase, results in the production of siRNAs. RNA interference (RNAi) silencing complexes use the antisense strand of siRNA to direct mRNA cleavage, thereby promoting mRNA degradation. To silence a particular gene using siRNAs, for example, in mammalian cells, the base pair region is selected to avoid opportunistic complementarity to unrelated mRNA. It has been known in the art such as, for example, by Fire et al, nature391:806-81 (1998) and McManus et al, nat. Rev. Genet.3 (10): 737-747 (2002) identified RNAi silencing complexes.

In the present application, the term "antisense strand" generally refers to a strand of an siRNA that includes a region substantially complementary to a target sequence. As used herein, the term "complementarity region" generally refers to a region on the antisense strand that is substantially complementary to a sequence defined herein (e.g., a target sequence). When the region of complementarity is not perfectly complementary to the target sequence, the mismatch may be in the interior or terminal region of the molecule. Typically, the most tolerated mismatches are within the terminal region, e.g., 5, 4, 3 or 2 nucleotides at the 5 'end and/or 3' end.

In the present application, the term "sense strand" generally refers to a strand of an siRNA that includes a region that is substantially complementary to a region that is the term antisense strand as defined herein. The "sense" strand is sometimes referred to as the "sense" strand, the "passenger" strand, or the "anti-guide" strand. By virtue of their sequences, the antisense strand targets the desired mRNA, while the sense strand targets a different target. Thus, if the antisense strand is incorporated into RISC, the correct target is targeted. Incorporation of the sense strand can lead to off-target effects. These off-target effects can be limited by the use of modifications on the sense strand or the use of 5' end caps.

In the present application, the term "complementary" when used to describe a first nucleotide sequence (e.g., sense strand or target mRNA) in reference to a second nucleotide sequence (e.g., antisense strand) refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize (form base pair hydrogen bonds) and form a duplex or duplex structure with an oligonucleotide or polynucleotide comprising the second nucleotide sequence under certain conditions. Complementary sequences include Watson-Crick base pairs (Watson-Crick base pairs) or non-Watson-Crick base pairs and include natural or modified nucleotides or nucleotide mimics, provided that the above requirements with respect to their hybridization ability are fulfilled. "complementary" does not necessarily have nucleobase complementarity on every nucleoside. Conversely, some mismatch may be tolerated.

In the present application, the term "target nucleic acid" or "target sequence" generally refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during transcription of a gene encoding an intron retention cleavage product of a DNA damage-inducible transcript 4-like transcript (DDILT gene), including mRNA that is the RNA processing product of the major transcript. The target portion of the sequence should be at least long enough to serve as a substrate for antisense oligonucleotide or siRNA directed cleavage at or near the position of that portion of the nucleotide sequence of the mRNA molecule formed during DDILT gene transcription. In one embodiment, the target sequence is within the protein coding region of the DIR and/or functional fragment thereof.

In the present application, the term "comprising" generally means including, summarizing, containing or comprising. In some cases, the meaning of "as", "consisting of … …" is also indicated.

The term "about" generally refers to a range of values that is 20% greater or less than a specified value. For example, "about X" includes a range of values of ± 20%, ±10%, ±5%, ±2%, ±1%, ±0.5%, ±0.2% or ± 0.1% of X, wherein X is a value.

Detailed Description

DIR and functional fragments thereof

An intron-retained form (DIR) resulting from aberrant cleavage of human DDIT 4L. Through sequence alignment, the nucleic acid sequence is found to be conserved in primates, where chimpanzees with closer relatedness to humans are highly conserved, whereas conservation in macaques is relatively poor. However, there is no conservation in the common experimental animals such as rats, mice, dogs, pigs, etc.

In the present application, the functional fragment generally refers to a polypeptide comprising an amino acid sequence that differs from the amino acid sequence of a parent or reference polypeptide (e.g., DIR) by at least one amino acid residue. In the present application, the functional fragment may have a higher (e.g., at least 80%) homology to the parent or reference polypeptide. The homology may include sequence similarity or identity. In the present application, the homology may be determined using standard techniques known in the art (see, for example, smith and Waterman, adv. Appl. Math, progress in applied mathematics); the percent identity shared by polynucleotide or polypeptide sequences is determined by direct comparison of sequence information between molecules, by sequence alignment and determination of identity using methods known in the art. An example of an algorithm suitable for determining sequence similarity is the BLAST algorithm (see Altschul et al, J.mol. Biol., J.Mol., 215:403-410[1990 ]). Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information (NCBI).

In the present application, the expression level of the DIR may include the expression level of the DIR gene, the transcription level of the DIR gene, and/or the expression level of the DIR protein. For example, the expression level may include the amount of a polynucleotide, mRNA, or amino acid product or protein of a particular gene (e.g., the human DDIT4L gene; and/or a gene encoding a human DIR and/or a functional fragment thereof (e.g., DIR-I, and/or DIR-II) (e.g., the human DIR gene)). The expression level may include the amount of a polynucleotide transcribed from a particular gene, a translated protein, or a fragment of a post-translationally modified protein. In the present application QDLIR may be used in place of DIR, which may be a form of intron retention resulting from aberrant cleavage upon expression of the human DDIT4L gene. In the present application, the gene encoding the DIR may be referred to as a DIR gene.

In the present application, the expression level of the functional fragment of DIR (e.g., DIR-I, and/or DIR-II) may include the expression level of the functional fragment gene encoding DIR, the transcription level of the functional fragment gene encoding DIR, and/or the expression level of the functional fragment protein of DIR. For example, the expression level may include the amount of a polynucleotide, mRNA, or amino acid product or protein of a particular gene (e.g., a gene encoding a functional fragment of human DIR (e.g., DIR-I, and/or DIR-II)). The expression level may include the amount of a polynucleotide transcribed from a particular gene (e.g., a gene encoding a functional fragment of human DIR (e.g., DIR-I, and/or DIR-II)), a translated protein, or a fragment of a post-translationally modified protein.

In the present application, the DIR-I may be an IR (i.e., the amino acid sequence encoded by the retained intron, the amino acid sequence of which is shown in SEQ ID NO. 3) consisting of the first 27 amino acids from the N-terminus. The amino acid sequence of DIR-I is shown as SEQ ID NO. 4. The DIR-II may be an amino acid sequence of IR consisting of the last 27 amino acids from the C-terminus. The amino acid sequence of DIR-II is shown in SEQ ID NO. 5.

In the present application, the reduction may include a reduction in the expression level of the DIR of at least about 10% as compared to the expression level of the original DIR in the subject. For example, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, 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 may be reduced.

In the present application, the expression level of the DIR may be measured by using a substance selected from the group consisting of: primers for specifically amplifying DIR genes, nucleic acid molecules that specifically bind to DIR proteins, small molecules that specifically bind to DIR proteins, probes that specifically bind to DIR proteins, and polypeptides that specifically bind to DIR proteins.

In the present application, the expression level of a functional fragment of DIR (e.g., DIR-I, and/or DIR-II) can be measured by using a substance selected from the group consisting of: primers for specifically amplifying a functional fragment gene of DIR, nucleic acid molecules that specifically bind to a functional fragment of DIR (e.g., DIR-I, and/or DIR-II), small molecules that specifically bind to a functional fragment of DIR (e.g., DIR-I, and/or DIR-II), probes that specifically bind to a functional fragment of DIR (e.g., DIR-I, and/or DIR-II), and polypeptides that specifically bind to a functional fragment of DIR (e.g., DIR-I, and/or DIR-II).

In the present application, the expression level of the DIR and/or functional fragment thereof may be measured by performing an assay selected from the group consisting of: reverse transcription and amplification assays (e.g., PCR, ligation RT-PCR, or quantitative RT-PCT), hybridization assays, northern blotting (Northern blotting), dot blotting, in situ hybridization, gel electrophoresis, capillary electrophoresis, column chromatography, western blotting, immunohistochemistry, immunostaining, or mass spectrometry. For example, the expression level of the DIR of the application may be measured by qPCR, qRT-PCR, northern hybridization, western hybridization and/or ELISA detection. The expression level of the DIR may also be measured by performing an analysis directly on a biological sample or on proteins/nucleic acids isolated from the sample.

In the present application, the activity of the DIR and/or functional fragment thereof may include the biological activity of a DIR protein. For example, the biological activity may include affecting excitability of the neuron and/or inhibiting activity of the neuron. For example, the biological activity may include inhibiting cognitive ability by inhibiting excitability of neurons and/or inhibiting activity of neurons.

In the present application, the biological activity may include being able to reduce the frequency of excitatory postsynaptic currents (EPSCs), and/or being able to reduce the amplitude of EPSCs. For example, the reducing may include administering the DIR and/or functional fragment thereof and/or a nucleic acid encoding the DIR and/or functional fragment thereof, may reduce the frequency of excitatory postsynaptic currents (EPSCs) in the subject and/or reduce the magnitude of EPSCs in the subject, as compared to the biological activity of the original DIR and/or functional fragment thereof in the subject.

For example, the functional fragment DIR-I of the DIR may reduce the frequency of EPSCs. For example, the reduction may include a reduction in frequency of EPSCs of at least about 10% after administration of the DIR-I as compared to the frequency of original EPSCs in the subject. For example, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, 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 may be reduced. For example, the functional fragment DIR-II of the DIR may reduce the frequency of EPSCs. For example, the reduction may include a reduction in the amplitude of EPSC of at least about 10% after administration of the DIR-II as compared to the amplitude of the original EPSC in the subject. For example, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, 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 may be reduced.

In the present application, the biological activity may include affecting cognitive ability. In the present application, the biological activity may include participation in a signal pathway associated with aβ deposition and/or participation in a signal pathway associated with Tau entanglement production. For example, the DIR and/or functional fragment thereof may inhibit cognitive ability. For example, the DIR and/or functional fragment thereof may inhibit cognitive ability by inhibiting signaling pathways associated with aβ deposition and/or inhibiting signaling pathways associated with Tau entanglement production.

In the present application, the reduction in the activity of the DIR and/or functional fragment thereof may comprise administering the DIR and/or functional fragment thereof and/or a nucleic acid encoding the DIR and/or functional fragment thereof to reduce the cognitive ability of the subject as compared to the original biological activity of the DIR and/or functional fragment thereof in the subject.

In the present application, the reduction may include a reduction in the biological activity of the DIR of at least about 10% as compared to the biological activity of the original DIR in the subject. For example, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, 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 may be reduced.

In the present application, the expression level of the DIR and/or functional fragment thereof (e.g., expression level in plasma) may be positively correlated with the aβ level (e.g., expression level in plasma). For example, the expression level of the DIR and/or functional fragment thereof may be positively correlated with the expression level of aβ40. For example, the expression level of the DIR and/or functional fragment thereof may be positively correlated with the expression level of aβ42.

In the present application, the expression level of the DIR and/or functional fragment thereof (e.g., the expression level in plasma) may be positively correlated with the degree of decrease in cognitive ability in a subject. For example, the expression level of the DIR and/or functional fragment thereof can be increased with progression of a cognitive disorder (e.g., disease progression of MCI and/or AD).

In the present application, the expression level of the DIR and/or functional fragment thereof (e.g., expression level in plasma) may be positively correlated with the aβ level (e.g., aβ -PET expression level in cortex). In the present application, the expression level of the DIR and/or the functional fragment thereof may be correlated with amyloid plaque formation in AD patients.

In the present application, the expression level of the DIR and/or functional fragment thereof may be related to the storage of the declarative memory. For example, the higher the expression level of the DIR and/or functional fragment thereof, the lower the ability to store declarative memory. In the present application, the expression level of the DIR and/or the functional fragment thereof may be related to the storage of associative learning. For example, the higher the expression level of the DIR and/or functional fragment thereof, the lower the associative learning ability to store.

In the present application, a decrease in the expression level of the DIR and/or a functional fragment thereof may increase cognitive ability. In the present application, the expression level of the DIR and/or the functional fragment thereof may be reduced by a gene editing method. For example, the method of gene editing can include knock-down (e.g., can be via a CRISPR/Cas system; e.g., can be via an antisense oligonucleotide). In the present application, the reduction in the expression level may include a reduction in the expression level of the DIR of at least about 10% as compared to the expression level of the original DIR in the subject. For example, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, 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 may be reduced.

In the present application, the lower the expression level of the DIR and/or functional fragment thereof, the higher the cognitive ability (e.g., as measured by a new object recognition behavioral experiment). For example, the cognitive ability can be increased by at least about 10% when the expression level is reduced by at least about 10% as compared to the expression level of the original DIR in the subject. For example, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, 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 may be increased.

In the present application, the DIR and/or functional fragment thereof may be of mammalian origin. For example, it may be derived from a primate. For example, it may be of human origin.

In the present application, the DIR may comprise the amino acid sequence shown in SEQ ID NO. 1.

In the present application, the functional fragment of the DIR may comprise an amino acid sequence encoded by an intron residing in DDIT 4L. In the present application, the functional fragment of DIR may comprise the amino acid sequence shown in SEQ ID NO. 3.

In the present application, the functional fragment of DIR may comprise the amino acid sequence shown in any one of SEQ ID NO. 4-5.

In the present application, the DIR and/or functional fragment thereof may be involved in aβ deposition. For example, the DIR and/or functional fragment thereof may induce aβ deposition by gelsolin.

Among them, the retained intron in DDIT4L (i.e., DIR-Intron (IR) whose amino acid sequence is shown in SEQ ID NO. 3) may be the main region where DIR interacts with Abeta. Aβ may contribute to the interaction between DIR and gelsolin. In the present application, the DIR-intron may be involved in Abeta deposition.

In the present application, the DIR and/or functional fragment thereof is capable of causing aβ deposition and amyloid plaque formation by binding to gelsolin under pathological conditions.

Subject and indication

In the present application, the subject may comprise a mammal. For example, the subject may comprise a rodent and/or primate, e.g., the subject may comprise a human.

In the present application, the subject may include a cognitive disorder patient and/or a neurodegenerative disease patient.

In the present application, the neurodegenerative disease may include acute neurodegenerative disease and chronic neurodegenerative disease. 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 functions, neurodegenerative diseases caused by abnormal intercellular communication, and/or neurodegenerative diseases caused by impaired cell movement.

In the present application, the subject may comprise a patient with a neurodegenerative disease. For example, the subject may comprise a patient with alzheimer's disease. For example, the Alzheimer's disease patient may be in the early, mid or late stages of Alzheimer's disease.

In the present application, the cognitive disorders may include early cognitive disorders (MCI), mid-term cognitive disorders, and late cognitive disorders. For example, the cognitive disorder may include cognitive disorder caused by normal aging, lewy Body Dementia (LBD), frontotemporal dementia, and/or vascular dementia. For example, the cognitive disorder-inducing disease may include Alzheimer's disease, multiple infarctions, parkinson's disease, AIDS and/or Creutzfeldt-Jakob disease (CJD). In the present application, the cognitive disorder may include amnestic MCI multi-cognitive domain impairment (aMCI-m).

In the present application, the subject may comprise a cognition impaired patient. For example, the subject may have early cognitive impairment (MCI) (e.g., loss of short term memory, difficulty in expressing or understanding abstract things, fanciful emotion or behavior, difficulty in learning new things and following complex instructions, impaired judgment and/or need for bystander alerting), mid-term cognitive impairment (e.g., confusion between long term memory and real-world memory, word inadequacy, behavioral character transition or mood instability, and/or need for bystander assistance to self-care), or late cognitive impairment (e.g., impaired memory, decline in physical activity and mental status, inability to effectively express or communicate, inability to self-care, and/or confusion in biological clocks). In the present application, the subject may have a disease capable of causing induction of the cognitive disorder. For example, the subject may have alzheimer's disease, multiple infarctions, parkinson's disease, aids, and/or creutzfeldt-jakob disease (CJD).

In the present application, the subject may be in an aging stage. For example, the subject has shown cognitive impairment due to normal aging. For example, the subject has exhibited symptoms of early cognitive impairment (MCI). For example, the subject has exhibited symptoms of a neurodegenerative disease (e.g., alzheimer's disease).

In the present application, the subject may have Alzheimer's disease. For example, the subject may be in early, mid and/or late stages of Alzheimer's disease.

Regulation agent

The present application provides a modulator that can reduce the expression level and/or biological activity of the DIR and/or a functional fragment thereof, and/or a nucleic acid encoding the DIR and/or a functional fragment thereof.

In the present application, the modulator may reduce the expression level and/or biological activity of the DIR and/or functional fragment thereof, and/or nucleic acid encoding the DIR and/or functional fragment thereof.

In the present application, the modulator may reduce the expression level of the DIR gene, the transcription level of the DIR gene, and/or the expression level of the DIR protein. For example, the modulator may reduce, for example, the human DDIT4L gene; and/or the amount of a polynucleotide, mRNA, or amino acid product or protein of a gene (e.g., a human DIR gene) encoding human DIR and/or a functional fragment thereof (e.g., DIR-I, and/or DIR-II). For example, the modulator may reduce, for example, the human DDIT4L gene; and/or, the amount of a polynucleotide transcribed from a gene encoding a human DIR and/or a functional fragment thereof (e.g., DIR-I, and/or DIR-II) (e.g., a human DIR gene), a translated protein, or a fragment of a post-translationally modified protein.

In the present application, the modulator may reduce the expression level of the functional fragment gene encoding DIR, the transcription level of the functional fragment gene encoding DIR, and/or the expression level of the functional fragment protein of DIR. For example, the modulator may reduce the amount of a polynucleotide, mRNA, or amino acid product or protein encoding a functional fragment of human DIR (e.g., DIR-I, and/or DIR-II) gene. For example, the modulator can reduce the amount of a polynucleotide transcribed, a translated protein, or a fragment of a post-translationally modified protein of a gene encoding a functional fragment of human DIR (e.g., DIR-I, and/or DIR-II).

In the present application, the reduction may include a reduction in the expression level of the functional fragment of the DIR of at least about 10% as compared to the expression level of the original DIR in the subject. For example, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, 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 may be reduced.

In the present application, the modulator may reduce the biological activity of the DIR and/or functional fragment thereof. In the present application, the reduction may comprise a reduction in the biological activity of the DIR or functional fragment thereof of at least about 10% as compared to the biological activity of the original DIR in the subject. For example, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, 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 may be reduced.

In the present application, the modulator may inhibit a signal pathway associated with aβ deposition. In the present application, the modulator may inhibit the signaling pathway associated with Tau entanglement production. In the present application, the inhibition may comprise a reduction in the level of expression and/or biological activity of molecules originally involved in the signal pathway associated with aβ deposition and/or the signal pathway associated with Tau entanglement production in the subject of at least about 10% as compared to the level of expression and/or biological activity of these molecules upon administration of the modulator. For example, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, 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 may be reduced. In the present application, the modulator may inhibit aβ deposition and/or amyloid plaque formation by gelsolin. For example, the modulator may inhibit aβ deposition and/or amyloid plaque formation by inhibiting binding of the DIR and/or functional fragment thereof to gelsolin.

In the present application, the modulator may reduce the binding of DIR-II to gelsolin. For example, the level of binding of DIR-II to gelsolin can be reduced by at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, 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 upon administration of the modulator.

In the present application, the modulator may comprise an inhibitor of the DIR and/or functional fragment thereof.

In the present application, the modulator may include small molecule compounds, polymers, and/or biological macromolecules. In the present application, the modulator may comprise an antibody or antigen-binding fragment thereof. In the present application, the modulator may comprise an antibody or antigen-binding fragment thereof that specifically binds to DIR and/or a functional fragment thereof. In the present application, the modulator may comprise an antibody or antigen binding fragment thereof that targets DIR-II.

In the present application, the modulator may comprise an antisense oligonucleotide. In certain embodiments, wherein the antisense oligonucleotide comprises a region of complementarity that is substantially complementary or fully complementary to at least a portion of an mRNA encoding a DIR and/or functional fragment thereof.

In the present application, the complementary region may be less than 19 nucleotides in length.

In the present application, the modulator may comprise dsRNA. In the present application, the modulator may include siRNA.

In the present application, the modulator may comprise an siRNA that specifically binds to an intron retention cleavage product DIR of a 4-like transcript of a DNA damage inducing transcript and/or a functional fragment thereof.

The siRNA of the application may further comprise one or more single stranded nucleotide overhangs, e.g., 1,2 or 3 nucleotides. The overhang may be on the sense strand, the antisense strand, or any combination thereof. In addition, the overhanging nucleotides can be present at the 5 '-end, 3' -end, or both ends of the antisense or sense strand of the siRNA. The overhang may be caused by one strand being longer than the other, or by interleaving two strands of the same length. The overhang may form a mismatch with the target mRNA or it may be complementary to the targeted gene sequence or may be another sequence.

In the present application, the siRNA may comprise a sense strand and an antisense strand, the antisense strand may comprise a region of complementarity substantially complementary to at least a portion of an mRNA encoding a DIR and/or functional fragment thereof, and the antisense strand may comprise at least 15, 16, 17, 18, or 19 consecutive nucleotides differing by no more than 3 nucleotides from any one of the following sequences: SEQ ID NO: xx; the sense strand and the antisense strand may be complementary together to form a region of complementarity ranging from 17 to 21 nucleotides in length.

In the present application, the siRNA may comprise a sense strand and an antisense strand, the antisense strand may comprise a region of complementarity substantially complementary to at least a portion of an mRNA encoding a DIR and/or functional fragment thereof, and the antisense strand may comprise any one of the sequences selected from the group consisting of seq id nos: SEQ ID NO 85, 87; the sense strand and the antisense strand may be complementary together to form a region of complementarity ranging from 17 to 21 nucleotides in length.

In the present application, the siRNA may comprise a sense strand and an antisense strand, the antisense strand may comprise a region of complementarity substantially complementary to at least a portion of an mRNA encoding a DIR and/or functional fragment thereof, and the antisense strand may comprise a nucleotide sequence set forth in any one of seq id nos. 85, 87; the sense strand and the antisense strand may be complementary together to form a region of complementarity ranging from 17 to 21 nucleotides in length.

In the present application, the siRNA may comprise a sense strand and an antisense strand, the sense strand may comprise a nucleotide sequence shown in any one of SEQ ID nos. 84, 86, and the antisense strand may comprise a nucleotide sequence shown in any one of SEQ ID nos. 85, 87.

In the present application, the 3 'end of the sense strand of the siRNA and/or the 3' end of the antisense strand of the siRNA may be linked to at least one (e.g., 2) nucleotide. For example, the linked nucleotide may be identical to the target gene. For another example, the linked nucleotide may be T, for example, TT. For example, the sense strand and the antisense strand may be selected from any one or more of the following combinations:

A sense strand comprising the nucleotide sequence set forth in SEQ ID NO. 84, and an antisense strand comprising the nucleotide sequence set forth in SEQ ID NO. 85; and, a sense strand comprising the nucleotide sequence set forth in SEQ ID NO. 86, and an antisense strand comprising the nucleotide sequence set forth in SEQ ID NO. 87;

In the present application, the length of each strand may be between 17 and 23 nucleotides.

In the present application, no more than 3 nucleotides in each strand may be replaced by other nucleotides, respectively, while substantially maintaining the ability to inhibit expression of DIR and/or functional fragments thereof in transfected cells.

In the present application, the siRNA may comprise at least one modified nucleotide.

In the present application, one or more nucleotides on the sense strand and/or the antisense strand may be modified to form modified nucleotides.

In the present application, all nucleotides of the sense strand and all nucleotides of the antisense strand may comprise modifications.

The antisense oligonucleotides or siRNAs of the present application can be introduced into brain nerve cells or glial cells by transfection methods well known in the art. These methods include sonication, electrical pulsing, electroporation, osmotic shock, calcium phosphate precipitation and DEAE dextran transfection, lipid-mediated delivery, passive delivery, and the like. The term "transfection" includes a variety of techniques that may be used to introduce nucleic acids into mammalian cells, including electroporation, calcium phosphate precipitation, DEAE-dextran treatment, lipofection, microinjection, and/or viral infection.

In another aspect, the application also provides the use of the modulator (e.g., antisense oligonucleotide or siRNA described herein) in the manufacture of a medicament for the prevention and/or treatment of a disease or disorder, wherein the disease or disorder comprises a cognitive disorder and/or a neurodegenerative disease.

In another aspect, the application also provides said modulator (e.g. antisense oligonucleotide or siRNA according to the application) for use in the prevention and/or treatment of cognitive disorders and/or neurodegenerative diseases.

In another aspect, the present application also provides a method for preventing and/or treating cognitive disorders and/or neurodegenerative diseases, comprising the steps of: administering to a subject in need thereof the modulator (e.g., antisense oligonucleotide or siRNA as described herein).

In the present application, the modulator may be formulated for oral administration and/or injection administration.

Drug screening method/system

In the present application, the drug candidate may comprise a modulator according to the present application. For example, the drug candidate may include an inhibitor of the DIR and/or a functional fragment thereof. The candidate drug may include small molecule compounds, polymers, and/or biological macromolecules.

In the present application, the drug candidate may be formulated for oral administration and/or injection administration.

The application also provides a system for screening for drugs capable of preventing and/or treating cognitive disorders and/or treating neurodegenerative diseases, which may include a detection module. The detection module can detect an effect of a candidate drug on the expression level and/or biological activity of a DIR and/or functional fragment thereof in a subject, wherein upon administration of the candidate drug, the expression level and/or biological activity of the DIR and/or functional fragment thereof is reduced, and the candidate drug is capable of preventing and/or treating a cognitive disorder and/or treating a neurodegenerative disease.

The detection module may comprise an instrument and/or reagent capable of detecting the expression level and/or biological activity of the DIR and/or functional fragment thereof in the subject.

For example, the detection module can include primers that specifically amplify the DIR gene, a nucleic acid molecule that specifically binds to the DIR protein, a small molecule that specifically binds to the DIR protein, a probe that specifically binds to the DIR protein, and/or a polypeptide that specifically binds to the DIR protein; and/or, primers that specifically amplify the functional fragment gene of DIR, nucleic acid molecules that specifically bind to the functional fragment of DIR (e.g., DIR-I, and/or DIR-II), small molecules that specifically bind to the functional fragment of DIR (e.g., DIR-I, and/or DIR-II), probes that specifically bind to the functional fragment of DIR (e.g., DIR-I, and/or DIR-II), and polypeptides that specifically bind to the functional fragment of DIR (e.g., DIR-I, and/or DIR-II).

For example, the detection module may include instrumentation and/or reagents that specifically detect aβ deposition and/or amyloid plaque formation. For example, the detection module may include primers capable of specifically detecting a target gene (e.g., tau gene) associated with aβ deposition and/or amyloid plaque formation, a nucleic acid molecule that specifically binds to the target gene, a small molecule that specifically binds to a protein encoded by the target gene, a probe that specifically binds to a protein encoded by the target gene, and/or a polypeptide that specifically binds to a protein encoded by the target gene.

The detection module may output a measured value (e.g., a quantitative value; e.g., a qualitative value compared to one or more thresholds) of the expression level and/or biological activity of the DIR and/or functional fragment thereof following administration of the candidate drug.

In the present application, the system may include a judgment module that may compare the expression level and/or quantitative value of biological activity of the DIR and/or functional fragment thereof after administration of the drug candidate outputted by the detection module; a magnitude of a quantitative value corresponding to the expression level and/or biological activity of the original DIR and/or functional fragment thereof in the subject. The determination module is capable of outputting a determination that the candidate drug is capable of preventing and/or treating a cognitive disorder and/or treating a neurodegenerative disease if the expression level and/or biological activity of the DIR and/or functional fragment thereof is reduced after administration of the candidate drug.

In the present application, the system may include a display module that may display the determination result of the determination module in a quantitative and/or qualitative manner.

In the present application, the system may comprise a memory; and a processor coupled to the memory, the processor configured to execute the step of detecting in the detection module and/or the determination module the effect of the candidate drug on the expression level and/or the biological activity of the DIR and/or the functional fragment thereof in the subject based on instructions stored in the memory.

The application also provides a detection kit capable of detecting the expression level and/or biological activity of DIR and/or functional fragments thereof. The detection kit may comprise instructions describing specific steps of how to use the detection kit to detect the expression level and/or biological activity of DIR and/or functional fragments thereof, and/or specific steps of using the detection results to determine whether the candidate drug is capable of preventing and/or treating cognitive disorders and/or treating a subject with neurodegenerative disease.

In the present application, the detection kit may further comprise an agent capable of detecting other targets for determining whether the candidate drug is capable of preventing and/or treating cognitive disorders and/or treating neurodegenerative diseases.

Antigen binding proteins

In one aspect, the application provides an isolated antigen binding protein.

In the present application, the isolated antigen binding protein has the following properties: in ELISA assays, specific binding to human DIR and/or functional fragments thereof was achieved at a working concentration of about 10ng/ml or more.

In the present application, the isolated antigen binding protein may comprise LCDR2, and the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 74. For example, an isolated antigen binding protein of the application may have DIR binding capacity. For example, the CDRs may be determined by an IMGT numbering scheme.

X ₁X₂ S (SEQ ID NO. 74), wherein X ₁ is A or Y and X ₂ is A or Y.

In the present application, the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 26 or 16.

In the present application, the isolated antigen binding protein may comprise LCDR1, and the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO. 73. For example, an isolated antigen binding protein of the application may have DIR binding capacity. For example, the CDRs may be determined by an IMGT numbering scheme. For example, the LCDR1 may comprise LCDR1 in the light chain variable region of IR-II-1 through IR-II-10.

Q X ₁X₂D X₃X₄X₅X₆X₇ Y (SEQ ID NO. 73), wherein X ₁ is deletion or S, X ₂ is deletion or V, X ₃ is deletion or Y, X ₄ is deletion or D, X ₅ is G or I, X ₆ is D, E or S, and X ₇ is N or S.

In the present application, the LCDR1 may comprise an amino acid sequence set forth in any one of SEQ ID NOs 25, 52, 15.

In the present application, the isolated antigen binding protein may comprise LCDR3, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 75. For example, an isolated antigen binding protein of the application may have DIR binding capacity. For example, the CDRs may be determined by an IMGT numbering scheme. For example, the LCDR3 may comprise LCDR3 in the light chain variable region of IR-II-1 through IR-II-10.

X ₁Q X₂ X₃ X₄ X₅P X₆ X₇ (SEQ ID NO. 75), wherein X ₁ is L or Q, X ₂ is S or Y, X ₃ is N, S or Y, X ₄ is D, E or K, X ₅ is D or L, X ₆ is F or R, and X ₇ is A or T.

In the present application, the LCDR3 may comprise an amino acid sequence set forth in any one of SEQ ID NOs 27, 53, 67, 17.

In the present application, the isolated antigen binding protein may comprise LCDR3, LCDR2 and LCDR1. For example, LCDR2 of the isolated antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO. 74, LCDR1 may comprise the amino acid sequence shown in SEQ ID NO. 73, and LCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 75. For example, an isolated antigen binding protein of the application may have DIR binding capacity. For example, the CDRs may be determined by an IMGT numbering scheme. For example, the LCDR1-3 may comprise LCDR1-3 in the light chain variable region of IR-II-1 through IR-II-10.

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

For example, LCDR1 of the isolated antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO. 52, LCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 26, and LCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 53.

For example, LCDR1 of the isolated antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO. 52, LCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 26, and LCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 67.

For example, LCDR1 of the isolated antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO. 15, LCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 16, and LCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 17.

For example, an isolated antigen binding protein of the application may have DIR binding capacity. For example, the CDRs may be determined by an IMGT numbering scheme. For example, the LCDR1-3 may comprise LCDR1-3 in the heavy chain variable region of IR-II-1 through IR-II-10.

In the present application, the isolated antigen binding protein may comprise HCDR1, and the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO. 70. For example, an isolated antigen binding protein of the application may have DIR binding capacity. For example, the CDRs may be determined by an IMGT numbering scheme. For example, the HCDR1 may comprise HCDR1 in the heavy chain variable region of IR-II-1 through IR-II-10.

GYTFX ₁X₂YX₃ (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.

In the present application, the HCDR1 may comprise an amino acid sequence set forth in any one of SEQ ID NOs 20, 35, 56, 10.

In the present application, the isolated antigen binding protein may comprise HCDR2, and the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 71. For example, an isolated antigen binding protein of the application may have DIR binding capacity. For example, the CDRs may be determined by an IMGT numbering scheme. For example, the HCDR1 may comprise HCDR2 in the heavy chain variable region of IR-II-1 through IR-II-10.

I X ₁P X₂ X₃ X₄ X₅ X₆ 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.

In the present application, the HCDR2 may comprise an amino acid sequence set forth in any one of SEQ ID NOs 21, 36, 42, 48, 11.

In the present application, the isolated antigen binding protein may comprise HCDR3, and the HCDR3 may comprise the amino acid sequence depicted in SEQ ID NO. 72. For example, an isolated antigen binding protein of the application may have DIR binding capacity. For example, the CDRs may be determined by an IMGT numbering scheme. For example, the HCDR3 may comprise HCDR3 in the heavy chain variable region of IR-II-1 through IR-II-10.

X₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀X₁₁X₁₂X₁₃X₁₄X₁₅DY(SEQ ID NO.72), Wherein X ₁ is deletion or a, X ₂ is deletion or R, X ₃ is deletion, S or T, X ₄ is deletion, D, G or Y, X ₅ is deletion, D, I, M or V, X ₆ is deletion, G or I, X ₇ is deletion, T or Y, X ₈ is deletion, A, L or T, X ₉ is deletion, R, S or T, X ₁₀ is deletion, G or T, X ₁₁ is deletion, D or E, X ₁₂ is D or Y, X ₁₃ is F, L or Y, X ₁₄ is A, T or V, and X ₁₅ is F or M.

In the present application, the HCDR3 may comprise an amino acid sequence set forth in any one of SEQ ID NOs 22, 30, 37, 43, 49, 57, 62, 12.

In the present application, the isolated antigen binding protein may comprise HCDR3, HCDR2 and HCDR1. For example, the isolated antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO. 71, HCDR1 may comprise the amino acid sequence shown in SEQ ID NO. 70, and HCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 72. For example, an isolated antigen binding protein of the application may have DIR binding capacity. For example, the CDRs may be determined by an IMGT numbering scheme. For example, the HCDR1-3 may comprise HCDR1-3 in the heavy chain variable region of IR-II-1 through IR-II-10.

For example, the isolated antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO. 20, HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 21, and HCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 22.

For example, the isolated antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO. 20, HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 21, and HCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 30.

For example, the isolated antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO. 35, HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 36, and HCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 37.

For example, the isolated antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO. 20, HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 42, and HCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 43.

For example, the isolated antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO. 20, HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 48, and HCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 49.

For example, the isolated antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO. 56, HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 36, and HCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 57.

For example, the isolated antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO. 35, HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 36, and HCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 62.

For example, the isolated antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO. 35, HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 42, and HCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 37.

For example, the isolated antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO. 10, HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 11, and HCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 12.

For example, an isolated antigen binding protein of the application may have DIR binding capacity. For example, the CDRs may be determined by an IMGT numbering scheme. For example, the HCDR1-3 may comprise HCDR1-3 in the heavy chain variable region of IR-II-1 through IR-II-10.

In the present application, the isolated antigen binding protein may comprise HCDR3, HCDR2, HCDR1, LCDR3, LCDR2 and LCDR1.

For example, an isolated antigen binding protein of the application HCDR1 may comprise the amino acid sequence shown in SEQ ID NO. 20, HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 21, HCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 22, LCDR1 may comprise the amino acid sequence shown in SEQ ID NO. 25, LCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 26, and LCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 27.

For example, an isolated antigen binding protein of the application HCDR1 may comprise the amino acid sequence shown in SEQ ID NO. 20, HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 21, HCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 30, LCDR1 may comprise the amino acid sequence shown in SEQ ID NO. 25, LCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 26, and LCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 27.

For example, an isolated antigen binding protein of the application HCDR1 may comprise the amino acid sequence shown in SEQ ID NO. 35, HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 36, HCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 37, LCDR1 may comprise the amino acid sequence shown in SEQ ID NO. 25, LCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 26, and LCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 27.

For example, an isolated antigen binding protein of the application HCDR1 may comprise the amino acid sequence shown in SEQ ID NO. 20, HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 42, HCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 43, LCDR1 may comprise the amino acid sequence shown in SEQ ID NO. 25, LCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 26, and LCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 27.

For example, an isolated antigen binding protein of the application HCDR1 may comprise the amino acid sequence shown in SEQ ID NO. 20, HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 48, HCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 49, LCDR1 may comprise the amino acid sequence shown in SEQ ID NO. 52, LCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 26, and LCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 53.

For example, an isolated antigen binding protein of the application HCDR1 may comprise the amino acid sequence shown in SEQ ID NO. 56, HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 36, HCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 57, LCDR1 may comprise the amino acid sequence shown in SEQ ID NO. 25, LCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 26, and LCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 27.

For example, an isolated antigen binding protein of the application HCDR1 may comprise the amino acid sequence shown in SEQ ID NO. 35, HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 36, HCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 62, LCDR1 may comprise the amino acid sequence shown in SEQ ID NO. 25, LCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 26, and LCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 27.

For example, an isolated antigen binding protein of the application HCDR1 may comprise the amino acid sequence shown in SEQ ID NO. 35, HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 42, HCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 37, LCDR1 may comprise the amino acid sequence shown in SEQ ID NO. 52, LCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 26, and LCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 53.

For example, an isolated antigen binding protein of the application HCDR1 may comprise the amino acid sequence shown in SEQ ID NO. 56, HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 36, HCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 57, LCDR1 may comprise the amino acid sequence shown in SEQ ID NO. 52, LCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 26, and LCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 67.

For example, an isolated antigen binding protein of the application HCDR1 may comprise the amino acid sequence shown in SEQ ID NO. 10, HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 11, HCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 12, LCDR1 may comprise the amino acid sequence shown in SEQ ID NO. 15, LCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 16, and LCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 17.

For example, an isolated antigen binding protein of the application may have DIR binding capacity. For example, the CDRs may be determined by an IMGT numbering scheme. For example, the HCDR1-3 may comprise HCDR1-3 in the heavy chain variable region of IR-II-1 through IR-II-10; and, the LCDR1-3 may comprise LCDR1-3 in the light chain variable region of IR-II-1 to IR-II-10.

In the present application, the isolated antigen binding protein may comprise H-FR1, and the C-terminus of the H-FR1 may be directly or indirectly linked to the N-terminus of the HCDR 1.

In the present application, the isolated antigen binding protein may comprise H-FR2, and the H-FR2 may be located between the HCDR1 and the HCDR 2.

In the present application, the isolated antigen binding protein may comprise H-FR3, and the H-FR3 may be located between the HCDR2 and the HCDR 3.

In the present application, the isolated antigen binding protein may comprise 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 comprise H-FR1, H-FR2, H-FR3 and H-FR4.

In the present application, the isolated antigen binding protein may comprise 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 isolated antigen binding protein may comprise L-FR2, and the L-FR2 may be located between the LCDR1 and the LCDR 2.

In the present application, the isolated antigen binding protein may comprise L-FR3, and the L-FR3 may be located between the LCDR2 and the LCDR 3.

In the present application, the isolated antigen binding protein may comprise L-FR4, and the N-terminus of L-FR4 may be linked to the C-terminus of LCDR 3.

In the present application, the isolated antigen binding protein may comprise L-FR1, L-FR2, L-FR3 and L-FR4.

In the present application, the isolated antigen binding protein may comprise a heavy chain variable region VH which may comprise an amino acid sequence as set out in any one of SEQ ID NOs 13, 23, 31, 38, 44, 50, 58, 63, 65. For example, an isolated antigen binding protein of the application may have DIR binding capacity.

In the present application, the isolated antigen binding protein may comprise a light chain variable region VL, which may comprise an amino acid sequence as set forth in any one of SEQ ID NOs 18, 28, 33, 40, 46, 54, 60, 68. For example, an isolated antigen binding protein of the application may have DIR binding capacity.

In the present application, the isolated antigen binding protein may comprise the VH and VL. In certain embodiments, the VH may comprise the amino acid sequence shown in SEQ ID NO. 23 and the VL may comprise the amino acid sequence shown in SEQ ID NO. 28.

In certain embodiments, the VH may comprise the amino acid sequence shown in SEQ ID NO. 31 and the VL may comprise the amino acid sequence shown in SEQ ID NO. 33.

In certain 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.

In certain 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.

In certain 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.

In certain 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.

In certain 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.

In certain 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.

In certain 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.

In certain 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, an isolated antigen binding protein of the application may have DIR binding capacity.

In the present application, the isolated antigen binding protein may comprise at least one CDR in a VH according to the present application. In the present application, the isolated antigen binding protein may comprise at least one CDR in the VL of the present application. The CDRs may be partitioned according to any partitioning scheme. In the present application, the CDR may cover a CDR sequence divided according to any CDR division manner; variants thereof are also contemplated.

In the present application, the isolated antigen binding protein may comprise HCDR1, HCDR2 and HCDR3 in the VH of the application.

In the present application, the isolated antigen binding protein may comprise HCDR1, HCDR2 and HCDR3 in the VH of the application. The VH may comprise an amino acid sequence as set out in any one of SEQ ID NOs 13, 23, 31, 38, 44, 50, 58, 63, 65. For example, an isolated antigen binding protein of the application may have DIR binding capacity. In the present application, the CDR may cover a CDR sequence divided according to any CDR division manner; variants thereof are also contemplated.

In the present application, the isolated antigen binding protein may comprise LCDR1, LCDR2 and LCDR3 in VL of the application. The VL may comprise an amino acid sequence as set forth in any one of SEQ ID NOs 18, 28, 33, 40, 46, 54, 60, 68. For example, an isolated antigen binding protein of the application may have DIR binding capacity. In the present application, the CDR may cover a CDR sequence divided according to any CDR division manner; variants thereof are also contemplated.

In the present application, the isolated antigen binding protein may comprise an antibody heavy chain constant region. The antibody heavy chain constant region may be derived from human IgG, igA, igD, igE and/or IgM heavy chain constant regions. The antibody heavy chain constant region may be derived from a human IgG heavy chain constant region. In certain embodiments, the isolated antigen binding protein may comprise 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 certain embodiments, the isolated antigen binding protein may comprise an antibody heavy chain constant region, and the antibody heavy chain constant region may be derived from a human IgG1 heavy chain constant region.

In the present application, the isolated antigen binding protein may comprise an antibody light chain constant region. The antibody light chain constant region may comprise an igκ -derived constant region or an igλ -derived constant region. The antibody light chain constant region may be derived from a human igκ constant region.

In the present application, the isolated antigen binding protein may comprise an antibody or antigen binding fragment thereof.

In certain embodiments, the antigen binding fragment can comprise a Fab, fab ', fv fragment, F (ab') ₂,F(ab)₂, scFv, di-scFv, VHH, and/or dAb.

In certain embodiments, the antibody may comprise a monoclonal antibody. In certain embodiments, the antibodies may include murine antibodies and/or chimeric antibodies.

Furthermore, it is noted that the isolated antigen binding proteins of the application may comprise heavy and/or light chain sequences to which one or more conservative sequence modifications exist. By "conservative sequence modifications" is meant amino acid modifications that do not significantly affect or alter the binding properties of the antibody. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications may be introduced into the isolated antigen binding proteins of the application by standard techniques known in the art, such as point mutations and PCR-mediated mutations. Conservative amino acid substitutions are substitutions of amino acid residues with amino acid residues having similar side chains. Groups of amino acid residues having similar side chains are known in the art. 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), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In certain embodiments, one or more amino acid residues in the CDR regions of the isolated antigen binding proteins of the application may be replaced with other amino acid residues of the same side chain set. Those skilled in the art will appreciate that some conservative sequence modifications do not result in the loss of antigen binding, see, e.g. ,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.

The antigen binding proteins of the application may be identified, screened or characterized by various assays known in the art.

For example, the antigen binding activity of an antigen binding protein or fusion protein of the application may be tested by known methods such as enzyme-linked immunosorbent assay (ELISA), immunoblotting (e.g., western blotting), flow cytometry (e.g., FACS), immunohistochemistry, immunofluorescence, and the like.

In the present application, the isolated antigen binding protein is capable of specifically binding to a DIR or functionally active fragment thereof. In the present application, the isolated antigen binding protein is capable of specifically binding DIR-II.

In certain embodiments, the DIR or functionally active fragment thereof may be a full-length DIR or functionally active fragment thereof, or a fragment that exhibits DIR functional activity (e.g., may be DIR-II). In certain embodiments, the DIR or functionally active fragment thereof may be an isolated DIR or functionally active fragment thereof, or a mixture of various forms of DIR or functionally active fragments thereof. For example, the DIR or functionally active fragment thereof may be a human DIR or functionally active fragment thereof.

In certain embodiments, the isolated antigen binding protein can be assayed for binding activity of the antigen binding protein to an antigen (e.g., DIR-II) by the Elisa method. For example, the isolated antigen binding protein may have the ability to bind the or a functional fragment thereof at a value of about 10ng/ml or greater (e.g., may be at least 15ng/ml, at least 20ng/ml, at least 25ng/ml, at least 30ng/ml, at least 35ng/ml, at least 40ng/ml, at least 45ng/ml, at least 50ng/ml, at least 55ng/ml, at least 60ng/ml, at least 65ng/ml, at least 70ng/ml, at least 75ng/ml, at least 80ng/ml, at least 85ng/ml, at least 90ng/ml, at least 95ng/ml, at least 100ng/ml, at least 500ng/ml, at least 1000ng/ml or greater). For example, the number of the cells to be processed,

In one aspect, the application provides a polypeptide molecule, nucleic acid molecule, vector, immunoconjugate, cell and pharmaceutical composition.

In another aspect, the application provides polypeptide molecules, which may comprise the isolated antigen binding proteins of the application.

In certain embodiments, the polypeptide molecule may comprise a fusion protein. In certain embodiments, the polypeptide molecule may be a fusion protein. In certain embodiments, the polypeptide molecule is a multispecific (e.g., bispecific, trispecific, or other multispecific) antibody. The multispecific antibody may comprise: 1) The application of the isolated antigen binding proteins, and 2) one or more binding to other antigens, and/or binding to the same antigen epitope of other targeting portion. In certain embodiments, the polypeptide molecules of the application may comprise structures other than amino acids, e.g., the polypeptide molecules of the application may comprise nucleic acids, polysaccharides, lipids, small molecules, and any combination of the foregoing.

In another aspect, the application provides isolated nucleic acid molecules that can encode the isolated antigen binding proteins of the application. For example, it may be produced or synthesized by: (i) Amplified in vitro, for example by Polymerase Chain Reaction (PCR) amplification; (ii) produced by clonal recombination; (iii) Purified, e.g., fractionated by cleavage and gel electrophoresis; or (iv) synthesized, for example by chemical synthesis.

In another aspect, the application provides a vector which may comprise a nucleic acid molecule according to the application. In addition, other genes may be included in the vector, such as marker genes that allow selection of the vector in an appropriate host cell and under appropriate conditions. In addition, the vector may also contain expression control elements that allow for proper expression of the coding region in an appropriate host. Such control elements are well known to those skilled in the art and may include, for example, promoters, ribosome binding sites, enhancers and other control elements which regulate gene transcription or mRNA translation, and the like. The vector may be expressed by transforming, transducing or transfecting a host cell such that the genetic element carried thereby is expressed within the host cell. The vector may include, for example, a plasmid, cosmid, virus, phage, or other vector commonly used in, for example, genetic engineering. For example, the vector is an expression vector. In addition, the vector may include components that assist it in entering the cell, such as viral particles, liposomes, or protein shells, but not exclusively.

In another aspect, the application provides a cell, which may comprise a nucleic acid molecule according to the application or a vector according to the application. In certain embodiments, each or each host cell may comprise one or more nucleic acid molecules or vectors of the application. In certain embodiments, each or each host cell may comprise a plurality (e.g., 2 or more) or a plurality (e.g., 2 or more) of the nucleic acid molecules or vectors of the application. For example, the vectors of the application may be introduced into such host cells, e.g., eukaryotic cells, such as cells from plants, fungal or yeast cells, and the like. In certain embodiments, the cell may be a bacterial cell (e.g., E.coli), a yeast cell, or other eukaryotic cell. The vectors of the application may be introduced into the host cell by methods known in the art, such as electroporation, lipofectine transfection, lipofectamine transfection, and the like.

In another aspect, the application also provides immunoconjugates which may comprise the isolated antigen binding proteins of the application.

In certain embodiments, the isolated antigen binding proteins or fragments thereof of the present application may be linked to another agent, such as a chemotherapeutic agent, toxin, immunotherapeutic agent, imaging probe, spectroscopic probe, or the like. The linkage may be through one or more covalent bonds, or non-covalent interactions, and may include chelation. A variety of linkers (which may be known in the art) may be used to form the immunoconjugate. Furthermore, the immunoconjugate may be provided in the form of a fusion protein, which may be expressed from a polynucleotide encoding the immunoconjugate.

In another aspect, the application also provides a pharmaceutical composition, which may comprise an isolated antigen binding protein of the application, a polypeptide molecule of the application, an immunoconjugate of the application, a nucleic acid molecule of the application, a vector of the application and/or a cell of the application, and optionally a pharmaceutically acceptable carrier.

Pharmaceutical compositions of the application include, but are not limited to, liquid, frozen and lyophilized compositions.

In certain embodiments, the pharmaceutical compositions may also contain more than one active compound, typically those active compounds having complementary activity that do not adversely affect each other. The type and effective amount of such drugs may depend, for example, on the amount and type of antagonist present in the formulation, as well as the clinical parameters of the subject.

In certain embodiments, the pharmaceutically acceptable carrier may include any and all solvents, dispersion media, coatings, isotonic agents, and absorption delaying agents compatible with drug administration, generally safe, non-toxic.

In certain embodiments, the pharmaceutical composition may comprise parenteral, transdermal, endoluminal, intra-arterial, intrathecal and/or intranasal administration or direct injection into tissue. For example, the pharmaceutical composition may be administered to a patient or subject by infusion or injection.

In a further aspect, the application provides the use of an isolated antigen binding protein of the application, a polypeptide molecule of the application, an immunoconjugate of the application, a nucleic acid molecule of the application, a vector of the application, a cell of the application and/or a pharmaceutical composition of the application for the preparation of a medicament for the prevention and/or treatment of a disease or disorder, wherein the disease or disorder comprises a cognitive disorder and/or a neurodegenerative disease.

In a further aspect, the application provides an isolated antigen binding protein according to the application, a polypeptide molecule according to the application, an immunoconjugate according to the application, a nucleic acid molecule according to the application, a vector according to the application, a cell according to the application and/or a pharmaceutical composition according to the application for use in the prevention and/or treatment of cognitive disorders and/or neurodegenerative diseases.

In another aspect, the present application also provides a method for preventing and/or treating cognitive disorders and/or neurodegenerative diseases, comprising the steps of: administering to a subject in need thereof an isolated antigen binding protein of the application, a polypeptide molecule of the application, an immunoconjugate of the application, a nucleic acid molecule of the application, a vector of the application, a cell of the application and/or a pharmaceutical composition of the application.

In the present application, the cognitive disorders may include early cognitive disorders (MCI), mid-term cognitive disorders, and late cognitive disorders. For example, the cognitive disorder may include cognitive disorder caused by normal aging, lews Body Dementia (LBD), frontotemporal dementia, and/or vascular dementia. For example, the cognitive disorder-inducing disease may include Alzheimer's disease, multiple infarctions, parkinson's disease, AIDS and/or Creutzfeldt-Jakob disease (CJD). In the present application, the cognitive disorder may include amnestic MCI multi-cognitive domain impairment (aMCI-m).

In the present application, the subject may comprise a patient with a neurodegenerative disease. In the present application, the subject may comprise a cognition impaired patient. In the present application, the subject may be in an aging stage. In the present application, the subject suffers from Alzheimer's disease.

Without intending to be limited by any theory, the following examples are meant to illustrate the protein fragments, methods of preparation, uses, and the like of the present application and are not intended to limit the scope of the application.

Examples

Participants of clinical study

The participants in this study were from the Shanghai Aging Study (SAS) project. Detailed study design and recruitment procedures for SAS have been published elsewhere (see Ding,D.et al.The Shanghai Aging Study:study design,baseline characteristics,and prevalence of dementia.Neuroepidemiology 43,114-122(2014)). in this study, following follow-up diagnosis from 2018 to 2021, selected participants are (1) aged 60 years or older, (2) able to coordinate physical examination and neuropsychological testing, and (3) consent to draw blood the exclusion criteria are (1) systemic diseases that are significantly unstable, such as advanced cancer, organ failure or severe chronic systemic disease complications, and (2) current severe alcohol or substance abuse, and (3) significant mental diseases, which make participation in the study difficult.

The study was approved by the ethical committee of the affiliated Huashan hospital at the complex denier university. Written informed consent was obtained from each participant.

Quantitative detection of Abeta 40, abeta 42, tau and pTau181

Plasma Aβ40, Aβ42, total Tau and pTau181 were quantified on an automated Simoa HD-X platform (GBIO, hangzhou, china) using ultrasensitive Simoa techniques (Quanterix, MA, US) according to manufacturer's instructions. Multiplex Neurology 3-Plex A (cat No. 101995) and pTau 181V 2 (cat No. 103714) detection kits were purchased from Quanterix and used accordingly. In all assays, plasma samples were taken at 1: 4. The calibrator and quality control were duplicated. All measurements were made on a single run basis. The operator is unaware of the disease condition of the participant.

Quantitative detection of DIR

Plasma DIR was quantified using an enzyme-linked immunosorbent assay (ELISA) kit developed by the company per se, according to the instructions. In all assays, plasma samples were taken at 1: 4. The calibrator and quality control were duplicated. All data are within the recommended range for ELISA kits.

Immunoprecipitation

U87 cells were lysed in pre-chilled RIPA buffer (50mM Tris,150mM NaCl,0.1%Triton-100, 10% glycerol, 0.5mg/mL BSA and protease inhibitor). After centrifugation of the lysate, the supernatant was mixed with the antibody at 4℃overnight, and then protein G-sepharose was added to bind the antibody at 4℃for 4h. Agarose is then resuspended in RIPA buffer, washed at least 3 times, SDS buffer is added, and incubated at 60 ℃ for 20 minutes. And performing immunoblotting experiments on the treated samples.

Mass Spectrum (MS)

For mass spectrometry, gel digestion was performed using the following procedure. The gel was treated with SDS equilibration buffer (50 mM Tris-Cl (pH 8.8), 6M urea, 30% glycerol, 2% SDS and bromophenol blue) containing 1%1, 4-dithio-trisaccharide alcohol (DTT) for 15min. After this step, the free thiol is alkylated with SDS equilibration buffer containing 2.5% iodoacetamide, which step requires 15 minutes of light protection. It was then cut into gel sections (each section being about 1.0cm in length). Each gel slice was washed three times alternately with acetonitrile and 100mm ammonium bicarbonate. In the last wash, the gel sheets were incubated in 100mM ammonium bicarbonate for 15 minutes, with the temperature maintained at 4 ℃. The gel pieces were dried by vacuum centrifugation and expanded in a trypsin solution containing trypsin (20. Mu.g/ml) and ammonium bicarbonate (50 mM) for 45min, the temperature being maintained at 4 ℃. The trypsin solution was added again and stored at 37℃for 20h. After centrifugation, the supernatant was transferred to another vial and the gel sheets were extracted 3 times for 15min each with 60% acetonitrile solution containing 0.1% formic acid. The recovered peptide solution was dried by vacuum centrifugation, desalted and washed with Ziptip (Millipore, corp.Bedford, MA).

The components of the peptide mixture were separated by reverse phase high performance liquid chromatography (RP-HPLC) and then subjected to tandem mass spectrometry. RP-HPLC was performed on a measurer LC system (Thermo Finnigan, san Jose, calif.). C18 chromatography columns (RP, 180 μm. Times.150 mm) were purchased from column Technology Inc. (Fremeont, CA). The pump flow rate was 1:120, reaching a column flow rate of 1.5. Mu.l/min. Mobile phase a was 0.1% aqueous formic acid and mobile phase B was 0.1% acetonitrile formic acid. The tryptic peptide mixture was eluted with a 2-98% gradient of B over 180 minutes.

Mass spectrometry was performed on an LTQ linear ion trap mass spectrometer (Thermo Finnigan, san Jose, CA), equipped with an electrospray interface, and operated in positive ion mode. The capillary temperature was 170℃and the spray voltage was 3.4kV. The normalized collision energy was 35%. The maximum signal for each scan is obtained using automatic gain control. The mass spectrometer was set up such that 10 MS/MS scans were performed on the 10 most intense ions after one complete MS scan. Dynamic exclusion was set to repeat count 2, repeat duration 30s, exclusion duration 90s.

The obtained MS/MS spectra were searched in IPI human database using BioWorks 3.0.0 software (Thermo Finnigan) on an 8-node Dell PowerEdge 2650 cluster. An accepted sequence result has a Δcn score of at least 0.1 (regardless of charge state), which has a high confidence in the sequence search. And combining all output results together by using Build Summary software, and deleting redundant data. To ensure that the MS/MS spectra have good quality, fragment ions are significantly higher than baseline noise, reference was made to parameters reported in previous studies, and more stringent peptide recognition criteria were applied. The peptides were validated after meeting the following criteria. For a +1 tryptic peptide, the SEQUEST cross-correlation score must be greater than or equal to 1.9; for +2 tryptic peptides, the score is greater than or equal to 2.2; for +3 tryptic peptides, the score was > 3.75. In addition, in the case of the optical fiber, the cut-off value of delta Cn is more than or equal to 0.1, the SP grade of the peptide is less than or equal to 4.

PET

All subjects were subjected to AV45-PET imaging using a PET/CT system (Biograph TruePoint HD 64 PET/CT, siemens; erlangen, germany). The subjects were injected intravenously with AV45 (average dose: 5.55MBq/kg [0.15mCi/kg ]), and then rested in a darkened room for 20 minutes. A 10 minute PET acquisition procedure was then performed and a low dose CT scan was performed. After acquisition, the filtered backprojection algorithm is used to reconstruct the PET image and correct for attenuation, normalization, dead time (dead time), photon attenuation, scattering, and random coincidence. The reconstructed PET image matrix size was 168×168×148, and the voxel size was 2.04×2.04×1.5mm ³.

Image analysis

The region of interest (ROI) is drawn on the lateral parietal, lateral temporal, medial temporal, posterior cingulate (posterior cingulate), frontal, occipital and anterior cuncial cortex. The Standard Uptake Value (SUV) is the radioactivity of the region of interest (ROI), referenced to the radioactivity of the cerebellar nodules. The total SUV score is calculated by a weighted average of these ROIs.

Antibodies to

DIR antibodies used in this study were produced by Jier Biochemical (Shanghai) Inc. (GL Biochem), which recognizes DIR (PPLPLCRRCHKIHLRRLLSKFSNIFSP) of the last 27 AAs, and by Santa Biomedicine (Shanghai) Inc. (Sanyoubio), and polyclonal antibodies for immunoprecipitation were derived from rabbits. Monoclonal antibodies for intraperitoneal injection and immunostaining are derived from hybridomas. Monoclonal antibodies for Elisa capture were prepared from hybridoma cells by gill biochemistry (Shanghai) limited and detection antibodies were constructed by phage display library screening by Santa biomedical (Shanghai) limited. Polyclonal antibodies for Western blotting were derived from rabbits and polyclonal antibodies for immunostaining were derived from guinea pigs.

To detect the specificity of the antibodies, the antibodies were incubated with antigen (10 ^-5 or 10 ^-6 M) at 4 ℃ for 12h for an absorption experiment, and then the absorbed and unabsorbed antibody solutions were incubated-nitrocellulose membranes containing the same lysate samples. And incubated with an hrp-linked secondary antibody and imaged. In addition, immunostaining was performed on brain sections of the same DIR-KI mice to detect the specificity of the antibodies.

Construction and expression of plasmids

Primers were designed to clone the full length CDS sequence. Purification of product and Carrier use HieffPlus One Step Cloning Kit (next san Biotech (Shanghai) Inc.) were recombined. DIR CDS was cloned into pCMV-flag vector gelsolin (NM-000177.5) was cloned into pEGFP-N3 and pcDNA3.1-myc-his plasmids. DDIT4L was constructed into pcDNA3.1-myc-his vector. The primers are shown below.

HEK293T or U87MG cells were transfected with PEI40000 reagent (next St. Biotech (Shanghai)). Further culturing was carried out for 48 hours, cells were lysed with HEPES lysis buffer (30mM HEPES,150mM NaCl,10mM NaF,1%Triton X-100,0.01% SDS), centrifuged at 12000rpm at 4℃for 10min, and the supernatant was transferred for Western blotting.

For pCMV-Flag-DIR:

Forward:5’-CCATGGAGGCCCGAATTCGGATGGTTGCAACTGGC-3’(SEQ ID NO.76),

Reverse:5’-ACTCATCAATGTATCTTATCTTATGGAGAGAAGATGTTAGAAAA-3’(SEQ ID NO.77)；

for pcDNA3.1-DDIT4L-myc-his:

Forward:5’-GCCACCATGGTTGCAACTGGCAGTTTGAGC-3’(SEQ ID NO.78),

Reverse:5’-GAATTCCACCACACTGGACTAGTGGATCCG-3’(SEQ ID NO.79)；

for gelsolin-pEGFP:

Forward:5’-TCAAGCTTCGAATTCATGGCTCCGCACCGCCC-3’(SEQ ID NO.80),

Reverse:5’-CGGGCCCGCGGTACCGGCAGCCAGCTCAGCCATGG-3’(SEQ ID NO.81)；

for pcDNA3.1-gelsolin-myc-his:

Forward:5’-GTGGAATTCGCCACCATGGCTCCGCACCGCCC-3’(SEQ ID NO.82),

Reverse:5’-CCCTCTAGACTCGAGGGCAGCCAGCTCAGCCATGG-3’(SEQ ID NO.83)；

In vitro polymerization experiments

DIR (31-84) and Abeta 42 were synthesized by Gill Biochemical (Shanghai) Inc. The pcDNA3.1-gelsolin-myc-his plasmid was transfected into HEK293 cells and the medium was collected for protein purification. Briefly, ni-NTA agarose resin was washed and transferred to a glass column to be used as an adsorption column. The medium was then mixed with an equal volume of lysis buffer (50 mM NaH ₂PO₄, 300mM NaCl,10mM imidazole, pH=8.0), and the mixture was passed through an adsorption column and washed (50 mM NaH ₂PO₄, 300mM NaCl,20mM imidazole, pH=8.0). The protein of interest was then released from the resin with elution buffer (50 mM NaH ₂PO₄, 300mM NaCl,250mM imidazole, pH=8.0). The eluate was further concentrated and replaced with PBS. The purified proteins were diluted with PBS and the different groups were set for the solutes (Abeta 42, abeta 42/DIR/gelsolin, etc.). 1/10 of the volume of the solution was used as the loading solution, and the remaining solution was spun overnight at 4 ℃. The next day was centrifuged, and the supernatant was immunoprecipitated and the pellet was dissolved with 8M urea. All components were further analyzed by immunoblotting.

Immunoblotting

Adult mice were anesthetized and perfused with PBS. Brain tissue was then extracted and lysed in pre-chilled RIPA buffer. After centrifugation, the brain lysate supernatant was collected and stored at low temperature. Human and mouse plasma was diluted 1:10 with PBS. All samples were separated by Sodium Dodecyl Sulfate (SDS) gel electrophoresis, transferred onto nitrocellulose membranes, and blocked with 5% skimmed milk in TBS buffer containing Tween-20 (TBST). Membranes were incubated in primary antibody overnight at 4 ℃. The next day, the membranes were washed three times with TBST and incubated with HRP-linked secondary antibody. After further washing, imaging was performed by adding ECL buffer. Protein bands were imaged with a GE imaging system. The primary antibodies used are as follows :flag(Sigma,SAB4200071),actin(Chemicon,MAB1501),GAPDH(Proteintech,10494),gelsolin(Invitrogen,PA518605),GFP(Roche,11814460001).

Immunohistochemistry

Mice were anesthetized and perfused with 4% Paraformaldehyde (PFA). Brain tissue was then harvested and fixed in 4% PFA for 1 hour at 4 ℃. After three washes in PBS, the tissues were placed in 30% sucrose/PBS overnight at 4 ℃ and then embedded with OCT compound. Brain sections (thickness 40 μm) were obtained by frozen sections. For immunostaining, tissue sections were blocked with PBST (0.1% Triton X-100/2.5% normal donkey serum/PBS) for 30 min at room temperature and then incubated overnight with primary antibody at 4 ℃. Tissue sections were washed 3 times with PBS, incubated with Alexa fluorescent conjugated secondary antibody (Invitrogen) at 37℃for 60min, washed 3 times with PBS, and loaded with DAPI-containing medium. The image acquisition used a Leica SP8 confocal microscope. The human brain tissue paraffin section is provided by the national academy of medicine human think tank, beijing covariant medical college of China. The distribution of DIR, gelsolin and Abeta was studied using immunofluorescent staining. In a single tissue section, primary antibodies generated in different species (mouse, rabbit or sheep) were used. Secondary antibodies (anti-mouse, anti-rabbit or anti-sheep) were conjugated to different fluorochromes (Alexa Fluor 488, cy3 or Cy 5). After staining, the fluorescently labeled fraction (Vector Labs) was covered with blocking solution.

Ortho-position connection technique (PLA)

The human brain tissue paraffin section is provided by human think tank at the national academy of medical science, beijing co-ordinates medical college. The samples were blocked and then incubated in a humidity chamber at 37℃for 60 minutes. Antibodies recognizing DIR, gelsolin and Abeta were then provided and samples were stained overnight at 4 ℃. The following day samples were incubated with positive and negative PLA probes (1:5; sigma; DUO92001 and DUO 92005), 37℃for 1 hour, then with 1 Xligation buffer (1:40; sigma; DUO 92008), and incubated for an additional 30 minutes at 37 ℃. In addition, the mixture was left to stand at 37℃for 100 minutes with an amplification-polymerase solution (1:80; sigma; DUO 92008). Finally, the samples were protected with a minimum volume Duolink of caplets containing DAPI dye (Sigma; DUO 82040). Finally, imaging with a Leica SP8 microscope.

Brain tissue slice preparation

Adult mice were anesthetized and the brains were removed rapidly and placed in ice-cold artificial cerebrospinal fluid (ACSF) containing the following compounds (unit: mM) for :117 NaCl,3.6 KCl,1.2 NaH₂PO₄·2H₂O,2.5 CaCl₂·2H₂O,1.2 MgCl₂·6H₂O,25NaHCO₃,11 glucose. The ice-cold ACSF had a pH of 7.4 when containing 95% o ₂ and 5% co ₂. Brains were glued on the table of a Leka VT1200S shaker and a 350 μm thick cross section of hippocampus was cut. The sections were incubated in an oxygen-containing (95% o ₂ and 5% co ₂) ACSF for at least 1 hour at room temperature.

Immunofluorescent staining of paraffin sections

The paraffin sections of the brain tissue are soaked in xylene for 10 minutes to dewax for two times; soaking the slices in pure ethanol for 10 minutes, and performing twice; and sequentially soaked in 95%, 90%, 80% and 70% ethanol for 5 minutes each for rehydration, followed by three washes with PBS. Finally, the sections were placed in 1x TE antigen retrieval buffer, repaired for 20 minutes at 100℃and then allowed to cool naturally to room temperature and washed 3 times with PBS. TE antigen retrieval solution (20 x): 4.84g Tris, 1.48g EDTA, 180mL distilled water, naOH to adjust pH to 9.0, adding 2mL Tween 20, constant volume to 200mL,4 ℃ preservation. When in use, the powder is diluted to 1x according to the requirement and stored at normal temperature.

Sections were added to PBS containing 3% sheep serum and blocked for 1 hour. A True Black working solution (380ul 70%EtOH,20ul pre-heated 20x True Black) was prepared, added dropwise to the tissue, incubated at room temperature for 1 min, and then aspirated off and washed 3 times with PBS. The sections were placed in a wet box, the diluted antibodies were covered on the specimen surface, and the wet box was incubated overnight in a refrigerator at 4 ℃.

The next day, the sections were washed three times with PBS and the diluted fluorescent secondary antibody was covered on the sample surface and incubated for 1 hour at room temperature. The sections were then washed three times with PBS, added with 50% ethanol solution containing 0.05% thioflavin S, incubated for 8 minutes in the dark, and washed twice with 80% ethanol for 10 seconds each. Finally, the sections were washed three times with PBS, and fluorescent capper cappers were dropped on and recorded using a microscope.

Preparation of DIR-KI mice

Cas9mRNA and sgRNA for knock-in were obtained by means of in vitro transcription (SEQ ID NO:88; then, a homologous recombination vector comprising a5 'homology arm of about 3kb (SEQ ID NO: 89), a nucleic acid sequence encoding the last 64 amino acids of human QDLIR (SEQ ID NO: 90), and a 3' homology arm of about 3kb (SEQ ID NO: 91) was constructed as a donor, followed by microinjection of the mRNA encoding Cas9, sgRNA and donor vector into fertilized eggs of C57BL/6J mice to obtain F0 mice.

New object identification experiment

Mice were acclimatized to the experimental room 30 minutes ahead of the day of the experiment. The experiment is divided into an adaptation period, a training period and an experimental period. The adaptation period is as follows: the mice were removed from the feeder cages and placed in the center of a plastic box to freely explore the experimental sites for 10 minutes. Training period is carried out after 12-24 hours of adaptation period; training period: 2 identical batteries were placed 5 cm from the wall. The mice were placed in the center of the field and were free to explore for 10 minutes. The experimental period is carried out within 3-4 hours after the training period is finished; experimental period: the toy randomly replaces 1 battery, and the placement position is the same as that of the original object. Mice were left in the center of the field for 10 minutes of free exploration. After the experiment is finished, the mice are put into cages. The whole process adopts a camera for video recording. 75% alcohol is adopted to clean the experimental device in the experimental interval. After the experiment is finished, the time of exploring new and old objects by the mice in the video is counted manually, and the time identification index of the new objects is calculated. When the nose tip of the mouse is within 2 cm from the object, and the mouse sniffs or touches the object to be regarded as exploring the behavior of the object, biting or climbing the object is not regarded as exploring the behavior. Identification index of the mouse on the new object= (time to explore the new object-time to explore the old object)/(time to explore the new object + time to explore the old object). Data from mice exploring total time less than 15 seconds will be excluded. The experimental operator and the data analyst were double blind.

Morris water maze experiment

Mice were acclimatized to the experimental room 30 minutes ahead of the day of the experiment. The experiment was divided into training days and experimental days. Training days are 5-6 days, and experimental days are 1 day. Before the experiment, water with the depth of 30 cm is filled into a water tank, the water temperature is maintained at 19-22 ℃, and talcum powder is added into the water to cause turbidity. The water tank is divided into four directions: n, S, E and W, the platform was placed in the NE quadrant, and mice were challenged daily with non-repeated combinations of S, W, NW and SE. The room setup was kept unchanged during the experiment, with specific patterns attached around the water maze as far-end cues. The room light is non-direct light. The whole experiment process adopts a camera to record, and the video of the experiment day uses the method Vision XT 14 software to analyze the result. Training day: the platform height was placed 0.5 cm under water. The mice were filled with water from the designated water inlet point, facing the tank wall, and timing was started for 1 minute. The timing was stopped when the mice reached the platform, and if the mice did not reach the platform within 1 minute, the mice were placed on the platform or guided to the upper platform. After the mice stay on the platform for 30 seconds, the mice are taken out, wiped dry and put into a cage. An interval of 30 minutes was required before the next water entry. The mice were placed at a new entry point and the above experiment was repeated 3 times. The experimenter recorded the time spent on the platform in each experiment. The next day, the above experiment was repeated. Training is carried out for 4 times per day for 5-6 days. The experimental day: experiments were performed 24 hours after the end of the training day. The platform is removed. The mice were water-filled from the SW quadrant, facing the tank wall. After 1 minute, the mice were removed, wiped dry, and returned to the cage. After the experiment is finished, ethoVision XT software is adopted to analyze the behavior video of the mice, and the acquisition data comprises: escape latency into the target quadrant, number of passes through the platform location, dwell time in the target and non-target quadrants.

Y maze experiment

Mice were acclimatized to the feeding environment for one week prior to the conduct of the behavioural experiments. Mice were acclimatized to the experimental room 30 minutes ahead of the day of the experiment. After the start of the experiment, the mice were placed face-wise on the Y-maze starting arm a, free to explore the maze for 10 minutes. When located in the center of the maze, the mice can choose to enter in any direction. Mice with normal cognition will tend to enter the unexplored arm during the course of the experiment. The whole process adopts a camera for video recording. After the experiment is finished, the mice are put into cages. 75% alcohol is adopted to clean the experimental device in the experimental interval. After the end of the experiment, the sequence in which the mice explored the maze arms was manually recorded for calculating the total arm-in number and the alternate ratio (alternate ratio = actual alternate arm-in number/(total arm-in number-2)). The experimental operator and the data analyst were double blind.

Statistical analysis

The differences between groups of numerical data (e.g., biomarker concentrations) were analyzed using the Welch's t test or one-way anova. And adopting a linear regression model to perform correlation analysis. Subject operating characteristics (ROC) curves were analyzed with area under the curve (AUC) as predictive value for biomarkers.

Statistical analysis was performed using PRISM (GraphPad Software). The box map and roc were generated using package ggplot in R (4.0.2 th edition). Differences were considered significant when p <0.05 (< p <0.05, < p <0.01, < p < 0.001).

Example 1 DIR identification

By analysis of transcripts from Alzheimer's disease patients (AD), it was found that a variable sheared form of the DDIT4L gene was highly expressed in patients, and that such shearing resulted in retention of the 2 nd intron of DDIT4L, thereby generating a novel transcript DIR.

Specifically, a normal cutter of the human DDIT4L gene will partially cleave the Intron (Intron) during the formation of mRNA and ligate adjacent two exons (Exon). In the course of aberrant cleavage, intron Retention (IR) is caused, forming an entirely new mRNA form of DIR (see fig. 1A, where NS represents normal cleavage and IR represents DIR formed by aberrant cleavage).

Identification of Normal (NC) and Alzheimer's disease patients (AD) DDIT4L and DIR mRNA: and taking partial brain tissues donated by normal people and senile dementia patients, extracting RNA, reversing the RNA into cDNA, and identifying DDIT4L and DIR mRNA by a PCR method. Primer design (forward primer: 5 '-TGCTGGACTGTGGCTATCAC' (SEQ ID NO. 6); reverse primer 5'-acaaggacctttgagcaacca-3' (SEQ ID NO. 7)) in both exons, the PCR product of DIR (about 2000 bp) was greater than that of normally sheared DDIT4L (about 200 bp) due to the retention of introns by aberrant cleavage. The mRNA identification results for DDIT4L and DIR are shown in fig. 1B.

The open reading frame of DIR is cloned and exogenously expressed, and the DIR can be normally translated into a corresponding DIR protein (the amino acid sequence of which is shown as SEQ ID NO. 1) in vitro, and the protein can be secreted out of cells.

Specifically, the open reading frame of DIR (SEQ ID NO. 8) was constructed into pCMV-Flag vector, and Flag-DIR plasmid and control Flag plasmid were transfected into HEK293 cells, respectively, after 48 hours, cell culture medium and cell lysate were taken as Western Blot, the expression was detected with DIR-specific antibody, and actin was used as control. The results are shown in FIG. 1C.

DIR in human blood was tested. Blood from 2 normal persons and 7 AD patients of different ages (ages 82, 55, 65, 53, 78, 68 and 58, respectively) was taken as Western Blot, and the expression was detected with DIR-specific antibodies, and ponceau stained bands were used as loading controls. The results are shown in FIG. 1D. It can be seen that DIR may be specifically present in AD patients.

Example 2 DIR biological Functions

2.1 DIR has interaction with gelsolin

The DIR-expressing plasmid is transfected into U87 cells, the cells are lysed after 48 hours, the cell lysate is divided into two parts averagely, the equal amount of IgG and DIR antibody are respectively added for an immune coprecipitation experiment, experimental products are separated by SDS-PAGE, separated PAGE gel is soaked in coomassie brilliant blue dye solution for 1 hour, and then dye solution which is non-specifically attached to the surface of the PAGE gel is washed off by decolorizing solution until bands can be clearly displayed in the PAGE gel. Then, specific bands are cut off and mass spectrometry analysis is performed to obtain candidate molecules gelsolin.

Then cloning gelsolin open reading frame (SEQ ID NO. 9) into pEGFP-N3 vector to obtain Gelsolin-GFP plasmid, co-transfecting gelsolin-GFP and Flag or Flag-DIR plasmid into HEK293 cells, lysing cells after 48 hours, obtaining lysate, performing co-immunoprecipitation experiment with Flag antibody, and finally detecting gelsolin binding to DIR by GFP antibody.

2.2 DIR inhibits excitatory postsynaptic current amplitude

Brain tissues of adult C57BL/6 mice are taken to prepare brain slices, and brain slices of sea horse parts are taken for electrophysiological recording. Post-synaptic excitatory current changes in brain slices are recorded before and after administration of IR peptide fragments.

The results are shown in FIG. 4. The results show that the current amplitude was reduced after the DIR was applied, while the frequency of the current emission was unchanged.

The functional fragment of example 3 DIR and its biological function

DIR (i.e., QDLIR, whose amino acid sequence is shown as SEQ ID NO. 1) comprises the amino acid sequence encoded by the first exon (whose amino acid sequence is shown as SEQ ID NO. 2) and the amino acid sequence encoded by the retained intron (whose amino acid sequence is shown as SEQ ID NO. 3).

Wherein the amino acid sequence (IR) encoded by the retained intron can be divided into 2 parts, namely an IR-I consisting of the first 27 amino acids from the N-terminus (namely DIR-I, the amino acid sequence of which is shown as SEQ ID NO. 4) and a DIR-II consisting of the last 27 amino acids (namely DIR-II, the amino acid sequence of which is shown as SEQ ID NO. 5). See in particular fig. 2 for the above structure.

The function of the above-mentioned IR fragment is verified by means of electrophysiological recording.

First, the IR fragment was synthesized, brain slices of wild-type mice were incubated, and excitatory postsynaptic currents (EPSCs) were recorded (see, for details, patel et al, curr biol, current Biol, 26:2194-2201[2016 ]). The results show that IR can reduce the amplitude of EPSC, although no significant drop in EPSC frequency was observed, a trend was seen to be decreasing.

The IR-I and DIR-II fragments were synthesized separately to screen functional regions. The results show that IR-I can reduce the frequency of EPSCs but does not affect the amplitude of EPSCs; although DIR-II has no significant effect on both the amplitude and frequency of the EPSC. However, it can be seen from the statistics that DIR-II has a tendency to decrease the amplitude of EPSC and a tendency to increase the frequency of EPSC (see FIG. 3).

From the above results, IR has two functional areas. Wherein IR-I can reduce the frequency of EPSCs, while DIR-II can reduce the amplitude of EPSCs.

Example 4 DIR participation in Abeta deposition

4.1 DIR induces Abeta deposition by gelsolin

In AD patient samples, DIR was mainly co-localized with aβ in the hippocampus (see fig. 5 a). Interactions between DIR and aβ were observed in transfected HEK293T cells, but DDIT4L (including DIR-exons) was unable to interact with aβ (see fig. 5b, fig. 6 a). These results indicate that the DIR-intron (i.e., the amino acid sequence encoded by the retained intron, the amino acid sequence of which is shown in SEQ ID NO. 3) may be the primary region of interaction of DIR with Abeta.

Proximity Ligation Analysis (PLA) showed that DIR did not interact directly with aβ (see fig. 5 c). Using the synthetic DIR-intron and Abeta protein, it was found that the DIR-intron was not able to bind directly to Abeta (see FIG. 6 b), indicating that the DIR had another binding target.

DIR was transfected into human brain cell lines and cell lysates were immunoprecipitated with rabbit DIR antibodies (purchased from Gill Biochemical (Shanghai) Inc.). The results showed that a single immunoreactive band of approximately 85kDa molecular weight was co-immunoprecipitated (see FIG. 5 d). The bands were extracted and further analyzed by mass spectrometry. This analysis determined 12-15 peptides that matched human gelsolin (85 kDa) and covered 35% of gelsolin sequence (see FIG. 5 e).

Gelsolin have been shown to interact with aβ17 to form a complex that is transported to the circulatory system. Studies have shown that DIR interacts with gelsolin in DIR knock-in mice, HEK293T cells and human tissue (see fig. 5f, g, fig. 6 c).

In HEK293T cell lysates transfected with DIR and endogenously expressed gelsolin, the addition of aβ42 enhanced the interaction of DIR with gelsolin in a dose-dependent manner, suggesting that aβ contributes to the interaction between DIR and gelsolin (see fig. 5 h.) furthermore, the addition of synthetic DIR-intron and aβ42 protein to purified gelsolin or HEK293T cell lysates significantly enhanced the formation of insoluble aβ42, whereas insoluble aβ42 is a major component of amyloid plaques (see fig. 5i, fig. 6 d).

It follows that DIR-introns are involved in the deposition of Abeta.

4.2 DIR mediated amyloid plaque formation of Abeta

Thioflavin S (thioflavine S) is a specific marker of aβ plaques in the brain of AD patients. However, thioflavin S cannot label amyloid plaques knocked in the hippocampus of mice (see fig. 7 a) because the mouse aβ sequence cannot be detected with thioflavin.

However, amyloid plaques in DIR knock-in mice hippocampal slices incubated for 6 hours with human aβ40 peptide could be stained with thioflavin S (see fig. 7 a), and are consistent with the results of thioflavin S staining human aβ plaques.

Also, triple immunostaining showed that co-localization of DIR with gelsolin and aβ was higher in dense core plaques than in diffuse plaques in hippocampal and cortical areas of AD patients, whereas no DIR or amyloid plaque staining was evident in control subjects without AD (see fig. 7b, fig. 8 a).

In the dense core plaques of the hippocampus and cortical areas of AD patients, aβ signals were stronger than thioflavins S and DIR, indicating that the dense core plaques are insoluble; furthermore, soluble aβ peptides are present in the peripheral region (see fig. 7 c).

From the above results, it is clear that an increase in DIR expression may lead to aβ deposition and subsequent amyloid plaque formation by binding to gelsolin under pathological conditions. DIR may be an initiator of aβ deposition and amyloid plaque formation (see fig. 11 e).

Example 5 DIR disease-related

5.1 Plasma DIR levels as diagnostic indicators

DIR levels in plasma were measured in 33 cognitive normal persons (as a control group), 44 non-amnestic MCI (non-AMNESTIC MCI, NAMCI) patients, 42 amnestic MCI (aMCI) patients and 31 patients clinically diagnosed with AD.

The DIR concentration in plasma was progressively higher in naMCI, acci and AD patients compared to the cognitively normal control group (see figure 9 a). Furthermore, DIR concentration in plasma was found to correlate positively with the concentration of pTau181, but negatively with the aβ42/40 ratio, and negatively with the severity of cognitive decline throughout the clinical range from normal cognition to naMCI, aci and AD.

However, this trend was not observed for the total Tau (tTau) concentration in plasma (see fig. 9 b-d).

DIR and pTau181 levels in plasma showed the potential to distinguish AD patients from cognitively normal individuals with AUC of AUC _DIR =0.86 and AUC _pTau181 =0.86, respectively (see fig. 9e and f);

AUC _Aβ42/40 ratio in plasma was only up to 71% for distinguishing AD patients from normal controls (see fig. 9 g). Whereas plasma tTau levels were not effective in distinguishing AD patients from control groups (auc=0.55) (see fig. 10 a). The integrated model combined with DIR, pTau, A β42/40 demonstrated the potential to distinguish AD patients from cognitive normal control groups with an AUC of 96% higher than that obtained with pTau and aβ42 (88%)/40 (see fig. 9 h).

DIR levels in plasma also showed better performance in distinguishing aMCI patients from cognitively normal patients (auc=0.73) compared to pTau181 (auc=0.63), aβ42/40 (auc=0.59) and tTau (auc=0.56) (fig. 9i-k, fig. 10 b). The combined DIR, pTau, A β42/40 model showed better effect in distinguishing aMCI individuals from the cognitive normal control group, with an AUC of 81% higher than that obtained for pTau and aβ42 (75%)/40 (fig. 9 l).

5.2 Plasma DIR levels are correlated with disease progression and plasma Aβ levels

Plasma from 12 MCI patients at cognitive normal (as baseline samples) and post-illness plasma were examined for DIR concentrations three years ago, respectively. DIR plasma concentrations in these MCI patient samples were found to be higher than their baseline samples (fig. 11 a).

Also, DIR levels in plasma were positively correlated with levels of aβ40 and aβ42 in both MCI patients and cognitive normal persons (as control group) (see fig. 11b, fig. 12 a).

It follows that an increase in DIR concentration in plasma occurs as cognitive dysfunction progresses and is correlated with levels of aβ.

5.3 Correlation of plasma DIR levels with Abeta-PET levels

The correlation between plasma DIR and Abeta-PET levels was examined. In AD/MCI patients, plasma DIR levels correlated significantly with aβ -PET levels throughout the cortex, and plasma DIR levels correlated closely with aβ -PET levels in the temporal lobes, indicating aβ accumulation (see fig. 11c, fig. 12 b). The DIR concentration in plasma was higher for aβ -positive subjects (n=11) than for aβ -negative subjects (n=26) (see fig. 11 d). DIR levels in plasma have good predictive value for aβ -PET positivity (auc=0.90, see fig. 11 d).

These results show that the presence of DIR in plasma strongly reflects amyloid plaque formation in AD patients. Thus, DIR is a potential biomarker for AD.

Example 6 DIR antibodies and their biological Functions

6.1 Preparation of DIR antibodies

1) Preparation of hybridomas and obtaining DIR antibodies

Mice immunized with antigen were taken and their spleens were digested into single cells. The B cells are then isolated and cultured in an in vitro system and fused with myeloma cells using a chemical inducer. The fused cells were cultured in 96-well plates by flow sorting techniques. Finally, the culture medium is collected and incubated into an antigen-coated well plate, and the specificity of the DIR antibody is detected.

2) Antibody sequencing

Total RNA was reverse transcribed into cDNA using antisense primers according to the technical manual of PRIMESCRIPT ^TM first strand cDNA Synthesis kit (Takara, catalog No. 6110A). Antibody fragments of VH and VL were then amplified according to the SOP of Biointron Biologic inc. And PCR fragments were cloned into TA/Blunt-Zero Cloning vector. Colony PCR was performed to screen clones and no less than 5 positive clones were sequenced.

The sequencing results of IR-II-1 to IR-II-10 (IR-II-10, A2D 10) were measured, respectively, and the results are shown below:

TABLE 1

6.2 Detection of binding Activity of DIR antibodies

The binding activity of the above-mentioned IR-II-1 to IR-II-10 antibodies to the DIR-II fragment was examined. The specific method comprises the following steps:

The fragment of interest DIR-II was coated on a 96-well plate using a coating buffer and blocked with a blocking buffer. After further equal dilution of monoclonal antibodies (1 mg/mL) respectively (1:2k, 1:6k,1:18k,1:54k,1:162k, 1:481 k,1:1458k,1:4374 k), the pipettes were added to the individual wells and shaken well for 2 hours at 37℃and washed three times with wash buffer. Then secondary antibodies (1:5 k) with horseradish peroxidase were added and incubated at 37 ℃. After three times of cleaning, adding a proper volume of TMB substrate solution, incubating for a certain time at 37 ℃, and then placing under an enzyme-labeled instrument to record the absorbance value of the sample at the wavelength of 450 nm.

The results are shown in Table 2 and FIG. 13, and it is found from the results that the above-mentioned IR-II-1 to IR-II-10 antibodies specifically bind to DIR-II fragments at a working concentration of about 10ng/ml or more.

TABLE 2

Example 7 detection of DIR Using DIR antibodies

7.1

Antibodies targeting 30 amino acids of the DIR exon 2 translational sequence were prepared and tested in patient tissues. Immunoblots of GBM tissues showed two bands (-12 kDa and (-22 kDa) (see FIG. 14 c). Since the molecular weight of DDIT4L is about 22kDa, it is speculated that the 12kDa band may be DIR.

Further, a 54 amino acid antibody (guinea pig-derived polyclonal antibody, available from gill biochemistry (Shanghai) limited) recognizing only the intron translation sequence unique to DIR was obtained and tested in patient tissues. Only a protein of about 12kDa was detected in GBM tissue (see fig. 14 d).

In addition, a heterologous expression plasmid containing the DIR open reading frame (the nucleotide sequence of which is shown in SEQ ID NO. 1) was constructed and transfected into HEK293T cells. DIR could be detected in cell lysates and conditioned medium (see FIG. 14 e), indicating that DIR can be translated and secreted in vivo.

7.2

Antibodies targeting 27 amino acids at the C-terminus of DIR (i.e., DIR-II) were prepared to detect the presence of DIR in the blood of GBM patients. Immunoblots showed the presence of high levels of DIR in the blood of patients with tumor invasion of the hippocampus (see fig. 14f, g). Specifically, DIR was present in 71.4% of blood samples of GBM patients (n=7) who affected the hippocampus, but only 15.4% of blood samples of GBM patients (n=13) who did not affect the hippocampus.

This suggests a correlation between the presence and generation of DIR and hippocampal tumor invasion, which is important for the storage of declarative memory and associative learning.

Example 8 DIR reduced levels can increase cognitive levels

8.1 Use of siRNA

(1) After transfecting Flag-DIR plasmids in HEK293 cells for 6 hours, 50nM siRNA (wherein the nucleotide sequence of the sense strand of DIR siRNA1 is shown as SEQ ID NO.84, the nucleotide sequence of the antisense strand of siRNA1 is shown as SEQ ID NO.85, the nucleotide sequence of the sense strand of DIR siRNA2 is shown as SEQ ID NO.86, and the nucleotide sequence of the antisense strand of siRNA2 is shown as SEQ ID NO. 87) was transfected respectively. After 48 hours of plasmid transfection, the supernatant was collected by centrifugation of the lysed cells and examined for DIR expression by immunoblotting.

The results are shown in fig. 15A, and as a result, it was found that the expression level of DIR was significantly decreased after siRNA administration.

(2) The hippocampus of 3 month old DIR-KI mice was administered by injection using a brain stereotactic apparatus, and siRNA2 (1.25 mM) and a control drug PBS were administered, respectively. The administration was divided into three doses of 4 μl each, three days apart. The mice were allowed to recover for three days after dosing was completed and a new object recognition behavior experiment was performed. In fig. 15B, O, N are the old object and the new object, respectively.

As shown in FIG. 15B, the results show that the behavioural recognition of new objects after siRNA injection into the hippocampus of DIR-KI mice shows a remarkable improvement in cognitive function (n.gtoreq.9).

8.2 Utilization of IR-II antibodies

To examine whether or not an IR-II antibody could improve the disease such as AD, A2D10 (0.5 mg/day) or IgG obtained in example 6 was intraperitoneally injected into homozygous DIR-KI mice for 5 days.

The mice after injection were tested by behavioral tests and the results are shown in fig. 16. Wherein O, N are the old object and the new object, respectively.

The results of fig. 16A-16B show that applying A2D10 improves working memory and new object recognition. The results of fig. 16C show that administration of A2D10 reduced escape latency during the Morris water maze test training phase. In the probe test (probe test) for evaluating spatial memory, the latency of A2D 10-administered mice into the target quadrant was shorter and the residence time was longer.

All documents mentioned in this disclosure are incorporated by reference in this disclosure as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the application as defined in the appended claims.

Claims

A regulator, which regulates the intron retention splicing product DIR and/or its functional fragment of a DNA damage-inducible transcript 4-like transcript, and its use in preparing an agent for preventing and/or treating a disease, wherein the disease includes cognitive impairment.

A regulator, which regulates the intron retention splicing product DIR and/or its functional fragment of a DNA damage-inducible transcript 4-like transcript, and its use in preparing an agent for preventing and/or treating a disease, wherein the disease includes a neurodegenerative disease.

The use according to any one of claims 1-2, wherein the modulator reduces the expression level and/or biological activity of the DIR and/or its functional fragment in the subject.

The use according to claim 3, wherein the reduction comprises a reduction of the expression level and/or biological activity of the DIR and/or its functional fragment by at least about 10% compared to the original expression level and/or biological activity of the DIR and/or its functional fragment in the subject.

The use according to any one of claims 3-4, wherein the expression level includes the expression level of the gene encoding the DIR/or its functional fragment, the transcription level of the gene encoding the DIR/or its functional fragment and/or the expression level of the DIR/or its functional fragment.

The use according to any one of claims 3 to 5, wherein the expression level is measured by performing an assay selected from the group consisting of qPCR, qRT-PCR, hybridization analysis, Northern blotting, dot blotting, in situ hybridization, gel electrophoresis, capillary electrophoresis, column chromatography, Western blotting, immunohistochemistry, immunostaining, and mass spectrometry.

The use according to any one of claims 3-6, wherein the expression level is measured by utilizing a substance selected from the following group: a primer capable of specifically amplifying the gene encoding the DIR/or its functional fragment, a nucleic acid molecule that specifically binds to the gene encoding the DIR/or its functional fragment, a nucleic acid molecule that specifically binds to the DIR/or its functional fragment, a small molecule that specifically binds to the DIR/or its functional fragment, a probe that specifically binds to the DIR/or its functional fragment, and a polypeptide that specifically binds to the DIR/or its functional fragment.

The use according to any one of claims 1-7, wherein the functional fragment of DIR retains at least part of the biological activity of DIR.

The use according to claim 8, wherein the biological activity comprises affecting the excitability of neurons and/or inhibiting the activity of neurons.

The use according to any one of claims 8-9, wherein the biological activity comprises the ability to reduce the frequency of excitatory postsynaptic current (EPSC), and/or the ability to reduce the amplitude of EPSC.

The use according to claim 10, wherein the reduction comprises administering the DIR and/or its functional fragment and/or a nucleic acid encoding the DIR and/or its functional fragment, thereby reducing the frequency of excitatory postsynaptic current (EPSC) in the subject and/or reducing the amplitude of EPSC in the subject, compared to the biological activity of the original DIR and/or its functional fragment in the subject.

The use according to any one of claims 8 to 11, wherein the biological activity comprises affecting cognitive abilities.

The use according to any one of claims 8 to 12, wherein the biological activity comprises participation in a signaling pathway associated with Aβ deposition, and/or participation in a signaling pathway associated with Tau tangle generation.

The use according to any one of claims 8 to 13, wherein the biological activity comprises inducing Aβ deposition and/or amyloid plaque formation by gelsolin.

The use according to claim 14, wherein the DIR and/or its functional fragment induces Aβ deposition and/or amyloid plaque formation by binding to gelsolin.

The use according to any one of claims 8 to 15, wherein the expression level of the DIR and/or its functional fragment is positively correlated with the expression level of Aβ.

The use according to any one of claims 8-16, wherein the reduction comprises administering the DIR and/or its functional fragment and/or the nucleic acid encoding the DIR and/or its functional fragment, thereby reducing the cognitive ability of the subject compared to the biological activity of the original DIR and/or its functional fragment in the subject.

The use according to any one of claims 1-17, wherein the DIR and/or its functional fragment is derived from a mammal.

The use according to any one of claims 1-18, wherein the DIR and/or its functional fragment is derived from a primate.

The use according to any one of claims 1-19, wherein the DIR and/or its functional fragment are derived from humans.

The use according to any one of claims 1 to 20, wherein the DIR comprises the amino acid sequence shown in SEQ ID NO.1.

The use according to any one of claims 1 to 21, wherein the functional fragment of DIR comprises an amino acid sequence encoded by a retained intron in DDIT4L.

The use according to any one of claims 1-22, wherein the functional fragment of DIR comprises the amino acid sequence shown in any one of SEQ ID NO.4-5.

The use according to any one of claims 1 to 23, wherein the cognitive impairment comprises cognitive impairment caused by normal aging, Lewis body dementia (LBD), frontotemporal dementia and/or vascular dementia.

The use according to any one of claims 1 to 24, wherein the inducing diseases of cognitive impairment include Alzheimer's disease, multi-infarct type, Parkinson's disease, AIDS and/or Creutzfeldt-Jakob disease (CJD).

The use according to any one of claims 1 to 25, wherein the cognitive impairment comprises early cognitive impairment (MCI), mid-term cognitive impairment and late-term cognitive impairment.

The use according to any one of claims 1-26, wherein the cognitive impairment comprises amnestic MCI with multiple cognitive domain impairment (aMCI-m).

The use according to any one of claims 2-27, wherein the neurodegenerative disease includes acute neurodegenerative disease and chronic neurodegenerative disease.

The use according to any one of claims 2-28, 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 communication and/or a neurodegenerative disease caused by impaired cell motility.

The use according to any one of claims 2 to 29, wherein the neurodegenerative disease comprises Alzheimer's disease, Parkinson's disease, multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS) and/or Huntington's disease (HD).

The use according to any one of claims 2 to 30, wherein the neurodegenerative disease comprises Alzheimer's disease.

The use according to any one of claims 2 to 31, wherein the neurodegenerative disease comprises early Alzheimer's disease, middle Alzheimer's disease and/or late Alzheimer's disease.

The use according to any one of claims 3-32, wherein the subject comprises a mammal.

The use according to any one of claims 3-33, wherein the subject comprises a human.

The use according to any one of claims 3-34, wherein the subject comprises a patient with a neurodegenerative disease and/or a patient with a cognitive disorder.

The use according to any one of claims 3-35, wherein the subject comprises an Alzheimer's disease patient.

The use according to any one of claims 3-36, wherein the subject is in the elderly stage.

The use according to any one of claims 1 to 37, wherein the agent is formulated for oral administration and/or injection administration.

The use according to any one of claims 1 to 38, wherein the modulator comprises a small molecule compound, a polymer and/or a biomacromolecule.

The use according to any one of claims 1 to 39, wherein the modulator comprises an antibody or an antigen-binding fragment thereof.

The use according to any one of claims 1 to 40, wherein the regulator comprises an antibody or an antigen-binding fragment thereof that specifically binds to the intron-retained splicing product DIR of the DNA damage-inducible transcript 4-like transcript and/or its functional fragment.

The use according to any one of claims 1-41, wherein the modulator comprises an antisense oligonucleotide.

The use according to any one of claims 1-42, wherein the modulator comprises siRNA.

The use according to any one of claims 1 to 43, wherein the regulator comprises an siRNA that specifically binds to the intron retained splicing product DIR and/or its functional fragment of the DNA damage-inducible transcript 4-like transcript.

The use according to any one of claims 1-44, wherein the modulator comprises the nucleotide sequence shown in any one of SEQ ID NOs. 84-87.

A method for preventing and/or treating cognitive impairment, comprising the following steps: reducing the expression level and/or biological activity of the intron retained splicing product DIR and/or its functional fragment of a DNA damage-inducible transcript 4-like transcript in a subject in need thereof.

A method for preventing and/or treating neurodegenerative diseases, comprising the following steps: reducing the expression level and/or biological activity of the intron retained splicing product DIR and/or its functional fragment of a DNA damage-inducible transcript 4-like transcript in a subject in need thereof.

The method according to any one of claims 46-47, wherein the reduction comprises a reduction of at least about 10% in the expression level and/or biological activity of the DIR and/or its functional fragment compared to the original expression level and/or biological activity of the DIR and/or its functional fragment in the subject.

The method according to any one of claims 46-48, wherein the expression level includes the expression level of the gene encoding the DIR/or its functional fragment, the transcription level of the gene encoding the DIR/or its functional fragment and/or the expression level of the DIR/or its functional fragment.

The method of any one of claims 46-49, wherein the expression level is measured by performing an assay selected from the group consisting of qPCR, qRT-PCR, hybridization analysis, Northern blotting, dot blotting, in situ hybridization, gel electrophoresis, capillary electrophoresis, column chromatography, Western blotting, immunohistochemistry, immunostaining, and mass spectrometry.

The method according to any one of claims 46-50, wherein the expression level is measured by utilizing a substance selected from the following group: a primer capable of specifically amplifying the gene encoding the DIR/or its functional fragment, a nucleic acid molecule that specifically binds to the gene encoding the DIR/or its functional fragment, a nucleic acid molecule that specifically binds to the DIR/or its functional fragment, a small molecule that specifically binds to the DIR/or its functional fragment, a probe that specifically binds to the DIR/or its functional fragment, and a polypeptide that specifically binds to the DIR/or its functional fragment.

The method according to any one of claims 46-51, wherein the functional fragment of DIR retains at least part of the biological activity of DIR.

The method according to any one of claims 46-52, wherein the biological activity comprises affecting the excitability of neurons and/or inhibiting the activity of neurons.

The method according to any one of claims 46-53, wherein the biological activity comprises the ability to reduce the frequency of excitatory postsynaptic currents (EPSCs), and/or the ability to reduce the amplitude of EPSCs.

The method according to claim 54, wherein the reduction comprises administering the DIR and/or its functional fragment and/or the nucleic acid encoding the DIR and/or its functional fragment, thereby reducing the frequency of excitatory postsynaptic current (EPSC) in the subject and/or reducing the amplitude of EPSC in the subject, compared to the biological activity of the original DIR and/or its functional fragment in the subject.

The method of any one of claims 46-55, wherein the biological activity comprises affecting cognitive abilities.

The method according to any one of claims 46-56, wherein the biological activity comprises participation in a signaling pathway associated with Aβ deposition, and/or participation in a signaling pathway associated with Tau tangle generation.

The method according to any one of claims 46-57, wherein the biological activity comprises induction of Aβ deposition and/or amyloid plaque formation by gelsolin.

The method according to claim 58, wherein the DIR and/or its functional fragment induces Aβ deposition and/or amyloid plaque formation by binding to gelsolin.

The method according to any one of claims 46-59, wherein the expression level of the DIR and/or its functional fragment is positively correlated with the expression level of Aβ.

The method according to any one of claims 46-60, wherein the reduction comprises administering the DIR and/or its functional fragment and/or the nucleic acid encoding the DIR and/or its functional fragment, thereby reducing the cognitive ability of the subject compared to the biological activity of the original DIR and/or its functional fragment in the subject.

The method according to any one of claims 46-61, wherein the DIR and/or its functional fragment are derived from a mammal.

The method according to any one of claims 46-62, wherein the DIR and/or its functional fragment is derived from a primate.

The method according to any one of claims 46-63, wherein the DIR comprises the amino acid sequence shown in SEQ ID NO.1.

The method according to any one of claims 46-64, wherein the functional fragment of DIR comprises an amino acid sequence encoded by a retained intron in DDIT4L.

The method according to any one of claims 46-65, wherein the functional fragment of DIR comprises the amino acid sequence shown in any one of SEQ ID NO.4-5.

The method according to any one of claims 46-66, wherein the cognitive impairment comprises cognitive impairment caused by normal aging, Lewis body dementia (LBD), frontotemporal dementia and/or vascular dementia.

The method according to any one of claims 46-67, wherein the inducing disease of cognitive impairment comprises Alzheimer's disease, multi-infarct type, Parkinson's disease, AIDS and/or Creutzfeldt-Jakob disease (CJD).

The method according to any one of claims 46-68, wherein the cognitive impairment comprises early cognitive impairment (MCI), mid-stage cognitive impairment and late stage cognitive impairment.

The method of any one of claims 46-69, wherein the cognitive impairment comprises amnestic MCI with impairment of multiple cognitive domains (aMCI-m).

The method according to any one of claims 47-70, wherein the neurodegenerative disease comprises an acute neurodegenerative disease and a chronic neurodegenerative disease.

The method according to any one of claims 47-71, 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 communication and/or a neurodegenerative disease caused by impaired cell motility.

The method of any one of claims 47-72, wherein the neurodegenerative disease comprises Alzheimer's disease, Parkinson's disease, multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS) and/or Huntington's disease (HD).

The method of any one of claims 47-73, wherein the neurodegenerative disease comprises Alzheimer's disease.

The method according to any one of claims 47-74, wherein the neurodegenerative disease comprises early Alzheimer's disease, middle Alzheimer's disease and/or late Alzheimer's disease.

The method of any one of claims 46-75, wherein the subject comprises a mammal.

The method of any one of claims 46-76, wherein the subject comprises a human.

The method according to any one of claims 46-77, wherein the subject comprises a patient with a neurodegenerative disease and/or a patient with a cognitive disorder.

The method according to any one of claims 46-78, wherein the subject comprises an Alzheimer's disease patient.

The method according to any one of claims 46-79, wherein the subject is elderly.

A method according to any one of claims 46-80, wherein the method comprises the following steps: administering a modulator to a subject in need thereof, wherein the modulator is capable of reducing the expression level and/or biological activity of the DIR and/or its functional fragment, and/or the nucleic acid encoding the DIR and/or its functional fragment.

The method according to claim 81, wherein the administration comprises oral administration and/or injection administration.

The method according to any one of claims 81-82, wherein the modulator comprises a small molecule compound, a polymer and/or a biological macromolecule.

The method of any one of claims 46-83, wherein the modulator comprises an antibody or an antigen-binding fragment thereof.

The method according to any one of claims 46-84, wherein the modulator comprises an antibody or an antigen-binding fragment thereof that specifically binds to the intron-retained splicing product DIR and/or its functional fragment of the DNA damage-inducible transcript 4-like transcript.

The method of any one of claims 46-85, wherein the modulator comprises an antisense oligonucleotide.

The method of any one of claims 46-86, wherein the modulator comprises siRNA.

The method according to any one of claims 46-87, wherein the modulator comprises an siRNA that specifically binds to the intron-retained splicing product DIR and/or its functional fragment of the DNA damage-inducible transcript 4-like transcript.

The method according to any one of claims 46-88, wherein the modulator comprises the nucleotide sequence shown in any one of SEQ ID NO.84-87.

A method for screening drugs capable of preventing and/or treating cognitive disorders and/or treating neurodegenerative diseases, comprising the following steps: detecting the effect of a candidate drug on the expression level and/or biological activity of DIR and/or its functional fragments in a subject, wherein after administration of the candidate drug, the expression level and/or biological activity of the DIR and/or its functional fragments decreases, and the candidate drug is capable of preventing and/or treating cognitive disorders and/or treating neurodegenerative diseases.

The method of claim 90, wherein the reduction comprises a reduction of at least about 10% in the expression level and/or biological activity of the DIR and/or its functional fragment compared to the original expression level and/or biological activity of the DIR and/or its functional fragment in the subject.

The method according to any one of claims 90-91, wherein the expression level includes the expression level of the gene encoding the DIR/or its functional fragment, the transcription level of the gene encoding the DIR/or its functional fragment and/or the expression level of the DIR/or its functional fragment.

The method of any one of claims 90-92, wherein the expression level is measured by performing an assay selected from the group consisting of qPCR, qRT-PCR, hybridization analysis, Northern blotting, dot blotting, in situ hybridization, gel electrophoresis, capillary electrophoresis, column chromatography, Western blotting, immunohistochemistry, immunostaining, and mass spectrometry.

The method according to any one of claims 90-93, wherein the expression level is measured by utilizing a substance selected from the following group: a primer capable of specifically amplifying the gene encoding the DIR/or its functional fragment, a nucleic acid molecule that specifically binds to the gene encoding the DIR/or its functional fragment, a nucleic acid molecule that specifically binds to the DIR/or its functional fragment, a small molecule that specifically binds to the DIR/or its functional fragment, a probe that specifically binds to the DIR/or its functional fragment, and a polypeptide that specifically binds to the DIR/or its functional fragment.

The method according to any one of claims 90-94, wherein the functional fragment of DIR retains at least part of the biological activity of the DIR.

The method of claim 95, wherein the biological activity comprises affecting the excitability of neurons and/or inhibiting the activity of neurons.

A method according to any one of claims 90-96, wherein the biological activity includes the ability to reduce the frequency of excitatory postsynaptic currents (EPSCs), and/or the ability to reduce the amplitude of EPSCs.

The method according to any one of claims 90-97, wherein the reduction comprises administering the DIR and/or its functional fragment and/or a nucleic acid encoding the DIR and/or its functional fragment, thereby reducing the frequency of excitatory postsynaptic currents (EPSCs) in the subject and/or reducing the amplitude of EPSCs in the subject, compared to the biological activity of the original DIR and/or its functional fragment in the subject.

The method of any one of claims 90-98, wherein the biological activity comprises affecting cognitive abilities.

The method according to any one of claims 90-99, wherein the biological activity comprises participation in a signaling pathway associated with Aβ deposition, and/or participation in a signaling pathway associated with Tau tangle generation.

The method according to any one of claims 90-100, wherein the biological activity comprises induction of Aβ deposition and/or amyloid plaque formation by gelsolin.

The method according to claim 101, wherein the DIR and/or its functional fragment induces Aβ deposition and/or amyloid plaque formation by binding to gelsolin.

The method according to any one of claims 90-102, wherein the expression level of the DIR and/or its functional fragment is positively correlated with the expression level of Aβ.

The method according to any one of claims 90-103, wherein the reduction comprises administering the DIR and/or its functional fragment and/or the nucleic acid encoding the DIR and/or its functional fragment, thereby reducing the cognitive ability of the subject compared to the biological activity of the original DIR and/or its functional fragment in the subject.

The method according to any one of claims 90-104, wherein the DIR and/or its functional fragment is derived from a mammal.

The method according to any one of claims 90-105, wherein the DIR and/or its functional fragment is derived from a primate.

The method according to any one of claims 90-106, wherein the DIR comprises the amino acid sequence shown in SEQ ID NO.1.

The method according to any one of claims 90-107, wherein the functional fragment of DIR comprises an amino acid sequence encoded by a retained intron in DDIT4L.

The method according to any one of claims 90-108, wherein the functional fragment of DIR comprises the amino acid sequence shown in any one of SEQ ID NO.4-5.

The method according to any one of claims 90-109, wherein the cognitive impairment comprises cognitive impairment caused by normal aging, Lewis body dementia (LBD), frontotemporal dementia and/or vascular dementia.

The method according to any one of claims 90-110, wherein the inducing disease of cognitive impairment comprises Alzheimer's disease, multi-infarct type, Parkinson's disease, AIDS and/or Creutzfeldt-Jakob disease (CJD).

The method according to any one of claims 90-111, wherein the cognitive impairment comprises early cognitive impairment (MCI), mid-term cognitive impairment and late cognitive impairment.

The method according to any one of claims 90-112, wherein the cognitive impairment comprises amnestic MCI with impairment of multiple cognitive domains (aMCI-m).

The method according to any one of claims 90-113, wherein the neurodegenerative disease comprises an acute neurodegenerative disease and a chronic neurodegenerative disease.

The method according to any one of claims 90-114, 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 communication and/or a neurodegenerative disease caused by impaired cell motility.

The method of any one of claims 90-115, wherein the neurodegenerative disease comprises Alzheimer's disease, Parkinson's disease, multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS) and/or Huntington's disease (HD).

The method of any one of claims 90-116, wherein the neurodegenerative disease comprises Alzheimer's disease.

The method according to any one of claims 90-117, wherein the neurodegenerative disease comprises early Alzheimer's disease, middle Alzheimer's disease and/or late Alzheimer's disease.

The method of any one of claims 90-118, wherein the subject comprises a mammal.

The method according to any one of claims 90-119, wherein the subject comprises a patient with a neurodegenerative disease and/or a patient with a cognitive disorder.

The method according to any one of claims 90-120, wherein the subject comprises an Alzheimer's disease patient.

The method according to any one of claims 90-121, wherein the subject is elderly.

The method according to any one of claims 90-122, wherein the administration comprises oral administration and/or injection administration.

The method according to any one of claims 90-123, wherein the candidate drug comprises a small molecule compound, a polymer and/or a biomacromolecule.

An isolated antigen-binding protein having the following properties: specifically binding to human DIR and/or a functional fragment thereof at a working concentration of about 10 ng/ml or more in an ELISA assay.

The antigen binding protein according to claim 125, comprising LCDR2, wherein the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:74.

The antigen binding protein according to claim 126, wherein the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:26 or 16.

The antigen binding protein according to any one of claims 125-127, comprising LCDR1, wherein the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:73.

The antigen binding protein according to claim 128, wherein the LCDR1 comprises the amino acid sequence shown in any one of SEQ ID NO: 25, 52, and 15.

The antigen binding protein according to any one of claims 125-129, comprising LCDR3, wherein the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:75.

According to the antigen binding protein of claim 130, the LCDR3 comprises an amino acid sequence shown in any one of SEQ ID NOs: 27, 53, 67, 17.

The antigen binding protein according to any one of claims 125-131, comprising 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 antigen binding protein according to any one of claims 125-132, comprising HCDR1, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:70.

The antigen binding protein according to claim 133, wherein the HCDR1 comprises the amino acid sequence shown in any one of SEQ ID NO: 20, 35, 56, 10.

The antigen binding protein according to any one of claims 125 to 134, comprising HCDR2, wherein the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 71.

The antigen binding protein according to claim 135, wherein the HCDR2 comprises the amino acid sequence shown in any one of SEQ ID NO: 21, 36, 42, 48, 11.

The antigen binding protein according to any one of claims 125-136, comprising HCDR3, wherein the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:70.

The antigen binding protein of claim 137, wherein the HCDR3 comprises the amino acid sequence shown in any one of SEQ ID NO: 22, 30, 37, 43, 49, 57, 62, 12.

The antigen binding protein according to any one of claims 125-138, comprising 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 antigen binding protein according to any one of claims 125-139, comprising a heavy chain variable region VH, wherein 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.

The antigen binding protein according to any one of claims 125-140, comprising a light chain variable region VL, wherein the VL comprises an amino acid sequence shown in any one of SEQ ID NO: 18, 28, 33, 40, 46, 54, 60, 68.

The antigen binding protein according to any one of claims 125 to 141, comprising 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 antigen binding protein according to any one of claims 125-142, comprising a heavy chain constant region, and the heavy chain constant region comprises a constant region derived from IgG.

The antigen binding protein of claim 143, 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.

The antigen binding protein according to any one of claims 125 to 144, comprising a light chain constant region, wherein the light chain constant region comprises a constant region derived from Igκ or a constant region derived from Igλ.

The antigen binding protein of claim 145, wherein the light chain constant region comprises a constant region derived from human Igκ.

The antigen binding protein according to any one of claims 125-146, which comprises an antibody or an antigen binding fragment thereof.

The antigen binding protein of claim 147, 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.

The antigen binding protein according to any one of claims 147-148, wherein the antibody is selected from the group consisting of a monoclonal antibody, a murine antibody, and a chimeric antibody.

A polypeptide comprising the isolated antigen binding protein of any one of claims 125-149.

An immunoconjugate comprising the isolated antigen binding protein of any one of claims 125-149 or the polypeptide of claim 150.

An isolated nucleic acid molecule encoding the isolated antigen binding protein of any one of claims 125-149, or the polypeptide of claim 150.

A vector comprising the isolated nucleic acid molecule of claim 152.

A cell comprising the isolated antigen binding protein of any one of claims 125-149, the polypeptide of claim 150, the immunoconjugate of claim 151, the isolated nucleic acid molecule of claim 152 and/or the vector of claim 153.

A method for preparing the isolated antigen-binding protein of any one of claims 125-149 or the polypeptide of claim 150, the method comprising culturing the cell of claim 154 under conditions such that the isolated antigen-binding protein of any one of claims 125-149 or the polypeptide of claim 150 is expressed.

A pharmaceutical composition comprising the isolated antigen-binding protein of any one of claims 125-149, the polypeptide of claim 150, the immunoconjugate of claim 151, the isolated nucleic acid molecule of claim 152, the vector of claim 153, the cell of claim 154, and/or a pharmaceutically acceptable adjuvant and/or excipient.

Use of the isolated antigen-binding protein of any one of claims 125 to 149 and/or the polypeptide of claim 150 in the preparation of a medicament for preventing and/or treating a disease or condition, wherein the disease or condition comprises cognitive impairment and/or neurodegenerative disease.

The use according to claim 157, wherein the neurodegenerative disease comprises acute neurodegenerative disease and chronic neurodegenerative disease.

The use according to any one of claims 157-158, wherein the cognitive impairment comprises early cognitive impairment (MCI), mid-term cognitive impairment and late-term cognitive impairment.