Use of discoidin domain receptor 2 in the diagnosis of neurodegenerative diseases and related computer readable media

Abstract

The present invention discloses the use of a reagent for measuring the expression level of discoidin domain receptor 2 (DDR2) in the manufacture of a kit for diagnosing a neurodegenerative disease in a subject, wherein a higher level of DDR2 in a sample derived from the subject than that of a control not affected by the disease indicates that the subject is affected by the neurodegenerative disease. The present invention also discloses a kit, method, and computer-readable medium for diagnosing neurodegenerative diseases. The present invention efficiently and accurately diagnoses neurodegenerative diseases by measuring DDR2.

Description

[Technical Field]

[0001] The present invention relates to kits, methods and computer-readable media for diagnosing neurodegenerative diseases, and more particularly to diagnosing neurodegenerative diseases by detecting discoidin domain receptor 2 (DDR2). [Background technology]

[0002] Neurodegenerative diseases are diseases in which degenerative changes in neurons in the central nervous system cause various symptoms, such as motor and sensory dysfunction, which impairs higher-order functions such as memory, learning, and computational reasoning. Examples of neurodegenerative diseases include, but are not limited to, Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, frontotemporal dementia, spinocerebellar ataxia, brain trauma, and genetic mutations that secondarily affect the function of central nervous system cells. Neurodegenerative diseases have long been considered incurable and complex, and currently, there are no effective drugs that target systemic pathological processes and improve the overall condition of neurodegenerative diseases.

[0003] Since the progression of neurodegenerative diseases is generally irreversible, the key to treatment is early diagnosis and early intervention to slow the progression of the disease. If we can quickly diagnose the onset of the disease in patients, or detect potential risks in patients early, and provide appropriate treatment or prevention, we can help stop the patient's progression at the stage of mild damage and postpone deterioration, thereby ensuring the patient's quality of life and reducing the burden on society.

[0004] Currently, diagnostic methods for various neurodegenerative diseases are primarily multidisciplinary, including neuropsychological assessment and cognitive impairment testing, brain imaging (e.g., brain PET scans and nuclear magnetic resonance imaging), cerebrospinal fluid marker testing, blood testing, and genetic risk analysis. However, commonly used diagnostic methods require the injection of a certain dose of radioactive material into the subject, or are prone to surgical infection due to significant surgical trauma. Therefore, the development of new markers for the early diagnosis of neurodegenerative diseases is an important direction for future diagnosis and treatment.

[0005] Discoidin domain receptor 2 (DDR2) is a receptor tyrosine kinase (RTK) that utilizes extracellular matrix collagens as its ligands. In addition to its kinase function, DDR2 activates β1-integrins to promote cell adhesion. The unique function of DDR2 is to mediate signaling from the extracellular matrix to the cytoplasm, thereby balancing extracellular matrix regulation, and to participate in the regulation of cell growth, differentiation, and metabolism. Activation of DDR2 by extracellular matrix collagens is essential for normal development and tissue homeostasis, whereas aberrant activation of these receptors after injury or disease is detrimental.

[0006] DDR2 is considered an important target for inflammation (e.g., arthritis, including osteoarthritis and rheumatoid arthritis) and fibrosis (e.g., pulmonary fibrosis, liver cirrhosis, renal fibrosis, and skin fibrosis). DDR2 is primarily expressed in interstitial cells, such as fibroblasts, myofibroblasts, and smooth muscle cells, in the kidney, skin, lung, heart, and connective tissue. A growing body of evidence suggests that abnormal DDR2 expression is involved in the progression of various diseases, including inflammation, liver fibrosis, renal fibrosis, pulmonary fibrosis, skin scarring, and atherosclerosis. Studies using mouse inflammation models have revealed elevated DDR2 expression in the knee joints of aging mice. Studies using a rat model of rheumatoid arthritis have revealed elevated DDR2 expression in the synovial cells.

[0007] However, the expression and function of DDR2 have never been reported in neuronal cells (especially glial cells), and currently, there are no studies or reports on the use of DDR2 in the diagnosis of neurodegenerative diseases. Summary of the Invention

[0008] The inventors of the present application have unexpectedly found that DDR2 expression is significantly elevated in samples related to neurodegenerative diseases compared to normal controls, and have utilized a reagent capable of binding to DDR2 to measure the presence and / or level of DDR2 in a sample, thereby efficiently and accurately diagnosing neurodegenerative diseases.

[0009] In a first aspect, the present invention provides a kit for diagnosing a neurodegenerative disease in a subject, comprising reagents for measuring the expression level of discoidin domain receptor 2 (DDR2), wherein a level of DDR2 in a sample derived from the subject that is higher than the level in a control that is not affected by the disease indicates that the subject is affected by the neurodegenerative disease.

[0010] In a second aspect, the present invention provides the use of a reagent for measuring the expression level of discoidin domain receptor 2 (DDR2) in the manufacture of a kit for diagnosing a neurodegenerative disease in a subject, wherein a level of DDR2 in a sample derived from the subject that is higher than the level in a control that does not suffer from the disease indicates that the subject is suffering from the neurodegenerative disease.

[0011] In a third aspect, the present invention provides a reagent for measuring the expression level of discoidin domain receptor 2 (DDR2) to diagnose a neurodegenerative disease in a subject, wherein a higher level of DDR2 in a sample derived from the subject indicates that the subject is suffering from a neurodegenerative disease when the level is higher than that of a control not suffering from the disease.

[0012] In a fourth aspect, the present invention provides methods for diagnosing a neurodegenerative disease in a subject, comprising measuring the presence and / or level of discoidin domain receptor 2 (DDR2) in a sample from the subject. In some embodiments, the method comprises contacting the sample from the subject with a reagent capable of binding to DDR2, detecting the presence of a complex formed by the reagent with DDR2 in the sample after contacting, and determining that the subject has or is at risk of having a neurodegenerative disease based on the presence and / or level of the complex.

[0013] In a fifth aspect, the present invention provides a computer-readable storage medium having stored thereon computer instructions that can be read and executed by a computer to perform a method of diagnosing whether a subject is affected by a neurodegenerative disease, the method comprising the steps of: (a) contacting a sample from the subject with a reagent capable of binding to discoidin domain receptor 2 (DDR2); (b) detecting and reading a signal from the sample after contacting to determine whether the reagent has formed a complex with DDR2 in the sample; and (c) determining whether the signal exceeds a predetermined threshold, and if the signal exceeds the predetermined threshold, determining that the subject is affected by the neurodegenerative disease, wherein the threshold is a median level from subjects not affected by the disease.

[0014] In a sixth aspect, the present invention provides a method of treating a neurodegenerative disease in a subject, the method comprising: (a) contacting a sample from the subject with a reagent capable of binding to discoidin domain receptor 2 (DDR2); (b) detecting and reading a signal from the sample after contacting to determine whether the reagent has formed a complex with DDR2 in the sample; (c) determining whether the signal exceeds a predetermined threshold, and if the signal exceeds the predetermined threshold, determining that the subject is afflicted with a neurodegenerative disease, wherein the threshold is a median level from subjects not afflicted with the disease; and (d) administering a neuroprotective or neurorestorative therapy to the subject determined to be afflicted with the neurodegenerative disease.

[0015] In any of the above aspects, the reagent for measuring the expression level of DDR2 comprises a reagent capable of binding to DDR2 to measure the level of DDR2 in the sample. In some embodiments, the reagent capable of binding to DDR2 comprises a protein, a nucleic acid, or a small molecule compound. In some embodiments, the reagent capable of binding to DDR2 is an anti-DDR2 monoclonal antibody or antigen-binding fragment thereof, or an anti-DDR2 polyclonal antibody. In some embodiments, the anti-DDR2 monoclonal antibody is an anti-DDR2 nanobody.

[0016] In some embodiments, the agent capable of binding to DDR2 is labeled with a detectable marker, hi some embodiments, the detectable marker is selected from a fluorescent label, a chemiluminescent label, a paramagnetic label, a radioisotope label, and an enzyme label.

[0017] In any of the above embodiments, the sample is brain tissue or cerebrospinal fluid. In any of the above embodiments, the neurodegenerative disease is a neurodegenerative disease mediated by astrocyte damage. In any of the above embodiments, the neurodegenerative disease is a neurodegenerative disease mediated by astrocytes with elevated DDR2 expression, and the elevated DDR2 expression is compared to the DDR2 expression level in astrocytes of a normal subject. Preferably, in any of the above-mentioned embodiments, the neurodegenerative disease is Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), Parkinson's disease, Huntington's disease, frontotemporal dementia, spinal muscular atrophy, prion disease, spinocerebellar axia, Friedreich's ataxia, primary lateral sclerosis, spinocerebellar atrophy, Machado-Joseph's disease, Lewy body dementia, progressive bulbar palsy, progressive supranuclear palsy, or the like. In a more preferred embodiment, the neurodegenerative disease is selected from the group consisting of supranuclear palsy and multiple system atrophy. In a more preferred embodiment, the neurodegenerative disease is Alzheimer's disease. In a more preferred embodiment, the neurodegenerative disease is amyotrophic lateral sclerosis.

[0018] In a seventh aspect, the present invention provides a kit for diagnosing a neurodegenerative disease in a subject, comprising exosomes derived from a subject and a reagent capable of binding to discoidin domain receptor 2 (DDR2) so as to measure the level of DDR2 expressed by said exosomes, wherein if the level of DDR2 expressed by said exosomes is higher than that of a control not affected by the disease, it indicates that said subject has the neurodegenerative disease.

[0019] In an eighth aspect, the present invention provides the use of exosomes derived from a subject in the manufacture of a kit for diagnosing a neurodegenerative disease in a subject, wherein a level of discoidin domain receptor 2 (DDR2) expressed by said exosomes higher than that of a control not affected by the disease indicates that said subject is affected with the neurodegenerative disease.

[0020] In a ninth aspect, the present invention provides exosomes derived from a subject for diagnosing a neurodegenerative disease in said subject, wherein a level of discoidin domain receptor 2 (DDR2) expressed by said exosomes is higher than that of a control not affected by the disease, indicating that said subject is affected by the neurodegenerative disease.

[0021] In a tenth aspect, the present invention provides a method for diagnosing a neurodegenerative disease in a subject, comprising isolating exosomes from the subject and measuring the presence and / or level of discoidin domain receptor 2 (DDR2) in exosomes derived from the subject. In some embodiments, the method comprises contacting exosomes derived from the subject with a reagent capable of binding to DDR2, detecting the presence of a complex formed by the reagent with DDR2 in the exosomes after contacting, and determining that the subject is affected by or at risk of being affected by the neurodegenerative disease based on the presence and / or level of the complex.

[0022] In an eleventh aspect, the present invention provides a computer-readable storage medium having stored thereon computer instructions that are readable and executed by a computer, the computer instructions being executed to perform a method for diagnosing whether a subject is affected by a neurodegenerative disease, the method comprising the steps of: (a) isolating exosomes from a subject; (b) contacting the isolated exosomes with a reagent capable of binding to discoidin domain receptor 2 (DDR2); (c) detecting and reading a signal from the exosomes after contact to determine whether the reagent has formed a complex with DDR2 in the exosomes; and (d) determining whether the signal exceeds a predetermined threshold, and determining that the subject is affected by the neurodegenerative disease if the signal exceeds the predetermined threshold, wherein the threshold is a median level derived from subjects not affected by the disease.

[0023] In a twelfth aspect, the present invention provides a method for treating a neurodegenerative disease in a subject, comprising the steps of: (a) isolating exosomes from a subject; (b) contacting the isolated exosomes with a reagent capable of binding to discoidin domain receptor 2 (DDR2); (c) detecting and reading a signal from the exosomes after contact, thereby determining whether the reagent has formed a complex with DDR2 in the exosomes; (d) determining whether the signal exceeds a predetermined threshold, and determining that the subject is affected with a neurodegenerative disease if the signal exceeds the predetermined threshold, wherein the threshold is a median level derived from subjects not affected with the disease; and (e) administering a neuroprotective or neurorestorative therapy to the subject determined to be affected with the neurodegenerative disease. In a preferred embodiment, the neuroprotective or neurorestorative therapy is, for example, a cholinesterase inhibitor (e.g., galantamine, donepezil, huperzine A, rivastigmine), an NMDA receptor antagonist (e.g., memantine), an inflammatory factor inhibitor (e.g., a nonsteroidal anti-inflammatory agent), a glutamate inhibitor (e.g., riluzole), or a free radical scavenger (e.g., edaravone).

[0024] In any of the above aspects, the exosomes are derived from a bodily fluid of the subject. In some embodiments, the bodily fluid comprises one or more of peripheral blood, serum, plasma, semen, sputum, synovial fluid, aqueous humor, amniotic fluid, milk, semen, prostatic fluid, Cowper's fluid, ejaculation fluid, sweat, feces, tears, cyst fluid, pleural fluid, ascites, pericardial fluid, chyle, bile, interstitial fluid, menstrual blood, pus, vomit, vaginal secretions, mucosal secretions, pancreatic juice, blastocyst cavity fluid, umbilical cord blood, urine, cerebrospinal fluid, saliva, lymph, mucus in stool, endobronchial aspirate, bronchoalveolar lavage fluid, and nasal wash. In some embodiments, the bodily fluid is serum or plasma. In some embodiments, the exosomes are isolated from the sample derived from the subject by size exclusion chromatography, density gradient centrifugation, differential centrifugation, nanomembrane ultrafiltration, immunoadsorption capture, affinity chromatography capture, microfluidic separation, or a combination thereof.

[0025] In any of the above aspects, the reagent capable of binding to DDR2 comprises a protein, a nucleic acid, or a small molecule compound. In some embodiments, the reagent capable of binding to DDR2 is an anti-DDR2 monoclonal antibody or antigen-binding fragment thereof, or an anti-DDR2 polyclonal antibody. In some embodiments, the reagent capable of binding to DDR2 is labeled with a detectable marker. In some embodiments, the detectable marker is selected from a fluorescent label, a chemiluminescent label, a paramagnetic label, a radioisotope label, and an enzyme label.

[0026] Other aspects and advantages of the present invention are described in detail in specific embodiments, and those skilled in the art will be able to discern other aspects and advantages not expressly set forth in this disclosure from the following specific description. [Brief explanation of the drawings]

[0027] [Figure 1] FIG. 1 shows the affinity constants of DDR2 nanobodies for the extracellular segment of the DDR2 antigen. [Figure 2]Figure 2 shows the imaging results of an ALS mouse model. [Figure 3] Figure 3 shows the imaging results of an AD mouse model. [Figure 4] FIG. 4 shows the results of Western blot analysis of exosomes in the plasma of ALS patients. [Figure 5] FIG. 5 shows the results of flow cytometry measurement of exosomes in the plasma of ALS patients. [Figure 6] Figure 6 shows the DDR2 expression status in ALS-related experimental data. [Figure 7] Figure 7 shows the DDR2 expression status in AD-related experimental data. [Figure 8] Figure 8 shows the DDR2 expression status in AD-related experimental data. [Figure 9] Figure 9 shows the correlation analysis of DDR2 with other diagnostic genes. [Figure 10] Figure 10 shows in vivo fluorescence imaging of the DDR2 nanobody 1A12-ICG. [Figure 11] FIG. 11 shows PET / CT imaging of AD mice. [Figure 12] FIG. 12 shows PET / CT imaging of ALS mice. DETAILED DESCRIPTION OF THE INVENTION

[0028] (definition) Throughout the specification and claims, unless the context dictates otherwise, the terms "comprise" and variations such as "comprise" and "contain" shall be understood to include said integers, steps, or components, but not to exclude any other integers, steps, or components. As used herein, the term "comprise" can be replaced with the term "contain" or may be replaced herein with the term "having."

[0029] As used herein, "about" refers to a numerical value that is within an acceptable error range of a specific value as determined by one of ordinary skill in the art, and the numerical value will depend to some extent on how it is measured or determined (i.e., the limitations of the measurement system). For example, in each practice in the art, "about" can mean within or more than one standard deviation. Alternatively, "about" or "substantially comprising" can mean a range of up to 20%. For biological systems or processes, the term can also mean up to an order of magnitude or up to five times the numerical value. Unless otherwise specified, when specific values ​​appear in the specification and claims, "about" or "substantially comprising" should be interpreted as meaning within an acceptable error range of the specific value.

[0030] As used herein, the terms "subject," "patient," or "individual" refer to any subject for whom diagnosis, prognosis, or treatment is desired, particularly a mammalian subject. Mammals include humans, livestock, farm animals, zoo animals, sport animals, or pets, such as dogs, cats, pigs, rabbits, rats, mice, horses, cattle, and dairy cows. Preferably, a subject as referred to herein is a human. In some embodiments, the subject is suffering from or susceptible to one or more diseases or conditions. The patient may exhibit one or more symptoms of a disease or condition, or may already be diagnosed with one or more diseases or conditions. In some embodiments, the patient is undergoing or has undergone a particular therapy to diagnose and / or treat such disease, disease, or condition.

[0031] As used herein, the term "detection" includes any means of detection, including direct and indirect detection, quantitative and qualitative detection, and refers to identifying the presence and / or level of a particular molecule (e.g., DDR2 protein) in a subject or a sample derived from a subject.

[0032] As used herein, the term "diagnosis" refers to the identification or classification of a molecular or pathological condition, disease, or condition. For example, "diagnosis" can refer to the identification of a neurodegenerative disease or the identification of a specific type thereof.

[0033] According to the present invention, the term "binding" preferably refers to specific binding. "Specific binding" refers to the stronger binding of a reagent to a specific target compared to binding to another target. The dissociation constant (K D It binds more strongly to a first target than to a second target if its dissociation constant (K) is less than its dissociation constant for the second target. Preferably, the dissociation constant (K) of the target to which the reagent does not specifically bind is less than the dissociation constant (K) of the target to which the reagent does not specifically bind. D ) compared to the dissociation constant (K D ) is 10 2 double, 10 3 double, 10 4 double, 10 5 double, 10 6 double, 10 7 double, 10 8 double, 10 9 double or 10 10 More than twice as low.

[0034] Preferably, a reagent (e.g., a protein or polypeptide) is specific for a given target if it can bind to said given target but cannot bind to other targets, i.e., if it has no significant affinity for and does not significantly bind to other targets by standard measurements. According to the present invention, a reagent is specific for DDR2 if it can bind to DDR2 but (substantially) cannot bind to other targets. Preferably, the K of a reagent binding to a given target is D binds to its nonspecific target, K D At least 10 of 2 times at least 10 3 times at least 10 4 times at least 10 5 times at least 10 6 times at least 10 7 times at least 10 8times at least 10 9 times or at least 10 10 A reagent is specific for said target if its activity is 2-fold lower.

[0035] Binding of a reagent to a target can be determined experimentally by any suitable method, which is within the ability of one skilled in the art. Affinity can be readily determined using conventional techniques, such as equilibrium dialysis, surface plasmon resonance analysis according to the manufacturer's general procedure outline, radioimmunoassay using radiolabeled target antigen, or other methods known to those skilled in the art. Affinity data can be analyzed, for example, by methods known in the art. The measured affinity for a particular interaction may change when measured under different conditions (e.g., salt concentration, pH). Therefore, affinity and other binding parameters (e.g., K D ,I C 50 ) is preferably measured using standardized solutions of binding agent, target and standardized buffer.

[0036] As used herein, the term "antibody" refers to any form of antibody that exhibits a desired biological activity (e.g., through inhibiting binding of a ligand to its receptor or inhibiting ligand-induced receptor signaling). "Antibody fragment" and "antigen-binding fragment" generally refer to antigen-binding fragments of antibodies and antibody analogs that contain at least a portion of the antigen-binding or variable region (e.g., one or more CDRs) of the parent antibody. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a polyclonal antibody.

[0037] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, each antibody in the population being identical except for possible natural variants that may be present in minor amounts. Monoclonal antibodies have high specificity, being able to target a single antigenic site. Furthermore, unlike conventional (polyclonal) antibody preparations that typically include several different antibodies directed against several different determinants (epitopes), each monoclonal antibody targets only a single determinant on the antigen. The modifier "monoclonal" refers to the character of the antibody as being obtained from a substantially homogeneous population of antibodies and should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies for use in the present invention can be produced by hybridoma or recombinant DNA technology.

[0038] Monoclonal antibodies may include "chimeric," humanized, or fully human antibodies. In some embodiments, an antibody is part of a larger biological molecule, such as a fusion protein or antibody-drug conjugate. Antibody fragments retain at least some of the binding specificity of the parent antibody. Generally, antibody fragments retain at least 10% of the binding activity of the parent, when activity is expressed in molar terms. Preferably, antibody fragments retain at least 20%, at least 50%, at least 70%, at least 80%, at least 90%, at least 95%, at least 100% or more of the binding affinity of the parent antibody for the target.

[0039] As used herein, the term "heavy chain antibody" refers to an antibody that lacks a light chain and consists of only a heavy chain, including two constant regions (CH2 and CH3), a hinge region, and a heavy chain variable region (VHH). Examples include, but are not limited to, naturally occurring heavy chain antibodies, antibodies naturally lacking a light chain, heavy chain antibodies derived from conventional four-chain antibodies, and artificial antibodies. Heavy chain antibodies may be derived from species of the Camelidae family, such as antibodies produced in camels, llamas, dromedaries, alpacas, and draft horses. Species other than Camelidae can also produce heavy chain antibodies naturally lacking a light chain, and such heavy chain antibodies are within the scope of the present invention.

[0040] As used herein, the term "nanobody" refers to a single-domain antibody consisting only of the heavy chain variable region, obtained by cloning the variable region of a heavy chain antibody. Also known as a VHH (variable domain of heavy chain antibody) or single-domain antibody, it is the smallest functional antigen-binding fragment. Nanobodies recognize antigens with high specificity and affinity similar to IgG antibodies, but their small size (approximately 15 kDa) allows them to penetrate tumor tissue more easily. Nanobodies are also resistant to harsh pH, ​​heat denaturation, protein hydrolysis, solvents, and detergents. They can be expressed and produced with high yield and solubility.

[0041] The terms "antibody fragment" and "antigen-binding fragment" generally refer to antigen-binding fragments of antibodies and antibody analogs that contain at least a portion of the antigen-binding or variable region (e.g., one or more CDRs) of the parent antibody. Antibody fragments retain at least some of the binding specificity of the parent antibody. Generally, antibody fragments retain at least 10% of the binding activity of the parent, when activity is expressed in molar terms. Preferably, antibody fragments retain at least 20%, at least 50%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% or more of the binding affinity of the parent antibody for the target. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, diabodies, linear antibodies, single-chain antibody molecules (e.g., scFv (single-chain variable fragment)), nanobodies, domain antibodies, and multispecific antibodies formed by antibody fragments. An antibody against DDR2 refers to an antibody that specifically binds to DDR2, and includes artificially designed antibodies and any form of antibody (for example, the antibody fragments and antigen-binding fragments defined above).

[0042] An "equivalent variant" of an antibody or polypeptide refers to an antibody or polypeptide having a degree of homology or sequence identity to the amino acid sequence of the antibody or polypeptide. In some embodiments, the sequence identity is 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 98%, or at least about 99%. In some embodiments, the equivalent variant has one, two, three, four, or five additions, deletions, substitutions, and combinations thereof, compared to the reference antibody or polypeptide. In some embodiments, an equivalent variant of an antibody or polypeptide retains the activity (e.g., epitope binding) or structure (e.g., salt bridges) of the reference sequence.

[0043] As used herein, the term "variant" of a sequence refers to a sequence that differs from the subject sequence in one or more amino acid residues, but retains the biological activity of the molecule of interest.

[0044] As used herein, the term "% identity" between two sequences refers to a function of the number of identical positions the sequences have (i.e., % homology = # of identical positions / total # of positions x 100), taking into account the number of gaps and the length of each gap, which are introduced during optimal alignment of the two sequences. The comparison of sequences and determination of % identity between two sequences can be accomplished using a mathematical algorithm.

[0045] As used herein, the term "nucleic acid" is intended to include deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), e.g., genomic DNA, cDNA, mRNA, recombinantly produced, and chemically synthesized molecules. Nucleic acids may be single-stranded or double-stranded. RNA includes in vitro transcribed or synthesized RNA.

[0046] The terms "control," "control sample," "standard control," or "standard" refer to a sample that serves as a reference (generally a known reference) against which a test sample is compared. For example, a test sample can be obtained from a patient suspected of having a given disease and compared to a sample from a patient with a known disease or a known normal (non-disease) individual. A control can represent an average value obtained from a population of similar individuals (e.g., patients with a disease or healthy individuals with a similar medical history, the same age, weight, etc.). A control value can come from a sample obtained previously from the same individual, for example, before the disease or before treatment. It will be apparent to those skilled in the art that controls can be designed to evaluate many parameters.

[0047] The term "body fluid" or "body fluid sample" as used herein generally refers to a flowing liquid, which generally resides in and / or can be produced from the body or tissues of a subject or patient. For example, a body fluid may include one or more of peripheral blood, serum, plasma, semen, sputum, synovial fluid, aqueous humor, amniotic fluid, milk, semen, prostatic fluid, Cowper's fluid, ejaculation fluid, sweat, feces, tears, cyst fluid, pleural fluid, ascites, pericardial fluid, chyle, bile, interstitial fluid, menstrual blood, pus, vomit, vaginal secretions, mucosal secretions, pancreatic juice, blastocyst fluid, umbilical cord blood, urine, cerebrospinal fluid, saliva, lymph, mucus, stool, endobronchial aspirate, bronchoalveolar lavage fluid, and nasal wash, including components or fractions thereof. Body fluid samples may be mixed or combined. The bodily fluid sample can be provided by removing the bodily fluid from the patient, but can also be provided using pre-separated bodily fluid sample material, hi some embodiments, the bodily fluid or bodily fluid sample used in the present invention is a serum or plasma sample.

[0048] As used herein, the term "exosome" refers to a vesicle approximately 30-150 nm in diameter that is secreted by various cells and contains specific proteins (e.g., exosome membranes are rich in the transmembrane protein family CD63, CD81, and CD9, which are involved in exosome trafficking), lipids, cytokines, or genetic material. Various cells can secrete exosomes under normal and pathological conditions. They are widely present in bodily fluids such as blood, saliva, urine, cerebrospinal fluid, and milk, and are considered to be specifically secreted vesicles involved in intercellular communication. In some embodiments, exosomes can be isolated and obtained from a bodily fluid sample derived from a subject by size exclusion chromatography, density gradient centrifugation, differential centrifugation, nanomembrane ultrafiltration, immunoadsorption capture, affinity chromatography capture, microfluidic separation, or a combination thereof.

[0049] (Reagent for measuring DDR2 expression levels) In one aspect, the present invention provides reagents for measuring the expression level of discoidin domain receptor 2 (DDR2) for diagnosing a neurodegenerative disease in a subject.

[0050] As used herein, the term "reagent for measuring the expression level of DDR2" refers to any reagent known in the art that can be used to measure DDR2, including, for example, targeting or affinity reagents for DDR2, and particularly reagents that can form a chemically, physically, or biologically detectable complex by binding (particularly, specifically binding) to DDR2.

[0051] In some embodiments, the reagent capable of binding to DDR2 comprises a protein, a nucleic acid, or a small molecule compound, and can target one or more epitopes of the DDR2 protein.

[0052] In some embodiments, the reagent capable of binding to DDR2 is an anti-DDR2 monoclonal antibody or antigen-binding fragment thereof, or an anti-DDR2 polyclonal antibody. Anti-DDR2 antibodies are available from commercial sources, such as GTX102526 (GeneTex), AF2538 (Novus Biologicals), and MAB2538 (R&D Systems). For more DDR2 antibodies, see https: / / www.antibodypedia.com / gene / 4177 / DDR2 (last accessed September 1, 2022). Alternatively, anti-DDR2 antibodies can be produced de novo using methods known in the art. In some embodiments, the anti-DDR2 antibody is any form of antibody or antibody fragment as defined herein.

[0053] In some embodiments, the anti-DDR2 monoclonal antibody is an anti-DDR2 nanobody. In some embodiments, the anti-DDR2 nanobody comprises CDR1, CDR2, and CDR3, where CDR1 comprises or is the sequence set forth in SEQ ID NO: 1 or an equivalent variant thereof, CDR2 comprises or is the sequence set forth in SEQ ID NO: 2 or an equivalent variant thereof, and CDR3 comprises or is the sequence set forth in SEQ ID NO: 3 or an equivalent variant thereof, and the CDRs are defined according to IMGT. In some embodiments, the nanobody comprises or is the sequence set forth in SEQ ID NO: 4 or an equivalent variant thereof.

[0054] In some embodiments, the equivalent variants of CDR1, CDR2, and CDR3 refer to those that have a single amino acid substitution, deletion, or insertion compared to the reference sequence.

[0055] In some embodiments, equivalent variants of the Nanobodies have at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 4 and have the same or equivalent CDR1, CDR2, and CDR3. In some embodiments, the CDR1, CDR2, and CDR3 are defined according to any one of the following definition systems: IMGT, Kabat, Chothia, Contact, or AbM. In some embodiments, the CDR1, CDR2, and CDR3 are defined according to the IMGT definition system.

[0056] In some embodiments, the anti-DDR2 nanobody comprises CDR1, CDR2, and CDR3, wherein CDR1 comprises the sequence set forth in SEQ ID NO: 1, CDR2 comprises the sequence set forth in SEQ ID NO: 2, and CDR3 comprises the sequence set forth in SEQ ID NO: 3, and the CDRs are defined according to IMGT.

[0057] In some embodiments, the anti-DDR2 nanobody comprises CDR1, CDR2, and CDR3, wherein CDR1 is the sequence set forth in SEQ ID NO: 1, CDR2 is the sequence set forth in SEQ ID NO: 2, and CDR3 is the sequence set forth in SEQ ID NO: 3, and the CDRs are defined according to IMGT.

[0058] In some embodiments, the substitutions described herein are conservative substitutions. A "conservative (amino acid) substitution" refers to a substitution in which an amino acid residue is replaced with an amino acid having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art and include 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), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, it is preferred that a non-essential amino acid residue in an immunoglobulin polypeptide be replaced with another amino acid residue from the same side chain family. In another embodiment, the amino acid string can be replaced with a string of structurally similar side chain family members that differ in order and / or composition.

[0059] In some embodiments, the reagent capable of binding to DDR2 is a peptide or nucleic acid aptamer. Such aptamers can be selected from oligonucleotide or peptide libraries by any method known in the art. Nucleic acid aptamers can be selected through SELEX (Systematic Evolution of Ligands by Exponential Enrichment). Peptide aptamers can be selected using yeast or bacterial two-hybrid systems.

[0060] In some embodiments, the agent capable of binding to DDR2 is labeled with a detectable marker, hi some embodiments, the detectable marker is selected from a fluorescent label, a chemiluminescent label, a paramagnetic label, a radioisotope label, and an enzyme label.

[0061] The choice of label depends on the detection method. For example, fluorescent labels (e.g., indocyanine green (ICG), rare earth chelates (e.g., europium chelates)), fluorescein-type labels (e.g., fluorescein, fluorescein isothiocyanate, 5-carboxyfluorescein, 6-carboxyfluorescein, dichlorotriazinylamine fluorescein), rhodamine-type labels (e.g., ALEXA568 (Invitrogen) or dansyl chloride), VIVOTAG 680 XLFLUOROCHROMETM (Perkin Elmer), phycoerythrin, 7-hydroxycoumarin, Lissamine, cyanine, phycoerythrin, Texas Red, BODIPY (Invitrogen), or analogs thereof are suitable for optical detection.

[0062] Chemiluminescent labels (e.g., luminol, luciferase, luciferin, aequorin) can also be employed. Such diagnosis and detection can be achieved by linking a reagent capable of binding to DDR2 to a detectable substance, including, but not limited to, various enzymes, including, but not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase, or by linking it to prosthetic group complexes, such as, but not limited to, streptavidin-biotin and avidin-biotin.

[0063] Paramagnetic and radioisotope labels may also be employed, preferably detected by Positron Emission Tomography (PET) or Single-Photon Emission Computed Tomography (SPECT). Radiolabels include bismuth ( 213 Bi), carbon ( 11 C. 13 C. 14 C), chromium ( 51 Cr), Cobalt ( 57 Co, 60 Co), copper( 64 Cu), Dysprosium ( 165 Dy), Erbium ( 169 Er), fluorine ( 18 F), gadolinium ( 153 Gd, 159 Gd), gallium ( 68 Ga, 67 Ga), germanium ( 68 Ge), gold ( 198 Au), holmium ( 166 Ho), hydrogen ( 3 H), indium ( 111 In, 112 In, 113 In, 115 In), iodine ( 121 I, 123 I, 125 I, 131 I), iridium ( 192 Ir), iron ( 59 Fe), krypton ( 81m Kr), Lanthanum ( 140 La), lutetium ( 177 Lu), manganese ( 54 Mn), molybdenum ( 99 Mo), nitrogen ( 13 N, 15 N), oxygen ( 15 O), palladium ( 103 Pd), phosphorus ( 32 P), potassium ( 42 K), praseodymium ( 142 Pr), promethium ( 149 Pm), rhenium (186 Re, 188 Re), rhodium ( 105 Rh), rubidium ( 81 Rb, 82 Rb), ruthenium ( 82 Ru, 97 Ru), samarium ( 153 Sm), Scandium ( 47 Sc), Selenium ( 75 Se), sodium ( 24 Na), strontium ( 85 Sr, 89 Sr, 92 Sr), Sulfur ( 35 S), technetium ( 99 Tc), thallium ( 201 Tl), tin ( 113 Sn, 117 Sn), xenon ( 133 Xe), Ytterbium ( 169 Yb, 175 Yb, 177 Yb), yttrium ( 90 Y), zinc ( 65 Zn), various positron-emitting metals for positron emission tomography and non-radioactive paramagnetic metal ions, such as paramagnetic aluminum (Al) ions, barium (Ba) ions, calcium (Ca) ions, cerium (Ce) ions, dysprosium (Dy) ions, erbium (Er) ions, europium (Eu) ions, gadolinium (Gd) ions, holmium (Ho) ions, iridium (Ir) ions, lithium (Li) ions, magnesium (Mg) ions, manganese (Man) ions, and the like. Gunn (Mn) ion, molybdenum (M) ion, neodymium (Nd) ion, osmium (Os) ion, oxygen (O) ion, palladium (Pd) ion, platinum (Pt) ion, rhodium (Rh) ion, ruthenium (Ru) ion, samarium (Sm) ion, sodium (Na) ion, strontium (Sr) ion, terbium (Tb) ion, thulium (Tm) ion, tin (Sn) ion, titanium (Ti) ion, tungsten (W) ion, zirconium (Zi) ion, especially Co +2 , C.R. +2 , Cr +3 , Cu+2 , Fe +2 , Fe +3 , Ga +3 , Mn +3 , Ni +2 , Ti +3 , V + , V +4 Methods for preparing radiolabeled amino acids and related peptide derivatives are known in the art. For example, radioisotopes can be conjugated by the chloramine-T method.

[0064] In some embodiments, the present invention provides a reagent for measuring the expression level of DDR2 for diagnosing a neurodegenerative disease in a subject, the reagent comprising an anti-DDR2 nanobody and a detectable label linked to the nanobody, wherein the detectable label is a fluorescent label. In some embodiments, the fluorescent label is indocyanine green (ICG).

[0065] In some embodiments, the present invention provides a reagent for measuring the expression level of DDR2 for diagnosing a neurodegenerative disease in a subject, the reagent comprising an anti-DDR2 Nanobody and a detectable label linked to said Nanobody, wherein said detectable label is a radioisotope. 68 Ga or 64 It is Cu.

[0066] (Disease to be diagnosed) The inventors have found that neurodegenerative diseases can be diagnosed by detecting discoidin domain receptor 2 (DDR2).

[0067] As used herein, the term "neurodegenerative disease" refers to any disease associated with degenerative changes in neurons in the central nervous system, including, but not limited to, Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease, Huntington's disease, frontotemporal dementia, spinal muscular atrophy, prion disease, spinocerebellar ataxia, Friedreich's ataxia, primary lateral sclerosis, spinocerebellar degeneration, Machado-Joseph disease, dementia with Lewy bodies, progressive bulbar palsy, progressive supranuclear palsy, and multiple system atrophy. In some embodiments, the neurodegenerative disease is associated with abnormal activation of astrocytes. In some embodiments, the neurodegenerative disease is Alzheimer's disease or amyotrophic lateral sclerosis.

[0068] (Alzheimer's disease (AD)) AD is an age-related progressive neurodegenerative disease. The core symptom of AD is memory impairment accompanied by damage to other cognitive domains, affecting normal daily life and work. Because AD is difficult to detect, progresses irreversibly, and has a high disability rate, early diagnosis and treatment are crucial for improving the survival rate and, in particular, the quality of life of AD patients.

[0069] Currently, amyloid β-protein 42 (Aβ) is a classic biomarker for detecting early AD. 42 ), total tau protein (T-tau), phosphorylated tau protein (P-tau), and the newly discovered neurogranin and microRNAs. Generally, T-tau and P-tau are significantly increased in the cerebrospinal fluid (CSF) of AD patients, and Aβ 42 The expression of miR-100, miR-1274a, and miR-146a in the CSF of AD patients is significantly different from that of other AD-related proteins. Since conventional biomarkers cannot meet clinical needs, there is an urgent need to find biomarkers for early AD.

[0070] Although there is no cure for AD, some FDA-approved drugs, such as cholinesterase inhibitors (which increase the amount of the neurotransmitter acetylcholine in the brain and promote intercellular signaling) and NMDA receptor antagonists (which alter brain cell signaling), can slow the progression of the disease. However, the effectiveness of these drugs remains poor, and the clinical needs of patients remain unmet, especially for those with moderate to severe AD.

[0071] More and more studies have shown that astrocytes affect the pathological changes of Aβ and tau proteins. Gene library studies have revealed that there are more than 40 gene sites associated with late-onset AD, most of which are expressed in astrocytes. Furthermore, astrocytes play a major role in the neuroinflammatory and neurodegenerative processes of AD. Therefore, astrocytes may be involved in the pathological development and progression of AD.

[0072] (Amyotrophic lateral sclerosis (ALS)) ALS is the most common type of motor neuron disease, affecting both upper and lower motor neurons. Pathologically, ALS patients exhibit a reduction in the number of motor neurons in the anterior horn of the spinal cord, brainstem, and motor cortex. The loss of motor neurons leads to muscle denervation and atrophy. Patients experience a gradual loss of motor function, beginning with muscle weakness and difficulty swallowing, and ultimately resulting in respiratory failure and death. Most ALS cases are sporadic, with 5-10% being familial. SOD1 was the first ALS-related gene identified, and mutations in this gene account for 20% of familial cases. To date, more than 20 genes have been identified that, when mutated, cause familial ALS, and gene mutations have also been found in sporadic cases. While genetic diversity exists, ALS is commonly characterized by protein deposits in the affected areas.

[0073] Although there is currently no cure for ALS, the drug riluzole (a glutamate release inhibitor) has shown a modest effect, extending survival by several months. Despite decades of research, there is still no consensus regarding the mechanism of ALS pathogenesis, which has hindered the development of effective therapies. Another approach is to improve motor neuron survival through activation of endogenous neurotrophic factor pathways, rather than specifically blocking the processes that cause motor neuron death.

[0074] In healthy individuals, astrocytes help protect and nourish surrounding motor neurons. However, recent findings in ALS patients suggest that changes in astrocytes may contribute to the disease. Researchers have found that in ALS, astrocytes lose important protective functions, specifically the ability to absorb glutamate. This leads to glutamate accumulation, damaging motor neurons. Another study found that astrocytes with different ALS gene mutations have distinct underlying molecular patterns, indicating mutation-dependent changes in astrocytes during ALS development.

[0075] (kit) In one aspect, the present invention provides a kit for diagnosing a neurodegenerative disease in a subject, comprising reagents for measuring the expression level of discoidin domain receptor 2 (DDR2), wherein a level of DDR2 in a sample derived from the subject that is higher than the level in a control that does not suffer from the disease indicates that the subject is suffering from the neurodegenerative disease.

[0076] In another aspect, the present invention provides a kit for diagnosing a neurodegenerative disease in a subject, comprising exosomes derived from a subject and a reagent capable of binding to discoidin domain receptor 2 (DDR2) such that the level of DDR2 expressed by the exosomes is measured, wherein a level of DDR2 expressed by the exosomes higher than that of a disease-free control indicates that the subject has the neurodegenerative disease.

[0077] In some embodiments, the reagent for measuring the expression level of DDR2 comprises a reagent capable of binding (particularly, specifically binding) to DDR2 to form a chemically, physically, or biologically detectable complex, such as a protein, nucleic acid, or small molecule compound, particularly an anti-DDR2 monoclonal antibody or an antigen-binding fragment thereof, or an anti-DDR2 polyclonal antibody.

[0078] In some embodiments, the anti-DDR2 monoclonal antibody is an anti-DDR2 nanobody. In some embodiments, the anti-DDR2 nanobody comprises CDR1, CDR2, and CDR3, wherein CDR1 comprises or is the sequence set forth in SEQ ID NO: 1 or an equivalent variant thereof, CDR2 comprises or is the sequence set forth in SEQ ID NO: 2 or an equivalent variant thereof, and CDR3 comprises or is the sequence set forth in SEQ ID NO: 3 or an equivalent variant thereof. In some embodiments, the nanobody comprises or is the sequence set forth in SEQ ID NO: 4 or an equivalent variant thereof.

[0079] In some embodiments, the reagent capable of binding to DDR2 is labeled with a detectable marker. In some embodiments, the detectable marker is selected from a fluorescent label, a chemiluminescent label, a paramagnetic label, a radioisotope label, and an enzyme label. In some embodiments, the marker is indocyanine green (ICG). In some embodiments, the marker is 68 Ga or 64 It is Cu.

[0080] In some embodiments, the kit further comprises different concentrations of DDR2 recombinant antigen controls for generating a standard curve for quantitative identification.

[0081] (Detection or diagnostic method and computer-readable storage medium) In one aspect, the present invention provides the use of discoidin domain receptor 2 (DDR2) as a marker in the diagnosis of a neurodegenerative disease in a subject.

[0082] In another aspect, the present invention provides a method for diagnosing a neurodegenerative disease in a subject in vitro or ex vivo, the method comprising measuring the presence and / or level of discoidin domain receptor 2 (DDR2) in a sample (e.g., brain tissue, cerebrospinal fluid, or exosomes) derived from the subject.

[0083] In another aspect, the present invention provides reagents capable of binding to discoidin domain receptor 2 (DDR2) for the in vivo diagnosis of a neurodegenerative disease in a subject.

[0084] In each of the above aspects, the reagent capable of binding to DDR2 is an anti-DDR2 monoclonal antibody or an antigen-binding fragment thereof, or an anti-DDR2 polyclonal antibody. In a preferred embodiment, the reagent capable of binding to DDR2 is an anti-DDR2 monoclonal antibody or an antigen-binding fragment thereof.

[0085] In some embodiments, the anti-DDR2 monoclonal antibody is an anti-DDR2 nanobody. In some embodiments, the anti-DDR2 nanobody comprises CDR1, CDR2, and CDR3, where CDR1 comprises or is the sequence set forth in SEQ ID NO: 1 or an equivalent variant thereof, CDR2 comprises or is the sequence set forth in SEQ ID NO: 2 or an equivalent variant thereof, and CDR3 comprises or is the sequence set forth in SEQ ID NO: 3 or an equivalent variant thereof, and the CDRs are defined according to IMGT. In some embodiments, the nanobody comprises or is the sequence set forth in SEQ ID NO: 4 or an equivalent variant thereof.

[0086] Preferably, in each of the above aspects, the reagent capable of binding to DDR2 is labeled with a detectable marker. In preferred embodiments, the detectable marker is selected from a fluorescent label, a chemiluminescent label, a paramagnetic label, a radioisotope label, and an enzyme label. In preferred embodiments, the detectable marker is selected from a fluorescent label or a chemiluminescent label. In some embodiments, the marker is indocyanine green (ICG). In some embodiments, the marker is 68 Ga or 64 It is Cu.

[0087] Preferably, in each of the above aspects, the neurodegenerative disease is selected from Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease, Huntington's disease, frontotemporal dementia, spinal muscular atrophy, prion disease, spinocerebellar ataxia, Friedreich's ataxia, primary lateral sclerosis, spinocerebellar degeneration, Machado-Joseph disease, dementia with Lewy bodies, progressive bulbar palsy, progressive supranuclear palsy, and multiple system atrophy. In some embodiments, the neurodegenerative disease is associated with abnormal activation of astrocytes. In preferred embodiments, the neurodegenerative disease is Alzheimer's disease or amyotrophic lateral sclerosis.

[0088] A variety of immunoassays can be used in diagnostic methods. In some embodiments, such immunoassays include the use of competitive and non-competitive assay systems, such as radioimmunoassays, immunochromatography, ELISAs, "sandwich" immunoassays, precipitation reactions, immunoblots, gel diffusion precipitation reactions, immunodiffusion, immunoagglutination assays, complement fixation assays, radioimmunoassays, and fluorescent immunoassays. Both in vitro and in vivo assays can be used.

[0089] Generally, the level of DDR2 in a sample is compared to a reference level, and a difference from the reference level indicates the presence and / or stage of a neurodegenerative disease in the subject. The reference level may be a level determined in a control sample (e.g., from a healthy tissue or subject) or a median level from healthy subjects. The presence of DDR2 and / or an increased amount of DDR2 in a sample compared to the reference level, e.g., compared to a subject not affected by the disease, can indicate the presence or risk of developing a neurodegenerative disease in the subject.

[0090] In some embodiments, DDR2 is present in an amount that is at least about 2-fold, at least about 5-fold, at least about 7.5-fold, at least about 10-fold, at least about 15-fold, or at least about 20-fold greater in a sample (e.g., brain tissue, cerebrospinal fluid, or exosomes) from a subject afflicted with a neurodegenerative disease compared to a subject not afflicted with the disease.

[0091] Methods for diagnosis allow for quantitative and / or qualitative assessment, for example absolute and / or relative measurement of target molecules, such as measuring the content of DDR2 in a sample.

[0092] In some embodiments of the methods of the present invention, determining the presence and / or amount of DDR2 in a sample comprises (i) contacting the sample (e.g., brain tissue, cerebrospinal fluid, or exosomes) with a reagent capable of binding to DDR2, and (ii) detecting the formation of a complex between said reagent and DDR2 and / or determining the amount of said complex.

[0093] In some embodiments, the detection / diagnosis method of the present invention can be used in combination with other detection / diagnosis methods for neurodegenerative diseases. For example, the detection / diagnosis method of the present invention can be used in combination with the detection of other biomarkers for neurodegenerative diseases (e.g., amyloid beta protein, tau, APOE, GFAP, AQP4, MAPT, SOD1).

[0094] In another aspect, the present invention provides a computer-readable storage medium having stored thereon computer instructions that can be read and executed by a computer to perform a method of diagnosing whether a subject has a neurodegenerative disease, the method comprising: (a) contacting a sample from a subject with a reagent capable of binding to discoidin domain receptor 2 (DDR2); (b) detecting and reading a signal from the sample after contact to determine whether the reagent has formed a complex with DDR2 in the sample; and (c) determining whether the signal exceeds a predetermined threshold, and if so, determining that the subject has a neurodegenerative disease. In a preferred embodiment, the threshold is a median level from subjects without the disease.

[0095] In another aspect, the present invention provides a computer-readable storage medium having stored thereon computer instructions that can be read and executed by a computer, the computer instructions being executed to perform a method of diagnosing whether a subject is affected by a neurodegenerative disease, the method comprising: (a) isolating exosomes from a subject; (b) contacting the isolated exosomes with a reagent capable of binding to discoidin domain receptor 2 (DDR2); (c) detecting and reading a signal from the exosomes after contacting to determine whether the reagent has formed a complex with DDR2 in the exosomes; and (d) determining whether the signal exceeds a predetermined threshold, and determining that the subject is affected by the neurodegenerative disease if the signal exceeds the predetermined threshold, wherein the threshold is a median level derived from subjects not affected by the disease.

[0096] In another aspect, the present invention provides a method of treating a neurodegenerative disease in a subject, comprising: (a) contacting a sample from the subject with a reagent capable of binding to discoidin domain receptor 2 (DDR2); (b) detecting and reading a signal from the sample after contacting to determine whether the reagent has formed a complex with DDR2 in the sample; (c) determining whether the signal exceeds a predetermined threshold, and if the signal exceeds the predetermined threshold, determining that the subject is afflicted with a neurodegenerative disease, wherein the threshold is a median level from subjects not afflicted with the disease; and (d) administering a neuroprotective or neurorestorative therapy to the subject determined to be afflicted with the neurodegenerative disease.

[0097] In another aspect, the present invention provides a method for treating a neurodegenerative disease in a subject, comprising the steps of: (a) isolating exosomes from a subject; (b) contacting the isolated exosomes with a reagent capable of binding to discoidin domain receptor 2 (DDR2); (c) detecting and reading a signal from the exosomes after contact, thereby determining whether the reagent has formed a complex with DDR2 in the exosomes; (d) determining whether the signal exceeds a predetermined threshold, and if the signal exceeds the predetermined threshold, determining that the subject is affected with a neurodegenerative disease, wherein the threshold is a median level derived from subjects not affected with the disease; and (e) administering a neuroprotective or neurorestorative therapy to the subject determined to be affected with the neurodegenerative disease.

[0098] In some embodiments, the reagent capable of binding to DDR2 comprises a protein, a nucleic acid, or a small molecule compound. In preferred embodiments, the reagent capable of binding to DDR2 is an anti-DDR2 monoclonal antibody or antigen-binding fragment thereof, or an anti-DDR2 polyclonal antibody. In some embodiments, the reagent capable of binding to DDR2 is labeled with a detectable marker. In some embodiments, the detectable marker is selected from a fluorescent label, a chemiluminescent label, a paramagnetic label, a radioisotope label, and an enzyme label. In some embodiments, the marker is indocyanine green (ICG). In some embodiments, the marker is 68 Ga or 64 It is Cu.

[0099] In a preferred embodiment, the neuroprotective or neurorestorative therapy is, for example, a cholinesterase inhibitor (e.g., galantamine, donepezil, huperzine A, rivastigmine), an excitatory amino acid receptor antagonist (e.g., memantine), an inflammatory factor inhibitor (e.g., a nonsteroidal anti-inflammatory agent), a glutamate inhibitor (e.g., riluzole), or a free radical scavenger (e.g., edaravone).

[0100] In some embodiments, the predetermined threshold value may be a reference level as described above, and a difference from the reference level indicates the presence and / or stage of the relevant disease in the subject. The reference level may be a level determined in a control sample (e.g., from healthy tissue or subject) or a median level from healthy subjects. The presence of DDR2 and / or an increased amount of DDR2 in the sample compared to the reference level, e.g., compared to a subject not affected by the disease, may indicate the presence or risk of developing a neurodegenerative disease in the subject.

[0101] In some embodiments, DDR2 is present in an amount that is at least about 2-fold, at least about 5-fold, at least about 7.5-fold, at least about 10-fold, at least about 15-fold, or at least about 20-fold greater in a sample (e.g., brain tissue or exosomes) from a subject afflicted with a neurodegenerative disease compared to a subject not afflicted with the disease. [Table 1]

[0102] (Example) Example 1: Antibody screening and affinity measurements 1. Antibody Screening Anti-DDR2 heavy chain antibodies were screened from alpacas immunized with the human DDR2 extracellular segment (UniProtKB / Swiss-Prot: Q16832.2 aa22-aa399). The antibodies were then sequenced to confirm their VHH moieties, resulting in a nanobody designated 1A12, the sequence of which is listed in the "Sequence Listing" section above. The nanobody was expressed and purified for further characterization and experimentation.

[0103] 2. Affinity of DDR2 nanobodies for the extracellular segment of the DDR2 antigen The experimental steps were as follows: For the above DDR2 nanobody 1A12, the affinity of the antibody to the antigen was measured using biolayer interferometry (BLI) in the molecular interaction analysis system ForteBio, and the affinity of the measured nanobody to the DDR2 antigen extracellular segment protein was analyzed.

[0104] The experimental materials were as follows: The antibody was dissolved in PBS (pH 7.4), the antigen was dissolved in PBS (pH 7.4), the sensor was Ni-NTA, the kinetics buffer was PBST (PBS + 0.02% Tween-20, pH 7.4), the regeneration buffer was 10 mM glycine-HCl (pH 1.7), and the re-charged buffer was 10 mM NiCl in water.

[0105] The operation steps were as follows: a. The probe was pre-wetted in kinetics buffer for 10 minutes. b. Set Baseline 1: The biosensor was baselined for 180 seconds in kinetics buffer. c. Immobilization: The His-tagged DDR2 antigen extracellular segment was diluted to 20 μg / mL in kinetics buffer and captured by the sensor until it reached 4 nM (300 seconds). d. Set baseline 2. A 60 second baseline was set for the biosensor in kinetics buffer. e. Binding: The antibody solution was diluted to a certain concentration with kinetics buffer (diluted two-fold from 100 nM to 3.125 nM), and the sensor was placed in the antibody solution to allow binding (600 seconds). f. Dissociation: The sensor was dissociated in kinetics buffer (600 seconds). g. Regenerate the sensor with 10 mM glycine-HCl (pH 1.7) for 5 seconds. h. Neutralization: After the sensor was regenerated, it was neutralized in kinetics buffer for 5 seconds. i. Repeat regeneration step g and neutralization step h for a total of three times (30 seconds). j. Set baseline 3. After the sensor was regenerated, 10 mM NiCl was added and held for 60 seconds.

[0106] Pharmacokinetic curves were generated and relevant parameters were calculated. Several binding / dissociation curves with suitable concentration gradients were selected, and all curves were fitted using the 1:1 binding mode. The three curves with the best fit were selected and graphed for analysis. Finally, important parameters such as affinity values, binding constants, and dissociation constants were obtained.

[0107] The results were analyzed as follows: The analysis results of the binding of DDR2 nanobody 1A12 to the DDR2 antigen are shown in Figure 1 and Table 1. [Table 2]

[0108] Example 2: In vitro imaging results of DDR2 nanobodies The DDR2 antibody used in this example was the nanobody 1A12 (Nb-DDR2) of Example 1, and its amino acid sequence was as shown in SEQ ID NO:4.

[0109] The experimental steps were as follows: 1. Labeling of DDR2 nanobodies with indocyanine green (ICG) Nb-DDR2 was dissolved in PBS at a concentration of 2 mg / mL and vortexed to mix uniformly.

[0110] ICG-NHS was dissolved in DMSO at a concentration of 2 mM and vortexed to mix evenly.

[0111] 500 μL of 2 mg / mL Nb-DDR2 solution was transferred to a 1.5 mL centrifuge tube, and 18 μL of 2 mM ICG-NHS solution was added to the Nb-DDR2 solution in nine portions, each containing 2 μL. After each addition, the solution was vortexed for several seconds to ensure uniform mixing.

[0112] The pH of the mixed solution was measured and adjusted to 8.5 to 9 with 2 M NaOH solution.

[0113] The centrifuge tube was placed on a shaker and reacted at 60 rpm at room temperature for 2 hours.

[0114] Unreacted ICG-NHS was removed by multiple centrifugations at 14,000 g for 10 min using a 0.5 mL ultracentrifugal filter, the solution was replaced with 0.9% NaCl, and the protein solution was filtered through a 0.22 μm membrane filter and then stored at 4°C.

[0115] 2. Imaging of the lateral ventricles in an amyotrophic lateral sclerosis (ALS) mouse model To construct an ALS mouse model, Optn-CKO mice were purchased and transfected with Optn from the Shanghai Model Organisms Center, Inc. flox / + C57BL / 6J mice were constructed and bred to Optn flox / flox Three-week-old Optn mice were obtained. flox / + After dividing the mice into several cages, they were ear-tagged and numbered, and their tails were cut. The genotypes of the mice were identified using PCR and agarose gel electrophoresis. flox / flox Mice were screened and, at 2 months of age, injected with adenovirus into the lateral ventricle (hAd5-Opt Cre virus, 6.58 × 10 10 After Cre expression (PFU / mL, 20 μL / animal), the OPTN gene was specifically knocked out through the loxp site. Images were taken 3 months after Cre virus injection.

[0116] The imaging method was as follows: DDR2 nanobody 1A12 labeled with indocyanine green (ICG) was injected into experimental mice (wild-type mice and ALS model mice) via the tail vein, and saline was injected into control ALS model mice (15 μg / mouse). Images were taken using a bioimaging device with excitation / emission wavelengths of 730 / 820 nm. The results are shown in Figure 2.

[0117] The results showed that the ICG-labeled DDR2 antibody specifically recognized DDR2, which was highly expressed in the brain tissue of ALS mice, but no significant uptake was observed in either the wild-type or saline controls. Therefore, detecting DDR2 expression can accurately diagnose ALS.

[0118] 3. Imaging of Alzheimer's Disease (AD) Mouse Models In this example, APP / PS1 double gene-modified mice were used as genetically modified mice commonly used in AD. The mice were 2 years old. The control group consisted of wild-type C57 mice.

[0119] The DDR2 nanobody 1A12 labeled with indocyanine green (ICG) was injected into experimental mice (wild-type mice and AD model mice) via the tail vein at a dose of 15 μg per mouse. Images were taken using a bioimaging device with excitation / emission wavelengths of 730 / 820 nm. The results are shown in Figure 3.

[0120] The results showed that the ICG-labeled DDR2 antibody specifically recognized DDR2, which was highly expressed in the brain tissue of AD mice, but no significant uptake was observed in wild-type controls. Therefore, detecting DDR2 expression can accurately diagnose AD.

[0121] Example 3: Measurement of exosomes 1. Western blot (WB) measurement of exosomal DDR2 in plasma of ALS patients The measurement method was as follows. Exosomes were extracted. 200 μL of plasma and 350 μL of PBS were mixed uniformly and then placed in an exosome isolation device (EXODUS Series H-300, Shenzhen Huixin Biomedical Technology Co., Ltd.). 400 μL of PBS was added to resuspend the exosomes. Exosomes were identified. The particle size range of exosomes was measured using a Resun (Shenzhen) Tech Co., Ltd. nanocoulter counter. 90% of the exosomes were between 30 and 120 nm, indicating that the extracted material was exosomes.

[0122] A. 10 μL of exosomes were added with SDS-PAGE sample loading buffer (5x, catalog no. RM00001), boiled, and then directly loaded into an SDS-PAGE gel preparation kit (Shosai Seibutsu, model JC-PE001).

[0123] B. The assembled electrophoresis apparatus was placed in a tank, and the marker and protein sample were loaded. Freshly prepared SDS electrophoresis buffer was poured into the tank. The voltage was 120 V for about 20 minutes, and then switched to 240 V. Electrophoresis was stopped when the smallest molecular band of the marker reached the bottom.

[0124] C. Transfer (transfer solution was prepared with 700 mL of ultrapure water, 200 mL of methanol, and 100 mL of fast transfer solution). The PVDF membrane was activated with methanol, and the electrophoresis gel was added to the "sandwich" device. To transfer the gel from the negative electrode to the positive electrode, the gel was positioned on the "black sandwich" side and the membrane was positioned on the "transparent sandwich" side. The device was then inserted into the transfer chamber, and the transfer solution (refrigerated) was poured into the electrophoresis chamber. The transfer was performed at 400 mA for 35 minutes.

[0125] D. Blocking. The membrane was blocked with 5% skim milk blocking solution or 5% BSA on a shaker for 2 hours.

[0126] E. Anti-hDDR2-biotin (RD, Cat. No. BAF2538, 1:2000) was used and incubated overnight at 4°C.

[0127] F. The secondary antibody was streptavidin-HRP (RD, Catalog No. DY998, 1:5000), incubated for 1 hour, and developed using ECL. The device was a chemiluminescence gel imaging system, model BoLuTeng, GELVLew 6000 Pro.

[0128] The results are shown in FIG.

[0129] The results were analyzed as follows: Figure 4 shows that significant DDR2 expression was detected in plasma exosomes from ALS patients at each stage compared to the negative control, indicating that ALS at each stage can be diagnosed by detecting DDR2 in exosomes.

[0130] 2. Flow cytometry measurement of exosomal DDR2 in plasma of ALS patients The steps of the flow cytometry experiment were as follows. Exosomes were extracted. 200 μL of plasma and 350 μL of PBS were mixed uniformly and then placed in an exosome isolation device (EXODUS Series H-300, Shenzhen Huixin Biomedical Technology Co., Ltd.). 400 μL of PBS was added to resuspend the exosomes. Exosomes were identified. The particle size range of exosomes was measured using a Resun (Shenzhen) Tech Co., Ltd. nanocoulter counter. 90% of the exosomes were between 30 and 120 nm, indicating that the extracted material was exosomes.

[0131] Flow cytometry experiments were as follows. (1) CD9 capture beads were resuspended, and 12.5 μL was aspirated and mixed evenly with 50 μL of sample, followed by incubation at 4° C. for 18 hours. (2) After washing twice with wash buffer, the supernatant was discarded and the cells were resuspended in 250 μL of PBS. (3) 25 μL of the resuspension was aspirated, 1 μg of 1A12 nanobody-FITC fluorescent antibody was added, the volume was adjusted to 100 μL with PBS, and the mixture was incubated in the dark for 1 hour. (4) After washing twice, the cells were resuspended in 200 μL of PBS, loaded, and measured. The results are shown in Figure 5.

[0132] The results were analyzed as follows: Figure 5 shows that the percentage of DDR2 positives in plasma exosomes of ALS patients at each stage was significantly increased compared to normal subjects, and was at least about twice that of normal controls, indicating that ALS at each stage can be diagnosed by detecting DDR2 in exosomes.

[0133] In this example (including WB measurements (FIG. 4) and flow cytometry measurements (FIG. 5)), the "early," "intermediate," and "late" stages of ALS patients were determined by scoring based on the ALSFRS-R (ALS Functional Rating Scale-Revised). The scoring criteria are well known in the art, and details can be found, for example, at https: / / neurotoolkit.com / alsfrs-r / or https: / / www.outcomes-umassmed.org / ALS / alsscale.aspx (last accessed September 19, 2022). The criteria for categorizing ALS into early, intermediate, and late stages are as follows: 49-40 points is early stage, 39-30 points is intermediate stage, and 29 points or less is late stage.

[0134] Example 4: Bioinformatics analysis of neurodegenerative disease-related experimental data 1. Amyotrophic lateral sclerosis (ALS) The data analyzed in this example are derived from brain tissue of 13 normal donors from the Allen brain map (https: / / portal.brain-map.org / atlases-and-data / rnaseq / human-m1-10x) and from motor cortex tissue of 16 ALS patients from NCBI GSE174332, totaling 239,531 cells.

[0135] After compiling the data, the expression status of DDR2 was analyzed, and the results are shown in Figure 6. Panels A, B, and C of Figure 6 show the cell distribution and expression level of DDR2 in normal brain tissue and ALS patient brain tissue, respectively, and the proportion and distribution of cells expressing DDR2.

[0136] The results were analyzed as follows: Figure 6 shows that DDR2 expression in astrocytes (Astro) in ALS samples was significantly increased compared to normal controls, indicating that ALS can be diagnosed by detecting DDR2.

[0137] 2. Alzheimer's disease (AD) The original data for this example was from https: / / www.ncbi.nlm.nih.gov / geo / query / acc.cgi?acc=GSE147528, and the quality-controlled data was from https: / / www.synapse.org / #!Synapse:syn21788402. The data included 20 samples collected from the entorhinal cortex (EC) and superior frontal gyrus (SFG) of 10 AD patients. The number of entorhinal cortex cells was 42,528, and the number of superior frontal gyrus cells was 63,508.

[0138] The upper and lower right panels of Figure 7 are cellular dimensionality reduction maps by tSNE for the entorhinal cortex (EC) and superior frontal gyrus (SFG) of 10 patients, respectively. The left panel is a feature plot of DDR2, where each dot represents a cell and the intensity of the color indicates the level of gene expression.

[0139] The results analysis was as follows: Figure 7 showed that DDR2 was highly expressed in astrocytes in AD samples.

[0140] The two boxplots in Figure 8(a) show the proportion of DDR2-expressing cells in different types of cells in the entorhinal cortex (EC) and superior frontal gyrus (SFG), respectively. The vertical axis represents the proportion of DDR2-expressing cells, and the horizontal axis represents the different cell types. Each point in the figure represents one sample.

[0141] The two bar graphs in Figure 8(b) show the percentage of DDR2-expressing astrocytes in different samples in the entorhinal cortex (EC) and superior frontal gyrus (SFG), respectively. The vertical axis represents the percentage of DDR2-expressing cells, and the horizontal axis represents the different samples. The numbers above each represent the number of cells of that cell type.

[0142] Patients with Braak stage 0 or 2 were placed in the mild group, and patients with Braak stage 6 were placed in the severe group. The two boxplots in Figure 8(c) show the proportion of DDR2-expressing astrocytes in the entorhinal cortex (EC) and superior frontal gyrus (SFG) of patients with different severity levels, respectively. The vertical axis represents the proportion of DDR2-expressing cells, and the horizontal axis represents the different severity levels. Each point in the figure represents one sample, and the Wilcoxon test (wlicox.test) was used.

[0143] The results were analyzed as follows: As can be seen from Figure 8, the proportion of DDR2-expressing astrocytes in the AD samples was high, and the proportion of DDR2-expressing astrocytes was significantly higher in most samples. Compared to mild AD patients, the proportion of DDR2-expressing astrocytes in severe AD patients was significantly higher.

[0144] Figure 9 shows the correlation analysis of the expression of DDR2 with APOE, GFAP, AQP4, and MAPT in astrocytes from the entorhinal cortex (EC) and superior frontal gyrus (SFG) of 10 patients. The test method was Spearman, and cells were screened to select cells that co-expressed the two genes targeted for correlation analysis.

[0145] The results were analyzed as follows: Figure 9 shows that DDR2 has a good correlation with both conventional AD detection markers.

[0146] In summary, DDR2 is significantly expressed in AD patient samples and is mainly concentrated in astrocytes. As an AD diagnostic marker, DDR2 has good consistency with conventional detection markers, so it can be used to diagnose AD.

[0147] Example 5: ICG-labeled DDR2 nanobody 1A12 was produced as described in Example 2.

[0148] APP / PS1 (C57BL / 6) genetically engineered mice (AD = Alzheimer's disease model) were used, and C57BL / 6 mice of the same background served as control mice. 15 μg of ICG-labeled DDR2 nanobody 1A12 probe was injected via the tail vein into both the AD model mice and control mice. After 15 minutes of in vivo circulation, the mice were subjected to in vivo imaging. The results are shown on the left side of Figure 10. Compared to the control group (C57BL / 6 normal mice), clear fluorescent signals were observed in the brains of the AD model mice. The AD model mice already had the disease.

[0149] SOD1 gene mutant mice were selected as a mouse model of amyotrophic lateral sclerosis (ALS), and C57BL / 6 mice served as normal controls. 15 μg of ICG-labeled DDR2 nanobody 1A12 probe was injected via the tail vein into both the ALS model and control mice. After 15 minutes of in vivo circulation, the mice were subjected to in vivo imaging. The results are shown on the right side of Figure 10. Compared to the control group (C57BL / 6 normal mice), clear fluorescent signals were observed in the brains of the ALS model mice. The ALS model mice already exhibited symptoms.

[0150] Example 6: Nuclides ( 64 Cu, 68 Labeling procedure for nanobody 1A12 with conventional nuclides such as Ga 1. Integration with NOTA (1) The antibody solution was centrifuged at 12,000 g, and the supernatant was collected. The solvent for the antibody solution was replaced with 0.1 M ammonium acetate (pH = 7). After the replacement, the concentration of nanobody 1A12-cys was adjusted to 1.33 mg / mL. The concentration was measured before and after the replacement and was used as the concentration after the replacement.

[0151] (2) 50 mM Mal-NOTA (dissolved in DMSO) was used, and the molar ratio of antibody to Mal-NOTA was 1:5. Mal-NOTA was diluted with DMSO to less than 10% of the reaction system volume (for example, if the reaction system was 1 mL, Mal-NOTA was diluted to 80-90 μL and shaken to mix uniformly, thereby dissolving Mal-NOTA sufficiently in DMSO). The Mal-NOTA solution was added to the antibody solution in several portions, 10 μL per addition, and after each addition, the solution was shaken for several tens of seconds to mix uniformly. The pH of the solution was adjusted to 7 (the reaction solvent was 0.1 M ammonium acetate, and the pH did not generally need to be adjusted; it could be confirmed by measurement). The solution was then placed on a shaker and reacted at room temperature for 2 hours. The shaker rotation speed was 150 rpm.

[0152] (3) After the reaction was completed, the antibody solution was centrifuged at 12,000 g, and the supernatant was collected. The antibody solution was purified and concentrated with 0.1 M ammonium acetate to a volume of 50 to 80 μL per 100 μg of antibody, sealed with a sealing film, and stored in a refrigerator at -80°C.

[0153] 2. 68 Ga, 64 Labeling with conventional nuclides such as Cu (1) 68 Ga, 64 Cu or other nuclides are added to the precursor solution ( 64 In the case of Cu, the concentration was set as high as possible, with the total radioactivity input for 100 μg of precursor being approximately 1.5 mCi, and the final reaction volume was 150 μL or less), and the mixture was mixed uniformly and the pH was adjusted to 4-5 (generally, the pH was 4-5 after uniform mixing without any adjustment).

[0154] (2) If you have a vibrating thermostat, place it in the vibrating thermostat and let it react at 37°C for 2 hours. If you don't have one, heat it to 37°C and let it react for 2 hours (in a water bath, oven, etc.).

[0155] (3) The sample was spotted on a filter paper for paper chromatography, developed with a developing solution, and the labeling rate was measured by iTLC (the developing solution was sodium citrate).

[0156] Example 7: Nucleotides of Nanobody 1A12 64 Labeling and imaging with Cu Based on the procedure described in Example 6, 64 Cu-labeled nanobody 1A12 was prepared.

[0157] The same AD model mice and ALS model mice as in Example 5 were used and compared with normal control mice. 64Cu-labeled DDR2 nanobody 1A12 probe was injected via the tail vein and allowed to circulate in vivo for 45 minutes. PET imaging data were collected and reconstructed to obtain a PET / CT image. The uptake signal is indicated by the circle.

[0158] The results are shown in Figures 11 and 12. The results showed that the AD model mice and ALS model mice had probe uptake signals in the cerebral cortex and motor cortex, respectively. There was no uptake signal in the normal control mice.

[0159] While the present invention has been specifically disclosed in terms of preferred embodiments and optional features, it is to be understood that those skilled in the art may make modifications, improvements, and variations to the invention disclosed herein, and that these modifications, improvements, and variations are considered to be within the scope of the present invention. The materials, methods, and examples provided herein are representative and illustrative of preferred embodiments and are not intended as limitations on the scope of the invention.

Claims

1. Use of a reagent for measuring the expression level of discoidin domain receptor 2 (DDR2) in the manufacture of a kit for diagnosing a neurodegenerative disease in a subject, wherein if the level of DDR2 in a sample derived from the subject is higher than the level in a control not affected by the disease, it indicates that the subject is affected by the neurodegenerative disease.

2. The use according to claim 1 , wherein the reagent for measuring the expression level of DDR2 comprises a reagent capable of binding to DDR2 so as to measure the level of DDR2 in the sample.

3. The use according to claim 2, wherein the reagent capable of binding to DDR2 is an anti-DDR2 monoclonal antibody or an antigen-binding fragment thereof, or an anti-DDR2 polyclonal antibody.

4. The use according to claim 3, wherein the anti-DDR2 monoclonal antibody is an anti-DDR2 nanobody.

5. 5. The use according to claim 4, wherein the anti-DDR2 Nanobody comprises CDR1, CDR2 and CDR3, wherein CDR1 comprises or is the sequence set forth in SEQ ID NO: 1 or an equivalent variant thereof, CDR2 comprises or is the sequence set forth in SEQ ID NO: 2 or an equivalent variant thereof, and CDR3 comprises or is the sequence set forth in SEQ ID NO: 3 or an equivalent variant thereof, and wherein the CDRs are defined according to IMGT.

6. 5. The use according to claim 4, wherein the Nanobody comprises or is the sequence shown in SEQ ID NO: 4 or an equivalent variant thereof.

7. The use according to claim 2, wherein said reagent capable of binding to DDR2 is labeled with a detectable marker.

8. The use according to claim 7, wherein the detectable marker is selected from a fluorescent label, a chemiluminescent label, a paramagnetic label, a radioisotope label and an enzyme label.

9. 2. The use according to claim 1, wherein the neurodegenerative disease is selected from Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease, Huntington's disease, frontotemporal dementia, spinal muscular atrophy, prion disease, spinocerebellar ataxia, Friedreich's ataxia, primary lateral sclerosis, spinocerebellar degeneration, Machado-Joseph disease, dementia with Lewy bodies, progressive bulbar palsy, progressive supranuclear palsy, and multiple system atrophy.

10. The use according to claim 1, wherein the neurodegenerative disease is Alzheimer's disease.

11. The use according to claim 1, wherein the neurodegenerative disease is amyotrophic lateral sclerosis.

12. A computer-readable storage medium having stored thereon computer instructions that can be read and executed by a computer, Execution of the computer instructions causes: (a) contacting a sample from a subject with a reagent capable of binding to discoidin domain receptor 2 (DDR2); (b) detecting and reading a signal from the sample after contact to determine whether the reagent has formed a complex with DDR2 in the sample; (c) determining whether the signal exceeds a predetermined threshold, and if the signal exceeds the predetermined threshold, determining that the subject is suffering from a neurodegenerative disease, wherein the threshold is a median level derived from subjects not suffering from the disease.

13. 13. The computer-readable storage medium of claim 12, wherein the neurodegenerative disease is Alzheimer's disease or amyotrophic lateral sclerosis.

14. Use of exosomes derived from a subject in the manufacture of a kit for diagnosing a neurodegenerative disease in a subject, wherein a level of discoidin domain receptor 2 (DDR2) expressed by the exosomes higher than that of a control not affected by the disease indicates that the subject is affected with the neurodegenerative disease.

15. 15. The use of claim 14, wherein the exosomes are derived from serum or plasma of the subject.

16. 15. The use of claim 14, wherein the kit further comprises a reagent capable of binding to DDR2 so as to measure the level of DDR2 expressed by the exosomes.

17. 15. The use according to claim 14, wherein the neurodegenerative disease is Alzheimer's disease or amyotrophic lateral sclerosis.

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