WO2024061128A1 - 盘状结构域受体2在诊断神经退行性疾病中的应用及相关的计算机可读介质 - Google Patents

盘状结构域受体2在诊断神经退行性疾病中的应用及相关的计算机可读介质 Download PDF

Info

Publication number
WO2024061128A1
WO2024061128A1 PCT/CN2023/119127 CN2023119127W WO2024061128A1 WO 2024061128 A1 WO2024061128 A1 WO 2024061128A1 CN 2023119127 W CN2023119127 W CN 2023119127W WO 2024061128 A1 WO2024061128 A1 WO 2024061128A1
Authority
WO
WIPO (PCT)
Prior art keywords
ddr2
disease
subject
binding
neurodegenerative disease
Prior art date
Application number
PCT/CN2023/119127
Other languages
English (en)
French (fr)
Inventor
苏金
李佳
蒋海山
魏新茹
Original Assignee
菲创生物医学技术(广州)有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 菲创生物医学技术(广州)有限公司 filed Critical 菲创生物医学技术(广州)有限公司
Publication of WO2024061128A1 publication Critical patent/WO2024061128A1/zh

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16H—HEALTHCARE INFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR THE HANDLING OR PROCESSING OF MEDICAL OR HEALTHCARE DATA
    • G16H50/00—ICT specially adapted for medical diagnosis, medical simulation or medical data mining; ICT specially adapted for detecting, monitoring or modelling epidemics or pandemics
    • G16H50/20—ICT specially adapted for medical diagnosis, medical simulation or medical data mining; ICT specially adapted for detecting, monitoring or modelling epidemics or pandemics for computer-aided diagnosis, e.g. based on medical expert systems
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
    • G01N2333/912—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2814—Dementia; Cognitive disorders
    • G01N2800/2821—Alzheimer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2878—Muscular dystrophy

Definitions

  • the present invention relates to kits, methods and computer-readable media for diagnosing neurodegenerative diseases, and more specifically, to diagnosing neurodegenerative diseases by detecting Discoid Domain Receptor 2 (DDR2).
  • DDR2 Discoid Domain Receptor 2
  • Neurodegenerative diseases are diseases that cause various symptoms due to degenerative changes in nerve cells in the central nervous system, such as impairment of motor and sensory functions, and suppression of high-level 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, traumatic brain injury and secondary effects on central nervous system cells Functional gene mutations, etc.
  • Neurodegenerative diseases have long been considered to be incurable and complex diseases, and there are currently no effective drugs to improve the overall condition of neurodegenerative diseases by targeting systemic pathological processes.
  • the course of neurodegenerative diseases is usually an irreversible process. Therefore, the key to treatment is early diagnosis, intervention at the early stage of the disease and delaying the progression of the disease. If we can quickly diagnose the patient in the early stages of the disease or enable potential patients to detect risks in advance and carry out targeted treatment or prevention, it can help the patient stay in the minor injury stage and slow down the deterioration, thus ensuring the patient's quality of life and reducing the burden on society. .
  • the current diagnostic method for various neurodegenerative diseases is mainly combined diagnosis, which mainly includes: neuropsychological assessment, cognitive impairment testing; brain imaging (such as brain PET scan, MRI); cerebrospinal fluid marker testing; and blood testing and genetic risk analysis, etc.
  • brain imaging such as brain PET scan, MRI
  • cerebrospinal fluid marker testing and blood testing and genetic risk analysis, etc.
  • commonly used diagnostic methods either require the subject to be injected with a certain dose of radioactive material, or the operation is very damaging and can easily cause surgical infection. Therefore, the development of new markers for early diagnosis of neurodegenerative diseases is one of the important directions for future diagnosis and treatment.
  • Discoidin domain receptor 2 is a receptor tyrosine kinase (RTK) that utilizes the extracellular matrix protein collagen as its ligand.
  • RTK receptor tyrosine kinase
  • DDR2 promotes cell adhesion through activation of ⁇ 1-integrin.
  • the unique function of DDR2 is to mediate the transmission of signals from the extracellular matrix into the cell, balancing the regulation of the extracellular matrix, and participating in the regulation of cell growth, differentiation and metabolism.
  • DDR2 is considered an important target in inflammation such as arthritis (eg osteoarthritis, rheumatoid arthritis) and fibrosis (eg pulmonary fibrosis, liver cirrhosis, renal fibrosis or skin fibrosis).
  • DDR2 is mainly expressed in interstitial cells such as fibroblasts, myofibroblasts, and smooth muscle cells in the kidney, skin, lung, heart, and connective tissue.
  • abnormal expression of DDR2 is associated with various disease processes, such as inflammation, liver fibrosis, renal fibrosis, pulmonary fibrosis, skin scarring, and atherosclerosis.
  • a mouse inflammation model study it was found that DDR2 expression was upregulated in the knee joints of aged mice.
  • DDR2 expression was found to be upregulated in synoviocytes.
  • the inventor of the present application unexpectedly found that the expression of DDR2 in relevant samples from patients with neurodegenerative diseases was significantly increased compared with normal controls.
  • the inventors use reagents capable of binding to DDR2 to detect the presence and/or level of DDR2 in samples, thereby efficiently and accurately diagnosing neurodegenerative diseases.
  • the present invention provides a kit for diagnosing a neurodegenerative disease in a subject, the kit comprising a reagent for detecting the expression level of Discoid Domain Receptor 2 (DDR2), wherein the Levels of DDR2 in the subject's sample that are higher than levels in controls without the disease indicate that the subject has a neurodegenerative disease.
  • DDR2 Discoid Domain Receptor 2
  • the present invention provides use of a reagent for detecting the expression level of Discoid Domain Receptor 2 (DDR2) in the preparation of a kit for diagnosing a neurodegenerative disease in a subject, wherein the Levels of DDR2 in the sample that are higher than levels in controls without the disease indicate that the subject has a neurodegenerative disease.
  • DDR2 Discoid Domain Receptor 2
  • the invention provides reagents for detecting expression levels of Discoid Domain Receptor 2 (DDR2) for use in diagnosing a neurodegenerative disease in a subject, wherein the level of DDR2 in a sample from the subject is higher than Levels in disease-free controls indicate that the subject has a neurodegenerative disease.
  • DDR2 Discoid Domain Receptor 2
  • the invention provides a method of diagnosing a neurodegenerative disease in a subject, said method comprising detecting the presence and/or level of Discoid Domain Receptor 2 (DDR2) in a sample from said subject.
  • the method includes contacting an agent capable of binding to DDR2 with a sample from the subject; detecting the presence of a complex formed between the agent and DDR2 in the sample upon contact; and based on the complex The presence and/or level determines that the subject has or is at risk of suffering from a neurodegenerative disease.
  • DDR2 Discoid Domain Receptor 2
  • the invention provides a computer-readable storage medium having computer instructions stored thereon for reading and execution by a computer, the computer instructions being executed to perform a method of diagnosing whether a subject suffers from a neurodegenerative disease,
  • the method includes: (a) contacting a sample from the subject with a reagent capable of binding to Discodomain Receptor 2 (DDR2); (b) detecting and reading the signal of the contacted sample to determine the whether the reagent forms a complex with DDR2 in the sample; and (c) determining whether the signal exceeds a predetermined threshold, and when the signal exceeds the predetermined threshold, it is determined that the subject suffers from a neurodegenerative disease, and wherein the The threshold is the median level from subjects without disease.
  • DDR2 Discodomain Receptor 2
  • the invention provides a method of treating a neurodegenerative disease in a subject, the method comprising: (a) combining a sample from the subject with a protein capable of binding to Discoid Domain Receptor 2 (DDR2) Contact the reagent; (b) detect and read the signal of the contacted sample to determine whether the reagent forms a complex with DDR2 in the sample; (c) determine whether the signal exceeds a predetermined threshold, and when the signal exceeds when predetermining a threshold, determining that the subject has a neurodegenerative disease, and wherein the threshold is a median level from subjects who do not have the disease; and (d) administering neuroprotection to the subject determined to have the neurodegenerative disease or neurorestorative therapy.
  • DDR2 Discoid Domain Receptor 2
  • the reagent for detecting the expression level of DDR2 includes a reagent capable of binding to DDR2 to detect the level of DDR2 in the sample.
  • the agent capable of binding to DDR2 includes a protein, nucleic acid or small molecule compound.
  • the agent capable of binding to DDR2 is an anti-DDR2 monoclonal antibody or an antigen-binding fragment thereof, or an anti-DDR2 polyclonal antibody.
  • the anti-DDR2 monoclonal antibody is an anti-DDR2 Nanobody.
  • the agent capable of binding to DDR2 is labeled with a detectable label.
  • the detectable label is selected from the group consisting of fluorescent labels, chemiluminescent labels, paramagnetic labels, radioisotope labels, and enzyme labels.
  • the sample is brain tissue or cerebrospinal fluid.
  • the neurodegenerative disease is a neurodegenerative disease mediated by damage to astrocytes.
  • the neurodegenerative disease is a neurodegenerative disease mediated by astrocytes with elevated DDR2 expression, the elevated DDR2 expression being consistent with DDR2 in astrocytes of normal subjects Expression levels are compared.
  • the neurodegenerative disease is selected from Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), Parkinson's disease (Parkinson's disease), Huntington's disease, frontotemporal dementia, spinal muscular atrophy, prion disease, spinocerebellar ataxia, Friedrich's ataxia Friedreichs axia, primary lateral sclerosis, spinocerebellar atrophy, Machado-hoseph's diease, Lewy Body dementia, progressive Bulbar palsy (progressive bulbar palsy), progressive supranuclear palsy (progressive supranuclear palsy) and multiple system atrophy (multiple system atrophy).
  • AD Alzheimer's disease
  • ALS amyotrophic lateral sclerosis
  • Parkinson's disease Huntington's disease
  • Huntington's disease frontotemporal dementia
  • spinal muscular atrophy prion disease
  • spinocerebellar ataxia Friedrich's ataxia Friedreichs axia
  • the present invention provides a kit for diagnosing neurodegenerative diseases in a subject, the kit comprising exosomes from the subject and capable of detecting Discoid Domain Receptor 2 (DDR2 ) binds a reagent to detect the level of DDR2 expressed by the exosomes, wherein a level of DDR2 expressed by the exosomes that is greater than a level in a control without the disease indicates that the subject suffers from a neurodegenerative disease.
  • DDR2 Discoid Domain Receptor 2
  • the present invention provides use of exosomes from a subject in the preparation of a kit for diagnosing a neurodegenerative disease in the subject, wherein the exosomes express Discoid Domain Receptor 2 ( Levels of DDR2) that are higher than levels in controls without the disease indicate that the subject has a neurodegenerative disease.
  • DDR2 Discoid Domain Receptor 2
  • the present invention provides exosomes from a subject for use in diagnosing a neurodegenerative disease in the subject, wherein the exosomes express a level of Discoid Domain Receptor 2 (DDR2) that is higher than that without the disease.
  • DDR2 Discoid Domain Receptor 2
  • the invention provides a method of diagnosing a neurodegenerative disease in a subject, the method comprising isolating exosomes from the subject and detecting a discoid domain receptor in the exosomes from the subject 2(DDR2) presence and/or levels.
  • the method includes contacting an agent capable of binding to DDR2 with exosomes from the subject; detecting the presence of a complex formed between the agent and DDR2 in the exosomes upon contact; and based on the The presence and/or level of the complex is used to determine that the subject has or is at risk of suffering from a neurodegenerative disease.
  • the present invention provides a computer-readable storage medium having computer instructions stored thereon for reading and execution by a computer, the computer instructions being executed to perform a method of diagnosing whether a subject suffers from a neurodegenerative disease , the method includes: (a) isolating exosomes from the subject; (b) contacting the isolated exosomes with a reagent capable of binding to Discoid Domain Receptor 2 (DDR2); (c) detecting and reading the signal of the contacted exosomes to determine whether the agent forms a complex with DDR2 in the exosomes; and (d) determining whether the signal exceeds a predetermined threshold, and when the signal exceeds the predetermined threshold , determining that the subject has a neurodegenerative disease, and wherein the threshold is a median level from subjects without the disease.
  • DDR2 Discoid Domain Receptor 2
  • the present invention provides a method of treating a neurodegenerative disease in a subject, the method comprising: (a) isolating exosomes from the subject; (b) combining the isolated exosomes with a substance capable of being combined with Contact with a reagent that binds Discodomain Receptor 2 (DDR2); (c) detect and read the signal of the contacted exosomes to determine whether the reagent forms a complex with DDR2 in the exosomes; (d) ) determine whether the signal exceeds a predetermined threshold, and when the signal exceeds the predetermined threshold, determine that the subject suffers from a neurodegenerative disease, and wherein the threshold is a median level from subjects who do not suffer from the disease; and ( e) administering a neuroprotective or neurorestorative therapy to a subject determined to have a neurodegenerative disease.
  • DDR2 Discodomain Receptor 2
  • the neuroprotective or neurorestorative therapy is, for example: a cholinesterase inhibitor, such as galantamine, donepezil, huperzine A, rivastigmine; an NMDA receptor antagonist, such as methacrylate Adamantine; inflammatory factor inhibitors, such as non-steroidal anti-inflammatory drugs; glutamate inhibitors, such as riluzole; free radical scavengers, such as edaravone.
  • a cholinesterase inhibitor such as galantamine, donepezil, huperzine A, rivastigmine
  • an NMDA receptor antagonist such as methacrylate Adamantine
  • inflammatory factor inhibitors such as non-steroidal anti-inflammatory drugs
  • glutamate inhibitors such as riluzole
  • free radical scavengers such as edaravone.
  • the exosomes are derived from body fluids of the subject.
  • the body fluids include peripheral blood, serum, plasma, serosal fluid, sputum, synovial fluid, aqueous humor, amniotic fluid, breast milk, semen, prostatic fluid, Cowper's fluid, female ejaculate, sweat, excreta , tears, cyst fluid, pleural effusion, ascites fluid, pericardial fluid, chyle, bile, interstitial fluid, menstrual blood, pus, vomitus, vaginal secretions, mucosal secretions, pancreatic juice, blastocoel fluid, umbilical cord blood, One or more of urine, cerebrospinal fluid, saliva, lymph fluid, loose stool, bronchopulmonary aspirate fluid, bronchoalveolar lavage fluid, and nasal lavage fluid.
  • the body fluid is serum or plasma.
  • the exosomes are isolated from the exosomes by size exclusion chromatography, density gradient centrifugation, differential centrifugation, nanomembrane ultrafiltration, immunosorbent capture, affinity capture, microfluidic separation, or a combination thereof. Sample separation of objects.
  • the agent capable of binding to DDR2 includes proteins, nucleic acids or small molecule compounds.
  • the agent capable of binding to DDR2 is an anti-DDR2 monoclonal antibody or an antigen-binding fragment thereof, or an anti-DDR2 polyclonal antibody.
  • the agent capable of binding to DDR2 is labeled with a detectable label.
  • the detectable label is selected from the group consisting of fluorescent labels, chemiluminescent labels, paramagnetic labels, radioisotope labels, and enzyme labels.
  • Figure 1 Affinity constant diagram of DDR2 Nanobodies and the extracellular segment of DDR2 antigen.
  • Figure 2 Imaging results of ALS mouse model.
  • Figure 3 Imaging results of AD mouse model.
  • Figure 4 Western blot detection results of exosomes in plasma of ALS patients.
  • Figure 5 Flow cytometric detection results of exosomes in plasma of ALS patients.
  • FIG. 7 DDR2 expression in AD-related experimental data.
  • FIG. 8 DDR2 expression in AD-related experimental data.
  • FIG. 9 Correlation analysis of DDR2 and other diagnostic genes.
  • Figure 10 In vivo fluorescence imaging of DDR2 Nanobody 1A12-ICG.
  • FIG. 11 PET/CT imaging of AD mice.
  • Figure 12 PET/CT imaging of ALS mice.
  • “about” means that the value is within the acceptable error range of the specific value determined by a person of ordinary skill in the art, and the value depends in part on how it is measured or determined (i.e., the limits of the measurement system). For example, in each practice in the art, “about” can mean within 1 or more than 1 standard deviation. Alternatively, “about” or “substantially including” can mean a range of up to 20%. In addition, for biological systems or processes, the term can mean up to an order of magnitude or up to 5 times the value. Unless otherwise stated, when a specific value appears in the application and claims, the meaning of "about” or “substantially including” should be assumed to be within the acceptable error range of the specific value.
  • the terms "subject,” “patient,” or “individual” refer to any subject, particularly a mammalian subject, for whom diagnosis, prognosis, or treatment is desired. Mammals include humans, livestock, farm animals, zoo animals, competitive animals, or pets, such as dogs, cats, pigs, rabbits, rats, mice, horses, cattle, cows, and the like. The object referred to herein is preferably a human being.
  • the subject suffers from or is susceptible to one or more disorders or conditions.
  • a patient may exhibit one or more symptoms of a disorder or condition, or may have been diagnosed with one or more disorders or conditions.
  • the patient is receiving or has received certain therapy for the diagnosis and/or treatment of such diseases, disorders or conditions.
  • detecting includes any means of detection, including direct and indirect detection, quantitative and qualitative detection, and means identifying the presence and/or level of a specific molecule (eg, DDR2 protein) in a subject or in a sample from a subject.
  • a specific molecule eg, DDR2 protein
  • diagnosis refers to the identification or classification of a molecular or pathological state, disease or disorder.
  • diagnosis may refer to the identification of a neurodegenerative disease or the identification of a specific type thereof.
  • the term "binding” preferably relates to specific binding.
  • “Specifically binds” means that an agent binds more strongly to a specific target than to another target.
  • An agent binds more strongly to the first target than to the second target if its dissociation constant ( KD ) for binding to the first target is less than the dissociation constant for the second target.
  • the dissociation constant (K D ) of the target to which the agent specifically binds is more than 10 2 times, 10 3 times, 10 4 times, or more than the dissociation constant (K D ) of the target to which the agent does not specifically bind. 10 5 times, 10 6 times, 10 7 times, 10 8 times, 10 9 times or 10 10 times lower.
  • an agent e.g., a protein or a polypeptide
  • a target if it is capable of binding to a predetermined target but it is not capable of binding to other targets, i.e. it has no significant affinity for and does not bind significantly to other targets in a standard assay.
  • an agent is DDR2 specific if it is able to bind to DDR2 but is (substantially) unable to bind to other targets.
  • the agent binds to the intended target with a KD that is at least 102- fold, 103 -fold, 104-fold, 105 -fold, 106 -fold, 107 - fold, 108-fold greater than the KD of binding to its non- specific target , 10 9 -fold or 10 10 -fold lower, the agent is specific for the target.
  • Binding of the agent to the target can be determined experimentally using any suitable method and is within the scope of one skilled in the art. Affinity can be readily determined using conventional techniques, such as by equilibrium dialysis; Surface plasmon resonance analysis is used by using general procedures outlined by the manufacturer; by radioimmunoassay using radiolabeled target antigen; or by other methods known to the skilled artisan. Affinity data can be analyzed, for example, by methods known in the art. The measured affinity for a particular interaction can vary if measured under different conditions (eg salt concentration, pH). Therefore, measurements of affinity and other binding parameters (eg, KD , IC50 ) are preferably performed with standardized solutions of binding agent and target and standardized buffers.
  • affinity and other binding parameters eg, KD , IC50
  • the term “antibody” refers to any form of antibody that exhibits a desired biological activity, such as inhibiting the binding of a ligand to its receptor or by inhibiting ligand-induced receptor signaling.
  • Antibody fragment and “antigen-binding fragment” refer to antigen-binding fragments of antibodies and antibody analogs, which generally include at least part of the antigen-binding region or variable region (eg, one or more CDRs) of the parent antibody.
  • the antibody is a monoclonal antibody. In other embodiments, the antibody is a polyclonal antibody.
  • the term "monoclonal antibody” as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, ie, the individual antibodies making up the population are identical except for possible natural mutations that may be present in minor amounts. Monoclonal antibodies are highly specific and target a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations, which typically include multiple different antibodies directed against multiple different determinants (epitopes), each monoclonal antibody is directed against only a single determinant on the antigen.
  • the modifier "monoclonal” indicates the character of an antibody obtained from a substantially homogeneous population of antibodies and is not to be understood as requiring that the antibody be prepared by any particular method. For example, monoclonal antibodies for use in the present invention can be prepared by hybridoma or recombinant DNA methods.
  • Monoclonal antibodies can include "chimeric" antibodies, humanized antibodies, or fully human antibodies.
  • the antibody constitutes part of a larger biomolecule, such as a fusion protein or an antibody drug conjugate.
  • Antibody fragments retain at least some of the binding specificity of the parent antibody. Typically, when the activity is expressed on a molar basis, the antibody fragment retains at least 10% of the parent binding activity. Preferably, the antibody fragment retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the binding affinity of the parent antibody to the target.
  • heavy chain antibody refers to an antibody lacking a light chain and consisting only of a heavy chain, which contains two constant regions (CH2 and CH3), a hinge region and a heavy chain variable region (i.e. VHH). Examples include, but are not limited to, native heavy chain antibodies, antibodies naturally lacking light chains, heavy chain antibodies derived from conventional 4-chain antibodies, and engineered antibodies. Heavy chain antibodies may be derived from species of the family Camelidae, such as those produced in camels, llamas, dromedaries, alpacas, and draft horses. Species other than Camelidae may produce heavy chain antibodies that naturally lack light chains; such heavy chain antibodies are within the scope of the invention.
  • nanobody refers to a single-domain antibody composed only of the heavy chain variable region obtained by cloning the variable region of a heavy chain antibody, also known as VHH (Variable domain of heavy chain). of heavy chain antibody) or single domain antibody, is the smallest functional antigen-binding fragment.
  • Nanobodies recognize antigens with high specificity and affinity similar to IgG antibodies, but can better penetrate tumor tissue due to their smaller size ( ⁇ 15 kD). In addition, nanobodies are resistant to extreme pH, thermal denaturation, proteolysis, solvents and detergents. They can be expressed and produced in high yields and high solubility.
  • Antibody fragment and “antigen-binding fragment” refer to antigen-binding fragments of antibodies and antibody analogs, which generally include at least part of the antigen-binding region or variable region (eg, one or more CDRs) of the parent antibody. Antibody fragments retain at least some of the binding specificity of the parent antibody. Typically, an antibody fragment retains at least 10% of the parent binding activity when activity is expressed on a molar basis. Preferably, the antibody fragment retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the binding affinity of the parent antibody for the target.
  • antibody fragments include, but are not limited to: Fab, Fab', F(ab') 2 and Fv fragments; diabodies; linear antibodies; single chain antibody molecules, such as scFv (single chain variable fragment); Nanobodies; domain antibodies ; and multispecific antibodies formed from antibody fragments.
  • Antibodies against DDR2 refer to antibodies that specifically bind to DDR2, including artificially designed antibodies, and any form of antibodies, such as antibody fragments and antigen-binding fragments as defined above.
  • an “equivalent variant” of an antibody or polypeptide refers to an antibody or polypeptide that has a certain degree of homology or sequence identity with the amino acid sequence of the antibody or polypeptide. In some aspects, the sequence identity is at least about 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%. In some aspects, equivalent variants have one, two, three, four or five additions, deletions, substitutions and combinations thereof compared to a reference antibody or polypeptide. In some aspects, equivalent variants of an antibody or polypeptide retain the activity (eg, epitope binding) or structure (eg, salt bridges) of the reference sequence.
  • a "variant" of a sequence refers to a sequence that differs from the sequence shown at one or more amino acid residues but retains the biological activity of the resulting molecule.
  • nucleic acid as used herein is intended to include deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), such as genomic DNA, cDNA, mRNA, recombinantly produced and chemically synthesized molecules. Nucleic acids can be single-stranded or double-stranded. RNA includes in vitro transcribed RNA or synthetic RNA.
  • control refers to a sample that serves as a reference (usually a known reference) for comparison with a test sample.
  • a test sample may be taken 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.
  • Controls may also represent averages collected from a population of similar individuals (eg, disease patients or healthy individuals with similar medical backgrounds, same age, weight, etc.). Control values may also be obtained from the same individual, for example from a sample obtained earlier, before disease, or before treatment.
  • controls can be designed to assess many parameters.
  • body fluid or “body fluid sample” may generally refer to a fluid that is typically present in the body or body tissue of a subject or patient and/or may be produced by the body of a subject or patient.
  • body fluids may include peripheral blood, serum, plasma, serosal fluid, sputum, synovial fluid, aqueous humor, amniotic fluid, breast milk, semen, prostatic fluid, Cowper's fluid, female ejaculate, sweat, excreta, tears, Cyst fluid, pleural effusion, ascites fluid, pericardial fluid, chyle, bile, interstitial fluid, menstrual blood, pus, vomitus, vaginal secretions, mucosal secretions, pancreatic juice, blastocoel fluid, umbilical cord blood, urine, One or more of cerebrospinal fluid, saliva, lymph fluid, loose stool, bronchopulmonary aspirate fluid, bronchoalveolar lavage fluid and nasal lavage fluid, including components or fraction
  • Bodily fluid samples can be mixed or combined.
  • the body fluid sample may be provided by removing the body fluid from the patient, but may also be provided by using previously isolated body fluid sample material.
  • the body fluid or body fluid sample used in the present invention is a serum or plasma sample.
  • exosome refers to tiny membrane vesicles with a diameter of approximately 30-150 nm, secreted by a variety of cells, and containing specific proteins (e.g., the exosome membrane is rich in proteins involved in exocytosis).
  • a variety of cells can secrete exosomes under normal and pathological conditions. They are widely found in body fluids such as blood, saliva, urine, cerebrospinal fluid, and breast milk. They are regarded as specifically secreted membrane vesicles and participate in intercellular communication.
  • the present invention provides reagents for detecting the expression level of Discoid Domain Receptor 2 (DDR2) for use in diagnosing a neurodegenerative disease in a subject.
  • DDR2 Discoid Domain Receptor 2
  • the term "reagent for detecting the expression level of DDR2" refers to any reagent known in the art that can be used to detect DDR2, such as targeting reagents or affinity reagents for DDR2, especially including those that can be used to detect DDR2.
  • DDR2 An agent that binds (especially specifically binds) thereby forming a chemically, physically or biologically detectable complex.
  • the agent capable of binding to DDR2 includes a protein, nucleic acid, or small molecule compound that can target one or more epitopes of the DDR2 protein.
  • the agent capable of binding to DDR2 is an anti-DDR2 monoclonal antibody or an 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), MAB2538 (R&D Systems). For more DDR2 antibodies, see https://www.antibodypedia.com/gene/4177/DDR2 (last accessed on September 1, 2022). Alternatively, anti-DDR2 antibodies can be generated de novo using methods known in the art.
  • the anti-DDR2 antibody is any form of antibody or antibody fragment as defined herein.
  • the anti-DDR2 monoclonal antibody is an anti-DDR2 Nanobody.
  • the anti-DDR2 Nanobody includes CDR1, CDR2, and CDR3, wherein CDR1 includes or is the sequence shown in SEQ ID NO: 1 or an equivalent variant thereof, and CDR2 includes or is the sequence shown in SEQ ID NO: 2.
  • the Nanobody comprises or is the sequence shown in SEQ ID NO: 4 or an equivalent variant thereof.
  • equivalent variants of CDR1, CDR2, and CDR3 refer to substitutions, deletions, or insertions of a single amino acid compared to the reference sequence.
  • an equivalent variant of the Nanobody is one that has at least 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO:4, And have the same or equivalent CDR1, CDR2 and CDR3.
  • the CDR1, CDR2 and CDR3 are defined based on any one of IMGT, Kabat, Chothia, Contact or AbM definition schemes.
  • the CDR1, CDR2 and CDR3 are defined based on the IMGT definition scheme.
  • the anti-DDR2 Nanobody includes CDR1, CDR2, and CDR3, wherein CDR1 includes the sequence shown in SEQ ID NO:1, CDR2 includes the sequence shown in SEQ ID NO:2, and CDR3 includes SEQ ID NO: The sequence shown in 3, where the CDRs are defined according to IMGT.
  • the anti-DDR2 Nanobody includes CDR1, CDR2, and CDR3, wherein CDR1 is the sequence shown in SEQ ID NO: 1, CDR2 is the sequence shown in SEQ ID NO: 2, and CDR3 is SEQ ID NO: The sequence shown in 3, where the CDRs are defined according to IMGT.
  • substitutions described herein are conservative substitutions.
  • Consative (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 (such as glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (such as alanine, valine acid, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), ⁇ -branched side chains (e.g., threonine, valine, isoleucine ) and aromatic side chains (e.g.
  • a non-essential amino acid residue in an immunoglobulin polypeptide is preferably replaced by another amino acid residue from the same side chain family.
  • a string of amino acids can be substituted with a structurally similar string that differs in the order and/or composition of the side chain family members.
  • the agent capable of binding to DDR2 is a peptide or nucleic acid aptamer.
  • 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, systematic evolution of ligands through exponential enrichment).
  • Peptide aptamers can be selected using yeast or bacterial two-hybrid systems.
  • the agent capable of binding to DDR2 is labeled with a detectable label.
  • the detectable label is selected from the group consisting of fluorescent labels, chemiluminescent labels, paramagnetic labels, radioisotope labels, and enzyme labels.
  • marker depends on the means of detection.
  • fluorescent labels such as indocyanine green (ICG), rare earth chelates (such as europium chelates)
  • fluorescein-type labels such as fluorescein, fluorescein isothiocyanate, 5- Carboxyfluorescein, 6-carboxyfluorescein, dichlorotriazinylamine fluorescein
  • rhodamine-type markers such as ALEXA568 (Invitrogen), or dansyl chloride (dansyl chloride)
  • VIVOTAG 680XLFLUOROCHROMETM Perkin Elmer
  • algae Red pigment 7-hydroxycoumarin, lissamine, cyanine, phycoerythrin, Texas Red, BODIPY (Invitrogen) or their analogs are suitable for optical detection.
  • Chemiluminescent labels eg, luminol, luciferase, luciferin, and aequorin
  • diagnosis and detection can also be accomplished by connecting reagents capable of binding to DDR2 to detectable substances, including but not limited to: various enzymes, including but not limited to horseradish peroxidase. , alkaline phosphatase, beta-galactosidase or acetylcholinesterase, or by linking it to prosthetic group complexes such as, but not limited to: streptavidin /biotin and avidin/biotin.
  • Radioactive labels include, but are not limited to, 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), lanthane ( 213 Bi), carbon ( 11 C, 13 C, 14 C), chromium ( 51 Cr), cobalt ( 57 Co, 60 Co), copper ( 64 Cu), dysprosium ( 165 Dy
  • the present invention provides reagents for detecting the expression level of DDR2 for use in diagnosing neurodegenerative diseases in a subject, and the reagent for detecting the expression level of DDR2 includes an anti-DDR2 Nanobody and a Nanobody with the Nanobody.
  • a detectable label is attached, wherein the detectable label is a fluorescent label.
  • the fluorescent label is indocyanine green (ICG).
  • the present invention provides a reagent for detecting the expression level of DDR2 for diagnosing a neurodegenerative disease in a subject, wherein the reagent for detecting the expression level of DDR2 comprises an anti-DDR2 nanobody and a detectable label linked to the nanobody, wherein the detectable label is a radioactive isotope.
  • the radioactive isotope is 68 Ga or 64 Cu.
  • DDR2 Discoid Domain Receptor 2
  • the term "neurodegenerative disease” refers to all diseases associated with degenerative changes in nerve cells of the central nervous system. In particular, it includes conditions selected from: Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease, Huntington's disease, frontotemporal dementia, spinal muscular atrophy, prion diseases, spinocerebellar ataxia, Friedrich's ataxia, primary lateral sclerosis, spinocerebellar atrophy, Machado-Joseph disease, Lewy body dementia, progressive bulbar palsy, progressive supranuclear palsy, and multiple system atrophy, but not limited to this.
  • the neurodegenerative disease is associated with abnormal activation of astrocytes.
  • the neurodegenerative disease is Alzheimer's disease or amyotrophic lateral sclerosis.
  • AD Alzheimer's disease
  • AD is an age-related progressive neurodegenerative disease.
  • the core symptom of AD is memory impairment accompanied by damage to other cognitive domains, which affects normal life and work.
  • the onset of AD is insidious, the course of the disease is irreversible, and the disability rate is high. Therefore, early diagnosis and treatment are of great significance to improve the survival rate of AD patients, especially to improve their quality of life.
  • the classic biomarkers for early AD detection include amyloid ⁇ -protein 42 (A ⁇ 42 ), total tau (T-tau) and phosphorylated tau (P-tau), as well as newly discovered neurogranulins and microRNAs.
  • a ⁇ 42 amyloid ⁇ -protein 42
  • T-tau total tau
  • P-tau phosphorylated tau
  • AD neurogranulins and microRNAs.
  • T-tau and P-tau in the cerebrospinal fluid (CSF) of AD patients are significantly increased
  • a ⁇ 42 is significantly reduced
  • neurogranulins in the CSF of AD patients are significantly increased
  • miR-100, miR-1274a and miR-146a are significantly differentially expressed in AD.
  • Existing biomarkers cannot meet clinical needs, making the search for early AD biomarkers imminent.
  • AD Alzheimer's disease
  • drugs can slow the progression of the disease, such as cholinesterase inhibitors (increase the amount of the neurotransmitter acetylcholine in the brain and promote intercellular signaling), NMDA receptor antagonists (change Brain cell signaling), but the efficacy of these drugs is still not good enough, especially for patients with moderate to severe AD, and they still fail to meet the clinical needs of patients.
  • astrocytes have an impact on the pathological changes of A ⁇ and tau protein.
  • Genetic gene bank research shows that there are more than 40 gene loci related to late-onset AD, most of which are expressed in astrocytes, and astrocytes play a role in neuroinflammation and neurodegeneration in AD. Plays a major role in the sexual transformation process. Therefore, astrocytes may be involved in the pathological development and progression of AD.
  • ALS Amyotrophic lateral sclerosis
  • ALS is the most common type of motor neuron disease, in which both upper and lower motor neurons are affected.
  • Pathologically ALS patients show reduced numbers of motor neurons in the anterior horn of their spinal cord, brainstem, and motor cortex. Loss of motor neurons leads to muscle denervation and atrophy. Patients are characterized by progressive loss of motor function, including muscle weakness and dysphagia, and ultimately die from respiratory failure.
  • Most ALS cases are sporadic, and 5 to 10% are familial.
  • SOD1 is the first ALS-related gene discovered, and its mutations account for 20% of familial cases. So far, more than 20 genes have been found to cause familial ALS when mutated, and genetic mutations have also been found in sporadic cases. Although genetic heterogeneity exists, protein deposition in affected areas is a common hallmark of ALS.
  • astrocytes When healthy, astrocytes help protect and nourish surrounding motor neurons.
  • ALS patients suggest that changes in astrocytes may contribute to the disease.
  • astrocytes with different ALS gene mutations also had different underlying molecular patterns. This suggests that during ALS, astrocytes acquire mutation-dependent changes.
  • the present invention provides a kit for diagnosing a neurodegenerative disease in a subject, the kit comprising a reagent for detecting the expression level of Discoid Domain Receptor 2 (DDR2), wherein from the Levels of DDR2 in a subject's sample that are higher than levels in controls without the disease indicate that the subject has a neurodegenerative disease.
  • DDR2 Discoid Domain Receptor 2
  • the present invention provides a kit for diagnosing neurodegenerative diseases in a subject, the kit comprising exosomes from the subject and capable of detecting Discoid Domain Receptor 2 (DDR2 ) binds a reagent to detect the level of DDR2 expressed by the exosomes, wherein a level of DDR2 expressed by the exosomes that is greater than a level in a control without the disease indicates that the subject suffers from a neurodegenerative disease.
  • DDR2 Discoid Domain Receptor 2
  • the reagent for detecting the expression level of DDR2 includes a reagent, such as a protein, that is capable of binding (especially specifically binding) to DDR2 to form a chemically, physically or biologically detectable complex.
  • a reagent such as a protein
  • nucleic acids or small molecule compounds especially anti-DDR2 monoclonal antibodies or antigen-binding fragments thereof, or anti-DDR2 polyclonal antibodies.
  • the anti-DDR2 monoclonal antibody is an anti-DDR2 Nanobody.
  • the anti-DDR2 Nanobody includes CDR1, CDR2, and CDR3, wherein CDR1 includes or is the sequence shown in SEQ ID NO: 1 or an equivalent variant thereof, and CDR2 includes or is the sequence shown in SEQ ID NO: 2.
  • the Nanobody comprises or is the sequence shown in SEQ ID NO: 4 or an equivalent variant thereof.
  • the agent capable of binding to DDR2 is labeled with a detectable label.
  • the detectable label is selected from the group consisting of fluorescent labels, chemiluminescent labels, paramagnetic labels, radioisotope labels, and enzyme labels.
  • the marker is indocyanine green (ICG).
  • the label is 68 Ga or 64 Cu.
  • the kit further comprises DDR2 recombinant antigen reference substances of different concentrations, so as to prepare a standard curve for quantitative identification.
  • the present invention provides use of discoidin domain receptor 2 (DDR2) as a marker for diagnosing a neurodegenerative disease in a subject.
  • DDR2 discoidin domain receptor 2
  • the invention provides a method for diagnosing a neurodegenerative disease in a subject in vitro or ex vivo, the method comprising detecting a sample from the subject (e.g., brain tissue, cerebrospinal fluid, or The presence and/or levels of Discoid Domain Receptor 2 (DDR2) in exosomes.
  • a sample from the subject e.g., brain tissue, cerebrospinal fluid, or The presence and/or levels of Discoid Domain Receptor 2 (DDR2) in exosomes.
  • DDR2 Discoid Domain Receptor 2
  • the present invention provides agents capable of binding to Discoid Domain Receptor 2 (DDR2) for use in diagnosing neurodegenerative diseases in a subject in vivo.
  • DDR2 Discoid Domain Receptor 2
  • 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.
  • the agent capable of binding to DDR2 is an anti-DDR2 monoclonal antibody or an antigen-binding fragment thereof.
  • the anti-DDR2 monoclonal antibody is an anti-DDR2 Nanobody.
  • the anti-DDR2 Nanobody includes CDR1, CDR2, and CDR3, wherein CDR1 includes or is the sequence shown in SEQ ID NO: 1 or an equivalent variant thereof, and CDR2 includes or is the sequence shown in SEQ ID NO: 2.
  • the Nanobody comprises or is the sequence shown in SEQ ID NO: 4 or an equivalent variant thereof.
  • the agent capable of binding to DDR2 is labeled with a detectable label.
  • the detectable label is selected from the group consisting of fluorescent labels, chemiluminescent labels, paramagnetic labels, radioisotope labels and enzyme labels.
  • the detectable label is selected from fluorescent labels or chemiluminescent labels.
  • the marker is indocyanine green (ICG).
  • the label is 68 Ga or 64 Cu.
  • 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, spinal cord disease Cerebellar ataxia, Friedrich's ataxia, primary lateral sclerosis, spinocerebellar atrophy, Machado-Joseph disease, Lewy body dementia, progressive bulbar palsy, progressive supranuclear palsy, and multiple system atrophy .
  • the neurodegenerative disease is associated with abnormal activation of astrocytes.
  • the neurodegenerative disease is Alzheimer's disease or amyotrophic lateral sclerosis.
  • immunoassays can be used in diagnostic methods.
  • such immunoassays include the use of, for example, radioimmunoassays, immunochromatography, ELISA, "sandwich” immunoassays, precipitation reactions, immunoblot analysis, gel diffusion precipitation reactions, immunodiffusion assays, agglutination Detection, complement fixation detection, immune radiation dose detection, fluorescence immunoassay and other competitive and non-competitive detection systems. Both in vitro and in vivo assays can be used.
  • the level of DDR2 in a sample is compared to a reference level, where deviation from the reference level is indicative of the presence and/or stage of the neurodegenerative disease in the subject.
  • the reference level may be a level determined in a control sample (eg, from healthy tissue or subjects) or a median level from healthy subjects.
  • the presence of DDR2 and/or an increased amount of DDR2 in a sample compared to a reference level, eg compared to a subject without the disease may be indicative of the presence or risk of developing a neurodegenerative disease in the subject.
  • the DDR2 in a sample (eg, brain tissue, cerebrospinal fluid, or exosomes) from a subject with a neurodegenerative disease is at least about 2-fold, at least about 5-fold, Present in an amount of at least about 7.5 times, at least about 10 times, at least about 15 times, or at least about 20 times.
  • the method used for diagnosis allows for quantitative and/or qualitative evaluation, such as absolute and/or relative measurement of the target molecule, for example measuring the content of DDR2 in the sample.
  • determining the presence and/or amount of DDR2 in the sample includes: (i) contacting the sample (e.g., brain tissue, cerebrospinal fluid, or exosomes) with an agent capable of binding to DDR2, and (ii) ) detects the formation of a complex between the agent and DDR2 and/or determines the amount of the complex.
  • the sample e.g., brain tissue, cerebrospinal fluid, or exosomes
  • the detection/diagnosis methods of the present invention can be used in conjunction with other methods of detection/diagnosis of neurodegenerative diseases.
  • the detection/diagnostic methods of the invention can be used in conjunction with detection of other biomarkers of neurodegenerative diseases (eg amyloid beta, tau, APOE, GFAP, AQP4, MAPT, SOD1).
  • biomarkers of neurodegenerative diseases eg amyloid beta, tau, APOE, GFAP, AQP4, MAPT, SOD1.
  • the invention provides a computer-readable storage medium having computer instructions stored thereon for reading and execution by a computer, the computer instructions being executed to perform a method of diagnosing whether a subject suffers from a neurodegenerative disease
  • the method includes: (a) contacting a sample from the subject with a reagent capable of binding to Discodomain Receptor 2 (DDR2); (b) detecting and reading the signal of the contacted sample to determine whether whether the reagent forms a complex with DDR2 in the sample; and (c) determining whether the signal exceeds a predetermined threshold, and when the signal exceeds the predetermined threshold, it is determined that the subject suffers from a neurodegenerative disease.
  • the threshold is the median level from subjects without the disease.
  • the invention provides a computer-readable storage medium having computer instructions stored thereon for reading and execution by a computer, the computer instructions being executed to perform a method of diagnosing whether a subject suffers from a neurodegenerative disease,
  • the method includes: (a) isolating exosomes from the subject; (b) contacting the isolated exosomes with a reagent capable of binding to Discodomain Receptor 2 (DDR2); (c) detecting and reading Taking the signal of the contacted exosomes to determine whether the agent forms a complex with DDR2 in the exosomes; and (d) determining whether the signal exceeds a predetermined threshold, and when the signal exceeds the predetermined threshold,
  • DDR2 Discodomain Receptor 2
  • the subject is determined to have a neurodegenerative disease, and wherein the threshold is a median level from subjects without the disease.
  • the invention provides a method of treating a neurodegenerative disease in a subject, the method comprising: (a) combining a sample from the subject with a protein capable of binding to Discoid Domain Receptor 2 (DDR2) Contact the reagent; (b) detect and read the signal of the contacted sample to determine whether the reagent forms a complex with DDR2 in the sample; (c) determine whether the signal exceeds a predetermined threshold, and when the signal exceeds determining the subject to have a neurodegenerative disease when predetermining a threshold, and wherein the threshold is a median level from subjects who do not have the disease; and (d) administering neuroprotection to the subject determined to have the neurodegenerative disease or neurorestorative therapy.
  • DDR2 Discoid Domain Receptor 2
  • the invention provides a method of treating a neurodegenerative disease in a subject, the method comprising: (a) isolating exosomes from the subject; (b) combining the isolated exosomes with a substance capable of interacting with the disc (c) detect and read the signal of the exosomes after contact to determine whether the reagent forms a complex with DDR2 in the exosomes; (d) Determining whether the signal exceeds a predetermined threshold, and when the signal exceeds the predetermined threshold, determining that the subject suffers from a neurodegenerative disease, and wherein the threshold is a median level from subjects without the disease; and (e ) administering a neuroprotective or neurorestorative therapy to a subject determined to have a neurodegenerative disease.
  • the agent capable of binding to DDR2 includes a protein, nucleic acid or small molecule compound.
  • the agent capable of binding to DDR2 is an anti-DDR2 monoclonal antibody or an antigen-binding fragment thereof, or an anti-DDR2 polyclonal antibody.
  • the agent capable of binding to DDR2 is labeled with a detectable label.
  • the detectable label is selected from the group consisting of fluorescent labels, chemiluminescent labels, paramagnetic labels, radioisotope labels, and enzyme labels.
  • the marker is indocyanine green (ICG).
  • the label is 68 Ga or 64 Cu.
  • the neuroprotective or neurorepair therapy is, for example: a cholinesterase inhibitor, such as galantamine, donepezil, huperzine A, rivastigmine; an excitatory amino acid receptor antagonist, For example, memantine; inflammatory factor inhibitors, such as nonsteroidal anti-inflammatory drugs; glutamate inhibitors, such as riluzole; free radical scavengers, such as edaravone.
  • a cholinesterase inhibitor such as galantamine, donepezil, huperzine A, rivastigmine
  • an excitatory amino acid receptor antagonist for example, memantine
  • inflammatory factor inhibitors such as nonsteroidal anti-inflammatory drugs
  • glutamate inhibitors such as riluzole
  • free radical scavengers such as edaravone.
  • the predetermined threshold may be a reference level as described above, wherein deviations from the reference level are indicative of the presence and/or stage of the relevant disease in the subject.
  • the reference level may be a level determined in a control sample (eg, from healthy tissue or subjects) or a median level from healthy subjects.
  • the presence of DDR2 and/or an increased amount of DDR2 in a sample compared to a reference level, eg compared to a subject without the disease may be indicative of the presence or risk of developing a neurodegenerative disease in the subject.
  • DDR2 is present in a sample (e.g., brain tissue or exosomes) of a subject with a neurodegenerative disease at least about 2 times, at least about 5 times, at least about 7.5 times, at least about 10 times, at least about 15 times, or at least about 20 times the amount in a subject without the disease.
  • a sample e.g., brain tissue or exosomes
  • Anti-DDR2 heavy chain antibodies were screened from alpacas immunized with the human DDR2 extracellular segment (UniProtKB/Swiss-Prot: Q16832.2aa 22 to aa 399). The antibody was then sequenced to confirm its VHH part, and the Nanobody was obtained, named 1A12. Its sequence is shown in the "Sequence Listing" section above. The Nanobody was expressed and purified for further characterization and experiments.
  • the above-mentioned DDR2 nanobody 1A12 was used to determine the affinity between the antibody and the antigen using the BLI method, and the affinity between the nanobody and the extracellular segment protein of the DDR2 antigen was analyzed and detected.
  • Antibody dissolved in PBS (pH 7.4); Antigen: dissolved in PBS (pH 7.4); Sensor: Ni-NTA; Kinetics Buffer: PBST (PBS+0.02% Tween-20, pH 7.4); Regeneration Buffer: 10mM Glycine-HCl, pH 1.7; Re-charged Buffer: 10mM NiCl in H2O.
  • Baseline 1 Baseline the Biosensors in kinetics buffer for 180s;
  • Fixation Use kinetics buffer to dilute the His-tagged antigen DDR2 extracellular segment to 20 ⁇ g/ml, and capture it with the sensor; dilute it to 4nM (300s);
  • Binding Dilute the antibody solution to a certain concentration with kinetics buffer (from 100nM, 2-fold dilution to 3.125nM), and the sensor is inserted into the antibody solution to bind (600s);
  • the DDR2 antibody used in this example is Nanobody 1A12 (Nb-DDR2) in Example 1, and its amino acid sequence is shown in SEQ ID NO: 4.
  • Nb-DDR2 was dissolved in PBS at a concentration of 2 mg/mL and vortexed to mix.
  • ALS Amyotrophic lateral sclerosis
  • ALS mouse model construction Purchase Optn-CKO mice, Optn flox/+ C57BL/6J mice were constructed by Southern Model Biology Company, and the mice were crossed to obtain Optnf lox/flox . The 3-week-old Optn flox/+ mice were divided into cages and ear-tagged and numbered. The tails of the mice were cut. The genotypes of the mice were identified using PCR and agarose gel electrophoresis. Optn flox/flox mice were screened out. At the age of 2 months, Adenovirus, hAd5-Opt Cre virus, 6.58*10 10 PFU/ml, 20ul/animal was injected into the lateral ventricle. After the Cre enzyme is expressed, the OPTN gene can be specifically knocked out through the loxp site. Imaging 3 months after Cre virus injection.
  • the imaging method is as follows: Indocyanine green (ICG)-labeled DDR2 nanobody 1A12 is injected into the tail vein of experimental mice (wild-type mice, ALS model mice), and the control group ALS model mice are injected with normal saline at a dose of 15 ⁇ g. /Only, use an in vivo imager for imaging, excitation light/emission light selection: 730/820nm. The results are shown in Figure 2.
  • ICG Indocyanine green
  • ICG-labeled DDR2 antibody can specifically recognize DDR2, which is highly expressed in the brain tissue of ALS mice, while there is no obvious uptake in wild-type and saline controls. Therefore, ALS can be accurately diagnosed by detecting the expression of DDR2.
  • AD Alzheimer's disease
  • This example uses APP/PS1 double transgenic mice, which are commonly used transgenic mice for AD. Age of the mouse: 2 years. Control group: wild-type C57 mice.
  • ICG-labeled DDR2 antibody can specifically recognize DDR2, which is highly expressed in the brain tissue of AD mice, while the wild-type control has no obvious uptake. Therefore, AD can be accurately diagnosed by detecting the expression of DDR2.
  • the detection method is as follows:
  • Extraction of exosomes Mix 200 ⁇ L of plasma and 350 ⁇ L of PBS, put it into an exosome separator (EXODUS series H-300 instrument from Huixin Biotech), and use 400 ⁇ L of PBS to resuspend the exosomes.
  • Exosome identification Rockchip-Nano Coulter particle size analyzer detects that 90% of the exosome particle size range is between 30-120nm, indicating that the extracted exosomes are exosomes.
  • the secondary antibody is streptavidin-HRP (RD, Cat. No. DY998, 1:5000); incubate for 1 hour and develop color by ECL.
  • Extraction of exosomes Mix 200 ⁇ L of plasma and 350 ⁇ L of PBS, put it into an exosome separator (EXODUS series H-300 instrument from Huixin Biotech), and use 400 ⁇ L of PBS to resuspend the exosomes.
  • Exosome identification Rockchip-Nano Coulter particle size analyzer detects that 90% of the exosome particle size range is between 30-120nm, indicating that the extracted exosomes are exosomes.
  • ALSFRS-R ALS Functional Rating Scale-Revised Rating determined. This scoring scale is well known in the art, details of which can be found, for example, at https://neurotoolkit.com/alsfrs-r/ or https://www.outcomes-umassmed.org/ALS/alsscale.aspx (last accessed 2022 September 19, 2019).
  • ALS Amyotrophic lateral sclerosis
  • the data analyzed in this example come from the brain tissue of 13 normal donors in Allen brain map (https://portal.brain-map.org/atlases-and-data/rnaseq/human-m1-10x), from NCBI GSE174332
  • the motor cortex tissue of the brains of 16 ALS patients contained a total of 239,531 cells.
  • FIG. 6A, B, and C show the cell distribution and expression level of DDR2 in normal brain tissue and ALS patient brain tissue, as well as the ratio and distribution of DDR2-expressing cells, respectively.
  • the data has 20 samples, taken from the entorhinal cortex (EC) and superior frontal gyrus (SFG) of ten AD patients. Among them, the number of cells in the entorhinal cortex: 42528, and the number of cells in the superior frontal gyrus: 63508.
  • the right pictures in the upper and lower parts of Figure 7 are the dimensionality reduction maps of tSNE cells in the entorhinal cortex (EC) and superior frontal gyrus (SFG) of ten patients respectively.
  • the left picture is the Feature plot of DDR2. Each point represents a cell.
  • the depth of color indicates the level of gene expression.
  • DDR2 is highly expressed in astrocytes in AD samples.
  • the two box plots in Figure 8(a) respectively represent the proportion of cells expressing DDR2 in different types of cells in the entorhinal cortex (EC) and superior frontal gyrus (SFG).
  • the vertical axis represents the proportion of cells expressing DDR2
  • the horizontal axis represents the proportion of cells expressing DDR2.
  • the axes represent different cell types, and each point on the graph represents a sample.
  • the two bar graphs in Figure 8(b) respectively represent the proportion of astrocytes expressing DDR2 in different samples in the entorhinal cortex (EC) and superior frontal gyrus (SFG).
  • the vertical axis represents the proportion of cells expressing DDR2.
  • the horizontal axis represents different samples. The numbers above indicate the number of cells of that cell type.
  • FIG. 8(c) The two box plots in Figure 8(c) represent different degrees of disease respectively.
  • the vertical axis represents the proportion of cells expressing DDR2, and the horizontal axis represents different degrees of disease.
  • Figure Each point in represents a sample, using the statistical test method of wlicox.test.
  • Figure 9 shows the correlation analysis of DDR2 expression with APOE, GFAP, AQP4, and MAPT in astrocytes of the entorhinal cortex (EC) and superior frontal gyrus (SFG) of ten patients.
  • the test method was Spearman, and the cells were screened. , cells in which both genes expressed for correlation analysis were selected.
  • DDR2 is significantly expressed in samples from AD patients and is mainly concentrated on astrocytes.
  • AD diagnostic marker DDR2 has good consistency with existing detection markers and can be used for diagnosing AD.
  • ICG-labeled DDR2 Nanobody 1A12 was prepared as described in Example 2.
  • AD model Alzheimer's disease model
  • C57BL/6 mice with the same background were used as control mice.
  • AD model mice and control mice were injected with 15 ⁇ g ICG-labeled into the tail vein.
  • the DDR2 nanobody 1A12 probe was circulated in the body for 15 minutes and then imaged in vivo in mice. The results are shown on the left side of Figure 10. Compared with the control group (C57BL/6 normal mice), obvious fluorescence signals can be observed in the brains of AD model mice. AD model mice have developed the disease.
  • SOD1 gene mutant mice were selected as the amyotrophic lateral sclerosis (ALS) mouse model, and C57BL/6 mice were used as normal control mice.
  • ALS model mice and control mice were injected with 15 ⁇ g of ICG-labeled DDR2 nanobody 1A12 probe into the tail vein.
  • in vivo imaging of mice was performed after 15 minutes of in vivo circulation. The results are shown on the right side of Figure 10. Compared with the control group (C57BL/6 normal mice), obvious fluorescence signals can be observed in the brains of ALS model mice. ALS model mice have developed the disease.
  • Example 6 Nanobody 1A12 labeled nuclide ( 64 Cu, 68 Ga and other conventional nuclide) process
  • the same AD model mice and ALS model mice as in Example 5 were used, and compared with control normal mice respectively.
  • the above-mentioned experimental mice were all injected with 200 ⁇ Ci 64 Cu-labeled DDR2 nanobody 1A12 probe into the tail vein. After circulating in the body for 45 minutes, PET imaging data were collected, and PET/CT images were obtained after reconstruction. The uptake signal is in the circle.

Landscapes

  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Biochemistry (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Pathology (AREA)
  • Medicinal Chemistry (AREA)
  • Medical Informatics (AREA)
  • Public Health (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Food Science & Technology (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Microbiology (AREA)
  • Data Mining & Analysis (AREA)
  • Databases & Information Systems (AREA)
  • Cell Biology (AREA)
  • Biotechnology (AREA)
  • Epidemiology (AREA)
  • Primary Health Care (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

本发明公开了用于检测盘状结构域受体2(DDR2)的表达水平的试剂在制备用于诊断对象的神经退行性疾病的试剂盒中的应用，其中来自所述对象的样品中的DDR2的水平高于未患疾病的对照的水平表示所述对象患有神经退行性疾病。本发明还公开了用于诊断神经退行性疾病的试剂盒、方法和计算机可读介质。本发明通过检测DDR2来高效、准确地诊断神经退行性疾病。

Description

盘状结构域受体2在诊断神经退行性疾病中的应用及相关的计算机可读介质 技术领域
本发明涉及用于诊断神经退行性疾病的试剂盒、方法和计算机可读介质，更具体地，涉及通过检测盘状结构域受体2(DDR2)来诊断神经退行性疾病。
背景技术
神经退行性疾病是一种由于中枢神经系统神经细胞的退行性变化而引起各种症状的疾病，例如运动和感觉功能受损、如记忆、学习和计算推理等高级功能受到抑制。神经退行性疾病的例子包括但不限于阿尔茨海默病、帕金森病、亨廷顿病、肌萎缩侧索硬化、额颞叶痴呆、脊髓小脑共济失调、脑外伤及继发影响中枢神经系统细胞功能的基因突变等。神经退行性疾病长期以来被认为是不可治愈的复杂疾病，目前还没有通过靶向全身性病理过程来改善神经退行性疾病总体状况的有效药物。
神经退行性疾病的病程通常是一个不可逆的过程，因此，治疗的关键是早期诊断，在疾病早期对其进行干预并延缓病程进展。如果能在病人发病早期进行快速诊断或使潜在病人提前发现风险，针对性地进行治疗或预防，则能有助于患者病程滞留在轻微损伤阶段而减缓恶化，从而保证病人生活质量和减轻社会负担。
目前对各种神经退行性疾病的诊断方法主要是联合诊断，主要包括：神经心理学评估，认知损伤测试；脑成像(例如脑部PET扫描、核磁共振)；脑脊液标志物检测；以及血液检测和遗传风险分析等。但是，常用的诊断方法要么需要对受检者注射一定剂量的放射性物质，要么操作损伤大，易造成外科感染。因此，对神经退行性疾病早期诊断的新标志物开发是未来诊疗的重要方向之一。
盘状结构域受体2(discoidin domain receptor 2)是一种受体酪氨酸激酶(RTK)，它利用细胞外基质蛋白胶原作为其配体。除了其激酶功能外，DDR2还通过激活β1-整合素来促进细胞粘附。DDR2的特有功能是介导细胞外基质的信号向胞内传递，使细胞外基质调节得到平衡，同时参与调控细胞的生长、分化和新陈代谢。虽然细胞外基质胶原蛋白激活DDR2是正常发育和组织稳态所必需的，但这些受体在损伤或疾病后的异常激活是有害的。
DDR2被认为是炎症例如关节炎(如骨关节炎、类风湿性关节炎)和纤维化(如肺纤维化、肝硬化、肾纤维化或皮肤纤维化)的重要靶点。DDR2主要在肾、皮肤、肺、心脏和结缔组织的纤维原细胞、成肌纤维细胞、平滑肌细胞等间质细胞中表达。很多证据表明DDR2的异常表达与多种疾病进程相关，如炎症、肝纤维化、肾纤维化、肺纤维化、皮肤瘢痕和动脉粥样硬化。在小鼠炎症模型研究中发现，老年鼠的膝关节中DDR2表达上调。在风湿性关节炎的大鼠模型研究中，发现其滑膜细胞中DDR2表达上调。
然而，DDR2的表达和功能在神经系统细胞(特别是胶质细胞)中从未被报道过。目前尚未见研究和报道DDR2在神经退行性疾病诊断中的应用。
发明内容
本申请的发明人意外地发现与正常对照相比，神经退行性疾病患者中相关样品的DDR2表达明显增高。发明人利用能够与DDR2结合的试剂检测样品中DDR2的存在和/或水平，从而高效、准确地诊断神经退行性疾病。
在第一方面，本发明提供一种用于诊断对象的神经退行性疾病的试剂盒，所述试剂盒包括用于检测盘状结构域受体2(DDR2)的表达水平的试剂，其中来自所述对象的样品中的DDR2的水平高于未患疾病的对照的水平表示所述对象患有神经退行性疾病。
在第二方面，本发明提供用于检测盘状结构域受体2(DDR2)的表达水平的试剂在制备用于诊断对象的神经退行性疾病的试剂盒中的应用，其中来自所述对象的样品中的DDR2的水平高于未患疾病的对照的水平表示所述对象患有神经退行性疾病。
在第三方面，本发明提供用于检测盘状结构域受体2(DDR2)的表达水平的试剂用于诊断对象的神经退行性疾病，其中来自所述对象的样品中的DDR2的水平高于未患疾病的对照的水平表示所述对象患有神经退行性疾病。
在第四方面，本发明提供一种诊断对象的神经退行性疾病的方法，所述方法包括检测来自所述对象的样品中的盘状结构域受体2(DDR2)的存在和/或水平。在一些实施方式中，所述方法包括将能够与DDR2结合的试剂与来自所述对象的样品接触；检测接触后所述试剂与样品中DDR2形成的复合物的存在；以及基于所述复合物的存在和/或水平来确定所述对象患有或有风险患有神经退行性疾病。
在第五方面，本发明提供一种计算机可读存储介质，其上存储有供计算机读取和执行的计算机指令，所述计算机指令被执行以进行诊断对象是否患有神经退行性疾病的方法，所述方法包括：(a)将来自所述对象的样品与能够与盘状结构域受体2(DDR2)结合的试剂接触；(b)检测并读取接触后的样品的信号，以确定所述试剂是否与样品中的DDR2形成复合物；以及(c)判断所述信号是否超过预定阈值，并且当所述信号超过预定阈值时，确定所述对象患有神经退行性疾病，并且其中所述阈值是来自未患疾病的对象的中值水平。
在第六方面，本发明提供一种治疗对象的神经退行性疾病的方法，所述方法包括：(a)将来自所述对象的样品与能够与盘状结构域受体2(DDR2)结合的试剂接触；(b)检测并读取接触后的样品的信号，以确定所述试剂是否与样品中的DDR2形成复合物；(c)判断所述信号是否超过预定阈值，并且当所述信号超过预定阈值时，确定所述对象患有神经退行性疾病，并且其中所述阈值是来自未患疾病的对象的中值水平；以及(d)向被确定患有神经退行性疾病的对象施用神经保护或神经修复疗法。
在上述任一方面，所述用于检测DDR2的表达水平的试剂包括能够与DDR2结合的试剂，以检测所述样品中的DDR2的水平。在一些实施方式中，所述能够与DDR2结合的试剂包括蛋白质、核酸或小分子化合物。在一些实施方式中，所述能够与DDR2结合的试剂是抗DDR2单克隆抗体或其抗原结合片段，或是抗DDR2多克隆抗体。在一些实施方式中，所述抗DDR2单克隆抗体是抗DDR2纳米抗体。
在一些实施方式中，所述能够与DDR2结合的试剂被可检测的标记物标记。在一些实施方式中，所述可检测的标记物选自荧光标记、化学发光标记、顺磁标记、放射性同位素标记和酶标记。
在上述任一方面，所述样品为脑组织或脑脊液。在上述任一方面，所述神经退行性疾病是星形胶质细胞受损介导的神经退行性疾病。在上述任一方面，所述神经退行性疾病是由DDR2表达升高的星形胶质细胞介导的神经退行性疾病，所述DDR2表达升高是与正常对象的星形胶质细胞的DDR2表达水平比较的。优选地，在上述任一方面，所述神经退行性疾病选自阿尔茨海默病(Alzheimer’s disease，AD)、肌萎缩侧索硬化(amyotrophic lateral sclerosis，ALS)、帕金森病(Parkinson's disease)、亨廷顿病(Huntington’s disease)、额颞叶痴呆(frontotemporal dementia)、脊髓性肌萎缩(spinal muscular atrophy)、朊病毒病(prion disease)、脊髓小脑共济失调(spinocerebella axia)、弗里德里希共济失调(Friedreichs ataxia)、原发性侧索硬化(primary lateral sclerosis)、脊髓小脑萎缩(spinocerebellar atrophy)、马查多-约瑟夫病(Machado-hoseph’s diease)、路易体痴呆(Lewy Body dementia)、进行性延髓麻痹(progressive bulbar palsy)、进行性核上性麻痹(progressive supranuclear palsy)和多系统萎缩(multiple system atrophy)。在更优选的实施方式中，所述神经退行性疾病是阿尔茨海默病。在更优选的实施方式中，所述神经退行性疾病是肌萎缩侧索硬化。
在第七方面，本发明提供一种用于诊断对象的神经退行性疾病的试剂盒，所述试剂盒包括来自所述对象的外泌体，以及能够与检测盘状结构域受体2(DDR2)结合的试剂以检测所述外泌体表达的DDR2的水平，其中所述外泌体表达的DDR2的水平高于未患疾病的对照的水平表示所述对象患有神经退行性疾病。
在第八方面，本发明提供来自对象的外泌体在制备用于诊断所述对象的神经退行性疾病的试剂盒中的应用，其中所述外泌体表达的盘状结构域受体2(DDR2)的水平高于未患疾病的对照的水平表示所述对象患有神经退行性疾病。
在第九方面，本发明提供来自对象的外泌体用于诊断所述对象的神经退行性疾病，其中所述外泌体表达的盘状结构域受体2(DDR2)的水平高于未患疾病的对照的水平表示所述对象患有神经退行性疾病。
在第十方面，本发明提供一种诊断对象的神经退行性疾病的方法，所述方法包括从所述对象分离外泌体以及检测来自所述对象的外泌体中的盘状结构域受体2(DDR2)的存在和/或水平。在一些实施方式中，所述方法包括将能够与DDR2结合的试剂与来自所述对象的外泌体接触；检测接触后所述试剂与外泌体中DDR2形成的复合物的存在；以及基于所述复合物的存在和/或水平来确定所述对象患有或有风险患有神经退行性疾病。
在第十一方面，本发明提供一种计算机可读存储介质，其上存储有供计算机读取和执行的计算机指令，所述计算机指令被执行以进行诊断对象是否患有神经退行性疾病的方法，所述方法包括：(a)从所述对象分离外泌体；(b)将分离的外泌体与能够与盘状结构域受体2(DDR2)结合的试剂接触；(c)检测并读取接触后的外泌体的信号，以确定所述试剂是否与外泌体中的DDR2形成复合物；以及(d)判断所述信号是否超过预定阈值，并且当所述信号超过预定阈值时，确定所述对象患有神经退行性疾病，并且其中所述阈值是来自未患疾病的对象的中值水平。
在第十二方面，本发明提供一种治疗对象的神经退行性疾病的方法，所述方法包括：(a)从所述对象分离外泌体；(b)将分离的外泌体与能够与盘状结构域受体2(DDR2)结合的试剂接触；(c)检测并读取接触后的外泌体的信号，以确定所述试剂是否与外泌体中的DDR2形成复合物；(d)判断所述信号是否超过预定阈值，并且当所述信号超过预定阈值时，确定所述对象患有神经退行性疾病，并且其中所述阈值是来自未患疾病的对象的中值水平；以及(e)向被确定患有神经退行性疾病的对象施用神经保护或神经修复疗法。在优选的实施方式中，所述神经保护或神经修复疗法例如是：胆碱酯酶抑制剂，例如加兰他敏、多奈哌齐、石杉碱甲、卡巴拉汀；NMDA受体拮抗剂，例如美金刚；炎性因子抑制剂，例如非甾体抗炎药；谷氨酸抑制剂，例如利鲁唑；自由基清除剂，例如依达拉奉。
在上述任一方面，所述外泌体来自所述对象的体液。在一些实施方式中，所述体液包括外周血、血清、血浆、浆膜液、痰、滑液、房水、羊水、乳汁、精液、前列腺液、考珀液、女性射出液、汗液、排泄物、泪液、囊液、胸腔积液、腹水液、心包液、乳糜、胆汁、间质液、经血、脓液、呕吐物、阴道分泌物、粘膜分泌物、胰液、囊胚腔液、脐带血、尿液、脑脊液、唾液、淋巴液、稀便、支气管肺抽吸液、支气管肺泡灌洗液和鼻腔灌洗液中的一种或多种。在一些实施方式中，所述体液为血清或血浆。在一些实施方式中，所述外泌体通过尺寸排阻色谱、密度梯度离心、差速离心、纳米膜超滤、免疫吸附捕获、亲和捕获、微流体分离或它们的组合而从来自所述对象的样本分离。
在上述任一方面，所述能够与DDR2结合的试剂包括蛋白质、核酸或小分子化合物。在一些实施方式中，所述能够与DDR2结合的试剂是抗DDR2单克隆抗体或其抗原结合片段，或是抗DDR2多克隆抗体。在一些实施方式中，所述能够与DDR2结合的试剂被可检测的标记物标记。在一些实施方式中，所述可检测的标记物选自荧光标记、化学发光标记、顺磁标记、放射性同位素标记和酶标记。
本发明的其他方面和优点将在具体实施方式中详细描述，本领域技术人员能够从下文的具体描述中意识到未在本公开中明确表述的其他方面和优点。
附图说明
图1：DDR2纳米抗体与DDR2抗原胞外段的亲和常数图。
图2：ALS小鼠模型成像结果图。
图3：AD小鼠模型成像结果图。
图4：ALS病人血浆中外泌体蛋白质印迹检测结果图。
图5：ALS病人血浆中外泌体流式检测结果图。
图6：ALS相关实验数据中的DDR2表达情况。
图7：AD相关实验数据中的DDR2表达情况。
图8：AD相关实验数据中的DDR2表达情况。
图9：DDR2与其他诊断性基因的相关性分析。
图10：DDR2纳米抗体1A12-ICG活体荧光成像。
图11：AD小鼠PET/CT成像。
图12：ALS小鼠PET/CT成像。
具体实施方式
定义
在整个说明书和权利要求书中，除非上下文另有表示，否则词语“包括”以及诸如“包含”和“含有”之类的变体将被理解为包括所述整数、步骤或成分，但不排除任何其他整数、步骤或者成分。当在本文中使用时，术语“包括”可以用术语“包含”或“含有”代替，或者有时在本文中使用术语“具有”代替。
在本发明中，“约”是指数值在由本领域一般技术人员所测定的具体值的可接受误差范围内，所述数值部分取决于怎样测量或测定(即测量体系的限度)。例如，在本领域每一次实践中，“约”可意味着在1以内或超过1的标准差。或者，“约”或“基本上包含”可意味着至多20％的范围。此外，对于生物学系统或过程而言，该术语可意味着至多一个数量级或数值的至多5倍。除非另外说明，否则当具体值在本申请和权利要求中出现时，“约”或“基本上包含”的含义应该假定为在该具体值的可接受误差范围内。
如本文所用，术语“对象”、“患者”或“个体”是指任何期望进行诊断、预后或治疗的对象，特别是哺乳动物对象。哺乳动物包括人、家畜、农畜、动物园动物、竞技动物或宠物，例如狗、猫、猪、兔、大鼠、小鼠、马、牛、奶牛等。本文所称的对象优选是人。在一些实施方案中，对象患有或易患一种或多种病症或病况。患者可表现出病症或病况的一种或多种症状，或可能已被诊断患有一种或多种病症或病况。在一些实施方案中，患者正在接受或已接受用于诊断和/或治疗此类疾病、病症或病况的某种疗法。
如本文中使用的，术语“检测”包括任意检测手段，包括直接和间接检测、定量和定性检测，意指在对象中或来自对象的样品中鉴定特定分子(例如DDR2蛋白)的存在和/或水平。
如本文中使用的，术语“诊断”是指分子或病理学状态、疾病或病症的鉴定或分类。例如，“诊断”可以指神经退行性疾病的鉴定或其具体类型的鉴定。
根据本发明，术语“结合”优选地涉及特异性结合。“特异性结合”意指与另一靶标的结合相比，试剂与特异性靶标更强地结合。如果试剂与第一靶标结合的解离常数(KD)小于对第二靶标的解离常数，则与第二靶标相比其与第一靶标更强地结合。优选地，与试剂不特异性结合的靶标的解离常数(KD)相比，试剂特异性结合的靶标的解离常数(KD)为超过102倍、103倍、104倍、105倍、106倍、107倍、108倍、109倍或1010倍更低。
优选地，如果试剂(例如蛋白或多肽)能够与预定靶标结合而其不能够与其他靶标结合，即在标准测定中对其他靶标不具有显著亲和力并且不与其他靶标显著地结合，则其是所述预定靶标特异性的。根据本发明，如果试剂能够与DDR2结合但是(基本上)不能够与其他靶标结合，则其是DDR2特异性的。优选地，如果试剂与预定靶标结合的KD是与其非特异性靶标结合的KD的至少102倍、103倍、104倍、105倍、106倍、107倍、108倍、109倍或1010倍更低，则试剂是所述靶标特异性的。
试剂与靶标的结合可以使用任何合适的方法经实验确定，这在本领域技术人员的范围之内。亲和力可以使用常规技术容易地确定，例如通过平衡透析； 通过使用制造商概述的一般操作来使用表面等离子体共振分析；通过使用经放射性标记的靶抗原的放射免疫测定；或通过技术人员已知的其他方法。亲和力数据可以例如通过本领域已知的方法分析。如果在不同条件(例如盐浓度、pH)下测量，特定相互作用的测量的亲和力可变化。因此，亲和力和其他结合参数(例如，KD、IC50)的测量优选用结合剂和靶标的标准化溶液和标准化缓冲液进行。
如本文中使用的，术语“抗体”是指表现所需生物学活性(例如抑制配体与其受体的结合或通过抑制配体诱导的受体信号转导)的抗体的任何形式。“抗体片段”和“抗原结合片段”是指抗体的抗原结合片段及抗体类似物，其通常包括至少部分母抗体的抗原结合区或可变区(例如一个或多个CDR)。在一些实施方式中，抗体是单克隆抗体。在另一些实施方式中，抗体是多克隆抗体。
本文所用术语“单克隆抗体”是指从基本上同种抗体群中获得的抗体，即除了可能少量存在的可能的天然突变体外，构成所述群的各个抗体是一致的。单克隆抗体具有高度特异性，可针对单个的抗原位点。此外，与通常包括针对多个不同的决定簇(表位)的多种不同抗体的常规(多克隆)抗体制备物相反，每种单克隆抗体仅针对抗原上的单个决定簇。修饰语“单克隆”表示从基本上同种抗体群获得的抗体的特性，不能理解为需要通过任何特定方法来制备所述抗体。例如，用于本发明的单克隆抗体可通过杂交瘤或重组DNA方法制备。
单克隆抗体可以包括“嵌合”抗体、人源化抗体或全人源抗体。在一些实施方式中，抗体构成更大的生物分子的一部分，例如融合蛋白或抗体药物偶联物。抗体片段保留亲本抗体的至少某些结合特异性。通常，当基于摩尔来表示活性时，抗体片段保留至少10％的亲本结合活性。优选地，抗体片段保留至少20％、50％、70％、80％、90％、95％或100％或更多的亲本抗体对靶标的结合亲和力。
如本文中使用的，术语“重链抗体”是指缺失轻链而只由重链组成的抗体，其包含两个恒定区(CH2和CH3)、一个铰链区和一个重链可变区(即VHH)。实例包括但不限于天然重链抗体、天然没有轻链的抗体、从常规4-链抗体衍生的重链抗体和工程化抗体。重链抗体可以来自骆驼科(Camelidae)物种，例如在骆驼、美洲驼、单峰骆驼、羊驼和驮马中产生的抗体。除了骆驼科之外的其他物种可以产生天然缺少轻链的重链抗体；这种重链抗体在本发明的范围内。
如本文中使用的，术语“纳米抗体(nanobody)”是指克隆重链抗体的可变区而得到的只由重链可变区组成的单域抗体，又称为VHH(Variable domain of heavy chain of heavy chain antibody)或单结构域抗体，是最小的功能性抗原结合片段。纳米抗体识别具有与IgG抗体相似的高特异性和亲和力的抗原，但由于尺寸较小(～15kD)可以更好地穿透肿瘤组织。此外，纳米抗体对极端pH、热变性、蛋白水解、溶剂和去污剂有抵抗作用。它们可以以高收率和高溶解度被表达和生产。
“抗体片段”和“抗原结合片段”是指抗体的抗原结合片段及抗体类似物，其通常包括至少部分母抗体的抗原结合区或可变区(例如一个或多个CDR)。抗体片段保留母抗体的至少某些结合特异性。通常，当基于摩尔来表示活性时，抗体片段保留至少10％的母体结合活性。优选地，抗体片段保留至少20％、50％、70％、80％、90％、95％或100％或更多的母体抗体对靶标的结合亲和力。抗体片段的例子包括但不限于：Fab、Fab'、F(ab')2和Fv片段；双抗体；线性抗体；单链抗体分子，例如scFv(single chain variable fragment)；纳米抗体；结构域抗体；和由抗体片段形成的多特异性抗体。针对DDR2的抗体是指与DDR2特异性结合的抗体，包括人工设计抗体，以及抗体的任何形式，例如如上所定义的抗体片段和抗原结合片段。
抗体或多肽的“等效变体”是指与该抗体或多肽的氨基酸序列具有一定程度的同源性或序列同一性的抗体或多肽。在一些方面，序列同一性为至少约70％、75％、80％、85％、90％、95％、98％或99％。在一些方面，与参考抗体或多肽相比，其等效变体具有一个、两个、三个、四个或五个添加、缺失、取代及它们的组合。在一些方面，抗体或多肽的等效变体保留了参考序列的活性(例如，表位结合)或结构(例如，盐桥)。
如本文中使用的，序列的“变体”是指在一个或多个氨基酸残基处不同于所示的序列但保留所得到的分子的生物学活性的序列。
本文所用的两个序列之间的“％同一性”是指所述序列共有的等同位置的数目的函数(即％同源性＝等同位置数/总位置数x 100)，其中会考虑到空位数目及各空位长度，所述空位需要在进行两个序列最佳比对时引入。序列比较和两个序列之间％同一性的确定可用数学算法来完成。
本文使用的术语“核酸”旨在包括脱氧核糖核酸(DNA)和核糖核酸(RNA)，例如基因组DNA、cDNA、mRNA、重组产生的和化学合成的分子。核酸可以是单链或双链的。RNA包括体外转录的RNA或合成RNA。
术语“对照”、“对照样品”、“标准对照”或“标准品”是指充当用于与测试样品比较的参照(通常是已知参照)的样品。举例来说，测试样品可取自怀疑患有给定疾病的患者，并且与来自已知疾病患者或已知正常(非疾病)个体的样品进行比较。对照也可代表自类似个体(例如疾病患者或具有类似医学背景、相同年龄、重量等的健康个体)的群体采集的平均值。对照值也可自同一个体，例如自较早获得的样品，在疾病之前，或在治疗之前获得。技术人员将认识到对照可被设计用于评估许多参数。
如本文中使用的，术语“体液”或“体液样品”通常可指流体，所述流体通常存在于对象或患者的身体或身体组织中，并且/或可由对象或患者的身体产生。举例来说，体液可以包括外周血、血清、血浆、浆膜液、痰、滑液、房水、羊水、乳汁、精液、前列腺液、考珀液、女性射出液、汗液、排泄物、泪液、囊液、胸腔积液、腹水液、心包液、乳糜、胆汁、间质液、经血、脓液、呕吐物、阴道分泌物、粘膜分泌物、胰液、囊胚腔液、脐带血、尿液、脑脊液、唾液、淋巴液、稀便、支气管肺抽吸液、支气管肺泡灌洗液和鼻腔灌洗液中的一种或多种，包括其组分或级分。体液样品可以混合或合并。体液样品可以通过从患者取出体液来提供，但也可以通过使用先前分离的体液样品材料来提供。在一些实施方式中，本发明使用的体液或体液样品是血清或血浆样品。
如本文中使用的，术语“外泌体(exosome)”是指直径大约为30-150nm的微小膜泡，由多种细胞分泌，含有特定的蛋白质(例如，外泌体膜上富含参与外泌体运输的跨膜蛋白家族CD63、CD81和CD9)、脂质、细胞因子或遗传物质。多种细胞在正常及病理状态下均可分泌外泌体，它们广泛存在于血液、唾液、尿液、脑脊液和乳汁等体液中，被视为特异性分泌的膜泡，参与细胞间通讯。在一些实施方式中，可以通过尺寸排阻色谱、密度梯度离心、差速离心、纳米膜超滤、免疫吸附捕获、亲和捕获、微流体分离或它们的组合而从来自对象的体液样本分离得到外泌体。
用于检测DDR2的表达水平的试剂
在一个方面，本发明提供用于检测盘状结构域受体2(DDR2)的表达水平的试剂用于诊断对象的神经退行性疾病。
如本发明所用，术语“用于检测DDR2的表达水平的试剂”是指本领域已知的任何能够用来检测DDR2的试剂，例如是针对DDR2的靶向试剂或亲和试剂，尤其包括能够与DDR2结合(特别是特异性结合)从而形成化学上、物理上或生物学上可检测的复合物的试剂。
在一些实施方式中，所述能够与DDR2结合的试剂包括蛋白质、核酸或小分子化合物，其可以靶向DDR2蛋白的一个或多个表位。
在一些实施方式中，所述能够与DDR2结合的试剂是抗DDR2单克隆抗体或其抗原结合片段，或是抗DDR2多克隆抗体。抗DDR2抗体可得自商业来源，例如GTX102526(GeneTex)、AF2538(Novus Biologicals)、MAB2538(R&D Systems)。更多的DDR2抗体参见https://www.antibodypedia.com/gene/4177/DDR2(最后一次访问日期2022年9月1日)。可替代地，使用本领域已知的方法，可以从头产生抗DDR2抗体。在一些实施方式中，所述抗DDR2抗体是任何形式的抗体或如本文所定义的抗体片段。
在一些实施方式中，所述抗DDR2单克隆抗体是抗DDR2纳米抗体。在一些实施方式中，所述抗DDR2纳米抗体包含CDR1、CDR2、CDR3,其中CDR1包含或为SEQ ID NO:1所示的序列或其等效变体，CDR2包含或为SEQ ID NO:2所示的序列或其等效变体，CDR3包含或为SEQ ID NO:3所示的序列或其等效变体，其中CDR根据IMGT定义。在一些实施方式中，所述纳米抗体包含或为SEQ ID NO:4所示的序列或其等效变体。
在一些实施方式中，所述CDR1、CDR2、CDR3的等效变体是指与参考序列相比具有单个氨基酸的取代、缺失或插入。
在一些实施方式中，所述纳米抗体的等效变体是指与SEQ ID NO:4具有至少75％、80％、85％、90％、95％、98％或99％的序列同一性，并且具有相同或等效的CDR1、CDR2和CDR3。在一些实施方式中，所述CDR1、CDR2和CDR3是基于IMGT、Kabat、Chothia、Contact或AbM中的任一种定义方案定义的。在一些实施方式中，所述CDR1、CDR2和CDR3是基于IMGT定义方案定义的。
在一些实施方式中，所述抗DDR2纳米抗体包含CDR1、CDR2、CDR3, 其中CDR1包含SEQ ID NO:1所示的序列，CDR2包含SEQ ID NO:2所示的序列，CDR3包含SEQ ID NO:3所示的序列，其中CDR根据IMGT定义。
在一些实施方式中，所述抗DDR2纳米抗体包含CDR1、CDR2、CDR3，其中CDR1为SEQ ID NO:1所示的序列，CDR2为SEQ ID NO:2所示的序列，CDR3为SEQ ID NO:3所示的序列，其中CDR根据IMGT定义。
在一些实施方案中，本文所述的取代是保守性取代。“保守性(氨基酸)取代”是指用具有相似侧链的氨基酸取代氨基酸残基的取代。具有相似侧链的氨基酸残基家族已在本领域中定义，包括碱性侧链(例如赖氨酸、精氨酸、组氨酸)、酸性侧链(例如天冬氨酸、谷氨酸)、不带电荷的极性侧链(例如甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸)、非极性侧链(例如丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸、色氨酸)、β-支链侧链(例如苏氨酸、缬氨酸、异亮氨酸)和芳香侧链(例如酪氨酸、苯丙氨酸、色氨酸、组氨酸)。因此，免疫球蛋白多肽中的非必需氨基酸残基优选被来自相同侧链家族的另一个氨基酸残基取代。在另一个实施方案中，氨基酸串可以用在侧链家族成员的顺序和/或组成上不同的结构相似的串取代。
在一些实施方式中，所述能够与DDR2结合的试剂是肽或核酸适体。通过本领域已知的任意方法，可以从寡核苷酸或肽文库中选择这样的适体。经由SELEX(Systematic Evolution of Ligands by Exponential Enrichment，通过指数富集对配体的系统进化)，可以选择核酸适体。使用酵母或细菌双杂交系统，可以选择肽适体。
在一些实施方式中，所述能够与DDR2结合的试剂被可检测的标记物标记。在一些实施方式中，所述可检测的标记物选自荧光标记、化学发光标记、顺磁标记、放射性同位素标记和酶标记。
标记的选择取决于检测的手段。例如，荧光标记(比如，吲哚菁绿(ICG)、稀土螯合物(例如铕螯合物))、荧光素(fluorescein)型标记(例如，荧光素、异硫氰酸荧光素、5-羧基荧光素、6-羧基荧光素、二氯三嗪基胺荧光素)、若丹明型标记(例如ALEXA568(Invitrogen)、或丹磺酰氯(dansyl chloride))、VIVOTAG 680XLFLUOROCHROMETM(Perkin Elmer)、藻红素、7-羟基香豆素、丽丝胺(Lissamine)、花菁、藻红素、德克萨斯红(Texas Red)、BODIPY (Invitrogen)或其类似物，是适合光学检测的。
也可采用化学发光标记(例如，鲁米诺(luminol)、荧光素酶、虫荧光素(luciferin)和水母发光蛋白)。这样的诊断和检测也可通过将能够与DDR2结合的试剂与可检测的物质连接来完成，所述可检测的物质包括但不限于：各种酶，酶包括但不限于辣根过氧化物酶、碱性磷酸酶、β-半乳糖苷酶或乙酰胆碱酯酶，或通过将其与辅基复合物(prosthetic groupcomplexes)连接来完成，所述辅基复合物例如但不限于：链霉抗生物素/生物素和抗生物素蛋白/生物素。
也可采用顺磁标记和放射性同位素标记，其优选地使用正电子放射断层成像术(Positron Emission Tomography)(PET)或单光子计算机断层成像术(Single-PhotonEmission Computed Tomography)(SPECT)被检测。放射性标记包括但不限于，铋(213Bi)、碳(11C、13C、14C)、铬(51Cr)、钴(57Co、60Co)、铜(64Cu)、镝(165Dy)、铒(169Er)、氟(18F)、钆(153Gd、159Gd)、镓(68Ga、67Ga)、锗(68Ge)、金(198Au)、钬(166Ho)、氢(3H)、铟(111In、112In、113In、115In)、碘(121I、123I、125I、131I)、铱(192Ir)、铁(59Fe)、氪(81mKr)、镧(140La)、镥(177Lu)、锰(54Mn)、钼(99Mo)、氮(13N、15N)、氧(15O)、钯(103Pd)、磷(32P)、钾(42K)、镨(142Pr)、钷(149Pm)、铼(186Re、188Re)、铑(105Rh)、铷(81Rb、82Rb)、钌(82Ru、97Ru)、钐(153Sm)、钪(47Sc)、硒(75Se)、钠(24Na)、锶(85Sr、89Sr、92Sr)、硫(35S)、锝(99Tc)、铊(201Tl)、锡(113Sn、117Sn)、氙(133Xe)、镱(169Yb、175Yb、177Yb)、钇(90Y)和锌(65Zn)；可以使用各种正电子放射断层成像术的正电子发射金属和非放射性顺磁金属离子，比如，顺磁性铝(Al)离子、钡(Ba)离子、钙(Ca)离子、铈(Ce)离子、镝(Dy)离子、铒(Er)离子、铕(Eu)离子、钆(Gd)离子、钬(Ho)离子、铱(Ir)离子、锂(Li)离子、镁(Mg)离子、锰(Mn)离子，钼(M)离子、钕(Nd)离子、锇(Os)离子、氧(O)离子、钯(Pd)离子、铂(Pt)离子、铑(Rh)离子、钌(Ru)离子、钐(Sm)离子、钠(Na)离子、锶(Sr)离子、铽(Tb)离子、铥(Tm)离子、锡(Sn)离子、钛(Ti)离子、钨(W)离子和锆(Zi)离子，尤其是Co+2、CR+2、Cr+3、Cu+2、Fe+2、Fe+3、Ga+3、Mn+3、Ni+2、Ti+3、V+和V+4。制备放射性标记的氨基酸和相关的肽衍生物的方法在本领域是已知的。例如，可通过氯胺T法缀合放射性同位素。
在一些实施方式中，本发明提供用于检测DDR2的表达水平的试剂用于诊断对象的神经退行性疾病，所述用于检测DDR2的表达水平的试剂包括抗 DDR2纳米抗体和与所述纳米抗体连接的可检测的标记，其中所述可检测的标记是荧光标记。在一些实施方式中，所述荧光标记为吲哚菁绿(ICG)。
在一些实施方式中，本发明提供用于检测DDR2的表达水平的试剂用于诊断对象的神经退行性疾病，所述用于检测DDR2的表达水平的试剂包括抗DDR2纳米抗体和与所述纳米抗体连接的可检测的标记，其中所述可检测的标记是放射性同位素。在一些实施方式中，所述放射性同位素为68Ga或64Cu。
诊断的疾病
发明人发现，可以通过检测盘状结构域受体2(DDR2)来诊断神经退行性疾病。
如本文所用，术语“神经退行性疾病”是指与中枢神经系统神经细胞的退行性变化相关的所有疾病。特别地，它包括选自以下的病症：阿尔茨海默病、肌萎缩侧索硬化、帕金森病、亨廷顿病、额颞叶痴呆、脊髓性肌萎缩、朊病毒病、脊髓小脑共济失调、弗里德里希共济失调、原发性侧索硬化、脊髓小脑萎缩、马查多-约瑟夫病、路易体痴呆、进行性延髓麻痹、进行性核上性麻痹和多系统萎缩等，但不限于此。在一些实施方式中，所述神经退行性疾病与星形胶质细胞的异常活化相关。在一些实施方式中，所述神经退行性疾病是阿尔茨海默病或肌萎缩侧索硬化。
阿尔茨海默病(AD)
AD是一种与年龄相关的进行性神经系统变性疾病。AD的核心症状是记忆障碍伴有其他认知域的损害，并对正常生活、工作造成影响。AD起病隐蔽、病程呈不可逆发展，致残率高，因此，早期诊治对提高AD病患生存率、尤其对提高其生存质量意义重大。
目前检测早期AD经典生物标志物有β淀粉样蛋白42(Amyloidβ-protein42，Aβ42)、总tau(T-tau)和磷酸化tau(P-tau)，以及新发现的神经颗粒蛋白及MicroRNA。通常AD患者脑脊液(CSF)中的T-tau和P-tau显著增加，Aβ42显著减少、AD患者CSF神经颗粒蛋白显著增加以及miR-100、miR-1274a和miR-146a在AD中具有显著差异表达。现有的生物标志物不能满足临床需求，使得寻找早期AD生物标志物已迫在眉睫。
尽管没有治愈AD的方法，但一些FDA批准的药物可以延缓疾病进展，如胆碱酯酶抑制剂(增加脑内神经递质乙酰胆碱的量，促进细胞间信号传导)、NMDA受体拮抗剂(改变脑细胞信号传导)，但是这些药物的疗效依然不够好，特别对中重度AD患者，依然未能满足患者的临床需求。
越来越多的研究表明星形胶质细胞对Aβ及tau蛋白的病理变化具有影响。而遗传基因库研究显示，与迟发性AD相关的基因位点达40多个，其中大多数相关基因在星形胶质细胞中表达，且星形胶质细胞在AD的神经炎症和神经退行性变过程中起主要作用。因此，星形胶质细胞可能与AD的病理发展和进展有关。
肌萎缩侧索硬化(ALS)
ALS是最常见的运动神经元疾病类型，这种疾病中，上和下运动神经元都受到影响。在病理学上，ALS患者显示在其脊髓前角、脑干和运动皮层中的运动神经元数量减少。运动神经元的损失导致肌肉去神经支配和萎缩。患者的特征是运动功能逐渐丧失，包括肌肉无力和吞咽困难，最后死于呼吸衰竭。大多数ALS病例是散发性的，5～10％是家族性病例。SOD1是首个发现的ALS相关基因，其突变占家族性病例的20％。到目前为止，发现超过20个基因在突变时引起家族性ALS，在散发病例中也发现了基因突变。虽然存在遗传异质性，但是受影响区域的蛋白质沉积是ALS的常见标志。
目前没有治愈ALS的方法，而利鲁唑(一种谷氨酸释放抑制剂)显示出了温和的效应，能将存活提升数月。经过数十年的研究，仍然没有关于ALS的致病机制的共识，这阻碍了有效疗法的开发。另一种策略试图通过激活内源性神经营养因子通路来提升运动神经元的存活，而不是特异性地阻断导致运动神经元死亡的致病过程。
健康时，星形胶质细胞有助于保护和滋养周围的运动神经元。然而，最近来自ALS患者的发现表明星形胶质细胞的变化可能导致这种疾病。研究人员发现在ALS中，星形胶质细胞失去了重要的保护功能，特别是吸收谷氨酸的能力。这会导致谷氨酸的积累，从而损害运动神经元。在另一项研究中，发现具有不同ALS基因突变的星形胶质细胞也具有不同的潜在分子模式。这表明，在ALS期间，星形胶质细胞获得突变依赖性变化。
试剂盒
在一个方面，本发明提供一种用于诊断对象的神经退行性疾病的试剂盒，所述试剂盒包括用于检测盘状结构域受体2(DDR2)的表达水平的试剂，其中来自所述对象的样品中的DDR2的水平高于未患疾病的对照的水平表示所述对象患有神经退行性疾病。
在另一方面，本发明提供一种用于诊断对象的神经退行性疾病的试剂盒，所述试剂盒包括来自所述对象的外泌体，以及能够与检测盘状结构域受体2(DDR2)结合的试剂以检测所述外泌体表达的DDR2的水平，其中所述外泌体表达的DDR2的水平高于未患疾病的对照的水平表示所述对象患有神经退行性疾病。
在一些实施方式中，所述用于检测DDR2的表达水平的试剂包括能够与DDR2结合(尤其是特异性结合)从而形成化学上、物理上或生物学上可检测的复合物的试剂，例如蛋白质、核酸或小分子化合物，特别是抗DDR2单克隆抗体或其抗原结合片段，或是抗DDR2多克隆抗体。
在一些实施方式中，所述抗DDR2单克隆抗体是抗DDR2纳米抗体。在一些实施方式中，所述抗DDR2纳米抗体包含CDR1、CDR2、CDR3,其中CDR1包含或为SEQ ID NO:1所示的序列或其等效变体，CDR2包含或为SEQ ID NO:2所示的序列或其等效变体，CDR3包含或为SEQ ID NO:3所示的序列或其等效变体。在一些实施方式中，所述纳米抗体包含或为SEQ ID NO:4所示的序列或其等效变体。
在一些实施方式中，所述能够与DDR2结合的试剂被可检测的标记物标记。在一些实施方式中，所述可检测的标记物选自荧光标记、化学发光标记、顺磁标记、放射性同位素标记和酶标记。在一些实施方式中，所述标记物是吲哚菁绿(ICG)。在一些实施方式中，所述标记物是68Ga或64Cu。
在一些实施方式中，所述试剂盒还包括不同浓度的DDR2重组抗原对照品，从而制备标准曲线以进行定量鉴定。
检测或诊断方法及计算机可读存储介质
在一个方面，本发明提供盘状结构域受体2(DDR2)作为标记物在诊断对象的神经退行性疾病中的应用。
在另一个方面，本发明提供一种体外(in vitro)或离体(ex vivo)诊断对象的神经退行性疾病的方法，所述方法包括检测来自所述对象的样品(例如脑组织、脑脊液或外泌体)中的盘状结构域受体2(DDR2)的存在和/或水平。
在另一个方面，本发明提供用于体内(in vivo)诊断对象的神经退行性疾病的能够与盘状结构域受体2(DDR2)结合的试剂。
在以上的各个方面，所述能够与DDR2结合的试剂是抗DDR2单克隆抗体或其抗原结合片段，或是抗DDR2多克隆抗体。在优选的实施方式中，所述能够与DDR2结合的试剂是抗DDR2单克隆抗体或其抗原结合片段。
在一些实施方式中，所述抗DDR2单克隆抗体是抗DDR2纳米抗体。在一些实施方式中，所述抗DDR2纳米抗体包含CDR1、CDR2、CDR3,其中CDR1包含或为SEQ ID NO:1所示的序列或其等效变体，CDR2包含或为SEQ ID NO:2所示的序列或其等效变体，CDR3包含或为SEQ ID NO:3所示的序列或其等效变体，其中CDR根据IMGT定义。在一些实施方式中，所述纳米抗体包含或为SEQ ID NO:4所示的序列或其等效变体。
优选地，在以上各个方面，其中所述能够与DDR2结合的试剂被可检测的标记物标记。在优选的实施方式中，其中所述可检测的标记物选自荧光标记、化学发光标记、顺磁标记、放射性同位素标记和酶标记。在优选的实施方式中，其中所述可检测的标记物选自荧光标记或化学发光标记。在一些实施方式中，所述标记物是吲哚菁绿(ICG)。在一些实施方式中，所述标记物是68Ga或64Cu。
优选地，在以上各个方面，所述神经退行性疾病选自阿尔茨海默病、肌萎缩侧索硬化、帕金森病、亨廷顿病、额颞叶痴呆、脊髓性肌萎缩、朊病毒病、脊髓小脑共济失调、弗里德里希共济失调、原发性侧索硬化、脊髓小脑萎缩、马查多-约瑟夫病、路易体痴呆、进行性延髓麻痹、进行性核上性麻痹和多系统萎缩。在一些实施方式中，所述神经退行性疾病与星形胶质细胞的异常活化相关。在优选的实施方式中，所述神经退行性疾病是阿尔茨海默病或肌萎缩侧索硬化。
在诊断方法中可以使用各种免疫检测法。在一些实施方式中，这样的免疫检测法包括使用例如放射免疫检测、免疫层析法、ELISA、“夹心法”免疫检测、沉淀反应、免疫印迹分析、凝胶扩散沉淀反应、免疫扩散检测、凝集检测、补体固定检测、免疫放射量检测、荧光免疫检测等竞争性和非竞争性检测体系。体外和体内检测都可以被使用。
通常，将样品中DDR2的水平与参考水平进行比较，其中与所述参考水平的偏差表示出对象中神经退行性疾病的存在和/或阶段。参考水平可以是在对照样品(例如，来自健康组织或对象)中确定的水平或来自健康对象的中值水平。与参考水平相比，例如与未患疾病的对象相比，样品中DDR2的存在和/或DDR2的量增加可以指示所述对象中神经退行性疾病的存在或发生的风险。
在一些实施方式中，与未患疾病的对象相比，患有神经退行性疾病的对象的样品(例如脑组织、脑脊液或外泌体)中的DDR2以至少约2倍、至少约5倍、至少约7.5倍、至少约10倍、至少约15倍、或至少约20倍的量存在。
用于诊断的方法允许定量和/或定性评价，例如靶分子的绝对和/或相对测量，例如测量样品中DDR2的含量。
在本发明方法的一些实施方案中，测定样品中DDR2的存在和/或量包括：(i)使样品(例如脑组织、脑脊液或外泌体)与能够与DDR2结合的试剂接触，以及(ii)检测所述试剂与DDR2之间的复合物的形成和/或确定所述复合物的量。
在一些实施方式中，本发明的检测/诊断方法可与其他检测/诊断神经退行性疾病的方法联合使用。例如，本发明的检测/诊断方法可以与神经退行性疾病的其他生物标记物(例如β淀粉样蛋白、tau、APOE、GFAP、AQP4、MAPT、SOD1)的检测联合使用。
在另一方面，本发明提供一种计算机可读存储介质，其上存储有供计算机读取和执行的计算机指令，所述计算机指令被执行以进行诊断对象是否患有神经退行性疾病的方法，所述方法包括：(a)将来自所述对象的样品与能够与盘状结构域受体2(DDR2)结合的试剂接触；(b)检测并读取接触后的样品的信号，以确定所述试剂是否与样品中的DDR2形成复合物；以及(c)判断所述信号是否超过预定阈值，并且当所述信号超过预定阈值时，确定所述对象患有神经退行性疾病。在优选的实施方式中，所述阈值是来自未患疾病的对象的中值水平。
在另一方面，本发明提供一种计算机可读存储介质，其上存储有供计算机读取和执行的计算机指令，所述计算机指令被执行以进行诊断对象是否患有神经退行性疾病的方法，所述方法包括：(a)从所述对象分离外泌体；(b)将分离的外泌体与能够与盘状结构域受体2(DDR2)结合的试剂接触；(c)检测并读取接触后的外泌体的信号，以确定所述试剂是否与外泌体中的DDR2形成复合物；以及(d)判断所述信号是否超过预定阈值，并且当所述信号超过预定阈值时，确定所述对象患有神经退行性疾病，并且其中所述阈值是来自未患疾病的对象的中值水平。
在另一方面，本发明提供一种治疗对象的神经退行性疾病的方法，所述方法包括：(a)将来自所述对象的样品与能够与盘状结构域受体2(DDR2)结合的试剂接触；(b)检测并读取接触后的样品的信号，以确定所述试剂是否与样品中的DDR2形成复合物；(c)判断所述信号是否超过预定阈值，并且当所述信号超过预定阈值时，确定所述对象患有神经退行性疾病，并且其中所述阈值是来自未患疾病的对象的中值水平；以及(d)向被确定患有神经退行性疾病的对象施用神经保护或神经修复疗法。
在另一方面，本发明提供一种治疗对象的神经退行性疾病的方法，所述方法包括：(a)从所述对象分离外泌体；(b)将分离的外泌体与能够与盘状结构域受体2(DDR2)结合的试剂接触；(c)检测并读取接触后的外泌体的信号，以确定所述试剂是否与外泌体中的DDR2形成复合物；(d)判断所述信号是否超过预定阈值，并且当所述信号超过预定阈值时，确定所述对象患有神经退行性疾病，并且其中所述阈值是来自未患疾病的对象的中值水平；以及(e)向被确定患有神经退行性疾病的对象施用神经保护或神经修复疗法。
在一些实施方式中，所述能够与DDR2结合的试剂包括蛋白质、核酸或小分子化合物。在优选的实施方式中，所述能够与DDR2结合的试剂是抗DDR2单克隆抗体或其抗原结合片段，或是抗DDR2多克隆抗体。在一些实施方式中，所述能够与DDR2结合的试剂被可检测的标记物标记。在一些实施方式中，所述可检测的标记物选自荧光标记、化学发光标记、顺磁标记、放射性同位素标记和酶标记。在一些实施方式中，所述标记物是吲哚菁绿(ICG)。在一些实施方式中，所述标记物是68Ga或64Cu。
在优选的实施方式中，所述神经保护或神经修复疗法例如是：胆碱酯酶抑制剂，例如加兰他敏、多奈哌齐、石杉碱甲、卡巴拉汀；兴奋性氨基酸受体拮抗剂，例如美金刚；炎性因子抑制剂，例如非甾体抗炎药；谷氨酸抑制剂，例如利鲁唑；自由基清除剂，例如依达拉奉。
在一些实施方式中，所述预定阈值可以是如上所述的参考水平，其中与 所述参考水平的偏差表示出对象中相关疾病的存在和/或阶段。参考水平可以是在对照样品(例如，来自健康组织或对象)中确定的水平或来自健康对象的中值水平。与参考水平相比，例如与未患疾病的对象相比，样品中DDR2的存在和/或DDR2的量增加可以指示所述对象中神经退行性疾病的存在或发生的风险。
在一些实施方式中，与未患疾病的对象相比，患有神经退行性疾病的对象的样品(例如脑组织或外泌体)中的DDR2以至少约2倍、至少约5倍、至少约7.5倍、至少约10倍、至少约15倍、或至少约20倍的量存在。
序列表
实施例
实施例1：抗体筛选和亲和力测定
(1)抗体筛选
从用人DDR2胞外段(UniProtKB/Swiss-Prot:Q16832.2aa 22至aa 399)免疫的羊驼中筛选出抗DDR2重链抗体。然后对抗体进行测序，确认其VHH部分，获得纳米抗体，命名为1A12，其序列见以上“序列表”部分。表达并纯化该纳米抗体，用于进一步表征和实验。
(2)DDR2纳米抗体与DDR2抗原胞外段的亲和力
实验步骤：
将上述DDR2纳米抗体1A12，基于分子互相作用分析平台ForteBio生物层干涉技术(BLI)，利用BLI法测定抗体与抗原的亲和力，分析检测纳米抗体与DDR2抗原胞外段蛋白的亲和力。
实验耗材：抗体：溶解于PBS(pH 7.4)；抗原：溶解于PBS(pH 7.4)；传感器：Ni-NTA；Kinetics Buffer：PBST(PBS+0.02％Tween-20，pH 7.4)；Regeneration Buffer：10mM Glycine-HCl，pH 1.7；Re-charged Buffer：10mM NiCl in H2O。
操作步骤：
a.探针在kinetics buffer中预湿10min；
b.Baseline 1：Baseline the Biosensors in kinetics buffer for 180s；
c.固定：用kinetics buffer将带His标签的抗原DDR2胞外段稀释到20μg/ml，传感器捕获；其至4nM(300s)；
d.Baseline 2：Baseline the Biosensors in kinetics buffer for 60s；
e.结合：将抗体溶液用kinetics buffer稀释到一定浓度(从100nM，2倍稀释到3.125nM)，传感器伸入抗体溶液中结合(600s)；
f.解离：传感器在kinetics buffer中解离(600s)；
g.传感器再生：10mM Glycine-HCl,pH 1.7for 5s；
h.中和：传感器再生后在kinetics buffer中和5s；
i.重复再生步骤g和中和步骤h：一共3次(30s)；
j.Baseline 3：传感器再生后，加入10mM NiCl中60s。
制作动力学曲线，计算各相关参数。选择合适的几个浓度梯度的结合解离曲线采用1：1binding的模式对所有曲线进行拟合，选取拟合度最好的三条曲线进行作图分析，最终得到亲和力数值及结合常数和解离常数等重要参数。
结果分析：DDR2纳米抗体1A12与DDR2抗原结合的分析结果如图1和表1所示。
表1：纳米抗体1A12的亲和力数据
实施例2：DDR2纳米抗体的体外成像结果
本实施例使用的DDR2抗体为实施例1中的纳米抗体1A12(Nb-DDR2)，其氨基酸序列如SEQ ID NO:4所示。
实验步骤：
(1)吲哚菁绿(ICG)标记DDR2纳米抗体
Nb-DDR2以2mg/mL的浓度溶解于PBS，涡旋混匀。
ICG-NHS以2mM的浓度溶解于DMSO，涡旋混匀。
取500μL 2mg/mL的Nb-DDR2溶液于1.5mL离心管中，将18μL 2mM的ICG-NHS溶液加入Nb-DDR2溶液中，分9次加入，每次2μL，每次加入均涡旋混匀数秒。
测量混合溶液的pH值，使用2M NaOH溶液调整pH至8.5-9。
将离心管置于60rpm摇床上室温反应2h。
使用0.5mL超滤管14000g 10min多次离心，去除未反应的ICG-NHS，并将溶液置换为0.9％NaCl，0.22μm滤膜过滤蛋白溶液后置于4℃保存。
(2)肌萎缩侧索硬化(ALS)小鼠模型(侧脑室)成像
ALS小鼠模型构建：购买Optn-CKO小鼠，Optnflox/+C57BL/6J小鼠交由南方模式生物公司构建，将小鼠杂交得到Optnflox/flox。将3周龄Optnflox/+小鼠分笼后打耳标编号，剪取鼠尾，利用PCR、琼脂糖凝胶电泳鉴定小鼠基因型，筛选出Optnflox/flox小鼠，在2月龄时侧脑室注射腺病毒，hAd5-Opt Cre病毒，6.58*1010PFU/ml，20ul/只，Cre酶表达之后可通过loxp位点进行OPTN基因的特异性敲除。注射Cre病毒后3个月成像。
成像方法如下：取吲哚菁绿(ICG)标记的DDR2纳米抗体1A12，尾静脉注射到实验小鼠(野生型小鼠，ALS模型小鼠)，对照组ALS模型小鼠注射生理盐水，剂量15μg/只，利用活体成像仪进行成像，激发光/发射光选择：730/820nm。结果如图2所示。
结果分析：ICG标记的DDR2抗体可特异识别ALS小鼠脑组织中高度表达的DDR2，而野生型和生理盐水对照均无明显摄取。因此，通过检测DDR2的表达，可精确诊断ALS。
(3)阿尔茨海默病(AD)小鼠模型成像
本实施例使用APP/PS1双转基因小鼠，是AD常用的转基因小鼠。该小鼠年龄：2年。对照组：野生型C57小鼠。
取吲哚菁绿(ICG)标记的DDR2纳米抗体1A12，尾静脉注射到实验小鼠(野生型小鼠，AD模型小鼠)，剂量15μg/只，利用活体成像仪进行成像，激发光/发射光选择：730/820nm。结果如图3所示。
结果分析：ICG标记的DDR2抗体可特异识别AD小鼠脑组织中高度表达的DDR2，而野生型对照无明显摄取。因此，通过检测DDR2的表达，可精确诊断AD。
实施例3：外泌体检测
(1)ALS病人血浆中外泌体DDR2蛋白质印迹(WB)检测
检测方法如下：
提取外泌体：将200μL血浆与350μL PBS混匀后放入外泌体分离仪(汇芯生物的EXODUS系列H-300仪器)，使用400μL PBS重悬外泌体。外泌体鉴定：瑞芯智造-纳米库尔特粒径仪检测外泌体粒径范围的90％在30-120nm处，说明提取得到的是外泌体。
A.外泌体10ul加SDS-PAGE Sample Loading Buffer(5x)，货号：RM00001；煮沸之后直接上样；选用SDS-PAGE凝胶制备试剂盒，晶彩生物，型号：JC-PE001。
B.把装好的电泳装置放入槽内，上样Marker和蛋白样品，槽内倒入新配置SDS电泳缓冲液，电压120V，20min左右，再换成240V，至Marker最小的分子条带到最底部，停止电泳。
C.转模：(转膜液配置：700ml超纯水+200ml甲醇+100ml快转液)PVDF膜甲醇活化；电泳胶放到“三明治”装置中，因转模是从负极转向正极，所以胶在“黑色三明治”面，膜在“透明三明治”面，卡入转模槽中，电泳槽倒入转膜液(冷藏)，400mA转模35min。
D.封闭：膜用5％牛奶封闭液或5％BSA摇床封闭2h。
E.Anti-hDDR2-生物素(RD，货号BAF2538，1:2000)，4℃孵育过夜。
F.二抗为链霉亲和素-HRP(RD，货号DY998，1:5000)；孵育1h，ECL显色。仪器化学发光凝胶成像系统，型号：博鹭腾；GELVLew 6000Pro。
结果如图4所示。
结果分析：从图4可以看出，与阴性对照相比，各个阶段ALS病人的血浆外泌体中均可以检测出显著的DDR2表达，表明可以通过检测外泌体中的DDR2来诊断各个阶段的ALS。
(2)ALS病人血浆中外泌体DDR2流式检测
流式实验步骤：
提取外泌体：将200μL血浆与350μL PBS混匀后放入外泌体分离仪(汇芯生物的EXODUS系列H-300仪器)，使用400μL PBS重悬外泌体。外泌体鉴定：瑞芯智造-纳米库尔特粒径仪检测外泌体粒径范围的90％在30-120nm处，说明提取得到的是外泌体。
流式：
(1)重悬CD9 capture beads，吸取12.5μL与50μL样本进行混匀，4℃孵育18h；
(2)使用wash buffer清洗2遍后，弃去上清，250μL PBS重悬；
(3)吸取25μL重悬液，加入1ug 1A12纳米抗体-FITC荧光抗体，并用PBS调整体积至100μL，避光孵育1h；
(4)清洗2遍，使用200μL PBS重悬，上机检测。结果如图5所示。
结果分析：从图5可以看出，与正常人相比，各个阶段ALS病人的血浆外泌体中DDR2阳性的百分比显著增加，至少为正常对照的约2倍，表明可以通过检测外泌体中的DDR2来诊断各个阶段的ALS。
在本实施例中(包括WB检测(图4)和流式检测(图5))，ALS病人的“早期”、“中期”和“晚期”是根据ALSFRS-R(ALS Functional Rating Scale-Revised)评分确定的。该评分标准是本领域公知的，其细节例如可以参见https://neurotoolkit.com/alsfrs-r/或https://www.outcomes-umassmed.org/ALS/alsscale.aspx(最后一次访问日期2022年9月19日)。早期、中期和晚期的划分标准：49-40分为早期，39-30为中期，29以下为晚期。
实施例4：神经退行性疾病相关实验数据的生物信息学分析
(1)肌萎缩侧索硬化(ALS)
本实施例分析的数据来自Allen brain map(https://portal.brain-map.org/atlases-and-data/rnaseq/human-m1-10x)的13个正常捐献者的脑组织，来自NCBI GSE174332的16个ALS病人的脑的马达皮层组织的总共239,531个细胞。
数据汇总后，对DDR2表达情况进行分析，结果如图6所示。图6A、B和C分别显示了正常脑组织、ALS患者脑组织中的细胞分布和DDR2的表达水平以及表达DDR2的细胞的比率及其分布。
结果分析：从图6可以看出，与正常对照相比，ALS样本中星形胶质细胞(Astro)的DDR2表达显著增加，表明可以通过检测DDR2来诊断ALS。
(2)阿尔茨海默病(AD)
本实施例原始数据：https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi？acc＝GSE147528，质控后的数据来源：https://www.synapse.org/#！Synapse:syn21788402。该数据有20个样本，取自十位AD患者的内嗅皮层(EC)和额上回(SFG)。其中内嗅皮层细胞数量：42528，额上回细胞数量：63508。
图7上部和下部的右侧图分别为十位患者内嗅皮层(EC)和额上回(SFG)的tSNE细胞降维图，左侧图为DDR2的Feature plot，每一个点表示一个细胞，颜色的深浅表示基因表达量的高低。
结果分析：从图7可以看出，DDR2在AD样本中星形胶质细胞高表达。
图8(a)的两个箱线图分别表示内嗅皮层(EC)和额上回(SFG)中在不同类型细胞中表达DDR2的细胞占比，纵轴为表达DDR2的细胞占比，横轴为不同的细胞类型，图中的每个点表示一个样本。
图8(b)的两个柱状图分别表示内嗅皮层(EC)和额上回(SFG)中不同样本的星形胶质细胞表达DDR2的细胞占比，纵轴为表达DDR2的细胞占比，横轴为不同的样本。上方的数字表示该细胞类型的细胞数目。
将braak stage为0、2的患者归为轻度组(mild)，将Braak stage为6的患者归为重度组(severe)，图8(c)的两个箱线图分别表示不同患病程度的患者的内嗅皮层(EC)和额上回(SFG)中的星形胶质细胞表达DDR2的细胞占比，纵轴为表达DDR2的细胞占比，横轴为不同的患病程度，图中每个点表示一个样本，采用wlicox.test的统计检验方法。
结果分析：从图8可以看出，AD样本星形胶质细胞中表达DDR2的细胞占比较大，并且绝大部分样本中均有显著比例的星形胶质细胞表达DDR2，并且与轻度AD患者相比，重度AD患者中星形胶质细胞表达DDR2的细胞占比显著增加。
图9显示了十位患者内嗅皮层(EC)和额上回(SFG)的星形胶质细胞中DDR2与APOE、GFAP、AQP4、MAPT表达的相关性分析，检验方法为Spearman，细胞经过筛选，选择了做相关性分析的两种基因都有表达的细胞。
结果分析：从图9可以看出，DDR2与现有的AD检测标志物均有良好的相关性。
综上所述，DDR2在AD患者的样本中有显著表达，且主要集中在星形胶质细胞上，并且作为AD诊断标志物，DDR2与现有的检测标志物具有良好的一致性，可以用于诊断AD。
实施例5
如实施例2所述制备ICG标记的DDR2纳米抗体1A12。
使用APP/PS1(C57BL/6)转基因小鼠(AD模型，老年痴呆模型)，以同背景的C57BL/6小鼠做对照小鼠，AD模型小鼠和对照小鼠尾静脉注射15μg ICG标记的DDR2纳米抗体1A12探针，体内循环15min后对小鼠进行活体成像。结果如图10左侧所示，与对照组(C57BL/6正常小鼠)相比，AD模型小鼠可在脑部观察到明显荧光信号。AD模型小鼠已发病。
肌萎缩侧索硬化(ALS)小鼠模型选择SOD1基因突变小鼠，C57BL/6小鼠做正常对照小鼠，ALS模型小鼠和对照小鼠尾静脉注射15μg ICG标记的DDR2纳米抗体1A12探针，体内循环15min后对小鼠进行活体成像。结果如图10右侧所示，与对照组(C57BL/6正常小鼠)相比，ALS模型小鼠可在脑部观察到明显荧光信号。ALS模型小鼠已发病。
实施例6：纳米抗体1A12标记核素(64Cu、68Ga等常规核素)流程
1.偶联NOTA
(1)将抗体溶液12000g离心，取上清，将抗体溶液置换溶剂至0.1M醋酸铵(pH＝7)中，置换后使纳米抗体1A12-cys浓度为1.33mg/mL，置换前后测量浓度，以置换后的浓度为准。
(2)取50mM Mal-NOTA(DMSO溶解)，使抗体和Mal-NOTA的摩尔量比为1：5，将Mal-NOTA用DMSO稀释，使其体积小于反应体系体积的10％(例如：若反应体系为1mL，则将Mal-NOTA稀释至80-90μL，震荡混匀，使Mal-NOTA充分溶解在DMSO中)，将Mal-NOTA溶液分次加入抗体溶液中，10μL/次，每次加入后震荡混匀数十秒，调整溶液pH至7(反应溶剂为0.1M醋酸铵，pH基本不用调整，测量确认即可)，放置在摇床上室温反应2h，摇床转速150rpm。
(3)反应结束后将抗体溶液12000g离心，取上清，用0.1M醋酸铵将抗体溶液纯化浓缩，每100μg抗体浓缩至50-80μL，封口膜封口，-80℃冰箱保存。
2.标记68Ga、64Cu等常规核素
(1)取68Ga或64Cu或其他核素加入前体溶液中(64Cu尽量浓一些，100μg前体投料活度大约1.5mCi，最终反应体积不超过150μL)，混匀，调整pH值至4-5(一般情况下不用调整，混匀后pH即为4-5)。
(2)如有震荡控温器置于震荡控温器37℃反应2小时，如没有，37℃加热反应2小时即可(水浴锅、烘箱等)。
(3)层析纸点样，展开剂展开，iTLC检测标记率(展开剂为柠檬酸钠)。
实施例7：纳米抗体1A12标记核素64Cu成像
根据实施例6中描述的流程，制备64Cu标记的纳米抗体1A12。
采用与实施例5相同的AD模型小鼠和ALS模型小鼠，将其分别与对照正常小鼠对比。上述实验小鼠均进行尾静脉注射200μCi 64Cu标记的DDR2纳米抗体1A12探针，体内循环45min后采集PET显像数据，经重建获得PET/CT图像。圆圈中为摄取信号。
结果如图11和12所示。结果显示AD模型小鼠、ALS模型小鼠分别在大脑皮层和运动皮层有探针摄取信号。正常对照组小鼠没有摄取信号。
应该理解的是，尽管已经通过优选实施方式和任选的特征具体公开了本发明，但是本领域技术人员可以对本文所公开的本发明进行修改、改进和变化，这些修改、改进和变化被认为在本发明的范围内。在此提供的材料、方法和实施例是优选的实施方式的代表和示例性的，并且不旨在作为对本发明范围的限制。

Claims (17)

  1. 用于检测盘状结构域受体2(DDR2)的表达水平的试剂在制备用于诊断对象的神经退行性疾病的试剂盒中的应用，其中来自所述对象的样品中的DDR2的水平高于未患疾病的对照的水平表示所述对象患有神经退行性疾病。
  2. 根据权利要求1所述的应用，其中所述用于检测DDR2的表达水平的试剂包括能够与DDR2结合的试剂，以检测所述样品中的DDR2的水平。
  3. 根据权利要求2所述的应用，其中所述能够与DDR2结合的试剂是抗DDR2单克隆抗体或其抗原结合片段，或是抗DDR2多克隆抗体。
  4. 根据权利要求3所述的应用，其中所述抗DDR2单克隆抗体是抗DDR2纳米抗体。
  5. 根据权利要求4所述的应用，其中所述抗DDR2纳米抗体包含CDR1、CDR2、CDR3，其中CDR1包含或为SEQ ID NO:1所示的序列或其等效变体，CDR2包含或为SEQ ID NO:2所示的序列或其等效变体，CDR3包含或为SEQ ID NO:3所示的序列或其等效变体，其中CDR根据IMGT定义。
  6. 根据权利要求4所述的应用，其中所述纳米抗体包含或为SEQ ID NO:4所示的序列或其等效变体。
  7. 根据权利要求2所述的应用，其中所述能够与DDR2结合的试剂被可检测的标记物标记。
  8. 根据权利要求7所述的应用，其中所述可检测的标记物选自荧光标记、化学发光标记、顺磁标记、放射性同位素标记和酶标记。
  9. 根据权利要求1所述的应用，其中所述神经退行性疾病选自阿尔茨海默病、肌萎缩侧索硬化、帕金森病、亨廷顿病、额颞叶痴呆、脊髓性肌萎缩、朊病毒病、脊髓小脑共济失调、弗里德里希共济失调、原发性侧索硬化、脊髓小脑萎缩、马查多-约瑟夫病、路易体痴呆、进行性延髓麻痹、进行性核上性麻痹和多系统萎缩。
  10. 根据权利要求1所述的应用，其中所述神经退行性疾病是阿尔茨海默病。
  11. 根据权利要求1所述的应用，其中所述神经退行性疾病是肌萎缩侧索硬化。
  12. 一种计算机可读存储介质，其上存储有供计算机读取和执行的计算机指令，所述计算机指令被执行以进行诊断对象是否患有神经退行性疾病的方法，所述方法包括：
    (a)将来自所述对象的样品与能够与盘状结构域受体2(DDR2)结合的试剂接触；
    (b)检测并读取接触后的样品的信号，以确定所述试剂是否与样品中的DDR2形成复合物；以及
    (c)判断所述信号是否超过预定阈值，并且当所述信号超过预定阈值时，确定所述对象患有神经退行性疾病，并且其中所述阈值是来自未患疾病的对象的中值水平。
  13. 根据权利要求12所述的计算机可读存储介质，其中所述神经退行性疾病是阿尔茨海默病或肌萎缩侧索硬化。
  14. 来自对象的外泌体在制备用于诊断所述对象的神经退行性疾病的试剂盒中的应用，其中所述外泌体表达的盘状结构域受体2(DDR2)的水平高于未患疾病的对照的水平表示所述对象患有神经退行性疾病。
  15. 根据权利要求14所述的应用，其中所述外泌体来自所述对象的血清或血浆。
  16. 根据权利要求14所述的应用，其中所述试剂盒还包括能够与DDR2结合的试剂，以检测所述外泌体表达的DDR2的水平。
  17. 根据权利要求14所述的应用，其中所述神经退行性疾病是阿尔茨海默病或肌萎缩侧索硬化。
PCT/CN2023/119127 2022-09-20 2023-09-15 盘状结构域受体2在诊断神经退行性疾病中的应用及相关的计算机可读介质 WO2024061128A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202211142267.5 2022-09-20
CN202211142267.5A CN116626294A (zh) 2022-09-20 2022-09-20 盘状结构域受体2在诊断神经退行性疾病中的应用及相关的计算机可读介质

Publications (1)

Publication Number Publication Date
WO2024061128A1 true WO2024061128A1 (zh) 2024-03-28

Family

ID=87596086

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2023/119127 WO2024061128A1 (zh) 2022-09-20 2023-09-15 盘状结构域受体2在诊断神经退行性疾病中的应用及相关的计算机可读介质

Country Status (2)

Country Link
CN (1) CN116626294A (zh)
WO (1) WO2024061128A1 (zh)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116027038A (zh) * 2022-09-16 2023-04-28 菲创生物医学技术(广州)有限公司 盘状结构域受体2在诊断胶质瘤中的应用及相关的计算机可读介质
CN116626294A (zh) * 2022-09-20 2023-08-22 菲创生物医学技术(广州)有限公司 盘状结构域受体2在诊断神经退行性疾病中的应用及相关的计算机可读介质

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100068207A1 (en) * 2005-04-07 2010-03-18 Abdallah Fanidi DDR2 in Cancer Diagnosis, Detection and Treatment
KR20150061285A (ko) * 2013-11-27 2015-06-04 한국과학기술연구원 디스코이딘 도메인 수용체 2 활성을 조절하는 물질의 동정 방법
US20190262323A1 (en) * 2016-10-31 2019-08-29 Georgetown University Compositions and methods for treating neurodegenerative disorders
CN116626294A (zh) * 2022-09-20 2023-08-22 菲创生物医学技术(广州)有限公司 盘状结构域受体2在诊断神经退行性疾病中的应用及相关的计算机可读介质

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011036297A2 (de) * 2009-09-27 2011-03-31 Ruhr-Universität-Bochum Verfahren zur therapie und diagnose von morbus alzheimer
US20150252075A1 (en) * 2009-12-11 2015-09-10 Copenhagen University Neuroplastin derived peptides
RU2013129860A (ru) * 2010-11-29 2015-01-10 Дженентек, Инк. Способы выявления нейродегенеративных заболеваний или нарушений
EP3060913A4 (en) * 2013-10-24 2018-04-18 Nanosomix Inc. Biomarkers and diagnostic methods for alzheimer's disease and other neurodegenerative disorders
US10203342B2 (en) * 2015-06-11 2019-02-12 Nanosomix, Inc. Biomarkers and differential diagnosis of alzheimer's disease and other neurodegenerative disorders

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100068207A1 (en) * 2005-04-07 2010-03-18 Abdallah Fanidi DDR2 in Cancer Diagnosis, Detection and Treatment
KR20150061285A (ko) * 2013-11-27 2015-06-04 한국과학기술연구원 디스코이딘 도메인 수용체 2 활성을 조절하는 물질의 동정 방법
US20190262323A1 (en) * 2016-10-31 2019-08-29 Georgetown University Compositions and methods for treating neurodegenerative disorders
CN116626294A (zh) * 2022-09-20 2023-08-22 菲创生物医学技术(广州)有限公司 盘状结构域受体2在诊断神经退行性疾病中的应用及相关的计算机可读介质

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
AHMED ELKAMHAWY ET AL.: "The Journey of DDR1 and DDR2 Kinase Inhibitors as Rising Stars in the Fight against Cancer", INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES, vol. 22, no. 12, 18 June 2021 (2021-06-18), XP055867412, ISSN: 1422-0067, DOI: 10.3390/ijms22126535 *
HEBRON MICHAELINE; PEYTON MARGO; LIU XIAOGUANG; GAO XIAOKONG; WANG RUOCHONG; LONSKAYA IRINA; MOUSSA CHARBEL E.-H.: "Discoidin domain receptor inhibition reduces neuropathology and attenuates inflammation in neurodegeneration models", JOURNAL OF NEUROIMMUNOLOGY, ELSEVIER SCIENCE PUBLISHERS BV., NL, vol. 311, 12 August 2017 (2017-08-12), NL , pages 1 - 9, XP085184842, ISSN: 0165-5728, DOI: 10.1016/j.jneuroim.2017.07.009 *

Also Published As

Publication number Publication date
CN116626294A (zh) 2023-08-22

Similar Documents

Publication Publication Date Title
US11124563B2 (en) Camelid single-domain antibody directed against phosphorylated tau proteins and methods for producing conjugates thereof
WO2024061128A1 (zh) 盘状结构域受体2在诊断神经退行性疾病中的应用及相关的计算机可读介质
US20150315267A1 (en) A method of reducing brain amyloid plaques using anti-ab antibodies
US20190315846A1 (en) Antibodies to Human Alpha-Synuclein
WO2011060182A1 (en) Monoclonal antibodies against gmf-beta antigens, and uses therefor
BR112020014940A2 (pt) Adrenomedulina (adm) para diagnóstico e/ou predição de demência e ligante antiadrenomedulina para uso em terapia e prevenção de demência
AU2017350947B2 (en) Anti-ApoE antibodies
JP6680873B2 (ja) 認知症の診断マーカー及びそれを用いた認知症罹患鑑別方法
WO2024056075A1 (zh) 盘状结构域受体2在诊断胶质瘤中的应用及相关的计算机可读介质
WO2024044637A2 (en) Anti-tau mtbr antibodies and methods to detect cleaved fragments of tau and uses thereof
US20220064271A1 (en) Methods for treating tauopathies
Düchs et al. AAV-mediated expression of a new conformational anti-aggregated α-synuclein antibody prolongs survival in a genetic model of α-synucleinopathies
US20230151089A1 (en) Novel alpha-synuclein binding antibodies, or antigen binding portions thereof
US20230241171A1 (en) Use of gdf11 to diagnose and treat anxiety and depression
WO2024107745A1 (en) Methods to detect csf mtbr-tau and uses thereof
JP2023504191A (ja) 内因性調節因子を標的とすることによる血液脳関門薬物輸送の増強
WO2022159766A1 (en) Methods for detecting csf tau species with stage and progression of alzheimer disease, and use thereof
US20180259516A1 (en) Means and methods for diagnosing and treating inflammatory disorders

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23867417

Country of ref document: EP

Kind code of ref document: A1