WO2024056075A1 - 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 - Google Patents

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 Download PDF

Info

Publication number
WO2024056075A1
WO2024056075A1 PCT/CN2023/119117 CN2023119117W WO2024056075A1 WO 2024056075 A1 WO2024056075 A1 WO 2024056075A1 CN 2023119117 W CN2023119117 W CN 2023119117W WO 2024056075 A1 WO2024056075 A1 WO 2024056075A1
Authority
WO
WIPO (PCT)
Prior art keywords
ddr2
subject
glioma
binding
sample
Prior art date
Application number
PCT/CN2023/119117
Other languages
English (en)
French (fr)
Inventor
苏金
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 WO2024056075A1 publication Critical patent/WO2024056075A1/zh

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4164—1,3-Diazoles
    • A61K31/4188—1,3-Diazoles condensed with other heterocyclic ring systems, e.g. biotin, sorbinil
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • 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
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer

Definitions

  • the present invention relates to kits, methods and computer-readable media for diagnosing glioma, and more particularly, to diagnosing glioma by detecting Discoid Domain Receptor 2 (DDR2).
  • DDR2 Discoid Domain Receptor 2
  • Gliomas are tumors that occur in the brain and spinal cord and account for approximately 30% of all primary brain tumors and 80% of all malignant brain tumors.
  • the clinical characteristics of glioma are high incidence, high postoperative recurrence rate, and low cure rate.
  • gliomas can be divided into astrocytomas (astrocytes), oligodendrogliomas (oligodendrocytes), and ventricular gliomas. myomas (ependymal cells) and mixed gliomas (such as oligodendrogliomas and astrocytomas, including mixed glial cells).
  • gliomas can be divided into WHO grades I to IV from low to high malignancy. Grade I gliomas are usually benign. The average survival for grade II (low-grade glioma) is about 7 years.
  • Grade II gliomas can progress to grade III (high-grade glioma) and finally grade IV (secondary glioblastoma), of which grade IV glioblastoma accounts for more than 50% and is the most malignant. , the prognosis is extremely poor.
  • Grade III and IV gliomas are collectively referred to as high-grade glioma (HGG), including anaplastic astrocytoma, anaplastic oligodendroglioma, and the most malignant glioblastoma.
  • Glioblastoma (GBM). HGG is the most common invasive primary intracranial tumor. In clinical practice, comprehensive treatment is often combined with surgery, radiotherapy and chemotherapy. However, the treatment effect is still not ideal, and recurrence is very easy to occur.
  • auxiliary diagnostic molecular markers for glioma include IDH, MGMT, EGFR, P53, PTEN, etc. Somatic mutations in IDH1/2, genomic amplification of EGFR, and co-deletion of chromosome arms 1p and 19q are usually examined for accurate diagnosis. IDH1/2 mutations are commonly found in low-grade gliomas and secondary glioblastomas and are known to have a profound impact on the prolonged survival of glioma patients. Furthermore, approximately 50% of glioblastomas show focal amplification of EGFR. Furthermore, EGFR gene amplification is known to promote cell proliferation, and therefore, clinical trials of EGFR inhibitors are often used in cancer treatment.
  • IHC immunohistochemistry
  • FISH fluorescence in situ hybridization
  • 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 inventors of the present application unexpectedly discovered that DDR2 expression in brain gliomas 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 brain tissue or exosomes, thereby efficiently and accurately diagnosing glioma.
  • the present invention provides a kit for diagnosing glioma in a subject, the kit comprising a reagent for detecting the expression level of Discoid Domain Receptor 2 (DDR2), wherein from Levels of DDR2 in a subject's sample that are higher than levels in controls without the disease indicate that the subject has glioma.
  • 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 glioma in a subject, wherein a sample from the subject Levels of DDR2 in are higher than levels in controls without the disease indicating that the subject has glioma.
  • DDR2 Discoid Domain Receptor 2
  • the present invention provides a reagent for detecting the expression level of discoidin domain receptor 2 (DDR2) for diagnosing glioma in a subject, wherein a level of DDR2 in a sample from the subject higher than that of a control without the disease indicates that the subject suffers from glioma.
  • DDR2 discoidin domain receptor 2
  • the invention provides a method of diagnosing glioma 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 glioma.
  • DDR2 Discoid Domain Receptor 2
  • the present invention provides a computer-readable storage medium on which are stored computer instructions for reading and executing by a computer, and the computer instructions are executed to perform a method of diagnosing whether a subject suffers from glioma, so
  • 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, determining that the subject suffers from glioma, and wherein the threshold is Median levels from subjects without disease.
  • DDR2 Discodomain Receptor 2
  • the invention provides a method of treating glioma in a subject, the method comprising: (a) combining a sample from the subject with an agent capable of binding to Discoid Domain Receptor 2 (DDR2) Contact; (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 the predetermined at a threshold, determining that the subject has glioma, and wherein the threshold is a median level from subjects without the disease; and (d) administering an anti-tumor therapy to the subject determined to have glioma.
  • the anti-tumor therapy is temozolomide.
  • 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.
  • the glioma is selected from the group consisting of astrocytoma, oligodendroglioma, ependymoma and mixed glioma.
  • the glioma is selected from the group consisting of anaplastic astrocytoma, anaplastic oligodendroglioma, and glioblastoma.
  • the glioma is glioblastoma.
  • the present invention provides a kit for diagnosing glioma in a subject, the kit comprising exosomes from the subject and capable of detecting Discoid Domain Receptor 2 (DDR2)
  • the combined reagent is used to detect the level of DDR2 expressed by the exosomes, wherein the level of DDR2 expressed by the exosomes is greater than the level of a control without disease indicating that the subject has glioma.
  • the invention provides use of exosomes from a subject in preparing a kit for diagnosing glioma in the subject, wherein the exosomes express Discoid Domain Receptor 2 (DDR2 ) levels that are higher than those in disease-free controls indicate that the subject has glioma.
  • DDR2 Discoid Domain Receptor 2
  • the invention provides exosomes from a subject for use in diagnosing glioma in said subject, wherein said exosomes express a higher level of Discoid Domain Receptor 2 (DDR2) than those without the disease.
  • DDR2 Discoid Domain Receptor 2
  • the invention provides a method of diagnosing glioma in a subject, the method comprising isolating exosomes from the subject and detecting Discoid Domain Receptor 2 in the exosomes from the subject (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 glioma.
  • the present invention provides a computer-readable storage medium on which are stored computer instructions for reading and executing by a computer, and the computer instructions are executed to perform a method of diagnosing whether a subject suffers from glioma
  • 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, The subject is determined to have glioma, and wherein the threshold is a median level from subjects without the disease.
  • DDR2 Discodomain Receptor 2
  • the present invention provides a method of treating glioma 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 has glioma, and wherein the threshold is a median level from subjects without the disease; and (e) Anti-tumor therapy is administered to a subject determined to have glioma.
  • the anti-tumor therapy is temozolomide.
  • 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 In vitro imaging results of glioma using DDR2 nanobody.
  • Figure 3 Flow cytometric detection results of plasma exosomes in primary glioma patients.
  • Figure 4 (a) DDR2 expression in glioma-related experimental data; (b) Violin diagram shows the distribution of DDR2-positive cells, and the histogram shows the DDR2 expression level.
  • FIG. 5 (a) UMAP cell cluster analysis. The left picture shows the distribution of various cell types, and the right picture shows the distribution of cells from various sample sources; (b) The corresponding expression of various marker genes in the UMAP cell clustering distribution diagram.
  • Figure 6 Expression distribution diagram of related genes in different types of cells.
  • “about” means that the value is within an acceptable error range for the specific value as determined by one of ordinary skill in the art, which value depends in part on how it is measured or determined (ie, the limits of the measurement system). For example, in every practice in the art, “about” can mean within 1 or more than 1 standard deviation. Alternatively, “about” or “substantially comprising” may mean a range of up to 20%. Furthermore, with respect to biological systems or processes, the term may mean up to one order of magnitude or up to five times a value. Unless stated otherwise, when a specific value appears in this application and claims, the meaning of "about” or “substantially comprising” should be assumed to be within an acceptable error range for that 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.
  • detection includes any means of detection, including direct and indirect detection, quantitative and qualitative detection, meaning the identification of the presence of a specific molecule (e.g., DDR2 protein) in a subject or in a sample from a subject and/or level.
  • a specific molecule e.g., 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 glioma or the identification of a specific type thereof.
  • diagnosis includes distinguishing between different grades of glioma or determining the grade of glioma.
  • 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; by using surface plasmon resonance analysis using general procedures outlined by the manufacturer; by radioimmunoassay using radiolabeled target antigen; or by methods known to the skilled artisan Other methods. 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 may include "chimeric" antibodies, humanized antibodies, or fully human antibodies.
  • the antibody forms 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, antibody fragments retain at least 10% of the binding activity of the parent 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.
  • 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, Cystic 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
  • Body 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 glioma 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.
  • 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 Liquinasamine
  • cyanine phycoerythrin
  • Texas Red Texas Red
  • BODIPY Invitrogen
  • 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 invention provides a kit for diagnosing glioma in a subject, the kit comprising a reagent for detecting the expression level of Discoid Domain Receptor 2 (DDR2), wherein from the subject Levels of DDR2 in a sample that are higher than levels in controls without the disease indicate that the subject has glioma.
  • DDR2 Discoid Domain Receptor 2
  • the present invention provides a kit for diagnosing glioma in a subject, the kit comprising exosomes from the subject and capable of detecting Discoid Domain Receptor 2 (DDR2)
  • the combined reagent is used to detect the level of DDR2 expressed by the exosomes, wherein the level of DDR2 expressed by the exosomes is greater than the level of a control without disease indicating that the subject has glioma.
  • 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 kit further includes DDR2 recombinant antigen control substances at different concentrations to prepare a standard curve for quantitative identification.
  • the present invention provides the use of Discoid Domain Receptor 2 (DDR2) as a marker in diagnosing glioma in a subject.
  • DDR2 Discoid Domain Receptor 2
  • the invention provides a method of diagnosing glioma in a subject in vitro or ex vivo, the method comprising detecting a sample (e.g., brain tissue or exosomes) from the subject. The presence and/or levels of Discoid Domain Receptor 2 (DDR2) in ).
  • a sample e.g., brain tissue or exosomes
  • DDR2 Discoid Domain Receptor 2
  • the present invention provides an agent capable of binding to Discoid Domain Receptor 2 (DDR2) for use in the diagnosis of glioma 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 glioma is selected from the group consisting of astrocytoma, oligodendrogliomas, ependymoma and mixed glioma.
  • the glioma is selected from the group consisting of anaplastic astrocytoma, anaplastic oligodendroglioma, and glioblastoma.
  • the glioma is glioblastoma.
  • 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 the sample is compared to a reference level, where deviation from the reference level is indicative of the presence and/or stage of glioma 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 occurrence of glioma in the subject.
  • the DDR2 in a sample (eg, brain tissue or exosomes) from a subject with glioma is at least about 3-fold, at least about 5-fold, at least about 7.5 times higher than that of a subject without the disease. 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 a sample includes: (i) contacting the sample (e.g., brain tissue or exosomes) with an agent capable of binding to DDR2, and (ii) detecting Formation of a complex between the agent and DDR2 and/or determination of the amount of the complex.
  • the detection/diagnosis methods of the present invention can be used in combination with other methods of detection/diagnosis of glioma.
  • the detection/diagnosis method of the present invention can be used in conjunction with the detection of other biomarkers of glioma (eg, IDH, MGMT, EGFR, P53, PTEN).
  • the present invention provides a computer-readable storage medium having computer instructions stored thereon for reading and execution by a computer, and the computer instructions are executed to perform a method of diagnosing whether a subject suffers from glioma, so
  • the method includes: (a) contacting a sample from the subject with an agent capable of binding to Discodomain Receptor 2 (DDR2); (b) detecting and reading the signal of the contacted sample to determine 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 When, it is determined that the subject suffers from glioma.
  • the threshold is the median level from subjects without the disease.
  • the present invention provides a computer-readable storage medium having computer instructions stored thereon for reading and execution by a computer, and the computer instructions are executed to perform a method of diagnosing whether a subject suffers from glioma, so
  • 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 a signal of the exosomes after contact to determine whether the agent forms a complex with DDR2 in the exosome; and (d) determine whether the signal exceeds a predetermined threshold, and when the signal exceeds the predetermined threshold, determine The subject has glioma, and wherein the threshold is a median level from subjects without the disease.
  • DDR2 Discodomain Receptor 2
  • the invention provides a method of treating glioma in a subject, the method comprising: (a) combining a sample from the subject with an agent capable of binding to Discoid Domain Receptor 2 (DDR2) Contact; (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 the predetermined at a threshold, determining that the subject has glioma, and wherein the threshold is a median level from subjects without the disease; and (d) administering an anti-tumor therapy to the subject determined to have glioma.
  • DDR2 Discoid Domain Receptor 2
  • the invention provides a method of treating glioma in a subject, the method comprising: (a) isolating exosomes from the subject; (b) combining the isolated exosomes with a substance capable of interacting with a discoid Contact with the reagent bound by domain receptor 2 (DDR2); (c) detect and read the signal of the exosomes after contact to determine whether the reagent forms a complex with DDR2 in the exosome; (d) judge whether the signal exceeds a predetermined threshold, and when the signal exceeds the predetermined threshold, determining that the subject has glioma, and wherein the threshold is a median level from subjects without the disease; and (e) to Subjects determined to have glioma are administered anti-tumor therapy.
  • DDR2 domain receptor 2
  • 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 anti-tumor therapy is surgical resection, chemotherapy, radiotherapy or a combination thereof.
  • the chemotherapy is temozolomide, lomustine, carmustine, nimustine, procarbazine, vinblastine, vincristine, teniposide, etoposide, ulcerin cisplatin, carboplatin, bevacizumab, or combinations thereof.
  • the chemotherapy is temozolomide.
  • 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 occurrence of glioma in the subject.
  • the DDR2 in a sample (eg, brain tissue or exosomes) from a subject with glioma is at least about 3-fold, at least about 5-fold, at least about 7.5 times higher than that of a subject without the disease. times, at least about 10 times, at least about 15 times, or at least about 20 times.
  • Anti-DDR2 heavy chain antibodies were screened from alpacas immunized with the human DDR2 extracellular segment (UniProtKB/Swiss-Prot: Q16832.2 aa 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.
  • DDR2 expression is significantly higher in brain gliomas.
  • Take sections of left fronto-parietal lobe intracranial tumors and adjacent normal tissue sections from patients with various grades of glioma add 2 ⁇ g of the above prepared ICG-labeled DDR2 nanobody 1A12, incubate at room temperature for 1 hour, rinse with PBS several times to remove unbound
  • the probe uses a small animal live imager to perform near-infrared imaging, and the results are shown in Figure 2.
  • Low-grade gliomas in the figure refer to grade I/II gliomas
  • high-grade gliomas refer to grade III/IV gliomas.
  • ICG-labeled DDR2 antibodies can specifically recognize DDR2, which is highly expressed in gliomas of all grades, without significant uptake in adjacent normal tissues, and can be used for precise in vitro imaging and diagnosis.
  • Example 3 Flow cytometric detection results of plasma exosomes from primary glioma patients
  • Extract exosomes Mix 200 ⁇ L plasma and 350 ⁇ L PBS, put it into the exosome separator, and use 400 ⁇ L PBS to resuspend the exosomes.
  • DDR2 expression filtering was performed on the summary data.
  • the conditions are: 200 ⁇ nFeature_RNA ⁇ 6000, percent.mt ⁇ 10, Percent.DDR2>0.01 (the following gene expression Positive screening is based on this condition), that is, in the single-cell sequencing results, 1 out of every 10,000 RNA sequencing results is DDR2 and all are selected as DDR2 positive.
  • Figure 4(b) shows the statistical results of the expression levels of DDR2-positive cells.
  • the DDR2 expression levels of DDR2-positive cells in both ndGBM and rGBM samples were higher than those of DDR2-positive cells in LGG samples, especially in rGBM.
  • the average DDR2 expression level of ndGBM was lower than that of rGBM, but the highest value of a few cells was higher, showing that the expression of DDR2-positive cells varied greatly, while rGBM was relatively balanced.
  • Table 1 Cell numbers and related gene expression positive rates in various GBM samples
  • gliomas can be divided into glioblast1, neuroblast and fibroblast1 of EGFR-positive epidermal origin, and glioblast1 and fibroblast2 of PDGFRA and IGFBP2-positive mesenchymal origin.
  • the specific gene expression distribution map can be seen in Figure 5(b).
  • Most of the cells with high DDR2 expression are glioblast1 and fibroblast1 of epidermal origin, while fibroblast2 of mesenchymal origin is also highly expressed, and its distribution mostly overlaps with that of FAP and is significantly higher than that of FAP.
  • Figure 6 shows the expression of each gene in different types of cell clusters. The results showed that there were fewer DDR2-positive cells in fibroblast1 than in fibroblast2, but the DDR2 expression level of its DDR2-positive cells was higher.
  • grade IV gliomas (including ndGBM and rGBM) have significantly higher proportions and expression levels of DDR2 positive cells (approximately 10 times).
  • EGFR can clearly target glioma, but its clinical application value for distinguishing different grades of glioma may not be as good as DDR2.
  • FAP can distinguish different grades of gliomas, its expression level and difference in gliomas are far lower than that of DDR2. Therefore, DDR2 may have better clinical significance in the differential diagnosis and targeted therapy of grade IV glioma.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Medicinal Chemistry (AREA)
  • Hematology (AREA)
  • Biomedical Technology (AREA)
  • Urology & Nephrology (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Microbiology (AREA)
  • Pathology (AREA)
  • General Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Analytical Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Biotechnology (AREA)
  • Physics & Mathematics (AREA)
  • Cell Biology (AREA)
  • Public Health (AREA)
  • Food Science & Technology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Organic Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Oncology (AREA)
  • Hospice & Palliative Care (AREA)
  • Epidemiology (AREA)
  • General Chemical & Material Sciences (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胶质瘤的试剂盒中的应用，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胶质瘤。本发明还公开了用于诊断胶质瘤的试剂盒、方法和计算机可读介质。本发明通过检测DDR2来高效、准确地诊断胶质瘤。

Description

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 技术领域
本发明涉及用于诊断胶质瘤的试剂盒、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)来诊断胶质瘤。
背景技术
胶质瘤是一种发生在大脑和脊髓中的肿瘤，约占所有原发性脑肿瘤的30％和所有恶性脑肿瘤的80％。胶质瘤的临床特点是发病率高、术后复发率高、治愈率低。
根据肿瘤细胞与正常脑胶质细胞在形态上的相似程度，胶质瘤可分为星形细胞瘤(星形胶质细胞)、少突胶质细胞瘤(少突胶质细胞)、室管膜瘤(室管膜细胞)和混合型胶质瘤(例如少突胶质细胞瘤和星形细胞瘤，包括混合型胶质细胞)。根据肿瘤分子病理学特征及生物学行为，胶质瘤恶性程度从低到高可分为WHO I～IV级。I级胶质瘤通常是良性的。II级(低级胶质瘤)的平均生存期约为7年。II级胶质瘤可以进展为III级(高级别胶质瘤)，最终是IV级(继发性胶质母细胞瘤)，其中IV级胶质母细胞瘤占比超过50％，恶性程度最高，预后效果极差。III级和IV级的胶质瘤统称为高级别胶质瘤(High-grade Glioma，HGG)，包括间变性星形细胞瘤、间变性少突胶质细胞瘤、以及恶性程度最高的胶质母细胞瘤(Glioblastoma，GBM)。HGG是最为常见的一种侵袭性颅内原发性肿瘤，临床上常联合应用手术以及放化疗进行综合治疗，但治疗效果仍不理想，并且极易出现复发的情况。
目前胶质瘤的辅助诊断分子标志物有IDH、MGMT、EGFR、P53、PTEN等。通常检查IDH1/2的体细胞突变、EGFR的基因组扩增以及染色体臂1p和19q的共缺失来进行准确诊断。IDH1/2突变常见于低级别胶质瘤和继发性胶质母细胞瘤，众所周知，它对胶质瘤患者的延长生存期产生深远影响。此外，约50％的胶质母细胞瘤显示EGFR的局灶性扩增。再者，已知EGFR基因扩增促进细胞增殖，因此，EGFR抑制剂的临床试验经常用于癌症治疗。
在此背景下，为了有效诊断胶质瘤，通常使用免疫组织化学(IHC)和荧光原位杂交(FISH)对被认为是胶质瘤增殖的必需基因IDH1的目标突变和EGFR的局灶性扩增进行病理诊断。然而，越来越需要以更简单的方式有效诊断胶质瘤。
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的表达和功能在胶质瘤细胞中从未被报道过。目前尚未见研究和报道DDR2在胶质瘤诊断中的应用。
发明内容
本申请的发明人意外地发现与正常对照相比，脑胶质瘤中的DDR2表达明显增高。发明人利用能够与DDR2结合的试剂检测脑组织或外泌体中DDR2的存在和/或水平，从而高效、准确地诊断胶质瘤。
在第一方面，本发明提供一种用于诊断对象的胶质瘤的试剂盒，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胶质瘤。
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胶质瘤的试剂盒中的应用，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胶质瘤。
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胶质瘤，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胶质瘤。
在第四方面，本发明提供一种诊断对象的胶质瘤的方法，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胶质瘤。
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胶质瘤的方法，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，确定所述对象患有胶质瘤，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胶质瘤的方法，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，确定所述对象患有胶质瘤，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)向被确定患有胶质瘤的对象施用抗肿瘤疗法。在优选的实施方式中，所述抗肿瘤疗法是替莫唑胺。
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。在上述任一方面，所述胶质瘤选自星形细胞瘤、少突胶质细胞瘤、室管膜瘤和混合型胶质瘤。在一些实施方式中，所述胶质瘤选自间变性星形细胞瘤、间变性少突胶质细胞瘤和胶质母细胞瘤。在一些实施方式中，所述胶质瘤是胶质母细胞瘤。
在第七方面，本发明提供一种用于诊断对象的胶质瘤的试剂盒，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胶质瘤。
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胶质瘤的试剂盒中的应用，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胶质瘤。
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胶质瘤，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胶质瘤。
在第十方面，本发明提供一种诊断对象的胶质瘤的方法，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胶质瘤。
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胶质瘤的方法，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，确定所述对象患有胶质瘤，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胶质瘤的方法，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，确定所述对象患有胶质瘤，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)向被确定患有胶质瘤的对象施用抗肿瘤疗法。在优选的实施方式中，所述抗肿瘤疗法是替莫唑胺。
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：DDR2纳米抗体的胶质瘤体外成像结果图。
图3：胶质瘤原代病人血浆外泌体流式检测结果图。
图4：(a)胶质瘤相关实验数据中的DDR2表达情况；(b)Violin图显示DDR2阳性细胞分布，柱状图为DDR2表达水平。
图5：(a)UMAP细胞聚类分析。左图为各种细胞类型的分布，右图显示各种样本来源的细胞分布情况；(b)各种标志物基因在UMAP细胞聚类分布图中对应的表达情况。
图6：相关基因在不同类型细胞中的表达分布图。
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”可以指胶质瘤的鉴定或其具体类型的鉴定。在本发明的一些实施方案中，“诊断”包括区分不同等级的胶质瘤或确定胶质瘤的等级。
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胶质瘤。
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。
试剂盒
在一个方面，本发明提供一种用于诊断对象的胶质瘤的试剂盒，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胶质瘤。
在另一方面，本发明提供一种用于诊断对象的胶质瘤的试剂盒，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胶质瘤。
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胶质瘤中的应用。
在另一个方面，本发明提供一种体外(in vitro)或离体(ex vivo)诊断对象的胶质瘤的方法，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)诊断对象的胶质瘤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。
优选地，在以上各个方面，所述胶质瘤选自星形细胞瘤、少突胶质细胞瘤、室管膜瘤和混合型胶质瘤。在一些实施方式中，所述胶质瘤选自间变性星形细胞瘤、间变性少突胶质细胞瘤和胶质母细胞瘤。在一些实施方式中，所述胶质瘤是胶质母细胞瘤。
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胶质瘤的存在和/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胶质瘤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，患有胶质瘤的对象的样品(例如脑组织或外泌体)中的DDR2以至少约3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/诊断胶质瘤的方法联合使用。例如，本发明的检测/诊断方法可以与胶质瘤的其他生物标记物(例如IDH、MGMT、EGFR、P53、PTEN)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胶质瘤的方法，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，确定所述对象患有胶质瘤。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胶质瘤的方法，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，确定所述对象患有胶质瘤，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胶质瘤的方法，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，确定所述对象患有胶质瘤，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)向被确定患有胶质瘤的对象施用抗肿瘤疗法。
在另一方面，本发明提供一种治疗对象的胶质瘤的方法，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，确定所述对象患有胶质瘤，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)向被确定患有胶质瘤的对象施用抗肿瘤疗法。
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。。
在优选的实施方式中，所述抗肿瘤疗法是手术切除、化疗、放疗或其组合。在优选的实施方式中，所述化疗是替莫唑胺、洛莫司汀、卡莫司汀、尼莫司汀、丙卡巴肼、长春碱、长春新碱、替尼泊苷、依托泊苷、伊立替康、顺铂、卡铂、贝伐珠单抗或它们的组合。在优选的实施方式中，所述化疗是替莫唑胺。
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胶质瘤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，患有胶质瘤的对象的样品(例如脑组织或外泌体)中的DDR2以至少约3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:Q16832.2 aa 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)胶质瘤原代病人样本DDR2-1A12-ICG探针成像
与正常脑组织相比，脑胶质瘤中的DDR2表达明显增高。取患有各个级别胶质瘤的病人的左侧额顶叶颅内肿瘤和癌旁正常组织切片，加入2μg以上制备的ICG标记的DDR2纳米抗体1A12室温孵育1h，PBS冲洗多次去除未结合的探针，使用小动物活体成像仪进行近红外成像，结果如图2所示。图中的低级别胶质瘤是指I/II级胶质瘤，高级别胶质瘤是指III/IV级胶质瘤。
结果分析：ICG标记的DDR2抗体可特异识别各个级别胶质瘤中高度表达的DDR2，而癌旁正常组织无明显摄取，可用于体外精准成像和诊断。
实施例3：胶质瘤原代病人血浆外泌体流式检测结果
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，使用400μL PBS重悬外泌体。
流式：
(1)重悬CD9capture 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 FITC标记的DDR2纳米抗体1A12，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3所示。
结果分析：从图3可以看出，与正常人相比，WHO II级和IV级胶质瘤患者的术前血浆外泌体中DDR2阳性的百分比显著增加，分别约为正常对照的4倍和7.5倍，表明可以通过检测外泌体中的DDR2来诊断各个级别的胶质瘤。
实施例4：胶质瘤相关实验数据的生物信息学分析
以下数据分析来源于三组胶质瘤相关数据：
GSE174554(40Human ndGBM+40rGBM)snRNAseq
+GSE182109(2grade II LGG,10grade IV ndGBM+5grade IV rGBM)scRNAseq
+GSE135045(7grade IV IDHwt ndGBM)scRNAseq
(1)DDR2表达情况分析
数据汇总后，对DDR2表达情况进行分析，结果如图4(a)所示。从图4(a)可以看出，IV级原发GBM(ndGBM)和复发GBM(rGBM)样本中表达DDR2的细胞数量和DDR2表达水平均显著高于II级低级别GBM(LGG)。
为了更方便显示表达DDR2的细胞(DDR2+)的数量和表达水平，对汇总数据进行 了DDR2表达筛选，条件为：200<nFeature_RNA<6000,percent.mt<10,Percent.DDR2>0.01(以下基因表达阳性筛选均为此条件)，即单细胞测序结果中每10000个RNA测序中有1个为DDR2均选出为DDR2阳性。
图4(b)显示了DDR2阳性细胞表达水平统计结果。ndGBM和rGBM样本中DDR2阳性细胞的DDR2表达水平均高于LGG样本中的DDR2阳性细胞，rGBM尤为显著。其中，ndGBM的DDR2平均表达水平低于rGBM，但少数细胞最高值偏高，显示其DDR2阳性细胞的表达差异较大，而rGBM相对均衡。
结合表1中细胞数量和DDR2阳性细胞比例，可看出，ndGBM和rGBM样本中DDR2表达阳性细胞比例分别为6.32％和5.36％，均显著高于LGG样本0.79％的DDR2阳性细胞比例。对比FAP阳性细胞比例，虽FAP亦可显著区分IV级GBM与II级LGG，但DDR2阳性细胞占比高出FAP三倍，且自身比较中DDR2似乎对ndGBM更为敏感，而FAP对rGBM较为敏感。
表1：各种GBM样本中细胞数量和相关基因表达阳性率
(2)UMAP细胞聚类分析
对单细胞测序数据进行UMAP细胞聚类分析可明确区分各样本细胞构成和各细胞类型之间的相关性。如图5(a)所示，胶质瘤可分为EGFR阳性epidermal origin的glioblast1、neuroblast和fibroblast1，及PDGFRA和IGFBP2阳性mesenchymal origin的glioblast1和fibroblast2。具体基因表达情况分布图可参见图5(b)。DDR2高表达细胞多为epidermal origin的glioblast1和fibroblast1，而mesenchymal origin的fibroblast2也有高表达，其分布情况与FAP多有重叠且明显高于FAP。图6显示了各基因在不同类型细胞聚类中的表达情况。结果显示fibroblast1中DDR2阳性细胞少于fibroblast2，但其DDR2阳性细胞的DDR2表达水平较高。
综上所述，IV级胶质瘤(包括ndGBM和rGBM)对比II级胶质瘤LGG，其DDR2阳性细胞比例和表达水平均显著升高(约10倍)。EGFR可明确靶向胶质瘤，但用于鉴别不同等级胶质瘤的临床应用价值可能不及DDR2。而FAP虽能区分不同等级胶质瘤，但其在胶质瘤中的表达水平和差异性均远不及DDR2。因此，DDR2在IV级胶质瘤的鉴别诊断和靶向治疗中可能具有更佳的临床意义。
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 (13)

  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胶质瘤的试剂盒中的应用，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胶质瘤。
  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，其中所述胶质瘤选自间变性星形细胞瘤、间变性少突胶质细胞瘤和胶质母细胞瘤。
  10. 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胶质瘤的方法，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，确定所述对象患有胶质瘤，并且其中所述阈值是来自未患疾病的对象的中值水平。
  11. 来自对象的外泌体在制备用于诊断所述对象的胶质瘤的试剂盒中的应用，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胶质瘤。
  12. 根据权利要求11所述的应用，其中所述外泌体来自所述对象的血清或血浆。
  13. 根据权利要求11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。
PCT/CN2023/119117 2022-09-16 2023-09-15 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 WO2024056075A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202211130381.6 2022-09-16
CN202211130381.6A CN116027038A (zh) 2022-09-16 2022-09-16 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

Publications (1)

Publication Number Publication Date
WO2024056075A1 true WO2024056075A1 (zh) 2024-03-21

Family

ID=86076506

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2023/119117 WO2024056075A1 (zh) 2022-09-16 2023-09-15 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

Country Status (2)

Country Link
CN (1) CN116027038A (zh)
WO (1) WO2024056075A1 (zh)

Families Citing this family (1)

* 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

Citations (9)

* 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
US20110287011A1 (en) * 2008-08-12 2011-11-24 Oncomed Pharmaceuticals, Inc. DDR1-Binding Agents and Methods of Use Thereof
WO2013034933A1 (en) * 2011-09-08 2013-03-14 Imperial Innovations Limited Anti ddr1 antibodies, their uses and methods identifying them
US20160340738A1 (en) * 2014-01-24 2016-11-24 Technische Universität Dresden New fusion gene as therapeutic target in proliferative diseases
EP3428647A1 (en) * 2017-07-12 2019-01-16 Consejo Superior de Investigaciones Científicas (CSIC) Expression signature for glioma diagnosis and/or prognosis in a subject
WO2022007283A1 (zh) * 2020-07-08 2022-01-13 深圳霁因生物医药转化研究院 用于诊断fap表达异常相关疾病的试剂盒、方法及计算机可读存储介质
WO2022040506A2 (en) * 2020-08-21 2022-02-24 Yale University Nanobody compositions and methods of use of the same
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

Patent Citations (9)

* 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
US20110287011A1 (en) * 2008-08-12 2011-11-24 Oncomed Pharmaceuticals, Inc. DDR1-Binding Agents and Methods of Use Thereof
WO2013034933A1 (en) * 2011-09-08 2013-03-14 Imperial Innovations Limited Anti ddr1 antibodies, their uses and methods identifying them
US20160340738A1 (en) * 2014-01-24 2016-11-24 Technische Universität Dresden New fusion gene as therapeutic target in proliferative diseases
EP3428647A1 (en) * 2017-07-12 2019-01-16 Consejo Superior de Investigaciones Científicas (CSIC) Expression signature for glioma diagnosis and/or prognosis in a subject
WO2022007283A1 (zh) * 2020-07-08 2022-01-13 深圳霁因生物医药转化研究院 用于诊断fap表达异常相关疾病的试剂盒、方法及计算机可读存储介质
WO2022040506A2 (en) * 2020-08-21 2022-02-24 Yale University Nanobody compositions and methods of use of the same
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

Also Published As

Publication number Publication date
CN116027038A (zh) 2023-04-28

Similar Documents

Publication Publication Date Title
JP6672383B2 (ja) 間葉及び上皮間葉形質転換循環腫瘍細胞のための特異的検出ツール
US8916204B2 (en) SPARC and methods of use thereof
JP6075881B2 (ja) 乳癌のバイオマーカー
AU2006249235B2 (en) Sparc and methods of use thereof
WO2013099925A1 (ja) Ｅｘｏｓｏｍｅ検出用モノクローナル抗体
JP5586368B2 (ja) 腫瘍マーカーとしてのｎ１，ｎ１２−ジアセチルスペルミン
JP5669757B2 (ja) 精巣癌診断および予後診断におけるｒｂｍ３タンパク質
CN105392800A (zh) 抗cxcl1、cxcl7和cxcl8抗体及其应用
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
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
JP6977105B2 (ja) Ｉｇｆ−１ｒ抗体および癌の診断のためのその使用
EA037027B1 (ru) Применение и способ для профилактики или лечения рака желудка с использованием композиции, содержащей моноклональное прогастринсвязывающее антитело
US20190137523A1 (en) Method and means for detecting the level of total vegf-a
WO2011090166A1 (ja) 食道癌マーカー
KR20160007625A (ko) Tk1 단백질의 발현이 항진된 결장직장암 환자에 대한 치료효과 예측 방법
JP6312700B2 (ja) 膀胱癌中のｐｏｄｘｌ
WO2018208122A1 (ko) 담도 세포에서 메티오닐-타알엔에이 합성효소를 이용한 담도암 진단 방법
US20140370525A1 (en) Alpha-hemoglobin stabilizing protein antibodies and methods of use thereof
CN116449010B (zh) Fam111b在胶质瘤的诊断或预后中的应用及相关的计算机可读介质
US20200271647A1 (en) Biomarkers for predicting prognosis of kidney disease
CN113759113A (zh) 用于诊断髓母细胞瘤的尿液蛋白标记物及其用途
JP2024003567A (ja) 膵癌又は前立腺癌の診断を補助する方法及び膵癌又は前立腺癌の診断用試薬
JP2023133869A (ja) 卵巣がん患者の予後予測用バイオマーカー
US20160326251A1 (en) Therapeutic agent for cancer, and method for determining prognosis of cancer
EA044968B1 (ru) Гуманизированное антитело и способ его применения

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: 23864804

Country of ref document: EP

Kind code of ref document: A1