CN116027038A

CN116027038A - Use of discoid domain receptor 2 in diagnosis of glioma and related computer readable medium

Info

Publication number: CN116027038A
Application number: CN202211130381.6A
Authority: CN
Inventors: 请求不公布姓名
Original assignee: Feichuang Biomedical Technology Guangzhou Co ltd
Current assignee: Feichuang Biomedical Technology Guangzhou Co ltd
Priority date: 2022-09-16
Filing date: 2022-09-16
Publication date: 2023-04-28
Also published as: WO2024056075A1

Abstract

The present invention discloses the use of a reagent for detecting the expression level of discoid domain receptor 2 (DDR 2) in the preparation of a kit for diagnosing glioma in a subject, wherein a level of DDR2 in a sample from the subject that is higher than a control without disease is indicative of the subject having glioma. Kits, methods, and computer-readable media for diagnosing glioma are also disclosed. The invention can efficiently and accurately diagnose glioma by detecting DDR 2.

Description

Use of discoid domain receptor 2 in diagnosis of glioma and related computer readable medium

Technical Field

The present invention relates to kits, methods and computer readable media for diagnosing glioma, more particularly to diagnosing glioma by detecting discoid domain receptor 2 (DDR 2).

Background

Gliomas are a tumor that occurs in the brain and spinal cord, accounting for about 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 classified into astrocytomas (astrocytes), oligodendrogliomas (oligodendrocytes), ependymomas (ependymal cells), and mixed gliomas (e.g., oligodendrogliomas and astrocytomas, including mixed gliomas) according to the degree of morphological similarity of tumor cells to normal brain glial cells. According to the molecular pathological characteristics and biological behaviors of tumors, glioma malignant degrees can be classified into WHO I-IV grades from low to high. Grade I gliomas are generally benign. The average survival of grade II (low grade glioma) is about 7 years. Grade II gliomas can progress to grade III (high grade glioma), and eventually grade IV (secondary glioblastoma), where the grade IV glioblastoma accounts for over 50%, with the highest malignancy, and very poor prognosis. Grade III and grade IV gliomas are collectively known as High-grade gliomas (HGGs), including anaplastic astrocytomas, anaplastic oligodendrogliomas, and Gliobastoma (GBM) which are the most malignant. HGG is the most common invasive primary intracranial tumor, and is often clinically used for comprehensive treatment by combining surgery and radiotherapy and chemotherapy, but the treatment effect is still not ideal, and the recurrence is very easy to occur.

The molecular markers for auxiliary diagnosis of glioma at present include IDH, MGMT, EGFR, P, PTEN and the like. Somatic mutations in IDH1/2, genomic amplification of EGFR, and co-deletion of chromosome arms 1p and 19q are typically examined for accurate diagnosis. IDH1/2 mutations are common in low grade gliomas and secondary glioblastomas, which are well known to have profound effects on the prolonged survival of glioma patients. In addition, approximately 50% of glioblastomas showed focal expansion of EGFR. Furthermore, EGFR gene amplification is known to promote cell proliferation, and thus clinical trials of EGFR inhibitors are often used in cancer treatment.

In this context, in order to effectively diagnose gliomas, immunohistochemistry (IHC) and Fluorescence In Situ Hybridization (FISH) are generally used to make pathological diagnosis of target mutations of IDH1, which is considered to be an essential gene for glioma proliferation, and focal amplification of EGFR. However, there is an increasing need for an effective diagnosis of gliomas in a simpler manner.

Discoid domain receptor 2 (discoidin domain receptor 2) is a Receptor Tyrosine Kinase (RTK) that uses extracellular matrix protein collagen as its ligand. In addition to its kinase function, DDR2 promotes cell adhesion by activating beta 1-integrins. The DDR2 has the special function of mediating the signal transmission of extracellular matrix to cells, so that the extracellular matrix is regulated to be balanced, and the DDR2 is involved in regulating and controlling the growth, differentiation and metabolism of cells. Although extracellular matrix collagen activation DDR2 is necessary for normal development and tissue homeostasis, abnormal activation of these receptors following injury or disease is detrimental.

DDR2 is considered an important target for inflammation such as arthritis (e.g., osteoarthritis, rheumatoid arthritis) and fibrosis (e.g., pulmonary fibrosis, cirrhosis, renal fibrosis, or skin fibrosis). DDR2 is mainly expressed in interstitial cells such as fibroblasts, myofibroblasts, smooth muscle cells, etc. of kidney, skin, lung, heart and connective tissue. Much evidence suggests that aberrant expression of DDR2 is associated with a variety of disease processes, such as inflammation, liver fibrosis, kidney fibrosis, lung fibrosis, skin scarring and atherosclerosis. In the mouse inflammation model study, DDR2 expression was found to be up-regulated in the knee joint of aged mice. In a rat model study of rheumatoid arthritis, it was found that DDR2 expression was up-regulated in synovial cells.

However, expression and function of DDR2 has never been reported in glioma cells. The application of DDR2 in glioma diagnosis is not yet studied and reported.

Disclosure of Invention

The inventors of the present application have unexpectedly found that DDR2 expression is significantly increased in gliomas compared to normal controls. The inventors detected the presence and/or level of DDR2 in brain tissue or exosomes using an agent capable of binding to DDR2, thereby diagnosing glioma with high efficiency and accuracy.

In a first aspect, the invention provides a kit for diagnosing glioma in a subject, the kit comprising reagents for detecting the expression level of discoid domain receptor 2 (DDR 2), wherein a level of DDR2 in a sample from the subject that is higher than a control that is not afflicted with a disease indicates that the subject has glioma.

In a second aspect, the invention provides the use of a reagent for detecting the expression level of discoid domain receptor 2 (DDR 2) in the manufacture of a kit for diagnosing glioma in a subject, wherein a level of DDR2 in a sample from the subject that is higher than a control that is not suffering from a disease is indicative that the subject has glioma.

In a third aspect, the invention provides a reagent for detecting the expression level of discoid domain receptor 2 (DDR 2) for diagnosing glioma in a subject, wherein a level of DDR2 in a sample from the subject that is higher than a control that is not afflicted with a disease indicates that the subject has glioma.

In a fourth aspect, the invention provides a method of diagnosing glioma in a subject comprising detecting the presence and/or level of discoid domain receptor 2 (DDR 2) in a sample from the subject. In some embodiments, the method comprises contacting an agent capable of binding to DDR2 with a sample from the subject; detecting the presence of complexes formed by the reagent and DDR2 in the sample after contact; and determining that the subject has or is at risk of having a glioma based on the presence and/or level of the complex.

In a fifth aspect, the present invention provides a computer readable storage medium having stored thereon computer instructions for computer reading and execution, the computer instructions being executable to perform a method of diagnosing whether a subject has glioma, the method comprising: (a) Contacting a sample from the subject with an agent capable of binding to discotic domain receptor 2 (DDR 2); (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 determining that the subject has glioma when the signal exceeds the predetermined threshold, and wherein the threshold is a median level from subjects not suffering from the disease.

In a sixth aspect, the present invention provides a method of treating glioma in a subject, the method comprising: (a) Contacting a sample from the subject with an agent capable of binding to discotic domain receptor 2 (DDR 2); (b) Detecting and reading the signal of the contacted sample to determine whether the reagent forms a complex with DDR2 in the sample; (c) Determining whether the signal exceeds a predetermined threshold, and determining that the subject has glioma when the signal exceeds a predetermined threshold, and wherein the threshold is a median level from subjects not suffering from the disease; and (d) administering an anti-tumor therapy to the subject determined to have glioma. In a preferred embodiment, the anti-tumor therapy is temozolomide.

In any of the above aspects, the reagent for detecting the expression level of DDR2 comprises a reagent capable of binding to DDR2 to detect the level of DDR2 in the sample. In some embodiments, the agent capable of binding to DDR2 comprises a protein, a nucleic acid, or a small molecule compound. In some embodiments, the agent capable of binding to DDR2 is an anti-DDR 2 monoclonal antibody or antigen-binding fragment thereof, or an anti-DDR 2 polyclonal antibody. In some embodiments, the anti-DDR 2 monoclonal antibody is an anti-DDR 2 nanobody.

In some embodiments, the agent capable of binding to DDR2 is labeled with a detectable label. In some embodiments, the detectable label is selected from the group consisting of a fluorescent label, a chemiluminescent label, a paramagnetic label, a radioisotope label, and an enzymatic label.

In any of the above aspects, the sample is brain tissue. In any of the above aspects, the glioma is selected from the group consisting of astrocytomas, oligodendrogliomas, ependymomas, and hybrid gliomas. In some embodiments, the glioma is selected from the group consisting of anaplastic astrocytoma, anaplastic oligodendroglioma, and glioblastoma. In some embodiments, the glioma is glioblastoma.

In a seventh aspect, the invention provides a kit for diagnosing glioma in a subject, the kit comprising an exosome from the subject, and a reagent capable of binding to detect discoid domain receptor 2 (DDR 2) to detect the level of DDR2 expressed by the exosome, wherein a level of DDR2 expressed by the exosome that is higher than a level of a control that does not have a disease indicates that the subject has glioma.

In an eighth aspect, the invention provides the use of an exosome from a subject in the manufacture of a kit for diagnosing glioma in the subject, wherein a level of discoid domain receptor 2 (DDR 2) expressed by the exosome that is higher than a control not suffering from a disease indicates that the subject has glioma.

In a ninth aspect, the invention provides an exosome from a subject for diagnosing glioma in the subject, wherein a level of discoid domain receptor 2 (DDR 2) expressed by the exosome that is higher than a control without disease indicates that the subject has glioma.

In a tenth aspect, the invention provides a method of diagnosing glioma in a subject, the method comprising isolating exosomes from the subject and detecting the presence and/or level of discoid domain receptor 2 (DDR 2) in exosomes from the subject. In some embodiments, the method comprises contacting an agent capable of binding to DDR2 with an exosome from the subject; detecting the presence of a complex formed by the agent and DDR2 in the exosomes after contact; and determining that the subject has or is at risk of having a glioma based on the presence and/or level of the complex.

In an eleventh aspect, the present invention provides a computer-readable storage medium having stored thereon computer instructions for computer reading and execution, the computer instructions being executable to perform a method of diagnosing whether a subject has glioma, the method comprising: (a) isolating exosomes from the subject; (b) Contacting the isolated exosomes with an agent capable of binding to discotic domain receptor 2 (DDR 2); (c) Detecting and reading the signal of the contacted exosomes to determine if the agent forms a complex with DDR2 in the exosomes; and (d) determining whether the signal exceeds a predetermined threshold, and determining that the subject has glioma when the signal exceeds the predetermined threshold, and wherein the threshold is a median level from subjects not suffering from the disease.

In a twelfth aspect, the present invention provides a method of treating glioma in a subject, the method comprising: (a) isolating exosomes from the subject; (b) Contacting the isolated exosomes with an agent capable of binding to discotic domain receptor 2 (DDR 2); (c) Detecting and reading the signal of the contacted exosomes to determine if the agent forms a complex with DDR2 in the exosomes; (d) Determining whether the signal exceeds a predetermined threshold, and determining that the subject has glioma when the signal exceeds a predetermined threshold, and wherein the threshold is a median level from subjects not suffering from the disease; and (e) administering an anti-tumor therapy to the subject determined to have glioma. In a preferred embodiment, the anti-tumor therapy is temozolomide.

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

In any of the above aspects, the agent capable of binding to DDR2 comprises a protein, a nucleic acid, or a small molecule compound. In some embodiments, the agent capable of binding to DDR2 is an anti-DDR 2 monoclonal antibody or antigen-binding fragment thereof, or an anti-DDR 2 polyclonal antibody. In some embodiments, the agent capable of binding to DDR2 is labeled with a detectable label. In some embodiments, the detectable label is selected from the group consisting of a fluorescent label, a chemiluminescent label, a paramagnetic label, a radioisotope label, and an enzymatic label.

Additional aspects and advantages of the invention will be described in the detailed description, and will be apparent to those skilled in the art from the detailed description that follows, other aspects and advantages not explicitly recited in the present disclosure.

Drawings

Fig. 1: affinity constant profile of DDR2 nanobodies with extracellular segments of DDR2 antigens.

Fig. 2: glioma in vitro imaging results for DDR2 nanobodies.

Fig. 3: and (3) a plasma exosome flow detection result diagram of the primary glioma patient.

Fig. 4: (a) DDR2 expression profile in glioma-related experimental data; (b) The Violin plot shows DDR2 positive cell distribution, and the histogram is DDR2 expression level.

Fig. 5: (a) UMAP cell cluster analysis. The left graph shows the distribution of various cell types, and the right graph shows the distribution of various sample-derived cells; (b) The corresponding expression of various marker genes in the UMAP cell cluster profile.

Fig. 6: expression profile of related genes in different cell types.

Detailed Description

Definition of the definition

Throughout the specification and claims, unless the context indicates otherwise, the word "comprise" and variations such as "comprises" and "comprising" will be understood to imply the inclusion of a stated integer, step or component but not the exclusion of any other integer, step or component. The term "comprising" when used herein may be replaced with the term "including" or "containing" or sometimes the term "having" is used herein.

In the present invention, "about" means that the value is within an acceptable error range for the particular value being determined by one of ordinary skill in the art, which value depends in part on how it is measured or determined (i.e., the limits of the measurement system). For example, "about" may mean within 1 or exceeding a standard deviation of 1 in each practice in the art. Alternatively, "about" or "substantially comprising" may mean a range of up to 20%. Furthermore, for a biological system or process, the term may mean at most one order of magnitude or at most 5 times the value. Unless otherwise indicated, when a particular value is found in this application and in the claims, the meaning of "about" or "consisting essentially of" should be assumed to be within the acceptable error range for that particular value.

As used herein, the term "subject," "patient," or "individual" refers to any subject, particularly a mammalian subject, for whom diagnosis, prognosis, or treatment is desired. Mammals include humans, domestic animals, farm animals, zoo animals, athletic animals or pets, such as dogs, cats, pigs, rabbits, rats, mice, horses, cows, etc. The subject referred to herein is preferably a human. In some embodiments, the subject suffers from or is susceptible to one or more disorders or conditions. The patient may exhibit one or more symptoms of the disorder or condition, or may have been diagnosed with the one or more disorders or conditions. In some embodiments, the patient is receiving or has received a certain therapy for diagnosing and/or treating such diseases, disorders or conditions.

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

As used herein, the term "diagnosis" refers to the identification or classification of a molecular or pathological state, disease or condition. For example, "diagnosis" may refer to the identification of glioma or the identification of a particular type thereof. In some embodiments of the invention, "diagnosing" includes distinguishing between different grades of glioma or determining the grade of glioma.

According to the present invention, the term "binding" preferably relates to specific binding. By "specific binding" is meant that the agent binds more strongly to the specific target than to another target. If the dissociation constant (K _D ) Less than the dissociation constant for the second target, it binds more strongly to the first target than to the second target. Preferably, the dissociation constant (K _D ) In contrast, reagentsDissociation constant (K) of specifically bound target _D ) Is more than 10 ² Multiple of 10 ³ Multiple of 10 ⁴ Multiple of 10 ⁵ Multiple of 10 ⁶ Multiple of 10 ⁷ Multiple of 10 ⁸ Multiple of 10 ⁹ Multiple or 10 ¹⁰ The times lower.

Preferably, an agent (e.g., a protein or polypeptide) is specific for a predetermined target if it is capable of binding to the predetermined target but not to other targets, i.e., does not have significant affinity for and does not significantly bind to other targets in a standard assay. According to the invention, an agent is DDR2 specific if it is capable of binding to DDR2 but is (substantially) incapable of binding to other targets. Preferably, if the reagent binds K to the predetermined target _D Is K bound to its non-specific target _D At least 10 of (2) ² Multiple of 10 ³ Multiple of 10 ⁴ Multiple of 10 ⁵ Multiple of 10 ⁶ Multiple of 10 ⁷ Multiple of 10 ⁸ Multiple of 10 ⁹ Multiple or 10 ¹⁰ Lower than the first, the reagent is specific for the target.

Binding of the agent to the target may be determined experimentally using any suitable method, which is within the purview of one skilled in the art. Affinity can be readily determined using conventional techniques, for example by equilibrium dialysis; surface plasmon resonance analysis is used by using the general procedure outlined by the manufacturer; by radioimmunoassay using a radiolabeled target antigen; or by other methods known to the skilled person. Affinity data may be analyzed, for example, by methods known in the art. The measured affinity of a particular interaction may vary if measured under different conditions (e.g., salt concentration, pH). Thus, affinity and other binding parameters (e.g., K _D 、IC ₅₀ ) Preferably with standardized solutions and standardized buffers of the binding agent and the target.

As used herein, the term "antibody" refers to any form of antibody that exhibits a desired biological activity (e.g., inhibits ligand binding to its receptor or receptor signaling induced by inhibition of ligand). "antibody fragment" and "antigen-binding fragment" refer to antigen-binding fragments of antibodies and antibody analogs, which generally comprise at least a portion of the antigen-binding or variable regions (e.g., one or more CDRs) of a parent antibody. In some embodiments, 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 population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible natural mutations that may be present in minor amounts. Monoclonal antibodies are highly specific and can be directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations, which typically include a plurality of different antibodies directed against a plurality of different determinants (epitopes), each monoclonal antibody is directed against only a single determinant on the antigen. The modifier "monoclonal" refers to the identity of the antibody as obtained from a population of substantially homogeneous antibodies and is not to be construed as requiring preparation of the antibody by any particular method. For example, monoclonal antibodies for use in the present invention may be prepared by hybridoma or recombinant DNA methods.

Monoclonal antibodies may include "chimeric" antibodies, humanized antibodies, or fully human antibodies. In some embodiments, the antibody forms part of a larger biomolecule, such as a fusion protein or antibody drug conjugate. The antibody fragment retains 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 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 to the target.

As used herein, the term "heavy chain antibody" refers to an antibody that lacks a light chain but consists of only a heavy chain, comprising two constant regions (CH 2 and CH 3), one hinge region, and one heavy chain variable region (i.e., VHH). Examples include, but are not limited to, natural heavy chain antibodies, antibodies that do not naturally have a light chain, heavy chain antibodies derived from conventional 4-chain antibodies, and engineered antibodies. Heavy chain antibodies may be from Camelidae (Camelidae) species, such as antibodies produced in camels, llamas, dromedaries, alpacas and piggyhorses. Other species than camelidae may produce heavy chain antibodies that naturally lack light chains; such heavy chain antibodies are within the scope of the invention.

As used herein, the term "nanobody" refers to a single domain antibody derived from cloning the variable region of a heavy chain antibody, which is the smallest functional antigen binding fragment, consisting of only the heavy chain variable region, also known as VHH (Variable domain of heavy chain of heavy chain antibody) or single domain antibody. Nanobodies recognize antigens with high specificity and affinity similar to IgG antibodies, but can penetrate tumor tissue better 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 comprise at least a portion of the antigen-binding or variable regions (e.g., one or more CDRs) of a parent antibody. The 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 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 to the target. Examples of antibody fragments include, but are not limited to: fab, fab ', F (ab') ₂ And Fv fragments; a diabody; a linear antibody; single chain antibody molecules, such as scFv (single chain variable fragment); a nanobody; domain antibodies; and multispecific antibodies formed from antibody fragments. Antibodies against DDR2 refer to antibodies that specifically bind to DDR2, including engineered antibodies, as well as 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 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, as compared to a reference antibody or polypeptide. In some aspects, an equivalent variant of an antibody or polypeptide retains the activity (e.g., epitope binding) or structure (e.g., salt bridge) of a reference sequence.

As used herein, 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.

"percent identity" between two sequences as used herein refers to a function of the number of identical positions shared by the sequences (i.e.,% homology = number of identical positions/number of total positions x 100), wherein the number of gaps, and the length of each gap, are taken into account, which need to be introduced when optimally aligning the two sequences. Sequence comparison and determination of% identity between two sequences can be accomplished using mathematical algorithms.

The term "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. The nucleic acid may be single-stranded or double-stranded. RNA includes in vitro transcribed RNA or synthetic RNA.

The terms "control", "control sample", "standard control" or "standard" refer to a sample that serves as a reference (typically a known reference) for comparison to a test sample. For example, a test sample may be taken from a patient suspected of having a given disease and compared to samples from known disease patients or known normal (non-disease) individuals. Controls may also represent averages collected from populations of similar individuals (e.g., disease patients or healthy individuals with similar medical background, same age, weight, etc.). The control value may also be obtained from the same individual, for example from an earlier obtained sample, prior to disease, or prior to treatment. The skilled artisan will recognize that controls may be designed to evaluate a number of parameters.

As used herein, the term "body fluid" or "body fluid sample" may generally refer to a fluid that is typically present in and/or may be produced by the body of a subject or patient. For example, the bodily fluid may include one or more of peripheral blood, serum, plasma, serosal fluid, sputum, synovial fluid, aqueous humor, amniotic fluid, milk, semen, prostatic fluid, cowper's fluid, female emissions fluid, sweat, fecal matter, tears, cyst fluid, pleural effusion, ascites fluid, pericardial fluid, chyle, bile, interstitial fluid, menstrual blood, pus, vomit, vaginal secretions, mucosal secretions, pancreatic juice, blastocyst fluid, umbilical cord blood, urine, cerebrospinal fluid, saliva, lymph, stool, bronchopulmonary aspirate, bronchoalveolar lavage fluid, and nasal lavage fluid, including components or fractions thereof. The body fluid samples may be mixed or pooled. The body fluid sample may be provided by taking body fluid from the patient, but may also be provided by using previously separated body fluid sample material. In some embodiments, the body fluid or body fluid sample used in the present invention is a serum or plasma sample.

As used herein, the term "exosome" refers to a tiny vesicle of about 30-150 a nm a diameter secreted by a variety of cells containing a specific protein (e.g., CD63, CD81, and CD9, which are transmembrane proteins involved in exosome transport), lipid, cytokine, or genetic material on the exosome membrane. A variety of cells secrete exosomes under normal and pathological conditions, and they are widely present in body fluids such as blood, saliva, urine, cerebrospinal fluid and milk, and are considered as specifically secreted vesicles, involved in intercellular communication. In some embodiments, the exosomes may be isolated from a body fluid sample from a subject by size exclusion chromatography, density gradient centrifugation, differential centrifugation, nanomembrane ultrafiltration, immunoadsorption capture, affinity capture, microfluidic separation, or a combination thereof.

Reagent for detecting DDR2 expression level

In one aspect, the invention provides reagents for detecting expression levels of discoid domain receptor 2 (DDR 2) for diagnosing glioma in a subject.

As used herein, the term "reagent for detecting expression levels of DDR 2" refers to any reagent known in the art that can be used to detect DDR2, such as targeting or affinity reagents for DDR2, including in particular reagents that can bind (specifically bind) to DDR2 to form a chemically, physically or biologically detectable complex.

In some embodiments, the agent capable of binding to DDR2 comprises a protein, nucleic acid, or small molecule compound that can target one or more epitopes of DDR2 protein.

In some embodiments, the agent capable of binding to DDR2 is an anti-DDR 2 monoclonal antibody or antigen-binding fragment thereof, or an anti-DDR 2 polyclonal antibody. anti-DDR 2 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 access date 2022, month 9, 1). Alternatively, anti-DDR 2 antibodies can be generated de novo using methods known in the art. In some embodiments, the anti-DDR 2 antibody is any form of antibody or antibody fragment as defined herein.

In some embodiments, the anti-DDR 2 monoclonal antibody is an anti-DDR 2 nanobody. In some embodiments, the anti-DDR 2 nanobody comprises CDR1, CDR2, CDR3, wherein CDR1 comprises or is the sequence shown in SEQ ID No. 1 or an equivalent variant thereof, CDR2 comprises or is the sequence shown in SEQ ID No. 2 or an equivalent variant thereof, and CDR3 comprises or is the sequence shown in SEQ ID No. 3 or an equivalent variant thereof, wherein CDR is defined according to IMGT. In some embodiments, the nanobody comprises or is a sequence set forth in SEQ ID NO. 4 or an equivalent variant thereof.

In some embodiments, the equivalent variants of CDR1, CDR2, CDR3 refer to substitutions, deletions or insertions of a single amino acid compared to a reference sequence.

In some embodiments, equivalent variants of the nanobody refer to having at least 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID No. 4 and having the same or equivalent CDR1, CDR2, and CDR3. In some embodiments, the CDR1, CDR2, and CDR3 are defined based on any one of the definition schemes IMGT, kabat, chothia, contact or AbM. In some embodiments, the CDR1, CDR2, and CDR3 are defined based on IMGT definition schemes.

In some embodiments, the anti-DDR 2 nanobody comprises a CDR1, a CDR2, a CDR3, wherein CDR1 comprises the sequence shown in SEQ ID NO. 1, CDR2 comprises the sequence shown in SEQ ID NO. 2, and CDR3 comprises the sequence shown in SEQ ID NO. 3, wherein CDR is defined according to IMGT.

In some embodiments, the anti-DDR 2 nanobody comprises a CDR1, a CDR2, a 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 the sequence shown in SEQ ID NO. 3, wherein CDR is defined according to IMGT.

In some embodiments, the substitutions described herein are conservative substitutions. "conservative (amino acid) substitution" refers to the substitution of an amino acid residue with an amino acid having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a non-essential amino acid residue in an immunoglobulin polypeptide is preferably substituted with another amino acid residue from the same side chain family. In another embodiment, the amino acid strings may be substituted with structurally similar strings that differ in the order and/or composition of the side chain family members.

In some embodiments, the agent capable of binding to DDR2 is a peptide or a nucleic acid aptamer. Such an aptamer may be selected from a library of oligonucleotides or peptides by any method known in the art. Nucleic acid aptamers can be selected via SELEX (Systematic Evolution of Ligands by Exponential Enrichment ). Peptide aptamers can be selected using yeast or bacterial two-hybrid systems.

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

Chemiluminescent labels (e.g., luminol, luciferase, luciferin, and aequorin) may also be employed. Such diagnosis and detection may also be accomplished by linking agents capable of binding to DDR2 with detectable substances including, but not limited to: various enzymes, including but not limited to horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase, or by ligating it with a prosthetic complex (prosthetic groupcomplexes), such as but not limited to: streptavidin/biotin and avidin/biotin.

Paramagnetic and radioisotope labels may also be employed, which are preferably detected using positron emission tomography (Positron Emission Tomography) (PET) or Single photon computed tomography (Single-PhotonEmission Computed Tomography) (SPECT). The radiolabel comprisesBut not limited to bismuth% ²¹³ Bi, C ¹¹ C、 ¹³ C、 ¹⁴ C) The chromium is ⁵¹ Cr, co% ⁵⁷ Co、 ⁶⁰ Co, cu% ⁶⁴ Cu and dysprosium% ¹⁶⁵ Dy and erbium ¹⁶⁹ Er, fluorine ] ¹⁸ F) Gadolinium% ¹⁵³ Gd、 ¹⁵⁹ Gd, gallium% ⁶⁸ Ga、 ⁶⁷ Ga, ge% ⁶⁸ Ge, gold% ¹⁹⁸ Au), holmium ¹⁶⁶ Ho, hydrogen ³ H) The indium is ¹¹¹ In、 ¹¹² In、 ¹¹³ In、 ¹¹⁵ In and iodine% ¹²¹ I、 ¹²³ I、 ¹²⁵ I、 ¹³¹ I) The iridium is% ¹⁹² Ir, iron ⁵⁹ Fe, krypton ^81m Kr, lanthanum% ¹⁴⁰ La, lutetium ] ¹⁷⁷ Lu and Mn ⁵⁴ Mn, mo ⁹⁹ Mo and N ¹³ N、 ¹⁵ N, oxygen% ¹⁵ O, pd% ¹⁰³ Pd and P ] ³² P and K ⁴² K) Praseodymium (praseodymium) ¹⁴² Pr and promethium ¹⁴⁹ Pm, re ] ¹⁸⁶ Re、 ¹⁸⁸ Re and rhodium% ¹⁰⁵ Rh and rubidium% ⁸¹ Rb、 ⁸² Rb, ru ] ⁸² Ru、 ⁹⁷ Ru and samarium ¹⁵³ Sm, scandium ⁴⁷ Sc and Se% ⁷⁵ Se, na ²⁴ Na, sr ⁸⁵ Sr、 ⁸⁹ Sr、 ⁹² Sr and S ³⁵ S, technetium ⁹⁹ Tc), thallium ²⁰¹ Tl, tin ] ¹¹³ Sn、 ¹¹⁷ Sn and xenon ¹³³ Xe, ytterbium% ¹⁶⁹ Yb、 ¹⁷⁵ Yb、 ¹⁷⁷ Yb and yttrium% ⁹⁰ Y) and Zinc% ⁶⁵ Zn); various positron-emitting and nonradioactive paramagnetic metal ions for positron emission tomography can be used, such as, for example, paramagnetic aluminum (Al) ions, barium (Ba) ions, calcium (Ca) ions, cerium (Ce) ions, dysprosium (Dy) ions, erbium (Er) ions, europium (Eu) ions, gadolinium (Gd) ions, holmium (Ho) ions, iridium (Ir) ions, lithium (Li) ions, magnesium (Mg) ions, manganese (Mn) ions, molybdenum (M) ions, neodymium (Nd) ions, osmium (Os) ions, oxygen (O) ions, palladium (Pd) ions, platinum (Pt) ions, rhodium (Rh) ions, ruthenium (Ru) ions, samarium (Sm) ions, sodium (Na) ions, strontium (Sr) ions, terbium (Tb) ions, thulium (Tm) ions, tin (Sn) ions, titanium (Ti) ions, tungsten (Sn) ions(W) ions and zirconium (Zi) ions, especially Co ⁺² 、CR ⁺² 、Cr ⁺³ 、Cu ⁺² 、Fe ⁺² 、Fe ⁺³ 、Ga ⁺³ 、Mn ⁺³ 、Ni ⁺² 、Ti ⁺³ 、V ⁺ And V ⁺⁴ . Methods for preparing radiolabeled amino acids and related peptide derivatives are known in the art. For example, the radioisotope may be conjugated by chloramine-T method.

Kit for detecting a substance in a sample

In one aspect, the invention provides a kit for diagnosing glioma in a subject, the kit comprising reagents for detecting the expression level of discoid domain receptor 2 (DDR 2), wherein a level of DDR2 in a sample from the subject that is higher than a control that is not afflicted with a disease indicates that the subject has glioma.

In another aspect, the invention provides a kit for diagnosing glioma in a subject, the kit comprising an exosome from the subject, and a reagent capable of binding to detect discoid domain receptor 2 (DDR 2) to detect the level of DDR2 expressed by the exosome, wherein a level of DDR2 expressed by the exosome that is higher than a level of a control that does not have a disease indicates that the subject has glioma.

In some embodiments, the reagent for detecting the expression level of DDR2 comprises a reagent capable of binding (particularly specifically binding) to DDR2 to form a chemically, physically or biologically detectable complex, such as a protein, nucleic acid or small molecule compound, particularly an anti-DDR 2 monoclonal antibody or antigen-binding fragment thereof, or an anti-DDR 2 polyclonal antibody.

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

In some embodiments, the agent capable of binding to DDR2 is labeled with a detectable label. In some embodiments, the detectable label is selected from the group consisting of a fluorescent label, a chemiluminescent label, a paramagnetic label, a radioisotope label, and an enzymatic label. In some embodiments, the label is indocyanine green (ICG).

In some embodiments, the kit further comprises DDR2 recombinant antigen controls at different concentrations, thereby preparing a standard curve for quantitative identification.

Detection or diagnosis method and computer readable storage medium

In one aspect, the invention provides the use of discoid domain receptor 2 (DDR 2) as a marker in diagnosing glioma in a subject.

In another aspect, the invention provides an in vitro methodin vitro) Or ex vivo%ex vivo) A method of diagnosing glioma in a subject, the method comprising detecting the presence and/or level of discoid domain receptor 2 (DDR 2) in a sample (e.g., brain tissue or exosomes) from the subject.

In another aspect, the present invention provides a method for treating a disease in a bodyin vivo) An agent capable of binding to discoid domain receptor 2 (DDR 2) that diagnoses glioma in a subject.

In each of the above aspects, the agent capable of binding to DDR2 is an anti-DDR 2 monoclonal antibody or antigen-binding fragment thereof, or an anti-DDR 2 polyclonal antibody. In a preferred embodiment, the agent capable of binding to DDR2 is an anti-DDR 2 monoclonal antibody or antigen-binding fragment thereof.

Preferably, in each of the above aspects, wherein the agent capable of binding to DDR2 is labeled with a detectable label. In a preferred embodiment, wherein the detectable label is selected from the group consisting of fluorescent labels, chemiluminescent labels, paramagnetic labels, radioisotope labels, and enzymatic labels. In a preferred embodiment, wherein the detectable label is selected from a fluorescent label or a chemiluminescent label.

Preferably, in each of the above aspects, the glioma is selected from the group consisting of astrocytomas, oligodendrogliomas, ependymomas, and hybrid gliomas. In some embodiments, the glioma is selected from the group consisting of anaplastic astrocytoma, anaplastic oligodendroglioma, and glioblastoma. In some embodiments, the glioma is glioblastoma.

Various immunoassays can be used in the diagnostic method. In some embodiments, such immunoassays include the use of competitive and non-competitive detection systems such as radioimmunoassays, immunochromatography, ELISA, "sandwich" immunoassays, precipitation reactions, immunoblot analysis, gel diffusion precipitation reactions, immunodiffusion assays, agglutination assays, complement fixation assays, immunoradiometric assays, fluorescent immunoassays, and the like. Both in vitro and in vivo assays may be used.

Typically, the level of DDR2 in the sample is compared to a reference level, wherein a 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 (e.g., from healthy tissue or subject) or a median level from a healthy subject. An increase in the presence of DDR2 and/or the amount of DDR2 in a sample compared to a reference level, e.g., compared to a subject not suffering from a disease, may be indicative of the risk of the presence or occurrence of glioma in the subject.

In some embodiments, DDR2 in a sample (e.g., brain tissue or exosomes) of a subject having a glioma is present in an amount of at least about 3-fold, at least about 5-fold, at least about 7.5-fold, at least about 10-fold, at least about 15-fold, or at least about 20-fold compared to a subject not having the disease.

Methods for diagnosis allow for quantitative and/or qualitative assessment, e.g. absolute and/or relative measurement of target molecules, e.g. measuring the content of DDR2 in a sample.

In some embodiments of the methods of the invention, determining the presence and/or amount of DDR2 in the sample comprises: (i) Contacting a sample (e.g., brain tissue or exosomes) with an agent capable of binding to DDR2, and (ii) detecting the formation of a complex between the agent and DDR2 and/or determining the amount of the complex.

In some embodiments, the detection/diagnostic methods of the present invention may be used in combination with other methods of detecting/diagnosing glioma. For example, the detection/diagnostic methods of the invention may be used in conjunction with the detection of other biomarkers of glioma (e.g., IDH, MGMT, EGFR, P53, PTEN).

In another aspect, the present invention provides a computer-readable storage medium having stored thereon computer instructions for computer reading and execution, the computer instructions being executable to perform a method of diagnosing whether a subject has glioma, the method comprising: (a) Contacting a sample from the subject with an agent capable of binding to discotic domain receptor 2 (DDR 2); (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 determining that the subject has glioma when the signal exceeds the predetermined threshold. In a preferred embodiment, the threshold is a median level from subjects not suffering from the disease.

In another aspect, the present invention provides a computer-readable storage medium having stored thereon computer instructions for computer reading and execution, the computer instructions being executable to perform a method of diagnosing whether a subject has glioma, the method comprising: (a) isolating exosomes from the subject; (b) Contacting the isolated exosomes with an agent capable of binding to discotic domain receptor 2 (DDR 2); (c) Detecting and reading the signal of the contacted exosomes to determine if the agent forms a complex with DDR2 in the exosomes; and (d) determining whether the signal exceeds a predetermined threshold, and determining that the subject has glioma when the signal exceeds the predetermined threshold, and wherein the threshold is a median level from subjects not suffering from the disease.

In another aspect, the invention provides a method of treating glioma in a subject, the method comprising: (a) Contacting a sample from the subject with an agent capable of binding to discotic domain receptor 2 (DDR 2); (b) Detecting and reading the signal of the contacted sample to determine whether the reagent forms a complex with DDR2 in the sample; (c) Determining whether the signal exceeds a predetermined threshold, and determining that the subject has glioma when the signal exceeds a predetermined threshold, and wherein the threshold is a median level from subjects not suffering from the disease; and (d) administering an anti-tumor therapy to the subject determined to have glioma.

In another aspect, the invention provides a method of treating glioma in a subject, the method comprising: (a) isolating exosomes from the subject; (b) Contacting the isolated exosomes with an agent capable of binding to discotic domain receptor 2 (DDR 2); (c) Detecting and reading the signal of the contacted exosomes to determine if the agent forms a complex with DDR2 in the exosomes; (d) Determining whether the signal exceeds a predetermined threshold, and determining that the subject has glioma when the signal exceeds a predetermined threshold, and wherein the threshold is a median level from subjects not suffering from the disease; and (e) administering an anti-tumor therapy to the subject determined to have glioma.

In some embodiments, the agent capable of binding to DDR2 comprises a protein, a nucleic acid, or a small molecule compound. In a preferred embodiment, the agent capable of binding to DDR2 is an anti-DDR 2 monoclonal antibody or antigen-binding fragment thereof, or an anti-DDR 2 polyclonal antibody. In some embodiments, the agent capable of binding to DDR2 is labeled with a detectable label. In some embodiments, the detectable label is selected from the group consisting of a fluorescent label, a chemiluminescent label, a paramagnetic label, a radioisotope label, and an enzymatic label. .

In a preferred embodiment, the anti-tumor therapy is surgical excision, chemotherapy, radiation therapy, or a combination thereof. In a preferred embodiment, the chemotherapy is temozolomide, lomustine, carmustine, nimustine, procarbazine, vinblastine, vincristine, teniposide, etoposide, irinotecan, cisplatin, carboplatin, bevacizumab, or a combination thereof. In a preferred embodiment, the chemotherapy is temozolomide.

In some embodiments, the predetermined threshold may be a reference level as described above, wherein a deviation from the reference level is 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 (e.g., from healthy tissue or subject) or a median level from a healthy subject. An increase in the presence of DDR2 and/or the amount of DDR2 in a sample compared to a reference level, e.g., compared to a subject not suffering from a disease, may be indicative of the risk of the presence or occurrence of glioma in the subject.

Sequence listing

Examples

Example 1: antibody screening and affinity assay

(1) Antibody screening

anti-DDR 2 heavy chain antibodies were selected from alpacas immunized with human DDR2 extracellular domain (UniProtKB/Swiss-Prot: Q16832.2 aa 22 to aa 399). Then the antibody is sequenced, the VHH part of the antibody is confirmed, and the nano antibody is obtained and named as 1A12, and the sequence of the nano antibody is shown in the sequence table part. The nanobody was expressed and purified for further characterization and experimentation.

(2) Affinity of DDR2 nanobody to extracellular segment of DDR2 antigen

The experimental steps are as follows:

the DDR2 nano antibody 1A12 is subjected to analysis and detection on the affinity of the nano antibody and DDR2 antigen extracellular domain protein by measuring the affinity of the antibody and the antigen by using a BLI method based on a molecular interaction analysis platform ForteBio biological layer interference technology (BLI).

Experiment consumable: antibody: dissolved in PBS (pH 7.4); antigen: dissolved in PBS (pH 7.4); a sensor: ni-NTA; kinetic Buffer: PBST (PBS+0.02% Tween-20, pH 7.4); regeneration Buffer:10 mM Glycine-HCl, pH 1.7; re-charged Buffer:10 mM NiCl in H2O.

The operation steps are as follows:

a. pre-wetting the probe in a kinetic buffer for 10 min;

b. Baseline 1：Baseline the Biosensors in kinetics buffer for 180 s；

c. fixing: diluting the His-tagged antigen DDR2 extracellular section to 20 mug/ml by using a kinetic buffer, and capturing by a sensor; to 4 nM (300 s);

d. Baseline 2：Baseline the Biosensors in kinetics buffer for 60 s；

e. combining: diluting the antibody solution to a certain concentration (from 100 nM to 3.125 nM by 2 times) by using a kinetic buffer, and stretching the sensor into the antibody solution for binding (600 s);

f. dissociation: the sensor dissociates in the tiles buffer (600 s);

g. sensor regeneration: 10 mM Glycine-HCl, pH 1.7 for 5 s;

h. and (3) neutralization: neutralizing 5 s in a kinetic buffer after the sensor is regenerated;

i. repeating the regenerating step g and the neutralizing step h: a total of 3 times (30 s);

j. baseline 3: after sensor regeneration 60 s in 10 mM NiCl was added.

And (5) making a dynamic curve and calculating each relevant parameter. The binding dissociation curve for selecting the appropriate several concentration gradients was 1: and (3) fitting all curves in a binding mode, and selecting three curves with the best fitting degree for drawing analysis to finally obtain important parameters such as affinity values, binding constants, dissociation constants and the like.

Analysis of results: the results of analysis of binding of DDR2 nanobody 1a12 to DDR2 antigen are shown in fig. 1 and table 1.

Table 1: affinity data for nanobody 1a12

Nanobody numbering	Affinity for
		1A12	4.359*10^-9

Example 2: in vitro imaging results of DDR2 nanobodies

The DDR2 antibody used in this example is nanobody 1A12 (Nb-DDR 2) of example 1, which has the amino acid sequence shown in SEQ ID NO. 4.

The experimental steps are as follows:

(1) Indocyanine green (ICG) marked DDR2 nano antibody

Nb-DDR2 was dissolved in PBS at a concentration of 2 mg/mL and vortexed.

ICG-NHS was dissolved in DMSO at a concentration of 2 mM and vortexed.

500. Mu.L of 2 mg/mL Nb-DDR2 solution was placed in a 1.5 mL centrifuge tube, 18. Mu.L of 2 mM ICG-NHS solution was added to the Nb-DDR2 solution in 9 portions, 2. Mu.L each, and vortexed for several seconds each addition.

The pH of the mixed solution was measured and adjusted to pH 8.5-9 using 2M NaOH solution.

The tube was placed on a 60 rpm shaker and reacted at room temperature 2 h.

The unreacted ICG-NHS was removed by centrifugation several times at 14000 g for 10 min using a 0.5. 0.5 mL ultrafiltration tube, and the solution was replaced with 0.9% NaCl and the protein solution was filtered through a 0.22 μm filter and stored at 4 ℃.

(2) DDR2-1A12-ICG probe imaging of glioma primary patient sample

DDR2 expression in brain gliomas is markedly elevated compared to normal brain tissue. Left frontal parietal intracranial tumor and paracancerous normal tissue sections of patients with gliomas of various grades were taken, 2 μg of ICG-labeled DDR2 nanobody 1a12 prepared above was added and incubated for 1 h at room temperature, pbs was washed multiple times to remove unbound probes, and near infrared imaging was performed using a small animal living imager, and the results are shown in fig. 2. The low grade glioma in the figure refers to grade I/II glioma, and the high grade glioma refers to grade III/IV glioma.

Analysis of results: the ICG marked DDR2 antibody can specifically identify the DDR2 highly expressed in glioma of each grade, and the normal tissue beside the cancer does not have obvious uptake, so that the ICG marked DDR2 antibody can be used for in-vitro accurate imaging and diagnosis.

Example 3: plasma exosome flow detection result of glioma primary patient

Flow type experiment steps:

extracting exosomes: 200. Mu.L of plasma was mixed with 350. Mu.L of PBS and placed in an exosome separator, and exosomes were resuspended using 400. Mu.L of PBS.

Flow type:

(1) Re-suspending the CD9 capture beads, sucking 12.5 mu L and mixing with 50 mu L sample, and incubating at 4deg.C for 18h;

(2) After washing 2 times with wash buffer, the supernatant was discarded and resuspended in 250 μl PBS;

(3) Sucking 25 mu L of heavy suspension, adding 1 ug of FITC-labeled DDR2 nano antibody 1A12, adjusting the volume to 100 mu L by using PBS, and incubating for 1h in a dark place;

(4) Wash 2 times, re-suspend with 200 μl PBS and run on-machine. The results are shown in FIG. 3.

Analysis of results: as can be seen from fig. 3, the percentage of DDR2 positives in preoperative plasma exosomes from WHO grade II and IV glioma patients increased significantly compared to normal persons, approximately 4-fold and 7.5-fold, respectively, of the normal controls, indicating that various grades of gliomas can be diagnosed by detecting DDR2 in exosomes.

Example 4: bioinformatics analysis of glioma-related experimental data

The following data analysis was derived from three sets of glioma-related data:

GSE174554 (40 Human ndGBM + 40 rGBM) snRNAseq

+ GSE182109 (2 grade II LGG, 10 grade IV ndGBM + 5 grade IV rGBM) scRNAseq

+ GSE135045 (7 grade IV IDHwt ndGBM) scRNAseq

(1) DDR2 expression profiling

After data were collected, DDR2 expression was analyzed, and the results are shown in FIG. 4 (a). As can be seen from fig. 4 (a), both the cell numbers expressing DDR2 and the DDR2 expression levels in grade IV primary GBM (ndGBM) and recurrent GBM (rGBM) samples were significantly higher than grade II low-grade GBM (LGG).

To more conveniently display the number and expression level of DDR2 expressing cells (DDR 2+), DDR2 expression screening was performed on the total data, provided that: 200 < nfeature_rna < 6000, percent.mt < 10, percent.ddr2 > 0.01 (this condition is the following gene expression positive selection), i.e., 1 out of every 10000 RNA sequencing in single cell sequencing results is DDR2 positive.

FIG. 4 (b) shows the statistical results of DDR2 positive cell expression levels. DDR2 expression levels of DDR2 positive cells in the ndGBM and rGBM samples were higher than those of DDR2 positive cells in the LGG samples, with rGBM being particularly pronounced. Wherein, the average DDR2 expression level of ndGBM is lower than rGBM, but the highest value of a few cells is higher, which shows that the expression difference of DDR2 positive cells is larger, and rGBM is relatively balanced.

Combining the cell numbers and DDR2 positive cell ratios in table 1, it can be seen that the DDR2 expression positive cell ratios in the ndGBM and rGBM samples were 6.32% and 5.36%, respectively, which were significantly higher than the DDR2 positive cell ratios in the LGG samples of 0.79%. Comparing the ratio of FAP positive cells, although FAP can also significantly differentiate grade IV GBM from grade II LGG, DDR2 positive cells are three times higher than FAP, and DDR2 appears to be more sensitive to ndGBM in self-comparison, whereas FAP is more sensitive to rGBM.

Table 1: cell number and related Gene expression Positive Rate in various GBM samples

	LGG	ndGBM	rGBM
				Total cell number	10176	182059	221215
DDR2+	80	11508	11850
				DDR2+%	0.79%	6.32%	5.36%
FAP+	22	2902	4319
				FAP+%	0.22%	1.59%	1.95%
EGFR+	1952	51883	55669
				EGFR+%	19.18%	28.50%	25.17%
EGFR+DDR2	25	6120	5946
				EGFR+DDR2% in EGFR+	1.28%	11.80%	10.68%
EGFR+DDR2% in DDR2+	31.25%	53.18%	50.18%

(2) UMAP cell cluster analysis

UMAP cell cluster analysis of single cell sequencing data clearly distinguishes correlations between individual sample cell constituents and individual cell types. As shown in FIG. 5 (a), gliomas can be classified into gliobast 1, neurobelast and fibribelast 1 of EGFR-positive epidermal origin, and gliobast 1 and fibribelast 2 of PDGFRA and IGFBP 2-positive mesenchymal origin. The specific gene expression profile can be seen in FIG. 5 (b). DDR2 high expression cells are mostly gliobast 1 and fibriobast 1 of the epidermal origin, while the fibriobast 2 of mesenchymal origin is also highly expressed, and the distribution situation is overlapped with FAP and is obviously higher than FAP. FIG. 6 shows the expression of the genes in different cell clusters. The results showed that the DDR2 positive cells in fiblatinst 1 were less than fiblatinst 2, but their DDR2 expression levels were higher for DDR2 positive cells.

Taken together, grade IV gliomas (including ndGBM and rGBM) showed significantly higher (about 10-fold) DDR2 positive cell ratios and expression levels than grade II glioma LGG. EGFR can specifically target gliomas, but clinical application value for identifying different grades of gliomas may be inferior to DDR2. While FAP can distinguish gliomas of different grades, the expression level and the difference of the FAP in gliomas are far less than those of DDR2. Therefore, DDR2 may have better clinical significance in differential diagnosis and targeted therapy of grade IV glioma.

It should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification, variation and variation of the inventions herein disclosed may be resorted to by those skilled in the art, and that such modifications, improvements and variations are considered to be within the scope of this invention. The materials, methods, and examples provided herein are representative and illustrative of the preferred embodiments and are not intended as limitations on the scope of the invention.

Claims

1. Use of an agent for detecting the expression level of discoid domain receptor 2 (DDR 2) in the preparation of a kit for diagnosing glioma in a subject, wherein a level of DDR2 in a sample from the subject that is higher than a control that is not afflicted with a disease indicates that the subject has glioma.

2. The use of claim 1, wherein the reagent for detecting the expression level of DDR2 comprises a reagent capable of binding to DDR2 to detect the level of DDR2 in the sample.

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

4. The use of claim 3, wherein the anti-DDR 2 monoclonal antibody is an anti-DDR 2 nanobody.

5. The use of claim 4, wherein the anti-DDR 2 nanobody comprises CDR1, CDR2, CDR3, wherein CDR1 comprises or is the sequence shown in SEQ ID No. 1 or an equivalent variant thereof, CDR2 comprises or is the sequence shown in SEQ ID No. 2 or an equivalent variant thereof, and CDR3 comprises or is the sequence shown in SEQ ID No. 3 or an equivalent variant thereof, wherein CDR is defined according to IMGT.

6. The use according to claim 4, wherein the nanobody comprises or is the sequence shown in SEQ ID No. 4 or an equivalent variant thereof.

7. The use of claim 2, wherein the agent capable of binding to DDR2 is labeled with a detectable label.

8. The use according to claim 7, wherein the detectable label is selected from the group consisting of fluorescent labels, chemiluminescent labels, paramagnetic labels, radioisotope labels and enzymatic labels.

9. The use of claim 1, wherein the glioma is selected from the group consisting of anaplastic astrocytoma, anaplastic oligodendroglioma, and glioblastoma.

10. A computer-readable storage medium having stored thereon computer instructions for computer reading and execution, the computer instructions being executed to perform a method of diagnosing whether a subject has glioma, the method comprising:

(a) Contacting a sample from the subject with an agent capable of binding to discotic domain receptor 2 (DDR 2);

(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 determining that the subject has glioma when the signal exceeds the predetermined threshold, and wherein the threshold is a median level from subjects not suffering from the disease.

11. Use of an exosome from a subject in the manufacture of a kit for diagnosing glioma in the subject, wherein a level of discoid domain receptor 2 (DDR 2) expressed by the exosome that is higher than a control without disease indicates that the subject has glioma.

12. The use of claim 11, wherein the exosomes are serum or plasma from the subject.

13. The use of claim 11, wherein the kit further comprises an agent capable of binding to DDR2 to detect the level of DDR2 expressed by the exosome.