Cancer examination method and cancer treatment agent

Abstract

Provided is a biomarker useful for the diagnosis of cancer and the development of new drugs. Further provided are a cancer examination method, a treatment agent, and a reagent for studies of cancer, in each of which the biomarker is used. A useful biomarker is provided by identifying a protein that is expressed on the surface of the membrane of an extracellular vesicle (EV) in a cancer-specific manner. The cancer examination method includes detecting a protein (biomarker) that is expressed, in a cancer-specific manner, on the surface of the membrane of an EV contained in a collected body fluid specimen to detect a cancer-specific EV. An antibody against a protein expressed on the surface of the membrane of an EV in a cancer-specific manner can be used in a cancer examination method, a cancer treatment agent, and a reagent for studies of cancer.

Description

Cancer testing method and cancer treatment agent

[0001] The present invention relates to a cancer testing method, a cancer therapeutic agent, and a reagent for cancer research.

[0002] This application claims priority from Japanese Patent Application No. 2023-107773, which is incorporated herein by reference.

[0003] Biomarkers are biological substances, such as proteins and genes, that serve as physiological indicators of the presence or progression of a disease. Biomarkers include a variety of data, including clinical test values ​​such as biochemical tests, blood tests, and tumor markers, as well as diagnostic imaging data such as computed tomography (CT) and magnetic resonance imaging (MRI). Examples include indicators that quantify biological information to quantitatively grasp biological changes within the body. In particular, biomarkers that indicate the presence or progression of disease as indicators of disease have become indispensable in current medical practice, including disease detection, diagnosis, and prognosis prediction. Biomarkers are also used in new drug development to select and evaluate development compounds.

[0004] Accurate evaluation of the malignancy and extent of cancer invasion is important for determining treatment strategies. In particular, for cancers that have few subjective symptoms in the early stages of the disease, there is a need for biomarkers that are useful for diagnosis and drug discovery.

[0005] Biopsies are crucial for determining diagnosis, prognosis, and treatment, especially for cancer patients. However, for some cancer types, such as non-small cell lung cancer (NSCLC), extracting sufficient tissue from a biopsy for biomarker testing can be challenging due to the patient's condition and tumor location. Liquid biopsies, which collect samples from bodily fluids, secretions, or excretions, such as blood, urine, saliva, cerebrospinal fluid, pericardial effusion, pleural effusion, ascites, and stool, have recently gained attention as an alternative sample acquisition method to complement or overcome the barriers to obtaining tumor tissue samples. Liquid biopsies are less invasive than tissue biopsies and can be performed repeatedly. Targets for liquid biopsy analysis include circulating tumor cells (CTCs), cell-free DNA (cfDNA), and circulating tumor DNA (ctDNA). However, because CTCs and ctDNA are expected to be detected when cancer cells metastasize, they are suitable for predicting cancer prognosis but cannot be used for early diagnosis.

[0006] In addition to cfDNA, ctDNA, and CTCs, extracellular vesicles (EVs) are also attracting attention as targets for liquid biopsy analysis. Depending on their size and origin, EVs are known by various names, including exosomes, ectosomes, and microvesicles (MVs). Collectively, they are called extracellular vesicles (EVs). Cells release EVs along with humoral factors, and cancer cells in particular release EVs. EVs have been reported to induce cancer cell metastasis and suppress immune function. Among EVs, exosomes in particular contain biomarkers such as microRNAs, lipids, and proteins that highly reflect the characteristics of the cells from which they originate. It is believed that cancer cells produce more exosomes than normal cells.

[0007] A lung cancer testing method has been disclosed that includes a step of analyzing a complex between CHL1 (cell adhesion molecule L1-like) present on the membrane of extracellular vesicles present in a blood sample from a subject and a CHL1 partner protein (Patent Document 1).Patent Document 1 also discloses various CHL1 partner proteins.

[0008] Patent Literature 2 discloses the isolation of tissue-exuded extracellular vesicles (te-EVs) from diseased tissues and the analysis of biomarkers. Patent Literature 2 reports that te-EVs derived from cancer tissues and normal tissues of cancer patients were used to identify tetraspanin molecules, such as CD63, CD81, and CD9, known as exosome markers, and their expression levels were analyzed by liquid chromatography-mass spectrometry (LC / MS). However, there is no disclosure of anti-tetraspanin antibodies.

[0009] It has been disclosed that analysis of urinary extracellular vesicles revealed that a specific group of membrane proteins is useful as a biomarker for urothelial cancer (Patent Document 3). It has also been disclosed that each marker may be detected using an immunological assay or an antibody.

[0010] To confirm the function of cancer-associated extracellular vesicles (EVs) in the human body, EVs were extracted directly from surgically resected viable renal cell carcinoma (ccRCC) tissue and adjacent normal kidney tissue (n520). 3,871 proteins in tissue-exuded EVs (Te-EVs) were identified by quantitative LC / MS analysis. Among these, azurocidin (AZU1) was found to be highly concentrated in tumor Te-EVs, and AZU1 content was significantly higher in serum EVs from CCRCC patients than in serum EVs from healthy donors. Te-EVs have been suggested as an ideal resource for investigating physiological EV functions (Non-Patent Document 1). Analysis of EV membrane proteins as effective biomarkers for disease diagnosis and drug development, as well as the development of selective antibodies against EV membrane proteins for diagnostic and therapeutic applications, are highly anticipated.

[0011] JP 2021-188970 A International Publication No. WO2018 / 079689 (Patent Publication No. 6712737) International Publication No. WO2020 / 071489

[0012] K. Jingushi et al., Int. J. Cancer: 142, 607-617 (2018)

[0013] The present invention relates to providing a biomarker useful for cancer diagnosis and new drug development, and further aims to provide a cancer testing method, a cancer therapeutic agent, and a reagent for cancer research that use the biomarker.

[0014] The present inventors focused on cancer-specific extracellular vesicles (hereinafter sometimes simply referred to as "EVs") and conducted extensive research to solve the above-mentioned problems. As a result, they succeeded in identifying proteins that are cancer-specifically expressed on the membrane surface of EVs, providing biomarkers useful for cancer diagnosis and new drug development. A cancer testing method involves detecting proteins (biomarkers) that are cancer-specifically expressed on the membrane surface of EVs contained in a collected body fluid sample. Antibodies against proteins that are cancer-specifically expressed on the membrane surface of EVs can be used in cancer testing methods, and can also be used as cancer therapeutics and cancer research reagents.

[0015] That is, the present invention comprises the following: 1. A method for detecting cancer, comprising detecting a protein expressed in a cancer-specific manner on the membrane surface of extracellular vesicles contained in a collected body fluid sample, thereby detecting cancer-specific extracellular vesicles. 2. The method for detecting cancer according to the preceding item 1, wherein the protein expressed in a cancer-specific manner on the membrane surface of extracellular vesicles is one or more proteins selected from ABCE1 (ATP binding cassette subfamily E member 1), STAT1 (Signal transducer and activator of transcription 1), and ZA2G (Alpha-2-glycoprotein 1, zinc-binding). 3. The method for detecting cancer according to the preceding item 1 or 2, wherein the cancer is one or more types of cancer selected from colorectal cancer, gastric cancer, pancreatic cancer, lung cancer, ovarian cancer, uterine cancer, renal cancer, and glioblastoma. 4. The method for detecting cancer according to the preceding item 1 or 2, wherein the collected body fluid sample is blood or saliva. 5. 5. The method for detecting cancer according to the preceding paragraph 1 or 2, wherein the cancer-specific extracellular vesicles are detected using an immunosandwich method or flow cytometry with an antibody against a protein specifically expressed on the membrane surface of the extracellular vesicles and an antibody against an extracellular vesicle-specific membrane surface marker protein or an antibody against a protein specifically expressed on the membrane surface of the extracellular vesicles. 6. The method for detecting cancer according to the preceding paragraph 1 or 2, wherein the cancer-specific extracellular vesicles are detected using an immunosandwich method or flow cytometry with an antibody against a protein specifically expressed on the membrane surface of the extracellular vesicles and an extracellular vesicle-specific membrane surface marker protein or a protein that binds to the extracellular vesicle membrane. 7. The method for detecting cancer according to the preceding paragraph 5, wherein the extracellular vesicle-specific membrane surface marker protein is one or more selected from CD9, CD63, CD81, integrin, selectin, and CD40, and the protein that binds to the extracellular vesicle membrane is Tim4. 8. 7. The method for testing according to 6 above, wherein the extracellular vesicle-specific membrane surface marker protein is one or more selected from CD9, CD63, CD81, integrin, selectin, and CD40, and the protein that binds to the extracellular vesicle membrane is Tim4.9. A cancer testing device comprising a substrate onto which an antibody against one or more proteins selected from ABCE1, STAT1, and ZA2G is immobilized. 10. A cancer testing kit for detecting cancer-specific extracellular vesicles from extracellular vesicles contained in a collected body fluid sample, comprising at least one configuration selected from the following 1) to 3): 1) a substrate onto which an antibody against a protein expressed specifically in cancer on the membrane surface of extracellular vesicles is immobilized, and a labeling reagent capable of specifically detecting extracellular vesicles; 2) a substrate onto which an antibody against an extracellular vesicle-specific membrane surface marker protein or a protein that binds to the extracellular vesicle membrane is immobilized, and a labeling reagent capable of detecting a protein expressed specifically in cancer on the membrane surface of extracellular vesicles; 3) a substrate onto which an antibody against a protein expressed specifically in cancer on the membrane surface of extracellular vesicles is immobilized, and a labeling reagent capable of detecting a protein expressed specifically in cancer on the membrane surface of extracellular vesicles. 11. 11. The cancer detection kit according to the preceding item 10, wherein the cancer-specific protein is one or more proteins selected from ABCE1, STAT1, and ZA2G. 12. The cancer detection kit according to the preceding item 10 or 11, wherein the extracellular vesicle-specific membrane surface marker protein is one or more proteins selected from CD9, CD63, CD81, integrin, selectin, and CD40, and the protein that binds to the extracellular vesicle membrane is Tim4. 13. A cancer therapeutic agent comprising, as an active ingredient, an antibody against a protein expressed on the membrane surface of cancer-specific extracellular vesicles. 14. The cancer therapeutic agent according to the preceding item 13, wherein the protein expressed on the membrane surface of cancer-specific extracellular vesicles is one or more proteins selected from ABCE1, STAT1, and ZA2G. 15. The cancer therapeutic agent according to the preceding item 13 or 14, wherein the antibody against a protein expressed on the membrane surface of cancer-specific extracellular vesicles is an IgG immunoglobulin and / or a scFv (single chain Fv).

[0016] Evaluations using pre- and postoperative plasma samples from cancer patients and plasma from healthy volunteers demonstrated the presence of cancer-specific EVs in bodily fluid samples, specifically blood samples and saliva, and confirmed the expression of cancer-specific proteins on the EV membrane surface. These levels were confirmed to decrease after surgery. Therefore, detecting cancer-specific proteins on the EV membrane surface as markers may be useful for early cancer diagnosis, stage determination, and treatment. Immune cells infiltrate cancer tissue to attack cancer cells. Among these immune cells, NK cells are responsible for early detection and attack. However, cancer cells release EVs as a means of evading these attacks, and various RNAs and proteins within these EVs are thought to ward off NK cell attack. Antibodies against proteins specifically expressed on the EV membrane surface can remove cancer-specific EVs. This can restore NK cell activity suppressed by cancer-specific EVs, potentially suppressing cancer cell survival. Therefore, antibodies against proteins specifically expressed on the EV membrane surface can be used as cancer therapeutics.

[0017] The following figures show the results of Western blotting using anti-ABCE1 antibodies to confirm ABCE1 expression on the membrane surface of small and large EVs obtained from tissue sections collected from cancerous (T) and normal (N) areas of stage I to stage IV colon cancer patients. (Example 1) The results of flow cytometry confirming the reactivity of EVs derived from colon cancer tissue culture supernatant with anti-ABCE1 antibodies. Binding of anti-ABCE1 antibodies was below the detection limit for EVs derived from non-cancerous tissues of colon cancer patients, but was significant for EVs derived from cancerous tissues. (Example 2) The results of ELISA confirming the reactivity of anti-ABCE1 antibodies in colon cancer patients using pre- and post-operative plasma from colon cancer patients and plasma from healthy individuals. (Example 3) The results of confirming the effect of EVs collected from non-cancerous and cancerous areas of colon cancer patients on the cytotoxic activity of NK cells against cancer cells. (Reference Example 1) The results of confirming the effect of anti-ABCE1 antibodies on the suppression of NK cell cytotoxicity by colon cancer-derived EVs. Figure 5a shows the protocol for treating HT29 cell-derived EVs with anti-ABCE1 antibody or control antibody. Figure 5b shows the results of Western blotting using anti-ABCE1 antibody on HT29 cell-derived SEV lysate. Figure 5c shows the effect of EVs with or without anti-ABCE1 antibody treatment on the cytotoxic activity of NK cells, as measured by HT29 cell viability. (Example 4) This shows the results of examining STAT1 expression in large and small EVs derived from cancer tissues (N, Stage I-IV) and non-cancerous tissues (N) of colorectal cancer patients using an anti-STAT1 antibody (Santacruz, sc-417). (Reference Example 2) This shows the results of examining ZA2G expression in large and small EVs derived from cancer tissues and non-cancerous tissues (Normal, 2 cases) of ovarian cancer patients using an anti-ZA2G antibody (Abcam, ab-180574). (Example 5) The reactivity of anti-ABCE1 antibody, anti-STAT1 antibody, and anti-ZA2G antibody in ovarian cancer patients was confirmed by ELISA using plasma from pre- and post-operative ovarian cancer patients and plasma from healthy individuals. (Example 6)

[0018] The present invention relates to a cancer testing method that detects cancer-specific EVs by detecting proteins that are expressed in a cancer-specific manner on the membrane surface of extracellular vesicles (EVs) contained in a collected body fluid sample.

[0019] As used herein, "extracellular vesicles (EVs)" refer to tiny vesicles with a membrane structure that are secreted from various types of cells, and examples include exosomes, ectosomes, microvesicles, microvesicles, and apoptotic bodies.

[0020] As used herein, "proteins expressed in a cancer-specific manner on the membrane surface of extracellular vesicles (EVs)" include, for example, ABCE1 (ATP-binding cassette subfamily E member 1), STAT1 (Signal transducer and activator of transcription 1), and ZA2G (Alpha-2-glycoprotein 1, zinc-binding). ABCE1, STAT1, and ZA2G are all known to function as cytoplasmic proteins, and their detection as various biomarkers required detection of gene expression. However, the present invention has for the first time discovered that these proteins are expressed in a cancer-specific manner on the membrane surface of EVs. That is, the present invention has for the first time discovered proteins expressed in a cancer-specific manner on the membrane surface of EVs, namely, one or more proteins selected from ABCE1, STAT1, and ZA2G, as biomarkers.

[0021] As used herein, "cancer-specific expression" means cancer tissue-specific expression, but also includes cases where the expression is selectively in cancer tissue compared to normal tissue, or cases where the expression is significantly higher in cancer tissue compared to normal tissue, making it clearly distinguishable from normal tissue.

[0022] ABCE1 is a member of the ATP-binding cassette (ABC) transporters. ABC proteins regulate the transport of various physiological substrates. ABC proteins convert the energy of ATP hydrolysis into structural changes in protein molecules, and by coupling this with the transport of drugs, they are believed to determine the pharmacokinetic profile of drugs (absorption, distribution, metabolism, excretion, and effective drug concentration at target sites), which in turn determines the overall pharmacological effect of the drug.

[0023] STAT1 is a member of the STAT protein family, which, in response to cytokines and growth factors, are phosphorylated by receptor-associated kinases, then form homodimers or heterodimers and translocate to the cell nucleus, where they act as transcriptional activators.

[0024] ZA2G, also known as AZGP1 (Alpha-2-Glycoprotein 1, Zinc-Binding), is a 41 kDa soluble glycoprotein with a structure similar to MHC class I and that binds to β2-microglobulin. It is known to exist in body fluids and is thought to function in lipid metabolism. It is speculated to be associated with the loss of adipose tissue during cachexia in cancer.

[0025] As used herein, the term "body fluid sample" refers to a sample collected by liquid biopsy, and is preferably, for example, blood or saliva. The biological species that can be subjected to the testing method of the present invention are not particularly limited and include various mammals such as humans, monkeys, mice, rats, dogs, cats, and rabbits, with humans being the most preferred.

[0026] In the cancer testing method of the present invention, the test sample to be used can be appropriately prepared from a body fluid sample. When the body fluid sample is blood, serum, plasma, etc. prepared by a method known per se or a method to be developed in the future can be used as the test sample, and test samples prepared for routine clinical testing can also be used. When the body fluid sample is saliva, a sample prepared by pretreatment using a method known per se or a method to be developed in the future can also be used. EVs separated and purified from a collected body fluid sample can also be used as the test sample. EVs can be purified, separated, concentrated, etc. from a body fluid sample using a method known per se or any method to be developed in the future. Methods for purifying, separating, concentrating, etc. of EVs include, for example, ultracentrifugation (e.g., pellet-down method, sucrose cushion method, density gradient centrifugation, etc.), methods using immunoaffinity carriers, gel filtration, field-flow fractionation, FACS, etc. EVs can also be purified, separated, concentrated, etc. using commercially available kits.

[0027] The "cancer testing method" of the present invention is a method for detecting cancer-specific EVs by detecting proteins expressed specifically in cancer on the membrane surface of EVs as biomarkers. Specifically, the method is not particularly limited as long as it can detect proteins expressed specifically in cancer on the membrane surface of EVs contained in a collected body fluid sample, i.e., one or more proteins selected from ABCE1, STAT1, and ZA2G as biomarkers. Cancer-specific EVs can be detected by detecting one or more proteins selected from ABCE1, STAT1, and ZA2G as biomarkers.

[0028] As used herein, examples of "proteins that are cancer-specifically expressed on the EV membrane surface" include one or more proteins selected from ABCE1, STAT1, and ZA2G. Examples of "EV-specific membrane surface marker proteins" include one or more proteins selected from CD9, CD63, CD81, integrin, selectin, and CD40, and examples of "EV membrane-binding proteins" include Tim4.

[0029] Herein, methods for detecting cancer-specific proteins expressed on the membrane surface of EVs include, for example, immunological detection methods, including known methods and methods to be developed in the future, such as immunosandwich methods, including ELISA, sandwich ELISA, EIA, RIA, and Western blotting.

[0030] In the immunosandwich assay, a test sample is reacted with an antibody against a protein specifically expressed in cancer on the EV membrane surface, and detection can be performed using a detection reagent that reacts with the test sample that has reacted with the antibody against the protein. Specifically, a substrate is used to immobilize an antibody against a protein specifically expressed in cancer on the EV membrane surface, and the test sample that has reacted with the antibody captured on the substrate can be detected using a labeled reagent that can be specifically detected in EVs, specifically a labeled antibody against an EV-specific membrane surface marker protein or a labeled protein that binds to the EV membrane. Alternatively, as another embodiment of the detection reagent, detection can be performed using a labeled antibody against a protein specifically expressed in cancer on the EV membrane surface. The antibodies of the present invention against proteins specifically expressed in cancer on the EV membrane surface can also be used as reagents for cancer research.

[0031] In another embodiment of the immunosandwich assay, a test sample is reacted with an antibody against an EV-specific membrane surface marker protein or a protein that binds to the EV membrane, and the test sample that reacted with the antibody against the EV-specific membrane surface marker protein or the EV membrane-binding protein can be detected using a labeled antibody against a protein that is cancer-specifically expressed on the EV membrane surface as a detection reagent. The labeled antibody can be a labeled anti-IgG antibody or a labeled anti-Fc antibody, as long as it can detect the desired target.

[0032] Another method for detecting proteins expressed on the membrane surface of EVs contained in collected body fluid samples in the present invention is mass spectrometry using a mass spectrometer. Ionization methods using an ion source can be selected appropriately, such as electron ionization (EI), chemical ionization (CI), field desorption (FD), fast atom bombardment (FAB), matrix-assisted laser desorption ionization (MALDI), and electrospray ionization (ESI). Separation methods for ionized peptide samples in the analytical section can be selected appropriately, such as magnetic deflection, quadrupole, ion trap, time-of-flight (TOF), and Fourier transform ion cyclotron resonance. Tandem mass spectrometry (MS / MS) and triple quadrupole mass spectrometry, which combine two or more mass spectrometry methods, can also be used. Furthermore, samples containing phosphorylated peptides can be concentrated using iron ion-immobilized affinity chromatography (Fe-IMAC) before being introduced into the mass spectrometer. Alternatively, the peptides constituting the target biomarker can be separated and purified by liquid chromatography (LC) or HPLC to prepare a test sample. However, the detection method of the present invention is characterized by its ease of use in that the above-mentioned immunological detection method can be applied.

[0033] The present invention also encompasses a testing device used in the cancer testing method of the present invention, i.e., a testing device capable of detecting cancer-specific proteins expressed on the membrane surface of EVs. Examples of testing devices include substrates on which antibodies against cancer-specific proteins expressed on the membrane surface of EVs are immobilized. Furthermore, substrates on which antibodies against EV marker proteins or proteins that bind to EV membranes are immobilized can also be used in conjunction with detection reagents. In the case of a testing device made of a substrate on which antibodies against EV marker proteins or proteins that bind to EV membranes are immobilized, a detection reagent capable of detecting cancer-specific proteins expressed on the membrane surface of EVs is required.

[0034] The present invention also encompasses a test kit capable of detecting a protein expressed specifically on the membrane surface of an EV. The test kit includes at least the test device described above and may further include a detection reagent. Specifically, examples of the test kit include a cancer test kit comprising at least one of the following components: 1) a substrate on which an antibody against a protein expressed specifically on the membrane surface of an EV is immobilized, and a labeled reagent capable of specifically detecting EVs; 2) a substrate on which an antibody against an EV surface marker protein or a protein that binds to the EV membrane is immobilized, and a reagent capable of detecting a protein expressed specifically on the membrane surface of an EV; or 3) a substrate on which an antibody against the cancer-specific protein expressed on the membrane surface of an EV is immobilized, and a labeled antibody capable of detecting a protein expressed specifically on the membrane surface of an EV.

[0035] The material and shape of the substrate used in the testing device of the present invention are not particularly limited, and any substrate capable of immobilizing antibodies and detecting proteins expressed on the surface of EV membranes can be used. Known substrates, commercially available substrates, and substrates to be developed in the future can be used. The antibodies immobilized on the testing device and contained in the detection reagent are not particularly limited as long as they are antigen-binding. For example, antibody fragments are also encompassed by the antibodies of the present invention. Examples include polyclonal antibodies, monoclonal antibodies, and chimeric antibodies. They may also be portions of the above antibodies that have antigen-binding properties, such as single-chain antibodies, Fab fragments, and fragments generated by Fab expression libraries. Labels that can be used in the detection reagent include fluorescent dyes, enzymes, proteins, radioisotopes, chemiluminescent substances, biotin, and the like. Target antigens, such as antibodies against biomarkers and immunogens used to generate antibodies contained in the detection reagent, can be obtained based on known gene sequence information by DNA cloning, construction of various plasmids, transfection into hosts, culturing transformants, and recovering proteins from the culture. These procedures can be performed using known methods or any methods to be developed in the future.

[0036] In addition to the substrate and detection reagent, the testing kit of the present invention may also include various reagents (e.g., secondary antibodies, buffer solutions, etc.), tools (e.g., tools for purifying, separating, and concentrating EVs, such as columns), etc.

[0037] The testing method of the present invention can provide the amount and / or concentration of a target biomarker, which is an indicator for detecting a target cancer. Target cancers for the testing method of the present invention include colorectal cancer, gastric cancer, pancreatic cancer, lung cancer, ovarian cancer, uterine cancer, renal cancer, and glioblastoma, with colorectal cancer, gastric cancer, ovarian cancer, and glioblastoma being particularly preferred.

[0038] The present invention also extends to cancer screening methods using the testing method of the present invention. Immune cells infiltrate cancer tissues to attack cancer cells. Among these immune cells, NK cells are known to detect and attack cancer cells early. However, cancer cells release EVs as a means of evading this attack. The cancer testing method of the present invention, using bodily fluid samples (e.g., blood, saliva), can easily detect the presence of EVs positive for cancer-specific proteins expressed on the membrane surface of EVs from test samples. For example, the presence of EVs positive for cancer-specific proteins expressed on the membrane surface of EVs can be easily detected in healthy individuals and cancer patients. Furthermore, the presence of EVs positive for cancer-specific proteins expressed in cancer patients can be easily detected preoperatively and postoperatively, and depending on the stage of cancer. Specifically, for example, the presence of ABCE1-positive EVs can be detected in the blood (plasma) of colorectal cancer patients, and confirmation tests can be performed to determine whether their levels decrease after surgery. It is believed that ABCE1-positive EVs are also released from other cancer types, such as ovarian cancer and renal cancer. The cancer testing method or cancer screening method of the present invention can be used to assist in the differentiation of cancer, such as cancer diagnosis, treatment effect assessment, pathological condition elucidation, prognosis prediction, patient stratification, and treatment method selection (personalized medicine, treatment responsiveness), and is therefore useful.

[0039] The present invention also extends to cancer therapeutic agents containing as an active ingredient an antibody against a protein expressed on the membrane surface of cancer-specific EVs. Alternatively, the present invention also encompasses cancer therapeutic agents containing as an active ingredient an antibody against a protein expressed on the membrane surface of cancer-specific EVs. Specifically, the present invention also encompasses cancer therapeutic agents containing as an active ingredient an antibody against one or more of the following: ABCE1, STAT1, and ZA2G. To reiterate, cancer cells are thought to release cancer-specific EVs and avoid attack by NK cells by utilizing the various RNAs and proteins contained within these EVs. Therefore, by using antibodies against proteins expressed on the membrane surface of cancer-specific EVs, specifically antibodies against one or more of the following selected from ABCE1, STAT1, and ZA2G, it is possible to remove ABCE1-positive, STAT1-positive, and / or ZA2G-positive cancer EVs from cancer EVs. As a result, antitumor effects, for example, by NK cells, are expected. Furthermore, cancer EVs are thought to possess the characteristics of cancer cells. Therefore, efficient delivery of these cancer characteristics (neoantigens) to antigen-presenting cells is expected to effectively activate adaptive immunity. By administering a cancer therapeutic agent containing as an active ingredient an antibody against one or more antibodies selected from ABCE1, STAT1, and ZA2G of the present invention, or by administering in vitro a reaction product of EVs derived from cancer patients with an antibody against one or more antibodies selected from ABCE1, STAT1, and ZA2G of the present invention, it is expected that the uptake of each antibody-bound cancer EV via the Fc receptor of antigen-presenting cells in the body will be promoted, and this may also be considered a potential new cancer immunity inducer.

[0040] The antibodies contained in the cancer therapeutic agents of the present invention, i.e., antibodies against proteins expressed specifically on the membrane surface of EVs (e.g., antibodies against one or more proteins selected from ABCE1, STAT1, and ZA2G), are not particularly limited as long as they specifically bind to EVs expressing cancer-specific proteins on their membrane surface. For example, antibody fragments are also encompassed within the scope of the antibodies of the present invention. Examples include polyclonal antibodies, monoclonal antibodies, and chimeric antibodies. They may also include antigen-binding fragments of the above antibodies, such as single-chain antibodies, Fab fragments, and fragments generated by Fab expression libraries. The antibody fragment may comprise a heavy chain variable region (VH-CDR) and / or a light chain variable region (VL-CDR) or fragments thereof. The antibody class refers to the type of constant domain or constant region in the heavy chain (H chain) of the antibody, and includes, for example, IgA, IgD, IgE, IgG, and IgM. The antibody class herein is not particularly limited, but IgG is most preferred. Examples of IgG subclasses include IgG1, IgG2, IgG3, and IgG4, with IgG1 or IgG2 being preferred. Antibody fragments can include, for example, Fv, Fab, Fab', Fab'-SH, F(ab')2, or a combination thereof. Antibodies can be obtained from antibody libraries. For example, a technique for obtaining human antibodies using a human antibody library by biopanning is known. For example, the variable regions of human antibodies can be expressed on the surface of phages as single-chain antibodies (scFv) using phage display, and phages that bind to antigens can be selected. Genetic analysis of the selected phages allows the DNA sequence encoding the variable regions of human antibodies that bind to antigens to be determined. Once the DNA sequence of the scFv that binds to an antigen is identified, an appropriate expression vector can be constructed based on that sequence to obtain human antibodies. These methods are already well known, and reference can be made to WO92 / 01047, WO92 / 20791, WO93 / 06213, WO93 / 11236, WO93 / 19172, WO95 / 01438, and WO95 / 15388.

[0041] Cancer treatment involves administering antibodies against proteins expressed on the membrane surface of cancer-specific EVs, specifically antibodies against one or more selected from ABCE1, STAT1, and ZA2G, in combination with known therapeutic methods, as appropriate. Examples of such therapeutic methods include chemotherapy, surgery, radiation therapy, and immunotherapy. These can be performed according to known methods.

[0042] The form of the cancer therapeutic agent of the present invention is not particularly limited, but a dosage form that can be administered as an antibody formulation is desirable. The amount of the composition of the cancer therapeutic agent of the present invention to be applied (e.g., administered, ingested, inoculated, etc.) is not particularly limited, as long as it is an effective amount that exhibits medicinal efficacy. Typically, the weight of the active ingredient is generally 0.1 to 1000 mg / kg body weight per day. The above dosage is preferably administered once or in 2 to 3 divided doses per day, and can be increased or decreased as appropriate depending on age, disease state, and symptoms.

[0043] The immunogens used to generate antibodies against proteins expressed on the membrane surface of cancer-specific EVs can be obtained based on all or part of known or newly discovered gene sequence information based on proteins expressed on the membrane surface of cancer-specific EVs by DNA cloning, constructing each plasmid, transfecting the host, culturing the transformant, and recovering the protein from the culture. These procedures can be performed using known methods or any methods developed in the future.

[0044] EXAMPLES In the following, the present invention will be described in more detail with reference to examples in order to deepen understanding of the present invention, but it goes without saying that the present invention is not limited to the descriptions of the examples.

[0045] (Example 1) Confirmation of ABCE1 expression in extracellular vesicles (EVs) derived from cancer tissues of colon cancer patients after surgery In this example, the expression of ABCE1 in extracellular vesicles (EVs) derived from cancer tissues of colon cancer patients was confirmed.

[0046] SEV (Small EV: 1.0 × 10) obtained from tissue specimens collected from cancerous (T) and normal (N) areas of stage I to stage IV colorectal cancer patients (12 patients each). 8ABCE1 expression in each EV was confirmed by Western blotting using anti-ABCE1 antibody (Abcam, Cat. No. ab185548, used at 1 / 1000 dilution) for LEV (large EV: 1.0 × 107 particles). Each EV was added to 1× Laemmli SDS sample buffer (TEFCO), and run on SDS-PAGE (12% gel). The gel was then transferred to a PVDF membrane. After blocking with 3% BSA / TBS-T for 1 hour at room temperature, the membrane was incubated with anti-ABCE1 antibody (used at 1 / 1000 dilution) overnight at 4°C. After washing the membrane with TBS-T, the membrane was incubated with HRP-conjugated anti-mouse IgG antibody (Cell Signaling Technology, Cat. No. 7076, used at 1 / 5000 dilution) for 1 hour at room temperature. After washing the membrane with TBS-T, the membrane was incubated with ECL. TM The antibody was reacted with Prime Western Blotting Detection Reagent (Cytiva, Cat. No. RPN2232) and purified by Amersham TM Bands were detected using an Imager 680 (Cytiva).

[0047] As a result, ABCE1 was detected in the cancerous tissues of both SEVs and LEVs derived from stage I to IV colon cancer tissues. The expression level was also found to be significantly higher than that of EVs derived from normal colon tissues. Furthermore, the amount of ABCE1 protein in LEVs tended to increase depending on the stage (Figure 1).

[0048] Example 2 Reactivity of anti-ABCE1 antibody with cancer EVs derived from colon cancer tissue culture supernatant In this example, the reactivity of EVs derived from colon cancer tissue culture supernatant with anti-ABCE1 antibody was confirmed.

[0049] PS capture as a sample for flow cytometry analysis TMEVs derived from colon cancer tissue culture supernatant were prepared using an exosome flow cytometry kit (Fujifilm Wako Pure Chemical Industries, Cat. No. 297-79701). 100 μl of culture supernatant from non-cancerous or cancerous colon cancer tissue was mixed with 10 μl of Tim4-conjugated EV capture beads (beads) and incubated at room temperature for 1 hour with agitation every 20 minutes. After washing the beads with 1× wash buffer (provided with the kit), anti-ABCE1 antibody (Abcam, Cat. No. ab185548, used at a 1 / 200 dilution) was added and incubated at room temperature for 1 hour with agitation every 20 minutes. After washing the beads with 1x wash buffer (supplied with the kit), FITC-labeled human IgG antibody (Sigma, Cat. No. F5512-1ML, used at a 1 / 400 dilution) and Alexa Fluor 568-labeled rabbit IgG antibody (Invitrogen, Cat. No. A10042, used at a 1 / 200 dilution) were added and incubated at room temperature for 1 hour with stirring every 20 minutes. After washing the beads with 1x wash buffer (supplied with the kit), the activity was measured using a MACS Qunat X (Miltenyi).

[0050] The results showed that the binding of EVs to the anti-ABCE1 antibody was below the detection limit for EVs derived from non-cancerous colon cancer tissues, but significant binding was observed for EVs derived from cancerous tissues, indicating that the ABCE1 protein recognized by the anti-ABCE1 antibody is expressed on the membrane of colon cancer EVs (Figure 2).

[0051] Example 3 Reactivity of anti-ABCE1 antibodies in plasma from colon cancer patients In this example, the reactivity of anti-ABCE1 antibodies was confirmed by ELISA using plasma from colon cancer patients before and after surgery.

[0052] Anti-ABCE1 antibody (Abcam, Cat No. ab185548, 0.1 μg) was added to a 96-well plate and coated overnight at 4°C. After washing three times with PBS-T (PBS containing 0.05% Tween-20), the plate was blocked at room temperature for 1 hour with PBS containing 1% BSA at 37°C. Then, 100 μl each of pre- and post-operative plasma from colorectal cancer patients (eight cases) and plasma from healthy individuals (two cases) was added and incubated for 2 hours with shaking at 37°C. After removing the plasma and washing three times with PBS-T, anti-CD9 antibody (Cosmo Bio, Cat No. SHI-EXO-M01, 0.1 μg) was added and incubated for 1 hour at 37°C. After removing the anti-CD9 antibody, the plate was washed three times with PBS-T. HRP-labeled anti-mouse IgG antibody (Cell signaling technology, Cat. No. 7076, used at a 1 / 5000 dilution) was added and incubated at 37°C for 1 hour. After washing with PBS-T, the plate was subjected to chemical color development using TMB Substrate Set (BioLegend, Cat. No. 421101). The color development and absorbance at 450 nm were measured using an iMark microplate reader (Biorad).

[0053] The constructed ELISA method for detecting ABCE1-expressing EVs in the plasma of colorectal cancer patients, shown in Figure 3 (top), showed that the measured values ​​in postoperative plasma were reduced to almost the same level as those in healthy subjects compared to preoperative plasma (Figure 3 (bottom)). This demonstrated that anti-ABCE1 antibodies can cancer-selectively detect colorectal cancer EVs, demonstrating that the constructed ELISA system can be applied to the diagnosis of colorectal cancer.

[0054] (Reference Example 1) Effect of colon cancer EVs on the cytotoxic activity of NK cells NK cells are cells that detect and attack cancer cells at an early stage, but cancer cells are said to release EVs as a means of evading this attack. Therefore, in this reference example, we investigated the effect of EVs derived from colon cancer cells on the cytotoxic activity of NK cells.

[0055] EVs derived from non-cancerous tissue of colon cancer postoperative specimens (SEV: 3.95 × 10 9 Particles, LEV: 1.70×10 8 particles) and tumor-derived EVs (SEV: 3.95 × 109 Particles, LEV: 1.70×10 8 particles) to NK cells (KHYG-1 cells, 2 x 10 5 The NK cells were added to colon cancer cell lines (human colon adenocarcinoma cells) and cultured for 48 hours. Then, the cells were co-cultured with colon cancer cell lines HT29 cells or SW620 cells (human colon adenocarcinoma) at a ratio of 3:1 for 48 hours. NK cells were stained with a CD45 antibody (Miltenyi Biotec, Cat. No. 130-108-020, used at a 1 / 200 dilution), and dead cells were stained with propidium iodide (PI). The percentage of viable colon cancer cell lines (CD45-negative, PI-negative) was measured using a flow cytometer.

[0056] As a result, the viability of HT29 and SW620 cells was higher when co-cultured with EVs derived from tumor-derived colon cancer cells than with EVs derived from non-tumor-derived colon cancer cells for both SEVs and LEVs (Figure 4). It was revealed that EVs derived from tumor-derived colon cancer cells have the effect of suppressing the cytotoxic activity of NK cells against tumor-derived colon cancer cells. These results suggest that tumor-derived colon cancer cells release EVs to maintain their own viability and suppress the cytotoxic activity of NK cells.

[0057] Example 4: Effect of human anti-ABCE1 antibody on NK activity of colorectal cancer EVs. In the above Reference Example, it was confirmed that cancer cell-derived EVs suppress the cytotoxic activity of NK cells. Therefore, in this Example, cancer cell-derived EVs were treated with anti-ABCE1 antibody, and the effect of this antibody on suppressing the cytotoxic activity of NK cells against cancer cells was confirmed.

[0058] 1) Removal of anti-ABCE1 antibody-reactive EVs from HT29 cell SEVs. 1.5 mg of Dynabeads Protein G (Life Technologies, Cat. No. 10003D) was mixed with 2 μg of anti-ABCE1 antibody or Normal Rabbit IgG (Fujifilm Wako Pure Chemical Industries, Cat. No. 148-09551) as a control antibody, and the mixture was incubated for 10 minutes with rotation at room temperature to bind the antibody to Dynabeads Protein G. The control antibody bound to Dynabeads Protein G was used as the control antibody-bound beads. After washing with PBS-T, HT29 cell SEVs (0.5 × 10 10 The ABCE1 particles were mixed with control antibody-conjugated beads and incubated for 10 minutes at room temperature with rotation. After incubation, the beads were washed three times with PBS-T, and 20 μl of 1× Laemmli SDS sample buffer was added to the beads and incubated at 70°C for 10 minutes. The supernatant (EV lysate) was used for Western blotting (see 2) below. Furthermore, for analysis (see 3) below, SEV was reacted with anti-ABCE1 antibody-conjugated beads or control antibody-conjugated beads, and the supernatant was removed using a magnet. The SEVs treated with anti-ABCE1 antibody-conjugated beads and control antibody-conjugated beads are referred to as colon cancer SEV low and colon cancer SEV high, respectively (Figure 5a).

[0059] 2) Western blotting with anti-ABCE1 antibody The EV lysate obtained in 1) above was developed by SDS-PAGE (10% gel) and transferred to a PVDF membrane. After blocking with 3% BSA / TBS-T for 1 hour at room temperature, the membrane was reacted with anti-ABCE1 antibody (used at 1 g / ml) overnight at 4°C. After washing the membrane with TBS-T, the membrane was reacted with HRP-labeled rabbit IgG antibody (Cell signaling technology, Cat. No. 7074, used at 1 / 10000 dilution) for 1 hour at room temperature. After washing the membrane with TBS-T, the membrane was subjected to ECL. TM The antibody was reacted with Prime Western Blotting Detection Reagent (Cytiva, Cat. No. RPN2232) and purified by AmershamTM Bands were detected using an Imager 680 (Cytiva) (Fig. 5b).

[0060] 3) Evaluation of cytotoxic activity of NK cells The colon cancer SEV high fraction and colon cancer SEV low fraction obtained in 1) above were cultured with NK cells (KHYG-1 cells, 20 × 10 4 The cells were added to HT29 cells and cultured for 48 hours. NK cells (KHYG-1 cells) treated with colon cancer SEV were co-cultured with HT29 cells at a ratio of 3:1 for 48 hours. NK cells were stained with CD45 antibody (Miltenyi Biotec, Cat. No. 130-108-020, used at a 1 / 200 dilution), and viable HT29 cells (CD45 negative) were counted using a flow cytometer (Fig. 5c).

[0061] As a result, in the SEVs collected after reacting anti-ABCE1 antibody with SEVs from the colon cancer cell line HT-29, an ABCE1 band was detected by Western blot analysis, which was not detected in the reaction with control IgG (Fig. 5b). Furthermore, the cytotoxic activity of HT29 cells induced by KHYG-1 cells was significantly restored in HT29 cell SEVs captured with anti-ABCE1 antibody (colon cancer SEV low) compared with HT29 cell SEVs captured with control antibody (colon cancer SEV high) (Fig. 5c). These results suggest that removal of anti-ABCE1 antibody-reactive colon cancer SEVs restores NK cell cytotoxic activity.

[0062] (Reference Example 2) Confirmation of STAT1 expression in EVs derived from cancerous tissues of colon cancer postoperative specimens In this example, the expression of STAT1 in extracellular vesicles (EVs) derived from cancer tissues of colon cancer patients was confirmed.

[0063] Colorectal cancer clinical specimen EVs (non-cancerous EVs: N, cancerous EVs: T; small EVs: 3.0 × 10 8 Particles, Large EV: 3.0×10 7The sample was prepared by adding 1x Laemmli SDS sample buffer to the cellulose membrane, which was then developed by SDS-PAGE (12% gel) and transferred to a PVDF membrane. After blocking with 3% BSA / TBS-T for 1 hour at room temperature, the sample was reacted with anti-STAT1 antibody (Santa Cruz Biotechnology, Cat No. sc-417, used at 1 / 1000 dilution) at 4°C overnight. After washing the membrane with TBS-T, the sample was reacted with HRP-labeled anti-mouse IgG antibody (Cell signaling technology, Cat No. 7076, used at 1 / 5000 dilution) at room temperature for 1 hour. After washing the membrane with TBS-T, the sample was subjected to ECL. TM The antibody was reacted with Prime Western Blotting Detection Reagent (Cytiva, Cat. No. RPN2232) and purified by Amersham TM Bands were detected using an Imager 680 (Cytiva).

[0064] As a result, it was revealed that there were cases in which STAT1 was significantly more abundant in EVs derived from the cancerous parts of colorectal cancer clinical specimens than in EVs derived from non-cancerous parts (Figure 6).

[0065] (Example 5) Confirmation of ZA2G Expression in EVs Derived from Cancerous Regions of Ovarian Cancer Post-Operative Specimens EVs from ovarian cancer clinical specimens (non-cancerous region EVs: Normal (1), (2); cancerous region EVs: Stage I (3), (4), Stage II (5), (6), Stage III (7), (8), Stage IV (9), (10); small EVs: 3.0 × 10 8 Particles, Large EV: 3.0×10 7The sample was prepared by adding 1x Laemmli SDS sample buffer to the cellulose membrane, which was then developed by SDS-PAGE (12% gel) and transferred to a PVDF membrane. After blocking with 3% BSA / TBS-T for 1 hour at room temperature, the sample was reacted with anti-ZA2G antibody (Abcam, Cat No. ab-180574) at 4°C overnight. After washing the membrane with TBS-T, the sample was reacted with HRP-labeled anti-mouse IgG antibody (Cell signaling technology, Cat No. 7076, diluted 1 / 5000) at room temperature for 1 hour. After washing the membrane with TBS-T, the sample was subjected to ECL. TM The antibody was reacted with Prime Western Blotting Detection Reagent (Cytiva, Cat. No. RPN2232) and purified by Amersham TM Bands were detected using an Imager 680 (Cytiva).

[0066] As a result, it was revealed that ZA2G, which was not detected in EVs derived from non-cancerous parts of ovarian cancer clinical specimens, was significantly expressed in EVs derived from the cancerous parts of ovarian cancer clinical specimens (Figure 7).

[0067] Example 6 Reactivity of Each Antibody in Plasma from Ovarian Cancer Patients In this example, the reactivity of anti-ABCE1 antibody, anti-STAT1 antibody, and anti-ZA2G antibody was confirmed by ELISA using plasma from ovarian cancer patients before and after surgery.

[0068] Anti-ABCE1 antibody (Abcam, Cat. No. ab185548, 0.1 μg), anti-STAT1 antibody (Santa Cruz Biotechnology, Cat. No. sc-417, used at a 1 / 1000 dilution, 0.1 μg), or anti-ZA2G antibody (Abcam, ab-180574, 0.05 μg) was added to a 96-well plate and coated overnight at 4°C. After washing three times with PBS-T (PBS containing 0.05% Tween-20), the plate was blocked with PBS containing 1% BSA at 37°C for 1 hour at room temperature. Then, 100 μl each of pre- and postoperative plasma from ovarian cancer patients (8 cases) and healthy volunteer plasma (2 cases) was added and incubated with shaking at 37°C for 2 hours. After removing the plasma and washing three times with PBS-T, anti-CD9 antibody (Cosmobio, Cat. No. SHI-EXO-M01, 0.1 μg) was added and incubated at 37°C for 1 hour. After removing the anti-CD9 antibody and washing three times with PBS-T, HRP-labeled anti-mouse IgG antibody (Cell signaling technology, Cat. No. 7076, used at a 1 / 5000 dilution) was added and incubated at 37°C for 1 hour. After washing with PBS-T, the plate was chemically developed using TMB Substrate Set (BioLegend, Cat. No. 421101), and the color development and absorbance were measured at 450 nm using an iMark microplate reader (Biorad).

[0069] Using the same method as in Example 3 (Figure 3, top), we performed an ELISA to detect ABCE1-expressing EVs, STAT1-expressing EVs, and ZA2G-expressing EVs in the plasma of ovarian cancer patients, and found that the measured values ​​in postoperative plasma were significantly lower than those in preoperative plasma (Figure 8). This demonstrates that anti-ABCE1, anti-STAT1, and anti-ZA2G antibodies can cancer-selectively detect ovarian cancer EVs, demonstrating that the constructed ELISA system can be applied to the diagnosis of ovarian cancer.

[0070] As described above, detecting proteins that are cancer-specifically expressed on the membrane surface of EVs as markers is expected to provide indicators for early cancer diagnosis, cancer stage determination, treatment, etc. The testing method of the present invention can be used to test body fluid samples such as blood and saliva, and is useful in that it can be performed without invasively obtaining biological samples from the subject, with little burden on the subject.

[0071] Furthermore, immune cells infiltrate cancer tissues to attack cancer cells. Among these immune cells, NK cells are responsible for early detection and attack of cancer cells. However, cancer cells release EVs as a means of evading this attack, and it is thought that the diverse RNAs and proteins contained in these EVs allow them to avoid attack by NK cells. Antibodies against proteins specifically expressed on the membrane surface of EVs discovered in this study can remove cancer-specific EVs. This can restore the activity of NK cells suppressed by cancer-specific EVs, potentially suppressing cancer cell survival. Furthermore, because cancer EVs are thought to contain neoantigens, which are characteristic of cancer cells, efficient delivery of these cancer characteristics to antigen-presenting cells can effectively activate adaptive immunity. Therefore, antibodies against proteins specifically expressed on the membrane surface of EVs can be used as cancer therapeutics and are extremely useful industrially.

Claims

1. A cancer testing method comprising detecting proteins that are expressed in a cancer-specific manner on the membrane surface of extracellular vesicles contained in a collected body fluid sample, thereby detecting cancer-specific extracellular vesicles.

2. The cancer testing method of claim 1, wherein the protein expressed in a cancer-specific manner on the membrane surface of the extracellular vesicles is one or more proteins selected from ABCE1 (ATP binding cassette subfamily E member 1), STAT1 (Signal transducer and activator of transcription 1), and ZA2G (Alpha-2-glycoprotein 1, zinc-binding).

3. The cancer testing method according to claim 1 or 2, wherein the cancer is one or more types of cancer selected from colorectal cancer, gastric cancer, pancreatic cancer, lung cancer, ovarian cancer, uterine cancer, renal cancer and glioblastoma.

4. The cancer testing method according to claim 1 or 2, wherein the collected body fluid sample is blood or saliva.

5. A method for testing cancer according to claim 1 or 2, characterized in that the cancer-specific extracellular vesicles are detected using an immunosandwich method or a flow cytometry method using an antibody against a protein that is expressed in a cancer-specific manner on the membrane surface of the extracellular vesicles and an antibody against an extracellular vesicle-specific membrane surface marker protein or an antibody against a protein that is expressed in a cancer-specific manner on the membrane surface of the extracellular vesicles.

6. A method for detecting cancer according to claim 1 or 2, characterized in that the cancer-specific extracellular vesicles are detected using an immunosandwich method or a flow cytometry method using an antibody against a protein that is expressed in a cancer-specific manner on the membrane surface of the extracellular vesicles and an extracellular vesicle-specific membrane surface marker protein or a protein that binds to the extracellular vesicle membrane.

7. The method of claim 5, wherein the extracellular vesicle-specific membrane surface marker protein is one or more selected from CD9, CD63, CD81, integrin, selectin, and CD40, and the protein that binds to the extracellular vesicle membrane is Tim4.

8. The method of claim 6, wherein the extracellular vesicle-specific membrane surface marker protein is one or more selected from CD9, CD63, CD81, integrin, selectin, and CD40, and the protein that binds to the extracellular vesicle membrane is Tim4.

9. A cancer testing device comprising a substrate having immobilized thereon antibodies against one or more proteins selected from ABCE1, STAT1 and ZA2G.

10. A cancer testing kit for detecting cancer-specific extracellular vesicles from extracellular vesicles contained in a collected body fluid sample, comprising at least one of the following configurations selected from 1) to 3): 1) a substrate on which an antibody against a protein expressed in a cancer-specific manner on the membrane surface of an extracellular vesicle is immobilized, and a labeling reagent capable of specifically detecting extracellular vesicles; 2) a substrate on which an antibody against an extracellular vesicle-specific membrane surface marker protein or a protein that binds to the extracellular vesicle membrane is immobilized, and a labeling reagent capable of detecting a protein expressed in a cancer-specific manner on the membrane surface of an extracellular vesicle; 3) a substrate on which an antibody against a protein expressed in a cancer-specific manner on the membrane surface of an extracellular vesicle is immobilized, and a labeling reagent capable of detecting a protein expressed in a cancer-specific manner on the membrane surface of an extracellular vesicle.

11. The cancer testing kit according to claim 10, wherein the protein specifically expressed in a cancer-specific manner is one or more proteins selected from the group consisting of ABCE1, STAT1 and ZA2G.

12. The cancer testing kit according to claim 10 or 11, wherein the extracellular vesicle-specific membrane surface marker protein is one or more selected from CD9, CD63, CD81, integrin, selectin, and CD40, and the protein that binds to the extracellular vesicle membrane is Tim4.

13. A cancer therapeutic agent comprising as an active ingredient an antibody against a protein expressed on the membrane surface of cancer-specific extracellular vesicles.

14. The cancer therapeutic agent according to claim 13, wherein the protein expressed on the membrane surface of the cancer-specific extracellular vesicles is one or more proteins selected from ABCE1, STAT1 and ZA2G.

15. A cancer therapeutic agent described in claim 13 or 14, wherein the antibody against a protein expressed on the membrane surface of cancer-specific extracellular vesicles is an IgG immunoglobulin and / or scFv (single chain Fv).

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