Capturing method and detection method for extracellular vesicles derived from cancer cells, and kit

Abstract

[Abstract] Disclosed is a technology for capturing or detecting extracellular vesicles derived from all types of cancer cells regardless of the types of the cancer. A method for capturing extracellular vesicles derived from cancer cells and contained in a biological sample includes a step for bringing the biological sample into contact with (A) a polypeptide that includes an ID1-ID2a domain of a VAR2CSA protein derived from a protozoan parasite that causes tropical malaria, (B) a polypeptide that has 90% or higher sequence identity with an amino acid sequence for the polypeptide mentioned in (A) and is capable of binding to an extracellular vesicle derived from a cancer cell, or (C) a polypeptide that is a portion of the polypeptide mentioned in (A) or the polypeptide mentioned in (B) and is capable of binding to an extracellular vesicle derived from a cancer cell, to cause the binding of the polypeptide to the extracellular vesicles derived from the cancer cells.

Description

Method for capturing and detecting cancer cell-derived extracellular vesicles, and kit

[0001] The present invention relates to a method for capturing extracellular vesicles derived from cancer cells, and a method and kit for detecting cancer-derived extracellular vesicles.

[0002] Malignant neoplasms (cancer) are the leading cause of death in Japan, and if cancer can be detected at an early stage, it is almost always curable. Therefore, as one of the important measures for cancer prevention, there is a need for the development of minimally invasive technology that can detect cancer at the earliest possible stage.

[0003] Liquid biopsy (body fluid diagnosis) is known as a minimally invasive technique for detecting cancer. Instead of performing a conventional biopsy in which tumor tissue is collected, liquid biopsy (body fluid diagnosis) is used as a specimen to detect cancer by measuring extracellular vesicles (hereinafter sometimes referred to as "EVs").

[0004] Cancer-derived EVs are expected to be difficult to detect because they are overwhelmingly rarer than EVs derived from organs and tissues in the body. However, it is thought that EVs derived from cancer cells can be detected by combining antibodies against proteins such as CD9, CD63, and CD81 that are specifically expressed in EVs with antibodies against proteins expressed in cancer cells, known as cancer markers.

[0005] However, it has been reported that the protein expression patterns of proteins contained in EVs are significantly different from those of the cells from which the EVs are derived (Non-Patent Document 1). As a specific example, the membrane surfaces of cells and the EVs produced by those cells were biotinylated, and the proteins were collected using avidin beads. The results of analyzing the proteins by LC-MS / MS showed that the membrane protein compositions of cells and their EVs were significantly different (Non-Patent Document 2).

[0006] Therefore, in order to detect EVs derived from cancer cells, it is not possible to simply use cancer cell markers; it is necessary to analyze the proteins in EVs derived from cancer cells and find proteins that can serve as markers.

[0007] In addition, several methods are known for detecting extracellular vesicles derived from cancer cells using markers expressed in specific cancer cells.

[0008] For example, in the case of colon cancer, it has been shown that EVs derived from colon cancer can be detected using antibodies targeting CD9, an EV marker, and CD147, which is expressed in colon cancer-derived EVs (Patent Document 1). In fact, by analyzing proteins contained in EVs of cancer cells, CD147 was found to be specifically expressed in EVs derived from colon cancer.

[0009] Furthermore, with regard to bladder cancer, proteome analysis of EVs expressed in EVs derived from the urine of bladder cancer patients was performed, and CD55, CD82, etc. were discovered. It was shown that bladder cancer-derived EVs can be detected using antibodies targeting CD9, a marker for EVs, and CD55 and CD82 expressed in bladder cancer-derived EVs (Patent Document 2).

[0010] Furthermore, a paper on pancreatic cancer EVs performed proteome analysis of EVs derived from the serum of pancreatic cancer patients and found GPRC5C and EPS8 as markers, demonstrating that the expression levels of these markers in cells and extracellular vesicles in various pancreatic cancer cell lines and non-cancerous pancreatic cells were not correlated (Non-Patent Document 3). This suggests that cancer markers discovered through expression analysis in cells cannot simply be applied as cancer markers for EVs.

[0011] On the other hand, it has been reported that the VAR2CSA protein derived from Plasmodium falciparum binds to chondroitin sulfate A in cancer cells (Patent Document 3, Non-Patent Document 4). These cancer cell-derived chondroitin sulfates are also known as oncofetal chondroitin sulfates or placental-like chondroitin sulfates. However, there is no evidence that these chondroitin sulfates are expressed on the surface of EVs. Furthermore, a method for measuring these placental-like chondroitin sulfates by ELISA has been reported (Non-Patent Document 5). However, these techniques detect soluble fetal chondroitin sulfates in cell lysates or body fluids, not EVs.

[0012] International Publication No. 2014 / 167969 International Publication No. 2016 / 143805 Special Publication No. 2015-513524

[0013] Jeppesen, DK. et al., 2019, Cell 177, 428-445)Kirkemo, LL. et al. eLife 2022;0:e73982.Yoshioka, Y.et al., Cancer Science. 2022;113:3498-3509Salanti, A. et al., Cancer Cell. 2015; 28(4): 500-514. Zhang, J. et al., Int J. Med. Sci. 2020; 17: 161-169.

[0014] Previously, to detect EVs derived from cancer cells, it was necessary to obtain markers specific to the EVs derived from each cancer type, and no technology had been developed to isolate or detect cancer-derived EVs that could be used for all cancer types. Therefore, for early cancer diagnosis, it was necessary to perform tests using multiple markers for each cancer type.

[0015] An object of the present invention is to provide a technology for capturing or detecting extracellular vesicles derived from all cancers, regardless of cancer type.

[0016] To solve the above problems, the present inventors conducted extensive research and developed a technology for capturing and detecting cancer-derived extracellular vesicles using the VAR2CSA polypeptide of Plasmodium falciparum, which has the property of binding to chondroitin sulfate on the surface of cancer cells. The present invention was completed based on these findings.

[0017] That is, the present invention provides the following: (1) A method for capturing cancer cell-derived extracellular vesicles contained in a biological sample, the method comprising the step of contacting the biological sample with any one of the following polypeptides (A) to (C) to bind the polypeptide to cancer cell-derived extracellular vesicles: (A) a polypeptide comprising the ID1-ID2a domain of the VAR2CSA protein derived from Plasmodium falciparum; (B) a polypeptide that has 90% or more sequence identity with the amino acid sequence of the polypeptide (A) above and binds to cancer cell-derived extracellular vesicles; (C) a polypeptide that is a portion of the polypeptide (A) or (B) above and binds to cancer cell-derived extracellular vesicles; (2) The method according to (1), in which the amino acid sequence of the ID1-ID2a domain of the VAR2CSA protein is set forth in SEQ ID NO: 1, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14. (3) The method according to (1) or (2), in which the polypeptides (A) to (C) are immobilized on a carrier. (4) The method according to (1) or (2), wherein the biological sample is a human cell culture supernatant, blood, serum, or plasma. (5) A method for detecting cancer cell-derived extracellular vesicles contained in a biological sample, the method comprising the steps of: contacting the biological sample with any one of the following polypeptides (A) to (C) to bind the polypeptide to cancer cell-derived extracellular vesicles; and detecting cancer cell-derived extracellular vesicles bound to any one of the polypeptides (A) to (C). (A) A polypeptide comprising the ID1-ID2a domain of the VAR2CSA protein derived from Plasmodium falciparum. (B) A polypeptide having 90% or more sequence identity with the amino acid sequence of the polypeptide of (A) above and binding to cancer cell-derived extracellular vesicles. (C) A polypeptide which is a portion of the polypeptide of (A) or (B) above and binds to cancer cell-derived extracellular vesicles. (6) The method described in (5), further comprising contacting the cancer cell-derived extracellular vesicles with a second binding substance which binds to the surface of the extracellular vesicles, and detecting the cancer cell-derived extracellular vesicles bound to the second binding substance and any of the polypeptides of (A) to (C).(7) The method according to (6), wherein either (i) the polypeptide of any one of (A) to (C) or (ii) the second binding substance is immobilized on a carrier, the other is labeled, and the detection is carried out by detecting the label. (8) The method according to (7), wherein the second binding substance is an antibody or an antigen-binding fragment thereof, or a phosphatidylserine-binding domain of Tim1, Tim3, Tim4, or beta-2-glycoprotein 1 (β2GP1), which is a protein that binds to phosphatidylserine located on the outside membrane of extracellular vesicles. (9) The method according to (8), wherein the antibody is an antibody against CD9, CD63, or CD81. (10) The method according to (6), wherein the biological sample is human cell culture supernatant, blood, serum, or plasma. (11) The method according to any one of (5) to (10), wherein the amino acid sequence of the ID1-ID2a domain of the VAR2CSA protein is set forth in SEQ ID NO: 1, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14. (12) A method for obtaining information on whether a subject is affected with cancer, the method being carried out by detecting cancer cell-derived extracellular vesicles contained in a biological sample obtained from the subject by the method of any one of (1) to (11). (13) A kit comprising any one of the following polypeptides (A) to (C) and a second binding substance that binds to the surface of extracellular vesicles, wherein either (i) the polypeptide of any one of (A) to (C) and (ii) the second binding substance is immobilized on a carrier, and the other is labeled. (A) A polypeptide comprising the ID1-ID2a domain of the VAR2CSA protein derived from Plasmodium falciparum. (B) A polypeptide having 90% or more sequence identity with the amino acid sequence of the polypeptide of (A) above and binding to cancer cell-derived extracellular vesicles. (C) A polypeptide that is a portion of the polypeptide of (A) or (B) above and binds to cancer cell-derived extracellular vesicles. (14) The kit described in (13), wherein the kit is for diagnosing whether a subject is suffering from cancer.

[0018] The present invention enables the capture and detection of cancer-derived extracellular vesicles. Also provided are a method and kit for diagnosing cancer based on the detection results of the cancer-derived extracellular vesicles.

[0019] FIG. 1 shows the structure of a polypeptide comprising the ID1-ID2a domain of the VAR2CSA protein used in the Examples of the present invention. FIG. 2 is a schematic diagram illustrating an example of the analytical method of the present invention. FIG. 3 shows the results of detecting purified EVs derived from U87-MG cells captured with VAR2CSA using an anti-CD63 antibody. FIG. 4 shows the results of detecting purified EVs derived from U87-MG cells captured with an anti-CD9 antibody using an anti-CD63 antibody. FIG. 5 shows the results of detecting purified EVs derived from WI-38-hTERT cells captured with VAR2CSA using an anti-CD63 antibody. FIG. 6 shows the results of detecting purified EVs derived from WI-38-hTERT cells captured with an anti-CD9 antibody using an anti-CD63 antibody. FIG. 7 shows the results of detecting purified EVs derived from pancreatic cancer cell lines KMP2 and PANC-1 cells captured with VAR2CSA using a mixed antibody of anti-CD9 and anti-CD81 antibodies. FIG. 8 shows the results of detecting purified EVs derived from pancreatic cancer cell lines KMP2 and PANC-1 cells captured with Tim4 using a mixed antibody of anti-CD9 and anti-CD81 antibodies. Figure 1 shows the results of detecting purified EVs derived from prostate cancer cell line PC-3 cells and prostate cancer cell line LNCap cells, normal prostate epithelial cell line HPrEC-TKD cells, and normal prostate fibroblast cell line HPrF-TKD cells captured with VAR2CSA using a combination of three antibodies: anti-CD9, anti-CD63, and anti-CD81. Figure 2 shows the results of detecting purified EVs derived from prostate cancer cell line PC-3 cells and prostate cancer cell line LNCap cells, normal prostate epithelial cell line HPrEC-TKD cells, and normal prostate fibroblast cell line HPrF-TKD cells captured with Tim4 using a combination of three antibodies: anti-CD9, anti-CD63, and anti-CD81. Figure 1 shows the results of detecting purified EVs derived from lung cancer cell lines H69AR3, NCI-H1703, and A549 cells, and normal fetal lung fibroblast cell line WI-38-hTERT cells captured with VAR2CSA, using a combination of three antibodies: anti-CD9, anti-CD63, and anti-CD81. Figure 2 shows the results of detecting purified EVs derived from lung cancer cell lines H69AR3, NCI-H1703, and A549 cells, and normal fetal lung fibroblast cell line WI-38-hTERT cells captured with Tim4, using a combination of three antibodies: anti-CD9, anti-CD63, and anti-CD81.Figure 1 shows the results of detecting purified EVs derived from osteosarcoma cell line MG-63 cells and normal osteoblast cell line NHOst-TKD cells captured with VAR2CSA using a combination of three antibodies: anti-CD9, anti-CD63, and anti-CD81. Figure 2 shows the results of detecting purified EVs derived from osteosarcoma cell line MG-63 cells and normal osteoblast cell line NHOst-TKD cells captured with Tim4 using a combination of three antibodies: anti-CD9, anti-CD63, and anti-CD81. Figure 3 shows the results of detecting purified EVs derived from acute myeloid leukemia cell line KG-1 cells captured with VAR2CSA using an anti-CD63 antibody. This figure also shows that the capture of extracellular vesicles by VAR2CSA is inhibited by chondroitin sulfate A (CSA). Figure 4 shows the results of detecting purified EVs derived from acute myeloid leukemia cell line KG-1 cells captured with VAR2CSA or Tim4 and then detected with an anti-CD63 antibody. This figure shows the results of detecting purified EVs from esophageal cancer cell lines KYSE150, KYSE510, and KYSE1240 cells captured with VAR2CSA using a mixed antibody mixture of anti-CD9, anti-CD63, and anti-CD81 antibodies. This figure shows the results of detecting purified EVs from esophageal cancer cell lines KYSE150, KYSE510, and KYSE1240 cells captured with Tim4 using a mixed antibody mixture of anti-CD9, anti-CD63, and anti-CD81 antibodies. This figure shows the results of detecting purified EVs from gastric cancer cell lines HGC-27, AGS, and MKN45 cells captured with VAR2CSA using a mixed antibody mixture of anti-CD9, anti-CD63, and anti-CD81 antibodies. This figure shows the results of detecting purified EVs from gastric cancer cell lines HGC-27, AGS, and MKN45 cells captured with Tim4 using a mixed antibody mixture of anti-CD9, anti-CD63, and anti-CD81 antibodies. This figure shows the results of detecting purified EVs from the colon cancer cell lines SW837, SW480, HTC-116, and Caco-2 cells captured with VAR2CSA using a combination of three antibodies: anti-CD9, anti-CD63, and anti-CD81. This figure shows the results of detecting purified EVs from the colon cancer cell lines SW837, SW480, HTC-116, and Caco-2 cells captured with Tim4 using a combination of three antibodies: anti-CD9, anti-CD63, and anti-CD81.Figure 1 shows the results of capturing purified EVs from the EpCAM-positive pancreatic cancer cell lines KMP2 and PANC-1 and the EpCAM-negative pancreatic cancer cell line MIA Paca-2 with VAR2CSA polypeptide and detecting them with a combination of three antibodies: anti-CD9, anti-CD63, and anti-CD81. Figure 2 shows the results of capturing purified EVs from the EpCAM-positive pancreatic cancer cell lines KMP2 and PANC-1 and the EpCAM-negative pancreatic cancer cell line MIA Paca-2 with Tim4 and detecting them with a combination of three antibodies: anti-CD9, anti-CD63, and anti-CD81. Figure 3 shows the results of serially diluting culture supernatants from glioblastoma cell line U87-MG and normal fetal lung fibroblast cell line WI-38-hTERT cells, adding them to an anti-CD63 antibody-coated plate to capture EVs in the sample, and detecting the captured EVs with VAR2CSA polypeptide. This figure shows the results of capturing purified EVs derived from lung cancer cell line NCI-H1703 cells and normal fetal lung fibroblast cell line WI-38-hTERT cells added to bovine serum using VAR2SCA and detecting them with a mixture of three antibodies: anti-CD9, anti-CD63, and anti-CD81. This figure shows a comparison of EV capture rates using different methods of immobilizing VAR2CSA polypeptide to a carrier. The values ​​in the figure are the ratios of values ​​obtained using a nickel chelate-coated plate to values ​​obtained using a streptavidin-coated plate. This figure shows the results of SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of purified VAR2CSA polypeptides from five strains. This figure shows the results of binding analysis (ELISA) of the VAR2CSA polypeptides from five strains to bovine decorin. This figure shows the results of binding analysis (ELISA) of the VAR2CSA polypeptides from five strains to EVs derived from leukemia-derived cell line KG-1 cells and normal fetal lung fibroblast cell line WI-38-hTERT cells. This figure shows the results of detecting purified EVs from renal cancer cell lines Caki-1 and Caki-2 cells and normal renal epithelial cell line HREC-TKD cells captured by VAR2CSA using a combination of three antibodies: anti-CD9, anti-CD63, and anti-CD81.Figure 1 shows the results of detecting purified EVs from renal cancer cell lines Caki-1 and Caki-2 cells and normal renal epithelial cell line HREC-TKD cells captured with Tim4 using a combination of three antibodies: anti-CD9, anti-CD63, and anti-CD81. Figure 2 shows the results of detecting purified EVs from breast cancer cell lines MDA-MB-231, MDA-MB-45, and T47D cells and normal mammary epithelial cell line HMEC-TKD cells captured with VAR2CSA using a combination of three antibodies: anti-CD9, anti-CD63, and anti-CD81. Figure 3 shows the results of detecting purified EVs from breast cancer cell lines MDA-MB-231, MDA-MB-45, and T47D cells and normal mammary epithelial cell line HMEC-TKD cells captured with Tim4 using a combination of three antibodies: anti-CD9, anti-CD63, and anti-CD81. This figure shows the results of detecting purified EVs from ovarian cancer cell line OVCAR3 cells and bladder cancer cell line T24 cells captured with VAR2CSA using a combination of three antibodies: anti-CD9, anti-CD63, and anti-CD81. This figure shows the results of detecting purified EVs from ovarian cancer cell line OVCAR3 cells and bladder cancer cell line T24 cells captured with Tim4 using a combination of three antibodies: anti-CD9, anti-CD63, and anti-CD81. This figure shows the results of detecting purified EVs from liver cancer cell line HepG2 cells, gallbladder cancer cell line TYGBK-8 cells, cholangiocarcinoma cell lines HuCCT1 cells, and TFK-1 cells captured with VAR2CSA using a combination of three antibodies: anti-CD9, anti-CD63, and anti-CD81. Figure 1 shows the results of detecting purified EVs from liver cancer cell line HepG2 cells, gallbladder cancer cell line TYGBK-8 cells, cholangiocarcinoma cell line HuCCT1 cells, and TFK-1 cells captured with Tim4 using a combination of three antibodies: anti-CD9, anti-CD63, and anti-CD81. Figure 2 shows the results of detecting purified EVs from acute lymphoblastic leukemia cell line NALM-6 cells and acute myeloid leukemia cell line KG-1 cells captured with VAR2CSA using a combination of three antibodies: anti-CD9, anti-CD63, and anti-CD81. Figure 3 shows the results of detecting purified EVs from acute lymphoblastic leukemia cell line NALM-6 cells and acute myeloid leukemia cell line KG-1 cells captured with Tim4 using a combination of three antibodies: anti-CD9, anti-CD63, and anti-CD81.Figure 1 shows the results of detecting purified EVs from tracheal smooth muscle cell line HBSMC-TKD cells, aortic endothelial cell line HAEC-TKD cells, and acute myeloid leukemia cell line KG-1 cells captured with VAR2CSA using a mixture of three antibodies: anti-CD9, anti-CD63, and anti-CD81. Figure 2 shows the results of detecting purified EVs from tracheal smooth muscle cell line HBSMC-TKD cells and aortic endothelial cell line HAEC-TKD cells captured with Tim4 using a mixture of three antibodies: anti-CD9, anti-CD63, and anti-CD81. Figure 3 shows the procedure for purifying EVs from healthy human plasma supplemented with extracellular vesicles derived from acute myeloid leukemia cell line KG-1 cells. Figure 4 shows the results of detecting purified EVs from healthy human plasma supplemented with extracellular vesicles derived from acute myeloid leukemia cell line KG-1 cells captured with VAR2CSA using a mixture of three antibodies: anti-CD9, anti-CD63, and anti-CD81. This figure shows the results of detecting purified EVs from human plasma of healthy donors supplemented with extracellular vesicles derived from the acute myeloid leukemia cell line KG-1 cells, captured with Tim4, using a mixture of three antibodies: anti-CD9, anti-CD63, and anti-CD81. This figure shows the results of detecting purified EVs from colorectal cancer patient plasma and healthy donor plasma captured with VAR2CSA, as well as buffer solution and purified EVs from the leukemia cell line KG-1 cells and the colorectal cancer cell line HTC-116 cells, using a mixture of three antibodies: anti-CD9, anti-CD63, and anti-CD81. This figure shows the results of detecting purified EVs from colorectal cancer patient plasma and healthy donor plasma captured with Tim4, as well as buffer solution and purified EVs from the leukemia cell line KG-1 cells and the colorectal cancer cell line HTC-116 cells, using a mixture of three antibodies: anti-CD9, anti-CD63, and anti-CD81.

[0020] The following detailed description of the embodiments of the present invention will be given, and it should be understood that the present invention can be practiced using conventional techniques of molecular biology and immunology within the skill of the art, such techniques being fully explained in the literature.

[0021] The present invention provides a method for capturing cancer cell-derived extracellular vesicles contained in a biological sample, comprising the step of contacting the biological sample with (A): a polypeptide comprising the ID1-ID2a domain of the VAR2CSA protein derived from Plasmodium falciparum; (B): a polypeptide that has 90% or more sequence identity with the amino acid sequence of the polypeptide (A) and binds to cancer cell-derived extracellular vesicles; or (C): a polypeptide that is a portion of the polypeptide (A) or (B) and binds to cancer cell-derived extracellular vesicles, thereby binding the polypeptide to the cancer cell-derived extracellular vesicles.

[0022] 1. Extracellular vesicles Extracellular vesicles (hereinafter sometimes referred to as EVs) are defined as particles released from cells that do not have a nucleus and are surrounded by a lipid bilayer membrane. Based on differences in their production mechanisms, they are classified into exosomes, microvesicles, and apoptotic bodies.

[0023] Exosomes are vesicles derived from the endosomal membrane and are formed during endocytosis, measuring approximately 30-200 nm in size. Microvesicles, which vary in size from 100-1000 nm, differ from exosomes in that they bud off directly from the cell membrane and are secreted extracellularly. Apoptotic bodies are particles measuring 50-5000 nm in size that bud off from the membrane of apoptotic cells, similar to microvesicles.

[0024] In addition, extracellular vesicles may contain proteins, RNA (mRNA, miRNA, non-coding RNA), DNA fragments, etc.

[0025] 2. Plasmodium falciparum VAR2CSA Polypeptide The methods of the present invention use Plasmodium falciparum VAR2CSA polypeptide or a polypeptide derived therefrom. The finding that VAR2CSA polypeptide binds to cancer cell-derived extracellular vesicles but not to normal cell-derived extracellular vesicles, as specifically described in the Examples below, is a new finding discovered by the present invention. First, the Plasmodium falciparum VAR2CSA polypeptide will be described below.

[0026] In pregnant women infected with Plasmodium falciparum, the accumulation of infected red blood cells in the placental villi disrupts placental function, resulting in miscarriage or stillbirth. Plasmodium-infected red blood cells bind to the placenta through the binding of a member of the Plasmodium falciparum multiple erythrocyte membrane protein 1 (PFEMP1) family of proteins expressed in Plasmodium-infected red blood cells to placental chondroitin sulfate proteoglycans. Plasmodium PFEMP1 is encoded by approximately 60 var genes, and the var2csa gene has been shown to be responsible for binding to placental chondroitin sulfate proteoglycans (Baruch, DI et al., Cell, 1995; 82, 77-87; Kraemer, SM & Smith, JD, Curr. Opin. Microbiol. 2006; 9, 374-380).

[0027] Furthermore, it has been shown that the VAR2CSA polypeptide encoded by the var2csa gene specifically binds to glycosaminoglycans composed of chondroitin sulfate A (Salanti, A. et al., Mol. Microbiol. 2003; 49, 179-191), and that the VAR2CSA polypeptide binds to chondroitin sulfate A in various cancer cell lines (Salanti, A. et al., Cancer Cell 2015; 28, 500-514).

[0028] Several strains of Plasmodium falciparum exist, including FCR3, K1, 3D7, Dd2, T9 / 94, Tak9 / 96, MAD20, K39, Honduras-1, HB3, 7G8, FCB, V1 / S, M0920, and M.Camp. These strains are available from the Biological Resources Laboratory of the Institute of Tropical Medicine, Nagasaki University.

[0029] The nucleotide sequence information and corresponding amino acid sequence information of the var2csa gene of isolates isolated from patients infected with Plasmodium falciparum are GenBank (trade name) accession nos. GU249598 and AAQ73926 for FCR3, JQ247428 for 3D7, XM001350379 for NF54, KOB63403 for HB3, EUR69480 for 7G8, OK346631 for M.Camp, OK346630 for M0920, QGY73032 for M200101, ABS79817 for WR80, and KOB85905 for Dd2, and can be obtained from databases of nucleic acid nucleotide sequences and protein amino acid sequences such as GenBank.

[0030] The VAR2CSA polypeptides of isolates isolated from patients infected with Plasmodium falciparum exhibit an average homology of 70-80% between Plasmodium falciparum strains. However, all strains can infect humans, resulting in symptoms such as placental dysfunction, miscarriage, and stillbirth. Given the aforementioned infection mechanism, it is believed that the VAR2CSA polypeptides of each strain possess binding affinity to chondroitin sulfate A. The VAR2CSA polypeptides of the present invention are preferably VAR2CSA polypeptides of isolates isolated from patients infected with Plasmodium falciparum. Therefore, as long as the VAR2CSA polypeptides have the original amino acid sequence among Plasmodium falciparum strains and no mutations are introduced into any amino acids, they are believed to retain the extracellular vesicle-trapping ability shown in the Examples of the present invention. On the other hand, it is also possible to introduce insertions, deletions, or mutations into any amino acids in the VAR2CSA polypeptides of isolates isolated from patients infected with Plasmodium falciparum, as long as they do not lose their binding affinity to chondroitin sulfate A.

[0031] VAR2CSA is a 350 kD protein consisting of six Duffy-Binding Like (DBL) domains and several inter-domain regions (IDs), ID2a and ID2b of which are also called cysteine-rich inter-domain regions (CIDRs).

[0032] Binding studies of each domain with chondroitin sulfate A have shown that polypeptides consisting of DBL1X-ID1-DBL2X-ID2a-ID2b (DBL1X-ID2b), DBL1X-ID1-DBL2X-ID2a (DBL1X-ID2a), and ID1-DBL2X-ID2a (ID1-ID2a) bind to the chondroitin sulfate proteoglycan of bovine articular cartilage-derived decorin (Clausen, TM et al., J. Biol. Chem. 2012; 287, 23332-23345). Furthermore, a comparative study of the VAR2CSA polypeptide between DBL1X-ID2a and ID1-ID2a demonstrated comparable efficacy in capturing cancer cells (Sand, NT, Int. J. Mol. Sci. 2020; 21, 2401-2416).

[0033] In the present invention, the VAR2CSA polypeptide can preferably be an ID1-ID2a polypeptide fragment derived from the Plasmodium falciparum FCR3, 7G8, M0920, 3D7, or M.Camp strain. The amino acid sequences of these polypeptides are set forth in SEQ ID NOs: 1, 9, 12, 13, and 14, respectively, in the Sequence Listing. Furthermore, the polynucleotide sequence encoding the polypeptide of SEQ ID NO: 1 is set forth in SEQ ID NO: 2, and the polynucleotide sequence encoding the polypeptide of SEQ ID NO: 9 is set forth in SEQ ID NO: 10. These polypeptide sequences are listed in GenBank under accession numbers GU249598, EUR69480, OK346630, JQ247428, and OK346631, respectively.

[0034] The method of the present invention uses: (A) a polypeptide comprising the ID1-ID2a domain of the VAR2CSA protein derived from Plasmodium falciparum; (B) a polypeptide that has 90% or more sequence identity with the amino acid sequence of the polypeptide (A) above and binds to cancer cell-derived extracellular vesicles; or (C) a polypeptide that is a portion of the polypeptide (A) or (B) above and binds to cancer cell-derived extracellular vesicles.

[0035] Although the full-length VAR2CSA protein can be used, because the portion that binds to oncofetal chondroitin sulfate A present on the surface of cancer cell-derived extracellular vesicles is the ID1-ID2a domain of the VAR2CSA protein, a polypeptide comprising the ID1-ID2a domain can be used. Furthermore, a polypeptide comprising this ID1-ID2a domain (e.g., the full-length VAR2CSA protein) also binds to oncofetal chondroitin sulfate A and can therefore be used in the method of the present invention.

[0036] Polypeptides that bind to cancer cell-derived extracellular vesicles and share 90% or more, preferably 95% or more, more preferably 97% or more, and even more preferably 99% or more, sequence identity with the amino acid sequence of the polypeptide (A) above can also be used in the methods of the present invention. Here, the sequence identity of an amino acid sequence is expressed as a percentage by aligning two amino acid sequences so that the number of identical amino acid residues is maximized (with gaps inserted if necessary), and dividing the number of identical amino acid residues by the total number of amino acid residues (or the number of amino acid residues in the shorter amino acid sequence if the two amino acid sequences differ in length). Sequence identity can be easily calculated by those skilled in the art using FASTA, which is available free of charge on the Internet. In the present invention, sequence identity is not defined for sequences with a total length of less than 20 amino acid residues.

[0037] The polypeptide of the present invention comprising the ID1-ID2aA domain of the VAR2CSA protein derived from Plasmodium falciparum is preferably a polypeptide comprising the ID1-ID2a domain of the VAR2CSA protein derived from a Plasmodium falciparum isolate isolated from a patient infected with Plasmodium falciparum, because, as described above, this amino acid sequence is thought to have binding affinity to chondroitin sulfate A.

[0038] Several amino acid sequences of the ID1-ID2a domain of the above-mentioned VAR2CSA protein are known, including those shown in Table 1 below.

[0039]

[0040]

[0041] As described above, among these, those represented by FCR3 (SEQ ID NO: 1), 7G8 (SEQ ID NO: 9), M0920 (SEQ ID NO: 12), 3D7 (SEQ ID NO: 13), or M. Camp (SEQ ID NO: 14) are preferred, and FCR3 (SEQ ID NO: 1) is most preferred.

[0042] Furthermore, a polypeptide that is a portion of the above polypeptide (A) or (B) and that binds to cancer cell-derived extracellular vesicles can also be used in the method of the present invention. The number of amino acid residues in this partial polypeptide is not particularly limited as long as it binds to cancer cell-derived extracellular vesicles, but is usually 20 or more, preferably 30 or more, more preferably 40 or more, even more preferably 50 or more, and most preferably 100 or more.

[0043] The "binding" in the "polypeptide that binds to cancer cell-derived extracellular vesicles" of the present invention may be any binding that allows cancer cell-derived extracellular vesicles to be detected by the detection method of the present invention.

[0044] A tag peptide sequence can be fused to the N-terminus and / or C-terminus of any of the above polypeptides (A) to (C) used in the method of the present invention (hereinafter referred to as "VAR2CSA polypeptide" unless otherwise specified and unless otherwise clear from the context) for the purpose of purifying or detecting the polypeptide.

[0045] Suitable examples of the tag peptide include FLAG-Tag, 3xFLAG-Tag, HA-Tag, Myc-Tag, HIS-Tag, V5-Tag, VSV-G-Tag, CBD-Tag, CBP-Tag, GST-Tag, MBP-Tag, Thioredoxin-Tag, SNAP-Tag, CLIP-Tag, Strep-Tag II, Twin-Strep-tag, SpyTag, and Avi-Tag.

[0046] Polypeptides comprising the VAR2CSA domain that binds to chondroitin sulfate proteoglycans can be produced in, but are not limited to, Escherichia coli or insect cells, and can also be produced in animal cells such as 293 cells. Furthermore, the polypeptides can be expressed in hosts such as yeast and filamentous fungi, which are used for expressing recombinant proteins. The polypeptides can also be obtained using a cell-free protein synthesis system.

[0047] For example, in the case of E. coli, a polynucleotide encoding a polypeptide consisting of DBL1X-ID2a or ID1-ID2a of VAR2CSA is synthesized by referring to the sequence registered in GenBank and cloned into a vector that expresses the protein using E. coli as a host. The codons of the introduced gene can also be mutated to codons preferentially used in the host species, E. coli.

[0048] Expression vectors for expressing the VAR2CSA polypeptide include the pET expression vector (Merck Millipore) with a T7 promoter, the pGEX expression vector (Cytiva) or pLEAD expression vector (Nippon Gene) with a tac promoter, and the pCold expression vector (Takara Bio) with a promoter for the E. coli cold shock gene cspA. The pET expression vector is preferred. In the following examples, pET-21b, a type of pET expression vector, was used. In the following examples, a DNA fragment consisting of a polynucleotide encoding a polypeptide in which a Twin-Strep tag was linked to the N-terminus of an ID1-ID2a domain polypeptide and a 3xFLAG tag was linked to the C-terminus, was cloned into pET-21b. As a result, a 6xHis tag was linked to the C-terminus of the final polypeptide sequence. The amino acid sequence of the VAR2CSA polypeptide derived from the FCR3 strain produced from the above expression vector is shown in SEQ ID NO:8.

[0049] The host Escherichia coli is transformed with a vector into which DNA encoding the VAR2CSA polypeptide used in the present invention has been cloned, and after cultivation, an appropriate inducer is added to express the protein, which is the cloned gene product. This can be achieved by well-known genetic engineering techniques, as described specifically in the Examples below. Taking the pET expression vector as an example, examples of hosts include BL21(DE3), Tuner (trade name) (DE3), Rosetta (trade name) (DE3), Origami (trade name) (DE3), and SHuffle T7 Express, and IPTG is a suitable inducer.

[0050] 3. Immobilization of VAR2CSA Polypeptide on a Support As described below, VAR2CSA polypeptide can be used by immobilizing it on a support. Known methods can be used to immobilize VAR2CSA polypeptide on a support. Polymer beads, magnetic beads, microplate wells, and other supports widely used in the field of immunoassays can be used. For example, VAR2CSA polypeptide diluted in an appropriate buffer can be added to the wells of a polystyrene plate designed for enzyme-linked immunosorbent assay (ELISA), allowing the VAR2CSA polypeptide to passively bind to the polystyrene surface.

[0051] For example, streptavidin can be bound to the surface of a carrier, and the VAR2CSA polypeptide can be immobilized (immobilized) on the surface of the carrier by binding between the streptavidin on the surface of the carrier and the Twin-Strep-tag arranged on the VAR2CSA polypeptide. Examples of plate-shaped carriers to which streptavidin can be bound include IMMOBILIZER STREPTAVIDIN (Thermo Scientific Nunc, 436022), Streptavidin plate C8 transparent (Nunc, 236004), and Strep-Tactin (trade name) XT coated 96-well plate (IBS).

[0052] Alternatively, the VAR2CSA polypeptide can be immobilized on a support surface by binding a nickel chelate to the support surface and then forming a bond between the nickel chelate on the support surface and a 6×His-Tag peptide attached to the VAR2CSA polypeptide. Examples of plate-shaped supports with nickel chelate binding include IMMOBILIZER NICKEL-CHELATE (Thermo Scientific Nunc, 436024) and Pierce (trade name) Nickel Coated Plates (Thermo Scientific).

[0053] More specifically, for example, VAR2CSA polypeptide is diluted with PBS to 5-20 μg / mL, added to an ELISA plate, and incubated for 1 to 24 hours. After washing with a washing solution such as PBST, an ELISA plate blocking agent is added and the plate is incubated at room temperature for 1 hour to immobilize the VAR2CSA polypeptide. Commercially available blocking agents, such as Blocking-one (Nacalai Tesque), Blocker (trade name) BLOTTO in TBS (Thermo Fisher Scientific), and ELISA Coating Buffer 1X (Abcam), can be used for blocking. PBS containing 0.1% Tween 20 (PBST, Thermo Fisher Scientific) is a suitable buffer for washing the carrier with bound extracellular vesicles.

[0054] Magnetic beads can be used as a carrier for immobilizing the VAR2CSA polypeptide. The VAR2CSA polypeptide can be directly immobilized on magnetic beads. Suitable beads for this purpose include Dynabeads (trade name) M-270 Epoxy (Thermo), Dynabeads M-280 Tosylactivated (Thermo), and Dynabeads M-270 Carboxylic Acid (Thermo).

[0055] Alternatively, streptavidin-coated beads can be used. Examples of streptavidin-coated magnetic beads include Dynabeads M-270 Streptavidin (Thermo), Dynabeads (trade name) MyOne (trade name) Streptavidin C1 (Thermo), Dynal CELLection Biotin Binder (Thermo), Sera-Mag (trade name) SpeedBeads (trade name) Blocked Streptavidin particles (Sigma), MagStrep "type 3" XT Beads (IBA), Magnosphere (trade name) MS300 / Streptavidin (MBL), and Strep-Tactin (trade name) AlphaLISA (trade name) Acceptor beads (Perkin Elmer). By mixing these magnetic beads with a VAR2CSA polypeptide having a Twin-Strep-tag peptide attached thereto, the Twin-Strep-tag peptide attached to the VAR2CSA polypeptide can be bound to streptavidin on the surface of the beads.

[0056] Alternatively, beads with nickel or cobalt chelates attached to their surfaces can be used. Examples of nickel-chelate-immobilized magnetic beads include His60 Ni Magnetic Beads (Takara Bio), TALON Magnetic Beads (Takara Bio), and HisPur Ni-NTA Magnetic Beads (Thermo). Mixing these magnetic beads with the VAR2CSA polypeptide allows the 6X His-Tag peptide attached to the VAR2CSA polypeptide to bind to the nickel or cobalt chelate on the bead surface.

[0057] Agarose and Sepharose can be used as carriers for immobilizing the VAR2CSA polypeptide. Examples of carriers for immobilizing streptavidin include streptavidin-agarose (Millipore), Streptavidin HP MultiTrap (Cytiva), Strep-Tactin (trade name) TACS Agarose (IBA), and Strep-Tactin Sepharose (IBA).

[0058] Furthermore, agarose or Sepharose immobilized with nickel or cobalt chelates can also be used. Examples of nickel or cobalt chelate-immobilized supports include Ni Sepharose (trade name) 6 Fast Flow (Cytiva), Ni-NTA Agarose (Thermo), and TALON (trade name) Metal Affinity Resin (Takara Bio).

[0059] When immobilizing the VAR2CSA polypeptide on a carrier, either the N-terminus or C-terminus of the VAR2CSA polypeptide can be used for binding to the carrier. The orientation of the VAR2CSA polypeptide immobilized on the carrier can be adjusted by positioning / inserting a tag peptide, such as the Twin-Strep-tag or 6xHis-Tag peptide, at either the N-terminus or C-terminus of the VAR2CSA polypeptide depending on the purpose and detection sensitivity. Because the amount of EVs captured by the VAR2CSA polypeptide increases when the VAR2CSA polypeptide is bound to the carrier at its C-terminus rather than its N-terminus, it is more preferable to use the C-terminus of the VAR2CSA polypeptide for immobilization on the carrier.

[0060] 4. Biological Samples Biological samples applicable to the methods of the present invention include cell line culture media and body fluids. Cell line culture media are the supernatants obtained by culturing cells. Cancer cell lines are cell lines that have been cultured from cancer tissue and can be subcultured. Normal cell lines are cell lines that have been cultured from healthy tissue and can be subcultured, primary cultures of healthy tissue, and even immortalized cell lines that have been immortalized by introducing immortalizing genes (e.g., hTERT, CDK4, CCND1, c-myc), which are known as non-cancer cell lines.

[0061] Examples of body fluids include blood (whole blood), plasma, serum, urine, stool, cerebrospinal fluid, pleural effusion, synovial fluid, bone marrow, gastric juice, feces, semen, prostatic fluid, saliva, sputum, pleural effusion, bile, pancreatic juice, intestinal fluid, sweat, tears, nasal discharge, amniotic fluid, milk, aqueous humor, ascites, serous cavity fluid, lymph, etc. Preferred body fluids in the present invention include blood, plasma, serum, urine, saliva, and sputum used as samples for liquid biopsy (body fluid diagnosis).

[0062] Furthermore, the biological sample may be derived from an animal that has developed cancer or is cancer-bearing, or from an animal that serves as a control for these, and is preferably a specimen derived from a mammal, more preferably a specimen derived from a human, mouse, rat, rabbit, cow, pig, horse, dog, or cat, even more preferably a specimen derived from a human, dog, or cat, and most preferably a specimen derived from a human.

[0063] 5. Capture Method The method for capturing cancer cell-derived extracellular vesicles according to the present invention includes the step of contacting a biological sample with a VAR2CSA polypeptide and binding the cancer cell-derived extracellular vesicles in the biological sample to the VAR2CSA polypeptide.

[0064] The binding step is preferably carried out by immobilizing the VAR2CSA polypeptide on a carrier as described above. The binding conditions between the cancer cell-derived extracellular vesicles and the VAR2CSA polypeptide are not particularly limited and can be appropriately set, but can be carried out, for example, by incubating them in a buffer solution at 4°C to room temperature for 1 to 18 hours.

[0065] The above-described capture method of the present invention can be applied to removing cancer-derived extracellular vesicles from the blood of cancer patients using an extracorporeal circulation device, and to treatments for suppressing cancer metastasis and improving the lifespan and quality of life of cancer patients.

[0066] It has been reported that cancer-derived extracellular vesicles are deeply involved in the metastasis and progression of cancer. Therefore, by using an extracorporeal circulation device to remove cancer-derived extracellular vesicles from the blood of cancer patients, it is possible to stop cancer metastasis and progression, prolong the patient's life, and improve their quality of life.

[0067] An example of such an extracorporeal circulation device is a device that removes cancer-derived extracellular vesicles from blood containing cells, extracellular vesicles, and plasma, in which a hollow fiber through which blood passes has a plurality of pores, the pores are configured so that the extracellular vesicles in the blood can flow through the pores from the inside of the hollow fiber to the outside of the hollow fiber, and the VAR2CSA polypeptide immobilized on the hollow fiber is designed to have a functionalized surface that binds to the extracellular vesicles and removes them from the blood.

[0068] 6. Detection Method The method for detecting cancer cell-derived extracellular vesicles according to the present invention comprises the steps of contacting a biological sample with a VAR2CSA polypeptide to allow the cancer cell-derived extracellular vesicles in the biological sample to bind to the VAR2CSA polypeptide, and detecting the cancer cell-derived extracellular vesicles bound to the VAR2CSA polypeptide. The detection method of the present invention can also be used to perform quantification or semi-quantification, but since quantification or semi-quantification necessarily involves detection, quantification and semi-quantification are also included within the scope of the detection method of the present invention.

[0069] This detection method utilizes the specific binding between the VAR2CSA polypeptide and cancer cell-derived extracellular vesicles (i.e., it binds to cancer cell-derived extracellular vesicles but not to normal cell-derived extracellular vesicles), and therefore the method itself can be applied to well-known immunoassay techniques. That is, techniques such as the sandwich method, agglutination method, competitive method, and immunochromatography can be applied. Of these, the sandwich method is preferred, and the sandwich method is also used in the Examples below. To perform the sandwich method, another substance (second binding substance) that binds to cancer cell-derived extracellular vesicles is required. Examples of this second binding substance include antibodies or antigen-binding fragments thereof, or aptamers that bind to the surface of extracellular vesicles. Proteins such as CD9, CD63, and CD81 are known to be expressed on the surface of extracellular vesicles, and antibodies or antigen-binding fragments thereof, or aptamers against these proteins can be used as second binding substances that bind to extracellular vesicles. The antibody may be a monoclonal or polyclonal antibody as long as it can detect extracellular vesicles that substantially express CD9, CD63, CD81, etc., and the animal species from which the antibody is obtained may be any of human, mouse, rabbit, goat, sheep, chicken, camel, llama, alpaca, shark, and ostrich.

[0070] In addition, as a second binding substance other than an antibody or its antigen-binding fragment, it is also possible to use the phosphatidylserine-binding domain of Tim1, Tim3, Tim4 or beta-2-glycoprotein 1 (β2GP1), which are proteins that bind to phosphatidylserine located on the outside of the membrane of extracellular vesicles.

[0071] In the sandwich method, either the VAR2CSA polypeptide or the second binding substance is immobilized on a carrier, and the other is labeled, and detection is performed by detecting the label bound to the carrier. The VAR2CSA polypeptide can be immobilized by the method described above. When the second binding substance is immobilized on a carrier, since the second binding substance is also a protein, it can be immobilized by the well-known method described above.

[0072] For example, when the VAR2CSA polypeptide is immobilized on a carrier, the sandwich method can detect cancer cell-derived extracellular vesicles in a biological sample by first contacting the biological sample with the carrier on which the VAR2CSA polypeptide is immobilized, washing the carrier, and then contacting the biological sample with a labeled second binding substance, washing the carrier, and then detecting the label bound to the carrier via the cancer cell-derived extracellular vesicles.

[0073] Alternatively, the biological sample may first be contacted with a labeled second binding substance, then with a carrier on which the VAR2CSA polypeptide has been immobilized, and after washing, the label bound to the carrier via cancer cell-derived extracellular vesicles may be detected (reverse method).

[0074] When the second binding substance is immobilized on a carrier, the sandwich method can detect cancer cell-derived extracellular vesicles in a biological sample by first contacting the biological sample with the carrier on which the second binding substance is immobilized, washing the carrier, and then contacting the biological sample with a labeled VAR2CSA polypeptide. After washing the carrier, the label bound to the carrier via the cancer cell-derived extracellular vesicles is detected.

[0075] Alternatively, cancer cell-derived extracellular vesicles in a biological sample can be detected by first contacting the biological sample with a labeled VAR2CSA polypeptide, then contacting the biological sample with a carrier on which a second binding substance is immobilized, washing the carrier, and then detecting the label bound to the carrier via the cancer cell-derived extracellular vesicles (reverse method).

[0076] Alternatively, the biological sample, the carrier on which the VAR2CSA polypeptide is immobilized, and the labeled second binding substance may be simultaneously contacted, and after washing, the label bound to the carrier via cancer cell-derived extracellular vesicles may be detected.

[0077] Alternatively, the biological sample, the carrier on which the second binding substance is immobilized, and the labeled VAR2CSA polypeptide may be simultaneously contacted, and after washing, the label bound to the carrier via cancer cell-derived extracellular vesicles may be detected.

[0078] As the label, well-known labels used in the field of immunoassays can be used. That is, well-known enzyme labels (e.g., HRP, ALP, etc.), fluorescent labels (e.g., FITC, PE, Alexa Fluor, etc.), etc. can be used. These labels are commercially available as kits, so commercially available kits can be used. In addition, methods for binding proteins or aptamers to labels are also well-known and can be easily performed using commercially available kits.

[0079] Alternatively, a label for Amplified Luminescent Proximity Homogeneous Assay (Alpha) can be used. This method uses a label (donor bead) that is excited by excitation light and a signal-generating label (acceptor bead) that generates a signal by singlet oxygen generated by the excitation, and the signal emitted from the signal-generating label is detected by the singlet oxygen generated from the excited label.

[0080] In an embodiment of the detection method of the present invention using Alpha, either the VAR2CSA polypeptide or the second binding substance is immobilized on donor beads, and the other is bound to acceptor beads. When the donor beads and acceptor beads come close to each other via the binding between the VAR2CSA polypeptide and cancer cell-derived extracellular vesicles and the binding between the cancer cell-derived extracellular vesicles and the second binding substance, the aforementioned signal is generated and detected.

[0081] The detection method using Alpha can be considered to be the donor bead as a carrier and the acceptor bead as a label, and is therefore included in the detection method of the present invention. This method is also well known, and kits for it are commercially available, so it can be easily performed using commercially available products.

[0082] In the capture method and detection method of the present invention, the conditions for each "contact" are not particularly limited and can be set appropriately, and can be, for example, performed by incubating in a buffer solution at 4° C. to room temperature for 1 to 18 hours. Furthermore, detection of the label can be performed by a well-known method appropriate for each label.

[0083] Examples of buffer solutions that can be used to dilute proteins or aptamers, such as antibodies, that detect extracellular vesicles include Blocking-one diluted 20-fold with PBS containing 0.1% Tween 20, as well as commercially available antibody dilutions such as Can Get Signal (TOYOBO), ChonBlock Detection Antibody Dilution ELISA Buffer (Chondrex), and Universal Antibody Dilution Buffer (trade name) (GeneTex).

[0084] Methods for detecting VAR2CSA polypeptide bound to extracellular vesicles can also utilize antibodies against VAR2CSA and tag sequences attached to the VAR2CSA polypeptide (e.g., Twin-Strep-tag peptide, 3 x FLAG-tag peptide, 6 x His-tag peptide, etc., particularly 3 x FLAG-tag peptide and 6 x His-tag peptide attached to the C-terminus in the examples of the present invention). When targeting Twin-Strep-tag peptide, streptavidin that binds to Twin-Strep-tag peptide can be used, with streptavidin labeled with HRP or the like being preferred. When targeting 3 x FLAG-tag peptide, anti-FLAG-tag antibodies can be used, with anti-FLAG-tag antibodies labeled with HRP or the like being preferred. When targeting 6 x His-tag peptide, anti-His-tag antibodies can be used, with anti-His-tag antibodies labeled with HRP being preferred.

[0085] 7. Cancers from which Extracellular Vesicles are Derived Examples of cancers in the present invention include pancreatic cancer, biliary tract cancer, gallbladder cancer, bile duct cancer, lung cancer, colon cancer, prostate cancer, breast cancer, gastric cancer, cervical cancer, bladder cancer, testicular cancer, ovarian cancer, endometrial cancer, liver cancer, kidney cancer, head and neck cancer, esophageal cancer, pharyngeal cancer, osteosarcoma, sarcoma, glioma, lymphoma, and leukemia, and the extracellular vesicles produced by these cancers may express oncofetal chondroitin sulfate A on their surface. Gliomas include glioblastomas, and leukemias include acute myeloid leukemia and acute lymphoid leukemia.

[0086] 8. Method for Obtaining Information on Whether or Not a Subject Has Cancer The detection method of the present invention described above can also be used to quantify cancer cell-derived extracellular vesicles in a biological sample. If the amount of VAR2CSA polypeptide-trapped extracellular vesicles quantified in a biological sample from a subject is significantly higher than the amount of VAR2CSA polypeptide-trapped extracellular vesicles in a biological sample from a healthy subject, the subject can be determined to have a high likelihood of cancer. Alternatively, data on the amount of VAR2CSA polypeptide-trapped extracellular vesicles in a biological sample from a healthy subject can be obtained in advance, and a threshold can be set in advance. If the amount of VAR2CSA polypeptide-trapped extracellular vesicles in a biological sample from a subject is higher than the threshold, the subject can be determined to have a high likelihood of cancer. Similar assessments can also be performed using patients with benign diseases (including inflammatory diseases) other than cancer as a control instead of healthy subjects.

[0087] The present invention also provides a kit for use in the above-described detection method of the present invention. The kit of the present invention comprises a VAR2CSA polypeptide and a second binding substance that binds to the surface of extracellular vesicles, in which either (i) any one of the polypeptides (A) to (C) and (ii) the second binding substance is immobilized on a carrier, and the other is labeled.

[0088] The kit of the present invention is preferably a kit for diagnosing whether or not a subject has cancer, comprising a VAR2CSA polypeptide and a second binding substance that binds to the surface of extracellular vesicles, wherein either (i) any one of the polypeptides (A) to (C) and (ii) the second binding substance is immobilized on a carrier, and the other is labeled.

[0089] Alternatively, the kit of the present invention can be used as a kit for capturing extracellular vesicles derived from cancer cells, in which the VAR2CSA polypeptide is immobilized on a carrier. For example, the kit can be used for functional analysis of captured extracellular vesicles derived from cancer cells, or for studies to identify cancer types or elucidate the state of cancer by analyzing molecules contained in the extracellular vesicles.

[0090] The kit of the present invention may contain a buffer solution for diluting a biological sample before contacting it with the VAR2CSA polypeptide or the like, instructions for use, and the like.

[0091] The present invention will be described in more detail below with reference to examples. However, these examples are merely illustrative examples for specifically explaining the present invention and are not intended to limit the technical scope of the present invention.

[0092] Reference Example 1: Preparation of VAR2CSA Polypeptide. A polynucleotide encoding a polypeptide (SEQ ID NO: 6) comprising the amino acid sequence (SEQ ID NO: 1) of the ID1-ID2a domain of VAR2CSA from the Plasmodium falciparum FCR3 strain (GenBank Accession No. GU249598), a Twin-Strep tag (SEQ ID NO: 3), a flexible linker (SEQ ID NO: 4) at the N-terminus, and a 3xFLAG tag (SEQ ID NO: 5) at the C-terminus, was synthesized and optimized for Escherichia coli codons (SEQ ID NO: 7). This polynucleotide was inserted between the NdeI and XhoI sites of the E. coli expression vector pET21b. This vector was designated pET21b-VAR2-FCR3. The amino acid sequence of the polypeptide expressed from pET21b-VAR2-FCR3 is the polypeptide of SEQ ID NO: 6, with a His-tag added to the C-terminus, and the sequence of this polypeptide is set forth in SEQ ID NO: 8.

[0093] A schematic diagram of the structure of the VAR2CSA polypeptide is shown in FIG.

[0094] The pET21b-VAR2-FCR3 plasmid DNA was transformed into the E. coli strain SHuffle T7 Express Competent E. coli (NEB; C3029J) to obtain transformants. A clone of this transformant was cultured in 10 mL of LB medium containing 100 μg / mL carbenicillin at 37°C for 18 hours with shaking. Six mL of the culture was inoculated into 150 mL of LB medium (containing 100 μg / mL carbenicillin) and cultured at 37°C with shaking until the OD600 reached approximately 0.6. 1 mol / L IPTG was added to the culture to a final concentration of 0.1 mmol / L, and the culture was cultured at 20°C for approximately 20 hours with shaking. After the culture was completed, the culture was centrifuged at 6,000 x g for 15 minutes at 4°C, and the E. coli pellet was collected and stored frozen at -80°C. Cryopreserved E. coli pellets were thawed at room temperature, and 5 mL of B-PER™ Bacterial Cell Lysis Reagent (Thermo Fisher Scientific; 89821) and 1 / 100 volume of protease inhibitor cocktail (Nacalai, 03969-21) were added per gram of wet weight, followed by suspension by pipetting. After gentle stirring at room temperature for 15 minutes on a rotator, the pellets were centrifuged at 12,000 x g for 15 minutes at 4°C, and the supernatant was transferred to a new tube.

[0095] Purification was performed using the Strep-Tactin XT 4Flow Starter Kit (IBA; 2-5998-000) according to the accompanying manual. The extract was loaded onto a 1 mL Strep-Tactin XT 4Flow column, washed with Buffer W provided with the kit, and eluted with Buffer BXT provided with the kit. The eluate was replaced with PBS by centrifugation using a PD MiniTrap G25 (Cytiva; 28918007). The protein content of purified VAR2CSA was measured using a TaKaRa BCA Protein Assay Kit (Takara; T9300A), and the molecular weight was verified by SDS-PAGE. The biological activity of VAR2CSA was confirmed by binding to bovine decorin (Sigma; D8428) immobilized on an ELISA plate.

[0096] Reference Example 2: Preparation of Extracellular Vesicles The cell lines, media, and additives used in the present invention are shown in Table 2 for cancer cell lines, and in Table 3 for normal cell lines (non-cancer cell lines). The normal cells used in the present invention were prepared by introducing an immortalization gene into primary cell lines obtained from ATCC or Lonza. Each cell line was cultured in a medium containing the additives listed in the table until it became 70% to 80% confluent. After removing the culture medium, the cells were cultured for 4 to 5 days in a medium containing 5% Exosome-Depleted FBS (ThermoFisher Scientific, A2720803).

[0097] Culture supernatants from each cell line were collected and centrifuged at 300 x g for 5 minutes. The supernatants were then centrifuged again at 8,000 x g for 30 minutes. The supernatants were filtered through a 0.22 μm syringe filter (Millipore). Next, 40 mL of the filtered culture supernatant was concentrated to 1 mL using a 100 kDa molecular weight cutoff centrifugal concentration tube, VIVASPIN 20 (Sartorius; VS2041) or Amicon Ultra-15 (Millipore; UFC910008). Extracellular vesicles from the concentrated samples were purified using the MagCapture Exosome Isolation Kit PS Ver. 2 (Fujifilm) according to the kit's instructions. Briefly, 60 μL of Tim4-coated magnetic beads were added to 1 mL of concentrated sample to adsorb extracellular vesicles. After washing, the extracellular vesicles were eluted with 100 μL of elution buffer. To this eluate was added 1 / 100 volume of an extracellular vesicle blocking reagent, EV-Save (trade name) (Fujifilm), and the mixture was dispensed and stored at -80°C.

[0098]

[0099]

[0100] Reference Example 3: Preparation of antibodies Antibodies that recognize CD9, CD63, and CD81 expressed on the surface of extracellular vesicles were used to detect extracellular vesicles. Specifically, the following antibodies were used: an anti-human CD9 rat monoclonal antibody (77B, Fujifilm), an anti-human CD63 mouse monoclonal antibody (3-13, Fujifilm), and an anti-human CD81 rat monoclonal antibody (9B, Fujifilm), which are not cross-reactive with bovine antibodies.

[0101] 100 μg of these antibodies were labeled with Peroxidase using the Peroxidase Labeling Kit-NH2 (Dojindo). For detection of extracellular vesicles, the labeled antibodies were diluted 500-fold and used.

[0102] Reference Example 4 Detection of extracellular vesicles by ELISA To detect extracellular vesicles, the ELISA plate included with the PS Capture (trade name) Exosome ELISA kit (Fujifilm) or the CD9 / CD63 ELISA kit (Cosmo Bio) was used, and the POD-labeled antibody described in Reference Example 3 above was used as the detection antibody.

[0103] Briefly, the Tim4-coated plate of the PS Capture™ Exosome ELISA Kit or the anti-CD9 antibody-coated plate of the CD9 / CD63 ELISA Kit was washed three times with the wash buffer provided with each kit. Extracellular vesicle samples diluted in the reaction buffer provided with each kit were added to the plate wells, vortexed for 30 seconds, and incubated overnight at 4°C. After five washes with the wash buffer provided with each kit, the POD-conjugated antibody diluted 500-fold in the reaction buffer provided with each kit was added, vortexed for 30 seconds, and incubated at room temperature. After two hours, the plate was washed five times with the wash buffer provided with each kit. The ELISA POD Substrate TMB Kit HYPER (Nacalai) colorimetric substrate solution was added, vortexed for 30 seconds, and incubated for 5 to 10 minutes. The reaction was stopped by adding stop solution (1M sulfuric acid) and vortexing for 10 seconds. The absorbance (450 nm) was immediately measured using a microplate reader. The absorbance was expressed as a value obtained by subtracting the blank value from each value.

[0104] [A. Method for capturing and detecting extracellular vesicles using VAR2CSA polypeptide in the examples] The VAR2CSA polypeptide prepared in Reference Example 1 was diluted with PBS to 10 μg / mL. 100 μL of the 10 μg / mL VAR2CSA polypeptide solution was added to each well of a streptavidin-coated plate (Nunc) and incubated overnight at 4°C. Alternatively, 100 μL of the 10 μg / mL VAR2CSA polypeptide solution was added to each well of Pierce (trade name) Nickel Coated Plates (Thermo Scientific) and incubated overnight at 4°C. After incubation, these plates were washed three times with PBST and blocked by adding 200 μL of Blocking One (Nacalai) diluted 5-fold with DW to each well and incubating for 1 hour.

[0105] After washing three times with PBST, extracellular vesicles diluted with Blocking One diluted 20-fold with PBST were added, and the mixture was stirred for 30 seconds and then incubated overnight at 4°C.

[0106] Detection of extracellular vesicles captured by the VAR2CSA polypeptide was performed as follows. After incubation, the plate was washed five times with PBST. The antibody from Reference Example 3, diluted 500-fold with antibody diluent, was added, stirred for 30 seconds, and then incubated for 2 hours. After washing five times with PBST, the ELISA POD Substrate TMB Kit HYPER (Nacalai) colorimetric substrate solution was added, stirred for 30 seconds, and allowed to react for 5–10 minutes. The reaction was stopped by adding a stop solution (1 M sulfuric acid) and stirring for 10 seconds. After stopping the reaction, the absorbance (450 nm) was immediately measured using a microplate reader. The absorbance was expressed as the value obtained by subtracting the blank value from each value. A schematic diagram illustrating the above analysis method is shown in Figure 2.

[0107] Example 1: Detection of extracellular vesicles derived from glioblastoma cell lines using VAR2CSA polypeptide. (1) Extracellular vesicles prepared from U87-MG cells, a glioblastoma cell line, using the method described in Reference Example 2 were serially diluted and captured and detected using the method described in Section A. Specifically, purified EV samples were serially diluted and added to each well of a VAR2CSA-coated plate. After overnight incubation at 4°C, the plate was washed five times with PBST. An antibody solution prepared by diluting an HRP-labeled anti-CD63 antibody 500-fold with diluent was added to each well and incubated at room temperature. After 2 hours, the plate was washed five times with PBST, TMB substrate was added, and upon color development, a stop solution was added. The absorbance at 450 nm was immediately measured using a microplate reader. The results are shown in Figure 3.

[0108] Comparative Example 1: Detection of extracellular vesicles from glioblastoma cell lines using anti-CD9 antibodies. For comparison, serially diluted purified extracellular vesicle samples from U87-MG cells were added to each well of an anti-CD9 antibody-coated plate. After overnight incubation at 4°C and washing five times with PBST, an antibody solution containing HRP-labeled anti-CD63 antibody diluted 500-fold with diluent was added to each well and incubated at room temperature. After 2 hours, the plate was washed five times with PBST, TMB substrate was added, and upon color development, stop solution was added. The absorbance at 450 nm was immediately measured using a microplate reader. The results are shown in Figure 4.

[0109] The results of Example 1 and Comparative Example 1 above show that purified extracellular vesicles derived from U87-MG cells are captured by VAR2CSA polypeptide and anti-CD9 antibody and detected by anti-CD63 antibody.

[0110] Comparative Example 2: Attempt to detect extracellular vesicles derived from a normal fibroblast cell line using VAR2CSA polypeptide. Extracellular vesicles prepared from the fetal lung fibroblast cell line WI-38-hTERT using the method described in Reference Example 2 were serially diluted and captured and detected using the method described in Section A. Specifically, purified EV samples were serially diluted and added to each well of a VAR2CSA-coated plate. After overnight incubation at 4°C, the plate was washed five times with PBST. An antibody solution containing an HRP-labeled anti-CD63 antibody diluted 500-fold with diluent was added to each well and incubated at room temperature. After 2 hours, the plate was washed five times with PBST, TMB substrate was added, and upon color development, a stop solution was added. The absorbance at 450 nm was immediately measured using a microplate reader. The results are shown in Figure 5.

[0111] Comparative Example 3: Detection of Extracellular Vesicles Derived from a Normal Fibroblast Cell Line with Anti-CD9 Antibody. For comparison, serially diluted purified extracellular vesicle samples derived from WI-38-hTERT cells were added to each well of an anti-CD9 antibody-coated plate. After overnight incubation at 4°C and washing five times with PBST, an antibody solution containing HRP-labeled anti-CD63 antibody diluted 500-fold with diluent was added to each well and incubated at room temperature. After 2 hours, the plate was washed five times with PBST, TMB substrate was added, and upon color development, stop solution was added. The absorbance at 450 nm was immediately measured using a microplate reader. The results are shown in Figure 6.

[0112] The results of Comparative Examples 2 and 3 above show that purified extracellular vesicles derived from WI-38-hTERT cells cannot be captured by VAR2CSA polypeptide, but are captured by anti-CD9 antibody and detected by anti-CD63 antibody.

[0113] Example 2: Detection of extracellular vesicles derived from pancreatic cancer cell lines using VAR2CSA polypeptide. (1) Culture supernatants were collected from two cultures of pancreatic cancer cell lines, PANC-1 and KMP2 cells (Preparation 1 and Preparation 2), and extracellular vesicles prepared as described in Reference Example 2 were diluted and captured and detected as described in Section A. Specifically, purified EV samples were diluted 4-fold and added to each well of a VAR2CSA-coated plate. After overnight incubation at 4°C, the plate was washed five times with PBST. An antibody cocktail solution containing HRP-labeled anti-CD9 antibody and HRP-labeled anti-CD81 antibody diluted 500-fold with diluent was added to each well and incubated at room temperature. After 2 hours, the plate was washed five times with PBST, TMB substrate was added, and upon color development, a stop solution was added. The absorbance at 450 nm was immediately measured using a microplate reader. The results are shown in Figure 7.

[0114] Comparative Example 4: Detection of extracellular vesicles from pancreatic cancer cell lines using Tim4 polypeptide. Furthermore, purified extracellular vesicle samples from PANC-1 cells and KMP2 cells, diluted 40-fold, were added to each well of the Tim4-coated plate. After overnight incubation at 4°C and washing five times with washing buffer, an antibody cocktail solution containing HRP-labeled anti-CD9 antibody and HRP-labeled anti-CD81 antibody, each diluted 500-fold with diluent, was added to each well and incubated at room temperature. After 2 hours, the plate was washed five times with washing buffer, and TMB substrate was added. Upon color development, a stop solution was added, and the absorbance at 450 nm was immediately measured using a microplate reader. The results are shown in Figure 8.

[0115] The results of Example 2 and Comparative Example 4 above show that purified extracellular vesicles derived from PANC-1 cells and KMP2 cells are captured by VAR2CSA polypeptide and Tim4 and detected with a cocktail of anti-CD9 and anti-CD81 antibodies.

[0116] Example 3: Detection of Extracellular Vesicles from Prostate Cancer Cell Lines Using VAR2CSA Polypeptide. Extracellular vesicles prepared from prostate cancer cell lines PC-3 and LNCap cells using the method described in Reference Example 2 were diluted and captured and detected using the method described in Section A. Specifically, purified EV samples were diluted 4-fold and added to each well of a VAR2CSA-coated plate. After overnight incubation at 4°C and washing five times with PBST, antibody cocktail solutions containing HRP-labeled anti-CD9 antibody, HRP-labeled anti-CD63 antibody, and HRP-labeled anti-CD81 antibody, each diluted 500-fold with diluent, were added to each well and incubated at room temperature. After 2 hours, the plates were washed five times with PBST, TMB substrate was added, and upon color development, a stop solution was added. The absorbance at 450 nm was immediately measured using a microplate reader. The results are shown in Figure 9.

[0117] Comparative Example 5: Attempt to detect extracellular vesicles derived from a normal prostate fibroblast cell line using VAR2CSA polypeptide An attempt was made to detect extracellular vesicles using normal cells, HPrEC-TKD cells and HPrF-TKD cells, instead of the prostate cancer cell line used in Example 3. The results are shown in Figure 9.

[0118] Comparative Example 6: Detection of extracellular vesicles from prostate cancer cell lines using Tim4 polypeptide. For comparison, purified extracellular vesicle samples from prostate cancer cell lines PC-3 and LNCap cells, diluted 40-fold, were added to each well of the Tim4-coated plate. After overnight incubation at 4°C and washing five times with washing buffer, an antibody cocktail solution containing HRP-labeled anti-CD9 antibody, HRP-labeled anti-CD63 antibody, and HRP-labeled anti-CD81 antibody, each diluted 500-fold with diluent, was added to each well and incubated at room temperature. After 2 hours, the plate was washed five times with washing buffer, and TMB substrate was added. Upon color development, stop solution was added, and the absorbance at 450 nm was immediately measured using a microplate reader. The results are shown in Figure 10.

[0119] Comparative Example 7: Detection of extracellular vesicles derived from a normal prostate fibroblast cell line using Tim4 polypeptide Extracellular vesicles were detected using normal cells, HPrEC-TKD cells and HPrF-TKD cells, instead of the prostate cancer cell line used in Comparative Example 6. The results are shown in Figure 10.

[0120] The results of Example 3 and Comparative Examples 5 to 7 above show that purified extracellular vesicles derived from PC-3 cells and LNCap cells were captured by VAR2CSA polypeptide and Tim4 and detected with a cocktail of anti-CD9, anti-CD63, and anti-CD81 antibodies, whereas purified extracellular vesicles derived from normal cells HPrEC-TKD and HPrF-TKD were captured by Tim4 and detected with a cocktail of anti-CD9, anti-CD63, and anti-CD81 antibodies, but were not captured by VAR2CSA polypeptide.

[0121] Example 4: Detection of extracellular vesicles derived from lung cancer cell lines using VAR2CSA polypeptide. Extracellular vesicles prepared from lung cancer cell lines A549, NCI-H1703, and N69AR cells using the method described in Reference Example 2 were diluted and captured and detected using the method described in Section A. Specifically, purified EV samples were diluted 4-fold and added to each well of a VAR2CSA-coated plate. After overnight incubation at 4°C and washing five times with PBST, antibody cocktail solutions containing HRP-labeled anti-CD9 antibody, HRP-labeled anti-CD63 antibody, and HRP-labeled anti-CD81 antibody, each diluted 500-fold with diluent, were added to each well and incubated at room temperature. After 2 hours, the plates were washed five times with PBST, TMB substrate was added, and upon color development, a stop solution was added. The absorbance at 450 nm was immediately measured using a microplate reader. The results are shown in Figure 11.

[0122] Comparative Example 8 Attempt to detect extracellular vesicles derived from a normal lung fibroblast cell line using VAR2CSA polypeptide An attempt was made to detect extracellular vesicles using normal cells, WI-38-hTERT cells, instead of the lung cancer cell line used in Example 4. The results are shown in Figure 11.

[0123] Comparative Example 9: Detection of extracellular vesicles from lung cancer cell lines using Tim4 polypeptide. For comparison, purified extracellular vesicle samples from A549, NCI-H1703, and N69AR cells, diluted 40-fold, were added to each well of the Tim4-coated plate. After overnight incubation at 4°C and washing five times with washing buffer, an antibody cocktail consisting of HRP-labeled anti-CD9 antibody, HRP-labeled anti-CD63 antibody, and HRP-labeled anti-CD81 antibody, each diluted 500-fold with diluent, was added to each well and incubated at room temperature. After 2 hours, the plate was washed five times with washing buffer, and TMB substrate was added. Upon color development, stop solution was added and the absorbance at 450 nm was immediately measured using a microplate reader. The results are shown in Figure 12.

[0124] Comparative Example 10: Detection of extracellular vesicles derived from a normal lung fibroblast cell line using Tim4 polypeptide. Extracellular vesicles were detected using normal WI-38-hTERT cells instead of the lung cancer cell line used in Comparative Example 9. The results are shown in Figure 12.

[0125] The results of Example 4 and Comparative Examples 8 to 10 above show that purified extracellular vesicles derived from A549 cells, NCI-H1703 cells, and H69AR cells were captured by VAR2CSA polypeptide and Tim4 and detected with a cocktail of anti-CD9, anti-CD63, and anti-CD81 antibodies, whereas purified extracellular vesicles derived from normal WI-38-hTERT cells were captured by Tim4 and detected with a cocktail of anti-CD9, anti-CD63, and anti-CD81 antibodies, but were not captured by VAR2CSA polypeptide.

[0126] Example 5: Detection of Extracellular Vesicles from Osteosarcoma Cell Lines Using VAR2CSA Polypeptide. Extracellular vesicles prepared from MG-63 cells, an osteosarcoma cell line, using the method described in Reference Example 2 were diluted and captured and detected using the method described in Section A. Specifically, the purified sample was diluted 4-fold and added to each well of a VAR2CSA-coated plate. After overnight incubation at 4°C and washing five times with PBST, an antibody cocktail solution containing HRP-labeled anti-CD9 antibody, HRP-labeled anti-CD63 antibody, and HRP-labeled anti-CD81 antibody, each diluted 500-fold with diluent, was added to each well and incubated at room temperature. After 2 hours, the plate was washed five times with PBST, TMB substrate was added, and upon color development, a stop solution was added. The absorbance at 450 nm was immediately measured using a microplate reader. The results are shown in Figure 13.

[0127] Comparative Example 11: Attempt to detect extracellular vesicles derived from a normal osteoblast cell line using VAR2CSA polypeptide An attempt was made to detect extracellular vesicles using normal osteoblastic NHOst-TKD cells instead of the osteosarcoma cell line used in Example 5. The results are shown in Figure 13.

[0128] Comparative Example 12: Detection of extracellular vesicles from osteosarcoma cell lines using Tim4 polypeptide. For comparison, a 40-fold diluted sample of purified extracellular vesicles from MG-63 cells, an osteosarcoma cell line, was added to each well of the Tim4-coated plate. After overnight incubation at 4°C and washing five times with washing buffer, an antibody cocktail consisting of HRP-labeled anti-CD9 antibody, HRP-labeled anti-CD63 antibody, and HRP-labeled anti-CD81 antibody, each diluted 500-fold with diluent, was added to each well and incubated at room temperature. After 2 hours, the plate was washed five times with washing buffer, and TMB substrate was added. Upon color development, a stop solution was added and the absorbance at 450 nm was immediately measured using a microplate reader. The results are shown in Figure 14.

[0129] Comparative Example 13: Detection of extracellular vesicles derived from normal osteoblast cell lines using Tim4 polypeptide Extracellular vesicles were detected using normal osteoblast NHOst-TKD cells instead of the osteosarcoma cell line used in Comparative Example 12. The results are shown in Figure 14.

[0130] The results of Example 5 and Comparative Examples 11 to 13 above show that purified extracellular vesicles derived from MG-63 cells were captured by VAR2CSA polypeptide and Tim4 and detected with a cocktail of anti-CD9, anti-CD63, and anti-CD81 antibodies, whereas purified extracellular vesicles derived from normal NHOst-TKD cells were captured by Tim4 and detected with a cocktail of anti-CD9, anti-CD63, and anti-CD81 antibodies, but were not captured by VAR2CSA polypeptide.

[0131] Example 6: Detection of extracellular vesicles from acute myeloid leukemia cell line by VAR2CSA polypeptide and its inhibition by chondroitin sulfate A. A serially diluted chondroitin sulfate (CSA) was added to an extracellular vesicle sample prepared from KG-1 cells, an acute myeloid leukemia cell line, using the method described in Reference Example 2, and the samples were captured and detected using the method described in Section A. Specifically, a 4-fold diluted purified EV sample was mixed with CSA at concentrations of 0.2 μg / mL, 1 μg / mL, 5 μg / mL, and 25 μg / mL, and the mixture was added to each well of a VAR2CSA-coated plate. After overnight incubation at 4°C and washing five times with PBST, an antibody solution prepared by diluting an HRP-labeled anti-CD63 antibody 500-fold with diluent was added to each well and incubated at room temperature. After 2 hours, the plate was washed five times with PBST, and TMB substrate was added. When color developed, a reaction stop solution was added, and the absorbance at 450 nm was immediately measured using a microplate reader. The results are shown in Figure 15.

[0132] Comparative Example 14: Detection of extracellular vesicles from acute myeloid leukemia-derived cell lines using Tim4 polypeptide. Extracellular vesicle samples prepared from KG-1 cells were diluted 40-fold and added to Tim4-coated wells. After overnight incubation at 4°C, the wells were washed five times with washing buffer. An HRP-labeled anti-CD63 antibody diluted 500-fold with diluent was then added to the wells and incubated at room temperature. After 2 hours, the wells were washed five times with washing buffer, and TMB substrate was added. Upon color development, a stop solution was added and the absorbance at 450 nm was immediately measured using a microplate reader. Evaluation was also performed using a VAR2CSA-coated plate, as in Example 6. The results are shown in Figure 16.

[0133] The results of Example 6 and Comparative Example 14 above demonstrate that purified extracellular vesicles derived from KG-1 cells are captured by the VAR2CSA polypeptide and Tim4 and detected with an anti-CD63 antibody. Furthermore, the capture of extracellular vesicles by the VAR2CSA polypeptide is inhibited by CSA, suggesting that the VAR2CSA polypeptide binds to the CSA of extracellular vesicles.

[0134] Example 7: Detection of extracellular vesicles derived from esophageal cancer cell lines using VAR2CSA polypeptide. Extracellular vesicles prepared from esophageal cancer cell lines with different differentiation types, KYSE150 cells (poorly differentiated), KYSE510 cells (well differentiated), and KYSE1240 cells (moderately differentiated), as described in Reference Example 2, were diluted and captured and detected using the method described in Section A. Specifically, purified EV samples were diluted 4-fold and added to each well of a VAR2CSA-coated plate. After overnight incubation at 4°C and washing five times with PBST, an antibody cocktail solution containing HRP-labeled anti-CD9 antibody, HRP-labeled anti-CD63 antibody, and HRP-labeled anti-CD81 antibody, each diluted 500-fold with diluent, was added to each well and incubated at room temperature. After 2 hours, the plate was washed five times with PBST, TMB substrate was added, and upon color development, a stop solution was added and the absorbance at 450 nm was immediately measured using a microplate reader. The results are shown in Figure 17.

[0135] Comparative Example 15: Detection of extracellular vesicles from esophageal cancer cell lines using Tim4 polypeptide. Purified extracellular vesicle samples from KYSE150, KYSE510, and KYSE1240 cells, diluted 40-fold, were added to each well of the Tim4-coated plate. After overnight incubation at 4°C and washing five times with washing buffer, an antibody cocktail solution containing HRP-labeled anti-CD9 antibody, HRP-labeled anti-CD63 antibody, and HRP-labeled anti-CD81 antibody, each diluted 500-fold with diluent, was added to each well and incubated at room temperature. After 2 hours, the plate was washed five times with PBST, and TMB substrate was added. Upon color development, stop solution was added and the absorbance at 450 nm was immediately measured using a microplate reader. The results are shown in Figure 18.

[0136] The results of Example 7 and Comparative Example 15 above demonstrate that purified extracellular vesicles derived from KYSE150, KYSE510, and KYSE1240 cells were captured with VAR2CSA polypeptide and Tim4 and detected with a cocktail of anti-CD9, anti-CD63, and anti-CD81 antibodies. Although less differentiated esophageal cancer cells tend to be more malignant, VAR2CSA polypeptide was also able to capture extracellular vesicles derived from esophageal cancer cell lines with different degrees of differentiation.

[0137] Example 8: Detection of Extracellular Vesicles from Gastric Cancer Cell Lines Using VAR2CSA Polypeptide. Extracellular vesicles prepared from gastric cancer cell lines HGC-27, AGS, and MKN45 cells using the method described in Reference Example 2 were diluted and captured and detected using the method described in Section A. Specifically, purified EV samples were diluted 2-fold and added to each well of a VAR2CSA-coated plate. After overnight incubation at 4°C and washing five times with PBST, antibody cocktail solutions containing HRP-labeled anti-CD9 antibody, HRP-labeled anti-CD63 antibody, and HRP-labeled anti-CD81 antibody, each diluted 500-fold with diluent, were added to each well and incubated at room temperature. After 2 hours, the plates were washed five times with PBST, TMB substrate was added, and upon color development, a stop solution was added. The absorbance at 450 nm was immediately measured using a microplate reader. The results are shown in Figure 19.

[0138] Comparative Example 16: Detection of extracellular vesicles from gastric cancer cell lines using Tim4 polypeptide. Purified extracellular vesicle samples from HGC-27, AGS, and MKN45 cells, diluted 20-fold, were added to each well of the Tim4-coated plate. After overnight incubation at 4°C and washing five times with washing buffer, an antibody cocktail consisting of HRP-labeled anti-CD9 antibody, HRP-labeled anti-CD63 antibody, and HRP-labeled anti-CD81 antibody, each diluted 500-fold with diluent, was added to each well and incubated at room temperature. After 2 hours, the plate was washed five times with PBST, and TMB substrate was added. Upon color development, stop solution was added and the absorbance at 450 nm was immediately measured using a microplate reader. The results are shown in Figure 20.

[0139] The results of Example 8 and Comparative Example 16 above show that purified extracellular vesicles derived from HGC-27 cells, AGS cells, and MKN45 cells are captured by VAR2CSA polypeptide and Tim4 and detected with a cocktail of anti-CD9, anti-CD63, and anti-CD81 antibodies.

[0140] Example 9 Detection of extracellular vesicles derived from colon cancer cell lines using VAR2CSA polypeptide

[0141] Extracellular vesicles prepared from colon cancer cell lines SW837, SW480, HTC-116, and Caco-2 cells using the method described in Reference Example 2 were diluted and captured and detected using the method described in Section A. Specifically, purified EV samples were diluted 2.5-fold and added to each well of a VAR2CSA-coated plate. After overnight incubation at 4°C and washing five times with PBST, an antibody cocktail solution containing HRP-labeled anti-CD9 antibody, HRP-labeled anti-CD63 antibody, and HRP-labeled anti-CD81 antibody, each diluted 500-fold with diluent, was added to each well and incubated at room temperature. After 2 hours, the plate was washed five times with PBST, TMB substrate was added, and upon color development, a stop solution was added. The absorbance at 450 nm was immediately measured using a microplate reader. The results are shown in Figure 21.

[0142] Comparative Example 17: Detection of extracellular vesicles from colon cancer cell lines using Tim4 polypeptide. Purified extracellular vesicle samples from SW837, SW480, HTC-116, and Caco-2 cells, diluted 25-fold, were added to each well of the Tim4-coated plate. After overnight incubation at 4°C and washing five times with washing buffer, an antibody cocktail consisting of HRP-labeled anti-CD9 antibody, HRP-labeled anti-CD63 antibody, and HRP-labeled anti-CD81 antibody, each diluted 500-fold with diluent, was added to each well and incubated at room temperature. After 2 hours, the plate was washed five times with PBST, and TMB substrate was added. Upon color development, stop solution was added and the absorbance at 450 nm was immediately measured using a microplate reader. The results are shown in Figure 22.

[0143] The results of Example 9 and Comparative Example 17 above demonstrate that purified extracellular vesicles derived from SW837, SW480, HTC-116, and Caco-2 cells can be captured with VAR2CSA polypeptide and Tim4 and detected with a cocktail of anti-CD9, anti-CD63, and anti-CD81 antibodies. These results suggest that the target of VAR2CSA polypeptide is particularly abundant in extracellular vesicles derived from SW480 cells. Furthermore, the colon cancer extracellular vesicle marker CD147 is not expressed in low-grade Caco-2 cells and cannot be detected in an assay system using anti-CD9 and anti-CD147 antibodies ( WO 2014 / 167969 ), but can be detected in a system capturing extracellular vesicles with VAR2 polypeptide.

[0144] Example 10: Detection of extracellular vesicles derived from EpCAM-negative pancreatic cancer cells using VAR2CSA polypeptide. Many epithelial cancers are EpCAM-positive, and EpCAM is often used as a marker for cancer-derived extracellular vesicles. However, many cancers are EpCAM-negative, and the inability to detect EpCAM-negative cancers with anti-EpCAM antibodies poses a problem.

[0145] We therefore investigated whether VAR2CSA polypeptide could capture extracellular vesicles from EpCAM-negative cell lines. Extracellular vesicle samples prepared from the culture supernatants of EpCAM-positive KMP2 and PANC-1 cells and EpCAM-negative MIA Paca-2 cells were diluted 2.5-fold and added to each well of a VAR2CSA polypeptide-coated plate. After overnight incubation at 4°C and washing five times with PBST, an antibody cocktail consisting of HRP-labeled anti-CD9 antibody, HRP-labeled anti-CD63 antibody, and HRP-labeled anti-CD81 antibody, each diluted 500-fold with diluent, was added to each well and incubated at room temperature. After 2 hours, the plate was washed five times with PBST, TMB substrate was added, and upon color development, stop solution was added and the absorbance at 450 nm was immediately measured using a microplate reader. The results are shown in Figure 23.

[0146] Comparative Example 18: Detection of extracellular vesicles from EpCAM-negative pancreatic cancer cells using Tim4 polypeptide. Purified extracellular vesicle samples from KMP2, PANC-1, and MIA Paca-2 cells, diluted 25-fold, were added to each well of the Tim4-coated plate. After overnight incubation at 4°C and washing five times with washing buffer, an antibody cocktail consisting of HRP-labeled anti-CD9 antibody, HRP-labeled anti-CD63 antibody, and HRP-labeled anti-CD81 antibody, each diluted 500-fold with diluent, was added to each well and incubated at room temperature. After 2 hours, the plate was washed five times with PBST, and TMB substrate was added. Upon color development, stop solution was added and the absorbance at 450 nm was immediately measured using a microplate reader. The results are shown in Figure 24.

[0147] The results of Example 10 and Comparative Example 18 above show that purified extracellular vesicles derived from EpCAM-positive KMP2 cells and PANC-1 cells and EpCAM-negative MIA Paca-2 cells were captured by VAR2CSA polypeptide and Tim4 and detected with a cocktail of anti-CD9, anti-CD63, and anti-CD81 antibodies.

[0148] Example 11 Detection of extracellular vesicles derived from glioblastoma cell lines captured with anti-CD63 antibodies using VAR2CSA polypeptides Culture supernatant of glioblastoma cell line U87-MG cells was serially diluted two-fold with sample dilution buffer, and each diluted sample was added to an anti-CD63 antibody-immobilized plate (non-cross-reactive with bovine antibodies) (CD63-Capture Human Exosome ELISA Kit; Fujifilm).

[0149] After incubation at 4°C for 18 hours and washing with a washing buffer, a 5 µg / mL VAR2CSA polypeptide solution was added and the mixture was incubated at room temperature for 2 hours.

[0150] After washing, HRP-anti-FLAG antibody (Sigma-Aldrich), which recognizes the 3xFLAG-tag peptide attached to the VAR2CSA polypeptide, was added and incubated for an additional 2 hours at room temperature. After washing, TMB solution, a peroxidase chromogenic substrate, was added and allowed to react for 10 minutes. The reaction was stopped by adding a color-development stop solution, and the absorbance at 450 nm of each well was immediately measured. The absorbance values ​​shown in the figure were calculated by subtracting the value obtained when culture medium was added. The results are shown in Figure 25.

[0151] Comparative Example 19: Attempt to detect extracellular vesicles derived from a normal fetal lung fibroblast cell line captured with an anti-CD63 antibody using VAR2CSA polypeptide An attempt was made to detect extracellular vesicles using WI-38-hTERT cells, which are normal fetal lung fibroblasts, instead of the glioblastoma cell line used in Example 11. The results are shown in Figure 25.

[0152] The results of Example 11 and Comparative Example 19 above show that, after capture of extracellular vesicles with antibodies against extracellular vesicle markers, extracellular vesicles derived from cancer cells can be detected with VAR2CSA polypeptide, but extracellular vesicles derived from normal cells cannot be detected with VAR2CSA polypeptide (only background level signals were detected regardless of the amount of extracellular vesicles added).

[0153] Example 12: Detection of extracellular vesicles derived from lung cancer cell lines added to serum. 25 μL of purified extracellular vesicles derived from the lung cancer cell line NCI-H1703 cells and 25 μL of purified extracellular vesicles derived from the normal fetal lung fibroblast cell line WI-38-hTERT cells were added to 75 μL of extracellular vesicle-removed FBS and mixed. The resulting samples were added to a plate on which the VAR2SCA polypeptide had been immobilized.

[0154] After incubation at 4°C for 18 hours and washing with washing buffer, a mixed antibody solution of HRP-labeled anti-CD9 antibody, HRP-labeled anti-CD63 antibody, and HRP-labeled anti-CD81 antibody diluted with antibody diluent was added and incubated at room temperature for 2 hours.

[0155] After washing, a peroxidase chromogenic substrate, TMB solution, was added and allowed to react for 10 minutes. The reaction was stopped by adding a color-development stop solution, and the absorbance of each well at 450 nm was immediately measured. The absorbance values ​​were calculated by subtracting the blank value from each value. The results are shown in Figure 26.

[0156] The results of Example 12 above demonstrate that extracellular vesicles derived from cancer cells added to serum can be detected.

[0157] The above results are summarized in Table 4. 〇 in the table indicates capture, and x indicates no capture. This indicates that extracellular vesicles derived from cancer cells are captured by VAR2CSA, but extracellular vesicles derived from normal cells are not.

[0158]

[0159] Example 13: Comparison of extracellular vesicle capture rates between different methods of immobilizing VAR2CSA polypeptide to a carrier. The capture efficiency of extracellular vesicles by VAR2CSA polypeptide was compared between a method in which the N-terminus of VAR2CSA polypeptide was immobilized on a plate (Figure 2A) and a method in which the C-terminus of VAR2CSA polypeptide was immobilized on a plate (Figure 2B). To capture the N-terminus of VAR2CSA polypeptide, the VAR2CSA polypeptide was added to a streptavidin-coated plate. To capture the C-terminus of VAR2CSA polypeptide, the VAR2CSA polypeptide was added to a nickel-chelate-coated plate (see Section A).

[0160] Serially diluted purified extracellular vesicles from the lung cancer cell line NCI-H1703 were added to each plate. After incubation for 18 hours at 4°C and washing with wash buffer, a mixed antibody solution of HRP-anti-CD9, HRP-anti-CD63, and HRP-anti-CD81 antibodies diluted in antibody diluent was added and incubated for 2 hours at room temperature. After washing, a peroxidase chromogenic substrate, TMB solution, was added and incubated for 10 minutes. The reaction was stopped by adding stop solution, and the absorbance at 450 nm in each well was immediately measured. The blank value was subtracted from the absorbance values ​​shown in the figure. The results are shown in Figure 27. The values ​​in the figure are the ratio of the values ​​obtained on the nickel chelate-coated plate to the values ​​obtained on the streptavidin-coated plate. The results showed that the immobilization of the C-terminus of VAR2CSA on the nickel chelate-coated plate resulted in a 1.2- to 2.2-fold improvement in extracellular vesicle capture efficiency.

[0161] Reference Example 4: Preparation of VAR2CSA Polypeptides from Strains Other Than the FCR3 Strain A polynucleotide encoding a polypeptide containing the amino acid sequence of the ID1-ID2a domain of VAR2CSA from Plasmodium falciparum FCR3 strains (GenBank accession numbers EUR69480 (7G8), OK346630 (M0920), JQ247428 (3D7), and OK346631 (sometimes abbreviated as M.Campaign; MC) (SEQ ID NOs: 9, 12, 13, and 14, respectively) with a Twin-Strep tag (SEQ ID NO: 3) and a flexible linker (SEQ ID NO: 4) at the N-terminus and a 3xFLAG tag (SEQ ID NO: 5) and a 6xHis tag (HHHHHH) at the C-terminus was synthesized and optimized for E. coli codons. This polynucleotide was inserted between the NdeI and NotI sites of the E. coli expression vector pET21b. The vectors were designated pET21b-VAR2-7G8, pET21b-VAR2-M0920, pET21b-VAR2-3D7, and pET21b-VAR2-M.Camp, respectively. The amino acid sequences of the polypeptides expressed from these vectors are shown in SEQ ID NOs: 64, 65, 66, and 67. Table 5 also shows the calculated molecular weights of these polypeptides.

[0162]

[0163] Expression of the genes encoded by these vectors and purification of the expressed proteins were performed according to Reference Example 1. The purified VAR2CSA polypeptides from the five strains were analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) at 2 μg / lane. The results are shown in Figure 28. These results demonstrate that the five VAR2CSA polypeptides were highly pure.

[0164] Reference Example 5: Binding Analysis of Five VAR2CSA Polypeptides. Bovine decorin (Sigma; D8428) was immobilized on an ELISA plate at concentrations of 2 μg / mL and 100 μg / mL. The five VAR2CSA polypeptides were serially diluted 2-fold starting from 100 nM. These diluted samples were added to the bovine decorin-immobilized ELISA plate. After washing, the samples were detected with an antibody against the 3xFLAG tag in the VAR2CSA polypeptide (HRP-conjugated anti-FLAFM2 antibody, Sigma-Aldrich; A8592). The results are shown in Figure 29. The binding strength to bovine decorin was in the following order: 7G8 > FCR3 > 3D7 > M. Camp (MC) > M0920.

[0165] Next, we examined the binding of VAR2CSA polypeptides derived from various Plasmodium falciparum strains to EVs. Various VAR2CSA polypeptides were immobilized on streptavidin plates (Nunc; 236004) at a concentration of 100 nM. 100 μL of purified EVs derived from leukemia-derived KG-1 cells and WI-38-hTERT cells, a normal fetal lung fibroblast cell line, were added to the wells of the plate at a 4-fold dilution. After incubation at 4°C overnight, the plate was washed five times with washing buffer. An antibody cocktail consisting of HRP-labeled anti-CD9, HRP-labeled anti-CD63, and HRP-labeled anti-CD81 antibodies, each diluted 500-fold in diluent, was added to each well and incubated at room temperature. After 2 hours, the plate was washed five times with PBST. TMB substrate was added. Upon color development, stop solution was added and the absorbance at 450 nm was immediately measured using a microplate reader. The blank value was subtracted from each absorbance value. The results are shown in Figure 30.

[0166] The VAR2CSA polypeptides from both strains captured EVs derived from cancer cell lines but not from normal cell lines. Furthermore, the VAR2CSA polypeptide from the M0920 strain showed low binding affinity to cancer-derived EVs, reflecting its binding affinity to decorin.

[0167] Reference Example 6 Preparation of Extracellular Vesicles The cell lines, media, and additives additionally used in the present invention are shown in Table 6 for cancer cell lines and in Table 7 for normal cell lines (non-cancer cell lines). Extracellular vesicles were prepared from the culture supernatant in the same manner as in Reference Example 2.

[0168]

[0169]

[0170] Using these purified EV samples, we analyzed the binding of VAR2CSA polypeptide to EVs derived from various cell lines.

[0171] Example 14: Detection of extracellular vesicles derived from renal cancer cell lines using VAR2CSA polypeptide. Extracellular vesicles prepared from renal cancer cell lines Caki-1 and Caki-2 cells using the method described in Reference Example 6 were diluted and captured and detected using the method described in Section A. Specifically, purified EV samples were diluted 2.5-fold and added to each well of a VAR2CSA-immobilized plate. VAR2CSA-immobilized plates were prepared by adding VAR2CSA polypeptide to nickel chelate-coated plates. After overnight incubation at 4°C, the plates were washed five times with PBST. An antibody cocktail solution containing HRP-labeled anti-CD9 antibody, HRP-labeled anti-CD63 antibody, and HRP-labeled anti-CD81 antibody, each diluted 500-fold with diluent, was added to each well and incubated at room temperature. After 2 hours, the plates were washed five times with PBST, TMB substrate was added, and upon color development, a stop solution was added. The absorbance at 450 nm was immediately measured using a microplate reader. The absorbance values ​​in the figure were calculated by subtracting the blank value from each value. The results are shown in Figure 31.

[0172] Comparative Example 20: Attempt to detect extracellular vesicles derived from HREC-TKD cells, a normal renal epithelial cell line, using VAR2CSA polypeptide An attempt was made to detect extracellular vesicles using HREC-TKD cells, a normal renal epithelial cell line, instead of the renal cancer cell line used in Example 14. The results are shown in Figure 31.

[0173] Comparative Example 21: Detection of extracellular vesicles from renal cancer cell lines using Tim4 polypeptide. Furthermore, purified extracellular vesicle samples from Caki-1 and Caki-2 cells, diluted 25-fold, were added to each well of a Tim4-coated plate. After overnight incubation at 4°C and washing five times with washing buffer, an antibody cocktail solution containing HRP-labeled anti-CD9 antibody, HRP-labeled anti-CD63 antibody, and HRP-labeled anti-CD81 antibody, each diluted 500-fold with diluent, was added to each well and incubated at room temperature. After 2 hours, the plate was washed five times with washing buffer, and TMB substrate was added. Upon color development, a stop solution was added and the absorbance at 450 nm was immediately measured using a microplate reader. The blank value was subtracted from each absorbance value. The results are shown in Figure 32.

[0174] Comparative Example 22: Detection of extracellular vesicles derived from HREC-TKD cells, a normal renal epithelial cell line, using Tim4 polypeptide. Extracellular vesicles were detected using HREC-TKD cells, a normal renal epithelial cell line, instead of the renal cancer cell line used in Comparative Example 21. The results are shown in Figure 32.

[0175] The results of Example 14 and Comparative Examples 20 to 22 above show that purified extracellular vesicles derived from Caki-1 cells and Caki-2 cells are captured by VAR2CSA polypeptide and Tim4 and detected with a cocktail of anti-CD9, anti-CD63, and anti-CD81 antibodies, whereas purified extracellular vesicles derived from HREC-TKD are captured by Tim4 and detected with a cocktail of anti-CD9, anti-CD63, and anti-CD81 antibodies, but are not captured by VAR2CSA polypeptide.

[0176] Example 15: Detection of Extracellular Vesicles from Breast Cancer Cell Lines Using VAR2CSA Polypeptide. Extracellular vesicles prepared from breast cancer cell lines MDA-MB-231 cells (also known as MM231 cells), MDA-MB-453 cells (also known as MM453 cells), and T47D cells using the method described in Reference Example 6 were diluted and captured and detected using the method described in Section A. Specifically, purified EV samples were diluted 2.5-fold and added to each well of a VAR2CSA-immobilized plate. The VAR2CSA-immobilized plate was prepared by adding VAR2CSA polypeptide to a nickel-chelate-coated plate. After overnight incubation at 4°C and washing five times with PBST, an antibody cocktail solution containing HRP-labeled anti-CD9 antibody, HRP-labeled anti-CD63 antibody, and HRP-labeled anti-CD81 antibody, each diluted 500-fold with diluent, was added to each well and incubated at room temperature. After 2 hours, the plate was washed five times with PBST, and TMB substrate was added. Upon color development, a stop solution was added and the absorbance at 450 nm was immediately measured using a microplate reader. The blank value was subtracted from each absorbance value. The results are shown in Figure 33.

[0177] Comparative Example 23: Attempt to detect extracellular vesicles derived from a normal mammary epithelial cell line using VAR2CSA polypeptide An attempt was made to detect extracellular vesicles using HMEC-TKD cells, a normal mammary epithelial cell line, instead of the breast cancer cell line used in Example 15. The results are shown in Figure 33.

[0178] Comparative Example 24: Detection of extracellular vesicles from breast cancer cell lines using Tim4 polypeptide. Purified extracellular vesicle samples from MDA-MB-231, MDA-MB-453, and T47D cells, diluted 25-fold, were added to each well of the Tim4-coated plate. After overnight incubation at 4°C and washing five times with washing buffer, an antibody cocktail solution containing HRP-labeled anti-CD9 antibody, HRP-labeled anti-CD63 antibody, and HRP-labeled anti-CD81 antibody, each diluted 500-fold with diluent, was added to each well and incubated at room temperature. After 2 hours, the plate was washed five times with washing buffer, and TMB substrate was added. Upon color development, a stop solution was added and the absorbance at 450 nm was immediately measured using a microplate reader. The blank value was subtracted from each absorbance value. The results are shown in Figure 34.

[0179] Comparative Example 25: Detection of extracellular vesicles derived from a normal mammary epithelial cell line using Tim4 polypeptide. Extracellular vesicles were detected using HMEC-TKD cells, a normal mammary epithelial cell line, instead of the breast cancer cell line used in Comparative Example 24. The results are shown in Figure 34.

[0180] The results of Example 15 and Comparative Examples 23 to 25 above show that purified extracellular vesicles derived from MDA-MB-231 cells, MDA-MB-45 cells, and T47D cells were captured by VAR2CSA polypeptide and Tim4 and detected with a cocktail of anti-CD9, anti-CD63, and anti-CD81 antibodies, whereas purified extracellular vesicles derived from HMEC-TKD cells were captured by Tim4 and detected with a cocktail of anti-CD9, anti-CD63, and anti-CD81 antibodies, but were not captured by VAR2CSA polypeptide.

[0181] Example 16: Detection of Extracellular Vesicles Derived from Ovarian Cancer Cell Lines Using VAR2CSA Polypeptide. Extracellular vesicles prepared from ovarian cancer cell line OVCAR3 cells using the method described in Reference Example 6 were diluted and captured and detected using the method described in Section A. Specifically, purified EV samples were diluted 2.5-fold and added to each well of a VAR2CSA-immobilized plate. VAR2CSA-immobilized plates were prepared by adding VAR2CSA polypeptide to nickel chelate-coated plates. After overnight incubation at 4°C, the plates were washed five times with PBST. An antibody cocktail solution containing HRP-labeled anti-CD9 antibody, HRP-labeled anti-CD63 antibody, and HRP-labeled anti-CD81 antibody, each diluted 500-fold with diluent, was added to each well and incubated at room temperature. After 2 hours, the plates were washed five times with PBST, TMB substrate was added, and upon color development, a stop solution was added. The absorbance at 450 nm was immediately measured using a microplate reader. The blank value was subtracted from each absorbance value in the figure. The results are shown in Figure 35.

[0182] Example 17 Detection of extracellular vesicles derived from bladder cancer cell line using VAR2CSA polypeptide Extracellular vesicles were detected using the bladder cancer cell line T24 cells instead of the ovarian cancer cell line used in Example 16. The results are shown in Figure 35.

[0183] Comparative Example 26: Detection of extracellular vesicles from ovarian cancer cell lines using Tim4 polypeptide. A 25-fold diluted purified extracellular vesicle sample from OVCAR3 cells was added to each well of the Tim4-coated plate. After overnight incubation at 4°C and washing five times with washing buffer, an antibody cocktail solution containing HRP-labeled anti-CD9 antibody, HRP-labeled anti-CD63 antibody, and HRP-labeled anti-CD81 antibody, each diluted 500-fold with diluent, was added to each well and incubated at room temperature. After 2 hours, the plate was washed five times with PBST, and TMB substrate was added. Upon color development, stop solution was added and the absorbance at 450 nm was immediately measured using a microplate reader. The blank value was subtracted from each absorbance value. The results are shown in Figure 36.

[0184] Comparative Example 27: Detection of extracellular vesicles derived from bladder cancer cell line using Tim4 polypeptide Extracellular vesicles were detected using the bladder cancer cell line T24 cells instead of the ovarian cancer cell line used in Comparative Example 26. The results are shown in Figure 36.

[0185] The results of Examples 16 and 17 and Comparative Examples 26 and 27 above show that purified extracellular vesicles derived from the ovarian cancer cell line OVCAR3 cells and the bladder cancer cell line T24 cells were captured by VAR2CSA polypeptide and Tim4 and detected with a cocktail of anti-CD9, anti-CD63, and anti-CD81 antibodies.

[0186] Examples 18-20: Detection of extracellular vesicles derived from liver cancer cell lines, gallbladder cancer cell lines, and cholangiocarcinoma cell lines using VAR2CSA polypeptide. Extracellular vesicles prepared from the liver cancer cell line HepG2 cells (Example 18), the gallbladder cancer cell line TYGBK-8 cells (Example 19), and the cholangiocarcinoma cell lines HuCCT1 and TFK-1 cells (all of which are Example 20) using the method described in Reference Example 6 were diluted and captured and detected using the method described above in Section A. Specifically, purified EV samples were diluted 2.5-fold and added to each well of a VAR2CSA-immobilized plate. The VAR2CSA-immobilized plate was prepared by adding VAR2CSA polypeptide to a nickel chelate-coated plate. After overnight incubation at 4°C and washing five times with PBST, an antibody cocktail consisting of HRP-labeled anti-CD9 antibody, HRP-labeled anti-CD63 antibody, and HRP-labeled anti-CD81 antibody diluted 500-fold with diluent was added to each well and incubated at room temperature. After 2 hours, the wells were washed five times with PBST, TMB substrate was added, and upon color development, stop solution was added and the absorbance at 450 nm was immediately measured using a microplate reader. The blank value was subtracted from each absorbance value. The results are shown in Figure 37.

[0187] Comparative Examples 28-30: Detection of extracellular vesicles from hepatoma, gallbladder, and cholangiocarcinoma cell lines using Tim4 polypeptide. Furthermore, purified extracellular vesicle samples from HepG2 cells (Comparative Example 28), TYGBK-8 cells (Comparative Example 29), HuCCT1 cells, and TFK-1 cells (Comparative Example 30) diluted 10-fold were added to each well of the Tim4-coated plate. After overnight incubation at 4°C and washing five times with washing buffer, an antibody cocktail solution containing HRP-labeled anti-CD9 antibody, HRP-labeled anti-CD63 antibody, and HRP-labeled anti-CD81 antibody, each diluted 500-fold with diluent, was added to each well and incubated at room temperature. After 2 hours, the plate was washed five times with PBST, and TMB substrate was added. Upon color development, a stop solution was added and the absorbance at 450 nm was immediately measured using a microplate reader. The blank value was subtracted from each absorbance value. The results are shown in Figure 38.

[0188] The results of Examples 18 to 20 and Comparative Examples 28 to 30 above demonstrate that purified extracellular vesicles derived from the liver cancer cell line HepG2 cells, the gallbladder cancer cell line TYGBK-8 cells, the cholangiocarcinoma cell line HuCCT1 cells, and the TFK-1 cells were captured by VAR2CSA polypeptide and Tim4 and detected with a cocktail of anti-CD9, anti-CD63, and anti-CD81 antibodies.

[0189] Example 21: Detection of Extracellular Vesicles from Acute Lymphoblastic Leukemia Cell Lines Using VAR2CSA Polypeptide. Extracellular vesicles prepared from the acute lymphoblastic leukemia cell line NALM-6 cells using the method described in Reference Example 6 were diluted and captured and detected using the method described in Section A. NALM-6 cell EVs were prepared using samples (1) and (2) prepared in two independent experiments. Specifically, these purified EV samples were diluted 2.5-fold and added to each well of a VAR2CSA-immobilized plate. The VAR2CSA-immobilized plate was prepared by adding VAR2CSA polypeptide to a nickel-chelate-coated plate. After overnight incubation at 4°C and washing five times with PBST, an antibody cocktail solution containing HRP-labeled anti-CD9 antibody, HRP-labeled anti-CD63 antibody, and HRP-labeled anti-CD81 antibody, each diluted 500-fold with diluent, was added to each well and incubated at room temperature. After 2 hours, the plate was washed five times with PBST, and TMB substrate was added. Upon color development, a stop solution was added and the absorbance at 450 nm was immediately measured using a microplate reader. The blank value was subtracted from each absorbance value. The results are shown in Figure 39.

[0190] Example 22 Detection of extracellular vesicles derived from acute myeloid leukemia cell line using VAR2CSA polypeptide An attempt was made to detect extracellular vesicles using acute myeloid leukemia cell line KG-1 cells instead of the acute lymphocytic leukemia cell line used in Example 21. The results are shown in Figure 39.

[0191] Comparative Example 31: Detection of extracellular vesicles from acute lymphocytic leukemia cell lines using Tim4 polypeptide. A 25-fold diluted sample of purified extracellular vesicles from NALM-6 cells was added to each well of a Tim4-coated plate. After overnight incubation at 4°C and washing five times with washing buffer, an antibody cocktail solution containing HRP-labeled anti-CD9 antibody, HRP-labeled anti-CD63 antibody, and HRP-labeled anti-CD81 antibody, each diluted 500-fold with diluent, was added to each well and incubated at room temperature. After 2 hours, the plate was washed five times with PBST, and TMB substrate was added. Upon color development, stop solution was added and the absorbance at 450 nm was immediately measured using a microplate reader. The blank value was subtracted from each absorbance value. The results are shown in Figure 40.

[0192] Comparative Example 32 Detection of extracellular vesicles derived from acute myeloid leukemia cell line using Tim4 polypeptide An attempt was made to detect extracellular vesicles using KG-1 cells instead of the NALM-6 cells used in Comparative Example 30. The results are shown in Figure 40.

[0193] The results of Examples 21 and 22 and Comparative Examples 31 and 32 above show that purified extracellular vesicles derived from NALM-6 cells and KG-1 cells are captured by VAR2CSA polypeptide and Tim4 and detected with a cocktail of anti-CD9, anti-CD63, and anti-CD81 antibodies.

[0194] Comparative Example 33: Attempt to detect extracellular vesicles derived from a normal tracheal smooth muscle cell line using VAR2CSA polypeptide. Extracellular vesicles prepared from the tracheal smooth muscle cell line HBSMC-TKD cells using the method described in Reference Example 6 were diluted and captured and detected using the method described in Section A. Specifically, purified EV samples were diluted 2.5-fold and added to each well of a VAR2CSA-immobilized plate. VAR2CSA-immobilized plates were prepared by adding VAR2CSA polypeptide to a nickel chelate-coated plate. After overnight incubation at 4°C, the plates were washed five times with PBST. An antibody cocktail solution containing HRP-labeled anti-CD9 antibody, HRP-labeled anti-CD63 antibody, and HRP-labeled anti-CD81 antibody, each diluted 500-fold with diluent, was added to each well and incubated at room temperature. After 2 hours, the plates were washed five times with PBST, TMB substrate was added, and upon color development, a stop solution was added and the absorbance at 450 nm was immediately measured using a microplate reader. The absorbance values ​​in the figure were calculated by subtracting the blank value from each value. The results are shown in Figure 41.

[0195] Comparative Example 34: Attempt to detect extracellular vesicles derived from a normal aortic endothelial cell line using VAR2CSA polypeptide An attempt was made to detect extracellular vesicles using the aortic endothelial cell line HAEC-TKD cells instead of the tracheal smooth muscle cell line HBSMC-TKD cells used in Comparative Example 33. The results are shown in Figure 41.

[0196] Example 23 Detection of extracellular vesicles derived from acute myeloid leukemia cell line using VAR2CSA polypeptide Extracellular vesicles were detected using acute myeloid leukemia cell line KG-1 cells instead of the tracheal smooth muscle cell line HBSMC-TKD cells used in Comparative Example 32. The results are shown in Figure 41.

[0197] Comparative Example 35: Detection of extracellular vesicles from a normal tracheal smooth muscle cell line using Tim4 polypeptide. A 10-fold diluted sample of purified extracellular vesicles from HBSMC-TKD cells was added to each well of a Tim4-coated plate. After overnight incubation at 4°C and washing five times with washing buffer, an antibody cocktail consisting of HRP-labeled anti-CD9 antibody, HRP-labeled anti-CD63 antibody, and HRP-labeled anti-CD81 antibody, each diluted 500-fold with diluent, was added to each well and incubated at room temperature. After 2 hours, the plate was washed five times with PBST, and TMB substrate was added. Upon color development, a stop solution was added and the absorbance at 450 nm was immediately measured using a microplate reader. The blank value was subtracted from each absorbance value. The results are shown in Figure 42.

[0198] Comparative Example 36 Detection of extracellular vesicles derived from a normal aortic endothelial cell line using Tim4 polypeptide Extracellular vesicles were detected using HAEC-TKD cells instead of the HBSMC-TKD cells used in Comparative Example 34. The results are shown in Figure 42.

[0199] The results of Example 23 and Comparative Examples 33 to 36 above show that purified extracellular vesicles derived from normal cells, HBSMC-TKD cells and HAEC-TKD cells, are not captured by VAR2CSA polypeptide, but are captured by Tim4 and detected with a cocktail of anti-CD9 antibody, anti-CD63 antibody, and anti-CD81 antibody.

[0200] Furthermore, the results of Examples 14 to 23 and Comparative Examples 20 to 36 are summarized in Table 8. In the table, ◯ indicates that the substance was captured, and × indicates that the substance was not captured. The results showed that extracellular vesicles derived from renal cancer cells (Caki-1 cells, Caki-2 cells), breast cancer cells (MDA-MB-231 cells, MDA-MB-453 cells, T47D cells), ovarian cancer cells (OVCAR3 cells), bladder cancer cells (T24 cells), liver cancer cells (HepG2 cells), gallbladder cancer cells (TYGBK-8 cells), cholangiocarcinoma cells (HuCCT1 cells, TFK-1 cells), acute lymphoblastic leukemia cell line (NALM-6 cells), and acute myeloid leukemia cell line (KG-1 cells) were captured by the antibody, but not those derived from normal renal epithelial cells (HREC-TKD cells), breast epithelial cells (HMEC-TDK cells), tracheal smooth muscle cells (HBSMC-TKD cells), or aortic endothelial cells (HAEC-TKD cells).

[0201]

[0202] Example 24: Detection of extracellular vesicles derived from acute myeloid leukemia cell line KG-1 cells added to healthy human plasma using VAR2CSA polypeptide. EVs were purified from 25 μL of EVs purified from KG-1 cell culture supernatant added to 1 mL of healthy human plasma using the MagCapture™ Exosome Isolation Kit PS Ver. 2 (Fujifilm). The procedure is shown in Figure 43. The VAR2 polypeptide was immobilized on a plate by adding VAR2 polypeptide derived from the 7G8 strain to a nickel chelate-coated plate (Thermo Scientific).

[0203] 90 μL of EVs purified from spiked-in plasma was added to a 7G8 strain VAR2 polypeptide-coated plate. The sample volume was adjusted to 100 μL by adding buffer. After incubation for 18 hours at 4°C and washing with wash buffer, a mixed antibody solution of HRP-anti-CD9, HRP-anti-CD63, and HRP-anti-CD81 antibodies diluted with antibody diluent was added and incubated for 2 hours at room temperature. After washing, a peroxidase chromogenic substrate, TMB solution, was added and incubated for 10 minutes. The reaction was stopped by adding color-development stop solution, and the absorbance at 450 nm of each well was immediately measured. The results are shown in Figure 44. Cancer-derived EVs spiked into healthy donor plasma were captured by VAR2CSA in the same way as 25 μL of purified EVs derived from KG-1 cells used for spike-in.

[0204] Comparative Example 37: Attempt to detect extracellular vesicles derived from healthy human plasma using VAR2CSA polypeptide. Instead of the plasma spiked with EVs in Comparative Example 35, an attempt was made to detect extracellular vesicles using 90 μL of EVs purified from plasma that had not been spiked with EVs. The results are shown in Figure 44.

[0205] Comparative Example 38: Detection of extracellular vesicles derived from acute myeloid leukemia cell line KG-1 cells spiked into healthy human plasma using Tim4 polypeptide. Next, we confirmed whether these extracellular vesicles could be captured by Tim4. Briefly, 10 μL of prepared EVs were diluted to 100 μL with buffer and added to a Tim4 polypeptide-coated plate. At the same time, 10 μL of purified EVs derived from KG-1 cells used for spike-in were also diluted to 100 μL with buffer and added to a Tim4 polypeptide-coated plate. After overnight incubation at 4°C and washing five times with washing buffer, an antibody cocktail solution containing HRP-labeled anti-CD9 antibody, HRP-labeled anti-CD63 antibody, and HRP-labeled anti-CD81 antibody, each diluted 500-fold with diluent, was added to each well and incubated at room temperature. After 2 hours, the wells were washed five times with PBST, TMB substrate was added, and upon color development, stop solution was added and the absorbance at 450 nm was immediately measured using a microplate reader. The results are shown in Figure 45. Tim4 was able to capture extracellular vesicles from all samples.

[0206] Comparative Example 39: Detection of extracellular vesicles derived from healthy human plasma using Tim4 polypeptide. Instead of the plasma spiked with EVs in Comparative Example 37, extracellular vesicles were detected using plasma without EVs spiked into it. The results are shown in Figure 45.

[0207] Example 25: Detection of extracellular vesicles in the plasma of colorectal cancer patients using VAR2CSA polypeptide. We investigated whether cancer cell-derived extracellular vesicles in the plasma of colorectal cancer patients could be captured by VAR2CSA polypeptide. VAR2 polypeptide was immobilized on a plate by adding VAR2 polypeptide derived from the 7G8 strain to a nickel chelate-coated plate (Thermo Scientific).

[0208] EVs were purified using 1 mL each of colon cancer patient plasma (stage IV, purchased from National BioServices) and healthy donor plasma (purchased from National BioServices) using the MagCapture™ Exosome Isolation Kit PS Ver. 2 (Fujifilm). 90 μL of the purified EVs were diluted to 100 μL with buffer and added to a 7G8 VAR2 polypeptide-coated plate. As controls, 100 μL of buffer, EVs purified from leukemia cell line KG-1 cell culture supernatant, and EVs purified from colon cancer cell line HTC-116 cell culture supernatant diluted 2.5-fold with buffer were added to the 7G8 VAR2 polypeptide-coated plate. After incubation for 18 hours at 4°C and washing five times with washing buffer, an antibody cocktail consisting of HRP-labeled anti-CD9 antibody, HRP-labeled anti-CD63 antibody, and HRP-labeled anti-CD81 antibody, each diluted 500-fold with diluent, was added to each well and incubated at room temperature. After 2 hours, the wells were washed five times with PBST, and TMB substrate was added. Upon color development, stop solution was added and the absorbance at 450 nm was immediately measured using a microplate reader. The results are shown in Figure 46.

[0209] Comparative Example 40: Detection of extracellular vesicles in colorectal cancer patient plasma using Tim4 polypeptide. Next, 10 μL of each purified EV was added to a 100 μL sample buffer solution and added to a Tim4-coated plate. After incubation at 4°C for 18 hours and washing five times with washing solution, an antibody cocktail solution containing HRP-labeled anti-CD9 antibody, HRP-labeled anti-CD63 antibody, and HRP-labeled anti-CD81 antibody, each diluted 500-fold with diluent, was added to each well and incubated at room temperature. After 2 hours, the plate was washed five times with PBST, and TMB substrate was added. Upon color development, stop solution was added and the absorbance at 450 nm was immediately measured using a microplate reader. The results are shown in Figure 47.

[0210] According to Examples 24 and 25 and Comparative Examples 37 to 40, extracellular vesicles purified from the plasma of colorectal cancer patients were captured by VAR2, but extracellular vesicles from the plasma of healthy individuals were not captured by VAR2. On the other hand, all extracellular vesicles were captured by Tim4.

[0211] As shown in the above examples, the method of the present invention can be used to detect glioblastoma cells (U87-MG cells), pancreatic cancer cells (KMP2 cells, PANC-1 cells, MIA Paca-2 cells), prostate cancer cells (PC-3 cells, LNCap cells), lung cancer cells (NCI-H1703 cells, H69AR cells, A549 cells), osteosarcoma (MG-63 cells), esophageal cancer cells (KYSE150 cells, KYSE510 cells, KYSE1240 cells), gastric cancer cells (HGC-27 cells, AGS cells, MKN45 cells), colon cancer cell lines (SW837 cells, SW480 cells, HTC-116 cells, Caco-2 cells), and renal cancer cells (Caki-1 cells). It was demonstrated that the method of the present invention can capture and detect extracellular vesicles derived from cancer cells (e.g., Caki-2 cells), breast cancer cells (MDA-MB-231 cells, MDA-MB-453 cells, T47D cells), ovarian cancer cells (OVCAR3 cells), bladder cancer cells (T24 cells), liver cancer cells (hepG2 cells), gallbladder cancer cells (TYGBK-8 cells), cholangiocarcinoma cells (HuCCT1 cells, TFK-1 cells), acute myeloid leukemia cells (KG-1 cells), and acute lymphoblastic leukemia cells (NALM-6 cells). Furthermore, it was demonstrated that the method of the present invention can capture and detect extracellular vesicles derived from biological samples of patients with cancer. Thus, the method of the present invention can detect extracellular vesicles derived from a wide range of cancer cell types, including epithelial cancers, sarcomas, and hematologic cancers, and is therefore reasonably considered to be applicable to cancer detection regardless of the type of cancer.

[0212] The method for isolating cancer-derived extracellular vesicles of the present invention provides a method and kit for capturing and detecting cancer cell-derived extracellular vesicles in biological samples, and can be used for cancer diagnosis.

Claims

1. A method for capturing cancer cell-derived extracellular vesicles contained in a biological sample, comprising the step of contacting the biological sample with any one of the following polypeptides (A) to (C) to bind the polypeptide to cancer cell-derived extracellular vesicles: (A) a polypeptide comprising the ID1-ID2a domain of the VAR2CSA protein derived from Plasmodium falciparum; (B) a polypeptide that has 90% or more sequence identity with the amino acid sequence of the polypeptide (A) above and binds to cancer cell-derived extracellular vesicles; (C) a polypeptide that is a portion of the polypeptide (A) or (B) above and binds to cancer cell-derived extracellular vesicles.

2. The method according to claim 1, wherein the amino acid sequence of the ID1-ID2a domain of the VAR2CSA protein is set forth in SEQ ID NO:1, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:13, or SEQ ID NO:

14.

3. The method according to claim 1 or 2, wherein the polypeptides (A) to (C) are immobilized on a carrier.

4. The method according to claim 1 or 2, wherein the biological sample is human cell culture supernatant, blood, serum or plasma.

5. A method for detecting cancer cell-derived extracellular vesicles contained in a biological sample, comprising the steps of contacting the biological sample with any one of the following polypeptides (A) to (C) to bind the polypeptide to cancer cell-derived extracellular vesicles, and detecting the cancer cell-derived extracellular vesicles bound to any one of the polypeptides (A) to (C): (A) a polypeptide comprising the ID1-ID2a domain of the VAR2CSA protein derived from Plasmodium falciparum; (B) a polypeptide that has 90% or more sequence identity with the amino acid sequence of the polypeptide (A) above and binds to cancer cell-derived extracellular vesicles; and (C) a polypeptide that is a portion of the polypeptide (A) or (B) above and binds to cancer cell-derived extracellular vesicles.

6. The method of claim 5, further comprising contacting the cancer cell-derived extracellular vesicles with a second binding substance that binds to the surface of the extracellular vesicles, and detecting the cancer cell-derived extracellular vesicles bound to the second binding substance and any one of the polypeptides (A) to (C).

7. The method according to claim 6, wherein either (i) any one of the polypeptides (A) to (C) or (ii) the second binding substance is immobilized on a carrier, and the other is labeled, and the detection is carried out by detecting the label.

8. The method of claim 7, wherein the second binding substance is an antibody or an antigen-binding fragment thereof, or a phosphatidylserine-binding domain of Tim1, Tim3, Tim4 or beta-2-glycoprotein 1 (β2GP1), a protein that binds to phosphatidylserine located on the outside of the membrane of extracellular vesicles.

9. The method of claim 8, wherein the antibody is an antibody against CD9, CD63 or CD81.

10. The method of claim 6, wherein the biological sample is human cell culture supernatant, blood, serum or plasma.

11. The method according to any one of claims 5 to 10, wherein the amino acid sequence of the ID1-ID2a domain of the VAR2CSA protein is set forth in SEQ ID NO:1, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:13, or SEQ ID NO:

14.

12. A method for obtaining information on whether or not a subject is suffering from cancer, the method being carried out by detecting cancer cell-derived extracellular vesicles contained in a biological sample obtained from the subject using a method according to any one of claims 1 to 11.

13. A kit comprising any one of the following polypeptides (A) to (C) and a second binding substance that binds to the surface of extracellular vesicles, in which (i) any one of the polypeptides (A) to (C) and (ii) the second binding substance are immobilized on a carrier, and the other is labeled: (A) a polypeptide comprising the ID1-ID2a domain of the VAR2CSA protein derived from Plasmodium falciparum, (B) a polypeptide that has 90% or more sequence identity with the amino acid sequence of the polypeptide (A) above and binds to cancer cell-derived extracellular vesicles, or (C) a polypeptide that is a portion of the polypeptide (A) or (B) above and binds to cancer cell-derived extracellular vesicles.

14. The kit according to claim 13, wherein the kit is for diagnosing whether or not a subject is suffering from cancer.

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