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  • G01N33/575—Immunoassay; Biospecific binding assay; Materials therefor for cancer
  • G01N33/57525—Immunoassay; Biospecific binding assay; Materials therefor for cancer of the liver or pancreas
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  • G01N33/575—Immunoassay; Biospecific binding assay; Materials therefor for cancer
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  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
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  • G01N33/57545—Immunoassay; Biospecific binding assay; Materials therefor for cancer of the ovaries
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
  • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

• the present invention relates to a method for capturing extracellular vesicles derived from cancer cells, and a method and kit for detecting extracellular vesicles derived from cancer.
• Liquid biopsy body fluid diagnosis
• body fluid diagnosis is known as a minimally invasive technique for detecting cancer, which does not require the conventional biopsy of tumor tissue, but uses body fluids as samples and can detect cancer by measuring extracellular vesicles (EVs).
• EVs extracellular vesicles
• cancer-derived EVs will be difficult to detect because there are far fewer of them than EVs derived from organs and tissues in the body.
• Non-Patent Document 1 the membrane surface of a cell and the EVs produced from the cell were biotin-labeled, and the proteins were collected using avidin beads, after which the proteins were analyzed by LC-MS/MS, showing that the composition of membrane proteins of the cell and its EVs is significantly different (Non-Patent Document 2).
• 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).
• CD147 was found to be specifically expressed in EVs derived from colon cancer.
• bladder cancer a proteome analysis was performed on EVs expressed in EVs derived from the urine of bladder cancer patients, and CD55, CD82, etc. were discovered. It was shown that bladder cancer-derived EVs can be detected using antibodies targeting CD9, an EV marker, and CD55 and CD82 expressed in bladder cancer-derived EVs (Patent Document 2).
• Non-Patent Document 3 a paper on pancreatic cancer EVs performed proteomic analysis of EVs derived from the serum of pancreatic cancer patients, identifying GPRC5C and EPS8 as markers, and showing that the expression levels of these markers in cells and extracellular vesicles in various pancreatic cancer cell lines and non-cancerous pancreatic cells are not correlated. This suggests that cancer markers discovered from expression analysis in cells cannot simply be applied as cancer markers for EVs.
• Non-Patent Document 4 chondroitin sulfates derived from cancer cells are also called oncofetal chondroitin sulfate and placenta-like chondroitin sulfate.
• chondroitin sulfate is expressed on the surface of EVs.
• Non-Patent Document 5 a method for measuring these placenta-like chondroitin sulfates by ELISA has been presented (Non-Patent Document 5).
• these techniques detect soluble fetal chondroitin sulfate in cell lysates or body fluids, and do not measure EVs.
• the inventors conducted extensive research and developed a technology to capture and detect 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.
• the present invention provides the following.
• 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 the cancer cell-derived extracellular vesicles.
• (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 that binds to cancer cell-derived extracellular vesicles.
• 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).
• (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 that 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 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).
• 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 (A) to (C) or (ii) the second binding substance is immobilized on a carrier, and the other is labeled.
• kits (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 that binds to cancer cell-derived extracellular vesicles.
• the present invention makes it possible to capture and detect cancer-derived extracellular vesicles. It also provides a method and kit for diagnosing cancer based on the results of the detection of the cancer-derived extracellular vesicles.
• FIG. 1 shows the structure of a polypeptide containing the ID1-ID2a domain of the VAR2CSA protein used in the examples of the present invention.
• FIG. 1 is a schematic diagram illustrating an example of an analysis method of the present invention.
• Fig. 1 shows the results of detecting purified EVs derived from U87-MG cells captured by VAR2CSA with an anti-CD63 antibody.
• Fig. 1 shows the results of detecting purified EVs derived from U87-MG cells using an anti-CD63 antibody after capture with an anti-CD9 antibody.
• Fig. 1 shows the results of detecting purified EVs derived from WI-38-hTERT cells captured by VAR2CSA with an anti-CD63 antibody.
• FIG. 1 shows the results of detecting purified EVs derived from WI-38-hTERT cells using an anti-CD63 antibody after capture with an anti-CD9 antibody.
• This figure shows the results of detecting purified EVs derived from pancreatic cancer cell lines KMP2 and PANC-1 cells captured by VAR2CSA using a mixed antibody of anti-CD9 and anti-CD81 antibodies.
• This figure shows the results of detecting purified EVs derived from pancreatic cancer cell lines KMP2 and PANC-1 cells captured by Tim4 using a mixed antibody of anti-CD9 and anti-CD81 antibodies.
• This figure shows the results of detecting purified EVs derived from prostate cancer cell lines PC-3 and LNCap cells, normal prostate epithelial cell line HPrEC-TKD cells, and normal prostate fibroblast cell line HPrF-TKD 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 derived from prostate cancer cell lines PC-3 and LNCap cells, normal prostate epithelial cell line HPrEC-TKD cells, and normal prostate fibroblast cell line HPrF-TKD 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 derived from lung cancer cell lines H69AR3, NCI-H1703, and A549 cells, and normal fetal lung fibroblast cell line WI-38-hTERT cells captured by VAR2CSA using a mixture of three antibodies: anti-CD9, anti-CD63, and anti-CD81.
• This figure 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 mixture of three antibodies: anti-CD9, anti-CD63, and anti-CD81.
• This figure shows the results of detecting purified EVs derived from osteosarcoma cell line MG-63 cells and normal osteoblast cell line NHOst-TKD cells captured by VAR2CSA using a mixture of three types of antibodies: anti-CD9 antibody, anti-CD63 antibody, and anti-CD81 antibody.
• This figure shows the results of detecting purified EVs derived from osteosarcoma cell line MG-63 cells and normal osteoblast cell line NHOst-TKD cells captured by Tim4 using a mixture of three types of antibodies: anti-CD9 antibody, anti-CD63 antibody, and anti-CD81 antibody.
• This figure shows the results of detecting purified EVs derived from acute myeloid leukemia cell line KG-1 cells captured by VAR2CSA with an anti-CD63 antibody, and further shows that the capture of the extracellular vesicles by VAR2CSA is inhibited by chondroitin sulfate A (CSA).
• This figure shows the results of capturing purified EVs derived from acute myeloid leukemia cell line KG-1 cells with VAR2CSA or Tim4 and detecting them with an anti-CD63 antibody.
• This figure shows the results of detecting purified EVs from esophageal cancer cell lines KYSE150, KYSE510, and KYSE1240 captured by VAR2CSA using a mixture of three antibodies: anti-CD9, anti-CD63, and anti-CD81.
• This figure shows the results of detecting purified EVs from esophageal cancer cell lines KYSE150, KYSE510, and KYSE1240 captured by Tim4 using a mixture of three antibodies: anti-CD9, anti-CD63, and anti-CD81.
• This figure shows the results of detecting purified EVs from gastric cancer cell lines HGC-27 cells, AGS cells, and MKN45 cells captured by VAR2CSA using a mixture of three types of antibodies: anti-CD9 antibody, anti-CD63 antibody, and anti-CD81 antibody.
• This figure shows the results of detecting purified EVs from gastric cancer cell lines HGC-27 cells, AGS cells, and MKN45 cells captured by Tim4 using a mixture of three types of antibodies: anti-CD9 antibody, anti-CD63 antibody, and anti-CD81 antibody.
• This figure shows the results of detecting purified EVs from colon cancer cell lines SW837 cells, SW480 cells, HTC-116 cells, and Caco-2 cells captured by VAR2CSA using a mixture of three antibodies: anti-CD9 antibody, anti-CD63 antibody, and anti-CD81 antibody.
• This figure shows the results of detecting purified EVs from colon cancer cell lines SW837, SW480, HTC-116, and Caco-2 cells captured with Tim4 using a mixture of three antibodies: anti-CD9, anti-CD63, and anti-CD81.
• This figure 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 mixture of three antibodies: anti-CD9, anti-CD63, and anti-CD81.
• This figure 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 mixture of three antibodies: anti-CD9, anti-CD63, and anti-CD81.
• This figure shows the results of serially diluting culture supernatants of glioblastoma cell line U87-MG and normal fetal lung fibroblast cell line WI-38-hTERT cells, adding them to an anti-CD63 antibody-immobilized 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 types of antibodies: anti-CD9 antibody, anti-CD63 antibody, and anti-CD81 antibody.
• FIG. 1 shows a comparison of EV capture rates using different methods of immobilizing VAR2CSA polypeptide onto a carrier.
• the values in the figure are the ratios of the values on a nickel chelate-coated plate to the values on a streptavidin-coated plate.
• FIG. 1 shows the results of SDS polyacrylamide gel electrophoresis (SDS-PAGE) of purified VAR2CSA polypeptides from five strains.
• FIG. 1 shows the results of binding analysis (ELISA) of VAR2CSA polypeptides of five different strains to bovine decorin.
• This figure shows the results of binding analysis (ELISA) of VAR2CSA polypeptides of five different strains to EVs derived from the leukemia-derived cell line KG-1 cells and the normal fetal lung fibroblast cell line WI-38-hTERT cells.
• ELISA binding analysis
• 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 mixture of three antibodies: anti-CD9, anti-CD63, and anti-CD81.
• This figure shows the results of detecting purified EVs from renal cancer cell lines Caki-1 and Caki-2, and normal renal epithelial cell line HREC-TKD cells captured by Tim4 using a mixture of three antibodies: anti-CD9, anti-CD63, and anti-CD81.
• This figure shows the results of detecting purified EVs from breast cancer cell lines MDA-MB-231 cells, MDA-MB-45 cells, and T47D cells, and from normal mammary epithelial cell line HMEC-TKD cells captured by VAR2CSA using a mixture of three antibodies: anti-CD9 antibody, anti-CD63 antibody, and anti-CD81 antibody.
• This figure shows the results of detecting purified EVs from breast cancer cell lines MDA-MB-231, MDA-MB-45, and T47D cells, and from normal mammary epithelial cell line HMEC-TKD cells captured by Tim4 using a mixture 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 by VAR2CSA using a mixture of three types of antibodies: anti-CD9 antibody, anti-CD63 antibody, and anti-CD81 antibody.
• 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 mixture of three types of antibodies: anti-CD9 antibody, anti-CD63 antibody, and anti-CD81 antibody.
• 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 by VAR2CSA using a mixture of three antibodies: anti-CD9 antibody, anti-CD63 antibody, and anti-CD81 antibody.
• 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 Tim4 using a mixture of three antibodies: anti-CD9 antibody, anti-CD63 antibody, and anti-CD81 antibody.
• This figure shows the results of detecting purified EVs from the acute lymphoblastic leukemia cell line NALM-6 cells and the acute myeloid leukemia cell line KG-1 cells captured by VAR2CSA using a mixture of three antibodies: anti-CD9 antibody, anti-CD63 antibody, and anti-CD81 antibody.
• This figure shows the results of detecting purified EVs from the acute lymphoblastic leukemia cell line NALM-6 cells and the acute myeloid leukemia cell line KG-1 cells captured with Tim4 using a mixture of three antibodies: anti-CD9 antibody, anti-CD63 antibody, and anti-CD81 antibody.
• This figure 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 antibody, anti-CD63 antibody, and anti-CD81 antibody.
• FIG. 1 shows a procedure for purifying EVs from plasma of healthy humans to which extracellular vesicles derived from acute myeloid leukemia cell line KG-1 cells have been added.
• This figure shows the results of detecting purified EVs from healthy human plasma supplemented with extracellular vesicles derived from the acute myeloid leukemia cell line KG-1 cells, captured by VAR2CSA, using a mixture of three antibodies: anti-CD9 antibody, anti-CD63 antibody, and anti-CD81 antibody.
• This figure shows the results of detecting purified EVs from plasma of healthy humans to which extracellular vesicles derived from the acute myeloid leukemia cell line KG-1 cells had been added, captured by Tim4, using a mixture of three antibodies: anti-CD9, anti-CD63, and anti-CD81.
• the figure shows the results of detecting purified EVs from colorectal cancer patient plasma and healthy donor plasma captured by VAR2CSA, as well as purified EVs from buffer solution, leukemia cell line KG-1 cells, and colorectal cancer cell line HTC-116 cells as controls, using a mixture of three antibodies: anti-CD9 antibody, anti-CD63 antibody, and anti-CD81 antibody.
• This figure shows the results of detecting purified EVs from the plasma of colorectal cancer patients and healthy donors captured by Tim4, as well as 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.
• the present invention relates to a method for capturing cancer cell-derived extracellular vesicles contained in a biological sample, the method comprising the steps of: collecting the biological sample from a biological sample; and collecting the biological sample from the biological sample; and collecting the collected biological sample from the biological sample.
• the method comprises the steps of: collecting the biological sample from a biological sample; and collecting the collected biological sample from the biological sample; and collecting the collected biological sample from the biological sample.
• the method comprises the steps of: collecting the biological sample from the biological sample; collecting the collected ...
• Extracellular vesicles are defined as particles released from cells that are surrounded by a lipid bilayer membrane and do not have a nucleus. Based on differences in their production mechanisms, they are classified into exosomes, microvesicles, and apoptotic bodies.
• Exosomes are small vesicles derived from the endosomal membrane that are formed during endocytosis and are approximately 30-200 nm in size.
• Microvesicles are vesicles that come in a wide range of sizes, from 100-1000 nm, and differ from exosomes in that they bud off directly from the cell membrane and are secreted outside the cell.
• Apoptotic bodies are particles 50-5000 nm in size that bud off from the membrane of cells that have undergone apoptosis, similar to microvesicles.
• Extracellular vesicles may also contain proteins, RNA (mRNA, miRNA, non-coding RNA), DNA fragments, etc.
• Plasmodium falciparum VAR2CSA polypeptide In the method of the present invention, a Plasmodium falciparum VAR2CSA polypeptide or a polypeptide derived therefrom is used.
• the Plasmodium falciparum VAR2CSA polypeptide will be described below.
• PFEMP1 Plasmodium falciparum-multiple erythrocyte membrane protein 1
• PFEMP1 in malaria parasites is encoded by approximately 60 different var genes, and it has been shown that the var2csa gene among them is responsible for binding to placental chondroitin sulfate proteoglycans (Baruch, D. I. et al., Cell, 1995; 82, 77-87; Kraemer, S. M. & Smith, J. D., Curr. Opin. Microbiol. 2006; 9, 374-380).
• 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).
• Plasmodium falciparum There are several strains of Plasmodium falciparum, specifically the FCR3, K1, 3D7, Dd2, T9/94, Tak9/96, MAD20, K39, Honduras-1, HB3, 7G8, FCB, V1/S, M0920 and M.Camp strains.
• the FCR3, K1, 3D7, Dd2, T9/94, Tak9/96, MAD20, K39, Honduras-1, HB3, 7G8, FCB and V1/S strains are stored and available at the Biological Resources Room of the Institute of Tropical Medicine, Nagasaki University.
• 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.
• the homology between the VAR2CSA polypeptides of isolates isolated from patients infected with Plasmodium falciparum malaria parasite and Plasmodium falciparum strains is 70-80% on average, but all strains can infect humans, causing symptoms such as placental dysfunction, miscarriage, and stillbirth. Considering the above-mentioned mechanism of infection, it is considered that all VAR2CSA polypeptides of each strain have binding properties with chondroitin sulfate A.
• the VAR2CSA polypeptide of the present invention is preferably a VAR2CSA polypeptide of an isolate isolated from a patient infected with Plasmodium falciparum malaria parasite.
• the VAR2CSA polypeptide has the original amino acid sequence between Plasmodium falciparum malaria parasite strains and has the extracellular vesicle capturing ability shown in the examples of the present invention as long as no mutation is introduced into any amino acid.
• VAR2CSA is a 350 kD protein consisting of six Duffy-Binding Like (DBL) domains and several inter-domain regions (ID).
• DBL Duffy-Binding Like
• ID2a and ID2b are also called Cysteine-rich Inter-Domain Regions (CIDR).
• the VAR2CSA polypeptide may preferably be an ID1-ID2a polypeptide fragment derived from Plasmodium falciparum FCR3, 7G8, M0920, 3D7, or M.Camp strain.
• the amino acid sequences of the polypeptides are shown in SEQ ID NO:1, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14, respectively, in the sequence listing.
• the polynucleotide sequence encoding the polypeptide of SEQ ID NO:1 is shown in SEQ ID NO:2, and the polynucleotide sequence encoding the polypeptide of SEQ ID NO:9 is shown in SEQ ID NO:10.
• GenBank under accession numbers GU249598, EUR69480, OK346630, JQ247428, and OK346631, respectively.
• 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; or (C) A polypeptide that is a portion of the polypeptide (A) or (B) above and that binds to cancer cell-derived extracellular vesicles is used.
• the full-length VAR2CSA protein can be used, but since the part 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 consisting of the ID1-ID2a domain can be used. Furthermore, a polypeptide containing 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.
• Polypeptides that have a sequence identity of 90% or more, preferably 95% or more, more preferably 97% or more, and even more preferably 99% or more with the amino acid sequence of the above polypeptide (A) and bind to cancer cell-derived extracellular vesicles can also be used in the method of the present invention.
• 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 as large as possible (inserting gaps if necessary), and dividing the number of identical amino acid residues by the number of amino acid residues in the full length (the number of amino acid residues in the shorter one if the two amino acid sequences have different lengths).
• Sequence identity can be easily calculated by a person skilled in the art using FASTA, which is available free of charge on the Internet. Note that in the present invention, sequence identity is not defined when the number of amino acid residues in the full length is less than 20.
• the polypeptide of the present invention containing the ID1-ID2aA domain of the VAR2CSA protein derived from Plasmodium falciparum is preferably a polypeptide containing the ID1-ID2a domain of the VAR2CSA protein derived from a Plasmodium falciparum isolate isolated from a patient infected with Plasmodium falciparum. As described above, this is because the amino acid sequence is considered to have binding properties with chondroitin sulfate A.
• FCR3 SEQ ID NO: 1
• 7G8 SEQ ID NO: 9
• M0920 SEQ ID NO: 12
• 3D7 SEQ ID NO: 13
• M. Camp SEQ ID NO: 14
• FCR3 SEQ ID NO: 1
• polypeptide (A) or (B) above 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.
• binding in the "polypeptide that binds to cancer cell-derived extracellular vesicles” of the present invention may be any binding that allows the detection of cancer cell-derived extracellular vesicles by the detection method of the present invention.
• VAR2CSA polypeptide any of the above polypeptides (A) to (C) used in the method of the present invention (hereinafter, unless otherwise specified and unless otherwise clear from the context, referred to as "VAR2CSA polypeptide") may be fused to the N-terminus and/or C-terminus of the polypeptide for the purpose of purifying or detecting the polypeptide.
• Preferred examples of the above tag peptides 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, Avi-Tag, etc.
• Polypeptides consisting of the domain of VAR2CSA that binds to chondroitin sulfate proteoglycans can be produced in, but are not limited to, E. coli and insect cells, and can also be produced in animal cells such as 293 cells.
• the polypeptides can also be expressed in yeast, filamentous fungi, and other hosts used for expressing recombinant proteins.
• the polypeptides can also be obtained by a cell-free protein synthesis system.
• 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 that are preferentially used in the host species, E. coli.
• Expression vectors for expressing the VAR2CSA polypeptide include the pET expression vector (Merck Millipore) with a T7 promoter, the pGEX expression vector (Cytiva) and the pLEAD expression vector (Nippon Gene) with a tac promoter, and the pCold expression vector (Takara Bio) with the promoter of the E. coli cold shock gene cspA.
• the pET expression vector is preferred.
• pET-21b a type of pET expression vector, was used.
• the host E. coli is transformed with a vector into which DNA encoding the VAR2CSA polypeptide used in the present invention is cloned, and after cultivation, an appropriate inducer is added to express the protein that is the cloned gene product.
• an appropriate inducer is added to express the protein that is the cloned gene product.
• hosts include BL21 (DE3), Tuner (trade name) (DE3), Rosetta (trade name) (DE3), Origami (trade name) (DE3), and SHuffle T7 Express, and the inducer is preferably IPTG.
• VAR2CSA polypeptide can be used by immobilizing it on a carrier.
• a known method can be used to immobilize the VAR2CSA polypeptide on a carrier.
• Polymer beads, magnetic beads, and wells of a microplate, which are widely used in the field of immunoassays, can be used as the carrier.
• the VAR2CSA polypeptide can be passively bound to the polystyrene surface by adding VAR2CSA polypeptide diluted with an appropriate buffer to the wells of a polystyrene plate made for enzyme-linked immunosorbent assay (ELISA).
• ELISA enzyme-linked immunosorbent assay
• streptavidin can be bound to the surface of the carrier, and the VAR2CSA polypeptide can be immobilized (solid-phased) on the carrier surface by binding between the streptavidin on the carrier surface and the Twin-Strep-tag located on the VAR2CSA polypeptide.
• plate-shaped carriers to which streptavidin is bound include IMMOBILIZER STREPTAVIDIN (Thermo Scientific Nunc, 436022), Streptavidin plate C8 transparent (Nunc, 236004), and Strep-Tactin (product name) XT coated 96-well plate (IBS).
• the VAR2CSA polypeptide can also be immobilized on the support surface by binding a nickel chelate to the support surface and forming a bond between the nickel chelate on the support surface and a 6X His-Tag peptide arranged on the VAR2CSA polypeptide.
• IMMOBILIZER NICKEL-CHELATE Thermo Scientific Nunc, 436024
• Pierce product name Nickel Coated Plates (Thermo Scientific) can be used as plate-shaped supports with nickel chelate bound thereto.
• VAR2CSA polypeptide is diluted with PBS to 5-20 ⁇ g/mL, added to an ELISA plate, and incubated for 1 to 24 hours. After washing with a washing solution such as PBST, a blocking agent for ELISA plates is added and incubated at room temperature for 1 hour to block and immobilize the VAR2CSA polypeptide.
• a washing solution such as PBST
• a blocking agent for ELISA plates is added and incubated at room temperature for 1 hour to block and immobilize the VAR2CSA polypeptide.
• commercially available blocking agents such as Blocking-one (Nacalai Tesque), Blocker (product name) BLOTTO in TBS (Thermo Fisher Scientific), and ELISA Coating Buffer 1X (Abcam) can be used.
• PBS containing 0.1% Tween 20 PBST, Thermo Fisher Scientific
• Magnetic beads can be used as a carrier for immobilizing VAR2CSA polypeptide.
• VAR2CSA polypeptide can be immobilized directly onto magnetic beads, and suitable beads include Dynabeads (product name) M-270 Epoxy (Thermo), Dynabeads M-280 Tosylactivated (Thermo), and Dynabeads M-270 Carboxylic Acid (Thermo).
• beads with streptavidin bound to the surface can be used.
• streptavidin-immobilized 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 "type3" XT Beads (IBA), Magnosphere (trade name) MS300/Streptavidin (MBL), and Strep-Tactin (trade name) AlphaLISA (trade name) Acceptor beads (Perkin Elmer).
• the Twin-Strep-tag peptide on the VAR2CSA polypeptide can be bound to the streptavidin on the surface of the beads.
• beads with nickel or cobalt chelates bound to their surfaces can be used.
• nickel chelate-immobilized magnetic beads include His60 Ni Magnetic Beads (Takara Bio), TALON (trade name) Magnetic Beads (Takara Bio), and HisPur (trade name) Ni-NTA Magnetic Beads (Thermo).
• Agarose or sepharose can be used as a carrier for immobilizing the VAR2CSA polypeptide.
• streptavidin immobilization carriers include streptavidin-agarose (Millipore), Streptavidin HP MultiTrap (Cytiva), Strep-Tactin (trade name), TACS Agarose (IBA), and Strep-Tactin Sepharose (IBA).
• agarose or sepharose with immobilized nickel or cobalt chelates can also be used.
• 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).
• either the N-terminus or the 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 arranging/inserting a tag peptide, such as the Twin-Strep-tag or 6xHis-Tag peptide, at either the N-terminus or the C-terminus of the VAR2CSA polypeptide depending on the purpose and detection sensitivity.
• VAR2CSA polypeptide Since the amount of EVs captured by the VAR2CSA polypeptide is increased 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.
• Biological samples applicable to the method of the present invention include cell line culture fluid and body fluids.
• Cell line culture fluid is the supernatant of cultured cells.
• Cancer cell lines are cell lines that have been cultured from cancer tissue and can be subcultured, while normal cell lines are cell lines that have been cultured from healthy tissue and can be subcultured, primary cultures of healthy tissue, and further cell lines that have been immortalized by introducing immortalization genes (hTERT, CDK4, CCND1, c-myc, etc.) into these cell lines, and are so-called non-cancer cell lines.
• immortalization genes hTERT, CDK4, CCND1, c-myc, etc.
• 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 fluid, bile, pancreatic juice, intestinal fluid, sweat, tears, nasal discharge, amniotic fluid, milk, aqueous humor, peritoneal fluid, serous cavity fluid, lymphatic fluid, 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).
• the biological sample may be derived from an animal that has developed or bears cancer, or from an animal that serves as a control for comparison therewith, and is preferably a specimen derived from a mammalian animal, 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.
• the method for capturing cancer cell-derived extracellular vesicles according to the present invention includes a 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.
• 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 set appropriately, but can be carried out, for example, by incubating in a buffer solution at 4°C to room temperature for 1 to 18 hours.
• the above-mentioned 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 inhibiting cancer metastasis and for extending the lives and improving the quality of life of cancer patients.
• 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 being configured so that the extracellular vesicles of 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.
• the method for detecting cancer cell-derived extracellular vesicles according to the present invention includes the steps of contacting a biological sample with a VAR2CSA polypeptide, binding the cancer cell-derived extracellular vesicles in the biological sample 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 perform quantification or semi-quantification, but since detection is inevitably performed when quantification or semi-quantification is performed, quantification and semi-quantification are also included in the scope of the detection method of the present invention.
• 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), so the method itself can be applied to well-known immunoassay methods. That is, it is possible to apply methods such as the sandwich method, the agglutination method, the competitive method, and the immunochromatography method. Of these, the sandwich method is preferable, and the sandwich method is also used in the following examples. In order to perform the sandwich method, another substance (second binding substance) that binds to cancer cell-derived extracellular vesicles is required.
• this second binding substance examples include antibodies or antigen-binding fragments 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 or aptamers against these proteins can be used as the second binding substance that binds 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.
• Tim1, Tim3, Tim4, or beta-2-glycoprotein 1 which are proteins that bind to phosphatidylserine located on the outside of the membrane of extracellular vesicles.
• either the VAR2CSA polypeptide or the second binding substance is immobilized on a carrier, 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.
• the second binding substance is immobilized on a carrier, it can also be immobilized by the well-known method described above, since the second binding substance is also a protein.
• 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 detecting the label bound to the carrier via the cancer cell-derived extracellular vesicles.
• the biological sample may first be contacted with a labeled second binding substance, then with a carrier on which a VAR2CSA polypeptide is immobilized, and after washing, the label bound to the carrier via the cancer cell-derived extracellular vesicles may be detected (reverse method).
• 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, washing the carrier, and detecting the label bound to the carrier via the cancer cell-derived extracellular vesicles.
• the biological sample can be first contacted with a labeled VAR2CSA polypeptide, then with a carrier on which a second binding substance is immobilized, and after washing the carrier, the label bound to the carrier via the cancer cell-derived extracellular vesicles can be detected in the biological sample (reverse method).
• 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 the cancer cell-derived extracellular vesicles may be detected.
• the biological sample, the carrier to which the second binding substance is immobilized, and the labeled VAR2CSA polypeptide may be contacted simultaneously, and after washing, the label bound to the carrier via the cancer cell-derived extracellular vesicles may be detected.
• label used in the field of immunoassays. 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, the method of binding the label to the protein or aptamer is also well-known, and can be easily performed using commercially available kits.
• enzyme labels e.g., HRP, ALP, etc.
• fluorescent labels e.g., FITC, PE, Alexa Fluor, etc.
• these labels are commercially available as kits, so commercially available kits can be used.
• the method of binding the label to the protein or aptamer is also well-known, and can be easily performed using commercially available kits.
• labels for Amplified Luminescent Proximity Homogeneous Assay can be used as labels.
• 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 excitation, and detects the signal emitted from the signal-generating label by the singlet oxygen generated from the excited label.
• either the VAR2CSA polypeptide or the second binding substance is immobilized on a donor bead, and the other is bound to an acceptor bead.
• the donor bead and the acceptor bead come close to each other via the binding between the VAR2CSA polypeptide and the 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.
• the detection method using Alpha is included in the detection method of the present invention, since the donor beads can be considered as the carrier and the acceptor beads as the label. This method is also well known, and kits for it are commercially available, so it can be easily performed using commercially available products.
• the conditions for each "contact” are not particularly limited and can be set appropriately, but 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 suitable for each label.
• 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 (product name) (GeneTex) can be used.
• TOYOBO Can Get Signal
• ChonBlock Detection Antibody Dilution ELISA Buffer ChonBlock Detection Antibody Dilution ELISA Buffer
• Universal Antibody Dilution Buffer product name
• VAR2CSA polypeptide bound to extracellular vesicles antibodies against VAR2CSA and tag sequences arranged on VAR2CSA polypeptide (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 bound to the C-terminus in the embodiment of the present invention) can be used.
• Twin-Strep-tag peptide is targeted, streptavidin that binds to Twin-Strep-tag peptide can be used, and streptavidin labeled with HRP or the like is preferred.
• anti-FLAG-tag antibody When 3 x FLAG-tag peptide is targeted, anti-FLAG-tag antibody can be used, and anti-FLAG-tag antibody labeled with HRP or the like is preferred. When 6 x His-tag peptide is targeted, anti-His-tag antibody can be used, and anti-His-tag antibody labeled with HRP is preferred.
• cancers from which extracellular vesicles are derived 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
• leukemias include acute myeloid leukemia and acute lymphatic leukemia.
• the above-mentioned detection method of the present invention 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 derived from a subject is significantly greater than the amount of VAR2CSA polypeptide-trapped extracellular vesicles in a biological sample derived from a healthy subject, it is also possible to determine that the subject has a high possibility of having cancer.
• data on the amount of VAR2CSA polypeptide-trapped extracellular vesicles in a biological sample from a healthy subject can be obtained in advance, a threshold can be set in advance, and if the amount of VAR2CSA polypeptide-trapped extracellular vesicles in a biological sample derived from a subject is greater than the threshold, the subject can be determined to have a high possibility of having cancer.
• a threshold can be set in advance, and if the amount of VAR2CSA polypeptide-trapped extracellular vesicles in a biological sample derived from a subject is greater than the threshold, the subject can be determined to have a high possibility of having cancer.
• the present invention also provides a kit for use in the above-mentioned detection method of the present invention.
• the kit of the present invention is a kit comprising 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.
• 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, in which 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.
• the kit of the present invention described above can also be used as a kit for capturing extracellular vesicles derived from cancer cells, and is a kit in which the VAR2CSA polypeptide is immobilized on a carrier.
• the VAR2CSA polypeptide is immobilized on a carrier.
• it can be used for functional analysis of the captured extracellular vesicles derived from cancer cells, or for analysis of molecules contained in the extracellular vesicles, in studies for identifying cancer types or for studies for elucidating the state of cancer.
• the kit of the present invention may include a buffer solution for diluting the biological sample before contacting it with the VAR2CSA polypeptide, etc., instructions for use, etc.
• VAR2CSA Polypeptide A polynucleotide encoding a polypeptide (SEQ ID NO: 6) having a Twin-Strep-tag (SEQ ID NO: 3) and a flexible linker (SEQ ID NO: 4) at the N-terminus of the amino acid sequence (SEQ ID NO: 1) of the ID1-ID2a domain of VAR2CSA of Plasmodium falciparum FCR3 strain of GenBank accession number GU249598 and a 3xFLAG-tag (SEQ ID NO: 5) at the C-terminus was synthesized by optimizing the codons of E. coli (SEQ ID NO: 7).
• This polynucleotide was inserted between NdeI and XhoI of E. coli expression vector pET21b.
• the 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 the polypeptide is shown in SEQ ID NO: 8.
• FIG. 1 A schematic diagram of the structure of this VAR2CSA polypeptide is shown in Figure 1.
• the pET21b-VAR2-FCR3 plasmid DNA was introduced into the E. coli strain SHuffle T7 Express Competent E. coli (NEB; C3029J) to obtain a transformant.
• a clone of this transformant was cultured in 10 mL of LB medium containing 100 ⁇ g/mL carbenicillin at 37°C for 18 hours with shaking. 6 mL of the above culture was inoculated into 150 mL of LB medium (containing 100 ⁇ g/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 cultured at 20°C for approximately 20 hours with shaking.
• the culture was centrifuged at 6,000xg for 15 minutes at 4°C, the E. coli pellet was collected and frozen and stored at -80°C.
• the frozen E. coli pellet was thawed at room temperature and 5 mL of B-PER (trade name) 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, and the pellet was suspended by pipetting. After gently stirring at room temperature for 15 minutes on a rotator, the pellet was centrifuged at 12,000xg for 15 minutes at 4°C, and the supernatant was transferred to a new tube.
• B-PER Bacterial Cell Lysis Reagent
• protease inhibitor cocktail Nacalai, 03969-21
• 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 in the present invention are cells 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, and after removing the culture medium, each cell was cultured in a medium containing 5% Exosome-Depleted FBS (ThermoFisher Scientific, A2720803) for 4 to 5 days.
• the culture supernatant of each cell was collected and centrifuged at 300 x g for 5 minutes to recover the supernatant. This supernatant was centrifuged again at 8,000 x g for 30 minutes. The centrifuged supernatant was filtered through a 0.22 ⁇ m syringe filter (Millipore). Next, 40 mL of this filtered culture supernatant was concentrated to 1 mL using a centrifugal concentration tube with a molecular weight cutoff of 100 kDa, VIVASPIN 20 (Sartorius; VS2041) or Amicon Ultra-15 (Millipore; UFC910008).
• Extracellular vesicles in the concentrated samples were purified using MagCapture (trade name) Exosome Isolation Kit PS Ver.2 (Fujifilm) according to the kit's attached manual. Briefly, 60 ⁇ L of Tim4-immobilized magnetic beads were added to 1 mL of concentrated sample to adsorb extracellular vesicles. After washing, the extracellular vesicles were eluted with 100 ⁇ L of elution solution. 1/100 volume of extracellular vesicle blocking reagent, EV-Save (product name) (Fujifilm), was added to this elution solution, which was then dispensed and stored at -80°C.
• MagCapture trade name
• Exosome Isolation Kit PS Ver.2 Flujifilm
• Antibodies that recognize CD9, CD63, and CD81 expressed on the surface of extracellular vesicles were used to detect extracellular vesicles. Specifically, anti-human CD9 rat monoclonal antibody (77B, Fujifilm), anti-human CD63 mouse monoclonal antibody (3-13, Fujifilm), and anti-human CD81 rat monoclonal antibody (9B, Fujifilm), which do not cross-react with bovine antibodies, were used.
• the Tim4-immobilized plate of the PS Capture Exosome ELISA Kit or the anti-CD9 antibody-immobilized plate of the CD9/CD63 ELISA Kit was washed three times with the washing buffer provided with each kit, and then the extracellular vesicle sample diluted with the reaction buffer provided with each kit was added to the wells of the plate, stirred for 30 seconds, and incubated overnight at 4°C. After washing five times with the washing buffer provided with each kit, the POD-labeled antibody diluted 500-fold with the reaction buffer provided with each kit was added, stirred for 30 seconds, and incubated at room temperature.
• the plate was washed five times with the washing buffer provided with each kit, and the color-developing substrate solution of the ELISA POD Substrate TMB Kit HYPER (Nacalai) was added, stirred for 30 seconds, and allowed to react for 5 to 10 minutes.
• the reaction was stopped by adding the reaction stop solution (1M sulfuric acid) and stirring for 10 seconds.
• 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.
• VAR2CSA polypeptide prepared in Reference Example 1 was diluted with PBS to 10 ⁇ g/mL. 100 ⁇ L of the VAR2CSA polypeptide solution at 10 ⁇ g/mL was added to each well of a streptavidin-coated plate (Nunc) and incubated overnight at 4° C. Alternatively, 100 ⁇ L of the VAR2CSA polypeptide solution at 10 ⁇ g/mL 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 200 ⁇ L of Blocking One (Nacalai) diluted 5-fold with DW was added to each well and incubated for 1 hour for blocking.
• the mixture was washed three times with PBST, and extracellular vesicles diluted with Blocking One diluted 20-fold in PBST were added. The mixture was stirred for 30 seconds and then incubated overnight at 4°C.
• Detection of extracellular vesicles captured by VAR2CSA polypeptide was performed as follows. After incubation, the plate was washed five times with PBST, and the antibody from Reference Example 3 diluted 500-fold with antibody diluent was added. After stirring for 30 seconds, the plate was incubated for 2 hours. The plate was washed five times with PBST, and the color-developing substrate solution from the ELISA POD Substrate TMB Kit HYPER (Nacalai) was added. The plate was stirred for 30 seconds and allowed to react for 5-10 minutes. A reaction stop solution (1M sulfuric acid) was added and the reaction was stopped by 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 explaining the above analysis method is shown in Figure 2.
• Example 1 Detection of extracellular vesicles derived from glioblastoma cell lines by VAR2CSA polypeptide
• Extracellular vesicles prepared from U87-MG cells, a glioblastoma cell line, by the method described in Reference Example 2 were serially diluted and captured and detected by the method described above in Section A. Specifically, the purified EVs sample was serially diluted and added to each well of the VAR2CSA solid-phase plate. After overnight incubation at 4°C, the wells were washed five times with PBST, and an antibody solution in which the HRP-labeled anti-CD63 antibody was diluted 500 times 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 when color developed, a reaction stop solution was added and the absorbance at 450 nm was immediately measured with a microplate reader. The results are shown in Figure 3.
• 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.
• Comparative Example 2 Attempt to detect extracellular vesicles derived from normal fibroblast cell line using VAR2CSA polypeptide
• Extracellular vesicles prepared from fetal lung fibroblast cell line WI-38-hTERT by the method described in Reference Example 2 were serially diluted and captured and detected by the method described above in Section A. Specifically, purified EVs samples were serially diluted and added to each well of the VAR2CSA solid-phase plate. After overnight incubation at 4°C, the wells were washed five times with PBST, and an antibody solution in which HRP-labeled anti-CD63 antibody was diluted 500 times 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 when color developed, a reaction stop solution was added and the absorbance at 450 nm was immediately measured with a microplate reader. The results are shown in Figure 5.
• Example 2 Detection of extracellular vesicles from pancreatic cancer cell lines by VAR2CSA polypeptide (1)
• the culture supernatant was collected from two cultures (Preparation 1 and Preparation 2), and the extracellular vesicles prepared by the method described in Reference Example 2 were diluted and captured and detected by the method described above in Section A. Specifically, the purified EVs sample was diluted 4-fold and added to each well of the VAR2CSA solid-phase plate.
• Comparative Example 4 Detection of extracellular vesicles derived from pancreatic cancer cell lines by Tim4 polypeptide.
• 40-fold diluted samples of purified extracellular vesicles derived from PANC-1 cells and KMP2 cells were added to each well of Tim4-coated plate. After overnight incubation at 4°C and washing five times with washing solution, an antibody cocktail solution of HRP-labeled anti-CD9 antibody and HRP-labeled anti-CD81 antibody, each diluted 500-fold with dilution solution, was added to each well and incubated at room temperature. After 2 hours, the wells were washed five times with washing solution, and TMB substrate was added. When color was developed, a reaction stop solution was added and the absorbance at 450 nm was immediately measured with a microplate reader. The results are shown in Figure 8.
• 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 are detected with a cocktail of anti-CD9 and anti-CD81 antibodies.
• Example 3 Detection of extracellular vesicles derived from prostate cancer cell lines using VAR2CSA polypeptide
• Extracellular vesicles prepared from prostate cancer cell lines PC-3 cells and LNCap cells by the method described in Reference Example 2 were diluted and captured and detected by the method described above in Section A.
• the purified EVs sample was diluted 4-fold and added to each well of the VAR2CSA solid-phase plate. After overnight incubation at 4°C and washing five times with PBST, an antibody cocktail solution in which HRP-labeled anti-CD9 antibody, HRP-labeled anti-CD63 antibody, and HRP-labeled anti-CD81 antibody were each diluted 500-fold with dilution solution 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 when color developed, a reaction stop solution was added and the absorbance at 450 nm was immediately measured with a microplate reader. The results are shown in Figure
• Comparative Example 5 Attempt to detect extracellular vesicles derived from 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 in Example 3. The results are shown in Figure 9.
• Comparative Example 7 Detection of extracellular vesicles derived from 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 in Comparative Example 6. The results are shown in Figure 10.
• Example 3 and Comparative Examples 5 to 7 above show that purified extracellular vesicles derived from PC-3 cells and LNCap 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 normal cells HPrEC-TKD and HPrF-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.
• Example 4 Detection of extracellular vesicles derived from lung cancer cell lines using VAR2CSA polypeptide
• Extracellular vesicles prepared from lung cancer cell lines A549 cells, NCI-H1703 cells, and N69AR cells by the method described in Reference Example 2 were diluted and captured and detected by the method described above in Section A. Specifically, the purified EVs sample was diluted 4-fold and added to each well of the VAR2CSA solid-phase plate. After overnight incubation at 4 ° C.
• Comparative Example 8 Attempt to detect extracellular vesicles derived from 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 in Example 4. The results are shown in Figure 11.
• Comparative Example 10 Detection of extracellular vesicles derived from normal lung fibroblast cell line using Tim4 polypeptide Extracellular vesicles were detected using normal cells, WI-38-hTERT cells, instead of the lung cancer cell line in Comparative Example 9. The results are shown in Figure 12.
• Example 4 and Comparative Examples 8 to 10 above show that purified extracellular vesicles derived from A549 cells, NCI-H1703 cells, and H69AR 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 normal cells WI-38-hTERT 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.
• Example 5 Detection of extracellular vesicles derived from osteosarcoma cell line using VAR2CSA polypeptide
• Extracellular vesicles prepared from MG-63 cells, an osteosarcoma cell line, by the method described in Reference Example 2 were diluted and captured and detected by the method described above in Section A. Specifically, the purified sample was diluted 4-fold and added to each well of the VAR2CSA solid-phase plate.
• Comparative Example 11 Attempt to detect extracellular vesicles derived from normal osteoblast cell line using VAR2CSA polypeptide An attempt was made to detect extracellular vesicles using normal osteoblast cells, NHOst-TKD cells, instead of the osteosarcoma cell line in Example 5. The results are shown in Figure 13.
• Example 5 and Comparative Examples 11 to 13 show that purified extracellular vesicles derived from MG-63 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 normal cells NHOst-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.
• Example 6 Detection of extracellular vesicles of acute myeloid leukemia cell line by VAR2CSA polypeptide and its inhibition by chondroitin sulfate
• CSA chondroitin sulfate
• the purified EVs sample was diluted 4 times and mixed with CSA to concentrations of 0.2 ⁇ g/mL, 1 ⁇ g/mL, 5 ⁇ g/mL, and 25 ⁇ g/mL, and added to each well of the VAR2CSA solid-phase plate.
• Comparative Example 14 Detection of extracellular vesicles of cell lines derived from acute myeloid leukemia cells using Tim4 polypeptide.
• extracellular vesicle samples prepared from KG-1 cells were diluted 40-fold and added to wells with Tim4 immobilization, incubated overnight at 4°C, washed five times with washing solution, and then HRP-labeled anti-CD63 antibody diluted 500-fold with dilution solution was added to the wells and incubated at room temperature. After 2 hours, the wells were washed five times with washing solution, and TMB substrate was added. When color was developed, a reaction stop solution was added and the absorbance at 450 nm was immediately measured using a microplate reader. Evaluation was also performed using a VAR2CSA immobilization plate as in Example 6. The results are shown in Figure 16.
• Example 6 and Comparative Example 14 above show that purified extracellular vesicles derived from KG-1 cells are captured by VAR2CSA polypeptide and Tim4 and detected with anti-CD63 antibody. Furthermore, the capture of extracellular vesicles by VAR2CSA polypeptide is inhibited by CSA, suggesting that VAR2CSA polypeptide binds to CSA of extracellular vesicles.
• Example 7 Detection of extracellular vesicles derived from esophageal cancer cell lines using VAR2CSA polypeptide
• Comparative Example 15 Detection of extracellular vesicles derived from esophageal cancer cell lines using Tim4 polypeptide.
• purified extracellular vesicle samples derived from KYSE150 cells, KYSE510 cells, 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 solution, an antibody cocktail solution of HRP-labeled anti-CD9 antibody, HRP-labeled anti-CD63 antibody, and HRP-labeled anti-CD81 antibody, each diluted 500-fold with dilution solution, 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 when color developed, a reaction stop solution was added and the absorbance at 450 nm was immediately measured with a microplate reader. The results are shown in Figure 18.
• Example 7 and Comparative Example 15 above show that purified extracellular vesicles derived from KYSE150 cells, KYSE510 cells, and KYSE1240 cells were captured by VAR2CSA polypeptide and Tim4, and detected with a cocktail of anti-CD9, anti-CD63, and anti-CD81 antibodies.
• VAR2CSA polypeptide was able to capture extracellular vesicles derived from them.
• Example 8 Detection of extracellular vesicles derived from gastric cancer cell lines using VAR2CSA polypeptide
• Extracellular vesicles prepared from gastric cancer cell lines HGC-27 cells, AGS cells, and MKN45 cells by the method described in Reference Example 2 were diluted and captured and detected by the method described above in Section A. Specifically, the purified EVs sample was diluted 2-fold and added to each well of the VAR2CSA solid-phase plate. After overnight incubation at 4 ° C.
• 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 are detected with a cocktail of anti-CD9, anti-CD63, and anti-CD81 antibodies.
• Extracellular vesicles prepared from colon cancer cell lines SW837, SW480, HTC-116, and Caco-2 cells by the method described in Reference Example 2 were diluted and captured and detected by the method described above in Section A. Specifically, the purified EVs sample was diluted 2.5 times and added to each well of the VAR2CSA solid-phase plate. After overnight incubation at 4°C, the wells were washed five times with PBST, and an antibody cocktail solution in which HRP-labeled anti-CD9 antibody, HRP-labeled anti-CD63 antibody, and HRP-labeled anti-CD81 antibody were each diluted 500 times with dilution solution was added to each well and incubated at room temperature.
• Comparative Example 17 Detection of extracellular vesicles derived from colon cancer cell lines by Tim4 polypeptide.
• 25-fold diluted samples of purified extracellular vesicles derived from SW837 cells, SW480 cells, HTC-116 cells, and Caco-2 cells were added to each well of Tim4 immobilization. After overnight incubation at 4°C and washing five times with washing solution, an antibody cocktail solution of HRP-labeled anti-CD9 antibody, HRP-labeled anti-CD63 antibody, and HRP-labeled anti-CD81 antibody, each diluted 500-fold with dilution solution, was added to each well and incubated at room temperature.
• Example 9 and Comparative Example 17 above show that purified extracellular vesicles derived from SW837 cells, SW480 cells, HTC-116 cells, and Caco-2 cells are captured by VAR2CSA polypeptide and Tim4, and detected with a cocktail of anti-CD9, anti-CD63, and anti-CD81 antibodies.
• the target of VAR2CSA polypeptide is highly expressed in extracellular vesicles derived from SW480 cells.
• 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 (WO2014/167969), but can be detected in a system that captures with VAR2 polypeptide.
• Example 10 Detection of extracellular vesicles derived from EpCAM-negative pancreatic cancer cells using VAR2CSA polypeptide
• EpCAM-positive EpCAM-positive
• EpCAM is often used as a marker for cancer-derived extracellular vesicles.
• many cancers are EpCAM-negative, and in these cases, the problem is that they cannot be detected with anti-EpCAM antibodies.
• VAR2CSA polypeptide could capture extracellular vesicles derived 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-immobilized plate. After overnight incubation at 4°C and washing five times with PBST, an antibody cocktail solution in which HRP-labeled anti-CD9 antibody, HRP-labeled anti-CD63 antibody, and HRP-labeled anti-CD81 antibody were each diluted 500-fold with diluent was added to each well and incubated at room temperature.
• Comparative Example 18 Detection of extracellular vesicles derived from EpCAM-negative pancreatic cancer cells by Tim4 polypeptide.
• 25-fold diluted purified extracellular vesicle samples derived from KMP2 cells, PANC-1 cells, and MIA Paca-2 cells were added to each well of Tim4 solid phase. After overnight incubation at 4°C and washing five times with washing solution, an antibody cocktail solution of HRP-labeled anti-CD9 antibody, HRP-labeled anti-CD63 antibody, and HRP-labeled anti-CD81 antibody, each diluted 500-fold with dilution solution, was added to each well and incubated at room temperature.
• 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.
• Example 11 Detection of glioblastoma cell line-derived extracellular vesicles captured with anti-CD63 antibody using VAR2CSA polypeptide.
• Culture supernatant of glioblastoma cell line U87-MG was serially diluted 2-fold with sample dilution buffer, and each diluted sample was added to an anti-CD63 antibody-immobilized plate (no cross-reactivity with bovine antibodies) (CD63-Capture Human Exosome ELISA Kit; Fujifilm).
• VAR2CSA polypeptide solution was added and incubated at room temperature for 2 hours.
• HRP-anti-FLAG antibody (Sigma-Aldrich), which recognizes the 3xFLAG-tag peptide attached to the VAR2CSA polypeptide, was added and incubated for a further 2 hours at room temperature. After washing, TMB solution, a peroxidase color-developing substrate, was added and allowed to react for 10 minutes. A color-developing stop solution was added to stop the reaction, 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 when culture medium was added. The results are shown in Figure 25.
• Comparative Example 19 Attempt to detect extracellular vesicles derived from normal fetal lung fibroblast cell line captured with anti-CD63 antibody using VAR2CSA polypeptide An attempt was made to detect extracellular vesicles using WI-38-hTERT cells, which are normal fetal lung fibroblast cells, instead of the glioblastoma cell line in Example 11. The results are shown in Figure 25.
• Example 11 and Comparative Example 19 above show that, after capture of extracellular vesicles with an antibody against an extracellular vesicle marker, 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).
• Example 12 Detection of extracellular vesicles derived from lung cancer cell lines added to serum 25 ⁇ L of purified extracellular vesicles derived from lung cancer cell line NCI-H1703 cells and purified extracellular vesicles derived from normal fetal lung fibroblast cell line WI-38-hTERT cells were added to 75 ⁇ L of extracellular vesicle-removed FBS and mixed. The samples were added to a plate on which VAR2SCA polypeptide was immobilized.
• Example 12 show that extracellular vesicles derived from cancer cells added to serum can be detected.
• Example 13 Comparison of extracellular vesicle capture rates by different methods of immobilizing VAR2CSA polypeptide to a carrier
• the capture efficiency of extracellular vesicles by VAR2CSA polypeptide was compared between the method of immobilizing the N-terminus of VAR2CSA polypeptide to a plate (Fig. 2A) and the method of immobilizing the C-terminus of VAR2CSA polypeptide to a plate (Fig. 2B).
• To capture the N-terminus of VAR2CSA polypeptide VAR2CSA polypeptide was added to a streptavidin-coated plate.
• To capture the C-terminus of VAR2CSA polypeptide VAR2CSA polypeptide was added to a nickel-chelate-coated plate (see Section A).
• the values in the figure are the ratio of the values on the nickel chelate-coated plate to the values on the streptavidin-coated plate. It was shown that the capture rate of extracellular vesicles was 1.2 to 2.2 times better when the C-terminal side of VAR2CSA was immobilized on the nickel chelate-coated plate.
• VAR2CSA polypeptides from strains other than FCR3 strains
• This polynucleotide was inserted between NdeI and NotI in 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. Furthermore, the calculated molecular weights of these polypeptides are shown in Table 5.
• VAR2CSA polypeptides derived from various P. falciparum strains were added to a streptavidin plate (Nunc; 236004) to immobilize the VAR2CSA polypeptides.
• VAR2CSA polypeptides derived from both lines captured EVs derived from cancer cell lines, but were unable to capture EVs derived from normal cell lines.
• VAR2CSA polypeptides derived from the M0920 line showed low binding affinity to EVs derived from cancer cells. This result reflected their binding affinity to decorin.
• 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 by the method of Reference Example 6 were diluted and captured and detected by the method described above in Section A. Specifically, the purified EVs sample was diluted 2.5 times and added to each well of the VAR2CSA solid-phase plate.
• the VAR2CSA solid-phase plate was prepared by adding VAR2CSA polypeptide to a nickel chelate-coated plate.
• the plate was washed five times with PBST, and an antibody cocktail solution in which HRP-labeled anti-CD9 antibody, HRP-labeled anti-CD63 antibody, and HRP-labeled anti-CD81 antibody were each diluted 500 times with dilution solution 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 when color developed, a reaction stop solution was added and the absorbance at 450 nm was immediately measured using a microplate reader. The absorbance in the figure was calculated by subtracting the blank value from each value. The results are shown in FIG.
• 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 in Example 14. The results are shown in Figure 31.
• Comparative Example 21 Detection of extracellular vesicles derived from renal cancer cell lines by Tim4 polypeptide.
• 25-fold diluted Caki-1 and Caki-2 cell purified extracellular vesicle samples were added to each well of Tim4 solid-phase. After overnight incubation at 4°C and washing five times with washing solution, an antibody cocktail solution of HRP-labeled anti-CD9 antibody, HRP-labeled anti-CD63 antibody, and HRP-labeled anti-CD81 antibody, each diluted 500-fold with dilution solution, was added to each well and incubated at room temperature.
• 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 in Comparative Example 21. The results are shown in Figure 32.
• 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.
• Example 15 Detection of extracellular vesicles derived from breast cancer cell lines using VAR2CSA polypeptide
• Extracellular vesicles prepared from breast cancer cell lines MDA-MB-231 cells (also called MM231 cells), MDA-MB-453 cells (also called MM453 cells) and T47D cells by the method of Reference Example 6 were diluted and captured and detected by the method described above in Section A. Specifically, the purified EVs sample was diluted 2.5 times and added to each well of the VAR2CSA solid-phase plate.
• the VAR2CSA solid-phase plate was prepared by adding the VAR2CSA polypeptide to a nickel chelate-coated plate.
• Comparative Example 23 Attempt to detect extracellular vesicles derived from 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 in Example 15. The results are shown in Figure 33.
• Comparative Example 24 Detection of extracellular vesicles derived from breast cancer cell lines by Tim4 polypeptide.
• 25-fold diluted samples of purified extracellular vesicles derived from MDA-MB-231 cells, MDA-MB-453 cells, and T47D cells were added to each well of the Tim4-coated plate.
• an antibody cocktail solution of HRP-labeled anti-CD9 antibody, HRP-labeled anti-CD63 antibody, and HRP-labeled anti-CD81 antibody each diluted 500-fold with dilution solution, was added to each well and incubated at room temperature.
• Comparative Example 25 Detection of extracellular vesicles derived from 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 in Comparative Example 24. The results are shown in Figure 34.
• 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 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 HMEC-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.
• Example 16 Detection of extracellular vesicles derived from ovarian cancer cell line by VAR2CSA polypeptide
• Extracellular vesicles prepared from ovarian cancer cell line OVCAR3 cells by the method described in Reference Example 6 were diluted and captured and detected by the method described above in Section A. Specifically, the purified EVs sample was diluted 2.5 times and added to each well of the VAR2CSA solid-phase plate.
• the VAR2CSA solid-phase plate was prepared by adding VAR2CSA polypeptide to a nickel chelate-coated plate. After overnight incubation at 4 ° C.
• Example 17 Detection of extracellular vesicles derived from bladder cancer cell line using VAR2CSA polypeptide Extracellular vesicles were detected using bladder cancer cell line T24 cells instead of the ovarian cancer cell line in Example 16. The results are shown in Figure 35.
• Comparative Example 26 Detection of extracellular vesicles derived from ovarian cancer cell lines by Tim4 polypeptide.
• 25-fold diluted purified extracellular vesicle samples derived from OVCAR3 cells were added to each well of Tim4 solid-phase. After overnight incubation at 4°C and washing five times with washing solution, an antibody cocktail solution of HRP-labeled anti-CD9 antibody, HRP-labeled anti-CD63 antibody, and HRP-labeled anti-CD81 antibody, each diluted 500-fold with dilution solution, was added to each well and incubated at room temperature.
• 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 in Comparative Example 26. The results are shown in Figure 36.
• 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.
• Examples 18 to 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 liver cancer cell line HepG2 cells (Example 18), gallbladder cancer cell line TYGBK-8 cells (Example 19), cholangiocarcinoma cell lines HuCCT1 cells, and TFK-1 cells (Example 20) by the method described in Reference Example 6 were diluted and captured and detected by the method described above in Section A. Specifically, the purified EVs sample was diluted 2.5 times and added to each well of the VAR2CSA solid-phase plate.
• the VAR2CSA solid-phase plate was prepared by adding the VAR2CSA polypeptide to a nickel chelate-coated plate. After overnight incubation at 4 ° C. and washing five times with PBST, an antibody cocktail solution in which HRP-labeled anti-CD9 antibody, HRP-labeled anti-CD63 antibody, and HRP-labeled anti-CD81 antibody were each diluted 500 times with diluent was added to each well and incubated at room temperature. After 2 hours, the plate was washed 5 times with PBST, TMB substrate was added, and when color developed, a reaction stop solution was added and the absorbance at 450 nm was immediately measured using a microplate reader. The absorbance in the figure was calculated by subtracting the blank value from each value. The results are shown in Figure 37.
• Comparative Examples 28 to 30 Detection of extracellular vesicles derived from hepatoma cell lines, gallbladder cancer cell lines, and cholangiocarcinoma cell lines using Tim4 polypeptides.
• purified extracellular vesicle samples derived 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 Tim4 solid-phased cells.
• Examples 18 to 20 and Comparative Examples 28 to 30 above show that purified extracellular vesicles derived from liver cancer cell line HepG2 cells, gallbladder cancer cell line TYGBK-8 cells, cholangiocarcinoma cell line HuCCT1 cells, and TFK-1 cells were captured by VAR2CSA polypeptide and Tim4, and detected with a cocktail of antibodies consisting of anti-CD9 antibodies, anti-CD63 antibodies, and anti-CD81 antibodies.
• Example 21 Detection of extracellular vesicles derived from acute lymphocytic leukemia cell line using VAR2CSA polypeptide
• Extracellular vesicles prepared from acute lymphocytic leukemia cell line NALM-6 cells by the method described in Reference Example 6 were diluted and captured and detected by the method described above in Section A.
• NALM-6 cell EVs were prepared using samples (1) and (2) prepared in two independent experiments. Specifically, these purified EVs samples were diluted 2.5 times and added to each well of the VAR2CSA solid-phase plate.
• the VAR2CSA solid-phase plate was prepared by adding the VAR2CSA polypeptide to a nickel chelate-coated plate.
• Example 22 Detection of extracellular vesicles derived from acute myeloid leukemia cell line using VAR2CSA polypeptide Detection of extracellular vesicles was attempted using acute myeloid leukemia cell line KG-1 cells instead of the acute lymphocytic leukemia cell line in Example 21 above. The results are shown in FIG.
• Comparative Example 31 Detection of extracellular vesicles derived from acute lymphocytic leukemia cell line by Tim4 polypeptide.
• 25-fold diluted purified extracellular vesicle samples derived from NALM-6 cells were added to each well of Tim4 solid-phase. After overnight incubation at 4°C and washing five times with washing solution, antibody cocktail solutions of HRP-labeled anti-CD9 antibody, HRP-labeled anti-CD63 antibody, and HRP-labeled anti-CD81 antibody, each diluted 500-fold with dilution solution, were added to each well and incubated at room temperature.
• 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 in Comparative Example 30. The results are shown in FIG.
• 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.
• Comparative Example 33 Attempt to detect extracellular vesicles derived from normal tracheal smooth muscle cell line using VAR2CSA polypeptide Extracellular vesicles prepared from the tracheal smooth muscle cell line HBSMC-TKD cells by the method described in Reference Example 6 were diluted and captured and detected by the method described above in Section A. Specifically, the purified EVs sample was diluted 2.5 times and added to each well of the VAR2CSA solid-phase plate. The VAR2CSA solid-phase plate was prepared by adding VAR2CSA polypeptide to a nickel chelate-coated plate.
• the plate was washed five times with PBST, and an antibody cocktail solution in which HRP-labeled anti-CD9 antibody, HRP-labeled anti-CD63 antibody, and HRP-labeled anti-CD81 antibody were each diluted 500 times with dilution solution 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 when color developed, a reaction stop solution was added and the absorbance at 450 nm was immediately measured with a microplate reader. The absorbance in the figure was calculated by subtracting the blank value from each value. The results are shown in FIG.
• Comparative Example 34 Attempt to detect extracellular vesicles derived from normal aortic endothelial cell line using VAR2CSA polypeptide An attempt was made to detect extracellular vesicles using aortic endothelial cell line HAEC-TKD cells instead of tracheal smooth muscle cell line HBSMC-TKD cells in the above Comparative Example 33. The results are shown in Figure 41.
• 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 in Comparative Example 32. The results are shown in FIG.
• Comparative Example 35 Detection of extracellular vesicles derived from normal tracheal smooth muscle cell line by Tim4 polypeptide.
• 10-fold diluted purified extracellular vesicle samples derived from HBSMC-TKD cells were added to each well of Tim4 solid-phase. After overnight incubation at 4°C and washing five times with washing solution, antibody cocktail solutions of HRP-labeled anti-CD9 antibody, HRP-labeled anti-CD63 antibody, and HRP-labeled anti-CD81 antibody, each diluted 500-fold with dilution solution, were added to each well and incubated at room temperature.
• Comparative Example 36 Detection of extracellular vesicles derived from normal aortic endothelial cell line by Tim4 polypeptide Extracellular vesicles were detected using HAEC-TKD cells instead of HBSMC-TKD cells in Comparative Example 34. The results are shown in Figure 42.
• 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 antibodies consisting of anti-CD9, anti-CD63, and anti-CD81 antibodies.
• Examples 14 to 23 and Comparative Examples 20 to 36 are summarized in Table 8.
• ⁇ indicates that the substance was captured, and ⁇ indicates that the substance was not captured.
• the results show 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) are captured, but extracellular vesicles derived from normal cells such as renal epithelial cells (HREC-TKD cells), breast epithelial cells (HMEC-TDK cells), tracheal smooth muscle cells (HBSMC-TKD
• Example 24 Detection of extracellular vesicles derived from acute myeloid leukemia cell line KG-1 cells added to human healthy plasma using VAR2CSA polypeptide EVs were purified from a sample in which 25 ⁇ L of EVs purified from KG-1 cell culture supernatant was added to 1 mL of healthy plasma using MagCapture (trade name) Exosome Isolation Kit PS Ver.2 (manufactured by Fujifilm). The procedure scheme is shown in Figure 43. In addition, the immobilization of VAR2 polypeptide on the plate was performed by adding VAR2 polypeptide derived from the 7G8 strain to a nickel chelate-coated plate (Thermo Scientific).
• Comparative Example 37 Attempt to detect extracellular vesicles derived from healthy human plasma using VAR2CSA polypeptide In Comparative Example 35, instead of the plasma spiked with EVs, an attempt was made to detect extracellular vesicles using 90 ⁇ L of EVs purified from plasma that had not been spiked with EVs. The results are shown in FIG.
• Comparative Example 39 Detection of extracellular vesicles derived from healthy human plasma using Tim4 polypeptide
• Comparative Example 37 Extracellular vesicles were detected using plasma without EVs spiked in, instead of the plasma spiked in with EVs. The results are shown in Figure 45.
• VAR2CSA polypeptide was immobilized on a plate by adding VAR2 polypeptide derived from the 7G8 strain to a nickel chelate-coated plate (Thermo Scientific).
• EVs were purified using 1 mL each of colorectal cancer patient plasma (stage IV, purchased from National BioService) and healthy donor plasma (purchased from National BioService) using MagCapture (product name) Exosome Isolation Kit PS Ver.2 (Fujifilm). 90 ⁇ uL of the purified EVs (100 ⁇ L) were diluted with buffer to 100 ⁇ L and added to a 7G8 VAR2 polypeptide-immobilized plate.
• buffer EVs purified from leukemia cell line KG-1 cell culture supernatant, and EVs purified from colorectal cancer cell line HTC-116 cell culture supernatant were diluted 2.5-fold with buffer, and 100 ⁇ L of the diluted samples were added to a 7G8 VAR2 polypeptide-immobilized plate. After incubation at 4°C for 18 hours and washing five times with washing solution, an antibody cocktail solution in which HRP-labeled anti-CD9 antibody, HRP-labeled anti-CD63 antibody, and HRP-labeled anti-CD81 antibody were each diluted 500-fold with dilution solution was added to each well and incubated at room temperature.
• the method of the present invention can effectively inhibit the expression of glioma 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, H It was shown that the method can capture and detect extracellular vesicles derived from human TC-116 cells, Caco-2 cells), 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
• the method of the present invention can capture and detect extracellular vesicles derived from biological samples of patients suffering from cancer.
• the method of the present invention it is possible to detect extracellular vesicles derived from a wide range of cancer cells of different categories, such as epithelial cancer, sarcoma, and blood cancer, and it is therefore reasonably believed that cancer can be detected regardless of the type of cancer.
• 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 diagnosing cancer.

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