Cancer stem cell-specific molecule

Abstract

To provide pharmaceutical compositions that can be used as anticancer agents, drug tolerant anti-cancer therapeutics, cancer recurrence inhibitors, cancer metastasis inhibitors, or postoperative adjuvant therapeutic agents which contain as an active ingredient a cell surface molecule that is specific to a Leucine-rich repeat-containing G-protein coupled receptor 5 (Lgr5) positive cancer stem cell with high proliferation potency and to a Lgr5 negative cancer stem cell with low proliferation potency, or an antibody to cell surface molecules thereof.SOLUTION: The present invention provides a pharmaceutical composition containing at least one antibody binding to a particular sequence of Lgr5.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to cell surface molecules specific to Lgr5-positive cancer stem cells with high proliferation potential and Lgr5-negative cancer stem cells with low proliferation potential, and to pharmaceutical compositions containing antibodies against these cell surface molecules as active ingredients.The present invention further relates to a reagent for detecting cancer stem cells and a method for selecting cancer patients using the antibodies. [Background technology]

[0002] Cancer stem cells (CSCs) are considered to be the origin of cancer because they have the ability to self-renew and differentiate into tumor hierarchies (Non-Patent Document 1). In addition, CSCs can migrate and can withstand anticancer drug treatment (Non-Patent Document 1). As they are considered a rare subset in tumors, efforts have been made to characterize CSCs by cell surface markers and tumor-initiating activity in xenografts. CSCs have been reported in several cancers, including acute myeloid leukemia (AML) (Non-Patent Documents 2 and 3), breast cancer (Non-Patent Document 4), glioma (Non-Patent Document 5), head and neck cancer (Non-Patent Document 6), pancreatic cancer (Non-Patent Documents 7 and 8), lung cancer (Non-Patent Document 9), prostate cancer (Non-Patent Documents 10 and 11), mesenchymal neoplasms (Non-Patent Document 12), and melanoma (Non-Patent Documents 13 and 14). In colorectal cancer, early studies by O'Brien et al. (Non-Patent Document 15) and Ricci-Vitiani et al. (Non-Patent Document 16) reported that CD133 is a CSC marker. Later, other research groups reported additional markers, such as CD44, EpCAM, CD166 (Non-Patent Document 17), and ALDH (Non-Patent Documents 18, 19). Recently, Pang et al. showed that CD26 is a marker for a subpopulation of CSCs with metastatic activity (Non-Patent Document 20).

[0003] To isolate CSCs, many studies have used EpCAM high / CD44 + / CD166 + (Non-patent document 17), CD133 + / CD44 + (Non-patent Document 21), CD44 high / ALDH + (Non-Patent Document 18), and ALDH1 + / CD133 + Cell sorting approaches using a combination of CSC markers such as those described above have been used (Non-Patent Document 19). In vitro spheroid (cell mass) cultures and direct xenotransplantation of cancer cells into immune-deficient mice have also been used to enrich for CSCs (Non-Patent Document 22). However, to further understand the properties of CSCs, it was necessary to obtain cancer stem cells in high purity and in large quantities.

[0004] One of the problems in isolating CSCs is thought to be due to the heterogeneity and / or instability of the CSC phenotype (Non-Patent Document 29). Three-dimensional spheroid cultures are often used as a source of CSCs. This spheroid culture can be directly adapted to tumor cells from clinically resected specimens, and the ability to maintain a heterogeneous population of CSCs may have certain advantages over xenografts. However, due to the heterogeneity, the results in biochemical analysis often show complex characteristics of CSCs. CSC selection using antibodies against cell surface marker proteins is widely used to isolate CSCs, but the number and purity of cells obtained by this method are limited. On the other hand, the use of xenografts as a source of CSCs is also a common approach, since the phenotype of xenografts is stably maintained even after multiple passages. However, there is also an argument that the passage of xenografts in mice can only select cells that can survive in mice, thereby eliminating cells that are less susceptible to such an environment. Needless to say, the CSCs present within the xenograft tumors will reflect the original characteristics of CSCs, as long as they maintain the capacity for self-renewal and production of the differentiated lineage of the original tumor.

[0005] Lgr5 (leucine-rich-repeat-containing G protein-coupled receptor 5) was initially identified as an orphan G protein-coupled receptor of the glycoprotein hormone receptor family (Non-Patent Documents 23, 24) and was shown to be a Wnt target gene whose expression is restricted in crypts (Non-Patent Document 25). The discovery that Lgr5-positive columnar cells can regenerate all epithelial lineages (Non-Patent Document 25) and that single Lgr5-positive cells can form crypto-viral organoids in vitro without a mesenchymal niche (Non-Patent Document 26) clearly demonstrated that Lgr5-positive cells are stem cells in the normal colon. Furthermore, it was reported that Lgr5-positive cells form adenomas under Apc deletion (Non-Patent Document 27) and that Lgr5 is expressed in colon cancer cell lines (Non-Patent Document 25). Taken together, these results indicate that Lgr5-positive cells are the origin of colon cancer (Non-Patent Document 25). It has been demonstrated that Wnt activity is essential for the proliferation of CSCs in vitro and in vivo, as in normal colonic stem cells, and that exogenous HGF stimulates Wnt activity (Non-Patent Document 28).

[0006] Lgr5 has been identified as a stem cell marker for normal colon and has been shown to be a marker for the origin of colon cancer (Patent Document 1, Non-Patent Document 30), and Lgr5 has also been shown to be a protein that is overexpressed in colon cancer stem cells (Patent Document 2). However, the physiological role of Lgr5 in the development of colon cancer remains unknown.

[0007] Although various cancer treatment methods and anticancer drugs have been developed, there are still problems to be solved, such as insufficient efficacy, side effects, and limited efficacy in patients. In recent years, cancer stem cell-targeting treatment methods have been attracting attention, but there are many unknowns regarding their efficacy and side effects (Non-Patent Document 31). [Prior art documents] [Patent documents]

[0008] [Patent Document 1] US20100275280 [Patent Document 2] WO09 / 005809 [Non-patent literature]

[0009] [Non-Patent Document 1] Reya T, Morrison SJ, Clarke MF, Weissman IL (2001) Stem cells, cancer, and cancer stem cells. Nature 414:105-111. [Non-Patent Document 2] Bonnet D, Dick JE (1997) Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nat Med 3:730-737. [Non-Patent Document 3] Lapidot T, et al. (1994) A cell initiating human acute myeloid leukaemia after transplantation into SCID mice. Nature 367:645-648. [Non-Patent Document 4] Al-Hajj M, Wicha MS, Benito-Hernandez A, Morrison SJ, Clarke MF (2003) Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci USA 100:3983-3988. [Non-Patent Document 5] Singh SK, et al. (2004) Identification of human brain tumor initiating cells. Nature 432:396-401. [Non-Patent Document 6] Prince ME, et al. (2007) Identification of a subpopulation of cells with cancer stem cell properties in head and neck squamous cell carcinoma. Proc Natl Acad Sci USA 104:973-978. [Non-Patent Document 7] Hermann PC, et al. (2007) Distinct populations of cancer stem cells determine tumor growth and metastatic activity in human pancreatic cancer. Cell Stem Cell 1: 313-323. [Non-Patent Document 8] Li C, et al. (2007) Identification of pancreatic cancer stem cells. Cancer Res 67:1030-1037. [Non-Patent Document 9] Eramo A, et al. (2008) Identification and expansion of the tumorigenic lung cancer stem cell population. Cell Death Differ 15:504-514. [Non-Patent Document 10] Collins AT, Berry PA, Hyde C, Stower MJ, Maitland NJ (2005) Prospective identification of tumorigenic prostate cancer stem cells. Cancer Res 65:10946-10951. [Non-Patent Document 11] Patrawala L, et al. (2006) Highly purified CD44+ prostate cancer cells from xenograft human tumors are enriched in tumorigenic and metastatic progenitor cells. Oncogene 25:1696-1708.

Non-Patent Document 12

Non-Patent Document 13

Non-Patent Document 14

Non-Patent Document 15

Non-Patent Document 16

Non-licensed Document 17

Non-licensed Document 18

Non-licensed Document 19

Non-licensed Document 20

Non-licensed Document 21

Non-licensed Document 22

[0010] The present invention has been made in view of these circumstances, and aims to provide a cancer therapeutic drug using antibodies against the molecules by obtaining two types of substantially homogeneous cancer stem cell populations characterized by the cell surface marker Lgr5 and identifying cell membrane molecules specifically expressed in these cancer stem cells.Another aim of the present invention is to provide a reagent for detecting cancer stem cells and a method for diagnosing and selecting cancer patients using antibodies against cell membrane molecules specifically expressed in cancer stem cells. [Means for solving the problem]

[0011] In order to prove the stem cell theory that can explain the carcinogenesis mechanism, great efforts have been made to identify, isolate and characterize cancer stem cells (CSCs). However, it has been difficult to obtain a sufficient amount of CSCs with high purity for characterization. Therefore, it has also been difficult to identify cell membrane molecules that are specifically expressed in cancer stem cells. The present inventors have conducted intensive research to solve the above problems.

[0012] The present inventors have established for the first time a method for obtaining a large amount of highly pure colon CSCs by applying in vitro monolayer culture (also simply referred to as adherent culture) using serum-free stem cell medium to isolate colon CSCs from human colon cancer xenografts maintained in NOG mice. Specifically, the present inventors found that a large amount of highly pure colon CSCs could be obtained by isolating and adhering cells derived from moderately differentiated human colon cancer xenografts maintained in NOD / Shi-scid, IL-2Rγnull (NOG) mice. Under these conditions, only colon CSCs could proliferate, survive, and expand, thereby obtaining highly pure and substantially homogeneous colon CSCs. The colon CSCs obtained by this method were stably maintained without phenotypic changes for more than one month by passaging adherent culture using serum-free stem cell medium. These cells expressed various colon cancer stem cell markers reported so far (CD133, CD44, EpCAM, CD166, CD24, CD26, and CD29), and showed tumor-initiating activity at a frequency of nearly 100%. They reconstituted tumors with the same histopathological characteristics (hierarchical structure) as the original primary tumor. These cells were also characterized as being highly proliferative under adherent culture conditions and positive for the cell surface marker Lgr5. Furthermore, when administered via the tail vein of mice, highly proliferative Lgr5-positive cancer stem cells formed tumor masses in organs such as the lungs and liver, indicating that they play an important role in cancer metastasis.

[0013] On the other hand, by treating cancer stem cells that showed high proliferation under adherent culture conditions and were positive for the cell surface marker Lgr5 with anticancer drugs such as irinotecan and 5-FU, it was possible to isolate cancer stem cells that were low proliferation and Lgr5 negative. Furthermore, it was shown that low proliferation and Lgr5 negative cancer stem cells were converted to high proliferation and Lgr5 positive cancer stem cells by culturing them again under adherent culture conditions. Therefore, it was shown that high proliferation and Lgr5 positive cancer stem cells and low proliferation and Lgr5 negative cancer stem cells are mutually convertible and have a self-replacing function. Due to this ability, when CSCs were cultured under changed culture conditions or exposed to anticancer drugs, actively proliferating Lgr5 positive colon CSCs were converted to a quiescent Lgr5 negative state. Alternatively, by isolating the cells and culturing them again under adherent culture, Lgr5 negative CSCs became actively proliferating Lgr5 positive CSCs. These cells also showed tumor-initiating activity at a frequency of almost 100%. Furthermore, when highly proliferative Lgr5-positive cancer stem cells were inoculated via the tail vein of mice, they formed tumor masses in organs such as the lungs and liver, indicating that they play an important role in cancer metastasis.

[0014] This self-alteration of CSCs between two states upon environmental changes may help explain drug resistance and recurrence of cancer. The presence of Lgr5-negative CSCs in carcinogenesis may be related to fundamental stem cell properties. One possible hypothesis is that CSCs can utilize endogenous means to turn into distinct subsets of cell populations under environmental changes such as aggressive anticancer drug treatment. It is considered that CSCs survive by changing into Lgr5-negative, low-proliferative CSCs upon stress, such as anticancer drugs or changes in the culture environment, to avoid the stress, and then change back into Lgr5-positive, high-proliferative CSCs and start proliferating when the stress is released. This means that CSCs have a self-protective ability to adapt to new environments by endogenous mechanisms (Figure 36).

[0015] From these research results by the inventors, both highly proliferative Lgr5-positive cancer stem cells and low proliferative Lgr5-negative cancer stem cells play important roles in the development, formation, metastasis, recurrence, drug resistance, etc. of cancer, and can be major target cells in the development of anticancer drugs. In particular, highly proliferative Lgr5-positive cancer stem cells are thought to be involved in the formation and metastasis of cancer, while low proliferative Lgr5-negative cancer stem cells are thought to be involved in the recurrence of cancer. Therefore, if the cell surface molecules specifically expressed in highly proliferative Lgr5-positive cancer stem cells and low proliferative Lgr5-negative cancer stem cells can be identified, treatment using antibodies will be possible, and new anticancer drugs and reagents for detecting cancer stem cells will be created.

[0016] More specifically, the present invention relates to the following: [1] A pharmaceutical composition comprising, as an active ingredient, at least one antibody that binds to a protein set forth in SEQ ID NO: 1 to 8; [2] The pharmaceutical composition according to [1], which is an anticancer agent. [3] The pharmaceutical composition according to [2], which is an agent for suppressing cancer recurrence. [4] The pharmaceutical composition according to [2], which is an agent for suppressing cancer metastasis or an agent for postoperative adjuvant therapy. [5] The pharmaceutical composition according to [4], which is an agent for suppressing metastasis of Lgr5-positive cancer or an agent for postoperative adjuvant therapy, comprising at least one antibody that binds to a protein set forth in SEQ ID NOs: 1 to 6 as an active ingredient. [6] The pharmaceutical composition according to [2], which is a therapeutic agent for drug-resistant cancer. [7] The pharmaceutical composition according to [6], which is a cancer therapeutic agent for Lgr5-negative cancer, comprising as an active ingredient at least one antibody that binds to a protein shown in SEQ ID NO: 1 to 8. [8] The pharmaceutical composition according to [7], wherein the Lgr5-negative cancer is drug-resistant. [9] The pharmaceutical composition according to any one of [2] to [8], which is a cancer stem cell proliferation inhibitor or a cancer stem cell disrupting agent.

[10] The pharmaceutical composition according to any one of [2] to [9], wherein the cancer is a solid cancer.

[11] The pharmaceutical composition according to any one of [2] to

[10] , wherein the cancer is a gastrointestinal cancer.

[12] The pharmaceutical composition according to any one of [2] to

[11] , wherein the cancer is colorectal cancer.

[13] The pharmaceutical composition according to any one of [1] to

[12] , wherein the antibody is a monoclonal antibody.

[14] The pharmaceutical composition according to any one of [1] to

[13] , wherein the antibody is a chimeric antibody, a humanized antibody, or a human antibody.

[15] The pharmaceutical composition according to any one of [1] to

[14] , wherein the antibody is an antibody fragment.

[16] The pharmaceutical composition according to

[15] , wherein a cytotoxic substance or a growth inhibitor is linked to the antibody.

[17] The pharmaceutical composition according to any one of [1] to

[14] , wherein the antibody has cytotoxic activity.

[18] The pharmaceutical composition according to

[17] , wherein the cytotoxic activity is ADCC activity.

[19] The pharmaceutical composition according to

[17] or

[18] , comprising an antibody whose sugar chain composition has been modified so that the proportion of fucose-deficient antibodies is increased, or the proportion of antibodies to which bisecting N-acetylglucosamine has been added is increased.

[20] The pharmaceutical composition according to

[17] , wherein the cytotoxic activity is CDC activity.

[21] The pharmaceutical composition according to any one of [1] to

[20] , wherein the antibody has neutralizing activity.

[22] The pharmaceutical composition according to any one of [2] to

[21] , which is used simultaneously with a chemotherapy agent or after chemotherapy treatment, or a pharmaceutical composition comprising a polypeptide represented by SEQ ID NO: 9 or a polypeptide in which one or more amino acids contained in the polypeptide have been added, deleted or substituted.

[23] A reagent for detecting cancer stem cells, comprising as an active ingredient at least one antibody that binds to a protein set forth in SEQ ID NO: 1 to 8.

[24] The reagent according to

[23] for detecting Lgr5-positive cancer stem cells, wherein the antibody is at least one antibody that binds to a protein set forth in SEQ ID NOs: 1 to 6.

[25] The reagent according to

[23] for detecting Lgr5-negative cancer stem cells, wherein the antibody is at least one antibody that binds to a protein set forth in SEQ ID NOs: 1 to 8.

[26] A method for diagnosing cancer or screening a cancer patient, comprising detecting the presence of at least one protein in a sample isolated from a cancer patient using at least one antibody that binds to a protein set forth in SEQ ID NO: 1 to 8 (a method for testing or screening for cancer);

[27] The method for diagnosing Lgr5-positive cancer or selecting a cancer patient according to

[26] , wherein the antibody is at least one antibody that binds to a protein set forth in SEQ ID NOs: 1 to 6.

[28] The method for diagnosing Lgr5-negative cancer or selecting a cancer patient according to

[26] , wherein the antibody is at least one antibody that binds to a protein set forth in SEQ ID NO: 1 to 8.

[29] A method for confirming the efficacy of the pharmaceutical composition according to any one of [1] to

[22] , comprising detecting the presence of any one or more of the proteins set forth in SEQ ID NOs: 1 to 8 and / or polynucleotides encoding said proteins in a sample isolated from a subject to which the pharmaceutical composition has been administered;

[30] The method according to

[29] , which uses at least one antibody that binds to a protein set forth in SEQ ID NO: 1 to 8; and

[31] The method according to

[29] , wherein a polynucleotide encoding a protein set forth in SEQ ID NO: 1 to 8 and / or a complementary strand thereof is used. This provides:

[0017] The present invention also provides the following: [A1] A method for treating cancer, comprising administering to a subject at least one antibody that binds to a protein set forth in SEQ ID NOs: 1 to 8; [A2] at least one antibody that binds to a protein set forth in SEQ ID NOs: 1 to 8 for use in treating cancer; [A3] use of at least one antibody that binds to a protein set forth in SEQ ID NOs: 1 to 8 for producing an anticancer agent; [A4] a process for producing an anticancer agent, comprising a step of using at least one antibody that binds to a protein set forth in SEQ ID NOs: 1 to 8; This provides: In one non-limiting aspect of the present invention, the cancer treatment is suppression of cancer recurrence, suppression of cancer metastasis, postoperative adjuvant therapy, drug-resistant cancer treatment, suppression of cancer stem cell proliferation, or destruction of cancer stem cells, and the anticancer agent is a cancer recurrence suppressor, a cancer metastasis suppressor, a postoperative adjuvant therapy agent, a drug-resistant cancer treatment agent, an inhibitor of cancer stem cell proliferation, or an agent for destroying cancer stem cells.

[0018] Further, the present invention relates to the following: [B1] A reagent for detecting the presence of any one or more of the proteins set forth in SEQ ID NOs: 1 to 8 and / or polynucleotides encoding said proteins, preferably a reagent for detecting cancer stem cells, a reagent for diagnosing cancer, a reagent for selecting cancer patients, or a reagent for confirming the effectiveness of the pharmaceutical compositions of [1] to

[22] , which comprises at least one antibody that binds to the proteins set forth in SEQ ID NOs: 1 to 8, or a polynucleotide encoding the proteins set forth in SEQ ID NOs: 1 to 8 and / or a portion of its complementary strand; [B2] A method for detecting cancer stem cells, diagnosing cancer, selecting a cancer patient, or confirming the effectiveness of a pharmaceutical composition according to any one of [1] to

[22] , comprising detecting the presence of any one or more of the proteins set forth in SEQ ID NOs: 1 to 8 and / or polynucleotides encoding said proteins in a sample isolated from a cancer patient, preferably using at least one antibody that binds to the proteins set forth in SEQ ID NOs: 1 to 8, or a polynucleotide encoding the proteins set forth in SEQ ID NOs: 1 to 8 and / or a portion of a complementary strand thereof; [B3] A reagent for detecting the presence of any one or more of the proteins set forth in SEQ ID NOs: 1 to 8 and / or polynucleotides encoding said proteins, for use in detecting cancer stem cells, diagnosing cancer, selecting cancer patients, or confirming the effectiveness of the pharmaceutical compositions of [1] to

[22] , preferably at least one antibody that binds to the proteins set forth in SEQ ID NOs: 1 to 8, or a portion of the polynucleotide encoding the proteins set forth in SEQ ID NOs: 1 to 8 and / or its complementary strand; [B4] A reagent for detecting the presence of any one or more of the proteins set forth in SEQ ID NOs: 1 to 8 and / or polynucleotides encoding said proteins, preferably at least one antibody that binds to the proteins set forth in SEQ ID NOs: 1 to 8, or a polynucleotide encoding the proteins set forth in SEQ ID NOs: 1 to 8 and / or a portion of its complementary strand, for the manufacture of a reagent for detecting cancer stem cells, a reagent for diagnosing cancer, a reagent for selecting cancer patients, or a reagent for confirming the effectiveness of the pharmaceutical compositions of [1] to

[22] ; and [B5] A process for producing a reagent for detecting the presence of any one or more of the proteins set forth in SEQ ID NOs: 1 to 8 and / or polynucleotides encoding said proteins, preferably at least one antibody that binds to the proteins set forth in SEQ ID NOs: 1 to 8, or a polynucleotide encoding the proteins set forth in SEQ ID NOs: 1 to 8 and / or a portion of its complementary strand, for use in detecting cancer stem cells, diagnosing cancer, selecting cancer patients, or validating the pharmaceutical compositions of [1] to

[22] ; This provides: [Brief description of the drawings]

[0019] [Figure 1]Photographs showing histology (HE staining) of colon cancer xenografts PLR59 and PLR123 derived from moderately differentiated colon cancer. Cells derived from PLR59 and PLR123 xenografts reconstituted tumors with morphology (hierarchical structure) quite similar to the original tumor, with ductal structures and budding clusters (arrows) containing goblet cells (inset), even after 15 passages. Original indicates surgically resected tumor, early passage indicates PLR59 xenografts and PLR123 xenografts passaged 4 times in NOG mice, and late passage indicates PLR59 xenografts and PLR123 xenografts passaged 15 times in MOG mice. Scale bar indicates 100 μm. [Diagram 2] Figure 1 shows the flow cytometry analysis of known CSC markers in cells of xenografts of PLR59 and PLR123 passaged in NOG mice. Cells were stained with antibodies against the indicated markers and analyzed by flow cytometry. Grey indicates the fluorescence intensity or ALDH activity after staining cells with the indicated antibodies, and white indicates the fluorescence intensity or ALDH activity after staining cells with control isotype antibodies or ALDH inhibitors. [Diagram 3] Photographs showing histological images (HE staining) of tumors formed by injection of 100 PLR59 and PLR123 cells. The morphology of tumors derived from 100 PLR59 and PLR123 cells was highly similar to that of the original tumor. Arrows indicate budding clusters. Scale bar indicates 100 μm. [Figure 4] Photographs showing the results of phase contrast microscopy of floating cells and adherent cells (PLR123 cells). The cells were cultured in serum-free medium supplemented with EGF and FGF. The floating cells tightly interacted with each other to form spheroid-like structures, whereas the adherent cells proliferated without forming cell clusters. The scale bar indicates 25 μm. [Diagram 5]Figure 1 shows proliferation of non-adherent and adherent CSCs (PLR123 cells). The number of viable cells after 3 days of culture (black column) is shown as a percentage of the number on day 0 (white column). Results are the average of three experiments. The bar above each column indicates the standard deviation. [Figure 6] Figure 1 shows the results of flow cytometry analysis of known CSC markers in non-adherent cells and adherent cells (PLR123 cells). Both cells were positive for known CSC markers such as CD133, CD44, EpCAM, CD166, CD24, CD26 and CD29, but only adherent cells were positive for Lgr5 and ALDH activity. Grey indicates fluorescence intensity or ALDH activity after staining cells with the indicated antibodies, and white indicates fluorescence intensity or ALDH activity after staining cells with control isotype antibodies or ALDH inhibitors. [Figure 7] Photographs showing the results of Western blot analysis of β-catenin, TCF1, TCF3, TCF4, and phosphorylated c-JUN proteins in primary cells, non-adherent CSCs, and adherent CSCs of PLR123 cells. Compared to primary cells, the expression of all proteins was upregulated in Lgr5-positive adherent CSCs. GAPDH was also visualized as a reference protein for protein loading. [Figure 8] Figure 1 shows the proliferation inhibition of Lgr5-positive adherent CSCs (PLR123 cells) by FH535 (50 μM) and cardamonin (50 μM). The number of viable cells after 3 days of culture with FH535 (gray column) and cardamonin (black column) is shown as a percentage of the number with DMSO alone (white column). Results are the average of three experiments. The bar on top of each column indicates the standard deviation. [Figure 9] Figure 1 shows the proliferation of PLR123 cells in the presence or absence of EGF and FGF. Adherent CSCs were cultured for 3 days in the presence or absence (black columns). The number of live cells is shown as a percentage of the number on day 0 (white columns). Results are the average of three experiments. The bar on top of each column indicates the standard deviation. [Figure 10]Figure 1 shows the effect of chemotherapy on the proliferation of Lgr5-positive adherent CSCs and Lgr5-negative non-adherent CSCs (PLR123 cells). The number of viable cells after treatment with 5-FU (10 μg / mL, grey column) or irinotecan (10 μg / mL, black column) is shown as a percentage of the number of viable cells cultured without chemotherapy (white column). Results are the average of three experiments. The bar above each column indicates the standard deviation. [Figure 11] This is a diagram showing the change in Lgr5 expression in adherent CSCs (PLR123 cells) after cells are treated with chemotherapy. The results of flow cytometry are shown. The upper part shows no chemotherapy (control), the middle part shows 5-FU-treated cells, and the lower part shows irinotecan-treated cells. Grey indicates the fluorescence intensity or ALDH activity after cells are stained with the indicated antibodies, and white indicates the fluorescence intensity or ALDH activity after cells are stained with a control isotype antibody with an ALDH inhibitor. [Figure 12] FIG. 1 shows the levels of Lgr5 mRNA in PLR123 cells before (designated as 1) and after transition between adherent and suspension culture. F→A indicates transition from suspension to adherent culture, and A→F indicates transition from adherent to suspension culture. Results are the average of three experiments. Bars on top of each column indicate standard deviation. [Figure 13] Photographs showing the results of Western blot analysis of E-cadherin and Snail in Lgr5-negative non-adherent CSCs and Lgr5-positive adherent CSCs (PLR123 cells). Non-adherent CSCs expressed high levels of E-cadherin, whereas adherent CSCs expressed high levels of Snail. GADPH was used as a loading control. [Figure 14] Photographs showing the results of immunocytochemistry of Lgr5-negative floating CSCs and Lgr5-positive adherent CSCs (PLR123 cells) using E-cadherin, Snail, and β-catenin antibodies. Floating CSCs were epithelial-like cells with high expression of E-cadherin and β-catenin on the cell surface, whereas adherent CSCs were mesenchymal-like cells with Snail and β-catenin localized in the nucleus. The scale bar indicates 25 μm. [Figure 15]Photographs showing the results of immunohistochemistry of xenograft tissues derived from PLR123 using anti-Lgr5 and anti-Snail antibodies. Co-expression of nuclear Snail and cytoplasmic Lgr5 was detected in EMT-like cells in the budding region (left panel), but not in the ducts (right panel). Arrows indicate Lgr5-positive budding cells. Scale bar indicates 10 μm. [Figure 16] Photographs showing the histopathological characteristics of xenograft tissues. Histopathology of poorly differentiated colorectal cancer (PDCC) xenografts. PDCC xenografts reconstituted nearly identical histopathological morphology to the original tumor, and PDCC xenografts did not contain distinct epithelial ductal structures (passages 4 and 13). Scale bar indicates 100 μm. [Figure 17] Photographs showing the histopathological results of xenograft tumors derived from 1 and 10 Lgr5-positive cells derived from PLR123 and 10 Lgr5-negative cells derived from PLR123. All tumors showed hierarchical structure, and their histopathological characteristics were very similar to those of the original tumors. The scale bar indicates 100 μm. [Figure 18] Photographs showing phase contrast microscopy of floating colon CSCs and adherent colon CSCs (PLR59 cells). The cells were cultured in serum-free medium supplemented with EGF and FGF. The floating cells tightly interacted with each other to form spheroid-like structures, whereas the adherent cells proliferated without forming cell clusters. The scale bar indicates 25 μm. [Figure 19] Figure 1 shows proliferation of floating and adherent CSCs (PLR59 cells). The number of viable cells after 3 days of culture (black column) is shown as a percentage of the number on day 0 (white column). Results are the average of three experiments. The bar above each column indicates the standard deviation. [Figure 20]Figure 1 shows the results of flow cytometry analysis of reported CSC markers in floating cells and adherent cells (PLR59 cells). Adherent cells were positive for all reported markers, while floating cells were negative for Lgr5 and ALDH. Grey indicates fluorescence intensity or ALDH activity after staining cells with the indicated antibodies, and white indicates fluorescence intensity or ALDH activity after staining cells with control isotype antibodies or ALDH inhibitors. [Figure 21] Photographs showing the results of Western blot analysis of β-catenin, TCF1, TCF3, TCF4, and phosphorylated c-JUN proteins in primary PLR59 cells, non-adherent CSCs, and adherent CSCs. Compared to primary cells, the expression of all proteins was upregulated in Lgr5-positive adherent CSCs. GAPDH was also visualized as a reference for protein loading. [Figure 22] Figure 1 shows the inhibition of proliferation of Lgr5-positive adherent CSCs (PLR59 cells) by FH535 (50 μm) and cardamonin (50 μm). The number of viable cells after 3 days of culture with FH535 (gray column) and cardamonin (black column) is shown as a percentage of the number on day 0 (white column). [Figure 23] Figure 1 shows the proliferation of PLR59 cells in the presence and absence of EGF and FGF. Adherent CSCs were cultured in the presence or absence for 3 days (black columns). The number of viable cells is shown as a percentage of the number on day 0 (white columns). [Figure 24] 1 shows the effect of 5-FU (10 μg / mL) and irinotecan (10 μg / mL) on the proliferation of Lgr5-positive adherent CSCs and Lgr5-negative non-adherent CSCs (PLR59 cells). The number of viable cells after treatment with 5-FU (gray column) or irinotecan (black column) is shown as a percentage of the number of cells cultured in the absence of the drug (white column). [Diagram 25]Figure 1 shows the results of flow cytometry analysis of CSC markers after treatment of adherent CSCs (PLR59 cells) with 5-FU or irinotecan. The top shows 5-FU-treated cells, and the bottom shows irinotecan-treated cells. Grey indicates the fluorescence intensity or ALDH activity after staining cells with the indicated antibodies, and white indicates the fluorescence intensity or ALDH activity after staining cells with control isotype antibodies or ALDH inhibitors. [Figure 26] Figure 1 shows the results of Western blot analysis of E-cadherin and Snail in Lgr5-negative non-adherent CSCs and Lgr5-positive adherent CSCs (PLR59 cells). Non-adherent CSCs expressed high levels of E-cadherin, whereas adherent CSCs expressed high levels of Snail. GADPH was used as a loading control. [Figure 27] Figure 1 shows the expression levels of Lgr5 mRNA in Lgr5-negative floating CSCs and Lgr5-positive CSCs (PLR59 cells). The amount of Lgr5 mRNA in adherent and floating CSCs is shown as a ratio to the amount of Lgr5 mRNA in primary cells of PLR59 and PLR123 xenograft tumors. The Lgr5 mRNA level in adherent CSCs (right: white column) was significantly increased compared to the level in primary cells of xenografts (left: black column), but not in floating CSCs (center: gray column). The amount of Lgr5 mRNA was measured by normalization of GAPDH and ACTB expression and quantitative PCR. All experiments were performed in triplicate. Error bars indicate standard deviation. [Figure 28]Photographs showing the specificity of anti-human Lgr5 monoclonal antibodies (mAbs), 2U2E-2 and 2T15E-2, in DG44 cells transfected with Lgr4, Lgr5, or Lgr6 cDNA, confirmed by immunofluorescence microscopy. Non-transfected parental cells and transfectants were fixed and treated with 5 μg / mL of antibody. Strong fluorescence (green signal on the right) was observed in cells containing Lgr5 cDNA, but not in parental cells or cells containing Lgr4 or Lgr6 cDNA. Scale bar indicates 5 μm. [Figure 29] This figure shows the specificity of anti-human Lgr5 monoclonal antibody (mAb), 2T15E-2, in DG44 cells transfected with Lgr4, Lgr5, or Lgr6 cDNA by flow cytometry. Non-transfected parental cells and transfectants were incubated with monoclonal 2T15E-2 antibody and analyzed by FACS. 2T15E-2 antibody reacted with cells containing Lgr5 cDNA, but not with parental cells or cells containing Lgr4 or Lgr6 cDNA. Expression of Lgr4, Lgr5, and Lgr6 in transfectants was confirmed by Western blot analysis. [Diagram 30] Figure 1 shows the results of flow cytometry analysis of reported cancer stem cell markers after one month of culture of adherent CSCs derived from PLR59 and PLR123 xenografts. Even after one month of in vitro culture, both adherent CSCs derived from PLR59 and PLR123 were positive for all reported cancer stem cell markers. Grey indicates the fluorescence intensity after staining cells with the indicated antibodies or activity of ALDH, white indicates the fluorescence intensity after staining cells with a control isotype antibody or treatment of cells with ALDH inhibitor for ALDH activity. [Diagram 31]After one month of culture, the adherent CSCs derived from PLR59 and PLR123 xenografts were analyzed by flow cytometry (Figure 30) and injected into NOG mice. The number of adherent CSCs shown in the figure was subcutaneously injected into the flank of NOG mice to examine the tumor-initiating activity in NOG mice. This figure shows the results of tumor formation determined 47 days after inoculation. Even subcutaneous injection of 10 adherent CSCs produced tumors at all injection sites, and the histopathological morphology of the tumors was highly similar to that of the original tumor. [Diagram 32] Photographs showing alternating changes in colon CSC phenotypes due to culture conditions and chemotherapy treatment. The sensitivity of Lgr5-positive CSCs to 5-FU and irinotecan was examined. Both 5-FU and irinotecan significantly inhibited the proliferation of Lgr5-positive colon CSCs. After 3 days of exposure to 5-FU or irinotecan, cells resistant to these chemotherapy drugs appeared. The morphology of the drug-resistant cells was densely aggregated. The scale bar indicates 25 μm. [Diagram 33] These photographs show alternating changes in CSC morphology. When Lgr5-negative colon CSCs were dissociated and cultured in flat-bottom plates, some of the cells adhered to the bottom of the plate, became Lgr5-positive, and exhibited a mesenchymal cell-like morphology (left). On the other hand, when Lgr5-positive adherent colon CSCs were cultured in ultra-low attachment plates, some of the cells stopped proliferating and formed spheroid-like structures. The scale bar indicates 10 μm. [Diagram 34] Figure 1 shows the tumor-initiating activity of adherent CSCs in multiple organs. 5x105 adherent CSCs of PLR 123 were injected into the tail vein (n=5). The incidence of tumor formation in various organs 40 days after administration is shown. [Diagram 35] Photographs showing the results of histopathological examination of tumors in the lung, liver, lymph node, and subcutaneous tissue. In the lung, tumor cells showed undifferentiated tumor nests, whereas in the liver and other organs, tumor cells showed ductal structures with multiple stages of differentiation. Scale bar indicates 100 μm. [Diagram 36]Schematic diagram of the proposed model of CSCs. The two distinct states of CSCs are endogenously alternated under environmental changes, such as the presence of anticancer drugs. Previous findings suggest that normal stem cells expressing Lgr5 can be transformed into CSCs by mutations in multiple genes. Highly proliferative CSCs express Lgr5 and undergo EMT. Under certain stress conditions, they can change to an Lgr5-negative quiescent state. Engagement by the niche environment stimulates the transition of CSCs to the differentiation stage. [Figure 37] Fig. 13 shows the results of flow cytometry analysis of EREG in PLR59 and PLR123 primary cells, Lgr5+ cancer stem cells, and Lgr5- cancer stem cells. [Figure 38] Fig. 13 is a graph showing the ADCC activity of anti-EREG antibodies against Lgr5-positive and Lgr5-negative PLR59 cells and Lgr5-positive and Lgr5-negative PLR123 cells. [Figure 39] Photographs showing immunostaining of Lgr5 in a surgically resected tumor (PLR123) and in xenograft models derived from PLR123 at passage 5 (FIG. 39B), passage 10 (FIG. 39C), and passage 15 (FIG. 39D). Tissue sections were stained with anti-Lgr5 antibody. "Original" shows the tumor surgically resected from the patient (FIG. 39A). Scale bar indicates 25 μm. [Diagram 40] Photographs showing histopathological results (HE staining) of xenograft tumors derived from Lgr5-positive cells of PLR59 (FIGS. 40A and B) and PLR123 (FIGS. 40C and D). 10 cells (FIGS. 40A and C) or 1 cell (FIGS. 40B and D) of Lgr5-positive cells from adherent cultures of PLR59 and PLR123 were subcutaneously injected into NOG mice. The histopathological characteristics of all tumors were quite similar to the original tumors. The scale bar indicates 50 μm. [Diagram 41]Photographs showing symmetric cell division of Lgr5-positive cells. Lgr5-positive cells stained with PKH67 dye and cultured for 72 hours were observed under a fluorescent microscope. Figures 41A, B, and C show stained images at 0 hours, 48 ​​hours, and 72 hours, respectively. The scale bar indicates 20 μm. [Diagram 42] Photographs showing symmetric division of Lgr5-positive cells in the absence of Matrigel and serum (FIGS. 42A and B) and asymmetric division of Lgr5-positive cells in the presence of Matrigel and serum (FIGS. 42C and D). FIGS. 42A and 42C show one division figure, and FIGS. 42B and 42D show two to three division figures. [Figure 43-1] The photographs show immunostaining images of colon CSCs that were exposed to irinotecan for three days and became Lgr5 negative, using specific antibodies against HLA-DMA (A) and TMEM173 (B). [Figure 43-2] The photographs show immunostaining images of colon CSCs that were exposed to irinotecan for 3 days and became Lgr5 negative, using specific antibodies against ZMAT3 (C) and GPR110 (D). [Diagram 44] Photographs showing immunostained images of Lgr5 in Lgr5-positive CSCs (PLR123) treated with irinotecan. Immunostained images are shown before irinotecan treatment (FIG. 44A), after irinotecan treatment (FIG. 44B), and from Lgr5-negative cells reseeded and cultured in the absence of irinotecan, Lgr5-positive cells appeared at the latest 4 days after reseeding (FIG. 44C), and increased by 8 days after reseeding (FIG. 44D). Scale bar indicates 50 μm. [Diagram 45] This is a graph showing the measurement of the transcription amount of the Lgr5 gene by quantitative real-time PCR. The mRNA level of Lgr5 was high in Lgr5-positive cells in adherent culture, but decreased under spheroid culture conditions, and was barely detectable in Lgr-negative cells after irinotecan treatment. On the other hand, the mRNA level of the CK20 gene was below the detection level in Lgr5-positive and -negative cells in adherent culture, but increased in Lgr5-positive cells under spheroid culture conditions. [Figure 46]Photographs showing the expression of Lgr5 and CK20 proteins confirmed by tissue immunostaining. Thin sections of Lgr5-positive CSCs (PLR59 (FIG. 46A) and PLR123 (FIG. 46B)) in fixed spheroid cultures were reacted with Lgr5 antibody (2L36) and CK20 antibody (DAKO). The spheroids contained a small number of Lgr5-positive cells and a large number of Lgr5-negative CK20-positive cells. [Figure 47] This is a graph showing the ratio of Lgr5-positive (black line) and Lgr5-negative (gray line) surviving cells to untreated control cells when Lgr5-positive and Lgr5-negative CSCs (PLR59 and PLR123) were cultured in the presence or absence of irinotecan or 5-FU for 3 days at the concentrations shown on the horizontal axis. Lgr5-negative cells were completely resistant to both proliferation inhibitors. [Figure 48] Figure showing the expression of CSC markers. "Before treatment" represents the expression of CSC markers in Lgr5-positive cells obtained from adherent culture derived from PLR123 xenograft model, "after irinotecan treatment" represents the expression of CSC markers in Lgr5-negative cells obtained by irinotecan treatment, and "reseeding after irinotecan removal" represents the expression of CSC markers in Lgr5-negative cells reseeded in medium from which irinotecan has been removed. Grey indicates ALDH activity or fluorescence intensity after staining cells with the indicated antibodies, and white indicates ALDH activity in the presence of ALDH inhibitors. [Figure 49] Figure showing the alternation of Lgr5 positive and negative cells. Lgr5 positive cells collected by FACS and subjected to limiting dilution were seeded and cultured under adherent culture conditions for 3 days in the presence of irinotecan. Meanwhile, Lgr5 negative cells treated with irinotecan and subjected to limiting dilution were seeded and cultured under adherent culture conditions for 4 days in the absence of irinotecan. Expression of Lgr5 was visualized using PE-labeled anti-mouse IgG antibody (shown in red) or AlexaFluo 488-labeled anti-mouse IgG antibody (shown in green). [Figure 50]A heat map of 7 genes (A) whose expression is significantly upregulated in Lgr5-negative cells compared to Lgr5-positive cells, and 20 genes (B) whose expression is elevated in Lgr5-positive and Lgr5-negative cells compared to primary cells derived from xenograft animals. RNA prepared from Lgr5-positive and Lgr5-negative CSCs derived from PLR59 and PLR123, and primary cells isolated from xenograft animals, was analyzed using Affymetrix U133. [Figure 51] Photographs showing the binding of anti-HLA-DMA antibody and anti-EREG antibody to Lgr5-positive CSCs and Lgr5-negative CSCs by tissue immunostaining. Fixed CSCs (PLR123) were treated with anti-HLA-DMA antibody (Dako) and anti-EREG antibody (EP27). Strong fluorescent signals (red for both HLA-DMA and EREG) were detected from Lgr5-negative cells treated with anti-HLA-DMA antibody, whereas faint or no fluorescence was detected from Lgr5-positive cells. Fluorescent signals were detected from both Lgr5-negative and Lgr5-positive cells treated with anti-EREG antibody. [Figure 52] Photographs showing the transition from Lgr5-negative CSCs to Lgr5-positive CSCs at the early stage of tumor formation. Tumors derived from Lgr5-negative CSCs in NOG mice injected with Lgr5-negative CSCs from PLR123 xenograft animals were stained with antibodies against Lgr5 (green), HLA-DMA (red) and EREG (green). On day 5, there were cells with low Lgr5 expression, HLA-DMA positive, and EREG positive, as well as cells with Lgr5 positive, HLA-DMA negative, and EREG positive. The scale bar indicates 10 μm. [Figure 53] Photographs showing reconstruction of tumor hierarchy from Lgr5-negative CSCs. Histological structure (FIG. 53A) and immunofluorescence microscopic observation images using anti-Lgr5 and anti-E-cadherin antibodies (FIG. 53B) are shown. Green indicates the presence of Lgr5, and red indicates the presence of E-cadherin. The scale bar indicates 50 μm. [Figure 54]Photographs showing histopathology (HE) of tumors after irinotecan treatment (first row), immunostaining with Lgr5 antibody (green) and E-cadherin antibody (red) (second row), immunostaining with Lgr5 antibody (green) and HLA-DMA antibody (red) (third row), and immunostaining with EREG antibody (red) (fourth row). Tumors after irinotecan treatment in mice bearing tumors derived from Lgr5-positive CSCs (PLR123) were observed. Irinotecan or vehicle was administered to the mice on days 12, 15, and 18 after tumor implantation. The scale bar indicates 25 μm. [Figure 55] Tumors derived from Lgr5-positive CSCs (PLR123) were implanted into NOG mice and administered irinotecan (120 mg / kg / day) on days 12, 15, and 18. The tumor volumes of vehicle-treated control mice and irinotecan-treated mice are shown by diamonds (◆) and squares (■) or triangles (▲), respectively. Each value represents the mean + standard deviation. [Figure 56] This is a graph showing the number of Lgr5-positive and HLA-DMA-positive cells in xenografted tumor tissues. After the tissue slices were treated with Lgr5 antibody and HLA-DMA antibody, the number of Lgr5-positive and HLA-DMA-positive cells was counted. The values ​​represent the total number of cells counted in each group (n=3). [Figure 57] 1 is a graph showing the antitumor effect of EREG antibody after irinotecan treatment. Irinotecan was administered at a dose of 100 mg / kg / day to SCID mice injected intraperitoneally with PLR123-derived Lgr5-positive cells 6, 10, and 13 days after injection of the Lgr5-positive cells. EREG antibody was then administered at a dose of 10 mg / kg / day 17 and 24 days after injection of the Lgr5-positive cells. Each value represents the mean + standard deviation (n=4). [Figure 58]A diagram showing the antitumor activity of EREG antibodies. FIG. 58A (first row) shows tissue (HE) staining of tumors in a xenograft model in which Lgr5-positive cells derived from PLR123 were intravenously injected into NOG mice, and FIG. 58A (second row) shows tissue immunostaining using EREG antibodies. The arrows in the figure indicate EREG-expressing cells in nodules (second row) and the corresponding HE staining images (first row). FIG. 58B and C show the number of tumors formed in the lungs of SCID-beige mice intravenously injected with Lgr5-positive cells derived from the PLR123 xenograft model. In the EREG antibody administration group, EREG antibodies were administered once a week for five times starting three days after the injection of Lgr5-positive cells. The number of tumor nodules per animal in thin sections of the animal lungs is shown in FIG. 58B. Each symbol (circle) indicates the number of tumor nodules in each animal in the tested group (FIG. 58B). The number of tumor nodules classified by size in the antibody-administered and control groups is shown in Figure 58C. The white columns represent tumors smaller than 100 μm, the gray columns represent tumors of 100-200 μm, and the black columns represent tumors of over 200 μm (Figure 58C). Histological staining (HE staining) of tumors in the EREG antibody-administered and non-administered groups is shown in Figure 58D. The scale bar represents 200 μm. [Figure 59] The same sections of primary and hepatic metastatic colorectal cancer tissues isolated from patients were stained with HE (rows 2 and 4) or antibodies against Lgr5 (green), HLA-DMA (red) and EREG (green) (rows 1 and 3). Lgr5 positivity represents proliferative CSCs, whereas EREG and HLA-DMA positivity represent Lgr5-negative quiescent CSCs. Lgr5-negative and -positive CSCs were detected in both ductal structures and budding regions of primary and hepatic metastatic tumors. Lgr5-positive CSCs were also found in the stromal region as single cells. Similar staining patterns were observed in multiple tumor tissues isolated from different patients. Arrows represent CSCs. Scale bar represents 10 μm. [Figure 60] 1 shows the antitumor effect of anti-CD70 antibody on irinotecan-untreated CSCs, with the horizontal axis showing antibody concentration and the vertical axis showing the percentage of cell proliferation inhibitory activity by anti-CD70 antibody. [Figure 61] 1 is a graph showing the antitumor effect of an anti-EDAR antibody on irinotecan-untreated CSCs, where the horizontal axis shows the antibody concentration and the vertical axis shows the percentage of cell proliferation inhibitory activity by the anti-EDAR antibody. [Figure 62] 1 shows the antitumor effect of anti-FAS antibody on irinotecan-untreated CSCs, with the horizontal axis showing antibody concentration and the vertical axis showing the percentage of cell proliferation inhibitory activity by anti-FAS antibody. [Figure 63] 1 shows the antitumor effect of anti-PVRL4 antibody on irinotecan-untreated CSCs, with the horizontal axis showing antibody concentration and the vertical axis showing the percentage of cell proliferation inhibitory activity by anti-PVRL4 antibody. [Figure 64] 1 shows the antitumor effect of anti-TNFSF9 antibody on irinotecan-untreated CSCs, with the horizontal axis showing antibody concentration and the vertical axis showing the percentage of cell proliferation inhibitory activity by anti-TNFSF9 antibody. [Figure 65] 1 shows the antitumor effect of anti-PROCR antibody on irinotecan-untreated CSCs, with the horizontal axis showing antibody concentration and the vertical axis showing the percentage of cell proliferation inhibitory activity by anti-PROCR antibody. [Figure 66] 1 shows the antitumor effect of anti-EPCAM antibody on irinotecan-untreated CSCs, with the horizontal axis showing antibody concentration and the vertical axis showing the percentage of cell proliferation inhibitory activity by anti-EPCAM antibody. [Figure 67] 1 shows the antitumor effect of anti-FAS antibody on CSCs treated with irinotecan, with the horizontal axis showing antibody concentration and the vertical axis showing the percentage of cell proliferation inhibitory activity by anti-FAS antibody. [Figure 68] 1 shows the antitumor effect of anti-PROM2 antibody on irinotecan-treated CSCs, with the horizontal axis showing antibody concentration and the vertical axis showing the percentage of cell proliferation inhibitory activity by anti-PROM2 antibody. [Figure 69] 1 shows the antitumor effect of anti-PVRL4 antibody on CSCs treated with irinotecan, with the horizontal axis showing antibody concentration and the vertical axis showing the percentage of cell proliferation inhibitory activity by anti-PVRL4 antibody. [Figure 70]1 shows the antitumor effect of anti-TNFSF9 antibody on CSCs treated with irinotecan, with the horizontal axis showing antibody concentration and the vertical axis showing the percentage of cell proliferation inhibitory activity by anti-TNFSF9 antibody. [Figure 71] 1 is a graph showing the antitumor effect of anti-PROCR antibody on CSCs treated with irinotecan, where the horizontal axis indicates antibody concentration and the vertical axis indicates the percentage of cell proliferation inhibitory activity by anti-PROCR antibody. [Figure 72] 1 shows the antitumor effect of anti-EPCAM antibody on CSCs treated with irinotecan, with the horizontal axis showing antibody concentration and the vertical axis showing the percentage of cell proliferation inhibitory activity by anti-EPCAM antibody. [Figure 73] 73A and 73B show the expression levels of the differentiation marker CK20 in CSCs cultured with the addition of BMP4, respectively showing the expression levels of CK20 in PLR59 and PLR123. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] The present invention relates to a cell surface molecule that is specifically expressed in cancer stem cells, and to a pharmaceutical composition (such as an anticancer agent) and a reagent for detecting cancer stem cells that use an antibody against the molecule.

[0021] In the present invention, the term "cancer" refers to a physiological state in a mammal that is typically characterized by uncontrolled cell proliferation, or refers to such a physiological state. In the present invention, the types of cancer are not particularly limited, but include the following: Carcinomas (epithelial cancers) include pancreatic cancer, prostate cancer, breast cancer, skin cancer, gastrointestinal cancer, lung cancer, hepatocellular carcinoma, cervical cancer, uterine cancer, ovarian cancer, fallopian tube cancer, vaginal cancer, liver cancer, bile duct cancer, bladder cancer, ureter cancer, thyroid cancer, adrenal cancer, kidney cancer, or other glandular tissue cancer. Sarcomas (non-epithelial cancers) include liposarcoma, leiomyosarcoma, rhabdomyosarcoma, synovial sarcoma, angiosarcoma, fibrosarcoma, malignant peripheral nerve tumors, gastrointestinal stromal tumors, desmoid tumors, Ewing's sarcoma, osteosarcoma, chondrosarcoma, leukemia, lymphoma, myeloma, and other solid organ tumors, such as melanoma or brain tumors (Kumar V, Abbas AK, Fausio N. Robbins and Cotran Pathologic Basis of Disease. 7th Ed. Unit I: General Pathology, 7: Neoplasia, Biology of tumor growth: Benign and malignant neoplasms. 269-342, 2005). As used herein, a "tumor" refers to any mass of tissue resulting from excessive cell growth or proliferation, whether benign (non-cancerous) or malignant (cancerous), including pre-cancerous lesions.

[0022] In the present invention, cancer stem cells (CSCs) refer to cells having the abilities described in i) and / or ii) below. i) Possess the ability to self-renew. The ability to self-renew refers to the ability of one or both of the two daughter cells produced by division to produce cells that retain the same abilities and degree of differentiation as the parent cell in terms of cell lineage. ii) They can differentiate into multiple types of cancer cells that make up a cancer cell mass. Similar to normal stem cells, multiple types of cancer cells differentiated from cancer stem cells form a hierarchical structure with cancer stem cells at the top of the lineage in terms of cell lineage. Cancer stem cells produce multiple types of cancer cells in stages, forming a cancer cell mass with diverse characteristics.

[0023] Cancer stem cells are cancer cells that have the ability to form cancer and have the pluripotency and self-renewal capabilities of normal stem cells. Cancer stem cells form a hierarchical structure with cancer stem cells at the top. Cancer stem cells produce a variety of cancer cells in stages, forming cancer cell masses with diverse characteristics. A cancer cell mass is a mass formed by cells adhering to each other rather than being separated, as in human tumor tissue, and is a mass constructed of cancer cells, non-cancer cells such as interstitial cells and blood cells, and extracellular matrices such as collagen and laminin.

[0024] The origin of the cancer stem cells to be treated with the pharmaceutical composition of the present invention is not particularly limited, and those derived from mammals such as humans, monkeys, chimpanzees, dogs, cows, pigs, rabbits, rats, and mice can be used, but those derived from humans are preferred, and those derived from human tumor tissue are even more preferred.

[0025] The cancer stem cells detected in the present invention are preferably a cell group that reproduces the hierarchical structure of cancer tissue, and for example, it is possible to confirm that an established cancer cell line produced by transplanting and subculturing a cancer tissue from which the detected cancer stem cells have been collected, preferably into a non-human animal, reproduces the hierarchical structure of such cancer tissue.More preferably, as a non-human animal, it is possible to confirm that an established NOG cancer cell line produced by transplanting and subculturing a cancer tissue into an immunodeficient animal, most preferably into a NOG mouse or the like lacking functional T cells, B cells, and natural killer cells, reproduces the hierarchical structure of such cancer tissue.

[0026] Furthermore, the cancer stem cells detected in the present invention may be spheroids (cell masses) formed by spheroid culture. Spheroid culture refers to seeding cells in a culture vessel such as a non-adhesive or low-adhesive culture flask, plate, or dish using a medium capable of culturing cancer stem cells, and then culturing the cells in a three-dimensional suspended state, and the cell masses formed by this method are called spheroids.

[0027] NOG established cancer cell lines can be prepared by methods known to those skilled in the art, for example, the method described in Fujii E. et al., Pathol int. 2008; 58: 559-567, and can be established by physically mincing human colon cancer, gastric cancer, lung cancer, breast cancer, pancreatic cancer, etc., excised by surgery with scissors, and subcutaneously implanting and passage in NOG mice. NOG established cancer cell lines maintain the characteristics of the original human cancer tissue even after passage.

[0028] In the present invention, cancer stem cells can be selected using cell markers, such as Lgr5 (leucine-rich repeat-containing G protein-coupled receptor 5), CD133, CD44, EpCAM, CD166, CD24, CD26, and CD29.

[0029] The present invention relates to a molecule expressed in cancer stem cells that are positive for the expression of the cell marker Lgr5 and are characterized by being adherent and highly proliferative under serum-free culture conditions. Hereinafter, such cancer stem cells may be referred to as "Lgr5-positive highly proliferative cancer stem cells."

[0030] The present invention also relates to a molecule expressed in cancer stem cells that are negative for the expression of the cell marker Lgr5, and that are non-adherent and low-proliferative under serum-free culture conditions. Hereinafter, such cancer stem cells may be referred to as "Lgr5-negative low-proliferative cancer stem cells."

[0031] The medium or culture solution used for culturing the cancer stem cells of the present invention is not particularly limited as long as it is serum-free and can be any medium or culture solution capable of culturing cancer stem cells. For example, a conventionally known basal culture solution containing EGF, bFGF, hLIF, HGF, NGF, NSF-1, TGFβ, TNFα, heparin, BSA, insulin, transferrin, putrescine, selenite, progesterone, hydrocortisone, D-(+)-glucose, sodium bicarbonate, HEPES, L-glutamine, or N-acetylcysteine ​​or a mixture thereof can be used as the culture solution. The concentration of EGF is not particularly limited, but is 0.1 to 100 ng / mL, preferably 0.5 to 50 ng / mL, and more preferably 1 to 20 ng / mL. The concentration of bFGF is not particularly limited, but is 0.1 to 100 ng / mL, preferably 0.5 to 50 ng / mL, and more preferably 1 to 20 ng / mL. The concentration of hLIF is not particularly limited, but is 0.1 to 100 ng / mL, preferably 0.5 to 50 ng / mL, more preferably 1 to 20 ng / mL. The concentration of HGF is not particularly limited, but is 0.1 to 100 ng / mL, preferably 1 to 50 ng / mL. The concentration of NGF is not particularly limited, but is 0.1 to 100 ng / mL, preferably 1 to 50 ng / mL. The concentration of NSF-1 is not particularly limited, but is 0.1 to 100 ng / mL, preferably 1 to 50 ng / mL. The concentration of TGFβ is not particularly limited, but is 0.1 to 100 ng / mL, preferably 1 to 50 ng / mL. The concentration of TNFα is not particularly limited, but is 0.1 to 100 ng / mL, preferably 1 to 50 ng / mL. The concentration of Heparin is not particularly limited, but is 10 ng / mL to 10 μg / mL, preferably 2 to 5 μg / mL. The concentration of BSA is not particularly limited, but is 0.1 to 10 mg / mL, preferably 1 to 8 mg / mL. The concentration of insulin is not particularly limited, but is 1 to 100 μg / mL, preferably 10 to 50 μg / mL. The concentration of transferrin is not particularly limited, but is 10 to 500 μg / mL, preferably 50 to 200 μg / mL.The concentration of putrescine is not particularly limited, but is 1 to 50 μg / mL, preferably 10 to 20 μg / mL. The concentration of selenite is not particularly limited, but is 1 to 50 nM, preferably 20 to 40 nM. The concentration of progesterone is not particularly limited, but is 1 to 50 nM, preferably 10 to 30 nM. The concentration of hydrocortisone is not particularly limited, but is 10 ng / mL to 10 μg / mL, preferably 100 ng / mL to 1 μg / mL. The concentration of D-(+)-glucose is not particularly limited, but is 1 to 20 mg / mL, preferably 5 to 10 mg / mL. The concentration of sodium bicarbonate is not particularly limited, but is 0.1 to 5 mg / mL, preferably 0.5 to 2 mg / mL. The concentration of HEPES is not particularly limited, but is 0.1 to 50 mM, preferably 1 to 20 mM. The concentration of L-Glutamine is not particularly limited, but is 0.1 to 10 mM, preferably 1 to 5 mM. The concentration of N-acetylcysteine ​​is not particularly limited, but is 1 to 200 μg / mL, preferably 10 to 100 μg / mL. The known basal culture medium is not particularly limited as long as it is suitable for culturing cancer cells that are the source of cancer stem cells, but examples thereof include DMEM / F12, DMEM, F10, F12, IMDM, EMEM, RPMI-1640, MEM, BME, Mocoy's 5A, and MCDB131. Among these, DMEM / F12 is preferred.

[0032] The most preferred stem cell medium is a DMEM / F12 medium supplemented with final concentrations of 20 ng / mL human EGF, 10 ng / mL human bFGF, 4 μg / mL heparin, 4 mg / mL BSA, 25 μg / mL human insulin, and 2.9 mg / mL glucose.

[0033] As described herein, Lgr5-positive highly proliferative cancer stem cells exhibit the properties of mesenchymal cells (mesenchymal cells). On the other hand, Lgr5-negative low proliferative cancer stem cells exhibit the properties of epithelial cells, as described herein. In the present invention, epithelial cells refer to cells that constitute epithelial tissue in vivo.

[0034] The origin of the cancer stem cells in the present invention is not particularly limited, but is preferably solid cancer, more preferably digestive system cancer, such as esophageal cancer, gastric cancer, duodenal cancer, pancreatic cancer, bile duct cancer, gallbladder cancer, biliary tract cancer, colon cancer, colon cancer, and rectal cancer, preferably colon cancer.

[0035] Furthermore, in the present invention, cancer stem cells are preferably positive for one or more of the cell markers CD133, CD44, EpCAM, CD166, CD24, CD26, and CD29, and more preferably positive for CD133, CD44, EpCAM, CD166, CD24, CD26, and CD29.

[0036] Furthermore, in the present invention, ALDH (acetaldehyde dehydrogenase) activity can be used as a cell marker. In the present invention, Lgr5-positive adherent cancer stem cells are positive for the cell marker ALDH activity, and Lgr5-negative cancer stem cells are negative for ALDH activity.

[0037] Furthermore, in the present invention, one or more of HLA-DMA, TMEM173, ZMAT3, or GPR110 can be used as a cell marker. Lgr5-positive adherent cancer stem cells are negative for any of the cell markers HLA-DMA, TMEM173, ZMAT3, or GPR110, and Lgr5-negative cancer stem cells are positive for any of the cell markers HLA-DMA, TMEM173, ZMAT3, or GPR110.

[0038] In the present invention, cancer stem cells are preferred that have the characteristic of recapitulating the hierarchical structure of cancer tissue. In the present invention, hierarchical structure refers to the histopathological detection of part of the characteristic inherent structure seen in normal tissue in the tumor structure originating from that tissue. Generally, this hierarchical structure is reproduced to a higher degree in well-differentiated cancers. For example, in the case of tumors of organs that form glandular cavities (gastric cancer, colon cancer, pancreatic cancer, liver cancer, bile duct cancer, breast cancer, lung adenocarcinoma, prostate cancer, etc.), lumen formation and the appearance of mucus cells are observed, and in the case of tumors with a squamous epithelial structure (squamous cell carcinoma of the lung, skin, vaginal mucosa, etc.), the formation of a stratified epithelial structure and a tendency to keratinization are observed. On the other hand, in poorly differentiated cancers, the hierarchical structure is not fully reproduced and is highly atypia-prone (Kumar V, Abbas AK, Fausio N. Robbins and Cotran Pathologic Basis of Disease. 7th Ed. Unit I: General Pathology, 7: Neoplasia, Biology of tumor growth: Benign and malignant neoplasms. 272-281, 2005). This hierarchical structure is thought to be reproduced as a result of various biological reactions of cancer, so the utility of cancer stem cells that reproduce this structure is considered to be high. "Reproducing the hierarchical structure" means that the characteristic inherent structure of the original cancer stem cells is observed in the same way even after isolating or inducing the cancer stem cells.

[0039] In the present invention, the cancer stem cells are preferably cancer stem cells having epithelial to mesenchymal transition (EMT). In the present invention, the term "epithelial to mesenchymal transition" includes both the transition of epithelial cells by acquiring the properties of mesenchymal cells, and the transition of mesenchymal cells by acquiring the properties of epithelial cells. EMT does not occur in normal cells except during embryogenesis. Epithelial cells that are tightly bound to each other and exhibit polarity give rise to mesenchymal cells that are more loosely bound to each other, exhibit loss of polarity, and have the ability to migrate. These mesenchymal cells can not only diffuse into tissues surrounding the primary tumor, but also detach from the tumor, invade blood vessels and lymphatic vessels, migrate to new locations, and divide there to form further tumors. The formation of further tumors helps to explain drug resistance or metastasis or recurrence of cancer.

[0040] The present invention also provides pharmaceutical compositions comprising, as an active ingredient, an antibody that binds to a molecule expressed in a substantially homogenous cancer stem cell population, including the above-mentioned cancer stem cells of the present invention. The term "substantially homogeneous" refers to a method such as that described in Hu Y & Smyth GK., J Immunol Methods. 2009 Aug 15;347(1-2):70-8 or Ishizawa K & Rasheed ZA. et al., Cell Stem Cell. 2010 Sep 3;7(3):279-82, in which 1000 cells, 100 cells, or 10 cells are transplanted into an immunodeficient animal and the frequency of formation of a cancer cell population is analyzed using Extreme Limiting Dilution Analysis (Hu Y & Smyth GK., J Immunol Methods. 2009 Aug 15;347(1-2):70-8), and the frequency of cancer stem cell formation is found to be 1 / 20 or more, preferably 1 / 10 or more, more preferably 1 / 5 or more, even more preferably 1 / 3 or more, even more preferably 1 / 2 or more, and most preferably 1 / 1.

[0041] In the present invention, a cancer stem cell population can be prepared, for example, by culturing cells containing the cancer stem cells described herein or a cell group containing cancer stem cells.

[0042] In the present invention, the term "adherent culture" refers to culturing and subculturing cells in an adherent state after seeding them in a culture vessel for adherent culture, and is a culture in which floating cells are removed. Cells that have proliferated confluently are detached using Accutase and subcultured in a new adherent culture flask, adherent culture plate, or adherent culture dish for continued culture. The culture vessel for adherent culture is not particularly limited as long as it is a vessel used for adherent culture, and can be appropriately selected from flasks for adherent culture or highly adhesive, plates for adherent culture or highly adhesive, flat-bottom plates for adherent culture or highly adhesive, dishes for adherent culture or highly adhesive, etc. The medium used for the adherent culture is not particularly limited, but it is preferable to use a serum-free stem cell medium. In the present invention, the term "adherent" refers to the property of cells adhering to a culture vessel when cultured in the culture vessel for adherent culture.

[0043] In the present invention, suspension culture refers to culturing and subculturing cells in a suspended state after seeding them in a culture vessel for suspension culture, and is a culture in which adherent cells are removed. Cells that have proliferated confluently are subcultured to a new low-adhesion cell culture flask, ultra-low-adhesion cell culture flask, low-adhesion plate, ultra-low-adhesion plate, low-adhesion dish, or ultra-low-adhesion dish to continue the culture. The culture vessel for suspension culture is not particularly limited as long as it is a vessel used for suspension culture, and low-adhesion cell culture flask, ultra-low-adhesion cell culture flask, low-adhesion plate, ultra-low-adhesion plate, low-adhesion dish, or ultra-low-adhesion dish can be appropriately selected and used. The medium used for suspension culture is not particularly limited, but it is preferable to use a serum-free stem cell medium. Note that it is preferable to proliferate the cell population containing cancer stem cells before performing adherent culture or suspension culture. In the present invention, the term "suspended" refers to the property of cells being able to be cultured in a suspended state without adhering to the culture vessel when the cells are cultured in a culture vessel for suspension culture.

[0044] Proliferating a cell population refers to, for example, proliferation by spheroid culture or transplantation into a non-human animal and subculturing, but is not particularly limited to these.

[0045] As described herein, for transplantation, immunodeficient animals can be used as non-human animals because they are less likely to cause rejection. As immunodeficient animals, non-human animals lacking functional T cells, such as nude mice and nude rats, and non-human animals lacking functional T cells and B cells, such as SCID mice and NOD-SCID mice, are preferred, and mice lacking T cells, B cells, and NK cells, which have excellent transplantability (including, for example, NOG mice, etc.) are more preferred. The age of the non-human animal is preferably 4 to 100 weeks in the case of, for example, an athymic nude mouse, a SCID mouse, a NOD / SCID mouse, or a NOG mouse. NOG mice can be produced, for example, by the method described in WO 2002 / 043477, and can also be obtained from the Central Institute for Experimental Animals or The Jackson Laboratory (NSG mice).

[0046] The cells to be transplanted may be any type, such as a cell mass, a tissue fragment, individual dispersed cells, isolated and cultured cells, or cells that have been transplanted into another animal and then isolated again from that animal. Dispersed cells are preferred. The number of cells to be transplanted is 10 6 The following numbers are sufficient, but more than these numbers may also be transplanted.

[0047] Subcutaneous transplantation is a preferred transplantation site because the transplantation procedure is simple, but the transplantation site is not particularly limited, and it is preferable to select an appropriate transplantation site depending on the animal to be used. The transplantation procedure for the NOG-established cancer cell line is not particularly limited, and can be performed according to a conventional transplantation procedure.

[0048] Cancer stem cells or cancer stem cell populations can be prepared, for example, by culturing cancer tissue collected from a patient in an adherent or floating culture using a serum-free stem cell medium. Alternatively, cancer tissue collected from a patient can be cultured as spheroids, and then cultured in an adherent or floating culture using a serum-free stem cell medium. Alternatively, cancer tissue collected from a patient can be transplanted and passaged into a non-human animal, and then cultured in an adherent or floating culture using a serum-free stem cell medium. Furthermore, a method can be used in which NOG-established cancer cell lines prepared by transplanting and passaged cancer tissue collected from a patient into NOG mice can be cultured in an adherent or floating culture using a serum-free stem cell medium.

[0049] The cancer stem cells or cancer stem cell population of the present invention can be used in methods for screening pharmaceuticals or anticancer agents. As one embodiment of the pharmaceutical screening method of the present invention, there is provided a method comprising the following steps (a) to (c): (a) preparing a substantially homogenous cancer stem cell population containing Lgr5-positive adherent cancer stem cells; (b) contacting the cancer stem cell population or cancer stem cells contained in the cancer stem cell population with a test substance; (c) detecting a change in the biological properties of the cancer stem cell population or cancer stem cells contacted with the test substance.

[0050] In this method, first, a substantially homogeneous cancer stem cell population containing Lgr5-positive adherent cancer stem cells or a substantially homogeneous cancer stem cell population containing Lgr5-negative cancer stem cells is prepared. Then, the prepared cancer stem cell population or the cancer stem cells contained in the cancer stem cell population are contacted with a test substance. In this method, the method of contacting the cancer stem cell population or the cancer stem cells contained in the cancer stem cell population with the test substance is not particularly limited. For example, the test substance can be contacted with cultured cells of a cancer stem cell population or cancer stem cells contained in a cancer stem cell population. This treatment can be performed by adding the test substance to a cell culture solution or a cell extract of the cells. When the test substance is a protein, for example, a vector containing DNA encoding the protein can be introduced into the cancer stem cell population or the cancer stem cells contained in the cancer stem cell population, or the vector can be added to a cell extract of the cancer stem cell population or the cancer stem cells contained in the cancer stem cell population. In addition, for example, a two-hybrid method using yeast or animal cells can be used.

[0051] In this method, the change in biological characteristics of the cancer stem cell population or cancer stem cells treated with the test substance is then detected. Here, examples of the change in biological characteristics include a change in proliferation ability, a change in the number of live cells, a change in tissue structure that is characteristically observed in the cancer progression process of the cancer stem cell population or cancer stem cells, and a change in the expression of DNA, RNA, protein, or metabolites contained in the cancer stem cell population or cancer stem cells. The change in biological characteristics can be detected, for example, by the following method.

[0052] The expression confirmation of DNA, RNA, protein, peptide, and metabolites is not particularly limited and can be performed according to a conventional expression confirmation method. Examples of RNA include microRNA, siRNA, tRNA, snRNA, mRNA, and non-coding RNA. For example, the mRNA of each gene can be extracted according to a standard method, and the transcription level of each gene can be measured by performing Northern hybridization or RT-PCR using this mRNA as a template. Furthermore, it is also possible to measure the expression level of each gene using DNA array technology. In addition, the translation level of the gene can be measured by recovering a fraction containing a protein encoded by each gene according to a standard method and detecting the expression of each protein by electrophoresis such as SDS-PAGE. In addition, it is also possible to measure the translation level of the gene by performing Western blotting using an antibody against each protein and detecting the expression of each protein. These methods can be used to screen pharmaceuticals (pharmaceutical compositions).

[0053] Preferred examples of DNA, RNA, and proteins contained in cancer stem cell populations or cancer stem cells that are characteristically found in the cancer progression process of cancer stem cells include proteins or polypeptides set forth in any of SEQ ID NOs:1 to 6, or polynucleotides encoding such proteins or polypeptides.

[0054] For example, when the biological characteristics of the cancer stem cell population or cancer stem cells after treatment with the test substance show no change compared to before treatment, or the rate of change is reduced, the test substance is considered to be useful as a pharmaceutical product (pharmaceutical composition) that has the function of suppressing cancer recurrence or metastasis (e.g., a cancer recurrence suppressor, a post-chemotherapy treatment adjuvant agent, a post-operative adjuvant therapy agent, an anticancer agent, or a cancer metastasis suppressor), and such test substances can be selected as active substances that have a therapeutic or preventive effect against cancer diseases. In this way, pharmaceutical products (pharmaceutical compositions) that have the function of suppressing the progression of cancer are used as cancer recurrence suppressors, post-chemotherapy treatment adjuvant agents, post-operative adjuvant therapy agents, anticancer agents, or cancer metastasis suppressors. The anticancer agent of the present invention may be used, for example, against cancers that are resistant to drugs or chemotherapeutic agents. In other words, the pharmaceutical product (pharmaceutical composition) of the present invention also includes drug-resistant or chemotherapeutic agent-resistant cancer treatment agents.

[0055] In the present invention, the pharmaceutical agent (pharmaceutical composition) is not particularly limited to an anticancer agent or a metastasis or recurrence inhibitor, and can also be used as an angiogenesis inhibitor, a cell proliferation inhibitor, etc. The pharmaceutical agent (pharmaceutical composition) of the present invention may be used at the same time as a chemotherapeutic agent or after treatment with a chemotherapeutic agent. The pharmaceutical agent is not particularly limited, and examples thereof include protein drugs, nucleic acid drugs, low molecular weight drugs, and cellular drugs.

[0056] As another embodiment of the screening method of the present invention, there is provided a screening method for a pharmaceutical product (pharmaceutical composition), comprising the following steps (a) to (c): (a) preparing a substantially homogenous cancer stem cell population containing Lgr5-negative non-adherent cancer stem cells; (b) contacting the cancer stem cell population or cancer stem cells contained in the cancer stem cell population with a test substance (c) detecting a change in the biological properties of the cancer stem cell population or cancer stem cells contacted with the test substance.

[0057] In this method, a substantially homogeneous cancer stem cell population containing Lgr5-negative floating cancer stem cells is first prepared.Then, the prepared cancer stem cell population or the cancer stem cells contained in the cancer stem cell population are treated with a test substance.Then, changes in the biological properties of the cancer stem cell population or the cancer stem cells treated with the test substance are detected.

[0058] Such DNA, RNA, and proteins contained in a cancer stem cell population or cancer stem cells that are characteristically found in the cancer progression process of the cancer stem cell population or cancer stem cells are preferably a protein or polypeptide set forth in any one of SEQ ID NOs: 1 to 8, or a polynucleotide encoding the protein or polypeptide. In a non-limiting embodiment of the present invention, a protein or polypeptide set forth in SEQ ID NOs: 1 to 6, or a polynucleotide encoding the protein or polypeptide, may be used. In a different non-limiting embodiment of the present invention, a protein or polypeptide set forth in SEQ ID NO: 7 or 8, or a polynucleotide encoding the protein or polypeptide, may be used.

[0059] The pharmaceutical (pharmaceutical composition) obtained by the screening method can be used as an anticancer drug, although it is not particularly limited. That is, when the biological properties of the cancer stem cell population or cancer stem cells after treatment with the test substance do not change compared to before the treatment, or the rate of change is reduced, the test substance is considered to be useful as a pharmaceutical having the function of suppressing cancer recurrence or metastasis (e.g., a cancer recurrence suppressor, a post-chemotherapy treatment adjuvant agent, a post-operative adjuvant therapy agent, an anticancer drug, or a cancer metastasis suppressor), and these test substances can be selected as effective substances having a therapeutic or preventive effect on cancer diseases. Thus, pharmaceuticals (pharmaceutical compositions) having the function of suppressing the progression of cancer are used as cancer recurrence suppressors, post-chemotherapy treatment adjuvant agents, post-operative adjuvant therapy agents, anticancer drugs, or cancer metastasis suppressors. Furthermore, the pharmaceuticals (pharmaceutical compositions) of the present invention include cancer therapeutic agents for Lgr5-negative cancers that contain as an active ingredient at least one antibody that binds to a protein set forth in SEQ ID NOs: 1 to 8. Here, Lgr5-negative cancers include cancers that are resistant to drugs or chemotherapeutic agents.

[0060] In yet another embodiment of the screening method of the present invention, a method is provided that uses a non-human animal administered with the cancer stem cell population of the present invention or cancer stem cells contained in the cancer stem cell population and a test substance. Specifically, the present invention provides a screening method for a pharmaceutical product (pharmaceutical composition) comprising the following steps (a) to (c): (a) preparing a substantially homogenous cancer stem cell population containing Lgr5-positive adherent cancer stem cells; (b) administering to a non-human animal the cancer stem cells contained in the cell population or the cancer stem cell population and a test substance. (c) detecting the formation of a tumor in said non-human animal.

[0061] In this method, a substantially homogeneous cancer stem cell population containing Lgr5-positive adherent cancer stem cells is first prepared.Then, the prepared cancer stem cell population or the cancer stem cells contained in the cancer stem cell population and a test substance are administered to a non-human animal.

[0062] In this method, the method of administering the test substance to the non-human animal is not particularly limited. Depending on the type of the test substance to be administered, oral administration or parenteral administration such as subcutaneous, intravenous, topical, transdermal or enteral (rectal) administration can be appropriately selected.

[0063] In addition, in this method, the method of administering a cancer stem cell population or cancer stem cells to a non-human animal is not particularly limited. An appropriate administration method can be selected depending on the cell population to be administered, but subcutaneous administration or intravenous administration is preferable.

[0064] The method then involves detecting the formation of a tumor in the non-human animal. The evaluation of the test substance can be performed by extracting the tissue administered with the cancer stem cell population or cancer stem cells and the test substance from the non-human animal, observing the histological characteristics of the administered tissue, and determining whether a tumor is formed. If no tumor is formed, the test substance is considered to be useful as a pharmaceutical agent (e.g., an anticancer agent or an agent for suppressing cancer metastasis or cancer recurrence) that has the function of suppressing the progression or metastasis of cancer, and the test substance can be selected as an effective substance for treating or preventing cancer disease. That is, the pharmaceutical agent (pharmaceutical composition) obtained by the screening method can be used as an anticancer agent or an agent for suppressing cancer metastasis or cancer recurrence, although it is not particularly limited.

[0065] The "test substance" in the method of the present invention is not particularly limited, and examples thereof include single compounds such as natural compounds, organic compounds, inorganic compounds, proteins, antibodies, peptides, and amino acids, as well as compound libraries, expression products of gene libraries, cell extracts, cell culture supernatants, fermentation microbial products, marine organism extracts, plant extracts, prokaryotic cell extracts, eukaryotic single cell extracts, and animal cell extracts. These may be purified products or crude products such as extracts of plants, animals, or microorganisms. The method for producing the test substance is also not particularly limited, and the test substance may be isolated from a natural product, chemically or biochemically synthesized, or prepared by genetic engineering. Antisense and RNAi molecules designed by known methods based on a partial sequence of a polynucleotide encoding any of the proteins described in SEQ ID NOs: 1 to 8 may also be used as appropriate. The test substance may be appropriately labeled as necessary. Examples of the label include radioactive labels and fluorescent labels. In addition to the test substances described above, mixtures of multiple types of these labels are also included in the test substances of the present invention.

[0066] Furthermore, the present invention also provides pharmaceuticals such as vaccines containing partial peptides of any of the proteins set forth in SEQ ID NOs: 1 to 8, and a method for screening the vaccine. A suitable example of such a screening method is a method for measuring the cytotoxic activity targeting the cancer stem cells disclosed in the present invention in a test tube using cytotoxic T cells (CTLs) induced by the cancer vaccine of the present invention. Specifically, human peripheral blood is centrifuged in a Ficoll-Conray density gradient to collect peripheral blood mononuclear cells (PBMCs), from which adherent cells and non-adherent cells are separated. The adherent cells are incubated in AIM-V (Gibco) with 100 ng / ml GM-CSF (Novartis) and 10 IU / ml IL-4 (Gibco BRL). These cells are used as antigen-presenting cells (APCs). The non-adherent cells are incubated in AIM-V with 30 to 100 IU / ml recombinant IL-4 (Ajinomoto). On days 7 to 10, a partial peptide (final concentration 30 μg / ml) of any of the proteins described in SEQ ID NOs: 1 to 8 provided by the present invention is added to the APC. One day later, recombinant TNF-α and IFN-α (Sumitomo Pharmaceuticals) are added to mature the APC. Then, the irradiated APC and CD8 positive cells separated from autologous non-adherent cells are mixed in AIM-V that does not contain IL-2. After two days of incubation, IL-2 (Takeda Pharmaceuticals) is added to the culture at a final concentration of 100 IU / ml. Every seven days, CD8 positive cells are stimulated using autologous PHA blasts (PHA-stimulated T cells) stimulated with PHA, a T cell mitogen, as APC. Fresh medium containing 100 IU / ml of IL-2 is added to the culture for each stimulation. CTLs on day 28 are used for activity testing. Lgr5-positive highly proliferative cancer stem cells and Lgr5-negative low-proliferative cancer stem cells provided by the present invention can be used as target cells for CTLs. Cytotoxicity was measured according to the ADCC activity measurement method. 51 It can be evaluated by measuring the uptake activity of Cr-sodium chromate.

[0067] Furthermore, the pharmaceuticals selected by the screening method of the present invention can be selected as more effective and practical preventive or therapeutic active substances by further conducting other efficacy tests, safety tests, etc., as necessary, and further by conducting clinical trials on human cancer patients. The pharmaceuticals selected in this way can also be industrially produced by chemical synthesis, biochemical synthesis (fermentation), or genetic engineering, based on the results of their structural analysis.

[0068] High proliferation ability means that when cultured in serum-free medium supplemented with EGF and FGF using the method described herein, the doubling time is 6 days or less, preferably 4 days or less, and more preferably 3 days or less. Low proliferation ability means that when cultured in serum-free medium supplemented with EGF and FGF using the method described herein, the doubling time is 7 days or more, preferably 14 days or more, and more preferably no significant proliferation is observed.

[0069] In preparing the Lgr5-positive highly proliferative cancer stem cells and the Lgr5-negative low-proliferative cancer stem cells, the cell marker Lgr5 can be used to separate them. The separation method includes a method of separating a cell population containing cancer stem cells using an Lgr5 antibody, a method of preparing a substantially homogeneous cancer stem cell population by first culturing the population containing cancer stem cells in adherent or suspension culture, and then separating the cancer stem cells using an Lgr5 antibody, and a method of preparing a substantially homogeneous cancer stem cell population by culturing the population containing cancer stem cells in a medium containing or not containing a proliferation inhibitor, and then separating the cancer stem cells using an Lgr5 antibody. Either method may be used in the present invention. Preferably, cells are separated from cancer tissues that have been passaged for three or more generations in NOG mice, and adherent culture is performed in a serum-free stem cell medium to prepare Lgr5-positive highly proliferative cancer stem cells. The obtained Lgr5-positive cancer stem cells can then be treated with irinotecan (adding 10 μg / ml of irinotecan to serum-free stem cell medium and culturing for 3 days) and maintained under various stresses, such as exposure to proliferation inhibitors, to prepare Lgr5-negative low-proliferative cancer stem cells.

[0070] Furthermore, the present invention provides a method for screening pharmaceutical products, which comprises contacting a test substance with cancer stem cells with different proliferative potential induced by the method provided by the present invention. That is, the present invention provides a method for screening pharmaceutical products, which comprises contacting a test substance with cancer stem cells with high proliferative potential or cancer stem cells with low proliferative potential induced by a method of inducing cancer stem cells with low proliferative potential into cancer stem cells with high proliferative potential, or inducing cancer stem cells with high proliferative potential into cancer stem cells with low proliferative potential, and detecting changes in the biological properties of the cancer stem cells.

[0071] That is, as described herein, cancer stem cells having high proliferation ability can be obtained by maintaining cancer stem cells having low proliferation ability under various stresses such as suspension culture and contact with a proliferation inhibitor. For example, cancer stem cells having high proliferation ability can be induced to cancer stem cells having low proliferation ability by suspension culture of cancer stem cells having high proliferation ability. Alternatively, cancer stem cells having high proliferation ability can be induced to cancer stem cells having low proliferation ability by culturing cancer stem cells having high proliferation ability in a low adhesion or ultra-low adhesion cell culture vessel such as a low adhesion plate, ultra-low adhesion plate, low adhesion dish, ultra-low adhesion dish, low adhesion flask, or ultra-low adhesion cell culture flask. That is, cancer stem cells having low proliferation ability can also be produced by culturing cancer stem cells having high proliferation ability in a low adhesion or ultra-low adhesion cell culture vessel such as a low adhesion plate, ultra-low adhesion plate, low adhesion dish, ultra-low adhesion dish, low adhesion flask, or ultra-low adhesion cell culture flask.

[0072] In a non-limiting embodiment, cancer stem cells with high proliferation ability can be induced to cancer stem cells with low proliferation ability by using a proliferation inhibitor such as 5-FU or irinotecan. That is, cancer stem cells with low proliferation ability can be produced by exposing cancer stem cells with high proliferation ability to a proliferation inhibitor such as 5-FU or irinotecan. Exposure to the proliferation inhibitor can be performed under any environment in a living body, such as culture in a test tube or in a transplanted non-human animal. In this case, a person skilled in the art can appropriately select the exposure amount of the cancer stem cells to the proliferation inhibitor. In addition, cancer stem cells with high proliferation ability can be produced by reseeding cancer stem cells with low proliferation ability in a medium that does not contain a proliferation inhibitor such as 5-FU or irinotecan. In another non-limiting embodiment, cancer stem cells with high proliferation ability can also be produced by discontinuing the administration of a proliferation inhibitor to a non-human animal that has cancer stem cells with low proliferation ability.

[0073] In addition, cancer stem cells with low proliferation ability can be induced to have high proliferation ability by adherent culture. Alternatively, cancer stem cells with low proliferation ability can be induced to have high proliferation ability by culturing them in a cell culture vessel with high adhesion that is not low adhesion, such as a flat-bottom plate, plate, adherent culture plate, adherent culture flask, dish, adherent culture dish, etc. That is, cancer stem cells with high proliferation ability can be produced by culturing cancer stem cells with low proliferation ability in a cell culture vessel with high adhesion that is not low adhesion, such as a flat-bottom plate, plate, adherent culture plate, adherent culture flask, dish, adherent culture dish, etc.

[0074] The present invention also relates to a reagent for detecting cancer cells. The reagent for detecting cancer cells of the present invention preferably contains, as an active ingredient, at least one antibody that binds to a protein shown in SEQ ID NO:1 to 8 (a protein consisting of an amino acid sequence shown in any one of SEQ ID NO:1 to 8). Another embodiment of the reagent of the present invention includes a reagent for detecting Lgr5-positive cancer cells, which preferably contains at least one antibody that binds to a protein shown in SEQ ID NO:1 to 6 (a protein consisting of an amino acid sequence shown in any one of SEQ ID NO:1 to 6). Yet another embodiment of the reagent of the present invention includes a reagent for detecting Lgr5-negative cancer cells, which preferably contains at least one antibody that binds to a protein shown in SEQ ID NO:1 to 8 (a protein consisting of an amino acid sequence shown in any one of SEQ ID NO:1 to 8).

[0075] <Proliferation inhibitors> In a non-limiting embodiment, the growth inhibitor may suitably include a DNA damaging agent, an antimitotic agent, and / or an antimetabolite.The DNA damaging agent may be an alkylating agent, a topoisomerase inhibitor, and / or a DNA intercalator.The growth inhibitor may suitably include, but is not limited to, carboplatin (DNA alkylating agent), etoposide (topoisomerase II inhibitor), doxorubicin (DNA intercalator), docetaxel (antimitotic agent), and Gemzar (gemcitabine, an antimetabolite).

[0076] The alkylating agent may be selected from at least one of the following: chlorambucil, cyclophosphamide, ifosfamide, mechlorethamine, melphalan, uracil mustard, thiotepa, busulfan, carmustine, lomustine, streptozocin, carboplatin, cisplatin, satraplatin, oxaliplatin, altretamine, ET-743, XL119 (becatecarin), dacarbazine, chlormethine, bendamustine, trofosfamide, uramustine, fotemustine, nimustine, prednimustine, ranimustine, semustine, nedaplatin, triplatin tetranitrate, mannosulfan, treosulfan, temozolomide, carboquone, triaziquone, triethylenemelamine, and procarbazine.

[0077] The topoisomerase inhibitor may be selected from at least one of the following: doxorubicin (Doxil), daunorubicin, epirubicin, idarubicin, anthracenedione (Novantrone), mitoxantrone, mitomycin C, bleomycin, dactinomycin, plicatomycin, irinotecan (Camptosar), camptothecin, rubitecan, belotecan, etoposide, teniposide, and topotecan (Hycamptin), etc. may be used.

[0078] The DNA intercalator may be at least one topoisomerase inhibitor selected from proflavine, doxorubicin (adriamycin), daunorubicin, dactinomycin, thalidomide, and the like.

[0079] The antimitotic agent may be selected from at least one of the following: At least one topoisomerase inhibitor selected from paclitaxel (Abraxane) / Taxol, docetaxel (Taxotere), BMS-275183, Xyotax, Tocosal, vinorlebine, vincristine, vinblastine, vindesine, vinzolidine, etoposide (VP-16), teniposide (VM-26), ixabepilone, larotaxel, ortataxel, tesetaxel, and ispinesib, etc. may be used.

[0080] The antimetabolite may be selected from at least one of the following: At least one topoisomerase inhibitor selected from fluorouracil (5-FU), floxuridine (5-FUdR), methotrexate, Xeloda, Arranon, leucovorin, hydroxyurea, thioguanine (6-TG), mercaptopurine (6-MP), cytarabine, pentostatin, fludarabine phosphate, cladribine (2-CDA), asparaginase, gemcitabine, pemetrexed, bortezomib, aminopterin, raltitrexed, clofarabine, enocitabine, sapacitabine, and azacitidine, etc. may be used.

[0081] Furthermore, the present invention relates to a screening method for an anticancer agent using cancer stem cells isolated and induced by the above-mentioned method of the present invention.

[0082] Furthermore, the present invention relates to a method for evaluating a compound, which uses cancer stem cells isolated / induced by the above-mentioned method of the present invention.

[0083] <Method for detecting cancer stem cells> The present invention also provides a method for detecting, identifying or quantifying the presence of the cancer stem cells of the present invention. Specifically, the present invention provides a method for detecting, identifying or quantifying the presence of the cancer stem cells of the present invention or a substantially homogeneous population of cancer stem cells, comprising the following steps (a) and (b): (a) providing a sample obtained from a cancer patient; (b) contacting the sample with an Lgr5 antibody.

[0084] In this method, a sample obtained from a cancer patient is first prepared. In the present invention, the "sample" is not particularly limited as long as it is preferably an organ or tissue derived from a cancer patient, and frozen or unfrozen organs or tissues can be used. For example, cancer (tumor) tissue obtained from a cancer patient can be mentioned. In this method, the sample is then contacted with an Lgr5 antibody.

[0085] The above-mentioned method of the present invention for detecting, identifying or quantifying the presence of cancer stem cells or a substantially homogeneous population of cancer stem cells can be used, for example, in diagnosing cancer, selecting cancer patients, predicting or confirming the effectiveness of drugs (pharmaceutical compositions), monitoring treatment, and imaging cancer.

[0086] Specifically, for example, an organ or tissue is removed from a cancer patient, a specimen is prepared, and the presence of cancer stem cells can be detected, identified, or quantified using the specimen. A known method can be appropriately used to prepare the specimen, for example, the PFA-AMeX-Paraffin method (WO 09 / 078386). For example, a frozen or unfrozen organ or tissue can be used as the sample. A sample from a cancer patient is first fixed with a PFA solution. The PFA solution is a cell fixation solution in which a buffer such as a phosphate buffer is added to a 1-6% aqueous solution of paraformaldehyde, and preferably a 4% PFA fixation solution (4% paraformaldehyde / 0.01 M PBS (pH 7.4)) is used. Fixation with a PFA fixation solution can be performed by immersing the target organ or tissue in a PFA fixation solution containing 1-6%, preferably 4%, paraformaldehyde at a temperature of 0-8°C, preferably about 4°C, for 2-40 hours, preferably 6-30 hours. The fixed organ or tissue is then washed with phosphate buffered saline or the like. At this time, the part of the organ or tissue to be observed may be excised and then washed.

[0087] The organ or tissue thus prepared is then embedded in paraffin by the AMeX method. The AMeX method is a paraffin embedding method that involves a series of operations: cold acetone fixation, dehydration with acetone, clearing with methyl benzoate and xylene, and paraffin embedding. Specifically, the tissue is immersed in acetone at -25 to 8°C, preferably -20 to 6°C, for 2 to 24 hours, preferably 4 to 16 hours, and then the acetone containing the tissue is returned to room temperature, or the organ or tissue is transferred to acetone at room temperature, and then dehydrated at room temperature for 0.5 to 5 hours, preferably 1 to 4 hours. Next, the tissue is immersed in methyl benzoate at room temperature for 0.5 to 3 hours, preferably 0.5 to 2 hours, and in xylene at room temperature for 0.5 to 3 hours, preferably 0.5 to 2 hours, for clearing, and then embedded in paraffin at 55 to 65°C, preferably 58 to 62°C, for 1 to 4 hours, preferably 1 to 3 hours. The paraffin blocks of organs or tissues obtained in this manner by the PFA-AMeX method are stored at low temperatures until use.

[0088] When in use, the paraffin block obtained above is sliced ​​into thin sections using a microtome or the like, and the thin sections are then deparaffinized and hydrophilized. Deparaffinization and hydrophilization can be performed by known methods. For example, deparaffinization can be performed using xylene or toluene, and hydrophilization can be performed using alcohol or acetone.

[0089] The thin sections thus obtained are subjected to, for example, tissue staining, immunohistochemical staining, or enzyme histochemical staining for detection, identification, or quantification.

[0090] When staining the prepared specimens with special stains, any stain that can be used on normal paraffin-embedded sections can be used (e.g., PAS stain, Giemsa stain, Toluidine Blue stain, etc.). For enzyme histochemical staining, any stain that can be used on sections can be used (e.g., various stains such as ALP, ACP, TRAP, esterase, etc.). For staining pathological tissues, general stains such as Hematoxylin-Eosin staining can be used; for collagen fibers, Van Gieson staining, Azan staining, Masson Trichrome staining can be used; for elastic fibers, Weigert staining, Elastica Van Gieson staining can be used; and for reticular fibers and basement membranes, Watanabe's silver staining and PAM staining (Periodic acid methenamine silver stain) can be used.

[0091] Immunohistochemical staining or enzyme histochemical staining can be performed using, but is not limited to, a direct method in which a primary antibody is labeled with an enzyme or a labeling substance, or an indirect method in which a secondary antibody is labeled without labeling the primary antibody. The antibody can be labeled by a commonly known method. Examples of labeling substances include radioisotopes, enzymes, fluorescent substances, biotin / avidin, etc. These labeling substances can be commercially available. Examples of radioisotopes include 32 P, 33 P, 131 I, 125 I, 3 H, 14 C. 35 Examples of the fluorescent substance include fluorescein isothiocyanate (FITC) and rhodamine. These are commercially available and can be labeled by known methods.

[0092] Furthermore, the thin sections are subjected to, for example, tissue staining, immunohistochemical staining, or enzyme histochemical staining for detection, identification, or quantification.

[0093] In addition, detection, identification, or quantification can be performed by quantifying DNA and RNA in cells in an organ or tissue specimen. There is no particular limitation on the expression confirmation, and it can be performed according to a conventional expression confirmation method. Examples of RNA include microRNA, siRNA, tRNA, snRNA, mRNA, and non-coding RNA. For example, the mRNA of Lgr5 can be extracted according to a standard method, and the transcription level of each gene can be measured by performing Northern hybridization or RT-PCR using this mRNA as a template. Furthermore, it is also possible to measure the expression level of Lgr5 using DNA array technology.

[0094] Microdissection, particularly laser microdissection (LMD), can be used to extract desired tissues or cells from a specimen. The LMD method can extract a target cell group from a living tissue, so that it is possible to know exactly how much of a specific gene is expressed in which cells among various cells that make up a tissue in the living body. For example, the AS-LMD system (manufactured by Leica Microsystems) can be used as a device for performing microdissection.

[0095] The present invention also provides a method for diagnosing cancer, detecting cancer stem cells, or selecting a cancer patient, comprising detecting the presence of at least one of the proteins in a sample isolated from a cancer patient using at least one antibody that binds to the protein set forth in SEQ ID NOs: 1 to 8. To detect the presence of cancer stem cells, at least one antibody that binds to the protein set forth in SEQ ID NOs: 1 to 8 can be used in place of the Lgr5 antibody.

[0096] In one non-limiting embodiment of the present invention, there is provided a method for diagnosing cancer, detecting cancer stem cells, or selecting a cancer patient, comprising detecting the presence of at least one protein in a sample isolated from a cancer patient using at least one antibody that binds to the protein set forth in SEQ ID NOs: 1 to 6. To detect the presence of Lgr5-positive cancer stem cells, it is possible to use at least one antibody that binds to the protein set forth in SEQ ID NOs: 1 to 6 instead of the Lgr5 antibody. By detecting the presence of the protein, it is possible to detect the presence of Lgr5-positive cancer stem cells, but the present invention does not exclude the simultaneous detection of the presence of Lgr5.

[0097] In one non-limiting embodiment of the present invention, there is provided a method for diagnosing cancer, detecting cancer stem cells, or selecting a cancer patient, comprising detecting the presence of at least one of the proteins set forth in SEQ ID NOs: 1 to 8 in a sample isolated from a cancer patient using at least one antibody that binds to the protein. To detect the presence of Lgr5-negative cancer stem cells, it is possible to use at least one antibody that binds to a protein set forth in SEQ ID NOs: 1 to 8 instead of the Lgr5 antibody. By detecting the presence of the protein, it is possible to detect the presence of Lgr5-negative cancer stem cells, but the present invention does not exclude the simultaneous detection of the presence of Lgr5.

[0098] The present invention also provides a method for confirming the efficacy of a pharmaceutical composition comprising at least one antibody binding to a protein set forth in SEQ ID NO: 1 to 8, comprising detecting the presence of any one or more of the proteins set forth in SEQ ID NO: 1 to 8 and / or polynucleotides encoding the proteins in a sample isolated from a subject to which the pharmaceutical composition has been administered. In the method, at least one antibody binding to a protein set forth in SEQ ID NO: 1 to 8, or a portion of the polynucleotide encoding a protein set forth in SEQ ID NO: 1 to 8 and / or its complementary strand may be used for the detection. In another non-limiting aspect, a method for evaluating the efficacy of a treatment for cancer in a subject is provided, comprising comparing the expression of at least one of the proteins set forth in SEQ ID NO: 1 to 8 and / or polynucleotides encoding the proteins in a first sample obtained from the subject before providing at least a portion of the treatment to the subject with the expression of at least one of the proteins set forth in SEQ ID NO: 1 to 8 and / or polynucleotides encoding the proteins in a second sample obtained from the subject after providing at least a portion of the treatment, wherein a significantly lower level of the protein and / or polynucleotide in the second sample relative to the first sample is an indication that the treatment is effective in inhibiting cancer in the subject.

[0099] In one non-limiting embodiment of the invention, there is provided a method for monitoring the efficacy of treatment in a subject with an antibody provided by the invention, comprising: (i) obtaining a pre-treatment sample from the subject prior to administration of the antibody; (ii) detecting the level of expression of at least one marker protein selected from the proteins set forth in SEQ ID NOs: 1 to 8, its mRNA, or its genomic DNA in the pre-administration sample; (iii) obtaining one or more post-dose samples from the subject; (iv) detecting the level of expression or activity of at least one marker protein selected from the proteins set forth in SEQ ID NOs: 1 to 8, its mRNA or its genomic DNA in the post-administration sample; (v) comparing the level of expression or activity of the marker protein, the mRNA, or the genomic DNA in the pre-administration sample with the marker protein, the mRNA, or the genomic DNA in the post-administration sample or samples; and (vi) Accordingly, methods are provided that include a step consisting of altering the administration of an antibody to a subject, for example, increasing the administration of an antibody of the invention can be used to decrease the expression or activity of a marker (the level of expression or activity of the marker protein, the mRNA, or the genomic DNA in a pre-administration sample) to a level higher than is detectable, i.e., to increase the effectiveness of the antibody.

[0100] In another non-limiting embodiment of the invention, there is provided a method for monitoring the efficacy of treatment in a subject with an antibody provided by the invention, comprising: (i) detecting Lgr5-positive cancer stem cells in a pre-administration sample obtained from a subject prior to administration of the antibody; (ii) detecting the level of expression of at least one marker protein selected from the proteins set forth in SEQ ID NOs: 1 to 6, its mRNA, or its genomic DNA in the pre-administration sample; (iii) obtaining one or more post-dose samples from the subject; (iv) detecting the level of expression or activity of at least one marker protein selected from the proteins set forth in SEQ ID NOs: 1 to 6, its mRNA or its genomic DNA in the post-administration sample; (v) comparing the level of expression or activity of the marker protein, the mRNA, or the genomic DNA in the pre-administration sample with the marker protein, the mRNA, or the genomic DNA in the post-administration sample or samples; and (vi) Accordingly, methods are provided that include a step consisting of altering the administration of an antibody to a subject, for example, increasing the administration of an antibody of the invention can be used to decrease the expression or activity of a marker (the level of expression or activity of the marker protein, the mRNA, or the genomic DNA in a pre-administration sample) to a level higher than is detectable, i.e., to increase the effectiveness of the antibody.

[0101] In another non-limiting embodiment of the present invention, there is provided a method for monitoring the efficacy of treatment in a subject with an antibody provided by the present invention, comprising: (i) detecting Lgr5-negative cancer stem cells in a pre-administration sample obtained from a subject prior to administration of the antibody; (ii) detecting the level of expression of at least one marker protein selected from the proteins set forth in SEQ ID NOs: 1 to 8, its mRNA, or its genomic DNA in the pre-administration sample; (iii) obtaining one or more post-dose samples from the subject; (iv) detecting the level of expression or activity of at least one marker protein selected from the proteins set forth in SEQ ID NOs: 1 to 8, its mRNA or its genomic DNA in the post-administration sample; (v) comparing the level of expression or activity of the marker protein, the mRNA, or the genomic DNA in the pre-administration sample with the marker protein, the mRNA, or the genomic DNA in the post-administration sample or samples; and (vi) Accordingly, methods are provided that include a step consisting of altering the administration of an antibody to a subject, for example, increasing the administration of an antibody of the invention can be used to decrease the expression or activity of a marker (the level of expression or activity of the marker protein, the mRNA, or the genomic DNA in a pre-administration sample) to a level higher than is detectable, i.e., to increase the effectiveness of the antibody.

[0102] <Cancer stem cell inhibitor> A cancer stem cell inhibitor refers to an agent that has effects such as inhibiting the proliferation of cancer stem cells, inhibiting the metastasis or recurrence of cancer stem cells, and killing cancer stem cells, and may have effects such as inhibiting the proliferation of cancer cells, inhibiting the metastasis or recurrence of cancer cells, and killing cancer cells.

[0103] The terms "suppress", "suppressing" and their literal equivalents, when used in the context of biological activities such as, but not limited to, cancer stem cell proliferation or metastasis, refer to the downregulation of biological activities, which can reduce or eliminate targeted functions such as the production of proteins or the phosphorylation of molecules. In certain embodiments, suppression can refer to a reduction of about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% of targeted activity. When used in the context of a disorder or disease, the term refers to the prevention of the onset of symptoms, the alleviation of symptoms, or the successful alleviation of a disease, condition or disorder.

[0104] "Metastasis" refers to the process by which cancer spreads or migrates from its primary site in the body to other areas, developing similar cancerous lesions in the new locations. "Metastatic" or "metastasizing" cells are cells that lose adhesive contacts with neighboring cells, migrate from the primary site of disease through the bloodstream or lymph, and invade nearby body structures. "Recurrence" refers to the reappearance of the same malignant tumor in a remaining organ after partial resection of an organ to remove a malignant tumor from a cancer patient, or after postoperative chemotherapy following such resection.

[0105] <Protein> The protein used in the present invention can be easily prepared by creating an expression vector containing a gene including DNA encoding the protein by any method known to those skilled in the art, culturing a transformant transformed with the expression vector, producing and accumulating the protein, and harvesting it.

[0106] The expression vector can be prepared according to a method known in the art. For example, (1) excising a DNA fragment containing a gene including DNA encoding a protein; (2) ligating the DNA fragment downstream of a promoter in an appropriate expression vector; It can be produced by the above method.

[0107] Vectors that can be used include plasmids derived from Escherichia coli (e.g., pBR322, pBR325, pUC18, pUC118), plasmids derived from Bacillus subtilis (e.g., pUB110, pTP5, pC194), plasmids derived from yeast (e.g., pSH19, pSH15), bacteriophages such as λ phage, retroviruses, and animal viruses such as vaccinia virus and baculovirus.

[0108] The promoter used in the present invention may be any suitable promoter corresponding to the host used for gene expression. For example, when the host is Escherichia coli, the trp promoter, the lac promoter, the recA promoter, the λPL promoter, the lpp promoter, etc. are preferred, when the host is Bacillus subtilis, the SPO1 promoter, the SPO2 promoter, the penP promoter, etc. are preferred, when the host is yeast, the PHO5 promoter, the PGK promoter, the GAP promoter, the ADH promoter, etc. are preferred. When an animal cell is used as the host, the SRα promoter, the SV40 promoter, the LTR promoter, the CMV promoter, the HSV-TK promoter, etc. are preferred.

[0109] In addition to the above, an enhancer, a splicing signal, a polyA addition signal, a selection marker, an SV40 replication origin, and the like, which are known in the art, can be added to the expression vector as desired. In addition, the protein used in the present invention can be expressed as a fusion protein with other proteins (e.g., glutathione S-transferase and protein A) as necessary. Such a fusion protein can be cleaved with an appropriate protease to separate the individual proteins.

[0110] Examples of host cells that can be used include Escherichia bacteria, Bacillus bacteria, yeast, insect cells, insects, and animal cells.

[0111] Specific examples of Escherichia bacteria that can be used include Escherichia coli K12 DH1 (Proc. Natl. Acad. Sci, USA, Vol. 60, 160 (1968)), JM103 (Nucleic Acids Research, Vol. 9, 309 (1981)), JA221 (Journal of Molecular Biology, Vol. 120, 517 (1978)), and HB101 (Journal of Molecular Biology, Vol. 41, 459 (1969)).

[0112] Examples of the Bacillus bacteria that can be used include Bacillus subtilis MI114 (Gene, Vol. 24, 255 (1983)), 207-21 [Journal of Biochemistry, Vol. 95, 87 (1984)].

[0113] Examples of yeast that can be used include Saccharomyces cerevisiae AH22, AH22R-, NA87-11A, DKD-5D, and 20B-12, Schizosaccaromyces pombe NCYC1913 and NCYC2036, and Pichia pastoris.

[0114] Examples of animal cells that can be used include monkey cells COS-7 and Vero, Chinese hamster cells CHO (hereinafter abbreviated as CHO cells), dhfr gene-deficient CHO cells, mouse L cells, mouse AtT-20 cells, mouse myeloma cells, rat GH3 cells, and human FL cells.

[0115] Transformation of these host cells can be carried out according to methods known in the art, for example, see the following documents: Proc. Natl. Acad. Sci. USA, vol. 69, 2110 (1972); Gene, vol. 17, 107 (1982); Molecular & General Genetics, vol. 168, 111 (1979); Methods in Enzymology, vol. 194, 182-187 (1991); Proc. Natl. Acad. Sci. USA), vol. 75, 1929 (1978); and Virology, vol. 52, 456 (1973).

[0116] The transformants thus obtained can be cultured according to methods known in the art. For example, when the host is a bacterium of the genus Escherichia, the culture is usually performed at about 15 to 43° C. for about 3 to 24 hours, and aeration or stirring can be performed as necessary. When the host is a bacterium of the genus Bacillus, the culture is usually performed at about 30 to 40° C. for about 6 to 24 hours, and aeration or stirring can be performed as necessary.

[0117] When culturing a transformant in which the host is yeast, the culture is usually carried out in a medium adjusted to a pH of about 5 to 8 at about 20°C to 35°C for about 24 to 72 hours, and aeration or stirring can be added as necessary.

[0118] When culturing a transformant in which the host is an animal cell, the culture is usually performed in a medium adjusted to a pH of about 6 to 8 at about 30°C to 40°C for about 15 to 60 hours, and aeration or stirring can be performed as necessary.

[0119] To separate and purify the protein used in the present invention from the above culture, for example, after the cultivation, the bacteria or cells are collected by a known method, suspended in an appropriate buffer, and the bacteria or cells are disrupted by ultrasonic waves, lysozyme, and / or freeze-thawing, and then a crude protein extract is obtained by centrifugation or filtration. TMThe culture medium may contain a surfactant such as . When the protein is secreted into the culture medium, after the culture is completed, the bacteria or cells are separated from the supernatant by a known method, and the supernatant is collected. The proteins contained in the culture supernatant or extract thus obtained can be purified by appropriately combining known separation and purification methods.

[0120] The protein thus obtained can be optionally modified or partially removed by treating the recombinantly produced protein with an appropriate protein modifying enzyme such as trypsin or chymotrypsin before or after purification using known methods or methods similar thereto. The presence of the proteins used in the present invention can be determined by various binding assays, enzyme immunoassays using specific antibodies, and the like.

[0121] <Antibody> The antibody used in the present invention is not particularly limited as long as it binds to the protein used in the present invention, and can be obtained as a polyclonal or monoclonal antibody by known means. As the antibody used in the present invention, a monoclonal antibody derived from a mammal is particularly preferred. Mammalian-derived monoclonal antibodies include those produced by hybridomas and those produced by hosts transformed with an expression vector containing an antibody gene by genetic engineering techniques. It is preferable that the antibody used in the present invention specifically binds to the protein used in the present invention.

[0122] Monoclonal antibody-producing hybridomas can basically be prepared using known techniques as follows. That is, the protein used in the present invention is used as a sensitizing antigen, and the antigen is immunized according to a conventional immunization method, and the resulting immune cells are fused with a known parent cell by a conventional cell fusion method, and monoclonal antibody-producing cells are screened by a conventional screening method. Specifically, monoclonal antibodies can be prepared as follows.

[0123] The gene sequence encoding the protein is inserted into a known expression vector system to transform an appropriate host cell, and the protein is then purified from the host cell or culture supernatant by a known method.

[0124] The protein is then used as a sensitizing antigen. Alternatively, a partial peptide of the protein can be used as a sensitizing antigen. In this case, the partial peptide can be obtained by chemical synthesis from the amino acid sequence of the protein by a general method known to those skilled in the art.

[0125] Here, the partial polypeptide of the protein may be, for example, a peptide having at least 10 or more, preferably 50 or more, more preferably 70 or more, more preferably 100 or more, and most preferably 200 or more amino acids from the amino acid sequence constituting the protein, and having a biological activity substantially equivalent to the function of the protein. The C-terminus of the partial peptide is usually a carboxyl group (-COOH) or a carboxylate (-COO-), but the C-terminus may be an amide (-CONH2) or an ester (-COOR). Furthermore, the partial peptide may include those in which the amino group of the N-terminal methionine residue is protected with a protecting group, those in which the glutamyl group generated by in vivo cleavage of the N-terminus is pyroglutamylated, those in which the substituents on the side chains of the amino acids in the molecule are protected with an appropriate protecting group, and conjugated peptides such as so-called glycopeptides to which a sugar chain is bound.

[0126] The mammal to be immunized with the sensitizing antigen is not particularly limited, but is preferably selected taking into consideration compatibility with the parent cells used in cell fusion, and generally rodents such as mice, rats, and hamsters are used.

[0127] Immunization of animals with a sensitizing antigen is carried out according to known methods. For example, a general method is to inject the sensitizing antigen intraperitoneally or subcutaneously into a mammal. Specifically, the sensitizing antigen is appropriately diluted and suspended in PBS (Phosphate-Buffered Saline) or physiological saline, etc., and mixed with an appropriate amount of a conventional adjuvant, such as Freund's complete adjuvant, if desired, and emulsified, and then administered to the mammal several times every 4 to 21 days. An appropriate carrier can also be used during immunization with the sensitizing antigen.

[0128] After immunizing a mammal in this manner and confirming that the desired antibody level is increased in the serum, immune cells are collected from the mammal and subjected to cell fusion, with preferred immune cells being, in particular, spleen cells.

[0129] The other parent cell to be fused with the immune cell is a mammalian myeloma cell. This myeloma cell may be any of various known cell lines, such as P3 (P3x63Ag8.653) (J. Immunol. (1979) 123, 1548-1550), P3x63Ag8U.1 (Current Topics in Microbiology and Immunology (1978) 81, 1-7), NS-1 (Kohler. G. and Milstein, C. Eur. J. Immunol. (1976) 6, 511-519), MPC-11 (Margulies. DH et al., Cell (1976) 8, 405-415), SP2 / 0 (Shulman, M. et al., Nature (1978) 276, 269-270), F0 (de St. Groth, S. F. et al., Nature (1978) 276, 269-270), and NS-1 (Kohler. G. and Milstein, C. Eur. J. Immunol. (1976) 6, 511-519). al., J. Immunol. Methods (1980) 35, 1-21), S194 (Trowbridge, ISJ Exp. Med. (1978) 148, 313-323), R210 (Galfre, G. et al., Nature (1979) 277, 131-133), etc. are preferably used.

[0130] The cell fusion between the immune cells and myeloma cells can basically be carried out according to known methods, for example, the method of Kohler and Milstein et al. (Kohler, G. and Milstein, C., Methods Enzymol. (1981) 73, 3-46).

[0131] More specifically, the cell fusion is carried out in a normal nutrient culture medium in the presence of a cell fusion promoter, such as polyethylene glycol (PEG) or Sendai virus (HVJ), and if desired, an auxiliary agent such as dimethyl sulfoxide can be added to enhance the fusion efficiency.

[0132] The ratio of immune cells to myeloma cells can be set arbitrarily. For example, it is preferable to use 1 to 10 times the immune cells relative to the myeloma cells. As the culture medium used for the cell fusion, for example, RPMI1640 culture medium suitable for the growth of the myeloma cell line, MEM culture medium, or other usual culture medium used for this type of cell culture can be used, and serum supplements such as fetal calf serum (FCS) can also be used in combination. In cell fusion, a predetermined amount of the immune cells and myeloma cells are thoroughly mixed in the culture medium, and a PEG solution (e.g., average molecular weight of about 1000-6000) preheated to about 37°C is added usually at a concentration of 30-60% (w / v), and mixed to form the desired fused cells (hybridomas). Next, a suitable culture medium is successively added, and the supernatant is centrifuged and removed, and the process is repeated to remove cell fusion agents and the like that are unfavorable to the growth of the hybridomas.

[0133] The hybridomas thus obtained are selected by culturing in a conventional selective culture medium, such as HAT culture medium (a culture medium containing hypoxanthine, aminopterin, and thymidine). The culture in the HAT culture medium is continued for a sufficient time (usually several days to several weeks) for cells other than the target hybridoma (non-fused cells) to die. Then, a conventional limiting dilution method is performed to screen and single-clone hybridomas that produce the antibodies used in the present invention.

[0134] In addition to obtaining the above-mentioned hybridomas by immunizing non-human animals with an antigen, human lymphocytes can be sensitized to the protein in vitro, and the sensitized lymphocytes can be fused with myeloma cells of human origin having the ability to divide forever to obtain a desired human antibody having binding activity to the protein (see JP-B-1-59878). Furthermore, the protein as an antigen can be administered to a transgenic animal having the entire repertoire of human antibody genes to obtain antibody-producing cells, which can then be immortalized to obtain human antibodies against the protein (see International Patent Application Publication Nos. WO 94 / 25585, WO 93 / 12227, WO 92 / 03918, and WO 94 / 02602).

[0135] A technique for obtaining a human antibody by panning using a human antibody library is also known. For example, the V region of a human antibody is expressed on the surface of a phage as a single chain antibody (scFv) by phage display. A phage expressing an scFv that binds to an antigen can be selected. By analyzing the genes of the selected phage, the DNA sequence encoding the V region of a human antibody that binds to an antigen can be determined. After determining the DNA sequence of the scFv that binds to an antigen, the V region sequence can be fused in frame with the sequence of the C region of a desired human antibody and then inserted into an appropriate expression vector to prepare an expression vector. The expression vector is introduced into a suitable expression cell as listed above, and the gene encoding the human antibody is expressed to obtain the human antibody. These methods are already known (see International Publications WO 1992 / 001047, WO 1992 / 020791, WO 1993 / 006213, WO 1993 / 011236, WO 1993 / 019172, WO 1995 / 001438, WO 1995 / 015388).

[0136] The hybridomas producing the monoclonal antibodies thus prepared can be subcultured in a normal culture medium and can be stored for a long period in liquid nitrogen.

[0137] To obtain a monoclonal antibody from the hybridoma, the hybridoma is cultured according to a conventional method and the culture supernatant is obtained, or the hybridoma is administered to a compatible mammal to grow and the ascites is obtained, etc. The former method is suitable for obtaining a highly pure antibody, while the latter method is suitable for mass production of the antibody.

[0138] The monoclonal antibodies used in the present invention may be recombinant antibodies produced by, for example, cloning an antibody gene from a hybridoma, incorporating it into an appropriate vector, introducing this into a host, and using genetic engineering techniques (see, for example, Vandamme, AM et al., Eur. J. Biochem. (1990) 192, 767-775, 1990).

[0139] Specifically, mRNA encoding the variable (V) region of an antibody is isolated from a hybridoma producing the antibody. The mRNA is isolated by a known method, such as guanidine ultracentrifugation (Chirgwin, JMet al., Biochemistry (1979) 18, 5294-5299) or AGPC (Chomczynski, P. et al., Anal. Biochem. (1987) 162, 156-159), to prepare total RNA, and the target mRNA is prepared using an mRNA Purification Kit (Pharmacia) or the like. Alternatively, mRNA can be directly prepared using a QuickPrep mRNA Purification Kit (Pharmacia).

[0140] From the obtained mRNA, cDNA of the antibody V region is synthesized using reverse transcriptase. cDNA synthesis is performed using AMV Reverse Transcriptase First-strand cDNA Synthesis Kit (Seikagaku Corporation) or the like. In addition, to synthesize and amplify cDNA, 5'-Ampli FINDER RACE Kit (Clontech) and the 5'-RACE method using PCR (Frohman, MA et al., Proc. Natl. Acad. Sci. USA (1988) 85, 8998-9002; Belyavsky, A. et al., Nucleic Acids Res. (1989) 17, 2919-2932) or the like can be used.

[0141] The desired DNA fragment is purified from the obtained PCR product and ligated to a vector DNA. A recombinant vector is then prepared from the fragment, and introduced into E. coli or the like to select colonies to prepare the desired recombinant vector. The base sequence of the desired DNA is then confirmed by a known method, such as the dideoxynucleotide chain termination method.

[0142] After obtaining DNA encoding the V region of the antibody of interest, it is inserted into an expression vector containing DNA encoding the constant region (C region) of the desired antibody.

[0143] To produce an antibody for use in the present invention, an antibody gene is incorporated into an expression vector so that it is expressed under the control of an expression control region, such as an enhancer or promoter. Then, a host cell is transformed with this expression vector to express the antibody.

[0144] For expression of antibody genes, DNAs encoding the antibody heavy chain (H chain) and light chain (L chain) may be separately incorporated into expression vectors and then co-transformed into host cells, or DNAs encoding the H chain and L chain may be incorporated into a single expression vector and then used to transform host cells (see WO 94 / 11523).

[0145] In addition to the above-mentioned host cells, transgenic animals can be used to produce recombinant antibodies. For example, an antibody gene is inserted into a gene encoding a protein (such as casein in goats) that is produced specifically in milk to prepare a fusion gene. A DNA fragment containing the fusion gene into which the antibody gene has been inserted is injected into a goat embryo, and this embryo is introduced into a female goat. The desired antibody is obtained from the milk produced by the transgenic goat born from the goat that received the embryo or its descendants. In order to increase the amount of milk containing the desired antibody produced by the transgenic goat, an appropriate hormone may be used in the transgenic goat (Ebert, KM et al., Bio / Technology (1994) 12, 699-702).

[0146] In the present invention, in addition to the above antibodies, recombinant antibodies artificially modified for the purpose of reducing heterologous antigenicity against humans, such as chimeric antibodies, humanized antibodies, and human antibodies, can be used. These modified antibodies can be produced by known methods. The monoclonal antibodies of the present invention include not only the above animal-derived monoclonal antibodies, but also recombinant antibodies artificially modified, such as chimeric antibodies, humanized antibodies, and bispecific antibodies.

[0147] A chimeric antibody can be obtained by linking the thus obtained DNA encoding the antibody V region to DNA encoding the human antibody C region, incorporating the resultant into an expression vector, and introducing the vector into a host for production. Useful chimeric antibodies can be obtained using this known method.

[0148] Humanized antibodies, also called reshaped human antibodies, are antibodies in which the complementarity determining regions (CDRs) of an antibody from a non-human mammal, such as a mouse, are grafted onto the complementarity determining regions of a human antibody, and general genetic recombination techniques for such antibodies are known (see European Patent Application Publication No. EP 125023 and WO 96 / 02576).

[0149] Specifically, a DNA sequence designed to link the CDR of a mouse antibody with the framework region (FR) of a human antibody is synthesized by PCR using as primers several oligonucleotides that have been engineered to have overlapping portions at the terminal regions of both the CDR and FR (see the method described in WO98 / 13388).

[0150] The framework region of the human antibody linked via the CDR is selected from those in which the complementarity determining region forms a good antigen-binding site. If necessary, amino acids in the framework region of the antibody variable region may be substituted so that the complementarity determining region of the reshaped human antibody forms a suitable antigen-binding site (Sato, K. et al., Cancer Res. (1993) 53, 851-856).

[0151] For the C region of a chimeric antibody or a humanized antibody, those of a human antibody are used, for example, CH1, CH2, CH3, and CH4 can be used for the H chain, and Cκ and Cλ can be used for the L chain. Furthermore, the C region of a human antibody may be modified to improve the stability of the antibody or its production.

[0152] A chimeric antibody is composed of a variable region of an antibody derived from a non-human mammal and a constant region derived from a human antibody. On the other hand, a humanized antibody is composed of a complementarity determining region of an antibody derived from a non-human mammal and a framework region and C region derived from a human antibody. Since the antigenicity of a humanized antibody in the human body is reduced, the humanized antibody is useful as an active ingredient of the therapeutic agent of the present invention.

[0153] The antibody used in the present invention is not limited to the whole antibody molecule, but may be an antibody fragment or a modified antibody fragment thereof, and includes both bivalent and monovalent antibodies, so long as it binds to the protein used in the present invention. For example, antibody fragments include Fab, F(ab')2, Fv, Fab / c having one Fab and a complete Fc, single chain Fv (scFv) in which the Fv of the H chain or L chain is linked with an appropriate linker, and diabody. Specifically, antibodies are treated with enzymes such as papain or pepsin to generate antibody fragments, or genes encoding these antibody fragments are constructed and introduced into expression vectors, which are then expressed in appropriate host cells (see, for example, Co, MS et al., J. Immunol. (1994) 152, 2968-2976; Better, M. & Horwitz, AH Methods in Enzymology (1989) 178, 476-496, Academic Press, Inc.; Plueckthun, A. & Skerra, A. Methods in Enzymology (1989) 178, 476-496, Academic Press, Inc.; Lamoyi, E., Methods in Enzymology (1989) 121, 652-663; Rousseaux, J. et al., Methods in Enzymology (1989) 121, 663-669; see Bird, RE et al., TIBTECH (1991) 9, 132-137).

[0154] An scFv can be obtained by linking the H chain V region and L chain V region of an antibody. In this scFv, the H chain V region and the L chain V region are linked via a linker, preferably a peptide linker (Huston, JS et al., Proc. Natl. Acad. Sci. USA (1988) 85, 5879-5883). The H chain V region and the L chain V region in the scFv may be derived from any of the antibodies described herein. As the peptide linker linking the V regions, any single-chain peptide consisting of 12-19 amino acid residues, such as (GGGGS)n, can be used.

[0155] DNA encoding an scFv can be obtained by using the DNA encoding the H chain or H chain V region of the antibody, and the DNA encoding the L chain or L chain V region, as a template, and amplifying all or a portion of the DNA encoding the desired amino acid sequence by PCR using a primer pair that specifies both ends of the DNA as a template, and then further amplifying the DNA by combining it with a DNA encoding a peptide linker portion and a primer pair that specifies such that both ends are linked to the H chain and L chain, respectively.

[0156] Furthermore, once DNA encoding scFv has been prepared, an expression vector containing it and a host transformed with the expression vector can be obtained in accordance with standard methods, and by using the host, scFv can be obtained in accordance with standard methods.

[0157] Diabodies are dimers formed by binding two fragments (e.g., scFvs) in which the variable regions are linked by a linker or the like, and usually contain two VLs and two VHs (P. Holliger et al., Proc. Natl. Acad. Sci. USA, 90, 6444-6448 (1993), EP 404097, WO 93 / 11161, Johnson et al., Method in Enzymology, 203, 88-98, (1991), Holliger et al., Protein Engineering, 9, 299-305, (1996), Perisic et al., Structure, 2, 1217-1226, (1994), John et al., Protein Engineering, 12(7), 597-604, (1999), Holliger et al., Proc.Natl.Acad.Sci.USA., 90, 6444-6448, (1993), Atwell et al., Mol.Immunol., 33, 1301-1312, (1996), etc.).

[0158] These antibody fragments can be produced by a host by obtaining and expressing the genes in the same manner as described above. In the present invention, the "antibody" also includes these antibody fragments.

[0159] The antibody of the present invention may be used as a modified antibody bound to various molecules such as polyethylene glycol (PEG). Radioisotopes, chemotherapeutic agents, bacterial toxins, and other cytotoxic substances may also be bound to the antibody. The "antibody" of the present invention includes these modified antibodies. Such modified antibodies can be obtained by chemically modifying the obtained antibody. Methods for modifying antibodies have already been established in this field.

[0160] Furthermore, the antibody used in the present invention may be a bispecific antibody. The bispecific antibody may be a bispecific antibody having antigen-binding sites that recognize different epitopes on the protein used in the present invention, or a bispecific antibody that recognizes the protein used in the present invention and another protein, or one of the antigen-binding sites may recognize the protein used in the present invention and the other antigen-binding site may recognize a cytotoxic substance such as a chemotherapeutic agent or a cell-derived toxin. In this case, it is possible to directly act a cytotoxic substance on a cancer stem cell expressing the protein used in the present invention to specifically damage the cancer stem cell and suppress the proliferation of the cancer stem cell. In addition, a bispecific antibody may be used in which one of the antigen-binding sites recognizes a molecule that constitutes a T cell receptor complex such as CD3 expressed on cytotoxic T cells and the other antigen-binding site recognizes an epitope present in any of the proteins described in SEQ ID NOs: 1 to 8 of the present invention. A bispecific antibody can be produced by binding the HL pairs of two types of antibodies, or can be obtained by fusing hybridomas that produce different monoclonal antibodies to produce a bispecific antibody-producing fusion cell. Furthermore, a bispecific antibody can be produced by a genetic engineering technique.

[0161] The antibody gene constructed as described above can be expressed and obtained by known methods. In the case of mammalian cells, expression can be achieved by functionally linking a commonly used useful promoter, the antibody gene to be expressed, and a polyA signal downstream of the 3' end of the promoter / enhancer. For example, the human cytomegalovirus immediate early promoter / enhancer can be used.

[0162] Other examples of promoters / enhancers that can be used for expressing the antibodies used in the present invention include viral promoters / enhancers such as those from retroviruses, polyoma viruses, adenoviruses, and simian virus 40 (SV40), as well as promoters / enhancers derived from mammalian cells, such as human elongation factor 1α (HEF1α).

[0163] When the SV40 promoter / enhancer is used, gene expression can be easily achieved by the method of Mulligan et al. (Nature (1979) 277, 108), and when the HEF1α promoter / enhancer is used, by the method of Mizushima et al. (Nucleic Acids Res. (1990) 18, 5322).

[0164] Replication origins that can be used include those derived from SV40, polyoma virus, adenovirus, bovine papilloma virus (BPV), etc., and further, in order to amplify the gene copy number in the host cell system, the expression vector can contain, as a selection marker, an aminoglycoside transferase (APH) gene, a thymidine kinase (TK) gene, an Escherichia coli xanthine guanine phosphoribosyltransferase (Ecogpt) gene, a dihydrofolate reductase (dhfr) gene, etc.

[0165] In the case of E. coli, a commonly used useful promoter, a signal sequence for antibody secretion, and an antibody gene to be expressed can be functionally linked to express the gene. Examples of promoters include the lacz promoter and the araB promoter. When the lacz promoter is used, expression can be achieved by the method of Ward et al. (Nature (1098) 341, 544-546; FASEB J. (1992) 6, 2422-2427), or when the araB promoter is used, expression can be achieved by the method of Better et al. (Science (1988) 240, 1041-1043).

[0166] As a signal sequence for antibody secretion, when producing the antibody in the periplasm of E. coli, the pelB signal sequence (Lei, SP et al., J. Bacteriol. (1987) 169, 4379) may be used. After isolating the antibody produced in the periplasm, the antibody structure is appropriately refolded for use.

[0167] Any expression system can be used for the production of the antibodies used in the present invention, e.g., eukaryotic or prokaryotic cell systems. Eukaryotic cells include animal cells such as established mammalian cell lines, insect cell lines, filamentous fungal cells and yeast cells, and prokaryotic cells include bacterial cells such as E. coli cells. Preferably, the antibodies used in the present invention are expressed in mammalian cells, e.g., CHO, COS, myeloma, BHK, Vero, HeLa cells.

[0168] Next, the transformed host cells are cultured in vitro or in vivo to produce the desired antibody. The host cells are cultured according to a known method. For example, DMEM, MEM, RPMI1640, or IMDM can be used as a culture medium, and serum supplements such as fetal calf serum (FCS) can also be used in combination.

[0169] The antibodies expressed and produced as described above can be separated from cells and host animals and purified to homogeneity. Separation and purification of the antibodies used in the present invention can be carried out using an affinity column. For example, columns using protein A columns include Hyper D, POROS, and Sepharose FF (Pharmacia). In addition, any separation and purification method used for ordinary proteins may be used, and is not limited thereto. For example, antibodies can be separated and purified by appropriately selecting and combining chromatography columns, filters, ultrafiltration, salting out, dialysis, and the like other than the above affinity columns (Antibodies A Laboratory Manual. Ed Harlow, David Lane, Cold Spring Harbor Laboratory, 1988).

[0170] The antigen-binding activity (Antibodies A Laboratory Manual. Ed Harlow, David Lane, Cold Spring Harbor Laboratory, 1988) and ligand-receptor binding inhibitory activity (Harada, A. et al., International Immunology (1993) 5, 681-690) of the antibodies used in the present invention can be measured by known means.

[0171] The antigen-binding activity of the antibody can be measured by ELISA (enzyme-linked immunosorbent assay), EIA (enzyme immunoassay), RIA (radioimmunoassay) or fluorescent antibody technique. For example, when using enzyme immunoassay, a sample containing the antibody, for example, culture supernatant of the antibody-producing cells or purified antibody, is added to a plate coated with the protein used in the present invention. A secondary antibody labeled with an enzyme such as alkaline phosphatase is added, the plate is incubated and washed, and then an enzyme substrate such as p-nitrophenyl phosphate is added and absorbance is measured to evaluate the antigen-binding activity.

[0172] The antibody used in the present invention may be appropriately linked to the growth inhibitor or a cytotoxic substance such as a toxic peptide or a radioactive chemical. Such an antibody modification (hereinafter referred to as an antibody conjugate) can be obtained by chemically modifying the obtained antibody. That is, the linker molecule binds the growth inhibitor to the antibody via a chemical bond (as described above) so that the growth inhibitor or cytotoxic substance and the antibody can be chemically conjugated (e.g., covalently bonded) to each other. Preferably, the linker is a cleavable linker. More preferably, the linker is cleaved under mild conditions (i.e., conditions within the cell where drug activity is not affected). Examples of suitable cleavable linkers include disulfide linkers, acid labile linkers, photolabile linkers, peptidase labile linkers, and esterase labile linkers. A disulfide-containing linker is a linker that is cleavable through disulfide exchange that can occur under physiological conditions. Acid-labile linkers are linkers that are cleavable at acidic pH. For example, certain intracellular compartments, such as endosomes and lysosomes, have an acidic pH (pH 4-5), providing suitable conditions for cleaving acid-labile linkers. Photolabile linkers are useful on body surfaces and in many body cavities that can be exposed to light. Additionally, infrared light can penetrate tissues. Peptidase-labile linkers can be used to cleave specific peptides inside or outside of cells (see, e.g., Trouet et al. (Proc. Natl. Acad. Sci. USA (1982) 79, 626-629, and Umemoto et al. Int. J. Cancer (1989) 43, 677-684).

[0173] In addition to the above-mentioned chemical modifications, such modified antibodies can be obtained in molecular forms such as bispecific antibodies designed using recombinant gene technology to recognize growth inhibitors, toxic peptides, radioactive chemicals, etc. The "antibody" of the present invention also encompasses these antibodies.

[0174] Further, examples of the antibody modification provided by the present invention include antibodies modified with toxic peptides such as ricin, abrin, ribonuclease, onconase, DNase I, Staphylococcal enterotoxin-A, pokeweed antiviral protein, gelonin, diphtheria toxin, Pseudomonas exotoxin, Pseudomonas endotoxin, L-asparaginase, and PEG L-asparaginase. In another embodiment, one or more growth inhibitors and toxic peptides can be combined and used to modify the antibody. As described above, the antibody that binds to at least one of the proteins set forth in SEQ ID NOs: 1 to 8 of the present invention can be bound to the above growth inhibitors, toxic peptides, or radioactive chemicals by covalent or non-covalent bonds. Methods for producing antibody modifications bound to these chemotherapeutic agents are known.

[0175] Furthermore, proteinaceous drugs or toxins can be bound to antibodies by genetic engineering techniques. Specifically, for example, a recombinant vector can be constructed by fusing in frame DNA encoding the toxic peptide with DNA encoding an antibody that binds to at least one of the proteins set forth in SEQ ID NOs: 1 to 8 of the present invention and incorporating the DNA in an expression vector. The vector can be introduced into an appropriate host cell to obtain a transformed cell, which can be cultured and the incorporated DNA can be expressed to obtain a modified antibody bound to a toxic peptide as a fusion protein. When obtaining a fusion protein with an antibody, the proteinaceous drug or toxin is generally placed on the C-terminus of the antibody. A peptide linker can also be interposed between the antibody and the proteinaceous drug or toxin.

[0176] The antibody used in the present invention may have cytotoxic activity. Examples of cytotoxic activity in the present invention include complement-dependent cytotoxicity (CDC) activity and antibody-dependent cell-mediated cytotoxicity (ADCC) activity. In the present invention, CDC activity means cytotoxic activity by the complement system, and ADCC activity means an activity in which, when a specific antibody adheres to a cell surface antigen of a target cell, Fcγ receptor-bearing cells (immune cells, etc.) bind to the Fc portion via the Fcγ receptor, thereby damaging the target cell.

[0177] Whether or not the antibody used in the present invention has ADCC activity or CDC activity can be determined by known methods (e.g., Current protocols in Immunology, Chapter 7. Immunologic studies in humans, Editor, John E, Coligan et al., John Wiley & Sons, Inc., (1993) etc.).

[0178] Specifically, for example, cytotoxic activity can be measured by the following method. Preparation of effector cells Spleens are removed from CBA / N mice and the like, and spleen cells are isolated in RPMI1640 medium (GIBCO). After washing with the same medium containing 10% fetal bovine serum (FBS, HyClone), the cell concentration is adjusted to 5 × 10 6 / ml and prepare effector cells.

[0179] Preparation of complement solution Baby Rabbit Complement (CEDARLANE) is diluted 10-fold with 10% FBS-containing medium (GIBCO) to prepare a complement solution.

[0180] Preparation of target cells The protein-expressing cells used in the present invention (such as cancer stem cells) were incubated with 0.2 mCi 51 The cells are radiolabeled by culturing them with Cr-sodium chromate (Amersham Pharmacia Biotech) in 10% FBS-containing DMEM medium at 37°C for 1 hour. After radiolabeling, the cells are washed three times with 10% FBS-containing RPMI1640 medium, and the cell concentration is adjusted to 2 x 10 5 / ml to prepare target cells.

[0181] -Measurement of ADCC activity 50 μl each of the target cells and the antibody used in the present invention are added to a 96-well U-bottom plate (Becton Dickinson) and reacted on ice for 15 minutes. Then, 100 μl of effector cells are added and cultured in a carbon dioxide incubator for 4 hours. The final concentration of the antibody is 0 or 10 μg / ml. After culture, 100 μl of the supernatant is collected and the radioactivity is measured using a gamma counter (COBRAIIAUTO-GMMA, MODEL D5005, Packard Instrument Company). The cytotoxic activity (%) is (AC) / (BC) x 100 A indicates the radioactivity (cpm) in each sample, B indicates the radioactivity (cpm) in a sample to which 1% NP-40 (manufactured by Nakarai Co., Ltd.) was added, and C indicates the radioactivity (cpm) in a sample containing only target cells.

[0182] Measurement of CDC activity 50 μl each of the target cells and the antibody used in the present invention are added to a 96-well flat-bottom plate (Becton Dickinson) and reacted on ice for 15 minutes. Then, 100 μl of complement solution is added and cultured in a carbon dioxide incubator for 4 hours. The final concentration of the antibody is 0 or 3 μg / ml. After culture, 100 μl of the supernatant is collected and the radioactivity is measured using a gamma counter. Cytotoxicity can be determined in the same manner as in the measurement of ADCC activity.

[0183] The antibodies provided by the present invention may also be used as appropriate with modified sugar chains. It is known that the cytotoxic activity of an antibody can be enhanced by modifying the sugar chain of the antibody. For example, the following antibodies are known as antibodies with modified sugar chains. - Antibodies with modified glycosylation (e.g. WO1999 / 054342) -Antibodies lacking fucose that is added to the sugar chain (WO2000 / 061739, WO2002 / 031140, etc.) -Antibodies having sugar chains with bisecting GlcNAc (bisecting N-acetylglucosamine) (WO2002 / 079255, etc.) The antibodies of the present invention preferably include antibodies whose sugar chain composition has been modified so that the proportion of fucose-deficient antibodies is increased, or the proportion of antibodies to which bisecting N-acetylglucosamine has been added is increased.

[0184] In the present invention, an antibody having neutralizing activity may also be used as appropriate. In general, neutralizing activity refers to the activity of inhibiting the biological activity of a ligand that has biological activity against cells, which is an exogenous molecule such as a virus or a toxin, or an endogenous molecule such as a hormone or a cytokine. That is, a substance having neutralizing activity refers to a substance that binds to the ligand or a receptor to which the ligand binds, and inhibits the binding of the ligand to the receptor. A receptor that has been prevented from binding to a ligand by neutralizing activity cannot exert biological activity through the receptor. When the antigen-binding molecule is an antibody, an antibody having such neutralizing activity is generally called a neutralizing antibody. The neutralizing activity of a certain test substance can be measured by comparing the biological activity in the presence of a ligand between the conditions in the presence or absence of the test substance.

[0185] An example of EREG, which is the target of the EP27 antibody described later in the Examples, is illustrated below. In the case of the EGF receptor, which is considered to be the main receptor of EREG and is represented by SEQ ID NO: 3, it forms a dimer by binding with a ligand and activates its own domain, tyrosine kinase, present in the cell. The activated tyrosine kinase forms peptides containing phosphorylated tyrosine by autophosphorylation, and associates various accessory molecules for signal transduction with them. These are mainly PLCγ (phospholipase Cγ), Shc, Grb2, etc. Of these accessory molecules, the first two are further phosphorylated by the tyrosine kinase of the EGF receptor. The main pathway for signal transduction from the EGF receptor is a pathway in which phosphorylation is transmitted in the order of Shc, Grb2, Sos, Ras, and Raf / MAPK kinase / MAP kinase. It is also believed that there is an alternative pathway from PLCγ to PKC. Since such intracellular signal cascades differ depending on the cell type, target molecules can be appropriately set for each target cell of interest, and are not limited to the factors mentioned above. A commercially available kit for measuring activation of in vivo signals can be used as appropriate (eg, Protein Kinase C Activity Measurement System (GE Healthcare Biosciences, Ltd.)).

[0186] In addition, activation of in vivo signals can be detected using the transcription induction effect on target genes present downstream of an in vivo signal cascade as an indicator. Changes in transcription activity can be detected by the principle of reporter assay. Specifically, a reporter gene such as GFP (Green Fluorescence Protein) or luciferase is placed downstream of a transcription factor or promoter region of a target gene, and the reporter activity is measured, thereby measuring changes in transcription activity as reporter activity.

[0187] Furthermore, since EGF receptors usually act to promote cell proliferation, activation of in vivo signal transduction can be evaluated by measuring the proliferation activity of target cells. In the present invention, the neutralizing activity of the neutralizing antibody of the present invention is evaluated by evaluating the latter cell proliferation activity, but the method is not limited to this method, and the above-mentioned methods can be suitably adopted for evaluation for each selected target cell.

[0188] That is, the neutralizing activity of an anti-EREG antibody can be evaluated or measured, for example, by measuring the cell proliferation activity as described below. 3 A method is used that measures the incorporation of [H]-labeled thymidine into viable cells as an index of DNA replication ability. As a simpler method, the dye exclusion method, which measures the ability of cells to exclude dyes such as trypan blue, under a microscope, and the MTT method are used. The latter method utilizes the ability of living cells to convert the tetrazolium salt MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) into a blue formazan product. More specifically, after a certain period of time has passed since the addition of a test antibody to the culture medium of the test cells, an MTT solution is added to the culture medium and allowed to stand for a certain period of time to allow the MTT to be taken up by the cells. As a result, the yellow compound MTT is converted into a blue compound by succinate dehydrogenase in the mitochondria inside the cells. This blue product is dissolved and colored, and its absorbance is measured to provide an index of the number of living cells.

[0189] In addition to MTT, reagents such as MTS, XTT, WST-1, and WST-8 are also commercially available (e.g., Nacalai Tesque) and can be used suitably. Furthermore, methods for evaluating cell proliferation activity using cellular ATP or cell culture impedance as an index are also known. When measuring activity, a binding antibody having the same isotype as the anti-EREG antibody and lacking the neutralizing activity is used as a control antibody in the same manner as the anti-EREG antibody, and activity can be determined by whether the anti-EREG antibody exhibits stronger neutralizing activity than the control antibody.

[0190] The cells whose proliferation is inhibited by anti-EREG antibodies are not particularly limited as long as they express EREG protein. Preferred EREG-expressing cells are, for example, cancer cells. Specifically, cells derived from colon cancer, lung adenocarcinoma, pancreatic cancer, gastric cancer, and renal cancer are suitable as EREG-expressing cells in the present invention. According to the present invention, an effective inhibitory effect on cell proliferation can be obtained for both primary and metastatic lesions of these cancers. More preferred cancer cells are primary colon cancer, metastatic colon cancer, lung adenocarcinoma, pancreatic cancer, gastric cancer, and renal cancer. Therefore, anti-EREG antibodies can be used for the purpose of treating and preventing diseases caused by cell proliferation, such as colon cancer, lung adenocarcinoma, pancreatic cancer, gastric cancer, and renal cancer. These cancers can be treated or prevented regardless of whether they are primary or metastatic lesions. More preferably, anti-EREG antibodies can be used for the purpose of treating and / or preventing primary colon cancer, metastatic colon cancer, and pancreatic cancer. Furthermore, among these cancers, cancers that proliferate in an EREG-dependent manner are preferred as targets for treatment and / or prevention in the present invention.

[0191] In the present specification and tables, when bases, amino acids, etc. are represented by abbreviations, they are based on the abbreviations specified by the IUPAC-IUB Commission on Biochemical Nomenclature or the abbreviations commonly used in the relevant field. In addition, when an amino acid may have optical isomers, it refers to the L-isomer unless otherwise specified.

[0192] <Cancer stem cell inhibitor of the present invention> The effective dose of the cancer stem cell inhibitor of the present invention is selected from the range of 0.001 mg to 1000 mg per kg of body weight per administration. Alternatively, a dose of 0.01 to 100,000 mg / body per patient can be selected. However, the inhibitor of the present invention is not limited to these doses. In addition, the inhibitor of the present invention can be administered before or after the clinical symptoms of the disease appear. The inhibitor of the present invention can be formulated according to a conventional method (Remington's Pharmaceutical Science, latest edition, Mark Publishing Company, Easton, USA), and may contain pharma- ceutical acceptable carriers and additives. Examples of such carriers and pharmaceutical additives include water, pharma- ceutical acceptable organic solvents, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymer, sodium carboxymethylcellulose, sodium polyacrylate, sodium alginate, water-soluble dextran, sodium carboxymethyl starch, pectin, methylcellulose, ethylcellulose, xanthan gum, gum arabic, casein, agar, polyethylene glycol, diglycerin, glycerin, propylene glycol, petrolatum, paraffin, stearyl alcohol, stearic acid, human serum albumin (HSA), mannitol, sorbitol, lactose, surfactants acceptable as pharmaceutical additives, etc. Actual additives are selected from the above alone or in appropriate combinations according to the dosage form of the inhibitor of the present invention, but are not limited to these. For example, when used as an injectable preparation, it can be dissolved in a solvent such as physiological saline, buffer solution, glucose solution, etc., and an adsorption inhibitor such as Tween 80, Tween 20, gelatin, human serum albumin, etc. can be added thereto. Alternatively, it may be freeze-dried to prepare a dosage form that is dissolved and reconstituted before use, and sugar alcohols and saccharides such as mannitol, glucose, etc., can be used as excipients for freeze-drying. The inhibitors of the present invention are usually administered parenterally, for example, by injection (subcutaneous injection, intravenous injection, intramuscular injection, intraperitoneal injection, etc.), transdermal, transmucosal, nasal, or pulmonary route, but oral administration is also possible.

[0193] In the present invention, the term "use in combination" of a cancer stem cell inhibitor with an anticancer agent means that these agents may be administered simultaneously, sequentially, or one of them may be administered first and then administered with an interval.

[0194] In the present invention, the cancer stem cell inhibitor can be used in various aspects, such as prevention of cancer recurrence, suppression of cancer recurrence, prevention of cancer metastasis, suppression of cancer metastasis, and adjuvant therapy for preventing postoperative recurrence. Any cancer stem cell inhibitor can be used as the cancer stem cell inhibitor of the present invention if used in the above-mentioned aspects, but non-limiting suitable examples include cancer stem cell proliferation inhibitors and cancer stem cell destroyers. The cancer stem cell proliferation inhibitor provided by the present invention does not matter by what mechanism the proliferation of cancer stem cells is suppressed if the proliferation of the target cancer stem cells is suppressed. Non-limiting examples of such cancer stem cell proliferation inhibitors include cancer stem cell proliferation inhibitors containing an antibody having neutralizing activity against the proliferation or growth of cancer stem cells or cytotoxic activity against cancer stem cells as an active ingredient. Similarly, the cancer stem cell destroyer provided by the present invention does not matter by what mechanism the cancer stem cells are destroyed if the target cancer stem cells are destroyed. Non-limiting examples of such cancer stem cell destroyers include cancer stem cell proliferation inhibitors containing an antibody having cytotoxic activity against cancer stem cells or apoptotic activity as an active ingredient. Apoptosis activity can be measured by known methods including TUNEL (Terminal deoxynucleotidyl Transferase Biotin-dUTP Nick End Labeling) assay, caspase activity (particularly caspase-3) assay, and fas ligand and annexin V assay, allowing a person skilled in the art to determine whether a test cancer stem cell disrupting agent has apoptosis activity. Further non-limiting preferred examples include cancer stem cell differentiation promoters. Non-limiting examples of such differentiation promoters include BMP4, i.e., the polypeptide represented by SEQ ID NO: 9, or a polypeptide equivalent in which one or more amino acids contained in the polypeptide are added, deleted, or substituted. It is preferable that the polypeptide equivalent has a differentiation-inducing activity on CSCs equivalent to that of the polypeptide represented by SEQ ID NO: 9.The differentiation-inducing activity can be defined as equivalent, for example, when it is 10%, preferably 20%, also preferably 30%, more preferably 40%, and even more preferably 50% of the CK20-inducing activity against CSCs of the polypeptide represented by SEQ ID NO: 9. In yet another non-limiting aspect, the differentiation-inducing activity can be defined as equivalent, for example, when it is 60%, preferably 70%, also preferably 80%, more preferably 90%, and even more preferably 95% of the CK20-inducing activity against CSCs of the polypeptide represented by SEQ ID NO: 9.

[0195] Anticancer drugs that can be used in combination with the cancer stem cell inhibitor of the present invention include alkylating agents, antimetabolites, natural products, platinum complexes, and other drugs. Alkylating agents include Nitrogen Mustards, Ethylenimines, Methylmelamines, Alkyl Sulfonates, Nitrosoureas, and Triazenes. Nitrogen Mustards include, for example, Mechlorethamine, Cyclophosphamide, Ifosfamide, Melphalan, and Chlorambucil. Ethylenimines and Methylmelamines include, for example, Hexamethylmelamine and Thiotepa. Examples of alkyl sulfonates include busulfan. Examples of nitrosoureas include carmustine (BCNU), lomustine (CCNU), semustine (methyl-CCNU), and streptozocin. Examples of triazenes include dacarbazine (DTIC). Examples of metabolic antagonists include folic acid analogues, pyrimidine analogues, and purine analogues. Examples of folic acid analogues include methotrexate. Examples of pyrimidine analogues include fluorouracil (5-FU), doxifluridine (5'-DFUR, trade name Furtulon), capecitabine (trade name Xeloda), floxuridine (FudR), and cytarabine.Examples of purine analogues include mercaptopurine (6-MP), thioguanine (TG), and pentostatin. Examples of natural products include vinca alkaloids, epipodophyllotoxins, and antibiotics. Examples of vinca alkaloids include vinblastine (VLB) and vincristine (VCR). Examples of epipodophyllotoxins include etoposide and teniposide. Examples of antibiotics include dactinomycin (actinomycin D), daunorubicin, doxorubicin, bleomycin, plicamycin, and mitomycin. Platinum complexes refer to platinum coordination complexes, and examples of such complexes include cisplatin (CDDP) and carboplatin. Other drugs include topoisomerase inhibitors such as irinotecan and camptothecin, taxols such as paclitaxel and docetaxel, anthracenediones such as mitoxantrone, urea substitutes such as hydroxyurea, methyl hydrazines such as procarbazine hydrochloride (trade name Natulan), and vitamin A metabolites such as tretinoin (trade name Vesanoid). In addition, rituximab, alemtuzumab, trastuzumab, bevacizumab, cetuximab, panitumumab, trastuzumab, gemtuzumab, etc. are also included.

[0196] All prior art documents cited in this specification are hereby incorporated by reference. EXAMPLES

[0197] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these.

[0198] Establishment of human colon cancer cell lines in immune-deficient NOG mice Colon cancer specimens were obtained from consenting patients under the approval of the ethical committees of PharmaLogicals Research (Singapore) and Parkway Laboratory Services (Singapore). Tumor pieces were minced with scissors and transplanted into the flanks of NOG mice. Human colon cancer xenografts were maintained by passaging in NOG mice provided by the Central Institute for Experimental Animals (Japan). Mice used in this study were treated in accordance with the animal experiment guidelines of PharmaLogicals Research. For histopathological examination, small pieces of human tissue surgical specimens and xenograft tumors were fixed in 4% paraformaldehyde for 16-24 hours at 4°C and embedded in paraffin by the AMeX method (Sato Y, et al., (1986) Am J Pathol, 125; 431-435., Sato Y, et al., (1992) Am J Pathol, 140; 775-779., Suzuki M, et al. (2002) J Toxicol Sci, 27; 165-172.). Thin sections were stained with eosin and hematoxylin and examined by microscopy.

[0199] Isolation and in vitro culture of colorectal CSCs Single-cell suspensions of cancer cells from xenografts were prepared by mincing the tissue with a razor and incubated in DPBS containing collagenase / dispase (Roche) and DNaseI (Roche) for 3 h at 37°C, then filtered through a 40 μm cell strainer (BD Biosciences) and suspended in lysis buffer (BD Biosciences) to remove red blood cells. The resulting xenograft-derived cells (such cells are referred to as primary cells) were cultured in DMEM / F12 medium (Invitrogen) containing N-2 supplement (Invitrogen), 20 ng / mL human EGF (Invitrogen), 10 ng / mL human basic fibroblast growth factor (Sigma), 4 μg / mL heparin (Sigma), 4 mg / mL BSA (Invitrogen), 20 μg / mL human insulin, zinc solution (Invitrogen), and 2.9 mg / mL glucose (Sigma) at 37°C under a 5% CO2 atmosphere (Todaro M, et al. (2007) Cell Stem Cell 1; 389-402.). Polystyrene-treated regular cell culture flasks (BD Biosciences) and ultra-low attachment cell culture flasks (Corning) were used to culture adherent and floating CSCs, respectively.

[0200] In vivo tumorigenesis analysis Cell suspensions were prepared by serial dilution. 100 μL of cancer cell suspension in Hanks' balanced salt solution (Invitrogen) was subcutaneously inoculated into the flank of mice with 50% Matrigel (BD Bioscience). Tumor development was monitored for 7 weeks. For single cell inoculation, cells were labeled with FITC-labeled mouse anti-human CD326 (EpCAM) antibody (Miltenyi Biotec) and seeded in Terasaki plates (Termo Fisher Scientific). Single cells were identified under a fluorescent microscope. Single cells were inoculated into the flank of mice with 50 μl of 50% Matrigel. Tumor development was monitored for 10 weeks.

[0201] Establishment of cells expressing full-length human Lgr4, Lgr5, and Lgr6 Full-length human Lgr4, Lgr5, and Lgr6 cDNAs were cloned by PCR based on the sequences of NM_018490 (Lgr4), NM_001017403 (Lgr6), and NM_003667 (Lgr5). The cloned genes were expressed with or without an HA tag at the N-terminus. The expression plasmids were transfected into Chinese hamster ovary cell line CHO DG44 (Invitrogen) using a Gene Pulser (BioRad). Stable cell lines HA-Lgr4 / DG, HA-Lgr5 / DG, and HA-Lgr6 / DG were selected using G418.

[0202] Preparation of soluble Lgr5-Fc protein Soluble Lgr5 (amino acids 1-555) protein was expressed as a fusion protein with the Fc portion of mouse IgG2a in CHO DG44. Transfectants were screened by sandwich ELISA using goat anti-mouse IgG2a (Bethyl laboratories) and HRP rat anti-mouse IgG2a Ab (Serotec). The clone that produced sLgr5-Fc most abundantly was named 2D3. 2D3 culture supernatant was collected, and Lgr5-Fc protein was affinity purified by protein A-Sepharose column (Pharmacia). Lgr5-Fc served as antigen for protein immunization and ELISA screening.

[0203] Generation of anti-Lgr5 monoclonal antibodies by Lgr5-Fc protein immunization Balb / c mice (Charles River Japan) were subcutaneously immunized with 50 μg of Lgr5-Fc emulsified in complete Freund's adjuvant. After 2 weeks, injections were repeated once a week for 2 weeks with the same amount in incomplete Freund's adjuvant. Three days before cell fusion, mice were intravenously injected with 25 μg of Lgr5Fc. Splenic lymphocytes from immunized mice were fused with P3-X63Ag8U1 mouse myeloma cells (ATCC) by conventional methods (Kremer L and Marquez G (2004) Methods Mol Biol., 239; 243 - 260). Hybridoma culture supernatants were screened for antibodies reactive with sLgr5-Fc using ELISA. Lgr5-specific mouse mAbs 2T15E-2 and 2U2E-2 were established.

[0204] Flow cytometry analysis Colon CSCs were incubated with labeled antibodies and analyzed using EPICS ALTRA (Beckman Coulter) and FACSCalibur (Becton Dickinson). The antibodies used were PE-labeled mouse anti-human CD133 antibody (Miltenyi Biotec), PE-labeled mouse anti-human CD44 antibody (BD Pharmingen), FITC-labeled mouse anti-human CD326 (EpCAM) antibody (Miltenyi Biotec), PE-labeled mouse anti-human CD166 antibody (R&D Systems), PE-labeled mouse anti-human CD24 antibody (BD Pharmingen), PE-labeled mouse anti-human CD26 antibody (BD Pharmingen), and PE-labeled mouse anti-human CD29 antibody (BD Pharmingen).

[0205] To stain Lgr5, colon CSCs were incubated with mouse anti-human Lgr5 antibody (2T15E-2) followed by PR-labeled rat anti-mouse IgG antibody (Invitrogen). Aldehyde dehydrogenase activity was measured using the AldeFluor Kit (Stemcell Technologies). Mouse cells and human colon CSCs were distinguished by staining with anti-mouse MHC class I antibody (Abcam) and PE- or APC-labeled goat anti-human IgG2a antibody (BioLegend). Dead cells were also removed with 7-AAD Viability Dye (Beckman Coulter).

[0206] Western blot analysis Proteins were extracted using RIPA buffer (Sigma) supplemented with Complete Mini protease inhibitor cocktail (Roche). Proteins were fractionated on NuPAGE gels (Invitrogen) and transferred to PVDF membranes. After blocking with 1% skim milk in PBS, the membranes were probed with rabbit anti-human β-catenin antibody (Sigma), rabbit anti-human phospho-c-JUN antibody (Sigma), rabbit anti-human TCF1 antibody (Cell Signaling), rabbit anti-human TCF3 antibody (Cell Signaling), rabbit anti-human TCF4 antibody (Cell Signaling), rabbit anti-human Lgr5 antibody (Abcam), mouse anti-human E-cadherin antibody (Abcam), rabbit anti-human Snail antibody (Abcam), and mouse anti-human GAPDH antibody (Santa Cruz). Reactive bands were detected using BCIP / NBT substrate (KPL).

[0207] Quantitative real-time polymerase chain reaction Total RNA was isolated using the RNeasy Mini Kit including DNAase treatment (Qiagen). cDNA was synthesized using the First-Strand cDNA Synthesis Kit (SABiosciences). Quantitative real-time PCR (QRT-PCR) analysis was performed using SYBR Green / Rox qPCR (SABiosciences) on an Mx3005P Real-Time PCR System (Stratagene). Fold induction values ​​were calculated using the 2-ΔΔCt method. GAPDH and ACTB were used as references. All experiments were performed in triplicate.

[0208] Primers for quantitative real-time PCR analysis The following primers were used to amplify the reactive transcripts: Lgr5: Forward primer 5'-AGTTTATCCTTCTGGTGGTAGTCC-3' (SEQ ID NO: 10), Reverse primer 5'-CAAGATGTAGAGAAGGGGATTGA-3' (SEQ ID NO: 11), GAPDH: Forward primer 5'-CTCTGCTCCTCCTGTTCGAC-3' (SEQ ID NO: 12), Reverse primer 5'-ACGACCAAATCCGTTGACTC-3' (SEQ ID NO: 13), ACTB: Forward primer 5'-AAGTCCCTTGCCATCCTAAAA-3' (SEQ ID NO: 14), Reverse primer 5'-ATGCTATCACCTCCCCTGTG-3' (SEQ ID NO: 15)

[0209] Cell proliferation assay Suspended CSCs and adherent CSCs were cultured at approximately 100 suspended CSCs and 1 × 10 adherent CSCs per well, respectively. 4The cells were seeded in a 96-well plate at 100 x 10 cells / well. On days 0 and 3, the viable cell count was measured by Cell Counting Kit-8 assay (Doujindo) according to the manufacturer's protocol. The average absorbance on day 0 was expressed as 100%. For chemosensitivity analysis, floating CSCs and adherent CSCs were seeded at approximately 100 floating CSCs and 1 x 10 adherent CSCs / well, respectively. 4 The cells were seeded in 96-well plates at 10 μg / mL and incubated for 24 h before adding 10 μg / mL 5-FU (Hospira), 10 μg / mL irinotecan (Hospira), 50 mM TCF inhibitor FH535 (Merck), and 50 mM β-catenin inhibitor cardamonin (Merck). After 3 days of culture in the presence of drugs, Cell Counting Kit-8 was added to the cells. The average absorbance of cells exposed to DMSO or medium alone was expressed as 100%. All experiments were performed in triplicate.

[0210] Immunofluorescence staining of cultured cells and xenograft tissues For immunofluorescence cytochemistry, cells fixed with 4% paraformaldehyde and methanol were incubated with mouse anti-human E-cadherin antibody (Abcam), rabbit anti-human Snail antibody (Abcam), or rabbit anti-human β-catenin antibody (Sigma), and then visualized with goat anti-mouse IgG antibody or goat anti-rabbit IgG antibody labeled with AlexaFluor 488, respectively. For immunofluorescence histochemistry, thin sections from paraffin blocks of the xenograft tumors were incubated with mouse anti-human Lgr5 antibody (2U2E-2) or rabbit anti-human Snail antibody (Abcam). After incubation with the primary antibody, Lgr5 protein was detected by goat anti-mouse antibody conjugated with polymer-HRP (DAKO) and visualized by AlexaFluor 488-labeled tyramide (Invitrogen). Snail protein was detected with biotinylated goat anti-rabbit antibody (VECTOR) and visualized with AlexaFluor 568-conjugated streptavidin (Invitrogen). The cells and specimens were also stained with DAPI (Invitrogen).

[0211] Example 1: Establishment of colon cancer xenografts As previously reported (Fujii E. et al. (2008) Establishment and characterization of in vivo human tumor models in the NOD / SCID / gamma(c)(null) mouse. Pathol Int 58:559-567.), the present inventors established 11 types of human colon cancer xenografts using NOD / Shi-scid, IL-2Rγnull (NOG) mice (Table 1; number of human colon cancer cell lines established in immune-deficient NOG mice).

[0212] [Table 1]

[0213] In Table 1 above, an asterisk indicates that the strain was established but not suitable for testing, and a dagger indicates that the strain was infected. As shown in Table 1, 17 colon cancer xenografts were established from samples from 53 human colon cancer patients. In addition to the 17 xenografts, 19 developed concomitant EBV-infected lymphoma cells (which exacerbated the condition of NOG mice), 14 developed other types of infection, and 3 did not develop tumor growth. Of these 17 xenografts, 11 survived even after freezing and thawing, retained tumor reconstitution potential, and showed histopathological characteristics similar to those of the original tumor. Of these 11 xenografts, 10 were derived from grade 2 moderately differentiated adenocarcinomas and 1 was derived from grade 3 poorly differentiated adenocarcinomas. Ten of the eleven xenografts were derived from moderately differentiated colorectal carcinoma (MDCC), and the remaining one was derived from a poorly differentiated colorectal carcinoma (PDCC) (Table 2; histopathological classification of the original human colorectal carcinomas used to establish the eleven xenografts).

[0214] [Table 2]

[0215] Both MDCC and PDCC xenografts reconstituted nearly identical histopathological morphology to the original tumors, with MDCC xenografts forming distinct epithelial ducts and small budding clusters with goblet cells that appeared to undergo epithelial-to-mesenchymal transition (EMT). In contrast, PDCC xenografts did not show distinct epithelial ductal structures (Figures 1 and 16).

[0216] [Example 2] Isolation of colon CSCs We used two types of MDCC xenografts, namely PLR59 and PLR123, to isolate colon CSCs. We selected these xenografts because they maintained the ability to rapidly proliferate and reconstitute tumors with epithelial ducts and small budding clusters even after 10 passages in NOG mice (Figure 1). Therefore, we believed that stable CSCs could be obtained from these xenografts.

[0217] Flow cytometry analysis of primary cells derived from PLR59 and PLR123 passaged in NOG mice showed that the levels of CD44, ALDH, CD26, and Lgr5 signals were lower than those of CD133, EpCAM, CD166, CD24, and CD29, indicating the presence of a small population of CSCs (Figure 2). Indeed, when primary cells derived from PLR59 and PLR123 were subcutaneously implanted into NOG mice at 100 cells per injection site, tumors were formed in approximately half of the injection sites (5 out of 12 injection sites; Table 3), and the histopathological morphology of these tumors was highly similar to that of the original tumor in terms of hierarchical structure (Figure 3). However, when 10 primary cells derived from PLR59 and PLR123 were injected subcutaneously per injection site, they failed to form tumors in NOG mice (Table 3). Table 3 shows the tumorigenic activity 49 days after inoculation.

[0218] [Table 3]

[0219] In Table 3 above, an asterisk indicates a tumor xenograft established in NOG mice, and a dagger indicates a cell preparation. "Primary" refers to cells (primary cells) prepared by harvesting xenograft tumor tissue grown in NOG mice and then removing red blood cells and mouse cells, "floating" refers to cells obtained by culturing primary cells in vitro under non-adherent conditions, and "adherent" refers to cells cultured in vitro under adherent conditions. A plus sign (1) indicates the number of tumors formed, and a plus sign (2) indicates the total number of inoculation sites. Brackets indicate the percentage of tumor formation (cancer formation). Lgr5 + indicates Lgr5 positivity, and Lgr5 - indicates Lgr5 negativity.

[0220] Primary cells derived from PLR59 and PLR123 were cultured in serum-free medium supplemented with EGF and FGF, resulting in adherent and floating cells. We collected the adherent and floating cells and cultured them separately. The adherent cells proliferated with a doubling time of about 2.5 days and showed mesenchymal cell-like morphology, whereas the floating cells showed no significant proliferation and formed spheroid-like cell clusters (Figures 4, 5, 18, and 19). After more than one week of culture, both the adherent and floating cells were highly homogenous, as judged by colon CSC markers, and the adherent cells were Lgr5. + , A.L.D.H. + , CD133 + , CD44 + , EpCAM + , CD166 + , CD24 + , CD26 + , and CD29 + On the other hand, the non-adherent cells were Lgr5 - , and ALDH - This was different from that of adherent cells (FIGS. 6 and 20). A significant level of Lgr5 mRNA was detected in adherent cells, but the level of Lgr5 mRNA in non-adherent cells was undetectable (FIG. 27).

[0221] [Example 3] Lgr5 protein expression analysis To investigate the expression of Lgr5 protein, we generated two Lgr5-specific monoclonal antibodies (2L36, 2T15E-2 and 2U2E-2) for immunohistochemical and flow cytometric analysis, respectively. Our antibodies were highly specific to Lgr5, but did not cross-react with Lgr4 and Lgr6, both of which have high homology to Lgr5 (Fig. 28, 29). Using these antibodies, we demonstrated that Lgr5 was expressed in adherent CSCs.

[0222] Lgr5-positive cells were detected throughout the passage of the original tumor tissues of PLR59 and PLR123 and their xenograft cancer tissues (Figure 39). The frequency of Lgr5-positive cells in the original tumor tissues was low (0.01% in PLR59 and 0.04% in PLR123). In the xenograft cancer tissues, the frequency of Lgr5-positive cells increased with passage, but remained unchanged after the 10th generation (Figure 39). Meanwhile, the tumor reconstitution ability of the initial cells from the PLR123 xenograft model also increased with passage. The estimated proportion of CSCs in the initial cells evaluated from the tumor reconstitution ability was approximately 0.1% after 5 passages, whereas it increased to approximately 0.4% after 14 passages.

[0223] [Example 4] Tumor reconstitution ability of Lgr5-positive and Lgr5-negative colon CSCs If Wnt signaling is a hallmark of colon cancer stem cell population, only Lgr5-positive adherent cells can form tumors in vivo. To verify whether this is true, we investigated the tumorigenicity of Lgr5-positive adherent cells and Lgr5-negative non-adherent cells.

[0224] As a result, tumorigenicity was stronger in Lgr5-positive adherent cells than in Lgr5-negative non-adherent cells, but both Lgr5-positive and Lgr5-negative cells retained tumorigenicity in NOG mice. Subcutaneous injection of 10 Lgr5-positive cells caused tumors at all injection sites (6 out of 6 sites), whereas Lgr5-negative cells caused tumors at 2 (PLR123-derived cells) or 1 (PLR59-derived cells) out of 6 injection sites (Table 3). Even when only 1 Lgr5-positive cell was injected per inoculation site, tumors were reconstructed at 2 (PLR123-derived cells) or 1 (PLR59-derived cells) out of 12 injection sites (Figure 7), and the histopathological morphology of the tumors derived from Lgr5-positive and Lgr5-negative cells was almost identical to the original tumors (Figures 17 and 40). Furthermore, the expression of cell surface markers and the tumor-initiating activity of Lgr5-positive CSCs did not change even after one month of subculture (FIGS. 30 and 31).

[0225] Lgr5+ cells divided symmetrically under adherent culture conditions (Fig. 41). On the other hand, in the presence of Matrigel and serum, Lgr5+ cells divided asymmetrically, as shown by the distribution of Lgr5 protein to one of the two daughter cells in culture under the same conditions (Fig. 42C and D). One characteristic of CSCs is symmetric cell division for self-renewal, and another characteristic of CSCs is asymmetric cell division. Lgr5+ adherent cells divided symmetrically under adherent culture conditions (Fig. 41), while in the presence of Matrigel and FBS, Lgr5+ cells divided asymmetrically to generate two different progeny, as shown by the distribution of Lgr5 protein to one daughter cell (Fig. 42).

[0226] These results demonstrated that Lgr5-positive and Lgr5-negative cells derived from PLR59 and PLR123 are highly purified colon CSCs, and that Lgr5-positive and Lgr5-negative cells represent two distinct states of CSCs in colon cancer.

[0227] [Example 5] Effects of TCF and β-catenin Consistent with Lgr5 expression, the levels of β-catenin, TCF1, TCF3, and TCF4 proteins were upregulated in Lgr5-positive cells but not in Lgr5-negative cells (FIGS. 7 and 21). On the other hand, phosphorylation of the N-terminal region of c-Jun was not detected in Lgr5-positive CSCs compared to Lgr5-negative CSCs (FIGS. 7 and 21).

[0228] To address the question of whether Wnt signaling drives the proliferation of colorectal CSCs, we investigated the effects of the β-catenin / TCF inhibitor FH535 and the Wnt / β-catenin inhibitor cardamonin (which induces the degradation of β-catenin) on the proliferation of colorectal CSCs.

[0229] As a result, 50 μM FH535 significantly reduced the proliferation of Lgr5-positive colon CSCs, but had no effect on the proliferation of Lgr5-negative colon CSCs (FIGS. 8 and 22). On the other hand, 50 μM cardamonin reduced the number of viable cells in Lgr5-positive colon CSCs by 70%, and reduced the number of viable cells in Lgr5-negative colon CSCs by approximately 50% (FIGS. 8 and 22).

[0230] These results suggest that TCF mediates the proliferation of Lgr5-positive cells and that β-catenin is involved in the survival of colon CSCs. Interestingly, Lgr5-positive cells proliferate even without the supply of EGF and FGF (FIGS. 9 and 23), indicating that colon CSCs contain an endogenous / autocrine mechanism to activate Wnt signaling for their proliferation.

[0231] [Example 6] Ability of colon CSCs to switch from Lgr5-positive to Lgr5-negative state One of the characteristics of CSCs is resistance to chemotherapy, so we investigated the sensitivity of colon CSCs to 5-FU and irinotecan. As mentioned above, Lgr5-positive cells proliferated with a doubling time of about 2.5 days, while Lgr5-negative CSCs were quiescent in terms of proliferation. When treated with 5-FU (10 μg / ml) and irinotecan (10 μg / ml), the proliferation of Lgr5-positive colon CSCs was significantly inhibited, but the proliferation and survival of Lgr5-negative colon CSCs were not affected (FIGS. 10 and 24). After exposing Lgr5-positive colon CSCs to 5-FU (10 μg / ml) or irinotecan (10 μg / ml) for 3 days, cells resistant to these chemotherapy drugs appeared. Surprisingly, the drug-resistant cells were Lgr5 negative and their morphology was changed (FIGS. 11, 32, and 25), indicating a change from an Lgr5-positive state to an Lgr5-negative state.

[0232] We selected HLA-DMA, TMEM173, ZMAT3, and GPR110 as markers to specifically detect these Lgr5-negative CSCs, and attempted immunostaining using specific antibodies against the same molecules. Specific staining images were obtained in colon CSCs that had been exposed to irinotecan for three days and had become Lgr5-negative (Figure 43). We also confirmed that this immunostaining method can be applied to tissue sections obtained from commonly used paraffin blocks (Figure 43). These results indicate that HLA-DMA, TMEM173, ZMAT3, and GPR110 can be specific markers for Lgr5-negative CSCs.

[0233] The fluorescence indicating Lgr5 positivity observed before irinotecan treatment (Figure 44A) disappeared by irinotecan treatment (Figure 44B). From Lgr5-negative cells that were reseeded and cultured in the absence of irinotecan, Lgr5-positive cells appeared at 4 days after reseeding (Figure 44C) and increased by 8 days after reseeding (Figure 44D). All of the drug-resistant Lgr5-negative cells were Lgr5-negative (Figures 44 and 45) and remained CK20-negative (Figure 46), suggesting that the transition of colon CSCs from an actively proliferating state to a quiescent state is associated with the loss of Lgr5 molecules. The above association was also confirmed by the results of an in vitro growth inhibitor resistance assay (Figure 47). In addition, ALDH activity was decreased, but no changes were observed in other CSC markers (Figure 48).

[0234] When the tumorigenic activity of Lgr5-negative cells obtained by irinotecan treatment was examined, subcutaneous injection of 10 cells derived from PLR59 and PLR123 formed tumors in 2 and 1 NOG mice, respectively (Table 4). Table 4 shows the tumorigenic activity of Lgr5-negative CSCs 49 hours after inoculation. In Table 4 below, asterisks indicate tumor xenografts established in NOG mice. Plus (1) indicates the number of animals that showed tumors, and plus (2) indicates the total number of animals.

[0235] [Table 4]

[0236] To confirm whether Lgr5-negative colon CSCs change to an Lgr5-positive state, the inventors also cultured Lgr5-negative colon CSCs prepared by irinotecan treatment in serum-free stem cell culture medium again, which resulted in Lgr5-positive cells, exhibiting mesenchymal cell-like morphology (FIGS. 12 and 33), and initiating cell proliferation. On the other hand, when Lgr5-positive adherent colon CSCs were cultured in ultra-low adhesion plates, the inventors observed that some of the cells stopped proliferating, formed spheroid-like structures, and showed very low levels of Lgr5 mRNA (FIGS. 12 and 33). The switch from an Lgr5-positive state to an Lgr5-negative state (and vice versa) was also confirmed by observations using single cells in culture. When single Lgr5-positive cells were cultured in multiwell plates, the cells transitioned to an Lgr5-negative state within 3 days after irinotecan treatment. When single Lgr5-negative cells obtained by irinotecan treatment were cultured in multiwell plates in the absence of irinotecan, 19 to 43% of the cells transitioned to an Lgr5-positive state within 4 days (Figure 49 and Table 5).

[0237] [Table 5]

[0238] Table 5 shows the ratio of Lgr5-positive and -negative cells stained by immunocytostaining using anti-Lgr5 antibody (2L36 antibody). The numbers in parentheses indicate the percentage of Lgr5-positive or -negative cells.

[0239] Therefore, the present inventors concluded that colorectal CSCs alternate between an Lgr5-positive state and an Lgr5-negative state, and such a change does not require exogenous factors or a niche environment.

[0240] [Example 7] EMT of Lgr5-positive colon CSCs in vitro and in vivo Mesenchymal-like cells expressing nuclear β-catenin are considered to be migrating CSCs and metastasis-forming CSCs undergoing EMT (Brabletz T, Jung A, Spaderna S, Hlubek F, Kirchner T (2005) Opinion: migrating cancer stem cells - an integrated concept of malignant tumour progression. Nat Rev Cancer 5:744-749.). Because the morphology of Lgr5-positive colonic CSCs resembles mesenchymal cells, we tested whether Lgr5-positive colonic CSCs represent migrating CSCs. Western blot analysis revealed low levels of cell surface E-cadherin, high levels of Snail, and nuclear-localized β-catenin expression, which is a hallmark of EMT, in Lgr5-positive colonic CSCs (Figure 13, Figure 14, and Figure 26). In contrast, Lgr5-negative colon CSCs did not show any signs of EMT, i.e., high expression of cell surface E-cadherin, low expression of Snail, and no nuclear localization of β-catenin. Furthermore, in xenograft tumor tissues, co-expression of Snail and Lgr5 was observed in cells undergoing EMT in the budding region (Figure 15), supporting the view that Lgr5-positive colon CSCs correspond to migratory stem cells.

[0241] Furthermore, the present inventors demonstrated that Lgr5-positive colon CSCs formed tumors in multiple tissues, including the lung, liver, lymph node, and subcutaneous tissue. Interestingly, at least 40 days after intravenous injection of tumor cells, tumors with epithelial ductal structures were reconstituted in the liver, lymph node, and subcutaneous tissue, but not in the lung (FIGS. 34 and 35).

[0242] Next, it was investigated whether Lgr5-negative CSCs directly generate the cancer hierarchy or whether they first transform into Lgr5-positive cells in vivo. To search for a marker to detect Lgr5-negative CSCs, gene expression profiling was performed using Lgr5-positive cells, Lgr5-negative cells, and primary cells of xenografted tumors. As a result, HLA-DMA was selected from among the molecules whose expression was detectable at a higher level in Lgr5-negative CSCs compared to Lgr5-positive CSCs and primary cells (Figure 50). HLA-DMA was confirmed to be specifically expressed in Lgr5-negative CSCs by tissue immunostaining using Lgr5, HLA-DMA, and EREG antibodies (Figure 51). Since HLA-DMA is also expressed in macrophages, another marker expressed in CSCs was also searched for to exclude the possibility that cells stained by HLA-DMA antibody in tissue immunostaining were macrophages. EREG is expressed in both Lgr5-positive and -negative CSCs (Fig. 50). Immunohistochemical staining of Lgr5-positive and -negative CSCs using an antibody against EREG confirmed that EREG is expressed in both Lgr5-positive and -negative CSCs (Fig. 51). By detecting both markers in combination, it was confirmed that Lgr5-negative CSCs can be identified as cells positive for both HLA-DMA and EREG. After a homogeneous population of Lgr-negative CSCs was injected into NOG mice, cells that weakly expressed Lgr5 but were still positive for HLA-DMA and EREG appeared for one day. By 5 days after injection, HLA-DMA-negative, Lgr5-positive and EREG-positive cells appeared (Fig. 52). Tumors derived from Lgr5-negative CSCs had a clear ductal structure and contained Lgr5-positive cells (Fig. 53).

[0243] To explore the possibility of conversion to a growth inhibitor-resistant state in vivo, irinotecan was administered intraperitoneally at a maximum tolerated dose (MTD) (120 mg / kg) to NOG mice containing tumors derived from Lgr5-positive CSCs. Tumor growth was almost completely inhibited (Figure 55), and ductal structures were severely destroyed (Figure 54). In this state, Lgr5-positive cells were extremely reduced (Figures 54 and 56). After irinotecan treatment, the number of Lgr5-negative and HLA-DMA-positive cells increased significantly. In contrast, in vehicle-treated control mice, approximately one-third of the cancer cells in both ducts and budding regions were Lgr5-positive (Figure 54). Both Lgr5-positive cells and HLA-DMA-positive and Lgr5-negative cells were EREG-positive, confirming that they were CSCs (Figure 54). After the end of irinotecan treatment, Lgr5-positive cells reappeared (Figure 54). Taken together, these results indicate that Lgr5-negative CSCs are the origin of colon cancer after treatment with growth inhibitors, and that they reconstitute the cancer hierarchy via Lgr5-positive cells.

[0244] [Example 8] Identification of molecules specifically expressed in cancer stem cells 1. Preparation of Lgr5-negative adherent cells by irinotecan treatment Lgr5-positive adherent cells were cultured in stem cell medium at 3 × 10 5The cells were seeded in a 6-well plate (BD, Cat. No. 353046). The next day, irinotecan (Hospira, 61703-349-09) was added to the cells at a final concentration of 10 μg / mL, and the cells were continued to be cultured. After 3 days of culture, cells resistant to irinotecan were observed. These cells were collected with Accutase and suspended in FACS buffer, and then 7-AAD Viability Dye was added as a dead cell stain, and FITC-labeled mouse mAb to human CD326 (EpCAM), PE-labeled mouse mAb to human CD133 / 1 (AC133), PE-labeled mouse mAb to human CD44, PE-labeled mouse mAb to human CD166, PE-labeled mouse mAb to human CD24, PE-labeled mouse mAb to human CD26, or PE-labeled mouse mAb to human CD29 were added as cancer stem cell markers, and the cells were incubated at 4°C for 30 minutes. For Lgr5, mouse mAb to human Lgr5 was added and incubated at 4℃ for 30 minutes, then washed once with FACS buffer, and PE-labeled goat Ab to mouse IgG2a was added and incubated at 4℃ for 30 minutes. The cells were then washed once with FACS buffer and subjected to flow cytometry analysis. ALDH activity was detected using the AldeFluor Kit by following the procedure recommended by the manufacturer. EPICS ALTRA was used for flow cytometry analysis, and cancer stem cell markers were analyzed for 7-AAD Viability Dye-negative cells. Irinotecan-resistant cells were observed to change from Lgr5-positive to Lgr5-negative.

[0245] 2. Identification of molecules specifically expressed in cancer stem cells Primary cells of PLR59 and PLR123, Lgr5-positive, highly proliferative cancer stem cells produced by adherent culture of primary cells, and Lgr5-negative, low-proliferative cancer stem cells produced by treating the cells with irinotecan in the same manner as above were physically disrupted using QIAshredder (Qiagen, Cat. No. 79654), and RNA was extracted using RNeasy Mini Kit (Qiagen, Cat. No. 74104) and RNase-Free DNase Set (Qiagen, Cat. No. 79254) according to the manufacturer's recommended procedures. The extracted RNA was analyzed for purity and quality using an Agilent 2100 Bioanalyzer. After cRNA synthesis, gene expression information was obtained using Affymetrix's GeneChip (HG-U133 plus2). Data analysis was performed using Microsoft Excel and Statistics software R. Three types of cells (primary cells, Lgr5-positive cells, and Lgr5-negative cells) were compared to each other, and a list of genes with significantly increased expression was created for each. In other words, the raw data from the GeneChip was normalized and log-based converted to base 2 using GCRMA, and expression differences were calculated between different sample types (three types: primary cells and Lgr5-positive cells, Lgr5-positive cells and Lgr5-negative cells, and Lgr5-negative cells and primary cells). The following three criteria were used to extract genes with differential expression:

[0246] (1) Genes that are more than twice as abundant in Lgr5-positive cells compared to primary cells and more than twice as abundant in Lgr5-negative cells compared to primary cells (highly expressed in both Lgr5-positive and -negative cancer stem cells) (Tables 6-1 to 6-10) (Partial amino acid sequences of proteins encoded by the genes are shown in SEQ ID NOs: 1 to 6 and 9.) (2) Genes that were increased by more than 2-fold in Lgr5-positive cells compared to primary cells and decreased by less than 2-fold in Lgr5-negative cells compared to primary cells (high expression only in Lgr5-positive cancer stem cells) (Tables 7-1 to 7-5) (3) Genes that are changed less than 2-fold in Lgr5-positive cells compared to primary cells and are changed more than 2-fold in Lgr5-negative cells compared to primary cells (high expression only in Lgr5-negative cancer stem cells) (Tables 8-1 to 8-2) (Partial amino acid sequences of proteins encoded by the genes are shown in SEQ ID NO: 7 or 8.)

[0247] To obtain genes that code for proteins that are specifically displayed on the cell membrane in cancer stem cells, we extracted genes with GO:0005886 [plasma membrane] in GeneOntology (GO). Furthermore, we extracted genes with GO:0005576 [extracellular region], GO:0009986 [cell surface], GO:0016020 [membrane], or genes predicted to have a transmembrane region by the membrane protein prediction software TMHMM, or genes predicted to have a signal peptide by the signal peptide prediction software SignalP, but not GO:0031090 [organelle membrane]. GeneChip data derived from normal colon tissues were also used to exclude genes with relatively high expression in normal tissues and primary cells, and genes with low change rates in Lgr5-positive and Lgr5-negative cells.

[0248] [Table 6-1]

[0249] Table 6-2 is a continuation of Table 6-1. [Table 6-2]

[0250] Table 6-3 is a continuation of Table 6-2. [Table 6-3]

[0251] Table 6-4 is a continuation of Table 6-3. [Table 6-4]

[0252] Table 6-5 is a continuation of Table 6-4. [Table 6-5]

[0253] Table 6-6 is a continuation of Table 6-5. [Table 6-6]

[0254] Table 6-7 is a continuation of Table 6-6. [Table 6-7]

[0255] Table 6-8 is a continuation of Table 6-7. [Table 6-8]

[0256] Table 6-9 is a continuation of Table 6-8. [Table 6-9]

[0257] Table 6-10 is a continuation of Table 6-9. [Table 6-10] (The numbers in Tables 6-1 to 6-10 above indicate expression differences (log2 ratios).)

[0258] [Table 7-1]

[0259] Table 7-2 is a continuation of Table 7-1. [Table 7-2]

[0260] Table 7-3 is a continuation of Table 7-2. [Table 7-3]

[0261] Table 7-4 is a continuation of Table 7-3. [Table 7-4]

[0262] Table 7-5 is a continuation of Table 7-4. [Table 7-5] (The numbers in Tables 7-1 to 7-5 above indicate expression differences (log2 ratios).)

[0263] [Table 8-1]

[0264] Table 8-2 is a continuation of Table 8-1. [Table 8-2] (The numbers in Tables 8-1 and 8-2 above indicate expression differences (log2 ratios).)

[0265] Furthermore, to obtain genes encoding proteins that are specifically displayed on the cell membrane in cancer stem cells, we extracted genes with GO:0005886 [plasma membrane] in GeneOntology (GO) selected by another criterion described below (Tables 9 and 10).

[0266] Markers common to proliferative CSCs and dormant CSCs: Genes with an average expression value greater than 64 in Lgr5-negative cells and Lgr5-positive cells, with a more than 4-fold change compared to primary cells in both Lgr5-negative cells and Lgr5-positive cells, and with a significant difference by t-test (Table 9).

[0267] [Table 9]

[0268] Dormant CSC-specific markers: Genes with an average expression value greater than 64 in Lgr5-negative cells, an average expression value less than 64 in both primary cells and Lgr5-positive cells, a greater than 20-fold change in Lgr5-negative cells compared to Lgr5-positive cells, and a significant difference in t-test (Table 10).

[0269] [Table 10]

[0270] 3. Expression analysis by flow cytometry 3.1.Flow cytometry analysis of NOG-established cancer cell lines NOG established cancer cell lines collected from mice were suspended in FACS buffer, and then rat mAb to mouse MHC I (Abcam, ab15680) and mAb to human EREG (EP27, WO2008 / 047723) were added and reacted at 4°C for 30 minutes, followed by washing once with FACS buffer, and 7-AAD Viability Dye (Beckman Coulter, A07704) as a dead cell stain, PE-labeled goat F(ab')2 fragment to mouse IgG (H+L) (Beckman Coulter, IM0855) and APC-labeled goat Ab to rat IgG (BioLegend, 405406) ​​as secondary antibodies, and reacted at 4°C for 30 minutes. The cells were then washed once with FACS buffer and subjected to flow cytometry analysis. EPICS ALTRA was used for flow cytometry analysis, and EREG expression was analyzed in 7-AAD Viability Dye-negative and mouse MHC-negative cells.

[0271] 3.2.Flow cytometry analysis of in vitro cultured cancer cell lines Lgr5-positive adherent cells and Lgr5-negative adherent cells treated with irinotecan were collected with Accutase and suspended in FACS buffer. After addition of mouse mAb to human EREG, the cells were incubated at 4°C for 30 minutes, washed once with FACS buffer, and 7-AAD Viability Dye was added as a dead cell stain and PE-labeled goat F(ab')2 fragment to mouse IgG (H+L) was added as a secondary antibody and incubated at 4°C for 30 minutes. The cells were then washed once with FACS buffer and subjected to flow cytometry analysis. EPICS ALTRA was used for flow cytometry analysis, and EREG expression was analyzed for 7-AAD Viability Dye-negative cells. The results showed that the corresponding protein was highly expressed on the cell membrane surface.

[0272] Primary cells of PLR59 and PLR123, Lgr5 + Cancer stem cells and Lgr5 -The results of flow cytometry analysis of EREG in cancer stem cells are shown in Figure 37. Cells were stained with an antibody against EREG and analyzed by flow cytometry. Primary cells were EREG-negative, Lgr5 + Cancer stem cells and Lgr5 - It was shown that cancer stem cells contain a homogenous population of EREG-positive cells. The gray area indicates the fluorescence intensity after staining the cells with the indicated antibodies, and the white area indicates the fluorescence intensity after staining the cells with a control isotype antibody.

[0273] 4. In vitro efficacy confirmation by measuring ADCC activity 4.1. Preparation of effector cell suspension Human-derived effector cells were mononuclear cell fractions collected from human peripheral blood. 50 mL of peripheral blood was collected from a healthy volunteer (adult male) in-house using a syringe pre-injected with 200 μL of 1000 units / mL heparin solution (Novo Heparin Injection 5000 Units, Novo Nordisk). This peripheral blood was diluted 2-fold with PBS(-) and added to a Leucosep lymphocyte separation tube (Greiner bio-one) that had been pre-injected with Ficoll-Paque PLUS and centrifuged. This was centrifuged (2150 rpm, 10 min, room temperature) and the mononuclear cell fraction layer was isolated. The cells were washed once with 10% FBS / D-MEM and then diluted to a cell density of 5 x 10 with 10% FBS / D-MEM. 6 / mL was used as an effector cell suspension.

[0274] 4.2. Preparation of target cell suspension The target cell suspension was prepared fresh at the time of the test. 1 x 10 6 The cancer cell lines were centrifuged (1200 rpm, 5 min, room temperature), and the cell pellet was suspended in 200 μL of 0.2 mg / mL calcein-AM (Nacalai Tesque) / DMEM (10% FBS) medium. The cell suspension in the calcein-AM solution was cultured for 2 hours in a CO2 incubator set at a CO2 concentration of 5% and a temperature of 37°C. After washing once with 10% FBS / D-MEM, the cell density was adjusted to 2 x 105 / mL to prepare a target cell suspension.

[0275] 4.3. Measurement of ADCC activity Anti-EREG antibody was prepared at a concentration of 0.5 mg / mL and further diluted with 10% FBS / D-MEM to prepare an antibody solution. The final concentrations were 0.4 μg / mL, 4 μg / mL, and 40 μg / mL. 50 μL of each antibody solution or 10% FBS / D-MEM at each concentration was injected into a 96-well U-bottom plate. Next, 50 μL of target cell suspension was added to each well and allowed to stand at room temperature for 15 minutes. Next, 100 μL of effector cell suspension was added to the wells into which the antibody solution and target cell suspension, or 10% FBS / D-MEM and target cell suspension were injected. 100 μL of 10% FBS / D-MEM or 2% NP-40 solution (NP-40 substitute, Wako Pure Chemical Industries, Ltd.) was added to the other wells into which the 10% FBS / D-MEM and target cell suspension were injected. The plate was centrifuged (1200 rpm, 5 min, room temperature) and incubated for 4 hours in a CO2 incubator set at 5% CO2 and 37°C. The plate was centrifuged (1200 rpm, 5 min, room temperature), and 100 μL of the supernatant was collected from each well and the fluorescence intensity (λ ex = 490 nm, λ em The specific calcein release rate: cytotoxicity (%) was calculated according to the following formula:

[0276] [Formula 1] cytotoxicity(%) = (AC) x 100 / (BC)

[0277] A shows the fluorescence intensity in each well, B shows the average fluorescence intensity in wells where 50 μL of 10% FBS / D-MEM was added with 50 μL of target cell suspension and 100 μL of NP-40 solution, and C shows the average fluorescence intensity in wells where 50 μL of 10% FBS / D-MEM was added with 50 μL of target cell suspension and 100 μL of 10% FBS / D-MEM. The test was performed with N=3, and the cytotoxicity (%) of each antibody concentration was calculated using Microsoft Office Excel 2007.

[0278] The ADCC activity of anti-EREG antibodies against Lgr5-positive and Lgr5-negative PLR59 cells and Lgr5-positive and Lgr5-negative PLR123 cells is shown in Figure 38. As a result, no cytotoxic activity was observed with the control antibody, but the anti-EREG antibodies showed dose-dependent cytotoxic activity against both Lgr5-positive and -negative cancer stem cells of both PRL59 and PLR123.

[0279] To confirm the expression of EREG in vivo, Lgr5-positive cells were administered subperitoneally to NOG mice. EREG was highly expressed in the early stages of tumor formation, but in the later stages when the tumor formed a clear ductal structure, EREG expression was somewhat restricted to the budding clusters compared to the ductal structures. EREG-positive cells were detected even after irinotecan was administered to tumor-bearing mice (Figure 54). Therefore, the antitumor activity of EREG antibodies after irinotecan treatment was examined. Since EREG antibodies require effector cells to exert ADCC activity, SCID mice were used as a model for evaluating the efficacy of EREG antibodies. Tumor growth was suppressed when the antibody was administered 4 and 11 days after the final administration of irinotecan (Figure 57).

[0280] To evaluate the efficacy of the drug in a metastatic model, EREG expression in the metastatic model was first examined. When Lgr5-positive cells were injected intravenously into NOG mice, tumors were formed in multiple tissues, including the lungs. Most of the tumor cells formed in the lungs were EREG-positive (Fig. 58A). The efficacy of the EREG antibody was examined using SCID-Beige mice, in which macrophages and monocytes can exert ADCC as effector cells. In mice administered with EREG antibody once a week for five times starting from 3 days after injection of Lgr5-positive cells, the number of tumor cells at distant sites was shown to be extremely reduced compared to the number in control mice (Fig. 58B). In addition, the size of each tumor was also shown to be extremely reduced in mice administered with the antibody (Fig. 58C and D).

[0281] [Example 9] Presence of Lgr5-negative and -positive CSCs in clinical tumor specimens Proliferative and dormant CSCs were determined by tissue immunostaining using Lgr5 antibody (2U2E-2), HLA-DMA antibody and EREG antibody (Figure 59 and Table 11). Proliferative CSCs represent Lgr5 positive cells, and dormant CSCs represent HLA-DMA positive and EREG positive cells (Table 11). Lgr5 positive cells positive for both HLA-DMA and EREG, and Lgr5 negative cells positive for both HLA-DMA and EREG, were present in small amounts in primary and metastatic colon cancer specimens isolated from colon cancer patients (Figure 59). Of the 12 specimens of human colon cancer tissue, both Lgr5 positive and Lgr5 negative cells were detected in 8 cases, and either Lgr5 positive or Lgr5 negative cells were observed in the remaining 4 cases. Of all samples, 0.003 to 1.864% were Lgr5-positive cells, and 0.001-10.243% were Lgr5-negative cells (Table 11).

[0282] [Table 11] (P†: indicates that proliferative or dormant CSCs were detected. N: indicates that proliferative or dormant CSCs were not detected.) (Frequency: Percentage of cells.)

[0283] Both Lgr5-positive and -negative CSCs were detected in the ducts and budding regions (Figure 59). In addition, Lgr5-positive and -negative CSCs in the ducts were not confined to specific regions but were observed randomly throughout the ducts.

[0284] [Example 10] Antitumor effects of various antibodies using Mab-ZAP and Rat-ZAP It was evaluated whether targeted therapy targeting highly expressed membrane proteins in irinotecan-treated or irinotecan-untreated PLR59 and PLR123 could show antitumor effects. The binding activity of the commercially available antibodies listed in Table 12 to antigens expressed on the cell surface of irinotecan-treated or irinotecan-untreated PLR59 or PLR123 was measured using flow cytometry (FCM). The results are summarized in Table 13.

[0285] [Table 12]

[0286] [Table 13] (NT stands for no test)

[0287] The internalization activity (into cells) of various antibodies confirmed to have binding activity was evaluated using Mab-ZAP and Rat-ZAP. Mab-ZAP and Rat-ZAP are anti-mouse IgG antibodies or anti-rat IgG antibodies bound to saporin, a toxin that inhibits protein synthesis (Advanced Targeting Systems). When evaluating the internalization activity of cells not treated with irinotecan, PLR59 and PLR123 cells were seeded in each well of a 96-well plate at a cell density of 30,000 cells / 80 μL / well, and the next day, various antibody solutions were added to each well to a final concentration of 0.01, 0.1, or 1 μg / mL. Subsequently, the plates to which Mab-ZAP or Rat-ZAP was added to each well at a final concentration of 1 μg / mL were cultured in a CO2 incubator at 37°C for 72 hours. When the internalization activity against cells treated with irinotecan was evaluated, PLR59 and PLR123 cells and irinotecan at a final concentration of 15 μM were added to each well of the 96-well plate, which were cultured for 72 hours in a CO2 incubator at 37°C. The internalization activity of various antibodies against cells cultured in the presence or absence of irinotecan as described above was evaluated. During the assay, the internalization activity of various antibodies against cells present in each well, which had been replaced with a medium that did not contain irinotecan, was evaluated, as in the case of cells not treated with irinotecan. After 72 hours from the addition of the antibodies, Mab-ZAP, and Rat-ZAP, the cells present in the plate to which 3% SDS (Nacalai Tesque) was added at a dose of 10 μL / well were thoroughly dissolved by stirring the plate with a plate mixer. Then, CellTiter-Glo (registered trademark) Luminescent Cell Viability Assay (Promega) was added at a dose of 100 μL / well, and the luminescence signal of the mixture in each well was measured. The antitumor activity obtained as a result is shown in Table 14 and Figures 60 to 72.The percentage of cell proliferation inhibition shown on the vertical axis in Figures 60 to 72 means the relative value of the difference between the luminescence signal value of the mixture in the well to which only the test antibody was added (without Mab-ZAP and Rat-ZAP) and the luminescence signal value of the mixture in the well to which the test antibody, Mab-ZAP, and Rat-ZAP were added, when the difference between the luminescence signal value of the mixture in the well to which only the test antibody was added (without Mab-ZAP and Rat-ZAP) and the luminescence signal value of the mixture in the well to which the test antibody, Mab-ZAP, and Rat-ZAP were added is taken as 100%. The symbols -, +, ++, and +++ in Table 14 represent the relative value of the internalization activity when the test antibody was tested at a concentration of 1 μg / μL. The relative value means the relative value of the difference between the luminescence signal value of the mixture in a well to which only the test antibody was added (without Mab-ZAP and Rat-ZAP added) and the luminescence signal value of the mixture in a well to which no cells were seeded, when the difference between the luminescence signal value of the mixture in a well to which only the test antibody was added (without Mab-ZAP and Rat-ZAP added) and the luminescence signal value of the mixture in a well to which the test antibody, Mab-ZAP, and Rat-ZAP were added is taken as 100%, and -, +, ++, and +++ indicate that the relative value is less than 5%, 5% to less than 15%, 15% to less than 25%, and 25% or more, respectively.

[0288] [Table 14]

[0289] As shown in Figure 66, under the condition where sufficient antitumor activity was confirmed with the anti-EPCAM antibody used as a positive control, anti-CD70 antibody and anti-FAS antibody showed internalization activity of 25% or more against irinotecan-untreated PLR59 and PLR123 (Figures 60 and 62). In addition, anti-EDAR antibody showed 15-25% internalization activity against PLR59, and anti-PVRL4 antibody and anti-PROCR antibody showed 5-15% internalization activity against PLR59 (Figures 61, 63 and 65). On the other hand, anti-FAS antibody and anti-TNFRSF9 antibody showed 25% or more internalization activity against irinotecan-treated PLR59 and PLR123, and anti-PROM2 antibody showed 5-15% internalization activity against irinotecan-treated PLR59 and PLR123 (Figures 67, 70 and 68). Both anti-PVRL4 and anti-PROCR antibodies showed internalization activity against irinotecan-treated PLR59 and PLR123, and the internalization activity against PLR59 was higher than that against PLR123 (Figures 69 and 71). These results demonstrated that all of the antibodies evaluated showed antitumor effects against PLR59 and PLR123.

[0290] The differentiation-promoting effect of BMP4 on irinotecan-untreated and irinotecan-treated PLR59 and PLR123 cells was evaluated. When evaluating the differentiation-promoting effect on irinotecan-untreated cells, PLR59 and PLR123 cells suspended in medium containing BMP4 (R&D Systems, final concentration 20 nM) or control buffer were cultured at 5 x 10 5 The cells were seeded in each well of a 12-well plate at a cell density of 1.5 mL / well. The cells were subcultured by replacing the medium with the same medium 2, 4, and 7 days after seeding. When evaluating the differentiation-promoting effect on cells treated with irinotecan, 17 x 10 5Irinotecan was added to a final concentration of 15 μM to a 12.5 mL culture flask seeded with PLR59 or PLR123 at a cell density of 5 mL / flask the day after seeding. The flasks were cultured in a CO2 incubator at 37°C for 72 hours. The medium in the flasks was then replaced with medium supplemented with BMP4 or control buffer, and was replaced with the same medium 2, 4, and 7 days after the replacement. cDNA was synthesized using the ThermoScript RT-PCR System (Invitrogen) with RNA extracted from cells isolated 4 and 9 days after the first medium replacement as a template using the RNeasy Plus Mini Kit and RNase-Free DNase Set (QIAGEN).

[0291] Quantitative real-time PCR was performed using the cDNA isolated as described above, and as shown in FIG. 73, an increase in CK20 was observed in PLR59 and PLR123 cells cultured with the addition of BMP4. [Industrial Applicability]

[0292] According to the present invention, a cell surface molecule that is specifically expressed in cancer stem cells has been identified, and by using an antibody against the molecule, new anticancer agents and reagents for detecting cancer stem cells have been provided.

Claims

1. A pharmaceutical composition that is an inhibitor of cancer metastasis of Lgr5-positive cancer stem cells, comprising an antibody against a PROCR molecule as an active ingredient, wherein the antibody is linked to a cytotoxic substance or a proliferation inhibitor.

2. A pharmaceutical composition which is an agent for suppressing cancer recurrence in Lgr5-negative cancer stem cells or an agent for treating drug-resistant cancer in Lgr5-negative cancer stem cells, comprising an antibody against a PROCR molecule as an active ingredient, wherein the antibody is linked to a cytotoxic substance or a proliferation inhibitor.

3. The pharmaceutical composition according to claim 1 or 2, wherein the cancer is a solid cancer.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the cancer is a gastrointestinal cancer.

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the cancer is colon cancer.

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein the antibody is a monoclonal antibody.

7. The pharmaceutical composition of claim 1 , wherein the antibody is a chimeric antibody, a humanized antibody, or a human antibody.

8. The pharmaceutical composition of claim 1 , wherein the antibody is an antibody fragment.

9. The pharmaceutical composition according to any one of claims 1 to 8, wherein the antibody is an antibody having neutralizing activity.

10. A pharmaceutical composition according to any one of claims 1 to 9, characterized in that it is used contemporaneously with or after chemotherapy.

11. A method for confirming the effectiveness of a pharmaceutical composition described in any one of claims 1 to 10, comprising detecting the presence of a PROCR molecule and / or a polynucleotide encoding a PROCR molecule in a sample isolated from a subject to which the pharmaceutical composition has been administered.

12. The method described in claim 11, which uses an antibody that binds to the PROCR molecule.

13. The method of claim 11, using a portion of a polynucleotide encoding a PROCR molecule and / or its complementary strand.

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