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 a cell surface molecule specific for Lgr5-positive cancer stem cells with high proliferative ability and Lgr5-negative cancer stem cells with low proliferative ability, and a pharmaceutical composition containing, as an active ingredient, an antibody against these cell surface molecules. Further, the present invention relates to a reagent for detecting cancer stem cells using the above antibody and a method for selecting cancer patients.

Background Art

[0002] Cancer stem cells (CSCs) are considered to be the origin of cancer. This is because they have self-renewal ability and the ability to differentiate into tumor hierarchies (Non-Patent Document 1). Furthermore, CSCs can move and can withstand anticancer agent treatment (Non-Patent Document 1). Since CSCs are considered to be a rare subset in tumors, efforts have been made to determine their properties based on cell surface markers and tumor-initiating activity in xenotransplantation. 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, in initial studies by O'Brien et al. (Non-Patent Document 15) and Ricci-Vitiani et al. (Non-Patent Document 16), it was reported that CD133 is a CSC marker. Subsequently, CD44, EpCAM, CD166 (Non-Patent Document 17), and ALDH (Non-Patent Documents 18 and 19) were reported as additional markers by other research groups. In recent years, Pang et al. showed that CD26 serves as a marker for a subpopulation of CSCs with metastatic activity (Non-Patent Document 20).

[0003] In order to isolate CSCs, in many studies, EpCAM high / CD44 + / CD166 + (Non-Patent Document 17), CD133 + / CD44 + (Non-Patent Document 21), CD44 high / ALDH + (Non-Patent Document 18), and ALDH1 + / CD133 + (Non-Patent Document 19) and other combinations of CSC markers have been used for cell sorting approaches. Also, for the enrichment of CSCs, in vitro spheroid (cell mass) cultures and direct xenotransplantation of cancer cells into immunodeficient mice have also been used (Non-Patent Document 22). However, in order to further understand the properties of CSCs, it was necessary to obtain cancer stem cells with high purity and in large quantities.

[0004] One of the problems in CSC isolation is thought to be due to the heterogeneity and / or instability of the CSC phenotype (Non-Patent Document 29). As a source of CSCs, three-dimensional spheroid cultures are often used. This spheroid culture can be directly applied to tumor cells of clinically resected specimens, and the ability to maintain a heterogeneous population of CSCs may have certain advantages over xenotransplantation. However, due to its heterogeneity, the results in biochemical analysis often show complex characteristics of CSCs. Antibodies against cell surface marker proteins are widely used for CSC selection to isolate CSCs, but the number and purity of cells obtained by this method are limited. On the other hand, since the phenotype of xenografts is stably maintained even after multiple passages, using xenografts as a source of CSCs is also a common approach. However, there is also a discussion that the passage of xenografts in mice can only select cells that can survive in mice, thereby excluding cells that are less affected by such an environment. Needless to say, CSCs present in xenograft tumors reflect the original characteristics of CSCs as long as they maintain their self-renewal ability and the ability to produce differentiated strains 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 and 24), and was shown to be a Wnt target gene with restricted expression 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 crypt-virus organoids in vitro without mesenchymal niche (Non-Patent Document 26) clearly demonstrated that Lgr5-positive cells are stem cells in the normal colon. Furthermore, Lgr5-positive cells form adenomas under deletion of Apc (Non-Patent Document 27), and it has been reported that Lgr5 is expressed in colorectal cancer cell lines (Non-Patent Document 25). Collectively, these results indicate that Lgr5-positive cells are the origin of colorectal cancer (Non-Patent Document 25). Similar to that in normal colonic stem cells, it has been demonstrated that Wnt activity is essential for the proliferation of CSCs in vitro and in vivo, and that exogenous HGF stimulates Wnt activity (Non-Patent Document 28).

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

[0007] To date, although various treatment methods and anticancer agents for cancer treatment have been developed, problems such as the efficacy not being always sufficient, side effects occurring, and the number of patients showing an effect being limited still remain as issues to be solved. In recent years, treatment methods targeting cancer stem cells have attracted attention, but there are many unclear points regarding their effects and side effects (Non-Patent Document 31).

Prior Art Documents

Patent Documents

[0008]

Patent Document 1

Patent Document 2

Non-Patent Document

[0009]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

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Non-Patent Document 8

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Non-Patent Document 10

Non-Patent Document 11

Non-Patent Document 12

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Non-Patent Document 15

Non-Patent Document 16

Non-Patent Document 17

Non-Patent Document 18

Non-Patent Document 19

Non-Patent Document 20

Non-Patent Document 21

Non-Patent Document 22

Non-Patent Document 23

Non-Patent Document 24

Non-Patent Document 25

Non-Patent Document 26

Non-Patent Document 27

[0010] The present invention has been made in view of such circumstances, and an object of the present invention is to obtain two substantially uniform cancer stem cell populations characterized by using Lgr5, a cell surface marker, and to identify cell membrane molecules specifically expressed in these cancer stem cells, thereby providing a cancer therapeutic agent using an antibody against these molecules. Another object of the present invention is to provide a reagent for detecting cancer stem cells, a diagnostic and screening method for cancer patients, using an antibody against a cell membrane molecule specifically expressed in cancer stem cells.

Means for Solving the Problems

[0011] To prove the stem cell theory that can explain the carcinogenesis mechanism, maximum efforts have been made for the identification, isolation, and characterization of cancer stem cells (CSCs). However, it has remained difficult to obtain a sufficient amount of highly pure CSCs for characterization. Therefore, it has also been difficult to identify cell membrane molecules specifically expressed in cancer stem cells. The present inventors have conducted intensive studies to solve the above problems.

[0012] In order to isolate colorectal CSCs from human colorectal cancer xenografts maintained in NOG mice, the inventors first established a method for obtaining a large amount of highly pure colorectal CSCs by applying in vitro monolayer culture (also simply referred to as adherent culture) using a serum-free stem cell medium. Specifically, the inventors found that a large amount of highly pure colorectal CSCs could be obtained by separating cells derived from moderately differentiated human colorectal cancer xenografts maintained in NOD / Shi-scid, IL-2Rγnull (NOG) mice and performing adherent culture. Under these conditions, only colorectal CSCs were able to proliferate, survive, and expand, thereby enabling the obtainment of highly pure and substantially homogeneous colorectal CSCs. The colorectal CSCs obtained by this method were stably maintained for over one month without changing their phenotype by performing subculture by adherent culture using a serum-free stem cell medium. These cells expressed various colorectal cancer stem cell markers (CD133, CD44, EpCAM, CD166, CD24, CD26, and CD29) that have been reported so far, and showed tumor-initiating activity at a frequency of almost 100%, reconstructing tumors with the same histopathological characteristics (hierarchical structure) as the original primary tumor. These cells were characterized by their high proliferative ability under adherent culture conditions and the positivity of the cell surface marker Lgr5. In addition, highly proliferative and Lgr5-positive cancer stem cells were shown to play an important role in cancer metastasis because they formed tumor masses in organs such as the lungs and liver when administered via the mouse tail vein.

[0013] On the one hand, cancer stem cells that show high proliferative ability under adherent culture conditions and are positive for the cell surface marker Lgr5 were treated with anticancer agents such as irinotecan and 5-FU, and cancer stem cells with low proliferative ability and negative for Lgr5 were successfully isolated. Furthermore, after isolating cancer stem cells with low proliferative ability and negative for Lgr5, culturing them again under adherent culture conditions showed that they could transform into cancer stem cells with high proliferative ability and positive for Lgr5. Therefore, it was demonstrated that cancer stem cells with high proliferative ability and positive for Lgr5 and cancer stem cells with low proliferative ability and negative for Lgr5 can be mutually converted and have self-alternating functions. Due to this ability, when CSCs are cultured by changing the culture conditions or exposed to anticancer agents and then cultured, the actively proliferating Lgr5-positive colorectal CSCs were converted into a quiescent Lgr5-negative state. Alternatively, by separating the cells and culturing them again under adherent culture conditions, the Lgr5-negative CSCs became actively proliferating Lgr5-positive CSCs. These cells also showed tumor-initiating activity at a frequency of almost 100%. In addition, since cancer stem cells with high proliferative ability and positive for Lgr5 formed tumor masses in organs such as the lungs and liver when inoculated from the mouse tail vein, it was shown that they play an important role in cancer metastasis.

[0014] This self-alternation of CSCs between two states due to environmental changes can help explain cancer drug resistance and recurrence. The existence of Lgr5-negative CSCs in carcinogenesis may be related to the basic properties of stem cells. One possible hypothesis is that CSCs can utilize an intrinsic means to alternate into distinct subsets of the cell population under environmental changes such as aggressive anticancer agent treatment. When CSCs are subjected to stress represented by anticancer agents or culture environment changes, they change into Lgr5-negative and low-proliferative CSCs to avoid stress and continue to survive, and when the stress is relieved, they change back into Lgr5-positive and high-proliferative CSCs to initiate proliferation. This means that CSCs have a self-defense ability to adapt to a new environment through an intrinsic mechanism (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 cancer occurrence, formation, metastasis, recurrence, drug resistance, etc., and can become major target cells in the development of anticancer drugs. In particular, highly proliferative Lgr5-positive cancer stem cells are involved in cancer formation and metastasis, and low-proliferative Lgr5-negative cancer stem cells are considered to be involved in cancer recurrence. Therefore, if 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 becomes possible, and it becomes possible to create new anticancer drugs and reagents for detecting cancer stem cells.

[0016] More specifically, the present invention is as follows: 〔1〕A pharmaceutical composition containing, as an active ingredient, at least one antibody that binds to the proteins described in SEQ ID NOs: 1 to 8, 〔2〕The pharmaceutical composition according to 〔1〕, which is an anticancer drug, 〔3〕The pharmaceutical composition according to 〔2〕, which is a cancer recurrence inhibitor, 〔4〕The pharmaceutical composition according to 〔2〕, which is a cancer metastasis inhibitor or a postoperative adjuvant therapy agent, 〔5〕The pharmaceutical composition according to 〔4〕, which is an Lgr5-positive cancer metastasis inhibitor or a postoperative adjuvant therapy agent containing, as an active ingredient, at least one antibody that binds to the proteins described in SEQ ID NOs: 1 to 6, 〔6〕The pharmaceutical composition according to 〔2〕, which is a drug-resistant cancer treatment agent, 〔7〕The pharmaceutical composition according to 〔6〕, which is an Lgr5-negative cancer treatment agent containing, as an active ingredient, at least one antibody that binds to the proteins described in SEQ ID NOs: 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 destroying 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 digestive tract cancer, 〔12〕The cancer is colorectal cancer, and the pharmaceutical composition according to any one of 〔2〕 to 〔11〕, 〔13〕The antibody is a monoclonal antibody, and the pharmaceutical composition according to any one of 〔1〕 to 〔12〕, 〔14〕The antibody is any one of a chimeric antibody, a humanized antibody, or a human antibody, and the pharmaceutical composition according to any one of 〔1〕 to 〔13〕, 〔15〕The antibody is an antibody fragment, and the pharmaceutical composition according to any one of 〔1〕 to 〔14〕, 〔16〕The antibody is conjugated with a cytotoxic substance or a growth inhibitor, and the pharmaceutical composition according to 〔15〕, 〔17〕The antibody is an antibody having cytotoxic activity, and the pharmaceutical composition according to any one of 〔1〕 to 〔14〕, 〔18〕The cytotoxic activity is ADCC activity, and the pharmaceutical composition according to 〔17〕, 〔19〕The sugar chain composition of the antibody is modified so that the proportion of fucose-deficient antibodies is increased, or the proportion of antibodies with bisecting N-acetylglucosamine added is increased, and the pharmaceutical composition according to 〔17〕 or 〔18〕, 〔20〕The cytotoxic activity is CDC activity, and the pharmaceutical composition according to 〔17〕, 〔21〕The antibody is an antibody having neutralizing activity, and the pharmaceutical composition according to any one of 〔1〕 to 〔20〕, 〔22〕The pharmaceutical composition according to any one of 〔2〕 to 〔21〕, or a polypeptide represented by SEQ ID NO: 9 or a polypeptide in which one or more amino acids among the amino acids contained in the polypeptide are added, deleted, or substituted, characterized in that it is used simultaneously with a chemotherapeutic agent or after treatment with a chemotherapeutic agent, 〔23〕A reagent for detecting cancer stem cells, containing at least one antibody that binds to the proteins described in SEQ ID NOs: 1 to 8 as an active ingredient, 〔24〕The reagent according to 〔23〕 for detecting Lgr5-positive cancer stem cells, wherein the antibody is at least one antibody that binds to the proteins described in SEQ ID NOs: 1 to 6, 〔25〕The reagent according to 〔23〕 for detecting Lgr5-negative cancer stem cells, which is at least one antibody that binds to the protein described in SEQ ID NOs: 1 to 8. 〔26〕A method for diagnosing cancer or screening cancer patients (cancer examination and screening method), which comprises detecting the presence of at least one such protein in a sample isolated from a cancer patient by using at least one antibody that binds to the protein described in SEQ ID NOs: 1 to 8. 〔27〕The method according to 〔26〕 for diagnosing Lgr5-positive cancer or screening cancer patients, wherein the antibody is at least one antibody that binds to the protein described in SEQ ID NOs: 1 to 6. 〔28〕The method according to 〔26〕 for diagnosing Lgr5-negative cancer or screening cancer patients, wherein the antibody is at least one antibody that binds to the protein described in SEQ ID NOs: 1 to 8. 〔29〕A method for confirming the efficacy of the pharmaceutical composition according to any one of 〔1〕 to 〔22〕, which comprises detecting the presence of any one or more of the protein described in SEQ ID NOs: 1 to 8 and / or the polynucleotide encoding the protein in a sample isolated from a subject administered with the pharmaceutical composition. 〔30〕The method according to 〔29〕, using at least one antibody that binds to the protein described in SEQ ID NOs: 1 to 8, and 〔31〕The method according to 〔29〕, using a polynucleotide encoding the protein described in SEQ ID NOs: 1 to 8 and / or a partial complementary strand thereof. is provided.

[0017] Further, the present invention provides the following: 〔A1〕A method for treating cancer, which comprises administering to a subject at least one antibody that binds to the protein described in SEQ ID NOs: 1 to 8. 〔A2〕At least one antibody that binds to the protein described in SEQ ID NOs: 1 to 8 for use in treating cancer. 〔A3〕Use of at least one antibody that binds to the protein described in SEQ ID NOs: 1 to 8 for manufacturing an anticancer agent. A process for manufacturing an anticancer agent, comprising the step of using at least one antibody that binds to the proteins described in SEQ ID NOs: 1 to 8; which is provided. In a non-limiting aspect of the present invention, the treatment of cancer is suppression of cancer recurrence, suppression of cancer metastasis, postoperative adjuvant therapy, treatment of drug-resistant cancer, suppression of cancer stem cell proliferation, or destruction of cancer stem cells, and the anticancer agent is a cancer recurrence inhibitor, a cancer metastasis inhibitor, a postoperative adjuvant therapy agent, a drug-resistant cancer treatment agent, a cancer stem cell proliferation inhibitor, or a cancer stem cell destruction agent.

[0018] Furthermore, the present invention provides the following: 〔B1〕A reagent for detecting the presence of any one or more of the proteins described in SEQ ID NOs: 1 to 8 and / or polynucleotides encoding the proteins, preferably at least one antibody that binds to the proteins described in SEQ ID NOs: 1 to 8, or a reagent for detecting cancer stem cells, a reagent for cancer diagnosis, a reagent for screening cancer patients, or a reagent for confirming the effectiveness of the pharmaceutical compositions of 〔1〕~〔22〕, which contains a polynucleotide encoding the proteins described in SEQ ID NOs: 1 to 8 and / or a part of its complementary strand; 〔B2〕Preferably, using at least one antibody that binds to the proteins described in SEQ ID NOs: 1 to 8, or a polynucleotide encoding the proteins described in SEQ ID NOs: 1 to 8 and / or a part of its complementary strand, to detect the presence of any one or more of the proteins described in SEQ ID NOs: 1 to 8 and / or polynucleotides encoding the proteins in a sample isolated from a cancer patient, a method for detecting cancer stem cells, diagnosing cancer, screening cancer patients, or confirming the effectiveness of the pharmaceutical compositions of 〔1〕~〔22〕; A reagent for detecting cancer stem cells, diagnosing cancer, selecting cancer patients, or confirming the effectiveness of the pharmaceutical compositions of [1] to

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

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

[22] , which includes using a reagent for detecting the presence of any one or more of the proteins described in SEQ ID NOs: 1 to 8 and / or polynucleotides encoding said proteins, preferably at least one antibody that binds to the proteins described in SEQ ID NOs: 1 to 8, or a polynucleotide encoding the proteins described in SEQ ID NOs: 1 to 8 and / or a portion of its complementary strand; is provided.

Brief Description of the Drawings

[0019]

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Mode for Carrying Out the Invention

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

[0021] In the present invention, "cancer" typically refers to, or means, a physiological state of a mammal characterized by unregulated cell growth. In the present invention, the types of cancer are not particularly limited, and examples include the following. As carcinomas (epithelial cancers), there are pancreatic cancer, prostate cancer, breast cancer, skin cancer, cancers of the digestive tract, lung cancer, hepatocellular carcinoma, cervical cancer, endometrial cancer, ovarian cancer, fallopian tube cancer, vaginal cancer, liver cancer, bile duct cancer, bladder cancer, ureteral cancer, thyroid cancer, adrenal cancer, kidney cancer, or cancers of other glandular tissues. As sarcomas (non-epithelial cancers), there are liposarcoma, leiomyosarcoma, rhabdomyosarcoma, synovial sarcoma, angiosarcoma, fibrosarcoma, malignant peripheral nerve sheath tumor, gastrointestinal stromal tumor, desmoid tumor, Ewing's sarcoma, osteosarcoma, chondrosarcoma, leukemia, lymphoma, myeloma, tumors of other parenchymal organs, such as melanoma or brain tumor (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). In the present invention, "tumor" refers to any mass of tissue resulting from excessive cell growth or proliferation that is either 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 the following i) and / or ii). i) They possess self-renewal ability. Self-renewal ability refers to the ability of one or both of the two daughter cells produced by division to produce cells that retain, in the cell lineage, the same ability and degree of differentiation as the parent cell. ii) They can differentiate into multiple types of cancer cells that make up a cancer cell mass. The multiple types of cancer cells differentiated from cancer stem cells form a hierarchical structure with cancer stem cells at the apex, similar to normal stem cells. By gradually producing various cancer cells from cancer stem cells, a cancer cell mass with diverse characteristics is formed.

[0023] Cancer stem cells are cancer cells that have the ability to form cancer and, like normal stem cells, have the ability to differentiate into multiple cell types and self-renew. Cancer stem cells form a hierarchical structure with cancer stem cells at the apex. A variety of cancer cell masses with diverse characteristics are formed by the stepwise production of various cancer cells from cancer stem cells. A cancer cell mass is a mass formed by cells adhering to each other rather than being scattered, like human tumor tissue, and is composed of cancer cells, cells other than cancer cells such as stromal cells and blood cells, and extracellular matrix 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, mice, etc. can be used, but those derived from humans are preferred, and those derived from human tumor tissue are 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. For example, it is possible to confirm that an established cancer cell line prepared by transplanting and subculturing the cancer tissue from which the detected cancer stem cells were collected, preferably into a non-human animal, reproduces such a hierarchical structure of cancer tissue. More preferably, as the non-human animal, an immunodeficient animal, most preferably a NOG mouse lacking functional T cells, B cells, and natural killer cells, can be used to confirm that an established NOG cancer cell line prepared by transplanting and subculturing cancer tissue reproduces such a hierarchical structure of cancer tissue.

[0026] Furthermore, the cancer stem cells detected in the present invention can also be spheroids (cell masses) formed by spheroid culture. Spheroid culture is a method of culturing cells in a three-dimensionally suspended state by seeding cells in a non-adherent or low-adhesion culture flask, plate, dish, or other culture container using a medium capable of culturing cancer stem cells, and the cell mass formed by this method is called a spheroid.

[0027] The NOG established cancer cell lines can be prepared by methods known to those skilled in the art. For example, the methods described in Fujii E. et al., Pathol int. 2008; 58: 559-567 can be used. Human colorectal cancer, gastric cancer, lung cancer, breast cancer, pancreatic cancer, etc. removed by surgery can be physically minced with scissors and subcutaneously transplanted and passaged into NOG mice to establish the cell lines. In the NOG established cancer cell lines, the characteristics of the original human cancer tissue are maintained even after passage.

[0028] In the present invention, cancer stem cells can be selected using cell markers. Examples of the cell markers used in the present invention include 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, which is characterized in that the expression of the cell marker Lgr5 is positive, adherent under serum-free culture conditions, and highly proliferative. Hereinafter, in this specification, the cancer stem cells may be referred to as "highly proliferative cancer stem cells positive for Lgr5".

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

[0031] The medium or culture solution used for culturing cancer stem cells of the present invention may be any medium or culture solution as long as it is serum-free and can culture cancer stem cells, and is not particularly limited. For example, a conventionally known basal culture solution added with EGF, bFGF, hLIF, HGF, NGF, NSF-1, TGFβ, TNFα, heparin, BSA, insulin, transferrin, putrescine, selenite, progesterone, hydrocortisone, D-(+)-glucose, sodium bicarbonate, HEPES, L-glutamine, 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, 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, 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. As a known basal medium, there is no particular limitation as long as it is suitable for culturing cancer cells that are the source of cancer stem cells. Examples include DMEM / F12, DMEM, F10, F12, IMDM, EMEM, RPMI-1640, MEM, BME, Mocoy's 5A, MCDB131, etc. Among these, DMEM / F12 is preferred.

[0032] As the most preferred stem cell medium, there may be mentioned a medium obtained by adding human EGF at a final concentration of 20 ng / mL, human bFGF at 10 ng / mL, heparin at 4 μg / mL, BSA at 4 mg / mL, human insulin at 25 μg / mL, and glucose at 2.9 mg / mL to a DMEM / F12 medium.

[0033] As described herein, Lgr5-positive highly proliferative cancer stem cells exhibit the properties of mesenchymal cells (mesenchymal cells). On the other hand, as described herein, Lgr5-negative low-proliferative cancer stem cells exhibit the properties of epithelial cells. The epithelial cells in the present invention 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 a solid cancer, more preferably derived from a digestive organ cancer. Examples of digestive organ cancers include esophageal cancer, gastric cancer, duodenal cancer, pancreatic cancer, bile duct cancer, gallbladder cancer, biliary tract cancer, colorectal cancer, colon cancer, rectal cancer, etc., and preferably colorectal cancer.

[0035] In the present invention, the cancer stem cells are preferably positive for one or more of the cell markers CD133, CD44, EpCAM, CD166, CD24, CD26, 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 of ALDH activity, and Lgr5-negative cancer stem cells are negative for ALDH activity.

[0037] 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 of HLA-DMA, TMEM173, ZMAT3 or GPR110, and Lgr5-negative cancer stem cells are positive for any of the cell markers of HLA-DMA, TMEM173, ZMAT3 or GPR110.

[0038] In the present invention, it is preferable that the cancer stem cells have characteristics that reproduce the hierarchical structure of the cancer tissue. In the present invention, the hierarchical structure means that a part of the characteristic intrinsic structure found in normal tissue is pathologically detected in the tumor structure originating from the tissue. Generally, this hierarchical structure is more highly reproduced in well-differentiated cancers. For example, in the case of tumors of lumen-forming organs (such as gastric cancer, colorectal cancer, pancreatic cancer, liver cancer, cholangiocarcinoma, breast cancer, lung adenocarcinoma, prostate cancer, etc.), lumen formation and the appearance of mucous cells are observed. In the case of tumors with a squamous epithelial structure (such as squamous cell carcinoma of the lung, skin, vaginal mucosa, etc.), the formation of a stratified epithelial structure and a tendency to keratinize are observed. On the other hand, in poorly differentiated cancers, the reproduction of this hierarchical structure is insufficient, and they are said to be highly atypical (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). Since this hierarchical structure is considered to be reproduced as a result of various biological reactions of cancer, the utility value of cancer stem cells that reproduce it is considered to be high. To reproduce the hierarchical structure means that the characteristic intrinsic structure originally possessed by the cancer stem cells can still be observed after the separation or induction of the cancer stem cells.

[0039] In the present invention, it is preferable that the cancer stem cells have the ability of epithelial to mesenchymal transition (EMT). In the present invention, the ability of epithelial to mesenchymal transition includes both the meaning that epithelial cells acquire the properties of mesenchymal cells and migrate, and that mesenchymal cells acquire the properties of epithelial cells and migrate. EMT does not occur in normal cells except during the process of 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, show loss of polarity, and have the ability to move. These mesenchymal cells can not only spread into the tissues around the primary tumor, but also separate from the tumor, invade blood vessels and lymphatic vessels, move to a new location, and divide there to form further tumors. The formation of further tumors helps to explain the drug resistance, metastasis or recurrence of cancer.

[0040] The present invention also provides a pharmaceutical composition containing, as an active ingredient, an antibody that binds to a molecule expressed in a substantially homogeneous cancer stem cell population containing the cancer stem cells of the present invention. "Substantially homogeneous" means that the frequency of cancer stem cells is 1 / 20 or more, preferably 1 / 10 or more, more preferably 1 / 5 or more, still more preferably 1 / 3 or more, still more preferably 1 / 2 or more, and most preferably 1 / 1 by analyzing the frequency of the formation of cancer cell populations by transplanting 1000 cells, 100 cells, or 10 cells into immunodeficient animals using methods described in Hu Y & Smyth GK., J Immunol Methods. 2009 Aug 15;347(1-2):70-8 and Ishizawa K & Rasheed ZA. et al., Cell Stem Cell. 2010 Sep 3;7(3):279-82 and using Extreme Limiting Dilution Analysis (Hu Y & Smyth GK., J Immunol Methods. 2009 Aug 15; 347(1-2): 70-8).

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

[0042] In the present invention, adherent culture means culturing and subculturing cells in an adherent state after seeding cells in a culture vessel for adherent culture, which is a culture excluding floating cells. Cells that have grown confluently are detached using Accutase and subcultured into a new adherent culture flask, adherent culture plate, or adherent culture dish to continue the culture. The culture vessel for adherent culture is not particularly limited as long as it is a vessel used for adherent culture, and an adherent culture flask or a flask with high adhesiveness, an adherent culture plate or a plate with high adhesiveness, an adherent culture flat-bottom plate or a flat-bottom plate with high adhesiveness, an adherent culture dish or a dish with high adhesiveness, etc. can be appropriately selected and used. The medium used for adherent culture is not particularly limited, but it is preferable to use a serum-free stem cell medium. In the present invention, adhesiveness refers to the property of cells to adhere to a culture vessel when cultured in a culture vessel for adherent culture.

[0043] In the present invention, suspension culture means culturing and subculturing cells in a suspended state after seeding cells in a culture vessel for suspension culture, which is a culture excluding adherent cells. Cells that have grown confluently are subcultured into 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 a low-adhesion cell culture flask, ultra-low adhesion cell culture flask, low-adhesion plate, ultra-low adhesion plate, low-adhesion dish, ultra-low adhesion dish, etc. 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 before performing adherent culture or suspension culture, it is preferable to proliferate the cell group containing cancer stem cells. In the present invention, "floatability" refers to the property that when cells are cultured in a culture vessel for suspension culture, they can be cultured in a floating state without adhering to the culture vessel.

[0044] To proliferate a cell group means, for example, to proliferate by spheroid culture or by transplanting into a non-human animal and subculturing, but is not particularly limited thereto.

[0045] As described in this specification, at the time of transplantation, as the non-human animal, an immunodeficient animal can be used in that rejection reaction hardly occurs. As the immunodeficient animal, a non-human animal lacking functional T cells, for example, a nude mouse or a nude rat, a non-human animal lacking functional T cells and B cells, for example, the use of a SCID mouse or a NOD-SCID mouse is preferable, and among them, the use of a mouse lacking T cells, B cells, and NK cells having excellent transplantability (for example, including NOG mice, etc.) is more preferable. For the age of the non-human animal, for example, in the case of a nude athymic mouse, a SCID mouse, a NOD / SCID mouse, or a NOG mouse, those aged 4 to 100 weeks are preferably used. The NOG mouse 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 mouse).

[0046] The cells to be transplanted may be any such as cell aggregates, tissue pieces, individually dispersed cells, cells once cultured after isolation, cells isolated again from an animal after going through the process of being transplanted into another animal, etc., but dispersed cells are preferred. Also, the number of cells to be transplanted may be a number of 10 6 or less, but a larger number of cells may also be transplanted.

[0047] Subcutaneous transplantation is a suitable transplantation site in that the transplantation technique is simple. However, the transplantation site is not particularly restricted, and it is preferable to appropriately select the transplantation site according to the animal to be used. In addition, the transplantation operation of the NOG established cancer cell line is not particularly restricted and can be performed according to conventional transplantation operations.

[0048] Cancer stem cells or a cancer stem cell population can be prepared, for example, by adherent culture or suspension culture of cancer tissue collected from a patient using a serum-free stem cell medium. It can also be prepared by spheroid culture of cancer tissue collected from a patient and then adherent culture or suspension culture using a serum-free stem cell medium. It can also be prepared by transplanting and subculturing cancer tissue collected from a patient into a non-human animal and then performing adherent culture or suspension culture using a serum-free stem cell medium. Furthermore, a method can also be used in which the NOG established cancer cell line prepared by transplanting and subculturing cancer tissue collected from a patient into a NOG mouse is subjected to adherent culture or suspension 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 screening methods for pharmaceuticals and anticancer agents and the like. As one aspect of the screening method for the pharmaceutical of the present invention, a method including the following steps (a) to (c) is provided; (a) A step of preparing a substantially homogeneous cancer stem cell population containing Lgr5-positive adherent cancer stem cells, (b) A step of contacting the cancer stem cell population or the cancer stem cells contained in the cancer stem cell population with a test substance, (c) A step of detecting a change in the biological characteristics 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. Next, the prepared cancer stem cell population or the cancer stem cells contained in the cancer stem cell population are brought into contact with a test substance. In this method, the method of bringing the cancer stem cell population or the cancer stem cells contained in the cancer stem cell population into contact with the test substance is not particularly limited. For example, the test substance can be brought into contact with the cultured cells of the cancer stem cell population or the cancer stem cells contained in the cancer stem cell population. The treatment can be carried out by adding the test substance to the cell culture medium or the cell extract. When the test substance is a protein, for example, it is also possible to introduce a vector containing the DNA encoding the protein into the cancer stem cell population or the cancer stem cells contained in the cancer stem cell population, or to add the vector to the cell extract of the cancer stem cell population or the cancer stem cells contained in the cancer stem cell population. Further, for example, a two-hybrid method using yeast or animal cells can also be used.

[0051] In this method, next, changes in the biological properties of the cancer stem cell population or cancer stem cells treated with the test substance are detected. Here, the changes in biological properties include, for example, changes in proliferative ability, changes in the number of living cells, changes in tissue structure characteristically observed in the cancer progression process of the cancer stem cell population or cancer stem cells, changes in the expression of DNA, RNA, proteins, or metabolites contained in the cancer stem cell population or cancer stem cells, and the like. Further, the detection of changes in biological characteristics can be carried out, for example, by the following methods.

[0052] There are no particular restrictions on the confirmation of the expression of DNA, RNA, proteins, peptides, and metabolites, and it can be carried out according to conventional expression confirmation methods. Examples of RNA include microRNA, siRNA, tRNA, snRNA, mRNA, or 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 fraction containing the protein encoded by each gene can be recovered according to a standard method, and the translation level of the gene can be measured by detecting the expression of each protein by electrophoresis such as SDS-PAGE. Also, it is 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. Screening of pharmaceuticals (pharmaceutical compositions) can be carried out by these methods.

[0053] As such DNA, RNA, and proteins contained in the cancer stem cell population or cancer stem cells that are characteristically observed in the cancer progression process of the cancer stem cell population or cancer stem cells, the protein or polypeptide described in any of SEQ ID NOs: 1 to 6, or a polynucleotide encoding the protein or polypeptide is preferably mentioned.

[0054] For example, when the biological characteristics of a cancer stem cell population or cancer stem cells after treatment with a test substance do not show a change or the rate of change decreases compared to before treatment, the test substance is considered useful as a pharmaceutical (pharmaceutical composition) having the function of suppressing cancer recurrence and metastasis (for example, a cancer recurrence inhibitor, an adjuvant agent after chemotherapy treatment, a postoperative adjuvant therapy agent, an anticancer agent or a cancer metastasis inhibitor), and these test substances can be selected as an active substance having a therapeutic or preventive effect on cancer diseases. Thus, a pharmaceutical (pharmaceutical composition) having the function of suppressing the progression of cancer is used as a cancer recurrence inhibitor, an adjuvant agent after chemotherapy treatment, a postoperative adjuvant therapy agent, an anticancer agent or a cancer metastasis inhibitor. The anticancer agent of the present invention may be used, for example, for cancers having resistance to drugs or chemotherapeutic agents. That is, the pharmaceutical (pharmaceutical composition) of the present invention also includes an agent for treating drug-resistant or chemotherapeutic agent-resistant cancers.

[0055] In the present invention, the pharmaceutical (pharmaceutical composition) is not particularly limited to an anticancer agent or a metastasis or recurrence inhibitor, and can also be used as, for example, an angiogenesis inhibitor, a cell growth inhibitor, etc. Further, the pharmaceutical (pharmaceutical composition) of the present invention may be used simultaneously with a chemotherapeutic agent or after treatment with a chemotherapeutic agent. Although the pharmaceutical is not particularly limited, examples include protein drugs, nucleic acid drugs, low molecular drugs, cell drugs, etc.

[0056] As another aspect of the screening method of the present invention, there is provided a screening method for a pharmaceutical (pharmaceutical composition) including the following steps (a) to (c); (a) A step of preparing a substantially homogeneous cancer stem cell population containing Lgr5-negative floating cancer stem cells, (b) A step of contacting the cancer stem cell population or the cancer stem cells contained in the cancer stem cell population with a test substance (c) A step of detecting a change in the biological characteristics of the cancer stem cell population or cancer stem cells contacted with the test substance.

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

[0058] As such DNA, RNA, and proteins that are characteristically recognized in the cancer stem cell population or cancer stem cells in the cancer progression process of the cancer stem cell population or cancer stem cells, proteins or polypeptides described in any of SEQ ID NOs: 1 to 8, or polynucleotides encoding the proteins or polypeptides are preferably mentioned. In a non-limiting aspect of the present invention, proteins or polypeptides described in SEQ ID NOs: 1 to 6 or polynucleotides encoding the proteins or polypeptides can be used. Further, in a different non-limiting aspect of the present invention, proteins or polypeptides described in SEQ ID NO: 7 or 8 or polynucleotides encoding the proteins or polypeptides can be used.

[0059] The pharmaceutical (pharmaceutical composition) obtained by the screening method is not particularly limited, but can be used as an anticancer agent. That is, when the biological characteristics of the cancer stem cell population or cancer stem cells after treatment with the test substance do not show a change or the rate of change decreases compared to before treatment, the test substance is considered useful as a pharmaceutical (for example, a cancer recurrence inhibitor, an adjuvant after chemotherapy treatment, a postoperative adjuvant therapy agent, an anticancer agent, or a cancer metastasis inhibitor) that has the function of suppressing cancer recurrence and metastasis, and those test substances can be selected as active substances having a therapeutic or preventive effect on cancer diseases. Thus, a pharmaceutical (pharmaceutical composition) having the function of suppressing cancer progression is used as a cancer recurrence inhibitor, an adjuvant after chemotherapy treatment, a postoperative adjuvant therapy agent, an anticancer agent, or a cancer metastasis inhibitor. In addition, the pharmaceutical (pharmaceutical composition) of the present invention includes a cancer therapeutic agent for Lgr5-negative cancer containing, as an active ingredient, at least one antibody that binds to the proteins described in SEQ ID NOs: 1 to 8. Here, Lgr5-negative cancers include cancers having drug resistance and resistance to chemotherapeutic agents.

[0060] As yet another aspect of the screening method of the present invention, there is provided a method using a non-human animal administered with a cancer stem cell population of the present invention or cancer stem cells contained in the cancer stem cell population and a test substance. Specifically, there is provided a screening method for a pharmaceutical (pharmaceutical composition) including the following steps (a) to (c); (a) A step of preparing a substantially homogeneous cancer stem cell population containing Lgr5-positive adherent cancer stem cells, (b) A step of administering the cell population or cancer stem cells contained in the cancer stem cell population and the test substance to a non-human animal (c) A step of detecting the formation of tumors in the non-human animal.

[0061] In this method, first, a substantially homogeneous cancer stem cell population containing Lgr5-positive adherent cancer stem cells is prepared. Next, the prepared cancer stem cell population or cancer stem cells contained in the cancer stem cell population and the 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 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] Also in this method, the method of administering the cancer stem cell population or cancer stem cells to the non-human animal is not particularly limited. Depending on the cell population to be administered, an appropriate administration method can be selected, but subcutaneous administration or intravenous administration is preferred.

[0064] In this method, then, the formation of tumors in the non-human animal is detected. The evaluation of the test substance can be carried out for the non-human animal by excising the cancer stem cell population or cancer stem cells and the tissue administered with the test substance, observing the histological characteristics of the administered tissue, and measuring whether a tumor is formed or not. Here, when no tumor is formed, the test substance is considered useful as a pharmaceutical having the function of suppressing cancer progression and metastasis (for example, an anticancer agent or a cancer metastasis or cancer recurrence inhibitor), and these test substances can be selected as active substances having a therapeutic or preventive effect on cancer diseases. That is, the pharmaceutical (pharmaceutical composition) obtained by the screening method is not particularly limited, but can be used as an anticancer agent or a cancer metastasis or cancer recurrence inhibitor.

[0065] In addition, the "test substance" in the method of the present invention is not particularly limited. For example, a single compound such as a natural compound, an organic compound, an inorganic compound, a protein, an antibody, a peptide, an amino acid, etc., and a compound library, an expression product of a gene library, a cell extract, a cell culture supernatant, a fermentation microbial product, a marine organism extract, a plant extract, a prokaryotic cell extract, a eukaryotic single cell extract or an animal cell extract, etc. can be mentioned. These may be purified products or crude purified products such as extracts of plants, animals or microorganisms, etc. Also, the production method of the test substance is not particularly limited, and it may be isolated from natural products, chemically or biochemically synthesized, or prepared by genetic engineering. In addition, 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 can also be appropriately used. The above test substance can be appropriately labeled and used as needed. Examples of the label include a radioactive label, a fluorescent label, etc. In addition, in addition to the above test substances, a mixture in which a plurality of these labels are mixed is 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 described in SEQ ID NOs: 1 to 8, and a screening method for vaccines. As such a screening method, a method of measuring the cytotoxic activity targeting cancer stem cells disclosed in the present invention using cytotoxic T lymphocytes (CTLs) induced by the cancer vaccine of the present invention in vitro is preferably mentioned. Specifically, peripheral blood mononuclear cells (PBMCs) collected by centrifuging human peripheral blood in a Ficoll-Conray density gradient are separated into adherent cells and non-adherent cells. The adherent cells are incubated in AIM-V (Gibco) with 100 ng / ml of GM-CSF (Novartis) and 10 IU / ml of IL-4 (Gibco BRL). These cells are used as antigen-presenting cells (APCs). The above non-adherent cells are incubated in AIM-V with 30 - 100 IU / ml of recombinant IL-4 (Ajinomoto). On the 7th to 10th day, a partial peptide of any of the proteins described in SEQ ID NOs: 1 to 8 provided by the present invention (final concentration 30 μg / ml) is added to the APCs. One day later, the APCs are matured by adding recombinant TNF-α and IFN-α (Sumitomo Pharmaceutical). Subsequently, the irradiated APCs and CD8-positive cells separated from autologous non-adherent cells are mixed in AIM-V without IL-2. Two days after incubation, IL-2 (Takeda Pharmaceutical Company Limited) is added to the culture at a final concentration of 100 IU / ml. Every 7 days, autologous PHA blasts (PHA-stimulated T cells) stimulated with the T cell mitogen PHA are used as APCs to stimulate the CD8-positive cells. For each stimulation, a fresh medium containing 100 IU / ml of IL-2 is added to the culture. The CTLs on the 28th day are used for the activity test. As the cells targeted by CTLs, the Lgr5-positive highly proliferative cancer stem cells and Lgr5-negative low proliferative cancer stem cells provided by the present invention can be used. The cytotoxic activity can be evaluated by measuring the 51 uptake activity of Cr-sodium chromate according to the measurement method of ADCC activity.

[0067] In addition, the pharmaceutical selected by the screening method of the present invention can be further selected as a more effective and highly practical preventive or therapeutic substance by conducting additional efficacy tests, safety tests, etc. as necessary, and further by conducting clinical trials on human cancer disease patients. The pharmaceutical thus selected can also be industrially manufactured by chemical synthesis, biochemical synthesis (fermentation), or genetic manipulation based on the results of its structural analysis.

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

[0069] In the preparation of the Lgr5-positive highly proliferative cancer stem cells and Lgr5-negative low proliferative cancer stem cells, separation can be carried out using Lgr5, a cell marker. This separation method includes a method of separating a cell population containing cancer stem cells using an Lgr5 antibody, a method of first subjecting a population containing cancer stem cells to adherent culture or suspension culture to prepare a substantially homogeneous cancer stem cell population and then separating it using an Lgr5 antibody, and a method of subjecting a population containing cancer stem cells to adherent culture in a medium with or without a growth inhibitor to prepare a substantially homogeneous cancer stem cell population and then separating it using an Lgr5 antibody. In the present invention, either method can be used. Preferably, cells can be separated from cancer tissues passaged in NOG mice for three or more generations and cultured adherently in a serum-free stem cell medium to prepare Lgr5-positive highly proliferative cancer stem cells. Then, the obtained Lgr5-positive cancer stem cells can be maintained under various stresses such as contact with a growth inhibitor (adding 10 μg / ml of irinotecan to the serum-free stem cell medium and culturing for 3 days) to prepare Lgr5-negative low proliferative cancer stem cells.

[0070] The present invention provides a method for screening a pharmaceutical, which comprises contacting a test substance with cancer stem cells having different proliferative abilities induced by the method provided by the present invention. That is, a method for screening a pharmaceutical is provided, which comprises detecting a change in the biological properties of the cancer stem cells by contacting a test substance with cancer stem cells having high proliferative ability or cancer stem cells having low proliferative ability, which are induced by a method of inducing cancer stem cells with low proliferative ability into cancer stem cells with high proliferative ability or inducing cancer stem cells with high proliferative ability into cancer stem cells with low proliferative ability.

[0071] That is, as described in the present specification, cancer stem cells having low proliferative ability can be obtained by maintaining cancer stem cells having high proliferative ability under various stresses such as suspension culture and contact with a growth inhibitor. For example, by culturing cancer stem cells having high proliferative ability in suspension, cancer stem cells having high proliferative ability can be induced into cancer stem cells having low proliferative ability. Alternatively, by culturing cancer stem cells having high proliferative ability in a low-adhesion or ultra-low-adhesion cell culture container such as a low-adhesion plate, an ultra-low-adhesion plate, a low-adhesion dish, an ultra-low-adhesion dish, a low-adhesion flask, or an ultra-low-adhesion cell culture flask, cancer stem cells having high proliferative ability can also be induced into cancer stem cells having low proliferative ability. That is, cancer stem cells having low proliferative ability can also be produced by culturing cancer stem cells having high proliferative ability in a low-adhesion or ultra-low-adhesion cell culture container such as a low-adhesion plate, an ultra-low-adhesion plate, a low-adhesion dish, an ultra-low-adhesion dish, a low-adhesion flask, or an ultra-low-adhesion cell culture flask.

[0072] In a non-limiting embodiment, cancer stem cells with high proliferative ability can also be induced into cancer stem cells with low proliferative ability by using growth inhibitors such as 5-FU or irinotecan. That is, cancer stem cells with high proliferative ability can be exposed to growth inhibitors such as 5-FU or irinotecan to produce cancer stem cells with low proliferative ability. The exposure to the growth inhibitor can be carried out in any environment, such as in vitro culture or in vivo in a transplanted non-human animal. In this case, those skilled in the art can appropriately select the exposure amount of the cancer stem cells to the growth inhibitor. Also, cancer stem cells with high proliferative ability can be produced by re-seeding cancer stem cells with low proliferative ability in a medium not containing a growth inhibitor such as 5-FU or irinotecan. In another non-limiting embodiment, cancer stem cells with high proliferative ability can also be produced by discontinuing the administration of a growth inhibitor to a non-human animal holding cancer stem cells with low proliferative ability.

[0073] In addition, cancer stem cells with low proliferative ability can be induced into cancer stem cells with high proliferative ability by adherent culture. Alternatively, cancer stem cells with low proliferative ability can be induced into cancer stem cells with high proliferative ability by culturing them in a highly adhesive cell culture container that is not low-adhesive, 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 proliferative ability can also be produced by culturing cancer stem cells with low proliferative ability in a highly adhesive cell culture container that is not low-adhesive, 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 the protein described in SEQ ID NOs: 1 to 8 (a protein consisting of the amino acid sequence described in any of SEQ ID NOs: 1 to 8). As another aspect of the reagent of the present invention, a reagent for detecting Lgr5-positive cancer cells is mentioned, and the reagent preferably contains at least one antibody that binds to the protein described in SEQ ID NOs: 1 to 6 (a protein consisting of the amino acid sequence described in any of SEQ ID NOs: 1 to 6). As yet another aspect of the reagent of the present invention, a reagent for detecting Lgr5-negative cancer cells is mentioned, and the reagent preferably contains at least one antibody that binds to the protein described in SEQ ID NOs: 1 to 8 (a protein consisting of the amino acid sequence described in any of SEQ ID NOs: 1 to 8).

[0075] <Proliferation inhibitor> In a non-limiting aspect, as the proliferation inhibitor, a DNA damaging agent, an anti-mitotic agent and / or an antimetabolite may be preferably mentioned. The DNA damaging agent can be an alkylating agent, a topoisomerase inhibitor and / or a DNA intercalator. Carboplatin (a DNA alkylating agent), etoposide (an inhibitor of topoisomerase II), doxorubicin (a DNA intercalator), docetaxel (an anti-mitotic agent) and Gemzar (gemcitabine, an antimetabolite), etc. can be exemplified as non-limiting preferred proliferation inhibitors.

[0076] The alkylating agent can be selected from at least one of the following. That is, chlorambucil, cyclophosphamide, ifosfamide, mechlorethamine, melphalan, uracil mustard, thiotepa, busulfan, carmustine, lomustine, streptozocin, carboplatin, cisplatin, satraplatin, oxaliplatin, altretamine, ET-743, XL119 (becatecarin), dacarbazine, chloromethine, bendamustine, trofosfamide, uramustine, homoharringtonine, nimustine, prednimustine, ranimustine, semustine, nedaplatin, triplatin tetranitrate, mannosulfan, treosulfan, temozolomide, carbocon, triazicon, triethylenemelamine, procarbazine, etc. At least one alkylating agent selected therefrom can be used.

[0077] The topoisomerase inhibitor can be selected from at least one of the following. Doxorubicin (Doxil), daunorubicin, epirubicin, idarubicin, anthracenedione (Novantrone), mitoxantrone, mitomycin C, bleomycin, dactinomycin, plicamycin, irinotecan (Camptosar), camptothecin, rubitecan, velotecan, etoposide, teniposide, and topotecan (Hycamptin), etc. At least one topoisomerase inhibitor selected therefrom can be used.

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

[0079] The anti-mitotic agent can be selected from at least one of the following: Paclitaxel (Abraxane) / Taxol, Docetaxel (Taxotere), BMS-275183, Xyotax, Tocosal, Vinorelbine, Vincristine, Vinblastine, Vindesine, Vinzolidine, Etoposide (VP-16), Teniposide (VM-26), Ixabepilone, Larotaxel, Ortataxel, Tesetaxel, and at least one topoisomerase inhibitor such as Ispinesib can be used.

[0080] The antimetabolite can be selected from at least one of the following: 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, and at least one topoisomerase inhibitor can be used.

[0081] Furthermore, the present invention relates to a method for screening anti-cancer agents using cancer stem cells separated and induced by the method of the present invention.

[0082] Furthermore, the present invention relates to a method for evaluating a compound using cancer stem cells separated and induced by the 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 cancer stem cells of the present invention. Specifically, a method for detecting, identifying, or quantifying the presence of cancer stem cells of the present invention or a substantially homogeneous population of cancer stem cells, which includes the following steps (a) and (b), is provided; (a) A step of preparing a sample obtained from a cancer patient. (b) Contacting the sample with an Lgr5 antibody.

[0084] In this method, first, a sample obtained from a cancer patient is 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. Next, in this method, the sample is contacted with an Lgr5 antibody.

[0085] The method for detecting, identifying, or quantifying the presence of the cancer stem cells or a substantially homogeneous population of cancer stem cells of the present invention can be used, for example, for cancer diagnosis, screening of cancer patients, prediction and confirmation of the efficacy of drugs (pharmaceutical compositions), monitoring of treatment, and cancer imaging.

[0086] Specifically, for example, an organ or tissue can be excised from a cancer patient, a specimen can be prepared, and the presence of cancer stem cells can be detected, identified, or quantified using the specimen. Known methods can be appropriately used for the preparation of the specimen, for example, the PFA-AMeX-Paraffin method (WO 09 / 078386) can be used. As the sample, for example, frozen or unfrozen organs or tissues can be used. The sample of the cancer patient is first fixed with a PFA solution. The PFA solution is a cell fixation solution obtained by adding a buffer solution such as a phosphate buffer 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 the 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. Next, the fixed organ or tissue is washed with, for example, phosphate buffered saline. At this time, after cutting out the observed part of the organ or tissue, it may be washed.

[0087] The thus-prepared organ or tissue is then paraffin-embedded by the AMeX method. The AMeX method is a paraffin-embedding method that involves a series of operations including fixation with cold acetone, dehydration with acetone, clearing with methyl benzoate and xylene, and paraffin embedding. Specifically, it 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 after transferring the organ or tissue to room temperature acetone, it is dehydrated at room temperature for 0.5 to 5 hours, preferably 1 to 4 hours. Next, it 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 infiltrated and 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 block of the organ or tissue obtained by the PFA-AMeX method in this way is stored at a low temperature until use.

[0088] At the time of use, the paraffin block obtained above is used to prepare thin sections using a microtome or the like, and further, deparaffinization and hydrophilic treatment of the thin sections are performed. Deparaffinization and hydrophilic treatment can be carried out by known methods. For example, deparaffinization can be carried out with xylene or toluene, and hydrophilic treatment can be carried out with 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 performing tissue staining (special staining) on the prepared specimens, any staining that can be performed on ordinary paraffin-embedded sections can be used (for example, PAS staining, Giemsa staining, toluidine blue staining, etc.). For enzyme histochemical staining, any staining that can be performed on the sections can be used (for example, various stainings such as ALP, ACP, TRAP, esterase, etc.). In addition, for staining pathological tissues, as general staining, hematoxylin and eosin (Hematoxylin-Eosin) staining; for collagen fibers, Van Gieson staining, Azan staining, Masson Trichrome staining; for elastic fibers, Weigert staining, Elastica Van Gieson staining; for reticular fibers and basement membranes, Watanabe's silver staining, PAM staining (Periodic acid methenamine silver stain), etc. can be used.

[0091] Immunohistochemical staining or enzyme histochemical staining can be performed using, but not limited to, direct methods using primary antibodies labeled with enzymes or labeling substances and indirect methods labeling secondary antibodies without labeling primary antibodies. Antibodies can be labeled by commonly known methods. Examples of labeling substances include, for example, radioisotopes, enzymes, fluorescent substances, biotin / avidin, etc. Commercially available labeling substances can be used for these. Examples of radioisotopes include 32 P, 33 P, 131 I, 125 I, 3 H, 14 C, 35 S. Examples of enzymes include, for example, alkaline phosphatase, horseradish peroxidase, β-galactosidase, β-glucosidase, etc. Examples of fluorescent substances include, for example, fluorescein isothiocyanate (FITC), rhodamine. These can be obtained commercially and are labeled by known methods.

[0092] The thin-section slices are subjected to, for example, tissue staining, immunohistochemical staining, or enzyme histochemical staining for detection, identification, or quantification.

[0093] Furthermore, detection, identification, or quantification can also be performed by quantifying DNA or RNA in cells in an organ or tissue specimen. There are no particular restrictions on these expression confirmation methods, and they can be carried out according to conventional expression confirmation methods. Examples of RNA include microRNA, siRNA, tRNA, snRNA, mRNA, or 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] To collect a desired tissue or cells from a specimen, a microdissection method, particularly the laser microdissection (LMD) method, can be used. Since the LMD method can collect a target cell population from a biological tissue, it is possible to accurately know in which cells and to what extent a specific gene is expressed among various cells constituting the tissue in vivo. As a device for performing microdissection, for example, an AS-LMD system (manufactured by Leica Microsystems) can be used.

[0095] In addition, in the present invention, there is provided a method for diagnosing cancer, detecting cancer stem cells, or selecting cancer patients, which includes detecting the presence of at least one of the proteins described in SEQ ID NOs: 1 to 8 in a sample isolated from a cancer patient using at least one antibody that binds to the proteins. To detect the presence of cancer stem cells, it is possible to use at least one antibody that binds to the proteins described in SEQ ID NOs: 1 to 8 instead of the aforementioned Lgr5 antibody.

[0096] In one non-limiting aspect of the present invention, there is provided a method for diagnosing cancer, detecting cancer stem cells, or selecting cancer patients, which includes detecting the presence of at least one such protein in a sample isolated from a cancer patient using at least one antibody that binds to the proteins 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 proteins set forth in SEQ ID NOs: 1 to 6 instead of the aforementioned 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 also detecting the presence of Lgr5.

[0097] In one non-limiting aspect of the present invention, there is provided a method for diagnosing cancer, detecting cancer stem cells, or selecting cancer patients, which includes detecting the presence of at least one such protein in a sample isolated from a cancer patient using at least one antibody that binds to the proteins set forth in SEQ ID NOs: 1 to 8. To detect the presence of Lgr5-negative cancer stem cells, it is possible to use at least one antibody that binds to the proteins set forth in SEQ ID NOs: 1 to 8 instead of the aforementioned 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 also detecting the presence of Lgr5.

[0098] In addition, the present invention provides a method for confirming the effectiveness of a pharmaceutical composition comprising at least one antibody that binds to the proteins described in SEQ ID NOs: 1 to 8, the method comprising detecting the presence of any one or more of the proteins described in SEQ ID NOs: 1 to 8 and / or polynucleotides encoding said proteins in a sample isolated from a subject to whom the pharmaceutical composition has been administered. In this method, at least one antibody that binds to the proteins described in SEQ ID NOs: 1 to 8, or a polynucleotide encoding the proteins described in SEQ ID NOs: 1 to 8 and / or a portion of its complementary strand can also be used for detection. In another non-limiting aspect, there is provided a method for evaluating the effectiveness of cancer treatment in a subject, the method comprising comparing the expression of at least one of the proteins described in SEQ ID NOs: 1 to 8 and / or polynucleotides encoding said proteins in a first sample obtained from the subject before providing at least a part of the treatment to the subject, with the expression of at least one of the proteins described in SEQ ID NOs: 1 to 8 and / or polynucleotides encoding said proteins in a second sample obtained from the subject after providing the part of the treatment, wherein a significantly lower level of said protein and / or said polynucleotide in the second sample compared to the first sample is an effective indicator that the treatment inhibits cancer in the subject.

[0099] In a non-limiting aspect of the present invention, there is provided a method for monitoring the effectiveness of treatment with an antibody provided by the present invention in a subject, (i) obtaining a pre-administration sample from the subject before administration of the antibody; (ii) detecting the level of expression in the pre-administration sample of at least one marker protein selected from the proteins described in SEQ ID NOs: 1 to 8, its mRNA, or its genomic DNA; (iii) obtaining from the subject one or more post-administration samples; (iv) detecting the level of expression or activity in a post - administration sample of at least one marker protein selected from the proteins described in SEQ ID NOs: 1 to 8, its mRNA, or its genomic DNA; (v) comparing the level of expression or activity in a pre - administration sample of the marker protein, the mRNA, or the genomic DNA with that of the marker protein, the mRNA, or the genomic DNA in the post - administration sample or samples; and, (vi) accordingly, a method is provided that comprises the step of altering the administration of the antibody to the subject. For example, an increase in the administration of the antibody of the present invention can be used to decrease the expression or activity of a marker (the level of expression or activity of the marker in the pre - administration sample of the marker protein, the mRNA, or the genomic DNA) to a higher level than that detected, i.e., to increase the effectiveness of the antibody.

[0100] In another non - limiting aspect of the present invention, a method for monitoring the effectiveness of treatment with an antibody provided by the present invention in a subject, (i) detecting Lgr5 - positive cancer stem cells in a pre - administration sample obtained from the subject prior to administration of the antibody; (ii) detecting the level of expression in a pre - administration sample of at least one marker protein selected from the proteins described in SEQ ID NOs: 1 to 6, its mRNA, or its genomic DNA; (iii) obtaining one or more post - administration samples from the subject; (iv) detecting the level of expression or activity in a post - administration sample of at least one marker protein selected from the proteins described in SEQ ID NOs: 1 to 6, its mRNA, or its genomic DNA; (v) comparing the level of expression or activity in a pre - administration sample of the marker protein, the mRNA, or the genomic DNA with that of the marker protein, the mRNA, or the genomic DNA in the post - administration sample or samples; and, (vi) A method is provided that comprises the step of changing the administration of the antibody to the subject accordingly. For example, an increase in the administration of the antibody of the present invention can be used to reduce the expression or activity of a marker (the level of expression or activity in the pre - administration sample of the marker protein, the mRNA, or the genomic DNA) to a level higher than that detected, i.e., to increase the efficacy of the antibody.

[0101] In a different non - limiting aspect of the present invention, a method for monitoring the efficacy of treatment with an antibody provided by the present invention in a subject, comprising: (i) Detecting Lgr5 - negative cancer stem cells in a pre - administration sample obtained 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 - administration 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) A method is provided that comprises the step of changing the administration of the antibody to the subject accordingly. For example, an increase in the administration of the antibody of the present invention can be used to reduce the expression or activity of a marker (the level of expression or activity in the pre - administration sample of the marker protein, the mRNA, or the genomic DNA) to a level higher than that detected, i.e., to increase the efficacy of the antibody.

[0102] <Cancer stem cell inhibitor> The cancer stem cell inhibitor refers to, for example, an agent having effects such as suppression of cancer stem cell proliferation, suppression of cancer stem cell metastasis or recurrence, and death of cancer stem cells, and may also have effects such as suppression of cancer cell proliferation, suppression of cancer cell metastasis or recurrence, and death of cancer cells.

[0103] The terms "suppress", "suppression", and phenomena that are linguistically equivalent thereto, when used in relation to a biological activity exemplified without limitation such as cancer stem cell proliferation or metastasis, refer to the down-regulation of the biological activity, which can reduce or eliminate the targeted function such as protein production or molecular phosphorylation. In certain embodiments, suppression can refer to a decrease of about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% of the targeted activity. When used in relation to a disorder or disease, the term refers to the prevention of the occurrence of symptoms, the alleviation of symptoms, or the success of alleviating the disease, condition or disorder.

[0104] "Metastasis" refers to the process by which cancer spreads from the primary site in the body to other regions or moves to develop similar cancerous lesions in new locations. "Metastatic" or "metastasizing" cells are cells that lose adhesive contact with adjacent cells and move through the bloodstream or lymph from the primary site of the disease to invade neighboring body structures. "Recurrence" refers to the reappearance of the same malignant tumor in the remaining organs after partial resection of the organ for removing the 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 encoding the protein by any method known to those skilled in the art, culturing a transformant transformed with the expression vector to produce and accumulate the protein, and collecting the protein.

[0106] The above expression vector can be created according to methods known in the art. For example, (1) Excise a DNA fragment containing a gene encoding the protein, (2) Link the DNA fragment downstream of a promoter in an appropriate expression vector. It can be produced thereby.

[0107] As the vector, 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, animal viruses such as retroviruses, vaccinia virus, baculovirus, etc. can be used.

[0108] As the promoter used in the present invention, any promoter appropriate for the host used for gene expression may be used. For example, when the host is Escherichia coli, the trp promoter, lac promoter, recA promoter, λPL promoter, lpp promoter, etc., when the host is Bacillus subtilis, the SPO1 promoter, SPO2 promoter, penP promoter, etc., when the host is yeast, the PHO5 promoter, PGK promoter, GAP promoter, ADH promoter, etc. are preferable. When animal cells are used as the host, the SRα promoter, SV40 promoter, LTR promoter, CMV promoter, HSV-TK promoter, etc. can be mentioned.

[0109] In addition to the above, enhancers, splicing signals, polyA addition signals, selection markers, SV40 replication origins, etc., known in the art can be added to the expression vector as desired. Further, if necessary, the protein used in the present invention can also be expressed as a fusion protein with other proteins (e.g., glutathione S-transferase and protein A). Such fusion proteins can be cleaved using appropriate proteases and separated into their respective proteins.

[0110] As host cells, for example, bacteria belonging to the genus Escherichia, bacteria belonging to the genus Bacillus, yeast, insect cells, insects, animal cells, etc. are used.

[0111] Specific examples of bacteria belonging to the genus Escherichia 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)), etc.

[0112] As bacteria belonging to the genus Bacillus, for example, Bacillus subtilis MI114 (Gene, Vol. 24, 255 (1983)), 207-21 [Journal of Biochemistry, Vol. 95, 87 (1984)], etc. are used.

[0113] As yeast, for example, Saccharomyces cerevisiae AH22, AH22R-, NA87-11A, DKD-5D, 20B-12, Schizosaccharomyces pombe NCYC1913, NCYC2036, Pichia pastoris, etc. are used.

[0114] As animal cells, for example, monkey cells COS-7, 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, human FL cells, etc. are used.

[0115] Transformation of these host cells can be carried out according to methods known in the art. For example, the following described literature can be referred to. 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 transformant 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 carried out at about 15 - 43 °C for about 3 - 24 hours, and aeration and agitation can be added if necessary. When the host is a bacterium of the genus Bacillus, the culture is usually carried out at about 30 - 40 °C for about 6 - 24 hours, and aeration and agitation can be added if necessary.

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

[0118] When culturing a transformant whose host is an animal cell, the culture is usually carried out at about 30 °C - 40 °C for about 15 - 60 hours using a medium adjusted to a pH of about 6 - 8, and aeration and agitation can be added as required.

[0119] To separate and purify the protein used in the present invention from the above culture, for example, after culturing, the cells or bacteria are collected by known methods, suspended in an appropriate buffer, and the cells or bacteria are disrupted by ultrasonic waves, lysozyme and / or freeze - thawing, etc., and then a crude extract of the protein is obtained by centrifugation or filtration. Protein denaturants such as urea and guanidine hydrochloride, and Triton X - 100 in the buffer TMSurfactants such as etc. may be included. When a protein is secreted into the culture medium, after the completion of the culture, the cells or cells and the supernatant are separated by a known method, and the supernatant is collected. The purification of the protein contained in the thus obtained culture supernatant or extract can be carried out by appropriately combining known separation and purification methods.

[0120] The protein thus obtained can be arbitrarily modified or a polypeptide can be partially removed by allowing an appropriate protein-modifying enzyme such as trypsin and chymotrypsin to act on the protein produced by the recombinant, before or after purification, by a known method or a method analogous thereto. The presence of the protein used in the present invention can be measured by various binding assays and enzyme immunoassays using specific antibodies.

[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 using known means. As the antibody used in the present invention, monoclonal antibodies derived from mammals are particularly preferred. Monoclonal antibodies derived from mammals include those produced by hybridomas and those produced by hosts transformed with an expression vector containing an antibody gene by genetic engineering techniques. In addition, the antibody used in the present invention preferably binds specifically to the protein used in the present invention.

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

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

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

[0125] Here, as the partial polypeptide of the protein, for example, it has an amino acid sequence of at least 10 or more, preferably 50 or more, more preferably 70 or more, still more preferably 100 or more, and most preferably 200 or more of the constituent amino acid sequences of the protein, and for example, a peptide having a biological activity substantially equivalent to the function of the protein is used. 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 includes those in which the amino group of the N-terminal methionine residue is protected by a protecting group, those in which the glutamyl group generated by cleavage in vivo at the N-terminal side is pyroglutaminated, those in which the substituents on the side chains of the amino acids in the molecule are protected by appropriate protecting groups, or complex peptides such as so-called glycopeptides to which sugar chains are bound.

[0126] The mammal immunized with the sensitizing antigen is not particularly limited, but it is preferably selected in consideration of compatibility with the parent cells used for cell fusion. Generally, rodents such as mice, rats, hamsters, etc. are used.

[0127] To immunize an animal with a sensitizing antigen, it is carried out according to a known method. For example, as a general method, it is carried out by injecting the sensitizing antigen into the abdominal cavity or subcutaneous tissue of a mammal. Specifically, the sensitizing antigen is diluted and suspended in an appropriate amount with PBS (Phosphate-Buffered Saline), physiological saline, etc., and an appropriate amount of a normal adjuvant, such as Freund's complete adjuvant, is mixed as desired. After emulsification, it is administered to the mammal several times every 4 to 21 days. Also, an appropriate carrier can be used during sensitizing antigen immunization.

[0128] After immunizing the mammal in this way and confirming that the desired antibody level has increased in the serum, immune cells are collected from the mammal and subjected to cell fusion. Preferred immune cells include, in particular, spleen cells.

[0129] As the other parent cell to be fused with the immune cells, mammalian myeloma cells are used. These myeloma cells are 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. D. H. 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., J. Immunol. Methods (1980) 35, 1-21), S194 (Trowbridge, I. S. J. Exp. Med. (1978) 148, 313-323), R210 (Galfre, G. et al., Nature (1979) 277, 131-133), etc., and are preferably used.

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

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

[0132] The usage ratio of the immune cells to the myeloma cells can be arbitrarily set. For example, it is preferable to make the immune cells 1-10 times that of the myeloma cells. As the culture medium used for the cell fusion, for example, RPMI1640 culture medium, MEM culture medium, which are suitable for the growth of the myeloma cell line, and other normal culture media used for this type of cell culture can be used, and further, a serum supplement such as fetal bovine serum (FCS) can also be used in combination. For cell fusion, a predetermined amount of the immune cells and the myeloma cells are well mixed in the culture medium, and a PEG solution (for example, with an average molecular weight of about 1000-6000) pre-warmed to about 37°C is added usually at a concentration of 30-60% (w / v) and mixed to form the target fusion cells (hybridomas). Subsequently, an appropriate culture medium is sequentially added, and the operation of centrifuging to remove the supernatant is repeated to remove cell fusion agents and the like that are not favorable for the growth of hybridomas.

[0133] The hybridomas thus obtained are selected by culturing them in a normal selection culture medium, for example, an HAT culture medium (a culture medium containing hypoxanthine, aminopterin, and thymidine). Culturing in the above HAT culture medium continues for a time sufficient for cells other than the target hybridomas (non-fused cells) to die (usually, several days to several weeks). Subsequently, the ordinary limiting dilution method is carried out to screen and single clone the hybridomas that produce the antibody used in the present invention.

[0134] In addition to immunizing animals other than humans with the antigen to obtain the above hybridomas, human lymphocytes can be sensitized to the protein in vitro, and the sensitized lymphocytes can be fused with myeloma cells having the ability to continuously divide derived from humans to obtain a desired human antibody having binding activity to the protein (see Japanese Patent Publication No. 1-59878). Further, the protein serving as an antigen may be administered to a transgenic animal having all repertoires of human antibody genes to obtain antibody-producing cells, and a human antibody against the protein may be obtained from the immortalized cells (see International Patent Application Publication Nos. WO 94 / 25585, WO 93 / 12227, WO 92 / 03918, and WO 94 / 02602).

[0135] Techniques for obtaining human antibodies by panning using a human antibody library are also known. For example, the V region of a human antibody is expressed on the surface of a phage by the phage display method as a single-chain antibody (scFv). Phages expressing an scFv that binds to an antigen can be selected. By analyzing the gene of the selected phage, a DNA sequence encoding the V region of a human antibody that binds to the antigen can be determined. After determining the DNA sequence of the scFv that binds to the antigen, an expression vector can be prepared by fusing the V region sequence in-frame with the sequence of a desired human antibody C region and then inserting it into an appropriate expression vector. The human antibody is obtained by introducing the expression vector into a suitable expression cell as described above and expressing the gene encoding 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] Hybridomas that produce monoclonal antibodies produced in this way can be subcultured in a normal culture medium and can also be stored long-term in liquid nitrogen.

[0137] To obtain a monoclonal antibody from the hybridoma, methods such as culturing the hybridoma according to a normal method and obtaining it as the culture supernatant, or administering the hybridoma to a compatible mammal for growth and obtaining it as ascites are adopted. The former method is suitable for obtaining a high-purity antibody, while the latter method is suitable for mass production of the antibody.

[0138] The monoclonal antibody used in the present invention may be a recombinant one 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 recombination technology (see, for example, Vandamme, A.M. et al., Eur.J.Biochem. (1990)192, 767-775, 1990).

[0139] Specifically, mRNA encoding the variable (V) region of the antibody is isolated from a hybridoma that produces the antibody. The isolation of mRNA is carried out by known methods, for example, the guanidine ultracentrifugation method (Chirgwin, J.M. et al., Biochemistry (1979)18,5294-5299), the AGPC method (Chomczynski, P. et al., Anal.Biochem. (1987)162, 156-159), etc. to prepare total RNA, and the target mRNA is prepared using an mRNA Purification Kit (manufactured by Pharmacia) or the like. Also, mRNA can be directly prepared by using the QuickPrep mRNA Purification Kit (manufactured by Pharmacia).

[0140] Using reverse transcriptase, cDNA of the antibody V region is synthesized from the obtained mRNA. The synthesis of cDNA is carried out using an AMV Reverse Transcriptase First-strand cDNA Synthesis Kit (manufactured by Seikagaku Corporation) or the like. Also, for the synthesis and amplification of cDNA, the 5'-Ampli FINDER RACE Kit (manufactured by Clontech) and the 5'-RACE method using PCR (Frohman, M.A. et al., Proc.Natl.Acad.Sci.USA (1988)85, 8998-9002, Belyavsky, A. et al., Nucleic Acids Res. (1989) 17, 2919-2932) etc. can be used.

[0141] The target DNA fragment is purified from the obtained PCR product and ligated to vector DNA. Further, a recombinant vector is prepared therefrom, introduced into Escherichia coli or the like, and colonies are selected to prepare the desired recombinant vector. Then, the nucleotide sequence of the target DNA is confirmed by a known method, for example, the dideoxynucleotide chain termination method or the like.

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

[0143] To produce the antibody used in the present invention, the antibody gene is incorporated into an expression vector so as to be expressed under the control of an expression control region, for example, an enhancer or a promoter. Next, host cells are transformed with this expression vector to express the antibody.

[0144] For the expression of the antibody gene, the DNA encoding the antibody heavy chain (H chain) or light chain (L chain) may be separately incorporated into an expression vector to co-transform host cells, or alternatively, the DNA encoding the H chain and L chain may be incorporated into a single expression vector to transform host cells (see WO 94 / 11523).

[0145] In addition to the above host cells, transgenic animals can be used for the production of recombinant antibodies. For example, the antibody gene is inserted into the middle of the gene encoding a protein specifically produced in milk (such as goat casein) 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 or its offspring born from the goat that received the embryo. Further, in order to increase the amount of milk containing the desired antibody produced by the transgenic goat, hormones may be appropriately used for the transgenic goat (Ebert, K.M. et al., Bio / Technology (1994) 12, 699-702).

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

[0147] A chimeric antibody can be obtained by ligating the DNA encoding the antibody V region obtained as described above to the DNA encoding the human antibody C region, incorporating this into an expression vector, introducing it into a host, and producing it. Using this known method, useful chimeric antibodies can be obtained.

[0148] A humanized antibody, also referred to as a reshaped human antibody, is one in which the complementarity determining regions (CDRs) of an antibody from a non-human mammal, such as a mouse antibody, are transplanted into the complementarity determining regions of a human antibody, and the general genetic engineering techniques thereof are also known (see European Patent Application Publication No. EP 125023, WO 96 / 02576).

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

[0150] The framework regions of human antibodies linked via CDR are selected such that the complementarity-determining regions form good antigen-binding sites. If necessary, the amino acids of the framework regions in the variable regions of the antibodies may be substituted so that the complementarity-determining regions of the recombinant human antibodies form appropriate antigen-binding sites (Sato, K. et al., Cancer Res. (1993) 53, 851-856).

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

[0152] Chimeric antibodies consist of the variable regions of antibodies derived from non-human mammals and the constant regions derived from human antibodies. On the other hand, humanized antibodies consist of the complementarity-determining regions of antibodies derived from non-human mammals and the framework regions and C regions derived from human antibodies. Since the antigenicity of humanized antibodies in the human body is reduced, they are useful as the active ingredient of the therapeutic agent of the present invention.

[0153] The antibodies used in the present invention are not limited to the whole antibody molecule, and may be antibody fragments or their modified products as long as they bind to the proteins used in the present invention, including both bivalent antibodies and monovalent antibodies. For example, antibody fragments include Fab, F(ab')2, Fv, Fab / c having one Fab and a complete Fc, or single-chain Fv (scFv) in which the Fv of the H chain or L chain is linked by an appropriate linker, and Diabody. Specifically, the antibody is treated with an enzyme, such as papain or pepsin, to generate an antibody fragment, or a gene encoding these antibody fragments is constructed, introduced into an expression vector, and then expressed in an appropriate host cell (for example, see Co, M. S. et al, J. Immunol. (1994) 152, 2968-2976, Better, M. & Horwitz, A. H. 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, Bird, R. E. et al., TIBTECH (1991) 9, 132-137).

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

[0155] The DNA encoding the scFv uses, as a template, the DNA encoding the H chain or the V region of the H chain of the antibody, and the DNA encoding the L chain or the V region of the L chain, and amplifies them by the PCR method using a primer pair that defines both ends thereof. Then, it is obtained by further combining and amplifying the DNA encoding the peptide linker portion and a primer pair that defines both ends thereof to be linked to the H chain and the L chain, respectively.

[0156] Also, once the DNA encoding the scFv is prepared, an expression vector containing them and a host transformed with the expression vector can be obtained according to a conventional method, and by using the host, the scFv can be obtained according to a conventional method.

[0157] A diabody is a dimer formed by linking two fragments (such as scFv) that each consist of a variable region linked to another variable region by a linker or the like, and usually contains 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 obtained and expressed in the same manner as described above and produced by a host. The "antibody" in the present invention includes these antibody fragments as well.

[0159] As a modification of the antibody, the antibody of the present invention conjugated with various molecules such as polyethylene glycol (PEG) can also be used. It is also possible to conjugate the antibody with cytotoxic substances such as radioisotopes, chemotherapeutic agents, and bacterial-derived toxins. The "antibody" in the present invention includes these antibody modifications as well. Such antibody modifications can be obtained by chemically modifying the obtained antibody. Note that 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. Alternatively, one antigen-binding site 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 specifically damage cancer stem cells by directly acting a cytotoxic substance on cancer stem cells expressing the protein used in the present invention, and to suppress the proliferation of cancer stem cells. Also, a bispecific antibody in which one antigen-binding site recognizes a molecule constituting 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 can also be used. The bispecific antibody can be prepared by binding the HL pairs of two types of antibodies, or by fusing hybridomas producing different monoclonal antibodies to produce bispecific antibody-producing fused cells. Furthermore, it is also possible to produce a bispecific antibody by genetic engineering techniques.

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

[0162] In addition, as promoters / enhancers that can be used for antibody expression in the present invention, there are virus promoters / enhancers such as retrovirus, polyomavirus, adenovirus, simian virus 40 (SV40), or promoters / enhancers derived from mammalian cells such as human elongation factor 1α (HEF1α).

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

[0164] As the origin of replication, those derived from SV40, polyomavirus, adenovirus, bovine papillomavirus (BPV), etc. can be used. Furthermore, for gene copy number amplification in the host cell line, the expression vector can contain the aminoglycoside transferase (APH) gene, thymidine kinase (TK) gene, Escherichia coli xanthine-guanine phosphoribosyltransferase (Ecogpt) gene, dihydrofolate reductase (dhfr) gene, etc. as selectable markers.

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

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

[0167] For the production of the antibody used in the present invention, any expression system, such as eukaryotic cells or prokaryotic cell lines, can be used. Examples of eukaryotic cells include animal cells such as established mammalian cell lines, insect cell lines, filamentous fungal cells, and yeast cells, and examples of prokaryotic cells include bacterial cells such as Escherichia coli cells. Preferably, the antibody used in the present invention is expressed in mammalian cells, such as CHO, COS, myeloma, BHK, Vero, and HeLa cells.

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

[0169] As described above, the expressed and produced antibody can be separated from cells or host animals and purified to homogeneity. The separation and purification of the antibody used in the present invention can be carried out using an affinity column. For example, as columns using Protein A columns, Hyper D, POROS, Sepharose F.F. (manufactured by Pharmacia), etc. can be mentioned. In addition, the separation and purification methods usually used for ordinary proteins may be used, and there is no limitation whatsoever. For example, by appropriately selecting and combining chromatography columns other than the above-mentioned affinity columns, filters, ultrafiltration, salting out, dialysis, etc., the antibody can be separated and purified (Antibodies A Laboratory Manual. Ed Harlow, David Lane, Cold Spring Harbor Laboratory, 1988).

[0170] Known means can be used for measuring 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 antibody used in the present invention.

[0171] As a method for measuring the antigen-binding activity of this antibody, ELISA (enzyme-linked immunosorbent assay), EIA (enzyme immunoassay), RIA (radioimmunoassay) or fluorescent antibody method can be used. For example, when using the enzyme immunoassay, a sample containing the antibody, for example, the culture supernatant of the antibody-producing cells or a 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 to measure the absorbance, thereby evaluating the antigen-binding activity.

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

[0173] Such antibody modifications can be obtained, in addition to the above-mentioned chemical modifications, as molecular forms such as bispecific antibodies designed using genetic recombination techniques so as to recognize growth inhibitors, toxic peptides, or radiochemicals. The "antibodies" in the present invention also include these antibodies.

[0174] In addition, 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 aspect, one or more growth inhibitors and toxic peptides can be combined and used for antibody modification respectively. As described above, covalent or non-covalent bonds can be used for the binding between the antibody that binds to the protein described in at least one of SEQ ID NOs: 1 to 8 of the present invention and the above growth inhibitor, toxic peptide, or radiochemical. Methods for preparing antibody modifications conjugated with these chemotherapeutic agents are known.

[0175] Furthermore, proteinaceous drugs and toxins can be bound to antibodies by genetic engineering techniques. Specifically, for example, a recombinant vector can be constructed by fusing in-frame the DNA encoding the above toxic peptide and the DNA encoding an antibody that binds to any of the proteins described in at least one of SEQ ID NOs: 1 to 8 of the present invention and incorporating it into an expression vector. By culturing the transformed cells obtained by introducing the vector into an appropriate host cell and expressing the incorporated DNA, an antibody modification conjugated with a toxic peptide can be obtained as a fusion protein. When obtaining a fusion protein with an antibody, generally, a proteinaceous drug or toxin is arranged on the C-terminal side 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 the cytotoxic activity in the present invention include complement-dependent cytotoxicity (CDC) activity, antibody-dependent cell-mediated cytotoxicity (ADCC) activity, and the like. In the present invention, the CDC activity means cytotoxic activity by the complement system, and the ADCC activity means the activity in which when a specific antibody adheres to the cell surface antigen of a target cell, an Fcγ receptor-bearing cell (immune cell, etc.) binds to the Fc portion thereof via the Fcγ receptor and damages the target cell.

[0177] Whether the antibody used in the present invention has ADCC activity or CDC activity can be measured by a known method (for example, 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, the measurement of cytotoxic activity can be performed by the following method. · Preparation of effector cells The spleen is removed from CBA / N mice or the like, and spleen cells are separated in RPMI1640 medium (manufactured by GIBCO). After washing with the same medium containing 10% fetal bovine serum (FBS, manufactured by HyClone), the cell concentration is adjusted to 5×10 6 / ml to prepare effector cells.

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

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

[0181] ·Measurement of ADCC activity To a 96-well U-bottom plate (manufactured by Becton Dickinson), 50 μl each of the target cells and the antibody used in the present invention are added 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 with a gamma counter (COBRAII AUTO-GMMA, MODEL D5005, manufactured by Packard Instrument Company). The cytotoxic activity (%) can be determined by (A - C) / (B - C)×100 where A represents the radioactivity (cpm) in each sample, B represents the radioactivity (cpm) in the sample with 1% NP-40 (manufactured by Hanrei), and C represents the radioactivity (cpm) in the sample containing only target cells.

[0182] ·Measurement of CDC activity To a 96-well flat-bottom plate (manufactured by Becton Dickinson), 50 μl each of the target cells and the antibody used in the present invention are added 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 with a gamma counter. The cytotoxic activity can be determined in the same manner as the measurement of ADCC activity.

[0183] The antibodies provided by the present invention may also be appropriately used antibodies 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. As antibodies with modified sugar chains, for example, the following antibodies are known. - Antibodies with modified glycosylation (such as WO1999 / 054342, etc.) - Antibodies lacking fucose added to the sugar chain (such as WO2000 / 061739, WO2002 / 031140, etc.) - Antibodies having a sugar chain with bisecting GlcNAc (bisecting N - acetylglucosamine) (such as WO2002 / 079255, etc.) The antibodies of the present invention preferably include antibodies whose sugar chain composition is modified such that the proportion of fucose - deficient antibodies is increased, or the proportion of antibodies with added bisecting N - acetylglucosamine is increased.

[0184] In the present invention, antibodies having neutralizing activity may also be appropriately used. Generally, neutralizing activity refers to the activity of inhibiting the biological activity of a ligand, which is an exogenous molecule such as a virus or a toxin, or an endogenous molecule such as a hormone or a cytokine, which has biological activity against cells. That is, a substance having neutralizing activity refers to a substance that binds to the ligand or the receptor to which the ligand binds and inhibits the binding between the ligand and the receptor. A receptor whose binding to the ligand is blocked by neutralizing activity cannot exert biological activity through the receptor. When the antigen - binding molecule is an antibody, such an antibody having neutralizing activity is generally called a neutralizing antibody. The neutralizing activity of a 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] Examples of EREG, which is the target of the EP27 antibody described later in the examples, are illustrated below. In the case of the EGF receptor, which is considered to be the main receptor for EREG represented by SEQ ID NO: 3, ligand binding forms a dimer and activates the tyrosine kinase, which is its own domain present inside the cell. The activated tyrosine kinase forms peptides containing phosphorylated tyrosine by autophosphorylation and associates various signal transduction accessory molecules with them. They are mainly PLCγ (phospholipase Cγ), Shc, Grb2, etc. Among these accessory molecules, the first two are further phosphorylated by the tyrosine kinase of the EGF receptor. The main pathway in signal transduction from the EGF receptor is the pathway in which phosphorylation is transmitted in the order of Shc, Grb2, Sos, Ras, Raf / MAP kinase / MAP kinase. Furthermore, there is thought to be a pathway from PLCγ, a secondary pathway, to PKC. Since such intracellular signal cascades vary depending on cell types, target molecules can be appropriately set for each target cell of interest and are not limited to the above factors. As the measurement kit for activation of in vivo signals, commercially available ones can be appropriately used (for example, Protein Kinase C Activity Measurement System (GE Healthcare Biosciences Corp.) etc.).

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

[0187] Furthermore, since the EGF receptor usually functions in the direction of promoting cell proliferation, the activation of in vivo signal transduction can be evaluated by measuring the proliferation activity of the 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 thereto, and the methods listed above can be preferably adopted and evaluated for each selected target cell.

[0188] That is, for example, the neutralizing activity of the anti-EREG antibody can be evaluated or measured by measuring cell proliferation activity as follows. For example, a method of measuring the uptake of 3 H]-labeled thymidine added to the medium by living cells as an index of DNA replication ability is used. As a more convenient method, a dye exclusion method for measuring the ability of a dye such as trypan blue to exclude extracellularly under a microscope or an MTT method is used. The latter 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 adding the test antibody to the culture solution of the test cells and allowing a certain period of time to elapse, an MTT solution is added to the culture solution and allowed to stand for a certain period of time to allow MTT to be taken up by the cells. As a result, MTT, which is a yellow compound, is converted into a blue compound by succinate dehydrogenase in the mitochondria inside the cell. After dissolving and coloring this blue product, the absorbance is measured to be used as 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 (such as nacalai tesque) and can be preferably used. Furthermore, methods for evaluating cell proliferation activity using cell ATP or cell culture impedance as an index are also known. When measuring the activity, a binding antibody having the same isotype as the anti-EREG antibody and having no such neutralizing activity is used as a control antibody in the same manner as the anti-EREG antibody, and the activity can be determined by showing that the anti-EREG antibody has a stronger neutralizing activity than the control antibody.

[0190] Cells in which anti-EREG antibody suppresses proliferation are not particularly limited as long as they are cells expressing EREG protein. Preferred EREG-expressing cells are, for example, cancer cells. Specifically, cells derived from colorectal 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, it is possible to obtain an inhibitory effect on cell proliferation that is effective against both the primary lesion and metastatic lesions of these cancers. More preferred cancer cells are primary colorectal cancer, metastatic colorectal cancer, lung adenocarcinoma, pancreatic cancer, gastric cancer, and renal cancer. Therefore, anti-EREG antibody can be used for the treatment and prevention of diseases caused by cell proliferation, such as colorectal cancer, lung adenocarcinoma, pancreatic cancer, gastric cancer, renal cancer, etc. These cancers can be targeted for treatment or prevention regardless of whether they are primary or metastatic lesions. More preferably, anti-EREG antibody can be used for the treatment and / or prevention of primary colorectal cancer, metastatic colorectal cancer, and pancreatic cancer. Furthermore, among these cancers, cancers that grow in an EREG-dependent manner are preferred as targets for treatment and / or prevention in the present invention.

[0191] In addition, in this specification and tables, when abbreviations are used to represent bases, amino acids, etc., they are based on the abbreviations by the IUPAC-IUB Commision on Biochemical Nomenclature or the conventional abbreviations in the art. When there may be optical isomers for amino acids, the L-form is shown unless otherwise specified.

[0192] <The cancer stem cell inhibitor of the present invention> The effective dosage of the cancer stem cell inhibitor of the present invention is selected in the range of 0.001 mg to 1000 mg per 1 kg of body weight per administration. Alternatively, a dosage of 0.01 to 100000 mg / body per patient can be selected. However, the inhibitor of the present invention is not limited to these dosages. Also, as for the administration timing of the inhibitor of the present invention, it can be administered regardless of whether it is before or after the clinical symptoms of the disease occur. The inhibitor of the present invention can be formulated according to conventional methods (Remington's Pharmaceutical Science, latest edition, Mark Publishing Company, Easton, USA) and may contain a pharmaceutically acceptable carrier and additives together. Examples of such carriers and pharmaceutical additives include water, pharmaceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinyl pyrrolidone, carboxyvinyl polymer, sodium carboxymethyl cellulose, sodium polyacrylate, sodium alginate, water-soluble dextran, sodium carboxymethyl starch, pectin, methyl cellulose, ethyl cellulose, 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. The actual additives are selected singly or in appropriate combinations from the above according to the dosage form of the inhibitor of the present invention, but of course, they 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 anti-adsorption agent such as Tween80, Tween20, gelatin, human serum albumin, etc. can be added thereto for use. Alternatively, it may be lyophilized to form a dosage form that is dissolved and reconstituted before use, and as an excipient for lyophilization, for example, sugar alcohols and saccharides such as mannitol and glucose can be used. The inhibitor of the present invention is usually administered by a parenteral administration route, such as an injection (subcutaneous injection, intravenous injection, intramuscular injection, intraperitoneal injection, etc.), transdermal, transmucosal, nasal, pulmonary, etc., but oral administration is also possible.

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

[0194] In the present invention, the cancer stem cell inhibitor can be used in various aspects such as, for example, prevention of cancer recurrence, suppression of cancer recurrence, prevention of cancer metastasis, suppression of cancer metastasis, and adjuvant therapy for prevention of postoperative recurrence. If used in the above aspects, any cancer stem cell inhibitor can be used as the cancer stem cell inhibitor of the present invention. Non-limiting preferred examples include cancer stem cell growth inhibitors, cancer stem cell destroyers, and the like. The cancer stem cell growth inhibitor provided by the present invention is not concerned with the mechanism by which the growth of the target cancer stem cells is suppressed as long as the growth of the cancer stem cells is suppressed. Non-limiting examples of such cancer stem cell growth inhibitors may include cancer stem cell growth inhibitors containing, as an active ingredient, an antibody having neutralizing activity against the growth or growth of cancer stem cells or cytotoxic activity against cancer stem cells. Similarly, the cancer stem cell destroyer provided by the present invention is not concerned with the mechanism by which the cancer stem cells are destroyed as long as the target cancer stem cells are destroyed. Non-limiting examples of such cancer stem cell destroyers may include cancer stem cell growth inhibitors containing, as an active ingredient, an antibody having cytotoxic activity or apoptosis activity against cancer stem cells. The method for measuring apoptosis activity can be used by those skilled in the art to measure whether the test cancer stem cell destroyer has apoptosis activity by using known methods including TUNEL (Terminal deoxynucleotidyl Transferase Biotin-dUTP Nick End Labeling) assay, caspase activity (especially caspase-3) assay, and assays of fas ligand and annexin V. Further non-limiting preferred examples may preferably include cancer stem cell differentiation promoters and the like. Non-limiting examples of such differentiation promoters include BMP4, that is, a polypeptide represented by SEQ ID NO: 9 or a polypeptide equivalent in which one or more amino acids among the amino acids contained in the polypeptide are added, deleted, or substituted. The polypeptide equivalent preferably has a differentiation-inducing activity equivalent to the differentiation-inducing activity of the polypeptide represented by SEQ ID NO: 9 against CSCs.Whether the differentiation-inducing activity is equivalent or not can be defined, for example, as being equivalent when it is 10%, preferably 20%, more preferably 30%, still more preferably 40%, and even more preferably 50% of the CK20-inducing activity against CSCs possessed by the polypeptide represented by SEQ ID NO: 9. In yet another non-limiting aspect, whether the differentiation-inducing activity is equivalent or not can be defined, for example, as being equivalent when it is 60%, preferably 70%, more preferably 80%, still more preferably 90%, and even more preferably 95% of the CK20-inducing activity against CSCs possessed by the polypeptide represented by SEQ ID NO: 9.

[0195] Anticancer agents used in combination with the cancer stem cell inhibitor of the present invention include alkylating agents, antimetabolites, natural products, platinum complexes, and other agents. Examples of alkylating agents include Nitrogen Mustards, Ethylenimines, Methylmelamines, Alkyl Sulfonates, Nitrosoureas, and Triazens. Examples of Nitrogen Mustards include Mechlorethamine, Cyclophosphamide, Ifosfamide, Melphalan, and Chlorambucil. Examples of Ethylenimines and Methylmelamines include Hexamethylmelamine and Thiotepa. An example of Alkyl Sulfonates is Busulfan. Examples of Nitrosoureas include Carmustine (BCNU), Lomustine (CCNU), Semustine (methyl-CCNU), and Streptozocin. An example of Triazens is Dacarbazine (DTIC). Examples of antimetabolites include folic acid analogs, pyrimidine analogs, and purine analogs. An example of folic acid analogs is Methotrexate. Examples of pyrimidine analogs include Fluorouracil (5-FU), Doxifluridine (5'-DFUR, trade name Furtulon), Capecitabine (trade name Xeloda), Floxuridine (FudR), and Cytarabine.Examples of purine analogs include, for example, Mercaptopurine (6-MP), Thioguanine (TG), and Pentostatin. Examples of natural products include Vinca Alkaloids, Epipodophyllotoxins, and antibiotics. Examples of Vinca Alkaloids include, for example, Vinblastine (VLB) and Vincristine (VCR). Examples of Epipodophyllotoxins include, for example, Etoposide and Teniposide. Examples of antibiotics include, for example, Dactinomycin (actinomycin D), Daunorubicin, Doxorubicin, Bleomycin, Plicamycin, and Mitomycin. Platinum complexes refer to platinum coordination complexes, and examples 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 substituents such as Hydroxyurea, Methyl Hydrazines such as Procarbazine Hydrochloride (trade name Natulan), vitamin A metabolites such as Tretinoin (trade name Vesanoid). Also included are Rituximab, Alemtuzumab, Trastuzumab, Bevacizumab, Cetuximab, Panitumumab, Trastuzumab, Gemtuzumab, etc.

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

Examples

[0197] Hereinafter, the present invention will be described in detail by way of examples, but the present invention is not limited thereto.

[0198] Establishment of human colorectal cancer cell lines in immunodeficient NOG mice Colorectal cancer specimens were obtained from patients who gave consent under the approval of the ethics committees of PharmaLogicals Research (Singapore) and Parkway Laboratory Services (Singapore). Tumor pieces were minced finely with scissors and transplanted into the flanks of NOG mice. Human colorectal cancer xenografts were maintained by serial passage in NOG mice provided by the Central Institute for Experimental Animals (Japan). The mice used in this experiment were treated according to the animal experiment guidelines of PharmaLogicals Research. For histopathological examination, small pieces of surgical specimens of human tissues and small pieces of xenograft tumors were fixed with 4% paraformaldehyde at 4 °C for 16 - 24 hours 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.). Sections were stained with eosin and hematoxylin and examined by microscopic observation.

[0199] Isolation and in vitro culture of colorectal CSCs A single-cell suspension of cancer cells from xenografts was prepared by mincing the tissue with a razor, incubated in DPBS containing collagenase / dispase (Roche) and DNase I (Roche) at 37 °C for 3 hours, filtered through a 40-μm cell strainer (BD Biosciences), suspended in lysis buffer (BD Biosciences) to remove red blood cells. The resulting xenograft-derived cells (such cells are called primary cells, initial cells or 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 in a 5% CO2 atmosphere (Todaro M, et al. (2007) Cell Stem Cell 1; 389-402.). To culture adherent CSCs and floating CSCs, a normal polystyrene-treated cell culture flask (BD Biosciences) and an ultra-low attachment cell culture flask (Corning) were used, respectively.

[0200] In vivo tumor formation assay A cell suspension was prepared by serial dilution. 100 μL of the cancer cell suspension in Hanks balanced salt solution (Invitrogen) was subcutaneously inoculated into the flank of mice using 50% Matrigel (BD Bioscience). Tumor development was monitored for 7 weeks. For single-cell inoculation, the cells were labeled with FITC-labeled mouse anti-human CD326 (EpCAM) antibody (Miltenyi Biotec) and seeded in a Terasaki plate (Termo Fisher Scientific). Single cells were confirmed under a fluorescence microscope. Single cells were inoculated into the flank of mice using 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 added to the N-terminus. The expression plasmids were transfected into the 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 labotratories) and HRP rat anti-mouse IgG2a mAb (Serotec). The clone that produced the most abundant sLgr5-Fc was named 2D3. The 2D3 culture supernatant was collected, and the Lgr5-Fc protein was affinity purified by a protein A-Sepharose column (Pharmacia). Lgr5-Fc served as an antigen for protein immunization and ELISA screening.

[0203] Generation of anti-Lgr5 monoclonal antibody by immunization with Lgr5-Fc protein Balb / c mice (Charles River Japan) were subcutaneously immunized with 50 μg of Lgr5-Fc emulsified in complete Freund's adjuvant. Two weeks later, the same amount in incomplete Freund's adjuvant was used to repeat the injection once a week for two weeks. Three days before cell fusion, the mice were intravenously injected with 25 μg of Lgr5Fc. Spleen lymphocytes derived from immunized mice were fused with P3-X63Ag8U1 mouse myeloma cells (ATCC) by the conventional method (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 Colorectal 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, colorectal CSCs were incubated with a mouse anti-human Lgr5 antibody (2T15E-2), followed by a PR-labeled rat anti-mouse IgG antibody (Invitrogen). Aldehyde dehydrogenase activity was measured using an AldeFluor Kit (Stemcell Technologies). Mouse cells were distinguished from human colorectal CSCs by staining with an anti-mouse MHC class I antibody (Abcam) and a 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 a Complete Mini protease inhibitor cocktail (Roche). Proteins were fractionated on a NuPAGE gel (Invitrogen) and transferred to a PVDF membrane. After blocking with PBS containing 1% skim milk, the membrane was probed with a rabbit anti-human β-catenin antibody (Sigma), a rabbit anti-human phospho-c-JUN antibody (Sigma), a rabbit anti-human TCF1 antibody (Cell Signaling), a rabbit anti-human TCF3 antibody (Cell Signaling), a rabbit anti-human TCF4 antibody (Cell Signaling), a rabbit anti-human Lgr5 antibody (Abcam), a mouse anti-human E-cadherin antibody (Abcam), a rabbit anti-human Snail antibody (Abcam), and a mouse anti-human GAPDH antibody (Santa Cruz). Reactive bands were detected using a 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 the Mx3005P Real-Time PCR System (Stratagene). The 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 Floating CSCs and adherent CSCs were plated at approximately 100 floating CSCs per well and 1×10 adherent CSCs per well, respectively. 4cells were seeded in a 96-well plate. On days 0 and 3, the number of viable cells 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 per well and 1×10 4 cells per well in a 96-well plate, incubated for 24 hours, and then 10 μg / mL of 5-FU (Hospira), 10 μg / mL of irinotecan (Hospira), 50 mM of the TCF inhibitor FH535 (Merck), and 50 mM of the β-catenin inhibitor cardamonin (Merck) were added. After culturing for 3 days in the presence of the 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 using goat anti-mouse IgG antibody or goat anti-rabbit IgG antibody labeled with AlexaFluor 488, respectively. For immunofluorescence histochemistry, sections from paraffin blocks of the above 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 with goat anti-mouse antibody conjugated with polymer-HRP (DAKO) and visualized with AlexaFluor 488-labeled tyramide (Invitrogen). Snail protein was detected with biotinylated goat anti-rabbit antibody (VECTOR) and visualized with AlexaFluor 568-labeled streptavidin (Invitrogen). These cells and specimens were also stained with DAPI (Invitrogen).

[0211] Example 1: Establishment of Colorectal 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 human colorectal cancer xenografts using NOD / Shi-scid, IL-2Rγnull (NOG) mice (Table 1; number of human colorectal cancer cell lines established in immunodeficient NOG mice).

[0212] [Table 1]

[0213] In Table 1 above, asterisks indicate those that were established but not suitable for experiments, and daggers indicate infections and the like. As shown in Table 1, 17 colorectal cancer xenografts were established from samples of 53 human colorectal cancer patients. In addition to the 17 xenografts, in 19 cases, accompanying EBV-infected lymphoma cells (which deteriorated the condition of NOG mice) occurred, in 14 cases other types of infections occurred, and in 3 cases tumor growth did not occur. Among these 17 xenografts, 11 xenografts survived even after freeze-thawing, retained the ability to reconstruct tumors, and showed histopathological features similar to the original tumors. Among these 11 xenografts, 10 were derived from grade 2 moderately differentiated adenocarcinoma, and 1 was derived from grade 3 poorly differentiated adenocarcinoma. Ten out of 11 xenografts were derived from moderately differentiated colorectal cancer (MDCC), and the remaining 1 was derived from poorly differentiated colorectal cancer (PDCC) (Table 2; histopathological classification of the original human colorectal cancer used for the establishment of 11 xenografts).

[0214] [Table 2]

[0215] Both MDCC xenografts and PDCC xenografts reconstructed almost the same histopathological morphology as the original tumors, but MDCC xenografts formed distinct epithelial tubes with goblet cells and small budding clusters (which seemed to undergo epithelial-to-mesenchymal transition (EMT)). In contrast, PDCC xenografts did not show distinct epithelial tube structures (Figure 1 and Figure 16).

[0216] [Example 2] Isolation of Colorectal CSCs The inventors used two types of MDCC xenografts, namely PLR59 and PLR123, to isolate colorectal CSCs. These xenografts were selected by the inventors because they retained the ability to rapidly proliferate and reconstruct tumors with epithelial tubes and small budding clusters even after 10 passages in NOG mice (Figure 1). Therefore, it was considered that stable CSCs could be obtained from these xenografts.

[0217] Flow cytometric analysis of primary cells derived from PLR59 and PLR123 passaged in NOG mice revealed that the levels of signals for CD44, ALDH, CD26, and Lgr5 were lower than those for CD133, EpCAM, CD166, CD24, and CD29, indicating that there was a small population of CSCs (Figure 2). In fact, when primary cells derived from PLR59 and PLR123 were subcutaneously transplanted into NOG mice at 100 cells per injection site, tumors occurred at 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 tumors in terms of having a hierarchical structure (Figure 3). However, when 10 primary cells derived from PLR59 and PLR123 were subcutaneously injected per injection site, tumors could not be formed in NOG mice (Table 3). Table 3 shows the carcinogenic activity 49 days after inoculation.

[0218]

Table 3

[0219] In Table 3 above, the asterisk indicates a tumor xenograft established in NOG mice, and the dagger indicates a cell preparation. "Primary" indicates cells (primary cells) prepared by removing red blood cells and mouse cells after collecting xenograft tumor tissues grown in NOG mice. "Suspension" indicates cells obtained by culturing primary cells in vitro under non-adherent conditions, and "adherent" indicates cells obtained by culturing in vitro under adherent conditions. The plus sign (one) indicates the number of tumors formed, and the plus sign (two) indicates the total number of inoculation sites. The parentheses indicate the percentage of tumor formation (carcinogenesis). Lgr5 + indicates Lgr5 positive, and Lgr5 - indicates Lgr5 negative.

[0220] When primary cells derived from PLR59 and PLR123 were cultured in a serum-free medium supplemented with EGF and FGF, adherent cells and suspension cells were generated. The inventors collected the adherent cells and suspension cells and cultured them separately. The adherent cells proliferated with a doubling time of approximately 2.5 days and showed a mesenchymal cell-like morphology, while the suspension cells did not show significant proliferation and formed spheroid-like cell clusters (Figs. 4, 5, 18, and 19). After culturing for more than one week and determination with colorectal CSC markers, both the adherent cells and suspension cells became highly homogeneous. The adherent cells were Lgr5 + , ALDH + , CD133 + , CD44 + , EpCAM + , CD166 + , CD24 + , CD26 + , and CD29 + . On the other hand, the suspension cells were different from the adherent cells in that they were Lgr5 - , and ALDH - (Figs. 6 and 20). A significant level of Lgr5 mRNA was detected in the adherent cells, but the Lgr5 mRNA in the suspension cells was at an undetectable level (Fig. 27).

[0221] [Example 3] Lgr5 Protein Expression Analysis To examine the expression of Lgr5 protein, the inventors prepared two types of Lgr5-specific monoclonal antibodies (2L36, 2T15E-2, and 2U2E-2) for immunohistochemical analysis and flow cytometry analysis, respectively. The inventors' antibodies were highly specific for Lgr5 and did not cross-react with Lgr4 and Lgr6, which both have high homology to Lgr5 (Figs. 28, 29). By using these antibodies, the inventors demonstrated that Lgr5 is expressed in adherent CSCs.

[0222] Lgr5-positive cells were detected through the passage of the tumor tissues that were the origin of PLR59 and PLR123 and their xenograft cancer tissues (Fig. 39). The frequency of Lgr5-positive cells in the original tumor tissues was low (0.01% for PLR59 and 0.04% for PLR123). In the xenograft cancer tissues, the frequency of Lgr5-positive cells increased with passage but did not change after 10 passages (Fig. 39). On the other hand, the tumor reconstruction ability of the primary cells from the PLR123 xenograft model also increased with passage. The estimated proportion of CSCs in the primary cells evaluated from the tumor reconstruction ability was approximately 0.1% after 5 passages, whereas it increased to approximately 0.4% at 14 passages.

[0223] 〔Example 4〕Tumor reconstruction ability of Lgr5-positive and Lgr5-negative colorectal CSCs If the characteristic of the stem cell population of colorectal cancer is Wnt signal transduction, then in vivo, only Lgr5-positive adherent cells can form tumors. To confirm whether this is true, the inventors examined the tumor-forming ability of Lgr5-positive adherent cells and Lgr5-negative floating cells.

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

[0225] Lgr5-positive cells divided symmetrically under adherent culture conditions (Figure 41). On the other hand, in the presence of Matrigel and serum, asymmetric cell division was shown by the distribution of Lgr5 protein to one of the two daughter cells in culture under the same conditions (Figures 42C and D). One characteristic of CSCs is symmetric cell division for self-renewal, and another prominent characteristic of CSCs is asymmetric cell division. Lgr5-positive adherent cells divided symmetrically under adherent culture conditions (Figure 41), while in the presence of Matrigel and FBS, it was shown that Lgr5-positive cells gave rise to two different progenies through asymmetric division, as indicated by the distribution of Lgr5 protein to one daughter cell (Figure 42).

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

[0227] [Example 5] Effects of TCF and β-catenin Consistent with the expression of Lgr5, 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, the inventors examined the effects of FH535, a β-catenin / TCF inhibitor, and cardamonin, a Wnt / β-catenin inhibitor (which induces the degradation of β-catenin), on the proliferation of colorectal CSCs.

[0229] As a result, 50 μM of FH535 significantly decreased the proliferation of Lgr5-positive colorectal CSCs but had no effect on the proliferation of Lgr5-negative colorectal CSCs (FIGS. 8 and 22). On the other hand, 50 μM of cardamonin decreased the viable cell number by up to 70% in Lgr5-positive colorectal CSCs and by approximately 50% in Lgr5-negative colorectal CSCs (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 colorectal CSCs. Interestingly, Lgr5-positive cells proliferated even in the absence of EGF and FGF supply (FIGS. 9 and 23), indicating that colorectal CSCs possess an intrinsic / autocrine mechanism for activating Wnt signaling for their proliferation.

[0231] [Example 6] Ability of colorectal CSCs to switch from an Lgr5-positive state to an Lgr5-negative state One of the characteristics of CSCs is their resistance to chemotherapeutic agents. Therefore, the inventors examined the sensitivity of colorectal CSCs to 5-FU and irinotecan. As described above, Lgr5-positive cells proliferated with a doubling time of approximately 2.5 days, while Lgr5-negative CSCs were in a quiescent state in terms of proliferation. When treated with 5-FU (10 μg / ml) and irinotecan (10 μg / ml), the proliferation of Lgr5-positive colorectal CSCs was significantly inhibited in both cases, but the proliferation and survival of Lgr5-negative colorectal CSCs were not affected (Figures 10 and 24). After exposing Lgr5-positive colorectal CSCs to 5-FU (10 μg / ml) or irinotecan (10 μg / ml) for 3 days, cells resistant to these chemotherapeutic agents appeared. Surprisingly, the drug-resistant cells were Lgr5-negative and had changed in morphology (Figures 11, 32, and 25), indicating that they had changed from the Lgr5-positive state to the Lgr5-negative state.

[0232] As specific markers for detecting these Lgr5-negative normalized CSCs, HLA-DMA, TMEM173, ZMAT3, and GPR110 were selected, and immunostaining using specific antibodies against these molecules was attempted. As a result, a specific staining pattern was obtained in colon CSCs that had been exposed to the above-mentioned irinotecan for 3 days and had become Lgr5-negative (Figure 43). It was also confirmed that this immunostaining method was applicable to tissue sections obtained from paraffin blocks, which are generally widely used (Figure 43). From the above, it is shown that HLA-DMA, TMEM173, ZMAT3, and GPR110 can be specific markers for CSCs with negative Lgr5.

[0233] The fluorescence representing Lgr5 positivity observed before irinotecan treatment (Figure 44A) disappeared upon irinotecan treatment (Figure 44B). Lgr5-positive cells emerged from Lgr5-negative cells that were reseeded and cultured in the absence of irinotecan at 4 days after reseeding (Figure 44C) and increased by 8 days after reseeding (Figure 44D). Since all of the drug-resistant cells that were Lgr5-negative were Lgr5-negative (Figure 44 and Figure 45) and remained CK20-negative (Figure 46), it was suggested that the transition of colon CSCs from an actively proliferating state to a quiescent state is associated with the disappearance of the Lgr5 molecule. The above-mentioned relevance was also confirmed by the results of the growth inhibitor resistance assay in vitro (Figure 47). Also, although ALDH activity decreased, no change was observed in other CSC markers (Figure 48).

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

[0235]

Table 4

[0236] To confirm whether Lgr5-negative colorectal CSCs change to the Lgr5-positive state, the inventors again adherently cultured Lgr5-negative colorectal CSCs prepared by irinotecan treatment in serum-free stem cell culture medium. As a result, they became Lgr5-positive, showed a mesenchymal cell-like morphology (Figs. 12 and 33), and initiated cell proliferation. On the other hand, when Lgr5-positive adherent colorectal CSCs were cultured in an ultra-low attachment plate, the inventors observed that some of the cells stopped proliferating, formed spheroid-like structures, and showed a very low level of Lgr5 mRNA (Figs. 12 and 33). The alternation between the Lgr5-positive state and the Lgr5-negative state (and vice versa) was also confirmed by observations using single cells during culture. When single Lgr5-positive cells were cultured in a multi-well plate, the cells shifted to the Lgr5-negative state within 3 days after irinotecan treatment. When single Lgr5-negative cells obtained by irinotecan treatment were cultured in a multi-well plate in the absence of irinotecan, 19 to 43% of the cells shifted to the Lgr5-positive state within 4 days (Figs. 49 and Table 5).

[0237]

Table 5

[0238] Table 5 shows the ratio of the number of Lgr5-positive and -negative cells stained as a result of immunocytostaining using an anti-Lgr5 antibody (2L36 antibody). The numbers in parentheses indicate the proportion of Lgr5-positive or -negative cells.

[0239] Therefore, the inventors concluded that colorectal CSCs alternate between the Lgr5-positive state and the Lgr5-negative state, and such changes do not require external factors and niche environments.

[0240] 〔Example 7〕EMT of Lgr5-Positive Colorectal CSCs In Vitro and In Vivo Mesenchymal-like cells expressing nuclear β-catenin are thought to be migratory CSCs and metastatic CSCs that undergo 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.). Since the morphology of Lgr5-positive colorectal CSCs resembles that of mesenchymal cells, the inventors tested whether Lgr5-positive colorectal CSCs correspond to migratory CSCs. Western blot analysis revealed the expression of low levels of cell surface E-cadherin, high levels of Snail, and nuclear-localized β-catenin in Lgr5-positive colorectal CSCs, which are characteristics of EMT (FIGS. 13, 14, and 26). In contrast, Lgr5-negative colorectal CSCs did not show any signs of EMT, that is, cell surface E-cadherin was highly expressed, Snail was lowly expressed, and nuclear localization of β-catenin was not observed. Furthermore, co-expression of Snail and Lgr5 was observed in cells undergoing EMT in the budding regions in xenograft tumor tissues (FIG. 15), which supports the view that Lgr5-positive colorectal CSCs correspond to migratory stem cells.

[0241] Furthermore, the inventors showed that Lgr5-positive colorectal CSCs form tumors in multiple tissues including the lung, liver, lymph nodes, and subcutaneous. Interestingly, tumors with epithelial duct structures were reconstituted in the liver, lymph nodes, and subcutaneous, but not in the lung, at least 40 days after intravenous injection of tumor cells (FIGS. 34, 35).

[0242] Next, it was investigated whether Lgr5-negative CSCs directly generate the cancer hierarchy or are first converted to Lgr5-positive cells in vivo. To search for markers used to detect Lgr5-negative CSCs, gene expression profiling was performed using Lgr5-positive cells, Lgr5-negative cells, and the primary cells of xenografted tumors. As a result, HLA-DMA was selected from among the molecules whose expression could be detected at a high level in Lgr5-negative CSCs compared with Lgr5-positive CSCs and primary cells (Figure 50). It was confirmed by tissue immunohistochemistry using Lgr5 antibody, HLA-DMA antibody, and EREG antibody that HLA-DMA is specifically expressed in Lgr5-negative CSCs (Figure 51). Since HLA-DMA is also expressed in macrophages, another marker expressed in CSCs was also searched to exclude the possibility that the cells stained in tissue immunohistochemistry with the HLA-DMA antibody are macrophages. By immunohistochemistry of Lgr5-positive CSCs and negative CSCs using an antibody against EREG, which is expressed in both Lgr5-positive CSCs and negative CSCs (Figure 50), it was also confirmed that EREG is expressed in both Lgr5-positive CSCs and Lgr5-negative CSCs (Figure 51). By detecting the combination of both markers, it was confirmed that Lgr5-negative CSCs can be identified as cells that are positive for both HLA-DMA and EREG. After injecting a homogeneous population of Lgr-negative CSCs into NOG mice, cells that slightly expressed Lgr5 but were still positive for HLA-DMA and EREG appeared for 1 day. By 5 days after injection, HLA-DMA-negative, Lgr5-positive, and EREG-positive cells appeared (Figure 52). Tumors derived from Lgr5-negative CSCs clearly had a tubular structure and contained Lgr5-positive cells (Figure 53).

[0243] To explore the possibility of conversion to a growth inhibitor-resistant state in vivo, irinotecan at the maximum tolerated dose (MTD) (120 mg / kg) was administered intraperitoneally to NOG mice containing tumors derived from Lgr5-positive CSCs. Tumor growth was almost completely inhibited (Figure 55), and the tubular structure was extremely disrupted (Figure 54). In this state, Lgr5-positive cells were extremely decreased (Figures 54 and 56). The number of Lgr5-negative and HLA-DMA-positive cells increased significantly after irinotecan treatment. In contrast, in control mice treated with vehicle, approximately one-third of the cancer cells in both the tubules and the budding regions were Lgr5-positive (Figure 54). Both Lgr5-positive cells and HLA-DMA-positive and Lgr5-negative cells were EREG-positive, and these were confirmed to be CSCs (Figure 54). After the completion of irinotecan treatment, Lgr5-positive cells reappeared (Figure 54). Collectively, these results indicated that Lgr5-negative CSCs are the origin of colorectal cancer after growth inhibitor treatment and reorganize 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 using stem cell medium at 3×10 5Cells were seeded in 6-well plates (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 culture was continued. After 3 days of culture, cells resistant to irinotecan were observed. These cells were collected with Accutase, suspended in FACS buffer, and then 7-AAD Viability Dye as a dead cell stain, FITC-labeled mouse mAb to human CD326 (EpCAM) as a cancer stem cell marker, 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 respectively, and reacted at 4°C for 30 minutes. For Lgr5, mouse mAb to human Lgr5 was added and reacted at 4°C for 30 minutes, then washed once with FACS buffer, and PE-labeled goat Ab to mouse IgG2a was added and reacted at 4°C for 30 minutes. Then the cells were washed once with FACS buffer and subjected to flow cytometry analysis. ALDH activity was detected using the AldeFluor Kit by performing the manufacturer's recommended operations. EPICS ALTRA was used for flow cytometry analysis, and the cancer stem cell markers were analyzed for 7-AAD Viability Dye-negative cells. Changes were observed in irinotecan-resistant cells 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 and highly proliferative cancer stem cells prepared by adherent culture of primary cells, and Lgr5-negative and low-proliferative cancer stem cells prepared by treating the cells with irinotecan by the same method as above were physically disrupted using QIAshredder (Qiagen, Cat. No. 79654), and RNA was extracted by performing the manufacturer's recommended operations using RNeasy Mini Kit (Qiagen, Cat. No. 74104) and RNase-Free DNase Set (Qiagen, Cat. No. 79254). 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 statistical analysis software R (Statistics softwere R). Three types of cells (primary cells, Lgr5-positive cells, Lgr5-negative cells) were compared with each other, and a list of genes with significantly enhanced expression was created for each. That is, the raw data of the GeneChip was standardized by GCRMA and logarithmized with base 2, and the expression differences were calculated among different sample types (three types: primary cells and Lgr5-positive cells, Lgr5-positive cells and Lgr5-negative cells, Lgr5-negative cells and primary cells). The following three types were used as criteria for extracting genes with expression variation.

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

[0247] In addition, in order to obtain genes encoding proteins specifically presented on the cell membrane in cancer stem cells, genes having GO:0005886 [plasma membrane] in Gene Ontology (GO) were extracted. Furthermore, genes having GO:0005576 [extracellular region], GO:0009986 [cell surface], GO:0016020 [membrane], or genes predicted to have transmembrane regions by the membrane protein prediction software TMHMM and genes predicted to have signal peptides by the signal peptide prediction software SignalP, and not having GO:0031090 [organelle membrane] were extracted. GeneChip data from normal colorectal tissues was also utilized, and genes with relatively high expression in normal tissues and primary cells, and genes with low change rates in Lgr5-positive and Lgr5-negative cells were excluded.

[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

[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

[0263]

Table 8-1

[0264] Table 8-2 is a continuation of Table 8-1.

Table 8-2

[0265] Furthermore, in order to obtain genes encoding proteins specifically presented on the cell membrane in cancer stem cells, genes having GO:0005886 [plasma membrane] in Gene Ontology (GO) selected according to another criterion described below were extracted (Tables 9 and 10).

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

[0267]

Table 9

[0268] Quiescent CSC-specific markers: Genes with an average expression value greater than 64 in Lgr5-negative cells, less than 64 in both primary cells and Lgr5-positive cells, more than a 20-fold change in Lgr5-negative cells compared to Lgr5-positive cells, and a significant difference recognized by 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 The NOG-established cancer cell line recovered from the mouse was 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. After that, it was washed once with FACS buffer, and 7-AAD Viability Dye (Beckman Coulter, A07704) was used as a dead cell stain, and 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) were added as secondary antibodies and reacted at 4°C for 30 minutes. Subsequently, the cells were washed once with FACS buffer and then subjected to flow cytometry analysis. For the flow cytometry analysis, EPICS ALTRA was used, and the analysis of EREG expression was performed on cells negative for 7-AAD Viability Dye and negative for mouse MHC.

[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 recovered with Accutase, suspended in FACS buffer, and then mouse mAb to human EREG was added and reacted at 4°C for 30 minutes. After that, it was washed once with FACS buffer, and 7-AAD Viability Dye was used 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 reacted at 4°C for 30 minutes. Subsequently, the cells were washed once with FACS buffer and then subjected to flow cytometry analysis. For the flow cytometry analysis, EPICS ALTRA was used, and the analysis of EREG expression was performed on cells negative for 7-AAD Viability Dye. As a result, it was shown 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 Fig. 37. Cells were stained with an antibody against EREG and analyzed by flow cytometry. In primary cells, EREG was negative and Lgr5 + Cancer stem cells and Lgr5 - In cancer stem cells, a homogeneous cell population positive for EREG was shown. Gray indicates the fluorescence intensity after staining cells with the described antibody, and white indicates the fluorescence intensity after staining cells with a control isotype antibody.

[0273] 4. In vitro drug efficacy confirmation by measuring ADCC activity 4.1. Preparation of effector cell suspension Human-derived effector cells were prepared using the mononuclear cell fraction collected from human peripheral blood. Using a syringe pre-injected with 200 μL of a 1000 unit / mL heparin solution (Novolin Heparin Injection 5000 units, Novo Nordisk), 50 mL of peripheral blood was collected from a healthy in-house volunteer (adult male). After diluting this peripheral blood 2-fold with PBS(-), it was added to a Leucosep lymphocyte separation tube (Greiner bio-one) pre-injected with Ficoll-Paque PLUS and centrifuged. After centrifugation (2150 rpm, 10 minutes, room temperature), the mononuclear cell fraction layer was collected. After washing the cells once with 10% FBS / D-MEM, the cell density was adjusted to 5 x 10 6 / mL to prepare an effector cell suspension.

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

[0275] 4.3. Measurement of ADCC activity The anti-EREG antibody was prepared at a concentration of 0.5 mg / mL and further diluted with 10% FBS / D-MEM to obtain an antibody solution. The final concentrations were 0.4 μg / mL, 4 μg / mL, and 40 μg / mL. 50 μL of the antibody solution at each concentration or 10% FBS / D-MEM was injected into each well of a 96-well U-bottom plate. Next, 50 μL of the target cell suspension was added to all wells and left standing at room temperature for 15 minutes. Subsequently, 100 μL of the effector cell suspension was added to the wells into which the antibody solution and the target cell suspension, or 10% FBS / D-MEM and the 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 another well into which 10% FBS / D-MEM and the target cell suspension were injected. The plate was centrifuged (1200 rpm, 5 minutes, room temperature) and cultured in a CO2 incubator set at a CO2 concentration of 5% and a temperature of 37 °C for 4 hours. The plate was centrifuged (1200 rpm, 5 minutes, room temperature), and 100 μL of the supernatant was collected from each well, and the fluorescence intensity (λ ex = 490 nm, λ em = 515 nm) was measured, and the specific calcein release rate: cytotoxicity (%) was determined by the following formula.

[0276] 〔Equation 1〕 cytotoxicity (%) = (A - C) x 100 / (B - C)

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

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

[0279] To confirm the expression of EREG in vivo, when Lgr5-positive cells were administered subcutaneously to NOG mice, EREG was highly expressed at the initial stage of tumor formation, but at the later stage when the tumor formed a distinct tubular structure, the expression of EREG was somewhat localized to budding clusters compared to the tubular structure. EREG-positive cells were still detected even after irinotecan was administered to the mice bearing the tumor (Fig. 54). Therefore, the antitumor activity of the EREG antibody after irinotecan treatment was investigated. Since effector cells are required for the anti-EREG antibody to exert ADCC activity, SCID mice were used as the model for evaluating the efficacy of the EREG antibody. Tumor growth was suppressed when the antibody was administered at 4 days and 11 days after the final administration of irinotecan (Fig. 57).

[0280] To evaluate the efficacy of drugs using the metastasis model, EREG expression in the metastasis model was first examined. When Lgr5-positive cells were injected intravenously into NOG mice, tumors were formed in multiple tissues including the lung. Most of the tumor cells formed in the lung showed EREG positivity (Figure 58A). Using SCID-Beige mice in which macrophages and monocytes can exert ADCC as effector cells, the efficacy of the EREG antibody was examined. In mice administered with the EREG antibody once a week for 5 times starting 3 days after the injection of Lgr5-positive cells, it was shown that the number of tumor cells at remote sites decreased extremely compared to that in control mice (Figure 58B). In addition, it was also shown that the size of each tumor decreased extremely in the mice administered with the antibody (Figures 58C and D).

[0281] Example 9. Presence of Lgr5-negative and positive CSCs in clinical tumor specimens Proliferating and quiescent CSCs were determined by tissue immunohistochemistry using an Lgr5 antibody (2U2E-2), an HLA-DMA antibody, and an EREG antibody (Figure 59 and Table 11). Proliferating CSCs represent Lgr5-positive cells, and quiescent CSCs represent HLA-DMA-positive and EREG-positive cells (Table 11). Lgr5-positive cells that were positive for both HLA-DMA and EREG, as well as Lgr5-negative cells that were positive for both HLA-DMA and EREG, were present but scarce in primary and metastatic colorectal cancer specimens isolated from colorectal cancer patients (Figure 59). Among 12 specimens of human colorectal cancer tissue, both Lgr5-positive cells and Lgr5-negative cells were detected in 8 cases, and either Lgr5-positive cells or Lgr5-negative cells were observed in the remaining 4 cases. From 0.003 to 1.864% of all specimens were Lgr5-positive cells, and 0.001 - 10.243% were Lgr5-negative cells (Table 11).

[0282] [Table 11] (P†: Indicates that proliferating or quiescent CSCs were detected. N: Indicates that proliferating or quiescent CSCs were not detected.) (Frequency: Represents the 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 restricted to specific regions but were randomly observed throughout the ducts.

[0284] [Example 10] Antitumor effects of various antibodies using Mab-ZAP and Rat-ZAP In PLR59 and PLR123 treated or untreated with irinotecan, it was evaluated whether antitumor effects could be shown by targeted therapy targeting highly expressed membrane proteins. The binding activities of the commercial antibodies listed in Table 12 to the antigens expressed on the cell surface of PLR59 or PLR123 treated or untreated with irinotecan were measured using flow cytometry (FCM). The results are summarized in Table 13.

[0285] [Table 12]

[0286] [Table 13] (NT represents no test)

[0287] The internalization activity of various antibodies confirmed to have binding activity (into cells) was evaluated using Mab-ZAP and Rat-ZAP. Mab-ZAP and Rat-ZAP are those obtained by binding saporin, a protein synthesis-inhibiting toxin, to an anti-mouse IgG antibody or an anti-rat IgG antibody (manufactured by Advanced Targeting Systems). When evaluating the internalization activity against irinotecan-untreated cells, on the day after seeding PLR59 and PLR123 cells at a cell density of 30,000 cells / 80 μL / well in each well of a 96-well plate, various antibody solutions were added to each well so as to have a final concentration of 0.01, 0.1, and 1 μg / mL. Subsequently, plates to which Mab-ZAP or Rat-ZAP was added to each well so as to have a final concentration of 1 μg / mL were cultured in a CO2 incubator at 37°C for 72 hours. When evaluating the internalization activity against cells treated with irinotecan, 96-well plates in which PLR59 and PLR123 cells and irinotecan were added to each well at a final concentration of 15 μM were cultured in a CO2 incubator at 37°C for 72 hours. The internalization activity of various antibodies against cells cultured in the presence or absence of irinotecan as described above was evaluated. At the time of the assay, the internalization activity of various antibodies against the cells present in each well replaced with a medium not containing irinotecan was evaluated in the same manner as for the irinotecan-untreated cells. After 72 hours of adding the antibody, Mab-ZAP, and Rat-ZAP, the cells present in the plates to which 3% SDS (Nacalai Tesque) was added at a dose of 10 μL / well were sufficiently lysed by stirring the plate with a plate mixer. Thereafter, the luminescence signal of the mixture in each well to which the CellTiter-Glo® Luminescent Cell Viability Assay (Promega) was added at a dose of 100 μL / well was measured. The resulting antitumor activity was shown in Table 14 and Figures 60 to 72.The percentage of cell growth inhibition indicated by the vertical axis in FIGS. 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 adding Mab-ZAP and Rat-ZAP) and the luminescence signal value of the mixture in the well without seeded cells, when the difference is taken as 100%, and the difference between the luminescence signal value of the mixture in the well to which only the test antibody was added (without adding 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. The symbols -, +, ++, and +++ in Table 14 represent the relative values 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 the well to which only the test antibody was added (without adding Mab-ZAP and Rat-ZAP) and the luminescence signal value of the mixture in the well without seeded cells, when the difference is taken as 100%, and the difference between the luminescence signal value of the mixture in the well to which only the test antibody was added (without adding 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, and -, +, ++, and +++ represent that the relative value is less than 5%, 5% or more and less than 15%, 15% or more and less than 25%, and 25% or more, respectively.

[0288]

Table 14

[0289] As shown in Fig. 66, under the conditions where sufficient antitumor activity was confirmed with the anti-EPCAM antibody used as a positive control, the anti-CD70 antibody and the anti-FAS antibody showed an internalization activity of 25% or more against irinotecan-untreated PLR59 and PLR123 (Figs. 60 and 62). Also, against PLR59, the anti-EDAR antibody showed an internalization activity of 15 - 25%, and the anti-PVRL4 antibody and the anti-PROCR antibody showed an internalization activity of 5 - 15% (Figs. 61, 63, and 65). On the other hand, against PLR59 and PLR123 treated with irinotecan, the anti-FAS antibody and the anti-TNFRSF9 antibody showed an internalization activity of 25% or more, and the anti-PROM2 antibody showed an internalization activity of 5 - 15% (Figs. 67, 70, and 68). The anti-PVRL4 antibody and the anti-PROCR antibody also both showed internalization activity against PLR59 and PLR123 treated with irinotecan, and the internalization activity against PLR59 was higher than that against PLR123 (Figs. 69 and 71). From the above results, it became clear that all the evaluated antibodies showed an antitumor effect against PLR59 and PLR123.

[0290] The effect of BMP4 on promoting differentiation of irinotecan-untreated and irinotecan-treated PLR59 and PLR123 was evaluated. When evaluating the effect of promoting differentiation on irinotecan-untreated cells, PLR59 and PLR123 cells suspended in a medium supplemented with BMP4 (R&D Systems, final concentration 20 nM) or control buffer were seeded into each well of a 12-well plate at a cell density of 5 x10 5 cells / 1.5 mL / well. Subculture and cultivation were performed by replacing the medium with the same medium 2, 4, and 7 days after seeding. When evaluating the effect of promoting differentiation on cells treated with irinotecan, 17 x10 5Cells were seeded at a cell density of cells / 5 mL / flask into 12.5 mL culture flasks with PLR59 or PLR123. On the day after seeding, irinotecan was added at a final concentration of 15 μM. The flasks were cultured in a 37°C CO2 incubator for 72 hours. Subsequently, the medium in the flasks was replaced with medium supplemented with BMP4 or control buffer, and the medium was replaced with the same medium 2, 4, and 7 days after the replacement. cDNA was synthesized using ThermoScript RT-PCR System (Invitrogen) with RNA extracted from cells isolated 4 days and 9 days after the first medium replacement as templates, 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. As shown in Figure 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, cell surface molecules specifically expressed in cancer stem cells have been identified, and by using antibodies against these molecules, new anticancer agents and reagents for detecting cancer stem cells have been provided.

Claims

1. A pharmaceutical composition which is a cancer recurrence inhibitor for Lgr5-negative cancer stem cells or a therapeutic agent for drug-resistant cancer of Lgr5-negative cancer stem cells, containing an antibody against the TNFSF9 molecule as an active ingredient, wherein a cytotoxic substance or a growth inhibitor is linked to the antibody.

2. The pharmaceutical composition according to Claim 1, wherein the cancer is a solid cancer.

3. The pharmaceutical composition according to Claim 1 or 2, wherein the cancer is a digestive tract cancer.

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

5. The pharmaceutical composition according to any one of Claims 1 to 4, wherein the antibody is a monoclonal antibody.

6. The pharmaceutical composition according to any one of Claims 1 to 5, wherein the antibody is any one of a chimeric antibody, a humanized antibody, or a human antibody.

7. The pharmaceutical composition according to any one of Claims 1 to 6, wherein the antibody is an antibody fragment.

8. The pharmaceutical composition according to any one of Claims 1 to 7, wherein the antibody is an antibody having neutralizing activity.

9. The pharmaceutical composition according to any one of Claims 1 to 8, which is characterized by being used simultaneously with a chemotherapeutic agent or after treatment with a chemotherapeutic agent.

10. A method for confirming the effectiveness of the pharmaceutical composition according to any one of Claims 1 to 9, which includes detecting the presence of the TNFSF9 molecule and / or a polynucleotide encoding the TNFSF9 molecule in a sample isolated from a subject to whom the pharmaceutical composition has been administered.

11. The method according to Claim 10, using an antibody that binds to the TNFSF9 molecule.

12. The method according to Claim 10, using a polynucleotide encoding the TNFSF9 molecule and / or a partial sequence of its complementary strand.

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