US20140302511A1 - Cancer stem cell-specific molecule - Google Patents

Cancer stem cell-specific molecule Download PDF

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Publication number
US20140302511A1
US20140302511A1 US14/354,517 US201214354517A US2014302511A1 US 20140302511 A1 US20140302511 A1 US 20140302511A1 US 201214354517 A US201214354517 A US 201214354517A US 2014302511 A1 US2014302511 A1 US 2014302511A1
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cells
cancer
antibody
lgr5
cancer stem
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Tatsumi Yamazaki
Hisafumi Okabe
Shinta Kobayashi
Takeshi Watanabe
Koichi Matsubara
Osamu Natori
Atsuhiko Kato
Masami Suzuki
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Chugai Pharmaceutical Co Ltd
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PHARMALOGICALS RESEARCH Pte Ltd
Chugai Pharmaceutical Co Ltd
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Assigned to CHUGAI SEIYAKU KABUSHIKI KAISHA reassignment CHUGAI SEIYAKU KABUSHIKI KAISHA ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: YAMAZAKI, TATSUMI, WATANABE, TAKESHI, KATO, ATSUHIKO, OKABE, HISAFUMI, SUZUKI, MASAMI, NATORI, OSAMU, KOBAYASHI, SHINTA, MATSUBARA, KOICHI
Assigned to CHUGAI SEIYAKU KABUSHIKI KAISHA reassignment CHUGAI SEIYAKU KABUSHIKI KAISHA ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: PHARMALOGICALS RESEARCH PTE. LTD.
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Definitions

  • the present invention relates to cell surface molecules specific to Lgr5-positive cancer stem cells with a high proliferative potential or Lgr5-negative cancer stem cells with a low proliferative potential; and pharmaceutical compositions that comprise as an active ingredient an antibody against such a cell surface molecule.
  • the present invention also relates to reagents for detecting cancer stem cells and methods for selecting cancer patients, which use an antibody described above.
  • CSCs Cancer stem cells
  • Non-patent Document 1 Cancer stem cells
  • Non-patent Documents 2 and 3 have been reported in several types of cancer, including acute myelocytic 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), prostatic cancer (Non-patent Documents 10 and 11), mesenchymal neoplasm (Non-patent Document 12), and melanoma (Non-patent Documents 13 and 14).
  • AML acute myelocytic leukemia
  • Non-patent Documents 2 and 3 breast cancer
  • Non-patent Document 5 glioma
  • Non-patent Document 6 head and neck cancer
  • pancreatic cancer Non-patent Documents 7 and 8
  • lung cancer Non-patent Document 9
  • prostatic cancer Non-patent Documents 10 and 11
  • Non-patent Document 16 reported that CD133 served as a CSC marker for colorectal cancer. Thereafter, different research groups have reported other markers: CD44, EpCAM, CD166 (Non-patent Document 17), and ALDH (Non-patent Documents 18 and 19). Recently, Pang et al. demonstrated that CD26 serves as a marker for a CSC subpopulation with metastatic capacity (Non-patent Document 20).
  • Non-patent Document 17 EpCAM high /CD44 + /CD166 +
  • Non-patent Document 21 CD133 + /CD44 +
  • CD44 high /ALDH + Non-patent Document 18
  • ALDH1 + /CD133 + Non-patent Document 19
  • In vitro spheroid (cell mass) cultures and direct cancer cell xenograft transplantation to immunodeficient mice have also been used to enrich CSCs (Non-patent Document 22).
  • Non-patent Document 22 there was a necessity to prepare a large number of cancer stem cells with a high purity for further understanding the properties of CSCs.
  • Non-patent Document 29 Three-dimensional spheroid cultures are often used as a CSC source. Spheroid cultures are applicable directly to tumor cells of clinically resected specimens and enable maintenance of heterogeneous CSC populations, and can have certain potential advantages compared to xenograft transplantations. Due to the heterogeneity, however, results of biochemical analyses often show complicated CSC characteristics. CSC selection using antibodies against cell surface marker proteins is commonly used to isolate CSCs, but the number and purity of cells obtained by this method is limited.
  • xenograft passages in mice only select cells viable in mice and result in elimination of cells that are hardly affected by such an environment. It goes without saying that CSCs in xenograft tumors reflect the characteristics of original CSCs, as long as they maintain the self-renewability and lineage differentiation capacity of the original tumor.
  • Lgr5 Leucine-rich repeat-containing G-protein-coupled receptor 5 (Lgr5) was originally identified as an orphan G-protein-coupled receptor of the glycoprotein hormone receptor family (Non-patent Documents 23 and 24) and was demonstrated to be a Wnt target gene whose expression is restricted to the crypt (Non-patent Document 25).
  • Non-patent Document 25 The discovery that Lgr5-positive columnar cells can regenerate all epithelial lineages (Non-patent Document 25) and that a single Lgr5-positive cell can form crypt-villus organoids in vitro without a mesenchymal niche (Non-patent Document 26), conclusively proves that Lgr5-positive cells are stem cells in the normal large intestine.
  • Non-patent Document 27 Lgr5-positive cells form adenomas in the absence of Apc
  • Non-patent Document 25 Lgr5-positive cells are an origin of colorectal cancer. It has been proven that, as in stem cells of the normal large intestine, Wnt activity is essential for in vitro and in vivo proliferation of CSCs and that exogenous HGF enhances Wnt activity (Non-patent Document 28).
  • Lgr5 was identified as a marker for normal large intestine stem cells, and has been demonstrated to serve as a marker for origins of colorectal cancer (Patent Document 1 and Non-patent Document 30). Furthermore, Lgr5 was reported to be a protein that is over-expressed in colorectal cancer stem cells (Patent Document 2). The biological role of Lgr5 in the development of large intestine cancer remains poorly understood.
  • Non-patent Document 31 Non-patent Document 31
  • the present invention was achieved in view of the circumstances described above.
  • the present invention isolates two types of cancer stem cell populations, which are substantially homogeneous and characterized with Lgr5, a cell surface marker, and identifies cell membrane molecules expressed specifically in the cancer stem cells.
  • An objective of the present invention is to provide agents for treating cancer which use antibodies to those cell membrane molecules.
  • Another objective of the present invention is to provide reagents for detecting cancer stem cells, methods for diagnosing and selecting cancer patients, which use antibodies to cell membrane molecules expressed specifically in cancer stem cells.
  • CSCs cancer stem cells
  • the present inventors established, for the first time, a method for preparing a large quantity of highly pure large intestine CSCs using an in vitro monolayer culture (also simply referred to as adherent culture) with serum-free stem cell media. Specifically, the present inventors demonstrated that a large number of large intestine CSCs can be obtained with high purity by performing an adherent culture of isolated cells derived from moderately-differentiated human colorectal cancer xenografts maintained in NOD/Shi-scid, IL-2R ⁇ null (NOG) mice.
  • NOG NOG
  • large intestine CSCs can grow, survive, and expand, and thus, the present inventors were able to obtain substantially homogeneous large intestine CSCs with high purity.
  • large intestine CSCs prepared by the above method were maintained stably without phenotypic alterations over a month or more.
  • the cells expressed various previously-reported colorectal cancer stem cell markers (CD133, CD44, EpCAM, CD166, CD24, CD26, and CD29), and exhibited tumor-initiating activity at a rate of almost 100%. Furthermore, the cells formed tumors having the same histopathological features (hierarchical organization) as the original primary tumor.
  • the cells are also characterized by being highly proliferative under adherent culture conditions and positive for Lgr5, a cell surface marker. Furthermore, the high proliferative Lgr5-positive cancer stem cells, when administered via the caudal vein to mice, formed tumor masses in organs such as lung and liver, which indicates that the cells play an essential role in cancer metastasis.
  • low proliferative Lgr5-negative cancer stem cells were isolated by treating with anti-cancer agents such as irinotecan and 5-FU cancer stem cells that are positive for cell surface marker Lgr5 and highly proliferative under adherent culture conditions. Furthermore, the isolated low proliferative Lgr5-negative cancer stem cells were revealed to be converted to high proliferative Lgr5-positive cancer stem cells by re-culturing under adherent culture conditions. This demonstrates that high proliferative Lgr5-positive cancer stem cells and low proliferative Lgr5-negative cancer stem cells are interconvertible to each other and thus have an intrinsic interchangeability.
  • Lgr5-positive large intestine CSCs when cultured under an altered culture condition or in the presence of an anti-cancer drug, were converted into the Lgr5-negative quiescent state.
  • Lgr5-negative CSCs when isolated and then cultured again under an adherent culture condition, were converted to Lgr5-positive CSCs that proliferate actively. These cells also exhibited tumor-initiating activity at a rate of almost 100%.
  • the high proliferative Lgr5-positive cancer stem cells when inoculated via the caudal vein to mice, formed tumor masses in organs such as lung and liver, which indicates that the cells play an essential role in cancer metastasis.
  • CSCs can use intrinsic means to convert into a different subset of a cell population under environmental changes such as by aggressive treatment with anti-cancer agents.
  • CSCs when exposed to stress such as anti-cancer drugs or changes in the culturing environment, convert themselves to low proliferative Lgr5-negative CSCs in order to survive avoiding the stress. Once the stress is removed, the cells can change again into high proliferative Lgr5-positive CSCs and start to proliferate. This implies that CSCs have a self-defense ability based on an intrinsic mechanism to adapt to new environments ( FIG. 36 ).
  • high proliferative Lgr5-positive and low proliferative Lgr5-negative cancer stem cells both play important roles in cancer development, formation, metastasis, recurrence, drug resistance, etc., and can be major target cells in the development of anti-cancer agents.
  • high proliferative Lgr5-positive cancer stem cells are considered to be involved in oncogenesis and metastasis while low proliferative Lgr5-negative cancer stem cells are thought to be involved in cancer recurrence.
  • the present invention provides:
  • [1] a pharmaceutical composition comprising as an active ingredient at least one antibody that binds to a protein of SEQ ID NOs: 1 to 8; [2] the pharmaceutical composition of [1], which is an anti-cancer agent; [3] the pharmaceutical composition of [2], which is an agent for inhibiting cancer recurrence; [4] the pharmaceutical composition of [2], which is an agent for inhibiting cancer metastasis or an agent for postoperative adjuvant therapy; [5] the pharmaceutical composition of [4], which is an agent for inhibiting cancer metastasis or an agent for postoperative adjuvant therapy against Lgr5-positive cancer, which comprises as an active ingredient at least one antibody that binds to a protein of SEQ ID NOs: 1 to 6; [6] the pharmaceutical composition of [2], which is a therapeutic agent against drug-resistant cancer; [7] the pharmaceutical composition of [6], which is a therapeutic agent against Lgr5-negative cancer, and which comprises as an active ingredient at least one antibody that binds to a protein of SEQ ID NOs
  • the present invention also provides:
  • [A1] a method for treating cancer, comprising administering to a subject at least one antibody that binds to a protein of SEQ ID NOs: 1 to 8; [A2] at least one antibody that binds to a protein of SEQ ID NOs: 1 to 8 for use in the treatment of cancer; [A3] use of at least one antibody that binds to a protein of SEQ ID NOs: 1 to 8 for producing an anti-cancer agent; and [A4] a process for manufacturing an anti-cancer agent, which comprises the step of using at least one antibody that binds to a protein of SEQ ID NOs: 1 to 8.
  • treatment of cancer includes inhibition of cancer recurrence, inhibition of cancer metastasis, postoperative adjuvant therapy, treatment of drug-resistant cancer, inhibition of cancer stem cell proliferation, and disruption of cancer stem cells; and anti-cancer agents include agents for inhibiting cancer recurrence, agents for inhibiting cancer metastasis, agents for postoperative adjuvant therapy, agents for treating drug-resistant cancer, agents for inhibiting cancer stem cell proliferation, and agents for disrupting cancer stem cells.
  • the present invention provides:
  • [B1] a reagent for detecting the presence of one or more of the proteins of SEQ ID NOs: 1 to 8 and/or polynucleotides encoding the proteins preferably, a reagent for detecting a cancer stem cell, a reagent for cancer diagnosis, a reagent for selecting a cancer patient, or a reagent for testing the effectiveness of the pharmaceutical compositions of [1] to [22], which contains at least one antibody that binds to a protein of SEQ ID NOs: 1 to 8, or a portion of a polynucleotide encoding the protein of SEQ ID NOs: 1 to 8 and/or the complementary strand thereof; [B2] a method for detecting a cancer stem cell, diagnosing cancer, selecting a cancer patient, or testing the effectiveness of the pharmaceutical compositions of [1] to [22], which comprises detecting the presence of one or more of the proteins of SEQ ID NOs: 1 to 8 and/or polynucleotides encoding the proteins in a sample
  • FIG. 1 shows photographs depicting histological images (HE stain) of colorectal cancer xenografts PLR59 and PLR123 derived from moderately-differentiated colorectal cancer. Even after 15 passages, cells derived from xenografts PLR59 and PLR123 formed tumors with a morphology (hierarchical organization) very similar to the original tumor, and had budding clusters (arrow) and ductal structures with goblet cells (inset).
  • HE stain histological images
  • “Original” indicates tumors obtained by surgical resection; “Early passage” indicates xenografts PLR59 and PLR123 after 4 passages in NOG mice; and “Late passage” indicates xenograft PLR59 after 15 passages and PLR123 after 19 passages in MOG mice. Scale bar represents 100 ⁇ m.
  • FIG. 2 is a diagram showing a result of flow cytometry analysis of cells from xenografts PLR59 and PLR123 passaged in NOG mice for known CSC markers. The cells were stained with antibodies against the markers indicated and then analyzed with flow cytometry. Gray area indicates the ALDH activity or fluorescence intensity of cells after staining with the indicated antibodies. White area indicates the ALDH activity in the presence of an ALDH inhibitor or the fluorescence intensity of cells after staining with isotype antibodies as a control.
  • FIG. 3 shows photographs depicting histological images (HE stain) of tumors formed by injection of 100 cells each of PLR59 and PLR123 cells.
  • the morphologies of the tumors derived from 100 cells each of PLR59 and PLR123 cells were highly similar to the original tumors. Arrows indicate budding clusters. Scale bar represents 100 ⁇ m.
  • FIG. 4 shows photographs depicting a result of phase contrast microscopic observation of non-adherent and adherent cells (PLR123 cells).
  • the cells were cultured in serum-free media supplemented with EGF and FGF.
  • the non-adherent cells closely interacted together to form a spheroid-like structure, whereas the adherent cells proliferated without forming cell clusters.
  • Scale bar represents 25 ⁇ m.
  • FIG. 5 is a diagram showing the proliferation of non-adherent and adherent CSCs (PLR123 cells).
  • the viable cell count after three days of culture (black column) is shown in percentage to the count on day 0 (white column). The results were averaged from three experiments. The bar at the top of each column represents standard deviation.
  • FIG. 6 is a diagram showing a result of flow cytometry analysis of non-adherent cells and adherent cells (PLR123 cells) for known CSC markers. Both types of cells were positive for known CSC markers such as CD133, CD44, EpCAM, CD166, CD24, CD26, and CD29, while the adherent cells alone were positive for Lgr5 and ALDH activity. Gray area indicates the ALDH activity or fluorescence intensity of cells after staining with the indicated antibodies. White area indicates the ALDH activity in the presence of an ALDH inhibitor or the fluorescence intensity of cells after staining with isotype antibodies as a control.
  • CSC markers such as CD133, CD44, EpCAM, CD166, CD24, CD26, and CD29
  • Gray area indicates the ALDH activity or fluorescence intensity of cells after staining with the indicated antibodies.
  • White area indicates the ALDH activity in the presence of an ALDH inhibitor or the fluorescence intensity of cells after staining with isotype antibodies as a control.
  • FIG. 7 is a photograph showing a result of Western blot analysis of primary cells of PLR123 cells, non-adherent CSCs, and adherent CSCs for ⁇ -catenin, TCF1, TCF3, TCF4, and phosphorylated c-JUN protein. Expression of all of the proteins was up-regulated in Lgr5-positive adherent CSCs as compared to the primary cells. GAPDH was also visualized as a reference protein for protein loading.
  • FIG. 8 is a diagram showing the inhibition of growth of Lgr5-positive adherent CSCs (PLR123 cells) by FH535 (50 ⁇ M) and Cardamonin (50 ⁇ M).
  • the viable cell count after three days of culture in the presence of FH535 (gray column) or Cardamonin (black column) is shown in percentage to the count in the presence of DMSO alone (white column). The results were averaged from three experiments. The bar at the top of each column represents standard deviation.
  • FIG. 9 is a diagram showing the proliferation of PLR123 cells in the presence or absence of EGF and FGF.
  • Adherent CSCs were cultured for three days in the presence or absence of EGF and FGF (black column). The viable cell count is shown in percentage to the count on day 0 (white column). The results were averaged from three experiments. The bar at the top of each column represents standard deviation.
  • FIG. 10 is a diagram showing the effect of chemotherapeutic agents on the proliferation of Lgr5-positive adherent CSCs and Lgr5-negative non-adherent CSCs (PLR123 cells).
  • the viable cell count after treatment with 5-FU (10 ⁇ g/ml; gray column) or irinotecan (10 ⁇ g/ml; black column) is shown in percentage to the viable cell count after culturing without the chemotherapeutic agents (white column).
  • the results were averaged from three experiments. The bar at the top of each column represents standard deviation.
  • FIG. 11 is a diagram showing a change in Lgr5 expression after treatment of adherent CSCs (PLR123 cells) with a chemotherapeutic agent.
  • This figure shows a result of flow cytometry.
  • the upper panels show the result in the absence of chemotherapeutic agent (control); the middle panels show cells treated with 5-FU; and the bottom panels show cells treated with irinotecan.
  • Gray area indicates the ALDH activity or fluorescence intensity of cells after staining with the indicated antibodies.
  • White area indicates the ALDH activity in the presence of an ALDH inhibitor or the fluorescence intensity of cells after staining with isotype antibodies as a control.
  • FIG. 12 is a diagram showing Lgr5 mRNA levels in PLR123 cells before and after switching to adherent culture or suspension culture (normalized to 1).
  • F ⁇ A represents the switching from suspension culture to adherent culture
  • a ⁇ F represents the switching from adherent culture to suspension culture. The results were averaged from three experiments. The bar at the top of each column represents standard deviation.
  • FIG. 13 shows photographs depicting a result of Western blot analysis of Lgr5-negative non-adherent CSCs and Lgr5-positive adherent CSCs (PLR123 cells) for E-cadherin and Snail.
  • Non-adherent CSCs expressed E-cadherin at a high level, while adherent CSCs expressed Snail at a high level.
  • GADPH was used as a loading control.
  • FIG. 14 shows photographs depicting a result of immunocytochemistry of Lgr5-negative non-adherent CSCs and Lgr5-positive adherent CSCs (PLR123 cells) using E-cadherin antibody, Snail antibody, and ⁇ -catenin antibody.
  • Non-adherent CSCs were epithelium-like cells expressing cell-surface E-cadherin and ⁇ -catenin at high levels, while adherent CSCs were mesenchyme-like cells with nuclear localization of Snail and ⁇ -catenin.
  • Scale bar represents 25 ⁇ m.
  • FIG. 15 shows photographs depicting a result of immunohistochemistry of PLR123-derived xenograft tissues using anti-Lgr5 antibody and anti-Snail antibody.
  • the concomitant expression of nuclear Snail and cytoplasmic Lgr5 was detected in EMT-like cells of budding areas (left panel), while such expression was not observed in the ducts (right panel).
  • Scale bar represents 10 ⁇ m.
  • FIG. 16 shows photographs depicting histopathological features of xenograft tissues. Histopathological PDCC xenografts derived from a poorly-differentiated colorectal cancer (PDCC) xenograft reconstructed almost the same histopathological morphology as the original tumor. The PDCC xenografts did not have apparent epithelial duct structures (4 and 13 passages). Scale bar represents 100 ⁇ m.
  • PDCC colorectal cancer
  • FIG. 17 shows photographs depicting a histopathological result on xenograft tumors that originated from a single or ten Lgr5-positive cells derived from PLR123, or ten Lgr5-negative cells derived from PLR123.
  • the hierarchical organization was observed in all tumors, and their histopathological features were highly similar to the original tumor.
  • Scale bar represents 100 ⁇ m.
  • FIG. 18 shows photographs depicting a result of phase contrast microscopic observation of non-adherent and adherent large intestine CSCs (PLR59 cells).
  • the cells were cultured in serum-free media supplemented with EGF and FGF.
  • the non-adherent cells closely interacted together to form a spheroid-like structure, whereas the adherent cells proliferated without forming cell clusters.
  • Scale bar represents 25 ⁇ m.
  • FIG. 19 is a diagram showing the proliferation of non-adherent and adherent CSCs (PLR59 cells).
  • the viable cell count after three days of culture (black column) is shown in percentage to the count on day 0 (white column). The results were averaged from three experiments. The bar at the top of each column represents standard deviation.
  • FIG. 20 is a diagram showing a result of flow cytometry analysis of non-adherent and adherent cells (PLR59 cells) for known CSC markers.
  • Adherent cells were positive for all markers reported, whereas non-adherent cells were negative for Lgr5 and ALDH.
  • Gray area indicates the ALDH activity or fluorescence intensity of cells after staining with the indicated antibodies.
  • White area indicates the ALDH activity in the presence of an ALDH inhibitor or the fluorescence intensity of cells after staining with isotype antibodies as a control.
  • FIG. 21 shows photographs depicting a result of Western blot analysis of primary cells of PLR59 cells, non-adherent CSCs, and adherent CSCs for ⁇ -catenin, TCF1, TCF3, TCF4, and phosphorylated c-JUN protein. Expression of all of the proteins was up-regulated in Lgr5-positive adherent CSCs as compared to the primary cells. GAPDH was also visualized as a reference for protein loading.
  • FIG. 22 is a diagram showing growth inhibition of Lgr5-positive adherent CSCs (PLR59 cells) by FH535 (50 ⁇ m) and Cardamonin (50 ⁇ m). The viable cell count after three days of culture in the presence of FH535 (gray column) or Cardamonin (black column) is shown in percentage to the count on day 0 (white column).
  • FIG. 23 is a diagram showing the proliferation of PLR59 cells in the presence or absence of EGF and FGF.
  • Adherent CSCs were cultured for three days in the presence or absence of EGF and FGF (black column). The viable cell count is shown in percentage to the count on day 0 (white column).
  • FIG. 24 is a diagram showing the effect of 5-FU (10 ⁇ g/ml) and irinotecan (10 ⁇ g/ml) on the growth of Lgr5-positive adherent CSCs and Lgr5-negative non-adherent CSCs (PLR59 cells).
  • the viable cell count after treatment with 5-FU (gray column) or irinotecan (black column) is shown in percentage to that after culturing without the agents (white column).
  • FIG. 25 is a diagram showing a result of flow cytometry analysis of adherent CSCs (PLR59 cells) for CSC markers after treatment with 5-FU or irinotecan.
  • the upper panels show cells treated with 5-FU, and the bottom panels show cells treated with irinotecan.
  • Gray area indicates the ALDH activity or fluorescence intensity of cells after staining with the indicated antibodies.
  • White area indicates the ALDH activity in the presence of an ALDH inhibitor or the fluorescence intensity of cells after staining with isotype antibodies as a control.
  • FIG. 26 shows photographs depicting a result of Western blot analysis of Lgr5-negative non-adherent CSCs and Lgr5-positive adherent CSCs (PLR59 cells) for E-cadherin and Snail.
  • Non-adherent CSCs expressed E-cadherin at a high level and adherent CSCs expressed Snail at a high level.
  • GADPH was used as a loading control.
  • FIG. 27 is a diagram showing the expression levels of Lgr5 mRNA in Lgr5-negative non-adherent CSCs and Lgr5-positive CSCs (PLR59 cells).
  • Levels of Lgr5 mRNA in adherent and non-adherent CSCs are shown as a ratio to the levels in primary cells from xenograft tumors PLR59 and PLR123.
  • the level of Lgr5 mRNA in adherent CSCs (right, white column) was remarkably increased as compared to the level in the primary cells from xenografts (left, black column), while the level was not increased in non-adherent CSCs (middle, gray column).
  • Lgr5 mRNA levels were determined by quantitative PCR and normalization with the expression of GAPDH and ACTB. All experiments were performed in triplicate. Error bars represent standard deviation.
  • FIG. 28 shows photographs depicting a result of specificity assessment of anti-human Lgr5 monoclonal antibodies (mAbs) 2U2E-2 and 2T15E-2 by immunofluorescence microscopy observation of DG44 cells transfected with Lgr4, Lgr5, or Lgr6 cDNA.
  • the transfectants and non-transfected parental cells were fixed and treated with 5 ⁇ g/ml antibodies.
  • Intense fluorescence (green signals at right) was observed in cells containing Lgr5 cDNA but not in parental cells and cells containing Lgr4 or Lgr6 cDNA.
  • Scale bar represents 5 ⁇ m.
  • FIG. 29 is a diagram showing a result of specificity assessment of anti-human Lgr5 monoclonal antibody (mAb) 2T15E-2 by flow cytometry of DG44 cells transfected with Lgr4, Lgr5, or Lgr6 cDNA.
  • the transfectants and non-transfected parental cells were incubated with monoclonal antibody 2T15E-2 and analyzed by FACS.
  • Antibody 2T15E-2 reacted with cells containing Lgr5 cDNA but not with parental cell and cells containing Lgr4 or Lgr6 cDNA.
  • the expression of Lgr4, Lgr5, and Lgr6 in the transfectants was assessed by Western blot analysis.
  • FIG. 30 is a diagram showing a result of flow cytometry analysis of adherent CSCs derived from xenografts PLR59 and PLR123.
  • the cells were cultured for one month and analyzed for known cancer stem cell markers. Even after one month of in vitro culture, adherent CSCs derived from PLR59 and PLR123 were positive for all of known cancer stem cell markers.
  • Gray area indicates the ALDH activity or fluorescence intensity of cells after staining with the indicated antibodies.
  • White area indicates the treatment of cells by the ALDH activity in the presence of an ALDH inhibitor or the fluorescence intensity of cells after staining with isotype antibodies as a control.
  • FIG. 31 After culturing for one month, adherent CSCs derived from xenografts PLR59 and PLR123 were analyzed by flow cytometry ( FIG. 30 ), and injected to NOG mice. The indicated numbers of adherent CSCs were injected subcutaneously in the lateral abdomen of NOG mice to assess the tumor-initiating activity in NOG mice.
  • This figure is a diagram showing the result of assessment of tumorigenesis 47 days after inoculation. Even subcutaneous injection of 10 adherent CSCs allowed tumor formation at all of the injection sites. The tumors were highly similar in histopathological morphology to the original tumors.
  • FIG. 32 shows photographs depicting the phenotypic interconversion of large intestine CSCs depending on the culture condition or chemotherapeutic treatment.
  • Lgr5-positive CSCs were tested for the sensitivity to 5-FU and irinotecan. Both 5-FU and irinotecan significantly inhibited the proliferation of Lgr5-positive large intestine CSCs.
  • After three days of exposure to the 5-FU or irinotecan cells resistant to the chemotherapeutic drugs arose.
  • the drug-resistant cells exhibited a dense, agglutinative morphology. Scale bar represents 25 ⁇ m.
  • FIG. 33 shows photographs depicting the morphological interconversion of CSCs.
  • Lgr5-negative large intestine CSCs were dispersed and then cultured in a flat-bottomed plate, some of the cells adhered to the plate bottom, became positive for Lgr5, and showed a mesenchymal cell-like morphology (at left).
  • Lgr5-positive adherent large intestine CSCs were cultured in an ultra-low adherent plate, some of the cells halted their growth and formed a spheroid-like structure.
  • Scale bar represents 10 ⁇ m.
  • FIG. 35 shows photographs depicting a result of histopathological experiments on tumors in the lungs, liver, lymph nodes, and subcutaneous tissues.
  • tumor cells formed undifferentiated tumor foci.
  • tumor cells formed a ductal structure involving multiple differentiation stages.
  • Scale bar represents 100 ⁇ m.
  • FIG. 36 is a schematic diagram for the proposed CSC model.
  • CSCs undergo an intrinsic interconversion between two types of independent states in response to environmental changes such as the presence of anti-cancer drugs.
  • normal stem cells expressing Lgr5 are assumed to transform into CSCs via mutation in multiple genes.
  • High proliferative CSCs express Lgr5, and undergo EMT. Under a specific stressful environment, the cells can change into the Lgr5-negative quiescent state.
  • Niche environment is involved in stimulating the transition of CSCs to the differentiation stage.
  • FIG. 37 is a diagram showing a result of flow cytometric analysis of primary cells from PLR59 and PLR123, Lgr5 + cancer stem cells, and Lgr5 ⁇ cancer stem cells with EREG.
  • FIG. 38 shows graphs depicting the ADCC activity of anti-EREG antibody against Lgr5-positive and Lgr5-negative cells derived from PLR59 and Lgr5-positive and Lgr5-negative cells derived from PLR123 cells.
  • FIG. 39 shows photographs depicting immunostaining of tumor (PLR123) obtained by surgical resection, and a PLR123-derived xenograft model (5 passages ( FIG. 39B ), 10 passages ( FIG. 39C ), and 15 passages ( FIG. 39D )) in terms of Lgr5. Tissue sections were stained with anti-Lgr5 antibody. “Original” indicates the tumor obtained from a patient by surgical resection ( FIG. 39A ). Scale bar represents 25 ⁇ m.
  • FIG. 40 shows photographs depicting a histopathological result (HE stain) on xenograft tumors derived from Lgr5-positive cells of PLR59 ( FIGS. 40A and 40B ) and PLR123 ( FIGS. 40C and 40D ).
  • NOG mice were subcutaneously injected with ten ( FIGS. 40A and 40C ) or a single ( FIGS. 40B and 40D ) Lgr5-positive cell(s) obtained from PLR59 or PLR123 by adherent culture. All tumors showed histopathological features highly similar to the original tumors. Scale bar represents 50 ⁇ m.
  • FIG. 41 shows photographs depicting symmetrical cell division of Lgr5-positive cells.
  • Lgr5-positive cells stained with PKH67 dye were cultured for 72 hours, and then observed under a fluorescent microscope.
  • FIG. 41A , B, and C show stained images of the cells for 0, 48, and 72 hours, respectively. Scale bar represents 20 ⁇ m.
  • FIG. 42 shows photographs depicting symmetrical cell division of Lgr5-positive cells in the absence of matrigel and serum ( FIGS. 42A and 42B ), and asymmetrical cell division of Lgr5-positive cells in the presence of matrigel and serum ( FIGS. 42C and 42D ).
  • FIGS. 42A and 42C show images after a single division
  • FIGS. 42B and 42D show images after second or third division.
  • FIG. 43-1 shows photographs depicting immunostained images of colon CSCs that varied to negative for Lgr5 after three days of exposure to irinotecan.
  • the cells were stained with antibodies specific to HLA-DMA (A) and TMEM173 (B).
  • FIG. 43-2 shows photographs depicting immunostained images of colon CSCs that varied to negative for Lgr5 after three days of exposure to irinotecan.
  • the cells were stained with antibodies specific to ZMAT3 (C) and GPR110 (D).
  • FIG. 44 shows photographs depicting immunostained images of irinotecan-treated Lgr5-positive CSCs (PLR123).
  • the cells were immunostained for Lgr5.
  • the immunostained images include those before irinotecan treatment ( FIG. 44A ) and after irinotecan treatment ( FIG. 44B ).
  • Scale bar represents 50 ⁇ m.
  • FIG. 45 shows graphs depicting transcript levels for the Lgr5 gene determined by quantitative real-time PCR.
  • the level of Lgr5 mRNA was high in Lgr5-positive cells prepared by adherent culture. The level was decreased under the spheroid culture condition and was almost undetectable in Lgr-negative cells after irinotecan treatment. Meanwhile, in Lgr5-positive and -negative cells prepared by adherent culture, the mRNA level for the CK20 gene was below the detection limit. The level was increased in Lgr5-positive cells of the spheroid culture condition.
  • FIG. 46 shows photographs depicting the expression of Lgr5 and CK20 proteins assessed by immunohistochemical staining.
  • Spheroid cultures of Lgr5-positive CSCs PLR59 ( FIG. 46A ) and PLR123 ( FIG. 46B ) were fixed and sliced into thin sections and then reacted with Lgr5 antibody (2L36) and CK20 antibody (DAKO).
  • the spheroids contained a small number of Lgr5-positive cells as well as a large number of CK20-positive cells that were negative for Lgr5.
  • FIG. 47 shows graphs depicting the percentage of viable cells, relative to non-treated control cells, of Lgr5-positive (black line) and -negative (gray line) CSCs (PLR59 and PLR123) cultured for three days in the absence or presence of irinotecan or 5-FU at each concentration indicated on the horizontal axis. Lgr5-negative cells were fully resistant to both growth inhibitors.
  • FIG. 48 is a diagram showing the expression of CSC markers.
  • Before treatment indicates the expression of CSC markers in Lgr5-positive cells prepared by an adherent culture, which are derived from a PLR123 xenograft model.
  • After irinotecan treatment indicates the expression of CSC markers in Lgr5-negative cells prepared via irinotecan treatment.
  • Re-inoculation after irinotecan removal indicates the expression of CSC markers in Lgr5-negative cells re-inoculated to an irinotecan-free medium.
  • Gray area indicates the ALDH activity or fluorescence intensity of cells after staining with the indicated antibodies.
  • White area indicates the ALDH activity in the presence of an ALDH inhibitor.
  • FIG. 49 is a diagram showing interconversion between Lgr5-positive and -negative cells.
  • Lgr5-positive cells were collected by FACS. After limiting dilution, the cells were inoculated and cultured for three days in the presence of irinotecan under adherent culture conditions. On the other hand, irinotecan-treated Lgr5-negative cells were diluted by limiting dilution, and then inoculated and cultured for four days in the absence of irinotecan under adherent culture conditions. Lgr5 expression was visualized with PE-labeled anti-mouse IgG antibody (indicated in red) or AlexaFluo 488-labeled anti-mouse IgG antibody (indicated in green).
  • FIG. 50 shows diagrams depicting heat maps for (A) seven genes the expression of which was significantly up-regulated in Lgr5-negative cells as compared to Lgr5-positive cells and for (B) 20 genes the expression levels of which were elevated in Lgr5-positive and Lgr5-negative cells as compared to primary cells derived from xenograft animals.
  • RNAs were prepared from Lgr5-positive and -negative CSCs derived from PLR59 and PLR123, and primary cells isolated from xenograft animals. RNAs were analyzed using Affymetrix U133.
  • FIG. 51 shows photographs depicting the binding of anti-HLA-DMA antibody and anti-EREG antibody to Lgr5-positive and Lgr5-negative CSCs with immunohistochemical staining.
  • CSCs PLR123
  • anti-HLA-DMA antibody Dako
  • anti-EREG antibody EP27
  • Intense fluorescence signals red for both HLA-DMA and EREG were observed on Lgr5-negative cells treated with anti-HLA-DMA antibody, whereas weak fluorescence (green) or no fluorescence was detected on Lgr5-positive cells. Fluorescence signals were detected on both Lgr5-negative and -positive cells treated with anti-EREG antibody.
  • FIG. 52 shows photographs depicting the transition from Lgr5-negative CSCs to Lgr5-positive CSCs at an early stage of tumor formation.
  • NOG mice injected with PLR123 xenograft animal-derived Lgr5-negative CSCs tumors derived from Lgr5-negative CSCs were stained with antibodies against Lgr5 (green), HLA-DMA (red), and EREG (green).
  • Lgr5-weakly-expressing, HLA-DMA-positive, EREG-positive cells, and Lgr5-positive, HLA-DMA-negative, EREG-positive cells were observed to be present on day 5.
  • Scale bar represents 10 ⁇ m.
  • FIG. 53 shows photographs depicting the reconstitution of tumor hierarchy from Lgr5-negative CSCs.
  • the tissue structure FIG. 53A
  • FIG. 53B An image obtained by immunofluorescence microscopic observation using anti-Lgr5 antibody and anti-E-cadherin antibody
  • Green and red indicate the presence of Lgr5 and E-cadherin, respectively.
  • Scale bar represents 50 ⁇ m.
  • FIG. 54 shows photographs depicting histopathology (HE) (first row), immunostaining using Lgr5 antibody (green) and E-cadherin antibody (red) (second row), immunostaining using Lgr5 antibody (green) and HLA-DMA antibody (red) (third row), and immunostaining using EREG antibody (red) (fourth row), of tumors after irinotecan treatment.
  • Mice bearing tumors derived from Lgr5-positive CSCs (PLR123) were treated with irinotecan, and their tumors were observed.
  • Irinotecan or vehicle was administered to mice at days 12, 15, and 18 after tumor grafting. Scale bar represents 25 ⁇ m.
  • FIG. 55 Irinotecan was administered at a dose of 120 mg/kg/day to NOG mice at days 12, 15, and 18 after grafting tumors derived from Lgr5-positive CSCs (PLR123).
  • This figure is a graph showing tumor volumes in control mice administered with vehicle (closed diamond) and mice administered with irinotecan (closed square or triangle). Each value represents mean+standard deviation.
  • FIG. 57 is a graph showing the anti-tumor effect of EREG antibody after irinotecan treatment.
  • PLR123-derived Lgr5-positive cells were injected to the peritoneal cavities of SCID mice, and irinotecan was administered at a dose of 100 mg/kg/day at days 6, 10, and 13 after injection of Lgr5-positive cells.
  • FIG. 58 shows photographs and diagrams depicting the anti-tumor effect of EREG antibody.
  • the first row shows histological staining (HE) of tumors from xenograft models prepared by intravenously injecting PLR123-derived Lgr5-positive cells to NOG mice, and the second row shows immunohistochemical staining using EREG antibody.
  • Arrows in the panels indicate EREG-expressing cells at nodules (second row) and corresponding HE stain images (first row).
  • FIGS. 58B and 58C show the number of tumors formed in the lungs of SCID-beige mice intravenously injected with Lgr5-positive cells derived from PLR123 xenograft models.
  • EREG antibody In the EREG antibody administration group, EREG antibody was administered once a week for five times from three days after injection of Lgr5-positive cells.
  • Each symbol (circle) indicates the number of tumor nodules in an animal in a group tested ( FIG. 58B ).
  • the number of tumor nodules categorized by size in the antibody administration group and control group are shown in FIG. 58C .
  • White columns indicate tumors that are smaller than 100 ⁇ m; gray columns indicate tumors of 100 to 200 ⁇ m; and black columns indicate tumors larger than 200 ⁇ m ( FIG. 58C ).
  • Tissue staining (HE stain) of tumors in the EREG antibody administration and non-administration groups is shown in FIG. 58D .
  • Scale bar represents 200 ⁇ m.
  • FIG. 59 shows photographs depicting the same tissue sections from primary and liver metastatic colorectal cancers isolated from patients, which were stained with HE (second and fourth rows), and antibodies against Lgr5 (green), HLA-DMA (red), and EREG (green) (first and third rows).
  • Positivity for Lgr5 indicates proliferating CSCs, while positivity for EREG and HLA-DMA implies Lgr5-negative quiescent CSCs.
  • Both Lgr5-negative and -positive CSCs were detected in ductal structures and budding areas of primary and liver metastatic tumors.
  • Lgr5-positive CSCs were also found as single cells in stromal regions. The same staining patterns were also observed in multiple tumor tissues isolated from different patients. Arrows indicate CSCs. Scale bar represents 10 ⁇ m.
  • FIG. 60 is a diagram showing the anti-tumor effect of anti-CD70 antibody on irinotecan non-treated CSCs.
  • the horizontal and vertical axes indicate the antibody concentration and % inhibition of cell growth by anti-CD70 antibody, respectively.
  • FIG. 61 is a diagram showing the anti-tumor effect of anti-EDAR antibody on irinotecan non-treated CSCs.
  • the horizontal and vertical axes indicate the antibody concentration and % inhibition of cell growth by anti-EDAR antibody, respectively.
  • FIG. 62 is a diagram showing the anti-tumor effect of anti-FAS antibody on irinotecan non-treated CSCs.
  • the horizontal and vertical axes indicate the antibody concentration and % inhibition of cell growth by anti-FAS antibody, respectively.
  • FIG. 63 is a diagram showing the anti-tumor effect of anti-PVRL4 antibody on irinotecan non-treated CSCs.
  • the horizontal and vertical axes indicate the antibody concentration and % inhibition of cell growth by anti-PVRL4 antibody, respectively.
  • FIG. 64 is a diagram showing the anti-tumor effect of anti-TNFSF9 antibody on irinotecan non-treated CSCs.
  • the horizontal and vertical axes indicate the antibody concentration and % inhibition of cell growth by anti-TNFSF9 antibody, respectively.
  • FIG. 65 is a diagram showing the anti-tumor effect of anti-PROCR antibody on irinotecan non-treated CSCs.
  • the horizontal and vertical axes indicate the antibody concentration and % inhibition of cell growth by anti-PROCR antibody, respectively.
  • FIG. 66 is a diagram showing the anti-tumor effect of anti-EPCAM antibody on irinotecan non-treated CSCs.
  • the horizontal and vertical axes indicate the antibody concentration and % inhibition of cell growth by anti-EPCAM antibody, respectively.
  • FIG. 67 is a diagram showing the anti-tumor effect of anti-FAS antibody on irinotecan treated CSCs.
  • the horizontal and vertical axes indicate the antibody concentration and % inhibition of cell growth by anti-FAS antibody, respectively.
  • FIG. 68 is a diagram showing the anti-tumor effect of anti-PROM2 antibody on irinotecan treated CSCs.
  • the horizontal and vertical axes indicate the antibody concentration and % inhibition of cell growth by anti-PROM2 antibody, respectively.
  • FIG. 69 is a diagram showing the anti-tumor effect of anti-PVRL4 antibody on irinotecan treated CSCs.
  • the horizontal and vertical axes indicate the antibody concentration and % inhibition of cell growth by anti-PVRL4 antibody, respectively.
  • FIG. 70 is a diagram showing the anti-tumor effect of anti-TNFSF9 antibody on irinotecan treated CSCs.
  • the horizontal and vertical axes indicate the antibody concentration and % inhibition of cell growth by anti-TNFSF9 antibody, respectively.
  • FIG. 71 is a diagram showing the anti-tumor effect of anti-PROCR antibody on irinotecan treated CSCs.
  • the horizontal and vertical axes indicate the antibody concentration and % inhibition of cell growth by anti-PROCR antibody, respectively.
  • FIG. 72 is a diagram showing the anti-tumor effect of anti-EPCAM antibody on irinotecan treated CSCs.
  • the horizontal and vertical axes indicate the antibody concentration and % inhibition of cell growth by anti-EPCAM antibody, respectively.
  • FIG. 73 is a diagram showing the expression levels of differentiation marker CK20 in CSCs cultured in the presence of BMP4.
  • FIGS. 73A and 73B show the expression levels of CK20 in PLR59 and PLR123, respectively.
  • the present invention relates to cell surface molecules that are specifically expressed on cancer stem cells, and pharmaceutical compositions (anti-cancer agents, etc.) and reagents for detecting cancer stem cells, which use antibodies against the cell surface molecules.
  • cancer refers to the physiological condition in mammals, which is typically characterized by unregulated cell growth, or such a physiological condition.
  • cancer types are not particularly limited, and include those listed below.
  • Carcinomas epistatic cancers
  • breast cancer skin cancer
  • cancers of the digestive tract lung cancer, hepatocellular carcinoma, cervical cancer, uterine cancer, ovary cancer, fallopian tube cancer, vaginal cancer, liver cancer, bile duct cancer, bladder cancer, ureter cancer, thyroid cancer, adrenal cancer, kidney cancer, and cancers of other glandular tissues.
  • Sarcomas include 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, for example, melanoma and brain tumor (Kumar V, Abbas A K, 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).
  • tumor refers to arbitrary benign (non-cancerous) and malignant (cancerous) tissue masses, including pre-cancerous lesions, which result from overgrowth or overexpansion of cells.
  • cancer stem cell refers to cells having the abilities described in (i) and/or (ii) below.
  • the ability to self-renew refers to the ability of either or both of the divided daughter cells to produce cells which maintain the same capacity and the degree of differentiation as the parental cell in terms of cell lineage.
  • the ability to differentiate into various types of cancer cells that constitute a cancer cell mass Like normal stem cells, various types of cancer cells differentiated from cancer stem cells generate a hierarchical organization with cancer stem cells at the top in terms of cell lineage. Various types of cancer cells are generated in a sequential manner from cancer stem cells. This results in the formation of a cancer cell mass that exhibits a variety of features.
  • Cancer stem cell refers to a cancer cell that has the ability to form cancers as well as, like normal stem cell, pluripotency and self-renewal ability. Cancer stem cells generate a hierarchical organization with cancer stem cells at the top. Various types of cancer cells are generated in a sequential manner from cancer stem cells. This results in the formation of a cancer cell mass that exhibits a variety of features. Cancer cell mass refers to, not a group of individual cells, but a mass formed by the adhesion of cells etc. as in human tumor tissue, which is built with cancer cells, and other cells such as stromal cells and blood cells, extracellular matrix such as collagen and laminin, and so on.
  • cancer stem cells which are the target in therapy using pharmaceutical compositions of the present invention, is not particularly limited; it is possible to use cancer stem cells derived from mammals such as humans, monkeys, chimpanzees, dogs, bovines, pigs, rabbits, rats, and mice. However, cancer stem cells derived from humans are preferred, and those derived from human tumor tissues are more preferred.
  • Cancer stem cells to be detected using the present invention are preferably those which reconstitute the hierarchical structure of cancer tissues.
  • cancer cell lines are prepared by grafting cancer tissues from which the detected cancer stem cells have been collected, into, preferably, nonhuman animals, and passaging them in such animals, and one can test whether the established cancer cell lines reconstitute the hierarchical structure of the cancer tissues.
  • One can test whether the hierarchical structure of cancer tissues is reproduced by NOG-established cancer cell lines prepared by grafting and passaging cancer tissues in nonhuman animals, more preferably immunodeficient animals, and still more preferably NOG mice which lack functional T cells, B cells, and natural killer cells.
  • cancer stem cells to be detected using the present invention can be a spheroid (cell mass) formed by spheroid culture.
  • Spheroid culture means that cancer stem cells are inoculated in a culture vessel such as non-adherent or low-adherent cell culture flasks, plates, or dishes using a medium capable of culturing cancer stem cells, and then the cells are cultured under a three-dimensionally floating condition. A cell mass formed by this method is called “spheroid”.
  • NOG-established cancer cell lines can be generated by a method known to those skilled in the art, for example, the method described in Fujii E. et al., Pathol int. 2008; 58: 559-567.
  • Human colorectal cancer, stomach cancer, lung cancer, breast cancer, pancreatic cancer, or the like is resected surgically. After mechanically mincing it with scissors, the cancer is grafted subcutaneously in NOG mice and passaged to establish cell lines. Even after passages, NOG-established cancer cell lines maintain the properties of the original human cancer tissues.
  • cancer stem cells can be selected by using cell markers.
  • Cell markers used in the present invention include, for example, leucine-rich repeat-containing G-protein-coupled receptor 5 (Lgr5), CD133, CD44, EpCAM, CD166, CD24, CD26, and CD29.
  • the present invention relates to molecules expressed in cancer stem cells that are positive for the expression of the cell marker Lgr5, and are adherent and highly proliferative under serum-free culture conditions.
  • cancer stem cells are also referred to as “high proliferative Lgr5-positive cancer stem cells”.
  • the present invention also relates to molecules expressed in cancer stem cells that are negative for the expression of the cell marker Lgr5, and non-adherent and poorly proliferative under serum-free culture conditions.
  • cancer stem cells are also referred to as “low proliferative Lgr5-negative cancer stem cells”.
  • Any culture media or liquids can be used to culture cancer stem cells of the present invention as long as they are serum-free media and capable of culturing cancer stem cells.
  • the culture media or liquids There is no particular limitation on the culture media or liquids.
  • the concentration of EGF is not particularly limited; however, it ranges from 0.1 to 100 ng/ml, preferably from 0.5 to 50 ng/ml, and more preferably from 1 to 20 ng/ml.
  • the concentration of bFGF is not particularly limited; however, it ranges from 0.1 to 100 ng/ml, preferably from 0.5 to 50 ng/ml, and more preferably from 1 to 20 ng/ml.
  • the concentration of hLIF is not particularly limited; however, it ranges from 0.1 to 100 ng/ml, preferably from 0.5 to 50 ng/ml, and more preferably from 1 to 20 ng/ml.
  • the concentration of HGF is not particularly limited; however, it ranges from 0.1 to 100 ng/ml, preferably from 1 to 50 ng/ml.
  • the concentration of NGF is not particularly limited; however, it ranges from 0.1 to 100 ng/ml, preferably from 1 to 50 ng/ml.
  • the concentration of NSF-1 is not particularly limited; however, it ranges from 0.1 to 100 ng/ml, preferably from 1 to 50 ng/ml.
  • the concentration of TGF ⁇ is not particularly limited; however, it ranges from 0.1 to 100 ng/ml, preferably from 1 to 50 ng/ml.
  • the concentration of TNF ⁇ is not particularly limited; however, it ranges from 0.1 to 100 ng/ml, preferably from 1 to 50 ng/ml.
  • the concentration of heparin is not particularly limited; however, it ranges from 10 ng/ml to 10 ⁇ g/ml, preferably from 2 to 5 ⁇ g/ml.
  • the concentration of BSA is not particularly limited; however, it ranges from 0.1 to 10 mg/ml, preferably from 1 to 8 mg/ml.
  • the concentration of insulin is not particularly limited; however, it ranges from 1 to 100 ⁇ g/ml, preferably from 10 to 50 ⁇ g/ml.
  • the concentration of transferrin is not particularly limited; however, it ranges from 10 to 500 ⁇ g/ml, preferably from 50 to 200 ⁇ g/ml.
  • the concentration of putrescine is not particularly limited; however, it ranges from 1 to 50 ⁇ g/ml, preferably from 10 to 20 ⁇ g/ml.
  • the concentration of selenite is not particularly limited; however, it ranges from 1 to 50 nM, preferably from 20 to 40 nM.
  • the concentration of progesterone is not particularly limited; however, it ranges from 1 to 50 nM, preferably from 10 to 30 nM.
  • the concentration of hydrocortisone is not particularly limited; however, it ranges from 10 ng/ml to 10 ⁇ g/ml, preferably from 100 ng/ml to 1 ⁇ g/ml.
  • the concentration of D-(+)-glucose is not particularly limited; however, it ranges from 1 to 20 mg/ml, preferably from 5 to 10 mg/ml.
  • the concentration of sodium bicarbonate is not particularly limited; however, it ranges from 0.1 to 5 mg/ml, preferably from 0.5 to 2 mg/ml.
  • the concentration of HEPES is not particularly limited; however, it ranges from 0.1 to 50 mM, preferably from 1 to 20 mM.
  • the concentration of L-glutamine is not particularly limited; however, it ranges from 0.1 to 10 mM, preferably from 1 to 5 mM.
  • the concentration of N-acetylcysteine is not particularly limited; however, it ranges from 1 to 200 ⁇ g/ml, preferably from 10 to 100 ⁇ g/ml.
  • Known basal culture liquids which are not particularly limited as long as they are suitable for culturing cancer cells from which cancer stem cells are derived, include, for example, DMEM/F 12, DMEM, F10, F12, IMDM, EMEM, RPMI-1640, MEM, BME, Mocoy's 5A, and MCDB131. Of them, DMEM/F12 is preferred.
  • the most preferred stem cell media include DMEM/F12 medium supplemented with 20 ng/ml human EGF, 10 ng/ml human bFGF, 4 ⁇ g/ml heparin, 4 mg/ml BSA, 25 ⁇ g/ml human insulin, and 2.9 mg/ml glucose where each concentration a final concentration.
  • high proliferative Lgr5-positive cancer stem cells have the properties of mesenchymal cells.
  • low proliferative Lgr5-negative cancer stem cells have the properties of epithelial cells.
  • epithelial cells refers to cells that constitute epithelial tissues in the living body.
  • the origin of cancer stem cells is not particularly limited; however, they are preferably derived from a solid cancer, more preferably a gastrointestinal cancer.
  • Gastrointestinal cancers include, for example, esophageal cancer, stomach cancer, duodenal cancer, pancreatic cancer, bile duct cancer, gallbladder cancer, biliary tract cancer, colorectal cancer, colon cancer, and rectal cancer.
  • a preferred gastrointestinal cancer is colorectal cancer.
  • cancer stem cells are preferably positive for one or more of the cell markers CD133, CD44, EpCAM, CD166, CD24, CD26, and CD29, more preferably positive for CD133, CD44, EpCAM, CD166, CD24, CD26, and CD29.
  • acetaldehyde dehydrogenase (ALDH) activity can be used as a cell marker.
  • ALDH acetaldehyde dehydrogenase
  • Lgr5-positive adherent cancer stem cells are positive for the ALDH activity cell marker, whereas Lgr5-negative cancer stem cells are negative for ALDH activity.
  • one or more of HLA-DMA, TMEM173, ZMAT3, and GPR110 can also be used as cell markers.
  • Lgr5-positive adherent cancer stem cells are negative for any of the cell markers HLA-DMA, TMEM173, ZMAT3, and GPR110, while Lgr5-negative cancer stem cells are positive for any of the cell markers HLA-DMA, TMEM173, ZMAT3, and GPR110.
  • cancer stem cells preferably have the feature of reconstituting the hierarchical structure of cancer tissues.
  • hierarchical structure means that some of the unique and characteristic structures observed in a normal tissue are detected histopathologically in the structure of a tumor originated from the tissue.
  • highly-differentiated cancers reconstitute the hierarchical structure to a high degree.
  • lumen formation and mucous cells are observed in the case of tumors of glandular lumen-forming organs (stomach cancer, colorectal cancer, pancreatic cancer, liver cancer, bile duct cancer, breast cancer, lung adenocarcinoma, prostatic cancer, etc.).
  • cancer stem cells preferably have the ability of epithelial-mesenchymal transition (EMT).
  • EMT epithelial-mesenchymal transition
  • the ability of epithelial-mesenchymal transition means both that epithelial cells transition into mesenchymal cells by obtaining their characteristics, and that mesenchymal cells transition into epithelial cells by obtaining their characteristics. EMT does not occur in normal cells except during the process of embryogenesis. Epithelial cells, which are bound together tightly and exhibit polarity, change into mesenchymal cells that are bound together more loosely, exhibit a loss of polarity, and have the ability to move.
  • mesenchymal cells can spread into tissues around the primary tumor, and also separate from the tumor, invade blood and lymph vessels, and move to new locations where they divide and form additional tumors. Drug resistance, metastasis, or recurrence of cancer can be explained by such additional tumor formation.
  • the present invention provides pharmaceutical compositions comprising as an active ingredient an antibody that binds to a molecule expressed in a substantially homogeneous cancer stem cell population comprising the above cancer stem cells of the present invention.
  • substantially homogeneous means that, when immunodeficient animals are grafted with 1000 cells, 100 cells, or 10 cells and analyzed for the frequency of formation of cancer cell populations using Extreme Limiting Dilution Analysis (Hu Y & Smyth G K., J Immunol Methods. 2009 Aug. 15; 347(1-2): 70-8) utilizing, for example, the method described in Hu Y & Smyth G K., J Immunol Methods. 2009 Aug.
  • the frequency of cancer stem cells is 1/20 or more, preferably 1/10 or more, more preferably 1/5 or more, even more preferably 1/3 or more, still more preferably 1/2 or more, and yet more preferably 1/1.
  • cancer stem cell populations can be prepared, for example, by culturing cells or a group of cells containing the cancer stem cells described herein.
  • adherent culture means that, after seeding cells into culture vessels for adherent culture, the adhered cells are cultured and passaged while non-adherent cells are removed. The cells grown to confluency are detached with Accutase and passaged into fresh adherent culture flasks, adherent culture plates, or adherent culture dishes for further culture. Culture vessels for adherent culture are not particularly limited as long as they are used for adherent culture.
  • flasks for adherent culture or highly adherent flasks it is possible to appropriately select and use flasks for adherent culture or highly adherent flasks, plates for adherent culture or highly adherent plates, flat-bottomed plates for adherent culture or highly adherent flat-bottomed plates, dishes for adherent culture or highly adherent dishes, etc.
  • Media used for adherent culture are not particularly limited; however, it is preferable to use serum-free stem cell culture media.
  • adherent refers to the property of cells to adhere to culture vessels for adherent culture when they are cultured in the vessels.
  • “suspension culture” means that, after seeding cells into culture vessels for suspension culture, the floating cells are cultured and passaged while adherent cells are removed. The cells grown to confluency are passaged into fresh low adherent cell culture flasks, ultra low adherent cell culture flasks, low adherent plates, ultra low adherent plates, low adherent dishes, or ultra low adherent dishes for further culture.
  • Culture vessels for suspension culture are not particularly limited as long as they are used for suspension culture. It is possible to appropriately select and use low adherent cell culture flasks, ultra low adherent cell culture flasks, low adherent plates, ultra low adherent plates, low adherent dishes, ultra low adherent dishes, etc.
  • Non-adherent refers to the property of cells to be cultured in a floating state without adherence to culture vessels for suspension culture when the cells are cultured in the vessels.
  • “Expansion of a cell group” means, for example, proliferation by spheroid culture or grafting and passaging in nonhuman animals, but is not particularly limited thereto.
  • immunodeficient animals can be used for grafting since they are unlikely to have rejection reactions.
  • Immunodeficient animals preferably used include nonhuman animals that lack functional T cells, for example, nude mice and nude rats, and nonhuman animals that lack both functional T and B cells, for example, SCID mice and NOD-SCID mice. It is more preferably to use mice that lack T, B, and NK cells and have excellent transplantability, including, for example, NOG mice.
  • NOG mice can be prepared, for example, by the method described in WO 2002/043477, and are available from the Central Institute for Experimental Animals or the Jackson Laboratory (NSG mice).
  • Cells to be grafted may be any cells, including cell masses, tissue fragments, individually dispersed cells, cells cultured after isolation, and cells isolated from a different animal into which the cells have been grafted; however, dispersed cells are preferred.
  • the number of grafted cells may be 10 6 or less; however, it is acceptable to graft more cells.
  • the grafting site is not particularly limited, and it is preferable to select an appropriate grafting site depending on the animal used. There is no particular limitation on the grafting operation of NOG-established cancer cell lines, and the cells can be grafted by conventional grafting operations.
  • Cancer stem cells or a cancer stem cell population can be prepared, for example, by collecting cancer tissues from patients and culturing the tissues in a serum-free stem cell culture medium under adherent or floating culture conditions.
  • cancer tissues collected from patients can be spheroid-cultured, and then cultured in a serum-free stem cell culture medium under adherent or floating culture conditions to prepare cancer stem cells or a cancer stem cell population
  • cancer tissues collected from patients can be grafted and passaged in nonhuman animals, and then cultured in a serum-free stem cell culture medium under adherent or floating culture conditions to prepare cancer stem cells or a cancer stem cell population.
  • Cancer stem cells and cancer stem cell populations of the present invention can be used in methods of screening for pharmaceutical agents, anti-cancer agents, or the like.
  • the present invention provides methods comprising the steps of:
  • a substantially homogeneous cancer stem cell population containing Lgr5-positive adherent cancer stem cells or Lgr5-negative cancer stem cells is prepared. Then, a test substance is contacted with the prepared cancer stem cell population or cancer stem cells contained in the cancer stem cell population.
  • a test substance there is no particular limitation on the method for contacting a test substance with a cancer stem cell population or cancer stem cells contained in the cancer stem cell population.
  • a test substance may be contacted with cultured cells of a cancer stem cell population or cancer stem cells contained in the cancer stem cell population. This treatment can be carried out by adding a test substance to a cell culture medium or cell extract.
  • a test substance is a protein
  • this treatment can be performed, for example, as follows: a vector comprising a DNA encoding the protein is introduced into a cancer stem cell population or cancer stem cells contained in the cancer stem cell population; or the vector is added to a cell extract of a cancer stem cell population or cancer stem cells contained in the cancer stem cell population.
  • a vector comprising a DNA encoding the protein is introduced into a cancer stem cell population or cancer stem cells contained in the cancer stem cell population; or the vector is added to a cell extract of a cancer stem cell population or cancer stem cells contained in the cancer stem cell population.
  • a change in a biological property of the cancer stem cell population or cancer stem cells treated with the test substance is detected.
  • a change in a biological property includes, for example, a change in the proliferation ability, a change in the viable cell count, a change in a tissue structure characteristic of the process of cancer progression of the cancer stem cell population or cancer stem cells, and a change in the expression of a DNA, RNA, protein, or metabolite in the cancer stem cell population or cancer stem cells.
  • a change in a biological property can be detected, for example, by the methods described below.
  • RNAs include microRNAs, siRNAs, tRNAs, snRNAs, mRNAs, and non-coding RNAs.
  • mRNAs of a gene are extracted according to a conventional method. Using the mRNAs as a template, the transcriptional level of the gene can be determined using the Northern hybridization or RT-PCR method. DNA array techniques can also be used to determine the expression level of the gene. Alternatively, fractions containing a protein encoded by a gene are collected according to a conventional method.
  • the translational level of the gene can be determined by detecting the protein expression by an electrophoresis method such as SDS-PAGE.
  • the translational level of a gene can also be determined by performing the Western blotting method using an antibody against a protein and detecting the protein expression. These methods can be used to screen for pharmaceutical agents (pharmaceutical compositions).
  • the DNAs, RNAs, and proteins that are contained in a cancer stem cell population or cancer stem cells and which are characteristic of the process of cancer progression of the cancer stem cell population or cancer stem cells preferably include the proteins or polypeptides of any one of SEQ ID NOs: 1 to 6, and polynucleotides encoding the proteins or polypeptides.
  • test substance when there is no change in a biological property of a cancer stem cell population or cancer stem cells, or the degree of the change is reduced after treatment with a test substance compared to before the treatment, the test substance is expected to be useful as a pharmaceutical agent (pharmaceutical composition) that has the activity of suppressing cancer recurrence or metastasis (for example, an agent for suppressing cancer recurrence, an agent for post-chemotherapy adjuvant therapy, an agent for postoperative adjuvant therapy, an anti-cancer agent, or an agent for suppressing cancer metastasis).
  • a pharmaceutical agent pharmaceutical composition
  • Such test substances can be selected as effective substances that have the therapeutic or preventive effect against cancerous diseases.
  • Such pharmaceutical agents (pharmaceutical compositions) having the activity of suppressing cancer progression are used as an agent for suppressing cancer recurrence, an agent for post-chemotherapy adjuvant therapy, an agent for postoperative adjuvant therapy, an anti-cancer agent, or an agent for suppressing cancer metastasis.
  • Anti-cancer agents of the present invention may be used against, for example, cancers resistant to pharmaceutical agents or chemotherapeutic agents.
  • pharmaceutical agents (pharmaceutical compositions) of the present invention also include therapeutic agents against drug-resistant or chemotherapeutic agent-resistant cancers.
  • the above pharmaceutical agents are not particularly limited to anti-cancer agents or agents for suppressing metastasis or recurrence, and they can also be used as an agent for inhibiting angiogenesis or cell growth.
  • the pharmaceutical agents (pharmaceutical compositions) of the present invention may be used simultaneously with chemotherapeutic agents or after treatment with chemotherapeutic agents.
  • the pharmaceutical agents are not particularly limited, and they include proteinaceous agents, nucleic acid agents, low-molecular-weight agents, and cellular agents.
  • the present invention provides methods of screening for pharmaceutical agents (pharmaceutical compositions), which comprise the steps of:
  • a substantially homogeneous cancer stem cell population containing Lgr5-negative non-adherent cancer stem cells is prepared. Then, the prepared cancer stem cell population or cancer stem cells contained in the cancer stem cell population are treated with a test substance. Next, a change in a biological property of the cancer stem cell population or cancer stem cells treated with the test substance is detected.
  • the DNAs, RNAs, and proteins that are contained in such a cancer stem cell population or cancer stem cells, and which are characteristic of the process of cancer progression of the cancer stem cell population or cancer stem cells preferably include the proteins or polypeptides of any one of SEQ ID NOs: 1 to 8, and polynucleotides encoding the proteins or polypeptides.
  • the proteins or polypeptides of SEQ ID NOs: 1 to 6, and polynucleotides encoding the proteins or polypeptides may be used.
  • the protein or polypeptide of SEQ ID NO: 7 or 8, and polynucleotides encoding the protein or polypeptide may be used.
  • Pharmaceutical agents that are obtained by the screening methods are not particularly limited, and they can be used as anti-cancer agents. Specifically, when there is no change in a biological property of a cancer stem cell population or cancer stem cells, or the degree of the change is reduced after treatment with a test substance compared to before the treatment, the test substance is expected to be useful as a pharmaceutical agent that has the activity of suppressing cancer recurrence or metastasis (for example, an agent for suppressing cancer recurrence, an agent for post-chemotherapy adjuvant therapy, an agent for postoperative adjuvant therapy, an anti-cancer agent, or an agent for suppressing cancer metastasis).
  • Such test substances may be selected as effective substances that have the therapeutic or preventive effect against cancerous diseases.
  • Such pharmaceutical agents having the activity of suppressing cancer progression are used as an agent for suppressing cancer recurrence, an agent for post-chemotherapy adjuvant therapy, an agent for postoperative adjuvant therapy, an anti-cancer agent, or an agent for suppressing cancer metastasis.
  • pharmaceutical agents include cancer therapeutic agents against Lgr5-negative cancers, which contain as an active ingredient at least an antibody that binds to a protein of SEQ ID NOs: 1 to 8.
  • the Lgr5-negative cancers include those resistant to drugs or chemotherapeutic agents.
  • Still another embodiment of the screening methods of the present invention includes methods that use nonhuman animals administered with a test substance, and a cancer stem cell population of the present invention or cancer stem cells contained in the cancer stem cell population.
  • the present invention provides methods of screening for pharmaceutical agents (pharmaceutical compositions), which comprise the steps of:
  • a substantially homogeneous cancer stem cell population containing Lgr5-positive adherent cancer stem cells is prepared. Then, nonhuman animals are administered with a test substance, and the cancer stem cell population prepared or cancer stem cells contained in the cancer stem cell population.
  • the method for administering a test substance to nonhuman animals is not particularly limited.
  • Oral administration, or parenteral administration such as subcutaneous, intravenous, local, transdermal, or transintestinal (transrectal) administration can be appropriately selected depending on the type of a test substance to be administered.
  • the method for administering a cancer stem cell population or cancer stem cells to nonhuman animals there is no particular limitation on the method for administering a cancer stem cell population or cancer stem cells to nonhuman animals, and an appropriate method can be selected depending on the cell population to be administered.
  • the preferred method is subcutaneous or intravenous administration.
  • tumor formation is detected in the nonhuman animals.
  • test substance can be performed as follows: tissues administered with a test substance and a cancer stem cell population or cancer stem cells are excised from nonhuman animals, and then histological features of the tissues are observed to determine the presence or absence of tumor formation.
  • the test substance is expected to be useful as a pharmaceutical agent having the activity of suppressing cancer progression or metastasis (for example, an anti-cancer agent or an agent for suppressing cancer metastasis or recurrence), and the test substance can be selected as an effective substance that has the therapeutic or preventive effect against cancerous diseases.
  • pharmaceutical agents (pharmaceutical compositions) obtained by the screening methods are not particularly limited, and can be used as an anti-cancer agent, or an agent for suppressing cancer metastasis or recurrence.
  • Test substances used in the methods of the present invention are not particularly limited, and include, for example, single compounds such as natural compounds, organic compounds, inorganic compounds, proteins, antibodies, peptides, and amino acids, as well as compound libraries, expression products of gene libraries, cell extracts, cell culture supernatants, products of fermenting microorganisms, extracts of marine organisms, plant extracts, prokaryotic cell extracts, unicellular eukaryote extracts, and animal cell extracts. These may be purified products or crude purified products such as extracts of plants, animals, and microorganisms. Also, methods for producing test substances are not particularly limited; test substances may be isolated from natural materials, synthesized chemically or biochemically, or prepared by genetic engineering.
  • RNAi molecules that are designed by known methods based on partial sequences of polynucleotides encoding the protein of any one of SEQ ID NOs: 1 to 8. If needed, the above test substances can be appropriately labeled and used. Labels include, for example, radiolabels and fluorescent labels. Mixtures of an above-mentioned test substance and multiple kinds of such labels are included in the test substances of the present invention.
  • the present invention provides pharmaceutical agents such as vaccines comprising a partial peptide of the protein of any one of SEQ ID NOs: 1 to 8, and methods of screening for vaccines.
  • screening methods preferably include methods for determining the cytotoxic activity targeted to cancer stem cells disclosed in the present application using cytotoxic T cells (CTL) or the like induced with a cancer vaccine of the present invention in vitro.
  • CTL cytotoxic T cells
  • adherent and non-adherent cells are separated from peripheral blood mononuclear cells (PBMCs) collected by centrifugation of human peripheral blood in Ficoll-Conray density gradient.
  • PBMCs peripheral blood mononuclear cells
  • the adherent cells are incubated with 100 ng/ml GM-CSF (Novartis) and 10 IU/ml IL-4 (GIBCO-BRL) in AIM-V (GIBCO), and then the cells are used as antigen-presenting cells (APC). Meanwhile, the non-adherent cells are incubated with 30 to 100 IU/ml recombinant IL-4 (Ajinomoto) in AIM-V.
  • a partial peptide of the protein of any one of SEQ ID NOs: 1 to 8 provided by the present invention is added (at a final concentration of 30 ⁇ g/ml) to APC.
  • TNF- ⁇ and IFN- ⁇ are added for APC maturation.
  • CD8-positive cells isolated from autologous non-adherent cells are mixed with irradiated APC in IL-2-free AIM-V.
  • IL-2 Takeda Pharmaceutical Company
  • IL-2 is added at a final concentration 100 IU/ml to the culture.
  • the CD8-positive cells are stimulated every seven days using, as APC, autologous PHA blasts (PHA-stimulated T cells) that have been stimulated with the T cell mitogen PHA.
  • a fresh medium containing 100 IU/ml IL-2 is added to the culture at every time point of stimulation.
  • CTL on day 28 is used for the activity assay.
  • High proliferative Lgr5-positive cancer stem cells and low proliferative Lgr5-negative cancer stem cells that are provided by the present invention can be used as target cells of CTL.
  • the cytotoxic activity can be assessed by determining 51 Cr-sodium chromate uptake activity by a measurement method similar to that of ADCC activity.
  • pharmaceutical agents selected by the screening methods of the present invention may be further screened as necessary for more effective and practical preventive or therapeutic active substances by conducting additional drug effectiveness tests and safety tests, and further conducting clinical tests in human cancer patients. Based on results of structural analysis of pharmaceutical agents thus selected, they can be industrially manufactured by chemical synthesis, biochemical synthesis (fermentation), or genetic engineering.
  • “High proliferative ability” means that the doubling time is 6 days or less, preferably 4 days or less, and more preferably 3 days or less when cells are cultured in a serum-free medium supplemented with EGF and FGF using the method described herein.
  • Low proliferative ability means that the doubling time is 7 days or more, preferably 14 days or more, and more preferably there is no significant proliferation when cells are cultured in a serum-free medium supplemented with EGF and FGF using the method described herein.
  • the cells can be separated using the cell marker Lgr5.
  • the separation methods include the following: methods in which a cell population containing cancer stem cells is isolated by using an anti-Lgr5 antibody;
  • a substantially homogeneous cancer stem cell population is first prepared by culturing a population containing cancer stem cells under adherent or suspension culture conditions, and then the population is isolated by using an anti-Lgr5 antibody; and methods in which a substantially homogeneous cancer stem cell population is first prepared by culturing a population containing cancer stem cells in a medium with or without a growth inhibitor under adherent culture conditions, and then the population is isolated by using an anti-Lgr5 antibody.
  • Any of the above methods may be used in the present invention.
  • cells are isolated from cancer tissues after three or more passages in NOG mice, and cultured in a serum-free stem cell culture media under adherent culture conditions to prepare high proliferative Lgr5-positive cancer stem cells.
  • low proliferative Lgr5-negative cancer stem cells can be prepared as follows.
  • the resulting Lgr5-positive cancer stem cells are maintained under various stresses such as a contact with a growth inhibitor, for example, treatment with irinotecan (culture for three days in a serum-free stem cell medium supplemented with 10 ⁇ g/ml irinotecan).
  • the present invention provides methods of screening for pharmaceutical agents, which comprise contacting a test substance with cancer stem cells that differ in the proliferation ability, which are induced by the methods provided by the present invention.
  • the present invention provides methods of screening for pharmaceutical agents, which comprises detecting a change in a biological property of cancer stem cells by contacting a test substance with high or low proliferative cancer stem cells induced by a method for converting low proliferative cancer stem cells to high proliferative cancer stem cells, or converting high proliferative cancer stem cells to low proliferative cancer stem cells.
  • low proliferative cancer stem cells can be prepared by maintaining high proliferative cancer stem cells under various stresses such as a suspension culture or in contact with a growth inhibitor.
  • high proliferative cancer stem cells can be converted to low proliferative cancer stem cells by culturing high proliferative cancer stem cells under suspension culture conditions.
  • high proliferative cancer stem cells can be converted to low proliferative cancer stem cells by culturing high proliferative cancer stem cells in low adherent or ultra low adherent cell culture vessels such as low adherent plates, ultra low adherent plates, low adherent dishes, ultra low adherent dishes, low adherent flasks, or ultra low adherent cell culture flasks.
  • low proliferative cancer stem cells can be prepared by culturing high proliferative cancer stem cells in low adherent or ultra low adherent cell culture vessels such as low adherent plates, ultra low adherent plates, low adherent dishes, ultra low adherent dishes, low adherent flasks, or ultra low adherent cell culture flasks.
  • high proliferative cancer stem cells can be converted to low proliferative cancer stem cells by using a growth inhibitor such as 5-FU or irinotecan.
  • a growth inhibitor such as 5-FU or irinotecan.
  • low proliferative cancer stem cells can be produced by exposing high proliferative cancer stem cells to a growth inhibitor such as 5-FU or irinotecan. Exposure to a growth inhibitor can be achieved under any condition such as in vitro culture or inside the body of grafted nonhuman animals. In this case, those skilled in the art can select an appropriate exposure dose of a cell growth inhibitor for cancer stem cells.
  • high proliferative cancer stem cells can be prepared by re-seeding low proliferative cancer stem cells into a medium without a growth inhibitor such as 5-FU or irinotecan.
  • high proliferative cancer stem cells can be produced by discontinuing administration of a growth inhibitor to nonhuman animals having low proliferative cancer stem cells.
  • low proliferative cancer stem cells can be cultured under adherent culture conditions to convert them to high proliferative cancer stem cells.
  • low proliferative cancer stem cells can be converted to high proliferative cancer stem cells by culturing low proliferative cancer stem cells in a non-low-adherent but highly adherent cell culture vessel such as a flat-bottomed plate, plate, adherent culture plate, adherent culture flask, dish, or adherent culture dish.
  • high proliferative cancer stem cells can be produced by culturing low proliferative cancer stem cells in a non-low-adherent but highly adherent cell culture vessel such as a flat-bottomed plate, plate, adherent culture plate, adherent culture flask, dish, or adherent culture dish.
  • a non-low-adherent but highly adherent cell culture vessel such as a flat-bottomed plate, plate, adherent culture plate, adherent culture flask, dish, or adherent culture dish.
  • the present invention also relates to cancer cell detection reagents.
  • Cancer cell detection reagents of the present invention preferably contain as an active ingredient at least one antibody that binds to a protein of SEQ ID NOs: 1 to 8 (protein composed of the amino acid sequence of any one of SEQ ID NOs: 1 to 8).
  • the reagents of the present invention include reagents for detecting Lgr5-positive cancer cells, which preferably contain at least one antibody that binds to a protein of SEQ ID NOs: 1 to 6 (protein composed of the amino acid sequence of any one of SEQ ID NOs: 1 to 6).
  • the reagents of the present invention include reagents for detecting Lgr5-negative cancer cells, which preferably contain at least one antibody that binds to the protein of any one of SEQ ID NOs: 1 to 8 (protein composed of the amino acid sequence of any one of SEQ ID NOs: 1 to 8).
  • preferred growth inhibitors include DNA-damaging agents, antimitotic agents, and/or anti-metabolites.
  • a DNA-damaging agent may be an alkylating reagent, a topoisomerase inhibitor, and/or a DNA intercalator.
  • preferred growth inhibitors include, but are not limited to, carboplatin (DNA alkylating reagent), etoposide (topoisomerase II inhibitor), doxorubicin (DNA intercalator), docetaxel (antimitotic agent), and Gemzar (gemcitabine; anti-metabolite).
  • Alkylating reagents can be at least one reagent selected from the following. Specifically, it is possible to use at least one alkylating reagent selected from: chlorambucil, cyclophosphamide, ifosfamide, mechlorethamine, melphalan, uracil mustard, thiotepa, busulfan, carmustine, lomustine, streptozocin, carboplatin, cisplatin, satraplatin, oxaliplatin, altretamine, ET-743, XL119 (becatecarin), dacarbazine, chlormethine, bendamustine, trofosfamide, uramustine, fotemustine, nimustine, prednimustine, ranimustine, semustine, nedaplatin, triplatin tetranitrate, mannosulfan, treosulfan, temozolomide, carboquone, triaziquone, tri
  • Topoisomerase inhibitors can be at least one inhibitor selected from the following. Specifically, it is possible to use at least one topoisomerase inhibitor selected from: doxorubicin (Doxil), daunorubicin, epirubicin, idarubicin, anthracenedione (Novantrone), mitoxantrone, mitomycin C, bleomycin, dactinomycin, plicatomycin, irinotecan (Camptosar), camptothecin, rubitecan, belotecan, etoposide, teniposide, topotecan (Hycamptin), etc.
  • doxorubicin Doxil
  • daunorubicin epirubicin
  • idarubicin anthracenedione
  • Novantrone novantrone
  • mitoxantrone mitoxantrone
  • mitomycin C bleomycin
  • dactinomycin plicatomycin
  • At least one topoisomerase inhibitor selected from the following can be used as a DNA intercalator: proflavin, doxorubicin (adriamycin), daunorubicin, dactinomycin, thalidomide, etc.
  • Antimitotic agents can be at least one agent selected from the following. Specifically, it is possible to use at least one topoisomerase inhibitor selected from: paclitaxel (Abraxane)/Taxol, docetaxel (Taxotere), BMS-275183, Xyotax, Tocosal, vinorlebine, vincristine, vinblastine, vindesine, vinzolidine, etoposide (VP-16), teniposide (VM-26), ixavepilone, larotaxel, ortataxel, tesetaxel, ispinesib, etc.
  • topoisomerase inhibitor selected from: paclitaxel (Abraxane)/Taxol, docetaxel (Taxotere), BMS-275183, Xyotax, Tocosal, vinorlebine, vincristine, vinblastine, vindesine, vinzolidine, etoposide (VP-16), teniposide (VM-
  • Anti-metabolites can be at least one inhibitor selected from the following. Specifically, it is possible to use at least one topoisomerase inhibitor selected from: fluorouracil (5-FU), floxuridine (5-FUdR), methotrexate, Xeloda, Arranon, leucovorin, hydroxyurea, thioguanine (6-TG), mercaptopurine (6-MP), cytarabine, pentostatin, fludarabine phosphate, cladribine (2-CDA), asparaginase, gemcitabine, pemetrexed, bortezomib, aminopterin, raltitrexed, clofarabine, enocitabine, sapacitabine, azacytidine, etc.
  • fluorouracil 5-FU
  • floxuridine 5-FUdR
  • methotrexate Xeloda
  • Arranon leucovorin
  • hydroxyurea thioguanine
  • the present invention relates to methods of screening for anti-cancer drugs, which use cancer stem cells isolated or induced by the above methods of the present invention.
  • the present invention also relates to methods for assessing compounds, which use cancer stem cells isolated or induced by the above methods of the present invention.
  • the present invention provides methods for detecting, identifying, or quantifying the presence of cancer stem cells of the present invention. Specifically, the present invention provides methods for detecting, identifying, or quantifying the presence of cancer stem cells or substantially homogeneous cancer stem cell populations of the present invention, which comprise the steps of:
  • samples obtained from cancer patients are prepared.
  • a “sample” is not particularly limited as long as it is preferably an organ or tissue derived from a cancer patient. It is possible to use a frozen or unfrozen organ or tissue. Such samples include, for example, cancer (tumor) tissues isolated from cancer patients. In these methods, a sample is then contacted with an anti-Lgr5 antibody.
  • Methods for detecting, identifying, or quantifying the presence of above-described cancer stem cells or substantially homogeneous cancer stem cell populations of the present invention can be used in, for example, cancer diagnosis, selection of cancer patients, prediction or assessment of the effectiveness of an agent (pharmaceutical composition), treatment monitoring, and cancer imaging.
  • organs or tissues are isolated from cancer patients, and specimens are prepared.
  • the specimens can be used to detect, identify, or quantify the presence of cancer stem cells.
  • Specimens can be appropriately prepared by using known methods, for example, the PFA-AMeX-Paraffin method (WO 09/078,386).
  • the samples include, for example, frozen or unfrozen organs or tissues.
  • PFA solution refers to a cell fixation solution which is an aqueous solution of 1 to 6% paraformaldehyde combined with a buffer such as phosphate buffer. It is preferable to use 4% PFA fixation solution (4% paraformaldehyde/0.01 M PBS (pH7.4)).
  • organs or tissues of interest are immersed in a PFA solution containing 1 to 6%, preferably 4% paraformaldehyde, at 0 to 8° C., preferably at about 4° C., for 2 to 40 hours, preferably for 6 to 30 hours. Then, fixed organs or tissues are washed with phosphate buffered saline or such. Washing may be carried out after excising portions from the observed organs or tissues.
  • the AMeX method is a paraffin embedding method with a series of the following steps: cold acetone fixation, dehydration with acetone, clearing in methylbenzoate and xylene, and paraffin embedding. Specifically, tissues are immersed in acetone at ⁇ 25 to 8° C., preferably at ⁇ 20 to 6° C., for 2 to 24 hours, preferably for 4 to 16 hours. Then, the tissues in acetone are warmed to room temperature. Alternatively, organs or tissues are transferred into acetone at room temperature. Then, dehydration is performed for 0.5 to 5 hours, preferably 1 to 4 hours at room temperature.
  • the organs or tissues are cleared by immersion in methylbenzoate at room temperature for 0.5 to 3 hours, preferably for 0.5 to 2 hours, followed by immersion in xylene at room temperature for 0.5 to 3 hours, preferably 0.5 to 2 hours.
  • the organs or tissues are embedded in paraffin by penetration at 55 to 65° C., preferably at 58 to 62° C. for 1 to 4 hours, preferably for 1 to 3 hours.
  • the paraffin blocks of organs or tissues prepared by the PFA-AMeX method are stored at low temperature before use.
  • the paraffin blocks thus prepared are sliced into thin sections using a microtome or the like. Then, the thin sections are deparaffinized and rehydrated. Deparaffinization and rehydration can be performed by known methods. For example, deparaffinization can be performed using xylene and toluene, while rehydration can be carried out using alcohol and acetone.
  • the resulting thin sections are stained, for example, by histochemistry, immunohistochemistry, or enzyme histochemistry for detection, identification, or quantitation.
  • the prepared samples are stained by histochemistry (special staining), it is possible to use any staining method commonly available for paraffin-embedded sections (for example, PAS staining, giemsa staining, and toluidine blue staining).
  • the sections may be stained by any staining method available for sections (for example, various staining such as with ALP, ACP, TRAP, or esterase).
  • histopathological tissues can be stained by the following: hematoxylin-eosin staining for general staining; van Gieson staining, azan staining, and Masson Trichrome staining for collagen fiber staining; Weigert staining and Elastica van Gieson staining for elastic fiber staining; Watanabe's silver impregnation staining and PAM staining (periodic acid methenamine silver stain) for reticular fibers/basal membrane staining, etc.
  • Staining with immunohistochemistry and enzyme histochemistry can be performed by direct methods using primary antibodies labeled with an enzyme or labeling substance, or indirect methods using non-labeled primary antibodies and labeled secondary antibodies. However, such methods are not limited thereto.
  • Antibodies can be labeled by conventional methods. Labeling substances include, for example, radioisotopes, enzymes, fluorescent substances, and biotin/avidin. The labeling substances may be those commercially available. Radioisotopes include, for example, 32 P, 33 P, 131 I, 125 I, 3 H, 14 C, and 35 S.
  • Enzymes include, for example, alkaline phosphatase, horse radish peroxidase, ⁇ -galactosidase, and ⁇ -glucosidase.
  • Fluorescent substances include, for example, fluorescein isothiocyanate (FITC) and rhodamine. These may be commercially available. Labeling can be carried out by known methods.
  • Thin sections are stained, for example, by histochemistry, immunohistochemistry, or enzyme histochemistry for detection, identification, or quantitation.
  • RNAs include microRNAs, siRNAs, tRNAs, snRNAs, mRNAs, and non-coding RNAs.
  • Lgr5 mRNA is extracted according to conventional methods. Using the mRNA as a template, the transcriptional level of each gene can be determined by the Northern hybridization or RT-PCR method. DNA array techniques can also be used to determine the expression level of Lgr5.
  • Desired tissues, cells, or such can be collected from samples by the microdissection method, in particular, laser microdissection (LMD) method.
  • LMD laser microdissection
  • the LMD method can collect a group of target cells from living tissues, and thus accurately determine which cells express a specific gene among various cells that constitute a tissue, and at what level the cells express the gene.
  • Devices used for microdissection include, for example, the AS-LMD system (Leica Microsystems).
  • the present invention provides methods for diagnosing cancer, detecting cancer stem cells, or selecting cancer patients, which comprise using at least one antibody that binds to a protein of SEQ ID NOs: 1 to 8 to detect the presence of at least one of the proteins in a sample isolated from a cancer patient.
  • the present invention provides methods for diagnosing cancer, detecting cancer stem cells, or selecting cancer patients, which comprise using at least one antibody that binds to a protein of SEQ ID NOs: 1 to 6 to detect the presence of at least one of the proteins in a sample isolated from a cancer patient.
  • at least one antibody that binds to a protein of SEQ ID NOs: 1 to 6 it is possible to use, instead of the anti-Lgr5 antibody described above, at least one antibody that binds to a protein of SEQ ID NOs: 1 to 6.
  • the presence of Lgr5-positive cancer stem cells can be detected by detecting the presence of the protein.
  • the present invention does not exclude detection of Lgr5 in addition to the protein.
  • the present invention provides methods for diagnosing cancer, detecting cancer stem cells, or selecting cancer patients, which comprise using at least one antibody that binds to a protein of SEQ ID NOs: 1 to 8 to detect the presence of at least one of the proteins in a sample isolated from a cancer patient.
  • at least one antibody that binds to a protein of SEQ ID NOs: 1 to 8 it is possible to use, instead of the anti-Lgr5 antibody described above, at least one antibody that binds to a protein of SEQ ID NOs: 1 to 8.
  • the presence of Lgr5-negative cancer stem cells can be detected by detecting the presence of the protein.
  • the present invention does not exclude detection of Lgr5 in addition to the protein.
  • the present invention provides methods for assessing the effectiveness of a pharmaceutical composition comprising at least one antibody that binds to a protein of SEQ ID NOs: 1 to 8, which comprise detecting one or more of the proteins of SEQ ID NOs: 1 to 8 and/or polynucleotides encoding the proteins in a sample isolated from a subject administered with the pharmaceutical composition.
  • detection may be carried out using at least one antibody that binds to a protein of SEQ ID NOs: 1 to 8, or a portion of a polynucleotide encoding the protein of any one of SEQ ID NOs: 1 to 8 and/or a complementary strand thereof.
  • the present invention provides methods for assessing the effectiveness of a cancer treatment in a test subject, which comprise comparing the expression of at least one of the proteins of SEQ ID NOs: 1 to 8 and/or polynucleotides encoding the proteins in a first sample obtained from a test subject before providing the subject with at least part of the treatment, to the expression of at least one of the proteins of SEQ ID NOs: 1 to 8 and/or polynucleotides encoding the proteins in a second sample obtained from the subject after providing the part of the treatment, wherein a significantly lower level of the protein and/or polynucleotide in the second sample than in the first sample is an indicator showing that the treatment is effective for inhibiting cancer in the test subject.
  • the present invention provides methods of monitoring in a test subject the effectiveness of a treatment with an antibody provided by the present invention, which comprise the steps of:
  • an increased dosage of an antibody of the present invention can be used to reduce the expression or activity of a marker towards a level higher than the detected level (the expression or activity level of the marker protein, mRNA, or genomic DNA in the sample before administration), i.e., to increase the effectiveness of the antibody.
  • the present invention provides methods of monitoring in a test subject the effectiveness of a treatment with an antibody provided by the present invention, which comprise the steps of:
  • an increased dosage of an antibody of the present invention can be used to reduce the expression or activity of a marker towards a level higher than the detected level (the expression or activity level of the marker protein, mRNA, or genomic DNA in the sample before administration), i.e., to increase the effectiveness of the antibody.
  • the present invention provides methods of monitoring in a test subject the effectiveness of a treatment with an antibody provided by the present invention, which comprise the steps of:
  • an increased dosage of an antibody of the present invention can be used to reduce the expression or activity of a marker towards a level higher than the detected level (the expression or activity level of the marker protein, mRNA, or genomic DNA in the sample before administration), i.e., to increase the effectiveness of the antibody.
  • Cancer stem cell inhibitor refers to, for example, an agent having the effect of suppressing the proliferation of cancer stem cells, suppressing the metastasis or recurrence of cancer stem cells, killing cancer stem cells, etc. It may have the effect of suppressing the proliferation of cancer cells, suppressing the metastasis or recurrence of cancer cells, killing cancer cells, etc.
  • the terms “suppress” and “suppressing”, and synonymous expressions refer to the down-regulation of the biological activity. This can reduce or eliminate a target function such as protein production and phosphorylation of a molecule, etc. In a specific embodiment, the suppression means a decrease in a target activity by about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%.
  • the terms refer to the prevention of development of a symptom, relief of a symptom, or successful alleviation of a disease, condition, or disorder.
  • Metalastasis refers to a process where cancer spreads or moves from the primary site to another site in the body, resulting in the development of similar cancer lesions at the new site.
  • Metalstatic cell or “metastasizing cell” refers to a cell that loses the adhesive contact with adjacent cells, and leaves the primary site of the disease and invades a nearby body structure via blood or lymphatic circulation.
  • Recurrence means that the same malignant tumor reappears in a remaining organ after partial resection of an organ for removing a malignant tumor from a cancer patient, or after postoperative chemotherapy following the resection.
  • Proteins for use in the present invention can be easily prepared by any method known to those skilled in the art as follows.
  • An expression vector containing a gene comprising a DNA encoding a protein is constructed.
  • the protein is produced and accumulated by culturing transformants transformed with the expression vector.
  • the transformants are harvested to prepare the protein.
  • Such an expression vector can be constructed according to methods known in the art, for example, by the following:
  • Such vectors used include E. coli -derived plasmids (for example, pBR322, pBR325, pUC18, and pUC118), Bacillus subtilis -derived plasmids (for example, pUB110, pTP5, and pC194), yeast-derived plasmids (for example, pSH19 and pSH15), bacteriophages such as ⁇ phage, and animal viruses such as retroviruses, vaccinia viruses, and Baculoviruses.
  • E. coli -derived plasmids for example, pBR322, pBR325, pUC18, and pUC118
  • Bacillus subtilis -derived plasmids for example, pUB110, pTP5, and pC194
  • yeast-derived plasmids for example, pSH19 and pSH15
  • bacteriophages such as ⁇ phage
  • animal viruses such as retroviruses, vaccini
  • Promoters for use in the present invention may be any promoters as long as they are appropriate and compatible with a host to be used for gene expression.
  • preferred promoters include the trp promoter, lac promoter, recA promoter, ⁇ PL promoter, and lpp promoter.
  • preferred promoters include the SPO1 promoter, SPO2 promoter, and penP promoter.
  • preferred promoters include the PHOS promoter, PGK promoter, GAP promoter, and ADH promoter.
  • promoters include the SR ⁇ promoter, SV40 promoter, LTR promoter, CMV promoter, and HSV-TK promoter.
  • a protein for use in the present invention can be expressed as a fusion protein with another protein (for example, glutathione-S-transferase or Protein A). Such a fusion protein can be cleaved into individual proteins by using an appropriate protease.
  • Host cells include, for example, bacteria of the genus Escherichia , bacteria of the genus Bacillus , yeasts, insect cells, insects, and animal cells.
  • bacteria of the genus Escherichia include Escherichia coli K12 DH1 (Proc. Natl. Acad. Sci, USA, 60, 160 (1968)), JM103 (Nucleic Acids Research, 9, 309 (1981)), JA221 (Journal of Molecular Biology, 120, 517 (1978)), and HB101 (Journal of Molecular Biology, 41, 459 (1969)).
  • Bacteria of the genus Bacillus include, for example, Bacillus subtilis MI114 (Gene, 24, 255 (1983)) and 207-21 (Journal of Biochemistry, 95, 87 (1984)).
  • Yeasts include, for example, Saccharomyces cerevisiae AH22, AH22R-, NA87-11A, DKD-5D, and 20B-12; Schizosaccaromyces pombe NCYC1913 and NCYC2036; and Pichia pastoris.
  • Animal cells include, for example, monkey COS-7 cells, Vero cells, Chinese hamster CHO cells (hereinafter abbreviated as CHO cells), dhfr gene-deficient CHO cells, mouse L cells, mouse AtT-20 cells, mouse myeloma cells, rat GH3 cells, and human FL cells.
  • CHO cells Chinese hamster CHO cells
  • dhfr gene-deficient CHO cells mouse L cells
  • mouse AtT-20 cells mouse myeloma cells
  • rat GH3 cells rat GH3 cells
  • human FL cells human FL cells.
  • Transformants thus prepared can be cultured according to methods known in the art.
  • hosts were bacteria of the genus Escherichia , in general, they are cultured at about 15 to 43° C. for about 3 to 24 hours. Aeration or stirring is performed as necessary.
  • hosts are bacteria of the genus Bacillus , in general, they are cultured at about 30 to 40° C. for about 6 to 24 hours. Aeration or stirring is performed as necessary
  • transformants are cultured at about 20° C. to 35° C. for about 24 to 72 hours in a medium adjusted to about pH 5 to 8. Aeration or stirring is performed as necessary.
  • transformants are cultured at about 30° C. to 40° C. for about 15 to 60 hours in a medium adjusted to about pH 6 to 8. Aeration or stirring is performed as necessary.
  • a protein for use in the present invention from the above culture, for example, cells or bacteria are harvested after culture by a known method, and this is suspended in an appropriate buffer. After disrupting the cells or bacteria by sonication, lysozyme, and/or freeze-thawing, a crude protein extract is prepared by centrifugation or filtration.
  • the buffer may contain protein denaturants such as urea and guanidine hydrochloride, and detergents such as Triton X-100TM.
  • the supernatant is separated from the cells or bacteria after culture by a known method, and the supernatant is collected.
  • a protein contained in the resulting culture supernatant or extract can be purified by appropriately combining known isolation/purification methods.
  • a protein prepared as described above can be arbitrarily modified or a polypeptide can be partially removed from the protein by treating the protein produced by recombinants with an appropriate protein modification enzyme such as trypsin and chymotrypsin before or after purification.
  • an appropriate protein modification enzyme such as trypsin and chymotrypsin before or after purification.
  • the presence of a protein for use in the present invention can be assessed by various binding assays, enzyme immunoassays using specific antibodies, etc.
  • Antibodies for use in the present invention are not particularly limited as long as they bind to proteins for use in the present invention.
  • the antibodies may be obtained as polyclonal or monoclonal antibodies using known methods.
  • Particularly preferred antibodies for use in the present invention include monoclonal antibodies derived from mammals
  • Monoclonal antibodies derived from mammals include those produced by hybridomas and those produced by hosts transformed with expression vectors carrying antibody genes using gene engineering technologies. It is preferable that antibodies for use in the present invention specifically bind to proteins for use in the present invention.
  • hybridomas producing monoclonal antibodies can be prepared using known techniques by the following procedure. Specifically, immunization is carried out using as a sensitizing antigen a protein for use in the present invention according to conventional immunization methods. The resulting immune cells are fused with known parental cells by conventional cell fusion methods. Monoclonal antibody-producing cells are screened using conventional screening methods. More specifically, monoclonal antibodies can be prepared by the following procedure.
  • a gene sequence encoding the protein is inserted into a known expression vector system, and this is transformed into appropriate host cells. Then, the protein is purified from the host cells or culture supernatant by known methods.
  • the protein is used as a sensitizing antigen.
  • a partial peptide of the protein is used as a sensitizing antigen.
  • the partial peptide can be prepared by chemical synthesis based on the amino acid sequence of the protein according to common methods known to those skilled in the art.
  • Such a partial polypeptide of the protein has, for example, at least 10 or more amino acids, preferably 50 or more amino acids, more preferably 70 or more amino acids, still more preferably 100 or more amino acids, and yet more preferably 200 or more amino acids of the amino acid sequence constituting the protein, and has, for example, a biological activity substantially equivalent to the function of the protein.
  • the C terminus of the partial peptide is generally a carboxyl group (—COOH) or carboxylate (—COO—); however, the C terminus may also be amide (—CONH 2 ) or ester (—COOR).
  • the partial peptides include those in which the amino group of the N-terminal methionine residue is protected with a protecting group, those in which a glutamyl residue resulting from in vivo N-terminal cleavage is pyroglutamine-oxidized, those in which a substituent group in the side chain of an amino acid in the molecule is protected with an appropriate protecting group, and conjugated peptides such as so-called glycopeptides linked with sugar chains
  • Mammals that are immunized with a sensitizing antigen are not particularly limited, though it is preferable to take into consideration compatibility with the parent cell used for cell fusion.
  • rodents such as mice, rats, or hamsters are generally selected.
  • Immunization of animals with a sensitizing antigen is performed according to known methods.
  • standard methods of delivering sensitizing antigen to mammals involve intraperitoneal or subcutaneous injection.
  • a sensitizing antigen is diluted to be an appropriate volume with PBS (phosphate-buffered saline), physiological saline, or the like. If desired, this may be mixed with an appropriate amount of a typical adjuvant, for example, Freund's complete adjuvant, made into an emulsion, and then administered to mammals several times every 4 to 21 days.
  • An appropriate carrier may also be used for immunization with sensitizing antigens.
  • Immunocytes that are preferably subjected to cell fusion are splenocytes in particular.
  • mammalian myeloma cells are used.
  • myeloma cells it is preferable to use various known cell lines, for example, 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.
  • immunocytes and myeloma cells can be fused according to known methods, examples of which are described by Kohler and Milstein et al. (Kohler, G. and Milstein, C., Methods Enzymol. (1981) 73, 3-46).
  • the above-described cell fusion is carried out, for example, in a typical nutrient culture medium in the presence of a cell fusion promoting agent.
  • a cell fusion promoting agent for example, polyethylene glycol (PEG), Sendai virus (HVJ), or such can be used as the fusion promoting agent.
  • PEG polyethylene glycol
  • HVJ Sendai virus
  • adjuvants such as dimethylsulfoxide can additionally be used to increase fusion efficiency.
  • the preferred ratio of myeloma cells to immunocytes is, for example, from 1:1 to 1:10.
  • the culture medium used for the above-described cell fusion may be, for example, RPMI1640 medium, MEM medium, which are suitable for proliferation of the above-described myeloma cell lines, or other kinds of culture medium commonly used for culturing such cells.
  • serum supplements such as fetal calf serum (FCS) may be used in combination.
  • the cell fusion is carried out by thoroughly mixing prescribed amounts of the above-described immunocytes and myeloma cells in the aforementioned culture medium, adding to the medium a PEG solution preheated to about 37° C. generally at a concentration of 30% to 60% (w/v), wherein the PEG has an average molecular weight of about 1,000 to 6,000, for example, and mixing them to form the desired fusion cells (hybridomas).
  • An appropriate culture medium is then successively added.
  • Cell fusing agents and such that are undesirable for the proliferation of hybridomas are removed by repeatedly removing the supernatant by centrifugation.
  • the hybridomas obtained in this manner are selected by culturing them in a common selection culture medium, for example, the HAT medium (a culture medium containing hypoxanthine, aminopterin, and thymidine). Culture in the HAT medium described above is continued for a sufficient time, usually from a few days to a few weeks, to allow death of all cells but the target hybridomas (the non-fused cells). The usual limiting dilution method is then performed to screen and clone hybridomas producing antibodies used the present invention.
  • a common selection culture medium for example, the HAT medium (a culture medium containing hypoxanthine, aminopterin, and thymidine). Culture in the HAT medium described above is continued for a sufficient time, usually from a few days to a few weeks, to allow death of all cells but the target hybridomas (the non-fused cells).
  • the usual limiting dilution method is then performed to screen and clone hybridomas producing antibodies used the present invention.
  • desired human antibodies having an activity of binding to the protein can also be obtained by in vitro sensitizing human lymphocytes with the protein and fusing the sensitized lymphocytes with human-derived myeloma cells having permanent cell division ability (see Japanese Patent Application Kokoku Publication No. (JP-B) H01-59878 (examined, approved Japanese patent application published for opposition)).
  • human antibodies against a protein may be obtained from immortalized antibody-producing cells that are prepared by administering the protein as an antigen to a transgenic animal having a full repertoire of human antibody genes (see, International Patent Applications WO 94/25585, WO 93/12227, WO 92/03918, and WO 94/02602).
  • the V regions of human antibodies can be expressed as single-chain antibodies (scFvs) on the surface of phages using a phage display methods, from which phages presenting scFv that binds to an antigen can be selected.
  • the DNA sequences encoding the V regions of human antibodies that bind to the antigen can be determined by analyzing the genes of selected phages. After identifying the DNA sequences of scFvs that bind to the antigen, the V region sequences are fused in frame with the C region sequences of a desired human antibody. Then, the resulting DNA is inserted into an appropriate expression vector to construct an expression vector.
  • the expression vector is introduced into suitable cells for expression, such as those described above.
  • the human antibody can be obtained by expressing the gene encoding the human antibody. These methods are already known (see WO 1992/001047, WO 1992/020791, WO 1993/006213, WO 1993/011236, WO 1993/019172, WO 1995/001438, and WO 1995/015388).
  • the hybridomas prepared in this manner that produce monoclonal antibodies can be passaged in a common culture medium and stored for a long time in liquid nitrogen.
  • Monoclonal antibodies may be obtained from the hybridomas using common techniques; for example, the hybridomas are cultured according to standard methods and the antibodies may be obtained from the culture supernatants. Alternatively, the hybridomas are administered to a compatible mammal for proliferation and then the antibodies may be obtained from the ascites fluid.
  • the former method is suitable for obtaining highly pure antibodies, while the latter method is suitable for mass production of antibodies.
  • Monoclonal antibodies used in the present invention may be recombinant antibodies produced by genetic engineering techniques. They can be produced, for example, by cloning an antibody gene from a hybridoma, incorporating the antibody gene into an appropriate vector, and introducing the resulting vector into a host (see, for example, Vandamme, A. M. et al., Eur. J. Biochem., (1990) 192, p. 767-775, 1990).
  • mRNAs encoding antibody variable (V) regions are isolated from hybridomas producing the antibodies.
  • mRNAs can be isolated by preparing total RNAs using known methods, for example, guanidine-ultracentrifugation method (Chirgwin, J. M. et al., Biochemistry (1979) 18, 5294-5299), AGPC method (Chomczynski, P. et al., Anal. Biochem. (1987) 162, 156-159), or such.
  • mRNAs of interest are prepared using the mRNA Purification Kit (Pharmacia) or such.
  • the mRNAs can be prepared directly by using the QuickPrep mRNA Purification Kit (Pharmacia).
  • cDNAs are synthesized using the AMV Reverse Transcriptase First-strand cDNA Synthesis Kit (Seikagaku Co.) or such.
  • cDNA may be synthesized and amplified following the 5′-RACE method (Frohman, M. A. et al., Proc. Natl. Acad. Sci. USA (1988) 85, 8998-9002; Belyaysky, A. et al., Nucleic Acids Res. (1989) 17, 2919-2932) using the 5′-Ampli FINDER RACE Kit (Clontech) and PCR, and such.
  • DNA fragments of interest are purified from the resulting PCR products, and ligated to vector DNAs. From this, a recombinant vector is produced. The recombinant vector is then introduced into E. coli or such, and the desired recombinant vector is prepared from a selected colony.
  • the nucleotide sequences of DNAs of interest are then determined by known methods, for example, the dideoxynucleotide chain termination method.
  • a DNA encoding the antibody V region of interest is obtained, and then incorporated into an expression vector carrying a DNA that encodes a desired antibody constant region (C region).
  • the antibody gene is incorporated into an expression vector so that the gene will be expressed under the control of an expression regulatory region, for example, an enhancer and a promoter. Then, host cells are transformed with the resulting expression vector to express the antibody.
  • an expression regulatory region for example, an enhancer and a promoter.
  • a DNA encoding an antibody heavy chain (H chain) or light chain (L chain) can be each separately incorporated into an expression vector to simultaneously transform the host cell, or alternatively DNAs encoding H and L chains can be incorporated into a single expression vector to transform the host cells (see, WO 94/11523).
  • transgenic animals can also be used to produce recombinant antibodies.
  • an antibody gene is prepared as a fusion gene by inserting the antibody gene into a gene encoding a protein that is specifically produced in milk, such as goat casein. DNA fragments containing the fusion gene to which the antibody gene has been inserted is injected into goat embryos, which are then introduced into female goats. The desired antibody is then obtained from the milk produced by the transgenic goats, which are born from the goats that received the embryos, or from their offspring. Hormones may be suitably given to the transgenic goat to increase the production of milk containing the antibody of interests (Ebert, K. M. et al., Bio/Technology (1994) 12, 699-702).
  • artificially modified genetically-recombinant antibodies such as chimeric, humanized, and human antibodies can be used to reduce heterologous antigenicity against humans and such.
  • modified antibodies can be produced using known methods.
  • Monoclonal antibodies of the present invention include not only those derived from animals described above but also artificially modified genetically-recombinant antibodies such as chimeric antibodies, humanized antibodies, and bispecific antibodies.
  • a chimeric antibody can be obtained by linking a DNA encoding the antibody V region obtained as described above to a DNA encoding the human antibody C region, incorporating this into an expression vector, and then introducing it into a host for production.
  • Useful chimeric antibodies can be obtained using this known method.
  • Humanized antibodies are also referred to as “reshaped human antibodies”, which are antibodies obtained by grafting the complementarity determining regions (CDRs) of an antibody from a non-human mammal (e.g., mouse antibody) to the complementarity determining regions of a human antibody.
  • CDRs complementarity determining regions
  • General gene recombination procedures are also known (see, European Patent Application Publication No. EP 125023; International Patent Application Publication No. WO 96/02576).
  • a DNA sequence designed to link a mouse antibody CDR to the framework region (FR) of a human antibody is synthesized by PCR, using as primers several oligonucleotides prepared to contain overlapping portions in both CDR and FR terminal regions (see methods described in WO 98/13388).
  • the human antibody framework region to be linked via CDR is selected such that complementarity determining region forms a favorable antigen-binding site.
  • amino acids of the framework region in the antibody variable region may be substituted so that the complementarity determining region of the reshaped human antibody forms a suitable antigen-binding site (Sato, K. et al., 1993, Cancer Res. 53, 851-856).
  • Human antibody C-regions are used as the C-regions of chimeric antibodies or humanized antibodies.
  • CH1, CH2, CH3, and CH4 can be used for the H chain, while C ⁇ and C ⁇ can be used for the L chain.
  • the human antibody C-region may be modified in order to improve stability of the antibody or its production.
  • a chimeric antibody is composed of the variable region of an antibody derived from a non-human mammal and the constant region derived from a human antibody.
  • a humanized antibody is composed of the complementarity determining region of an antibody derived from a non-human mammal, and the framework region and C region derived from a human antibody. Since the antigenicity of humanized antibodies is low in the human body, and humanized antibodies are useful as an active ingredient in therapeutic agents of the present invention.
  • Antibodies used in the present invention are not limited to whole antibody molecules, and as long as they bind to proteins used in the present invention, antibody fragments and modification products thereof as well as divalent and monovalent antibodies are also included.
  • Antibody fragments include, for example, Fab, F(ab′)2, Fv, Fab/c having an Fab and the whole Fc, single chain Fv (scFv) in which Fv fragments from H and L chains are ligated via an appropriate linker, and Diabody.
  • antibody fragments are prepared by treating antibodies with an enzyme, for example, papain or pepsin.
  • the antibody fragments are expressed in appropriate host cells using the vector (see, for example, Co, M.
  • scFv is obtained by ligating antibody H-chain V region with an antibody L-chain V region.
  • the H-chain and L-chain V regions are ligated via a linker, preferably via a peptide linker (Huston, J. S. et al., Proc. Natl. Acad. Sci. U.S.A. (1988) 85, 5879-5883).
  • the H-chain V region and L-chain V region of an scFv may be derived from any of the antibodies described herein.
  • any single-chain peptides consisting of 12 to 19 amino acid residues such as (GGGGS)n may be used as a peptide linker for ligating the V regions.
  • a DNA encoding an scFv can be obtained by using, among DNAs encoding the antibody H chain or H chain V region and the antibody L chain or L chain V region mentioned above, all or DNA portion encoding amino acid sequence of interest as a template, amplifying by PCR using a primer pair that defines its two ends; and then carrying out a subsequent amplification using a combination of a DNA encoding the peptide linker portion, and primer pairs that define both ends of the linker DNA to be ligated to the H chain and L chain, respectively.
  • expression vectors carrying the DNAs and hosts transformed with the expression vectors can be obtained according to conventional methods. Furthermore, scFvs can be obtained using these hosts according to conventional methods.
  • Diabodies are dimers formed by linking two fragments (for example, scFv) in which a variable region is linked to another variable region via a linker or such, and typically have two VLs and two VHs (P. Holliger et al., Proc. Natl. Acad. Sci.
  • antibody fragments can be produced, in a similar manner as described above, by obtaining their genes and expressing them in hosts.
  • antibody comprises such antibody fragments.
  • antibodies of the present invention linked to various molecules such as polyethylene glycol (PEG) may be used.
  • antibodies can also be linked to radioisotopes, chemotherapeutic agents, cytotoxic substances such as bacteria-derived toxins, or such.
  • antibody includes such modified antibodies. Modified antibodies can be obtained by chemically modifying the prepared antibodies. Such antibody modification methods are already established in the art.
  • antibodies used in the present invention may be bispecific antibodies.
  • Bispecific antibodies of the present invention may be those having antigen-binding sites that each recognizes different epitopes in the protein used in the present invention or those which recognize the protein used in the present invention and a different protein.
  • bispecific antibodies of the present invention may be those in which one antigen-binding domain recognizes the protein used in the present invention and the other recognizes a chemotherapeutic agent or a cytotoxic substance such as a cell-derived toxin.
  • proliferation of cancer stem cells can be suppressed by allowing a cytotoxic substance to act directly on cancer stem cells expressing a protein used in the present invention and specifically damaging the cancer stem cells.
  • bispecific antibodies in which one antigen-binding domain recognizes a molecule that constitutes the T cell receptor complex such as CD3, expressing on cytotoxic T cells, and the other recognizes an epitope in the protein of any one of SEQ ID NOs: 1 to 8 of the present invention.
  • the bispecific antibodies may be prepared by linking pairs of H and L chains from two types of antibodies, or by fusing hybridomas that produce different monoclonal antibodies to yield a fusion cell producing bispecific antibodies.
  • the bispecific antibodies can be prepared using genetic engineering techniques.
  • Antibody genes constructed as described above can be expressed and obtained according to known methods.
  • antibody genes can be expressed using a DNA in which a common useful promoter, an antibody gene to be expressed, and a poly A signal positioned downstream of the antibody gene on the 3′ side are operably linked
  • Promoter/enhancer includes, for example, human cytomegalovirus immediate early promoter/enhancer.
  • the expression vector may include, as a selection marker, the aminoglycoside transferase (APH) gene, thymidine kinase (TK) gene, E. coli xanthine-guanine phosphoribosyltransferase (Ecogpt) gene, dihydrofolate reductase (dhfr) gene, and such.
  • APH aminoglycoside transferase
  • TK thymidine kinase
  • Ecogpt E. coli xanthine-guanine phosphoribosyltransferase
  • dhfr dihydrofolate reductase
  • the antibody gene can be expressed by an operably linked common useful promoter, a signal sequence for antibody secretion, and the antibody gene to be expressed.
  • promoters include, for example, the lacz promoter and araB promoter.
  • the gene can be expressed by the method of Ward et al. (Nature (1098) 341, 544-546; FASEB J. (1992) 6, 2422-2427) or the method of Better et al. (Science (1988) 240, 1041-1043), respectively.
  • the pel B signal sequence (Lei, S. P. et al., J. Bacteriol. (1987) 169, 4379) may be used as a signal sequence for antibody secretion. After antibodies produced into the periplasm is separated, the antibody structure is appropriately refolded and then used.
  • Eukaryotic cells include, for example, animal cells such as established mammalian cell systems, insect cell systems, cells of filamentous fungi, and yeast cells.
  • Prokaryotic cells include, for example, bacterial cells such as E. coli cells.
  • Antibodies used in the present invention are preferably expressed in mammalian cells, for example, CHO, COS, myeloma, BHK, Vero, and HeLa cells.
  • transformed host cells are cultured in vitro or in vivo to produce antibodies of interest.
  • Host cells are cultured according to known methods.
  • DMEM, MEM, RPMI1640, or IMDM may be used as a culture medium, and this may also be used with serum supplements such as fetal calf serum (FCS).
  • FCS fetal calf serum
  • Antibodies expressed and produced as described above can be isolated from cells or host animals and purified to be homogeneous.
  • Antibodies used in the present invention can be isolated/purified by using affinity columns.
  • Protein A columns include Hyper D, POROS, and Sepharose F. F. (Pharmacia). It is also possible to use other common protein isolation/purification methods. Such methods are not particularly limited.
  • antibodies may be isolated/purified by appropriately selecting/combining chromatography columns other than the above-described affinity columns, filters, ultrafiltration, salting-out, dialysis, and such (Antibodies A Laboratory Manual. Ed Harlow, David Lane, Cold Spring Harbor Laboratory, 1988).
  • 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 an antibody used in the present invention can be determined by using known methods.
  • Enzyme-linked immunosorbent assays ELISAs
  • enzyme immunoassays EIAs
  • radioimmunoassays RIAs
  • fluorescent antibody methods can be used to determine the antigen-binding activity of the antibody of the present invention.
  • an enzyme immunoassay samples containing an antibody of the present invention such as a culture supernatant of cells producing the antibody or the purified antibody are added to plates coated with a protein used in the present invention.
  • a secondary antibody labeled with an enzyme such as alkaline phosphatase is added, and the plates are incubated. After washing, an enzyme substrate such as p-nitrophenyl phosphate is added and the absorbance is measured to evaluate the antigen-binding activity.
  • An antibody used in the present invention may appropriately be linked to a cytotoxic substance described above such as a proliferation inhibitor, toxic peptide, or radioactive chemical substance.
  • a cytotoxic substance described above such as a proliferation inhibitor, toxic peptide, or radioactive chemical substance.
  • modified antibodies hereinafter referred to as antibody conjugates
  • a linker molecule links a growth inhibitor to an antibody via chemical bonding so that the antibody and growth inhibitor or cytotoxic substance can chemically conjugate with each other (for example, can bind covalently).
  • Preferred binders (linkers) are cleavable linkers. It is more preferable that the linkers are cleaved under mild conditions (specifically, intracellular conditions that do not affect the activity of inhibitors).
  • cleavable linkers examples include disulfide linkers, acid-labile linkers, photo-labile linkers, peptidase-labile linkers, and esterase-labile linkers.
  • Disulfide-containing linkers can be cleaved via disulfide exchange, which can occur under physiological conditions.
  • Acid-labile linkers can be cleaved at acid pH. For example, certain intracellular compartments such as endosomes and lysosomes have an acidic pH (pH 4 to 5), and provide conditions suitable for cleaving acid-labile linkers.
  • Photo-labile linkers are useful on the body surface and in many body cavities, which can be exposed to light. Furthermore, infrared light can penetrate tissues.
  • Peptidase-labile linkers can be used to cleave certain peptides inside or outside cells (for example, see Trouet et al., Proc. Natl. Acad. Sci. USA (1982) 79, 626-629; Umemoto et al., Int. J. Cancer (1989) 43, 677-684).
  • modified antibodies can be prepared not only by chemical modification as described above, but also in a molecular form such as a bispecific antibody designed to recognize a growth inhibitor, toxic peptide, radioactive chemical substance, or the like using genetic recombination techniques.
  • Antibody of the present invention also comprises such antibodies.
  • modified antibodies that are provided by the present invention also include those modified with a toxic peptide such as ricin, abrin, ribonuclease, onconase, DNase I, Staphylococcal enterotoxin-A, pokeweed antiviral protein, gelonin, diphtheria toxin, Pseudomonas exotoxin, Pseudomonas endotoxin, L-asparaginase, or PEG L-Asparaginase.
  • a toxic peptide such as ricin, abrin, ribonuclease, onconase, DNase I, Staphylococcal enterotoxin-A, pokeweed antiviral protein, gelonin, diphtheria toxin, Pseudomonas exotoxin, Pseudomonas endotoxin, L-asparaginase, or PEG L-Asparaginase.
  • antibodies may
  • the linkage between the antibody of the present invention that binds to at least one protein described in SEQ ID NOs: 1 to 8 and an above-described growth inhibitor, toxic peptide, or radioactive chemical substance may be a covalent or non-covalent bond.
  • Methods for preparing modified antibodies linked to such chemotherapeutic agents are known.
  • proteinaceous pharmaceutical agents and toxins can be linked to an antibody by using a genetic engineering procedure.
  • a recombinant vector into which a DNA encoding a toxic peptide described above and a DNA encoding an antibody that binds to any of proteins of at least one of SEQ ID NOs: 1 to 8 of the present invention are fused in frame and incorporated into an expression vector is constructed.
  • Transformed cells obtained by introducing the vector into appropriate host cells are cultured to express the incorporated DNA.
  • the modified antibody linked to the toxic peptide is obtained as a fusion protein.
  • a proteinaceous pharmaceutical agent or toxin is placed at the C terminus of the antibody.
  • a peptide linker may be interposed between the antibody and a proteinaceous pharmaceutical agent or toxin.
  • Antibodies used in the present invention may have a cytotoxic activity.
  • the cytotoxic activity includes, for example, complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC).
  • CDC refers to a cytotoxic activity mediated by the complement system
  • ADCC refers to an activity of damaging target cells, which is caused by binding of Fc ⁇ receptor-carrying cells (immunocytes, etc.) via Fc ⁇ receptor to the Fc portion of specific antibody upon binding of the antibody to cell-surface antigens on target cells.
  • ADCC Whether an antibody used in the present invention has ADCC or CDC can be measured by known methods (see, for example, Current protocols in Immunology, Chapter 7. Immunologic studies in humans, Editor, John E, Coligan et al., John Wiley & Sons, Inc., (1993)).
  • cytotoxicity can be measured, for example, by the following method.
  • Spleen is removed from a CBA/N mouse or the like, and spleen cells are dispersed in RPMI1640 medium (GIBCO). After washing with the same medium containing 10% fetal bovine serum (FBS, HyClone), effector cells with a cell concentration adjusted to 5 ⁇ 10 6 cells/ml were prepared.
  • RPMI1640 medium GIBCO
  • FBS fetal bovine serum
  • Baby Rabbit Complement (CEDARLANE) is diluted 10-fold with a medium (GIBCO) containing 10% FBS to prepare a complement solution.
  • a medium GIBCO
  • Cells expressing a protein used in the present invention are radiolabeled by incubating them with 0.2 mCi of 51 Cr-sodium chromate (Amersham Pharmacia Biotech) in DMEM medium containing 10% FBS for one hour at 37° C. After radiolabeled, the cells are washed three times with RPMI1640 medium containing 10% FBS, and the target cells with a cell concentration adjusted to 2 ⁇ 10 5 cells/ml were prepared.
  • the target cells and 50 ⁇ l of the antibody used in the present invention are each added to a 96-well U-bottom plate (Becton Dickinson), and reacted for 15 minutes on ice. Thereafter, 100 ⁇ l of effector cells are added and incubated in a carbon dioxide incubator for four hours. The final antibody concentration is adjusted to 0 or 10 ⁇ g/ml. After incubation, 100 ⁇ l of the supernatant is collected and the radioactivity is measured with a gamma counter (COBRAIIAUTO-GMMA, MODEL D5005, Packard Instrument Company). The cytotoxic activity (%) can be calculated according to:
  • A represents the radioactivity (cpm) of each sample
  • B represents the radioactivity (cpm) of a sample where 1% NP-40 (nacalai tesque) has been added
  • C represents the radioactivity (cpm) of a sample containing the target cells alone.
  • 50 ⁇ l of the target cells and 50 ⁇ l of the antibody used in the present invention are each added to a 96-well flat-bottom plate (Becton Dickinson), and reacted for 15 minutes on ice. Thereafter, 100 ⁇ l of the complement solution is added, and incubated in a carbon dioxide incubator for four hours. The antibody final concentration is adjusted to 0 or 3 ⁇ g/ml. After incubation, 100 ⁇ l of the supernatant is collected to measure the radioactivity with a gamma counter. The cytotoxic activity can be calculated by the similar way as in the ADCC determination.
  • Antibodies with modified sugar chains can appropriately be used in the antibodies provided by the present invention. It is known that cytotoxic activity of antibodies can be increased by modifying its sugar chains. Known antibodies with modified sugar chains include, for example:
  • Antibodies of the present invention preferably include antibodies with modified sugar chains whose sugar chain composition has been altered to increase the ratio of defucosylated antibody or to increase the ratio of antibody attached with bisecting N-acetylglucosamine.
  • Antibodies having a neutralizing activity can also be used appropriately in the present invention.
  • neutralizing activity refers to the activity of a foreign molecule such as a toxin or virus, or an internal molecule such as a hormone or cytokine to inhibit a ligand's biological activity on cells.
  • substance having a neutralizing activity refers to a substance that binds to a ligand or a receptor to which the ligand binds, thereby inhibiting the ligand-receptor binding.
  • the receptor whose ligand binding is inhibited by the neutralizing activity cannot exert their receptor-mediated biological activity.
  • the antigen-binding molecule is an antibody, in general, such an antibody with a neutralizing activity is called a neutralizing antibody.
  • the neutralizing activity of a test substance can be assessed by comparing biological activities in the presence of a ligand, in the condition of when the test substance is present or not present.
  • EGF receptor which is believed to be a main receptor for the EREG represented by SEQ ID NO: 3, dimerizes upon ligand binding and activates its own cytoplasmic tyrosine kinase domain.
  • the activated tyrosine kinase causes a peptide having phosphotyrosine by autophosphorylation, which allows association of various signal transduction accessory molecules.
  • the molecules are mainly phospholipase C ⁇ (PLC ⁇ ), Shc, Grb2, and such. Of these accessory molecules, the former two are further phosphorylated by the tyrosine kinase of the EGF receptor.
  • the main signaling pathway from the EGF receptor is the one in which phosphorylation occurs in order of Shc, Grb2, Sos, Ras, and Raf/MAPK kinase/MAP kinase. It is believed that there is also an alternative pathway from PLC ⁇ to PKC. Since such intracellular signal cascades vary depending on cell type, a target molecule can appropriately be selected for each target cell type of interest and is not limited to the factors described above. It is possible to use an appropriate in vivo signal activation assay kit available on the market (for example, protein kinase C activation measurement system (GE Healthcare Bioscience, etc.)).
  • the in vivo signal activation can be detected by using as an indicator the transcriptional induction of a target gene downstream in the in vivo signal cascade.
  • Changes in the transcriptional activity can be detected based on the principle of reporter assay. Specifically, a reporter gene such as GFP (Green Fluorescence Protein) or luciferase is placed downstream of the transcriptional factor or promoter region of the target gene. A change in the transcriptional activity can be determined as reporter activity by measuring the reporter activity.
  • GFP Green Fluorescence Protein
  • the EGF receptor typically functions to promote cell proliferation, and thus the activation of in vivo signal transduction can be assessed by measuring the proliferative activity of the target cell.
  • the neutralizing activities of neutralizing antibodies of the present invention are assessed by measuring the cell proliferative activity.
  • methods are not limited thereto, and methods described above can suitably be used to assess the activity depending on the type of selected target cells.
  • the neutralizing activity of an anti-EREG antibody can be assessed or determined by measuring the cell proliferative activity, for example, using the method described below.
  • the method where the incorporation of [ 3 H]-labeled thymidine, which is added to a culture medium, into viable cells is measured as an indicator for the DNA replication ability is used.
  • Simpler methods include the MTT method and dye exclusion tests in which the ability of cells to exclude dyes such as Trypan Blue outside is measured under a microscope.
  • the MTT method utilizes the ability of viable cells to convert MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide), a tetrazolium salt, into a blue formazan product. More specifically, the MTT method is performed as follows: a test antibody is added to a culture medium containing test cells; after a certain period, an MTT solution is added to the culture medium; the mixture is allowed to stand for a certain period so that MMT is incorporated into the cells. As a result, a yellow compound MTT is converted into a blue compound by succinate dehydrogenase in cytoplasmic mitochondria. After the blue product is dissolved for color development, the absorbance is measured as an indicator for the viable cell count.
  • MTT MTT
  • WST-1 WST-8
  • nacalai tesque, etc. a control antibody that is of the same isotype as the anti-EREG antibody of interest but does not have the neutralizing activity is used in the same manner as for the anti-EREG antibody. The activity can be assessed whether the anti-EREG antibody exhibits the neutralizing activity greater than that of the control antibody.
  • Cells whose proliferation is inhibited by the anti-EREG antibody are not particularly limited, as long as they express EREG protein.
  • Examples of preferred EREG-expressing cells include cancer cells. Specifically, cells derived from colorectal cancer, lung adenocarcinoma, pancreatic cancer, stomach cancer, or kidney cancer are preferable EREG-expressing cells in the present invention. A cell proliferation-inhibitory effect against both primary and metastatic lesions of all these cancers can be achieved by the present invention. More preferred cancer cells include those of primary colorectal cancer, metastatic colorectal cancer, lung adenocarcinoma, pancreatic cancer, stomach cancer, and kidney cancer.
  • anti-EREG antibodies can be used to treat/prevent diseases caused by cell proliferation, for example, colorectal cancer, lung adenocarcinoma, pancreatic cancer, stomach cancer, and kidney cancer. These cancers can be targets of treatment or prevention, regardless of primary or metastatic lesions. More preferably, anti-EREG antibodies are used to treat and/or prevent primary colorectal cancer, metastatic colorectal cancer, and pancreatic cancer. Furthermore, among these cancers, those which grow in an EREG-dependent manner are preferred as a target of treatment and/or prevention in the present invention.
  • nucleotides and amino acids are represented by abbreviations, these abbreviations are based on the abbreviations by IUPAC-IUB Commission on Biochemical Nomenclature, or the conventional abbreviations in the art.
  • amino acids when an optical isomer exists, it represents L form, unless otherwise specified.
  • the effective dosage of cancer stem cell inhibitors of the present invention is selected within the range of 0.001 to 1,000 mg/kg weight for each administration. Alternatively, the dosage may be selected within the range of 0.01 to 100,000 mg/body for each patient. However, the dosage of the inhibitors of the present invention is not limited to these doses. Meanwhile, with respect to the timing of administration, an inhibitor of the present invention may be administered before or after manifestation of clinical symptoms of diseases.
  • the inhibitors of the present invention can be formulated according to conventional methods (Remington's Pharmaceutical Science, latest edition, Mark Publishing Company, Easton, US), and may contain both pharmaceutically acceptable carriers and additives.
  • Such carriers and medical additives include, for example, water, pharmaceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymer, carboxymethylcellulose sodium, sodium polyacrylate, sodium alginate, water-soluble dextran, carboxymethyl starch sodium, pectin, methyl cellulose, ethyl cellulose, xanthan gum, gum arabic, casein, agar, polyethylene glycol, diglycerin, glycerin, propylene glycol, vaseline, paraffin, stearyl alcohol, stearic acid, human serum albumin (HSA), mannitol, sorbitol, lactose, and surfactants that are acceptable as a medical additive.
  • water pharmaceutically acceptable organic solvents
  • collagen collagen
  • polyvinyl alcohol polyvinylpyrrolidone
  • carboxyvinyl polymer carboxymethylcellulose sodium, sodium polyacrylate, sodium alginate, water-soluble dextran, carboxymethyl star
  • additives are selected alone or in appropriate combination from those listed above depending on the dosage form of an inhibitor of the present invention; but are not limited thereto.
  • an inhibitor of the present invention when used as a preparation for injection, it can be used in a form in which the inhibitor is dissolved in a medium such as physiological saline, buffer, or glucose solution and an adsorption inhibitor such as Tween80, Tween20, gelatin, or human serum albumin is added; alternatively, the inhibitor of the preset invention may be in a lyophilized form for dissolution and reconstitution before use.
  • excipients for lyophilization for example, sugar alcohols and saccharides such as mannitol and glucose can be used.
  • the inhibitors of the present invention are generally administered by a parenteral route, for example, via injection (subcutaneous, intravenous, intramuscular, intraperitoneal, etc.), transdermal, transmucosal, intranasal, or pulmonary administration; however, the inhibitor can be administered orally.
  • cancer stem cell inhibitor and an anti-cancer agent combined use means that these agents may be administered at the same time or in succession; alternatively, one is administered at an interval after administration of the other.
  • cancer stem cell inhibitors can be used as various embodiments such as, for example, prevention of cancer recurrence, suppression of cancer recurrence, prevention of cancer metastasis, suppression of cancer metastasis, and adjuvant therapy for preventing postoperative recurrence for application.
  • any cancer stem cell inhibitor can be used as a cancer stem cell inhibitor of the present invention.
  • non-limiting preferred examples include agents for inhibiting cancer stem cell proliferation or agents for disrupting cancer stem cell. As long as agents for inhibiting cancer stem cell proliferation that are provided by the present invention can suppress the proliferation of target cancer stem cells, mechanism of suppressing cancer stem cell proliferation is not relevant.
  • agents for inhibiting cancer stem cell proliferation include those comprising as an active ingredient an antibody having neutralizing activity against cancer stem cell proliferation or growth or an antibody having cytotoxicity against cancer stem cells.
  • agents for disrupting cancer stem cells that are provided by the present invention can destroy target cancer stem cells, mechanism of disrupting cancer stem cells is not relevant.
  • agents for disrupting cancer stem cells include agents for inhibiting cancer stem cell proliferation comprising as an active ingredient an antibody having cytotoxicity or apoptotic activity against cancer stem cells.
  • a test agent for disrupting cancer stem cells has apoptotic activity by using known methods including terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) assay, caspase activity (in particular, caspase-3) assay, fas ligand assay, and Annexin V assay as apoptotic activity assay methods.
  • TUNEL terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling
  • caspase activity in particular, caspase-3
  • fas ligand assay fas ligand assay
  • Annexin V assay as apoptotic activity assay methods.
  • Non-limiting preferred examples include cancer stem cell differentiation enhancers.
  • differentiation enhancers include BMP4, i.e., the polypeptide of SEQ ID NO: 9, or polypeptide equivalents having one or several amino acid addition(s), deletion(s), and/or substitution
  • Such polypeptide equivalents preferably have a CSC differentiation-inducing activity equivalent to that of the polypeptide of SEQ ID NO: 9.
  • the equivalency of the differentiation-inducing activity can be defined, for example, by whether the CK20-inducing activity for CSCs is 10%, preferably 20%, more preferably 30%, even more preferably 40%, and still more preferably 50% of that of the polypeptide of SEQ ID NO: 9.
  • the equivalency of the differentiation-inducing activity can be defined, for example, by whether the CK20-inducing activity for CSCs is 60%, preferably 70%, more preferably 80%, even preferably 90%, and still preferably 95% of that of the polypeptide of SEQ ID NO: 9.
  • Anti-cancer agents that are used in combination with a cancer stem cell inhibitor of the present invention include alkylating agents, metabolic antagonists, natural products, platinum complexes, and other pharmaceutical agents.
  • Alkylating agents include nitrogen mustards, ethylenimines, methylmelamines, alkyl sulfonates, nitrosoureas, and triazens.
  • Nitrogen mustards include, for example, mechlorethamine, cyclophosphamide, ifosfamide, melphalan, and chlorambucil.
  • Ethylenimines and methylmelamines include, for example, hexamethylmelamine and thiotepa.
  • Alkyl Sulfonates include busulfan.
  • Nitrosoureas include, for example, carmustine (BCNU), lomustine (CCNU), semustine (methyl-CCNU), and streptozocin.
  • Triazens include dacarbazine (DTIC).
  • Metabolic antagonists include folic acid analogs, pyrimidine analogs, and purine analogs.
  • Folic acid analogs include methotrexate.
  • Pyrimidine analogs include, for example, fluorouracil (5-FU), doxifluridine (5′-DFUR; trade name: FURTULON), capecitabine (trade name: Xeloda), floxuridine (FudR), and cytarabine.
  • Purine analogs include, for example, mercaptopurine (6-MP), thioguanine (TG), and pentostatin.
  • Natural products include vinca alkaloids, epipodophyllotoxins, and antibiotics.
  • Vinca alkaloids include, for example, vinblastine (VLB) and vincristine (VCR).
  • Epipodophyllotoxins include, for example, etoposide and teniposide.
  • Antibiotics include, for example, dactinomycin (actinomycin D), daunorubicin, doxorubicin, bleomycin, plicamycin, and mitomycin.
  • Platinum complex refers to platinum coordination complex, and includes, for example, cisplatin (CDDP) and carboplatin.
  • topoisomerase inhibitors such as irinotecan and camptothecin
  • taxols for example, paclitaxel, docetaxel
  • anthracenediones for example, mitoxantrone
  • urea-substituted derivatives for example, hydroxyurea
  • methyl hydrazines for example, procarbazine hydrochloride (trade name: Natulan)
  • vitamin A metabolites for example, tretinoin (trade name: VESANOID)
  • rituximab alemtuzumab, trastuzumab, bevacizumab, cetuximab, panitumumab, trastuzumab, and gemutuzumab.
  • Colorectal cancer specimens were obtained from patients with their consent under approval of the ethical committees of PharmaLogicals Research (Singapore) and Parkway Laboratory Services (Singapore). Tumor blocks were cut into small pieces with a razor blade, and grafted in the lateral region of NOG mice. Human colorectal cancer xenografts were maintained by passaging them in NOG mice provided by the Central Institute for Experimental Animals (Japan). Mice used in this experiment were treated in accordance with the animal experiment guidelines of PharmaLogicals Research. For histopathological examination, small blocks of xenograft tumors and surgical human tissue samples were fixed with 4% paraformaldehyde at 4° C.
  • Tissues of xenograft were cut with a razor blade in order to prepare single cancer cell suspensions. After the suspensions were incubated at 37° C. for 3 hours in DPBS containing collagenase/dispase (Roche) and DNaseI (Roche), the suspensions were filtered through 40- ⁇ m Cell Strainers (BD Biosciences). The cells were suspended in the lysis buffer (BD Biosciences) to remove erythrocytes. The prepared xenograft-derived cells (such cells are referred to as primary cells) were cultured under 5% CO 2 atmosphere at 37° C.
  • Cell suspensions were prepared by serial dilution. 100 ⁇ l of cancer cell suspensions in Hanks' balanced salt solution (Invitrogen) were subcutaneously inoculated into lateral region of mice using 50% matrigel (BD Bioscience). The tumor development was monitored over seven weeks. In order to inoculate single cell, cells were labeled using an FITC-labeled mouse anti-human CD326 (EpCAM) antibody (Miltenyi Biotec), and plated in a terasaki plate (Termo Fisher Scientific). Cell singularity was confirmed under a microscope. Single cells were inoculated into lateral region of mice using 50 ⁇ l of 50% matrigel. The tumor development was monitored over 10 weeks.
  • EpCAM mouse anti-human CD326
  • 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 adding HA tag to their N termini Cells of Chinese hamster ovary cell line CHO DG44 (Invitrogen) were transfected with expression plasmids using Gene Pulser (BioRad).
  • Stable cell lines HA-Lgr4/DG, HA-Lgr5/DG, and HA-Lgr6/DG were selected using G418.
  • Soluble Lgr5 protein (amino acids 1 to 555) was expressed as a fusion protein with the Fc domain of mouse IgG2a in CHO DG44. Transfectants were screened by sandwich ELISA using a goat anti-mouse IgG2a (Bethyl laboratories) and HRP-rat anti-mouse IgG2a mAb (Serotec). A clone that produces sLgr5-Fc at the highest level was named 2D3. A culture supernatant of 2D3 was collected, and Lgr5-Fc protein was affinity-purified using a Protein A-Sepharose column (Pharmacia). Lgr5-Fc was used as an antigen in protein immunization and ELISA screening.
  • mice (Charles River Japan) were immunized subcutaneously with 50 ⁇ g of Lgr5-Fc emulsified in Freund's complete adjuvant. After two weeks, the mice were injected with the same amount of Lgr5-Fc in Freund's incomplete adjuvant once a week over two weeks. Three days before cell fusion, 25 ⁇ g of Lgr5Fc was intravenously injected to the mice. Spleen lymphocytes derived from the immunized mice were fused with cells of mouse myeloma line P3-X63Ag8U1 (ATCC) using a conventional method (Kremer L and Marquez G (2004) Methods Mol. Biol., 239: 243-260). Hybridoma culture supernatants were screened for antibodies reactive to sLgr5-Fc by ELISA to establish Lgr5-specific mouse mAb 2T15E-2 and 2U2E-2.
  • Protein was extracted using RIPA buffer (Sigma) supplemented with the Complete Mini Protease Inhibitor Cocktail (Roche). Proteins were fractionated by the NuPAGE Gel (Invitrogen) and transferred onto PVDF membrane. After blocking with PBS containing 1% skimmed milk, the membrane was probed with rabbit anti-human ⁇ -catenin antibody (Sigma), rabbit anti-human phospho-c-JUN antibody (Sigma), rabbit anti-human TCF1 antibody (Cell Signaling), rabbit anti-human TCF3 antibody (Cell Signaling), rabbit anti-human TCF4 antibody (Cell Signaling), rabbit anti-human Lgr5 antibody (Abcam), mouse anti-human E-cadherin antibody (Abcam), rabbit anti-human Snail antibody (Abcam), and mouse anti-human GAPDH antibody (Santa Cruz). Reactive bands were detected using BCIP/NBT substrate (KPL).
  • RNAs were isolated using the RNeasy Mini Kit including DNAase treatment (Qiagen). cDNAs were synthesized using the First-Strand cDNA Synthesis Kit (SABiosciences). Quantitative real-time PCR (QRT-PCR) analysis was performed with the SYBR Green/Rox qPCR(SABiosciences) using the Mx3005P Real-Time PCR System (Stratagene). The fold induction value was calculated according to the 2- ⁇ Ct method. GAPDH and ACTB were used as a reference. All experiments were performed in triplicate.
  • QRT-PCR Quantitative real-time PCR
  • the following primers were used to amplify reactive transcripts.
  • Lgr5 forward primer: (SEQ ID NO: 10) 5′-AGTTTATCCTTCTGGTGGTAGTCC-3′; reverse primer: (SEQ ID NO: 11) 5′-CAAGATGTAGAGAAGGGGATTGA-3′; GAPDH: forward primer: (SEQ ID NO: 12) 5′-CTCTGCTCCTCCTGTTCGAC-3′; reverse primer: (SEQ ID NO: 13) 5′-ACGACCAAATCCGTTGACTC-3′; ACTB: forward primer: (SEQ ID NO: 14) 5′-AAGTCCCTTGCCATCCTAAAA-3′; reverse primer: (SEQ ID NO: 15) 5′-ATGCTATCACCTCCCCTGTG-3′
  • Floating and adherent CSCs were plated at about 100 and 1 ⁇ 10 4 cells/well in 96-well plates, respectively. On days 0 and 3, viable cell counts were determined by the Cell Counting Kit-8 Assay (Doujindo) according to the manufacturer's protocol. Average absorbance on day 0 was taken as 100%. For chemosensitivity assay, floating and adherent CSCs were plated at about 100 and 1 ⁇ 10 4 cells/well in 96-well plates, respectively.
  • immunofluorescent cytochemistry cells were fixed with 4% paraformaldehyde and methanol, and incubated with a mouse anti-human E-cadherin antibody (Abcam), rabbit anti-human Snail antibody (Abcam), or rabbit anti-human ⁇ -catenin antibody (Sigma)). Then, the cells were visualized using the AlexaFluor 488-labeled goat anti-mouse IgG antibody or goat anti-rabbit IgG antibody.
  • thin sections derived from paraffin blocks of xenograft tumors described above were incubated with a mouse anti-human Lgr5 antibody (2U2E-2) or rabbit anti-human Snail antibody (Abcam).
  • Lgr5 protein was detected with a goat anti-mouse antibody conjugated with the polymer-HRP (DAKO) and visualized with the AlexaFluor 488-labeled tyramide (Invitrogen), while Snail protein was detected with biotinylated goat anti-rabbit antibody (VECTOR) and visualized with the AlexaFluor 568-labeled streptavidin (Invitrogen). These cells and samples were also stained with DAPI (Invitrogen).
  • 17 types of colorectal cancer xenografts were established from samples of 53 human colorectal cancer patients. Except for the 17 types of xenografts, associated EBV-infected lymphoma cells occurred in 19 cases (which aggravated the condition of NOG mice); other infections were found in 14 cases; and no tumor growth was observed in three cases. Of the 17 types, 11 xenografts survived even after freeze-thawing, and had the capacity to reconstitute tumor, and showed similar histopathological features as those of the original tumors. Of the 11 types, 10 xenografts were derived from grade-2 moderately-differentiated adenocarcinomas, and the remaining one was derived from a grade-3 poorly-differentiated adenocarcinoma.
  • xenografts were derived from moderately-differentiated colorectal cancer (MDCC), and the remaining one was derived from poorly-differentiated colorectal cancer (PDCC) (Table 2; histopathological classification of the original human colorectal cancers that were used to establish the 11 xenografts).
  • MDCC moderately-differentiated colorectal cancer
  • PDCC poorly-differentiated colorectal cancer
  • MDCC and PDCC xenografts reconstituted histopathological morphologies almost equivalent to those of the original tumors.
  • MDCC xenografts formed specific epithelial ducts which contained goblet cells, and small budding clusters (may undergo epithelial-mesenchymal transition (EMT)).
  • EMT epithelial-mesenchymal transition
  • PDCC xenografts did not form such specific epithelial duct structures ( FIGS. 1 and 16 ).
  • the present inventors used two types of MDCC xenografts, i.e., PLR59 and PLR123. These xenografts were chosen by the present inventors because they grew rapidly even after 10 passages in NOG mice while maintaining the capacity to reconstitute tumors with epithelial ducts and small budding clusters ( FIG. 1 ). Thus, the present inventors predicted that stable CSCs can be obtained from the xenografts.
  • asterisk indicates tumor xenografts established with NOG mice
  • dagger indicates cell preparations.
  • Primary indicates cells (primary cells) prepared by harvesting xenograft tumor tissues grown in NOG mice and removing erythrocytes and mouse cells.
  • Floating indicates cells prepared by in vitro culturing primary cells under a non-adherent culture condition.
  • Adherent indicates cells prepared by in vitro culturing primary cells under adherent culture conditions. Plus symbol (single) indicates the number of formed tumors, while plus symbol (double) indicates the total number of inoculation sites. Parenthesis indicates percent tumor (cancer) formation.
  • Lgr5 + represents Lgr5 positive
  • Lgr5 ⁇ represents Lgr5 negative.
  • adherent and non-adherent cells were highly homogeneous.
  • the adherent cells were of Lgr5 + , ALDH + , CD133 + , CD44 + , EpCAM + , CD 166 + , CD24 + , CD26 + , and CD29 + .
  • the non-adherent cells were of Lgr5 ⁇ and ALDH ⁇ , and thus were different from the adherent cells ( FIGS. 6 and 20 ).
  • Lgr5 mRNA was detected at a significant level in the adherent cells, while it was undetectable in non-adherent cells ( FIG. 27 ).
  • Lgr5-specific monoclonal antibodies 2L36, 2T15E-2, and 2U2E-2) respectively for immunohistochemistry and flow cytometry analysis.
  • the antibodies produced by the present inventors were highly specific to Lgr5 without any cross-reactivity to Lgr4 or Lgr6, both of which are highly homologous to Lgr5 ( FIGS. 28 and 29 ).
  • the present inventors demonstrated the expression of Lgr5 in the adherent CSCs.
  • Lgr5-positive cells were detected in tumor tissues that were the origins of PLR59 and PLR123 as well as in xenograft cancer tissues therefrom through all passages ( FIG. 39 ).
  • the frequency of Lgr5-positive cells was low in the original tumor tissues (0.01% and 0.04% for PLR59 and PLR123, respectively).
  • the frequency of Lgr5-positive cells was increased as passage number increased; however, there was no further change after tenth generation ( FIG. 39 ).
  • the tumor-reconstituting capacity of primary cells from PLR123 xenograft model was also potentiated as passage number increased.
  • the ratio of CSCs in the primary cells which was estimated based on the capacity to reconstitute tumor, was about 0.1% after 5 passages, and was increased to about 0.4% after 14 passages.
  • Lgr5-positive adherent cells alone can form tumors in vivo.
  • the present inventors assessed the tumor-forming capacities of Lgr5-positive adherent cells and Lgr5-negative non-adherent cells.
  • Lgr5-positive adherent cells were more potent than Lgr5-negative non-adherent cells in the capacity to form tumors.
  • both Lgr5-positive and Lgr5-negative cells had the capacity to form tumors in NOG mice.
  • Subcutaneous injection of ten Lgr5-positive cells caused tumor formation at every injection site (six of six sites), while Lgr5-negative cells formed tumors at two of six injection sites (PLR123-derived cells) or at a single site (PLR59-derived cells) (Table 3).
  • Lgr5-positive cells even when injected at only one cell per inoculation site, reconstituted tumors at two of 12 injection sites (PLR123-derived cells) or at a single site (PLR59-derived cells) ( FIG. 7 ).
  • the histopathological morphologies of tumors derived from the Lgr5-positive and Lgr5-negative cells were almost the same as those of the original tumors ( FIGS. 17 and 40 ). Furthermore, there was no change in the expression of cell surface markers and tumor-initiating activity of the Lgr5-positive CSCs even after one month of passage culture ( FIGS. 30 and 31 ).
  • the Lgr5-positive cells Under adherent culture conditions, the Lgr5-positive cells underwent symmetric cell division ( FIG. 41 ). Meanwhile, in the presence of matrigel and serum, Lgr5 protein was distributed to one of two daughter cells under the same culture conditions ( FIGS. 42C and 42D ), demonstrating that the Lgr5-positive cells undergo asymmetrical cell division.
  • One of CSC's properties is symmetrical cell division for self-renewal, and another characteristic property is asymmetrical cell division.
  • the Lgr5-positive adherent cells divided symmetrically under adherent culture conditions ( FIG. 41 ) whereas, in the presence of matrigel and FBS, as seen from the fact that Lgr5 protein was distributed to one of daughter cells ( FIG. 42 ), the Lgr5-positive cells underwent asymmetrical cell division, which resulted in two distinct progenies.
  • Lgr5-positive and Lgr5-negative cells derived from PLR59 and PLR123 are highly pure large intestine CSCs, and that the Lgr5-positive and Lgr5-negative cells correspond to two independent CSC states in colorectal cancer.
  • the present inventors assessed the effects of ⁇ -catenin/TCF inhibitor FH535 and Wnt/ ⁇ -catenin inhibitor cardamonin (which induces ⁇ -catenin degradation) on the proliferation of large intestine CSCs.
  • CSC's properties is resistance to chemotherapeutic agents.
  • the present inventors tested large intestine CSCs for the sensitivity to 5-FU and irinotecan.
  • the Lgr5-positive cells proliferated with a doubling time of about 2.5 days.
  • the Lgr5-negative CSCs were in a quiescent state in terms of growth.
  • Both 5-FU (10 ⁇ g/ml) and irinotecan (10 ⁇ g/ml) treatments significantly inhibited the proliferation of Lgr5-positive large intestine CSCs, while they did not affect the proliferation and survival of Lgr5-negative large intestine CSCs ( FIGS. 10 and 24 ).
  • HLA-DMA, TMEM173, ZMAT3, and GPR110 were chosen as markers for use in specific detection of such CSCs stably negative for Lgr5. Immunostaining performed using specific antibodies against the above molecules yielded a specific staining pattern with colon CSCs that converted to negative for Lgr5 after three days of irinotecan exposure ( FIG. 43 ). Furthermore, this immunostaining method was demonstrated to be applicable to tissue sections prepared from paraffin blocks, which are used commonly ( FIG. 43 ). These findings suggest that HLA-DMA, TMEM173, ZMAT3, GPR110 can serve as specific markers for CSCs that converted to negative for Lgr5.
  • the Lgr5-negative cells prepared via irinotecan treatment were assessed for the tumor-forming activity.
  • Subcutaneous injection of ten cells derived from PLR59 and PLR123 resulted in formation of tumors in two and one NOG mice (Table 4), respectively.
  • Table 4 shows the tumor-forming activity of Lgr5-negative CSCs 49 hours after inoculation.
  • asterisk indicates tumor xenografts established in NOG mice; plus symbol (single) indicates the number of animals bearing tumors, and plus symbol (double) indicates the total number of animals.
  • the present inventors cultured adherently Lgr5-negative large intestine CSCs prepared via irinotecan treatment again in a serum-free stem cell culture medium. The cells became positive for Lgr5 and exhibited mesenchymal cell-like morphology ( FIGS. 12 and 33 ), and started to proliferate.
  • Table 5 shows cell count ratios of Lgr5-positive and -negative cells stained by immunocytochemistry using an anti-Lgr5 antibody (antibody 2L36). Number in parenthesis represents the ratio of Lgr5-positive or -negative cell count.
  • the present inventors demonstrated that the Lgr5-positive large intestine CSCs formed tumors in multiple tissues including lung, liver, lymph node, and subcutaneous tissues. Interestingly, in the liver, lymph node, and subcutaneous tissues, tumors with epithelial ductal structures were reconstituted by at least 40 days after intravenous injection of tumor cells, whereas such structures were not reconstituted in the lung ( FIGS. 34 and 35 ).
  • Lgr5-negative CSCs directly form the hierarchical organization of cancer or first undergo transition to Lgr5-positive cells in vivo.
  • gene expression profiling was carried out using Lgr5-positive cells, Lgr5-negative cells, and primary cells from xenograft tumors.
  • HLA-DMA was selected from molecules whose expression can be detected at high level in the Lgr5-negative CSCs as compared to the Lgr5-positive CSC and primary cells ( FIG. 50 ).
  • HLA-DMA was demonstrated to be specifically expressed in the Lgr5-negative CSCs ( FIG. 51 ). HLA-DMA is also expressed in macrophages. Then, to rule out the possibility that cells stained by immunohistochemistry using the anti-HLA-DMA antibody are macrophages, the present inventors tested not only HLA-DMA but also other markers expressed in CSCs. Immunohistochemistry using an antibody against EREG expressed in both Lgr5-positive and -negative CSCs ( FIG. 50 ) confirmed that EREG was expressed in both of Lgr5-positive and Lgr5-negative CSCs ( FIG. 51 ).
  • Lgr5-negative CSCs could be identified as cells that are positive for both HLA-DMA and EREG by detection using both markers in combination.
  • cells expressing Lgr5 only weakly for one day after the injection, which however remained positive for HLA-DMA and EREG, appeared.
  • cells that are negative for HLA-DMA but remain positive for Lgr5 and EREG appeared by five days after the injection ( FIG. 52 ).
  • Tumors derived from Lgr5-negative CSCs had specific ductal structures and included Lgr5-positive cells ( FIG. 53 ).
  • irinotecan was administered at the maximum tolerated dose (MTD) (120 mg/kg) into the peritoneal cavities of NOG mice bearing tumors derived from Lgr5-positive CSCs. Tumor growth was inhibited almost completely ( FIG. 55 ), and the ductal structures were collapsed extensively ( FIG. 54 ). This condition resulted in a dramatic decrease of Lgr5-positive cells ( FIGS. 54 and 56 ). The number of Lgr5-negative and HLA-DMA-positive cells increased significantly after irinotecan treatment. By contrast, in vehicle-treated control mice, about one third of cancer cells were positive for Lgr5 in both ductal and budding areas ( FIG.
  • MTD maximum tolerated dose
  • Lgr5-positive cells and HLA-DMA-positive and Lgr5-negative cells were positive for EREG, and were identified to be CSCs ( FIG. 54 ).
  • Lgr5-positive cells appeared again ( FIG. 54 ). The results described above, when considered together, suggest that Lgr5-negative CSCs can be the origin of colorectal cancer after growth inhibitor treatment and reconstitute cancer hierarchy via transition to Lgr5-positive cells.
  • Lgr5-positive adherent cells were seeded at 3 ⁇ 10 5 cells/well in a 6-well plate (BD, Cat. No. 353046).
  • irinotecan Hospira, 61703-349-09
  • FACS buffer After three-day culture, irinotecan-resistant cells were detected.
  • the cells were harvested using Accutase, and suspended in FACS buffer. Then, the cells were incubated at 4° C. for 30 minutes with 7-AAD Viability Dye as dead cell staining and each of the following antibodies as cancer stem cell markers:
  • FITC-labeled mouse mAb to human CD326 EpCAM
  • PE-labeled mouse mAb to human CD133/1 AC133
  • PE-labeled mouse mAb to human CD44 PE-labeled mouse mAb to human CD166
  • PE-labeled mouse mAb to human CD24 PE-labeled mouse mAb to human CD26
  • PE-labeled mouse mAb to human CD29 PE-labeled mouse mAb to human CD29.
  • Lgr5 the cells were incubated with the mouse mAb to human Lgr5 at 4° C. for 30 minutes. After washing once with FACS buffer, the cells were incubated with the PE-labeled goat Ab to mouse IgG2a at 4° C. for 30 minutes.
  • RNAs were extracted from them using RNeasy Mini Kit (Qiagen, Cat. No. 74104) and RNase-Free DNase Set (Qiagen, Cat. No. 79254) according to the procedure recommended by the manufacturer.
  • the extracted RNAs were analyzed for the purity and quality using Agilent 2100 Bioanalyzer.
  • GeneChip HG-U133 plus2 of Affymetrix. Data analysis was performed with Microsoft Excel and Statistics software R. The three types of cells (primary cells, Lgr5-positive cells, and Lgr5-negative cells) were compared to each other to make a list of genes whose expression levels are significantly increased in each cell type. Specifically, raw data from GeneChip were normalized and log 2 transformed by GCRMA to calculate differences in the expression level between distinct sample types (three types: primary cells and Lgr5-positive cells, Lgr5-positive cells and Lgr5-negative cells, and Lgr5-negative cells and primary cells). The criteria used for selecting differently expressed genes were:
  • genes of GO:0005886 [plasma membrane] were extracted from GeneOntology (GO). Then, the present inventors extracted genes with GO:0005576 [extracellular region], GO:0009986 [cell surface], and GO:0016020 [membrane], or genes which are predicted to have a transmembrane region by membrane protein prediction software TMHMM and to have a signal peptide by signal peptide prediction software SignalP, and which do not have GO:0031090 [organelle membrane].
  • the present inventors exclude genes whose expression levels are relatively high in normal tissues or primary cells as well as gene only showing a small fold-change in Lgr5-positive or Lgr5-negative cells.
  • Table 6-2 is a continuation of Table 6-1.
  • NP_066924.1 CLDN1 claudin 1 1.4 0.7 3.4 3.5
  • NP_066939.1 ADCY1 adenylate cyclase 1 (brain) 2.9 2.2 2.1 1.4 —
  • Table 6-3 is a continuation of Table 6-2.
  • elegans 0.8 1.6 1.1 3.1 — NP_658985.2 APOA1BP apolipoprotein A-1 binding protein 1.8 1.3 2.2 1.6 — NP_940852.3 APOOL apolipoprotein O-like 1.6 1.6 2.3 2.3 — NP_647537.1 ATRN attractin 1.5 2.1 1.1 1.8 — NP_001193.2 BMP4 bone morphogenetic protein 4 2.9 4.2 1.9 2.8 9 NP_001720.1 BTC betacellulin 1.4 2.5 1.7 3.2 — NP_001224.1 CAV2 caveolin 2 1.9 2.9 3.7 4.2 — NP_001788.2 CDH11 cadherin 11, type 2, OB-cadherin (osteoblast) 6.2 2.4 3.8 0.6 — NP_857592.1 CKLF chemokine-like factor 0.9 1.6 ⁇ 0.8 1.1 — NP_612419.1 CMTM7 CKLF-like MARVEL trans
  • Table 6-4 is a continuation of Table 6-3.
  • Table 6-5 is a continuation of Table 6-4.
  • Table 6-6 is a continuation of Table 6-5.
  • Table 6-7 is a continuation of Table 6-6.
  • Table 6-8 is a continuation of Table 6-7.
  • Table 6-9 is a continuation of Table 6-8.
  • NP_002949.2 RYK RYK receptor-like tyrosine kinase 1.5 1.0 1.2 1.2 — NP_116250.3 SERAC1 serine active site containing 1 1.8 0.8 2.7 1.5 — NP_005016.1 SERPINI1 serpin peptidase inhibitor, clade I (neuroserpin), 5.5 8.7 2.1 4.2 — member 1 NP_008927.1 SLC19A2 solute carrier family 19 (thiamine transporter), member 2 3.8 2.7 2.0 1.2 — NP_065075.1 SLC39A10 solute carrier family 39 (zinc transporter), member 10 2.9 3.1 2.6 2.7 — NP_060306.3 SLC41A3 solute carrier family 41, member 3 1.9 1.5 1.3 0.8 —
  • Table 6-10 is a continuation of Table 6-9.
  • Table 7-2 is a continuation of Table 7-1.
  • Table 7-3 is a continuation of Table 7-2.
  • Table 7-4 is a continuation of Table 7-3.
  • NP_055452.3 MTFR1 mitochondrial fission regulator 1 1.6 0.9 0.2 ⁇ 0.1 — NP_005947.3 MTHFD1 methylenetetrahydrofolate dehydrogenase (NADP+ 1.7 1.6 0.3 0.1 — dependent) 1, methenyltetrahydrofolate cyclohydrolase, formyltetrahydrofolate synthetase NP_005366.2 MYB v-myb myeloblastosis viral oncogene homolog (avian) 2.7 2.3 ⁇ 4.4 0.2 — NP_078938.2 NAT10 N-acetyltransferase 10 (GCN5-related) 1.9 1.3 0.1 ⁇ 0.1 — NP_777549.1 NDUFAF2 NADH dehydrogenase (ubiquinone) 1 alpha 1.0 1.4 ⁇ 0.9 0.1 — subcomplex, assembly factor 2
  • Table 7-5 is a continuation of Table 7-4.
  • Table 8-2 is a continuation of Table 8-1.
  • genes that meet a criterion described below and have GO:0005886 [plasma membrane] from GeneOntology (GO) were extracted in order to obtain genes encoding proteins that are specifically presented on cell membrane of cancer stem cells.
  • Quiescent CSC-specific markers genes whose expression levels are in average greater than 64 in Lgr5-negative cells and are in average less than 64 in both primary cells and Lgr5-positive cells; which show a greater than 20 fold change in Lgr5-negative cells relative to Lgr5-positive cells; and which show a significant difference by t-test (Table 10).
  • Lgr5-positive adherent cells and Lgr5-negative adherent cells resulting from induction by irinotecan treatment were harvested using Accutase.
  • the cells were suspended in FACS buffer, and then incubated at 4° C. for 30 minutes with mouse mAb to human EREG. After the cells were washed once with FACS buffer, 7-AAD Viability Dye as dead cell staining and a PE-labeled goat F(ab′)2 fragment to mouse IgG (H+L) as a secondary antibody were added thereto. The cells were incubated at 4° C. for 30 minutes. Then, the cells were washed once with FACS buffer, and subjected to flow cytometry analysis. Flow cytometry analysis was performed using EPICS ALTRA. Cells negative for 7-AAD Viability Dye were analyzed for EREG expression. The result showed that the corresponding protein was expressed at a high level on cell membrane surface.
  • FIG. 37 The result obtained by EREG flow cytometry analysis of primary cells from PLR59 and PLR123, and Lgr5 + and Lgr5 ⁇ cancer stem cells is shown in FIG. 37 .
  • the cells were stained using an antibody against EREG and analyzed by flow cytometry. It was demonstrated that primary cells were negative for EREG while Lgr5 + and Lgr5 ⁇ cancer stem cells were homogeneous EREG-positive cell populations. Gray indicates fluorescence intensity after cell staining with an indicated antibody; and white indicates fluorescence intensity after cell staining with a control isotype antibody.
  • a mononuclear cell fraction collected from human peripheral blood was used as human effector cells.
  • Fifty ml of peripheral blood was collected from a healthy volunteer (adult male) of the inventors' company using a syringe loaded in advance with 200 ⁇ l of 1000 units/ml heparin solution (Novo-Heparin 5,000 units/5 ml for Injection; Novo Nordisk).
  • the peripheral blood was diluted twofold with PBS( ⁇ ), and then introduced into a Leucosep lymphocyte separation tube (Greiner bio-one) in advance loaded with Ficoll-Paque PLUS and subjected to centrifugation. After centrifugation (2150 rpm, room temperature, 10 minutes), the monocyte fraction layer was collected from the tube.
  • the cells were washed once with 10% FBS/D-MEM, and then suspended at a cell density of 5 ⁇ 10 6 /ml in 10% FBS/D-MEM. The suspension was used as an effector cell suspension.
  • Target cell suspensions were prepared at the time of use.
  • One ⁇ 10 6 cells of cancer lines were centrifuged (1200 rpm, room temperature, 5 minutes).
  • the cell pellets were suspended in 200 ⁇ l of 0.2 mg/ml calcein-AM (Nacalai Tesque)/DMEM (10% FBS) medium.
  • Cell suspensions in calcein-AM solution were incubated for two hours in a CO 2 incubator set to 37° C. and to a CO 2 concentration of 5%. After washing once with 10% FBS/D-MEM, the cells were adjusted to a cell density of 2 ⁇ 10 5 /ml with 10% FBS/D-MEM to prepare target cell suspensions.
  • Anti-EREG antibody was prepared at a concentration of 0.5 mg/ml, which was further diluted with 10% FBS/D-MEM to give antibody solutions. The final concentration was adjusted to 0.4, 4, and 40 ⁇ g/ml.
  • the antibody solutions of respective concentrations or 10% FBS/D-MEM were each added at 50 ⁇ l/well to a 96-well round-bottomed plate. Then, the target cell suspensions were added at 50 ⁇ l/well to every well. The plate was incubated at room temperature for 15 minutes. Next, 100 ⁇ l of the effector cell suspension was added to each well containing target cell suspension, and antibody solution or 10% FBS/D-MEM.
  • cytotoxicity(%) ( A ⁇ C ) ⁇ 100/( B ⁇ C ) Formula 1:
  • A represents the fluorescence intensity in each well
  • B represents the mean value of fluorescence intensity in a well where 50 ⁇ l of target cell suspension and 100 ⁇ l of NP-40 solution were added to 50 ⁇ l of 10% FBS/D-MEM
  • C represents the mean value of fluorescence intensity in a well where 50 ⁇ l of target cell suspension and 100 ⁇ l of 10% FBS/D-MEM were added to 50 ⁇ l of 10% FBS/D-MEM.
  • the anti-EREG antibody-mediated ADCC activities against Lgr5-positive and -negative cells derived from PLR59 cells, and those against Lgr5-positive and -negative cells derived from PLR123 cells are shown in FIG. 38 .
  • the result showed that the anti-EREG antibody exerted cytotoxic activity against both Lgr5-positive and -negative cancer stem cells from PRL59 or PLR123 in a dose dependent manner whereas the control antibody had no cytotoxic activity.
  • EREG Lgr5-positive cells were administered into the peritoneal cavities of NOG mice.
  • EREG was expressed at a high level.
  • EREG expression was somewhat localized to the budding clusters rather than ductal structures.
  • EREG-positive cells were detected even after irinotecan administration to tumor-bearing mice ( FIG. 54 ).
  • the anti-EREG antibody was assessed for anti-tumor activity after irinotecan treatment. Effector cells are essential for the anti-EREG antibody to mediate ADCC activity.
  • SCID mice were used as a model to assess the pharmacological efficacy of the anti-EREG antibody. Tumor growth was suppressed when the antibody was administered at the time points of days 4 and 11 after the final irinotecan administration ( FIG. 57 ).
  • EREG EREG is expressed in the metastasis model.
  • Lgr5-positive cells were intravenously injected into NOG mice, tumors were formed in multiple tissues including lung. Cells of the tumors formed in lungs are mostly positive for EREG ( FIG. 58A ).
  • the pharmacological efficacy of the anti-EREG antibody was assessed using SCID-Beige mice where macrophages and mononuclear cells can serve as effector cells to mediate ADCC.
  • the anti-EREG antibody was administered to mice once a week for a total of five times starting at three days after the injection of Lgr5-positive cells.
  • the number of tumor cells in distal locations was demonstrated to be markedly reduced as compared to that in control mice ( FIG. 58B ).
  • the size of each tumor was also shown to be remarkably reduced in mice administered with the antibody ( FIGS. 58C and 58D ).
  • Proliferating and quiescent CSCs were identified by immunohistochemistry using anti-Lgr5 antibody (2U2E-2), anti-HLA-DMA antibody, and anti-EREG antibody ( FIG. 59 and Table 11).
  • Proliferative CSC represents Lgr5-positive cell
  • quiescent CSC represents HLA-DMA-positive and EREG-positive cell
  • Lgr5-positive cells which are positive for both HLA-DMA and EREG and Lgr5-negative cells which are positive for both HLA-DMA and EREG were present in a very small number in primary and metastatic colorectal cancer specimens isolated from colorectal cancer patients ( FIG. 59 ).
  • Lgr5-positive and -negative cells were detected in eight of 12 specimens of human colorectal cancer tissues. Meanwhile, either Lgr5-positive or Lgr5-negative cells were observed in the remaining four specimens. Throughout all specimens, Lgr5-positive cells accounted for 0.003 to 1.864%, and Lgr5-negative cells accounted for 0.001 to 10.243% (Table 11).
  • Lgr5-positive and -negative CSCs were detected in the ductal and budding areas ( FIG. 59 ). Furthermore, in ducts, Lgr5-positive and -negative CSCs were not limited to particular areas but distributed at random over the entire ducts.
  • the present inventors tested whether an anti-tumor effect can be expected with target therapy using a membrane protein expressed at a high level as the target in irinotecan-treated or non-treated PLR59 and PLR123.
  • Commercially available antibodies shown in Table 12 were assessed by flow cytometry (FCM) for the binding activity to antigens expressed on the cell surface of irinotecan-treated or non-treated PLR59 or PLR123. The result is summarized in Table 13.
  • Mab-ZAP and Rat-ZAP are anti-mouse IgG antibody and anti-rat IgG antibody, respectively, conjugated with saporin, a toxin that inhibits protein synthesis (Advanced Targeting Systems).
  • PLR59 and PLR123 cells were seeded at a cell density of 30000 cells/80 ⁇ l/well to respective wells of a 96-well plate. Following day, each antibody solution was added at a final concentration of 0.01, 0.1, or 1 ⁇ g/ml to the respective wells.
  • Mab-ZAP or Rat-ZAP was added at a final concentration 1 ⁇ g/ml to the respective wells, and the plate was incubated at 37° C. for 72 hours in a CO 2 incubator.
  • PLR59 and PLR123 cells were seeded in a 96-well plate, and irinotecan was added at a final concentration of 15 ⁇ M to each well. The plate was incubated at 37° C. for 72 hours in a CO 2 incubator.
  • Various antibodies were assessed for the activity of internalization to cells cultured as described above in the presence or absence of irinotecan.
  • the various antibodies were each assessed for the internalization activity into cells contained in each well where the medium was replaced with the same irinotecan-free medium as used for irinotecan-non-treated cells. Seventy-two hours after addition of antibodies and Mab-ZAP or Rat-ZAP, 10 ⁇ l of 3% SDS (Nacalai Tesque) was added to each well of the plate. The cells in the plate were lysed thoroughly by stirring the plate using a plate mixer. Then, 100 ⁇ l of CellTiter-GloTM Luminescent Cell Viability Assay (Promega) was added to each well. The mixture in each well was assayed to determine its luminescent signal.
  • SDS Nacalai Tesque
  • the determined anti-tumor activity is shown in Table 14 and FIGS. 60 to 72 .
  • the percent suppression of cell proliferation indicated by the vertical axis represents a relative value for the difference in the luminescence signal value between the mixtures in wells, one of which contained a test antibody alone (without Mab-ZAP and Rat-ZAP) and the other contained a test antibody, and Mab-ZAP or Rat-ZAP, when taking as 100% the difference in the luminescence signal value between the mixtures in wells, one of which contained a test antibody alone (without Mab-ZAP and Rat-ZAP) and the other did not contain any cells.
  • symbols, ⁇ , +, ++, and +++ represent relative values for the internalization activity when a test antibody was assayed at a concentration of 1 ⁇ g/ ⁇ l.
  • the relative value refers to a relative value for the difference in the luminescence signal value between the mixtures in wells, one of which contained a test antibody alone (without Mab-ZAP and Rat-ZAP) and the other contained a test antibody, and Mab-ZAP or Rat-ZAP, when taking as 100% the difference in the luminescence signal value between the mixtures in wells, one of which contained a test antibody alone (without Mab-ZAP and Rat-ZAP) and the other did not contain any cells.
  • Symbols, ⁇ , +, ++, and +++ indicate 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.
  • anti-CD70 antibody and anti-FAS antibody showed the internalization activity level of 25% or more under a condition where the anti-EPCAM antibody used as a positive control had been demonstrated to exhibit a sufficient anti-tumor activity ( FIGS. 60 and 62 ).
  • anti-EDAR antibody showed the internalization activity level of 15 to 25%
  • anti-PVRL4 antibody and anti-PROCR antibody exhibited the internalization activity level of 5 to 15% ( FIGS. 61 , 63 , and 65 ).
  • anti-FAS antibody and anti-TNFRSF9 antibody showed the internalization activity level of 25% or more, and anti-PROM2 antibody exhibited the internalization activity level of 5 to 15% ( FIGS. 67 , 70 , and 68 ).
  • Both anti-PVRL4 antibody and anti-PROCR antibody showed the internalization activity for irinotecan-treated PLR59 and PLR123, and the activity for PLR59 was greater than that for PLR123 ( FIGS. 69 and 71 ).
  • the result described above demonstrates that all antibodies tested have an anti-tumor effect against PLR59 and PLR123.
  • BMP4 was assessed for the differentiation-enhancing effect on irinotecan-non-treated and irinotecan-treated PLR59 and PLR123.
  • PLR59 and PLR123 cells suspended in the media where BMP4 (R&D Systems; a final concentration of 20 nM) or a control buffer was added to culture media were seeded at a cell density of 5 ⁇ 10 5 cells/1.5 ml/well to each well of a 12-well plate. The cells were passaged while changing the culture media with the same type of medium 2, 4, and 7 days after seeding.
  • PLR59 or PLR123 was seeded at a cell density of 17 ⁇ 10 5 cells/5 ml/flask to a 12.5-ml culture flask. Following day, irinotecan was added at a final concentration of 15 ⁇ M. The flask was incubated at 37° C. for 72 hours in a CO 2 incubator. Then, the medium in the flask was changed with a medium containing BMP4 or a control buffer. The medium was further changed with the same type of medium 2, 4, and 7 days after the initial medium change.
  • RNAs were extracted using RNeasy Plus Mini Kit and RNase-Free DNase Set (QIAGEN). cDNAs were synthesized with ThermoScript RT-PCR System (Invitrogen) using the extracted RNAs as a template.
  • Quantitative real-time PCR was carried out using the cDNAs isolated as described above. As shown in FIG. 73 , elevated CK20 levels were observed in PLR59 and PLR123 cells cultured in the presence of BMP4.
  • the present inventors identified cell surface molecules that are expressed specifically on cancer stem cells.
  • the present invention provides novel anti-cancer drugs and reagents for detecting cancer stem cells, which use antibodies against the cell surface molecules.

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