WO2011105551A1

WO2011105551A1 - Method for detection of cancer stem cell, and therapeutic agent or recurrence-preventing agent for cancer

Info

Publication number: WO2011105551A1
Application number: PCT/JP2011/054287
Authority: WO
Inventors: 正樹森; 直紹原口; 秀始石井
Original assignee: 国立大学法人大阪大学
Priority date: 2010-02-26
Filing date: 2011-02-25
Publication date: 2011-09-01

Abstract

A functional molecule involved in the regulation of cancer stem cells is newly identified. Thus, disclosed are: a technique for detecting cancer stem cells accurately; and a technique for inducing the apoptosis of cancer stem cells to completely cure cancer. CD13 is involved in the regulation of an active oxygen species removal pathway in a cancer stem cell, and is therefore a molecule essential to the maintenance of cancer stem cells. Therefore, cancer stem cells can be detected accurately using CD13 as an indicator. Further, the radical treatment of cancer (particularly liver cancer and gastrointestinal cancer) can be achieved using a CD13 inhibitor and an anti-cancer agent or a radiation therapy in combination.

Description

Cancer stem cell detection method and cancer therapeutic or recurrence preventive agent

The present invention relates to a method for detecting cancer stem cells and a drug for treating or preventing cancer recurrence.

Cancer cells have the property of being capable of self-proliferation and being able to wet to surrounding tissues or metastasize to distant tissues. However, not all of the cancer cells forming the cancer tissue have these characteristics, and there are very few cancer cells that develop cancer or advance cancer. It is known to be a cancer stem cell. Cancer stem cells exhibit undifferentiated surface characteristics like normal stem cells, have self-renewal ability and differentiation ability, and have the property of producing all cancer cells in various differentiation stages constituting cancer tissue. That is, cancer stem cells are thought to be responsible for generating the majority of cancer cells by differentiation while maintaining the same cells as themselves by self-replication in cancer tissues.

In order to embody the purpose of cancer suppression, it is considered important to clarify the properties of cancer stem cells and to suppress cancer stem cells. Therefore, if a technique capable of accurately detecting cancer stem cells can be established, it will be extremely useful not only for the cure of cancer but also for the prevention of cancer and the prediction of risk, and the impact on the medical community will be strong.

Conventionally, molecules that have been used as indicators for detecting cancer stem cells have no functionality as cancer stem cells, but are merely indicators (those that have functionality have nothing to do with functions as cancer stem cells) There are also functional molecules involved in the control of cancer stem cells. Several candidate molecules have been reported for the former, but no firm candidate molecules have been reported for the latter. For example, although it has been reported that CD133, CD44, CD90 and the like are indicators of cancer stem cells (see, for example, Non-Patent Documents 1 to 6), in the technology for detecting only these molecules as indicators of cancer stem cells, The detection accuracy of cancer stem cells is insufficient. For this reason, it is eagerly desired to establish a technique for accurately detecting cancer stem cells by newly identifying functional molecules involved in the control of cancer stem cells.

An object of the present invention is to provide a technique for accurately detecting cancer stem cells by newly identifying functional molecules involved in the control of cancer stem cells. Another object of the present invention is to provide a technique for leading to DNA fragmentation and apoptosis for cancer stem cells and curing the cancer, that is, a radical therapeutic agent for cancer (cancer radical treatment) and a treatment method (cancer radical treatment method). To do. A further object of the present invention is to provide a technique for preventing recurrence of cancer by leading to DNA fragmentation and apoptosis of cancer stem cells and curing the cancer, that is, a cancer recurrence preventive agent and a recurrence preventive method. .

The inventors of the present invention have made extensive studies to solve the above-mentioned problems. As a result, CD13 is involved in the control of the reactive oxgen species (ROS) removal pathway in cancer stem cells and maintains cancer stem cells. It was found that cancer stem cells are CD13-positive cells (CD13 ⁺ cells), and that cancer stem cells can be accurately detected by using CD13 as an index. Furthermore, by combining conventional cancer treatments such as chemotherapy and ionizing radiation with treatment with a compound having an action of inhibiting CD13 (hereinafter referred to as “CD13 inhibitor”), the ROS concentration is low and the cell cycle is reduced. It is possible to target cancer stem cells that have the characteristic of being in the dormant or late state and exhibit extremely high treatment resistance, and as a result, the therapeutic effect of cancer can be significantly improved In particular, it was found to be effective in the cure of cancer and prevention of recurrence. In other words, this means that a combination of a CD13 inhibitor and an anticancer agent or ionizing radiation treatment can fundamentally treat cancer and prevent cancer recurrence, in other words, a CD13 inhibitor and cancer therapy ( It means that combined use with anticancer drug administration or ionizing radiation treatment is effective as a cancer radical treatment method or a cancer recurrence prevention therapy. Further, CD13 ⁺ cells are suppressed in DNA double-strand damage or have activated double-strand damage repair ability (see FIG. 14), so a CD13 inhibitor and, for example, 5-fluorouracil, etc. Thus, it is considered that a higher cancer curative effect or cancer recurrence preventing effect can be obtained by using in combination with an anticancer agent having a DNA synthesis inhibitory action. Similarly, an anthracycline anticancer agent doxorubicin having a very high ROS increase effect is used in combination to enhance the ROS excretion effect of the CD13 inhibitor, thereby increasing the ROS by the CD13 inhibitor. It is possible to damage cancer stem cells in the induced state with very high efficiency, and to lead to the cure of cancer.

The present invention has been completed by further studies based on such knowledge.

That is, this invention provides the invention of the aspect hung up below.
(I) Cancer stem cell detection method (I-1). A method for detecting cancer stem cells, comprising measuring a cell expressing CD13 for a test cell derived from a cancer tissue or a tissue after cancer treatment.
(I-2). The detection method according to (I-1), wherein the measurement of cells expressing CD13 is performed using an antibody capable of specifically binding to CD13.
(I-3). The detection method according to (I-2), wherein the antibody is labeled with a labeling substance selected from a fluorescent substance and an enzyme.
(I-4). The detection method according to any one of (I-1) to (I-3), which comprises a step of measuring cells expressing CD90 together with CD13.
(I-5). The detection method according to (I-4), wherein measurement of cells expressing CD90 is performed using an antibody capable of specifically binding to CD90.
(I-6). The detection method according to (I-5), wherein the antibody is labeled with a labeling substance selected from a fluorescent substance and an enzyme.
(I-7). The detection method according to any one of (I-1) to (I-6), wherein measurement of cells expressing CD13 or CD90 is performed by a flow cytometry method.

(II) Method of measuring the degree or risk of cancer symptoms (II-1). A method of measuring the degree of cancer symptoms (cancer severity) or cancer risk of a subject,
(A) a step of measuring a cell expressing CD13 for a test cell derived from a cancer tissue of a subject or a tissue after cancer treatment, and (B) a CD13-expressing cell measured in the step (A) Determining the cancer severity or cancer risk according to the number of
The above-mentioned measuring method characterized by including.
(II-2). In the step (B), when the number of CD13-expressing cells measured in the step (A) is large, the cancer severity and cancer risk are high, and when the number of CD13-expressing cells is small, the cancer severity and The measurement method according to (II-1), which is a step of determining that the cancer risk is low.
(II-3). The measurement method according to (II-1) or (II-2), wherein the measurement of CD13-expressing cells is performed using an antibody capable of specifically binding to CD13.
(II-4). The measurement method according to (II-3), wherein the antibody is labeled with a labeling substance selected from a fluorescent substance and an enzyme.
(II-5). (A) The measuring method according to any one of (II-1) to (II-4), wherein the step is a step of measuring cells expressing CD90 together with CD13.
(II-6). The measurement method according to (II-5), wherein the measurement of cells expressing CD90 is performed using an antibody capable of specifically binding to CD90.
(II-7). The measurement method according to (II-6), wherein the antibody is labeled with a labeling substance selected from a fluorescent substance and an enzyme.
(II-8). The measurement method according to any one of (II-1) to (II-7), wherein measurement of cells expressing CD13 or CD90 is performed by a flow cytometry method.
(II-9). The measurement method according to any one of (II-1) to (II-8), wherein the cancer severity or cancer risk is the probability that the cancer will recur.

(III) Method for measuring the therapeutic effect of cancer (III-1). A method for measuring the therapeutic effect of cancer on a cancer patient,
(A) a step of measuring a cell expressing CD13 in a test cell derived from a tissue after cancer treatment of a cancer patient, and (b) the presence or absence of a CD13-expressing cell measured in the step (a) Depending on the step of determining the therapeutic effect of the cancer,
The above-mentioned measuring method characterized by including.
(III-2). The step (b) is a step of determining that the cancer treatment effect is poor when CD13-expressing cells are detected in the step (a) and that the cancer treatment effect is good when CD13-expressing cells are not detected. The prediction method described in (III-1).
(III-3). The measurement method according to (III-1) or (III-2), wherein measurement of CD13-expressing cells is performed using an antibody capable of specifically binding to CD13.
(III-4). The measurement method according to (III-3), wherein the antibody is labeled with a labeling substance selected from a fluorescent substance and an enzyme.
(III-5). (A) The measuring method according to any one of (III-1) to (III-4), wherein the step is a step of measuring cells expressing CD90 together with CD13.
(III-6). The measurement method according to (III-5), wherein the measurement of cells expressing CD90 is performed using an antibody capable of specifically binding to CD90.
(III-7). The measurement method according to (III-6), wherein the antibody is labeled with a labeling substance selected from a fluorescent substance and an enzyme.
(III-8). The measurement method according to any one of (III-1) to (III-7), wherein measurement of cells expressing CD13 or CD90 is performed by a flow cytometry method.

(IV) Method of measuring the risk of cancer recurrence (IV-1). A method for measuring the risk of cancer recurrence after cancer treatment for a cancer patient,
(A ′) a step of measuring a cell expressing CD13 in a test cell derived from a tissue after cancer treatment of a cancer patient, and (b ′) CD13 expression measured in the step (a ′) Determining the cancer recurrence risk of the cancer patient according to the presence or absence of cells,
The above-mentioned measuring method characterized by including.
(IV-2). In the above step (b ′), when CD13-expressing cells are detected in the step (a ′), the cancer patient is determined to have a high risk of cancer recurrence, and when CD13-expressing cells are not detected, The prediction method according to (IV-1), which is a step of determining that the cancer patient has a low risk of cancer recurrence.
(IV-3). The measurement method according to (IV-1) or (IV-2), wherein measurement of CD13-expressing cells is performed using an antibody capable of specifically binding to CD13.
(IV-4). The measurement method according to (IV-3), wherein the antibody is labeled with a labeling substance selected from a fluorescent substance and an enzyme.
(IV-5). (A) The measurement method according to any one of (IV-1) to (IV-4), wherein the step is a step of measuring cells expressing CD90 together with CD13.
(IV-6). The measurement method according to (IV-5), wherein measurement of cells expressing CD90 is performed using an antibody capable of specifically binding to CD90.
(IV-7). The measurement method according to (IV-6), wherein the antibody is labeled with a labeling substance selected from a fluorescent substance and an enzyme.
(IV-8). The measurement method according to any one of (IV-1) to (IV-7), wherein measurement of cells expressing CD13 or CD90 is performed by a flow cytometry method.

(V) Cancer stem cell detection reagent and detection kit (V-1). A cancer stem cell detection reagent comprising a substance that specifically binds to CD13 (hereinafter referred to as “CD13-binding substance”) as an active ingredient.
(V-2). At least one selected from the group consisting of an antibody (hereinafter referred to as “anti-CD13 antibody”), a microRNA, an RNA aptamer, and a dominant negative mutant, wherein the CD13 binding substance can specifically bind to CD13. The detection reagent according to (V-1).
(V-3). A cancer stem cell detection kit comprising a CD13 binding substance as a cancer stem cell detection reagent.
(V-4). The detection kit according to (V-3), further comprising a substance that specifically binds to CD90 (hereinafter referred to as “CD90-binding substance”).
(V-5). At least one selected from the group consisting of an antibody (hereinafter referred to as “anti-CD90 antibody”), a microRNA, an RNA aptamer, and a dominant negative mutant, wherein the CD90-binding substance can specifically bind to CD90. The detection kit according to (V-4).

(VI) Cancer therapeutic agents, drug kits for cancer treatment, and cancer radical treatment methods (VI-1). A cancer therapeutic agent comprising a CD13 inhibitor and an anticancer agent.
(VI-2). The cancer therapeutic agent according to (VI-1), wherein the CD13 inhibitor is at least one selected from the group consisting of a CD13 neutralizing antibody and Ubenimex.
(VI-3). The cancer therapeutic agent according to (VI-1) or (VI-2), wherein the anticancer agent is a DNA synthesis inhibitor or an anticancer agent having an ROS increasing action.
(VI-4). The cancer therapeutic agent according to (VI-3), wherein the DNA synthesis inhibitor is 5-fluorouracil, a prodrug thereof, doxorubicin or a salt thereof.
(VI-5). The cancer therapeutic agent according to (VI-3), wherein the anticancer agent having an action of increasing ROS is an anthracycline anticancer agent, preferably doxorubicin or a pharmaceutically acceptable salt thereof.
(VI-6). The cancer therapeutic agent according to any one of (VI-1) to (VI-5), which is a radical therapeutic agent for cancer.
(VI-7). Any of (VI-1) to (VI-6), wherein the cancer to be treated is a solid cancer expressing CD13, preferably liver cancer, lung cancer and gastrointestinal cancer, more preferably liver cancer and colon cancer The anticancer agent as described in.
(VI-8). A pharmaceutical kit for treating cancer, comprising a first agent containing a CD13 inhibitor and a second agent containing an anticancer agent in separate packaging forms.
(VI-9). The kit according to (VI-8), wherein the CD13 inhibitor is at least one selected from the group consisting of a CD13 neutralizing antibody and Ubenimex.
(VI-10). The kit according to (VI-8) or (VI-9), wherein the anticancer agent is a DNA synthesis inhibitor or an anticancer agent having an ROS increasing action.
(VI-11). The kit according to any one of (VI-8) to (VI-10), which is a pharmaceutical kit for radical treatment of cancer.
(VI-12). Any of (VI-7) to (VI-11), wherein the cancer to be treated is a solid cancer expressing CD13, preferably liver cancer, lung cancer and gastrointestinal cancer, more preferably liver cancer and colon cancer The kit according to 1.
(VI-13). Having a step of administering a CD13 inhibitor and an anticancer agent (preferably a DNA synthesis inhibitor or an anticancer agent having an ROS-raising action) to a cancer patient, or a step of performing a CD13 inhibitor administration treatment and an ionizing radiation treatment to a cancer patient , A radical cure for cancer.
(VI-14). The method according to (VI-13), wherein the CD13 inhibitor is at least one selected from the group consisting of a CD13 neutralizing antibody and Ubenimex.
(VI-15). The method according to (VI-13) or (VI-14), wherein the anticancer agent is a DNA synthesis inhibitor or an anticancer agent having an ROS increasing action.
(VI-16). Use of a combination of a CD13 inhibitor and an anticancer agent, preferably a combination of a CD13 inhibitor and a DNA synthesis inhibitor or an anticancer agent having an ROS-elevating action, for the preparation of a radical therapeutic agent for cancer.
(VI-17). A combination of a CD13 inhibitor and an anticancer agent, preferably a combination of a CD13 inhibitor and a DNA synthesis inhibitor or an anticancer agent having an ROS-raising action, which is used for radically treating cancer.
(VI-18). A combination preparation comprising a combination of a CD13 inhibitor and an anticancer agent (preferably a DNA synthesis inhibitor or an anticancer agent having an ROS-raising action) containing a CD13 inhibitor and an anticancer agent (preferably a DNA synthesis inhibitor or an anticancer agent having an ROS-raising action). Or a combination according to (VI-17), which is a pharmaceutical kit containing a CD13 inhibitor and an anticancer agent (preferably a DNA synthesis inhibitor or an anticancer agent having a ROS-elevating action) in a separate packaging form.

(VII) Cancer recurrence preventive agent, cancer recurrence prevention pharmaceutical kit and cancer recurrence prevention method (VII-1). A cancer recurrence preventive agent comprising a CD13 inhibitor and an anticancer agent.
(VII-2). The cancer recurrence preventive agent according to (VII-1), wherein the CD13 inhibitor is at least one selected from the group consisting of a CD13 neutralizing antibody and Ubenimex.
(VII-3). The cancer recurrence preventive agent according to (VII-1) or (VII-2), wherein the anticancer agent is a DNA synthesis inhibitor or an anticancer agent having an ROS increasing action.
(VII-4). The cancer recurrence preventive agent according to (VII-3), wherein the DNA synthesis inhibitor is 5-fluorouracil, a prodrug thereof, doxorubicin or a salt thereof.
(VII-5). The cancer recurrence preventive agent according to (VII-3), wherein the anticancer agent having an ROS-elevating action is an anthracycline-based anticancer agent, preferably doxorubicin or a pharmaceutically acceptable salt thereof.
(VII-6). The target cancer is a solid cancer expressing CD13, preferably liver cancer, lung cancer or gastrointestinal cancer, more preferably liver cancer or colon cancer, any of (VII-1) to (VII-5) For preventing cancer recurrence.
(VII-7). A pharmaceutical kit for preventing cancer recurrence, comprising a first agent containing a CD13 inhibitor and a second agent containing an anticancer agent.
(VII-8). The kit according to (VII-6), wherein the CD13 inhibitor is at least one selected from the group consisting of a CD13 neutralizing antibody and Ubenimex.
(VII-9). The kit according to (VII-7) or (VII-8), wherein the anticancer agent is a DNA synthesis inhibitor or an anticancer agent having an ROS increasing action.
(VII-10). The kit according to any one of (VII-6) to (VII-9), wherein the target cancer is a solid cancer expressing CD13, preferably liver cancer, lung cancer and gastrointestinal cancer, more preferably liver cancer and colon cancer.
(VII-11). Having a step of administering a CD13 inhibitor and an anticancer agent (preferably a DNA synthesis inhibitor or an anticancer agent having an ROS-raising action) to a cancer patient, or a step of performing a CD13 inhibitor administration treatment and an ionizing radiation treatment to a cancer patient How to prevent cancer recurrence.
(VII-12). The method according to (VII-11), wherein the CD13 inhibitor is at least one selected from the group consisting of a CD13 neutralizing antibody and Ubenimex.
(VII-13). The method according to (VII-11) or (VII-12), wherein the anticancer agent is a DNA synthesis inhibitor or an anticancer agent having an ROS increasing action.
(VII-14). Use of a combination of a CD13 inhibitor and an anticancer agent, preferably a combination of a CD13 inhibitor and a DNA synthesis inhibitor or an anticancer agent having an ROS-raising action, for the preparation of an agent for preventing cancer recurrence.
(VII-15). A combination of a CD13 inhibitor and an anticancer agent, preferably a combination of a CD13 inhibitor and a DNA synthesis inhibitor or an anticancer agent having an ROS-raising action, used for preventing cancer recurrence.
(VII-16). A combination preparation comprising a combination of a CD13 inhibitor and an anticancer agent (preferably a DNA synthesis inhibitor or an anticancer agent having an ROS-raising action) containing a CD13 inhibitor and an anticancer agent (preferably a DNA synthesis inhibitor or an anticancer agent having an ROS-raising action). Or a combination described in (VII-15), which is a pharmaceutical kit containing a CD13 inhibitor and an anticancer agent (preferably a DNA synthesis inhibitor or an anticancer agent having a ROS-elevating action) in a separate packaging form.

According to the detection method of the present invention, cancer stem cells can be accurately detected. Therefore, the present invention can contribute to the improvement of cancer treatment technology, and is an innovative that is extremely useful for predicting the degree of cancer symptoms such as cancer occurrence, progression, metastasis, and recurrence or the risk of cancer. Can be technology.

In addition, according to the cancer therapeutic agent or cancer treatment pharmaceutical kit of the present invention, by using a CD13 inhibitor in combination with an anticancer agent, preferably a DNA synthesis inhibitor, not only cancer cells but also cancer stem cells are eliminated, Since cancer can be suppressed, it is also possible to provide a cancer root treatment method and a cancer root treatment method that exhibit an unprecedented excellent therapeutic effect. Further, according to the cancer recurrence preventive agent or the cancer recurrence prevention pharmaceutical kit of the present invention, cancer recurrence is prevented by eradicating cancer cells or cancer stem cells survived by conventional cancer therapy, and thus the survival rate of cancer patients. Can be significantly improved.

A: It is a flowchart which shows the operation procedure which performed "1. Confirmation that CD13 is a marker candidate of a cancer stem cell" in an Example. B: It is a figure which shows the expression intensity | strength in SP fraction in a human hepatoma cell, and a non- SP fraction about two marker candidates of CD13 and CD31, respectively. C and D: shows the results of analyzing the expression of CD13, CD133, and CD90 in hepatitis infection negative cell line (HuH7) and hepatitis infection positive cell line (PLC / PRF / 5: described as “PLC” in the figure) FIG. It is a figure which shows the result of the term of "1. Confirmation that CD13 is a cancer stem cell marker candidate" in an Example. A: The results of examining the expression status of CD13 and CD31 in HuH7, Hep3B, and PLC / PRF / 5 are shown. B: shows the relationship between CD13 and Hu fraction in HuH7. It can be seen that CD31 ⁺ cells are mainly present in the G2 / M / SP fraction. It is a figure which shows the result of the term of "2. Confirmation that CD13 is a marker of the hepatocellular carcinoma (HCC) cell in a dormant stage" in an Example. A: The result of examining the relationship between the expression of CD13 of HuH7 and PLC (PLC / PRF / 5) and the cell cycle of HuH7 and PLC (PLC / PRF / 5). B: Results of comparing cell proliferation of CD13 ⁺ CD133 ⁺ cells, CD13 ⁻ CD133 ⁺ cells, and CD13 ⁻ CD133 ⁻ cells in order to evaluate the relationship between the cell cycle and the expression of CD13. C: Results of analysis of cell fate and dye-retaining capacity of HuH7 CD13 ⁺ cells. D: Results of multicolor analysis and cell cycle analysis by 7-Amino-Actinomycin D (7-AAD) DNA labeling for CD13 ⁺ CD90 ⁻ cells, CD13 ⁺ CD90 ⁺ cells, and CD13 ⁻ CD90 ⁺ cells. FIG. 4 is a view showing the results in the section “3. Confirmation that CD13 is specifically expressed in moderately to poorly differentiated colorectal cancer cells” in Examples. Specifically, the results of immunostaining a moderately to poorly differentiated human colon cancer tissue with an anti-CD13 antibody (anti-human CD13 mouse monoclonal antibody) are shown. (B) is an enlarged view of (A). In the section “4. Confirmation that CD13 ⁺ cells form spheres and create a CD90 ⁺ phenotype” in the Examples section, CD13 in spheres derived from HuH7, PLC / PRF / 5 and clinically obtained HCC It is a figure which shows the result of having examined the expression of. Potentially dormant CD13 ⁺ CD90 ⁻ cells (Dormant Cancer stem Cells: Dormant CSCs) to proliferating CD13 ⁺ CD90 ⁺ cells (Activated CSCs) and CD13 ⁻ CD90 ⁺ cells (Progesitors of Cancer Cells) (these collectively referred to as "CD90 ⁺ cells") is generated, and from several CD90 ⁺ cells with these proliferative, potentially in dormant CD13 ⁺ CD90 ^- schematic diagram showing that the cells have been generated It is. It should be noted that CD13 ⁺ CD90 ⁻ cells are also associated with a decrease in intracellular ROS due to an increase in antioxidant capacity. Therefore, conventional cancer treatments (genotoxic chemotherapy and radiation It shows resistance to radiation therapy. It is a figure which shows the result of the section of "5. Confirmation that CD13 ⁺ cell shows resistance to cancer chemotherapy and radiotherapy" in an Example. A and B: shows that CD13 ⁺ cells are resistant to an anticancer drug (an anticancer drug having a ROS-raising action: doxorubicin hydrochloride (DXR)). C: Indicates that CD13 ⁺ cells are resistant to irradiation. In FIG. 7C, “RT 4G 24h” means 24 hours after irradiation (4 Gray), and “RT 4G 48h” means 48 hours after irradiation (4 Gray). It is a figure which shows the result of the term of "6. Confirmation that CD13 is selectively expressing in the cell which shows treatment tolerance" in an Example. A: In order to identify the expression of CD13 in clinically obtained HCC cells, the results of digesting an HCC sample and analyzing the hematopoietic CD45 (Lin / CD45) negative fraction by multicolor flow cytometry are shown. B: shows the result of confirming the expression of CD13 in a fresh cryosurgical specimen. In FIG. 8, “After TAE” means a sample subjected to hepatic artery embolization therapy, and “Non-TAE” and “Not treatment” mean samples not subjected to hepatic artery embolization therapy. In FIG. 8B, in the left end and middle column images, CD13 is stained red and DAPI is blue. It is a figure which shows the result of the term of "7. Confirmation that CD13 inhibition leads to a cell apoptosis" in an Example. In FIG. 9, “CD13 Ab” means a CD13 neutralizing antibody. In FIG. 9B, “Ube” means Ubenimex. It is a figure which shows the result of the term of "7. Confirmation that CD13 inhibition leads to a cell apoptosis" in an Example. In FIG. 10, “DOX” is synonymous with DXR. It is a figure which shows the result of the term of "8. Confirmation that CD13 inhibition induces tumor regression" in an Example. In FIG. 11, “Ube” means Ubenimex. In the image of A in FIG. 11 and the image in the right column of B, CD13 is stained red, Ki67 is stained green, and DAPI is blue. The results of the "confirmation that the double-stranded DNA damage strand is kept low in 10.CD13 ⁺ cells""9.CD13 ⁺ cells that confirmation containing low levels of ROS" and in Examples FIG. In FIG. 12, “CD13 Ab” means a CD13 neutralizing antibody. In FIG. 12B, “Ube” means Ubenimex. The results of the "confirmation that the double-stranded DNA damage strand is kept low in 10.CD13 ⁺ cells""9.CD13 ⁺ cells that confirmation containing low levels of ROS" and in Examples FIG. In FIG. 13, “RT 4Gy” means a group irradiated with radiation, and “RT4Gy with Tempol” means a group irradiated after tempol treatment. In the sections of “9. Confirmation that CD13 ⁺ cells contain low levels of ROS” and “10. Confirmation that double strand damage to DNA strands is kept low in CD13 ⁺ cells” in the Examples, HuH7 And the result of having measured the intracellular ROS density | concentration after chemotherapy (DNA synthesis inhibitor: 5-FU and the anticancer agent which has a ROS raise effect | action: DXR) using PLC / PRF / 5 with time is shown. In the figure, “dCSC” means “dormant cancer stem cell (s)”. In the sections of “9. Confirmation that CD13 ⁺ cells contain low levels of ROS” and “10. Confirmation that double strand damage to DNA strands is kept low in CD13 ⁺ cells” in the Examples, HuH7 And PLC / PRF / 5 to determine the double strand damage of DNA strands caused by free radicals after radioactive irradiation (4Gy) over time using γ-H2AX as an index The measurement results are shown. The red numbers in the figure are the percentage of γ-H2AX in CD13 ⁺ cells (PLC / PRF / 5: CD13 ⁺ CD90 ⁻ cells, HuH7: CD13 ⁺ CD133 ⁺ cells), and the blue numbers are CD13 ⁻ cells The ratio (%) of γ-H2AX in (PLC / PRF / 5: CD13 ⁻ CD90 ⁺ cells, HuH7: CD13 ⁻ CD133 ⁺ cells) is shown.

1. Method for Detecting Cancer Stem Cell The method for detecting a cancer stem cell of the present invention is characterized by measuring a cell expressing CD13 in a test cell derived from a cancer tissue of a cancer patient or a tissue after cancer treatment. . Hereinafter, the detection method of the present invention will be described in detail.

In the present invention, “cancer stem cell” means a cell that has self-replicating ability and maintains an undifferentiated state and can produce cancer cells by differentiation.

In the present invention, the “test cell” is a cell that is a target of determination of whether or not it expresses CD13, that is, whether or not it is a cancer stem cell. The test cell may be a single cell isolated from cancer tissue of a cancer patient or tissue after cancer treatment, or may be a group of cells isolated from cancer tissue or tissue after cancer treatment. .

The cancer tissue from which the test cell is derived is not particularly limited, and examples thereof include liver cancer, colorectal cancer, esophageal cancer, gastric cancer, bile duct cancer (bile duct includes intrahepatic bile duct and extrahepatic bile duct), Gallbladder cancer (the left two or three are collectively called the biliary tract), pancreatic cancer (the pancreas is divided into duct and secretory gland tissue, the latter includes endocrine and exocrine systems), duodenal cancer, colon cancer (colon Includes ascending, transverse, descending, sigmoid colon, rectum), breast cancer, and solid tumor tissues such as brain tumors; and hematopoietic tumors (blood cancers) such as leukemia, malignant lymphoma, and multiple myeloma Hematopoietic tissue involved is exemplified. Since the present invention is suitable for detection of cancer stem cells contained in liver cancer, among these cancer tissues, examples of preferable cancer tissues include solid cancer tissues, particularly preferably liver cancer tissues.

The tissue after cancer treatment from which the test cells are derived is a tissue after the above cancer treatment, and the cancer treatment treatment is performed by at least one selected from surgical therapy, chemotherapy, and radiation therapy. Any organization after being applied may be used. In addition, when a cancer tissue is excised by surgical therapy, a tissue near the excision site can be targeted.

The test cells to be detected in the detection method of the present invention may be those extracted from the cancer tissue of the cancer patient, or the tissue after cancer treatment of the cancer patient, or the cancer tissue of the cancer patient. Or the thing of the state which exists in the tissue after cancer treatment of a cancer patient may be sufficient. In the latter case, the target tissue may be in the body of the cancer patient, but is preferably in a state of being removed from the body of the cancer patient.

Note that cancer patients targeted by the present invention are mammals including humans. Humans are preferred, but rodents such as mice, rats, and guinea pigs; and laboratory animals such as rabbits, cats, dogs, and monkeys can also be used.

In the detection method of the present invention, cells expressing CD13 are measured in test cells. By the present inventors, CD13 is a molecule present on the surface of cancer stem cells and is involved in the control of the ROS elimination pathway, reducing ROS-induced DNA damage that occurs after genotoxic chemoradiation stress, and apoptosis. (Non-patent document 10: Haraguchi 保護 N, Ishii H, Mimori K, Tanaka F, Ohkuma M, Kim HM, Akita H, Takiuchi D, Hatano H, Nagano H , Barnard GF, Doki Y, Mori M. CD13 is a therapeutic target in human liver cancer stem cells. J Clin Invest. 2010; 120 (9): 3326-3339.).

In the detection method of the present invention, a substance that specifically binds to CD13 (hereinafter referred to as “CD13-binding substance”) is brought into contact with a test cell, and the presence or absence of binding of the substance to the test cell is measured. Among the cells, cells expressing CD13 can be measured. That is, for the test cell, a cell expressing CD13 can be detected using the binding of the CD13 binding substance as an index. Here, the CD13 binding substance is not particularly limited as long as it can specifically bind to CD13. For example, an antibody capable of specifically binding to CD13 (hereinafter referred to as “anti-CD13 antibody”). MicroRNA, RNA aptamer, dominant negative mutant and the like. Among these, an anti-CD13 antibody is exemplified as a preferable one.

Also, it is desirable that the CD13 binding substance is labeled with a fluorescent substance such as FITC or a labeling substance such as an enzyme from the viewpoint of easy detection. Measurement of a test cell bound with a CD13 binding substance can be performed by a method known in the art. For example, if the CD13 binding substance is labeled with a fluorescent substance, the cells to which the substance is bound can be measured qualitatively and quantitatively by using flow cytometry.

In addition, in the detection method of the present invention, it is preferable to measure the expression of CD90 in addition to the expression of CD13, thus further improving the accuracy of detection of cancer stem cells. Measurement of test cells expressing CD90 can also be performed by contacting a test cell with a substance that specifically binds to CD90 (hereinafter referred to as “CD90-binding substance”), as in the case of CD13. it can. The CD90-binding substance is not particularly limited as long as it can specifically bind to CD90. For example, an antibody that can specifically bind to CD90 (hereinafter referred to as “anti-CD90 antibody”), Examples include microRNA, RNA aptamer, and dominant negative mutant. An anti-CD90 antibody is preferred. Also, the CD90 binding substance is desirably labeled with a fluorescent substance such as FITC or a labeling substance such as an enzyme, as in the case of the measurement of CD13. The method for measuring a test cell bound with a CD90-binding substance can also be performed by a method known in the art, such as flow cytometry, as in the case of CD13.

Thus, the measured test cells expressing CD13 or the test cells expressing CD13 and CD90 thus measured are detected and identified as cancer stem cells.

The cancer stem cells detected by the method of the present invention can be isolated, if necessary, and used for screening for a therapeutic drug for cancer or for evaluating the efficacy of a therapeutic drug for cancer. For example, cancer stem cells detected by the method of the present invention are used to advance drug discovery specifically for clones in the cell cycle stationary phase or drug-resistant clones, thereby acting only on resistant cancer cells against cancer treatment. Therefore, it is possible to develop a cancer therapeutic agent that has no side effects. Furthermore, by developing drugs using cancer stem cells detected by the method of the present invention, the development of drugs that prevent cancer recurrence or cancer recurrence, and the development of drugs whose drug efficacy spectrum is specialized for cancer stem cells Is also expected.

2. Method of measuring the degree of cancer symptoms or the risk of cancer The presence of cancer stem cells in cancer tissue is used to determine the degree of cancer symptoms (cancer severity) and the risk of cancer progression, recurrence or metastasis The detection method of the present invention can be used as a method for measuring cancer severity or cancer risk.

Therefore, the present invention also provides a method for measuring cancer seriousness or cancer risk using the above-mentioned “cancer stem cell detection method”.

The method includes the following steps (A) and (B):
(A) a step of measuring cells expressing CD13 (CD13-expressing cells) for test cells derived from cancer tissues of cancer patients or tissues after cancer treatment;
(B) A step of determining the cancer severity or cancer risk of the cancer patient according to the number of CD13-expressing cells detected in the step (A).

Here, “measuring CD13-expressing cells” means both qualitative analysis for measuring the presence or absence of CD13-expressing cells in a test cell and quantitative analysis for measuring the abundance thereof. The CD13-expressing cells measured in step (A) correspond to cancer stem cells. For this reason, the step (B) corresponds to a step of predicting cancer severity or cancer risk using the number of cancer stem cells as an index.

In the step (A), as described above in the section of “1. Detection method of cancer stem cells” with respect to the detection method of the present invention, the CD13 binding property may be labeled with a labeling substance such as a fluorescent substance or an enzyme. It can be carried out by bringing a substance into contact with a test cell and measuring the presence or absence of binding of the substance to the test cell. Thus, CD13-expressing cells, that is, cancer stem cells, can be detected from test cells using the binding of a CD13-binding substance as an indicator. Detection of CD13-expressing cells using the binding of the CD13-binding substance as an index can be performed by a method known in the art as described above. For example, when the CD13-binding substance is labeled with a fluorescent substance Can be performed by using flow cytometry. In addition, when flow cytometry is used, the number (amount) of CD13-expressing cells (cancer stem cells) can be measured simultaneously based on the level of fluorescence intensity, so CD13-expressing cells (cancer stem cells) can be qualitatively and quantitatively determined. Can be measured.

In the measurement in the step (B), specifically, the more CD13-expressing cells (cancer stem cells) measured in the step (A) using the detection method of the present invention, the more severe the cancer symptoms, It is determined that the cancer risk is high. In addition, it is determined that the smaller the CD13-expressing cells (cancer stem cells) measured in step (A) using the detection method of the present invention, the lighter the cancer symptoms and the lower the risk of cancer.

In addition, in the measurement method of the present invention, it is preferable to measure the expression of CD90 in addition to the expression of CD13. In this way, the accuracy of detection of cancer stem cells can be further increased, and the cancer severity or cancer risk can be increased. It can be determined more accurately. CD90 expression can be measured in the same manner as CD13 expression.

3. Method for Measuring Cancer Treatment Effect or Risk of Cancer Recurrence Furthermore, the presence of cancer stem cells in tissues after cancer treatment can be used as an evaluation standard for cancer treatment effect and can be used to predict cancer recurrence. it can. Therefore, the “1. Method for detecting cancer stem cells” of the present invention can also be used as a method for measuring the therapeutic effect of cancer and a method for measuring the risk of cancer recurrence.

Therefore, the present invention also provides a method for measuring the effect of cancer treatment or the risk of cancer recurrence using the above-described “method for detecting cancer stem cells”.

The method includes the following steps (a) and (b):
(A) measuring CD13-expressing cells (CD13-expressing cells) for test cells derived from tissues after cancer treatment of cancer patients;
(B) A step of determining a cancer therapeutic effect or cancer recurrence risk for the cancer patient according to the presence or absence of the CD13-expressing cells measured in the step (a).

Here, “measuring CD13-expressing cells” means both qualitative analysis for measuring the presence or absence of CD13-expressing cells in a test cell and quantitative analysis for measuring the abundance thereof.

The CD13-expressing cells measured in step (a) are cancer stem cells as described above, and step (b) determines the cancer therapeutic effect or cancer recurrence risk for cancer patients using the presence or absence of the cancer stem cells as an index. It corresponds to the process to do.

The step (a) is a CD13 binding substance which may be labeled with a fluorescent substance or a labeling substance such as an enzyme, as described in the section of “1. Detection method of cancer stem cells” above for the detection method of the present invention. Can be carried out by contacting the test cells with the test cells and measuring the presence or absence of binding of the substance to the test cells. Thus, CD13-expressing cells, that is, cancer stem cells, can be detected from test cells using the binding of a CD13-binding substance as an indicator. Detection of CD13-expressing cells using the binding of the CD13-binding substance as an index can be performed by a method known in the art as described above. For example, when the CD13-binding substance is labeled with a fluorescent substance Can be performed by using flow cytometry. In addition, using flow cytometry, the number of CD13-expressing cells (cancer stem cells) can also be measured simultaneously by the level of fluorescence intensity, so CD13-expressing cells (cancer stem cells) can be measured qualitatively and quantitatively. it can.

Specifically, in step (b), CD13-expressing cells (cancer stem cells) were detected from the tissue after cancer treatment in step (a) using the “method for detecting cancer stem cells” of the present invention. In some cases, it is determined that the therapeutic effect of cancer is poor and there is a possibility of cancer recurrence (high risk of cancer recurrence). If cancer stem cells are not detected, it is determined that the cancer treatment effect is good and the possibility of cancer recurrence is low (the risk of cancer recurrence is low).

Further, in the measurement method of the present invention, it is preferable to measure the expression of CD90 in addition to the expression of CD13. This can further improve the accuracy of detection of cancer stem cells, and can increase the cancer therapeutic effect or the risk of cancer recurrence. It can be determined more accurately. CD90 expression can be measured in the same manner as CD13 expression.

4). Cancer Stem Cell Detection Reagent and Detection Kit The present invention is intended to perform the “cancer stem cell detection method” described in 1 above, and to qualitatively or alternatively detect CD13 expressing cells (cancer stem cells) in the measurement methods described in 2 and 3 above A reagent for detecting cancer stem cells, which is used for quantitative measurement, is also provided. Specifically, the detection reagent of the present invention includes a substance that specifically binds to CD13 (CD13-binding substance).

In addition, the present invention is for performing the “cancer stem cell detection method” described in 1 above, and for qualitatively or quantitatively measuring CD13-expressing cells (cancer stem cells) in the measurement methods described in 2 and 3 above. Also provided is a kit for detecting cancer stem cells used in the above. Specifically, the detection kit of the present invention is characterized by containing a CD13 binding substance as a detection reagent for cancer stem cells. The detection kit of the present invention may further contain a substance that specifically binds to CD90 (CD90-binding substance) as a detection reagent for cancer stem cells. Further, the detection kit of the present invention further includes “reagent for detecting cancer stem cells” described in 1 above, or other reagents and instruments required for performing the measurement methods described in 2 and 3 above. Further, the detection kit of the present invention may include a procedure manual for performing the above detection method.

Specific examples of the CD13 binding substance and the CD90 binding substance used in the cancer stem cell detection reagent and detection kit of the present invention are as described in “1. Detection method of cancer stem cells” above.

In addition, as described above, the “cancer stem cell detection method” is a method for measuring the degree of cancer symptoms (cancer severity) or cancer risk, or a method for measuring cancer therapeutic effect or cancer recurrence risk. Therefore, the detection reagent and detection kit of the present invention can be used as a diagnostic agent and a diagnostic kit for measuring the degree of cancer symptoms (cancer severity) or cancer risk, and further the therapeutic effect of cancer or cancer. It can also be used as a diagnostic agent and a diagnostic kit for measuring the risk of recurrence.

5. Therapeutic agent for cancer and preventive agent for cancer recurrence The present invention further provides a therapeutic agent for cancer and a preventive agent for cancer recurrence , which further comprises a CD13 inhibitor and an anticancer agent. Hereinafter, the cancer therapeutic agent and cancer recurrence preventing agent of the present invention will be described in detail. The cancer therapeutic agent of the present invention can be used for the purpose of curing cancer, and in this sense, it can also be referred to as a cancer curative. Moreover, the cancer therapeutic agent of this invention can prevent the recurrence of cancer as a result of curing cancer.

The CD13 inhibitor used in the cancer therapeutic agent and cancer recurrence preventive agent of the present invention is pharmaceutically acceptable and can inhibit the function of CD13, that is, can inhibit the control function of the ROS removal pathway. As long as it is not particularly limited. Specific examples of CD13 inhibitors include CD13 neutralizing antibody, ubenimex (bestatin), aminopeptidase N (APN / CD13) inhibitor 24F (Novel aminopeptidase N (APN / CD13) inhibitor 24F can suppress invasion of hepatocellular carcinoma cells as . Biosci Trends. 2010 2010 Apr; 4 (2): 56-60).

These CD13 inhibitors may be used alone or in combination of two or more. These CD13 inhibitors are commercially available (for example, as for the CD13 neutralizing antibody, “mouse monoclonal anti-human CD13 antibody (clone WM15)” is available from Gene Tex, and Ubenimex is available from Nippon Kayaku). For this reason, although a commercial item may be used for a CD13 inhibitor, it may be manufactured and used by a known method.

Examples of anticancer agents used in the cancer therapeutic agent and cancer recurrence preventive agent of the present invention include conventionally known alkylating agents (mustard drugs, nitroureas), antimetabolites (folic acid antimetabolites, pyrimidine antimetabolites, Purine antimetabolite, hydroxycarbamide), antitumor antibiotics (anthracycline drugs, others, mitomycin C, etc.), platinum preparations, topoisomerase inhibitors (topoisomerase I inhibitors, topoisomerase II inhibitors), etc. it can. Preferred are anticancer agents having an activity of inhibiting DNA synthesis (in the present invention, such anticancer agents are collectively referred to as “DNA synthesis inhibitors”) and anticancer agents having an activity of increasing ROS. These anticancer agents may be used individually by 1 type, and may be used in combination of 2 or more type.

Examples of DNA synthesis inhibitors include alkylating agents such as ifosfamide, melphalan, nimustine hydrochloride, ranimustine, and procarbazine hydrochloride; 5-fluorouracil (5-FU), 5-FU prodrugs (eg, doxyfluridine, tegafur, carmofur) , 5'-doxyfluridine prodrugs (eg capecitabine), cytarabine, cytarabine prodrugs (eg cytarabine ocphosphate phosphate hydrate), pyrimidine antagonists such as enocitabine, gemcitabine hydrochloride; mercaptopurine hydrate, fludarabine phosphate Purine antimetabolites such as esters and cladribine, and other antimetabolites such as levofolinate calcium and hydroxycarbamide; doxorubicin hydrochloride, daunorubicin hydrochloride, pirarubicin, and mitoxantrone hydrochloride Examples include anthracycline antibiotics; other antibiotics such as bleomycin hydrochloride; platinum preparations such as cisplatin, carboplatin, and nedaplatin; and topoisomerase inhibitors such as etoposide. The above pyrimidine antagonists such as 5-fluorouracil (5-FU) and prodrugs thereof are preferable, and 5-fluorouracil is more preferable. These DNA synthesis inhibitors may be used individually by 1 type, and may be used in combination of 2 or more type.

An example of an anticancer agent having a ROS-raising action is an anthracycline anticancer agent belonging to an antitumor antibiotic. Specific examples of the anthracycline anticancer agent include doxorubicin, daunorubicin, pirarubicin, epirubicin, idarubicin, aclarubicin, amrubicin, mitoxantrone, or a pharmaceutically acceptable salt thereof. These anticancer agents may be used alone or in combination of two or more. Moreover, you may use together with the said DNA synthesis inhibitor.

In the cancer therapeutic agent and cancer recurrence preventing agent of the present invention, the content of the CD13 inhibitor and the anticancer agent (preferably a DNA synthesis inhibitor or an anticancer agent having a ROS-elevating action) depends on the type of cancer to be treated, the degree of symptoms, A therapeutically effective amount according to the age, sex, etc. of the cancer patient is sufficient. Although it cannot be defined uniformly, for example, it is desirable that the content satisfy the following range as a dose per adult.
・ Dose of CD13 inhibitor per adult:
Usually 10 to 600 mg, preferably 15 to 90 mg, more preferably 30 to 60 mg
・ Dose of adult anticancer drug (preferably DNA synthesis inhibitor) per adult:
Usually 100 to 3000 mg, preferably 200 to 1000 mg, more preferably 250 to 750 mg.

In the cancer therapeutic agent and cancer recurrence preventive agent of the present invention, the ratio of the CD13 inhibitor to the anticancer agent (preferably a DNA synthesis inhibitor or an anticancer agent having an ROS-raising action) is appropriately determined within the range satisfying the therapeutically effective amount. The ratio of the anticancer agent (preferably a DNA synthesis inhibitor or an anticancer agent having an ROS-raising action) to 1 part by weight of the CD13 inhibitor is usually 1 to 100 parts by weight, preferably 2 to 50 parts by weight, more preferably Examples are 4 to 30 parts by weight.

The cancer therapeutic agent and cancer recurrence preventive agent of the present invention include pharmaceutically acceptable carriers and additives in addition to a CD13 inhibitor and an anticancer agent (preferably a DNA synthesis inhibitor or an anticancer agent having an ROS-raising action). May be. Examples of such carriers and bases include excipients, extenders, binders, disintegrants, surfactants, lubricants, solubilizers, plasticizers, pH adjusters, buffers, chelating agents, Preservatives, antioxidants, solvents, disintegrants and the like can be mentioned.

In addition, the dosage forms of the cancer therapeutic agent and cancer recurrence preventing agent of the present invention are not particularly limited, and may be appropriately set according to the administration form. Specific examples of the dosage form of the cancer therapeutic agent and cancer recurrence preventing agent of the present invention include tablets, pills, powders, solutions, suspensions, emulsions, granules, capsules and the like.

The dosage form of the cancer therapeutic agent and cancer recurrence preventing agent of the present invention may be appropriately set according to the type of cancer to be treated, and may be oral administration such as buccal administration, sublingual administration, In addition, parenteral administration such as intravenous administration, intramuscular administration, subcutaneous administration, pulmonary administration, and rectal administration may be used.

In addition, the cancer to be treated by the cancer therapeutic agent and the cancer recurrence preventing agent of the present invention is not particularly limited as long as it is a cancer tissue expressing CD13. Examples of cancer include gastrointestinal cancers such as esophageal cancer, gastric cancer, duodenal cancer, colorectal cancer, and colon cancer (the large intestine includes the ascending, transverse, descending, sigmoid colon, and rectum); liver cancer; lung cancer Breast cancer; brain tumor; bile duct cancer (the bile duct includes the intrahepatic and extrahepatic bile ducts); gallbladder cancer (the left two or three are collectively referred to as the biliary tract); pancreatic cancer (the pancreas is the duct and secretory tissue) The latter includes solid cancers such as the endocrine system and exocrine system); and hematopoietic tumors (blood cancers) such as leukemia, malignant lymphoma, and multiple myeloma. The cancer targeted by the invention is a cancer expressing CD13. Among these, solid cancer is preferable, preferably liver cancer, lung cancer, and gastrointestinal cancer, and particularly preferably liver cancer and colon cancer.

The cancer therapeutic agent of the present invention can be applied as a first-choice therapy to cancer patients suffering from the above cancer. That is, cancer treatment (radical treatment of cancer) can be performed using the cancer therapeutic agent before performing surgical treatment such as cancer tissue resection surgery or radiation treatment on the cancer patient. In addition, the cancer therapeutic agent of the present invention is also applied to cancer patients who are resistant to cancer therapy, and the cancer does not disappear by other cancer chemotherapy, surgical treatment such as cancer tissue resection surgery, or radiation treatment. be able to.

On the other hand, the cancer recurrence-preventing agent of the present invention is used for cancer patients who have achieved a temporary cancer therapeutic effect by other cancer chemotherapy, surgical treatment such as cancer tissue resection surgery, or radiation treatment. It can be applied for the purpose of preventing.

6). A pharmaceutical kit for treating cancer or preventing cancer recurrence Further, the present invention provides a second agent comprising a first agent containing a CD13 inhibitor and an anticancer agent (preferably a DNA synthesis inhibitor or an anticancer agent having a ROS-elevating action). A pharmaceutical kit for treating cancer or preventing cancer recurrence (hereinafter, both are collectively referred to as “pharmaceutical kit”).

Regarding the CD13 inhibitor used in the first agent of the pharmaceutical kit of the present invention, the type, the content in the first agent, and the dose are as shown in the above "5. Cancer therapeutic agent and cancer recurrence preventive agent". is there. In addition, regarding the anticancer agent (preferably a DNA synthesis inhibitor or an anticancer agent having an ROS-raising action) used in the second agent of the pharmaceutical kit of the present invention, the type, the content in the second agent, and the dosage are also described above. As described in "5. Cancer therapeutic agent and cancer recurrence preventive agent".

Each of the first agent and the second agent may further contain a pharmaceutically acceptable carrier or additive, and examples of such carriers and additives are also described in “5. And the same as those used in the “cancer recurrence preventive agent”. Further, the formulation forms of the first agent and the second agent are not particularly limited, and specific examples thereof are the same as those in the case of “5. Cancer therapeutic agent and cancer recurrence preventing agent” above.

Furthermore, the cancer to be treated by the pharmaceutical kit of the present invention is the same as the cancer to which the above-mentioned “5. Cancer therapeutic agent and cancer recurrence preventing agent” is applied.

In addition to the first agent and the second agent, the pharmaceutical kit of the present invention may include an administration manual showing these administration methods and the like.

7). The present invention provides a radical cancer treatment method and a cancer recurrence prevention method (hereinafter, these methods are collectively referred to as "cancer radical cure and recurrence suppression therapy").

The cancer curative and recurrence-suppressing therapy of the present invention includes (1) a step of administering a CD13 inhibitor and an anticancer agent to a cancer patient, or (2) a CD13 inhibitor administration treatment and ionizing radiation irradiation for a cancer patient. It has the process of performing treatment, It is characterized by the above-mentioned.

In the method of (1), the administration of the CD13 inhibitor and the anticancer agent to the cancer patient may be performed simultaneously, that is, in parallel, or may be performed at different administration timings, or a drug withdrawal period may be provided in the middle. good. Regarding the CD13 inhibitor and anticancer agent, the type, content, added carrier and additive, dose, formulation form, and administration route are as described in “5. Cancer therapeutic agent and cancer recurrence preventive agent” above. The anticancer agent is preferably a DNA synthesis inhibitor or an anticancer agent having an ROS increasing action. The formulation forms and administration routes of the CD13 inhibitor and anticancer agent may be the same or different.

In the method of (2), administration of a CD13 inhibitor to a cancer patient may be performed simultaneously with the ionizing radiation irradiation treatment for the patient, that is, in parallel, or at a different time, and may be suspended during the course. A period or a rest period may be provided. Regarding the CD13 inhibitor, the type, content, carrier and additive to be added, dosage, formulation form, and administration route are as described in “5. Cancer therapeutic agent and cancer recurrence preventing agent” above. The treatment conditions in the ionizing radiation irradiation treatment can be appropriately selected according to a standard method according to the type of cancer of the cancer patient, the seriousness such as the size and progression of the cancer, the age, sex and weight of the patient.

Furthermore, the cancer patient that is the subject of the cancer cure and the recurrence-suppressing therapy of the present invention is a cancer patient suffering from a cancer to which the above-mentioned “5. cancer therapeutic agent and cancer recurrence preventive agent” is applied, preferably Solid cancer patients, more preferably liver cancer, lung cancer, or gastrointestinal cancer patients, particularly preferably liver cancer or colon cancer patients.

Cancer radical therapy can be performed on such cancer patients before surgical treatment such as cancer tissue resection surgery or radiation treatment, and other cancer chemotherapy, surgery such as cancer tissue resection surgery, etc. The present invention can also be applied to cancer patients who are resistant to cancer therapy and whose cancer has not disappeared even by treatment or irradiation treatment. On the other hand, the method for preventing cancer recurrence according to the present invention provides cancer recurrence to a cancer patient who has achieved a temporary cancer therapeutic effect by other cancer chemotherapy, surgical treatment such as cancer tissue resection surgery, or radiation treatment. It is applied for the purpose of preventing.

In order to clarify the configuration and effects of the present invention, experiments will be described below, but the present invention is not limited thereto.

<Experiment method>
1. Cell culture human hepatoma cells HuH7 and PLC / PRF5 (obtained from Tohoku University Institute of Aging Medicine, Medical Cell Resource Center) in RPMI 1640 medium (Invitrogen) containing 10% FBS (fetal bovine serum; Equitech-Bio) Culture was performed. The culture was performed in an atmosphere of 37 ° C. and 5% CO ₂ .

2. Commercially available products were used for the flow cytometry analysis and cell sorting antibodies. Cells were harvested using trypsin and EDTA. Doublet cells were removed using FSC-A / FSC-H and SSC-A / SSC-H. Dead and damaged cells were removed using 7-AAD (BD Pharmingen). An isotype control (BD Biosciences) was used. FcR blocking was performed using FcR blocking reagent (Miltenyi-Biotec, BergischGladbach, Germany). Hematopoietic cells in clinical samples were removed using FITC-conjugated Lineage Cocktail (Lin1; BD Pharmingen) and FITC-conjugated anti-human CD45 antibody (BD Pharmingen, CA, USA).

3. Cell cycle analysis In order to analyze the characteristics of the SP (side population) fraction, 1 × 10 ⁶ cells were preincubated at 37 ° C. for 30 minutes in 2% FCS / 1 mM HEPES buffer / DMEM. Cells were then labeled with 10 μg / ml Hoechst 33342 (Molecular Probes) by incubating at 37 ° C. for 70 minutes in a staining medium. In the Hoechst staining step, 15 μg / ml reserpine (Sigma Aldrich) was used. Cells were first stained with Hoechst 33342 at 37 ° C. for analysis of the cell cycle of pyronin Y (PY) staining. After 50 minutes, 1 μg / ml PY was added and the cells were incubated at 37 ° C. for 20 minutes. Analysis and cell sorting were performed using FACSVantage SE DiVa (Becton Dickinson) and FACS SORP Aria (Becton Dickinson). The cell cycle was further analyzed by staining with 10 μg / ml 7-AAD (BD Pharmingen).

4). Analysis of gene expression Total RNA was prepared using TRIzol reagent (Invitrogen). Reverse transcription was performed using SuperScript III (Invitrogen). Quantitative real-time RT-PCR was performed using a LightCycler TaqMan Masterkit (Roche Diagnostics; Tokyo, Japan). The expression of the mRNA copy was determined based on the expression level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. The PCR primers used for amplification are as follows.
GCLM: 5'-TTGTGTGATGCCACCAGATTT-3 '(SEQ ID NO: 1) and 5'-TTCACAATGACCGAATACCG-3' (SEQ ID NO: 2)
GAPDH: 5′-TTGGTATCGTGGAAGGACTCA-3 ′ (SEQ ID NO: 3) and 5′-TGTCATCATATTTGGCAGGTTT-3 ′ (SEQ ID NO: 4).

5. Analysis of cell proliferation and drug resistance For analysis of cell proliferation, the isolated cells were seeded in a 96-well culture plate at 5 × 10 ³ cells / well. 72 hours later, live cells were measured by ATP bioluminescence assay (CellTiter-GloLuminescent Cell Viability Assay; Promega), and luminescence signal was detected using a luminometer (ARVO MX; Perkin Elmer, Turku, Finland).

For analysis of drug resistance, cells were seeded in a 96-well culture plate at 5 × 10 ³ cells / well. After 24 hours, doxorubicin was added to the culture (0.01, 0.05 and 0.1 μg / ml). 72 hours after the addition of the cancer chemotherapeutic agent, live cells were measured in the same manner as in the analysis of cell proliferation.

6). Measurement of cell fate Cells were labeled with 20 μM PKH26GL (Sigma). The purified cell group was isolated and placed at 5 × 10 ³ cells per well of CultureSlide (BD Falcon) 4 Chamber. Cells were cultured in RPMI 1640 medium (Invitrogen) containing 20% fetal bovine serum (FBS, Equitech-Bio). Cell fate was analyzed every 30 minutes for 238 hours using a time-lapse fluorescence microscope (BZ-9000 Biorevo; Keyence). Data was analyzed using a BZ-II analyzer (Keyence).

7). Cells for sphere analysis were seeded in serum-free medium on Ultra Low Attachment culture dishes (Corning). DMEM / F-12 serum-free medium (Invitrogen) consists of 2 mM L-glutamine, 1% sodium pyruvate (Invitrogen), 1% MEM non-essential amino acids (Invitrogen), 1% insulin-transferrin-selenium-X supplement (Invitrogen) , 1 μM dexamethasone (Wako), 200 μM L-ascorbic acid 2-phosphate (Sigma), 10 mM nicotinamide (Wako), 100 μg / ml penicillin G, and 100 U / ml streptomycin, 20 ng / ml epidermal growth factor And 10 ng / ml fibroblast growth factor 2 (PeproTech). Cell passage was performed every 3 days.

8). Analysis of differentiation from spheres Each single sphere established from normal hepatocytes was seeded in a culture chamber (BD bioscience). The spheres were cultured in sphere medium containing 10% FBS and entered the differentiation process. Three days after the spheres adhered to the bottom of the chamber and spread out cells appeared, the cells were fixed, anti-human CD13 mouse monoclonal antibody (clone WM15, dilution 1:50; Santa Cruz Biotechnology), FITC-labeled anti-anti Human albumin goat polyclonal antibody (dilution 1: 500; Bethyl Laboratories), anti-human cytokeratin 19 mouse monoclonal antibody (clone RCK108, dilution 1:50; Dako), and anti-human α-fetoprotein mouse monoclonal antibody (clone 189502, concentration 5 μg / ml; R & D Systems).

9. A fragment with an immunohistochemical thickness of 4 μm was obtained with a cryostat and fixed with 4% paraformaldehyde for 15 minutes. After 1 hour blocking, the fragments were incubated with primary antibody overnight at 4 ° C. in a humidified chamber. Primary antibodies include anti-human CD13 mouse monoclonal antibody (clone WM15, dilution 1:50; Santa Cruz Biotechnology), anti-human carbonic anhydrase IX (CA9) rabbit polyclonal antibody (dilution 1: 1000; Novus Biologicals), anti-human CD90 rabbit monoclonal antibody (dilution 1: 1000; Epitomics) and anti-human Ki-67 rabbit polyclonal antibody (dilution 1: 100; Santa Cruz Biotechnology) were used. As secondary antibodies, goatanti-mouse IgG1, Alexa-Fluor 546-conjugate DNAd highly cross-adsorbe DNAtibody (Molecular Probes) and goat anti-rabbit IgG, Alexa-Fluor 488-conjugate DNAd highly cross-adsorbe DNAtibody (Molecular Probes) were used. Coverslip was installed using ProLong Gold and SlowFade GolDNAtifadeReagent (Molecular Probe), and observed using a fluorescence microscope (BZ-9000 Biorevo). Continuous cryostat fragments were stained with modified Hematoxylin and Eosin (H & E).

10. Preparation of tumor cells With the consent of the patient and approval from the ethics review committee of Osaka University, main tumor tissue (liver cancer tissue) samples were obtained from Osaka University. The tumor tissue was cut into small pieces of 2 mm or less, further chopped with a sterile scalpel, and then washed twice with DMEM / 10% FBS. The tumor tissue slices were then placed in DMEM / 10% FBS containing 2 mg / ml collagenase A (Roche Diagnostics) solution. Incubation was performed with stirring at 7 ° C. until the tumor tissue slices were completely digested. Cells were collected by passing through a 40 μm nylon mesh, washed twice, removed by Ficoll (GE Healthcare) concentration gradient centrifugation, cell fragments and cell debris were removed, and staining for flow cytometry was performed.

11. Inhibition of CD13 5 × 10 ³ cells were seeded in a 96-well plate containing 200 μl of medium. After 24 hours, the culture broth was diluted with 1, 5, 10 and 20 μg / ml CD13 neutralizing antibody (mouse monoclonal anti-human CD13 antibody, clone WM15; Gene Tex) or 25, 50, 100, 250 and 500 μg / ml Ubenimex. It was replaced with fresh medium containing (Nihon Kayaku). Cell survival was analyzed at 24, 48 and 72 hours using cell counting kit-8 (Dojindo). Absorbance at 450 nm was measured using a 680 XR microplate reader (Bio-Rad). As a negative control, 10 μg IgG1 mouse monoclonal antibody (Gene Tex) was used. Doxorubicin resistant (DXR-R) HuH7 cells were established by sequential treatment with 1 μg / ml doxorubicin (DXR) and selection of resistant clones. Cell apoptosis was measured using Propidium Iodide (PI) and APC-Annexin V (BD Pharmingen) together with Apoptosis Detection Kit (Bio Vision).

12 In vivo analysis 1 x 10 ⁵ cells of HuH7 and PLC / PRF / 5 were injected into NOD / SCID mice under anesthesia to produce xenografted model mice. In the cell injection step, cells were resuspended in a mixture containing medium and Matrigel (BD Biosciences) in a 1: 1 ratio. In mice xenografted with HuH7 cells, 5-FU (30 mg / kg; intraperitoneal administration) as a DNA synthesis inhibitor or Ubenimex (20 mg / kg; oral administration) as a CD13 inhibitor was administered for 3 days. The next day, mice were dissected and tumors removed for immunochemical histological analysis. In the study of model mice xenotransplanted with PLC / PRF / 5 cells, 5-FU (30 mg / kg, intraperitoneal administration for 5 days and discontinuation of drug administration for 2 days, 2 courses, 14 days), Ubenimex (20 mg / kg, 14-day gavage), Ubenimex and 5-FU (a combination of 2 courses of 30 mg / kg of 5-FU and 20 mg / kg of Ubenimex for 14 days), respectively. Tumor size and relative tumor volume were calculated according to the following formula.

The day after the 14-day treatment, the mice were dissected and the tumor was removed for immunochemical histological analysis. Relative tumor volume was estimated as follows.

After 14 days of 5-FU treatment, the remaining tumor was excised, shredded to a size of 2 mm, and subcutaneously administered with Matrigel to the second NOD / SCID. Mice were treated with ubenimex (20 mg / kg) the next day 7 days after transplantation. Tumor growth was observed for 3 weeks. In order to statistically analyze the results, each experiment was performed with 4 or more mice.

13. ROS analysis To examine intracellular ROS levels, cells were loaded into 10 μM 2 ′, 7′-dichlorofluorescein diacetate (DCF-DA) at 37 ° C. for 30 minutes. ROS was activated when treated with 100 μM H ₂ O _{2 at} 37 ° C. for 120 minutes. To examine the effect of CD13 inhibition on ROS levels, cells were pretreated for 4 hours at 37 ° C. with 5 μg / ml CD13 neutralizing antibody (mouse monoclonal anti-human CD13 antibody, clone WM15) or 25 μg / ml Ubenimex. Stained with DCF-DA. For mitochondrial ROS detection, cells were loaded into 5 μM MitoSOX (Molecular Probes) at 37 ° C. for 20 minutes.

14 DNA fragmentation analysis For alkaline comet analysis, 5000 isolated cells were irradiated (4 Gray) on ice, suspended in 0.6% low melting point agarose, seeded on a slide wall and placed in alkaline solution. Soaked for 30 minutes. Subsequently, alkaline electrophoresis was performed. Slides were stained with silver for visualization. For Tempol experiments, cells were treated with 10 mM Tempol (Sigma) for 15 minutes prior to irradiation. For in situ hybridization for fragmented DNA detection, 10 μm thick sections obtained from fresh frozen samples were terminal deoxynucleotide using the Tumor TACS in situ Apoptosis Detection Kit (Trevigen). Hybridized with transferase (TdT).

<Experimental result>
1. Confirmation that CD13 is a candidate marker for cancer stem cells After culturing HuH7 and PLC / PRF5 human hepatoma cells and staining them with Hoechst Red and Hoechst Blue, it is divided into SP (side population) and non-SP fractions. It was.

In order to identify specific cell surface markers related to the SP cell fraction, the gene expression profile data of the SP fraction and the non-SP fraction were used by microarray analysis (Non-patent Document 7). In SP cells, from the list of 268 genes that are up-regulated more than twice, using UniProtKB database (http://www.uniprot.org/) to potentially encode cell surface proteins 56 genes were selected. Furthermore, the surface marker highly expressed by SP fraction was identified using the antibody (commercial item) couple | bonded with the surface protein which these genes code (refer the operation procedure described in A of FIG. 1).

In this screening, two marker candidates, CD13 and CD31, were identified. The expression analysis results of CD13 were 1.64 ± 0.45 (relative value) in the SP fraction and 0.51 ± 0.03 (relative value) in the non-SP fraction (P <0.01) (B in FIG. 1). CD31 expression was high in the G2 / M / SP fraction (Fig. 2B) and was not common for hepatoma cells (HuH7, PLC / PRF / 5 and Hep3B). (B in FIG. 1).

The expression of CD13, CD133, and CD90 in hepatitis infection negative (HuH7) and positive (PLC / PRF / 5) cell lines was analyzed. In liver cancer, the SP fraction of CD133 ⁺ and CD90 ⁺ has been reported as cancer stem cells or cancer / tumor initiating cells (Non-patent Document 11: Ma S, et al. Identification and characterization of tumorigenic liver cancer stem / progenitor cells. Gastroenterology. 2007; 132 (7): 2542-2556, Non-Patent Document 12: Zhu Z, et al. Cancer stem / progenitor cells are highly enriched in CD133 (+) CD44 (+) population in hepatocellular carcinoma. Int J Cancer 2009; 126 (9): 2067-2078, Non-Patent Document 13: Yang ZF, et al. Significance of CD90 + cancer stem cells in human liver cancer. Cancer Cell. 2008; 13 (2): 153-166. Patent Document 14: Yang ZF, et al. Identification of local and circulating cancer stem cells in human liver cancer. Hepatology. 2008; 47 (3): 919-928.). CD133 expression was detected in HuH7 but not in PLC / PRF / 5. CD90 expression was detected in PLC / PRF / 5 but not in HuH7. The expression of CD13 was confirmed in any of Hep3B, HuH7, and PLC / PRF / 5 (C in FIG. 1 and A in FIG. 2). In particular, in HuH7, many CD13 ⁺ cells were observed in the fraction (CD13 ⁺ CD133 ⁺ ) overexpressing CD133.

Multi-color analysis with Hoechst staining revealed clear localization of CD13 ⁺ cells in the SP fractions of HuH7 and PLC / PRF / 5. The CD13 ⁻ CD133 ⁺ and CD13 ⁻ CD90 ⁺ fractions were localized in the G1 to G2 fractions, but not in the SP fraction (D in FIG. 1).

From these results, it was confirmed that CD13 is a universal marker candidate closely related to the hepatoma cell SP fraction. In addition, no single marker showing a stronger association with the SP fraction than CD13 was found.

2. Confirmation that CD13 is a marker of potentially dormant hepatocellular carcinoma (HCC) cells Considering hematopoiesis and leukemic stem cells are in G0 phase, dormant or poorly proliferating cancer cells are resistant to anticancer drugs It is thought to be related to resistance and cancer recurrence. Therefore, the identification and characterization of cancer cell populations that are dormant or have a low proliferative ability are very important in examining resistance to anticancer drugs and cancer recurrence. CD13 expression of HuH7 and PLC / PRF / 5 and the relationship between the cell cycle of HuH7 and PLC / PRF / 5 were examined. Most of the CD13 ⁺ fractions were present in the G1 / G0 phase, and CD13 was strongly expressed. The apparent population was localized in the G0 phase. The cell cycle was analyzed using a DNA binding stain (Hoechst blue: Hoechst 33342) and an RNA binding stain (Pyronin Y). The population of CD133 ⁺ cells in HuH7 and the fraction of CD90 ⁺ cells in PLC / PRF / 5 were distributed in G1 / G0 and G2 / M phases. A relationship between the SP fraction and the G0 cell cycle was also observed, and the SP fraction was clearly localized in the G0 phase even in the absence of reserpine (ABC transporter blocker) (A in FIG. 3).

Cell proliferation analysis was performed to evaluate the relationship between cell cycle and CD13 expression. The results of the HuH7 population revealed that CD13 ⁺ CD133 ⁺ cells had a slower growth rate than CD13 ⁻ CD133 ⁺ cells (B in FIG. 3). The CD13 ⁻ CD133 ⁻ cell population also grew slowly but remained viable for one week. In order to analyze the cell fate and dye-retaining capacity of HuH7 CD13 ⁺ cells, the cell surface was labeled with PKH26GL reagent, and the cell fate was observed for 238 hours. Equal amounts of cells were seeded for each cell population. The CD13 ⁺ CD133 ⁺ fraction showed very slow growth compared to the CD13 ⁻ CD133 ⁺ fraction. Cells that retained the dye were observed only in the CD13 ⁺ CD133 ⁺ fraction (indicated as “CD13 ⁺ ” in FIG. 3C), even 238 hours after cell seeding (see arrow C in FIG. 3). CD13 ^- CD133 ^- fractions showed cell fragmentation and apoptotic changes during culture.

In order to observe the state of the cell cycle of CD13 ⁺ cells in PLC / PRF / 5, multi-color analysis and cell cycle analysis by 7-Amino-Actinomycin D (7-AAD) DNA labeling were performed. The CD13 ⁺ CD90 ⁻ population was mainly in the G0 / G1 phase, and the CD13 ⁺ CD90 ⁺ population was clearly in the S–G2 / M phase. CD13 ⁻ CD90 ⁺ cells were present in all cell cycles, but clearly had a higher proportion in the G2 / M and S phases than the CD13 ⁺ CD90 ⁻ population (D in FIG. 3).

From this result, it was revealed that CD13 ⁺ cells in HuH7 and PLC / PRF / 5 are in a proliferative state with a slow cell cycle from the dormant phase.

3. Confirmation that CD13 is specifically expressed in moderately differentiated to poorly differentiated colorectal cancer cells Immunostaining of moderately differentiated to poorly differentiated human colon cancer tissue with anti-CD13 antibody (anti-human CD13 mouse monoclonal antibody) The results are shown in FIG. An enlarged view of FIG. 4A is shown in FIG. As shown in FIG. 17, it was confirmed that cells expressing CD13 (CD13 ⁺ cells) were isolated in a moderately or poorly differentiated human colon cancer tissue. On the other hand, well-differentiated colorectal cancer tissue among human colorectal cancer tissues does not show staining with anti-CD13 antibody (results not shown), and it is considered that CD13 ⁺ cells are absent or very little even if they are present. It was. Thus, since CD13 is specifically expressed in moderately to poorly differentiated colorectal cancer, which is characterized by failure of differentiation control, CD13 ⁺ cells are also used in colorectal cancer, which is a type of digestive cancer. It can be seen that it is a cancer stem cell positioned higher in the differentiation hierarchy and a cell resistant to an anticancer agent.

4). Confirmation that CD13 ⁺ cells form spheres and create a CD90 ⁺ phenotype The formation of spheres is a common feature of stem cells. In order to evaluate CD13 as a marker for cancer stem cells, expression of CD13 in spheres derived from HuH7, PLC / PRF / 5 and clinically obtained HCC was examined. CD13 expression is both HuH7 (67.0%, 33.5-fold increase in the sphere versus control 2.0%) and PLC / PRF / 5 (83.8%, 5.51-fold increase in the sphere versus control 15.2%). It increased (A in FIG. 5). There was no significant difference in the expression of CD133 in HuH7. In PLC / PRF / 5, CD90 expression was decreased in the sphere (2.5% in the sphere, a 14.28-fold increase in the sphere compared to 35.7% in the control). In this sphere study, CD13 expression was more immature stem-like than CD133 and CD90, suggesting a dormant population. The sphere obtained from the clinically obtained HCC sample was localized in the CD13 ⁺ CD90 ⁻ CD133 ⁻ fraction as in the case of PLC / PRF / 5 (FIG. 5B).

The time course of CD13 and CD90 expression in PLC / PRF / 5 was examined. When the CD13 ⁺ CD90 ⁻ fraction was isolated from the PLC / PRF / 5 sphere and cultured in a serum-containing medium, the CD13 ⁺ CD90 ⁺ fraction was observed after 96 hours (C in FIG. 5). The isolated CD13 ⁻ CD90 ⁻ fraction induced cell death within a few days and was unable to maintain viability. The isolated CD13 ⁻ CD90 ⁻ fraction rapidly produced a CD13 ⁺ CD90 ⁻ fraction within 24 hours (FIG. 5C). These findings indicate that potentially dormant CD13 ⁺ cells produce proliferative CD90 ⁺ cells, and some proliferative CD90 ⁺ cells produce potentially dormant CD13 ⁺ cells. (See FIG. 6).

5. Confirmation that CD13 ⁺ cells are resistant to cancer chemotherapy and radiation therapy Cell surface markers before and after treatment with doxorubicin hydrochloride (DXR), an anticancer drug with an inhibitory effect on DNA synthesis, using HuH7 and PLC / PRF / 5 Changes in expression were examined. In HuH7, CD13 expression (control 2.0%, 40.3% with DXR treatment) and SP fraction (0.7% control, 58.6% with DXR treatment) were significantly increased after DXR treatment. (A in FIG. 7), the expression of CD133 (88.0% in DXR treatment compared to 87.1% in control) was hardly changed. Next, drug resistance of CD13 ⁺ CD133 ⁺ , CD13 ⁻ CD133 ⁺ and CD13 ⁻ CD133 ⁻ was confirmed in HuH7. The CD13 ⁺ CD133 ⁺ fraction was more resistant to DXR than the CD13 ⁻ CD133 ⁺ and CD13 ⁻ CD133 ⁻ fractions (FIG. 7B). The CD13 ⁻ CD133 ⁻ fraction showed a high drug resistance, although the cell growth rate was slow in proliferation tests and cell fate studies (FIGS. 7A and C). In PLC / PRF / 5, the CD13 ⁻ CD90 ⁺ fraction shifted to the CD13 ⁺ fraction and the CD90 ⁺ fraction decreased (A in FIG. 7).

Next, HuH7 and PLC / PRF / 5 cells were irradiated with radiation, and cell surface markers expressed in the cells remaining after irradiation were analyzed. After 24 hours of irradiation, the remaining cells were localized in the CD13 ⁺ fraction in HuH7 and in the CD13 ⁺ CD90 ^- fraction in PLC / PRF / 5. After 48 hours of irradiation, the remaining cells started to grow, and CD13 ⁻ CD133 ⁺ cells were created with HuH7, and CD13 ⁺ CD90 ⁺ cells were created with PLC / PRF / 5 (C in FIG. 7). These results corroborate the examination results of changes with time (C in FIG. 6), and show that CD13 ⁺ cells exist as a core fraction of the cellular hierarchy.

6). Confirmation that CD13 is selectively expressed in resistant cells To identify CD13 expression in clinically obtained HCC cells, digest HCC samples and be negative for hematopoietic CD45 (Lin / CD45) Fractions were analyzed by multicolor flow cytometry. 12 clinically obtained HCC samples [3 are non-hepatitis-derived HCC (1 is recurrence after hepatic artery embolization), 9 are hepatitis-derived HCC (4 are recurrence after hepatic artery embolization)] In all, expression of CD133 was not observed. In all samples, expression of CD13 and CD90 was observed as four groups of CD13 ⁺ CD90 ⁺ , CD13 ⁺ CD90 ⁻ , CD13 ⁻ CD90 ⁺ and CD13 ⁻ CD90 ⁻ . Samples with recurrence after hepatic artery embolization had a higher CD13 ⁺ CD90 ^- fraction than those without hepatic artery embolization (as compared to 48 ± 12% in samples with recurrence after hepatic artery embolization) (8 ± 4%; 6-fold increase in the sample without hepatic artery embolization). The CD13 ^- CD90 ⁺ fraction was higher in the relapsed sample after hepatic artery embolization than in the sample without hepatic artery embolization (40 in the sample without hepatic artery embolization). Compared to ± 18%, 12 ± 5%: 3.3-fold increase in the sample of recurrence after hepatic artery embolization therapy) (A in FIG. 8). In all 12 clinically obtained HCC samples, the expression pattern was very close to PLC / RLF / 5. Here, the percentage of cells indicates the percentage (%) of cells surviving after mechanical and enzymatic digestion. Most hepatocellular carcinoma cells retain liver cell function, accumulate fat and glycogen, and produce bilirubin. Hepatocyte cancer cells are relatively large compared to other cancer cells and are more susceptible to damage by mechanical and enzymatic digestion.

CD13 expression was confirmed in fresh cryosurgical specimens. In cases after hepatic artery embolization, CD13 ⁺ HCC cells were present along fibrous capsules forming cell clusters. In cases where hepatic artery embolization was not performed, CD13 ⁺ HCC cells usually formed small cell clusters inside the cancer lesion (FIG. 8B). CD13 was expressed on the cell surface in the HCC case. In normal liver samples, CD13 was expressed in sinusoids and bile ducts, clearly different from CD13 expression in HCC samples. HCC recurrence after hepatic artery embolization usually occurs in fibrotic capsules, and drug-resistant viable HCC (chemo-resistant viable HCC) is mainly present around fibrotic capsules, so immunity in samples after hepatic artery embolization Histochemical findings fully support clinical experience.

7). Confirmation that inhibition of CD13 leads to apoptosis Apoptosis was analyzed for the effect of inhibition of CD13 on cell proliferation. Cell proliferation was suppressed in a concentration-dependent manner when exposed to CD13 neutralizing antibody (CD13 Ab) for 72 hours. Apparently, treatment with the CD13 neutralizing antibody concentrations of 10 μg / ml and 20 μg / ml suppressed cell growth to about 80% in 24 hours and to about 95% in 72 hours (A in FIG. 9). Apoptosis analysis revealed that both the CD13 neutralizing antibody (CD13 Ab) and the CD13 inhibitor ubenimex (Ube) induced apoptosis in both HuH7 and PLC / PRF / 5 after 24 hours. (B in FIG. 9).

From this result, although the CD13 ⁺ CD90 ⁺ fraction was mainly present in the G2 / M / S phase (A in FIG. 3), this fraction increased after treatment with DXR, which is a DNA synthesis inhibitor. In addition to ABC (ATP-binding Cassette) transporter, CD13 is presumed to be responsible for cytoprotection against anticancer drugs (resistance to anticancer drugs). DXR is also known as an ABC transporter-dependent anticancer agent. A DXR resistant HuH7 clone was established that was able to survive 90% of cells with 0.5 μg / ml DXR (FIG. 10). Note that 99% of the HuH7 parental cells can be killed by the above concentration of DXR. Inhibition of CD13 was able to suppress cell proliferation to 50% or less in the DXR resistant HuH7 clone (C in FIG. 9). This finding can inhibit the multidrug resistance of cancer cells by inhibiting CD13, resulting in a reduction in the number of cancer cells that can remain after treatment with conventional anticancer drugs by inhibiting CD13 It suggests that you can.

8). Confirmation that CD13 inhibition induces tumor regression Each population of HuH7 cells (CD13 ⁺ CD133 ⁺ , CD13 ⁻ CD133 ⁺ , CD13 ⁻ CD133 ⁻ ) was treated with NOD / SCID mice (5 weeks old) at the cell amounts shown in Table 1. ) Was administered subcutaneously under anesthesia. Tumor formation was analyzed after 4 weeks. The number of mice that formed tumors per mouse used is shown in Table 1.

From this result, as shown in Table 1, it was confirmed that CD13 ⁺ HuH7 cells have high tumorigenicity. In NOD / SCID mice, the CD13 ⁺ CD133 ⁺ fraction formed tumors from 100 cells, the CD13 ⁻ CD133 ⁺ fraction formed tumors from 1,000 cells, and the CD13 ⁻ CD133 ⁻ fraction failed to form tumors even from 5,000 cells. .

Then, in the preliminary study, transplanted HuH7 cells in NOD / SCID mice, and anti-cancer agents with DNA synthesis inhibitory activity (5-FU), treated respectively out with ubenimex (Ube) a CD13 inhibitor, CD13 ⁺ fraction Confirmed whether or not. The results are shown in FIG. By 3 days after intraperitoneal administration of 5-FU (30 mg / kg), Ki67 ⁺ active cells were almost gone and remained only in small lesions. And most of the tumor was replaced by CD13 ⁺ Ki67 ^- cells. In the control, CD13 expression was limited to a small fraction with cell clusters, and most cells expressing CD13 were Ki67 ⁻ . On the other hand, in the mouse (20 mg / kg, 3days) treated with Ubenimex (Ube), most of CD13 ⁺ cells were lost and replaced with Ki67 ⁺ active cells (A in FIG. 11).

Further analysis was then performed using PLC / PRF / 5. Marker expression in this cell line is similar to clinically obtained HCC and is useful as an HCC model. The results are shown in FIG. In control mice, CD13 expression was limited to a small fraction and most of the cells expressed CD90. After administration of 5-FU (30 mg / kg, 5 days injection and 2 days of withdrawal, 2 courses), most of CD90 ⁺ cells disappeared, and most of the tumor was replaced by CD13 ⁺ cells. After Ubeimex (Ube) treatment (20 mg / kg, every day for 14 days), not only CD90 ⁺ cells but also CD13 ⁺ cells were detected. In the case treated with both 5-FU and Ubenimex (Ube), most of the tumor cells were lost. The expression of CD13 was confirmed to be irregular and non-specific (FIG. 11B). Considering that CD90 ⁺ cells rapidly create CD13 ⁺ cells within 24 hours, and that most of CD13 ⁺ cells have been lost by both 5-FU and Ubeimex (Ube) treatment, Ubenimex (Ube) It is believed that CD13 ⁺ cells generated by treatment can be made fresh from the remaining CD90 ⁺ cells.

In the treatment examples of Ubenimex and 5-FU, high nuclear degeneration was observed, suggesting that DNA fragmentation occurred. The state of DNA fragmentation was analyzed by in situ hybridization using terminal deoxynucleotidyl transferase (TdT). There were several DNA fragmentations in both control and 5-FU treated cases, but more DNA fragmentation was observed in Ubenimex treated cases. In particular, in the treatment example with both 5-FU and Ubenimex, considerable DNA fragmentation was observed in the remaining tumor cells (FIG. 11B).

In the group treated with both 5-FU and Ubenimex (Ube + 5-FU), compared to the control group, the group treated with 5-FU alone (5-FU), or the group treated with Ubenimex alone (Ube), After 14 days of treatment, tumor volume was significantly reduced (FIGS. 11C and D).

Next, the CD13 inhibitory effect was examined. The CD13 ⁺ rich fractions obtained from 5-FU treated mice were serially transplanted into NOD / SCID mice. After transplantation, the mice were treated with ubenimex (20 mg / kg) for 7 days. After 3 weeks of observation, no tumor formation was observed in Ubenimex-treated mice (Ube) (n = 0/6). On the other hand, in the absence of Ubenimex treatment (Control), 60% of the mice had increased tumors (n = 6/10) (E in FIG. 11).

Based on the above, it is possible to more effectively perform DNA fragmentation by treating cancer stem cells, which are CD13 ⁺ cells, with a combination of a CD13 inhibitor such as Ubenimex and an anticancer agent such as 5-FU (an anticancer agent based on DNA synthesis inhibition). It was confirmed that it induces apoptosis of cancer stem cells. From this, it is possible to eliminate not only cancer cells but also cancer stem cells that are resistant to cancer treatment by using a combination of a CD13 inhibitor and an anticancer agent, preferably an anticancer agent based on an inhibitory action on DNA synthesis. Treatment is expected to be possible. Furthermore, the treatment of cancer stem cells, which are CD13 ⁺ cells, with a combination of a CD13 inhibitor and an anticancer agent (preferably an anticancer agent based on DNA synthesis inhibitory action) suppresses the tumorigenicity of cancer stem cells, in other words, the ability of self-renewal. It was confirmed that it was possible. From this, it is considered that the recurrence of cancer caused by cancer stem cells can be prevented by using a CD13 inhibitor in combination with an anticancer agent.

9. CD13 ⁺ confirmed 10 of that cell contains a low level of ROS. Confirmation that DNA strand double strand damage is kept low in CD13 ⁺ cells We focused on the relationship between DNA fragmentation and apoptosis induced by inhibiting CD13 and the ROS removal pathway. Dormant stem cells that are capable of self-renewal usually have low levels of ROS because intracellular ROS is low, or low ROS is beneficial for cell survival even if ROS levels are not reached immediately It has been reported that the operating principle of vital functions is shifted in the direction of maintaining the above, and that dysregulation of ROS levels impairs the function of stem cells (Non-patent Document 8). Intracellular ROS was measured by 2 ', 7'-dichlorofluorescein diacetate (DCF-DA) staining. In both HuH7 and PLC / PRF / 5, the CD13 ⁺ fraction had a lower ROS concentration than the CD133 strong expression fraction and the CD90 ⁺ fraction. After stimulation of oxidative stress with H ₂ O ₂ , ROS was clearly lower in the CD13 ⁺ fraction than in the CD13 ⁻ fraction. Treatment with a CD13 neutralizing antibody or a CD13 inhibitor such as Ubenimex significantly increased the ROS concentration in CD13 ⁺ cells to the same extent as the ROS concentration in the CD13 ⁻ fraction (FIG. 12A). In clinically obtained HCC, like PLC / PRF / 5, the CD13 ⁺ CD90 ⁻ fraction showed a lower ROS concentration than the CD13 ⁻ CD90 ⁺ and CD13 ⁺ CD90 ⁺ fraction (FIG. 12B). The CD13 ⁺ fraction contained MitoSOX, another ROS indicator (FIG. 12C). In order to examine the correlation between CD13 and the ROS removal pathway, the expression of GCLM, a gene capable of encoding glutamine-cysteine ligase, was analyzed by RT-PCR. The expression of GCLM was overexpressed in the CD13 ⁺ CD90 ⁻ fraction compared to the CD13 ⁺ CD90 ⁺ , CD13 ⁻ CD90 ⁺ and CD13 ⁻ CD90 ⁻ fractions (p <0.001) (C in FIG. 12).

It is known that cell death after cytotoxic chemotherapy and ionizing radiation is partly caused by free radicals (Non-patent Document 9). In this study, a low ROS concentration was shown in the CD13 ⁺ cell population, so it was verified whether the CD13 ⁺ fraction caused DNA damage. For this verification, purified CD13 ⁺ CD90 ⁻ , CD13 ⁺ CD90 ⁺ , CD13 ⁻ CD90 ⁺ , and CD13 ⁻ CD90 ⁻ HCC cells were irradiated and subjected to an alkaline comet assay. There was no significant difference in the extent of DNA damage in cells that were not irradiated after ionizing radiation, but when ionizing radiation was applied, DNA in CD13 ⁺ CD90 ^- cells was higher than in the other three fractions. There was little chain breakage (P <0.01) (A in FIG. 13). In HuH7 cells, the CD13 ⁺ fraction had a lower level of DNA damage compared to the CD13 ⁻ fraction. In the CD13 ⁻ CD133 ⁻ fraction, no significant difference was observed between the irradiated group and the tempol treated group (FIG. 13B). These findings demonstrate that enhanced ROS protection in the CD13 ⁺ fraction contributes to reduced DNA damage after genotoxic cancer treatment.

Furthermore, by using HuH7 and PLC / PRF / 5, the intracellular ROS concentration after chemotherapy (DNA synthesis inhibitor: 5-FU and anticancer agent with an ROS-raising effect [Anthracycline anticancer agent]: DXR) was changed over time. The measurement results are shown in FIG. As can be seen from FIG. 14, the ROS concentration in CD13 ⁺ cells was suppressed to be lower than that in CD13 ⁻ cells, and the ROS concentration once increased by chemotherapy was quickly restored to a low level. This fact is also supported by the results in FIG. 7 showing that CD13 ⁺ cells are resistant to chemotherapy.

Using HuH7 and PLC / PRF / 5, DNA strand double-strand damage caused by free radicals after ionizing irradiation (4Gy) was measured over time using the appearance of γ-H2AX as an index. The result of quantitative measurement is shown in FIG. The red numbers in the figure are the percentage of γ-H2AX in CD13 ⁺ cells (PLC / PRF / 5: CD13 ⁺ CD90 ⁻ cells, HuH7: CD13 ⁺ CD133 ⁺ cells), and the blue numbers are CD13 ⁻ cells The ratio (%) of γ-H2AX in (PLC / PRF / 5: CD13 ⁻ CD90 ⁺ cells, HuH7: CD13 ⁻ CD133 ⁺ cells) is shown. As shown in FIG. 15, double strand damage to DNA strands was suppressed to a lower level in CD13 ⁺ cells than in CD13 ⁻ cells. This fact is also supported by the results in FIG. 7, which show that CD13 ⁺ cells are resistant to radiation therapy. Thus, even with ionizing radiation treatment or chemotherapy, double strand damage to DNA strands is suppressed in CD13 ⁺ cells, and the ability to repair double strand damage to DNA strands is activated. Suggests that.

11. Summary of the results Based on the above results, CD13 is specifically expressed on the surface of cancer stem cells, especially solid stem cancer stem cells of human liver cancer and gastrointestinal cancer (colon cancer), and is involved in the regulation of ROS elimination pathway Thus, it has an essential function for maintaining cancer stem cells. This revealed that CD13 is a specific marker for cancer stem cells, particularly cancer stem cells of solid cancers such as liver cancer and gastrointestinal cancer (colon cancer). In particular, cancer stem cells expressing CD13 (CD13 ⁺ cells) are in the cell cycle quiescence, ROS concentration is suppressed low, and DNA double-strand damage is suppressed or double. Since the ability to repair chain damage was activated, it was confirmed that the solid cancer stem cells were extremely resistant to conventional treatments such as chemotherapy and ionizing radiation. This suggests that problems in cancer treatment such as conventional chemotherapy and ionizing radiation (incurable, recurrence, metastasis) may be caused by CD13 ⁺ cells. In other words, it is considered that by using CD13 ⁺ cells as a target for cancer treatment, problems in conventional cancer treatment can be solved, cancer can be cured, and cancer recurrence and metastasis can be prevented.

In hypoxia, the tissue falls into acidosis, and the effects of anticancer drugs and anticancer treatment with radiation are diminished. Hypoxia is considered to cause cancer cells with higher malignancy by further causing genetic changes in tumor cells. Furthermore, in a hypoxic state, cell division is likely to stop and the number of stationary cells increases. Such quiescent cells have high resistance to cancer treatment by anticancer agents and radiation. Hypoxia also occurs when tumor angiogenesis does not catch up with tumor growth (in part because the tumor itself moves its surrounding environment in a direction that favors cancer). It is also known that the anticancer agent itself does not reach the target site at such sites.

As described above, since the cancer stem cells which are CD13 ⁺ cells are in a hypoxic state (ROS concentration is suppressed to a low level), they have high resistance to cancer treatment by anticancer agents and radiation irradiation. According to the knowledge of the present invention, by treating the cells with a CD13 inhibitor, the ROS concentration in CD13 ⁺ cells can be increased, and as a result, the sensitivity to anticancer agents and radiation is increased, and the therapeutic effect of cancer is increased. Can be improved. In other words, the present invention uses a combination of a CD13 inhibitor such as a CD13 neutralizing antibody or ubenimex and a cancer treatment with an anticancer agent or radiation to induce apoptosis in cancer stem cells remaining in the conventional therapy. It is effective as a treatment method for cancer cure (cancer radical treatment method) and as a treatment method for preventing cancer recurrence (cancer recurrence prevention therapy).

In addition, as described above, CD13 ⁺ cells have DNA double strand damage suppressed or DNA double strand damage repair ability is activated. From this, it is possible to improve sensitivity to anticancer agents by increasing ROS by treating with CD13 inhibitors, and preferably by using anticancer agents having an inhibitory action on DNA synthesis such as 5-fluorouracil as anticancer agents, DNA fragmentation and apoptosis can be guided, and as a result, cancer can be cured and cancer recurrence can be prevented.

SEQ ID NOs: 1 and 2 are the nucleotide sequences of PCR primers used for the amplification of the glutamine-cysteine ligase (GCLM) gene, and SEQ ID NOs: 1 and 2 are glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene sequences. It means the base sequence of PCR primer used for amplification.

Claims

A cancer therapeutic agent or a cancer recurrence preventive agent for solid cancer expressing CD13, comprising a CD13 inhibitor and an anticancer agent.
The cancer therapeutic agent or cancer recurrence preventing agent according to claim 1, wherein the CD13 inhibitor is at least one selected from the group consisting of a CD13 neutralizing antibody and Ubenimex.
The anticancer agent according to claim 1 or 2, wherein the anticancer agent is an anticancer agent having a DNA synthesis inhibitory action or an ROS increasing action.
The anticancer agent or cancer recurrence preventing agent according to any one of claims 1 to 3, wherein the solid cancer is any one selected from liver cancer, lung cancer and gastrointestinal cancer.
A pharmaceutical kit for treating cancer or preventing cancer recurrence for a solid cancer expressing CD13, comprising a first agent containing a CD13 inhibitor and a second agent containing an anticancer agent.
A method of radically treating cancer or preventing recurrence, comprising a step of administering a CD13 inhibitor and an anticancer agent to a cancer patient, or a step of administering a CD13 inhibitor and ionizing radiation to a cancer patient.
A method for measuring a therapeutic effect after cancer treatment for a cancer patient,
(A) a step of measuring cells expressing CD13 in a test cell derived from a tissue after cancer treatment of a cancer patient, and (b) depending on the number of CD13-expressing cells detected in the step Measuring the therapeutic effect of cancer,
The above-mentioned measuring method characterized by including.
A method of measuring the risk of cancer recurrence after cancer treatment for a cancer patient,
(A ′) a step of measuring cells expressing CD13 in a test cell derived from a tissue after cancer treatment of a cancer patient, and (b ′) the number of CD13-expressing cells detected in the step According to the process of measuring the risk of cancer recurrence,
The above-mentioned measuring method characterized by including.
A method for detecting cancer stem cells comprising a step of measuring cells expressing CD13, targeting test cells derived from cancer tissue or tissue after cancer treatment.
The detection method according to claim 1, wherein the measurement of cells expressing CD13 is performed using an antibody capable of specifically binding to CD13.
The detection method according to claim 1 or 2, comprising a step of measuring cells expressing CD90 together with CD13.
A cancer stem cell detection reagent comprising a substance that specifically binds to CD13.
A cancer stem cell detection kit comprising a substance that specifically binds to CD13.
The detection kit according to claim 8, further comprising a substance that specifically binds to CD90.
A method for measuring the degree of cancer symptoms or cancer risk for cancer patients,
(A) measuring cells expressing CD13, targeting test cells derived from cancer tissue of cancer patients or tissue after cancer treatment, and (B) the number of CD13-expressing cells measured in the step In response, measuring the degree of cancer symptoms or cancer risk,
The above-mentioned measuring method characterized by including.