Dental pulp-derived stem cells expressing sodium-iodine cotransporters

Abstract

The present invention addresses the problem of providing cells that accumulate in a cancer tissue, have secured safety for humans, and can be prepared stably. Further, the present invention addresses the problem of providing a cancer diagnosis method and a cancer therapy method using the aforesaid cells that express sodium iodide symporter (NIS), and providing a composition to be used in the diagnosis and therapy methods. More specifically, the present invention relates to: cells that are dental pulp-derived pluripotent stem cells expressing NIS; a composition for cancer therapy or diagnosis, characterized by comprising a combination of the aforesaid cells with a radionuclide; and a cancer therapy method and a cancer diagnosis method using the composition.

Description

[Technical Field]

[0001] The present invention relates to dental pulp-derived stem cells expressing a sodium iodide cotransporter (NIS), and to a method for diagnosing and treating cancer using said dental pulp-derived stem cells. [Background technology]

[0002] Currently, cancer treatment involves a combination of various methods, including surgical treatments such as surgery, drug therapy using anticancer drugs, radiation therapy, and immune checkpoint inhibitors, and the survival rate of cancer patients has improved. However, for intractable cancers such as scirrhous gastric cancer, pancreatic cancer, and triple-negative breast cancer, as well as gastrointestinal cancers such as gastric cancer, pancreatic cancer, and colorectal cancer, and peritoneal dissemination resulting from metastases from ovarian cancer and ureteral cancer, existing treatments often fail to produce satisfactory results. In particular, peritoneal dissemination is difficult to remove surgically, and treatment with conventional chemotherapy alone is ineffective, resulting in an extremely poor prognosis.

[0003] Attempts are being made to develop new cancer treatment methods that focus on the tumor microenvironment (TME). Cancer, as it is commonly known, consists of tumor tissue composed of tumor cells, and cancer stroma, which comprises stromal fibroblasts, cells that form blood vessels and lymphatic vessels, invasive inflammatory cells, extracellular matrix such as collagen, and bioactive substances. Cancer stroma grows through the high accumulation and proliferation of stromal cells, and mesenchymal stem cells (MSCs) are incorporated during this growth process. MSCs are characterized by their high proliferative capacity and the ability to differentiate into cells that make up connective tissues such as bone, fat, cartilage, and muscle, playing a crucial role in the maintenance and regeneration of various tissues.

[0004] In recent years, cancer treatment methods have been proposed that utilize the ability of bone marrow-derived MSCs to accumulate in the tumor stroma to deliver cancer therapeutic genes and other substances to the tumor microenvironment (see, for example, Non-Patent Document 1). In particular, radiotherapy combining bone marrow-derived MSCs with radionuclides has attracted attention. Bone marrow-derived MSCs expressing sodium iodide symporter (NIS) have been reported to be usable to deliver radionuclides into the tumor micromembrane and kill cancer cells (see, for example, Non-Patent Document 2). NIS is a transmembrane glycoprotein with 13 transmembrane domains and is a radionuclide. 131 I, 123 I, 125 I, 124 I, 99m Tc, 188 Re and 211 It can transport radioactive nuclides such as At. For example, Non-Patent Document 2 describes MSCs expressing NIS, 131 It has been reported that administering I to a mouse model of liver cancer resulted in tumor reduction.

[0005] As described above, the treatment method of using bone marrow-derived MSCs expressing NIS as a carrier to deliver radionuclides to cancer lesions is a method that is highly expected to have excellent therapeutic effects. However, there are problems that need to be solved in the stable preparation of bone marrow-derived MSCs, due to reasons such as the decrease in the number of stem cells in the bone marrow with age and the heavy burden on the body of bone marrow aspiration to obtain bone marrow fluid. [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] WO2020 / 027163 [Patent Document 2] WO2002 / 007679 [Patent Document 3] Japanese Patent Publication No. 2010-252778 [Patent Document 4] Special Publication 2008-507962 [Patent Document 5] WO2009 / 072527 [Patent Document 6] Japanese Patent Publication No. 2004-201612 [Non-patent literature]

[0007] [Non-Patent Document 1] Loebinger et al., Cancer Res. 69:4134-4142 2009. [Non-Patent Document 2] Knoop et al., Molecular Therapy 19:1704-1713 2011. [Non-Patent Document 3] Gronthos et al., Proc Natl Acad Sci USA, 97:13625-13630 2000. [Non-Patent Document 4] Spitzweg and Morrist, Clin Endocrinol 57:559-574 2002. [Non-Patent Document 5] Yamamoto et al., Cancer Res. 109:1480-1492 2018. [Overview of the Initiative] [Problems that the invention aims to solve]

[0008] In view of the above circumstances, the present invention aims to provide cells that accumulate in cancer tissue and can be prepared more stably than bone marrow-derived MSCs. Furthermore, the present invention aims to provide a method for diagnosing and treating cancer using cells expressing NIS, as well as a composition to be used in said diagnostic and therapeutic methods. [Means for solving the problem]

[0009] The inventors earnestly studied cells that can be used as carriers for delivering radionuclides to cancer tissues in place of bone marrow-derived MSCs and contribute to solving the above problems. As a result, they focused on dental pulp stem cells (DPCs). It is already known that pluripotent stem cells can be obtained from dental pulp (see, for example, Patent Documents 1 to 6 and Non-Patent Document 3, etc.), and it has been reported that the cell proliferation ability is higher than that of bone marrow-derived MSCs (Non-Patent Document 3). In addition, if there is consent from the provider, dental pulp stem cells contained in deciduous teeth or permanent teeth can be easily collected non-invasively, and it is considered that there are few ethical and safety problems.

[0010] First, the inventors prepared DPCs (hereinafter referred to as "hNIS-DPCs") that stably express human NIS. When comparing the iodine uptake amounts of hNIS-DPCs and hNIS-MSCs, it was revealed that the iodine uptake amount by hNIS-DPCs was about 2 to 4 times higher than that of hNIS-MSCs. Therefore, it was suggested that hNIS-DPCs take up more radionuclides than hNIS-MSCs and have a very high therapeutic effect on cancer. Furthermore, when the inventors administered hNIS-DPCs into the abdominal cavity or vein of peritoneal seeding model mice, they found that the cells accumulated near the peritoneal seeding of gastric cancer. Furthermore, the inventors administered the cells to peritoneal seeding model mice and then 131 I or 211 When radionuclides such as At were administered, it was found that the cells accumulated near the peritoneal seeding took up these radionuclides, thereby delivering the radionuclides to the peritoneal seeding tissue, and the radionuclides damaged and killed the tumor cells. From the above results, it was shown that cancer such as peritoneal seeding can be detected and treated by using DPCs expressing NIS (also referred to as "NIS-DPCs").

[0011] That is, the present invention is as follows in (1) to (12). (1) Dental pulp-derived stem cells that express sodium iodide symporter (NIS). (2) The cells described in (1) above, wherein the cells are derived from human dental pulp. (3) The cells described in (1) above, wherein the NIS is introduced from an external source. (4) A composition comprising the cells described in any of (1) to (3) above, characterized in that it is used in combination with a radionuclide. (5) The composition described in (4) above, wherein the composition is a pharmaceutical composition and is for the treatment of cancer. (6) The composition according to (5) above, characterized in that the composition is administered intratumorally, intraperitoneally, or intravenously, and the radionuclide is administered intratumorally, intraperitoneally, intravenously, or orally. (7) The radioactive nuclide is 131 I, 211 Tom or 188 The composition described in (5) above, wherein it is any of Re. (8) The composition according to (5) above, wherein the cancer is peritoneal dissemination. (9) The composition according to (4) above, wherein the composition is a composition for the diagnosis of cancer. (10) The composition according to (9) above, characterized in that the composition is administered intratumorally, intraperitoneally, or intravenously, and the radionuclide is administered intratumorally, intraperitoneally, intravenously, or orally. (11) The radioactive nuclide is 131 I, 123 I, 125 I, 124 I or 99m The composition described in (9) above, wherein it is any of Tc. (12) The composition according to (9) above, wherein the cancer is peritoneal dissemination. In this specification, the symbol "~" indicates a numerical range that includes the values ​​to its left and right. [Effects of the Invention]

[0012] This invention provides NIS-DPC, which can exhibit higher cancer treatment efficacy than NIS-MSC. This makes it possible to provide a more effective treatment method than conventional cancer treatments using radionuclides. [Brief explanation of the drawing]

[0013] [Figure 1] Figure 1 shows the trend in the doubling time of hNIS-DPC after infection of DPC with lentivirus. The vertical axis represents the doubling time of hNIS-DPC, and the horizontal axis represents the cumulative PDL of hNIS-DPC. [Figure 2] Figure 2 shows the changes in the hNIS positivity rate when hNIS-DPCs, prepared by infecting DPCs with lentivirus using protamine or polyblen, were continuously cultured. The vertical axis represents the hNIS positivity rate, and the horizontal axis represents the number of passages of the hNIS-DPC. [Figure 3] Figure 3 shows the relationship between the MOI (multiplicity of infection) at the time of infection and the hNIS positivity rate when cells were harvested after two passages of lentivirus containing the hNIS coding sequence (3 lots). The vertical axis represents the hNIS positivity rate, and the horizontal axis represents the degree of multiple infection (MOI). [Figure 4] Figure 4 shows the relationship between the NIS positivity rate of hNIS-DPC and the amount of iodine uptake. The vertical axis represents the amount of iodine uptake (pmol) per 1 × 10⁶ cells, and the horizontal axis represents the NIS positivity rate. [Figure 5] Figure 5 shows a comparison of iodine uptake by hNIS-DPC and hNIS-mouse MSCs. The vertical axis represents the amount of iodine uptake (pmol) per 1 × 10⁶ cells. [Figure 6] Figure 6 shows the results of immunostaining of peritoneal tissue with anti-human Lamin B1 antibody after intraperitoneal administration of DPC (intact) to a mouse peritoneal dissemination model. The area enclosed by the dashed line is cancerous tissue. Arrows indicate immunostaining-positive areas originating from DPC. ip: intraperitoneal (intraperitoneal administration) [Figure 7]Figure 7 shows the results of immunostaining of peritoneal tissue with anti-human Lamin B1 antibody after intravenous administration of DPC to a mouse peritoneal dissemination model. The area enclosed by the dashed line is cancerous tissue. Arrows indicate immunostaining-positive areas originating from DPC. iv: intravenous administration [Figure 8] Figure 8 shows an experimental schedule for evaluating the migration and accumulation of hNIS-DPC in cancer tissue after intraperitoneal administration in a mouse peritoneal dissemination model. [Figure 9] Figure 9 shows the results of PCR evaluation of the migration of hNIS-DPC to cancer tissue after intraperitoneal administration in a mouse peritoneal dissemination model. The band indicated by the arrow is the band (203 bp) that is specifically amplified by human GAPDH. [Figure 10] Figure 10 shows the results of immunostaining of peritoneal tissue with anti-human Lamin B1 antibody after intraperitoneal administration of hNIS-DPC to a mouse peritoneal dissemination model. In this experiment, frozen cells were administered directly. The area enclosed by the dashed line is cancerous tissue. The arrows indicate immunostaining-positive areas derived from hNIS-DPC. ip: intraperitoneal (intraperitoneal administration) [Figure 11] Figure 11 shows the results of immunostaining of peritoneal tissue with anti-human Lamin B1 antibody after intraperitoneal administration of hNIS-DPC to a mouse peritoneal dissemination model. In this experiment, frozen cells were thawed, pre-cultured, and then administered. The area enclosed by the dashed line is cancerous tissue. The arrows indicate immunostaining-positive areas derived from hNIS-DPC. ip: intraperitoneal (intraperitoneal administration) [Figure 12] Figure 12 shows the results of immunostaining of peritoneal tissue with anti-human Lamin B1 antibody after intravenous administration of hNIS-DPC to a mouse peritoneal dissemination model. In this experiment, frozen cells were thawed and administered without a culture process. The area enclosed by the dashed line is cancerous tissue. The arrows indicate immunostaining-positive areas derived from hNIS-DPC. iv: intravenous administration [Figure 13]Figure 13 shows an experimental schedule for evaluating the migration of hNIS-DPC to cancer tissue and the accumulation of 125I in a mouse peritoneal dissemination model after intraperitoneal administration of hNIS-DPC and oral administration of 125I. [Figure 14] Figure 14 shows the gamma radiation dose measurements in major organs of a mouse peritoneal dissemination model after intraperitoneal administration of hNIS-DPC and oral administration of 125I. The vertical axis represents CPM (counts per minute). [Figure 15] Figure 15 shows the experimental schedule for optimizing the dosage of 211At. [Figure 16] Figure 16 shows the results of visually evaluating and scoring the state of peritoneal dissemination in mice administered hNIS-DPC and 211At. [Figure 17] Figure 17 shows representative images of peritoneal dissemination in mice administered hNIS-DPC and 211At. The image shows the condition of the right peritoneum. The numbers in the image are scores from visual evaluation. 4; Large tumor particles present throughout the area; 3; Tumor particles present but not throughout the area; 2; Tumor particles clearly present but small; 1; Tumor particles only slightly visible upon close observation; 0; No tumor particles. [Figure 18] Figure 18 shows the results of evaluating the state of peritoneal dissemination in mice administered hNIS-DPC and 211At, using the expression level of the FGFR4 gene derived from cancer cells as an indicator. [Figure 19] Figure 19 shows the experimental schedule for confirming the dose (number of administrations) dependence of the antitumor effect on peritoneal dissemination in mice using hNIS-DPC. [Figure 20] Figure 20 shows the results of visually evaluating and scoring the state of peritoneal dissemination in mice administered 211At and hNIS-DPC (1, 2, or 3 times). [Figure 21] Figure 21 shows the results of evaluating the survival time of peritoneal dissemination model mice administered with hNIS-DPC and 211At using Kaplan-Meier curves and the Log-rank test. [Figure 22]Figure 22 shows the results of evaluating the survival rate of peritoneal dissemination model mice administered with hNIS-DPC and 211At. [Figure 23] Figure 23 shows the results of visually evaluating and scoring the state of peritoneal dissemination in the peritoneal dissemination model mice used for evaluation in Figures 21 and 22. [Modes for carrying out the invention]

[0014] Embodiments of the present invention will be described below. The first embodiment is a dental pulp stem cell (DPC) that expresses a sodium iodide cotransporter (NIS), and is hereinafter also referred to as "NIS-DPC". NIS is an intrinsic membrane protein with 13 transmembrane domains that functions as an ion pump, containing one iodide ion (I). - )It has the function of simultaneously transporting two sodium ions (Na+) into the cell. Reports have been made on its potential as a new tool in disease treatment (see, for example, Non-Patent Document 4). The NIS-DPC according to this embodiment may be prepared by introducing exogenous NIS from an external source into DPC. The exogenous NIS used in this embodiment may be selected from any animal species depending on the purpose of use, for example, human NIS consisting of the amino acid sequence represented by Sequence ID No. 1 is preferred, but is not limited to this sequence. In addition to naturally occurring NIS, a modified or homolog thereof may also be used (for example, a modified or homolog of NIS consisting of an amino acid sequence that has 90% or more identity with the amino acid sequence of naturally occurring NIS and has the activity to simultaneously transport iodide ions and sodium ions into the cell), for example, the NIS modified disclosed in US8852576B2 may be used.

[0015] These are stem cells discovered by Gronthos et al. as stem cells present in dental pulp tissue (Non-Patent Document 3). It is already known that pluripotent stem cells can be obtained from dental pulp, for example, by the methods disclosed in Patent Documents 1 to 6. DPC can be isolated by culturing cells collected from dental pulp, selecting them using DPC surface antigen markers as indicators, and finally isolating them. The cell population collected from dental pulp contains a mixture of DPC and other cells, but because DPC proliferates faster than other cells, the proportion of DPC increases by the end of the culture. Therefore, by repeatedly culturing the cell population collected from dental pulp, the proportion of DPC increases, and cells containing almost only DPC can be obtained.

[0016] In selecting DPC from a population of dental pulp-derived cells, there are no particular limitations, but for example, cells may be selected using at least one of the following characteristics (1) to (4) as indicators, and these may ultimately be used as DPC in this embodiment: (1) The patient is positive for CD73, CD90, CD105, and CD166, and negative for CD34, CD40, CD45, CD80, CD86, and MHC-class II antigen, and becomes positive for MHC-class II antigen when stimulated with interferon-γ, and expresses prostaglandin E2 and / or vascular endothelial growth factor, with the expression level of prostaglandin E2 increasing when stimulated with TNFα; (2) At least one of CD73, CD90, CD105, and CD166 is positive, and CD34 and CD45 are negative; (3) At least one of CD47, CD81, and CD147 is positive, and at least one of CD19, CD34, and CD206 is negative; (4) At least one of CD47, CD81, and CD147 is positive, and at least one of CD19, CD31, CD33, CD34, CD38, CD45, CD206, CD235a, and SSEA-1 is negative.

[0017] For details regarding the preparation method of the DPC exemplified above, please refer to Patent Document 1. In this embodiment, the DPC may be modified depending on the intended use, and may be immortalized using, for example, SV40 large T antigen. Furthermore, the animal from which the DPC used in this embodiment is derived is not particularly limited, but human-derived DPC is most preferred.

[0018] The expression of exogenous NIS within a DPC can be easily carried out based on common technical knowledge in the field. While not particularly limited, vectors that can be used to introduce exogenous NIS into a DPC include viral vectors such as retroviral vectors (e.g., lentiviral vectors), adenovirus vectors, adeno-associated virus vectors, Simian virus 40 (SV40) vectors, and herpesvirus vectors, as well as non-viral plasmid vectors. Gene transfer methods using non-viral vectors include, for example, calcium phosphate methods, electroporation methods, and particle gun methods. Furthermore, promoters that can be used to express NIS within a DPC may be either constitutive or inductive promoters, such as the E2F promoter, telomerase (hTERT) promoter, cytomegalovirus-derived promoter (CMV IE promoter), and EF1-α promoter, but are not limited to these. Additionally, enhancers may be ligated to the promoter to make the transcriptional activity by the promoter more efficient.

[0019] The expression of exogenous NIS within a DPC using a lentiviral vector can be easily carried out based on common technical knowledge in the art. The selection of auxiliary reagents used for lentiviral infection and their optimal usage amounts can be appropriately determined through preliminary experiments. For example, when preparing an NIS-DPC according to this embodiment, polybren or protamine can be used as the infection reagent, although this is not particularly limited, and protamine can be used in particular.

[0020] The second embodiment is a composition comprising a DPC that expresses NIS (NIS-DPC), characterized in that it is used in combination with a radionuclide (hereinafter also referred to as "the composition according to this embodiment"). The composition according to this embodiment can be used, for example, for the treatment of cancer or for the diagnosis of cancer, and is characterized by being administered separately to a radionuclide in combination with the composition. As described above, NIS-DPC accumulates in the cancer stroma and takes up radionuclides into cells, enabling the delivery of radionuclides to cancer tissue (cancer lesions). By identifying the location of the radionuclide, the site of the cancer lesion can be identified. Furthermore, by using a radionuclide with cytotoxic activity, radiation from NIS-DPC can damage and even kill surrounding tumor cells. Therefore, the composition according to this embodiment can be used for the diagnosis or treatment of cancer.

[0021] The radionuclides used in combination with the composition according to this embodiment may be any radionuclides that can be incorporated by NIS-DPC, and are not particularly limited, but for example, 131 I, 123 I, 125 I, 124 I, 99m Tc, 188 Re and 211 Examples include At. When the composition according to this embodiment is used for the treatment of cancer, the radioactive nuclide is preferably one that emits alpha or beta rays, which have a high cell-killing ability, for example, 131 I, 188 Re or 211At is preferred. Furthermore, when the composition according to this embodiment is used for cancer diagnosis, the radioactive nuclide is preferably one that can be detected by, for example, SPECT (Single Photon Emission Computed Tomography) or PET (Positron Emission Tomography), such as a nuclide that emits gamma rays, for example, 123 I, 125 I, 124 I or 99m Tc and similar are preferred.

[0022] When the composition according to this embodiment is used for the treatment of cancer, the cancers (malignant tumors / malignant neoplasms) to be treated are not particularly limited, but include, for example, hepatocellular carcinoma, cholangiocarcinoma, renal cell carcinoma, squamous cell carcinoma, basal cell carcinoma, transitional cell carcinoma, adenocarcinoma, malignant gastrinoma, malignant melanoma, fibrosarcoma, myxosarcoma, liposarcoma, leiomyosarcoma, rhabdomyosarcoma, malignant teratoma, angiosarcoma, Kaposi's sarcoma, osteosarcoma, chondrosarcoma, lymphangiosarcoma, malignant meningioma, non-Hodgkin lymphoma, Hodgkin lymphoma, and leukemia. Other examples include brain tumors, neoplasms derived from epithelial cells (epithelial carcinomas), basal cell carcinomas, adenocarcinomas, lip cancers, oral cancers, gastrointestinal cancers such as esophageal cancer, small intestine cancer and stomach cancer, colon cancer, rectal cancer, liver cancer, bladder cancer, pancreatic cancer, ovarian cancer, cervical cancer, lung cancer, breast cancer, skin cancers such as squamous cell carcinoma and basal cell carcinoma, prostate cancer, and renal cell carcinoma, as well as cancers that have metastasized from these cancers, such as peritoneal dissemination resulting from metastases from digestive tract cancers such as stomach cancer, pancreatic cancer, and colorectal cancer, or from ovarian cancer and ureteral cancer.

[0023] The NIS-DPC, which is the active ingredient of the composition according to this embodiment, may be suspended in a solution that can suspend it, such as physiological saline or phosphate-buffered saline (PBS). Furthermore, the NIS-DPC suspension may contain pharmaceutically acceptable additives. When the composition according to this embodiment is used for the treatment or diagnosis of cancer, possible routes of administration to the body include, for example, intratumoral administration (in the tumor or its vicinity, for example, intraperitoneal administration if the target of treatment is peritoneal dissemination) or intravenous administration. Furthermore, possible routes of administration of radionuclides administered in combination include, for example, intratumoral administration (in the tumor or its vicinity, for example, intraperitoneal administration if the target of treatment is peritoneal dissemination), intravenous administration, or oral administration.

[0024] The NIS-DPC, which is the active ingredient of the composition according to this embodiment, may be provided in a frozen state. In this case, the solution containing the frozen NIS-DPC may be thawed several days before administration, for example 10 days, and administered after being grown to the required number of cells, or it may be thawed immediately before administration and administered without going through the culture process.

[0025] The administration order of the composition and radionuclide according to this embodiment is not particularly limited, but it is preferable to administer the composition first, followed by the radionuclide. The administration interval between the composition and radionuclide according to this embodiment is not particularly limited, but for example, 1 to 7 days is preferred. Furthermore, the number of administrations of the composition and radionuclide according to this embodiment may be one administration or multiple administrations. The dosage of the composition and radionuclide according to this embodiment is not particularly limited, as long as it does not place an excessive burden on the body and can achieve the therapeutic effect or diagnostic purpose, and can be determined by the judgment of a specialist such as a physician.

[0026] The compositions according to this embodiment (therapeutic compositions and diagnostic compositions) may be provided in the form of a kit along with instructions for use describing the method of administration, etc. The compositions contained in the kit are supplied separately for each active ingredient in containers made of a material that effectively maintains the activity of the active ingredient (i.e., DPC or NIS-DPC, radionuclides, etc.) for a long period of time, prevents the ingredients from adsorbing to the inside of the container, and does not alter the ingredients. Radionuclides must be supplied in a form suitable to prevent radiation leakage. Furthermore, the instructions for using this kit may be provided printed on paper or on an electronically readable medium such as a CD-ROM or DVD-ROM.

[0027] The third embodiment is a method for treating cancer (hereinafter also referred to as "the method for treating cancer according to this embodiment"), which includes administering the composition and radionuclide according to this embodiment to a patient. The radionuclide used in this embodiment is preferably one that is incorporated into NIS-DPC and emits alpha or beta rays with high cell-killing ability, for example, 131 I, 188 Re or 211 Examples include "At," etc. Here, "treatment" refers to preventing or mitigating the progression and worsening of the disease in patients who have already been diagnosed with cancer, and is a procedure aimed at preventing or mitigating the progression and worsening of cancer. Furthermore, the treatment is not limited to humans, but may also apply to other mammals such as mice, rats, dogs, and cats, as well as livestock such as cattle, horses, and sheep, and primates such as monkeys, chimpanzees, and gorillas, with humans being particularly preferred. The cancer treatment method according to this embodiment includes the steps of administering NIS-DPC and administering a radionuclide. The interval between the administration of NIS-DPC and the radionuclide is not particularly limited, but for example, 1 to 7 days is preferred. The method of administering the radionuclide may be administration to tumor tissue (cancer lesion) or tissue containing tumor cells, intravenous administration, intraperitoneal administration, or oral administration. Furthermore, the number of administrations of NIS-MSC and the radionuclide may be a single administration or multiple administrations. The cancer treatment method according to this embodiment may be carried out in combination with other chemotherapy or radiotherapy, etc.

[0028] The cancers (malignant tumors / malignant neoplasms) targeted by the cancer treatment method according to this embodiment are not particularly limited, but include, for example, hepatocellular carcinoma, cholangiocarcinoma, renal cell carcinoma, squamous cell carcinoma, basal cell carcinoma, transitional cell carcinoma, adenocarcinoma, malignant gastrinoma, malignant melanoma, fibrosarcoma, myxosarcoma, liposarcoma, leiomyosarcoma, rhabdomyosarcoma, malignant teratoma, angiosarcoma, Kaposi's sarcoma, osteosarcoma, chondrosarcoma, lymphangiosarcoma, malignant meningioma, non-Hodgkin lymphoma, Hodgkin lymphoma, leukemia, brain tumors, and epithelial tumors. Examples of cancers that can be classified as neoplasms include cell-derived neoplasms (epithelial carcinomas), basal cell carcinomas, adenocarcinomas, lip cancers, oral cancers, gastrointestinal cancers such as esophageal cancers, small intestine cancers and stomach cancers, colon cancers, rectal cancers, liver cancers, bladder cancers, pancreatic cancers, ovarian cancers, cervical cancers, lung cancers, breast cancers, skin cancers such as squamous cell carcinomas and basal cell carcinomas, prostate cancers, and renal cell carcinomas. Other examples include cancers that have metastasized from these cancers, such as peritoneal dissemination resulting from metastases from digestive tract cancers such as stomach cancer, pancreatic cancer, and colorectal cancer, as well as ovarian cancer and ureteral cancer.

[0029] The fourth embodiment is a method for diagnosing cancer (hereinafter also referred to as "the method for diagnosing cancer according to this embodiment") which includes administering the composition and radionuclide according to this embodiment to a subject. The cancer diagnostic method according to this embodiment includes the steps of administering NIS-DPC to a subject, administering a radionuclide that can be taken up by NIS-DPC to the subject, and identifying the site in the body where the radionuclide accumulates. The radionuclide used in the cancer diagnostic method according to this embodiment is preferably one that can be taken up by NIS-DPC and detected by a device capable of identifying the location of the radionuclide, such as SPECT (Single Photon Emission Computed Tomography) or PET (Positron Emission Tomography). For example, a radionuclide that emits gamma rays is preferred. 123 I, 125 I, 124 I or 99m Tc and similar are preferred.

[0030] Where this specification is translated into English and contains the singular forms of "a," "an," and "the," they shall be considered to include both singular and plural forms unless the context clearly indicates otherwise. The present invention will be further explained below with reference to examples, but these examples are merely illustrative of embodiments of the present invention and do not limit the scope of the present invention. [Examples]

[0031] [Example 1] Fabrication of NIS-DPC 1. Production and quality testing of lentiviruses 1.1. Construction of pLVSIN-EF1α-hNIS-Pur The self-inactivating (SIN) lentiviral vector pLVSIN-EF1α-hNIS-Pur was created by inserting the nucleic acid sequence encoding human NIS (human NIS, hereinafter "hNIS", SEQ ID NO: 2, NCBI accession number: NM_000453.3) into the multi-cloning site of the pLVSIN-EF1α-Pur vector (Takara Bio). Specifically, the DNA encoding hNIS was artificially synthesized with a Not I site added to the 5' end and a BamH I site added to the 3' end, and then treated with the corresponding restriction enzymes. Next, the pLVSIN-EF1α-Pur vector was cleaved with the restriction enzymes Not I and BamH I to linearize the vector, and pLVSIN-EF1α-hNIS-Pur was produced by ligation with the restriction enzyme-treated hNIS using Ligation high Ver.2 (TOYOBO).

[0032] 1.2. Packaging of lentiviruses For lentivirus packaging, in addition to the SIN-type lentiviral vector (pLVSIN-EF1α-hNIS-Pur) prepared in 1.1 above, we used Lentiviral High Titer Packaging Mix (hereinafter referred to as "Packaging Mix") (Takara Bio), which is a plasmid mixture containing the components necessary for lentiviral vector preparation (HIV-1 derived Gag, Pol, Tat, Rev lentiviral proteins and VSV-G envelope protein), and prepared the product by slightly modifying the standard protocol. Specifically, 4 × 10¹⁶ samples were placed on a 10¹⁶ cm dish treated with Poly-L-Lysine. 6 Cells were seeded, and the day after seeding, a DNA mix was prepared by adding 7 μL of Packaging Mix and 11 μL of pLVSIN-EF1α-hNIS-Pur (prepared to 0.5 μg / μL) to 1.5 mL of OptiMEM I Reduced Serum Medium (Gibco). Then, Fugene HD (Promega) or TransIT-Lenti Transfection Reagent (Takara Bio) was added to the prepared DNA mix, and transfection was performed. The cells were then cultured at 37°C in a 5% CO2 incubator. Approximately 24 hours after transfection, the supernatant was aspirated and removed, and the medium was changed to 10 mL of DMEM medium. The cells were then cultured at 37°C in a 5% CO2 incubator for approximately 24 hours. After that, the culture supernatant was collected, centrifuged at 500×G for 10 minutes, passed through a 0.45 μm filter, and then pipetted into microcentrifuge tubes and stored at -80°C. Furthermore, a portion of the recovered culture supernatant was concentrated using Lenti-X Concentrator (Takara Bio).

[0033] 1.3. Titer measurement of lentiviruses The prepared lentiviruses were measured for three parameters: p24 protein content (ELISA), viral genome content (qPCR), and biological infectivity titer (flow cytometry). p24 protein content was measured using the Lenti-X p24 Rapid Titer Kit (Takara Bio), and viral genome content was measured using the Lenti-X qRT-PCR Titration Kit (Takara Bio). Biological infectivity titer was calculated by infecting cultured HEK293T with lentivirus and measuring the hNIS positivity rate by flow cytometry two days after infection. The measured titers of the prepared lentiviruses ranged from approximately 20 ng / mL to 200 ng / mL (p24 protein content), and 1 × 10⁻⁶ titers were also measured. 7 VG / mL ~ 1 × 10 9 VG / mL (viral genome amount), 1 × 10⁻⁶ 6 TU / mL ~ 6 × 10 6 The TU / mL (biological infectivity titer) was approximately 200 ng / mL to 400 ng / mL (p24 protein content), 1 × 10⁻⁶. 8 VG / mL ~ 6 × 10 9 VG / mL (viral genome amount), 1 × 10⁻⁶ 7 TU / mL ~ 3 × 10 7 The concentration was approximately TU / mL. The concentrated solution was used as the hNIS-LV stock solution.

[0034] 2. Examination of methods for modifying DPC functions using lentiviruses We investigated infection conditions that would allow for functional modification using lentiviruses without compromising the characteristics of DPC. 2.1. Examination of conditions for infection of DPC by lentiviruses 2.1.1. Examination of auxiliary reagents We compared and evaluated the effects of polybren and protamine, commonly known auxiliary reagents for promoting lentivirus infection of cells, on post-infection cell proliferation and the positive rate of introduced hNIS. 2.1.1.1. Method DPC at 20,000 cells / cm 2 The day after sowing, approximately 90 μL / cm³2 The hNIS-LV stock solution was added in the following manner. At that time, the virus stock solution was supplemented with either a polyblen solution with a concentration of 8 μg / mL or a protamine sulfate solution with a concentration of 100 μg / mL after addition. The hNIS-LV stock solution was removed the day after addition, and an equal volume of DMEM medium was added. Subsequently, the culture was continued until 22 days after infection, and the doubling days and cumulative PDL (Population doubling level) values ​​were calculated and compared. Furthermore, we compared the effect of passage number on the hNIS positivity rate (hNIS positivity rate was confirmed up to 6 passages).

[0035] 2.1.1.2.Results Figure 1 shows the changes in doubling days for DPC after lentivirus infection. The doubling days immediately after viral infection were 6.75 days for the group infected with polybren and 8.36 days for the group infected with protamine. Compared to 1.6 days for DPC, which was measured in parallel as a control, both groups showed higher values, indicating a tendency for a temporary decrease in proliferation. Subsequent continuous cultures showed that although proliferation was lower than that of DPC, it was thought that proliferation could be maintained for about 6 passages after infection. Furthermore, it was thought that using protamine as an auxiliary reagent suppressed the decrease in proliferation.

[0036] Figure 2 shows the changes in the hNIS positivity rate during continuous culture. Measurements were also taken for DPC cells that were not infected as a control. The hNIS positivity rate immediately after the start of culture was 89.0% in the group infected with protamine and 79.0% in the group infected with polybren, indicating that the DPC cells infected with protamine had a higher hNIS positivity rate. Subsequently, when the hNIS positivity rate was checked during continuous culture and passage, there was a tendency for the positivity rate to gradually decrease, but it was thought that a high positivity rate could be maintained for 2 or 3 passages. Furthermore, it was found that the protamine group, which had a higher positivity rate, was able to maintain the positivity rate better than the group infected with polybren. When the positivity rate was low, it was assumed that the positivity rate decreased as the number of uninfected cells gradually became significant. Therefore, it was considered necessary to aim for an hNIS positivity rate of 90% or higher after infection. Based on the above, it was concluded that using protamine as an auxiliary reagent during infection would enable more efficient gene transfer by lentivirus and maintain the hNIS positivity rate even after subculturing. Therefore, protamine was chosen as an auxiliary reagent for lentivirus-based infection of DPC.

[0037] 2.1.2. Examination of titer at time of infection We conducted optimization studies on the viral titer at the time of infection (Moment of Infection: MOI), which is necessary when adding lentivirus to DPC. 2.1.2.1. Method The hNIS-LV stock solution prepared in 1.3 was used. DPC was applied at 20,000 cells / cm². 2 The hNIS-LV stock solution was added the day after sowing. At that time, 100 μg / mL of protamine sulfate solution was added to the hNIS-LV stock solution as a reagent to promote viral infection of cells. The hNIS-LV stock solution was removed the day after addition, and an equal volume of DMEM medium was added. Three lots of hNIS-LV stock solution with different titers were used, and the stock solution was diluted 2-fold four times to infect DPC with five different viral loads (see Table 1). [Table 1] Four days after lentivirus infection, the cells were passaged and cultured in DMEM supplemented with puromycin at a concentration of 1 μg / mL. Subsequently, each cell under each condition was passaged again at the time of confluence (cultivated in DMEM without puromycin), and after a total of two passages, the hNIS positivity rate of the harvested cells was measured by flow cytometry.

[0038] 2.1.2.2.Results Figure 3 shows the relationship between MOI at the time of infection and the hNIS positivity rate when cells were collected after two passages. A strong correlation was observed between MOI and hNIS positivity, and it was confirmed that the hNIS positivity rate increased as the MOI of the added virus increased. Furthermore, it was suggested that a positivity rate of 80% could be achieved when the MOI exceeded 8, and nearly 100% of cells could be introduced with the target gene, hNIS, when the MOI reached 15. Although selection was performed using antibiotics to obtain only positive cells during culture after lentivirus addition, the hNIS positivity rate did not increase as expected. Therefore, it was considered important to add a viral load (= MOI) that can ensure a sufficient positivity rate at the time of lentivirus infection. Considering the results of the previous studies and the currently envisioned hNIS-DPC manufacturing process, the hNIS positivity rate at the time of infection should be targeted at around 90%. Therefore, the target MOI of the lentivirus added to DPC should be 10 or higher.

[0039] 3. Creation of hNIS implementation DPC The hNIS-LV stock solution prepared in 1.3 was added to DPC to create hNIS-introduced DPC (hNIS-DPC). The infection conditions were those found in the previous study. 3.1. Creation of hNIS-DPC The preparation of hNIS-DPC was carried out in general as follows. DPC at 20,000 cells / cm 2The day after sowing, the hNIS-LV stock solution was added to achieve an MOI of 10. At that time, protamine sulfate solution was added to the virus stock solution to promote viral infection of DPC, so that the concentration after addition was 100 μg / mL. The hNIS-LV stock solution was removed the day after addition, and an equal volume of DMEM medium was added.

[0040] 3.1.1. DPC culture Thaw the frozen DPC and place 20,000 cells / cm in a 10 cm dish. 2 Cells were seeded in this manner. After gene transfer using a lentiviral vector with protamine sulfate solution according to the conditions shown in 2.1, the seeded cells were harvested on day 4 of culture and stored in a T225 flask at a rate of 7,000 cells / cm³. 2 Cells were seeded. On the fourth day of culture, the culture medium was replaced with DMEM to which puromycin was added to a concentration of 1 μg / mL. Cells were harvested on day 6 of culture and stored in two T1000 flasks at a rate of 8,000 cells / cm². 2 Cells were seeded. Cells were harvested on day 6 of culture, with 3 × 10⁶ cells per tube. 6 The cells were dispensed into vials and stored frozen.

[0041] 4. Quality check of hNIS-DPC We performed a quality evaluation of the hNIS-DPC fabricated in section 3.1. 4.1. Method The evaluation items included (1) doubling time during freezing, (2) PDL (number of cell divisions) and cumulative PDL, (3) percentage of hNIS-positive cells, and (4) the relationship between hNIS positivity and iodine uptake. The doubling days and PDL were calculated using the following formulas. [Doubling days] Culture days × Log10 2 / (Log10 (number of cells recovered) - Log10 (number of cells seeded)) [PDL (Number of Cell Divisions)] (Log10 (number of cells recovered) - Log10 (number of cells seeded)) × Log2 10 hNIS-positive cells were confirmed by flow cytometry analysis. The harvested cells were suspended in a blocking agent prepared by adding 30% BSA (Fujifilm Wako Pure Chemical Industries) to DPBS (Gibco) to achieve a 3% BSA concentration. The cells were left to stand at room temperature for at least 30 minutes or at 4°C until the next day. Subsequently, the cells were washed by centrifugation with Stain Buffer (BD), and then 1 × 10⁶ hNIS antibodies (Human SLC5A5C2 antibody (R&D)) were used. 6 Cells were suspended in an antibody solution prepared by adding hNIS antibody to Stain Buffer to an antibody level of 0.25 μg to 0.5 μg per cell, and allowed to stand at room temperature for at least 30 minutes to perform the primary antibody reaction. After that, the cells were centrifuged three times with Stain Buffer, and then suspended in an antibody solution prepared by diluting Alexa Fluor 488 goat anti Mouse IgG antibody (invitrogen) 400-fold in Stain Buffer. The cells were allowed to stand at room temperature and in the dark for at least 30 minutes to perform the secondary antibody reaction. After that, the cells were centrifuged three times with Stain Buffer, and then flow cytometry measurements were performed. The hNIS positivity rate was calculated by measuring samples processed with DPC according to the above procedure, and adjusting the gate position so that the cell population within the hNIS-positive gate in the FITC-A histogram was approximately 0.1-1%. The amount of iodine uptake was measured by the method described in 4.3.1 below.

[0042] 4.2.Results The doubling time for the prepared hNIS-DPC was 2.1 days, the PDL was 2.9, the cumulative PDL was 24.0, and the percentage of hNIS-positive cells was 94.1%. Since the doubling time was around 1.5 to 2.0 days in DPC without gene transfer, it was considered that the effect of lentivirus-mediated hNIS gene transfer on cell proliferation was minimal. Furthermore, the relationship between iodine uptake and the hNIS positivity rate is shown in Figure 4. In both cases, where infection was performed using protamine and polybren, a linear positive correlation was observed between the NIS positivity rate and the iodine uptake rate, confirming that the NIS expressed in DPC was functioning normally.

[0043] 4.3. Quality comparison with hNIS-mouse MSC We compared the quality of hNIS-DPC cells produced by introducing hNIS into mouse-derived MSCs (hereinafter referred to as hNIS-mouse MSCs) prepared separately, with hNIS-DPC cells prepared using this method. The comparison was evaluated based on the amount of iodine uptake via transporters, which are considered important for drug efficacy.

[0044] 4.3.1. Method 50,000 hNIS-mouse MSCs or hNIS-DPCs per 96-well or 48-well plate 2 Seeds were seeded in the specified manner and culture was started at 37°C and 5% CO2. The day after the start of culture, the cells were washed twice with 10 mM HEPES-HBSS, and then a NaI solution prepared by mixing NaI (Fujifilm Wako Pure Chemical Industries) with 10 mM HEPES-HBSS to a concentration of 5 μM was added. The cells were incubated at 37.0°C and 5.0% CO2. After 4 hours, the cells were washed twice with 10 mM HEPES-HBSS, and then sterile water for injection was added. The cells were allowed to stand for up to 1 day until NaI was eluted from the cells, and the cell eluate was collected. Subsequently, the amount of iodine uptake by the cells was calculated by measuring the iodine concentration in the eluate using a Non-Radioactive Iodide Assay Kit (Bertin Pharma).

[0045] 4.3.2.Results The amount of iodine taken up is 1 × 10⁻⁶ 6The amount was calculated per cell. Measurements were performed on three lots of hNIS-DPC prepared in 4.1.2. As a control, measurements were also performed on intact DPC without the introduction of hNIS. The results are shown in Figure 5. Almost no iodine uptake was observed in intact DPC without the introduction of hNIS. On the other hand, in DPC with the introduction of hNIS, it was confirmed that more iodine was taken up in all lots compared to hNIS-mouse MSCs. Therefore, it was concluded that the hNIS-DPC prepared using lentivirus in this study maintains its function as an iodine transporter and can take up a large amount of iodine depending on a high positive rate.

[0046] [Example 2] In vivo study of hNIS-DPC 1. Creation of a peritoneal dissemination model All animal experiments were approved by the University of Tokyo Animal Experimentation Committee and conducted in accordance with its guidelines. Five-week-old C57BL / 6 mice (Charles River Japan Co., Ltd.) were transplanted with the mouse gastric cancer cell line YTN 16 (1 × 10⁻¹⁶). 7 Cells (per animal) were administered intraperitoneally to induce peritoneal dissemination. YTN16 is a cell line established by the inventors (Non-Patent Literature 5). One to three weeks after administration of YTN16, macroscopically visible peritoneal dissemination was confirmed to have formed on the peritoneum.

[0047] 2. Evaluation of migration and accumulation around cancer tissue after intraperitoneal and intravenous administration of DPC. 2.1. Method 2.1.1. Administration of intact DPC The DPC was thawed, cultured, and expanded, and the harvested cells were frozen (using two cryoprotective agents, TC protector and Cell Banker). Before administration to animals, the DPC was thawed, pre-cultured, and the required amount of cells were suspended in the medium. The number of cells administered was 5 × 10⁶ for both intraperitoneal and intravenous administration. 6 cells / body, 1×10 6 cells / body, 0.5 × 10 6Three doses were used for cells / body. Each dose was administered to three peritoneal dissemination model mice (intravenous and intraperitoneal administration, using 3 doses x 3 mice for a total of 9 peritoneal dissemination model mice). DPC was administered on days 15, 17, and 19, with the day of YTN16 administration being day 1.

[0048] 2.1.2. Evaluation Methods for DPC Migration and Accumulation Cell migration and accumulation were evaluated by immunostaining of tissue sections. Mice were euthanized on day 21 after YTN16 administration (Day 21), and the peritoneum containing peritoneal dissemination was excised. The peritoneum was pinned onto a rubber plate and fixed overnight in 4% neutral buffered paraformaldehyde. The fixed peritoneum was transferred to a 50 mL Falcon tube, washed with PBS for 20 minutes three times or more on a shaker, and then washed again with PBS overnight. Paraffin blocks were then prepared by Advantech Co., Ltd. Sections were prepared from the paraffin blocks, and immunostaining was performed using anti-human Lamin B1 antibody (Anti-human Lamin B1: HS-404 017 (Rat monoclonal purified IgG), HistoSure) to evaluate the presence or absence of stained cells.

[0049] 2.2.Results When the number of cells administered for each route of administration was evaluated, it was found to be 5 × 10 6 In the group that received intravenous administration of cells / body, 3 animals were found to have 1 × 10⁶ bodies. 6 One death was observed in the group administered cells / body intravenously. On the other hand, no deaths were observed in the group administered intraperitoneally. When tissue sections prepared from peritoneal tissue were examined for nonspecific staining in cancerous areas, no nonspecific staining was observed in either normal mouse peritoneal tissue or peritoneal tissue with peritoneal dissemination when DPC was not administered. Therefore, it was determined that the antibody (anti-human Lamin B1 antibody) can be used to appropriately detect the presence or absence of human cells in peritoneal tissue.

[0050] Figure 6 shows representative immunohistochemical staining results for peritoneal tissue in mice that received intraperitoneal cell administration. In all mouse peritoneal tissues, staining suggestive of the administered DPC was observed, with DPC accumulation observed surrounding the cancerous tissue (the area enclosed by the dashed line in the figure is the cancerous tissue). Furthermore, differences in the degree of staining were observed depending on the cell number, with 1 × 10⁻⁶ cells being the most effective. 6 When administering a cell count greater than or equal to cells / body, there did not appear to be any significant difference in the staining pattern. Representative results from individuals administered intravenously are shown in Figure 7. 5×10 6 All individuals that received intravenous administration of cells / body died, therefore 1 × 10 6 cells / body and 0.5×10 6 Only the results for individuals administered cells / body are shown. Specific staining derived from DPC was observed even with intravenous administration, similar to intraperitoneal administration, suggesting that DPC migrates to cancer tissue even with this administration route. Regarding the degree of accumulation, intravenous administration appeared to be less than intraperitoneal administration. The results above suggest that DPC migrates to cancer tissue not only through intraperitoneal administration but also through intravenous administration. Furthermore, based on the results obtained in this study, the number of cells to be administered in future studies will be 1 × 10⁶. 6 The upper limit was set at cells / body.

[0051] 3. Evaluation of migration and accumulation of hNIS-DPC around cancer tissue after intraperitoneal administration. Previous studies have confirmed the migration of DPC to the vicinity of cancer tissue. Therefore, we evaluated whether a similar trend could be observed in DPC introduced using a lentivirus to create a human sodium-iodine cotransporter (NIS) (hereinafter referred to as hNIS-DPC).

[0052] 3.1. Evaluation Method The evaluation was performed using the same method as described in 2 above. Figure 8 shows the experimental schedule. This evaluation used two types of NIS-DPC: cells that had been pre-cultured before administration (cultured cells) and frozen cells (frozen cells). The administration routes were intraperitoneal and intravenous. The number of cells administered was 1 × 10⁶ for intravenous administration. 6 cells / body and 0.5×10 6 cells / body), intraperitoneal administration (1 × 10⁻¹⁰ 6 The procedure was performed using cells / body. Additionally, DPC (frozen product only) was set up as a control, and intravenous administration (0.5 × 10⁻¹⁰) was performed. 6 cells / bod), intraperitoneal administration (1 x 10) 6 Cells were evaluated (24 individuals total) per body. Migratory cells were detected by PCR and immunostaining. PCR evaluation was performed as follows: Peritoneal tissue was collected from euthanized mice, homogenized, RNA was extracted, reverse transcription was performed, and PCR was carried out using the resulting cDNA as a template. Three tissue samples were collected from each individual. Cell accumulation was evaluated by the expression of the human gene GAPDH. Mouse GAPDH was used as a control. The conditions for PCR are as follows: Mold quantity 1 / 50th the amount of cDNA created from 1 μgRNA Thermal cycler settings 94℃ 5min, (94℃ 30sec, 60℃ 30sec, 72℃ 30sec) × 40 cycles, 72℃ 30sec Primer used Mouse GAPDH; amplification size: 177 bp, Human GAPDH; amplification size: 203 bp

[0053] 3.2.Results 3.2.1. Evaluation by PCR The results of the PCR evaluation are shown in Figure 9. In the group administered DPC (frozen product), human GAPDH expression was detected in both intravenous and intraperitoneal administration. There was variability in detection among individuals, which was presumed to be due to the location of the peritoneal tissue sample. In hNIS-DPC, human GAPDH expression was confirmed regardless of whether it was a frozen or cultured product, similar to DPC. Furthermore, expression was observed regardless of the administration route, and there were no significant differences between doses.

[0054] 3.2.2. Evaluation by immunohistochemical staining Figure 10 shows representative immunohistochemical staining results when frozen hNIS-DPC was thawed and administered intraperitoneally without pre-culture, and Figure 11 shows representative immunohistochemical staining results when it was administered intraperitoneally after pre-culture. Figure 12 shows representative immunohistochemical staining results when frozen hNIS-DPC was thawed and administered intravenously without pre-culture. In individuals administered hNIS-DPC, regardless of whether it was frozen or cultured, staining originating from human cells was observed around the peritoneal tissue. Comparing intravenous and intraperitoneal administration, more cell accumulation appeared to occur with intraperitoneal administration (for example, compare the areas indicated by arrows in Figures 10, 11, and 12).

[0055] 3.2.3.Summary PCR and immunohistochemical staining results suggested that even in hNIS-DPC functionally modified with lentivirus, cells accumulated and infiltrated around cancerous tissue regardless of the administration route. Although not quantitative, the amount of accumulated cells appeared to be greater with intraperitoneal administration than with intravenous administration. Furthermore, in the case of intraperitoneal administration, there did not appear to be a significant difference in the degree of accumulation between frozen and cultured hNIS-DPC. When considering conducting clinical trials, administering cells into the peritoneal cavity does not place a significant burden on the patient, as many patients already have ports for drug administration. Moreover, intraperitoneal administration allows for localized cell delivery, which is expected to lead to higher efficacy. Therefore, in future studies, intraperitoneal administration will be selected as the first-line method of administration.

[0056] 4. 125Evaluation of migration and accumulation of hNIS-DPC around cancer tissue using I The administered hNIS-DPC accumulated in cancer tissue, and we evaluated whether the hNIS expressed in DPC could take up radionuclides. The radionuclides used were those that emit gamma rays. 125 I used I.

[0057] 4.1. Method The evaluation was carried out using the same method as described in item 2 above. To shorten the evaluation period, the model creation period after YTN16 cell administration was changed from 14 days to 7 days. 125 To suppress the accumulation of I, 2 μg of L-T4 / d levothyroxine (thyroid hormone) was administered from day 4 after YTN16 transplantation. Only cells that had been pre-cultured before administration (cultured cells) were used in the study, the administration route was intraperitoneal, and the number of cells administered was 1 × 10⁶. 6 The experiment was performed using cells / body samples. A control group (DPC, also cultured) was also established. The experimental schedule is shown in Figure 13. Two groups were formed: two hNIS-DPC administered and one DPC administered. Cells were administered in three separate doses. A total of seven cell administrations were performed. 125 I was administered three times (oral, 4 MBq). Day 1 was the day of YTN16 administration, and tissue was collected on day 28. The gamma radiation dose of each tissue was measured using a gamma counter to determine cell migration to each tissue and radioactive iodine. 125 The presence or absence of uptake of γ-ray I was evaluated. The gamma radiation dose was measured in the following tissues: peritoneum, liver, stomach, intestines, colon, pancreas, spleen, kidneys, heart, lungs, brain, ovaries, thyroid gland, submandibular gland, and bone.

[0058] 4.2.Results 4.2.1. Gamma ray dose results in each tissue Figure 14 shows the results of gamma ray dose measurements in major organs. Gamma ray doses in peritoneal tissue were higher in individuals treated with hNIS-DPC compared to individuals treated with DPC. Therefore, it was suggested that the hNIS-infused DPC was localized in the peritoneal tissue and incorporated radioactive iodine. In other tissues, there was a high accumulation of radioactive iodine in the stomach, lungs, thyroid gland, and submandibular gland. In mice, since the expression level of NIS was high in the order of the stomach, thyroid gland, submandibular gland, colon, testis, and lungs, it was considered that the accumulation amount increased accordingly. Also, the accumulation in the lungs was considered to be affected by the accumulation in the bronchus. Also, 125 Since I was administered orally (intragastric administration) instead of intraperitoneally, radiation remained in the digestive tract, and a large amount of residue in the stomach was observed, which may have led to variations among individuals.

[0059] 4.2.2. Parentheses From the previous results, it was suggested that hNIS-DPC administered to the peritoneal seeding model accumulated around the cancer tissue and took up the administered 125 I through NIS expressed in DPC. Therefore, it was confirmed that by using NIS-DPC as a radiation delivery carrier, it is possible to accumulate cells around the cancer tissue.

[0060] 5. 211 Evaluation of the effect on cancer tissue when using At as a radionuclide (1) 211 At is a nuclide that emits α-rays. α-rays are heavy particle rays that double-strand break DNA and cause significant damage to cancer cells, and this effect is thought to be exerted regardless of the cell cycle compared to β-rays. Also, since α-rays are heavy particle rays, their flight distance is short, the damage to surrounding normal cells other than cancer cells is small, the side effects are few because they penetrate only a few levels of cells, the half-life is short when assuming administration to patients, they are excreted mixed in urine and feces, and there is no risk of exposure from patients to people around them. Therefore, it is considered to be much superior to the conventional treatment methods using β nuclides without the need for long-term isolation hospitalization like 131 I currently used in the treatment of thyroid cancer. Therefore, next, 211 To examine the possibility of peritoneal seeding treatment using At, hNIS-DPC was 211 used as a delivery carrier for At, and for peritoneal seeding 211The cytotoxicity of At was evaluated using a mouse peritoneal seeding model. administer 211 To optimize the At dosage, cytotoxicity evaluations were performed with target dosages of 0.2 MBq and 0.5 MBq, referring to the dosages previously administered to humans.

[0061] 5.1. Method The experimental schedule is shown in Figure 15. As for the group composition, (1) untreated group (n = 10), (2) 211 At (0.2 MBq / body) administration group (n = 10), (3) hNIS-DPC / 211 At (0.2 MBq / body) administration group (n = 10), (4) 211 At (0.5 MBq / body) administration group (n = 10), (5) hNIS-DPC / 211 At (0.5 MBq / body) administration group (n = 10) were set up as five groups. The administered cells were thawed frozen cells, the number of administered cells was 1×10 6 cells / body, and after administering the cells three times, 211 At was administered once. The administration route was intraperitoneal for both the cells and 211 At. 211 After administering At, observations were made for 21 days, and after euthanasia, evaluations were conducted. The evaluation visually scored the degree of peritoneal seeding, and the score was on a five-point scale (4; large tumor granules are present throughout, 3; there are tumor granules but not throughout, 2; tumor granules are clearly present but small, 1; it can be confirmed that there are slightly some tumor granules upon careful observation, 0; no tumor granules), with 4 when a large amount of peritoneal seeding remained and 0 when it had almost disappeared. Also, to quantify the amount of cancer cells remaining in the peritoneal tissue, the FGFR4 gene specifically expressed in YTN16 was measured by qPCR to quantitatively evaluate the cytotoxicity. Specifically, after collecting and homogenizing tissue from the peritoneum, RNA was extracted, reverse transcription was performed, and qPCR was carried out using the obtained cDNA as a template. Two specimens were collected per individual for the tissue. Mouse GAPDH was used as a control. The evaluation was calculated as the relative value when the expression level of the FGFR4 gene in the untreated group was set as 1. The conditions for PCR are as follows: Mold quantity 1 / 50th the amount of cDNA created from 1 μgRNA Quantitative Thermal Cycler Settings 94℃ 10 min, (95℃ 10 sec, 60℃ 30 sec, 72℃ 15 sec) × 40 cycles Primer used Mouse FGFR4; amplification size: 219 bp; Mouse GAPDH; amplification size: 177 bp

[0062] 5.2.Results The evaluation results obtained by visual scoring are shown in Figure 16. Representative images of visual evaluations of peritoneal dissemination in individual mice are shown in Figure 17. 211 Although the amount of At could not be measured due to a malfunction of the calibrator, it was estimated that approximately 0.3 and 0.6 MBq were administered to each individual based on calculations of half-lives, etc. (Hereafter, the administered amounts will be referred to as 0.3 and 0.6 MBq). Compared to the untreated group, 211 In the group receiving At as monotherapy, neither low dose (0.3 MBq) nor high dose (0.6 MBq) showed sufficient efficacy. On the other hand, in the hNIS-DPC / 211 In the At-administered group, sufficient efficacy was not observed at 0.3 MBq, but a reduction in peritoneal dissemination was observed at 0.6 MBq (mean score of 1.4 compared to 3.2 in the untreated group). Therefore, hNIS-DPC accumulates around cancer tissue. 211 It was thought that the incorporation of At resulted in damage to surrounding cells (i.e., regression and death of cancer cells), which affected the score (i.e., the score decreased). From these results, 211 Compared to administering only At, hNIS-DPC 211 It was observed that A functioned as a delivery carrier and exerted sufficient damaging effects against cancer.

[0063] Furthermore, hNIS-DPC and FGFR4 expression levels determined by qPCR were used as indicators. 211Figure 18 shows the results of the evaluation of the damage to cancer tissue caused by administering A. A similar trend to that observed in the visual evaluation was found. That is, 211 At alone did not show sufficient cancer degeneration effects, while hNIS-DPC / 211 In the group administered 0.6 MBq, a significant reduction in cancer was observed compared to the untreated group.

[0064] Based on the above results, hNIS-DPC 211 It has been revealed that it can be used as a transport carrier for At to peritoneal dissemination. Furthermore, when administered alone, doses that do not show cancer regression effect were found. 211 Even at, when administered together with hNIS-DPC, it efficiently accumulates near peritoneal dissemination and exerts a cancer regression effect; in other words, hNIS-DPC 211 It has been revealed that At enhances the cancer regression effect.

[0065] 6. 211 Evaluation of the effects of using At as a radionuclide on cancer tissue (2) Next, we investigated whether the antitumor effect of hNIS-DPC was enhanced in a dose (number of administrations)-dependent manner. In addition to the three administrations performed in step 5 above, we also set up one and two administrations and compared their effects on cancer tissue. The number of cells administered was 0.5 × 10⁶. 6 Even when using cells / body, a significant difference was observed compared to the untreated group (data not shown), so the cell count was 1 × 10⁻¹⁰ as in step 5 above. 6 0.5 × 10, half the amount of cells / body 6 We decided to administer cells / body from this experiment onward. 211 The At amount was set to 0.6 MBq / body. 6-1. Method The group composition for the peritoneal dissemination model was as follows: (1) Untreated group (n=10), (2) 211 At (0.6 MBq / body) administration group (n=10), (3) hNIS-DPC (1 dose) + 211 At (0.6 MBq / body) administration group (n=10), (4) hNIS-DPC (2 times) +211 At (0.6 MBq / body) administration group (n=10), (5) hNIS-DPC (3 times) + 211 Five groups were established for administration of At (0.6 MBq / body) (n=10). The timing of cell administration is shown in Figure 19. The administered cells were thawed frozen cells, and the number of administered cells was 0.5 × 10⁶. 6 cells / body, after administering cells 1-3 times 211 At was administered once. The route of administration was via cells. 211 Both At and the surrounding tissue were considered to be in the abdominal cavity. 211 The day of at administration was designated as day 0, and after euthanasia 21 days later, the amount of tumor dissemination in the peritoneal cavity was evaluated and scored. The score was on a 5-point scale (4: large tumor particles present throughout, 3: tumor particles present but not throughout, 2: clearly present but small tumor particles, 1: only a few tumor particles can be confirmed upon close observation, 0: no tumor particles). A score of 4 indicated a large amount of residual peritoneal dissemination, and a score of 0 indicated almost complete disappearance. Box plots were created based on the scores of each group, and the presence or absence of significant differences between the three groups was evaluated using the Tukey test.

[0066] 6.2.Results 211 After administration of At, each individual was observed for 21 days, and the evaluation results based on visual scoring are shown in Figure 20. Compared to the untreated group, 211 No significant difference was observed between the At monotherapy group (211At) and the single cell administration group (NIS-DPC / x1+At). However, differences were observed in the two-dose group (NIS-DPC / x2+At) and the three-dose group (NIS-DPC / x3+At), indicating an enhanced antitumor effect in a dose-dependent manner. This indicates that the antitumor effect depends on the number of NIS-DPC administrations (number of cells administered), confirming that NIS-DPC is essential for the antitumor effect to be exerted. Furthermore, it was suggested that a certain level of NIS-DPC accumulation near tumor tissue is necessary for the antitumor effect to be achieved.

[0067] 7. 211 Drug efficacy evaluation using survival curves in hNIS-DPC with At. 7.1. Method The group composition for the peritoneal dissemination model was (1) untreated group (n=16), (2) 211 At (0.6 MBq / body) administration group (n=17), (3) hNIS-DPC / 211 Three groups were established: one receiving At (0.6 MBq / body) (n=17), and another group receiving At (0.6 MBq / body). Additionally, (1) was performed on normal mice. 211 At administration group (n=3), (2) hNIS-DPC / 211 Two groups were set up at, 211 At standalone and 211 The impact of At / hNIS-DPC on survival rates was also evaluated. The administered cells were thawed frozen cells, and the number of administered cells was 0.5 × 10⁶. 6 cells / body, after administering cells three times 211 At was administered once. The route of administration was via cells. 211 Both At and the surrounding tissue were considered to be in the abdominal cavity. 211 The day of At administration was designated as day 0, and individuals in each group were observed to check for death. The observation period was 49 days. Kaplan-Meier curves were created from the survival time (days) of each individual, and the presence or absence of significant differences between groups was evaluated using the Log-rank test. After euthanasia at the time of death and at the end of the observation period, the amount of dissemination present in the peritoneal cavity was evaluated and scored. The score was on a 5-point scale (4: large tumor particles present throughout, 3: tumor particles present but not throughout, 2: clearly present but small tumor particles, 1: only a few tumor particles can be confirmed upon close observation, 0: no tumor particles), with a score of 4 indicating a large amount of residual peritoneal dissemination and a score of 0 indicating almost complete disappearance. Box plots were created based on the scores of each group, and the presence or absence of significant differences between the three groups was evaluated using the Tukey test.

[0068] 7.2.Results 211 After administration of At, each individual was observed for 49 days, and survival time was evaluated and analyzed using a Log-rank test with Kaplan-Meier curves. The results are shown in Figure 21. In the untreated group (#1: untreated), deaths were observed from day 21, and the median survival time, which corresponds to a 50% survival rate, was 35.5 days (solid line). 211 The group that received At alone (#2:211 In group At, deaths were observed from day 25. The number of deaths increased more slowly than in the untreated group, with a median of 44 days (large dotted line), but at the end of the observation period, the survival rate was not significantly different from that of the untreated group. On the other hand, 211 At and NIS-DPC administration group (#3: 211 In At+NIS-DPC), the untreated group and 211 Compared to the group treated with At monotherapy, an extension of survival rate was observed, and a statistically significant difference was found between the two groups (vs untreated group: P=0.00583, vs 211 At: P=0.02). Also, to normal mice. 211 At standalone and 211 In At / hNIS-DPC, no toxicity or findings affecting survival rates were observed in any case, and survival was confirmed in all individuals during the observation period.

[0069] The survival rates after the end of the observation period for each of the above groups are summarized in Figure 22. 211 In the At+NIS-DPC administration group, the untreated group and the untreated group were 211 Compared to the group treated with At as monotherapy, it showed a higher survival rate.

[0070] Figure 23 shows the results of visual scoring for peritoneal dissemination in the above peritoneal dissemination model. Compared to the untreated group, 211 No significant difference was observed between the group receiving At monotherapy (211At) and the other group. 211 A significant difference was observed between the At and NIS-DPC treatment groups (211At + NIS-DPC). Similarly, a significant difference was observed when compared to 211At, although it was not as strong as when compared to the untreated group.

[0071] Based on these results, the administered hNIS-DPC accumulated around the cancer tissue. 211 It was thought that the incorporation of At resulted in damage to surrounding cells (i.e., regression and death of cancer cells), which affected the score (i.e., the score decreased). Furthermore, it was suggested that the survival rate was extended due to the regression and death of cancer cells. From these results, it was concluded that hNIS-DPC 211It was observed that it functioned as a delivery carrier for A and exerted sufficient damaging effects against cancer, and was thought to contribute to extending the survival rate.

[0072] 8. Summary qPCR and immunohistochemical staining results suggested that DPC migrates to and accumulates in the tissues surrounding cancer cells, regardless of the administration route, such as intraperitoneal or intravenous administration. Similarly, DPC modified by introducing hNIS using a lentivirus (hNIS-DPC) was also confirmed to migrate to and accumulate around cancer cells, confirming that it maintains the characteristics of DPC. Furthermore, it is a radioactive isotope. 125 I and 211 Since the uptake of At into hNIS-DPC was confirmed, it was considered that the iodine transporter introduced into DPC also maintained its function. Furthermore, alpha radiation is emitted. 211 At was evaluated in a cancer cell damage assessment test, and hNIS-DPC and 211 The observed reduction of cancer cells with the At combination confirmed the proof of concept (PoC) for internal radiation therapy using hNIS-DPC and alpha rays. This treatment method is expected to be a new treatment option for refractory cancers, such as those with peritoneal dissemination. [Industrial applicability]

[0073] The therapeutic composition according to the present invention is effective in treating cancer, particularly intractable cancers (e.g., peritoneal carcinomatosis). Therefore, the present invention is expected to be used in the medical field (especially in the field of cancer treatment).

Claims

1. A therapeutic composition for peritoneal dissemination, characterized by comprising human dental pulp-derived stem cells expressing an externally introduced sodium iodide cotransporter (NIS), and used in combination with a radionuclide.

2. The therapeutic composition for peritoneal dissemination according to claim 1, characterized in that the composition is administered intraperitoneally or intravenously, and the radionuclide is administered intratumorally, intraperitoneally, intravenously, or orally.

3. The aforementioned radioactive nuclide is 131 I, 211 At or 188 The therapeutic composition for peritoneal dissemination according to claim 2, wherein Re is one of the following.

4. The therapeutic composition for peritoneal dissemination according to any one of Claims 1 to 3, wherein the human dental pulp-derived stem cells are not immortalized.

5. A diagnostic composition for peritoneal dissemination, characterized by comprising human dental pulp-derived stem cells expressing NIS introduced from an external source, and used in combination with a radionuclide.

6. The diagnostic composition for peritoneal dissemination according to claim 5, characterized in that the composition is administered intraperitoneally or intravenously, and the radionuclide is administered intratumorally, intraperitoneally, intravenously, or orally.

7. The aforementioned radioactive nuclide is 131 I, 123 I, 125 I, 124 I or 99 The diagnostic composition for peritoneal dissemination according to claim 6, wherein the mTc is one of the mTc.

8. The diagnostic composition for peritoneal dissemination according to any one of claims 5 to 7, wherein the human dental pulp-derived stem cells are not immortalized.

Images