Therapeutic agent for cancer

Abstract

Provided is an exceptional therapeutic agent for cancer. This therapeutic agent for cancer includes an immune checkpoint inhibitor and mesenchymal stem cells.

Description

Cancer treatment agents

[0001] The present invention relates to a cancer therapeutic agent.

[0002] Cancer has been the leading cause of death in Japan since 1981, and by 2022, approximately one in four people will die from cancer. It is estimated that approximately one million people were diagnosed with cancer in 2019, and approximately one in two people will develop cancer in their lifetime. By age group, cancer accounts for approximately 10% of deaths among children and approximately 36% of deaths among people aged 20 to 64. According to the "2022 Vital Statistics (Definitive Figures)" published by the Ministry of Health, Labor and Welfare in September 2023, 385,797 deaths will be due to cancer (malignant neoplasms, or tumors), accounting for 24.6% of all deaths. Furthermore, the number of cancer deaths is expected to continue to increase due to the increasing number of elderly cancer patients associated with the rapid growth of the elderly population. Therefore, the development of new cancer treatments with excellent efficacy is desired.

[0003] Currently, there are four cancer treatments: (1) surgery, (2) radiation therapy, (3) chemotherapy (cancer treatment drugs), and (4) immunotherapy, collectively known as the four major cancer treatments. Immunotherapy utilizes the immune system to attack cancer. Immune checkpoint inhibitors are one of the immunotherapies with scientifically proven efficacy and safety. Immune checkpoint inhibitors have been applied to various malignant tumors and their use is expanding. Immune checkpoint inhibitors inhibit immune checkpoints, which suppress the activity of immune cells, thereby enhancing their ability to attack cancer cells and demonstrating cancer treatment efficacy. However, immune checkpoint inhibitors are not effective in all cancer patients; the response rate is approximately 10% to 30%, depending on the type of cancer. Furthermore, even if immune checkpoint inhibitors are effective after the initial administration, acquired resistance has been reported, in which continued administration leads to a loss of cancer treatment efficacy (see Non-Patent Document 1). Therefore, complete tumor elimination in subcutaneous tumor model mice is generally considered difficult.

[0004] Meanwhile, in recent years, cell culture of tissues, cells, fertilized eggs, etc. from various parts of the human body has been put to practical use, and the cultured cells have been used in regenerative medicine, etc. One such example is mesenchymal stem cells. Mesenchymal stem cells are multipotent progenitor cells first isolated from bone marrow by Friedenstein (see Non-Patent Document 2). Mesenchymal stem cells have been shown to exist in various tissues, such as bone marrow, umbilical cord, and adipose tissue, and mesenchymal stem cell transplantation is expected to be a new treatment method for various intractable diseases (see Patent Documents 1 and 2). Recently, it has been discovered that cells with equivalent functions exist in interstitial cells of adipose tissue, placenta, umbilical cord, fetal membrane, etc. Therefore, mesenchymal stem cells are sometimes referred to as mesenchymal stromal cells.

[0005] Japanese Patent Publication No. 2012-157263 Publication of Japanese Special Publication No. 2012-508733

[0006] Padmanee S. et al. , Cell, 2017, 168(4), pp. 707-723 Pittenger F. M. et al. , Science, 1999, 284, pp. 143-147

[0007] In view of the above circumstances, an object of the present invention is to provide an excellent cancer therapeutic agent.

[0008] As a result of intensive research to solve the above problems, the present inventors discovered that a composition containing an immune checkpoint inhibitor and mesenchymal stem cells has a high cancer therapeutic effect, and completed the present invention. According to the present invention, a novel therapeutic agent having a high cancer therapeutic effect can be provided. That is, the gist of the present invention is as follows.

[0009] [1] A cancer therapeutic agent comprising an immune checkpoint inhibitor and mesenchymal stem cells. [2] The cancer therapeutic agent according to [1], wherein the cancer therapeutic agent is used in the treatment of cancer resistant to the immune checkpoint inhibitor. [3] The cancer therapeutic agent according to [1] or [2], wherein the mesenchymal stem cells are allogeneic to a subject to which the cancer therapeutic agent is administered. [4] The cancer therapeutic agent according to any of [1] to [3], wherein the mesenchymal stem cells are adipose tissue-derived mesenchymal stem cells, umbilical cord-derived mesenchymal stem cells, or bone marrow-derived mesenchymal stem cells. [5] A cancer treatment kit comprising an immune checkpoint inhibitor and mesenchymal stem cells, the cancer treatment kit comprising the cancer therapeutic agent according to any of [1] to [4] and a container containing the cancer therapeutic agent.

[0010] The cancer therapeutic agent of the present invention can provide a high therapeutic effect even against tumors for which the therapeutic effect has been low until now.

[0011] FIG. 1 is a graph showing the tumor volume of mice administered mesenchymal stem cells (MSCs) and PD-L1 antibody (ICI). FIG. 2 is a graph showing the survival rate of mice administered mesenchymal stem cells (MSCs) and PD-L1 antibody (ICI). FIG. 3 is a graph showing the tumor disappearance rate of mice administered mesenchymal stem cells (MSCs) and PD-L1 antibody (ICI). FIG. 4 is a graph showing the tumor volume of mice administered mesenchymal stem cells (MSCs) and PD-L1 antibody (ICI). FIG. 5 is a graph showing the tumor volume of mice administered mesenchymal stem cells (MSCs) and PD-L1 antibody (ICI). FIG. 6 is a graph showing the tumor volume of mice administered only PD-L1 antibody (ICI).

[0012] The cancer therapeutic agent of the present invention will be described in detail below. The matters described below in this specification can be incorporated into each of the above-mentioned gist either alone or in combination. <1. Cancer therapeutic agent> The cancer therapeutic agent of the present invention comprises an immune checkpoint inhibitor and mesenchymal stem cells.

[0013] (Immune checkpoint inhibitors) Immune checkpoint inhibitors are cancer therapeutic drugs that target "immune checkpoints" that suppress the function of immune cells. Immune checkpoint inhibitors are not particularly limited as long as they inhibit immune checkpoints, and examples include drugs that inhibit the binding of PD-1 to its ligands (PD-L1, PD-L2), drugs that inhibit the binding of PD-L1 to its receptor PD-1, and drugs that inhibit the binding of CTLA-4 to its ligands, B7.1 (CD80) and B7.2 (CD86) molecules on antigen-presenting cells.

[0014] Examples of immune checkpoint inhibitors include PD-1 antibodies, PD-L1 antibodies, and cytotoxic T-lymphocyte antigen-4 (CTLA-4) antibodies. More specifically, examples of immune checkpoint inhibitors include nivolumab (Opdivo (registered trademark), OPDIVO (registered trademark), Ono Pharmaceutical Co., Ltd.), pembrolizumab (Keytruda (registered trademark), KEYTRUDA (registered trademark), MSD), spartalizumab (Novartis Pharma), cemiplimab (Sanofi), avelumab (BAVENCIO (registered trademark), BAVENCIO (registered trademark)), and others. Examples include atezolizumab (Tecentriq (registered trademark), Merck Biopharmaceuticals), atezolizumab (Tecentriq (registered trademark), TECENTRIQ (registered trademark), Chugai Pharmaceutical), durvalumab (Imfinzi (registered trademark), IMFINZI (registered trademark), AstraZeneca), ipilimumab (Yervoy (registered trademark), YERVOY (registered trademark), Bristol-Myers Squibb), and tremelimumab (AstraZeneca).

[0015] (Mesenchymal stem cells) In the present invention, mesenchymal stem cells refer to cells that have the ability to differentiate into cells belonging to the mesenchymal system (e.g., bone cells, cardiomyocytes, chondrocytes, tendon cells, adipocytes, etc.) and can proliferate while maintaining this ability. The term mesenchymal stem cells used in the present invention refers to the same cells as stromal cells, and does not particularly distinguish between the two.

[0016] Examples of tissues containing mesenchymal stem cells include adipose tissue, umbilical cord, bone marrow, umbilical cord blood, placenta, and dental pulp. Therefore, for example, adipose tissue-derived mesenchymal stem cells refer to mesenchymal stem cells contained in adipose tissue and may also be referred to as adipose tissue-derived stromal cells. Among mesenchymal stem cells derived from these various tissues, from the viewpoints of the efficacy of the cancer therapeutic agent of the present invention and the ease of obtaining mesenchymal stem cells, adipose tissue-derived mesenchymal stem cells, umbilical cord-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, umbilical cord blood-derived mesenchymal stem cells, placenta-derived mesenchymal stem cells, and dental pulp-derived mesenchymal stem cells are preferred, with adipose tissue-derived mesenchymal stem cells, umbilical cord-derived mesenchymal stem cells, and bone marrow-derived mesenchymal stem cells being more preferred, and adipose tissue-derived mesenchymal stem cells and umbilical cord-derived mesenchymal stem cells being even more preferred. From the viewpoint of the efficacy of the cancer therapeutic agent, umbilical cord-derived mesenchymal stem cells are particularly preferred. From the viewpoint of the ease of obtaining mesenchymal stem cells, adipose tissue-derived mesenchymal stem cells are particularly preferred.

[0017] The mesenchymal stem cells of the present invention are preferably allogeneic or allogeneic to the recipient of the cancer therapeutic agent. Even when administered to an allogeneic recipient, mesenchymal stem cells are less likely to cause rejection. Therefore, donor mesenchymal stem cells can be expanded and cryopreserved in advance and used as the mesenchymal stem cells in the cancer therapeutic agent of the present invention. Thus, using donor mesenchymal stem cells in a cancer therapeutic agent has the advantage of being easier to commercialize and more likely to produce consistent, consistent effects compared to preparing autologous mesenchymal stem cells and using them in a cancer therapeutic agent. Therefore, it is more preferable that the mesenchymal stem cells of the present invention be allogeneic to the recipient of the cancer therapeutic agent.

[0018] The subject of administration of the cancer therapeutic agent is not particularly limited as long as it is an animal suffering from cancer, but is preferably a mammal, more preferably a primate, and even more preferably a human. Therefore, the mesenchymal stem cells are preferably human mesenchymal stem cells, more preferably human adipose tissue-derived mesenchymal stem cells, human umbilical cord-derived mesenchymal stem cells, or human bone marrow-derived mesenchymal stem cells.

[0019] In the present invention, mesenchymal stem cells may be a cell population consisting solely of mesenchymal stem cells, or any cell population containing mesenchymal stem cells. In the latter cell population, the proportion of mesenchymal stem cells is at least 20% or more, preferably 30% or more, more preferably 40% or more, even more preferably 50% or more, even more preferably 60% or more, even more preferably 70% or more, even more preferably 75% or more, even more preferably 80% or more, even more preferably 85% or more, even more preferably 90% or more, even more preferably 93% or more, even more preferably 96% or more, even more preferably 97% or more, even more preferably 98% or more, even more preferably 99% or more.

[0020] In the present invention, adipose tissue refers to tissue containing adipocytes and stromal cells (including microvascular cells, etc.), and is, for example, tissue obtained by surgical resection or aspiration of subcutaneous fat from a mammal. Adipose tissue can be obtained from subcutaneous fat. It is preferable that adipose tissue be obtained from subcutaneous fat of an animal of the same species as the target of administration of the cancer therapeutic agent. Considering the administration of the cancer therapeutic agent to humans, adipose tissue is more preferably obtained from human subcutaneous fat. The individual providing the subcutaneous fat may be living or dead, but the adipose tissue used in the present invention is preferably tissue collected from a living individual. When collecting adipose tissue from an individual, it can be performed by liposuction. Examples of liposuction include PAL (power-assisted) liposuction, Erchoria laser liposuction, and body jet liposuction. When collecting adipose tissue from an individual, it is preferable not to use ultrasound in order to maintain the state of the cells.

[0021] In the present invention, the umbilical cord is a white tubular tissue that connects the fetus and the placenta, and is composed of the umbilical vein, umbilical artery, gelatinous tissue (Wharton's jelly), the umbilical cord matrix itself, etc., and is rich in mesenchymal stem cells. The umbilical cord is preferably obtained from an animal of the same species as the subject to which the cancer therapeutic agent is to be administered. Considering that the cancer therapeutic agent will be administered to a human, the umbilical cord is more preferably obtained from a human.

[0022] In the present invention, bone marrow refers to the soft tissue that fills the cavity of bone and is a hematopoietic organ. Bone marrow contains bone marrow fluid, and the cells present therein are called bone marrow cells. Bone marrow cells include erythrocytes, granulocytes, megakaryocytes, lymphocytes, adipocytes, mesenchymal stem cells, hematopoietic stem cells, endothelial progenitor cells, etc. Bone marrow cells can be collected, for example, from the ilium, long bones, or other bones. Bone marrow is preferably obtained from an animal of the same species as the subject to which the cancer therapeutic agent is to be administered. Considering the administration of the cancer therapeutic agent to humans, bone marrow is more preferably obtained from humans.

[0023] As described above, mesenchymal stem cells can be derived from various tissues, such as adipose tissue, umbilical cord, bone marrow, etc. Mesenchymal stem cells may be characterized, for example, by growth characteristics (e.g., population doubling capacity from passage to senescence, doubling time), karyotype analysis (e.g., normal karyotype, maternal lineage or neonatal lineage), surface marker expression by flow cytometry (e.g., FACS analysis), immunohistochemistry and / or immunocytochemistry (e.g., epitope detection), gene expression profiling (e.g., gene chip arrays; polymerase chain reaction such as reverse transcription PCR, real-time PCR, conventional PCR), miRNA expression profiling, protein arrays, protein secretion such as cytokines (e.g., plasma clotting analysis, ELISA, cytokine arrays), metabolites (metabolomic analysis), or other methods known in the art.

[0024] (Method for Preparing Mesenchymal Stem Cells) Mesenchymal stem cells can be prepared by methods well known to those skilled in the art. As an example, a method for preparing adipose tissue-derived mesenchymal stem cells will be described below. Adipose tissue-derived mesenchymal stem cells can be obtained, for example, by the production method described in U.S. Patent No. 6,777,231, and can be produced, for example, by a method comprising the following steps (i) to (iii): (i) obtaining a cell suspension by enzymatic digestion of adipose tissue; (ii) sedimenting the cells and resuspending them in an appropriate medium; and (iii) culturing the cells on a solid surface and removing cells that do not bind to the solid surface.

[0025] To confirm that the selected cells are adipose tissue-derived mesenchymal stem cells of the present invention, surface antigens may be analyzed by conventional methods such as flow cytometry. Furthermore, the ability to differentiate into each cell lineage may be examined, and such differentiation can be carried out by conventional methods.

[0026] Mesenchymal stem cells can be prepared as described above, but may also be defined as cells that have all of the following properties (1) to (3): (1) exhibiting adhesion to plastic when cultured in a standard medium; (2) being positive for positive markers specific to mesenchymal stem cells (e.g., cell surface markers CD44 and CD29) and negative for negative markers specific to mesenchymal stem cells (e.g., cell surface markers CD19, CD11b, and CD45); and (3) being capable of differentiating into osteocytes, adipocytes, and chondrocytes under differentiation culture conditions.

[0027] Mesenchymal stem cells may be in any state, for example, cells recovered by detaching cells from a culture vessel during adherent culture, or cells frozen in a cryopreservation solution. Mesenchymal stem cells are expanded and then aliquoted from the same lot and cryopreserved. The use of cryopreserved cells is preferred in terms of stable effects and ease of handling. Cryopreserved mesenchymal stem cells may be thawed immediately before use and administered directly while suspended in a cryopreservation solution, or they may be administered after being suspended in an infusion solution or medium. Alternatively, the cryopreserved mesenchymal stem cells may be mixed with an infusion solution or medium after removing the cryopreservation solution by centrifugation or other methods.

[0028] (Dosage and Administration) From the viewpoint of efficacy, the administration of the cancer therapeutic agent of the present invention is preferably intravenous, intraarterial, intramuscular, subcutaneous, intralymph node, intradermal, intracranial, intrathoracic, intratumoral, or intraperitoneal administration. From the viewpoint of safety, the administration of the cancer therapeutic agent is preferably carried out by intravenous, intramuscular, or subcutaneous administration.

[0029] In the cancer therapeutic agent of the present invention, the dose of mesenchymal stem cells may vary depending on the patient's condition (body weight, age, symptoms, physical condition, etc.), the administration route of the cancer therapeutic agent, the dosage form of the cancer therapeutic agent, etc. When the cancer therapeutic agent of the present invention is administered to an adult, the dose of mesenchymal stem cells (number of cells) is, for example, 1 x 10 ３ ~1 x 10 １２ Cells / time, preferably 1 x 10 ４ ~1 x 10 １１ cells / time, more preferably 1 x 10 ５ ~1 x 10 １０ cells / time, more preferably 5 x 10 ６ ~1 x 10 ９ cells / time, most preferably 8 x 10 ７ ~2 x 10 ８ The mesenchymal stem cells may be administered once at the above dose, or multiple times at the above dose.

[0030] The dose (number of cells) of mesenchymal stem cells per patient's body weight is, for example, 1×10 to 5×10 １０ cells / kg, preferably 1 x 10 ２ ~5 x 10 ９ cells / kg, more preferably 1 x 10 ３ ~5 x 10 ８ cells / kg, more preferably 1 x 10 ４ ~5 x 10 ７ The mesenchymal stem cells may be administered multiple times at the above-mentioned dosage (dosage per body weight) as a single dose, or the above-mentioned dosage (dosage per body weight) may be administered in multiple divided doses.

[0031] In administering the cancer therapeutic agent of the present invention, the immune checkpoint inhibitor and mesenchymal stem cells may be administered simultaneously, or separately, or the mesenchymal stem cells may be administered after the administration of the immune checkpoint inhibitor, or the immune checkpoint inhibitor may be administered after the administration of the mesenchymal stem cells.

[0032] Furthermore, the cancer therapeutic agent of the present invention may be used after the use of a cancer therapeutic agent containing an immune checkpoint inhibitor, or may be used before the use of a cancer therapeutic agent containing an immune checkpoint inhibitor.

[0033] When the immune checkpoint inhibitor and the mesenchymal stem cells are administered separately, examples of the method of administering the mesenchymal stem cells include intravenous administration, intra-arterial administration, intramuscular administration, subcutaneous administration, intralymph node administration, intradermal administration, intracranial administration, intrathoracic administration, intratumoral administration, and intraperitoneal administration, and preferably intravenous administration, intramuscular administration, or subcutaneous administration.

[0034] When the immune checkpoint inhibitor and the mesenchymal stem cells are administered separately, the immune checkpoint inhibitor can be administered to a subject according to the usage and dosage described in the package insert. Examples of administration methods include intravenous administration, intra-arterial administration, intramuscular administration, subcutaneous administration, intralymph node administration, intradermal administration, intracranial administration, intrathoracic administration, intratumoral administration, and intraperitoneal administration, and intravenous administration and subcutaneous administration are preferred.

[0035] The dosage of the immune checkpoint inhibitor may vary depending on the type of drug, the route of administration, the patient's condition (body weight, age, symptoms, physical condition, etc.), etc., but when administered to an adult, it is, for example, 10 to 5000 mg / dose, preferably 100 to 3000 mg / dose, and for example, 0.01 to 1000 mg / kg, preferably 0.1 to 100 mg / kg. The administration interval of the immune checkpoint inhibitor is, for example, every 2 weeks, every 3 weeks, every 4 weeks, or every 6 weeks.

[0036] When the cancer therapeutic agent of the present invention is administered together with one or more other drugs, the cancer therapeutic agent of the present invention and the other drugs may be administered simultaneously, or the other drugs may be administered after a certain time has elapsed since the administration of the cancer therapeutic agent of the present invention, or the other drugs may be administered after a certain time has elapsed since the administration of the other drugs, or these administrations may be performed in combination.

[0037] (Applicable Diseases) Target tumors for the cancer therapeutic agent of the present invention include, for example, malignant melanoma, non-small cell lung cancer, renal cell carcinoma, Hodgkin's lymphoma, head and neck cancer, gastric cancer, urothelial cancer, and Merkel cell carcinoma.

[0038] The cancer therapeutic agent of the present invention can also be used in the treatment of cancers resistant to immune checkpoint inhibitors. The cancer therapeutic agent of the present invention may be used for subjects who have not previously received treatment with the single-agent administration of an immune checkpoint inhibitor, or may be used for subjects who have previously received treatment with the single-agent administration of an immune checkpoint inhibitor.

[0039] (Method for preparing cancer therapeutic agents) The cancer therapeutic agents of the present invention can be prepared according to the respective dosage forms by mixing mesenchymal stem cells prepared by the above-mentioned preparation method with an appropriate cell suspension liquid (including pharmaceutically acceptable carriers and additives) according to standard methods. Pharmaceutically acceptable carriers include, for example, cryopreservation solutions, infusion solutions, and culture media. Additives include, for example, isotonicity agents, stabilizers, preservatives, buffers, solubilizers, and soothing agents.

[0040] <2. Other Aspects> (Cancer Therapeutic Agent) According to another aspect, there is provided a cancer therapeutic agent for administration in combination with an immune checkpoint inhibitor, the cancer therapeutic agent comprising mesenchymal stem cells. For specific details of this invention, please refer to the description in the section <1. Cancer Therapeutic Agent>.

[0041] (Cancer Treatment Kit) According to another aspect, a cancer treatment kit is provided. The cancer treatment kit according to a first embodiment includes a cancer treatment agent containing an immune checkpoint inhibitor and mesenchymal stem cells, and a container containing the cancer treatment agent. The cancer treatment kit according to a second embodiment is used for administration in combination with an immune checkpoint inhibitor, and includes a cancer treatment agent containing mesenchymal stem cells, and a container containing the cancer treatment agent.

[0042] For the cancer therapeutic agent included in the kit, please refer to the description in the section <1. Cancer therapeutic agent>. The container included in the kit is not particularly limited, and examples thereof include cryotubes for freezing mesenchymal stem cells, containers for suspending mesenchymal stem cells, vials, test tubes, etc. These containers may be formed from a variety of materials such as glass, metal, plastic, or a combination thereof. The outer surface of the container usually has a description of the contents written on it. For example, a label describing the contents may be affixed to the container.

[0043] The present kits may further include other materials desirable from a commercial and user standpoint, including other additives, other agents, diluents, filters, needles, syringes, and package inserts with instructions for use.

[0044] (Cancer Treatment Method) According to yet another aspect, there is provided a cancer treatment method comprising administering to a subject a cancer treatment agent comprising an immune checkpoint inhibitor and mesenchymal stem cells. According to yet another aspect, there is provided a cancer treatment method comprising administering a cancer treatment agent comprising mesenchymal stem cells in combination with an immune checkpoint inhibitor. These inventions are inventions that capture the cancer treatment agent invention from different aspects, and therefore, for specific content, please refer to the description in the section <1. Cancer Treatment Agent>.

[0045] (Mesenchymal stem cells for use in cancer treatment) According to yet another aspect, there is provided a composition comprising an immune checkpoint inhibitor and mesenchymal stem cells for use in cancer treatment. According to yet another aspect, there is provided mesenchymal stem cells for use in cancer treatment in combination with an immune checkpoint inhibitor. These inventions are inventions that capture the invention of a cancer therapeutic agent from a different perspective, and therefore, for specific content, please refer to the description in the section <1. Cancer therapeutic agent>.

[0046] (Use of mesenchymal stem cells in the manufacture of a cancer therapeutic agent) According to yet another aspect, there is provided the use of a composition comprising an immune checkpoint inhibitor and mesenchymal stem cells in the manufacture of a cancer therapeutic agent. According to yet another aspect, there is provided the use of mesenchymal stem cells in the manufacture of a cancer therapeutic agent for administration in combination with an immune checkpoint inhibitor. These inventions are inventions that capture the invention of a cancer therapeutic agent from different aspects, and therefore, for specific content, please refer to the description in the section <1. Cancer therapeutic agent>.

[0047] The items described in this specification can be incorporated into each of the above aspects either alone or in combination.

[0048] 3. Preferred Embodiments Preferred embodiments are summarized below. [A1] A cancer therapeutic agent comprising an immune checkpoint inhibitor and mesenchymal stem cells. [A2] The cancer therapeutic agent according to [A1], wherein the cancer therapeutic agent is used to treat cancer resistant to the immune checkpoint inhibitor. [A3] The cancer therapeutic agent according to [A1] or [A2], wherein the mesenchymal stem cells are allogeneic to the subject to which the cancer therapeutic agent is administered. [A4] The cancer therapeutic agent according to any one of [A1] to [A3], wherein the mesenchymal stem cells are adipose tissue-derived mesenchymal stem cells, umbilical cord-derived mesenchymal stem cells, or bone marrow-derived mesenchymal stem cells. [A5] The cancer therapeutic agent according to any one of [A1] to [A4], wherein the mesenchymal stem cells are adipose tissue-derived mesenchymal stem cells or umbilical cord-derived mesenchymal stem cells.

[0049] [A6] The cancer therapeutic agent according to any one of [A1] to [A5], wherein the mesenchymal stem cells are umbilical cord-derived mesenchymal stem cells. [A7] The cancer therapeutic agent according to any one of [A1] to [A5], wherein the mesenchymal stem cells are adipose tissue-derived mesenchymal stem cells. [A8] The cancer therapeutic agent according to any one of [A1] to [A7], wherein the mesenchymal stem cells are non-genetically modified mesenchymal stem cells. [A9] The cancer therapeutic agent according to any one of [A1] to [A8], wherein the immune checkpoint inhibitor is a PD-1 antibody, a PD-L1 antibody, or a cytotoxic T-lymphocyte antigen-4 (CTLA-4) antibody. [A10] The cancer therapeutic agent according to any one of [A1] to [A9], wherein the immune checkpoint inhibitor is a PD-L1 antibody.

[0050] [A11] The mesenchymal stem cells are 1×10 ３ ~1 x 10 １２ Cells / time, preferably 1 x 10 ４ ~1 x 10 １１ cells / time, more preferably 1 x 10 ５ ~1 x 10 １０ cells / time, more preferably 5 x 10 ６ ~1 x 10 ９ cells / time, more preferably 8 x 10 ７ ~2 x 10 ８ The cancer therapeutic agent according to any one of [A1] to [A10], wherein the agent is administered at a dose of 1 x 10 to 5 x 10 cells / dose. [A12] The mesenchymal stem cells are administered at a dose of 1 x 10 to 5 x 10 cells / dose. １０ cells / kg, preferably 1 x 10 ２ ~5 x 10 ９ cells / kg, more preferably 1 x 10 ３ ~5 x 10 ８ cells / kg, more preferably 1 x 10 ４ ~5 x 10 ７ The cancer therapeutic agent according to any one of [A1] to [A11], wherein the immune checkpoint inhibitor is administered at a dose of 10 to 5000 mg / dose, preferably 100 to 3000 mg / dose. [A13] The cancer therapeutic agent according to any one of [A1] to [A12], wherein the immune checkpoint inhibitor is administered at a dose of 10 to 5000 mg / dose, preferably 100 to 3000 mg / dose. [A14] The cancer therapeutic agent according to any one of [A1] to [A13], wherein the immune checkpoint inhibitor is administered at a dose of 0.01 to 1000 mg / kg, preferably 0.1 to 100 mg / kg. [A15] The cancer therapeutic agent according to any one of [A1] to [A14], wherein the cancer therapeutic agent is used in the treatment of a cancer whose tumor volume does not decrease by single-agent administration of the immune checkpoint inhibitor.

[0051] [B1] A cancer treatment kit comprising the cancer therapeutic agent according to any one of [A1] to [A15] and a container containing the cancer therapeutic agent.

[0052] [C1] A cancer therapeutic agent for administration in combination with an immune checkpoint inhibitor, the cancer therapeutic agent comprising mesenchymal stem cells. [C2] The cancer therapeutic agent according to [C1], wherein the cancer therapeutic agent is used in the treatment of cancer resistant to the immune checkpoint inhibitor. [C3] The cancer therapeutic agent according to [C1] or [C2], wherein the mesenchymal stem cells are allogeneic to the subject to which the cancer therapeutic agent is administered. [C4] The cancer therapeutic agent according to any one of [C1] to [C3], wherein the mesenchymal stem cells are adipose tissue-derived mesenchymal stem cells, umbilical cord-derived mesenchymal stem cells, or bone marrow-derived mesenchymal stem cells. [C5] The cancer therapeutic agent according to any one of [C1] to [C4], wherein the mesenchymal stem cells are adipose tissue-derived mesenchymal stem cells or umbilical cord-derived mesenchymal stem cells.

[0053] [C6] The cancer therapeutic agent according to any one of [C1] to [C5], wherein the mesenchymal stem cells are umbilical cord-derived mesenchymal stem cells. [C7] The cancer therapeutic agent according to any one of [C1] to [C5], wherein the mesenchymal stem cells are adipose tissue-derived mesenchymal stem cells. [C8] The cancer therapeutic agent according to any one of [C1] to [C7], wherein the mesenchymal stem cells are non-genetically modified mesenchymal stem cells. [C9] The cancer therapeutic agent according to any one of [C1] to [C8], wherein the immune checkpoint inhibitor is a PD-1 antibody, a PD-L1 antibody, or a cytotoxic T-lymphocyte antigen-4 (CTLA-4) antibody. [C10] The cancer therapeutic agent according to any one of [C1] to [C9], wherein the immune checkpoint inhibitor is a PD-L1 antibody.

[0054] [C11] The mesenchymal stem cells are 1×10 ３ ~1 x 10 １２ Cells / time, preferably 1 x 10 ４ ~1 x 10 １１ cells / time, more preferably 1 x 10 ５ ~1 x 10 １０ cells / time, more preferably 5 x 10 ６ ~1 x 10 ９ cells / time, more preferably 8 x 10 ７ ~2 x 10 ８The cancer therapeutic agent according to any one of [C1] to [C10], wherein the agent is administered at a dose of 1 x 10 to 5 x 10 cells / dose. １０ cells / kg, preferably 1 x 10 ２ ~5 x 10 ９ cells / kg, more preferably 1 x 10 ３ ~5 x 10 ８ cells / kg, more preferably 1 x 10 ４ ~5 x 10 ７ The cancer therapeutic agent according to any one of [C1] to [C11], wherein the immune checkpoint inhibitor is administered at a dose of 10 to 5000 mg / dose, preferably 100 to 3000 mg / dose. [C13] The cancer therapeutic agent according to any one of [C1] to [C12], wherein the immune checkpoint inhibitor is administered at a dose of 10 to 5000 mg / dose, preferably 100 to 3000 mg / dose. [C14] The cancer therapeutic agent according to any one of [C1] to [C13], wherein the immune checkpoint inhibitor is administered at a dose of 0.01 to 1000 mg / kg, preferably 0.1 to 100 mg / kg. [C15] The cancer therapeutic agent according to any one of [C1] to [C14], wherein the cancer therapeutic agent is used in the treatment of a cancer whose tumor volume does not decrease by single-agent administration of the immune checkpoint inhibitor.

[0055] [D1] A cancer treatment kit used for administration in combination with an immune checkpoint inhibitor, comprising the cancer therapeutic agent according to any one of [C1] to [C15] and a container containing the cancer therapeutic agent. [D2] A cancer treatment kit comprising: a first medicament comprising the cancer therapeutic agent according to any one of [C1] to [C15] and a first container containing the cancer therapeutic agent; and a second medicament comprising an immune checkpoint inhibitor and a second container containing the immune checkpoint inhibitor.

[0056] [E1] A method for treating cancer, comprising administering to a subject the cancer therapeutic agent according to any one of [A1] to [A15]. [E2] A method for treating cancer, comprising administering to a subject the cancer therapeutic agent according to any one of [C1] to [C15] in combination with an immune checkpoint inhibitor.

[0057] [F1] Mesenchymal stem cells for use in cancer treatment in combination with an immune checkpoint inhibitor. [F2] The mesenchymal stem cells according to [F1], which are mesenchymal stem cells contained in the cancer therapeutic agent according to any one of [C1] to [C15].

[0058] [G1] Use of mesenchymal stem cells in the manufacture of a cancer therapeutic agent for administration in combination with an immune checkpoint inhibitor. [G2] The use according to [G1], wherein the mesenchymal stem cells are mesenchymal stem cells contained in the cancer therapeutic agent according to any one of [C1] to [C15].

[0059] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.

[0060] <1> Example 1: Preparation of Mesenchymal Stem Cells (MSCs) Adipose tissue was isolated from the subcutaneous fat of the inguinal region of male C57BL / 6J mice (7 weeks old) and washed with physiological saline. After washing, collagenase (Roche) was added to the adipose tissue, and the cells were dispersed by shaking at 37°C for 90 minutes. The cells were then collected, and the cell suspension was centrifuged at 800g for 5 minutes to obtain a precipitate of stromal vascular cells. Serum-free medium for mesenchymal stem cells (Rohto) was added to the obtained cell precipitate, and the cell suspension was centrifuged at 400g for 5 minutes. After removing the supernatant, the cell precipitate was resuspended in serum-free medium for mesenchymal stem cells (Rohto), and the cells were seeded into cell culture flasks. The cells were incubated at 37°C for several days in 5% CO ２ The cells were cultured in a PBS flask. After incubation, the cultures were washed with phosphate-buffered saline (PBS) to remove blood cells and residual adipose tissue. Cells adhering to the cell culture flask were used to identify adipose tissue-derived mesenchymal stem cells (MSCs). MSCs were confirmed to differentiate into adipocytes, chondrocytes, and osteocytes, to express CD44, CD29, and Sca1, and to lack expression of CD19, CD11b, and CD45.

[0061] <2> Example 2: Examination of the additive effect of combined administration of MSCs and immunotherapy against LLC. Mice (C57BL / 6J mice, male, 6 weeks old) were subcutaneously injected with 5 × 10 ５On days 7, 11, and 15 after administration of the low immunogenic mouse lung cancer cell line LLC (LLC), 200 μg / mouse anti-mouse PD-L1 antibody (ICI) was administered subcutaneously (ICI administration group) at 100 μg / mouse. In the MSC+ICI administration group, in addition to administration of 200 μg / mouse ICI on days 7, 11, and 15 after tumor administration, 1 × 10 6 MSCs obtained in Example 1 were administered subcutaneously to the side opposite to the LLC inoculation site 3 days before tumor administration. ６ MSCs were administered at a dose of 1000 cells / mouse. A control group received LLC alone. Tumor volumes were measured using digital calipers from the day of tumor administration until day 21 (Figure 1). Survival rates were also monitored from the day of tumor administration until day 61 (Figure 2).

[0062] On day 21 after tumor administration, tumor volume was reduced in the MSC+ICI and ICI groups compared with the control group, but no difference in tumor volume was observed between the MSC+ICI and ICI groups. On day 61 after tumor administration, tumors completely disappeared in one of nine mice in the MSC+ICI group, whereas tumors enlarged and all nine mice died in the ICI group.

[0063] The above results demonstrate that administration of ICI and MSCs can provide a high therapeutic effect on cancer.

[0064] <3> Example 3: Examination of the additional effect of combined administration of MSCs and immunotherapy against MC38. Instead of LLC in Example 2, 5 × 10 ５ The highly immunogenic mouse colon cancer cell line MC38 (MC38) was subcutaneously administered to mice (C57BL / 6J mice, male, 6 weeks old) at a dose of 1 × 10 cells / mouse. ICI and MSCs were administered in the same manner as in Example 2, and the tumor disappearance rate was calculated (Figure 3). The MSC+ICI administration group was administered with 1 × 10 cells obtained in Example 1. ４ MSC cells / animal were mixed with MC38 and administered subcutaneously.

[0065] On the 20th day after tumor administration, 3 out of 9 mice in the MSC + ICI group had completely disappeared and survived. In the ICI group, 1 out of 9 mice had completely disappeared and survived. In the control group, all 9 mice died due to tumor growth.

[0066] The above results demonstrate that administration of ICI and MSCs has a high therapeutic effect on cancer.

[0067] <4> Example 4: Examination of changes in ICI reactivity due to MSC administration in cases where the efficacy assessment of immune checkpoint inhibitors showed partial response (PR) or progressive disease (PD) Mice (C57BL / 6J mice, male, 6 weeks old) were subcutaneously injected with 5 × 10 ５ The highly immunogenic murine colon cancer cell line MC38 (MC38) was administered at 1x10 cells / mouse. 7, 10, 17, 20, and 23 days after tumor administration, 200 μg / mouse anti-mouse PD-L1 antibody (ICI) was administered. 14 days after tumor administration, the therapeutic effect of ICI was assessed and mice were classified into a progressive disease group (PD group) of 4 mice, a partial response group (PR group) of 4 mice, and a complete response group (CR group) of 1 mouse (Figure 4). For the MSC administration group, 14 days after tumor administration, each mouse was administered 1x10 ６ MSCs were administered at a dose of 1000 cells / mouse, and tumor volume was measured using digital calipers (Figure 5). The control group received no MSCs, but received MC38 in the same manner as the MSC-administered group. 200 μg / mouse of ICI was administered 7, 10, and 14 days after tumor administration, and tumor volume was measured (Figure 6). In Figures 5 and 6, the tumor volumes for each group except the CR group are the average tumor volumes.

[0068] In the CR group, no tumor expansion was observed in either the MSC-administered group or the control group. In the PR group, the control group showed an expansion of tumor volume after day 14, but the MSC-administered group showed no tumor expansion after day 17. In the PD group, the control group showed a large increase in tumor volume after day 14, but the MSC-administered group suppressed the rapid increase in tumor volume.

[0069] The above results indicated that administration of MSCs and ICI to the PR and PD groups resulted in a high cancer treatment effect.

Claims

1. A cancer treatment containing immune checkpoint inhibitors and mesenchymal stem cells.

2. The cancer therapeutic agent according to claim 1, which is used in the treatment of cancer resistant to the immune checkpoint inhibitor.

3. The cancer therapeutic agent according to claim 1, wherein the mesenchymal stem cells are allogeneic to the subject to which the cancer therapeutic agent is administered.

4. The cancer therapeutic agent according to claim 1, wherein the mesenchymal stem cells are adipose tissue-derived mesenchymal stem cells, umbilical cord-derived mesenchymal stem cells, or bone marrow-derived mesenchymal stem cells.

5. A cancer treatment kit comprising an immune checkpoint inhibitor and mesenchymal stem cells, the cancer treatment kit comprising the cancer treatment agent according to any one of claims 1 to 4 and a container containing the cancer treatment agent.

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