Use of combination therapy for enhancing NK cells in treatment of malignant tumors

Abstract

The present invention belongs to the field of biotechnology and provides use of a pharmaceutical combination in the preparation of a kit for treating tumors (including low immune infiltration tumors (also referred to as "cold tumors")). Specifically, the present invention relates to treatment of tumors (including low immune infiltration tumors) with immune cells in combination with a chemotherapeutic drug. More specifically, the present invention relates to synergistic treatment of tumors (including low immune infiltration tumors) with NK cells in combination with TMZ.

Description

Application of combined enhancement of NK cells in the treatment of malignant tumors Technical Field

[0001] This invention belongs to the field of biotechnology and relates to the treatment of tumors with immune cells combined with chemotherapy drugs, specifically to the synergistic effect of NK cells combined with TMZ in the treatment of tumors. Background Technology

[0002] Natural killer (NK) cells are part of the innate immune system and play a crucial role in immune surveillance, host defense against viral infections, and the fight against tumor cells. Recent studies have also indicated that NK cells are potential therapeutic targets for cancer treatment. In the human body, NK cells comprise 8-20% of lymphocytes. Unlike other lymphocytes (including B cells, T cells, and natural killer T (NKT) cells), NK cells do not express antigen-specific receptors such as clonal B cell receptors or the T cell receptor / CD3ε complex. Instead, NK cells function in an antigen-independent manner (typically without generating immune memory or long-term protective immunity). NK cells regulate their own cytotoxicity through activating and inhibitory receptors on their surface. Unlike T cells, NK cells retain their natural ability to recognize and target tumor cells. At the same time, compared with T cells, NK cells do not require strict HLA (human leukocyte antigen) matching, so the risk of GVHD (graft-versus-host disease) is lower. Furthermore, in current clinical trials, they have not shown safety issues similar to cytokine release syndrome associated with T cell therapy, thus opening up the possibility of producing "off-the-shelf" allogeneic cell therapy products.

[0003] Immunotherapy, a novel approach that utilizes adoptive immune cells or the body's own immune system to fight tumors, is transforming the treatment of various cancers. At its core, immunotherapy involves triggering the patient's immune system to fight tumor cells by correcting effector cell dysfunction and reducing suppressive immune cell populations. Research indicates that the tumor's immune environment can predict responsiveness to immunotherapy; favorable tumor environments ("hot" tumors) typically respond well to treatment, while "cold" tumors with minimal immune infiltration often exhibit unsatisfactory responses. For example, cold tumors may lack innate anti-tumor immunity, or existing innate immune responses may be ineffective due to immune cell rejection; compared to hot tumors, cold tumors show limited responsiveness to immune checkpoint inhibitors, etc.

[0004] To improve the efficacy of immunotherapy, there is an increasing emphasis on integrating multiple cancer treatment modalities to overcome tumor drug resistance and enhance the responsiveness of tumors that are not very sensitive to conventional immunotherapy. The key to this approach lies in regulating the expression of chemokines during tumor treatment, which is crucial for both treatment efficacy and patient tolerability (see, for example, Wu B, et al. Cold and hot tumors: from molecular mechanisms to targeted therapy. Signal Transduct Target Ther. 2024 Oct 18; 9(1):274.).

[0005] Therefore, there is a need in the field for novel therapeutic strategies that utilize combinations of cancer treatments to enhance the regulation of tumor immune infiltration and the chemotactic capacity of immune cells by immunotherapies (such as those involving NK cells). Summary of the Invention

[0006] In a first aspect, the present invention provides the use of a pharmaceutical combination in the preparation of a kit for treating tumors, wherein the pharmaceutical combination comprises an active ingredient comprising natural killer (NK) cells and a chemotherapeutic agent, wherein the chemotherapeutic agent comprises temozolomide (TMZ).

[0007] In some implementations, the tumors include, but are not limited to, nervous system tumors, neuroendocrine tumors, hematologic tumors, ovarian cancer, prostate cancer, pancreatic cancer, osteosarcoma, thyroid cancer, soft tissue sarcoma, insulinoma, and testicular cancer.

[0008] In some embodiments, the nervous system tumors include brainstem glioma (DIPG), supratentorial and infratentorial gliomas, astrocytoma, oligodendroglioma, ependymoma, medulloblastoma, schwannoma, meningioma, pituitary adenoma, neuroblastoma, spinal cord glioma, meningioma, metastatic spinal tumors, peripheral nerve sheath tumors, neurofibroma, brain metastasis, and neuroectodermal tumors.

[0009] In some embodiments, the hematologic malignancies include leukemia (e.g., acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML)), lymphoma (e.g., Hodgkin lymphoma (HL), non-Hodgkin lymphoma (NHL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma (MCL), Burkitt lymphoma (BL)), and bone marrow tumors (e.g., multiple myeloma (MM), myelodysplastic syndrome (MDS), myeloproliferative neoplasm (MPN), polycythemia vera (PV), essential thrombocythemia (ET), myelofibrosis (MF)).

[0010] In a preferred embodiment, the tumor is a nervous system tumor.

[0011] In a second aspect, the present invention provides the use of a pharmaceutical combination in the preparation of a kit for treating low-immune-invasive tumors, wherein the pharmaceutical combination comprises an active ingredient comprising natural killer (NK) cells and a chemotherapeutic agent, wherein the chemotherapeutic agent comprises temozolomide (TMZ).

[0012] In some implementations, the low-immune-invasive tumors include, but are not limited to, low-immune-invasive nervous system tumors, neuroendocrine tumors, non-small cell lung cancer, small cell lung cancer, colorectal cancer, melanoma, breast cancer, esophageal cancer, gastric cancer, bladder cancer, endometrial cancer, head and neck cancer, cervical cancer, liver cancer, and kidney cancer.

[0013] In some embodiments, the low-immune-infiltrating nervous system tumors include low-immune-infiltrating brainstem gliomas (DIPG), supratentorial and infratentorial gliomas, astrocytomas, oligodendrogliomas, ependymomas, medulloblastomas, schwannomas, meningiomas, pituitary adenomas, neuroblastomas, spinal cord gliomas, meningiomas, metastatic spinal tumors, peripheral nerve sheath tumors, neurofibromas, brain metastasis, and neuroectodermal tumors.

[0014] In a preferred embodiment, the low-immune-invasive tumor is low-immune-invasive cervical cancer.

[0015] In a preferred embodiment, the low-immune-infiltrating tumor is a low-immune-infiltrating nervous system tumor.

[0016] In some embodiments, the NK cells in the first and second aspects are unmodified NK cells or modified NK cells. In a preferred embodiment, the NK cells in the first and second aspects are unmodified NK cells.

[0017] In some embodiments, the NK cells of the first and second aspects include, but are not limited to, NK-92 cells, NK cells derived from peripheral blood or umbilical cord blood, and NK cells derived from induced pluripotent stem cells (iPSCs) or embryonic stem cells. In a preferred embodiment, the NK cells of the first and second aspects are NK cells derived from induced pluripotent stem cells (iPSCs).

[0018] In some implementations, the NK cells in the first and second aspects are unmodified NK cells derived from induced pluripotent stem cells.

[0019] In some embodiments, the NK cells of the first and second aspects are prepared using a kit for inducing stem cell differentiation into natural killer cells, the kit comprising a second culture medium, a fourth culture medium, and a fifth culture medium; wherein the second culture medium is a basal culture medium containing a BMP signaling pathway activator; the fourth culture medium is a basal culture medium containing at least one, at least two, at least three, or at least four of the following: BMP signaling pathway activator, vascular endothelial growth factor, colony-stimulating factor, and interleukin; and the fifth culture medium is a basal culture medium containing at least one, at least two, at least three, at least four, at least five, or at least six of the following: BMP signaling pathway activator, vascular endothelial growth factor, colony-stimulating factor, interleukin, stem cell agonist, and stem cell expansion agent.

[0020] In some embodiments, the kit for inducing stem cell differentiation into natural killer cells further comprises a third culture medium, which is a basal culture medium containing at least one, at least two, or at least three of the following: BMP signaling pathway activator, growth factor, and TGFβ / ALK inhibitor.

[0021] In some embodiments, the kit for inducing stem cell differentiation into natural killer cells further comprises a first culture medium, which is a basal culture medium containing a ROCK inhibitor.

[0022] In some implementations, the NK cells of the first and second aspects are prepared by a method of inducing stem cells to differentiate into natural killer (NK) cells, said method including the step of using a kit for inducing stem cells to differentiate into natural killer cells as defined above.

[0023] In some embodiments, the method includes: step (1) culturing stem cells to obtain embryoid bodies; step (2) culturing the embryoid bodies for mesodermal differentiation to obtain mesodermal cells; step (3) culturing the mesodermal cells for hematopoietic endothelial differentiation to obtain hematopoietic endothelial cells; and step (4) culturing the hematopoietic endothelial cells in the fourth and fifth culture media of the kit for inducing stem cell differentiation into natural killer cells as defined above to obtain NK cells. In a preferred embodiment, the culture in step (1) is performed in the first culture media of the kit for inducing stem cell differentiation into natural killer cells as defined above. In a preferred embodiment, the culture in step (2) is performed in the second culture media of the kit for inducing stem cell differentiation into natural killer cells as defined above. In a preferred embodiment, the culture in step (3) is performed in the third culture media of the kit for inducing stem cell differentiation into natural killer cells as defined above. In a preferred embodiment, the culture time in step (1) is 12-36 hours. In a preferred embodiment, the culture time in step (2) is 24-72 hours. In a preferred embodiment, the culture time in step (3) is 48-144 hours. In a preferred embodiment, the step (4) of culturing the hematopoietic endothelial cells in the fourth and fifth culture media of the kit for inducing stem cell differentiation into natural killer cells as defined above means culturing the hematopoietic endothelial cells sequentially in the fourth and fifth culture media of the kit for inducing stem cell differentiation into natural killer cells as defined above. In a preferred embodiment, the culture time of the hematopoietic endothelial cells in the fourth culture medium is 96-192 hours. In a preferred embodiment, the culture time of the hematopoietic endothelial cells in the fifth culture medium is 144-432 hours.

[0024] In some embodiments, the drug combination further comprises other chemotherapeutic agents selected from anticancer agents, metabolic antagonists, DNA demethylators, plant-derived antitumor agents, alkylating agents, antimetabolites, anticancer antibiotics, topoisomerase inhibitors, mitotic inhibitors, differentiation agents, and hormone therapy agents. In a preferred embodiment, the other chemotherapeutic agents are selected from carmustine, lomustine, vorinostat, mebendazole, imatinib mesylate, AG119, or any therapeutic derivative thereof.

[0025] In one embodiment, the active ingredient does not contain trimethoprim.

[0026] In one embodiment, the active ingredient does not contain immune checkpoint inhibitors.

[0027] In one implementation, the chemotherapeutic agent is administered before, after, or simultaneously with the administration of the first and second aspects of NK cells.

[0028] In one embodiment, the chemotherapeutic agent is administered 8 to 72 hours before, 8 to 72 hours after, or both before and 8 to 72 hours after the administration of the first and second aspects of NK cells. Attached Figure Description

[0029] Figures 1A-1F show microscopic images, cell counts, and statistical results of inhibition rates for detecting the killing effects of NK92 cells alone, TMZ alone, and NK92 cells in combination with TMZ on SKOV3 and NALM6 cells.

[0030] Figure 2 shows the results of detecting the chemotactic activity of NK92 cells alone, TMZ alone, and NK92 cells in combination with TMZ using the Tranwell assay.

[0031] Figures 3A-3E show the cell count and lysis rate (%) of HELA cells, SKOV3 cells, NALM6 cells, IOMM-Lee cells, and MDA-MB-361 cells after treatment with iNK cells alone, TMZ alone, and iNK cells in combination with TMZ.

[0032] Figures 4A and 4B show the cell number and lysis rate (%) of MDA-MB-361 cells and IOMM-Lee cells after treatment with NK92 cells alone, TMZ alone, and NK92 cells in combination with TMZ.

[0033] Figures 5A-5C show the cell count and lysis rate (%) of SKOV3 cells, NALM6 cells, and IOMM-Lee cells after treatment with PBNK cells alone, TMZ alone, and PBNK cells in combination with TMZ.

[0034] Figures 6A-6D show the results of detecting the chemotactic ability of iNK cells alone, TMZ alone, and iNK cells in combination with TMZ using the Tranwell assay. Detailed Implementation

[0035] General definitions and terms

[0036] All patents, patent applications, scientific publications, manufacturers' specifications and guidelines, etc., cited herein, are incorporated herein in their entirety, whether mentioned above or below. Nothing herein should be construed as an admission that this disclosure is not entitled to precede such disclosure.

[0037] Unless otherwise stated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, terms related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, and microbiology used herein are all widely used terms in their respective fields (see, for example, *Molecular Cloning: A Laboratory Manual*, 2008). nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989. Meanwhile, to better understand this invention, definitions and explanations of relevant terms are provided below.

[0038] As used herein, the expressions “comprising,” “including,” “containing,” and “having” are open-ended, meaning they include the listed elements, steps, or components but do not exclude other unlisted elements, steps, or components. The expression “composed of” excludes any unspecified elements, steps, or components. The expression “substantially composed of” means that the scope is limited to the specified elements, steps, or components, plus optional elements, steps, or components that do not significantly affect the essential and novel nature of the claimed subject matter. It should be understood that the expressions “substantially composed of” and “composed of” are encompassed within the meaning of the expression “including.”

[0039] As used herein, unless the context otherwise indicates, the singular expressions “a,” “an,” or “this” include plural references. The terms “one or more” or “at least one” cover 1, 2, 3, 4, 5, 6, 7, 8, 9, or more.

[0040] The listing of ranges of values ​​in this document is merely a shorthand method for individually referring to each distinct value falling within the range. Unless otherwise stated herein, each individual value is included in this specification as it is individually listed herein. Unless explicitly stated otherwise, all numerical values ​​or ranges shown herein are modified with “about”, indicating that the listed or claimed numerical values ​​or ranges are ±20%, ±10%, ±5%, or ±3%.

[0041] The term "pluripotent cell" refers to a cell that can self-renew and proliferate while remaining undifferentiated, and can be induced to differentiate into specialized cell types under appropriate conditions.

[0042] As used herein, the term “pluripotent stem cell” refers to a stem cell capable of differentiating into all cell types in the following three germ layers: endoderm (e.g., gastric junction, gastrointestinal tract, lung, etc.), mesoderm (e.g., muscle, bone, blood, urogenital tissue, etc.), or ectoderm (e.g., epidermal tissue and nervous system tissue). The term “pluripotent stem cell” as used herein also includes “induced pluripotent stem cells” or “iPSCs,” which are pluripotent stem cells derived from non-pluripotent cells and reprogrammed by introducing specific transcription factors. Exemplary human pluripotent stem cell lines include the H1 and H9 human pluripotent stem cell lines. Other exemplary pluripotent stem cell lines include those available through the National Institutes of Health Human Embryonic Stem Cell Registry and the Howard Hughes Medical Institute HUES collection (e.g., Cowan CA, et al. Derivation of embryonic stem-cell lines from human blastocysts. N Engl J Med. 2004 Mar 25; 350(13):1353-6.). Pluripotent stem cells (iPSCs) can be induced to differentiate into various cell types for different preventative or therapeutic purposes, to prevent or treat different diseases. As those skilled in the art will understand, the differentiation method depends on the desired cell type using known techniques. For example, cells can be differentiated in suspension and then formulated into a gel matrix form, such as matrix gelatin, gelatin, or fibrin / thrombin, to promote cell survival. Differentiation can typically be determined by assessing the presence of cell-specific markers, as is known in the art. For example, cells can be differentiated under specific differentiation conditions into cardiomyocytes, nerve cells, glial cells, endothelial cells, T cells, NK cells, NKT cells, macrophages, hematopoietic progenitor cells, mesenchymal cells, pancreatic islet cells, chondrocytes, retinal pigment epithelial cells, kidney cells, hepatocytes, thyroid cells, skin cells, blood cells, or epithelial cells. In some embodiments, iPSCs are differentiated into NK cells.

[0043] NK cells derived from iPSCs are also referred to herein as “iNK cells,” “iPS-NK cells,” or “iPSC-NK cells.” In this document, the terms “iNK cells,” “iPS-NK cells,” “iPSC-NK cells,” and “NK cells derived from induced pluripotent stem cells (iPSCs)” are used interchangeably.

[0044] As used herein, the term "pluripotent" refers to the ability of a cell to form a complete organism. For example, in mammals, only the fertilized egg and the first cleavage blastomeres are totipotent. In some embodiments, the pluripotent stem cells described herein are not totipotent and do not form a complete organism.

[0045] The cells can be derived from, for example, humans or non-human mammals. Exemplary non-human mammals include, but are not limited to, mice, rats, cats, dogs, rabbits, guinea pigs, hamsters, sheep, pigs, horses, cattle, and non-human primates. In some embodiments, the cells are derived from adult humans or non-human mammals. In some embodiments, the cells are derived from neonatal humans, adult humans, or non-human mammals.

[0046] As used herein, the terms “subject” or “patient” refer to any animal, such as a domesticated animal, a zoo animal, or a human. A “subject” or “patient” can be a mammal, such as a dog, cat, bird, livestock, or human. Specific examples of “subject” and “patient” include, but are not limited to, individuals (especially humans) having a disease or condition related to the liver, heart, lungs, kidneys, pancreas, brain, nervous tissue, blood, bones, bone marrow, etc. In some embodiments, “subject” and “patient” refer to an individual (especially a human) suffering from a tumor. In some embodiments, “subject” and “patient” refer to an individual (especially a human) suffering from a low-immune-invasive tumor.

[0047] In this paper, the terms “reduction” and “reduction” are generally used to indicate a reduction that is statistically significant. However, to avoid ambiguity, “reduction” or “reduction” includes a reduction of at least 10% compared to a reference level, such as a reduction of at least about 20% or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to and including a 100% reduction (i.e., a level that does not exist compared to the reference sample), or any reduction between 10% and 100%.

[0048] In this document, the term “increase” is generally used to indicate an increase that is statistically significant; to avoid any ambiguity, the term “increase” means an increase of at least 10% compared to a reference level, such as an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to and including 100% increase or any increase between 10 and 100%, or at least about 2 times, or at least about 3 times, or at least about 4 times, or at least about 5 times or at least about 10 times increase compared to a reference level, or any increase between 2 and 10 times or greater than 10 times.

[0049] In this article, the terms “NK cell” or “natural killer cell” are used interchangeably. NK cells are effector cells of the innate immune system, belonging to the innate lymphocyte (ILC) family, characterized by the presence of CD56 and the absence of the CD3 surface marker. Most NK cells are located in the peripheral blood, lymph nodes, spleen, and bone marrow. NK cells protect the host by killing infected, foreign, stressed, or transformed cells, and can kill target cells in the absence of specific antigenic stimuli and without restriction according to MHC class, where target cells can be tumor cells or virus-carrying cells. Current preclinical and clinical development strategies enhance the antitumor function of NK cells through adoptive immunotherapy, direct stimulation, recruitment of NK cells to the tumor microenvironment (TME), and blocking inhibitory receptors that limit NK cell function (see, for example, Bald T, et al. The NK cell-cancer cycle: advances and new challenges in NK cell-based immunotherapies. Nat Immunol. 2020 Aug; 21(8):835-847.).

[0050] Cytotoxic lymphocytes include cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. Although these two cell types differ significantly in their activation patterns and target recognition mechanisms, key pathways mediating target cell death are conserved. Once bound to a target cell, cytotoxic secretory granules are transported to the immune synapse, releasing protein cargo, including perforin, granzymes, and granzymes, into the synaptic cleft. Specifically, cytotoxic lymphocytes recognize their target cells and form an immune synapse. The microtubule organizing center (MTOC) of the cytotoxic lymphocyte polarizes, and the secretory granules move toward the presynaptic membrane. The secretory granules then fuse with the presynaptic membrane, releasing perforin and granzymes into the synaptic cleft. On the postsynaptic membrane, perforin forms a large transmembrane pore, allowing granzymes to diffuse into the target cell cytoplasm. Granzymes then initiate apoptosis in target cells, detaching cytotoxic lymphocytes from dying cells and enabling them to interact with another target cell for successive killing (see, for example, Voskoboinik I, et al. Perforin and granzymes: function, dysfunction and human pathology. Nat Rev Immunol. 2015 Jun; 15(6):388-400.). As used herein, the term "perforin" refers to a protein that can insert into the target cell membrane to form pores. These pores disrupt the integrity of the target cell membrane, leading to cell lysis and destruction. As used herein, the term "granzyme" refers to a class of serine proteases, primarily including granzymes A, B, and K. They can induce apoptosis (programmed cell death) in target cells by activating intracellular apoptosis-related molecules such as caspase, thereby triggering DNA fragmentation and cell death in target cells.

[0051] As used herein, the terms "Adoptive Cell Transfer Therapy," "Adoptive Immunotherapy," "Adoptive Immunotherapy," or "Adoptive Cell Therapy" refer to an immunotherapy method that modifies and expands immune cells in vitro to enhance their immune function before infusing them into the patient to exert an anti-tumor effect. These immune cells can be derived from allogeneic or allogeneic sources. Allogeneic adoptive immunotherapy refers to the transfer of immune cells from an allogeneic source (a different individual) to the patient. Allogeneic adoptive immunotherapy refers to the collection, expansion, and transfer of the patient's own immune cells. These immune cells can be natural killer cells, T cells, or lymphocytes, etc.

[0052] As used herein, the term "chemotherapeutic agent" refers to a compound or derivative thereof that can interact with cancer cells to reduce their proliferative state and / or kill them, for example by impairing cell division or DNA synthesis, or by effectively targeting rapidly dividing cells by disrupting DNA. Examples of chemotherapeutic agents include, but are not limited to, anticancer agents, metabolic antagonists, DNA demethylators, plant-derived antitumor agents, alkylating agents, antimetabolites, anticancer antibiotics, topoisomerase inhibitors, mitotic inhibitors, differentiation agents, hormone therapy agents, and combinations of the above.

[0053] As used herein, the term "alkylating agent" or "alkylating agent" refers to a class of highly reactive compounds with active alkyl groups. These alkyl groups can be converted into electron-deficient, reactive intermediates, which rapidly form covalent bonds with many cellular substances that have affinity for them. Alkylating agents can covalently bind to electron-rich groups (such as amino, thiol, hydroxyl, carboxyl, and phosphate groups) in cellular macromolecules (such as DNA, RNA, and enzymes), causing them to lose activity, break DNA molecules, or cause base mismatches during DNA replication. This disrupts the structure and function of DNA, leading to tumor cell death.

[0054] As used herein, the term "temozolomide (TMZ)" is an oral alkylating prodrug that transfers a methyl group to a purine base (O6-guanine; N7-guanine and N3-adenine) in DNA and is frequently used in conjunction with radiotherapy as part of first-line treatment for malignant gliomas. TMZ has the chemical formula C6H6N6O2, its chemical structure is shown below, and its chemical name is 3,4-dihydro-3-methyl-4-oxoimidazo[5,1-d]-asymmetric-tetrazolium-8-amide. The main advantages of TMZ are its high oral bioavailability (almost 100%, although the concentration found in cerebrospinal fluid is about 20% of the plasma concentration of TMZ), its lipophilicity, and its small size, which allows it to cross the blood-brain barrier (see, for example, Ortiz R, et al. Temozolomide: An Updated Overview of Resistance Mechanisms, Nanotechnology Advances and Clinical Applications. Curr Neuropharmacol. 2021; 19(4):513-537.).

[0055] As used herein, any form of administration of “combination,” “combination therapy,” and / or “combination treatment regimen” means at least two therapeutically active pharmaceutical ingredients or combinations thereof, which may be administered simultaneously as individual or combined formulations, or sequentially at different time intervals of minutes, hours, or days, but in some way work together to provide the desired therapeutic response.

[0056] The term "differentiation" is a process by which less specialized cells form offspring of at least one more specialized new cell type.

[0057] The term "embryoid body," also known as an embryoid body or aggregate, refers to a homogeneous or heterogeneous cluster of cells containing differentiated cells, partially differentiated cells, and / or pluripotent stem cells in suspension culture. To summarize some of the inherent clues of differentiation in vivo, certain aspects of the invention can use three-dimensional embryoid bodies as an intermediate step. Differentiation can be initiated at the beginning of cell aggregation, and cells can begin to reproduce embryonic development to a limited extent. While they cannot form trophoblastic ectoderm tissue, almost all other cell types present in the organism can develop. The invention can further promote hematopoietic progenitor cell differentiation after embryoid body formation.

[0058] The term "basal medium" refers to media with a defined chemical composition, including but not limited to IMDM (Iscove's Modified Dulbecco's Medium), Eagle's Basal Medium (BME), MEM, DMEM, Ham's F-12, RPMI 1640, Fischer's, E8, mTESR, StemFit Basic 03, StemFit Basic 04, NutriStem hPSC XF, StemMACS iPS Brew, Stem-Partner ACF, TeSR-AOF, TeSR2, and other basic cell media.

[0059] The term "multiple colony-stimulating factor" is also known as interleukin-3. It is primarily produced by activated Z-cell or T-cell clones. Containing 13 amino acid residues, it acts as an immunomodulator, stimulating the proliferation and differentiation of pluripotent stem cells and various progenitor cells.

[0060] The term "stem cell factor," also known as mast cell growth factor (MGF), Kit ligand (KL), and Steel factor (SLF), is an acidic glycoprotein produced by stromal cells in the bone marrow microenvironment. Its glycosyl groups are linked to the N and O groups of the peptide bond, with a relative molecular mass of 31,000-36,000, and it consists of two non-covalently linked identical subunits. Its isoelectric point (PI) is 3.8. SCF contains 273 amino acids. The signal peptide is defined as -25 to -1, the extracellular functional region as +1 to +189, the transmembrane region as +190 to +216, and the cytoplasmic functional region as +217 to +248. Mouse and human SCF share 83% homology.

[0061] The term "vitamin C" includes vitamin C or its various salts or derivatives thereof.

[0062] The term “UM171” includes UM171 (CAS No.: 1448724-09-1) or its various salts or its various derivatives.

[0063] The term "SR1" includes SR1 (StemRegenin1, CAS No.: 1227633-49-9) or its various derivatives.

[0064] The term "glutamine" refers to L-glutamine, an amino acid that encodes proteins. It is a non-essential amino acid for mammals and is used as an essential additive for cell culture in this invention.

[0065] The term "SB431542" also includes SB431542 and its salts, especially pharmaceutically acceptable salts.

[0066] The term "CHIR99021" includes CHIR99021 and its salts, especially pharmaceutically acceptable salts.

[0067] The term "Y-27632" also includes Y-27632 and its salts, especially pharmaceutically acceptable salts. A preferred pharmaceutically acceptable salt is Y-27632 2HCl.

[0068] The term "fibroblast growth factor" refers to a polypeptide secreted by the pituitary gland and hypothalamus that promotes fibroblast mitosis, mesodermal cell growth, and angiogenesis. It plays a role in wound healing and limb regeneration. There are two types: acidic (pI 5.6) and basic (pI 9.6), namely aFGF and bFGF.

[0069] The term "bone morphogenetic protein-4" (BMP4) plays a regulatory role in the proliferation and differentiation of various cells during embryonic development.

[0070] The term "vitamin A-free B27 culture medium supplement" is custom-designed. The culture medium is vitamin A-free. Vitamin A (retinol) can be converted into retinoic acid, which can induce stem cells to differentiate into nerve cells. Vitamin A-free formulations are ideal for stem cell culture.

[0071] Cell detection markers

[0072] The developmental stages of NK cells are primarily based on the expression levels of CD117, CD45, CD56, and CD94. Increased CD56 expression is crucial for NK cell maturation, followed by CD94 / NKG2A expression. Therefore, during NK cell differentiation, CD45 expression is typically selected. + CD56 + The expression of CD3 indicates the stage of differentiation, and CD3-negative expression also indicates whether there is interference from T cells in the differentiation system.

[0073] CD45: The cell surface phosphatase CD45 plays a key role in intracellular signal transduction for NK cell development and receptor activation, and is a crucial enzyme for inducing NK cell responses.

[0074] CD56, also known as the neural cell adhesion molecule, is a 175-185 kDa glycoprotein belonging to the immunoglobulin superfamily C2. It is primarily expressed on human NK cells and a small number of MHC-mediating T lymphocytes, with over 95% expression on the surface of human NK cells, making it an important marker of NK cell surface activity. NK cells in the human body are mainly characterized by CD3+. - CD56 + Lymphocyte population.

[0075] CD3: A signaling component of the T cell receptor (TCR) complex, used to indicate whether there is interference from T cells during NK cell differentiation, as some T cells can also express NK cell surface markers.

[0076] CD94: CD94 exists mainly on the surface of NK cells in the form of isotype or heterotype of NKG2A, and is a marker of the maturation of differentiated NK cells.

[0077] NKp30: NKp30 belongs to the CD28 family and is a member of the immunoglobulin superfamily (IgSF). It is expressed only on the surface of NK cells and is a unique marker of NK cells. Its ligand is B7-H6, and its function is to activate NK cells. As an NK cell activation receptor, it plays an important role in immune surveillance and killing of tumor and pathogen-infected cells by binding to its corresponding ligand.

[0078] NKp44: NKp44 is a member of the immunoglobulin superfamily (IgSF) and is expressed only on the surface of activated NK cells. It is a specific marker of activated NK cells, and its extracellular region contains only a V-shaped domain. The cytoplasmic region of NKp44 lacks the ITAM motif, while its transmembrane region contains positively charged lysine residues. This allows it to non-covalently bind to the negatively charged aspartic acid residue in the transmembrane region and the DAP12 homodimer containing the ITAM motif in the cytoplasmic region, thereby acquiring the function of transducing activation signals. NKp44 is only expressed after NK cells are activated, further amplifying the signal.

[0079] NKp46: NKp46 is expressed on the surface of all NK cells (including mature, immature, quiescent, and activated NK cells). The extracellular region of NKp46 contains two Ig-like domains. The cytoplasmic region of NKp46 is relatively short, and the transmembrane region contains positively charged arginine residues, which can non-covalently bind to the CD3ζζ homodimer containing negatively charged aspartic acid residues in the transmembrane region and the ITAM motif in the cytoplasmic region, thereby acquiring the function of transducing activation signals.

[0080] As used herein, the terms "first culture medium" and similar terms such as "second culture medium," "third culture medium," "fourth culture medium," and "fifth culture medium" are used for distinguishing purposes only and are not intended to limit the order in which these culture media are used. The above terms may be the same or different in different schemes.

[0081] Unless otherwise stated, all methods described herein may be performed in any suitable order.

[0082] Drug combination

[0083] In a first aspect, the present invention provides a pharmaceutical combination for treating tumors, the pharmaceutical combination comprising an active ingredient comprising natural killer (NK) cells and a chemotherapeutic agent, wherein the chemotherapeutic agent includes temozolomide.

[0084] As used herein, the term "active ingredient" refers to any single substance or mixture of substances in a pharmaceutical combination that has pharmacological activity, either directly or indirectly, in the diagnosis, treatment, symptom relief, prevention of relapse, or prevention of disease.

[0085] In some implementations, the tumor includes, but is not limited to, nervous system tumors, neuroendocrine tumors, hematologic tumors, ovarian cancer, prostate cancer, pancreatic cancer, osteosarcoma, thyroid cancer, soft tissue sarcoma, insulinoma, and testicular cancer.

[0086] In some implementations, nervous system tumors include brainstem gliomas (DIPG), supratentorial and infratentorial gliomas, astrocytomas, oligodendrogliomas, ependymomas, medulloblastomas, schwannomas, meningiomas, pituitary adenomas, neuroblastomas, spinal cord gliomas, meningiomas, metastatic spinal tumors, peripheral nerve sheath tumors, neurofibromas, brain metastasis, and neuroectodermal tumors.

[0087] In some implementations, hematologic malignancies include leukemia (e.g., acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML)), lymphoma (e.g., Hodgkin lymphoma (HL), non-Hodgkin lymphoma (NHL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma (MCL), Burkitt lymphoma (BL)), and bone marrow malignancies (e.g., multiple myeloma (MM), myelodysplastic syndrome (MDS), myeloproliferative neoplasm (MPN), polycythemia vera (PV), essential thrombocythemia (ET), myelofibrosis (MF)).

[0088] In a preferred embodiment, the tumor is a nervous system tumor.

[0089] In some embodiments, the tumor includes low-immune-invasive tumors. In some embodiments, the tumor includes low-immune-invasive neurological tumors, neuroendocrine tumors, hematologic malignancies, ovarian cancer, prostate cancer, pancreatic cancer, osteosarcoma, thyroid cancer, soft tissue sarcoma, insulinoma, and testicular cancer. In some embodiments, low-immune-invasive neurological tumors include low-immune-invasive brainstem gliomas (DIPG), supratentorial and infratentorial gliomas, astrocytomas, oligodendrogliomas, ependymomas, medulloblastomas, schwannomas, meningiomas, pituitary adenomas, neuroblastomas, spinal cord gliomas, spinal cord meningiomas, spinal cord metastases, peripheral schwannomas, neurofibromas, brain metastases, and neuroectodermal tumors. In some implementations, low-immune-infiltrating hematologic malignancies include low-immune-infiltrating leukemias (e.g., acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML)), low-immune-infiltrating lymphomas (e.g., Hodgkin lymphoma (HL), non-Hodgkin lymphoma (NHL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma (MCL), Burkitt lymphoma (BL)), and low-immune-infiltrating bone marrow tumors (e.g., multiple myeloma (MM), myelodysplastic syndrome (MDS), myeloproliferative neoplasm (MPN), polycythemia vera (PV), essential thrombocythemia (ET), myelofibrosis (MF)).

[0090] In another aspect, the present invention provides a pharmaceutical combination for treating low-immune-invasive tumors, the pharmaceutical combination comprising an active ingredient comprising natural killer (NK) cells and a chemotherapeutic agent, wherein the chemotherapeutic agent includes temozolomide.

[0091] In some implementations, the low-immune-invasive tumors include, but are not limited to, low-immune-invasive nervous system tumors, neuroendocrine tumors, non-small cell lung cancer, small cell lung cancer, colorectal cancer, melanoma, breast cancer, esophageal cancer, gastric cancer, bladder cancer, endometrial cancer, head and neck cancer, cervical cancer, liver cancer, and kidney cancer.

[0092] In some implementations, low-immune-infiltrating nervous system tumors include low-immune-infiltrating brainstem gliomas (DIPG), supratentorial and infratentorial gliomas, astrocytomas, oligodendrogliomas, ependymomas, medulloblastomas, schwannomas, meningiomas, pituitary adenomas, neuroblastomas, spinal cord gliomas, spinal cord meningiomas, spinal cord metastases, peripheral schwannomas, neurofibromas, brain metastases, and neuroectodermal tumors.

[0093] In a preferred embodiment, the low-immune-invasive tumor is low-immune-invasive cervical cancer.

[0094] In a preferred embodiment, the low-immune-infiltrating tumor is a low-immune-infiltrating nervous system tumor.

[0095] As used herein, the terms “highly immune-infiltrating tumor,” “hot tumor,” or “immunoinflammatory tumor” are used interchangeably and refer to tumors exhibiting high T-cell infiltration, enhanced interferon-γ (IFN-γ) signaling, programmed death-ligand 1 (PD-L1) expression, and high tumor mutational burden (TMB). Furthermore, such tumors with an immune-inflammatory phenotype typically show a higher response to immune checkpoint inhibitors (ICIs). The terms “lowly immune-infiltrating tumor” or “cold tumor” are used interchangeably and encompass immune-excluded, immune-suppressed, and immune-desert tumors. In immune-excluded tumors, CD8… +T lymphocytes remain confined to the periphery of invasion, failing to effectively penetrate the tumor body. This "immune exclusion" phenotype reflects the host's intrinsic ability to effectively initiate a T cell-mediated immune response, and the tumor's ability to evade this response by physically hindering T cell penetration. In immunosuppressive tumors, the tumor site exhibits low-level immune infiltration, indicating that the lack of physical barriers and the presence of an immunosuppressive environment limit further recruitment and expansion of immune cells. Immune desert tumors, on the other hand, lack CD8+ T lymphocytes both within and around the tumor. Low mutational burden, low major histocompatibility complex (MHC) class I expression, and low PD-L1 expression are also key characteristics of this type of tumor. Furthermore, "low-immune-infiltrating tumors" or "cold tumors" may also contain immunosuppressive cell populations, such as tumor-associated macrophages (TAMs), regulatory T cells (Tregs), and myeloid-derived suppressor cells (MDSCs). "Low-immune-infiltrating tumors" or "cold tumors" may lack innate anti-tumor immunity, or existing innate immune responses may be ineffective due to immune cell rejection. Compared with "highly immune-infiltrating tumors" or "hot tumors", immune cells have difficulty recognizing and killing "lowly immune-infiltrating tumors" or "cold tumors", and immunosuppressants are also less effective, resulting in poor immunotherapy efficacy and limited responsiveness to immune checkpoint inhibitors (see, for example, Khosravi GR, et al. Immunologic tumor microenvironment modulators for turning cold tumors hot. Cancer Commun (Lond). 2024 May; 44(5):521-553. and Galon J, et al. Approaches to treat immune hot, altered and cold tumors with combination immunotherapies. Nat Rev Drug Discov. 2019 Mar; 18(3):197-218.).

[0096] Common methods used in this field to differentiate between hot and cold tumors include the "Immunoscore," which assesses the type, density, and location of immune cells at the tumor site. This scoring system has been validated globally in colorectal cancer and has significantly surpassed the currently accepted classic TNM staging system (the standard method for staging malignant tumors in clinical practice; TNM tumor staging: T for primary tumor status; N for regional lymph node involvement; M for distant metastasis) in terms of prognostic accuracy for colorectal cancer patients. It is also superior to assessment methods such as tumor differentiation degree and tumor microsatellite instability (MSI) (see, for example, Galon J, et al. Approaches to treat immune hot, altered and cold tumors with combination immunotherapies. Nat Rev Drug Discov. 2019 Mar; 18(3):197-218.).

[0097] Specifically, the immune score is a standardized scoring system that quantifies two lymphocyte populations (CD3 and CD8) at the tumor center and invasive margin. The immune score ranges from I0 (indicating low density and no immune cell infiltration; absence of both cell types in both areas indicates a cold tumor) to I4 (high immune cell infiltration; high immune cell density in both locations indicates a hot tumor), a total of five levels. The tumor tissue examined includes the tumor core (CT) and the invasive margin (IM). Immunohistochemical staining was performed using CD3 and CD8 monoclonal antibodies. The highest immune score was I4, which required high infiltration of CD3 and CD8 T cells in both the tumor core and peripheral regions. I3 was also classified as a high immune score group along with I4. Of the four indicators of CD3 and CD8 staining in the tumor core and peripheral regions, three showed high T cell infiltration. I2 was a moderate immune score group, where half of the four indicators of the above-mentioned detection sites and cell types showed no obvious T cell infiltration. I1 and I0 belonged to the low immune score groups. I1 showed no T cell infiltration in three-quarters of the above-mentioned indicators, while I0 showed no T cell infiltration (see, for example, Taube JM, et al. Implications of the tumor immune microenvironment for staging and therapeutics. Mod Pathol. 2018 Feb; 31(2):214-234.).

[0098] Currently, "low immune infiltration tumors" or "cold tumors" have been found to be more common in many tumor types. For example, ovarian cancer is often classified as a cold tumor because its cold tumor immune microenvironment makes it less responsive to immunotherapy (see, for example, Yang Y, et al. Nanomedicine Strategies for Heating "Cold" Ovarian Cancer (OC): Next Evolution in Immunotherapy of OC. Adv Sci (Weinh). 2022 Oct; 9(28):e2202797. and Le Saux O, et al. Challenges for immunotherapy for the treatment of platinum resistant ovarian cancer. Semin Cancer Biol. 2021 Dec; 77:127-143.).

[0099] Previous studies have found that cervical cancer includes cold tumors. Researchers retrospectively analyzed the expression of CD8, FoxP3, HLA-1, PD-L1, and XRCC4 in 100 cases of cervical cancer. The observed tumor immune microenvironment included a cold tumor-type immune microenvironment. Moreover, this cold tumor-type immune microenvironment is considered a poor prognostic factor, and the prognosis of cold tumors is significantly worse than other types (such as inflammatory hot tumors) (see, for example, Someya M, et al. Prediction of treatment response from the microenvironment of tumor immunity in cervical cancer patients treated with chemoradiotherapy. Med Mol Morphol. 2021 Sep; 54(3):245-252.).

[0100] In addition, the cold tumor phenotype is also present in many solid tumors. For example, pancreatic cancer (see, e.g., Ullman NA, et al. Immunologic Strategies in Pancreatic Cancer: Making Cold Tumors Hot. J Clin Oncol. 2022 Aug 20; 40(24):2789-2805. and Zheng W, et al. Combination of radiotherapy and vaccination overcomes checkpoint blockade resistance. Oncotarget. 2016 Jul 12; 7(28):43039-43051.), renal cell carcinoma (see, e.g., Brück O, et al. Spatial immunoprofiling of the intratumoral and peritumoral tissue of renal cell carcinoma patients. Mod Pathol. 2021 Dec; 34(12):2229-2241.), non-small cell lung cancer (see, e.g., Yu H, et al. Targeting METTL3 reprograms the tumor microenvironment to improve cancer immunotherapy. Cell Chem. Biol. 2024 Apr 18; 31(4):776-791.e7.), colorectal cancer (see, for example, Pagès F, et al. International validation of the consensus Immunoscore for the classification of colon cancer: a prognostic and accuracy study. Lancet. 2018 May 26; 391(10135):2128-2139.), prostate cancer (see, for example, Qi Z, et al. Overcoming resistance to immune checkpoint therapy in PTEN-null prostate cancer by intermittent anti-PI3Kα / β / δ treatment. Nat Commun. 2022 Jan 10; 13(1):182.), breast cancer (see, for example, Yang L, et al. Breast Cancer Treatment Strategies Targeting the Tumor Microenvironment: How to Convert "Cold" Tumors to "Hot" Tumors. Int J Mol Sci. 2024 Jun 29; 25(13):7208.), hepatocellular carcinoma (see, for example, Feng GS, et al. Improving the Efficacy of Liver Cancer Immunotherapy: The Power of Combined Preclinical and Clinical Studies. Hepatology. 2021 Jan; 73 Suppl 1(Suppl 1):104-114.), osteosarcoma (see, for example, Wu C, et al. A tumor microenvironment-based prognostic index for osteosarcoma. J Biomed Sci. 2023 Apr 13; 30(1):23.), endometrial tumor (see, for example, Jeffrey A. How, et al. Predictors of innate resistance). to pembrolizumab in patients with microsatellite instability-high endometrial cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022; 82 (12_Suppl): Abstract nr 1248.), gastric cancer (see, e.g., Zhao Z, et al. al. Necroptosis-Related lncRNAs: Predicting Prognosis and the Distinction between the Cold and Hot Tumors in Gastric Cancer. J Oncol. 2021 Nov 8; 2021:6718443.), melanoma (see, e.g., Gajewski TF, et al.Cancer Immunotherapy Targets Based on Understanding the T Cell-Inflamed Versus Non-T Cell-Inflamed Tumor Microenvironment. Adv Exp Med Biol. 2017; 1036:19-31.), meningiomas (see, for example, Brastianos PK, et al. Phase 2 study of pembrolizumab in patients with recurrent and residual high-grade meningiomas. Nat Commun. 2022 Mar 14; 13(1):1325.), brain metastases (see, for example, Uceda-Castro R, et al. Re-purposing the pro-senescence properties of doxorubicin to introduce immunotherapy in breast cancer brain metastasis. Cell Rep Med. 2022 Nov 15; 3(11):100821.), etc.

[0101] Among hematologic malignancies (such as lymphoma and leukemia), "low-immune-infiltrating tumors" or "cold tumors" are also quite common. For example, acute B-precursor lymphoblastic leukemia (see, for example, Willier S, et al. Leukemia escape in immune desert: intraocular relapse of pediatric pro-B-ALL during systemic control by CD19-CAR T cells. J Immunother Cancer. 2020 Sep; 8(2):e001052.); diffuse large B-cell lymphoma (see, for example, Higashi M, et al. CD24 is a surrogate for 'immune-cold' phenotype in aggressive large B-cell lymphoma. J Pathol Clin Res. 2022 Jul; 8(4):340-354.); acute myeloid leukemia (see, for example, Khaldoyanidi S, et al. Immune Biology of Acute Myeloid Leukemia: Implications for Immunotherapy. J Clin Oncol. 2021 Feb 10; 39(5):419-432.), etc.

[0102] In some embodiments, the NK cells are unmodified NK cells or modified NK cells. In a preferred embodiment, the NK cells are unmodified NK cells.

[0103] As used herein, the term "unmodified" means that there has been no genetic modification by intentionally adding foreign genetic material, such as DNA or RNA, to the total genetic material of a cell, inserting genes, or editing (directed modification) genes. For example, the term "unmodified" means that there has been no intentional genetic modification that results in the insertion or deletion of DNA fragments, or the deletion or substitution of DNA bases.

[0104] In some implementations, NK cells include, but are not limited to, NK-92 cells, NK cells derived from peripheral blood or umbilical cord blood, NK cells derived from induced pluripotent stem cells (iPSCs) or embryonic stem cells.

[0105] In some implementations, the embryonic stem cells are commercially available human embryonic stem cells (hESCs) (e.g., H1, H9).

[0106] In some implementations, embryonic stem cells are stem cells isolated or obtained from human embryos that have not undergone in vivo development and are within 14 days of fertilization.

[0107] In some implementations, induced pluripotent stem cells (iPSCs) are human induced pluripotent stem cells (hiPSCs) (e.g., WC50, IMR90).

[0108] In a preferred embodiment, the NK cells are NK cells derived from induced pluripotent stem cells (iPSCs).

[0109] In a preferred embodiment, the NK cells are unmodified NK cells derived from induced pluripotent stem cells.

[0110] In some embodiments, NK cells (preferably NK cells derived from induced pluripotent stem cells) are prepared using a kit for inducing stem cell differentiation into natural killer cells, the kit comprising: a second culture medium, a fourth culture medium, and a fifth culture medium; wherein the second culture medium is a basal culture medium containing a BMP signaling pathway activator; the fourth culture medium is a basal culture medium containing at least one, at least two, at least three, or at least four of the following: BMP signaling pathway activator, vascular endothelial growth factor, colony-stimulating factor, and interleukin; and the fifth culture medium is a basal culture medium containing at least one, at least two, at least three, at least four, at least five, or at least six of the following: BMP signaling pathway activator, vascular endothelial growth factor, colony-stimulating factor, interleukin, stem cell agonist, and stem cell expansion agent.

[0111] In some implementations, the second culture medium is a basal medium containing BMP signaling pathway activators and GSK3 inhibitors.

[0112] In some embodiments, the BMP signaling pathway activator of the second culture medium comprises at least one selected from BMP2, BMP4, SB4, SJ000291942, SJ000063181, SJ000370178, isoglycyrrhizin, geraniol, apigenin, and chickpea sprout extract. In a preferred embodiment, the BMP signaling pathway activator of the second culture medium comprises BMP4.

[0113] In some embodiments, the GSK-3 inhibitor of the second culture medium comprises at least one of a GSK-3α inhibitor and a GSK-3β inhibitor. In a preferred embodiment, the GSK-3 inhibitor of the second culture medium comprises at least one of B216763, TWS119, NP031112, SB216763, CHIR-98014, AZD2858, AZD1080, SB415286, LY2090314, and CHIR-99021. In a more preferred embodiment, the GSK-3 inhibitor of the second culture medium comprises CHIR-99021.

[0114] In some embodiments, the concentration of the BMP signaling pathway activator in the second culture medium is 1-100 ng / mL; preferably 2-50 ng / mL; more preferably 20 ng / mL.

[0115] In some embodiments, the concentration of the GSK-3 inhibitor in the second culture medium is 0.1-10 μM; preferably 1-10 μM; more preferably 3 μM.

[0116] In some embodiments, the basal medium of the second culture medium comprises at least one of IMDM (Iscove's Modified Dulbecco's Medium), Eagle's Basal Medium (BME), MEM, DMEM, Ham's F-12, RPMI 1640, Fischer's, and DMEM / F12. In a preferred embodiment, the basal medium of the second culture medium comprises RPMI 1640.

[0117] In some embodiments, the basal medium of the second culture medium contains at least one, at least two, at least three, or at least four of the following: non-essential amino acids, glutamine, vitamin C, and B27 culture medium supplement. In a preferred embodiment, the basal medium of the second culture medium contains non-essential amino acids, glutamine, vitamin C, and B27 culture medium supplement.

[0118] In some implementations, the B27 medium supplement is a vitamin A-free B27 medium supplement.

[0119] In some embodiments, the concentration of non-essential amino acids in the second culture medium is 0.2-5% (v / v); preferably 0.5-2% (v / v), more preferably 1% (v / v).

[0120] In some embodiments, the concentration of glutamine in the second culture medium is 0.2-5% (v / v); preferably 0.5-2% (v / v), more preferably 1% (v / v).

[0121] In some embodiments, the concentration of vitamin C in the second culture medium is 10-100 μg / mL; preferably 20-50 μg / mL, more preferably 50 μg / mL.

[0122] In some embodiments, the concentration of the B27 culture medium additive in the second culture medium is 1%-10% (v / v), preferably 2% (v / v).

[0123] The second culture medium, as defined above, is used to induce embryoid differentiation into mesodermal cells.

[0124] In some embodiments, the BMP signaling pathway activator in the fourth and / or fifth culture media comprises at least one selected from BMP2, BMP4, SB4, SJ000291942, SJ000063181, SJ000370178, isoglycyrrhizin, geraniol, apigenin, and chickpea spore extract. In a preferred embodiment, the BMP signaling pathway activator in the fourth and / or fifth culture media comprises BMP4.

[0125] In some embodiments, the vascular endothelial growth factor in the fourth and / or fifth culture media comprises at least one of VEGF-A, VEGF-165, VEGF-183, VEGF-110, VEGF-121, VEGF-B, VEGF-C, VEGF-D, and VEGF-E. In a preferred embodiment, the vascular endothelial growth factor in the fourth and / or fifth culture media comprises VEGF-165.

[0126] In some embodiments, the colony-stimulating factor in the fourth culture medium includes at least one selected from G-CSF (granulocyte colony-stimulating factor), M-CSF (macrophage colony-stimulating factor), GM-CSF (recombinant human granulocyte-macrophage colony-stimulating factor), multi-CSF (also known as IL-3), EPO (erythropoietin), TPO, SCF, and FLT3L. In a preferred embodiment, the colony-stimulating factor in the fourth culture medium includes at least one selected from IL-3, SCF, and FLT3L. In a more preferred embodiment, the colony-stimulating factor in the fourth culture medium includes IL-3, SCF, and FLT3L.

[0127] In some embodiments, the colony-stimulating factor in the fifth culture medium includes at least one selected from G-CSF (granulocyte colony-stimulating factor), M-CSF (macrophage colony-stimulating factor), GM-CSF (recombinant human granulocyte-macrophage colony-stimulating factor), multi-CSF (also known as IL-3), EPO (erythropoietin), TPO, SCF, and FLT3L. In a preferred embodiment, the colony-stimulating factor in the fifth culture medium includes at least one selected from SCF and FLT3L. In a more preferred embodiment, the colony-stimulating factor in the fifth culture medium includes both SCF and FLT3L.

[0128] In some embodiments, the interleukins in the fourth and / or fifth culture media comprise at least one of IL-1, IL-2, IL-6, IL-7, IL-12, IL-15, IL-18, IL-21, and IL-27. In a preferred embodiment, the interleukins in the fourth and / or fifth culture media comprise at least one of IL-7 and IL-15. In a more preferred embodiment, the interleukins in the fourth and / or fifth culture media comprise both IL-7 and IL-15.

[0129] In some embodiments, the stem cell agonist in the fifth culture medium comprises at least one of UM171 and UM729. In a preferred embodiment, the stem cell agonist comprises UM171.

[0130] In some embodiments, the stem cell expansion agent in the fifth culture medium contains at least one of SR1 and PD98059. In a preferred embodiment, the stem cell expansion agent contains SR1.

[0131] In some embodiments, the concentration of the BMP signaling pathway activator in the fourth culture medium is 1-50 ng / mL; preferably 1-10 ng / mL; more preferably 5 ng / mL.

[0132] In some embodiments, the concentration of vascular endothelial growth factor in the fourth culture medium is 5-50 ng / mL; preferably 5-20 ng / mL; more preferably 10 ng / mL.

[0133] In some embodiments, the concentration of colony-stimulating factor in the fourth culture medium is 7-200 ng / mL; preferably 16-80 ng / mL; more preferably 35 ng / mL.

[0134] In some embodiments, the concentration of IL-3 in the fourth culture medium is 1-50 ng / mL; preferably 1-10 ng / mL; more preferably 5 ng / mL.

[0135] In some embodiments, the concentration of SCF in the fourth culture medium is 5-100 ng / mL; preferably 10-50 ng / mL; more preferably 20 ng / mL.

[0136] In some embodiments, the concentration of FLT3L in the fourth culture medium is 1-50 ng / mL; preferably 5-20 ng / mL; more preferably 10 ng / mL.

[0137] In some embodiments, the concentration of interleukin in the fourth culture medium is 2-100 ng / mL; preferably 15-70 ng / mL; more preferably 30 ng / mL.

[0138] In some embodiments, the concentration of IL15 in the fourth culture medium is 1-50 ng / mL; preferably 5-20 ng / mL; more preferably 10 ng / mL.

[0139] In some embodiments, the concentration of IL7 in the fourth culture medium is 1-50 ng / mL; preferably 10-50 ng / mL; more preferably 20 ng / mL.

[0140] In some embodiments, the concentration of the BMP signaling pathway activator in the fifth culture medium is 1-50 ng / mL; preferably 5-10 ng / mL; more preferably 5 ng / mL.

[0141] In some embodiments, the concentration of vascular endothelial growth factor in the fifth culture medium is 5-50 ng / mL; preferably 5-20 ng / mL; more preferably 10 ng / mL.

[0142] In some embodiments, the concentration of colony-stimulating factor in the fifth culture medium is 6-150 ng / mL; preferably 15-70 ng / mL; more preferably 30 ng / mL.

[0143] In some embodiments, the concentration of SCF in the fifth culture medium is 5-100 ng / mL; preferably 10-50 ng / mL; more preferably 20 ng / mL.

[0144] In some embodiments, the concentration of FLT3L in the fifth culture medium is 1-50 ng / mL; preferably 5-20 ng / mL; more preferably 10 ng / mL.

[0145] In some embodiments, the concentration of interleukin in the fifth culture medium is 2-100 ng / mL; preferably 15-70 ng / mL; more preferably 30 ng / mL.

[0146] In some embodiments, the concentration of IL15 in the fifth culture medium is 1-50 ng / mL; preferably 5-20 ng / mL; more preferably 10 ng / mL.

[0147] In some embodiments, the concentration of IL7 in the fifth culture medium is 1-50 ng / mL; preferably 10-50 ng / mL; more preferably 20 ng / mL.

[0148] In some embodiments, the concentration of the stem cell agonist in the fifth culture medium is 0.5-10 μM; preferably 0.5 μM-5 μM; more preferably 1 μM.

[0149] In some embodiments, the concentration of the stem cell expansion agent in the fifth culture medium is 0.5-10 μM; preferably 0.5 μM-5 μM; more preferably 1 μM.

[0150] In some embodiments, the basal medium of the fourth culture medium comprises at least one of IMDM (Iscove's Modified Dulbecco's Medium), Eagle's Basal Medium (BME), MEM, DMEM, Ham's F-12, RPMI 1640, Fischer's, and DMEM / F12. In a preferred embodiment, the basal medium of the fourth culture medium comprises IMDM (Iscove's Modified Dulbecco's Medium).

[0151] In some embodiments, the basal medium of the fourth culture medium contains at least one, at least two, at least three, at least four, or at least five of the following: non-essential amino acids, glutamine, vitamin C, B27 medium supplement, and N-acetyl-L-cysteine ​​(NAC). In a preferred embodiment, the basal medium of the fourth culture medium contains non-essential amino acids, glutamine, vitamin C, B27 medium supplement, and N-acetyl-L-cysteine.

[0152] In some implementations, the B27 medium supplement is a vitamin A-free B27 medium supplement.

[0153] In some embodiments, the concentration of non-essential amino acids in the fourth culture medium is 0.2-5% (v / v); preferably 0.5-2% (v / v), more preferably 1% (v / v).

[0154] In some embodiments, the concentration of glutamine in the fourth culture medium is 0.2-5% (v / v); preferably 0.5-2% (v / v), more preferably 1% (v / v).

[0155] In some embodiments, the concentration of vitamin C in the fourth culture medium is 10-100 μg / mL; preferably 20-50 μg / mL, more preferably 50 μg / mL.

[0156] In some embodiments, the concentration of the B27 medium additive in the fourth medium is 1-10% (v / v), preferably 2% (v / v).

[0157] In some embodiments, the concentration of N-acetyl-L-cysteine ​​in the fourth culture medium is 1-100 μM; preferably 10-50 μM, more preferably 30 μM.

[0158] In some embodiments, the basal medium of the fifth culture medium comprises at least one of IMDM (Iscove's Modified Dulbecco's Medium), Eagle's Basal Medium (BME), MEM, DMEM, Ham's F-12, RPMI 1640, Fischer's, and DMEM / F12. In a preferred embodiment, the basal medium of the fifth culture medium comprises IMDM (Iscove's Modified Dulbecco's Medium).

[0159] In some embodiments, the basal medium of the fifth culture medium contains at least one, at least two, at least three, at least four, or at least five of the following: non-essential amino acids, glutamine, vitamin C, B27 medium supplement, and N-acetyl-L-cysteine ​​(NAC). In a preferred embodiment, the basal medium of the fifth culture medium contains non-essential amino acids, glutamine, vitamin C, B27 medium supplement, and N-acetyl-L-cysteine ​​(NAC).

[0160] In some implementations, the B27 medium supplement is a vitamin A-free B27 medium supplement.

[0161] In some embodiments, the concentration of non-essential amino acids in the fifth culture medium is 0.2-5% (v / v); preferably 0.5-2% (v / v), more preferably 1% (v / v).

[0162] In some embodiments, the concentration of glutamine in the fifth culture medium is 0.2-5% (v / v); preferably 0.5-2% (v / v), more preferably 1% (v / v).

[0163] In some embodiments, the concentration of vitamin C in the fifth culture medium is 10-100 μg / mL; preferably 20-50 μg / mL, more preferably 50 μg / mL.

[0164] In some embodiments, the concentration of the B27 medium additive in the fifth medium is 1-10% (v / v), preferably 2% (v / v).

[0165] In some embodiments, the concentration of N-acetyl-L-cysteine ​​in the fifth culture medium is 1-100 μM; preferably 10-50 μM, more preferably 30 μM.

[0166] The fourth and fifth culture media, as defined above, are used to induce the differentiation of hematopoietic endothelial cells into NK cells and to activate (mature and / or expand) NK cells.

[0167] In some implementations, the kit for inducing stem cell differentiation into natural killer cells also includes a third culture medium, which is a basal culture medium containing at least one, at least two, or at least three of the following: BMP signaling pathway activator, growth factor, and TGFβ / ALK inhibitor.

[0168] In some embodiments, the BMP signaling pathway activator of the third culture medium comprises at least one selected from BMP2, BMP4, SB4, SJ000291942, SJ000063181, SJ000370178, isoglycyrrhizin, geraniol, apigenin, and chickpea sprout extract. In a preferred embodiment, the BMP signaling pathway activator of the third culture medium comprises BMP4.

[0169] In some embodiments, the growth factors in the third culture medium include at least one selected from epidermal growth factor (EGF), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), hepatocyte growth factor (HGF), insulin-like growth factor-I (IGF-1), IGF-II, leukemia inhibitory factor (LIF), nerve growth factor (NGF), oncogene M (OSM), platelet-derived endothelial growth factor (PDECGF), transforming growth factor-α (TGF-α), and vascular endothelial growth factor (VEGF). In a preferred embodiment, the growth factors in the third culture medium include at least one selected from fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF). In a more preferred embodiment, the growth factors in the third culture medium include both fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF).

[0170] In some embodiments, the vascular endothelial growth factor comprises at least one of VEGF-A, VEGF-165, VEGF-183, VEGF-110, VEGF-121, VEGF-B, VEGF-C, VEGF-D, and VEGF-E. In a preferred embodiment, the vascular endothelial growth factor comprises VEGF-165.

[0171] In some embodiments, the fibroblast growth factor comprises at least one of acidic fibroblast growth factor and basic fibroblast growth factor. In a preferred embodiment, the fibroblast growth factor comprises basic fibroblast growth factor.

[0172] In some embodiments, the TGFβ / ALK inhibitor of the third culture medium comprises at least one of SB431542, SB-505, A-83-01, GW6604, IN-1130, Ki26894, LY2157299, LY364947 (HTS-466284), LY550410, LY573636, LY580276, NPC-30345, SB-505124, SD-093, Sm16, SM305, SX-007, Antp-Sm2A, and LY2109761. In a preferred embodiment, the TGFβ / ALK inhibitor of the third culture medium comprises SB431542.

[0173] In some embodiments, the concentration of the BMP signaling pathway activator in the third culture medium is 1-50 ng / mL; preferably 1-10 ng / mL; more preferably 5 ng / mL.

[0174] In some embodiments, the concentration of vascular endothelial growth factor in the third culture medium is 1-100 ng / mL; preferably 20-50 ng / mL; more preferably 50 ng / mL.

[0175] In some embodiments, the concentration of fibroblast growth factor in the third culture medium is 1-100 ng / mL; preferably 5-50 ng / mL; more preferably 50 ng / mL.

[0176] In some embodiments, the concentration of the TGFβ / ALK inhibitor in the third culture medium is 1-50 μM; preferably 5-20 μM; more preferably 10 μM.

[0177] In some embodiments, the basal medium of the third culture medium comprises at least one of IMDM (Iscove's Modified Dulbecco's Medium), Eagle's Basal Medium (BME), MEM, DMEM, Ham's F-12, RPMI 1640, Fischer's, and DMEM / F12. In a preferred embodiment, the basal medium of the third culture medium comprises RPMI 1640.

[0178] In some embodiments, the basal medium of the third culture medium contains at least one, at least two, at least three, or at least four of the following: non-essential amino acids, glutamine, vitamin C, and B27 culture medium supplement. In a preferred embodiment, the basal medium of the third culture medium contains non-essential amino acids, glutamine, vitamin C, and B27 culture medium supplement.

[0179] In some implementations, the B27 medium supplement is a vitamin A-free B27 medium supplement.

[0180] In some embodiments, the concentration of non-essential amino acids in the third culture medium is 0.2-5% (v / v); preferably 0.5-2% (v / v), more preferably 1% (v / v).

[0181] In some embodiments, the concentration of glutamine in the third culture medium is 0.2-5% (v / v); preferably 0.5-2% (v / v), more preferably 1% (v / v).

[0182] In some embodiments, the concentration of vitamin C in the third culture medium is 10-100 μg / mL; preferably 20-50 μg / mL, more preferably 50 μg / mL.

[0183] In some embodiments, the concentration of the B27 medium additive in the third medium is 1%-10% (v / v), preferably 2% (v / v).

[0184] The third culture medium, as defined above, is used to induce mesodermal cells to differentiate into hematopoietic endothelial cells.

[0185] In some implementations, the kit for inducing stem cell differentiation into natural killer cells also includes a first culture medium, which is a basal culture medium containing ROCK inhibitors.

[0186] In some embodiments, the ROCK inhibitor comprises at least one of Blebbistatin, HA-100, Y-27632, HA-1077, KD-025, Y-33075, and Narciclasine. In a preferred embodiment, the ROCK inhibitor comprises Y-27632.

[0187] In some embodiments, the concentration of the ROCK inhibitor in the first culture medium is 1-50 μM; preferably 5 μM-20 μM; more preferably 10 μM.

[0188] In some embodiments, the basal medium of the first culture medium comprises at least one selected from E8 medium, mTESR medium, StemFit Basic O3, StemFit Basic O4, NutriStem hPSC XF medium, StemMACS iPS Brew medium, Stem-Partner ACF medium, TeSR-AOF medium, and TeSR2 medium. In a preferred embodiment, the basal medium of the first culture medium comprises StemFit Basic O3.

[0189] The first culture medium, as defined above, is used to obtain embryoids.

[0190] In some implementations, the stem cells are stem cells with multi-directional differentiation potential.

[0191] In some embodiments, the stem cells with multipotent differentiation potential include at least one of embryonic stem cells, parthenogenetic stem cells, induced pluripotent stem cells, mesenchymal stem cells, adipose stem cells, and umbilical cord blood stem cells. In a preferred embodiment, the stem cells with multipotent differentiation potential include induced pluripotent stem cells.

[0192] In some implementations, the stem cells are derived from mammals; preferably, they are derived from primates; more preferably, they are derived from humans.

[0193] In some implementations, the stem cells are commercially available human embryonic stem cells (hESCs) (e.g., H1, H9) and / or human induced pluripotent stem cells (hiPSCs) (e.g., WC50, IMR90).

[0194] In some implementations, embryonic stem cells are stem cells isolated or obtained from human embryos that have not undergone in vivo development and are within 14 days of fertilization.

[0195] In one specific embodiment, the kit for inducing stem cell differentiation into natural killer cells comprises a first culture medium, a second culture medium, a third culture medium, a fourth culture medium, and a fifth culture medium; the first culture medium is StemFit Basic 03 medium containing 10 μM Y276322HCl; the second culture medium is RPMI 1640 medium containing 1% (v / v) non-essential amino acids, 1% (v / v) glutamine, 50 μg / mL vitamin C, 2% (v / v) vitamin A-free B27 medium supplement, 20 ng / mL BMP4, and 3 μM CHIR-99021; the third culture medium is RPMI 1640 medium containing 1% (v / v) non-essential amino acids, 1% (v / v) glutamine, 50 μg / mL vitamin C, 2% (v / v) vitamin A-free B27 medium supplement, 5 ng / mL BMP4, 50 ng / mL VEGF-165, 50 ng / mL bFGF, and 10 μM The first medium was RPMI 1640 medium containing SB431542; the second medium was IMDM medium containing 1% (v / v) non-essential amino acids, 1% (v / v) glutamine, 50 μg / mL vitamin C, 2% (v / v) vitamin A-free B27 medium supplement, 30 μM NAC, 5 ng / mL BMP4, 10 ng / mL VEGF-165, 20 ng / mL SCF, 5 ng / mL IL3, 10 ng / mL IL15, 20 ng / mL IL7, and 10 ng / mL FLT3L; the third medium was IMDM medium containing 1% (v / v) non-essential amino acids, 1% (v / v) glutamine, 50 μg / mL vitamin C, 2% (v / v) vitamin A-free B27 medium supplement, 30 μM NAC, 5 ng / mL BMP4, 10 ng / mL VEGF, 20 ng / mL SCF, and 10 ng / mL FLT3L. IMDM medium containing IL15, 20 ng / mL IL7, 10 ng / mL FLT3L, 1 μM UM171, and 1 μM SR1.

[0196] In one specific embodiment, the kit for inducing stem cell differentiation into natural killer cells comprises a first culture medium, a second culture medium, a third culture medium, a fourth culture medium, and a fifth culture medium; the first culture medium is StemFit Basic 03 medium containing 10 μM Y27632 or Y27632 2HCl; the second culture medium is RPMI 1640 medium containing 1% non-essential amino acids, 1% glutamine, 50 μg / mL vitamin C, 2% (v / v) vitamin A-free B27 medium supplement, 20 ng / mL BMP4, and 3 μM CHIR-99021; the third culture medium is RPMI 1640 medium containing 1% non-essential amino acids, 1% glutamine, 50 μg / mL vitamin C, 2% (v / v) vitamin A-free B27 medium supplement, 5 ng / mL BMP4, 50 ng / mL VEGF, 50 ng / mL bFGF, and 10 μM The first medium was RPMI 1640 medium containing SB431542; the second medium was IMDM medium containing 1% non-essential amino acids, 1% glutamine, 50 μg / mL vitamin C, 2% (v / v) vitamin A-free B27 medium supplement, 5 ng / mL BMP4, 10 ng / mL VEGF, 20 ng / mL SCF, 5 ng / mL IL3, 10 ng / mL IL15, 20 ng / mL IL7, and 10 ng / mL FLT3L; the third medium was IMDM medium containing 1% non-essential amino acids, 1% glutamine, 50 μg / mL vitamin C, 2% (v / v) vitamin A-free B27 medium supplement, 5 ng / mL BMP4, 10 ng / mL VEGF, 20 ng / mL SCF, 10 ng / mL IL15, 20 ng / mL IL7, 10 ng / mL FLT3L, 1 μM UM171, 1 μM SR1, and 30 μM... IMDM medium for NAC.

[0197] In some implementations, NK cells are prepared by inducing stem cells to differentiate into natural killer (NK) cells, the method including the step of using a kit for inducing stem cells to differentiate into natural killer cells as defined above.

[0198] In some embodiments, the method includes: step (1) culturing stem cells to obtain embryoid bodies; step (2) culturing the embryoid bodies for mesodermal differentiation to obtain mesodermal cells; step (3) culturing the mesodermal cells for hematopoietic endothelial differentiation to obtain hematopoietic endothelial cells; and step (4) culturing the hematopoietic endothelial cells in the fourth and fifth culture media of the kit for inducing stem cell differentiation into natural killer cells as defined above to obtain NK cells.

[0199] In some implementations, the culture in step (1) is carried out in the first culture medium of a kit for inducing stem cell differentiation into natural killer cells as defined above.

[0200] In some implementations, the culture in step (2) is carried out in a second culture medium of a kit for inducing stem cell differentiation into natural killer cells as defined above.

[0201] In some implementations, the culture in step (3) is carried out in a third culture medium of a kit for inducing stem cell differentiation into natural killer cells as defined above.

[0202] In some implementations, the culture time in step (1) is 12-36 h; preferably 20-28 h; more preferably 20-24 h.

[0203] In some implementations, the culture time in step (2) is 24-72h; preferably 36-60h; more preferably 42-48h.

[0204] In some implementations, the culture time in step (3) is 48-144h; preferably 72-120h; more preferably 90-96h.

[0205] In some implementations, step (4) involves culturing the hematopoietic endothelial cells in the fourth and fifth culture media of the kit for inducing stem cell differentiation into natural killer cells as defined above.

[0206] In some embodiments, the culture time of hematopoietic endothelial cells in the fourth culture medium is 96-192 h; preferably 120-192 h; more preferably 144-168 h.

[0207] In some embodiments, the culture time of hematopoietic endothelial cells in the fifth culture medium is 144-432 h; preferably 216-360 h; more preferably 312-336 h.

[0208] In some implementations, the culture medium for hematopoietic endothelial cells is changed every 5-7 days in the fifth culture medium.

[0209] In some implementations, the above culture conditions are 35-38°C and 4-6% CO2.

[0210] In some implementations, the culture in steps (1), (2), and (3) is a suspension culture.

[0211] In some implementations, the cultivation in steps (1), (2), and (3) is carried out in a low-attachment plate.

[0212] In some implementations, the culture in step (4) is adherent culture.

[0213] In some implementations, the culture in step (4) is carried out in a normal adherent plate.

[0214] In some implementations, the stem cells are stem cells with multi-directional differentiation potential.

[0215] In some embodiments, the stem cells with multipotent differentiation potential include at least one of embryonic stem cells, parthenogenetic stem cells, induced pluripotent stem cells, mesenchymal stem cells, adipose stem cells, and umbilical cord blood stem cells. In a preferred embodiment, the stem cells with multipotent differentiation potential include induced pluripotent stem cells.

[0216] In some implementations, the stem cells are derived from mammals; preferably, they are derived from primates; more preferably, they are derived from humans.

[0217] In some implementations, the stem cells are commercially available human embryonic stem cells (hESCs) (e.g., H1, H9) and / or human induced pluripotent stem cells (hiPSCs) (e.g., WC50, IMR90).

[0218] In some implementations, human embryonic stem cells are stem cells isolated or obtained from human embryos that have not undergone in vivo development and are within 14 days of fertilization.

[0219] In some implementations, the drug combination also includes other chemotherapeutic agents. These other chemotherapeutic agents include, but are not limited to, anticancer agents, metabolic antagonists, DNA demethylators, plant-derived antitumor agents, alkylating agents, antimetabolites, anticancer antibiotics, topoisomerase inhibitors, mitosis inhibitors, differentiation agents, and hormone therapy agents. For example, antitumor agents (such as actinomycin D) D) Vincristine, alkylating agents, platinum compounds, antimetabolites, and nucleoside analogs such as methotrexate and purine and pyrimidine analogs; anti-hypertrophic agents (e.g., vincristine, doxorubicin, mitoxantrone, camptothecin, cisplatin, bleomycin, cyclophosphamide, methotrexate, and streptozotocin); antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, and 5-fluorouracil dacarbazine); alkylating agents (e.g., mechlorethamine, thiotepa). (thiotepa), chlorambucil, lactamase (CC-1065), melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C and cis-dichlorodiamine cycloplatin(II) (DDP), cisplatin), anthracyclines (e.g., daunomycin (formerly known as daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly known as actinomycin), bleomycin, mithramycin and antramycin (AMC)), antimitotic agents (e.g., vincristine, vinblastine, paclitaxel and levodopa). In some implementations, the other chemotherapeutic agents are selected from anticancer agents, metabolic antagonists, DNA demethylators, plant-derived antitumor agents, alkylating agents, antimetabolites, anticancer antibiotics, topoisomerase inhibitors, mitotic inhibitors, differentiation agents, and hormone therapy agents. These other chemotherapeutic agents may further include, but are not limited to: carmustine, lomustine, vorinostat, mebendazole, imatinib mesylate, AG119, or any therapeutic derivative thereof.

[0220] In some implementations, the other chemotherapeutic agents are selected from carmustine, lomustine, vorinostat, mebendazole, imatinib mesylate, AG119, or any therapeutic derivative thereof.

[0221] In some implementations, the active ingredient does not include trimethoprim.

[0222] In some implementations, the active ingredient does not contain immune checkpoint inhibitors.

[0223] In some implementations, the chemotherapy agent is only temozolomide.

[0224] In some implementations, the active ingredient consists of natural killer cells and chemotherapeutic agents.

[0225] In some implementations, the active ingredient consists of natural killer cells and temozolomide.

[0226] In some embodiments, the active ingredients are NK-92 cells and temozolomide, NK cells derived from peripheral blood or umbilical cord blood and temozolomide, NK cells derived from induced pluripotent stem cells (iPSCs) and temozolomide, or NK cells derived from embryonic stem cells and temozolomide.

[0227] In some implementations, the active ingredient consists of NK cells derived from induced pluripotent stem cells and temozolomide.

[0228] In some implementations, the active ingredient consists of unmodified NK cells derived from induced pluripotent stem cells and temozolomide.

[0229] In some implementations, the active ingredient consists of NK cells derived from induced pluripotent stem cells and temozolomide, wherein the NK cells derived from induced pluripotent stem cells are prepared using a kit for differentiating induced stem cells into natural killer cells as defined above.

[0230] In some embodiments, the active ingredient consists of NK cells derived from induced pluripotent stem cells and temozolomide, wherein the NK cells derived from induced pluripotent stem cells are prepared by a method for differentiating induced stem cells into natural killer cells as defined above.

[0231] In some embodiments, the active ingredient consists of unmodified NK cells derived from induced pluripotent stem cells and temozolomide, the NK cells being prepared using a kit for differentiating induced stem cells into natural killer cells as defined above.

[0232] In some embodiments, the active ingredient consists of unmodified NK cells derived from induced pluripotent stem cells and temozolomide, wherein the NK cells derived from induced pluripotent stem cells are prepared by a method for differentiating induced stem cells into natural killer cells as defined above.

[0233] In some embodiments, the active ingredient in the drug combination, natural killer cells (NK cells), is prepared by a kit for inducing stem cell differentiation into natural killer cells as defined above and / or by a method for inducing stem cell differentiation into natural killer cells as defined above.

[0234] In some implementations, CD45 in the NK cells + CD56 + The cell content is 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, or 96% or more; preferably 96% or more.

[0235] In some implementations, CD45 in the NK cells + CD3 - The cell content is 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, or 98% or more; preferably 98% or more.

[0236] In some implementations, CD56 in the NK cells + CD3 - The cell content is 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, or 97% or more; preferably 97% or more.

[0237] In some implementations, CD56 in the NK cells + CD94 + The cell content is 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 51% or more, 52% or more, or 53% or more; preferably 53% or more.

[0238] In some implementations, CD45 in the NK cells + CD94 + The cell content is 30% or more, 35% or more, 40% or more, 45% or more, 46% or more, or 47% or more; preferably 47% or more.

[0239] In some implementations, CD3 in the NK cells - CD16 - The cell content is 90% or more, 95% or more, or 96% or more; preferably 96% or more.

[0240] In some implementations, CD3 in the NK cells - CD45 + CD56+ The cell content is 80% or more, 85% or more, or 90% or more; preferably 90% or more.

[0241] In some implementations, CD3 in the NK cells - Nkp46 + The cell content is 60% or more, 65% or more, or 70% or more; preferably 70% or more.

[0242] In some implementations, CD3 in the NK cells - CD56 + Nkp44 + The cell content is 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, or 97% or more; preferably 93% or more.

[0243] In some implementations, CD3 in the NK cells - CD56 + Nkp30 + The cell content is 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, or 97% or more; preferably 85% or more.

[0244] In some implementations, CD3 in the NK cells - CD56 + Nkp44 + Nkp30 + The cell content is 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, or 97% or more; preferably 85% or more.

[0245] In some implementations, the NK cells comprise a population of NK cells.

[0246] In some embodiments, the active ingredients in the drug combination may be used simultaneously or separately (e.g., sequentially). In some embodiments, the active ingredients in the drug combination may be mixed or exist independently.

[0247] In some implementations, the drugs are combined as a kit.

[0248] In some embodiments, the kit contains natural killer cells and chemotherapeutic agents as defined above, or a combination of drugs as defined above, along with instructions for use. The kit may also contain a suitable container. In some embodiments, the kit further includes an administration (or delivery) device. Kits generally include a label indicating the intended use and / or method of use of the kit contents. The term "label" includes any written or recorded material provided on or with the kit or otherwise accompanied by the kit.

[0249] In some embodiments, the active ingredient in the drug combination, natural killer (NK) cells, is contained in the drug composition. In some embodiments, the drug composition also contains a pharmaceutically acceptable carrier.

[0250] In some embodiments, the pharmaceutical composition is a liquid formulation. In some embodiments, the pharmaceutical composition is an intravenous injection reagent. In some embodiments, the pharmaceutically acceptable carrier includes, but is not limited to, saline, buffer solution, glucose, water, DMSO, and combinations thereof.

[0251] In some embodiments, the concentration of NK cells in the pharmaceutical composition is 1 × 10⁻⁶. 3 -1×10 7 Cells / μL; preferably 1×10 4 -1×10 6 Cells / μL; more preferably 1×10 5 -9.9×10 5 per μL.

[0252] Use of drug combinations in the preparation of reagent kits

[0253] In a first aspect, the present invention provides the use of natural killer (NK) cells and chemotherapeutic agents as defined above, or drug combinations as defined above, in the preparation of a kit for treating tumors. Specifically, the present invention provides the use of a drug combination in the preparation of a kit for treating tumors, wherein the drug combination comprises an active ingredient comprising natural killer (NK) cells and a chemotherapeutic agent, wherein the chemotherapeutic agent includes temozolomide (TMZ).

[0254] In some implementations, the tumor includes, but is not limited to, nervous system tumors, neuroendocrine tumors, hematologic tumors, ovarian cancer, prostate cancer, pancreatic cancer, osteosarcoma, thyroid cancer, soft tissue sarcoma, insulinoma, and testicular cancer.

[0255] In some embodiments, the nervous system tumors include brainstem gliomas (DIPG), supratentorial and infratentorial gliomas, astrocytomas, oligodendrogliomas, ependymomas, medulloblastomas, schwannomas, meningiomas, pituitary adenomas, neuroblastomas, spinal cord gliomas, spinal cord meningiomas, spinal cord metastatic tumors, peripheral schwannomas, neurofibromas, brain metastases, and neuroectodermal tumors.

[0256] In some embodiments, the hematologic malignancies include leukemia (e.g., acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML)), lymphoma (e.g., Hodgkin lymphoma (HL), non-Hodgkin lymphoma (NHL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma (MCL), Burkitt lymphoma (BL)), and bone marrow tumors (e.g., multiple myeloma (MM), myelodysplastic syndrome (MDS), myeloproliferative neoplasm (MPN), polycythemia vera (PV), essential thrombocythemia (ET), myelofibrosis (MF)).

[0257] In a preferred embodiment, the tumor is a nervous system tumor.

[0258] In some embodiments, the tumor includes low-immune-invasive tumors. In some embodiments, the tumor includes low-immune-invasive neurological tumors, neuroendocrine tumors, hematologic malignancies, ovarian cancer, prostate cancer, pancreatic cancer, osteosarcoma, thyroid cancer, soft tissue sarcoma, insulinoma, and testicular cancer. In some embodiments, low-immune-invasive neurological tumors include low-immune-invasive brainstem gliomas (DIPG), supratentorial and infratentorial gliomas, astrocytomas, oligodendrogliomas, ependymomas, medulloblastomas, schwannomas, meningiomas, pituitary adenomas, neuroblastomas, spinal cord gliomas, spinal cord meningiomas, spinal cord metastases, peripheral schwannomas, neurofibromas, brain metastases, and neuroectodermal tumors. In some implementations, low-immune-infiltrating hematologic malignancies include low-immune-infiltrating leukemias (e.g., acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML)), low-immune-infiltrating lymphomas (e.g., Hodgkin lymphoma (HL), non-Hodgkin lymphoma (NHL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma (MCL), Burkitt lymphoma (BL)), and low-immune-infiltrating bone marrow tumors (e.g., multiple myeloma (MM), myelodysplastic syndrome (MDS), myeloproliferative neoplasm (MPN), polycythemia vera (PV), essential thrombocythemia (ET), myelofibrosis (MF)).

[0259] In a second aspect, the present invention provides the use of natural killer (NK) cells and chemotherapeutic agents as defined above, or drug combinations as defined above, in the preparation of a kit for treating low-immune-invasive tumors. Specifically, the present invention provides the use of a drug combination in the preparation of a kit for treating low-immune-invasive tumors, wherein the drug combination comprises an active ingredient comprising natural killer (NK) cells and a chemotherapeutic agent, wherein the chemotherapeutic agent includes temozolomide (TMZ).

[0260] As used herein, the term "treatment" means improving or reversing at least one measurable physical parameter associated with cancer (e.g., a low-immune-invasive tumor), which need not be identifiable in the subject, but may be identifiable in the subject. The term "treatment" can also mean causing regression, preventing progression, or at least slowing the progression of a disease, symptom, or condition. In one particular embodiment, "treatment" means reducing or preventing the development or onset of one or more symptoms (such as a tumor or, more preferably, cancer, e.g., a low-immune-invasive tumor) associated with a disease, symptom, or condition, or reducing its duration. In one particular embodiment, "treatment" means preventing the recurrence of a disease, symptom, or condition. In one particular embodiment, "treatment" means increasing the survival of a subject suffering from a disease, symptom, or condition. In one particular embodiment, "treatment" means eliminating the disease, symptom, or condition in a subject.

[0261] In some implementations, the low-immune-invasive tumors include, but are not limited to, low-immune-invasive nervous system tumors, neuroendocrine tumors, non-small cell lung cancer, small cell lung cancer, colorectal cancer, melanoma, breast cancer, esophageal cancer, gastric cancer, bladder cancer, endometrial cancer, head and neck cancer, cervical cancer, liver cancer, and kidney cancer.

[0262] In some embodiments, the low-immune-infiltrating nervous system tumors include low-immune-infiltrating brainstem gliomas (DIPG), supratentorial and infratentorial gliomas, astrocytomas, oligodendrogliomas, ependymomas, medulloblastomas, schwannomas, meningiomas, pituitary adenomas, neuroblastomas, spinal cord gliomas, spinal cord meningiomas, spinal cord metastases, peripheral schwannomas, neurofibromas, brain metastases, and neuroectodermal tumors.

[0263] In a preferred embodiment, the low-immune-invasive tumor is low-immune-invasive cervical cancer.

[0264] In a preferred embodiment, the low-immune-infiltrating tumor is a low-immune-infiltrating nervous system tumor.

[0265] In some embodiments, the NK cells in the first and second aspects are unmodified NK cells or modified NK cells. In a preferred embodiment, the NK cells in the first and second aspects are unmodified NK cells.

[0266] In some embodiments, the NK cells of the first and second aspects include, but are not limited to, NK-92 cells, NK cells derived from peripheral blood or umbilical cord blood, and NK cells derived from induced pluripotent stem cells (iPSCs) or embryonic stem cells. In a preferred embodiment, the NK cells of the first and second aspects are NK cells derived from induced pluripotent stem cells (iPSCs).

[0267] In some implementations, the NK cells in the first and second aspects are unmodified NK cells derived from induced pluripotent stem cells.

[0268] In some implementations, the NK cells of the first and second aspects are prepared using a kit for inducing stem cell differentiation into natural killer cells, said kit being as defined herein.

[0269] In some embodiments, the NK cells of the first and second aspects are prepared using a kit for inducing stem cell differentiation into natural killer cells, the kit comprising a second culture medium, a fourth culture medium, and a fifth culture medium; wherein the second culture medium is a basal culture medium containing a BMP signaling pathway activator; the fourth culture medium is a basal culture medium containing at least one, at least two, at least three, or at least four of the following: BMP signaling pathway activator, vascular endothelial growth factor, colony-stimulating factor, and interleukin; and the fifth culture medium is a basal culture medium containing at least one, at least two, at least three, at least four, at least five, or at least six of the following: BMP signaling pathway activator, vascular endothelial growth factor, colony-stimulating factor, interleukin, stem cell agonist, and stem cell expansion agent.

[0270] In some embodiments, the kit for inducing stem cell differentiation into natural killer cells further comprises a third culture medium, which is a basal culture medium containing at least one, at least two, or at least three of the following: BMP signaling pathway activator, growth factor, and TGFβ / ALK inhibitor.

[0271] In some embodiments, the kit for inducing stem cell differentiation into natural killer cells further comprises a first culture medium, which is a basal culture medium containing a ROCK inhibitor.

[0272] In some implementations, the NK cells of the first and second aspects are prepared by a method of inducing stem cells to differentiate into natural killer (NK) cells, the method of inducing stem cells to differentiate into natural killer (NK) cells as defined herein.

[0273] In some implementations, the NK cells of the first and second aspects are prepared by a method of inducing stem cells to differentiate into natural killer (NK) cells, said method including the step of using a kit for inducing stem cells to differentiate into natural killer cells as defined above.

[0274] In some embodiments, the method includes: step (1) culturing stem cells to obtain embryoid bodies; step (2) culturing the embryoid bodies for mesodermal differentiation to obtain mesodermal cells; step (3) culturing the mesodermal cells for hematopoietic endothelial differentiation to obtain hematopoietic endothelial cells; and step (4) culturing the hematopoietic endothelial cells in the fourth and fifth culture media of the kit for inducing stem cell differentiation into natural killer cells as defined above to obtain NK cells. In a preferred embodiment, the culture in step (1) is performed in the first culture media of the kit for inducing stem cell differentiation into natural killer cells as defined above. In a preferred embodiment, the culture in step (2) is performed in the second culture media of the kit for inducing stem cell differentiation into natural killer cells as defined above. In a preferred embodiment, the culture in step (3) is performed in the third culture media of the kit for inducing stem cell differentiation into natural killer cells as defined above. In a preferred embodiment, the culture time in step (1) is 12-36 hours. In a preferred embodiment, the culture time in step (2) is 24-72 hours. In a preferred embodiment, the culture time in step (3) is 48-144 hours. In a preferred embodiment, the step (4) of culturing the hematopoietic endothelial cells in the fourth and fifth culture media of the kit for inducing stem cell differentiation into natural killer cells as defined above means culturing the hematopoietic endothelial cells sequentially in the fourth and fifth culture media of the kit for inducing stem cell differentiation into natural killer cells as defined above. In a preferred embodiment, the culture time of the hematopoietic endothelial cells in the fourth culture medium is 96-192 hours. In a preferred embodiment, the culture time of the hematopoietic endothelial cells in the fifth culture medium is 144-432 hours.

[0275] In some embodiments, the drug combination further comprises other chemotherapeutic agents, including but not limited to anticancer agents, metabolic antagonists, DNA demethylators, plant-derived antitumor agents, alkylating agents, antimetabolites, anticancer antibiotics, topoisomerase inhibitors, mitotic inhibitors, differentiation agents, and hormone therapy agents. For example, antitumor agents (e.g., actinomycin D, vincristine, vinblastine, alkylating agents, platinum compounds, antimetabolites, and nucleoside analogs such as methotrexate and purine and pyrimidine analogs), anti-hypertrophic agents (e.g., vincristine, doxorubicin, mitoxantrone, camptothecin, cisplatin, bleomycin, cyclophosphamide, methotrexate, and streptozotocin), antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, and 5-fluorouracil dacarbazine), alkylating agents (e.g., ... Examples of chemotherapeutic agents include nitrogen mustard, thiotepa, chlorambucil, lactamazole (CC-1065), mefenamic acid, carmustine, lomustine, cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C and cis-dichlorodiamine cycloplatin (II) (DDP), cisplatin), anthracyclines (e.g., daunorubicin and doxorubicin), antibiotics (e.g., daunorubicin, bleomycin, sclerosomycin, and atrazodine), and antimitotic agents (e.g., vincristine, vinblastine, paclitaxel, and maytansine-like drugs). In some embodiments, other chemotherapeutic agents are selected from anticancer agents, metabolic antagonists, DNA demethylators, plant-derived antitumor agents, alkylating agents, antimetabolites, anticancer antibiotics, topoisomerase inhibitors, mitotic inhibitors, differentiation agents, and hormone therapy agents. These other chemotherapeutic agents may further include, but are not limited to: carmustine, lomustine, vorinostat, mebendazole, imatinib mesylate, AG119, or any therapeutic derivative thereof.

[0276] In some implementations, the other chemotherapeutic agents are selected from carmustine, lomustine, vorinostat, mebendazole, imatinib mesylate, AG119, or any therapeutic derivative thereof.

[0277] In one embodiment, the active ingredient does not contain trimethoprim. In another embodiment, the active ingredient does not contain an immune checkpoint inhibitor.

[0278] In some implementations, chemotherapeutic agents are administered before, after, or simultaneously with the administration of NK cells for the first and second aspects.

[0279] In some implementations, the chemotherapeutic agent is administered 8 to 72 hours before, 8 to 72 hours after, or both before and 8 to 72 hours after the administration of the first and second aspect of NK cells.

[0280] In some implementation schemes, a therapeutically effective amount of chemotherapeutic agents is administered before, after, or simultaneously with the administration of therapeutically effective amounts of first- and second-party NK cells.

[0281] In some implementations, a therapeutically effective dose of chemotherapeutic agent is administered 8 to 72 hours before, 8 to 72 hours after, or both before and 8 to 72 hours after the administration of therapeutically effective doses of first and second aspect NK cells.

[0282] The term "therapeutic effective dose" refers to the amount of an agent (e.g., a combination of drugs, reagents in a kit) sufficient to prevent or inhibit the occurrence of a disease or symptom and / or slow, reduce, or delay the development or severity of a disease or symptom. Therapeutic effective doses are influenced by factors including, but not limited to, the rate and severity of disease or symptom development, the subject's age, sex, weight, and physiological condition, the duration of treatment, and the specific route of administration. Therapeutic effective doses may be administered in one or more doses. Therapeutic effective doses can be achieved through continuous or intermittent administration.

[0283] According to a specific implementation scheme, a therapeutically effective dose may refer to a dose sufficient to achieve one, two, three, four, or more of the following effects: (i) reducing or improving the severity of the disease, condition, or illness to be treated, or related symptoms; (ii) reducing the duration of the disease, condition, or illness to be treated, or related symptoms; (iii) preventing the progression of the disease, condition, or illness to be treated, or related symptoms; (iv) causing the remission of the disease, condition, or illness to be treated, or related symptoms; and (v) preventing the development or onset of the disease, condition, or illness to be treated, or related symptoms. (vi) To prevent the recurrence of a disease, condition, or illness to be treated, or related symptoms; (vii) To reduce hospitalization of subjects suffering from a disease, condition, or illness to be treated, or related symptoms; (viii) To reduce the length of hospital stay of subjects suffering from a disease, condition, or illness to be treated, or related symptoms; (ix) To increase the survival of subjects suffering from a disease, condition, or illness to be treated, or related symptoms; (xi) To suppress or reduce a disease, condition, or illness to be treated, or related symptoms in subjects; and / or (xii) To enhance or improve the preventive or therapeutic effect of another therapy.

[0284] NK cells as defined above and / or drug combinations as defined above can be administered in any physiologically acceptable carrier. A cell population containing NK cells as defined above may comprise a purified cell population. Those skilled in the art can readily determine the cells in the cell population using various well-known methods, with a purity ranging from about 50% to about 55%, from about 55% to about 60%, from about 60% to about 65%, from about 65% to about 70%, from about 70% to about 75%, from about 75% to about 80%, from about 80% to about 85%, from about 85% to about 90%, from about 90% to about 95%, or from about 95% to about 100%. Those skilled in the art can readily adjust the dosage; for example, a decrease in purity may require an increase in dosage.

[0285] NK cells and / or combinations of drugs containing NK cells, as defined above, can be administered to subjects at a dose based on cells / kg (cells / kg) of subject weight. Generally, the cell dose is approximately 10... 4 To about 10 10 Within the range of cells / kg body weight, for example, approximately 10 5 To about 10 9 Approximately 10 5 To about 10 8 Approximately 10 5 To about 10 7 Or about 10 5 To about 10 6 The dosage depends on the method and site of application. Exemplary dosage ranges include, but are not limited to, 1 × 10⁻⁶. 4 -1×10 8 2×10 4 -1×10 8 3×10 4 -1×10 8 4×10 4 -1×10 8 5×10 4 -1×10 8 6×10 4 -1×10 8 7×10 4 -1×10 8 8×10 4 -1×10 8 9×10 4 -1×10 8 1×10 5 -1×10 8 1×10 5 -9×10 7 1×10 5 -8×10 7 1×105 -7×10 7 、1×10 5 -6×10 7 、1×10 5 -5×10 7 、1×10 5 -4×10 7 、1×10 5 -3×10 7 、1×10 5 -2×10 7 、1×10 5 -1×10 7 、1×10 5 -9×10 6 、1×10 5 -8×10 6 、1×10 5 -7×10 6 、1×10 5 -6×10 6 、1×10 5 -5×10 6 、1×10 5 -4×10 6 、1×10 5 -3×10 6 、1×10 5 -2×10 6 、1×10 5 -1×10 6 、2×10 5 -9×10 7 、2×10 5 -8×10 7 、2×10 5 -7×10 7 、2×10 5 -6×10 7 、2×10 5 -5×10 7 、2×10 5 -4×10 7 、2×10 5 -3×10 7 、2×10 5 -2×10 7 、2×10 5 -1×10 7 、2×10 5 -9×10 6 、2×10 5 -8×10 6 、2×10 5 -7×10 62×10 5 -6×10 6 2×10 5 -5×10 6 2×10 5 -4×10 6 2×10 5 -3×10 6 2×10 5 -2×10 6 2×10 5 -1×10 6 3×10 5 -3×10 6 Cells / kg, etc. Furthermore, the dosage can be adjusted to consider whether to administer a single dose or multiple doses. What is considered an effective dose can be accurately determined based on each subject's individual factors.

[0286] As used herein, the term "effect-to-target ratio (E:T)" refers to the ratio of effector cells to target cells. In some embodiments herein, the effect-to-target ratio refers to the ratio of NK cells used to tumor cells (e.g., low-immune-invasive tumors). Exemplary effect-to-target ratio ranges include, but are not limited to, 100:1, 90:1, 85:1, 80:1, 75:1, 70:1, 65:1, 60:1, 55:1, 50:1, 45:1, 40:1, 35:1, 30:1, 25:1, 20:1, 19:1, 18:1, 17:1, 16:1, 15:1, 14:1, 13:1, 12: 1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20. In some implementations, the effective-to-target ratio is 1:1, 1:4, 1:8. In some implementations, the effective-to-target ratio is 1:1.

[0287] TMZ and / or combinations of drugs containing TMZ, as defined above, may be administered to subjects at a dose of mg / kg of the subject's body weight. Exemplary doses include, but are not limited to, about 0.01, about 0.05, about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.8, about 1, about 2, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 150, about 200, about 250, about 300, about 350, about 400, about 450, about 500, about 550, about 600, about 650, about 700, about 750, about 800, about 850, about 900, or about 1000 mg / kg of subject.

[0288] It can be expressed in mg / m 2 The dose per body surface area of ​​the subject is administered to the subject as defined above for TMZ and / or combinations of drugs containing TMZ. Exemplary dosages include, but are not limited to, about 0.01, about 0.05, about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.8, about 1, about 2, about 5, about 10, about 15, about 20, about 25, about 30, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 150, about 200, about 250, about 300, about 350, about 400, about 450, about 500, about 550, about 600, about 650, about 700, about 750, about 800, about 850, about 900, or about 1000 mg / m². 2 Subjects.

[0289] The amount of TMZ defined above can be, for example, 0.001-5000μM, for example, 10-1000μM, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 650, 700, 750, 800, 850, 900, 950, 1000μM.

[0290] Methods of treating tumors

[0291] In one aspect, the present invention also provides a method for treating tumors in a subject in need, comprising the following steps:

[0292] Administering natural killer (NK) cells to subjects; and

[0293] The subjects were treated with chemotherapy drugs.

[0294] Among them, chemotherapy agents include temozolomide (TMZ).

[0295] In some implementations, chemotherapy is administered before, after, or concurrently with the administration of NK cells to the subject, or any combination thereof.

[0296] In some implementations, the tumor includes, but is not limited to, nervous system tumors, neuroendocrine tumors, hematologic tumors, ovarian cancer, prostate cancer, pancreatic cancer, osteosarcoma, thyroid cancer, soft tissue sarcoma, insulinoma, and testicular cancer.

[0297] In some embodiments, the nervous system tumors include brainstem gliomas (DIPG), supratentorial and infratentorial gliomas, astrocytomas, oligodendrogliomas, ependymomas, medulloblastomas, schwannomas, meningiomas, pituitary adenomas, neuroblastomas, spinal cord gliomas, spinal cord meningiomas, spinal cord metastatic tumors, peripheral schwannomas, neurofibromas, brain metastases, and neuroectodermal tumors.

[0298] In some embodiments, the hematologic malignancies include leukemia (e.g., acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML)), lymphoma (e.g., Hodgkin lymphoma (HL), non-Hodgkin lymphoma (NHL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma (MCL), Burkitt lymphoma (BL)), and bone marrow tumors (e.g., multiple myeloma (MM), myelodysplastic syndrome (MDS), myeloproliferative neoplasm (MPN), polycythemia vera (PV), essential thrombocythemia (ET), myelofibrosis (MF)).

[0299] In some implementations, the tumor is a nervous system tumor.

[0300] In some embodiments, the tumor includes low-immune-invasive tumors. In some embodiments, the tumor includes low-immune-invasive neurological tumors, neuroendocrine tumors, hematologic malignancies, ovarian cancer, prostate cancer, pancreatic cancer, osteosarcoma, thyroid cancer, soft tissue sarcoma, insulinoma, and testicular cancer. In some embodiments, low-immune-invasive neurological tumors include low-immune-invasive brainstem gliomas (DIPG), supratentorial and infratentorial gliomas, astrocytomas, oligodendrogliomas, ependymomas, medulloblastomas, schwannomas, meningiomas, pituitary adenomas, neuroblastomas, spinal cord gliomas, spinal cord meningiomas, spinal cord metastases, peripheral schwannomas, neurofibromas, brain metastases, and neuroectodermal tumors.

[0301] In some implementations, low-immune-infiltrating hematologic malignancies include low-immune-infiltrating leukemias (e.g., acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML)), low-immune-infiltrating lymphomas (e.g., Hodgkin lymphoma (HL), non-Hodgkin lymphoma (NHL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma (MCL), Burkitt lymphoma (BL)), and low-immune-infiltrating bone marrow tumors (e.g., multiple myeloma (MM), myelodysplastic syndrome (MDS), myeloproliferative neoplasm (MPN), polycythemia vera (PV), essential thrombocythemia (ET), myelofibrosis (MF)).

[0302] In some embodiments, the NK cells are unmodified NK cells or modified NK cells. In a preferred embodiment, the NK cells are unmodified NK cells.

[0303] In some implementations, NK cells include, but are not limited to, NK-92 cells, NK cells derived from peripheral blood or umbilical cord blood, and NK cells derived from induced pluripotent stem cells (iPSCs) or embryonic stem cells.

[0304] In some implementations, the embryonic stem cells are commercially available human embryonic stem cells (hESCs) (e.g., H1, H9).

[0305] In some implementations, embryonic stem cells are stem cells isolated or obtained from human embryos that have not undergone in vivo development and are within 14 days of fertilization.

[0306] In some implementations, induced pluripotent stem cells (iPSCs) are human induced pluripotent stem cells (hiPSCs) (e.g., WC50, IMR90).

[0307] In some implementations, the NK cells are NK cells derived from induced pluripotent stem cells (iPSCs).

[0308] In some implementations, the NK cells are unmodified NK cells derived from induced pluripotent stem cells.

[0309] In some embodiments, NK cells are prepared using a kit for inducing stem cell differentiation into natural killer cells, the kit comprising a second culture medium, a fourth culture medium, and a fifth culture medium; wherein the second culture medium is a basal culture medium containing a BMP signaling pathway activator; the fourth culture medium is a basal culture medium containing at least one, at least two, at least three, or at least four of the following: BMP signaling pathway activator, vascular endothelial growth factor, colony-stimulating factor, and interleukin; and the fifth culture medium is a basal culture medium containing at least one, at least two, at least three, at least four, at least five, or at least six of the following: BMP signaling pathway activator, vascular endothelial growth factor, colony-stimulating factor, interleukin, stem cell agonist, and stem cell expansion agent.

[0310] In some embodiments, the kit for inducing stem cell differentiation into natural killer cells further comprises a third culture medium, which is a basal culture medium containing at least one, at least two, or at least three of the following: BMP signaling pathway activator, growth factor, and TGFβ / ALK inhibitor.

[0311] In some embodiments, the kit for inducing stem cell differentiation into natural killer cells further comprises a first culture medium, which is a basal culture medium containing a ROCK inhibitor.

[0312] In some embodiments, the NK cells are prepared by a method for inducing stem cell differentiation into natural killer (NK) cells as defined above, the method including the step of using a kit for inducing stem cell differentiation into natural killer cells as defined above.

[0313] In some embodiments, the method for inducing stem cell differentiation into NK cells includes: step (1) culturing stem cells to obtain embryoid bodies; step (2) culturing the embryoid bodies for mesodermal differentiation to obtain mesodermal cells; step (3) culturing the mesodermal cells for hematopoietic endothelial differentiation to obtain hematopoietic endothelial cells; and step (4) culturing the hematopoietic endothelial cells in the fourth and fifth culture media of the kit for inducing stem cell differentiation into natural killer cells as defined above to obtain NK cells. In a preferred embodiment, the culture in step (1) is performed in the first culture media of the kit for inducing stem cell differentiation into natural killer cells as defined above. In a preferred embodiment, the culture in step (2) is performed in the second culture media of the kit for inducing stem cell differentiation into natural killer cells as defined above. In a preferred embodiment, the culture in step (3) is performed in the third culture media of the kit for inducing stem cell differentiation into natural killer cells as defined above. In a preferred embodiment, the culture time in step (1) is 12-36 hours. In a preferred embodiment, the culture time in step (2) is 24-72 hours. In a preferred embodiment, the culture time in step (3) is 48-144 hours. In a preferred embodiment, the step (4) of culturing the hematopoietic endothelial cells in the fourth and fifth culture media of the kit for inducing stem cell differentiation into natural killer cells as defined above means culturing the hematopoietic endothelial cells sequentially in the fourth and fifth culture media of the kit for inducing stem cell differentiation into natural killer cells as defined above. In a preferred embodiment, the culture time of the hematopoietic endothelial cells in the fourth culture medium is 96-192 hours. In a preferred embodiment, the culture time of the hematopoietic endothelial cells in the fifth culture medium is 144-432 hours.

[0314] In some implementations, the method of treating a tumor in a subject in need further includes administering other chemotherapeutic agents to the subject. Other chemotherapeutic agents include, but are not limited to, anticancer agents, metabolic antagonists, DNA demethylators, plant-derived antitumor agents, alkylating agents, antimetabolites, anticancer antibiotics, topoisomerase inhibitors, mitotic inhibitors, differentiation agents, and hormone therapy agents. Examples include antitumor agents (e.g., actinomycin D, vincristine, vinblastine, alkylating agents, platinum compounds, antimetabolites, and nucleoside analogs such as methotrexate and purine and pyrimidine analogs), anti-hypertrophic agents (e.g., vincristine, doxorubicin, mitoxantrone, camptothecin, cisplatin, bleomycin, cyclophosphamide, methotrexate, and streptozotocin), antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, and 5-fluorouracil dacarbazine), and alkylating agents (e.g., [examples not provided]). Examples of chemotherapeutic agents include nitrogen mustard, thiotepa, chlorambucil, lactamazole (CC-1065), mefenamic acid, carmustine, lomustine, cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C and cis-dichlorodiamine cycloplatin (II) (DDP), cisplatin), anthracyclines (e.g., daunorubicin and doxorubicin), antibiotics (e.g., daunorubicin, bleomycin, sclerosomycin, and atrazodine), and antimitotic agents (e.g., vincristine, vinblastine, paclitaxel, and maytansine-like drugs). In some embodiments, other chemotherapeutic agents are selected from anticancer agents, metabolic antagonists, DNA demethylators, plant-derived antitumor agents, alkylating agents, antimetabolites, anticancer antibiotics, topoisomerase inhibitors, mitotic inhibitors, differentiation agents, and hormone therapy agents. These other chemotherapeutic agents may further include, but are not limited to: carmustine, lomustine, vorinostat, mebendazole, imatinib mesylate, AG119, or any therapeutic derivative thereof.

[0315] In some implementations, the other chemotherapeutic agents are selected from carmustine, lomustine, vorinostat, mebendazole, imatinib mesylate, AG119, or any therapeutic derivative thereof.

[0316] In some implementations, other chemotherapeutic agents may be administered to the subject before, after, or concurrently with the administration of NK cells or temozolomide (TMZ), or any combination thereof.

[0317] In some implementations, the method of treating a tumor in a subject of need does not include administering trimethoprim to the subject. In some implementations, the method of treating a tumor in a subject of need does not include administering an immune checkpoint inhibitor to the subject.

[0318] In some implementations, the chemotherapeutic agent is temozolomide (TMZ).

[0319] In some implementations, the method for treating a tumor in a subject of need includes the following steps:

[0320] Administering natural killer (NK) cells to subjects; and

[0321] Treatment of a subject with a chemotherapy agent, wherein the chemotherapy agent is administered before, after, or concurrently with the administration of NK cells to the subject, or any combination thereof, wherein the chemotherapy agent is temozolomide.

[0322] In some implementations, the method for treating a tumor in a subject of need includes the following steps:

[0323] NK cells derived from induced pluripotent stem cells were administered to the subjects; and

[0324] The subject is treated with a chemotherapy agent, wherein the chemotherapy agent is administered before, after, or concurrently with the administration of NK cells derived from induced pluripotent stem cells to the subject, or any combination thereof, wherein the chemotherapy agent is temozolomide.

[0325] In some implementations, the method for treating a tumor in a subject of need includes the following steps:

[0326] Administering unmodified NK cells derived from induced pluripotent stem cells to subjects; and

[0327] The subject is treated with a chemotherapy agent, wherein the chemotherapy agent is administered before, after, or concurrently with the administration of unmodified NK cells derived from induced pluripotent stem cells, or any combination thereof, wherein the chemotherapy agent is temozolomide (TMZ).

[0328] In some implementations, the method for treating a tumor in a subject of need includes the following steps:

[0329] NK cells derived from induced pluripotent stem cells were administered to the subjects; and

[0330] The subject is treated with a chemotherapy agent, wherein the chemotherapy agent is administered before, after, or concurrently with the administration of NK cells derived from induced pluripotent stem cells, or any combination thereof, wherein the chemotherapy agent is temozolomide (TMZ), and wherein the NK cells derived from induced pluripotent stem cells are prepared using a kit for inducing stem cell differentiation into natural killer cells as defined herein.

[0331] In some implementations, a method for treating a tumor in a subject of need includes the following steps:

[0332] NK cells derived from induced pluripotent stem cells were administered to the subjects; and

[0333] The subject is treated with a chemotherapy agent, wherein the chemotherapy agent is administered before, after, or concurrently with the administration of NK cells derived from induced pluripotent stem cells, or any combination thereof, wherein the chemotherapy agent is temozolomide (TMZ), and wherein the NK cells derived from induced pluripotent stem cells are prepared by a method for inducing stem cell differentiation into natural killer cells as defined herein.

[0334] In some implementations, the chemotherapeutic agent is administered to the subject 8 to 72 hours before, 8 to 72 hours after, or both before and 8 to 72 hours after the administration of NK cells.

[0335] In some implementations, the chemotherapy agent is administered to the subject 8, 16, 24, 32, 40, 48, 56, 64, or 72 hours before administering NK cells, 8, 16, 24, 32, 40, 48, 56, 64, or 72 hours after administering NK cells, or both before and after administering NK cells.

[0336] In some implementations, temozolomide is administered to the subject 8, 16, 24, 32, 40, 48, 56, 64, or 72 hours before administering NK cells, 8, 16, 24, 32, 40, 48, 56, 64, or 72 hours after administering NK cells, or both before and after administering NK cells.

[0337] In some implementations, temozolomide is administered to the subject 8, 16, 24, 32, 40, 48, 56, 64, or 72 hours before administering NK cells derived from induced pluripotent stem cells, 8, 16, 24, 32, 40, 48, 56, 64, or 72 hours after administering NK cells derived from induced pluripotent stem cells, or 8, 16, 24, 32, 40, 48, 56, 64, or 72 hours before administering NK cells derived from induced pluripotent stem cells and 8, 16, 24, 32, 40, 48, 56, 64, or 72 hours after administering NK cells derived from induced pluripotent stem cells.

[0338] Treatment methods for low-immune-infiltrating tumors

[0339] In one aspect, the present invention also provides a method for treating a subject with a low immune-invasive tumor, comprising the following steps:

[0340] Administering natural killer (NK) cells to subjects; and

[0341] The subjects were treated with chemotherapy drugs.

[0342] Among them, chemotherapy agents include temozolomide (TMZ).

[0343] In some implementations, chemotherapy is administered before, after, or concurrently with the administration of NK cells to the subject, or any combination thereof.

[0344] In some implementations, the low-immune-invasive tumors include, but are not limited to, low-immune-invasive nervous system tumors, neuroendocrine tumors, non-small cell lung cancer, small cell lung cancer, colorectal cancer, melanoma, breast cancer, esophageal cancer, gastric cancer, bladder cancer, endometrial cancer, head and neck cancer, cervical cancer, liver cancer, and kidney cancer.

[0345] In some embodiments, the low-immune-infiltrating nervous system tumors include low-immune-infiltrating brainstem gliomas (DIPG), supratentorial and infratentorial gliomas, astrocytomas, oligodendrogliomas, ependymomas, medulloblastomas, schwannomas, meningiomas, pituitary adenomas, neuroblastomas, spinal cord gliomas, spinal cord meningiomas, spinal cord metastases, peripheral schwannomas, neurofibromas, brain metastases, and neuroectodermal tumors.

[0346] In a preferred embodiment, the low-immune-invasive tumor is low-immune-invasive cervical cancer.

[0347] In a preferred embodiment, the low-immune-infiltrating tumor is a low-immune-infiltrating nervous system tumor.

[0348] In some embodiments, the NK cells are unmodified NK cells or modified NK cells. In a preferred embodiment, the NK cells are unmodified NK cells.

[0349] In some implementations, NK cells include, but are not limited to, NK-92 cells, NK cells derived from peripheral blood or umbilical cord blood, and NK cells derived from induced pluripotent stem cells (iPSCs) or embryonic stem cells.

[0350] In some implementations, the embryonic stem cells are commercially available human embryonic stem cells (hESCs) (e.g., H1, H9).

[0351] In some implementations, embryonic stem cells are stem cells isolated or obtained from human embryos that have not undergone in vivo development and are within 14 days of fertilization.

[0352] In some implementations, induced pluripotent stem cells (iPSCs) are human induced pluripotent stem cells (hiPSCs) (e.g., WC50, IMR90).

[0353] In some implementations, the NK cells are NK cells derived from induced pluripotent stem cells (iPSCs).

[0354] In some implementations, the NK cells are unmodified NK cells derived from induced pluripotent stem cells.

[0355] In some embodiments, NK cells are prepared using a kit for inducing stem cell differentiation into natural killer cells, the kit comprising a second culture medium, a fourth culture medium, and a fifth culture medium; wherein the second culture medium is a basal culture medium containing a BMP signaling pathway activator; the fourth culture medium is a basal culture medium containing at least one, at least two, at least three, or at least four of the following: BMP signaling pathway activator, vascular endothelial growth factor, colony-stimulating factor, and interleukin; and the fifth culture medium is a basal culture medium containing at least one, at least two, at least three, at least four, at least five, or at least six of the following: BMP signaling pathway activator, vascular endothelial growth factor, colony-stimulating factor, interleukin, stem cell agonist, and stem cell expansion agent.

[0356] In some embodiments, the kit for inducing stem cell differentiation into natural killer cells further comprises a third culture medium, which is a basal culture medium containing at least one, at least two, or at least three of the following: BMP signaling pathway activator, growth factor, and TGFβ / ALK inhibitor.

[0357] In some embodiments, the kit for inducing stem cell differentiation into natural killer cells further comprises a first culture medium, which is a basal culture medium containing a ROCK inhibitor.

[0358] In some embodiments, the NK cells are prepared by inducing stem cells to differentiate into natural killer (NK) cells, the method including the step of using a kit for inducing stem cells to differentiate into natural killer cells as defined above.

[0359] In some embodiments, the method for inducing stem cell differentiation into NK cells includes: step (1) culturing stem cells to obtain embryoid bodies; step (2) culturing the embryoid bodies for mesodermal differentiation to obtain mesodermal cells; step (3) culturing the mesodermal cells for hematopoietic endothelial differentiation to obtain hematopoietic endothelial cells; and step (4) culturing the hematopoietic endothelial cells in the fourth and fifth culture media of the kit for inducing stem cell differentiation into natural killer cells as defined above to obtain NK cells. In a preferred embodiment, the culture in step (1) is performed in the first culture media of the kit for inducing stem cell differentiation into natural killer cells as defined above. In a preferred embodiment, the culture in step (2) is performed in the second culture media of the kit for inducing stem cell differentiation into natural killer cells as defined above. In a preferred embodiment, the culture in step (3) is performed in the third culture media of the kit for inducing stem cell differentiation into natural killer cells as defined above. In a preferred embodiment, the culture time in step (1) is 12-36 hours. In a preferred embodiment, the culture time in step (2) is 24-72 hours. In a preferred embodiment, the culture time in step (3) is 48-144 hours. In a preferred embodiment, the step (4) of culturing the hematopoietic endothelial cells in the fourth and fifth culture media of the kit for inducing stem cell differentiation into natural killer cells as defined above means culturing the hematopoietic endothelial cells sequentially in the fourth and fifth culture media of the kit for inducing stem cell differentiation into natural killer cells as defined above. In a preferred embodiment, the culture time of the hematopoietic endothelial cells in the fourth culture medium is 96-192 hours. In a preferred embodiment, the culture time of the hematopoietic endothelial cells in the fifth culture medium is 144-432 hours.

[0360] In some implementations, the method of treating a subject with a low-immune-invasive tumor further includes administering other chemotherapeutic agents to the subject. Other chemotherapeutic agents include, but are not limited to, anticancer agents, metabolic antagonists, DNA demethylators, plant-derived antitumor agents, alkylating agents, antimetabolites, anticancer antibiotics, topoisomerase inhibitors, mitotic inhibitors, differentiation agents, and hormone therapy agents. Examples include antitumor agents (e.g., actinomycin D, vincristine, vinblastine, alkylating agents, platinum compounds, antimetabolites, and nucleoside analogs such as methotrexate and purine and pyrimidine analogs), anti-hypertrophic agents (e.g., vincristine, doxorubicin, mitoxantrone, camptothecin, cisplatin, bleomycin, cyclophosphamide, methotrexate, and streptozotocin), antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, and 5-fluorouracil dacarbazine), and alkylating agents (e.g., [examples not provided]). Examples of chemotherapeutic agents include nitrogen mustard, thiotepa, chlorambucil, lactamazole (CC-1065), mefenamic acid, carmustine, lomustine, cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C and cis-dichlorodiamine cycloplatin (II) (DDP), cisplatin), anthracyclines (e.g., daunorubicin and doxorubicin), antibiotics (e.g., daunorubicin, bleomycin, sclerosomycin, and atrazodine), and antimitotic agents (e.g., vincristine, vinblastine, paclitaxel, and maytansine-like drugs). In some embodiments, other chemotherapeutic agents are selected from anticancer agents, metabolic antagonists, DNA demethylators, plant-derived antitumor agents, alkylating agents, antimetabolites, anticancer antibiotics, topoisomerase inhibitors, mitotic inhibitors, differentiation agents, and hormone therapy agents. These other chemotherapeutic agents may further include, but are not limited to: carmustine, lomustine, vorinostat, mebendazole, imatinib mesylate, AG119, or any therapeutic derivative thereof.

[0361] In some implementations, the other chemotherapeutic agents are selected from carmustine, lomustine, vorinostat, mebendazole, imatinib mesylate, AG119, or any therapeutic derivative thereof.

[0362] In some implementations, other chemotherapeutic agents may be administered to the subject before, after, or concurrently with the administration of NK cells or temozolomide (TMZ), or any combination thereof.

[0363] In some implementations, the method of treating a subject with low immune-invasive tumors does not include administering trimethoprim to the subject. In some implementations, the method of treating a subject with low immune-invasive tumors does not include administering an immune checkpoint inhibitor to the subject.

[0364] In some implementations, the chemotherapeutic agent is temozolomide (TMZ).

[0365] In some implementations, the method for treating a subject with a low immune-invasive tumor includes the following steps:

[0366] Administering natural killer (NK) cells to subjects; and

[0367] Treatment of a subject with a chemotherapy agent, wherein the chemotherapy agent is administered before, after, or concurrently with the administration of NK cells to the subject, or any combination thereof, wherein the chemotherapy agent is temozolomide.

[0368] In some implementations, the method for treating a subject with a low immune-invasive tumor includes the following steps:

[0369] NK cells derived from induced pluripotent stem cells were administered to the subjects; and

[0370] The subject is treated with a chemotherapy agent, wherein the chemotherapy agent is administered before, after, or concurrently with the administration of NK cells derived from induced pluripotent stem cells to the subject, or any combination thereof, wherein the chemotherapy agent is temozolomide.

[0371] In some implementations, the method for treating a subject with a low immune-invasive tumor includes the following steps:

[0372] Administering unmodified NK cells derived from induced pluripotent stem cells to subjects; and

[0373] The subject is treated with a chemotherapy agent, wherein the chemotherapy agent is administered before, after, or concurrently with the administration of unmodified NK cells derived from induced pluripotent stem cells, or any combination thereof, wherein the chemotherapy agent is temozolomide (TMZ).

[0374] In some implementations, the method for treating a subject with a low immune-invasive tumor includes the following steps:

[0375] NK cells derived from induced pluripotent stem cells were administered to the subjects; and

[0376] The subject is treated with a chemotherapy agent, wherein the chemotherapy agent is administered before, after, or concurrently with the administration of NK cells derived from induced pluripotent stem cells, or any combination thereof, wherein the chemotherapy agent is temozolomide (TMZ), and wherein the NK cells derived from induced pluripotent stem cells are prepared using a kit for inducing stem cell differentiation into natural killer cells as defined herein.

[0377] In some implementations, a method for treating a subject with a low-immune-invasive tumor includes the following steps:

[0378] NK cells derived from induced pluripotent stem cells were administered to the subjects; and

[0379] The subject is treated with a chemotherapy agent, wherein the chemotherapy agent is administered before, after, or concurrently with the administration of NK cells derived from induced pluripotent stem cells, or any combination thereof, wherein the chemotherapy agent is temozolomide (TMZ), and wherein the NK cells derived from induced pluripotent stem cells are prepared by a method for inducing stem cell differentiation into natural killer cells as defined herein.

[0380] In some implementations, the chemotherapeutic agent is administered to the subject 8 to 72 hours before, 8 to 72 hours after, or both before and 8 to 72 hours after the administration of NK cells.

[0381] In some implementations, the chemotherapy agent is administered to the subject 8, 16, 24, 32, 40, 48, 56, 64, or 72 hours before administering NK cells, 8, 16, 24, 32, 40, 48, 56, 64, or 72 hours after administering NK cells, or both before and after administering NK cells.

[0382] In some implementations, temozolomide is administered to the subject 8, 16, 24, 32, 40, 48, 56, 64, or 72 hours before administering NK cells, 8, 16, 24, 32, 40, 48, 56, 64, or 72 hours after administering NK cells, or both before and after administering NK cells.

[0383] In some implementations, temozolomide is administered to the subject 8, 16, 24, 32, 40, 48, 56, 64, or 72 hours before administering NK cells derived from induced pluripotent stem cells, 8, 16, 24, 32, 40, 48, 56, 64, or 72 hours after administering NK cells derived from induced pluripotent stem cells, or 8, 16, 24, 32, 40, 48, 56, 64, or 72 hours before administering NK cells derived from induced pluripotent stem cells and 8, 16, 24, 32, 40, 48, 56, 64, or 72 hours after administering NK cells derived from induced pluripotent stem cells. Beneficial effects

[0384] The NK cell combined with TMZ defined in this invention can exhibit excellent effects, such as, but not limited to: (1) stronger tumor (including low immune infiltration tumors) killing ability compared to NK cells alone or TMZ alone; (2) enhanced secretion of chemokines in vitro, improving the ability to recruit immune cells, including T cells, to infiltrate tumors; (3) activated other immune cells in vivo and recruited them to infiltrate tumors, turning "cold" tumors into "hot" tumors; and (4) prolonged survival.

[0385] Example

[0386] The invention is further described with reference to the following embodiments. It should be understood that these embodiments are merely examples and do not constitute a limitation on the invention. Unless otherwise specified, the following materials and instruments are commercially available or prepared according to methods known in the art. And unless otherwise specified, the following experiments were performed according to the manufacturer's instructions or according to methods and procedures known in the art.

[0387] The induced pluripotent stem cells (iPSCs) used in this embodiment are reprogrammed iPSCs from our company, processed via CTS. TM CytoTune TM The virus was prepared using the iPS 2.1 Sendai Virus Reprogramming Kit (catalog number: A34546) (see kit instructions for details), with human peripheral blood cells as the initial cells.

[0388] In this embodiment, the low-attachment 96-well plate was purchased from Corning; BMP4 was purchased from StemImmune LLC, Cat#HST-B4-0100; CHIR-99021 was purchased from Selleck; non-essential amino acids were purchased from Gibco, Cat#11140050; glutamine was purchased from Gibco; vitamin C was purchased from Sigma; and vitamin A-free B27 medium supplement was purchased from Gibco.

[0389] Example 1: NK cells combined with TMZ can enhance the ability to kill tumors (especially cold tumors).

[0390] A. Preparation of various NK cell and tumor cell lines.

[0391] iPS-NK cells were provided by Shize Biotechnology, NK92 cell line was purchased from Zhejiang Meisen Cell Technology Co., Ltd., and peripheral blood-derived PB-NK cells were purchased from Shanghai Heyousheng Biotechnology Co., Ltd. Cell culture and differentiation methods can be found in CN118546880A.

[0392] Specifically, iPS-NK cells can be prepared by the following methods:

[0393] Step (1) Culture human induced pluripotent stem cells (iPSCs) in low-attachment cell culture plates to obtain embryoid bodies: at 2×10 5 Cell / well cell quantity: iPSCs were seeded into low-adhesion six-well plates using the first medium (StemFit Basic 03 medium containing 10 μM Y27632·2HCl (company: Ajinomoto, catalog number: AK03N)) and cultured at 37°C and 5% CO2 for one day to obtain embryoid bodies, which were denoted as D0.

[0394] Step (2) The embryoids were cultured for mesodermal differentiation to obtain mesodermal cells: One day later, the first culture medium was replaced with the second culture medium (differentiation medium containing 20 ng / mL BMP4 (bone morphogenetic protein 4) and 3 μM CHIR-99021, wherein the differentiation medium is RPMI1640 medium containing 1% (v / v) non-essential amino acids, 1% (v / v) glutamine, 50 μg / mL vitamin C and 2% (v / v) vitamin A-free B27 medium supplement), and cultured at 37℃ and 5% CO2 for two days. This time was recorded as D2, and mesodermal cells (ME cells) were obtained.

[0395] Step (3) The mesodermal cells were cultured for hematopoietic endothelial differentiation to obtain hematopoietic endothelial cells: Two days later, the second culture medium was replaced with the third culture medium (a differentiation medium containing 5 ng / mL BMP4, 50 ng / mL VEGF-165 (vascular endothelial growth factor), 50 ng / mL bFGF (basic fibroblast growth factor), and 10 μM SB431542, wherein the differentiation medium was RPMI1640 medium containing 1% (v / v) non-essential amino acids, 1% (v / v) glutamine, 50 μg / mL vitamin C and 2% (v / v) vitamin A-free B27 medium supplement), and cultured at 37℃ and 5% CO2 for four days to obtain hematopoietic endothelial cells (HE cells);

[0396] Step (4) NK cell differentiation culture was performed on the hematopoietic endothelial cells to obtain NK cells: Four days later, the embryoid bodies (hematopoietic endothelial cells obtained after four days of culture in step (3)) were collected from the low-attachment 96-well plate into a 50 mL centrifuge tube. The cells were allowed to settle naturally for 1-2 min, and then the supernatant was aspirated. About 5 mL of supernatant remained in the centrifuge tube. Then, 14 mL of the fourth culture medium (containing 5 ng / mL BMP4, 10 ng / mL VEGF-165, 20 ng / mL SCF (stem cell factor), 5 ng / mL IL3 (multi-CSF), 10 ng / mL IL15 (interleukin-15), 20 ng / mL IL7 (interleukin-7) and 10 ng / mL The differentiation medium for FLT3L (Fms-associated tyrosine kinase 3 ligand) was prepared as follows: the differentiation medium consisted of IMDM (Iscove's Modified Dulbecco's Medium) containing 1% (v / v) non-essential amino acids, 1% (v / v) glutamine, 50 μg / mL vitamin C, 2% (v / v) vitamin A-free B27 medium supplement, and 30 μM N-acetyl-L-cysteine. Cell spheroids were gently pipetted and evenly distributed into three normally adherent 6-well plates. The plates were then incubated at 37°C and 5% CO2 for 7 days. After 7 days, approximately 1 mL of supernatant was gently aspirated from each well. Then, 3 mL of the fifth medium was added to each well of the plate, and the plates were incubated at 37°C and 5% CO2 for another 7 days. After 7 days, 2 mL of the fifth medium was added to each well of the plate, and the plates were incubated at 37°C and 5% CO2 for another 7 days.

[0397] Step (5) Collection of differentiated NK cells: On days 13, 16, and 20 of NK cell differentiation culture from hematopoietic endothelial cells, the supernatant (suspension cells) from the six-well plates was collected into centrifuge tubes and centrifuged at 1500 rpm for 10 minutes. Then, 5 mL of differentiation medium (containing 5 ng / mL BMP4, 10 ng / mL VEGF-165, 20 ng / mL SCF, 10 ng / mL IL15, 20 ng / mL IL7, 10 ng / mL FLT3L, 1 μM UM171 (CAS No.: 1448724-09-1) and 1 μM SR1 (CAS No.: 1227633-49-9) was used. The differentiation medium contained 1% (v / v) non-essential amino acids, 1% (v / v) glutamine, 50 μg / mL vitamin C, 2% (v / v) vitamin A-free B27 medium supplement and 30 μM Cells were resuspended in IMDM medium containing N-acetyl-L-cysteine, and then the cell suspension was filtered through a 70 μm cell sieve. The filtered cell suspension was centrifuged again at 1500 rpm for 5 minutes. The cells were then resuspended in 5 mL of the fifth medium for AO / PI cell counting. Cell viability and cell number were recorded, and subsequent cell tests were performed as needed.

[0398] Currently, "low-immune-infiltrating tumors" or "cold tumors" are relatively common in many tumor types, such as ovarian cancer, cervical cancer, lymphoma, leukemia, meningioma, and brain metastases; ovarian cancer, in particular, is often classified as a cold tumor. SK-OV-3 cells (human ovarian cancer cells) exhibit low tumor mutational burden (TMB), low T cell infiltration, and high levels of immunosuppressive factors (such as IL-10). In the following examples, SK-OV-3 cells, HeLa cells (human cervical cancer cells), NALM6 cells (human B-lymphoblastic leukemia cells), IOMM-Lee cells (human meningioma cells), and MDA-MB-361 cells (a cell line derived from human breast cancer brain metastases) were used to test the effects of various NK cell lines combined with TMZ on different tumors (especially cold tumors). SK-OV-3 cells, HeLa cells, and NALM6 cells were cultured in 1640 medium containing 10% FBS. IOMM-Lee cells and MDA-MB-361 cells were cultured in DMEM medium containing 10% FBS.

[0399] B. NK cells from different sources, combined with TMZ, kill tumors (especially cold tumors).

[0400] This embodiment describes the in vitro detection of the killing effect of various NK cells (such as iPS-NK, NK92, PB-NK cells) combined with TMZ on different tumor cells (such as ovarian cancer, cervical cancer, human B-lymphoblastic leukemia, meningioma, and brain metastases). The detection method is as follows.

[0401] Experimental methods: Target cells (SKOV3 cells, HeLa cells, NALM6 cells, IOMM-Lee cells, and MDA-MB-361 cells) were harvested and seeded at a density of 5E+3 / well to 2E+4 / well. After overnight plating and cell adhesion, cells were treated with 1 mM TMZ for 24-48 hours according to the groupings in Table 1. The TMZ treatment method can be found in CN118546880A. Then, NK cells from different sources (e.g., NK92 cells, plated at 2E+4 / well) were added and co-cultured for 4-24 hours. After 4-24 hours, the wells were photographed to detect the number of remaining tumor cells. (For HELA and SKOV3 cells, a cell counter (Rigel S2; manufacturer: Countstar) and AO / PI staining solution (RE010212, Countstar) were used for counting. For NALM6, IOMM-Lee, and MDA-MB-361 cells, flow cytometry (Agilent, Novocyte3000) and software (Agilent, Novoexpress) were used for counting. Cells were pre-treated with CellTrace Violet (Thermo).) Fisher) was used to label the samples, and the inhibition rate (or cleavage rate) and q value were statistically analyzed (microscopic images of 5E+3 / well were recorded, and the data were statistically analyzed based on the 1E+4 / well plate density).

[0402] q = E(A+B) / (EA+EB-EA·EB), where E(A+B) is the inhibition rate of the two drugs used together, and EA and EB are the inhibition rates of each drug used alone. q<1 indicates that the two drugs have an antagonistic effect when used together, and q>1 indicates that the two drugs have a synergistic effect when used together.

[0403] Table 1. Experimental Groups

[0404] Table 2. Statistical analysis of q values ​​for the TMZ + effector cell combination regimen

[0405] The results are shown in Figures 1A-5C and Table 2. The combination of NK cells from different sources (such as NK92 cells, iPS-NK cells, and PB-NK cells) with TMZ can effectively kill tumor cells SKOV3 cells, HeLa cells, NALM6 cells, IOMM-Lee cells, and MDA-MB-361 cells. Moreover, its tumor-killing ability is further enhanced compared with the use of TMZ alone and effector cells alone, showing a synergistic effect.

[0406] C. NK cells from different sources combined with TMZ-secreted granzyme and perforin.

[0407] This embodiment examines the effects of various NK cells (such as iPS-NK, NK92, and PB-NK cells) combined with TMZ on granzyme and perforin in vitro. The detection methods are as follows, and the experimental groups are shown in Table 1.

[0408] Experimental Methods: Target cells (SKOV3 cells, HeLa cells, NALM6 cells, IOMM-Lee cells, MDA-MB-361 cells) were harvested and seeded into 96-well plates at a density of 100 μL per well (5E+3 / well to 2E+4 / well). After overnight seeding and cell adhesion, cells were pretreated with 1000 μM TMZ for 48 hours according to the groupings in Table 1. For TMZ treatment instructions, please refer to CN118546880A. After 48 hours, 100 μL of NK cells from different sources (seeding density: 5E+3 / well to 2E+4 / well) were added to the corresponding effector cell wells, and the cells were gently mixed by pipetting. The 96-well plates containing effector and target cells were placed in a cell culture incubator and incubated for 1 hour. Golgi inhibitor (GolgiStop) was then added to all wells. TM After treatment, cells were incubated for another 3 hours. After incubation, cells were transferred from the wells to corresponding 1.5 mL centrifuge tubes, centrifuged at 800 g for 5 minutes, and the supernatant was discarded. Cells were resuspended in 100 μL PBS, and 1 μL of CD56 flow cytometry antibody (BD, catalog number: 5555518; channel APC) was added to each tube. Cells were incubated at room temperature in the dark for 20 minutes, followed by centrifugation at 800 g for 5 minutes. After discarding the supernatant, cell permeabilization fixation solution was added for 15 minutes, followed by staining with granzyme B (catalog number: 3129195, BD) and perforin (catalog number: B358136, Biolegend). Finally, cells were resuspended in 100 μL PBS and flow cytometry analysis was performed.

[0409] The results showed that NK cells from different sources could secrete granzymes and perforin when co-cultured with tumor cells. Furthermore, the ability of NK cells from different sources to secrete granzymes and perforin was enhanced after combination with TMZ, indicating that the combination of NK cells and TMZ enhances their cytotoxicity, thereby further strengthening their tumor-killing ability.

[0410] Example 2: NK cells combined with TMZ can chemotactically attract immune cells in vitro.

[0411] This embodiment examines the effects of the combined use of NK cells and TMZ on chemokines and other immune cells in vitro.

[0412] Tranwell assay for the chemotactic activity of NK cells combined with TMZ on immune cells

[0413] Refer to Table 3 to set up the groups. Culture SKOV3, HeLa, NALM6, and IOMM-Lee tumor cells at appropriate densities. After overnight plating and cell adhesion, collect the cells and wash them 2-3 times with PBS to remove residual serum or other interfering substances. Count the cell concentration and adjust it to the appropriate concentration for the experiment (1E+5 / well). Add tumor cells to the lower chamber (bottom compartment) of the Transwell and, according to the group, add 1000 μM TMZ as needed for treatment for 12-48 hours. After treatment, change the culture medium and add CellTrace to the lower chamber (bottom compartment) of the Transwell according to the group. TM Violet-labeled NK cell suspension or empty culture medium. Simultaneously, add CFSE-labeled PBMCs (usually about 200 μl) to the upper chamber (upper compartment) of the Transwell. Incubate the Transwell containing cells in a 37°C, 5% CO2 cell culture incubator for 8-24 hours.

[0414] Cells from the lower chamber were collected, and the number of CFSE-labeled PBMCs in the lower chamber was measured for statistical analysis.

[0415] Table 3. Experimental Groups

[0416] Table 4. Results of Tranwell assay for the chemotactic effect of NK92 cells combined with TMZ on immune cells.

[0417] Table 5. Results of Tranwell assay for the chemotactic effect of iNK cells combined with TMZ on immune cells.

[0418] The results are shown in Tables 4-5, Figure 2, and Figures 6A-6D. Compared with TMZ alone and NK alone, NK cells from different sources (such as NK92 cells and iPS-NK cells) combined with TMZ can chemotact more immune cells from the upper chamber to the lower chamber of Transwell. This indicates that the combination of NK cells and TMZ enhances the ability to recruit immune cells, which is beneficial for immune cell infiltration into tumors.

[0419] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be defined by the claims.

Claims

1. Use of a pharmaceutical combination in the preparation of a kit for treating tumors, wherein the pharmaceutical combination comprises an active ingredient comprising natural killer (NK) cells and a chemotherapeutic agent, wherein the chemotherapeutic agent comprises temozolomide (TMZ).

2. The use of claim 1, wherein the tumor comprises nervous system tumors, neuroendocrine tumors, hematologic tumors, ovarian cancer, prostate cancer, pancreatic cancer, osteosarcoma, thyroid cancer, soft tissue sarcoma, insulinoma, and testicular cancer; Preferably, the nervous system tumors include brainstem glioma (DIPG), supratentorial and infratentorial gliomas, astrocytomas, oligodendrogliomas, ependymomas, medulloblastomas, schwannomas, meningiomas, pituitary adenomas, neuroblastomas, spinal cord gliomas, spinal cord meningiomas, spinal cord metastatic tumors, peripheral schwannomas, neurofibromas, brain metastases, and neuroectodermal tumors. Preferably, the hematologic malignancies include acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), Hodgkin lymphoma (HL), non-Hodgkin lymphoma (NHL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma (MCL), Burkitt lymphoma (BL), multiple myeloma (MM), myelodysplastic syndrome (MDS), myeloproliferative neoplasm (MPN), polycythemia vera (PV), essential thrombocythemia (ET), and myelofibrosis (MF).

3. Use of a pharmaceutical combination in the preparation of a kit for treating low-immune-invasive tumors, wherein the pharmaceutical combination comprises an active ingredient comprising natural killer (NK) cells and a chemotherapeutic agent, wherein the chemotherapeutic agent includes temozolomide (TMZ).

4. The use of claim 3, wherein the low-immune-invasive tumor includes low-immune-invasive nervous system tumors, neuroendocrine tumors, non-small cell lung cancer, small cell lung cancer, colorectal cancer, melanoma, breast cancer, esophageal cancer, gastric cancer, bladder cancer, endometrial cancer, head and neck cancer, cervical cancer, liver cancer, and kidney cancer. Preferably, the low-immune-infiltrating nervous system tumors include low-immune-infiltrating brainstem gliomas (DIPG), supratentorial and infratentorial gliomas, astrocytomas, oligodendrogliomas, ependymomas, medulloblastomas, schwannomas, meningiomas, pituitary adenomas, neuroblastomas, spinal cord gliomas, spinal cord meningiomas, spinal cord metastatic tumors, peripheral schwannomas, neurofibromas, brain metastases, and neuroectodermal tumors. Preferably, the low-immune-infiltrating tumor is a low-immune-infiltrating cervical cancer or a low-immune-infiltrating nervous system tumor.

5. The use according to any one of claims 1-4, wherein the NK cell is an unmodified NK cell or a modified NK cell; Preferably, the NK cells are unmodified NK cells.

6. The use of any one of claims 1-5, wherein the NK cells comprise NK-92 cells, NK cells derived from peripheral blood or umbilical cord blood, NK cells derived from induced pluripotent stem cells (iPSCs) or embryonic stem cells. Preferably, the NK cells are NK cells derived from induced pluripotent stem cells (iPSCs).

7. The use of any one of claims 1-6, wherein the NK cells are prepared by a kit for inducing stem cell differentiation into natural killer cells, the kit comprising a second culture medium, a fourth culture medium, and a fifth culture medium; wherein, The second culture medium is a basal culture medium containing BMP signaling pathway activators; The fourth culture medium is a basal culture medium containing at least one, at least two, at least three, or at least four of the following: BMP signaling pathway activator, vascular endothelial growth factor, colony-stimulating factor, and interleukin. The fifth culture medium is a basal culture medium containing at least one, at least two, at least three, at least four, at least five, or at least six of the following: BMP signaling pathway activator, vascular endothelial growth factor, colony-stimulating factor, interleukin, stem cell agonist, and stem cell expansion agent.

8. The use of claim 7, wherein the kit for inducing stem cell differentiation into natural killer cells further comprises a third culture medium, said third culture medium being a basal culture medium comprising at least one, at least two, or at least three of the following: a BMP signaling pathway activator, a growth factor, and a TGFβ / ALK inhibitor.

9. The use of claim 7 or 8, wherein the kit for inducing stem cell differentiation into natural killer cells further comprises a first culture medium, the first culture medium being a basal culture medium containing a ROCK inhibitor.

10. Use according to any one of claims 1-6, wherein the NK cells are prepared by a method of inducing stem cells to differentiate into natural killer (NK) cells, the method comprising the step of using a kit for inducing stem cells to differentiate into natural killer cells as defined in any one of claims 7-9.

11. The use of claim 10, wherein the method comprises: Step (1) The stem cells are cultured to obtain embryoid bodies; Step (2) The embryoid body is subjected to mesodermal differentiation culture to obtain mesodermal cells; Step (3) The mesodermal cells were cultured for hematopoietic endothelial differentiation to obtain hematopoietic endothelial cells; Step (4) Hematopoietic endothelial cells are cultured in the fourth and fifth culture media of the kit for inducing stem cell differentiation into natural killer cells as defined in claim 7 to obtain NK cells; Preferably, the culture in step (1) is carried out in the first culture medium of the kit for inducing stem cell differentiation into natural killer cells as defined in claim 9; Preferably, the culture in step (2) is carried out in the second culture medium of the kit for inducing stem cell differentiation into natural killer cells as defined in claim 7; Preferably, the culture in step (3) is carried out in the third culture medium of the kit for inducing stem cell differentiation into natural killer cells as defined in claim 8; Preferably, the culture time in step (1) is 12-36 hours; Preferably, the culture time in step (2) is 24-72 hours; Preferably, the culture time in step (3) is 48-144 hours; Preferably, the step (4) of culturing hematopoietic endothelial cells in the fourth and fifth culture media of the kit for inducing stem cell differentiation into natural killer cells as defined in claim 7 is to culture hematopoietic endothelial cells sequentially in the fourth and fifth culture media of the kit for inducing stem cell differentiation into natural killer cells as defined in claim 7. Preferably, the hematopoietic endothelial cells are cultured in the fourth culture medium for 96-192 hours; Preferably, the hematopoietic endothelial cells are cultured in the fifth culture medium for 144-432 hours.

12. The use of any one of claims 1-11, wherein the pharmaceutical combination further comprises other chemotherapeutic agents selected from anticancer agents, metabolic antagonists, DNA demethylators, plant-derived antitumor agents, alkylating agents, antimetabolites, anticancer antibiotics, topoisomerase inhibitors, mitotic inhibitors, differentiation agents, and hormone therapy agents. Preferably, the other chemotherapeutic agents are selected from carmustine, lomustine, vorinostat, mebendazole, imatinib mesylate, AG119, or any therapeutic derivative thereof.

13. Use according to any one of claims 1-12, wherein the active ingredient does not contain trimethoprim, and / or immune checkpoint inhibitors.

14. The use of any one of claims 1-13, wherein the chemotherapeutic agent is administered before, after, or simultaneously with the administration of NK cells; Preferably, the chemotherapeutic agent is administered 8 to 72 hours before, 8 to 72 hours after, or both before and 8 to 72 hours after the administration of NK cells.

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