Kit for inducing stem cells to differentiate into NK cells, application of kit and application of NK cells and TMZ in treatment of TMZ drug-resistant GBM

Abstract

The invention belongs to the technical field of biology, and provides a kit and a method for inducing stem cells to be differentiated into natural killer cells and application of the kit and the method. The kit adopts a culture medium with definite chemical components, small chemical molecules and cytokines, does not involve serum and other substances which are easy to cause unstable factors such as batch difference, is lower in price compared with a known commercial differential culture medium, and is reduced in differentiation period at the same time. Moreover, the present invention provides a pharmaceutical composition and a method for treating glioblastoma, particularly drug-resistant glioblastoma, and uses of the pharmaceutical composition. Specifically, the invention relates to the treatment of glioblastoma by combining immune cells with chemotherapeutic drugs. More specifically, the invention relates to treatment of TMZ drug-resistant glioblastoma by synergistic interaction of NK cells and TMZ.

Description

[0001] This application is a divisional application of Chinese patent application No. 202410202295.4, filed on February 23, 2024, with the invention name of "Kit for inducing stem cells to differentiate into NK cells and its application, and the application of NK cells combined with TMZ in the treatment of TMZ-resistant GBM". Technical Field

[0002] The present invention belongs to the field of biotechnology, and relates to a kit for inducing stem cells to differentiate into natural killer cells and its application. The present invention also relates to the treatment of solid tumors by combining immune cells with chemotherapy drugs, and specifically to the synergistic treatment of TMZ-resistant GBM by combining NK cells with TMZ. Background Art

[0003] Natural killer cells are part of the innate immune system and play an important role in immune surveillance and host defense against viral infections, as well as against tumor cells. Recent studies have also shown that NK cells are potential therapeutic targets for cancer treatment. In the human body, NK cells account for 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 T cell receptor / CD3ε complexes. Instead, NK cells act in an antigen-independent manner (usually without generating immune memory or long-term protective immunity). NK cells regulate their own cytotoxicity through activating and inhibitory receptors on their own surface. Unlike T cells, NK cells retain their natural receptor recognition and intrinsic ability to 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, and in multiple current clinical trials, they have not shown safety issues such as cytokine release syndrome similar to T cell therapy, thus opening the possibility of producing "off-the-shelf" allogeneic cell therapy products.

[0004] NK cells derived from peripheral blood and umbilical cord blood are affected by the donor and have limited cell sources. The state, quality and yield of the obtained cells are also different. In addition, T cell contamination may occur during the cell expansion process. And after the amplified NK cells are frozen, the cell state will also deteriorate. However, there are many problems with the current methods of inducing stem cells to differentiate into natural killer cells: the method for producing NK cells derived from iPS cell differentiation includes MEK inhibitors, GSK3 inhibitors and ROCK inhibitors, etc., but the efficiency of differentiated NK cells is low and the number of NK cells obtained is limited; the method for producing NK cells derived from iPS cell differentiation in patent document CN107429230A is: hematopoietic progenitor cells are differentiated in the form of embryoid bodies 3D, and then CD34 needs to be enriched. + CD45 + The cells are then differentiated into NK cells, and commercial culture medium with uncertain composition and heterologous substances such as serum are used, which makes the operation process complicated and is not conducive to the preparation of subsequent clinical-grade cell products; Patent document CN The method for NK cells derived from iPS cell differentiation in 112608895A is as follows: first, EB (embryoid body) is differentiated into permanent hematopoietic HPCs (hematopoietic progenitor cells). The subsequent process is to inoculate the EB into a culture vessel coated with matrix protein to obtain permanent hematopoietic HPCs and then differentiate them into NK cells. The efficiency of permanent hematopoietic HPCs obtained by this method is low, and the differentiation of NK cells is greatly affected by the cell state of the previous HPCs and the efficiency of the differentiated HPCs. In addition, the cells need to be inoculated into a culture dish containing matrix protein, and the differentiation of NK cells will also be affected by the matrix protein. The method for NK cells derived from iPS cell differentiation in patent document CN113801846A is as follows: human induced pluripotent stem cells are inoculated into a wave bioreactor and a cell factory to obtain endothelial progenitor cells, and then the endothelial progenitor cells are induced to differentiate into NK progenitor cells, and the NK progenitor cells are further differentiated into NK cells. In this differentiation system, endothelial progenitor cells have the potential to differentiate into a variety of endothelial cells, increasing the risk that the efficiency of differentiation into NK cells is affected by the external environment (such as the parameter settings of the bioreactor).

[0005] Gliomas are malignant tumors that form in the brain or in nerves originating from the brain. Based on pathological grading, gliomas are divided into grades I-IV, with grade IV being glioblastoma (GBM). GBM is the most common and most aggressive central nervous system tumor in the brain. For the treatment of newly diagnosed GBM, the current standard therapy includes maximal safe tumor resection, followed by radiotherapy (RT) and temozolomide (TMZ) combined with chemotherapy.

[0006] However, GBM is divided into sensitive and resistant types to TMZ. Patients with sensitive GBM have a relatively better prognosis with standard TMZ treatment, but almost all GBM will relapse. The reason is that sensitive GBM tumor cells develop various strategies to escape the effects of TMZ and transform into resistant GBM. Ultimately, like the primary resistant GBM, they resist the effects of TMZ, relapse, and metastasis, endangering life (Weller M, et al. Standards of care for treatment of recurrent glioblastoma--are we there yet? Neuro Oncol. 2013 Jan; 15 (1): 4-27.). Therefore, new treatment strategies are urgently needed to treat TMZ-resistant GBM.

[0007] Immunotherapy is a new type of therapy that uses adoptive immune cells or the autoimmune system to fight tumors. It is changing the treatment of many cancers. However, so far, due to the high heterogeneity of GBM, the heterogeneity of targets, and the low utilization rate of protein drugs caused by the blood-brain barrier, immunotherapy has not made a breakthrough in the treatment of GBM. This field needs a new treatment strategy that uses adoptive immune cells combined with TMZ to improve the treatment of TMZ-resistant GBM. Summary of the invention

[0008] In a first aspect, the present invention provides a 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 comprising a BMP signaling pathway activator; the fourth culture medium is a basal culture medium comprising at least one, at least two, at least three or at least four of the following: a BMP signaling pathway activator, a vascular endothelial growth factor, a colony stimulating factor, an interleukin; the fifth culture medium is a basal culture medium comprising at least one, at least two, at least three, at least four, at least five or at least six of the following: a BMP signaling pathway activator, a vascular endothelial growth factor, a colony stimulating factor, an interleukin, a stem cell agonist, a stem cell expander.

[0009] In one embodiment, the kit further comprises a third culture medium, which is a basic 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.

[0010] In one embodiment, the kit further comprises a first culture medium, which is a basal culture medium comprising a ROCK inhibitor.

[0011] In one embodiment, the second culture medium is a basal culture medium comprising a BMP signaling pathway activator and a GSK3 inhibitor.

[0012] In a second aspect, the present invention provides the use of the kit of the first aspect of the present invention in preparing embryoid bodies, preparing mesoderm cells, preparing hemogenic endothelial cells, preparing natural killer (NK) cells, preparing products for inducing stem cells to differentiate into embryoid bodies, preparing products for inducing stem cells to differentiate into mesoderm cells, preparing products for inducing stem cells to differentiate into hemogenic endothelial cells, and / or preparing products for inducing stem cells to differentiate into NK cells.

[0013] In a third aspect, the present invention provides a method for inducing stem cells to differentiate into natural killer (NK) cells, the method comprising the steps of using the kit of the first aspect of the present invention.

[0014] In one embodiment, the method comprises: step (1) culturing stem cells to obtain embryoid bodies; step (2) subjecting the embryoid bodies to mesoderm differentiation culture to obtain mesoderm cells; step (3) subjecting the mesoderm cells to hemogenic endothelial differentiation culture to obtain hemogenic endothelial cells; step (4) culturing the hemogenic endothelial cells in the fourth culture medium and the fifth culture medium of the kit of the first aspect of the present invention to obtain NK cells.

[0015] In a fourth aspect, the present invention provides a natural killer (NK) cell, which is prepared by the kit of the first aspect of the present invention and / or the method of the third aspect of the present invention.

[0016] In a fifth aspect, the present invention provides a pharmaceutical composition comprising the NK cells of the fourth aspect of the present invention.

[0017] In a sixth aspect, the present invention provides use of the NK cell of the fourth aspect of the present invention or the pharmaceutical composition of the fifth aspect of the present invention in the preparation of a drug for preventing and / or treating tumors.

[0018] In a seventh aspect, the present invention provides a pharmaceutical combination for treating glioblastoma, comprising an active ingredient comprising natural killer (NK) cells and a chemotherapeutic agent, wherein the chemotherapeutic agent comprises temozolomide (TMZ).

[0019] In one embodiment, the glioblastoma is resistant to TMZ.

[0020] In one embodiment, the NK cells are unmodified NK cells.

[0021] In one embodiment, the 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 (iPSC) or embryonic stem cells.

[0022] In one embodiment, the NK cells are NK cells derived from induced pluripotent stem cells (iPSCs).

[0023] In one embodiment, the NK cells are unmodified NK cells derived from induced pluripotent stem cells.

[0024] In one embodiment, NK cells derived from induced pluripotent stem cells (iPSCs) are prepared by the kit of the first aspect of the present invention or the method of the third aspect of the present invention.

[0025] In one embodiment, NK cells derived from induced pluripotent stem cells (iPSC) are prepared by the method described herein for inducing stem cell differentiation into natural killer cells. In one embodiment, the drug combination further comprises other chemotherapeutic agents selected from anticancer agents, metabolic antagonists, DNA demethylating agents, antitumor agents derived from plants, alkylating agents, antimetabolites, anticancer antibiotics, topoisomerase inhibitors, mitotic inhibitors, differentiation agents, hormone therapy agents.

[0026] In one embodiment, the additional chemotherapeutic agent is selected from carmustine, lomustine, vorinostat, mebendazole, imatinib mesylate, AG119, or any therapeutic derivatives thereof.

[0027] In one embodiment, the active ingredient does not comprise trimetrexate.

[0028] In one embodiment, the active ingredient does not comprise an immune checkpoint inhibitor.

[0029] In one embodiment, the pharmaceutical combination is a kit.

[0030] In an eighth aspect, the present invention further provides use of the drug combination of the seventh aspect of the present invention in preparing a drug for treating glioblastoma.

[0031] In one embodiment, the chemotherapeutic agent is administered before, after, or simultaneously with the administration of NK cells.

[0032] In one embodiment, the chemotherapeutic agent is administered 8 to 72 hours before, 8 to 72 hours after, or 8 to 72 hours before and 8 to 72 hours after administration of the NK cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1The diagrams of inducing human induced pluripotent stem cells to differentiate into natural killer cells and cell differentiation diagrams in Example 2 are as follows: A is a diagram of inducing human induced pluripotent stem cells to differentiate into natural killer cells in Example 2; B is a cell morphology diagram of hemogenic endothelial cells after 2 days of NK cell differentiation culture (iNK-D2, EB adherence D2) in Example 2 (observed under a 4x objective lens); C is a cell morphology diagram of hemogenic endothelial cells after 6 days of NK cell differentiation culture (iNK-D20) in Example 2 (observed under a 10x objective lens).

[0034] Figure 2 13, 16, and 20 days of NK cell differentiation culture of hemogenic endothelial cells in Example 2: A is the CD45 + CD56 + Cell detection result diagram; B is the CD45 of NK cells collected on the 16th day of NK cell differentiation culture of hemogenic endothelial cells in Example 2 + CD56 + Cell detection result diagram; C is the CD45 of NK cells collected on the 20th day of NK cell differentiation culture of hemogenic endothelial cells in Example 2 + CD3- cell detection result diagram; D is the CD56 of NK cells collected on the 20th day of NK cell differentiation culture of hemogenic endothelial cells in Example 2. + CD3- cell detection result diagram; E is the CD45 of NK cells collected on the 20th day of NK cell differentiation culture of hemogenic endothelial cells in Example 2. + CD56 + Cell detection result diagram; F is the CD45 of NK cells collected on the 20th day of NK cell differentiation culture of hemogenic endothelial cells in Example 2 + CD94 + Cell detection result diagram; G is the CD56 of NK cells collected on the 20th day of NK cell differentiation culture of hemogenic endothelial cells in Example 2 + CD94 + Cell detection results.

[0035] Figure 3 is a flow cytometry result diagram of NK cells collected on the 20th day of NK cell differentiation culture of hemogenic endothelial cells in Example 2: wherein A is the CD3-CD45 + CD56 +Cell detection result diagram; B is a CD3-CD16- cell detection result diagram of NK cells collected on the 20th day of NK cell differentiation culture of hemogenic endothelial cells in Example 2; C is a CD3-Nkp46- cell detection result diagram of NK cells collected on the 20th day of NK cell differentiation culture of hemogenic endothelial cells in Example 2 + Cell detection result diagram; D is the CD3-CD56 in NK cells collected on the 20th day of NK cell differentiation culture of hemogenic endothelial cells in Example 2. + Nkp44 + Cell detection result diagram; E is the CD3-CD56 in NK cells collected on the 20th day of NK cell differentiation culture of hemogenic endothelial cells in Example 2. + Nkp30 + Cell detection result diagram; F is the CD3-CD56 in NK cells collected on the 20th day of NK cell differentiation culture of hemogenic endothelial cells in Example 2. + Nkp44 + Nkp30 + Cell detection results.

[0036] Figure 4 2 is a flow cytometry result of NK cells collected on the 20th day of NK cell differentiation culture of hemogenic endothelial cells in comparative example 2: A is a flow cytometry result of NK cells collected on the 20th day of NK cell differentiation culture of hemogenic endothelial cells in comparative example 2. + CD3- cell cell detection result diagram; B is the CD43 in NK cells collected on the 20th day of NK cell differentiation culture of hemogenic endothelial cells in Example 2; + CD3- cell cell detection result diagram; C is the CD45 in NK cells collected on the 20th day of NK cell differentiation culture of hemogenic endothelial cells in Example 2; + CD56 + Cell cell detection result diagram; D is the CD45 in NK cells collected on the 20th day of NK cell differentiation culture of hemogenic endothelial cells in Example 2. + CD94 + Cell cell detection result diagram; E is the CD56 in NK cells collected on the 20th day of NK cell differentiation culture of hemogenic endothelial cells in Example 2. + CD94 + Cell cell detection results diagram.

[0037] Figure 5 20 days after the NK cell differentiation culture of hemogenic endothelial cells in comparative example 4: A is the flow cytometry result of NK cells collected on the 20th day after the NK cell differentiation culture of hemogenic endothelial cells in comparative example 4: +CD3 - Cell cell detection result diagram; B is the CD43 in NK cells collected on the 20th day of NK cell differentiation culture of hemogenic endothelial cells in Example 4. + CD3 - Cell cell detection result diagram; C is the CD45 in NK cells collected on the 20th day of NK cell differentiation culture of hemogenic endothelial cells in Example 4. + CD56 + Cell cell detection result diagram; D is the CD45 in NK cells collected on the 20th day of NK cell differentiation culture of hemogenic endothelial cells in Example 4. + CD94 + Cell cell detection result diagram; E is the CD56 in NK cells collected on the 20th day of NK cell differentiation culture of hemogenic endothelial cells in Example 4. + CD94 + Cell cell detection results diagram.

[0038] Figure 6 Flow cytometry results of NK cells collected on the 20th day of NK cell differentiation culture of hemogenic endothelial cells in Example 6, Example 4-1, Example 4-3, Example 4-2, and Example 2: wherein A is a flow cytometry result of NK cells collected on the 20th day of NK cell differentiation culture of hemogenic endothelial cells in Example 6; B is a flow cytometry result of NK cells collected on the 20th day of NK cell differentiation culture of hemogenic endothelial cells in Example 4-1; C is a flow cytometry result of NK cells collected on the 20th day of NK cell differentiation culture of hemogenic endothelial cells in Example 4-3; D is a flow cytometry result of NK cells collected on the 20th day of NK cell differentiation culture of hemogenic endothelial cells in Example 4-2; E is a CD45+ / -100% expression of NK cells in NK cells collected on the 20th day of NK cell differentiation culture of hemogenic endothelial cells in Example 6, Example 4-1, Example 4-3, Example 4-2, and Example 2. + CD56 + Bar graph of cell proportions.

[0039] Figure 7These are bright field images of NK cells collected on the 20th day of NK cell differentiation culture of hemogenic endothelial cells in Example 6, Example 4-1, Example 4-3, and Example 4-2: wherein A is a bright field image of NK cells collected on the 20th day of NK cell differentiation culture of hemogenic endothelial cells in Example 6; B is a bright field image of NK cells collected on the 20th day of NK cell differentiation culture of hemogenic endothelial cells in Example 4-1; C is a bright field image of NK cells collected on the 20th day of NK cell differentiation culture of hemogenic endothelial cells in Example 4-3; D is a bright field image of NK cells collected on the 20th day of NK cell differentiation culture of hemogenic endothelial cells in Example 4-2.

[0040] Figure 8 This is a graph showing the results of IFN-γ expression by the NK cells in Example 2 and peripheral blood-derived NK cells (PB-NK) treated under the same conditions.

[0041] Fig. 9 This is a graph showing the killing effect of NK cells in Example 2 and peripheral blood-derived NK cells (PB-NK) treated under the same conditions on Hela cells.

[0042] Fig.10 It is a schematic diagram of the NK cell in vivo killing experiment process in Example 4.

[0043] Fig.11 This is a graph showing the results of the NK cell in vivo killing experiment in Example 4.

[0044] Fig.12 This is a graph showing the expansion multiples of different batches of NK cells after differentiation in Example 5.

[0045] Fig.13 This is a graph comparing the cell viability of NK cells before and after cryopreservation in Effect Example 5.

[0046] Fig.14 (AD) show the sensitivity responses of different GBM cell lines to TMZ.

[0047] Fig.15 (AB) show the effect of TMZ on cell cycle arrest in different GBM cell lines.

[0048] Fig.16 The results showed that TMZ treatment induced upregulation of various immune ligands in drug-resistant GBM tumor cells after 48 hours of treatment.

[0049] Fig.17 The figure shows the characterization results of NK cells differentiated from iPSCs in vitro.

[0050] Fig.18(AB) show the results of the degranulation activity test of iPSC-NK combined with TMZ in killing GBM.

[0051] Fig.19 The results show that iPSC-NK combined with TMZ has a killing activity against drug-resistant GBM (T98G).

[0052] Fig. 20 Shown are the characterization results of NK92 cell line and PB-NK cells.

[0053] Fig.21 The results show that NK cells from different sources combined with TMZ have a cytotoxic effect on drug-resistant GBM. Specific implementation plan

[0054] General Definitions and Terminology

[0055] All patents, patent applications, scientific publications, manufacturer's instructions and guidelines, etc., cited herein, whether supra or infra, are hereby incorporated by reference in their entirety. Nothing herein should be construed as an admission that the present disclosure is not entitled to antedate such publication.

[0056] Unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the terms related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, and microbiology used herein are terms widely used in the corresponding fields (see, for example, Molecular Cloning: A Laboratory Manual, 2 nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989). Meanwhile, in order to better understand the present invention, the definitions and explanations of relevant terms are provided below.

[0057] As used herein, the expressions "comprises," "comprising," "containing," and "having" are open ended, meaning the inclusion of the listed elements, steps, or components but not the exclusion of other unlisted elements, steps, or components. The expression "consisting of" excludes any element, step, or component not specified. The expression "consisting essentially 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 basic and novel properties of the claimed subject matter. It should be understood that the expressions "consisting essentially of" and "consisting of" are encompassed within the meaning of the expression "comprising."

[0058] As used herein, the singular forms "a", "an" or "the" include plural references unless the context indicates otherwise. The terms "one or more" or "at least one" encompass 1, 2, 3, 4, 5, 6, 7, 8, 9 or more.

[0059] The recitation of ranges of values ​​herein is intended merely to serve as a shorthand method of referring individually to each different value falling within the range. Unless otherwise indicated herein, each separate value is incorporated into the specification as if it were individually recited herein. Unless expressly indicated to the contrary, values ​​or ranges set forth herein are modified by "about", meaning ±20%, ±10%, ±5%, or ±3% of the recited or claimed value or range.

[0060] The term "pluripotent cell" refers to a cell that is capable of self-renewal and proliferation while remaining in an undifferentiated state and that can be induced to differentiate into a specialized cell type under appropriate conditions.

[0061] As used herein, the term "pluripotent stem cell" has the potential to differentiate into any of 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 constructed by reprogramming non-pluripotent cells by introducing specific transcription factors derived from non-pluripotent cells. Exemplary human pluripotent stem cell lines include H1 human pluripotent stem cell lines and H9 human pluripotent stem cell lines. Additional exemplary pluripotent stem cell lines include those available through the National Institutes of Health Human Embryonic Stem Cell Registry and Howard Hughes Medical Institute HUES collection (such as Cowan CA, et al. Derivation of embryonic stem-cell lines from human blastocysts. N Engl J Med. 2004 Mar 25; 350 (13): 1353-6. described). Pluripotent stem cells can be induced to differentiate into different cells, which can be used for different prevention or treatment purposes, to prevent or treat different diseases. As will be appreciated by those skilled in the art, differentiation methods depend on the desired cell type using known techniques. For example, cells can be suspended for differentiation and then made into a gel matrix form, such as matrigel, gelatin or fibrin / thrombin form, to promote cell survival. Differentiation can usually be determined as known in the art by assessing the presence of cell-specific markers. For example, cells can be differentiated into cardiomyocytes, neurons, glial cells, endothelial cells, T cells, NK cells, NKT cells, macrophages, hematopoietic progenitor cells, mesenchymal cells, islet cells, chondrocytes, retinal pigment epithelial cells, kidney cells, hepatocytes, thyroid cells, skin cells, blood cells or epithelial cells under certain differentiation conditions. In some embodiments, iPSC is differentiated into NK cells.

[0062] NK cells derived from iPSCs are also referred to herein as iNK cells. Herein, the terms "iNK cells", "iPSC-NK cells" and "NK cells derived from induced pluripotent stem cells (iPSCs)" are used interchangeably.

[0063] As used herein, the term "totipotency" refers to the ability of a cell to form a complete organism. For example, in mammals, only the fertilized egg and the first cleavage stage blastomere are totipotent. In some embodiments, the pluripotent stem cells described herein do not have totipotency and will not form a complete organism.

[0064] The cell can be from, for example, a human or non-human mammal. 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 cell is from an adult or a non-human mammal. In some embodiments, the cell is from a newborn human, an adult, or a non-human mammal.

[0065] As used herein, the term "subject" or "patient" refers 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, a cat, a bird, a livestock, or a human. Specific examples of "subjects" and "patients" include, but are not limited to, individuals (particularly humans) with diseases or conditions associated with the liver, heart, lungs, kidneys, pancreas, brain, neural tissue, blood, bones, bone marrow, etc. In some embodiments, "subjects" and "patients" refer to individuals (particularly humans) suffering from glioblastoma. In some preferred embodiments, "subjects" and "patients" refer to individuals (particularly humans) suffering from TMZ-resistant glioblastoma.

[0066] In this article, the terms "reduce" and "reduce" are generally used to represent a statistically significant amount of reduction. However, for the avoidance of doubt, "reduce", "reduce" 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% compared to a reference level, 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 a reference sample), or any reduction between 10-100%.

[0067] As used herein, the term "increase" is generally used to indicate an increase by a statistically significant amount; to avoid any doubt, the term "increase" refers to 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 a 100% increase or any increase between 10-100%, or at least about 2-fold, or at least about 3-fold, or at least about 4-fold, or at least about 5-fold or at least about 10-fold compared to a reference level, or any increase between 2-fold and 10-fold or greater than 10-fold.

[0068] In this article, the term "NK cell" or "natural killer cell (natural killer cell)" is used interchangeably. NK cells are effector cells of the innate immune system, belonging to the family of innate lymphocytes (ILC), characterized by the presence of CD56 and the absence of CD3 surface markers. Most NK cells are located in peripheral blood, lymph nodes, spleen and bone marrow. NK cells can protect the host by killing infected, foreign, stressed or transformed cells, which can kill target cells in the absence of specific antigen stimulants and not restricted by MHC categories, wherein the target cells can be tumor cells or cells carrying viruses. At present, in the strategies of preclinical and clinical development, adoptive immunotherapy, direct stimulation, NK cells are recruited to the tumor microenvironment (TME), and the methods such as blocking inhibitory receptors that limit NK cell function enhance the anti-tumor function of NK cells (see, for example, Bald T, et al. The NK cell-cancer cycle: advances and new challenges in NK cell-based immunotherapies. Nat Immunol. 2020Aug; 21 (8): 835-847.).

[0069] As used herein, the terms "Adoptive Cell Transfer Therapy", "Adoptive Immune Cell Therapy", "Adoptive Immune Cell Therapy" or "Adoptive Cell Therapy" are an immune cell therapy method that improves the immune function of immune cells by in vitro modification and amplification, and then injects them into the patient's body to exert an anti-tumor effect. These immune cells can be from allogeneic or homologous. Allogeneic adoptive immunotherapy refers to the transfer of immune cells from an allogeneic (different individual) to a patient. Allogeneic adoptive immunotherapy refers to the collection, amplification and transfer of the patient's own immune cells. These immune cells can be natural killer cells, T cells or lymphocytes, etc.

[0070] As used herein, the term "glioblastoma" or "glioblastoma" is the most common malignant primary brain tumor, accounting for approximately 57% of gliomas and 48% of primary malignant central nervous system (CNS) tumors (see, e.g., Tan AC, et al. Management of glioblastoma: State of the art and future directions. CA Cancer J Clin. 2020 Jul; 70 (4): 299-312.), which has problems such as drug resistance, poor prognosis and short survival during treatment. There are currently few clinical drugs for the treatment of GBM, mainly temozolomide (TMZ), carmustine, lomustine and bevacizumab. Among them, the most commonly used first-line drug in clinical practice is TMZ. However, the effect of GBM on TMZ is divided into sensitive and resistant types. The term "sensitive GBM" means that GBM is sensitive to standard TMZ treatment and has a relatively better prognosis. However, tumors that were originally sensitive to chemotherapy usually gradually become resistant to the drug, so sensitive GBM tumor cells will also develop various strategies to escape the effects of TMZ and eventually turn into resistant GBM. The term "resistant GBM" means that GBM is resistant to TMZ and can resist the effects of TMZ. Drug resistance is not caused by a single factor, but may be related to many aspects such as the DNA mismatch repair system, the expression of O6-methylguanine-DNA methyltransferase, and blood-brain barrier drug penetration (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.).

[0071] As used herein, the term "chemotherapeutic agent" refers to a compound or derivative thereof that can interact with cancer cells, thereby reducing the proliferation state of the cells and / or killing the cells, for example by impairing cell division or DNA synthesis, or effectively targeting rapidly dividing cells by damaging DNA. Examples of chemotherapeutic agents include, but are not limited to, anticancer agents, metabolic antagonists, DNA demethylating agents, antitumor agents derived from plants, alkylating agents, antimetabolites, anticancer antibiotics, topoisomerase inhibitors, mitotic inhibitors, differentiation agents, hormone therapeutic agents, and combinations of the above agents.

[0072] As used herein, the term "alkylating agent" or "alkylating agent" refers to a class of chemically highly active compounds with active alkyl groups that can be converted into electron-deficient active intermediates that quickly form covalent bonds with many substances with affinity in cells. Alkylating agents can covalently bind to electron-rich groups (such as amino, sulfhydryl, hydroxyl, carboxyl, phosphate, etc.) in biological macromolecules (such as DNA, RNA, enzymes, etc.) in cells, causing them to lose their activity, break DNA molecules, or cause base mismatches during DNA replication, thereby destroying the structure and function of DNA and leading to tumor cell death.

[0073] As used herein, the term "temozolomide (TMZ)" is an oral alkylating prodrug that transfers a methyl group to the purine bases (O6-guanine; N7-guanine and N3-adenine) of DNA, and is often used together with radiotherapy as part of the first-line treatment of malignant glioma. The chemical formula of TMZ is C6H6N6O2, and its chemical structure is shown below, and its chemical name is 3,4-dihydro-3-methyl-4-oxoimidazolo[5,1-d]-unsymmetrical-tetrazolyl-8-amide. The main advantages of TMZ are its high oral bioavailability (almost 100%, although the concentration found in cerebrospinal fluid is approximately 20% of the plasma concentration of TMZ), its lipophilicity and small size, which enable it to cross the blood-brain barrier (see, e.g., Ortiz R, et al. Temozolomide: An Updated Overview of Resistance Mechanisms, Nanotechnology Advances and Clinical Applications. Curr Neuropharmacol. 2021; 19(4): 513-537.).

[0074]

[0075] As used herein, administration in any form of "combination," "combination therapy," and / or "combination therapeutic regimen" refers to at least two therapeutically active drugs or compositions, or a combination of drugs comprising at least two therapeutically active ingredients, which may be administered simultaneously in separate or combined formulations, or sequentially at different times separated by minutes, hours, or days, but act together in some manner to provide the desired therapeutic response.

[0076] The term "differentiation" is the process by which a less specialized cell forms progeny of at least one new, more specialized cell type.

[0077] The term "embryoid body", i.e., embryoid body or aggregate, refers to a homogeneous or heterogeneous cell cluster comprising differentiated cells, partially differentiated cells and / or pluripotent stem cells cultured in suspension. In order to summarize some clues inherent to in vivo differentiation, certain aspects of the present invention can use three-dimensional embryoid bodies as an intermediate step. At the beginning of cell aggregation, differentiation can be initiated and cells can begin to reproduce embryonic development to a limited extent. Although they cannot form trophectoderm tissue, almost all other types of cells present in the organism can develop. The present invention can further promote the differentiation of hematopoietic progenitor cells after embryoid body formation.

[0078] The term "basal culture medium" refers to a culture medium with defined chemical composition, including but not limited to IMDM (Iscove's Modified Dulbecco's Medium) medium, Eagle's Basal Medium (BME) medium, MEM medium, DMEM medium, Ham's F-12 medium, RPMI1640 medium, Fischer's medium, E8 medium, mTESR medium, StemFit Basic 03, StemFit Basic 04, NutriStem hPSC XF medium, StemMACS iPSBrew medium, Stem-Partner ACF medium, TeSR-AOF medium, TeSR2 medium and other basic cell culture media.

[0079] The term "multiple colony stimulating factor" is also known as interleukin-3. It is mainly produced by activated Z cells or T cell clones. It contains 13 amino acid residues and can act as an immunomodulator to stimulate the proliferation and differentiation of pluripotent stem cells and various progenitor cells.

[0080] The term "stem cell factor" is also known as mast cell growth factor (MGF), Kit ligand (KL) and Steel factor (SLF). It is an acidic glycoprotein produced by stromal cells in the bone marrow microenvironment. Its sugar group is linked to the N and O groups of the peptide bond, with a relative molecular mass of 31000-36000, and is composed of two identical subunits that are non-covalently bound. The isoelectric point PI = 3.8. SCF has a total of 273 amino acids. From -25 to -1 is the signal peptide, +1-+189 is the extracellular functional region, +190-+216 is the transmembrane region, and +217-+248 is the cytoplasmic functional region. Mouse and human SCF have 83% homology.

[0081] The term "vitamin C" includes vitamin C or its various forms of salts or its various forms of derivatives.

[0082] The term "UM171" includes UM171 (CAS No.: 1448724-09-1) or various forms of salts thereof or various forms of derivatives thereof.

[0083] The term "SR1" includes SR1 (StemRegenin 1, CAS No.: 1227633-49-9) or its various forms of derivatives.

[0084] The term "glutamine" refers to L-glutamine, which is an amino acid encoded in protein synthesis and a non-essential amino acid for mammals. In the present invention, it is used as an essential supplement for cell culture.

[0085] The term "SB431542" also includes SB431542 and salts thereof, especially pharmaceutically acceptable salts.

[0086] The term "CHIR99021" includes CHIR99021 and salts thereof, especially pharmaceutically acceptable salts.

[0087] The term "Y-27632" also includes Y-27632 and its salts, especially pharmaceutically acceptable salts. A preferred pharmaceutically acceptable salt is Y-27632 2HCl.

[0088] The term "fibroblast growth factor" refers to a polypeptide secreted by the pituitary gland and hypothalamus that can promote fibroblast mitosis, mesoderm cell growth, and stimulate angiogenesis. It plays a role in wound healing and limb regeneration. There are two types: acidic (pI 5.6) and alkaline (pI 9.6), namely aFGF and bFGF.

[0089] The term "bone morphogenetic protein 4" refers to bone morphogenetic protein-4, BMP4, which regulates the proliferation and differentiation of various cells during embryonic development.

[0090] The term "B27 Medium Supplement without Vitamin A" is custom Culture medium supplement, without vitamin A. Vitamin A (retinol) can be converted into retinoic acid, which can induce the differentiation of stem cells into neural cells. The vitamin A-free formula is ideal for stem cell culture.

[0091] Cell detection markers

[0092] The development stage of NK cells is mainly based on the expression levels of CD117, CD45, CD56 and CD94. The increase in CD56 expression is the key to NK maturation, followed by CD94 / NKG2A expression. Therefore, CD45 is usually selected during the differentiation process of NK. + CD56 +The expression of indicates the stage of differentiation, and the negative expression of CD3 will also indicate whether there is T cell interference in the differentiation system.

[0093] CD45: The cell surface phosphatase CD45 plays a role in NK cell development and intracellular signal transduction of activating receptors and is a key enzyme in inducing NK cell responses.

[0094] CD56: Also known as neural cell adhesion molecule, it is a 175-185kDa glycoprotein belonging to the immunoglobulin superfamily C2 group. It is mainly expressed on human NK cells and a few MHC-mediated T lymphocytes. It is expressed on the surface of human NK cells at more than 95%, and is an important marker on the surface of NK cells. NK cells are mainly characterized by CD3 - CD56 + Lymphocyte population.

[0095] CD3: The signal transduction component of the T cell receptor (TCR) complex, used to indicate whether there is T cell interference in the NK differentiation process, because some T cells can also express NK cell surface markers.

[0096] CD94: CD94 exists on the surface of NK cells mainly in the form of homotypic or heterotypic forms with NKG2A, and is a sign of the maturation of differentiated NK cells.

[0097] Nkp30: NKp30 belongs to the CD28 family and is a member of the immunoglobulin superfamily (IgSF). It is only expressed 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 activating receptor, it plays an important role in immune surveillance and killing of tumor and pathogen-infected cells by binding to its corresponding ligand.

[0098] Nkp44: NKp44 is a member of the immunoglobulin superfamily (IgSF) and is only expressed on the surface of activated NK cells. It is a specific marker for activated NK cells and has only one V-type domain in its extracellular region. The NKp44 cytoplasmic region does not contain ITAM motifs, and the transmembrane region contains positively charged lysine, which can non-covalently bind to the DAP12 homodimer with negatively charged aspartic acid in the transmembrane region and ITAM motifs in the cytoplasmic region, thereby obtaining the function of transducing activation signals. NKp44 is only expressed after NK cells are activated, and plays a role in further amplifying the signal.

[0099] Nkp46: NKp46 is expressed on the surface of all NK cells (including mature, immature, resting and activated NK cells). The extracellular region of NKp46 contains two Ig-like domains. The cytoplasmic region of NKp46 is shorter, and the transmembrane region contains positively charged arginine, which can non-covalently bind to CD3ζζ homodimers containing negatively charged aspartic acid in the transmembrane region and ITAM motifs in the cytoplasmic region, thereby obtaining the function of transducing activation signals.

[0100] As used herein, the term "first culture medium" and similarly used "second culture medium", "third culture medium", "fourth culture medium", "fifth culture medium", etc. are used only for the purpose of distinction and are not intended to limit the order in which these culture media are used. The above terms used in different protocols may be the same or different.

[0101] Unless otherwise stated, all methods described herein can be performed in any suitable order.

[0102] Reagent test kit

[0103] In a first aspect, the present invention provides a 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 comprising a BMP signaling pathway activator; the fourth culture medium is a basal culture medium comprising at least one, at least two, at least three or at least four of the following: a BMP signaling pathway activator, a vascular endothelial growth factor, a colony stimulating factor, an interleukin; and the fifth culture medium is a basal culture medium comprising at least one, at least two, at least three, at least four, at least five or at least six of the following: a BMP signaling pathway activator, a vascular endothelial growth factor, a colony stimulating factor, an interleukin, a stem cell agonist, a stem cell expander.

[0104] In some embodiments, the kit is a kit for inducing stem cells to differentiate into natural killer cells.

[0105] In one embodiment, the second culture medium is a basal culture medium comprising a BMP signaling pathway activator and a GSK3 inhibitor.

[0106] In one embodiment, the BMP signaling pathway activator of the second culture medium comprises at least one of BMP2, BMP4, SB4, SJ000291942, SJ000063181, SJ000370178, isoliquiritigenin, diosmetin, apigenin and biochanin. In a preferred embodiment, the BMP signaling pathway activator of the second culture medium comprises BMP4.

[0107] In one embodiment, 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.

[0108] In one embodiment, 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.

[0109] In one embodiment, 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.

[0110] In one embodiment, the basal medium of the second culture medium comprises at least one of IMDM (Iscove's Modified Dulbecco's Medium) medium, Eagle's Basal Medium (BME) medium, MEM medium, DMEM medium, Ham's F-12 medium, RPMI1640 medium, Fischer's medium, and DMEM / F12 medium. In a preferred embodiment, the basal medium of the second culture medium comprises RPMI1640 medium.

[0111] In one embodiment, the basal medium of the second culture medium comprises at least one, at least two, at least three or at least four of the following: non-essential amino acids, glutamine, vitamin C, B27 culture medium supplement. In a preferred embodiment, the basal medium of the second culture medium comprises non-essential amino acids, glutamine, vitamin C and B27 culture medium supplement.

[0112] In one embodiment, the B27 medium supplement is a B27 medium supplement without vitamin A.

[0113] In one embodiment, 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).

[0114] In one embodiment, 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).

[0115] In one embodiment, 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.

[0116] In one embodiment, the concentration of the B27 medium supplement in the second culture medium is 1%-10% (v / v), preferably 2% (v / v).

[0117] In the present invention, the second culture medium is used to induce embryoid bodies to differentiate into mesodermal cells.

[0118] In one embodiment, the BMP signaling pathway activator of the fourth culture medium and / or the fifth culture medium comprises at least one of BMP2, BMP4, SB4, SJ000291942, SJ000063181, SJ000370178, isoliquiritigenin, diosmetin, apigenin and biochanin. In a preferred embodiment, the BMP signaling pathway activator of the fourth culture medium and / or the fifth culture medium comprises BMP4.

[0119] In one embodiment, the vascular endothelial growth factor of the fourth culture medium and / or the fifth culture medium 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 of the fourth culture medium and / or the fifth culture medium comprises VEGF-165.

[0120] In one embodiment, the colony stimulating factor of the fourth culture medium comprises at least one of G-CSF (granulocyte colony stimulating factor), M-CSF (macrophage colony stimulating factor), GM-CSF (recombinant human granulocyte-macrophage colony stimulating factor), multi-CSF (multiple colony stimulating factor, also known as IL-3), EPO (erythropoietin), TPO, SCF, and F1T3L. In a preferred embodiment, the colony stimulating factor of the fourth culture medium comprises at least one of IL-3, SCF, and F1T3L. In a more preferred embodiment, the colony stimulating factor of the fourth culture medium comprises IL-3, SCF, and F1T3L.

[0121] In one embodiment, the colony stimulating factor of the fifth culture medium comprises at least one of G-CSF (granulocyte colony stimulating factor), M-CSF (macrophage colony stimulating factor), GM-CSF (recombinant human granulocyte-macrophage colony stimulating factor), multi-CSF (multiple colony stimulating factor, also known as IL-3), EPO (erythropoietin), TPO, SCF, and F1T3L. In a preferred embodiment, the colony stimulating factor of the fifth culture medium comprises at least one of SCF and F1T3L. In a more preferred embodiment, the colony stimulating factor of the fifth culture medium comprises SCF and F1T3L.

[0122] In one embodiment, the interleukin of the fourth culture medium and / or the fifth culture medium comprises 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 interleukin of the fourth culture medium and / or the fifth culture medium comprises at least one of IL-7 and IL-15. In a more preferred embodiment, the interleukin of the fourth culture medium and / or the fifth culture medium comprises IL-7 and IL-15.

[0123] In one embodiment, the stem cell agonist of the fifth culture medium comprises at least one of UM171 and UM729. In a preferred embodiment, the stem cell agonist comprises UM171.

[0124] In one embodiment, the stem cell expander of the fifth culture medium comprises at least one of SR1 and PD98059. In a preferred embodiment, the stem cell expander comprises SR1.

[0125] In one embodiment, 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.

[0126] In one embodiment, 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.

[0127] In one embodiment, the concentration of the colony stimulating factor in the fourth culture medium is 7-200 ng / mL; preferably 16-80 ng / mL; more preferably 35 ng / mL.

[0128] In one embodiment, 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.

[0129] In one embodiment, the concentration of SCF in the fourth culture medium is 5-100 ng / mL; preferably 10-50 ng / mL; more preferably 20 ng / mL.

[0130] In one embodiment, the concentration of FLT3L in the fourth culture medium is 1-50 ng / mL; preferably 5-20 ng / mL; more preferably 10 ng / mL.

[0131] In one embodiment, the concentration of interleukin in the fourth culture medium is 2-100 ng / mL; preferably 15-70 ng / mL; more preferably 30 ng / mL.

[0132] In one embodiment, the concentration of IL15 in the fourth culture medium is 1-50 ng / mL; preferably 5-20 ng / mL; more preferably 10 ng / mL.

[0133] In one embodiment, the concentration of IL7 in the fourth culture medium is 1-50 ng / mL; preferably 10-50 ng / mL; more preferably 20 ng / mL.

[0134] In one embodiment, 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.

[0135] In one embodiment, 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.

[0136] In one embodiment, the concentration of the colony stimulating factor in the fifth culture medium is 6-150 ng / mL; preferably 15-70 ng / mL; more preferably 30 ng / mL.

[0137] In one embodiment, the concentration of SCF in the fifth culture medium is 5-100 ng / mL; preferably 10-50 ng / mL; more preferably 20 ng / mL.

[0138] In one embodiment, the concentration of FLT3L in the fifth culture medium is 1-50 ng / mL; preferably 5-20 ng / mL; more preferably 10 ng / mL.

[0139] In one embodiment, the concentration of interleukin in the fifth culture medium is 2-100 ng / mL; preferably 15-70 ng / mL; more preferably 30 ng / mL.

[0140] In one embodiment, the concentration of IL15 in the fifth culture medium is 1-50 ng / mL; preferably 5-20 ng / mL; more preferably 10 ng / mL.

[0141] In one embodiment, the concentration of IL7 in the fifth culture medium is 1-50 ng / mL; preferably 10-50 ng / mL; more preferably 20 ng / mL.

[0142] In one embodiment, 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.

[0143] In one embodiment, the concentration of the stem cell expander in the fifth culture medium is 0.5-10 μM; preferably 0.5 μM-5 μM; more preferably 1 μM.

[0144] In one embodiment, the basal medium of the fourth culture medium comprises at least one of IMDM (Iscove's Modified Dulbecco's Medium) medium, Eagle's Basal Medium (BME) medium, MEM medium, DMEM medium, Ham's F-12 medium, RPMI1640 medium, Fischer's medium, and DMEM / F12 medium. In a preferred embodiment, the basal medium of the fourth culture medium comprises IMDM (Iscove's Modified Dulbecco's Medium) medium.

[0145] In one embodiment, the basal medium of the fourth culture medium comprises at least one, at least two, at least three, at least four, at least five, or at least six of the following: non-essential amino acids, glutamine, vitamin C, B27 medium supplement, N-acetyl-L-cysteine ​​(NAC). In a preferred embodiment, the basal medium of the fourth culture medium comprises non-essential amino acids, glutamine, vitamin C, B27 medium supplement, and N-acetyl-L-cysteine ​​(NAC).

[0146] In one embodiment, the B27 medium supplement is a B27 medium supplement without vitamin A.

[0147] In one embodiment, 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).

[0148] In one embodiment, 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).

[0149] In one embodiment, 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.

[0150] In one embodiment, the concentration of the B27 medium supplement in the fourth culture medium is 1-10% (v / v), preferably 2% (v / v).

[0151] In one embodiment, the concentration of N-acetyl-L-cysteine ​​in the fourth culture medium is 1-100 μM; preferably 10-50 μM, more preferably 30 μM.

[0152] In one embodiment, the basal medium of the fifth culture medium comprises at least one of IMDM (Iscove's Modified Dulbecco's Medium) medium, Eagle's Basal Medium (BME) medium, MEM medium, DMEM medium, Ham's F-12 medium, RPMI1640 medium, Fischer's medium, and DMEM / F12 medium. In a preferred embodiment, the basal medium of the fifth culture medium comprises IMDM (Iscove's Modified Dulbecco's Medium) medium.

[0153] In one embodiment, the basal medium of the fifth culture medium comprises at least one, at least two, at least three, at least four, at least five, or at least six of the following: non-essential amino acids, glutamine, vitamin C, B27 medium supplement, N-acetyl-L-cysteine ​​(NAC). In a preferred embodiment, the basal medium of the fifth culture medium comprises non-essential amino acids, glutamine, vitamin C, B27 medium supplement, and N-acetyl-L-cysteine ​​(NAC).

[0154] In one embodiment, the B27 medium supplement is a B27 medium supplement without vitamin A.

[0155] In one embodiment, 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).

[0156] In one embodiment, 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).

[0157] In one embodiment, 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.

[0158] In one embodiment, the concentration of the B27 medium supplement in the fifth medium is 1-10% (v / v), preferably 2% (v / v).

[0159] In one embodiment, the concentration of N-acetyl-L-cysteine ​​in the fifth culture medium is 1-100 μM; preferably 10-50 μM, more preferably 30 μM.

[0160] In the present invention, the fourth culture medium and the fifth culture medium are used to induce differentiation of hemogenic endothelial cells into NK cells and activation (maturation and / or expansion) of NK cells.

[0161] In one embodiment, the kit further comprises a third culture medium, which is 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.

[0162] In one embodiment, the BMP signaling pathway activator of the third culture medium comprises at least one of BMP2, BMP4, SB4, SJ000291942, SJ000063181, SJ000370178, isoliquiritigenin, diosmetin, apigenin and biochanin. In a preferred embodiment, the BMP signaling pathway activator of the third culture medium comprises BMP4.

[0163] In one embodiment, the growth factor of the third culture medium comprises at least one of 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), oncostatin M (OSM), platelet-derived endothelial cell growth factor (PDECGF), transforming growth factor-α (TGF-α), and vascular endothelial cell growth factor (VEGF). In a preferred embodiment, the growth factor of the third culture medium comprises at least one of fibroblast growth factor (FGF) and vascular endothelial cell growth factor (VEGF). In a more preferred embodiment, the growth factor of the third culture medium comprises fibroblast growth factor (FGF) and vascular endothelial cell growth factor (VEGF).

[0164] In one embodiment, 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.

[0165] In one embodiment, 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.

[0166] In one embodiment, 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.

[0167] In one embodiment, 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.

[0168] In one embodiment, 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.

[0169] In one embodiment, 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.

[0170] In one embodiment, the concentration of the TGFβ / ALK inhibitor in the third culture medium is 1-50 μM; preferably 5-20 μM; more preferably 10 μM.

[0171] In one embodiment, the basal medium of the third culture medium comprises at least one of IMDM (Iscove's Modified Dulbecco's Medium) medium, Eagle's Basal Medium (BME) medium, MEM medium, DMEM medium, Ham's F-12 medium, RPMI1640 medium, Fischer's medium, and DMEM / F12 medium. In a preferred embodiment, the basal medium of the third culture medium comprises RPMI1640 medium.

[0172] In one embodiment, the basal medium of the third culture medium comprises at least one, at least two, at least three or at least four of the following: non-essential amino acids, glutamine, vitamin C, B27 culture medium supplement. In a preferred embodiment, the basal medium of the third culture medium comprises non-essential amino acids, glutamine, vitamin C and B27 culture medium supplement.

[0173] In one embodiment, the B27 medium supplement is a B27 medium supplement without vitamin A.

[0174] In one embodiment, 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).

[0175] In one embodiment, 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).

[0176] In one embodiment, 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.

[0177] In one embodiment, the concentration of the B27 medium supplement in the third culture medium is 1%-10% (v / v), preferably 2% (v / v).

[0178] In the present invention, the third culture medium is used to induce mesodermal cells to differentiate into hemogenic endothelial cells.

[0179] In one embodiment, the kit further comprises a first culture medium, which is a basal culture medium comprising a ROCK inhibitor.

[0180] In one embodiment, 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.

[0181] In one embodiment, the concentration of the ROCK inhibitor in the first culture medium is 1-50 μM; preferably 5 μM-20 μM; more preferably 10 μM.

[0182] In one embodiment, the basal medium of the first culture medium comprises at least one of E8 culture medium, mTESR culture medium, StemFitBasic 03, StemFit Basic 04, NutriStem hPSC XF culture medium, StemMACS iPS Brew culture medium, Stem-Partner ACF culture medium, TeSR-AOF culture medium, and TeSR2 culture medium. In a preferred embodiment, the basal medium of the first culture medium comprises StemFit Basic 03.

[0183] In the present invention, the first culture medium is used to obtain embryoid bodies.

[0184] In one embodiment, the stem cells described herein are stem cells with multidirectional differentiation potential.

[0185] In one embodiment, the stem cells with multidirectional 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 multidirectional differentiation potential include induced pluripotent stem cells.

[0186] In one embodiment, the stem cells are derived from mammals; preferably, they are derived from primates; more preferably, they are derived from humans.

[0187] In one embodiment, the stem cells are commercial human embryonic stem cells (hESCs) (eg, H1, H9) and / or human induced pluripotent stem cells (hiPSCs) (eg, WC50, IMR90).

[0188] In one embodiment, embryonic stem cells are stem cells isolated or obtained from human embryos within 14 days of fertilization that have not undergone in vivo development.

[0189] In a specific embodiment, the kit 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 a StemFit Basic 03 culture medium containing 10 mM Y27632 2HCl; the second culture medium is an RPMI1640 culture medium containing 1% (v / v) non-essential amino acids, 1% (v / v) glutamine, 50 mg / mL vitamin C, 2% (v / v) B27 culture medium supplement without vitamin A, 20 ng / mL BMP4 and 3 mM CHIR-99021; the third culture medium is an RPMI1640 culture medium containing 1% (v / v) non-essential amino acids, 1% (v / v) glutamine, 50 mg / mL vitamin C, 2% (v / v) B27 culture medium supplement without vitamin A, 5 ng / mL BMP4, 50 ng / mL VEGF-165, 50 ng / mL bFGF and 10mM SB431542; the fourth culture medium is IMDM medium containing 1% (v / v) non-essential amino acids, 1% (v / v) glutamine, 50mg / mL vitamin C, 2% (v / v) B27 medium supplement without vitamin A, 30mM N-acetyl-L-cysteine ​​(NAC), 5ng / mL BMP4, 10ng / mL VEGF-165, 20ng / mL SCF, 5ng / mL IL3, 10ng / mL IL15, 20ng / mL IL7, and 10ng / mL FLT3L; the fifth culture medium is IMDM medium containing 1% (v / v) non-essential amino acids, 1% (v / v) glutamine, 50mg / mL vitamin C, 2% (v / v) B27 medium supplement without vitamin A, 30mM NAC, 5ng / mL BMP4, 10ng / mL VEGF, 20ng / mL IMDM medium containing SCF, 10 ng / mL IL15, 20 ng / mL IL7, 10 ng / mL FLT3L, 1 mM UM171, and 1 mM SR1.

[0190] In a specific embodiment, the kit 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 a StemFit Basic03 culture medium comprising 10 mM Y27632 or Y27632 2HCl; the second culture medium is an RPMI1640 culture medium comprising 1% non-essential amino acids, 1% glutamine, 50 mg / mL vitamin C, 2% (v / v) B27 culture medium supplement without vitamin A, 20 ng / mL BMP4 and 3 mM CHIR-99021; the third culture medium is a RPMI1640 culture medium comprising 1% non-essential amino acids, 1% glutamine, 50 mg / mL vitamin C, 2% (v / v) B27 culture medium supplement without vitamin A, 5 ng / mL BMP4, 50 ng / mL VEGF, 50 ng / mL bFGF and 10 mM The fourth culture medium is RPMI1640 medium containing SB431542; the fourth culture medium is IMDM medium containing 1% non-essential amino acids, 1% glutamine, 50 mg / mL vitamin C, 2% (v / v) B27 culture medium supplement without vitamin A, 5 ng / mL BMP4, 10 ng / mL VEGF, 20 ng / mL SCF, 5 ng / mL IL3, 10 ng / mL IL15, 20 ng / mL IL7, 10 ng / mL FLT3L; the fifth culture medium is IMDM medium containing 1% non-essential amino acids, 1% glutamine, 50 mg / mL vitamin C, 2% (v / v) B27 culture medium supplement without vitamin A, 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 mM UM171, 1 mM SR1, 30 mM NAC-containing IMDM medium.

[0191] In a second aspect, the present invention provides the use of the kit of the present invention in preparing embryoid bodies, preparing mesoderm cells, preparing hemogenic endothelial cells, preparing natural killer (NK) cells, preparing products for inducing stem cells to differentiate into embryoid bodies, preparing products for inducing stem cells to differentiate into mesoderm cells, preparing products for inducing stem cells to differentiate into hemogenic endothelial cells, and preparing products for inducing stem cells to differentiate into NK cells.

[0192] In one embodiment, the stem cells described herein are stem cells with multidirectional differentiation potential.

[0193] In one embodiment, the stem cells with multidirectional 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 multidirectional differentiation potential include induced pluripotent stem cells.

[0194] In one embodiment, the stem cells are derived from mammals; preferably, they are derived from primates; more preferably, they are derived from humans.

[0195] In one embodiment, the stem cells are commercial human embryonic stem cells (hESCs) (eg, H1, H9) and / or human induced pluripotent stem cells (hiPSCs) (eg, WC50, IMR90).

[0196] In one embodiment, human embryonic stem cells are stem cells isolated or obtained from human embryos within 14 days of fertilization that have not undergone in vivo development.

[0197] Method for inducing stem cells to differentiate into natural killer cells

[0198] In a third aspect, the present invention provides a method for inducing stem cells to differentiate into natural killer cells, the method comprising the steps of using the kit of the first aspect of the present invention.

[0199] In one embodiment, the method comprises:

[0200] Step (1) culturing stem cells to obtain embryoid bodies;

[0201] Step (2) performing mesodermal differentiation culture on the embryoid bodies to obtain mesodermal cells;

[0202] Step (3) differentiating and culturing the mesoderm cells into hemogenic endothelial cells to obtain hemogenic endothelial cells;

[0203] Step (4) The hemogenic endothelial cells are placed in the fourth culture medium and the fifth culture medium of the kit of the first aspect of the present invention and cultured to obtain NK cells.

[0204] In one embodiment, the culturing in step (1) is performed in the first culture medium of the kit of the first aspect of the present invention.

[0205] In one embodiment, the culturing in step (2) is performed in the second culture medium of the kit of the first aspect of the present invention.

[0206] In one embodiment, the culturing in step (3) is performed in the third culture medium of the kit of the first aspect of the present invention.

[0207] In one embodiment, the culturing time in step (1) is 12-36 h, preferably 20-28 h, and more preferably 20-24 h.

[0208] In one embodiment, the culturing time in step (2) is 24-72 h, preferably 36-60 h, and more preferably 42-48 h.

[0209] In one embodiment, the culturing time in step (3) is 48-144 h, preferably 72-120 h, and more preferably 90-96 h.

[0210] In one embodiment, in step (4), culturing the hemogenic endothelial cells in the fourth culture medium and the fifth culture medium of the kit of the first aspect of the present invention is culturing the hemogenic endothelial cells in the fourth culture medium and the fifth culture medium of the kit of the first aspect of the present invention in sequence.

[0211] In one embodiment, the culture time of the hemogenic endothelial cells in the fourth culture medium is 96-192 h, preferably 120-192 h, and more preferably 144-168 h.

[0212] In one embodiment, the culture time of the hemogenic endothelial cells in the fifth culture medium is 144-432 h, preferably 216-360 h, and more preferably 312-336 h.

[0213] In one embodiment, the hemogenic endothelial cells are cultured in the fifth culture medium with a culture medium change every 5-7 days.

[0214] In one embodiment, the culture conditions are 35-38°C and 4-6% CO2.

[0215] In one embodiment, the cultivation in steps (1), (2) and (3) is suspension cultivation.

[0216] In one embodiment, the culturing in steps (1), (2) and (3) is performed in a low attachment plate.

[0217] In one embodiment, the culturing in step (4) is adherent culturing.

[0218] In one embodiment, the culturing in step (4) is performed in a normal adherent well plate.

[0219] In one embodiment, the stem cells described herein are stem cells with multidirectional differentiation potential.

[0220] In one embodiment, the stem cells with multidirectional 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 multidirectional differentiation potential include induced pluripotent stem cells.

[0221] In one embodiment, the stem cells are derived from mammals; preferably, they are derived from primates; more preferably, they are derived from humans.

[0222] In one embodiment, the stem cells are commercial human embryonic stem cells (hESCs) (eg, H1, H9) and / or human induced pluripotent stem cells (hiPSCs) (eg, WC50, IMR90).

[0223] In one embodiment, human embryonic stem cells are stem cells isolated or obtained from human embryos within 14 days of fertilization that have not undergone in vivo development.

[0224] NK cells

[0225] In a fourth aspect, the present invention provides a natural killer (NK) cell, which is prepared by the kit of the first aspect of the present invention and / or the method of the third aspect of the present invention.

[0226] In one embodiment, the NK cells have CD45 + 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; and more preferably 96.72%.

[0227] In one embodiment, the NK cells have CD45 + The content of CD3- cells 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; more preferably 98.87%.

[0228] In one embodiment, the NK cells have CD56 + The content of CD3- cells 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; more preferably 97.12%.

[0229] In one embodiment, the NK cells have CD56 + 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; more preferably 53.45%.

[0230] In one embodiment, the NK cells have CD45 + 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; more preferably 47.90%.

[0231] In one embodiment, the content of CD3-CD16- cells in the NK cells is 90% or more, 95% or more, or 96% or more; preferably 96% or more; more preferably 96.47%.

[0232] In one embodiment, the NK cells have CD3-CD45 + CD56 + The cell content is 80% or more, 85% or more, or 90% or more; preferably 90% or more; more preferably 90.64%.

[0233] In one embodiment, the NK cells are CD3-Nkp46 + The cell content is 60% or more, 65% or more, or 70% or more; preferably 70% or more; more preferably 73.96%.

[0234] In one embodiment, the NK cells are CD3-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; more preferably 93.35%.

[0235] In one embodiment, the NK cells are CD3-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; more preferably 87.96%.

[0236] In one embodiment, the NK cells are CD3-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; more preferably 85.77%.

[0237] In one embodiment, the NK cells comprise a population of NK cells.

[0238] Pharmaceutical composition

[0239] In a fifth aspect, the present invention provides a pharmaceutical composition comprising the NK cells of the fourth aspect of the present invention.

[0240] In one embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0241] In one embodiment, the pharmaceutical composition is a liquid preparation.

[0242] In one embodiment, the pharmaceutical composition is an intravenous injection agent.

[0243] In one embodiment, the pharmaceutically acceptable carrier includes but is not limited to: saline, buffer, glucose, water, DMSO, and combinations thereof.

[0244] In one embodiment, the concentration of NK cells in the pharmaceutical composition is 1×10 3 -1×10 7 / μL; preferably 1×10 4 -1×10 6 / μL; more preferably 1×10 5 -9.9×10 5 Pieces / μL.

[0245] In one embodiment, the pharmaceutical composition further comprises an anti-tumor drug.

[0246] In one embodiment, the anti-tumor drug comprises at least one of camptothecin, temozolomide, PARP inhibitor, ATR inhibitor, CDK4 / 6 inhibitor, cyclophosphamide, doxorubicin, cisplatin, and paclitaxel; preferably, the drug comprises temozolomide.

[0247] In one embodiment, the NK cells and the anti-tumor drug are mixed or exist independently.

[0248] In one embodiment, the tumor comprises at least one of a blood tumor and a solid tumor; preferably, the tumor comprises at least one of 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, kidney cancer, and glioma; more preferably, the tumor comprises cervical cancer and glioma.

[0249] Drug combinations

[0250] In a seventh aspect, the present invention provides a pharmaceutical combination for treating glioblastoma, the pharmaceutical combination comprising an active ingredient comprising natural killer (NK) cells and a chemotherapeutic agent, wherein the chemotherapeutic agent comprises temozolomide.

[0251] As used herein, the term "active ingredient" refers to any substance or mixture of substances in a pharmaceutical composition that has pharmacological activity in the diagnosis, treatment, symptom relief, prevention of recurrence or prevention of disease, and can act directly or indirectly.

[0252] In one embodiment, the glioblastoma is resistant to TMZ.

[0253] In one embodiment, the glioblastoma is resistant to TMZ, and TMZ induces upregulation of expression of one or more immune ligands selected from CD112, CD155, CD54, CD70, B7-H6, CD48, PD-L1, and CD1d in the glioblastoma.

[0254] In one embodiment, the NK cells are unmodified NK cells.

[0255] As used herein, the term "unmodified" means that there is no genetic modification by intentionally adding exogenous genetic material in the form of DNA or RNA to the total genetic material of the cell to insert genes or edit the genes (directed modification). For example, the term "unmodified" means that there is no intentional genetic modification that results in the insertion or deletion of DNA fragments, or the deletion or substitution of DNA bases.

[0256] In one embodiment, NK cells include but are not limited to NK-92 cells, peripheral blood or umbilical cord blood-derived NK cells, NK cells derived from induced pluripotent stem cells (iPSCs) or embryonic stem cells.

[0257] In one embodiment, the embryonic stem cells are commercial human embryonic stem cells (hESCs) (eg, H1, H9).

[0258] In one embodiment, embryonic stem cells are stem cells isolated or obtained from human embryos within 14 days of fertilization that have not undergone in vivo development.

[0259] In one embodiment, the induced pluripotent stem cells (iPSCs) are human induced pluripotent stem cells (hiPSCs) (eg, WC50, IMR90).

[0260] In one embodiment, the NK cells are NK cells derived from induced pluripotent stem cells (iPSCs).

[0261] In one embodiment, the NK cells are unmodified NK cells derived from induced pluripotent stem cells.

[0262] In one embodiment, NK cells derived from induced pluripotent stem cells are prepared by the kit of the first aspect of the present invention or the method of the third aspect of the present invention.

[0263] In one embodiment, the drug combination further comprises other chemotherapeutic agents. Other chemotherapeutic agents include, but are not limited to, anticancer agents, metabolic antagonists, DNA demethylating agents, antitumor agents derived from plants, alkylating agents, antimetabolites, anticancer antibiotics, topoisomerase inhibitors, mitotic inhibitors, differentiation agents, and hormone therapeutic agents. For example, antitumor agents (e.g., actinomycin D) D), vincristine, vinblastine, alkylating agents, platinum compounds, antimetabolites and nucleoside analogs, such as methotrexate and purine and pyrimidine analogs), antineoplastic 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 The invention relates to thiotepa, chlorambucil, razithromycin (CC-1065), melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C and cis-dichlorodiamine platinum (II) (DDP, cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin and anthramycin (AMC)), antimitotic agents (e.g., vincristine, vinblastine, paclitaxel and maytansine). In one embodiment, other chemotherapeutic agents are selected from anticancer agents, metabolic antagonists, DNA demethylating agents, antitumor agents derived from plants, alkylating agents, antimetabolites, anticancer antibiotics, topoisomerase inhibitors, mitotic inhibitors, differentiation agents, 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 derivatives thereof.

[0264] In one embodiment, the additional chemotherapeutic agent is selected from carmustine, lomustine, vorinostat, mebendazole, imatinib mesylate, AG119, or any therapeutic derivatives thereof.

[0265] In one embodiment, the active ingredient does not comprise trimetrexate.

[0266] In one embodiment, the active ingredient does not comprise an immune checkpoint inhibitor.

[0267] In one embodiment, the chemotherapeutic agent is only temozolomide.

[0268] In one embodiment, the active ingredients consist of natural killer cells and a chemotherapeutic agent.

[0269] In one embodiment, the active ingredients consist of natural killer cells and temozolomide.

[0270] In one embodiment, the active ingredients are NK-92 cells and temozolomide, peripheral blood-derived or cord blood-derived NK cells and temozolomide, induced pluripotent stem cell (iPSC)-derived NK cells and temozolomide, or embryonic stem cell-derived NK cells and temozolomide.

[0271] In one embodiment, the active ingredients consist of NK cells derived from induced pluripotent stem cells and temozolomide.

[0272] In one embodiment, the active ingredients consist of unmodified NK cells derived from induced pluripotent stem cells and temozolomide.

[0273] In one embodiment, the active ingredients consist of NK cells derived from induced pluripotent stem cells and temozolomide, and the NK cells derived from induced pluripotent stem cells are prepared by the kit of the first aspect of the present invention.

[0274] In one embodiment, the active ingredients consist of NK cells derived from induced pluripotent stem cells and temozolomide, and the NK cells derived from induced pluripotent stem cells are prepared by the method for inducing stem cells to differentiate into natural killer cells according to the third aspect of the present invention.

[0275] In one embodiment, the active ingredients consist of unmodified NK cells derived from induced pluripotent stem cells and temozolomide, and the NK cells derived from induced pluripotent stem cells are prepared by the kit of the first aspect of the present invention.

[0276] In one embodiment, the active ingredients consist of unmodified NK cells derived from induced pluripotent stem cells and temozolomide, and the NK cells derived from induced pluripotent stem cells are prepared by the method for inducing stem cells to differentiate into natural killer cells according to the third aspect of the present invention.

[0277] In one embodiment, the drug combination is used to treat glioblastoma, wherein the glioblastoma is resistant to TMZ, wherein the drug combination comprises an active ingredient consisting of NK cells derived from induced pluripotent stem cells and temozolomide.

[0278] In one embodiment, the drug combination is used to treat glioblastoma, wherein the glioblastoma is resistant to TMZ, wherein the drug combination comprises an active ingredient consisting of unmodified NK cells derived from induced pluripotent stem cells and temozolomide.

[0279] In one embodiment, the active ingredients of the pharmaceutical combination may be used simultaneously or separately (eg, sequentially).

[0280] In one embodiment, the pharmaceutical combination is a kit.

[0281] In the tenth aspect, the present invention provides a kit comprising the natural killer cells and chemotherapeutic agents defined in the seventh aspect of the present invention or the drug combination of the seventh aspect, and instructions for use. The kit may also include a suitable container. In certain embodiments, the kit also includes a device for administration. The kit generally includes a label indicating the intended use and / or method of use of the contents of the kit. The term "label" includes any written or recorded material provided on or with the kit or otherwise provided with the kit.

[0282] Methods of treating glioblastoma

[0283] In an eleventh aspect, the present invention also provides a method for treating glioblastoma in a subject in need thereof, comprising the following steps:

[0284] administering natural killer (NK) cells to the subject; and

[0285] administering a chemotherapeutic agent to the subject,

[0286] Among them, chemotherapeutic agents include temozolomide (TMZ).

[0287] In one embodiment, the chemotherapeutic agent is administered prior to administering NK cells to the subject, after administering NK cells to the subject, or simultaneously with administering NK cells to the subject, or any combination thereof.

[0288] In one embodiment, the glioblastoma is resistant to TMZ.

[0289] In one embodiment, the glioblastoma is resistant to TMZ, and TMZ induces upregulation of expression of one or more immune ligands selected from CD112, CD155, CD54, CD70, B7-H6, CD48, PD-L1, and CD1d in the glioblastoma.

[0290] In one embodiment, the NK cells are unmodified NK cells.

[0291] In one embodiment, NK cells include, but are not limited to, NK-92 cells, peripheral blood-derived or umbilical cord blood-derived NK cells, NK cells derived from induced pluripotent stem cells (iPSCs) or embryonic stem cells.

[0292] In one embodiment, the embryonic stem cells are commercial human embryonic stem cells (hESCs) (eg, H1, H9).

[0293] In one embodiment, embryonic stem cells are stem cells isolated or obtained from human embryos within 14 days of fertilization that have not undergone in vivo development.

[0294] In one embodiment, the induced pluripotent stem cells (iPSCs) are human induced pluripotent stem cells (hiPSCs) (eg, WC50, IMR90).

[0295] In one embodiment, the NK cells are NK cells derived from induced pluripotent stem cells (iPSCs).

[0296] In one embodiment, the NK cells are unmodified NK cells derived from induced pluripotent stem cells.

[0297] In one embodiment, NK cells derived from induced pluripotent stem cells are prepared by the kit of the first aspect of the present invention.

[0298] In one embodiment, NK cells derived from induced pluripotent stem cells are prepared by the method for inducing stem cells to differentiate into natural killer cells according to the third aspect of the present invention.

[0299] In one embodiment, the method further comprises administering to the subject other chemotherapeutic agents. Other chemotherapeutic agents include, but are not limited to, anticancer agents, metabolic antagonists, DNA demethylating agents, antitumor agents derived from plants, alkylating agents, antimetabolites, anticancer antibiotics, topoisomerase inhibitors, mitotic inhibitors, differentiation agents, hormone therapy agents. For example, antitumor agents (e.g., actinomycin D) D), vincristine, vinblastine, alkylating agents, platinum compounds, antimetabolites and nucleoside analogs, such as methotrexate and purine and pyrimidine analogs), antineoplastic 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 The invention relates to thiotepa, chlorambucil, razithromycin (CC-1065), melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C and cis-dichlorodiamine platinum (II) (DDP, cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin and anthramycin (AMC)), antimitotic agents (e.g., vincristine, vinblastine, paclitaxel and maytansine). In one embodiment, other chemotherapeutic agents are selected from anticancer agents, metabolic antagonists, DNA demethylating agents, antitumor agents derived from plants, alkylating agents, antimetabolites, anticancer antibiotics, topoisomerase inhibitors, mitotic inhibitors, differentiation agents, 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 derivatives thereof.

[0300] In one embodiment, the additional chemotherapeutic agent is selected from carmustine, lomustine, vorinostat, mebendazole, imatinib mesylate, AG119, or any therapeutic derivatives thereof.

[0301] In one embodiment, other chemotherapeutic agents are administered before, after, or simultaneously with the administration of NK cells or temozolomide (TMZ) to the subject, or any combination thereof.

[0302] In one embodiment, the method does not comprise administering trimetrexate to the subject.

[0303] In one embodiment, the method does not comprise administering an immune checkpoint inhibitor to the subject.

[0304] In one embodiment, the chemotherapeutic agent is temozolomide (TMZ).

[0305] In one embodiment, the method comprises the following steps:

[0306] administering natural killer (NK) cells to the subject; and

[0307] The subject is administered a chemotherapeutic agent, wherein the chemotherapeutic agent is administered before the subject is administered NK cells, after the subject is administered NK cells, or simultaneously with the subject is administered NK cells, or any combination thereof, wherein the chemotherapeutic agent is temozolomide.

[0308] In one embodiment, the method comprises the following steps:

[0309] administering to the subject NK cells derived from induced pluripotent stem cells; and

[0310] The subject is treated with a chemotherapeutic agent, wherein the chemotherapeutic agent is administered before the subject is treated with NK cells derived from induced pluripotent stem cells, after the subject is treated with NK cells derived from induced pluripotent stem cells, or simultaneously with the subject being treated with NK cells derived from induced pluripotent stem cells, or any combination thereof, wherein the chemotherapeutic agent is temozolomide.

[0311] In one embodiment, a method for treating glioblastoma in a subject in need thereof comprises the following steps:

[0312] administering to the subject NK cells derived from induced pluripotent stem cells; and

[0313] administering a chemotherapeutic agent to the subject, wherein the chemotherapeutic agent is administered before administering the NK cells derived from induced pluripotent stem cells to the subject, after administering the NK cells derived from induced pluripotent stem cells to the subject, or simultaneously with administering the NK cells derived from induced pluripotent stem cells to the subject, or any combination thereof,

[0314] Among them, the chemotherapy agent is temozolomide (TMZ),

[0315] Among them, glioblastoma is resistant to TMZ.

[0316] In one embodiment, a method for treating glioblastoma in a subject in need thereof comprises the following steps:

[0317] administering to the subject unmodified NK cells derived from induced pluripotent stem cells; and

[0318] administering a chemotherapeutic agent to the subject, wherein the chemotherapeutic agent is administered before administering unmodified NK cells derived from induced pluripotent stem cells to the subject, after administering unmodified NK cells derived from induced pluripotent stem cells to the subject, or simultaneously with administering unmodified NK cells derived from induced pluripotent stem cells to the subject, or any combination thereof,

[0319] Among them, the chemotherapy agent is temozolomide (TMZ),

[0320] Among them, glioblastoma is resistant to TMZ.

[0321] In one embodiment, a method for treating glioblastoma in a subject in need thereof comprises the following steps:

[0322] administering to the subject NK cells derived from induced pluripotent stem cells; and

[0323] administering a chemotherapeutic agent to the subject, wherein the chemotherapeutic agent is administered before administering the NK cells derived from induced pluripotent stem cells to the subject, after administering the NK cells derived from induced pluripotent stem cells to the subject, or simultaneously with administering the NK cells derived from induced pluripotent stem cells to the subject, or any combination thereof,

[0324] Among them, the chemotherapy agent is temozolomide (TMZ),

[0325] Among them, glioblastoma is resistant to TMZ.

[0326] Wherein, NK cells derived from induced pluripotent stem cells are prepared by the kit of the first aspect of the present invention.

[0327] In one embodiment, a method for treating glioblastoma in a subject in need thereof comprises the following steps:

[0328] administering to the subject NK cells derived from induced pluripotent stem cells; and

[0329] administering a chemotherapeutic agent to the subject, wherein the chemotherapeutic agent is administered before administering the NK cells derived from induced pluripotent stem cells to the subject, after administering the NK cells derived from induced pluripotent stem cells to the subject, or simultaneously with administering the NK cells derived from induced pluripotent stem cells to the subject, or any combination thereof,

[0330] Among them, the chemotherapy agent is temozolomide (TMZ),

[0331] Among them, glioblastoma is resistant to TMZ.

[0332] The NK cells derived from induced pluripotent stem cells are prepared by the method for inducing stem cells to differentiate into natural killer cells according to the third aspect of the present invention.

[0333] In one embodiment, the chemotherapeutic agent is administered to the subject 8 to 72 hours before the NK cells are administered to the subject, 8 to 72 hours after the NK cells are administered to the subject, or both before the NK cells are administered to the subject and 8 to 72 hours after the NK cells are administered to the subject.

[0334] In one embodiment, the subject is treated with a chemotherapeutic agent 8, 16, 24, 32, 40, 48, 56, 64, 72 hours before, 8, 16, 24, 32, 40, 48, 56, 64, 72 hours after, or both before and after administration of the NK cells to the subject.

[0335] In one embodiment, temozolomide treatment is administered to the subject 8, 16, 24, 32, 40, 48, 56, 64, 72 hours before, 8, 16, 24, 32, 40, 48, 56, 64, 72 hours after, or both before and after administration of NK cells to the subject.

[0336] In one embodiment, temozolomide treatment is administered to the subject 8, 16, 24, 32, 40, 48, 56, 64, 72 hours before, 8, 16, 24, 32, 40, 48, 56, 64, 72 hours after, or both before and after administration of NK cells derived from induced pluripotent stem cells to the subject.

[0337] Applications, Uses and Treatments

[0338] In a sixth aspect, the present invention provides use of the NK cell of the fourth aspect of the present invention or the pharmaceutical composition of the fifth aspect of the present invention in the preparation of a drug for preventing and / or treating tumors.

[0339] In a ninth aspect, the present invention provides a method for treating tumors, the method comprising: administering the NK cells of the fourth aspect of the present invention or administering the pharmaceutical composition of the fifth aspect of the present invention to a subject.

[0340] In one embodiment, the subject is a mammal, preferably a primate, more preferably a human.

[0341] In one embodiment, the site of administration is a vein of the subject.

[0342] In one embodiment, the tumor comprises at least one of a blood tumor and a solid tumor; preferably, the tumor comprises at least one of 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, kidney cancer, and glioma; more preferably, the tumor comprises cervical cancer and glioma.

[0343] In an eighth aspect, the present invention provides use of the natural killer (NK) cells and chemotherapeutic agents defined in the seventh aspect of the present invention, the drug combination of the seventh aspect, or the kit of the tenth aspect in the preparation of a drug for treating glioblastoma.

[0344] In one embodiment, the glioblastoma is resistant to TMZ.

[0345] In one embodiment, the glioblastoma is resistant to TMZ, and TMZ induces upregulation of expression of one or more immune ligands selected from CD112, CD155, CD54, CD70, B7-H6, CD48, PD-L1, and CD1d in the glioblastoma.

[0346] In one embodiment, the chemotherapeutic agent is administered before, after, or simultaneously with the administration of NK cells.

[0347] In one embodiment, the chemotherapeutic agent is administered 8 to 72 hours before, 8 to 72 hours after, or 8 to 72 hours before and 8 to 72 hours after administration of the NK cells.

[0348] In a twelfth aspect, the present invention provides the natural killer (NK) cells and chemotherapeutic agents defined in the seventh aspect of the present invention, the drug combination of the seventh aspect of the present invention, or the kit of the tenth aspect of the present invention for treating glioblastoma.

[0349] In one embodiment, the glioblastoma is resistant to TMZ.

[0350] In one embodiment, the glioblastoma is resistant to TMZ, and TMZ induces upregulation of expression of one or more immune ligands selected from CD112, CD155, CD54, CD70, B7-H6, CD48, PD-L1, and CD1d in the glioblastoma.

[0351] In one embodiment, the method of the present invention for treating glioblastoma in a subject in need thereof comprises the following steps:

[0352] administering to a subject a therapeutically effective amount of NK cells as defined in the seventh aspect of the present invention; and

[0353] A therapeutically effective amount of a chemotherapeutic agent as defined in the seventh aspect of the invention is administered to a subject, wherein the chemotherapeutic agent is administered before administering NK cells to the subject, after administering NK cells to the subject, or simultaneously with administering NK cells to the subject, or any combination of the foregoing.

[0354] As used herein, the term "treatment" means to improve or reverse at least one measurable physical parameter associated with cancer (e.g., glioblastoma or glioblastoma resistant to TMZ), which is not necessarily discernible in the subject, but can be discernible in the subject. The term "treatment" can also refer to causing regression, preventing progression or at least slowing down the progression of a disease, disorder or condition. In a specific embodiment, "treatment" refers to alleviating, preventing the development or onset of one or more symptoms associated with a disease, disorder or condition (such as a tumor or more preferably cancer, for example, glioblastoma or glioblastoma resistant to TMZ), or reducing the duration. In a specific embodiment, "treatment" refers to preventing the recurrence of a disease, disorder or condition. In a specific embodiment, "treatment" refers to increasing the survival of a subject suffering from a disease, disorder or condition. In a specific embodiment, "treatment" refers to eliminating a disease, disorder or condition of a subject.

[0355] The term "therapeutically effective amount" refers to an amount of an agent (e.g., a drug, a drug combination, or an agent in a kit) sufficient to prevent or inhibit the occurrence of a disease or symptom and / or slow down, alleviate, or delay the development or severity of a disease or symptom. The therapeutically effective amount is affected by factors including, but not limited to, the speed and severity of the development of the disease or symptom, the age, sex, weight, and physiological condition of the subject, the duration of treatment, and the specific route of administration. The therapeutically effective amount can be administered in one or more doses. The therapeutically effective amount can be achieved by continuous or intermittent administration.

[0356] According to specific embodiments, a therapeutically effective amount may refer to a therapeutic amount sufficient to achieve one, two, three, four or more of the following effects: (i) reduce or ameliorate the severity of the disease, disorder or condition being treated, or symptoms associated therewith; (ii) reduce the duration of the disease, disorder or condition being treated, or symptoms associated therewith; (iii) prevent the progression of the disease, disorder or condition being treated, or symptoms associated therewith; (iv) cause the regression of the disease, disorder or condition being treated, or symptoms associated therewith; (v) prevent the development or onset of the disease, disorder or condition being treated, or symptoms associated therewith. ; (vi) prevent recurrence of the disease, disorder or condition being treated, or symptoms associated therewith; (vii) reduce hospitalization of a subject suffering from the disease, disorder or condition being treated, or symptoms associated therewith; (viii) reduce the length of hospital stay of a subject suffering from the disease, disorder or condition being treated, or symptoms associated therewith; (ix) increase survival of a subject suffering from the disease, disorder or condition being treated, or symptoms associated therewith; (xi) inhibit or reduce the disease, disorder or condition being treated, or symptoms associated therewith in a subject; and / or (xii) enhance or improve the prophylactic or therapeutic effect of another therapy.

[0357] In one embodiment, the natural killer (NK) cells and chemotherapeutic agents defined in the seventh aspect of the present invention, the drug combination of the seventh aspect of the present invention, the method of the eleventh aspect of the present invention, the kit of the tenth aspect of the present invention, or the drug defined in the eighth aspect of the present invention can also be used to treat sensitive GBM that gradually develops resistance to the drug, develops various strategies to escape the effects of TMZ, and finally transforms into resistant GBM.

[0358] The NK cell of the present application and / or the drug combination of the present application can be administered in any physiologically acceptable carrier. The cell group comprising the NK cell of the present application can include a purified cell group. Those skilled in the art can easily determine the cell in the cell group using various well-known methods, and its purity range can be 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 easily adjust dosage, for example, purity reduction may require dosage increase.

[0359] The NK cells of the present application can be administered at a dose based on cells / kilogram (cell / kg) of subject body weight, and NK cells and / or a drug combination comprising NK cells can be administered to a subject. Generally, the cell dose is about 10 4 to about 10 10cells / kg body weight, for example, about 10 5 to about 10 9 , about 10 5 to about 10 8 , about 10 5 to about 10 7 or about 10 5 to about 10 6 , depending on the route and site of administration. Exemplary dosage ranges include, but are not limited to, 1×10 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 -6×10 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 , 1×10 5 -7×10 7 , 1×10 5 -6×10 7 , 1×10 5 -5×10 7 , 1×10 5 -4×10 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 -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 -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 6 、2×10 5 -6×10 6 、2×10 5 -5×10 6 、2×10 5 -4×10 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 6Cells / kg, etc. In addition, the dose can be adjusted to take into account whether a single dose is administered or whether multiple doses are administered. What is considered an effective dose can be accurately determined based on individual factors for each subject.

[0360] As used herein, the term "effect-target ratio (E:T)" refers to the ratio of effector cells to target cells. In some embodiments herein, the effect-target ratio refers to the ratio of NK cells used to glioblastoma cells. Exemplary effect-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, 1 2:1, 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, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20. In some embodiments, the effector-target ratio is 1:1, 1:4, 1:8. In one embodiment, the effector-target ratio is 1:1.

[0361] The TMZ of the present application can be administered at a dose of mg / kg of subject body weight, and TMZ and / or a drug combination comprising TMZ can be administered to a subject. 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 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 the subject.

[0362] The TMZ of this application can be expressed in mg / m 2The subject is dosed with TMZ and / or a drug combination comprising 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 / m 2 Subjects.

[0363] The dosage of TMZ in the present application can be, for example, 0.001-5000 μM, for example 10-1000 μM, 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, 1000 μM.

[0364] Beneficial Effects

[0365] The present invention provides a kit for inducing stem cells to differentiate into natural killer cells. The kit uses a culture medium with clear chemical composition, chemical small molecules and cytokines, and does not involve substances such as serum that are prone to cause unstable factors such as batch differences. In addition, the kit is cheaper than known commercial differentiation culture media, and the differentiation cycle is shortened.

[0366] The present invention provides an efficient and clear NK cell differentiation strategy based on the above-mentioned kit, wherein stem cells are cultured to obtain embryoid bodies, then the embryoid bodies are cultured for mesoderm differentiation to obtain mesodermal cells, then the mesodermal cells are cultured for hemogenic endothelial differentiation to obtain hemogenic endothelial cells, and finally the hemogenic endothelial cells are differentiated into NK cells to obtain NK cells; since the kit uses a culture medium, chemical small molecules and cytokines with clear chemical components, the entire differentiation process does not involve the influence of serum (human AB serum) and matrix proteins and matrix cells, making the induction differentiation method more standardized, and providing a large number of uniform functional NK cells for clinical treatment transformation; at the same time, the EB differentiation method is used in the hemogenic endothelial cell differentiation stage, which shortens the early differentiation cycle and has a high efficiency in obtaining hemogenic endothelial cells, thereby improving the subsequent differentiation quantity, differentiation quality and differentiation purity of NK cells; the CD45 + CD94 +Cells and CD56 + CD94 + The cell content is significantly increased, and it has a strong function of secreting IFN-γ and has a good function of killing tumor cells. And the obtained NK cells can be combined with the current CAR treatment method to improve the targeting and killing effect of NK cells on tumor cells. What is even more surprising is that the mature NK cells finally prepared by the present invention have an amplification multiple of more than 500 times relative to the initial iPSC cells, and the usual NK cell amplification step is no longer required, which greatly facilitates cell transplantation and related disease treatment. At the same time, the induction method can also be applied to the current universal iPSC, thereby inducing the differentiation of universal NK cells, which are suitable for a variety of patients.

[0367] The NK cells combined with TMZ of the present invention, and the corresponding drug combination or kit can show excellent effects, such as but not limited to: (1) NK cells show stronger degranulation activity; (2) have stronger tumor cell killing activity against drug-resistant GBM; (3) can resist the immunosuppressive effect of drug-resistant GBM; (4) prolong survival. In particular, NK cells derived from induced pluripotent stem cells combined with TMZ have the most excellent tumor cell killing activity against drug-resistant GBM.

[0368] Exemplary embodiments

[0369] Embodiment 1. A 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 comprising a BMP signaling pathway activator; the fourth culture medium is a basal culture medium comprising at least one, at least two, at least three or at least four of the following: a BMP signaling pathway activator, a vascular endothelial growth factor, a colony stimulating factor, an interleukin; the fifth culture medium is a basal culture medium comprising at least one, at least two, at least three, at least four, at least five or at least six of the following: a BMP signaling pathway activator, a vascular endothelial growth factor, a colony stimulating factor, an interleukin, a stem cell agonist, a stem cell expander.

[0370] Embodiment 2. The kit of embodiment 1, wherein the stem cell agonist comprises at least one of UM171 and UM729; preferably, the stem cell agonist comprises UM171; and / or the stem cell amplifier comprises at least one of SR1 and PD98059; preferably, the stem cell amplifier comprises SR1.

[0371] Embodiment 3. The kit of embodiment 1 or 2, wherein the kit further comprises a third culture medium, wherein the third culture medium is a basic 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.

[0372] Embodiment 4. The kit according to any one of Embodiments 1 to 3, further comprising a first culture medium, wherein the first culture medium is a basal culture medium comprising a ROCK inhibitor.

[0373] Embodiment 5. The kit according to any one of Embodiments 1 to 4, wherein the second culture medium is a basal culture medium comprising a BMP signaling pathway activator and a GSK3 inhibitor.

[0374] Embodiment 6. Use of the kit of any one of Embodiments 1-5 in preparing embryoid bodies, preparing mesoderm cells, preparing hemogenic endothelial cells, preparing natural killer (NK) cells, preparing products for inducing stem cells to differentiate into embryoid bodies, preparing products for inducing stem cells to differentiate into mesoderm cells, preparing products for inducing stem cells to differentiate into hemogenic endothelial cells, and preparing products for inducing stem cells to differentiate into NK cells.

[0375] Embodiment 7. A method for inducing stem cells to differentiate into natural killer (NK) cells, the method comprising the steps of using the kit of any one of Embodiments 1-5.

[0376] Embodiment 8. The method of embodiment 7, the method comprising: step (1) culturing stem cells to obtain embryoid bodies; step (2) subjecting the embryoid bodies to mesodermal differentiation culture to obtain mesodermal cells; step (3) subjecting the mesodermal cells to hemogenic endothelial differentiation culture to obtain hemogenic endothelial cells; step (4) culturing the hemogenic endothelial cells in the fourth culture medium and the fifth culture medium of the kit defined in any one of embodiments 1-5 to obtain NK cells; preferably, the culturing in step (1) is carried out in the first culture medium of the kit defined in embodiment 4; preferably, the culturing in step (2) is carried out in the second culture medium of the kit defined in embodiment 5; preferably, the culturing in step (3) is carried out in the test kit defined in embodiment 3 Preferably, the culturing time in step (1) is 12-36 h; preferably, the culturing time in step (2) is 24-72 h; preferably, the culturing time in step (3) is 48-144 h; preferably, the step (4) of culturing the hemogenic endothelial cells in the fourth culture medium and the fifth culture medium of the kit defined in any one of embodiments 1-5 is to sequentially culture the hemogenic endothelial cells in the fourth culture medium and the fifth culture medium of the kit defined in any one of embodiments 1-5; preferably, the culturing time of the hemogenic endothelial cells in the fourth culture medium is 96-192 h; preferably, the culturing time of the hemogenic endothelial cells in the fifth culture medium is 144-432 h.

[0377] Embodiment 9. A natural killer (NK) cell prepared by the kit of any one of embodiments 1-5 or the method of embodiment 7 or 8; preferably, the NK cell has CD3 - CD45 + CD56 + The content of cells is more than 90%; preferably, the CD3 - CD56 + Nkp44 + The content of cells is more than 90%; preferably, the CD3 - CD56 + Nkp30 + The cell content is more than 85%.

[0378] Embodiment 10. A pharmaceutical composition comprising the NK cells described in Embodiment 9; preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0379] Embodiment 11. The pharmaceutical composition of Embodiment 10, further comprising an anti-tumor drug.

[0380] Embodiment 12. Use of the NK cells described in Embodiment 9 or the pharmaceutical composition of any one of Embodiments 10-11 in the preparation of drugs for preventing and / or treating tumors.

[0381] Embodiment 13. A pharmaceutical combination for treating glioblastoma, comprising an active ingredient comprising natural killer (NK) cells and a chemotherapeutic agent, wherein the chemotherapeutic agent comprises temozolomide (TMZ).

[0382] Embodiment 14. The pharmaceutical combination of embodiment 13, wherein the glioblastoma is resistant to TMZ.

[0383] Embodiment 15. The pharmaceutical combination of Embodiment 13 or 14, wherein the NK cells are unmodified NK cells.

[0384] Embodiment 16. The pharmaceutical combination of any one of Embodiments 13-15, wherein the NK cells are NK cells derived from induced pluripotent stem cells (iPSCs).

[0385] Embodiment 17. The pharmaceutical combination of any one of Embodiments 13-16, wherein the NK cells are prepared by the kit of any one of Embodiments 1-5 or the method of Embodiment 7 or 8.

[0386] Embodiment 18. The drug combination of any one of Embodiments 13-17, which further comprises other chemotherapeutic agents selected from anticancer agents, metabolic antagonists, DNA demethylating agents, plant-derived antitumor agents, alkylating agents, antimetabolites, anticancer antibiotics, topoisomerase inhibitors, mitotic inhibitors, differentiation agents, and hormone therapeutic agents.

[0387] Embodiment 19. The pharmaceutical combination of embodiment 18, wherein the other chemotherapeutic agent is selected from carmustine, lomustine, vorinostat, mebendazole, imatinib mesylate, AG119, or any therapeutic derivatives thereof.

[0388] Embodiment 20. The pharmaceutical combination according to any one of embodiments 13-19, wherein the active ingredient does not comprise trimetrexate.

[0389] Embodiment 21. The pharmaceutical combination of any one of Embodiments 13-20, wherein the active ingredient does not comprise an immune checkpoint inhibitor.

[0390] Embodiment 22. The pharmaceutical combination according to any one of embodiments 13-21, which is a kit.

[0391] Embodiment 23. Use of the pharmaceutical combination of any one of Embodiments 13-22 in the preparation of a medicament for treating glioblastoma.

[0392] Embodiment 24. The use of embodiments 13-23, wherein

[0393] The chemotherapeutic agent is administered before, after, or simultaneously with the administration of the NK cells.

[0394] Embodiment 25. The use of embodiments 13-24, wherein the chemotherapeutic agent is administered 8 to 72 hours before, 8 to 72 hours after, or 8 to 72 hours before and 8 to 72 hours after the administration of the NK cells.

[0395] Example

[0396] The present invention is further described by reference to the following examples. It should be understood that these examples are intended to be illustrative only and not to limit the present 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 are performed according to the manufacturer's instructions or according to methods and procedures known in the art.

[0397] The human induced pluripotent stem cells (iPSCs) used in this example are iPSCs reprogrammed by our company and obtained through CTS TM CytoTune TM -iPS2.1 Sendai virus reprogramming kit (Cat. No.: A34546) was prepared (see the kit manual for specific methods), and the initial cells were human peripheral blood cells.

[0398] In this example, low-attachment 96-well plates were 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 B27 medium supplement without vitamin A was purchased from Gibco.

[0399] Example 1 A kit for inducing human induced pluripotent stem cells to differentiate into natural killer cells

[0400] A kit for inducing human induced pluripotent stem cells to differentiate into natural killer cells, comprising: a first culture medium, a second culture medium, a third culture medium, a fourth culture medium and a fifth culture medium;

[0401] The first culture medium is StemFit Basic 03 culture medium (company: Ajinomoto, catalog number: AK03N) containing 10 μM Y27632·2HCl;

[0402] The second culture medium is a differentiation medium containing 20 ng / mL BMP4 (bone morphogenetic protein 4) and 3 μM CHIR-99021, wherein the differentiation medium is an RPMI1640 medium containing 1% (v / v) non-essential amino acids, 1% (v / v) glutamine, 50 μg / mL vitamin C and 2% (v / v) B27 medium supplement without vitamin A;

[0403] The third culture medium is 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 is an RPMI1640 medium containing 1% (v / v) non-essential amino acids, 1% (v / v) glutamine, 50 μg / mL vitamin C, and 2% (v / v) B27 medium supplement without vitamin A;

[0404] The fourth culture medium is a differentiation medium containing 5 ng / mL BMP4, 10 ng / mL VEGF-165, 20 ng / mL SCF (stem cell factor), 5 ng / mL IL3 (multiple colony stimulating factor, multi-CSF), 10 ng / mL IL15 (interleukin 15), 20 ng / mL IL7 (interleukin 7) and 10 ng / mL FLT3L (Fms-related tyrosine kinase 3 ligand), wherein the differentiation medium is an IMDM (Iscove's Modified Dulbecco's Medium) medium containing 1% (v / v) non-essential amino acids, 1% (v / v) glutamine, 50 μg / mL vitamin C, 2% (v / v) B27 medium supplement without vitamin A and 30 μM N-acetyl-L-cysteine;

[0405] The fifth culture medium is a 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), wherein the differentiation medium is an IMDM medium containing 1% (v / v) non-essential amino acids, 1% (v / v) glutamine, 50 μg / mL vitamin C, 2% (v / v) B27 medium supplement without vitamin A and 30 μM N-acetyl-L-cysteine.

[0406] Example 2 A method for inducing human induced pluripotent stem cells to differentiate into natural killer cells

[0407] A method for inducing human induced pluripotent stem cells to differentiate into natural killer cells, comprising the steps of using the kit of Example 1, comprising the following steps (differentiation diagram as shown in Figure 1 (shown in A):

[0408] Step (1) culturing human induced pluripotent stem cells in a low-attachment 96-well plate to obtain embryoid bodies:

[0409] Press 2×10 5 Cells / well iPSCs were seeded into low-attachment six-well plates using the first culture medium and cultured at 37°C and 5% CO2 for one day to obtain embryoid bodies, which were recorded as D0;

[0410] Step (2) performing mesodermal differentiation culture on the embryoid bodies to obtain mesodermal cells:

[0411] One day later, the first culture medium was replaced with the second culture medium and cultured at 37°C and 5% CO2 for two days, which was recorded as D2 to obtain mesoderm cells (ME cells);

[0412] Step (3) performing hemogenic endothelial differentiation culture on the mesoderm cells to obtain hemogenic endothelial cells:

[0413] Two days later, the second culture medium was replaced with the third culture medium and cultured at 37°C and 5% CO2 for four days to obtain hemogenic endothelial cells (HE cells);

[0414] Step (4) performing NK cell differentiation culture on hemogenic endothelial cells to obtain NK cells:

[0415] After four days, the embryoid bodies (the hemogenic endothelial cells obtained after four days of culture in step (3)) in the low-attachment 96-well plate were collected and placed in a 50 mL centrifuge tube. The cells were allowed to settle naturally for 1-2 minutes, and then the supernatant was removed. About 5 mL of the supernatant remained in the centrifuge tube. Then 14 mL of the fourth culture medium was added, and the cell balls were gently blown and mixed. The cells were evenly distributed into 3 normal adherent 6-well plates and returned to the incubator for culture at 37°C and 5% CO2 for 7 days. The cell morphology after 2 days of culture (iNK-D2, EB adherent D2) is shown in the figure below. Figure 1 As shown in B;

[0416] After seven days, gently remove about 1 mL of the supernatant from the six-well plate, then add 3 mL of the fifth culture medium to each well of the six-well plate, and return to the incubator for culture at 37°C and 5% CO2 for 7 days;

[0417] After seven days, 2 mL of the fifth culture medium was added to each well of the six-well plate and the plate was returned to the incubator for 7 days at 37°C and 5% CO2. The cell morphology of iNK-D20 cells after 6 days of culture is shown in the figure below. Figure 1 As shown in C;

[0418] Step (5) collecting differentiated NK cells:

[0419] On the 13th, 16th and 20th days of NK cell differentiation culture of hemogenic endothelial cells, the supernatant (suspended cells) in the six-well plate was collected into a centrifuge tube, and the tube was centrifuged at 1500 rpm for 10 minutes. Then, 5 mL of the fifth culture medium was used to resuspend the cells. The cell suspension was then passed through a 70 μm cell sieve to filter the cells. The filtered cell suspension was centrifuged again at 1500 rpm for 5 minutes. The cells were resuspended in 5 mL of the fifth culture medium for AO / PI cell counting, and the cell viability and cell number were recorded. Subsequent cell testing and cell freezing experiments were performed as needed.

[0420] Example 3-1 A kit for inducing human induced pluripotent stem cells to differentiate into natural killer cells

[0421] A kit for inducing human induced pluripotent stem cells to differentiate into natural killer cells is the same as Example 1, except that the concentration of BMP4 (bone morphogenetic protein 4) in the second culture medium is 2 ng / mL.

[0422] Example 3-2 A kit for inducing human induced pluripotent stem cells to differentiate into natural killer cells

[0423] A kit for inducing human induced pluripotent stem cells to differentiate into natural killer cells is the same as Example 1, except that the concentration of BMP4 (bone morphogenetic protein 4) in the second culture medium is 10 ng / mL.

[0424] Example 3-3 A kit for inducing human induced pluripotent stem cells to differentiate into natural killer cells

[0425] A kit for inducing human induced pluripotent stem cells to differentiate into natural killer cells is the same as Example 1, except that the concentration of BMP4 (bone morphogenetic protein 4) in the second culture medium is 5 ng / mL.

[0426] Example 4-1 A method for inducing human induced pluripotent stem cells to differentiate into natural killer cells

[0427] A method for inducing human induced pluripotent stem cells to differentiate into natural killer cells is the same as that of Example 2, except that the kit of Example 3-1 is used.

[0428] Example 4-2 A method for inducing human induced pluripotent stem cells to differentiate into natural killer cells

[0429] A method for inducing human induced pluripotent stem cells to differentiate into natural killer cells is the same as that of Example 2, except that the kit of Example 3-2 is used.

[0430] Example 4-3 A method for inducing human induced pluripotent stem cells to differentiate into natural killer cells

[0431] A method for inducing human induced pluripotent stem cells to differentiate into natural killer cells is the same as that of Example 2, except that the kit of Example 3-3 is used.

[0432] Comparative Example 1 A kit for inducing human induced pluripotent stem cells to differentiate into natural killer cells

[0433] A kit for inducing human induced pluripotent stem cells to differentiate into natural killer cells, comprising: a first culture medium, a second culture medium, a third culture medium, a fourth culture medium and a fifth culture medium;

[0434] The first culture medium contains 10 μM Y27632·2HCl. Basic 03 medium;

[0435] The second culture medium is a differentiation medium containing 20 ng / mL BMP4 (bone morphogenetic protein 4) and 3 μM CHIR-99021, wherein the differentiation medium is an RPMI1640 medium containing 1% (v / v) non-essential amino acids, 1% (v / v) glutamine, 50 μg / mL vitamin C and 2% (v / v) B27 medium supplement without vitamin A;

[0436] The third culture medium is 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 is an RPMI1640 medium containing 1% (v / v) non-essential amino acids, 1% (v / v) glutamine, 50 μg / mL vitamin C, and 2% (v / v) B27 medium supplement without vitamin A;

[0437] The fourth culture medium is a differentiation medium containing 20 ng / mL SCF (stem cell factor), 5 ng / mL IL3 (interleukin 3), 10 ng / mL IL15 (interleukin 15), 10 ng / mL IL7 (interleukin 7) and 10 ng / mL FLT3L (Fms-related tyrosine kinase 3 ligand), wherein the differentiation medium is X-VIVO15 medium (X-VIVO15 medium) containing 20% ​​(v / v) Human AB-FBS (human AB serum);

[0438] The fifth culture medium is a differentiation medium containing 20 ng / mL SCF (stem cell factor), 10 ng / mL IL15 (interleukin 15), 10 ng / mL IL7 (interleukin 7) and 10 ng / mL FLT3L (Fms-related tyrosine kinase 3 ligand), wherein the differentiation medium is X-VIVO15 medium (X-VIVO15 medium) containing 20% ​​(v / v) Human AB-FBS (human AB serum).

[0439] Comparative Example 2 A method for inducing human induced pluripotent stem cells to differentiate into natural killer cells

[0440] A method for inducing human induced pluripotent stem cells to differentiate into natural killer cells is the same as Example 2, except that: the kit of Comparative Example 1 is used; and step (5) is only to collect the supernatant (suspended cells) in the six-well plate into a centrifuge tube on the 20th day of culturing hemogenic endothelial cells for NK cell differentiation.

[0441] Comparative Example 3 A kit for inducing human induced pluripotent stem cells to differentiate into natural killer cells

[0442] A kit for inducing human induced pluripotent stem cells to differentiate into natural killer cells, comprising: a first culture medium, a second culture medium, a third culture medium, a fourth culture medium and a fifth culture medium;

[0443] The first culture medium contains 10 μM Y27632·2HCl. Basic 03 medium;

[0444] The second culture medium is a differentiation medium containing 20 ng / mL BMP4 (bone morphogenetic protein 4) and 3 μM CHIR-99021, wherein the differentiation medium is an RPMI1640 medium containing 1% (v / v) non-essential amino acids, 1% (v / v) glutamine, 50 μg / mL vitamin C and 2% (v / v) B27 medium supplement without vitamin A;

[0445] The third culture medium is 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 is an RPMI1640 medium containing 1% (v / v) non-essential amino acids, 1% (v / v) glutamine, 50 μg / mL vitamin C, and 2% (v / v) B27 medium supplement without vitamin A;

[0446] The fourth culture medium is a differentiation medium containing 20 ng / mL SCF (stem cell factor), 5 ng / mL IL3 (interleukin 3), 10 ng / mL IL15 (interleukin 15), 10 ng / mL IL7 (interleukin 7), 10 ng / mL FLT3L (Fms-related tyrosine kinase 3 ligand), 5 ng / mL sodium selenite, 50 μM Ethanolamine, 1 μM β-Mercaptoethanol and 20 μg / mL ascorbic acid, wherein the differentiation medium is a DMEM / Ham's F-12 (2:1) (DF-12) medium containing 20% ​​Human AB-FBS (human AB serum) (DMEM high glucose medium:Ham's F-12 medium=2:1 (v / v));

[0447] The fifth culture medium is a differentiation medium containing 20 ng / mL SCF (stem cell factor), 5 ng / mL IL3 (interleukin 3), 10 ng / mL IL15 (interleukin 15), 10 ng / mL IL7 (interleukin 7), 10 ng / mL FLT3L (Fms-related tyrosine kinase 3 ligand), 5 ng / mL sodium selenite, 50 μM Ethanolamine, 1 μM β-Mercaptoethanol and 20 μg / mL ascorbic acid, wherein the differentiation medium is a DMEM / Ham's F-12 (2:1) (DF-12) medium containing 20% ​​Human AB-FBS (human AB serum) (DMEM high glucose medium: Ham's F-12 medium = 2:1 (v / v)).

[0448] Comparative Example 4 A method for inducing human induced pluripotent stem cells to differentiate into natural killer cells

[0449] A method for inducing human induced pluripotent stem cells to differentiate into natural killer cells is the same as Example 2, except that: the kit of Comparative Example 3 is used; and step (5) is to collect the supernatant (suspended cells) in the six-well plate into a centrifuge tube only on the 20th day of NK cell differentiation culture of hemogenic endothelial cells.

[0450] Comparative Example 5 A kit for inducing human induced pluripotent stem cells to differentiate into natural killer cells

[0451] A kit for inducing human induced pluripotent stem cells to differentiate into natural killer cells is the same as Example 1, except that the concentration of BMP4 (bone morphogenetic protein 4) in the second culture medium is 0 ng / mL.

[0452] Comparative Example 6 A method for inducing human induced pluripotent stem cells to differentiate into natural killer cells

[0453] A method for inducing human induced pluripotent stem cells to differentiate into natural killer cells is the same as that of Example 2, except that the kit of Comparative Example 5 is used.

[0454] Effect Example 1

[0455] The NK cells collected on the 13th, 16th, and / or 20th day of NK cell differentiation culture of hemogenic endothelial cells in Example 2, Example 4-1, Example 4-2, Example 4-3, Comparative Example 2, Comparative Example 4, and Comparative Example 6 were collected into centrifuge tubes, centrifuged at 1500 rpm for 5 minutes, the supernatant was discarded, and the cells were resuspended with PBS. The cells resuspended with PBS were centrifuged again for 3 minutes at 1500 rpm, the supernatant was discarded, and the cells were resuspended with 100 μL of PBS containing 0.5% bovine serum albumin (BSA), and the corresponding volume of antibodies was added according to the antibody instructions (the antibodies are shown in Table 1), incubated at room temperature in the dark for 20 minutes, and then the cells were washed three times with PBS, and then the cells were resuspended with 200 μL of PBS and placed in a flow cytometer for flow detection. The results are as follows. Figure 2 , 3 , 4, 5, and 6 show that in Example 2, in the cells collected on the 13th day of NK cell differentiation culture of hemogenic endothelial cells, CD45 + CD56 + The cell ratio was 72.18%; in the cells collected on the 16th day of NK cell differentiation culture of hemogenic endothelial cells, CD45 + CD56 + The cell ratio was 89.53%; in the cells collected on the 20th day of NK cell differentiation culture of hemogenic endothelial cells, CD45 + CD56 + The cell ratio was 96.72%, CD45 + The proportion of CD3- cells was 98.87%, CD56 + The proportion of CD3- cells was 97.12%, CD56 + CD94 + The cell ratio was 53.45%, CD45 + CD94 + The cell ratio was 47.90%, CD3-CD45 + CD56 +The proportion of cells was 90.64%, the proportion of CD3-CD16- cells was 96.47%, and the proportion of CD3-Nkp46 + The cell ratio was 73.96%, CD3-CD56 + Nkp44 + The cell ratio was 93.35%, CD3-CD56 + Nkp30 + The cell ratio was 87.96%, CD3-CD56 + Nkp44 + Nkp30 + The cell ratio was 85.77%; in the cells collected on the 20th day of NK cell differentiation culture of hemogenic endothelial cells in Comparative Example 2, CD45 + CD56 + The cell ratio was 95.10%, CD45 + The proportion of CD3- cells was 99.24%, CD43 + The proportion of CD3- cells was 97.95%, CD56 + CD94 + The cell ratio was 29.37%, CD45 + CD94 + The cell ratio was 24.44%; in the cells collected on the 20th day of NK cell differentiation culture of hemogenic endothelial cells in Comparative Example 4, CD45 + CD56 + The cell ratio was 92.17%, CD45 + The proportion of CD3- cells was 99.88%, CD43 + The proportion of CD3- cells was 99.14%, CD56 + CD94 + The cell ratio was 14.92%, CD45 + CD94 + The cell ratio was 13.35%. It can be seen that on the 20th day of NK cell differentiation culture of hemogenic endothelial cells, NK cell differentiation was basically completed. In Comparative Examples 2 and 4, in the cells collected on the 20th day of NK cell differentiation culture of hemogenic endothelial cells, CD45 + CD56 + The cell ratio is slightly lower than that in Example 2, and the fourth and fifth culture media in Comparative Examples 2 and 4 contain serum, and the fourth and fifth culture media in Comparative Example 4 are mixed with two culture media, which is complicated to operate; in Comparative Examples 2 and 4, in the cells collected on the 20th day of NK cell differentiation culture of hemogenic endothelial cells, CD45 + CD94 + Cells and CD56 + CD94 +The cell ratio is much smaller than that in Example 2, and the differentiation effect is not good; in the differentiated NK cells collected on the 20th day of NK cell differentiation culture of hemogenic endothelial cells under different concentrations of BMP4 (0, 2, 20, 10, 5 ng / mL, corresponding to Example 6, Example 4-1, Example 2, Example 4-2, Example 4-3, respectively), CD45 + CD56 + The cell ratios were 34.95%, 58.86%, 96.72%, 90.29%, and 74.29%, respectively; it can be seen that 20 ng / mL of BMP4 is more conducive to NK cell differentiation. Similarly, the bright field cell observation of differentiated NK cells collected on the 20th day of NK cell differentiation culture of hemogenic endothelial cells under different concentrations of BMP4 (0, 2, 10, 5 ng / mL, corresponding to Example 6, Example 4-1, Example 4-2, and Example 4-3, respectively) is shown in Figure 2 Figure 7 As shown: under low concentration (0-2ng / mL) of BMP4, the cells hardly differentiated and no suspension cell secretion occurred, while under high concentration (10ng / mL) of BMP4, the cells had suspension cell secretion; it can be seen that 5-20ng / mL of BMP4 is more conducive to NK cell differentiation.

[0456] Table 1 Antibody details

[0457] Antibody Fluorescent labeling source Part Number CD45 PECY7 Biolegend 304016 CD56 PE Biolegend 304606 CD3 FITC biolegend 300306 CD94 FITC BD Biosciences 555888 CD16 APC Biolegend 360706 Nkp44 PECY7 Biolegend 325116 Nkp30 APC Biolegend 325210 Nkp46 APC Biolegend 331918

[0458] Effect Example 2

[0459] The NK cells collected on the 20th day of NK cell differentiation culture of hemogenic endothelial cells in Example 2 were transferred to a medium containing IL2 (a basic medium containing 1 μM UM171, 1 μM SR1, 10 ng / ml IL15, 20 ng / ml SCF and 50 ng / ml IL2 (interleukin 2), wherein the basic medium is an IMDM medium containing 1% (v / v) non-essential amino acids, 1% (v / v) glutamine, 50 μg / mL vitamin C and 2% (v / v) B27 medium supplement without vitamin A) and cultured for 5 days. The cultured medium containing NK cells (iPS-NK) was collected into a centrifuge tube and centrifuged at 1500 rpm for 5 minutes. The supernatant of NK cells was collected into a centrifuge tube and ELISA kit for IFN-γ (purchased from Stemcell, Cat. No.: 7664-9) was used. 3-9) The cell supernatant was detected according to the instructions, and then the secretion of IFN-γ was detected in an ELISA instrument (the PB-NK cells were treated with the same conditions (refer to the treatment method of iPS-NK cells in this effect example to complete the post-culture treatment of PB-NK cells: NK cells were transferred to a culture medium containing IL2 and cultured for 5 days, and the culture medium containing NK cells (iPS-NK) was collected into a centrifuge tube, centrifuged at 1500 rpm for 5 minutes, and the NK cell supernatant was collected into a centrifuge tube) and the supernatant was collected as a control). The results are as follows Figure 8 As shown: the NK cells collected on the 20th day of NK cell differentiation culture of hemogenic endothelial cells in Example 2 have a concentration of IFN-γ secreted by the NK cells after 5 days of culture as mentioned above, which is similar to the secretion amount of IFN-γ secreted by peripheral blood-derived NK (PB-NK) after the same treatment. It can be seen that the NK cells obtained by the present invention have a strong function of secreting IFN-γ.

[0460] Effect Example 3

[0461] The tumor cells (Hela cells) with a cell density of 80% were digested and counted by AO / PI. 5×10 cells were added to each well of a 96-well plate. 4 Cells were seeded and allowed to adhere for 5 hours.

[0462] After 5 hours, the NK cells collected on the 20th day of NK cell differentiation culture of the hemogenic endothelial cells in Example 2 were cultured in the IL2-containing medium described in Example 2 for 5 days, and the NK cell-containing medium (IMDM-iNK) was collected into a centrifuge tube (the peripheral blood-derived NK cells (PB-NK, Sai Li Bio) treated with the same conditions (refer to the IL2-containing medium described in Example 2) were used as a control), and centrifuged at 1500 rpm for 5 minutes, the supernatant was discarded, and the cells were resuspended in the IL2-containing medium in Example 2, and then AO / PI counting was performed. The NK cells and tumor cells after counting were divided into different ratios (cancer cell:NK The cells were inoculated at 1:0, 1:1, 1:3, 1:5, and 1:10, and the cells were co-cultured for 18 hours. The supernatant was removed, and 100 mL of fresh IL2-containing medium described in Example 2 was added to each well. 10× CCK8 was added to the medium in advance. After incubation at 37°C and 5% CO2 for 1.5 hours, the 96-well plate was taken out of the incubator, and the fluorescence value was detected using an ELISA instrument to detect the killing effect of NK cells on tumor cells. The results are shown in Fig. 9 As shown in the figure: when the cancer cell:NK cell ratio is 1:1, 1:3, 1:5, and 1:10, the killing rates of the NK cells in Example 2 against Helacell are 17%, 45%, 67%, and 80%, respectively, which are comparable to the killing rates of NK cells derived from peripheral blood (PB-NK) (33%, 57%, 76%, and 76%). It can be seen that the NK cells obtained by the present invention have a better function of killing tumor cells.

[0463] Effect Example 4

[0464] The flow chart of this embodiment is as follows: Fig.10 As shown, the details are as follows: 5 days in advance (D-5), 1.3×10 U87 glioma cells were injected 5 -1.5×10 5 After the mouse tumor model was successfully constructed (D0), the mice were divided into 6 groups, 4 mice in each group, and each group was given three times, as follows: ①. PBS group (mouse tumor model mice, injected with an equal amount of PBS), ②. TMZ group (mouse tumor model, injected with temozolomide, once every seven days, 5 mpk each time, positive control), ③. Negative control group (mouse tumor model, injected with an equal amount of PBS containing 10 μM IL15 and 20 μM IL12, once every seven days), ④. i-NK group (mouse tumor model, each mouse was injected with 5×10 5 / μL of iPS-NK cells, once every seven days, 5μL each time. The preparation method of i-NK cells is as follows: the culture medium containing NK cells collected on the 20th day of NK cell differentiation culture of hemogenic endothelial cells in Example 2 is collected into a centrifuge tube, placed in a centrifuge and centrifuged at 1500 rpm for 5 minutes, the supernatant is discarded, and then 10mL of PBS containing 10μM IL15 and 20μM IL12 is used to resuspend the cells, the cell suspension is passed through a 40μM cell sieve to ensure that the cells are in a single cell state, the cells are placed in a centrifuge and centrifuged at 1500 rpm for 5 minutes, the supernatant is discarded, and then 5mL of PBS containing 10μM IL15 and 20μM IL12 is used to resuspend the cells, and the cells are counted by AO / PI), ⑤.PB-NK group (mouse tumor model, each mouse is injected with 5×10 5 / mL of PB-NK cells, once every seven days, 5μL each time. The preparation method of PB-NK cells is as follows: the culture medium containing peripheral blood-derived NK cells (PB-NK) is collected into a centrifuge tube, centrifuged at 1500 rpm for 5 minutes, the supernatant is discarded, and then the cells are resuspended in 10mL of PBS containing 10μM IL15 and 20μM IL12, the cell suspension is passed through a 40μM cell sieve to ensure that the cells are in a single cell state, the cells are centrifuged at 1500 rpm for 5 minutes, the supernatant is discarded, and then the cells are resuspended in 5mL of PBS containing 10μM IL15 and 20μM IL12, and the cells are counted by AO / PI), ⑥.i-NK+TMZ drug group (mouse tumor model, each mouse is injected with 5×10 5 / μL of i-NK cells and 5mpk of TMZ, once every seven days, 5μL each time), the survival results of mice are shown in Fig.11 As shown: the mice in the PBS control group and the negative control group basically all died around D40-D50 days, the survival rate of mice in the i-NK group was significantly higher than that in the chemical small molecule drug TMZ group, and slightly lower than that in the natural NK cell group (PB-NK group), and the survival rate of mice in the i-NK+TMZ combined medication group was significantly better than that in the natural NK group. It should also be noted that when the i-NK cells acted alone, one of the four mice in the group survived until the end of the experiment (D150), indicating that the mouse was basically cured. It can be seen that the NK cells prepared by the present invention have a good anti-tumor effect, and when used in combination with other anti-tumor drugs, they have a synergistic effect, further enhancing the anti-tumor effect of the NK cells prepared by the present invention.

[0465] Effect Example 5

[0466] During the differentiation process of stem cells into hemogenic endothelial cells, low-attachment 96-well plates were used to ensure the uniformity of EB spheres and thus improve the differentiation efficiency. According to the method of Example 2, 2×10 cells were initially plated per 96-well plate. 5 The number of iPSC cells seeded / well is 1.009×10 8 -1.0465×10 8 NK cells (NK cells were collected on the 20th day of NK cell differentiation culture of hemogenic endothelial cells. The results are as follows Fig.12 As shown in the figure, from the initial iPSC cells to the final harvested mature NK cells, the cell number has expanded by more than 500 times, and the usual NK cell expansion step is no longer required, which greatly facilitates cell transplantation and disease treatment). In addition, the differentiated NK cells are cryopreserved using a cryopreservation solution ( CS10, purchased from stem cell technologies) after freezing the cells (cell concentration was 1×10 7 / mL, that is, 1mL of freezing solution contains 1×10 7 cells, the freezing method is that the freezing solution containing cells is cooled to -80℃ through a program cooling box, placed in a -80℃ refrigerator for 24 hours, and then placed in a liquid nitrogen tank for 1 week), and still has a high cell viability ( Fig.13 ).

[0467] Example 5. Evaluation of the sensitivity response of different GBM cell lines to TMZ

[0468] Clinically, GBM tumor cells will produce sensitive and resistant responses to TMZ. In this example, the sensitivity response of different GBM tumor cell lines to TMZ was evaluated. TMZ-sensitive GBM (U87) and TMZ-resistant GBM (T98G) were purchased from Zhejiang Meisen Cell Technology Co., Ltd. U87 and T98G cells were cultured in DMEM medium (Gibco, 11965084) containing 10% FBS (cellmax, SA211.02).

[0469] 5.1. Sensitivity of GBM to different concentrations of TMZ

[0470] A. Cell plating

[0471] U87 and T98G cells were cultured and grown to 80%-90% fusion rate. Trypsin (Biyuntian, C0201-100mL) digestion solution was added and incubated at 37°C for 5 min. Two volumes of DMEM medium (Gibco, 11965084) were added to terminate the digestion. The cell suspension was transferred to a centrifuge tube and centrifuged at 300g for 5 min. The cell pellet was resuspended in DMEM medium. AOPI staining (AO (acridine orange) / PI (propidium iodide)) was used for counting. According to the counting results, the cell suspension density was adjusted to 1×10 5 Cells / mL. According to the grouping requirements, the cell suspension was added to a 96-well plate at 100 mL / well, and then the 96-well plate was placed in a 37°C incubator for overnight culture.

[0472] B.TMZ Configuration

[0473] Take TMZ powder (Sigma, T2577-25MG), add appropriate volume of DMSO to dissolve, and prepare a TMZ stock solution with a concentration of 100mM; first prepare the highest concentration of TMZ (5000mM): take 225mL TMZ stock solution, dilute it with 4.5mL DMEM culture medium, and then use serial dilution method to obtain TMZ working solutions with concentrations of 1000mM, 500mM, 200mM, 100mM and 10mM, respectively.

[0474] C. TMZ drug treatment

[0475] After overnight culture of U87 and T98G cells, it was observed that they were attached to the wall and in the logarithmic growth phase. According to the grouping settings, TMZ working solution of corresponding working concentration was added, 100 mL / well, and then the cells were returned to the incubator. Cell viability and number were detected after 72 hours.

[0476] D. Cell Viability Assay

[0477] use Luminescent Cell Viability Assay Kit (Promega, G7572) was used according to the manufacturer's instructions. The solution was mixed with DMEM medium in a 1:1 ratio to prepare the working solution. The working solution was added to a 96-well plate, 50 mL / well, and incubated at room temperature in the dark for 10 min. 200PRO) to detect chemiluminescence.

[0478] Test results such as Fig.14As shown in (A), as the concentration gradient of TMZ increases, the number of U87 cells decreases significantly, and the reaction is sensitive. The number of T98G cells begins to decrease significantly after exceeding 500mM, and the reaction is slow. When TMZ is at 200mM, the number of U87 live cells is about 50%, while the number of T98G live cells is maintained at more than 80%; when TMZ is at 500mM, the number of U87 live cells is about 40%, while the number of T98G live cells is maintained at 70%. The sensitivity difference range is 100mM-500mM.

[0479] 5.2. Sensitivity of GBM to TMZ at different treatment times

[0480] The test and detection scheme were the same as in Example 5.1, and the TMZ concentration was selected to be the sensitive interval concentration of 300 mM in Example 5.1.

[0481] Test results such as Fig.14 As shown in (B), as the TMZ treatment time prolonged, the number of viable cells of U87 cells decreased significantly and gradually, less than 40% at 72 hours; while T98G was less affected by TMZ, and the number of viable cells remained above 80% at 72 hours. The above results indicate that U87 is a TMZ-sensitive GBM and T98G is a TMZ-resistant GBM.

[0482] Fig.14 (C) shows the cell microscopic results of drug-resistant GBM (T98G) treated with TMZ (300 mM) for 48 hours. Fig.14 (D) shows the cell microscopic results of sensitive GBM (U87) treated with TMZ (300 mM) for 48 hours. The microscopic results show that compared with the control DMSO treatment, after 48 hours of treatment with TMZ (300 mM), the growth of T98G cells was not much different, while the growth of U87 cells was significantly inhibited.

[0483] Example 6. Drug-resistant GBM (T98G) is able to resist TMZ-induced cell cycle arrest

[0484] After TMZ is absorbed by the human body and enters tumor cells, it acts on DNA, causing DNA alkylation damage, which in turn forms DNA double-strand breaks or cross-links, blocks the cell cycle, and causes tumor cell proliferation stagnation or apoptosis. In order to explore the mechanism by which drug-resistant GBM cell lines resist TMZ, a cell cycle assay was used to detect the effect of TMZ on the cell cycle of different GBM cell lines.

[0485] A. Cell culture and treatment: The experimental procedures for U87 and T98G cell culture and TMZ treatment refer to Example 5. The TMZ concentration used in this example is 300 mM.

[0486] B. Preparation of cell samples: Use the Bio-Tech Cell Cycle Kit (Bi-Tech, C1052) according to the manufacturer's instructions. Take 1×10 6 Place the cells in a centrifuge tube for later use, centrifuge at 1000g for 3-5 minutes to precipitate the cells. Aspirate the supernatant, add 1mL of pre-cooled PBS, resuspend the cells, and transfer to a 1.5mL centrifuge tube. Centrifuge the precipitated cells and aspirate the supernatant.

[0487] C. Cell fixation: Add 1 mL of pre-cooled 70% ethanol, mix by pipetting, and fix at 4°C for 30 minutes. Centrifuge at 1000g for 3-5 minutes to precipitate the cells. Remove the supernatant, add about 1 mL of ice-cold PBS, and resuspend the cells. Centrifuge again to precipitate the cells and remove the supernatant.

[0488] D. Preparation and staining of propidium iodide (PI) staining solution:

[0489] Refer to Table 2 and prepare an appropriate amount of PI staining solution according to the number of samples to be tested.

[0490] Table 2. Preparation of PI staining solution

[0491]

[0492] Note: In this example, PI staining solution is prepared based on the number of 12 samples.

[0493] Staining: Add 0.5 mL of PI staining solution to each tube of cell sample, slowly and fully resuspend the cell pellet, and warm it at 37°C in the dark for 30 minutes. Store at 4°C or on ice in the dark. Complete flow cytometry within 24 hours after staining.

[0494] E. Flow cytometric detection and analysis: Red fluorescence was detected at an excitation wavelength of 488 nm using a flow cytometer (Agilent, Novocyte 3000) and light scattering was detected simultaneously. Cellular DNA content analysis and light scattering analysis were performed using appropriate analysis software (Agilent, Novoexpress).

[0495] After PI enters the cell nucleus, it binds to DNA and shows fluorescence. The intensity of PI is positively correlated with the DNA content in the cell. The peak on the left of each flow chart represents the DNA in the G1 phase of the cell cycle 2N, the peak on the right represents the DNA in the G2 / M phase of the cell cycle 4N, and the middle represents the DNA being synthesized in the S phase (between 2N-4N). Fig.15As shown in (A), after TMZ treatment, DNA in the G2 / M phase of sensitive GBM (U87) gradually accumulated over time. Compared with the cells treated with control DMSO, the DNA in the G2 / M phase increased (16.86% at 24 hours, 23.22% at 48 hours, and 40.01% at 72 hours), indicating that the cell cycle was arrested in the G2 / M phase. Fig.15 As shown in (B), after TMZ treatment, the DNA content in the G2 / M phase of drug-resistant GBM (T98G) was not significantly changed, and was similar to that of the control DMSO-treated group. The above data indicate that TMZ can cause cell cycle arrest in GBM cells, and drug-resistant GBM (T98G) can resist this cell cycle arrest.

[0496] Example 7. TMZ induces upregulation of a series of immune activation-related ligands in drug-resistant GBM tumor cells

[0497] Immune surveillance is an important mechanism for identifying and eliminating tumor cells, and the dynamic expression of proteins related to immune activation on the tumor cell membrane is also the key to its recognition by immune cells or escape recognition. This example further studies the expression changes of immune-related proteins while GBM resists the effects of TMZ, accumulating a theoretical basis for immunotherapy.

[0498] A. GBM cell culture and treatment: T98G cell culture steps refer to Example 5. The cells were grown to 80%-90% confluency, and trypsin digestion solution was added. The cells were incubated at 37°C for 5 minutes, and two volumes of DMEM medium were added to terminate digestion. The cell suspension was transferred to a centrifuge tube and centrifuged at 300g for 5 minutes. The cell pellet was resuspended in DMEM medium and counted by AOPI. The cell suspension density was adjusted to 1×10 5 cells / mL, and then add an appropriate volume of TMZ stock solution (100 mM) to the cell suspension to make the concentration of TMZ in the cell suspension 1000 mM. The cells were seeded into a 24-well plate, 500 ml / well.

[0499] B. Antibody incubation and flow cytometry detection of immune-related ligands

[0500] The cells treated with TMZ for 48 hours were dissociated with trypsin digestion solution, centrifuged, the supernatant was discarded, and the corresponding antibodies shown in Table 3 were added and incubated at room temperature for 30 minutes; the cells were washed with PBS, centrifuged, resuspended, and detected by flow cytometry.

[0501] Table 3. Antibodies against immune activation-related targets

[0502]

[0503]

[0504] GBM tumor cell immune activation ligands are important targets for immune cell surveillance and can even activate NK cells. Fig.16 As shown in the figure, after 48 hours of TMZ treatment, flow cytometry results showed that various immune activation ligands were upregulated to varying degrees. The above data suggest that the upregulated expression of a series of immune activation ligands may induce stronger NK killing, providing a theoretical basis for the treatment of drug-resistant GBM with TMZ combined with immune cells.

[0505] Example 8: TMZ induces upregulation of GBM immune-related ligand expression, thereby triggering a stronger immune response of iPSC-NK

[0506] Further in vitro killing studies were conducted to detect the effects of NK cells combined with TMZ on GBM tumor cells to explore the possibility of combined medication. The NK cells used in this example were derived from NK cells differentiated from human induced pluripotent stem cells (iPSCs).

[0507] 8.1. In vitro differentiation of human induced pluripotent stem cells (iPSCs) into NK cells

[0508] In this example, the human induced pluripotent stem cells (iPSCs) used were prepared using CTS TM CytoTune TM -iPS2.1 Sendai virus reprogramming kit (CTS TM CytoTune TM -iPS2.1SendaiReprogramming Kit, Catalog No.: A34546) is a human induced pluripotent stem cell (iPSC) prepared from human peripheral blood cells, but the human induced pluripotent stem cells and their preparation in the present invention are not limited thereto, and may include human induced pluripotent stem cells and their preparation methods from common known sources in the art.

[0509] A. iPSCs were cultured in a low-adhesion 96-well plate to obtain embryoid bodies. 5 iPSC cells were evenly divided into 96-well plates and inoculated into low-adhesion 96-well plates (coning, 3474) using StemFit Basic 03 medium (Ajinomoto, StemFitBasic03Liquid A / B / rHu BFGF) (first culture medium) containing 10 mM Y27632·2HCl (selleck, S6390) and cultured for 1 day to form embryoid bodies.

[0510] B. Mesoderm differentiation: After one day, the culture medium was changed to mesoderm differentiation medium and cultured for 2 days.

[0511] Mesoderm differentiation medium (second medium): RPMI1640 (Gibico, 22400-089), supplemented with 1% non-essential amino acids (Gibco, 11140050), 1% glutamine (Gibco, 35050-061), 50 mg / mL vitamin C (Sigma, S4226), 2% (v / v) B27 medium supplement without vitamin A (Gibco, 12587-010), 20 ng / mL BMP4 (R&D, 314-BP) and 3 mM CHIR-99021 (selleck, S1263).

[0512] C. Hemogenic endothelial cell differentiation: After 2 days, the mesoderm differentiation medium was replaced with hemogenic endothelial differentiation medium and cultured for 2 days.

[0513] Hemogenic endothelial differentiation medium (third medium): RPMI1640 (Gibico, 22400-089), supplemented with 1% non-essential amino acids (Gibco, 11140050), 1% glutamine (Gibco, 35050-061), 50 mg / mL vitamin C (Sigma, S4226), 2% (v / v) B27 medium supplement without vitamin A (Gibco, 12587-010), 5 ng / mL BMP4 (R&D, 314-BP), 50 ng / mL VEGF (Novoprotein, C083), 50 ng / mL bFGF (Sino Biological, 10014-HNAE) and 10 mM SB431542 (selleck, S1067).

[0514] D. NK cell differentiation-1. After 2 days, collect the embryoid bodies in the low-adsorption 96-well plate into a 50 mL centrifuge tube, allow the cells to settle naturally for 1-2 minutes, discard the supernatant, and there is about 10 mL of supernatant remaining in the centrifuge tube. Then add about 40 mL of NK cell differentiation medium 1, gently blow and mix the cell balls, and transfer them to a T225 culture flask and culture them for 7 days.

[0515] NK cell differentiation medium 1 (fourth medium): IMDM medium (Gibico, 12400-053), supplemented with 1% non-essential amino acids (Gibco, 11140050), 1% glutamine (Gibco, 35050-061), 50 mg / mL vitamin C (Sigma, S4226), 2% (v / v) B27 medium supplement without vitamin A (Gibco, 12587-010), 5 ng / mL BMP4 (R&D, 314-BP), 10 ng / mL VEGF (Novoprotein, C083), 20 ng / mL SCF (Novoprotein, GMP-CD53), 5 ng / mL IL3 (PeproTech, 200-03), 10 ng / mL IL15 (Sino Biological, GMP-10360-HNCE), 20 ng / mL IL7 (Sino Biological, GMP-11821-HNCE), 10 ng / mL FLT3L (PeproTech, 300-19).

[0516] E. NK cell differentiation-2. After 7 days of culture, NK cell differentiation medium 2 was added and cultured for 7 days.

[0517] NK cell differentiation medium 2 (fifth medium): IMDM medium (Gibico, 12400-053), supplemented with 1% non-essential amino acids (Gibco, 11140050), 1% glutamine (Gibco, 35050-061), 50 mg / mL vitamin C (Sigma, S4226), B27 medium supplement without vitamin A (Gibco, 12587-010), 5 ng / mL BMP4 (R&D, 314-BP), 10 ng / mL VEGF (Novoprotein, C083), 20 ng / mL SCF (Novoprotein, GMP-CD53), 10 ng / mL IL15 (Sino Biological, 10360-HNCE), 20 ng / mL IL7 (Sino Biological, 11821-HNCE), 10 ng / mL FLT3L (PeproTech, 300-19), 1 mM UM171 (APExBIO, A8950), 1mM SR1 (selleck, S2858), 30mMNAC (Sigma, A9165).

[0518] F. NK cell differentiation-3. After 7 days of culture, NK cell differentiation medium 2 was added again and cultured for 7 days.

[0519] G. NK cell activation and expansion. Collect differentiated NK cells and centrifuge them at 1500 rpm for 10 minutes. Then resuspend the cells with 5 mL of NK cell activation and expansion medium. Pass the cell suspension through a 70 mm cell sieve to filter the cells. Centrifuge the filtered cell suspension again at 1500 rpm for 5 minutes. Resuspend the cells with 5 mL of NK cell activation and expansion medium. Adjust the starting density to 1×10 6 cells / mL, change medium every 3-4 days, and culture for 7-10 days. Used for subsequent functional testing.

[0520] Activation expansion medium: CTS NK-Xpander Medium (GIBICO, A5019001), supplemented with 500 IU of IL-2 (PeproTech, 200-02), 10 ng / mL of IL-15 (Sino Biological, 10360-HNCE), 10 ng / mL of IL-18 (Sino Biological, 10119-HNCE), and 5% of hAB (Shanghai Hengyuan Biotechnology, HY120-AB).

[0521] Finally, the harvested iPSC-NK (iNK) was characterized by flow cytometry. The characterization results are shown in Fig.17 As shown, the NK cells differentiated from iPSC in vitro have ideal cell viability (91.51%), purity (97.68%) and expression of functional receptors (purity is greater than 75%). The information of antibodies related to NK cell characterization is shown in Table 4.

[0522] Table 4. Antibodies against immune activation-related targets

[0523] Antibody Targets Fluorescence channels factory Part Number CD3 BV421 Biolegend 317344 CD56 APC BD Biosciences 555518 NKG2D PE Biolegend 320806 NKp30 PE Biolegend 325208 NKp46 PE BD Biosciences 557991 NKp44 PE BD Biosciences 558563

[0524] 8.2. iPSC-NK combined with TMZ showed stronger degranulation activity

[0525] The iPSC-NK cells differentiated in Example 8.1 were co-cultured with GBM tumor cells, or co-cultured with TMZ-treated GBM tumor cells to detect changes in immune response intensity (degranulation activity). The experimental design is shown in Table 5.

[0526] Table 5. Degranulation activity test design

[0527]

[0528] Preparation of target cells: The culture methods of T98G and U87 cells refer to Example 5. The target cells in the TMZ pretreatment group were treated with 1000 μM TMZ 48 hours in advance and set aside.

[0529] Target cell plating: Centrifuge the target cells of each group in advance, resuspend and count, and dilute to 5×10 5 The density of cells / mL was set at 100 μL / well of the corresponding target cells in the corresponding well plate according to the grouping settings.

[0530] Effector cell delivery: iNK cells were centrifuged and resuspended for counting, and the cell density was diluted to 5×10 5 cells / mL, inoculate 100 μL / well of effector cells in the corresponding well plate, and mix by gently pipetting.

[0531] Blank control: 100 μL of activation amplification medium was added to the blank control wells.

[0532] Positive control: Add PMA / io (Biolegend, 423301) to 5 mL of activation amplification medium at a ratio of 1:500, pipette to mix, and add to the positive control wells, 100 μL / well.

[0533] Incubation with CD107a antibody (BD, 560664): prepare an antibody suspension at a ratio of 1 μL per well, dilute the antibody suspension with 10-fold volume of activation amplification medium, pipette to mix, and add 10 μL of the antibody suspension to each well.

[0534] Place the culture plate configured above in an incubator and incubate for 1 hour.

[0535] Add Golgi blockers: Dilute the required golgistop (BD, 51-2092KZ) and golgiplug (BD, 51-2301KZ) into 10 times the volume of complete culture medium at a ratio of 0.2 μL / well, and add 10 μL to each well. Place the culture plate in the incubator and continue to culture for 3 hours.

[0536] After the culture, the cells in the well plate were transferred to the corresponding 1.5 mL centrifuge tubes, centrifuged at 800 g for 5 minutes, the supernatant was discarded, and the cells were resuspended in 100 μL PBS. 1 μL CD56 flow cytometry antibody was added to each tube, incubated at room temperature in the dark for 20 minutes, centrifuged at 800 g for 5 minutes, the supernatant was discarded, and the cells were resuspended in 100 μL PBS for flow cytometry detection.

[0537] like Fig.18 As shown in (A), for the drug-resistant strain T98G, co-culture of iNK and target cells triggered a certain proportion of degranulation activation, and the expression of CD107a increased from the background of 11.98% to 16.50%. In combination with TMZ, the expression of CD107a was significantly increased to 30.85%, indicating that iNK combined with TMZ enhanced the immune activation ability of NK cells.

[0538] like Fig.18As shown in (B), for the sensitive strain U87, co-culture of iNK and target cells triggered a stronger degranulation activation, and the expression of CD107a increased from the background of 12.83% to 38.29%, and the combination of TMZ further increased the expression of CD107a to 60.22%.

[0539] The above results indicate that TMZ can induce a stronger immune response of iNK cells to GBM tumor cells.

[0540] Example 9: iPSC-NK combined with TMZ has stronger tumor cell killing activity against drug-resistant GBM (T98G)

[0541] Furthermore, this example explores the in vitro efficacy of iPSC-NK combined with TMZ by monitoring the killing of tumor target cells.

[0542] A. For GBM tumor cell T98G culture, digestion and plating methods, see Example 5.

[0543] B. TMZ configuration, the method of treating cells is shown in Example 5. In this example, 1000 μM was used for pretreatment for 48 hours.

[0544] C. For the preparation of iPSC-NK cells, see Example 8.

[0545] D. Co-culture killing. According to the effector-target ratio (1:8, 1:4 and 1:1), take an appropriate volume of iNK cells that are being expanded and cultured (note that the viability of iNK cells should be maintained above 90%), and count them by AOPI; according to the counting results, adjust the cell suspension density to 1×10 5 Cells / mL. 1×10 4 After the 96-well T98G plate was attached to the wall (6 hours after re-digestion and inoculation), the NK cell suspension was added to the T98G plate according to the experimental grouping design. After incubation for 18 hours, The viability of T98G cells was detected by luminescence cell viability detection kit. Each group was repeated 3 times.

[0546] E. Cell viability assay. At the set time points (8, 16, 24, 48 hours), cells were collected. Luminescent Cell Viability Assay Kit was used according to the manufacturer's instructions. The solution was mixed with DMEM medium in a 1:1 ratio to prepare the working solution. The working solution was added to a 96-well plate, 50 μL / well, incubated at room temperature in the dark for 10 min, and then the chemiluminescence was detected using an enzyme reader.

[0547] F. Calculation of killing rate. Killing rate (%) = (fluorescence intensity of the unkilled group - fluorescence intensity of the killed group) / fluorescence intensity of the unkilled group * 100%. t test: p < 0.05 is marked with *, p < 0.01 is marked with **.

[0548] The results are as follows Fig.19 As shown in the figure, TMZ had a weak killing effect on drug-resistant GBM (T98G) at 8, 16, 24, and 48 hours after co-culture, with only about 10% killing at 48 hours (blank column). iNK has a significant killing effect on target cells, and it increases with time and the effect-target ratio increases (gray column). Importantly, the killing of target cells by the iNK combined with TMZ group was significantly improved at the corresponding time points and the effect-target ratio (black slash column). The above results show that although drug-resistant GBM (T98G) has a resistance effect to TMZ, TMZ combined with iNK cells will have a stronger tumor killing activity against it.

[0549] Example 10: Synergistic effect of NK cells from other sources (NK92, PB-NK) combined with TMZ on drug-resistant GBM

[0550] Since immune target recognition and tumor immune killing are basic tumor immune mechanisms, not limited to iPSC-NK, this example also uses NK cells from other sources for in-depth research and comparison. NK92 cell line was purchased from Zhejiang Meisen Cell Technology Co., Ltd., and PB-NK from peripheral blood was purchased from Shanghai Heyousheng Biotechnology Co., Ltd.

[0551] A. NK92 cell line culture and expansion. NK92 culture medium: CTS NK-Xpander Medium (GIBICO, A5019001), supplemented with 500 IU of IL-2 (PeproTech, 200-02) and 5% hAB (Shanghai Hengyuan Biotechnology, HY120-AB). 5 -5×10 5 The cells were inoculated at a density of 10 cells / mL, and the medium was changed halfway every 48 hours. The cells were cultured in a 37°C incubator.

[0552] B. PB-NK cell culture and expansion. PB-NK culture medium: CTS NK-Xpander Medium (GIBICO, A5019001), supplemented with 500 IU of IL-2 (PeproTech, 200-02), 10 ng / mL of IL-15 (Sino Biological, 10360-HNCE), 10 ng / mL of IL-18 (Sino Biological, 10119-HNCE), and 5% hAB (Shanghai Hengyuan Biotechnology, HY120-AB). 6The cells were inoculated at a density of 10 cells / mL, and the medium was changed every 2-3 days. The cells were cultured in a 37°C incubator and expanded for 14 days for subsequent functional testing.

[0553] Finally, the harvested NK92 cells and PB-NK cells were characterized by flow cytometry. The specific operation and reagents used are shown in Example 8. Fig. 20 As shown, the harvested amplified NK92 cell line and PB-NK cells have a viability of more than 85%, a purity of more than 85%, and a high expression of functional receptors. Among them, the NKp44 expression of PB-NK is about 14% lower, which may be because different functional receptors are dynamically expressed in NK cells from different peripheral blood sources, with certain individual differences, which is a normal phenomenon.

[0554] C. NK cells from different sources combined with TMZ to kill GBM in vitro. The efficacy of iPSC-NK, NK92, and PB-NK cells combined with TMZ was studied. The in vitro killing method is shown in Example 9. The effector-target ratio E:T=1:1, and the co-culture time with GBM tumor cells was set to 8 hours. The groups were set as shown in Table 6.

[0555] Table 6. Experimental design of NK cells combined with TMZ to kill drug-resistant GBM

[0556]

[0557] Test results such as Fig.21 As shown in the figure, 8 hours after co-culture, TMZ had almost no killing effect on drug-resistant GBM (T98G) (blank column). iNK, NK92 and PB-NK all had killing effects on target cells (gray columns), with a killing rate of 40-60%, and the killing of target cells by NK cells from various sources combined with TMZ groups was significantly improved (black slash columns), greater than 60% killing rate. Further analysis and comparison of the killing activity of iNK, NK92 and PB-NK showed that the in vitro killing activity of iNK combined with TMZ was better than that of NK cells from other sources combined with TMZ. The above results show that compared with NK cells from other sources combined with TMZ, iPSC-derived NK cells combined with TMZ have stronger tumor killing activity against drug-resistant GBM (T98G).

[0558] Although the present invention has been disclosed as above in the form of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A pharmaceutical combination for treating glioblastoma, comprising an active ingredient comprising natural killer (NK) cells and a chemotherapeutic agent, wherein the chemotherapeutic agent comprises temozolomide (TMZ).

2. The pharmaceutical combination of claim 1, wherein the glioblastoma is resistant to TMZ.

3. The pharmaceutical combination of claim 1 or 2, wherein the NK cells are unmodified NK cells.

4. The pharmaceutical combination according to any one of claims 1 to 3, wherein the NK cells are NK cells derived from induced pluripotent stem cells (iPSCs).

5. The pharmaceutical combination according to any one of claims 1 to 4, wherein the NK cells are prepared by a 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 basic culture medium comprising at least one, at least two, at least three or at least four of the following: a BMP signaling pathway activator, a vascular endothelial growth factor, a colony stimulating factor, and an interleukin; The fifth culture medium is a basic 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, stem cell expander.

6. The pharmaceutical combination of claim 5, wherein the kit further comprises a third culture medium, which is a basic culture medium comprising at least one, at least two or at least three of the following: a BMP signaling pathway activator, a growth factor, a TGFβ / ALK inhibitor.

7. The pharmaceutical combination of claim 5, wherein the kit further comprises a first culture medium, and the first culture medium is a basal culture medium comprising a ROCK inhibitor.

8. The pharmaceutical combination according to any one of claims 5 to 7, wherein the NK cells are prepared by a method of inducing stem cells to differentiate into natural killer (NK) cells, the method comprising the steps of using the kit defined in any one of claims 5 to 7.

9. The pharmaceutical combination of claim 8, wherein the method comprises: Step (1) culturing stem cells to obtain embryoid bodies; Step (2) performing mesodermal differentiation culture on the embryoid bodies to obtain mesodermal cells; Step (3) performing hemogenic endothelial differentiation culture on the mesoderm cells to obtain hemogenic endothelial cells; Step (4) culturing the hemogenic endothelial cells in the fourth culture medium and the fifth culture medium of the kit defined in claim 5 to obtain NK cells; Preferably, the culturing in step (1) is carried out in the first culture medium of the kit defined in claim 7; Preferably, the culturing in step (2) is carried out in the second culture medium of the kit defined in claim 5; Preferably, the culturing in step (3) is carried out in the third culture medium of the kit defined in claim 6; Preferably, the culturing time in step (1) is 12-36 hours; Preferably, the culturing time in step (2) is 24-72 hours; Preferably, the culturing time in step (3) is 48-144h; Preferably, the step (4) of placing the hemogenic endothelial cells in the fourth culture medium and the fifth culture medium of the kit defined in claim 5 for culturing is placing the hemogenic endothelial cells in the fourth culture medium and the fifth culture medium of the kit defined in claim 5 for culturing in sequence; Preferably, the hemogenic endothelial cells are cultured in the fourth culture medium for 96-192 hours; Preferably, the hemogenic endothelial cells are cultured in the fifth culture medium for 144-432 hours.

10. The pharmaceutical combination of any one of claims 1 to 9, further comprising other chemotherapeutic agents selected from anticancer agents, metabolic antagonists, DNA demethylating agents, antitumor agents derived from plants, alkylating agents, antimetabolites, anticancer antibiotics, topoisomerase inhibitors, mitotic inhibitors, differentiation agents, hormone therapeutic agents; Preferably, the other chemotherapeutic agent is selected from carmustine, lomustine, vorinostat, mebendazole, imatinib mesylate, AG119, or any therapeutic derivatives thereof.

11. The pharmaceutical combination according to any one of claims 1 to 10, wherein the active ingredient does not contain trimetrexate, and / or an immune checkpoint inhibitor.

12. The pharmaceutical combination according to any one of claims 1 to 11, which is a kit.

13. Use of the drug combination according to any one of claims 1 to 12 in the preparation of a drug for treating glioblastoma; Preferably, the chemotherapeutic agent is administered before, after, or simultaneously with the administration of the NK cells; Preferably, the chemotherapeutic agent is administered 8 to 72 hours before, 8 to 72 hours after, or 8 to 72 hours before and after administration of the NK cells.

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