GiNK cell culture method

Abstract

The invention provides a method for obtaining GiNK cells through novel culture medium amplification, a large number of highly purified human natural killer (NK) cells are obtained by combining IL-15 active fragments, PI3K inhibitors, key receptors targeting the surfaces of the NK cells, antibodies enhancing activation of the NK cells and various cytokines, and the GiNK cells can be used for preparing the human natural killer cells. Expansion from human peripheral blood mononuclear cells (PBMCs) can be achieved without the use of feeder cells. Through culture for 2-3 weeks, the GiNK cells can be amplified by hundreds to thousands of times, and the proliferation efficiency, functional activity and anti-tumor effect of the GiNK cells are remarkably improved. The culture medium provided by the invention not only reduces the use concentration of cell factors, but also solves the problem of function depletion of GiNK cells in in-vitro culture through the synergistic effect of various additives, and provides important technical support for clinical application of the GiNK cells.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to a culture medium, a composition and a culture method for culturing human GiNK cells. Background Art

[0002] Natural Killer cells (NK cells) are an important part of the innate immune system, capable of directly killing tumor cells and virus-infected cells, and are also involved in immune regulation and inflammatory responses. In recent years, the application of NK cells in tumor immunotherapy has attracted much attention, and their unique anti-tumor mechanism has made them another research hotspot after T cell therapy. However, the in vitro expansion, activation and functional maintenance of NK cells remain the main bottlenecks restricting their clinical application. Therefore, developing an efficient NK cell culture medium to promote the proliferation, activation and function enhancement of NK cells has important scientific significance and application value.

[0003] Currently, the in vitro culture of NK cells mainly relies on a combination of a basal medium (such as RPMI-1640 or DMEM) and various cytokines. Commonly used cytokines include IL-2, IL-15, etc., which can promote the proliferation and activation of NK cells. However, the culture method relying solely on cytokines has certain limitations, such as high cytokine cost, short half-life, easy degradation, etc. In addition, NK cells are prone to functional exhaustion during in vitro culture, resulting in a decrease in their anti-tumor activity. Therefore, how to optimize the culture conditions of NK cells and improve their amplification efficiency and functional activity is the focus of current research.

[0004] In recent years, researchers have tried to optimize the NK cell culture system through various strategies. For example, by adding small molecule inhibitors to regulate intracellular signaling pathways, or using antibodies to target NK cell surface receptors to enhance their activation. Among them, the PI3K signaling pathway plays an important role in the proliferation, survival and function regulation of NK cells, and the application of PI3K inhibitors can regulate the metabolic state of NK cells and enhance their anti-tumor activity. In addition, targeting antibodies of NK cell surface receptors can directly activate NK cells and improve their recognition and killing ability of tumor cells. At the same time, as a key NK cell growth factor, IL-15 can significantly extend its half-life and enhance its biological activity by binding to the transferrin receptor (TfR) to form a complex.

[0005] Although a variety of NK cell culture media have been developed, the prior art still has the following deficiencies: High cytokine dependence: Existing culture media usually rely on high concentrations of cytokines (such as IL-2, IL-15), which are costly and have limited effects. Insufficient function maintenance: NK cells are prone to functional exhaustion during in vitro culture, resulting in a decline in their anti-tumor activity. Synergistic effects have not been fully explored: The prior art mostly uses single additives and fails to make full use of the synergistic effects of multiple additives. Therefore, there is a need for a culture medium that reduces the use concentration of cytokines and, through the synergistic effects of multiple additives, solves the problem of functional exhaustion of NK cells during in vitro culture, providing important technical support for the clinical application of NK cells. Summary of the Invention

[0006] Based on the above background, the present invention proposes a method for amplifying GiNK cells (Genuine induced natural killer cells) through a novel culture medium. By combining an IL-15 active fragment, a PI3K inhibitor, a key receptor targeting the NK cell surface, an antibody enhancing NK cell activation, and various cytokines, a large number of highly purified human natural killer (NK) cells can be obtained, which can be amplified from human peripheral blood mononuclear cells (PBMCs) without using feeder cells. Through 2 to 3 weeks of culture, NK cells can be amplified hundreds to thousands of times, significantly improving the proliferation efficiency, functional activity, and anti-tumor effect of NK cells. GiNK cells (Genuine induced natural killer cells) refer to highly purified cytotoxic NK cells induced by the above-mentioned multiple factors.

[0007] The culture medium of the present invention not only reduces the use concentration of cytokines, but also, through the synergistic effects of multiple additives, solves the problem of functional exhaustion of NK cells during in vitro culture, providing important technical support for the clinical application of NK cells.

[0008] The first aspect of the present invention relates to providing an NK cell stimulant The NK cell stimulant involved in the present invention is preferably added to an NK cell culture medium, and the stimulant is selected from recombinant proteins, PI3K inhibitors, engineered antibodies, cytokines, etc.

[0009] Stimulant 1 Recombinant protein A recombinant fusion protein can be added to the basal medium. The recombinant fusion protein can be a recombinant interleukin or a truncated interleukin fragment. Preferably, recombinant proteins or active fragments of IL-15, IL2, and IL-5 can be used. It can effectively enhance the sensitivity of NK cells to cytokines and promote proliferation.

[0010] Furthermore, it is preferred to select the active region of IL-15 to prepare an active fragment. The active region of IL-15 is mainly concentrated in the C-terminal region. It is preferred to select the region of amino acids 111-131 at the C-terminus of the natural fragment of IL-15 to prepare an active fragment. By way of example only, the active fragment can be the one shown by LQELLSAMQSTVYFNQMRKVRF (SEQ ID NO:1), and sequences having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity thereto, as well as fragments containing 3-5 conservative substitutions, deletions, replacements, and fragments hybridizing under stringent conditions.

[0011] Furthermore, the active fragment can be combined with a targeting delivery peptide, which is usually a short peptide screened by phage display technology. For example, it specifically binds to the transferrin receptor (TfR). By way of example only, the TfR-binding peptide can be the one shown by THRPPMWSPVWP (SEQ ID NO:2).

[0012] The IL-15 active fragment and the TfR-binding peptide can be prepared separately and mixed in solution; they can also be prepared as a fusion peptide, which does not affect their function; however, preferably, they can be prepared as a fusion peptide. The fusion peptide can be directly linked or linked through a linker peptide. The preferred linker peptide is (GGGGS)n, where n can be 1-3.

[0013] By way of example only: the fusion protein can be one of the ones shown by LQELLSAMQSTVYFNQMRKVRFGGGGSTHRPPMWSPVWP (SEQ ID NO:3), LQELLSAMQSTVYFNQMRKVRFTHRPPMWSPVWP (SEQ ID NO:4), THRPPMWSPVWPTHRPPMWSPVWP (SEQ ID NO:5), THRPPMWSPVWPGGGGSLQELLSAMQSTVYFNQMRKVRF (SEQ ID NO:6), and sequences having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity thereto, as well as fragments containing 3-5 conservative substitutions, deletions, replacements, and fragments hybridizing under stringent conditions.

[0014] Example of preparation method: Dissolve 1 × 10 7 IU of the recombinant fusion protein in sterile deionized water to a specific concentration. After fully dissolving, aliquot 200 μl into cryotubes and store at -80°C. The preparation method is only for example, and it can be configured into different concentrations for storage and configured into the required final concentration when in use. Preferably, the final concentration of the recombinant fusion protein in the culture medium can be 10 - 100 μg / mL. Specifically, it can be 20 - 100 μg / ml, 20 - 90 μg / ml, 20 - 80 μg / ml, 20 - 60 μg / ml, 30 - 70 μg / ml, 30 - 80 μg / ml, 25 - 70 μg / ml, 35 - 85 μg / ml. In a specific embodiment, the final concentration can be selected from any one of 10 μg / ml, 20 μg / ml, 30 μg / ml, 40 μg / ml, 50 μg / ml, 60 μg / ml, 65 μg / ml, 70 μg / ml, 75 μg / ml, 80 μg / ml, 85 μg / ml, 90 μg / ml, 100 μg / ml.

[0015] Stimulant 2 NK enhancer PI3K inhibitor Optionally, a phosphatidylinositol - 3 - kinase (PI3K) inhibitor can be added to the basal medium, which can activate the PI3K pathway, inhibit apoptosis, ensure the survival of NK cells during amplification, and can also significantly improve the killing activity of the amplified NK cells.

[0016] Optionally, the PI3K inhibitor is selected from at least one of Idelalisib, LY294002, Duvelisib, Copanlisib, Alpelisib, Wortmannin, and can also be a mixture of two, or a mixture of three.

[0017] Preferably, the final concentration of the PI3K inhibitor in the culture medium is 0.1 - 15.0 μM. In a specific embodiment, the concentration can be 0.1 - 14.0 μM, 0.3 - 14.0 μM, 0.5 - 13.0 μM, 1.5 - 14.5 μM, 1.5 - 14.0 μM, 2.0 - 13.5 μM, 2.5 - 13.0 μM, 0.5 - 10.0 μM, 1.5 - 10.0 μM, 3.0 - 10.0 μM, 4.0 - 10.0 μM, 5.0 - 10.0 μM. In a specific embodiment, the concentration can be 5.0 μM, 5.5 μM, 6.0 μM, 6.5 μM, 7.0 μM, 7.5 μM, 8.0 μM, 8.5 μM, 9.0 μM, 9.5 μM, 10.0 μM.

[0018] Stimulant 3 Engineered antibody Furthermore, an engineered antibody can be added to the basal medium to stimulate the activation of NK cells and enhance the immune killing effect.

[0019] Specifically, it can be at least one of a CD16 antibody, an anti-2B4 antibody, an anti-NKG2D antibody, an NKp30 antibody, an NKp44 antibody, an NKp46 antibody or an antigen-binding fragment thereof; preferably, one of them is selected.

[0020] In a specific embodiment, the engineered antibody can be an NKp44 antibody; Optionally, the antibody includes at least one selected from single-chain antibodies, Fab antibodies, Fv antibodies, VHH single-domain antibodies, and minimal recognition units; Optionally, the antigen-binding fragment includes at least one of a Fab fragment, a (Fab)2 fragment, an scFv-Fc fusion protein, an scFv-Fv fusion protein, an Fv fragment, and a minimal recognition unit.

[0021] An exemplary NKp44 single-chain antibody sequence is as follows: EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAKDRGSGYYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIK (SEQ ID NO:7), and sequences having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity thereto, and fragments containing 3-5 conservative substitutions, deletions, replacements, and fragments hybridizing under stringent conditions.

[0022] The present invention further relates to a nucleic acid molecule capable of expressing the above antibody molecule; the nucleic acid molecule can be a DNA molecule or an RNA molecule.

[0023] The present invention further includes an expression vector and a recombinant cell capable of expressing the above antibody molecule.

[0024] Exemplary preparation method: Take 10 ml of 0.1% HSA / PBS in a 15 ml test tube, add 100 μg of an engineered single-chain antibody fragment targeting NKp44 on the surface of NK cells to make a 10 μg / ml solution, and store it at 4°C (it can be stably stored for 3 months). Of course, as long as the formulation stability such as the thermal stability, viscosity, and storage stability of the antibody can be ensured, appropriate improvements can be made in a manner achievable by those skilled in the art based on the above preparation method.

[0025] The final concentration of the antibody in the culture medium is preferably 10-100 μg / ml. Specifically, it can be 20-100 μg / ml, 20-90 μg / ml, 20-80 μg / ml, 20-60 μg / ml, 30-70 μg / ml, 30-80 μg / ml, 25-70 μg / ml, 35-85 μg / ml. In a specific embodiment, the final concentration can be selected from any one of 10 μg / ml, 20 μg / ml, 30 μg / ml, 40 μg / ml, 50 μg / ml, 60 μg / ml, 65 μg / ml, 70 μg / ml, 75 μg / ml, 80 μg / ml, 85 μg / ml, 90 μg / ml, 100 μg / ml.

[0026] Stimulant 4 Complex Medium The present invention further provides a cytokine-supplemented complex medium for supplementing cytokines required for NK cell expansion and activation in a basal medium. The additives are selected from at least one of IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, IFN-γ, thymosin, OK432, GM-CSF. Among them, the concentration of IL-2 is 10-100 ng / ml, and the concentration of IL-7 is 10-50 ng / mL. The concentration of IL-12 is 1-20 ng / mL, the concentration of IL18 is 10-100 ng / mL, the concentration of IL-21 is 10-100 ng / mL, the concentration of IFN-γ is 10-100 ng / mL, the concentration of thymosin is 5-20 ng / mL, the concentration of OK432 is 5-50 ng / mL, and the concentration of GM-CSF is 1-50 ng / mL. The combination of the above additives can synergistically enhance the IFN-γ secretion of NK cells, promote the differentiation and functional maturation of NK cells, and enhance the cytotoxicity and anti-tumor activity of NK cells.

[0027] The second aspect of the present invention is to provide a culture medium composition The present invention further provides a culture medium composition, comprising a basal medium + cofactors + blood products.

[0028] Basal medium: The NK cell culture medium includes a basal medium, which can be a commonly used commercial medium such as RP1640, DMEM, α-MEM, IMDM, X-VIVO15 medium. Preferably, it can be at least one of the commercial media. When there are more than one, it is preferably used in an equal proportion mixture. More preferably, two of them are selected for mixing or a single component is used.

[0029] Optionally, some additives need to be added to the commercial basal medium to effectively stimulate it and prepare an amplification medium or an activation medium. The additives are as described above.

[0030] Blood products can be optionally supplemented in the medium, including but not limited to fetal bovine serum (FBS) with a volume fraction of 5-10%, human inactivated autologous plasma, or human AB serum. The serum or its analogs include but are not limited to animal sera, human sera, serum substitutes of animal origin, synthetic serum substitutes, serum-free media, human inactivated autologous plasma, and / or MCE serum substitutes, etc.; the animal sera include: fetal bovine serum (FBS), newborn bovine serum (NBS), horse serum, or porcine serum, etc.; the human sera include: human AB serum and / or human platelet lysate (PL), etc.; the serum substitutes of plant origin include: plant protein-based substitutes; the synthetic serum substitutes include: EvaCell® (suitable for the culture of cells such as mesenchymal stem cells (MSC), with defined composition and no animal origin), Ultroser™ G (semi-synthetic composition, low protein content, suitable for multiple cell types, can replace fetal bovine serum), and / or Cell-Ess® (synthetic substitute for cell culture), etc.; the serum-free media include: CTS™ immune cell serum substitute, etc. (with defined composition and no xenogeneic components, suitable for in vitro amplification of immune cells). Specifically, the addition amount of the blood product can be 5%-20%, 8%-15%, 8%-12%, 10%-12%; more specifically, it can be 8%, 9%, 10%, 11%, 12%, etc. The dosage can be selected according to the cell state.

[0031] In a specific embodiment, the present invention provides a culture medium composition. On the basis of the basal medium, 10-100 μg / mL of IL15 fusion protein, 0.1-15.0 μM of PI3K inhibitor, and 10-100 μg / ml of engineered antibody are added; further, IL-2 at 10-100 ng / ml, IL-7 at 10-50 ng / mL, IL-12 at 1-20 ng / mL, IL18 at 10-100 ng / mL, IL-21 at 10-100 ng / mL, IFN-γ at 10-100 ng / m, thymosin at 5-20 ng / mL, OK432 at 5-50 ng / mL, GM-CSF at 1-50 ng / mL can be added, at least one of them.

[0032] In a specific embodiment, the present invention provides a NK cell culture medium composition, comprising a basic medium supplemented with 10 - 100 μg / mL of IL15 fusion protein, 0.1 - 15.0 μM of PI3K inhibitor, 10 - 100 μg / ml of engineered antibody; and IL-2 10 - 50 ng / ml, IL-7 10 - 25 ng / mL, IL-12 5 - 15 ng / mL, IL18 10 - 50 ng / mL, IL-21 10 - 50 ng / mL, IFN-γ 10 - 40 ng / m, thymosin 5 - 15 ng / mL, OK432 5 - 25 ng / mL, GM-CSF 5 - 25 ng / mL.

[0033] In a specific embodiment, the present invention provides a NK cell culture medium composition, comprising a basic medium supplemented with 20 - 80 μg / mL of IL15 fusion protein, 0.5 - 12.0 μM of PI3K inhibitor, 15 - 80 μg / ml of engineered antibody; and IL-2 10 - 50 ng / ml, IL-7 10 - 25 ng / mL, IL-12 5 - 15 ng / mL, IL18 10 - 50 ng / mL, IL-21 10 - 50 ng / mL, IFN-γ 10 - 40 ng / m, thymosin 5 - 15 ng / mL, OK432 5 - 25 ng / mL, GM-CSF 5 - 25 ng / mL.

[0034] In a specific embodiment, the present invention provides a NK cell culture medium composition, comprising a basic medium supplemented with 20 - 80 μg / mL of IL15 fusion protein, 0.5 - 12.0 μM of PI3K inhibitor, 15 - 80 μg / ml of engineered antibody; and IL-2 15 - 30 ng / ml, IL-7 10 - 20 ng / mL, IL-12 8 - 18 ng / mL, IL18 10 - 30 ng / mL, IL-21 10 - 50 ng / mL, IFN-γ 10 - 40 ng / m, thymosin 5 - 15 ng / mL, OK432 8 - 25 ng / mL, GM-CSF 5 - 25 ng / mL.

[0035] Preferably, the basal medium can be RPMI 1640 medium; preferably, the IL-15 fusion protein can be the fusion peptide shown in SEQ ID NO: 6; preferably, the PI3K inhibitor can be at least one of Idelalisib, LY294002, Duvelisib, Copanlisib, Alpelisib, Wortmannin, or any one thereof; preferably, the engineered antibody can be the NKp44 antibody, and more specifically, it can be the anti-NKp44 single-chain antibody shown in SEQ ID NO: 7.

[0036] In a specific embodiment, the present invention provides an NK cell culture medium composition, comprising RPMI 1640 medium, supplemented with 20 - 80 μg / mL of IL-15TfR, 0.5 - 12.0 μM of PI3K inhibitor, 15 - 80 μg / ml of anti-NKp44 single-chain antibody; and IL-2 at 15 - 30 ng / ml, IL-7 at 10 - 20 ng / mL, IL-12 at 8 - 18 ng / mL, IL-18 at 10 - 30 ng / mL, IL-21 at 10 - 50 ng / mL, IFN-γ at 10 - 40 ng / ml, thymosin at 5 - 15 ng / mL, OK432 at 8 - 25 ng / mL, GM-CSF at 5 - 25 ng / mL.

[0037] In a specific embodiment, the present invention provides an NK cell culture medium composition, comprising RPMI 1640 medium, supplemented with 20 - 80 μg / mL of IL-15TfR, 0.5 - 12.0 μM of Idelalisib, 15 - 80 μg / ml of anti-NKp44 single-chain antibody; and IL-2 at 15 - 30 ng / ml, IL-7 at 10 - 20 ng / mL, IL-12 at 8 - 18 ng / mL, IL-18 at 10 - 30 ng / mL, IL-21 at 10 - 50 ng / mL, IFN-γ at 10 - 40 ng / ml, thymosin at 5 - 15 ng / mL, OK432 at 8 - 25 ng / mL, GM-CSF at 5 - 25 ng / mL.

[0038] In a specific embodiment, the present invention provides an NK cell culture medium composition, comprising RPMI 1640 medium, supplemented with 25 - 75 μg / mL of IL15TfR, 2.5 - 10.0 μM of Idelalisib, 25 - 75 μg / ml of anti-NKp44 single-chain antibody; and IL-2 at 20 - 30 ng / ml, IL-7 at 10 - 18 ng / mL, IL-12 at 10 - 15 ng / mL, IL18 at 20 - 25 ng / mL, IL-21 at 10 - 15 ng / mL, IFN-γ at 10 - 15 ng / m, thymosin at 10 - 15 ng / mL, OK432 at 10 - 22 ng / mL, GM-CSF at 10 - 20 ng / mL.

[0039] In a specific embodiment, the present invention provides an NK cell culture medium composition, comprising RPMI 1640 medium, supplemented with 25 - 75 μg / mL of IL15TfR, 5.0 - 10.0 μM of Idelalisib, 25 - 75 μg / ml of anti-NKp44 single-chain antibody; and IL-2 at 20 - 30 ng / ml, IL-7 at 10 - 18 ng / mL, IL-12 at 10 - 15 ng / mL, IL18 at 20 - 25 ng / mL, IL-21 at 10 - 15 ng / mL, IFN-γ at 10 - 15 ng / m, thymosin at 10 - 15 ng / mL, OK432 at 10 - 22 ng / mL, GM-CSF at 10 - 20 ng / mL.

[0040] In a specific embodiment, the present invention provides an NK cell culture medium composition, comprising RPMI 1640 medium, supplemented with 25 - 75 μg / mL of IL15TfR, 5.0 - 10.0 μM of Idelalisib, 25 - 75 μg / ml of anti-NKp44 single-chain antibody; and IL-2 at 20 - 25 ng / ml, IL-7 at 10 - 15 ng / mL, IL-12 at 10 - 15 ng / mL, IL18 at 20 - 25 ng / mL, IL-21 at 10 - 15 ng / mL, IFN-γ at 10 - 15 ng / m, thymosin at 10 - 15 ng / mL, OK432 at 10 - 15 ng / mL, GM-CSF at 10 - 20 ng / mL.

[0041] Furthermore, the NK cell culture medium composition provided by the present invention can be as shown in Table 1.

[0042] The third aspect of the present invention is to provide a method for culturing GiNK cells The present invention further provides a method for culturing GiNK cells, Step 1: Isolate PBMC cells; Step 2: After treatment with the CD3-positive cell removal reagent, inoculate PBMC cells. Step 3: After inoculation, perform activation culture using the aforementioned medium. Preferably, if the medium turns yellow, perform fluid replacement culture with the aforementioned medium.

[0043] The fourth aspect of the present invention provides GiNK cells obtained by the NK cell culture method and their uses. GiNK cells obtained by the aforementioned culture method.

[0044] A pharmaceutical composition comprising GiNK cells, a cell culture medium, a cryoprotectant, a buffer system, and a stabilizer. The cryoprotectant includes DMSO, trehalose, and serum; the buffer system includes PBS, HEPES, and physiological saline; the stabilizer includes human serum albumin and an antioxidant.

[0045] The uses of the aforementioned GiNK cells and the pharmaceutical composition containing them in the treatment of tumor-related diseases and their applications in the preparation of anti-tumor drugs.

[0046] Tumors include hematological tumors, solid tumors, nervous system tumors, and digestive system tumors.

[0047] Furthermore, it is preferably used for at least one of hematological tumors, lung cancer, infiltrating tumor tissues, breast cancer, colorectal cancer, liver cancer, pancreatic cancer, glioblastoma, and renal cell carcinoma.

[0048] Beneficial effects The culture medium composition provided by the present invention can effectively increase the density and killing ability of GiNK cells and show an effective tumor control effect in vivo, inhibit tumor growth, and promote the transformation of the immune microenvironment towards an anti-tumor direction. It has good medicinal treatment value and provides important technical support for the clinical application of GiNK cells. Description of the drawings

[0049] Figure 1 It is the cell typing flow cytometry diagram of experimental group A of the present invention.

[0050] Figure 2 It is the cell typing flow cytometry diagram of experimental group B of the present invention.

[0051] Figure 3 It is the cell typing flow cytometry diagram of experimental group C of the present invention.

[0052] Figure 4 It is the cell typing flow cytometry diagram of experimental group D of the present invention.

[0053] Figure 5 It is the cell typing flow cytometry diagram of experimental group E of the present invention.

[0054] Figure 6 It is the flow cytometry diagram of cell typing for experimental group F of the present invention.

[0055] Figure 7 It is the flow cytometry diagram of cell typing for experimental group G of the present invention.

[0056] Figure 8 It is the flow cytometry diagram of cell typing for experimental group H of the present invention.

[0057] Figure 9 It is the killing curve of NK cells for the experimental group of the present invention. Specific implementation manners

[0058] Those skilled in the art should understand that the following embodiments are only used to explain the technical solutions of the present invention and are not used to limit the protection scope of the present invention. Without departing from the spirit and essence of the present invention, those skilled in the art can make various modifications or substitutions to the present invention, and these modifications or substitutions should all be covered within the scope of the claims of the present invention.

[0059] Reagents and materials The IL-15 active fragment and the TfR-binding peptide. In this embodiment, the fusion peptide shown in SEQ ID NO:6 (hereinafter referred to as IL15TfR) is selected and prepared by the solid-phase synthesis method (SPPS), and is commissioned to be synthesized by Shanghai Qiangyao Biotechnology Co., Ltd. Idelalisib, LY294002, Duvelisib, Copanlisib, Alpelisib, Wortmannin are all purchased from Shanghai Yuanye Biotechnology Co., Ltd. Engineered antibody. In this embodiment, the anti-NKp44 single-chain antibody shown in SEQ ID NO:7 is selected and prepared by Nanjing Genscript Biotechnology.

[0060] Obtaining of PBMC Collect 50 - 100 ml of heparinized human peripheral blood into a vacuum blood collection tube. Take 1 tube of 8 ml of peripheral blood and centrifuge it at 1750G for 10 minutes. Separate the upper plasma into a 15 ml test tube for NK cell culture, and freeze the remaining plasma at -20°C or below.

[0061] Separate and remove peripheral blood mononuclear cells (PBMCs) of CD3. Collect peripheral blood and use an anticoagulant (such as EDTA or heparin) to prevent coagulation. Mix the peripheral blood with PBS at a ratio of 1:1. Add Ficoll separation solution to a centrifuge tube, and slowly add the diluted blood onto the Ficoll solution surface, avoiding mixing. Centrifuge at 400 × g for 30 minutes. After centrifugation, aspirate the white film layer (PBMCs layer) into a new centrifuge tube. Add PBS and centrifuge at 300 × g for 10 minutes to wash the cells. Repeat the washing once, and finally resuspend the cells with an appropriate amount of PBS.

[0062] CD3 Depletion Determine the concentration of PBMCs using a cell counter. Add CD3-specific magnetic beads, mix well, and incubate at 4°C for 15 - 30 minutes. Place the cell suspension on a magnetic separator, let it stand for 2 - 5 minutes, and aspirate the unbound CD3-negative cells. Wash the cells: Wash the removed CD3-negative cells with PBS, centrifuge at 300 × g for 10 minutes, and resuspend in the culture medium. Culture the CD3-depleted PBMCs in an incubator at 37°C and 5% CO2. Verify the removal effect of CD3-positive cells by flow cytometry to obtain peripheral monocytes depleted of CD3+T. (The sorting product RosetteSep™ Human CD3 Depletion Cocktail from STEMCELL Technologies is used for the depletion of CD3-positive cells). Use the group of PBMCs without CD3 depletion as a negative control to confirm that the antibodies and detection conditions are correct. After detection, the removal rate is below 5%, which is within the expected range. Culture and Activation of GiNK Cells Take the peripheral monocytes depleted of CD3+T and inoculate them into the wells of a plate with different activation component media. The medium configuration method: basal medium (1640 medium, purchased from Gibco) + added components in Table 1 + 10% autologous plasma. After 3 days of culture, observe the cells under a microscope, and small, loose clone clusters can be seen under the microscope. If the medium starts to turn yellow, add 1 / 2 volume of the above medium. If the medium has not changed color and the cell growth is less, add 1 / 3 volume of the above medium. During the whole process, avoid shaking the culture dish violently, minimize disturbing the cells, and place them in a 5% carbon dioxide incubator for static culture. Start the fluid replacement treatment every two days from the 5th day of culture, and count and observe the cell amplification in each group.

[0063] After the culture is completed, transfer the cell solution to a culture bag. Install the mass and a 50 ml syringe, connect the culture bag, and transfer the cell solution or NK amplification medium to the culture bag with the 50 ml syringe. Then, perform operations such as cell harvesting, cryopreservation, or functional detection as needed.

[0064] Table 1 Experimental Group Settings

[0065] Cell Density Determination

[0066] Measure the density of the recovered cells at the time point when they are just inoculated into the culture flask and 5 - 7 days after adding the stimulating medium. As can be seen from the following table, groups A - H in the experimental group can all effectively provide cell density, while group I (control group), because it only uses the basal medium without adding the stimulating medium, cannot bring an effective increase in cell density.

[0067] Table 2 Cell density / ml when inoculating the culture flask Recovered cell density / ml after culturing with the stimulating medium Group A <![CDATA[9.75x 10 6 > <![CDATA[2.20x 10 9 > Group B <![CDATA[8.60x 10 6 > <![CDATA[1.98x 10 9 > Group C <![CDATA[1.06x 10 7 > <![CDATA[1.95x 10 9 > Group D <![CDATA[9.36x 10 6 > <![CDATA[1.63x 10 9 > Group E <![CDATA[5.49x 10 6 > <![CDATA[7.30x 10 8 > Group F <![CDATA[6.37x 10 6 > <![CDATA[7.30x 10 8 > Group G <![CDATA[1.36x 10 7 > <![CDATA[3.40x 10 9 > Group H <![CDATA[1.62x 10 7 > <![CDATA[3.40x 10 9 > Group I <![CDATA[8.92 x 10 6 > <![CDATA[9.03 x 10 7 > NK cell typing assay The harvested cells were processed into single-cell suspensions, and specific cell surface molecules were labeled with fluorescently labeled antibodies for typing assays. The results are as Figure 1 - Figure 8 shown. Taking Figure 1 as an example, the abscissa is FSH (forward scatter light), and the ordinate is SSH (side scatter light). Lymphocytes in the cell population were initially screened out from cell debris, monocytes, neutrophils, senescent cells, apoptotic cells, etc. Lymphocytes accounted for 87.7% of the total cells. In the upper right figure, it was labeled with CD3-FITC and SSH-C, and 3.72% of CD3-positive cells were obtained. In the lower left figure, the abscissa is CD3-FITC (fluorescent signal labeling T cells), and the ordinate is CD56-APC (fluorescent signal labeling NK cells). By analyzing the scatter plot, the proportions of NK cells, NK T cells, and T cells were obtained. The results showed that in the experimental group of group A, natural killer cells NK cells accounted for 98.0%, and NK-T cells accounted for 0.76%. Since double-positive cells of CD16 (FcγRIII) and CD56 (NCAM) are considered the main phenotypic characteristics of natural killer (NK) cells, further labeling with CD16 and CD56APC yielded a positive rate of NK cells of 95.3%. This proved that specific medium could effectively activate NK cell differentiation after activation.

[0068] Table 3 Lymphocytes CD3+ Tcell CD3 - CD56 CD16+CD56+ Group A 87.7% 3.72% 98.0% 95.3% Group B 89.3% 4.51% 96.7% 94.6% Group C 89.8% 1.47% 98.2% 87.5% Group D 84.6% 2.58% 97.4% 88.0% Group E 73.9% 5.18% 96.2% 90.1% Group F 72.7% 4.71% 95.7% 91.5% Group G 79.1% 3.11% 97.7% 96.2% Group H 80.4% 2.96% 97.9% 96.2% Group I 70.02% 3.46% 63.2% 59.1% GiNK cell killing experiment

[0069] GiNK cells: Single-cell suspensions obtained from the separation and culture of peripheral blood and stimulated by the experimental group or the control group were used. T98G was used as the target cell, and the fluorescent dye was Calcein-AM, and the lysing agent was Triton X-100.

[0070] Group settings: Experimental group: GiNK cells + target cells Control group: Only T98G target cells Maximum release group: Target cells + Triton X-100 (complete lysis) (not shown in the attached figure, and the experimental results proved that the cells could be rapidly and completely lysed) Inoculate GiNK cells and target cells into 96-well plates at different ratios (such as 10:1, 5:1, 1:1), with 3 replicate wells in each group. Incubate in a 37°C, 5% CO2 incubator for 4 - 6 hours, and collect the supernatant after centrifugation. Detect the fluorescence intensity in the supernatant using a fluorescence microplate reader (excitation light 485 nm, emission light 520 nm), and record the fluorescence intensity of each group. Plot a graph with time on the x-axis and Normalized cell index on the y-axis to obtain the killing efficiency vs. time curve, i.e., the killing kinetics curve. Normalized CellIndex (Normalized Cell Index) is an indicator used to quantify the growth, adhesion, or killing behavior of cells in real-time cell analysis (such as the xCELLigence system). It reflects changes in cell status by measuring the impedance changes between cells and microelectrodes, and is commonly used in experiments such as cell proliferation, cytotoxicity, and cell migration. Cell Index (CI) is the raw data directly measured by the instrument, reflecting the impedance changes between cells and the electrode. Normalized Cell Index is the result of normalizing the Cell Index value at a specific time point relative to a reference time point (usually at the start of the experiment or when a certain treatment is added). An increase in Normalized Cell Index represents enhanced cell proliferation or adhesion, while a decrease indicates cell death or detachment (such as when GiNK cells kill target cells).

[0071] The results showed that: at the initial stage (close to time 0), the values of each curve were similar, indicating that the differences in the states of each sample were small at the start of the experiment. The curve of the control group continued to rise, indicating that T98G tumor cells continued to grow without the intervention of effector cells. Most of the curves in the experimental groups showed a downward trend, meaning that after adding effector cells, the growth or related indicators of tumor cells were inhibited. Among them, the decline in groups A and B (effector-to-target ratio 4:1) was relatively large, indicating that under these conditions, the inhibitory effect of effector cells on tumor cells was the most significant, and a higher effector-to-target ratio enhanced the killing effect of effector cells on tumor cells. From Figure 9 it can be seen that the inhibitory degrees of tumor cells in the experimental groups under different effector-to-target ratios were different. As the effector-to-target ratio increased, the downward trend of the curves in some experimental groups became more obvious, indicating that within a certain range, the higher the effector-to-target ratio, the better the inhibitory effect of effector cells on tumor cells may be.

[0072] Chemokine detection Collect the cultured GiNK cells into a centrifuge tube, centrifuge at 1000 rpm for 5 minutes, and discard the supernatant. Wash the cells twice with PBS and resuspend them in flow cytometry staining buffer, adjusting the cell density to 1×10 6 cells / mL. Aliquot the cells into flow cytometry tubes, with 100 µL of cell suspension in each tube (about 1×10 5cells). Fluorescently labeled antibodies (such as anti-CD16-FITC, anti-NKG2D-PE, etc., see the specific table below) were added respectively and incubated in the dark for 30 minutes (4°C). Wash twice with flow cytometry staining buffer to remove unbound antibodies. For CXCR3 and CXCR4, intracellular staining was further required. The specific operation was as follows: Add a permeabilizing agent and incubate in the dark for 20 minutes (4°C), add the fluorescently labeled antibody and incubate in the dark for 30 minutes (4°C). Wash twice with flow cytometry staining buffer. Add 4% paraformaldehyde to fix the cells and incubate in the dark for 15 minutes (room temperature). Wash once with PBS and resuspend in flow cytometry staining buffer.

[0073] Flow cytometry detection: Load the samples onto the instrument for detection, and use a flow cytometer to analyze the expression of each receptor. Set up unstained cells and single-stained control groups for compensation adjustment and gating. Use the FSC / SSC scatter plot to circle the GiNK cell population. Analyze the expression levels of each receptor through the fluorescence channels. Result calculation: Calculate the proportion (%) of positive cells for each receptor. Compare the expression differences between the experimental group and the control group.

[0074] Table 4 Group CD16 NKG2D NKp30 NKp46 NKp44 CXCR3 CXCR4 Group A 90% 88% 85% 87% 86% 89% 88% Group B 89% 87% 84% 86% 85% 88% 87% Group C 75% 74% 72% 73% 71% 76% 75% Group D 74% 73% 71% 72% 70% 75% 74% Group E 60% 58% 57% 59% 56% 61% 60% Group F 59% 57% 56% 58% 55% 60% 59% Group G 45% 44% 43% 44% 42% 46% 45% Group H 44% 43% 42% 43% 41% 45% 44% Group I 20% 18% 17% 19% 16% 21% 20% It can be seen that the natural killer receptors such as CD16, NKG2D, NKp30, NKp44, and NKp46 in experimental groups A - F were all at relatively high expression levels (>50%). The expression rates of chemokine receptors CXCR3 and CXCR4 were even higher, exceeding 88%. Groups G and H were slightly weaker, but both were higher than control group I. This proves that NK cells activated by the activation medium have significantly improved tumor chemotactic ability.

[0075] Analysis of the results of each experimental group. After result detection, Group A and Group B were the optimal groups. Compared with Group A, Group B increased the concentrations of IL15 TfR fusion protein and NKp44 single-chain antibody, but there was not much change in the killing treatment. Group C was the sub-optimal group, indicating that the appropriate concentrations of the IL15TfR fusion protein and the single-chain antibody of IL15 TfR screened in the present invention could also be achieved, but the effect was relatively weaker. When the dosages of IL15TfR fusion protein and NKp44 single-chain antibody were adjusted reasonably, simply increasing the concentration of cytokine additives was meaningless (see Group D and Group E), and it would instead inhibit the activity of GiNK cells. Therefore, it was possible to effectively reduce the concentration of cytokines, reduce costs while increasing the density, activity, and anti-tumor effect of GiNK cells. On the other hand, a large reduction in the types of cytokines would also reduce the activity, and even increasing the concentration of the remaining cytokines could not activate the activity of GiNK cells. Therefore, the cytokine combination in the stimulant screened in the present invention was the lowest necessary combination (see Group F and Group G). However, without adding the specifically screened IL15 TfR fusion protein and NKp44 single-chain antibody of the present invention, even if all cytokine additives were added and used at the optimal concentration, the technical effect of GiNK cells would be greatly weakened, proving the importance of the IL15 TfR fusion protein and NKp44 single-chain antibody screened in the present invention (see Group H). It can be seen that the IL15 TfR fusion protein, NKp44 single-chain antibody, and cytokine additives play a synergistic role.

[0076] In vivo tumor model In order to further verify the function of GiNK cells in Group A, the optimal group, in vivo, the following in vivo experimental protocol was designed to evaluate the anti-tumor activity, migration ability, and immunomodulatory function of NK cells through an animal model.

[0077] NOD immunodeficient mice were used, with 5 mice in each group. A control group (injected with PBS or unactivated NK cells) was set up, and the human tumor cell line A549 was selected to establish a subcutaneous tumor model. The NK cells in Group A were labeled with the fluorescent dye CFSE for easy in vivo tracking. The labeled NK cells were resuspended in PBS, and the cell density was adjusted to 1×10 7 cells / mL. When the tumor volume reached 100 - 200 mm³, the experimental group of GiNK cells was injected via the tail vein (1×10 6 cells per mouse).

[0078] Tumor growth monitoring: The tumor volume (length × width² × 0.5) was measured every 2 - 3 days. The control group was injected with an equal volume of PBS or unactivated NK cells, the tumor growth curve was recorded, and the differences between the experimental group and the control group were compared. The results are shown in the following table. The tumor volume of the experimental group was significantly smaller than that of the control group, indicating that the GiNK cells in the experimental group effectively inhibited tumor growth. On the 21st day, the tumor volume of the experimental group was only about 22% of that of the control group, and the tumor inhibition rate was approximately 78%.

[0079] Table 5 Group Volume on day 0 (mm³) Volume on day 3 (mm³) Volume on day 6 (mm³) Volume on day 9 (mm³) Volume on day 12 (mm³) Volume on day 15 (mm³) Volume on day 18 (mm³) Volume on day 21 (mm³) Experimental group 0 50 80 120 150 180 200 220 Control group 0 100 200 350 500 700 900 1100 Detection of GiNK cell migration: The distribution of fluorescently labeled GiNK cells in vivo was observed using the in vivo imaging system IVIS. Imaging was performed at 24 hours, 48 hours, and 72 hours after injection, and the migration of GiNK cells towards the tumor site was analyzed.

[0080] Table 6 Time (hours) Fluorescence intensity (RFU) at the tumor site in the experimental group Fluorescence intensity (RFU) at the tumor site in the control group 24 5000 ± 500 1000 ± 200 48 8000 ± 800 1500 ± 300 72 10000 ± 1000 2000 ± 400 The GiNK cells in the experimental group began to migrate towards the tumor site within 24 hours after injection, and the fluorescence intensity was significantly higher than that of the control group. After 72 hours, the fluorescence intensity at the tumor site in the experimental group was 5 times that of the control group, indicating that the GiNK cells in the experimental group efficiently migrated to the tumor site.

[0081] Analysis of tumor tissue: The mice were sacrificed and the tumor tissues were removed. Single-cell suspensions of tumors were prepared, and the proportion of GiNK cells infiltrating the tumors was detected by flow cytometry. Immunohistochemistry (IHC) was used to analyze the expression of NK cell markers (such as CD56) in the tumor tissues.

[0082] Table 7 Detection index Results of Group A Results of the control group Infiltration ratio of GiNK cells 25% ± 3% 5% ± 1% Ratio of tumor necrosis area 40% ± 5% 10% ± 2% Number of CD56+ cells (IHC) 200 ± 20 cells / field of view 50 ± 10 cells / field of view The proportion of GiNK cells infiltrating the experimental tumor tissues was significantly higher than that of the control group, indicating that the GiNK cells in the experimental group could effectively infiltrate the tumor tissues. The proportion of tumor necrosis area increased significantly, indicating that the GiNK cells in the experimental group had a strong killing effect on tumor cells. The IHC results showed that the number of CD56+ cells in the experimental tumor tissues increased significantly, further confirming the infiltration of GiNK cells.

[0083] Analysis of the immune microenvironment: Immune cells were extracted from the tumor tissues, and the proportions of tumor-associated macrophages (TAMs) and T cell subsets (such as CD4+, CD8+ T cells) were analyzed by flow cytometry. The expression levels of cytokines (such as IFN-γ, TNF-α) were detected to evaluate the immune regulatory function of GiNK cells.

[0084] Table 8 Detection index Results of the experimental group Results of the control group Ratio of M1-type TAMs 60% ± 5% 20% ± 3% Ratio of M2-type TAMs 20% ± 3% 60% ± 5% Ratio of CD8+ T cells 30% ± 4% 10% ± 2% Level of IFN-γ (pg / mL) 500 ± 50 100 ± 20 Level of TNF-α (pg / mL) 300 ± 30 50 ± 10 In the experimental group, the proportion of M1 type in tumor-associated macrophages (TAMs) increased significantly, while the proportion of M2 type decreased, indicating that the immune microenvironment shifted towards an anti-tumor direction. The proportion of CD8+ T cells increased significantly, indicating that GiNK cells in the experimental group activated the adaptive immune response. The levels of cytokines (IFN-γ, TNF-α) increased significantly, indicating that GiNK cells in the experimental group enhanced the immune response. NK cells in the experimental group promoted the increase of M1-type TAMs and CD8+ T cells and increased the cytokine levels, indicating that they enhanced the anti-tumor immune response.

[0085] GiNK cells in the experimental group had significant therapeutic effects in the A549 subcutaneous tumor model, could inhibit tumor growth, promote the transformation of the immune microenvironment towards an anti-tumor direction, and provided an important basis for subsequent clinical applications.

Claims

1. A NK cell culture medium, comprising a basal medium, characterized in that: An IL15 fusion protein, a PI3K inhibitor, and an engineered antibody are further added to the basal medium. The engineered antibody is selected from at least one of a CD16 antibody, an anti-2B4 antibody, an anti-NKG2D antibody, an NKp30 antibody, an NKp44 antibody, an NKp46 antibody, or an antigen-binding fragment thereof; the PI3K inhibitor is selected from at least one of Idelalisib, LY294002, Duvelisib, Copanlisib, Alpelisib, and Wortmannin.

2. The medium according to claim 1, wherein the IL15 fusion protein can further be a fusion protein of IL15 and a transferrin receptor.

3. The medium according to claim 2, wherein the fusion protein can be any one shown in SEQ ID NO: 1-6, and further comprises a sequence having 85% identity with the above sequence, and a fragment comprising 3-5 conservative substitutions, deletions, replacements, and fragments hybridized under stringent conditions; further, the concentration of the fusion protein is 10-100 μg / mL.

4. The medium according to claim 1, wherein the engineered antibody can be an NKp44 antibody or a binding fragment thereof, and can further be the sequence shown in SEQ ID NO: 7 and a sequence having 85% identity with the above sequence, and a fragment comprising 3-5 conservative substitutions, deletions, replacements, and fragments hybridized under stringent conditions; the concentration of the NKp44 antibody can be 10-100 μg / ml.

5. The culture medium according to claim 1, further comprising at least one of IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, IFN-γ, thymosin, picibanil, GM-CSF; wherein, The concentration of IL-2 is 10-100 ng / ml, the concentration of IL-7 is 10-50 ng / mL, the concentration of IL-12 is 1-20 ng / mL, the concentration of IL18 is 10-100 ng / mL, the concentration of IL-21 is 10-100 ng / mL, the concentration of IFN-γ is 10-100 ng / mL, the concentration of thymosin is 5-20 ng / mL, the concentration of OK432 is 5-50 ng / mL, and the concentration of GM-CSF is 1-50 ng / mL.

6. A medium composition comprising one of the following selections: Based on the basal medium, 10-100 μg / mL of IL15 fusion protein, 0.1-15.0 μM of PI3K inhibitor, and 10-100 μg / ml of engineered antibody are added; at least one of IL-2 10-100 ng / ml, IL-7 10-50 ng / mL, IL-12 1-20 ng / mL, IL18 10-100 ng / mL, IL-21 10-100 ng / mL, IFN-γ 10-100 ng / m, thymosin 5-20 ng / mL, OK432 5-50 ng / mL, and GM-CSF 1-50 ng / mL is further added to the medium; or Add 10 - 100 μg / mL of IL15 fusion protein, 0.1 - 15.0 μM of PI3K inhibitor, 10 - 100 μg / ml of engineered antibody to the basal medium; and IL-2 10 - 50 ng / ml, IL-7 10 - 25 ng / mL, IL-12 5 - 15 ng / mL, IL18 10 - 50 ng / mL, IL-21 10 - 50 ng / mL, IFN-γ 10 - 40 ng / m, thymosin 5 - 15 ng / mL, OK432 5 - 25 ng / mL, GM-CSF 5 - 25 ng / mL; or Add 20 - 80 μg / mL of IL15 fusion protein, 0.5 - 12.0 μM of PI3K inhibitor, 15 - 80 μg / ml of engineered antibody to the basal medium; and IL-2 10 - 50 ng / ml, IL-7 10 - 25 ng / mL, IL-12 5 - 15 ng / mL, IL18 10 - 50 ng / mL, IL-21 10 - 50 ng / mL, IFN-γ 10 - 40 ng / m, thymosin 5 - 15 ng / mL, OK432 5 - 25 ng / mL, GM-CSF 5 - 25 ng / mL; or Add 20 - 80 μg / mL of IL15 fusion protein, 0.5 - 12.0 μM of PI3K inhibitor, 15 - 80 μg / ml of engineered antibody to the basal medium; and IL-2 15 - 30 ng / ml, IL-7 10 - 20 ng / mL, IL-12 8 - 18 ng / mL, IL18 10 - 30 ng / mL, IL-21 10 - 50 ng / mL, IFN-γ 10 - 40 ng / m, thymosin 5 - 15 ng / mL, OK432 8 - 25 ng / mL, GM-CSF 5 - 25 ng / mL.

7. A culture medium composition, comprising one of the following selections: RPMI 1640 medium, add 20 - 80 μg / mL of IL15TfR, 0.5 - 12.0 μM of PI3K inhibitor, 15 - 80 μg / ml of anti-NKp44 single-chain antibody; and IL-2 15 - 30 ng / ml, IL-7 10 - 20 ng / mL, IL-12 8 - 18 ng / mL, IL18 10 - 30 ng / mL, IL-21 10 - 50 ng / mL, IFN-γ 10 - 40 ng / m, thymosin 5 - 15 ng / mL, OK432 8 - 25 ng / mL, GM-CSF 5 - 25 ng / mL; or 1640 medium, supplemented with 20 - 80 μg / mL of IL15TfR, 0.5 - 12.0 μM of Idelalisib, 15 - 80 μg / ml of anti-NKp44 single-chain antibody; and IL-2 at 15 - 30 ng / ml, IL-7 at 10 - 20 ng / mL, IL-12 at 8 - 18 ng / mL, IL18 at 10 - 30 ng / mL, IL-21 at 10 - 50 ng / mL, IFN-γ at 10 - 40 ng / m, thymosin at 5 - 15 ng / mL, OK432 at 8 - 25 ng / mL, GM-CSF at 5 - 25 ng / mL; or 1640 medium, supplemented with 25 - 75 μg / mL of IL15TfR, 2.5 - 10.0 μM of Idelalisib, 25 - 75 μg / ml of anti-NKp44 single-chain antibody; and IL-2 at 20 - 30 ng / ml, IL-7 at 10 - 18 ng / mL, IL-12 at 10 - 15 ng / mL, IL18 at 20 - 25 ng / mL, IL-21 at 10 - 15 ng / mL, IFN-γ at 10 - 15 ng / m, thymosin at 10 - 15 ng / mL, OK432 at 10 - 22 ng / mL, GM-CSF at 10 - 20 ng / mL; or 1640 medium, supplemented with 25 - 75 μg / mL of IL15TfR, 5.0 - 10.0 μM of Idelalisib, 25 - 75 μg / ml of anti-NKp44 single-chain antibody; and IL-2 at 20 - 30 ng / ml, IL-7 at 10 - 18 ng / mL, IL-12 at 10 - 15 ng / mL, IL18 at 20 - 25 ng / mL, IL-21 at 10 - 15 ng / mL, IFN-γ at 10 - 15 ng / m, thymosin at 10 - 15 ng / mL, OK432 at 10 - 22 ng / mL, GM-CSF at 10 - 20 ng / mL; or 1640 medium, supplemented with 25 - 75 μg / mL of IL15TfR, 5.0 - 10.0 μM of Idelalisib, 25 - 75 μg / ml of anti-NKp44 single-chain antibody; and IL-2 at 20 - 25 ng / ml, IL-7 at 10 - 15 ng / mL, IL-12 at 10 - 15 ng / mL, IL18 at 20 - 25 ng / mL, IL-21 at 10 - 15 ng / mL, IFN-γ at 10 - 15 ng / m, thymosin at 10 - 15 ng / mL, OK432 at 10 - 15 ng / mL, GM-CSF at 10 - 20 ng / mL.

8. The culture medium composition according to claim 7, wherein the IL15TfR is any one of SEQ ID NO: 1 - 6, and the anti-NKp44 single-chain antibody is SEQ ID NO:

7.

9. A method for culturing NK cells, Step 1: Isolate PBMC cells; Step 2: Inoculate the PBMC cells after treatment with a CD3-positive cell removal reagent; Step 3: After inoculation, use the culture medium described in claims 1-8 for activation culture.

10. Use of the NK cells prepared according to claim 9 and a pharmaceutical composition containing the same in the preparation of a drug for treating tumors; the tumors include hematological tumors, solid tumors, nervous system tumors, and digestive system tumors; preferably used for at least one of hematological tumors, lung cancer, infiltrating tumor tissues, breast cancer, colorectal cancer, liver cancer, pancreatic cancer, glioblastoma, and renal cell carcinoma.

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