A method to enhance the function of natural killer cells using interleukin-9

Abstract

This invention relates to the field of cell culture technology, specifically to a method for enhancing the function of natural killer cells using interleukin-9. This invention provides an expansion culture medium for NK cells and its uses. By treating NK cells with IL-9 during NK cell culture, or by culturing NK cells using a medium containing IL-9, the toxic function of NK cells can be significantly improved.

Description

Technical Field

[0001] This invention relates to the field of cell culture technology, and specifically to a method for enhancing the function of natural killer cells using interleukin-9. Background Technology

[0002] NK cells are innate immune cells with antiviral and antitumor functions. The STAT family is a family of DNA-binding proteins, currently comprising seven types. They typically respond to various extracellular cytokines and growth signals, with STAT5 being crucial for NK cell function. IL-15 forms a trimer with IL-15Ra and its γ-chain receptor outside NK cells. Within NK cells, it is phosphorylated by STAT5 / STAT3 / STAT1, enters the nucleus, and promotes NK cell proliferation, synthesis of toxic proteins, and increased BCL2 expression. The IL-15-STAT / Jak pathway is extremely important for NK cells.

[0003] IL-9 was discovered over 20 years ago, initially linked to T cell proliferation. Later studies showed that IL-9 does not promote the proliferation of primary T cells. Extracellularly, IL-9 forms a trimer with IL-9Ra and its γ-chain receptor, subsequently phosphorylating STAT5 / STAT3 / STAT1 intracellularly, thereby promoting T cell proliferation and function. However, the effects of IL-9 on other immune cells remain unknown. Summary of the Invention

[0004] This invention aims to at least partially solve one of the technical problems in the related art. Therefore, one object of this invention is to provide a culture medium for the expansion of NK cells and its use therein, which significantly enhances the toxicity of NK cells by treating them with IL-9 during NK cell culture or by culturing NK cells in a medium containing IL-9.

[0005] A first aspect of the present invention provides an amplification culture medium for NK cells. According to an embodiment of the present invention, the amplification culture medium comprises IL-9.

[0006] The inventors discovered that culturing iPSC-derived NK cells with IL-9 during the culture process enhances the toxicological function of iPSC-NK. Using IL-9 during the culture process eliminates the need for new gene editing of iPSC-NK, simplifying the complexity of gene editing. Pretreatment with IL-9 enhances iPSC-NK function, and the elimination of the need for additional IL-9 supplementation in vivo further facilitates translation into clinical applications.

[0007] According to an embodiment of the present invention, the amplification medium further comprises IL-2 and / or IL-15.

[0008] According to an embodiment of the present invention, the amplification culture medium includes IL-9, IL-2 and IL-15.

[0009] According to an embodiment of the present invention, the working concentration of IL-9 is 1-50 ng / mL.

[0010] According to an embodiment of the present invention, the working concentration of IL-2 is 50-1000 IU / mL.

[0011] According to an embodiment of the present invention, the working concentration of the IL-15 is 0.1 - 50 ng / mL.

[0012] According to an embodiment of the present invention, the amplification medium further comprises a complete culture medium for lymphocyte culture; According to an embodiment of the present invention, the complete culture medium for lymphocyte culture is KBM-581 serum-free lymphocyte culture medium.

[0013] According to an embodiment of the present invention, the amplification culture medium further comprises AB serum.

[0014] According to an embodiment of the present invention, the working concentration of the AB serum is 0.1-10% (v / v).

[0015] The second aspect of the present invention provides the use of the expansion culture medium for NK cells described in the first aspect in the preparation of NK cells with enhanced cytotoxicity.

[0016] A third aspect of the present invention provides the use of IL-9 in the preparation of NK cells with enhanced cytotoxicity.

[0017] A fourth aspect of the present invention provides a method for preparing NK cells with enhanced cytotoxicity. According to an embodiment of the present invention, the preparation method includes: The NK cells were treated with IL-9.

[0018] According to an embodiment of the present invention, the working concentration of IL-9 is 1-50 ng / mL.

[0019] A fifth aspect of the present invention provides another method for preparing NK cells with enhanced cytotoxicity. According to an embodiment of the present invention, the preparation method includes: The NK cells are expanded and cultured using the expansion medium for NK cells described in the first aspect.

[0020] According to an embodiment of the present invention, the preparation method further includes: During the expansion culture of the NK cells, a stimulant targeting the NK cells is added to the culture system.

[0021] According to an embodiment of the present invention, the stimulant for NK cell amplification is a genetically engineered antigen-presenting cell.

[0022] According to an embodiment of the present invention, the stimulant for NK cell expansion is mIL21 and 4-1 BBL modified antigen-presenting cells.

[0023] According to an embodiment of the present invention, during amplification culture, the seeding density of the NK cells in the amplification medium is 0.7-1.5 × 10⁻⁶. 6 per mL.

[0024] According to an embodiment of the present invention, during amplification culture, the ratio of the genetically engineered antigen-presenting cells to the NK cells is 1:(0.7-2).

[0025] A sixth aspect of the present invention provides an NK cell with enhanced cytotoxicity. According to embodiments of the present invention, the NK cell is prepared by the preparation method described in the fourth or fifth aspect.

[0026] The seventh aspect of the present invention provides the use of the NK cells with enhanced killing function described in the sixth aspect in the preparation of antiviral drugs and / or tumor immunotherapy drugs.

[0027] The eighth aspect of this invention provides an antiviral drug or a tumor immunotherapy drug. According to embodiments of the invention, the antiviral drug or tumor immunotherapy drug comprises the NK cells with enhanced killing function described in the sixth aspect. The beneficial effects of the technical solution of this invention are as follows: Adding IL-9 to the culture medium did not alter the phenotype of NK cells, and there were no significant differences in NK cell purity and functional markers compared to the control group. The iNK cells in the IL-9-treated group had higher granzyme B levels than the untreated group, indicating that IL-9 treatment can increase the content of NK cytotoxic proteins. Furthermore, the cytotoxic activity against target cells was significantly higher in the IL-9-treated iNK cells than in the untreated group. This demonstrates that the cytotoxic function of iNK cells is enhanced after IL-9 treatment.

[0028] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0029] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which: Figure 1 This demonstrates the pathway from iPSC to mature iNK; Figure 2 The growth curves of iNK cells treated with different concentrations of IL-9 are shown. Figure 3 The results of toxicity testing of cells cultured in Example 3 are shown. The horizontal axis in the figure represents the ratio of effector cells to target cells. Figure 4 The results of cytotoxicity testing of cells cultured in Example 3 are shown. Figure 5 The flow cytometry phenotypic bar chart of IL-9-treated iNK cells is shown. Figure 6 The expression of GraB in iNK after IL-9 treatment is shown; Figure 7 This shows the differences in GraB expression abundance in iNK cells under different treatments; Figure 8 The curves showing the killing effect of iNK on K562 tumor cells under different effector-to-target ratios after IL-9 pretreatment are displayed. Figure 9 The study demonstrated the killing effect of iNK cells on Nalm-6 tumor cells after IL-9 pretreatment. Figure 10 The study demonstrated the cytotoxic activity of IL-9 pretreated Anti-GPRC5D CAR iNK against GPRC5D-MM.1S tumor cells. Figure 11 The study demonstrated the cytotoxic activity of IL-9 against GPRC5D5-MM.1S by iNK after pretreatment in a rechallenge. Figure 12 The study demonstrates the toxicity effects of IL-9 on K562 after treatment with iPSC-NK with different modifications. Detailed Implementation

[0030] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0031] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0032] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0033] To facilitate understanding of the invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains.

[0034] In this document, the terms “comprising” or “including” are open-ended expressions, meaning that they include the contents specified in this invention, but do not exclude other aspects.

[0035] In this document, the terms “optionally,” “optionally,” or “optionally” generally refer to an event or condition that may, but may not, occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0036] According to a more specific embodiment of the present invention, the present invention provides an amplification culture medium for NK cells, which includes IL-9.

[0037] The NK cell amplification culture medium provided by the present invention contains IL-9 cytokine. It should be noted that the components of the NK cell amplification culture medium provided by the present invention include, but are not limited to, IL-9, and may also contain other components. All of these components of the NK cell amplification culture medium are covered within the scope of protection of the present invention.

[0038] According to a specific embodiment of the present invention, the amplification medium further comprises IL-2 and / or IL-15.

[0039] According to a specific embodiment of the present invention, the amplification medium may contain IL-9 and IL-2, or IL-9 and IL-15, or the amplification medium may contain IL-9, IL-2 and IL-15.

[0040] According to a preferred embodiment of the present invention, the amplification culture medium comprises IL-9, IL-2 and IL-15.

[0041] According to a specific embodiment of the present invention, the working concentration of IL-9 is 1-50 ng / mL. For example, the working concentration of IL-9 can be 1 ng / mL, 5 ng / mL, 10 ng / mL, 15 ng / mL, 20 ng / mL, 25 ng / mL, 30 ng / mL, 35 ng / mL, 40 ng / mL, 45 ng / mL, 50 ng / mL, etc. Preferably, the working concentration of IL-9 is 1-20 ng / mL.

[0042] According to a specific embodiment of the present invention, the working concentration of IL-2 is 50-1000 IU / mL. For example, the working concentration of IL-2 is 50 IU / mL, 100 IU / mL, 200 IU / mL, 300 IU / mL, 400 IU / mL, 500 IU / mL, 600 IU / mL, 700 IU / mL, 800 IU / mL, 900 IU / mL, or 1000 IU / mL. Preferably, the working concentration of IL-2 is 1000 IU / mL. It should be noted that the unit "IU" here refers to the biological activity unit of IL-2, determined according to the CTLL-2 cell proliferation method.

[0043] According to a specific embodiment of the present invention, the working concentration of IL-15 is 0.1-50 ng / mL. For example, the working concentration of IL-15 is 0.1 ng / mL, 5 ng / mL, 10 ng / mL, 15 ng / mL, 20 ng / mL, 25 ng / mL, 30 ng / mL, 35 ng / mL, 40 ng / mL, 45 ng / mL, 50 ng / mL, etc. Preferably, the working concentration of IL-15 is 10 ng / mL.

[0044] According to one specific embodiment of the present invention, the amplification medium further comprises a complete culture medium for lymphocyte culture.

[0045] According to a preferred embodiment of the present invention, the complete culture medium for lymphocyte culture is KBM-581 serum-free lymphocyte culture medium.

[0046] According to a specific embodiment of the present invention, the amplification culture medium further comprises AB serum.

[0047] According to a specific embodiment of the present invention, the working concentration of the AB serum is 0.1-10% (v / v). For example, the working concentration of the AB serum is 0.1% (v / v), 1% (v / v), 2% (v / v), 3% (v / v), 4% (v / v), 5% (v / v), 6% (v / v), 7% (v / v), 8% (v / v), 9% (v / v), 10% (v / v), etc. Preferably, the working concentration of the AB serum is 5% (v / v).

[0048] According to a specific embodiment of the present invention, the present invention provides the use of the aforementioned expansion culture medium for NK cells in the preparation of NK cells with enhanced cytotoxic function.

[0049] In this invention, the "NK cells" in the "expansion culture medium for NK cells" can be mature NK cells or immature iNK cells. For example, during the differentiation of immature iNK cells into mature iNK cells, the aforementioned expansion culture medium for NK cells can be used for cell culture, which can enhance the killing function of mature iNK cells. This enhancement of killing activity includes both broad-spectrum killing activity (cells without gene modification) and specific killing activity (cells with CAR structural modifications).

[0050] According to a specific embodiment of the present invention, the present invention provides the use of IL-9 in the preparation of NK cells with enhanced cytotoxic function.

[0051] According to a specific embodiment of the present invention, the present invention provides a method for preparing NK cells with enhanced cytotoxicity, comprising: The NK cells were treated with IL-9.

[0052] According to a specific embodiment of the present invention, the working concentration of IL-9 is 1-50 ng / mL. For example, the working concentration of IL-9 can be 1 ng / mL, 5 ng / mL, 10 ng / mL, 15 ng / mL, 20 ng / mL, 25 ng / mL, 30 ng / mL, 35 ng / mL, 40 ng / mL, 45 ng / mL, 50 ng / mL, etc. Preferably, the working concentration of IL-9 is 1-20 ng / mL.

[0053] According to a specific embodiment of the present invention, the present invention provides another method for preparing NK cells with enhanced cytotoxicity, the preparation method comprising: The NK cells were expanded and cultured using the expansion medium for NK cells described above.

[0054] According to a specific embodiment of the present invention, the preparation method further includes: During the expansion culture of the NK cells, a stimulant targeting the NK cells is added to the culture system.

[0055] According to a specific embodiment of the present invention, the stimulant for NK cell amplification is a genetically engineered antigen-presenting cell.

[0056] It should be noted that the stimulants for NK cell expansion are primarily used to efficiently activate and massively expand NK cells, while simultaneously enhancing their effector function. Various genetically engineered antigen-presenting cell types known in the art to possess this function are all covered within the scope of this invention.

[0057] According to a preferred embodiment of the present invention, the stimulant for NK cell expansion is an antigen-presenting cell modified with mIL21 and 4-1BBL.

[0058] According to a specific embodiment of the present invention, during amplification culture, the seeding density of the NK cells in the amplification medium is 0.7-1.5 × 10⁻⁶. 6 Cells / mL. For example, the seeding density of the NK cells in the amplification medium is 0.7 × 10⁻⁶ cells / mL. 6 cells / mL, 0.8×10 6 cells / mL, 0.9×10 6 cells / mL, 1.0×10 6 cells / mL, 1.1×10 6 cells / mL, 1.2×10 6 cells / mL, 1.3×10 6 cells / mL, 1.4 × 10 6 cells / mL, 1.5×10 6 per mL.

[0059] According to a specific embodiment of the present invention, during amplification culture, the ratio of the genetically engineered antigen-presenting cells to the NK cells is 1:(0.7-2). For example, the ratio of the genetically engineered antigen-presenting cells to the NK cells can be 1:0.7, 1:1, 1:1.2, 1:1.5, 1:1.8, 1:2, etc. Preferably, the ratio of the genetically engineered antigen-presenting cells to the NK cells is 1:1.

[0060] According to a specific embodiment of the present invention, the present invention provides an NK cell with enhanced killing function, wherein the NK cell is prepared by the preparation method described above.

[0061] According to a specific embodiment of the present invention, the present invention provides the use of the aforementioned enhanced NK cells in the preparation of antiviral drugs and / or tumor immunotherapy drugs.

[0062] According to a specific embodiment of the present invention, the present invention provides an antiviral drug or a tumor immunotherapy drug, wherein the antiviral drug or tumor immunotherapy drug includes, but is not limited to, the aforementioned NK cell-enhancing drugs. The antiviral drug or tumor immunotherapy drug may also include other carriers or excipients, and may also be used in combination with other drugs with similar functions to achieve a synergistic effect. According to a specific embodiment of the present invention, the testing process for pretreating iNK cells with IL-9 is as follows: Remove the differentiation medium from iPSC-differentiated iNK cells, then add complete NK cell culture medium (composed of KBM-581 medium, 5% AB plasma, and 1000 IU / mL IL-2 + 10 ng / mL IL-15) to adjust the viable cell density to 1E6 / mL, and add an equal amount of APC ( Figure 1 The control group received no IL-9, while the experimental groups received 1-100 ng / mL of IL-9. After thorough mixing, the cells were incubated statically at 37°C with 5% CO2. On the third day, cells were harvested and replenished with an equal volume of IL-9. The IL-9-treated groups received an additional 1-100 ng / mL of IL-9. Subsequently, cell counting and replenishment were performed every 2-3 days, adjusting the cell density to 1E6 / mL. The IL-9-treated groups received replenishment based on the amount of IL-9 replenished each time. Cell toxicity and function were assessed on days 10-14 after cell expansion.

[0063] There was no difference in CD56 purity between the IL-9 treated and untreated groups of iNK. Figure 3 There was no significant difference in cell activation markers. Figure 3 However, significant differences were observed in granzyme B (GraB), a representative indicator of cytotoxicity. Figure 4 The content of granzyme B in iNK pretreated with IL-9 was significantly higher than that in untreated iNK. Figure 5 In subsequent direct toxicity tests, iNK cells pretreated with IL-9 showed stronger killing ability against tumor cell lines. Figure 6-9 ). Furthermore, IL-9 treatment not only enhances the killing ability of iNK cells against various tumor cell lines ( Figure 6-7 ), and also have similar effects on iNKs with different gene modifications ( Figure 8This indicates that the enhancement of iNK cells by IL-9 pretreatment is not specific to any particular tumor cell or cell line. IL-9 pretreatment enhances iNK cytotoxicity without requiring gene editing, reducing the difficulty and cost of iNK cell preparation. Furthermore, it eliminates the need for continuous use of IL-9 to enhance iNK function, making it more feasible for clinical translational applications.

[0064] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0065] Example 1: iPSC differentiates into immature iNK Revive a single iPSC cell and differentiate it into NK cells according to patent CN 118667760 A (see patent CN 118667760 A for specific differentiation pathways). Figure 1 iPSCs formed EB spheres via Aggre Wells. These EB spheres were cultured in StemPro34 SFM medium supplemented with BMP4 and GSK-3β inhibitors. After 9-14 days, highly CD34+CD43+ positive HSCs were formed, which were then transferred to the second-stage differentiation medium. The differentiation medium was DMEM / F12, supplemented with IL15, SCF, Flt3L, IL-7, and IL-3. IL-3 was only used during the first week. Cells were cultured in the differentiation medium with the medium changed every 3-5 days. Immature iNK cells were tested for CD56+CD3+ at 28-35 days of differentiation. Afterward, the test medium was changed for amplification culture.

[0066] Example 2: iNK amplification at different IL-9 concentrations The immature iNK cells from Example 1 were centrifuged to remove the differentiation medium and replaced with complete NK cell culture medium. The complete medium consisted of KBM-581 supplemented with 1000 IU / mL IL-2, 10 ng / mL IL-15, and 5% AB serum. The cell density was adjusted to 1E6 / mL using the complete NK cell culture medium. Then, mIL21 and 4-1 BBL-modified APC were added as NK cell expansion stimulants at a 1:1 ratio. No IL-9 was added to the vehicle group, while 1-20 ng / mL IL-9 was added to the experimental groups according to the culture volume. Cells were then cultured in an incubator. After 3 days, cells were harvested and an equal volume of complete NK cell culture medium was added. The experimental groups were supplemented with IL-9 according to the replenishment volume. Cell counting and replenishment were performed every 2-3 days to adjust the cell density to 1E6 / mL. The expansion curve was observed until day 14. Figure 2 . Figure 2 The results showed that the amplification capacity of iNK was slightly higher than that of the Vehicle group under different concentrations of IL-9 treatment.

[0067] Example 3: Effect of IL-9 on iNK function at different concentrations of IL2 / IL15 The iNK culture medium from Example 1 was removed by centrifugation and replaced with complete NK cell culture medium. The complete culture medium consisted of KBM-581 supplemented with 500 IU / mL IL-2, 1 ng / mL IL-15, and 5% AB serum. The culture procedure was the same as in Example 2. Cytotoxicity was assessed on day 14; short-term toxicity assays are shown below. Figure 3 (The horizontal axis in the figure represents the effector cell / target cell ratio): Cells treated with IL9 were used in a challenge experiment (i.e., 1E5 Nalm-6 cells were added to each well every 24 hours), and cytotoxicity was assessed in the fifth round. Results are shown below. Figure 4 The results showed that adding 500 IU / mL IL-2 and 1 ng / mL IL-15 to the culture medium had little effect on enhancing the iNK function of IL-9, but this effect needed to be within a certain range (working concentration of IL-2: 50-1000 IU / mL; working concentration of IL-15: 0.1-50 ng / mL). The results in the figure also indicate that IL-9 treatment significantly enhanced the cytotoxic activity of iNK cells in challenge experiments.

[0068] Example 4: Flow cytometry phenotype of iNK treated with IL-9 In Example 2, iNK amplification was performed on day 12, followed by cell phenotype analysis. Flow cytometry was conducted using a 5E5 / Marker method. Cells were removed from the incubator, mixed, and counted. The cell count was determined based on the number of markers detected. The cells were centrifuged to remove the supernatant, then washed once with DPBS to remove culture medium components. After washing, 100 μL of DPBS was added for resuspending, followed by the addition of flow cytometry antibodies against CD56 / CD3 / CD69 / NKG2D / HLA-DR / GraB, and their corresponding isotype antibodies. The results are shown in [Figure number missing]. Figure 5 This indicates that IL-9-treated iNK cells showed no significant phenotypic difference compared to untreated iNK cells. However, differences were observed in the expression abundance of GraB. Figure 6 iNK cells pretreated with IL-9 showed nearly 1-fold higher expression abundance in GraB. Figure 7 This indicates that IL-9 pretreatment enhances the toxicity of iNK cells.

[0069] Example 5: Killing effect of IL9-treated iNK cells on hematologic malignancy cell lines In Example 2, the cytotoxicity of the iNK cells cultured was assessed on day 10 of expansion. The hematologic malignancy cell line (K562 Luci / Nalm-6 Luci) was removed, mixed, and counted. Based on the count, 1E6 / mL of tumor cells was collected, centrifuged, and the culture supernatant was removed. The cells were resuspended in 1 mL of 1640 + 10% FBS medium. After resuspending, the cells were seeded at 50 μL / well into 96-well opaque white culture plates. The iNK cells were then removed from the incubator, mixed, and counted. Based on the count, corresponding amounts of cells were collected according to different effector-to-target ratios. After centrifugation and removal of the supernatant, 1 mL of 1640 + 10% FBS was added as co-incubation medium. Based on different effector-to-target ratios, 50 μL / well was added to each well of the 96-well plate containing tumor cells and mixed thoroughly. The plates were incubated for 4 hours. Four hours later, the culture plates were removed and toxicity was assessed using the Vazyme Luciferase Assay System. 50 μL of substrate enzyme was added per well according to the instructions, and after standing for 10 minutes, the signal was detected using a chemiluminescence immunoassay reader. The cytotoxic activity was calculated using the formula: Killing efficiency % = (Control group - Killed group) / Cell control group * 100%. The results showed that IL-9 treatment significantly enhanced the ability of iNK cells to kill hematologic malignancies. Figure 8 Furthermore, this enhancement is not only effective for a single tumor cell line, but also shows significant improvement for multiple tumor cell lines. Figure 9 This indicates that IL-9 pretreatment of iNK cells significantly enhances their antitumor activity.

[0070] Example 6: Killing of specific tumor cells by CAR-iNK after IL-9 treatment To test whether IL-9 pretreatment had a similar enhancing effect on CAR-iNK, we used GPRC5D-targeted CAR-iNK (preparation procedure referred to Efficacy of Human iPSC-Derived CAR-NK Cells Targeting Multiple Myeloma Cells, 2023, Blood https: / / doi.org / 10.1182 / blood-2023-181613) for testing. The CAR-iNK culture and expansion protocol was the same as in Examples 1-2, and the cell function testing method was the same as in Example 4. The target cells used were myeloma cells MM.1S overexpressing GPRC5D. The results showed that even with a low target-to-cell ratio, there was still an enhancing effect on GPRC5D-MM.1S. Figure 10 In subsequent Rechallenge tests, it was further observed that IL-9-treated iNK cells maintained stronger killing activity in the third round of killing activity. Figure 11 This indicates that the toxicity of iNK cells pretreated with IL-9 was enhanced.

[0071] Example 7: Killing of tumor cells by multiple iNK cells after IL-9 treatment To test whether IL-9 pretreatment had a similar enhancing effect on multiple iNK cells, we tested different modified iNK cells. The iNK cell culture and expansion protocols were the same as in Examples 1-2, and the cell function assay methods were the same as in Example 4. K562 cells were used as the target cells. The cytotoxicity of two different genetically modified iPSC-NK cells (Anti-GPRC5D / IL15 iNK and CD16 / IL15 iNK) to K562 was enhanced after IL-9 pretreatment. Figure 12 This demonstrates that IL-9 pretreatment is not only effective for a single cell line, but has a broad spectrum of effects.

[0072] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," "some implementations," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0073] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A culture medium for expanding NK cells, characterized in that, Including IL-9.

2. The amplification culture medium according to claim 1, characterized in that, The amplification medium further includes IL-2 and / or IL-15; Optionally, the amplification medium includes IL-9, IL-2, and IL-15; Optionally, the working concentration of the IL-9 is 1-50 ng / mL; Optionally, the working concentration of the IL-2 is 50-1000 IU / mL; Optionally, the working concentration of the IL-15 is 0.1 - 50 ng / mL.

3. The amplification culture medium according to claim 1, characterized in that, The amplification medium further includes a complete culture medium for lymphocyte culture; Optionally, the complete culture medium for lymphocyte culture is KBM-581 serum-free lymphocyte culture medium; Optionally, the amplification medium further comprises AB serum; Optionally, the working concentration of the AB serum is 0.1-10% (v / v).

4. Use of the expansion medium for NK cells according to any one of claims 1-3 in the preparation of NK cells with enhanced killing function.

5. Use of IL-9 in the preparation of NK cells with enhanced cytotoxicity.

6. A method for preparing NK cells with enhanced cytotoxicity, characterized in that, This includes treating the NK cells with IL-9.

7. The preparation method according to claim 6, characterized in that, During the treatment, the working concentration of IL-9 is 1-50 ng / mL.

8. A method for preparing NK cells with enhanced cytotoxicity, characterized in that, include: The NK cells are expanded and cultured using the expansion medium for NK cells according to any one of claims 1-3.

9. The preparation method according to claim 8, characterized in that, The preparation method further includes: During the expansion and culture of the NK cells, a stimulant targeting the NK cells is added to the culture system; Optionally, the stimulant for NK cell expansion is a genetically engineered antigen-presenting cell; Optionally, the stimulant for NK cell expansion is mIL21 and 4-1 BBL-modified antigen-presenting cells.

10. The preparation method according to claim 9, characterized in that, During amplification culture, the NK cells were seeded at a density of 0.7-1.5 × 10⁻⁶ cells / day in the amplification medium. 6 cells / mL; Optionally, during amplification culture, the ratio of the genetically engineered antigen-presenting cells to the NK cells is 1:(0.7-2).

11. An NK cell with enhanced killing function, characterized in that, It is prepared by the preparation method according to any one of claims 6-10.

12. Use of the NK cells with enhanced killing function as described in claim 11 in the preparation of antiviral drugs and / or tumor immunotherapy drugs.

13. An antiviral drug or a tumor immunotherapy drug, characterized in that, Including the NK cells with enhanced killing function as described in claim 11.

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