A NK cell and a method for amplifying the same

Abstract

The present invention discloses a NK cell and a method for amplifying the same, and belongs to the field of biotechnology. The amplification method comprises: culturing genetically modified trophoblastic cells, crushing the trophoblastic cells to obtain liposomes, and using the liposomes to activate NK cells for amplification and culture; the genetically modified trophoblastic cells overexpress IL-21. The genetically modified trophoblastic cells used in the method of the present invention can express cytokines that stimulate the growth of NK cells, and the liposome extraction method used does not require the additional addition of a nutrient solution that promotes liposome secretion, and will not introduce new substances into the culture system. The operation is simple and the safety is high.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a NK cell and a method for amplifying the same. Background Art

[0002] Natural killer cells (NK cells) are the main immune cells known to kill tumor cells that have undergone tumor immunoediting. They are one of the important means of clinical tumor cell immunotherapy. Clinical studies have shown that allogeneic and autologous NK cell immunotherapy is safe. In 2009, the Ministry of Health issued the "Technical Management Specifications for Autologous Immune Cell T Cells and NK Cell Therapy (Draft for Comments)", which included NK cell immunotherapy technology as a third-category medical new technology in the scope of clinical treatment. However, over the past ten years, NK cell therapy technology has not been widely used in hospitals. In some early clinical trials, the efficacy of autologous NK cell therapy in clinical application was not obvious. The fundamental reason is that the inability to obtain sufficient number and activity of NK immune cells affects the therapeutic effect of NK cells. However, if the amount of NK cells infused is much smaller than the amount of tumor cells in the body, the therapeutic effect will be weakened. Therefore, how to achieve large-scale expansion and cultivation of NK cells in vitro is currently a key issue in NK cell therapy.

[0003] Liposomes are vesicles formed by a phospholipid bilayer membrane, which encloses an aqueous core. Due to the amphiphilicity of liposomes, both hydrophilic and hydrophobic drugs can be efficiently loaded into liposomes. Liposomes tend to encapsulate water-soluble drugs in the central aqueous phase and fat-soluble drugs in the area between the bilayer membranes. At the same time, since the basic structure of biological membranes is also a phospholipid bilayer membrane, liposomes have good biocompatibility and biodegradability.

[0004] Two commonly used methods for large-scale in vitro expansion of NK cells include trophoblast (K562 cell) stimulation method and factor stimulation method. The trophoblast (K562 cell) stimulation method uses genetic engineering to construct trophoblast cells, and then uses the modified trophoblast cells to stimulate NK cell expansion. The NK cells obtained by this method have higher purity and larger multiples, but because (1) it involves gene transfection, the cost is high and the steps are relatively complicated; (2) K562 cells (a type of tumor cell) are required, and users have certain concerns about their safety; (3) when NK cells or CAR-NK cells are registered as drugs, they need to be evaluated for safety. Trophoblast cells are complete cells, and the evaluation content involved will be very complicated, which greatly increases the difficulty and cost of safety evaluation. The factor stimulation method is a traditional NK cell in vitro expansion method, which uses some cytokines or factor combinations to stimulate peripheral blood mononuclear cells or purified NK cells in vitro. The NK cells obtained by this method are generally not of high purity (low NK cell purity means that the content of other miscellaneous cells is high, such as T cells, and T cells are specific cells. If they are re-infused into the human body, they are prone to GVHD reactions), and the multiples are not large (low multiples mean that the number of NK cells in the final product is small, and a small number cannot achieve the purpose of treating or killing target cells). There are also extreme ones that can obtain extremely high-purity effector cells, but the expansion multiples are extremely low, and even if the culture time is extended, the number required for clinical practice cannot be reached.

[0005] Therefore, there is an urgent need to provide an expansion and culture method that can effectively improve the proliferation ability of NK cells, increase the purity of NK cells and has high safety. Summary of the invention

[0006] In view of the deficiencies in the prior art and actual needs, the present invention provides a NK cell and a method for amplifying the same, which solves the problems in the prior art, such as the limited proliferation ability and low cell purity of NK cells cultured by the factor stimulation method, the introduction of new genetic material into NK cells cultured by the trophoblast cell method, and safety concerns.

[0007] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides a method for expanding NK cells, the method comprising: culturing genetically modified trophoblastic cells, disrupting the trophoblastic cells to obtain liposomes, and using the liposomes to activate NK cells for expansion and culture.

[0009] The genetically modified trophoblast cells used in the method of the present invention can express cytokines that stimulate NK growth. The liposome extraction process in the present invention does not require the addition of a nutrient solution that promotes liposome secretion, and no new substances are introduced into the culture system. The method of the present invention cultured NK cells with less additional growth factors, and the operation is simple. The method of obtaining liposomes in the present invention is clearer and simpler, and physical crushing is used, so no new substances are introduced into the culture system, and the safety is high, and there is no risk of pollution.

[0010] Liposomes with IL-21 can stimulate NK cells, promote their maturation, enhance cell cytotoxicity, and increase the killing rate of target cells. IL-21 can induce the maturation of NK cells and enhance their cytotoxicity. IL-21R is expressed on activated NK cells, inducing killer cells to produce a variety of cytokines, such as IFN-γ, perforin, etc., thereby increasing the killing rate of NK cells against target cells.

[0011] IL-21, as an inducing factor for NK cell maturation, is crucial in NK cell culture, and there is a certain requirement for its concentration during the culture process. During the culture process, liposomes with IL-21 can continuously stimulate NK cells, while other growth factors have a low substitution ability for IL-21 and cannot achieve the same stimulation effect.

[0012] It can be understood that the genetically modified trophoblast cells described in the present invention overexpress IL-21, and the genetically modified trophoblast cells with the same function in the art are all suitable for the present invention, and can be prepared by genetic engineering methods or directly purchased, for example, K562 mother cells can be purchased from the ATCC Biological Standard Resource Center and the National Biomedical Experimental Cell Resource Bank in the United States, and then the IL-21 gene can be inserted into the cells by liposome transfection, lentiviral transfection, or electrofection.

[0013] Preferably, the genetically modified trophoblast cells overexpress IL-21; the trophoblast cells include myeloid cells; the myeloid cells include K562 cells; the NK cells include CAR-NK cells; the culturing of the genetically modified trophoblast cells includes culturing the genetically modified trophoblast cells using a basal medium containing 8-12% fetal bovine serum at 35-37° C. and a CO2 concentration of 3-5%, replenishing the basal medium containing 8-12% fetal bovine serum every 2-3 days, and controlling the cell concentration to be (0.5-2)×10 6 Pieces / mL.

[0014] The specific point values ​​in the above 8-12% can be 8%, 9%, 10%, 11%, 12% etc.

[0015] The point values ​​in the range of 35-37°C mentioned above may specifically be 35°C, 35.1°C, 35.2°C, 35.4°C, 36°C, 36.4°C, 37°C and the like.

[0016] The specific point values ​​in the above 3-5% can be 3%, 3.1%, 3.2%, 3.6%, 4%, 4.2%, 4.6%, 5%, etc.

[0017] The point values ​​in the above 0.5-2 can be specifically selected as 0.5, 0.6, 0.8, 1, 1.4, 1.6, 1.8, 2, etc.

[0018] Preferably, the method for disrupting trophoblast cells includes filtration disruption; the filtration disruption includes collecting the cultured trophoblast cells, filtering them 10-100 times using a filter membrane, and after filtration, performing differential centrifugation on the disrupted trophoblast cell suspension to obtain nucleus-free liposomes; the pore size of the filter membrane is 0.2-50 μm; the differential centrifugation includes centrifugation at 800-1200 g for 8-15 min, removing the precipitate, and taking the supernatant and centrifuging it at 60000-100000 g for 0.5-2 h.

[0019] The point values ​​in the above 10-100 times can be specifically selected as 10 times, 11 times, 12 times, 15 times, 20 times, 30 times, 50 times, 80 times, 100 times, etc.

[0020] The point values ​​in the above 0.2-50 μm can specifically be selected as 0.2 μm, 0.3 μm, 0.4 μm, 1 μm, 10 μm, 20 μm, 30 μm, 40 μm, 45 μm, 50 μm, etc.

[0021] The point values ​​in the above 800-1200 g can be specifically selected as 800 g, 810 g, 820 g, 900 g, 1000 g, 1100 g, 1150 g, 1200 g, etc.

[0022] The point values ​​in the above 8-15 min can be specifically selected as 8 min, 9 min, 10 min, 11 min, 12 min, 13 min, 14 min, 15 min, etc.

[0023] The point values ​​in the above 60000-100000 g can be specifically selected as 60000 g, 61000 g, 62000 g, 70000 g, 80000 g, 85000 g, 90000 g, 95000 g, 100000 g, etc.

[0024] The point values ​​in the above 0.5-2 h can be specifically selected as 0.5 h, 0.6 h, 0.7 h, 1.0 h, 1.2 h, 1.5 h, 1.8 h, 2 h, etc.

[0025] Preferably, the activation of NK cells for expansion culture comprises: extracting mononuclear cells from peripheral blood or umbilical cord blood, culturing the mononuclear cells in a serum-free medium supplemented with liposomes, replenishing the serum-free medium supplemented with liposomes every 2-3 days, replenishing the liposomes every 1-2 days, and controlling the cell concentration to be (0.5-2) × 10 6 / mL, and culture for 4-12 days.

[0026] The point values ​​in the above 0.5-2 can be specifically selected as 0.5, 0.6, 0.8, 1, 1.4, 1.6, 1.8, 2, etc.

[0027] The point values ​​in the above 4-12 days can be specifically selected as 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, etc.

[0028] Preferably, the extraction of mononuclear cells from peripheral blood or umbilical cord blood includes centrifuging the peripheral blood or umbilical cord blood sample at 150-450 g for 8-15 min, removing the upper layer of light yellow plasma, diluting the remaining blood with a buffer solution, adding the diluted blood dropwise to the upper layer of lymphocyte separation solution, and centrifuging at 350-400 g for 25-35 min to obtain mononuclear cells in the middle layer; the volume ratio of the buffer solution to the remaining blood is (1-2):1.

[0029] The point values ​​in the above 150-450 g can be specifically selected as 150 g, 160 g, 180 g, 200 g, 250 g, 270 g, 300 g, 350 g, 400 g, 450 g, etc.

[0030] The point values ​​in the above 8-15 min can be specifically selected as 8 min, 9 min, 10 min, 11 min, 12 min, 13 min, 14 min, 15 min, etc.

[0031] The point values ​​in the above 350-400 g can be specifically selected as 350 g, 360 g, 370 g, 380 g, 390 g, 395 g, 398 g, 400 g, etc.

[0032] The point values ​​in the above 25-35 min can be specifically selected as 25 min, 26 min, 27 min, 28 min, 30 min, 32 min, 34 min, 35 min, etc.

[0033] Preferably, the serum-free medium contains any one of IL-15, IL-18 or IL-2; the concentration of IL-15 in the serum-free medium is 50-1000 ng / mL; the concentration of IL-18 in the serum-free medium is 50-1000 ng / mL; the concentration of IL-2 in the serum-free medium is 50-5000 IU / mL.

[0034] IL-2 can stimulate NK cell proliferation, enhance NK cell killing activity and produce cytokines, and induce LAK cell production. In addition, IL-2 can also activate the killing function of NK cells by binding to receptors on NK cells, thereby enhancing the body's killing effect on tumor cells.

[0035] IL-15 is essential for the maturation and activation of NK cells. It can enhance the function of NK cells, promote the growth and differentiation of NK cells, and play an important role in maintaining the persistence of NK cells, restoring mitochondrial integrity, and reducing cell apoptosis. It also has low toxic side effects. Preclinical studies have shown that IL-15 can enhance the function of NK cells after exposure to liver cancer.

[0036] IL-18 is also an important cytokine that promotes NK cell function. It produces NK cells with enhanced cytokine response through short-term in vitro priming and activates receptor stimulation that lasts for weeks to months. This combination can promote the anti-tumor effect of NK cells and significantly prolong survival time.

[0037] The combined effects of IL-15, IL-18 and IL-2 can significantly enhance the growth and differentiation of NK cells, and comprehensively enhance their killing ability, thus promoting the anti-tumor effect of NK cells.

[0038] The point values ​​in the above 50-1000 ng / mL may specifically be 50 ng / mL, 60 ng / mL, 70 ng / mL, 80ng / mL, 100 ng / mL, 500 ng / mL, 700 ng / mL, 900 ng / mL, 1000 ng / mL, etc.

[0039] The point values ​​in the above 50-5000 IU / mL may specifically be 50 IU / mL, 60 IU / mL, 70 IU / mL, 80 IU / mL, 100 IU / mL, 1000 IU / mL, 3000 IU / mL, 4000 IU / mL, 5000 IU / mL, etc.

[0040] Preferably, the concentration of the liposomes in serum-free medium is 10-50000 ng / mL.

[0041] The point values ​​in the above 10-50000 ng / mL can specifically be selected as 10 ng / mL, 20 ng / mL, 50 ng / mL, 100ng / mL, 1000 ng / mL, 5000 ng / mL, 10000 ng / mL, 20000 ng / mL, 40000 ng / mL, 50000 ng / mL, etc.

[0042] Preferably, the co-cultivation temperature is 35-37°C and the CO2 concentration is 3-5%.

[0043] The point values ​​in the range of 35-37°C mentioned above may specifically be 35°C, 35.1°C, 35.2°C, 35.4°C, 36°C, 36.4°C, 37°C and the like.

[0044] The specific point values ​​in the above 3-5% can be 3%, 3.1%, 3.2%, 3.6%, 4%, 4.2%, 4.6%, 5%, etc.

[0045] As a preferred technical solution, the method for amplifying NK cells of the present invention comprises the following steps:

[0046] (1) Genetically modified K562 trophoblast cells and / or myeloid cells are cultured at 35-37°C and 3-5% CO2 using a basal medium containing 8-12% fetal bovine serum, wherein the gene-modified K562 trophoblast cells and / or myeloid cells overexpress IL-21, and the basal medium containing 8-12% fetal bovine serum is supplemented every 2-3 days to control the cell concentration to (0.5-2)×10 6 Pieces / mL;

[0047] (2) collecting the cultured trophoblast cells, filtering them through a filter membrane for 10-100 times, and subjecting the broken trophoblast cell suspension to differential centrifugation to obtain nucleus-free liposomes, wherein the pore size of the filter membrane is 0.2-50 μm, and the differential centrifugation includes centrifugation at 800-1200 g for 8-15 min, removing the precipitate, and taking the supernatant and centrifuging it at 60,000-100,000 g for 0.5-2 h;

[0048] (3) The peripheral blood or umbilical cord blood sample was centrifuged at 150-450 g for 8-15 min, the upper layer of light yellow plasma was removed, the remaining blood was diluted with buffer, the diluted blood was added to the upper layer of lymphocyte separation solution, and centrifuged at 350-400 g for 25-35 min to obtain the mononuclear cells in the middle layer. The volume ratio of the buffer to the remaining blood was (1-2):1. The mononuclear cells were cultured in a serum-free medium supplemented with liposomes. The serum-free medium supplemented with liposomes was supplemented every 2-3 days, and the liposomes were supplemented every 1-2 days. The cell concentration was controlled at (0.5-2) × 10 6 / mL, co-culture for 4-12 days, the serum-free medium with added liposomes contains any one of IL-15, IL-18 or IL-2, the concentration of IL-15 in the serum-free medium is 50-1000 ng / mL, the concentration of IL-18 in the serum-free medium is 50-1000 ng / mL, the concentration of IL-2 in the serum-free medium is 50-5000 IU / mL, the concentration of liposomes in the serum-free medium is 10-50000 ng / mL, the temperature of the co-culture is 35-37°C, and the CO2 concentration is 3-5%.

[0049] In a second aspect, the present invention provides a NK cell, wherein the NK cell is amplified by the method for amplifying NK cells described in the first aspect.

[0050] Compared with the prior art, the present invention has the following beneficial effects:

[0051] (1) The genetically modified K562 trophoblast cells used in the method of the present invention can express cytokines that stimulate NK growth. The liposome extraction process does not require the addition of additional nutrient solution that promotes liposome secretion, and no impurities are introduced into the NK cell suspension. The method of the present invention requires less additional growth factors to culture NK cells, and the operation is simple. The liposomes with IL-21 can stimulate NK cells, promote their maturation, enhance their cytotoxicity, and increase the killing rate of target cells. IL-21 can induce the maturation of NK cells and enhance their cytotoxicity. IL-21R is expressed on activated NK cells, inducing killer cells to produce a variety of cytokines, such as IFN-γ, perforin, etc., thereby increasing the killing rate of NK cells against target cells.

[0052] (2) The method for obtaining liposomes of the present invention is clearer and simpler, and adopts physical disruption, does not introduce new substances into the cell suspension, is highly safe, and has no pollution risk;

[0053] (3) The liposomes obtained by the present invention do not contain cell nucleus components, and compared with the trophoblast method, the cost of safety evaluation is greatly reduced. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 This is a graph showing the purity test results of NK cells amplified by the method of Example 1 of the present invention;

[0055] Figure 2 This is a graph showing the detection result of the natural death rate of target cells in the control group of the present invention;

[0056] Figure 3 This is a graph showing the result of detecting the mortality rate of target cells after the target cells were treated with NK cells obtained in Example 1 of the present invention. DETAILED DESCRIPTION

[0057] To further illustrate the technical means and effects of the present invention, the present invention is further described below in conjunction with the embodiments and drawings. It should be understood that the specific implementation methods described herein are only used to explain the present invention, rather than to limit the present invention.

[0058] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0059] Example 1

[0060] The present invention provides a method for expanding NK cells, which is as follows:

[0061] (1) Obtaining liposomes: K562 cells purchased from the ATCC Biological Standard Resource Center in the United States were genetically modified, and the IL-21 gene fragment was inserted into the K562 cells using the lentiviral transfection method to obtain genetically modified IL21_K562 trophoblast cells. The cells were expanded and cultured in 1640 medium + 10% fetal bovine serum at 37°C and 5% CO2. The cells were replenished every 2 days and cultured for 12 days. The cell suspension was collected and concentrated to 5×10 6 / mL concentration.

[0062] Use a 5 μm pore filter cloth, clamp it with a filter clamp, and connect a 10 mL syringe at each end of the filter clamp. Push and pull the syringe back and forth 50 times to obtain a broken cell suspension. Centrifuge the cell suspension at 1000 g for 10 min to remove the precipitate, take the supernatant and centrifuge it at 80,000 g for 1 h to obtain liposomes without cell nuclei, and use a freeze dryer to make freeze-dried powder.

[0063] (2) Isolation of mononuclear cells from peripheral blood: Take a peripheral blood sample, centrifuge at 300 g for 10 min, remove the upper yellowish plasma, and dilute the remaining portion with PBS at a volume ratio of 2:1 PBS: blood. Slowly drip the diluted blood onto the upper layer of Ficoll to separate it into two layers, blood and Ficoll. Centrifuge at 390 g for 30 min, and remove the mononuclear cells in the middle layer. The mononuclear cells with an initial NK purity of 12.5% ​​were obtained.

[0064] (3) Cultivation of NK cells: Mononuclear cells were cultured at a rate of 5×10 5The cells were cultured in 581 medium at an initial concentration of 5 × 10 cells / mL, and IL-15 (100 ng / mL), IL-18 (100 ng / mL), IL-2 (500 IU / mL) and liposomes (100 ng / mL) were initially added. On the 4th, 6th, 8th and 10th days thereafter, the cells were replenished with 581 medium to a cell concentration of 5 × 10 cells / mL. 5 / mL, and liposomes (100 ng / mL) were added at the same time as the rehydration, and the cells were cultured for 12 days.

[0065] Example 2

[0066] The present invention provides a method for expanding NK cells, which is as follows:

[0067] (1) Obtaining liposomes: Genetically modified trophoblasts were cultured in 1640 medium + 8% fetal bovine serum at 37°C and 5% CO2. The culture medium was replenished every 2 days and cultured for 12 days. The cell suspension was collected and concentrated to 5×10 6 / mL concentration.

[0068] Use a filter cloth with a pore size of 0.2 μm, clamp it with a filter membrane clamp, and connect a 10 mL syringe at each end of the filter membrane clamp. Push and pull the syringe back and forth 100 times to obtain a broken cell suspension. Centrifuge the cell suspension at 1200 g for 8 min to remove the precipitate, take the supernatant and centrifuge it at 60,000 g for 2 h to obtain liposomes without cell nuclei, and use a freeze dryer to make freeze-dried powder.

[0069] (2) Isolation of mononuclear cells from peripheral blood: Take a peripheral blood sample, centrifuge it at 300 g for 10 min, remove the upper yellowish plasma, and dilute the remaining portion with PBS at a volume ratio of 2:1 PBS: blood. Slowly add the diluted blood to the upper layer of Ficoll to separate it into two layers: blood and Ficoll. Centrifuge at 390 g for 30 min, and aspirate the mononuclear cells in the middle layer to obtain mononuclear cells with an initial NK purity of 12.5%.

[0070] (3) Cultivation of NK cells: Mononuclear cells were cultured at a rate of 5×10 5 The cells were cultured in 581 medium at an initial concentration of 5 × 10 cells / mL, and IL-15 (1000 ng / mL), IL-18 (1000 ng / mL), IL-2 (5000 IU / mL) and liposomes (10 ng / mL) were initially added. On the 4th, 6th, 8th and 10th days thereafter, the cells were replenished with 581 medium to a cell concentration of 5 × 10 cells / mL after replenishment. 5 / mL, and liposomes (10 ng / mL) were added at the same time as the rehydration, and the culture was carried out for 12 days.

[0071] Example 3

[0072] The present invention provides a method for expanding NK cells, which is as follows:

[0073] (1) Obtaining liposomes: Genetically modified IL21_K562 trophoblast cells were cultured in 1640 medium + 12% fetal bovine serum at 37°C and 3% CO2. The culture medium was replenished every 2 days and cultured for 12 days. The cell suspension was collected and concentrated to 5×10 5 / mL concentration.

[0074] Use a 50 μm pore size filter cloth, clamp it with a filter clamp, and connect a 10 mL syringe at each end of the filter clamp. Push and pull the syringe back and forth 10 times to obtain a broken cell suspension. Centrifuge the cell suspension at 1000 g for 10 min to remove the precipitate, take the supernatant and centrifuge it at 80,000 g for 1 h to obtain liposomes without cell nuclei, and use a freeze dryer to make freeze-dried powder.

[0075] (2) Isolation of mononuclear cells from peripheral blood: Take a peripheral blood sample, centrifuge at 300 g for 10 min, remove the upper yellowish plasma, and dilute the remaining portion with PBS at a volume ratio of 2:1 PBS: blood. Slowly drip the diluted blood onto the upper layer of Ficoll to separate it into two layers, blood and Ficoll. Centrifuge at 390 g for 30 min, and remove the mononuclear cells in the middle layer. The mononuclear cells with an initial NK purity of 12.5% ​​were obtained.

[0076] (3) Cultivation of NK cells: Mononuclear cells were cultured at a rate of 5×10 5 The cells were cultured in 581 medium at an initial concentration of 5 × 10 cells / mL, and IL-15 (1000 ng / mL), IL-18 (1000 ng / mL), IL-2 (5000 IU / mL) and liposomes (50000 ng / mL) were initially added. On the 4th, 6th, 8th and 10th days thereafter, the cells were replenished with 581 medium to a cell concentration of 5 × 10 cells / mL after replenishment. 5 / mL, and liposomes (50000 ng / mL) were added at the same time as the rehydration, and the culture was carried out for 12 days.

[0077] Example 4

[0078] The only difference from Example 1 is that the concentration of liposomes in the 581 medium for culturing NK cells in step (3) is 5 ng / mL.

[0079] Example 5

[0080] The only difference from Example 1 is that the concentration of liposomes in the 581 medium for culturing NK cells in step (3) is 70000 ng / mL.

[0081] Example 6

[0082] The only difference from Example 1 is that in step (3), the 581 medium for culturing NK cells does not contain IL-15, and its mass fraction is proportionally distributed to IL-18 and IL-2.

[0083] Example 7

[0084] The only difference from Example 1 is that the 581 medium for culturing NK cells in step (3) does not contain IL-18, and its mass fraction is proportionally distributed to IL-15 and IL-2.

[0085] Example 8

[0086] The only difference from Example 1 is that the 581 medium for culturing NK cells in step (3) does not contain IL-2, and its mass fraction is proportionally distributed to IL-18 and IL-15.

[0087] Example 9

[0088] The only difference from Example 1 is that in step (3), the 581 medium for culturing NK cells does not contain IL-18 and IL-2, and their mass fractions are allocated to IL-15.

[0089] Example 10

[0090] The only difference from Example 1 is that the 581 culture medium for culturing NK cells in step (3) does not contain IL-15, IL-18 and IL-2.

[0091] Embodiment 11

[0092] The only difference from Example 1 is that the growth factors in the 581 medium for culturing NK cells in step (3) are IL-15, IL-18 and IL-16, among which IL-15 (100 ng / mL), IL-18 (100 ng / mL) and IL-16 (500 IU / mL).

[0093] Example 12

[0094] The only difference from Example 1 is that the growth factors in the 581 medium for culturing NK cells in step (3) are IL-15, IL-2 and IL-21, including IL-15 (100 ng / mL), IL-21 (100 ng / mL), and IL-2 (500 IU / mL).

[0095] Comparative Example 1

[0096] The only difference from Example 1 is that the 581 culture medium for culturing NK cells in step (3) does not contain liposomes.

[0097] Comparative Example 2

[0098] The only difference from Example 1 is that the method for crushing trophoblast cells to obtain liposomes in step (1) is to treat IL21_K562 trophoblast cells with sodium dodecyl sulfate (SDS) cell lysis solution. The specific steps are: take an appropriate amount of SDS lysis solution, add phenylmethylsulfonyl fluoride (PMSF) within a few minutes before use, so that the final concentration of PMSF is 1 mM. Collect the cells by centrifugation, and add lysis solution at a ratio of 250 μL lysis solution per well of the 6-well plate. Then flick with your fingers to fully lyse the cells. If the amount of cells is large, divide them into 1 million cells / tube and then lyse them. There should be no obvious cell precipitation after sufficient lysis. Thereafter, extract the liposome components by the same centrifugation step.

[0099] Comparative Example 3

[0100] The only difference from Example 1 is that the method for crushing trophoblast cells to obtain liposomes in step (1) is a repeated freeze-thaw method, and the specific steps are: collecting the cell suspension, centrifuging at 4°C (450 g, 5 min), discarding the supernatant, adding a certain amount of PBS, gently blowing and mixing, freezing at -20°C for 30 min, thawing at 37°C, and repeating this process 5 times. Thereafter, the liposome components are extracted by the same centrifugation step.

[0101] Test Example 1

[0102] Detect the purity of NK cells. The detection method is as follows:

[0103] The cultured cells were centrifuged (300 g, 3 min), the supernatant was discarded, PBS was added, and the cells were resuspended to 1×10 7 / mL; take 40 μL of cells into an EP tube or flow tube, add CD3 antibody and CD56 antibody, add 2 μL of each antibody; add the corresponding antibody and mix well, incubate at 25℃ in the dark for 15 min; after staining, add 0.5 mL of PBS to wash the cells, centrifuge at 300 g for 3 min; discard the supernatant, add 0.5 mL of PBS to resuspend, and use flow cytometer for detection. The test results are as follows Figure 1 , the upper left quadrant shows that the CD3-negative and CD56-positive cell population is NK cells, and the proportion of this cell population is the purity of NK cells.

[0104] The results are shown in Table 1.

[0105] Table 1

[0106]

[0107] From the above data, it can be seen that in Example 1-Example 3, the method of the present invention is used to expand NK cells, and the purity of the NK cells collected on the 12th day can reach up to 95.81%. Example 4-Example 5 shows that the concentration of liposomes needs to be precisely controlled to facilitate the reduction of impurities in the process of expanding NK cells. Examples 6-10 show that IL-15, IL-18 and IL-2 in the method of the present invention are beneficial to promote the expansion of NK cells, and no impurities affecting the purity of NK cells will be generated during the expansion process. Example 11-Example 12 shows that the selection of the three specific interleukins IL-15, IL-18 and IL-2 of the present invention can ensure that no impurities will be generated during the effective expansion of NK cells. Comparative Example 1 shows that the liposomes in the method of the present invention can effectively activate the expansion of NK cells, and no additional nutrient solution is required to promote the secretion of liposomes, and no impurities will be introduced into the NK cell suspension. Comparative Example 2 shows that the method of the present invention uses a physical filtration and crushing method that can not introduce impurities, has high safety, and no pollution risk. Comparative Example 3 shows that although other physical crushing methods do not enter impurities, the product effect obtained is not as good as the present invention, and the process has high energy consumption, takes longer, and repeatedly transfers the cell position and has a pollution risk.

[0108] Test Example 2

[0109] Detect NK cell expansion multiples. The number of NK cells is an important guarantee for their killing performance, so sufficient cell expansion multiples is also an important reference indicator for evaluating a NK cell culture method.

[0110] To measure the expansion multiple of NK cells, the initial PBMCs before culture and the number of cells after culture were counted. The counting method was as follows: 50 μL of the cell suspension was mixed and dropped onto a hemacytometer plate. The cell concentration was converted under an optical microscope and then multiplied by the total culture volume to calculate the number of NK cells.

[0111] Then, the method in Test Example 1 was used to test the NK purity of the initial PBMC before culture and the number of cells after culture. Finally, the expansion multiple of NK cells was calculated according to the following formula:

[0112] Expansion factor = (final cell amount × final NK purity) ÷ (initial PBMC amount × initial NK purity)

[0113] The results are shown in Table 2.

[0114] Table 2

[0115]

[0116] From the above data, it can be seen that in Example 1-Example 3, the NK cells were amplified by the method of the present invention on the 12th day, and the NK cells were amplified up to 2464 times. Example 4-Example 5 shows that the concentration of liposomes needs to be precisely controlled to facilitate the amplification of NK cells. Examples 6-10 show that IL-15, IL-18 and IL-2 in the method of the present invention have a synergistic effect in promoting the expansion of NK cells. Example 11-Example 12 shows that the selection of the three specific interleukins IL-15, IL-18 and IL-2 of the present invention can ensure the effective amplification of NK cells. Comparative Example 1 shows that the liposomes in the method of the present invention can effectively activate the amplification of NK cells. Comparative Example 2 shows that the method of the present invention uses a physical filtration and crushing method to effectively remove the damaging effects of impurities on NK cell amplification. Comparative Example 3 shows that other physical crushing methods have high energy consumption and take a long time, but the effect is not better than the present invention, and the cost performance is not as good as the present invention.

[0117] Test Example 3

[0118] Detection of NK cell killing performance.

[0119] NK cells are mainly used to kill cancer cells in the body, and their killing performance is the most direct evaluation of their function. K562 cells (purchased from ATCC, without any modification) were selected as target cells for detecting the killing activity of human NK cells. The death rate of target cells after being acted on by NK cells was detected by flow cytometry to reflect the killing activity of NK cells. K562 target cells in the exponential growth phase were taken and mixed with NK cells in a test tube at a ratio of 20:1. A control group of natural death of K562 cells alone was set up. Placed in a 37℃ carbon dioxide incubator, after 6 hours of action, propidium iodide (PI, 50 μg / mL) and phosphatidyl binding protein V (Annexin V, 50 μg / mL) were added to the above test tubes, and the death rate of K562 target cells was detected by flow cytometry, and then the killing activity of NK cells was calculated. K562 cells stained by both PI and Annexin V were considered dead cells, and the killing activity of NK cells was based on the death rate of target cells. Calculate the killing activity of NK cells (%) = the target cell death rate (%) of the NK cell experimental group - the natural death rate (%) of the target cells. Figure 2 , Figure 3 . Figure 2 The upper right corner Q1 is the natural death rate of target cells in the control group. Figure 3 The upper right corner Q1 is the target cell death rate of the experimental group. The results are shown in Table 3:

[0120] Table 3

[0121]

[0122] From the above data, it can be seen that since the number of NK cells used for killing detection is the same, the changing trend of the killing performance of NK cells produced in each embodiment and the comparative example is basically the same as the changing trend of purity. It shows that when the number of cells is equal, the killing performance of NK cells is closely related to purity. The use of the best culture method, that is, the method described in Examples 1-3 of the present invention, can improve the product purity of NK cells, thereby improving the killing performance.

[0123] In summary, the genetically modified trophoblast cells used in the method of the present invention can express cytokines that stimulate NK growth, and the liposome extraction process does not require the addition of additional nutrient solution that promotes liposome secretion, and will not introduce new substances into the culture system. The operation is simple and safe.

[0124] The applicant declares that the present invention illustrates the detailed method of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned detailed method, that is, it does not mean that the present invention must rely on the above-mentioned detailed method to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A method for expanding NK cells, characterized in that: The method comprises: culturing genetically modified trophoblastic cells, crushing the trophoblastic cells to obtain liposomes, and using the liposomes to activate NK cells for expansion and culture; The genetically modified trophoblast cells overexpress IL-21; The trophoblasts include myeloid cells; The myeloid cells include K562 cells; The NK cells include CAR-NK cells; The culturing of the gene-modified trophoblast cells comprises culturing the gene-modified trophoblast cells using a basic culture medium containing 8-12% fetal bovine serum at 35-37° C. and a CO2 concentration of 3-5%, replenishing the basic culture medium containing 8-12% fetal bovine serum every 2-3 days, and controlling the cell concentration to be (0.5-2)×10 6 Pieces / mL; The method for crushing trophoblast cells includes filtration crushing; the filtration crushing includes collecting the cultured trophoblast cells, filtering them with a filter membrane for 10-100 times, and then performing differential centrifugation on the crushed trophoblast cell suspension to obtain liposomes without cell nuclei; the pore size of the filter membrane is 0.2-50 μm; the differential centrifugation includes centrifugation at 800-1200 g for 8-15 min, removing the precipitate, and taking the supernatant and centrifuging it at 60000-100000 g for 0.5-2 h; The activated NK cells for expansion culture include: extracting mononuclear cells from peripheral blood or umbilical cord blood, culturing the mononuclear cells in a serum-free medium supplemented with liposomes, replenishing the serum-free medium supplemented with liposomes every 2-3 days, replenishing the liposomes every 1-2 days, and controlling the cell concentration to be (0.5-2)×10 6 / mL, cultured for 4-12 days; The serum-free culture medium contains a combination of IL-15, IL-18 and IL-2; the concentration of the liposome in the serum-free culture medium is 10-50000 ng / mL.

2. The method for expanding NK cells according to claim 1, characterized in that: The method of extracting mononuclear cells from peripheral blood or umbilical cord blood includes centrifuging the peripheral blood or umbilical cord blood sample at 150-450 g for 8-15 min, removing the upper layer of light yellow plasma, diluting the remaining blood with a buffer solution, dripping the diluted blood on the upper layer of lymphocyte separation solution, and centrifuging at 350-400 g for 25-35 min to obtain mononuclear cells in the middle layer; the volume ratio of the buffer solution to the remaining blood is (1-2):

1.

3. The method for expanding NK cells according to claim 1, characterized in that: The concentration of IL-15 in serum-free medium is 50-1000 ng / mL; The concentration of IL-18 in serum-free medium is 50-1000 ng / mL; The concentration of IL-2 in serum-free medium is 50-5000 IU / mL.

4. The method for expanding NK cells according to claim 1, characterized in that: The co-culture temperature is 35-37° C., and the CO 2 concentration is 3-5%.

5. The method for expanding NK cells according to claim 1, characterized in that: The method comprises the following steps: (1) Genetically modified K562 trophoblast cells were cultured at 35-37°C and 3-5% CO2 using a basal medium containing 8-12% fetal bovine serum. The genetically modified K562 trophoblast cells overexpressed IL-21. The basal medium containing 8-12% fetal bovine serum was supplemented every 2-3 days to control the cell concentration to (0.5-2)×10 6 Pieces / mL; (2) collecting the cultured trophoblast cells, filtering them through a filter membrane for 10-100 times, and subjecting the broken trophoblast cell suspension to differential centrifugation to obtain nucleus-free liposomes, wherein the pore size of the filter membrane is 0.2-50 μm, and the differential centrifugation includes centrifugation at 800-1200 g for 8-15 min, removing the precipitate, and taking the supernatant and centrifuging it at 60,000-100,000 g for 0.5-2 h; (3) The peripheral blood or umbilical cord blood sample was centrifuged at 150-450 g for 8-15 min, the upper layer of light yellow plasma was removed, the remaining blood was diluted with buffer, the diluted blood was added to the upper layer of lymphocyte separation solution, and centrifuged at 350-400 g for 25-35 min to obtain the mononuclear cells in the middle layer. The volume ratio of the buffer to the remaining blood was (1-2):

1. The mononuclear cells were cultured in a serum-free medium supplemented with liposomes. The serum-free medium supplemented with liposomes was supplemented every 2-3 days, and the liposomes were supplemented every 1-2 days. The cell concentration was controlled at (0.5-2)×10 6 / mL, co-culture for 4-12 days, the serum-free medium with added liposomes contains a combination of IL-15, IL-18 and IL-2, the concentration of IL-15 in the serum-free medium is 50-1000 ng / mL, the concentration of IL-18 in the serum-free medium is 50-1000 ng / mL, the concentration of IL-2 in the serum-free medium is 50-5000 IU / mL, the concentration of liposomes in the serum-free medium is 10-50000 ng / mL, the temperature of the co-culture is 35-37°C, and the CO2 concentration is 3-5%.

6. A NK cell, characterized in that: The NK cells are amplified by the method for amplifying NK cells according to any one of claims 1-5.

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