Method for preparing NK cells

Abstract

Provided are a method for preparing NK cells and a composition comprising NK cells prepared by the method.

Description

Methods for preparing NK cells

[0001] Citation of relevant applications

[0002] This application claims priority and benefit to Chinese Patent Application No. 202410804937.8, filed on June 20, 2024, the entire contents of which are incorporated herein by reference for all purposes. Technical Field

[0003] This application relates to the fields of biotechnology and medical technology, and specifically to a method for preparing NK cells. Background Technology

[0004] Natural killer (NK) cells originate from bone marrow lymphoid stem cells and are mainly distributed in the bone marrow, peripheral blood, liver, spleen, lungs, and lymph nodes. NK cells are a type of lymphocyte that is not MHC-restricted, does not require prior sensitization, and does not require antibody involvement; they can directly and non-specifically kill tumor cells and virus-infected cells. NK cells typically contain large amounts of perforin and granzyme B. When activated NK cells encounter target cells, the perforin and granzyme B released by the NK cells attack the target cells. NK cells can also secrete cytokines such as IFN-γ, TNF-α, GM-CSF, and IL-3. These cytokines can act directly on target cells or activate other types of immune cells to attack target cells.

[0005] NK cells are the first line of defense in the immune system, capable of recognizing and killing cancer cells as well as cells infected by viruses.

[0006] NK cells exert their cytotoxic effect primarily through the following pathways:

[0007] 1. Direct lysis of target cells: NK cells release cytotoxic particles such as perforin and granzymes through exocytosis, which activate the caspase pathway to induce necrosis or apoptosis of target cells;

[0008] 2. Secretion of cytokines: Cytokine-mediated killing effect. NK cells can synthesize and secrete a variety of cytokines, such as IFN-γ, TNF-α, IL-1, IL-5, IL-8, IL-10 and G-CSF, which induce apoptosis of target cells.

[0009] 3. Induction of apoptosis: Activation of NK cells to express Fas(CD95) ligand and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) molecules, inducing CD95... +Target cells and TRAIL receptor-positive target cells undergo apoptosis through a cascade of endogenous enzyme reactions;

[0010] 4. ADCC: Antibody-dependent cell-mediated cytotoxicity;

[0011] 5. Immune checkpoint pathway: Activate NK cells using immune checkpoint blocking antibodies.

[0012] Because NK cells have these important functions in killing target cells, and the main factors secreted by NK cells are IL-3 and GM-CSF, NK cells without cytokine storm (CRS) and allogeneic NK cells almost never cause graft-versus-host disease (GvHD). Therefore, adoptive NK cell immunotherapy has become an important means of tumor cell immunotherapy in clinical practice.

[0013] However, there is an urgent need in this field to improve the NK cell preparation process, increase NK cell production capacity, reduce NK cell preparation costs, achieve clinical accessibility, and benefit more patients. Summary of the Invention

[0014] In a first aspect, this application provides a method for preparing NK cells, the method comprising an activation phase and an amplification phase, wherein both the activation phase and the amplification phase are performed in a serum-free culture system and neither phase uses feeder cells, wherein in the amplification phase, NK cells are amplified using a rocking bioreactor, and the culture process parameters include: total aeration rate of 10-40 mL / min, rocking speed of 8-30 rpm (number of times the reactor is rocked per minute), rocking angle of 5-12°, dissolved oxygen (DO) ≥10%, and pH of 6.8-7.2.

[0015] In some embodiments, the serum-free culture medium used in the amplification phase comprises basal culture medium, interleukin-type NK cell amplification factor, and serum substitute.

[0016] In some embodiments, the serum-free culture medium used in the amplification phase also contains fat-soluble vitamins.

[0017] In some embodiments, the serum-free culture medium used in the amplification phase also contains trace elements.

[0018] In some embodiments, the interleukin-type NK cell amplification factor is selected from any combination of IL2, IL15, IL12, IL18, IL21, or more.

[0019] In some embodiments, the serum-free culture medium used in the amplification phase does not contain nicotinamide and / or dimercaprol.

[0020] In some embodiments, the serum-free culture medium used in the amplification phase also contains heparin sodium.

[0021] In some embodiments, the serum-free culture medium used in the amplification phase also contains one or more of reduced glutathione, human transferrin, and human serum albumin.

[0022] In some embodiments, NK cell expansion is performed using a swing bioreactor during the expansion phase, and the culture process parameters include:

[0023] Total ventilation is 20-40 mL / min; and / or

[0024] The oscillation speed is 8-10 rpm; and / or

[0025] The swing angle is 6°; and / or

[0026] Dissolved oxygen (DO) ≥ 20%.

[0027] In some implementations, the interleukin-like NK cell amplification factors are IL2 and IL15.

[0028] In some embodiments, the fat-soluble vitamin is selected from one or more of the following: vitamin A, vitamin E, and vitamin K.

[0029] In some embodiments, the trace element is selected from one or more compounds including: sodium selenite, ferrous chloride, zinc chloride, copper chloride, nickel chloride, cobalt chloride hexahydrate, manganese chloride tetrahydrate, chromium chloride, silver chloride, potassium iodide, sodium metavanadate, ammonium molybdate tetrahydrate, rubidium chloride, germanium tetrachloride, and stannous chloride.

[0030] In some embodiments, the serum substitute is platelet lysate.

[0031] In some embodiments, the basal medium is RPMI-1640 basal medium.

[0032] In some embodiments, the carbon source cultured during the amplification phase includes glucose, and the glucose concentration of the culture in the bioreactor is controlled at 1.00-2.00 g / L.

[0033] In some implementations, the culture temperature during the amplification phase is 37.0 ± 0.5 °C.

[0034] In some embodiments, the pH of the culture in the swing bioreactor is controlled at 7.0 ± 0.2.

[0035] In some embodiments, the method further includes equilibrating the swaying bioreactor at the beginning of the amplification phase, wherein the process parameters for equilibration include: an aeration rate of 100-1000 mL / min, a heater temperature of 41.0±2.0℃, an exhaust gas temperature of 55.0±5.0℃, a swaying speed of 12-20 rpm, a swaying angle of 6-12°, and a culture temperature of 37.0±1.0℃.

[0036] In some implementations, the process parameters for the balancing process include:

[0037] The ventilation rate is 300 mL / min; and / or

[0038] The heater temperature is 41.0℃; and / or

[0039] The exhaust gas temperature is 55.0℃; and / or

[0040] The oscillation speed is 12-20 rpm; and / or

[0041] The swing angle is 6-12°; and / or

[0042] The incubation temperature was 37.0±0.5℃.

[0043] In some embodiments, when the medium change operation is performed during the amplification phase, the volume of the medium changed each time is 5% to 70% of the total volume of the culture system.

[0044] In some implementations, the medium change operation is performed using dynamic culture for intermittent or continuous medium change operations.

[0045] In some embodiments, the serum-free culture medium used in the activation phase comprises basal culture medium, interleukin-type NK cell activating factors, fat-soluble vitamins, trace elements, and serum substitutes.

[0046] In some embodiments, the serum-free culture medium used in the activation phase also contains nicotinamide.

[0047] In some embodiments, the serum-free culture medium used in the activation phase also contains dimercaptoethanol.

[0048] In some embodiments, the serum-free culture medium used in the activation phase also contains sodium heparin.

[0049] In some embodiments, the serum-free culture medium used in the activation phase further comprises one or more of reduced glutathione, human transferrin, and human serum albumin.

[0050] In some embodiments, the serum-free culture medium used in the activation phase also contains quercetin.

[0051] In some embodiments, the interleukin-type NK cell activating factor is selected from any combination of IL2, IL7, IL15, IL12, IL18, IL21, or more.

[0052] In some embodiments, the fat-soluble vitamin is selected from one or more of the following: vitamin A, vitamin E, and vitamin K.

[0053] In some embodiments, the trace element is selected from one or more compounds including: sodium selenite, ferrous chloride, zinc chloride, copper chloride, nickel chloride, cobalt chloride hexahydrate, manganese chloride tetrahydrate, chromium chloride, silver chloride, potassium iodide, sodium metavanadate, ammonium molybdate tetrahydrate, rubidium chloride, germanium tetrachloride, and stannous chloride.

[0054] In some embodiments, the serum substitute is platelet lysate.

[0055] In some embodiments, the basal medium is RPMI-1640 basal medium.

[0056] In some implementations, the cultivation process parameters for the activation phase include a temperature of 37.0 ± 0.5 °C and a CO2 concentration of 5.0 ± 0.5%.

[0057] In some embodiments, the activation phase further includes coating the bottom of the cell culture vessel with an antibody targeting one or more of the following molecules: CD3, CD25, CD33, CD52, CD4, CD8, 4-1BB, CD19, CLL1, CD38, and CD16.

[0058] In some implementations, NK cells are derived from peripheral blood mononuclear cells (PBMCs).

[0059] In some implementations, NK cells are obtained from NK cells derived from umbilical cord blood.

[0060] In some implementations, NK cells are obtained from in vitro culture of NK cell lines.

[0061] In some implementations, NK cells are obtained from the in vitro induction and culture of induced pluripotent stem cells (iPSCs) or mesenchymal stem cells (MSCs).

[0062] In a second aspect, this application provides a composition comprising NK cells prepared by the method described in the first aspect.

[0063] In some embodiments, the composition contains 100 billion or more NK cells.

[0064] In some embodiments, CD3 in the composition - CD56 + The purity of NK cells is greater than or equal to 90%.

[0065] In some embodiments, CD3 in the composition - CD56 + The purity of NK cells is greater than or equal to 95%.

[0066] In some embodiments, CD3 in the composition - CD56 + NKG2D + The proportion of NK cells is greater than or equal to 95% and CD3 - CD56 + CD16 + The proportion of NK cells is greater than or equal to 95%.

[0067] In some embodiments, the proportion of non-NK cells in the composition is less than 10%.

[0068] In some embodiments, the proportion of non-NK cells in the composition is less than 5%.

[0069] In some embodiments, the composition is formulated as an injectable solution. Attached Figure Description

[0070] Figure 1 shows the growth changes of NK cells during NK cell expansion culture using a 50L reactor in Example 5.

[0071] Figure 2 shows a graph illustrating the change in NK cell viability during NK cell expansion culture using a 50L reactor in Example 5.

[0072] Figure 3 shows the change curve of NK cell purity during NK cell expansion culture using a 50L reactor in Example 5.

[0073] Figure 4 shows the growth changes of NK cells in NK cell expansion culture using a 100L reactor in Example 5.

[0074] Figure 5 shows the change curve of NK cell viability during NK cell expansion culture using a 100L reactor in Example 5.

[0075] Figure 6 shows the change curve of NK cell purity during NK cell expansion culture using a 100L reactor in Example 5.

[0076] Figure 7 shows a bar chart of the in vitro cytotoxic activity of natural killer cells (obtained from Example 5).

[0077] Figure 8 shows the flow cytometry analysis results (CD56) of natural killer cells (Example 6). + CD3 - NKG2D + CD19 + ).

[0078] Figure 9 shows the flow cytometry analysis results (CD56) of natural killer cells (Example 6). + CD3 - NKG2D + CD19 + ).

[0079] Figure 10 shows a bar chart of the in vitro cytotoxic activity of NK cell injection (prepared in Example 4, IBR900).

[0080] Figure 11 shows the cell growth changes of NK cells cultured using the process of Comparative Example 1 and a 50L reactor for NK cell expansion.

[0081] Figure 12 shows a graph illustrating the changes in cell viability of NK cells cultured using the process of Comparative Example 1 and a 50L reactor for NK cell expansion.

[0082] Figure 13 shows the NK purity change curve of NK cells cultured using the process of Comparative Example 1 and a 50L reactor for NK cell expansion. Detailed Implementation

[0083] This application employs a fully enclosed, large-scale NK cell culture and preparation process using a pure factor method, non-feeder layer culture, serum-free NK cell culture kit, and NK cell preparation technology. The method for preparing NK cells in this application reduces the risk of formulation contamination and cross-contamination, ensuring product safety, and also achieves higher NK cell purity (over 95%) and high NK cell viability density (8.5 × 10⁻⁶). 6 This application describes a method for preparing NK cells with a single-batch system of 25-50 L and a production capacity of up to 2.1 × 10⁻⁶ cells / mL or higher. The method yields NK cells with high cytotoxicity (over 80% at an effector-to-target ratio of 10:1). 11 One live cell, sufficient for 42 infusions (5.0 × 10⁶ per infusion). 9 (Each live cell) ensures product compliance, meets clinical needs, and significantly reduces production and testing costs.

[0084] The method for preparing NK cells using this application can culture NK cells into allogeneic natural killer cells with a purity greater than 95% (FACS), an expansion fold greater than 100, and good cytotoxic activity. The method for preparing NK cells using this application can obtain ready-to-use allogeneic natural killer cells (raNK).

[0085] serum-free culture medium

[0086] Serum-free culture medium for preparing NK cells is a serum-free and heterologous component-free reagent for in vitro culture of NK cells. It is suitable for, but not limited to, the culture of fresh or frozen NK cells derived from peripheral blood, leukopak, umbilical cord blood (UCB), umbilical cord, human induced pluripotent stem cells (hiPSC), and NK cell lines. After activation and expansion phases, high-purity NK cells are obtained. The individual differences between batches of NK cell culture are small, and no additional autologous plasma or human serum needs to be added during use.

[0087] Serum-free culture media include serum-free media used in the activation phase and serum-free media used in the amplification phase.

[0088] Activation stage

[0089] NK cells mainly originate from: 1. peripheral blood, 2. umbilical cord blood, umbilical cord, placenta, etc., and 3. iPSCs.

[0090] Cell Culture: NK cells frozen at low temperature were removed, thawed, centrifuged, and the supernatant discarded. They were then added to the serum-free medium used in the activation phase for resuscitation culture. During this process, the medium was continuously replenished and the cells were transferred to bags for further culture. On days 7-9, samples were taken for cell counting, and serum-free medium used in the expansion phase was added based on the viable cell density. Serum-free medium used in the expansion phase was continuously replenished during this period. On days 9-12, the total number of viable cells, NK cell purity, and cell viability were used to determine whether to transfer the cells to a reactor for further culture.

[0091] Amplification Phase

[0092] Pre-vaccination preparation

[0093] The serum-free culture medium used in the amplification phase was equilibrated in the culture bag. After equilibration, the NK cell suspension was transferred to a rocking reactor for further culture. The culture conditions of the rocking reactor were as follows: total aeration rate of 20-40 mL / min, rocking speed of 8 rpm, rocking angle of 6°, rocking temperature of 37.0±0.5℃, pH of 7.0±0.2, and DO≥20%.

[0094] NK cell expansion culture (Method 1)

[0095] During the expansion culture, samples are taken for counting and biochemical analysis. Based on the viable cell density and glucose concentration, 5%-70% of the cell supernatant is pumped out once or multiple times. Serum-free medium (used in the expansion phase) is then added one or more times, and glucose solution is added to maintain a final glucose concentration of 1.00-2.00 g / L. Once the culture volume reaches the maximum working volume, continuous or intermittent dynamic cell culture is performed, using fresh NK cell expansion medium to change 5%-70% of the medium once or multiple times, ensuring the NK cells remain in the exponential growth phase.

[0096] NK cell expansion culture (Method 2)

[0097] During the amplification culture, samples were taken for counting and biochemical analysis. Serum-free medium used in the amplification phase was replenished according to the viable cell density and glucose concentration. Glucose solution was also added after replenishment to a final glucose concentration of 1.00-2.00 g / L. Once the culture volume reached the maximum working volume, continuous or intermittent dynamic culture was performed. 5%-70% of the cell supernatant was pumped out once or multiple times, and then 5%-70% of the medium was replaced with fresh NK cell amplification medium once or multiple times to keep the NK cells in the exponential growth phase.

[0098] NK cell culture was terminated after 16-28 days.

[0099] During the NK cell activation culture stage and the NK cell expansion culture stage, NK cell-related cytokines, including but not limited to IL2 and long-acting IL15, are continuously added to the cell suspension to maintain their proliferation capacity and killing activity.

[0100] After the NK cell expansion phase, NK cell stock solution was obtained, concentrated, washed, resuspended, and aliquoted. The NK cell stock solution was then filled into disposable cell cryopreservation bags to obtain NK cell injection solution (DP).

[0101] Typically, the purity of NK cells in both NK stock solution and cell injection solution can reach over 95%.

[0102] NK cell injection solution can be further prepared into a cryopreservation solution for the formulation.

[0103] In a first aspect, this application provides a method for preparing NK cells, the method comprising an activation phase and an amplification phase, wherein both the activation phase and the amplification phase are performed in a serum-free culture system and neither phase uses feeder cells, wherein in the amplification phase, NK cells are amplified using a rocking bioreactor, and the culture process parameters include: total aeration rate of 10-40 mL / min, rocking speed of 8-30 rpm, rocking angle of 5-12°, dissolved oxygen (DO) ≥10%, and pH of 6.8-7.2.

[0104] In some embodiments, NK cell expansion is performed using a swing bioreactor during the expansion phase, and the culture process parameters include:

[0105] Total ventilation is 20-40 mL / min; and / or

[0106] The oscillation speed is 8-10 rpm; and / or

[0107] The swing angle is 6°; and / or

[0108] Dissolved oxygen (DO) ≥ 20%.

[0109] In some embodiments, NK cell amplification is carried out using a swing bioreactor during the amplification phase, and the culture process parameters include a total aeration rate of 20-40 mL / min.

[0110] In some embodiments, NK cell amplification is carried out using a rocking bioreactor during the amplification phase, and the culture process parameters include a rocking speed of 8-10 rpm.

[0111] In some embodiments, NK cell amplification is performed using a swing-type bioreactor during the amplification phase, and the culture process parameters include a swing angle of 6°.

[0112] In some embodiments, NK cell expansion is carried out using a swing bioreactor during the expansion phase, and the culture process parameters include dissolved oxygen (DO) ≥ 20%.

[0113] In some implementations, the total ventilation rate is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 mL / min, or a range between any of the above values.

[0114] In some implementations, the oscillation speed is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 rpm, or a range between any of the above values.

[0115] In some implementations, the swing angle is 5°, 6°, 7°, 8°, 9°, 10°, 11°, or 12°, or any range between the above values.

[0116] In some embodiments, the dissolved oxygen (DO) is ≥10%, ≥11%, ≥12%, ≥13%, ≥14%, ≥15%, ≥16%, ≥17%, ≥18%, ≥19%, or ≥20%.

[0117] In some embodiments, the serum-free culture medium used in the amplification phase comprises basal culture medium, interleukin-type NK cell amplification factor, and serum substitute.

[0118] In some embodiments, the serum-free culture medium used in the amplification phase also contains fat-soluble vitamins.

[0119] In some embodiments, the serum-free culture medium used in the amplification phase also contains trace elements.

[0120] In some embodiments, the interleukin-type NK cell amplification factor is selected from any combination of IL2, IL15, IL12, IL18, IL21, or more.

[0121] In some embodiments, the serum-free culture medium used in the amplification phase does not contain nicotinamide and / or dimercaprol.

[0122] In some embodiments, the serum-free culture medium used in the amplification phase also contains heparin sodium.

[0123] In some embodiments, the serum-free culture medium used in the amplification phase also contains one or more of reduced glutathione, human transferrin, and human serum albumin.

[0124] In some implementations, the interleukin-like NK cell amplification factors are IL2 and IL15.

[0125] In some embodiments, the fat-soluble vitamin is selected from one or more of the following: vitamin A, vitamin E, and vitamin K.

[0126] In some embodiments, the trace element is selected from one or more compounds including: sodium selenite, ferrous chloride, zinc chloride, copper chloride, nickel chloride, cobalt chloride hexahydrate, manganese chloride tetrahydrate, chromium chloride, silver chloride, potassium iodide, sodium metavanadate, ammonium molybdate tetrahydrate, rubidium chloride, germanium tetrachloride, and stannous chloride.

[0127] In some embodiments, the serum substitute is platelet lysate.

[0128] In some embodiments, the basal medium is RPMI-1640 basal medium.

[0129] In some embodiments, the serum-free culture medium contains 100-2050 IU / mL of interleukin-type NK cell amplification factor and 0.05-1 v / v% serum substitute, based on the total volume of the serum-free culture medium.

[0130] In some embodiments, the serum-free culture medium contains 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1010, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, or 2050 IU / mL or any of the above values, based on the total volume of the serum-free culture medium.

[0131] In some embodiments, the serum-free culture medium comprises, by total volume, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95 or 1 v / v% or any range of the above values ​​as a serum substitute.

[0132] In some embodiments, the serum-free culture medium contains 1-2250 μg / L of fat-soluble vitamins, based on the total volume of the serum-free culture medium.

[0133] In some embodiments, the serum-free culture medium comprises, based on the total volume of the serum-free culture medium, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1 Fat-soluble vitamins of 000, 1050, 1100, 1135, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950, 2000, 2050, 2100, 2150, 2200 or 2250 μg / L or any value between the above.

[0134] In some embodiments, the serum-free culture medium contains 1000 IU / mL of IL2 and 10 ng / mL of IL15, based on the total volume of the serum-free culture medium.

[0135] In some embodiments, the serum-free culture medium contains 35 μg / L of vitamin A, 1000 μg / L of vitamin E and 100 μg / L of vitamin K1, based on the total volume of the serum-free culture medium.

[0136] In some embodiments, the vitamin A is vitamin A1 or vitamin A2.

[0137] In some embodiments, the vitamin E is α, β, γ, or δ-tocopherol.

[0138] In some embodiments, the serum-free culture medium comprises, by total volume, 6.7 μg / L sodium selenite, 20.2 μg / L ferrous chloride, 19.2 μg / L zinc chloride, 16.9 μg / L copper chloride, 0.073 μg / L nickel chloride, 0.044 μg / L cobalt chloride hexahydrate, 0.123 μg / L manganese chloride tetrahydrate, 0.073 μg / L chromium chloride, 0.133 μg / L silver chloride, 13.07 μg / L potassium iodide, 0.012193 μg / L sodium metavanadate, 0.107 μg / L ammonium molybdate tetrahydrate, 11.32 μg / L rubidium chloride, 5.874 μg / L germanium tetrachloride, and 0.3194 μg / L stannous chloride.

[0139] In some embodiments, the serum-free culture medium contains 0.1-20 μg / L, preferably 0.5-10 μg / L, and more preferably 6.7 μg / L, sodium selenite, based on the total volume of the serum-free culture medium.

[0140] In some embodiments, the serum-free culture medium contains 1-50 μg / L, preferably 20.2 μg / L, of ferrous chloride, based on the total volume of the serum-free culture medium.

[0141] In some embodiments, the serum-free culture medium contains 1-50 μg / L, preferably 19.2 μg / L, of zinc chloride, based on the total volume of the serum-free culture medium.

[0142] In some embodiments, the serum-free culture medium contains 1-50 μg / L, preferably 16.9 μg / L, of copper chloride, based on the total volume of the serum-free culture medium.

[0143] In some embodiments, the serum-free culture medium contains 0.0001-0.1 μg / L, preferably 0.073 μg / L, of nickel chloride, based on the total volume of the serum-free culture medium.

[0144] In some embodiments, the serum-free culture medium contains 0.0001-0.1 μg / L, preferably 0.044 μg / L, of cobalt chloride hexahydrate, based on the total volume of the serum-free culture medium.

[0145] In some embodiments, the serum-free culture medium contains 0.0001-0.5 μg / L, preferably 0.123 μg / L, of manganese chloride tetrahydrate, based on the total volume of the serum-free culture medium.

[0146] In some embodiments, the serum-free culture medium contains 0.0001-0.1 μg / L, preferably 0.073 μg / L, chromium chloride, based on the total volume of the serum-free culture medium.

[0147] In some embodiments, the serum-free culture medium contains 0.0001-0.5 μg / L, preferably 0.133 μg / L, of silver chloride, based on the total volume of the serum-free culture medium.

[0148] In some embodiments, the serum-free culture medium contains 1-50 μg / L, preferably 13.07 μg / L, of potassium iodide, based on the total volume of the serum-free culture medium.

[0149] In some embodiments, the serum-free culture medium contains 0.0001-0.1 μg / L, preferably 0.012193 μg / L, sodium metavanadate, based on the total volume of the serum-free culture medium.

[0150] In some embodiments, the serum-free culture medium contains 0.0001-0.5 μg / L, preferably 0.107 μg / L, of ammonium molybdate tetrahydrate, based on the total volume of the serum-free culture medium.

[0151] In some embodiments, the serum-free culture medium contains 1-50 μg / L, preferably 11.32 μg / L, of rubidium chloride, based on the total volume of the serum-free culture medium.

[0152] In some embodiments, the serum-free culture medium contains 0.1-10 μg / L, preferably 5.874 μg / L, germanium tetrachloride, based on the total volume of the serum-free culture medium.

[0153] In some embodiments, the serum-free culture medium contains 0.0001-0.5 μg / L, preferably 0.3194 μg / L stannous chloride, based on the total volume of the serum-free culture medium.

[0154] In some embodiments, the serum-free culture medium contains 0.1 v / v% platelet lysis buffer based on the total volume of the serum-free culture medium.

[0155] In some embodiments, the serum-free culture medium contains 1-10 U / mL of heparin sodium, based on the total volume of the serum-free culture medium.

[0156] In some embodiments, the serum-free culture medium contains heparin sodium at concentrations of 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10 U / mL or any of the above values, based on the total volume of the serum-free culture medium.

[0157] In some embodiments, the serum-free culture medium contains 2 U / mL of heparin sodium, based on the total volume of the serum-free culture medium.

[0158] In some embodiments, the serum-free culture medium contains 10-1000 μg / L of reduced glutathione, 1000-10000 μg / L of human transferrin, and 1-10 g / L of human serum albumin, based on the total volume of the serum-free culture medium.

[0159] In some embodiments, the serum-free culture medium contains 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 768.3, 800, 850, 900, 950 or 1000 μg / L or any of the above values, based on the total volume of the serum-free culture medium.

[0160] In some embodiments, the serum-free culture medium comprises, based on the total volume of the serum-free culture medium, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950, 2000, 2050, 2100, 2150, 2200, 2250, 2300, 2350, 2400, 2450, 2500, 2550, 2600, 2650, 2700, 2750, 2800, 2850, 2900, 2950, ​​3000, 30 50, 3100, 3150, 3200, 3250, 3300, 3350, 3400, 3450, 3500, 3550, 3600, 3650, 3700, 3750, 3800, 3850, 3900, 3950, 4000, 4050, 4100, 4150, 4200, 4250, 4300, 4350, 4400, 4450, 4500, 4550, 4600, 4650, 4700, 4750, 4800, 4850, 4900, 4950, 5000, 5050, 5100, 5150, 5200, 5250, 5300, 5350, 5400, 54 50, 5500, 5550, 5600, 5650, 5700, 5750, 5800, 5850, 5900, 5950, 6000, 6050, 6100, 6150, 6200, 6250, 6300, 6350, 6400, 6450, 6500, 6550, 6600, 6650, 6700, 6750, 6800, 6850, 6900, 6950, 7000, 7050, 7100, 7150, 7200, 7250, 7300, 7350, 7400, 7450, 7500, 7550, 7600, 7650, 7700, 7750, 7800, 78 Human transferrin in the range of 50, 7900, 7950, 8000, 8050, 8100, 8150, 8200, 8250, 8300, 8350, 8400, 8450, 8500, 8550, 8600, 8650, 8700, 8750, 8800, 8850, 8900, 8950, 9000, 9050, 9100, 9150, 9200, 9250, 9300, 9350, 9400, 9450, 9500, 9550, 9600, 9650, 9700, 9750, 9800, 9850, 9900, 9950 or 10000 μg / L or any value between the above.

[0161] In some embodiments, the serum-free culture medium contains 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10 g / L or any of the above values, based on the total volume of the serum-free culture medium.

[0162] In some embodiments, the serum-free culture medium contains 768.3 μg / L of reduced glutathione, 10000 μg / L of human transferrin, and 5 g / L of human serum albumin, based on the total volume of the serum-free culture medium.

[0163] In some embodiments, the serum-free culture medium, based on its total volume, comprises 1000 IU / mL IL-2, 10 ng / mL IL-15, 35 μg / L vitamin A, 1000 μg / L vitamin E, 100 μg / L vitamin K1, 6.7 μg / L sodium selenite, 20.2 μg / L ferrous chloride, 19.2 μg / L zinc chloride, 16.9 μg / L copper chloride, 0.073 μg / L nickel chloride, 0.044 μg / L cobalt chloride hexahydrate, and 0.123 μg / L manganese chloride. Chromium chloride tetrahydrate, 0.073 μg / L chromium chloride, 0.133 μg / L silver chloride, 13.07 μg / L potassium iodide, 0.012193 μg / L sodium metavanadate, 0.107 μg / L ammonium molybdate tetrahydrate, 11.32 μg / L rubidium chloride, 5.874 μg / L germanium tetrachloride, 0.3194 μg / L stannous chloride, 0.1 v / v% platelet lysis buffer, 2 U / mL sodium heparin, 768.3 μg / L reduced glutathione, 10000 μg / L human transferrin, and 5 g / L human serum albumin.

[0164] In some embodiments, the carbon source cultured during the amplification phase includes glucose, and the glucose concentration of the culture in the bioreactor is controlled at 1.00-2.00 g / L.

[0165] In some implementations, the glucose concentration of the culture in the bioreactor is controlled to be 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2.00 g / L, or any range between the above values.

[0166] In some implementations, the culture temperature during the amplification phase is 37.0 ± 0.5 °C.

[0167] In some implementations, the culture temperature is 36.5, 36.6, 36.7, 36.8, 36.9, 37, 37.1, 37.2, 37.3, 37.4 or 37.5°C, or a range between any of the above values.

[0168] In some implementations, the pH of the culture in the swing bioreactor is controlled at 7.0 ± 0.2.

[0169] In some implementations, the pH of the culture in the swing bioreactor is controlled at 6.8, 6.9, 7, 7.1 or 7.2, or any range between these values.

[0170] In some embodiments, the method further includes equilibrating the swaying bioreactor at the beginning of the amplification phase, wherein the process parameters for equilibration include: an aeration rate of 100-1000 mL / min, a heater temperature of 41.0±2.0℃, an exhaust gas temperature of 55.0±5.0℃, a swaying speed of 12-20 rpm, a swaying angle of 6-12°, and a culture temperature of 37.0±1.0℃.

[0171] In some embodiments, the method further includes equilibrating the swing bioreactor at the beginning of the amplification phase, wherein the process parameters for the equilibration treatment include:

[0172] The ventilation rate is 300 mL / min; and / or

[0173] The heater temperature is 41.0℃; and / or

[0174] The exhaust gas temperature is 55.0℃; and / or

[0175] The oscillation speed is 12-20 rpm; and / or

[0176] The swing angle is 6-12°; and / or

[0177] The incubation temperature was 37.0±0.5℃.

[0178] In some implementations, the ventilation rate is 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 mL / min, or a range between any of the above values.

[0179] In some implementations, the oscillation speed is 12, 13, 14, 15, 16, 17, 18, 19 or 20 rpm, or a range between any of the above values.

[0180] In some implementations, the swing angle is 6°, 7°, 8°, 9°, 10°, 11°, or 12°, or any range between the above values.

[0181] In some embodiments, when the medium change operation is performed during the amplification phase, the volume of the medium changed each time is 5% to 70% of the total volume of the culture system.

[0182] In some implementations, the volume of medium changed each time is 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36% of the total volume of the culture system. %, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, or 70%, or any range between the above values.

[0183] In some implementations, the medium change operation is performed using dynamic culture for intermittent or continuous medium change operations.

[0184] In some embodiments, the serum-free culture medium used in the activation phase comprises basal culture medium, interleukin-type NK cell activating factors, fat-soluble vitamins, trace elements, and serum substitutes.

[0185] In some embodiments, the serum-free culture medium used in the activation phase also contains nicotinamide.

[0186] In some embodiments, the serum-free culture medium used in the activation phase also contains dimercaptoethanol.

[0187] In some embodiments, the serum-free culture medium used in the activation phase also contains sodium heparin.

[0188] In some embodiments, the serum-free culture medium used in the activation phase further comprises one or more of reduced glutathione, human transferrin, and human serum albumin.

[0189] In some embodiments, the serum-free culture medium used in the activation phase also contains quercetin.

[0190] In some embodiments, the interleukin-type NK cell activating factor is selected from any combination of IL2, IL7, IL15, IL12, IL18, IL21, or more.

[0191] In some embodiments, the fat-soluble vitamin is selected from one or more of the following: vitamin A, vitamin E, and vitamin K.

[0192] In some embodiments, the trace element is selected from one or more compounds including: sodium selenite, ferrous chloride, zinc chloride, copper chloride, nickel chloride, cobalt chloride hexahydrate, manganese chloride tetrahydrate, chromium chloride, silver chloride, potassium iodide, sodium metavanadate, ammonium molybdate tetrahydrate, rubidium chloride, germanium tetrachloride, and stannous chloride.

[0193] In some embodiments, the serum substitute is platelet lysate.

[0194] In some embodiments, the basal medium is RPMI-1640 basal medium.

[0195] In some embodiments, the serum-free culture medium contains 100-2100 IU / mL of interleukin-type NK cell activating factor, 1-2250 μg / L of fat-soluble vitamins, and 0.05-10 v / v% of serum substitute, based on the total volume of the serum-free culture medium.

[0196] In some embodiments, the serum-free culture medium contains 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, or 2100 IU / mL or any of the above values ​​as a range of interleukin-type NK cell activating factors, based on the total volume of the serum-free culture medium.

[0197] In some embodiments, the serum-free culture medium comprises, based on the total volume of the serum-free culture medium, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 10 Fat-soluble vitamins in the range of 00, 1050, 1100, 1135, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950, 2000, 2050, 2100, 2150, 2200 or 2250 μg / L or any range between the above values.

[0198] In some embodiments, the serum-free culture medium comprises, by total volume, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 v / v%, or any of the values ​​listed above, as a serum substitute.

[0199] In some embodiments, the serum-free culture medium contains 1000 IU / mL of IL2 and 10 ng / mL of IL15, based on the total volume of the serum-free culture medium.

[0200] In some embodiments, the serum-free culture medium contains 35 μg / L of vitamin A, 1000 μg / L of vitamin E and 100 μg / L of vitamin K1, based on the total volume of the serum-free culture medium.

[0201] In some embodiments, the serum-free culture medium comprises, by total volume, 6.7 μg / L sodium selenite, 20.2 μg / L ferrous chloride, 19.2 μg / L zinc chloride, 16.9 μg / L copper chloride, 0.073 μg / L nickel chloride, 0.044 μg / L cobalt chloride hexahydrate, 0.123 μg / L manganese chloride tetrahydrate, 0.073 μg / L chromium chloride, 0.133 μg / L silver chloride, 13.07 μg / L potassium iodide, 0.012193 μg / L sodium metavanadate, 0.107 μg / L ammonium molybdate tetrahydrate, 11.32 μg / L rubidium chloride, 5.874 μg / L germanium tetrachloride, and 0.3194 μg / L stannous chloride.

[0202] In some embodiments, the serum-free culture medium contains 0.1-20 μg / L, preferably 0.5-10 μg / L, and more preferably 6.7 μg / L, sodium selenite, based on the total volume of the serum-free culture medium.

[0203] In some embodiments, the serum-free culture medium contains 1-50 μg / L, preferably 20.2 μg / L, of ferrous chloride, based on the total volume of the serum-free culture medium.

[0204] In some embodiments, the serum-free culture medium contains 1-50 μg / L, preferably 19.2 μg / L, of zinc chloride, based on the total volume of the serum-free culture medium.

[0205] In some embodiments, the serum-free culture medium contains 1-50 μg / L, preferably 16.9 μg / L, of copper chloride, based on the total volume of the serum-free culture medium.

[0206] In some embodiments, the serum-free culture medium contains 0.0001-0.1 μg / L, preferably 0.073 μg / L, of nickel chloride, based on the total volume of the serum-free culture medium.

[0207] In some embodiments, the serum-free culture medium contains 0.0001-0.1 μg / L, preferably 0.044 μg / L, of cobalt chloride hexahydrate, based on the total volume of the serum-free culture medium.

[0208] In some embodiments, the serum-free culture medium contains 0.0001-0.5 μg / L, preferably 0.123 μg / L, of manganese chloride tetrahydrate, based on the total volume of the serum-free culture medium.

[0209] In some embodiments, the serum-free culture medium contains 0.0001-0.1 μg / L, preferably 0.073 μg / L, chromium chloride, based on the total volume of the serum-free culture medium.

[0210] In some embodiments, the serum-free culture medium contains 0.0001-0.5 μg / L, preferably 0.133 μg / L, of silver chloride, based on the total volume of the serum-free culture medium.

[0211] In some embodiments, the serum-free culture medium contains 1-50 μg / L, preferably 13.07 μg / L, of potassium iodide, based on the total volume of the serum-free culture medium.

[0212] In some embodiments, the serum-free culture medium contains 0.0001-0.1 μg / L, preferably 0.012193 μg / L, sodium metavanadate, based on the total volume of the serum-free culture medium.

[0213] In some embodiments, the serum-free culture medium contains 0.0001-0.5 μg / L, preferably 0.107 μg / L, of ammonium molybdate tetrahydrate, based on the total volume of the serum-free culture medium.

[0214] In some embodiments, the serum-free culture medium contains 1-50 μg / L, preferably 11.32 μg / L, of rubidium chloride, based on the total volume of the serum-free culture medium.

[0215] In some embodiments, the serum-free culture medium contains 0.1-10 μg / L, preferably 5.874 μg / L, germanium tetrachloride, based on the total volume of the serum-free culture medium.

[0216] In some embodiments, the serum-free culture medium contains 0.0001-0.5 μg / L, preferably 0.3194 μg / L stannous chloride, based on the total volume of the serum-free culture medium.

[0217] In some embodiments, the serum-free culture medium contains 0.25 v / v% platelet lysis buffer based on the total volume of the serum-free culture medium.

[0218] In some embodiments, the serum-free culture medium contains 1-10 mM of nicotinamide, based on the total volume of the serum-free culture medium.

[0219] In some embodiments, the serum-free culture medium contains nicotinamide in the range of 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10 mM or any of the above values, based on the total volume of the serum-free culture medium.

[0220] In some embodiments, the serum-free culture medium contains 2.5 mM of nicotinamide based on the total volume of the serum-free culture medium.

[0221] In some embodiments, the serum-free culture medium contains 0.001-0.055 mM of dimercaptoethanol, based on the total volume of the serum-free culture medium.

[0222] In some embodiments, the serum-free culture medium comprises 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.011, 0.012, 0.013, 0.014, 0.015, 0.016, 0.017, 0.018, 0.019, 0.02, 0.021, 0.022, 0.023, 0.024, 0.025, 0.026, 0. Dimercaptoethanol at concentrations of 0.027, 0.028, 0.029, 0.03, 0.031, 0.032, 0.033, 0.034, 0.035, 0.036, 0.037, 0.038, 0.039, 0.04, 0.041, 0.042, 0.043, 0.044, 0.045, 0.046, 0.047, 0.048, 0.049, 0.05, 0.051, 0.052, 0.053, 0.054 or 0.055 mM or any value between the above.

[0223] In some embodiments, the serum-free culture medium contains 0.011 mM of dimercaptoethanol, based on the total volume of the serum-free culture medium.

[0224] In some embodiments, the serum-free culture medium contains 1-10 U / mL of heparin sodium, based on the total volume of the serum-free culture medium.

[0225] In some embodiments, the serum-free culture medium contains heparin sodium at concentrations of 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10 U / mL or any of the above values, based on the total volume of the serum-free culture medium.

[0226] In some embodiments, the serum-free culture medium contains 2 U / mL of heparin sodium, based on the total volume of the serum-free culture medium.

[0227] In some embodiments, the serum-free culture medium contains 10-1000 μg / L of reduced glutathione, 1000-10000 μg / L of human transferrin, and 1-10 g / L of human serum albumin, based on the total volume of the serum-free culture medium.

[0228] In some embodiments, the serum-free culture medium contains 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 768.3, 800, 850, 900, 950 or 1000 μg / L or any of the above values, based on the total volume of the serum-free culture medium.

[0229] In some embodiments, the serum-free culture medium comprises, based on the total volume of the serum-free culture medium, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950, 2000, 2050, 2100, 2150, 2200, 2250, 2300, 2350, 2400 2450, 2500, 2550, 2600, 2650, 2700, 2750, 2800, 2850, 2900, 2950, ​​3000, 3050, 3100, 3150, 3200, 3250, 3300, 3350, 3400, 3450, 3500, 3550, 3600, 3650 3700, 3750, 3800, 3850, 3900, 3950, 4000, 4050, 4100, 4150, 4200, 4250, 4300, 4350, 4400, 4450, 4500, 4550, 4600, 4650, 4700, 4750, 4800, 4850, 4900 4950, 5000, 5050, 5100, 5150, 5200, 5250, 5300, 5350, 5400, 5450, 5500, 5550, 5600, 5650, 5700, 5750, 5800, 5850, 5900, 5950, 6000, 6050, 6100, 6150 6200, 6250, 6300, 6350, 6400, 6450, 6500, 6550, 6600, 6650, 6700, 6750, 6800, 6850, 6900, 6950, 7000, 7050, 7100, 7150, 7200, 7250, 7300, 7350, 7400 7450, 7500, 7550, 7600, 7650, 7700, 7750, 7800, 7850, 7900, 7950, 8000, 8050, 8100, 8150, 8200, 8250, 8300, 8350, 8400, 8450, 8500, 8550, 8600, 8650Human transferrin in concentrations of 8700, 8750, 8800, 8850, 8900, 8950, 9000, 9050, 9100, 9150, 9200, 9250, 9300, 9350, 9400, 9450, 9500, 9550, 9600, 9650, 9700, 9750, 9800, 9850, 9900, 9950, or 10000 μg / L, or any value within the range of the above.

[0230] In some embodiments, the serum-free culture medium contains 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10 g / L or any of the above values, based on the total volume of the serum-free culture medium.

[0231] In some embodiments, the serum-free culture medium contains 768.3 μg / L of reduced glutathione, 10000 μg / L of human transferrin, and 5 g / L of human serum albumin, based on the total volume of the serum-free culture medium.

[0232] In some embodiments, the serum-free culture medium, based on its total volume, comprises 1000 IU / mL IL-2, 10 ng / mL IL-15, 35 μg / L vitamin A, 1000 μg / L vitamin E, 100 μg / L vitamin K1, 6.7 μg / L sodium selenite, 20.2 μg / L ferrous chloride, 19.2 μg / L zinc chloride, 16.9 μg / L copper chloride, 0.073 μg / L nickel chloride, 0.044 μg / L cobalt chloride hexahydrate, 0.123 μg / L manganese chloride tetrahydrate, and 0.073 μg / L sodium selenite. The following concentrations were added: L of chromium chloride, 0.133 μg / L of silver chloride, 13.07 μg / L of potassium iodide, 0.012193 μg / L of sodium metavanadate, 0.107 μg / L of ammonium molybdate tetrahydrate, 11.32 μg / L of rubidium chloride, 5.874 μg / L of germanium tetrachloride, 0.3194 μg / L of stannous chloride, 0.25 v / v% platelet lysis buffer, 2.5 mM nicotinamide, 0.011 mM dimercaptoethanol, 2 U / mL of sodium heparin, 768.3 μg / L of reduced glutathione, 10000 μg / L of human transferrin, and 5 g / L of human serum albumin.

[0233] In some implementations, the cultivation process parameters for the activation phase include a temperature of 37.0 ± 0.5 °C and a CO2 concentration of 5.0 ± 0.5%.

[0234] In some implementations, the temperature in the culturing process parameters during the activation phase is 36.5, 36.6, 36.7, 36.8, 36.9, 37, 37.1, 37.2, 37.3, 37.4, or 37.5°C, or a range between any of the above values.

[0235] In some implementations, the CO2 concentration in the cultivation process parameters during the activation phase is 4.5%, 4.6%, 4.7%, 4.8%, 4.9%, 5%, 5.1%, 5.2%, 5.3%, 5.4%, or 5.5%, or any range between the above values.

[0236] In some embodiments, the activation phase further includes coating the bottom of the cell culture vessel with an antibody (coating agent) targeting one or more of the following molecules: CD3, CD25, CD33-CLL1, CD52, CD4, CD8, 4-1BB, CD19, CD38, and CD16.

[0237] In some embodiments, the coating agent for the NK cell culture vessel comprises an anti-CD38 antibody and an antibody or combination of antibodies targeting one or more of the following antigens: CD3, CD25, CD33, CD52, CD4, CD8, 4-1BB, CD19, CLL1, and CD16.

[0238] In some embodiments, the concentration of the anti-CD38 antibody is 1000 μg / L.

[0239] In some implementations, the anti-CD38 antibody is diluted with physiological saline or phosphate buffer.

[0240] In some embodiments, the coating agent further comprises an anti-CD52 antibody.

[0241] In some embodiments, the concentration of the anti-CD52 antibody is 10,000 μg / L.

[0242] In some implementations, the anti-CD52 antibody is diluted with physiological saline or phosphate buffer.

[0243] In some embodiments, the coating agent further comprises an anti-CD33-CLL1 bispecific antibody.

[0244] In some embodiments, the concentration of the anti-CD33-CLL1 bispecific antibody is 1000 μg / L.

[0245] In some embodiments, the anti-CD33-CLL1 bispecific antibody is diluted with physiological saline or phosphate buffer.

[0246] In some embodiments, the coating agent comprises anti-CD52 antibody and anti-CD38 antibody.

[0247] In some embodiments, the coating agent comprises an anti-CD38 antibody, an anti-CD52 antibody, and an anti-CD33-CLL1 bispecific antibody.

[0248] In some implementations, NK cells are derived from peripheral blood mononuclear cells (PBMCs).

[0249] In some implementations, NK cells are obtained from NK cells derived from umbilical cord blood.

[0250] In some implementations, NK cells are obtained from in vitro culture of NK cell lines.

[0251] In some implementations, NK cells are obtained from the in vitro induction and culture of induced pluripotent stem cells (iPSCs) or mesenchymal stem cells (MSCs).

[0252] In a second aspect, this application provides a composition comprising NK cells prepared by the method described in the first aspect.

[0253] In some embodiments, the composition contains 100 billion or more NK cells.

[0254] In some embodiments, CD3 in the composition - CD56 + The purity of NK cells is greater than or equal to 90%.

[0255] In some embodiments, CD3 in the composition - CD56 + The purity of NK cells is greater than or equal to 90%, 91%, 92%, 93%, 94%, or 95%.

[0256] In some embodiments, CD3 in the composition - CD56 + The purity of NK cells is greater than or equal to 95%.

[0257] In some embodiments, CD3 in the composition - CD56 + NKG2D + The proportion of NK cells is greater than or equal to 95% and CD3 - CD56 + CD16 + The proportion of NK cells is greater than or equal to 95%.

[0258] In some embodiments, the proportion of non-NK cells in the composition is less than 10%.

[0259] In some embodiments, the proportion of non-NK cells in the composition is less than 5%.

[0260] In some embodiments, the proportion of non-NK cells in the composition is less than 10%, 9%, 8%, 7%, 6%, or 5%.

[0261] In some embodiments, the composition is formulated as an injectable solution.

[0262] In some implementations, the NK cells are derived from peripheral blood mononuclear cells (PBMCs).

[0263] In some implementations, the NK cells are derived from umbilical cord blood NK cells.

[0264] In some implementations, the NK cells are obtained from in vitro culture of NK cell lines.

[0265] In some embodiments, the NK cells are obtained from in vitro induction and culture of induced pluripotent stem cells (iPSCs) or mesenchymal stem cells (MSCs).

[0266] Example

[0267] This application will be described in more detail by way of specific examples. The following embodiments are provided for illustrative purposes only and are not intended to limit this application in any way. Those skilled in the art will readily recognize that various non-critical parameters can be changed or modified to produce substantially the same results.

[0268] Example 1: Preparation of serum-free culture medium

[0269] Prepare serum-free culture medium (activation medium) used in the activation phase and serum-free culture medium (amplification medium) used in the amplification phase.

[0270] The serum-free culture medium (activation medium) used in the activation phase contains RPMI-1640 basal medium, interleukin-type NK cell activating factors (e.g., IL2 and IL15), fat-soluble vitamins (e.g., vitamin A, vitamin E, and vitamin K1), trace elements (e.g., sodium selenite, ferrous chloride, zinc chloride, copper chloride, nickel chloride, cobalt chloride hexahydrate, manganese chloride tetrahydrate, chromium chloride, silver chloride, potassium iodide, sodium metavanadate, ammonium molybdate tetrahydrate, rubidium chloride, germanium tetrachloride, and stannous chloride), serum substitutes (e.g., platelet lysate), nicotinamide, dimercaptoethanol, sodium heparin, reduced glutathione, human transferrin, and human serum albumin.

[0271] The serum-free medium (amplification medium) used in the amplification phase contains RPMI-1640 basal medium, interleukin-type NK cell activating factors (e.g., IL2 and IL15), fat-soluble vitamins (e.g., vitamin A, vitamin E, and vitamin K1), trace elements (e.g., sodium selenite, ferrous chloride, zinc chloride, copper chloride, nickel chloride, cobalt chloride hexahydrate, manganese chloride tetrahydrate, chromium chloride, silver chloride, potassium iodide, sodium metavanadate, ammonium molybdate tetrahydrate, rubidium chloride, germanium tetrachloride, and stannous chloride), serum substitutes (e.g., platelet lysate), sodium heparin, reduced glutathione, human transferrin, and human serum albumin.

[0272] The serum-free culture protocol includes a coating agent (for coating the culture vessel), an activation medium, and an amplification medium. The coating agent is diluted with physiological saline. Both the activation and amplification media use RPMI-1640 as the basal medium and supplemented with activators and amplifiers. The formulation of the RPMI-1640 basal medium is shown in Table 1. The sources of the reagents used in the examples are shown in Table 2.

[0273] The serum-free culture medium (amplification medium) used in the amplification phase includes RPMI-1640 basal medium and amplification reagents. Examples of amplification reagents are shown in Table 3.

[0274] The serum-free culture medium (activation medium) used in the activation phase includes RPMI-1640 basal medium and activators. Examples of activators are shown in Table 3.

[0275] Examples of coating agents are shown in Table 3.

[0276] Table 1: Formulation of RPMI-1640 basal medium

[0277] Table 2: Reagent Sources

[0278] Table 3: Components and concentrations of exemplary coating agents, activation media, and amplification media

[0279] Example 2: NK cell activation stage

[0280] I) Cell resuscitation and culture

[0281] (1) Coating of culture flasks: Take the coating agent into the culture flask and mix well. Place the culture flask at 37±0.5℃ for coating and set aside for later use.

[0282] (2) Cell resuscitation: First, take out the cryopreserved cells and thaw them. Then, transfer the cell suspension in the cryopreservation bag to a centrifuge tube and make up to 45 mL with a 10 mL pipette. Place the centrifuge tube in a centrifuge, set the centrifugal force to 600 g, and centrifuge at room temperature for 8 ± 2 min.

[0283] (3) After centrifugation, discard the supernatant, use a 10mL pipette to draw up the NK cell activation culture medium, repeatedly pipette to resuspend the cell pellet, mix well, and then take a sample for counting.

[0284] (4) Based on the counting results, the cell suspension is inoculated and cultured.

[0285] (5) After vaccination, T225cm 2 The culture flasks were placed in a carbon dioxide incubator at 37.0±0.5℃ and a CO2 concentration of 5.0±0.5% for static incubation.

[0286] II) Culture flask replenishment culture

[0287] Add NK cell activation medium to the culture flask and continue to incubate in a carbon dioxide incubator at 37.0±0.5℃ and CO2 concentration of 5.0±0.5%.

[0288] III) Culture flask transfer bag culture

[0289] (1) After mixing the cell suspension in the culture flask, take samples and count them.

[0290] (2) Based on the counting results, calculate the total number of viable cells to determine whether to transfer the cells to a culture bag. If transfer is necessary, determine the volume of replenishment fluid. Transfer the NK cell suspension and NK cell activation medium to the cell culture bag.

[0291] (3) After replenishing the solution, continue to place it in a carbon dioxide incubator at 37.0±0.5℃ and CO2 concentration of 5.0±0.5% for static incubation.

[0292] IV) Cell culture bag replenishment culture

[0293] (1) Sampling and counting: After mixing the cell suspension in the cell culture bag, sampling and counting are performed.

[0294] (2) Determine the volume of fluid to be added based on the density of live cells before fluid addition. If the volume does not meet the requirements for fluid addition, postpone the fluid addition operation.

[0295] (3) Fluid replenishment: Add NK cell expansion medium to the cell culture bag.

[0296] (4) After replenishing the solution, continue to place it in a carbon dioxide incubator at 37.0±0.5℃ and CO2 concentration of 5.0±0.5% for static incubation.

[0297] V) Cell culture bag sampling and central control result confirmation

[0298] (1) Sampling and counting: After mixing the cell suspension in the culture bag, sampling and counting are performed.

[0299] (2) Control standard for entering the reactor: total cell count ≥ 2.0 × 10⁻⁶ 9 Live cells, cell viability ≥80.0%, NK cell purity ≥70.0%, total T cell impurities (CD3+) + ≤9.0%, B cell impurities (CD19) + If the concentration of α is ≤6.0%, and all the above control criteria are met, the culture can be transferred to the reactor for amplification; otherwise, the culture should continue and the transfer to the reactor should be postponed.

[0300] Example 3: NK cell expansion stage

[0301] I) Bioreactor inoculation and culture: Days 9-12

[0302] (1) Calculate and add NK cell amplification medium to a disposable cell culture bag based on the total number of live cells, the volume of NK cell suspension, and the density of live cells after replenishment.

[0303] (2) Equilibration: Set the air flow rate of the equilibration reactor to 300 mL / min, heater temperature to 41.0℃, exhaust gas temperature to 55.0℃, rotation speed to 12-20 rpm, angle to 6-12°, and incubation temperature to 37.0±0.5℃, and start operation. During operation, record the pH and DO information according to the information on the bag. After the reactor is equilibrated, calibrate the DO and pH.

[0304] (3) After calibration, set the relevant parameters of the reactor and determine the culture conditions: total aeration rate of 20-40 mL / min, rocking speed of 8 rpm, rocking angle of 6°, rocking temperature of 37.0±0.5℃, pH of 7.0±0.2, and DO≥20%. Prepare for cell inoculation.

[0305] (4) Cell inoculation: Connect the cell culture bag to the 50L culture bag tubing using a sterile connector, and pump the NK cell suspension into the disposable cell culture bag for culture. After inoculation, mix the NK cell suspension, take a sample to test the pH value and perform biochemical analysis, and add glucose solution to the blood glucose concentration after inoculation until the glucose concentration is 1.00-2.00 g / L.

[0306] (5) Reconfirm the culture conditions: total aeration rate of 20-40 mL / min, shaking speed of 8 rpm, shaking angle of 6°, shaking temperature of 37.0±0.5℃, pH of 7.0±0.2, and DO≥20%.

[0307] II) Bioreactor expansion culture (days 10-21)

[0308] (1) After entering the reactor, record the monitoring information on the display interface of the swing reactor control unit before sampling and counting. And take samples.

[0309] (2) Detection items: live cell density, cell viability, pH value and biochemical analysis.

[0310] (3) NK cell expansion culture uses two methods:

[0311] (i) Culture Method 1: Sampling, counting, and biochemical detection. Based on the viable cell density and glucose concentration, pump out 5%-70% of the cell supernatant once or multiple times. Add 10%-100% NK cell expansion medium once or multiple times. Once the culture volume reaches the maximum working volume (25L for a 50L reactor, 50L for a 100L reactor, and 100L for a 200L reactor), conduct continuous or intermittent dynamic cell culture, changing 5%-70% of the medium once or multiple times using fresh NK cell expansion medium.

[0312] (ii) Culture Method Two: Sampling, counting, and biochemical detection. NK cell expansion medium is added based on viable cell density and glucose concentration. Once the culture volume reaches the maximum working volume (25L for a 50L reactor, 50L for a 100L reactor, and 100L for a 200L reactor), continuous or intermittent dynamic culture is performed. 5%-70% of the cell supernatant is pumped out once or multiple times daily, followed by one or more exchanges of 5%-70% of the medium with fresh NK cell expansion medium.

[0313] (4) Replenishment of culture medium: If the working volume is <25L, determine whether to replenish the medium based on the counting results. Replenishment procedure: Connect the NK cell expansion medium to the disposable cell culture bag using a sterile connector, add the NK cell expansion medium, and add glucose solution according to the glucose concentration after replenishment until the glucose concentration is 1.00-2.00g / L.

[0314] (5) Dynamic culture operation: Use continuous or intermittent dynamic culture to replace 5%-70% of the fresh NK cell expansion medium, and add glucose solution to the glucose concentration after the medium change until the glucose concentration is 1.00-2.00 g / L. Replace 500-2000 mL of culture supernatant in each cycle, and repeat this cycle until the total medium volume reaches the dynamic operation volume.

[0315] (6) Culture conditions: total aeration rate of 20-40 mL / min, shaking speed of 8-10 rpm, shaking angle of 6°, shaking temperature of 37.0±0.5℃, pH of 7.0±0.2, and DO≥20%.

[0316] (7) Central control sampling: During culture, samples are taken, counted, and sent for flow cytometry testing. The central control standard is cell viability ≥ 85.0% and NK purity (CD3+). - CD56 + ≥85.0%, total T cell impurities (CD3) + ≤6.0%, double-negative cell impurities (CD3) - CD56 - If the concentration of microorganisms (≤6.0%) does not meet the above intermediate control standards, the culture shall be terminated. If the intermediate control results meet the above standards, the culture shall continue. Samples taken during the culture period must be sterile, and the test results must show no detectable microorganisms.

[0317] The process parameters for NK cell expansion culture (days 9-25) are shown in Table 4 below.

[0318] Table 4: NK cell expansion and culture process parameters

[0319] Example 4: Preparation of NK cell injection solution

[0320] Following the steps of Example 3, an NK cell suspension was obtained after the NK cell expansion phase. The NK cell suspension was concentrated, washed, and resuspended to obtain NK cell stock solution (DS). The NK stock solution was then filled into disposable cell cryopreservation bags and cryopreserved at ultra-low temperature to obtain NK cell injection solution (DP).

[0321] Example 5: Detection of NK cell culture process

[0322] NK cells were amplified according to the methods described in Examples 1-3, and changes in cell growth, cell viability, and purity of the NK cell suspension were monitored in real time. Cell viability and viable cell density (corresponding to changes in cell growth) were detected using cell counting, while NK cell purity and the proportion of contaminating cells were detected using flow cytometry.

[0323] Figures 1 to 3 show the growth changes of NK cells cultured in a 50L reactor (Figure 1), the cell viability changes (Figure 2), and the purity changes (Figure 3), respectively.

[0324] Figures 4 to 6 show the growth changes of NK cells cultured in a 100L reactor (Figure 4), the cell viability changes (Figure 5), and the purity changes (Figure 6), respectively.

[0325] The results showed that the total number of viable cells after the NK cell activation and expansion phases could reach hundreds of billions. After 10 days of expansion culture, high-purity NK cells with high viable cell density were obtained, with NK cell viability maintained at over 90%, even exceeding 95%, and NK cell purity reaching over 95%.

[0326] Example 6: NK cell flow cytometry analysis

[0327] NK cells were amplified according to the methods described in Examples 1-4, and NK cell stock solution and NK cell injection solution were prepared. Flow cytometry analysis of the NK cell stock solution and NK cell injection solution was performed. As shown in Figure 8, the NK cell purity of the NK cell stock solution obtained after concentration, washing, and resuspension was as high as 97% or more. Among them, CD3... - CD56 + NKG2D + The proportion of NK cells is greater than or equal to 95%, even as high as 99%, and CD3 - CD56 + CD16 + The proportion of NK cells is greater than or equal to 99%. As shown in Figure 9, the NK cell purity of the NK cell injection solution is greater than or equal to 98%, and CD3... - CD56 + NKG2D + The proportion of NK cells is greater than or equal to 99%, and CD3 - CD56 + CD16 + The proportion of NK cells is greater than or equal to 99%.

[0328] Example 7: In vitro cytotoxic activity of NK cell preparations

[0329] The NK cells obtained in Example 5 were used to test the in vitro killing activity of NK cell suspensions with different effector-to-target ratios against Daudi tumor cells (Figure 7).

[0330] The results showed that when the effector-target ratio was 1:1, the in vitro killing activity of NK cells against Daudi tumor cells reached 48%, and when the effector-target ratio was 10:1 or 30:1, the in vitro killing rate of NK cells against Daudi tumor cells was as high as 85% or more.

[0331] The in vitro killing activity was tested using the NK cell injection solution described in Example 4.

[0332] 1) Stain Daudi tumor cells with Calcein AM for 30 min, then wash three times; adjust the tumor cell concentration to 1.5 × 10⁻⁶. 5NK cell injection solution was seeded at 100 μl / well in a 96-well cell culture plate. The concentration of NK cell injection solution was adjusted to 9.0 × 10⁻⁶ cells / ml with cell diluent. 6 cells / ml, 4.5×10 6 cells / ml, 1.5×10 6 cells / ml, 5×10 5 cells / ml, 1.7×10 5 cells / ml, 5.6×10 4 cells / ml, 1.9×10 4 cells / ml, 6.2×10 3 Cells / ml were analyzed, with 6 replicates per gradient. 100 μl was added per well to a 96-well plate to achieve final effector-to-target ratios of 60:1, 30:1, 10:1, 3:1, 1:1, and 1:3. 100 μl of cell lysis buffer was added to each well in column 10 (BG row), and the cells were incubated at 37.0 ± 0.5℃ and 5.0 ± 0.5% CO2 for 4–6 hours. Tumor cells were stained with Calcein AM for 30 min, washed three times, and the tumor cell concentration was adjusted to 1.5 × 10⁻⁶ cells / ml. 5 NK cell injection solution was seeded at 100 μl / well in a 96-well cell culture plate. The concentration of NK cell injection solution was adjusted to 9.0 × 10⁻⁶ cells / ml with cell diluent. 6 cells / ml, 4.5×10 6 cells / ml, 1.5×10 6 cells / ml, 5.0×10 5 cells / ml, 1.7×10 5 cells / ml, 5.6×10 4 cells / ml, 1.9×10 4 cells / ml, 6.1×10 3 cells / ml, 2.0×10 3 Cells / ml were analyzed, with 12 replicates per gradient. 100 μl was added per well to a 96-well plate to achieve the following effector-to-target ratios: 60:1, 30:1, 10:1, 3:1, 1:1, 1:3, 1:9, 1:27, and 1:81. Cells were incubated at 37.0±0.5℃ and 5±0.5% CO2 for 3.5 hours. 100 μl of cell lysis buffer was added to each well in column 10 (BG row), and the plates were incubated at 37.0±0.5℃ and 5±0.5% CO2 for another 0.5 hours.

[0333] 2) Detection on the plate: Transfer 120 μl of supernatant per well to a completely black 96-well microplate. Measure the fluorescence intensity using 490 nm as the excitation wavelength and 535 nm as the emission wavelength. Analyze the changes in fluorescence intensity and calculate the cell killing rate.

[0334] As shown in Figure 10, the cell killing rate can reach 52% when the effector-to-target ratio is 10:1, and the cell killing rate can be greater than 70% when the effector-to-target ratio is 30:1 or 60:1. This indicates that the NK cell injection has a significant killing effect on the tumor cells Daudi.

[0335] Comparative Example 1: Comparison of Culture System Processes

[0336] The parameters set in Example 3 were adjusted to study the effect of parameter changes on NK cell expansion.

[0337] The same steps and operations as in Example 3 were performed on Comparative Example 1, except that the two parameters, the swing speed and the swing angle, were adjusted in the following steps (see the underlined part for specific parameter changes):

[0338] I) Bioreactor inoculation and culture: Days 9-12

[0339] (3) After calibration, set the relevant parameters of the reactor and determine the culture conditions: total aeration rate of 20-40 mL / min, rocking speed of 3 rpm, rocking angle of 2°, rocking temperature of 37.0±0.5℃, pH of 7.0±0.2, and DO≥20%. Prepare for cell inoculation.

[0340] II) Bioreactor expansion culture (days 10-21)

[0341] (6) Culture conditions: total aeration rate of 20-40 mL / min, shaking speed of 3-6 rpm, shaking angle of 2-4°, shaking temperature of 37.0±0.5℃, pH of 7.0±0.2, and DO≥20%.

[0342] Other culture conditions and process controls in Comparative Example 1 were the same as in Example 3. Changes in cell growth, cell viability, and purity of the NK cell suspension are shown in Figures 11, 12, and 13.

[0343] Regarding cell growth changes, Figure 1 shows the culture results of cells cultured in a 50L reactor using the process of Example 3. Figure 11 shows the culture results of cells cultured in a 50L reactor using the process of Comparative Example 1. The results show that after 18 days of culture, the total number of cells cultured using Example 3 (Figure 1) reached approximately 600 × 10⁻⁶. 8 In Comparative Example 1, it only reached approximately 300 × 10 8 (Figure 11).

[0344] Regarding changes in cell viability, Figure 2 shows the culture results of cells cultured using Example 3. Figure 12 shows the culture results of cells cultured using Comparative Example 1. The results indicate that the cell viability of cells cultured using Example 3 (Figure 2) remained above 90% throughout the 12-day culture period and subsequent culture periods. In contrast, in Comparative Example 1, the cell viability was lower during the 12-day culture period and subsequent culture periods, fluctuating around 90%, and even falling below 80% in some cases (Figure 12).

[0345] Regarding the changes in NK purity, Figure 3 shows the culture results of cells cultured using Example 3. Figure 13 shows the culture results of cells cultured using Comparative Example 1. The results indicate that the NK purity of cells cultured using Example 3 (Figure 3) reached 95% during the 9-11 day culture period, and maintained a NK purity greater than 95% during subsequent culture. In contrast, in Comparative Example 1, the NK purity only reached approximately 90% during the 9-11 day culture period, and the NK purity fluctuated significantly during subsequent culture (Figure 13).

Claims

1. A method for preparing NK cells, the method comprising an activation phase and an expansion phase, wherein both the activation phase and the expansion phase are performed in a serum-free culture system and neither phase uses feeder cells, wherein... During the expansion phase, NK cells were expanded using a swing-type bioreactor, and the culture process parameters included: The total ventilation rate is 10-40 mL / min, the swing speed is 8-30 rpm, the swing angle is 5-12°, the dissolved oxygen (DO) is ≥10%, and the pH is 6.8-7.

2.

2. The method of claim 1, wherein the serum-free culture medium used in the amplification phase comprises basal culture medium, interleukin-type NK cell amplification factor, and serum substitute.

3. The method of claim 2, wherein the serum-free culture medium used in the amplification phase further comprises fat-soluble vitamins.

4. The method of claim 2 or 3, wherein the serum-free culture medium used in the amplification phase further contains trace elements.

5. The method according to any one of claims 2 to 4, wherein the interleukin-type NK cell amplification factor is selected from any combination of IL2, IL15, IL12, IL18, IL21 or above.

6. The method according to any one of claims 2 to 5, wherein the serum-free culture medium used in the amplification phase does not contain nicotinamide and / or dimercaprol.

7. The method according to any one of claims 2 to 6, wherein the serum-free culture medium used in the amplification phase further comprises one or more of heparin sodium, reduced glutathione, human transferrin, and human serum albumin.

8. The method according to any one of claims 2 to 7, wherein in the amplification phase, NK cell amplification is performed using a swing-type bioreactor, and the culture process parameters include: Total ventilation is 20-40 mL / min; and / or The oscillation speed is 8-10 rpm; and / or The swing angle is 6°; and / or Dissolved oxygen (DO) ≥ 20%.

9. The method of claim 5, wherein the interleukin-type NK cell amplification factors are IL2 and IL15.

10. The method of claim 3, wherein the fat-soluble vitamin is selected from one or more of the following: vitamin A, vitamin E, and vitamin K.

11. The method of claim 4, wherein the trace element is selected from one or more compounds including: sodium selenite, ferrous chloride, zinc chloride, copper chloride, nickel chloride, cobalt chloride hexahydrate, manganese chloride tetrahydrate, chromium chloride, silver chloride, potassium iodide, sodium metavanadate, ammonium molybdate tetrahydrate, rubidium chloride, germanium tetrachloride, and stannous chloride.

12. The method of any one of claims 2 to 11, wherein the serum substitute is platelet lysis buffer.

13. The method according to any one of claims 2 to 12, wherein the basal culture medium is RPMI-1640 basal culture medium.

14. The method of any one of claims 1 to 13, wherein in the amplification phase, the carbon source cultured comprises glucose, and the glucose concentration of the culture in the bioreactor is controlled at 1.00-2.00 g / L.

15. The method according to any one of claims 1 to 14, wherein the culture temperature during the amplification phase is 37.0 ± 0.5 °C.

16. The method of any one of claims 1 to 15, further comprising, at the beginning of the amplification phase, equilibrating the swing-type bioreactor, wherein the process parameters for the equilibration treatment include: The aeration rate was 100-1000 mL / min, the heater temperature was 41.0±2.0℃, the exhaust gas temperature was 55.0±5.0℃, the swaying speed was 12-20 rpm, the swaying angle was 6-12°, and the culture temperature was 37.0±1.0℃.

17. The method of any one of claims 1 to 16, wherein during the amplification phase, the volume of the medium exchanged each time is 5% to 70% of the total volume of the culture system.

18. The method of claim 17, wherein the medium change operation is performed by using dynamic culture for intermittent or continuous medium change operations.

19. The method of claim 1, wherein the serum-free culture medium used in the activation phase comprises basal culture medium, interleukin-type NK cell activating factors, fat-soluble vitamins, trace elements, and serum substitutes.

20. The method of claim 19, wherein the serum-free culture medium used in the activation phase further comprises nicotinamide.

21. The method of claim 19, wherein the serum-free culture medium used in the activation phase further comprises dimercaprol.

22. The method of claim 19, wherein the serum-free culture medium used in the activation phase further comprises one or more of sodium heparin, reduced glutathione, human transferrin, and human serum albumin.

23. The method of claim 16, wherein the process parameters for the balancing process include: The ventilation rate is 300 mL / min; and / or The heater temperature is 41.0℃; and / or The exhaust gas temperature is 55.0℃; and / or The oscillation speed is 12-20 rpm; and / or The swing angle is 6-12°; and / or The incubation temperature was 37.0±0.5℃.

24. The method of claim 19, wherein the serum-free culture medium used in the activation phase further comprises quercetin.

25. The method of claim 19, wherein the interleukin-type NK cell activating factor is selected from any combination of IL2, IL7, IL15, IL12, IL18, IL21 or more.

26. The method of claim 19, wherein the fat-soluble vitamin is selected from one or more of the following: vitamin A, vitamin E, and vitamin K.

27. The method of claim 19, wherein the trace element is selected from one or more compounds including: sodium selenite, ferrous chloride, zinc chloride, copper chloride, nickel chloride, cobalt chloride hexahydrate, manganese chloride tetrahydrate, chromium chloride, silver chloride, potassium iodide, sodium metavanadate, ammonium molybdate tetrahydrate, rubidium chloride, germanium tetrachloride, and stannous chloride.

28. The method of any one of claims 19 to 27, wherein the serum substitute is platelet lysis buffer.

29. The method of any one of claims 19 to 28, wherein the basal culture medium is RPMI-1640 basal culture medium.

30. The method of claim 1, wherein the culture process parameters during the activation phase include: The temperature was 37.0±0.5℃ and the CO2 concentration was 5.0±0.5%.

31. The method of claim 1, wherein the activation phase further comprises coating the bottom of a cell culture vessel with an antibody targeting one or more of the following molecules: CD3, CD25, CD33, CD52, CD4, CD8, 4-1BB, CD19, CLL1, CD38, and CD16.

32. The method of claim 1, wherein, The NK cells were obtained from peripheral blood mononuclear cells (PBMCs); or The NK cells are obtained from NK cells derived from umbilical cord blood; or The NK cells were obtained from in vitro culture of NK cell lines; or The NK cells were obtained from in vitro induction and culture of induced pluripotent stem cells (iPSCs) or mesenchymal stem cells (MSCs).

33. A composition comprising NK cells prepared by any one of claims 1 to 32.

34. The composition of claim 33, wherein the NK cells in the composition are greater than or equal to 100 billion.

35. The composition of claim 33 or 34, wherein CD3 in the composition - CD56 + The purity of NK cells is greater than or equal to 95%.

36. The composition of any one of claims 33 to 35, wherein CD3 in the composition - CD56 + NKG2D + The proportion of NK cells is greater than or equal to 95% and CD3 - CD56 + CD16 + The proportion of NK cells is greater than or equal to 95%.

37. The composition of any one of claims 33 to 36, wherein the proportion of non-NK cells in the composition is less than 5%.

38. The composition of any one of claims 33 to 37, wherein the composition is formulated as an injection solution.

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