A method for improving NK cell viability under high-density culture conditions

Abstract

The present invention relates to a method for improving the viability of NK cells under high-density culture conditions. Specifically, the present invention can significantly improve the viability of NK cells by adding dasatinib to the NK cell culture system, without affecting cell expansion, and the cell's killing activity is better after cryopreservation and resuscitation. The method of the present invention solves the common problems of decreased NK cell viability during high-density expansion and decreased tumor cell killing activity after cryopreservation and resuscitation without changing the process route. It has the characteristics of simple operation, low cost, and no dependence on process methods, and has broad application prospects in the field of producing NK products.

Description

Technical Field

[0001] The invention relates to the technical field of cell culture, and in particular to a method for improving the viability of NK cells under high-density culture conditions. Background Art

[0002] As a general-purpose off-the-shelf cell therapy product, NK cell production has the characteristics of mass production and high-density amplification to meet the needs of batch cell production of dozens to hundreds of people. Similar to other cells, during the high-density culture of NK cells, competition will occur between cells, nutrients and space will become limited, resulting in a slowdown in cell growth rate, deterioration of cell status, and a decrease in cell viability, which is manifested as a decrease in cell plasma membrane integrity. Cell viability is an important detection indicator for cell biology experiments and is the basis for cell culture, cytotoxicity and physiological research. The decrease in cell viability during the culture process will directly affect the quality of NK cells, especially after cryopreservation and resuscitation, the killing activity of NK against tumor cells will be significantly reduced.

[0003] In the process of cell culture, cell growth rate and cell viability are important indicators of healthy cell culture. In order to improve the damage of high-density culture to cell viability and activity, and thus obtain better cell products, in the process of production process development, this can be achieved by changing the cell density, the size of the culture container, and the supply and supply method of cell nutrients. Although these methods can improve the situation to a certain extent, they have high requirements for the developed process route and need to adjust the process according to the batch production output and the expected key quality attributes of the cells, which makes it difficult to be universal.

[0004] For cell therapy drugs, the ability of cells to kill tumor cells after cryopreservation and resuscitation is one of the difficulties to be overcome, especially NK drug products are particularly sensitive to cryopreservation, and maintaining high tumor killing activity of cells after cryopreservation is still one of the important difficulties in the industry. High-density amplification is a necessary step in the production of NK drug products, but this process will cause the viability of NK cells to decrease, which will further reduce the killing activity of NK cells against tumor cells after cryopreservation and resuscitation. Therefore, improving the viability of NK cells under high-density conditions and the killing activity of cells after cryopreservation and resuscitation has great research value.

[0005] Dasatinib is a tyrosine kinase inhibitor. Sheng Lixia et al. (2020) reported the scheme of adding dasatinib during NK cell culture and its effect (Dasatinib's regulatory effect on in vitro expansion, cell subsets, receptor expression and cytotoxic function of NK cells, Sheng Lixia et al., "Chinese Journal of Experimental Hematology", Issue 5, 2020). In the scheme disclosed in the article, dasatinib was added throughout the NK culture process. The results showed that 5-50 nmol / L of dasatinib can improve the expansion efficiency of NK cells, and 20 nmol / L of dasatinib can enhance the cytotoxicity of NK cells. However, the article also found that when the concentration of dasatinib added during the whole culture process was 50 nM, the cell apoptosis rate was as high as about 25%, that is, the addition of dasatinib under this scheme was not conducive to the viability of NK cells.

[0006] Therefore, there is an urgent need in the art to develop a culture method that can improve the viability of NK cells and their killing activity after cryopreservation and resuscitation under high-density culture conditions. Summary of the invention

[0007] The purpose of the present invention is to provide a culture method for improving the viability of NK cells and the killing activity of NK cells after cryopreservation and resuscitation under high-density culture conditions.

[0008] In a first aspect of the present invention, a method for culturing NK cells under high density conditions is provided, comprising the steps of:

[0009] A. Cultivating NK cells in a culture system, wherein the culture system does not contain dasatinib (Dasa), and the culture comprises:

[0010] (1) Add activation factors, culture medium and starting NK cells to the first culture container and perform activation culture for 4 to 6 days;

[0011] (2) Placing the activated cultured NK cells into a second culture container, adding culture medium, and performing amplification culture for 1 to 5 days until the density of NK cells in the culture system reaches 3×10 6 / mL~1.5×10 7 Pieces / mL;

[0012] B. Add 5-1000nM dasatinib (Dasa) to the culture system and continue culturing for 2-4 days. The cell culture is terminated.

[0013] C. Harvesting of cultured NK cells.

[0014] In another preferred embodiment, the NK cells are NK cells differentiated from iPSCs.

[0015] In another preferred embodiment, the NK cells are NK cells differentiated from gene-edited iPSCs.

[0016] In another preferred embodiment, in step A, the density of NK cells in the culture system reaches 4×10 6 / mL~1.2×10 7 Pieces / mL.

[0017] In another preferred embodiment, in step A, the density of NK cells in the culture system reaches 4×10 6 / mL~1×10 7 Pieces / mL.

[0018] In another preferred embodiment, in step A, the density of NK cells in the culture system reaches 5×10 6 / mL~8×10 6 Pieces / mL.

[0019] In another preferred embodiment, in step B, 50-1000 nM dasatinib is added to the culture system.

[0020] In another preferred embodiment, in step B, 100-1000 nM dasatinib is added to the culture system.

[0021] In another preferred embodiment, in step B, 100-500 nM dasatinib is added to the culture system.

[0022] In another preferred embodiment, in step C, the cell viability is ≥85%, preferably ≥90%, and more preferably ≥93%.

[0023] In another preferred embodiment, in step C, the expansion multiple of the cultured cells is ≥600.

[0024] In another preferred embodiment, the cultured NK cells harvested in step C are cryopreserved, and after recovery, the killing ability of the NK cells against tumor cells is significantly better than that of the NK cells obtained by culturing without adding Dasa.

[0025] In another preferred embodiment, step A further comprises:

[0026] A1. In a first culture container, culture medium and starting NK cells are added for activation culture; the first culture container is coated with an activation factor;

[0027] A2. In a second culture container, add culture medium and the cells activated and cultured in step A1 to perform expansion culture.

[0028] In another preferred embodiment, the activating factor is selected from antibodies against the following proteins: natural cytotoxicity receptor, Fc receptor CD16, NKG2D.

[0029] In another preferred embodiment, the natural cytotoxicity receptors include: NKp30 and NKp46.

[0030] In another preferred embodiment, the coating treatment includes the steps of: preparing a coating solution, using DPBS as a diluent, dissolving the activating factor in a solvent to a concentration of 0.5-10 ug / mL; adding the coating solution to a culture container, covering the surface with the liquid, and incubating at 4°C±1°C overnight.

[0031] In another preferred embodiment, the culture solution comprises the following components: NK cell basal culture medium and cytokine combination.

[0032] In another preferred embodiment, the NK cell basal medium is selected from: OptiVitro NK, CTS TM NK-Xpander, Irvine TM PRMIE-XV NK Cell CDM, or a combination thereof.

[0033] In another preferred embodiment, the cytokine combination is IL-2, IL-12, IL-15, IL-18 and IL-21.

[0034] In another preferred embodiment, the concentrations of the cytokines in the culture medium are: IL-2 at 100 IU / ml~2000 IU / ml, IL-12 at 5 ng / ml~100 ng / ml, IL-15 at 5 ng / ml~100 ng / ml, IL-18 at 5 ng / ml~100 ng / ml, and IL-21 at 10 ng / ml~200 ng / ml.

[0035] In another preferred embodiment, the culture medium in step A1 and the culture medium in step A2 are the same.

[0036] In another preferred embodiment, in step A1, the first culture container is selected from the following group: a culture dish, a multi-well plate, a culture bottle, or a combination thereof.

[0037] In another preferred example, in step A1, the first culture container is a culture dish.

[0038] In another preferred embodiment, in step A1, the activation culture is static culture.

[0039] In another preferred embodiment, in step A1, the activation culture time is 5-7 days, wherein the culture medium is replenished every 1-3 days.

[0040] In another preferred embodiment, in step A2, before the expansion culture begins, the cells are diluted to 2×10 5 ~1×10 6 The density of pcs / mL.

[0041] In another preferred embodiment, in step A2, the second culture container is selected from the following group: a culture bottle, a culture dish, a multi-well plate, or a combination thereof.

[0042] In another preferred embodiment, in step A2, the second culture container is a culture bottle.

[0043] In another preferred embodiment, in step A2, the expansion culture is a dynamic culture, such as a dynamic culture on a shaker.

[0044] In another preferred embodiment, in step A1, the amplification culture time is 9-14 days, wherein the culture medium is replenished every 1-3 days.

[0045] In the second aspect of the present invention, a NK cell culture fluid containing the tyrosine kinase inhibitor dasatinib is provided, wherein the concentration of dasatinib in the culture fluid is 5-1000nM, and the culture fluid is used in the later stage of NK cell culture to improve the cell viability during NK cell culture and the killing activity after cryopreservation and resuscitation.

[0046] In another preferred embodiment, the late stage of NK culture refers to 2-4 days before cell harvesting.

[0047] In another preferred embodiment, the late stage of NK culture refers to the density of NK cells in the culture system reaching 3×10 6 / mL~1×10 7 / mL, preferably 4×10 6 / mL~9×10 6 / mL, more preferably 5×10 6 / mL~8×10 6 Pieces / mL.

[0048] In another preferred embodiment, the concentration of dasatinib in the culture medium is 50-1000 nM.

[0049] In another preferred embodiment, the concentration of dasatinib in the culture medium is 100-1000 nM.

[0050] In another preferred embodiment, the concentration of dasatinib in the culture medium is 100-500 nM.

[0051] In another preferred embodiment, the cell viability is ≥87%, preferably ≥90%, and more preferably ≥93%.

[0052] In another preferred embodiment, the NK cell culture medium further comprises: NK cell basal culture medium and cytokine combination.

[0053] In another preferred embodiment, the NK cell basal medium is selected from: OptiVitro NK, CTS TM NK-Xpander, Irvine TM PRMIE-XV NK Cell CDM, or a combination thereof.

[0054] In another preferred embodiment, the cytokine combination is IL-2, IL-12, IL-15, IL-18 and IL-21.

[0055] In another preferred example, the concentrations of the cytokines in the NK cell culture medium are: IL-2 at 100 IU / ml~2000 IU / ml, IL-12 at 5 ng / ml~100 ng / ml, IL-15 at 5 ng / ml~100 ng / ml, IL-18 at 5 ng / ml~100 ng / ml, and IL-21 at 10 ng / ml~200 ng / ml.

[0056] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here.

[0057] The beneficial effects of the present invention include:

[0058] 1. The culture method of the present invention can significantly improve the cell viability during NK cell culture, does not affect the cell expansion multiple, and can enhance the target cell killing ability of the cells.

[0059] 2. The culture method of the present invention is suitable for culturing NK cells under high-density culture conditions, and significantly reduces the adverse effects of high-density conditions on NK cells.

[0060] 3. The NK cells cultured by the culture method of the present invention have a higher cell killing ability than the control group after cryopreservation and resuscitation.

[0061] 4. The culture method of the present invention has excellent effects on iNK cells from different sources, including iNK cells differentiated from iPSCs that have not been gene-edited or have been gene-edited, so the culture method of the present invention has universal applicability.

[0062] 5. The culture method of the present invention is simple and easy to operate, does not require major adjustments to existing processes, and is universal. Dasatinib is an approved tumor drug with high safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1 The results of cell viability detection of each group of cells cultured for 12 days according to the protocol of Example 1 are shown.

[0064] Figure 2 The total expansion fold of each group of cells cultured for 12 days according to the protocol of Example 1 is shown.

[0065] Figure 3 The results show the killing ability of each group of cells cultured for 12 days according to the protocol of Example 1 on tumor cells K562.

[0066] Figure 4 The results of cell viability testing of each group of cells cultured for 12 days according to the protocol of Example 5 are shown.

[0067] Figure 5 The total expansion fold of each group of cells cultured for 12 days according to the protocol of Example 5 is shown.

[0068] Figure 6 The results show the killing ability of each group of cells cultured for 12 days according to the protocol of Example 5 on tumor cells K562.

[0069] Figure 7 The results show that the cells obtained by culturing for 12 days according to the protocol of Example 5 have the ability to kill tumor cells K562 after being frozen and revived.

[0070] Figure 8 The purity of cells after treatment with 200 nM dasatinib in the protocol of Example 5 is shown.

[0071] Fig. 9 The viable cell count density of each group of cells cultured for 12 days during the culture process in the protocol of Example 5 is shown.

[0072] Fig.10 The flow cytometry results of cell viability of each group of cells in the scheme of Example 12 are shown.

[0073] Fig.11 The results of cell viability analysis of each group of cells in the protocol of Example 12 are shown.

[0074] Fig.12 The figure shows the changes in cell proliferation folds of each group of cells in the scheme of Example 12 after the addition of dasatinib and dexamethasone.

[0075] Fig.13 The killing ability of each group of cells in the scheme of Example 12 on tumor cells K562 is shown.

[0076] Fig.14 The figure shows the expansion trend of NK cells in the experimental group and the control group in the scheme of comparative example 1.

[0077] Fig.15 The figure shows the expansion multiples of NK cells cultured to the 11th day in the experimental group and the control group in the scheme of comparative example 1. DETAILED DESCRIPTION

[0078] After extensive and in-depth research, the inventors have provided a method for culturing NK cells under high-density culture conditions, by adding Dasatinib (Dasa) in the culture system, the viability of NK cells is improved, and the cell proliferation is not affected, and the cell killing activity is better. The method of the present invention can solve the common problems of decreased NK cell viability and activity during high-density proliferation and decreased tumor cell killing activity after cryopreservation and resuscitation with simple operations without changing the process route, and has the characteristics of simple operation, low cost, and no dependence on process methods.

[0079] On this basis, the present invention has been completed.

[0080] Dasatinib

[0081] Dasatinib (Dasa) is an oral tyrosine kinase inhibitor that was approved by the FDA in 2006. Dasatinib is a potent, sub-nanomolar BCR-ABL kinase inhibitor that can inhibit BCR-ABL kinase and SRC family kinases as well as many other selective oncogenic kinases.

[0082] In the present invention, the inventors unexpectedly discovered that directly adding dasatinib into the NK cell culture system can significantly improve the viability of NK cells, especially under high-density cell culture conditions, without changing other culture steps and conditions.

[0083] This provides a new use of dasatinib, namely, adding it to the NK cell culture system to improve the viability of NK cells under high-density culture conditions.

[0084] The results of the present invention show that directly adding dasatinib into the culture system can not only significantly improve the viability of cells under high-density culture conditions, but also does not affect cell proliferation, and the cell killing activity is better, which is conducive to the development of better NK cell therapy products.

[0085] NK cell culture method of the present invention

[0086] The present invention provides a method for improving the viability of NK cells in a culture process by adding dasatinib, comprising the steps of: adding dasatinib to a culture system 2-4 days before harvesting during the NK cell culture process.

[0087] In the present invention, the source of NK cells is not limited, and can be NK cells differentiated from induced pluripotent stem cells (iPSCs), or NK cells derived from PBMCs. Preferably, the NK cells of the present invention are NK cells differentiated from iPSCs.

[0088] In the present invention, the culture process of NK cells is not limited. Generally, the culture of NK cells is divided into two stages, an activation culture stage and an expansion culture stage. In the activation culture stage, in order to make the NK cells have a higher expansion rate, an activation factor can be added to activate the NK cells. In a preferred embodiment, the activation factor includes but is not limited to: natural cytotoxicity receptors, Fc receptors, activated killer cell Ig-like receptors (KIR).

[0089] In the present invention, the activation factor in the activation culture stage can be coated into the culture container by coating treatment. For example, the specific operation is: prepare a coating solution, use DPBS as a diluent, and dissolve the activation factor to a concentration of 0.5-10ug / mL. Add the coating solution to the culture dish with an untreated (NTC) surface, cover the surface with the liquid, and incubate at 4°C overnight.

[0090] In the present invention, the culture medium for NK cell culture is not limited and may contain components known in the art that are beneficial to NK cell culture. In a preferred embodiment, the culture medium for NK cells includes: NK cell culture medium and cytokine combination. Among them, the NK cell culture medium can be homemade or commercial; the cytokine combination includes: IL-2 (concentration 100IU / ml~2000IU / ml), IL-12 (concentration 5ng / ml~100 ng / ml), IL-15 (concentration 5ng / ml~100ng / ml), IL-18 (concentration 5ng / ml~100ng / ml), IL-21 (concentration 10ng / ml~200ng / ml).

[0091] Add cell culture medium and NK cells to the coated culture plate and place it in a 37°C, 5% CO2 incubator for activation culture. Preferably, the activation culture is static culture, and the number of days for activation culture is 5-7 days, wherein the culture medium is supplemented every 1-3 days according to the cell growth situation.

[0092] After the activation culture stage is completed, the activated NK cells are transferred to a culture container without an activation factor for expansion culture. In a preferred embodiment, the culture medium components of the expansion culture are the same as those of the activation culture stage. Preferably, the expansion culture is a dynamic culture, such as a dynamic culture in a shaking table. The total number of days for activation culture and expansion culture is 9-14 days, for example 12 days, wherein the culture medium is supplemented every 1-3 days according to the cell growth.

[0093] In the present invention, 5-1000 nM of dasatinib is added to the culture system 4 to 2 days before the end of NK cell culture, and the other culture conditions remain unchanged. Preferably, the amount of dasatinib added is 100-1000 nM, more preferably 100-500 nM. After the culture is completed, the NK product is harvested by washing and freezing.

[0094] The cell culture method of the present invention is particularly suitable for use under high-density culture conditions. For example, in the expansion culture stage, the density of NK cells is 3×10 6 / mL~1.5×10 7 / mL, preferably 4×10 6 / mL~1.2×10 7 / mL, more preferably 5×10 6 / mL~8×10 6 In the high-density culture process, the quality of cell culture will decline, especially in the decrease of cell viability. In the culture scheme of adding dasatinib of the present invention, under the high-density culture condition, the viability of NK cells can still be significantly improved without changing other culture steps, and it has general applicability.

[0095] For cell therapy drugs, the ability of cells to kill tumor cells after cryopreservation and resuscitation is one of the difficulties to be overcome, especially for NK drug products. Studies have found that NK cells are extremely sensitive to cryopreservation and resuscitation, and the survival rate and cytotoxicity of NK cells after resuscitation are significantly reduced. Therefore, exploring how to improve the ability of NK cells to kill tumor cells after cryopreservation and resuscitation is an important part of the development of NK drug products.

[0096] The NK cells obtained by culturing with the culture scheme of adding dasatinib of the present invention have a higher cell killing ability than the control group after being revived by cryopreservation.

[0097] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples that do not specify specific conditions are usually performed under conventional conditions, such as the conditions described in Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise indicated, percentages and fractions are weight percentages and weight fractions.

[0098] Example 1: NK cell culture protocol with immunosuppressant added

[0099] In this implementation, a NK cell culture protocol with the addition of different types and concentrations of immunosuppressants is provided, including tyrosine kinase inhibitor dasatinib (Dasa), tyrosine kinase inhibitor nilotinib (Nilo) and glucocorticoid dexamethasone (DEX). The specific implementation steps are as follows:

[0100] (1) Prepare the coating solution. Use DPBS as the diluent to dissolve the activating factor CD16 antibody (1 μg / μl) to a concentration of 0.5-10 μg / ml. Add the coating solution to the culture dish with the untreated (NTC) surface, spread the liquid over the surface, and incubate at 4°C overnight.

[0101] (2) Obtain iNK cells derived from iPSCs through differentiation and resuspend iNK cells to 2×10 5 ~1×10 6 IL-2 (concentration 200 IU / ml), IL-12 (concentration 20 ng / ml), IL-15 (concentration 5 ng / ml), IL-18 (concentration 10 ng / ml), and IL-21 (concentration 50 ng / ml) were added to the culture medium at a density of 100 / mL.

[0102] (3) Take out the culture plate that has been coated overnight, remove the coating solution, add the iNK cell suspension with stimulating factors to the coated culture plate, incubate in a 37°C, 5% CO2 incubator for 3 days, and activate by static activation.

[0103] (4) After 3 days, add half the volume of fresh culture medium relative to the previous culture system, and continue to add IL-2 (concentration 200 IU / ml), IL-12 (concentration 20 ng / ml), IL-15 (concentration 5 ng / ml), IL-18 (concentration 10 ng / ml), and IL-21 (concentration 50 ng / ml), and then let it rest for activation for another 2 days.

[0104] (5) After a total of 5 days of activation, the activated iNK cells were transferred to a shake flask. At the same time, fresh culture medium was added to dilute the activated cells to 2×10 5 ~1×10 6 / mL, add IL-2 (concentration 200IU / ml), IL-12 (concentration 20ng / ml), IL-15 (concentration 5ng / ml), IL-18 (concentration 10ng / ml), IL-21 (concentration 50ng / ml). Place the shake flask in a shaker and perform dynamic culture at 45rpm.

[0105] (6) Thereafter, culture medium and IL-2 (concentration 200 IU / ml), IL-12 (concentration 20 ng / ml), IL-15 (concentration 5 ng / ml), IL-18 (concentration 10 ng / ml), and IL-21 (concentration 50 ng / ml) were added every 1 to 3 days and the culture was continued for 12 days.

[0106] Among them, before harvesting, iNK cells were grouped, and each group was added with the following concentrations of immunosuppressants in the culture system at 4 days before harvest (i.e., day 8 of culture) or 2 days before harvest (i.e., day 10 of culture) respectively: 5nM, 10nM, 50nM, 100nM of dasatinib (Dasa), 10nM, 100nM of nilotinib (Nilo), and 500nM, 1000nM of dexamethasone (DEX), and the other culture conditions remained unchanged. No immunosuppressants were added to the control group (control). After four days, the iNK products were harvested after washing and freezing.

[0107] Example 2: Cell viability detection

[0108] The viability of NK cells cultured in each scheme in Example 1 was detected by flow cytometry. The NK cells harvested after 12 days of expansion in Example 1 were stained with Annexin V and 7AAD, and Annexin V-7AAD- were surviving target cells. The viability of NK cells was analyzed by analyzing the proportion of Annexin V-7AAD- cell population.

[0109] The results are as follows Figure 1 As shown in the figure, the cell viability of dasatinib-treated cells was higher than that of the control group, and the effect of 4-day treatment was greater than that of 2-day treatment.

[0110] Nilotinib and dexamethasone treatment groups had a negative effect on NK cell viability, with cell viability below 80%, while for the 4-day group treated with dasatinib, cell viability exceeded 80%, and there was a concentration-dependent effect.

[0111] The experimental results of this example show that in this experiment, the optimal condition is 100 nM dasatinib concentration, and the NK cell viability can be maintained at more than 90% after 4 days of treatment. At the same time, the results show that the cell viability has a dose-dependent effect. Under the same 4-day treatment condition, the higher the dasatinib concentration, the higher the cell viability.

[0112] Example 3: Cell proliferation rate detection

[0113] In steps (5) and (6) of Example 1, the proliferation of NK cells during the entire growth process was recorded, and the proliferation multiples of NK cells after 12 days of expansion were calculated by cell counting.

[0114] The results are as follows Figure 2As shown, whether treated for 2 days or 4 days, dasatinib did not significantly inhibit NK expansion relative to the control group, and there was no gradient dependence.

[0115] The results of Example 2 and Example 3 show that the group adding dasatinib significantly improved the viability of NK cells without affecting the expansion multiple of NK cells.

[0116] Example 4: Cell killing ability detection

[0117] The killing ability of NK cells cultured in each scheme in Example 1 was detected.

[0118] Tumor cells K562 were co-cultured with NK cells cultured for 12 days, and flow cytometry was used to detect the killing effect of NK on target cells (Annexin V-7AAD- represents surviving target cells) at different E:T effector-target ratios (NK cells: target cells K562).

[0119] The results are as follows Figure 3 As shown, the killing effect of NK cells treated with Dasatinib on tumor cells K562 is better than that of the control group, and the concentration of 100nM is better than 50nM, showing a dose-dependent effect. At the low efficiency target ratio, E:T=0.3:1, the Dasa (100nM) group has a more significant advantage over the control group, and the killing ability of tumor cells K562 is increased by 1.5 times.

[0120] The above results indicate that when there are fewer local NK cells than tumor cells, such as in vivo, NK cells treated with dasatinib have a greater advantage in killing tumors.

[0121] Example 5: High-density culture protocol

[0122] In order to further explore the optimal concentration of dasatinib, in this example, four concentration gradients of 100 nM, 200 nM, 500 nM and 1000 nM were further established under the condition of 4 days of immunosuppressant treatment. At the same time, in order to further test whether dasatinib can be used at the pilot production level, a relatively high-density culture mode was set up, that is, the density of NK cells was maintained at 5×10 6 / ml~1×10 7 / ml. On the 8th day of cell expansion, 5×10 6 / ml density, and supplement fresh culture medium, cytokines and dasatinib by centrifugation every day. Dasatinib is added according to the set concentration gradient. The specific steps are as follows:

[0123] (1) Prepare the coating solution, using DPBS as the diluent to dissolve the activating factor to a concentration of 0.5-10ug / ml. Add the coating solution to the culture dish with the untreated (NTC) surface, spread the liquid over the surface, and incubate at 4°C overnight.

[0124] (2) Obtain iNK cells derived from iPSCs through differentiation and resuspend iNK cells to 2×10 5 ~1×10 6 IL-2 (concentration 200 IU / ml), IL-12 (concentration 20 ng / ml), IL-15 (concentration 5 ng / ml), IL-18 (concentration 10 ng / ml), and IL-21 (concentration 50 ng / ml) were added to the culture medium at a density of 100 / mL.

[0125] (3) Take out the culture plate that has been coated overnight, remove the coating solution, add the iNK cell suspension with stimulating factors to the coated culture plate, incubate in a 37°C, 5% CO2 incubator for 3 days, and activate by static activation.

[0126] (4) After 3 days, add half the volume of fresh culture medium relative to the previous culture system, and continue to add IL-2 (concentration 200 IU / ml), IL-12 (concentration 20 ng / ml), IL-15 (concentration 5 ng / ml), IL-18 (concentration 10 ng / ml), and IL-21 (concentration 50 ng / ml), and then let it rest for activation for another 2 days.

[0127] (5) After a total of 5 days of activation, the activated iNK cells were transferred to a shake flask. At the same time, fresh culture medium was added to dilute the activated cells to 2×10 5 ~1×10 6 / mL, add IL-2 (concentration 200IU / ml), IL-12 (concentration 20ng / ml), IL-15 (concentration 5ng / ml), IL-18 (concentration 10ng / ml), IL-21 (concentration 50ng / ml). Place the shake flask in a shaker and perform dynamic culture at 45rpm.

[0128] (6) On the 8th day of culture, the cell density increased to 5e6 / ml, and the medium was centrifuged and replaced with fresh medium and IL-2 (concentration 200IU / ml), IL-12 (concentration 20ng / ml), IL-15 (concentration 5ng / ml), IL-18 (concentration 10ng / ml), and IL-21 (concentration 50ng / ml) were added. They were divided into six groups, five groups were added with five concentration gradients of dasatinib 0nM, 100nM, 200nM, 500nM and 1000nM, and one group was added with 100nM nilotinib.

[0129] (7) After that, each group was centrifuged and the medium was replaced with fresh culture medium every day, and IL-2 (concentration 200 IU / ml), IL-12 (concentration 20 ng / ml), IL-15 (concentration 5 ng / ml), IL-18 (concentration 10 ng / ml), and IL-21 (concentration 50 ng / ml) were added. In addition, 0 nM, 100 nM, 200 nM, 500 nM and 1000 nM of dasatinib and 100 nM of nilotinib were added to the corresponding groups, and the culture was continued until the 12th day. After 4 days of dasatinib treatment, the iNK products were harvested by washing and freezing. During the culture process, the density of NK cells was maintained at 5×10 6 / ml, the highest is 1.1×10 7 / ml.

[0130] Example 6: Cell viability under high density culture conditions

[0131] The viability of NK cells cultured in each of the schemes in Example 5 was detected. After 12 days of expansion and 4 days of treatment with dasatinib or nilotinib, the cell viability was detected by flow cytometry.

[0132] The results are as follows Figure 4 The results showed that the cell viability of the 4-day treatment with dasatinib was higher than that of the control group. The tyrosine kinase inhibitor nilotinib had a negative effect on the viability of NK cells, with the cell viability being less than 80%. However, for the 4-day treatment group with dasatinib, the cell viability exceeded 90%.

[0133] The results of this example show that under high-density culture conditions (5×10 6 / ml~1.1×10 7 / ml), the optimal condition was when Dasa concentration was 200nM and treatment time was 4 days, which could keep NK cell viability above 90%.

[0134] Example 7: Amplification under high-density culture conditions

[0135] For the culture scheme in Example 5, the proliferation of NK cells was recorded throughout the growth process, and the proliferation multiples of NK cells after 12 days of expansion were calculated by cell counting.

[0136] The results are as follows Figure 5 Dasatinib had no significant effect on NK cell proliferation.

[0137] The results of Example 6 and Example 7 show that the group with the addition of dasatinib significantly improved the viability of NK cells under high-density culture conditions without affecting the expansion multiple of NK cells.

[0138] Example 8: Cell killing ability under high-density culture conditions

[0139] The killing ability of NK cells cultured in each scheme in Example 5 was detected.

[0140] Tumor cells K562 were co-cultured with NK cells cultured for 12 days in Example 5 to detect the killing ability of tumor cells.

[0141] The results are as follows Figure 6 As shown. The test showed that the killing effect of NK cells treated with dasatinib (4 days) on tumor cells K562 was better than that of the control group, and 200nM concentration was the optimal condition. The results showed that under high-density culture conditions, dasatinib treatment increased the viability of NK cells and slightly increased the killing activity of cells before freezing.

[0142] Example 9: Killing activity of cells after cryopreservation and thawing

[0143] In the examples, the ability of NK cells cultured in the scheme of Example 5 to kill tumor cells after freezing and thawing was further verified. The cells treated with dasatinib or nilotinib in Example 5 were frozen and the killing activity after thawing was detected.

[0144] The results are as follows Figure 7 The results showed that when the concentration of dasatinib was 200nM and the treatment lasted for 4 days, the maximum killing activity against tumor cells could be obtained, which was significantly higher than that of the control group. When the effect-target ratio E:T=1:1, the killing ability of the Dasa (200nM) group against the tumor cell K562 was increased by about 2 times compared with the control group.

[0145] The results of Example 8 and Example 9 jointly show that although the tumor killing effect of NK cells treated with nilotinib before cryopreservation was slightly improved compared with the control group, the tumor killing effect was significantly reduced after cryopreservation, while the killing activity of the dasatinib group was improved after cryopreservation, indicating that NK cells treated with dasatinib are more adapted to cryopreservation conditions.

[0146] Example 10: Cell purity detection

[0147] The purity of NK cells harvested after 4-day treatment with 200 nM dasatinib in Example 5 was analyzed by detecting the expression of marker proteins on the surface of NK cells using flow cytometry to characterize the purity of the cells (the proportion of CD3-CD56+ cell population to all cells).

[0148] The results are as follows Figure 8 Compared with the control group, dasatinib treatment did not affect the purity of NK cells, which was maintained above 99%.

[0149] Example 11: Changes in cell density during high-density culture

[0150] The density changes of the NK cells cultured in Example 5 during the culture process were counted using NC-200, and the counting results showed the density of live cells (DAPI-).

[0151] The results are as follows Fig. 9 As shown in the figure, the results showed that there was no significant difference in the viable cell density between the dasatinib-treated group and the control group during the culture process. From Day 8 to Day 12, the NK density was maintained at 5×10 6 / ml, the highest is 1.1×10 7 / ml, indicating that the cell culture density was always maintained at a high level during the experiment.

[0152] Example 12: Culture protocol for gene-edited NK cells (CD16-iNK)

[0153] In this implementation, a culture scheme for shear-resistant CD16 NK cells (CD16-iNK) with the addition of different types and concentrations of immunosuppressants is provided, including tyrosine kinase inhibitor dasatinib (Dasa), and glucocorticoid dexamethasone (DEX). The specific implementation steps are as follows:

[0154] (1) Prepare the coating solution. Use DPBS as the diluent to dissolve the activating factor CD16 antibody (1 μg / μl) to a concentration of 0.5-10 μg / ml. Add the coating solution to the culture dish with the untreated (NTC) surface, spread the liquid over the surface, and incubate at 4°C overnight.

[0155] (2) Obtain CD16-iNK cells differentiated from iPSCs edited with the shear-resistant CD16 gene by differentiation, and resuspend iNK cells to 2×10 in serum-free NK-specific culture medium. 5 ~1×10 6 IL-2 (concentration 200 IU / ml), IL-12 (concentration 20 ng / ml), IL-15 (concentration 5 ng / ml), IL-18 (concentration 10 ng / ml), and IL-21 (concentration 50 ng / ml) were added to the culture medium at a density of 100 / mL.

[0156] (3) Take out the culture plate that has been coated overnight, remove the coating solution, add the CD16-iNK cell suspension with stimulating factors to the coated culture plate, and incubate in a 37°C, 5% CO2 incubator for 3 days for static activation.

[0157] (4) After 3 days, add half the volume of fresh culture medium relative to the previous culture system, and continue to add IL-2 (concentration 200 IU / ml), IL-12 (concentration 20 ng / ml), IL-15 (concentration 5 ng / ml), IL-18 (concentration 10 ng / ml), and IL-21 (concentration 50 ng / ml), and then let it rest for activation for another 2 days.

[0158] (5) After a total of 5 days of activation, the activated iNK cells were transferred to a shake flask. At the same time, fresh culture medium was added to dilute the activated cells to 2×10 5 ~1×10 6 / mL, add IL-2 (concentration 200IU / ml), IL-12 (concentration 20ng / ml), IL-15 (concentration 5ng / ml), IL-18 (concentration 10ng / ml), IL-21 (concentration 50ng / ml). Place the shake flask in a shaker and perform dynamic culture at 45rpm.

[0159] (6) Thereafter, culture medium and IL-2 (concentration 200 IU / ml), IL-12 (concentration 20 ng / ml), IL-15 (concentration 5 ng / ml), IL-18 (concentration 10 ng / ml), and IL-21 (concentration 50 ng / ml) were added every 1 to 3 days and the culture was continued for 12 days.

[0160] Among them, on the 11th day of culture, iNK cells were divided into groups, and the following concentrations of immunosuppressants were added to the culture system for each group: a control group without immunosuppressants, a group with 50nM dasatinib (Dasa), a group with 100nM dasatinib (Dasa), and a group with 500nM dexamethasone (DEX), and the other culture conditions remained unchanged. Three days after the addition, CD16-iNK products were harvested by washing and freezing.

[0161] Example 13: Cell viability detection

[0162] The viability of CD16-iNK cells cultured in each scheme in Example 12 was detected by flow cytometry. The NK cells harvested after 14 days of expansion in Example 12 were stained with Annexin V and 7AAD, Annexin V-7AAD- were surviving target cells, and the viability of NK cells was analyzed by analyzing the proportion of Annexin V-7AAD- cell population.

[0163] The results are as follows Fig.10 and Fig.11 As shown in the figure, the cell viability of dasatinib-treated cells was significantly increased compared with the control group, from 72.1% to 95.5%. The dexamethasone-treated group had no significant effect on the viability of NK cells, with a cell viability of 75.5%.

[0164] The experimental results of this example show that before cell harvesting, treatment with 50 nM dasatinib and 100 nM dasatinib for 3 days can maintain the viability of CD16-iNK cells at more than 95% (Annexin-7AAD-), which is significantly higher than the control group without dasatinib treatment.

[0165] Example 14: Cell proliferation rate detection

[0166] In step (6) of Example 12, the expansion fold of CD16-iNK cells three days after the addition of dasatinib and dexamethasone was calculated by cell counting.

[0167] The results are as follows Fig.12 As shown, dasatinib treatment had no significant effect on the expansion fold of CD16-iNK cells compared with the control group or dexamethasone treatment group.

[0168] The results of Example 13 and Example 14 show that the group to which dasatinib is added can significantly improve the viability of CD16-iNK cells without affecting the proliferation rate.

[0169] Example 15: Cell killing ability detection

[0170] The killing ability of NK cells cultured in each scheme in Example 12 was detected.

[0171] Tumor cells K562 were co-cultured with CD16-iNK cells cultured for 14 days, and flow cytometry was used to detect the killing effect of NK on target cells (Annexin V-7AAD- represents surviving target cells) at an E:T effector-target ratio of 0.3:1 (NK cells: target cells K562).

[0172] The results are as follows Fig.13 As shown, the killing effect of NK cells treated with dasatinib on tumor cells K562 was better than that of the control group, and the cells in the dexamethasone-treated group had the weakest killing effect on tumor cells K562.

[0173] The above results indicate that dasatinib treatment of CD16-iNK can not only increase the viability of NK cells, but also enhance the killing of tumor cells K562.

[0174] Comparative Example 1: Scheme of adding dasatinib throughout the NK cell culture and viability detection

[0175] The culture scheme for adding dasatinib to the culture medium of NK cells starting from the 0th day of culture and the method for detecting the expansion multiple of iNK cells after culture are as follows:

[0176] (1) Prepare the coating solution. Use DPBS as the diluent to dissolve the activating factor CD16 antibody (1 μg / μl) to a concentration of 0.5-10 μg / ml. Add the coating solution to the culture dish with the untreated (NTC) surface, spread the liquid over the surface, and incubate at 4°C overnight.

[0177] (2) Obtain iNK cells derived from iPSCs through differentiation and resuspend iNK cells to 2×10 5 ~1×10 6 IL-2 (concentration 200 IU / ml), IL-12 (concentration 20 ng / ml), IL-15 (concentration 5 ng / ml), IL-18 (concentration 10 ng / ml), and IL-21 (concentration 50 ng / ml) were added to the culture medium at a density of 100 / mL.

[0178] (3) The iNK cell suspension with added cytokines was equally divided into an experimental group and a control group. Dasatinib (Dasa) was added to the experimental group at a concentration of 50 ng / ml.

[0179] (4) Take out the culture plate that has been coated overnight, remove the coating solution, add the iNK cell suspension of the experimental group and the control group to the coated culture plate, incubate in a 37°C, 5% CO2 incubator for 3 days, and activate by static activation.

[0180] (5) After 3 days of activation, the experimental and control groups were supplemented with half the volume of fresh culture medium relative to the previous culture system, and IL-2 (concentration 200 IU / ml), IL-12 (concentration 20 ng / ml), IL-15 (concentration 5 ng / ml), IL-18 (concentration 10 ng / ml), and IL-21 (concentration 50 ng / ml) were added. Dasatinib was added to the experimental group at a concentration of 50 ng / ml. The cells were left to activate for another 2 days.

[0181] (6) After a total of 5 days of activation, the activated iNK cells were transferred to a shake flask. At the same time, fresh culture medium was added to dilute the activated cells to 2×10 5 ~1×10 6 The density of cells / mL was set, and IL-2 (concentration 200IU / ml), IL-12 (concentration 20ng / ml), IL-15 (concentration 5ng / ml), IL-18 (concentration 10ng / ml), and IL-21 (concentration 50ng / ml) were added at the same time. At the same time, dasatinib (concentration 50ng / ml) was added to the experimental group. The shake flask was placed in a shaker and dynamic culture was performed at 45rpm.

[0182] (7) Thereafter, culture medium and IL-2 (concentration 200 IU / ml), IL-12 (concentration 20 ng / ml), IL-15 (concentration 5 ng / ml), IL-18 (concentration 10 ng / ml), and IL-21 (concentration 50 ng / ml) were added every 1 to 3 days, and the cells were diluted to 2 × 10 5 ~1×10 6 Similarly, dasatinib (concentration 50 ng / ml) was added to the experimental group. This operation was repeated and iNK was cultured until day 12.

[0183] During the culture process, the cells were counted and the expansion times were calculated. Figure 14-15 The results showed that adding dasatinib during the entire cell culture process would lead to a decrease in the proliferation rate of NK cells, which is not conducive to the culture of NK cells.

[0184] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A method for culturing NK cells under high density conditions, characterized in that: Includes steps: A. Cultivating NK cells in a culture system, wherein the culture system does not contain dasatinib, and the culture comprises: (1) Add activation factors, culture medium and starting NK cells to the first culture container and perform activation culture for 4 to 6 days; (2) Placing the activated cultured NK cells into a second culture container, adding culture medium, and performing amplification culture for 1 to 5 days until the density of NK cells in the culture system reaches 4×10 6 / mL~1.5×10 7 Pieces / mL; B. Add dasatinib at a final concentration of 100-1000 nM to the culture system, continue culturing for 2-4 days, and terminate the cell culture; C. Harvesting of cultured NK cells.

2. The method according to claim 1, characterized in that In step B, dasatinib is added to the culture system at a final concentration of 100-500 nM.

3. The method according to claim 1, characterized in that In step A, the density of NK cells in the culture system reaches 4×10 6 / mL~1.2×10 7 Pieces / mL.

4. The method according to claim 1, characterized in that The cultured NK cells harvested in step C are cryopreserved, and after recovery, the killing ability of the NK cells against tumor cells is significantly better than that of the NK cells obtained by culturing without adding dasatinib.

5. The method according to claim 1, characterized in that In step A, the first culture container is coated with an activation factor.

6. The method according to claim 5, characterized in that The activating factor is selected from the group consisting of antibodies to the following proteins: Fc receptor CD16, NKp30, NKp46, and NKG2D.

7. The method according to claim 1, characterized in that The culture solution comprises the following components: NK cell basal culture medium and cytokine combination.

8. The method according to claim 7, characterized in that The cytokine combination is IL-2, IL-12, IL-15, IL-18 and IL-21.

9. The method according to claim 8, characterized in that The concentrations of the cytokines in the culture medium are: IL-2 concentration is 100IU / ml~2000IU / ml, IL-12 concentration is 5ng / ml~100ng / ml, IL-15 concentration is 5ng / ml~100ng / ml, IL-18 concentration is 5ng / ml~100ng / ml, and IL-21 concentration is 10ng / ml~200ng / ml.

10. The method according to claim 1, characterized in that The NK cells are NK cells differentiated from iPSCs.

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