Medium, composition and culture method for human NK cells

Abstract

The present invention relates to the field of biotechnology, and particularly to a culture medium, a composition and a culture method for human NK cells. Through optimization, the present invention provides a culture medium, a composition and a culture method for NK cell culture. The culture medium, the composition and the culture method are used for NK cell culture, with clear culture components, simple culture steps, low cost, good amplification multiple of the cultured NK cells and high cell purity. At the same time, the cultured NK cells have good tumor cell killing ability and have good market promotion value.

Description

Technical Field

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

[0002] Currently, there are mainly two NK cell culture processes: the feeder cell process and the non-feeder cell process. Among them, the feeder cell process has a significant effect on the activation and amplification of NK cells. However, since the feeder cells are irradiated tumor cells, there is a risk of tumor cell residue. In addition, the feeder cells require the construction, banking and cell bank verification of engineered cell lines, resulting in high costs.

[0003] The non-feeder cell process uses some cytokines or cytokine combinations to stimulate PBMC or purified NK cells in vitro for NK cell culture. However, the NK cells obtained by this method generally have low purity and low amplification multiples, making it difficult to meet clinical requirements. Moreover, the components of some non-feeder cell process culture media are complex and not well-defined.

[0004] Therefore, it is particularly necessary to develop a non-feeder cell process with a new culture medium having well-defined components, good amplification multiples and high cell purity, which can be used for large-scale preparation of NK cells. Summary of the Invention

[0005] In view of this, the technical problem to be solved by the present invention is to provide a culture medium, a composition and a culture method for culturing human NK cells.

[0006] The present invention provides a culture medium, which comprises: a basal medium, a culture medium additive, cytokines and / or serum or its analogues;

[0007] The basal medium is selected from at least one of IMDM, DMEM, RPM1640, a medium obtained by mixing DMEM and F12 in a volume ratio of 1:1 (DMEM / F12 (1:1)), a medium obtained by mixing IMDM and DMEM in a volume ratio of 1:1 (IMDM + DMEM (1:1)), a medium obtained by mixing IMDM and RPM1640 in a volume ratio of 1:1 (IMDM + RPM1640 (1:1)), or a medium obtained by mixing IMDM, DMEM and RPM1640 in a volume ratio of 1:1:1 (IMDM + DMEM + RPM1640 (1:1:1));

[0008] The culture medium additive is selected from ITS-A, lipid mixture and / or nicotinamide;

[0009] The cytokines include IL-2, IL-15, IL-18 and / or IL-21;

[0010] The serum or its analogs include, but are not limited to, animal serum, human serum, serum substitutes of animal origin, synthetic serum substitutes, serum-free media, human inactivated autologous plasma, and / or MCE serum substitutes, etc.; the animal serum includes: fetal bovine serum (FBS), newborn bovine serum (NBS), horse serum, or porcine serum, etc.; the human serum includes: human AB serum and / or human platelet lysate (PL), etc.; the serum substitutes of plant origin include: plant protein-based substitutes; the synthetic serum substitutes include: EvaCell® (suitable for the culture of cells such as mesenchymal stem cells (MSC), with defined composition and no animal origin), Ultroser™ G (semi-synthetic composition, low protein content, suitable for multiple cell types, can replace fetal bovine serum), and / or Cell-Ess® (synthetic substitute for cell culture), etc.; the serum-free media includes: CTS™ Immune Cell Serum Substitute, etc. (with defined composition, no xenogeneic components, suitable for the in vitro expansion of immune cells).

[0011] In specific embodiments of the present invention,

[0012] The basal medium is preferably IMDM;

[0013] The medium additives are preferably ITS-A and / or nicotinamide;

[0014] The cytokines include IL-2, IL-15, IL-18, and IL-21;

[0015] For different usage scenarios, the present invention uses fetal bovine serum (FBS), human inactivated autologous plasma, or human AB serum on the basis of the above-mentioned medium components; specifically, using fetal bovine serum is suitable for laboratory research environments; using human inactivated autologous plasma is suitable for clinical human reinfusion applications; using human AB serum is suitable for clinical human reinfusion applications;

[0016] According to different usage scenarios, the medium of the present invention includes:

[0017] IMDM, ITS-A, nicotinamide, IL-2, IL-15, IL-18, IL-21, and fetal bovine serum; and / or

[0018] IMDM, ITS-A, nicotinamide, IL-2, IL-15, IL-18, IL-21, and human inactivated autologous plasma; and / or

[0019] IMDM, ITS-A, nicotinamide, IL-2, IL-15, IL-18, IL-21, and human AB serum.

[0020] Furthermore, the medium of the present invention includes:

[0021] IMDM, 0.5 vt% - 1.5 vt% ITS-A, 3 - 7 mM nicotinamide, 200 IU / mL - 2000 IU / mL IL-2, 5 - 50 ng / mL IL-15, 1 - 40 ng / mL IL-18, 5 - 20 ng / mL IL-21, and 5 vt% - 10 vt% fetal bovine serum; and / or

[0022] IMDM, 0.5 vt% - 1.5 vt% ITS-A, 3 - 7 mM nicotinamide, 200 IU / mL - 2000 IU / mL IL-2, 5 - 50 ng / mL IL-15, 1 - 40 ng / mL IL-18, 5 - 20 ng / mL IL-21, and 0.5 vt% - 10 vt% heat-inactivated autologous human plasma; and / or

[0023] IMDM, 0.5 vt% - 1.5 vt% ITS-A, 3 - 7 mM nicotinamide, 200 IU / mL - 2000 IU / mL IL-2, 5 - 50 ng / mL IL-15, 1 - 40 ng / mL IL-18, 5 - 20 ng / mL IL-21, and 0.5 vt% - 5 vt% human AB serum.

[0024] Specifically, in the specific embodiments of the present invention, when the culture medium is as follows, the NK cells have a high expansion multiple, good NK cell purity, and strong NK cell killing ability:

[0025] IMDM, 1 vt% ITS-A, 5 mM nicotinamide, 500 IU / mL IL-2, 10 ng / mL IL-15, 20 ng / mL IL-18, 15 ng / mL IL-21, and 10 vt% fetal bovine serum; and / or

[0026] IMDM, 1 vt% ITS-A, 5 mM nicotinamide, 500 IU / mL IL-2, 10 ng / mL IL-15, 20 ng / mL IL-18, 15 ng / mL IL-21, and 5 vt% heat-inactivated autologous human plasma; and / or

[0027] IMDM, 1 vt% ITS-A, 5 mM nicotinamide, 500 IU / mL IL-2, 10 ng / mL IL-15, 20 ng / mL IL-18, 15 ng / mL IL-21, and 2.5 vt% human AB serum.

[0028] In the present invention, the ITS-A is obtained by purchase. Specifically, the ITS-A is purchased from Shanghai Yuanpei, product number #S451J7;

[0029] The present invention provides a composition, which comprises an activating antibody and the culture medium of the present invention;

[0030] The activating antibodies include CD16 antibody, NKG2D antibody, and / or 4-1BB antibody; experiments in the present invention show that better effects are achieved when the activating antibodies are CD16 antibody and NKG2D antibody; in the present invention, the concentration of the CD16 antibody is 0.5 μg / mL to 15 μg / mL, and the concentration of the NKG2D antibody is 0.5 μg / mL to 15 μg / mL; further, the concentration of the CD16 antibody is 3 μg / mL to 7 μg / mL; the concentration of the NKG2D antibody is 3 μg / mL to 7 μg / mL; the concentration of the NKG2D antibody is 3 μg / mL to 7 μg / mL; in specific embodiments of the present invention, the concentration of the CD16 antibody is 5 μg / mL; the concentration of the NKG2D antibody is 5 μg / mL; specifically, the CD16 antibody is anti-human CD16 antibody, the NKG2D antibody is anti-human NKG2D antibody, and the 4-1BB antibody is anti-human 4-1BB antibody;

[0031] Further, the composition of the present invention further includes at least one of a CD3 positive cell removal reagent and / or a supplemented medium;

[0032] The supplemented medium includes IMDM, ITS-A, nicotinamide, IL-2, IL-15, IL-18, and serum or serum analog;

[0033] The serum or serum analog is selected from fetal bovine serum FBS, human inactivated autologous plasma, and / or human AB serum;

[0034] Further, the supplemented medium includes:

[0035] IMDM, 0.5 vt% to 1.5 vt% ITS-A, 3 to 7 mM nicotinamide, 200 IU / mL to 2000 IU / mL IL-2, 5 to 50 ng / mL IL-15, 1 to 40 ng / mL IL-18, and 5 vt% to 10 vt% fetal bovine serum; and / or

[0036] IMDM, 0.5 vt% to 1.5 vt% ITS-A, 3 to 7 mM nicotinamide, 200 IU / mL to 2000 IU / mL IL-2, 5 to 50 ng / mL IL-15, 1 to 40 ng / mL IL-18, and 0.5 vt% to 10 vt% human inactivated autologous plasma; and / or

[0037] IMDM, 0.5 vt% - 1.5 vt% ITS-A, 3 - 7 mM nicotinamide, 200 IU / mL - 2000 IU / mL IL-2, 5 - 50 ng / mL IL-15, 1 - 40 ng / mL IL-18, and 0.5 vt% - 5 vt% human AB serum.

[0038] Furthermore, when the supplemented medium is as follows, the NK cells have a good expansion multiple and high cell purity:

[0039] IMDM, 1 vt% ITS-A, 5 mM nicotinamide, 500 IU / mL IL-2, 10 ng / mL IL-15, 20 ng / mL IL-18, and 10 vt% fetal bovine serum; and / or

[0040] IMDM, 1 vt% ITS-A, 5 mM nicotinamide, 500 IU / mL IL-2, 10 ng / mL IL-15, 20 ng / mL IL-18, and 5 vt% heat-inactivated autologous plasma; and / or

[0041] IMDM, 1 vt% ITS-A, 5 mM nicotinamide, 500 IU / mL IL-2, 10 ng / mL IL-15, 20 ng / mL IL-18, and 2.5 vt% human AB serum.

[0042] The CD3-positive cell depletion reagent includes an antibody mixture reagent and / or sorting magnetic beads;

[0043] The antibody mixture reagent includes CD3 Depletion Cocktail; in some specific embodiments of the present invention, Stemcell's RosetteSep™ Human CD3 Depletion Cocktail is used for the depletion of CD3-positive cells;

[0044] The sorting magnetic beads include positive sorting magnetic beads and / or negative sorting magnetic beads; in some embodiments of the present invention, Dynabeads CD3 / CD28 sorting magnetic beads, CytoSinct™ CD3 Nanobead sorting magnetic beads and Human CD3Positive Selection Kit matching sorting magnetic beads are selected for sorting, and the results show that the selection of Dynabeads CD3 / CD28 has poor effect, and the sorting effect of other magnetic beads is equivalent to that of antibody mixed reagent sorting. However, RosetteSep™ HumanCD3 Depletion Cocktail is suitable for starting with peripheral blood or single blood collection or cord blood as raw materials, and is more suitable for large-scale preparation of NK cells, so RosetteSep™ Human CD3 Depletion Cocktail is preferred.

[0045] The present invention provides the use of at least one of the following I) to II) in NK cell culture:

[0046] 1), the culture medium of the present invention;

[0047] II), the composition of the present invention.

[0048] The present invention provides a method for culturing genetically unmodified and / or genetically modified NK cells, which comprises using at least one of the following i) to ii):

[0049] i), the culture medium of the present invention;

[0050] ii), the composition of the present invention.

[0051] Furthermore, the culture method of the present invention comprises the following steps:

[0052] Step 1, coating a culture container with the activated antibody in the composition of the present invention;

[0053] Step 2, the PBMC cells are treated with the CD3 positive cell removal reagent in the composition of the present invention and then inoculated into the activation antibody coated culture container of step 1, and then cultured using the culture medium of the present invention;

[0054] Step 3: If the culture medium turns yellow, replenish the liquid using the supplementary culture medium in the composition of the present invention.

[0055] Further, in the culture method, the coating time is 1 to 3 days; in the specific embodiments of the present invention, PBMCs with CD3-positive cells removed are inoculated 1 day after the culture container is coated with the activating antibody; that is, the coating time is 1 day; those skilled in the art can adjust the length of the coating time according to actual needs, and the present invention does not make any limitation in this regard;

[0056] The culture medium for the fluid replacement is the supplementary culture medium in the composition of the present invention; the fluid replacement includes primary fluid replacement and subsequent fluid replacement; in the primary fluid replacement, the initial added volume of the supplementary culture medium is 1 / 2 of the volume of the culture medium of the present invention during the culture in step 2; in the subsequent fluid replacement, the added volume of the supplementary culture medium is 1 / 3 of the total culture medium volume after the primary fluid replacement;

[0057] In the present invention, the primary fluid replacement occurs on the third day after the culture in step 2, and the subsequent fluid replacement is carried out every two days; during the actual test process, those skilled in the art can adjust the fluid replacement time and the number of fluid replacement times according to the growth status of the cells or the situation of the culture medium, and the present invention does not make any limitation in this regard.

[0058] In the present invention, the difference between the supplementary culture medium and the culture medium is that the supplementary culture medium does not contain IL-21; the test results of the present invention show that when only IL-21 is added to the culture medium and IL-21 is not added to the supplementary culture medium for subsequent fluid replacement, the amplification multiple of the cultured NK cells is better and the cell purity is higher.

[0059] The unmodified and / or genetically modified NK cells of the present invention are a type of cells with the functional characteristics of NK cells; the genetically modified NK cells include but are not limited to CAR-NK cells, and the present invention does not make any limitation in this regard; the unmodified NK cells corresponding to the genetically modified ones are natural NK cells.

[0060] The present invention provides NK cells obtained by culturing using the culture method described above.

[0061] Further, the present invention provides the use of the NK cells in the preparation of anti-tumor drugs.

[0062] The tumors include but are not limited to lung cancer, colorectal cancer, liver cancer, and / or pancreatic cancer, etc., and the present invention does not make any limitation in this regard.

[0063] The present invention provides anti-tumor drugs, the raw materials of which include the NK cells of the present invention.

[0064] The present invention provides a culture medium, a culture composition and a culture method for culturing NK cells; the culture medium and the composition used for culture in the present invention are obtained through multiple optimizations; specifically, the present invention screens and optimizes the types of basal media, culture medium additives and cytokines in the culture medium, and obtains a culture medium with the highest NK cell amplification multiple and the best cell purity; on this basis, through the screening of methods for removing CD3-positive cells, a culture composition more suitable for large-scale culture of NK cells starting from peripheral blood, apheresis blood or cord blood as raw materials while ensuring relatively high cell purity is obtained; secondly, through the optimization of the addition timing of IL-21, the amplification multiple and cell purity of NK cells after culture are further improved; therefore, the various parameters in the culture medium, the culture composition and the culture method of the present invention interact with each other, cooperate with each other, and jointly affect the culture effect of NK cells.

[0065] The culture medium and composition described in the present invention have clear components, avoiding the risks of tumor cell contamination and random insertion of tumor genes in the trophoblast culture process; compared with non-trophoblast culture media and processes, the large-scale amplification and purity of NK cells in the culture medium, composition and culture method of the present invention are significantly improved; in addition, the culture medium described in the present invention has clear and simple components, is suitable for the culture preparation and large-scale amplification of NK cells or CAR-NK cells, and also has good NK cell culture effects in different usage scenarios.

[0066] Furthermore, the culture medium and composition described in the present invention are also applicable to the trophoblast culture process and can be used in combination with other parameters of the trophoblast culture process for culturing NK cells.

[0067] Through optimization, the present invention provides a culture medium, a composition and a culture method for culturing NK cells. The culture medium, the composition and the culture method are used for culturing NK cells, with clear culture components, simple culture steps, low cost, good NK cell amplification multiple and high cell purity. In the culture of NK cells in the scenario of using fetal bovine serum (FBS) as the culture medium, the amplification multiple on Day 19 is 7221.26 times, and the CD3 - CD56 + % NK cells is 97.84%; at the same time, the cultured NK cells have good tumor cell killing ability and have good market promotion value. BRIEF DESCRIPTION OF THE DRAWINGS

[0068] Figure 1 Shows the cell culture process of the present invention;

[0069] Figure 2 Shows the killing efficiency of the NK cells prepared by the present invention at different effector-to-target ratios;

[0070] Figure 3Show the expression of natural killer receptors of NK cells prepared by the present invention;

[0071] Figure 4 Show the expression of chemokine receptors of NK cells prepared by the present invention. Detailed implementation manners

[0072] The present invention provides a culture medium, a composition and a culture method for culturing human NK cells. Those skilled in the art can draw on the content of this article and appropriately modify the process parameters to achieve. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments. Those skilled in the art can obviously make changes or appropriate changes and combinations to the methods and applications in this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0073] Explanation of related terms and source of materials:

[0074] Ficoll: A polysucrose-diatrizoate cell separation solution, composed of sucrose polymer (Ficoll) and diatrizoate (urografin). The Ficoll described in the present invention is purchased from GE Company, and the product number is 17-1440-03.

[0075] Cocktail is a mixture of specific antibodies used for the removal of CD3-positive cells; in some specific embodiments of the present invention, the sorting product RosetteSep™ Human CD3 Depletion Cocktail from STEMCELL Technologies is used for the removal of CD3-positive cells;

[0076] NK: Natural killer cell

[0077] CAR: Chimeric antigen receptor;

[0078] CAR-NK: NK cells with chimeric antigen receptors;

[0079] IL-2: Interleukin 2;

[0080] IL-15: Interleukin 15;

[0081] IL-18: Interleukin 18;

[0082] IL-21: Interleukin 21;

[0083] PBMC: Human peripheral blood mononuclear cells;

[0084] DPBS: Phosphate buffered saline without calcium and magnesium ions;

[0085] HSA: Human Serum Albumin;

[0086] HPBS: DPBS solution containing 1 vt% HSA

[0087] anti-human CD16: anti-human CD16 antibody;

[0088] anti-human NKG2D: anti-human NKG2D antibody

[0089] anti-human 4-1BB: anti-human 4-1BB antibody;

[0090] Activating receptors such as CD16, NKG2D, NKp30, NKp44, and NKp46 play a key role in the natural tumor killing of NK cells, and the clearance of virus-infected cells and senescent cells, mainly promoting the rapid activation response of NK cells.

[0091] The low purity of NK cells means that the content of other miscellaneous cells is high, such as T cells. T cells are specific cells, and if transfused back into the human body, it is easy to produce GVHD reactions;

[0092] The low expansion multiple of the NK cells means that the number of NK cells in the final product is small, and a small number cannot achieve the purpose of treating or killing target cells, and even extending the culture time cannot reach the number required clinically.

[0093] The test materials used in the present invention are all ordinary commercially available products and can be purchased on the market. The present invention will be further described below in conjunction with the examples:

[0094] Example 1 Screening of methods for removing CD3-positive cells

[0095] I. Treatment of PBMC cells

[0096] 1. Isolation of PBMC from apheresis blood

[0097] Peripheral blood from healthy individuals was used. After blood collection, the blood was diluted with physiological saline at a ratio of 1:1, and then the diluted blood was added to an equal volume of Ficoll and centrifuged at 800 g at 20 °C for 30 min. After centrifugation, most of the diluted plasma and platelets were aspirated and discarded, and the buffy coat cells were aspirated and transferred to a new centrifuge tube. DPBS was added for washing, and centrifuged at 500 g for 15 minutes, and the supernatant was discarded. This was repeated twice. According to the actual experimental requirements, the washed cells were added to the corresponding medium, pipetted and mixed well, and counted by AO / PI for the next experiment.

[0098] 2. Resuscitation of cryopreserved PBMC

[0099] Take a 50 mL centrifuge tube, add 25 mL of blank medium, and label it with the number corresponding to the PBMC to be revived. Turn on the water bath and set the temperature to 37 °C. After the temperature of the water bath stabilizes at 37 °C, take out the PBMC stored in the liquid nitrogen tank, quickly transfer it to the clean area, put it into the 37 °C water bath for revival. The transfer time should be less than 1 min. When reviving the cells, the water level in the water bath should be below the cryopreservation tube. Keep shaking the cryopreservation tube to accelerate cell revival. The revival ends when there are ice cubes the size of mung beans in the cryopreservation tube. Take out the cryopreservation tube from the water bath and wipe off the excess water on the tube wall with a dust-free cloth. The entire revival time is 3 - 5 min. Wipe the surface of the cryopreservation tube with 75% (v / v) disinfected alcohol, and transfer the cryopreservation tube of revived PBMC into the biosafety cabinet. Aspirate the PBMC in the cryopreservation tube into the cryopreservation tube labeled with the corresponding number of PBMC, centrifuge at 500 g for 10 min, discard the supernatant, resuspend with the corresponding medium according to the actual experimental requirements, pipette and mix well, and perform AO / PI counting. Specifically, the corresponding medium can be the medium in the screening of the following method for removing CD3-positive cells.

[0100] II. Screening of Methods for Removing CD3-Positive Cells

[0101] To ensure the stable production of high-purity NK cells, a process for removing CD3-positive cells was introduced before cell culture expansion, and four CD3-positive cell removal reagents were screened. Among them, RosetteSep™ Human CD3 Depletion Cocktail removes T cells by coupling CD3-positive cells and red blood cells, while Dynabeads CD3 / CD28, CytoSinct™ CD3 Nanobeads, and Human CD3 Positive Selection Kit use magnetic beads to bind CD3-positive cells in PBMC and remove T cells by magnetic sorting.

[0102] 1. Removal of CD3-Positive Cells with RosetteSep™ Human CD3 Depletion Cocktail

[0103] The specific operation is as follows: After blood collection, dilute the blood with physiological saline at a ratio of 1:1, add Cocktail to the diluted blood (to make its concentration 50 μL / mL), and incubate at room temperature for 20 minutes; then add the incubated product to Ficoll with the same volume as the diluted blood, and centrifuge at 800 g and 20 °C for 30 min. After centrifugation, aspirate and discard most of the diluted plasma and platelets, and aspirate the buffy coat cells and transfer them to a new centrifuge tube. Add HPBS for washing, centrifuge at 500 g for 15 minutes, and discard the supernatant. Repeat twice. Resuspend the cells with HPBS, pipette and mix well, perform AO / PI counting, and then proceed to the next experimental operation.

[0104] 2. Dynabeads CD3 / CD28

[0105] Take the required PBMC cells (here, to keep the experimental conditions as consistent as possible, use the PBMC that has not been cryopreserved and has undergone the AO / PI counting step of "1. Single blood collection and separation of PBMC"; in specific experiments, cryopreserved and resuscitated PBMC can also be used), centrifuge at 500g for 10 min, discard the supernatant, and resuspend with 3 - 5 mL of DPBS. Magnetic bead washing: Calculate the required magnetic bead volume according to the magnetic bead instruction manual and the CD3 + T cell content in PBMC: Magnetic bead volume = number of cells × (CD3 + T)% × magnetic bead - cell incubation ratio ÷ 4 × 10 7 . In a biosafety cabinet, take a 15 - mL centrifuge tube, add an appropriate amount of HPBS, gently pipette and mix the magnetic beads, aspirate the required volume of magnetic beads into a cryotube containing HPBS, mix well, place it on a magnetic stand, let it stand for 2 min, then aspirate and discard the supernatant (note not to touch the tube wall), remove the cryotube from the magnetic stand, add 1 - 5 mL of DPBS, and mix well. Magnetic bead - cell co - incubation: Add the mixed cell suspension to the washed magnetic bead cryotube and mix well (the cell density is 1×10 7 ~10×10 8 cells / mL), place it in a four - dimensional mixer, and incubate with shaking at room temperature for 30 min. Then remove the cryotube containing the cell and magnetic bead mixture from the four - dimensional mixer, immediately insert the cryotube into the magnetic stand, let it stand for 2 min, aspirate the supernatant (note not to touch the cryotube) and transfer it to a new centrifuge tube. Add a certain amount of HPBS to the centrifuge tube containing the supernatant, insert the tube into the magnetic stand, after timing for 2 min, aspirate the supernatant (note not to touch the cryotube) and transfer it to a new centrifuge tube. Repeat twice. Take the cell suspension, dilute it, pipette and mix well, and perform AO / PI counting.

[0106] 3. CytoSinct™ CD3 Nanobead

[0107] Take the required number of PBMC cells (here, to keep the experimental conditions as consistent as possible, use the PBMC that has not been cryopreserved and has undergone the AO / PI counting step of "1. Single blood collection and separation of PBMC"; in specific experiments, cryopreserved and resuscitated PBMC can also be used), centrifuge at 500g for 10 min, discard the supernatant, and resuspend with HPBS to 1×10 8 / mL. Add 100 μl / mL of CytoSinct™ CD3 Nanobeads (GenScript, #L00896) to the cells, and incubate at room temperature for 30 min. Supplement HPBS to 25 mL, gently pipette 2 or 3 times with a pipette. Centrifuge at 500g, with an acceleration of 8 and a deceleration of 6 for 10 min, discard the supernatant, and resuspend with HPBS to approximately 3×10 7 / mL. Conditioning the sorting column: Rinse the CytoSinctTM gL column (GenScript, #D00008) with 3 mL of HPBS. Loading the sample: Transfer the sample bound to magnetic beads to the CytoSinctTM gL column using a pipette and collect the unlabeled cells. Washing: Wash the CytoSinctTM gL column with 3 mL of Buffer and collect the unlabeled cells. Take the cell suspension, dilute it, pipette to mix well, and count using AO / PI.

[0108] 4. Human CD3 Positive Selection Kit

[0109] Take the required number of PBMC cells (here, to keep the experimental conditions as consistent as possible, use PBMC that has not been cryopreserved and has undergone the AO / PI counting step in "1. Isolate PBMC by apheresis"; in specific experiments, cryopreserved and thawed PBMC can also be used), centrifuge at 500 g for 10 min, discard the supernatant, and resuspend in HPBS to a concentration of 1×10 8 / mL. Add 100 μl / mL of Selection Cocktail to the cells and incubate at room temperature for 3 min. Vortex the magnetic beads for 30 s. Add 100 μl / mL of Releasable Rapid Spheres magnetic beads to the sample and incubate at room temperature for 3 min. Add HPBS to make a final volume of 2.5 mL and gently pipette 2 or 3 times. Place on a magnet and let stand for 5 min; pick up the magnet and pour the supernatant into a new tube. Repeat three times. Take the cell suspension, dilute it, pipette to mix well, and count using AO / PI.

[0110] The specific grouping of CD3-positive cell depletion is shown in Table 1:

[0111] Table 1. Experimental grouping

[0112]

[0113] III. Experimental results

[0114] Take the cells treated by the above four methods and perform flow cytometry analysis. The specific operation method is as follows: Take 1×10 6Cells were centrifuged to remove the supernatant, washed once with FACS buffer (DPBS containing 1% v / v FBS), and then 1 μL of CD3 antibody (FITC anti-human CD3, Biolegend, #317306) and CD56 antibody (PE anti-human CD56, Biolegend, #362524) were added to each sample. After mixing, the samples were stained at 2 - 8°C for 20 min. After staining, the samples were washed once with FACS buffer, resuspended in 200 μL of FACS buffer, and then subjected to flow cytometry analysis. The yields of NK cells and the depletion rates of T cells were analyzed, and the results are as follows:

[0115] Table 2. CD3 + Results of T cell depletion

[0116]

[0117] From the experimental results, the T cell depletion efficiencies of G1, G3, and G4 were relatively high. Considering the yields of NK cells, the yield of NK cells in the G1 group was the highest. Moreover, the RosetteSep™ Human CD3 Depletion Cocktail in the G1 group was suitable for starting with peripheral blood, apheresis blood, or cord blood as raw materials and was more suitable for the large-scale preparation process of NK cells. Therefore, the magnetic separation reagent for CD3-positive T cell (CD3 + T cell) depletion was selected as RosetteSep™ Human CD3 Depletion Cocktail.

[0118] Example 2. Study on activating antibodies

[0119] According to literature research, the activation and expansion of NK cells require multiple external signal stimulations, including activating receptor and cytokine signals. In this invention, three activating receptors, CD16, NKG2D, and 4-1BB, were selected as targets for the study of the activating antibody addition process. The specific steps are as follows:

[0120] I. Treatment of PBMC cells and removal of CD3-positive cells

[0121] Peripheral blood from healthy donors was used to remove CD3-positive cells by the RosetteSep™ Human CD3 Depletion Cocktail method in Example 1 to obtain peripheral mononuclear cells depleted of CD3+T cells.

[0122] II. Antibody coating

[0123] One day before cell seeding, the activating antibodies were coated in 6-well plates or culture flasks. The specific operation is as follows:

[0124] Take anti-human CD16 antibody (Jiangwan protein, #GMP-A091), anti-human NKG2D antibody (Jiangwan protein, #GMP-A075) and anti-human 4-1BB antibody (Jiangwan protein, #GMP-A037), dilute to working concentration with DPBS according to the instructions, add to the 6-well plate, mix well, make the antibody solution evenly spread on the bottom of the well plate, and then place it at 2~8℃ overnight before use. The antibody concentration and experimental grouping are as follows:

[0125] Table 3. Optimization grouping of activation antibodies

[0126]

[0127] 3. Cell inoculation and culture

[0128] The peripheral mononuclear cells obtained in step 1 after CD3+T removal were inoculated into the 6-well plates coated with different activation antibodies in step 2 at a density of 2×10 6 cells / mL, the culture medium included: basal medium (NK serum-free medium NH01 (Suzhou Yikesai, #NH000-N012) and other supplementary components (10vt% FBS (Corning, #35-081-CV) + 200IU / mL IL-2 (Shuanglu Pharmaceutical, trade name #Xingjier)).

[0129] After 3 days of culture, observe the cells under a microscope. Usually, small, loose clones can be observed. If the culture medium begins to turn yellow, add 1 / 2 volume of the above culture medium. If the culture medium has not changed color and the cells grow less, add 1 / 3 volume of the above culture medium. During the whole process, avoid vigorous shaking of the culture dish, minimize disturbance of the cells, and place in a 5% carbon dioxide incubator for static culture. Replenish the liquid every two days starting from Day 5, and adjust the cell density to 1×10 6 The cells were cultured at 100 cells / mL and the cell proliferation in each group was observed by counting.

[0130] IV. Experimental Results

[0131] NK cell viability is shown in Table 4, NK cell expansion multiples are shown in Table 5, NK cell purity (CD3 - CD56 + %) as shown in Table 6:

[0132] Table 4. NK cell viability

[0133]

[0134] Table 5. NK cell expansion multiples

[0135]

[0136] Table 6. NK cell purity

[0137]

[0138] Considering the data of NK cell viability, amplification fold, and purity comprehensively, the NK cells in the group supplemented with anti-human CD16 had an advantage in proliferation, and the addition of anti-human NKG2D could further promote the amplification of NK cells. Therefore, anti-human CD16 (5 μg / mL) and anti-human NKG2D (5 μg / mL) were selected to co-activate NK cells.

[0139] Example 3 Screening of basal media

[0140] I. PBMC cell treatment and removal of CD3-positive cells

[0141] Peripheral blood from healthy donors was used, and CD3-positive cells were removed according to the RosetteSep™ Human CD3 Depletion Cocktail method in Example 1.

[0142] II. Antibody coating

[0143] The antibody coating method was the same as that in Example 2, and the specific antibody concentrations were anti-human CD16 (5 μg / mL) and anti-human NKG2D (5 μg / mL).

[0144] III. Cell seeding and culture

[0145] The cell seeding and culture method was the same as that in Example 2; in addition, the basal media in the culture medium were replaced respectively as shown in the groups in Table 7, and other added components were kept consistent. The other added components were 5% FBS (Corning, # 35-081-CV) + 200 IU / mL IL-2 (Shuanglu Pharmaceutical, trade name # Xing'er). The optimized groups of basal media in Table 7 are as follows:

[0146] Table 7. Grouping of culture medium composition

[0147]

[0148] IV. Results of basal media screening

[0149] The NK cell viability is shown in Table 8, the NK cell amplification fold is shown in Table 9, and the NK cell purity (CD3 - CD56 + %) is shown in Table 10:

[0150] Table 8. NK cell viability

[0151]

[0152] Table 9. NK cell expansion multiples

[0153]

[0154] Table 10. NK cell purity

[0155]

[0156] Based on the above experimental results, G13, i.e., IMDM medium, is significantly superior to other basic medium in terms of NK cell viability, cell proliferation, and purity. Therefore, IMDM was selected as the basic medium.

[0157] Example 4 Screening of culture medium additives

[0158] The normal proliferation of NK cells not only depends on the basic nutrients provided by the basal culture medium, but also requires the addition of additional ingredients. Different additional ingredients will result in different metabolism, activity and growth of NK cells. Therefore, a comparative study of these added ingredients was conducted.

[0159] 1. Removal of CD3-positive cells

[0160] PBMCs isolated from single blood samples were treated with RosetteSep™ Human CD3 Depletion Cocktail. The specific treatment steps are shown in Example 1.

[0161] 2. Antibody Coating

[0162] The antibody coating method was the same as in Example 2, with specific antibody concentrations of anti-human CD16 (5 μg / mL) and anti-human NKG2D (5 μg / mL).

[0163] 3. Cell inoculation and culture

[0164] The cell inoculation and culture method was the same as in Example 2; in addition, IMDM medium was used as the basic culture medium, and other supplementary components were added in groups as shown in Table 11, wherein the other supplementary components included common components of different groups (10% FBS (Corning, #35-081-CV) + 200 IU / mL IL-2 (Shuanglu Pharmaceutical, trade name #Xingjier) + 10 ng / mL IL-15 (Beirong Biotechnology, #GE07.1) and the additives shown in Table 11.

[0165] Table 11. Experimental groups of medium supplements

[0166]

[0167] The main components and concentration information of ITS-A (Shanghai Yuanpei, catalog number #S451J7) are as follows: 1.0 g / L recombinant human insulin, 550 mg / L recombinant human transferrin, 0.67 mg / L sodium selenite, 11.0 g / L sodium pyruvate; nicotinamide (Sigma, #N0636-100G); lipid mixture (Sigma, #L0288), and the specific components of the lipid mixture include 2 μg / mL arachidonic acid, 10 μg / mL (linoleic acid, linolenic acid, myristic acid, oleic acid, palmitic acid, and stearic acid), 0.22 mg / mL cholesterol, 2.2 mg / mL Tween 80, 70 μg / mL tocopheryl acetate, and 100 mg / mL Pluronic F-68.

[0168] IV. Results of additive screening

[0169] The viability of NK cells is shown in Table 12, the amplification fold of NK cells is shown in Table 13, and the purity of NK cells (CD3 - CD56 + )% is shown in Table 14:

[0170] Table 12. Viability of NK cells

[0171]

[0172] Table 13. Amplification fold of NK cells

[0173]

[0174] Table 14. Purity of NK cells

[0175]

[0176] In the table, "-" indicates that cell amplification stops and the experiment ends. Considering the data of NK cell viability, amplification, and purity, the G21 group has obvious advantages compared with other experimental groups, indicating that ITS-A and nicotinamide play important roles in the culture of NK cells. Compared with the G19, G21, G22, and G23 groups, and considering the G20 and G24 groups at the same time, the addition of ITS-A is essential for the survival of NK cells.

[0177] Example 5 IL-2 concentration study

[0178] To optimize the amplification effect of NK cells, the concentration of IL-2 was studied to determine the optimal addition concentration.

[0179] I. Removal of CD3-positive cells

[0180] The single blood collection separated PBMC was treated with RosetteSep™ Human CD3 Depletion Cocktail, and the specific treatment steps refer to Example 1.

[0181] II. Antibody Coating

[0182] The antibody coating method was the same as that in Example 2, and the specific antibody concentrations were anti-human CD16 (5 μg / mL) and anti-human NKG2D (5 μg / mL).

[0183] III. Cell Seeding and Culture

[0184] The cell seeding and culture method was the same as that in Example 2; in addition, the other components of the culture medium were kept the same except for the IL-2 concentration as shown in Table 15; the other components were: IMDM + 1 vt% ITS-A + 5 mM nicotinamide + 10 vt% FBS + 10 ng / mL IL-15, and their respective manufacturers and catalog numbers were the same as those in the previous examples; IL-2 was purchased from Shuanglu Pharmaceutical Co., Ltd., and the trade name was #Xingji'er. The specific grouping was as follows:

[0185] Table 15. Added Concentration of IL-2

[0186]

[0187] IV. Screening Results of IL-2 Concentration

[0188] Table 16. NK Cell Viability

[0189]

[0190] Table 17. NK Cell Expansion Fold

[0191]

[0192] Table 18. NK Cell Purity

[0193]

[0194] Based on the comprehensive data of NK cell viability, expansion fold, and purity, there was no difference in purity among the groups. When the IL-2 concentration was 500 IU / mL in Group G26, NK had a higher expansion fold. Therefore, 500 IU / mL of IL-2 was selected as the added concentration.

[0195] Example 6 IL-15 Concentration Study

[0196] In order to optimize the expansion effect of NK cells, the concentration of IL-15 was optimized to determine the optimal added concentration.

[0197] I. Removal of CD3-Positive Cells

[0198] The single blood collection separated PBMC was treated with RosetteSep™ Human CD3 Depletion Cocktail, and the specific treatment steps refer to Example 1.

[0199] II. Antibody Coating

[0200] The antibody coating method was the same as that in Example 2, and the specific antibody concentrations were anti-human CD16 (5 μg / mL) and anti-human NKG2D (5 μg / mL).

[0201] III. Cell Seeding and Culture

[0202] The cell seeding and culture method was the same as that in Example 2; in addition, the components of the culture medium were kept the same except for the IL-15 concentration as shown in Table 19; the other components were: IMDM + 1 vt% ITS-A + 5 mM nicotinamide + 10 vt% FBS + 500 IU / mL IL-2, and their respective manufacturers and catalog numbers were the same as those in the previous examples; IL-15 was purchased from Bairong Biotechnology, catalog number GE07.1, and the specific grouping was as follows:

[0203] Table 19. Added Concentration of IL-15

[0204]

[0205] IV. Screening Results of IL-15 Concentration

[0206] Table 20. Viability Rate of NK Cells

[0207]

[0208] Table 21. Expansion Fold of NK Cells

[0209]

[0210] Table 22. Purity of NK Cells

[0211]

[0212] Based on the comprehensive data of the viability rate, expansion fold, and purity of NK cells, there was no significant difference in purity among the groups. When the IL-15 concentration was 10 ng / mL in Group G31, NK cells had a higher expansion fold. Therefore, 10 ng / mL of IL-15 was selected as the added concentration.

[0213] Example 7 Study on IL-18 Concentration

[0214] In order to improve the activation and expansion effects of NK cells, IL-18 was added to the culture medium, and its concentration was optimized to determine the optimal added concentration.

[0215] I. Removal of CD3-positive cells

[0216] The PBMC separated by apheresis was treated with RosetteSep™ Human CD3 Depletion Cocktail. For the specific treatment steps, refer to Example 1.

[0217] II. Antibody coating

[0218] The antibody coating method was the same as that in Example 2. The specific antibody concentrations were anti-human CD16 (5 μg / mL) and anti-human NKG2D (5 μg / mL).

[0219] III. Cell seeding and culture

[0220] The cell seeding and culture method was the same as that in Example 2. In addition, the components of the culture medium were kept the same except for the IL-18 concentration as shown in Table 23. The other components were: IMDM + 1 vt% ITS-A + 5 mM nicotinamide + 10 vt% FBS + 500 IU / mL IL-2 + 10 ng / mL IL-15. The manufacturers and catalog numbers of each component were the same as those in the previous examples. IL-18 was purchased from Sino Biological Inc., catalog number LC17DE2813. The specific grouping was as follows:

[0221] Table 23. Added concentration of IL-18

[0222]

[0223] IV. Screening results of IL-18 concentration

[0224] Table 24. Viability of NK cells

[0225]

[0226] Table 25. Expansion fold of NK cells

[0227]

[0228] Table 26. Purity of NK cells

[0229]

[0230] Based on the comprehensive data of the viability, expansion fold, and purity of NK cells, there was no significant difference in purity among groups. When the IL-18 concentration was 20 ng / mL in Group G36, NK cells had a relatively high expansion fold, reaching 730.15 times, and maintained a relatively high cell viability. Therefore, 20 ng / mL of IL-18 was selected as the added concentration.

[0231] Example 8 Study on IL-21 concentration

[0232] To improve the activation and expansion of NK cells, IL-21 was added to the culture medium. Considering the cytotoxicity of long-term addition of IL-21, we compared the effects of adding on Day 0 and continuous addition, and determined the addition method. Then, we optimized the concentration study to determine the optimal addition concentration.

[0233] I. Removal of CD3-positive cells

[0234] PBMC separated from apheresis blood was treated with RosetteSep™ Human CD3 Depletion Cocktail, and the specific treatment steps refer to Example 1.

[0235] II. Antibody coating

[0236] The antibody coating method was the same as that in Example 2, and the specific antibody concentrations were anti-human CD16 (5 μg / mL) and anti-human NKG2D (5 μg / mL).

[0237] III. Cell seeding and culture

[0238] The cell seeding and culture method was the same as that in Example 2; in addition, the other components of the culture medium were kept the same except for the frequency at a fixed concentration or the IL-21 concentration as shown in Tables 27 and 28; the other components were: IMDM + 1 vt% ITS-A + 5 mM nicotinamide + 10 vt% FBS + 500 IU / mL IL-2 + 10 ng / mL IL-15 + 20 ng / mL IL-18, and their respective manufacturers and catalog numbers were the same as those in the previous examples; IL-21 was purchased from Sino Biological Inc., catalog number 10584-HNAE, and the specific grouping was as follows:

[0239] Table 27. Comparison of IL-21 addition frequencies

[0240]

[0241] Table 28. IL-21 addition concentration

[0242]

[0243] IV. Research results of IL-21

[0244] 1. Research results of comparison of IL-21 addition frequencies

[0245] Table 29. Viability of NK cells

[0246]

[0247] Table 30. Expansion fold of NK cells

[0248]

[0249] Table 31. NK cell purity

[0250]

[0251] In the table, "-" indicates that the cells did not expand and the experiment was stopped. Considering the comprehensive data of NK cell viability, amplification multiple, and purity, adding IL-21 only on Day 0 has significant advantages in NK cell amplification, viability, and purity, especially in viability and amplification. The viability of cells with continuous addition of IL-21 continuously decreases, showing negative proliferation. It is speculated that the continuous presence of IL-21 in the culture medium causes toxicity to the cells. Therefore, IL-21 was added once on Day 0.

[0252] 2. Research results of IL-21 concentration

[0253] Table 32. NK cell viability

[0254]

[0255] Table 33. NK cell amplification multiple

[0256]

[0257] Table 34. NK cell purity

[0258]

[0259] Considering the comprehensive data of NK cell viability, amplification multiple, and purity, there is no obvious difference in purity among groups. When the IL-21 concentration is 15 ng / mL in Group G42, NK has a relatively high amplification multiple, reaching 403.16 times, and maintains a relatively high cell viability. Therefore, 15 ng / mL of IL-21 was selected as the addition concentration.

[0260] Based on the comprehensive research results of the addition frequency and addition concentration of IL-21, it was finally determined that IL-21 was added once on Day 0 at a concentration of 15 ng / mL.

[0261] Example 9 Verification of NK culture medium and culture process

[0262] Based on the results of the previous studies on NK basal medium, medium additives, activating antibodies, cytokines, etc., the basal medium for NK was determined to be IMDM, the medium additives were 1vt% ITS-A and 5 mM nicotinamide, the activating antibodies were anti-human CD16 (5 μg / mL) and anti-human NKG2D (5 μg / mL), and the cytokines were IL-2 (500 IU / mL), IL-15 (10 ng / mL), IL-18 (20 ng / mL), and IL-21 (15 ng / mL). Depending on the application scenario and experimental purpose, the additives were replaced. The added serum could be fetal bovine serum FBS, human autologous plasma, or other serum analogs (such as human AB serum). Therefore, according to different application scenarios and experimental purposes, the above NK medium and culture process were verified under fetal bovine serum, human autologous plasma, and human AB serum conditions, and their amplification effects were observed.

[0263] The culture process flow chart is as Figure 1 shown. The specific operation steps are as follows:

[0264] I. PBMC cell treatment and removal of CD3-positive cells

[0265] Peripheral blood from healthy individuals was used, and CD3-positive cells were removed according to the RosetteSep™ Human CD3 Depletion Cocktail method in Example 1 to obtain peripheral mononuclear cells depleted of CD3+T.

[0266] II. Antibody coating

[0267] One day before cell seeding, i.e., Day-1, the activating antibodies were coated in a 6-well plate or culture flask. The specific operation was as follows:

[0268] Anti-human CD16 antibody (5 μg / mL) and anti-human NKG2D antibody (5 μg / mL) were separately taken and diluted to the working concentration with DPBS according to the instructions, added to the 6-well plate or culture flask, mixed well to make the antibody solution evenly cover the bottom of the well plate, and then placed at 2-8 °C overnight for use.

[0269] III. Cell seeding and culture

[0270] On Day0, the peripheral mononuclear cells depleted of CD3+T obtained in Step I were seeded into the 6-well plate coated with the activating antibody in Step II, and the seeding density was 2×10 6cells / mL. The culture medium used contains: IMDM, 1vt% ITS-A, 5 mM nicotinamide, 500 IU / mL IL-2, 10 ng / mL IL-15, 20 ng / mL IL-18, 15 ng / mL IL-21 (where IL-21 is added only once on Day 0), and serum; the serum is fetal bovine serum FBS or human inactivated autologous plasma or other serum analogues such as human AB serum.

[0271] After 3 days of culture, the cells are observed under a microscope, and usually small, loose clone clusters can be observed. If the culture medium starts to turn yellow, add 1 / 2 volume of the above-mentioned culture medium without IL-21 and with other components as above. If the culture medium has not changed color and the cell growth is less, add 1 / 3 volume of the above-mentioned culture medium without IL-21 and with other components as above. During the whole process, violent shaking of the culture dish should be avoided to minimize disturbing the cells, and place them in a 5% carbon dioxide incubator for static culture. Starting from Day 5, replenish the liquid every two days and adjust the cell density to 1×10 6 cells / mL for amplification culture, and count and observe the cell amplification in each group (the culture medium in this liquid replenishment process is the same, and the components do not contain IL-21 and other components are as above. The specific culture medium for the liquid replenishment process is IMDM, 1vt% ITS-A, 5 mM nicotinamide, 500 IU / mL IL-2, 10 ng / mL IL-15, 20 ng / mL IL-18).

[0272] IV. Test Results

[0273] 1. Amplification multiple and cell purity

[0274] (1) Culture with human inactivated autologous plasma (addition amount 5vt%)

[0275] The amplification multiple on Day 13 is 3257 times, CD3 - CD56 + % NK = 99.33%, CD3 + CD56 - % T cells = 0.01%.

[0276] Table 35. Amplification multiple

[0277]

[0278] Table 36. NK cell purity

[0279]

[0280] (2) Culture with human AB serum (addition amount 2.5vt%)

[0281] Amplification multiple on Day 18 was 2221.36 - fold, CD3 - CD56 + % NK cells = 98.34%, CD3 + CD56 - % T cells < 0.05%.

[0282] Table 37. Amplification multiple

[0283]

[0284] Table 38. NK cell purity

[0285]

[0286] (3)Fetal bovine serum FBS culture (addition amount 10 vt%)

[0287] Amplification multiple on Day 19 was 7221.26 - fold, CD3 - CD56 + % NK cells was 97.84%, CD3 + CD56 - % T cells < 0.05%.

[0288] Table 39. Amplification multiple

[0289]

[0290] Table 40. NK cell purity

[0291]

[0292] 2. Verification of the in - vitro killing function of the cultured NK

[0293] The NK prepared by the above - mentioned human AB serum culture process was subjected to in - vitro killing verification and detection of killing phenotypes: Lung cancer, pancreatic cancer, liver cancer, and colorectal cancer cell lines were selected. 1×10 4 target cells were added to each well of a 96 - well white plate, and 3 replicate wells were set for each group. After co - culturing NK cells and target cells at different effector - to - target ratios (E:T) for 20 h, the luciferase substrate (one - glo) working solution was added, and the killing efficiency was detected in an enzyme - linked immunosorbent assay (ELISA) reader. The in - vitro killing results are as Figure 2 shown. Under different effector - to - target ratio conditions, NK showed high killing efficiency (Lysis %) on lung cancer (HCC827 - Luc, human non - small cell lung cancer cell line), colorectal cancer (HCT116 - Luc, human colorectal cancer cell line), liver cancer (Huh - 7 - Luc, human liver cancer cell line), and pancreatic cancer (PANC - 1 - Luc, human pancreatic cancer cell line).

[0294] Detection of receptors related to killing and tumor chemotaxis: The cultured NK cells were taken, and the expression levels of natural killer receptors CD16, NKG2D, NKp30, NKp46, NKp44 and tumor chemotaxis-related receptor CXCR3, CXCR4 were detected by flow cytometry. It can be seen that the natural killer receptors CD16, NKG2D, NKp30, NKp44 and NKp46, etc. were all at relatively high expression levels (>50%) ( Figure 3 ). The expression rate of chemokine receptor CXCR3 was higher than that of CXCR4, exceeding 90% ( Figure 4 ).

[0295] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A culture medium, characterized in that include: IMDM, 1vt% ITS-A, 5mM nicotinamide, 5μg / mL CD16 antibody, 5μg / mL NKG2D antibody, 500IU / mL IL-2, 10ng / mL IL-15, 20ng / mL IL-18, 15ng / mL IL-21, and 5vt% to 10vt% fetal bovine serum; or IMDM, 1vt% ITS-A, 5mM nicotinamide, 5μg / mL CD16 antibody, 5μg / mL NKG2D antibody, 500IU / mL IL-2, 10ng / mL IL-15, 20ng / mL IL-18, 15ng / mL IL-21, and 0.5vt% to 10vt% human inactivated autologous plasma; or IMDM, 1 vt% ITS-A, 5 mM nicotinamide, 5 μg / mL CD16 antibody, 5 μg / mL NKG2D antibody, 500 IU / mL IL-2, 10 ng / mL IL-15, 20 ng / mL IL-18, 15 ng / mL IL-21 and 0.5 vt% to 5 vt% human AB serum.

2. A composition, characterized in that include: CD3-positive cell removal reagent and supplemented culture medium; The CD3 positive cell removal reagent includes CD3 Depletion Cocktail; The supplemented culture medium comprises: IMDM, 1vt% ITS-A, 5mM nicotinamide, 5μg / mL CD16 antibody, 5μg / mL NKG2D antibody, 500IU / mL IL-2, 10ng / mL IL-15, 20ng / mL IL-18, and 5vt% to 10vt% fetal bovine serum; or IMDM, 1vt% ITS-A, 5mM nicotinamide, 5μg / mL CD16 antibody, 5μg / mL NKG2D antibody, 500IU / mL IL-2, 10ng / mL IL-15, 20ng / mL IL-18, and 0.5vt% to 10vt% human inactivated autologous plasma; or IMDM, 1 vt% ITS-A, 5 mM nicotinamide, 5 μg / mL CD16 antibody, 5 μg / mL NKG2D antibody, 500 IU / mL IL-2, 10 ng / mL IL-15, 20 ng / mL IL-18 and 0.5 vt% to 5 vt% human AB serum.

3. A method for culturing non-genetically modified and / or genetically modified NK cells, characterized in that: The method comprises culturing NK cells using at least one of the following i) to ii): i), the culture medium according to claim 1; ii) The composition according to claim 2.

4. The culture method according to claim 3, characterized in that The steps include: Step 1, coating a culture container with the CD16 antibody and the NKG2D antibody in the culture medium of claim 1; Step 2, the PBMC cells are treated with the CD3-positive cell removal reagent in the composition of claim 2 and then inoculated into the culture container obtained in step 1, and cultured using the other components of the culture medium of claim 1 except the CD16 antibody and the NKG2D antibody; Step 3: If the culture medium turns yellow, replenish the liquid using the supplementary culture medium in the composition of claim 2.

5. NK cells obtained by culturing the method according to claim 3 or 4.

6. Use of the NK cells according to claim 5 in the preparation of tumor therapeutic drugs.

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