Method for in vitro induction and amplification of highly pure and highly cytotoxic NK cells

Abstract

The present invention relates to a method for in vitro induction and amplification of highly pure and highly cytotoxic NK cells. The method includes the steps of seeding isolated mononuclear cells into a pre-coated culture flask or petri dish for culture treatment, activating and inducing culture with a medium containing a monoclonal antibody and a cytokine, and finally obtaining NK cells. The coating is treated in a buffer solution containing a monoclonal antibody against CD16 and IL-21. The present invention solves at least to some extent one of the technical problems in the related art. Therefore, the present invention provides a method for culturing NK cells. By seeding isolated mononuclear cells into a pre-coated culture flask or petri dish for culture treatment and performing induction and proliferation treatment, highly pure and highly cytotoxic NK cells can be obtained.

Description

Technical Field

[0001] The present invention belongs to the field of biopharmaceuticals. Specifically, the present invention relates to the preparation of highly pure and highly cytotoxic active NK cells by in vitro induction amplification. More specifically, the present invention relates to a method for culturing NK cells, NK cells, a pharmaceutical composition, and the use of the pharmaceutical composition in the preparation of drugs.

Background Art

[0002] In recent years, biotherapy has become the fifth major therapy following surgery, chemoradiotherapy, and endocrine therapy, and has gradually attracted attention. Adoptive cell immunotherapy (ACI) is one of the cell biological therapies, which refers to injecting immune cells with anti-tumor activity into tumor patients to directly kill tumor cells or stimulate the body's immune response to kill tumor cells to achieve the purpose of tumor treatment. Currently, the adoptive immune cells used clinically include DC-CIK cells, TIL cells, LAK cells, and NK cells. CIK cells, LAK cells, and A-NK cells are all anti-cancer systems mainly composed of natural killer cells.

[0003] Natural killer cells, also called NK cells, mainly originate from lymphocytes of bone marrow CD34+ and are distributed in the bone marrow, peripheral blood, and spleen. NK cells account for 5%-15% of peripheral blood lymphocytes and play an important role in tumor immunity and the removal of non-self cells. It is the main component of natural immune defense and the first line of defense of the body's defense system. The killing activity of NK cells is not restricted by MHC (major histocompatibility complex), does not depend on antibodies, can be recognized without prior sensitization of antigens, kills tumor and virus-infected cells, and directly exerts cytotoxic effects on tumor cells through the perforin-granzyme pathway and the Fas-FasL pathway to kill tumor cells. At the same time, it can secrete various cytokines and chemokines such as TNF-α, IFN-γ, and IL-I in the early stage of onset. Since these cytokines are involved in the regulation of anti-cancer reactions and adaptive immune response reactions, NK cells are also a bridge connecting natural immunity and adaptive immunity.

[0004] The safety and therapeutic effects of the anti-cancer effects of NK cells have been affirmed. However, since they only account for 5%-15% of peripheral blood lymphocytes, how to obtain high-purity and high-quality NK cell products is the key to NK treatment. In recent years, it has been found that relatively large-scale preparation of NK cells is possible through in vitro stimulation culture. From research, cytokines such as IL-2 are the most commonly used cytokines in the in vitro amplified NK cell system. Within a 24-hour reaction period, they enhance the killing activity of NK cells to activate NK cells and promote the proliferation of NK cells and the production of cytokines. Cytokines such as IL-15, IL-18, and IL-12 also play a very important role in the activation and amplification of NK cells. The synergistic effect of IL-15 and IL-2 also acts together in the in vitro amplification of NK cells. At the same time, by binding to the complex receptor gamma chain expressed on the surface of NK cells, they promote the oriented differentiation of hematopoietic stem cells into NK cells and play an important role in the development, differentiation, and maintenance of long-term in vitro survival of NK cells.

[0005] Currently, there are the following industrial bottlenecks in the large-scale preparation of NK cells: 1. The addition of conventional NK cell culture methods such as soluble recombinant growth factors, IL-2, IL-15, etc. has the disadvantages of limited amplification multiples and low killing activity. 2. Using K562 as a feeder layer cell (K562IL15-4-IBB ligand) during NK cell amplification culture and used after gamma-ray irradiation, it does not meet the GMPP standard and raises safety questions. At the same time, although using cells fed with allogeneic (different individuals or tissues of the same species) from different sources has an excellent amplification effect, the current means do not meet the national clinical use standards. Also, feeding cells with allogeneics may lead to the spread of other diseases and cause medical ethics problems. Note that these methods are not effective in eliminating CD3+ T lymphocytes, and their proportion in the amplification system remains high. An attempt has been made to add an anti-CD3 monoclonal antibody to a culture system supplemented with feeder cells, but while this maximally activates the feeder cells, it poses a risk of amplifying and activating residual CD3+ cells, and similarly there is a risk of graft-versus-host disease (GVHD) associated with an allogeneic feeder environment. 3. In order to increase the purity of NK cells, after obtaining peripheral blood mononuclear cells, the proportion of NK cells is increased by sorting and isolating NK cell precursors or CD56+ using immunomagnetic beads. However, doing so increases the cell sorting steps, and the cost of the sorting and isolation method is high, increasing the overall cost of cell culture. Next, NK cells are also lost during the sorting process, reducing the cell volume, decreasing the cell amplification efficiency, and being disadvantageous for the application of large-scale culture.

[0006] Therefore, it is necessary to develop a preparation method capable of culturing NK cells with high purity and high cytotoxic activity.

Summary of the Invention

Problems to be Solved by the Invention

[0007] The present invention solves at least to some extent one of the technical problems in the related art. For this reason, the present invention provides a method for culturing NK cells, in which the isolated mononuclear cells are seeded into a pre-coated culture flask or petri dish for culture treatment, and by performing an induction and proliferation treatment, NK cells with high purity and high cytotoxic activity can be obtained.

Means for Solving the Problems

[0008] The present invention has been completed based on the following findings of the inventors.

[0009] According to a certain study, cytokine is the most commonly used cytokine in the in vitro amplified NK cell line. It enhances the killing activity of NK cells in a short time, activates NK cells, and promotes the proliferation of NK cells and the production of cytokines. However, due to defects such as limited amplification multiples, low killing activity, the potential to cause graft-versus-host disease and the spread of other diseases by allogeneic feeder cells, the large-scale production of NK cells has become a difficulty in the industry.

[0010] Therefore, the present invention proposes a method for culturing NK cells, and by changing the culture conditions, NK cells are cultured on a large scale to produce NK cells with high purity, high cytotoxic activity, low risk of graft-versus-host disease (GVHD), and no possibility of causing the spread problem of other diseases by allogeneic feeder cells.

[0011] Therefore, in the first aspect of the present invention, the present invention proposes a method for culturing NK cells. According to an embodiment of the present invention, the method includes the step of seeding and culturing the isolated mononuclear cells (PBMC) in a pre-coated culture flask or petri dish, and the coating is treated with a monoclonal antibody containing anti-CD16 and a buffer containing IL-21. According to an embodiment of the present invention, since the coating solution contains CD16, the ADCC effect mediated by NK cells can be triggered, different cytotoxic molecules can be released by NK cells, causing the death of target cells. In addition, the inventor further added IL-21 to the coating solution to regulate the proliferation and apoptosis of B cells, promote the production of immunoglobulins and isotype conversion, enhance the cytotoxicity of CD8+ T cells, natural killer cells and NKT cells, and not cause apoptosis of cells due to activation. One of the simple methods to enhance ADCC activity is to stimulate NK cells with pro-inflammatory cytokines, and the combination of a monoclonal antibody against CD16 and IL-21 can significantly enhance the activation and ADCC activity of NK cells.

[0012] The NK cell culture method used in the present invention is characterized by simple operation, fast amplification speed, high cell activity, high cell purity, high safety, and low cost. It has no medical ethics and GVHD risk problems and can be applied to the preparation of large-scale NK cells and clinical applications.

[0013] According to an embodiment of the present invention, the method for culturing the NK cells may further include at least one of the following additional technical features.

[0014] According to an embodiment of the present invention, the buffer solution is selected from PBS buffer solutions.

[0015] According to an embodiment of the present invention, the concentration of the anti-CD16 monoclonal antibody in the PBS buffer solution is 1 to 5 mg / ml, preferably 2 mg / ml. The inventor discovered that in the process of concentration optimization, when the CD16 gradient concentration is 0.5 to 5 mg / ml and is 2 mg / ml or more, it shows better cell proliferation ability and higher NK cell purity. Therefore, preferably, the CD16 concentration is 2 mg / ml.

[0016] According to an embodiment of the present invention, the concentration of the IL-21 in the PBS buffer solution is 20 to 100 ng / ml, preferably 50 ng / ml.

[0017] According to an embodiment of the present invention, the mononuclear cells are derived from peripheral blood.

[0018] According to an embodiment of the present invention, the culture treatment includes an activation treatment and an induction treatment.

[0019] According to an embodiment of the present invention, the activation treatment is performed in a first medium containing IL-2.

[0020] According to an embodiment of the present invention, the concentration of the IL-2 in the first medium is 3.5 to 6.5 ng / ml, preferably 5 ng / ml.

[0021] According to an embodiment of the present invention, the induction treatment is performed in a second medium containing a monoclonal antibody against CD3 and interleukin.

[0022] According to an embodiment of the present invention, the interleukin is selected from at least one of IL-2, IL-12, and IL-15.

[0023] According to an embodiment of the present invention, the interleukin is IL-2. IL-2 is widely applied to promote the activation and proliferation of T cells and NK cells. IL-2 stimulates the proliferation of NK cells, increases cytotoxicity, and stimulates NK cells to secrete various cytokines.

[0024] According to an embodiment of the present invention, the concentration of the monoclonal antibody against CD3 in the second medium is 0.5 - 5 ng / ml, preferably 1 ng / ml. The inventor discovered that the combined use of IL-2 and a monoclonal antibody against CD3 helps amplify PBMC-derived NK cells. Note that a monoclonal antibody against the CD3 molecule can stimulate or block the activation signal transduction of T cells, clear effector T cells, or induce the production of regulatory T cells. As a result of verifying the concentration gradient of 0.5 - 5 ng / ml in the experiment, it was found that at least 1 ng / ml of the monoclonal antibody against CD3 can enable NK cells to obtain a high amplification effect.

[0025] According to an embodiment of the present invention, the concentration of the IL-2 in the second medium is 3.5 - 6.5 ng / ml, preferably 5 ng / ml. The inventor discovered that IL-2 has a positive regulatory effect on the activation and differentiation of NK cells. As a result of experimental verification, 3.5 ng / ml of IL-2 can preferably induce the differentiation and proliferation of NK cells. During the culture process, through the verification of the cell number and the detection of NK purity by flow cytometry, it was comprehensively confirmed that a high proportion of NK cells can be obtained at 5 ng / ml.

[0026] According to the embodiments of the present invention, the basal media of the first medium and the second medium are each independently selected from at least one of CTS AIM V serum-free medium, general-purpose DMEM F12 serum-free medium, KBM581 serum-free medium, X-vivo15, GT-551, and TBD.

[0027] According to the embodiments of the present invention, the basal media of the first medium and the second medium are KBM581 serum-free medium. The inventor selected the aforementioned media and found that the KBM581 serum-free medium has advantages in terms of cell amplification number, NK purity, etc.

[0028] In the second aspect of the present invention, the present invention provides NK cells. According to the embodiments of the present invention, the NK cells are prepared by the method described in the first aspect of the present invention.

[0029] The NK cells prepared according to the embodiments of the present invention are low in cost, high in cell activity, high in cell purity, exhibit good killing activity against tumors, can be prepared and applied on a large scale clinically, and do not cause the occurrence of other diseases.

[0030] In the third aspect of the present invention, the present invention provides a pharmaceutical composition. According to the embodiments of the present invention, the pharmaceutical composition contains the NK cells described in the second aspect of the present invention.

[0031] According to the embodiments of the present invention, the pharmaceutical composition further contains a pharmaceutically acceptable adjuvant.

[0032] As described above, the aforementioned NK cells have advantages such as high cell activity, high cytotoxicity, good biocompatibility, strong delivery ability, and good immune activation effect. The delivery of the aforementioned NK cells into the body helps to exert the killing effect of NK cells on tumor cells.

[0033] In the fourth aspect of the present invention, the present invention provides the use of a pharmaceutical composition in the preparation of a drug. According to the embodiments of the present invention, the drug is used for the immunotherapy of solid tumors or hematological tumors.

[0034] According to an embodiment of the present invention, the solid tumor includes at least one selected from solid tumors occurring in organs, such as pancreatic cancer, ovarian cancer, mesothelioma, liver cancer, cholangiocarcinoma, gastric cancer, colorectal cancer, esophageal cancer, lung cancer, head and neck cancer, cervical cancer, glioblastoma, renal cancer, breast cancer, prostate cancer, melanoma, and the like.

[0035] According to an embodiment of the present invention, the hematological tumor includes at least one selected from acute myeloid leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, and multiple myeloma in blood cells and the hematopoietic system.

[0036] In a fifth aspect of the present invention, the present invention proposes a method for treating or preventing a solid tumor or a hematological tumor. According to an embodiment of the present invention, the method includes the step of administering to a subject at least one of the NK cells described in the second aspect or the pharmaceutical composition described in the third aspect. According to some specific embodiments of the present invention, by administering an appropriate dosage of NK cells or a pharmaceutical composition to a subject, the growth of solid tumor and / or hematological tumor cells is suppressed.

[0037] According to an embodiment of the present invention, the above method further includes at least one of the following technical features.

[0038] According to an embodiment of the present invention, the solid tumor includes at least one selected from solid tumors occurring in organs, such as pancreatic cancer, ovarian cancer, mesothelioma, liver cancer, cholangiocarcinoma, gastric cancer, colorectal cancer, esophageal cancer, lung cancer, head and neck cancer, cervical cancer, glioblastoma, renal cancer, breast cancer, prostate cancer, melanoma, and the like.

[0039] According to an embodiment of the present invention, the hematological tumor includes at least one selected from acute myeloid leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, and multiple myeloma in blood cells and the hematopoietic system.

[0040] In a sixth aspect of the present invention, the present invention proposes the use of the NK cells described in the second aspect or the pharmaceutical composition described in the third aspect in the treatment or prevention of solid tumors or hematological tumors. According to some specific examples of the present invention, different treatment means including any one or a combination of NK cells or the pharmaceutical composition can be selected and used for different types of solid tumors or hematological tumors by utilizing the above products, so as to enhance the targeting and effectiveness of the treatment of solid tumors and / or hematological tumors.

[0041] According to an embodiment of the present invention, the above method further includes at least one of the following technical features.

[0042] According to an embodiment of the present invention, the solid tumor includes at least one selected from tangible tumors occurring in organs, such as pancreatic cancer, ovarian cancer, mesothelioma, liver cancer, cholangiocarcinoma, gastric cancer, colorectal cancer, esophageal cancer, lung cancer, head and neck cancer, cervical cancer, glioblastoma, renal cancer, breast cancer, prostate cancer, melanoma, etc.

[0043] According to an embodiment of the present invention, the hematological tumor includes at least one selected from acute myeloid leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, multiple myeloma in blood cells and the hematopoietic system.

[0044] Additional aspects and advantages of the present invention are partially shown in the following description, partially become apparent from the following description, or can be understood through the practice of the present invention. The above and / or additional aspects and advantages of the present invention will become apparent and easy to understand from the description of the embodiments combined with the following drawings.

Brief Description of the Drawings

[0045]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Modes for Carrying Out the Invention

[0046] Hereinafter, the embodiments of the present invention shown in the drawings will be described in detail. In all the drawings, the same or similar reference numerals indicate the same or similar elements or elements having the same or similar functions. The embodiments described through the following reference drawings are exemplary and are for explaining the present invention, and should not be understood as a limitation to the present invention.

[0047] In the process of explaining the present invention, the related terms in this specification have been interpreted and explained, but these interpretations and explanations are only for facilitating the understanding of the means and are not considered as limiting the protection means of the present invention.

[0048] In this specification, the term "including" or "comprising" is a non-limiting expression, that is, it includes the content shown by the present invention but does not exclude the content of other aspects.

[0049] In this specification, the terms "optionally", "any", or "optional" generally mean that the events or situations described hereinafter may occur, but do not necessarily occur, and the description includes both the cases where the events or situations occur and the cases where the events or situations do not occur.

[0050] Method for culturing NK cells

[0051] In one aspect of the present invention, a method for culturing NK cells is proposed. According to an embodiment of the present invention, the method includes a step of seeding and culturing isolated mononuclear cells (PBMC) in a culture flask pre-coated with a monoclonal antibody against CD16 at 1 to 5 mg / ml and IL-21 at 20 to 100 ng / ml. Next, it is induced and cultured with a monoclonal antibody against CD3 at 0.5 to 5 mg / ml and a cytokine (at least one of IL-2, IL-12, and IL-15) at 3.5 to 6.5 ng / ml to obtain highly active and highly pure NK cells. The inventor has discovered that a first medium containing a monoclonal antibody against CD16 and IL-21 is selected to coat the culture flask or petri dish, which has a strong function of activating NK cells and enhancing ADCC.

[0052] In this specification, antibody-dependent cell-mediated cytotoxicity (ADCC) is an effective cytotoxic mechanism mainly mediated by natural killer (NK) cells. The ADCC effect mainly activates NK cells by antibodies that bind to Fc receptors (FcRs), and the most characteristic FcR on the NK cell membrane is FcγRIII (CD16). CD16 mainly plays a role in triggering NK cell-mediated ADCC. When CD16a recognizes the target of IgG conditioning, NK cells release different cytotoxic molecules, causing the death of target cells. Such a mechanism depends on the formation of immunological synapses and the degranulation of lytic granules containing perforin and granule enzymes. In addition to degranulation, NK cells can also clear target cells by binding target death receptors (such as DR4, DR5, or Fas) to their death receptor ligands (such as FasL and TRAIL). The main effects of the various biological effects of IL-21 are manifested in the direct regulation of the proliferation and apoptosis of B cells, the promotion of immunoglobulin production and isotype conversion, and the enhancement of the cytotoxicity of CD8+ T cells, natural killer cells, and NKT cells without causing cell apoptosis by activation. One of the simple ways to improve ADCC activity is to stimulate NK cells with pro-inflammatory cytokines, and the combination of monoclonal antibodies against CD16 and IL-21 can significantly enhance the activation of NK cells and ADCC activity.

[0053] Specifically, it is necessary to explain that the mononuclear cells (PBMCs) in the present invention are collected from the blood cells of a subject, and the blood cells are collected from the peripheral blood, umbilical cord blood, bone marrow, and / or lymph nodes of the subject.

[0054] The present invention creates an efficient and stable method for in vitro amplification of NK cells and has the following advantages: 1. The amplification method is simple. After treating PBMC obtained from peripheral blood isolation with a combination of antibodies and cytokines, it can be directly used for subsequent culture without the step of increasing magnetic beads to select NK cells. The selection of NK cells by magnetic beads increases the cell amplification cost and reduces the acquisition amount of NK cells and the NK amplification efficiency. 2. The culture flask is coated with a combination of two antibodies and cytokines to activate the cells, reduce heterologous cells in PBMC, and screen out most T cells, making NK cells the dominant cells, stimulating the induced amplification of NK cells, and significantly improving the purity of NK cells. 3. The combination of CD16 monoclonal antibody and interleukin cytokine IL-21 is used for culture to induce the activity of NK cell differentiation and proliferation. 4. During the culture process, the culture medium is supplemented according to the growth status of the cells and the culture system is expanded to prevent the cell growth concentration from being too high. In this way, the cell amplification efficiency can be improved and the cell activity can be maintained.

[0055] In a further aspect of the present invention, the present invention proposes a pharmaceutical composition. According to an embodiment of the present invention, the pharmaceutical composition contains the aforementioned prepared NK cells.

[0056] According to an embodiment of the present invention, the pharmaceutical composition further contains a pharmaceutically acceptable adjuvant.

[0057] As used herein, the term "pharmaceutically acceptable" indicates that the pharmaceutical composition can be administered to a subject without causing adverse physiological reactions that interfere with the administration of the pharmaceutical composition. For example, "pharmaceutically acceptable adjuvants" refer to adjuvants that are generally safe, non-toxic, and useful for the preparation of desirable pharmaceutical compositions. Preferably, examples of these adjuvants or diluents include water, physiological saline, Ringer's solution, glucose, mannitol, dextrose, lactose, starch, magnesium stearate, cellulose, magnesium carbonate, 0.3% glycerin, hyaluronic acid, ethanol, polyalkylene glycols such as polypropylene glycol, triglycerides, 5% human serum albumin, but are not limited thereto, and media such as liposomes and non-aqueous media of non-volatile oils can also be used.

[0058] Hereinafter, the embodiments of the present invention shown in the accompanying drawings will be described in more detail. Hereinafter, the embodiments described with reference to the accompanying drawings are exemplary and are for interpreting the present invention and are not to be understood as limiting the present invention.

Example

[0059] Example 1: Method for Amplifying NK Cells

[0060] Culture Flask Coating Treatment

[0061] The culture flask coating solution (PBS buffer containing a monoclonal antibody against CD16 and IL-21) used in this example is diluted to a preferred concentration using an antibody or cytokine to which a monoclonal antibody against CD16 (Anti-CD16mAb, 1 to 5 mg / ml, 2 mg / ml in this example) and IL-21 (Interleukin 21, 20 to 100 ng / ml, 50 ng / ml in this example) are added.

[0062] 1. Absorb 15 ml of PBS buffer (commercialized PBS can be used, free of pyrogen and endotoxin) and add it to a 50 mL centrifuge tube. 2. Add anti-human CD16 monoclonal antibody dissolved in PBS buffer and human IL-21 to the 50 mL centrifuge tube in Step 1, mix well (pay attention not to blow air bubbles), obtain the coating solution, 3. Add the coating solution prepared in Step 2 evenly to two sterile T-75 culture flasks, and cover the bottom surface of the culture flasks evenly with the coating liquid, 4. Incubate the T-75 culture flasks coated in Step 3 in a 4°C refrigerator overnight (about 12 - 16 hours) for coating to obtain the "NK initial culture flasks" (it is recommended to prepare the initial culture flasks the day before use and use them within 12 - 36 hours after preparation is completed), 5. Take out the NK initial culture flasks from the 4°C refrigerator before use, let them return to room temperature (10 - 20 minutes), discard the coating solution with a pipette before use, discard it as clean as possible (note: it is not necessary to wash the initial culture flasks), and make sure the pipette does not touch the bottom of the flask so as not to affect the coating effect. Obtain a T-75 culture flask without the coating solution and cells can be seeded.

[0063] Isolation of PBMC

[0064] The NK cells used in this example are derived from human peripheral blood and are isolated using the following steps and methods to obtain peripheral blood mononuclear cells (PBMC), 1. Preparation of the GMP laboratory: Before entering the GMP laboratory, it is necessary to turn on the air conditioner in the GMP laboratory 30 minutes in advance. Disinfect the biological safety cabinet with an ultraviolet lamp for 30 minutes before use. Wipe the test table with 75% alcohol before use. After turning on the biological safety cabinet, check that it is operating normally 20 minutes later and then start operating. 2. Centrifuge the donor's peripheral blood to isolate cells and plasma. Use a centrifuge to centrifuge at a rotation speed of 800g at room temperature for 10 minutes, lower the speed 1, and obtain layered blood cells and plasma. 3. Use a sterile straw to aspirate the plasma from Step 2 in a biological safety cabinet, place it in another sterile 50 ml centrifuge tube, inactivate it in a water bath at 56°C for 30 minutes, leave it at 4°C for 15 minutes, then centrifuge at 800 g for 10 minutes. Transfer the upper plasma to a new sterile 50 ml centrifuge tube and store it at 4°C for standby use. Add 0.9% injectable physiological saline to the remaining blood cell precipitate to a total volume 1 - 1.2 times the total blood volume, mix it upside down evenly, slowly inject 15 ml - 20 ml of the blood cell suspension into a 50 ml centrifuge tube containing 15 ml of lymphocyte separation solution, and centrifuge at 700 g for 20 minutes at room temperature (increase speed 2 and decrease speed 1) to obtain the separation solution. 4. Aspirate the mononuclear cell layer (lymphocytes and monocytes) from the separation solution obtained in Step 3 into a new 50 ml sterile centrifuge tube, with a volume of 15 milliliters or less per tube. 5. Supplement the centrifuge tube containing the mononuclear cells from Step 4 with 0.9% injectable physiological saline to 45 - 50 ml, mix it upside down evenly, centrifuge at 700 g for 10 minutes at room temperature, increase speed 9 and decrease speed 9 to wash once. Discard the supernatant, resuspend it with another 45 - 50 ml of 0.9% injectable physiological saline, mix it upside down, centrifuge at 500 g for 10 minutes at room temperature, increase speed 9 and decrease speed 9 to wash once, and discard the supernatant. Obtain the prepared peripheral blood mononuclear cells.

[0065] Initial culture of NK cells

[0066] According to the examples of the present invention, the initial culture of NK cells is initially cultured in the first medium (KBM581 medium containing IL - 2). 1. On the 0th day of culture: Resuspend the prepared mononuclear cells in KBM581 medium (Corning Lymphocyte Serum - Free Medium KBM581, product number #88 - 581 - CM, 1000 mL) containing 5% autologous plasma, and adjust the cell density to 1.3×10 6 / ml. Transfer the cells to the prepared "NK initial culture flask" (divide them evenly into 2 flasks on average, 40 - 45 ml / flask), and add IL - 2 to a final concentration of 5 ng / ml. 2. Transfer the cells into an incubator at 37°C, saturated humidity, and 5.0% CO2 and continue culturing.

[0067] Induction (amplification) culture of NK cells

[0068] For the induction and amplification culture of cells in the present invention, an NK amplification culture medium (second medium) is used, which is a serum-free medium of KBM581 supplemented with Anti-CD3mAb and IL-2. 1. On the 3rd day of culture: Supplement IL-2 to a final concentration of 5 ng / ml, and place the cells in an incubator at 37°C, saturated humidity, and 5.0% CO2 to continue culturing. 2. On the 5th day of culture, perform the first subculture of the cells. Liquid supplementation method: Supplement "NK amplification culture medium" (preparation method: Add one amplification factor to 1 L of KBM581 medium and mix well) to a cell density of 1.2×10 6 / ml, and supplement IL-2 at a total amount of 5 ng / ml and 2% autologous plasma. 3. On the 7th day of culture: For the cells at the bottom of the flask (just blow up the cells, then take out a small amount of cell suspension and mix well alone in an EP tube), after mixing well, count. If the cell density is 1.5×10 6 / ml or less, subculture is not required, but it is necessary to supplement IL-2 at a total amount of 5 ng / ml and 2% autologous plasma. If the cell density exceeds 1.5×10 6 / ml, perform subculture operation, supplement "NK amplification culture medium", adjust the cell density to 1.4×10 6 / ml, and continue culturing at 37°C and 5.0% CO2. 4. Culture until the 9th to 33rd day. Generally, the cells need to be subcultured once every two days. The subculture method is as in step 3, using "NK amplification culture medium", IL-2, and autologous plasma (Note: According to observation, if the cell density is still 1.5×10 6 / ml or less, subculture is not required, and supplement IL-2 at a total amount of 5 ng / ml and 2% autologous plasma. If the cell density exceeds 1.5×10 6 / ml. At the time of subculture, adjust the cell density to 1.5×10 6Adjust to / ml. If the culture liquid volume exceeds 60 ml, transfer it to a T175 flask, and if it exceeds 200 ml, transfer it to a culture bag for culture (TAKARA, 1.8 L specification). 5. During the initial culture of NK cells and the amplification culture process of NK cells, record the cell growth status, and at different time points of culture (from the seeding time, on the 1st, 5th, 9th, 13th, 17th, 21st, 25th, 29th, 33rd days), count the cells and plot the NK cell growth curve (Figure 2).

[0069] Harvest of NK cells 1. When the cultured NK cells reach the required number, take the cell suspension and verify and evaluate it according to the items and methods stipulated in the "Finished Product Verification Criteria" of the product quality control standards. 2. Transfer the cultured cells to a sterile centrifuge bottle (250 ml), centrifuge at 850 g for 20 minutes to collect the cells, wash them twice with 0.9% injection physiological saline, centrifuge at 850 g for 8 minutes to obtain NK cells. 3. Resuspend the NK cells in 100 ml of 0.9% injection physiological saline, collect 5 ml of 20% human blood albumin with a 5 ml syringe, inject it into the cell suspension until the final concentration reaches 1%, and mix well. Filter with a 70 μm cell filter to remove cell clumps. Count the cells.

[0070] Detection of NK cell purity and biological activity 1) Experimental materials Flow cytometry antibodies: Fluorescently labeled isotype control mouse monoclonal antibodies: mouse IgG1-FITC (product number: 400108), mouse IgG1-Alexa Flour 647 (product number: 362514), mouse IgG1-Percp cy5.5 (product number: 400150), mouse IgG1-APC (product number: 400120), mouse IgG1 -BV785 (product number: 400170), all purchased from Biolegend, surface molecule labeling antibodies: CD3-Percpcy5.5 (product number: 300430), CD56-BV785 (product number: 362550), FITC-CD3 (product number: 300406), CD56-Alexa Flour 647 (product number: 362514), NKG2D-APC (product number: 320808), Perforin-FITC (product number: 353310) and Granzyme B-Alexa Flour 647 (product number: 515406), all purchased from Biolegend, DAPI (product number: 422801) purchased from Biolegend, CFSE (product number: 51-9010817) purchased from BD Biosciences. Blocking antibody: mouse serum, purchased from Future, product number is F001008. Flow cytometry tubes: 5 ml round-bottom capped tubes made of clear polystyrene or polypropylene. Monensin: purchased from sigma, product number is 22373-78-0. Fixation / permeabilization Diluent: purchased from ebioscience, product number is 4298341. Fixation / permeabilization concentrate: purchased from ebioscience, product number is 4311034. Permeabilization buffer: purchased from ebioscience, product number is 4314840. DAPI (4’,6-Diamidino-2-Phenylindole, Dilactate): purchased from Biolegend, product number is 422801. Flow cytometry washing solution: PBS. In addition, related reagents from equivalent companies and flow cytometry antibodies with the same clone number can also be used. 2) Main instruments and equipment Centrifuge, clean bench, flow cytometer (such as FACS Calibur, Beckman CytoFlex, etc.), RTCA detection system. 3) Detection method for related molecules of cell viability 1 Preparation of single-cell suspension of intracellular molecule-labeled cells (monensin induction): Take 4×10 6 cells (2 ml) cultured and place them in a 6-well plate. Add 10 μl of monensin (0.5 μg / μl) and induce for 4 hours in a 37°C, 5% CO2 constant temperature incubator. After the induction of intracellular molecule-labeled cells with monensin is completed, add 5 ml of 1×PBS and wash twice. Finally, resuspend in 180 μl of 1×PBS to obtain a single-cell suspension. 2 Preparation of single-cell suspension of surface molecule-labeled cells: Take 9.0×10 6 cultured NK cells and place them in a 15-ml centrifuge tube. Centrifuge at 400 g for 8 minutes to collect the cells. Wash twice with 10 ml of 1×PBS. Finally, resuspend in 0.81 ml of 1×PBS to obtain a single-cell suspension. 3 Blocking: Add 90 μl of mouse serum to the surface molecule-labeled cells obtained in 2, mix well. Add 20 μl of mouse serum to the intracellular molecule-labeled cells obtained in 1, mix well, and let stand at room temperature for 15 - 30 minutes. 4 Labeling antibody: Dispense the surface molecule-labeled cells after blocking obtained in step 3 into 9 flow cytometry tubes at 0.09 ml / tube. When multiple batches of cells are detected simultaneously, the cells in tubes 1 - 8 can be mixed in equal amounts with several batches of cells and then labeled. The amount of cells used in each tube is 0.8 - 1×10 6 cells. Sample tube 1 contains surface molecule-labeled cells, and the amount of cells used is 0.8 - 1×10 6 cells. Sample tube 2 contains intracellular molecule-labeled cells, and the amount of cells used is 3 - 4×10 6Add the corresponding fluorescently labeled antibody in the amount of [[ID=]], mix well, and then let it stand in the dark at 4°C for 30 minutes (mix well once every 15 minutes during this period). The specific labeling method is shown in Table 1.

[0071]

Table 1-1

Table 1-2

[0072] 5 Washing: Add 1 ml of 1×PBS to each sample tube 1 (surface molecule-labeled cells), centrifuge at 400 g for 8 minutes at 4°C to collect the cells, and repeat the washing twice with 1 ml of 1×PBS. Finally, add 200 μl of 1×PBS to each tube to resuspend the labeled cells, add 5 μl of DAPI (50 μg / ml, 40×) to the experimental group, transfer to the tube and detect online. After completing the labeling of the external standard molecules CD3 and CD56 in sample tube 2 (intracellular molecule-labeled cells), first add 5 μl of DAPI, label for 3 min, add 1 ml of PBS to each tube and wash twice, and discard the supernatant (Note: Discard as clean as possible). Add 300 μl of Fixation Buffermix (Fixation / permeabilization concentrate : Fixation / permeabilization Diluent = 1:3) to the tube, fix in a 4°C freezer for 1 h (mix well once every 30 min). After completion, add 1 ml of Perm Wash Buffer (the liquid storage is 10× and diluted to 1× with double-distilled water) at 500 g, at 4°C for 5 min, and discard the supernatant. Next, resuspend the cells with 180 μl of Perm Wash Buffer, add 20 μl of mouse serum, and block in a 4°C freezer for 20 min. After blocking, divide the cells into two equal parts and label with antibodies. The specific labeling method is shown in Table 2. Label in the dark in a 4°C freezer for 1 h (mix well once during that time). After the antibody labeling is completed, add 1 ml of Perm Wash Buffer, at 500 g, 4°C, for 5 min. Wash again once. Finally, add 200 μl of Perm Wash Buffer to resuspend the cells and detect online. 6 Detection: Calibrate and adjust the flow cytometer according to the instrument manual. The blank control tube sample is used to adjust the voltages of forward scatter and side scatter. The isotype control sample adjusts the voltage of each fluorescence channel so that its fluorescence is within the negative range. The single-label control sample is used to adjust the fluorescence compensation of each channel. When detecting, after defining the cell gate, collect 1×10 4 cells in each sample gate.

[0073]

Table 2

[0074] 4) Validity determination of experimental results The positive rate of fluorescence staining of the blank control tube sample < 1%, the absolute cell number is less than 100, and the isotype controls of the five different fluorescently labeled antibodies each have a positive rate < 1% and an absolute cell number of less than 100 cells. Percentage of positive cells (positive cells %) = (number of positive cells ÷ total number of counted cells 1 × 10 4 ) × 100%.

[0075] The results of the experiment are considered qualified if they meet the above requirements, and conversely, unqualified.

[0076] Detection of amplification results As can be seen from the NK cell flow cytometer detection results (Figure 1), NK cells reached 95.9% and 92.2% after amplification from 11.67% and 11.88% before amplification, and the purity of the NK cells obtained after amplification is high. From the statistical results (Figure 2) obtained after 5 experiments (N = 5), it can be seen that the cell growth efficiency is high, the proportion increase of NK% and CD56+% is fast, and the number of NK cells increases 1000 - 2000 times within the amplification cycle, and CD56+ is more than 90%. From the results of detecting the expression of activating receptors on the surface of NK cells after amplification (Figure 3), when detecting the expression of activating receptors on the surface of NK cells after amplification by flow cytometry, it can be seen that the expression of some activating receptors is clearly enhanced compared with that before amplification.

[0077] Example 2, Detection method of cell killing activity

[0078] Use real-time label-free cell analysis technology to detect the killing activity of NK cells against solid tumors (suitable for adherent cells, and the solid tumors verified in this example include ovarian cancer cell line HO8910 and melanoma A375 cell line).

[0079] Preparation of target cell suspension (Day 1) 1. In the clean bench, under aseptic conditions, suck out the old culture medium in the petri dish. 2. Next, wash twice with 1×PBS, and add 1 mL of trypsin solution containing EDTA into the petri dish. Digest at room temperature for 2 - 6 min. 3. Discard the trypsin solution, add appropriate medium into the petri dish, gently blow the cells to form a cell suspension, and centrifuge at 140 g for 5 min. After centrifugation, resuspend the cells with 1 mL of complete medium, take 20 μl for cell counting, and adjust to the required cell concentration of 1.6×10 5 cells / ml.

[0080] RTCA DP System Calibration and Target Cell Culture 1. Add 50 μl of medium into the wells of E-Plate 16. 2. Place E-Plate 16 on the RTCA Station. 3. The RTCA system can automatically scan (「Scan Plate」) -> check whether the contact is good (display Connection OK on the 「Message」 page). 4. Start background detection (Background) -> confirm that the selected wells are in normal contact. 5. Take out E-Plate 16, add 100 μl of the above-prepared uniformly mixed target cell suspension into the wells, and make the number of cells in each well 1.6×10 4 cells / well (Note: After adding cells to E-Plate 16, it is not necessary to mix the cells well with the original medium in the wells). 6. Leave E-Plate 16 in the clean bench for 20 min to allow the cells to settle to the bottom of the plate. 7. Place E-Plate 16 on the RTCA Station in the incubator. 8. After the system automatically scans 「Scan Plate」, start Step 2 to perform real-time dynamic detection of cell growth. 9. When the target cells grow for about 12 hours, at this time, the cell index reaches about 2.0, and the preparation for adding NK cells begins.

[0081] Preparation of NK cells to be detected and killing experiment (Day 2) 1. Adjust the prepared NK cells to an appropriate cell density according to the effector-target ratio (the ratio of effector cells to target cells). 2. Take out the E-Plate 16 and place it on the clean bench. 3. Add 50 μL of the NK cell suspension (or an equal volume of medium: blank control) into the culture plate at different effector-target ratios (1:1, 2:1, 4:1, 8:1). In addition, control wells with only target cells and only NK cells should be set up. 4. Place the E-Plate 16 detection plate on the RTCA DP detection platform and monitor it in real time to observe the real-time killing effect of NK cells on HO8910 cells. 5. Result calculation method: (Average value of cell index in control group - Average value of cell index in experimental group) / Average value of cell index in control group × 100%.

[0082] As shown by the results, the amplified NK cells have a high tumor cell killing function. Against HO8910 cells, when the effector-target ratio is 4:1, a killing efficiency of about 80% (Figure 4) can be achieved. Against A375 cells, when the effector-target ratio is 4:1, a killing efficiency of about 90% (Figure 4) can be achieved. The NK cell preparation shows a dose-effect relationship for the killing of two types of tumor cells, and the killing efficiency gradually improves with the increase of the effector-target ratio.

[0083] Detection of the killing ability of NK cells against hematological tumors (suitable for floating cells, and the hematological tumors verified in this example include leukemia K562 cell line and MOLT4 cell line) by flow cytometry

[0084] Preparation of the target cell suspension: 1. Count the target cells (K562 cells): Resuspend the cells in 1640 medium containing 0.5% FBS (the medium used for target cells), count them, and adjust the cell density to 1×10 6 / mL. 2. Fluorescent dye CFSE (hydroxyfluorescein diacetate succinimidyl lipid) staining: Add the cell suspension prepared in Step 1 to CFSE (working concentration 5 μM), immediately stir with a 1 mL pipette, vortex to mix well, incubate in the dark in a 37 °C incubator for 15 min, take it out every 5 min and vortex once to mix well, and obtain the stained target cells. 3. Stop staining: Add 5 volumes of 4 °C pre-cooled complete medium (the medium used for target cells) to the stained target cell solution in Step 2 to stop the staining, and perform a 5-minute ice bath. Centrifuge at 140 g at 4 °C for 5 min. 4. Wash the target cells: Resuspend with 4 °C pre-cooled complete medium (the medium used for target cells), centrifuge the cells at 140 g at 4 °C for 5 min, and repeat the washing 2 times. 5. Count: Resuspend the target cells, count them, and adjust the cell density to 2×10 5 / mL. 6. Seed the plate: Add 100 μl of the target cell suspension to each well of a 96-well round bottom plate so that the final number of cells per well is 20,000 cells / well. 7. Adjust to an appropriate NK cell density. 8. Take out the E-Plate 16 and place it on the clean bench. 9. Add 100 μl of the NK cell suspension to the culture plate at different effector-target ratios (5:1, 2.5:1, 1.25:1). In addition, three groups of controls should be set: only the target cells stained with CFSE (to detect the natural mortality rate of the target cells), only the effector cells (to prove that there is no non-specific staining of CFSE in the effector cells), and only the target cells + Tween (registered trademark)-20 (as a positive control for target cell apoptosis). 10. Co-incubate and add NK cells to each well in the pre-designed order. Centrifuge at 120 g at room temperature for 2 min to allow the effector cells and target cells to contact sufficiently. Return to the 37 °C incubator and incubate for 4 hours. 11. After the incubation is completed, add 1 μl of 7-AAD (7-aminoactinomycin D), mix well, incubate in the dark for 5 min, and detect online. 12. Result analysis: Killing percentage = [((Experimental group target cell mortality rate (%) - Target cell mortality rate (%)) / (100% - Target cell natural mortality rate (%))) × 100%].

[0085] As shown by the results, NK cells and tumor cell lines were co-incubated at different ratios of effector cells to target cells, and the mortality rate of target cells was detected 4 hours later. Since K562 cells are sensitive to killing by NK cells, when the effector-to-target ratio is 8:1, a killing efficiency of approximately 80% (Figure 5) can be achieved. For MOLT4 cells, when the effector-to-target ratio is 8:1, a killing efficiency of approximately 60% (Figure 5) can be achieved. NK cells show a dose-effect relationship for killing two types of tumor cells, and as the effector-to-target ratio increases, the killing efficiency gradually improves.

[0086] Note that the terms "first" and "second" are used only for the purpose of explanation and cannot be understood as indicating or implying relative importance or suggesting the number of technical features shown. Therefore, the features defined by "first" and "second" can implicitly include at least one such feature. In the description of the present invention, "a plurality" means at least two, for example, two, three, etc., unless otherwise specifically limited.

[0087] In the description of this specification, descriptions referring to terms such as "one embodiment", "several embodiments", "example", "specific example", or "several examples" mean that the specific features, structures, materials, or characteristics described with reference to the corresponding embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the general expressions of the above terms do not necessarily target the same embodiment or example. In addition, the specific features, structures, materials, or characteristics described can be incorporated in any one or more embodiments or examples in an appropriate manner. Furthermore, those skilled in the art can combine and combine the various embodiments or examples described in this specification and the features related to the various embodiments or examples without contradiction.

[0088] Embodiments of the present invention have been presented and described. However, the above embodiments are exemplary and should not be understood as limiting the present invention. Those skilled in the art can understand that various changes, modifications, substitutions, and variations can be made to the above embodiments within the scope of the present invention.

Claims

1. A method for culturing NK cells, comprising: seeding and culturing the isolated mononuclear cells in a pre-coated culture flask or petri dish, wherein the coating is treated in a buffer solution containing a monoclonal antibody against CD16 and IL-21. A method for culturing NK cells, characterized in that.

2. The method according to claim 1, wherein the buffer solution is selected from PBS buffer solution.

3. The method according to claim 1 or 2, wherein the concentration of the monoclonal antibody against CD16 in the PBS buffer solution is 1 to 5 mg / ml.

4. The method according to claim 3, wherein the concentration of the monoclonal antibody against CD16 in the PBS buffer solution is 2 mg / ml.

5. The method according to claim 1 or 2, wherein the concentration of IL-21 in the PBS buffer solution is 20 to 100 ng / ml.

6. The method according to claim 5, wherein the concentration of IL-21 in the PBS buffer solution is 50 ng / ml.

7. The method according to claim 1, wherein the mononuclear cells are derived from peripheral blood.

8. The method according to claim 1, wherein the culture treatment includes an activation treatment and an induction treatment.

9. The method according to claim 8, wherein the activation treatment is performed in a first medium containing IL-2.

10. The method according to claim 9, wherein the concentration of IL-2 in the first medium is 3.5 to 6.5 ng / ml.

11. The method according to claim 10, wherein the concentration of IL-2 in the first medium is 5 ng / ml.

12. The method according to claim 8, wherein the induction treatment is performed in a second medium containing a monoclonal antibody against CD3 and an interleukin.

13. The method according to claim 12, wherein the interleukin includes at least one selected from IL-2, IL-12, and IL-15.

14. The method according to claim 13, wherein the interleukin is IL-2.

15. The method according to claim 12, wherein the concentration of the monoclonal antibody against CD3 in the second medium is 0.5 to 5 ng / ml.

16. The method according to claim 15, wherein the concentration of the anti-CD3 monoclonal antibody in the second medium is 1 ng / ml.

17. The method according to claim 12 or 14, wherein the concentration of IL-2 in the second medium is 3.5 to 6.5 ng / ml.

18. The method according to claim 17, wherein the concentration of IL-2 in the second medium is 5 ng / ml.

19. The method according to claim 9 or 12, wherein the basal media of the first medium and the second medium are each independently selected from at least one of CTS AIM V serum-free medium, general-purpose DMEM F12 serum-free medium, KBM581 serum-free medium, X-vivo15, GT-551, and TBD.

20. The method according to claim 19, wherein the basal media of the first medium and the second medium are KBM581 serum-free medium.

21. An NK cell characterized by being prepared by the method according to any one of claims 1 to 20.

22. A pharmaceutical composition comprising the NK cell according to claim 21.

23. The pharmaceutical composition according to claim 22, further comprising a pharmaceutically acceptable adjuvant.

24. Use of the NK cell according to claim 21 or the pharmaceutical composition according to any one of claims 22 to 23 in the preparation of a drug for immunotherapy of solid tumors or hematological tumors.

25. The use according to claim 24, wherein the solid tumor comprises at least one selected from solid tumors occurring in organs, including pancreatic cancer, ovarian cancer, mesothelioma, liver cancer, cholangiocarcinoma, gastric cancer, colorectal cancer, esophageal cancer, lung cancer, head and neck cancer, cervical cancer, glioblastoma, renal cancer, breast cancer, prostate cancer, melanoma, etc.

26. The use according to claim 24, wherein the hematological tumor comprises at least one selected from acute myeloid leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, multiple myeloma in blood cells and the hematopoietic system.

27. A method for treating or preventing a solid tumor or a hematological tumor, comprising the step of administering to a subject at least one of the NK cell according to claim 21 or the pharmaceutical composition according to any one of claims 22 to 23.

28. The method according to claim 27, wherein the solid tumor comprises at least one selected from solid tumors occurring in organs, including pancreatic cancer, ovarian cancer, mesothelioma, liver cancer, cholangiocarcinoma, gastric cancer, colorectal cancer, esophageal cancer, lung cancer, head and neck cancer, cervical cancer, brain glioma, renal cancer, breast cancer, prostate cancer, melanoma, and the like.

29. The method according to claim 27, wherein the hematological tumor comprises at least one selected from acute myeloid leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, multiple myeloma in blood cells and the hematopoietic system.

30. Use of the NK cells according to claim 21 or the pharmaceutical composition according to any one of claims 22 to 23 for the treatment or prevention of solid tumors or hematological tumors.

31. The use according to claim 30, wherein the solid tumor comprises at least one selected from solid tumors occurring in organs, including pancreatic cancer, ovarian cancer, mesothelioma, liver cancer, cholangiocarcinoma, gastric cancer, colorectal cancer, esophageal cancer, lung cancer, head and neck cancer, cervical cancer, brain glioma, renal cancer, breast cancer, prostate cancer, melanoma, and the like.

32. The use according to claim 30, wherein the hematological tumor comprises at least one selected from acute myeloid leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, multiple myeloma in blood cells and the hematopoietic system.

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