Culture method for efficiently amplifying NK (Natural Killer) cells in vitro

Abstract

The invention discloses a culture method for efficiently amplifying NK (Natural Killer) cells in vitro. According to the application, the types of used factors are few, the use amount of autologous plasma is gradually reduced in the amplification stage, and IL-2 and IL-15 with lower concentrations are used, so that the cost is reduced, and meanwhile, the requirement of large-scale amplification of NK is met. The culture period is short, the NK cells can be amplified by more than 700 times in only 14 days, and the NK cells can be amplified by more than 3000 times in the 20th day. According to the method, sorting is not needed, all mononuclear cells are used for culture, and the operation procedure is simplified. Feeder layer cells, serum and blood replacement are not needed, risks caused by the feeder layer cells and external blood are avoided, and the safety performance is high. The method is simple in process operation and low in cost, the experimental process is simplified, and meanwhile a large number of high-purity and high-cytotoxicity NK cells are obtained.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular relates to a method for culturing NK cells for efficiently amplifying NK cells in vitro. Background Art

[0002] In the past decade, immunotherapy has made great progress and has become a promising method for treating many diseases. T cell-based immunotherapy has been developed and successfully applied to the clinical treatment of various cancers. It is very important to study anti-tumor effector cells and immunotherapy strategies other than T cells. Natural killer cells (NK), as innate immune cells with the ability to directly kill malignant cells, are considered to be a promising immunotherapy tool. Various NK cell sources, such as peripheral blood mononuclear cells (PBMCs), umbilical cord blood (UCB) cells, induced pluripotent stem cells (iPSCs), and NK cell lines, have been used to generate NK cells in vitro, but the proportion of NK cells in peripheral lymphocytes is low. When infused clinically, NK cells need to reach a certain amount of cell number and high cell purity. Therefore, how to obtain higher cell therapy doses and higher purity when expanding peripheral blood NK cells in vitro has become the key to tumor treatment.

[0003] At present, most NK cell clinical trials are still in the early stages. In the future, we need to continuously improve the effectiveness and safety of NK cell therapy, optimize production processes, reduce costs, shorten production cycles, and expand the scope of treatment based on existing technologies. Therefore, it is necessary to further study a culture medium and culture method for in vitro NK cell expansion that is time-saving, easy to prepare, and low-cost. Summary of the invention

[0004] To make up for the deficiencies of the prior art, the present invention provides a method for efficiently amplifying NK cells in vitro.

[0005] To achieve the above purpose, the present invention adopts the following technical solution.

[0006] A first aspect of the present invention provides a composition for culturing NK cells, the composition comprising an activation medium comprising interleukin, OK432, NKp46, a serum replacement, and a serum-free medium.

[0007] Furthermore, the interleukin is selected from one or more of IL-12, IL-15, IL-2, IL-18, and IL-21.

[0008] Furthermore, the serum substitute is selected from CTS immune cell serum substitute.

[0009] Further, the final concentration of IL-12 was 1-10 ng / ml, the final concentration of IL-15 was 0.001-0.01 μg / ml, the final concentration of IL-2 was 100-200 U, the final concentration of IL-18 was 1-5 ng / ml, the final concentration of IL-21 was 1-5 ng / ml, the final concentration of OK432 was 0.01-0.1 KE / ml, the final concentration of NKp46 was 0.05-0.5 μg / ml, and the concentration of CTS immune cell serum replacement was 2.5%-10%.

[0010] Further, the final concentration of IL-12 was 10 ng / ml, the final concentration of IL-15 was 0.01 μg / ml, the final concentration of IL-2 was 200 U, the final concentration of IL-18 was 5 ng / ml, the final concentration of IL-21 was 5 ng / ml, the final concentration of OK432 was 0.02 KE / ml, the final concentration of NKp46 was 0.05 μg / ml, and the concentration of CTS immune cell serum replacement was 2.5%.

[0011] Furthermore, the composition also includes an expansion medium.

[0012] Furthermore, the expansion medium includes IL-2 and / or IL-15 and serum-free medium.

[0013] Furthermore, the final concentration of IL-2 is 200 U.

[0014] Furthermore, the final concentration of IL-15 is 0.01 μg / ml.

[0015] Furthermore, the composition also includes a coating solution.

[0016] Furthermore, the components of the coating solution include a buffer solution and an antibody.

[0017] Furthermore, the buffer is selected from PBS.

[0018] Furthermore, the antibody is selected from anti-CD16 antibody.

[0019] Furthermore, the final concentration of the anti-CD16 antibody is 0.01-0.05 μg / ml.

[0020] Furthermore, the final concentration of the anti-CD16 antibody is 0.05 μg / ml.

[0021] The second aspect of the present invention provides a method for expanding NK cells, which comprises culturing a NK cell population using the composition described in the first aspect of the present invention.

[0022] Furthermore, the method specifically comprises: 1) Coating a cell culture vessel with the coating solution described in the first aspect of the present invention; 2) activating the NK cell population using the activation medium described in the first aspect of the present invention; 3) Expanding the NK cell population activated in 2) using the expansion medium described in the first aspect of the present invention.

[0023] Furthermore, steps 2) and 3) further comprise adding autologous plasma.

[0024] Furthermore, the concentration of the autologous plasma is 1%-10%.

[0025] Furthermore, the NK cell population is obtained from peripheral blood mononuclear cells.

[0026] The third aspect of the present invention provides use of the composition described in the first aspect of the present invention or the method described in the second aspect of the present invention in culturing NK cells.

[0027] The fourth aspect of the present invention provides a NK cell, wherein the NK cell is cultured by the composition described in the first aspect of the present invention or the method described in the second aspect of the present invention.

[0028] The fifth aspect of the present invention provides a cell population, which includes the NK cells described in the fourth aspect of the present invention.

[0029] The sixth aspect of the present invention provides a pharmaceutical composition, which comprises the NK cells described in the fourth aspect of the present invention or the cell population described in the fifth aspect of the present invention.

[0030] Furthermore, the pharmaceutical composition also includes pharmaceutically acceptable excipients.

[0031] The seventh aspect of the present invention provides the use of the NK cells described in the fourth aspect of the present invention or the cell population described in the fifth aspect of the present invention in the preparation of a pharmaceutical composition for preventing / treating diseases, wherein the diseases include autoimmune diseases, blood system diseases or cancer.

[0032] Furthermore, the cancer includes solid cancer and non-solid cancer.

[0033] The eighth aspect of the present invention provides the use of the NK cells described in the fourth aspect of the present invention or the cell population described in the fifth aspect of the present invention in the preparation of a product having a killing effect on tumor cells.

[0034] Furthermore, the tumor cells are selected from K562.

[0035] Advantages and beneficial effects of the present invention: The present application uses a small number of factors, gradually reduces the amount of autologous plasma used in the expansion stage, and uses lower concentrations of IL-2 and IL-15 to reduce costs while meeting the needs of large-scale expansion of NK. The present application has a short culture cycle, and NK cells can be expanded more than 700 times in only 14 days, and more than 3000 times on the 20th day. The present application does not require sorting, and all mononuclear cells are used for culture, simplifying the operating procedures. The present application does not require feeder cells, serum, and blood replacement, avoids the risks brought by feeder cells and foreign blood, and has high safety performance. The present application process is simple to operate, low cost, and simplifies the experimental process, while also obtaining a large number of high-purity and high-cytotoxic NK cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 is a schematic diagram of NK cell expansion in Example 1; Figure 2 This is a schematic diagram of the results of flow cytometry detection of CD3-CD56+ of NK cells cultured for 14 days in Example 1; Figure 3 is a schematic diagram of the NK cell proliferation multiples in Example 2; Figure 4 This is a schematic diagram of the CD3-CD56+ results of flow cytometry detection of NK cells after 14 days of culture in Example 2; Figure 5 This is a graph showing the results of the killing experiment of NK cells cultured for 19 days against tumor cells K562 in Example 2; Figure 6 is a schematic diagram of NK cell expansion in Example 3; Figure 7 This is a schematic diagram of the results of flow cytometry detection of CD3-CD56+ of NK cells cultured for 14 days in Example 3; Figure 8 This is a schematic diagram of the results of flow cytometry detection of CD3-CD56+ of NK cells cultured for 20 days in Example 3; Fig. 9 This is a graph showing the results of the killing experiment of NK cells cultured for 14 days against tumor cells K562 in Example 3. DETAILED DESCRIPTION

[0037] The definitions of some terms used in this specification are provided below. Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0038] The present invention provides a composition for culturing NK cells. The composition comprises an activation culture medium, and the activation culture medium comprises interleukin, OK432, NKp46, a serum replacement, and a serum-free culture medium.

[0039] In some embodiments, natural killer cells (NK cells) are defined as large granular lymphocytes (LGL) and constitute the third type of cell differentiated from a common lymphoid progenitor cell that gives rise to B and T lymphocytes. NK cells are known to differentiate and mature in the bone marrow, lymph nodes, spleen, tonsils, and thymus, where they then enter the circulation. NK cells are phenotypically different from natural killer T cells (NKT) by origin and by their respective effector functions; typically, NKT cell activity promotes NK cell activity by secreting IFNγ. In contrast to NKT cells, NK cells do not express the T cell antigen receptor (TCR) or the pan T marker CD3 or the surface immunoglobulin (Ig) B cell receptor, but they typically express the surface markers CD16 (FcyRIII) and CD56 in humans and NK1.1 or NK1.2 in C57BL / 6 mice.

[0040] In some embodiments, the culture includes providing chemical and physical conditions (e.g., temperature, gas), and growth factors required for NK cell maintenance. Typically, culturing NK cells includes providing conditions for activation and amplification (proliferation) of NK cells. Examples of chemical conditions that can support NK cell expansion include, but are not limited to, buffers, serum, nutrients, vitamins, antibiotics, cytokines, and other growth factors, which are regularly provided in (or can be manually administered) a cell culture medium suitable for NK cell expansion.

[0041] The interleukin is selected from one or more of IL-12 (interleukin 12), IL-15 (interleukin 15), IL-2 (interleukin 2), IL-18 (interleukin 18), and IL-21 (interleukin 21).

[0042] In a specific embodiment, the interleukin is selected from a combination of IL-12, IL-15, IL-2, IL-18 and IL-21.

[0043] In some embodiments, interleukins also include derivatives of interleukins.

[0044] In some embodiments, serum replacement refers to an agent used as a substitute for serum (e.g., FBS) in cell culture to maintain the non-differentiated state of cells and their culture. Examples of serum replacements include KNOCKOUTTM SR (KnockOutTM serum replacement or KSR; Gibco), StemSure serum replacement (SSR; WakoPure Chemical Industries, Ltd.), N-2 supplement (Wako Pure Chemical Industries, Ltd.), CTS immune cell serum replacement.

[0045] In a specific embodiment, the serum replacement is selected from CTS immune cell serum replacement.

[0046] The present invention provides a method for expanding NK cells, which comprises culturing a NK cell population using the above composition.

[0047] The method specifically comprises: 1) Use the above coating solution to coat the cell culture vessel; 2) activating the NK cell population using the above activation medium; 3) Expand the NK cell population activated in 2) using the above expansion medium.

[0048] In some embodiments, expansion or proliferation refers to cell growth and an increase in cell number. Expansion or proliferation as used herein relates to an increased number of NK cells that occurs during culture.

[0049] In some embodiments, the cell culture container used in the culture of NK cells is not particularly limited as long as it can culture NK cells, and includes bottles, tissue culture bottles, dishes, flat dishes, tissue culture dishes, multi-dishes, microplates, microwell plates, multi-plates, multi-well plates, microscope slides, chamber slides, culture dishes, tubes, trays, culture bags, and roller bottles.

[0050] The cell culture device may be cell-adhesive or cell-non-adhesive, and may be appropriately selected according to the purpose. The cell-adhesive culture device may be coated with any cell-supporting matrix such as an extracellular matrix (ECM) for the purpose of increasing the adhesion of the surface of the culture device to the cells. The cell-supporting matrix may be any substance for the purpose of adhesion of NK cells.

[0051] Other culture conditions can be set appropriately. For example, the culture temperature is not particularly limited, but can be about 30-40° C., preferably about 37° C. The CO 2 concentration can be about 1-10%, preferably about 5%.

[0052] In some embodiments, the NK cell population can be obtained from peripheral blood mononuclear cells (PBMC), umbilical cord blood mononuclear cells, stem cells or bone marrow. In a preferred embodiment, the NK cell population is obtained from peripheral blood mononuclear cells.

[0053] The present invention provides a pharmaceutical composition, which comprises the above-mentioned NK cells or the above-mentioned cell population.

[0054] The pharmaceutical composition also includes pharmaceutically acceptable excipients.

[0055] In some embodiments, pharmaceutically acceptable refers to molecular entities and compositions that do not produce adverse, allergic or other inappropriate reactions when administered to animals such as humans. In view of the disclosure of this application, the preparation of pharmaceutical compositions containing antibodies or other active ingredients is known to those skilled in the art.

[0056] In some embodiments, pharmaceutically acceptable excipients include any and all aqueous solvents (e.g., water, alcohol / water solutions, saline solutions, parenteral vehicles such as sodium chloride, Ringer's dextrose, etc.), non-aqueous solvents (e.g., propylene glycol, polyethylene glycol, vegetable oils and injectable organic esters such as ethyl oleate), dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial or antifungal agents, antioxidants, chelating agents and inert gases), isotonic agents, absorption delaying agents, salts, drugs, drug stabilizers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, fluids and nutritional supplements, such similar materials and combinations thereof, which will be known to those of ordinary skill in the art. The pH and exact concentrations of the various components in the pharmaceutical composition are adjusted according to well-known parameters.

[0057] In some embodiments, the pharmaceutical composition for in vivo administration is usually provided in the form of a sterile preparation. Sterilization is achieved by filtering through a sterile filter membrane. When the composition is lyophilized, this method can be used for sterilization before or after lyophilization, reconstitution, dilution. The pharmaceutical composition of the present application can be selected for parenteral delivery. The composition for parenteral administration can be stored in a lyophilized form or in a solution. For example, it is prepared by conventional methods with physiological saline or an aqueous solution containing glucose and other adjuvants. Parenteral compositions are usually placed in a container with a sterile access hole, such as an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic injection needle. Alternatively, the composition can be selected for inhalation or delivery through the digestive tract (such as oral). The preparation of pharmaceutical compositions is within the technical scope of the art. Other pharmaceutical compositions will be apparent to those skilled in the art, including formulations containing antibodies in sustained or controlled release delivery formulations. Technologies for preparing a variety of other sustained or controlled delivery methods (such as liposome carriers, bio-erodible microparticles or porous beads and accumulated injections) are also known to those skilled in the art.

[0058] Once formulated, the pharmaceutical composition is stored in a sterile vial in the form of a solution, suspension, gel, emulsion, solid, crystal or lyophilized powder. The formulation can be stored in a ready-to-use form or in a form that is reconstituted (e.g., lyophilized) prior to administration.

[0059] In some embodiments, prevention and similar words, such as prevention, etc., indicate methods for preventing, inhibiting or reducing the likelihood of occurrence or recurrence of a condition. As used herein, prevention and similar words also include reducing the intensity, effects, symptoms and / or burden of a disease or condition before onset or recurrence.

[0060] The present invention provides the use of the above-mentioned NK cells or the above-mentioned cell population in preparing a pharmaceutical composition for preventing / treating diseases, wherein the diseases include autoimmune diseases, blood system diseases or cancers.

[0061] In some embodiments, treatment refers to the implementation of a regimen that may include administering one or more drugs to a patient in an effort to alleviate the signs or symptoms of a disease. Desirable therapeutic effects include reducing the rate of disease progression, improving or alleviating the disease state, and alleviating / or improving prognosis. Alleviation may occur before the onset of signs or symptoms of a disease or condition as well as after their onset. Thus, treatment may include the prevention of a disease or an undesirable condition. In addition, treatment does not require complete relief of signs or symptoms, does not require a cure, and particularly includes regimens that have only a marginal effect on a patient.

[0062] In some embodiments, cancer encompasses any type of cancer, including solid cancer and non-solid cancer. Specifically, the cancer includes cervical cancer, seminoma, testicular lymphoma, prostate cancer, ovarian cancer, lung cancer, rectal cancer, breast cancer, skin squamous cell carcinoma, colon cancer, liver cancer, pancreatic cancer, gastric cancer, esophageal cancer, thyroid cancer, bladder transitional epithelial carcinoma, leukemia, brain tumor, gastric cancer, peritoneal cancer, head and neck cancer, endometrial cancer, kidney cancer, female reproductive tract cancer, carcinoma in situ, neurofibroma, bone cancer, skin cancer, gastrointestinal stromal tumor, mast cell tumor, multiple myeloma, melanoma, glioma.

[0063] In some embodiments, the autoimmune disease described herein refers to a disease that results from an inappropriate immune response in a subject to substances and tissues that are normally present in the body. In other words, the immune system mistakes a part of the body for a pathogen and attacks its own cells. This may be limited to certain tissues (e.g., in autoimmune thyroiditis) or include specific tissues in different parts (e.g., Goodpasture's disease, which may affect the basement membranes of both the lungs and kidneys). Immunosuppressants, e.g., drugs that reduce immune responses, are commonly used to treat autoimmune diseases. Exemplary autoimmune diseases include, but are not limited to, glomerulonephritis, Goodpasture's syndrome, necrotizing vasculitis, lymphadenitis, periarteritis nodosa, systemic lupus erythematosus, rheumatoid disease, arthritis, psoriatic arthritis, systemic lupus erythematosus, psoriasis, ulcerative colitis, systemic sclerosis, dermatomyositis / polymyositis, antiphospholipid antibody syndrome, scleroderma, pemphigus vulgaris, ANCA-associated vasculitis (e.g., Wegener's granulomatosis, microscopic polyangiitis), uveitis, Sjogren's syndrome, Crohn's disease, Reiter's syndrome, ankylosing spondylitis, Lyme arthritis, Guillain-Barré syndrome, Hashimoto's thyroiditis, and cardiomyopathy.

[0064] The invention will be further described below in conjunction with specific examples. It should be understood that the specific embodiments described herein are presented by way of example and are not intended to limit the invention. The main features of the invention may be used in a variety of embodiments without departing from the scope of the invention.

[0065] Example Example 1

[0066] 1. Experimental Materials Reagents: immune cell serum-free medium, anti-CD16, OK432, NKp46, IL-2, IL-12, IL15, IL18, IL21, immune cell serum replacement (CTS immune cell serum replacement, brand Gibco, product number A2596101), immune cell serum-free medium (brand TBD, product number ANDLTBD-G).

[0067] 2. Experimental methods (1) Coat the cells with coating solution and incubate at 4°C overnight. The coating solution consists of PBS (phosphate buffered saline) and anti-CD16 antibody at a final concentration of 0.05 μg / ml.

[0068] (2) Using frozen peripheral blood mononuclear cells, resuspend the mononuclear cells in NK cell activation medium under different conditions, add 10% autologous plasma, and adjust the cell density to 2×10 6 The cells were cultured in a 37°C, 5% CO2 incubator.

[0069] a. The components of condition 1 activation medium are: IL-12 with a final concentration of 10 ng / ml; IL-15 with a final concentration of 0.01 μg / ml; IL-2 with a final concentration of 200 U; OK432 with a final concentration of 0.02 KE / ml; IL-18 with a final concentration of 0 ng / ml; IL-21 with a final concentration of 0 ng / ml; NKp46 with a final concentration of 0.05 μg / ml; 2.5% immune cell serum replacement; the rest is TBD-immune cell serum-free medium.

[0070] b. The components of condition 2 activation medium are: IL-12 with a final concentration of 10 ng / ml; IL-15 with a final concentration of 0.01 μg / ml; IL-2 with a final concentration of 200 U; OK432 with a final concentration of 0.02 KE / ml; IL-18 with a final concentration of 1 ng / ml; IL-21 with a final concentration of 0 ng / ml; NKp46 with a final concentration of 0.05 μg / ml; 2.5% immune cell serum replacement; the rest is TBD-immune cell serum-free medium.

[0071] c. The components of conditional three activation medium are: IL-12 with a final concentration of 10 ng / ml; IL-15 with a final concentration of 0.01 μg / ml; IL-2 with a final concentration of 200 U; OK432 with a final concentration of 0.02 KE / ml; IL-18 with a final concentration of 2 ng / ml; IL-21 with a final concentration of 0 ng / ml; NKp46 with a final concentration of 0.05 μg / ml; 2.5% immune cell serum replacement; the rest is TBD-immune cell serum-free medium.

[0072] d. The components of condition IV activation medium are: IL-12 with a final concentration of 10 ng / ml; IL-15 with a final concentration of 0.01 μg / ml; IL-2 with a final concentration of 200 U; OK432 with a final concentration of 0.02 KE / ml; IL-18 with a final concentration of 5 ng / ml; IL-21 with a final concentration of 0 ng / ml; NKp46 with a final concentration of 0.05 μg / ml; 2.5% immune cell serum replacement; the rest is TBD-immune cell serum-free medium.

[0073] e. The components of condition five activation medium are: IL-12 with a final concentration of 10 ng / ml; IL-15 with a final concentration of 0.01 μg / ml; IL-2 with a final concentration of 200 U; OK432 with a final concentration of 0.02 KE / ml; IL-18 with a final concentration of 10 ng / ml; IL-21 with a final concentration of 0 ng / ml; NKp46 with a final concentration of 0.05 μg / ml; 2.5% immune cell serum replacement; the rest is TBD-immune cell serum-free medium.

[0074] f. The components of condition six activation medium are: IL-12 with a final concentration of 10 ng / ml; IL-15 with a final concentration of 0.01 μg / ml; IL-2 with a final concentration of 200 U; OK432 with a final concentration of 0.02 KE / ml; IL-18 with a final concentration of 0 ng / ml; IL-21 with a final concentration of 1 ng / ml; NKp46 with a final concentration of 0.05 μg / ml; 2.5% immune cell serum replacement; the rest is TBD-immune cell serum-free medium.

[0075] g. The components of condition seven activation medium are: IL-12 with a final concentration of 10 ng / ml; IL-15 with a final concentration of 0.01 μg / ml; IL-2 with a final concentration of 200 U; OK432 with a final concentration of 0.02 KE / ml; IL-18 with a final concentration of 0 ng / ml; IL-21 with a final concentration of 2 ng / ml; NKp46 with a final concentration of 0.05 μg / ml; 2.5% immune cell serum replacement; the rest is TBD-immune cell serum-free medium.

[0076] h. The components of condition eight activation medium are: IL-12 with a final concentration of 10 ng / ml; IL-15 with a final concentration of 0.01 μg / ml; IL-2 with a final concentration of 200 U; OK432 with a final concentration of 0.02 KE / ml; IL-18 with a final concentration of 0 ng / ml; IL-21 with a final concentration of 5 ng / ml; NKp46 with a final concentration of 0.05 μg / ml; 2.5% immune cell serum replacement; the rest is TBD-immune cell serum-free medium.

[0077] i. The components of condition nine activation medium are: IL-12 with a final concentration of 10 ng / ml; IL-15 with a final concentration of 0.01 μg / ml; IL-2 with a final concentration of 200 U; OK432 with a final concentration of 0.02 KE / ml; IL-18 with a final concentration of 0 ng / ml; IL-21 with a final concentration of 10 ng / ml; NKp46 with a final concentration of 0.05 μg / ml; 2.5% immune cell serum replacement; the rest is TBD-immune cell serum-free medium.

[0078] j. The components of condition ten activation medium are: IL-12 with a final concentration of 10 ng / ml; IL-15 with a final concentration of 0.01 μg / ml; IL-2 with a final concentration of 200 U; OK432 with a final concentration of 0.02 KE / ml; IL-18 with a final concentration of 2 ng / ml; IL-21 with a final concentration of 2 ng / ml; NKp46 with a final concentration of 0.05 μg / ml; 2.5% immune cell serum replacement; the rest is TBD-immune cell serum-free medium.

[0079] k. The components of condition 11 activation medium are: IL-12 with a final concentration of 10 ng / ml; IL-15 with a final concentration of 0.01 μg / ml; IL-2 with a final concentration of 200 U; OK432 with a final concentration of 0.02 KE / ml; IL-18 with a final concentration of 5 ng / ml; IL-21 with a final concentration of 5 ng / ml; NKp46 with a final concentration of 0.05 μg / ml; 2.5% immune cell serum replacement; the rest is TBD-immune cell serum-free medium.

[0080] l. The components of condition twelve activation medium are: IL-12 with a final concentration of 10 ng / ml; IL-15 with a final concentration of 0.01 μg / ml; IL-2 with a final concentration of 200 U; OK432 with a final concentration of 0.02 KE / ml; IL-18 with a final concentration of 10 ng / ml; IL-21 with a final concentration of 10 ng / ml; NKp46 with a final concentration of 0.05 μg / ml; 2.5% immune cell serum replacement; the rest is TBD-immune cell serum-free medium.

[0081] (3) On the third day of culture, add NK cell expansion medium 1 and 10% autologous plasma, and continue to incubate in a 37°C, 5% CO2 incubator. The expansion medium 1 components are: IL-2 with a final concentration of 200 U; IL-15 with a final concentration of 0.01 μg / ml; the rest is immune cell serum-free medium.

[0082] (4) On the fifth day of culture, count the cells, add NK cell expansion medium 1, add 10% autologous plasma, and adjust the cell density to 1×10 6 The cells were cultured in a 37°C, 5% CO2 incubator.

[0083] (5) On the 7th day of culture, count the cells, add NK cell expansion medium 1, add 5% autologous plasma, and adjust the cell density to 1×10 6 The cells were cultured in a 37°C, 5% CO2 incubator.

[0084] (6) On the 9th day of culture, count the cells, add NK cell expansion medium 1, add 5% autologous plasma, and adjust the cell density to 1×10 6 The cells were cultured in a 37°C, 5% CO2 incubator.

[0085] (7) On the 12th day of culture, count the cells, add NK cell expansion medium 1, add 1% autologous plasma, and adjust the cell density to 1×10 6 The cells were cultured in a 37°C, 5% CO2 incubator.

[0086] (8) On the 14th day of culture, count the cells, add NK cell expansion medium 2, add 1% autologous plasma, and adjust the cell density to 1×10 6 / mL, and flow cytometry was performed. The components of expansion medium 2 were: IL-2 with a final concentration of 200 U.

[0087] (9) Count cells every 2-3 days and replenish expansion medium 2 to adjust the cell density to 1×10 6 Pieces / mL.

[0088] 3. Experimental results The results are as follows Figure 1 As shown, under condition 11 combination, after 20 days of culture, the NK cell expansion ability was the strongest, reaching 2014 times.

[0089] The results are as follows Figure 2 As shown, flow cytometry was used to detect NK cell surface markers CD3, CD56, and CD45. The experimental results showed that after 20 days of cell culture, under condition 11, the NK cell expansion purity was the highest, reaching 94%.

[0090] The experimental results show that the present invention provides a culture medium and method for efficiently expanding NK cells in vitro. The culture method of the present invention uses a monoclonal antibody coating of NK-specific activation receptors to stimulate NK cells. After the stimulation and expansion stages, the NK cells of the present invention are expanded by 2014 times and the cell purity is 94%.

[0091] Example 2 1. Experimental Materials Reagents: Immune cell serum-free medium, anti-CD16, OK432, NKp46, IL-2, IL-12, IL15, IL18, IL21.

[0092] 2. Experimental methods (1) Conditions 1 to 3: Incubate the cells with coating solution at 4°C overnight. The components of the coating solution are: PBS (phosphate buffered saline) with a final concentration of 0.05 μg / ml anti-CD16 antibody. Condition 4: Incubate the cells with coating solution at 4°C overnight. The components of the coating solution are: PBS (phosphate buffered saline) with a final concentration of 0.02 μg / ml anti-CD16 antibody.

[0093] (2) Using frozen peripheral blood mononuclear cells, resuspend the mononuclear cells in NK cell activation medium under different conditions, add 10% autologous plasma, and adjust the cell density to 2×10 6 The cells were cultured in a 37°C, 5% CO2 incubator.

[0094] a. The components of condition 1 activation medium are: IL-12 with a final concentration of 10 ng / ml; IL-15 with a final concentration of 0.01 μg / ml; IL-2 with a final concentration of 200 U; OK432 with a final concentration of 0.02 KE / ml; IL-18 with a final concentration of 5 ng / ml; IL-21 with a final concentration of 5 ng / ml; NKp46 with a final concentration of 0.05 μg / ml; 2.5% immune cell serum replacement; the rest is TBD-immune cell serum-free medium.

[0095] b. The components of condition 2 activation medium are: IL-12 with a final concentration of 10 ng / ml; IL-15 with a final concentration of 0.01 μg / ml; IL-2 with a final concentration of 200 U; OK432 with a final concentration of 0.02 KE / ml; IL-18 with a final concentration of 5 ng / ml; IL-21 with a final concentration of 5 ng / ml; NKp46 with a final concentration of 0.05 μg / ml; 1% immune cell serum replacement; the rest is TBD-immune cell serum-free medium.

[0096] c. The components of conditional three activation medium are: IL-12 with a final concentration of 10 ng / ml; IL-15 with a final concentration of 0.01 μg / ml; IL-2 with a final concentration of 200 U; OK432 with a final concentration of 0.02 KE / ml; IL-18 with a final concentration of 5 ng / ml; IL-21 with a final concentration of 5 ng / ml; NKp46 with a final concentration of 0.05 μg / ml; and the rest is TBD-immune cell serum-free medium.

[0097] f. The components of condition IV activation medium are: IL-12 with a final concentration of 10 ng / ml; IL-15 with a final concentration of 0.01 μg / ml; IL-2 with a final concentration of 200 U; OK432 with a final concentration of 0.02 KE / ml; IL-18 with a final concentration of 5 ng / ml; IL-21 with a final concentration of 5 ng / ml; NKp46 with a final concentration of 0.05 μg / ml; and the rest is TBD-immune cell serum-free medium.

[0098] (3) On the third day of culture, add NK cell expansion medium 1 and 10% autologous plasma, and continue to incubate in a 37°C, 5% CO2 incubator. The expansion medium 1 components are: IL-2 with a final concentration of 200 U; IL-15 with a final concentration of 0.01 μg / ml; the rest is immune cell serum-free medium.

[0099] (4) On the fifth day of culture, count the cells, add NK cell expansion medium 1, add 10% autologous plasma, and adjust the cell density to 1×10 6 The cells were cultured in a 37°C, 5% CO2 incubator.

[0100] (5) On the 7th day of culture, count the cells, add NK cell expansion medium 1, add 5% autologous plasma, and adjust the cell density to 1×10 6 The cells were cultured in a 37°C, 5% CO2 incubator.

[0101] (6) On the 9th day of culture, count the cells, add NK cell expansion medium 1, add 5% autologous plasma, and adjust the cell density to 1×10 6 The cells were cultured in a 37°C, 5% CO2 incubator.

[0102] (7) On the 12th day of culture, count the cells, add NK cell expansion medium 1, add 1% autologous plasma, and adjust the cell density to 1×10 6 The cells were cultured in a 37°C, 5% CO2 incubator.

[0103] (8) On the 14th day of culture, count the cells, add NK cell expansion medium 2, and adjust the cell density to 1×10 6 / mL, and continue to incubate in a 37°C, 5% CO2 incubator. The two components of the expansion medium are: IL-2 with a final concentration of 200 U; the rest is immune cell serum-free medium.

[0104] (9) Count cells every 2-3 days and replenish expansion medium 2 to adjust the cell density to 1×10 6Pieces / mL.

[0105] 3. Experimental results The results are as follows Figure 3 As shown, under condition 1, after 19 days of culture, the NK cell expansion capacity was the strongest, reaching 8280 times.

[0106] The results are as follows Figure 4 As shown, flow cytometry was used to detect NK cell surface markers CD3, CD56, and CD45. The experimental results showed that after 14 days of cell culture, the CD3-CD56+ of NK cells were: 94.49% under condition 1; 91.15% under condition 2; 92.39% under condition 3; and 90.84% ​​under condition 4.

[0107] The results are as follows Figure 5 As shown, after NK cells and K562 cells were co-cultured at different ratios for 4 hours, the death rate of K562 was detected by flow cytometry. The test results showed that as the effector-target ratio increased, the killing rate of NK cells against tumor cells gradually increased, among which the killing rate of K562 tumor cells could reach 82.25%.

[0108] Example 3 1. Experimental Materials Reagents: Immune cell serum-free medium, anti-CD16, OK432, NKp46, IL-2, IL-12, IL15, IL18, IL21.

[0109] 2. Experimental methods In order to verify whether the present invention has the same effect when applied to large-volume culture, the following experiment was conducted.

[0110] (1) Coat the cells with coating solution and incubate at 4°C overnight. The coating solution consists of PBS (phosphate buffered saline) and anti-CD16 antibody at a final concentration of 0.05 μg / ml.

[0111] (2) Using frozen peripheral blood mononuclear cells, resuspend the mononuclear cells in NK cell activation medium, add 10% autologous plasma, and adjust the cell density to 2×10 6 / mL, incubated in a 37°C, 5% CO2 incubator. The components of the activation medium are IL-12 with a final concentration of 10ng / ml; IL-15 with a final concentration of 0.01μg / ml; IL-2 with a final concentration of 200 U; OK432 with a final concentration of 0.02 KE / ml; IL-18 with a final concentration of 5ng / ml; IL-21 with a final concentration of 5ng / ml; NKp46 with a final concentration of 0.05μg / ml; 2.5% immune cell serum replacement; the rest is immune cell serum-free medium.

[0112] (3) On the third day of culture, add NK cell expansion medium 1 and 10% autologous plasma, and continue to incubate in a 37°C, 5% CO2 incubator. The expansion medium 1 components are: IL-2 with a final concentration of 200 U; IL-15 with a final concentration of 0.01 μg / ml; the rest is immune cell serum-free medium.

[0113] (4) On the fifth day of culture, count the cells, add NK cell expansion medium 1, add 10% autologous plasma, and adjust the cell density to 1×10 6 The cells were cultured in a 37°C, 5% CO2 incubator.

[0114] (5) On the 7th day of culture, count the cells, add NK cell expansion medium 1, add 5% autologous plasma, and adjust the cell density to 1×10 6 The cells were cultured in a 37°C, 5% CO2 incubator.

[0115] (6) On the 9th day of culture, count the cells, add NK cell expansion medium 1, add 5% autologous plasma, and adjust the cell density to 1×10 6 The cells were cultured in a 37°C, 5% CO2 incubator.

[0116] (7) On the 12th day of culture, count the cells, add NK cell expansion medium 1, add 1% autologous plasma, and adjust the cell density to 1×10 6 The cells were cultured in a 37°C, 5% CO2 incubator.

[0117] (8) On the 14th day of culture, count the cells, add NK cell expansion medium 2, and adjust the cell density to 1×10 6 / mL, and continue to incubate in a 37°C, 5% CO2 incubator. The two components of the expansion medium are: IL-2 with a final concentration of 200 U; the rest is immune cell serum-free medium.

[0118] (9) Count cells every 2-3 days and replenish expansion medium 2 to adjust the cell density to 1×10 6 Pieces / mL.

[0119] 3. Experimental results The results are as follows Figure 6 As shown, under amplified culture conditions, NK cells expanded 3707 times after 20 days of culture.

[0120] The results are as follows Figure 7 and Figure 8As shown, flow cytometry was used to detect NK cell surface markers CD3, CD56, and CD45. The experimental results showed that after 14 days of cell culture, the CD3-CD56+ of NK cells was 93.86% ( Figure 7 ); When the culture was continued for 20 days, the CD3-CD56+ of NK cells was 99.40% ( Figure 8 ).

[0121] The results are as follows Fig. 9 As shown in the figure, after NK cells and K562 cells were co-cultured at different ratios for 4 hours, the death ratio of K562 was detected by flow cytometry. The test results showed that as the effector-target ratio increased, the killing rate of NK cells against tumor cells gradually increased, among which the killing rate of K562 tumor cells could reach 89.82%.

[0122] The description of the above embodiments is only used to understand the method and core idea of ​​the present invention. It should be pointed out that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principle of the present invention, and these improvements and modifications will also fall within the scope of protection of the claims of the present invention.

Claims

1. A composition for culturing NK cells, characterized in that: The composition comprises an activation medium, wherein the activation medium comprises interleukin, OK432, NKp46, serum replacement, and serum-free medium; Preferably, the interleukin is selected from one or more of IL-12, IL-15, IL-2, IL-18, and IL-21; Preferably, the serum replacement is selected from CTS immune cell serum replacement; Preferably, the final concentration of IL-12 is 1-10 ng / ml, the final concentration of IL-15 is 0.001-0.01 μg / ml, the final concentration of IL-2 is 100-200 U, the final concentration of IL-18 is 1-5 ng / ml, the final concentration of IL-21 is 1-5 ng / ml, the final concentration of OK432 is 0.01-0.1 KE / ml, the final concentration of NKp46 is 0.05-0.5 μg / ml, and the concentration of CTS immune cell serum replacement is 2.5%-10%; Preferably, the final concentration of IL-12 is 10 ng / ml, the final concentration of IL-15 is 0.01 μg / ml, the final concentration of IL-2 is 200 U, the final concentration of IL-18 is 5 ng / ml, the final concentration of IL-21 is 5 ng / ml, the final concentration of OK432 is 0.02 KE / ml, the final concentration of NKp46 is 0.05 μg / ml, and the concentration of CTS immune cell serum replacement is 2.5%.

2. The composition according to claim 1, characterized in that The composition also includes an expansion medium; Preferably, the expansion medium comprises IL-2 and / or IL-15, serum-free medium; Preferably, the final concentration of IL-2 is 200 U; Preferably, the final concentration of IL-15 is 0.01 μg / ml.

3. The composition according to claim 2, characterized in that The composition further comprises a coating fluid; Preferably, the components of the coating solution include a buffer and an antibody; Preferably, the buffer is selected from PBS; Preferably, the antibody is selected from anti-CD16 antibody; Preferably, the final concentration of the anti-CD16 antibody is 0.01-0.05 μg / ml; Preferably, the final concentration of the anti-CD16 antibody is 0.05 μg / ml.

4. A method for expanding NK cells, characterized in that: The method comprises culturing a population of NK cells using the composition of any one of claims 1 to 3; Preferably, the method specifically comprises: 1) Coating a cell culture vessel with the coating solution according to claim 3; 2) activating the NK cell population using the activation medium according to claim 1; 3) using the expansion medium of claim 2 to expand the activated NK cell population in 2); Preferably, steps 2) and 3) further comprise adding autologous plasma; Preferably, the concentration of the autologous plasma is 1%-10%; Preferably, the NK cell population is obtained from peripheral blood mononuclear cells.

5. Use of the composition according to any one of claims 1 to 3 or the method according to claim 4 in culturing NK cells.

6. A NK cell, characterized in that: The NK cells are obtained by culturing the composition according to any one of claims 1 to 3 or the method according to claim 4.

7. A cell population, characterized in that The cell population includes the NK cells described in claim 6.

8. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the NK cell of claim 6 or the cell population of claim 7; Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

9. Use of the NK cell according to claim 6 or the cell population according to claim 7 in the preparation of a pharmaceutical composition for preventing / treating a disease, wherein the disease comprises an autoimmune disease, a blood system disease or a cancer; Preferably, the cancer includes solid cancers and non-solid cancers.

10. Use of the NK cell according to claim 6 or the cell population according to claim 7 in preparing a product having a killing effect on tumor cells; Preferably, the tumor cells are selected from K562.

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