Preparation and application of NK cells

Abstract

The present invention provides the preparation and application of NK cells. Specifically, the present invention provides a culture medium for culturing NK cells, comprising the following components: IL-2, IL-15, IL-12, IL-21, CD137 antibody, CD3 antibody, vitamins, basal culture medium, serum substitute. NK cells cultured with the culture medium have high activity and have good killing activity against tumor cells.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to the preparation and application of NK cells. Background Art

[0002] In recent years, research on chimeric antigen receptor (CAR)-based cell therapy for cancer has been very popular. CAR-T cells have achieved great success in the treatment of blood cancer, but there are still some defects in clinical application that need to be overcome, such as severe cytokine release syndrome (CRS), neurotoxicity and high treatment costs. As an important effector cell in innate immunity, NK cells are a type of MHC-independent lymphocytes. Compared with T cells, NK cells have more mechanisms to kill target cells and have a broad spectrum of anti-tumor effects. The main factors secreted by NK cells are IL-3 and GM-CSF, so it is almost impossible to cause cytokine storm (CRS). In addition, allogeneic NK cells rarely cause graft-versus-host disease (GvHD), which is conducive to the preparation of universal CAR-NK, thereby reducing clinical costs. Based on these advantages, NK cells are regarded as effector cells with greater potential to enhance their anti-tumor ability through CAR modification and are safer and more suitable for clinical application. According to data on the ClinicalTrials.gov website, many clinical trials of CAR-NK products are currently underway, most of which are in Phase I and Phase II clinical stages. The anti-tumor activity and safety in clinical applications are receiving much attention.

[0003] NK cells have a wide range of sources and can be obtained from peripheral blood (PB), umbilical cord blood (UCB), human induced pluripotent stem cells (hiPSC) and NK cell lines. iPSC-derived NK cells have many advantages, including low immunogenicity, unlimited proliferation, and low cost, but allogeneic iPSCs may have problems such as tumorigenicity and heterogeneity; NK cell lines such as NK-92 are derived from tumor tissues. Although they are easy to genetically modify, they need to be irradiated before being introduced into the human body, which increases safety risks and reduces the durability and efficacy of NK cells in patients; NK cells from umbilical cord blood and peripheral blood have high proliferation capacity, but studies have shown that PB-NK is more capable of killing tumors in vitro than UCB-NK and is more convenient to obtain. Therefore, PB-NK has good application prospects in tumor immunotherapy compared with NK cells from other sources.

[0004] Although the development and application of PB-NK cells have made great progress in recent years, the production technology of PB-NK cells still faces many challenges. PBMC can be obtained by directly collecting fresh blood from the donor's vein or by obtaining it from a single blood collection source. PBMC obtained by directly collecting fresh peripheral blood from the vein has good activity, which is beneficial for inducing the expansion of NK cells. However, the number of PBMCs from a single donor that can be obtained at one time is limited. The activation and expansion of PBMCs are greatly affected by individual differences of donors. There are also differences in blood collected from the same donor at different times. If the expansion of NK cells is directly induced after collection, it is easy to cause failure, which is not conducive to the stability and uniformity of the final cell product. In addition, the open and manual blood collection steps cannot meet the safety requirements of clinical trials, and there are some barriers that cannot be overcome in ethical requirements. PBMCs from single blood collection can obtain a larger number at one time, and can be frozen for the preparation of multiple batches. They are suitable for screening donors, improving the stability and uniformity of different batches of cell products, and more in line with the requirements of clinical trials. However, the induction and expansion of NK cells from frozen single-collected blood PBMCs faces a very challenging problem, that is, NK cells are extremely sensitive to the freezing and thawing process, and the activity of NK cells in thawed PBMCs is reduced, making it difficult to activate and expand them. Therefore, solving the problem of activating NK cells in thawed PBMCs is the top priority for preparing highly active single-collected blood NK cells.

[0005] The present invention describes in detail how to activate and massively expand NK cells obtained by sorting PBMCs from a single blood sample after resuscitation in vitro.

[0006] References:

[0007] 1.Martha Luevano et al, The unique profile of cord blood natural killercells balances incomplete maturation and effective killing function upon activation, Human Immunology 73 (2012) 248-257.

[0008] 2. Jacob A. Myers, Jeffrey S. Miller; Exploring the NK cell platform for cancer immunotherapy, Nat Rev Clin Oncol, 2021February, 18(2):85-100.

[0009] 3. Ying Gong et al, Chimeric antigen receptor natural killer (CAR-NK) cell design and engineering for cancer therapy, J Hematol Oncol (2021) 14:73. Summary of the invention

[0010] The purpose of the present invention is to establish a set of schemes suitable for PBMC from a single blood collection to be effectively activated and amplified in large quantities after freezing and thawing, and finally to harvest highly active NK cells. This scheme is also suitable for the amplification and culture of fresh PB-NK.

[0011] To achieve the above object, the present invention provides the following embodiments:

[0012] In the first aspect, the present invention provides a composition for culturing NK cells, the composition comprising an activation medium composition, and the activation medium composition is composed of the following components: IL-2, IL-15, IL-12, IL-21, CD137 antibody, CD3 antibody, multivitamins, basal culture medium, and serum replacement.

[0013] The term "natural killer cell (NK cell)" is defined as a large granular lymphocyte (LGL) and constitutes a third type of cell differentiated from a common lymphoid progenitor cell that produces B and T lymphocytes. It is known that NK cells differentiate and mature in the bone marrow, lymph nodes, spleen, tonsils, and thymus, where they then enter the circulation. According to origin and according to respective effector functions, NK cells are phenotypically different from natural killer T cells (NKT); generally, NKT cell activity promotes NK cell activity by secreting IFNγ. Compared with NKT cells, NK cells do not express T cell antigen receptors (TCR) or pan T markers (pan T marker) CD3 or surface immunoglobulin (Ig) B cell receptors, but they generally express surface markers CD16 (FcyRIII) and CD56 in humans, and express NK1.1 or NK1.2 in C57BL / 6 mice.

[0014] The term "culture" as used herein includes providing the chemical and physical conditions (e.g., temperature, gas), and growth factors required for NK cell maintenance. Conventionally, NK cells are cultured including providing conditions for NK cells to be used for expansion (proliferation). 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 given) a cell culture medium suitable for NK cell expansion.

[0015] As used herein, the terms "IL-2", "IL-15", "IL-12", "IL-21" refer to the cytokines interleukin-2, interleukin-15, interleukin-12, interleukin-21 and their derivatives.

[0016] As used herein, the term "antibody" includes reference to glycosylated and non-glycosylated immunoglobulins of any isotype or subclass or to antigen binding regions thereof that compete with intact antibodies for specific binding, including human, humanized, chimeric, multispecific, monoclonal, polyclonal antibodies and oligomers or antigen binding fragments thereof. Also included are proteins with antigen binding fragments or regions, such as Fab, Fab', F(ab')2, Fv, diabodies, Fd, dAb, large antibodies, single-chain antibody molecules, complementary determining region (CDR) fragments, scFv, diabodies, triabodies, tetrabodies and polypeptides, which contain at least a portion of an immunoglobulin sufficient to confer specific antigen binding to a target polypeptide. The term "antibody" includes, but is not limited to, antibodies prepared, expressed, produced or isolated by recombinant means, such as antibodies isolated from host cells transfected to express the antibody.

[0017] In one embodiment of the present invention, the CD137 antibody and the CD3 antibody are CD137 monoclonal antibody and CD3 monoclonal antibody.

[0018] The term "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 KNOCKOUT™ SR (KnockOut™ serum replacement or KSR; Gibco), StemSure serum replacement (SSR; WakoPure Chemical Industries, Ltd.), N-2 supplement (Wako Pure Chemical Industries, Ltd.), CTS TM Immune cell serum substitute. In a specific embodiment of the present invention, the serum substitute is CTS TM Immune cell serum replacement.

[0019] In one embodiment of the present invention, in the activation medium composition, the concentration of IL-2 in the medium is 1000-10000 IU / mL, the concentration of IL-15 in the medium is 50-2000 IU / mL, the concentration of IL-12 in the medium is 50-500 IU / mL, the concentration of IL-21 in the medium is 0.1-10 IU / mL, the concentration of CD137 antibody in the medium is 5-15 μg / mL, the concentration of CD3 antibody in the medium is 5-15 μg / mL, the concentration of multivitamins in the medium is 0-1000 ng / mL, and the concentration of serum replacement in the medium is 3-10%.

[0020] As a more preferred embodiment, in the activation medium composition, the concentration of IL-2 in the medium is 1000 IU / mL, the concentration of IL-15 in the medium is 300 IU / mL, the concentration of IL-12 in the medium is 100 IU / mL, the concentration of IL-21 in the medium is 5 IU / mL, the concentration of CD137 antibody in the medium is 5 μg / mL, the concentration of CD3 antibody in the medium is 5 μg / mL, the concentration of multivitamins in the medium is 100 ng / mL, and the concentration of serum substitute in the medium is 5%.

[0021] In one embodiment of the present invention, the basal culture medium of the activation culture medium composition is selected from NKMACS, X-VIVO15, GT-T551H3, cellgrowSCGM, and AIM-V. Preferably, the basal culture medium of the activation culture medium composition is X-VIVO15.

[0022] In one embodiment of the present invention, the complex vitamins of the activation medium composition are selected from one or more of vitamin A / retinol, vitamin B2 / vitamin B6 / vitamin B12, vitamin C / L-ascorbic acid, and vitamin E. Preferably, the complex vitamins of the activation medium composition contain vitamin B2 and vitamin E.

[0023] In one embodiment of the present invention, the composition of the present invention further comprises a first amplification medium composition, and the first amplification medium composition consists of the following components: serum replacement, IL-2, IL-15, IL-12, multivitamins, and basal culture medium.

[0024] In one embodiment of the present invention, in the first expansion medium composition, the concentration of serum replacement in the medium is 3-10%, the concentration of IL-2 in the medium is 1000-10000 IU / mL, the concentration of IL-15 in the medium is 50-2000 IU / mL, the concentration of IL-12 in the medium is 50-500 IU / mL, and the concentration of multivitamins in the medium is 0-1000 ng / mL.

[0025] As a more preferred embodiment, in the first amplification medium composition, the concentration of serum replacement in the medium is 5%, the concentration of IL-2 in the medium is 1000 IU / mL, the concentration of IL-15 in the medium is 300 IU / mL, the concentration of IL-12 in the medium is 100 IU / mL, and the concentration of multivitamins in the medium is 100 ng / mL.

[0026] In one embodiment of the present invention, the composition of the present invention further comprises a second amplification medium composition, and the second amplification medium composition consists of the following components: serum replacement, IL-2, IL-15, vitamins, and basal culture medium.

[0027] In one embodiment of the present invention, in the second amplification medium composition, the concentration of serum replacement in the medium is 3-10%, the concentration of IL-2 in the medium is 1000-10000 IU / mL, the concentration of IL-15 in the medium is 50-2000 IU / mL, and the concentration of multivitamins in the medium is 0-1000 ng / mL.

[0028] As a more preferred embodiment, in the second amplification medium composition, the concentration of serum replacement in the medium is 5%, the concentration of IL-2 in the medium is 1000 IU / mL, the concentration of IL-15 in the medium is 300 IU / mL, and the concentration of multivitamins in the medium is 100 ng / mL.

[0029] In one embodiment of the present invention, the basal culture medium of the first amplification medium composition and the second amplification medium composition are each independently selected from NK MACS, X-VIVO15, GT-T551H3, cellgrowSCGM, and AIM-V. Preferably, the basal culture medium of the first amplification medium composition and the second amplification medium composition are each independently X-VIVO15.

[0030] In one embodiment of the present invention, the complex vitamins of the first amplification medium composition and the second amplification medium composition are each independently selected from one or more of vitamin A / retinol, vitamin B2 / vitamin B6 / vitamin B12, and vitamin C / L-ascorbic acid. Preferably, the complex vitamins of the first amplification medium composition and the second amplification medium composition each independently include vitamin B2 and vitamin E.

[0031] In one embodiment of the present invention, the composition of the present invention further comprises a coating fluid composition, and the coating fluid composition comprises CD137 antibody, CD28 antibody, and CD16 antibody.

[0032] In one embodiment of the present invention, in the coating solution composition, the concentration of CD137 antibody in the coating solution is 8 μg / mL;

[0033] In one embodiment of the present invention, in the coating solution composition, the concentration of CD28 antibody in the coating solution is 8 μg / mL;

[0034] In one embodiment of the present invention, in the coating solution composition, the concentration of the CD16 antibody in the coating solution is 8 μg / mL.

[0035] In one embodiment of the present invention, the CD137 antibody, CD28 antibody, and CD16 antibody are CD137 monoclonal antibody, CD28 monoclonal antibody, and CD16 monoclonal antibody.

[0036] In a second aspect, the present invention provides a method for expanding NK cells, the method comprising: (1) pretreating a cell culture container with the coating solution composition described in the first aspect of the present invention;

[0037] (2) obtaining a population of NK cells;

[0038] (3) in the cell culture container obtained in 1), using the activation medium composition of the first aspect of the present invention to activate the NK cell population obtained in 2) to obtain an activated NK cell population;

[0039] (4) The first expansion medium composition and the second expansion medium composition described in the first aspect of the present invention are used to expand the activated NK cell population.

[0040] As used herein, the term "expansion" or "proliferation" refers to cell growth and an increase in cell number. As used herein, expansion or proliferation relates to an increased number of NK cells that occurs during a culture process, such as disclosed herein.

[0041] The cell culture container used in the culture of NK cells is not particularly limited as long as it can culture NK cells, but examples include 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.

[0042] The cell culture device can be cell adhesive or cell non-adhesive, and is suitably selected according to the purpose. The cell adhesive culture device can be coated with any cell support matrix such as an extracellular matrix (ECM) etc. for the purpose of increasing the adhesion of the surface of the culture device to the cell. The cell support matrix can be any material for the purpose of adhesion of NK cells.

[0043] Other culture conditions can be set appropriately. For example, the culture temperature is not particularly limited, but can be about 30 to 40° C., preferably about 37° C. The CO 2 concentration can be about 1 to 10%, preferably about 2 to 5%. The oxygen partial pressure can be 1 to 10%.

[0044] In one embodiment of the present invention, the NK cell population is obtained from peripheral blood mononuclear cells, umbilical cord blood mononuclear cells, stem cells or bone marrow. Preferably, the NK cell population is obtained from peripheral blood mononuclear cells. More preferably, the peripheral blood mononuclear cells are peripheral blood mononuclear cells that have been cryopreserved and resuscitated.

[0045] In one embodiment of the present invention, the method for obtaining a NK cell population comprises subjecting the peripheral blood mononuclear cells to magnetic bead sorting. Preferably, the magnetic bead sorting comprises magnetic bead negative sorting or magnetic bead positive sorting.

[0046] More preferably, the magnetic beads used in the magnetic bead negative selection include CD3 magnetic beads, CD14 magnetic beads, and CD19 magnetic beads. Most preferably, the amount of the magnetic beads is 20 μL / 10 7 cell.

[0047] More preferably, the magnetic beads used in the magnetic bead positive selection include CD56 magnetic beads.

[0048] In one embodiment of the present invention, the method of using the first expansion medium composition and the second expansion medium composition described in the first aspect of the present invention to expand the activated NK cell population comprises:

[0049] a. Day 3-Day 5, culturing the activated NK cell population obtained in step 3) of the second aspect using the first expansion medium composition of the present invention;

[0050] b. Day 6-Day 24, culturing the NK cell population described in a using the second expansion medium composition of the present invention;

[0051] c. Harvest NK cells on Day 25.

[0052] In a third aspect, the present invention provides a NK cell product, wherein the NK cell product is cultured using the method described in the second aspect of the present invention.

[0053] In one embodiment of the present invention, the purity of NK cells in the NK cell product reaches more than 99%.

[0054] In a fourth aspect, the present invention provides a pharmaceutical composition comprising the NK cell product described in the third aspect of the present invention and a pharmaceutically acceptable carrier.

[0055] The phrase "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 present disclosure, the preparation of pharmaceutical compositions comprising antibodies or other active ingredients is known to those skilled in the art. In addition, for animal (e.g., human) administration, it should be understood that the preparation should meet the sterility, pyrogenicity, general safety and purity standards required by the FDA Office of Biological Standards.

[0056] As used herein, "pharmaceutically acceptable carrier" includes any and all aqueous solvents (e.g., water, alcohol / aqueous 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 a fifth aspect, the present invention provides any of the following uses:

[0058] (1) Use of the product according to the third aspect of the present invention or the pharmaceutical composition according to the fourth aspect of the present invention in the preparation of an agent having a killing effect on tumor cells;

[0059] (2) Use of the product described in the third aspect of the present invention or the pharmaceutical composition described in the fourth aspect of the present invention in the preparation of a medicament for treating autoimmune diseases, blood system diseases and / or cancer.

[0060] In a specific embodiment of the present invention, the tumor cells include K562 and Raji.

[0061] "Treatment" refers to the implementation of a regimen that may include the administration of 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, ameliorating or alleviating the disease state, and alleviating or improving prognosis. Relief can occur before signs or symptoms of a disease or condition appear as well as after they appear. Thus, "treatment" may include "preventing" a disease or an undesirable condition. In addition, "treatment" does not require complete relief of signs or symptoms, does not require a cure, and specifically includes regimens that have only a marginal effect on the patient.

[0062] The cancer described in the present invention covers 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] Autoimmune diseases of the present invention refer to diseases caused by improper immune responses to substances and tissues normally present in the body, which are generated in the subject. 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 are commonly used to treat autoimmune diseases, for example, drugs that reduce immune responses. 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] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0065] NK cells have limited proliferation potential, and in vitro expansion is a challenge. In recent years, studies on NK cell expansion and culture generally use two methods: adding trophoblast cells and pure factor culture. Adding trophoblast cells can better activate NK cells and expand them in large quantities, but even if the added trophoblast cells have been irradiated, there is still a great risk and they are not suitable for clinical use; using the pure factor method to induce the expansion of NK cells in PBMC is safer and more suitable for clinical use, but the expansion multiples are generally not ideal. In addition, the reinfusion of allogeneic NK cells requires complete elimination of T cells to avoid GVHD; and for the resuscitated PBMC, it is undoubtedly an extremely difficult task to use the pure factor method to expand them in large quantities and obtain highly active NK cells with a purity greater than 99%.

[0066] Compared with the above-mentioned prior art, the present invention provides a method for culturing NK cells, and the expansion multiple of the NK cells obtained by the method can eventually reach 1000-10000 times, and the purity is above 99%. The present invention can effectively solve the problem of difficulty in obtaining high-purity NK cells, and provides the possibility for the allogeneic use of NK cells separated from peripheral blood from a single blood collection source. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] Figure 1 Figures A and B are the flow cytometry results of NK cells before and after magnetic bead sorting on day 0; Figures C and D are the percentages of CD14 and CD19 in total cells after magnetic bead sorting on day 0;

[0068] Figure 2 Graphs showing the expression ratio of CD16 in NK cells, wherein Graph A is a graph showing the expression ratio of CD16 in NK cells on day 0, and Graph B is a graph showing the expression ratio of CD16 in NK cells on day 25 of culture;

[0069] Figure 3 This is the flow cytometry result of NK cells after 25 days of culture;

[0070] Figure 4 Figure 1 is a graph showing the killing ability of NK cells against tumors on the 15th and 25th days of culture, wherein Figure A is a graph showing the killing ability of NK cells against tumors on the 15th day of culture, and Figure B is a graph showing the killing ability of NK cells against tumors on the 25th day of culture;

[0071] Figure 5 This is the proliferation curve of cells cultured to day 25;

[0072] Figure 6 This is a comparison of the flow cytometry results of NK cells after 25 days of culture;

[0073] Figure 7 This is a comparison chart of the proliferation curves of cells cultured to day 25. DETAILED DESCRIPTION

[0074] The present invention will be described in detail below in conjunction with embodiments and drawings, so that those skilled in the art can understand and implement the present invention and further recognize the advantages of the present invention.

[0075] Unless otherwise defined in the specification of the present invention, all technical terms herein are used according to the customary definitions commonly used and understood by those skilled in the art. The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0076] Example 1 NK cell culture method

[0077] 1. Experimental steps

[0078] 1. Pretreatment of cell culture flasks

[0079] Phosphate buffer containing 8 μg / mL CD137 monoclonal antibody, 8 μg / mL CD28 monoclonal antibody, and 8 μg / mL CD16 monoclonal antibody was added to a 6-well cell culture plate at 850 μL / well, and the liquid was fully dispersed in the wells to cover the entire bottom, and placed flat at 4°C overnight.

[0080] 2. Thawing and Recovery of Cryopreserved PBMCs

[0081] Preheat the RPMI 1640 medium containing 10% FBS in a 37°C water bath, remove the frozen PBMC cells from the liquid nitrogen, loosen the cap of the cryotube by 1 / 4 to release the pressure, and then immediately tighten the cap. Place the cryotube in a 37°C water bath for about 1.5 minutes, rotating it continuously during the period. When there are still trace amounts of small ice crystals remaining in the tube, remove the cryotube, wipe the outside of the cryotube with 75% ethanol, and place it in a biosafety cabinet. Pipette 9mL of the preheated RPMI 1640 medium into a 15mL centrifuge tube, transfer the cell suspension in the cryotube to the above centrifuge tube with a 1mL pipette, gently rinse the cryotube with 1mL of medium, and transfer the rinse solution to the above centrifuge tube, place the centrifuge tube in a centrifuge at 400g / min, centrifuge for 8 minutes, remove the supernatant after centrifugation, gently tap the bottom of the tube to loosen the cell pellet, and then immediately add 10mL of preheated medium to resuspend the cells. PBMC thawing is complete.

[0082] 3. Magnetic bead sorting

[0083] The thawed PBMC suspension was filtered through a 70 μm cell mesh to remove cell clusters, and samples were counted. The amount of MACS-Buffer and magnetic beads was calculated based on the counting results, and magnetic bead negative selection of CD3, CD14, and CD19 was performed. The amount of magnetic beads was 20 μL / 10 7Cells were collected from the flow cytometry + NK cells, discard the supernatant after centrifugation, resuspend in 2 mL X-VIVO15 basal medium, take a sample and count, and take a small amount of cell suspension for flow cytometry detection. NK cells (CD3 - CD56 + ) ratio is 99.17%.

[0084] 4. Day 0 vaccination

[0085] The NK cells obtained in step 1.3 were centrifuged and washed again, and then resuspended in X-VIVO15 medium containing IL-2 1000 IU / mL, IL-15 300 IU / mL, IL-12 100 IU / mL, IL-2 15 IU / mL, CD13 75 μg / mL, CD3 5 μg / mL, and multivitamins 100 ng / mL (vitamin B2: vitamin E = 1:1) and counted. The density was 1.26 × 10 6 / mL, inoculate into the 6-well plate coated in 1.1, 1.8mL / well, add 5% serum replacement (CTS TM Immune cell serum replacement), replenished to 2 mL, and cultured in an incubator (37°C, CO2 concentration of 5%, humidity of 45%-55%).

[0086] 5. Rehydration on Day 3

[0087] On the third day after cell inoculation, the supplementation volume was 2 mL. At this time, the supplemented medium contained 5% serum replacement (CTS TM Expansion medium 1 (basal medium is X-VIVO15) containing 1000 IU / mL immune cell serum replacement), IL-2 1000 IU / mL, IL-15 300 IU / mL, IL-12 100 IU / mL, and complex vitamins 100 ng / mL (vitamin B2: vitamin E = 1:1).

[0088] 6. Rehydration on Day 6

[0089] On the 6th day of culture, the second supplementation was performed with a volume of 4 mL. At this time, the supplemented medium contained 5% serum replacement (CTS TM Expansion medium 2 (basal medium is X-VIVO15) containing 1000 IU / mL immune cell serum replacement), IL-2 1000 IU / mL, IL-15 300 IU / mL, and complex vitamins 100 ng / mL (vitamin B2: vitamin E = 1:1).

[0090] 7. Rehydration on Day 7

[0091] On the 7th day of culture, the cell clusters were dispersed and cultured at 1×10 6The cells were supplemented with expansion medium 2 at a density of 1 cell / mL.

[0092] 8. Day 9-Day 23

[0093] After rehydration on Day 7, cells were counted and rehydrated every other day, that is, cells were counted and rehydrated on Day 9, Day 11, Day 13, Day 15, Day 17, Day 19, Day 21, and Day 23, respectively, at a rate of 1×10 6 / mL density was used to supplement expansion medium 2, and samples were taken for flow cytometry detection of NK purity.

[0094] 9. End of culture and testing on the 25th day

[0095] On Day 25, samples were taken to test the purity of NK cells, the ability of cells to kill tumors, and the expression of activating and inhibitory receptors by cells, and the culture was terminated on the 25th day.

[0096] 2. Experimental Results

[0097] The cell suspension was harvested on the 25th day of culture, and the overall cell number was expanded by more than 5000 times compared with the inoculation on DAY0. The expanded cells were also subjected to flow cytometry detection and their killing function against tumor cells (K562 and Raji).

[0098] Example 2 Comparison of the effects of different culture schemes

[0099] 1. Experimental plan

[0100] After cryopreserved single-blood mononuclear cells were thawed, they were cultured using the following schemes:

[0101] Control 1: After cell recovery on Day 0, NK cells were sorted and then cultured with a medium containing only the combination of IL-2 and IL-15 and serum replacement (IL-2 1000 IU / mL, IL-15 300 IU / mL, and 5% serum replacement);

[0102] P19 (experimental group): After the cells were revived on Day 0, NK cells were sorted and then cultured. The culture scheme was the same as in the example (the culture medium components were IL-2 1000 IU / mL, IL-15 300 IU / mL, IL-12 100 IU / mL, IL-2 15 IU / mL, CD13 75 ug / mL, CD3 5 ug / mL, complex vitamins 100 ng / mL (vitamin B2: vitamin E = 1:1), and 5% CTS was added TM Immune cell serum replacement);

[0103] Control 2 group: cells were directly cultured after recovery on Day 0 (without sorting), and the culture scheme was the same as that of the experimental group.

[0104] two, Experimental Results

[0105] A) By Figure 6 It can be seen that after 25 days of culture, the purity of NK cells in the Control1 and Control2 groups was low (<60%), while the purity in the experimental group reached more than 99%.

[0106] B) By Figure 7 It can be seen that the NK cell proliferation ability of the experimental group is significantly stronger than that of the control group. Therefore, it is believed that the experimental group is superior to the control group.

[0107] Although the present invention has been described with reference to the accompanying drawings and preferred embodiments, various modifications and variations of the present invention are apparent to those skilled in the art. Various modifications, variations, and equivalents of the present invention are covered by the appended claims.

Claims

1. A method for expanding NK cells, characterized in that: The method comprises: 1) pretreating a cell culture vessel with a coating solution composition; The coating solution composition consists of 8 μg / mL CD137 antibody, 8 μg / mL CD28 antibody, and 8 μg / mL CD16 antibody; 2) Obtain NK cell population from frozen peripheral blood mononuclear cells; 3) In the cell culture container obtained in step 1), using an activation medium composition to activate the NK cell population obtained in step 2) to obtain an activated NK cell population; the activation medium composition consists of the following components: IL-2 1000 IU / mL, IL-15 300 IU / mL, IL-12 100 IU / mL, IL-21 5 IU / mL, CD137 antibody 5 μg / mL, CD3 antibody 5 μg / mL, multivitamins 100 ng / mL, 5% serum replacement and basal culture medium; 4) expanding the activated NK cell population obtained in step 3) using the first expansion medium composition and the second expansion medium composition; The first expansion medium composition consists of the following components: IL-2 1000 IU / mL, IL-15 300 IU / mL, IL-12 100 IU / mL, multivitamins 100 ng / mL, 5% serum replacement and basal medium; The second expansion medium composition consists of the following components: IL-2 1000 IU / mL, IL-15 300 IU / mL, multivitamins 100 ng / mL, 5% serum replacement and basal medium; The basic culture medium is X-VIVO15; the vitamin complex consists of vitamin B2 and vitamin E, and the ratio of vitamin B2 to vitamin E is 1:1; The serum replacement is CTS™ immune cell serum replacement; The step 2) of obtaining the NK cell population includes thawing cells and magnetic bead negative selection; The magnetic beads used in the magnetic bead negative selection are CD3 magnetic beads, CD14 magnetic beads and CD19 magnetic beads, and the amount of magnetic beads is 20 μL / 10 7 cell.

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