NK cell efficient separation and multiplication culture scheme and application thereof

Abstract

The invention discloses an NK cell efficient separation and multiplication culture scheme and application thereof. The invention provides an efficient cord blood NK cell separation and multiplication culture scheme for the field, multiplication culture is carried out for 19 days, the amplification multiple can reach 1000 times, the cell purity is greater than 95%, the tumor cell killing efficiency is greater than 70%, and the problems of high T cell proportion, slow amplification, low purity and low killing activity in the cord blood NK cells are effectively solved.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology. Specifically, this invention relates to a highly efficient isolation and expansion culture scheme for NK cells and its application. Background Technology

[0002] Natural killer (NK) cells are important effector cells of the innate immune system, accounting for 5% to 15% of mononuclear cells in blood and lymphatic organs. NK cells are the body's first line of defense against viral infections and malignant cells. They can directly kill cancerous or infected cells and indirectly improve antibody- and T-cell-mediated responses. In addition, NK cells also have immunomodulatory functions, regulating dendritic cells, macrophages, neutrophils, and antigen-specific T-cell and B-cell responses.

[0003] NK cells have a wide range of sources, including peripheral blood, umbilical cord blood, induced pluripotent stem cells (iPSCs), and cell lines such as NK92. Among these sources, umbilical cord blood, as a readily available heterologous resource, has the advantages of wide availability and no ethical issues. The immature immune cells it contains are less likely to cause graft-versus-host disease (GVHD). In addition, umbilical cord blood contains more NK precursor cells than adult peripheral blood, making it the most promising non-invasive source of therapeutic NK cells.

[0004] In recent years, cytokine-based culture systems and feeder cell-based co-culture systems have become the main methods for culturing NK cells. The core objective of these methods is to achieve large-scale, high-purity, and highly active expansion of NK cells. However, due to limitations in isolation conditions, the initial umbilical cord lymphomonocytes (UCBMCs) often have an excessively high proportion of T cells, resulting in limited expansion and low purity of the final NK cells. The biggest concern with co-culturing with feeder cells is the safety risk; although feeder cells are irradiated, there is still a slight risk of introducing them into the final product, posing a potential risk of tumorigenesis. Therefore, providing an efficient and stable umbilical cord blood NK cell preparation protocol is crucial. Summary of the Invention

[0005] To overcome the shortcomings of existing technologies, the purpose of this invention is to provide an efficient and stable method for preparing umbilical cord blood NK cells.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] The first aspect of the present invention provides a composition for isolating and culturing NK cells.

[0008] In this invention, the term "natural killer cell (NK cell)" is defined as a large granular lymphocyte (LGL) and constitutes a third type of cell that differentiates from common lymphoid progenitor cells that produce 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 circulation. According to their origin and according to their respective effector functions, NK cells differ phenotype from natural killer T cells (NKT); typically, NKT cell activity promotes NK cell activity by secreting IFNγ. Compared to NKT cells, NK cells do not express T cell antigen receptor (TCR) or panT marker CD3 or surface immunoglobulin (Ig) B cell receptor, but they typically express surface markers CD16 (FcyRIII) and CD56 in humans, and NK1.1 or NK1.2 in C57BL / 6 mice.

[0009] In this invention, the term "culture" includes providing the chemical and physical conditions (e.g., temperature, gases) and growth factors necessary for the maintenance of NK cells. Generally, culturing NK cells involves providing conditions for their 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 periodically provided (or may be manually administered) in a cell culture medium suitable for NK cell expansion.

[0010] Furthermore, the composition includes CD3. + Magnetic beads.

[0011] Furthermore, the CD3 + The amount of magnetic beads used is 0.5 to 1.5 times the number of T cells in the blood sample to be separated.

[0012] Preferably, the CD3 + The amount of magnetic beads used is 1.5 times the number of T cells in the blood sample to be separated.

[0013] In this invention, the applicant proposes a method for utilizing CD3 during the PBMC separation stage. + The gravitational effect of micron-sized magnetic beads, during centrifugation using a Ficoll density gradient, causes the CD3 molecules coupled to the magnetic beads to... + T lymphocytes and NKT cells detach from the white membrane layer, thereby reducing the content of T lymphocytes and NKT cells. Further, a high-purity, high-activity umbilical cord blood NK cell preparation method is developed through an expansion culture system.

[0014] In this invention, the applicant relates to CD3. + The amount of magnetic beads used was studied, and it was found that when CD3... +When the amount of magnetic beads used is 1.5 times the number of T cells in the blood sample to be separated, the proportion of NK cells obtained is higher and the content of T lymphocytes is lower.

[0015] Furthermore, the composition further includes a first activation medium, which comprises the following components: IL 2. IL 15. IL 21. IL18, platelet lysate, human serum albumin, group A streptococci for injection, basal culture medium.

[0016] Furthermore, in the first activation medium, IL The concentration of 2 is 100 1000 IU / mL, IL The concentration of 15 is 50. 1000 IU / mL, IL The concentration of 21 is 5 10 IU / mL, the concentration of IL18 is 5 The concentration of platelet lysate was 20 ng / mL, the concentration of human serum albumin was 2%-10%, and the concentration of group A streptococci for injection was 0.01-1 KE / mL.

[0017] Preferably, in the first activation medium, IL The concentration of 2 is 500 IU / mL, IL The concentration of 15 is 100 IU / mL, IL The concentrations of IL-21 were 2 IU / mL, IL-18 were 10 ng / mL, platelet lysate was 5%, human serum albumin was 2%, and group A streptococci for injection were 0.01 KE / mL.

[0018] Furthermore, the basal culture medium is a serum-free culture medium.

[0019] Furthermore, the composition further includes any one or more of the following: 1) Second activation medium, which consists of the following components: IL 2. IL 15. IL 21. IL18, platelet lysate, human serum albumin, basal culture medium; Preferably, in the second activation medium, IL The concentration of 2 is 100 1000 IU / mL, IL The concentration of 15 is 50. 1000 IU / mL, IL The concentration of 21 is 5 10 IU / mL, the concentration of IL18 is 5 20 ng / mL, platelet lysate concentration of 2%-10%, human serum albumin concentration of 2%-10%; 2) First amplification medium, which consists of the following components: IL 2. IL 15. Platelet lysate, human serum albumin, basal culture medium; Preferably, in the first amplification medium, IL The concentration of 2 is 100 1000 IU / mL, IL The concentration of 15 is 50. 1000 IU / mL, platelet lysate concentration of 2%-10%, human serum albumin concentration of 2%-10%; 3) Second amplification medium, which consists of the following components: IL 2. IL 15. Human serum albumin, basal culture medium; Preferably, in the second amplification medium, IL The concentration of 2 is 100 1000 IU / mL, IL The concentration of 15 is 50. 1000 IU / mL, the concentration of human serum albumin is 2%-10%; 4) Coating solution, wherein the coating solution is composed of the following components: CD16 antibody and 4-1BB antibody; Preferably, the concentration of CD16 antibody and 4-1BB antibody in the coating solution is 5.0-20 μg / mL.

[0020] Preferably, in the second activation medium, IL The concentration of 2 is 500 IU / mL, IL The concentration of 15 is 100 IU / mL, IL The concentrations of IL-21 were 2 IU / mL, IL-18 were 10 ng / mL, platelet lysate was 5%, and human serum albumin was 2%.

[0021] Preferably, in the first amplification medium, IL The concentration of 2 is 500 IU / mL, IL The concentration of 15 was 100 IU / mL, the concentration of platelet lysate was 5%, and the concentration of human serum albumin was 2%.

[0022] Preferably, in the second amplification medium, IL The concentration of 2 is 500 IU / mL, IL The concentration of 15 is 100 IU / mL, and the concentration of human serum albumin is 2%.

[0023] Preferably, the concentration of CD16 antibody and 4-1BB antibody in the coating solution is 10 μg / mL.

[0024] Furthermore, the basal culture media for the second activation medium, the first amplification medium, and the second amplification medium are serum-free media.

[0025] Furthermore, the blood samples to be separated can be sourced from umbilical cord blood, peripheral blood, or bone marrow.

[0026] Preferably, the blood sample to be separated is umbilical cord blood.

[0027] In this invention, the term "IL" 2” “IL” 15” “IL” 21”, “IL” "18" refers to the cytokine interleukin. 2. Interleukins 15. Interleukins 21. Interleukin 18 and its derivatives.

[0028] A second aspect of the present invention provides a method for isolating and amplifying NK cells.

[0029] Furthermore, the method includes: a) Pretreatment of cell culture containers using the coating solution described in the first aspect of the present invention; b) Add CD3 to the blood sample to be separated + Magnetic beads are incubated, and mononuclear cells are obtained by separating the incubated blood sample. c) In the cell culture vessel obtained in a), the mononuclear cells obtained in b) are cultured using the first activation medium as described in the first aspect of the present invention; d) Supplement the second activation medium, the first amplification medium and the second amplification medium as described in the first aspect of the present invention.

[0030] Furthermore, the cell culture containers include T225 culture flasks, T75 culture flasks, and cell culture bags.

[0031] The cell culture containers used in NK cell culture are not particularly limited as long as they can be used to culture NK cells, but examples include bottles, tissue culture bottles, dishes, petri dishes, tissue culture dishes, multi-plate dishes, microplates, microwell plates, multi-plate trays, multiwell plates, microscope slides, chamber slides, culture dishes, tubes, trays, culture bags, and roller bottles.

[0032] Cell culture vessels can be either cell-adhesive or cell-non-adhesive, depending on the intended purpose. Cell-adhesive culture vessels are designed to increase the adhesion between the culture vessel surface and cells, and are coated with any cell support matrix such as extracellular matrix (ECM). Cell support matrices can be any material intended to promote NK cell adhesion.

[0033] 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 CO2 concentration can be about 1–10%, preferably about 2–5%. The oxygen partial pressure can be 1–10%.

[0034] Furthermore, the CD3 + The amount of magnetic beads used is 0.5 to 1.5 times the number of T cells in the blood sample to be separated.

[0035] Furthermore, the method for detecting the number of T cells in the blood sample to be separated is flow cytometry.

[0036] Furthermore, the addition of CD3 + The magnetic beads are incubated for 20-60 minutes.

[0037] Furthermore, the mononuclear cells were obtained by Ficoll density gradient centrifugation.

[0038] Furthermore, the seeding density of the mononuclear cells is 5.0 × 10⁻⁶. 5 / mL-2.0×10 6 per mL.

[0039] Furthermore, the second activation medium is added on days 1-3 after inoculation.

[0040] Furthermore, the first amplification medium was added on days 3-7 after inoculation.

[0041] Furthermore, the second amplification medium was added on days 7-19 after inoculation.

[0042] Furthermore, the volume increases by 2, 3, 4 or more times with each addition of culture medium.

[0043] Preferably, the CD3 + The amount of magnetic beads used is 1.5 times the number of T cells in the blood sample to be separated.

[0044] Preferably, the addition of CD3 + The magnetic beads were incubated for 30 minutes.

[0045] Preferably, the seeding density of the mononuclear cells is 1.0 × 10⁻⁶. 6 per mL.

[0046] Preferably, the second activation medium is added on the third day after inoculation.

[0047] Preferably, the first amplification medium is added on day 5 and day 7 after inoculation.

[0048] Preferably, the second amplification medium is added on days 9, 11, 13, 15 and 17 after inoculation.

[0049] Preferably, the volume doubles after each addition of culture medium.

[0050] Preferably, the blood sample to be separated comes from umbilical cord blood, peripheral blood, or bone marrow.

[0051] Preferably, the blood sample to be separated is umbilical cord blood.

[0052] The third aspect of the present invention provides NK cells prepared by the method described in the second aspect of the present invention.

[0053] In some implementations, the purity of the NK cells in the NK cell product reaches 95% or higher.

[0054] A fourth aspect of the present invention provides a pharmaceutical composition.

[0055] Furthermore, the pharmaceutical composition includes NK cells as described in the third aspect of the present invention.

[0056] Furthermore, the pharmaceutical composition also includes a pharmaceutically acceptable carrier, diluent, or excipient.

[0057] In this invention, the term "pharmaceutically acceptable" means a molecular entity and composition that, when properly administered to animals such as humans, does not produce an adverse, allergic, or other undesirable reaction. In view of this disclosure, the preparation of pharmaceutical compositions comprising antibodies or other active ingredients is known to those skilled in the art. Furthermore, 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.

[0058] In this invention, "pharmaceutically acceptable carriers" include any and all aqueous solvents (e.g., water, alcohol / aqueous solutions, saline solutions, parenteral media such as sodium chloride, Ringer's glucose, 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 delay agents, salts, pharmaceuticals, pharmaceutical stabilizers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavorings, dyes, fluids and nutritional supplements, and similar materials and combinations thereof, as will be known to those skilled in the art. The pH and exact concentration of the various components in the pharmaceutical composition are adjusted according to known parameters.

[0059] The fifth aspect of the present invention provides the use of the composition described in the first aspect of the present invention in the preparation of products containing NK cells.

[0060] The sixth aspect of this invention provides the use of the NK cells described in the third aspect of this invention or the pharmaceutical compositions described in the fourth aspect of this invention in the preparation of medicaments for cell therapy, medicaments for antiviral infection, and medicaments for treating cancer or autoimmune diseases.

[0061] Furthermore, the applications also include combined use with antibody drugs, nucleic acid drugs, small molecule drugs, oncolytic virus drugs, and cell drugs; and combined use of NK cells with radiotherapy, chemotherapy drugs, stem cell transplantation, interventional therapy, ablation therapy, and other treatment methods.

[0062] Furthermore, the cancers mentioned include solid cancers and non-solid cancers.

[0063] In this invention, "treatment" refers to a protocol that may include administering one or more medications to a patient with the aim of alleviating the symptoms or signs of a disease. Ideal therapeutic effects include slowing the rate of disease progression, improving or alleviating the disease state, and mitigating or improving prognosis. Relief can occur before or after the onset of symptoms or signs of a disease or condition. Therefore, "treatment" can include "preventing" a disease or an undesirable condition. Furthermore, "treatment" does not require complete relief of symptoms or signs, does not require a cure, and particularly includes protocols that have only a marginal effect on the patient.

[0064] The cancers described in this invention encompass any type of cancer, including solid and non-solid cancers. Specifically, the cancers include cervical cancer, seminoma, testicular lymphoma, prostate cancer, ovarian cancer, lung cancer, rectal cancer, breast cancer, squamous cell carcinoma of the skin, colon cancer, liver cancer, pancreatic cancer, stomach cancer, esophageal cancer, thyroid cancer, transitional cell carcinoma of the bladder, 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, and glioma.

[0065] The autoimmune diseases described in this invention refer to diseases in which an inappropriate immune response arises in the body against normally present substances and tissues. In other words, the immune system mistakenly identifies a part of the body as 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 locations (e.g., Goodpasseur disease, which may affect the basement membranes of both the lungs and kidneys). Treatment of autoimmune diseases typically involves immunosuppressants, such as drugs that reduce the immune response. Exemplary autoimmune diseases include, but are not limited to, glomerulonephritis, Goodpasser syndrome, necrotizing vasculitis, lymphadenitis, periarteritis nodosa, systemic lupus erythematosus, rheumatoid arthritis, arthritis, psoriatic arthritis, psoriasis, ulcerative colitis, systemic sclerosis, dermatomyositis / polymyositis, antiphospholipid antibody syndrome, scleroderma, pemphigus vulgaris, ANCA-associated vasculitis (e.g., Wegener's granulomatosis, microscopic polyangiitis), uveitis, Schogren's syndrome, Crohn's disease, Wright syndrome, ankylosing spondylitis, Lyme arthritis, Gullen-Barré syndrome, Hashimoto's thyroiditis, and cardiomyopathy.

[0066] Advantages and beneficial effects of the present invention: This invention provides a highly efficient method for isolating and expanding NK cells from umbilical cord blood. After 19 days of expansion culture, the expansion multiple can reach 1000 times, with cell purity >95% and tumor cell killing efficiency >70%. This invention effectively solves the problems of high T cell proportion, slow expansion, low purity, and low killing activity in umbilical cord blood NK cells. Attached Figure Description

[0067] Figure 1 The process CD3 of this invention + Comparison of magnetic bead dosage with NK cell flow cytometry results; Figure 2 The flow cytometry results of NK cells before and after separation on day 0 of the process of this invention; Figure 3 Flow cytometry results of NK cells before and after separation on day 0 of the conventional umbilical cord blood NK cell separation process; Figure 4The flow cytometry results of NK cells after 19 days of amplification and culture using the process of this invention; Figure 5 Flow cytometry results of NK cells cultured for 19 days using standard umbilical cord blood NK cell culture technology; Figure 6 This is the umbilical cord blood NK cell amplification curve in the process of this invention; Figure 7 Amplification curves for conventional umbilical cord blood NK cell culture process; Figure 8 The results are from flow cytometry analysis before and after umbilical cord blood NK cell separation. Figure 9 NK cell flow cytometry results after culturing in different culture media for 19 days; Figure 10 The results are from flow cytometry analysis of umbilical cord blood UCBMCs after separation. Figure 11 The results of flow cytometry analysis of UCBMCs cultured for 19 days after activation with different antibodies. Detailed Implementation

[0068] The present invention will now be described in detail with reference to the accompanying drawings and embodiments, so as to enable those skilled in the art to understand and implement the present invention, and to further recognize the advantages of the present invention. Unless otherwise defined in the specification of the present invention, all technical terms herein are used according to conventional definitions commonly used and understood by those skilled in the art. Unless otherwise specified, the experimental methods described in the following embodiments are conventional methods; the reagents and materials described are commercially available unless otherwise specified.

[0069] Example 1: Method for separating umbilical cord blood PBMCs in this invention I. Experimental Methods Isolation of umbilical cord blood mononuclear cells (UCBMCs): (1) Taking 100mL of umbilical cord blood (purchased from Bokang Bioengineering Shandong Co., Ltd.) as an example, the proportion of T cells detected by flow cytometry in 1mL of umbilical cord blood was 80.48%. Another 1mL of umbilical cord blood was taken, and the red blood cells were lysed and counted. The result was 5.0×10 6 / ml; (2) Divide the remaining umbilical cord blood into 4 groups, each with 24.5 mL; (3) Calculate the number of T cells in each group of umbilical cord blood: T cell count = Count result T cell ratio Umbilical cord blood volume = 5.0 × 10 6 80.48% 24.5 = 9.86 × 10 7 (4) Set different magnetic bead addition amounts: CD3 of 4 groups of blood samples + The magnetic beads (Tongli Haiyuan, TL-803L-1000) were added at amounts of 0, 0.5, 1, and 1.5 times the number of T cells, respectively. The specific calculation method is as follows: The quantity of magnetic beads in group 1 is 0; The amount of magnetic beads in the second group is 0.5. T cell count = 4.93 × 10 7 ; The amount of magnetic beads in group 3 = 1 T cell count = 9.86 × 10 7 ; The quantity of magnetic beads in group 4 is 1.5. T cell count = 1.48 × 10 8 ; (5) After adding the corresponding number of magnetic beads to each blood sample, place it on a sample mixer, set the rotation speed to 10 rpm, and incubate at room temperature for 30 min. (6) Slowly add the incubated blood sample to a 50ml centrifuge tube containing 15ml Ficoll, centrifuge for 30min, 750g, increase 9 and decrease 0; (7) Aspirate white membrane cells (UCBMC) and wash twice with 45 ml of sodium chloride injection solution; (8) Resuspend the cells in sodium chloride injection solution. Take 500 μl of cell suspension from each group for counting and detect cell viability and cell yield. At the same time, take 1.0 × 10⁻⁶ cells. 6 / mL cell flow cytometry was used to detect the proportion of NK cells.

[0070] II. Experimental Results As shown in Table 1, after CD3 + The cell viability of UCBMCs obtained by magnetic bead sorting was not significantly different from that of UCBMCs obtained by normal separation. Cell yield increased with CD3 concentration. + The amount of magnetic beads used decreased with increasing dosage. Flow cytometry analysis revealed that the proportion of NK cells decreased with increasing CD3 concentration. + The proportion of T cells increases with the increase in the amount of magnetic beads used, and the proportion of T cells increases with CD3. + The amount of magnetic beads used increases and decreases; when the amount of magnetic beads is 1.5 times the number of T cells, the proportion of NK cells can increase to more than 15%, and the proportion of T cells can decrease to about 2%. Figure 1 Therefore, it can be concluded that when the amount of magnetic beads is 1.5 times the number of T cells, UCBMCs can be effectively enriched, and cell viability is not affected by magnetic bead sorting. Therefore, the process of this invention selects 1.5 times the number of T cells for CD3... + The amount of magnetic beads is used as the amount added for sorting.

[0071] Table 1. Statistics on PBMC cell viability, yield, and proportion of each cell type obtained by sorting with different amounts of magnetic beads.

[0072] Example 2: Expansion and culture of umbilical cord blood NK cells in this invention I. Experimental Design 1. Pretreatment of cell culture flasks in this invention: Add 16 mL of DPBS containing 10 μg / mL humanized antibody CD16 (Tonglihaiyuan, GMP-TL201-0500) and 10 μg / mL humanized antibody 4-1BB (Baipusaisi, GMP-41LH26) to a T225 culture flask and incubate at 37°C for 2 hours.

[0073] 2. Isolation of umbilical cord blood mononuclear cells (UCBMCs): (1) Taking 100ml of cord blood as an example, if the blood volume is different, the operation can be adjusted accordingly; take 1mL of cord blood, lyse the red blood cells and count them. The counting result is 4.9×10 6 ; An additional 1 mL of umbilical cord blood was collected for flow cytometry analysis to determine the proportion of T cells, which was 64.49% (cell number UC032, flow cytometry results are as follows). Figure 2 ).

[0074] (2) Based on the number of T cells, calculate the amount of CD3 that needs to be added to 100 mL of umbilical cord blood. + Amount of magnetic beads used: CD3 + Number of magnetic beads = 1.5 T cell ratio Counting results Umbilical cord blood volume =1.5 × 64.49% × 4.9 × 10 6 ×100 =4.74×10 8 .

[0075] (3) Add the CD3 calculated in step (2) above. + For the magnetic beads, set the sample mixer speed to 10 rpm, place the blood sample with the added magnetic beads on the sample mixer, and incubate at room temperature for 30 minutes.

[0076] (4) Slowly add the incubated blood sample to a 50ml centrifuge tube containing 15ml of Ficoll, add 30ml of blood sample to each tube, centrifuge for 30min, 750g, increase 9 and decrease 0.

[0077] (5) Absorb white membrane cells (UCBMC) and wash twice with 45ml sodium chloride injection solution.

[0078] (6) Resuspend the cells in 40 mL of NK cell basal culture medium (Tongli Haiyuan, AS01-2), take 500 μL of the cell suspension for counting, and at the same time take 1.0 × 10 6 Flow cytometry analysis of cells at / mL showed that NK cell purity was 9.22% (cell number UC032). Figure 2 ).

[0079] 3. Vaccination: Based on the above counting results, take 1.5 × 10 7 Cells at 1.0 × 10 6 Cells were seeded at a density of 15 ml / mL into T225 culture flasks obtained in Step 1 containing 500 IU / mL IL-2 (Sihuan Bio, National Drug Approval Number S20040018), 100 IU / mL IL-15 (Jinyan, GMP-C016), 2 IU / mL IL-21 (Jinyan, GMP-CC45), 10 ng / mL IL-18 (Jinyan, GMP-CH29), 0.01 KE / mL Group A Streptococcus for Injection (Sinopharm Group Luya), 5% platelet lysate (BI, PLTGOLD500R), 2% human serum albumin (Jetbelin, S20170005), and 8.25 mL of NK cell basal medium (Tongli Haiyuan, AS01-2). The total volume was 15 ml / flask. The flasks were then incubated at 37°C in an incubator with a CO2 concentration of 5%.

[0080] 4. First fluid resuscitation: On the third day of culture, add 15 mL of activation medium containing 500 IU / mL IL-2, 100 IU / mL IL-15, 2 IU / mL IL-21, 10 ng / mL IL-18, 5% platelet lysate (HPL), and 2% human serum albumin to the culture flask, for a final volume of 30 mL.

[0081] 5. Second fluid resuscitation: On the 5th day of culture, add 15 mL of amplification medium containing 500 IU / mL IL-2, 100 IU / mL IL-15, 5% platelet lysate (HPL), and 2% human serum albumin to the culture flask (basal medium is Tongli Haiyuan, AS01-2), for a final volume of 45 mL.

[0082] 6. Third fluid resuscitation and bottle rotation: On the 7th day of culture, add 90 mL of amplification medium (basal medium is Tongli Haiyuan, AS01-2) containing 500 IU / mL IL-2, 100 IU / mL IL-15, 5% platelet lysate (HPL), and 2% human serum albumin to the culture flask. Mix well and transfer to one T225 culture flask, 135 mL / flask, for a total of 1 flask.

[0083] 7. Fourth fluid resuscitation: On day 9 of culture, the cell concentration was measured to be 1.63 × 10⁻⁶. 6 / mL, at 1.0×10 6 At a cell density of / mL, add 28mL of amplification medium containing IL-2 500IU / mL, IL-15 100IU / mL, and 2% human serum albumin (basal medium is Tongli Haiyuan, AS01-2), resulting in 163mL / bottle after replenishment.

[0084] 8. Fifth fluid resuscitation: On day 11 of culture, the cell concentration was measured to be 3.77 × 10⁻⁶. 6 / mL, at 1.0×10 6 At a cell density of / mL, 452mL of amplification medium containing IL-2 500IU / mL, IL-15 100IU / mL, and 2% human serum albumin was added (basal medium was Tongli Haiyuan, AS01-2). After adding the medium, the cells were transferred to three T225 culture flasks, 205mL per flask.

[0085] 9. Sixth fluid resuscitation: On day 13 of culture, the cell concentration was measured to be 2.93 × 10⁻⁶. 6 / mL, at 1.0×10 6 At a cell density of / mL, 1187mL of expansion medium containing IL-2 500IU / mL, IL-15 100IU / mL, and 2% human serum albumin was added (basal medium was Tongli Haiyuan, AS01-2). After replenishment, the medium was transferred to 3 cell culture bags, 600mL / bag.

[0086] 10. Seventh rehydration session: On day 15 of culture, the cell concentration was measured to be 2.03 × 10⁻⁶. 6 / mL, at 1.0×10 6 At a cell density of / mL, add 1854mL of amplification medium containing IL-2 500IU / mL, IL-15 100IU / mL, and 2% human serum albumin (basal medium is Tongli Haiyuan, AS01-2), ensuring 1218mL / bag, for a total of 3 bags.

[0087] 11. 8th fluid resuscitation On day 17 of culture, the cell concentration was measured to be 2.01 × 10⁻⁶. 6 / mL, at 1.0×10 6 At a cell density of / mL, supplement with 3691mL of amplification medium containing IL-2 500IU / mL, IL-15 100IU / mL, and 2% human serum albumin (basal medium is Tongli Haiyuan, AS01-2), ensuring 1800mL / bag, for a total of 4 bags.

[0088] 12. Testing: On day 19 of culture, the cell concentration was measured to be 1.68 × 10⁻⁶. 6 / mL, the purity of NK cells as determined by flow cytometry was 96.83% ( Figure 4 The cell number was UC032. Simultaneously, the number of expanded cells, flow cytometry analysis, and their tumor cell killing function were measured.

[0089] Example 3: Isolation and culture of fresh umbilical cord blood using conventional separation techniques. 1. Pretreatment of cell culture flasks: Add 6.5 mL of DPBS (Baipusaisi, GMP-41LH26) containing 10 μg / mL humanized antibody CD16 (Tonglihaiyuan, GMP-TL201-0500) and 10 μg / mL humanized antibody 4-1BB to a T75 culture flask and incubate overnight at 2-8°C.

[0090] 2. Isolation of umbilical cord blood mononuclear cells (UCBMCs): (1) Taking 100ml of umbilical cord blood as an example, if the blood volume is different, the operation can be adjusted accordingly; slowly add the blood sample to a 50ml centrifuge tube containing 15ml of Ficoll, add 30ml of blood sample to each tube, centrifuge for 30min, 750g, increase 9 and decrease 0.

[0091] (2) Absorb white membrane cells (UCBMC) and wash twice with 45ml sodium chloride injection solution.

[0092] (3) Resuspend the cells in 40 ml of NK cell basal culture medium (Tongli Haiyuan, AS01-2), take 500 μl of the cell suspension for counting, and at the same time take 1.0 × 10 6 The flow cytometry analysis showed that the purity of NK cells was 6.49% (cell number UC029). Figure 3 ).

[0093] 3. Vaccination: Based on the above counting results, take 1.5 × 10 7 Cells at 1.0 × 10 6The culture medium was inoculated into 6.12 ml of NK cell basal medium (Tongli Haiyuan, AS01-2) containing 500 IU / mL IL-2, 100 IU / mL IL-15, 3 IU / mL IL-21, and 10 ng / mL IL-18 in the T75 culture flask obtained in step 1. Simultaneously, 0.01 KE / mL group A streptococci for injection, 5% platelet lysate (HPL), and 2% human serum albumin were added, for a total volume of 15 ml / flask. The culture was incubated at 37°C in a 5% CO2 incubator.

[0094] 4. First fluid resuscitation: On the third day of culture, 15 mL of activation medium containing 500 IU / mL IL-2, 100 IU / mL IL-15, 3 IU / mL IL-21 and 10 ng / mL IL-18 was added to the culture flask (basal medium was Tongli Haiyuan, AS01-2), along with 5% platelet lysate (HPL) and 2% human serum albumin, for a final volume of 30 mL.

[0095] 5. Second fluid resuscitation: On the 5th day of culture, add 15 mL of amplification medium containing 500 IU / mL IL-2, 100 IU / mL IL-15, 5% platelet lysate (HPL), and 2% human serum albumin to the culture flask (basal medium is Tongli Haiyuan, AS01-2), for a final volume of 45 mL.

[0096] 6. Third fluid resuscitation and bottle rotation: On day 7 of culture, the cell concentration was measured to be 1.91 × 10⁻⁶. 6 Add 135 mL of amplification medium (basal medium is Tongli Haiyuan, AS01-2) containing 500 IU / mL IL-2, 100 IU / mL IL-15, 5% platelet lysate (HPL, AS01-2), and 2% human serum albumin to the culture flask. Mix well and transfer to one T225 culture flask, 180 mL / flask, for a total of one flask.

[0097] 7. Fourth fluid resuscitation: On day 9 of culture, the cell concentration was measured to be 9.4 × 10⁻⁶. 5 / mL, because the cell density did not reach 1.0×10 6 / mL, no additional culture medium is needed.

[0098] 8. Fifth fluid resuscitation: On day 11 of culture, the cell concentration was measured to be 2.96 × 10⁻⁶. 6 / mL, at 1.0×10 6At a cell density of / mL, 353mL of amplification medium containing IL-2 500IU / mL, IL-15 100IU / mL, and 2% human serum albumin was added (basal medium was Tongli Haiyuan, AS01-2), 177mL / bottle, for a total of 3 bottles.

[0099] 9. Sixth fluid resuscitation: On day 13 of culture, the cell concentration was measured to be 2.95 × 10⁻⁶. 6 / mL, at 1.0×10 6 At a cell density of / mL, add 1035mL of amplification medium (basal medium is Tongli Haiyuan, AS01-2) containing IL-2 500IU / mL, IL-15 100IU / mL and 2% human serum albumin, mix well and transfer to 3 culture bags, 522mL / bag.

[0100] 10. Seventh rehydration session: On day 15 of culture, the cell concentration was measured to be 1.91 × 10⁻⁶. 6 / mL, at 1.0×10 6 At a cell density of / mL, add 1425mL of amplification medium containing IL-2 500IU / mL, IL-15 100IU / mL, and 2% human serum albumin (basal medium is Tongli Haiyuan, AS01-2), 997mL / bag, for a total of 3 bags.

[0101] 11. Eighth fluid resuscitation: On day 17 of culture, the cell concentration was measured to be 2.08 × 10⁻⁶. 6 / mL, at 1.0×10 6 At a cell density of / mL, add 3230mL of amplification medium containing IL-2 500IU / mL, IL-15 100IU / mL, and 2% human serum albumin (basal medium is Tongli Haiyuan, AS01-2), 1555mL / bag, for a total of 4 bottles.

[0102] 12. Testing: On day 19 of culture, the cell concentration was measured to be 1.84 × 10⁻⁶. 6 / mL, the purity of NK cells as determined by flow cytometry was 90.93% (e.g., Figure 5 The expanded cells were counted, analyzed by flow cytometry, and their tumor cell killing function was detected.

[0103] Example 4: Detection of the cytotoxic function of the expanded NK cells I. Testing Plan: 1. After staining the target cells K562 with Calcein-AM, seed them in 96-well plates at a rate of 20,000 cells / 100 μl. 2. Add the NK cells prepared in Example 2 and Example 3 at an effector-to-target ratio of 5:1 and incubate in the dark for 4 hours; 3. Use a multi-functional microplate reader to detect the OD value and calculate the killing efficiency of each group.

[0104] II. Experimental Results: 1. Multiple batches of cell cultures were performed according to the process of this invention and the conventional umbilical cord blood NK cell culture process (6 samples were randomly collected and divided into 2 groups, cultured according to the process of this invention and the conventional culture process respectively, with 3 blood samples in each group, and each blood sample was numbered). The results showed that on day 19 of culture, the expansion of umbilical cord blood NK cells using the process of this invention was more than 1000-fold (as shown in Table 2), and the purity of NK cells was more than 95% (as shown in Table 2). Figure 4 ), amplification curve as shown Figure 6 As shown in Table 2, multiple batches of cells cultured using conventional umbilical cord blood technology achieved a maximum expansion fold of approximately 700-fold, while the purity of NK cells remained between 80-90%, none exceeding 95% (as shown in Table 2). Figure 5 ), amplification curve as shown Figure 7 As shown.

[0105] Table 2. Statistics on the overall expansion fold of D19 cells

[0106] 2. The NK cells amplified by the process of this invention have a killing efficiency of over 80% against K562 tumor cells. In contrast, the killing efficiency of NK cells separated from fresh umbilical cord blood without magnetic bead sorting is only 19.11%~69.46% against K562 tumor cells (as shown in Table 3).

[0107] Table 3. Killing efficiency of umbilical cord blood NK cells against K562 cells after 19 days of culture.

[0108] 3. The expression of NK cell activation receptors DNAM-1, NKG2D, NKp30, NKp44, and NKp46 at different time points was detected by flow cytometry. The results showed that the process of the present invention could achieve high levels of expression of multiple activation receptors as early as day 9, such as NKp30 reaching over 90% and NKp44 reaching 30%~50%, and the expression of each activation receptor could still be maintained at a high level after 19 days of culture (as shown in Table 4).

[0109] Table 4. Detection of NK cell activation receptor expression level on day 19 of culture (%)

[0110] Example 5: The activation medium was optimized to improve its effect on NK cell culture. I. Experimental Design: 1. Prepare the activation culture medium according to Table 5 below: Table 5 Optimization scheme for activation culture medium

[0111] 2. Pretreatment of cell culture flasks in this invention: Add 16 mL of DPBS containing 10 μg / mL humanized antibody CD16 (Tonglihaiyuan, GMP-TL201-0500) and 10 μg / mL humanized antibody 4-1BB (Baipusaisi, GMP-41LH26) to a T225 culture flask and incubate at 37°C for 2 hours.

[0112] 3. Isolation of umbilical cord blood mononuclear cells (UCBMCs): (1) Taking 100ml of umbilical cord blood as an example, the procedure can be adjusted accordingly if the blood volume is different. Take 1mL of umbilical cord blood, lyse the red blood cells and count them. Take another 1mL of umbilical cord blood for flow cytometry to detect the proportion of T cells (cell number UC034, such as...). Figure 8 ).

[0113] (2) Based on the number of T cells, calculate the amount of CD3 that needs to be added to 100 mL of umbilical cord blood. + Amount of magnetic beads used: CD3 + Number of magnetic beads = 1.5 T cell ratio Umbilical cord blood cell density Umbilical cord blood volume =1.5 × 80.48% × 4.52 × 10 6 ×100 =5.46×10 8 .

[0114] (3) Add the above amount of CD3 + For the magnetic beads, set the sample mixer speed to 10 rpm, place the blood sample with the added magnetic beads on the sample mixer, and incubate at room temperature for 30 minutes.

[0115] (4) Slowly add the incubated blood sample to a 50ml centrifuge tube containing 15ml Ficoll, add 30ml of blood sample to each tube, centrifuge for 30min, 750g, increase 9 and decrease 0.

[0116] (5) Absorb white membrane cells (UCBMC) and wash twice with 45ml sodium chloride injection solution.

[0117] (6) Resuspend the cells in 40 ml of NK cell basal culture medium (Tongli Haiyuan), take 500 μl of the cell suspension for counting, and at the same time take 1.0 × 10 6The flow cytometry analysis showed that the purity of NK cells was 23.95% (cell number UC034) (as shown in the figure). Figure 8 ).

[0118] 4. Vaccination: Based on the above counting results, 1.5 × 10⁻⁶ is taken for each group. 7 Cells at 1.0 × 10 6 / mL, inoculated into T225 culture flasks containing different activation media prepared in step 1, and 0.01KE / mL of group A streptococci for injection were added at the same time, with a total volume of 15ml / flask, for a total of 4 flasks; cultured in an incubator at 37℃ and CO2 concentration of 5%.

[0119] 5. First fluid resuscitation: On the third day of incubation, each group was supplemented with 15 mL of the activation medium prepared in step 1, for a final volume of 30 mL.

[0120] 6. Second fluid resuscitation: On day 5 of culture, 15 mL of amplification medium containing 500 IU / mL IL-2 and 100 IU / mL IL-15 (basal medium is Tongli Haiyuan) was added to the culture flask, along with 5% platelet lysate (HPL) and 2% human serum albumin, for a final volume of 45 mL.

[0121] 7. Third fluid resuscitation and bottle rotation: On day 7 of culture, the cell concentration of each group was measured, and the results are shown in Table 6. Each group was cultured at 1.0 × 10⁻⁶. 6 At a cell density of / mL, add expansion medium containing 500 IU / mL IL-2 and 100 IU / mL IL-15 (basal medium is Tongli Haiyuan), and add 5% platelet lysate (HPL) and 2% human serum albumin. After mixing, transfer to one T225 culture flask, 135mL / flask, for a total of one flask.

[0122] 8. Fourth fluid resuscitation: On day 9 of culture, cell concentration was measured, and the results are shown in Table 6. 3.0 × 10⁶ cells were retained in each group. 6 Cells and 1.0 × 10 6 At a cell density of / mL, add expansion medium (basal medium is Tongli Haiyuan) containing IL-2 500 IU / mL, IL-15 100 IU / mL and 2% human serum albumin, 30mL / bottle per group after replenishment.

[0123] 9. Fifth fluid resuscitation: On day 11 of culture, cell concentration was measured, and the results are shown in Table 6. 3.0 × 10⁻⁶ cells were retained in each group. 6 Cells and 1.0 × 10 6At a cell density of / mL, add expansion medium (basal medium is Tongli Haiyuan) containing IL-2 500 IU / mL, IL-15 100 IU / mL and 2% human serum albumin, 30mL / bottle per group after replenishment.

[0124] 10. Sixth rehydration session: On day 13 of culture, cell concentration was measured, and the results are shown in Table 6. 3.0 × 10⁶ cells were retained in each group. 6 Cells and 1.0 × 10 6 At a cell density of / mL, add expansion medium (basal medium is Tongli Haiyuan) containing IL-2 500 IU / mL, IL-15 100 IU / mL and 2% human serum albumin, 30mL / bottle per group after replenishment.

[0125] 11. 7th rehydration: On day 15 of culture, cell concentration was measured, and the results are shown in Table 6. 3.0 × 10⁶ cells were retained in each group. 6 Cells and 1.0 × 10 6 At a cell density of / mL, 1854mL of amplification medium containing IL-2 500IU / mL, IL-15 100IU / mL, and 2% human serum albumin was added (the basal medium was Tongli Haiyuan). After replenishment, 30mL was added to each group per bottle.

[0126] 12. Eighth fluid resuscitation On day 17 of culture, the cell concentration of each group was measured, and the results are shown in Table 6. Each group retained 3.0 × 10⁻⁶ cells. 6 Cells and 1.0 × 10 6 At a cell density of / mL, add expansion medium (basal medium is Tongli Haiyuan) containing IL-2 500 IU / mL, IL-15 100 IU / mL and 2% human serum albumin, 30mL / bottle per group after replenishment.

[0127] 13. Testing: On day 19 of culture, the cell concentration of each group was measured, and the results are shown in Table 6. NK cell purity was determined by flow cytometry from samples of each group, with results as follows: UC034-1: 96.82%; UC034-2: 95.35%; UC034-3: 96.1% (e.g., ...). Figure 9 Meanwhile, the number, viability, and tumor cell killing function of the expanded cells were detected, and the results are shown in Table 7.

[0128] Table 6. Cell concentration (cells / mL) of each group as measured every other day.

[0129] Table 7. Cell viability, expansion, purity, and killing efficiency against K562 cells after 19 days of culture in different activation media.

[0130] II. Experimental Results: Previous research has shown that using Tongli Haiyuan NK cell culture medium as the basal medium, supplementing only with platelet lysate (HPL) cannot maintain normal cell proliferation. Therefore, this experiment added 5% HPL during the activation phase, along with different concentrations of human serum albumin.

[0131] The results on Day 19 showed that the total cell expansion in the group with 2% human serum albumin was 943.96-fold, significantly higher than that in the groups with 1% and 0% human serum albumin. Regarding cell purity, the 2% human serum albumin group had a purity of 96.82%, while the other two groups had purities of 95.35% and 96.1%, respectively, with no significant difference in purity among the three groups. The killing efficiency of the three groups of cells against K562 tumor cells showed that the 2% human serum albumin group had a killing efficiency of 88.23%, the 1% human serum albumin group had a killing efficiency of 80.78%, and the group without human serum albumin had a killing efficiency of 83.33%.

[0132] Therefore, the activation medium was determined to be Tongli Haiyuan NK cell culture medium with 500 IU / mL IL-2 and 100 IU / mL IL-15, and 5% HPL and 2% human serum albumin were added. This activation medium can maintain the overall expansion and purity of umbilical cord blood NK cells, while also having a high killing efficiency against tumor K562 cells.

[0133] Example 6: Screening and comparison of antibodies used in the coated culture flasks of this invention I. Experimental Design: 1. Pretreatment of cell culture flasks: Group A: Add 16 mL of DPBS containing 10 μg / mL humanized antibody CD16 (Tongli Haiyuan, GMP-TL201-0500) and 10 μg / mL humanized antibody 4-1BB (Baipusaisi, GMP-41LH26) to a T75 culture flask and incubate overnight at 2-8°C.

[0134] Group B: Add 16 mL of DPBS containing 10 μg / mL humanized antibody CD16 (Tonglihaiyuan, GMP-TL201-0500) and 10 μg / mL humanized antibody CD28 (Tonglihaiyuan, GMP-TL102) to a T75 culture flask and incubate overnight at 2-8°C.

[0135] Group C: Add 16 mL of DPBS containing 10 μg / mL humanized antibody 4-1BB (Baipusaisi, GMP-41LH26) and 10 μg / mL humanized antibody CD28 (Tonglihaiyuan, GMP-TL102) to a T75 culture flask and incubate overnight at 2-8℃.

[0136] 2. Isolation of umbilical cord blood mononuclear cells (UCBMCs): (1) Slowly add 100mL of blood sample to a 50ml centrifuge tube containing 15ml of Ficoll. Add 30ml of blood sample to each tube, centrifuge for 30min, 750g, and adjust the pressure by 9°C and 0°C.

[0137] (2) Absorb white membrane cells (UCBMC) and wash twice with 45ml sodium chloride injection solution.

[0138] (3) Resuspend the cells in 40 ml of NK cell basal culture medium (Tongli Haiyuan), take 500 μl of the cell suspension for counting, and at the same time take 1.0 × 10 6 The flow cytometry analysis showed that the purity of NK cells was 5.94% (cell number UC019) (as shown in the figure). Figure 10 ).

[0139] 3. Vaccination: Based on the above counting results, 1.5 × 10⁻⁶ is taken for each group. 7 Cells at 1.0 × 10 6 / mL, inoculated into culture flasks A, B, and C obtained in step 1 containing 500 IU / mL IL-2, 100 IU / mL IL-15, 2 IU / mL IL-21, 10 ng / mL IL-18, 0.01 KE / mL Group A Streptococcus for injection, 5% platelet lysate (HPL), 2% human serum albumin, and 4.28 mL of NK cell basal medium (Tongli Haiyuan), with a total volume of 15 mL / flask. Incubated at 37°C in a 5% CO2 incubator.

[0140] 4. First fluid resuscitation: On the third day of culture, add 15 mL of activation medium (basal medium is Tongli Haiyuan) containing 500 IU / mL IL-2, 100 IU / mL IL-15, 2 IU / mL IL-21, 10 ng / mL IL-18, 5% platelet lysate (HPL), and 2% human serum albumin to the culture flask, for a final volume of 30 mL.

[0141] 5. Second fluid resuscitation: On the 5th day of culture, add 15 mL of amplification medium (basal medium is Tongli Haiyuan) containing 500 IU / mL IL-2, 100 IU / mL IL-15, 5% platelet lysate (HPL), and 2% human serum albumin to the culture flask, for a final volume of 45 mL.

[0142] 6. Third fluid resuscitation: On day 7 of culture, 3.0 × 10⁻⁶ cells were retained from each group. 7Cells and 1.0 × 10 6 At a cell density of / mL, amplification medium containing 500 IU / mL IL-2 and 100 IU / mL IL-15 was added (basal medium was Tongli Haiyuan). At the same time, 5% platelet lysate (HPL) and 2% human serum albumin were added. After replenishment, 30mL / bottle was added to each group, one bottle for each group.

[0143] 7. Fourth fluid resuscitation: On day 9 of culture, the cell concentration in each group was measured. Each group retained 3.0 × 10⁶ cells. 6 Cells and 1.0 × 10 6 At a cell density of / mL, add expansion medium (basal medium is Tongli Haiyuan) containing IL-2 500 IU / mL, IL-15 100 IU / mL and 2% human serum albumin, 30mL / bottle per group after replenishment.

[0144] 8. Fifth fluid resuscitation: On day 11 of culture, the cell concentration in each group was measured. Each group retained 3.0 × 10⁶ cells. 6 Cells and 1.0 × 10 6 At a cell density of / mL, add expansion medium (basal medium is Tongli Haiyuan) containing IL-2 500 IU / mL, IL-15 100 IU / mL and 2% human serum albumin, 30mL / bottle per group after replenishment.

[0145] 9. Sixth fluid resuscitation: On day 13 of culture, the cell concentration in each group was measured. Each group retained 3.0 × 10⁶ cells. 6 Cells and 1.0 × 10 6 At a cell density of / mL, add expansion medium (basal medium is Tongli Haiyuan) containing IL-2 500 IU / mL, IL-15 100 IU / mL and 2% human serum albumin, 30mL / bottle per group after replenishment.

[0146] 10. Seventh rehydration session: On day 15 of culture, the cell concentration in each group was measured. Each group retained 3.0 × 10⁶ cells. 6 Cells and 1.0 × 10 6 At a cell density of / mL, 1854mL of amplification medium containing IL-2 500IU / mL, IL-15 100IU / mL, and 2% human serum albumin was added (the basal medium was Tongli Haiyuan). After replenishment, 30mL was added to each group per bottle.

[0147] 11. 8th fluid resuscitation On day 17 of culture, the cell concentration in each group was measured. Each group retained 3.0 × 10⁶ cells. 6 Cells and 1.0 × 10 6At a cell density of / mL, add expansion medium (basal medium is Tongli Haiyuan) containing IL-2 500 IU / mL, IL-15 100 IU / mL and 2% human serum albumin, 30mL / bottle per group after replenishment.

[0148] 12. Testing: On day 19 of culture, the cell concentration of each group was measured; samples from each group were analyzed by flow cytometry to determine the purity of NK cells. The results were as follows: UC019-A: 94.12%; UC019-B: 90.04%; UC019-C: 91.72% (e.g., ...). Figure 11 Meanwhile, the number, viability, and activation receptors of the expanded cells were detected, and the results are shown in Tables 8-9.

[0149] II. Experimental Results: The activation effect on UCBMCs varied when different antibody combinations were used to coat the culture flasks in the early stages. After 19 days of culture, the results showed that the highest fold increase in total NK cell count was observed when culture flasks coated with DPBS containing 10 μg / mL CD16 and 10 μg / mL 4-1BB, with the two donors showing increases of 738.05-fold and 7342.64-fold, respectively, both higher than the other two antibody combinations. Flow cytometry analysis of NK cell purity showed differences in purity enhancement among different donors, but when both CD16 and 4-1BB concentrations were 10 μg / mL, NK cell purity was higher than the other two groups, with purities of 72.75% and 94.12%, respectively. However, there were no significant differences among the three groups in cell viability and activation receptors NKG2D, NKp30, and NKp46, all remaining at high levels.

[0150] Therefore, using 10 μg / mL of CD16 humanized antibody and 10 μg / mL of 4-1BB humanized antibody as activation antibody-coated culture flasks can effectively activate umbilical cord blood UCBMCs and improve NK cell purity and expansion capacity.

[0151] Table 8. Statistics on cell viability, expansion, and purity after culturing UCBMCs in culture flasks coated with different antibodies for 19 days.

[0152] Table 9. Results of receptor activation detection after UCBMCs were cultured for 19 days using culture flasks coated with different antibodies.

[0153] The above description of the embodiments is only for understanding the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. A composition for isolating and culturing NK cells, characterized in that, The composition includes CD3 + Magnetic beads; Preferably, the CD3 + The amount of magnetic beads used is 0.5 to 1.5 times the number of T cells in the blood sample to be separated; Preferably, the composition further includes a first activation medium, the first activation medium comprising the following components: IL 2. IL 15. IL 21. IL18, platelet lysate, human serum albumin, group A streptococci for injection, basal culture medium; Preferably, in the first activation medium, IL The concentration of 2 is 100 1000 IU / mL, IL The concentration of 15 is 50. 1000 IU / mL, IL The concentration of 21 is 5 10 IU / mL, the concentration of IL18 is 5 20 ng / mL, platelet lysate concentration of 2%-10%, human serum albumin concentration of 2%-10%, and group A streptococcus for injection concentration of 0.01-1 KE / mL; Preferably, the basal culture medium is a serum-free culture medium.

2. The composition according to claim 1, characterized in that, The CD3 + The amount of magnetic beads used is 1.5 times the number of T cells in the blood sample to be separated; Preferably, in the first activation medium, IL The concentration of 2 is 500 IU / mL, IL The concentration of 15 is 100 IU / mL, IL The concentrations of IL-21 were 2 IU / mL, IL-18 were 10 ng / mL, platelet lysate was 5%, human serum albumin was 2%, and group A streptococci for injection were 0.01 KE / mL. Preferably, the composition further includes any one or more of the following: 1) Second activation medium, which consists of the following components: IL 2. IL 15. IL 21. IL18, platelet lysate, human serum albumin, basal culture medium; Preferably, in the second activation medium, IL The concentration of 2 is 100 1000 IU / mL, IL The concentration of 15 is 50. 1000 IU / mL, IL The concentration of 21 is 5 10 IU / mL, the concentration of IL18 is 5 20 ng / mL, platelet lysate concentration of 2%-10%, human serum albumin concentration of 2%-10%; 2) First amplification medium, which consists of the following components: IL 2. IL 15. Platelet lysate, human serum albumin, basal culture medium; Preferably, in the first amplification medium, IL The concentration of 2 is 100 1000 IU / mL, IL The concentration of 15 is 50. 1000 IU / mL, platelet lysate concentration of 2%-10%, human serum albumin concentration of 2%-10%; 3) Second amplification medium, which consists of the following components: IL 2. IL 15. Human serum albumin, basal culture medium; Preferably, in the second amplification medium, IL The concentration of 2 is 100 1000 IU / mL, IL The concentration of 15 is 50. 1000 IU / mL, the concentration of human serum albumin is 2%-10%; 4) Coating solution, wherein the coating solution is composed of the following components: CD16 antibody and 4-1BB antibody; Preferably, the concentration of CD16 antibody and 4-1BB antibody in the coating solution is 5.0-20 μg / mL.

3. The composition according to claim 1 or 2, characterized in that, In the second activation medium, IL The concentration of 2 is 500 IU / mL, IL The concentration of 15 is 100 IU / mL, IL The concentrations of IL-21 were 2 IU / mL, IL-18 was 10 ng / mL, platelet lysate was 5%, and human serum albumin was 2%. Preferably, in the first amplification medium, IL The concentration of 2 is 500 IU / mL, IL The concentration of 15 was 100 IU / mL, the concentration of platelet lysate was 5%, and the concentration of human serum albumin was 2%. Preferably, in the second amplification medium, IL The concentration of 2 is 500 IU / mL, IL The concentration of 15 was 100 IU / mL, and the concentration of human serum albumin was 2%. Preferably, the concentration of CD16 antibody and the concentration of 4-1BB antibody in the coating solution are 10 μg / mL. Preferably, the basal culture medium for the second activation medium, the first amplification medium, and the second amplification medium is a serum-free medium; Preferably, the blood sample to be separated comes from umbilical cord blood, peripheral blood, or bone marrow; Preferably, the blood sample to be separated is umbilical cord blood.

4. A method for isolating and expanding NK cells, characterized in that, The method includes: a) Pretreatment of cell culture containers with the coating solution according to claim 2 or 3; b) Add CD3 to the blood sample to be separated + Magnetic beads are incubated, and mononuclear cells are obtained by separating the incubated blood sample. c) In the cell culture vessel obtained in a), the mononuclear cells obtained in b) are cultured using the first activation medium as described in claim 1 or 2; d) Supplement the second activation medium, the first amplification medium, and the second amplification medium as described in claim 2 or 3.

5. The method according to claim 4, characterized in that, The cell culture containers include T225 culture flasks, T75 culture flasks, and cell culture bags; Preferably, the CD3 + The amount of magnetic beads used is 0.5 to 1.5 times the number of T cells in the blood sample to be separated; Preferably, the method for detecting the number of T cells in the blood sample to be separated is flow cytometry; Preferably, the addition of CD3 + The incubation time for magnetic beads is 20-60 minutes; Preferably, the mononuclear cells are obtained by Ficoll density gradient centrifugation. Preferably, the seeding density of the mononuclear cells is 5.0 × 10⁻⁶. 5 / mL-2.0×10 6 cells / mL; Preferably, the second activation medium is added on days 1-3 after inoculation; Preferably, the first amplification medium is added on days 3-7 after inoculation; Preferably, the second amplification medium is added on days 7-19 after inoculation; Preferably, the volume increases by 2, 3, 4 or more times after each addition of culture medium.

6. The method according to claim 5, characterized in that, The CD3 + The amount of magnetic beads used is 1.5 times the number of T cells in the blood sample to be separated; Preferably, the addition of CD3 + The magnetic beads were incubated for 30 minutes. Preferably, the seeding density of the mononuclear cells is 1.0 × 10⁻⁶. 6 cells / mL; Preferably, the second activation medium is added on the third day after inoculation; Preferably, the first amplification medium is added on day 5 and day 7 after inoculation; Preferably, the second amplification medium is added on days 9, 11, 13, 15, and 17 after inoculation; Preferably, the volume doubles after each addition of culture medium; Preferably, the blood sample to be separated comes from umbilical cord blood, peripheral blood, or bone marrow; Preferably, the blood sample to be separated is umbilical cord blood.

7. NK cells prepared by the method according to any one of claims 4-6.

8. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the NK cells of claim 7; Preferably, the pharmaceutical composition further includes a pharmaceutically acceptable carrier, diluent, or excipient.

9. Use of the composition according to any one of claims 1-3 in the preparation of products containing NK cells.

10. The use of the NK cells of claim 7 or the pharmaceutical composition of claim 8 in the preparation of medicaments for cell therapy, medicaments for antiviral infection, and medicaments for treating cancer or autoimmune diseases; Preferably, the cancer includes solid cancer and non-solid cancer.

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