Construction method and application of gene modified K562 cell line

Abstract

The invention provides a construction method and application of a gene modified K562 cell line, and belongs to the technical field of biological medicines. According to the invention, two viruses CD86-4-1BBL and mbIL-21 are respectively subjected to gene transduction packaging by adopting a third-generation lentivirus four-plasmid packaging system, a K562 cell line is infected by the lentiviruses CD86-4-1BBL and mbIL-21, and a monoclonal cell line capable of stably expressing CD86, 4-1BBL and mbIL-21 proteins is obtained through screening. According to the method, cell lines with the cloning numbers of V5-E9, V10-A5, V5-D6 and V6-D7 are obtained through screening, the four cloning cell lines can serve as feeder cells for stimulating NK cell amplification after being amplified and subjected to irradiation treatment, NK cell amplification can be effectively promoted, the amplification multiple of the NK cells reaches up to 6000-8000, and large-scale preparation and production of the NK cells for treatment are met.

Description

Technical Field The present invention belongs to the technical field of biomedicine, and particularly relates to a method for constructing a genetically modified K562 cell line and its application. Background Art Natural killer (NK) cells, as effector cells of the innate immune system, play an important role in antiviral and anti-tumor activities. NK cell culture in vitro can promote cell proliferation through in vitro stimulation such as cytokines (a combination of cytokines or cytokines with other matrices / antibodies), antibody-coupled magnetic beads, etc. However, the amplification multiples of these stimulation methods are relatively low and cannot meet the needs of large-scale universal NK cell preparation. [1] . According to multiple studies, genetically modified K562 cells have a very significant ability to promote NK cell proliferation. K562 cells are an HLA I-deficient erythroleukemia cell line and are commonly used target cells for NK cell in vitro cytotoxicity analysis. They can provide contact-dependent proliferation stimulation for NK cells. [2] According to the inquiry, the NK cell process currently used in clinical research uses this genetically modified K562 as feeder cells to produce NK cells in large quantities. [3] Some researchers used irradiated K562Clone9.mbIL-21 [4] , used to massively expand NK cells and combine with bone marrow transplantation to treat AML patients, and also used to prepare CD19 CAR-NK to treat B cell tumors. There are also reports of K562 cells using mbIL-15 and 4-1BBL genetic modification for NK cell expansion. [5] , or use K562-derived aAPC to expand CD19 CAR-T to treat B cell tumors [6] Although these have promoted the expansion of NK cells to a certain extent, there is still a certain gap between them and the number of cells required for CAR-NK therapy. In addition, there are currently no studies using K562 expressing CD86, 4-1BBL, and IL-21 to promote BCMA-targeted NK cell expansion.

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[0006] Singh, H., et al., A new approach to gene therapy using Sleeping Beauty to genetically modify clinical - grade T cells to target CD19. Immunol Rev, 2014. 257(1):p. 181 - 90. Summary of the Invention In response to the above-mentioned shortcomings, the present invention provides a method for constructing a genetically modified K562 cell line and its application. The present invention adopts a third-generation lentiviral four-plasmid packaging system to carry out gene transduction and packaging of two viruses, CD86-4-1BBL and mbIL-21, respectively. The K562 cell line is infected with the lentivirus CD86-4-1BBL and mbIL-21, and a monoclonal cell line stably expressing CD86, 4-1BBL and mbIL-21 proteins is obtained through screening. Cell lines with clone numbers V5-E9, V10-A5, V5-D6 and V6-D7 were screened and obtained. After amplification and irradiation, the above four clonal cell lines can be used as feeder cells to stimulate NK cell amplification, which can effectively promote NK cell amplification. The amplification multiple of NK cells is as high as 6000-8000 times, which meets the requirements of large-scale NK cell preparation and production for treatment. The technical solution of the present invention includes: In one aspect, the present invention provides a method for constructing a genetically modified K562 cell line, characterized in that the construction method comprises the following steps: 1) Constructing lentivirus 1 containing CD86 and 4-1BBL genes and lentivirus 2 containing mbIL-21 gene; 2) Transfect K562 cells with lentivirus 1 and lentivirus 2 to obtain polyclonal cell lines; 3) The polyclonal cell line was cultured into a monoclonal cell line to obtain the genetically modified K562 cell line. Specifically, the lentivirus 1 in step 1) is the lentivirus CD86-4-1BBL, and the lentivirus 2 is the lentivirus mbIL-21; Specifically, the preparation process of lentivirus 1 or lentivirus 2 in step 1) is as follows: (1) Inoculate the host cells into the culture medium and culture until the viable cell density is 5.0×10 6 -8.0×10 6 cells / mL for lentiviral transfection; (2) transiently transfecting the shuttle plasmid, packaging plasmid, and envelope plasmid into host cells in proportion; (3) Culture for 24-54 hours, collect the supernatant, concentrate, resuspend, and filter to obtain lentivirus 1 or lentivirus 2. Preferably, the culture in step (1) is carried out until the viable cell density is 7.31×10 6 cells / mL for lentiviral transfection. Preferably, in step (1), PEIpro is used for lentiviral transfection, and the ratio of PEIpro to DNA is 2:1. More specifically, the culture medium in step (1) includes, but is not limited to, one or more of CHO medium, MEM medium, DMEM medium, RPMI1640 medium, SMM293TII serum-free medium, or OPM-293CD05 medium. Preferably, the culture medium in step (1) is a 293TS culture medium obtained by mixing SMM293TII serum-free culture medium and OPM-293CD05 medium in a ratio of 1:1. More specifically, the host cells in step (1) and step (2) are mammalian cells. Preferably, the host cells described in step (1) and step (2) include but are not limited to human embryonic kidney cells (HEK293) or 293T cells. More preferably, the host cells in step (1) and step (2) are 293TS cells. Preferably, the shuttle plasmids described in step (2) are Pre-Lenti-EF1-CD86-4-1BBL-CAR V2Wpmut and Pre-Lenti-EF1-mbIL-21-CAR V2 WPmut. Further preferably, the nucleotide sequence of the target gene of the shuttle plasmid in step (2) is as shown in SEQ ID NO: 1 or SEQ ID NO: 2; The sequence of the (Pre-Lenti-EF1-CD86-4-1BBL-CAR V2 Wpmut) SEQ ID NO: 1 is: The sequence of (Pre-Lenti-EF1-mbIL-21-CAR V2 WPmut) SEQ ID NO: 2 is: More specifically, the ratio of the shuttle plasmid, packaging plasmid and envelope plasmid in step (2) is shuttle plasmid: ZL004: ZL003: ZL006 = 7:5:3:5. Preferably, the culture in step (3) is fed after 6-8 hours, and the cells are harvested 48 hours after the 293TS cells are transfected. Preferably, the supernatant collected in step (3) is a supernatant collected by centrifugation. Preferably, the concentration in step (3) is carried out by centrifugation at 25,000 rpm, 10° C., and 2.5 h. Preferably, the resuspension in step (3) is performed using 0.9% sodium chloride injection. Specifically, the K562 cells described in step 2) were cultured using 1640 medium containing 10% FBS. More specifically, the K562 cells described in step 2) were cultured to a viable cell density of 5.0×10 6 -8.0×10 6 cells / mL for lentiviral transfection. Preferably, the K562 cells described in step 2) are cultured to a viable cell density of 7.31×10 6 cells / mL for lentiviral transfection. Specifically, the ratio of lentivirus 1 or lentivirus 2 to K562 cells in step 2) is 1.5 μL, 2.5 μL, 3.0 μL, 5.0 μL, 7.5 μL, 10.0 μL, 12.5 μL or 15.0 μL lentivirus / 2×10 4 cells K562. Preferably, the ratio of lentivirus 1 or lentivirus 2 to K562 cells in step 2) is 5.0 μL lentivirus / 2×10 4 cells K562. Specifically, the culture medium used for the monoclonal culture in step 3) is a serum-free medium X-VIVO 15 medium. On the other hand, the present invention provides a genetically modified K562 cell line constructed by the above construction method. In another aspect, the present invention provides use of the genetically modified K562 cell line in culturing NK cells. Preferably, the application is application in promoting NK cell expansion. In another aspect, the present invention provides a NK cell culture process, which comprises stimulating NK cells with a genetically modified K562 cell line and amplifying and culturing the NK cells. Specifically, the culturing process comprises the following steps: S1, genetically modified K562 cell line irradiated and ready for use; S2, purification of NK cells; S3, stimulating purified NK cells with the irradiated genetically modified K562 cell line for expansion culture; S4. On days 5-8 of expansion culture, NK cells are stimulated for a second time using the irradiated genetically modified K562 cell line; S5. Collect NK cells after expansion and culture. Preferably, the genetically modified K562 cell lines described in step S1 include V5-E9, V10-A5, V5-D6 and V6-D7 monoclonal clones. Preferably, the irradiation in step S1 is 100 Gy. Preferably, the purification of NK cells in step S2 comprises the following steps: collecting peripheral blood, separating PBMC, obtaining PBMC cells, separating and selecting and enriching CD3 - CD56 + of NK cells. Preferably, the ratio of the genetically modified K562 cell line to NK cells in step S3 and step S4 is 10-1:1. Further preferably, the ratio of the genetically modified K562 cell line to the NK cells in step S3 and step S4 is 1:1. Preferably, the cell concentration is maintained at 1×10 5 -1×10 6 cells / mL. More preferably, the cell concentration is maintained at 1.25×10 5 cells / mL. Preferably, step S4 is to perform a secondary stimulation of NK cells using the irradiated genetically modified K562 cell line on the 7th day of the expansion culture. Preferably, step S4 is to collect NK cells on the 14th day of expansion culture. In another aspect, the present invention provides a NK cell population prepared using the above-mentioned genetically modified K562 cell line. Specifically, the NK cell population includes but is not limited to: NK cells, CAR-NK cells, and NK92 cells. In another aspect, the present invention provides use of the above-mentioned NK cell population in preparing medicines. Specifically, the drug is a drug for treating tumors or autoimmune diseases. More specifically, the tumor is but not limited to a T-cell malignancy or a B-cell malignancy. Preferably, the T-cell malignancy includes but is not limited to acute lymphoblastic leukemia, T-cell large granular lymphocytic leukemia, adult T-cell leukemia / lymphoma, T-cell prolymphocytic leukemia or peripheral T-cell lymphoma. Preferably, the B-cell malignancy includes but is not limited to non-Hodgkin's lymphoma or chronic lymphocytic leukemia. The autoimmune diseases include, but are not limited to, systemic lupus erythematosus, rheumatoid arthritis, Sjögren's syndrome, systemic sclerosis, myositis, dermatomyositis, ankylosing spondylitis, hyperthyroidism, juvenile diabetes, idiopathic thrombocytic purpura, autoimmune hemolytic anemia, ulcerative colitis, Crohn's disease or psoriatic arthritis. The beneficial effects of the present invention are: (1) The present invention adopts the third-generation lentiviral four-plasmid packaging system to carry out gene transduction and package two viruses CD86-4-1BBL and mbIL-21 respectively, and transduces CD86, 4-1BBL and mbIL-21 genes into K562 cells through lentiviral CD86-4-1BBL and mbIL-21 infection to establish a stable co-expression K562 monoclonal cell line, which contributes to NK cell activation and proliferation. (2) The present invention uses clones V5-E9, V10-A5, V5-D6 and V6-D7 as feeder cells for NK cell culture and expansion stimulation, which can effectively promote NK cell expansion. The expansion multiple of NK cells is as high as 6000-8000 times, which meets the requirements of large-scale NK cell preparation and production. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 This is the flow cytometry detection of K562-mbIL-21 polyclonal. Figure 2 Flow cytometry was used to detect the expression of transduced genes in K562-mbIL-21 monoclonal cells. Figure 3 The figure shows the fold change of K562-mbIL-21 monoclonal clone during serial passage. Figure 4 The figure shows the statistical diagram of the expansion times of NK cells at different times under the stimulation of feeder cells of four clone numbers. Figure 5 Phenotypic analysis of the expansion process of NK cells stimulated by feeder cells for four clones. DETAILED DESCRIPTION The present invention is described below with reference to specific examples. The following examples are not intended to limit the present invention but are merely intended to illustrate the present invention so that the technical solutions of the present invention are more easily understood and grasped. 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. The material information used in the present invention is shown in Table 1: Table 1 Experimental materials Example 1 Construction of CD86-4-1BBL and mbIL-21 vectors and lentiviral packaging 1. Plasmid extraction The transformed bacteria containing Pre-Lenti-EF1-mbIL-21-CAR V2 WPmut and Pre-Lenti-EF1-CD86-4-1BBL-CAR V2 WPmut plasmids were cultured in 500 mL LB medium and shaken for 17 h. The OD value of the bacterial solution was 0. 600nm They are 3.25 and 2.98 respectively. The bacterial suspension was collected by centrifugation, and the plasmid was extracted using an endotoxin-free plasmid extraction kit. After elution, the harvest volume and plasmid concentration were measured. Plasmid samples were subjected to agarose gel electrophoresis for purity assessment, and samples were sent to Guangzhou Aiki Biotechnology Co., Ltd. for sequencing. The nucleotide sequence of the target gene of the plasmid Pre-Lenti-EF1-mbIL-21-CAR V2 WPmut is shown in SEQ ID NO: 1, and the nucleotide sequence of the target gene of the plasmid Pre-Lenti-EF1-CD86-4-1BBL-CAR V2 WPmut is shown in SEQ ID NO: 2. After qualified purity and sequencing, lentiviral packaging was performed. 2. Lentiviral packaging 293TS cells were seeded into 293TS medium and cultured to a cell density of 7.31×10 6 When the cell suspension was 1:1, 100 mL of cell suspension was used, PEIpro was used as the transfection reagent, the plasmid dosage was 2 mg / L (shuttle plasmid: ZL004: ZL003: ZL006 = 7:5:3:5), and the mixture was transfected into 293TS cells at a ratio of DNA: PEIpro = 1:2. The specific dosage is shown in Table 2. Table 2 Lentivirus packaging material information After adding the DNA / PEIpro mixture to the cells, continue shaking the culture and feed for 6-8 hours. Terminate the culture 48 hours after transfection, harvest all cells, collect the supernatant by centrifugation, filter, and concentrate at 25,000 rpm at 10°C for 2.5 hours. Resuspend the concentrated lentivirus in 3 mL of 0.9% sodium chloride injection and filter through a 0.22 μm filter. Aliquot 0.1 mL / vial and store at -80°C. Example 2 Construction of K562-mbIL-21 polyclonal cells 1. Culture medium preparation K562 cells were cultured in 1640 medium containing 10% FBS. The 1640 complete medium was prepared as follows: 450 mL RPMI Modified Medium (1640 medium) + 50 mL FBS. Shake well and store at 4°C until ready for use. 2. K562 cell transduction of target gene (1) K562 cells were passaged and expanded to establish a cell bank, and STR identification was performed on the K562 cells in the bank. K562 cells (lot number: WCB180605P04-28#) were obtained for establishment of the bank, and the cells were quickly thawed in a 37°C water bath, added to PBS, centrifuged at 400g for 5 minutes, and the supernatant was removed. The cells were resuspended in complete culture medium and transferred to a culture flask, which was placed in a 37°C CO2 incubator for culture and passage. (2) Cell counting, preparation density is 2×10 4 A K562 cell suspension of 10 cells / mL was seeded into 8 wells in a 24-well plate at 1 mL / well. Equal volumes (5 μL, 7.5 μL, and 10 μL) of CD86-4-1BBL and mbIL-21 lentivirus were added to each well. Finally, 1 μL of protamine was added to each well. The suspension was gently aspirated to mix well and cultured in a CO2 incubator at 37°C. (3) After 3 days of culture in T-25 flasks, samples were taken for flow cytometry detection of IL-21, CD86, and 4-1BBL expression. The results are shown in Table 3. Based on the positive rate of each gene detected by flow cytometry, cells with a lentiviral load of 5 μL were selected for amplification and used in subsequent experiments. Table 3 Gene expression in K562 cells infected with double lentivirus The assay results showed that K562-mbIL-21 polyclonal cells significantly increased the expression of IL-21, CD86, and 4-1BBL in K562 cells. (4) K562-mbIL-21 polyclonal cells with lentivirus added at a volume of 5 μL / well were collected, and the expression of the three genes HLA ABC, HAL AB, and OX40L expressed by the 5 uL lentivirus group and the mother K562 cells themselves was detected by flow cytometry. The results of the test are shown in Figure 1K562 cells were weakly positive when tested with anti-HLAABC antibodies but negative when tested with anti-HLAAB antibodies, indicating that K562 cells express HLAABC but not HLAAB. Similarly, transduced K562-mbIL-21 polyclonal cells were also weakly positive for HLAABC. Furthermore, transduced K562-mbIL-21 polyclonal cells expressed OX40L approximately 20% more strongly than K562 cells. Example 3: Comparison of K562-mbIL-21 polyclonal cells with prior art (1) FC cells: IL 21NK cell expansion reagent was purchased from Hangzhou Zhongying, with the product number ZY NKZ 0104. The FC cells are K562 cells that express IL21, 41BBL, CD14, CD19, and CD86, and have no proliferation activity after irradiation. (2) The K562-mbIL-21 polyclonal cells of the present invention were irradiated with 100 Gy, aliquoted and frozen for later use. After FC cells and K562-mbIL-21 polyclonal cells of the present invention were revived, NK cells were stimulated for expansion at a ratio of 2:1 (FC cells or K562-mbIL-21 polyclonal cells:NK cells) (Day 1, designated D0). Secondary stimulation was performed on Day 7 and harvested. The effectiveness of monoclonal feeder cell expansion of NK cells was evaluated by comparing NK cell density, viability, and expansion efficiency following FC cell and K562-mbIL-21 polyclonal cell stimulation. The key steps are as follows: NK cell purification: 10 mL of fresh single-collected blood (lot number: 0003-0006-FLWE0375) was transferred to a 50 mL centrifuge tube and diluted to 20 mL with 0.9% sodium chloride injection. 15 mL of lymphocyte separation fluid (Ficoll) was added to a new 50 mL centrifuge tube for separation. 20 mL of the diluted single-collected blood was slowly added to the Ficoll along the side of the tube, 400 × g, 20 ° C, centrifuged for 30 min (3 up, 3 down), the top plasma layer was discarded with a pipette, the middle buffy coat cells were aspirated, and transferred to a new 50 ml centrifuge tube to obtain 5.72 × 10 8 PBMC cells. PBMC were first negatively selected using CD3 magnetic beads to remove CD3 positive T cells, and then positively selected using CD56 magnetic beads to enrich CD3 - CD56 + NK cells, and finally 1.65E+07 NK cells were obtained. 1. Day 0 Feeder Cell Stimulation 9E+06 NK cells were obtained, and K562-mbIL-21 polyclonal cells, frozen and irradiated, were resuscitated and added to the K562-mbIL-21 polyclonal cells at a ratio of 1:2 for stimulation and expansion. The final NK cell density was adjusted to a viable cell density of 5E+05 cells / mL in 18 mL of culture medium. Culture was performed in a CO2 incubator (37°C, 5% CO2). Separately, 1E+06 NK cells were obtained, and irradiated, frozen FC cells were resuscitated and added to the FC cells at a ratio of 1:2 for stimulation and expansion. The final NK cell density was adjusted to a viable cell density of 5E+05 cells / mL in a 2 mL culture medium. Culture was performed in a CO2 incubator (37°C, 5% CO2). 2. Harvest cells on Day 7 During the culture process, cells were counted on Day 3 and the medium was exchanged at an equal volume. On Day 5, the medium was exchanged at a double volume. Cells were harvested on Day 7, and cell density, viability, and expansion efficiency were evaluated (see Table 4). Table 4 Comparison of polyclonal feeder cells The assay results showed that compared with the prior art, the K562-mbIL-21 polyclonal cells prepared in the present invention had a better expansion effect. Example 4 Monoclonal Cell Construction 1. Monoclonal Line Preparation (1) Collect K562-mbIL-21 polyclonal cells with lentivirus added at a volume of 5 μL / well and count the cell density to obtain 2.87×10 5 cells / mL. (2) Take X-VIVO 15 medium and resuspend the polyclonal cells to 500 cells / 15 mL, blow evenly, and then add 135 mL of X-VIVO 15 medium to make a 500 cells / 150 mL cell suspension. (3) Add 150 μL of cell suspension to each well of a 96-well plate, i.e., 0.5 cells / well. Plant 10 96-well plates and number them 1-10. Culture in a CO2 incubator at 37°C. Transfer the remaining polyclonal cells to a T-175 flask, add 35 mL of 1640 complete medium, and continue to expand the culture. After 5 days, collect the cells and freeze 3 tubes (5×10) in a freezing solution containing 10% DMSO (composition: 11.2 mL of 1640 complete medium + 3.2 mL of FBS + 1.6 mL of DMSO). 5 cells / tube). (4) After the 96-well plate was cultured in a 37°C, 5% carbon dioxide incubator for 7 days, the wells with green fluorescent cells were observed under a fluorescence microscope and replenished with 150 μL / well of X-VIVO 15 medium. (5) Continue culturing until the culture medium turns yellow. Name the corresponding wells and transfer them to a 24-well plate. Add 500 μL / well of X-VIVO 15 medium to continue expansion. The naming convention is V+plate number+well position, for example, V2-B3 refers to the cells in well B3 of plate 2 of the X-VIVO 15 medium seed plate. (6) When the cells in the 24-well plate expanded to a confluence of 80%-90%, they were transferred to a 6-well plate and supplemented with 2 mL / well of X-VIVO 15 medium to continue expansion. (7) When the cells in the 6-well plate are expanded to a high confluence, they are transferred to T-25 flasks and supplemented with 5 mL / flask of X-VIVO 15 medium for further expansion. At this time, the cells in the flask are recorded as Pn generation cells. 2. Monoclonal detection (1) A certain amount of cells were taken from each well of the T-25 flask for flow cytometry detection of IL-21, CD86 and 4-1BBL expression, and the wells expressing all three transduced genes were preliminarily screened. When the number of cells was sufficient, three tubes of seed cells were frozen (marked as Pn generation, with a freezing density of 1-2×10 6 cells / tube, the freezing solution formula is: FBS:DMSO=9:1), and the remaining cells are cultured in X-VIVO 15 medium at a rate of 1-2×10 5 cells / mL density for subculture. (2) 16 wells were selected for testing. After comparing the flow cytometry results, 4 monoclonal wells were selected for more comprehensive flow cytometry comparison, including the transduction genes IL-21, CD86 and 4-1BBL. (3) The results showed that the positive rates of IL-21, CD86 and 4-1BBL of the four monoclonal antibodies V5-E9, V10-A5, V5-D6 and V6-D7 were over 90%, as shown in Table 5. The four monoclonal antibodies V5-E9, V10-A5, V5-D6 and V6-D7 were tested for sterility, mycoplasma and STR identification. Table 5 Summary of positive rates of fluorescence detection of three transduced genes in monoclonal wells Note: The clone names in the table consist of the plate number and the well position. For example, clone V1-B10 represents well 10 in row B of the first plate. The bold clones in the table are the four single clones identified in subsequent screening. By comparing the positive rates of transduction genes in each monoclonal well in Table 5, when there was no significant difference in the positive rates of 4-1BBL and CD86 genes, 9 clones with high transduction gene expression rates were selected from high to low according to the positive rate of IL-21. At the same time, samples were taken for flow cytometry to compare the expression of transduction genes. The results are shown in Figure 5. Figure 2 Flow cytometry results for each clone are presented in a bar graph. Expression intensity (horizontal axis) and expression uniformity (peak width) of the four transduced genes varied among the clones. The results showed that four clones, V5-E9, V10-A5, V5-D6, and V6-D7, exhibited high expression intensity and good uniformity. Further comparisons were performed with clones V5-E9, V10-A5, V5-D6, and V6-D7 (Table 6). These clones were all negative for sterility and mycoplasma, and STR analysis confirmed that they were K562 cells. Table 6 Four monoclonal clones obtained in the first round of screening and their related information Note: The clone name in the table consists of plate number + well position. 5. Analysis of the proliferation capacity of monoclonal continuous culture and the stability of transduced gene passage Take 4×10 of each of the four monoclonal clones V5-E9, V10-A5, V5-D6 and V6-D7 6 The cells were serially subcultured in 20 mL of X-VIVO 15 medium, with each generation lasting 2 days. The density was maintained at 2 × 10 5 cells / mL and the culture volume remained unchanged at 20 mL (i.e., after counting the cells during passage, 4 × 10 6 Each generation of cells was supplemented with culture medium to 20 mL, and the excess cells were frozen. Cells were counted and cell concentration and viability were recorded at each generation, and the expansion times of each clone were calculated ( Figure 3 After 10 passages, the expression of transduced genes IL-21, CD86, and 4-1BBL was detected. The results are shown in Table 7. Table 7 Monoclonal phenotypic detection results of serial passages Note: The clone name in the table consists of plate number + well position. The results showed that 4 single clones were selected and serially passaged, and the cell density of each passage was recorded, so that the proliferation times after each passage were calculated and analyzed graphically. Figure 3As can be seen, the expansion folds of the four monoclonal cells did not decrease significantly during serial passage, indicating stable proliferation. Flow cytometry analysis of the transduced genes was performed after serial passage, and the results of the positive rate determination are shown in Table 6. After continuous culture, the expression of 4-1BBL and CD86 was greater than 95%, while IL-21 expression was greater than 93% in all clones except the V10-A5 clone. The positive rates did not vary by more than 10% compared to the Pn generation of each monoclonal cell (Table 3), indicating that the expression of the transduced genes did not decrease significantly after serial passage, indicating stable expression. Example 5 Comprehensive screening of K562-mbIL-21 monoclonal activity Four monoclonal cells were irradiated and cryopreserved. After thawing, NK cells were stimulated and expanded at a 1:1 ratio (total K562-mbIL-21:NK) (Day 1, designated D0). Secondary stimulation was performed on D7, and harvest was performed on D14. The effectiveness of monoclonal feeder cell expansion of NK cells was comprehensively evaluated by comparing NK cell density, viability, expansion efficiency, and NK cell surface phenotype expression under stimulation with each monoclonal cell. The key steps are as follows: Monoclonal irradiation and cryopreservation: After monoclonal clones (K562-mbIL-21V5-E9, K562-mbIL-21V10-A5, K562-mbIL-21V5-D6, K562-mbIL-21V6-D7) were expanded and cultured, the cells were collected and irradiated with 100 Gy, and the cells were cryopreserved at 1×10 6 cells / 1ml / tube and freeze 2 tubes for future use. NK cell purification: thaw a single blood sample (lot number: 0003-0006-LQME4247) one day in advance, resuspend in X-VIVO15 medium, and adjust the cell density to 1.5×10 6 cells / mL, with a total volume of 455 mL, and transferred to a GT610 cell culture bag for culture. After 24 hours, the cells were collected and separated using a lymphocyte separation medium (Ficoll) to obtain 1.64×10 8 CD3 positive cells were removed by negative selection using CD3 magnetic beads, and then CD56 magnetic beads were used for positive selection to enrich CD3 - CD56 + NK cells were cultured to obtain 8 mL of NK cell suspension with a viable cell density of 9.42 × 10 5 cells / mL. Take 1.25×10 6 cells NK cells / group for the experiment. 1. Day 0 Feeder Cells First Stimulation Four irradiated and frozen K562-mbIL-21 monoclonal clones were revived and added with feeder cells of each clone at a ratio of NK cells:feeder cells = 1:1. The cells were cultured in a CO2 incubator (37°C, 5% CO2). 2. Day 7 NK cell phenotype identification and secondary stimulation with feeder cells During the culture process, the medium was changed with equal volume after cell counting on Day 3, and with double volume after cell counting on Day 5. After cell counting on Day 7, NK cell samples were taken for phenotype identification (detection marker antibody information is shown in Table 8), and then 5×10 6 NK cells were again added with corresponding monoclonal feeder cells (NK cells: feeder cells = 1:1) for secondary stimulation and expansion, and the final NK cell density was adjusted to 1.25×10 5 cells / mL, the total culture volume was 40 mL, and the culture was continued in a CO2 incubator (37°C, 5% CO2). 3. Harvest NK cells on D14 During the culture process, the medium was replaced with 2 times the volume of D7 after counting the cells on D10, 3 times the volume of D10 after counting the cells on D12, and the cells were harvested after counting the cells on D14. Table 8 Information on antibodies to marker molecules related to phenotypic detection Note: 7-AAD was added to the sample 5 minutes before loading onto the instrument and analyzed after incubation. NK cells were prepared by a 14-day culture process and expanded under the stimulation of feeder cells. The viable cell density (Table 9), total viable cell count (Table 10), and cell expansion multiple (Table 11, Figure 4 ). Table 9 Changes in NK cell density and viability during NK cell expansion stimulated by feeder cells Table 10 Statistical results of total number of NK living cells (unit: cells) Table 11 Statistical results of NK cell expansion fold (unit: fold) After 14 days of culture, the cell density, expansion multiple (D1-D14) and total number of viable cells of NK cells using V5-E9 clone as feeder cells were higher than those of the other three clones, indicating that the feeder cells of V5-E9 clone can better promote the proliferation of NK cells. The NK cells obtained by amplification were stimulated by feeder cells of four clone numbers. The NK purity analysis (Table 12) showed that CD3 - CD56 + The positive rate can reach more than 99.5%, and the residual T cell detection amount at the time of harvest is less than 1%. Therefore, there is no significant difference in the phenotype and T cell residues among the NK cells of each batch, indicating that there is no significant difference in the purity and T cell residues of the NK cells obtained by stimulating and expanding the feeder cells of the four clones. Table 12CD3 - CD56 + Positive rate test results 3. Day 14 NK cell phenotype identification On Day 10, after counting cells, the medium was exchanged twice (80 mL). On Day 12, after counting cells, the medium was exchanged three times (120 mL). On Day 14, the cells were counted and NK cells were sampled again for phenotypic analysis (same as Day 7), and the culture was terminated. The results are shown in Table 13. Table 13 NK cell phenotype analysis The phenotype of NK cells at D7 and D14 was analyzed. Figure 5 As shown in Table 12, the activating receptors NKG2D and NKp46 are expressed steadily and at high levels, with a characteristic of sustained expression; NKp44 and NKp30 increase with increasing activation (or culture) time. The inhibitory receptors KIR2DL2 / 3, KIR2DL1, KIR3DL1, and PD1 are expressed at low levels throughout the culture process, with a slight increase as the culture time increases. The inhibitory receptor NKG2A is highly expressed and increases with increasing culture time, and the trends in each group are consistent. It can be seen that the phenotype of NK cells obtained by stimulating and expanding the K562-mbIL-21 feeder cells of the four clones is basically the same. Comparative Example 1mbIL21-86-41BBL lentivirus transfected K562 cells 1. Plasmid extraction The transformed strain of Pre-Lenti-EF1-CAR V2 WPmut-mbIL21-86-41BBL plasmid was cultured in 500 mL LB medium and cultured on a shaking platform for 17 h. The OD value of the bacterial solution was 0. 600nm They are 3.25 and 2.98 respectively. The bacterial suspension was collected by centrifugation, and the plasmid was extracted using an endotoxin-free plasmid extraction kit. After elution, the harvest volume and plasmid concentration were measured. Plasmid samples were subjected to agarose gel electrophoresis for purity assessment, and additional samples were sent to Guangzhou Aiki Biotechnology Co., Ltd. for sequencing. The nucleotide sequence of the plasmid Pre-Lenti-EF1-CAR V2 WPmut-mbIL21-86-41BBL is shown in SEQ ID NO: 3. After satisfactory purity and sequencing, lentiviral packaging was performed. SEQ ID NO: 3 2. Lentiviral packaging 293TS cells were seeded into 293TS medium and cultured to a cell density of 7.31×10 6 Transfection was performed when the cell suspension was 1:1 cells / mL. 100 mL of cell suspension was taken, PEIpro was used as the transfection reagent, the plasmid dosage was 2 mg / L (shuttle plasmid: ZL004: ZL003: ZL006 = 7:5:3:5), and the 293TS cells were transfected with a mixture of DNA: PEIpro = 1:2. The specific dosage is shown in Table 14. Table 14 Lentivirus packaging dosage After adding the DNA / PEIpro mixture to the cells, continue shaking the culture and feed for 6-8 hours. Terminate the culture 48 hours after transfection, harvest all cells, collect the supernatant by centrifugation, filter, and concentrate at 25,000 rpm at 10°C for 2.5 hours. Resuspend the concentrated lentivirus in 3 mL of 0.9% sodium chloride injection, filter through a 0.22 mm filter, and add 20% HSA at a 1:9 volume ratio. Aliquot 0.1 mL / vial and store at -80°C. 2. Culture medium preparation K562 cells were cultured in 1640 medium containing 10% FBS. The 1640 complete medium was prepared as follows: 450 mL RPMI Modified Medium (1640 medium) + 50 mL FBS. Shake well and store at 4°C until ready for use. 3. K562 cell transduction of target gene (1) K562 cells were passaged and expanded to establish a cell bank, and STR identification was performed on the K562 cells in the bank. K562 cells (lot number: WCB180605P04-28#) were obtained for establishment of the bank, and the cells were quickly thawed in a 37°C water bath, added to PBS, centrifuged at 400g for 5 minutes, and the supernatant was removed. The cells were resuspended in complete culture medium and transferred to a culture flask, which was placed in a 37°C CO2 incubator for culture and passage. (2) Cell counting, preparation density is 2×10 4 A K562 cell suspension of 10 cells / mL was seeded into 8 wells of a 24-well plate at 1 mL / well. 10 μL of CD86-4-1BBL-mbIL-21 lentivirus was added to each well. Finally, 1 μL of protamine was added to each well. Gently aspirate to mix well and incubate in a CO2 incubator at 37°C. 3. Preparation of K562 Monoclonal Line (1) After culturing in T-25 flasks for 3 days, K562 polyclonal cells were collected and the cell density was counted to obtain 2.87×10 5 cells / mL. (2) Take X-VIVO 15 medium and resuspend the polyclonal cells to 500 cells / 15 mL, blow evenly, and then add 135 mL of X-VIVO 15 medium to make a 500 cells / 150 mL cell suspension. (3) Add 150 μL of cell suspension to each well of a 96-well plate, i.e., 0.5 cells / well. Plant 10 96-well plates and number them 1-10. Culture in a CO2 incubator at 37°C. Transfer the remaining polyclonal cells to a T-175 flask, add 35 mL of 1640 complete medium, and continue to expand the culture. After 5 days, collect the cells and freeze 3 tubes (5×10) in a freezing solution containing 10% DMSO (composition: 11.2 mL of 1640 complete medium + 3.2 mL of FBS + 1.6 mL of DMSO). 5 cells / tube). (4) After the 96-well plate was cultured in a 37°C, 5% carbon dioxide incubator for 7 days, the wells with green fluorescent cells were observed under a fluorescence microscope and replenished with 150 μL / well of X-VIVO 15 medium. (5) Continue culturing until the culture medium turns yellow. Name the corresponding wells and transfer them to a 24-well plate. Add 500 μL / well of X-VIVO 15 medium to continue expansion. The naming convention is V+plate number+well position, for example, V2-B3 refers to the cells in well B3 of plate 2 of the X-VIVO 15 medium seed plate. (6) When the cells in the 24-well plate expanded to a confluence of 80%-90%, they were transferred to a 6-well plate and supplemented with 2 mL / well of X-VIVO 15 medium to continue expansion. (7) When the cells in the 6-well plate are expanded to a high confluence, they are transferred to T-25 flasks and supplemented with 5 mL / flask of X-VIVO 15 medium for further expansion. At this time, the cells in the flask are recorded as Pn generation cells. (8) Select a clone with high expression intensity and good uniformity. This single clone is negative for sterility and mycoplasma testing, and STR identification shows it is K562 cells. Obtain a K562 single clone. 4. Monoclonal activity assay Referring to the method described in Example 5, the monoclonal clones prepared in Comparative Example 1 were irradiated and cryopreserved. After thawing, NK cells were stimulated for expansion at a 1:1 ratio (total K562 monoclonal lines: NK cells) (Day 1, designated D0). A secondary stimulation was performed on D7, and the cells were harvested on D14. The effectiveness of monoclonal feeder cell expansion of NK cells was comprehensively evaluated by comparing NK cell density, viability, and expansion efficiency under stimulation with each monoclonal clone. The results are shown in Table 15. Table 15 Comparative Example 1 Effect of Monoclonal Feeder Cell Amplification on NK The above detailed description is a specific description of one feasible embodiment of the present invention and is not intended to limit the scope of the present invention. It should be noted that any equivalent implementation or modification that does not depart from the present invention should be included within the scope of the technical solution of the present invention. Therefore, the scope of protection of the patent of this invention should be based on the attached requirements.

Claims

1. A method for constructing a genetically modified K562 cell line, characterized in that, The construction method includes the following steps: 1) Construct lentivirus 1 containing CD86 and 4-1BBL genes and lentivirus 2 containing mbIL-21 gene; 2) Transfect lentivirus 1 and lentivirus 2 into K562 cells to obtain a polyclonal cell line; 3) Perform monoclonal culture on the polyclonal cell line to obtain a genetically modified K562 cell line.

2. The construction method according to claim 1, wherein The preparation process of lentivirus 1 or lentivirus 2 described in step 1) is as follows: (1) Inoculate host cells into a culture medium and culture them until the viable cell density reaches 5.0×10 6 -8.0×10 6 cells / mL, and then perform lentiviral transfection; (2) Co-transfect the shuttle plasmid, packaging plasmid and envelope plasmid into the host cell in proportion; (3) Culture for 24-54 hours, collect the supernatant, concentrate, resuspend, and filter to prepare lentivirus 1 or lentivirus 2.

3. The construction method according to claim 2, characterized in that The host cells described in step (1) and step (2) include mammalian cells.

4. The construction method according to claim 3, characterized in that, The nucleotide sequence of the target gene of the shuttle plasmid described in step (2) is as shown in SEQ ID NO: 1 or SEQ ID NO:

2.

5. The genetically modified K562 cell line constructed by the construction method described in any one of claims 1-4.

6. The genetically modified K562 cell line according to claim 5, characterized in that, The genetically modified K562 cell line described above expresses CD86, 4-1BBL and mbIL-21 genes.

7. Use of the genetically modified K562 cell line according to any one of claims 5-6 in culturing NK cells, characterized in that, The application described above is the application in promoting NK cell expansion.

8. The NK cell population prepared from the genetically modified K562 cell line described in any one of claims 5-6.

9. The application of the NK cell population described in claim 8 in the preparation of drugs.

10. Use of the NK cell population according to claim 9 in the preparation of a medicament, characterized in that, The drug described above is a drug for treating tumors or autoimmune diseases.

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