Feeder cell for amplifying NK (Natural Killer) cells as well as preparation method and application of feeder cell

Abstract

The invention provides a feeder cell for amplifying NK (Natural Killer) cells as well as a preparation method and application of the feeder cell. The feeder cell is a human cell with over-expression of IL-15, IL-21, 4-1BBL and IL-12p35 on a cell membrane. The human cells are selected from any one of immune cells, immortalized immune cells, lymphoma cells or leukemia cells. When the feeder cells are used for amplifying the NK cells, the amplification efficiency is high; the amplified NK cells are stronger in killing function; the feeder cell has very high clinical application value in amplification of the NK cells and large-scale clinical application of the NK cells.

Description

Technical Field

[0001] The invention belongs to the technical field of cell culture, and in particular relates to a feeder cell for amplifying NK cells and a preparation method and application thereof. Background Art

[0002] Natural killer (NK) cells are important members of innate immunity and are mainly distributed in peripheral blood, accounting for 5%-15% of the total number of lymphocytes. - CD56 + As a specific surface molecule of human NK cells, NK cells can destroy target cells (such as virus-infected cells, certain tumor cells and damaged cells) without antigen pre-sensitization, and their killing is not MHC-restricted, the response speed is fast, and it can play a role in the early stage of immune response.

[0003] The activity of NK cells is regulated in a unique way by receptors with activation or inhibition functions. NK cell receptors mainly include killer immunoglobulin-like receptors (KIR) and killer lectin-like receptors (KLR). KIR regulates the activation and inhibition of NK cells by binding to HLA molecules.

[0004] Important activating KIRs include KIR2DS and KIR3DS, and KLRs include CD94 / NKG2C and NKG2D. NKG2D is an important activating molecule of NK, and its recognized ligands include MICA / B and cytomegalovirus component UL16 binding protein (ULBP1 / 2 / 3 / 4). Other activated NK receptors include natural cytotoxicity receptors (NCR), such as NKp30, NKp44, and NKp46, which can recognize ligands on tumor cells or virus-infected cells. In addition, there is the IgG Fc receptor (FcγRⅢ), namely the CD16 molecule, which can mediate NK cell killing of tumor and virus-infected target cells through antibody-dependent cell-mediated cytotoxicity (ADCC). NK cell inhibition is mainly mediated by inhibitory KIR, such as KIR2DL and KIR3DL, and the interaction with their cognate human leukocyte antigen (HLA) ligands.

[0005] Common inhibitory KLRs include CD94 / NKG2A. NK cell expansion requires multiple cell signals for survival, proliferation, and activation. Activated NK cells can also produce direct cytotoxic effects through perforin and granzyme release or through death receptor signaling, and regulate other immune responses through the production of cytokines and chemokines.

[0006] NK cells have a strong therapeutic potential in cancer immunotherapy. NK cells for adoptive transfer therapy can be obtained from a variety of sources, including autologous or allogeneic human peripheral blood mononuclear cells (PBMCs), NK-92 cell lines, primary NK cells from umbilical cord blood, or CD34 + Induced differentiation from hematopoietic stem cells, pluripotent stem cells, and embryonic stem cells. Umbilical cord blood or stem cells can produce highly pure and functional NK cells, but their expansion time is long, the cell source is limited, and the maturity is low compared to NK cells derived from peripheral blood, so their cytotoxicity and ADCC effects are relatively weak. The NK-92 cell line has good stability and a simple purification procedure, but because it does not express or low-expresses CD16 molecules on its surface, its ADCC effect is limited, and it needs to be irradiated before administration, which limits its in vivo expansion ability and persistence. Its tumorigenicity is still a problem that cannot be ignored; the pluripotent stem cell (iPSC) technology has a wide range of donor cell sources, providing a potential universal cell source for all types of cell therapies, including tumor immunotherapy using NK cells. iPSC-derived NK cells are of uniform quality and easy to modify to target and kill tumor cells, but the required technology is complex and expensive, and its potential immunogenicity and risk of malignant transformation need to be considered in clinical applications. Therefore, it is relatively simple and easy to obtain PBMC-derived NK cells, with simple operation, rapid results, low cost, high safety, high amplification multiples, and high NK cell purity and stronger functions, which have high clinical application value.

[0007] Currently, there are two strategies for expanding PBMC-derived NK cells, the pure factor stimulation method and the irradiated feeder cell stimulation method. The pure factor method generally uses CD16 antibodies in combination with cytokines, such as IL-2, IL-15, IL-18 and IL-21, to expand and culture NK cells. The pure factor method is easier to comply with the GMP production process, but the individual differences of donors lead to large differences in expansion efficiency. The residual stimulation factors must also be considered, and it often requires a large number of initial cells for stimulation to reach the dose used in clinical practice. These methods generally require the use of single-collection products to ensure a large number of PBMC or NK cells, and even multiple single collections and expansions to ensure enough NK cells for reinfusion, indicating that their expansion capacity is still relatively low. In addition, the expansion of pure factor-stimulated NK generally requires CD3 to ensure its purity. + One-step magnetic bead negative selection or CD56 +Magnetic bead positive selection is a two-step process. Positive selection usually produces higher NK cell purity at the expense of a lower final cell count. The feeder cell expansion method uses irradiated, modified autologous or allogeneic PBMCs or cell lines (LCL, K562, etc.) to stimulate NK cell activation and proliferation. This method is simpler, produces higher purity NK cells, and the expansion effect is more stable than the pure factor method, with a higher expansion multiple, and is the mainstream method for expanding NK cells.

[0008] At present, the modification of feeder cells mainly uses lentivirus-infected cell lines to express membrane-bound IL-15, IL-18, IL-21 and 4-1BBL, etc., combined with soluble IL-2, to produce high-expansion multiples and high-cytotoxic NK cells. It is reported that the combination of multiple factors, such as K562 cells expressing membrane IL-21, IL-15 and 4-1BBL, can further enhance the expansion of NK cells and produce highly cytotoxic NK cells. However, the expansion efficiency and killing ability of NK cells using this feeder cell expansion still cannot meet the growing clinical needs, so it is urgent to develop a feeder cell that can enhance the expansion efficiency and killing ability of NK cells.

[0009] IL-12 is a proinflammatory cytokine secreted by antigen-presenting cells (such as dendritic cells, macrophages, and neutrophils) after antigen stimulation. IL-12 is a heterodimer composed of two subunits, p35 (i.e., IL-12α) and p40 (i.e., IL-12β). The two subunits are encoded by separate chromosomes and their expression is independently regulated. Among them, p40 can form the cytokine IL-23 with p19, and p35 can form the cytokine IL-35 with Ebi3. These cytokines are collectively referred to as IL-12 family cytokines. In addition to functioning as heterodimers, individual subunits can also function autonomously as monomers or homodimers. For example, in experimental models of arthritis, the IL-12p35 subunit has been shown to be a negative regulator of the IL-27 response. IL-12p40 is secreted independently of IL-12 in the serum of patients with pulmonary sarcoidosis and is considered a useful clinical marker of disease activity in pulmonary sarcoidosis. Similarly, IL-12p40 and disulfide-linked p40-p40 homodimers are secreted in the serum of patients with multiple sclerosis and are associated with the suppression of neurological dysfunction or endotoxemia by antagonizing IL-12 signaling and Th1 expansion. During the immune response, IL-12 activates the downstream STAT4 signaling pathway by binding to its receptor (IL-12R, composed of IL-12Rb1 and IL-12Rb2) to promote the activation of NK cells and T cells. IL-12 can serve as a growth factor for NK cells. IL-12 can enhance the cytokine that induces NK cells to produce IFN-γ to enhance the lytic activity of NK cells. In addition, some studies have shown that NK cells can exhibit memory-like properties through short-term pre-activation with IL-12, IL-15, and IL-18, becoming cytokine-induced memory-like NK cells, which subsequently show enhanced cell function when restimulated. Studies have shown that excessive or systemic IL-12 anti-tumor therapy has shown excessive toxic side effects.

[0010] At present, the pure factor method for amplifying NK cells has unstable amplification effect, low purity and poor amplification efficiency. The feeder cell amplification method is simpler to operate, has a more stable amplification effect and a higher amplification efficiency than the pure factor method. However, with the growing demand for the clinical application of NK cells, the traditional method of amplifying NK cells by overexpressing membrane-bound IL-21, IL-15 and 4-1BBL by feeder cells still cannot meet the growing clinical needs. It is urgent to develop a new feeder cell for a large number of NK cells to improve the killing function of the amplified NK cells, so as to meet large-scale clinical applications and improve the clinical application value of NK cells. Summary of the invention

[0011] In view of the shortcomings of the prior art, the object of the present invention is to provide a feeder cell for amplifying NK cells and a preparation method and application thereof. The present invention constructs a feeder cell that overexpresses membrane-bound IL-15, IL-21, 4-1BBL and IL-12, and the feeder cell has high efficiency in amplifying NK cells, and the amplified NK cells have strong killing function, which is conducive to the large-scale clinical application of NK cells and has higher clinical application value.

[0012] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0013] In a first aspect, the present invention provides a feeder cell for expanding NK cells, wherein the feeder cell is a human cell that overexpresses IL-15, IL-21, 4-1BBL and IL-12p35 on the cell membrane;

[0014] The human cells are selected from any one of immune cells, immortalized immune cells, lymphoma cells or leukemia cells.

[0015] Preferably, the human-derived cells include PBMC cells, LCL cells or K562 cells.

[0016] In the natural state, 4-1BBL is a membrane-expressed protein, and cytokines IL-15, IL-21 and IL-12 are secreted cytokines. In the present invention, the p35 subunits of IL-15, IL-21 and IL-12 are expressed on the membrane surface of PBMC cells, LCL cells or K562 cells, thereby constructing novel feeder cells. The above-mentioned PBMC cells, LCL cells or K562 cells are irradiated, and the PBMC cells can be autologous or allogeneic.

[0017] The present invention constructs a feeder cell expressing IL-15, IL-21, 4-1BBL and IL-12p35 on the membrane, which further improves the expansion multiple of NK cells and enhances the killing function of NK cells compared with feeder cells expressing IL-15, IL-21, 4-1BBL and IL-12p40 on the membrane and feeder cells expressing IL-15, IL-21 and 4-1BBL on the membrane. The method is simple to operate, fast in effect, and low in cost. After culturing for 14 days, NK cells can be expanded in vitro by more than 1000 times, and NK cells have high purity, higher expansion multiples, stronger functions, and reduced initial cell dosage, and have higher clinical application value.

[0018] Preferably, the coding sequence of IL-21 includes that shown in SEQ ID NO.1.

[0019] Preferably, the coding sequence of 4-1BBL includes that shown in SEQ ID NO.2.

[0020] Preferably, the coding sequence of IL-15 includes that shown in SEQ ID NO.3.

[0021] Preferably, the coding sequence of IL-12p35 includes that shown in SEQ ID NO.4.

[0022] Preferably, the IL-21, IL-15 and IL-12p35 are expressed by fusion with membrane proteins to form membrane-expressed IL-21, IL-15 and IL-12p35, and are localized on the cell membrane surface.

[0023] Preferably, the membrane protein comprises any one of type I membrane protein, type II membrane protein, multi-transmembrane protein or glycosylphosphatidylinositol anchored protein or a combination of at least two or more thereof.

[0024] Preferably, the membrane protein is selected from a full-length membrane protein, or a partial structure comprising a transmembrane domain / membrane anchoring domain.

[0025] Preferably, the membrane expressed IL-21 coding sequence includes that shown in SEQ ID NO.6.

[0026] Preferably, the membrane expressed IL-15 coding sequence includes that shown in SEQ ID NO.7.

[0027] Preferably, the membrane expressed IL-12p35 coding sequence includes that shown in SEQ ID NO.8.

[0028] In a second aspect, the present invention provides a method for preparing feeder cells for expanding NK cells as described in the first aspect, the preparation method comprising: constructing a vector containing a cytokine coding sequence, wherein the cytokine coding sequence is located on one or more vectors, and the cytokines include IL-21, 4-1BBL, IL-15 and IL-12p35, introducing the vector into human cells, sorting positive cells, and then expanding the culture to establish a feeder cell bank; before being used for NK cell expansion, irradiating the feeder cells in the feeder cell bank.

[0029] In the present invention, the IL-21, 4-1BBL, IL-15 and IL-12p35 can be on the same vector or on different vectors. When on the same vector, they can be connected by 2A peptide, IRES element, multiple promoters, etc.

[0030] Preferably, the vector comprises DNA and / or RNA.

[0031] Preferably, the method of introducing the vector into human cells comprises: any one or a combination of at least two or more of viral vector transduction, transposon insertion, homologous recombination insertion, RNA transfection or plasmid transfection.

[0032] Preferably, the viral vector comprises a lentiviral vector and / or a retroviral vector.

[0033] Preferably, the method for lentiviral vector transduction includes constructing a vector plasmid containing IL-21, 4-1BBL, IL-15 and IL-12p35 coding sequences, co-transfecting the vector plasmid with a lentiviral packaging plasmid into a virus packaging cell to obtain the virus, infecting human cells with the virus, sorting positive cells, and then expanding the culture to establish a feeder cell bank; before being used for NK cell amplification, the feeder cells in the feeder cell bank are irradiated.

[0034] In the present invention, the recombinant plasmid includes a lentiviral plasmid or a retroviral plasmid.

[0035] In a specific embodiment of the present invention, the lentiviral plasmid is pCDH-EF1α-MCS plasmid.

[0036] In a specific embodiment of the present invention, the virus packaging cells are 293T cells.

[0037] Preferably, the dose of the irradiation treatment is 5 Gy-500 Gy, for example, it can be 5 Gy, 10 Gy, 50 Gy, 100 Gy, 150 Gy, 200 Gy, 250 Gy, 300 Gy, 350 Gy, 400 Gy, 450 Gy or 500 Gy.

[0038] Preferably, the radiation source includes X-rays and gamma rays.

[0039] The present invention constructs a feeder cell whose membrane expresses IL-15, IL-21, 4-1BBL and IL-12p35, which further improves the expansion multiple of NK cells and enhances the killing function of NK cells compared with feeder cells whose membrane expresses IL-15, IL-21, 4-1BBL and IL-12p40 and feeder cells whose membrane expresses IL-15, IL-21 and 4-1BBL. The method is simple to operate, has rapid effect and low cost, can expand NK cells in vitro by more than 1000 times after culturing for 14 days, and the NK cells have high purity, higher expansion multiple and stronger function, reduce the initial cell dosage and have higher clinical application value.

[0040] In a third aspect, the present invention provides a nucleic acid molecule comprising any one or a combination of at least two or more of a membrane expressed IL-12p35 coding sequence, a membrane expressed IL-21, a membrane expressed IL-15 or a 4-1BBL coding sequence.

[0041] In a fourth aspect, the present invention provides a recombinant plasmid, wherein the recombinant plasmid contains the nucleic acid molecule described in the third aspect.

[0042] In a fifth aspect, the present invention provides a virus, wherein the virus is obtained by co-transfecting a virus packaging cell with the recombinant plasmid described in the fourth aspect and a lentiviral packaging plasmid.

[0043] In a sixth aspect, the present invention provides a method for improving the expansion efficiency of NK cells, the method comprising: using an NK cell culture medium to mix PBMC and the feeder cells for expanding NK cells described in the first aspect and adjusting the cell concentration to (0.2-5)×10 6 The ratio of PBMC to feeder cells is 100:1 to 1:100; NK cell expansion culture is performed, and the cell concentration is maintained at (0.2-5)×10 6 Pieces / mL.

[0044] Among them, the cell concentration is (0.2-5)×10 6 For example, 0.2×10 6 / mL, 0.5×10 6 / mL, 1×10 6 / mL, 1.5×10 6 / mL, 2×10 6 / mL, 2.5×10 6 / mL, 3×10 6 / mL, 3.5×10 6 / mL, 4×10 6 / mL, 4.5×10 6 / mL or 5×10 6 Pieces / mL, etc.

[0045] Preferably, the NK cell culture medium is selected from any one of OptiVitro NK cell culture medium, SCGM NK cell culture medium, Gibco CTS NK-Xpander culture medium or NK MACS culture medium, or a combination of at least two or more thereof.

[0046] In the present invention, the above culture medium is more suitable for amplification culture and has higher amplification efficiency.

[0047] Preferably, the NK cell culture medium is also supplemented with any one of AB serum, autologous plasma or serum substitutes, or a combination of at least two or more thereof, in an amount of 0.5-20 vol%, for example, 0.5 vol%, 1 vol%, 5 vol%, 10 vol%, 15 vol% or 20 vol%, etc.

[0048] Preferably, the PBMC cells and / or feeder cells are further revived before use.

[0049] In the seventh aspect, the present invention provides the use of the feeder cells for amplifying NK cells described in the first aspect, the nucleic acid molecules described in the third aspect, the recombinant plasmid described in the fourth aspect, the virus described in the fifth aspect, or the method for improving the amplification efficiency of NK cells described in the sixth aspect in amplifying NK cells.

[0050] The numerical range described in the present invention not only includes the point values ​​listed above, but also includes any point values ​​between the above numerical ranges that are not listed. Due to space limitations and for the sake of simplicity, the present invention no longer exhaustively lists the specific point values ​​included in the range.

[0051] Compared with the prior art, the present invention has the following beneficial effects:

[0052] (1) The present invention expands NK cells by constructing feeder cells that overexpress membrane-bound IL-21, IL-15, 4-1BBL and IL-12p35, feeder cells that overexpress IL-21, IL-15, 4-1BBL and IL-12p40, and feeder cells that overexpress IL-21, IL-15 and 4-1BBL; the results show that feeder cells that overexpress membrane-bound IL-21, IL-15, 4-1BBL and IL-12p35 have the best comprehensive effect, are highly efficient in expanding NK cells, and have stronger killing function of the expanded NK cells; this is conducive to the large-scale clinical application of NK cells and has a higher clinical application value.

[0053] (2) The expansion method of the present invention is simple to operate, has rapid results, and is low in cost. After 14 days of culture, NK cells can be expanded in vitro by more than 1,000 times. In addition, the NK cells are of high purity, have a higher expansion multiple, and have stronger functions. The initial cell dosage is reduced, and the clinical application value is higher. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 The results of functional detection of TIL overexpressing membrane-bound IL-12. A is the expression of TIL-mp35 and TIL-mp40IL-12 detected by flow cytometry. B is the expression level of IFN-γ in TIL overexpressing membrane-bound IL-12 by flow cytometry (the statistical method of this experiment uses paired t-test, * indicates p<0.05, significant difference; ns indicates p>0.05, no significant difference).

[0055] Figure 2 Figure 2 shows the molecular structures of SCCNK1, SCCNK1-mp35 and SCCNK1-mp40 and their expression results in K562 cells. A shows the molecular structures of SCCNK1, SCCNK1-mp35 and SCCNK1-mp40. B shows the expression of SCCNK1, SCCNK1-mp35 and SCCNK1-mp40 in K562 cells detected by flow cytometry.

[0056] Figure 3 Figure 1 shows the results of NK cell expansion multiples, cell viability, cell purity and CD16 expression. A shows the statistical analysis of NK cell expansion multiples, cell viability and cell purity. B shows the expression of CD3, CD56 and CD16 in NK cells detected by flow cytometry (the statistical method of this experiment uses one-way ANOVA, * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001, and the difference is significant; ns indicates p>0.05, and the difference is not significant).

[0057] Figure 4 This is the result of CD107a expression detection on NK cells.

[0058] Figure 5 These are the results of NK cell killing of senescent cells and tumor cells (the statistical method of this experiment was one-way analysis of variance, * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001, **** indicates p<0.0001 and there is a significant difference; ns indicates p>0.05 and there is no significant difference). DETAILED DESCRIPTION

[0059] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0060] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0061] Comparative Example 1

[0062] This comparative example refers to the method in patent CN118725140A to perform functional detection on membrane-expressed IL-12p35 and IL-12p40 molecules.

[0063] (1) Lentivirus preparation

[0064] The expression sequences of membrane-expressed IL-12p35 and IL-12p40 molecules (referred to as mp35 and mp40, with sequences of SEQ ID NO.8 and SEQ ID NO.9, respectively) were synthesized by whole gene, and then connected to the lentiviral vector pCDH-EF1α-MCS plasmid by molecular cloning to obtain membrane-expressed IL-12p35 and IL-12p40 expression plasmids, respectively. Using the transfection reagent Lipofectamine 3000, the above-mentioned lentiviral vector expression plasmids were co-transfected with lentiviral packaging plasmids pRSV-Rev, pMDLg / pRRE and pMD2.G in 293T cells according to the relevant instructions. Complete medium was replaced 6 hours after transfection. After 72 hours of culture, the virus supernatant was collected, centrifuged at 3000 rpm at 4°C for 10-15 minutes, filtered through a 0.45 μm pore size filter membrane, and finally resuspended in an appropriate volume after ultracentrifugation at 25000 rpm at 4°C for 2-3 hours. The obtained virus concentrate was stored at -80°C.

[0065] Jurkat cells were used as materials for virus titer detection. 300 μL complete medium (RPMI 1640 medium + 10% fetal bovine serum + 100 U / mL penicillin + 100 μg / mL streptomycin) and Jurkat cells were added to each well of a 24-well plate to a cell density of 1×10 5 cells / well. Take the lentiviral concentrate and dilute it 5 times with culture medium. Add the lentivirus of each dilution gradient to the above 24-well plate at a dosage of 200 μL / well, infect Jurkat cells with lentivirus (only culture medium is added to Jurkat cells in the negative control group), and culture them in a cell culture incubator (culture temperature is 37°C, carbon dioxide concentration is 5%). After 3 days of culture, gently mix the cells in each well and transfer them to a 1.5mL centrifuge tube, and measure the virus titer by qPCR. Table 1 is the statistical results of lentiviral titer detection of different batches.

[0066] Table 1

[0067] Structure Name PCR titer (IU / mL) mp35 6.90E+08 mp40 2.17E+08

[0068] (2) Preparation of TIL overexpressing membrane-bound IL-12

[0069] The tumor infiltrating lymphocytes (TIL) isolated and amplified from colorectal cancer tissue were adjusted to 2×10 6 / mL cell density, and cultured in TIL expansion medium for 24 h (the culture temperature of the cell culture incubator was 37°C and the carbon dioxide concentration was 5%). TILs were collected and the cell density was adjusted to 4×10 6 / mL. Add lentivirus at an MOI of 10 for transduction and culture in TIL expansion medium. After 4 hours, adjust the cell density to 2×10 6 / mL. After 24h, the cell density was maintained at (1-2)×10 6 / mL. The obtained TILs were named TIL-mp35 and TIL-mp40, and the TIL cells not transduced with lentivirus were named Mock TIL.

[0070] Wash the Mock TIL, TIL-mp35 and TIL-mp40 cells to be tested twice with PBS and resuspend them with MACS buffer. According to the antibody instructions, the primary antibody of TIL-mp35 was IL-12p35 antibody, and the secondary antibody was anti-Rat IgG-APC; the primary antibody of TIL-mp40 was biotinylated IL-12p40 antibody, and the secondary antibody was Streptavidin APC. The primary and secondary antibodies were incubated at 4°C for 1 hour. After the primary antibody incubation was completed, the supernatant was removed by centrifugation at 500g for 5min. Resuspend with MACS buffer, wash once by centrifugation at 500g for 5min, and then incubate with the secondary antibody. After the secondary antibody incubation, the supernatant was removed by centrifugation at 500g for 5min. Wash once with MACS buffer at 500g for 5min. Resuspend in 200μL MACS buffer and detect on a flow cytometer. Using Mock TIL cells not transfected with lentivirus as negative control, flow cytometry was used to detect the expression rates of IL-12p35 and IL-12p40 on the cell membrane surface of TIL-mp35 and TIL-mp40, and FlowJo software was used for analysis.

[0071] The results are as follows Figure 1 As shown in A, the expression rate of IL-12p35 on the cell membrane surface of TIL-mp35 is 35.5%, the expression rate of IL-12p40 on the cell membrane surface of TIL-mp40 is 66.5%, and the expression rates of IL-12p35 and IL-12p40 on the cell membrane surface of Mock TIL are both less than 1%, indicating that the overexpression of membrane-bound IL-12 TIL was successfully constructed.

[0072] (3) Flow cytometry was used to detect the intracellular expression level of the cytokine IFN-γ in TILs overexpressing membrane-bound IL-12

[0073] Mock TIL, TIL-mp35 and TIL-mp40 were co-cultured with SK-Hep-1 hepatoma cell line expressing liver cancer-associated antigen and hepatitis B virus-associated antigen in X-vivo medium without IL-2 (effect-target ratio = 4:1). After 12 hours, cytokine secretion blocker (BD GolgiPlug TMProtein Transport Inhibitor, BD biscience), cultured for 4 hours at 37°C and 5% carbon dioxide concentration. After completion of the culture, the Mock TIL, TIL-mp35 and TIL-mp40 to be tested were washed twice with PBS and resuspended with MACS buffer. According to the antibody instructions, AF700-labeled anti-human CD8 antibody, BV650-labeled anti-human CD4 antibody, BV421-labeled anti-human CD3 antibody and PE-Cy7-labeled anti-human PD-1 antibody were added to the cell suspension to be tested and incubated at 4°C for 30 minutes. The Mock TIL to be tested and each group of TIL were washed twice with MACS buffer, and the cells were fixed and permeabilized using the BD Cytofix / Cytoperm kit. FITC-labeled anti-human IFN-γ antibody was added and incubated at 4°C for 30 minutes. Flow cytometry was used to detect IFN-γ in each group of TIL. + CD8 + The proportion of TIL cells was analyzed using FlowJo software.

[0074] The results are as follows Figure 1 As shown in B, IFN-γ in the TIL-mp40 group + CD8 + The proportion of TIL cells was significantly higher than that of the Mock TIL and TIL-mp35 groups, indicating that the activation effect of mp40 on TIL was significantly stronger than that of mp35 (p<0.05).

[0075] Example 1

[0076] In this example, three different feeder cells were constructed: feeder cell SCCNK1, feeder cell SCCNK1-mp35 and feeder cell SCCNK1-mp40.

[0077] (1) Lentivirus preparation

[0078] like Figure 2As shown, mbIL-21-4-1BBL-mbIL-15 is composed of the following structures connected in series by 2A peptides: human membrane-expressed IL-21 (mbIL-21), human 4-1BBL, and membrane-expressed IL-15 (mbIL-15). The above molecular expression sequence was fully synthesized and then connected to the lentiviral vector pCDH-EF1α-MCS plasmid by molecular cloning to obtain the mbIL-21-4-1BBL-mbIL-15 expression plasmid. Using the transfection reagent Lipofectamine 3000, the above lentiviral vector expression plasmids were co-transfected into 293T cells with the lentiviral packaging plasmids pRSV-Rev, pMDLg / pRRE, and pMD2.G according to the relevant instructions. 48h after transfection, the virus supernatant was collected, centrifuged at 3000rpm for 10-15min at 4℃, filtered through a 0.45μm pore size filter membrane, and finally resuspended in an appropriate volume after ultracentrifugation at 25000rpm at 4℃ for 2-3h. The obtained virus concentrate was stored at -80℃ and named SCCNK1. Finally, Jurkat cells were used as materials for virus titer detection. The SCCNK1 virus qPCR titer was 3.90E+08IU / mL.

[0079] IL-21 amino acid sequence SEQ ID NO.1:

[0080] MLLLVTSLLLCELPHPAFLLIPQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS.

[0081] 4-1BBL amino acid sequence SEQ ID NO.2:

[0082] MEYASDASLDPEAPWPPARARACRVLPWALVAGLLLLLLLAAACAVFLACPWAVSGARASPGSAASPRLREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYK EDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPPSPRSE.

[0083] IL-15 amino acid sequence SEQ ID NO.3:

[0084] MLLLVTSLLLCELPHPAFLLIPIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS.

[0085] IL-12p35 amino acid sequence SEQ ID NO.4:

[0086] MCPARSLLLVATLVLLDHLSLARNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKL.

[0087] IL-12p40 amino acid sequence SEQ ID NO.5:

[0088] MCHQQLVISWFSLVFLASPLVAIWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS.

[0089] mbIL-21 (membrane-expressed IL-21) amino acid sequence SEQ ID NO.6:

[0090] MLLLVTSLLLCELPHPAFLLIPQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDSASESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKMALIVLGGVAGLLLFIGLGIFF。

[0091] Amino acid sequence of mbIL-15 SEQ ID NO.7:

[0092] MLLLVTSLLLCELPHPAFLLIPIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRN。

[0093] Amino acid sequence of mp35 SEQ ID NO.8:

[0094] MCPARSLLLVATLVLLDHLSLARNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNASFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRN。

[0095] Amino acid sequence of mp40, SEQ ID NO.9:

[0096] MCHQQLVISWFSLVFLASPLVAIWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSGGGGGGSASESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKIYIWAPLAGTCGVLLLSLVITLYCNHRN。

[0097] (2) Preparation of feeder cells

[0098] K562 cells in good condition were transduced with lentivirus at an MOI of 10 and cultured in a cell culture incubator (culture temperature was 37°C and carbon dioxide concentration was 5%). The cell density was maintained at (0.5 -1)×10 6 / mL. On the third day after transduction, cells were collected for expression measurement, and positive cells were sorted using a flow sorter. Subsequently, the culture was expanded to establish a feeder cell bank. Before harvesting the cells for freezing, cells were taken for expression measurement, and the expression rate was greater than 90%. Thus, the modified feeder cells SCCNK1 were obtained. Before being used for NK cell expansion, the frozen feeder cells were irradiated with a dose of 100Gy.

[0099] Based on SCCNK1 cells, mp35 and mp40 were transduced to obtain feeder cells SCCNK1-mp35 and SCCNK1-mp40, respectively. Before being used for NK cell expansion, the frozen feeder cells were irradiated with a dose of 100 Gy.

[0100] (3) Expression detection of membrane-expressed molecules in feeder cells

[0101] Wash the K562 cells to be tested once with MACS buffer, 500g, 5min. According to the antibody instructions, SCCNK1 was incubated with 4-1BBL-APC antibody at 4℃ in the dark for 15min. Wash once with MACS buffer, 500g, 5min, resuspend in 200μL MACS buffer, and detect on the flow cytometer. SCCNK1-mp35 used IL-12p35 antibody as the primary antibody, and anti-RatIgG-APC as the secondary antibody; SCCNK1-mp40 used biotin-labeled IL-12p40 antibody as the primary antibody, and Streptavidin APC as the secondary antibody. Both the primary and secondary antibodies were incubated at 4℃ for 1h. After the primary antibody incubation was completed, the supernatant was removed by centrifugation at 500g, 5min, and the supernatant was removed by centrifugation, and the secondary antibody was incubated after washing once with MACS buffer at 500g, 5min. After the secondary antibody incubation, the supernatant was removed by centrifugation at 500g, 5min, and the supernatant was removed, and the secondary antibody was washed once with MACS buffer at 500g, 5min. Resuspend in 200 μL MACS buffer and detect on flow cytometer.

[0102] The results are as follows Figure 2 As shown in B, with K562 cells as negative control, the expression rates of 4-1BBL in SCCNK1 were 99.6%, SCCNK1-mp35 was 94.4%, and SCCNK1-mp40 was 99.9%, all of which were greater than 90%, indicating that the feeder cells were successfully constructed.

[0103] Example 2

[0104] NK cell culture

[0105] PBMCs were purchased from Miaoshun (Shanghai) Biotechnology Co., Ltd. PBMCs were rewarmed in a 37°C water bath, resuspended in OptiVitro NK cell culture medium, and centrifuged at 300g for 10 min for counting. Feeder cells (feeder cells in Example 1) were rewarmed in a 37°C water bath, resuspended in IMDM + 10% FBS culture medium, and centrifuged at 500g for 5 min. The cell concentration was adjusted to 1×10 using OptiVitro NK cell culture medium (supplemented with 1% AB serum) according to the ratio of PBMC:feeder cells = 1:5. 6 cells / mL, and cultured for 14 days. The cell concentration was maintained at (0.5-3)×10 6 The NK cells obtained after culture were grouped and named as ①SCCNK1-NK, ②SCCNK1-mp35-NK, and ③SCCNK1-mp40-NK.

[0106] Collect cells on day 14 for cell counting and cell viability testing. Add 1 mL of MACS buffer and centrifuge at 500 g for 5 min. Use flow cytometry antibodies CD3-BV421, CD56-APC, and CD16-PE-CY7 to detect the purity of NK cells and incubate at 4°C in the dark for 15 min. Add 1 mL of MACS buffer and wash once at 500 g for 5 min. Add 200 μL of MACS buffer and test by flow cytometry.

[0107] The results are as follows Figure 3 As shown, the viability of NK cells in each group was greater than 90%, and the purity of NK cells was also greater than 90%. Among them, SCCNK1-mp35-NK cells and SCCNK1-mp40-NK cells achieved an expansion of more than 1000 times in a 14-day expansion cycle, which was 1.5 times and 1.3 times higher than that of SCCNK1-NK cells, respectively (p<0.05). This result shows that feeder cells that further express mp35 or mp40 on the basis of membrane expression of IL-15, IL-21, and 4-1BBL have a better effect in promoting NK expansion, and can be used to produce NK cells with high viability, high purity, and higher expansion times.

[0108] Example 3

[0109] 1. Detection of CD107a expression on NK cells

[0110] Tumor cells K562 were collected as target cells for the killing experiment, and PBS was added, 500g, 5min, washed twice, and the supernatant was removed and resuspended in serum-free medium. 1mL of tumor cells resuspended in serum-free medium were taken out and placed in a 15mL centrifuge tube, and CFSE with a final concentration of 2μmol / L was added, and incubated at 37°C for 10min. 3mL of medium containing 10% FBS was added to terminate the staining. PBS was added, 500g, 5min, washed twice, and serum-free medium was added to resuspend and count. NK cells were mixed with target cells at an effector-target ratio of 4:1, placed in a round 96-well plate for co-culture for 5h, and then CD107a-BV421 was added for incubation for half an hour. Collect the sample, 500g, 5min and wash once. During flow cytometry, the CFSE-negative cell population was used as NK cells, and its CD107a expression was detected.

[0111] When NK cells kill target cells, toxic granules will reach the serosal surface and fuse with the cell membrane, causing the release of granule contents, ultimately leading to the death of target cells. As degranulation occurs, CD107a molecules are transported to the cell membrane surface, and the upregulation of CD107a molecule expression is positively correlated with the secretion of toxic granules. Therefore, NK cells with positive expression of CD107a molecules can represent NK cells with killing activity.

[0112] The results are as follows Figure 4 As shown, SCCNK1-mp35-NK cells expressed higher CD107a than SCCNK1-mp40-NK cells and SCCNK1-NK cells, indicating that SCCNK1-mp35-NK cells have stronger degranulation ability and higher killing activity.

[0113] 2. NK killing senescent cells and tumor cells detection (ELISA)

[0114] Studies have shown that NK cells can not only kill tumor cells and resist viruses, but also kill and remove senescent cells in vivo and in vitro, enhance the body's immunity, and play an important role in anti-aging. IMR-90 is a fibroblast isolated from human embryonic lung tissue. 5 Resuspend senescent cells IMR-90, tumor cells HCT-116 and SK-OV-3 cells at a density of 100 μL / mL and inoculate them in a 96-well plate at a volume of 100 μL per well. Add NK cells at an effector-target ratio of 8:1 for co-culture overnight. Centrifuge at 600g for 5 minutes at room temperature and collect the supernatant. Use the BD OptEIA Human IFN-γ ELISA Set kit to detect IFN-γ in the co-culture supernatant. The steps include:

[0115] (1) Reagent preparation: coating solution is 0.1 M sodium carbonate (pH 9.5); blocking solution is PBS + 10% FBS; elution solution is PBS + 0.05% Tween; color development substrate is BD Pharmingen TM TMB Substrate Reagent Set.

[0116] (2) Coating antibody: According to the requirements of the kit, the coating antibody (Capture Antibody Purified Anti-Human IFN-γ) was diluted 1:250 with coating solution, 100 μL / well, gently shaken and placed in a 4°C refrigerator for overnight coating.

[0117] (3) Washing: Pour off the coating solution and wash the plate with elution solution, 200 μL / well, 3 × 1 min (shake the plate on a micro-oscillator each time).

[0118] (4) Blocking: Block with blocking solution, 200 μL / well, and leave at room temperature for 1 hour.

[0119] (5) Washing: Pour off the blocking solution and wash the plate with elution solution, 200 μL / well, 3×1 min (shake the plate on a microplate oscillator each time you wash).

[0120] (6) Dilution of standards and samples: Add 1 mL of deionized water to the standard lyophilized bottle (Recombinant Human IFN-γLyophilized Standard), let stand for 15-20 min, vortex to mix, and obtain the standard stock solution. Dilute with blocking solution to obtain a standard solution with a theoretical concentration of 600 pg / mL. Then use the two-fold dilution method to dilute to obtain another 7 dilution levels (300 pg / mL, 150 pg / mL, 75 pg / mL, 37.5 pg / mL, 18.8 pg / mL, 9.4 pg / mL, 4.7 pg / mL) of standard solutions. Use the blocking solution as the standard solution with a theoretical concentration of 0 pg / mL. Use the blocking solution to dilute the test samples.

[0121] (7) Sample addition: Start adding standards and samples at 100 μL / well, mix well, and leave at room temperature for 2 hours.

[0122] (8) Washing the plate: Pour away the sample solution and wash the plate with the eluent, 200 μL / well, 5 × 1 min (shake the plate on a micro-oscillator each time you wash it).

[0123] (9) Add antibodies and SAv-HRP: Mix the antibodies 15 min in advance (Detection Antibody Biotin Anti-Human IFN-γ and SAv-HRP are diluted 1:250, respectively), 100 μL / well, mix well and leave at room temperature for 1 hour.

[0124] (10) Washing the plate: Pour away the antibody and wash the plate with elution buffer, 200 μL / well, 7 × 1 min (shake the plate on a microplate oscillator each time you wash).

[0125] (11) Color development and termination: Prepare the color development substrate immediately, A:B = 1:1 (volume ratio), 100 μL / well, add the color development agent and let stand at room temperature in the dark. Start timing from the addition of the substrate. After 30 minutes, add 1M H 2 SO 4 Stop solution (50 μL / well) stops color development.

[0126] (12) Read the plate and save the original results: Use an ELISA reader to detect the absorbance at OD450 (570).

[0127] Result analysis: Make a standard curve based on the gradient dilution standard sample wells. 2 If the value is ≥0.99, the standard curve fitting meets the requirements, and the concentration of each well is calculated using the microplate reader operating software.

[0128] The results are as follows Figure 5 As shown, in the co-culture of NK cells with senescent cells IMR-90, tumor cells HCT-116 and SK-OV-3 cells, higher concentrations of IFN-γ were detected in the co-culture supernatant of SCCNK1-mp35-NK cells and SCCNK1-mp40-NK cells than SCCNK1-NK cells (p<0.05), indicating that NK cells stimulated and expanded by SCCNK1-mp35 and SCCNK1-mp40 feeder cells have stronger ability to kill senescent cells and tumor cells. In addition, we unexpectedly found that the IFN-γ release level of SCCNK1-mp35-NK cells was also significantly higher than that of SCCNK1-mp40-NK cells (p<0.05). This result shows that compared with mp40, the activation effect of NK cells is stronger when mp35 is stimulated by feeder cells.

[0129] In comparative example 1, IFN-γ was expressed in the TIL group overexpressing mp40. + CD8 +The ratio of TIL cells is significantly higher than that of the TIL group overexpressing mp35, indicating that mp40 is stronger than mp35 in activating immune cells. However, compared with Comparative Example 1 and the prior art (fusion protein, engineered immune cells and preparation methods and applications thereof, CN118725140A), Examples 2 and 3 obtain different results. In the in vitro culture and functional detection of NK, we found that the amplification multiple of SCCNK1-mp35-NK cells was higher than that of SCCNK1-mp40-NK cells, and SCCNK1-mp35-NK cells expressed higher CD107a than SCCNK1-mp40-NK cells, indicating that SCCNK1-mp35-NK cells have stronger degranulation ability and higher killing activity. In the co-culture of NK cells with senescent cells IMR-90, tumor cells HCT-116 and SK-OV-3 cells, SCCNK1-mp35-NK cells also have a higher expression level of IFN-γ than SCCNK1-mp40-NK cells. Therefore, feeder cells overexpressing mp35 are more effective in the proliferation and activation of NK cells than feeder cells overexpressing mp40, and the obtained NK cells have stronger proliferation capacity and stronger ability to kill tumor cells and senescent cells.

[0130] In summary, the present invention constructs a feeder cell that overexpresses membrane-bound IL-15, IL-21, 4-1BBL and IL-12p35, and unexpectedly finds that compared with feeder cells that overexpress membrane-bound IL-21, IL-15 and 4-1BBL and feeder cells that overexpress membrane-bound IL-15, IL-21, 4-1BBL and IL-12p40, the efficiency of amplifying NK cells is higher, the amplified NK cells have stronger killing function, which is conducive to the large-scale clinical application of NK cells and has higher clinical application value.

[0131] The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention shall fall within the protection scope and disclosure scope of the present invention.

Claims

1. A feeder cell for expanding NK cells, characterized in that The feeder cells are human cells that overexpress IL-15, IL-21, 4-1BBL and IL-12p35 on the cell membrane; The human-derived cells are selected from any one of immune cells, immortalized immune cells, lymphoma cells or leukemia cells.

2. The feeder cell for expanding NK cells according to claim 1, characterized in that The human cells include PBMC cells, LCL cells or K562 cells; Preferably, the coding sequence of IL-21 includes that shown in SEQ ID NO.1; Preferably, the coding sequence of 4-1BBL includes that shown in SEQ ID NO.2; Preferably, the coding sequence of IL-15 includes that shown in SEQ ID NO.3; Preferably, the coding sequence of IL-12p35 includes that shown in SEQ ID NO.4; Preferably, the IL-21, IL-15 and IL-12p35 are expressed by fusion with membrane proteins to form membrane-expressed IL-21, IL-15 and IL-12p35, and are localized on the cell membrane surface; Preferably, the membrane protein comprises any one of type I membrane protein, type II membrane protein, multi-transmembrane protein or glycosylphosphatidylinositol anchored protein or a combination of at least two or more thereof; Preferably, the membrane protein is selected from a full-length membrane protein, or a partial structure comprising a transmembrane domain / membrane anchoring domain; Preferably, the membrane expressed IL-21 coding sequence includes that shown in SEQ ID NO.6; Preferably, the membrane expressed IL-15 coding sequence includes that shown in SEQ ID NO.7; Preferably, the membrane expressed IL-12p35 coding sequence includes that shown in SEQ ID NO.

8.

3. The method for preparing feeder cells for expanding NK cells according to claim 1 or 2, characterized in that: The preparation method comprises: constructing a vector containing a cytokine coding sequence, wherein the cytokine coding sequence is located on one or more vectors, wherein the cytokines include IL-21, 4-1BBL, IL-15 and IL-12p35, introducing the vector into human cells, sorting positive cells, and then expanding the culture to establish a feeder cell bank; and irradiating the feeder cells in the feeder cell bank before being used for the amplification of NK cells; Preferably, the vector comprises DNA and / or RNA; Preferably, the method of introducing the vector into human cells comprises: any one or a combination of at least two or more of viral vector transduction, transposon insertion, homologous recombination insertion, RNA transfection or plasmid transfection; Preferably, the viral vector comprises a lentiviral vector and / or a retroviral vector; Preferably, the method for lentiviral vector transduction comprises constructing a vector plasmid containing IL-21, 4-1BBL, IL-15 and IL-12p35 coding sequences, co-transfecting the vector plasmid with a lentiviral packaging plasmid into a virus packaging cell to obtain the virus, infecting human cells with the virus, sorting positive cells, and then expanding the culture to establish a feeder cell bank; and irradiating the feeder cells in the feeder cell bank before using them for NK cell amplification.

4. The preparation method according to claim 3, characterized in that: The radiation treatment dose is 5 Gy-500 Gy; Preferably, the radiation source includes X-rays and gamma rays.

5. A nucleic acid molecule, characterized in that The nucleic acid molecule contains any one or a combination of at least two or more of the membrane-expressed IL-12p35 coding sequence, the membrane-expressed IL-21, the membrane-expressed IL-15 or the 4-1BBL coding sequence.

6. A recombinant plasmid, characterized in that: The recombinant plasmid contains the nucleic acid molecule according to claim 5.

7. A viral vector, characterized in that The virus is obtained by co-transfecting the recombinant plasmid according to claim 6 with a lentiviral packaging plasmid into a virus packaging cell.

8. A method for improving the expansion efficiency of NK cells, characterized in that: The method comprises: mixing PBMC and the feeder cells for expanding NK cells according to claim 1 or 2 using NK cell culture medium and adjusting the cell concentration to (0.2-5)×10 6 The ratio of PBMC to feeder cells is 100:1 to 1:100; NK cell expansion culture is performed, and the cell concentration is maintained at (0.2-5)×10 6 Pieces / mL.

9. The method for improving the expansion efficiency of NK cells according to claim 8, characterized in that: The NK cell culture medium is selected from: any one of OptiVitro NK cell culture medium, SCGM NK cell culture medium, Gibco CTS NK-Xpander culture medium or NK MACS culture medium, or a combination of at least two or more thereof; Preferably, the NK cell culture medium is further added with any one of AB serum, autologous plasma or serum substitute or a combination of at least two or more thereof, with the added amount being 0.5-20 vol%; Preferably, the PBMC cells and / or feeder cells are further revived before use.

10. Use of the feeder cells for expanding NK cells according to claim 1 or 2, the nucleic acid molecule according to claim 5, the recombinant plasmid according to claim 6, the virus according to claim 7 or the method for improving the expansion efficiency of NK cells according to claim 8 or 9 in expanding NK cells.

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