Method for amplifying NK (Natural Killer) cells in vitro by utilizing extracellular vesicles of artificial antigen presenting cells

Abstract

The invention belongs to the field of medical biology, and provides a method for in-vitro amplification of NK (Natural Killer) cells by utilizing extracellular vesicles of artificial antigen presenting cells. A feeder cell, namely an artificial antigen presenting cell, used in the invention carries four active molecules, namely CD48, anti-NKp46, mIL-15 and mIL-21. The structure of the exosome secreted by the exosome is complete, the particle size is 100-200 nanometers, and the exosome carries the four active molecules. The separated and purified exosome aAPC-Exo is effectively amplified in vitro to obtain high-purity NK cells, and the high-purity NK cells can be well proliferated in the later stage and have a relatively strong killing effect on tumor cells, so that a new methodology and material basis is provided for immune cell biological treatment.

Description

Technical Field

[0001] The present invention belongs to the field of medical biology, and more particularly, relates to a method for in vitro expansion of NK cells by using extracellular vesicles of artificial antigen-presenting cells. Background Art

[0002] Natural killer cells (NK) are an important part of the innate immune system, capable of directly killing tumor cells and virus-infected cells through non-specific mechanisms, and can also regulate adaptive immune responses by secreting cytokines. In recent years, adoptive immune cell drugs, including CAR-T, have developed rapidly and become a new type of biotherapy. Different from T and B cells, NK cells can function without prior sensitization and are not restricted by the major histocompatibility complex MHC. Therefore, NK cells can be used allogeneically in adoptive immune cell therapy and have important application prospects. However, the number of NK cells is usually limited, the in vitro expansion efficiency is restricted, and the killing ability of in vitro expanded NK cells is insufficient, etc., which have become the main obstacles in the application of immune cell therapy.

[0003] There are currently two common methods for NK cell in vitro expansion: pure cytokine culture and feeder cell + cytokine culture. The former is highly safe, but the cultured cells have low purity, high cost, and limited expansion multiples; the latter has high cell expansion efficiency, fast speed, and high purity, but there are potential risks in safety issues. Adding feeder cells to the in vitro culture system is an effective means to promote cell proliferation. Among them, artificial antigen presenting cells aAPC carrying various active molecules have been shown to effectively activate and expand NK cells. However, the stimulation specificity and potential safety issues of the active molecules carried by aAPC have always been issues that need to be urgently addressed. In recent years, extracellular vesicles, especially exosomes (Exo), can carry intracellular information such as proteins, RNA, and other active molecules, and transmit this information to target cells through fusion with target cells to regulate the behavior of target cells; this signal transmission can affect immune response, cell proliferation, etc. In addition, exosomes have natural biocompatibility and can be used as carriers of drugs or therapeutic molecules. Their membrane structure can effectively protect the active molecules therein from degradation, making them an emerging carrier tool. Genetically engineered aAPC-derived exosomes can accurately carry membrane-bound cytokines (such as mIL-15 / mIL-21) and co-stimulatory molecules (such as 4-1BBL, CD48), while retaining the three-dimensional topological characteristics of the natural membrane structure. This new nanoscale carrier can not only avoid the biosafety risks of trophoblast cells, but also efficiently deliver activation signals to NK cells through membrane fusion mechanisms. aAPC-derived exosomes (aAPC-Exo) have similar functions to aAPCs, and can carry co-stimulatory molecules and immune-active molecules to regulate the activity of immune cells. Therefore, aAPC-Exo is expected to become a more efficient, safer, and low-cost NK cell expansion tool. However, there are currently few studies on the application of aAPC-Exo in NK cell expansion, and no effective solution has yet been formed. Therefore, the development of a technology based on aAPC-derived exosomes for inducing NK cell expansion has important scientific value and broad clinical application prospects. Summary of the invention

[0004] The purpose of the present invention is to provide a method for amplifying NK cells in vitro using extracellular vesicles of artificial antigen presenting cells.

[0005] In order to achieve the purpose of the present invention, in a first aspect, the present invention provides an artificial antigen presenting cell 4aAPC, which is an artificial antigen presenting cell expressing CD48, anti-NKp46, mIL-15 and mIL-21.

[0006] Preferably, it is a K562 cell expressing CD48, anti-NKp46, mIL-15 and mIL-21.

[0007] In a second aspect, the present invention provides a method for constructing the artificial antigen-presenting cell 4aAPC, comprising: separately constructing lentiviral expression vectors containing CD48, anti-NKp46, mIL-15, and mIL-21, and sequentially transfecting the artificial antigen-presenting cells with the aid of lentiviral packaging helper plasmids.

[0008] Preferably, for the lentiviral expression vector containing mIL-15, the full sequence of the vector is as shown in SEQ ID NO:1; Preferably, for the lentiviral expression vector containing mIL-21, the full sequence of the vector is as shown in SEQ ID NO:2; Preferably, for the lentiviral expression vector containing anti-NKp46, the full sequence of the vector is as shown in SEQ ID NO:3; Preferably, for the lentiviral expression vector containing CD48, the full sequence of the vector is as shown in SEQ ID NO:4.

[0009] In a third aspect, the present invention provides the use of the artificial antigen-presenting cell 4aAPC in the in vitro expansion of NK cells.

[0010] In a fourth aspect, the present invention provides a method for in vitro expanding NK cells using extracellular vesicles of artificial antigen-presenting cells, and uses exosomes (sized between 100 - 200 nm) secreted by the artificial antigen-presenting cell 4aAPC for in vitro expansion of NK cells.

[0011] Specifically, the method comprises the following steps: S1. Cultivate the artificial antigen-presenting cell 4aAPC, and isolate and purify exosomes from the cell culture supernatant; S2. Prepare human peripheral blood mononuclear lymphocytes, inoculate the mononuclear cells into NK cell culture medium, and then add autologous plasma and the exosomes for in vitro culture.

[0012] Further, step S2 comprises: (1) Inoculate 1.0×10 6 peripheral blood mononuclear cells into a cell culture plate containing 1 ml of NK cell culture medium; subsequently add 5% autologous plasma and 100 μg / ml exosomes; finally place the culture plate in an incubator at 37°C and 5% CO2 for culture; (2) From day 1 to day 7, supplement 5% or 10% autologous plasma and 100 μg / ml exosomes to the culture plate every other day; (3) From day 9 to day 13, change the culture medium every other day, replace it with freshly prepared ALyS505NK-EX medium containing IL-2 at a final concentration of 700 IU / ml, and maintain the cell density at 1.0×10 6 cells / mL; On the 14th day, stop the culture, centrifuge to collect the cells, discard the supernatant, and resuspend the cell pellet with PBS.

[0013] Preferably, the NK cell culture medium is a serum-free ALyS505NK-EXNK cell expansion culture medium.

[0014] In a fifth aspect, the present invention provides any one of the following applications of NK cells prepared according to the described method: 1) For the preparation of anti-tumor drugs or compositions; 2) For the preparation of drugs or compositions for tumor cell immunotherapy.

[0015] In a sixth aspect, the present invention provides an anti-tumor drug or composition, the active ingredient of which is NK cells prepared according to the described method.

[0016] By virtue of the above technical solutions, the present invention has at least the following advantages and beneficial effects: The present invention provides a novel artificial antigen-presenting cell 4aAPC-derived exosome capable of inducing a large expansion of NK cells in vitro and its preparation method, as well as its application in NK cell expansion. The method includes: construction of 4aAPC and isolation and purification of exosomes: constructing a novel artificial antigen-presenting cell 4aAPC carrying CD48, anti-NKp46, mIL-15, and mIL-21 by lentiviral transfection of the K562 cell line. The above four active molecules can be stably expressed for a long time, and the exosomes secreted by them also carry the above specific active molecules.

[0017] The present invention also provides the application of 4aAPC exosomes in NK cell expansion: adding 4aAPC exosomes to the in vitro culture system of peripheral blood lymphocytes to specifically activate NK cells and promote their large proliferation. The NK cells amplified by this method have good proliferation, high purity, and obvious tumor cell killing effects.

[0018] The 4aAPC-derived exosomes of the present invention have the characteristics of high efficiency, stability, and controllability, solving the problems of low purity, limited cell expansion quantity, and weak cell killing activity faced by the NK cell preparation method, meeting the clinical requirements for NK cell therapy products and the NK source requirements in the development of CAR-NK. The active molecules carried by 4aAPC can specifically expand NK cells, improve the purity of NK cell culture, and effectively avoid the residual effects caused by directly adding feeder cells 4aAPC.

[0019] The present invention first provides a highly efficient, low-cost, high-purity, and safer method for in vitro expansion of NK cells. The NK cells amplified by the above method have CD3 - CD56 +The efficiency > 75%, and it can amplify more NK cells (247 times vs 187 times) than the active molecules in the commercial kits in the market. Description of the Drawings

[0020] Figure 1 It is the comparison of the mIL-21 gene sequencing results in the preferred embodiment of the present invention.

[0021] Figure 2 It is the comparison of the mIL-15 gene sequencing results in the preferred embodiment of the present invention.

[0022] Figure 3 It is the comparison of the anti-NKp46 gene sequencing results in the preferred embodiment of the present invention.

[0023] Figure 4 It is the comparison of the CD48 gene sequencing results in the preferred embodiment of the present invention.

[0024] Figure 5 It is the map of the lentiviral vector transfected with mIL-21 in the preferred embodiment of the present invention.

[0025] Figure 6 It is the map of the lentiviral vector transfected with mIL-15 in the preferred embodiment of the present invention.

[0026] Figure 7 It is the map of the lentiviral vector transfected with anti-NKp46 in the preferred embodiment of the present invention.

[0027] Figure 8 It is the map of the lentiviral vector transfected with CD48 in the preferred embodiment of the present invention.

[0028] Figure 9 It is the Western blot detection results of IL-21, IL-15, Myc-tag (anti-NKp46) and CD48 expressed by K562 and 4aAPC in the preferred embodiment of the present invention.

[0029] Figure 10 It is the detection results of transmission electron microscopy, nanoparticle tracking analysis (NTA) and Western blot (exosomes express IL-21, IL-15, Myc-tag (anti-NKp46) and CD48 proteins) of exosomes derived from 4aAPC in the preferred embodiment of the present invention. Among them, A: Protein blot verification of 4aAPC-Exo markers; B: Nanoparticle size analysis of 4aAPC-Exo; C: Transmission electron microscopy of 4aAPC-Exo.

[0030] Figure 11Detection results of transmission electron microscopy, nanoparticle tracking analysis (NTA), and Western blotting (exosomes express IL-21, IL-15, Myc-tag (anti-NKp46), and CD48 proteins) of K562-derived exosomes in the preferred embodiment of the present invention. Among them, A: Western blot verification of K562-Exo marker proteins; B: Analysis of the particle size of K562-Exo nanoparticles; C: Transmission electron microscopy of K562-Exo.

[0031] Figure 12 Proliferation curve of NK cells cultured in vitro with exosomes derived from K562 and 4aAPC in the preferred embodiment of the present invention.

[0032] Figure 13 Flow cytometry detection results of the purity of NK cells cultured in vitro with exosomes derived from K562 and 4aAPC in the preferred embodiment of the present invention.

[0033] Figure 14 Detection results of the amplification fold of NK cells cultured in vitro with exosomes derived from K562 and 4aAPC in the preferred embodiment of the present invention.

[0034] Figure 15 Killing ability of NK cells cultured in vitro with exosomes derived from K562 and 4aAPC against lung adenocarcinoma cells and lymphoma cells in the preferred embodiment of the present invention.

[0035] Figure 16 ELISA analysis of interferon γ (IFN-γ) production by NK cells in the preferred embodiment of the present invention.

[0036] Figure 17 ELISA analysis of granzyme B production by NK cells in the preferred embodiment of the present invention.

[0037] Figure 18 Comparison results of the amplification of NK cells in an in vitro culture system with exosomes derived from aAPC constructed with different active molecules in the preferred embodiment of the present invention.

[0038] Figure 19 Proliferation curve of NK cells cultured in vitro with exosomes derived from 4aAPC and the commercial kit Beso Baso ® NK High-Efficiency Induction Kit.

[0039] Figure 20 Exosomes derived from 4aAPC and Beso Baso ® Flow cytometry detection results of NK cells cultured in vitro with the NK High-Efficiency Induction Kit.

[0040] Figure 21In the preferred embodiment of the present invention, the exosomes derived from 4aAPC and Beso Baso ® The detection results of the amplification fold of NK cells cultured in vitro by the NK highly efficient induction kit.

[0041] Figure 22 In the preferred embodiment of the present invention, the exosomes derived from 4aAPC and Beso Baso ® The killing ability of NK cells cultured in vitro by the NK highly efficient induction kit against lung adenocarcinoma cells, breast cancer cells, myeloid leukemia cells and lymphoma cells. Detailed implementation manners

[0042] In view of the problems that the number of NK cells in the body is limited and their functions are easily lost during the amplification process; the NK cells cultured with pure cytokines have low purity, high cost and limited amplification fold; adding feeder cells such as traditional artificial antigen-presenting cells is difficult to provide spatiotemporally coordinated multi-signal stimulation due to the lack of a natural membrane structure, resulting in limited NK cell activation efficiency; and the residual effects generated affect the application safety of NK cells, the present invention provides a method for stably inducing the large-scale amplification of NK cells in vitro using extracellular vesicles of artificial antigen-presenting cells. The feeder cells used in the present invention, artificial antigen-presenting cells, carry 4 active molecules, namely CD48, anti-NKp46, mIL-15 and mIL-21. The exosomes secreted by them have a complete structure, with a particle size between 100 and 200 nanometers, and carry the above 4 active molecules. The isolated and purified exosomes aAPC-Exo can effectively amplify high-purity NK cells in vitro, and can proliferate well in the later stage, and also have a strong killing effect on tumor cells, providing a new methodology and material basis for immunocyte biotherapy.

[0043] In a first aspect, an artificial antigen-presenting cell, the artificial antigen-presenting cell is CD48 / anti-NKp46 / mIL-15 / mIL-21-aAPC, and its cells stably express CD48, anti-NKp46, membrane-fixed interleukin 15, and membrane-fixed interleukin 21.

[0044] Preferably, the artificial antigen-presenting cell is CD48 / anti-NKp46 / mIL-15 / mIL-21-K562 cells. The full sequences of the lentiviral expression vectors containing mIL-15, mIL-21, anti-NKp46, and CD48 are shown in SEQ ID NO: 1-4 respectively.

[0045] The present invention also provides a method for constructing the above artificial antigen-presenting cell, including the following steps: (1) Respectively construct lentiviral expression vectors containing CD48, anti-NKp46, mIL-15, and mIL-21; (2) Transfect the corresponding cells with the lentiviral expression vector containing mIL-21, screen the cell population stably expressing the above molecule with puromycin resistance, pick single cell clones, verify the molecular expression by Western blot, and obtain the cells with stable expression of mIL-21, mIL-21-aAPC; (3) Based on the mIL-21-aAPC obtained in step (2), introduce mIL-15 by transfecting with the lentiviral expression vector containing mIL-15, screen the cell population stably expressing the above molecule with puromycin resistance, pick single cell clones, verify the molecular expression by Western blot, and establish mIL-21 / mIL-15-aAPC; (4) Based on the mIL-21 / mIL-15-aAPC obtained in step (3), introduce anti-NKp46 by transfecting with the lentiviral expression vector containing anti-NKp46, screen the cell population stably expressing the above molecule with puromycin resistance, pick single cell clones, verify the molecular expression by Western blot, and establish anti-NKp46 / mIL-15 / mIL-21-aAPC; (5) Based on the anti-NKp46 / mIL-15 / mIL-21-aAPC obtained in step (4), introduce CD48 by transfecting with the lentiviral expression vector containing CD48, screen the cell population stably expressing the above molecule with puromycin resistance, pick single cell clones, verify the molecular expression by Western blot, and establish CD48 / anti-NKp46 / mIL-15 / mIL-21-aAPC (4aAPC); Preferably, the corresponding cells described in step (2) are K562 cells, but are not limited thereto.

[0046] The plasmid map used in the present invention is shown in Figures 5 - 6 and was constructed by GeneChem Co., Ltd.

[0047] In the second aspect, the present invention provides a method for large-scale induction of NK cell expansion, which uses the exosomes secreted by the above artificial antigen-presenting cells for expansion, and includes the following steps: (1) Collect the cell culture suspension of 4aAPC cells, and collect the supernatant after centrifugation; (2) Ultrafilter the supernatant with an Amicon Ultra-15 100KDa centrifuge tube, and separate the exosomes from the ultrafiltered supernatant through a commercial exosome extraction kit.

[0048] (3) Filter with a 0.22 mm filter head to further purify the exosomes.

[0049] (4) The particle size of exosomes was measured by nanoparticle tracking analysis (NTA), the morphology of exosomes was detected by transmission electron microscopy, and the expression of exosome markers and specific molecules, including CD63, CD9, CD81, TSG101, mIL-15, mIL-21, anti-NKp46, CD48, was detected by Western blot.

[0050] (5) Human peripheral blood mononuclear lymphocytes were prepared, and 1.0×10 6 mononuclear cells were seeded into a 24-well plate containing 1 ml of NK cell culture medium (serum-free ALyS505NK-EXNK cell expansion culture medium); subsequently, 5% autologous plasma and 100 μg / ml exosomes were added; finally, the culture flask was placed in an incubator at 37 °C and 5% CO2.

[0051] (6) From day 1 to day 7, 5% or 10% autologous plasma and 100 μg / ml exosomes were supplemented into the culture flask every other day.

[0052] (7) From day 9 to day 13, the medium was changed every other day, and fresh ALyS505NK-EX medium containing IL-2 at a final concentration of 700 IU / ml was replaced, and the cell density was maintained at 1.0×10 6 cells / mL.

[0053] (8) On day 14, the culture was stopped, the cells were collected, centrifuged at 1000 rpm for 5 min, and the supernatant was removed.

[0054] (9) The cell pellet was resuspended with PBS, the cell number was detected by a cell counter, the expansion rate and purity (CD3−CD56+) of NK cells were detected by flow cytometry, and the expression of surface NK cell receptors (such as NKG2D and NKp46) was evaluated.

[0055] (10) The killing activity of expanded NK cells was detected by tumor killing experiments and cytokine secretion experiments.

[0056] Thirdly, the present invention provides highly pure NK cells with killing activity against tumor cells prepared by the above method.

[0057] Fourthly, the present invention provides any one of the following applications of the NK cells: 1) For the preparation of anti-tumor drugs or compositions; 2) For tumor cell immunotherapy.

[0058] Fifthly, the present invention provides an anti-tumor drug or composition, the active ingredient of which is the NK cells cultured by the above method.

[0059] Four active molecules CD48, anti-NKp46, mIL-15, and mIL-21 constructed by the present invention can enhance the amplification rate, purity, activity, cytotoxicity of NK cells, and the production of interferon γ (IFN-γ). On this basis, the exosomes secreted by aAPC constructed have a higher purity of NK cells stimulated than the exosomes of aAPC constructed by other active molecules (CD86, CD137L, CD64, and mIL-15) (the former CD3 - CD56 + rate is greater than 75.0%, and the latter is approximately equal to 38.8%).

[0060] Using aAPC exosomes as active stimulating molecules in the in vitro culture system avoids the residue of feeder cells and unsafe factors. Exosomes are smaller in volume and more homogeneous; they are easy to produce and store; they have a long circulating half-life; and they are safer than aAPC.

[0061] The following examples are used to illustrate the present invention, but not to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0062] The ALyS505NK-EX medium (product number T20153P3) and serum-free ALyS505NK-EX NK cell amplification culture medium (product number: 01400P10) used in the following examples were all purchased from Baso ® Company. Example 1 Construction of artificial antigen-presenting cell 4aAPC expressing CD48, anti-NKp46, mIL-15, and mIL-21

[0063] 1. Lentivirus packaging (taking the recombinant lentivirus carrying mIL-21 as an example) 1.1 Prepare 293T cells: Use DMEM medium (Gibco), add 10% fetal bovine serum (Fetal Bovine Serum, FBS, Biological Industries), and 1% double antibody (100 U / ml penicillin and 0.1 mg / ml streptomycin (VivaCell)), and culture at 37°C and 5% CO2.

[0064] 1.2 Plate 293T cells: 24 hours before transfection, inoculate 293T cells at a density of 5×10 6 cells / dish (diameter 10 cm), add 10 mL of DMEM complete medium, and the cell density before transfection needs to grow to 80-90%.

[0065] 1.3 Medium replacement before transfection: 1-2 hours before transfection, replace the medium of the cells to be transfected with DMEM basal medium without fetal bovine serum and antibiotics.

[0066] 1.4 Transfection: Take a sterile 1.5 mL EP tube and prepare the reaction system according to the following components:

[0067] Among them, the target plasmid sequences (the complete sequences of the lentiviral expression vectors containing mIL-15, mIL-21, anti-NKp46, and CD48 are shown in SEQ ID NO: 1-4 respectively) were constructed by GeneChem Co., Ltd. The sequencing results showed that the amplified plasmid sequence was consistent with the designed plasmid sequence ( Figures 1 - 4 ).

[0068] After mixing well, mix centrifuge tube 1 and centrifuge tube 2. After incubating the transfection mixture at room temperature for 15 minutes, evenly drop it into the culture dish that has been replaced with fresh medium in advance. (psPAX2 is a packaging plasmid, product number 12260, Addgene; VSVG is an envelope plasmid, product number 12259, Addgene; PEI is a transfection reagent, product number 24765-100, Polysciences) 1.5 Medium replacement: Replace the medium 12 hours later, add 10 mL of fresh DMEM (containing 10% FBS and 1% double antibiotics) to each dish and continue culturing.

[0069] 1.6 Virus collection: Collect the cell culture supernatant at 48 hours, 60 hours, and 72 hours after transfection into a 50 mL centrifuge tube and add 10 mL of fresh DMEM (containing 10% FBS) again to continue culturing.

[0070] 1.7 Filtration and aliquoting: Filter the virus supernatant with a 0.22 μm filter head and aliquot it into new centrifuge tubes or EP tubes to obtain the lentiviral expression vector containing the target gene (such as mIL-12) (see the vector structure in Figures 5 - 8 ), make good marks, and store it at -80 °C for later use.

[0071] 2. Construction of 4aAPC 2.1 Preparation before transfection a. Take out the lentivirus containing the specific gene and polybrene from the -80 °C refrigerator and melt them on ice.

[0072] b. Transfer the K562 cells from the culture flask to a 50 mL centrifuge tube and centrifuge at 800 rpm for 5 minutes.

[0073] c. Discard the culture supernatant after centrifugation, add RPMI1640 complete medium (10% FBS and 1% double antibody), and pipette to resuspend the cells.

[0074] d. Use a cell counting chamber to count the viable cells, and dilute the cell suspension to a cell density of 1×10 5 cells per milliliter.

[0075] e. Inoculate 1 ml of cells into a 24-well plate, ensuring that 1 ml of the cell suspension contains 1×10 5 cells.

[0076] 2.2 Lentivirus transfection a. According to the reference cell transfection MOI of 20, add the lentivirus to the 24-well plate, add 8 μg of polybrene to each well to improve the transfection efficiency, and then place it in an incubator at 37°C and 5% CO2 for 24 hours.

[0077] b. Observe the cell morphology 8 to 12 hours later. If the cell state shows no obvious difference from that of non-infected cells, it indicates that the lentivirus has no obvious toxic effect on the cells, and continue the culture.

[0078] c. After 72 hours, add puromycin at a final concentration of 1.5 μg / mL to each well of the 24-well plate to screen for K562 cells stably expressing the target gene.

[0079] 2.3 Monoclonal cell selection a. Take 1×10 5 cells cultured and screened in the above steps. Resuspend them in 10 mL of fresh RPMI1640 complete medium.

[0080] b. Centrifuge at 1000 rpm for 5 minutes and discard the supernatant.

[0081] c. Resuspend the cells again in 10 mL of fresh RPMI1640 complete medium and count the cell concentration.

[0082] d. According to the cell concentration, take out 200 cells by the limited dilution method with RPMI1640 complete medium and add them to a 50 mL centrifuge tube.

[0083] e. Add 20 mL of fresh 1640 complete medium to the centrifuge tube and pipette to mix the cells evenly to obtain a cell mixture.

[0084] f. Take the cell mixture and add it to a 96-well plate, 100 μL per well.

[0085] g. Observe under an inverted microscope to find the wells with single cells and mark them.

[0086] h. After 72 hours, replenish 100 µL of fresh RPMI 1640 into the labeled wells.

[0087] i. After 7 days, transfer the cell population from the 96-well plate to a 24-well plate and continue to expand the cells to establish a monoclonal cell line.

[0088] 2.4 Western blot was used to verify the expression of the target molecule. A Myc-tag sequence was designed in the Anti-NKp46 construct, and the detection of the Myc-tag was used to analyze whether anti-NKp46 was successfully transfected. The antibodies used for Western blot verification included: recombinant Anti-IL-15 antibody [EPR1542Y] (Abcam, catalog number: ab134177), Rabbit polyclonal Anti-IL-21 antibody (Abcam, catalog number: ab5978), MYC tag Rabbit Polyclonal antibody (Proteintech, catalog number: 16286-1-AP), CD48 Polyclonal antibody (Proteintech, catalog number: 27519-1-AP).

[0089] 2.5 According to the above steps, CD48 / anti-NKp46 / mIL-15 / mIL-21-aAPC (4aAPC) was obtained by transfection using lentiviral expression vectors containing mIL-21, mIL-15, anti-NKp46, CD48 respectively.

[0090] The Western blot results showed that: 4aAPC cells could fully express the following active molecules: IL-21, IL-15, Myc-tag (anti-NKp46), CD48 ( Figure 9 ), which could be used for subsequent isolation, purification, verification and the use of its exosomes.

[0091] 3. Isolation and purification of 4aAPC-Exo 3.1 Collection of supernatant and ultrafiltration of 4aAPC culture medium a. Culture 4aAPC cells in a culture flask until the density reaches about 60%-70%. In a biosafety cabinet, collect all the cell suspension in the bottle and centrifuge at 800 rpm for 5 minutes.

[0092] b. After centrifugation, discard the supernatant, wash the cells 3 times with PBS, add RPMI 1640 basal medium without FBS (containing 1% double antibody), and culture in an incubator at 37°C and 5% CO2 for 48 hours.

[0093] c. Collect all the cell suspension in the culture flask and centrifuge in a centrifuge at a speed of 300 gCentrifuge for 10 minutes to remove the cells and take the supernatant. Then centrifuge again at 2000 g Centrifuge for another 10 minutes to remove cell debris and collect the supernatant.

[0094] d. Prepare an Amicon Ultra-15 100KDa centrifugal ultrafiltration tube (Millipore, MW 100000, catalog number: UFC91000). Rinse the ultrafiltration tube once with sterile distilled water or PBS before use. (Note: Once the filter membrane in the ultrafiltration tube is wetted, avoid drying it again and keep it in a wet state. After use, the ultrafiltration tube needs to be cleaned with 0.5M NaOH prepared and filtered through a 0.22μm filter head and then soaked.) e. Preliminarily filter the freshly collected cell supernatant with a 0.22μm filter head.

[0095] f. In a biosafety cabinet, use a pipette to transfer the filtered supernatant into the filter in the centrifugal ultrafiltration tube (add no more than 15 mL of liquid each time). Place the capped ultrafiltration tube in a centrifuge, adjust the centrifuge speed to 3000 g , centrifuge for 15 minutes, and after centrifugation, recover the remaining liquid in the upper filter, which is the required concentrated supernatant.

[0096] g. To improve the recovery efficiency, ultrafilter and centrifuge the lower liquid after ultrafiltration again, and repeat this process 3 times in total.

[0097] 3.2 Exosome isolation a. Collect the above-mentioned concentrated supernatant into a new 15mL centrifuge tube and add Exo-spin™ precipitant (Cell guidance system, catalog number: EX06-30) at a ratio of 2:1. For example, if the volume of the supernatant is 10 mL, then add 5 mL of Exo-spin™ precipitant.

[0098] b. Invert the centrifuge tube up and down to mix it well and incubate it overnight in a 4°C refrigerator.

[0099] c. Take out the mixture the next day and centrifuge it at 16000 g rpm for 1 hour at 4°C.

[0100] d. After centrifugation, carefully aspirate and discard the supernatant, resuspend the exosome-containing precipitate with 100µL - 300µL PBS, and further purify it through a 0.22μm filter head. Make a label (4aAPC-Exo) and store it in an -80°C refrigerator for later use.

[0101] 3.3 Exosome identification a. Measure the particle size of exosomes using nanoparticle tracking analysis (NTA).

[0102] b. Detect the morphology of exosomes by transmission electron microscopy.

[0103] c. Detect the expression of exosome markers and specific molecules by Western blot, such as CD63, CD9, CD81, TSG101, mIL-15, mIL-21, anti-NKp46, CD48. The antibodies used for Western blot verification include: C9 Rabbit Polyclonal antibody (Proteintech, catalog number: 20597-1-AP), CD63 Rabbit Polyclonal antibody (Proteintech, catalog number: 25682-1-AP), TSG101 Rabbit Polyclonal antibody (Proteintech, catalog number: 28283-1-AP), CD81 Rabbit Polyclonal antibody (Proteintech, catalog number: 27855-1-AP); the remaining antibodies used are the same as those in step 2.4.

[0104] 3.4 To verify the role of 4aAPC-Exo in the in vitro culture system, isolate and purify K562-Exo as the control group according to steps 3.1 to 3.3 (replace 4aAPC cells with K562 cells).

[0105] Nanoparticle tracking analysis (NTA) measures the particle size range of exosomes between 50 - 150 nm. Transmission electron microscopy detection shows that the exosomes have a complete structure and an obvious membrane. Western blot detection results show that the isolated and purified 4aAPC-Exo and K562-Exo possess exosome markers CD63, CD9, CD81, TSG101; and 4aAPC-Exo also carries the 4 active molecules we constructed: IL-21, IL-15, Myc-tag (anti-NKp46), CD48 ( Figure 10 and Figure 11 ). Therefore, this exosome can be used for subsequent in vitro expansion experiments of NK cells. Example 2 In vitro expansion of NK cells using exosome 4aAPC-Exo

[0106] 1. Isolate peripheral blood to obtain peripheral blood mononuclear cells PBMC a. Draw 20 mL of peripheral blood and place it in a sterile heparin sodium blood collection tube.

[0107] b. Carefully pour 20 mL of blood into a centrifuge tube containing 15 mL of Lymphoprep™ lymphocyte separation fluid (STEMCELL Technologies, catalog number 07851).

[0108] c. At room temperature, 800 g Centrifuge for 20 minutes at an acceleration rate of 3 and a deceleration rate of 2. If the blood has been stored for more than 2 hours, increase the centrifugation time to 30 minutes.

[0109] d. After centrifugation, the blood is separated into 4 layers, from top to bottom: the plasma layer (layer 1), the mononuclear cell layer (layer 2), the separation liquid layer (layer 3), and the red blood cell layer (layer 4).

[0110] e. Use a pipette to collect the mononuclear cells in layer 2 into a new centrifuge tube.

[0111] f. Add 35 mL of PBS to dilute the cell suspension, and centrifuge at 500 g for 10 minutes.

[0112] g. Remove the supernatant, add 35 mL of PBS to dilute the cell suspension, mix well, take 100 µL of the suspension for cell counting, and centrifuge the remaining suspension at 500 g for 10 minutes.

[0113] 8) After centrifugation, discard the supernatant, resuspend the pellet with 1 mL of ALyS505NK-EX serum-free NK cell expansion culture medium (Baso, catalog number: 01400P10). The resulting suspension is the peripheral blood mononuclear cells; set aside for later use.

[0114] 2. Preparation of inactivated autologous plasma a. Use a pipette to collect the plasma from layer 1 obtained in step 4.1d and collect it into a centrifuge tube.

[0115] b. Heat the plasma at 56 °C for 30 minutes; at room temperature, centrifuge at 1200 g for 10 minutes.

[0116] d. Use a pipette to collect the supernatant into a new centrifuge tube and store it at 4 °C for later use.

[0117] 3. Induction and expansion of NK cells a. On day 0, inoculate 1.0×10 6 cells of the above-obtained PBMC cell suspension into a 24-well plate containing 1 mL of ALyS505NK-EX serum-free cell expansion culture medium, then add 5% autologous inactivated plasma and 100 µg / ml 4aAPC-Exo, and place it in an incubator at 37 °C and 5% CO2.

[0118] b. On day 1, supplement the culture medium with 5% autologous inactivated plasma and 100 µg / ml 4aAPC-Exo.

[0119] c. On the 3rd day, supplement the culture medium with 10% autologous inactivated plasma and 100 μg / ml 4aAPC-Exo.

[0120] d. From the 5th day to the 7th day, add the culture medium according to the cell density, and supplement with 5% autologous inactivated plasma and 100 μg / ml 4aAPC-Exo.

[0121] e. From the 9th day to the 13th day, change the medium every two days to fresh serum-free expansion culture medium for ALyS505NK-EX cells containing IL-2 at a final concentration of 700 IU / ml, and maintain the cell density at 1.0×10 6 cells / mL.

[0122] f. On the 14th day, stop the culture, collect the cells, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, resuspend the cells with PBS, and obtain NK cells expanded in vitro.

[0123] g. Cell expansion quantity counting: Detect and record the cell quantity using a cell counter on the 1st, 3rd, 5th, 7th, 9th, 11th, 13th, and 14th days of cell culture respectively.

[0124] 4. Detection of NK cell phenotype by flow cytometry Take the NK cells expanded in vitro, prepare them into cell suspensions, detect the expansion rate and purity (CD3-CD56+) of NK cells by flow cytometry, and detect the expression of their surface receptors and killing function ligands (CD94, NKG2A, KIR, NKG2D, NKp44, NKp46, FasL, TraiL). The specific operations are as follows: a. Collect the cells, count them, and centrifuge at 1000 rpm for 5 minutes. Discard the supernatant and wash once with 2 ml PBS.

[0125] b. Resuspend the cells with sheath fluid, adjust the cell density to 1×10 5 cells / 100 μl, and dispense every 100 μl of cell suspension into 1 round-bottom flow cytometry tube.

[0126] c. Under light - avoiding conditions, add an appropriate amount of the corresponding antibodies to each flow tube according to the instructions (BD Pharmingen™: PerCP - Cy™5.5 Mouse Anti - Human CD3, FITC mouse anti - human CD56(NCAM - 1), PE mouse anti - human CD335 (NKp46), FITC mouse anti - human CD94; R&D: PE - conjugated anti - human NKG2A / CD159a; Biolegend TM : PE anti - human CD314(NKG2D), PE anti - human CD336 (NKp44), PE anti - human CD158 (KIR2DL1 / S1 / S3 / S5), PE anti - human CD253 (TRAIL), PE anti - human CD178 (Fas - L)), gently shake to mix well, and incubate at 4°C in the dark for 30 minutes.

[0127] d. After incubation, directly add 1 ml of sheath fluid to each tube, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, and wash away the excess antibodies.

[0128] e. Add 200 - 300 µl of sheath fluid to each flow tube, gently vortex with an oscillator, and wait for loading onto the instrument.

[0129] f. Use a BD FasCantoⅡ flow cytometer to obtain the expression data of each cell marker, record the data and analyze it.

[0130] 5. At the same time, according to the above steps 1 to 4, use exosome K562 - Exo to perform in vitro expansion of NK cells, and this experimental group serves as the control group.

[0131] The cell counting results show that the cell number proliferates from 1.3×10 6 on day 0 to 12.25×10 6 cells on day 14 (K562 - Exo group), and 22.53×10 6 cells (4aAPC - Exo group) ( Figure 12 ). Flow cytometry detection shows that the exosomes secreted by 4aAPC used in the present invention (4aAPC - Exo) compared with K562 - Exo, the ratio of lymphocyte population (the former is 34.4%, the latter is 7.10%) and the purity of NK cells (the former CD3 - CD56 +The rates were 59.8% for the former and 21.9% for the latter), NK cell receptor CD94 (41.1% for the former and 24.1% for the latter), and NK cell killing-related ligand FasL (14.8% for the former and 3.69% for the latter) were all higher ( Figure 13 ). The NK cell amplification fold of 4aAPC-Exo compared to K562-Exo was 103.6-fold vs 20.6-fold ( Figure 14 ). Example 3 Killing effect experiment of expanded and cultured NK cells on tumor cells

[0132] To further verify that the in vitro cultured NK cells have a killing effect on tumor cells, the survival rates of the above two types of tumor cells were evaluated by detecting the Luciferase luciferin carried by suspended tumor cells and detecting the oxidation ability of adherent tumor cells to WST–8 in the CCK8 reagent, respectively, so as to analyze the killing effect of NK cells on the above tumor cells. WST–8 in the CCK8 reagent can be oxidized by dehydrogenases in cell mitochondria into a highly water-soluble yellow formazan product, and the amount of formazan produced is proportional to the number of living cells. In addition, ELISA was used to analyze the production of interferon γ (IFN-γ) and granzyme (Granzyme B), which was also used as an index to analyze the NK cell effect. Granzyme is an enzyme with a killing effect on target cells released when NK cells encounter target cells. Under conditions such as pathogen infection or tumor cell stimulation in the body, NK cells can be rapidly activated, synthesize and secrete a large amount of IFN-γ. IFN-γ secreted by NK cells plays an important role in both the innate immunity and adaptive immunity of the body. It can not only act directly on target cells, but also activate other immune cells. Therefore, detecting the production of IFN-γ and granzyme can be an effective means to analyze the NK cell effect.

[0133] 1. Killing effect experiment of NK cells on tumor cells Twenty-four hours before the completion of NK cell culture, 5,000 lung adenocarcinoma cells SPC-A-1 were seeded in each well of a flat-bottom transparent 96-well plate. On the day when NK cell culture was completed, 5,000 K562-Luciferase cells were seeded in each well of a white-bottom 96-well plate. The NK cells detected by flow cytometry were co-incubated with target cells (tumor cells) at four ratios of effector cells (NK cells): target cells (tumor cells) = 0:1, 1:1, 2:1, and 4:1 for 24 hours. After removing the culture medium and suspended cells and washing the well plates twice with PBS solution, the viability of the remaining adherent tumor cells SPC-A-1 was quantitatively detected using a CCK-8 reagent (Proteintech, catalog number: PF00004). For the suspended tumor cells K562-Luciferase, 50 μL of luciferase substrate (ONE-Glo™ Luciferase Assay System, Promega, USA, catalog number: E6120) was added to the culture wells, and after gently shaking, the chemiluminescence readings of each well were immediately measured using a microplate reader.

[0134] 2. ELISA analysis of partial killing effector molecules The removed culture medium was analyzed by ELISA for the amounts of IFN-γ (Sinobestbio, catalog number: EHC102g.96) and granzyme (Sinobestbio, catalog number: EHC117.96) produced.

[0135] 3. The NK cells cultured with K562-Exo were used as a control group, and the above steps 1 to 2 for the killing effect experiment of tumor cells and ELISA analysis were also carried out.

[0136] The results of the CCK-8 experiment and ELISA detection showed that the killing effect of NK cells cultured with 4aAPC-Exo on lung adenocarcinoma SPC-A-1 and K562 cells was significantly enhanced compared with that of NK cells cultured with K562-Exo, and the amounts of interferon γ and granzyme produced were also significantly increased ( Figures 15 - 17 ). Note: The calculation method for the CCK8 experiment: Cell viability (%) = (OD450nm of the test sample – OD450nm of the blank) / OD450nm of the sample with an effector-to-target ratio of 0 × 100%; The calculation method for the luciferase experiment: Cell viability (%) = (chemiluminescence value of the sample – chemiluminescence value of the blank) / chemiluminescence value with an effector-to-target ratio of 0 × 100%. Example 4 Effects of exosomes secreted by aAPC constructed with different active molecules on the proliferation efficiency and purity of NK cells

[0137] Different active molecules have different effects on the in vitro expansion efficiency and purity of NK cells. The present invention examines the effect of exosomes secreted by aAPC constructed with different active molecules on the expansion of NK cells. The steps of constructing aAPC, the steps of separating and purifying exosomes, and the steps of in vitro culture and expansion are the same as those in steps 2.1 to 2.5, steps 3.1 to 3.3 in Example 1, and steps 1 to 4 in Example 2. The results show that the exosomes (4aAPC-Exo) secreted by 4aAPC constructed with CD48, anti-NKp46, mIL-15, and mIL-21 used in the present invention have a higher ratio of lymphocyte population (69.7% for the former and 11.6% for the latter) and a higher purity of NK cells (the CD3 - CD56 + rate is 76.4% for the former and 38.8% for the latter) after 14 days of in vitro stimulation and culture compared to the exosomes of aAPC constructed with other active molecules CD86, CD137L, CD64, and mIL-15( Figure 18 ). Therefore, the method for promoting the in vitro culture of NK cells using 4aAPC-Exo (CD48 / anti-NKp46 / mIL-15 / mIL-21-aAPC) used in the present invention is the result of optimization.

[0138] Furthermore, since K562 cells naturally express IL-21 ( Figure 9 ), the exosomes K562-Exo secreted by them contain IL-21 ( Figure 11 ). The cell count results of NK cells cultured with 4aAPC-Exo and K562-Exo show an increase in cell number ( Figure 12 ). Flow cytometry detection also shows that the purity of NK cells, the NK cell receptor CD94, and the NK cell killing-related ligand FasL are all higher in the NK cells cultured by the former ( Figure 13 ). The amplification multiple of NK cells is 103.6 times for 4aAPC-Exo compared to 20.6 times for K562-Exo ( Figure 14 ), and the specific data can be seen in Example 2. The above results show that the combination of four active molecules, CD48 + anti-NKp46 + mIL-15 + mIL-21, is at least optimal in promoting the in vitro culture of NK cells compared to cells containing only the m-IL-21 molecule.

[0139] Example 5 Effects of 4aAPC-Exo and commercial kit Beso ® NK High-Efficiency Induction Kit on the Proliferation Efficiency and Purity of NK Cells The present invention examines the exosomes secreted by 4aAPC and Beso ®Effect of NK High-efficiency Induction Kit (Product No.: T20153P3, including: ALyS505NK-A NK cell coating solution, ALyS505NK-B NK cell high-efficiency induction solution and ALyS505NK-C NK cell high-efficiency activator) on the expansion of NK cells.

[0140] 1. Antibody Coating of Culture Plate a. Add 900 μL of PBS and 100 μL of ALyS505NK-A (PBS: ALyS505NK-A = 9:1) coating solution to each well of a 24-well culture plate. Gently shake to evenly cover the bottom of the plate with the solution.

[0141] b. Incubate at room temperature for 40 - 60 minutes or store overnight at 2 - 8 °C until taken out before use, then remove the coating solution.

[0142] c. Wash the coated wells of the plate once with 1 mL of PBS. The washed culture plate should be used immediately. Note that when washing, do not scratch the coated bottom of the plate.

[0143] 2. Blood Separation and Preparation of PBMC The steps are the same as those in Step 1 of Example 2.

[0144] 3. Preparation of Inactivated Autologous Plasma The steps are the same as those in Step 2 of Example 2.

[0145] 4. Induction of NK Cells by Baso® NK High-efficiency Induction Kit (Day 0 to Day 3) a. Preparation of High-efficiency Induction Medium (EIM): Add 25 μL of ALyS505NK-B to 1 mL of ALyS505NK-EX serum-free expansion culture medium (Baso, Product No.: 01400P10) (ALyS505NK-B: ALyS505NK-EX = 40:1) to prepare high-efficiency induction medium (EIM); b. Resuspend the prepared PBMC with 1 mL of high-efficiency induction medium containing 5% autologous inactivated plasma to make the cell density approximately 1×10 6 cells / mL.

[0146] c. Add the above cell suspension to the antibody-coated culture plate gently, and do not scratch the bottom of the plate with the pipette. Incubate at 37 °C and 5% CO2.

[0147] 5. Activation of NK Cells by Baso® NK High-efficiency Induction Kit (Day 3 to Day 9) a. On Day 3, transfer the cell solution in the well plate to a 10 mL centrifuge tube, take 100 μL of the cell suspension for cell counting; centrifuge the cells in the centrifuge tube at 500g Centrifuge for 10 minutes; remove the supernatant and collect the cells.

[0148] b. Preparation of activation medium (EX-NKC): Add 100 µL of ALyS505NK-C to 50 mL of ALyS505NK-EX serum-free expansion culture medium (ALyS505NK-C:ALyS505NK-EX = 1:500) to prepare the activation medium (EX-NKC).

[0149] c. According to the cell counting results, resuspend the cells with 1 mL of activation medium (EX-NKC) containing 10% autologous inactivated plasma to keep the cell density at 1.3 - 1.5×10 6 cells / mL, and place them in an incubator at 37°C and 5% CO2 for culture.

[0150] d. On the 5th day, perform cell counting, transfer the cell suspension to a 6-well plate, and supplement the activation medium (EX-NKC) containing 5% autologous inactivated plasma to the plate to keep the cell density at 1.3 - 1.5×10 6 cells / mL; e. After 7 days of activation culture, transfer the cell suspension in the well plate to a T25 cm 2 culture flask, and supplement the EX-NKC culture medium to the culture flask to continue keeping the cell density at 1.3 - 1.5×10 6 cells / mL.

[0151] 6. Expansion of NK cells using the Beso Baso® NK High-Efficiency Induction Kit (from the 9th day to the 14th day) a. Preparation of the expansion EX culture medium: Add IL-2 to the ALyS505NK-EX serum-free expansion culture medium to make the final concentration of IL-2 in the culture medium 700 IU / mL.

[0152] b. From the 9th day to the 14th day, replace half of the expansion EX culture medium according to the cell quantity to keep the cell density at 1.3 - 1.5×10 6 cells / mL.

[0153] c. On the 14th day of culture, stop the culture, collect the cells, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, and resuspend the cells with PBS to obtain the in vitro expanded and cultured NK cells.

[0154] g. Cell expansion quantity counting Detect and record the cell quantity using a cell counter on the 1st, 3rd, 5th, 7th, 9th, 11th, 13th, and 14th days of cell culture respectively.

[0155] 7. Detection of NK cell phenotype by flow cytometry The steps are the same as those in Step 4 of Example 2.

[0156] 8. At the same time, according to Steps 1 to 4 in Example 2, exosome 4aAPC-Exo was used for in vitro expansion of NK cells, and the expansion quantity, phenotype, and tumor killing activity of NK cells obtained by the two culture methods were compared.

[0157] 9. Killing effect experiment of NK cells on tumor cells Twenty-four hours before the completion of NK cell culture, 5,000 lung adenocarcinoma cells SPC-A-1 or breast cancer cells MDA-MB-231 and SKBR-3 were respectively inoculated in a flat-bottom transparent 96-well plate; on the day when NK cell culture was completed, 5,000 K562-Luciferase and NAMALWA-Luciferase cells were respectively inoculated in a white-bottom 96-well plate. The NK cells detected by flow cytometry were co-incubated for 24 hours at six ratios of effector cells (NK cells): target cells (tumor cells) = 0:1, 1:1, 2:1, 4:1, 8:1, and 16:1. The culture medium and suspended cells were removed, and the well plate was washed twice with PBS solution, and then the viability of the remaining adherent tumor cells (SPC-A-1, MDA-MB-231, SKBR-3) was quantitatively detected with CCK-8 reagent. For the suspended tumor cells K562-Luciferase and NAMALWA-Luciferase, 50 μL of luciferase substrate was added to the culture wells, and after gently shaking, the chemiluminescence readings of each well were immediately measured with a microplate reader.

[0158] The cell counting results showed that the cell number increased from 1.3×10 6 on Day 0 to 48.34×10 6 cells (Baso® NK High-Efficiency Induction Kit group), and 54.24×10 6 cells (4aAPC-Exo group) ( Figure 19 ). The flow cytometry results showed that: the exosomes secreted by 4aAPC used in the present invention (4aAPC-Exo) had higher lymphocyte population ratios (the former was 69.7% and the latter was 56.7%), NK cell purity (the former CD3 - CD56 + rate was 76.4% and the latter was 65.0%), NK cell receptor CD94 (the former was 69.7% and the latter was 56.7%), and NK cell killing-related ligand TraiL (the former was 69.7% and the latter was 56.7%) than the Baso® NK High-Efficiency Induction Kit after 14 days of in vitro stimulation culture ( Figure 20). The NK cell expansion fold of 4aAPC-Exo was 247.1 times compared to 187.4 times for the Baso® kit ( Figure 21 ). The NK cells cultured with 4aAPC-Exo had a better tumor killing effect on MDA-MB-231 breast cancer than the NK cells cultured with the Baso® NK highly efficient induction kit, and the killing effects of the NK cells cultured by both on other tumor cells were comparable ( Figure 22 ). Therefore, the method for promoting the in vitro culture of NK cells using 4aAPC-Exo of the present invention is the result of optimization.

[0159] Although the present invention has been described in detail above with general descriptions and specific embodiments, based on the present invention, some modifications or improvements can be made, which are obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope claimed by the present invention.

Claims

1. An artificial antigen-presenting cell 4aAPC, characterized in that, It is an artificial antigen-presenting cell expressing CD48, anti-NKp46, mIL-15 and mIL-21.

2. The artificial antigen-presenting cell 4aAPC according to claim 1, characterized in that, It is a K562 cell expressing CD48, anti-NKp46, mIL-15 and mIL-21.

3. The method for constructing the artificial antigen-presenting cell 4aAPC according to claim 1 or 2, characterized in that, It includes: Construct lentiviral expression vectors containing CD48, anti-NKp46, mIL-15, and mIL-21 respectively, and with the aid of lentiviral packaging helper plasmids, sequentially transfect the artificial antigen-presenting cells; Among them, the lentiviral expression vector containing mIL-15, the full vector sequence is as shown in SEQ ID NO:1; The lentiviral expression vector containing mIL-21, the full vector sequence is as shown in SEQ ID NO:2; The lentiviral expression vector containing anti-NKp46, the full vector sequence is as shown in SEQ ID NO:3; The lentiviral expression vector containing CD48, the full vector sequence is as shown in SEQ ID NO:

4.

4. Use of the artificial antigen-presenting cell 4aAPC described in claim 1 or 2 in the in vitro expansion of NK cells.

5. A method for in vitro expanding NK cells using extracellular vesicles of artificial antigen-presenting cells, characterized in that, Use the exosomes secreted by the artificial antigen-presenting cell 4aAPC described in claim 1 or 2 for the in vitro expansion of NK cells.

6. The method according to claim 5, wherein It includes the following steps: S1. Culture the artificial antigen-presenting cell 4aAPC, and isolate and purify exosomes from the cell culture supernatant; S2. Prepare human peripheral blood mononuclear lymphocytes, inoculate the mononuclear cells in the NK cell culture medium, and then add autologous plasma and the exosomes for in vitro culture.

7. The method according to claim 6, wherein Step S2 includes: (1) Inoculate 1.0×10 6 peripheral blood mononuclear cells into a cell culture plate containing 1 ml of NK cell culture medium; then add 5% autologous plasma and 100 μg / ml exosomes; finally, place the culture plate in an incubator at 37°C and 5% CO2 for culture; (2) From the 1st day to the 7th day, supplement and add 5% or 10% autologous plasma and 100 μg / ml exosomes to the culture plate every other day; (3)From the 9th day to the 13th day, change the culture medium every other day, replace it with freshly prepared ALyS505NK-EX medium containing IL-2 with a final concentration of 700 IU / ml, and maintain the cell density at 1.0×10 6 cells / mL; (4) On the 14th day, stop the culture, centrifuge to collect the cells, remove the supernatant, and resuspend the cell pellet with PBS.

8. The method according to claim 6 or 7, characterized in that The NK cell culture medium is a serum-free ALyS505NK-EXNK cell expansion culture medium.

9. Any of the following uses of the NK cells prepared by the method described in any one of claims 5-8: 1) For preparing an anti-tumor drug or composition; 2) For preparing a drug or composition for tumor cell immunotherapy.

10. An anti-tumor drug or composition, characterized in that, The active ingredient is the NK cells prepared by the method described in any one of claims 5-8.

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