A method for in vitro expansion of NK cells using extracellular vesicles of artificial antigen-presenting cells

Abstract

The present invention belongs to the field of medical biology and provides a method for in vitro expansion of NK cells using extracellular vesicles of artificial antigen-presenting cells. The feeder cells used in the present invention, artificial antigen-presenting cells, carry four 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 four active molecules. The isolated and purified exosomes aAPC-Exo can effectively expand 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.

Description

Technical Field

[0001] The present invention belongs to the field of medical biology, and specifically 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, which can directly kill 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] Currently, there are two common methods for in vitro expansion of NK cells: pure cytokine culture and feeder cell + cytokine culture. The former offers high safety, but the resulting cells are of lower purity, are expensive, and have limited expansion times. The latter offers high efficiency, rapid expansion, and high purity, but carries potential safety risks. Adding feeder cells to in vitro culture systems is an effective means of promoting cell proliferation. Among them, artificial antigen-presenting cells (aAPCs), which carry various active molecules, have been shown to effectively activate and expand NK cells. However, the stimulatory specificity and potential safety issues of the active molecules carried by aAPCs remain pressing issues. In recent years, extracellular vesicles, particularly exosomes (Exosomes), have been shown to carry intracellular information such as proteins, RNA, and other active molecules. By fusing with target cells, they deliver this information to target cells and modulate their behavior. This signaling can influence immune responses, cell proliferation, and other aspects. Furthermore, exosomes possess inherent biocompatibility, making them suitable carriers for drugs or therapeutic molecules. Their membrane structure effectively protects the active molecules contained within from degradation, making them an emerging delivery tool. Genetically engineered aAPC-derived exosomes can precisely carry membrane-bound cytokines (such as mIL-15 / mIL-21) and costimulatory molecules (such as 4-1BBL and CD48) while retaining the three-dimensional topological features of their native membrane structure. This novel nanoscale carrier not only circumvents the biosafety risks of trophoblast cells but also efficiently delivers activation signals to NK cells through a membrane fusion mechanism. aAPC-derived exosomes (aAPC-Exo) exhibit similar functions to aAPCs, carrying costimulatory and immunoreactive molecules to modulate immune cell activity. Therefore, aAPC-Exo has the potential to become a more efficient, safer, and cost-effective tool for NK cell expansion. However, current research on the application of aAPC-Exo in NK cell expansion is limited, and an effective solution has yet to be established. Therefore, developing a technology based on aAPC-derived exosomes to induce 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 expanding NK cells in vitro using extracellular vesicles of artificial antigen-presenting cells.

[0005] 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, they are K562 cells 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 complete vector sequence is as shown in SEQ ID NO:1;

[0009] Preferably, for the lentiviral expression vector containing mIL-21, the complete vector sequence is as shown in SEQ ID NO:2;

[0010] Preferably, for the lentiviral expression vector containing anti-NKp46, the complete vector sequence is as shown in SEQ ID NO:3;

[0011] Preferably, for the lentiviral expression vector containing CD48, the complete vector sequence is as shown in SEQ ID NO:4.

[0012] 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.

[0013] 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 (with a size between 100 - 200 nm) secreted by the artificial antigen-presenting cell 4aAPC for in vitro expansion of NK cells.

[0014] Specifically, the method includes the following steps:

[0015] S1. Cultivate the artificial antigen-presenting cell 4aAPC, and isolate and purify exosomes from the cell culture supernatant;

[0016] 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.

[0017] Further, step S2 includes:

[0018] (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;

[0019] (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;

[0020] (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;

[0021] (4) On the 14th day, stop the culture, centrifuge to collect the cells, remove the supernatant, and resuspend the cell pellet with PBS.

[0022] Preferably, the NK cell culture medium is serum-free ALyS505NK-EX NK cell expansion culture medium.

[0023] In the fifth aspect, the present invention provides any one of the following applications of the NK cells prepared according to the described method:

[0024] 1) For the preparation of anti-tumor drugs or compositions;

[0025] 2) For the preparation of drugs or compositions for tumor cell immunotherapy.

[0026] In the 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.

[0027] By virtue of the above technical solutions, the present invention has at least the following advantages and beneficial effects:

[0028] The present invention provides a novel artificial antigen-presenting cell 4aAPC-derived exosome capable of inducing a large amount of NK cell expansion 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 K562 cell line. The above 4 active molecules can be stably expressed for a long time, and the exosomes secreted by them also carry the above specific active molecules.

[0029] 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-scale proliferation. The NK cells amplified by this method have good proliferation, high purity, and obvious tumor cell killing effect.

[0030] The 4aAPC-derived exosomes of the present invention are efficient, stable, and controllable, solving the problems of low purity, limited cell expansion quantity, and weak cell killing activity faced by NK cell preparation methods, and meeting the clinical requirements for NK cell therapy products and the NK source requirements in CAR-NK research and development. The active molecules carried by 4aAPC can specifically expand NK cells, improve the culture purity of NK cells, and effectively avoid the residual effects caused by directly adding feeder cells 4aAPC.

[0031] The present invention first provides an efficient, low-cost, high-purity, and safer method for in vitro expansion of NK cells. The NK cells amplified by the above method have a CD3 - CD56 + rate > 75%, and can expand more NK cells (247 times vs 187 times) than the active molecules in the market commercial kits. Description of the Drawings

[0032] Figure 1 It is the comparison of the mIL-21 gene sequencing results in the preferred embodiment of the present invention.

[0033] Figure 2 It is the comparison of the mIL-15 gene sequencing results in the preferred embodiment of the present invention.

[0034] Figure 3 It is the comparison of the anti-NKp46 gene sequencing results in the preferred embodiment of the present invention.

[0035] Figure 4 It is the comparison of the CD48 gene sequencing results in the preferred embodiment of the present invention.

[0036] Figure 5 It is the map of the lentiviral vector transfected with mIL-21 in the preferred embodiment of the present invention.

[0037] Figure 6 It is the map of the lentiviral vector transfected with mIL-15 in the preferred embodiment of the present invention.

[0038] Figure 7 It is the map of the lentiviral vector transfected with anti-NKp46 in the preferred embodiment of the present invention.

[0039] Figure 8 It is the map of the lentiviral vector transfected with CD48 in the preferred embodiment of the present invention.

[0040] Figure 9 It is the Western blot detection results of K562 and 4aAPC expressing IL-21, IL-15, Myc-tag (anti-NKp46), and CD48 in the preferred embodiment of the present invention.

[0041] Figure 10 These are the test results of transmission electron microscopy, nanoparticle tracking analysis (NTA), and western blotting (exosomes expressing IL-21, IL-15, Myc-tag (anti-NKp46), and CD48 proteins) of 4aAPC-derived exosomes in the preferred embodiment of the present invention. Among them, A: Western blot verification of 4aAPC-Exo marker proteins; B: Nanoparticle size analysis of 4aAPC-Exo; C: Transmission electron microscopy of 4aAPC-Exo.

[0042] Figure 11 These are the test results of transmission electron microscopy, nanoparticle tracking analysis (NTA), and western blotting (exosomes expressing 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: Nanoparticle size analysis of K562-Exo; C: Transmission electron microscopy of K562-Exo.

[0043] Figure 12 This is the proliferation curve of NK cells cultured in vitro with exosomes derived from K562 and 4aAPC in the preferred embodiment of the present invention.

[0044] Figure 13 This is the flow cytometry test result of the purity of NK cells cultured in vitro with exosomes derived from K562 and 4aAPC in the preferred embodiment of the present invention.

[0045] Figure 14 This is the test result of the amplification multiple of NK cells cultured in vitro with exosomes derived from K562 and 4aAPC in the preferred embodiment of the present invention.

[0046] Figure 15 This is the 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.

[0047] Figure 16 This is the ELISA analysis of interferon-γ (IFN-γ) production by NK cells in the preferred embodiment of the present invention.

[0048] Figure 17 This is the ELISA analysis of granzyme B production by NK cells in the preferred embodiment of the present invention.

[0049] Figure 18 This is the comparison result 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.

[0050] Figure 19In the preferred embodiment of the present invention, the exosomes derived from 4aAPC and the Beso Baso ® NK high-efficiency induction kit for the proliferation curve of NK cells cultured in vitro.

[0051] Figure 20 In the preferred embodiment of the present invention, the exosomes derived from 4aAPC and Beso Baso ® NK high-efficiency induction kit for the detection results of flow cytometry of NK cells cultured in vitro.

[0052] Figure 21 In the preferred embodiment of the present invention, the exosomes derived from 4aAPC and Beso Baso ® NK high-efficiency induction kit for the detection results of the amplification multiple of NK cells cultured in vitro.

[0053] Figure 22 In the preferred embodiment of the present invention, the exosomes derived from 4aAPC and Beso Baso ® NK high-efficiency induction kit for the killing ability of NK cells cultured in vitro against lung adenocarcinoma cells, breast cancer cells, myeloid leukemia cells and lymphoma cells. Detailed implementation mode

[0054] In view of the limited number of NK cells in vivo and the easy loss of function during the amplification process; the NK cells cultured with pure cytokines have low purity, high cost and limited amplification multiple; adding feeder cells such as traditional artificial antigen-presenting cells, due to the lack of natural membrane structure, it is difficult to provide spatiotemporal coordinated multi-signal stimulation, resulting in limited NK cell activation efficiency; and the residual effects generated affect the application safety of NK cells and other problems, the present invention provides a method for large-scale and stable induction of NK cell amplification in vitro using extracellular vesicles of artificial antigen-presenting cells. The feeder cell used in the present invention - artificial antigen-presenting cell carries 4 active molecules of CD48, anti-NKp46, mIL-15 and mIL-21. The exosomes secreted by it have a complete structure, the particle size is between 100-200 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.

[0055] In the 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.

[0056] Preferably, the artificial antigen-presenting cell is CD48 / anti-NKp46 / mIL-15 / mIL-21-K562 cell. 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.

[0057] The present invention also provides a method for constructing the above artificial antigen-presenting cell, comprising the following steps:

[0058] (1) Construct lentiviral expression vectors containing CD48, anti-NKp46, mIL-15, and mIL-21 respectively;

[0059] (2) Transfect the lentiviral expression vector containing mIL-21 into the corresponding cells, screen the cell population stably expressing the above molecules 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;

[0060] (3) Based on the mIL-21-aAPC obtained in step (2), introduce mIL-15 by transfection with the lentiviral expression vector containing mIL-15, screen the cell population stably expressing the above molecules with puromycin resistance, pick single cell clones, verify the molecular expression by Western blot, and establish mIL-21 / mIL-15-aAPC;

[0061] (4) Based on the mIL-21 / mIL-15-aAPC obtained in step (3), introduce anti-NKp46 by transfection with the lentiviral expression vector containing anti-NKp46, screen the cell population stably expressing the above molecules with puromycin resistance, pick single cell clones, verify the molecular expression by Western blot, and establish anti-NKp46 / mIL-15 / mIL-21-aAPC;

[0062] (5) Based on the anti-NKp46 / mIL-15 / mIL-21-aAPC obtained in step (4), introduce CD48 by transfection with the lentiviral expression vector containing CD48, screen the cell population stably expressing the above molecules 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);

[0063] Preferably, the corresponding cells in step (2) are K562 cells, but are not limited thereto.

[0064] The plasmid map used in the present invention is shown in Figures 5 - 6 and was constructed by GeneChem.

[0065] Second aspect, the present invention provides a method for large-scale induction of NK cell expansion, which uses exosomes secreted by the above-mentioned artificial antigen-presenting cells for expansion, and includes the following steps:

[0066] (1) Collect the 4aAPC cell culture suspension, and collect the supernatant after centrifugation;

[0067] (2) Ultrafilter the supernatant with an Amicon Ultra-15 100KDa centrifuge tube, and isolate exosomes from the ultrafiltered supernatant through a commercial exosome extraction kit.

[0068] (3) Filter with a 0.22 mm filter head to further purify the exosomes.

[0069] (4) Measure the particle size of exosomes by nanoparticle tracking analysis (NTA), detect the morphology of exosomes by transmission electron microscopy, and detect the expression of exosome markers and specific molecules by Western blot, including CD63, CD9, CD81, TSG101, mIL-15, mIL-21, anti-NKp46, CD48.

[0070] (5) Prepare human peripheral blood mononuclear lymphocytes, and inoculate 1.0×10 6 mononuclear cells into a 24-well plate containing 1 ml of NK cell culture medium (serum-free ALyS505NK-EXNK cell expansion culture medium); then add 5% autologous plasma and 100 μg / ml exosomes; finally, place the culture flask in an incubator at 37°C and 5% CO2.

[0071] (6) From day 1 to day 7, add 5% or 10% autologous plasma and 100 μg / ml exosomes to the culture flask every other day.

[0072] (7) From day 9 to day 13, change the 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.

[0073] (8) On day 14, stop the culture, collect the cells, centrifuge at 1000 rpm for 5 min, and remove the supernatant.

[0074] (9) Resuspend the cell pellet with PBS, detect the cell number by a cell counter, detect the expansion rate and purity (CD3-CD56+) of NK cells by flow cytometry, and evaluate the expression of surface NK cell receptors (such as NKG2D and NKp46).

[0075] (10) Detect the killing activity of the expanded NK cells through tumor killing experiments and cytokine secretion experiments.

[0076] In a third aspect, the present invention provides highly pure NK cells with killing activity against tumor cells prepared by the above method.

[0077] In a fourth aspect, the present invention provides any one of the following applications of the NK cells:

[0078] 1) For preparing anti-tumor drugs or compositions;

[0079] 2) For tumor cell immunotherapy.

[0080] In a fifth aspect, the present invention provides an anti-tumor drug or composition, the active ingredient of which is the NK cells cultured by the above method.

[0081] The 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 are purer than the exosomes of aAPC constructed by other active molecules (CD86, CD137L, CD64, and mIL-15) in terms of the NK cells stimulated (the former CD3 - CD56 + rate is greater than 75.0%, and the latter is approximately equal to 38.8%).

[0082] Using aAPC exosomes as active stimulating molecules in an in vitro culture system avoids the residues of feeder cells and unsafe factors. Exosomes are smaller in volume, more homogeneous; easy to produce and store; have a long circulating half-life; and are safer than aAPC.

[0083] The following examples are used to illustrate the present invention, but do not 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.

[0084] The ALyS505NK-EX medium (product number T20153P3) and serum-free ALyS505NK-EX NK cell expansion culture medium (product number: 01400P10) used in the following examples are both purchased from Baso ® Company.

[0085] Example 1 Construction of artificial antigen-presenting cell 4aAPC expressing CD48, anti-NKp46, mIL-15, and mIL-21

[0086] 1. Lentivirus packaging (taking the recombinant lentivirus carrying mIL-21 as an example)

[0087] 1.1 Preparation of 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 under 5% CO2 conditions.

[0088] 1.2 Seeding of 293T cells: 24 hours before transfection, seed 293T cells at a density of 5×10 6 cells per culture dish (10 cm in diameter), add 10 mL of complete DMEM medium, and the cell density needs to grow to 80 - 90% before transfection.

[0089] 1.3 Medium change before transfection: 1 - 2 hours before transfection, change the medium of the cells to be transfected to DMEM basal medium without fetal bovine serum and antibiotics.

[0090] 1.4 Transfection: Take a sterile 1.5 mL EP tube and prepare the reaction system according to the following components:

[0091]

[0092] 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 as SEQ ID NO:1 - 4 respectively) were constructed by GeneChem Co., Ltd. The sequencing results showed that the amplified plasmid sequences were consistent with the designed plasmid sequences ( Figures 1 - 4 ).

[0093] After mixing well, mix the 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 changed with medium in advance. (psPAX2 is the packaging plasmid, product number 12260, Addgene; VSVG is the envelope plasmid, product number 12259, Addgene; PEI is the transfection reagent, product number 24765 - 100, Polysciences)

[0094] 1.5 Medium change: Change the medium 12 hours later, add 10 mL of fresh DMEM (containing 10% FBS and 1% double antibody) to each dish and continue culturing.

[0095] 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.

[0096] 1.7 Filtration and Sub-packaging: The virus supernatant is filtered through a 0.22 μm filter head and then sub-packaged into new centrifuge tubes or EP tubes to obtain a lentiviral expression vector containing the target gene (such as mIL-12) (the vector structure is shown in Figures 5 - 8 ), make good marks, and store at -80 °C for later use.

[0097] 2. Construction of 4aAPC

[0098] 2.1 Preparation before Transfection

[0099] a. Take out the lentivirus containing the specific gene and polybrene from the -80 °C refrigerator and place them on ice to melt.

[0100] b. Transfer the K562 cells from the culture flask to a 50 mL centrifuge tube and centrifuge at 800 rpm for 5 minutes.

[0101] c. After centrifugation, discard the culture supernatant, add RPMI1640 complete medium (10% FBS and 1% double antibody) and resuspend the cells by pipetting.

[0102] 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.

[0103] e. Inoculate 1 ml of cells into a 24-well plate, ensuring that 1 ml of the cell suspension contains 1×10 5 cells.

[0104] 2.2 Lentiviral Transfection

[0105] a. According to the reference cell transfection MOI of 20, add the lentivirus to the 24-well plate, add 8 μg 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.

[0106] b. Observe the cell morphology 8 to 12 hours later. If the cell state shows no obvious difference from that of uninfected cells, it indicates that the lentivirus has no obvious toxic effect on the cells, and continue the culture.

[0107] c. After 72 hours, add puromycin with 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.

[0108] 2.3 Selection of Monoclonal Cells

[0109] a. Take 1×10 5 cells cultured and screened in the above steps. Add 10 mL of fresh RPMI1640 complete medium to resuspend.

[0110] b. Centrifuge at 1000 rpm for 5 minutes and discard the supernatant.

[0111] c. Re-suspend the cells in 10 mL of fresh RPMI 1640 complete medium and count the cell concentration.

[0112] d. According to the cell concentration, take out 200 cells by the limited dilution method with RPMI 1640 complete medium and add them into a 50 mL centrifuge tube.

[0113] 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.

[0114] f. Take the cell mixture and add it to a 96-well plate, 100 µL per well.

[0115] g. Observe under an inverted microscope to find the wells with single cells and mark them.

[0116] h. After 72 hours, supplement 100 µL of fresh RPMI 1640 to the marked wells.

[0117] 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.

[0118] 2.4 Western blot was used to verify the expression of the target molecule. A Myc-tag sequence was designed in the Anti-NKp46 structure, and detecting 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).

[0119] 2.5 According to the above steps, CD48 / anti-NKp46 / mIL-15 / mIL-21-aAPC (4aAPC) was obtained by transfection with lentiviral expression vectors containing mIL-21, mIL-15, anti-NKp46, CD48 respectively.

[0120] 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 can be used for subsequent isolation, purification, verification, and the use of its exosomes.

[0121] 3. Isolation and purification of 4aAPC-Exo

[0122] 3.1 Collection of supernatant and ultrafiltration of 4aAPC culture medium

[0123] 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 flask and centrifuge at 800 rpm for 5 minutes.

[0124] b. After centrifugation, discard the supernatant, wash the cells 3 times with PBS, add RPMI1640 basal medium without FBS (containing 1% double antibody), and culture in an incubator at 37°C and 5% CO2 for 48 hours.

[0125] c. Collect all the cell suspension in the culture flask and centrifuge at a speed of 300 g for 10 minutes to remove the cells, and take the supernatant. Then centrifuge again at a speed of 2000 g for 10 minutes to remove cell debris, and collect the supernatant.

[0126] d. Prepare an Amicon Ultra-15 100KDa centrifugal ultrafiltration tube (Millipore, MW 100000, product number: UFC91000). Before use, rinse the ultrafiltration tube once with sterile distilled water or PBS. (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.)

[0127] e. Preliminarily filter the freshly collected cell supernatant with a 0.22μm filter head.

[0128] 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 the 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.

[0129] g. To improve the recovery efficiency, ultrafilter and centrifuge the lower liquid after ultrafiltration again, and repeat 3 times in total.

[0130] 3.2 Isolation of exosomes

[0131] a. Collect the above - concentrated supernatant into a new 15 - mL 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.

[0132] b. Invert the centrifuge tube up and down to mix well and incubate overnight in a 4°C refrigerator.

[0133] c. Take out the mixture the next day and centrifuge at 16000 g rpm for 1 hour at 4°C.

[0134] d. After centrifugation, carefully aspirate and discard the supernatant. Resuspend the exosome - containing pellet with 100 µL - 300 µL of PBS and further purify it through a 0.22 - µm filter tip. Make a label (4aAPC - Exo) and store it in an - 80°C refrigerator for later use.

[0135] 3.3 Exosome identification

[0136] a. Measure the particle size of exosomes using nanoparticle tracking analysis (NTA).

[0137] b. Detect the morphology of exosomes by transmission electron microscopy.

[0138] c. Detect the expression of exosome markers and specific molecules, such as CD63, CD9, CD81, TSG101, mIL - 15, mIL - 21, anti - NKp46, CD48, by Western blotting. The antibodies used for Western blotting 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 antibodies used for the rest are the same as those in step 2.4.

[0139] 3.4 To verify the role of 4aAPC - Exo in the in vitro culture system, isolate and purify K562 - Exo as a control group according to steps 3.1 to 3.3 (replace 4aAPC cells with K562 cells).

[0140] Nanoparticle tracking analysis (NTA) measured the particle size range of exosomes to be between 50 - 150 nm. Transmission electron microscopy detection showed that the exosomes had a complete structure with an obvious membrane. Western blot detection results showed that the isolated and purified 4aAPC-Exo and K562-Exo had exosome markers CD63, CD9, CD81, and TSG101; and 4aAPC-Exo also carried the 4 active molecules we constructed: IL-21, IL-15, Myc-tag (anti-NKp46), CD48 ( Figure 10 and Figure 11 ). Therefore, these exosomes can be used for subsequent in vitro expansion experiments of NK cells.

[0141] Example 2 In vitro expansion of NK cells using exosome 4aAPC-Exo

[0142] 1. Isolate peripheral blood to obtain peripheral blood mononuclear cells (PBMC)

[0143] a. Draw 20 mL of peripheral blood and place it in a sterile heparin sodium blood collection tube.

[0144] b. Carefully pour 20 mL of blood into a centrifuge tube containing 15 mL of Lymphoprep™ lymphocyte separation solution (STEMCELL Technologies, catalog number 07851).

[0145] c. At room temperature, centrifuge at 800 g for 20 minutes, with 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.

[0146] d. After centrifugation, the blood is divided into 4 layers, from top to bottom: plasma layer (layer 1), mononuclear cell layer (layer 2), separation solution layer (layer 3), and red blood cell layer (layer 4).

[0147] e. Use a pipette to collect the mononuclear cells in layer 2 into a new centrifuge tube.

[0148] f. Add 35 mL of PBS to dilute the cell suspension and centrifuge at 500 g for 10 minutes.

[0149] 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.

[0150] 8) After centrifugation, discard the supernatant, and resuspend the pellet with 1 mL of ALyS505NK-EX NK cell serum-free expansion medium (Baso Biosciences, catalog number: 01400P10). The suspension at this time is peripheral blood mononuclear cells; set aside for later use.

[0151] 2. Preparation of inactivated autologous plasma

[0152] a. Use a pipette to collect the plasma from the first layer obtained in step 4.1d and collect it into a centrifuge tube.

[0153] b. Heat the plasma at 56 °C for 30 minutes; at room temperature, centrifuge at a speed of 1200 g for 10 minutes.

[0154] d. Use a pipette to collect the supernatant into a new centrifuge tube and store it at 4 °C for later use.

[0155] 3. Induction and expansion of NK cells

[0156] a. On day 0, take 1.0×10 6 cells of the above-obtained PBMC cell suspension and inoculate them into a 24-well plate containing 1 mL of ALyS505NK-EX cell serum-free expansion 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.

[0157] b. On day 1, supplement the medium with 5% autologous inactivated plasma and 100 μg / ml 4aAPC-Exo.

[0158] c. On day 3, supplement the medium with 10% autologous inactivated plasma and 100 μg / ml 4aAPC-Exo.

[0159] d. From day 5 to day 7, add medium according to the cell density, and supplement with 5% autologous inactivated plasma and 100 μg / ml 4aAPC-Exo.

[0160] e. From day 9 to day 13, change the medium every two days to fresh ALyS505NK-EX cell serum-free expansion medium containing a final concentration of 700 IU / ml IL-2, and maintain the cell density at 1.0×10 6 cells / mL.

[0161] f. On day 14, stop the culture, collect the cells, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, and resuspend the cells with PBS to obtain in vitro expanded and cultured NK cells.

[0162] 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.

[0163] 4. Detection of NK cell phenotype by flow cytometry

[0164] Collect the in vitro expanded and cultured NK cells, 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:

[0165] a. Collect the cells, count them, and centrifuge at 1000 rpm for 5 minutes. Discard the supernatant and wash once with 2 ml PBS.

[0166] b. Resuspend the cells with sheath fluid and adjust the cell density to 1×10 5 cells / 100 μl. Aliquot every 100 μl of cell suspension into 1 round-bottom flow tube.

[0167] c. Under light-proof conditions, add appropriate 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 and mix well, and incubate at 4°C in the dark for 30 minutes.

[0168] 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.

[0169] e. Add 200 - 300 μl of sheath fluid to each flow tube, gently vortex with an oscillator, and wait to be loaded onto the machine.

[0170] f. Use a BD FasCantoⅡ flow cytometer to obtain the expression data of each cell marker, record the data and analyze it.

[0171] 5. At the same time, follow the above steps 1 to 4 to amplify NK cells in vitro using exosomes K562-Exo, and this experimental group serves as the control group.

[0172] The cell counting results showed that the cell number increased from 1.3×10 6 on day 0 to 12.25×10 6 cells (K562-Exo group), and 22.53×10 6 cells (4aAPC-Exo group) ( Figure 12 ). Flow cytometry detection showed that the exosomes secreted by 4aAPC used in the present invention (4aAPC-Exo) had higher lymphocyte population ratios (34.4% for the former and 7.10% for the latter), NK cell purity (CD3 - CD56 + rate of 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) than K562-Exo after 14 days of in vitro stimulation culture ( Figure 13 ). The NK cell amplification fold of 4aAPC-Exo compared to K562-Exo was 103.6-fold vs 20.6-fold ( Figure 14 ).

[0173] Example 3 Killing effect experiment of amplified and cultured NK cells on tumor cells

[0174] To further verify that 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 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 effect of NK cells. Granzyme is an enzyme with a killing effect on target cells released when NK cells encounter target cells. In cases such as when the body is infected by pathogens or stimulated by tumor cells, 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 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 effect of NK cells.

[0175] 1. Experiment on the killing effect of NK cells on tumor cells

[0176] Twenty-four hours before the completion of NK cell culture, 5000 lung adenocarcinoma cells SPC-A-1 were inoculated into flat-bottom transparent 96-well plates respectively; on the day when NK cell culture was completed, 5000 K562-Luciferase were inoculated into white-bottom 96-well plates respectively. The NK cells detected by flow cytometry were co-incubated for 24 hours at four ratios of effector cells (NK cells): target cells (tumor cells) = 0:1, 1:1, 2:1, 4:1. After removing the culture medium and suspended cells, and washing the wells twice with PBS solution, the CCK-8 reagent (Proteintech, catalog number: PF00004) was used to quantitatively detect the viability of the remaining adherent tumor cells SPC-A-1. 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.

[0177] 2. ELISA analysis of some killing effect molecules

[0178] 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.

[0179] 3. The NK cells cultured with K562-Exo were used as the control group, and the killing effect experiment and ELISA analysis of tumor cells in the above steps 1 to 2 were also carried out.

[0180] The results of CCK-8 experiment and ELISA detection showed that: compared with the NK cells cultured with K562-Exo, the killing effect of the NK cells cultured with 4aAPC-Exo on lung adenocarcinoma SPC-A-1 and K562 cells was significantly enhanced, and the amounts of interferon γ and granzyme produced were also significantly increased ( Figures 15 - 17 ) Note: Calculation method of CCK8 experiment: Cell survival rate (%) = (OD450nm of test sample – OD450nm of blank) / OD450nm of sample with effector-to-target ratio of 0 × 100%; Calculation method of luciferase experiment: Cell survival rate (%) = (chemiluminescence value of sample – chemiluminescence value of blank) / chemiluminescence value of sample with effector-to-target ratio of 0 × 100%.

[0181] Example 4 Effects of exosomes secreted by aAPC constructed with different active molecules on the proliferation efficiency and purity of NK cells

[0182] Different active molecules have different effects on the in vitro expansion efficiency and purity of NK cells. The present invention examined the expansion effect of exosomes secreted by aAPC constructed with different active molecules on NK cells. The construction steps of aAPC, the steps of exosome isolation and purification, and the steps of in vitro culture and expansion were the same as 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 showed that: the lymphocyte population ratio (the former was 69.7%, the latter was 11.6%) and the NK cell purity (the CD3 - CD56 + rate of the former was 76.4%, the latter was 38.8%) obtained after in vitro stimulation and culture for 14 days by the exosomes (4aAPC-Exo) secreted by 4aAPC constructed with CD48, anti-NKp46, mIL-15, mIL-21 used in the present invention were both higher ( 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 preferred result.

[0183] Furthermore, since K562 cells naturally express IL-21 ( Figure 9 ), the exosomes K562-Exo secreted by them contain IL-21 ( Figure 11). The NK cells cultured with 4aAPC-Exo and K562-Exo showed an increase in cell count as shown by the cell counting results ( Figure 12 ). Flow cytometry also indicated that the NK cells cultured with the former had higher purity, NK cell receptor CD94, and NK cell killing-related ligand FasL ( Figure 13 ). The amplification fold of NK cells with 4aAPC-Exo was 103.6 times compared to 20.6 times with K562-Exo ( Figure 14 ). See Example 2 for specific data. The above results show that the combination of the four active molecules CD48 + anti-NKp46 + mIL-15 + mIL-21 is at least superior to the cells containing only the m-IL-21 molecule in promoting the in vitro culture of NK cells.

[0184] Example 5 Effects of 4aAPC-Exo and the commercial kit Beso ® NK High-Efficiency Induction Kit on the Proliferation Efficiency and Purity of NK Cells

[0185] The present invention examined the effects of exosomes secreted by 4aAPC and Beso ® NK High-Efficiency Induction Kit (product number: 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 amplification of NK cells.

[0186] 1. Coating of the culture plate with antibodies

[0187] a. In a 24-well culture plate, add 900 μL of PBS and 100 μL of ALyS505NK-A (PBS:ALyS505NK-A = 9:1) coating solution to each well. Gently shake to evenly cover the bottom of the well with the solution.

[0188] b. Incubate at room temperature for 40 - 60 minutes or store overnight at 2 - 8 °C until taken out before use, and then remove the coating solution.

[0189] c. Wash the coated well 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.

[0190] 2. Blood separation and preparation of PBMC

[0191] The steps are the same as those in Step 1 of Example 2.

[0192] 3. Preparation of inactivated autologous plasma

[0193] The steps are the same as those in Step 2 of Example 2.

[0194] 4. Baso® NK High-Efficiency Induction Kit NK Cell Induction (Day 0 to Day 3)

[0195] a. Preparation of High-Efficiency Induction Medium (EIM): Add 25 µL of ALyS505NK-B to 1 mL of ALyS505NK-EX Serum-Free Expansion Medium (Baso, Catalog No.: 01400P10) (ALyS505NK-B:ALyS505NK-EX = 40:1) to prepare High-Efficiency Induction Medium (EIM);

[0196] 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.

[0197] c. Add the above cell suspension to the antibody-coated culture plate gently, and avoid scratching the bottom of the plate with the pipette. Incubate at 37°C and 5% CO2.

[0198] 5. Baso® NK High-Efficiency Induction Kit NK Cell Activation (Day 3 to Day 9)

[0199] a. On Day 3, transfer the cell suspension 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 500 g for 10 minutes; remove the supernatant and collect the cells.

[0200] b. Preparation of Activation Medium (EX-NKC): Add 100 µL of ALyS505NK-C to 50 mL of ALyS505NK-EX Serum-Free Expansion Medium (ALyS505NK-C:ALyS505NK-EX = 1:500) to prepare Activation Medium (EX-NKC).

[0201] c. According to the cell counting result, 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 incubate at 37°C and 5% CO2.

[0202] d. On Day 5, perform cell counting, transfer the cell suspension to a 6-well plate, and supplement the well plate with Activation Medium (EX-NKC) containing 5% autologous inactivated plasma to keep the cell density at 1.3 - 1.5×10 6 cells / mL;

[0203] e. After 7 days of activation culture, transfer the cell suspension in the well plate to a T25cm 2Culture flask, add EX-NKC culture medium to the culture flask to keep the cell density at 1.3 - 1.5×10 6 cells / mL.

[0204] 6. Baso® NK High-efficiency Induction Kit for NK cell expansion (from day 9 to day 14)

[0205] a. Preparation of expanded EX culture medium: Add IL-2 to ALyS505NK-EX serum-free expansion culture medium to make the final concentration of IL-2 in the medium 700 IU / mL.

[0206] b. From day 9 to day 14, replace half of the expanded EX culture medium according to the cell number to keep the cell density at 1.3 - 1.5×10 6 cells / mL.

[0207] c. On day 14 of culture, stop the culture, collect the cells, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, resuspend the cells with PBS to obtain in vitro expanded and cultured NK cells.

[0208] g. Cell expansion quantity counting

[0209] Detect and record the cell quantity using a cell counter on days 1, 3, 5, 7, 9, 11, 13, and 14 of cell culture respectively.

[0210] 7. Detection of NK cell phenotype by flow cytometry

[0211] The steps are the same as step 4 in Example 2.

[0212] 8. At the same time, according to steps 1 to 4 in Example 2, use exosome 4aAPC-Exo for in vitro expansion of NK cells, and compare the expansion quantity, phenotype, and tumor killing activity of NK cells obtained by the two culture methods.

[0213] 9. Experiment on the killing effect of NK cells on tumor cells

[0214] 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 into flat-bottom transparent 96-well plates; on the day when NK cell culture was completed, 5,000 K562-Luciferase and NAMALWA-Luciferase cells were respectively inoculated into white-bottom 96-well plates. The NK cells detected by flow cytometry were co-incubated with target cells (tumor cells) at six ratios of effector cells (NK cells):target cells = 0:1, 1:1, 2:1, 4:1, 8:1, 16:1 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, MDA-MB-231, SKBR-3) was quantitatively detected using 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 using a microplate reader.

[0215] 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), 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 CD3 - CD56 + rate of the former 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 those of the Baso® NK High-Efficiency Induction Kit after 14 days of in vitro stimulation and culture ( Figure 20 ). The NK cell amplification fold of 4aAPC-Exo was 247.1-fold vs 187.4-fold compared with 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 High-Efficiency 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 preferred result.

[0216] 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 thereto, 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 of protection required by the present invention.

Claims

1. An artificial antigen-presenting cell 4aAPC, characterized in that, It is K562 cells expressing CD48, anti-NKp46, mIL-15 and mIL-21.

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

4.

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

4. A method for in vitro expansion of 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 for in vitro expansion of NK cells.

5. The method according to claim 4, characterized in that Including 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 into the NK cell culture medium, then add autologous plasma and the exosomes, and perform in vitro culture.

6. The method according to claim 5, characterized in that, 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 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 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.

7. The method according to claim 5 or 6, characterized in that, The NK cell culture medium is serum-free ALyS505NK-EXNK cell expansion culture medium.

8. Any of the following applications of the NK cells prepared by the method according to any one of claims 4-7: 1) For the preparation of anti-tumor drugs or compositions; 2) For the preparation of drugs or compositions for tumor cell immunotherapy.

9. An anti-tumor drug or composition, characterized in that, The active ingredient is NK cells prepared by the method according to any one of claims 4-7.

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