Preparation method and application of chimeric antigen receptor NK cell

Abstract

The invention discloses a preparation method and application of a chimeric antigen receptor NK cell, and relates to the technical field of immunization, and the preparation method comprises the following steps: (1) recovery of a peripheral blood mononuclear cell (PBMC); (2) sorting NK cells; (3) carrying out multiplication culture on the sorted NK cells; (4) adding cell factors into the NK cells subjected to multiplication culture, and performing activation culture; the cell factor is a combination of IL-2 (Interleukin-2), IL-15 and IL-21 (Interleukin-21); and (5) adding Polybrene into the activated and cultured NK cells, infecting for 2-24 hours by using lentivirus carrying CAR molecules and having MOI of 1-10 virus amount according to the cell density of 2 * 10 < 5 >-1 * 10 < 6 > / mL, changing the liquid after infection, and continuously culturing for 54-72 hours to obtain the chimeric antigen receptor NK cells. According to the invention, the optimized conditions are utilized to prepare the CAR-NK cells, and the CAR positive rate can reach 93.87%.

Description

Technical Field

[0001] The present invention relates to the field of immunotechnology, and in particular to a preparation method of chimeric antigen receptor NK cells and applications thereof. Background Art

[0002] The information disclosed in this background technology section is only intended to increase some understanding of the overall background of the invention, and should not be necessarily regarded as an admission or any form of suggestion that the information constitutes the prior art already known to ordinary technicians in this field.

[0003] Chimeric antigen receptor T cells (CAR-T) therapy is one of the most important breakthroughs in the field of hematological malignancies. As a genetically engineered immune cell therapy, CAR-T therapy can kill tumors accurately, quickly and efficiently. In recent years, it has made great breakthroughs and progress in the treatment of malignant tumors, especially hematological tumors. However, there are side effects such as cytokine storm (CRS) and neurotoxicity during the treatment process ['Off-the-shelf' allogeneic CAR T cells: development and challenges. NatRev Drug Discov. 2020.Mar;19(3):185-199.doi:10.1038 / s41573-019-0051-2. Epub2020 Jan 3. PMID: 31900462]. Chimeric antigen receptor Natural Killer cells (CAR-NK) therapy is based on the same principle as CAR-T therapy. Compared with CAR-T cell therapy, CAR-NK therapy can maintain the therapeutic effect while having better tolerance to allogeneic infusion, will not cause graft-versus-host disease, and rarely causes CRS and neurotoxicity, with good safety. Due to the allogeneic tolerance of NK cells, it is expected to be developed into a spot product, which is expected to significantly reduce the cost of cell therapy drugs. In addition, studies have shown that solid tumor cells are more sensitive to NK cell killing than T cells, so CAR-NK therapy has more advantages in the treatment of solid tumors [Forks in the road for CAR T and CAR NK cell cancer therapies. Nat Immunol. 2023 Dec;24(12):1994-2007. doi: 10.1038 / s41590-023-01659-y. Epub 2023 Nov 27. PMID: 38012406].

[0004] However, the lentiviral pseudotyped envelope protein traditionally used for T cell preparation is the VSV-G glycoprotein from Indiana vesicular stomatitis virus [Expert Opinion Biol. Therapy 2020, 1–15]. The receptor of VSVG-LV is LDL-R[Proc. Natl. Acad. Sci. USA 2013, 110, 7306–7311]. The abundance of LDL-R on the surface of NK cells is low, and the expression level cannot be significantly increased after activation, resulting in poor efficiency of infecting NK cells[A DistinctSubset of Highly Proliferative and Lentiviral Vector (LV)-Transducible NKCells Define a Readily Engineered Subset for Adoptive Cellular Therapy. FrontImmunol. 2019 Aug 22;10:2001. doi: 10.3389 / fimmu.2019.02001. Erratum in:Front Immunol. 2019 Dec 04;10:2784. doi: 10.3389 / fimmu.2019.02784. PMID:31507603; PMCID: PMC6713925]. Therefore, the low positive infection rate of NK cells has become a major problem in the preparation of CAR-NK cells.

[0005] Some studies have increased the NK infection positivity rate to 60% at an MOI of 10 by modifying the lentiviral envelope and optimizing the conditions of the transfection reagent [High Cytotoxic Efficiency of Lentivirally and AlpharetrovirallyEngineered CD19-Specific Chimeric Antigen Receptor Natural Killer CellsAgainst Acute Lymphoblastic Leukemia. Front Immunol. 2020 Jan 24;10:3123.doi: 10.3389 / fimmu.2019.03123. PMID: 32117200; PMCID: PMC7025537]. Summary of the invention

[0006] In order to solve the technical problems existing in the prior art, the present invention discloses a method for preparing chimeric antigen receptor NK cells (CAR-NK). In order to overcome the low efficiency of the lentiviral infection of primary NK cells commonly used in the prior art, the present invention greatly improves the CAR-NK positive rate level by optimizing the NK cell culture medium, the combination of cytokines required for activation, the transfection reagent, the infection liquid replacement time, the cell density, the infection multiplicity and other conditions, and solves the difficult problem of CAR molecular transduction and low positive rate in the preparation process of CAR-NK. In the present invention, when the MOI is 7, the NK infection positive rate can reach 80%, and when the MOI is 10, the infection positive rate can reach 93.87%.

[0007] The technical solution adopted by the present invention is as follows:

[0008] In a first aspect of the present invention, a method for preparing a chimeric antigen receptor NK cell is provided, the method comprising the following steps:

[0009] (1) Recovery of peripheral blood mononuclear cells (PBMCs);

[0010] (2) NK cell sorting;

[0011] (3) Proliferating and culturing the selected NK cells;

[0012] (4) adding activation cytokines to the expanded NK cells for activation culture; the activation cytokines are a combination of IL-2, IL-15 and IL-21;

[0013] (5) Polybrene was added to the activated NK cells, and the lentivirus carrying the CAR molecule was used at an MOI of 1 to 10 viral loads at a rate of 2 × 10 5 ~1×10 6 The cells were infected at a density of 100 cells / mL for 2-24 h, and the medium was changed after infection. Chimeric antigen receptor NK cells were obtained 54-72 h after the infection was completed.

[0014] In one or some embodiments of the present invention, the step (1) also includes the separation and cryopreservation of peripheral blood mononuclear cells (PBMCs), which is not specifically limited here and can be based on existing technologies.

[0015] In one or some embodiments of the present invention, in step (1), the recovery of peripheral blood mononuclear cells PBMC is not specifically limited herein, and existing technologies can be adopted. The recovery medium used in the recovery process can be Corning KBM 581 Serum-free Cell Medium, RPMI 1640, X-VIVO-15 Serum-free Hematopoietic Cell Medium or MACS NK expander Medium.

[0016] Corning KBM 581 Serum-free Cell Medium is preferred as the culture medium during the recovery process.

[0017] In one or some embodiments of the present invention, in step (2), NK cell sorting is not specifically limited herein, and existing technologies may be adopted.

[0018] Preferably, the present invention provides a method for sorting NK cells, which comprises the following steps: labeling PBMC cells with CD3 Microbeads, sorting CD3 - Cells; CD56 Microbeads labeled CD3 - PBMC cells were sorted again using the sorting column and magnetic rack to sort out CD3 - CD56 + cells; CD3 - CD56 + The cells were cultured in a sorting medium, to which IL-2 was added so that its concentration in the medium was 300 IU / mL.

[0019] In one or some embodiments of the present invention, in step (3), the NK cells are expanded and cultured until the number of NK cells is 5×10 5 ~1×10 6 Pieces / mL.

[0020] During the expansion culture process, the culture medium used is preferably Corning KBM 581 Serum-free Cell Medium.

[0021] Through experiments, it was found that different culture media have an impact on the positive infection rate of NK cells. In order to improve the positive infection rate, Corning KBM 581 Serum-free Cell Medium is preferred as the culture medium during the expansion culture process.

[0022] In one or some embodiments of the present invention, in step (4), the final concentrations of IL-2, IL-15 and IL-21 are 500 IU / mL, 10 ng / mL and 10 ng / mL, respectively.

[0023] In step (4), the activation culture time is 24 to 48 hours.

[0024] In one or some embodiments of the present invention, in step (5), the MOI is 4 to 10;

[0025] Preferably, the MOI is 7 to 10;

[0026] More preferably, the MOI is 10.

[0027] In step (5), the cell density is 2×10 5 ~1×10 6 Pieces / mL.

[0028] Preferably, the cell density is 8×10 5 Pieces / mL.

[0029] In step (5), a lentivirus carrying a CAR molecule with an MOI of 1 to 10 viral load was used at a rate of 2 × 10 5 ~1×10 6 The cells were infected at a density of 100 cells / mL for 24 h.

[0030] In step (5), the culture time after the medium is changed is continued for 72 hours.

[0031] In a second aspect of the present invention, a method for preparing chimeric antigen receptor NK cells and its application in preparing immunotherapy agents are provided.

[0032] Wherein, the diseases treated by the immunotherapy agent are one or more of the following: benign tumors, malignant tumors, infectious diseases, autoimmune diseases and allergic diseases.

[0033] Preferably, the disease treated by the immunotherapeutic agent is an autoimmune disease including systemic lupus erythematosus, rheumatoid arthritis, scleroderma, hyperthyroidism, juvenile diabetes, idiopathic thrombocytic purpura, autoimmune hemolytic anemia, ulcerative colitis, skin disease or chronic liver disease. One or more of the following.

[0034] Compared with the related art known to the inventor, one of the technical solutions of the present invention has the following beneficial effects:

[0035] Based on the existing technology of lentivirus infection of NK cells, the present invention optimizes the culture medium, cytokine combination, transfection-promoting reagent, infection time, cell density and MOI used before the infection process to form infection conditions suitable for NK cells. CAR-NK cells are prepared under the optimized conditions, and the CAR positivity rate can reach 93.87%. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] The accompanying drawings constituting a part of the present invention are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their description are used to explain the present invention and do not constitute improper limitations on the present invention.

[0037] Figure 1 The positive rate of NK cells cultured in different culture media 72 hours after infection.

[0038] Figure 2 The positive rate of NK cells activated by different cytokine combinations 72 hours after infection was completed.

[0039] Figure 3 The positive rate 72h after infection under different transfection-promoting reagent conditions.

[0040] Figure 4 The positive rate 72 hours after infection under different infection time conditions.

[0041] Figure 5 The positive rate 72h after infection under different cell density conditions.

[0042] Figure 6 The positive rate at 72h after infection under different MOI conditions. DETAILED DESCRIPTION

[0043] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0044] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates the presence of features, steps, operations and / or combinations thereof.

[0045] Terminology Note:

[0046] The cells used in the examples are NK cells, and the infection of the present invention is carried out in a 24-well cell culture plate.

[0047] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0048] Example 1

[0049] 1. Isolation and cryopreservation of peripheral blood mononuclear cells (PBMCs)

[0050] 1. Dilute the single-collected blood with an equal volume of phosphate buffered saline (PBS) and mix thoroughly by inverting.

[0051] 2. Slowly add 30 mL of diluted blood into a centrifuge tube containing 15 mL of lymphocyte separation fluid so that the blood and lymphocyte separation fluid form a distinct layer and centrifuge.

[0052] 3. Centrifugation parameters: 800 g for 20 min, ascent rate 7, descent rate 3, temperature 25°C.

[0053] 4. After centrifugation, gently aspirate the buffy coat (mononuclear cells) and transfer to a new 200 ml centrifuge tube.

[0054] 5. Add 2 to 3 times the volume of PBS (to 150 ml) to the obtained peripheral blood mononuclear cells PBMCs, mix well (avoid the formation of small bubbles), and the height of the liquid column should not exceed 2 / 3 to 3 / 4 of the centrifuge tube.

[0055] 6. After mixing, centrifuge at 400 g for 5 min, increase the speed to level 9, decrease the speed to level 7, and discard the supernatant.

[0056] 7. Use a small amount of liquid at the bottom of the tube to resuspend the cells, transfer to a new 50 ml centrifuge tube, add 2 to 3 times PBS to 45 ml, and centrifuge.

[0057] 8. Centrifugation parameters: 400 g, 5 min, 9th gear of the ascending speed, 7th gear of the descending speed, discard the supernatant.

[0058] 9. Resuspend the cells in 10 ml PBS and count.

[0059] 10. Calculate the volume of cryopreservation solution according to the number of cells to be frozen and the cell density. Use a program-controlled cooling instrument to freeze the cells. The cryopreservation density is 5 ~ 10×10 7 Pieces / ml.

[0060] 2. Recovery of peripheral blood mononuclear cells (PBMCs)

[0061] 1. Prepare recovery medium: Corning KBM 581 Serum-free Cell Medium, RPMI 1640, X-VIVO-15 Serum-free Hematopoietic Cell Medium and MACS NK expander Medium. Add 10% of the volume of the culture medium to each culture medium (SUPERGROW® Cell Culture Supplement (SGR-SM), brand: Dayu), and add IL-2 to a final concentration of 300 IU / mL.

[0062] 2. Take one tube of peripheral blood mononuclear cells (PBMCs) prepared in step 1, thaw it in a 37°C water bath, and add it to 10 ml of preheated culture medium.

[0063] 3. Centrifuge at 400 g for 5 min, with the speed increasing at gear 9 and the speed decreasing at gear 7.

[0064] 4. Discard the supernatant, resuspend the cells with 10 ml of the above culture medium, take samples, count and centrifuge.

[0065] 5. Discard the supernatant, resuspend with the above four culture media, and adjust the cell concentration to 1×10 6 / ml, and cultured in an incubator for 24 h.

[0066] 3. NK Cell Isolation

[0067] 1. Buffer preparation: phosphate buffered saline (PBS) containing 1 (v / v)% human serum albumin (HSA).

[0068] 2. Harvest the PBMCs from step 2, centrifuge at 400 g for 5 min, and discard the supernatant. Wash the PBMCs once with the buffer from step 1. Resuspend the cells in 80 μL buffer, add CD3 Microbeads to label the PBMCs, and use a separation column and magnetic rack to remove CD3 + Cells, harvest CD3 - cell.

[0069] 3. CD3 in step 2 - PBMCs were centrifuged at 400 g for 5 min, the supernatant was discarded, the cells were resuspended in 80 μL buffer, and CD56 Microbeads were added to label CD3 - PBMCs cells were sorted again using the sorting column and magnetic rack to sort out CD3 - CD56 + cell.

[0070] 4. Replace CD3 in step 3 -、CD56 + The cells were cultured with the above four culture media, and the final concentration of IL-2 was 300 IU / mL. Culture conditions: 37°C, 5% carbon dioxide incubator overnight.

[0071] 4. Preparation of chimeric antigen receptor NK cells by lentiviral infection

[0072] 1. Day 0: Peripheral blood mononuclear cells (PBMCs) were resuscitated and stably cultured in the above four culture media for 24 hours.

[0073] 2. On the first day, NK cells were sorted and expanded to a culture density of 1×10 6 / ml.

[0074] 3. On the second day, NK cells were activated by adding cytokine IL-2 (final concentration of 500 IU / mL) and cultured in an incubator at 37°C and 5% carbon dioxide for 48 h.

[0075] 4. On the 4th day, the lentivirus carrying the CAR molecule (product source: Shandong Lishan Biotechnology Co., Ltd., the public can purchase it through commercial channels) infects NK cells. According to MOI=1, calculate the required amount of virus. The calculation formula is as follows: Required amount of virus (mL) = (MOI×number of cells) / virus titer. Add the above calculated amount of virus to the culture flask. According to 2×10 5 / mL cell density, the above cells were incubated with the virus solution for 6 hours, and then replaced with the above four culture media added with 10 (v / v)% cell supplement and a final concentration of 300 IU / mL IL-2.

[0076] 5. On the 7th day, the NK cells infected with the above lentivirus were harvested.

[0077] 5. Detection of positive infection rate of chimeric antigen receptor NK cells

[0078] 1. Transfer the above-harvested lentivirus-infected NK cells to a flow cytometry tube and centrifuge at 400 g for 5 minutes.

[0079] 2. Discard the supernatant, add 50 μl of diluted Anti-CD3 flow cytometry antibody (manufacturer: BD, catalog number: 555339), 50 μl of diluted Anti-CD56 (manufacturer: BD, catalog number: 555518) flow cytometry antibody and 50 μl of PE-CD19 (manufacturer: BD, catalog number: 555413) flow cytometry detection reagent, and incubate for 1 hour.

[0080] 3. Add 1 ml of PBS solution to the above flow tube and centrifuge at 400 g for 5 minutes.

[0081] 4. Discard the supernatant and add 1 ml PBS solution again, centrifuge at 400 g for 5 min to wash away unbound antibodies.

[0082] 5. Using flow cytometry analysis, CD3 negative and CD56 positive cells are NK cells, PE positive cells are CAR positive cells, and the ratio of PE positive cells to NK cells is the CAR positivity rate.

[0083] Example 2

[0084] 1. The isolation and cryopreservation of peripheral blood mononuclear cells (PBMC) are the same as in step 1.

[0085] 2. The culture medium used for PBMCs recovery and NK cell sorting, expansion culture and infection process is Corning KBM581 Serum-free Cell Medium.

[0086] 3. During the process of preparing chimeric antigen receptor NK cells infected with lentivirus, the cytokine combinations were IL-2; IL-2 and IL-15; IL-2 and IL18; IL-2 and IL-21; IL-2, IL-15 and IL-18; IL-2, IL-15 and IL-21; IL-2, IL-18 and IL-21, among which the final concentrations of IL-2, IL-15, IL-18 and IL-21 were 500 IU / mL, 10 ng / mL, 10 ng / mL and 10 ng / mL. The above cytokine combinations were used to activate NK cells for 48 hours. After 48 hours, the virus with MOI=1 was used at a dose of 2×10 5 The cells were infected at a cell density of 100 cells / mL for 6 hours, and the medium was changed after 6 hours (Corning KBM 581 Serum-free Cell Medium with 10 (v / v)% cell supplement and a final concentration of 300 IU / mL IL-2).

[0087] 4. After changing the medium, continue culturing for 72 hours and detect the infection positive rate.

[0088] Example 3

[0089] 1. The isolation and cryopreservation of peripheral blood mononuclear cells (PBMC) are the same as in step 1.

[0090] 2. The culture medium used for PBMCs recovery and NK cell sorting, expansion culture and infection process is Corning KBM581 Serum-free Cell Medium.

[0091] 3. During the preparation of chimeric antigen receptor NK cells infected with lentivirus, the cytokine combination used for activation was IL-2, IL-15 and IL-21, with final concentrations of 500 IU / mL, 10 ng / mL and 10 ng / mL, respectively. The cytokine combination activated NK cells for 48 hours. After 48 hours, the virus amount used at MOI=1 was 2×10 5 The cells were infected at a cell density of 100 / mL for 6 hours. Before infection, protamine, Polybrene, Vectofusion and Retronectin (Retronectin needs to be coated on the culture plate in advance) were added to promote transfection. After 6 hours, the medium was changed (Corning KBM 581 Serum-free Cell Medium with 10 (v / v)% cell additives and a final concentration of 300 IU / mL IL-2 was added).

[0092] 4. After changing the medium, continue culturing for 72 hours and detect the infection positive rate.

[0093] Example 4

[0094] 1. The isolation and cryopreservation of peripheral blood mononuclear cells (PBMC) are the same as in step 1.

[0095] 2. The culture medium used for PBMCs recovery and NK cell sorting, expansion culture and infection process is Corning KBM581 Serum-free Cell Medium.

[0096] 3. During the preparation of chimeric antigen receptor NK cells infected with lentivirus, the activation cytokine combination used was IL-2, IL-15 and IL-21, with final concentrations of 500 IU / mL, 10 ng / mL and 10 ng / mL, respectively. The cytokine combination activated NK cells for 48 hours. After 48 hours, Polybrene was used as a transfection reagent, and the virus amount at MOI=1 was 2×10 5 The cells were infected at a cell density of 1:100, 1:100, 1:100 and 1:100 cells / mL for 2 h, 6 h, 12 h and 24 h, respectively. The medium was changed after infection at each time point (Corning KBM 581 Serum-free Cell Medium with 10 (v / v)% cell supplement and a final concentration of 300 IU / mL IL-2).

[0097] 4. After changing the medium, continue culturing for 72 hours and detect the infection positive rate.

[0098] Example 5

[0099] 1. The isolation and cryopreservation of peripheral blood mononuclear cells (PBMC) are the same as in step 1.

[0100] 2. The culture medium used for PBMCs recovery and NK cell sorting, expansion culture and infection process is Corning KBM581 Serum-free Cell Medium.

[0101] 3. During the preparation of chimeric antigen receptor NK cells infected with lentivirus, the cytokine combination used for activation was IL-2, IL-15 and IL-21, with final concentrations of 500 IU / mL, 10 ng / mL, and 10 ng / mL, respectively. The cytokine combination activated NK cells for 48 hours. After 48 hours, Polybrene was used as a transfection reagent, and the virus amount at MOI=1 was 2×10 5 / mL, 4×10 5 / mL, 6×10 5 / mL, 8×10 5 / mL and 1×10 6 The cells were infected at a cell density of 100 / mL for 24 hours, and the medium was changed after infection (Corning KBM 581 Serum-free Cell Medium with 10 (v / v)% cell supplement and a final concentration of 300 IU / mL IL-2).

[0102] 4. After changing the medium, continue culturing for 72 hours and detect the infection positive rate.

[0103] Example 6

[0104] 1. The isolation and cryopreservation of peripheral blood mononuclear cells (PBMC) are the same as in step 1.

[0105] 2. The culture medium used for PBMCs recovery and NK cell sorting, expansion culture and infection process is Corning KBM581 Serum-free Cell Medium.

[0106] 3. During the preparation of chimeric antigen receptor NK cells infected with lentivirus, the cytokine combination used for activation was IL-2, IL-15 and IL-21, with final concentrations of 500 IU / mL, 10 ng / mL, and 10 ng / mL, respectively. The cytokine combination activated NK cells for 48 hours. After 48 hours, Polybrene was used as a transfection reagent, and the virus amount was 8×10 at MOI=1, 3, 5, 7, and 10, respectively. 5 The cells were infected at a cell density of 100 cells / mL for 24 hours, and the medium was changed after infection (Corning KBM 581 Serum-free Cell Medium with 10 (v / v)% cell supplement and a final concentration of 300 IU / mL IL-2).

[0107] 4. After changing the medium, continue culturing for 72 hours and detect the infection positive rate.

[0108] Experimental results: Figure 1 The positive rate of NK cells cultured in different culture media 72 hours after infection in Example 1, wherein the positive rate was the highest when the culture medium was Corning KBM 581 Serum-free Cell Medium, reaching 5.89%. Figure 2 Based on the optimization of Example 1, the positive rate of NK cells activated by different cytokine combinations in Example 2 at 72 hours after infection is completed, among which the infection positive rate is the highest when the cytokine combination is IL-2, IL-15 and IL-21, which can reach 7.01%. Figure 3 Based on the optimization of Example 3, the positive rate 72 hours after the infection is completed under the conditions of different transfection-promoting reagents in Example 3 is shown. When the transfection-promoting reagent is Polybrene, the infection positive rate is the highest, which can reach 13.47%. Figure 4 Based on the optimization of Example 3, the positivity rate at 72 hours after infection under different infection time conditions in Example 4 was obtained, among which the CAR positivity rate was the highest when the infection time was 24 hours, reaching 26.25%. Figure 5 Based on the optimization of Example 4, the positive rate at 72 h after infection under different cell density conditions in Example 5 was obtained, where the cell density was 8×10 5 The CAR positive rate was the highest when the number of cells / mL was 28.30%. Figure 6 Based on the optimization of Example 5, the positive rate at 72h after infection under different MOI conditions in Example 6 is shown, among which when MOI=10, the CAR positive rate is the highest, reaching 93.87%.

[0109] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for preparing a chimeric antigen receptor NK cell, characterized in that: The method comprises the following steps: (1) Recovery of peripheral blood mononuclear cells (PBMCs); (2) NK cell sorting; (3) Proliferating and culturing the selected NK cells; (4) adding activation cytokines to the expanded NK cells for activation culture; the cytokines are a combination of IL-2, IL-15 and IL-21; (5) Polybrene was added to the activated NK cells, and the lentivirus carrying the CAR molecule was used at an MOI of 1 to 10 viral loads at a rate of 2 × 10 5 ~1×10 6 The cells were infected at a density of 100 cells / mL for 2-24 hours, the medium was changed after infection, and the culture was continued for 54-72 hours to obtain chimeric antigen receptor NK cells.

2. The method for preparing chimeric antigen receptor NK cells according to claim 1, characterized in that: Before step (1), the method also includes the separation and cryopreservation of peripheral blood mononuclear cells (PBMC).

3. The method for preparing chimeric antigen receptor NK cells according to claim 1, characterized in that: In step (1), the recovery medium used in the recovery process is Corning KBM 581 Serum-free Cell Medium.

4. The method for preparing chimeric antigen receptor NK cells according to claim 1, characterized in that: In step (2), the NK cell sorting method comprises the following steps: labeling PBMC cells with CD3 Microbeads, and sorting CD3 cells using a sorting column and a magnetic rack. - Cells; CD56 Microbeads labeled CD3 - PBMC cells were sorted again using the sorting column and magnetic rack to sort out CD3 - CD56 + cells; CD3 - CD56 + The cells were cultured in a sorting medium supplemented with a set amount of IL-2; the sorting medium was Corning KBM 581 Serum-free Cell Medium.

5. The method for preparing chimeric antigen receptor NK cells according to claim 1, characterized in that: In step (3), the NK cells were expanded and cultured until the number of NK cells reached 5×10 5 ~1×10 6 Pieces / mL.

6. The method for preparing chimeric antigen receptor NK cells according to claim 1, characterized in that: In step (4), the dosage and final concentration of IL-2, IL-15 and IL-21 are 500 IU / mL, 10 ng / mL and 10 ng / mL respectively; the activation culture time is 24 to 48 h.

7. The method for preparing chimeric antigen receptor NK cells according to claim 1, characterized in that: In step (5), the MOI is 4-10; the cell density is 2×10 5 ~1×10 6 Pieces / mL.

8. The method for preparing chimeric antigen receptor NK cells according to claim 1, characterized in that: In step (5), a lentivirus carrying a CAR molecule with an MOI of 1 to 10 viral load was used at a rate of 2 × 10 5 ~1×10 6 The cells were infected at a density of 100 cells / mL for 24 hours; the culture time after medium replacement was 72 hours.

9. Use of the method for preparing chimeric antigen receptor NK cells according to any one of claims 1 to 8 in preparing immunotherapy agents.

10. The use according to claim 9, characterized in that: Diseases treated with immunotherapeutic agents include one or more of the following: benign tumors, malignant tumors, infectious diseases, autoimmune diseases, and allergic diseases.

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