Preparation method and application of chimeric antigen receptor NK cells

Abstract

The present invention discloses a method for preparing chimeric antigen receptor NK cells and its application, which relates to the field of immunotechnology and includes: (1) resuscitation of peripheral blood mononuclear cells PBMC; (2) NK cell sorting; (3) amplifying and culturing the sorted NK cells; (4) adding cytokines to the amplified and cultured NK cells for activation culture; the cytokines are a combination of IL-2, IL-15 and IL-21; (5) adding Polybrene to the activated and cultured NK cells, and infecting with lentivirus carrying CAR molecules at a virus dose of MOI 1-10 for 2-24 h at a cell density of 2×10 5 ~1×10 6 cells / mL, changing the medium after infection, and continuing to culture for 54-72 h to obtain chimeric antigen receptor NK cells. The present invention uses the optimized conditions to prepare CAR-NK cells, and the CAR positive rate can reach 93.87%.

Description

Technical Field

[0001] The present invention relates to the field of immunology, and particularly to a method for preparing chimeric antigen receptor NK cells and its application. Background Art

[0002] Disclosing the information of this background art section is only intended to increase some understanding of the overall background of the present invention, and it is not necessarily regarded as an admission or an implication in any form that this information constitutes the prior art already known to those of ordinary skill in the art.

[0003] Chimeric antigen receptor T cells (CAR-T) therapy is one of the most important breakthroughs in the field of hematological malignancies. As a gene-engineered immunotherapy method, chimeric antigen receptor T cell therapy can accurately, rapidly, and efficiently kill tumors. In recent years, it has made great breakthroughs and progress in the treatment of malignancies, especially hematological tumors. However, there are side effects such as cytokine release syndrome (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 has the same principle as CAR-T therapy. Compared with CAR-T cell therapy, CAR-NK therapy can maintain the therapeutic effect while having better allogeneic infusion tolerance, not causing graft-versus-host disease, and rarely causing CRS and neurotoxicity, with good safety. Due to the allogeneic tolerance of NK cells, it is expected to be developed into an off-the-shelf product, which is expected to significantly reduce the cost of cell therapy drugs. And there is research showing that compared with T cells, solid tumor cells are more sensitive to NK cell killing. Therefore, CAR-NK therapy has more advantages in the treatment of solid tumors [Forks in the road forCAR 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 traditional lentiviral pseudotype envelope protein used for T cell preparation is the VSV-G glycoprotein from vesicular stomatitis virus Indiana [Expert Opinion Biol. Therapy 2020, 1–15]. The VSVG-LV receptor is LDL-R [Proc. Natl. Acad. Sci. USA 2013, 110, 7306–7311]. The expression 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 Distinct Subset of Highly Proliferative and Lentiviral Vector (LV)-Transducible NK Cells Define a Readily Engineered Subset for Adoptive Cellular Therapy. Front Immunol. 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 rate of NK cell infection has become a major problem in the preparation of CAR-NK cells.

[0005] Some studies have improved the positive rate of NK infection to 60% at an MOI of 10 by modifying the lentiviral envelope and optimizing the conditions of transfection-promoting reagents [High Cytotoxic Efficiency of Lentivirally and Alpharetrovirally Engineered CD19-Specific Chimeric Antigen Receptor Natural Killer Cells Against 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] To address the technical problems existing in the prior art, the present invention discloses a method for preparing chimeric antigen receptor NK cells (CAR-NK). To overcome the low efficiency of the commonly used lentiviral infection of primary NK cells, the present invention significantly improves the CAR-NK cell positivity rate by optimizing the NK cell culture medium, the combination of cytokines required for activation, the transfection reagent, the infection medium change time, the cell density, and the multiplicity of infection (MOI). This solves the challenges of difficult CAR molecule transduction and low positivity during CAR-NK cell preparation. At an MOI of 7, the NK cell infection positivity rate reaches 80%, and at an MOI of 10, the infection positivity rate reaches 93.87%.

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

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

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

[0010] (2) NK cell sorting;

[0011] (3) Expanding and culturing the sorted 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 cultured 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, and the medium was changed after infection. Chimeric antigen receptor NK cells were obtained 54-72 hours after infection.

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

[0015] In one or some embodiments of the present invention, in step (1), the resuscitation of peripheral blood mononuclear cells (PBMCs) is not specifically limited herein, and existing techniques can be adopted. The resuscitation medium used during the resuscitation 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] During the resuscitation process, the medium is preferably Corning KBM 581 Serum-free Cell Medium.

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

[0018] Preferably, the present invention provides a method for sorting NK cells, which includes the following steps: labeling PBMC cells with CD3 Microbeads, and sorting out CD3 - cells using a sorting column and a magnetic stand; labeling CD3 - PBMC cells with CD56 Microbeads, and again using a sorting column and a magnetic stand to sort out CD3 - CD56 + cells; culturing CD3 - CD56 + cells in a sorting medium, and adding IL-2 to the medium so that its concentration in the medium is 300 IU / mL.

[0019] In one or some embodiments of the present invention, in step (3), the amplification culture is carried out until the number of NK cells reaches 5×10 5 ~1×10 6 cells / mL.

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

[0021] It has been found through experiments that different media have an impact on the positive infection rate of NK cells. In order to increase the positive infection rate, during the amplification culture process, the medium is preferably Corning KBM 581 Serum-free Cell Medium.

[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 - 48 h.

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

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

[0026] More preferably, the MOI is 10.

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

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

[0029] In step (5), lentivirus carrying CAR molecules with a virus amount of MOI of 1 - 10 infects at a cell density of 2×10 5 ~1×10 6 cells / mL for 24 h.

[0030] In step (5), the continued culture time after medium replacement is 72 h.

[0031] In the second aspect of the present invention, there is provided the use of the method for preparing the chimeric antigen receptor NK cells in the preparation of an immunotherapeutic reagent.

[0032] Among them, the diseases treated by the immunotherapeutic reagent are one or more of the following: benign tumors, malignant tumors, infectious diseases, autoimmune diseases and allergic diseases.

[0033] Preferably, the diseases treated by the immunotherapeutic reagent include one or more of autoimmune diseases such as systemic lupus erythematosus, rheumatoid arthritis, scleroderma, hyperthyroidism, juvenile diabetes, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, ulcerative colitis, skin diseases or chronic liver diseases.

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

[0035] Based on the existing technology of lentivirus infecting NK cells, the present invention optimizes the culture medium, cytokine combination, transfection reagent, infection time, cell density and MOI used before the infection process, forms the infection conditions suitable for NK cells, and uses the optimized conditions to prepare CAR-NK cells, and the positive rate of CAR can reach 93.87%. Description of the Drawings

[0036] The attached drawings forming a part of this invention are used to provide a further understanding of the present invention. The schematic embodiments and descriptions of the present invention are used to explain the present invention and do not constitute an improper limitation to the present invention.

[0037] Figure 1 Positive rate of NK cells cultured with different culture media 72 hours after infection completion.

[0038] Figure 2 Positive rate of NK cells 72 hours after infection completion after activation with different cytokine combinations.

[0039] Figure 3 Positive rate of infection 72 hours after completion under different transfection reagent conditions.

[0040] Figure 4 Positive rate of infection 72 hours after completion under different infection time conditions.

[0041] Figure 5 Positive rate of infection 72 hours after completion under different cell density conditions.

[0042] Figure 6 Positive rate of infection 72 hours after completion under different MOI conditions. Detailed Description of the Invention

[0043] It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field 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 the 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, they indicate the presence of features, steps, operations and / or combinations thereof.

[0045] Term Explanation:

[0046] The cells used in the examples are NK cells, and all infections of the present invention are carried out in 24-well cell culture plates.

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

[0050] 1. Add an equal volume of phosphate buffer solution (PBS) to the apheresis blood for dilution, and invert and mix well.

[0051] 2. Slowly add 30 mL of the diluted blood to a centrifuge tube containing 15 mL of lymphocyte separation medium, so that the blood and the lymphocyte separation medium form an obvious layer, and then centrifuge.

[0052] 3. Centrifugation parameters: Centrifuge at 800 g for 20 min, acceleration rate 7, deceleration rate 3, temperature 25°C.

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

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

[0055] 6. After mixing, centrifuge at 400 g for 5 min, acceleration rate 9, deceleration rate 7, and discard the supernatant.

[0056] 7. Resuspend the cells with a small amount of liquid at the bottom of the tube, transfer it to a new 50 ml centrifuge tube, and supplement with 2 - 3 times the volume of PBS to 45 ml, and then centrifuge.

[0057] 8. Centrifugation parameters: 400 g, 5 min, acceleration rate 9, deceleration rate 7, and discard the supernatant.

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

[0059] 10. Calculate the volume of cryopreservation solution to be prepared according to the number of cells to be cryopreserved and the cell density, and use a programmable cryopreservation instrument for cryopreservation. The cryopreservation density is 5 - 10×10 7 cells / ml.

[0060] II. Thawing of peripheral blood mononuclear cells (PBMCs)

[0061] 1. Prepare the resuscitation 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 cell culture additive (product name: SUPERGROW® Cell Culture Additive (SGR-SM), brand: Dayou) to each medium, and add IL-2 with a final concentration of 300 IU / mL.

[0062] 2. Take a 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 pre-warmed medium.

[0063] 3. Centrifuge at 400 g for 5 min, with an acceleration rate of 9 and a deceleration rate of 7.

[0064] 4. Discard the supernatant, resuspend the cells with 10 ml of the above medium respectively, take a sample for counting, and then centrifuge.

[0065] 5. Discard the supernatant, resuspend with the above four media respectively, adjust the cell concentration to 1×10 6 cells / ml according to the counting results, and let it stand in the incubator for 24 h.

[0066] III. NK Cell Sorting

[0067] 1. Prepare the buffer: Phosphate Buffered Saline (PBS) containing 1 (v / v)% Human Serum Albumin (HSA).

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

[0069] 3. For the CD3 - PBMCs in step 2, centrifuge at 400 g for 5 min, discard the supernatant, resuspend the cells with 80 μL of buffer, add CDCD56 Microbeads to label the CD3 - PBMCs cells, and use the sorting column and the magnetic stand again to sort out CD3 - CD56 + cells.

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

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

[0072] 1. Day 0: Resuscitate peripheral blood mononuclear cells PBMCs and culture them stably in the above 4 kinds of culture media for 24 hours.

[0073] 2. Day 1, sort NK cells, amplify and culture them until the culture density reaches 1×10 6 / ml.

[0074] 3. Day 2, after adding the cytokine IL-2 (final concentration of 500 IU / mL) to NK cells, activate and culture them in an incubator at 37°C with 5% carbon dioxide for 48 h.

[0075] 4. Day 4, infect NK cells with lentivirus carrying the CAR molecule (product source: Shandong Lishan Biotechnology Co., Ltd., which can be obtained by the public through commercial channels). Calculate the required amount of virus according to MOI = 1. The calculation formula is as follows: Required amount of virus (mL) = (MOI × number of cells) / virus titer. Add the calculated amount of virus to the culture flask. According to the cell density of 2×10 5 / mL, incubate the above cells with the virus solution for 6 hours, and then replace them with the above four kinds of culture media added with 10 (v / v)% cell additives and IL-2 with a final concentration of 300 IU / mL respectively.

[0076] 5. Day 7, harvest the NK cells after the above lentiviral infection.

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

[0078] 1. Transfer the harvested NK cells after lentiviral infection to a flow tube and centrifuge at 400 g for 5 min.

[0079] 2. Discard the supernatant, add 50 μl of diluted Anti-CD3 flow antibody (manufacturer: BD, product number: 555339), 50 μl of diluted Anti-CD56 (manufacturer: BD, product number: 555518) flow antibody, and 50 μl of PE-CD19 (manufacturer: BD, product number: 555413) flow 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 min.

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

[0082] 5. Analyze using a flow cytometer. NK cells are those that are CD3 negative and CD56 positive. Among them, cells that are PE positive are CAR-positive cells, and the proportion of PE-positive cells among NK cells is the CAR positive rate.

[0083] Example 2

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

[0085] 2. The medium used for the resuscitation of PBMCs, sorting, amplification culture, and infection of NK cells is Corning KBM581 Serum-free Cell Medium.

[0086] 3. During the preparation of chimeric antigen receptor NK cells by lentiviral infection, the cytokine combinations are IL-2; IL-2 and IL-15; IL-2 and IL-18; 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. The final concentrations of IL-2, IL-15, IL-18, and IL-21 used are 500 IU / mL, 10 ng / mL, 10 ng / mL, and 10 ng / mL respectively. Activate NK cells with the above cytokine combinations for 48 hours. After 48 hours, infect at a cell density of 2×10 5 cells / mL with a virus amount of MOI = 1 for 6 hours. After 6 hours, change the medium (to Corning KBM 581 Serum-free Cell Medium supplemented with 10 (v / v)% cell additives and IL-2 at a final concentration of 300 IU / mL).

[0087] 4. Continue culturing for 72 hours after changing the medium and detect the infection positive rate.

[0088] Example 3

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

[0090] 2. The medium used for the resuscitation of PBMCs, sorting, amplification culture, and infection of NK cells is Corning KBM581 Serum-free Cell Medium.

[0091] 3. During the preparation of chimeric antigen receptor NK cells by lentiviral infection, the cytokine combination used for activation is 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 activates NK cells for 48 hours. After 48 hours, the virus amount with MOI = 1 is used to infect at a cell density of 2×10 5 cells / mL for 6 hours. Before infection, transfection reagents are added respectively, including protamine, Polybrene, Vectofusion, and Retronectin (Retronectin needs to coat the culture plate in advance). After 6 hours, the medium is changed (which means adding Corning KBM 581 Serum-free Cell Medium containing 10 (v / v)% cell additives and IL-2 with a final concentration of 300 IU / mL).

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

[0093] Example 4

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

[0095] 2. The medium used in the resuscitation of PBMCs, NK cell sorting, amplification culture, and infection processes is Corning KBM581 Serum-free Cell Medium.

[0096] 3. During the preparation of chimeric antigen receptor NK cells by lentiviral infection, the cytokine combination used for activation is 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 activates NK cells for 48 hours. After 48 hours, Polybrene is used as the transfection reagent, and the virus amount with MOI = 1 is used to infect at a cell density of 2×10 5 / mL for 2h, 6h, 12h, and 24h respectively. After the infection at each time point is completed, the medium is changed (which means adding Corning KBM 581 Serum-free Cell Medium containing 10 (v / v)% cell additives and IL-2 with a final concentration of 300 IU / mL).

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

[0098] Example 5

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

[0100] 2. The medium used for the resuscitation of PBMCs, sorting, amplification, culture and infection of NK cells is Corning KBM581 Serum-free Cell Medium.

[0101] 3. During the preparation of chimeric antigen receptor NK cells by lentiviral infection, the cytokine combination used for activation is IL-2, IL-15 and IL-21, and the final concentrations are 500 IU / mL, 10 ng / mL, 10 ng / mL respectively. The cytokine combination activates NK cells for 48 hours. After 48 hours, Polybrene is used as a transfection promoting reagent, and the virus amounts with MOI = 1 are used to infect at cell densities of 2×10 5 cells / mL, 4×10 5 cells / mL, 6×10 5 cells / mL, 8×10 5 cells / mL and 1×10 6 cells / mL for 24 hours. After the infection is completed, the medium is changed (which means adding Corning KBM 581 Serum-free Cell Medium with 10 (v / v)% cell additives and IL-2 with a final concentration of 300 IU / mL).

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

[0103] Example 6

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

[0105] 2. The medium used for the resuscitation of PBMCs, sorting, amplification, culture and infection of NK cells is Corning KBM581 Serum-free Cell Medium.

[0106] 3. During the preparation of chimeric antigen receptor NK cells by lentiviral infection, the cytokine combination used for activation is IL-2, IL-15 and IL-21, and the final concentrations are 500 IU / mL, 10 ng / mL, 10 ng / mL respectively. The cytokine combination activates NK cells for 48 hours. After 48 hours, Polybrene is used as a transfection promoting reagent, and the virus amounts with MOI = 1, 3, 5, 7, 10 are used to infect at a cell density of 8×10 5 cells / mL for 24 hours. After the infection is completed, the medium is changed (which means adding Corning KBM 581 Serum-free Cell Medium with 10 (v / v)% cell additives and IL-2 with a final concentration of 300 IU / mL).

[0107] 4. Continue culturing for 72 hours after changing the liquid, and detect the positive infection rate.

[0108] Experimental results: Figure 1 This is the positive rate at 72 h after the infection of NK cells cultured with different culture media in Example 1. Among them, when the culture medium is Corning KBM 581 Serum-free Cell Medium, the positive rate is the highest, reaching 5.89%. Figure 2 Based on the optimization of Example 1, this is the positive rate at 72 h after the infection of NK cells activated with different cytokine combinations in Example 2. Among them, when the cytokine combination is IL-2, IL-15, and IL-21, the positive infection rate is the highest, reaching 7.01%. Figure 3 Based on the optimization of Example 3, this is the positive rate at 72 h after the infection under different transfection reagent conditions in Example 3. Among them, when the transfection reagent is Polybrene, the positive infection rate is the highest, reaching 13.47%. Figure 4 Based on the optimization of Example 3, this is the positive rate at 72 h after the infection under different infection time conditions in Example 4. Among them, when the infection time is 24 h, the CAR positive rate is the highest, reaching 26.25%. Figure 5 Based on the optimization of Example 4, this is the positive rate at 72 h after the infection under different cell density conditions in Example 5. Among them, when the cell density is 8×10 5 cells / mL, the CAR positive rate is the highest, reaching 28.30%. Figure 6 Based on the optimization of Example 5, this is the positive rate at 72 h after the infection under different MOI conditions in Example 6. Among them, when MOI = 10, the CAR positive rate is the highest, reaching 93.87%.

[0109] The above embodiments are preferred embodiments of the present invention. However, the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A method for preparing chimeric antigen receptor NK cells, characterized in that, The method comprises the following steps: (1) Resuscitation of peripheral blood mononuclear cells (PBMCs): During the resuscitation process, Corning KBM 581 Serum-free Cell Medium is used as the culture medium; (2) NK cell sorting: The NK cell sorting method includes the following steps: Label PBMC cells with CD3 Microbeads, and use a sorting column and a magnetic stand to sort out CD3 - negative cells; Label CD3 - negative PBMC cells with CD56 Microbeads, and use the sorting column and the magnetic stand again to sort out CD3 - negative CD56 + positive cells; Culture the CD3 - negative CD56 + positive cells in a sorting medium, and add a set amount of IL-2 to the medium; The sorting medium is Corning KBM 581 Serum-free Cell Medium; (3) Amplification culture of the sorted NK cells: The culture medium used for the amplification culture is Corning KBM 581 Serum-free Cell Medium; (4) Adding activation cytokines to the NK cells after the amplification culture for activation culture; the cytokines are a combination of IL-2, IL-15, and IL-21, and the dosages and final concentrations 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 - 48 h; (5) Add Polybrene to the activated and cultured NK cells, and infect them at a cell density of 8×10 5 cells / mL with lentivirus carrying the CAR molecule at a virus dose of MOI = 10 for 24 h. After infection, change the medium and continue culturing for 72 h to obtain chimeric antigen receptor NK cells.

2. The preparation method of the chimeric antigen receptor NK cells according to claim 1, characterized in that, Before step (1), it also includes the isolation and cryopreservation of peripheral blood mononuclear cells (PBMCs).

3. The preparation method of the chimeric antigen receptor NK cells according to claim 1, characterized in that, In step (3), the NK cells are amplified and cultured until the number of NK cells reaches 5×10 5 ~1×10 6 cells / mL.

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