WO2022007665A1

WO2022007665A1 - Non-viral method for preparing stable nk cells with high chimeric receptor expression

Info

Publication number: WO2022007665A1
Application number: PCT/CN2021/103235
Authority: WO
Inventors: 肖�琳; 裘新红; 彭群武; 张亮; 朱苏闽; 徐芳芳
Original assignee: 杭州优凯瑞医药科技有限公司
Priority date: 2020-07-09
Filing date: 2021-06-29
Publication date: 2022-01-13
Also published as: CN113913458A

Abstract

Provided are a method for preparing stable natural killer (NK) cells with high chimeric receptor expression by means of a non-viral method and the use thereof, specifically comprising: (1) transferring the coding gene of the chimeric receptor into a natural killer cell using a transposon system to obtain an initial chimeric receptor-modified natural killer cell; and (2) amplifying the initial chimeric receptor-modified natural killer cell using an artificial antigen-presenting cell. The chimeric receptor-modified NK cell prepared by means of the method has a relatively strong tumor killing ability, and shows a relatively good anti-tumor effect in vitro.

Description

Preparation of NK cells with stable and high expression of chimeric receptors by non-viral method

technical field

The present application belongs to the technical field of medical bioengineering, and relates to a method for preparing NK cells modified by stable and high expression of chimeric receptors by using a non-viral method and its application.

Background technique

Natural killer (NK) cells are an important part of the innate immune system. The proportion of NK cells in peripheral blood lymphocytes is about 10%, and they are the third largest lymphocyte population after B cells and T cells. NK cells have multiple biological functions and are the body's first line of defense against infections and tumors. NK cell function is regulated by a sophisticated and complex network of activating and inhibitory receptors. Most circulating NK cells are in a quiescent phase and can be activated by cytokines and infiltrate pathogen-infected or malignant cells-containing tissues. When the receptors of NK cells are combined with the corresponding ligands, NK cells can also secrete some cytokines, such as interferon (IFN)-γ, to play an immunoregulatory role. One of the main functions of NK cells is to supervise the immune system of the body. Researchers have found that NK cells are involved in controlling the occurrence and development of various diseases. Several in vitro studies on mammalian cells, including human cells, and in vivo studies in mice and rats have shown that NK cells can recognize tumor cells as targets and control tumor cell growth, metastasis, and spread in vivo. Decreased peripheral blood NK cell activity increases the risk of cancer in adults, an 11-year follow-up epidemiological survey showed. Not only that, the role of NK cells as host immunity has also been studied in various cases of flavivirus infection such as Japanese encephalitis virus, yellow fever virus, dengue virus, tick-borne encephalitis virus and West Nile virus (WNV) ; their role in viral hepatitis, influenza virus and HIV-1 infection has also been well documented in several studies; likewise, their role in preventing respiratory tract infections by viruses such as bacteria, respiratory syncytial virus (RSV) and influenza The role of Aspects has also been described in detail in mouse studies. (For details, please refer to: Mandal A, Viswanathan C. et al., Natural killer cells: In health and diseas[J]. Hematology/oncology & Stem Cell Therapy, 2015, 8(2): 47-55.).

Unlike MHC-restricted T cells, NK cells do not require HLA matching, so they can be used as heterologous off-the-shelf cell drugs for immunotherapy of patients, which has important clinical application prospects. Adoptive therapy based on autologous or allogeneic NK cells has been clinically applied to anti-tumor and anti-viral infection, especially allogeneic NK cells have shown good therapeutic effects in the treatment of hematological cancers (Reference: Lupo K B, Matosevic S. et al., Natural Killer Cells as Allogeneic Effectors in Adoptive Cancer Immunotherapy [J]. Cancers, 2019, 11(6).

Although NK cells have powerful immune effector functions, many cancer cells and viruses suppress the function of NK cells through mechanisms such as antigen escape and immunosuppression. For these reasons, over the past decade, scientists have been exploring genetic engineering approaches to enhance the functional activity of NK cells and avoid immunosuppression, such as by expressing endogenous cytokines to enhance NK after adoptive reinfusion Cell proliferation ability, inhibiting immunosuppressive signals or enhancing the killing function of NK cells, etc. The latter approach currently redirects NK cells to tumor cells mainly by modifying chimeric antigen receptors (CARs). A chimeric antigen receptor is a fusion of an antibody's scFv with an intracellular signal transduction structure, and the scFv is used to mediate the recognition and binding of tumor cell surface antigens.

However, primary NK cells are more resistant to gene transfection, which results in a low rate of uptake by NK cells to various vector systems, low transgene expression in NK cells, and only viral vectors achieve high levels in primary NK cells gene transfer efficiency. At present, the modification of chimeric receptors in primary NK cells is generally achieved by electrotransformation of mRNA encoding genes of chimeric receptors or by lentivirus/retrovirus infection. mRNA electrotransformation can only make the gene encoding the chimeric receptor transiently expressed, and the cost of synthesizing mRNA in vitro is relatively high, requiring multiple injections in clinical practice, and the treatment cost is high. Lentiviral/retroviral transfection can produce NK cells with stable expression of chimeric receptor genes. However, compared with NK cell lines such as NK92, primary NK cells are extremely difficult to infect. So far, only a few scientific research teams have successfully applied NK cells. Virus/retrovirus prepares chimeric antigen receptor-modified primary NK cells. After repeated infection, the expression ratio of its transgene in the final product is between 20% and 80%. Among them, retrovirus transfection The efficiency is higher than that of lentivirus (see literature: Sandro et al., Viral and Nonviral Engineering of Natural Killer Cells as Emerging Adoptive Cancer Immunotherapies [J]. Journal of Immunology Research, 2018.). However, because retroviruses and/or lentiviruses are themselves pathogenic and have the potential for insertional mutagenesis, they pose a significant regulatory hurdle to the implementation of human clinical trials. Not only that, the large-scale production of viral vectors in cell lines has the problems of high cost and low efficiency, which constitutes an obstacle to the clinical transformation of chimeric receptor-modified NK cells and significantly increases the production cost.

To overcome the above limitations of viral vector systems, the researchers further explored the feasibility of non-viral vectors. Generally, the non-viral vector system is an artificial synthesis system, which does not depend on any viral components or mammalian cells for production, and is low in cost, and at the same time, it is beneficial to avoid the immune problems caused by the use of viral vectors. DNA transposon is a typical non-viral vector system with the advantages of sustainable gene expression, low immunogenicity, low cost and high efficiency.

In nature, DNA transposons are discrete DNA segments containing transposase genes flanked by inverted terminal repeats (ITRs) containing transposase binding sites. The process of transposition is that the transposase binds to the ITRs, "cuts" the transposition from one location, and "pastes" it to another new location. Transposon-based vector systems, such as Sleeping Beauty (SB) system, PiggyBac (PB) system, and Tol transposon system, use the method of transposition to introduce transgenes into the host genome. Similar to retroviral vectors, stable genome integration into the host can achieve long-term and efficient transgene expression (see references: Munoz-Lopez M, Garcia-Perez J L., et al. DNA Transposons: Nature and Applications in Genomics [J]. Current Genomics, 2010.). Both SB and PB transposons have been successfully used to modify T cells to express CD19-specific CARs (see: Harjeet S et al., Manufacture of Clinical-Grade CD19-Specific T Cells Stably Expressing Chimeric Antigen Receptor Using Sleeping Beauty System and Artificial Antigen Presenting Cells[J].Plos One,2013,8(5):e64138.; and David C. et al., PiggyBac-Engineered T Cells Expressing CD19-Specific CARs that Lack IgG1 Fc Spacers Have Potent Activity again B-ALL Xenografts [J], Molecular Therapy Volume 26,

Issue

8, 1 August 2018, Pages 1883-1895). However, there is no report on the use of transposon system to prepare transgenic primary NK cells.

SUMMARY OF THE INVENTION

In order to overcome the problems of low efficiency of chimeric receptor modification of primary NK cells by current technical means, as well as the problems of regulatory barriers and high production costs caused by the use of retroviruses, the present application provides a non-viral preparation of chimeric receptors The receptor-modified NK cell method uses a transposon system to introduce stable and highly expressed chimeric receptors into NK cells to achieve efficient genetic modification of NK cells.

For this purpose, the present invention adopts the following technical solutions:

The present invention provides a method for preparing natural killer cells modified by chimeric receptors by utilizing a non-viral transposon system and artificial antigen-presenting cells, comprising:

(1) Using the transposon system to transfer the gene encoding the chimeric receptor into NK cells;

(2) Using artificial antigen-presenting cells to expand chimeric receptor-modified NK cells.

Wherein, the NK cells are primary human NK cells, which can be derived from human peripheral blood, umbilical cord blood, placental tissue or induced pluripotent stem cells (iPSCs).

The chimeric receptors include chimeric antigen receptors, chimeric switching receptors, or other chimeric receptors synthesized by recombinant DNA technology.

Preferably, the transposon system of step (1) includes the PiggyBac transposon system, the Sleeping Beauty transposon system, the Tol2 transposon system or other transposon systems using transposase.

Preferably, the transposon system in step (1) includes a transposon element and a transposase element; the transposon element includes a transposon 5' inverted terminal repeat (5' ITR) and a 3' inverse terminal repeat. To the terminal repeat (3'ITR), there are two insulators between the 5'ITR and the 3'ITR, and between the two insulators include a promoter and a chimeric receptor encoding gene, the transposon can Is a plasmid or a linearized nucleic acid fragment; the transposase element can be encoded by a plasmid or a linearized nucleic acid fragment, or can be encoded by mRNA, or can be a protein.

Preferably, the nucleic acid sequence of the transposase and the nucleic acid sequence of the transposon may be on one vector or on two different vectors.

Preferably, genes encoding cytokines that promote the survival of NK cells, including IL-15 and/or IL-21, can be added to the transposon element.

The cytokine can be secreted, membrane-bound, or a combination of cytokine-linked receptors; the cytokine encoding gene can be in the same transposon expression vector with the chimeric receptor encoding gene , also in different transposon expression vectors.

More preferably, the IL-15 encoding gene that promotes the survival of NK cells is added to the transposon element, and is in the same transposon expression vector as the encoding gene of the chimeric receptor.

Preferably, the method for using the transposon system to transfer the encoding gene of the chimeric receptor into NK cells includes electroporation, chemical transfection, and other non-viral transfection methods.

More preferably, the relevant vectors of the transposon system are transferred into primary NK cells by electroporation.

Preferably, before the transposon system is used to transfer the transgene into the NK cells in step (1), the NK cells may or may not be activated in advance.

More preferably, before using the transposon system to transfer the transgene into the NK cells in step (1), the NK cells are activated in advance.

Preferably, if the NK cells are activated in advance, it can be 0 to 14 days after the activation of the NK cells, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14. Today, the gene encoding the chimeric receptor was transferred into NK cells using the transposon system.

More preferably, 1 to 7 days, eg, 1, 2, 3, 4, 5, 6 or 7 days after the activation of the NK cells, the gene encoding the chimeric receptor is transferred into the NK cells using a transposon system.

More preferably, 2 days after the activation of the NK cells, the gene encoding the chimeric receptor is transferred into the NK cells using a transposon system.

Preferably, if NK cells are activated in advance, artificial antigen-presenting cells, feeder cells, cytokines, antibodies or compounds can be used for activation.

In the present invention, the artificial antigen-presenting cells include cell-based, artificially synthesized or exosome-based artificial antigen-presenting cells.

The cell-based artificial antigen presenting cells are selected from human myeloid leukemia K562 cells, human Burkitt lymphoma Daudi cells, EBV transformed B lymphoblastoid cells (EBV-LCL) cells or mouse embryonic fibroblasts Cell line NIH/3T3 cells.

Preferably, the cell-based artificial antigen presenting cells are selected from K562 cells.

Preferably, the cell-based artificial antigen presenting cell is an engineered cell that expresses an antigen recognized by a chimeric receptor.

Preferably, the engineered cells express ligand molecules, cytokines, etc. required for NK cell activation, wherein the cytokines may be secreted or membrane-bound.

More preferably, the ligand molecules required for NK cell activation include CD137L and/or OX40L, and the cytokines required for NK activation include any one of human IL-12, human IL-15, human IL-18 or human IL-21 or a combination of at least two.

Preferably, the cell-based artificial antigen presenting cells and feeder cells are treated with gamma radiation (100 Gy) or mitomycin C (20 μg/mL).

More preferably, both NK cell activation and CAR-NK cell expansion can be performed using artificial antigen presenting cells.

Preferably, in step (1), 0 to 14 days after the gene encoding the chimeric receptor is transferred into NK cells using the transposon system, for example, 1, 2, 3, 4, 5, 6, 7, 8 , 9, 10, 11, 12, 13, or 14 days, using artificial antigen-presenting cells to stimulate the proliferation of chimeric receptor-modified NK cells.

Preferably, the chimeric receptor-modified NK cells in step (2) are expanded multiple times, and each expansion cycle is 3 to 14 days, such as 3, 4, 5, 6, 7, 8, 9, 10 , 11, 12, 13 or 14 days.

Preferably, in the method of the present invention, magnetic sorting can be used to enrich NK cells or CD3+ cells can be removed before step (1); or 0 to 14 days after step (1), magnetic sorting can be used to enrich NK cells cells or remove CD3+ cells; magnetic sorting can also be used to enrich NK cells or remove CD3+ cells after step (2).

Preferably, after step (1), the method further comprises the step of sorting CAR+ cells.

Preferably, after step (2), the method further includes the step of sorting CAR+ cells.

More preferably, after step (2), that is, after one round of co-culture with artificial antigen-presenting cells, sorting of CAR+ cells is performed.

Preferably, a cytokine composition, such as a combination of hrIL-12 and hrIL-18 or a combination of hrIL-12, hrIL-15 and hrIL-18, can be added during the preparation of chimeric receptor-modified NK cells using the transposon system The combination.

Preferably, the cytokine composition can be added on any day during the preparation of the chimeric receptor-modified NK cells.

More preferably, the cytokine composition can be removed by centrifugation one day after being added during the production of chimeric receptor-modified NK cells.

More preferably, the final concentration of hrIL-12 is 1-100 ng/mL, the final concentration of hrIL-15 is 1-100 ng/mL, the final concentration of hrIL-18 is 1-100 ng/mL, and the final concentration of hrIL-12 is 1-100 ng/mL. , the final concentration of hrIL-15 or hrIL-18 is the same or different, for example, it can be 1ng/mL, 10ng/mL, 20ng/mL, 30ng/mL, 40ng/mL, 50ng/mL, 60ng/mL, 70ng/mL, 80ng/mL, 90ng/mL or 100ng/mL.

In this application, the chimeric receptor-modified NK cell culture is carried out using a liquid medium, and the liquid medium can be a cell culture medium commonly used in the art, such as AIM V ^™ (Gibco), X-VIVO ^™ 15 ( Lonza), SCGM ^™ (CellGenix), RPMI, NK MACS ^™ (Miltenyi), OpTmizer ^™ (Gibco), ImmunoCult ^™ -XF T Cell Expansion Medium (STEMCELL) or StemSpan ^™ (STEMCELL).

As a preferred technical solution, the present application provides a method for preparing chimeric receptor-modified NK cells using the PiggyBac transposon system, the method comprising:

(1) Co-culturing the isolated peripheral blood mononuclear cells (PBMCs) with engineered K562 cells treated with γ-rays;

(2) After 2 days, using the Lonza 4D-Nucleofector device, the helper plasmid encoding the PiggyBac (PB) transposon system and the donor plasmid encoding the chimeric acceptor were electroporated into NK cells;

(3) After electroporation for 5 to 9 days (eg, 5, 6, 7, 8, or 9 days), the chimeric receptor-modified NK cells were co-cultured with γ-ray-treated engineered K562 cells, and added at the beginning of the culture. hrIL-12, hrIL-15 and hrIL-18;

Wherein, the cell culture medium is AIMV medium supplemented with 1-5% human AB serum/human autologous plasma, and hrIL-2 is present during the culture.

Further preferably, the method for preparing chimeric receptor-modified NK cells using the PiggyBac transposon system according to the present invention is:

(1) Use CD3 magnetic beads to remove CD3+ cells from mononuclear cells (MNCs);

(2) co-culturing the sorted mononuclear cells (MNCs) with engineered K562 cells treated with γ-rays;

(3) After 2 days, using the Lonza 4D-Nucleofector device, the helper plasmid encoding the PiggyBac (PB) transposon system and the donor plasmid encoding the chimeric acceptor were electroporated into NK cells;

(3) 5-9 days after electroporation, CAR+ cells were sorted;

(4) Co-culturing the sorted chimeric receptor-modified NK cells with γ-ray-treated engineered K562 cells, adding hrIL-12, hrIL-15 and hrIL-18 at the beginning of the culture;

Compared with the prior art, the present application has the following beneficial effects:

(1) At present, the modification of chimeric receptors in primary NK cells mainly relies on lentivirus or retrovirus-mediated gene integration or mRNA electroporation-mediated gene transient expression. During use, there are potential safety hazards, uneven virus quality, transient mRNA expression, and high production costs, which hinder the clinical exploration of chimeric receptor-modified NK cells in tumor treatment. This application proposes the application of transposon systems and artificial antigens. Presenting cells to prepare chimeric receptor-modified primary NK cells, in which the combination of transposon system and artificial antigen presenting cells is necessary, and this application also discovers and proposes the use of transposon system to transfer target genes into NK cells The time point is very important for the expansion efficiency, purity, and positive rate of chimeric receptors of NK cells. Through theoretical and experimental research, this application optimizes the sequence of each step, time point, concentration of reagents used or the ratio of co-culture. Etc., significantly improved the positive rate of chimeric receptors in NK cells, the amplification fold, purity and tumor killing ability of NK cells;

(2) Currently, in the process of preparing chimeric receptor-modified NK cells, the target gene mainly relies on lentiviral vectors or retroviral vectors for delivery. The production cost of gene-modified NK cells is increased, and the high price makes many tumor patients unaffordable. Therefore, the need to reduce the production cost of chimeric receptor-modified immune cells is particularly urgent. This application uses a transposon system to prepare chimeric receptors. Body-modified primary NK cells, the transposon system only needs GMP-grade plasmids, the raw material preparation process is simple, and the production cost is greatly reduced;

(3) The use of lentiviral vectors or retroviral vectors also brings certain safety hazards to operators. Not only that, when using lentiviral vectors or retroviral vectors to prepare chimeric receptor-modified NK cells, There is also a potential risk of contamination of NK cell products by replicating retrovirus (RCR) or replicating lentivirus (RCL). Although the viral vector design and production system are constantly improving, this risk has been greatly reduced, but it cannot be completely ruled out. , the transposon system used in this application is a non-viral method, which is very safe and will not have these hidden dangers;

(4) By applying the method of the present application, the integration efficiency of the gene encoding the chimeric receptor in NK cells is between 40% and 80%, which has reached the same level as that of using retrovirus to modify primary NK cells in the prior art.

Description of drawings

FIG. 1A is the map of plasmid 2, and FIG. 1B is the map of plasmid 4;

Figure 2A is the expression of membrane-bound human interleukin 15 on the surface of K562-NK1 cells, Figure 2B is the expression of membrane-bound human interleukin 21 on the surface of K562-NK1 cells, and the abscissa is the membrane-bound human interleukin IL-15 or IL-21 The expression intensity of , the ordinate is the cell count;

Fig. 3 is the flow chart that utilizes PiggyBac transposon system to prepare CAR-NK;

Figure 4A shows the proportion of NK cells in the cell population on the 10th, 17th, and 24th days of culture for different CAR-NK preparation methods, and Figure 4B shows the proportion of CAR+ cells in the cell population for different CAR-NK preparation methods on the 10th, 17th, and 24th days of culture. the proportion of NK cells;

Figure 5 shows the cumulative expansion fold of the total cell number on the 10th, 17th, and 24th days of culture for different CAR-NK preparation methods;

Figure 6A shows the proportion of NK cells in the cell population of mononuclear cells (MNCs) from different sources at the end of the 1st, 2nd, 3rd, and 4th rounds of co-culture on day 0, and Figure 6B shows the ratios of MNCs from different sources in the first At the end of 2, 3, and 4 rounds of co-culture, the proportion of CAR+ cells in NK cells;

Figure 7 is the cumulative expansion fold of the total cell number of MNCs from different sources at the end of the first, second, third, and fourth rounds of co-culture;

Figure 8A shows the real-time killing map of NK and NKG2D CAR-NK cells on the human colorectal cancer cell line HCT116 on the 21st day after the third round of co-culture; Figure 8B shows the NK and NKG2D CAR on the 21st day after the third round of co-culture - The real-time killing map of NK cells to human gastric cancer cell line MGC803; wherein, the abscissa is the time after tumor cells are plated (that is, after the experiment starts), in hours, and the ordinate is the cell index;

Figure 9 shows the overnight co-culture of NK and NKG2D CAR-NK cells with human colorectal cancer cell line HCT116, human ovarian cancer cell line SKOV3, and human gastric cancer cell line MGC803 on the 28th day after the fourth round of co-culture (E:T= 10:1) Secretion of IFNγ; wherein, the ordinate is the concentration of IFNγ in the co-culture supernatant (pg/mL), *: P<0.05, **: P<0.01;

Figure 10A shows the expression of GFP in NK cells 1 day after the pmax-GFP plasmid was electroporated. The NK cells used were NK cells on the 17th day of expansion. The abscissa is the expression intensity of GFP, the ordinate is the cell count, and Figure 10B is pmax -Number of NK cells before and after 1 day of electroporation of GFP plasmid, the NK cells used are NK cells on the 17th day of expansion, the abscissa is before and 1 day after electroporation, respectively, and the ordinate is the number of cells;

Figure 11A shows the proportion of NK cells in the cell population on the 10th, 17th, 24th and 31st days of culture by different CAR-NK preparation methods, and Figure 11B shows the ratio of NK cells in the cell population by different CAR-NK preparation methods on the 10th, 17th, 24th and 31st days of culture , the proportion of CAR+ cells in NK cells;

Figure 12 shows the cumulative expansion fold of the total cell number on the 10th, 17th, 24th, and 31st days of culture for different CAR-NK preparation methods;

Figure 13A is the real-time killing map of human ovarian cancer cell line SKOV3 by NK, CAR-NK and eCAR-NK cells on the 17th day. The coordinate is the cell index, and Figure 13B shows the data analysis after adding effector cells for 24 hours to calculate the tumor growth inhibition rate, the abscissa is the different experimental groups, and the ordinate is the tumor growth inhibition rate;

Figure 14A is the detection of the survival of NK, NKG2D CAR-NK and NKG2D eCAR-15NK on the 22nd day of culture in the presence or absence of IL-2 for one week in vitro; Figure 14B is the detection of the NK on the 21st day of culture , NKG2D eCAR-15NK and NKG2D eCAR-15 NK(sorted) were cultured in vitro for one week in the presence or absence of IL-2; Figure 14C is the detection of NK, NKG2D eCAR-NK ( sorted) and NKG2D eCAR-mbIL15 NK(sorted) in vitro cultured without IL-2 for one week; ) Survival of one week in vitro culture with or without IL-2; among them, "-IL2" in the abscissa is the culture group without IL-2, and "+IL2" is the culture group with IL-2 added , the ordinate is the percentage change relative to the number of cells before one week of culture;

Figure 15A shows the killing results of NK, NKG2D CAR-mbIL15 NK, and NKG2D eCAR-mbIL15 NK (sorted) on the 24th day after the third round of co-culture on human acute myeloid leukemia cell line KG1, the abscissa is the effect-target ratio (E : T), the killing percentage on the ordinate; Figure 15B is the killing result of NK and NKG2D eCAR-mbIL15 NK (sorted) on the human colorectal cancer cell line HCT116 on the 24th day after the third round of co-culture, the abscissa is the tumor cell plating The time after (that is, after the start of the experiment), in hours, the ordinate is the cell index;

Figure 16 is the expression of BCMA on the surface of K562-NK2 cells;

Figure 17A shows the proportion of NK cells in the cell population of peripheral blood mononuclear cells (PBMC) derived from a healthy donor at the end of the 1st, 2nd and 3rd rounds of co-culture on day 0; Figure 17B shows a healthy donor The proportion of CAR+ cells in NK cells at the end of the 1st, 2nd and 3rd rounds of co-culture of the derived PBMC;

Figure 18 is the expansion fold of the total cell number in each round of PBMC derived from a healthy donor at the end of the 1st, 2nd, and 3rd rounds of co-culture;

Figure 19 shows the killing results of human multiple myeloma cell line U266 by NK and BCMA eCAR-NK on the 36th day at the end of the fifth round of co-culture, wherein the abscissa is the effect-target ratio (E:T), and the ordinate is the killing percentage.

detailed description

In order to further illustrate the technical means adopted in the present application and its effects, the present application will be further described below with reference to the embodiments and the accompanying drawings. It should be understood that the specific embodiments described herein are only used to explain the present application, but not to limit the present application.

If no specific technique or condition is indicated in the examples, the technique or condition described in the literature in the field or the product specification is used. The reagents or instruments used without the manufacturer's indication are conventional products that can be purchased through regular channels.

Unless otherwise stated, cells were cultured at 37 ℃, 5% CO _2, of the tide (95% relative humidity) conditions.

The experimental materials used in the examples are as follows:

PBMC were extracted from peripheral blood of solid tumor patients or healthy donors; MNC derived from umbilical cord blood was purchased from Maishun Biotechnology Co., Ltd.; human chronic myeloid leukemia cell line wild-type K562 cells were purchased from ATCC, human multiple myeloma cell line U266 was purchased from Wuhan Proceeds Life Technology Co., Ltd.; the other reagents or consumables not mentioned were all from conventional reagent manufacturers in the field or prepared by conventional means in the field;

The medium of human ovarian cancer cell line SKOV3 and human colorectal cancer cell line HCT116 is McCoy 5A (Gibco) + 10% FBS (Gibco), and the medium of human gastric cancer cell lines MGC803 and U266 is RPMI (Gibco) + 10% FBS (Gibco), the medium of K562 and human AML cell line KG1 is IMDM (Gibco) + 10% FBS (Gibco);

The K562-NK1 cells used in the examples are γ-ray-treated (100 Gy) genetically engineered K562 encoding membrane-bound human IL-15 (mbIL15), membrane-bound human IL-21 (mbIL21), eGFP and Puromycin cell. Since K562 cells naturally express NKG2D ligands, the antigens recognized by NKG2D CAR are not additionally loaded into K562-NK1 cells here. Membrane-bound human IL-15 is composed of the GM-CSF Ra signal peptide (amino acids 1-22 of UniprotKB P15509), human IL-15 (amino acids 30-162 of UniprotKB P40933) and the hinge transmembrane region of human CD8 (UniprotKB P01732 Amino acids 128-213) are fused together; membrane-bound human IL-21 is composed of GM-CSF Rα signal peptide (amino acids 1-22 of UniprotKB P15509), human IL-21 (amino acids 25-162 of UniprotKB Q9HBE4) ), the hinge constant region of human IGHG4 (the 99th to 327th amino acids of UniprotKB P01861), the transmembrane region of human CD4 (the 397th to 418th amino acids of UniprotKB P01730) are fused together, and the amino acid sequence of eGFP is as shown in SEQ ID No:3 Shown, the amino acid sequence of Puromycin is shown in SEQ ID No:4;

The K562-NK2 cells used in the examples were genetically engineered to encode membrane-bound human IL-15 (mbIL15), membrane-bound human IL-21 (mbIL21), truncated BCMA ( tBCMA), eGFP and Puromycin K562 cells. The sequence of tBCMA is the amino acid sequence of positions 1-77 of UniProt KB-Q02223.

Human recombinant IL-2 (hrIL2) was purchased from Beijing Shuanglu Company, and human recombinant IL-12 (hrIL12), human recombinant IL-15 (hrIL15), and human recombinant IL-18 (hrIL18) were purchased from Nearshore Protein Company;

NK cell culture medium: AIM medium (Gibco company) + 5% human AB serum (Gemini company);

P3 Primary Cell 4D-Nucleofector X Kit was purchased from Lonza Company;

PE-conjugated anti-human CD3 antibody, APC-conjugated anti-human CD56 antibody were purchased from BD, FITC-conjugated anti-Strep-tag II antibody was purchased from GenScript, Biotin-conjugated anti-human IL-15 antibody and APC Conjugated Streptavidin was purchased from Biolegend Company, Biotin-conjugated anti-human IL-21 antibody was purchased from eBioscience Company, anti-Biotin microbeads and Streptavidin micobeads were purchased from Miltenyi Company;

Flow cytometer was purchased from BD Company, model C6 Sampler; real-time killing detector was purchased from ACEA Bio Company, model xCELLigence RTCA DP; human IFNγ ELISA detection kit was purchased from Biolegend Company; Calcein-AM was purchased from Yisheng Biotechnology; pmax-GFP was purchased from Lonza Company;

The amino acid sequence of PiggyBac transposase is from GenBank: AAA87375.2; the NKG2D CAR is: NKG2D-CD28-4-1BB-CD3ζ, the antigen-binding domain is from amino acids 83-216 of NKG2D (UniProtKB-P26718), and the hinge region is from Amino acids 99-110 of IgG4 (UniProtKB-P01861), transmembrane region from amino acids 153-179 of CD28 (UniProtKB-P10747), intracellular domain from 209-255 of 4-1BB (UniProtKB-Q07011) BCMA CAR is: anti-BCMA ScFv-CD28-4-1BB-DAP12ζ, the antigen binding domain is from the scFv, hinge of Anti-BCMA antibody C11D5.3 The region is derived from amino acids 99-110 of IGHG4 (UniProtKB-P01861), the transmembrane region is derived from amino acids 153-179 of CD28 (UniProtKB-P10747), and the intracellular domain is derived from the 209th amino acid of 4-1BB (UniProtKB-Q07011). Amino acids ~255 and amino acids 62-133 of DAP12 (UniprotKB-O43914).

Example 1 Vector construction

(1) Construction of a vector for preparing engineered K562-NK1 cells

DNA fragment 1: CMV promoter nucleotide sequence, puromycin coding nucleotide sequence, T2A, EGFP coding nucleotide sequence were synthesized by conventional biotechnology service companies in the field;

DNA fragment 2: CMV promoter nucleotide sequence, mbIL-15 coding nucleotide sequence, P2A coding nucleotide sequence, mbIL-21 coding nucleotide sequence are synthesized by conventional biotechnology service companies in the field;

DNA Fragment 1 and DNA Fragment 2 were cloned into pFastBac1 plasmid (purchased from Thermofisher) and designated as plasmid 1 .

(2) Construction of PiggyBac transposase vector (auxiliary vector)

In order to construct a plasmid encoding PiggyBac transposase, AgeI and SacI restriction sites were added to both ends of the PiggyBac transposase gene (GenBank: EF587698.1). Point-cloned into the pmaxCloning vector, named plasmid 2, as shown in Figure 1A, the CMV promoter in the pmaxCloning vector controls the expression of the PiggyBac transposase.

(3) Construction of piggyBac transposon vector (donor vector)

The 5' inverted terminal repeat of the PiggyBac transposon (SEQ ID NO: 1), the chicken β-globin chromatin insulator cHS4 (GenBank: AY040835.1), the EF1α promoter, the EcoRI and SalI restriction site sequences , SV40 polyA sequence, reverse complementary cHS4 sequence and 3' reverse terminal repeat sequence (SEQ ID NO: 2) were fused, and synthesized by an outsourced service company, cloned into pmaxCloning vector (purchased from Lonza company) through BsaI, named For pZTS4, the expression of the fusion gene is controlled by the EF1α promoter;

SEQ ID NO: 1: 5'-ccctagaaagataatcatattgtgacgtacgttaaagataatcatgtgtaaaattgacgcatg-3';

SEQ ID NO: 2: 5'-catgcgtcaattttacgcagactatctttctaggg-3';

(4) Construction of transposon donor vector containing NKG2D CAR

In order to construct the NKG2D CAR vector, the extracellular antigen-binding domain (ED) of NKG2D was fused with the IGHG4 hinge region, CD28 transmembrane region, 4-1BB and CD3ζ signaling domain to generate the second-generation NKG2D CAR vector; CAR expression was detected by cytometry, and 3 repeats of Strept-tag II (ST2) were added to the sequence of the CAR; the CAR fragment was synthesized by a conventional biotechnology service company in the field, and the 5' and 3' ends of the sequence included restriction Sexual restriction sites EcoRI and SalI; after the synthesized DNA fragment was digested by EcoRI and SalI, it was cloned into pFastBac1 plasmid (purchased from Thermofisher) and named as plasmid 3.

In order to construct a transposon donor vector containing the NKG2D CAR gene, the CAR sequence in plasmid 2 was digested with EcoRI and SalI and cloned into pZTS4, named plasmid 4. As shown in Figure 1B, the expression of the CAR gene is controlled by the EF1α promoter. control.

In order to construct the NKG2D CAR transposon donor vector containing the human IL-15 gene, the sequence of IRES and human IL-15 (UniprotKB: P40933) was fused, and the 5' and 3' ends of the sequence included the restriction site SalI . The synthesized DNA fragment was digested with SalI and cloned into plasmid 4, which was named plasmid 5.

To construct the NKG2D CAR transposon donor vector containing the membrane-bound human IL-15 (mbIL-15) gene, the IRES and the sequence of membrane-bound human IL-15 were fused, including restriction at the 5' and 3' ends of the sequence The cleavage site SalI. The synthesized DNA fragment was digested with SalI and cloned into plasmid 4, which was named plasmid 6.

(5) Construction of transposon donor vector containing anti-BCMA CAR

In order to construct an anti-BCMA CAR vector, the anti-BCMA scFv (C11D5.3) was fused with the IGHG44 hinge region, CD28 transmembrane region, 4-1BB and CD3ζ signaling domains to generate a second-generation anti-BCMA CAR vector; in order to To facilitate the detection of CAR expression by flow cytometry, 3 repeats of Strept-tag II (ST2) are added to the CAR sequence; the CAR fragment is synthesized by an outsourced service company, and the 5' and 3' ends of the sequence include restriction Sex restriction sites EcoRI and SalI; the synthesized DNA fragment was digested by EcoRI and SalI, and then cloned into pFastBac1 plasmid (purchased from Thermofisher), named plasmid 7.

In order to construct a transposon donor vector containing the anti-BCMA CAR gene, the CAR sequence in plasmid 7 was digested with EcoRI and SalI and cloned into pZTS4, which was named plasmid 8. The expression of the CAR gene was controlled by the EF1α promoter.

(6) Construction of a vector for preparing engineered K562-NK2 cells

In order to construct a transposon donor vector containing the tBCMA gene, the DNA sequence of tBCMA was synthesized by an outsourcing company, and cloned into pZTS4 by EcoRI and SalI, named plasmid 9.

Example 2 Preparation of engineered K562-NK1 cells

The wild-type K562 cells were resuspended in 10 mL Opti-MEM, centrifuged at 200 × g for 5 min, the cell pellet was resuspended in 100 μL P3 buffer, 10 μg plasmid 1 was added, and after mixing, it was transferred to the Lonza electric shock cup;

Place the electroporation cup in the Lonza 4D-NucleofectorTM X Unit (single electroporation cup module) for electroporation. After electroporation, slowly transfer the K562 cell suspension in a cuvette to a well of a 6-well plate. K562 medium (IMDM+10%FBS);

On the 5th day after electroporation, puromycin screening was performed at a concentration of 200 μg/mL for 1 month, and the medium was changed every 2 days;

One month later, single cells were sorted into 96-well plates using a cell sorter BD FACSAria (BD Biosciences), and the amplified single cell clones were analyzed by flow cytometry to detect fusion gene expression and antibody detection. APC-conjugated Streptavidin, biotin-conjugated anti-human IL-15 antibody and biotin-conjugated anti-human IL-21 antibody, respectively, and the screened single cell clone was named K562-NK1.

As shown in Figure 2A and Figure 2B, K562-NK1 cells expressed high levels of human IL-15 and human-IL21 on the surface, with positive rates of 100.0% and 78.9%, respectively.

Example 3 Preparation of NKG2D CAR-NK using piggyBac transposon system

The preparation process is shown in Figure 3:

On

day

0, 5×10 ⁶ PBMCs and 5×10 ⁶ K562-NK1 (100Gy γ-irradiated) were resuspended in 10 mL of NK cell culture medium, seeded into T25 cell culture flasks, and hrIL-2 was added to make the final concentration is 100IU/mL;

On the second day, the suspended cells were taken out and counted, and centrifuged at 300 × g for 10 min; resuspended the cell pellet with 10 mL Opti-MEM and centrifuged at 300 × g for 10 min; resuspended the cell pellet in 100 μL P3 buffer, and added 5

μg plasmid

2 and 10 μg Plasmid 4, mix well and transfer to the electroporation Lonza electroporation cup; place the electroporation cup in the Lonza 4D-NucleofectorTM X Unit (in a single electroporation cup module), and perform electroporation. After the electroporation, slowly transfer the NK cell suspension in one electroporation cup Transfer to a new T25 cell culture flask, add 10 mL of NK cell culture medium containing a final concentration of 100IU/mL hrIL-2, mix gently, and place the T25 cell culture flask in a 37°C cell incubator for cultivation;

From the 3rd day to the 9th day, according to the cell growth, add an appropriate amount of fresh NK cell culture medium containing hrIL-2;

On the 10th day, all cells were collected and counted, and 2 × 10 ⁶ cells were taken for flow cytometric phenotype analysis (anti-human CD3 antibody, anti-human CD56 antibody, anti-Strep-tag II antibody); 2 × 10 ⁶ cells were Resuspend with 2×10 ⁶ K562-NK1 (100Gy γ-ray irradiation) in 10mL medium, add hrIL-2 to make the final concentration 100IU/mL;

From the 10th day to the 16th day, according to the cell growth, add an appropriate amount of fresh NK cell culture medium containing hrIL-2;

On the 17th day, all cells were collected and counted, and 2×10 ⁶ cells were taken for flow cytometric phenotype analysis (anti-human CD3 antibody, anti-human CD56 antibody, anti-Strep-tag II antibody); 2×10 ⁶ cells were Resuspend with 2×10 ⁶ K562-NK1 (100Gy γ-ray irradiation) in 10mL medium, add hrIL-2 to make the final concentration 100IU/mL;

From the 17th day to the 23rd day, according to the cell growth, add an appropriate amount of fresh NK cell culture medium containing hrIL-2;

On the 24th day, all cells were collected and counted, and 2 × 10 ⁶ cells were taken for flow cytometric phenotype analysis (anti-human CD3 antibody, anti-human CD56 antibody, anti-Strep-tag II antibody); 2 × 10 ⁶ cells were Resuspend with 2×10 ⁶ K562-NK1 (100Gy γ-ray irradiation) in 10mL medium, add hrIL-2 to make the final concentration 100IU/mL;

From the 24th day to the 30th day, according to the cell growth, add an appropriate amount of fresh NK cell culture medium containing hrIL-2;

On the 31st day, all cells were collected and counted, and 2×10 ⁶ cells were taken for flow cytometric phenotype analysis (anti-human CD3 antibody, anti-human CD56 antibody, and anti-Strep-tag II antibody).

Example 4 Effects of different electroporation conditions on the expansion fold and cell phenotype of CAR-NK cells

This example compares the ratio of NK cells in CAR-NK, the ratio of CAR expression in NK cells, and the total cells obtained by electroporation before NK cell activation or on the 2nd or 4th day after NK cell activation. the amplification fold.

Electrically At day 2 activated NK cells transfected is the procedure of Example 3 is referred to as Day2 EP; method of electroporation prior to activation of NK cells is different from the embodiment 3 in that the 5 × 10 ⁶ PBMC were as described in embodiment The method of Example 3 was electroporated. Immediately after electroporation, it was ^{co-cultured with 5×10 6} K562-NK1 (100Gy γ-ray irradiation), which was recorded as Day0 EP; electroporation was performed on the 4th day after the activation of NK cells, which was different from that in Example 3. The point is that after 5×10 ⁶ PBMC and 5×10 ⁶ K562-NK1 (100 Gy γ-ray irradiation) were co-cultured for 4 days, the suspended cells were taken out and electroporated, which was recorded as Day4 EP.

The above three methods were used to prepare CAR-NK cells from a normal donor PBMC, and the obtained NK cell purity (CD3-CD56+) was compared. The results are shown in Figure 4A. The purity of NK cells was 68%, 77%, and 85% on the 10th, 17th, and 24th days of culture, respectively, while the purity of NK cells in the CAR-NK cells prepared by electroporation before NK cell activation was on the 10th, 17th day of culture. were 11% and 13%, respectively, and the purity of NK cells in CAR-NK cells prepared by electroporation 4 days after NK cell activation were 46%, 81%, and 88% on

culture days

10, 17, and 24, respectively. It shows that the purity of NK cells is higher in the CAR-NK cells prepared by electroporation after NK cell activation than that prepared by electroporation before NK activation.

Further, the expression ratio of CAR in the obtained CAR-NK cells was compared, and the results were shown in Figure 4B. were 14%, 30%, and 45%, while the proportion of CAR+ NK cells in CAR-NK cells prepared by electroporation before NK cell activation was 14% and 27% on the 10th and 17th days of culture, respectively. The proportion of CAR+ NK cells in the CAR-NK cells prepared by electroporation on day 4 was 2%, 15%, and 19% on the 10th, 17th, and 24th days of culture, respectively. It shows that the earlier electroporation is performed, the higher the proportion of CAR+ NK cells.

This example also compares the amplification multiples of the total number of cells obtained. The results are shown in Figure 5. The amplification multiples of the total number of cells obtained by the method of Example 3 for 24 days is 3022 times; After a total of 17 days of expansion, the total number of cells was expanded by 133 times; 4 days after the activation of NK cells, electroporated for 24 days, the total number of cells was expanded by 736 times. This indicated that the cells obtained by electroporation on the second day after the activation of NK cells had the highest expansion fold.

Based on the comparison of these three parameters, the method of Example 3, that is, electroporation was performed 2 days after the activation of NK cells, the prepared CAR-NK cells, the purity of NK cells, the proportion of CAR+ NK cells, and the amplification fold of the total number of cells. Relatively best.

Example 5 Mononuclear cells from multiple sources can use the PiggyBac transposon system to prepare NKG2D CAR-NK

In this example, 7 mononuclear cells (MNCs) from different sources were used to prepare NKG2D CAR-NK according to the method described in Example 3, wherein HD001, HD002, HD003, HD004, HD005, and HD006 were extracted from peripheral blood of healthy donors. PBMC, CB001 was derived from MNC extracted from umbilical cord blood.

As shown in Figure 6A, at the end of the first round of co-culture, the proportion of CD3-CD56+ NK cells reached about 90%, and after the next few rounds of co-culture with K562-NK1 (100Gy γ-ray irradiation), the proportion of NK cells remained basically unchanged. , the individual donors decreased slightly. As shown in Figure 6B, at the end of the first round of co-culture, the proportion of CAR+ NK cells ranged from 18% to 50% (median: 19.5%). With the increase of the number of co-cultures, the proportion of CAR+ NK cells also increased. Gradually increased, and at the end of the fourth round of co-culture, the proportion of CAR+ NK cells ranged from 30% to 90% (median: 64.7%).

As shown in Figure 7, the fourth round of co-culture was completed, and the cumulative expansion fold of the total number of cells was in the range of 3382-24257 (median: 13580).

The in vitro killing of tumor cell lines by NKG2D CAR-NK prepared from HD006 PBMC was detected by RTCA real-time killing detector. At the 0th hour of the RTCA experiment, human colorectal cancer cell line HCT116 or human gastric cancer cell line MGC803 cells were plated in a 16-well electrode plate (ACEA Bio Company), 5000 cells/well; about 24 hours later, NKG2D CAR- NK cells and NK cells (effector cells) were seeded in the electrode plate at a ratio of effector cells:target cells (E:T)=1:1, with a total volume of 200 μL, and each group of experiments set 2 replicate wells; The method of Example 3 was prepared, but electroporation was not required; xCELLigence RTCA was used to detect the killing effect of NKG2D CAR-NK cells on HCT116 and MGC803 in vitro, and NKG2D CAR-NK prepared by HD006 PBMC also showed significant tumor killing effect on HCT116 and MGC803 in vitro (Fig. 8A and Fig. 8B). Not only that, after overnight co-culture of NKG2D CAR-NK with HCT116 cells, MGC803 cells and human SKOV3 cells (E:T=10:1) in vitro, the IFNγ content in the culture supernatant was significantly increased (Figure 9).

Example 6 Direct electroporation of NK cells, transgene expression efficiency and NK cell survival rate

In this example, the green fluorescent protein GFP gene was used as the reporter gene, and the pmax-GFP plasmid was directly used to electroporate the NK cells amplified for 17 days. The NK cell expansion procedure is as follows: On

day

0, 2×10 ⁶ PBMCs and 2×10 ⁶ K562-NK1 (100Gy γ-ray irradiated) were resuspended in 10 mL of NK cell culture medium, and seeded in T75 cell culture flasks (vertical). hrIL-2 was added to make the final concentration 100IU/mL; from the 2nd day to the 10th day, according to the cell growth, an appropriate amount of fresh NK cell culture medium containing hrIL-2 was added; on the 10th day, all cells were collected Counting, taking 2 × 10 ⁶ cells for flow cytometric phenotype analysis (anti-human CD3 antibody, anti-human CD56 antibody); 2 × 10 ⁶ cells and 2 × 10 ⁶ K562-NK1 (100Gy γ-ray irradiation) ) was resuspended in 10 mL of medium, and hrIL-2 was added to make its final concentration 100 IU/mL; from the 10th day to the 17th day, according to the cell growth, an appropriate amount of fresh NK cell culture medium containing hrIL-2 was added; the 17th day On the next day, all cells were collected and counted, and 2×10 ⁶ cells were taken for flow cytometric phenotype analysis (anti-human CD3 antibody, anti-human CD56 antibody). The NK cells on the 17th day of expansion were taken, and the electroporation method in Example 3 was used. The number of electroporated NK cells was 2×10 ⁶ , and the plasmid was replaced by the pmax-GFP plasmid. On the second day after electroporation, cell counting and flow cytometry were performed to detect the proportion of GFP in NK cells. As shown in Figure 10A and Figure 10B, on the second day of electroporation, although the expression ratio of GFP was 17.99%, the survival rate of NK cells was only 3.2%.

The above results show that if only electroporation of NK cells is performed without artificial antigen-presenting cells enrichment and expansion, the viability of NK cells and the expression rate of transgenes are very low. Therefore, the combination of transposon system and artificial antigen-presenting cells is necessary, which can significantly improve the survival and expansion fold of NK cells, and significantly increase the proportion of NK cells modified by chimeric receptors.

Example 7 Preparation of NKG2D eCAR-NK using piggyBac transposon system

On

day

0, 5×10 ⁶ PBMCs and 5×10 ⁶ K562-NK1 (100 Gy γ-irradiated) were resuspended in 10 mL of NK cell culture medium, seeded into T25 cell culture flasks, and hrIL-2 was added to make the final concentration is 100IU/mL;

On the second day, the suspended cells were taken out for counting, and centrifuged at 300 × g for 10 min; resuspended the cell pellet with 10 mL Opti-MEM and centrifuged at 300 × g for 10 min; resuspended the cell pellet in 100 μL P3 buffer, and added 5

μg plasmid

2 and 10 μg Plasmid 4, mix well and transfer to the Lonza electroporation cup; place the electroporation cup in the Lonza 4D-NucleofectorTM X Unit (in a single electroporation cup module) and perform electroporation. After the electroporation, slowly transfer the NK cell suspension in one electroporation cup To a new T25 cell culture flask, add 10 mL of NK cell culture medium containing hrIL-2 with a final concentration of 100IU/mL, mix gently, and place the T25 cell culture flask in a 37°C cell incubator for cultivation;

On the 10th day, all cells were collected and counted, and 2 × 10 ⁶ cells were taken for flow cytometric phenotype analysis (anti-human CD3 antibody, anti-human CD56 antibody, anti-Strep-tag II antibody); 2 × 10 ⁶ cells were Resuspend with 2×10 ⁶ K562-NK1 (100Gy γ-ray irradiation) in 10mL medium, add hrIL-12, hrIL-15, hrIL-18 to make the final concentrations of 10ng/mL, 50ng/mL and 50ng, respectively /mL;

On the 11th day, all cells were collected and centrifuged at 300 × g for 10 min, resuspended in 20 mL of fresh NK cell culture medium, and hrIL-2 was added to make the final concentration 100 IU/mL;

From the 12th day to the 16th day, according to the cell growth, add an appropriate amount of fresh NK cell culture medium containing hrIL-2;

Example 8 Effects of different cytokine compositions on the expansion fold and cell phenotype of CAR-NK cells

This example compares the effect of adding cytokine compositions (IL-12, IL-15, IL-18) on the growth of CAR-NK cells during the second co-culture with K562-NK1 on the 10th day of CAR-NK preparation. Impact.

CAR-NK group, that is, when CAR-NK was co-cultured with K562-NK1 for the second time on the 10th day of preparation, only hrIL-2 was added (Example 3);

In the eCAR-NK group, that is, the second co-culture with K562-NK1 on the 10th day of CAR-NK preparation, cytokine compositions IL-12, IL-15, and IL-18 were added (Example 7).

The above two methods were used to prepare CAR-NK cells from the PBMC of the same normal peripheral blood donor, and the obtained NK cell purity (CD3-CD56+) was compared. The purity of NK cells in NK cells was 79%, 81%, 90%, and 93% on the 10th, 17th, 24th, and 31st days of culture, respectively, while the purity of NK cells in the eCAR-NK cells prepared in Example 4 was on the 1st day of culture. At 10, 17, 24, and 31 days, they were 79%, 87%, 93%, and 93%, respectively. This indicates that the addition of cytokines IL-12, IL-15 and IL-18 can slightly increase the proportion of NK cells during the second co-culture with K562-NK1 on the 10th day of CAR-NK preparation.

Further, the ratio of NK cells of CAR+ in the obtained cells was compared. The results are shown in Figure 11B. The ratio of NK cells of CAR+ in the CAR-NK cells prepared in Example 3 was on the 10th, 17th, 24th, and 31st days of culture, respectively. were 34%, 47%, 49%, and 74%, while the proportion of CAR+ NK cells in the eCAR-NK cells prepared in Example 7 was 34%, 57%, and 58% on the 10th, 17th, 24th, and 31st days of culture, respectively. , 85%. This indicated that the addition of cytokine compositions IL-12, IL-15 and IL-18 could significantly increase the expression ratio of CAR in NK cells during the second co-culture with K562-NK1 on the 10th day of CAR-NK preparation.

In this example, the total cell expansion folds obtained in the preparation process of CAR-NK and eCAR-NK cells are compared. The results are shown in Figure 12. The total expansion fold of the CAR-NK cells prepared in Example 3 is in 0-10 days, 10-17 days, 17-24 days and 24-31 days were 27, 21, 9, and 8 times respectively; while the eCAR-NK cells prepared in Example 7 had a total expansion fold at the 0- 10 days, 10-17 days, 17-24 days and 24-31 days were 27, 27, 9, and 7 times, respectively. This indicated that the addition of cytokine compositions IL-12, IL-15 and IL-18 could increase the expansion fold of NK cells during the second co-culture with K562-NK1 on the 10th day of CAR-NK preparation.

Comprehensively comparing the data of the above three aspects, the method described in Example 7, that is, adding the cytokine compositions IL-12, IL-15, IL- 18. Contribute to the expansion of CAR+ NK cells.

Example 9 The effect of adding cytokine composition on the 10th day of CAR-NK preparation on the killing of human ovarian cancer cell line SKOV3 in vitro (RTCA)

This example compares the effect of adding cytokine compositions (IL-12, IL-15, IL-18) on the in vitro effects of CAR-NK on the 10th day of NKG2D CAR-NK preparation, when it was co-cultured with K562-NK1 for the second time. impact on lethality.

At the 0th hour of the RTCA experiment, SKOV3 cells were plated in a 16-well electrode plate (ACEA Bio Company), 5000 cells/well; about 24 hours later, the NKG2D CAR-NK cells, NKG2D eCAR-NK cells and NKG2D eCAR-NK cells were cultured on the 17th day. Cells and NK cells (effector cells) were seeded in the electrode plate at the ratio of effector cells:target cells=1:1 or 2:1 respectively, with a total volume of 200 μL, and 2 duplicate wells were set for each group of experiments; NK cells were used in the example 3, but without electroporation; the cytotoxic effect of NKG2D CAR-NK cells on SKOV3 cells in vitro was detected by xCELLigence RTCA.

As shown in Figure 13A and Figure 13B, without adding effector cells (shown as "SKOV3" in the figure), tumor cells continued to grow for 70 hours; when effector cells:tumor cells=1:1, NK cells were added (Figure 13B). (shown as "SKOV3:NK 1:1" in the figure) or CAR-NK cells (shown as "SKOV3:CAR-NK 1:1" in the figure), the growth of tumor cells can be significantly inhibited, in which the killing of CAR-NK cells The tumor effect was significantly better than that of NK cells, which indicated the effect of CAR in NKG2D CAR-NK cells; while adding eCAR-NK cells (shown as "SKOV3:eCAR-NK 1:1" in the figure) or CAR-NK cells (Fig. shown as "SKOV3:CAR-NK 1:2"), the tumor cells were almost completely killed.

The data 24h after adding effector cells (indicated by the vertical dotted line in Figure 8A) were analyzed, and the tumor growth inhibition rate was calculated according to the following formula:

100%×(SKOV3 cell index-experimental group cell index)/SKOV3 cell index

It can be found that adding the eCAR-NK cell group (shown as "SKOV3:eCAR-NK 1:1" in the figure) and adding the CAR-NK cell group (shown as "SKOV3:CAR-NK 1:2" in the figure), the tumor The inhibition rates were all around 80%, which indicated that the addition of cytokine compositions (IL-12, IL-15, IL-18) during the second co-culture with K562-NK1 on the 10th day of NKG2D CAR-NK preparation could Significantly improved the growth inhibitory effect of CAR-NK cells on tumor cells, which may be due to the increase in the proportion of NK cells with CAR+ in NK cells and the enhancement of NK cells by cytokine compositions (IL-12, IL-15, IL-18). its own killing effect.

Example 10 Preparation of high-purity NKG2D eCAR-NK using the PiggyBac transposon system

The specific steps include: on the 0th day, 2×10 ⁷ PBMC cells were taken, and CD3+ cells were removed with CD3 magnetic beads (Miltenyi) according to the manufacturer’s recommended procedure; 5×10 ⁶ sorted PBMCs and 5×10 ^{6 cells were removed} K562-NK1 (100Gy γ-ray irradiation) was resuspended in 10mL of NK cell culture medium, inoculated into a T25 cell culture flask, and hrIL-2 was added to make the final concentration 50IU/mL;

μg plasmid

2 and 10 μg Plasmid 4, mix well and transfer to the Lonza electroporation cup; place the electroporation cup in the Lonza 4D-NucleofectorTM X Unit (in a single electroporation cup module) and perform electroporation. After the electroporation, slowly transfer the NK cell suspension in one electroporation cup To a new T25 cell culture flask, add 10 mL of NK cell culture medium containing hrIL-2 with a final concentration of 50IU/mL, mix gently, and place the T25 cell culture flask in a 37°C cell incubator for cultivation;

On the 10th day, all cells were collected and counted, and 2×10 ⁶ cells were taken for flow cytometric phenotype analysis (anti-human CD3 antibody, anti-human CD56 antibody, anti-Strep-tag II antibody); 1×10 ⁷ NKG2D was taken out CAR-NK cells were resuspended in 80 μL of DPBS+1% AB serum buffer, added with 20 μL of anti-Strep tag II antibody, mixed and incubated at 2-8°C for 20 min; after incubation, 5 mL of buffer was added and centrifuged at 300g 10min, discard the supernatant and resuspend in 80μL DPBS+1% AB serum buffer, add 20μL anti-Biotin magnetic beads, mix well, incubate at 2-8°C for 15min; add 5mL buffer after incubation, centrifuge at 300g for 10min , discard the supernatant and resuspend in 500 μL of buffer, and then follow the steps recommended by the Miltenyi manufacturer to sort the positive cells labeled with magnetic beads. Take 2×10 ⁶ sorted magnetic bead-labeled CAR-NK cells and 4×10 ⁶ K562-NK1 (100Gy γ-ray irradiated) and resuspend in 20 mL of medium, and add hrIL-2 to make the final concentration 50IU/ mL.

From the 11th day to the 16th day, according to the cell growth, add an appropriate amount of fresh NK cell culture medium containing hrIL-2;

On the 17th day, all cells were collected and counted, and 2×10 ⁶ cells were taken for flow cytometric phenotype analysis (anti-human CD3 antibody, anti-human CD56 antibody, anti-Strep-tag II antibody); 2×10 ⁶ cells were Resuspend with 2×10 ⁶ K562-NK1 (100Gy γ-ray irradiation) in 10mL medium, add hrIL-12, hrIL-15, hrIL-18 to make the final concentrations of 10ng/mL, 50ng/mL and 50ng, respectively /mL;

On the 18th day, all cells were collected, centrifuged at 300 × g for 10 min, resuspended in 20 mL of fresh NK cell culture medium, and hrIL-2 was added to make the final concentration 50 IU/mL;

From the 18th day to the 23rd day, according to the cell growth, add an appropriate amount of fresh NK cell culture medium containing hrIL-2;

On the 24th day, all cells were collected and counted, and 2×10 ⁶ cells were taken for flow cytometric phenotype analysis (anti-human CD3 antibody, anti-human CD56 antibody, and anti-Strep-tag II antibody).

Example 11 CAR+ cell sorting can significantly increase the proportion of CAR+ NK cells

In this example, the sorting of CAR+ cells and the effect of different sorting methods on the preparation of CAR-NK cells are compared.

The NKG2D CAR-NK cells on the 10th day after the first round of co-culture were taken and divided into three groups: in the first group, 2 × 10 ⁶ cells were co-cultured with 2 × 10 ⁶ K562-NK1 (100Gy γ-ray irradiation) cells1 Week, named as the unsorted group; in the second group, 1×10 ⁷ cells were sorted according to the method described in Example 10. After sorting, the remaining cells were selected as NK cells:artificial antigen-presenting cells=1:2 and K562 -NK1 (100Gy γ-ray irradiation) was co-cultured for 1 week, named Biotin Anti-Strep tag II Ab+anti-Biotin microbeads group; 1×10 ⁷ cells were taken from the third group and resuspended in 90 μL DPBS+1% AB serum Add 10 μL of Streptavidin microbeads to the buffer, mix well and incubate at 2-8 °C for 20 min; after incubation, add 5 mL of buffer, centrifuge at 300 × g for 10 min, discard the supernatant and resuspend in 500 μL of buffer, and then proceed with magnetic beads The labeled positive cells were sorted, and the remaining cells were taken after sorting and co-cultured with K562-NK1 (100Gy γ-ray irradiation) for 1 week according to NK cells:artificial antigen-presenting cells=1:2, named Streptavidin microbeads group.

The results are shown in Table 1 below. On the 17th day after the end of the second round of co-culture in the unsorted group, the proportion of CAR+ cells in the NK cells was 33.1%, and the expansion fold of the second round was 10.2 times; Biotin Anti-Strep tag II Ab+anti -Biotin cell yield of 66% after the component is selected from microbeads (100% × after sorting the resulting cell ^{/ (1 × 10 7 × CAR} +%)), the end of the second round of co-culture on day 17, NK cells are CAR + cells The ratio was 95.3%, and the expansion fold in the second round was 44.64 times; the cell yield of the Streptavidin microbeads group was 10.6%, and on the 17th day after the second round of co-culture, the proportion of CAR+ cells in the NK cells was 97.8%. The round amplification factor was 20.65 times.

Table 1

According to the above results, the sorting yield of Biotin Anti-Strep tag II Ab+anti-Biotin microbeads is higher, and the proportion of CAR+ cells in NK cells after one round of expansion can be increased to more than 90%.

Example 12 Adding secreted or membrane-bound human IL-15 to the CAR carrier can promote the survival of CAR-NK cells in vitro

In this example, the gene of human IL-15 was added to the transposon donor containing NKG2D CAR, plasmid 5 was constructed, and NK, NKG2D CAR-NK and NKG2D eCAR-NK that could secrete IL-15 were prepared from PBMC. NK (NKG2D eCAR-IL15 NK).

The preparation of NKG2D CAR-NK refers to Example 3; the preparation of NKG2D eCAR-IL15 NK refers to Example 7, and plasmid 4 is replaced with plasmid 5. Since the CAR vector contains IL-15, the cytokine composition on the 17th day IL-15 was removed. ^{Take 2 × 10 6} cells in each group on the 22nd day after the third round of co-culture, and count them after 1 week of in vitro culture with and without addition of hrIL-2 (50 IU/mL), and calculate the percentage change of cells ( 100%×the number of cells after 1 week of culture/2×10 ⁶ ).

As shown in Figure 14A, the percentage of cell change in NK and NKG2D CAR-NK was 3% and 7% without the addition of IL-2; after the addition of IL-15, the percentage of cell change in CAR-NK increased to 37% ; After the addition of IL-2, cell survival was improved in all groups.

In this example, NK, NKG2D eCAR-IL15 NK and NKG2D eCAR-IL15 NK (sorted) were prepared from umbilical cord blood, respectively. Among them, for the preparation of NKG2D eCAR-IL15 NK, refer to Example 7, and replace plasmid 4 with plasmid 5. Since the CAR vector contains IL-15, IL-15 was removed from the cytokine composition on day 17; NKG2D eCAR-IL15 NK (sorted) Preparation Refer to Example 10, replace plasmid 4 with plasmid 5, since IL-15 is contained in the CAR vector, IL-15 is removed from the cytokine composition on day 17.

^{Take 5 × 10 5} cells in each group on the 21st day after the third round of co-culture, and count them after 1 week of in vitro culture with and without hrIL-2 (50 IU/mL), and calculate the percentage change of cells ( 100%×the number of cells after 1 week of culture/5×10 ⁵ ).

As shown in Figure 14B, in the absence of IL-2 addition, the percentage of cell change in NK, NKG2D eCAR-IL15 NK, and NKG2D eCAR-IL15 NK(sorted) were 25%, 47%, and 316%, respectively, with IL- 15 and sorted high-purity CAR-NK cells even exhibited proliferation. With the addition of IL-2, cell survival was improved in all groups.

In this example, we also tried to add the membrane-bound human IL-15 gene to the transposon donor containing NKG2D CAR, constructed plasmid 6, and prepared NK, NKG2D eCAR-NK(sorted) and NKG2D respectively from umbilical cord blood eCAR-mbIL15 NK(sorted). The preparations of NKG2D eCAR-NK (sorted) and NKG2D eCAR-mbIL15 NK (sorted) refer to Example 10. Plasmid 4 was replaced with plasmid 6 in the preparation of NKG2D eCAR-mbIL15 NK (sorted), since the CAR vector contained mbIL-15, IL-15 was removed from the cytokine composition on day 17.

^{Take 2×10 6} cells in each group on the 21st day after the third round of co-culture, culture them in vitro for 1 week without adding hrIL-2 (50 IU/mL), and count the percentage of cell change (100%× The number of cells/2×10 ⁶ after culturing for 1 week).

As shown in Figure 14C, in the absence of IL-2 addition, the percentage of cell change in NK, NKG2D eCAR-NK(sorted), NKG2D eCAR-mbIL15 NK(sorted) were 6%, 13%, and 140%, respectively, and the membrane Binding of IL-15 (mbIL-15) can also promote the survival of CAR-NK in vitro.

In this example, NK, NKG2D CAR-mbIL15 NK and NKG2D eCAR-mbIL15 NK (sorted) were prepared from PBMCs, respectively. The preparation of NKG2D CAR-mbIL15 NK refers to Example 3, and plasmid 4 is replaced with plasmid 6. Since the CAR vector contains mbIL-15, IL-15 is removed from the cytokine composition on day 17; NKG2D eCAR-mbIL15 NK (sorted ) Preparation of ) Refer to Example 10, replace plasmid 4 with plasmid 6, since mbIL-15 is contained in the CAR vector, IL-15 is removed from the cytokine composition on day 17.

^{Take 2×10 6} cells in each group on the 16th day after the second round of co-culture, and count them after 1 week of in vitro culture without adding hrIL-2 (50 IU/mL), and calculate the percentage change of cells (100%× The number of cells/2×10 ⁶ after culturing for 1 week).

As shown in Figure 14D, in the absence of IL-2, the percentage of cell change in NK, NKG2D CAR-mbIL15 NK, NKG2D eCAR-mbIL15 NK(sorted) was 27%, 62%, and 114%, respectively, and membrane-bound IL -15 (mbIL-15) can also promote the survival of CAR-NK in vitro, and the sorted high-purity CAR-NK containing mbIL-15 even showed a small amount of proliferation in vitro.

Example 13 High-purity NKG2D CAR-mbIL15 NK after sorting showed strong killing effect on tumor cell lines in vitro

In this example, NK, NKG2D eCAR-mIL15 NK and NKG2D eCAR-mbIL15 NK (sorted) were prepared respectively with PBMC as the source, wherein the preparation of NKG2D eCAR-mbIL15 NK refers to Example 7, and plasmid 4 was replaced with plasmid 6, because CAR The vector contains mbIL-15, and IL-15 is removed from the cytokine composition on day 17; the preparation of NKG2D eCAR-mbIL15 NK (sorted) refers to Example 10, and plasmid 4 is replaced with plasmid 6, because the CAR vector contains mbIL -15, Depletion of IL-15 in the cytokine composition on day 17.

The cells on the 16th day after the second round of co-culture were taken for in vitro killing test. The human AML cell line KG1 cells ^{were taken and resuspended in DPBS at 1×10 6} cells/ml, and Calcein-AM (final concentration 0.2 μM) was added for staining at 37° C. for 15 min. After staining, FBS was added in an equal volume to stop the staining, and washed three times with DPBS. The stained KG1 was resuspended in the medium, the cell density was adjusted to 2×10 ⁵ cells/ml, and KG1 was plated in 100 μL per well in a U-shaped 96-well plate. Effector cells were seeded in 96-well plates according to E:T=2:1, 1:1 and 0.5:1, 100 μL per well, and the total volume per well was 200 μL. After overnight incubation at 37°C, the number of GFP+ cells per well was determined by flow cytometry the next day. The tumor growth inhibition rate was calculated according to the following formula:

100%×(the number of GFP+ cells in the single KG1 cell group - the number of GFP+ cells in the experimental group)/the number of GFP+ cells in the single KG1 cell group

As shown in Figure 15A, compared with NK, both NKG2D eCAR-mIL15 NK and NKG2D eCAR-mbIL15 NK(sorted) showed significant tumor killing effect on KG1, and NKG2D eCAR-mbIL15 NK(sorted) was slightly stronger on KG1 .

In this example, the killing effect of NKG2D eCAR-mbIL15 NK (sorted) on human colorectal cancer cell line HCT116 in vitro was also detected by RTCA real-time killing detector (refer to Example 5 for specific experimental information);

As shown in Figure 15B, when E:T=0.5:1, NK could only slow down the growth of tumor, and NKG2D eCAR-mbIL15 NK(sorted) showed a very significant killing effect on HCT116.

Example 14 Preparation of engineered K562-NK2 cells

The K562-NK1 cells were resuspended in 10 mL Opti-MEM, centrifuged at 300 × g for 10 min, the cell pellet was resuspended in 100 μL P3 buffer, 5

μg plasmid

2 and 10 μg plasmid 9 were added, and the cells were transferred to the Lonza electric shock cup; The cups were placed in a Lonza 4D-NucleofectorTM X Unit (in a single shock cup module) for electroporation.

After the electroporation, the K562 cell suspension in an electroporation cup was slowly transferred to a well of a 6-well plate, and K562 medium (IMDM+10% FBS) was pre-added to the well; on the 7th day after electroporation, the cells were used The sorting instrument BD Fusion (BD Biosciences) sorts single cells into 96-well plates, and analyzes the amplified single cell clones by flow cytometry to detect the expression of BCMA. The detection antibody is APC-conjugated anti-human BMCA. Antibody, the screened single cell clone was named K562-NK2.

As shown in Figure 16, K562-NK2 cells expressed high levels of BCMA on the surface, with a positive rate of 99.4%.

Example 15 Preparation of BCMA eCAR-NK using piggyBac transposon system

On

day

0, 2 × 10 ⁷ PBMC cells were taken, and CD3+ cells were removed using CD3 magnetic beads (Miltenyi) according to the manufacturer's recommended procedure, and about 1 × 10 ⁷ PBMCs remained; 5 × 10 ⁶ sorted PBMCs were removed and 5×10 ⁶ K562-NK2 (100Gy γ-ray irradiation) were resuspended in 10mL NK cell culture medium, inoculated into T25 cell culture flask, and hrIL-2 was added to make the final concentration 50IU/mL;

μg plasmid

2 and 10 μg Plasmid 8, after mixing, transfer to the electroporation Lonza electroporation cup; place the electroporation cup in the Lonza 4D-NucleofectorTM X Unit (in a single electroporation cup module), and perform electroporation. After the electroporation, slowly transfer the NK cell suspension in one electroporation cup Transfer to a new T25 cell culture flask, add 10 mL of NK cell culture medium containing hrIL-2 with a final concentration of 50IU/mL, mix gently, and place the T25 cell culture flask in a 37°C cell incubator for cultivation;

On the 10th day, all cells were collected and counted, and 2×10 ⁶ cells were taken for flow cytometric phenotype analysis (anti-human CD3 antibody, anti-human CD56 antibody, anti-Strep-tag II antibody); 1×10 ⁷ BCMA was taken out CAR-NK cells were resuspended in 80 μL DPBS+1% AB serum buffer, 20 μL anti-Strep tag II antibody was added, mixed and incubated at 2-8 °C for 20 min; after incubation, 5 mL buffer was added, 300× Centrifuge at g for 10 min, discard the supernatant and resuspend in 80 μL DPBS+1% AB serum buffer, add 20 μL anti-Biotin magnetic beads, mix well and incubate at 2-8 °C for 15 min; after the incubation, add 5 mL buffer, 300 After centrifugation at ×g for 10 min, the supernatant was discarded and resuspended in 500 μL buffer to sort the positive cells labeled with magnetic beads according to the procedure recommended by the manufacturer (Miltenyi). Take 2 × 10 ⁶ magnetic beads-labeled CAR-NK cells and 4 × 10 ⁶ K562-NK2 (100Gy γ-ray irradiated) and resuspend them in 10 mL of medium after sorting, and add hrIL-2 to make the final concentration 50IU/mL .

On the 17th day, all cells were collected and counted, and 2×10 ⁶ cells were taken for flow cytometric phenotype analysis (anti-human CD3 antibody, anti-human CD56 antibody, anti-Strep-tag II antibody); 2×10 ⁶ cells were Resuspend in 10 mL medium with 2×10 ⁶ K562-NK2 (100 Gy γ-irradiated), and add hrIL-12, hrIL-15 and hrIL-18 to make the final concentrations 10ng/mL, 50ng/mL and 50ng, respectively /mL;

As shown in Figure 17A, after the first round of co-culture, the proportion of CD3-CD56+ NK cells reached about 90%, and after the next few rounds of co-culture with K562-NK2 (100Gy γ-ray irradiation), the proportion of NK cells remained basically unchanged. . As shown in Figure 17B, the first round of co-culture ended, the proportion of CAR+ cells was 16%; the second round of co-culture ended, the proportion of CAR+ cells was 57.6%; the third round of co-culture ended, the proportion of CAR+ cells was 72.6% . As shown in Figure 17C, the expansion fold of the first round of co-culture was 13 times, the second round of co-culture after sorting was 60 times, and the third round of co-culture was 46.5 times.

Figure 18 shows the expansion fold of the total number of cells in each round at the end of the first, second, and third rounds of co-culture during the preparation of BCMA eCAR-NK from a healthy donor-derived PBMC, and the expansion of the first round of co-culture. The multiplication factor was 13 times, the second round of co-culture was 60 times, and the third round of co-culture was 46 times.

In this example, the killing of human MM cell line U266 in vitro by BCMA eCAR-NK cells on the 22nd day after the third round of co-culture was also detected.

U266 cells were taken and resuspended in DPBS at 1×10 ⁶ cells/mL, and Calcein-AM (final concentration 0.2 μM) was added for staining at 37° C. for 15 min. After staining, FBS was added in an equal volume to stop the staining, and washed three times with DPBS. The stained U266 cells were resuspended in the medium, the cell density was adjusted to 2×10 ⁵ cells/mL, and 100 μL per well of U266 was plated in a U-shaped 96-well plate. Effector cells were seeded in 96-well plates according to E:T=5:1, 2.5:1 and 1.25:1, 100 μL per well, and the total volume per well was 200 μL. After overnight incubation at 37°C, the number of GFP+ cells per well was detected by flow cytometry the next day. Tumor growth inhibition rate was calculated according to the following formula:

100%×(the number of GFP+ cells in the single U266 cell group - the number of GFP+ cells in the experimental group)/the number of GFP+ cells in the single U266 cell group

As shown in Figure 19, compared to NK, BCMA eCAR-NK (sorted) exhibited significant tumor killing effect on U266.

To sum up, the present application adopts a non-viral method and uses a transposon system to introduce a stable and highly expressed transgene into NK cells to achieve efficient genetic modification of NK cells. The production cycle is short and the production cost is low. The prepared transgenic modification NK cells have high purity and good safety, and have significant killing and/or inhibitory effects on tumor cells.

The applicant declares that the present application illustrates the detailed method of the present application through the above-mentioned embodiments, but the present application is not limited to the above-mentioned detailed method, which does not mean that the present application must rely on the above-mentioned detailed method for implementation. Those skilled in the art should understand that any improvement to the application, the equivalent replacement of each raw material of the product of the application, the addition of auxiliary components, the selection of specific methods, etc., all fall within the scope of protection and disclosure of the application.

List of gene sequences covered in this paper

Claims

A method for preparing natural killer cells modified by chimeric receptors using a non-viral transposon system and artificial antigen-presenting cells, comprising:

(1) using the transposon system to transfer the encoding gene of the chimeric receptor into natural killer cells to obtain the original natural killer cells modified by the chimeric receptor;

(2) Using artificial antigen-presenting cells to expand the original chimeric receptor-modified natural killer cells.
The method of claim 1, wherein the natural killer cells comprise human primary natural killer cells;

The primary human natural killer cells are derived from peripheral blood, umbilical cord blood, placental tissue or induced pluripotent stem cells.
The method of claim 1, wherein the chimeric receptor comprises a chimeric antigen receptor and/or a chimeric switch receptor.
The method according to claim 1, wherein the transposon system of step (1) comprises a PiggyBac transposon system, a Sleeping Beauty transposon system or a Tol2 transposon system.
The method according to claim 1, wherein the transposon system in step (1) comprises a transposon element and a transposase element.
The method of claim 5, wherein the transposon element comprises a transposon 5' inverted terminal repeat and a 3' inverted terminal repeat;

There are two insulators between the 5' inverted terminal repeat and the 3' inverted terminal repeat;

A gene encoding a promoter and a chimeric receptor is contained between the two insulators;

The transposon element is a plasmid or a linearized nucleic acid fragment.
The method of claim 5, wherein the transposase element comprises a transposase protein or a nucleic acid molecule encoding a transposase;

The nucleic acid molecules encoding transposases include plasmids, linearized nucleic acid fragments, or mRNA.
The method of claim 5, wherein the transposon element and the transposase element are linked to the same expression vector.
6. The method of claim 5, wherein the transposon element and the transposase element are on different expression vectors.
The method of claim 6, wherein the transposon element further comprises a gene encoding a cytokine that promotes NK cell survival, the cytokine comprising IL-15 or IL-21;

The cytokines include secreted, membrane-bound or a combination of cytokine-linked receptors;

The encoding gene of the cytokine and the encoding gene of the chimeric receptor are in the same transposon expression vector or in different transposon expression vectors.
The method according to claim 1, wherein the transposon system in step (1) is transferred into natural killer cells by electroporation and/or chemical reagents.
The method according to claim 1, further comprising a step of activating natural killer cells before step (1).
The method according to claim 12, wherein the transposon system is transferred into natural killer cells after 0-14 days of activation of the natural killer cells.
The method of claim 12, wherein the activation of natural killer cells is performed using artificial antigen presenting cells, feeder cells, cytokines, antibodies or compounds.
The method according to claim 1 or 14, wherein the artificial antigen-presenting cells comprise cell-based artificial antigen-presenting cells, artificially synthesized artificial antigen-presenting cells or exosome-based artificial antigen-presenting cells.
The method of claim 15, wherein the cell-based artificial antigen presenting cells comprise human myeloid leukemia K562 cells, human Burkitt lymphoma Daudi cells, EBV transformed B lymphoblastoid cells or small cells The mouse embryonic fibroblast cell line NIH/3T3 cells.
The method of claim 16, wherein the cell-based artificial antigen presenting cell is an engineered cell;

the engineered cells express an antigen recognized by the chimeric receptor;

The engineered cells express ligand molecules and/or cytokines for activating natural killer cells;

The ligand molecule includes CD137L and/or OX40L;

The cytokine comprises any one or a combination of at least two of human IL-21, human IL-15 or human IL-18;

The cytokines include secreted cytokines and/or membrane-bound cytokines;

The engineered cells were treated with gamma rays or mitomycin C.
The method according to claim 1, wherein the initial chimeric receptor-modified natural killer cells are expanded by using artificial antigen-presenting cells 0 to 14 days after the gene encoding the chimeric receptor is transferred into the natural killer cells.
The method according to claim 1, wherein the amplification is performed multiple times, and each round of amplification cycle is 3-14 days.
The method according to claim 1, wherein step (1) further comprises a step of magnetic sorting to enrich NK cells or remove CD3+ cells.
The method according to claim 20, wherein the step of magnetic sorting is before the transfer of the chimeric receptor-encoding gene into natural killer cells and/or before the initial chimeric receptor-modified natural killer cells The culture was carried out after 0 to 14 days.
The method according to claim 1, wherein after step (1), the operation of CAR+ cell sorting is further included.
The method according to claim 1, wherein after step (2), it further comprises the step of enriching NK cells or removing CD3+ cells by magnetic sorting.
The method according to claim 1, wherein after step (2), the operation of sorting CAR+ cells is further included.
The method according to claim 1, wherein, during the preparation of the chimeric receptor-modified natural killer cells, a cytokine composition is added to the culture medium, and the cytokine composition is hrIL-12 and hrIL- 18, or a combination of hrIL-12, hrIL-15 and hrIL-18.
Chimeric receptor-modified natural killer cells, which are prepared by the method described in any one of claims 1-25.
The application of the chimeric receptor-modified natural killer cell according to claim 26 in the preparation of a tumor therapeutic drug and/or a tumor preventive drug.