Method for differentiating chimeric antigen receptor pluripotent stem cells into immune cells and application

Abstract

The invention relates to a method for differentiating chimeric antigen receptor pluripotent stem cells into immune cells and application. The CAR-hPSCs with anti-FITC scFv are obtained by means of a CRISPR / Cas9 technology, then the CAR-hPSCs are differentiated into CAR-PMNs or CAR-NK by clearly combining chemical components with morphogenetic factors in a specific stage, various immune cells with excellent performance are obtained more simply, more accurately and more reliably, the production cycle can be shortened, the yield of target products is increased, the production cost is effectively reduced, and the immunotherapy effect is improved. The CAR-PMNs or CAR-NK can specifically recognize tumor cells highly expressed by folic acid receptors in a targeted manner through the bridging effect of the FITC-folic acid dual aptamers, the tumor cells are effectively killed, meanwhile, off-target toxic and side effects on normal tissues are avoided, and the effect of improving the treatment accuracy and the treatment effect of immune cells is achieved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biomedical engineering, in particular to a method for differentiating chimeric antigen receptor pluripotent stem cells into immune cells and application thereof. BACKGROUND

[0002] Immune therapy based on adoptive immune cells has emerged as a powerful and potentially curative approach in treating various cancer types. Despite showing benefits in clinical and preclinical studies, especially for hematological malignancies, its effectiveness in treating solid tumors is still limited due to the immunosuppressive nature of the tumor microenvironment (TME). To address this challenge, immune modulators such as cytokines, initiators, and checkpoint inhibitors are often combined with adoptive immune cells in the immunotherapy of solid tumors. However, these agents have significant limitations. For example, repeated administration of cytokines can trigger serious toxic reactions such as cytokine release syndrome and vascular leakage syndrome. In addition, due to the short duration of action of most agents, frequent and long-term use of cytokines and checkpoint inhibitors can lead to immune cell exhaustion.

[0003] One promising strategy to improve therapeutic efficacy is to use a combination of multiple immune cell types (i.e., immune cell "cocktails") to address the limitations of immune modulators. Each immune cell type has unique anti-tumor functions, and their combination can enhance the elimination of tumor cells through complementary mechanisms. This approach takes advantage of the unique advantages of various immune cells, promotes stronger anti-tumor responses in the TME by addressing issues such as immune suppression, drug resistance, and limited efficacy of monotherapy. It is particularly worth mentioning that these diverse immune cells can release a broad spectrum of cytokines and chemokines, enhancing therapeutic efficacy without the need for high doses of exogenous cytokines, which are often associated with side effects.

[0004] However, major challenges in implementing immune cell cocktail therapy include the following three points: 1. Difficulty in obtaining sufficient amounts of multiple immune cells from the same donor; 2. Complex genetic engineering requirements specific to each immune cell type; 3. Risk of immune imbalance or drug resistance due to accidental inhibition or excessive stimulation. SUMMARY

[0005] The present application aims to disclose a method for differentiating chimeric antigen receptor pluripotent stem cells into immune cells and application thereof, to solve one or more technical problems existing in the prior art, and to provide at least one beneficial option or create conditions.

[0006] To achieve the above-mentioned purpose, the present application is realized by the following technical solutions: The first aspect of the present application provides an expression gene construct of a chimeric antigen receptor (CAR) targeting fluorescein isothiocyanate (FITC).

[0007] The second aspect of the present application provides a chimeric antigen receptor pluripotent stem cell (CAR-hPSC) and a method for constructing the same.

[0008] The third aspect of the present application provides a method for preparing a chimeric antigen receptor polymorphonuclear cell population (CAR-PMN) from the CAR-hPSCs of the second aspect of the present application.

[0009] The fourth aspect of the present application provides a method for preparing a chimeric antigen receptor natural killer cell (CAR-NK) from the CAR-hPSCs of the second aspect of the present application.

[0010] The CAR expression gene construct of the first aspect of the present application comprises a signal peptide CD8a-SP, an extracellular anti-FITC single chain variable fragment (anti-FITC scFv), an extracellular hinge transmembrane domain of CD8a (CD8a-hg-tm), a costimulatory domain 4-1BB, and an intracellular signal transduction domain CD3ζ.

[0011] In some embodiments of the first aspect of the present application, the nucleotide sequence of the CAR is shown in SEQ ID No: 1.

[0012] The anti-FITC scFv is designed to enable the CAR to specifically target and recognize FITC.

[0013] The method for constructing the CAR-hPSCs of the second aspect of the present application comprises the following steps: (1) introducing the expression gene construct of the CAR into an AAVS1 safe harbor site in human pluripotent stem cells (hPSCs) via CRISPR / Cas9-mediated homologous recombination and an adeno-associated virus S1 (AAVS1) plasmid; (2) isolating successfully targeted single cell-derived human pluripotent stem cell colonies or human pluripotent stem cell mixtures to obtain a stable human pluripotent stem cell line expressing the CAR; (3) extracting the CAR-hPSCs from the cell line.

[0014] In some embodiments of the second aspect of the present application, the hPSCs comprise human embryonic stem cells (hESCs) and / or induced pluripotent stem cells (iPSCs).

[0015] In some embodiments of the second aspect of the present invention, the hESCs are selected from H1, H7, H9, H13, or H14. These hESCs possess a normal, stable diploid karyotype, with H1, H13, and H14 possessing a normal XY karyotype, and H7 and H9 possessing a normal XX karyotype. The H1, H7, H13, and H14 cell lines maintain a normal karyotype after repeated freezing and thawing for 5-6 months.

[0016] The method for preparing CAR-PMNs according to the third aspect of the present invention comprises the following steps: (a) stimulating the CAR-hPSCs according to the second aspect of the present invention using a culture medium containing a glycogen synthase kinase 3β (GSK3β) inhibitor; (b) Replace the culture medium with vascular endothelial growth factor (VEGF) to achieve hemogenic endothelial cell differentiation; (c) Replace the culture medium with a transforming growth factor β (TGFβ) inhibitor, stem cell factor (SCF), FMS-like tyrosine kinase 3 ligand (FLT3L), and / or granulocyte macrophage colony-stimulating factor (GM-CSF) to achieve hematopoietic progenitor cell commitment; (d) The culture medium containing GM-CSF and retinoic acid receptor agonist was replaced to obtain the CAR-PMNs.

[0017] In some embodiments of the third aspect of the present invention, the GSK3β inhibitor is selected from CHIR99021 or CHIR98014.

[0018] In some embodiments of the third aspect of the present invention, the TGFβ inhibitor is selected from SB431542 or A83-01.

[0019] In some embodiments of the third aspect of the present invention, the retinoic acid receptor agonist is selected from AM80 or AM50.

[0020] In some embodiments of the third aspect of the present invention, a pretreatment step is further included: dissociating the CAR-hPSCs using ethylenediaminetetraacetic acid (EDTA), seeding the cells at an adjusted cell density into a 24-well plate coated with iMatrix 511, and culturing them in mTeSR medium containing Y27632 for 24 hours. Preferably, mTeSR medium can be replaced with mTeSR Plus medium.

[0021] In some embodiments of the third aspect of the present invention, the culture medium in step (a) is DMEM culture medium; preferably, it further contains ascorbic acid (Vc).

[0022] In some embodiments of the third aspect of the present invention, the culture medium in step (b) is LasR basal medium.

[0023] In some embodiments of the third aspect of the application, the medium of step (c) and / or step (d) is Stemline II; preferably, the Stemline II medium of step (d) further comprises 1x GlutaMAX.

[0024] The method for preparing the CAR-NK of the fourth aspect of the application comprises the following steps: (I) stimulating the CAR-hPSCs of the second aspect of the application using a medium containing a GSK3β inhibitor; (II) replacing the medium containing VEGF to realize differentiation of hemogenic endothelial cells; (III) replacing the medium containing a TGFβ inhibitor, SCF and / or FLT3L to realize directional differentiation of hemogenic progenitor cells; (IV) replacing the medium containing interleukin-7 (IL-7), interleukin-15 (IL-15), SCF, FLT3L and a lineage reconstitution enhancer to obtain the CAR-NK by co-culturing with OP9-DLL4 monolayer cells.

[0025] In some embodiments of the fourth aspect of the application, the GSK3β inhibitor is selected from CHIR99021 or CHIR98014.

[0026] In some embodiments of the fourth aspect of the application, the TGFβ inhibitor is selected from SB431542 or A83-01.

[0027] In some embodiments of the fourth aspect of the application, the lineage reconstitution enhancer is selected from UM171.

[0028] In some embodiments of the fourth aspect of the application, the method further comprises a pretreatment step: dissociating the CAR-hPSCs using EDTA, inoculating the cells at an adjusted cell density into a 24-well plate coated with iMatrix 511, and culturing in a mTeSR medium containing Y27632 for 24 hours. Preferably, the mTeSR medium can be replaced by mTeSR Plus medium.

[0029] In some embodiments of the fourth aspect of the application, the medium of step (I) is a DMEM medium; preferably, the DMEM medium further comprises Vc.

[0030] In some embodiments of the fourth aspect of the application, the medium of step (II) is a LasR basal medium.

[0031] In some embodiments of the fourth aspect of the application, the medium of step (III) is a Stemline II medium.

[0032] In some embodiments of the fourth aspect of the application, the culture medium in step (IV) is α-MEM medium. Preferably, the medium further comprises 20% fetal bovine serum (FBS).

[0033] The present application has the following beneficial effects compared with the prior art: (1) The present application uses CRISPR / Cas9 technology to obtain CAR-hPSCs with anti-FITC scFv, and then differentiates into CAR-PMNs or CAR-NK by chemically combining specific morphogenesis factors at specific stages, which is simpler, more accurate and more reliable to obtain various immune cells with superior performance, can shorten the production cycle, increase the yield of target products, effectively reduce the production cost, and improve the immunotherapy effect.

[0034] (2) The CAR-PMNs or CAR-NK obtained by gene editing technology containing anti-FITC scFv can specifically target and recognize tumor cells with high expression of folate receptors through the bridging effect of FITC-folate bisaptamer, effectively killing tumor cells without off-target toxicity to normal tissues, and improving the treatment accuracy and efficacy of immune cells.

[0035] (3) The combination of CAR-PMNs and CAR-NK can be used for immune cell cocktail therapy, which breaks the limitations of single immune cell action and poor effect. BRIEF DESCRIPTION OF DRAWINGS

[0036] Figure 1 is a schematic diagram of the construction of the CAR described in Example 1; Figure 2 is an AAVS1 vector plasmid map in which the CAR described in Example 1 is inserted; Figure 3 is an electrophoresis map of the detection of CAR-hPSCs described in Example 1; Figure 4 is a schematic diagram of the differentiation of CAR-hPSCs into CAR-PMNs described in Example 2; Figure 5 is a bright field microscopic image at different differentiation time points in Example 2; Figure 6 is a flow cytometry analysis result diagram of the CAR expression at different differentiation time points in Example 2; Figure 7 is a schematic diagram of the differentiation of CAR-hPSCs into CAR-NK described in Example 3; Figure 8 is a fluorescence image of CAR-PMNs and tumor cells in Example 4; Figure 9 is a quantification analysis chart of CAR-PMNs and tumor cells in Example 4; Figure 10 is a cytotoxicity assay line chart in Example 4. DETAILED DESCRIPTION

[0037] The molecular biology test methods not specifically described in the following examples are all carried out according to the Molecular Cloning: A Laboratory Manual (3rd Edition) or according to the method and product instructions; the biological materials, if not specifically described, can be obtained from commercial channels.

[0038] Example 1: Construction of the CAR-hPSCs.

[0039] The CAR with the ability of specific target recognition of FITC was designed, and the schematic diagram of the target knock-in strategy at the AAVS1 safe site and construction is shown in Figure 1 The CAR structure comprises the following key elements: CD8a signal peptide (CD8a-SP), extracellular anti-isothiocyanate fluorescein single-chain variable fragment (anti-FITC scFv), extracellular hinge and transmembrane domain of CD8a (CD8a-hg-tm), costimulatory domain 4-1BB, and intracellular signal transduction domain CD3ζ.

[0040] Figure 2 An NK FITC CAR plasmid map shown as an exemplary embodiment is depicted.

[0041] The specific steps include: S1. The hPSCs are selected from H9 human embryonic stem cells (purchased from WiCell) and are cultured on Matrigel-coated culture plates added with mTeSR medium. In order to improve the survival rate of hPSCs, 10 μM of Y27632 is used for pretreatment of the cells 3-4 hours before nucleofection or during overnight. Then, the cells are treated with cell digestion solution for 8-10 minutes to achieve single cellization. 1-2.5 × 10 6 6 μg of SpCas9 AAVS1 gRNA T2 (Addgene; #79888) and 6 μg of CAR donor plasmid are mixed in 100 μL of human stem cell nucleofection solution (Lonza; #VAPH-5012), and nucleofection is performed by Nucleofector 2b equipment and using B-016 program.

[0042] S2. Cells after nucleofection were seeded into individual wells of a 6-well plate pre-coated with Matrigel and cultured in 3 mL of pre-warmed mTeSR plus or mTeSR1 medium containing 10 μM Y27632. After 24 hours, the medium was replaced with fresh mTeSR plus or mTeSR1 medium containing 5 μM Y27632 and the medium was changed daily. When the cells reached a confluency of more than 80%, a 24-hour drug selection was performed using 1 μg / mL of puromycin (Puro). After the cells recovered from the drug selection, the selective culture was continued with 1 μg / mL of Puro for about 1 week. Subsequently, individual clones were picked under a microscope inside a tissue culture hood and transferred into individual wells of a 96-well plate pre-coated with Matrigel. After 2-5 days of culture, the clones were subjected to PCR genotyping. For this, genomic DNA was extracted by scraping the cells into 40 μL of QuickExtract™ DNA Extraction Solution (Epicentre; #QE09050) and subjected to PCR amplification using 2x GoTaq Green Master Mix (Promega; #7123). For positive genotyping, the following primer pair was used with an annealing temperature Tm of 65°C, forward primer 5'-CTGTTTCCCCTTCCCAGGCAGGTCC-3' (SEQ ID No: 2) and reverse primer 5'-TCGTCGCGGGTGGCGAGGCGCACCG-3' (SEQ ID No: 3). For homozygosity screening, the following primer pair was used with an annealing temperature Tm of 60°C, forward primer 5'-CGGTTAATGTGGCTCTGGTT-3' (SEQ ID No: 4) and reverse primer 5'-GAGAGAGATGGCTCCAGGAA-3' (SEQ ID No: 5).

[0043] Results Figure 3 As shown, the expected PCR product size for correct targeting of the AAVS1 site was 991 bp (red arrow) and all 13 clones successfully targeted the site (100% efficiency). In addition, the knock-in clones were subjected to homozygosity testing and the results showed that clones that did not amplify a PCR product of about 204 bp were homozygous (blue arrow).

[0044] Example 2: Induction of differentiation of CAR-hPSCs into CAR-PMNs.

[0045] A schematic diagram of the induction of differentiation is shown in Figure 4 The specific steps include: S0. Pretreatment: To achieve differentiation of CAR neutrophils, the CAR-hPSCs obtained from Example 1 were first dissociated using 0.5 mM EDTA. Subsequently, the cells were seeded at a density of 1-8 x 10 4 cells / cm2 into 24-well plates coated with iMatrix 511 and cultured in mTeSR plus medium containing 5 μM Y27632 for 24 hours (defined as day -1).

[0046] S1. The cells were transferred to Dulbecco's modified Eagle medium (DMEM / Vc) supplemented with 6 μM CHIR99021 and 100 μg / mL ascorbic acid, and the formal induction of differentiation was started, which is defined as day 0.

[0047] S2. After 24 h of CHIR99021 treatment, the medium was exchanged for LasR basal medium at the end of day 1. During days 2-3, 50 ng / mL vascular endothelial growth factor (VEGF) was supplemented to the medium, and full medium exchange was performed daily.

[0048] S3. At the beginning of day 4, the medium was exchanged for Stemline II medium supplemented with 10 μM SB431542, 25 ng / mL SCF, and 25 ng / mL FLT3L.

[0049] S4. At the beginning of day 6, the old medium was aspirated, and the new medium was Stemline II medium containing 50 ng / mL SCF and 50 ng / mL FLT3L.

[0050] S5. At the beginning of day 9, the upper half of the medium was aspirated and supplemented with 0.5 mL fresh Stemline II medium containing 50 ng / mL SCF, 50 ng / mL FLT3L, and 25 ng / mL granulocyte macrophage colony-stimulating factor (GM-CSF). The same was performed at the beginning of day 12.

[0051] S6. At day 15, the suspended cells were gently collected, filtered through a 70 μm cell strainer, and transferred to the final differentiation medium consisting of Stemline II medium supplemented with 1 x GlutaMAX, 150 ng / mL GM-CSF, and 2.5 μM retinoic acid agonist AM580. Thereafter, the medium was exchanged half every 3 days. From day 21, mature CAR-PMNs could be harvested for further analysis.

[0052] Representative bright-field microscopic images of cell morphology at different differentiation time points are shown in Figure 5Figure 1 shows the differentiation process of CAR-hPSCs into CAR-NKs. The indicated are human pluripotent stem cells (hPSCs) on day 0 (D0), mesoderm cells on day 3 (D3), hemogenic endothelial cells on day 6 (D6), hematopoietic stem and progenitor cells (HSPCs) on day 12 (D12), myeloid progenitor cells on day 15 (D15), and neutrophil granulocytes on day 21 (D21). The results of flow cytometric analysis during the corresponding differentiation process are shown in Figure 2. Figure 6

[0053] Example 3: Inducing differentiation of CAR-hPSCs into CAR-NKs.

[0054] The flowchart of the inducing differentiation process is shown in Figure 1. Figure 7 The specific steps include: S0. Pretreatment: In order to realize the differentiation of CAR neutrophil granulocytes, the CAR-hPSCs obtained in Example 1 were first subjected to dissociation treatment using 0.5 mM EDTA. Subsequently, the cells were inoculated into 24-well plates coated with iMatrix 511 at a density of 1-8 x 10 4 cells per square centimeter, and were cultured in mTeSR plus medium containing 5 μM Y27632 for 24 hours (defined as day -1).

[0055] S1. The cells were transferred to Dulbecco's modified Eagle's medium (DMEM / Vc) supplemented with 6 μM CHIR99021 and 100 μg / mL ascorbic acid, and the formal induction differentiation treatment was started, which was recorded as day 0.

[0056] S2. After 24 h of CHIR99021 treatment, the medium was replaced with LasR basal medium at the end of day 1. During days 2-3, 50 ng / mL vascular endothelial growth factor (VEGF) was supplemented to the medium, and full-volume replacement was performed daily.

[0057] S3. At the beginning of day 4, the medium was replaced with Stemline II medium, and 10 μM SB431542, 25 ng / mL SCF, and 25 ng / mL FLT3L were added thereto.

[0058] S4. At the beginning of day 6, the old medium was aspirated, and the new medium was Stemline II medium containing 50 ng / mL SCF and 50 ng / mL FLT3L.

[0059] S5. At the beginning of day 9, the upper half of the medium was aspirated, and 0.5 mL of fresh Stemline II medium containing 50 ng / mL SCF and 50 ng / mL FLT3L was supplemented. The same operation was performed at the beginning of day 12.​

[0060] S6. At day 15, the suspension cells were collected gently and filtered through a 70 μm cell strainer to establish the co-culture system. The cells were seeded on OP9-DLL4 monolayer cells at a density of 2 x 10 4 cells / mL; NK differentiation culture was performed using a-MEM medium containing 20% fetal bovine serum (FBS), 5 ng / mL IL-7, 5 ng / mL FLT3L, 25 ng / mL SCF, 5 ng / mL IL-15 and 35 nM UM171. The NK differentiation medium was replaced every 3 days; the suspension cells were transferred to fresh OP9-DLL4 monolayer cells every 6 days.

[0061] Example 4: Folate receptor high expression tumor cell killing experiment.

[0062] The survival rate of human breast cancer cells MDA-MB-231 expressing luciferase was evaluated by D-luciferin detection method. The specific experimental steps are as follows: in a 96-well plate, 100 μL of MDA-MB-231 cell suspension (concentration of 5 x 10 3 cells / mL) was mixed with 100 μL of CAR-PMNs with concentrations of 1.5 x 10 4 , 2.5 x 10 4 and 5 x 10 4 cells / mL, respectively, and experimental groups with or without 10 nmol / L fluorescein isothiocyanate-labeled folic acid (FITC-folic acid) were set. The above mixtures were incubated in a 37°C, 5% CO2 incubator for 24 hours. After incubation, centrifugation was performed at 1000 rpm for 5 minutes, the supernatant was discarded, and 100 μL of culture medium containing 150 μg / mL D-luciferin was added to each well, and incubation was continued under the same conditions for 30 minutes. The bioluminescence signal was measured using a SpectraMax iD3 multifunctional microplate reader. The survival rate of MDA-MB-231 cells was calculated according to the following formula: Cell survival rate = (sample reading - blank control) / (negative control - blank control) * 100%.

[0063] CAR-PMNs bridged with FITC-folic acid bispecific antibody (purchased from MedChemExpress company, CAS number: 583037-91-6, catalog number: HY-13615) showed enhanced anti-tumor cytotoxicity. As Figure 8The immunological synapses formed between CAR-PMNs bridged by 10 nM FITC-folate bispecific aptamer and human breast cancer cells MDA-MB-231 are shown in FIG. 6. It can be found by quantitative analysis that there is no significant difference between CAR-PMNs and untreated neutrophil population without adding FITC-folate bispecific aptamer; but after adding FITC-folate bispecific aptamer, the immunological synapses between CAR-PMNs and MDA-MB-231 cells account for 60% of the total, which is significantly different from the untreated neutrophil population (as shown in FIG. 6). Figure 9

[0064] The results of cytotoxicity assay of CAR-PMNs with different proportions of MDA-MB-231 cells treated or untreated with 10 nM FITC-folate bispecific aptamer are shown in FIG. 7. MDA-MB-231 cells naturally express folate receptor a on the surface, while normal tissue organs hardly express it, so the CAR-PMNs can specifically target and recognize MDA-MB-231 cells through the bridging of FITC-folate bispecific aptamer. Figure 10

[0065] It is obvious to those skilled in the art that the present application is not limited to the details of the above exemplary embodiments, and the present application can be implemented in other specific forms without departing from the spirit or essential characteristics of the present application. Therefore, the embodiments should be regarded as exemplary and non-limiting, the scope of the present application is defined by the appended claims rather than the above description, and all changes falling within the meaning and scope of the equivalent elements of the claims are intended to be included in the present application.​​

Claims

1. A chimeric antigen receptor expression gene construct characterized in that, The chimeric antigen receptor comprises a signal peptide CD8a-SP, an extracellular anti-fluorescein diethylamide single-chain variable fragment, an extracellular hinge transmembrane domain CD8a-hg-tm of CD8a, a costimulatory domain 4-1BB and an intracellular signal transduction domain CD3ζ.

2. The chimeric antigen receptor expressing genetic construct of claim 1, wherein, The nucleotide sequence of the chimeric antigen receptor is shown as SEQ ID No:

1.

3. A method of constructing a chimeric antigen receptor pluripotent stem cell, comprising, The method comprises the steps of: (1) knocking in the chimeric antigen receptor expression gene construct of claim 1 or 2 into the AAVS1 safe harbor site in human pluripotent stem cells by CRISPR / Cas9-mediated homologous recombination; (2) isolating successfully targeted single-cell derived human pluripotent stem cell colonies or human pluripotent stem cell mixtures to obtain a stable human pluripotent stem cell line expressing the chimeric antigen receptor; (3) extracting the chimeric antigen receptor pluripotent stem cells from the cell line.

4. The method for constructing chimeric antigen receptor pluripotent stem cells according to claim 3, characterized in that: The human pluripotent stem cells comprise human embryonic stem cells and / or induced pluripotent stem cells.

5. The method for constructing chimeric antigen receptor pluripotent stem cells according to claim 4, characterized in that: The human embryonic stem cells are selected from H1, H7, H9, H13 or H14.

6. A chimeric antigen receptor pluripotent stem cell, characterized in that, The method is obtained by the construction method of any one of claims 3 to 5.

7. A method of preparing a population of chimeric antigen receptor neutrophils, characterized in that, The method comprises the steps of: (a) stimulating the chimeric antigen receptor pluripotent stem cells of claim 6 using a medium containing a glycogen synthase kinase 3β inhibitor; (b) replacing the medium containing vascular endothelial growth factor to achieve hematopoietic endothelial cell differentiation; (c) replacing the medium containing a transforming growth factor β inhibitor, stem cell factor, FMS-like tyrosine kinase 3 ligand and / or granulocyte macrophage colony-stimulating factor to achieve hematopoietic progenitor cell orientation; (d) replacing the medium containing granulocyte macrophage colony-stimulating factor and retinoic acid receptor agonist to obtain the chimeric antigen receptor neutrophil population.

8. The method of claim 7, wherein the chimeric antigen receptor neutrophil population is prepared by, The glycogen synthase kinase 3β inhibitor is selected from CHIR99021 or CHIR98014; the transforming growth factor β inhibitor is selected from SB431542 or A83-01; and the retinoic acid receptor agonist is selected from AM80 or AM50.

9. A method of preparing chimeric antigen receptor natural killer cells, characterized in that, The method comprises the steps of: (I) stimulating the chimeric antigen receptor pluripotent stem cells of claim 6 using a medium containing a glycogen synthase kinase 3β inhibitor; (II) replacing the medium containing vascular endothelial growth factor to achieve hematopoietic endothelial cell differentiation; (III) replacing the medium containing a transforming growth factor β inhibitor, stem cell factor and / or FMS-like tyrosine kinase 3 ligand to achieve hematopoietic progenitor cell orientation; (IV) replacing the medium containing interleukin-7, interleukin-15, stem cell factor, FMS-like tyrosine kinase 3 ligand and lineage reconstitution enhancer for co-culture with OP9-DLL4 monolayer cells to obtain the chimeric antigen receptor natural killer cell.

10. The method for preparing chimeric antigen receptor natural killer cells according to claim 9, characterized in that: The glycogen synthase kinase 3β inhibitor is selected from CHIR99021 or CHIR98014; the transforming growth factor β inhibitor is selected from SB431542 or A83-01; the retinoic acid receptor agonist is selected from AM80 or AM50; and the lineage reconstitution enhancer is UM171.

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