Method for producing iPS cell-derived natural killer cells

Abstract

The present invention provides a method for producing natural killer cells from iPS (induced pluripotent stem) cells, the method comprising: (i) contacting iPS cells with a composition comprising a GSK-3 inhibitor and a ROCK inhibitor to obtain embryoid bodies; (ii) contacting the embryoid bodies with a composition comprising a TGFβ receptor inhibitor to obtain hematopoietic progenitor cells; (iii) culturing the hematopoietic progenitor cells to obtain lymphoid progenitor cells; and (iv) differentiating the lymphocyte precursor cells into natural killer cells and then proliferating them Includes.

Description

[Technical Field]

[0001] The present invention relates to a method for producing natural killer cells from iPS (induced pluripotent stem) cells, natural killer cells or populations, pharmaceutical compositions containing the natural killer cells or populations, and methods for treating cancer comprising administering pharmaceutical compositions containing the natural killer cells or populations. [Background technology]

[0002] Natural killer (NK) cells are cytotoxic lymphocytes that constitute a major component of the innate immune system. NK cells are a subset of innate lymphoid cells (ILCs) that exert a direct cytotoxic effect on pathogenic cells by inducing apoptosis.

[0003] NK cells are activated in response to interferon or macrophage-derived cytokines. Their cytotoxic activity is primarily regulated by two types of surface receptors, which are considered "activating receptors" or "inhibitory receptors," although some receptors, such as CD94 and 2B4 (CD244), can function in either way upon ligand interaction.

[0004] NK cells have been shown to play a role in host rejection of tumors and to have the ability to kill virus-infected cells, among other activities. Natural killer cells can be activated by cells lacking or exhibiting reduced levels of major histocompatibility complex (MHC) proteins. Cancer cells with altered or reduced levels of self-class I MHC protein expression induce NK cell sensitivity. Activated and expanded NK cells and, in some cases, LAK cells from peripheral blood have been used in both ex vivo and in vivo treatment of patients with advanced cancer, with some success against bone marrow-related diseases such as leukemia, breast cancer, and certain types of lymphoma. Recently, anti-GPC3 CAR-expressing NK / ILC cells have been reported to be effective cell therapy for disseminated ovarian tumors (Cancer Sci. 2020 May;111(5):1478-1490. doi: 10.1111 / cas.14374. Epub 2020 March 31).

[0005] Despite the advantageous properties of NK cells in killing tumor cells and virus-infected cells, their application in immunotherapy remains challenging due to the difficulty in maintaining tumor targeting and tumor-killing ability, especially during culture and expansion. Therefore, there is a need in the art to develop efficient methods for producing and expanding natural killer cells that maintain tumor-killing ability. Detailed Description of the Invention

[0006] The present invention provides a method for producing natural killer cells from iPS (induced pluripotent stem) cells, natural killer cells or populations, pharmaceutical compositions containing the natural killer cells or populations, and methods for treating cancer comprising administering the pharmaceutical compositions containing the natural killer cells or populations.

[0007] Specifically, the following inventions are provided: 1. A method for producing natural killer cells from iPS (induced pluripotent stem) cells, comprising: (i) contacting iPS cells with a composition comprising a GSK-3 inhibitor and a ROCK inhibitor to obtain embryoid bodies; (ii) contacting the embryoid bodies with a composition comprising a TGFβ receptor inhibitor to obtain hematopoietic progenitor cells; (iii) culturing the hematopoietic progenitor cells to obtain lymphoid progenitor cells; and (iv) differentiating the lymphocyte precursor cells into natural killer cells and then proliferating them A method comprising: 2. The method of claim 1, wherein the iPS cells express a chimeric antigen receptor (CAR) specific for a tumor antigen. 3. The method of claim 2, wherein the CAR expression is maintained or selected throughout the differentiation process using a tracer gene, and the CAR is stably expressed at the natural killer cell stage. 4. The method of paragraph 2, wherein the tumor antigen is selected from the group consisting of GPC3, BCMA, PSMA, MUC1, HER2, mesothelin, Lewis-Y, AXL, EGFR, claudin 18.2, B7-H3, NKG2D, GD2, EpCAM, ROBO-1, CD19, CD20, CD22, CD30, CD33, CD38, CD123, CD276, and CD269. 5. The method according to item 2, wherein the iPS cells are undifferentiated from an iPS cell colony containing a CAR. 6. The method of claim 2, wherein the CAR is introduced into iPS cells using a viral vector, a non-viral vector, an artificial chromosome, or gene editing. 7. The method of claim 6, wherein the viral vector is a lentiviral vector, retroviral vector, adenoviral vector, or AAV vector, and the non-viral vector is a piggyBac vector. 8. The method of claim 6, wherein the gene editing comprises the use of CRISPAR / CAS9, TAL effector nucleases, homologous recombination, or other gene editing tools. 9. The method of claim 1, wherein the GSK-3 inhibitor is CHIR99021 and the ROCK inhibitor is Y-27632. 10. The method of claim 1, wherein the TGFβ receptor inhibitor is SB431542. 11. The method according to claim 1, wherein the composition of step (ii) further comprises VEGF, hbFGF and SCF. 12. The method of claim 1, wherein the hematopoietic progenitor cells are cultured in a composition comprising 2-mercaptoethanol, insulin-transferrin-selenium, ascorbic acid-2-phosphate, SCF, TPO, IL-7, hFlt3L, SDF1α, and a p38 inhibitor. 13. The method of claim 1, wherein the p38 inhibitor is SB203580. 14. The lymphocyte progenitor cells are CD7 + CD45 + 2. The method of claim 1, wherein the cell is a cell. 15. The method of claim 1, wherein the lymphoid progenitor cells are expanded on feeder cells comprising human PBMCs. 16. The method of claim 15, wherein the human PBMCs are autologous or allogeneic. 17. Natural killer cells or populations thereof produced by the method according to any one of items 1 to 16. 18.CD7 + CD45 + Natural killer cell populations, including cells that are cells. 19. CD7 in the natural killer cells + CD45 + 19. The natural killer cell population according to item 18, wherein the proportion of cells is greater than 60% by cell number. 20. The cells are CD3 - , CD4 - , CD5 - , CD8 - , CD117 + , CD337 + , CD159a + , CD161 + , CD336 + , CD226 + , and CD314 + 19. The natural killer cell population according to item 18, wherein 21. The natural killer cell population described in item 18, wherein the natural killer cells are contaminated with undifferentiated iPSCs at a cell count of less than 0.01%. 22. A pharmaceutical composition comprising the natural killer cells or a population thereof described in any one of items 17 to 21. 23. The pharmaceutical composition according to item 22, comprising a cryoprotectant. 24. The pharmaceutical composition according to item 22, comprising glucose, saline, dextran D, albumin, and dimethyl sulfoxide. 25. A method for treating cancer, comprising administering the pharmaceutical composition according to any one of items 22 to 24. 26. The method of claim 25, wherein the cancer is liver cancer, ovarian cancer, gastric cancer, lung cancer, prostate cancer, breast cancer, glioblastoma, colorectal cancer, esophageal cancer, head and neck cancer, cervical cancer, kidney cancer, pediatric solid tumors, osteosarcoma, germ cell tumors, neuroblastoma, hematopoietic malignancies, or multiple myeloma. [Brief explanation of the drawings]

[0008] Figure 1 shows the cytotoxic activity of iCAR-ILC / N101. The cytotoxic activity of iCAR-ILC / N101 was examined using a 51Cr release assay. Ovarian cancer KOC7c cells, which endogenously express GPC3, were used as target cells. Significant effector / target ratio-dependent cytotoxic activity was observed against KOC7c cells.

[0009] Figure 2 shows cytokine-dependent proliferation of iCAR-ILC / N101 cells. iCAR-ILC / N101 cells were stimulated with PHA-P (1 μg / ml) and irradiated with PBMCs (iCAR-ILC / N101 cells:PBMC = 1:10) for 3 days. The cells were cultured in the presence or absence of IL7 (10 ng / ml) and IL15 (5 ng / ml). Cell counts were determined every 3 days. As shown in Figure 2, iCAR-ILC / N101 cells exhibited significant cytokine-dependent proliferation. Cytokine-independent proliferation was not observed.

[0010] Figure 3 shows the pharmacokinetics of iCAR-ILC / N101. Luciferase-transduced ICAR-ILC / N101 cells were inoculated intraperitoneally into NOG mice, and chemiluminescence was monitored at different time points using an in vivo luminescence imaging device. ICAR-ILC / N101 chemiluminescence was detected up to day 7 after inoculation. No significant chemiluminescence was observed on days 14, 21, and 28.

[0011] Figure 4 shows the iPS cell contamination in iCAR-ILC / N101. iPSC contamination was examined using qPCR to detect LIN28A RNA. Cell lysates were prepared from 1x10 6 iCAR-ILC-N101, 1x10 6 1x10 human MSCs (hMSCs), and 1x10 6 iPS cells and 1x10 6 The lysates were prepared from a mixture of iPS cells and human MSCs. The mixture of iPS cells and human MSCs was serially diluted 10-fold with human MSC lysate. The following standard lysates were prepared: iPSC:hMSC = 1:10 1:100 1:1000 1:10000 qPCR was performed using total RNA prepared from lysates of iCAR-ILC-N101, human MSCs, and a mixture of iPS cells and human MSCs. A standard curve was constructed based on the C values of the mixture of iPS cells and human MSCs. The amount was calculated for the iCAR-ILC-N101 sample. Because the value was outside the range (0.01%–10%), we concluded that the contamination rate was <0.01. BEST MODE FOR CARRYING OUT THE INVENTION

[0012] In one embodiment, the present invention discloses a method for producing natural killer cells from iPS (induced pluripotent stem) cells, the method comprising the steps of: (step i) contacting iPS cells with a composition comprising a GSK-3 inhibitor and a ROCK inhibitor to obtain embryoid bodies; (step ii) contacting the embryoid bodies with a composition containing a TGFβ receptor inhibitor to obtain hematopoietic progenitor cells; (step iii) culturing the hematopoietic progenitor cells to obtain lymphoid progenitor cells; and (Step iv) differentiating the lymphocyte precursor cells into natural killer cells and then proliferating them.

[0013] The iPS cells are transduced with a chimeric antigen receptor (CAR) specific for a tumor antigen. CAR expression is maintained / selected throughout differentiation using a tracer gene, and the CAR is stably expressed at the natural killer cell stage.

[0014] The tumor antigen is GPC3, BCMA, PSMA, MUC1, HER2, mesothelin, Lewis-Y, AXL, EGFR, claudin 18.2, B7-H3, NKG2D, GD2, EpCAM, ROBO-1, CD19, CD20, CD22, CD30, CD33, CD38, CD123, CD276, or CD269.

[0015] The iPS cells are undifferentiated CAR-transduced iPSC colonies. The CAR is introduced into the iPS cells using a viral vector, a non-viral vector, an artificial chromosome, or gene editing.

[0016] Examples of the viral vector include a lentiviral vector, a retroviral vector, an adenoviral vector, or an AAV vector, and the non-viral vector is a piggyBac vector.

[0017] Examples of gene editing include CRISPAR / CAS9, TAL effector nuclease, homologous recombination, or other gene editing tools. GSK-3 inhibitors can maintain or increase the differentiation potential (potency) of cells compared to cells cultured in the absence of GSK-3 inhibitors. Examples of GSK-3 inhibitors include SB216763, AT7519, CHIR-98014, TWS119, SB415286, NP031112, and BIO, preferably CHIR99021. ROCK inhibitors can increase cell proliferation compared to cells cultured in the absence of ROCK inhibitors. Examples of ROCK inhibitors include ZINC00881524, thiazovivin, fasudil, GSK429286A, RKI-1447, NSC 33669, GSK269962, AR-13324, TC-S 7001, Y-33075, KD025, HA-1100, H-1152 dihydrochloride, and AT13148, preferably Y-27632. Examples of TGFβ receptor inhibitors include LY2157299, LY2109761, SB525334, SB505124, GW788388, and LY364947, preferably SB431542.

[0018] In one embodiment, the present invention discloses a method for producing natural killer cells from iPS (induced pluripotent stem) cells further comprising VEGF, hbFGF and SCF in step (ii).

[0019] In one embodiment, the present invention discloses a method for producing natural killer cells from iPS (induced pluripotent stem) cells, wherein the hematopoietic progenitor cells are cultured in a composition comprising 2-mercaptoethanol, insulin-transferrin-selenium, ascorbic acid-2-phosphate, SCF, TPO, IL-7, hFlt3L, SDF1α, and a p38 inhibitor, preferably SB203580. The lymphoid progenitor cells are CD7 + CD45 + It is a cell.

[0020] In one embodiment, the present invention discloses a method for producing natural killer cells from iPS (induced pluripotent stem) cells, wherein the lymphoid progenitor cells are expanded on feeder cells comprising human PBMCs, preferably autologous or allogeneic.

[0021] In one embodiment, the present invention discloses a natural killer cell or population thereof produced by the method.

[0022] In one embodiment, the present invention provides a method for detecting CD7 + CD45 + The present invention discloses a natural killer cell population comprising cells, preferably CD7 + CD45 + The proportion of cells exceeds 60% by cell number.

[0023] In one embodiment, the present invention provides a method for the detection of CD3 - , CD4 - , CD5 - , CD8 - , CD117 + , CD337 + , CD159a + , CD161 + , CD336 + , CD226 + , and CD314 + Disclosed is a natural killer cell population containing cells which are

[0024] In one embodiment, the present invention discloses a natural killer cell population, preferably wherein the natural killer cells are contaminated with undifferentiated iPSCs at less than 0.01% by cell number.

[0025] In one embodiment, the present invention discloses a pharmaceutical composition comprising the natural killer cells or a population thereof, the pharmaceutical composition further comprising a cryoprotectant such as glucose, saline, dextran D, albumin, and dimethyl sulfoxide.

[0026] In one embodiment, the present invention discloses a method for treating cancer comprising administering a pharmaceutical composition comprising said natural killer cells or a population thereof.

[0027] The cancer is liver cancer, ovarian cancer, gastric cancer, lung cancer, prostate cancer, breast cancer, glioblastoma, colon cancer, esophageal cancer, head and neck cancer, cervical cancer, kidney cancer, pediatric solid tumors, osteosarcoma, germ cell tumors, neuroblastoma, hematopoietic malignancies, or multiple myeloma.

[0028] The following examples of the present invention are provided merely as examples and do not limit the technical scope of the present invention. The technical scope of the present invention is limited only by the scope of the claims. The present invention may be modified, for example, by adding elements to the present invention, deleting elements from the present invention, or even substituting elements therein, without departing from the spirit of the present invention.

[0029] Conditions not specified in the examples will be those common in the art or those recommended by the manufacturer of the raw materials. Reagents for which the origin is not indicated will be commercially available, general-purpose reagents.

[0030] Method for producing natural killer cells (NK) / innate lymphoid cells (ILC) (method for producing iPSC-derived NK / ILC)

[0031] iPS cells transduced with a tumor antigen-specific chimeric antigen receptor (CAR) were differentiated into hematopoietic progenitor cells using the feeder-free embryoid body (EB) formation method described below.

[0032] [Process 1] Undifferentiated CAR-transduced iPSC colonies were treated with TrypLE select (Gibco) for 4 minutes (maximum 10 minutes, depending on the cell dispersion speed), transferred to low-adhesion plates, and incubated overnight in medium A (StemFit AK03N supplemented with 10 μmol / L ROCK inhibitor (Y-27632) and 10 μmol / L GSK3b inhibitor (CHIR99021)) to allow EB formation.

[0033] Table 1: Medium A JPEG2025526532000001.jpg20132

[0034] [Process 2] The EBs were collected, centrifuged, and resuspended in medium B [StemPro-34 supplemented with 2 mmol / L L-glutamine (1% GlutaMAX), 400 μmol / L monothioglycerol, 50 μg / mL ascorbic acid-2-phosphate, 1% insulin-transferrin-selenium supplement, 50 ng / mL hBMP-4, 50 ng / mL hbFGF, and 50 ng / mL VEGF] and incubated at 37°C in a 5% CO2 atmosphere.

[0035] Table 2: Medium B JPEG2025526532000002.jpg42139 * StemPro-34 does not contain TGF-β, IL-3, GSK3 inhibitors, or ROCK inhibitors.

[0036] [Process 3] On day 2, 6 μmol / L TGFβ receptor inhibitor (SB431542) was added to the culture.

[0037] [Step 4] On day 4, the EBs were collected, centrifuged, resuspended in medium C (StemPro-34 supplemented with 2 mmol / L L-glutamine, 400 μmol / L monothioglycerol, 50 μg / mL ascorbic acid-2-phosphate, 1% insulin-transferrin-selenium supplement, 50 ng / mL hbFGF, 50 ng / mL VEGF, and 50 ng / mL SCF) and incubated at 37°C in a 5% CO atmosphere.

[0038] Table 3: Medium C JPEG2025526532000003.jpg42139

[0039] [Step 5] On days 6, 8, 11, and 13, the cells in the culture were harvested, centrifuged, and resuspended in medium D (StemPro-34 supplemented with 2 mmol / L L-glutamine, 400 μmol / L monothioglycerol, 50 μg / mL ascorbic acid-2-phosphate, 1% insulin-transferrin-selenium supplement, 50 ng / mL hbFGF, 50 ng / mL VEGF, 50 ng / mL SCF, 100 ng / mL TPO, and 50 ng / mL hFlt3L) and incubated at 37°C under a 5% CO atmosphere.

[0040] Table 4: Medium D JPEG2025526532000004.jpg51139

[0041] [Step 6] On day 14, single-cell suspensions were prepared using a cell strainer and transferred to FcDLL4-coated plates. The cells were cultured in medium E (α-MEM supplemented with 15% FBS, 55 μM 2-mercaptoethanol, 1% insulin-transferrin-selenium, 50 μg / mL ascorbic acid-2-phosphate, 50 ng / mL SCF, 100 ng / mL TPO, 10 ng / mL IL-7, 50 ng / mL hFlt3L, 240 ng / mL SDF1α, and 15 μM p38 inhibitor (SB203580).

[0042] Table 5: Medium E JPEG2025526532000005.jpg56139

[0043] On days 15, 18, 22, 25, 29, and 32, the cells in the culture were harvested, centrifuged, resuspended in fresh Medium E, and returned to the same culture vessel. On days 21 and 28, the cells were harvested, centrifuged, resuspended in fresh Medium E, and transferred to new FcDLL4-coated plates.

[0044] [Step 7] On day 35, after 21 days of culture, the hematopoietic cells differentiated into lymphoid progenitor cells that were positive for CD7 and CD45.

[0045] [Step 8] The floating cells were collected and passed through a cell strainer, and the plate on which the cells remained was washed with PBS. The floating cells were mixed with the PBS solution containing the cells, centrifuged, and resuspended in STEM-CELLBANKER (registered trademark). The cells were cryopreserved.

[0046] [Step 9] The cells and cryopreserved irradiated human peripheral mononuclear cells (PBMCs) were thawed, centrifuged, and resuspended in medium F (α-MEM supplemented with 15% FBS, 1x (1%) insulin-transferrin-selenium, 50 μg / mL ascorbic acid-2-phosphate, 10 ng / mL IL-7, 5 ng / mL IL-15, and 2 μg / mL phytohemagglutinin (PHA). The cells and PBMCs were mixed at a 1:14 ratio and cultured for 10–16 days. IL-15 and IL-7 are used as important sources for NK cell activation and proliferation.

[0047] Table 6: Medium F JPEG2025526532000006.jpg38139

[0048] During the 10–16 days of culture, the culture medium was replaced every 2–3 days with fresh Medium G [α-MEM supplemented with 15% FCBS, 1x (1%) insulin-transferrin-selenium, 50 μg / mL ascorbic acid-2-phosphate, 10 ng / mL IL-7, and 105 ng / mL IL-15]. If cells were proliferating sufficiently, the culture was split into two and replenished with fresh Medium G. If sufficient proliferation was not observed, half of the culture was harvested, centrifuged, resuspended in fresh Medium G, and returned to the original plate.

[0049] Table 7: Medium G JPEG2025526532000007.jpg33139After culturing for 10 to 16 days, the cells were harvested, washed three times with PBS by centrifugation, and resuspended in cryoprotectant A as the final product (iCAR-ILC / N101), which was then frozen and stored until immediately before use.

[0050] Table 8: Cryoprotectant A JPEG2025526532000008.jpg33139

[0051] Stability of cryopreserved iCAR-ILC / N101 Table 9 shows the long-term stability of cryopreserved iCAR-ILC / N101. Table 9 JPEG2025526532000009.jpg217102JPEG2025526532000010.jpg21762

[0052] 2x10 iCAR-ILC / N101 cells 7 The cells were dispensed into tubes and frozen in the vapor phase of a liquid nitrogen tank for 137 days. On days 0, 43, 57, 71, and 137, the viability, cell concentration, CAR-positive rate, product (NK) purity, IFN-γ production, endotoxin / mycoplasma / bacteria and other pathogen detection, and appearance were examined. As shown in Table 9, all test results met the quality standards.

[0053] Table 10 shows the transport stability of cryopreserved iCAR-ILC / N101. Table 10 JPEG2025526532000011.jpg89134

[0054] Aliquot the same production batch of iCAR-ILC / N101 (2x10 7The cells were then frozen and stored. Three randomly selected frozen tubes were transferred to a MEDi STAR frozen shipping box at the cell processing facility and shipped to a shipper's facility 50 miles away. At this facility, the frozen tubes were transferred to the vapor phase of a liquid nitrogen tank. The frozen tubes were then returned to the frozen shipping box and air-shipped to another shipper's facility 300 miles away, and then returned to the cell processing facility via another air shipment. The total distance traveled exceeded 600 miles. The cells were tested for viability, cell concentration, CAR-positive rate, product purity, IFN-γ production, pathogen detection (e.g., endotoxin, mycoplasma, and bacteria), and appearance. As shown in Table 10, all test results met quality standards.

[0055] Table 11 shows the post-thaw stability of cryopreserved iCAR-ILC / N101. Table 11 JPEG2025526532000012.jpg93135

[0056] Aliquot the same production batch of iCAR-ILC / N101 (2x10 7 The cells were frozen (1000 x 1000 cells / tube). Three randomly selected frozen tubes were thawed and stored at room temperature. One tube each was tested for viability, cell concentration, CAR positivity, product purity, IFN-γ production, endotoxin / mycoplasma / bacteria pathogen detection, and appearance at 15, 30, and 90 minutes. At 15 minutes, all test results met the quality standards. However, at 30 minutes, although other test results met the standards, the ability to produce IFN-γ was significantly reduced.

[0057] Table 12 shows the stability of iCAR-ILC / N101 diluted in saline after thawing. Table 12 JPEG2025526532000013.jpg47134

[0058] Cryopreserved iCAR-ILC / N101 cells were thawed, diluted with saline, and stored at room temperature. These cells were tested for viability and viability. Even after 90 minutes, viability and viability did not decrease significantly.

[0059] Table 13 shows the standard tests for the final product (iCAR-ILC / N101). Table 13 JPEG2025526532000014.jpg54135

[0060] Standard tests included sterility, cell count, cell viability, cell phenotyping by flow cytometry, and IFN-γ secretion, and the functional assays shown in Table 13 were used as control tests.

Claims

1. A method for producing natural killer cells from iPS (induced pluripotent stem) cells, comprising: (i) contacting iPS cells with a composition comprising a GSK-3 inhibitor and a ROCK inhibitor to obtain embryoid bodies; (ii) contacting the embryoid bodies with a composition comprising a TGFβ receptor inhibitor to obtain hematopoietic progenitor cells; (iii) culturing the hematopoietic progenitor cells to obtain lymphoid progenitor cells; and (iv) differentiating the lymphocyte precursor cells into natural killer cells and then proliferating them A method comprising:

2. 2. The method of claim 1, wherein the iPS cells express a chimeric antigen receptor (CAR) specific for a tumor antigen.

3. 3. The method of claim 2, wherein the CAR expression is maintained or selected throughout the differentiation process using a tracer gene, and the CAR is stably expressed at the natural killer cell stage.

4. 3. The method of claim 2, wherein the tumor antigen is selected from the group consisting of GPC3, BCMA, PSMA, MUC1, HER2, mesothelin, Lewis-Y, AXL, EGFR, claudin 18.2, B7-H3, NKG2D, GD2, EpCAM, ROBO-1, CD19, CD20, CD22, CD30, CD33, CD38, CD123, CD276, and CD269.

5. 3. The method of claim 2, wherein the iPS cells are undifferentiated from an iPS cell colony containing a CAR.

6. 3. The method of claim 2, wherein the CAR is introduced into the iPS cells using a viral vector, a non-viral vector, an artificial chromosome, or gene editing.

7. 7. The method of claim 6, wherein the viral vector is a lentiviral vector, a retroviral vector, an adenoviral vector, or an AAV vector, and the non-viral vector is a piggyBac vector.

8. 7. The method of claim 6, wherein the gene editing comprises the use of CRISPAR / CAS9, TAL effector nucleases, homologous recombination, or other gene editing tools.

9. 2. The method of claim 1, wherein the GSK-3 inhibitor is CHIR99021 and the ROCK inhibitor is Y-27632.

10. 2. The method of claim 1, wherein the TGFβ receptor inhibitor is SB431542.

11. The method of claim 1, wherein the composition of step (ii) further comprises VEGF, hbFGF, and SCF.

12. The method of claim 1, wherein the hematopoietic progenitor cells are cultured in a composition comprising 2-mercaptoethanol, insulin-transferrin-selenium, ascorbic acid-2-phosphate, SCF, TPO, IL-7, hFlt3L, SDF1α, and a p38 inhibitor.

13. 2. The method of claim 1, wherein the p38 inhibitor is SB203580.

14. The lymphocyte progenitor cells are CD7 + CD45 + The method of claim 1, wherein the cell is a cell.

15. The method of claim 1, wherein the lymphoid progenitor cells are expanded on feeder cells comprising human PBMCs.

16. 16. The method of claim 15, wherein the human PBMCs are autologous or allogeneic.

17. A natural killer cell or a population thereof produced by the method according to any one of claims 1 to 16.

18. CD7 + CD45 + Natural killer cell populations, including cells that are cells.

19. CD7 in the natural killer cells + CD45 + The natural killer cell population of claim 18, wherein the proportion of cells is greater than 60% by cell number.

20. The cells are CD3 - , CD4 - , CD5 - , CD8 - , CD117 + , CD337 + , CD159a + , CD161 + , CD336 + , CD226 + , and CD314 + The natural killer cell population of claim 18, wherein

21. The natural killer cell population of claim 18, wherein the natural killer cells are contaminated with undifferentiated iPSCs at a cell count of less than 0.01%.

22. A pharmaceutical composition comprising the natural killer cells or a population thereof according to any one of claims 17 to 21.

23. 23. The pharmaceutical composition of claim 22, comprising a cryoprotectant.

24. 23. The pharmaceutical composition of claim 22, comprising glucose, saline, dextran D, albumin, and dimethyl sulfoxide.

25. A method for treating cancer, comprising administering the pharmaceutical composition according to any one of claims 22 to 24.

26. 26. The method of claim 25, wherein the cancer is liver cancer, ovarian cancer, gastric cancer, lung cancer, prostate cancer, breast cancer, glioblastoma, colorectal cancer, esophageal cancer, head and neck cancer, cervical cancer, kidney cancer, pediatric solid tumors, osteosarcoma, germ cell tumors, neuroblastoma, hematopoietic malignancies, or multiple myeloma.

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