Method for producing iPS cell-derived natural killer cells

Abstract

The present invention provides a method for producing natural killer cells from iPS cells (induced pluripotent stem cells) comprising the steps of: (i) contacting the iPS cells with a composition comprising a GSK-3 inhibitor and a ROCK inhibitor to obtain embryoids, (ii) contacting the embryoids with a composition comprising a TGF [beta] receptor inhibitor to obtain hematopoietic progenitor cells, (iii) culturing the hematopoietic progenitor cells to obtain lymphocyte progenitor cells, and (iv) differentiating and expanding the lymphocyte progenitor cells into natural killer cells.

Description

Technical Field

[0001] The present invention relates to a method for producing natural killer cells from iPS cells (induced pluripotent stem cells), natural killer cells or a population thereof, a pharmaceutical composition comprising natural killer cells or a population thereof, and a method for treating cancer, the method comprising administering a pharmaceutical composition comprising natural killer cells or a population thereof. Background Art

[0002] Natural killer (NK) cells are cytotoxic lymphocytes that constitute the main component of the innate immune system. Natural killer (NK) cells belong to a subpopulation of innate lymphoid cells (ILCs) and have direct cytotoxic effects on pathogenic cells by inducing apoptosis.

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

[0004] Among other activities, NK cells play a role in host rejection of tumors and have been shown to be able to kill virus-infected cells. Natural killer cells can be activated by cells that lack or display reduced levels of major histocompatibility complex (MHC) proteins. Cancer cells with altered or reduced expression levels of self-class I MHC lead to induced sensitivity of NK cells. Activated and expanded NK cells from peripheral blood, and in some cases LAK cells, have been used for 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.

[0005] Recently, NK / ILC cells expressing anti-GPC3 CAR have been reported, which showed effective cell therapy against disseminated ovarian tumors (Cancer Sci. 2020 May; 111(5): 1478-1490. doi: 10.1111 / cas.14374. Epub 2020 Mar 31.). Summary of the invention

[0006] Technical issues

[0007] Despite the favorable properties of NK cells in killing tumor cells and virus-infected cells, they are still difficult to apply in immunotherapy, mainly due to the difficulty in maintaining their tumor targeting and tumor killing abilities during culture and expansion. Therefore, there is a need in the art to develop an effective method to produce and expand natural killer cells that retain tumor killing functions.

[0008] Technical solutions for solving technical problems

[0009] The present invention provides a method for producing natural killer cells from iPS cells (induced pluripotent stem cells), natural killer cells or a population thereof, a pharmaceutical composition comprising natural killer cells or a population thereof, and a method for treating cancer, the method comprising administering a pharmaceutical composition comprising natural killer cells or a population thereof.

[0010] Specifically, the present application provides the following inventions.

[0011] 1. A method for producing natural killer cells from iPS cells (induced pluripotent stem cells), comprising the following steps:

[0012] (i) contacting iPS cells with a composition comprising a GSK-3 inhibitor and a ROCK inhibitor to obtain embryoid bodies,

[0013] (ii) contacting the embryoid bodies with a composition comprising a TGFβ receptor inhibitor to obtain hematopoietic progenitor cells,

[0014] (iii) culturing the hematopoietic progenitor cells to obtain lymphocyte progenitor cells, and

[0015] (iv) differentiating and expanding the lymphocyte progenitor cells into natural killer cells.

[0016] 2. The method according to item 1 above, wherein the iPS cells express a tumor antigen-specific chimeric antigen receptor (CAR).

[0017] 3. The method according to item 2 above, wherein a tracer gene is used to maintain or select CAR expression during differentiation, and CAR is stably expressed at the natural killer cell stage.

[0018] 4. The method according to item 2, wherein the tumor antigen is selected from GPC3, BCMA, PSMA, MUC1, HER2, mesothelin, Lewis-Y, AXL, EGFR, Claudin18.2, B7-H3, NKG2D, GD2, EpCAM, ROBO-1, CD19, CD20, CD22, CD30, CD33, CD38, CD123, CD276 and CD269.

[0019] 5. The method according to the above item 2, wherein the iPS cells are not cloned and differentiated from iPS cells having CAR.

[0020] 6. The method according to item 2 above, wherein the CAR is transduced into iPS cells using a viral vector, a non-viral vector, an artificial chromosome or gene editing.

[0021] 7. The method according to item 6 above, 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.

[0022] 8. The method according to item 6 above, wherein the gene editing comprises the use of CRISPAR / CAS9, Talen, homologous recombination or other gene editing tools.

[0023] 9. The method according to item 1 above, wherein the GSK-3 inhibitor is CHIR99021, and the ROCK inhibitor is Y-27632.

[0024] 10. The method according to the preceding item 1, wherein the TGFβ receptor inhibitor is SB431542.

[0025] 11. The method according to item 1 above, wherein the composition in step 2 further comprises VEGF, hbFGF and SCF.

[0026] 12. The method according to item 1 above, wherein the hematopoietic progenitor cells are cultured with a composition comprising 2-mercaptoethanol, insulin-transferrin-selenium, ascorbic acid-2-phosphate, SCF, TPO, IL-7, hFlt3L, SDF1α and a p38 inhibitor.

[0027] 13. The method according to item 1 above, wherein the p38 inhibitor is SB203580.

[0028] 14. The method according to item 1 above, wherein the lymphocyte progenitor cells are CD7+CD45+ cells.

[0029] 15. The method according to the preceding item 1, wherein the lymphocyte progenitor cells are expanded on feeder cells comprising human PBMCs.

[0030] 16. The method according to item 15 above, wherein the human PBMCs are autologous or allogeneic.

[0031] 17. Natural killer cells or a population thereof produced according to the method according to any one of the preceding items 1 to 16.

[0032] 18. A natural killer cell population comprising cells, wherein the cells are CD7 + CD45 + cell.

[0033] 19. The natural killer cell population according to item 18 above, wherein the percentage of CD7+CD45+ cells in the natural killer cells is greater than 60% of the cell number.

[0034] 20. The natural killer cell population according to item 18 above, wherein the cells are CD3 - 、CD4 - 、CD5 - 、CD8 - 、CD117 + 、CD337 + 、CD159a + 、CD161 + 、CD336 + 、CD226 + and CD314 + .

[0035] 21. The natural killer cell population according to item 18 above, wherein the contamination of undifferentiated iPSCs is less than 0.01% of the number of cells in the natural killer cells.

[0036] 22. A pharmaceutical composition comprising the natural killer cell or a population thereof according to any one of the preceding items 17 to 21.

[0037] 23. The pharmaceutical composition according to the above item 22, comprising a cryoprotectant.

[0038] 24. The pharmaceutical composition according to the above item 22, comprising glucose, saline, dextran D, albumin and dimethyl sulfoxide.

[0039] 25. A method for treating cancer, comprising administering the pharmaceutical composition according to any one of items 22 to 24 above.

[0040] 26. The method according to item 25 above, 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, hematological malignancies or multiple myeloma. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1The cytotoxic activity of iCAR-ILC / N101 is shown. The cytotoxic activity of iCAR-ILC / N101 was detected using a 51Cr release assay. Ovarian cancer KOC7c cells that endogenously express GPC3 were used as target cells. Significant effector cell / target cell ratio-dependent cytotoxic activity was observed for KOC7c.

[0042] Figure 2 Cytokine-dependent growth of iCAR-ILC / N101 is shown. iCAR-ILC / N101 cells (iCAR-ILC / N101 cells: PBMC = 1:10) were stimulated with PHA-P (1 μg / ml) and irradiated PBMC for three days. Cells were cultured with or without IL7 (10 ng / ml) and IL15 (5 ng / ml). Cell numbers were measured every three days. iCAR-ILC / N101 cells showed significant cytokine-dependent growth, as shown in Figure 2. Figure 2 No cytokine-independent growth was observed.

[0043] Figure 3 and Figure 4 The pharmacokinetics of iCAR-ILC / N101 are shown. ICAR-ILC / N101 cells transduced with luciferase gene are inoculated in the peritoneal cavity of NOG mice, and their chemiluminescence is monitored at different time points (rimepoints) using an in vivo bioluminescent imaging instrument. The chemiluminescence of ICAR-ILC / N101 is detected within up to 7 days after inoculation. No significant chemiluminescence is observed on the 14th, 21st and 28th days.

[0044] Figure 5 The iPS cell contamination in iCAR-ILC / N101 is shown. iPSC (iPS cell) contamination was detected using qPCR for LIN28A RNA. 6 iCAR-ILC-N101, 1x10 6 Human MSC (hMSC) and 1x10 6 iPS cells and 1x10 6 Prepare cell lysate from a mixture of iPS cells and human MSCs. Serially dilute the lysate of the mixture of iPS cells and human MSCs 10-fold in human MSC lysate. Prepare the following standard lysate solutions:

[0045] iPSC:hMSC=1:10 1:100 1:1000 1:10000

[0049] 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 line was drawn based on the CT value of a mixture of iPS cells and human MSCs. The amount of the iCAR-ILC-N101 sample was calculated. The value was outside the range (0.01% to 10%). Therefore, a contamination rate of <0.01 was concluded. DETAILED DESCRIPTION

[0050] Best Mode for Carrying Out the Invention

[0051] In one embodiment, the present invention discloses a method for producing natural killer cells from iPS cells (induced pluripotent stem cells), comprising the following steps:

[0052] (step i) contacting the iPS cells with a composition comprising a GSK-3 inhibitor and a ROCK inhibitor to obtain embryoid bodies,

[0053] (Step ii) contacting the embryoid body with a composition comprising a TGFβ receptor inhibitor to obtain hematopoietic progenitor cells,

[0054] (step iii) culturing the hematopoietic progenitor cells to obtain lymphocyte progenitor cells, and

[0055] (Step iv) differentiating and expanding the lymphocyte progenitor cells into natural killer cells.

[0056] iPS cells are iPS cells transduced with a tumor antigen-specific chimeric antigen receptor (CAR). CAR expression is maintained / selected using a tracer gene during differentiation, and CAR is stably expressed at the natural killer cell stage.

[0057] The tumor antigen is GPC3, BCMA, PSMA, MUC1, HER2, Mesothelin, Lewis-Y, AXL, EGFR, Claudin18.2, B7-H3, NKG2D, GD2, EpCAM, ROBO-1, CD19, CD20, CD22, CD30, CD33, CD38, CD123, CD276 or CD269.

[0058] iPS cells are undifferentiated CAR-transduced iPSC clones. The CAR is transduced into iPS cells using viral vectors, non-viral vectors, artificial chromosomes or gene editing.

[0059] Examples of viral vectors are lentiviral vectors, retroviral vectors, adenoviral vectors or AAV vectors, and the non-viral vector is a piggyBac vector.

[0060] Examples of gene editing are CRISPAR / CAS9, TALEN, homologous recombination or other gene editing tools.

[0061] Compared with cells cultured in the absence of GSK-3 inhibitors, GSK-3 inhibitors can maintain or increase the differentiation ability (efficacy) of cells to a greater extent. Examples of GSK-3 inhibitors include SB216763, AT7519, CHIR-98014, TWS119, SB415286, NP031112, BIO, preferably CHIR99021.

[0062] Compared with cells cultured in the absence of ROCK inhibitors, ROCK inhibitors can increase cell proliferation to a greater extent. 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, AT13148, preferably Y-27632.

[0063] Examples of TGFβ receptor inhibitors include LY2157299, LY2109761, SB525334, SB505124, GW788388, LY364947, preferably SB431542.

[0064] In one embodiment, the present invention discloses a method for producing natural killer cells from iPS cells (induced pluripotent stem cells), further comprising VEGF, hbFGF and SCF in step 2.

[0065] In one embodiment, the present invention discloses a method for producing natural killer cells from iPS cells (induced pluripotent stem cells), wherein the hematopoietic progenitor cells are cultured with a composition comprising 2-mercaptoethanol, insulin-transferrin-selenium, ascorbic acid-2-phosphate, SCF, TPO, IL-7, hFlt3L, SDF1α and a p38 inhibitor, preferably the p38 inhibitor is SB203580. The lymphocyte progenitor cells are CD7 + CD45 + cell.

[0066] In one embodiment, the present invention discloses a method for producing natural killer cells from iPS cells (induced pluripotent stem cells), wherein the lymphocyte progenitor cells are expanded on feeder cells comprising human PBMCs. Preferably, the human PBMCs are autologous or allogeneic.

[0067] In one embodiment, the present invention discloses natural killer cells or populations thereof produced by the present method.

[0068] In one embodiment, the present invention discloses a natural killer cell population comprising CD7 + CD45 + cells, preferably CD7 in natural killer cells + CD45 + The percentage of cells is greater than 60% of the cell number.

[0069] In one embodiment, the present invention discloses a natural killer cell population comprising cells, wherein the cells are CD3 - 、CD4 - 、CD5 - 、CD8 - 、CD117 + 、CD337 + 、CD159a + 、CD161 + 、CD336 + 、CD226 + and CD314 + .

[0070] In one embodiment, the present invention discloses a population of natural killer cells, preferably, the contamination of undifferentiated iPSCs is less than 0.01% of the number of cells in said natural killer cells.

[0071] In one embodiment, the present invention discloses a pharmaceutical composition comprising natural killer cells or a population thereof. The pharmaceutical composition further comprises a cryoprotectant, such as glucose, saline, dextran D, albumin and dimethyl sulfoxide.

[0072] In one embodiment, the present invention discloses a method of treating cancer comprising administering a pharmaceutical composition comprising natural killer cells or a population thereof.

[0073] 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 tumor, osteosarcoma, germ cell tumor, neuroblastoma, hematological malignancy or multiple myeloma.

[0074] Example

[0075] The embodiments of the present invention described below are provided as examples only and should not limit the technical scope of the present invention. The technical scope of the present invention is limited only by the description in the claims. Without departing from the scope of the gist of the present invention, the present invention may be modified, for example, elements may be added to the present invention, or elements of the present invention may be deleted or even replaced.

[0076] The conditions not specifically described in the examples are general conditions in the art or raw material conditions recommended by product manufacturers. Reagents without indicating their sources are commercially available conventional reagents.

[0077] Method for producing natural killer cells (NK) / innate lymphoid cells (ILC) (method for producing iPSC-derived NK / ILC)

[0078] Tumor antigen-specific chimeric antigen receptor (CAR)-transduced iPS cells were differentiated into hematopoietic cell precursors by the feeder-free embryoid body (EB) formation method as described below.

[0079] Step 1

[0080] Undifferentiated CAR-transduced iPSC clones were treated with TrypLE select (Gibco) for 4 min (up to 10 min, depending on how quickly the cells dispersed), transferred to low attachment 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.

[0081] Table 1: Culture medium A

[0082]

[0083]

[0084] (Step 2)

[0085] 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 then incubated at 37°C with 5% CO 2 incubated in an environment.

[0086] Table 2: Medium B

[0087] serial number Contents Final concentration 1 StemPro-34* - 2 GlutaMAX (100x) 1x(1％) 3 Monothioglycerol 400μmol / L 4 Ascorbic acid 2-phosphate 50 μg / mL 5 Insulin-Transferrin-Selenium (100x) 1x(1％) 6 hBMP-4 50ng / mL 7 hbFGF 50ng / mL 8 VEGF 50ng / mL

[0088] *StemPro-34 does not contain TGF-β, IL-3, GSK3 inhibitors or ROCK inhibitors.

[0089] (Step 3)

[0090] On day 2, 6 μmol / L TGFβ receptor inhibitor (SB431542) was added to the culture.

[0091] (Step 4)

[0092] On day 4, EBs were collected, centrifuged, and 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 then incubated at 37°C with 5% CO 2 incubated in an environment.

[0093] Table 3: Medium C

[0094] serial number Contents Final concentration 1 StemPro-34 - 2 GlutaMAX (100x) 1x(1％) 3 Monothioglycerol 400μmol / L 4 Ascorbic acid 2-phosphate 50 μg / mL 5 Insulin-Transferrin-Selenium (100x) 1x(1％) 6 hbFGF 50ng / mL 7 VEGF 50ng / mL 8 SCF 50ng / mL

[0095] (Step 5)

[0096] On days 6, 8, 11, and 13, cells in culture were collected, 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 then incubated at 37°C with 5% CO 2 incubated in an environment.

[0097] Table 4: Medium D

[0098] serial number Contents Final concentration 1 StemPro-34 - 2 GlutaMAX (100x) 1x(1％) 3 Monothioglycerol 400μmol / L 4 Ascorbic acid 2-phosphate 50 μg / mL 5 Insulin-Transferrin-Selenium (100x) 1x(1％) 6 hbFGF 50ng / mL 7 VEGF 50ng / mL 8 SCF 50ng / mL 9 TPO 100ng / mL 10 HkDJ 50ng / mL

[0099] (Step 6)

[0100] On day 14, single cell suspensions were prepared using a cell strainer and transferred to FcDLL4-coated plates. 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)).

[0101] Table 5: Medium E

[0102] serial number Contents Final concentration 1 α-MEM - 2 FBS 15％ 3 2-Mercaptoethanol 55μM 4 Insulin-Transferrin-Selenium (100x) 1x(1％) 5 Ascorbic acid 2-phosphate 50 μg / mL 6 SCF 50ng / mL 7 TPO 100ng / mL 8 IL-7 10 ng / mL 9 HkDJ 50ng / mL 10 SDF1α 240 ng / mL 11 p38i(SB203580) 15μM

[0103] On days 15, 18, 22, 25, 29, and 32, cells in culture were harvested, centrifuged, resuspended in fresh medium E, and transferred back to the same culture vessel. On days 21 and 28, cells were harvested, centrifuged, resuspended in fresh medium E, and transferred to new FcDLL4-coated plates.

[0104] (Step 7)

[0105] On day 35, after 21 days of culture, the hematopoietic cells differentiated into CD7, CD45 positive lymphocyte progenitor cells.

[0106] (Step 8)

[0107] The floating cells were collected and passed through a cell strainer, and the plate where the cells remained was washed with PBS. The floating cells and the PBS solution containing the cells were mixed, centrifuged, and resuspended in STEM-CELLBANKER (registered trademark). The cells were cryopreserved.

[0108] (Step 9)

[0109] 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)]. Cells were mixed with PBMCs at a ratio of 1:14 and cultured for 10 to 16 days. IL-15 and IL-7 were used as key raw materials for NK cell activation and expansion.

[0110] Table 6: Medium F

[0111] serial number Contents Final concentration 1 α-MEM - 2 FBS 15％ 3 Insulin-Transferrin-Selenium (100x) 1x(1％) 4 Ascorbic acid 2-phosphate 50 μg / mL 5 IL-7 10 ng / mL 6 IL-15 5ng / mL 7 PHA 2 μg / mL

[0112] During the 10-16 day culture period, the culture medium was replaced every 2-3 days with fresh culture 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]. When the cells grew well, the culture was divided into two parts and supplemented with fresh culture medium G. When sufficient growth was not observed, half of the culture was collected, centrifuged, resuspended in fresh culture medium G and returned to the initial plate.

[0113] Table 7: Medium G

[0114] serial number Contents Final concentration 1 α-MEM - 2 FBS 15％ 3 Insulin-Transferrin-Selenium (100x) 1x(1％) 4 Ascorbic acid 2-phosphate 50 μg / mL 5 IL-7 10 ng / mL 6 IL-15 5ng / mL

[0115] After 10-16 days of culture, the cells were harvested, washed three times with PBS by centrifugation, resuspended in cryoprotectant A as the final product (iCAR-ILC / N101), and stored frozen until use.

[0116] Table 8: Cryoprotectant A

[0117]

[0118] Stability of cryopreserved iCAR-ILC / N101

[0119] Table 9 shows the long-term stability of cryopreserved iCAR-ILC / N101.

[0120] Table 9

[0121]

[0122] iCAR-ILC / N101 cells were equally divided into 2×10 7 Cells / tube were collected and frozen in the vapor phase of a liquid nitrogen tank for 137 days. Viability, cell concentration, CAR positivity, product (NK) purity, IFN-γ production, endotoxin / mycoplasma / bacterial pathogen detection, and appearance were tested on days 0, 43, 57, 71, and 137. All test results passed the quality standards in Table 9.

[0123] Table 10 shows the transport stability of cryopreserved iCAR-ILC / N101.

[0124] Table 10

[0125]

[0126]

[0127] The same production batch of iCAR-ILC / N101 was divided into 2 × 10 7 / cell / tube) and frozen. Three randomly selected cryovials were transferred to the MEDi STAR refrigerated transport box of the cell processing facility and transported to the transporter facility 50 miles away. In the facility, the cryovials were transferred to the gas phase of the liquid nitrogen tank. The cryovials were transferred back to the refrigerated transport box, airlifted to another transporter facility 300 miles away, and returned to the cell processing facility as another airlift. The total distance is more than 600 miles. The cells were tested for viability, cell concentration, CAR positivity, product purity, IFN-γ production, endotoxin / mycoplasma / bacterial pathogen detection and appearance. All test results passed the quality standards shown in Table 10.

[0128] Table 11 shows the resuscitation stability of cryopreserved iCAR-ILC / N101.

[0129] Table 11

[0130]

[0131] The same production batch of iCAR-ILC / N101 was aliquoted (2 x 10 7 / cell / tube) and stored frozen. Three randomly selected frozen tubes were thawed and kept at room temperature. Cell viability, cell concentration, CAR positivity, product purity, IFN-γ production, endotoxin / mycoplasma / bacterial pathogen detection, and appearance were tested one tube at a time at 15, 30, and 90 minutes. All test results passed the quality standards at 15 minutes, however, at 30 minutes, although other test results passed the standards, the IFN-γ production capacity was significantly reduced.

[0132] Table 12 shows the stability of saline-diluted iCAR-ILC / N101 after thawing.

[0133] Table 12

[0134]

[0135]

[0136] Cryopreserved iCAR-ILC / N101 cells were thawed, diluted in saline, and kept at room temperature. The viable cell concentration and viability of the cells were tested. Even at 90 minutes, the viable cell concentration and viability did not decrease significantly.

[0137] Table 13 shows the standard tests of the final product (iCAR-ILC / N101).

[0138] Table 13

[0139]

[0140] As standard assays, sterility, cell number, cell viability, cell phenotype and secretion of IFN-γ were measured by flow cytometry, and functional assays as shown in Table 13 were used as reference assays.

Claims

1. A method for producing natural killer cells from iPS cells (induced pluripotent stem cells), comprising the following steps: (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 lymphocyte progenitor cells, and (iv) differentiating and expanding the lymphocyte progenitor cells into natural killer cells.

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

3. The method of claim 2, wherein a tracer gene is used to maintain or select CAR expression during differentiation, and CAR is stably expressed at the natural killer cell stage.

4. 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, Claudin18.2, B7-H3, NKG2D, GD2, EpCAM, ROBO-1, CD19, CD20, CD22, CD30, CD33, CD38, CD123, CD276, and CD269.

5. The method of claim 2, wherein the iPS cells are not cloned and differentiated from iPS cells having CAR.

6. The method of claim 2, wherein the CAR is transduced 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, a retroviral vector, an adenoviral vector or an AAV vector, and the non-viral vector is a piggyBac vector.

8. The method of claim 6, wherein the gene editing comprises using CRISPAR / CAS9, Talen, homologous recombination or other gene editing tools.

9. The method according to 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 in step 2 further comprises VEGF, hbFGF and SCF.

12. The method of claim 1, wherein the hematopoietic progenitor cells are cultured with 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 method of claim 1, wherein the lymphocyte progenitor cells are CD7 + CD45 + cell.

15. The method of claim 1, wherein the lymphocyte 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 according to the method of any one of claims 1 to 16.

18. A natural killer cell population comprising cells, wherein the cells are CD7 + CD45 + cell.

19. The natural killer cell population according to claim 18, wherein the natural killer cells contain CD7 + CD45 + The percentage of cells is greater than 60% of the cell number.

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

21. The natural killer cell population of claim 18, wherein contamination of undifferentiated iPSCs is less than 0.01% of the number of cells in the natural killer cells.

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

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

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

25. A method of treating cancer comprising administering the pharmaceutical composition according to any one of claims 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, hematological malignancies, or multiple myeloma.

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