Induced NK cell, and preparation method therefor and use thereof

Abstract

Provided are an induced NK cell, and a preparation method therefor and the use thereof. The method comprises mixing a CD34+ cell with a stromal cell, co-culturing the mixture to obtain an NK precursor cell, and subjecting the NK precursor cell to induction and amplification culture to obtain a mature NK cell, wherein the stromal cell comprises any one or a combination of at least two of an AFT024 cell, an MS5 cell, an OP9 cell, an HS-5 cell, an MSC cell, an MUTZ-3 cell, a stromal cell derived from tissues such as bone marrow or the liver, or a primary cell. Provided is the development of a brand-new method for inducing an NK cell on the basis of a CD34+ cell, which enables the induction of the NK cell with a high efficiency. The efficiency of outputting NK cells from a single CD34+ cell is improved, and the property of the induced NK cell is very close to that of a natural mature NK cell. They have uniform anti-tumor effects, can effectively kill tumor cells, and have broad application prospects.

Description

Induced NK cell, preparation method and application thereof TECHNICAL FIELD

[0001] The present application belongs to the technical field of stem cell biology, and particularly relates to an induced NK cell, a preparation method and application thereof. BACKGROUND

[0002] Human natural killer (NK) cells are an important subpopulation of innate lymphoid cells (ILC) and an indispensable component of the innate immune system. NK cells, T cells and B cells all originate from the same lymphoid progenitor cells and are very similar in lineage differentiation. NK cells exert their functions mainly in four aspects: first, they release perforin, granzyme and other cytokines to directly kill abnormal cells; second, they eliminate abnormal cells through antibody-dependent cell-mediated killing mechanism (ADCC); third, they regulate the immune system by synthesizing and releasing cytokines to trigger adaptive immune regulation and thereby enhance the body's resistance systemically; and fourth, they can express chimeric antigen receptors (CAR) or T cell antigen receptors (TCR) in NK cells through gene editing or genetic engineering, so as to precisely target tumor cells for killing. Unlike T cells, NK cells have no HLA restriction requirement, so they can be allogeneic transfused and will not cause GvHD and obvious side effects after allogeneic NK infusion, thus they are increasingly valued in tumor adoptive therapy.

[0003] At present, NK cells need to be transfused in large doses and multiple doses to achieve a therapeutic effect, so in NK cell therapy, how to obtain NK cells with strong uniformity and large quantity is a great challenge. In existing reports, the following three methods are mainly used to obtain NK cells in large quantities in vitro: (1) using cytokine combinations or feeder cells to expand mature NK cells in large quantities, the mature NK cells mainly come from adult peripheral blood or umbilical cord blood, but due to individual differences, these mature NK cells are highly heterogeneous and are easily contaminated with T cells, so they face the problems of uneven therapeutic effect and threat to patient safety due to T cell residues in clinical use; (2) using NK cell lines (NK-92) to prepare therapeutic NK cells, but the NK-92 cell line is a tumor cell line, its safety is challenged, and it needs to be irradiated before use, which greatly reduces its activity; (3) using stem cells (pluripotent stem cells, hematopoietic stem progenitor cells, etc.) to generate functional NK cells through in vitro induction, this method can standardize the production of sufficient NK cells, meet the requirements of unlimited source and uniformity, and has the potential to become an "off-the-shelf" product.

[0004] Although great progress has been made in the technology of inducing NK cells from pluripotent stem cells (PSCs), the safety of PSCs has not been definitively determined, so it is difficult for NK cells induced from PSCs to obtain the recognition of regulatory agencies and clinicians. In recent years, great progress has been made in the technology of inducing NK cells from hematopoietic stem progenitor cells (CD34 + cells), and the current method is mainly single-layer induction, that is, hematopoietic stem progenitor cells are added with different cytokines and compounds in a culture dish to direct them to differentiate into mature NK cells. CN113046314A discloses a method for inducing and expanding decidual-like natural killer cells from human umbilical cord blood or bone marrow hematopoietic stem cells in vitro. CD34 + hematopoietic stem cells are cultured in vitro for four stages, stage I is the expansion of CD34+ hematopoietic stem cells, stage II is the induction of CD34+ hematopoietic stem cells to differentiate into NK precursor cells, stage III is the induction of NK precursor cells to differentiate into NK cells, and stage IV is the induction of NK cell expansion and maturation. This method does not report the specific yield and tumor killing activity; US11118165B2 discloses a method for inducing CD34 + cells into NK cells. This method adds different cytokines and compounds to a culture dish to direct CD34 + cells to differentiate into mature NK cells. After 6 weeks of induction, each CD34 + cell can produce 2000 NK cells; US20180237749A1 discloses a method for inducing CD34 + cells into NK cells. After 25 days of induction and differentiation, one CD34 + cell can produce 2500 NK cells. The current literature reports that the technology of inducing NK cells from CD34 + cells in a GMP environment has been realized. The best yield in a culture vessel after 35 days of induction is that each CD34 + cell can produce 4450 NK cells (https: / / doi.org / 10.1007 / s00262-023-03492-6). However, the induction efficiency seriously restricts the yield of induced NK cells, and the number of CD34 + cells from the same tissue source is still limited, which limits the actual clinical application of NK cells.

[0005] In summary, developing new high-efficiency induction technology to improve the induction efficiency of NK cells will greatly improve the efficiency of CD34 + cells and enhance the uniformity of induced NK cells, thereby promoting the application of NK cells.

[0006] SUMMARY

[0007] The present application provides an induced NK cell and a preparation method and application thereof, and establishes a new CD34 + cell technology for inducing NK cells, thereby efficiently inducing NK cells.

[0008] In a first aspect, the present application provides a method for preparing NK cells (Natural Killer cells) in vitro, comprising:

[0009] mixing CD34 + cells with stromal cells, co-culturing with an induction expansion medium to obtain NK precursor cells, and further culturing the NK precursor cells to obtain mature NK cells; the stromal cells include any one or a combination of at least two of AFT024 cells, MS5 cells, OP9 cells, HS-5 cells, MSC cells, MUTZ-3 cells, tissue-derived stromal cells or primary cells.

[0010] The present application designs a new method for inducing NK cells from hematopoietic stem and progenitor cells (HSPC, CD34 + cells), which efficiently induces CD34 + cells into NK precursor cells by stromal cells, and further induces the precursor cells to mature, thereby obtaining a large number of NK cells, significantly improving the efficiency of inducing CD34 + cells to produce NK cells, 10 6 CD34 + cells can obtain 10 8 -10 16 NK cells, which can meet the NK cell needs of multiple patients, greatly improving the uniformity of NK cells, and the obtained NK cells have high maturity and high CD16 expression ratio, transcriptome level analysis shows that the induced NK cells are similar to human natural NK cells, and the anti-tumor effect is uniform and can effectively kill tumor cells.

[0011] In the present application, the NK cells induced based on CD34 + cells are further named as hematopoietic stem and progenitor cell-induced natural killer cells (HiNK cells).

[0012] It can be understood that the CD34 + cells described in the present application are hematopoietic stem and progenitor cells, and the sources include but are not limited to umbilical cord blood, placenta, mobilized peripheral blood, bone marrow, CD34 + cells induced from pluripotent stem cells, or CD34 + cells obtained by reverse differentiation or transdifferentiation of blood cells, etc.

[0013] Preferably, the tissue comprises bone marrow and / or liver tissue.

[0014] Preferably, the induction expansion medium comprises any one or a combination of at least two of KBM581 medium, Opti-MEM medium, APEL2 medium, Essential 6 medium, DMEM-high glucose medium, DMEM / F12 medium, a-MEM medium, F-12 medium, EBM2 medium, MEM medium, BME medium, RPMI 1640 medium, G-MEM medium or a basal medium containing active substances.

[0015] Preferably, the active substances comprise any one or a combination of at least two of serum replacement, Insulin-like growth factor 1 (IGF-1), non-essential amino acids, glutamine, stabilized dipeptide of L-alanyl-L-glutamine, β-mercaptoethanol, sodium selenite, ethanolamine, Aryl hydrocarbon receptor antagonist, ascorbic acid or cell growth factors.

[0016] Preferably, the cytokines comprise any one or a combination of at least two of IL-3, IL-7, IL-2, SCF, IL-12, IL-15, IL-21, hTPO, IL-12, IL-18 or Flt3L.

[0017] Preferably, the CD34 + The culture mode of co-culturing the cells with the stromal cells comprises 3D culture.

[0018] It can be understood that the 3D culture refers to the general three-dimensional cell culture method in the art.

[0019] Preferably, the culture medium of the induction expansion culture is also the induction expansion medium.

[0020] Preferably, the stromal cells can also overexpress factors that facilitate the development, maturation, expansion or survival of lymphoid cells.

[0021] Preferably, the factors comprise human Notch ligands and / or human cytokines.

[0022] Preferably, the human Notch ligands comprise any one or a combination of at least two of DLL1, an active fragment of DLL1, DLL4 or an active fragment of DLL4.

[0023] Preferably, the human cytokine comprises any one of IL-15, IL-12, IL-2 or IL-21 or a combination of at least two thereof.

[0024] Preferably, the induction expansion culture can further comprise a step of adding feeder cells.

[0025] Preferably, the feeder cells comprise any one of K562-mbIL-21 expansion cell line, K562-mbIL-15-mbIL-21 expansion cell line, K562-mbIL-15-4-1BBL expansion cell line or K562-mbIL-21-4-1BBL expansion cell line or a combination of at least two thereof.

[0026] The time point of adding feeder cells is any time point during the induction expansion culture stage.

[0027] Preferably, the CD34 + cells are in a number of 5 x 10 2 - 1 x 10 6 , including but not limited to 10 x 10 2 , 10 x 10 3 or 10 x 10 4 , etc.

[0028] Preferably, the ratio of CD34 + cells to stromal cells is 1 : (10-500), including but not limited to 1 : 12, 1 : 15, 1 : 20, 1 : 30, 1 : 50, 1 : 80, 1 : 100, 1 : 200, 1 : 300, 1 : 350, 1 : 400 or 1 : 450, etc.

[0029] Preferably, the time of co-culture and induction expansion culture is independently 5-30 days, including but not limited to 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 22, 25, 26, 27, 28 or 29 days, etc.

[0030] Preferably, the mixing of CD34 + cells with stromal cells can further comprise a step of expanding CD34 + cells.

[0031] Preferably, the expanding of CD34 + cells comprises:

[0032] seeding CD34 + cells into CD34 + cell expansion medium for culture, or,

[0033] seeding CD34 +The cells are co-cultured with AFT024 cells, AFT024-DLL1 cells, AFT024-DLL4 cells, or AFT024-DLL1-DLL4 cells.

[0034] Preferably, the CD34 + The cell expansion medium comprises StemMACS medium, StemSpan TM SFEMII medium, IMDM medium, Opti-MEM medium, Stemline medium, StemPro medium, QBSF-60 medium, XVIVO-15 medium, HPGM medium, RPMI medium, Hematopoietic Progenitor Expansion Medium XF medium (PromoCell), hPSC XF medium, Human Embryonic Hematopoietic Stem Cell Serum Free Expansion media (CELPROGEN) medium any one of, or a combination of at least two of, the base medium containing active substances.

[0035] Preferably, the active substances comprise any one of, or a combination of at least two of, a serum replacement, insulin-like growth factor 1, non-essential amino acids, glutamine, a stabilizing dipeptide of L-alanyl-L-glutamine, beta-mercaptoethanol, sodium selenite, ethanolamine, an aryl hydrocarbon receptor antagonist, ascorbic acid, or a cell growth factor.

[0036] Preferably, the cell growth factor comprises any one of, or a combination of at least two of, IL-2, IL-3, IL-7, SCF, hTPO, IL-6, IL-10, IL-11, IL-12, or Flt3L.

[0037] As a preferred technical solution, the method for preparing NK cells in vitro comprises the following steps:

[0038] (1) inoculating the CD34 + cells into an expansion medium for culture, or

[0039] inoculating the CD34 + cells into an expansion medium for culture, or

[0040] (2) mixing the CD34 + cells obtained in step (1) with stromal cells for co-culture to obtain HiNK precursor cells;

[0041] The stromal cells include any one of AFT024 cells, MS5 cells, OP9 cells, HS-5 cells, MSC cells, MUTZ-3 cells, stromal cells or primary cells derived from tissues such as bone marrow or liver, or a combination of at least two of them; and

[0042] (3) inducing and expanding the NK precursor cells obtained in step (2) to obtain NK cells.

[0043] In a second aspect, the present application provides an induced NK cell, which is prepared by the method for preparing NK cells in vitro of the first aspect.

[0044] In a third aspect, the present application provides the use of the method for preparing NK cells in vitro of the first aspect or the induced NK cell of the second aspect in the preparation of a drug for treating tumors.

[0045] Preferably, the tumor includes, but is not limited to, lymphoma (including Hodgkin's and non-Hodgkin's lymphoma), chronic or acute leukemia (including acute myeloid leukemia, chronic myeloid leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia), breast cancer, ovarian cancer, liver cancer, glioma, pancreatic cancer, lung cancer, colon cancer, rectal cancer, melanoma, renal cancer, bladder cancer, head and neck cancer, gastric cancer, nasopharyngeal cancer, laryngeal cancer, cervical cancer, uterine body tumor, osteosarcoma, bone cancer, skin cancer, prostate cancer, uterine cancer, anal cancer, testicular cancer, fallopian tube cancer, endometrial cancer, vaginal cancer, vulvar cancer, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, pediatric solid tumor, bladder cancer, kidney or ureter cancer, renal pelvis cancer, central nervous system (CNS) tumor, primary CNS lymphoma, tumor angiogenesis, spinal tumor, brain stem neuroglioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, various cancers induced by the environment, including asbestos-induced cancer, and combinations of the above cancers.

[0046] Compared with the prior art, the present application has at least the following beneficial effects:

[0047] (1) The present application designs a new method for inducing HiNK cells from hematopoietic stem / progenitor cells (CD34 + cells), which has a high efficiency of inducing CD34 + cells to produce HiNK cells and a large number of HiNK cells obtained, i.e., 10 6 HiNK cells can be obtained from 10 + CD34 8 cells. 16A single HiNK cell can meet the HiNK cell needs of multiple patients, greatly improving the uniformity of HiNK cells, and meeting the on-demand and general requirements of clinical immune cell preparations at the same time;

[0048] (2) The HiNK cells obtained by the application have high maturity, high CD16 expression ratio, similar performance to natural NK cells, uniform anti-tumor effect, and can effectively kill tumor cells. BRIEF DESCRIPTION OF DRAWINGS

[0049] Figure 1 is a flowchart of the induction and differentiation of CD34 + cells into HiNK cells.

[0050] Figure 2 is a graph of the expansion ratio of CD34 + cells.

[0051] Figure 3 is a flow cytometric phenotype result graph of HiNK precursor cells (CD45 + CD56 - CD34 + CD7 + , CD45 + CD56 - CD34 + CD7 - , CD45 + CD34 - CD7 + CD56 + ) at day 14 of induction by the 3D system.

[0052] Figure 4 is a flow cytometric phenotype (CD56, CD16) result graph of HiNK cells at day 7, day 14 and day 21 of the maturation and expansion stage.

[0053] Figure 5A is a graph of the proportion of HiNK cells (CD56 + ) in the maturation and expansion stage of HiNK cells.

[0054] Figure 5B is a graph of the number of HiNK cells obtained from 10 6 CD34 + cells.

[0055] Figure 6 is a graph of the in vitro killing of solid tumor cell line A1847 (ovarian cancer) by HiNK cells.

[0056] Figure 7A is a graph of the in vitro killing of hematological tumor cell line K562 (myeloid leukemia) by HiNK cells.

[0057] Figure 7B is a graph of the in vitro killing of hematological tumor cell line MOLM-13 (myeloid leukemia) by HiNK cells.

[0058] Figure 7C is a graph of the results of in vitro killing of the hematological tumor cell line HL-60 (myeloid leukemia) by HiNK cells.

[0059] Figure 7D is a graph of the results of in vitro killing of the hematological tumor cell line THP1 (myeloid leukemia) by HiNK cells.

[0060] Figure 7E is a graph of the results of in vitro killing of the hematological tumor cell line Nalm-6 (B-cell leukemia) by HiNK cells.

[0061] Figure 8 is a graph of the results of in vivo killing of A1847 (ovarian cancer) by HiNK cells. DETAILED DESCRIPTION

[0062] The technical solutions of the present application are further described below in conjunction with the accompanying drawings and through specific embodiments. However, the following examples are merely simple examples of the present application and do not represent or limit the protection scope of the present application, and the protection scope of the present application is subject to the claims.

[0063] If a specific technology or condition is not specified in the embodiments, the technology or condition is performed according to the technology or condition described in the literature in the art or according to the product manual. If the reagent or instrument used is not specified by the manufacturer, it is a conventional product that can be purchased through a regular channel.

[0064] The present application designs a brand new technology for inducing NK cells from hematopoietic stem / progenitor cells (CD34 + cells) to achieve efficient induction of NK cells and improve the efficiency of NK cell output from a single CD34 + cell. The flowchart is shown in Figure 1.

[0065] Step 1: Expand human hematopoietic stem / progenitor cells (CD34 + cells), and expand the CD34 + cells using expansion medium and / or feeder cells for 1-30 days.

[0066] Sources of CD34 + cells include, but are not limited to, umbilical cord blood, placenta, mobilized peripheral blood, bone marrow, CD34 + cells obtained by inducing pluripotent stem cells, CD34 + cells obtained by reverse differentiation or transdifferentiation of blood cells.

[0067] Expansion medium for CD34 + cells:

[0068] The CD34 + cell expansion medium can be selected from StemMACS, StemSpan TMSFEMII, IMDM, Opti-MEM, Stemline, StemPro, QBSF-60, XVIVO-15, HPGM, RPMI, Hematopoietic Progenitor Expansion Medium XF, hPSC XF Medium, Human Embryonic Hematopoietic Stem Cell Serum Free Expansion Media and any combination thereof. Can be basal medium supplemented with one or more of serum replacement, insulin-like growth factor 1, non-essential amino acids, glutamine, stabilized dipeptide of L-alanyl-L-glutamine, beta-mercaptoethanol, sodium selenite, ethanolamine, aryl hydrocarbon receptor antagonist, ascorbic acid, UM171, and cell growth factors including any one or a combination of at least two of TPO, IL-7, IL-10, IL-2, IL-3, SCF, Flt3L, IL-11, IL12, IL-6.

[0069] Expansion of CD34 + Cells in a manner comprising, but not limited to, the following:

[0070] 1. CD34 + Cells are expanded in culture using a medium for a period of 1-30 days.

[0071] 2. CD34 + Cells are co-cultured with AFT024 cells or AFT024-DLL1 cells or AFT024-DLL4 cells or AFT024-DLL1-DLL4 cells to expand CD34 + Cells for a period of 1-30 days.

[0072] Step 2: 3D induction of CD34 + Cells to differentiate into HiNK cell precursors and HiNK cells. CD34 + Cells are mixed with stromal cells in a ratio to make a 3D induction system, which is cultured on exogenous stromal or scaffolds for a period of 5-30 days.

[0073] 1. CD34 + Cells can be freshly prepared, unexpanded, or expanded fresh, or unexpanded, or expanded cryopreserved.

[0074] 2. The stromal cell species include, but not limited to, at least one of AFT024, MS5, OP9, HS-5, MSC, MUTZ-3, bone marrow-derived stromal cell lines or primary cells; the stromal cells can be modified to express factors that favor the development, maturation, expansion, and survival of lymphoid cells, including, but not limited to, at least one of human Notch ligands: DLL1 (Delta Like Canonical Notch Ligand 1) and / or DLL4 (Delta Like Canonical Notch Ligand 4), or active fragments thereof, human cytokines: IL-15, IL-2, IL-12, or IL-21, in a secreted form or anchored to the cell membrane (Membrane-bound).

[0075] 3. CD34 + cells are mixed at a ratio of 1: (10-500). + The number of cells can be in the range of 5 x 10 2 - 1 x 10 6 cells.

[0076] 4. The exogenous stromal or scaffold is a filter membrane with a pore size of no more than 3 pm made of any material, including, but not limited to, polycarbonate or polyester film.

[0077] 5. The 3D induction time is 5-30 days, and the induced cell phenotype is at least one of CD34 + , CD34 + CD7 + , CD34 - CD7 + , CD7 + CD56 + , or CD7 - CD56 + .

[0078] 6. The HiNK induction medium: the base medium can be selected from KBM581, Opti-MEM, APEL2, Essential 6, DMEM-high glucose, DMEM / F12, a-MEM, F-12, EBM2, MEM, BME, RPMI 1640, G-MEM, and any combination thereof. The base medium can be supplemented with one or more of the following: one or more serum substitutes, one or more non-essential amino acids, glutamine or stabilized dipeptide of L-alanyl-L-glutamine, β-mercaptoethanol, sodium selenite, ethanolamine, ascorbic acid, and cell growth factors including any one or a combination of at least two of IL-3, IL-7, IL-2, SCF, IL-15, IL-21, IL-12, IL-18, and Flt3L.

[0079] Step 3: Inducing maturation of HiNK precursor cells and expanding HiNK cells. The 3D system-induced cells are prepared into a single cell suspension, and after centrifugation, the cell supernatant is discarded, and the cell pellet is resuspended in an induction maturation and expansion medium, transferred into a culture vessel, and induced to mature and expand HiNK cells using the HiNK induction medium. The culture time is 5-30 days.

[0080] The induction maturation and expansion methods include, but are not limited to, direct induction maturation and expansion using the induction medium or combined induction maturation and expansion with feeder cells, which include, but are not limited to, K562-mbIL-21 expansion cell lines, K562-mbIL-15-mbIL-21 expansion cell lines, K562-mbIL-15-4-1BBL expansion cell lines, and / or K562-mbIL-21-4-1BBL expansion cell lines. The feeder cells are added at any time point during the induction maturation and expansion stage.

[0081] The culture vessels used for induction maturation and expansion include, but are not limited to, any size / model of culture dishes, any size / model of culture plates, any size / model of culture bottles, any size of culture bags, and any size of fermenters.

[0082] Example 1

[0083] This example expands CD34 + cells.

[0084] Alternatively, first, irradiated AFT024 cells, AFT024-DLL1 cells, and AFT024-DLL4 cells are seeded in 24-well plates at a density of 100,000 cells / well, and then CD34 + cells (10,000) are plated on the above-mentioned stromal cells for co-culture, and the culture medium is a base medium.+ Cells were counted for CD34 + cells at Day 7 and Day 14 of expansion, and the results are shown in Figure 2.

[0085] The results show that CD34 + cells expanded 54-fold in basal medium and 68-69-fold in the stromal cell co-culture environment. After 14 days of expansion, CD34 + cells expanded 522-fold in basal medium and 915-941-fold in the stromal cell co-culture environment.

[0086] Example 2

[0087] This example generated HiNK precursor cells based on a 3D culture system.

[0088] CD34 + cells obtained in Example 1 were mixed with OP9 (ATCC, Cat. No. CRL-2749) feeder cells at a ratio of 1:20, and the total cell amount was 210,000, to prepare a 3D induction system. Each 3D system occupied a culture surface area of 0.5 cm 2 . The prepared 3D system was placed on a filter membrane made of polycarbonate, and the filter membrane was supported in a culture plate. The induction expansion medium (KBM581 basal medium supplemented with 15% SUPERGROW cell culture supplement, 2 mM GlutaMAX TM , 1 mM beta-mercaptoethanol, 5 ng / mL sodium selenite, 50 mM ethanolamine, 20 pg / mL ascorbic acid, 1% Penicillin-Streptomycin Solution, 5 ng / mL IL-3, 20 ng / mL SCF, 20 ng / mL IL-7, 10 ng / mL IL-15, and 10 ng / mL Flt3L) was added to the culture plate, and induction was performed for 14 days, with medium replacement every 2 days. Flow cytometry was performed to detect HiNK cell precursor cells (CD45 + CD56 - CD34 + CD7 + , CD45 + CD56 - CD34 + CD7 - , CD45 + CD34 - CD7 + CD56 + ) at Day 14 of induction, and the results are shown in Figure 3.

[0089] The results showed that after 14 days of 3D culture, CD34 + The cells can effectively induce NK precursor cells (CD45). + CD56 - CD34 + / - CD7 + ) and NK cells (CD45) + CD56 + ).

[0090] Example 3

[0091] This embodiment describes the induction, maturation, and expansion of NK precursor cells.

[0092] The cells cultured in the 3D system to day 14 in Example 2 were prepared into a single-cell suspension. The cells were then transferred to a culture bag and cultured in an induction amplification medium (KBM581 basal medium supplemented with 15% SUPERGROW cell culture additive and 2 mM GlutaMAX). TM Cells were cultured with 1 μM β-mercaptoethanol, 5 ng / mL sodium selenite, 50 μM ethanolamine, 20 μg / mL ascorbic acid, 1% Penicillin-Streptomycin Solution, 5 ng / mL IL-3, 20 ng / mL SCF, 20 ng / mL IL-7, 10 ng / mL IL-15, and 10 ng / mL Flt3L. The solution was replenished every 3 days to maintain a cell density of 1.5 × 10⁻³ L / mL. 6 This step can effectively induce HiNK cells (CD45). + CD3 - CD56 + The flow cytometry results are shown in Figure 4. The flow cytometry phenotypes (CD56, CD16) of HiNK cells in the maturation and expansion phases at Day 7, Day 14, and Day 21 were analyzed. The results showed that CD56... + The proportion gradually increased, eventually reaching 100%, CD56 + The number gradually increased, eventually reaching 10 6 CD34 + Cells can obtain 10 13 -10 14 One HiNK cell.

[0093] As shown in Figures 5A and 5B, the maturity of HiNK cells (CD56) generated after induction is [data missing]. + The proportion of cells increased over time, eventually reaching nearly 100%, and the number of HiNK cells produced also increased over time. 6CD34 + cells can be obtained 10 13 -10 14 HiNK cells.

[0094] Example 4

[0095] This example detects the protein expression of the HiNK cells induced in Example 3.

[0096] In this example, the classical activating receptors and inhibitory receptors of NK cells, the flow cytometry antibodies NKp30 (Biolegend, P30-15), NKp44 (Biolegend, P44-8), NKG2D (Biolegend, 1D11), CD94 (BD Biosciences, HP-3D9), NKG2A (Biolegend, S19004C) are used to perform cell membrane staining on HiNK cells and natural NK cells (umbilical cord blood-derived NK, UCB-NK), and then the corresponding antigen expression amount is detected by flow cytometry after antibody incubation and cell combination. The results are shown in Table 1, and FMO refers to the negative control sample, which is used to define the fluorescence intensity range of receptor expression. The results show that HiNK cells can express activating receptors and inhibitory receptors of NK cells as well as umbilical cord blood naturally matured NK cells.

[0097] Table 1

[0098] Example 5

[0099] In this example, it is verified that the induced HiNK cells can kill solid tumor cells and blood tumor cells in vitro.

[0100] The HiNK cells obtained in Example 3 are co-incubated with A1847 (an ovarian cancer cell line) to evaluate the ability of HiNK cells to kill tumor cells (Cytotoxicity). The specific results are shown in Figure 6, where the X-axis represents different E:T ratios (HiNK cells (Effector, E): tumor cells (Target, T)); the Y-axis shows the percentage of dead tumor cells in the number of tumor cells; after 4h of co-incubation, the proportion of dead tumor cells in the tumor cells in each sample is detected by flow cytometry, which is the killing ability of HiNK cells (Cytotoxicity %). Each E:T repeat sample is 4, and the killing ability of HiNK cells is: A1847: 21.0% (1:1), 60.7% (5:1), 76.2% (10:1), indicating that the killing ability of HiNK cells is not weaker than that of natural NK cells (UCB-NK).

[0101] Example 6

[0102] This embodiment utilizes the induced HiNK cells to kill blood tumor cells in vitro.

[0103] The HiNK cells obtained in Example 3 were co-incubated with K562 (myeloid leukemia tumor), MOLM-13 (myeloid leukemia tumor), HL-60 (myeloid leukemia tumor), THP1 (myeloid leukemia tumor), and Nalm-6 (B-cell leukemia tumor), respectively, to evaluate the cytotoxicity of the HiNK cells, and the results are shown in FIGS. 7A-7E, which show the results of the killing of tumor cell lines such as K562, MOLM-13, HL-60, THP1, and Nalm-6 by the HiNK cells in vitro; wherein the X axis represents different E:T ratios (HiNK cells (Effector, E): tumor cells (Target, T)); the Y axis shows the percentage of dead tumor cells in the tumor cells; after co-incubation for 4 h, the flow cytometer was used to detect the proportion of dead tumor cells in the tumor cells in each sample, and this proportion was the cytotoxicity of the HiNK cells (%); each E:T sample was repeated 4 times, and the cytotoxicity of the HiNK cells was as follows: K562: 37.4% (1:1), 64.0% (5:1), and 74.1% (10:1); MOLM-13: 49.6% (1:1), 78.1% (5:1), and 81.2% (10:1); HL-60: 28.7% (1:1), 71.6% (5:1), and 74.6% (10:1); THP2: 55.0% (1:1), 87.1% (5:1), and 89.0% (10:1); and Nalm-6: 51.3% (1:1), 78.4% (5:1), and 85.5% (10:1), indicating that the cytotoxicity of the HiNK cells was not weaker than that of natural NK cells.

[0104] Example 7

[0105] This embodiment verifies the tumor-killing effect of the HiNK cells in vivo.

[0106] To evaluate the anti-tumor function of the HiNK cells in vivo, a tumor model was constructed in 8-week-old female B-NDG mice (severe immunodeficient mice, Boyo Setu) using A1847 cells (an ovarian cancer cell line, 200,000 cells were injected intraperitoneally into each mouse), and after the tumor load was observed by a small animal imaging system, the mice were randomly divided into 3 groups, 4 mice in each group, and the HiNK cells were injected intraperitoneally into the tumor model, 10 million cells were injected intraperitoneally into each mouse, and an equal amount of UCB-NK cells were injected as a control, and then the tumor load of the treated mice was detected by the small animal imaging system every week.

[0107] As shown in Figure 8, similar to UCB-NK cells, HiNK cells can effectively alleviate the tumor burden of mice, while the tumor burden of mice without injection is increasing, indicating that HiNK cells can kill tumor cells in vivo and reduce the tumor burden of mice.

[0108] In summary, the present application develops a new method for inducing NK cells based on hematopoietic stem / progenitor cells (CD34 + cells), which can efficiently induce NK cells, improve the efficiency of NK cells from a single CD34 + cell, and obtain NK cells with performance close to that of naturally mature cells, uniform anti-tumor effect, and effective killing of tumor cells, which has broad application prospects.

[0109] The applicant states that the above is only a specific embodiment of the present application, but the protection scope of the present application is not limited thereto. It should be understood by those skilled in the art that any changes or replacements within the technical scope disclosed by the present application can be easily thought of by any person skilled in the art, and falls within the protection scope and disclosure scope of the present application.

Claims

1. A method of in vitro induced production of NK cells, comprising: CD34 + The CD34 + The CD34 + The CD34 + The CD34 + The CD34 + The CD34 + The CD34 + The CD34 + The CD34 + The CD34 + The CD34 + The CD34 + The CD34 <000 wherein the stromal cells comprise any one of or a combination of at least two of AFT024 cells, MS5 cells, OP9 cells, HS-5 cells, MSC cells, MUTZ-3 cells, tissue-derived stromal cells, or primary cells.

2. The method for preparing NK cells in vitro by induction according to claim 1, wherein, CD34 + Sources of cells include umbilical cord blood, placenta, mobilized peripheral blood, bone marrow, CD34 + cells obtained by re-differentiation, transdifferentiation of blood cells. Any one or a combination of at least two of the CD34 + cells.

3. The method for preparing NK cells in vitro by induction according to claim 1 or 2, wherein, the tissue comprises bone marrow and / or liver tissue; preferably, the induction expansion medium comprises any one of or a combination of at least two of KBM581 medium, Opti-MEM medium, APEL2 medium, Essential 6 medium, DMEM-high glucose medium, DMEM / F12 medium, a-MEM medium, F-12 medium, EBM2 medium, MEM medium, BME medium, RPMI 1640 medium, G-MEM medium, or a basal medium containing active substances; preferably, the active substances comprise any one of or a combination of at least two of serum replacement, insulin-like growth factor 1, non-essential amino acids, glutamine, L-alanyl-L-glutamine stabilizing dipeptide, beta-mercaptoethanol, sodium selenite, ethanolamine, aryl hydrocarbon receptor antagonist, ascorbic acid, or a cell growth factor; preferably, the cell growth factor comprises any one of or a combination of at least two of IL-3, IL-7, IL-2, SCF, IL-12, IL-15, IL-21, hTPO, IL-12, IL-18, or Flt3L.

4. The method for producing NK cells in vitro according to any one of claims 1 to 3, wherein, the co-culturing culture mode comprises 3D culture.

5. The method for producing NK cells in vitro according to any one of claims 1 to 4, wherein, the stromal cells further overexpress a factor that facilitates development, maturation, expansion, or survival of lymphoid cells; preferably, the factor comprises a human Notch ligand and / or a human cytokine; preferably, the human Notch ligand comprises any one of or a combination of at least two of DLL1, DLL1 active fragment, DLL4, or DLL4 active fragment; preferably, the human cytokine comprises any one of or a combination of at least two of IL-15, IL-12, IL-2, or IL-21.

6. The method for producing NK cells in vitro according to any one of claims 1 to 5, wherein, the induction expansion culture further comprises a step of adding feeder cells; preferably, the feeder cells comprise any one of or a combination of at least two of K562-mbIL-21 expansion cell line, K562-mbIL-15-mbIL-21 expansion cell line, K562-mbIL-15-4-1BBL expansion cell line, or K562-mbIL-21-4-1BBL expansion cell line.

7. The method for producing NK cells in vitro according to any one of claims 1 to 6, wherein, The CD34 + The number of cells was 5 x 10 2 -1 x 10 6 cells; Preferably, said CD34 + The ratio of cells to stromal cells is 1: (10-500); preferably, the time of the co-culturing and the induction expansion culture is independently 5-30 days.

8. The method for producing NK cells in vitro according to any one of claims 1 to 7, wherein, CD34 + cells are mixed with stromal cells also includes a step of expanding the CD34 + cells prior to mixing with stromal cells. Preferably, the expansion of CD34 + cells comprises: CD34 + Cells seeded into CD34 + Cultured in cell expansion medium, or... CD34 + cells were co-cultured with AFT024 cells, AFT024-DLL1 cells, AFT024-DLL4 cells, or AFT024-DLL1-DLL4 cells; Preferably, said CD34 + Cell expansion media include StemMACS media, StemSpan media, StemPro media, X-VIVO 15 media, RPMI media, Hematopoietic Progenitor Expansion Medium XF media, Iscove's Modified Dulbecco's Medium (IMDM) media, Opti-MEM media, Stemline media, QBSF-60 media, HPGM media, and SFEM II media. TM Cell expansion media include StemMACS media, StemSpan media, StemPro media, X-VIVO 15 media, RPMI media, Hematopoietic Progenitor Expansion Medium XF media, Iscove's Modified Dulbecco's Medium (IMDM) media, Opti-MEM media, Stemline media, QBSF-60 media, HPGM media, and SFEM II media. any one of or a combination of at least two of hPSC XF medium, Human Embryonic Hematopoietic Stem Cell Serum Free Expansion Media medium, basal medium containing active substances. Preferably, the active substance comprises any one or a combination of at least two of serum replacement, insulin-like growth factor 1, non-essential amino acids, glutamine, stabilized dipeptide of L-alanyl-L-glutamine, β-mercaptoethanol, sodium selenite, ethanolamine, aryl hydrocarbon receptor antagonist, ascorbic acid or a cell growth factor. Preferably, the cell growth factor comprises any one or a combination of at least two of IL-2, IL-12, IL-3, IL-7, SCF, IL-6, IL-10, IL-11, hTPO or Flt3L.

9. The method for producing NK cells in vitro by induction according to any one of claims 1 to 8, wherein, The method comprises the following steps: (1) CD34 + cells are seeded into expansion medium for culture, or, CD34 + cells were co-cultured with AFT024 cells, AFT024-DLL1 cells, AFT024-DLL4 cells, or AFT024-DLL1-DLL4 cells; (2) obtaining CD34 + The cells are mixed with stromal cells for co-culture to obtain NK precursor cells; The stromal cells comprise any one or a combination of at least two of AFT024 cells, MS5 cells, OP9 cells, HS-5 cells, MSC cells, MUTZ-3 cells, stromal cells or primary cells derived from bone marrow or liver tissue; and (3) inducing and expanding the NK precursor cells obtained in step (2) to obtain NK cells. 10.Induced NK cells prepared by the method for preparing NK cells in vitro of any one of claims 1-9.

11. Use of the method for preparing NK cells in vitro of any one of claims 1-9 or the induced NK cells of claim 10 in the preparation of a medicament for treating tumors.

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