An inducible NK cell, a preparation method thereof and an application thereof

Abstract

The present invention relates to an inducible NK cell and its preparation method and application. The method includes mixing CD34+ cells with stromal cells for co-culture to obtain NK precursor cells, and taking the NK precursor cells for induced expansion culture 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, stromal cells or primary cells derived from tissues such as bone marrow or liver. The present invention develops a brand-new method for inducing NK cells based on CD34+ cells, which can efficiently induce the production of NK cells. It improves the efficiency of outputting NK cells from a single CD34+ cell. The performance of the induced NK cells is very close to that of naturally mature NK cells, with a uniform anti-tumor effect, can effectively kill tumor cells, and has broad application prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of stem cell biology, and relates to an inducible NK cell, a preparation method thereof, and an application thereof. Background Art

[0002] Human natural killer (NK) cells are an important subset of innate lymphoid cells (ILCs) and an indispensable part of the innate immune system. NK cells, T cells, and B cells all originate from the same lymphoid progenitor cell and are very similar in lineage differentiation. NK cells exert their functions mainly in four aspects. First, they directly release cytokines such as perforin and granzyme after directly contacting abnormal cells, thereby directly killing them. Second, they eliminate abnormal cells through the antibody-dependent cell-mediated killing mechanism (ADCC). Third, they regulate the immune system by synthesizing and releasing cytokines, triggering adaptive immune regulation, and thus systematically enhancing the body's resistance. Fourth, chimeric antigen receptors (CARs) or T cell antigen receptors (TCRs) can be expressed in NK cells by gene editing or genetic engineering methods, thereby precisely targeting tumor cells for killing. Different from T cells, NK cells do not have HLA restriction requirements, so allogeneic infusion can be performed, and allogeneic NK will not cause GvHD and obvious side effects after infusion, so it has been paid more and more attention in adoptive tumor therapy.

[0003] At present, large doses and multiple infusions of NK cells are required to achieve therapeutic effects. Therefore, in NK cell therapy, how to obtain NK cells with strong homogeneity and large quantity is a major challenge. Currently, there are mainly three methods for obtaining NK cells on a large scale in vitro: First, mature NK cells are massively expanded using cytokine combinations or feeder cells. Mature NK cells mainly come from adult peripheral blood or cord blood. However, due to individual differences, these mature NK cells are highly heterogeneous and are easily contaminated with T cells. Therefore, in clinical use, there are problems such as uneven therapeutic effects and threats to patient safety due to T cell residues. Second, therapeutic NK cells are prepared by massively culturing the NK cell line (NK-92). However, as a tumor cell line, the safety of the NK-92 cell line is challenged, and it needs to be irradiated before use, which greatly reduces its activity. Third, functional NK cells are generated from stem cells (pluripotent stem cells, hematopoietic stem and progenitor cells, etc.) through an in vitro induction approach. This method can standardize the production of sufficient NK cells, meet the requirements of unlimited sources and homogeneity, and has the potential to become an "off-the-shelf" product.

[0004] Although the technology of inducing NK cells from pluripotent stem cells (PSCs) has made great progress, the safety issue of PSCs has not been clearly defined. Therefore, NK cells induced from PSCs are difficult to be recognized by regulatory agencies and clinicians. In recent years, great progress has been made in the technology of inducing NK cells from hematopoietic stem and progenitor cells (CD34 + cells). The current methods mainly involve monolayer induction, that is, hematopoietic stem and progenitor cells are directed to differentiate into mature NK cells in a culture dish by adding different cytokines and compounds. For example, CN113046314A discloses a method for in vitro inducing and amplifying decidual-like natural killer cells from human umbilical cord blood or bone marrow hematopoietic stem cells. The CD34 + hematopoietic stem cells are cultured in vitro in four stages. Stage I is the amplification of CD34+ hematopoietic stem cells, stage II is the induction of CD34+ hematopoietic stem cells to differentiate into NK progenitor cells, stage III is the induction of NK progenitor cells to differentiate into NK cells, and stage IV is the induction of NK cell amplification and maturation. This method does not report the specific yield and tumor killing activity. For example, US11,118,165B2 discloses a method for inducing CD34 + cells into NK cells. This method directs CD34 + cells to differentiate into mature NK cells in a culture dish by adding different cytokines and compounds. After 6 weeks of induction, each CD34 + cell can produce 2,000 NK cells. For example, US20180237749A1 discloses a method for inducing CD34 + cells into NK cells. After 25 days of induced differentiation, one CD34 + cell can produce 2,500 NK cells. Currently, the literature reports that the technology of inducing NK cells from CD34 + cells has been realized in a GMP environment. The best yield after 35 days of induction in a culture container is that each CD34 + cell can produce 4,450 NK cells (https: / / doi.org / 10.1007 / s00262-023-03492-6). However, the induction efficiency severely restricts the yield of induced NK cells. The number of CD34 + cells from the same tissue source is still limited, which restricts the actual clinical application of NK cells.

[0005] In summary, developing a new highly efficient induction technology to improve the induction efficiency of NK cells will greatly improve the utilization efficiency of CD34 + cells, enhance the uniformity of induced NK cells, and promote the application of NK cells. Summary of the Invention

[0006] To solve the above technical problems, the present invention provides an induced NK cell, a preparation method and an application thereof, and establishes a technology for inducing NK cells with new CD34 + cells, and efficiently induces the production of NK cells.

[0007] To achieve this purpose, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides a method for in vitro inducing and preparing NK cells (natural killer cells), and the method includes:

[0009] Mix CD34 + cells with stromal cells, and perform co-culture with an induction and amplification medium to obtain NK precursor cells, and take the NK precursor cells to continue to perform induction and amplification culture to obtain mature NK cells; the stromal cells include any one or at least two combinations 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 invention designs a new method for inducing NK cells with human hematopoietic stem and progenitor cells (HSPC, CD34 + cells). Through stromal cells, CD34 + cells are efficiently induced into NK precursor cells, and then the precursor cells are further induced to mature, and a large number of NK cells are amplified, 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 requirements of multiple patients, greatly improve the homogeneity of NK cells, the obtained NK cells have a high degree of maturity, a high expression ratio of CD16, and the induced NK cells are similar to human natural NK cells in transcriptome level analysis, and the anti-tumor effect is uniform and can effectively kill tumor cells.

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

[0012] It can be understood that the CD34 + cells described in the present invention are hematopoietic stem and progenitor cells, and the sources include but are not limited to cord blood, placenta, mobilized peripheral blood, bone marrow, and CD34 +Obtaining CD34 by reverse differentiation or transdifferentiation of cells or blood cells + such as cells.

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

[0014] Preferably, the induction and amplification medium includes 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, α-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 include any one or a combination of at least two of serum substitute, 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 include 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 method of co-culturing the cells with stromal cells includes 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 medium for induction and amplification culture is also the induction and amplification medium.

[0020] Preferably, the stromal cells can also overexpress factors beneficial to the development, maturation, amplification, or survival of lymphoid cells.

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

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

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

[0024] Preferably, the induced amplification culture may further include the step of adding feeder cells.

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

[0026] The time point for adding the feeder cells is any time point during the induced amplification culture stage.

[0027] Preferably, the number of the CD34 + cells is 5×10 2 ~1×10 6 cells, including but not limited to 10×10 2 、10×10 3 or 10×10 4 and so on.

[0028] Preferably, the ratio of the CD34 + cells to the 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 for the co-culture and the induced amplification 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, and so on.

[0030] Preferably, before mixing the CD34 + cells with the stromal cells, it may further include the step of amplifying the CD34 + cells.

[0031] Preferably, the amplification of the CD34 + cells includes:

[0032] Inoculating the CD34 + cells into a CD34 + cell amplification medium for culture, or,

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

[0034] Preferably, the CD34 + cell expansion medium includes StemMACS medium, StemSpan TM SFEM II 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, or any one or a combination of at least two of the basal media containing active substances.

[0035] Preferably, the active substances include 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, aromatic hydrocarbon receptor antagonist, ascorbic acid, or cell growth factors.

[0036] Preferably, the cell growth factors include any one 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 in vitro inducing and preparing NK cells includes the following steps:

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

[0039] co-culture CD34 + cells with AFT024 cells, AFT024-DLL1 cells, AFT024-DLL4 cells, or AFT024-DLL1-DLL4 cells;

[0040] (2) Mix the CD34 + cells obtained in step (1) with stromal cells and conduct co-culture to obtain HiNK progenitor cells;

[0041] 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, stromal cells or primary cells derived from tissues such as bone marrow or liver.

[0042] (3) Perform induced expansion culture on the NK precursor cells obtained in step (2) to obtain NK cells.

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

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

[0045] Preferably, the tumors include but are not limited to lymphoma (including Hodgkin's and non-Hodgkin's lymphoma), chronic or acute leukemia (including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia), breast cancer, ovarian cancer, liver cancer, glioma, pancreatic cancer, lung cancer, colon cancer, rectal cancer, melanoma, kidney cancer, bladder cancer, head and neck cancer, gastric cancer, nasopharyngeal cancer, laryngeal cancer, cervical cancer, uterine body cancer, osteosarcoma, bone cancer, skin cancer, prostate cancer, uterine cancer, anal canal 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 tumors, bladder cancer, kidney or ureteral cancer, renal pelvic cancer, central nervous system (CNS) tumors, primary CNS lymphoma, tumor angiogenesis, spinal tumors, brainstem glioma, 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 invention has at least the following beneficial effects:

[0047] (1) The present invention designs a new method for inducing HiNK cells from hematopoietic stem and progenitor cells (CD34 + cells), and the efficiency of inducing CD34 + cells to produce HiNK cells is high, and the number of induced HiNK cells is large. 10 6 CD34 + cells can obtain 10 8 -10 16A single HiNK cell can meet the HiNK cell requirements of multiple patients, greatly improving the homogeneity of HiNK cells and meeting the clinical demand for off-the-shelf and universal immune cell preparations.

[0048] (2) The HiNK cells obtained in this invention have a high degree of maturity, a high proportion of CD16 expression, similar performance to natural NK cells, uniform anti-tumor effects, and can effectively kill tumor cells. Brief Description of the Drawings

[0049] Figure 1 For inducing CD34 + cells to differentiate into HiNK cells;

[0050] Figure 2 For CD34 + cell amplification fold result diagram;

[0051] Figure 3 For the flow cytometry phenotype result diagram of HiNK precursor cells (CD45 + CD56 - CD34 + CD7 + , CD45 + CD56 - CD34 + CD7 - , CD45 + CD34 - CD7 + CD56 + ) on the 14th day of 3D system induction;

[0052] Figure 4 For the flow cytometry phenotype (CD56, CD16) result diagram of HiNK cells on the 7th day (Day 7), 14th day (Day 14), and 21st day (Day 21) of the maturation and amplification stages;

[0053] Figure 5A For the proportion result diagram of HiNK cells (CD56 + ) during the maturation and amplification stages of HiNK cells;

[0054] Figure 5B For 10 6 CD34 + cell-derived HiNK cell quantity result diagram;

[0055] Figure 6 For the result diagram of HiNK cells killing solid tumor cell line A1847 (ovarian cancer) in vitro;

[0056] Figure 7AIt is the result diagram of the in vitro killing of the hematological tumor cell line K562 (myeloid leukemia) by HiNK cells;

[0057] Figure 7B It is the result diagram of the in vitro killing of the hematological tumor cell line MOLM-13 (myeloid leukemia) by HiNK cells;

[0058] Figure 7C It is the result diagram of the in vitro killing of the hematological tumor cell line HL-60 (myeloid leukemia) by HiNK cells;

[0059] Figure 7D It is the result diagram of the in vitro killing of the hematological tumor cell line THP1 (myeloid leukemia) by HiNK cells;

[0060] Figure 7E It is the result diagram of the in vitro killing of the hematological tumor cell line Nalm-6 (B-cell leukemia) by HiNK cells;

[0061] Figure 8 It is the result diagram of the in vivo killing of A1847 (ovarian cancer) by HiNK cells. Detailed implementation manners

[0062] The technical solution of the present invention will be further described below in conjunction with the accompanying drawings and through specific implementation manners. However, the following examples are only simple examples of the present invention and do not represent or limit the scope of the protection of the rights of the present invention. The scope of protection of the present invention shall be subject to the claims.

[0063] For those technical or conditions not specified in the examples, they shall be in accordance with the technologies or conditions described in the literature in this field or in accordance with the product specifications. For the reagents or instruments whose manufacturers are not specified, they are all conventional products that can be obtained through regular channels.

[0064] The present invention designs a technology for inducing NK cells from brand-new hematopoietic stem and progenitor cells (CD34 + cells) to achieve high-efficiency induction of NK cells and improve the efficiency of NK cell output per CD34 + cell. The process schematic diagram is as Figure 1 shown.

[0065] The first step: Amplify human hematopoietic stem and progenitor cells (CD34 + cells), and amplify the CD34 + cells for 1-30 days using an amplification medium or / and feeder cells.

[0066] CD34 + Cell sources: including but not limited to cord blood, placenta, mobilized peripheral blood, bone marrow, CD34 + cells obtained by inducing pluripotent stem cells, CD34 +Cells.

[0067] CD34 + Cell expansion medium:

[0068] CD34 + The cell expansion medium can be selected from: StemMACS, StemSpan TM SFEM II, 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. It can be a basal medium supplemented with the following substances: one or more serum substitutes, insulin-like growth factor 1, non-essential amino acids, glutamine, a stabilized dipeptide of L-alanyl-L-glutamine, β-mercaptoethanol, sodium selenite, ethanolamine, an aryl hydrocarbon receptor antagonist, ascorbic acid, UM171, and a cell growth factor such as one, two or more of TPO, IL-7, IL-10, IL-2, IL-3, SCF, Flt3L, IL-11, IL12, IL-6.

[0069] Amplify CD34 + The methods for amplifying CD34

[0070] 1. Culture and amplify CD34 + cells using the medium for 1 - 30 days.

[0071] 2. Co-culture CD34 + cells with AFT024 cells or AFT024-DLL1 cells or AFT024-DLL4 cells or AFT024-DLL1-DLL4 cells to amplify CD34 + cells for 1 - 30 days.

[0072] Second step: 3D induction of CD34 + cells to differentiate into HiNK cell precursors and HiNK cells. Mix CD34 + cells with stromal cells in a certain ratio to prepare a 3D induction system, and culture the 3D induction system on an exogenous matrix or scaffold for 5 - 30 days.

[0073] 1. CD34 + The cells can be freshly prepared and unamplified, or amplified and fresh, or cryopreserved and unamplified, or amplified and cryopreserved.

[0074] 2. The types of stromal cells include but are 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 above stromal cells can be modified in any way to express factors beneficial to the development, maturation, amplification, and survival of lymphoid cells, including but not limited to human Notch ligands: DLL1 (Delta Like Canonical Notch Ligand 1) and / or DLL4 (Delta Like Canonical Notch Ligand 4), or active fragments thereof, and human cytokines: at least one of IL-15, IL-2, IL-12, or IL-21. The cytokines are expressed in a secreted form or anchored to the cell membrane (membrane-bound) for expression.

[0075] 3. CD34 + The mixing ratio of CD34 cells to stromal cells is 1:(10 - 500). The number of CD34 cells + can be in the range of 5×10 2 ~1×10 6 cells.

[0076] 4. The exogenous matrix or scaffold is a filter membrane with a pore size not exceeding 3 μm prepared from any material, and the materials include but are not limited to polycarbonate or polyester film.

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

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

[0079] Step 3: Induce the maturation of HiNK precursor cells and expand HiNK cells. Prepare the cells induced by the 3D system into a single-cell suspension, collect it in a centrifuge tube, discard the cell supernatant after centrifugation, resuspend the cell pellet with the induction maturation and expansion medium, transfer it into a culture container, and use the HiNK induction medium to induce the maturation and expand HiNK cells. The culture time is 5 - 30 days.

[0080] The methods of induction maturation and expansion include, but are not limited to: directly using the induction medium for induction maturation and expansion or combining with feeder cells for induction maturation and expansion. The feeder cells include, but are not limited to: K562-mbIL-21 expansion cell line, K562-mbIL-15-mbIL-21 expansion cell line, K562-mbIL-15-4-1BBL expansion cell line, and / or K562-mbIL-21-4-1BBL expansion cell line; The addition time point of the feeder cells is any time point during the induction maturation and expansion stage.

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

[0082] Example 1

[0083] In this example, CD34 + cells were expanded.

[0084] Optionally, first, irradiated AFT024 cells, AFT024-DLL1 cells, and AFT024-DLL4 cells were seeded in a 24-well plate at a density of 100,000 cells / well for culture, and then CD34 + cells (10,000) were plated on the above stromal cells for co-culture. The medium was the basal medium, with an equal amount of CD34 +Cells were used as controls, and the number of CD34 cells was counted on the 7th day (Day 7) and 14th day (Day 14) of amplification, and the amplification fold was calculated. The results are as + shown. The results showed that after 7 days of amplification, CD34 cells were amplified 54-fold in the basal medium and 68 - 69-fold in the co-culture environment with the above-mentioned stromal cells. After 14 days of amplification, CD34 cells were amplified 522-fold in the basal medium and 915 - 941-fold in the co-culture environment with the above-mentioned stromal cells. Figure 2 + +

[0085] Example 2

[0086] In this example, HiNK progenitor cells were generated based on a 3D culture system.

[0087] The CD34 cells obtained in Example 1 were mixed with OP9 (ATCC, catalog number CRL - 2749) feeder cells at a mixing ratio of 1:20, and the total number of cells was 210,000, and a 3D induction system was prepared. 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. Induction amplification medium (KBM581 basal medium supplemented with 15% SUPERGROW cell culture additive, 2 mM GlutaMAX TM , 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) was added to the culture plate, and the cells were induced for 14 days, with the medium changed every 2 days. Flow cytometry was performed on the 14th day of induction to detect HiNK cell progenitor cells (CD45 + CD56 - CD34 + CD7 + , CD45 + CD56 - CD34 + CD7 - , CD45 + CD34 - CD7 + CD56 + ), and the results are as Figure 3 shown. The results showed that after 14 days of 3D culture, CD34 + ​​​​Cells can effectively induce NK precursor cells (CD45 + CD56 - CD34 + / - CD7 + ) and NK cells (CD45 + CD56 + ).

[0088] Example 3

[0089] In this example, NK precursor cells were induced to mature and amplified.

[0090] The cells cultured in the 3D system for 14 days in Example 2 were prepared into a single-cell suspension, and the cells were transferred to a culture bag. The cells were cultured using an induction and amplification medium (KBM581 basal medium supplemented with 15% SUPERGROW cell culture additive, 2 mM GlutaMAX TM , 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 medium was replenished every 3 days to ensure a cell density of 1.5×10 6 cells per milliliter. Through this step, HiNK cells (CD45 + CD3 - CD56 + ) could be effectively induced. The flow cytometry detection results are as Figure 4 shown. The flow cytometry phenotypes (CD56, CD16) of HiNK cells at Day 7, Day 14, and Day 21 in the maturation and amplification stages were analyzed and detected. The results showed that the proportion of CD56 + gradually increased and finally reached 100%, and the number of CD56 + gradually increased. Finally, 10 6 CD34 + cells could obtain 10 13 -10 14 HiNK cells.

[0091] Specifically, it can be seen from Figure 5A and Figure 5B that the maturity (proportion of CD56 + cells) of HiNK cells produced by the induction process increases with time and finally reaches nearly 100%. The number of HiNK cells produced increases with time. 10 6 CD34+ The cells can obtain 10 13 -10 14 HiNK cells.

[0092] Example 4

[0093] This example detects the protein expression of the induced HiNK cells in Example 3.

[0094] In this example, the classical activating receptors and inhibitory receptors of NK cells, namely the flow antibodies NKp30 (Biolegend, P30 - 15), NKp44 (Biolegend, P44 - 8), NKG2D (Biolegend, 1D11), CD94 (BD Biosciences, HP - 3D9), and NKG2A (Biolegend, S19004C), were used to stain the cell membranes of HiNK cells and natural NK cells (umbilical cord blood - derived NK, UCB - NK). After antibody incubation and cell binding, the expression levels of the corresponding antigens were detected by flow cytometry. The results are shown in Table 1. 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 the activating receptors and inhibitory receptors of NK cells just like umbilical cord blood - derived mature NK cells.

[0095] Table 1

[0096] NKp30 NKp44 NKG2D CD94 FMO 0.5 0.0 0.0 0.2 UCB-NK 97.4 99.1 93.7 86.1 HiNK 98.9 99.8 99.0 88.3

[0097] Example 5

[0098] This example verifies that the induced HiNK cells can kill solid tumor cells and hematological tumor cells in vitro.

[0099] The HiNK cells obtained in Example 3 were co - incubated with A1847 (ovarian cancer cell line) to evaluate the ability of HiNK cells to kill tumor cells (Cytotoxicity). The specific results are as Figure 6 shown. 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 among the total number of tumor cells. After 4 hours of co - incubation, flow cytometry was used to detect the proportion of dead tumor cells in each sample among the total number of tumor cells, and this proportion is the killing ability (Cytotoxicity%) of HiNK cells (4 replicates for each E:T sample. 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).

[0100] Example 6

[0101] In this example, the induced HiNK cells can kill hematological tumor cells in vitro.

[0102] 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 ability of HiNK cells to kill tumor cells (Cytotoxicity). The results are as Figure 7A - Figure 7E shown, showing the results of HiNK cells killing tumor cell lines such as K562, MOLM-13, HL-60, THP1, and Nalm-6 in vitro; where the X-axis represents different E:T ratios (HiNK cells (Effector, E): tumor cells (Target, T)); the Y-axis shows the percentage of the number of dead tumor cells among the number of tumor cells; after co-incubation for 4 h, flow cytometry was used to detect the proportion of dead tumor cells in each sample among the tumor cells, and this proportion is the killing ability of HiNK cells (Cytotoxicity%) (4 replicates for each E:T sample, and the killing ability of HiNK cells is: K562: 37.4% (1:1), 64.0% (5:1), 74.1% (10:1); MOLM-13: 49.6% (1:1), 78.1% (5:1), 81.2% (10:1); HL-60: 28.7% (1:1), 71.6% (5:1), 74.6% (10:1); THP2: 55.0% (1:1), 87.1% (5:1), 89.0% (10:1); Nalm-6: 51.3% (1:1), 78.4% (5:1), 85.5% (10:1)), indicating that the killing ability of HiNK cells is not weaker than that of natural NK cells.

[0103] Example 7

[0104] This example verifies the effect of HiNK cells killing tumors in vivo.

[0105] To evaluate the anti-tumor function of HiNK cells in vivo, a tumor model was established in 8-week-old female B-NDG mice (severely immunodeficient mice, Beijing Biocytogen) using A1847 cells (ovarian cancer cell line, 200,000 cells were intraperitoneally injected into each mouse). After observing the tumor burden of A1847 on the mice through a small animal imaging system, the mice were randomly divided into 3 groups with 4 mice in each group. HiNK cells were intraperitoneally injected into the tumor model, 10 million cells were intraperitoneally injected into each mouse, and an equal amount of UCB-NK cells were injected as a control. Subsequently, the tumor burden of the treated mice was detected weekly through a small animal imaging system.

[0106] The results are as Figure 8 shown. HiNK cells are similar to UCB-NK cells and can effectively relieve the tumor burden in mice, while the tumor burden in the uninjected mice has been increasing, indicating that HiNK cells can kill tumor cells in vivo and reduce the tumor burden in mice.

[0107] In summary, the present invention develops a new method for inducing NK cells based on hematopoietic stem and progenitor cells (CD34 + cells), which can efficiently induce the generation of NK cells, improve the efficiency of NK cell output from a single CD34 + cell, and the properties of the induced NK cells are very close to those of naturally mature cells. The anti-tumor effect is uniform, and it can effectively kill tumor cells, showing broad application prospects.

[0108] The applicant declares that the above description is only the specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of within the technical scope disclosed by the present invention fall within the protection scope and the disclosure scope of the present invention.

Claims

1. A method for inducing and preparing NK cells in vitro, characterized in that: The method comprises: CD34 + The cells were mixed with matrix cells at a mixing ratio of 1:20, with a total number of 210,000 cells, and 3D cultured for 14 days using an induction expansion medium to obtain NK precursor cells, and the NK precursor cells were further induced and expanded for 21 days to obtain mature NK cells; the matrix cells were OP9 cells; CD34 + Before the cells are mixed with stromal cells, CD34 + Cell steps; The amplified CD34 + Cells include: CD34 + Cells were cocultured with AFT024 cells, AFT024-DLL1 cells, or AFT024-DLL4 cells for 14 days; CD34 + Cell sources include umbilical cord blood, placenta, mobilized peripheral blood, bone marrow, and CD34 derived pluripotent stem cells. + Cells or blood cells reverse differentiation, transdifferentiation to obtain CD34 + Any one or a combination of at least two in the cell; The induction expansion medium includes any one of KBM581 medium, Opti-MEM medium, APEL2 medium, Essential 6 medium, DMEM-high glucose medium, DMEM / F12 medium, α-MEM medium, F-12 medium, EBM2 medium, MEM medium, BME medium, RPMI 1640 medium, G-MEM medium or a basic medium containing active substances, or a combination of at least two thereof; 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 cell growth factor; The cell growth factor includes any one of IL-3, IL-7, IL-2, SCF, IL-12, IL-15, IL-21, hTPO, IL-12, IL-18 or Flt3L, or a combination of at least two thereof.

2. The method for inducing and preparing NK cells in vitro according to claim 1, characterized in that: The stromal cells also overexpress factors that facilitate the development, maturation, expansion or survival of lymphoid cells.

3. The method for inducing and preparing NK cells in vitro according to claim 2, characterized in that: The factors include human Notch ligands and / or human cytokines.

4. The method for inducing and preparing NK cells in vitro according to claim 3, characterized in that: The human Notch ligand includes any one of DLL1, DLL1 active fragment, DLL4 or DLL4 active fragment, or a combination of at least two thereof.

5. The method for inducing and preparing NK cells in vitro according to claim 3, characterized in that: The human cytokine includes any one of IL-15, IL-12, IL-2 or IL-21, or a combination of at least two of them.

6. An inducible NK cell, characterized in that: The induced NK cells are prepared by the method for preparing NK cells by in vitro induction according to any one of claims 1-5.

7. The method for inducing NK cells in vitro according to any one of claims 1 to 5 or the use of the induced NK cells according to claim 6 in preparing a drug for treating tumors; The tumor is at least one of ovarian cancer, myeloid leukemia tumor or B-cell leukemia tumor.

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