Inducers for reprogramming T cells into NK-like cells and uses thereof

Abstract

The present application provides an inducer for reprogramming T cells into NK-like cells and its use. The inducer includes one or a combination of at least two of a DNA methyltransferase inhibitor, a histone deacetylase inhibitor, or a histone methyltransferase EZH2 inhibitor. The present application uses the inducer to induce a decrease in the methylation level of T cells, inhibit histone deacetylation and histone methylation, and express NK cell receptors and cytokines, thereby achieving the goal of reprogramming T cells into NK-like cells in vitro, with a simple method, high efficiency, and short cycle, and the prepared NK-like cells have a significant in vitro killing effect, which is of great significance in the field of cellular immunotherapy.

Description

[Technical Field]

[0001] The present application belongs to the field of biomedical technology and relates to an inducer for reprogramming T cells into NK-like cells and its use. [Background technology]

[0002] NK cells are an important component of the natural immune system, capable of killing abnormal cells without activation and without histocompatibility complex (MHC) restriction. NK cells are among the most effective and dynamic immune cells in the human body. They recognize viruses and virus-infected cells through NK cell receptors (NCRs), such as NKp46, and are involved in immune responses to multiple viruses. However, the limited number and regenerative capacity of naturally occurring NK cells in the human body poses a major obstacle to the application of NK cells in tumor therapy. T cell receptors (TCRs) on the surface of T cells recognize and respond to virus-infected cells or foreign antigens. NK-like cells can express not only NCR receptors, such as NKp46, NKp30, NKp44, and NKG2D, but also fully functional TCR receptors, thus possessing the functions of both T cells and NK cells. NK-like cells have stronger killing activity and broader antitumor effects than normal NK cells. They can also be expanded in large numbers in vitro, and in vitro expanded NK-like cells can survive in vivo for more than three weeks. Reprogramming T cells into NK-like cells provides an entirely new cell source for immunotherapy of tumors and related diseases, greatly promoting the development of tumor cell immunotherapy.

[0003] Currently, T cell reprogramming methods are mainly based on transgenic means, including lentiviral transfection, PB system electrotransfection, and CRISPR / Cas9 system knockout. However, these methods have many drawbacks. For example, lentiviral transfection requires a long processing cycle and is complicated; electrotransfection results in the loss of a large number of primary T cells, has low efficiency, and has the possibility of off-target effects during gene overexpression or knockout. Furthermore, all three of these methods are difficult to implement on a large scale, and are still fundamentally unable to achieve the effect of large-scale in vitro generation of NK cells.

[0004] There have been no relevant research reports in the prior art as to whether compound drugs can be used to reprogram T cells in vitro to prepare NK-like cells on a large scale. Summary of the Invention [Problem to be solved by the invention]

[0005] The present application provides an inducer for reprogramming T cells into NK-like cells and its use, and by treating T cells with the inducer, the demethylation level of T cells is reduced, the processes of histone acetylation and methylation are suppressed, and the T cells express NK cell surface receptors and secrete cytokines, thereby achieving the goal of reprogramming T cells into NK-like cells in vitro. [Means for solving the problem]

[0006] In aspect 1, the present application provides: a combination of at least one of a DNA methyltransferase inhibitor, a histone deacetylase inhibitor, or a histone methyltransferase EZH2 inhibitor; An inducer for reprogramming T cells into NK-like cells is provided.

[0007] In this application, we treat T cells with a small molecule DNA methyltransferase inhibitor to induce T cells to express NK cell receptors, achieving the goal of reprogramming T cells into NK-like cells in vitro.

[0008] Preferably, the DNA methyltransferase inhibitor comprises a DNA methyltransferase 1 inhibitor, preferably decitabine and / or GSK-3484862.

[0009] In this application, DNA methyltransferase 1 (DNMT1) is the most important methyltransferase in the human body and is also capable of regulating the cell cycle and the expression of tumor suppressor genes. Decitabine (DAC), a cytosine analogue, belongs to a specific methyltransferase inhibitor class, covalently binding to DNA methyltransferase to inhibit its enzymatic activity, resulting in DNA demethylation and the re-expression of tumor suppressor genes. GSK-3484862 is another non-covalent inhibitor of DNMT1, which exerts its anti-cancer effect by inducing DNA hypomethylation.

[0010] In this application, decitabine and / or GSK-3484862 are used to induce a decrease in the methylation level of T cells, which can express NK cell killing related molecules, achieving the goal of reprogramming T cells into NK-like cells in vitro.

[0011] Preferably, the histone deacetylase inhibitor comprises any one or a combination of at least two of Mocetinostat, Givinostat, or Entinostat.

[0012] Histone deacetylases (HDACs) play an important role in chromosome structural modification and gene expression regulation. By inhibiting nucleosome relaxation, they can suppress the binding of individual transcription factors and cooperating transcription factors to DNA sites, and can interact with other chromatin modulators to regulate epigenetic processes. Mocetinostat, Givinostat (ITF2357), and Entinostat (MS-275) are all novel heterogeneous selectivity inhibitors of HDACs.

[0013] Preferably, the histone methyltransferase inhibitor comprises tazemetostat and / or GSK126.

[0014] The histone methyltransferase enhancer of zeste homolog 2 (EZH2), a catalytic subunit of the polycomb repressive complex 2 (PRC2), methylates lysine 27 of nucleosomal histone H3 (H3K27), resulting in the silencing of downstream target genes and playing an important role in important biological processes such as cell apoptosis, cell cycle, and cell differentiation. Tazemetostat is an oral small molecule inhibitor of EZH2, and GSK126 (GSK2816126) is a novel, selective inhibitor of EZH2 enzyme activity.

[0015] Preferably, the inducer further comprises a pharmaceutically acceptable adjuvant.

[0016] Preferably, the auxiliary agent includes any one or a combination of at least two of a carrier, a diluent, an excipient, a filler, an adhesive, a wetting agent, a disintegrant, an emulsifier, a dissolution aid, a solubilizer, an osmotic pressure adjuster, a surfactant, a coating material, a colorant, a pH adjuster, an antioxidant, an antibacterial agent, or a buffer.

[0017] In aspect 2, the present application provides: co-culturing activated T cells with the inducer of embodiment 1 to obtain NK-like cells. A method for reprogramming T cells into NK-like cells is provided.

[0018] Preferably, the inducer comprises any one or a combination of at least two of Decitabine, GSK-3484862, Mocetinostat, Givinostat, Entinostat, Tazemetostat, or GSK126.

[0019] Preferably, the final concentration of decitabine is 0.05 to 0.5 μM, and may be, for example, 0.05 μM, 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM, or 0.5 μM.

[0020] Preferably, the final concentration of GSK-3484862 is 0.5 to 8 μM, and may be, for example, 0.5 μM, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, or 8 μM.

[0021] Preferably, the final concentration of Mocetinostat is 0.1 to 0.5 μM, and may be, for example, 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM, or 0.5 μM.

[0022] Preferably, the final concentration of Givinostat is 0.05 to 1 μM, and may be, for example, 0.05 μM, 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, 0.6 μM, 0.7 μM, 0.8 μM, 0.9 μM, or 1 μM.

[0023] Preferably, the final concentration of Entinostat is 0.05 to 1 μM, and may be, for example, 0.05 μM, 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, 0.6 μM, 0.7 μM, 0.8 μM, 0.9 μM, or 1 μM.

[0024] Preferably, the final concentration of tazemetostat is 0.1 to 5 μM, and may be, for example, 0.1 μM, 0.5 μM, 1 μM, 2 μM, 3 μM, 4 μM, or 5 μM.

[0025] Preferably, the final concentration of GSK126 is 0.05 to 1 μM, and may be, for example, 0.05 μM, 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, 0.6 μM, 0.7 μM, 0.8 μM, 0.9 μM, or 1 μM.

[0026] Preferably, the co-culture time is 3 to 10 days, for example, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days, and is preferably 5 days.

[0027] Preferably, the method for reprogramming T cells into NK-like cells comprises: The method comprises adding to the activated T cells one or a combination of at least two of decitabine at a final concentration of 0.05 to 0.5 μM, GSK-3484862 at a final concentration of 0.5 to 8 μM, GSK126 at a final concentration of 0.05 to 1 μM, mocetinostat at a final concentration of 0.1 to 0.5 μM, givinostat at a final concentration of 0.05 to 1 μM, entinostat at a final concentration of 0.05 to 1 μM, or tazemetostat at a final concentration of 0.1 to 5 μM, culturing the cells for 3 to 10 days, replacing half of the solution every day, and obtaining NK-like cells.

[0028] In embodiment 3, the present application provides NK-like cells prepared by the method of embodiment 2, which express an NK cell receptor and a T cell receptor.

[0029] Preferably, the NK cell receptor comprises any one or a combination of at least two of NKp46, NKp30, NKp44, or NKG2D.

[0030] In a fourth aspect, the present application provides a pharmaceutical composition comprising the NK-like cells of the third aspect.

[0031] Preferably, the pharmaceutical composition further comprises any one or a combination of at least two of a pharmaceutically acceptable carrier, excipient, or diluent.

[0032] In Aspect 5, the present application provides use of the inducer according to Aspect 1, the NK-like cell according to Aspect 3, or the pharmaceutical composition according to Aspect 4 in the preparation of a cellular immunotherapeutic agent. [Effects of the Invention]

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] (1) The present application achieves the objective of treating T cells with any one or a combination of at least two of small molecule drug DNA methyltransferase inhibitors, histone deacetylase inhibitors, or histone methyltransferase EZH2 inhibitors to induce the expression of NK cell receptors in T cells and reprogram the T cells into NK-like cells in vitro.

[0035] (2) The present application describes that reprogrammed T cells induced using the above-mentioned inducer express significantly higher NK cell receptors NKp46 and NKp30, have significant in vitro killing effects, and have stable dual functions of T cells and NK cells. The reprogrammed T cells can also secrete cytokines such as IFN-γ and granzyme B, and produce immune effects.

[0036] (3) The present method for preparing NK-like cells has a simple process and high reprogramming efficiency, significantly shortening the in vitro reprogramming cycle of T cells, increasing the utilization rate of primary T cells, and is suitable for large-scale process popularization, which is of great significance in the field of cellular immunotherapy. [Brief explanation of the drawings]

[0037] [Figure 1A] FIG. 1 shows the expression of NKp30 and NKp46 by CD4 T cells among T cells treated with DMSO. [Figure 1B]FIG. 1 shows the expression of NKp30 and NKp46 by CD4 T cells among T cells induced by DAC. [Figure 2A] FIG. 1 shows the expression of NKp30 and NKp46 by CD8 T cells among T cells treated with DMSO. [Figure 2B] FIG. 1 shows the expression status of NKp30 and NKp46 by CD8 T cells among T cells induced by DAC. [Figure 3A] FIG. 1 shows the expression of NKp30 and NKp46 by CD4 T cells among T cells treated with DMSO. [Figure 3B] FIG. 1 shows the expression of NKp30 and NKp46 by CD4 T cells among T cells induced by GSK-3484862. [Figure 4A] FIG. 1 shows the expression of NKp30 and NKp46 by CD8 T cells among T cells treated with DMSO. [Figure 4B] FIG. 1 shows the expression of NKp30 and NKp46 by CD8 T cells among T cells induced by GSK-3484862. [Figure 5A] FIG. 1 shows the expression of NKp30 and NKp46 by CD4 T cells among T cells treated with DMSO. [Figure 5B] FIG. 1 shows the expression status of NKp30 and NKp46 by CD4 T cells among T cells co-induced by DAC and Mocetinostat. [Figure 6A] FIG. 1 shows the expression of NKp30 and NKp46 by CD8 T cells among T cells treated with DMSO. [Figure 6B] FIG. 1 shows the expression of NKp30 and NKp46 by CD8 T cells among T cells co-induced by DAC and Mocetinostat. [Figure 7A] FIG. 1 shows the expression of NKp30 and NKp46 by CD4 T cells among T cells treated with DMSO. [Figure 7B]FIG. 1 shows the expression status of NKp30 and NKp46 by CD4 T cells among T cells co-induced by GSK-3484862 and Mocetinostat. [Figure 8A] FIG. 1 shows the expression of NKp30 and NKp46 by CD8 T cells among T cells treated with DMSO. [Figure 8B] FIG. 1 shows the expression status of NKp30 and NKp46 by CD8 T cells among T cells co-induced by GSK-3484862 and Mocetinostat. [Figure 9A] FIG. 1 shows the expression of NKp30 and NKp46 by CD4 T cells among T cells treated with DMSO. [Figure 9B] FIG. 1 shows the expression of NKp30 and NKp46 by CD4 T cells among T cells induced by GSK-3484862. [Figure 10A] FIG. 1 shows the expression of NKp30 and NKp46 by CD8 T cells among T cells treated with DMSO. [Figure 10B] FIG. 1 shows the expression of NKp30 and NKp46 by CD8 T cells among T cells induced by GSK-3484862. [Figure 11A] FIG. 1 shows the expression of NKp30 and NKp46 by CD4 T cells among T cells treated with tazemetostat. [Figure 11B] FIG. 1 shows the expression of NKp30 and NKp46 by CD4 T cells among T cells induced by GSK126. [Figure 12A] FIG. 1 shows the expression status of NKp30 and NKp46 by CD8 T cells among T cells treated with tazemetostat. [Figure 12B] FIG. 1 shows the expression of NKp30 and NKp46 by CD8 T cells among T cells induced by GSK126. [Figure 13A]FIG. 10 is a diagram showing the expression status of NKp30 and NKp46 by CD4 T cells among T cells co-induced by tazemetostat and GSK-3484862. [Figure 13B] This is a diagram showing the expression status of NKp30 and NKp46 by CD4 T cells among T cells co-induced by GSK126 and GSK-3484862. [Figure 14A] FIG. 10 is a diagram showing the expression status of NKp30 and NKp46 by CD8 T cells among T cells co-induced by tazemetostat and GSK-3484862. [Figure 14B] This is a diagram showing the expression status of NKp30 and NKp46 by CD8 T cells among T cells co-induced by GSK126 and GSK-3484862. [Figure 15A] FIG. 1 shows in vitro killing of T cells induced by GSK-3484862, induced by DAC, induced by Mocetinostat, co-induced by GSK-3484862 and Mocetinostat, co-induced by DAC and Mocetinostat, and DMSO-treated against K562. [Figure 15B] FIG. 1 shows in vitro killing of T cells induced by GSK-3484862, induced by Tazemetostat, induced by GSK126, co-induced by GSK-3484862 and Tazemetostat, co-induced by GSK-3484862 and GSK126, and DMSO-treated against K562. [Figure 16A] This is a graph showing the results of IFN-γ secretion levels when DAC-induced T cells were co-cultured with K562. [Figure 16B] This is a graph showing the results of granzyme B secretion levels when DAC-induced T cells were co-cultured with K562. [Figure 17A] This is a graph showing the results of IFN-γ secretion levels when T cells induced by GSK-3484862 were co-cultured with K562. [Figure 17B] This is a graph showing the results of the level of granzyme B secretion when T cells induced by GSK-3484862 were co-cultured with K562. [Figure 18A] This is a graph showing the results of IFN-γ secretion levels when T cells co-induced by DAC and Mocetinostat were co-cultured with K562. [Figure 18B] This is a graph showing the results of granzyme B secretion levels when T cells co-induced with DAC and mocetinostat were co-cultured with K562 cells. [Figure 19A] This is a graph showing the results of IFN-γ secretion levels when T cells co-induced by GSK-3484862 and Mocetinostat were co-cultured with K562 cells. [Figure 19B] This is a graph showing the results of the level of granzyme B secretion when T cells co-induced by GSK-3484862 and Mocetinostat were co-cultured with K562. DETAILED DESCRIPTION OF THE INVENTION

[0038] In order to further explain the technical means used in the present application and its effects, the present application will be further described below with reference to examples and drawings. It is understood that the specific examples described here are only for the purpose of interpreting the present application and are not intended to limit the present application.

[0039] Unless specific techniques or conditions are specified in the examples, they are carried out according to the techniques or conditions described in the literature in this field or in accordance with the product instructions. Reagents or equipment used without a manufacturer's name are all ordinary products that can be purchased through official channels.

[0040] material: The cord blood was sourced from the Guangdong Cord Blood Hematopoietic Stem Cell Bank. Pan T cell isolation kit was purchased from STEMCELL Technologies (Canada). Transact was purchased from Miltenyi Biotec, Inc. Decitabine was purchased from Selleck (Shanghai Selleck Biotech Co., Ltd., China); GSK-3484862, GSK126, Mocetinostat, Givinostat, Entinostat, and Tazemetostat were purchased from MCE (MedChemExpress). CD3 PE-Cy7, CD4 APC-Cy7, CD8 FITC, NKp30 PE, and NKp46 APC were all purchased from BioLegend (USA). K562 cells were from ATCC. ELISA detection reagents were purchased from Dakewe Biotech Co., Ltd.

[0041] Example 1 Isolation and in vitro culture of primary T cells from human umbilical cord blood In this example, umbilical cord blood mononuclear cells (UCBMC) were isolated by Ficoll-hypaque density gradient centrifugation, and 4 × 10 7 CD3-positive UCBMCs were cultured overnight at a cell density of 2 × 10 6 The remaining UCBMCs were cryopreserved, and T cells were sorted using a Pan T cell isolation kit. After culturing for a while, 4 × 10 7 pieces The above CD3-positive T cells were obtained, with a viability of ≥70% and no contamination with foreign microorganisms such as bacteria, fungi, or mycoplasma.

[0042] Example 2: Reprogramming of T cells was induced by small molecule drugs (1) T cells activated for 36 hours in Transact were taken and centrifuged at 300 g for 5 min. The T cells were resuspended in IMDM + 5% FBS + 1% double antibiotics (100x penicillin-streptomycin mixture) + IL2 (300 U) to a T cell density of (2–3) × 10. 5 The concentration was adjusted to 1 / mL.

[0043] (2) Each group of small molecule inhibitor drugs was added to the resuspended T cells. The final concentrations of each drug are shown in Table 1. Half of the fluid was replaced daily, and new drugs were added based on the volume after fluid replacement. The first dose was on Day 0, and drugs were continuously administered until Day 5. The cells were centrifuged at 300g for 5 minutes to replace the fluid, and then cultured in IMDM + 5% FBS + 1% dual antibiotic (100x penicillin-streptomycin mixture).

[0044] [Table 1]

[0045] Example 3: Phenotype of reprogrammed T cells was detected by flow cytometry (1) 200 μL of T cells were taken from each of the following T cells on Day 6: T cells induced with DAC, induced with GSK-3484862, co-induced with DAC and Mocetinostat, and co-induced with GSK-3484862 and Mocetinostat, as well as untreated T cells. These T cells were centrifuged at 400 g for 4 minutes, the supernatant was discarded, and the cells were resuspended in 50 μL of phosphate buffered saline (PBS). 0.5 μL each of PE-Cy7, CD4 APC-Cy7, CD8 FITC, NKp30 PE, and NKp46 APC was added and incubated at 4°C for 30 min in the dark. 500 μL of PBS was added to dilute the antibodies, and the mixture was centrifuged at 400 g for 4 min. The supernatant was carefully discarded, and 300 μL of PBS was added to resuspend the cells. The cells were transferred to a flow tube and detected by the instrument.

[0046] (2) Data analysis was performed using BD-flowcytometry software. 10,000 cells were collected per tube. Successful reprogramming was marked by a significant increase in NKp30 and NKp46-positive cells, and the percentage of positive T cells was calculated. Ta.

[0047] As shown in Figures 1A, 1B, 2A, and 2B, the NKp30 expression rate of CD4 T cells in the decitabine-induced group was approximately 83.2%, while the NKp30 expression rate of CD4 T cells in the DMSO group was approximately 7.74%. The NKp46 expression rate of CD8 T cells in the decitabine-induced group was approximately 11.2% and the NKp30 expression rate was approximately 60.5%, while the NKp46 expression rate of CD8 T cells in the DMSO group was approximately 0.26% and the NKp30 expression rate was approximately 12.2%. These results indicate that decitabine can induce T cells to express NKp30 and NKp46.

[0048] As shown in Figures 3A, 3B, 4A, and 4B, the CD4 T cell NKp46 expression rate was approximately 5.16% and the NKp30 expression rate was approximately 81.4% in the GSK-3484862-induced group, while the CD4 T cell NKp30 expression rate was approximately 7.74% in the DMSO group. The CD8 T cell NKp46 expression rate was approximately 21.4% and the NKp30 expression rate was approximately 50.8% in the GSK-3484862-induced group, while the CD8 T cell NKp46 expression rate was approximately 0.26% and the NKp30 expression rate was approximately 12.2% in the DMSO group. This indicates that GSK-3484862 can induce T cells to express NKp30 and NKp46.

[0049] As shown in Figures 5A, 5B, 6A, and 6B, compared with T cells induced with DMSO, the expression level of NKp30 in CD4 T cells co-induced with DAC and Mocetinostat was 7.84%, but NKp46 expression was almost nonexistent. Furthermore, the expression level of NKp30 in CD8 T cells co-induced with DAC and Mocetinostat was 20.4%, but NKp46 expression was also almost nonexistent.

[0050] As shown in Figures 7A, 7B, 8A, and 8B, among T cells co-induced with GSK-3484862 and Mocetinostat, the expression levels of NKp30 in CD4 T cells were 59.5%, and the expression level of NKp46 in CD8 T cells was 49.6%, and the expression level of NKp46 in CD8 T cells was 37.2%, indicating that the expression levels of NKp30 and NKp46 in T cells induced with GSK-3484862 and Mocetinostat were significantly elevated compared to the results of co-induction with DAC and Mocetinostat.

[0051] The same procedure was also performed on T cells at day 5 after induction with GSK-3484862, tazemetostat, GSK126, co-induction with tazemetostat and GSK-3484862, and co-induction with GSK-3484862 and GSK126, as well as untreated T cells. The phenotype of the reprogrammed T cells was identified by flow cytometry. The results are shown in Figures 9A, 9B, 10A, 10B, 11A, 11B, 12A, 12B, 13A, 13B, 14A, and 14B. As can be seen from the figure, compared with DMSO, the expression of NKp46 and NKp30 in CD4 and CD8 T cells induced by a single inhibitor was significantly higher. Furthermore, after the combination of GSK-3484862 with Tazemetostat and GSK126, the expression of NKP46 in CD4 and CD8 T cells was significantly increased. These results indicate that the use of single inhibitors (Tazemetostat, GSK126, GSK-3484862) or the combination of dual inhibitors (Tazemetostat + GSK-3484862, GSK-3484862 + GSK126) can induce the reprogramming of T cells into NK-like cells.

[0052] Example 4 Detection of in vitro killing of reprogrammed T cells induced by small molecule drugs (1) T cells induced by GSK-3484862, induced by DAC, induced by Mocetinostat, co-induced by GSK-3484862 and Mocetinostat, co-induced by DAC and Mocetinostat, and DMSO-treated T cells were cultured at 1 × 10 cells per 1000 cells at different effector:target cell ratios (16:1, 8:1, 4:1, 2:1, 1:1, 1:2, 1:4). 4 The cells were mixed with K562 cells (human chronic myeloid leukemia cells) and added to a 96-well cell culture plate, with three wells per group. A group containing tumor cells alone was also included as a positive control. The cells were centrifuged at 250 × g for 5 minutes and then placed in a 37°C, 5% CO2 incubator for 24 hours.

[0053] (2) After 24 hours, 100 μL / well of luciferase substrate (1×) was added to the 96-well cell culture plate, and the cells were resuspended and mixed evenly. Immediately afterwards, the RLU (relative light unit) was measured using a multi-function microplate reader. The measurement time was 0.1 seconds. The formula for calculating the killing rate of the quantitative killing efficiency evaluation method of luciferase was as follows: [Number 1] 100% x (control well reading - experimental well reading) / control well reading (the reading of the blank group to which no cells are added can be ignored)

[0054] The results, as shown in Figure 15A, showed that T cells induced by GSK-3484862, induced by DAC, induced by Mocetinostat, co-induced by GSK-3484862 and Mocetinostat, and co-induced by DAC and Mocetinostat could all effectively kill human chronic myeloid leukemia cells.

[0055] The same method was also used to detect the killing ability of reprogrammed T cells against K562 cells using T cells induced by GSK-3484862, induced by tazemetostat, induced by GSK126, co-induced by GSK-3484862 and tazemetostat, and co-induced by GSK-3484862 and GSK126, respectively, and using DMSO-treated T cells as a control. The results are shown in Figure 15B. The results showed that T cells induced by GSK-3484862, tazemetostat, induced by GSK126, co-induced by GSK-3484862 and tazemetostat, and co-induced by GSK-3484862 and GSK126 could all effectively kill human chronic myeloid leukemia cells.

[0056] Example 5: Cytokine secretion of reprogrammed T cells induced by small molecule drugs T cells induced by DAC, T cells induced by GSK-3484862, T cells co-induced by DAC and Mocetinostat, T cells co-induced by GSK-3484862 and Mocetinostat, and T cells treated with DMSO were co-cultured with human chronic myeloid leukemia cells K562 for 48 hours, and the expression levels of the cytokines IFN-γ and granzyme B in the supernatants were detected by ELISA.

[0057] The results, as shown in Figures 16A, 16B, 17A, 17B, 18A, 18B, 19A and 19B, showed that T cells induced by DAC, GSK-3484862 These results showed that T cells induced by DAC, T cells co-induced by DAC and Mocetinostat, and T cells co-induced by GSK-3484862 and Mocetinostat were all capable of producing immune effects.

[0058] In summary, this application uses decitabine and / or GSK-3484862 to induce T cells into NK-like cells, which is a simple method with high efficiency, a short cycle, and is suitable for large-scale preparation. The resulting NK-like cells have remarkable in vitro killing effects and stable dual functions of T cells and NK cells, and have important application prospects in the field of cellular immunotherapy.

[0059] Although the present application has described the detailed method of the present application through the above examples, the applicant declares that the present application is not limited to the above detailed method, i.e., the present application must not be carried out depending on the above detailed method. Those skilled in the art should understand that any improvements to the present application, equivalent replacement of each raw material of the product of the present application, addition of auxiliary ingredients, selection of specific forms, etc. are all within the scope of protection and disclosure of the present application.

Claims

1. 1) A DNA methyltransferase inhibitor, 2) a combination of a DNA methyltransferase inhibitor and a histone deacetylase inhibitor, or 3) Combinations of DNA methyltransferase inhibitors with histone methyltransferase EZH2 inhibitors; An inducer for reprogramming T cells into NK-like cells, comprising: the DNA methyltransferase inhibitor is selected from decitabine and GSK-3484862, the histone deacetylase inhibitor is mocetinostat, and the histone methyltransferase EZH2 inhibitor is selected from tazemetostat and GSK126; The NK-like cells express an NK cell receptor and a functionally intact TCR receptor, and have both the functions of a T cell and an NK cell, and the NK cell receptor is any one or a combination of at least two of NKp46, NKp30, NKp44, and NKG2D. The inducer.

2. The inducer further comprises a pharmaceutically acceptable adjuvant. The inducer according to claim 1 .

3. The auxiliary agent includes any one or a combination of at least two of a carrier, a diluent, an excipient, a filler, an adhesive, a wetting agent, a disintegrant, an emulsifier, a dissolution aid, a solubilizer, an osmotic pressure adjuster, a surfactant, a coating material, a colorant, a pH adjuster, an antioxidant, an antibacterial agent, or a buffering agent. The inducer according to claim 2 .

4. co-culturing activated T cells with the inducer according to any one of claims 1 to 3 to obtain NK-like cells, wherein the NK-like cells express NK cell receptors and functionally intact TCR receptors, and have both the functions of T cells and NK cells, and the NK cell receptors are any one or a combination of at least two of NKp46, NKp30, NKp44, and NKG2D; A method for reprogramming T cells into NK-like cells.

5. The final concentration of decitabine is 0.05 to 0.5 μM, the final concentration of GSK-3484862 is 0.5 to 8 μM; The final concentration of Mocetinostat is 0.1 to 0.5 μM, The final concentration of the tazemetostat is 0.1 to 5 μM, The final concentration of GSK126 is 0.05 to 1 μM. The method of claim 4.

6. The co-cultivation period is 3 to 10 days.

6. The method according to claim 4 or 5.

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