A method of inducing reprogramming of t cells to nk-like cells

Abstract

The application discloses a method for inducing T cell reprogramming into NK-like cells, comprising the following steps: (1) culturing the T cells in a culture system to obtain activated T cells; (2) adding small molecule inducers twice to the activated T cells to obtain NK-like cells; wherein the culture system in step (1) contains 1-10 v / v % serum substitute and / or platelet lysate, and does not contain fetal bovine serum. By optimizing the culture process and precisely adding drugs twice, the small molecule accumulation toxicity caused by multiple drug additions is avoided, the damage to the structure and function of the mitochondrial membrane is reduced, the mitochondrial membrane potential is significantly improved, the energy metabolism capacity of the mitochondria is enhanced, sufficient energy support is provided for the long-term survival and function maintenance of the cITNK cells, and the physiological activity of the cells is ensured from the energy metabolism level.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a method for inducing T cells to reprogram into NK cell-like cells. Background Technology

[0002] Natural killer (NK) cells are important immune cells in the body, involved not only in anti-tumor activity, anti-viral infection, and immune regulation, but also, in certain cases, in the development of hypersensitivity reactions and autoimmune diseases. NK cells are a crucial component of the innate immune system; their killing activity is not MHC (major histocompatibility complex)-restricted and does not depend on antibodies. NK cells are among the most effective and vigorous immune cells in the human body, recognizing viruses and virus-infected cells through NK cell receptors (NCRs) such as NKp30, and participating in immune responses to various viruses. However, the limited number and regenerative capacity of naturally occurring NK cells in the human body make cell availability a major obstacle to their application in cancer treatment. T cell receptors (TCRs) on the surface of T cells recognize virus-infected cells or exogenous antigens and respond accordingly.

[0003] cITNK cells (compound-induced T-to-NK cells) are T cells reprogrammed into ITNK cells using small chemical molecules. They combine the high proliferative capacity of T cells with the highly efficient killing ability of NK cells, making them a key direction for solving the cell source shortage problem. However, current cITNK cell induction and culture technologies still have many shortcomings that urgently need to be addressed, severely restricting their industrial production and clinical translation. Under traditional induction and culture systems, the proliferation and survival of cITNK cells are highly dependent on the stability of the culture operation and microenvironment. Existing drug administration strategies lack precise adaptation to the physiological state of the cells, failing to achieve a balance between epigenetic reprogramming and cell viability protection. Insufficient drug administration can lead to low reprogramming efficiency, while repeated drug administration can cause cell viability decline. The resulting cITNK cells not only fail to meet the needs of large-scale applications in quantity but also suffer from impaired mitochondrial function and a high apoptosis rate, resulting in poor overall cell viability and functional stability, thus affecting their in vivo anti-tumor killing and immunomodulatory effects.

[0004] Therefore, there is an urgent need to develop an optimized cITNK cell induction and culture method that can effectively improve mitochondrial function, reduce apoptosis rate, significantly enhance cell proliferation efficiency in vitro, and improve the survival stability and expansion capacity of cITNK cells in vitro. This would provide technical support for the large-scale industrial production of cITNK cells and promote their clinical application in immunotherapy for diseases such as tumors. Summary of the Invention

[0005] To address the technical problems existing in the induction and culture of cITNK cells, such as small molecule cumulative toxicity, mitochondrial dysfunction, high apoptosis rate, and low proliferation efficiency, this invention proposes a method for inducing T cells to reprogram into NK cell-like cells.

[0006] This invention provides a method for inducing T cells to reprogram into NK cell-like cells, comprising the following steps: (1) The T cells are activated and cultured in a culture system to obtain activated T cells; (2) Small molecule inducers were added twice to activated T cells for culture, and NK-like cells were obtained by reprogramming. The culture system described in step (1) contains 1-10 v / v% serum substitute and / or platelet lysis buffer, but does not contain fetal bovine serum; such as 1 v / v%, 2 v / v%, 3 v / v%, 4 v / v%, 5 v / v%, 6 v / v%, 7 v / v%, 8 v / v%, 9 v / v%, 10 v / v%, etc.

[0007] In this invention, "NK-like cells" or "cITNK cells" refer to engineered immune cells that are epigenetically reprogrammed by chemical small molecules, causing CD3-positive T cells to lose some of their inherent T cell phenotypes and acquire the core characteristics of natural killer cells (NK cells) (such as expressing NK cell-specific receptors such as NKp30 and NKp46), thus possessing both the high proliferative capacity of T cells and the non-MHC-restricted killing function of NK cells.

[0008] In this invention, "DNA methyltransferase inhibitor" refers to a class of small molecule compounds that can specifically inhibit the activity or expression of DNA methyltransferase (DNMT), thereby reducing the level of DNA methylation in cells and relieving epigenetic silencing of genes.

[0009] In this invention, "histone deacetylase inhibitor" refers to a class of small molecule compounds that can inhibit the activity of histone deacetylase (HDAC), thereby increasing histone acetylation levels, loosening chromatin structure, and promoting the transcriptional activation of target genes.

[0010] This invention involves only two additions of the inducer, precisely matching the critical time window for T cell epigenetic reprogramming. This ensures both the demethylation activation of T cell lineage genes by DNA methyltransferase inhibitors and the transcriptional initiation of NK cell-related genes by histone deacetylase inhibitors, while avoiding the continuous accumulation of small molecules intracellularly, reducing persistent damage to the mitochondrial membrane, significantly improving mitochondrial membrane potential stability, enhancing mitochondrial energy metabolism, and providing sufficient energy support for long-term cell survival. The stable enhancement of mitochondrial membrane potential effectively inhibits the activation of endogenous apoptosis pathways, reduces the release of pro-apoptotic proteins and the expression of apoptosis-related genes, thereby significantly reducing the total apoptosis rate of cITNK cells during long-term in vitro culture, minimizing cell loss, and improving the survival stability and effective yield of cells in vitro. Furthermore, this effect is consistently achieved in different drug concentration systems, demonstrating strong universality.

[0011] In this invention, the active ingredients in the serum substitute / platelet lysate can upregulate the expression of intracellular aquaporins and ion transporters and enhance their functional activity. Compared with the fetal bovine serum culture system, it enhances the cells' ability to rapidly sense and regulate intracellular and extracellular osmotic pressure, enabling cells to maintain intracellular osmotic pressure homeostasis rapidly through ion transport and water exchange in a culture medium environment with fluctuating osmotic pressure. This significantly expands the range of culture medium osmotic pressure that cells can tolerate and improves cell survival and activity in non-ideal culture environments.

[0012] Abandoning the FBS culture mode can effectively eliminate the problems of poor experimental reproducibility and unstable large-scale culture caused by large differences in nutrient composition between FBS batches. At the same time, it avoids risks such as foreign protein contamination, transmission of animal-derived pathogens, and ethical and safety concerns, making the cITNK cell culture system more in line with the safety standards for clinical applications and improving the safety and compliance of cell clinical translation.

[0013] In some embodiments, the culture system further comprises recombinant human interleukin-2, serum-free lymphocyte culture medium, and T-cell activator.

[0014] In some implementations, step (2) specifically involves adding a small molecule inducer to the activated T cells on day 0 and day 2 for culture, and reprogramming them to obtain NK-like cells.

[0015] In some embodiments, the small molecule inducer in step (2) comprises any three or more of the following: DNA methyltransferase inhibitor, histone deacetase inhibitor, histone methyltransferase EZH2 inhibitor, and vitamin C or its derivatives.

[0016] In some embodiments, the DNA methyltransferase inhibitor is any one or more of GSK-3484862, GSK-3685032, 5-aza-2-deoxycytidine, azacitidine, CC-486, RG108, SGI-1027, and SGI-110; different inhibitors have similar mechanisms of action and can be used alone or in combination to achieve stable demethylation regulation effects.

[0017] In some embodiments, the final concentration of the DNA methyltransferase inhibitor is 0.05~8 μM, for example 0.05μM, 0.1μM, 0.25μM, 0.5μM, 1μM, 3μM, 5μM, 6μM, 7μM, 8μM, etc.

[0018] In some embodiments, the histone deacetylase inhibitor is any one or more of Mocetinostat, gemivostazol, entenostat, romidabenamine, and temoxistat; when used in combination with a DNA methyltransferase inhibitor, it can form a dual epigenetic regulation, significantly improve reprogramming efficiency, and synergistically improve cell survival, antifreeze and antiaggregation properties.

[0019] In some embodiments, the final concentration of the histone deacetylase inhibitor is 0.05~1 μM, for example 0.05μM, 0.1μM, 0.2μM, 0.5μM, 0.75μM, 1μM, etc.

[0020] In some embodiments, the vitamin C or its derivative is any one or more of vitamin C, ascorbic acid-2-phosphate, magnesium ascorbic acid-2-phosphate, and sodium ascorbic acid-2-phosphate; when used in combination with DNA methyltransferase inhibitors and / or histone deacetase inhibitors, it significantly improves reprogramming efficiency, reduces damage to mitochondrial membrane structure and function, and synergistically improves cell survival and expansion.

[0021] In some embodiments, the final concentration of the vitamin C or its derivative is 0.2 to 1000 μM, such as 0.2 μM, 0.5 μM, 1 μM, 3 μM, 5 μM, 10 μM, 50 μM, 100 μM, 300 μM, 500 μM, 750 μM, 1000 μM, etc.

[0022] In some embodiments, the histone methyltransferase EZH2 inhibitor is any one or more of Tazemetostat, CPI-1205, EPZ005687, GSK126, PF-06821497, and UNC1999.

[0023] In some embodiments, the final concentration of the histone methyltransferase EZH2 inhibitor is 0.05~5 μM; such as 0.05 μM, 0.1 μM, 0.5 μM, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, etc.

[0024] In some implementations, the culture time in step (2) is not less than 2 days, preferably 2 to 14 days.

[0025] In some embodiments, the T cells are derived from any one of human peripheral blood, human umbilical cord blood, bone marrow, spleen tissue, lymph node tissue, and thymus tissue; preferably, human peripheral blood. All of these sources are rich in mature or precursor T lymphocytes, possessing a complete epigenetic regulatory system and signal transduction pathways, and can be reprogrammed into NK-like cells under the same induction system.

[0026] In summary, compared with the prior art, the present invention achieves the following technical effects: 1. This invention optimizes the culture process and uses two precise drug additions to avoid the cumulative toxicity of small molecules caused by multiple drug additions, reduces damage to mitochondrial membrane structure and function, significantly improves mitochondrial membrane potential, enhances mitochondrial energy metabolism capacity, and provides sufficient energy support for the long-term survival and functional maintenance of cITNK cells, thus ensuring the physiological activity of cells from the perspective of cellular energy metabolism.

[0027] 2. This invention replaces FBS with serum substitutes / platelet lysate, optimizes cell membrane structure and intracellular osmotic pressure regulation system, significantly broadens the range of culture medium osmotic pressure that cells can tolerate, reduces cell loss caused by osmotic pressure fluctuations during culture, improves cell in vitro expansion efficiency and culture controllability, is suitable for industrial large-scale culture processes, and reduces preparation costs.

[0028] 3. The induction method of the present invention avoids repeated disturbances to the culture microenvironment caused by multiple drug additions, maintains intracellular metabolic homeostasis and proliferation activity, significantly accelerates the in vitro proliferation rate of cITNK cells, shortens the cell doubling time, improves the efficiency of in vitro cell expansion, and greatly increases the cell yield during the culture period. Attached Figure Description

[0029] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0030] Figure 1 This is a cITNK cell proliferation curve diagram from Example 3 of the present invention.

[0031] Figure 2 This is the osmotic pressure-survival rate change curve for group a in Example 5 of the present invention.

[0032] Figure 3 This is the osmotic pressure-survival rate change curve for group b in Example 5 of the present invention.

[0033] Figure 4 This is the osmotic pressure-survival rate change curve for group c in Example 5 of the present invention.

[0034] Figure 5 This is the osmotic pressure-OD value change curve for group a in Example 5 of the present invention.

[0035] Figure 6 This is the osmotic pressure-OD value change curve for group b in Example 5 of the present invention.

[0036] Figure 7 This is the osmotic pressure-OD value change curve of group c in Example 5 of the present invention.

[0037] Figure 8 This is a graph showing the expression results of NKp30 and NKp46 in group a of Example 5 of the present invention.

[0038] Figure 9 This is a graph showing the expression results of NKp30 and NKp46 in group b of Example 5 of the present invention.

[0039] Figure 10 This is a graph showing the expression results of NKp30 and NKp46 in group c of Example 5 of the present invention.

[0040] Figure 11 This is a cITNK cell proliferation curve diagram from Example 6 of the present invention. Detailed Implementation

[0041] To enable those skilled in the art to better understand the present invention, the technical solutions in the embodiments of the present invention are clearly and completely described. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0042] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, all materials and reagents used are commercially available.

[0043] The materials used in the following embodiments are sourced from the following sources: The peripheral blood enrichment obtained by apheresis can be from a cancer patient who will be treated with the cell population generated by the method described in this invention (i.e., an autologous donor), or from an individual who donates a lymphocyte sample.

[0044] CD4 cell sorting beads (CliniMACS CD4 reagent), CD8 cell sorting beads (CliniMACS CD8 reagent), and T cell activation reagent (T cell Transact) were purchased from Miltenyi Biotechnology Co., Ltd., Germany.

[0045] GSK 3484862, Mocetinostat, and Tazemetostat were all purchased from MCE (MedChemExpress).

[0046] Vitamin C (Vc) was purchased from Sigma. Aldrich (USA).

[0047] The serum substitute (CTS immune cell SR) was purchased from Thermo Fisher (USA).

[0048] Platelet lysis buffer (PLTGold) was purchased from Mill Creek (USA).

[0049] CD3 PE Cy7, CD4 APC The Cy7, CD8 FITC, NKp30 PE, and NKp46 APC were all purchased from BioLegend (USA).

[0050] Unless otherwise specified, the cell culture conditions in the following experiments are 37°C and 5% CO2.

[0051] Example 1: Acquisition, activation, and small molecule compound-induced reprogramming of T cells (1) Isolation of peripheral blood mononuclear cells (PBMCs) and T cell sorting and enrichment from apheresis peripheral blood enrichment PBMCs were isolated from apheresis peripheral blood using a density gradient centrifugation with lymphocyte separation medium. An appropriate amount of PBMCs was cultured overnight at a cell density of 4 × 10⁻⁶. 6 T cells were sorted using CD4 and CD8 cell sorting magnetic beads to obtain CD3-positive T cells.

[0052] (2) Small molecule compounds induce T cell reprogramming The obtained CD3-positive T cells were resuspended in GT-T551 H3 medium (serum-free lymphocyte culture medium, TAKARA) + 5 v / v % FBS (fetal bovine serum) + 500 IU / ml rh-IL2 (recombinant human interleukin-2) and the T cell density was adjusted to 1×10⁻⁶ cells / mL. 6 Cells / mL, using T cell activation reagent (30 μL / 1×10⁶ cells / mL). 6 (One cell) was activated and cultured for 24 hours.

[0053] (3) After activation, small molecule compounds were added to the resuspended T cells, and the final concentrations of each drug were adjusted as shown in Table 1. Culture medium was added every two days to adjust the T cell density to 1×10⁻⁶. 6 The number of compounds added per mL was increased, and the corresponding small molecule inducer was added according to the volume after replenishment. The first addition of the compound was recorded as Day 0. The compound was continuously added until the specified number of days. After that, the culture was continued using GT-T551 H3 medium (TAKARA) + 5% v / v FBS + 500 IU / mL rh-IL2 replenishment solution.

[0054] Table 1 Grouping of different induction schemes

[0055] Example 2: Effects of different induction regimens on mitochondrial membrane potential and apoptosis rate of cITNK cells This embodiment evaluates the effects of different small molecule inducer addition methods on the stability of mitochondrial function and apoptosis level in cITNK cells, and improves the survival stability of cells during long-term culture.

[0056] Induction was performed according to the grouping in Example 1. The culture was continued for 14 days using GT-T551 H3 medium (TAKARA) + 5 v / v %FBS + 500 IU / mL rh-IL2 supplementation solution, and then used for subsequent detection.

[0057] On day 14 of culture, cITNK cells were collected from each group, with approximately 1 × 10⁶ cells taken from each group. 6 Transfer the cells to centrifuge tubes and centrifuge at 400 g for 5 min. Discard the supernatant and resuspend the cells in pre-chilled phosphate-buffered saline (PBS). Then, add JC-1 staining working solution to the cell suspension to a final concentration of 2 μM and incubate at 37°C in the dark for 20 min. After incubation, wash the cells with 1 mL of JC-1 staining buffer, centrifuge at 400 g for 5 min, discard the supernatant, resuspend the cells in 500 μL of PBS, and immediately transfer to flow cytometry tubes.

[0058] Fluorescence signals of cells in each group were detected by flow cytometry. The intensities of red fluorescence (aggregate state) and green fluorescence (monomer) of JC-1 cells were recorded, and the ratio of red fluorescence to green fluorescence was calculated as an indicator of mitochondrial membrane potential. The data were processed using flow cytometry software to statistically analyze the mean fluorescence intensity (MFI) and red / green fluorescence ratio of each group of cells. The relative change in mitochondrial membrane potential of the experimental groups was calculated with the control group as a baseline.

[0059] After mitochondrial membrane potential was measured, apoptosis in each group of cells was further assessed using Annexin V-FITC / PI double staining. Specifically, approximately 1 × 10⁶ cells from each group were collected. 6 Cells were transferred to centrifuge tubes, washed twice with PBS, and centrifuged at 400 g for 5 min each time. After discarding the supernatant, the cells were resuspended in 100 μL of Annexin V binding buffer, followed by the addition of 5 μL Annexin V-FITC and 5 μL propidium iodide (PI) dye, and incubated at room temperature in the dark for 15 min. After incubation, 400 μL of binding buffer was added and mixed thoroughly. Flow cytometry was immediately used to detect the cells, recording the proportions of early apoptotic cells, late apoptotic cells, and surviving cells, and calculating the total apoptosis rate for each group.

[0060] All experiments were repeated at least three times. The results were expressed as mean ± standard deviation (mean ± SD). Statistical methods were used to analyze the differences between the experimental group and the control group. P < 0.05 was used as the criterion for statistical significance.

[0061] The test results are shown in Table 2: Table 2 Statistical results of mitochondrial membrane potential and apoptosis rate

[0062] Note: Compared with group 1, P <0.05, # Compared with group 3, P <0.05, compared with group 5, P <0.05.

[0063] Table 2 shows that, under the same drug system, simply changing the timing of small molecule compound addition significantly affected the functional state of cITNK cells. In the high-dose system, compared with group 1, the group with small molecule compounds added only on Day 0 and Day 2 (group 2) showed a significant increase in mitochondrial membrane potential of cITNK cells, while the total apoptosis of cells at day 14 of in vitro culture was significantly reduced. These results indicate that, under the same drug system, reducing the number of small molecule compound additions can effectively avoid the cumulative toxicity of small molecules in cells, thereby significantly improving mitochondrial membrane potential stability and reducing apoptosis levels. This effect can be repeatedly verified in different concentration systems, indicating that the two-time precise dosing strategy has good stability and universality.

[0064] Example 3: Effects of different induction regimens on cITNK cell proliferation To verify the regulatory effect of the optimized inducer combination, concentration, and addition time on the proliferation capacity of cITNK cells, cITNK cells reprogrammed as shown in Table 1 were selected. The cell count at different culture times was detected by trypan blue staining, cell proliferation curves were plotted, and cell proliferation characteristics were analyzed. The specific operations and results are as follows: The cITNK cells obtained from reprogramming groups 1-6 in Table 1 were used as experimental groups, with each group having 3 replicates. The cITNK cells from each group were then cultured at 1×10⁶ cells / well. 6 The cells were initially inoculated at a density of 10 cells / mL and cultured in a culture system containing GT-T551 H3 medium + 5 v / v %FBS + 500 IU / mL rh-IL2. Samples were taken and tested on days 2, 4, 6, 8 and 10 of culture.

[0065] At each sampling time point, 10 μL of cITNK cell suspension was taken from each well of the culture system, placed in a sterile EP tube, and an equal volume (10 μL) of 0.4% trypan blue dye was added. The mixture was gently blown and mixed, and allowed to stand at room temperature for 3 min. The mixed staining solution was then dropped onto a hemocytometer and placed under an inverted microscope. The number of live cells (anti-stain, colorless and transparent) and dead cells (stained, blue) were counted separately, and the number of live cells per milliliter was calculated.

[0066] Proliferation curve plotting: The x-axis represents the culture time (days), and the x-axis represents the number of viable cells per milliliter (×10⁻⁶). 6 Using the cell count (cells / mL) as the ordinate, the in vitro proliferation curves of cITNK cells were plotted based on the live cell counts at different time points for each group.

[0067] The results are as follows Figure 1 As shown, the number of cITNK cells in group 1 increased to 8 × 10⁻⁶. 6 The number of cITNK cells in group 2 increased to 2.7 × 10⁻⁶. 7The number of cITNK cells in group 3 increased to 2.4 × 10⁻⁶. 7 The number of cITNK cells in group 4 increased to 6.4 × 10⁻⁶. 7 The number of cITNK cells in group 5 increased to 1.4 × 10⁻⁶. 7 The number of cITNK cells in group 6 increased to 4.2 × 10⁻⁶. 7 After optimizing the combination, concentration, and addition time of the inducer, the proliferation rate of cITNK cells was significantly accelerated, indicating that the present invention, through synergistic optimization of the composition and concentration of small molecule drugs, combined with the precise administration method of adding the drugs twice on Day 0 and Day 2, can significantly promote the in vitro proliferation of T cells reprogrammed into cITNK cells and greatly improve the cell expansion efficiency.

[0068] Example 4: Effects of different culture medium systems on the osmotic pressure tolerance of cITNK cells 1. Acquisition, activation, and small molecule compound-induced reprogramming of T cells (1) Isolation of peripheral blood mononuclear cells (PBMCs) and T cell sorting and enrichment from apheresis peripheral blood enrichment PBMCs were isolated from apheresis peripheral blood using a density gradient centrifugation with lymphocyte separation medium. An appropriate amount of PBMCs was cultured overnight at a cell density of 4 × 10⁻⁶. 6 T cells were sorted using CD4 and CD8 cell sorting magnetic beads to obtain CD3-positive T cells.

[0069] (2) Small molecule compounds induce T cell reprogramming The obtained CD3-positive T cells were resuspended in the culture medium corresponding to Table 3 and the T cell density was adjusted to 1×10⁶. 6 Cells / mL, using T cell activation reagent (30 μL / 1×10⁶ cells / mL). 6 (1 cell) activated and cultured for 36 hours.

[0070] Table 3 Different culture medium systems

[0071] (3) After activation, small molecule compounds in the ratio of group 4 were added to the resuspended T cells. The final concentration of each drug was GSK-3484862 + Mocetinostat + Vc (0.2 μM + 0.2 μM + 500 μM). The T cell density was adjusted to 1 × 10⁶ cells / day by adding culture medium every two days. 6 The compound was added at a rate of 1 / mL, and new compounds were added according to the volume after replenishment. The first addition of compound was recorded as Day 0, and the compound was added continuously until Day 2. After that, the appropriate replenishment solution was used to continue the culture.

[0072] 2. Preparation of culture media with different osmotic pressures Using the optimal osmotic pressure for conventional cell culture as a baseline, sterile GT-T551 H3 basal medium was used as the base medium. Sterile sodium chloride and mannitol were added to adjust the osmotic pressure, and complete osmotic pressure gradient complete mediums with osmotic pressure gradients of 240, 260, 280, 300, 320, 340, and 360 mOsm / L were prepared. Each gradient medium was supplemented with 5 v / v % FBS / 5 v / v % serum substitute / 5 v / v % platelet lysis buffer + 500 IU / mL rh-IL2 according to the corresponding group. After preparation, the actual osmotic pressure of each gradient medium was calibrated using an osmoremeter to ensure accurate values. After calibration, the media were stored at 4℃ for later use.

[0073] 3. Detection of cITNK cell osmotic pressure tolerance (1) Cell pretreatment: cITNK cells from each group induced and cultured for 10 days were centrifuged at 250×g at room temperature for 8 min. After discarding the supernatant, the cells were resuspended in the corresponding serum-free GT-T551 H3 medium and washed twice to remove residual inducers and metabolic impurities. Finally, the cell density of each group was adjusted to 2×10⁻⁶. 6 Quantity / mL, for later use.

[0074] (2) Cell seeding and culture: The pretreated cITNK cells in each group were seeded into 24-well sterile culture plates containing the above gradient osmotic pressure medium. 1 mL of cell suspension was added to each well. Three replicates were set for each osmotic pressure gradient. The plates were placed in a 37℃, 5% CO2 saturated humidity incubator and cultured for 48 h. Vigorous shaking was avoided during the culture period.

[0075] (3) Cell viability detection: After culture, 10 μL of cell suspension was taken from each well and thoroughly mixed with an equal volume of 0.4% trypan blue dye by pipetting. The mixture was then allowed to stand at room temperature for 3 min. The mixed staining solution was then added to a hemocytometer and placed under an inverted microscope. Three fields of view were randomly selected, and the number of live cells (anti-stain, colorless and transparent) and the total number of cells were counted. The cell viability was calculated. Cell viability = (number of live cells / total number of cells) × 100%.

[0076] (4) Cell viability verification: After completing the viability count, add 10 μL of CCK-8 detection reagent to each culture well, mix gently, and place in a 37℃, 5% CO2 incubator for 2 h. After the culture is completed, use an ELISA reader to detect the absorbance (OD value) of each well at a wavelength of 450 nm. Zero the culture with blank medium and record the OD value of each well.

[0077] 4. Determination of osmotic pressure tolerance range Plot the osmotic pressure of the culture medium on the x-axis and the cell viability and OD value on the y-axis to obtain the osmotic pressure-viability and osmotic pressure-OD value curves for each group of cITNK cells. The osmotic pressure range in which the cell viability is ≥80% and the OD value is maintained above 90% of the normal osmotic pressure (300 mOsm / L) of the group is defined as the osmotic pressure range that the cITNK cells of the group can tolerate. Statistical software was used to analyze the viability and OD value of each group under the same osmotic pressure gradient.

[0078] The test results are shown in Tables 4-6 and Figures 2-7 As shown: Table 4. Statistical results of survival rate and OD value of group a under different osmotic pressures (mean±SD)

[0079] Table 5. Statistical results of survival rate and OD value of group b under different osmotic pressures (mean±SD)

[0080] Table 6. Statistical results of survival rate and OD value of group c under different osmotic pressures (mean±SD)

[0081] Depend on Figures 2-7 The results showed that all three groups of cells maintained high activity, indicating that all three culture media systems could provide a good culture environment for cITNK cells under normal osmotic pressure conditions, maintaining high cell viability and activity. Under low osmotic gradient, the survival rate and OD value of group a cells decreased significantly, while groups b and c cells maintained high tolerance and no significant decrease in cell activity. Under high osmotic gradient, the tolerance of group a cells decreased sharply, while groups b and c maintained excellent high osmotic tolerance. Groups b to c could maintain cell activity in the range of 250-350 mOsm / L, and the survival rate and OD value of group c were slightly higher than those of group b under each osmotic pressure gradient. This indicates that the culture media system of this invention, which uses serum substitutes and platelet lysis buffer instead of FBS, can significantly improve the osmotic pressure tolerance of cITNK cells and greatly expand the range of osmotic pressures that cells can tolerate.

[0082] Example 5: Effects of different culture medium systems on cITNK cell phenotype Referring to the induction protocol in Example 3, the effect of the culture medium system on the cITNK cell phenotype was detected by flow cytometry. The specific steps are as follows: Collect 400 μL of each of the induced cITNK cells obtained from the culture media systems of groups a to c in Table 3. Centrifuge at 400g for 4 min, discard the supernatant, and resuspend the cells in 50 μL of phosphate-buffered saline (PBS). Then add 0.5 μL each of CD3PE-Cy7, CD4 APC-Cy7, CD8 FITC, NKp30 PE, and NKp46 APC, and incubate at 4°C in the dark for 30 min. Add 500 μL of PBS to dilute the antibody, centrifuge at 400g for 4 min, carefully discard the supernatant, resuspend the cells in 300 μL of PBS, transfer to a flow cytometer, and run on the instrument. Data analysis was performed using BD-flowcyto analysis software. 10,000 cells were collected from each tube, and the percentage of NKp46-positive T cells to NKp30-positive T cells was calculated.

[0083] The results are as follows Figures 8-10 As shown, in group a, the expression rates of NKp30 and NKp46 in CD3 T cells were 29.8% and 2.77%, respectively; in group b with added serum substitute, the expression rates of NKp30 and NKp46 in CD3 T cells were 41.3% and 1.96%, respectively; and in group c with added platelet lysis buffer, the expression rates of NKp30 and NKp46 in CD3 T cells were 62.9% and 2.96%, respectively. This indicates that the present invention significantly improved the expression rate of NKp30 in cITNK cells by adjusting the culture medium system.

[0084] Example 6: Effects of different culture medium systems on cITNK cell proliferation Referring to the induction protocol of Example 3, 10 μL of cITNK cells obtained from the culture medium systems of groups a to c in Table 3 were cultured for 2, 4, 6, 8, and 10 days, respectively, and mixed with 10 μL of trypan blue dye at a 1:1 ratio for counting and plotting cell proliferation curves.

[0085] The results are as follows Figure 11 As shown, the cITNK cell count in the fetal bovine serum-added group increased to 3.25 × 10⁻⁶. 7 The number of cITNK cells in the serum substitute group increased to 5.78 × 10⁻⁶. 7 Cell proliferation was increased by 78% compared to the group supplemented with fetal bovine serum; the cITNK cell count in the group supplemented with platelet lysis buffer increased to 7.40 × 10⁻⁶. 7 Cell proliferation was increased by 128% compared to the group with added fetal bovine serum, indicating that the serum substitute group and platelet lysate promoted the proliferation of T cells reprogrammed into cITNK cells induced by small molecule drugs.

[0086] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for inducing T cells to reprogram into NK cell-like cells, characterized in that, Includes the following steps: (1) The T cells are activated and cultured in a culture system to obtain activated T cells; (2) Small molecule inducers were added twice to activated T cells for culture, and NK-like cells were obtained by reprogramming. The culture system described in step (1) contains 1-10 v / v % serum substitute and / or platelet lysis buffer, but does not contain fetal bovine serum.

2. The method according to claim 1, characterized in that, The culture system also includes recombinant human interleukin-2, serum-free lymphocyte culture medium, and T cell activator.

3. The method according to claim 1, characterized in that, Step (2) specifically involves: Small molecule inducers were added to activated T cells on day 0 and day 2 for culture, and NK-like cells were obtained by reprogramming.

4. The method according to claim 1, characterized in that, The small molecule inducer in step (2) comprises any three or more of the following: DNA methyltransferase inhibitor, histone deacetase inhibitor, histone methyltransferase EZH2 inhibitor, and vitamin C or its derivatives.

5. The method according to claim 4, characterized in that, The DNA methyltransferase inhibitor is any one or more of GSK-3484862, GSK-3685032, 5-aza-2-deoxycytidine, azacitidine, CC-486, RG108, SGI-1027, and SGI-110.

6. The method according to claim 4, characterized in that, The histone deacetylase inhibitor is any one or more of Mocetinostat, gemivestine, entenostat, romidesin, chidamide, and temoxistat.

7. The method according to claim 4, characterized in that, The final concentration of the histone deacetylase inhibitor is 0.05~1 μM; Preferably, the final concentration of the DNA methyltransferase inhibitor is 0.05~8 μM; Preferably, the final concentration of the vitamin C or its derivative is 0.2~1000 μM.

8. The method according to claim 4, characterized in that, The vitamin C derivative is any one or more of ascorbic acid-2-phosphate, magnesium ascorbic acid-2-phosphate, and sodium ascorbic acid-2-phosphate.

9. The method according to claim 4, characterized in that, The histone methyltransferase EZH2 inhibitor is any one or more of Tazemetostat, CPI-1205, EPZ005687, GSK126, PF-06821497, and UNC1999; Preferably, the final concentration of the histone methyltransferase EZH2 inhibitor is 0.05~5 μM.

10. The method according to claim 1, characterized in that, The incubation time in step (2) shall not be less than 2 days.

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