Application of PRMT5-inhibited NK-92 cells in the preparation of drugs for treating colorectal cancer

Abstract

This invention relates to the field of biomedical technology, specifically disclosing the application of PRMT5-inhibited NK-92 cells in the preparation of drugs for treating colorectal cancer. The preparation process of PRMT5-inhibited NK-92 cells is as follows: NK-92 cells are seeded into a culture medium containing 1 μmol / L to 3 μmol / L EPZ015666 and cultured to obtain PRMT5-inhibited NK-92 cells. Compared with NK-92 cells, the PRMT5-inhibited NK-92 cells provided by this invention have significantly improved tumor cell killing ability and can significantly inhibit tumor volume and weight.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of PRMT5-inhibited NK-92 cells in the preparation of drugs for treating colorectal cancer. Background Technology

[0002] Cancer is a major public health problem worldwide and the second leading cause of death. Colorectal cancer (CRC) is the most common malignant tumor of the digestive tract. According to research reports, colorectal cancer is one of the three most common cancers worldwide, after breast cancer and lung cancer, and its mortality rate is the second highest, after lung cancer.

[0003] Colorectal cancer has an insidious onset, often presenting initially only with a positive fecal occult blood test. Subsequently, the following clinical manifestations may appear: changes in bowel habits and stool shape, abdominal pain, rectal and abdominal masses, and a range of systemic conditions. Complications occur in later stages, primarily including intestinal obstruction, intestinal bleeding, and complications related to peritoneal metastasis of the cancer.

[0004] The development of modern biomedicine has brought more options to the treatment of colorectal cancer, including surgical resection, radiotherapy, chemotherapy, immunotherapy, targeted therapy, and extensive surgical and local ablation treatments for metastatic lesions. These treatments have effectively inhibited the development of CRC and prolonged patient survival. However, due to its insidious onset, the vast majority of colorectal cancer patients are already in the middle or late stages at the time of initial diagnosis, missing the valuable opportunity for radical treatments such as surgical resection and local ablation.

[0005] With the rapid advancements in tumor immunotherapy in recent years, research has shown that immune checkpoints exist not only on T lymphocytes but also on natural killer (NK) cells. NK cells are an important component of the body's innate immune system, possessing both cytotoxic and immunomodulatory functions, and playing a crucial role in the body's immune response against tumors. Numerous studies have shown that compared to the normal colonic epithelial microenvironment, the number and killing function of NK cells are significantly downregulated in colorectal cancer (CRC). CRC patients with a higher number of tumor-infiltrating NK cells have a better prognosis, suggesting that NK cells play an important anti-tumor immune role in CRC, but are mostly in a state of functional exhaustion, resulting in poor anti-tumor efficacy. Therefore, it is necessary to explore a way to improve the efficacy of NK-92 cell therapy for colorectal cancer. Summary of the Invention

[0006] To explore ways to improve the efficacy of NK-92 cell therapy for colorectal cancer, this invention provides the application of PRMT5-inhibited NK-92 cells in the preparation of drugs for treating colorectal cancer. Compared to regular NK-92 cells, the PRMT5-inhibited NK-92 cells provided by this invention exhibit significantly enhanced tumor cell killing ability and can significantly inhibit tumor volume and weight.

[0007] This invention provides the application of PRMT5-inhibited NK-92 cells in the preparation of drugs for treating colorectal cancer.

[0008] This invention successfully modified NK-92 cells by inhibiting the expression of the PRMT5 gene in NK-92 cells to obtain PRMT5-inhibited NK-92 cells (E-NK-92 cells). Compared with NK-92 cells, E-NK-92 cells change the functional exhaustion state of NK cells, undergo phenotypic transformation, and reduce the expression of inhibitory receptors NKG2A and IL-1R8. This can prevent NK cell exhaustion, maintain NK cell activity, significantly improve the killing ability against tumor cells, and significantly inhibit tumor volume and weight.

[0009] Furthermore, RMT5-inhibited NK-92 cells were obtained by targeting and inhibiting the expression of the PRMT5 gene in NK-92 cells.

[0010] Furthermore, the method of targeting and inhibiting the expression of the PRMT5 gene in NK-92 cells is as follows: By adding a PRMT5 inhibitor during the culture of NK-92 cells, the PRMT5 gene in NK-92 cells is targeted and inhibited, thereby obtaining modified PRMT5-inhibited NK-92 cells.

[0011] Furthermore, the PRMT5 inhibitor is EPZ015666.

[0012] Furthermore, the preparation process of PRMT5-inhibited NK-92 cells is as follows: NK-92 cells are seeded into a culture medium containing 1 μmol / L to 3 μmol / L EPZ015666 to obtain PRMT5-inhibited NK-92 cells, namely E-NK-92 cells.

[0013] Furthermore, the concentration of EPZ015666 in the culture medium was 1 μmol / L.

[0014] Furthermore, the E-NK-92 cells are used to prepare drugs that inhibit tumor volume and weight.

[0015] Furthermore, the gene editing method for targeting and inhibiting the expression of the PRMT5 gene in NK-92 cells is as follows: interfering RNA that targets and inhibits or knocks out the PRMT5 gene is designed, and NK-92 cells are modified using the interfering RNA to obtain PRMT5-inhibited NK-92 cells.

[0016] This invention also provides the application of a PRMT5 inhibitor in the preparation of a drug synergist for NK-92 cell therapy of colorectal cancer, wherein the PRMT5 inhibitor is EPZ015666.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0018] This invention successfully modified NK-92 cells by inhibiting the expression of the PRMT5 gene in NK-92 cells to obtain PRMT5-inhibited NK-92 cells (E-NK-92 cells). Compared with NK-92 cells, E-NK-92 cells change the functional exhaustion state of NK cells, undergo phenotypic transformation, and reduce the expression of inhibitory receptors NKG2A and IL-1R8, effectively preventing NK cell exhaustion, maintaining NK cell activity, and significantly improving the killing ability against tumor cells, as well as significantly inhibiting tumor volume and weight.

[0019] PRMT5-inhibited NK cells exhibit enhanced antitumor activity against colorectal cancer. PRMT5-inhibited NK cells release more cytotoxic molecules, such as granzyme B or IFN-γ, which directly kill colorectal cancer cells. In addition, in the colorectal cancer microenvironment, PRMT5-inhibited NK cells show increased expression of activating receptors and decreased expression of inhibitory receptors on their surface, which enhances NK cell activity.

[0020] The development of PRMT5 inhibitors holds immense market potential, particularly in the field of tumor immunotherapy, aiming to improve treatment efficacy and reduce costs. Furthermore, by enhancing the effectiveness of existing immunotherapies, it could potentially lower the overall cost and healthcare burden of cancer treatment. If PRMT5-related NK cell therapy strategies are successfully translated into clinical applications, they will bring substantial economic benefits and commercial opportunities to the biopharmaceutical industry. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 The effect of PRMT5 inhibitors on NK-92 cell proliferation;

[0023] In the figure, A represents the PRMT5 inhibition level in NK-92 cells after two days of treatment with different concentrations of the PRMT5 inhibitor (EPZ015666) as detected by Western Blot.

[0024] Figure B is a statistical graph showing the effect of PRMT5 inhibitor on NK-92 cell proliferation, ****P<0.0001.

[0025] Figure 2The effect of PRMT5 inhibitor on NK-92 cell phenotype; Isotype is the isotype control, NK-92 cell is the control group, and E-NK-92 is the PRMT5-inhibited NK-92 cell, which is the experimental group.

[0026] In the figure, A is the flow cytometry plot of the effect of PRMT5 inhibition on CD56 expression in NK-92 cells;

[0027] B is the flow cytometry plot showing the effect of PRMT5 inhibition on CD16 expression in NK-92 cells;

[0028] C represents the flow cytometry plot of the effect of PRMT5 inhibition on NKG2D expression in NK-92 cells;

[0029] D is the flow cytometry plot showing the effect of PRMT5 inhibition on NKG2A expression in NK-92 cells;

[0030] E represents the flow cytometry plot of PRMT5 inhibition on IL-1R8 expression in NK-92 cells;

[0031] F is a statistical graph showing the effect of PRMT5 inhibition on the expression of CD16, CD56, NKG2A, NKG2D, and IL-1R8 in NK-92 cells.

[0032] Figure 3 The effect of PRMT5-inhibited NK-92 cells on the killing effect of colorectal cancer was *P<0.05,**P<0.01,****P<0.0001;

[0033] In the figure, A represents the killing effect of NK-92 cells and PRMT5-inhibited NK-92 cells on SW620 under different NK-92 cell numbers and SW620 co-culture conditions detected by CCK-8 assay; SW620 with NK-92 indicates NK-92 cells co-cultured with SW620, and sw620 with E-NK-92 indicates PRMT5-inhibited NK-92 cells co-cultured with SW620.

[0034] B represents the killing effect of NK-92 cells and PRMT5-inhibited NK-92 cells on HCT116 under different numbers of NK-92 cells co-cultured with SW620 cells as detected by CCK-8 assay; HCT116 with NK-92 indicates NK-92 cells co-cultured with HCT116 cells, and HCT116 with E-NK-92 indicates PRMT5-inhibited NK-92 cells co-cultured with HCT116 cells.

[0035] Figure 4To investigate the effect of PRMT5 inhibition on phenotype transformation of NK-92 cells after co-culturing with colorectal cancer cells SW620; Isotype was the isotype control, NK-92 cell was the control group, and E-NK-92 was the PRMT5-inhibited NK-92 cell, which was the experimental group.

[0036] In the figure, A is a flow cytometry plot showing the effect of PRMT5 inhibition on CD56 expression in NK-92 cells after co-culturing with colorectal cancer cells SW620;

[0037] B is a flow cytometry diagram showing the effect of PRMT5 inhibition on CD16 expression in NK-92 cells after co-culturing with colorectal cancer cells SW620.

[0038] C is a flow cytometry plot showing the effect of PRMT5 inhibition on NKG2D expression in NK-92 cells after co-culturing with colorectal cancer cells SW620;

[0039] D is a flow cytometry plot showing the effect of PRMT5 inhibition on NKG2A expression in NK-92 cells after co-culturing with colorectal cancer cells SW620;

[0040] E is a flow cytometry plot showing the effect of PRMT5 inhibition on IL-1R8 expression in NK-92 cells after co-culturing with colorectal cancer cells SW620;

[0041] F is a flow cytometry plot showing the effect of PRMT5 inhibition on the expression of various markers (CD16, CD56, NKG2A, NKG2D and IL-1R8) in NK-92 cells after co-culturing with colorectal cancer cells SW620.

[0042] Figure 5 To investigate the effect of PRMT5 inhibition on phenotypic transformation of NK-92 cells after co-culturing with colorectal cancer cells HCT116; Isotype was the isotype control, NK-92 cell was the control group, and E-NK-92 was the PRMT5-inhibited NK-92 cell, which was the experimental group.

[0043] In the figure, A is a flow cytometry plot showing the effect of PRMT5 inhibition on CD56 expression in NK-92 cells after co-culturing with colorectal cancer cells HCT116;

[0044] B is a flow cytometry diagram showing the effect of PRMT5 inhibition on CD16 expression in NK-92 cells after co-culturing with colorectal cancer cells HCT116.

[0045] C is a flow cytometry plot showing the effect of PRMT5 inhibition on NKG2D expression in NK-92 cells after co-culturing with colorectal cancer cells HCT116;

[0046] D is a flow cytometry plot showing the effect of PRMT5 inhibition on NKG2A expression in NK-92 cells after co-culturing with colorectal cancer cells HCT116;

[0047] E is a flow cytometry plot showing the effect of PRMT5 inhibition on IL-1R8 expression in NK-92 cells after co-culturing with colorectal cancer cells HCT116.

[0048] F is a flow cytometry plot showing the effect of PRMT5 inhibition on the expression of various markers (CD16, CD56, NKG2A, NKG2D and IL-1R8) in NK-92 cells after co-culturing with colorectal cancer cells HCT116.

[0049] Figure 6 The in vivo killing effect of PRMT5-inhibited NK-92 cells (E-NK cells) on colorectal cancer;

[0050] In the figure, A represents the effect of PRMT5-inhibited NK-92 cells (E-NK cells) on the size of colorectal cancer tumors in vivo;

[0051] B represents the effect of PRMT5-inhibited NK-92 cells (E-NK cells) on colorectal cancer tumor weight in vivo. *P<0.05 indicates a significant difference.

[0052] C represents the effect of PRMT5-inhibited NK-92 cells (E-NK cells) on colorectal cancer tumor volume in vivo. **P<0.01 indicates a highly significant difference.

[0053] Figure 7 Maps of the control plasmid and the plasmid overexpressing PRMT5;

[0054] In the figure, A is the map of the control plasmid (pCDH-ef1a-COGFP-PURO-SFFV-MCS-WPRE);

[0055] B is a map of the PCDH-EF1a-CoGFP-P2A-Puro-SFFV-PRMT5-3MYC-WPRE vector (a plasmid overexpressing PRMT5).

[0056] Figure 8 To establish a PRMT5-overexpressing NK92 cell line and its effect on NK cell proliferation;

[0057] A shows the cell states of NK92 cells after transfection with lentivirus containing control plasmid and plasmid overexpressing PRMT5 under bright field and dark field conditions.

[0058] B represents the Western spectroscopy analysis of PRMT5 protein levels in NK92-Vector and NK92-PRMT5 cells.

[0059] C represents the proliferation of NK-92 cells at different times of PRMT5 overexpression; ***P<0.001.

[0060] Figure 9 The effect of PRMT5 overexpression on the phenotype of NK92 cells;

[0061] In the figure, A is a histogram of the expression of CD16, CD56, SIGIRR, NKG2A, NKG2D, and TIGIT in NK92 cells detected by flow cytometry; the green line represents "IsoType", the blue line represents "NK92-Vector", and the purple line represents "NK92-PRMT5".

[0062] B is a statistical graph showing the expression of CD16, CD56, SIGIRR, NKG2A, NKG2D, and TIGIT in NK92 cells as detected by flow cytometry; *P<0.05, **P<0.01.

[0063] Figure 10 Overexpression of PRMT5 in vitro inhibits the killing effect of NK92 cells on CRC cells;

[0064] In the figure, A represents the CCK8 assay used to measure the killing activity of NK92-Vector or NK92-PRMT5 cells against HCT116 cells at a specified NK:HCT116 (1:1) ratio at 6h, 12h, and 24h.

[0065] B represents the CCK8 assay used to measure the cytotoxic activity of NK92-Vector or NK92-PRMT5 cells against SW620 cells at a specified NK:SW620 (1:1) ratio at 6h, 12h, and 24h. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0066] Figure 11 To detect phenotypic changes in NK92 cells after co-culturing with cancer cells by flow cytometry;

[0067] In the figure, A is the flow cytometry plot of CD56, CD16, NKG2D, NKG2A, IL-1R8 and TIGIT in NK-92 cells after co-culturing with colorectal cancer HCT116; Isotype is the isotype control, NK92-vector is the control group, and NK92-PRMT5 is the NK-92 cells overexpressing PRMT5, which is the experimental group;

[0068] B is a flow cytometry plot of CD56, CD16, NKG2D, NKG2A, IL-1R8 and TIGIT in NK-92 cells after co-culturing with colorectal cancer HCT116, *P<0.05, **P<0.001;

[0069] C is a flow cytometry plot of CD56, CD16, NKG2D, NKG2A, IL-1R8, and TIGIT in NK-92 cells after co-culturing with colorectal cancer SW620 cells; Isotype is the isotype control, NK92-vector is the control group, and NK92-PRMT5 is the experimental group of NK-92 cells overexpressing PRMT5.

[0070] D is a flow cytometry plot of CD56, CD16, NKG2D, NKG2A, IL-1R8 and TIGIT in NK-92 cells after co-culturing with colorectal cancer SW620 cells. *P<0.05, **P<0.001.

[0071] Figure 12 The effect of PRMT5 overexpression on the anti-tumor activity of NK92 cells;

[0072] In the figure, A shows the appearance of tumors dissected in nude mice injected with PBS, nude mice injected with NK-92 cells, and nude mice injected with PRMT5-overexpressing NK-92 cells.

[0073] B is a statistical graph showing the weight of dissected tumors in nude mice injected with PBS, nude mice injected with NK-92 cells, and nude mice injected with PRMT5-overexpressing NK-92 cells.

[0074] C represents the change in tumor volume over days in nude mice injected with PBS, nude mice injected with NK-92 cells, and nude mice injected with PRMT5-overexpressing NK-92 cells. *P<0.05. Detailed Implementation

[0075] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0076] Example 1: An NK cell, its preparation method and application.

[0077] I. Effects of PRMT5 inhibitors on regulating NK cell phenotypic transformation and enhancing NK cell tumorigenesis in the colorectal cancer microenvironment

[0078] 1. Effects of PRMT5 inhibitors on NK-92 cell proliferation.

[0079] NK cell culture: Purchase human NK cell line NK-92 cells (catalog number: CL-0530) from Pronosa. After the cells arrive, loosen the cap and stand the bottle upright for 5 hours until the cells settle to the bottom. Carefully aspirate the supernatant and transfer it to a 15mL centrifuge tube, leaving 8mL of culture medium in the bottle. Incubate at 37℃ with 5% CO2. During the culture process, replenish with 40% volume of fresh NK-92 medium (pronosa NK-92 cell-specific medium, catalog number: CM-0530) every 60 hours. If the turbidity of the culture medium increases significantly and the cell density reaches 85%, use the centrifugation optimized passage method for immediate passage. First, collect the cells along with the culture medium into a 15mL centrifuge tube, then centrifuge at 800rpm / min for 3 minutes at room temperature. After removing the supernatant, resuspend the cells in complete culture medium and adjust the density as needed. Then, aliquot the cells into new culture containers at an appropriate ratio, add fresh culture medium, and continue culturing.

[0080] NK-92 cells in the logarithmic growth phase were collected and divided into a control group and an experimental group. The control group consisted of normally cultured NK-92 cells, while the experimental group consisted of NK-92 cells cultured in NK-92 cell culture medium (purchasing NK-92 cell culture medium, catalog number: CM-0530, purchased from Pronosai) containing 1 μmol / L, 3 μmol / L, and 5 μmol / L of the PRMT5 enzyme activity inhibitor (EPZ015666). After two days of culture, the cells were collected, lysed, and proteins were extracted. The inhibitory effect of different concentrations of EPZ015666 on the PRMT5 enzyme activity in NK-92 cells was detected by Western blotting.

[0081] Protein extraction: After two days of treatment with PRMT5 enzyme activity inhibitor, cells were collected into 15 mL centrifuge tubes, centrifuged at 800 rpm for 3 minutes, the supernatant was discarded, and the cells were resuspended in PBS. After resuspending, the cells were pipetted and transferred to EP tubes, centrifuged at 2500 rpm for 5 minutes, the supernatant was discarded, and excess PBS was aspirated (without aspirating cells). 60 mL of lysis buffer was added to each EP tube, and lysis was performed for 50 minutes, mixing by pipetting every 10 minutes. The cells were then centrifuged at 13000 rpm for 5 minutes in a pre-cooled 4°C centrifuge. The supernatant was transferred to a new 1.5 mL EP tube, and the sample information and date were labeled. Protein concentration was determined using the BCA reagent kit method. Based on the quantity of standards and samples, BCA reagent and Cu reagent were mixed at a ratio of 50:1 to prepare the BCA working solution (prepared fresh for immediate use). In a 96-well plate, 200 µL of BCA working solution, 18 µL of buffer, and 2 µL of sample were added to each well as the experimental group, while 200 µL of BCA working solution and 18 µL of buffer were added as the control group. The plate was incubated at 37°C for 25 minutes, and the absorbance was measured at 562 nm using a microplate reader. The protein concentration was calculated according to the standard curve and marked on the EP tube. After protein concentration determination, a boiling treatment was performed. 6× luding buffer was mixed with protein at a ratio of 1:5, and after thorough mixing, the mixture was boiled at 100°C for 10 minutes, then cooled to room temperature and stored at -20°C for long-term storage.

[0082] Western blotting: Prepared samples were subjected to SDS-PAGE electrophoresis at 100V until phenol blue ran off the gel. The separated proteins were then transferred to a PVDF membrane via a constant current of 220mA for 2 hours, with the entire process performed on ice. The PVDF membrane was then blocked by immersing it in 5% skim milk powder at room temperature for 2 hours. The membrane was incubated overnight at 4°C with primary antibody; washed with TBST for 5 minutes x 5 times, followed by incubation with HRP-labeled secondary antibody at room temperature for 1 hour; washed with TBST for 5 minutes x 5 times, then ECL luminescence was added, and the membrane was detected using a UVP chemiluminescence imaging system.

[0083] The results are as follows Figure 1 As shown, 1 μmol / L, 3 μmol / L, and 5 μmol / L LEPZ015666 significantly inhibited PRMT5 activity in NK-92 cells. Figure 1 (A), and with the continuous effect of 1 μmol / LEPZ015666 on NK-92, the proliferation of NK-92 cells slowed down (A). Figure 1 B).

[0084] 2. Effects of PRMT5 inhibitors on NK-92 cell phenotype.

[0085] Flow cytometry was used to detect the effects of CD56 and CD16, the inhibitory receptors SIGIRR (IL-1R8) and NKG2A, and the activating receptor NKG2D in NK-92 cells. NK-92 cells in the logarithmic growth phase were used, and the cells were divided into groups of 5 × 10⁶ cells per well. 5 Cells were seeded at a density of 1000 cells per well in 6-well plates, and the experiment was then divided into three groups: the experimental group (containing 1 μmol / L of...) Cells were cultured for 2 days in EPZ015666 NK-92 cell-specific medium, a blank control group (with an equal volume of inhibitor-free NK-92 cell-specific medium), and an isotype control group (with an equal volume of inhibitor-free NK-92 cell-specific medium). Cells from each group were collected, centrifuged, and the supernatant was discarded. Cells were washed with PBS, centrifuged again, and resuspended. CD16 (1:600), CD56 (1:500), SIGIRR (1:600), NKG2A (1:600), and NKG2D (1:500) were diluted according to the manufacturer's instructions. The corresponding specific primary antibodies were added to the experimental and control groups. Cells were incubated at 4°C in the dark for 35 minutes. After incubation, the supernatant was discarded, and cells were washed again with PBS and collected by centrifugation. Secondary antibody dilutions were prepared according to the manufacturer's instructions (prepared fresh and stored in the dark). 500 µL of the secondary antibody dilution was added to each of the experimental, blank, and isotype control groups, and incubated in the dark for 30 minutes. After incubation, centrifuge again and discard the supernatant. Wash the cells with PBS, discard the supernatant, resuspend the cells in PBS, filter the cells through a 70μm cell filter, and each 1.5 ml centrifuge tube should contain approximately 150 mL of cell suspension. Label the samples and inject them into a flow cytometer for detection. Collect the data and use professional analysis software to statistically analyze and process the collected data.

[0086] The results are as follows Figure 2 As shown, the results indicated that the expression of effector molecules CD16, inhibitory receptors NKG2A and IL-1R8, and activating receptor NKG2D in NK-92 cells after PRMT5 inhibition was higher than that in wild-type NK-92 cells, but the expression of effector molecule CD56 in NK-92 cells after PRMT5 inhibition was lower than that in wild-type NK-92 cells.

[0087] 0. Effect of PRMT5-inhibited NK-92 cells on the killing effect of colorectal cancer.

[0088] Treatment of NK-92 cells: NK-92 cells in the logarithmic growth phase were taken and divided into two groups: the first group consisted of normally cultured NK-92 cells, and the second group consisted of NK-92 cells cultured for two days in NK-92 cell-specific medium containing 1 μmol / L EPZ015666.

[0089] Culture of human colorectal cancer cell lines SW620 and HCT116: The colorectal cancer cell lines of this invention are SW620 (catalog number: CL-0225) and HCT116 (catalog number: CL-0096) purchased from Pronos. After receiving the cells at room temperature, take photos to record any leakage or damage to the culture flasks. Do not open the culture flask caps. Place the cells in a cell culture incubator and incubate statically for 3 hours to stabilize the cell state. If the cell growth density is below 60% under a microscope, aspirate the culture medium from the culture flasks until only 7 mL of complete culture medium remains in the culture flasks. Place the flasks in a cell culture incubator and continue culturing. If the cell growth density reaches 70% or higher, the cells can be passaged. Aspirate the original culture medium, add about 2 mL of PBS, gently shake the culture flask to rinse the cells, aspirate and discard the PBS, add 7 mL of complete culture medium (SW620 cells use SW620 cell-specific culture medium purchased from Pronosa, catalog number: CM-0225A; HCT116 cells use HCT116 cell-specific culture medium, catalog number: CM-0096), shake well, and incubate in a 37℃ constant temperature, 5% CO2 incubator.

[0090] Two colorectal cancer cell lines, HCT116 and SW620, were selected as target cells. HCT116 and SW620 cells were seeded into 96-well plates. When the cells reached 55% confluence, the colorectal cancer cell lines were co-cultured with NK-92 cells or PRMT5-inhibited NK92 cells. First, 1×10⁻⁶ cells were... 3 3×10 3 5×10 3 7×10 3 9×10 3 11×10 3 After NK-92 cells and PRMT5 inhibition, NK-92 cells were co-cultured with HCT116 and SW620 cells for 6 hours, respectively. The activity of tumor cells was then detected using CCK8 reagent. NK-92 cells were aspirated, washed with PBS, and replaced with complete medium containing 10% CCK8. The 96-well plates were placed in a constant temperature incubator at 37°C and 5% CO2 and incubated in the dark for 2.5 hours. After incubation, the absorbance (OD value) of each well was measured at 450 nm using a microplate reader.

[0091] The results are as follows Figure 3 As shown, for SW620 cells, PRMT5-inhibited NK-92 cell killing activity was stronger than that of NK-92 cells ( Figure 3 The same result was observed in HCT116 cells (A); Figure 3 B).

[0092] 4. Effects of PRMT5 inhibition on NK-92 cell phenotype after co-culturing with colorectal cancer cells.

[0093] The expression of CD56, CD16, SIGIRR (IL-1R8), NKG2A, and NKG2D in NK-92 cells co-cultured with colorectal cancer cells and in cells inhibited by PRMT5 was detected by flow cytometry.

[0094] The results are as follows Figure 4 As shown, after co-culturing with SW620 cells: the results showed that the expression of CD56 and CD16 was not significantly different in NK-92 cells and NK-92 cells after PRMT5 inhibition. The expression of the NK cell activating receptor NKG2D was increased in NK-92 cells after PRMT5 inhibition, while the expression of the inhibitory receptors NKG2A and IL-1R8 was decreased in NK-92 cells after PRMT5 inhibition.

[0095] The results are as follows Figure 5 As shown, after co-culturing with HCT116 cells: the expression of CD56, CD16, and IL-1R8 showed no significant difference between NK-92 cells and those after PRMT5 inhibition. The expression of the NK cell activating receptor NKG2D was increased in NK-92 cells after PRMT5 inhibition, while the expression of the inhibitory receptor NKG2A was decreased. This indicates that PRMT5 inhibition induces a phenotypic change in NK-92 cells after co-culturing with colorectal cancer cells.

[0096] 5. PRMT5-inhibited NK-92 cells (E-NK-92 cells) enhance their killing effect against colorectal cancer in vivo.

[0097] Mouse xenograft model: Male BALB / c-nu nude mice were selected as experimental subjects. Tumor cells from each group were harvested and cultured under aseptic conditions, resuspended in PBS, and then subjected to 5×10⁻⁶ cells / mL. 6 Personal HCT116 colorectal cancer cells (total volume 0.2 mL) were injected subcutaneously into the groin area of ​​mice. After solid tumor formation, the short and long diameters of the tumor were measured daily using the following formula: V = d 2 The tumor volume is calculated using xD / 2(d: tumor short diameter, D: tumor long diameter).

[0098] Immunotherapy in nude mice: When the largest tumor in nude mice reached approximately 25 mm in size... 3 Nude mice were randomly divided into two groups (n=3). Cellular immunotherapy was initiated with NK-92 cells injected via the tail vein. The control group received wild-type NK-92 cells, while the experimental group received NK-92 cells treated with EPZ015666 for two days. Cells were collected aseptically, resuspended in PBS, and cultured at 5 × 10⁻⁶ cells / mL. 6Each nude mouse was injected with 200 μL of the tumor cells every three days for a total of four injections. The mice were observed during the injections, and the short and long diameters of the tumor were measured. The tumor volume was calculated using a formula. The tumor volume was considered reached when it reached 400 mm². 3 Nude mice were anesthetized, euthanized by cervical dissection, and the tumors were dissected and weighed. A tumor growth curve was plotted with time on the horizontal axis and tumor volume on the vertical axis.

[0099] The results are as follows Figure 6 As shown, tumors in nude mice injected with PRMT5-inhibited NK-92 grew more slowly than those in nude mice injected with NK-92 (e.g., Figure 6 (C), after dissection, the tumor was removed, and the tumors in nude mice injected with PRMT5-inhibited NK-92 were significantly smaller than those in nude mice injected with NK-92 cells (e.g., C). Figure 6 A, Figure 6 (B), which also proves that inhibiting PRMT5 enhances the tumor-killing effect of NK-92 cells in vivo.

[0100] II. Effects of PRMT5 overexpression on NK cell phenotypic transformation and attenuation of NK cell tumor activity in the colorectal cancer microenvironment.

[0101] 1. Establishment of PRMT5-overexpressing NK92 cell line and its effect on NK cell proliferation.

[0102] Purchased empty vector plasmid Lenti-SFFV-CoGFP-Puro from Qinke Biotechnology Co., Ltd. Figure 7 The PRMT5 overexpression plasmids PCDH-EF1a-CoGFP-P2A-Puro-SFFV-PRMT5-3MYC-WPRE (A) and PRMT5 overexpression plasmids Figure 7 (B).

[0103] Packaging plasmids pMD2.G and psPAX2, and target plasmids (empty vector plasmid or PRMT5 overexpression plasmid) were transfected into 293T cells with the aid of Lipofectamine 3000 reagent to prepare lentiviruses carrying the empty vector plasmid and the PRMT5 overexpression plasmid, respectively. The prepared lentivirus solutions carrying the empty vector plasmid and the PRMT5 overexpression plasmid were then transfected into NK92 cells to obtain NK92 cell lines with GFP fluorescence and PRMT5 overexpression (NK92-PRMT5) and NK92 cell lines transfected with the empty vector plasmid (NK92-Vector).

[0104] The results are as follows Figure 8As shown, Western blot was used to detect the protein expression level of PRMT5 in NK92-PRMT5 cells and NK92-Vector cells. It was evident that the protein expression level of PRMT5 in NK92-PRMT5 cells was significantly higher than that in NK92-Vector cells.

[0105] The results are as follows Figure 8 As shown, the effect of PRMT5 overexpression on the long-term proliferation of NK92-Vector and NK92-PRMT5 cells was detected by the CCK8 kit. It was found that the cell proliferation rate of NK92-PRMT5 cells was significantly higher than that of NK92-Vector cells after days 1, 2 and 3.

[0106] 2. Effects of PRMT5 overexpression on the phenotype of NK92 cells.

[0107] To investigate whether overexpression of PRMT5 in NK92 cells affects the phenotype of NK92 cells, this invention uses flow cytometry to detect the expression of CD56, CD16, CD56, SIGIRR (IL-1R8), NKG2A, NKG2D, and TIGIT in NK92-Vector cells and NK92-PRMT5 cells, respectively.

[0108] The results are as follows Figure 9 As shown, the expression of CD56, CD16, and NKG2A did not differ significantly between NK92-PRMT5 and NK92-Vector cells. However, the expression of TIGIT, NKG2D, and SIGIRR was significantly higher in PRMT5-overexpressing NK92-PRMT5 cells than in the control group NK92-Vector. This indicates that PRMT5 overexpression alters the phenotype of NK92 cells.

[0109] 3. In vitro, overexpression of PRMT5 inhibited the killing effect of NK92 cells on CRC cells.

[0110] To investigate the effect of PRMT5 overexpression on the cytotoxicity of NK92 cells, this invention selected two cell lines (HCT116 and SW620) as target cells and co-cultured them with NK92 cells. First, NK92-Vector cells and NK92-PRMT5 cells were co-cultured with HCT116 and SW620 cells, respectively, for 6 hours, and then the activity of the tumor cells was detected using the CCK8 reagent.

[0111] The results are as follows Figure 10As shown, for HCT116 cells, the cytotoxic effect of NK92-PRMT5 cells was significantly reduced compared to NK92-Vector cells; the same result was observed in SW620 cells.

[0112] 4. Flow cytometry analysis of phenotypic changes in NK92 cells after co-culture.

[0113] To investigate whether co-culturing NK92 cells with tumor cells after overexpressing PRMT5 has any effect on the phenotype of NK92 cells, this invention uses flow cytometry to detect the expression of CD56, CD16, CD56, SIGIRR (IL-1R8), NKG2A, NKG2D, and TIGIT in NK92-Vector cells and NK92-PRMT5 cells, respectively.

[0114] The results are as follows Figure 11 As shown, after co-culturing with HCT116 cells: the expression of CD56, CD16, and NKG2A did not differ significantly between NK92-PRMT5 and NK92-Vector cells. However, the expression of TIGIT and SIGIRR was significantly higher in PRMT5-overexpressing NK92-PRMT5 cells than in the control group NK92-Vector. Conversely, the expression of NKG2D was significantly lower in PRMT5-overexpressing NK92-PRMT5 cells than in the control group NK92-Vector.

[0115] Co-culture with SW620 cells: The results showed no significant difference in the expression of CD56, CD16, NKG2A, TIGIT, and SIGIRR between NK92-PRMT5 and NK92-Vector cells. However, the expression of NKG2D was significantly higher in PRMT5-overexpressing NK92-PRMT5 cells than in the control group NK92-Vector.

[0116] 5. Overexpression of PRMT5 in vivo inhibits the anti-tumor function of NK92 cells.

[0117] To investigate whether PRMT5 overexpression affects the antitumor function of NK92 in vivo, BALB / c nude mice were subcutaneously injected with HCT116 tumor cells on day 0. The average tumor size was 50 mm. 3 At that time, the treatment group began to receive tail vein injections of NK92 cells.

[0118] The results are as follows Figure 12As shown, the tumor volume of mice inoculated with NK92-Vector cells was significantly lower than that of mice in the PBS group (P<0.01); however, there was no difference in tumor volume and weight between mice inoculated with NK92-PRMT5 cells and mice in the PBS group, and the tumor volume and weight of mice inoculated with NK92-Vector cells were significantly lower than those of mice inoculated with NK92-PRMT5 cells (P<0.5). The same results were observed for tumor size. Therefore, tail vein injection of PRMT5-overexpressing NK92 cells (NK92-PRMT5 cells) significantly inhibited the original anti-tumor function of NK92 cells.

[0119] Although preferred embodiments of the invention have been described, those skilled in the art, once they have learned the basic inventive concept, can make other changes and modifications to these embodiments.

[0120] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. Use of PRMT5-inhibited NK-92® cells in the manufacture of a medicament for treating colorectal cancer, characterized in that, The PRMT5-inhibited NK-92 cells are obtained by targeting and inhibiting the expression of the PRMT5 gene of the NK-92 cells; The PRMT5 gene of the NK-92 cells is targeted and inhibited by adding a PRMT5 inhibitor in the NK-92 cell culture process, so that the modified PRMT5-inhibited NK-92 cells are obtained; The PRMT5-inhibited NK-92 cells are obtained by inoculating the NK-92 cells into a culture medium containing 1 μmol / L-3 μmol / L EPZ015666.

2. The PRMT5-inhibited NK-92® cells of claim 1 for use in the manufacture of a medicament for the treatment of colorectal cancer, wherein, The concentration of the EPZ015666 in the culture medium is 1 μmol / L.

3. The use of E-NK-92 cells according to claim 2 for the preparation of a medicament for the treatment of colorectal cancer, characterized in that, The E-NK-92 cells are used for inhibiting the tumor volume and weight.

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