Application of Nat10 inhibitor to promotion of anti-tumor immunity by targeted destruction of tumor infiltration Treg function

Abstract

The invention relates to application of a Nat10 inhibitor to promotion of anti-tumor immunity through targeted destruction of a tumor infiltration Treg function. Specifically, the invention relates to an application of a Nat10 inhibitor in preparation of drugs or kits for targeted destruction of a tumor infiltration Treg function. The invention proposes that the Nat10 inhibitor can recover the tumor killing function of CD8 + T cells by destroying the functions of Treg cells in a tumor immune microenvironment for the first time, so that the tumor immune microenvironment is improved, a positive influence is generated in the aspect of improving the tumor immune effect, and a theoretical foundation is laid for improving the clinical tumor treatment effect.

Description

Technical Field

[0001] This invention relates to the field of tumor immunotherapy, specifically to the application of Nat10 inhibitors in promoting anti-tumor immunity by targeting and disrupting the function of tumor-infiltrating Treg cells. This invention applies Nat10 inhibitors to target and disrupt the function of tumor-infiltrating Treg cells, effectively destroying Treg cell function in the tumor immune microenvironment and restoring CD8+. + T cells enhance the tumor-killing function and improve the tumor immune microenvironment, thus having a positive impact on improving tumor immunity. Background Technology

[0002] Melanoma is the deadliest form of skin cancer, originating from the malignant transformation of melanocytes. Melanoma has long been considered a malignant tumor with few treatment options. The low efficacy and unavoidable drug resistance of currently available targeted therapies have hindered further improvements in melanoma treatment.

[0003] Mounting evidence suggests that an elevated proportion of tumor-infiltrating regulatory T cells is associated with poor patient prognosis. Regulatory T cells (Tregs) are a subset of CD4+ T cells with immunosuppressive functions, characterized by the expression of the forkhead box transcription factor Foxp3, essential for maintaining immune homeostasis and preventing excessive tissue damage. While Tregs are necessary to limit autoimmunity and maintain immune tolerance, they can negatively impact cancer treatment by suppressing antitumor immunity. Tregs maintain a homeostatic proportion of 5%–10% in blood and lymphoid tissues, but they accumulate in the tumor microenvironment (TME) of humans and mice, typically exceeding 50% of all T cells. Tumor-infiltrating (TI) Tregs are highly suppressive and effectively inhibit antitumor immunity. A higher proportion of TI Tregs is associated with worse prognosis in patients with various cancer types. Therefore, the function of TI Tregs can be a significant obstacle to successful immunotherapy. Given that systemic Treg suppression can lead to life-threatening autoimmune or inflammatory complications, immunotherapy should target only TI Tregs. Targeting TI Treg cell-specific markers, such as the co-receptor Nrp1, chemokine receptor CXCR3, the signaling complex CARMA1-BCL10-MALT1 (CBM), transcription factor NF-κB, and epigenetic regulator EZH2, which maintain TI Treg cell stability, is effective and synergistic with anti-PD-1 immunotherapy. Therefore, targeting and disrupting the stability and immunosuppressive capacity of TI Treg cells may help improve the effectiveness of anti-tumor therapy.

[0004] In recent years, RNA epigenetics, through post-transcriptional modifications that affect RNA structure and function, has played a crucial role in the development of various diseases and is known as epitranscriptomics, becoming one of the cutting-edge research areas in biomedicine. Currently, more than 100 chemical RNA modifications have been identified, with studies reporting that m1A, m5C, m6A, m6Am, m3C, m7G, and ac4C primarily regulate RNA stability and translation efficiency. m6A is the most studied mRNA modification. However, little is known about other mRNA modifications, particularly ac4C. N4-acetylgycidine (ac4C) is a highly conserved RNA modification and the only acetylation event described to date in eukaryotic RNA, occurring widely on different types of eukaryotic RNA, including tRNA, rRNA, and mRNA. Its formation in the human body is catalyzed by N-acetyltransferase 10 (Nat10), the first known ac4C mRNA-modified acetyltransferase. Nat10 is a single enzyme with both acetyltransferase and RNA-binding activities, catalyzing the modification of ac4C at the wobbling cytidine site, thereby stabilizing mRNA and enhancing mRNA translation. Ac4C modification has been confirmed to be involved in several biological processes, including oocyte development, aging, myocardial infarction, systemic lupus erythematosus, and cancer. Nat10's catalysis of ac4C modification at the wobbling cytidine site, thereby stabilizing mRNA and enhancing mRNA translation, is closely related to the disease progression of various human tumors. In particular, numerous studies have reported a close association between Nat10 and the disease progression of various human tumors, including hepatocellular carcinoma, melanoma, bladder cancer, pancreatic cancer, and gastric cancer. However, previous research on the impact of ac4C mRNA modification on tumor progression has been limited to tumor cells, rather than immune cells such as Treg cells. Therefore, it remains unclear whether mRNA acetylation modification is essential for maintaining Treg cell homeostasis and function, and whether Nat10 can be considered a novel target for TI Treg immunotherapy. Summary of the Invention

[0005] Our study found that conditional loss of Nat10 in TI Treg cells enhances anti-tumor immunity by reducing TI Treg cell stability and inhibitory capacity. This can be achieved through synergistic anti-PD-1 immunotherapy and drug inhibition of Nat10 protein. Remodelin, a known Nat10 inhibitor, was found to impair TI Treg cell activation and immunosuppressive function, thereby affecting the fate of immune cells in the tumor microenvironment and promoting anti-tumor immunity.

[0006] This invention specifically relates to the following aspects:

[0007] On one hand, the present invention relates to the use of the Nat10 inhibitor Remodelin in promoting anti-tumor immunity by targeting and disrupting the function of tumor-infiltrating Tregs.

[0008] Specifically, this invention relates to the Nat10 inhibitor Remodelin, which can restore CD8. + T-cell tumor-killing effect.

[0009] Specifically, this invention relates to the Nat10 inhibitor Remodelin, which can improve the tumor immune microenvironment.

[0010] On the other hand, the present invention relates to the use of the Nat10 inhibitor Remodelin in the treatment of tumor diseases by targeting and disrupting the function of tumor-infiltrating Tregs.

[0011] Specifically, the present invention relates to the aforementioned tumor diseases, including melanoma, lung cancer, etc.

[0012] On the other hand, the present invention relates to the use of Nat10 inhibitors in the preparation of drugs or kits that target and disrupt the function of tumor-infiltrating Tregs.

[0013] Specifically, the Nat10 inhibitor described in this invention is Remodelin.

[0014] Specifically, this invention relates to TI Treg cells as the target cells for Remodelin.

[0015] Specifically, the drugs described in this invention include oral medications, injections, inhalers, etc.

[0016] Specifically, the drug or test kit described in this invention includes Remodelin and antiPD-1.

[0017] This invention is the first to propose a novel strategy for applying Nat10 inhibitors to tumor immunotherapy by disrupting the function of tumor-infiltrating Treg cells.

[0018] This invention also proposes for the first time that Remodelin can disrupt the function of tumor-infiltrating Treg cells.

[0019] This invention also proposes for the first time that Remodelin can restore CD8 function by disrupting the function of tumor-infiltrating Treg cells. + T cells enhance the tumor-killing function, thereby improving the tumor immune microenvironment, delaying tumor progression, and increasing the survival time of mice.

[0020] To achieve the above objectives, the present invention provides the following solution:

[0021] In some experimental protocols, the mouse tumor models are lung cancer and melanoma.

[0022] In some experimental protocols, the Nat10 inhibitor is Remodelin.

[0023] In some experimental protocols, the concentration of the drug administered to animals is 100-200 mg / kg, which is dose-dependent. The concentration used is 200 mg / kg, and the drug is prepared fresh for use and administered orally by gavage.

[0024] In some experimental protocols, B16F10 melanoma cells were used to construct both lung cancer and melanoma models, with tumor models established via tail vein injection and subcutaneous injection, respectively. B16F10 cells resemble fibroblasts under a microscope, exhibiting strong proliferative and invasive capabilities and a high tumorigenesis rate, making them significant for tumor treatment research.

[0025] This invention utilizes Nat10 inhibitors to improve the tumor immune microenvironment, which can delay tumor progression to a certain extent.

[0026] In some experimental protocols, treatment with the Nat10 inhibitor Remodelin was initiated on day 4 after tumor cell injection, at a dose of 200 mg / kg per day for a total of 6 consecutive doses. Mice were then sacrificed on day 14 after tumor cell injection for analysis or observation and statistical analysis of mouse survival curves.

[0027] The lungs are the body's respiratory organs, located in the chest cavity. Because pulmonary circulation is crucial for gas exchange and oxygenation, and the blood flow in pulmonary and systemic circulation is comparable, the lungs are one of the sites where tumors easily metastasize. Late-stage malignant tumors can metastasize to the lungs; once metastasis to the lungs occurs, the tumor is considered to be in an advanced stage.

[0028] Subcutaneous tumor models are an important method for preclinical drug efficacy evaluation in vivo and are widely used in biological research and drug development.

[0029] This invention utilizes Remodelin to improve the immune microenvironment of lung tumors and uses it to treat subcutaneous tumors, further verifying the universality of the conclusion that Remodelin can improve the tumor immune microenvironment.

[0030] This invention applies Nat10 inhibitors to target and disrupt the function of tumor-infiltrating Treg cells, effectively destroying Treg cell function in the tumor immune microenvironment and restoring CD8+. + T cells enhance the tumor-killing function and improve the tumor immune microenvironment, thus having a positive impact on improving tumor immunity.

[0031] Beneficial effects

[0032] This invention explores in depth the application of Nat10 inhibitors in improving the tumor immune microenvironment. It is the first discovery that using Nat10 inhibitors can restore CD8+ function by disrupting Treg cell function in the tumor immune microenvironment. + The tumor-killing function of T cells improves the tumor immune microenvironment and has a positive impact on enhancing tumor immunity, laying a theoretical foundation for improving the clinical efficacy of tumor treatment. Attached Figure Description

[0033] Figure 1 a. Experimental protocol. b. Tumor size in control, C57BL / 6 mice (n=8 / group) treated with 100 mg / Kg or 200 mg / Kg Remodelin, and on day 14 after subcutaneous injection of B16F10 tumor cells. c. Tumor growth rate and mass in control, C57BL / 6 mice (n=8 / group) treated with 100 mg / Kg or 200 mg / Kg Remodelin, and on day 14 after subcutaneous injection of B16F10 tumor cells. d. Survival rate of B16F10-bearing C57BL / 6 mice (n=8 / group) after treatment with control, 100 mg / Kg or 200 mg / Kg Remodelin.

[0034] Figure 2 :a, Figure 1 Figure a shows representative flow cytometry plots and statistical graphs of the percentage and absolute number of TI Treg cells in C57BL / 6 mice subcutaneously injected with B16F10 tumor cells after treatment with control and 100 mg / Kg or 200 mg / Kg Remodelin. Figures b and c are also shown. Figure 1 In the experiment shown in a, tumor-infiltrating Treg cells (PD-1+) and CD8+ cells... + The ratio of T cells (PD-1+). d, Figure 1 The rate of weight gain in mice after administration is shown in experiment a.

[0035] Figure 3 :ac, Figure 1 Figure a shows representative histograms, quantitative percentages, and MFI statistics of CD25(a), PD-1(b), and CXCR3(c) in TI Treg cells of C57BL / 6 mice subcutaneously injected with B16F10 tumor cells in the experiment.

[0036] Figure 4 :ad, Figure 1 In the experiment shown in a, C57BL / 6 mice subcutaneously injected with B16F10 tumor cells TI CD8 +Representative flow cytometry plots and percentages of Granzyme B(a), IFNγ(b), Perforin(c), and TNFα(d) in T cells, along with MFI plots.

[0037] Figure 5 a. Experimental protocol. b, c. On day 14 after tail vein injection of B16F10 tumor cells, the mRNA expression levels of NAT10 (b) and MHCI-related molecules (c) in tumor cells of control, anti-PD-1, cisplatin and paclitaxel-treated C57BL / 6 mice (n=7 per group) were observed.

[0038] Figure 6 a. Experimental protocol. b. Distribution of lung tumor nodules in C57BL / 6 mice (n=7 per group) treated with control, anti-PD-1, remodelin, and anti-PD-1 & Remodelin, on day 14 after tail vein injection of B16F10 tumor cells. c. Statistical graph of the number of lung tumor nodules in C57BL / 6 mice (n=7 per group) treated with control, anti-PD-1, remodelin, and anti-PD-1 & Remodelin, on day 14 after tail vein injection of B16F10 tumor cells. d. Survival status of B16F10 tumor-bearing C57BL / 6 mice treated with control (n=12), anti-PD-1 (n=12), remodelin (n=13), and anti-PD-1 & Remodelin (n=13).

[0039] Figure 7 :a, Figure 6 Figure a shows representative flow cytometry and quantitative plots of the percentage of TI Treg cells in C57BL / 6 mice treated with B16F10 tumor cells via tail vein injection in the experiment, after treatment with control, anti-PD-1, remodelin, and anti-PD-1 & remodelin. Figures b and c are also shown. Figure 6 In the experiment shown in a, tumor-infiltrating Treg cells (PD-1+) and CD8+ cells... + The ratio of T cells (PD-1+).

[0040] Figure 8 a, b, Figure 6 Figure a shows the representative histograms and quantitative percentages of CD25(a) and PD-1(b) in TI Treg cells of C57BL / 6 mice injected with B16F10 tumor cells via the tail vein in the experiment, as well as the MFI statistical plot.

[0041] Figure 9 : Figure 6Image a shows a representative immunofluorescence staining image of lung cancer tissue from C57BL / 6 mice injected with B16F10 tumor cells via the tail vein and a quantitative image of the percentage of Nat10+Foxp3+Treg cells within the tumor.

[0042] Figure 10 :ad, Figure 6 In the experiment shown in a, C57BL / 6 mice injected with B16F10 tumor cells via tail vein TI CD8 were... + Representative flow cytometry plots and percentages of Granzyme B(a), IFNγ(b), Perforin(c), and TNFα(d) in T cells, along with MFI plots. Detailed Implementation

[0043] The mice used in this experiment were Wild-type (WT) C57BL / 6 mice. They were housed under normal conditions, with a 12-hour light and dark cycle, and had free access to food and water.

[0044] B16F10 melanoma cells were cultured in RPMI-1640 (C3010-0500, VivaCell) medium containing 10% fetal bovine serum (C04001-020, BI) and 1% triple antibody (S120JV, BasalMedia) at 37°C in a 5% CO2 incubator. Cells were passaged at approximately 90% confluence. All subsequent experiments were performed using cells exhibiting exponential growth.

[0045] The Nat10 inhibitor used in this experiment was Remodelin (selleck), which was dissolved in dimethyl sulfoxide (DMSO) and diluted with sterile Tween 80 (Sigma Aldrich) and 45% 2-hydroxypropyl-β-cyclodextrin solution (Merck) to a final concentration of 20% DMSO, 15% Tween 80 and 65% (45% 2-hydroxypropyl-β-cyclodextrin solution).

[0046] Example 1: Phenotypic observation of the effects of different concentrations of Nat10 inhibitors in the treatment of melanoma

[0047] To determine whether Nat10 inhibitors are effective in treating tumors, wild-type mice bearing tumors were first treated with different concentrations of Nat10 inhibitors, and the phenotypic changes of the tumors in the mice after treatment with Nat10 inhibitors were observed.

[0048] 1. Experimental materials and treatment

[0049] Remodelin working solutions: The final concentrations of Remodelin were 20% DMSO, 15% Tween 80, and 65% (45% 2-hydroxypropyl-β-cyclodextrin solution). The mice were administered different volumes of working solutions orally by gavage according to their body weight and grouping. The working solutions were prepared fresh for each use.

[0050] 2. Experimental Methods

[0051] 2.1 Establishing a tumor model

[0052] The fur on the abdomen and back of the mice was shaved off with a shaver. B16F10 tumor cells were then subcutaneously injected using a 1mL insulin syringe, at a rate of 2 × 10⁶ cells per mouse. 5 Each injection volume is 100 μL.

[0053] 2.2 Use of Remodelin for tumor treatment

[0054] Starting from day 4 of tumor establishment, mice were treated with Remodelin via oral gavage once a day for a total of 6 times. The injection dose was divided into two groups: 100 mg / Kg and 200 mg / Kg, and a control group of 0 mg / Kg.

[0055] 2.3 Observation of anti-tumor effects

[0056] Starting from the third day after tumor formation, the tumor was measured every 2 to 3 days using vernier calipers. The tumor volume formula is V = (length × width × width) / 2.

[0057] After treatment, on day 14 of tumor establishment, the mice were sacrificed. Before sacrifice, the mice were weighed, subcutaneous tumors were photographed, and the tumors were weighed to observe changes in tumor size.

[0058] 3. Results Analysis

[0059] The results are as follows Figure 1 As shown, compared with the control treatment, Remodelin treatment delayed tumor growth and improved survival in a dose-dependent manner.

[0060] Example 2: Analysis of the effects of different concentrations of Nat10 inhibitors on the tumor immune microenvironment

[0061] As observed in Experiment 1, Nat10 inhibitors have good anti-tumor effects. In order to analyze their impact on the tumor immune microenvironment, this experiment conducted flow cytometry analysis on lymphocytes in the tumor environment.

[0062] 1. Experimental materials and treatment

[0063] Related flow cytometry antibodies:

[0064] FITC: anti-Foxp3 (FJK-16s)

[0065] PE:anti-CD25(PC61.5), anti-Granzyme B(QA16A02)

[0066] PerCP / Cy5.5:anti-IFNγ(XMG1.2)

[0067] PE-Cy7:anti-CXCR3 / CD183(CXCR3-173)

[0068] APC: anti-PD-1 / CD279(J43), anti-Perforin(eBioOMAK-D)

[0069] BV421: anti-CD4 (RM4-5)

[0070] BV510: anti-CD8 (53-6.7)

[0071] RP10 medium: Add 10% fetal bovine serum to 1640 medium to obtain RP10 medium.

[0072] 100% Percoll: Mix 100% Percoll by mixing Percoll stock solution (17089109, Cytiva) with 10×PBS buffer (BL316A, Biosharp) at a ratio of 9:1.

[0073] 42% Percoll: Mix 42 mL of 100% Percoll with 58 mL of 1×PBS to prepare a 42% Percoll solution. Prepare and use immediately.

[0074] 70% Percoll: Mix 70 mL of 100% Percoll with 30 mL of 1×PBS to prepare a 70% Percoll solution. Prepare and use immediately.

[0075] FACS buffer: Add 5% fetal bovine serum to 1×PBS buffer to obtain FACS buffer.

[0076] Tumor digestion fluid: DMEM + 1 mg / mL collagenase IV + 10 U / mL DNase I.

[0077] 2. Experimental Methods

[0078] 2.1 Isolation of tumor-infiltrating lymphocytes (subcutaneous tumor)

[0079] On day 14 after tumor formation, the mice were weighed and euthanized, and the intact tumor nodules were removed.

[0080] Weigh the tumor nodules and photograph them according to their size;

[0081] Place the tumor against the wall of a 50mL centrifuge tube, add a small amount of digestive fluid to keep the tumor moist, and then cut the tumor into pieces of approximately 1mm. 3 Add a suitable amount of digestive juice (500ml) to small pieces. 3 Add 5 mL to the tumor sample, and shake at 37°C and 200 rpm for 1 hour.

[0082] Add 10 mL of RP10 to stop digestion, and centrifuge at 500 g for 5 min;

[0083] Discard the supernatant, resuspend the precipitate in 3 mL of 42% Percoll, slowly add it to 3 mL of 70% Percoll (15 mL centrifuge tube), and centrifuge at 750 g for 30 min (6 up, 2 down).

[0084] Carefully remove the white film layer between the two layers of percoll and add it to a new 15mL centrifuge tube. Then fill the tube with RP10 and centrifuge at 500g for 5 minutes to wash away the percoll.

[0085] Discard the supernatant, resuspend the cell pellet in 1 mL of FACS Buffer, and pass it through a 70-micron sieve into a new 1.5 mL centrifuge tube;

[0086] Centrifuge at 500g for 5 min, discard the supernatant, resuspend the precipitated cells in FACS Buffer, filter and transfer to a new 1.5mL EP tube for subsequent cell counting and flow cytometry staining.

[0087] 2.2 Flow cytometry detection of cell surface molecules

[0088] Add 100 μL of the cell suspension obtained in step 2.1 to each well of a 96-well plate;

[0089] Centrifuge at 4℃, 1500 rpm for 5 min, discard the supernatant, and collect the cell pellet at the bottom of a 96-well plate;

[0090] After mixing the external standard antibody with FACS buffer at a certain ratio, add 100 μL to each well of a 96-well plate, resuspend the precipitated cells, incubate at 4°C in the dark for 40 min, and blow the mixture once every 20 minutes.

[0091] After incubation, add 100 μL of FACS buffer to each well of a 96-well plate, centrifuge at 1500 rpm for 5 min at 4°C, discard the supernatant, and the cell pellet will be at the bottom of the 96-well plate.

[0092] Resuspend the cell pellet in FACS buffer, add 200 μL to each well, mix well, filter and transfer to flow cytometry tubes;

[0093] Adjust the parameters according to the flow cytometer cell detection instructions and perform the detection.

[0094] 2.3 Flow cytometry detection of intracellular molecules

[0095] Add 100 μL of the cell suspension obtained in step 2.1 to each well of a 96-well plate;

[0096] Centrifuge at 4℃, 1500 rpm for 5 min, discard the supernatant, and collect the cell pellet at the bottom of a 96-well plate;

[0097] After mixing the external standard antibody with FACS buffer at a certain ratio, add 100 μL to each well of a 96-well plate, resuspend the precipitated cells, incubate at 4°C in the dark for 40 min, and blow the mixture once every 20 minutes.

[0098] Using the Transcription Factor Staining Buffer Set (2511819, ThermoFisher Scientific), prepare the nucleation buffer fresh according to the instructions, add 100 μL to each well of a 96-well plate, resuspend the precipitated cells, and incubate at 4°C in the dark for 1 hour, mixing once every 30 minutes.

[0099] After nucleolysis, add 100 μL of diluted Wash Buffer from the kit to each well in a 96-well plate. Centrifuge at 2500 rpm for 5 min at 4°C, discard the supernatant, and the cell pellet will settle at the bottom of the 96-well plate.

[0100] Mix the internal standard antibody with FACS buffer at a certain ratio and add 100 μL to each well of a 96-well plate. Resuspend the precipitated cells and incubate at 4°C in the dark for 40 min. Mix the cells by blowing once every 20 minutes.

[0101] After incubation, add 100 μL of FACS buffer to each well of a 96-well plate, centrifuge at 1500 rpm for 5 min at 4°C, discard the supernatant, and the cell pellet will be at the bottom of the 96-well plate.

[0102] Resuspend the cell pellet in FACS buffer, add 200 μL to each well, mix well, filter and transfer to flow cytometry tubes;

[0103] Adjust the parameters according to the flow cytometer cell detection instructions and perform the detection.

[0104] 2.4 Flow cytometry detection of transcription factors

[0105] Add 250 μL of cell suspension to a flow cytometer, then add 250 μL of RP10 medium to make a 500 μL system. Add PMA (final concentration 30 ng / mL), lonomycin (final concentration 1 μg / mL), Monensin (final concentration 2.5 μg / mL), and BFA (final concentration 0.6 μL / mL), mix well, and incubate at 37°C in a 5% CO2 incubator for 5-6 hours.

[0106] After stimulation, centrifuge at 4°C and 1500 rpm for 5 min, discard the supernatant, and collect the cell pellet at the bottom of the flow cytometry tube;

[0107] Resuspend the cell pellet in FACS buffer, add 200 μL to each tube, mix well and transfer to a 96-well plate. Centrifuge at 1500 rpm for 5 min at 4 °C, discard the supernatant, and the cell pellet will remain at the bottom of the 96-well plate.

[0108] After mixing the external standard antibody with FACS buffer at a certain ratio, add 100 μL to each well of a 96-well plate, resuspend the precipitated cells, incubate at 4°C in the dark for 40 min, and blow the mixture once every 20 minutes.

[0109] Using the Transcription Factor Staining Buffer Set kit, prepare the nucleation buffer fresh according to the instructions, add 100 μL to each well of a 96-well plate, resuspend the precipitated cells, and incubate at 4°C in the dark for 1 hour, mixing once every 30 minutes.

[0110] After the membrane was broken up, add 100 μL of the diluted Wash Buffer from the kit to each well in a 96-well plate. Centrifuge at 2500 rpm for 5 min at 4°C, discard the supernatant, and the cell pellet will be at the bottom of the 96-well plate.

[0111] Mix the internal standard antibody with FACS buffer at a certain ratio and add 100 μL to each well of a 96-well plate. Resuspend the precipitated cells and incubate at 4°C in the dark for 40 min. Mix the cells by blowing once every 20 minutes.

[0112] After incubation, add 100 μL of FACS buffer to each well of a 96-well plate, centrifuge at 1500 rpm for 5 min at 4°C, discard the supernatant, and the cell pellet will be at the bottom of the 96-well plate.

[0113] Resuspend the cell pellet in FACS buffer, add 200 μL to each well, mix well, filter and transfer to flow cytometry tubes;

[0114] Adjust the parameters according to the flow cytometer cell detection instructions and perform the detection.

[0115] 3. Results Analysis

[0116] Figure 2 As shown, on day 14 after injection of B16F10 tumor cells, the proportion of TI Treg cells in mice treated with high-dose (200 mg / kg) Remodelin was lower than that in mice treated with low-dose (100 mg / kg) Remodelin and the control group. Nat10 inhibitor treatment reduced the ratio of Treg cells to CD8+ cells. + T cell ratio and PD-1 + Treg and PD-1 + CD8 +

[0117] The ratio of T cells. However, Nat10 inhibitors did not affect the weight gain of mice. Figure 3 As shown, Remodelin treatment of mouse TI Treg cells resulted in lower expression of Treg cell markers (CD25, PD-1, and CXCR3), most of which were dose-dependent. Figure 4 As shown, Remodelin treatment of mouse TI CD8 + T cells express more tumor-killing molecules, most of which are dose-dependent. Therefore, the Nat10 inhibitor (Remodelin) impairs TI Treg cell function in a dose-dependent manner, and we used a dose of 200 mg / Kg for subsequent studies.

[0118] Example 3: Detection of the effects of immunotherapy and chemotherapy on the expression of TI Treg NAT10 and tumor cell MHCI and MHCII molecules in a lung cancer model

[0119] To investigate the mechanisms of drug resistance and low universality of immunotherapy and chemotherapy in the treatment of lung cancer in clinical practice, wild-type mice bearing tumors were first treated with anti-PD-1, cisplatin and paclitaxel. Changes in NAT10 expression in TI Treg and changes in the expression of MHCI and MHCII-related molecules in tumor cells were detected.

[0120] 1. Experimental materials and treatment

[0121] Mice were intraperitoneally injected with 200 μg / mouse of anti PD-1 antibody (clone29F.1A12, Bio X Cell, BE0273) or rat IgG2a isotype control (clone 2A3, Bio X Cell, BE0089) at specified time points.

[0122] Cisplatin working solution: Cisplatin (selleck) is dissolved and diluted with physiological saline. 5 mg / kg of cisplatin is injected intraperitoneally at specified time points according to the mouse's body weight. The working solution is prepared fresh for each use.

[0123] Paxlitaxel working solution: Paclitaxel (selleck) was prepared to a final concentration of 5% DMSO, 40% PEG300, 5% Tween 80 and 50% ddH2O. 10 mg / kg paclitaxel was injected intraperitoneally into mice at specified time points according to their body weight. The working solution was prepared fresh for each use.

[0124] 2. Experimental Methods

[0125] 2.1 Establishing a tumor model

[0126] Mice were injected via tail vein with B16F10 tumor cells at a rate of 2 × 10⁶ cells per mouse. 5 Each injection volume is 500 μL.

[0127] 2.2 Tumor treatment using anti-PD-1, cisplatin, and paclitaxel

[0128] Starting from day 6 after tumor formation, mice were treated with intraperitoneal injection of 200 μg anti PD-1 antibody, 5 mg / kg cisplatin, and 10 mg / kg paclitaxel.

[0129] 2.3 Detection of TI Treg NAT10 and MHCI molecular expression in tumor cells

[0130] After treatment, on day 14 of tumor establishment, the mice were sacrificed. Following the method in Example 2, TITreg cells were sorted by flow cytometry, RNA was extracted, and NAT10 expression in TITreg cells was detected by qPCR. Simultaneously, some tumor nodules were collected, RNA was extracted, and the expression of MHCII-related molecules (H2D, H2K) and MHCII-related molecules (H2A, H2E) in tumor cells was detected by qPCR.

[0131] 3. Results Analysis

[0132] The results are as follows Figure 5 As shown, compared with the control treatment, anti-PD-1, cisplatin, and paclitaxel treatments all increased the expression level of NAT10 in TITreg cells to some extent, which partly explains why immunotherapy and chemotherapy lead to poor treatment efficacy by enhancing TITreg function. On the other hand, compared with the control treatment, anti-PD-1, cisplatin, and paclitaxel treatments also increased the expression levels of MHCII and MHCII-related molecules in tumor cells to some extent, indicating that immunotherapy and chemotherapy not only recruit CD8+, but also... + T cells exert anti-tumor functions and can also recruit CD4. + T cells promote the activation and functional maturation of Tregs, leading to poor treatment efficacy or drug resistance.

[0133] Example 4: Detecting the effect of combined treatment of Nat10 inhibitor and anti-PD-1 on controlling tumor growth and improving the tumor immune microenvironment in a lung cancer model.

[0134] Examples 1 and 2 have demonstrated the therapeutic effect of the Nat10 inhibitor Remodelin on improving the immune microenvironment of subcutaneous melanoma. To verify the generalizability of the therapeutic effect of Remodelin, a lung cancer model was constructed by tail vein injection of B16F10 tumor cells into wild-type mice, and the therapeutic effect of Remodelin in lung cancer and its improvement on the tumor immune microenvironment were observed.

[0135] To verify whether the combined use of the Nat10 inhibitor Remodelin and anti-PD-1 immunotherapy has a synergistic effect and a better anti-tumor effect, Example 3 included four experimental groups: control, anti-PD-1, Remodelin, and anti-PD-1 & Remodelin. Mice were intraperitoneally injected with 200 μg / mouse of anti-PD-1 antibody (clone29F.1A12, Bio X Cell, BE0273) or rat IgG2a isotype control (clone 2A3, Bio X Cell, BE0089) at specified time points.

[0136] 1. Experimental Methods

[0137] 1.1 Establishing a tumor model

[0138] Mice were injected via tail vein with B16F10 tumor cells at a rate of 2 × 10⁶ cells per mouse. 5 Each injection volume is 500 μL.

[0139] 1.2 Tumor treatment using Remodelin and anti-PD-1

[0140] Starting on day 4 of tumor establishment, mice were administered 200 mg / kg Remodelin orally via gavage; starting on day 7, they were treated with 200 μg anti PD-1 antibody via intraperitoneal injection.

[0141] 1.3 Phenotypic observation of the effects of Nat10 inhibitors and anti-PD-1 therapy on tumor treatment

[0142] On day 14 of tumor establishment, mice were sacrificed, and lungs were harvested to count the number of tumor nodules and photographed.

[0143] 1.4 Flow Cytometry Analysis of the Tumor Immune Microenvironment

[0144] Following the method in Example 2, flow cytometry was used to detect the proportions and expression of surface molecules, internal molecules, and transcription factors of lymphocytes in a tumor environment.

[0145] 1.5 Immunofluorescence detection of TI Nat10 + FoxP3 + Treg cells

[0146] 2. Results Analysis

[0147] Figure 6 As shown, anti-PD-1 therapy alone cannot effectively control tumor growth and prolong survival compared to control therapy, while Nat10 inhibitors or combined anti-PD-1 therapy can better control tumor growth and prolong survival, and the efficacy of combination therapy is superior to that of the Nat10 inhibitor group. Figure 7 As shown, on day 14 after B16F10 cell injection, the proportion of TI Treg cells in the Remodelin and anti-PD-1 & Remodelin groups was lower than that in the control and anti-PD-1 alone groups. Simultaneously, Nat10 inhibitor treatment reduced the ratio of Treg cells to CD8+ cells. + T cell ratio and PD-1 + Treg and PD-1 + CD8 + The ratio of T cells indicates that combination therapy has a more effective PD-1 targeted immunotherapy effect. Figure 8 As shown, in TI Treg cells of mice treated with Remodelin in combination with anti-PD-1, the expression levels of Treg cell activation markers (CD25, PD-1) were the lowest. Figure 9 Immunofluorescence staining clearly showed that, compared with the control and anti-PD-1 treatment groups, the TI Nat10 levels in the Remodelin and Remodelin & anti-PD-1 treatment groups were significantly lower. + FoxP3 + The proportion of Treg cells was significantly reduced, but anti-PD-1 treatment did not affect Nat10. + FoxP3 + Tumor infiltration by Treg cells. Figure 10 As shown, the TI CD8 group of mice treated with Remodelin combined with anti-PD-1 showed... + T cells secrete the most tumor-killing molecules.

[0148] In conclusion, the combination of Nat10 inhibitors and antiPD-1 immunotherapy impairs the activation and immunosuppressive function of TI Treg cells, while enhancing effector CD8. + The cytotoxic function of T cells improves the tumor immune microenvironment and has a positive impact on enhancing tumor immunity, laying an important theoretical foundation for improving the clinical efficacy of tumor treatment.

Claims

1. Use of Nat10 inhibitors in the preparation of drugs or kits that target and disrupt the function of tumor-infiltrating Tregs.

2. The use according to claim 1, characterized in that, The Nat10 inhibitor is Remodelin.

3. The use according to claim 1 or 2, characterized in that, The targeted cells are TI Treg cells.

4. The use according to any one of claims 1-3, characterized in that, The drugs include oral medications, injections, and inhalers.

5. The use according to any one of claims 1-4, characterized in that, The drug or kit includes Remodelin and a PD-1 antibody.

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