Method for enhancing curative effect of immune checkpoint inhibitor by targeting DUSP9

Abstract

The invention discloses a method for enhancing the curative effect of an immune checkpoint inhibitor by targeting DUSP9, and application of the inhibitor of targeting DUSP9 and the immune checkpoint inhibitor (ICI) in preparation of a medicine for treating tumors through combined action, the immune checkpoint inhibitor is a PD-L1 antibody or a PD-1 antibody, and the inhibitor can reduce expression or enzyme activity of a DUSP9 gene or protein. According to the invention, siRNA targeting DUSP9 is designed, so that DUSP9 is effectively targeted, and the expression level of DUSP9 is remarkably reduced; siRNA and a PD-1 antibody are combined for use, so that the anti-tumor effect is improved; the action mechanism of the DUSP9 is related to PD-L1 expression regulation, and the DUSP9 acts with STAT3 to dephosphorylate the STAT3 so as to inhibit PD-L1 expression; pD-L1 expression can be up-regulated by using siRNA to interfere with DUSP9 expression, so that the ICI curative effect is improved; the DUSP9 can be used as an ICI curative effect prediction marker.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a method for targeting DUSP9 to enhance the efficacy of immune checkpoint inhibitors. Background Technology

[0002] Immune checkpoint inhibitors (ICIs), especially PD-1 / PD-L1 blocking antibodies, have attracted widespread attention due to their significant efficacy; however, their overall clinical response rate remains low (approximately 10-30%). Therefore, identifying responsive individuals to ICI treatment and developing combination therapy strategies to improve ICI efficacy are significant challenges. PD-1 / PD-L1 target deficiency is a major cause of ICI non-responsiveness. PD-L1 expression level is a widely used predictive biomarker for ICI efficacy in clinical practice, and patients with high PD-L1 expression are more likely to benefit from anti-PD-1 / PD-L1 therapy. Low or absent PD-L1 expression is one of the important factors contributing to ICI non-responsiveness.

[0003] Based on this, this application proposes a combined application of a DUSP9-targeting inhibitor and an immune checkpoint inhibitor, as well as related biomarkers. Summary of the Invention

[0004] The technical problem to be solved by this invention is to provide a method to enhance the efficacy of immune checkpoint inhibitors. This application starts with PD-L1 mutually exclusive expression genes and systematically analyzes PD-L1 mutually exclusive expression genes from transcriptome data through innovative bioinformatics analysis methods. Through experimental verification, it was finally found that siRNA targeting DUSP9 can increase the efficacy of ICI treatment. At the same time, DUSP9 itself can be used as a marker molecule for predicting ICI treatment response.

[0005] The technical problem to be solved by the present invention is achieved through the following technical solution: The application of a combination of a DUSP9-targeting inhibitor and an immune checkpoint inhibitor in the preparation of a drug for treating tumors, characterized in that the immune checkpoint inhibitor is a PD-L1 antibody or a PD-1 antibody, and the inhibitor is capable of reducing the expression of the DUSP9 gene or protein or the enzyme activity.

[0006] Preferably, in the above technical solution, the tumor is melanoma, mesothelioma, hepatocellular carcinoma, head and neck squamous cell carcinoma, colon cancer, lung cancer, or cervical cancer, and the inhibitor enhances the anti-tumor efficacy of the immune checkpoint inhibitor by upregulating the expression of PD-L1 on the surface of tumor cells; the inhibitor is a small molecule compound, protein, polypeptide, siRNA, or shRNA, and the siRNA or shRNA sequence is as shown in any one of SEQ ID No. 1-2.

[0007] A pharmaceutical composition or combination for treating tumors, wherein the active pharmaceutical ingredient of the pharmaceutical composition or combination comprises: The first active ingredient is any of the above-mentioned inhibitors targeting DUSP9; The second active ingredient is an immune checkpoint inhibitor, which is a PD-L1 antibody or a PD-1 antibody.

[0008] Preferably, in the above technical solution, the inhibitor targeting DUSP9 is a small molecule compound, protein, polypeptide, siRNA or shRNA, and the siRNA or shRNA sequence is shown in SEQ ID No. 1-2; the pharmaceutical composition or combination of drugs also includes a pharmaceutically acceptable carrier.

[0009] The application of DUSP9-targeting inhibitors in the preparation of products for enhancing the efficacy of immune checkpoint inhibitors, wherein the inhibitors are small molecule compounds, proteins, peptides, siRNAs, or shRNAs that increase STAT3 phosphorylation levels and upregulate PD-L1 expression by inhibiting DUSP9 expression or activity.

[0010] Preferably, in the above technical solution, the product contains siRNA or shRNA, the sequence of which is shown in SEQ ID No. 1-2, and the immune checkpoint inhibitor is a PD-L1 antibody.

[0011] Preferably, in the above technical solution, the product is used to inhibit the expression of DUSP9 enzyme, thereby increasing the phosphorylation level of STAT3 protein Y705 and S727 sites, thereby upregulating the expression of PD-L1 on the surface of tumor cells, and thus enhancing the anti-tumor efficacy of immune checkpoint inhibitors.

[0012] The application of reagents for detecting the DUSP9 gene or protein in the preparation of products for predicting the response of cancer patients to anti-PD-1 or anti-PD-L1 antibody therapy, assessing the efficacy of combination immunotherapy, determining prognosis, and monitoring minimal residual disease (MRD).

[0013] Preferably, in the above technical solution, the product includes gene detection reagents based on qPCR, digital PCR, and molecular POCT, or protein detection reagents based on immunohistochemistry, ELISA, and flow cytometry.

[0014] Application of the DUSP9 gene or protein as a biomarker in the preparation of kits for predicting the antitumor efficacy of immune checkpoint inhibitors, assessing the prognosis of combination immunotherapy, and monitoring minimal residual disease (MRD).

[0015] The above-described technical solution of the present invention has the following beneficial effects: (1) A siRNA targeting DUSP9 was designed, which can effectively target DUSP9 and significantly reduce the expression level of DUSP9.

[0016] (2) The combination of DUSP9 siRNA and PD-1 antibody increases the anti-tumor effect of the latter.

[0017] (3) The mechanism of action of DUSP9 is related to its regulation of PD-L1 expression. DUSP9 inhibits PD-L1 expression by interacting with STAT3 and dephosphorylating STAT3. By using siRNA to intervene in DUSP9 expression, PD-L1 expression can be upregulated, thereby increasing the efficacy of ICI.

[0018] (4) DUSP9 can be used as a predictive biomarker for the efficacy of ICI. Attached Figure Description

[0019] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments of the invention and, together with their description, serve to explain the principles of the invention.

[0020] Figure 1 DUSP9 negatively regulates PD-L1 expression in mouse tumor cells. (AC) Western blotting results showed PD-L1 expression in DUSP9 stably knocked-down (DUSP9-KD) Hepa1-6 cells (A), MC38 cells (B), and LLC cells (C). (DF) qPCR results showed PD-L1 mRNA expression in DUSP9-KD cells from Hepa1-6 cells (D), MC38 cells (E), and LLC cells (F). Data are presented as mean ± SEM; n=3; p-values ​​were determined using Dunnett's post-hoc two-way ANOVA; ns, not significant. p<0.05; p<0.01; , p<0.0001.

[0021] Figure 2DUSP9 negatively regulates PD-L1 expression in tumor cells. (AC) Western blot was used to detect PD-L1 expression in DUSP9-overexpressing (DUSP9-OE) tumor cell lines (including A549 cell line (A), FaDu cell line (B), and HeLa cell line (C)). (DF) Western blot was used to detect PD-L1 expression in DUSP9-KD Huh7 cells. (GI) Flow cytometry results showed PD-L1 expression in DUSP9 knocked-down Hepa1-6 cells (G), MC38 cells (H), and LLC cells (I). Data are expressed as mean ± standard error; n=3; two-way ANOVA combined with Sidak method (AC) or Dunnett method (D, F) was used for statistical analysis; ns, not significant; p<0.01; p < 0.001; , p<0.0001.

[0022] Figure 3 To investigate how DUSP9 reduces PD-L1 expression by catalyzing STAT3 dephosphorylation (A), immunoprecipitation-mass spectrometry (IP-MS) was used to compare the abundance of DUSP9 and STAT3 in the IgG control group and the anti-DUSP9 group. (B) Western blotting results showed PD-L1 expression and STAT3 phosphorylation levels in DUSP9-knockdown Huh7 cells. (C) Immunofluorescence results showed STAT3 phosphorylation in DUSP9-knockdown Huh7 cells. (D) Western blotting was used to detect PD-L1 expression and STAT3 phosphorylation levels in DUSP9 and STAT3-knockdown Huh7 cells. (E) Western blotting was used to detect PD-L1 expression and STAT3 phosphorylation levels in DUSP9-knockdown Huh7 cells treated with the p-STAT3-S727 specific inhibitor S3I-201. (F, G) Immunoprecipitation results showed the endogenous interaction between DUSP9 and STAT3 when using DUSP9 antibody (F) and STAT3 antibody (G). (H) In vitro dephosphorylation experiments showed that DUSP9 has a direct dephosphorylation function at the Y705 and S727 sites of p-STAT3. Positive control: calf intestinal alkaline phosphatase (CIAP). Data are expressed as mean ± standard error; n=3; p-values ​​were determined by Student's t-test (A), two-way ANOVA combined with Dunnett's post-hoc test (B), two-way ANOVA combined with Sidak (for PD-L1) or Tukey (for p-STAT3) post-hoc tests (D, E), and one-way ANOVA combined with Dunnett's post-hoc test (H); ns, no significant difference; p<0.05; p<0.01; p < 0.001; , p<0.0001.

[0023] Figure 4 To increase PD-L1 expression on the surface of tumor cells in mice by stable DUSP9 knockdown. A) Gating strategy for flow cytometry experiments. B) Flow cytometry results showing PD-L1 expression levels in DUSP9-KD Hepa1-6, MC38, and LLC-derived tumor cells (GFP+) in a BALB / c nude mouse xenograft model. C) Flow cytometry results showing PD-L1 expression levels in DUSP9-KD MC38 and LLC-derived tumor cells (GFP+) in an immunocompetent C57BL / 6 mouse xenograft model. MFI, mean fluorescence intensity. Data are presented as mean ± SEM; n = 4 (Hepa1-6 and MC38 tumors in BALB / c nude mice) or 6 (LLC tumors in BALB / c nude mice, and LLC tumors in MC38 and C57BL / 6 mice); p-values ​​were determined by Student t-test. p<0.05; p<0.01; p < 0.001; , p<0.0001.

[0024] Figure 5 To investigate the inhibitory effect of DUSP9 stable knockdown on tumor growth in vitro and in vivo. (A) Colony formation assays were performed on DUSP9-KD Hepa1-6 cells, MC38 cells, and LLC cells. Data are presented as mean ± SEM; n=2; p-values ​​were determined by two-way ANOVA using Dunnett's post-hoc test. p<0.05; p<0.01. (BJ) Overview of tumors in BALB / c nude mice subcutaneously transplanted with DUSP9-KDHepa1-6 cells (B), MC38 cells (E), and LLC cells (H). Tumor growth (C, F, I) and tumor weight (D, G, J) are also shown. Data are presented as mean ± SEM; n=4 (BG) or 6 (HJ); p-values ​​were determined by Student's t-test; p<0.01; , p<0.001.

[0025] Figure 6DUSP9 stably knocked down inhibited cytotoxic CD8+ T cells by inducing PD-L1 expression. (AE) The syngeneic tumor model of DUSP9-KD MC38 cells in C57BL / 6 mice included the following: (A) tumor size; (B) tumor growth curve; (C) tumor weight; (D) percentage statistics of tumor-infiltrating T cell subsets; (E) flow cytometry detection of GZMB+CD8+ T cell and TNF-α+CD8+ T cell subsets. (FH) Co-culture of primary T cells and DUSP9-KD Hepa1-6 cells and anti-PD-1 antibody blocking experiment: (F) experimental flowchart; (G) flow cytometry detection of GZMB+CD8+ T cell subsets; (H) percentage statistics of GZMB+CD8+ T cell subsets. Data are presented as mean ± SEM; n = 6 (AD) or 4 (FH); p-values ​​were determined by two-way ANOVA using Student's t-test (BD) or Dunnett's post-hoc test (H); ns, not significant; p<0.05; p<0.01; , p<0.001.

[0026] Figure 7 DUSP9 stably knocked-down combined with PD-1 antibody therapy increased intratumoral cytotoxic CD8+ T cell infiltration. (A) Immunofluorescence results in an MC38 syngeneic xenograft model showed that DUSP9 stably knocked-down combined with PD-1 antibody therapy increased CD8+GZMB+ T cell infiltration. (B) Flow cytometry results in an LLC syngeneic xenograft model showed that the proportions of GZMB+CD8+ T cells and IFN-γ+CD8+ T cells increased after DUSP9 stably knocked-down combined with PD-1 antibody therapy. (CD) Flow cytometry results in MC38 cells (C) or LLC cells (D) syngeneic xenograft models showed that the proportions of GZMB+CD8+ T cells and IFN-γ+CD8+ T cells increased after treatment with Dusp9 siRNA and PD-1 antibody. Data are presented as mean ± SEM; n=6; p-values ​​were determined using a one-way ANOVA with post-hoc Sidak test; ns, not significant; p<0.05; p<0.01; p < 0.001; , p<0.0001.

[0027] Figure 8Stable knockdown of DUSP9 combined with anti-PD-1 antibody therapy can improve anti-tumor efficacy. A C57BL / 6 mouse syngeneic tumor model was inoculated with various cell lines and treated according to the following steps: (A) DUSP9-KD MC38 cells treated with anti-PD-1 antibody; (B) DUSP9-KD LLC cells treated with anti-PD-1 antibody; (C) wild-type MC38 cells treated with siDusp9 and anti-PD-1 antibody; (D) wild-type LLC cells treated with siDusp9 and anti-PD-1 antibody. Data are presented as mean ± SEM; n=6; p-values ​​were determined by one-way ANOVA and Sidak post-hoc test; ns, not significant; p<0.05; p<0.01; p < 0.001; , p<0.0001.

[0028] Figure 9 DUSP9 can be used as a clinical predictor of the efficacy of immune checkpoint inhibitor therapy. (AC) The mRNA expression of DUSP9 and PD-L1 in responders and non-responders in various tumors was investigated: (A) melanoma treated with anti-PD-L1 (GSE172320 dataset); (B) mesothelioma treated with anti-CTLA4 and anti-PD-L1 (GSE117358); (C) HCC treated with anti-PD-L1 antibody (GSE279750). Scatter plots show the correlation between the mRNA expression levels of DUSP9 and PD-L1 according to each dataset. (D) Immunohistochemical results showed that DUSP9 was expressed in both responders and non-responders of anti-PD-1 / PD-L1 therapy in clinical head and neck squamous cell carcinoma (HNSC). Data are presented as mean ± SEM; p-values ​​were determined by Student's t-test (A, B, D) or Mann-Whitney's test (C); ns, not significant; p<0.01; , p<0.0001.

[0029] Figure 10The negative regulation of STAT3 phosphorylation by DUSP9 depends on its phosphatase activity. (A) Schematic diagram of the mutation at the DUSP9 phosphatase active site. (B) HeLa cells were transfected with a negative control vector, wild-type DUSP9 plasmid, and mutant DUSP9-C290A plasmid. After 24 h, IFN-γ (50 ng / ml) was added for 24 h of treatment. The expression level of PD-L1 and the phosphorylation level of STAT3 were detected by Western blotting. The experiment was independently repeated three times. The left figure is a representative result of one experiment; the right figure is a statistical graph of the gray values ​​of the three Western blotting experiments. Data are expressed as mean ± SEM; the p-value was tested by one-way ANOVA. p < 0.01, p < 0.001, p < 0.0001, no significant difference in ns. Detailed Implementation

[0030] Various exemplary embodiments of the present invention will now be described in detail with reference to the accompanying drawings. It should be noted that, unless otherwise specifically stated, the relative arrangement, numerical expressions, and values ​​of the components and steps set forth in these embodiments do not limit the scope of the invention.

[0031] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the materials and reagents used are commercially available. Unless otherwise specified, the equipment used in the experiments is well known to those skilled in the art.

[0032] Example 1: Construction and Validation of Gene Knockout Tools

[0033] This invention designed and screened multiple nucleic acid sequences targeting human and mouse DUSP9 (see Table 1). Among them, the human DUSP9-specific siRNAs shown in SEQ ID NO: 1 and SEQ ID NO: 2 showed the best performance in terms of gene knockdown efficiency and specificity, as verified by experiments. However, those skilled in the art can also reduce DUSP9 expression or enzyme activity through small molecules, antibodies, CRISPR, antisense oligonucleotides, and other equivalent functional means, all of which fall within the scope of protection of this invention.

[0034] 1. siRNA transfection and validation: The small interfering RNA (siRNA) sequences designed in this study are shown in Table 1.

[0035] Table 1. Names, sequences, and uses of small interfering RNAs

[0036] Table 2. Plasmid construction information for shRNA lentiviral vectors

[0037] All siRNAs were transfected using Lipofectamine RNAiMAX reagent. The experimental procedures are as follows, with the dosages shown for a 6-well plate.

[0038] (1) Cells in good growth condition, such as A549 (human lung adenocarcinoma cell lines si-DUSP9-1#, si-DUSP9-2#), HeLa (human cervical cancer cell lines si-DUSP9-1#, si-DUSP9-2#), Huh7 (human liver cancer cell lines si-DUSP9-1#, si-DUSP9-2#), Hepa1-6 (mouse liver cancer cell lines sh-Dusp9-1#, sh-Dusp9-3#), MC38 (mouse colon cancer cell lines sh-Dusp9-1#, sh-Dusp9-3#), LLC (mouse lung cancer cell lines sh-Dusp9-1#, sh-Dusp9-3#), etc., were seeded into 6-well cell culture plates at a seeding density of 5-8 × 10⁻⁶ cells / well. 5 / hole.

[0039] (2) Dissolve siRNA in sterile, enzyme-free water to 20 μmol / L, aliquot and freeze at -80℃.

[0040] (3) On the second day, after the cells have grown to about 60%-70%, prepare the transfection working solution.

[0041] (4) Further dilute the siRNA to be used to 10 μmol / L. Dilute 2.5 μl of siRNA with 125 μl of Opti-MEM in a sterile enzyme-free Eppendorf tube.

[0042] (5) Dilute 3.75 μl of RNAiMAX reagent with 125 μl of Opti-MEM in another sterile, enzyme-free Eppendorf tube.

[0043] (6) Add the diluted siRNA dropwise to the diluted RNAiMAX reagent, mix gently, and let stand at room temperature for 5 minutes.

[0044] (7) Add 250 μl of the prepared transfection working solution dropwise to the cell culture plate, shake gently to mix, and place in a cell incubator for culture.

[0045] (8) Harvest cells in which the target gene is knocked down and detect the interference effect by real-time quantitative PCR and Western blot.

[0046] 2. Construction of tumor cell lines stably knocked down Dusp9 (shDusp9): The operation is as follows: (1) Tumor cells were seeded into 6-well cell culture plates at a cell density of approximately 6 × 10⁻⁶ cells / well. 5 / hole.

[0047] (2) After the cells adhered on the second day, the supernatant was discarded and replaced with 1 ml of fresh complete culture medium. 2 μl of Polybrene (final concentration 1:1000) was added. 1 ml of virus supernatant was added to the cell culture plate (virus supernatant: complete culture medium volume ratio 1:1) and placed in a virus-specific incubator for culture.

[0048] (3) After 24 hours, discard the culture medium, wash with PBS, digest and passage to 100 mm culture dish.

[0049] (4) GFP-positive cell populations were sorted by flow cytometry and seeded into culture dishes of appropriate size. Puromycin was added to screen positive cells (500 ng / ml for Hepa1-6, 1 μg / ml for LLC, and 3 μg / ml for MC38). After drug screening, a portion of the cells were collected to detect the knockdown of the target gene.

[0050] The results show that the nucleic acid molecule designed in this application can effectively knock down DUSP9 expression. The successful construction of this tool lays the foundation for subsequent functional studies.

[0051] Example 2: Validation of the in vitro and in vivo tumor-suppressing effects of DUSP9 knockdown

[0052] 1. Plate colony formation assay Dusp9 Effects of stable knockdown on tumor growth in vitro: The operation is as follows: Hepa1-6, MC38, and LLC tumor cell lines from the shNC control group and the shDusp9 group were cultured at a density of 1×10⁻⁶ cells per well. 3 Cells were seeded at a density of 1000 cells per well in six-well plates. After two weeks, cells were fixed with 4% paraformaldehyde and stained with crystal violet. Colony counts were quantified using ImageJ (NIH).

[0053] 2. Observation of the effect of stable Dusp9 knockdown on tumor growth in nude mice: For the immunodeficient mouse model, Hepa1-6 cells (3×10⁻⁶) from the shNC control group and the shDusp9 group were used. 6 1, MC38 cells (3×10) 6 (number) and LLC cells (2×10) 6 (Number of mice) were subcutaneously injected into BALB / c nude mice. Tumor volume in the mice was measured every three days.

[0054] To investigate the effect of DUSP9 on tumor growth, we evaluated the proliferative capacity of DUSP9 stably knocked-down cells in vitro using a plate colony formation assay. In vivo, the cells were seeded subcutaneously in BALB / c nude mice, and tumor volume and weight were observed and measured regularly to assess their tumorigenicity in vivo.

[0055] The results showed that DUSP9 knockdown significantly inhibited the colony-forming ability of tumor cells in vitro and significantly delayed tumor growth in nude mice in vivo, indicating that DUSP9 has a tumor-promoting effect.

[0056] Example 3: Study on the mechanism of DUSP9 in regulating tumor immunity and its combination therapy

[0057] 1. Subcutaneous tumorigenesis model of tumor cells in immune-intact mice and PD-1 antibody therapy: The operation is as follows: (1) After obtaining enough tumor cells in vitro, digest and count them, then resuspend them in PBS to 2-5×107 / ml and place them on ice for later use.

[0058] (2) Add matrix gel at a 1:1 ratio, mix well with a pipette, and draw up the cell suspension with a 1 ml syringe. Inject approximately 100 μl (2-5×10⁶) of cells subcutaneously into SPF grade C57BL / 6J mice using a syringe.

[0059] (3) Put the mice back into the cage and observe whether the mice develop tumors after about 4 days. Then observe the mice’s survival and tumor formation every day thereafter.

[0060] (4) Once the mouse tumors reached a specific size, they were divided into a control group and a treatment group according to tumor volume. After grouping, anti-PD-1 (product number BE0146, BioXcell) or isotype (product number BE0089, BioXcell) control antibody was injected intraperitoneally every 2-3 days. Mouse weight and tumor growth curves were recorded. Mice were sacrificed when the tumor grew to 1000 mm3, and tumor tissue was collected for subsequent experiments.

[0061] 2. Flow cytometry detection of PD-L1 expression and immune cell infiltration in tumor tissue: The operation is as follows: (1) Digesting tumor tissue: Prepare RPMI-1640 tumor tissue digestion solution containing 1.5 mg / ml collagenase D and 2% hiFBS. Take 3 ml of digestion solution / tumor tissue into a centrifuge tube, cut the tissue into pieces in the digestion solution, and place it on a shaker at 37℃ and 100 rpm for 30 minutes.

[0062] (2) Grinding tumor tissue: Prepare a 100 mm culture dish, add 10 ml PBS + 2% hiFBS, add the deionized tumor tissue to the culture dish, grind it through a filter, and filter the homogenized tissue into a 15 ml centrifuge tube. Centrifuge at 1600 rpm for 5 minutes at 4℃, discard the supernatant, and resuspend the precipitate in 3 ml of 1640 containing 10% hiFBS.

[0063] (3) Separation of tumor cells and infiltrating immune cells from tumor tissue: Add 3 ml of 80% Percoll and mix thoroughly. Then, slowly add 3 ml of 70% Percoll to the bottom of the tube to separate the solution into layers. Centrifuge at 800 g for 30 minutes at 4°C. The cells will separate into three layers: upper (tumor cells), middle (immune cells), and lower (red blood cells).

[0064] (4) Aspirate the upper layer of tumor cells into a 15 ml centrifuge tube containing 10 ml PBS + 2% hiFBS; aspirate the middle layer of immune cells into a 15 ml centrifuge tube containing 10 ml PBS + 2% hiFBS. Centrifuge at 1600 rpm for 10 minutes at 4℃.

[0065] (5) Tumor cell staining: After centrifugation, discard the supernatant of tumor cells, resuspend them with antibody APC-anti-mPD-L1 (100 μl / sample), stain at 4°C in the dark for 30 minutes, wash and resuspend with PBS, add 2.5 μl / sample of 7-AAD, and perform instrument detection.

[0066] (6) Immunocyte staining: After centrifugation, discard the supernatant of the immunocellular cells, resuspend them with membrane molecular antibodies (see example) (100 μl / sample), stain at 4°C in the dark for 30 minutes, wash and resuspend with PBS, add 100 μl of fixative, mix well, fix at room temperature in the dark for 30 minutes, centrifuge at 2500 rpm and discard the supernatant. Dilute the perforation buffer 1:9 with ultrapure water and resuspend the cells at 300 μl / sample, centrifuge at 2500 rpm, perforate and discard the supernatant. Use the diluted perforation buffer to prepare cytokine antibodies and single positive tubes, resuspend the sample and corresponding single positive tube at 100 μl / sample, stain at room temperature in the dark for 40 minutes. Wash and resuspend with PBS, filter into flow cytometry tubes, and perform flow cytometry analysis.

[0067] 3. Preparation of frozen sections of tumor tissue and detection of immune cell infiltration by immunofluorescence staining: The operation is as follows: (1) Cut the freshly removed tumor tissue into appropriate sizes and place it in 4% paraformaldehyde and fix it at 4°C for 24-48 hours.

[0068] (2) Remove the tissue, wash it with PBS, place it in 20% sucrose at 4°C for 24 hours to dehydrate, and then place it in 30% sucrose at 4°C for another 24 hours to dehydrate.

[0069] (3) Place the tissue block into a mold made of tin foil, add an appropriate amount of OCT embedding agent to cover the tissue block, and place it in a -80℃ refrigerator to solidify and store.

[0070] (4) After trimming the section to the tissue position with a 40 μm microtome, cut the section to a thickness of 10 μm, attach it to an adhesive glass slide, bake the section at 60℃ for 1 hour, and then store it at -20℃.

[0071] (5) Immunofluorescence staining: First, remove the embedding agent from the tissue sections, i.e., soak them in PBS for 15 minutes. Then, cover the tissue with 10% normal goat serum and block at room temperature for 1 hour. Prepare the working solution by diluting the primary antibody with goat serum at a ratio of 1:200-1:500 according to the instructions, incubate overnight in a humidified chamber at 4°C, wash three times with PBS for 5 minutes each time, add the secondary fluorescent antibody (1:100 PBS) to cover the tissue, and incubate at room temperature in the dark for 1 hour. After washing with PBS, stain the nuclei by adding DAPI dye (1:100 PBS) and incubate at room temperature in the dark for 15 minutes. After washing with PBS, add anti-fluorescence attenuation mounting medium. Store at -20°C in the dark and observe under a fluorescence microscope.

[0072] The results showed that in immunocompetent mice, DUSP9 knockdown inhibited the infiltration and function of cytotoxic CD8+ T cells by upregulating PD-L1 expression in tumor cells, while anti-PD-1 treatment could reverse this inhibitory effect and exert a synergistic anti-tumor effect.

[0073] Example 4: In vivo combination therapy strategy based on siRNA

[0074] The procedure for administering siRNA to mice for tumor treatment is as follows: (1) Add 20 μL of ENT siRNA trans 001 (10T specification) to the animal experiment-specific liposome transfection reagent. Dissolve in L of anhydrous ethanol by sonication at room temperature for 5 minutes.

[0075] (2) Dissolve siRNA in sterile, enzyme-free water to 13 μg / μL.

[0076] (3) Preparation of transfection system: Add the dissolved siRNA (2 μL per mouse) to the bottom of a sterile, enzyme-free Eppendorf tube, add the transfection optimization solution GenOpti (48 μL per mouse), and mix gently. Add the dissolved transfection reagent (2 μL per mouse) close to the surface of the liquid, and add the transfection optimization solution GenOpti (48 μL per mouse) again. Do not vortex, centrifuge, or repeatedly pipette.

[0077] (4) The prepared transfection system was sonicated at 4KHz for 10 minutes at 70℃.

[0078] (5) Inject the transfection system after ultrasonic mixing into the tumor of mice, with an injection volume of 100 μL per mouse.

[0079] The results showed that intratumoral injection of siDusp9 could mimic the effect of genetic knockout, and its combination with anti-PD-1 antibody showed a significant synergistic anti-tumor effect, demonstrating the clinical translational potential of this strategy.

[0080] Example 5: Study on the interaction mechanism between DUSP9 and STAT3

[0081] 1. Detection of the interaction between DUSP9 and STAT3 using co-immunoprecipitation (CO-IP) is performed as follows: (1) After stimulating the cultured Huh7 cells with 50 ng / ml IFN-γ for 4 h, the culture medium was discarded, the cells were washed with PBS, and 1 ml of IP-specific lysis buffer + protease inhibitor + phosphatase inhibitor was added to each 100 mm culture dish. The cells were then lysed on ice for 30 minutes.

[0082] (2) After cell lysis, the supernatant was collected, centrifuged at 12000 g for 10 minutes at 4°C, and the supernatant was transferred to a new Eppendorf tube. Protein quantification was performed using a BCA quantitative assay kit. IP antibody was added at a ratio of 1 μg antibody / 500 μg protein, and the tube was incubated on a turntable at 4°C for 2 h.

[0083] (3) After adding protein G (60 μl / sample), incubate overnight at 4°C on a turntable.

[0084] (4) Wash 5 times with pre-cooled PBS, add 20 μl of lysis buffer, then add 10 μl of 6× protein electrophoresis loading buffer, denature at 99℃ for 10 minutes, and perform Western blot electrophoresis together with the retained input sample, following the standard procedure.

[0085] 2. The procedure for the in vitro dephosphorylation of STAT3 by DUSP9 is as follows: (1) HA-STAT3 was overexpressed in cultured HeLa cells by transfection with the plasmid using Polyplus. 24 h after transfection, the cells were stimulated with 50 ng / ml IFN-γ for 4 h, then the culture medium was discarded and the cells were washed with PBS. 1 ml of IP-specific lysis buffer + protease inhibitor + phosphatase inhibitor was added to each 100 mm culture dish, and the cells were lysed on ice for 30 minutes. The cells were then centrifuged at 12000 g for 10 minutes at 4°C, and the supernatant was collected. Protein quantification was performed using a BCA quantitative assay kit.

[0086] (2) Add HA-tagged IP antibody at a ratio of 1 μg antibody / 500 μg protein, incubate at 4°C for 2 hours, then add protein G and incubate at 4°C overnight.

[0087] (3) Take out the sample, wash with PBS, resuspend in 300 μl of in vitro dephosphorylation buffer, and divide into 3 Eppendorf tubes, 100 μl in each tube. No treatment was given to the NC group, 2.5 μg of recombinant DUSP9 protein was added to the DUSP9 group, and 30 U of nonspecific calf intestinal alkaline phosphatase (CIAP) was added to the positive control group. Incubate at 37°C for 1 hour.

[0088] (4) After washing with PBS, discard the supernatant to make the remaining volume about 30 μl. Add 6 μl of 6× protein electrophoresis loading buffer, denature at 99℃ for 10 minutes, and load all samples for Western blot detection.

[0089] To clarify whether DUSP9's dephosphorylation of STAT3 depends on its phosphatase activity, we constructed a DUSP9 phosphatase site mutant plasmid pcDNA3.1-DUSP9-C290A-Myc-His, in which the DUSP9 phosphatase active site (cysteine ​​at position 290) was mutated to alanine. Wild-type and mutant DUSP9 plasmids were transfected into HeLa cells, and IFN-γ was added to induce PD-L1 expression. Simultaneously, STAT3 phosphorylation levels were measured. The results showed that overexpression of wild-type DUSP9 significantly inhibited STAT3 phosphorylation and IFN-γ-induced upregulation of PD-L1 expression, while mutant DUSP9 did not exhibit this function. Figure 10 ).

[0090] The results showed that DUSP9 can bind directly to STAT3 and catalyze the dephosphorylation of its key phosphorylation sites (Y705, S727) through its phosphatase activity, thereby inhibiting its transcriptional activity and ultimately negatively regulating the expression of PD-L1.

[0091] Although the present invention has been disclosed above with reference to embodiments, it is not intended to limit the present invention. Any person skilled in the art can make various different choices and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention is defined by the claims and their equivalents.

Claims

1. Use of an inhibitor targeting DUSP9 in combination with an immune checkpoint inhibitor for the manufacture of a medicament for the treatment of a tumor, characterized in that, The immune checkpoint inhibitor is a PD-L1 antibody or a PD-1 antibody, and the inhibitor can reduce the expression or enzyme activity of the DUSP9 gene or protein.

2. Use according to claim 1, characterized in that, The tumor is a melanoma, mesothelioma, hepatocellular carcinoma, head and neck squamous cell carcinoma, colon cancer, lung cancer or cervical cancer, the inhibitor enhances the anti-tumor effect of the immune checkpoint inhibitor by up-regulating the expression of PD-L1 on the surface of tumor cells; the inhibitor is a small molecule compound, a protein and a polypeptide, siRNA or shRNA, and the siRNA or shRNA sequence is shown in any one of SEQ ID No. 1-2.

3. A pharmaceutical composition or combination for use in the treatment of a tumor, characterized in that, The pharmaceutical composition or the pharmaceutical active ingredient of the combination drug comprises: The first active ingredient is the DUSP9-targeted inhibitor according to any one of claims 1-2; The second active ingredient is an immune checkpoint inhibitor, which is a PD-L1 antibody or a PD-1 antibody.

4. The pharmaceutical composition or combination according to claim 3, characterized in that, The DUSP9-targeted inhibitor is a small molecule compound, a protein and a polypeptide, siRNA or shRNA, and the siRNA or shRNA sequence is shown in SEQ ID No. 1-2; the pharmaceutical composition or the combination drug further comprises a pharmaceutically acceptable carrier.

5. Use of a DUSP9-targeted inhibitor in the preparation of a product for enhancing the effect of an immune checkpoint inhibitor, wherein the inhibitor is a small molecule compound, a protein, a polypeptide, siRNA or shRNA, and the inhibitor can increase the phosphorylation level of STAT3 and up-regulate the expression of PD-L1 by inhibiting the expression or activity of DUSP9.

6. Use according to claim 5, characterized in that, The siRNA or shRNA has a sequence shown in SEQ ID No. 1-2, and the immune checkpoint inhibitor is a PD-L1 antibody.

7. Use according to claim 5, characterized in that, The product is used for inhibiting the expression of the DUSP9 gene or protein, increasing the phosphorylation level of STAT3 at Y705 and S727 sites, up-regulating the expression of PD-L1 on the surface of tumor cells, and enhancing the anti-tumor effect of the immune checkpoint inhibitor.

8. Use of a reagent for detecting the DUSP9 gene or protein in the preparation of a product for predicting the responsiveness of a tumor patient to anti-PD-1 antibody or anti-PD-L1 antibody treatment, evaluating the effect of combined immunotherapy, judging the prognosis and monitoring minimal residual disease (MRD).

9. Use according to claim 8, characterized in that, The product comprises a gene detection reagent based on qPCR, digital PCR or molecular POCT, or a protein detection reagent based on immunohistochemistry, ELISA or flow cytometry.

10. Use of the DUSP9 gene or protein as a biomarker in the preparation of a kit for predicting the anti-tumor effect of an immune checkpoint inhibitor, evaluating the prognosis of combined immunotherapy and monitoring minimal residual disease (MRD).

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