Application of USP15 inhibitor combined with PD-1 monoclonal antibody in preparation of medicine for treating colorectal cancer

Abstract

The invention discloses application of a USP15 inhibitor combined with a PD-1 monoclonal antibody in preparation of a medicine for treating colorectal cancer, relates to the technical field of tumor treatment, and aims to solve the problems that an existing immune checkpoint inhibitor is poor in MSS type intestinal cancer treatment effect and has drug resistance. The invention provides application of a USP15 inhibitor combined with a PD-1 monoclonal antibody in preparation of a medicine for treating colorectal cancer. The invention also provides a pharmaceutical composition for resisting colorectal cancer. The pharmaceutical composition comprises the USP15 inhibitor and the PD-1 monoclonal antibody. The USP15 inhibitor is combined for use, so that the immunotherapy effect of the PD-1 monoclonal antibody is remarkably improved, and the growth of subcutaneous transplanted tumors of mice is inhibited. MDSC recruitment is obviously inhibited, infiltration of CD8 + T cells is promoted, and the killing ability of the CD8 + T cells is enhanced. The invention is used for treating MSS type colorectal cancer.

Description

Technical Field

[0001] The present invention relates to the technical field of tumor treatment, and specifically to the use of a USP15 inhibitor combined with a PD-1 monoclonal antibody in the preparation of a drug for treating colorectal cancer. Background Art

[0002] According to statistics, the incidence and mortality of colorectal cancer (CRC) are among the highest among the major malignant tumors in the world. At present, immune checkpoint inhibitors (ICIs) have become the main immunotherapy drugs for colorectal cancer. Their mechanism of action is to restore the immune system's ability to kill tumor cells by blocking immune checkpoint molecules, thereby playing a role in tumor treatment. However, its treatment effect is not ideal for pMMR / MSS type colorectal cancer, and there is a problem of drug resistance. Therefore, it is urgent to develop more new ICIs combination strategies by exploring its specific mechanism.

[0003] Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of bone marrow cells that are involved in regulating immune responses and promoting tumor immune escape. MDSC inhibit the anti-tumor response of T cells and NK cells through multiple mechanisms, including the expression and production of immunosuppressive molecules, consumption of essential amino acids, activation of other immunosuppressive cells, and inhibition of T cell trafficking. A deeper understanding of the generation, recruitment, and functional mechanisms of MDSC will help discover immunotherapy targets based on MDSC subpopulations and provide support for biomarkers of cancer treatment response, thereby overcoming resistance to immune checkpoint inhibitors (ICIs) and other immunotherapies. CD8+T cells play a vital role in tumor immunotherapy. However, due to factors such as dysregulation of T cells during transport to tumor tissues and weakened killing ability, tumor T cell infiltration is insufficient and immunotherapy is not effective. Therefore, it is urgent to explore new treatment options to improve the efficacy of immunotherapy in colorectal cancer. Summary of the invention

[0004] The present invention aims to solve the problem that existing immune checkpoint inhibitors are not effective in treating MSS-type colorectal cancer and have drug resistance, and provides the use of USP15 inhibitors combined with PD-1 monoclonal antibodies in the preparation of drugs for treating colorectal cancer.

[0005] The present invention provides use of a USP15 inhibitor in combination with a PD-1 monoclonal antibody in the preparation of a drug for treating colorectal cancer.

[0006] Furthermore, the USP15 inhibitor is USP15-IN-1.

[0007] Furthermore, the colorectal cancer is MSS type colorectal cancer.

[0008] Furthermore, the PD-1 monoclonal antibody is CD279.

[0009] The present invention provides use of a USP15 inhibitor in preparing a drug for treating colorectal cancer.

[0010] Furthermore, the USP15 inhibitor is USP15-IN-1.

[0011] Furthermore, the colorectal cancer is MSS type colorectal cancer.

[0012] The present invention also provides a pharmaceutical composition for treating colorectal cancer, comprising a USP15 inhibitor and a PD-1 monoclonal antibody.

[0013] Beneficial effects of the present invention:

[0014] The present invention provides an application of the USP15 gene target in anti-tumor therapy, and proposes a dual combination regimen of a USP15 inhibitor and a PD-1 monoclonal antibody. USP15, as a new target for tumor therapeutic drugs, stabilizes the target protein by deubiquitination, affects downstream signaling pathways, inhibits the recruitment of myeloid-derived suppressor cells (MDSCs), reverses the tumor immunosuppressive microenvironment, inhibits the exhaustion of effector T cells, restores the function of effector T cells, enhances anti-tumor immunity, and enhances the efficacy of immunotherapy.

[0015] In preclinical animal models, intraperitoneal injection of USP15 inhibitors alone can inhibit colorectal cancer tumor growth compared with the control group. At the same time, the combination of USP15 inhibitors for sensitization immunotherapy also has a significant therapeutic effect. The combined use of USP15 inhibitors significantly improved the effect of PD-1 monoclonal antibody immunotherapy and inhibited the growth of subcutaneous transplanted tumors in mice. In addition, this regimen significantly inhibited MDSC recruitment, promoted the infiltration of CD8+T cells, and enhanced the killing ability of CD8+T cells.

[0016] Experiments have shown that subcutaneous transplanted tumors are ineffective when treated with PD-1 alone, indicating that drug resistance exists. Compared with the anti-PD-1 alone group, the tumors were significantly reduced after anti-PD-1 combined with USP15 inhibitors, indicating that the drug resistance problem has been solved.

[0017] The present invention is the first to prove that USP15 is a potential molecular marker and important therapeutic target for immunotherapy of MSS colorectal cancer, providing a new marker for immunotherapy of MSS colorectal cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a flowchart of the grouping and drug administration of MSS-type intestinal cancer model mice;

[0019] Figure 2 Comparison of the volume of syngeneic mouse subcutaneous transplanted tumors of CT26 cells in different treatment groups;

[0020] Figure 3 Comparison of the weight of syngeneic mouse subcutaneous transplanted tumors of CT26 cells in different treatment groups;

[0021] Figure 4 The changes of subcutaneous tumor volume of CT26 cells in different treatment groups with the administration time;

[0022] Figure 5 Comparison of tumor volumes of syngeneic mouse intestinal cancer orthotopic tumors of CT26 cells in different treatment groups;

[0023] Figure 6 is the tumor weight of syngeneic mouse intestinal cancer orthotopic tumors of CT26 cells in different treatment groups;

[0024] Figure 7 The fluorescence signal intensity of the syngeneic mouse intestinal cancer orthotopic tumors of CT26 cells in different treatment groups;

[0025] Figure 8 The proportion of MDSCs in different treatment groups was determined by flow cytometry;

[0026] Fig. 9 The proportion of cytotoxic T cells (CTL) in different treatment groups was determined by flow cytometry. DETAILED DESCRIPTION

[0027] To make the purpose, technical scheme and beneficial effects of the present invention clearer, the technical scheme of the present invention is described in detail below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Detailed implementation schemes and specific operation processes are given below, but the protection scope of the present invention is not limited to the following embodiments.

[0028] Example 1: In vivo targeting of USP15 combined with anti-PD-1 to treat CT26 cell-derived syngeneic mouse colorectal cancer subcutaneous xenografts.

[0029] The USP15 inhibitor used in this example is USP15-IN-1 (MCE, HY-148046), which is a commonly used ubiquitin-specific protease inhibitor in the field of colorectal cancer research. Other USP15 inhibitors can also be selected according to experimental or clinical needs.

[0030] The PD-1 monoclonal antibody (immune checkpoint inhibitor) used in this example is InVivoMAb anti-mouse PD-1 (CD279), which is a commonly used PD-1 monoclonal antibody in the field of colorectal cancer research. Other PD-1 monoclonal antibodies can also be selected according to experimental or clinical needs.

[0031] 1. Construction of mouse MSS-type colorectal cancer model.

[0032] (1) Female BALB / c mice aged 5-6 weeks were used to construct tumor-bearing models;

[0033] (2) After digestion and counting, CT26 cells (mouse MSS colon cancer cells) were counted at 1×10 6 pcs / 100 μL were inoculated subcutaneously into mice;

[0034] (3) The size of the transplanted tumor grows to 100 mm 3 Left and right, group treatment was performed;

[0035] 2. The constructed MSS-type colorectal cancer model mice were divided into 4 groups, with 7 mice in each group.

[0036] The reagent configuration scheme is as follows: CD279 is prepared using physiological saline; USP15-IN-1 is prepared using DMSO to form a 10 mg / ml storage solution, which is prepared immediately upon use, in a ratio of 10% DMSO + 90% (20% SBE-β-CD in saline).

[0037] The specific groups are as follows (the grouping and drug administration process of MSS type intestinal cancer model mice is as follows Figure 1 shown):

[0038] ①PBS group: intraperitoneal injection of 1M sterile PBS, 100 μL / mouse, once a day;

[0039] ② USP15 inhibitor group: intraperitoneal injection of USP15-IN-1, 100 μg / mouse, three times a week, for a total of five times;

[0040] ③Anti-PD-1 group: intraperitoneal injection of CD279, 200 μg / mouse, three times a week, for a total of five times;

[0041] ④USP15 inhibitor combined with anti-PD-1 group: intraperitoneal injection of USP15-IN-1, 100 μg / mouse, three times a week, for a total of five times; intraperitoneal injection of CD279, 200 μg / mouse, three times a week, for a total of five times; the two drugs were treated separately at the same time.

[0042] 3. Detection of tumor volume:

[0043] Starting from the 10th day after inoculation of CT26 cells, the length and width of the subcutaneous transplanted tumors of mice were measured with a vernier caliper every 2-3 days, and the tumor volume was calculated. Figure 2-Figure 4 As shown, Figure 3 The black circle in the middle represents the PBS group, the red circle represents the anti-PD-1 group, the blue circle represents the USP15 inhibitor group, and the green circle represents the USP15 inhibitor combined with anti-PD-1 group.

[0044] Results: Figure 2-Figure 4 As shown, the USP15 inhibitor alone inhibited the growth of mouse tumor volume, while the anti-PD-1 alone group showed no significant change in tumor volume. In addition, the dual-drug combination treatment group significantly inhibited the growth of mouse tumor volume compared with the single-drug treatment group, and did not cause obvious side effects. The above results suggest that targeting USP15 effectively enhances the therapeutic efficacy of anti-PD-1.

[0045] Figure 2 There is no difference in tumor size between the first and second rows, indicating that the use of single-drug PD-1 is ineffective for subcutaneous transplanted tumors, that is, drug resistance exists. Comparing the USP15 inhibitor combined with anti-PD-1 group (fourth row) with the single-drug anti-PD-1 group (second row), the tumors were significantly reduced after anti-PD-1 combined with USP15, indicating that the problem of drug resistance has been solved.

[0046] Example 2: In vivo targeting of USP15 combined with anti-PD-1 to treat orthotopic colorectal cancer in mice.

[0047] 1. Construction of an in situ model of MSS-type colorectal cancer in mice.

[0048] (1) Female BALB / c mice aged 5-6 weeks were used to construct tumor-bearing models;

[0049] (2) After digestion and counting, CT26 cells (mouse MSS colon cancer cells) carrying the fluorescent marker gene (Luc) were counted at 1×10 6 pcs / 100 μL were inoculated into the cecum of mice;

[0050] (3) The IVIS small animal in vivo imaging system was used to detect the tumor formation of the in situ model of colorectal cancer. After the in situ model of colorectal cancer was successfully constructed (about 14 days), group treatment was performed.

[0051] 2. The constructed MSS-type in situ colorectal cancer model mice were divided into 4 groups, with 3 mice in each group.

[0052] The reagent configuration scheme is as follows: CD279 is prepared using physiological saline; USP15-IN-1 is prepared using DMSO to form a 10 mg / mL storage solution, which is prepared immediately upon use, according to the ratio of 10% DMSO + 90% (20% SBE-β-CD in saline).

[0053] The specific groups are as follows (the grouping and drug administration process of MSS type intestinal cancer model mice is as follows Figure 1 shown):

[0054] ①PBS group: intraperitoneal injection of 1M sterile PBS, 100 μL / mouse, once a day;

[0055] ② USP15 inhibitor group: intraperitoneal injection of USP15-IN-1, 100 μg / mouse, three times a week, for a total of six times;

[0056] ③ Anti-PD-1 group: intraperitoneal injection of CD279, 200 μg / mouse, three times a week, for a total of six times;

[0057] ④USP15 inhibitor combined with anti-PD-1 group: intraperitoneal injection of USP15-IN-1, 100 μg / mouse, three times a week, for a total of six times; intraperitoneal injection of CD279, 200 μg / mouse, three times a week, for a total of six times; the two drugs were treated separately at the same time.

[0058] 3. Detection of tumor growth:

[0059] After the in situ model of intestinal cancer was successfully constructed, the fluorescence imaging system was used to detect tumor growth. After the four groups completed six doses of medication, the in situ tumor growth was detected again. The statistical results are as follows: Figure 5-Figure 7 As shown, Figure 6 and Figure 7 The black circle in the middle represents the PBS group, the red circle represents the anti-PD-1 group, the blue circle represents the USP15 inhibitor group, and the green circle represents the USP15 inhibitor combined with anti-PD-1 group.

[0060] Results: Figure 5-Figure 7 As shown, the USP15 inhibitor alone inhibited the growth of mouse cecal in situ tumors, while the anti-PD-1 alone group had no significant change in tumor volume. In addition, the dual-drug combination treatment group significantly inhibited the growth of mouse tumor volume compared with the single-drug treatment group. The above results suggest that targeting USP15 effectively enhances the therapeutic effect of anti-PD-1.

[0061] Example 3: Flow cytometry was used to determine the proportions of MDSC (CD45+, CD11b+, Gr-1+) and CTL (CD3+, CD8+, IFN-γ+) in subcutaneous transplanted tumor tissues of each group.

[0062] (1) Sampling: Tumor tissues were collected from BALB / c mice with subcutaneous transplanted tumors in the PBS group and the USP15 inhibitor combined with anti-PD-1 group in Example 1 on day 21, and the cells were dissociated and analyzed by flow cytometry.

[0063] (2) Dissociation of cells: The tumor tissues of both groups were minced with surgical scissors, digested with collagenase for 1.5 h, and centrifuged at 1500 rpm for 5 min.

[0064] (3) Washing: Wash the cells with pre-cooled PBS, centrifuge at 1500 rpm for 5 min, and wash twice.

[0065] (4) Surface staining: Resuspend the cells and perform surface staining (CD45, CD3, CD8, CD11b, Gr-1), incubate at 4°C in the dark for 30 min (all flow cytometry antibodies were purchased from BioLegend, USA).

[0066] (5) Cell fixation: Wash the cells twice with PBS, add 4% PFA fixative, and fix at 4°C in the dark for 30 min.

[0067] (6) Cell membrane permeabilization: Wash the cells twice with PBS, add permeabilization solution (True NuclearTM 10X Perm, Part: 73162, purchased from biolegend, USA), and permeabilize the membrane at 4°C in the dark for 30 min.

[0068] (7) Intracellular staining: Wash the cells with permeabilization buffer, centrifuge at 1500 rpm for 5 min, and wash twice. Perform intracellular staining (IFN-γ) and incubate at 4°C in the dark for 30 min (flow cytometry antibodies were purchased from BioLegend, USA).

[0069] (8) Wash the cells twice with permeabilization buffer, resuspend the cells in PBS, and load onto the analyzer.

[0070] (9) Cell flow cytometry analysis: Flowjo (V10).

[0071] Results: Flow cytometry was used to determine the proportion of MDSC and CTL in the double-drug combination treatment group and the control group. Figure 8 and Fig. 9 The results showed that the proportion of MDSC in the tumor tissue of the double-drug combination treatment group was significantly lower than that of the PBS group, and the proportion of CTL was significantly higher than that of other groups, indicating that the double-drug combination treatment group inhibited MDSC recruitment, promoted CTL infiltration in tumor tissue, and enhanced anti-tumor activity.

Claims

1. Application of USP15 inhibitors combined with PD-1 monoclonal antibodies in the preparation of drugs for the treatment of colorectal cancer.

2. The use according to claim 1, characterized in that: The USP15 inhibitor is USP15-IN-1.

3. The use according to claim 1, characterized in that: The colorectal cancer is MSS type colorectal cancer.

4. The use according to claim 1, characterized in that: The PD-1 monoclonal antibody is CD279.

5. Application of USP15 inhibitors in the preparation of drugs for the treatment of colorectal cancer.

6. The use according to claim 5, characterized in that: The USP15 inhibitor is USP15-IN-1.

7. The use according to claim 5, characterized in that: The colorectal cancer is MSS type colorectal cancer.

8. A pharmaceutical composition for treating colorectal cancer, characterized in that: The pharmaceutical composition comprises a USP15 inhibitor and a PD-1 monoclonal antibody.

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