Use of ctgf mab in combination with csf1r inhibitor and pd-1 mab for treating colorectal cancer and pharmaceutical composition

Abstract

The application of CTGF monoclonal antibody combined with CSF1R inhibitor and PD-1 monoclonal antibody in the preparation of a drug for treating colorectal cancer and a pharmaceutical composition, relates to the technical field of tumor treatment, and aims to solve the problem that the effect of the existing CSF1R inhibitor combined with immunotherapy is poor.The application provides the application of CTGF monoclonal antibody combined with CSF1R inhibitor and PD-1 monoclonal antibody in the preparation of a drug for treating colorectal cancer.The application also provides an anti-colorectal cancer pharmaceutical composition, which comprises CTGF monoclonal antibody, CSF1R inhibitor and PD-1 monoclonal antibody.The combination of CTGF monoclonal antibody and CSF1R inhibitor significantly improves the effect of PD-1 monoclonal antibody immunotherapy, and the combination of the three drugs significantly promotes the infiltration of CD8+ T cells and CTL in tumor tissues and enhances the anti-tumor activity.The application 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 CTGF monoclonal antibody in combination with CSF1R inhibitor and PD-1 monoclonal antibody in the preparation of drugs for treating colorectal cancer and a pharmaceutical composition. Background Art

[0002] Colorectal cancer (CRC) is the third most common malignancy in the world. Approximately 30% of CRC patients have distant metastasis at the time of diagnosis, and the 5-year survival rate is only 14%. Anti-tumor immunotherapy, especially immune checkpoint blockade (ICB) therapy, has become one of the most promising cancer treatment strategies. Immune checkpoint inhibitors (ICIs) have been approved for advanced CRC with high microsatellite instability or mismatch repair deficiency. However, due to the heterogeneity of tumor cells and the complexity of the tumor microenvironment, tumor immunotherapy still faces many obstacles. 85%-95% of microsatellite stable CRC patients in colorectal cancer are resistant to ICB, which highlights the urgent need to further study the relationship between tumor cell intrinsic events and host immune responses in CRC.

[0003] Tumor-associated macrophages (TAMs) are the most abundant immune cells in the tumor microenvironment and play a crucial role in the immunosuppressive microenvironment of colorectal cancer. Their high concentration in the tumor microenvironment, resulting in an immunosuppressive microenvironment, may be one of the causes of resistance to immunotherapy. Therefore, strategies targeting TAMs in combination with immune checkpoint inhibitors may enhance the efficacy of immunotherapy in colorectal cancer. The CSF1 receptor (CSF1R) is expressed on circulating monocytes and tissue macrophages. The CSF1 / CSF1R signaling axis is overexpressed in various tumor tissues, and its overexpression is associated with poor prognosis. Monoclonal antibodies or small molecule inhibitors of CSF1R can block CSF1R signaling, targeting TAMs and controlling the survival, proliferation, differentiation, and chemotaxis of these cells, successfully depleting TAMs. However, in preclinical animal models of colorectal cancer and early clinical studies, the efficacy of CSF1R inhibitors combined with immunotherapy has been poor. Therefore, there is an urgent need to explore more precise macrophage-targeted therapies to improve the efficacy of immunotherapy in colorectal cancer. Summary of the Invention

[0004] The present invention aims to solve the problem of poor effect of existing CSF1R inhibitor combined with immunotherapy, and provides the use of CTGF monoclonal antibody combined with CSF1R inhibitor and PD-1 monoclonal antibody in the preparation of drugs for treating colorectal cancer and a pharmaceutical composition.

[0005] To achieve the above objectives, the present invention provides the following technical solutions:

[0006] The present invention provides use of a CTGF monoclonal antibody in combination with a CSF1R inhibitor and a PD-1 monoclonal antibody in the preparation of a drug for treating colorectal cancer.

[0007] Furthermore, the CTGF monoclonal antibody is SHR1906 or Pamrevlumab, both of which are anti-fibrosis and anti-tumor monoclonal antibodies.

[0008] Furthermore, the CSF1R inhibitors are Pexidartinib PLX-3397, Emactuzumab RG7155, and Cabiralizumab FPA008, all of which are drugs targeting tumor-associated macrophages.

[0009] The present invention provides use of CTGF monoclonal antibody in preparing a drug for treating colorectal cancer.

[0010] Furthermore, the CTGF monoclonal antibody is SHR1906 or Pamrevlumab.

[0011] The present invention also provides a pharmaceutical composition for treating colorectal cancer, which comprises a CTGF monoclonal antibody, a CSF1R inhibitor and a PD-1 monoclonal antibody.

[0012] Beneficial effects of the present invention:

[0013] The present invention provides applications of the CTGF gene target in anti-tumor therapy and proposes a triple-combination regimen targeting CTGF in combination with a CSF1R inhibitor for enhanced immunotherapy. The CTGF gene plays an important role in extracellular matrix remodeling, cell adhesion, migration, and proliferation. As a target for new tumor immunotherapy drugs, it blocks the binding of the target protein to various cytokines and growth factors, affecting downstream signaling pathways. Combining this with a CSF1R inhibitor, a drug targeting tumor-associated macrophages, can effectively reverse and inhibit the exhaustion of effector T cells, restore effector T cell function, and enhance the efficacy of immunotherapy. Compared with a control group, intraperitoneal injection of a CTGF monoclonal antibody alone inhibited colorectal cancer tumor growth in mice.

[0014] This triple-drug regimen, combining CTGF-targeted immunotherapy with a CSF1R inhibitor for enhanced immunotherapy, demonstrated significant therapeutic efficacy in animal models. Combining a CTGF monoclonal antibody with a CSF1R inhibitor significantly enhanced the efficacy of PD-1 monoclonal antibody immunotherapy and prolonged survival in mice. The combination of anti-CTGF, a CSF1R inhibitor, and an anti-PD-1 agent significantly promoted the infiltration of CD8+ T cells and CTLs into tumor tissues, enhancing anti-tumor activity.

[0015] The present invention is the first to demonstrate that CTGF may be a potential molecular marker and important therapeutic target for the failure of immunotherapy for MSS colorectal cancer, providing a new marker for immunotherapy of MSS colorectal cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 To compare the volume of subcutaneous transplanted tumors in mice of different treatment groups;

[0017] Figure 2 The immunofluorescence results of mouse tumors showed that the combination of CTGF monoclonal antibody, CSF1R inhibitor and PD-1 antibody promoted CD8+ T cell infiltration in tumor tissues;

[0018] Figure 3 The CTL ratios in different treatment groups were determined by flow cytometry;

[0019] Figure 4 The proportion of macrophages in different treatment groups was determined by flow cytometry. DETAILED DESCRIPTION

[0020] To make the purpose, technical solution and beneficial effects of the present invention more clear, the technical solution of the present invention is described in detail below. Obviously, the embodiments described are only some embodiments of the present invention, rather than all embodiments. Detailed implementation schemes and specific operating procedures are given below, but the scope of protection of the present invention is not limited to the following embodiments.

[0021] CD8+ T cells play a key role in tumor immunotherapy. Disturbances in T cell trafficking to tumor tissues and reduced cytotoxicity are among the factors contributing to insufficient T cell infiltration in tumors. Therefore, promoting the recruitment of effector T cells to the tumor center may offer new possibilities for improving the efficacy of ICB therapy.

[0022] Connective tissue growth factor (CTGF) is a multifunctional protein in the CCN family that can regulate various biological processes such as cell proliferation, differentiation, and adhesion.

[0023] Combining a CTGF monoclonal antibody and a CSF1R inhibitor with a PD-1 monoclonal antibody can significantly enhance the efficacy of PD-1 monoclonal immunotherapy. The strategy of combining a CTGF monoclonal antibody with a CSF1R inhibitor to enhance immunotherapy does not involve the pharmacological toxicity and other experimental issues associated with newly developed drugs.

[0024] In addition, the use of CTGF monoclonal antibodies alone can also inhibit the growth of colorectal cancer cells.

[0025] Preferably, the CTGF monoclonal antibody in this embodiment is SHR1906. SHR1906 is a domestically produced monoclonal antibody targeting connective tissue growth factor (CTGF), which is in Phase 2 clinical research in China and is currently used to treat idiopathic pulmonary fibrosis, malignant tumors and other indications.

[0026] Preferably, the CSF1R inhibitor is PLX3397, which is a drug targeting tumor-associated macrophages.

[0027] Example 1: In vivo targeting of CTGF and CSF1R combined with anti-PD-1 to treat colorectal cancer in mice

[0028] 1. Construction of the MSS-type colorectal cancer model in mice:

[0029] (1) 5-6 week-old female BALB / c mice were used to construct tumor-bearing models;

[0030] (2) After digestion and counting, CT26 cells (mouse MSS colon cancer cells) were counted according to 1×10 6 pcs / 100 μL were inoculated subcutaneously into mice;

[0031] (3) The transplanted tumor grows to 100 mm 3 Around (7 days), group treatment was performed;

[0032] 2. The constructed MSS-type intestinal cancer model mice were divided into 8 groups, with 6 mice in each group:

[0033] The reagent configuration scheme is as follows: anti-PD1 and SHR1906 are prepared using normal saline; PLX3397 is prepared into a 50 mg / mL stock solution using DMSO, which is prepared immediately upon use in a ratio of 10% DMSO + 40% PEG300 + 5% Tween 80 + 45% ddH2O.

[0034] The specific groups are as follows:

[0035] ①PBS group: intraperitoneal injection of 1M sterile PBS, 100 μL / mouse, once a day;

[0036] ②CSF1R inhibitor group: PLX3397 was administered orally, 20 mg / kg, once a day;

[0037] ③Anti-CTGF group: intraperitoneal injection of SHR1906, 400 μg / mouse, once every three days, for a total of five times;

[0038] ④Anti-PD-1 group: intraperitoneal injection of anti-PD-1, 200 μg / mouse, once every three days, for a total of five times;

[0039] ⑤ CSF1R inhibitor combined with anti-PD1 group: PLX3397 was administered orally at 20 mg / kg once daily; anti-PD1 was injected intraperitoneally at 200 μg / mouse once every three days for a total of five times; the two drugs were administered at the same time;

[0040] ⑥Anti-CTGF combined with anti-PD-1 group: SHR1906, 400 μg / mouse, and anti-PD1, 200 μg / mouse, were intraperitoneally injected. Both drugs were administered once every three days for a total of five times. The two drugs were administered at the same time.

[0041] ⑦CSF1R inhibitor combined with anti-CTGF group: PLX3397 was administered orally at 20 mg / kg once a day; SHR1906 was injected intraperitoneally at 400 μg / mouse once every three days for a total of five times; the two drugs were administered at the same time.

[0042] ⑧CSF1R inhibitor, anti-CTGF and anti-PD-1 combination treatment group: PLX3397 was administered orally at 20 mg / kg once a day; SHR1906 was injected intraperitoneally at 400 μg / mouse; anti-PD1 was injected intraperitoneally at 200 μg / mouse. The two drugs were injected once every three days, for a total of five times; the three drugs were treated at the same time.

[0043] 3. Detection of tumor volume:

[0044] Starting from the 7th day after inoculation of CT26 cells, the length and width of the subcutaneous transplanted tumors in mice were measured every 3 days using a vernier caliper, and the average tumor diameter was calculated. Figure 1 shown.

[0045] Results: As Figure 1 As shown, single-agent treatment with anti-CTGF, CSF1R inhibitors, and anti-PD-1 all inhibited tumor growth in mice, indicating that CTGF monoclonal antibodies alone can also inhibit the growth of colorectal cancer cells. The triple-drug combination significantly inhibited tumor growth in mice compared to the single-agent and dual-drug combination treatments, without causing significant side effects. These results suggest that combining CTGF-targeted therapy with anti-macrophage therapy effectively enhances the therapeutic efficacy of anti-PD-1.

[0046] Example 2: Immunofluorescence analysis of the effect of the triple-drug combination therapy group on promoting T cell infiltration in tumor tissue compared to other groups

[0047] (1) Tumor tissues were collected from the BALB / c mice with subcutaneous transplanted tumors in Example 1 on day 19, fixed, and then subjected to immunofluorescence staining.

[0048] (2) Immunofluorescence was used to study the T cell infiltration in tumor tissues of different groups. Tumor tissue sections were labeled with CD8, CD163, and SMA (CD8 antibody, catalog number ab217344, purchased from abcam, USA; CD163 antibody, catalog number ab289979, purchased from abcam, USA; SMA antibody, catalog number 14395-1-AP, purchased from proteintech, China), and the tumor tissue infiltration in the three-drug combination treatment group and the control group (PBS group) was observed.

[0049] (3) Red, yellow, and green fluorescence represent CD8 / CD163 / SMA, and blue (DAPI fluorescence) represents stained cell nuclei.

[0050] Results: The immunofluorescence results of mouse tumors were as follows: Figure 2 As shown in the results, the total CD8+T cells in the tumor tissue of the triple-drug combination treatment group increased significantly compared with the control group; at the same time, CD163, a marker of the monocyte / macrophage system, and SMA, a marker of tumor-associated fibroblasts, decreased significantly compared with the control group; the above suggests that the triple-drug combination treatment group can effectively reverse the exhaustion of CD8+T cell function in tumor tissue and promote the infiltration of CD8+T cells in tumor tissue.

[0051] Example 3: Flow cytometry determination of the CTL (CD3+, CD8+, IFN-γ+) ratio and macrophage (CD45+, CD11b+, F4 / 80+) ratio in each group

[0052] (1) Sampling: Tumor tissue was collected from the subcutaneous transplanted tumors of C57BL / 6 mice in Example 1 on day 19, and the cells were dissociated and analyzed by flow cytometry.

[0053] (2) Dissociation of cells: 8 groups of tumor tissues were minced with surgical scissors, digested with collagenase for 1.5 h, and centrifuged at 1500 rpm for 5 min.

[0054] (3) Washing: Wash the cells with pre-cooled PBS, centrifuge at 1500 rpm for 5 minutes, and wash twice.

[0055] (4) Surface staining: Resuspend the cells and perform surface staining (CD3, CD8, CD45, CD11b, F4 / 80), incubate at 4°C in the dark for 30 min (CD3 antibody, Cat#100203; CD8 antibody, Cat#100711; CD45 antibody, Cat#157207; CD11b, Cat#101205; F4 / 80, Cat#157305. All the above antibodies were purchased from BioLegend, USA).

[0056] (5) Cell fixation: Wash the cells twice with PBS, add 4% PFA fixative, and fix at 4°C in the dark for 30 min.

[0057] (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.

[0058] (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).

[0059] (8) Wash the cells twice with permeabilization buffer, resuspend the cells with PBS, and load them onto the microscope.

[0060] (9) Cell flow cytometry analysis: Flowjo (V10).

[0061] Results: The ratio of CTL and macrophage in different treatment groups was determined by flow cytometry. Figure 3 and Figure 4 As shown, the proportion of cytotoxic T cells (CTL) in tumor tissue of the triple-drug combination treatment group was significantly increased compared with other groups, while the proportion of macrophages was significantly decreased compared with other groups, suggesting that the triple-drug combination treatment group promoted the infiltration of CTL in tumor tissue and enhanced anti-tumor activity.

Claims

1. Use of a CTGF monoclonal antibody in combination with a CSF1R inhibitor and a PD-1 monoclonal antibody in the preparation of a drug for the treatment of colorectal cancer; wherein the CTGF monoclonal antibody is SHR1906 and the CSF1R inhibitor is Pexidartinib PLX-3397.

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

3. A pharmaceutical composition for treating colorectal cancer, characterized in that: The pharmaceutical composition comprises CTGF monoclonal antibody, CSF1R inhibitor and PD-1 monoclonal antibody; wherein the CTGF monoclonal antibody is SHR1906, and the CSF1R inhibitor is Pexidartinib PLX-3397.

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