Application of CTGF monoclonal antibody combined with CSF1R inhibitor and PD-1 monoclonal antibody in preparation of medicine for treating colorectal cancer and medicine composition
Through the combined treatment of CTGF monoclonal antibody combined with CSF1R inhibitor and PD-1 monoclonal antibody, the problem of poor efficacy of existing CSF1R inhibitor combined with immunotherapy was solved, significantly improved the efficacy of immunotherapy in colorectal cancer, and promoted T cells in Infiltration in tumor tissue.
Patent Information
- Application Number
- CN202411574872.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-06
- Publication Date
- 2025-02-14
- Estimated Expiration
- 2044-11-06
AI Technical Summary
The existing CSF1R inhibitor combined with immunotherapy is poor in colorectal cancer, resulting in the problem of immunotherapy resistance.
The drug combination of CTGF monoclonal antibody combined with CSF1R inhibitor and PD-1 monoclonal antibody is used to target CTGF genes and tumor-associated macrophages to enhance the efficacy of immunotherapy.
It significantly improved the effect of PD-1 monoclonal antibody immunotherapy, extended the survival of mice, promoted the infiltration of CD8+ T cells and CTL in tumor tissues, and enhanced anti-tumor activity.
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Figure CN119424656A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of tumor treatment, and specifically to 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. Background Art
[0002] Colorectal cancer (CRC) is the third most common malignancy in the world. About 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 deeply study the relationship between tumor cell intrinsic events and host immune response in CRC.
[0003] Tumor-associated macrophages (TAMs) are the most abundant immune cells in the tumor microenvironment and play an important role in the immunosuppressive microenvironment of colorectal cancer. The immunosuppressive microenvironment caused by their high aggregation in the tumor microenvironment may be one of the reasons for immunotherapy resistance. Therefore, the strategy of targeting TAMs combined with immune checkpoint inhibitors may sensitize the efficacy of immunotherapy for colorectal cancer. CSF1 receptor (CSF1R) is expressed on circulating monocytes and tissue macrophages. The CSF1 / CSF1R signaling axis is overexpressed in a variety of tumor tissues, and its overexpression is associated with poor prognosis. Monoclonal antibodies or small molecule inhibitors of CSF1R can block CSF1R signaling, target TAMs, and control the survival, proliferation, differentiation, and chemotaxis of such cells, successfully consuming TAMs. However, in preclinical animal models of colorectal cancer and early clinical studies, the effect of CSF1R inhibitors combined with immunotherapy was not good. Therefore, it is urgent 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 that the existing CSF1R inhibitor combined with immunotherapy has poor effect, 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 object, the present invention provides the following technical solutions:
[0006] The present invention provides use of CTGF monoclonal antibody combined with CSF1R inhibitor and PD-1 monoclonal antibody in preparing 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 application of CTGF monoclonal antibody in preparing medicine 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, comprising 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 the application of CTGF gene target in anti-tumor treatment, and proposes a triple scheme of targeting CTGF combined with CSF1R inhibitor sensitization immunotherapy. The CTGF gene plays an important role in extracellular matrix remodeling, cell adhesion, migration, proliferation, etc. As a target of new tumor immunotherapy drugs, it blocks the binding of target protein with various cytokines and growth factors, affects downstream signaling pathways, and can effectively reverse and inhibit the exhaustion of effector T cells, restore the function of effector T cells, and enhance the efficacy of immunotherapy by combining CSF1R inhibitors, i.e., drugs targeting tumor-associated macrophages. Compared with the control group, intraperitoneal injection of CTGF monoclonal antibody alone inhibited the growth of colorectal cancer tumors in mice.
[0014] The triple combination of CTGF-targeted combined with CSF1R inhibitor sensitization immunotherapy has a significant therapeutic effect in animal models. The combination of CTGF monoclonal antibody and CSF1R inhibitor significantly improved the effect of PD-1 monoclonal antibody immunotherapy and prolonged the survival of mice. The combination of anti-CTGF, CSF1R inhibitor and anti-PD-1 significantly promoted the infiltration of CD8+T cells and CTLs in tumor tissues and enhanced 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-type 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 results of mouse tumor immunofluorescence showed that the combination of CTGF monoclonal antibody, CSF1R inhibitor and PD-1 antibody promoted CD8+T cell infiltration in tumor tissue;
[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 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.
[0021] CD8+T cells play a key role in tumor immunotherapy. The dysregulation of T cell trafficking to tumor tissues and the weakening of killing ability are one of the reasons for insufficient tumor T cell infiltration. Therefore, promoting the recruitment of effector T cells to the tumor center may provide a new possibility for improving the efficacy of ICB treatment.
[0022] Connective tissue growth factor (CTGF) is a multifunctional protein in the CCN family that can regulate multiple biological processes such as cell proliferation, differentiation, and adhesion.
[0023] The combination of CTGF monoclonal antibody and CSF1R inhibitor with PD-1 monoclonal antibody can significantly promote the effect of PD-1 monoclonal antibody immunotherapy. The strategy of combining CTGF monoclonal antibody with CSF1R inhibitor to enhance immunotherapy does not involve experimental issues such as pharmacological toxicity involved in 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 the inhibitor PLX3397. The inhibitor PLX3397 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 mouse MSS-type colorectal cancer model:
[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 at 1×10 6 pcs / 100 μL were inoculated subcutaneously into mice;
[0031] (3) The size of the transplanted tumor grows to 100 mm 3 Around (7 days), group treatment was carried out;
[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 physiological saline; PLX3397 is configured with DMSO into a 50 mg / mL storage solution, which is prepared immediately upon use, according to the 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 gavage, 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-PD1, 200 μg / mouse, once every three days, for a total of five times;
[0039] ⑤CSF1R inhibitor combined with anti-PD1 group: PLX3397 was administered orally, 20 mg / kg, once a day; anti-PD1 was injected intraperitoneally, 200 μg / mouse, once every three days, for a total of five times; the two drugs were administered at the same time;
[0040] ⑥ Anti-CTGF combined with anti-PD-1 group: intraperitoneal injection of SHR1906, 400 μg / mouse; intraperitoneal injection of anti-PD1, 200 μg / mouse, the two 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, 20 mg / kg, once a day; SHR1906 was injected intraperitoneally, 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 combined 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 of mice were measured every 3 days using a vernier caliper, and the average tumor diameter was calculated. Figure 1 shown.
[0045] Results: Figure 1 As shown in the figure, the single-drug groups of anti-CTGF group, CSF1R inhibitor group and anti-PD-1 group were able to inhibit the growth of mouse tumor volume, indicating that the use of CTGF monoclonal antibody alone can also inhibit the growth of colorectal cancer cells. Among them, the three-drug combination treatment group significantly inhibited the growth of mouse tumor volume compared with the single-drug treatment group and the double-drug combination treatment group, and did not cause obvious side effects. The above results suggest that targeting CTGF combined with anti-macrophage therapy effectively enhances the therapeutic efficacy of anti-PD-1.
[0046] Example 2: Using immunofluorescence technology to study the effect of the three-drug combination treatment group on promoting T cell infiltration in tumor tissue compared with 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) The immunofluorescence method 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 was observed in the three-drug combination treatment group and the control group (PBS group).
[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, the total CD8+T cells in the tumor tissue of the triple drug combination treatment group were significantly increased 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, were significantly reduced 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 was used to determine the proportion of CTL (CD3+, CD8+, IFN-γ+) and macrophage (CD45+, CD11b+, F4 / 80+) cells 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: The 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 min, and wash twice.
[0055] (4) Surface staining: Resuspend the cells and perform surface staining (CD3, CD8, CD45, CD11b, F4 / 80), and 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 in PBS, and load onto the analyzer.
[0060] (9) Cell flow cytometry analysis: Flowjo (V10).
[0061] Results: Flow cytometry was used to determine the ratio of CTL and macrophage in different treatment groups. 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. Application of CTGF monoclonal antibody combined with CSF1R inhibitor and PD-1 monoclonal antibody in the preparation of drugs for the treatment of colorectal cancer.
2. The use according to claim 1, characterized in that: The CTGF monoclonal antibody is SHR1906 or Pamrevlumab.
3. The use according to claim 1, characterized in that: The CSF1R inhibitors are Pexidartinib PLX-3397, Emactuzumab RG7155, and Cabiralizumab FPA008.
4. The use according to claim 1, characterized in that: The colorectal cancer is MSS type colorectal cancer.
5. Application of CTGF monoclonal antibody in the preparation of drugs for the treatment of colorectal cancer.
6. The use according to claim 5, characterized in that: The CTGF monoclonal antibody is SHR1906 or Pamrevlumab.
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 CTGF monoclonal antibody, CSF1R inhibitor and PD-1 monoclonal antibody.