Use of an immune checkpoint inhibitor in the manufacture of a medicament for prophylactic treatment of cancer in a subject

Abstract

The application discloses application of an immune checkpoint inhibitor. One of the applications is the application in the preparation of a drug for preventing and treating cancer in a subject. The embodiments of the application show that the application of the immune checkpoint inhibitor to the early cancer stage (high-grade intraepithelial neoplasia stage (HGIN)) of esophageal squamous cell carcinoma can reverse the progression of esophageal squamous cell carcinoma lesions, effectively control the recurrence and metastasis of cancer.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology. Specifically, this invention relates to the use of immune checkpoint inhibitors in the preparation of medicaments for the preventive treatment of cancer in subjects. Background Technology

[0002] Esophageal cancer is one of the most common malignant tumors of the upper gastrointestinal tract worldwide and one of the leading causes of cancer-related deaths. Esophageal cancer is mainly divided into two subtypes: esophageal adenocarcinoma and esophageal squamous cell carcinoma (ESCC). In my country, esophageal squamous cell carcinoma accounts for more than 90% of all esophageal cancer patients.

[0003] Currently, surgery remains the primary treatment for advanced esophageal squamous cell carcinoma. However, with advancements in medical technology and the emergence of various cancer screening methods, the early detection rate of esophageal cancer is gradually increasing. For patients with early-stage esophageal squamous cell carcinoma and high-grade intraepithelial neoplasia, endoscopic treatment has become the main treatment method due to its minimally invasive advantages. However, even with timely surgical treatment, the mortality rate of esophageal cancer has not decreased significantly, and the five-year survival rate has remained below 20% for many years. Patients with early-stage esophageal squamous cell carcinoma undergoing endoscopic treatment often experience severe impacts on their quality of life due to complications. Furthermore, because the esophagus lacks a serosal layer for protection and has abundant submucosal lymph node tissue, distant metastasis can occur even in the early stages of the disease. Even with timely endoscopic resection of lesions in the early stages, it is still impossible to effectively eliminate scattered tumor cells in the body, leading to tumor recurrence and distant metastasis.

[0004] With a deeper understanding of immune cells in the tumor microenvironment and the development of immunology and related disciplines, numerous tumor-killing therapies based on enhancing the immune system have emerged. Among them, immunotherapy, represented by immune checkpoint inhibitors, has brought new options and hope to cancer patients. During tumor development and progression, T cells located in the tumor microenvironment express receptors (also known as immune checkpoints, such as cytotoxic T-lymphocyte-associated protein 4 (CTLA4)) that inhibit anti-tumor immune function. These receptors can bind to ligands expressed on the surface of tumor cells, leading to a state of T cell exhaustion in the tumor microenvironment. Monoclonal antibodies targeting the PD-1 / PD-L1 axis or the CTLA-4 / B7 axis can effectively alleviate the immunosuppressive state in the tumor microenvironment. By restoring the body's normal anti-tumor immunity, these antibodies have a good killing effect on tumor lesions, as well as sporadic lesions and lymph node metastases that are difficult to eradicate. Summary of the Invention

[0005] One of the objectives of this invention is to provide the application of immune checkpoint inhibitors.

[0006] This invention provides the use of immune checkpoint inhibitors in the preparation of medicaments for the preventive treatment of cancer in subjects.

[0007] Optionally, according to the above application, the subject is in the early stage of cancer.

[0008] Alternatively, according to the above application, the cancer is esophageal squamous cell carcinoma.

[0009] Optionally, according to the above application, the early cancer stage is the high-grade intraepithelial neoplasia stage of the esophagus.

[0010] This invention also provides the use of immune checkpoint inhibitors in the preparation of drugs for preventing, delaying, or reversing the progression of early esophageal squamous cell carcinoma. The early esophageal squamous cell carcinoma may be high-grade intraepithelial neoplasia (HGIN) stage esophageal squamous cell carcinoma.

[0011] This invention also provides the use of immune checkpoint inhibitors in the preparation of products for increasing the infiltration of tumor-infiltrating lymphocytes (TILs) with tumor-killing activity. TILs include, for example, CD4+ T cells, CD8+ T cells, M1 tumor-associated macrophages, M2 tumor-associated macrophages, and / or regulatory T cells with FOXP3 as a surface marker.

[0012] Optionally, according to the above application, the immune checkpoint is selected from at least one of cytotoxic T lymphocyte-associated antigen-4 (CTLA4), programmed cell death-1 (PD-1), and programmed cell death-ligand 1 (PD-L1).

[0013] Optionally, according to the above application, the immune checkpoint is selected from at least one of CTLA4 antibody, PD-1 antibody and PD-L1 antibody.

[0014] The embodiments of the present invention demonstrate that applying immune checkpoint inhibitors to the early stage of esophageal squamous cell carcinoma (high-grade intraepithelial neoplasia (HGIN)) can reverse the progression of esophageal squamous cell carcinoma, effectively control cancer recurrence and metastasis, and free patients from the risks and discomforts of surgery. Attached Figure Description

[0015] Figure 1 The results are from the experiment "I. Mouse Modeling" in Example 1.

[0016] Figure 2 The results are from the experiment "I. Mouse Modeling" in Example 1.

[0017] Figure 3The results are from Example 1, "II. Application of PD-1 monoclonal antibody".

[0018] Figure 4 The results are from the experiment in Example 1, "III. Analysis of the Immune Microenvironment".

[0019] Figure 5 The results are from the experiment in Example 1, "III. Analysis of the Immune Microenvironment". Detailed Implementation

[0020] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0021] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0022] The mice used in the following examples are as follows:

[0023] All animal experiments were conducted in accordance with protocols approved by the Animal Management and Use Committee of the Chinese Academy of Sciences. C57BL / 6J mice were kept in a pathogen-free environment at 23-24°C, with free access to normal food and water under 12-hour light and 12-hour dark conditions. Cage bedding was changed weekly.

[0024] The chemical carcinogen 4-NQO was purchased from Sigma-Aldrich in the United States, catalog number N4181.

[0025] The anti-mouse PD-1 monoclonal antibody was purchased from BioCell, Inc., USA, catalog number BE0146.

[0026] The anti-mouse IgG monoclonal antibody was purchased from BioCell, Inc., USA, catalog number BE0089.

[0027] Example 1: PD-1 monoclonal antibody treatment of esophageal squamous cell carcinoma in mice

[0028] I. Mouse Model

[0029] The development of esophageal squamous cell carcinoma in mice was induced using the chemical carcinogen 4-NQO, and the pathological conditions of the esophagus at different time points during the induction process (mainly including LGIN, HGIN, and ESCC stages) were determined. The expression of major immune checkpoints (mainly PD-1 and PD-L1) under different pathological conditions was also observed.

[0030] Fifty-six female C57BL / 6J mice, 6 weeks old and weighing 16±1g, were randomly divided into a control group (7 mice) and a chemical carcinogen 4-NQO-induced group (49 mice). In the 4-NQO group, 7 mice were sacrificed at weeks 4, 8, 12, 16, 20, 24 and 28 of the treatment process.

[0031] like Figure 1 The processing flow is shown in section A.

[0032] Control group: 5 ml of acetone solution was added to 245 ml of sterile MilliQ water (250 ml / mouse bottle, final concentration of acetone solution was 2%) as drinking water for mice for 16 weeks. Then the acetone solution treatment was stopped, and the drinking water of the mice was replaced with sterile MilliQ water. The mice were raised normally until 28 weeks and then sacrificed.

[0033] 4-NQO induction group (chemical carcinogen): Prepare 50ml centrifuge tubes (wrapped in aluminum foil to protect from light), add 50ml acetone, then weigh 250mg of 4-NQO using a microbalance, add it to the bottle, tighten the cap, and shake repeatedly until completely dissolved to prepare a 5mg / ml 4-NQO stock solution. Mix 5ml of the 4-NQO stock solution (5mg / ml) with 245ml of sterile MilliQ water to prepare a 100μg / ml 4-NQO solution as drinking water for mice, continuing for 16 weeks. Then, stop 4-NQO treatment and raise the mice normally until 28 weeks, at which point they are sacrificed.

[0034] After euthanizing the mice, the esophagus was completely removed, cut into segments approximately 5 mm in size, and fixed in formalin for 24 hours. It was then transferred to 70% ethanol, embedded in paraffin along its vertical axis, and sliced ​​into 4 μm thick sections for Hematologic and Escherichia coli (H&E) staining to determine the condition of the esophagus. The definitions of LGIN, HGIN, and ESCC were based on the WHO Digestive System Tumors definition. H&E-stained sections were examined by a pathologist to determine the lesion condition.

[0035] Esophageal sections used for immunohistochemical analysis were baked in a 60°C oven for 1 hour, dewaxed with xylene, rehydrated with graded alcohols, and then boiled in EDTA antigen retrieval solution (ZLI-9066, ZsgbBio, Beijing, China) for approximately 30 minutes. After cooling, the tissues were treated with endogenous peroxidase inhibitors for 10 minutes, followed by blocking with goat serum for 1 hour. Next, rabbit anti-PD-1 (purchased from Cell Signaling Technology, catalog number D7DW) and PD-L1 (purchased from PROTEINTECH, catalog number 66248-1-lg) primary antibodies were added and incubated overnight. The next day, the primary antibody was washed away with TBST, followed by incubation with secondary antibody for 30 minutes, and then developed with DAB. Finally, the tissues were counterstained with hematoxylin and mounted with neutral resin. The staining intensity of PD-1 and PD-L1 at the above immune checkpoints was assessed using a semi-quantitative immunoreactivity scoring system (IRS).

[0036] The results are as follows Figure 1 and Figure 2 As shown. Figure 1 Figure B shows the proportion of esophageal pathological changes in mice in the 4-NQO group at different time points. Low-grade intraepithelial neoplasia began to appear in mice at week 4; high-grade intraepithelial neoplasia began to appear in mice at week 8; and esophageal squamous cell carcinoma began to appear in mice at week 20. C represents the representative disease stages (including normal esophageal epithelium, low-grade intraepithelial neoplasia (LGIN), high-grade intraepithelial neoplasia (HGIN), and esophageal squamous cell carcinoma (ESCC)) during 4-NQO-induced esophageal squamous cell carcinoma development. At week 4, 2 out of 7 mice showed LGIN; at week 8, 5 out of 7 mice were in LGIN and 1 in HGIN; at week 12, 2 out of 7 mice were in LGIN and 5 in HGIN; at week 16, 6 out of 7 mice showed HGIN; at week 20, 1 mouse developed ESCC; at week 24, 4 out of 7 mice were in the ESCC stage; and at week 28, all mice developed ESCC. D shows partial H&E stained sections and esophageal photographs; the arrows indicate irregular bulges or thickenings in the esophagus. E shows statistical data on mouse weight changes. Figure 2 The study investigated the expression of immune checkpoints (mainly cytotoxic T-lymphocyte-associated protein 4, CTLA4, programmed death receptor 1, PD-1, and programmed death receptor ligand 1, PD-L1) in representative disease stages (including normal esophageal epithelium, low-grade intraepithelial neoplasia, high-grade intraepithelial neoplasia, and esophageal squamous cell carcinoma) during 4-NQO-induced esophageal squamous cell carcinoma development. The results showed that from the HGIN stage onwards, the expression of PD-1 and PD-L1 proteins in the diseased esophagus of mice was significantly increased.

[0037] II. Administration of PD-1 monoclonal antibody

[0038] The experimental procedure is as follows: Figure 3 As shown in Figure A.

[0039] Twenty-one female C57BL / 6J mice, 6 weeks old and weighing 16±1g, were randomly divided into three groups: an IgG treatment group, a PD-1 prophylactic treatment group, and a PD-1 treatment group, with seven mice in each group. 5ml of the prepared 4-NQO stock solution (5mg / ml) and 245ml of sterile MilliQ water were mixed to prepare a 100μg / ml 4-NQO solution, which was used as drinking water for the mice for 16 weeks. Afterward, the 4-NQO treatment was stopped, and the mice were switched to drinking sterile MilliQ water.

[0040] PD-1 treatment group: Mice were normally raised until week 24. On week 24 (as defined as week 24), days 3, 6, 9, 12, 15, 18, and 21, mice were intraperitoneally injected with PD-1 monoclonal antibody at a dose of 100 μl (concentration of 200 μg / 100 μl). Mice were then raised until week 27 and euthanized.

[0041] PD-1 prevention and treatment group: Mice were normally raised to week 18, and then intraperitoneally injected with PD-1 monoclonal antibody on the day of week 18, day 3, day 6, day 9, day 12, day 15, day 18, and day 21. The injection dose was 100 μl, and the concentration was 200 μg / 100 μl. Mice were raised until week 27 and then sacrificed.

[0042] Control group: Mice were normally raised until week 24. On week 24, they were intraperitoneally injected with IgG monoclonal antibody at a dose of 100 μl (concentration of 200 μg / 100 μl) on day 24, day 3, day 6, day 9, day 12, day 15, day 18, and day 21. They were then raised until week 27 and euthanized.

[0043] HE staining was used to observe esophageal lesions in mice. The HE staining method was the same as described in section 1.

[0044] The results are as follows Figure 3 As shown, Figure 3 In this context, B represents the mouse's body weight. Figure 3In the figures, C represents H&E stained sections and gross images of the esophagus of mice in the control group, PD-1 prophylactic treatment group, and PD-1 treatment group after the end of treatment. The arrows indicate irregular bulges or thickenings in the esophagus. It is clearly visible that, compared to the control group and the PD-1 treatment group, esophageal lesions were significantly reduced after PD-1 prophylactic treatment, and H&E sections showed signs of complete reversal of esophageal lesions. Furthermore, in the gross images of the esophagus, the irregular bulges and thickenings in the prophylactic treatment group were significantly reduced compared to the PD-1 treatment group and the control group.

[0045] Comparing the esophageal lesions and body weight changes in mice after prophylactic administration of PD-1 monoclonal antibody during the HGIN phase and after treatment with PD-1 monoclonal antibody and control IgG during the ESCC phase, it was found that the body weight of mice in both the PD-1 prophylactic treatment group and the PD-1 treatment group increased compared to the control group. However, the body weight increase was most significant in the PD-1 prophylactic treatment group at the end of the experiment.

[0046] The results showed that PD-1 monoclonal antibody treatment in the HGIN stage of esophageal cancer in mice could more effectively reverse the progression of esophageal squamous cell carcinoma.

[0047] III. Analysis of the Immune Microenvironment

[0048] Esophageal sections of mice treated with IgG for immunohistochemical analysis of the esophageal immune microenvironment in the prophylactic treatment group and the control group were baked in a 60°C oven for 1 hour, dewaxed with xylene, rehydrated with graded alcohols, and then boiled in EDTA antigen retrieval solution (ZLI-9066, ZsgbBio, Beijing, China) for approximately 30 minutes. After cooling, the tissues were treated with endogenous peroxidase inhibitor for 10 minutes, followed by blocking with goat serum for 1 hour. Next, rabbit anti-CD4 (abcam, catalog number ab183685) and CD8 primary antibody (abcam, catalog number ab217344) were added and incubated overnight. The next day, the primary antibody was washed away with TBST, followed by incubation with secondary antibody for 30 minutes, and then developed with DAB (Zhongshan Jinqiao, catalog number ZLI-9017). Finally, the tissues were counterstained with hematoxylin and mounted with neutral resin. The staining intensity of CD4 and CD8 was assessed using a semi-quantitative immunoreactivity scoring system (IRS).

[0049] The results are as follows Figure 4 As shown, A represents the expression of CD4 and CD8+ T cells in mice after IgG injection, immediately after mice were sacrificed following the end of PD-1 prophylactic treatment, and 6 weeks after mice were sacrificed following the end of PD-1 prophylactic treatment. B represents the immunohistochemical score of the expression of CD4 and CD8+ T cells.

[0050] Compared to the control group injected with IgG, the prophylactic treatment group injected with PD-1 monoclonal antibody showed a significant increase in both CD4+ T cells and CD8+ T cells after the end of treatment. Comparing the duration of these cells' infiltration in the tumor microenvironment, even 6 weeks after the end of treatment, the PD-1 prophylactic treatment group still showed a certain number of CD4+ T cells and CD8+ T cells infiltrating the tumor microenvironment. These results indicate that prophylactic treatment with PD-1 monoclonal antibody in early esophageal squamous cell carcinoma can increase the infiltration of tumor-killing intraepithelial endothelial cells (TILs) and maintain this effect for a period of time.

[0051] Other immune cells infiltrating the immune microenvironment were analyzed, primarily including M1 / M2 tumor-associated macrophages and regulatory T cells with FOXP3 as a surface marker. The analytical methods are as described above. Rabbit anti-CD86 was purchased from Abcam (catalog number ab220188), rabbit anti-CD163 from Abcam (catalog number ab182422), and rabbit anti-FoxP3 from Abcam (catalog number ab215206).

[0052] The results are as follows Figure 5 As shown, A represents the expression of M1 macrophages (CD86), M2 macrophages (CD163), and regulatory T cells (FoxP3) in mice after IgG injection, immediately after the mice were sacrificed following the end of PD-1 prophylactic treatment, and 6 weeks after the mice were sacrificed following the end of PD-1 prophylactic treatment. B represents the immunohistochemical scoring of the expression of M1 macrophages (CD86), M2 macrophages (CD163), and regulatory T cells (FoxP3). C represents the analysis of the esophageal microenvironment of mice after IgG injection and immediately after the mice were sacrificed following the end of PD-1 prophylactic treatment using immunofluorescence. The results indicate that compared with mice injected with IgG, the expression of M1 macrophages, which play a tumor-killing role, was significantly upregulated after the end of PD-1 prophylactic treatment, while the expression of M2 macrophages, which have an immunosuppressive function, was significantly downregulated. This suggests that the effect of PD-1 monoclonal antibody in delaying tumor progression may be partly attributed to its influence on macrophage polarization.

[0053] In conclusion, applying immune checkpoint inhibitors to the early stages of esophageal squamous cell carcinoma can effectively delay or even reverse disease progression, providing a new direction for treatment options for early-stage cancer patients.

[0054] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. The use of an immune checkpoint inhibitor in the preparation of a drug for the prophylactic treatment of high-grade esophageal intraepithelial neoplasia in subjects; wherein the immune checkpoint inhibitor is an anti-PD-1 monoclonal antibody.

2. The application of immune checkpoint inhibitors in the preparation of drugs for the prevention, delay or reversal of high-grade esophageal intraepithelial neoplasia; wherein the immune checkpoint inhibitor is an anti-PD-1 monoclonal antibody.

Images