Application of Vipr2 receptor agonist BAY 55-9837 in tumor treatment

Abstract

The invention relates to the technical field of biological medicines, in particular to application of a Vipr2 receptor stimulant BAY 55-9837 in tumor treatment. Comprising an application of a Vipr2 receptor stimulant in tumor treatment and at least one of the following applications: (1) enhancing reception of a vasoactive intestinal peptide signal by immunosuppressive Treg cells; (2) reducing the number of immunosuppressive function Treg cells in tumors; and (3) application of the Vipr2 receptor agonist BAY 55-9837 in tumor treatment, wherein the Vipr2 receptor agonist BAY 55-9837 can be used for treating tumors or assisting in treating tumors. The number of Treg cells in a tumor is reduced by injecting a vipr2 receptor stimulant in the tumor, and a stronger anti-tumor effect on a solid tumor is shown.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to the application of the Vipr2 receptor agonist BAY 55-9837 in tumor treatment. Background Art

[0002] Worldwide, cancer remains one of the main causes of death. According to the statistical data in 2020, the number of newly diagnosed cancer cases globally was approximately 19.3 million, and the number of death cases was close to 10 million. Cancer has become one of the primary factors threatening public health.

[0003] Currently, the conventional treatment methods for cancer mainly include surgical resection, chemotherapy, radiotherapy, targeted therapy, and interventional therapy, etc. In recent years, tumor immunotherapy, as an emerging treatment strategy, has gradually become a research hotspot in the field of cancer treatment. Immunotherapy regulates the patient's own immune system, enhances its ability to recognize and kill tumor cells, so as to achieve the effect of controlling or even eliminating tumors. Different from traditional treatment methods, immunotherapy can not only directly attack tumor cells, but also prevent the recurrence and metastasis of tumors by activating the immune memory function of the body. The main methods of tumor immunotherapy include immune checkpoint inhibitors, CAR-T cell therapy, tumor vaccines, and cytokine therapy, etc. These methods help the immune system to re-recognize and attack tumor cells through different mechanisms, significantly improving the treatment effect and bringing new hope to many patients with advanced cancer. With the in-depth research and technological progress, the application prospect of tumor immunotherapy in clinical practice is broad, and it is expected to become one of the important means for future cancer treatment.

[0004] Vasoactive intestinal peptide (VIP) receptor 2 (VIPR2) is a member of the G protein-coupled receptor family, regulating multiple downstream signaling molecules such as protein kinase A (PKA), phosphatidylinositol 3-kinase (PI3K), and phospholipase C. It is involved in regulating cell proliferation, immune response, and angiogenesis. Currently, the drug development targeting VIPR2 mainly focuses on antagonists for inflammatory or metabolic diseases. Bay55-9837 is an effective and highly selective agonist of VIPR2. The prior art mainly uses it for type 2 diabetes research, but the application of this agonist in tumor treatment has not been explored.

[0005] We have deeply studied the agonist of vasoactive intestinal peptide (VIP) receptor 2 (VIPR2) to target regulatory T (Treg) cells in the tumor immunosuppressive network as an auxiliary strategy for tumor immunotherapy, and proposed the application of the Vipr2 receptor agonist BAY 55-9837 in tumor treatment. Summary of the Invention

[0006] The object of the present invention is to provide the use of the Vipr2 receptor agonist BAY 55-9837 in tumor treatment, and to provide an agonist of vasoactive intestinal peptide (VIP) receptor 2 (VIPR2) to target regulatory T (Treg) cells in the tumor immunosuppressive network as an adjuvant strategy for tumor immunotherapy.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] The present invention provides the use of a Vipr2 receptor agonist in tumor treatment.

[0009] Further, the Vipr2 receptor agonist is BAY 55-9837.

[0010] Further, the tumor is a solid tumor.

[0011] The present invention also provides the use of the Vipr2 receptor agonist BAY 55-9837 in at least one of the following:

[0012] (1) enhancing the reception of vasoactive intestinal peptide signals by immunosuppressive Treg cells;

[0013] (2) reducing the number of immunosuppressive functional Treg cells in the tumor;

[0014] (3) preparing a product for treating tumors or assisting in the treatment of tumors.

[0015] The present invention has at least the following beneficial effects:

[0016] The present invention provides a Vipr2 receptor agonist BAY 55-9837 that can treat tumors; and by injecting the vipr2 receptor agonist into the tumor, the present invention reduces the number of Treg cells in the tumor and shows a stronger anti-tumor effect on solid tumors. Description of the Drawings

[0017] To more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the following drawings are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0018] Figure 1 It is a flow chart for the immunotherapy of mice with the Vipr2 receptor agonist BAY 55-9837;

[0019] Figure 2 It is a graph of the subcutaneous tumor volume of the mice after treatment with the Vipr2 receptor agonist BAY 55-9837 and the control mice;

[0020] Figure 3 Expression profiles of vasoactive intestinal peptide (VIP) receptor 2 (VIPR2) on various immune cells

[0021] Figure 4 It is a result diagram of the analysis of tumor-infiltrating immune cells in tumor-bearing mice and control mice after treatment with the agonist BAY 55-9837 Detailed implementation manners

[0022] In order to make the objectives, technical solutions and advantages of the present invention clearer and more understandable, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention

[0023] Example 1

[0024] This example first provides an agonist BAY 55-9837 of the vipr2 receptor. The agonist BAY 55-9837 is a polypeptide, and the vipr2 receptor is a receptor on Treg cells that receives the vasoactive intestinal peptide signal Vip

[0025] The amino acid sequence of the said BAY 55-9837 is shown as follows

[0026] SEQ ID NO:1

[0027] HSDAVFTDNYTRLRKQVAAKKYLQSIKNKRY-NH2

[0028] It should be additionally noted that BAY 55-9837 is an existing synthetic polypeptide, and the prior art mainly uses it for researching or treating diseases related to type 2 diabetes 「Tsutsumi M,et,al.A potent and highly selective VPAC2 agonist enhances glucose-induced insulin release and glucos e disposal:a potential therapy for type 2diabetes.Diabetes.2002May；51(5):1453-60」 The present invention mainly studies its application in tumor treatment, as shown in the following examples

[0029] Example 2

[0030] This example is mainly used to provide the application of the agonist BAY 55-9837 of the vipr2 receptor in tumor treatment. As follows

[0031] (1) Construction of a subcutaneous melanoma mouse model and implementation of a treatment plan

[0032] Conventionally culture B6F10 melanoma cells. When the confluence reaches 80%, digest the cells with trypsin, collect the cells, wash them three times with PBS, and then resuspend the cells. Inject 0.5 million cells subcutaneously into C57BL / 6 mice. Divide the tumor-bearing mice into a control group and a treatment group evenly. Four days after tumor inoculation, the mice in the treatment group received the vipr2 receptor agonist BAY 55-9837 (10 μg / kg), and were injected once every other day (the flow chart of the immunotherapy of the vipr2 receptor agonist BAY 55-9837 in mice is shown in Figure 1 ). After 21 days of treatment, the samples were taken, and the results were as shown in Figure 2 . The subcutaneous tumor volume of the mice in the treatment group was significantly smaller than that of the mice in the control group.

[0033] We obtained the expression profiles of vasoactive intestinal peptide (VIP) receptor 2 (VIPR2) on various immune cells from the schmiedel dataset of THE HUMAN PROTEIN ATLAS database (https: / / www.proteinatlas.org / ENSG00000106018-VIPR2 / single+cell) , as shown in Figure 3 , and it can be seen that there is high expression in T-reg.

[0034] (2) Analysis of tumor-infiltrating immune cells in the mouse subcutaneous tumor model

[0035] Take mouse tumor tissues, cut the tissues into small pieces as much as possible, dilute the tissue lysate with DMEM, and make the diluted solution cover the tumor tissues. Digest for 2 h on a shaker at 37 °C. Grind and filter the digested tissues through a filter screen with a pore size of 40 μm, and collect the filtrate. Dilute the Percoll separation solution to 30% and 70%. Add 2 mL of 70% Percoll separation solution into a 15 mL centrifuge tube, and then carefully pipette 2 mL of 30% Percoll solution onto the top of the 70% Percoll solution. Finally, carefully pipette the filtrate onto the top of the 30% Percoll solution. Set the parameters of the centrifuge, with an acceleration of 1 during acceleration, an acceleration of 0 during deceleration, a rotation speed of 800 - 1000 g, and centrifuge for 30 min. Carefully aspirate the white cell layer in the middle layer with a pipette, then add 5 volumes of complete medium to resuspend the cells, centrifuge and discard the supernatant, and resuspend the cells with an appropriate volume of complete culture medium.Prepare single-cell suspensions. After centrifugation, discard the supernatant. Usually, retain approximately 100 μL of PBS. Add 0.5 μL of directly conjugated fluorescent primary antibody, and mix well by pulsed vortexing. Incubate in the dark at room temperature for 20 - 60 min. Add 3 mL of PBS, centrifuge, and discard the supernatant. Resuspend the stained cells in 200 μL of PBS, and analyze the sample by flow cytometry or continue with intracellular / nuclear antigen labeling: ① Intracellular antigen labeling: After the last wash, discard the supernatant and pulse-vortex the sample to completely dissociate cell clumps. Usually, retain approximately 100 μL of residual volume. Add 100 μL of IC fixation solution to fix the cells, then mix well by pulsed vortexing. Incubate in the dark at room temperature for 20 - 60 min. Add 2 mL of 1x permeabilization solution, centrifuge at 400 - 600 g for 5 min at room temperature, discard the supernatant, resuspend the cell pellet in 100 μL of 1x permeabilization solution, add 0.5 μL of directly conjugated primary antibody to bind intracellular antigens, and incubate overnight at 4°C. The next day, add 3 mL of PBS, centrifuge, and discard the supernatant. Resuspend the stained cells in 200 μL of PBS, and analyze the sample by flow cytometry or continue with fluorescent secondary antibody labeling; ② Nuclear antigen labeling: Centrifuge the collected cell suspension (1500 rpm, 5 min), discard the supernatant, resuspend the cells in 100 μL of Foxp3 fixation and permeabilization working solution, incubate in the dark at room temperature for 30 min, add 2 mL of 1x permeabilization solution, centrifuge (1500 rpm, 5 min), discard the supernatant, add the corresponding flow antibody, incubate overnight at 4°C in the dark, add 3 mL of PBS to resuspend and then centrifuge (1500 rpm, 5 min), discard the supernatant, resuspend the cells in 200 μL of PBS, and detect by flow cytometry or continue with secondary antibody labeling; ③ Fluorescent secondary antibody labeling: Centrifuge the cell suspension treated with non-directly labeled primary antibody (1500 rpm, 5 min), discard the supernatant, add the corresponding fluorescently labeled secondary antibody, incubate for 30 min at room temperature in the dark, add 3 mL of PBS to resuspend and then centrifuge, discard the supernatant, resuspend the cells in 200 μL of PBS, filter through a 200-mesh filter, and detect by flow cytometry. Analyze the results using FlowJo software. The results are as follows. Figure 4 As shown Figure 4 the number of tumor-infiltrating CD8+ T cells in mice treated with BAY 55-9837 was higher than that in the untreated group; the number of tumor-infiltrating Treg cells in mice treated with BAY 55-9837 was lower than that in the untreated group.

[0036] Combined with the results shown in the figure, we can clearly find that:

[0037] By intratumoral injection of the vipr2 receptor agonist BAY 55-9837, the present invention can reduce the number of Treg cells in tumors, enhance the reception of vasoactive intestinal peptide signals by immunosuppressive Treg cells, and exhibit a stronger anti-tumor effect on solid tumors. Thus, BAY 55-9837 can be used for tumor treatment to achieve a new method for inhibiting tumor growth, regulating the immune composition of the tumor microenvironment, or enhancing the anti-tumor immune response in monotherapy or combination therapy.

[0038] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments. The above embodiments and the principles described in the specification are only the principles of the present invention. Without departing from the spirit and scope of the present invention, various changes and improvements will occur to the present invention, and these changes and improvements all fall within the scope of the present invention claimed. The scope of protection required by the present invention is defined by the appended claims and their equivalents.

Claims

1. Use of a Vipr2 receptor agonist in tumor treatment.

2. The application according to claim 1, characterized in that, The Vipr2 receptor agonist is BAY 55-9837.

3. The application according to claim 1, characterized in that The tumor is a solid tumor.

4. Use of the Vipr2 receptor agonist BAY 55-9837 in at least one of the following: (1) Enhancing the reception of vasoactive intestinal peptide signals by immunosuppressive Treg cells; (2) Reducing the number of immunosuppressive functional Treg cells in tumors; (3) Preparing a product for treating or assisting in treating tumors.

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