Application of Venotog in preparation of cGAS-STING agonist

Abstract

The invention provides an application of Vinetrog in preparation of a cGAS-STING agonist. The cGAS-STING agonist can be used for enhancing the curative effect of solid tumor immunotherapy. A research proves that the ABT-199 can be used for remarkably activating a cGAS-STING signal channel, enhancing an IFN-I signal, increasing the expression and secretion of chemotactic factors CXCL9 and CXCL10, and enhancing the tumor treatment effect of a PD-L1-free monoclonal antibody. The invention provides a marketed small molecule medicine capable of activating a cGAS-STING signal channel, and a theoretical basis is provided for converting a cold tumor into a hot tumor by the ABT-199 and sensitizing colorectal cancer immunotherapy.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and relates to the use of Venetoclax (ABT-199) in the preparation of cGAS-STING agonists. Background Art

[0002] Immune checkpoint inhibitors represented by PD-1 / PD-L1 monoclonal antibodies have made significant progress in the treatment of colorectal cancer and are used for the first-line treatment of patients with microsatellite instability-high (MSI-H) colorectal cancer. However, the response rate of this part of patients to PD-1 / PD-L1 monoclonal antibodies is only 40%. In addition, microsatellite stable (MSS) colorectal cancer patients, accounting for 85-90%, basically have no response to PD-1 / PD-L1 monoclonal antibodies. Due to reasons such as the lack of tumor-associated antigens and weak antigen presentation, such tumors form a "cold" tumor microenvironment with less infiltration of immune cells and thus cannot respond to PD-1 / PD-L1 monoclonal antibodies. The activation of the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) signaling pathway can promote the release and presentation of tumor antigens, initiate and activate T cells, and increase the infiltration and killing function of T cells, playing a key role in the anti-tumor immune process. Therefore, activating the cGAS-STING signaling pathway in colorectal cancer can enhance its immunogenicity, improve the tumor microenvironment, and convert "cold" tumors into "hot" tumors, which is an important strategy to improve its responsiveness to immune checkpoint inhibitors.

[0003] Currently, the research and development of STING agonists mainly start from three ideas: 1) Cyclic dinucleotides (CDNs) agonists. As natural ligand analogs of STING, CDNs directly interact with STING. However, their metabolic stability and tissue permeability are poor, and they are mostly administered by intratumoral injection, which limits their clinical application to a certain extent; 2) Non-CDNs. Many small molecule agonists of this type only showed limited anti-tumor effects in early clinical trials, and it is difficult to control the activation degree of the STING signaling pathway, which may cause severe side effects such as cytokine storms; 3) Discovering STING agonists from existing drugs and screening clinical small molecule drugs that can activate the STING signaling pathway are expected to discover STING agonists that can be quickly clinically translated, which is of great significance for discovering combined strategies to enhance the efficacy of colorectal cancer immunotherapy.

[0004] Venetoclax (ABT-199) is a drug currently used in clinical practice for the treatment of specific types of hematological malignancies. Its pharmacological mechanism is to selectively inhibit the activity of B-cell lymphoma-2 protein (BCL-2), relieve its inhibitory effect on pro-apoptotic proteins, restore the apoptosis function, and thus effectively induce the death of tumor cells. ABT-199 has a significant killing effect on blood cancer cells expressing high levels of BCL-2, has relatively little impact on normal cells and solid tumor cells, and has a low risk of severe side effects during treatment. Common adverse reactions include mild neutropenia and gastrointestinal discomfort. Currently, this drug has been widely used in the treatment of diseases such as chronic lymphocytic leukemia (CLL) and acute myeloid leukemia (AML), showing good efficacy and safety. Summary of the Invention

[0005] The object of the present invention is to provide the use of Venetoclax (ABT-199) in the preparation of a cGAS-STING agonist, wherein the cGAS-STING agonist sensitizes the efficacy of solid tumor immunotherapy, and the solid tumor is caused by the activation of the cGAS-STING pathway in tumor cells. ABT-199, chemical name (4-{4-[2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-en-1-yl]-1H-1,2,3-triazol-1-yl}phenyl)[(3R)-1-(4-fluorophenyl)-3-methyl-2-oxopropyl]carbamate, molecular formula: C45H50ClF3N7O7S, molecular weight is 868.44.

[0006] It has been experimentally confirmed that in in vitro experiments, the said ABT-199 (5 μM) can significantly activate P-STING, P-TBK1 and P-IRF3 in colorectal cancer by Western blotting, and can significantly increase the release of type I interferon (IFN-I) IFNβ, C-X-C motif chemokine ligand 10 (CXCL10) and CXCL9 by qRT-PCR. And the synergistic effect of ABT-199 activating the cGAS-STING signaling pathway and PD-L1 monoclonal antibody was investigated through a colorectal cancer-bearing mouse model.

[0007] The drug is made of ABT-199 and pharmaceutically acceptable excipients, and the dosage form of the drug is tablet, capsule, oral solution, injection or lyophilized powder for injection. It is administered once a day, and the specific dosage is adjusted according to the patient's condition and treatment response.

[0008] ABT-199 is a selective inhibitor of B-cell lymphoma-2 protein (BCL-2), which is mainly used in the treatment of CLL and AML clinically. The in vitro experimental study of the present invention proves that ABT-199 can activate the cGAS-STING signaling pathway in colorectal cancer cells; the in vivo experimental study of the tumor-bearing mouse model proves that ABT-199 exerts a synergistic anti-tumor effect with anti-PD-L1 monoclonal antibody by activating the cGAS-STING signaling pathway. The significance of the present invention lies not only in clarifying the new function of ABT-199 in activating the cGAS-STING signaling pathway, but also in providing a theoretical basis for ABT-199 to convert "cold" tumors into "hot" tumors and sensitize the immunotherapy of colorectal cancer.

[0009] In the previous research of the inventors, an evaluation model of the cGAS-STING signaling pathway was established, and a systematic screening of the clinical anti-tumor drug library was carried out. It was found that the BCL-2 selective inhibitor ABT-199 could significantly activate the cGAS-STING signaling pathway, enhance the IFN-I signal, increase the expression and secretion of chemokines CXCL9 and CXCL10, and enhance the anti-tumor effect of anti-PD-L1 monoclonal antibody. At present, there is no good marketed small molecule drug that exerts anti-tumor effects by activating the cGAS-STING signaling pathway clinically, and there is no relevant research proof of ABT-199 activating the cGAS-STING signaling pathway. The present invention provides a marketed small molecule drug that can activate the cGAS-STING signaling pathway. The present invention provides a new medical use for ABT-199. Description of the Drawings

[0010] Figure 1 It is the Western blot and qRT-PCR results of ABT-199 (1.25, 2.5 and 5 μM) acting on human colorectal cancer HT-29 cells and murine colorectal cancer CT26 cells.

[0011] Figure 2 It is the immunofluorescence image of ABT-199 (5 μM) acting on human colorectal cancer HT-29 cells.

[0012] Figure 3 It is the schematic diagram of the experimental process, tumor growth curve and tumor photo of the treatment of CT26 tumor-bearing mice with ABT-199 (100 mg / kg) and anti-PD-L1 (10 mg / kg) monoclonal antibody alone or in combination.

[0013] Figure 4 The effects of single or combined treatment with ABT-199 (100 mg / kg) and anti-PD-L1 monoclonal antibody (10 mg / kg) on the infiltration and tumor-killing activity of CD3 + and CD8 + T cells. Detailed implementation manners

[0014] The present invention will be further described in conjunction with the accompanying drawings and embodiments.

[0015] Example 1 After ABT-199 acts on human colorectal cancer HT-29 cells and murine colorectal cancer CT26 cells for 24 h, the cGAS-STING signaling pathway is significantly activated. The specific steps are as follows:

[0016] Select non-human colorectal cancer HT-29 cells and murine colorectal cancer CT26 cells and seed them in 6-well plates at 1.0×10 6 cells / well, and culture them overnight in an incubator at 37 °C and 5% CO2; the next day, treat them with ABT-199 (1.25, 2.5, and 5 μM) for 24 h, collect and lyse the cells, extract proteins, and detect the phosphorylation levels of STING, IRF3, and TBK1 in the cells by Western blotting. The phosphorylation levels of STING, IRF3, and TBK1 in human colorectal cancer HT-29 cells and murine colorectal cancer CT26 cells at specific concentrations of ABT-199 are shown in Figure 1 A and Figure 1 C; at the same time, lyse the cells with Trizol to extract mRNA, and use the qRT-PCR method to investigate the changes in the levels of IFNβ and chemokines CXCL9 and CXCL10 in human colorectal cancer HT-29 cells and murine colorectal cancer CT26 cells after treatment with ABT-199. The changes in the levels of IFNβ and chemokines CXCL9 and CXCL10 in human colorectal cancer HT-29 cells and murine colorectal cancer CT26 cells at specific concentrations of ABT-199 are shown in Figure 1 B and Figure 1 D.

[0017] Example 2 ABT-199 promotes an increase in the cytoplasmic dsDNA level of human colorectal cancer HT-29 cells. The specific steps are as follows:

[0018] Select human colorectal cancer HT-29 cells and seed them in a confocol chamber at 1.0×10 5Cells / well were cultured overnight in an incubator at 37 °C with 5% CO2. The next day, ABT-199 (5 μM) was added and incubated for 24 h. dsDNA was labeled with a dsDNA dye, and the changes in cytoplasmic dsDNA were examined by confocal immunofluorescence. The specific cumulative effect of ABT-199 on cytoplasmic dsDNA in human colorectal cancer HT-29 cells is shown in Figure 2 , ABT-199 can significantly increase the level of cytoplasmic dsDNA in colorectal cancer cells.

[0019] Example 3 ABT-199 synergistically inhibits tumor growth with anti-PD-L1 monoclonal antibody. The specific steps are as follows:

[0020] Murine colorectal cancer CT26 cells were inoculated into the left axilla of BALB / c mice to establish a tumor-bearing mouse model. When the tumor grew to approximately 100 mm 3 , the mice were randomly divided into 4 groups: a control group, an ABT-199 100 mg / kg group, an anti-PD-L1 monoclonal antibody group, and an ABT-199 100 mg / kg + anti-PD-L1 monoclonal antibody group, with 6 mice in each group. ABT-199 was administered by gavage once a day or an equal volume of normal saline, or anti-PD-L1 monoclonal antibody was intravenously injected twice a week. The tumor volume and mouse body weight were measured daily. On the 11th day after ABT-199 administration, the mice were sacrificed and the tumor tissues were isolated. The specific effect is shown in Figure 3 , ABT-199 can synergistically inhibit tumor growth with anti-PD-L1 monoclonal antibody.

[0021] Example 4 ABT-199 synergistically increases the infiltration and tumor-killing activity of CD3 + and CD8 + T cells. The specific steps are as follows:

[0022] Freshly dissected tumor tissues were selected, and the tissues were lysed into single cells using an automatic tissue processor. The infiltration levels of CD3 + CD45 + T cells and CD8 + CD45 + T cells in the tumor tissues were detected by flow cytometry, and the contents of IFNγ, IL-2, and TNF-α in the tumor tissues were detected by ELISA. The specific effect is shown in Figure 4 , ABT-199 synergistically increases the infiltration and tumor-killing activity of CD3 + and CD8 + T cells.

Claims

1. Use of venetoclax in the preparation of a cGAS-STING agonist, wherein the chemical name of venetoclax is (4-{4-[2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-en-1-yl]-1H-1,2,3-triazol-1-yl}phenyl)[(3R)-1-(4-fluorophenyl)-3-methyl-2-oxopropyl]carbamate, with the molecular formula: C45H50ClF3N7O7S and a molecular weight of 868.

44.

2. The application according to claim 1, characterized in that, The cGAS-STING agonist can activate the cGAS-STING signaling pathway, enhance IFN-I signaling, increase the expression and secretion of chemokines CXCL9 and CXCL10, thereby sensitizing the efficacy of solid tumor immunotherapy, and the solid tumor is caused by significant activation of the tumor cGAS-STING signaling pathway.

3. The application according to claim 2, wherein The solid tumor is colorectal cancer caused by significant activation of the tumor cGAS-STING signaling pathway.

4. The application according to claim 1, characterized in that, The drug is made of venetoclax and pharmaceutically acceptable excipients, and the preparation form of the drug is tablets, capsules, oral solutions, injections or lyophilized powder injections.

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