New application of medicine for intervening HIF2alpha target spot

Abstract

Relates to a novel application of a medicine for intervening an HIF2alpha target spot. According to the invention, the influence of HIF2alpha on Th2 cell differentiation is found, so that the HIF2alpha plays an important role in diseases and senescence caused by type II immune diseases, tumors and Th2 / ILC2 differentiation disorder, and it is verified that the prevention and treatment of the diseases caused by type II immune diseases and Th2 / ILC2 differentiation disorder and the anti-tumor, anti-senescence and anti-senescence-related diseases can be carried out by intervening the HIF2alpha target. A new potential target spot is provided for clinically treating II-type immune diseases, tumors, diseases caused by Th2 / ILC2 differentiation disorder, aging resistance and related diseases, and good application prospects are achieved.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and particularly relates to a new use of a drug for intervening in the HIF2α target. Background Art

[0002] Type II immune diseases are inflammations mediated mainly by Th2 cells, ILC2 cells and related cytokines. At present, type 2 inflammation has been recognized as the immunopathological mechanism of various diseases, which involve multiple systems such as the skin, respiratory, and digestive systems. Such reactions are characterized by sensitivity to a variety of allergens, rapid response, and distribution in a variety of barrier tissues.

[0003] Asthma is a common type II immune disease. Approximately 300 million people worldwide suffer from asthma, which is mainly manifested by the narrowing of the airways in the lungs, the overproduction of mucus, the remodeling of the tracheal wall, and the disorders of the innate and adaptive immune systems, resulting in hyperreactivity of the bronchi to stimuli such as harmless antigens like pollen or cold air. Asthma patients often have symptoms such as shortness of breath, wheezing, and chest tightness. Currently, the conventional clinical treatment methods mainly utilize glucocorticoid drugs and short-acting or long-acting β2-adrenergic agonists. Among them, the role of corticosteroid drugs is mainly to inhibit the inflammatory reaction in the bronchi, while the role of β2-adrenergic agonists is mainly to open the constricted bronchial smooth muscle. However, these treatment methods for asthma can only control the symptoms to a certain extent and cannot cure asthma fundamentally. Therefore, further studying the pathogenesis of asthma and finding new targets for treating asthma are important research topics in this field.

[0004] Th2 cells play a central role in allergic asthma. They are the main source of type 2 cytokines such as interleukin (IL)-4 and IL-13, which can drive the immune and physiological characteristics of asthma, such as immunoglobulin E (IgE)-mediated inflammation, eosinophilia, airway hyperreactivity, and goblet cell hyperplasia. Th2 cells also interact with other T cell subsets such as Th17 cells and Th1 cells and jointly participate in the pathogenesis of asthma. In addition, the role of Th2 cells in asthma is not limited to immune responses. They can also serve as a bridge between inflammation and neuronal regulatory behavioral responses and play an important role in epithelial differentiation and injury repair. Therefore, Th2 cells are key factors in the pathogenesis of allergic asthma, and understanding their regulatory mechanisms and biological characteristics is crucial for developing new treatment strategies.

[0005] Hypoxia-inducible factor 2-alpha (HIF2α) is an important member of the HIF family and plays a role in various physiological and pathological processes. HIF2α is a transcription factor composed of two subunits, HIF-α and ARNT (HIF-β), and is mainly involved in the regulation of erythropoiesis and the vascular system. Under normal oxygen conditions, HIF2α is hydroxylated by prolyl hydroxylase (PHD) and then marked for ubiquitination and degradation by the VHL protein, thus maintaining low-level expression. Under hypoxic conditions, the activity of PHD decreases, HIF2α accumulates, forms a dimer with ARNT, enters the nucleus, binds to hypoxia response elements (HREs), and activates the expression of downstream genes. HIF2α plays a key role in the development of various cancers, especially in clear cell renal cell carcinoma (ccRCC), where its abnormal activity is a key carcinogenic factor. In addition, HIF2α is also involved in regulating processes such as brain development, immune response, and metabolism. However, there is currently no relevant report on the relationship between HIF2α and type II immune diseases. Summary of the Invention

[0006] In view of the problems of the prior art, the present invention provides a new use of a drug for intervening in the HIF2α target.

[0007] Use of a drug for intervening in the HIF2α target in the preparation of a drug for preventing and / or treating type II immune diseases and their complications, anti-tumor, anti-aging or anti-aging related diseases, wherein the drug for intervening in the HIF2α target is selected from drugs that interfere with the transcriptional activity of HIF2α, drugs that inhibit the expression of the HIF2α gene, drugs that knockout or knockdown the HIF2α gene, and drugs that degrade HIF2α.

[0008] Preferably, the drug that interferes with the transcriptional activity of HIF2α is selected from HIF2α inhibitors or drugs that interfere with the formation of a dimer between HIF2α and HIF1β.

[0009] Preferably, the HIF2α inhibitors are selected from inhibitors targeting the HIF2α TAD domain and inhibitors targeting the PAS-B domain.

[0010] Preferably, the HIF2α inhibitors or drugs that interfere with the formation of a dimer between HIF2α and HIF1β include small molecule compounds, polypeptides, and antibodies.

[0011] Preferably, the HIF2α inhibitor is a compound shown in the following formula I or its salt or its isomer:

[0012]

[0013] Wherein,

[0014] R1 is selected from halogen, hydroxyl, C1-C 10 alkyl, C1-C10 haloalkyl, C1-C 10 alkoxy;

[0015] R2 and R3 are each independently selected from halogen, C1-C 10 alkyl, C1-C 10 haloalkyl, C1-C 10 alkoxy;

[0016] n is selected from 0, 1, 2, 3, 4, 5, 6;

[0017] m is selected from 0, 1, 2;

[0018] k is selected from 0, 1, 2, 3.

[0019] Preferably, the HIF2α inhibitor is compound PT-2385, compound PT2977, or a salt or isomer thereof:

[0020]

[0021] Preferably, the drug for inhibiting HIF2α gene expression is selected from circular RNA, antisense nucleic acid, small interfering nucleic acid, nucleic acid aptamer, small activating nucleic acid, microRNA, mRNA drug, ribozyme.

[0022] Preferably, the drug for knocking out or knocking down the HIF2α gene can be the CRISPR / Cas9 gene editing system.

[0023] Preferably, the drug for degrading HIF2α includes proteolysis agents, and preferably the proteolysis agents are selected from PROTAC and molecular glue.

[0024] Preferably, the drug for intervening in the HIF2α target is an antibody-drug conjugate, and the antibody-drug conjugate is formed by connecting an antibody targeting the target cell with an active ingredient intervening in the HIF2α target through a linker; preferably, the target cell is a Th2 cell and / or an ILC2 cell.

[0025] Preferably, the drug for treating type II immune diseases is a drug for treating immune diseases mediated by Th2 cells and / or immune diseases mediated by ILC2 cells.

[0026] Preferably, the drug is a drug that inhibits the differentiation of stem cells into Th2 cells, inhibits the differentiation of stem cell-like Th2 cell subsets into pathogenic Th2 cell subsets, and inhibits the secretion of effector cytokines by Th2 cells.

[0027] Preferably, the type II immune diseases include atopic dermatitis, chronic spontaneous urticaria, prurigo nodularis, bullous pemphigoid, chronic rhinosinusitis with or without nasal polyps, allergic rhinitis, asthma, allergic bronchopulmonary aspergillosis, chronic obstructive pulmonary disease, eosinophilic granulomatosis with polyangiitis, food allergy, eosinophilic esophagitis, allergic conjunctivitis, ulcerative colitis, lichen planus, lymphedema.

[0028] Preferably, the complications of the type II immune diseases include tissue fibrosis and organ fibrosis.

[0029] Preferably, the tumors include prostate cancer, colon cancer, rectal cancer, pancreatic cancer, cervical cancer, gastric cancer, endometrial cancer, uterine cancer, brain cancer, liver cancer, bladder cancer, ovarian cancer, testicular cancer, head cancer, neck cancer, skin cancer, mesothelioma, leukemia, esophageal cancer, breast cancer, muscle cancer, connective tissue cancer, intestinal cancer, lung cancer, adrenal cancer, thyroid cancer, kidney cancer or bone cancer; or glioblastoma, mesothelioma, renal cell carcinoma, gastric cancer, sarcoma, choriocarcinoma, cutaneous basal cell carcinoma, testicular seminoma.

[0030] Preferably, the aging includes nervous system aging, immune aging, tissue aging, cardiovascular system aging, skin aging.

[0031] Preferably, the aging-related diseases include sarcopenia, chronic low-grade inflammation, Alzheimer's disease, Parkinson's disease, neuromyelitis optica, chronic infection, senescent obesity, cardiac fibrosis.

[0032] Use of a drug overexpressing HIF2α in the preparation of a drug for preventing and / or treating diseases caused by dysregulation of Th2 / ILC2 differentiation.

[0033] Preferably, the drugs overexpressing HIF2α include drugs overexpressed by T cell orientation, drugs overexpressed by adeno-associated virus mediation, drugs overexpressed by lentivirus mediation, drugs overexpressed by adenovirus mediation, drugs overexpressed by retrovirus mediation, drugs overexpressed by regulation of enhancer induction, drugs overexpressed by regulation of transcription induction, drugs overexpressed by regulation of transcription element induction, small molecule drug response systems for overexpressing genes, Cre-loxp systems, Flp-frt systems, Dre-rox systems.

[0034] Preferably, the drug overexpressing HIF2α is an antibody-drug conjugate, and the antibody-drug conjugate is formed by connecting an antibody targeting target cells with an active ingredient overexpressing HIF2α through a linker; preferably, the target cells are Th2 cells and / or ILC2 cells.

[0035] Preferably, the diseases caused by Th2 / ILC2 dysdifferentiation include worm infection, obesity, obesity-related complications, primary immune thrombocytopenia, rheumatoid arthritis, systemic lupus erythematosus, rheumatoid arthritis, type I diabetes, multiple sclerosis, chronic thyroiditis, and tumors.

[0036] Preferably, the obesity-related complications include diseases caused by abnormal lipid droplet accumulation.

[0037] Preferably, the diseases caused by abnormal lipid droplet accumulation include fatty liver, insulin resistance, diabetes, cardiovascular and cerebrovascular diseases, hyperlipidemia, chronic kidney diseases, atherosclerosis, coronary heart disease, heart failure, myocardial lipotoxicity, and metabolic syndrome.

[0038] Preferably, the tumors include prostate cancer, colon cancer, rectal cancer, pancreatic cancer, cervical cancer, gastric cancer, endometrial cancer, uterine cancer, brain cancer, liver cancer, bladder cancer, ovarian cancer, testicular cancer, head cancer, neck cancer, skin cancer, mesothelioma, leukemia, esophageal cancer, breast cancer, muscle cancer, connective tissue cancer, intestinal cancer, lung cancer, adrenal cancer, thyroid cancer, kidney cancer, or bone cancer; or glioblastoma, mesothelioma, renal cell carcinoma, gastric cancer, sarcoma, choriocarcinoma, cutaneous basal cell carcinoma, testicular seminoma.

[0039] The present invention for the first time discovers that HIF2α plays an important role in type II immune diseases, tumors, diseases caused by Th2 / ILC2 dysdifferentiation, and aging and related diseases through its influence on Th2 cell differentiation. The present invention also for the first time verifies that the prevention and treatment of type II immune diseases, diseases caused by Th2 / ILC2 dysdifferentiation, anti-tumor, anti-aging, and anti-aging related diseases can be achieved by intervening in the HIF2α target. The present invention provides a new potential target for clinically preventing and treating type II immune diseases, anti-tumor, treating diseases caused by Th2 / ILC2 dysdifferentiation, and anti-aging and related diseases. The present invention also provides new uses of old drugs such as PT-2385 and PT2977 for preventing and treating type II immune diseases, especially for asthma and allergic rhinitis.

[0040] Definition:

[0041] "A drug for preventing and / or treating type II immune diseases and their complications" means that the drug can prevent type II immune diseases alone, treat type II immune diseases alone, prevent the complications of type II immune diseases alone, and treat the complications of type II immune diseases alone, can prevent type II immune diseases and the complications of type II immune diseases simultaneously, can treat type II immune diseases and the complications of type II immune diseases simultaneously, or can treat and prevent type II immune diseases or the complications of type II immune diseases.

[0042] Drugs that inhibit the expression of the HIF2α gene: refer to any substances with the function of inhibiting the expression of the HIF2α gene, including antisense nucleic acids (ASO), small interfering nucleic acids (siRNA), nucleic acid aptamers, small activating nucleic acids, microRNAs, mRNA drugs, ribozymes, etc.

[0043] Drugs that knockout or knockdown the HIF2α gene: refer to any substances with the function of knocking out or knocking down the HIF2α gene. For example, the CRISPR / Cas9 gene editing system.

[0044] Drugs that degrade HIF2α: refer to any substances with the function of degrading the HIF2α protein. For example, proteolysis agents. Proteolysis agents include Protac, molecular glues, etc.

[0045] Drugs that interfere with the transcriptional activity of HIF2α: refer to any substances that can prevent HIF2α from forming the HIF complex or prevent the HIF complex from binding to the gene promoter to interfere with transcription. It includes HIF2α inhibitors that can inhibit the activity of HIF2α, such as PT-2385, PT2977, and any other molecules with the activity of inhibiting HIF2α disclosed in the prior art. It also includes any substances that can interfere with the binding of HIF2α and HIF1β to form a dimer. These substances can be small molecule compounds, polypeptides, antibodies, etc.

[0046] Drugs that overexpress HIF2α: refer to drugs that can make the expression level of HIF2α in cells or organisms higher than the normal state. These substances can be small molecule compounds, polypeptides, enzymes, nucleic acid drugs, and systems composed of them with gene editing or gene expression functions.

[0047] Type II immune diseases: refer to immune diseases mainly mediated by Th2 cells, ILC2 cells and related cytokines.

[0048] Th2 cells: refer to helper T cells type 2.

[0049] ILC2 cells: type 2 innate lymphoid cells.

[0050] Obviously, based on the above content of the present invention, according to the common general knowledge and conventional means in the art, without departing from the above basic technical idea of the present invention, various other forms of modifications, substitutions or changes can be made.

[0051] The above content of the present invention will be further described in detail below through specific embodiments in the form of examples. However, this should not be construed as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 The experimental results showing that HIF2α is highly expressed in Th2 cells compared with other types of CD4+ helper T cells.

[0053] Figure 2 The experimental results showing that overexpression of HIF2α positively regulates the differentiation efficiency of Th2 cells during in vitro differentiation compared with the control group.

[0054] Figure 3 The experimental results of clustering and visualization of helper T cells in asthma patients and sinusitis patients.

[0055] Figure 4 The experimental results showing the heterogeneity of Th2 cells in asthma and sinusitis patients.

[0056] Figure 5 The experimental results showing that the expression of Epas1 is positively correlated with the expression of pathogenic Th2 genes.

[0057] Figure 6 The experimental results showing that deletion of HIF2α inhibits the in vitro differentiation of Th2 cells.

[0058] Figure 7 The experimental results showing that deletion of HIF2α reduces the infiltrating cells in the lungs of asthmatic mice.

[0059] Figure 8 The experimental results showing that deletion of HIF2α causes a decrease in the total number of cells in BALF and the lungs.

[0060] Figure 9 The experimental results showing that deletion of HIF2α causes a decrease in the infiltration of immune cells in the BALF of asthmatic mice.

[0061] Figure 10 The experimental results showing that deletion of HIF2α causes a decrease in the infiltration of immune cells in the lungs of asthmatic mice.

[0062] Figure 11 The experimental results showing that deletion of HIF2α inhibits the differentiation of Th2 cells in the BALF of asthmatic mice.

[0063] Figure 12 The experimental results showing that deletion of HIF2α inhibits the differentiation of Th2 cells in the lungs of asthmatic mice.

[0064] Figure 13 The experimental results showing that deletion of HIF2α inhibits the expression of Th2 cell-related effector molecules.

[0065] Figure 14 The experimental results showing that deletion of HIF2α causes a decrease in the infiltration of eosinophils in the BALF.

[0066] Figure 15 Experimental results showing that HIF2α deficiency leads to a decrease in eosinophil infiltration in the lung.

[0067] Figure 16 Experimental results for defining cell types in asthma based on single-cell transcriptomics.

[0068] Figure 17 Experimental results showing that single-cell analysis reveals that HIF2α deficiency reduces the proportion of inflammation-related cells in asthma.

[0069] Figure 18 Experimental results showing that HIF2α deficiency inhibits the function of Th2 cells.

[0070] Figure 19 Experimental results for defining Th2 cell subsets in asthma based on single-cell transcriptomics.

[0071] Figure 20 Experimental results showing that HIF2α deficiency leads to changes in the proportion of Th2 cell subsets in asthma.

[0072] Figure 21 Experimental results for TCR clonotype analysis of Th2 cell subsets in asthma.

[0073] Figure 22 Experimental results for the lineage differentiation relationship of Th2 cell subsets.

[0074] Figure 23 Experimental results showing that HIF2α deficiency leads to changes in the proportion of stem cell-like Th2 cells and pathogenic Th2 cell subsets in the BALF of asthma.

[0075] Figure 24 Experimental results showing that HIF2α deficiency leads to changes in the proportion of stem cell-like Th2 cells and pathogenic Th2 cell subsets in the lung of asthma.

[0076] Figure 25 Experimental results showing that HIF2α deficiency leads to changes in the proportion of stem cell-like Th2 cells in the peripheral lymphoid organs of asthma.

[0077] Figure 26 Experimental results showing that HIF2α deficiency inhibits the differentiation of stem cell-like Th2 cells into pathogenic Th2 cells in vitro.

[0078] Figure 27 Schematic diagram of the transplantation protocol for stem cell-like Th2 cells.

[0079] Figure 28 Experimental results showing that HIF2α deficiency inhibits the differentiation of stem cell-like Th2 cells into pathogenic Th2 cells in vivo.

[0080] Figure 29Schematic diagram of the adoptive transfer protocol of pathogenic Th2 cells.

[0081] Figure 30 Experimental results showing that the deletion of HIF2α reduces asthma inflammation induced by pathogenic Th2 cells.

[0082] Figure 31 Experimental results showing that the deletion of HIF2α reduces immune cells in the BALF of asthma induced by pathogenic Th2 cells.

[0083] Figure 32 Experimental results showing that the deletion of HIF2α reduces immune cells in the lungs of asthma induced by pathogenic Th2 cells.

[0084] Figure 33 Experimental results showing that the deletion of HIF2α reduces the secretion of effector cytokines of pathogenic Th2 cells in the BALF.

[0085] Figure 34 Experimental results showing that the deletion of HIF2α reduces the secretion of effector cytokines of pathogenic Th2 cells in the lungs.

[0086] Figure 35 Experimental results showing that the deletion of HIF2α reduces eosinophils in the BALF of asthma induced by pathogenic Th2 cells.

[0087] Figure 36 Experimental results showing that the deletion of HIF2α reduces eosinophils in the lungs of asthma induced by pathogenic Th2 cells.

[0088] Figure 37 Experimental results showing that HIF2α is no longer induced in T cells of GATA3 knockout mice after stimulation with IL-4 compared with control mice.

[0089] Figure 38 Experimental results showing that GATA3 can bind to the DNA sequence of the HIF2α promoter as demonstrated by the luciferase reporter system and EMSA experiments.

[0090] Figure 39 Experimental results showing that GATA3 protein can bind to the DNA sequence of the HIF2α promoter as demonstrated by chromatin immunoprecipitation q-PCR and CUT&Tag experiments.

[0091] Figure 40 Experimental results showing that co-overexpression of HIF2α and GATA3 in 293T cells increases the expression levels of both.

[0092] Figure 41 Experimental results showing that the expression level of GATA3 in T cells is reduced by specific knockout of HIF2α in T cells compared with control mice as demonstrated by immunoblotting experiments.

[0093] Figure 42 Experimental results demonstrating that HIF2α protein can bind to the GATA3 promoter DNA sequence in a luciferase reporter system.

[0094] Figure 43 Experimental results demonstrating that HIF2α protein can bind to the GATA3 promoter DNA sequence by chromatin immunoprecipitation q-PCR and CUT&Tag experiments.

[0095] Figure 44 Experimental results demonstrating that HIF2α and GATA3 mainly bind to the promoter region of genes by CUT&Tag experiments.

[0096] Figure 45 Experimental results demonstrating that HIF2α and GATA3 jointly maintain phospholipid metabolism and regulate T cell activation and differentiation.

[0097] Figure 46 Experimental results demonstrating that the deletion of HIF2α leads to a weakened PI3K-AKT signaling pathway intensity when T cells respond to TCR stimulation.

[0098] Figure 47 Experimental results demonstrating that both HIF2α and GATA3 can bind to the Ipmk promoter region to mediate its transcriptional regulation.

[0099] Figure 48 Experimental results demonstrating that the expression of IPMK and the level of AKT phosphorylation are significantly higher during Th2 differentiation compared to other CD4 + helper T cells.

[0100] Figure 49 Experimental results demonstrating that Ipmk is specifically highly expressed in pathogenic Th2 cells and the deletion of HIF2α leads to a decrease in the expression level of Ipmk in pathogenic Th2 cells.

[0101] Figure 50 Experimental results demonstrating that overexpression of IPMK in HIF2α-deficient T cells can rescue the blocked Th2 differentiation.

[0102] Figure 51 Experimental results demonstrating that overexpression of IPMK in HIF2α-deficient T cells can rescue the weakened PI3K-AKT signaling and phosphatidylinositol levels.

[0103] Figure 52 Experimental results demonstrating that PT-2385 inhibits the in vitro differentiation of Th2 cells.

[0104] Figure 53 Experimental results demonstrating that PT-2385 treatment reduces the infiltration of cells in the lungs of asthmatic mice.

[0105] Figure 54 Experimental results of the reduction in the total number of BALF cells after PT-2385 treatment.

[0106] Figure 55 Experimental results of PT-2385 inhibiting the differentiation of Th2 cells in the BALF of asthmatic mice.

[0107] Figure 56 Experimental results of PT-2385 treatment inhibiting the expression of Th2 cell-related effector molecules.

[0108] Figure 57 Experimental results of clustering the BALF cells of asthmatic mice after PT-2385 treatment based on single-cell transcriptomics.

[0109] Figure 58 Experimental results of the change in the proportion of CD4+ T cells in the BALF of asthmatic mice after PT-2385 treatment.

[0110] Figure 59 Experimental results of PT-2385 treatment hindering the differentiation and function of pathogenic Th2 cell subsets in asthma.

[0111] Figure 60 Experimental results of PT2977 inhibiting the in vitro differentiation of Th2 cells.

[0112] Figure 61 Experimental results of PT2977 treatment reducing the infiltrating cells in the lungs of asthmatic mice.

[0113] Figure 62 Experimental results of the reduction in the total number of BALF cells after PT2977 treatment in an OVA-induced asthma model.

[0114] Figure 63 Experimental results of PT2977 treatment inhibiting the expression of Th2 cell-related effector molecules in an OVA-induced asthma model.

[0115] Figure 64 Experimental results of the reduction in the total number of BALF cells after PT2977 treatment in an HDM-induced asthma model.

[0116] Figure 65 Experimental results of PT2977 treatment reducing CD45 + cells and eosinophils in an HDM-induced asthma model.

[0117] Figure 66 Experimental results of PT2977 treatment inhibiting the expression of Th2 cell-related effector molecules in an HDM-induced asthma model. Specific implementation mode

[0118] In the following examples and experimental examples, reagents and raw materials not specifically described are commercially available products.

[0119] Example 1 Research on the Mechanism of Action of HIF2α in Asthma

[0120] I. Experimental Methods

[0121] 1. Explore the function of HIF2α in helper T cells

[0122] (1) Detect the expression of HIF2α gene in Th0, Th1, Th2, Th17, and Treg cells:

[0123] Isolate the above cells, extract total RNA by the conventional phenol-chloroform method and reverse transcribe it into cDNA, and detect the difference in the expression level of HIF2α mRNA in these cells by qPCR.

[0124] (2) Overexpress HIF2α during in vitro differentiation and detect the differentiation efficiency of various CD4+ helper T cells:

[0125] In vitro, use a retroviral vector to coat HIF2α to infect various CD4+ T cells during differentiation, including Th1 (IL-2, IL-12, anti-IL-4), Th2 (IL-2, IL-4, anti-IFNgama), Th17 (IL-6, TGFβ, anti-IL-4, anti-IFNgama), and Treg (IL-2, TGFβ, anti-IL-4, anti-IFNgama). Then, stimulate the cells with PMA, inomycin, monensin, etc. to produce effector cytokines, stain with corresponding flow antibodies, and use a flow cytometer to detect the differentiation efficiency of various CD4+ helper T cells.

[0126] (3) Explore the function of HIF2α in Th2 cells in type II immune diseases:

[0127] Integrate single-cell sequencing data of patients with allergic asthma and patients with chronic rhinosinusitis with nasal polyps, cluster Th2 cells into different subsets by UMAP dimensionality reduction, and analyze the characteristics of each Th2 subset and the functional relationship between HIF2α and Th2 cell subsets.

[0128] (4) Compare the differentiation efficiency of various CD4+ helper T cells in vitro between the control group and mice with T cell-specific deletion of HIF2α:

[0129] Two types of natural CD4-positive T cells in mouse spleen and lymph nodes were obtained by magnetic bead sorting, and in vitro differentiation experiments were carried out under specific cytokine and antibody conditions, Th1 (IL-2, IL-12, anti-IL-4), Th2 (IL-2, IL-4, anti-IFNgama), Th17 (IL-6, TGFβ, anti-IL-4, anti-IFNgama), Treg (IL-2, TGFβ, anti-IL-4, anti-IFNgama). Then, cells were stimulated with PMA, ionomycin, monensin, etc. to produce effector cytokines, stained with corresponding flow antibodies, and the differentiation efficiency of various CD4-positive helper T cells was detected by flow cytometry.

[0130] 2. In vivo study of the function of HIF2α in a mouse asthma model

[0131] (1) Construction of a mouse asthma model:

[0132] An allergen OVA and adjuvant alum were mixed and dissolved in PBS, and mice were intraperitoneally injected on days 0, 7, and 14. From day 28 to day 34, mice were nebulized with 3% OVA.

[0133] (2) After inducing asthma in T cell HIF2α knockout mice, the pulmonary inflammation was detected:

[0134] The lungs were sectioned, and after H&E staining, the pulmonary cell infiltration, bronchial wall morphology, and mucus production were observed.

[0135] (3) After inducing asthma in T cell HIF2α knockout mice, the number and composition of pulmonary infiltrating immune cells were detected:

[0136] The lungs of mice were lavaged with PBS, and the immune cells infiltrating at the alveolar bronchi of the lungs were taken out, the cell number was counted, and the immune cell types were detected by flow cytometry.

[0137] (4) After inducing asthma in T cell HIF2α knockout mice, the expression levels of Th2-related cytokines were detected:

[0138] CD4-positive helper T cells in the bronchoalveolar lavage fluid of the lungs were obtained by magnetic bead sorting, RNA was extracted and reverse transcribed into cDNA, and the expression levels of GATA3, IL-4, IL-5, and IL-13 were detected. The expression levels of IL-4, IL-5, and IL-13 in the supernatant of the bronchoalveolar lavage fluid of the lungs were detected by ELISA.

[0139] 3. The interleukin-4-GATA3 signaling pathway induces the expression of HIF2α

[0140] (1) Detect how HIF2α is induced:

[0141] Interleukin-4 stimulates normal T cells and GATA3-knockout T cells, extracts total cellular RNA and total protein, and detects the induction of HIF2α by real-time fluorescence quantitative PCR and immunoblotting.

[0142] (2) Explore the interaction between GATA3 and HIF2α:

[0143] Transfect a reporter plasmid containing the HIF2α promoter sequence and a plasmid expressing GATA3 or a GATA3 protein lacking DNA-binding function into 293T cells, and detect the relative luciferase activity using a microplate reader.

[0144] Overexpress GATA3 in the Jurkat cell line, extract nuclear proteins, incubate them with the conserved sequence or mutant sequence of GATA3 in the HIF2α promoter region, and use gel electrophoresis mobility shift assay to study the binding of GATA3 to the HIF2α promoter DNA.

[0145] Use CUT&Tag experiment to enrich the regulatory targets of GATA3, and use IGV to explore the binding peaks of GATA3 on the HIF2α gene.

[0146] 4. Single-cell sequencing reveals the molecular mechanism after HIF2α deletion in T cells in asthma

[0147] (1) Identify cell clusters by gene expression levels:

[0148] Determine the cell clusters to which a single cell belongs based on its gene expression level, and compare the cell cluster ratios and gene expression profiles between mice with HIF2α knockout in T cells and control mice.

[0149] (2) Detect the molecular mechanism effects of HIF2α knockout in T cells:

[0150] Perform gene pathway enrichment analysis (GSEA) through the gene expression matrix of Th2 cells to determine the cell functions affected after HIF2α knockout in T cells.

[0151] 5. Explore the genes regulated by HIF2α and GATA3 and the functions enriched by these genes

[0152] (1) Use CUT&Tag to study the genes regulated by HIF2α and GATA3:

[0153] Sort stem-like Th2 and pathogenic Th2, incubate them with antibodies against HIF2α and GATA3 respectively, use transposase to cut the chromatin regions bound by HIF2α and GATA3, perform DNA sequencing after digesting the proteins, and then compare the sequencing results to the reference genome to obtain the genes regulated by HIF2α and GATA3.

[0154] (2) Explore the functions of the genes regulated by HIF2α and GATA3:

[0155] Take the intersection of the genes regulated by HIF2α and GATA3, detect whether there is a synergistic effect between them, perform pathway analysis on the genes regulated by them alone and the genes regulated synergistically, and determine the functions exerted by HIF2α and GATA3 alone and the functions exerted synergistically.

[0156] 6. Explore the mechanism by which HIF2α regulates GATA3

[0157] (1) Use the sequencing data of CUT&Tag to explore the peaks enriched by HIF2α on the GATA3 gene:

[0158] Use IGV to explore the binding peaks of HIF2α on the GATA3 gene.

[0159] (2) Construct expression vectors containing mouse HIF2α and GATA3 respectively, and explore whether HIF2α affects the expression of GATA3:

[0160] Transfect the overexpression plasmids of HIF2α and GATA3 by the calcium phosphate method to express them separately and co-express them in 293T cells. Collect cell samples and extract proteins, and explore the expression of HIF2α and GATA3 by immunoblotting.

[0161] (3) Detect the expression of GATA3 between control mice and mice with HIF2α knocked out in T cells:

[0162] Use magnetic beads to sort CD4-positive T cells from control mice and mice with HIF2α knocked out in T cells. After stimulation with interleukin-4, extract proteins and explore the expression of GATA3 between the two groups of cells by immunoblotting.

[0163] (4) Detect the binding of HIF2α to the GATA3 promoter:

[0164] Transfect a reporter plasmid containing the GATA3 promoter sequence and a plasmid expressing HIF2α or a protein of HIF2α lacking the binding domain in 293T cells, and use an enzyme-linked immunosorbent assay (ELISA) reader to detect the relative luciferase activity.

[0165] Sort stem-like Th2 and pathogenic Th2, incubate with anti-HIF2α antibody, digest the protein to obtain DNA, and detect the enrichment of the GATA3 promoter by real-time fluorescence quantitative PCR.

[0166] 7. Explore the co-regulation of IPMK by HIF2α and GATA3

[0167] (1) Detect the effect of HIF2α deficiency on the PI3K-AKT signal during T cell activation:

[0168] In vitro, isolate WT and CD4Cre + Epas1 f / f mice CD4 + T cells are first stimulated with TCR and IL-4 for 24 hours to upregulate HIF2α expression, and then the cells are restimulated with TCR for 0, 5, and 10 minutes. Total cell protein is extracted, and the phosphorylation levels of key molecules in the PI3K-AKT signaling pathway such as AKT, S6K, and FOXO1 are detected by WB.

[0169] (2) Verify the regulation of IPMK by HIF2α and GATA3 in single-cell sequencing data and CUT&Tag sequencing data:

[0170] Use IGV to verify the binding peaks of HIF2α and GATA3 on the Ipmk promoter. Verify the expression levels of Ipmk in each Th2 subset in the presence and absence of HIF2α in single-cell sequencing data.

[0171] (3) Verify the expression level of IPMK in Th2 cells:

[0172] In vitro, induce the differentiation of CD4 + T cells. During the in vitro differentiation process, add the following to each culture medium: Th1 (IL-2, IL-12, anti-IL-4), Th2 (IL-2, IL-4, anti-IFNgama), Th17 (IL-6, TGFβ, anti-IL-4, anti-IFNgama), Treg (IL-2, TGFβ, anti-IL-4, anti-IFNgama). After 5 days of culture, collect the cells and perform TCR stimulation for 0, 5, and 10 minutes. Stain with IPMK and pAKT flow antibodies, and use flow cytometry to detect the expression of IPMK and the phosphorylation level of AKT in various CD4 + T cells.

[0173] From WT and CD4Cre + Epas1 f / fSorting of stem-like Th2, pathogenic Th2, and Ikzf2 in murine dLNs + Th2 cells. Cellular RNA was extracted and reverse transcribed into cDNA, and qPCR was used to detect the expression levels of Ipmk in different Th2 cell subsets upon HIF2α deletion.

[0174] (4) Investigate whether overexpression of IPMK can rescue the impaired Th2 differentiation caused by HIF2α deletion:

[0175] In vitro, various CD4-positive T cells in the process of differentiation, WT or HIF2α-deficient, were infected with IPMK-coated retroviral vectors, Th1 (IL-2, IL-12, anti-IL-4), Th2 (IL-2, IL-4, anti-IFNgama), Th17 (IL-6, TGFβ, anti-IL-4, anti-IFNgama), Treg (IL-2, TGFβ, anti-IL-4, anti-IFNgama), and then the cells were stimulated with PMA, inomycin, monensin, etc. to produce effector cytokines, stained with corresponding flow antibodies, and the differentiation efficiencies of various CD4-positive helper T cells and Th2 subset cells were detected using a flow cytometer.

[0176] (5) Investigate whether overexpression of IPMK can rescue the weakened PI3K-AKT signaling caused by HIF2α deletion:

[0177] In vitro, CD4 + T cells, WT or HIF2α-deficient, were infected with IPMK-coated retroviral vectors, the cells were stimulated with TCR for 10 minutes, total cellular proteins were extracted, and the phosphorylation levels of AKT, S6K, and FOXO1 were detected using WB.

[0178] II. Experimental Results

[0179] 1. HIF2α is highly expressed in pathogenic Th2 cells

[0180] Compared with Th0, Th1, Th17, and Treg cells, the expression level of the HIF2α gene in Th2 cells is relatively the highest (as Figure 1 shown). Overexpression of HIF2α during in vitro differentiation can promote the differentiation of Th2 cells, but has no effect on the differentiation of other helper T cells (as Figure 2 shown). The expression level of the HIF2α gene in Th2 cells is relatively the highest in type II immune response diseases (as Figure 3 shown). Th2 cells are heterogeneous in type II immune response diseases, and the expression level of the HIF2α gene is the highest in pathogenic Th2 cells (as Figure 4as shown). The expression level of the HIF2α gene was positively correlated with the enrichment score of the pathogenic gene set (as Figure 5 shown).

[0181] The above experimental results showed that the HIF2α gene had the highest expression level in pathogenic Th2 cells.

[0182] 2. HIF2α deficiency can inhibit the differentiation of Th2 cells

[0183] To further verify the effect of the HIF2α gene on Th2 cells, the differentiation efficiency of various CD4-positive helper T cells after knocking out HIF2α in T cells was investigated. The results showed that the in vitro differentiation of Th2 cells was inhibited, while the in vitro differentiation of other helper T cells was not affected (as Figure 6 shown).

[0184] The above experimental results showed that the HIF2α gene played a positive regulatory role in the differentiation of Th2 cells. When the function of Th2 cells was disordered, it would lead to the occurrence of the autoimmune disease asthma. Therefore, the effect of HIF2α deficiency on asthma was further investigated below.

[0185] 3. HIF2α deficiency can effectively relieve the inflammatory symptoms in a mouse asthma model

[0186] The in vivo experimental results showed that the number of immune cells infiltrating the lungs of mice with HIF2α-deficient T cells after inducing asthma was lower than that of the control group mice, and the degree of tracheal wall thickening was reduced (as Figures 7 to 10 shown). The relative proportion and absolute number of Th2 cells and eosinophils infiltrating the lungs of mice with HIF2α-deficient T cells after inducing asthma were lower than those of the control group mice (as Figures 11 to 12 shown). The expression levels of the Th2 transcription factor GATA3, cytokines IL-4, IL-5, and IL-13 in CD4-positive T cells in the lungs of mice with HIF2α-deficient T cells after inducing asthma were lower than those of the control group mice, and the concentrations of IL-4, IL-5, and IL-13 in the supernatant of bronchoalveolar lavage fluid were lower than those of the control group mice (as Figure 13 shown). The relative proportion and absolute number of Th2 cells and eosinophils infiltrating the lungs of mice with HIF2α-deficient T cells after inducing asthma were lower than those of the control group mice (as Figures 14 to 15 shown).

[0187] The above experimental results showed that HIF2α deficiency could reduce the infiltration of immune cells in the lungs of asthmatic mice and alleviate the asthmatic inflammatory symptoms.

[0188] 4. HIF2α promotes the differentiation of stem cell-like Th2 cells into pathogenic Th2 cells

[0189] Single cells revealed that the proportions of Th2 cells and eosinophils in asthmatic mice lacking HIF2α in T cells were lower than those in control mice (as Figures 16 to 17 shown). Deletion of HIF2α inhibited the function of Th2 cells (as Figure 18 shown). After deletion of HIF2α in T cells, the proportion of pathogenic Th2 cells was lower than that in control mice (as Figures 19 to 20 shown). Deletion of HIF2α hindered the differentiation of stem cell-like Th2 cells into pathogenic Th2 cells (as Figure 21 to attached Figure 28 ).

[0190] The above experimental results indicate that HIF2α promotes the differentiation of stem cell-like Th2 cells into pathogenic Th2 cells.

[0191] 5. HIF2α regulates the function of pathogenic Th2 cells

[0192] Compared with the control group, the ability of pathogenic Th2 cells lacking HIF2α to induce pulmonary inflammation was reduced (as Figures 29 to 32 shown). Compared with the control group, the ability of pathogenic Th2 cells lacking HIF2α to secrete effector cytokines was reduced (as Figures 33 to 34 shown). Compared with the control group, the ability of pathogenic Th2 cells lacking HIF2α to recruit eosinophils was reduced (as Figures 35 to 36 shown).

[0193] The above experimental results indicate that the ability of pathogenic Th2 cells lacking HIF2α to induce pulmonary inflammation, secrete effector cytokines, and recruit eosinophils is reduced, which helps to alleviate the inflammatory symptoms of asthma.

[0194] 6. Interleukin-4-GATA3 signaling pathway induces the expression of HIF2α gene

[0195] Compared with control mice, after knocking out GATA3, HIF2α in T cells could no longer be induced by interleukin-4 (as Figure 37 shown). GATA3 can bind to the promoter region of HIF2α and transcriptionally regulate the expression of HIF2α (as Figures 38 to 39 ).

[0196] The experimental results indicate that HIF2α is induced by interleukin-4 and GATA3 pathways during Th2 differentiation.

[0197] 7. HIF2α positively regulates Th2 differentiation by transcriptionally regulating the expression of GATA3

[0198] Overexpression of both HIF2α and GATA3 in 293T cells would increase their expression levels (as Figure 40)。Compared with the control group of mice, after T cell-specific knockout of HIF2α, the expression level of GATA3 in T cells decreased (as Figure 41 )。The results of the CUT&Tag experiment showed that the luciferase reporter system proved that HIF2α could bind to the GATA3 gene (as Figures 42 to 43 )。

[0199] 8. The co-regulatory function of HIF2α and GATA3

[0200] The CUT&Tag experiment proved that HIF2α and GATA3 mainly bind to the promoter region of the gene, transcriptionally regulate the expression of the gene, HIF2α and GATA3 have a co-regulatory function, and they co-regulate genes related to inflammation and immune response (Il1r2, Il10, Ifngr1, Ipmk, Peli1, Socs3, Fos, Jun, Il2ra, Ccr1, Tnfrsf8, Il10ra, Flt3l), genes related to T cell development and differentiation (Aff4, Gata3, Foxp1, Maf, Grap2, Tmem109, Rps6ka1), genes related to cell metabolism (Capn2, Npc2, Hmgcs1, Anxa11, Dgat1, Pmm1, Srebf2, Insig1, Cyp11a1, Fabp5, Fdps, Pparg), genes related to cell signaling and transduction (Rgs1, Adarb1, Gm2a, 2610507B11Rik, Vmp1, Ormld3, Rps6ka5, Dusp1, Rasgrp1, Tasp1, Ppp3ca, Dusp16, Crebl2, Itpr2, Dctn6), and some genes with less research reports (2610507B11Rik, AI467606, 1700061F12Rik, B4galnt4, Smco4, Cnot6l, Hip1, Nt5c3, Ttc39c). Through pathway enrichment analysis, it was found that these co-regulated genes were mainly enriched in phospholipid metabolism, T cell activation and differentiation, and signal transduction pathways (as Figures 44 to 45 )。

[0201] The above experimental results showed that HIF2α and GATA3 co-regulate phospholipid metabolism in Th2 cells.

[0202] 9. HIF2α and GATA3 mediate the differentiation of pathogenic Th2 cells by co-regulating IPMK expression

[0203] The deletion of HIF2α inhibited the PI3K-AKT signaling pathway, and the phosphorylation levels of AKT, S6K, and FOXO1 induced by TCR stimulation decreased significantly after the deletion of HIF2α (as Figure 46)。CUT&Tag sequencing analysis showed that HIF2α and GATA3 co - bind to the promoter region of Ipmk in pathogenic Th2 cells (as Figure 47 )。During Th2 differentiation, the expression level of IPMK and the phosphorylation level of AKT are significantly higher than those in other CD4 + helper T cells (as Figure 48 )。Ipmk is specifically highly expressed in pathogenic Th2 cells, and the deletion of HIF2α leads to a decrease in the expression level of Ipmk in pathogenic Th2 cells (as Figure 49 )。Overexpression of IPMK in HIF2α - deficient T cells can rescue the blocked Th2 differentiation and weakened PI3K - AKT signaling and phosphatidylinositol levels (as Figures 50 to 51 )。The above experimental results indicate that HIF2α and GATA3 jointly regulate the IPMK and PI3K - AKT signaling pathways to mediate the differentiation of pathogenic Th2 cells.

[0204] In summary, through in vitro and in vivo experiments in this example, it was found that the HIF2α gene has the highest expression level in pathogenic Th2 cells and plays a positive regulatory role in the differentiation of Th2 cells. After knocking out the HIF2α gene, the infiltration of immune cells in the lungs of asthmatic mice decreased, and the asthmatic inflammatory symptoms were alleviated. This indicates that the HIF2α gene is a potential target for the prevention and / or treatment of asthma.

[0205] Example 2 The HIF2α - specific inhibitor PT - 2385 can alleviate asthmatic inflammatory symptoms

[0206] I. Experimental methods

[0207] The therapeutic effect of the HIF2α - specific inhibitor PT - 2385 on asthma was explored according to the following steps:

[0208] (1) Add the inhibitor PT - 2385 during in vitro differentiation to detect the differentiation efficiency of various CD4 - positive helper T cells:

[0209] Add PT - 2385 to each culture medium during in vitro differentiation for Th1 (IL - 2, IL - 12, anti - IL - 4), Th2 (IL - 2, IL - 4, anti - IFNγ), Th17 (IL - 6, TGFβ, anti - IL - 4, anti - IFNγ), Treg (IL - 2, TGFβ, anti - IL - 4, anti - IFNγ), and then stimulate the cells with PMA, ionomycin, monensin, etc. to produce effector cytokines, stain with corresponding flow antibodies, and use a flow cytometer to detect the differentiation efficiency of various CD4 - positive helper T cells.

[0210] (2) Construction of mouse asthma model and treatment with PT-2385:

[0211] The allergen OVA and adjuvant alum were mixed and dissolved in PBS, and mice were intraperitoneally injected on days 0, 7, and 14. From day 28 to day 34, mice were nebulized with 3% OVA, and before each nebulization, mice were given the drug PT-2385 by gavage at a dose of 10 mg / kg based on the body weight of the mice.

[0212] (3) Detection of pulmonary inflammation in mice after treatment with PT-2385:

[0213] Lung tissue sections were made, and after H&E staining, the infiltration of lung cells, the morphology of the bronchial wall, and mucus production were observed.

[0214] (4) Detection of the number and composition of infiltrating immune cells in the lungs of mice after treatment with PT-2385:

[0215] The lungs of mice were lavaged with PBS, and the infiltrating immune cells at the alveolar bronchi of the lungs were taken out, the cell number was counted, and the types of immune cells were detected by flow cytometry.

[0216] (5) Detection of the expression levels of Th2-related cytokines in mice after treatment with PT-2385:

[0217] ELISA was used to detect the expression levels of IL-4, IL-5, and IL-13 in the supernatant of the bronchoalveolar lavage fluid of the lungs.

[0218] II. Experimental Results

[0219] The experimental results showed that adding PT-2385 during in vitro differentiation could inhibit the differentiation of Th2 cells, but had no effect on the differentiation of other helper T cells (as Figure 52 shown).

[0220] After inducing asthma in mice treated with PT-2385, the number of infiltrating immune cells in the lungs decreased compared with that in the control group mice, and the degree of thickening of the tracheal wall decreased (as Figures 53 to 54 ). After inducing asthma in mice treated with PT-2385, the relative proportion and absolute number of Th2 cells infiltrating the lungs decreased compared with those in the control group mice (as Figure 55 ). After inducing asthma in mice treated with PT-2385, the concentrations of IL-4, IL-5, and IL-13 in the supernatant of the bronchoalveolar lavage fluid decreased compared with those in the control group mice (as Figure 56 ). Single-cell analysis revealed that the proportion of Th2 cells in asthmatic mice after treatment with PT-2385 decreased compared with that in the control group mice (as Figures 57 to 58 ). Single-cell analysis revealed that treatment with PT-2385 would limit the differentiation and function of pathogenic Th2 cells in asthmatic mice (as Figure 59 ).

[0221] In summary, this example demonstrated that the HIF2α specific inhibitor PT-2385 has a therapeutic effect on asthma.

[0222] Example 3 The HIF2α specific inhibitor PT2977 (Belzutifan) can alleviate asthma inflammatory symptoms

[0223] I. Experimental methods

[0224] Explore the therapeutic effect of the HIF2α specific inhibitor PT2977 on asthma according to the following steps:

[0225] (1) Add the inhibitor PT2977 during in vitro differentiation and detect the differentiation efficiency of helper type 2 T cells:

[0226] Add PT2977 to the culture medium during in vitro differentiation. After Th2 (IL-2, IL-4, anti-IFNgama) differentiation is completed, part of the cells are stimulated with PMA, inomycin, monensin, etc. to produce effector cytokines, and are stained with corresponding flow antibodies. Part of the cells are used to detect the differentiation of pathogenic Th2 cells, and the differentiation efficiency of helper type 2 T cells is detected using a flow cytometer.

[0227] (2) Construction of a mouse OVA-induced asthma model and treatment with PT2977:

[0228] Mix the allergen OVA and adjuvant alum and dissolve them in PBS. Inject the mice intraperitoneally on days 0, 7, and 14. Nebulize the mice with 3% OVA from day 28 to day 34, and administer the drug PT2977 to the mice by gavage at a dose of 10 mg / kg of body weight every day before nebulization.

[0229] (3) Construction of a mouse HDM-induced asthma model and treatment with PT2977:

[0230] Dissolve HDM in PBS at a concentration of 0.5 μg / μl. Administer nasal drops to the mice at a dose of 25 μg / animal on days 0 to 2 and days 14 to 17. Starting from day 14, administer the drug PT2977 to the mice by gavage at a dose of 10 mg / kg of body weight every day before nasal drops.

[0231] (4) After treatment with PT2977, detect the pulmonary inflammation in mice:

[0232] Make tissue sections of the lungs, observe the pulmonary cell infiltration, bronchial wall morphology, and mucus production after H&E staining.

[0233] (5) After PT2977 treatment, the number and composition of infiltrating immune cells in the lungs of mice were detected:

[0234] The lungs of mice were lavaged with PBS, and the infiltrating immune cells at the alveolar bronchi of the lungs were taken out. The cell number was counted, and the types of immune cells were detected by flow cytometry.

[0235] II. Experimental Results

[0236] The experimental results showed that adding PT2977 during the in vitro differentiation process could inhibit the differentiation of Th2 cells and limit the differentiation of pathogenic Th2 cells (as Figure 60 shown).

[0237] After inducing asthma in mice with OVA after treatment with PT2977, the number of infiltrating immune cells in the lungs decreased compared with that in the control group of mice, and the degree of tracheal wall thickening decreased (as Figures 61 to 62 ). After inducing asthma in mice with PT2977 treatment, the relative proportion and absolute number of Th2 cells infiltrating in the lungs decreased compared with those in the control group of mice (as Figure 63 ).

[0238] After inducing asthma in mice with HDM after treatment with PT2977, the number of infiltrating immune cells in the lungs decreased compared with that in the control group of mice (as Figure 64 ), and the relative proportion and absolute number of CD45 + cells and eosinophils decreased compared with those in the control group of mice (as Figure 65 ). After inducing asthma in mice with PT2977 treatment, the relative proportion and absolute number of Th2 cells infiltrating in the lungs decreased compared with those in the control group of mice (as Figure 66 ).

[0239] In summary, this example proved that the HIF2α specific inhibitor PT2977 has a therapeutic effect on asthma.

[0240] In short, through single-cell sequencing data, the present invention found that Th2 cells are heterogeneous in patients with allergic asthma and patients with chronic rhinosinusitis with nasal polyps, and the transcription factor HIF2α is highly expressed in pathogenic Th2 cells. When interleukin-4 stimulates T cells, it can strongly induce the expression of the HIF2α gene (manifested as an increase in the mRNA level). Specific knockout of HIF2α in T cells can inhibit the differentiation of natural CD4-positive T cells into Th2 cells in vitro, while having no effect on the differentiation of other helper T cells.

[0241] Based on the above experimental results, it can be known that targeting HIF2α can treat and prevent diseases mediated mainly by Th2 cells, ILC2 cells and related cytokines, including but not limited to: type II immune diseases, tumors, prevention and treatment of diseases caused by Th2 / ILC2 dysregulation, and anti-aging and related diseases.

[0242] To further investigate the effect of HIF2α on asthma, a mouse asthma model was constructed to observe the physiological function of HIF2α in vivo. Compared with the control group of mice, the mice with T cell-specific knockout of HIF2α had fewer infiltrating immune cells in the lungs, a reduced degree of tracheal wall thickening, and a reduced overall inflammatory response.

[0243] To further verify whether HIF2α can be targeted for the treatment of asthma, the specific inhibitors of HIF2α, PT-2385 and PT2977, were used to treat asthmatic mice, and it was found that the inflammatory symptoms of the mice treated with the drugs were alleviated.

[0244] Thus, the present invention proves that type II immune diseases, prevention and treatment of diseases caused by Th2 / ILC2 dysregulation, anti-tumor, anti-aging and anti-aging related diseases can be achieved by intervening in the HIF2α target.

Claims

1. Use of a drug that intervenes in the HIF2α target in the preparation of a drug for preventing and / or treating type II immune diseases and their complications, anti-tumor, anti-aging or anti-aging related diseases, characterized in that: The drugs that intervene in the HIF2α target are selected from drugs that interfere with the transcriptional activity of HIF2α, drugs that inhibit the gene expression of HIF2α, drugs that knockout or knockdown the HIF2α gene, and drugs that degrade HIF2α.

2. The use according to claim 1, characterized in that: The drugs that interfere with the transcriptional activity of HIF2α are selected from HIF2α inhibitors or drugs that interfere with the formation of dimers between HIF2α and HIF1β.

3. The use according to claim 2, wherein: The HIF2α inhibitors are selected from inhibitors targeting the HIF2α TAD domain and inhibitors targeting the HIF2α PAS-B domain.

4. The use according to claim 2 or 3, characterized in that: The HIF2α inhibitors or drugs that interfere with the formation of dimers between HIF2α and HIF1β are selected from small molecule compounds, polypeptides, and antibodies.

5. The use according to claim 4, characterized in that: The HIF2α inhibitor is a compound represented by the following formula I or its salt or its isomer: Wherein, R1 is selected from halogen, hydroxy, C1-C 10 alkyl, C1-C 10 haloalkyl, C1-C 10 alkoxy; R2 and R3 are each independently selected from halogen, C1-C 10 alkyl, C1-C 10 haloalkyl, C1-C 10 alkoxy; n is selected from 0, 1, 2, 3, 4, 5, 6; m is selected from 0, 1, 2; k is selected from 0, 1, 2, 3.

6. The use according to claim 4, characterized in that: The HIF2α inhibitor is compound PT-2385, compound PT2977, or its salt or its isomer:

7. The use according to claim 1, characterized in that: The drugs that inhibit the gene expression of HIF2α are selected from circular RNAs, antisense nucleic acids, small interfering nucleic acids, nucleic acid aptamers, small activating nucleic acids, microRNAs, mRNA drugs, and ribozymes.

8. The use according to claim 1, characterized in that: The drugs that knockout or knockdown the HIF2α gene can be the CRISPR / Cas9 gene editing system.

9. The use according to claim 1, characterized in that: The drugs that degrade HIF2α include proteolysis agents, and preferably the proteolysis agents are selected from PROTACs and molecular glues.

10. The use according to claim 1, wherein: The drugs that intervene in the HIF2α target are antibody-drug conjugates, and the antibody-drug conjugates are formed by linking an antibody targeting target cells with an active ingredient that intervenes in the HIF2α target through a linker; preferably, the target cells are Th2 cells and / or ILC2 cells.

11. The use according to any one of claims 1 to 10, characterized in that: The drugs for treating type II immune diseases are drugs for treating immune diseases mediated by Th2 cells and / or immune diseases mediated by ILC2 cells.

12. According to the use described in claim 11, it is characterized in that: The drugs are drugs that inhibit the differentiation of stem cells into Th2 cells, inhibit the differentiation of stem cell-like Th2 cell subsets into pathogenic Th2 cell subsets, and inhibit the secretion of effector cytokines by Th2 cells.

13. According to the use described in any one of claims 1 to 10, it is characterized in that: The type II immune diseases include atopic dermatitis, chronic spontaneous urticaria, prurigo nodularis, bullous pemphigoid, chronic rhinosinusitis with or without nasal polyps, allergic rhinitis, asthma, allergic bronchopulmonary aspergillosis, chronic obstructive pulmonary disease, eosinophilic granulomatosis with polyangiitis, food allergy, eosinophilic esophagitis, allergic conjunctivitis, ulcerative colitis, lichen planus, and lymphedema.

14. The use according to any one of claims 1 to 10, characterized in that: The complications of the type II immune diseases include tissue fibrosis and organ fibrosis.

15. The use according to any one of claims 1 to 10, characterized in that: The tumors include prostate cancer, colon cancer, rectal cancer, pancreatic cancer, cervical cancer, gastric cancer, endometrial cancer, uterine cancer, brain cancer, liver cancer, bladder cancer, ovarian cancer, testicular cancer, head cancer, neck cancer, skin cancer, mesothelioma, leukemia, esophageal cancer, breast cancer, muscle cancer, connective tissue cancer, intestinal cancer, lung cancer, adrenal cancer, thyroid cancer, kidney cancer or bone cancer; or glioblastoma, mesothelioma, renal cell carcinoma, gastric cancer, sarcoma, choriocarcinoma, basal cell carcinoma of the skin, testicular seminoma.

16. The use according to any one of claims 1 to 10, characterized in that: The aging includes nervous system aging, immune aging, tissue aging, cardiovascular system aging, skin aging.

17. The use according to any one of claims 1 to 10, characterized in that: The aging-related diseases include sarcopenia, chronic low-grade inflammation, Alzheimer's disease, Parkinson's disease, neuromyelitis optica, chronic infection, senescent obesity, cardiac fibrosis.

18. Use of a drug overexpressing HIF2α in the preparation of a drug for preventing and / or treating diseases caused by Th2 / ILC2 differentiation disorder.

19. The use according to claim 18, wherein: The drugs overexpressing HIF2α include drugs overexpressed by T cell orientation, drugs overexpressed mediated by adeno-associated virus, drugs overexpressed mediated by lentivirus, drugs overexpressed mediated by adenovirus, drugs overexpressed mediated by retrovirus, drugs overexpressed induced by regulatory enhancer, drugs overexpressed induced by regulatory transcription, drugs overexpressed induced by regulatory transcription element, small molecule drug response system for overexpressing genes, Cre-loxp system, Flp-frt system, Dre-rox system.

20. The use according to claim 18, characterized in that: The drug overexpressing HIF2α is an antibody-drug conjugate, and the antibody-drug conjugate is formed by connecting an antibody targeting target cells with an active ingredient overexpressing HIF2α through a linker; preferably, the target cells are Th2 cells and / or ILC2 cells.

21. The use according to claim 18, characterized in that: The diseases caused by Th2 / ILC2 differentiation disorder include worm infection, obesity, obesity-related complications, primary immune thrombocytopenia, rheumatoid arthritis, systemic lupus erythematosus, rheumatoid arthritis, type I diabetes, multiple sclerosis, chronic thyroiditis, tumors.

22. The use according to claim 21, wherein: The obesity-related complications include diseases caused by abnormal lipid droplet accumulation.

23. The use according to claim 22, characterized in that: The diseases caused by abnormal lipid droplet accumulation include fatty liver, insulin resistance, diabetes, cardiovascular and cerebrovascular diseases, hyperlipidemia, chronic kidney disease, atherosclerosis, coronary heart disease, heart failure, myocardial lipotoxicity, metabolic syndrome.

24. The use according to claim 21, wherein: The tumors include prostate cancer, colon cancer, rectal cancer, pancreatic cancer, cervical cancer, gastric cancer, endometrial cancer, uterine cancer, brain cancer, liver cancer, bladder cancer, ovarian cancer, testicular cancer, head cancer, neck cancer, skin cancer, mesothelioma, leukemia, esophageal cancer, breast cancer, muscle cancer, connective tissue cancer, intestinal cancer, lung cancer, adrenal cancer, thyroid cancer, kidney cancer or bone cancer; or glioblastoma, mesothelioma, renal cell carcinoma, gastric cancer, sarcoma, choriocarcinoma, basal cell carcinoma of the skin, testicular seminoma.

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