PHB2 as tumor immunotherapy target and use thereof in drug development

Abstract

Disclosed is the use of a PHB2 gene or a PHB2 protein as a new immune checkpoint. Targeting PHB2 can inhibit mitochondrial fusion, thereby increasing mitochondrial fragmentation, elevating mitochondrial metabolic levels, and enhancing the anti-tumor function of cells in vivo and in vitro. Moreover, targeting PHB2 can reduce the expression of PD-1, and when used in combination with a CTLA-4 monoclonal antibody, can enhance anti-tumor effects. On this basis, further disclosed is the use of a drug having an inhibitory effect on a PHB2 gene or a PHB2 protein in the preparation of a PD-1 inhibitor, or the use of same in combination with a PD-1 monoclonal antibody in the preparation of a therapeutic product or a preventive product for treating or preventing tumors. Further disclosed is the use of a drug having an inhibitory effect on a PHB2 gene or a PHB2 protein in combination with a CTLA-4 monoclonal antibody in the preparation of a therapeutic product or a preventive product for treating or preventing tumors. Further disclosed is the use of a drug having an inhibitory effect on a PHB2 gene or a PHB2 protein alone, in combination with a PD-1 monoclonal antibody or in combination with a CTLA-4 monoclonal antibody in immunotherapy, especially tumor immunotherapy. It has been found in experiments that tumors in PHB2+ / - tumor-bearing mice are significantly reduced compared with those in the control. It has been further found that targeting mitochondrial PHB2 can increase mitochondrial fragmentation by means of inhibiting mitochondrial fusion, so that the mitochondrial metabolism is enhanced; moreover, after PHB2 knockout, PD-1 expression is reduced, and when used in combination with a CTLA-4 monoclonal antibody, can enhance anti-tumor effects.

Description

PHB2 as a target for tumor immunotherapy and its use in drug development Technical Field

[0001] The present application relates to PHB2 as a target for tumor immunotherapy and its use, and belongs to the field of biochemical technology. Background Art

[0002] Malignant tumors pose a serious threat to human health. The latest data from the WHO shows that in 2020, there were 19.29 million new cases of cancer and 9.96 million deaths worldwide. It is estimated that by 2040, the number of new cases of cancer worldwide will reach 28.4 million. my country is a severely affected area, with the predicted data of new cancer cases and deaths ranking first in the world. At present, the main treatments for tumors include surgery, radiotherapy, chemotherapy and targeted therapy, but these therapies cannot benefit the long-term survival of most patients with advanced solid tumors. With the continuous development and cross-penetration of related disciplines such as oncology and immunology, tumor immunotherapy is regarded as the most promising new therapy to conquer advanced solid tumors. T cell immunotherapy is the most promising treatment technology in the current field of tumor treatment, among which immune checkpoint inhibitors (ICB) have shown good efficacy in the treatment of tumors. Currently, representative immune checkpoint blockade therapies include anti-programmed death receptor 1 (PD-1), anti-programmed death ligand 1 (PD-L1), and anti-cytotoxic T lymphocyte-associated protein 4 (CTLA-4) antibody treatments. In 2018, my country launched immune checkpoint drugs targeting PD-1 / PD-L1, which brought long-term survival benefits to patients with various solid tumors such as melanoma and non-small cell lung cancer.

[0003] However, tumor immunotherapy still faces significant challenges. While an effective and important cancer treatment, its clinical application remains subject to numerous uncertainties. For example, the response rate for PD1 / PD-L1 therapy is only approximately 20%, with partial responders still accounting for the majority. Furthermore, one-third of responding patients develop acquired resistance after treatment, ultimately leading to disease relapse. Consequently, the diverse responses to tumor immunotherapy and the wide inter-individual variability in efficacy present numerous challenges for anti-tumor immunotherapy.

[0004] Regulation of mitochondrial energy metabolism is a crucial mechanism of cell development. Under normal physiological conditions, the degree of mitochondrial fragmentation depends on a dynamic balance between fission and fusion. Fission is the process of breaking up the mitochondrial network, while fusion is the reverse process. Mitochondrial dynamics is highly plastic. If either fusion or fission is inhibited, the other can over-accumulate, leading to excessive mitochondrial fragmentation or elongation. Currently, several small molecule drugs that inhibit mitochondrial fission have been reported, but all are still in the laboratory development stage. For example, Mdivi-1 inhibits the expression of mitochondrial fission-related proteins, resulting in increased mitochondrial length; and Vemurafenib acts on both mitochondrial fission and outer membrane fusion proteins. To date, no drugs have been specifically identified that target mitochondrial fusion. Therefore, developing drugs that can effectively regulate mitochondrial dynamics is key to promoting mitochondrial-targeted therapies and holds significant theoretical and practical value.

[0005] PHB is an important molecule in maintaining mitochondrial function and energy metabolism. It is a highly conserved group of proteins that are widely present in various cell types. The human PHB gene comprises two isoforms, PHB1 and PHB2. The PHB1 gene is located on chromosome 17q21 and encodes the 32kD PHB1 protein; the PHB2 gene is located on chromosome 12p13 and encodes the 37kD PHB2 protein. Studies have shown that PHB expression is associated with the proliferation, metastasis, and metabolism of various tumors, including melanoma, breast cancer, and prostate cancer. Inhibiting PHB expression can inhibit tumor cell growth and metabolism, assess tumor risk, and enhance sensitivity to the chemotherapeutic drug paclitaxel. However, it is unknown whether PHB2 is involved in regulating immune function, particularly tumor immunity. Furthermore, the effects and mechanisms of PHB2 on mitochondrial morphology and function remain unclear.

[0006] Summary of the Invention

[0007] Based on the above background, this patent proposes through experiments that PHB2 can become a new immune checkpoint. Targeting PHB2 can reduce the degree of cell exhaustion and provide a new target for tumor immunotherapy.

[0008] The first purpose of the present application is to propose the use of PHB2 gene or PHB2 protein as an immune checkpoint to solve the problem in the prior art that whether PHB2 is involved in regulating immune function is still unknown.

[0009] Furthermore, the use of the PHB2 gene or PHB2 protein as a target for tumor immunotherapy can solve the problem in the prior art that whether PHB2 is involved in regulating tumor immune function is still unknown.

[0010] Furthermore, the PHB2 gene or PHB2 protein can serve as a tumor immune checkpoint.

[0011] Furthermore, inhibiting the expression of the PHB2 gene or PHB2 protein can inhibit mitochondrial fusion in cells, increase mitochondrial fragmentation, increase mitochondrial metabolic levels, and enhance the anti-tumor function of cells in vivo or in vitro.

[0012] Furthermore, the cells are tumor cells or T cells.

[0013] Furthermore, the T cells are CD8+ T cells.

[0014] Furthermore, the tumor cells are HeLa cell line.

[0015] Tumor immunity primarily relies on adaptive immunity. Among the many immune cells, T cells are the primary executors of specific tumor cell killing and resistance to bacterial infection. Currently, a growing number of studies indicate that exhaustion of tumor-killing cells (CD8+ T cells, CTLs) in the tumor microenvironment is key to tumors' ability to evade immune responses and a major factor limiting the effectiveness of tumor immunotherapy. Exhausted CD8+ T cells highly express PD-1. Effectively mitigating the degree of CD8+ T cell exhaustion in patients and mastering in vivo T cell activation techniques could enhance tumor immunity, offering hope for overcoming the current challenges in tumor immunotherapy. Mitochondrial metabolism is a key determinant of CD8+ T cell development and differentiation. During initial activation by antigen stimulation, CD8+ T cells alter their metabolic pathways to generate more energy to support subsequent cell proliferation and functional differentiation. This is manifested by increased mitochondrial fragmentation and elevated oxidative phosphorylation levels in mitochondrial metabolism. Mitochondrial fragmentation not only influences cellular homeostasis, bioenergetics, and redox processes but is also closely linked to T cell fate determination, functional activation, and immune synapse formation.

[0016] The second object of the present application is to propose the use of the PHB2 gene or PHB2 protein as a drug target in the in vitro synthesis and screening of tumor immunotherapy drugs, wherein the drug has an inhibitory effect on the PHB2 gene or its expression product;

[0017] or,

[0018] Use of a drug having an inhibitory effect on the PHB2 gene or PHB2 protein in the preparation of a therapeutic product or a preventive product for treating or preventing tumors.

[0019] In one preferred embodiment, the drug is a product that can produce beneficial effects of treating, alleviating, inhibiting, and regulating the occurrence and development of tumors; the drug is a single preparation or a composition containing an effective amount of preparation components.

[0020] In another preferred embodiment, the drug has the function of down-regulating PHB2 gene replication, transcription, translation or its protein expression product.

[0021] In another preferred embodiment, the drug targets mitochondrial PHB2 and can play one or more of the following roles: 1) improving the level of mitochondrial metabolism in cells; 2) increasing the degree of mitochondrial fragmentation in cells; 3) inhibiting mitochondrial fusion in cells.

[0022] In another preferred embodiment, the drug targets mitochondrial PHB2 to enhance the mitochondrial metabolic level of CD8+T cells, which can play one or more of the following roles: 1) enhancing the tumor-killing activity of CD8+T cells; 2) enhancing the effector function of CD8+T cells; 3) promoting the proliferation of CD8+T cells; 4) reducing the degree of exhaustion of CD8+T cells; 5) downregulating PD-1 expression of CD8+T cells; 6) increasing the expression level of IFN-γ cytokine; 7) increasing the expression level of TNF-α cytokine; 8) increasing the mitochondrial metabolic level of CD8+T cells: increased oxygen consumption rate, or increased acid production rate in vitro; 9) increasing the degree of mitochondrial fragmentation in CD8+T cells; 10) inhibiting mitochondrial fusion in CD8+T cells.

[0023] In another preferred embodiment, the drug includes one or more of: a PHB2 nucleic acid inhibitor, a PHB2 protein inhibitor, and a PHB2 gene defective construct.

[0024] The third object of the present application is to propose the use of a drug that has an inhibitory effect on the PHB2 gene or PHB2 protein in the preparation of a PD-1 inhibitor;

[0025] or,

[0026] Use of a drug that has an inhibitory effect on the PHB2 gene or PHB2 protein in combination with a PD-1 monoclonal antibody in the preparation of a therapeutic product or preventive product for treating or preventing tumors.

[0027] Furthermore, the PD-1 monoclonal antibody is tislelizumab, nivolumab, panlevuzumab, atezolizumab, durvalumab, avelumab or toripalimab.

[0028] It should be understood that in clinical applications, PD-1 monoclonal antibodies can be either the aforementioned products that are currently in clinical use or newly developed products in the future.

[0029] The fourth object of the present application is to propose the use of a drug that has an inhibitory effect on the PHB2 gene or PHB2 protein in combination with a CTLA-4 monoclonal antibody in the preparation of a therapeutic product or preventive product for treating or preventing tumors.

[0030] It should be understood that in clinical applications, CTLA-4 monoclonal antibodies can be products that are currently in clinical use, such as ipilimumab, or they can be newly developed products in the future.

[0031] The fifth object of the present application is to propose the use of a drug that has an inhibitory effect on the PHB2 gene or PHB2 protein in the preparation of a mitochondrial fusion inhibitor.

[0032] The sixth object of the present application is to propose the use of drugs that have an inhibitory effect on the PHB2 gene or PHB2 protein in immunotherapy.

[0033] The seventh object of the present application is to propose the use of drugs that have an inhibitory effect on the PHB2 gene or PHB2 protein in tumor immunotherapy.

[0034] The eighth object of the present application is to propose the use of a drug that has an inhibitory effect on the PHB2 gene or PHB2 protein in combination with a PD-1 monoclonal antibody in immunotherapy.

[0035] The ninth object of the present application is to propose the use of a drug that has an inhibitory effect on the PHB2 gene or PHB2 protein in combination with a PD-1 monoclonal antibody in tumor immunotherapy.

[0036] The tenth object of the present application is to propose the use of a drug that has an inhibitory effect on the PHB2 gene or PHB2 protein in combination with a CTLA-4 monoclonal antibody in immunotherapy.

[0037] The eleventh objective of the present application is to propose the use of a drug that has an inhibitory effect on the PHB2 gene or PHB2 protein in combination with a CTLA-4 monoclonal antibody in tumor immunotherapy.

[0038] Furthermore, in the above uses, tumors include melanoma, cervical cancer, colorectal cancer, lung cancer, brain tumor, and lymphoma.

[0039] In order to achieve the aforementioned invention objectives, this application adopts the following technical solutions to verify them:

[0040] S1: PHB2 was semi-knocked out on the basis of immune normal mice to obtain PHB2+ / - heterozygous gene knockout mice; multiple PHB2+ / - heterozygous gene knockout mice were mated with immune normal mice and genetic identification was performed to obtain multiple PHB2+ / - heterozygous gene knockout mice; CD8+ in the spleen and lymph nodes of immune normal mice and PHB2+ / - heterozygous gene knockout mice were sorted respectively The cells were induced to differentiate into mature CD8+ T cells after in vitro co-stimulation with CD3 and CD28 antibodies;

[0041] S2: After the mature CD8+ T cells in step S1 are stimulated with the stimulator cooktail, the expression level of IFN-γ cytokine expressed in the T cells is detected by flow cytometry, and the expression difference is statistically analyzed;

[0042] S3: a) Construct subcutaneous tumor-bearing models in immune-normal mice and PHB2+ / - heterozygous knockout mice, and observe tumor growth and mouse survival; b) Measure mouse body weight and tumor volume every two days in the subcutaneous tumor model, with the experimental endpoint set at a tumor volume of 2000 mm 3 ; Record the size of the tumors in immune-normal mice and PHB2+ / - heterozygous gene knockout mice at each observation and draw a tumor growth curve; c) After killing the mice at the end of the experiment, remove the tumors, photograph them, and measure their size; d) Take part of the tumor tissue to isolate tumor-infiltrating lymphocytes, isolate the mouse inguinal lymph nodes and spleen lymphocytes, and perform flow cytometric analysis to detect the anti-tumor function of CD8+ T cells infiltrating the tumor microenvironment. The killing function is evaluated by the ability to express cytokines. The detection indicators of cytokine expression ability include IFN-γ and TNF-α.

[0043] Furthermore, step S3-a) includes: a1) constructing a subcutaneous melanoma tumor-bearing model in immune normal mice and PHB2+ / - heterozygous gene knockout mice: the tumor cells inoculated are mouse-derived B16-F10 melanoma cells, the inoculated cell number is 1*10^5 cells / mouse, the inoculated mice are 8-week-old female mice, and the tumor growth and mouse survival are observed; or, a2) constructing a subcutaneous colon cancer tumor-bearing model in immune normal mice and PHB2+ / - heterozygous gene knockout mice: the tumor cells inoculated are mouse-derived MC38 colon cancer cells, the inoculated cell number is 2.5*10^5 cells / mouse, the inoculated mice are 8-week-old female mice, and the tumor growth and mouse survival are observed.

[0044] Furthermore, the method further includes step S4: culturing a Hela cell line, constructing a shRNA knockout lentiviral vector targeting the knockout of PHB2, transfecting the Hela cell line to obtain a PHB2KO Hela cell line, inserting Mito-dendra2 into the control group and the PHB2KO Hela cell line, respectively, wherein Mito-dendra2 can emit red light after being irradiated with ultraviolet light of 380-400nm, marking mitochondria with immunofluorescence live cell staining TOM20, and observing the intensity and distribution changes of the red signal emitted by Mito-dendra2 by fluorescence microscopy at 0h and 1h, respectively.

[0045] Furthermore, step S5 is included: using PHB2-mCherry to insert into Hela cell mitochondria to obtain a Hela cell line with overexpression of PHB2, fixing the cell pellet with 0.1% Triton, using immunofluorescence staining TOM20 to mark mitochondria, and DAPI to mark cell nuclei, and observing the changes in mitochondrial morphology after PHB2 overexpression by fluorescence microscopy imaging.

[0046] Furthermore, the method further includes step S6: collecting the mature CD8+ T cells in step S1, fixing the cell pellet with 0.1% Triton, using immunofluorescence staining to mark mitochondria with TOM20, and using DAPI to mark cell nuclei, and observing the changes in mitochondrial morphology after PHB2 knockout by fluorescence microscopy imaging.

[0047] Furthermore, the method further includes step S7: collecting the mature CD8+ T cells in step S1, measuring the in vitro acid production rate and oxygen consumption rate of the cell mitochondria using a cell energy metabolism meter, and determining the mitochondrial energy metabolism level of the CD8+ T cells.

[0048] Furthermore, the method further includes step S8: collecting the mature CD8+ T cells in step S1, lysing the cell precipitates using 1X SDS loading buffer and boiling them in a metal bath at 100°C for 8 minutes, detecting the expression level of PD-1 in CD8+ T cells using SDS-PAGE immunoprotein electrophoresis, and using β-actin as an internal reference to detect changes in PD-1 expression after PHB2 knockout.

[0049] Furthermore, the method further includes step S9: collecting the mature CD8+ T cells in step S1, fixing the cell pellet with 0.1% Triton, using immunofluorescence staining to mark the distribution of PD-1 in the cells, using DAPI to mark the cell nucleus, and observing the changes in PD-1 expression after PHB2 knockout by fluorescence microscopy imaging.

[0050] Furthermore, the method also includes step S10: constructing a subcutaneous melanoma tumor model of immune normal mice and PHB2+ / - heterozygous gene knockout mice: the inoculated tumor cells are mouse-derived B16-F10 melanoma cells, the inoculated cell number is 1*10^5 cells / mouse, and the inoculated mice are 8-week-old female mice; 4 days after tumor inoculation, all tumor-bearing mice are evenly divided into 4 groups, the first group is an immune normal mouse control group, the second group is an immune normal mouse CTLA-4 treatment group alone, the third group is a PHB2+ / - heterozygous gene knockout mouse group, and the fourth group is a PHB2+ / - heterozygous gene knockout mouse combined with CTLA-4 monoclonal antibody treatment group; the second and fourth groups are given 100ug of CTLA-4 monoclonal antibody to each mouse by intraperitoneal injection on days 4, 7, 10, 13, and 16 after tumor inoculation; starting from day 6, the tumor size is measured every two days, the tumor growth curve is plotted, and the tumor is peeled and observed.

[0051] Furthermore, the immune normal mice include C57BL / 6WT wild-type mice and BALB / c mice.

[0052] Through multifaceted analyses including animal experiments, biochemistry and molecular biology, transcriptomics, proteomics, and metabolomics, this application found that tumors in PHB2+ / - tumor-bearing mice were significantly smaller than those in controls. Both in vivo and in vitro experiments demonstrated that targeting PHB2+ / - cells enhanced their cytotoxicity. Further studies revealed that targeting mitochondrial PHB2 inhibited mitochondrial fusion, increased mitochondrial fragmentation, elevated mitochondrial metabolism, and enhanced cellular function. Furthermore, studies have shown that PHB2 knockout reduced PD-1 expression, and that targeting PHB2 in combination with CTLA-4 monoclonal antibodies was more effective than CTLA-4 monoclonal antibodies alone. Therefore, this application identified PHB2 as a potential cellular target that can reduce cellular exhaustion and enhance anti-tumor immunity. The development of inhibitors targeting mitochondrial PHB2 is expected to provide a novel approach and effective technology to overcome the current challenges of tumor immunotherapy. PHB2 inhibitors may become an alternative to PD-1 inhibitors or a combination therapy option. Targeting PHB2 can enhance the therapeutic effect of CTLA-4 monoclonal antibodies, and combined treatment with PHB2-targeted CTLA-4 monoclonal antibodies could provide greater benefits to clinical cancer patients.

[0053] The advantages of the present invention are:

[0054] [1] Through multiple mouse tumor models and a series of in vitro experiments, it was found that PHB2 is a new immune checkpoint. Targeting PHB2 can enhance the anti-tumor immunity of cells in vivo and in vitro.

[0055] [2] Through immunofluorescence, immunoprotein electrophoresis and other experiments, it was found that PHB2 regulates mitochondrial fusion. After targeting PHB2KO, mitochondrial fusion was inhibited, the degree of mitochondrial fragmentation increased, the mitochondrial metabolic level increased, and cell function was enhanced.

[0056] [3] Through a series of in vitro and in vivo experiments, it was found that targeted inhibition of PHB2 can reduce PD-1 expression, and PHB2 inhibitors can be used as a substitute or combination preparation for PD-1 monoclonal antibodies.

[0057] [4] At the animal level, it was found that targeted inhibition of PHB2 can be combined with CTLA4 monoclonal antibody to enhance the anti-tumor effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1: Comparison of IFN-γ secretion levels of CD8+ T cells in C57BL / 6 wild-type mice and PHB2+ / - heterozygous knockout mice;

[0059] Figure 2: Tumor growth curves (A) and excised tumors (B) of C57BL / 6 wild-type mice and PHB2+ / - heterozygous knockout mice in a subcutaneous melanoma tumor model.

[0060] Figure 3: Tumor growth curves (A) and excised tumors (B) of C57BL / 6 wild-type mice and PHB2+ / - heterozygous knockout mice in a subcutaneous colon cancer tumor model.

[0061] Figure 4: Comparison of IFN-γ secretion levels, i.e., ratios and infiltration density, of CD8+ T cells in C57BL / 6 wild-type mice and PHB2+ / - heterozygous knockout mice in a subcutaneous melanoma tumor model.

[0062] Figure 5: Comparison of TNF-α secretion levels, i.e., proportions and infiltration density, of CD8+ T cells in C57BL / 6WT wild-type mice and PHB2+ / - heterozygous knockout mice in a subcutaneous melanoma tumor model.

[0063] Figure 6: In Hela cell lines, mitochondrial fusion was inhibited after PHB2KO;

[0064] Figure 7: Overexpression of PHB2 in Hela cells resulted in elongated mitochondria.

[0065] Figure 8: Comparison of mitochondrial morphology of CD8+ T cells from C57BL / 6WT wild-type mice and PHB2 knockout mice;

[0066] Figure 9: Comparison of mitochondrial metabolic levels in CD8+ T cells from C57BL / 6WT wild-type mice and PHB2+ / - heterozygous knockout mice;

[0067] Figure 10: Comparison of PD-1 protein expression levels in CD8+ T cells of C57BL / 6 wild-type mice and PHB2KO knockout mice. Immunoprotein electrophoresis results showed decreased PD-1 expression in the KO group.

[0068] Figure 11: Comparison of PD-1 protein expression in CD8+ T cells of C57BL / 6 wild-type mice and PHB2KO knockout mice. Immunofluorescence results showed decreased PD-1 expression in the KO group.

[0069] Figure 12: In the subcutaneous melanoma tumor model, C57BL / 6WT wild-type mice and PHB2+ / - heterozygous gene knockout mice, as well as both treated with CTLA-4, were used to remove the tumor (A) and plot the tumor growth curve (B).

[0070] In the figure, OCR: oxygen consumption rate; ECAR: in vitro acid production rate; Ctrl: control group mice (C57BL / 6WT wild-type mice); PHB2 + / - : heterozygotes obtained by semi-knockout of PHB2 in C57BL / 6 mice; aCTLA-4: immune normal mice treated with CTLA-4 alone; aCTLA-4+PHB2 + / - : PHB2+ / - heterozygous gene knockout mice combined with CTLA-4 monoclonal antibody treatment group; KO: abbreviation for knock out; TOM20: a mitochondrial characteristic protein marker, localized in the mitochondrial outer membrane; Mito-drendra2: a red-green photoconvertible protein localized in mitochondria; DAPI: a characteristic protein marker for the nucleus; WT: abbreviation for wildtype. DETAILED DESCRIPTION

[0071] The present application is further described below with reference to the embodiments, but is not limited thereto.

[0072] The experimental methods in the following examples, unless otherwise specified, were all conventional methods or performed according to the kit instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, were all commercially available.

[0073] In the examples: OCR: oxygen consumption rate; ECAR: in vitro acid production rate; Ctrl: control group mice (C57BL / 6WT wild-type mice); PHB2 + / - : heterozygotes obtained by semi-knockout of PHB2 in C57BL / 6 mice; aCTLA-4: immune normal mice treated with CTLA-4 alone; aCTLA-4+PHB2 + / -: PHB2+ / - heterozygous knockout mice combined with CTLA-4 monoclonal antibody treatment group; HeLa cell line: HeLa cell line; KO: abbreviation for “knock out”; TOM20: a mitochondrial characteristic protein marker, localized in the mitochondrial outer membrane; Mito-drendra2: a red-green photoconvertible protein localized in mitochondria; DAPI: a nuclear characteristic protein marker; WT: abbreviation for “wild type”.

[0074] Example 1:

[0075] A method for detecting the PHB2 target for regulating tumor immunity is intended to validate the use of PHB2 as a tumor immunity target, especially as an immune checkpoint, and includes the following steps:

[0076] S1: PHB2 was semi-knocked out in immune-normal mice to obtain PHB2+ / - heterozygous knockout mice; multiple PHB2+ / - heterozygous knockout mice were mated with C57BL / 6WT wild-type mice and genetic identification was performed to obtain multiple PHB2+ / - heterozygous knockout mice; CD8+ The cells were co-stimulated with CD3 and CD28 antibodies in vitro for 2 days and then differentiated into mature CD8+ T cells.

[0077] S2: After stimulating the mature CD8+ T cells from step S1 with the stimulatory agent cooktail, flow cytometry was used to measure the expression of the IFN-γ cytokine in the T cells, and the expression differences were statistically analyzed. As shown in Figure 1, it was found that the CD8+ T cells of the PHB2+ / - heterozygous knockout mice had enhanced secretion of cytotoxic cytokines.

[0078] S3: a1) Construction of a subcutaneous melanoma tumor-bearing model in C57BL / 6WT wild-type mice and PHB2+ / - heterozygous gene knockout mice: The inoculated tumor cells were mouse-derived B16-F10 melanoma cells, the inoculated cell number was 1*10^5 per mouse, and the inoculated mice were 8-week-old female mice. The tumor growth and mouse survival were observed. a2) Construction of a subcutaneous colon cancer tumor-bearing model in C57BL / 6WT wild-type mice and PHB2+ / - heterozygous gene knockout mice: The inoculated tumor cells were mouse-derived MC38 colon cancer cells, the inoculated cell number was 2.5*10^5 per mouse, and the inoculated mice were 8-week-old female mice. The tumor growth and mouse survival were observed. b) In the subcutaneous tumor model, the mouse body weight and tumor volume were measured every two days. The experimental endpoint was set when the tumor volume reached 2000mm 3; Record the size of the tumors in the wild-type and PHB2+ / - group mice at each observation and draw a tumor growth curve; c) After the mice were sacrificed at the end of the experiment, the tumors were removed, photographed, and measured; d) Part of the melanoma tumor tissue was taken to isolate tumor-infiltrating lymphocytes, isolate the inguinal lymph nodes and spleen lymphocytes of the mice, and perform flow cytometric analysis to detect the anti-tumor function of CD8+ T cells infiltrating the tumor microenvironment. The killing function was evaluated by the ability to express cytokines. The detection indicators of cytokine expression ability include IFN-γ and TNF-α.

[0079] As shown in Figures 2 and 3, tumors in PHB2+ / - heterozygous knockout mice were reduced in subcutaneous melanoma and colon cancer models. As shown in Figures 4 and 5, in this subcutaneous melanoma model, the ability of CD8+ T cells in the tumor microenvironment to express the cytokines IFN-γ and TNF-α was enhanced. Furthermore, the infiltration density, i.e., the absolute number of CD8+ T cells expressing the cytokines IFN-γ and TNF-α per unit tumor mass, was increased. These results clearly demonstrate enhanced anti-tumor activity in PHB2+ / - heterozygous knockout mice compared to tumors in wild-type mice.

[0080] Example 2:

[0081] A detection method for PHB2 targeting tumor immunity regulation aims to verify that PHB2 regulates tumor immunity by inhibiting mitochondrial fusion in tumor cells, thereby increasing mitochondrial fragmentation and thereby improving mitochondrial metabolism. The method includes the following steps:

[0082] Step S4: Cultivate Hela cell lines, construct shRNA knockout lentiviral vectors targeting PHB2 knockout, transfect Hela cell lines to obtain PHB2KO Hela cell lines, and insert Mito-dendra2 into control and PHB2KO Hela cell lines, respectively. Mito-dendra2 can emit red light after irradiation with ultraviolet light at 380-400nm. Mito-dendra2 is labeled with immunofluorescent live cell stain TOM20. The intensity and distribution changes of the red signal emitted by Mito-dendra2 are observed by fluorescence microscopy imaging at 0h and 1h, respectively. As shown in Figure 6, the degree of mitochondrial fusion in PHB2KO Hela cells is inhibited compared with the control group.

[0083] Step S5: PHB2-mCherry was inserted into Hela cell mitochondria to obtain a Hela cell line with PHB2 overexpression. The cell pellet was fixed with 0.1% Triton, and the mitochondria were labeled with TOM20 and the cell nucleus was labeled with DAPI using immunofluorescence staining. The changes in mitochondrial morphology after PHB2 overexpression were observed by fluorescence microscopy. The results are shown in Figure 7. After overexpression of PHB2, the cell mitochondria became elongated.

[0084] Example 3:

[0085] A detection method for PHB2 targeting tumor immunity regulation aims to verify that PHB2 regulates tumor immunity by inhibiting T cell mitochondrial fusion, thereby increasing mitochondrial fragmentation and improving mitochondrial metabolism. The method includes the following steps:

[0086] The sample preparation is the same as step S1 of Example 1, and further includes steps S6-S7:

[0087] Step S6: The mature CD8+ T cells from step S1 were collected, the cell pellets were fixed with 0.1% Triton, and mitochondria were labeled with TOM20 and nuclei were labeled with DAPI using immunofluorescence staining. The changes in mitochondrial morphology after PHB2 knockout were observed by fluorescence microscopy. The results are shown in Figure 8. The degree of mitochondrial fragmentation is increased in PHB2 gene knockout mice.

[0088] Step S7: The mature CD8+ T cells from step S1 were collected and their mitochondrial acid production and oxygen consumption rates were measured using a cell energy metabolism instrument to determine the mitochondrial energy metabolism level of the CD8+ T cells. As shown in Figure 9, the results showed that compared with C57BL / 6WT wild-type mice, the in vitro acid production and oxygen consumption rates of CD8+ T cells from PHB2+ / - heterozygous knockout mice were increased, indicating that the mitochondrial metabolism level of CD8+ T cells in C57BL / 6WT wild-type mice was elevated and the degree of activation was enhanced.

[0089] Example 4: A detection method for regulating tumor immunity by targeting PHB2, aiming to verify that targeted inhibition of PHB2 can reduce PD-1 expression.

[0090] The sample preparation is the same as step S1 of Example 1, and further includes steps S8-S9:

[0091] Step S8: Mature CD8+ T cells from step S1 were collected, and the cell pellets were lysed using 1X SDS loading buffer and boiled in a metal bath at 100°C for 8 minutes. PD-1 expression levels in CD8+ T cells were then assessed by SDS-PAGE immunoprecipitation. β-actin was used as an internal control to examine changes in PD-1 expression after PHB2 knockout. As shown in Figure 10, PD-1 protein expression levels were decreased in PHB2 knockout mice.

[0092] Step S9: Mature CD8+ T cells from Step S1 were collected, the cell pellets were fixed with 0.1% Triton, and intracellular PD-1 distribution was assessed using immunofluorescence staining. DAPI was used to label cell nuclei. Fluorescence microscopy was used to observe changes in PD-1 expression following PHB2 knockout. As shown in Figure 11, PD-1 protein expression and distribution levels decreased in PHB2 knockout mice during cell activation.

[0093] Example 5:

[0094] A detection method for PHB2-targeted tumor immunity regulation aims to explore the therapeutic effect of PHB2-targeted combined with CTLA-4 monoclonal antibody, including the following steps:

[0095] Step S10: Construct a subcutaneous melanoma tumor model of immune normal mice and PHB2+ / - heterozygous gene knockout mice: the inoculated tumor cells are mouse-derived B16-F10 melanoma cells, the inoculated cell number is 1*10^5 cells / mouse, and the inoculated mice are 8-week-old female mice; 4 days after tumor inoculation, all tumor-bearing mice are evenly divided into 4 groups, the first group is an immune normal mouse control group, the second group is an immune normal mouse CTLA-4 treatment group alone, the third group is a PHB2+ / - heterozygous gene knockout mouse group, and the fourth group is a PHB2+ / - heterozygous gene knockout mouse combined with CTLA-4 monoclonal antibody treatment group; the second and fourth groups are given 100ug of CTLA-4 monoclonal antibody to each mouse by intraperitoneal injection on days 4, 7, 10, 13, and 16 after tumor inoculation; starting from day 6, the tumor size is measured every two days. The observation is terminated on day 20 of tumor inoculation, the tumor growth curve is plotted, and the tumor is removed for observation. The results are shown in Figure 12. + / - The tumors in the knockout combined with CTLA-4 monoclonal antibody treatment group were significantly smaller than those in the CTLA-4 monoclonal antibody alone or the PHB2 targeting alone group. + / - The combined treatment of the two groups had a stronger anti-tumor effect.

[0096] In this application, CTLA-4 monoclonal antibody was used (clone 9H10, BioXcell BE0131).

[0097] Example 6:

[0098] The use of the CD8+T cell PHB2 gene or PHB2 protein as a target for melanoma immunotherapy, inhibiting the expression of the PHB2 gene or PHB2 protein can inhibit mitochondrial fusion, increase mitochondrial fragmentation, increase mitochondrial metabolic levels, and enhance the anti-tumor function of cells in vivo and in vitro.

[0099] Example 7:

[0100] 1) Use of the PHB2 gene or PHB2 protein as a drug target in CD8+ T cells for in vitro synthesis and screening of melanoma immunotherapy drugs, wherein the drug has an inhibitory effect on the PHB2 gene or its expression product.

[0101] 2) Use of a drug that has an inhibitory effect on the PHB2 gene or PHB2 protein in the preparation of a therapeutic product or a preventive product for treating or preventing melanoma.

[0102] The drug is a product that can treat, alleviate, inhibit, or regulate the development and progression of melanoma. The drug is a single agent or a composition comprising effective amounts of the agent's components. The drug has the function of downregulating the expression, transcription, or expression product of the PHB2 gene. The drug can be one or more of a PHB2 nucleic acid inhibitor, a PHB2 protein inhibitor, or a PHB2 gene-deficient construct.

[0103] The drug can have the following effects: 1) enhance the killing activity of CD8+T cells against tumors; 2) enhance the effector function of CD8+T cells; 3) promote the proliferation of CD8+T cells; 4) reduce the degree of exhaustion of CD8+T cells; 5) downregulate the expression of PD-1 in CD8+T cells; 6) increase the expression level of IFN-γ cytokine; 7) increase the expression level of TNF-α cytokine; 8) increase the mitochondrial metabolism level of CD8+T cells: increase the oxygen consumption rate or increase the acid production rate in vitro; 9) increase the degree of mitochondrial fragmentation in CD8+T cells; 10) inhibit mitochondrial fusion in CD8+T cells.

[0104] Example 8:

[0105] 1) Use of a drug that has an inhibitory effect on the PHB2 gene or PHB2 protein in the preparation of a PD-1 inhibitor.

[0106] 2) Use of a drug that inhibits the PHB2 gene or PHB2 protein in combination with a PD-1 monoclonal antibody in the preparation of a therapeutic or preventive product for treating or preventing tumors, where the PD-1 monoclonal antibody is tislelizumab, nivolumab, panlevuzumab, atezolizumab, durvalumab, avelumab, or toripalimab. The tumor is melanoma, cervical cancer, colorectal cancer, lung cancer, brain tumor, or lymphoma.

[0107] Example 9:

[0108] Use of a drug that inhibits the PHB2 gene or PHB2 protein in combination with a CTLA-4 monoclonal antibody in the preparation of a therapeutic product or preventive product for treating or preventing tumors, wherein the tumor is melanoma, cervical cancer, colorectal cancer, lung cancer, brain tumor, or lymphoma.

[0109] Example 10:

[0110] Use of a drug having an inhibitory effect on the PHB2 gene or PHB2 protein in the preparation of a mitochondrial fusion inhibitor.

[0111] Example 11:

[0112] The use of drugs that have an inhibitory effect on the PHB2 gene or PHB2 protein in immunotherapy, such as in the immunotherapy of tumors such as melanoma, cervical cancer, colorectal cancer, lung cancer, brain tumor or lymphoma, or autoimmune system diseases.

[0113] Example 12:

[0114] Use of a drug that has an inhibitory effect on the PHB2 gene or PHB2 protein in tumor immunotherapy, wherein the tumor is melanoma, cervical cancer, colorectal cancer, lung cancer, brain tumor or lymphoma.

[0115] Example 13:

[0116] The use of drugs that have an inhibitory effect on the PHB2 gene or PHB2 protein in combination with PD-1 monoclonal antibodies in immunotherapy, such as in the immunotherapy of tumors or autoimmune system diseases such as melanoma, cervical cancer, colorectal cancer, lung cancer, brain tumor or lymphoma.

[0117] Example 14:

[0118] The invention relates to the use of a drug that has an inhibitory effect on the PHB2 gene or PHB2 protein in combination with a PD-1 monoclonal antibody in tumor immunotherapy, wherein the tumor is melanoma, cervical cancer, colorectal cancer, lung cancer, brain tumor or lymphoma.

[0119] Example 15:

[0120] The use of drugs that have inhibitory effects on the PHB2 gene or PHB2 protein in combination with CTLA-4 monoclonal antibodies in immunotherapy, such as in the immunotherapy of tumors or autoimmune system diseases such as melanoma, cervical cancer, colorectal cancer, lung cancer, brain tumor or lymphoma.

[0121] Example 16:

[0122] The invention relates to the use of a drug that inhibits the PHB2 gene or PHB2 protein in combination with a CTLA-4 monoclonal antibody in tumor immunotherapy, wherein the tumor is melanoma, cervical cancer, colorectal cancer, lung cancer, brain tumor or lymphoma.

[0123] Although the present application has been described with reference to illustrative embodiments, it will be understood by those skilled in the art that various other changes, omissions, and / or additions may be made and that substantial equivalents may be substituted for elements of the described embodiments without departing from the spirit and scope of the present application. In addition, many modifications may be made to adapt a particular situation or material to the teachings of the present application without departing from the scope of the present application. Therefore, it is not intended that the present application be limited to the particular embodiments disclosed for carrying out the present application, but rather that the present application will encompass all embodiments falling within the scope of the appended claims.

Claims

1. Use of the PHB2 gene or PHB2 protein as an immune checkpoint.

2. Use according to claim 1, use of the PHB2 gene or PHB2 protein as a tumor immunotherapy target.

3. Use according to claim 2, the PHB2 gene or PHB2 protein can be used as a tumor immune checkpoint.

4. Use according to claim 3, inhibiting the expression of the PHB2 gene or PHB2 protein can inhibit mitochondrial fusion in cells, increase mitochondrial fragmentation, increase mitochondrial metabolic levels, and enhance the anti-tumor function of cells in vivo or in vitro.

5. Use according to claim 4, the cells are tumor cells or T cells.

6. Use according to claim 5, the T cells are CD8+ T cells.

7. Use according to claim 5, the tumor cells are the HeLa cell line.

8. Use of the PHB2 gene or PHB2 protein as a drug target in the in vitro synthesis and screening of tumor immunotherapy drugs, the drug having an inhibitory effect on the PHB2 gene or its expression product; Or, use of a drug having an inhibitory effect on the PHB2 gene or PHB2 protein in the preparation of a therapeutic product or prophylactic product for treating or preventing tumors.

9. The use according to claim 8, characterized in that, The drug is a product that can have beneficial effects of treating, alleviating, inhibiting, or regulating the occurrence and development of tumors; the drug is a single preparation or a composition containing an effective amount of a preparation ingredient.

10. The use according to claim 8, characterized in that, The drug has the function of down-regulating the replication, transcription, translation of the PHB2 gene or its protein expression product.

11. The use according to claim 8, characterized in that, The drug targets mitochondrial PHB2 and can play one or more of the following roles: 1) increasing the mitochondrial metabolic level in cells; 2) increasing the degree of mitochondrial fragmentation in cells; 3) inhibiting mitochondrial fusion in cells.

12. The use according to claim 8, characterized in that, The drug targeting mitochondrial PHB2 enhances the mitochondrial metabolic level of CD8+ T cells and can play one or more of the following roles: 1) enhancing the killing activity of CD8+ T cells against tumors; 2) enhancing the effector function of CD8+ T cells; 3) promoting the proliferation of CD8+ T cells; 4) reducing the degree of exhaustion of CD8+ T cells; 5) down-regulating the expression of PD-1 in CD8+ T cells; 6) increasing the expression level of IFN-γ cytokine; 7) increasing the expression level of TNF-α cytokine; 8) increasing the mitochondrial metabolic level of CD8+ T cells: an increase in oxygen consumption rate or an increase in the in vitro acid production rate; 9) increasing the degree of mitochondrial fragmentation in CD8+ T cells; 10) inhibiting mitochondrial fusion in CD8+ T cells.

13. The use according to claim 8, characterized in that, The drug includes one or more of: a PHB2 nucleic acid inhibitor, a PHB2 protein inhibitor, and a PHB2 gene-deficient construct.

14. Use of a drug having an inhibitory effect on the PHB2 gene or PHB2 protein in the preparation of a PD-1 inhibitor; Or, use of a drug having an inhibitory effect on the PHB2 gene or PHB2 protein in combination with a PD-1 monoclonal antibody in the preparation of a therapeutic product or prophylactic product for treating or preventing tumors.

15. The use according to claim 14, characterized in that, The PD-1 monoclonal antibody described above is tislelizumab, nivolumab, pembrolizumab, atezolizumab, durvalumab, avelumab or toripalimab.

16. Use of a drug having an inhibitory effect on the PHB2 gene or PHB2 protein in combination with a CTLA-4 monoclonal antibody in the preparation of a therapeutic product or a prophylactic product for treating or preventing tumors.

17. Use of a drug having an inhibitory effect on the PHB2 gene or PHB2 protein in the preparation of a mitochondrial fusion inhibitor.

18. Use of a drug having an inhibitory effect on the PHB2 gene or PHB2 protein in immunotherapy.

19. Use of a drug having an inhibitory effect on the PHB2 gene or PHB2 protein in tumor immunotherapy.

20. Use of a drug having an inhibitory effect on the PHB2 gene or PHB2 protein in combination with a PD-1 monoclonal antibody in immunotherapy.

21. Use of a drug having an inhibitory effect on the PHB2 gene or PHB2 protein in combination with a PD-1 monoclonal antibody in tumor immunotherapy.

22. Use of a drug having an inhibitory effect on the PHB2 gene or PHB2 protein in combination with a CTLA-4 monoclonal antibody in immunotherapy.

23. Use of a drug having an inhibitory effect on the PHB2 gene or PHB2 protein in combination with a CTLA-4 monoclonal antibody in tumor immunotherapy.

24. The use according to any one of claims 1-23, characterized in that, The tumors include melanoma, cervical cancer, colorectal cancer, lung cancer, brain tumor, lymphoma.

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