Drug development and immunotherapy application of targeted SLC45A2

Abstract

The invention relates to the field of biological medicine, in particular to drug development and immunotherapy application of targeted SLC45A2. According to the invention, the SLC45A2 on the surface of the T cell is taken as a target spot of immunotherapy, the acid-base environment balance of the T cell is regulated by regulating the SLC45A2 gene or an expression product thereof, the capability of secreting killer cell factors of the T cell is regulated, the depletion process of the T cell is controlled, and the regulation of the immune function of the T cell is further realized. Based on this, the invention further develops a borate bioactive material targeting SLC45A2, and the material can significantly reduce the expression of SLC45A2 and enhance the anti-tumor immune effect of T cells, and shows a significant anti-tumor effect both in vivo and in vitro.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and specifically relates to the drug development targeting SLC45A2 and its application in immunotherapy. Background Art

[0002] As a T cell immunotherapy method, immune checkpoint inhibitor therapy has opened up a new treatment path for the field of cancer treatment and has broad clinical application prospects and market potential. Currently, the main immune checkpoint inhibitors include Cytotoxic T Lymphocyte Antigen 4 (CTLA4), Programmed Cell Death 1 (PD-1), and Programmed Cell Death Ligand 1 (PD-L1), which enhance the anti-tumor immune response of T cells through different mechanisms.

[0003] The immune checkpoint inhibitor targeting CTLA4, ipilimumab, was approved by the US FDA in 2011 for the treatment of unresectable stage III / IV melanoma, and significantly improved the survival of 22% of patients, extending it to 3 years or longer. Drugs targeting PD-1, including pembrolizumab and nivolumab, became the first PD-1 inhibitors approved by the FDA for the treatment of refractory and unresectable melanoma in 2014, and clinical data showed that their efficacy was superior to traditional treatment regimens. In addition, atezolizumab targeting PD-L1 was approved for the treatment of urothelial cancer in 2014, significantly improving the survival prognosis of 15% of patients. In 2018, nivolumab was first launched in China as a PD-1 inhibitor for the treatment of advanced non-small cell lung cancer, successfully bringing obvious survival benefits to such patients.

[0004] Although T cell immunotherapy has made significant progress in the field of cancer treatment, its clinical application still has certain limitations. In particular, among advanced cancer patients, only a few can benefit from it. The objective response rate of immune monotherapy is only about 20%, and most of these patients are only partial responders. In addition, even if some patients achieve efficacy initially, about one-third of the patients develop drug resistance during the treatment process, ultimately leading to tumor recurrence. The main reason for this phenomenon can be attributed to the immunosuppressive state in the tumor microenvironment.

[0005] The immune state of the tumor microenvironment is a complex and multi-dimensional system involving multiple cell types, molecular factors, and signaling pathways that jointly influence tumor growth and metastasis through their interactions. Among these factors, T cell exhaustion is considered one of the key mechanisms of immune suppression in the tumor microenvironment. T cell exhaustion refers to a severe decline in T cell function, characterized by a significant decrease in effector function, upregulation of immunosuppressive receptor expression, weakened proliferative capacity, and programmed changes in cell fate. Specifically, T cell exhaustion is manifested by a reduction in the secretion of cytotoxic cytokines (such as IFN-γ, TNF-α, and IL-2), resulting in a significant weakening of the cytotoxic effect of T cells on tumors, thereby limiting the effectiveness of immunotherapy.

[0006] Studies have shown that T cell exhaustion is closely related to the acidic microenvironment of tumors. The tumor microenvironment is usually weakly acidic, which is closely related to the metabolic characteristics of tumors, especially the process of aerobic glycolysis. High levels of glycolysis are usually associated with poor tumor prognosis and poor efficacy of immune checkpoint inhibitor therapy. During glycolysis, tumor cells produce a large amount of hydrogen ions, lactate, and pyruvate, leading to an increase in the acidity of the microenvironment. The acidic tumor environment not only inhibits the function of T cells but also promotes T cell exhaustion, increasing the proportion of exhausted T cells, which provides favorable conditions for tumor cells to escape immune surveillance and immune clearance, thereby exacerbating tumor immune escape and resulting in poor prognosis. Therefore, the acidic microenvironment of tumors is one of the important mechanisms triggering T cell exhaustion and tumor immune suppression, and it is closely related to the therapeutic effect, prognosis, and response to immunotherapy of tumors. Developing drugs that can effectively regulate the acidity of the tumor microenvironment to enhance the anti-tumor immune response is particularly urgent, yet no such drugs have entered clinical application currently.

[0007] Currently, research on the regulation of cellular acid-base balance mainly focuses on the GPCR proton transport family (such as four subtypes including GPCR4, GPCR65, GPCR68, and GPCR132). Although these transport families play a key role in regulating cellular acid-base balance, little is known about their potential role in tumor immune regulation, and relevant research is still scarce. Therefore, developing new targeted drugs to enhance the anti-tumor immune effect by regulating cellular acid-base balance has important theoretical significance and clinical application value. Summary of the Invention

[0008] The present invention takes SLC45A2 as a target for immunotherapy and provides a drug development and immunotherapy application.

[0009] In a first aspect, the present invention provides the use of a preparation having a regulatory effect on the SLC45A2 gene or its expression product in the preparation of an immunotherapeutic drug, including: using the preparation having a regulatory effect on the SLC45A2 gene or its expression product as the active ingredient of the drug, and adding pharmaceutically acceptable excipients.

[0010] In some embodiments, the preparation includes any one or more of an SLC45A2 nucleic acid inhibitor, an SLC45A2 protein inhibitor, an SLC45A2 gene-deficient construct, an SLC45A2 nucleic acid agonist, an SLC45A2 protein agonist, an SLC45A2 nucleic acid activator, an SLC45A2 protein activator, an SLC45A2 nucleic acid antagonist, an SLC45A2 protein antagonist, an SLC45A2 nucleic acid promoter, an SLC45A2 protein promoter, an SLC45A2 nucleic acid enhancer, an SLC45A2 protein enhancer, an SLC45A2 nucleic acid sensitizer, and an SLC45A2 protein sensitizer.

[0011] In some embodiments, the preparation regulates the SLC45A2 gene or its expression product, and can play one or more of the following roles: 1) regulating the acid-base balance of the environment where T cells are located; 2) regulating the ability of CD8+ T cells or CD4+ T cells to secrete cytotoxic cytokines; 3) regulating the proliferation of CD8+ T cells or CD4+ T cells; 4) regulating the exhaustion degree of CD8+ T cells or CD4+ T cells; 5) regulating the immune function of T cells; 6) regulating the efficacy or prognosis of immunotherapy.

[0012] In some embodiments, the immunotherapeutic drug includes a tumor immunotherapeutic drug.

[0013] In some embodiments, the preparation regulates the SLC45A2 gene or its expression product, and can play one or more of the following roles: 1) enhancing the anti-tumor immune function of T cells; 2) inhibiting the proliferation or migration of tumor cells; 3) inhibiting tumorigenesis or development; 4) inhibiting tumor progression, recurrence or metastasis; 5) enhancing the efficacy or prognosis of tumor immunotherapy.

[0014] In a second aspect, the present invention provides the use of a preparation having a regulatory effect on the SLC45A2 gene or its expression product in the preparation of a disease detection kit, including: using the preparation having a regulatory effect on the SLC45A2 gene or its expression product as the detection reagent of the kit, the detection reagent detecting the expression level of the SLC45A2 gene or its expression product, and the diseases including tumors, autoimmune diseases, connective tissue diseases, infectious diseases, allergic diseases, chronic inflammatory diseases, and diseases related to immune balance disorders.

[0015] In some embodiments, the preparation includes any one or more of an SLC45A2 nucleic acid inhibitor, an SLC45A2 protein inhibitor, an SLC45A2 gene-deficient construct, an SLC45A2 nucleic acid agonist, an SLC45A2 protein agonist, an SLC45A2 nucleic acid activator, an SLC45A2 protein activator, an SLC45A2 nucleic acid antagonist, an SLC45A2 protein antagonist, an SLC45A2 nucleic acid promoter, an SLC45A2 protein promoter, an SLC45A2 nucleic acid enhancer, an SLC45A2 protein enhancer, an SLC45A2 nucleic acid sensitizer, and an SLC45A2 protein sensitizer.

[0016] In a third aspect, the present invention provides a borate bioactive material. In terms of molar percentage, the borate bioactive material includes 54 parts of boric anhydride, 22 parts of calcium oxide, 8 parts of magnesium oxide, 6 parts of sodium oxide, 8 parts of potassium oxide, and 2 parts of phosphorus pentoxide.

[0017] In some embodiments, the average diameter of the particles in the borate bioactive material is 1-10 μm.

[0018] In a fourth aspect, the present invention provides a method for preparing a borate bioactive material, which is characterized by including: S1. Mixing H 3 BO 3 , CaCO 3 , Na 2 CO 3 , 4MgCO 3 ·Mg(OH) 2 ·5H 2 O, K 2 CO 3 and NaH 2 PO 4 ·2H 2 O, and melting at 1150-1200 °C for 1-2 h to obtain a melt; S2. Quenching the melt in ice water to obtain a crude material; and S3. Adjusting the particle size of the crude material to obtain the borate bioactive material.

[0019] In a fifth aspect, the present invention provides a kit, including a detection reagent and an operation manual. The detection reagent is the above-mentioned borate bioactive material or is prepared by the method for preparing the above-mentioned borate bioactive material.

[0020] In a sixth aspect, the present invention provides an application of the borate bioactive material in screening or preparing a preparation having an inhibitory effect on the SLC45A2 gene or its expression product. The borate bioactive material is the above-mentioned borate bioactive material or is prepared by the method for preparing the above-mentioned borate bioactive material.

[0021] In a seventh aspect, the present invention provides an immunotherapy method, comprising: administering to a subject a preparation that regulates the SLC45A2 gene or its expression product, or administering to the subject a drug having as an active ingredient a preparation that regulates the SLC45A2 gene or its expression product.

[0022] In some embodiments, the preparation includes any one or more of an SLC45A2 nucleic acid inhibitor, an SLC45A2 protein inhibitor, an SLC45A2 gene-deficient construct, an SLC45A2 nucleic acid agonist, an SLC45A2 protein agonist, an SLC45A2 nucleic acid activator, an SLC45A2 protein activator, an SLC45A2 nucleic acid antagonist, an SLC45A2 protein antagonist, an SLC45A2 nucleic acid promoter, an SLC45A2 protein promoter, an SLC45A2 nucleic acid enhancer, an SLC45A2 protein enhancer, an SLC45A2 nucleic acid sensitizer, and an SLC45A2 protein sensitizer.

[0023] In an eighth aspect, the present invention provides a disease detection method, comprising: detecting a disease detection marker using a kit having as a detection reagent a preparation that regulates the SLC45A2 gene or its expression product, wherein the detection reagent detects the expression level of the SLC45A2 gene or its expression product, and the diseases include tumors, autoimmune diseases, connective tissue diseases, infectious diseases, allergic diseases, chronic inflammatory diseases, and diseases related to immune balance disorders.

[0024] In some embodiments, the preparation includes any one or more of an SLC45A2 nucleic acid inhibitor, an SLC45A2 protein inhibitor, an SLC45A2 gene-deficient construct, an SLC45A2 nucleic acid agonist, an SLC45A2 protein agonist, an SLC45A2 nucleic acid activator, an SLC45A2 protein activator, an SLC45A2 nucleic acid antagonist, an SLC45A2 protein antagonist, an SLC45A2 nucleic acid promoter, an SLC45A2 protein promoter, an SLC45A2 nucleic acid enhancer, an SLC45A2 protein enhancer, an SLC45A2 nucleic acid sensitizer, and an SLC45A2 protein sensitizer.

[0025] In a ninth aspect, the present invention provides the use of the SLC45A2 gene or its expression product as an immune checkpoint.

[0026] The beneficial effects of the present invention are as follows: SLC45A2 on the surface of T cells is used as a target for immunotherapy. By regulating the SLC45A2 gene or its expression products (proteins or RNAs), the acid-base environmental balance of T cells is regulated, the ability of T cells to secrete cytotoxic cytokines is regulated, the process of T cell exhaustion is controlled, and thus the regulation of the immune function of T cells is achieved. This drug development path provides new ideas for immunotherapy, especially for cancer immunotherapy. Based on this, the present invention further develops a borate bioactive material targeting SLC45A2. This material can significantly reduce the expression of SLC45A2, enhance the anti-tumor immune effect of T cells, and show significant anti-tumor activity both in vitro and in vivo. As an SLC45A2 inhibitor, it is expected to become an alternative to immune checkpoint inhibitors or an important component in combination therapy regimens. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 It shows the expression and localization of SLC45A2 in cells detected by immunofluorescence in one embodiment;

[0028] Figure 2 It shows the expression of the cytotoxic cytokine IFN-γ in one embodiment;

[0029] Figure 3 It shows the expression of the cytotoxic cytokine IFN-γ under acidic culture conditions in one embodiment;

[0030] Figure 4 It shows the pH change curve in one embodiment;

[0031] Figure 5 It shows the expression of the SLC45A2 gene under different concentration conditions in one embodiment;

[0032] Figure 6 It shows the expression of the SLC45A2 gene at different intervention times in one embodiment;

[0033] Figure 7 It shows the Bulk RNA-seq sequencing results in one embodiment;

[0034] Figure 8 It shows the tumor growth curve in one embodiment;

[0035] Figure 9 It shows the tumor photos at the experimental end point in one embodiment;

[0036] Figure 10 It shows the situation of CD8+ T cells secreting IFN-γ cytokine in one embodiment;

[0037] Figure 11The situation of CD4+ T cells secreting IFN-γ cytokine in one embodiment;

[0038] Figure 12 The cell activity situation of B16 melanoma cells in one embodiment;

[0039] Figure 13 The cell activity situation of K7M2 osteosarcoma cells in one embodiment;

[0040] Figure 14 The cell proliferation inhibition situation of B16 melanoma cells in one embodiment;

[0041] Figure 15 The cell proliferation inhibition situation of K7M2 osteosarcoma cells in one embodiment;

[0042] Figure 16 The cell migration inhibition situation of B16 melanoma cells in one embodiment;

[0043] Figure 17 The mass percentage content of each element in the borate bioactive material in one embodiment. Detailed implementation manners

[0044] The technical solutions of this patent will be further described in detail below in conjunction with the specific implementation manners. It should be noted that the following detailed descriptions are all exemplary and are intended to provide further explanations for this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which this application belongs.

[0045] Example 1: Detection of the expression location of SLC45A2

[0046] Construct a stable transfection plasmid of SLC45A2 with a flag tag on pcDNA3.1 through Crisper-Cas9, and transfect the EC109 tumor cell line to obtain an EC109 tumor cell line overexpressing SLC45A2. After 24 hours, collect the cell precipitate. After fixing the cell precipitate with 0.1% Triton, label SLC45A2 (green) with anti-flag primary antibody and corresponding secondary antibody, label the cell nucleus (blue) with DAPI, and observe the distribution state of SLC45A2 after overexpression through confocal fluorescence microscopy imaging. As a control (CTRL), the EC109 cell line transfected with the empty vector pcDNA3.1 is processed in the same way.

[0047] Results: Figure 1 The immunofluorescence detection results are shown. It can be seen that the expression of SLC45A2 is distributed at multiple positions on the cell membrane and in the cytoplasm.

[0048] Example 2: Effect of conditional knockout of SLC45A2 on the expression of cytotoxic cytokine IFN-γ

[0049] (1) Wild-type mice C57BL6 / j were used to construct SLC45A2 gene knockout mice and CD4Cre+ genetically engineered mice. By breeding and genotyping the SLC45A2 gene knockout mice and CD4Cre genetically engineered mice, SLC45A2 mice with conditional knockout of the SLC45A2 gene on T cells were obtained. + / - CD4 Cre+ mice.

[0050] (2) CD4+ T cells in the spleens and lymph nodes of SLC45A2 + / - CD4 Cre+ mice were sorted and co-stimulated in vitro with CD3 and CD28 antibodies and IL-2 for 2 days. Then, the differentiated helper T cells (Th0 cells) were stimulated with a stimulant cocktail, and the expression level of the cytotoxic cytokine IFN-γ in T cells was detected by flow cytometry. As a control, CD4+ T cells in SLC45A2 mice were sorted and treated in the same way. + / - CD4 Cre- mice were sorted and treated in the same way. CD4+ T cells were treated in the same way.

[0051] Results: Figure 2 The expression of the cytotoxic cytokine IFN-γ is shown. It can be seen that, compared with the control group, the function of CD4+ T cells in SLC45A2 conditional knockout mice to secrete cytotoxic cytokines is enhanced, indicating that conditional knockout of SLC45A2 can enhance the anti-tumor function of T cells.

[0052] Example 3: Effect of conditional knockout of SLC45A2 on the expression of cytotoxic cytokine IFN-γ under acidic culture conditions

[0053] The operating steps of this example are basically the same as those of Example 2, except that in the 2-day in vitro co-stimulation induction, media with different gradient pH values were used for culture, that is, media with pH 6.0, 6.5 or 7.2 were used for culture.

[0054] Results: Figure 3 The expression of the cytotoxic cytokine IFN-γ under acidic culture conditions is shown. It can be seen that the level of IFN-γ secreted by T cells under acidic culture conditions decreases, while the ability of T cells to secrete IFN-γ after targeted knockout of SLC45A2 is restored to a certain extent, indicating that conditional knockout of SLC45A2 can reverse the inhibition of immune function by acidity.

[0055] Example 4: Preparation of Borate Bioactive Material 3B that Inhibits SLC45A2

[0056] The borate bioactive material (1393 - B3, hereinafter referred to as 3B) was prepared by the standard melt quenching method. Specifically, H 3 BO 3 , CaCO 3 , Na 2 CO 3 , 4MgCO 3 ·Mg(OH) 2 ·5H 2 O, K 2 CO 3 and NaH 2 PO 4 ·2H 2 O were uniformly mixed and placed in a platinum / rhodium crucible in a muffle furnace, and melted at 1150 °C for 120 min. Then, the glass melt was poured into an ice - water bath to be cooled into glass raw materials, and then crushed, ground and sieved through an antioxidant steel sieve to obtain the borate bioactive material 3B. Among them, the addition amounts of each component during mixing were: Na 2 CO 3 3.59 g, K 2 CO 3 9.362 g, 4MgCO 3 ·Mg(OH) 2 ·5H 2 O 6.58 g, CaCO 3 18.654 g, H 3 BO 3 58.237 g, NaH 2 PO 4 ·2H 2 O 5.284 g.

[0057] The obtained borate bioactive material 3B was fully dissolved in PBS buffer solution. During the dissolution process, a solid - liquid interface would be formed due to sedimentation. The pH values of the solid - liquid interface and the precipitate were measured at 1 min, 5 min, 10 min, 15 min, 20 min, 30 min, and at each whole - hour moment from 1 h to 24 h, and the initial pH value of the solution was 7.4.

[0058] Results: By molar percentage, the borate bioactive material 3B obtained in this example included 54 parts of boron trioxide, 22 parts of calcium oxide, 8 parts of magnesium oxide, 6 parts of sodium oxide, 8 parts of potassium oxide and 2 parts of diphosphorus pentoxide, and the average diameter was 10 μm. In addition, Figure 17 showed the mass percentage content of each element in the borate bioactive material 3B.

[0059] Figure 4 It shows the law of the change of pH at the interface and in the solution when the borate bioactive material 3B is dissolved in the PBS solution. It can be seen that the pH of the borate bioactive material 3B is stable at about 9.1 to 9.2, which indicates that the prepared borate bioactive glass 3B can produce an alkaline environment, that is, this material can regulate the acid-base environmental balance of T cells.

[0060] In addition, the network structure of the borate bioactive material is mainly composed of boron trioxide, and its basic structural unit is a planar triangle. These planar triangles are interconnected through six-membered boroxyl rings to form a glass network. In this embodiment, by adding alkali metal or alkaline earth metal ions as network modifiers and introducing modified oxides, the planar triangle can be transformed into a tetrahedral structure, enhancing the connectivity of the glass network. The modification of this network structure further affects the degradation rate of the borate bioactive material and the release rate of metal ions. When the network connectivity is enhanced, the degradation rate of the material slows down, and the surface ion release rate decreases, which helps to extend the duration of the therapeutic effect, maintain the stability of the local environment, provide a more adaptable drug release, and thus provide more effective therapeutic support for tumor immunotherapy.

[0061] Example 5: Effect of borate bioactive material 3B on the expression of SLC45A2 gene in vitro

[0062] The B16 melanoma cells were intervened in vitro with the borate bioactive material 3B prepared in Example 4 at 0.5 mg / mL and 1 mg / mL. After 24 h, the cell precipitate was collected, RNA was extracted and reverse transcribed into cDNA, and finally the expression of the SLC45A2 gene was detected by qRT-PCR. As a control, the B16 melanoma cells were intervened in vitro with PBS and treated in the same way.

[0063] Furthermore, the B16 melanoma cells were intervened with the borate bioactive material 3B prepared in Example 4 at 1 mg / mL for 3 h and 6 h. As a control, the expression of the SLC45A2 gene before the intervention was detected.

[0064] Results: Figure 5 and Figure 6The expression of the SLC45A2 gene under different conditions is shown. It can be seen that compared with the control group, after the intervention of the borate bioactive material 3B, the expression level of the SLC45A2 gene decreased, and with the increase of the concentration and action time of the borate bioactive material 3B, the degree of decrease in the expression level of the SLC45A2 gene was greater. This indicates that the borate bioactive material 3B can effectively inhibit the expression of SLC45A2 and has an anti-tumor effect in vitro.

[0065] Example 6: Effect of borate bioactive material 3B on the expression of SLC45A2 gene in vivo

[0066] (1) Female mice at 8 weeks of age were selected. After adaptive feeding, they were inoculated with mouse-derived B16-F10 melanoma cells at an inoculation number of 10 5 cells / mouse to construct a subcutaneous tumor-bearing model of wild-type mice. Starting from the 7th day after tumor inoculation, 100 μL of suspension was injected into the tumor every other day. This suspension was formed by the borate bioactive material 3B prepared in Example 4 premixed in PBS, with a concentration of 10 mg / mL. The tumor growth and mouse survival were continuously observed, and the experimental end point was set when the tumor volume reached 2000 mm 3 .

[0067] As a control, the constructed subcutaneous tumor-bearing model of wild-type mice was evenly divided into a drug administration group and a control group according to the size of subcutaneous tumor formation, with at least 5 mice in each group. The control group was injected with 100 μL of PBS solution into the tumor at the same time and under the same operation background.

[0068] (2) At the experimental end point, after euthanizing the mice, the melanoma tissues were taken out and dissociated to enrich the immune cells in the tumor tissues. Then, specific antibodies were used to label CD45+ immune cells and sorted by a flow cytometer. After that, the sorted CD45+ tumor-infiltrating immune cells were lysed with Trizol reagent, and then RNA was extracted and bulk RNA-seq library construction and sequencing analysis were performed. As a control, the control group mice were subjected to the same experimental operations.

[0069] Results: Figure 7 The bulk RNA-seq sequencing results are shown. It can be seen that the expression level of the SLC45A2 gene in the drug administration group was lower than that in the control group, indicating that the administration of the borate bioactive material 3B can reduce the expression of SLC45A2.

[0070] Example 7: Effect of borate bioactive material 3B on B16 melanoma

[0071] From the 7th day after tumor inoculation, the tumor volume of the mice in step (1) of Example 6 was measured every two days. At the end of the experiment, the tumor tissues were collected, photographed, the size of the tumors was measured, and the tumor growth curves were plotted based on the measurement results.

[0072] Results: Figure 8 The tumor growth curves are shown, Figure 9 and the tumor photos at the end of the experiment are shown. It can be seen that compared with the control group, the tumors of the mice in the administration group grew slower and the tumor volume at the end of the experiment was smaller, indicating that the application of borate bioactive material 3B can inhibit the growth of B16 melanoma.

[0073] Example 8: Effect of borate bioactive material 3B on the secretion of IFN-γ by T cells

[0074] At the end of the experiment, after euthanizing the mice constructed in step (1) of Example 6, part of the tumor tissue was taken to isolate tumor-infiltrating lymphocytes, and at the same time, lymphocytes in the inguinal draining lymph nodes and spleens of the mice were isolated. Through flow cytometry analysis, the proportions and cell numbers of CD4+ T cells and CD8+ T cells in the tumor microenvironment and their ability to secrete the cytokine IFN-γ were detected.

[0075] Results: Figure 10 and Figure 11 respectively show the secretion of IFN-γ cytokine by CD8+ T cells and CD4+ T cells. It can be seen that compared with the control group, the T cells of the mice in the administration group had a higher IFN-γ expression level, indicating that borate bioactive material 3B can enhance the ability of tumor-infiltrating T cells to secrete IFN-γ.

[0076] Example 9: Effect of borate bioactive material 3B on the activity of tumor cells

[0077] After digesting B16 melanoma cells and K7M2 osteosarcoma cells, the cells were seeded in a 96-well plate at a density of 20,000 cells per well, with a culture system of 200 μL per well. After the cells adhered, different concentrations of the borate bioactive material 3B solution prepared in Example 4 were added for incubation, and the concentration range was 0, 0.1, 0.5, 1.0, 2.5, 5, 10, 25, 50, 100 mg / mL. After 24 hours, the cell viability was detected using a CCK-8 kit.

[0078] Results: Figure 12 and Figure 13The cell viability of B16 melanoma cells and K7M2 osteosarcoma cells was shown respectively. It can be seen that the application of borate bioactive material 3B can reduce the cell viability of B16 melanoma cells and K7M2 osteosarcoma cells, and with the increase of the administration concentration, the cell viability decreases, that is, the greater the toxicity of borate bioactive material 3B to tumor cells. Among them, in the toxicity experiment of borate bioactive material 3B on B16 melanoma cells, the IC 50 value was 25 mg / mL, and in the toxicity experiment of borate bioactive material 3B on K7M2 osteosarcoma cells, the IC 50 value was 10 mg / mL.

[0079] Example 10: Effect of borate bioactive material 3B on tumor cell proliferation

[0080] After digestion, B16 melanoma cells and K7M2 osteosarcoma cells were inoculated into 96-well plates at a density of 3000 cells per well, with 200 μL of culture system in each well. After the cells adhered, different concentrations of the extraction solution of borate bioactive material 3B were added. This extraction solution was obtained by extracting borate bioactive material 3B with DMEM medium containing 10% FBS for 24 hours, and the concentration range was 0, 0.1, 0.25, 0.5, 1, 2, 3, 4, 6, 8, 10, 12, 14 mg / mL. Then, through the Sartorius Incucyte long-term real-time live cell analysis system, the dynamic changes during the whole cell growth process were visualized and quantitatively analyzed.

[0081] Results: Figure 14 and Figure 15 respectively showed the inhibition of cell proliferation of B16 melanoma cells and K7M2 osteosarcoma cells. It can be seen that the inhibitory effect of borate bioactive material 3B on the proliferation of B16 melanoma cells and K7M2 osteosarcoma cells increased with the increase of concentration. Among them, in the proliferation inhibition experiment of borate bioactive material 3B on B16 melanoma cells, the IC 50 value was 4.5 mg / mL, and in the proliferation inhibition experiment of borate bioactive material 3B on K7M2 osteosarcoma cells, the IC 50 value was 3.4 mg / mL.

[0082] Example 11: Effect of borate bioactive material 3B on tumor cell migration

[0083] After digestion, B16 melanoma cells were seeded in a 96-well plate at a density of 20,000 cells per well, with a culture system of 200 μL per well. After the cells adhered, different concentrations of the leaching solution of borate bioactive material 3B were added. The leaching solution was obtained by leaching borate bioactive material 3B with DMEM medium containing 10% FBS for 24 hours, and the concentration range was 0, 0.25, 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, 7.5, 10.0 mg / mL. Then, through the Sartorius Incucyte long-term real-time live cell analysis system, the dynamic changes during the whole cell growth process were visualized and quantitatively analyzed.

[0084] Results: Figure 16 The inhibition of cell migration of B16 melanoma cells was demonstrated. It can be seen that the inhibitory effect of borate bioactive material 3B on the migration of B16 melanoma cells increased with the increase in concentration. Among them, in the experiment of the inhibitory effect of borate bioactive material 3B on the proliferation of B16 melanoma cells, the EC 50 value was 4.0 mg / mL.

[0085] It should be understood that the above embodiments take the inhibitory effects of borate bioactive material 3B on B16 melanoma and K7M2 osteosarcoma as examples to introduce the specific implementation manners of controlling T cell immune function by regulating the SLC45A2 gene or its expression product. However, the technical solutions provided by the present invention have wide applicability in the treatment mechanism. In practical applications, it can also be extended to other melanoma cells, or other types of tumors, such as epithelial tissue tumors such as squamous cell carcinoma and adenocarcinoma, mesenchymal tissue tumors such as fibrosarcoma, liposarcoma, and leiomyosarcoma, lymphohematopoietic tissue tumors such as malignant lymphoma and various types of leukemia, nerve tissue tumors such as glioma and schwannoma, sex cord tumors such as sertoli cell tumor and stromal cell tumor, and germ cell tumors such as seminoma and dysgerminoma. It can also be used for the immunotherapy and drug development of autoimmune diseases, connective tissue diseases, infectious diseases, allergic diseases, and chronic inflammatory diseases, or other diseases related to immune function disorder or immune imbalance. This should not be a limitation to this application.

[0086] In addition, other drugs or biological materials can be developed based on the above treatment mechanisms to achieve immunotherapy targeting the SLC45A2 gene or its expression product, which should not be considered a limitation to this application. Meanwhile, the developed drugs or biological materials can also have regulatory effects such as agonist, activation, antagonism, promotion, enhancement, and sensitization on the SLC45A2 gene or its expression product to achieve the following effects: 1) regulating the acid-base balance of the environment where T cells are located; 2) regulating the ability of CD8+ T cells or CD4+ T cells to secrete cytotoxic cytokines; 3) regulating the proliferation of CD8+ T cells or CD4+ T cells; 4) regulating the exhaustion degree of CD8+ T cells or CD4+ T cells; 5) regulating the immune function of T cells; 6) regulating the efficacy or prognosis of immunotherapy, which should not be considered a limitation to this application.

[0087] Furthermore, based on the technical solutions provided by the present invention, other immunotherapy methods can be combined, or non-immunotherapy methods such as surgery, chemotherapy, radiotherapy, ablation, and intervention can be combined for treatment and drug development, which should not be considered a limitation to this application.

[0088] Related terms:

[0089] SLC45A2: It belongs to the solute carrier transporter membrane protein superfamily. It is mainly composed of a single double transmembrane fragment hairpin structure that undergoes three replications to produce six double transmembrane fragment units, and then is replicated into a 12 double transmembrane fragment protein. Current research has found that SLC45A2 encodes a transport protein that mediates melanin synthesis. Under normal conditions, SLC45A2 can promote the excretion of H+ in melanosomes and maintain the acid-base balance of melanosomes, thus ensuring the normal function of the activity of related enzymes; when the expression of SlC45A2 decreases, it can inhibit the activity of tyrosinase by acidifying the pH of melanosomes, resulting in blocked melanin synthesis.

[0090] Cell Counting Kit-8 (CCK-8): A detection reagent based on WST-8 that is widely used in the detection of cell proliferation and cytotoxicity. In the presence of the electron carrier 1-methoxy-5-methylphenazinium methyl sulfate, it is reduced by dehydrogenases in mitochondria to produce a highly water-soluble orange-yellow formazan product. The greater the cytotoxicity, the lighter the color. For the same cells, the depth of the color is proportional to the number of live cells.

[0091] The above are only some embodiments of the present invention. For those skilled in the art, without departing from the inventive concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention.

Claims

1. Use of a preparation having a regulatory effect on the SLC45A2 gene or its expression product in the preparation of an immunotherapy drug.

2. The use according to claim 1, characterized in that: The preparation includes any one or more of a SLC45A2 nucleic acid inhibitor, a SLC45A2 protein inhibitor, a SLC45A2 gene defect construct, a SLC45A2 nucleic acid agonist, a SLC45A2 protein agonist, a SLC45A2 nucleic acid activator, a SLC45A2 protein activator, a SLC45A2 nucleic acid antagonist, a SLC45A2 protein antagonist, a SLC45A2 nucleic acid promoter, a SLC45A2 protein promoter, a SLC45A2 nucleic acid enhancer, a SLC45A2 protein enhancer, a SLC45A2 nucleic acid sensitizer, and a SLC45A2 protein sensitizer.

3. The use according to claim 1, characterized in that: The preparation regulates the SLC45A2 gene or its expression product, and can play one or more of the following roles: 1) Regulate the acid-base balance of the environment in which T cells are located; 2) Regulate the ability of CD8+T cells or CD4+T cells to secrete cytokines; 3) Regulate the proliferation of CD8+T cells or CD4+T cells; 4) Regulate the degree of exhaustion of CD8+T cells or CD4+T cells; 5) Regulate T cell immune function; 6) Regulate the efficacy or prognosis of immunotherapy.

4. The use according to any one of claims 1 to 3, characterized in that: The immunotherapy drugs include tumor immunotherapy drugs.

5. The use according to claim 4, characterized in that: The preparation regulates the SLC45A2 gene or its expression product, and can play one or more of the following roles: 1) Improve the anti-tumor immune function of T cells; 2) Inhibit the proliferation or migration of tumor cells; 3) Inhibit the occurrence or development of tumors; 4) Inhibit tumor progression, recurrence or metastasis; 5) Improve the efficacy or prognosis of tumor immunotherapy.

6. Use of a preparation having a regulatory effect on the SLC45A2 gene or its expression product in the preparation of a disease detection kit, including: A preparation having a regulatory effect on the SLC45A2 gene or its expression product is used as a detection reagent of the kit, and the detection reagent detects the expression level of the SLC45A2 gene or its expression product. The diseases include tumors, autoimmune diseases, connective tissue diseases, infectious diseases, allergic diseases, chronic inflammatory diseases and diseases related to immune balance disorder.

7. The use according to claim 6, characterized in that: The preparation includes any one or more of a SLC45A2 nucleic acid inhibitor, a SLC45A2 protein inhibitor, a SLC45A2 gene defect construct, a SLC45A2 nucleic acid agonist, a SLC45A2 protein agonist, a SLC45A2 nucleic acid activator, a SLC45A2 protein activator, a SLC45A2 nucleic acid antagonist, a SLC45A2 protein antagonist, a SLC45A2 nucleic acid promoter, a SLC45A2 protein promoter, a SLC45A2 nucleic acid enhancer, a SLC45A2 protein enhancer, a SLC45A2 nucleic acid sensitizer, and a SLC45A2 protein sensitizer.

8. A borate bioactive material, characterized in that: In terms of molar percentage, the borate bioactive material includes 54 parts of boron trioxide, 22 parts of calcium oxide, 8 parts of magnesium oxide, 6 parts of sodium oxide, 8 parts of potassium oxide and 2 parts of phosphorus pentoxide.

9. The borate bioactive material according to claim 8, characterized in that The average diameter of the particles in the borate bioactive material is 1-10 μm.

10. A method for preparing a borate bioactive material, characterized in that: include: S1. Mix H3BO3, CaCO3, Na2CO3, 4MgCO3·Mg(OH)2·5H2O, K2CO3 and NaH2PO4·2H2O, and melt them at 1150-1200°C for 1-2h to obtain a melt; S2, quenching the molten body in ice water to obtain a crude material; and S3. Controlling the particle size of the crude material to obtain the borate bioactive material.

11. A kit, characterized in that: The invention comprises a detection reagent and an operating instruction, wherein the detection reagent is the borate bioactive material according to claim 8 or 9, or is prepared by the preparation method of the borate bioactive material according to claim 10.

12. Use of a borate bioactive material in screening or preparing a preparation having an inhibitory effect on the SLC45A2 gene or its expression product, wherein the borate bioactive material is the borate bioactive material according to claim 8 or 9, or is prepared by the method for preparing the borate bioactive material according to claim 10.

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