Drug development targeting SLC45a2 and application in immunotherapy

Abstract

The present invention relates to the field of biomedicine, and specifically relates to drug development targeting SLC45A2 and application in immunotherapy. In the present invention, SLC45A2 on the surface of T cells is used as a target for immunotherapy. By regulating the SLC45A2 gene or an expression product thereof, the acid-base environment balance of the T cells is regulated and controlled, the ability of the T cells to secrete cytotoxic cytokines is regulated, and the exhaustion process of the T cells is controlled, thereby achieving the regulation and control of the immune function of the T cells. Based on this, in the present invention, a borate bioactive material targeting SLC45A2 is further developed. The material can significantly reduce the expression of SLC45A2, enhance the anti-tumor immune effect of the T cells, and exhibit significant anti-tumor effects both in vivo and in vitro.

Description

Drug development and immunotherapy applications targeting SLC45A2 Technical Field

[0001] This invention relates to the field of biomedicine, specifically to the development of drugs targeting SLC45A2 and their application in immunotherapy. Background Technology

[0002] Immune checkpoint inhibitor therapy, as a T-cell immunotherapy approach, has opened up new treatment pathways in the field of cancer treatment, with 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] Ipilimumab, an immune checkpoint inhibitor targeting CTLA4, was approved by the FDA in 2011 for the treatment of unresectable stage III / IV melanoma, significantly improving survival by 22% of patients, extending it to 3 years or longer. PD-1-targeting drugs, including pembrolizumab and nivolumab, became the first FDA-approved PD-1 inhibitors for the treatment of refractory and unresectable melanoma in 2014, with clinical data showing superior efficacy compared to traditional treatment regimens. Furthermore, atezolizumab, targeting PD-L1, was approved in 2014 for the treatment of urothelial carcinoma, significantly improving survival outcomes in 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 significant survival benefits to these patients.

[0004] Despite significant progress in cancer treatment, T-cell immunotherapy still faces limitations in clinical application. In particular, only a small percentage of patients with advanced cancer benefit from it. The objective response rate for immunotherapy monotherapy is only about 20%, with most patients being only partial responders. Furthermore, even among those who initially respond, approximately one-third develop resistance during treatment, ultimately leading to tumor recurrence. This phenomenon can be primarily attributed to the immunosuppressive state within the tumor microenvironment.

[0005] The immune status of the tumor microenvironment is a complex and multidimensional system involving various cell types, molecular factors, and signaling pathways, which interact to influence tumor growth and metastasis. Among these factors, T cell exhaustion is considered one of the key mechanisms of immunosuppression 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 altered cell fate programming. Specifically, T cell exhaustion manifests as a reduction in the secretion of cytotoxic cytokines (such as IFN-γ, TNF-α, and IL-2), leading to a significant weakening of the cytotoxic effect of T cells against tumors, thereby limiting the efficacy of immunotherapy.

[0006] Studies have shown that T cell exhaustion is closely related to the acidic tumor microenvironment. The tumor microenvironment is typically weakly acidic, which is closely related to the metabolic characteristics of tumors, especially aerobic glycolysis. High levels of glycolysis are often associated with poor tumor prognosis and poor response to immune checkpoint inhibitor therapy. During glycolysis, tumor cells produce large amounts of hydrogen ions, lactic acid, and pyruvate, leading to increased acidity in the microenvironment. This acidic tumor environment not only inhibits T cell function but also promotes T cell exhaustion, increasing the proportion of exhausted T cells. This provides favorable conditions for tumor cells to evade immune surveillance and clearance, thereby exacerbating tumor immune escape and leading to poor prognosis. Therefore, the acidic tumor microenvironment is one of the important mechanisms leading to T cell exhaustion and tumor immunosuppression, and it is closely related to tumor treatment efficacy, prognosis, and response to immunotherapy. Developing drugs that can effectively regulate the acidity of the tumor microenvironment, thereby enhancing anti-tumor immune responses, is particularly urgent; however, no such drugs have yet entered clinical application.

[0007] Currently, research on cellular acid-base homeostasis regulation mainly focuses on the GPCR proton transport family (such as the four subtypes GPCR4, GPCR65, GPCR68, and GPCR132). Although these transport families play a crucial role in regulating cellular acid-base homeostasis, their potential role in tumor immune regulation is poorly understood, and related research remains scarce. Therefore, developing new targeted drugs to enhance anti-tumor immune responses by regulating cellular acid-base homeostasis has significant theoretical and clinical application value. Summary of the Invention

[0008] This invention uses SLC45A2 as a target for immunotherapy, providing a method for drug development and immunotherapy applications.

[0009] In a first aspect, the present invention provides the use of a formulation having a regulatory effect on the SLC45A2 gene or its expression product in the preparation of an immunotherapeutic drug, comprising: using the formulation 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 formulation comprises any one or more of the following: SLC45A2 nucleic acid inhibitor, SLC45A2 protein inhibitor, SLC45A2 gene defect construct, SLC45A2 nucleic acid agonist, SLC45A2 protein agonist, SLC45A2 nucleic acid activator, SLC45A2 protein activator, SLC45A2 nucleic acid antagonist, SLC45A2 protein antagonist, SLC45A2 nucleic acid promoter, SLC45A2 protein promoter, SLC45A2 nucleic acid enhancer, SLC45A2 protein enhancer, SLC45A2 nucleic acid sensitizer, and SLC45A2 protein sensitizer.

[0011] In some embodiments, the formulation regulates the SLC45A2 gene or its expression product, and may perform one or more of the following functions: 1) regulating the acid-base balance of the environment in which T cells reside; 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 degree of exhaustion of CD8+ T cells or CD4+ T cells; 5) regulating T cell immune function; 6) regulating the efficacy or prognosis of immunotherapy.

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

[0013] In some embodiments, the formulation regulates the SLC45A2 gene or its expression product, and may have one or more of the following effects: 1) enhancing the anti-tumor immune function of T cells; 2) inhibiting tumor cell proliferation or migration; 3) inhibiting tumor occurrence or development; 4) inhibiting tumor progression, recurrence or metastasis; 5) improving the efficacy or prognosis of tumor immunotherapy.

[0014] Secondly, the present invention provides the application of a formulation that has a regulatory effect on the SLC45A2 gene or its expression product in the preparation of a disease detection kit, comprising: using the formulation that has a regulatory effect on the SLC45A2 gene or its expression product as a detection reagent of the kit, wherein the detection reagent detects the expression level of the SLC45A2 gene or its expression product, and the disease includes tumors, autoimmune diseases, connective tissue diseases, infectious diseases, allergic diseases, chronic inflammatory diseases, and diseases related to immune imbalance.

[0015] In some embodiments, the formulation comprises any one or more of the following: SLC45A2 nucleic acid inhibitor, SLC45A2 protein inhibitor, SLC45A2 gene defect construct, SLC45A2 nucleic acid agonist, SLC45A2 protein agonist, SLC45A2 nucleic acid activator, SLC45A2 protein activator, SLC45A2 nucleic acid antagonist, SLC45A2 protein antagonist, SLC45A2 nucleic acid promoter, SLC45A2 protein promoter, SLC45A2 nucleic acid enhancer, SLC45A2 protein enhancer, SLC45A2 nucleic acid sensitizer, and SLC45A2 protein sensitizer.

[0016] Thirdly, the present invention provides a borate bioactive material, which, by molar percentage, comprises 54 parts boron trioxide, 22 parts calcium oxide, 8 parts magnesium oxide, 6 parts sodium oxide, 8 parts potassium oxide, and 2 parts phosphorus pentoxide.

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

[0018] Fourthly, the present invention provides a method for preparing borate bioactive materials, characterized by comprising: S1, mixing H3BO3, CaCO3, Na2CO3, 4MgCO3·Mg(OH)2·5H2O, K2CO3 and NaH2PO4·2H2O, and melting at 1150-1200℃ for 1-2 hours to obtain a melt; S2, quenching the melt in ice water to obtain a coarse material; and S3, adjusting the particle size of the coarse material to obtain the borate bioactive material.

[0019] Fifthly, the present invention provides a kit comprising a detection reagent and an instruction manual, wherein the detection reagent is the above-mentioned borate bioactive material, or is prepared by the above-mentioned method for preparing borate bioactive material.

[0020] In a sixth aspect, the present invention provides the use of borate bioactive materials in screening or preparing formulations that have an inhibitory effect on the SLC45A2 gene or its expression product, wherein the borate bioactive material is the aforementioned borate bioactive material, or is prepared by the aforementioned method for preparing borate bioactive materials.

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

[0022] In some embodiments, the formulation comprises any one or more of the following: SLC45A2 nucleic acid inhibitor, SLC45A2 protein inhibitor, SLC45A2 gene defect construct, SLC45A2 nucleic acid agonist, SLC45A2 protein agonist, SLC45A2 nucleic acid activator, SLC45A2 protein activator, SLC45A2 nucleic acid antagonist, SLC45A2 protein antagonist, SLC45A2 nucleic acid promoter, SLC45A2 protein promoter, SLC45A2 nucleic acid enhancer, SLC45A2 protein enhancer, SLC45A2 nucleic acid sensitizer, and SLC45A2 protein sensitizer.

[0023] Eighthly, the present invention provides a disease detection method, comprising: detecting a disease biomarker using a kit that uses a preparation having a regulatory effect on the SLC45A2 gene or its expression product as a detection reagent, wherein the detection reagent detects the expression level of the SLC45A2 gene or its expression product, and the disease includes tumors, autoimmune diseases, connective tissue diseases, infectious diseases, allergic diseases, chronic inflammatory diseases, and diseases related to immune imbalance.

[0024] In some embodiments, the formulation comprises any one or more of the following: SLC45A2 nucleic acid inhibitor, SLC45A2 protein inhibitor, SLC45A2 gene defect construct, SLC45A2 nucleic acid agonist, SLC45A2 protein agonist, SLC45A2 nucleic acid activator, SLC45A2 protein activator, SLC45A2 nucleic acid antagonist, SLC45A2 protein antagonist, SLC45A2 nucleic acid promoter, SLC45A2 protein promoter, SLC45A2 nucleic acid enhancer, SLC45A2 protein enhancer, SLC45A2 nucleic acid sensitizer, and 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 this invention are that by using SLC45A2 on the surface of T cells as a target for immunotherapy, and by regulating the SLC45A2 gene or its expression products (protein or RNA), the acid-base balance of T cells is controlled, thereby regulating the ability of T cells to secrete cytotoxic cytokines, controlling the T cell exhaustion process, and thus achieving regulation of T cell immune function. This drug development pathway provides a new approach to immunotherapy, especially tumor immunotherapy. Based on this, this 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 exhibit significant anti-tumor activity both in vivo and in vitro. As an SLC45A2 inhibitor, it is expected to become an alternative to immune checkpoint inhibitors or an important component of combination therapy regimens. Attached Figure Description

[0027] Figure 1 shows the immunofluorescence detection of SLC45A2 expression localization in cells in one embodiment;

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

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

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

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

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

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

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

[0035] Figure 9 is a tumor photograph at the experimental endpoint in one embodiment;

[0036] Figure 10 shows the secretion of IFN-γ cytokines by CD8+ T cells in one embodiment;

[0037] Figure 11 shows the secretion of IFN-γ cytokines by CD4+ T cells in one embodiment;

[0038] Figure 12 shows the cell viability of B16 melanoma cells in one embodiment;

[0039] Figure 13 shows the cell viability of K7M2 osteosarcoma cells in one embodiment;

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

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

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

[0043] Figure 17 shows the mass percentage of each element in the borate bioactive material in one embodiment. Detailed Implementation

[0044] The technical solution of this patent will be further described in detail below with reference to specific embodiments. It should be noted that the following detailed descriptions are exemplary and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0045] Example 1: Detection of SLC45A2 expression sites

[0046] A flag-tagged SLC45A2 stable transfection plasmid was constructed on pcDNA3.1 using Crisper-Cas9, and transfected into the EC109 tumor cell line to obtain an EC109 tumor cell line overexpressing SLC45A2. After 24 hours, cell pellet was collected, fixed with 0.1% Triton, and labeled with anti-flag primary antibody and corresponding secondary antibody (green). Cell nuclei were labeled with DAPI (blue), and the distribution of SLC45A2 after overexpression was observed using confocal fluorescence microscopy. As a control (CTRL), EC109 cell lines transfected with the empty vector pcDNA3.1 underwent the same treatment.

[0047] Results: Figure 1 shows the results of immunofluorescence detection. It can be seen that SLC45A2 expression is distributed in multiple locations in the cell membrane and cytoplasm.

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

[0049] (1) SLC45A2 gene knockout mice and CD4Cre+ genetically engineered mice were constructed using wild-type mouse C57BL6 / j. Through mating and gene identification of the SLC45A2 gene knockout mice and CD4Cre genetically engineered mice, SLC45A2 mice with conditional SLC45A2 gene knockout on T cells were obtained. + / -CD4 Cre+ Mice.

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

[0051] Results: Figure 2 shows the expression of the cytotoxic cytokine IFN-γ. It can be seen that, compared to the control group, CD4+ T cells in SLC45A2 conditional knockout mice exhibited enhanced secretion of cytotoxic cytokines, 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 the cytotoxic cytokine IFN-γ under acidic culture conditions.

[0053] The operation steps in this embodiment are basically the same as those in Example 2. The difference is that during the two-day in vitro co-stimulation induction, culture media with different gradient pH values ​​are used, that is, culture media with pH values ​​of 6.0, 6.5 or 7.2 are used.

[0054] Results: Figure 3 shows the expression of the cytotoxic cytokine IFN-γ under acidic culture conditions. It can be seen that the level of IFN-γ secreted by T cells is reduced under acidic culture conditions, while the ability of T cells to secrete IFN-γ is restored to some extent after targeted knockout of SLC45A2. This indicates that conditional knockout of SLC45A2 can reverse the inhibitory effect of acidity on immune function.

[0055] Example 4: Preparation of borate bioactive material 3B for inhibiting SLC45A2

[0056] Borate bioactive materials (1393-B3, abbreviated as 3B) were prepared using a standard melt-quenching method. Specifically, H3BO3, CaCO3, Na2CO3, 4MgCO3·Mg(OH)2·5H2O, K2CO3, and NaH2PO4·2H2O were uniformly mixed using a ternary mixer and placed in a platinum / rhodium crucible in a muffle furnace, where they were melted at 1150℃ for 120 min. The glass melt was then cooled in an ice-water bath to obtain a glass raw material, which was subsequently pulverized, ground, and sieved through an antioxidant steel sieve to obtain borate bioactive material 3B. The amounts of each component added during mixing were: Na2CO3 3.59 g, K2CO3 9.362 g, 4MgCO3·Mg(OH)2·5H2O 6.58 g, CaCO3 18.654 g, H3BO3 58.237 g, and NaH2PO4·2H2O 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 was formed due to sedimentation. The pH of the solid-liquid interface and the precipitate was measured at each hour from 1 min, 5 min, 10 min, 15 min, 20 min, 30 min, and from 1 h to 24 h. The initial pH of the solution was 7.4.

[0058] Results: By molar percentage, the borate bioactive material 3B obtained in this embodiment comprises 54 parts boron trioxide, 22 parts calcium oxide, 8 parts magnesium oxide, 6 parts sodium oxide, 8 parts potassium oxide, and 2 parts phosphorus pentoxide, with an average diameter of 10 μm. Furthermore, Figure 17 shows the mass percentage content of each element in the borate bioactive material 3B.

[0059] Figure 4 shows the changes in pH over time at the interface and in the solution of borate bioactive material 3B dissolved in PBS. It can be seen that the pH of borate bioactive material 3B is stable at around 9.1 to 9.2, indicating that the prepared borate bioactive glass 3B can generate an alkaline environment, meaning that this material can regulate the acid-base balance of T cells.

[0060] Furthermore, the network structure of borate bioactive materials is mainly composed of boron trioxide, and its basic structural unit is... Planar triangles, interconnected by six-membered boron oxide rings, form a glass network. This embodiment introduces modified oxides by adding alkali metal or alkaline earth metal ions as network modifiers, which can... Plane triangle transformed into The tetrahedral structure enhances the connectivity of the glass network. This modification of the network structure further affects the degradation rate and metal ion release rate of the borate bioactive material. Increased network connectivity slows down the material's degradation rate and reduces surface ion release, helping to prolong the duration of the therapeutic effect, maintain local environmental stability, and provide more adaptive drug release, thus offering more effective therapeutic support for tumor immunotherapy.

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

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

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

[0064] Results: Figures 5 and 6 show the expression of the SLC45A2 gene under different conditions. It can be seen that, compared to the control group, the expression level of the SLC45A2 gene decreased after intervention with borate bioactive material 3B. Furthermore, the decrease in SLC45A2 gene expression level increased with increasing concentration and duration of borate bioactive material 3B, indicating that borate bioactive material 3B can effectively inhibit the expression of SLC45A2 and has in vitro antitumor effects.

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

[0066] (1) Select 8-week-old female mice and, after acclimatization, feed them at 10 5 A wild-type mouse subcutaneous tumor-bearing model was established by inoculating mice with B16-F10 melanoma cells at a seeding density of 100 μL / mouse. Starting from day 7 after tumor implantation, 100 μL of a suspension, consisting of borate bioactive material 3B prepared in Example 4 premixed in PBS at a concentration of 10 mg / mL, was injected into the tumor every other day. Tumor growth and mouse survival were continuously monitored, with the experimental endpoint set at a tumor volume of 2000 mm². 3 .

[0067] As a control, the established wild-type mouse subcutaneous tumor-bearing model was evenly divided into a treatment group and a control group according to the size of the subcutaneous tumor, 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 operating conditions.

[0068] (2) At the experimental endpoint, mice were euthanized, melanoma tissue was removed and dissociated, and immune cells in the tumor tissue were enriched. CD45+ immune cells were then labeled with specific antibodies and sorted using a flow cytometry system. Subsequently, the sorted CD45+ tumor-infiltrating immune cells were lysed using Trizol reagent, and RNA was extracted and subjected to bulk RNA-seq library construction and sequencing analysis. As a control, control mice underwent the same experimental procedures.

[0069] Results: Figure 7 shows the Bulk RNA-seq sequencing results. It can be seen that the SLC45A2 gene expression level in the treatment group was lower than that in the control group, indicating that the application of borate bioactive material 3B can reduce the expression of SLC45A2.

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

[0071] Starting from day 7 of tumor implantation, the tumor volume of the mice in step (1) of Example 6 was measured every two days. At the end of the experiment, tumor tissue was collected and photographed to measure the size of the tumor and to plot the tumor growth curve based on the measurement results.

[0072] Results: Figure 8 shows the tumor growth curve, and Figure 9 shows the tumor photograph at the experimental endpoint. It can be seen that, compared with the control group, the tumors in the treated mice grew more slowly and the tumor volume at the experimental endpoint 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 IFN-γ secretion by T cells

[0074] At the end of the experiment, the mice constructed in step (1) of Example 6 were euthanized, and tumor-infiltrating lymphocytes were isolated from a portion of the tumor tissue. At the same time, lymphocytes from the inguinal lymph nodes and spleen of the mice were isolated. The proportion, number and ability of CD4+ T cells and CD8+ T cells to secrete the cytokine IFN-γ in the tumor microenvironment were detected by flow cytometry sorting analysis.

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

[0076] Example 9: Effects of borate bioactive material 3B on tumor cell activity

[0077] After digesting B16 melanoma cells and K7M2 osteosarcoma cells, the cells were seeded at a density of 20,000 cells per well in 96-well plates, with 200 μL of culture medium per well. Once the cells adhered, they were incubated with different concentrations of the borate bioactive material 3B solution prepared in Example 4, ranging from 0, 0.1, 0.5, 1.0, 2.5, 5, 10, 25, 50, and 100 mg / mL. Cell viability was assessed using a CCK-8 assay kit after 24 hours.

[0078] Results: Figures 12 and 13 show the cell viability of B16 melanoma cells and K7M2 osteosarcoma cells, respectively. It can be seen that application of borate bioactive material 3B can reduce the cell viability of both B16 melanoma cells and K7M2 osteosarcoma cells, and the cell viability decreases with increasing concentration, indicating that the toxicity of borate bioactive material 3B to tumor cells is increasing. Specifically, in the toxicity experiment of borate bioactive material 3B on B16 melanoma cells, the IC50 value was... 50 The value was 25 mg / mL. In the toxicity experiment of borate bioactive material 3B against K7M2 osteosarcoma cells, the IC50 value was... 50 The value is 10 mg / mL.

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

[0080] B16 melanoma cells and K7M2 osteosarcoma cells were digested and seeded at a density of 3000 cells per well in 96-well plates, with 200 μL of culture medium per well. After cell attachment, different concentrations of borate bioactive material 3B extract were added. This extract was obtained by extracting borate bioactive material 3B in DMEM medium containing 10% FBS for 24 hours, with concentrations ranging from 0, 0.1, 0.25, 0.5, 1, 2, 3, 4, 6, 8, 10, 12, and 14 mg / mL. Subsequently, the dynamic changes throughout the cell growth process were visualized and quantitatively analyzed using the Sartorius Incucyte long-term real-time live cell analysis system.

[0081] Results: Figures 14 and 15 show the cell proliferation inhibition of B16 melanoma cells and K7M2 osteosarcoma cells, respectively. It can be seen that the inhibitory effect of borate bioactive material 3B on the proliferation of B16 melanoma cells and K7M2 osteosarcoma cells increases with increasing concentration. Specifically, in the experiment on the proliferation inhibition of B16 melanoma cells by borate bioactive material 3B, IC50... 50 The value was 4.5 mg / mL. In the experiment on the inhibition of K7M2 osteosarcoma cell proliferation by borate bioactive material 3B, the IC50 value was 4.5 mg / mL. 50 The value was 3.4 mg / mL.

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

[0083] B16 melanoma cells, after digestion, were seeded at a density of 20,000 cells per well in 96-well plates, with 200 μL of culture medium per well. After cell attachment, different concentrations of borate bioactive material 3B extract were added. This extract was obtained by extracting borate bioactive material 3B in DMEM medium containing 10% FBS for 24 hours, with concentrations ranging from 0, 0.25, 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, 7.5, and 10.0 mg / mL. Subsequently, the dynamic changes throughout the cell growth process were visualized and quantitatively analyzed using the Sartorius Incucyte long-term real-time live cell analysis system.

[0084] Results: Figure 16 shows the inhibition of B16 melanoma cell migration. It can be seen that the inhibitory effect of borate bioactive material 3B on the migration of B16 melanoma cells increases with increasing concentration. Specifically, in the experiment on the inhibition of B16 melanoma cell proliferation by borate bioactive material 3B, EC... 50 The value is 4.0 mg / mL.

[0085] Example 12: Application of formulations that regulate the SLC45A2 gene or its expression product

[0086] This embodiment provides the application of a formulation that regulates the SLC45A2 gene or its expression product in the preparation of an immunotherapeutic drug. The formulation, which regulates the SLC45A2 gene or its expression product, is used as the active ingredient and mixed with physiologically acceptable excipients to obtain an immunotherapeutic drug. This drug regulates the acid-base balance of T cells, modulates the ability of T cells to secrete cytotoxic cytokines, controls the T cell exhaustion process, and achieves regulation of T cell immune function, thus providing a new drug development strategy for immunotherapy.

[0087] Furthermore, this embodiment provides an immunotherapy method comprising: administering to a subject an agent that modulates the SLC45A2 gene or its expression product, or administering to a subject a drug having an agent that modulates the SLC45A2 gene or its expression product as an active ingredient. This method treats various diseases caused by abnormal or disordered immune system function by modulating the SLC45A2 gene or its expression product in vitro and in vivo.

[0088] Furthermore, this embodiment provides the application of a formulation that regulates the SLC45A2 gene or its expression product in the preparation of a disease detection kit. This includes using the formulation as a detection reagent, along with an instruction manual, to obtain a disease monitoring kit. This kit can be used for the diagnosis and early screening of target diseases, providing a reliable detection tool for clinical use.

[0089] Furthermore, this embodiment provides a disease detection method, comprising: using a kit that uses a preparation having a regulatory effect on the SLC45A2 gene or its expression product as a detection reagent to detect disease biomarkers, and detecting the expression level of the SLC45A2 gene or its expression product. Through analysis of the detection results, this method can clarify the occurrence, development status, or treatment effect of the target disease, thereby providing data support for personalized treatment.

[0090] Meanwhile, this embodiment provides the use of the SLC45A2 gene or its expression product as an immune checkpoint. This immune checkpoint offers an innovative direction for the development of targeted immunotherapies and may significantly improve the efficacy and applicability of existing immunotherapies.

[0091] It should be understood that the above embodiments, using the inhibitory effect of borate bioactive material 3B on B16 melanoma and K7M2 osteosarcoma as an example, illustrate a specific implementation method for controlling T-cell immune function by regulating the SLC45A2 gene or its expression product. However, the technical solution provided by this invention has broad applicability in terms of treatment mechanism. In practical applications, it can also be extended to other melanomas or other types of tumors, such as epithelial tissue tumors like squamous cell carcinoma and adenocarcinoma, mesenchymal tissue tumors like fibrosarcoma, liposarcoma, and leiomyosarcoma, lymphohematopoietic tissue tumors like malignant lymphoma and various types of leukemia, neurological tissue tumors like glioma and schwannoma, sex cord tumors like Sertoli cell tumors and stromal cell tumors, and germ cell tumors like seminoma and dysgerminoma. It can also be used for immunotherapy, disease detection, and drug development for autoimmune diseases, connective tissue diseases, infectious diseases, allergic diseases, and chronic inflammatory diseases, or other diseases related to immune dysfunction or immune imbalance. This should not be construed as a limitation of this application.

[0092] Furthermore, other drugs or biomaterials can be developed based on the above-mentioned treatment mechanisms to achieve immunotherapy targeting the SLC45A2 gene or its expression product, which should not be considered a limitation of this application. At the same time, the developed drugs or biomaterials can also exert regulatory effects on the SLC45A2 gene or its expression product, such as inhibition, activation, antagonism, promotion, enhancement, and sensitization, to achieve the following effects: 1) regulating the acid-base balance of the T cell environment; 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 degree of CD8+ T cell or CD4+ T cell exhaustion; 5) regulating T cell immune function; 6) regulating the efficacy or prognosis of immunotherapy, etc., which should also not be considered a limitation of this application.

[0093] Furthermore, based on the technical solutions provided by this invention, other immunotherapy methods can be combined, or non-immunotherapy methods such as surgery, chemotherapy, radiotherapy, ablation, intervention, and targeted therapy can be combined for treatment and drug development. This should not be construed as a limitation of this application.

[0094] Related terms:

[0095] SLC45A2 belongs to the solute carrier transporter membrane protein superfamily. It mainly consists of a single transmembrane hairpin fragment that undergoes triple replication to produce six transmembrane fragment units, which are then replicated into 12 transmembrane fragment proteins. Current research has found that SLC45A2 encodes a transporter protein that mediates melanin synthesis. Under normal conditions, SLC45A2 promotes the expulsion of H+ from melanosomes, maintaining the acid-base balance of melanosomes and thus ensuring the normal function of related enzymes. However, when SLC45A2 expression decreases, it can inhibit the activity of tyrosinase by acidifying the pH of melanosomes, thereby hindering melanin synthesis.

[0096] Cell Counting Kit-8 (CCK-8): A widely used assay based on WST-8 for cell proliferation and cytotoxicity. In the presence of the electron carrier 1-methoxy-5-methylphenazineonium sulfate dimethyl ester, it is reduced by mitochondrial dehydrogenases to a highly water-soluble orange-yellow formazan product. The greater the cytotoxicity, the lighter the color; for the same number of cells, the intensity of the color is directly proportional to the number of viable cells.

[0097] The above descriptions are merely some embodiments of the present invention. Those skilled in the art can make various modifications and improvements without departing from the inventive concept of the present invention, and these all fall within the scope of protection of the present invention.

Claims

1. Application of formulations that regulate the SLC45A2 gene or its expression product in the preparation of immunotherapeutic drugs.

2. Use according to claim 1, characterized in that, The formulation comprises any one or more of the following: SLC45A2 nucleic acid inhibitor, SLC45A2 protein inhibitor, SLC45A2 gene defect construct, SLC45A2 nucleic acid agonist, SLC45A2 protein agonist, SLC45A2 nucleic acid activator, SLC45A2 protein activator, SLC45A2 nucleic acid antagonist, SLC45A2 protein antagonist, SLC45A2 nucleic acid promoter, SLC45A2 protein promoter, SLC45A2 nucleic acid enhancer, SLC45A2 protein enhancer, SLC45A2 nucleic acid sensitizer, and SLC45A2 protein sensitizer.

3. Use according to claim 1, characterized in that, The formulation regulates the SLC45A2 gene or its expression product, and may achieve one or more of the following effects: 1) Regulate the acid-base balance of the environment in which T cells reside; 2) Regulate the ability of CD8+ T cells or CD4+ T cells to secrete cytotoxic 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 the immune function of T cells; 6) Regulate the efficacy or prognosis of immunotherapy.

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

5. Use according to claim 4, characterized in that, The formulation regulates the SLC45A2 gene or its expression product, and may achieve one or more of the following effects: 1) Enhance T cell anti-tumor immune function; 2) Inhibit tumor cell proliferation or migration; 3) Inhibit tumor occurrence or development; 4) Inhibit tumor progression, recurrence or metastasis; 5) Improve the efficacy or prognosis of tumor immunotherapy.

6. Use of an agent having a modulating effect on the SLC45A2 gene or its expression product in the manufacture of a disease detection kit, comprising: The kit uses a formulation that regulates the SLC45A2 gene or its expression product as a detection reagent. 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 imbalance.

7. Use according to claim 6, characterized in that, The formulation comprises any one or more of the following: SLC45A2 nucleic acid inhibitor, SLC45A2 protein inhibitor, SLC45A2 gene defect construct, SLC45A2 nucleic acid agonist, SLC45A2 protein agonist, SLC45A2 nucleic acid activator, SLC45A2 protein activator, SLC45A2 nucleic acid antagonist, SLC45A2 protein antagonist, SLC45A2 nucleic acid promoter, SLC45A2 protein promoter, SLC45A2 nucleic acid enhancer, SLC45A2 protein enhancer, SLC45A2 nucleic acid sensitizer, and SLC45A2 protein sensitizer.

8. A borate biologically active material, characterized by, According to the molar percentage, the borate bioactive material comprises 54 parts boron trioxide, 22 parts calcium oxide, 8 parts magnesium oxide, 6 parts sodium oxide, 8 parts potassium oxide and 2 parts phosphorus pentoxide.

9. The borate biologically active material of claim 8, wherein, The average diameter of the particles in the borate bioactive material is 1-10 μm.

10. A method for producing a borate bioactive material, characterized by, include: S1. Mix H3BO3, CaCO3, Na2CO3, 4MgCO3·Mg(OH)2·5H2O, K2CO3 and NaH2PO4·2H2O, and melt at 1150-1200℃ for 1-2 hours to obtain a melt. S2. Quench the melt in ice water to obtain a coarse material; and S3. The particle size of the coarse material is controlled to obtain the borate bioactive material.

11. A kit characterized in that, It includes a test reagent and an instruction manual, wherein the test reagent is the borate bioactive material as described in claim 8 or 9, or is prepared by the method for preparing the borate bioactive material as described in claim 10.

12. The use of borate bioactive materials in screening or preparing formulations that inhibit 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.

13. A method of immunotherapy comprising: The administration of a formulation that regulates the SLC45A2 gene or its expression product to a subject, or the administration of a drug that uses a formulation that regulates the SLC45A2 gene or its expression product as an active ingredient to a subject.

14. The immunotherapy method of claim 13, wherein, The formulation comprises any one or more of the following: SLC45A2 nucleic acid inhibitor, SLC45A2 protein inhibitor, SLC45A2 gene defect construct, SLC45A2 nucleic acid agonist, SLC45A2 protein agonist, SLC45A2 nucleic acid activator, SLC45A2 protein activator, SLC45A2 nucleic acid antagonist, SLC45A2 protein antagonist, SLC45A2 nucleic acid promoter, SLC45A2 protein promoter, SLC45A2 nucleic acid enhancer, SLC45A2 protein enhancer, SLC45A2 nucleic acid sensitizer, and SLC45A2 protein sensitizer.

15. A method of disease detection comprising: A kit is used to detect disease biomarkers by using a preparation that has a regulatory effect on the SLC45A2 gene or its expression product as a detection reagent. 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 imbalance.

16. The disease detection method according to claim 13, characterized in that, The formulation comprises any one or more of the following: SLC45A2 nucleic acid inhibitor, SLC45A2 protein inhibitor, SLC45A2 gene defect construct, SLC45A2 nucleic acid agonist, SLC45A2 protein agonist, SLC45A2 nucleic acid activator, SLC45A2 protein activator, SLC45A2 nucleic acid antagonist, SLC45A2 protein antagonist, SLC45A2 nucleic acid promoter, SLC45A2 protein promoter, SLC45A2 nucleic acid enhancer, SLC45A2 protein enhancer, SLC45A2 nucleic acid sensitizer, and SLC45A2 protein sensitizer.

17. The use of the SLC45A2 gene or its expression product as an immune checkpoint.

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