Application of bisphosphonate compound in preparation of antigen-specific activated T cell preparation

Abstract

The invention discloses an application of a bisphosphonate compound in preparation of an antigen-specific activated T cell preparation. The bisphosphonate compound is etidronate sodium as shown in a formula I; the clodronate sodium is shown as a formula II; the pamidronate sodium is shown as a formula III; the alendronate sodium trihydrate is as shown in a formula IV; zoledronic acid is as shown in formula V; the ibandronic acid is as shown in a formula VI; the risedronate sodium is shown as a formula VII; minodronic acid is shown as a formula VIII. The bisphosphonate compound can specifically activate T cells and remove target cells, and the target cells comprise various tumor cells, aging cells and infected cells.

Description

Technical Field

[0001] This application relates to the field of biomedical technologies, in particular to the use of bisphosphonate compounds in the preparation of antigen-specific T cell activating agents. Background Art

[0002] In the field of modern medicine, immunotherapy has become an important research direction for combating tumors, aging-related diseases, and infectious diseases. As a key component of the immune system, T cells play a crucial role in identifying and clearing abnormal cells in the body (such as tumor cells, senescent cells, infected cells, etc.). However, how to effectively activate T cells and enable them to exert antigen-specific killing functions has always been a key issue in immunotherapy research.

[0003] T cell-mediated specific activation and clearance of target cells rely on the antigen signal processed and presented by MHC mediated by the TCR receptor on the cell surface. Only when TCR-pMHC is correctly recognized can T cells successfully receive specific antigen signals; MHC class I molecules consist of the heavy chain α domain and β2-microglobulin (β2M), where β2M is encoded by the B2M gene and is crucial for stabilizing the spatial conformation of MHC I. Only when the complementarity-determining regions (CDRs) of TCR match the antigen peptide of pMHC and the polymorphic region of the MHC molecule simultaneously can the first signal for T cell activation be triggered. At the same time, the second signal provided by co-stimulatory molecules (such as CD28 / B7) further ensures the specificity and effectiveness of the immune response.

[0004] In the absence of TCR-pMHC recognition, even in the presence of co-stimulatory signals, T cells still cannot be activated and may even become dysfunctional. When the B2M gene is inhibited, the absence of β2M leads to the inability of MHC I to correctly assemble and present antigen peptides, TCR cannot recognize target cells, and the specific killing function of CTL is lost. Therefore, the integrity of the MHC class I antigen presentation pathway is an important prerequisite for the effectiveness of T cell immunotherapy.

[0005] Tumor cells express tumor-associated antigens through gene mutations or epigenetic alterations; virus-infected cells synthesize viral proteins intracellularly, which are degraded into antigen peptides by proteasomes and then presented to the cell surface by MHC I. After recognition by CTL, killing is rapidly initiated, and cytokines such as IFN-γ are secreted to inhibit virus replication; senescent cells accumulate due to DNA damage or telomere shortening, express senescence-associated secretory phenotype molecules on the surface, and secrete chemokines to recruit T cells. However, target cells can escape T cell killing through multiple mechanisms: tumor cells downregulate antigen presentation, virus-infected cells enter the latent period, and senescent cells activate anti-apoptosis pathways.

[0006] For these mechanisms, emerging therapies such as gene editing technology, bispecific antibodies, or oncolytic viruses are under development to enhance the specific clearance ability of T cells.

[0007] Bisphosphonate compounds are a class of drugs widely used in the treatment of bone diseases such as osteoporosis. For example, CN201380025570.X discloses: "An oral solution of bisphosphonate compounds such as zoledronic acid for the treatment or relief of pain or related conditions." In recent years, studies have found that bisphosphonate compounds not only have the effect of regulating bone metabolism but also have immunomodulatory functions.

[0008] For example, CN202310051620.7 discloses that PBMCs are first co-stimulated with bisphosphonate and coating solution to activate T cells, and then transfected with a vector containing CAR. The obtained CAR-T cells not only show strong killing effects on tumor cells with high expression of target antigens but also show very strong killing effects on tumor cells with significantly reduced expression of target antigens, which can improve the efficacy of CAR-T cell therapy, reduce drug resistance and recurrence of CAR-T cell therapy. This method is mainly used for the immunocyte therapy of hematological tumors and solid tumors. However, this method does not disclose how to reduce senescent cells to achieve the effect of delaying aging, let alone how to reduce infected cells to achieve the effect of anti-infection. Moreover, this method non-specifically activates T cells and cannot be applied to an antigen-specific activation environment.

[0009] The discovery that bisphosphonate compounds can antigen-specifically activate T cells and kill target cells has important clinical significance. In anti-tumor therapy, it provides a new strategy for tumor immunotherapy, promising to overcome the limitations of traditional treatment methods and improve the effect of tumor treatment.

[0010] The information disclosed in the background art section is only intended to increase the understanding of the overall background of the present invention and should not be regarded as an admission or any form of implication that this information constitutes the prior art already known to those of ordinary skill in the art. Summary of the Invention

[0011] The present application provides an application of a bisphosphonate compound in the preparation of an antigen-specific T cell activation preparation. The bisphosphonate compound can specifically activate T cells and eliminate target cells, and the target cells include various tumor cells, senescent cells, and infected cells.

[0012] The present application provides the use of a bisphosphonate compound in the preparation of an antigen-specific T cell activating preparation. The bisphosphonate compound is etidronate disodium as shown in Formula I; clodronate disodium as shown in Formula II; pamidronate disodium as shown in Formula III; alendronate sodium trihydrate as shown in Formula IV; zoledronic acid as shown in Formula V; ibandronic acid as shown in Formula VI; risedronate sodium as shown in Formula VII; minodronic acid as shown in Formula VIII:

[0013]

[0014] The bisphosphonate compound can antigen-specifically activate T cells and kill target cells; the target cells include: tumor cells, senescent cells, and infected cells.

[0015] Preferably, the tumor cells are at least one of lung cancer cell A549, glioblastoma U87, liver cancer cell MHCC97H, breast cancer cell BT549, breast cancer cell MDA-MB-231, colorectal cancer cell HCT116, SW620, pancreatic cancer cell BXPC-3, gastric cancer cell MKN-28, prostate cancer cell PC-3, melanoma cell B16F10, ovarian cancer cell SKOV3, cervical cancer cell Hela, osteosarcoma cell U2OS, renal cancer cell ACHN, and leukemia cell MOLT-4.

[0016] Preferably, the tumor cells are breast cancer cells MDA-MB-231.

[0017] Preferably, the infected cells are at least one of bacteria-infected cells, fungi-infected cells, and virus-infected cells.

[0018] Preferably, the bacteria-infected cells are Listeria-infected cells.

[0019] Preferably, the fungi-infected cells are Candida albicans-infected cells.

[0020] Preferably, the virus-infected cells are herpes simplex virus type 1-infected cells.

[0021] Preferably, the senescent cells are X-ray irradiation-induced senescent cells or H2O2-induced senescent cells.

[0022] Preferably, the dosage form of the preparation is an injection.

[0023] Preferably, after the bisphosphonate compound antigen-specifically activates T cells, it can reduce the proportion of tumor metastatic lung nodules in mice and reduce the positive rate of SA-β-gal in mice.

[0024] The beneficial effects that can be produced by the present application include:

[0025] 1) The application of the bisphosphonate compounds provided by the present application in the preparation of antigen-specific T cell activating preparations, based on the immunotherapy strategy of bisphosphonate compounds, provides new ideas and methods for the treatment of various diseases containing "non-self" antigens and has broad clinical application prospects. In the field of anti-aging, by clearing senescent cells, the aging process can be delayed and the symptoms of aging-related diseases can be improved. In the aspect of anti-infection treatment, the immune response of the body to pathogens can be enhanced and the cure rate of infectious diseases can be increased.

[0026] 2) The application of the bisphosphonate compounds provided by the present application in the preparation of antigen-specific T cell activating preparations found through animal experiments and cell experiments that bisphosphonate compounds can effectively reduce the number of various types of tumor cells, and at the same time can reduce the senescent cells induced by H2O2 oxidation and radiation induction, and can also reduce the number of cells infected by bacteria, fungi, and viruses; on the other hand, it can also reduce the proportion of metastatic lung nodules in mice, reduce the positive rate of SA-β-gal in mice, and can also reduce the viral load in the lungs of mice and reduce the number of fungi and bacteria infected in the livers of mice. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 : The results obtained in the bisphosphonate compound treatment group and the control group in Example 1; among them, the treatment object in A549-Con is lung cancer cells; the treatment object in A549-B2M ko is A549 lung cancer cells with the B2M gene knocked out; through this experiment, it is proved that bisphosphonate compounds activate T cells through an antigen-specific mechanism;

[0028] Figure 2 : The result diagram of zoledronic acid regulating T cell killing of various tumor cells obtained in Example 2;

[0029] Figure 3 : The result diagram of bisphosphonate regulating T cell clearance of senescent cells induced by H2O2 oxidation and radiation induction obtained in Example 3;

[0030] Figure 4 : The result diagram of bisphosphonate regulating T cell clearance of various infected cells obtained in Examples 4 to 6; among them: the result of listeria-infected HUVEC is the result of Example 4; the result of candida albicans-infected HUVEC is the result of Example 5; the result of herpes virus-infected 293T is the result of Example 6;

[0031] Figure 5 : The result diagram of zoledronic acid regulating T cell anti-tumor in mice obtained in Example 7; on the left are the tumor photos of each mouse in the administration group and the control group; on the right are the results of the proportion of metastatic lung nodules of the tumors of the mice in the administration group and the control group;

[0032] Figure 6: Results graph of zoledronic acid regulating T cells against mouse aging obtained in Example 8; on the left is the SA-β-gal staining result graph of mice in the blank group, aging model group, and drug administration group; on the left is the bar graph of the SA-β-gal positive rate of mice in the blank group, aging model group, and drug administration group;

[0033] Figure 7 : Results graph of zoledronic acid regulating T cells to reduce the liver bacterial load of mice infected with Listeria monocytogenes obtained in Example 9;

[0034] Figure 8 : Results graph of zoledronic acid regulating T cells to reduce the liver bacterial load of mice infected with Candida albicans obtained in Example 10;

[0035] Figure 9 : Results graph of zoledronic acid regulating T cells to reduce the pulmonary virus load of mice infected with HSV-1 obtained in Example 11;

[0036] Figure 10 : Chemical structural formulas of bisphosphonates used in each example. Detailed implementation manners

[0037] The present invention will be further described in detail below in conjunction with the drawings and embodiments, but the present invention is not limited in any way. Any transformation or improvement made based on the teachings of the present invention falls within the protection scope of the present invention.

[0038] Embodiment

[0039] In the following embodiments, the materials and instruments used are obtained from commercial channels without special instructions; the detection methods used are existing methods without special instructions.

[0040] In the following embodiments, the cell lines used in the experiments are all purchased from ATCC (American Type Culture Collection) and the relevant cells preserved in the seed cell bank of the Kunming Institute of Zoology, Chinese Academy of Sciences; the Listeria monocytogenes 19115 strain and Candida albicans strain (ATCC 14053) are purchased from ATCC in the United States, and HSV-1 (ab124004) is purchased from abcam.

[0041] The chemical structural formula of the bisphosphonate compound is as Figure 10As shown, all were purchased from MedChemExpress, USA: etidronate disodium (7414-83-7), formula I; clodronate disodium (22560-50-5), formula II; pamidronate disodium (57248-88-1), formula III; alendronate sodium trihydrate (121268-17-5), formula IV; zoledronic acid (118072-93-8), formula V; ibandronic acid (114084-78-5), formula VI; risedronate sodium (115436-72-1), formula VII; minodronic acid (180064-38-4), formula VIII.

[0042] Example 1: Verification of the activation of T cells by bisphosphonates through an antigen-specific mechanism

[0043] The pLenti-B2M vector was co-transfected with the lentiviral packaging plasmids psPAX2 and pMD2.G into 293T cells for lentiviral packaging. After culturing for 48 hours, the cell supernatant containing lentiviral particles was collected to infect cells (A549); after 72 hours, 3 mg / ml puromycin was added to screen out the cells with successful B2M knockout; T lymphocytes were isolated and cultured from human peripheral blood. The isolation used magnetic bead negative separation technology, and the kit used was Rosette Sep Human T Cell Enrichment Cocktail (#15061). The separation steps were carried out strictly according to the requirements of the kit instructions. The in vitro culture medium for T cells consisted of: RPMI 1640 + 10% FBS + 1% P / S (penicillin / streptomycin, Gibco, #15140-122) + 200 U / ml IL-2 (Gibco, #PHC0026); normal lung cancer cells (A549), B2M gene knockout lung cancer cells (A549-B2Mko), and T lymphocytes were counted respectively; after mixing evenly at a ratio of T cells: tumor cells = 100000:1000, they were evenly inoculated in 24-well plates; the above 8 bisphosphonates were respectively added to the cell culture system at a dose of 100 ng / ml, and the control group (CON) was added with the same volume of solvent (DMSO), and then placed in a 37°C cell culture incubator for 3 - 5 days. Observation was carried out through an inverted fluorescence microscope and the obtained results were photographed. The obtained test results are as Figure 1 shown. T cells treated with each bisphosphonate could effectively kill lung cancer cells (A549-Con), but could not kill A549 lung cancer cells with the B2M gene knocked out (A549-β2M ko ), proving that the above 8 bisphosphonate compounds all activate T cells and kill target cells through the mechanism of pMHC-specific antigen signals.

[0044] Example 2: Zoledronic acid activates T cells to kill various tumor cells

[0045] Glioblastoma U87, hepatocarcinoma cell line MHCC97H, breast cancer cell lines BT549 and MDA-MB-231, colorectal cancer cell lines HCT116 and SW620, pancreatic cancer cell line BXPC-3, gastric cancer cell line MKN-28, prostate cancer cell line PC-3, melanoma cell line B16F10, ovarian cancer cell line SKOV3, cervical cancer cell line Hela, osteosarcoma cell line U2OS, renal cancer cell line ACHN, and leukemia cell line MOLT-4 were cultured in vitro by existing methods. For convenient observation of test results, the tumor cells were fluorescently labeled with GFP or RFP.

[0046] The above-mentioned various tumor cells and T lymphocytes obtained by culturing were counted, and mixed according to 100,000 / well for T cells and 1,000 / well for tumor cells, and then evenly inoculated on 24-well plates; the above 8 bisphosphonate compounds were added to the wells of the cell culture system containing T cells and tumor cells at a concentration of 100 ng / ml. The control group (CON) was added with 100 ng / ml of solvent (DMSO) in the same cell culture system, and placed in a 37°C cell culture incubator for 2 days; the T cells were collected by centrifugation, washed 3 times with PBS, and then the T cells were inoculated in 24-well plates and cultured for another 24 h. Then, each tumor cell was inoculated into the 24-well plates containing T cells (the inoculation amount was 1×10*4 / well), and placed in a 37°C cell culture incubator for 3 - 5 days to observe the test results.

[0047] The results obtained were as Figure 2 shown, see Figure 2 . Compared with the control group, the number of tumor cells in the above 8 bisphosphonate treatment groups decreased significantly, indicating that T cells treated with bisphosphonates can effectively kill various tumor cells.

[0048] Example 3: Bisphosphonates activate T cells to eliminate senescent cells

[0049] Treatment group: After inoculating HUVEC (human umbilical vein endothelial cells) into 6-well plates (inoculation density 1×10^5 / well), DMEM complete medium containing 150 uM H2O2 was added to each well, and cultured at 37°C for 24 hours to induce senescent cells caused by reactive oxygen species damage;

[0050] In addition, HUVEC was inoculated in a 6-well plate containing DMEM complete medium at the same density. HUVEC was irradiated with 6 Gy of X-rays to induce cell senescence caused by radiation, and then continued to be cultured at 37 °C for 72 hours. The senescent cells induced by H2O2, the senescent cells induced by radiation, and T lymphocytes were counted respectively. According to the ratio of T cells: the number of each type of senescent cells being 100000 / well:1000 / well, T lymphocytes were added into the wells of each type of senescent plate, evenly inoculated in a 24-well plate, and then the above 8 bisphosphonates were respectively added to the cell culture system at a dose of 100 ng / ml;

[0051] The difference between the control group (CON) and the treatment group was only that the same volume of solvent (DMSO) was added instead of bisphosphonate;

[0052] After the control group and the treatment group were inoculated, they were placed in a cell culture incubator at 37 °C for 2 days; the T cells of each group were collected by centrifugation, washed 3 times with PBS, and then the T cells were inoculated in a 24-well plate and continued to be cultured. After 24 hours, the senescent HUVEC cells induced by H2O2 and radiation were respectively inoculated in a 24-well plate (1×10^4 / well), and placed in a cell culture incubator at 37 °C for 3 - 5 days to observe the test results. The obtained results are as Figure 3 shown.

[0053] See Figure 3 , compared with the control group, the T cells treated with bisphosphonate could effectively eliminate the senescent cells induced by H2O2 oxidation and radiation.

[0054] Example 4: Bisphosphonate activates T cells to eliminate bacteria-infected cells

[0055] HUVEC was cultured in vitro by the existing method, and Listeria was cultured overnight to the logarithmic growth phase by the existing method. After the cultured HUVEC and Listeria were washed 3 times with PBS, they were resuspended in serum-free medium; HUVEC was inoculated in a 24-well plate (density 5×10^4 / well), cultured at 37 °C until 80% confluence, Listeria was added (MOI = 10), and infected at 37 °C for 1 hour. The medium containing 50 ug / ml gentamicin was replaced, and continued to be cultured for 3 hours to remove extracellular bacteria. After washing 3 times with PBS, the complete medium was replaced and the culture was resumed for 12 hours. The infected cells and T lymphocytes were counted respectively. According to the ratio of T cells: the number of infected cells being 100000 / well:1000 / well, they were mixed evenly and inoculated in a 24-well plate to obtain a cell culture system.

[0056] In the bisphosphonate treatment group, the above 8 bisphosphonates (100 ng / ml) were added to the cell culture system; in the control group (CON), an equal volume of the solvent (DMSO) of the bisphosphonate was added; the bisphosphonate treatment group and the control group were placed in a cell culture incubator at 37°C for 2 days. T cells were collected by centrifugation, washed 3 times with PBS, and then the T cells were seeded in a 24-well plate and cultured for another 24 h. The HUVECs infected with bacteria were seeded in a 24-well plate (1×10^4 / well), and cultured in a cell culture incubator at 37°C for 3 - 5 days. The test results were observed as Figure 4 shown in Figure 4 . Compared with the control group, the number of Listeria in the results of the bisphosphonate treatment group was significantly reduced, indicating that the 8 bisphosphonates could all activate T cells to effectively clear bacteria-infected cells.

[0057] Example 5: Bisphosphonates activate T cells to clear fungal-infected cells

[0058] HUVECs were cultured in vitro. Candida albicans was cultured overnight to the logarithmic growth phase, washed 3 times with PBS, and then resuspended in serum-free medium; HUVECs were seeded in a 24-well plate (at a density of 5×10^4 / well), cultured at 37°C until 80% confluence, Candida albicans (MOI = 5) was added, and infected at 37°C for 2 hours. The medium containing 2 μg / ml amphotericin B was replaced, and the extracellular bacteria were removed by continued culture for 4 hours. After washing 3 times with PBS, the complete medium was replaced and the culture was resumed for 24 hours. The infected cells and T lymphocytes were counted respectively, and mixed according to the ratio of T cells: infected cells = 100000 / well: 1000 / well, and evenly seeded in a 24-well plate; bisphosphonate (100 ng / ml) was added to the cell culture system, and the control group (CON) was added with an equal volume of the solvent (DMSO), and cultured in a cell culture incubator at 37°C for 2 days; T cells were collected by centrifugation, washed 3 times with PBS, and then the T cells were seeded in a 24-well plate and cultured. After 24 h, the HUVECs infected with fungi were seeded in a 24-well plate (1×10^4 / well), and cultured in a cell culture incubator at 37°C for 3 - 5 days. The test results were observed as Figure 4 shown in Figure 4 . Compared with the control group, the number of Candida albicans in the results of the bisphosphonate treatment group was significantly reduced, indicating that the 8 bisphosphonates could all activate T cells to effectively clear Candida albicans-infected cells.

[0059] Example 6: Bisphosphonates regulate T cells to clear virus-infected cells

[0060] Vero cells were infected with HSV-1 (herpes simplex virus type 1) at an MOI of 0.1 to expand it, and the supernatant was collected after 48 h. 293T cells were seeded in 24-well plates (1×10^5 cells / well). When the confluence reached 80%, the cells were infected with HSV-1 at an MOI of 5. After 24 hours of infection at 37°C, the medium was replaced with a medium containing ganciclovir to block secondary infection, and then the fresh medium was replaced and the cells were cultured for another 24 h. The virus-infected cells and T lymphocytes were counted respectively, and they were mixed at a ratio of T cells:infected cells = 100000 / well:1000 / well and evenly seeded in 24-well plates. The above 8 bisphosphonates (100 ng / ml) were added to the cell culture system, and the control group (CON) was added with the same volume of solvent (DMSO). The cells were placed in a 37°C cell culture incubator and cultured for 2 days. The T cells were collected by centrifugation, washed 3 times with PBS, and then seeded in 24-well plates for further culture. After 24 h, the virus-infected 293T cells were seeded in 24-well plates (1×10^4 / well) and placed in a 37°C cell culture incubator for 3 - 5 days to observe the test results as Figure 4 shown, see Figure 4 . Compared with the control group, the number of herpes simplex virus type 1 in the results of the bisphosphonate treatment group was significantly reduced, indicating that the 8 bisphosphonates could activate T cells to effectively clear herpes simplex virus type 1-infected cells.

[0061] Example 7: Zoledronic acid regulates T cell anti-mouse tumor

[0062] 2×10^5 MDA-MB-231 cells in the logarithmic growth phase were injected into the left fourth mammary fat pad of female immunodeficient mice (NOG). After injection, the mice were randomly divided into a control group and a drug administration group, with 5 mice in each group.

[0063] Human-derived T cells (1×10*7 cells / mouse) were transfused into the mice in the drug administration group and the control group via the tail vein. The mice in the drug administration group were injected with the above 8 bisphosphonic acid compounds (at a dose of 0.2 mg / kg, 7 days /

[0064] time) via the tail vein, and the control group was injected with an equal volume of normal saline. T cells were infused again after 10 days and the drug administration continued.

[0065] When the tumors on the mice's mammary glands were visible, the length (L) and width (W) of the tumors were measured with vernier calipers. The tumor volume was calculated using the formula V = L×W2 / 2 (the tumor volume was calculated every three days). On the 30th day, all the mice were sacrificed. The in-situ tumors and lung tissues were dissected. The tumors of each mouse were as Figure 5 shown. And the proportion of tumor metastatic lung nodules in each mouse was counted, and the results were as Figure 5 shown.

[0066] See Figure 5It can be seen that there is no significant difference in the in-situ tumor size between the mice in the above 8 bisphosphate compound administration groups and the control group ( Figure 5 b), while the number of lung tumor metastases in the mice administered with the 8 bisphosphonate compounds is significantly less than that in the control group (78), and the average number of lung metastasis nodules in the above 8 bisphosphonate treatment groups is 19, 14, 15, 15, 16, 14, 15, and 14 respectively ( Figure 5 a, 5c).

[0067] Example 8: Zoledronic acid regulates T cells to resist mouse aging

[0068] Fifteen healthy BALB / c mice, with half males and half females, were randomly divided into 3 groups: blank group, aging model group, and administration group. Except for the blank group, the remaining mice were intraperitoneally injected with D-galactose at a dose of 200 mg / kg per day for 6 consecutive weeks. After the successful establishment of the aging model, the aging model group and the administration group were respectively intravenously injected with normal saline and the above 8 bisphosphonate compounds such as zoledronic acid (dose of 0.2 mg / kg, once every 7 days) through the tail vein every other day. The administration was continued for 2 weeks. After the end, the kidneys were taken for SA-β-gal staining to observe the proportion of senescent cells, and the results are as Figure 6 shown.

[0069] See Figure 6 a. It can be seen that the number of SA-β-gal increases in the aging model group, indicating that the modeling is successful. At the same time, it is found that the number of SA-β-gal cells in the above 8 bisphosphonate administration groups is significantly lower than that in the aging model group. By quantitatively counting the SA-β-gal positive rate, it can be known that the above 8 bisphosphonate compounds such as zoledronic acid can reduce the number and positive rate of SA-β-Gal cells in the renal cortex of senescent mice (6b).

[0070] Example 9: Zoledronic acid regulates T cells to reduce the liver bacterial load of mice infected with Listeria

[0071] Inject the Listeria monocytogenes cultured in Example 4 (2×10^4 CFU / mouse) into BALB / c mice (half male and half female) via the tail vein. Then randomly divide the mice into two groups, namely the control group and the drug-administered group, with 5 mice in each group. 12 hours after infection, the above 8 bisphosphonate compounds are injected via the tail vein (at a dose of 0.2 mg / kg), and the control group is injected with an equal volume of normal saline. 3 days after infection, after sacrificing the mice by cervical dislocation, soak the mice in 75% alcohol for 5 min, and then transfer the mice to a sterile operating table; expose the abdomen of the mice, separate the skin with sterile ophthalmic scissors and forceps, take 100 mg of the liver tissue of the mice, and place it in a cell culture dish containing 1 ml of sterile PBS: thoroughly grind the liver of the mice with the end of a sterile syringe; thoroughly grind the liver of the mice using a grinder; use the prepared 0.025% Tritonx-100 solution to perform gradient dilution on the ground liver tissue of the mice, suck 200 ul of the tissue suspension obtained after gradient dilution onto a BHI solid medium, spread the plate and culture it at 37°C for 24 h, count the number of colonies growing on the medium, and the tissue bacterial load (CFU) = the number of colonies on each medium on average / 0.2 × dilution factor. The results are as Figure 7 shown. See Figure 7 It can be seen that the average liver bacterial loads of the mice in the treatment groups of the above 8 bisphosphonate compounds are 1.99×10^3 CFU / g, 1.58×10^3 CFU / g, 2.63×10^3 CFU / g, 3.02×10^3 CFU / g, 1.58×10^3 CFU / g, 1.20×10^3 CFU / g, 1.20×10^3 CFU / g, and 1.99×10^3 CFU / g respectively, which are significantly lower than those of the control group mice (1.1×10^5 CFU / g).

[0072] Example 10: Zoledronic acid regulates T cells to reduce the liver fungal load in mice infected with Candida albicans

[0073] Inject the prepared Candida albicans (1×10^6 CFU / mouse) into BALB / c mice (half male and half female) via the tail vein. Then randomly divide the mice into two groups, namely the control group and the drug-administered group, with 5 mice in each group. 12 hours after infection, the mice in the drug-administered group are injected with the above 8 bisphosphate compounds via the tail vein (at a dose of 0.2 mg / kg), and the control group is injected with an equal volume of normal saline. 3 days after infection, sacrifice the mice by cervical dislocation and take the liver, perform tissue homogenization at 100 mg tissue / mL PBS, perform serial gradient dilution on the tissue homogenate, spread the plate on a YPD solid culture plate, and perform colony counting after culturing at 37°C for 24 h to analyze the fungal load in the tissue. The results are as Figure 8 shown. See Figure 8It can be seen that the average fungal loads in the livers of the mice in the treatment groups of the above 8 bisphosphonate compounds were 1.25×10^4CFU / g, 1.58×10^4CFU / g, 7.94×10^3CFU / g, 6.30×10^3CFU / g, 1.25×10^4CFU / g, 7.94×10^3CFU / g, 1.0×10^4CFU / g, and 5.021×10^3CFU / g respectively, which were significantly lower than those of the control group mice (1.58×10^8CFU / g).

[0074] Example 11: Zoledronic acid regulates T cells to reduce the pulmonary viral load in mice infected with HSV-1

[0075] Take the HSV-1 virus and thaw it flat on ice. Dilute the virus solution to 1×10^7 / 40ul; Anesthetize BALB / c mice (half male and half female) with a mixture of chloral hydrate and xylazine hydrochloride. Use a pipette to aspirate 40ul of the evenly mixed virus solution and slowly drip it into the nasal cavity of the mice. Then randomly divide the mice into two groups, namely the control group and the drug-added group, with 5 mice in each group. 24 hours after infection, the mice in the drug administration group were injected with the above 8 bisphosphonate compounds (at a dose of 0.2mg / kg) via the tail vein, and the control group was injected with an equal volume of normal saline. The mice were sacrificed on the 3rd day after infection. First, take 100mg of the mouse lung tissue and place it in a centrifuge tube containing 1mL of pre-cooled sterile PBS. Homogenize it on ice until the tissue is completely broken, and then centrifuge it at 4℃ and 3000rpm for 10 minutes. Collect the supernatant as the original virus solution. Dilute the supernatant 10-fold serially with cell culture medium. Use a 6-well plate filled with a monolayer of Vero E6 cells. After aspirating the original culture medium, add 100μL of virus solution at different dilution factors to each well, and adsorb it at 37℃ for 1 hour. Gently shake it during this period to ensure uniform adsorption. Subsequently, add a maintenance medium overlay containing 1.5% agarose, and incubate it in an incubator at 37℃ and 5% CO2 for 2-5 days. After plaque formation, develop the image by crystal violet staining. Count the number of clear plaques in each dilution well, and the results are as Figure 9 shown. See Figure 9 It can be seen that compared with the average number of 32 plaques in the control group, the average number of plaques in the treatment groups of the above 8 bisphosphate compounds were 13, 13, 12, 11, 11, 9, 11, and 5 respectively, and the treatment groups were significantly reduced.

[0076] Example 12

[0077] Using etidronate disodium as the raw material, an injection with an etidronate disodium concentration of 0.25% was prepared by mixing it with sterile pure water.

[0078] Example 13

[0079] Using clodronate disodium as the raw material, an oral liquid with an etidronate disodium concentration of 10% was prepared by mixing it with sterile pure water.

[0080] Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions recorded in the foregoing embodiments or perform equivalent replacements for some of the technical features. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. Use of a bisphosphonate compound in the preparation of an antigen-specific T cell activating preparation, characterized in that, Bisphosphonate compounds are etidronate disodium as formula I; clodronate disodium salt as formula II; pamidronate disodium salt as formula III; alendronate sodium trihydrate as formula IV; zoledronic acid as formula V; ibandronic acid as formula VI; sodium risedronate as formula VII; minidronic acid as formula VIII: Bisphosphonate compounds can antigen-specifically activate T cells and kill target cells; target cells include: tumor cells, senescent cells, infected cells. Tumor cells are at least one of lung cancer cell A549, glioblastoma U87, liver cancer cell MHCC97H, breast cancer cell BT549, breast cancer cell MDA-MB-231, colorectal cancer cells HCT116, SW620, pancreatic cancer cell BXPC-3, gastric cancer cell MKN-28, prostate cancer cell PC-3, melanoma cell B16F10, ovarian cancer cell SKOV3, cervical cancer cell Hela, osteosarcoma cell U2OS, renal cancer cell ACHN, leukemia cell MOLT-4.

2. Use of the bisphosphonate compound according to claim 1 in the preparation of an antigen-specific T cell activating preparation, characterized in that, Tumor cells are breast cancer cell MDA-MB-231.

3. Use of the bisphosphonate compound according to claim 1 in the preparation of an antigen-specific T cell activating preparation, characterized in that, Infected cells are at least one of bacteria-infected cells, fungi-infected cells, virus-infected cells.

4. Use of the bisphosphonate compound according to claim 1 in the preparation of an antigen-specific T cell activating preparation, characterized in that, Bacteria-infected cells are Listeria-infected cells.

5. Use of the bisphosphonate compound according to claim 1 in the preparation of an antigen-specific T cell activating preparation, characterized in that, Fungi-infected cells are Candida albicans-infected cells.

6. Use of the bisphosphonate compound according to claim 1 in the preparation of an antigen-specific T cell activating preparation, characterized in that, Virus-infected cells are herpes simplex virus type 1-infected cells.

7. Use of the bisphosphonate compound according to claim 1 in the preparation of an antigen-specific T cell activating preparation, characterized in that, Senescent cells are X-ray irradiation-induced senescent cells or H2O2-induced senescent cells.

8. Use of the bisphosphonate compound according to claim 1 in the preparation of an antigen-specific T cell activating preparation, characterized in that, The dosage form of the preparation is injection or oral liquid.

9. Use of the bisphosphonate compound according to claim 1 in the preparation of an antigen-specific T cell activating preparation, characterized in that, After bisphosphonate compounds antigen-specifically activate T cells, they can reduce the proportion of tumor metastatic lung nodules in mice and reduce the positive rate of SA-β-gal in mice.

10. Use of the bisphosphonate compound according to claim 1 in the preparation of an antigen-specific T cell activating preparation, characterized in that, ​

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