Combination therapy of Clostridium ghonii and tumor angiogenesis inhibitors

Abstract

This invention relates to the application of Clostridium ghonii in combination with tumor angiogenesis inhibitors in cancer treatment. This invention is the first to discover that the combination of Clostridium ghonii with low-dose tumor angiogenesis inhibitors can reduce the infiltration of M2-type macrophages, MDSCs, etc. in tumors, reduce the number of TGFβ in tumors, and reduce the suppressive effect of anti-tumor immune responses in TME. At the same time, the combination of low-dose tumor angiogenesis inhibitors can also suppress the CD8 + , CD3 + T, F4 / 80 + It can promote the infiltration of immune cells such as IL-1, IL-2, and IL-1 into tumors, improve the immune microenvironment in tumors, and enhance the antitumor therapeutic effect. The present invention can be applied to advanced malignant solid tumor types, and by combining Clostridium ghonii with low doses of Aitan, it can reduce the suppressive effect of antitumor immune response in the TME, changing the TME from an immunosuppressive state to an immune activated state, thereby achieving highly efficient antitumor treatment.

Description

[Technical Field]

[0001] [CROSS-REFERENCE TO RELATED APPLICATIONS] This application claims priority from a Chinese patent application filed with the China Patent Office on October 9, 2021, bearing application number CN202111177878.9 and entitled "Application of the combined use of Clostridium ghonii bacteria and tumor angiogenesis inhibitors," the entire contents of which are incorporated herein by reference.

[0002] The present invention belongs to the field of oncology technology and relates to the combination of Clostridium ghonii and tumor angiogenesis inhibitors, and in particular to the application of the combination of Clostridium ghonii and tumor angiogenesis inhibitors to relieve the tumor immunosuppressive microenvironment, improve the tumor microenvironment, and enhance the tumor therapeutic effect. [Background technology]

[0003] The tumor microenvironment (TME) is the local, steady-state environment of tumor growth, consisting of tumor cells, stromal cells, the extracellular matrix, and biomolecules infiltrated therein. The TME provides the material foundation necessary for tumor initiation, development, and invasion, and regulates various biological processes such as tumor metastasis and recurrence. At the same time, the TME can increase tumor drug resistance and radiation resistance, reducing therapeutic efficacy. Immune regulation within the TME plays an important role in tumor initiation and growth and can form local tumor immunosuppression through various mechanisms. How to control the TME immunotherapeutic strategies and reconstruct a positive immune microenvironment are both key and challenging points in antitumor therapy.

[0004] Malignant tumors evade host immune surveillance through various mechanisms, including lymphocyte infiltration damage, increased expression of immune checkpoint proteins due to hypoxia, recruitment of Tregs, and the creation of an immunosuppressive tumor microenvironment that impairs the function of resident and trafficking immune effector cells. Medullary cells that infiltrate the tumor microenvironment play an important role in regulating tumor progression, including immune escape and metastasis.

[0005] Solid tumors typically infiltrate large amounts of immune inhibitors, such as tumor-associated macrophages (TAMs), myeloid-derived suppressor cells (MDSCs), and regulatory T cells (Tregs). TGFβ plays an important role in the immune response of the TME, promoting antitumor immunosuppression and potentially causing resistance to antiangiogenic therapy.

[0006] Anoxia or hypoxia is a typical characteristic of solid tumors. Oxygen deficiency in solid tumors can directly increase the expression of the immune checkpoint protein PD-L1 in MDSCs, dendritic cells, and tumor cells through activation of HIF-1α, thereby supporting immune suppression and evasion. At the same time, tumor hypoxia can increase the invasive potential of tumor cells by promoting the induction of migration-promoting proteins (e.g., SDF1A and HGF) and the production of invasive extracellular matrix molecules. Oxygen deficiency also enhances tumor resistance to radiochemotherapy.

[0007] Clostridium ghonii is an anaerobic bacterium that germinates and grows abundantly only in anoxic or necrotic tumor areas, effectively destroying the TME without discriminating between tumor tissues. After Clostridium ghonii lyses tumors, it alters TME immunogenicity, modulates the immunosuppressive TME, and induces tumor immune responses. Currently, there are no reports suggesting that the combination of Clostridium ghonii with tumor angiogenesis inhibitors can significantly alter the tumor immunosuppressive microenvironment and enhance antitumor effects. Summary of the Invention [Problem to be solved by the invention]

[0008] In response to the shortcomings of the prior art, the present invention provides a safer and more targeted application of the anaerobic bacterium Clostridium ghonii in combination with a tumor angiogenesis inhibitor to treat solid tumors.

[0009] Application of Clostridium ghonii in combination with tumor angiogenesis inhibitors in the manufacture of pharmaceutical products for tumor treatment. It is a drug for treating tumors whose active ingredients include Clostridium ghonii and a tumor angiogenesis inhibitor. According to a preferred embodiment of the present invention, the Clostridium ghonii bacterium is Clostridium ghonii strain MW-DCG-LCv-26 (Australian National Metrology Research Institute). institution The strain is a domesticated strain of Clostridium ghonii (stored at the Australian National Metrology Research Institute under the strain collection number V12 / 001486). institution The strain is stored in the Australian National Metrology Research Center (ANCRC) under the strain storage number V12 / 001485, or the MW-DCG-CCv-17 strain (Australian National Metrology Research Center). institution and the strain collection number is V12 / 001487). According to a preferred embodiment of the present invention, the Clostridium ghonii bacteria are in the spore form. According to a preferred embodiment of the present invention, the tumor angiogenesis inhibitor is selected from Aitan, sunitinib, pazopanib, bevacizumab, ramucirumab, conbercept, aflibercept, sorafenib tosylate, and regorafenib. According to a preferred embodiment of the present invention, the Clostridium ghonii bacterium and the tumor angiogenesis inhibitor, which are the medicinal ingredients, are administered in the order of successive administration or simultaneous administration. According to a preferred embodiment of the present invention, the drug combination is 1×10 7 The optimal dose of Aitan was 60mg / kg / d in combination with freeze-dried powder of CFU Clostridium ghonii spores. According to a preferred embodiment of the present invention, the tumor includes, but is not limited to, colon cancer, Lewis lung cancer, nasopharyngeal carcinoma, non-small cell lung cancer, fibrosarcoma, or melanoma. The present invention allows for random combination of tumor angiogenesis inhibitors. The Clostridium ghonii spores must be pure microorganisms, i.e., they must be free of any other bacteria other than the spores, and the tumor angiogenesis inhibitors must also meet the requirements for sterility. The Clostridium ghonii spores are prepared and purified according to methods known in the art to obtain drugs that meet the relevant quality standards. [Effects of the Invention]

[0010] 1. The present invention is the first to discover that the combination of safer, more targeted Clostridium ghonii with tumor angiogenesis inhibitors can achieve highly effective antitumor therapy. 2. This invention is the first to discover that the combination of Clostridium ghonii and low-dose tumor angiogenesis inhibitors reduces the infiltration of M2 macrophages, MDSCs, and other cells in tumors, reduces the number of TGFβ in tumors, and reduces the suppressive effect of anti-tumor immune responses in the TME. At the same time, the combination of low-dose tumor angiogenesis inhibitors also reduces the CD8 + , CD3 + T, F4 / 80 + This can promote the infiltration of immune cells such as IL-1 and IL-2 into tumors, improve the immune microenvironment in tumors, and enhance the anti-tumor therapeutic effect. 3. In this invention, the optimal dose of Clostridium ghonii combined with a tumor angiogenesis inhibitor was clarified. 4. This invention can be applied to advanced malignant solid tumors. By combining Clostridium ghonii with low-dose Aitan, the inhibitory effect of anti-tumor immune responses in the TME can be reduced, thereby transforming the TME from an immunosuppressive to an immunoactivated state, achieving highly effective anti-tumor treatment. Its unique therapeutic effect is not due to the highly effective anti-tumor effect of using high-dose Aitan to exacerbate tumor anoxia, creating an oxygen-deficient environment in which Clostridium ghonii can proliferate, but rather to the change in the anti-tumor immune microenvironment. 5. The composition of the present invention is safer and more targeted when treating tumors, and can germinate only in the oxygen-deficient environment of the targeted tumor, but cannot germinate in the oxygen-deficient environment of non-tumor tissue. [Brief explanation of the drawings]

[0011] [Figure 1] FIG. 1 is a histogram of the net weight in Example 2. [Figure 2] FIG. 2 is a TTC stained image of cerebral infarction tissue in Example 2. [Figure 3] FIG. 3 shows a Gram stain of the tissue slice in Example 2. [Figure 4] FIG. 4 is a TTC stained image of myocardial infarction tissue in Example 2. [Figure 5] FIG. 5 shows a Gram stain of the tissue slice in Example 2. [Figure 6] FIG. 6 is a histogram of the net weight of the experimental mice in Example 3. [Figure 7] FIG. 7 is a graph showing the tumor volume curve of the experimental mice in Example 3. [Figure 8] FIG. 8 is a graph showing tumor weights of experimental mice in Example 3. [Figure 9] FIG. 9 is an anatomical diagram of the tumor in the experimental mouse in Example 3. [Figure 10] FIG. 10 shows the percentage of T cell infiltration in tumor tissues in Example 4. [Figure 11] Figure 11 shows the cytokine expression status of T cells in tumor tissues. [Figure 12] FIG. 12 shows the cytokine expression status in mouse tumors in Example 4. [Figure 13] 13 and 14 show the proportions of myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs) in mouse tumors in Example 4. [Figure 14] [Figure 15] 15 and 16 show immunohistochemical staining images of mouse tumor tissues and the percentage of CD163 cells in Example 4. [Figure 16] DETAILED DESCRIPTION OF THE INVENTION

[0012] Hereinafter, the present invention will be described in more detail with reference to specific embodiments, and the described embodiments are part of the enumerated embodiments for the purpose of facilitating public understanding, and are only used to explain the present invention, rather than limiting the protection scope of the present invention, which is defined by the claims. Example 1

[0013] Materials and Methods

[0014] Clostridium ghonii spores freeze-dried powder for injection. The strain is MW-DCG-LCv-26. This strain was approved by the Australian National Metrology Research Institute. institution The strain is stored under the strain number V12 / 001486 and the batch number is 202003001-1. The freeze-dried powder for injection of Clostridium ghonii spores is developed by Shandong Xinchuang Bioscience and Technology Co., Ltd. The active ingredient is Clostridium ghonii spores, and 1% sucrose is added. The freeze-dried powder is prepared by the following freeze-drying steps: -40°C for 4 hours, -35°C for 10 minutes with simultaneous vacuum application, -30°C for 10 minutes, -25°C for 10 minutes, -20°C for 26 hours, -15°C for 2 hours, -10°C for 10 minutes, -5°C for 10 minutes, 0°C for 10 minutes, 10°C for 2 hours, 15°C for 10 minutes, 20°C for 3 hours, and 27°C for 3 hours. The specifications are 1 x 10 8CFU / cell; control freeze-dried powder, lot number: 201910002F, developed by Shandong Xinchuang Bioscience and Technology Co., Ltd. and produced by the above freeze-drying process with 1mL of 1% sucrose solution; 0.9% sodium chloride injection, lot number: 2005062146, sold by Chenxin Pharmaceutical Co., Ltd.; sterile water for injection, lot number: 190221262, sold by Chenxin Pharmaceutical Co., Ltd.

[0015] Cell lines and cell culture

[0016] CT26.WT colon cancer cells, product code: 3131C0001000800037, Cell Resources Center, Shanghai Institutes for Life Sciences, Chinese Academy of Sciences. RPMIMedium 1640 basic medium was supplemented with 10% (volume percentage) fetal bovine serum (floating particles may be present in the serum after low-temperature lysis; remove the floating particles by centrifugation at 2000 rpm for 3 minutes) and a 1.1% (volume percentage) penicillin-streptomycin mixture. The cells were resuspended in a 75cm tube. 2 The cells were inoculated into cell culture bottles, 25 mL of cell culture medium was added to each bottle, and the bottle was placed in a 5% CO2 cell culture box and cultured at 37°C.

[0017] reagent

[0018] RPMI Medium 1640 basic medium was purchased from Gibco, special grade fetal bovine serum and penicillin-streptomycin were purchased from BI, Aitan (apatinib) was purchased from Jiangsu Hengrui Pharmaceutical Co., Ltd., and Clostridium ghonii spores were prepared and purified according to conventional methods to obtain drugs that met the relevant quality standards.

[0019] Model Building

[0020] CT26 tumor cells were subcutaneously inoculated into BALB / c mice to establish a colon cancer subcutaneous tumor model. The extract concentration was 7.5 × 10 6 / mL (tolerance: 6.75 x 10 6pieces / mL~8.25×10 6 0.2 mL of the cell suspension (cells / mL) was slowly injected subcutaneously into the axilla of the right forelimb of a mouse (previously disinfected with 75% alcohol). After the injection was completed, the needle site was gently pressed with a dry cotton ball.

[0021] Observation and testing

[0022] Clinical observation: During the administration period, the animals were observed once every morning and afternoon with the naked eye to check their behavior, whether they were dead or in the moribund state, etc. Tumor measurement: The maximum length (L) and width (W) of the tumor (including the thickness of the mouse skin) were measured with a vernier caliper.

[0023]

number

[0024] Tumor volume was calculated by: Body weight: During the administration period, body weight was measured from the day of treatment to the day of tumor measurement. Tumor weight: Tumor tissue was isolated at dissection and tumor weight was weighed and recorded. Tumor inhibition rate (IR TW %) = (mean tumor weight of control group - mean tumor weight of experimental group) / mean tumor weight of control group × 100%.

[0025] Flow cytometry analysis

[0026] Tumors and spleens from the colon cancer model were harvested at appropriate times and subjected to enzymatic digestion in DMEM medium containing type IV collagen (1 mg / mL, Sigma), hyaluronidase (1 mg / mL, Sigma), and DNase I (20 U / mL, Sigma) at 37°C for 1 hour to collect single tumor and spleen cells. Isolated single cells were washed with PBS containing 2% FCS and then surface stained with the relevant antibodies. After extensive washing, cells were acquired on a BDFACS Calibur (Becton Dickinson). Flow cytometry data were analyzed using Novo Express™ (ACEA Biosciences, Inc.).

[0027] Cytokine detection by Multi-ELISA

[0028] Peripheral blood and tumor tissues from experimental mice were collected and cytokines, including IL-10, TNF-α, GM-CSF, and TGF-β, were evaluated. Multi-ELISA kits were purchased from Qiagen (Australia) and performed according to the instructions provided with the kit. ELISA results were read at 450 nm using an ELISA process board (PolarstarOmega 96-well microplate reader, BMG Labtech GmbH, Germany).

[0029] TCC staining

[0030] The tumor tissue was removed, flash-frozen in liquid nitrogen, and then cut into five slices of equal thickness using a slicing knife. The tumor tissue slices were placed on a glass plate, covered with a drop of 2% TTC solution, and incubated in the dark for 30 minutes before being photographed with a digital camera.

[0031] immunohistochemistry

[0032] After harvesting, tumor tissue was rapidly fixed in 10% neutral formalin solution, embedded in paraffin, and sliced. Paraffin slices were typically dewaxed in water. They were then incubated in 3% H2O2 deionized water at room temperature for 10 minutes, protected from light, to remove endogenous hydrogen peroxidase activity, followed by three 5-minute washes with PBS. The slices were immersed in EDTA repair solution (1x), heated to boiling in a microwave oven, turned off, and repaired 1-2 more times at 5-10 minute intervals, then cooled. A 5% BSA blocking solution was added, incubated at 37°C for 30 minutes, and then rock-dried. Appropriately diluted primary antibodies, anti-CD163 and HIF-1α mouse monoclonal antibodies, were added, incubated at 37°C for 1-2 hours or overnight at 4°C, followed by three 5-minute washes with PBS. Biotin-labeled goat anti-rabbit IgG (secondary antibody) was added, followed by 30 minutes of incubation at 37°C. The specimen was washed with PBS for 5 minutes three times. SABC was added dropwise and incubated at 37°C for 30 minutes. The specimen was washed with PBS for 5 minutes three times. One drop each of color developers A, B, and C was added to 1 mL of distilled water, mixed, and added to the specimen. Color was developed for 1 to 10 minutes, and the reaction was stopped by thorough washing with distilled water. Hematoxylin staining was repeated. The specimen was dehydrated and became transparent. The specimen was sealed with neutral rubber and observed under a microscope.

[0033] Gram stain of tumor tissue slices

[0034] After harvesting, tumor tissue was rapidly fixed in 10% neutral formalin solution, embedded in paraffin, and sliced. The slices were dried for 30 minutes, then washed with xylene I for 5 minutes, xylene II for 5 minutes, 100% ethanol I for 2 minutes, 95% ethanol I for 2 minutes, and 80% ethanol I for 2 minutes, followed by running water and a brief drying period. Then, they were washed with Gram's reagent I for 1 minute, Gram's reagent II for 1 minute, Gram's reagent III for 20 seconds, followed by eosin II for 20 seconds, followed by running water and a subsequent rinsing period. Finally, they were washed with 95% ethanol II for 30 seconds, 95% ethanol III for 1 minute, 100% ethanol II for 2 minutes, xylene III for 3 minutes, and xylene IV for 3 minutes. Then, the slices were sealed with neutral rubber bands and observed.

[0035] Quantitative analysis of Clostridium ghonii

[0036] Tissue RNA was extracted using the Trizol method and PrimeScript TM RNA was reverse transcribed into cDNA using the RT reagent kit with gDNA Eraser (Perfect real time) kit. Using the cDNA as a template, Clostridium ghonii thioredoxin-specific primers were used to detect Clostridium ghonii bacteria. The primer sequences are as follows: Trx Forward primer(SEQID NO:1):5'--AATACAGGGAATTTTAGAGGTGCAG-3' Trx Reverse primer(SEQID NO:2):5'--GCTAACATCTTACAAGGCCCACA-3'

[0037] statistical analysis

[0038] Statistical analysis was performed using a double-tailed T-test or Prism 6.0 Mann-Whitney test (Graphpad Software, San Diego), with p < 0.05 indicating statistical significance. Example 2

[0039] High safety and potent targeting of Clostridium ghonii in solid tumor models

[0040] Model Building

[0041] A subcutaneous colon cancer tumor model was established. A middle cerebral artery occlusion (MCAO) model was established in SD rats. An acute myocardial infarction (MI) model was established in C57BL / 6 mice.

[0042] Animal Grouping

[0043] Tumor volume 0.35-0.60cm 3Experimental animals were selected for the study on the day of screening for tumor-bearing animals. Animals that met the requirements were randomly screened and divided into four groups by lottery: an intravenous control group, an intratumoral control group, an intravenous Clostridium ghonii spore administration group, and an intratumoral Clostridium ghonii spore administration group, each of which consisted of eight mice.

[0044] The SD rat middle cerebral artery occlusion (MCAO) model was randomly divided into four groups: a healthy rat control group, a cerebral infarction model control group, a cerebral infarction model intravenous administration group, and a cerebral infarction model intracranial administration group, each of which consisted of five rats.

[0045] C57BL / 6 mice with acute myocardial infarction (MI) model were randomly divided into three groups: a TTC stained myocardial infarction model group, a control myocardial infarction model group, and a tail vein administration myocardial infarction model group, each group consisting of five mice.

[0046] Administration

[0047] Colon cancer model: 1 x 10 spores 7 cfu / tumor / injection, intratumoral administration. The control group received the same volume of 0.9% sodium chloride injection (mass / volume percentage); spore dose: 1 x 10 8 cfu / tumor / time, administered via tail vein; the control group received the same volume of 0.9% sodium chloride injection; SD rat middle cerebral artery occlusion (MCAO) model: Tail vein administration of 5 × 10 7 CFU, intracranial dose 1 × 10 6 CFU. C57BL / 6 mouse acute myocardial infarction (MI) model: Tail vein administration of 2 × 10 7 CFU.

[0048] Observation and Detection

[0049] During the experiment, the colon cancer model was observed for the death or moribundity of the mice, their behavior, and their body weight. Brain and heart tissues from the MCAO and MI models were collected and stained with TTC, and Clostridium ghonii was detected by bacterial qPCR and spore culture. The tissues were sliced ​​and Clostridium ghonii was detected by Gram stain.

[0050] result

[0051] In the colon cancer model, no animal deaths were observed during the experimental period, regardless of whether the drug was administered intratumorally or intravenously. Furthermore, there was no significant difference in the net weight of the mice compared to the control group (Figure 1). In the MCAO model test, TTC staining was performed on brain tissue from the healthy rat control group, cerebral infarction model control group, cerebral infarction model intravenous administration group, and cerebral infarction model intracranial administration group. TTC staining of brain tissue slices from normal rats was red, while TTC staining of brain tissue slices from MCAO model rats showed that some areas of the brain tissue from rats with cerebral infarction were pale (Figure 2).

[0052] In the MCAO model test, Clostridium ghonii bacteria and spores were detected in brain tissue, and after a single tail vein administration of injectable Clostridium ghonii spores and a single intracranial administration of injectable Clostridium ghonii spores, Clostridium ghonii spores were detected in brain tissue where cerebral infarction occurred, but in both cases Clostridium ghonii bacteria were not detected.

[0053] Brain tissue slices were prepared and Gram stained. A digital pathology scanning system was used to scan the brain tissue slices from each group of rats to detect the distribution of Clostridium ghonii bacteria. Gram-stained tumor tissue slices from the positive control group revealed short, rod-shaped, blue-purple Clostridium ghonii bacteria (Figure 3a). Gram-stained tumor tissue slices from the negative control group showed no bacteria (Figure 3b). After middle cerebral artery occlusion in rats, a single dose of injectable Clostridium ghonii spores was administered via the tail vein, and a single dose of injectable Clostridium ghonii spores was administered intracranially. No Clostridium ghonii bacteria were detected in any of the brain tissue slices that developed cerebral infarction (Figures 3c and 3d).

[0054] TTC staining of the cardiac tissue of mice in the MI model TTC staining group showed that the myocardial tissue of the mice was white, and the cardiac tissue of the MI model mice exhibited obvious infarction (Figure 4). After a single injection of injectable Clostridium ghonii spores into the tail vein of MI model mice, Clostridium ghonii spores were detected in the myocardial tissue that developed myocardial infarction, but Clostridium ghonii bacteria were not detected.

[0055] Heart tissue was sliced ​​and Gram-stained. Mouse heart tissue slices from each group were scanned using a digital pathology scanning system to detect the distribution of Clostridium ghonii bacteria. Short, rod-shaped, blue-purple Clostridium ghonii bacteria were observed in Gram-stained tumor tissue slices from the positive control group (Figure 3a). No bacteria were observed in Gram-stained tumor tissue slices from the negative control group (Figure 3b). After a single tail vein injection of injectable Clostridium ghonii spores into MI model mice, no Clostridium ghonii bacteria were detected in myocardial tissue slices with cardiac infarction (Figure 5). Example 3

[0056] Antitumor effects of Clostridium ghonii spores in combination with different doses of Aitan in the CT26 mouse model

[0057] Mice that met the requirements (tumor volume 0.31-0.41 cm) 3 ) were randomly divided into eight groups by lottery: control group, Clostridium ghonii spore group, high-dose Aitan group, medium-dose Aitan group, low-dose Aitan group, spore and low-dose Aitan group, spore and medium-dose Aitan group, and spore and high-dose Aitan group.

[0058] Control group: the same volume of 0.9% sodium chloride injection was administered into the tumor; Clostridium ghonii fungus spore group: Usage amount 1×10 7 cfu / tumor / dose, intratumoral administration, 2 doses Aitan high dose group: 180 mg / kg / d, intragastrically administered once a day, a total of 7 times. Aitan medium dose group: Aitan 120 mg / kg / d was administered intragastrically once a day for a total of 7 doses. Aitan low-dose group: the dose was 60 mg / kg / d, administered by gastric irrigation once daily for a total of 7 doses; Clostridium ghonii and Aitan combination group: Clostridium ghonii spore count 1×10 7 The spores were administered once every other day for three days, for a total of two doses. The groups were administered high, medium, or low doses of Aitan in combination with the spores, and high, medium, or low doses of Aitan alone.

[0059] result During the treatment period, none of the mice in each group died or became moribund. There was no significant difference in the body weight of the mice in each group before treatment (p>0.05). There was no significant difference in the net weight of the mice in each group compared with the control group (p>0.05). The net weight of the tumor-bearing mice is shown in Table 1 and Figure 6.

[0060] [Table 1]

[0061] There was no significant difference in tumor volume between the groups before treatment (p>0.05). At the end of treatment, tumor volumes in the single spore group, the Clostridium ghonii + low-dose Aitan group, and the Clostridium ghonii + medium-dose Aitan group were all significantly smaller than the control group (p=0.045, p=0.000, p=0.026). The tumor volumes in tumor-bearing mice are shown in Table 2 and Figure 7.

[0062] [Table 2]

[0063] The tumor weights in the single spore group, the Clostridium ghonii and low-dose Aitan group, the Clostridium ghonii and medium-dose Aitan group, and the Clostridium ghonii and high-dose Aitan group were significantly smaller than the control group (p=0.000, p=0.000, p=0.000, p=0.000, p=0.000). The tumor weights in each group are shown in Table 3 and Figure 8.

[0064] [Table 3]

[0065] The tumor inhibition rate was calculated based on tumor weight. The tumor inhibition rates were as follows, as shown in Table 4: Clostridium ghonii combined with low-dose Aitan > spore combined with medium-dose Aitan > Clostridium ghonii combined with high-dose Aitan > spore alone > high-dose Aitan > low-dose Aitan > medium-dose Aitan. The tumor anatomical morphology is shown in Figure 9.

[0066] [Table 4]

[0067] In the group treated with Clostridium ghonii and low-dose Aitan, 28.6% of mouse tumors were completely eliminated, and no tumor growth was observed until the end of the experiment. The cure rate was significantly higher than the groups treated with Clostridium ghonii and high-dose Aitan (14.3%) and Clostridium ghonii and medium-dose Aitan (14.3%). However, in the high- and neutral-low-dose Aitan-only groups and the spore-only group, no tumor disappearance was observed, and the cure rate was 0%. As shown in Table 5, the combination of Clostridium ghonii and low-dose Aitan demonstrated a significant antitumor effect, clearly superior to the other groups.

[0068] [Table 5]

[0069] As can be seen from the above results, all dose treatment groups showed an inhibitory effect on tumor growth, with the most significant antitumor effect being achieved by the combination of spores and low-dose Aitan. Treatment with Clostridium ghonii spores in combination with anti-angiogenesis inhibitors such as Aitan showed a significant positive effect, with the most effective antitumor effect being achieved by the combination of Clostridium ghonii spores and low-dose Aitan. Example 4

[0070] Antitumor mechanism of action of Clostridium ghonii in combination with high-dose Aitan and low-dose Aitan

[0071] The antitumor effect of the combination of Clostridium ghonii and low-dose Aitan was clearly superior to that of the combination of Clostridium ghonii and high-dose Aitan, and we investigated the mechanism of the antitumor effect of the combination of Clostridium ghonii and low-dose Aitan.

[0072] Tumor tissues from mice treated with the control group, single spore group, Clostridium ghonii combined with low-dose Aitan group, and Clostridium ghonii combined with high-dose Aitan group were collected and analyzed using flow cytometry. Compared to the control group, CD45 expression in tumor tissues from the single spore group, Clostridium ghonii combined with low-dose Aitan group, and Clostridium ghonii combined with high-dose Aitan group was significantly higher. + CD3 + T, CD45 + CD3 + CD8 + T and F4 / 80 + The proportion of tumor cells infiltrating the cells was significantly increased in both groups, and CD45 expression in tumor tissues of mice treated with Clostridium ghonii and low-dose Aitan was significantly higher than in the single-spore group. + CD3 + T, CD45 + CD3 + CD8 + T and F4 / 80 + The percentage of tumors with cellular invasion increased significantly in both groups. However, no significant difference was observed between the Clostridium ghonii and high-dose Aitan combination groups (Figure 10).

[0073] Cytokine expression in intratumoral T cells was investigated using intracellular staining. Compared with the spore-only group, the intratumoral CD3 + IL-10 + T, CD3 + IFN-γ + T cells were significantly increased (p<0.05). However, the group receiving Clostridium ghonii and high-dose Aitan showed no significant increase in CD3 + IL-10 + T, CD3 + IFN-γ + No obvious changes were observed in T cells (Fig. 11).

[0074] Multi-ELISA analysis of intratumoral cytokines, including IL-10, TNF-α, GM-CSF, and TGF-β, showed increased levels of IL-10, TNF-α, and GM-CSF in the spore-free group, the Clostridium ghonii + high-dose Aitan group, and the Clostridium ghonii + low-dose Aitan group compared with the control group. Compared with the other groups, the Clostridium ghonii + low-dose Aitan group showed significantly reduced intratumoral TGF-β expression (Figure 12). Clostridium ghonii tumor lysis can induce cytokine and chemokine expression in the TME. At the same time, the combination of Clostridium ghonii + low-dose Aitan significantly reduced TGF-β expression, improving the immunosuppressive tumor microenvironment, while the combination with high-dose Aitan did not significantly suppress TGF-β expression.

[0075] The number of cells, such as myeloid-derived immunosuppressive cells (MDSCs) and tumor-associated macrophages (TAMs), in the tumors was analyzed by flow cytometry. Compared to the control group, the CD11b expression level in the tumors was significantly higher after treatment with Clostridium ghonii and low-dose Aitan. + Ly6G-Ly6C high Monocytic MDSCs and CD11b + Ly6G + Ly6C low No significant difference was observed in the number of PMN-MDSCs. Compared with the combination of Clostridium ghonii and high-dose Aitan, the proportions of Mo-MDSCs and PMN-MDSCs were both reduced after treatment with Clostridium ghonii and low-dose Aitan (Figure 13). Furthermore, CD11b expression was significantly higher in the Clostridium ghonii and low-dose Aitan group compared with the other treatment groups. + F4 / 80 + The proportion of TAMs among all viable cells was significantly reduced (Figure 14). IHC staining revealed that CD163 + (M2 macrophage marker), and the results showed that CD163 in tumors in the group treated with Clostridium ghonii and low-dose Aitan+ The cell count decreased, demonstrating that treatment with Clostridium ghonii in combination with low-dose Aitan can effectively reduce the number of TAMs in tumors (Figure 15).

[0076] HIF-1α immunohistochemical staining of tumor tissue showed that compared with the control group, low-dose Aitan combined with Clostridium ghonii significantly reduced tumor tissue hypoxia, while the level of hypoxia in tumor tissue increased in the group treated with Clostridium ghonii and high-dose Aitan (Figure 16). High-dose Aitan exacerbated tumor tissue hypoxia, creating an oxygen-deficient environment favorable for the proliferation of Clostridium ghonii, but its antitumor effect was lower than that of the group treated with Clostridium ghonii and low-dose Aitan. While the significantly reduced tumor hypoxia in the group treated with Clostridium ghonii and low-dose Aitan could theoretically accelerate tumor growth, tumor volume indicated that tumor growth was significantly inhibited.

[0077] In conclusion, the combination of Clostridium ghonii and low-dose Aitan inhibits CD45 + CD3 + T, CD45 + CD3 + CD8 + T, F4 / 80 +It promotes the infiltration of immune cells such as IFN-γ, TNF-α, and GM-CSF, and induces increased expression of cytokines and chemokines. At the same time, it effectively reduces the expression of TGF-β in tumor tissue, reducing the number of immune inhibitors such as tumor-associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs), thereby reducing immunosuppression. Thus, the highly effective antitumor effect of the combination of Clostridium ghonii and low-dose Aitan induces tumor vascular normalization, thereby transforming the TME from an immunosuppressive to an immunoactive state. This unique therapeutic effect is not due to the use of high-dose Aitan to exacerbate tumor anoxia, creating an oxygen-deficient environment in which Clostridium ghonii can thrive, but rather to changes in the antitumor immune microenvironment.

[0078] The above examples have described the present invention in detail, but they are only some examples of the present invention, not all examples, and other embodiments can be obtained according to the present embodiments without inventive steps, and all of these examples belong to the protection scope of the present invention.

Claims

[Claim 1] A pharmaceutical product for treating Lewis lung cancer or nasopharyngeal carcinoma, comprising: The pharmaceutical product comprises Clostridium ghonii and a tumor angiogenesis inhibitor; the tumor angiogenesis inhibitor is apatinib; 1 x 10 Clostridium ghonii bacteria in spore form ７ The combined apatinib dose per CFU is 60 mg / kg / d; The pharmaceutical product is characterized in that the Clostridium ghonii is Clostridium ghonii MW-DCG-LCv-26 strain (stored at the Australian National Metrology Institute, strain number V12 / 001486).

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