WO2023056972A1 - 戈氏梭菌联合肿瘤血管生成抑制剂的应用 - Google Patents

戈氏梭菌联合肿瘤血管生成抑制剂的应用 Download PDF

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WO2023056972A1
WO2023056972A1 PCT/CN2022/124089 CN2022124089W WO2023056972A1 WO 2023056972 A1 WO2023056972 A1 WO 2023056972A1 CN 2022124089 W CN2022124089 W CN 2022124089W WO 2023056972 A1 WO2023056972 A1 WO 2023056972A1
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clostridium
gordii
tumor
strain
dcg
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French (fr)
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王勇
刘园园
张文华
邢艳秋
王少鹏
王丹
朱红
徐兴鲁
姜圣彪
李晓楠
郑嘉辉
张蓉
杨冬霞
郜玉霞
邵石丽
韩停
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山东新创生物科技有限公司
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Priority to KR1020247011331A priority Critical patent/KR20240083179A/ko
Priority to EP22877986.4A priority patent/EP4393500A1/en
Priority to AU2022358834A priority patent/AU2022358834A1/en
Priority to US18/546,553 priority patent/US20240131086A1/en
Priority to CA3232787A priority patent/CA3232787A1/en
Publication of WO2023056972A1 publication Critical patent/WO2023056972A1/zh

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    • A—HUMAN NECESSITIES
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    • C12R2001/145—Clostridium

Definitions

  • the invention belongs to the technical field of oncology, and relates to the application of Clostridium gordii combined with tumor angiogenesis inhibitors, in particular to the use of Clostridium gordii combined with tumor angiogenesis inhibitors to release the tumor immunosuppressive microenvironment, improve the tumor microenvironment and enhance the treatment of tumors application of the effect.
  • TME tumor microenvironment
  • TME Tumor microenvironment
  • the tumor microenvironment is a local homeostatic environment composed of tumor cells, stromal cells, extracellular matrix, and biomolecules infiltrated in the process of tumor growth.
  • TME provides the necessary material basis for tumor occurrence, development, and invasion, and regulates various biological behaviors such as tumor metastasis and recurrence.
  • TME increases tumor drug resistance and radiation resistance, reducing the therapeutic effect.
  • the immune regulation in the TME plays an important role in the occurrence and development of tumors, and it can form local tumor immunosuppression through various mechanisms. This has an important impact on tumor proliferation, invasion, and angiogenesis. How to regulate TME immunotherapy strategies and reshape a positive immune microenvironment is the focus and difficulty of anti-tumor therapy.
  • Malignant tumors evade host immune surveillance through multiple mechanisms, including impaired lymphocyte infiltration, upregulation of immune checkpoint protein expression through hypoxia, recruitment of Tregs, and establishment of an immunosuppressive tumor microenvironment that impairs resident and transit immune effects cell function.
  • Myeloid cells infiltrating into the tumor microenvironment can regulate the key links of tumor development such as immune escape and tumor metastasis of tumor cells in the tumor microenvironment.
  • TGF ⁇ tumor-associated macrophages
  • MDSCs myeloid-derived suppressor cells
  • Tregs regulatory T cells
  • TGF ⁇ plays an important role in the immune response of TME, has the effect of promoting anti-tumor immunosuppression, and TGF ⁇ is considered to produce drug resistance to anti-angiogenic therapy.
  • Hypoxia or low oxygen content is a typical feature of solid tumors.
  • Hypoxia in solid tumors can directly upregulate the expression of immune checkpoint protein PD-L1 in MDSCs, dendritic cells and tumor cells through HIF-1 ⁇ activation to help immune suppression and escape.
  • tumor hypoxia can also increase the invasive potential of tumor cells by inducing the production of pro-migration proteins (such as SDF1A and HGF) and pro-invasive extracellular matrix molecules. Hypoxia also enhances tumor resistance to chemotherapy and radiotherapy.
  • Clostridium gordii is an obligate anaerobic bacterium, which can only germinate specifically and proliferate in large quantities in hypoxic or necrotic areas of tumors, effectively and indiscriminately dissolve tumor tissues and destroy TME. Clostridium gordii oncolytically alters TME immunogenicity, modulates immunosuppressive TME, and induces antitumor immune responses. At present, there are no related reports that Clostridium gordii combined with tumor angiogenesis inhibitors can significantly change the tumor immunosuppressive microenvironment and enhance the anti-tumor effect.
  • the present invention provides a safer and more targeted application of the anaerobic bacteria Clostridium gordii in combination with tumor angiogenesis inhibitors for the treatment of solid tumors.
  • Clostridium gordii combined with tumor angiogenesis inhibitors in the preparation of pharmaceutical products for treating tumors.
  • a drug for treating tumors the active ingredients of which include Clostridium gordii and tumor angiogenesis inhibitors.
  • the Clostridium gordii MW-DCG-LCv-26 The bacterial strain is preserved in the Australian National Institute of Metrology, and the bacterial strain preservation number is V12/001486;
  • the bacterial strain obtained after the domestication of Clostridium gordii includes MW-DCG-HNCv-18 strain or MW-DCG-CCv-17 bacterial strain;
  • the MW- The DCG-HNCv-18 strain is preserved in the Australian National Metrology Institute with the strain number V12/001485;
  • the MW-DCG-CCv-17 strain is preserved in the Australian National Metrology Institute with the strain preservation number V12/001487.
  • the Clostridium gordii is in the form of spores.
  • the tumor angiogenesis inhibitor is selected from: including but not limited to apatinib (Aitan), sunitinib, pazopanib, bevacizumab, ramucirumab , Conbercept, Aflibercept, Sorafenib, Regorafenib, etc.
  • the administration sequence of the active ingredient Clostridium gordii and the tumor angiogenesis inhibitor is sequential administration or simultaneous administration.
  • the preferred drug combination according to the present invention is 1 ⁇ 10 7 CFU lyophilized powder of Clostridium gordii spores combined with the optimal dose of Eitan 60 mg/kg/d.
  • the tumor includes but not limited to colon cancer, Lewis lung cancer, nasopharyngeal cancer, non-small cell lung cancer, fibrosarcoma, melanoma and the like.
  • tumor angiogenesis inhibitors can be used in random combination.
  • Clostridium gordii spores must be pure microorganisms, that is, they do not contain any other bacteria except spores, and tumor angiogenesis inhibitors should also meet the sterility requirements.
  • Clostridium gordii spores are prepared and purified according to existing methods in the art, and drugs meeting relevant quality standards are obtained.
  • the present invention discovers for the first time that the combination of safer and stronger targeting Clostridium gordii and tumor angiogenesis inhibitors can effectively anti-tumor.
  • Clostridium gordii combined with low-dose tumor angiogenesis inhibitors reduces the infiltration of M2-like macrophages and MDSCs in tumors, reduces the number of TGF ⁇ in tumors, and reduces the inhibitory effect of anti-tumor immune response in TME.
  • low-dose tumor angiogenesis inhibitors it can promote the infiltration of CD8 + , CD3 + T, F4/80 + and other immune cells into the tumor, improve the immune microenvironment in the tumor, and enhance the anti-tumor efficacy.
  • Clostridium gordii combined with low-dose Aitan reduces the inhibitory effect of anti-tumor immune response in TME, transforms TME from immunosuppression to immune activation state, and realizes high-efficiency anti-tumor.
  • the unique therapeutic effect should be attributed to the changes in the anti-tumor immune microenvironment, rather than the use of high doses of Etan to aggravate tumor hypoxia to create a hypoxic environment for the proliferation of Clostridium gordii to effectively anti-tumor.
  • composition of the present invention is safer and more targeted when treating tumors, and only targets tumors to germinate in hypoxic environments, but cannot germinate into bacteria in non-tumor hypoxic environments.
  • Fig. 1 is the net body weight histogram in embodiment 2;
  • Fig. 2 is the TTC staining figure of cerebral infarction tissue in embodiment 2;
  • Fig. 3 is the gram staining figure of tissue section in embodiment 2;
  • Fig. 4 is the TTC staining figure of embodiment 2 myocardial stem tissue
  • Fig. 5 is the gram staining figure of tissue section in embodiment 2;
  • Fig. 6 is the histogram of net body weight of experimental mice in embodiment 3.
  • Fig. 7 is a curve diagram of experimental mouse tumor volume in embodiment 3.
  • Fig. 8 is experiment mouse tumor weight figure in embodiment 3.
  • Fig. 9 is anatomical tumor figure of experimental mouse in embodiment 3.
  • Fig. 10 is the ratio of T cell infiltration in tumor tissue in embodiment 4.
  • Figure 11 shows the expression of cytokines in T cells in tumor tissues
  • Figure 12 is the mouse intratumoral cytokine expression situation in embodiment 4.
  • Example 14 are the ratios of myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs) in mouse tumors in Example 4;
  • MDSCs myeloid-derived suppressor cells
  • TAMs tumor-associated macrophages
  • 15 and 16 are immunohistochemical staining diagrams of mouse tumor tissues and CD163 cell ratio diagrams in Example 4.
  • the strain is MW-DCG-LCv-26 strain, which is preserved in the Australian National Metrology Institute, strain preservation number V12/001486, batch number: 202003001-1, Shandong Xinchuang Biotechnology Ltd. developed.
  • Clostridium gordii spore freeze-dried powder for injection uses Clostridium gordii spores as the active ingredient and 1% sucrose as the excipient, after -40°C for 4h; -35°C for 10min while vacuuming; , -20°C 26h, -15°C 2h, -10°C 10min, -5°C 10min, 0°C 10min, 10°C 2h, 15°C 10min, 20°C 3h, 27°C 3h freeze-drying procedures, the specification is 1 ⁇ 10 8 CFU/tube; reference substance freeze-dried powder, batch number: 201910002F, developed by Shandong Xinchuang Biotechnology Co., Ltd., prepared with 1mL of 1% sucrose solution through the above freeze-drying procedure; 0.9% sodium chloride injection, batch number: 2005062146, Chenxin Available from Pharmaceutical Co., Ltd.; Sterile Water for Injection, batch number: 1902212162, available from Chenxin Pharmaceutical Co., Ltd.
  • CT26.WT colon cancer cells number: 3131C0001000800037, Cell Resource Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.
  • RPMI Medium1640 basic basal medium was added with volume percentage concentration of 10% special grade fetal bovine serum (serum after low temperature thawing may have floating matter, 2000rpm centrifuged for 3min to remove floating matter) and volume percentage concentration of 1.1% penicillin and streptomycin mixture, Mix well, resuspend the cells, inoculate the cell suspension into 75cm 2 cell culture flasks, add 25mL cell culture medium to each flask, and place in a 5% CO 2 cell incubator at 37°C for static culture.
  • RPMI Medium1640 basic was purchased from Gibco
  • special-grade fetal bovine serum and penicillin streptomycin were purchased from BI
  • Aitan (apatinib) was purchased from Jiangsu Hengrui Medicine Co., Ltd. Methods Preparation and purification were carried out to obtain the drug that met the relevant quality standards.
  • BALB/c mice were subcutaneously inoculated with CT26 tumor cells to establish a subcutaneous xenograft model of colon cancer.
  • Clinical observation During the dosing period, observe with naked eyes every morning and afternoon, and observe animal behavior, death or near-death conditions once a day.
  • Tumor measurement Measure the maximum long diameter (L) and maximum transverse diameter (W) of the tumor (including the thickness of the mouse skin) with a vernier caliper, and according to Calculate the tumor volume.
  • Body weight During the administration period, body weight was measured on the day of tumor measurement from the day of treatment.
  • Tumor weight the tumor tissue was dissected and weighed and the tumor weight was recorded.
  • the tumor and spleen in the colon cancer model were collected at the corresponding time, and cultured in DMEM containing type IV collagenase (1mg/mL, Sigma), hyaluronidase (1mg/mL, Sigma) and DNaseI (20U/mL, Sigma) Enzyme digestion was carried out at 37°C for 1 hour, and single tumor cells and spleen cells were collected. Separated single cells were washed with PBS containing 2% FCS, respectively, and then surface stained with relevant antibodies. Cells were extensively washed and acquired on a BD FACS Calibur (Becton Dickinson). Flow cytometry data were analyzed by Novo ExpressTM (ACEA Biosciences, Inc.).
  • cytokines such as IL-10, TNF-a, GM-CSF and TGF ⁇ .
  • the Multi-ELISA kit was purchased from Qiagen (Australia), and the experiment was performed according to the instructions provided in the kit.
  • ELISA results were read by an ELISA program plate at 450 nm (Polarstar Omega 96-well microplate reader BMG Labtech GmBH, Germany).
  • the tumor tissues were taken out, frozen in liquid nitrogen, and cut into 5 slices with the same thickness using a microtome.
  • the tumor tissue slices were placed on glass slides, 2% TTC solution was added dropwise to cover the tissue slices, reacted in the dark for 30 minutes, and a digital camera was used to take pictures.
  • Tumor tissues were quickly fixed in 10% neutral formalin solution, embedded in paraffin, and sectioned. Paraffin sections were routinely dewaxed to water. Incubate in 3% H 2 O 2 deionized water at room temperature in the dark for 10 min to eliminate endogenous catalase activity, wash with PBS, 5 min x 3 times. Immerse the slices in EDTA repair solution (1 ⁇ ), heat in the microwave until boiling, then turn off the power, repair 1-2 times at intervals of 5-10min, and cool down. Add 5% BSA blocking solution dropwise, incubate at 37°C for 30min, and shake dry.
  • Tumor tissues were quickly fixed in 10% neutral formalin solution, embedded in paraffin, and sectioned. Bake slices for 30 minutes, xylene I for 5 minutes, xylene II for 5 minutes, 100% ethanol I for 2 minutes, 95% ethanol I for 2 minutes, 80% ethanol for I2 minutes, rinse with running water, and dry slightly; Rinse reagent 2 for 1 min, rinse with running water, Gram reagent 3 for about 20 s, rinse with running water, eosin for about 20 s, rinse with running water; 95% ethanol II for 30 s, 95% ethanol III for 1 min, 100% ethanol II for 2 min, xylene III for 3 min , Xylene IV 3min, neutral gum sealing, observation.
  • the tissue RNA was extracted by the Trizol method, and the RNA was reverse-transcribed into cDNA using the PrimeScript TM RT reagent Kit with gDNA Eraser (Perfect real time) kit.
  • cDNA as a template, Clostridium gordii thioredoxin-specific primers are used to detect Clostridium gordii bacteria.
  • the primer sequence is as follows:
  • Trx Forward primer SEQ ID NO:1:5'--AATACAGGGAATTTTAGAGGT GCAG-3'
  • mice On the day when the tumor-bearing animals were screened, experimental animals with a tumor volume of 0.35-0.60 cm 3 were selected for the experiment. According to the random principle, the screened animals that meet the requirements were divided into 4 groups by lottery: intravenous administration control group, intratumoral administration control group, intravenous administration of Clostridium gordoi spore group, intratumoral administration of Clostridium gordoi spore group , 8 mice per group.
  • SD rat middle cerebral artery occlusion (MCAO) model was randomly divided into 4 groups, healthy rat control group, cerebral infarction model control group, cerebral infarction model intravenous administration group, cerebral infarction model intracranial administration group, 5 in each group. Only.
  • the acute myocardial infarction (MI) model of C57BL/6 mice was randomly divided into 3 groups, the TTC staining group of the myocardial infarction model, the control group of the myocardial infarction model, and the tail vein administration group of the myocardial infarction model, with 5 mice in each group.
  • Colon cancer model the dose of spores was 1 ⁇ 10 7 cfu/tumor/time, administered intratumorally, and the control group was given the same volume of 0.9% sodium chloride injection with a mass volume percentage concentration; the dose of spores was 1 ⁇ 10 8 cfu/tumor / time, tail vein administration, the control group was given the same volume of mass volume percentage concentration 0.9% sodium chloride injection;
  • Middle cerebral artery occlusion (MCAO) model in SD rats a dose of 5 ⁇ 10 7 CFU was administered into the tail vein, and a dose of 1 ⁇ 10 6 CFU was administered into the brain.
  • MI myocardial infarction
  • brain tissue and heart tissue were taken respectively for TTC staining, Clostridium gordii qPCR and spore culture detection, and tissue section Clostridium gordii Gram stain detection.
  • TTC staining was performed on the brain tissues of the healthy rat control group, the cerebral infarction model control group, the cerebral infarction model intravenous administration group, and the cerebral infarction model intracranial administration group, and the brain tissue sections of normal rats were stained with TTC. It is red, and TTC staining of brain tissue sections of MCAO model rats shows that some areas of brain tissue of rats with cerebral infarction are pale ( Figure 2).
  • Clostridium gordoi bacteria and spores were detected in the brain tissue in the MCAO model experiment. Clostridium gordii spores were detected in the infarcted brain tissue, but none of the Clostridium gordii bacteria were detected.
  • the brain tissue was sliced and Gram stained, and the digital pathological scanning system was used to scan the brain tissue slices of rats in each group to detect the distribution of Clostridium gordii bacteria.
  • the tumor tissue slices of the positive control group showed short rods after Gram staining. , blue-purple Clostridium gordoi (Figure 3a); no bacteria were found in the negative control group after Gram staining of the tumor tissue sections ( Figure 3b), and rats were given a single administration of Ge Clostridium gordoi spores and intracranial single-administration Clostridium gordoi spores were not detected in brain tissue sections with cerebral infarction (Fig. 3c, 3d).
  • TTC staining of the heart tissue of the mice in the MI model group TTC staining showed that the myocardial tissue of the mice was white, and there was obvious infarction in the heart tissue of the MI model mice ( FIG. 4 ).
  • Clostridium gordoi spores for injection were administered to the tail vein of MI model mice once, and Clostridium gordoi spores were detected in myocardial tissue with myocardial infarction, but Clostridium gordoi bacteria were not detected.
  • the heart tissue was sliced and Gram stained, and the digital pathological scanning system was used to scan the heart tissue slices of mice in each group to detect the distribution of Clostridium gordii bacteria.
  • Fig. 3a short rod-shaped, blue-purple Clostridium gordoi
  • Fig. 3b no bacteria were found in the Gram-staining sections of the tumor tissue sections of the negative control group
  • Fig. 5 after a single administration of Clostridium gordoi spores for injection in the tail vein of MI model mice, no Clostridium gordoi bacteria were detected in myocardial tissue sections in which myocardial infarction occurred (Fig. 5).
  • mice meeting the requirements were randomly divided into 8 groups by lottery: control group, Clostridium gordoi spore group, high-dose Aitan group, middle-dose Aitan group, low-dose Aitan group Dosage group, spore combined with Aitan low dose group, spore combined with Aitan medium dose group, spore combined with Aitan high dose group.
  • Control group intratumoral administration of the same volume of 0.9% sodium chloride injection;
  • Clostridium gordii spore group the dose was 1 ⁇ 10 7 cfu/tumor/time, administered intratumorally, twice;
  • Aitan high-dose group the dose is 180mg/kg/d, intragastric administration, once a day, 7 times in total;
  • Aitan medium dose group the dose is 120mg/kg/d, intragastric administration, once a day, 7 times in total;
  • Aitan low-dose group the dose is 60mg/kg/d, intragastric administration, once a day, 7 times in total;
  • Clostridium gordii combined with Eitan group the dose of Clostridium gordii spores was 1 ⁇ 10 7 cfu/tumor/time, administered intratumorally, first gave Eitan for 3 days, once a day, and then administered spores, every 1 day Dosing once a day, 2 times in total. Combined high, medium and low doses with Altan high, medium and low doses alone;
  • n is the number of animals used in the experiment in each group, the net body weight of the mice is the body weight of the tumor-bearing mice after the tumor was dissected, and the data are expressed as mean ⁇ standard deviation.
  • the tumor volume of the tumor-bearing mice is shown in Table 2 and Figure 7.
  • n is the number of animals used in each group, and the data are expressed as mean ⁇ standard deviation. Compared with the control group, *p ⁇ 0.05, **p ⁇ 0.01.
  • the tumor inhibition rate was calculated according to the tumor weight, and the tumor inhibition rate was as follows: Clostridium gordii combined with low-dose Itan group > spores combined with medium-dose Itan group > Clostridium gordii combined with high-dose group > single spore group > high-dose Itan group>low-dose Aitan group>middle-dose Aitan group, as shown in Table 4.
  • Tumor inhibition rate Tumor anatomy is shown in Figure 9.
  • Clostridium gordii combined with low-dose Etan group 28.6% of the mouse tumors were completely eliminated, and no tumor growth was seen at the end of the experiment.
  • Clostridium combined with middle dose of Itan group (14.3%), while the high, medium and low dose of Itan group and the single spore group did not appear tumor disappearance, the cure rate was 0%. It can be seen that Clostridium gordii combined with low-dose Aitan showed a significant anti-tumor effect, which was significantly better than other groups, as shown in Table 5.
  • Clostridium gordii combined with low-dose Aitan is significantly better than that of Clostridium gordii combined with high-dose Aitan.
  • the anti-tumor mechanism of Clostridium gordii combined with low-dose Aitan and high-dose Aitan was studied.
  • mice treated with the control group, the spore group alone, the Clostridium gordii combined with low-dose Itan group, and the Clostridium gordii combined with high-dose Itan group were collected, and the above tumor tissue samples were analyzed by flow cytometry. Cytometry analysis.
  • Intracellular staining was used to study the cytokine expression of T cells in the tumor. Compared with the spore group alone, CD3 + IL-10 + T, CD3 + IFN- ⁇ + T cells in the tumor of Clostridium gordii combined with low-dose Etan group significantly increased (p ⁇ 0.05), while CD3 + IL-10 + T and CD3 + IFN- ⁇ + T cells did not change significantly in the Clostridium gordii combined with high-dose Etan group (Figure 11).
  • Multi-ELISA detected intratumoral cytokines such as IL-10, TNF-a, GM-CSF and TGF ⁇ .
  • cytokines such as IL-10, TNF-a, GM-CSF and TGF ⁇ .
  • IL-10, TNF-a, GM-CSF were all increased in dose Eitan group.
  • the expression of TGF ⁇ in the tumor was significantly decreased in the Clostridium gordii combined with low-dose Aitan group (Fig. 12). It can be seen that oncolysis of Clostridium gordii can induce the expression of cytokines and chemokines in TME.
  • Clostridium gordii combined with low dose of Itan can significantly reduce the expression of TGF ⁇ and improve the immunosuppressive tumor microenvironment, while the combination of high dose of Itan has no obvious inhibitory effect on the expression of TGF ⁇ .
  • the number of myeloid-derived suppressor cells (MDSCs), tumor-associated macrophages (TAMs) and other cells in the tumor was analyzed by flow cytometry. Compared with the control group, there was no significant difference in the number of intratumoral CD11b + Ly6G - Ly6C high mononuclear MDSCs and CD11b + Ly6G + Ly6C low polymorphonuclear-MDSCs after Clostridium gordii combined with low-dose Aitan treatment. Compared with Clostridium gordii combined with high-dose Itan, the proportions of Mo-MDSCs and PMN-MDSCs were both reduced after Clostridium gordii combined with low-dose Itan (Fig. 13).
  • CD11b + F4/80 + -TAMs in total viable cells was significantly decreased in the Clostridium gordii combined with low-dose Altan group compared with other treatment groups (Fig. 14).
  • CD163 + (M2-like macrophage marker) in the tumor was detected by IHC staining. The results showed that the number of CD163 + cells in the tumor of the Clostridium gordii combined with low-dose Etan group decreased, indicating that the treatment of Clostridium gordii combined with low-dose Etan Can effectively reduce the number of TAMs in tumors ( Figure 15).
  • Tumor tissue HIF-1 ⁇ immunohistochemical staining showed that compared with the control group, the tumor tissue hypoxia of Clostridium gordii combined with low-dose Aitan was significantly reduced, while the degree of tumor tissue hypoxia was increased in the Clostridium gordii combined with high-dose Aitan group. High ( Figure 16). It showed that high dose of Etan aggravated the hypoxia of tumor tissue, although it created a hypoxic environment conducive to the reproduction of Clostridium gordii, but its anti-tumor effect was lower than that of Clostridium gordii combined with low dose of Etan. However, although the tumor hypoxia in the Clostridium gordii combined with low-dose Etan group was significantly reduced, which may theoretically accelerate tumor growth, the tumor volume showed that tumor growth was significantly inhibited.
  • Clostridium gordii combined with low-dose Aitan can promote the infiltration of immune cells such as CD45 + CD3 + T, CD45 + CD3 + CD8 + T, F4/80 + , and induce IFN- ⁇ , TNF- ⁇ , GM - Enhanced expression of cytokines and chemokines such as CSF, while effectively reducing the expression of TGF ⁇ in tumor tissue, reducing the number of immunosuppressive tumor-associated macrophages (TAM), myeloid-derived suppressor cells (MDSC), etc., and reducing immunosuppression .
  • TAM immunosuppressive tumor-associated macrophages
  • MDSC myeloid-derived suppressor cells
  • Clostridium gordii combined with low-dose Aitan is to induce the normalization of tumor blood vessels and transform the TME from immunosuppressive to immune-activated state to achieve efficient anti-tumor effect. Changes in the microenvironment, rather than the use of high doses of Altan to aggravate tumor hypoxia to create a hypoxic environment for the proliferation of Clostridium gordii to effectively fight tumors.

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Abstract

提供了戈氏梭菌联合肿瘤血管生成抑制剂在制备治疗肿瘤的医药制品中的应用。还提供了一种治疗肿瘤的药物，该药物中的有效成分包括戈氏梭菌和肿瘤血管生成抑制剂。

Description

戈氏梭菌联合肿瘤血管生成抑制剂的应用
本申请要求于2021年10月09日提交中国专利局、申请号为CN202111177878.9、发明名称为“戈氏梭菌联合肿瘤血管生成抑制剂的应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明属于肿瘤学技术领域，涉及戈氏梭菌联合肿瘤血管生成抑制剂的应用，特别涉及利用戈氏梭菌联合肿瘤血管生成抑制剂解除肿瘤免疫抑制性微环境，改善肿瘤微环境增强治疗肿瘤效果的应用。
背景技术
肿瘤微环境(Tumor microenvironment，TME)是在肿瘤生长过程中，由肿瘤细胞、基质细胞和细胞外基质以及浸润在其中的生物分子等共同构成局部稳态环境。TME为肿瘤发生、发展、侵袭等提供了必要物质基础，调控肿瘤转移、复发等多种生物学行为。同时TME增加肿瘤耐药性和耐辐射性，降低治疗效果。TME内的免疫调节在肿瘤发生发展中具有重要功能，其可通过多种机制，形成肿瘤局部免疫抑制。而这对于肿瘤的增殖、侵袭及新生血管的形成具有重要影响，如何调控TME免疫治疗策略，重塑积极的免疫微环境是抗肿瘤治疗的重点和难点。
恶性肿瘤通过多种机制逃避宿主免疫监视，包括淋巴细胞浸润受损、通过缺氧上调免疫检查点蛋白表达、募集Treg，以及建立免疫抑制性肿瘤微环境，该微环境损害常驻和转运免疫效应细胞的功能。浸润到肿瘤微环境的髓样细胞在肿瘤微环境中能够调控肿瘤细胞的免疫逃逸、肿瘤转移等肿瘤发展的关键环节。
实体瘤通常浸润大量免疫抑制剂，如肿瘤相关巨噬细胞(TAM)、髓源性抑制细胞(MDSC)和调节性T细胞(Treg)。在TME的免疫反应中TGFβ发挥重要作用，具有促进抗肿瘤免疫抑制的作用，且TGFβ被认为对抗血管生成治疗产生耐药性。
乏氧或氧含量低是实体肿瘤典型特征，实体肿瘤的缺氧可通过HIF-1α激活直接上调MDSC、树突状细胞和肿瘤细胞的免疫检查点蛋白PD-L1的表达，以帮助免疫抑制和逃避。同时，肿瘤缺氧还可通过诱导促迁移蛋白(如SDF1A和 HGF)和促侵袭性细胞外基质分子的产生，增加肿瘤细胞的侵袭潜能。缺氧也增强了肿瘤对放化疗的抵抗力。
戈氏梭菌为专性厌氧菌，仅可在肿瘤乏氧或坏死区中特异性萌发和大量增殖，有效而不加区分溶解肿瘤组织，破坏TME。戈氏梭菌溶瘤后，改变TME免疫原性，调节免疫抑制性TME，诱导抗肿瘤免疫反应。目前并没有戈氏梭菌联合肿瘤血管生成抑制剂可明显改变肿瘤免疫抑制性微环境，增强抗肿瘤效果的相关报道。
发明内容
针对现有技术的不足，本发明提供一种更安全、强靶向性的治疗实体肿瘤的厌氧菌--戈氏梭菌联合肿瘤血管生成抑制剂的应用。
戈氏梭菌联合肿瘤血管生成抑制剂在制备治疗肿瘤医药制品中的应用。
一种治疗肿瘤药物，药物中的有效成分包括戈氏梭菌和肿瘤血管生成抑制剂。
根据本发明优选的，所述的戈氏梭菌为戈氏梭菌MW-DCG-LCv-26菌株或者戈氏梭菌驯化后获得的菌株；所述戈氏梭菌MW-DCG-LCv-26菌株保藏于澳大利亚国家计量研究院，菌株保藏号V12/001486；所述戈氏梭菌驯化后获得的菌株包括MW-DCG-HNCv-18菌株或MW-DCG-CCv-17菌株；所述MW-DCG-HNCv-18菌株保藏于澳大利亚国家计量研究院，菌株编号为V12/001485；所述MW-DCG-CCv-17菌株保藏于澳大利亚国家计量研究院，菌株保藏号V12/001487。
根据本发明优选的，所述的戈氏梭菌为芽孢形式。
根据本发明优选的，所述肿瘤血管生成抑制剂选自：包括但不限于阿帕替尼(艾坦)、舒尼替尼、帕唑帕尼、贝伐珠单抗、雷莫芦单抗、康柏西普、阿柏西普、索拉非尼、瑞戈非尼等。
根据本发明优选的，所述药效成分戈氏梭菌与肿瘤血管生成抑制剂的给药顺序为先后给药或同时给药。
根据本发明优选的药物组合是1×10 7CFU戈氏梭菌芽孢冻干粉联合艾坦最优剂量60mg/kg/d。
根据本发明优选的，所述肿瘤包括但不限于结肠癌、Lewis肺癌、鼻咽癌、非小细胞肺癌、纤维肉瘤、黑色素瘤等。
本发明可随机联合使用肿瘤血管生成抑制剂。戈氏梭菌芽孢必须是纯的微生物，即除含有芽孢外，不含任何其它细菌，肿瘤血管生成抑制剂应同样符合无菌要求。戈氏梭菌芽孢按照本领域已有的方法进行制备、纯化，获得符合相关质量标准的药物。
有益效果
1、本发明首次发现更安全、强靶向性的戈氏梭菌与肿瘤血管生成抑制剂联用高效抗肿瘤。
2、本发明首次发现戈氏梭菌联合低剂量肿瘤血管生成抑制剂降低肿瘤中M2样巨噬细胞、MDSC等浸润，并减少肿瘤中TGFβ数量，降低了TME中抗肿瘤免疫响应的抑制作用。同时联合低剂量肿瘤血管生成抑制剂可促进CD8 +、CD3 +T、F4/80 +等免疫细胞向肿瘤浸润，改善肿瘤中免疫微环境，增强抗肿瘤疗效。
3、本发明中明确了肿瘤血管生成抑制剂联合戈氏梭菌的最优剂量。
4、本发明可应用于晚期恶性实体肿瘤类型，戈氏梭菌联合低剂量艾坦通过降低TME中抗肿瘤免疫响应抑制作用，使TME由免疫抑制转变为免疫激活状态，实现高效抗肿瘤，其独特的治疗效果应归因于抗肿瘤免疫微环境的变化，而非利用高剂量艾坦加重肿瘤缺氧创造戈氏梭菌繁殖的缺氧环境高效抗肿瘤。
5、本发明中的组合物在治疗肿瘤时更为安全、靶向性更高，仅靶向肿瘤乏氧环境萌发，而在非肿瘤乏氧环境中不能萌发为细菌。
附图说明
图1是实施例2中的净体重柱状图；
图2是实施例2中脑梗组织TTC染色图；
图3是实施例2中组织切片革兰氏染色图；
图4是实施例2中心梗组织TTC染色图；
图5是实施例2中组织切片革兰氏染色图；
图6是实施例3中实验小鼠净体重柱状图；
图7是实施例3中实验小鼠肿瘤体积曲线图；
图8是实施例3中实验小鼠肿瘤重量图；
图9是实施例3中实验小鼠解剖肿瘤图；
图10是实施例4中肿瘤组织中T细胞浸润的比例；
图11是肿瘤组织中T细胞的细胞因子表达情况；
图12是实施例4中小鼠瘤内细胞因子表达情况；
图13，14是实施例4中小鼠肿瘤内髓系来源的抑制细胞(MDSC)、肿瘤相关巨噬细胞(TAM)的比例；
图15，16是实施例4中小鼠肿瘤组织免疫组织化学染色图及CD163细胞比例图。
具体实施方式
下面将结合具体实施例对本发明做进一步的详细说明，所描述的实施例仅仅是本发明为了使公众便于理解所列举的一部分技术方案，这些实施例仅用于说明本发明而不用于限制本发明的保护范围，本发明的保护范围由权利要求限定。
实施例1
材料和方法
戈氏梭菌芽孢冻干粉
注射用戈氏梭菌芽孢冻干粉，菌株为MW-DCG-LCv-26菌株，该菌株保藏于澳大利亚国家计量研究院，菌株保藏号V12/001486，批号：202003001-1，山东新创生物科技有限公司研制。注射用戈氏梭菌芽孢冻干粉以戈氏梭菌芽孢为活性成分，以1％蔗糖为辅料，经过-40℃4h；-35℃10min同时抽真空、-30℃10min、-25℃10min、-20℃26h、-15℃2h、-10℃10min、-5℃10min、0℃10min、10℃2h、15℃10min、20℃3h、27℃3h冻干程序制备，规格为1×10 8CFU/支；对照品冻干粉，批号：201910002F，山东新创生物科技有限公司研制，以1mL1％蔗糖溶液经过上述冻干程序制备；0.9％氯化钠注射液，批号：2005062146，辰欣药业股份有限公司有售；灭菌注射用水，批号：1902212162，辰欣药业股份有限公司有售；
细胞系及细胞培养
CT26.WT结肠癌细胞，编号：3131C0001000800037，中国科学院上海生命科学研究院细胞资源中心。RPMI Medium1640 basic基础培养基中加入体积百分比浓度10％特级胎牛血清(低温融化后的血清中可能有漂浮物，2000rpm离心3min以去除漂浮物)和体积百分比浓度1.1％青链霉素混合液，充分混匀，重悬细胞，将细胞悬液接种到75cm 2细胞培养瓶中，每瓶加入25mL细胞培养 基，置5％CO 2细胞培养箱37℃静置培养。
试剂
RPMI Medium1640 basic基础培养基购自Gibco，特级胎牛血清和青链霉素购自BI，艾坦(阿帕替尼)购自江苏恒瑞医药股份有限公司，戈氏梭菌芽孢按照已有的方法进行制备、纯化，获得符合相关质量标准的药物。
模型建立
采用BALB/c小鼠皮下接种CT26肿瘤细胞，建立结肠癌皮下移植瘤模型。以1mL一次性无菌注射器抽取0.2mL浓度为7.5×10 6个/mL(可接受范围：6.75×10 6个/mL～8.25×10 6个/mL)的细胞悬液，缓慢皮下注射接种于小鼠右前肢腋窝皮下(75％酒精预先消毒处理)。注射完毕后以干棉球轻轻按压针眼部位。
观察和检查
临床观察：给药期每天上午和下午肉眼观察，动物行为、死亡或濒死情况等1次。
肿瘤测量：以游标卡尺测量肿瘤最大长径(L)和最大横径(W)(包括小鼠皮肤厚度在内)，并根据
Figure PCTCN2022124089-appb-000001
计算肿瘤体积。
体重：给药期，自治疗之日起于肿瘤测量日称量体重。
肿瘤重量：解剖出肿瘤组织称量并记录肿瘤重量。
抑瘤率(IR TW％)＝(对照组平均瘤重-实验组平均瘤重)/对照组平均瘤重×100％。
流式细胞术分析
在相应的时间采集结肠癌模型中肿瘤和脾脏，在含有IV型胶原酶(1mg/mL，Sigma)、透明质酸酶(1mg/mL，Sigma)和DNaseI(20U/mL，Sigma)的DMEM培养基中进行37℃酶消化1小时，收集单个肿瘤细胞和脾脏细胞。分别以含2％FCS的PBS冲洗分离的单个细胞，然后用相关抗体进行表面染色。细胞经大量洗涤后，在BD FACS Calibur(Becton Dickinson)上获得。流式细胞术数据由Novo Express TM(ACEA Biosciences,Inc.)分析。
Multi-ELISA检测细胞因子
收集实验小鼠的外周血和肿瘤组织，评估IL-10、TNF-a、GM-CSF和TGFβ等细胞因子。Multi-ELISA试剂盒购自Qiagen(Australia)，按照试剂盒内提供的说明书进行实验。
ELISA结果由ELISA程序板在450nm处读取(Polarstar Omega 96-wellmicroplatereader BMG Labtech GmBH,Germany)。
TCC染色
取出肿瘤组织，液氮速冻后，用切片刀同样厚度均切为5片。将肿瘤组织切片置于玻片上，滴加2％的TTC溶液覆盖组织切片，避光反应30min，数码相机拍照。
免疫组织化学
肿瘤组织取材后快速置于10％中性福尔马林溶液中固定，石蜡包埋，切片。石蜡切片常规脱蜡至水。3％H 2O 2去离子水室温避光孵育10min，以消除内源性过氧化氢酶活性，PBS冲洗，5min×3次。将切片浸入到EDTA修复液(1×)中，微波炉加热到沸腾后断电，间隔5-10min再修复1-2次，冷却。滴加5％BSA封闭液37℃孵育30min，甩干。滴加适当稀释的抗CD163和HIF-1α的小鼠单克隆抗体一抗，37℃孵育1-2h或4℃过夜。PBS冲洗，5min×3次。滴加生物素标记山羊抗兔IgG(二抗)，37℃孵育30min。PBS冲洗，5min×3次。滴加SABC，37℃孵育30min。PBS冲洗，5min×3次。按1mL蒸馏水加显色剂A，B，C各1滴，混匀，加至标本上，显色1-10min，蒸馏水充分洗涤以终止反应。苏木素复染。脱水，透明。中性树胶封片，显微镜观察。
肿瘤组织切片Gram stain
肿瘤组织取材后快速置于10％中性福尔马林溶液中固定，石蜡包埋，切片。烤片30min，二甲苯I 5min，二甲苯II 5min，100％乙醇I 2min，95％乙醇I 2min，80％乙醇I2min，流水冲洗干净，晾稍干；革兰氏试剂一1min，流水冲洗，革兰氏试剂二1min，流水冲洗，革兰氏试剂三20s左右，流水冲洗，伊红20s左右，流水冲洗；95％乙醇II 30s，95％乙醇III 1min，100％乙醇II 2min，二甲苯III 3min，二甲苯IV 3min，中性树胶封片，观察。
戈氏梭菌定量分析
采取Trizol法提取组织RNA，利用PrimeScript TM RT reagent Kit with gDNA Eraser(Perfect real time)试剂盒将RNA反转录成cDNA。以cDNA为模板， 采取戈氏梭菌硫氧还蛋白特异性引物进行检测戈氏梭菌细菌，引物序列如下：
Trx Forward primer(SEQ ID NO:1):5'--AATACAGGGAATTTTAGAGGT GCAG-3'
Trx Reverse primer(SEQ ID NO:2):5'--GCTAACATCTTACAAGGCCCA CA-3'
统计分析
采用双尾T检验或Prism 6.0 Mann-Whitney检验(Graphpad Software,SanDiego)进行统计分析。p<0.05差异有统计学意义。
实施例2
戈氏梭菌在实体肿瘤模型中应用的高安全性和强靶向性
模型建立
建立结肠癌皮下移植瘤模型。SD大鼠大脑中动脉栓塞(MCAO)模型建立。C57BL/6小鼠急性心肌梗死(MI)模型建立。
动物分组
在筛选荷瘤动物当日，选择肿瘤体积0.35～0.60cm 3的实验动物用于试验。采用随机原则将筛选出的符合要求的动物以抽签法分为4组：静脉给药对照组、瘤内给药对照组、静脉给予戈氏梭菌芽孢组、瘤内给予戈氏梭菌芽孢组，每组8只小鼠。
SD大鼠大脑中动脉栓塞(MCAO)模型，随机分为4组，健康大鼠对照组，脑梗模型对照组，脑梗模型静脉给药组，脑梗模型颅内给药组，每组5只。
C57BL/6小鼠急性心肌梗死(MI)模型，随机分3组，心梗模型TTC染色组，心梗模型对照组，心梗模型尾静脉给药组，每组5只。
给药
结肠癌模型：芽孢剂量为1×10 7cfu/瘤/次，瘤内给药，对照组给予相同体积的质量体积百分比浓度0.9％氯化钠注射液；芽孢剂量为1×10 8cfu/瘤/次，尾静脉给药，对照组给予相同体积的质量体积百分比浓度0.9％氯化钠注射液；
SD大鼠大脑中动脉栓塞(MCAO)模型：尾静脉给药剂量5×10 7CFU，颅内给药剂量1×10 6CFU。
C57BL/6小鼠急性心肌梗死(MI)模型：尾静脉给药剂量2×10 7CFU。
观察与检查
结肠癌模型在实验期间观察小鼠死亡或濒死情况，行为、小鼠体重情况。
MCAO模型和MI模型，分别取脑组织和心脏组织，进行TTC染色、戈氏梭菌细菌qPCR和芽孢细菌培养法检测，以及组织切片戈氏梭菌Gram stain检测。
结果
结肠癌模型在实验期间，无论瘤内给药或静脉给药，均未出现动物死亡。且小鼠净体重与对照组相比，均无显著性差异(图1)。
MCAO模型试验中，分别对健康大鼠对照组，脑梗模型对照组，脑梗模型静脉给药组，脑梗模型颅内给药组的脑组织进行TTC染色，正常大鼠大脑组织切片TTC染色为红色，MCAO模型大鼠大脑组织切片TTC染色显示，发生脑梗死的大鼠脑组织部分区域呈苍白色(图2)。
对MCAO模型试验中的脑组织进行戈氏梭菌细菌和芽孢检测，尾静脉单次给药注射用戈氏梭菌芽孢和颅内单次给药注射用戈氏梭菌芽孢后，在发生脑梗死的脑组织中检测到戈氏梭菌芽孢，但均未检测到戈氏梭菌细菌。
脑组织进行切片制作和Gram stain，并采用数字病理扫描系统扫描各组大鼠脑组织切片检测戈氏梭菌细菌分布情况，阳性对照组肿瘤组织切片革兰氏染色后切片中可见呈短杆状、蓝紫色的戈氏梭菌(图3a)；阴性对照组肿瘤组织切片革兰氏染色后切片中未见细菌(图3b)，大鼠大脑中动脉栓塞后尾静脉单次给药注射用戈氏梭菌芽孢和颅内单次给药注射用戈氏梭菌芽孢，在发生脑梗死的脑组织切片中均未检测到戈氏梭菌细菌(图3c，3d)。
MI模型TTC染色组小鼠心脏组织TTC染色显示，小鼠心肌组织呈白色，MI模型小鼠心脏组织出现明显梗死(图4)。MI模型小鼠尾静脉单次给药注射用戈氏梭菌芽孢，发生心肌梗死的心肌组织中检测到戈氏梭菌芽孢，但未检测到戈氏梭菌细菌。
心脏组织进行切片制作和Gram stain，并采用数字病理扫描系统扫描各组小鼠心脏组织切片检测戈氏梭菌细菌分布情况。阳性对照组肿瘤组织切片革兰氏染色后切片中可见呈短杆状、蓝紫色的戈氏梭菌(图3a)；阴性对照组肿瘤组织切片革兰氏染色后切片中未见细菌(图3b)，MI模型小鼠尾静脉单次给药注射用戈氏梭菌芽孢后在发生心脏梗死的心肌组织切片中未检测到戈氏梭菌细菌(图5)。
实施例3
戈氏梭菌芽孢联合不同剂量艾坦在CT26小鼠模型中的抗肿瘤作用
采用随机原则将符合要求小鼠(肿瘤体积0.31～0.41cm 3)以抽签法分为8组：对照组、戈氏梭菌芽孢组、艾坦高剂量组、艾坦中剂量组、艾坦低剂量组、芽孢联合艾坦低剂量组、芽孢联合艾坦中剂量组、芽孢联合艾坦高剂量组。
对照组：瘤内给予相同体积的0.9％氯化钠注射液；
戈氏梭菌芽孢组：剂量为1×10 7cfu/瘤/次，瘤内给药，给药2次；
艾坦高剂量组：剂量为180mg/kg/d，灌胃给药，每天给药1次，共7次；
艾坦中剂量组：剂量为120mg/kg/d，灌胃给药，每天给药1次，共7次；
艾坦低剂量组：剂量为60mg/kg/d，灌胃给药，每天给药1次，共7次；
戈氏梭菌联合艾坦组：戈氏梭菌芽孢剂量为1×10 7cfu/瘤/次，瘤内给药，先给予艾坦3天，每天1次，再进行芽孢给药，隔1天给药1次，共给药2次。联合高、中、低剂量同单独艾坦高、中、低剂量组；
结果
治疗期间，各组均未出现死亡或濒死情况。治疗前，各组小鼠体重无显著差异(p>0.05)。与对照组相比，各组小鼠净体重无显著差异(p>0.05)。荷瘤鼠净体重见表1和图6。
表1.实验小鼠净体重
Figure PCTCN2022124089-appb-000002
注：n为各组试验用动物数，小鼠净体重为荷瘤鼠剖出肿瘤后的小鼠体重，数据以均数±标准差表示。
治疗前，各组荷瘤鼠肿瘤体积无显著差异(p>0.05)。给药末期，与对照组相比，单独芽孢组、戈氏梭菌联合低剂量艾坦组和戈氏梭菌联合中剂量艾坦组肿瘤体积均显著小于对照组(p＝0.045，p＝0.000，p＝0.026)。荷瘤鼠肿瘤体积见表2和图7。
表2.荷瘤鼠肿瘤体积
Figure PCTCN2022124089-appb-000003
注：n为各组试验用动物数，数据以均数±标准差表示。与对照组相比，*p<0.05，**p<0.01。
与对照组相比，单独芽孢组、戈氏梭菌联合低剂量艾坦组、戈氏梭菌联合中剂量艾坦组和戈氏梭菌联合高剂量艾坦组肿瘤重量显著小于对照组(p＝0.000，p＝0.000，p＝0.000，p＝0.000)。各组肿瘤重量见表3，图8。
表3.肿瘤重量
Figure PCTCN2022124089-appb-000004
根据肿瘤重量计算抑瘤率，抑瘤率表现为：戈氏梭菌联合低剂量艾坦组>芽孢联合中剂量艾坦组>戈氏梭菌联合高剂量组＞单独芽孢组＞高剂量艾坦组＞低剂量艾坦组＞中剂量艾坦组，如表4所示。抑瘤率肿瘤解剖形态如图9所示。
表4.抑瘤率
Figure PCTCN2022124089-appb-000005
Figure PCTCN2022124089-appb-000006
戈氏梭菌联合低剂量艾坦组有28.6％小鼠肿瘤完全消除，至实验终点也未见肿瘤生长，治愈率明显高于戈氏梭菌联合高剂量艾坦组(14.3％)和戈氏梭菌联合中剂量艾坦组(14.3％)，而高、中和低剂量单独艾坦组和单独芽孢组均未出现肿瘤消失情况，治愈率为0％。可见，戈氏梭菌联合低剂量艾坦表现出显著的抗肿瘤效果，并明显优于其他组别，如表5。
表5.治愈率
Figure PCTCN2022124089-appb-000007
由上述结果可见，各剂量治疗组均表现出对肿瘤生长的抑制作用，并以芽孢联合低剂量艾坦组抗肿瘤作用最明显。戈氏梭菌芽孢与抗血管生成抑制剂如艾坦联合治疗显示出超叠加作用，其中戈氏梭菌芽孢联合低剂量艾坦治疗的抗肿瘤效果最佳。
实施例4
戈氏梭菌联合高剂量艾坦和低剂量艾坦抗肿瘤作用机制研究
戈氏梭菌联合低剂量艾坦抗肿瘤作用明显优于高于戈氏梭菌联合高剂量艾坦，对戈氏梭菌联合低剂量艾坦和高剂量艾坦抗肿瘤的作用机制进行研究。
分别取对照组、单独芽孢组、戈氏梭菌联合低剂量艾坦组和戈氏梭菌联合高剂量艾坦组处理的小鼠肿瘤组织，采用流式细胞仪对上述肿瘤组织样品进行流式细胞术分析。与对照组相比，单独芽孢组、戈氏梭菌联合低剂量艾坦组和戈氏梭菌联合高剂量艾坦组肿瘤组织中的CD45 +CD3 +T、CD45 +CD3 +CD8 +T和F4/80 +细胞浸润肿瘤的比例均显著升高；与单独芽孢组相比，戈氏梭菌联合低剂量艾坦组小鼠肿瘤组织中CD45 +CD3 +T、CD45 +CD3 +CD8 +T和F4/80 +细胞浸 润的比例均显著升高，而戈氏梭菌联合高剂量艾坦组未见显著差异(图10)。
采用细胞内染色法研究肿瘤内T细胞的细胞因子表达，与单独芽孢组相比，戈氏梭菌联合低剂量艾坦组肿瘤内CD3 +IL-10 +T、CD3 +IFN-γ +T细胞显著升高(p<0.05)，而戈氏梭菌联合高剂量艾坦组CD3 +IL-10 +T、CD3 +IFN-γ +T细胞未见明显变化(图11)。
Multi-ELISA检测瘤内IL-10、TNF-a、GM-CSF和TGFβ等细胞因子，与对照组相比，单独芽孢组、戈氏梭菌联合高剂量艾坦组和戈氏梭菌联合低剂量艾坦组IL-10、TNF-a、GM-CSF均有增高。与其它组相比，戈氏梭菌联合低剂量艾坦组肿瘤内TGFβ表达明显降低(图12)。可见，戈氏梭菌溶瘤可诱导TME中细胞因子、趋化因子表达。同时戈氏梭菌联合低剂量艾坦可显著降低TGFβ的表达，改善免疫抑制性肿瘤微环境，而联合高剂量艾坦对TGFβ表达无明显抑制作用。
通过流式细胞术分析了肿瘤内髓系来源的抑制细胞(MDSC)、肿瘤相关巨噬细胞(TAM)等细胞的数量。与对照组相比，戈氏梭菌联合低剂量艾坦治疗后，瘤内CD11b +Ly6G -Ly6C high单核MDSCs和CD11b +Ly6G +Ly6C low多形核-MDSCs的数量未见明显差异。与戈氏梭菌联合高剂量艾坦相比，戈氏梭菌联合低剂量艾坦治疗后Mo-MDSCs和PMN-MDSCs比例均降低(图13)。此外，与其他治疗组相比，戈氏梭菌联合低剂量艾坦组的CD11b +F4/80 +-TAMs在总活细胞中的比例显著降低(图14)。IHC染色检测肿瘤中CD163 +(M2样巨噬细胞标记物)，研究结果显示，戈氏梭菌联合低剂量艾坦组肿瘤中CD163 +细胞数量下降，表明戈氏梭菌联合低剂量艾坦治疗可有效减少肿瘤中TAM的数量(图15)。
肿瘤组织HIF-1α免疫组织化学染色显示，与对照组相比，戈氏梭菌联合低剂量艾坦肿瘤组织缺氧明显减轻，而戈氏梭菌联合高剂量艾坦组肿瘤组织缺氧程度升高(图16)。表明高剂量艾坦加重了肿瘤组织缺氧，虽然创造了一个利于戈氏梭菌繁殖的缺氧环境，但其抗肿瘤效果低于戈氏梭菌联合低剂量艾坦组。而戈氏梭菌联合低剂量艾坦组的肿瘤缺氧虽明显降低，理论上可能加速肿瘤生长，但肿瘤体积显示肿瘤生长受到明显抑制。
综上所述，戈氏梭菌联合低剂量艾坦可促进CD45 +CD3 +T、CD45 +CD3 +CD8 +T、F4/80 +等免疫细胞的浸润，诱导IFN-γ、TNF-α、GM-CSF 等细胞因子和趋化因子表达增强，同时有效降低肿瘤组织中TGFβ的表达，降低免疫抑制剂肿瘤相关巨噬细胞(TAM)、髓源性抑制细胞(MDSC)等的数量，减少免疫抑制。可见，戈氏梭菌联合低剂量艾坦高效抗肿瘤是通过诱导肿瘤血管正常化，使TME由免疫抑制转变为免疫激活状态，实现高效抗肿瘤，其独特的治疗效果应归因于抗肿瘤免疫微环境的变化，而非利用高剂量艾坦加重肿瘤缺氧创造戈氏梭菌繁殖的缺氧环境高效抗肿瘤。
尽管上述实施例对本发明做出了详尽的描述，但它仅仅是本发明一部分实施例，而不是全部实施例，人们还可以根据本实施例在不经创造性前提下获得其他实施例，这些实施例都属于本发明保护范围。

Claims (21)

  1. 戈氏梭菌联合肿瘤血管生成抑制剂在制备治疗肿瘤的医药制品中的应用。
  2. 根据权利要求1所述的应用，其特征在于，所述医药制品包括促进CD45 +CD3 +T、CD45 +CD3 +CD8 +T和F4/80 +浸润，诱导IFN-γ、TNF-α和GM-CSF表达增强，降低TGFβ的表达，降低巨噬细胞TAM、髓源性抑制细胞MDSC的数量，减少免疫抑制的医药制品。
  3. 根据权利要求1所述的应用，其特征在于，所述医药制品包括诱导肿瘤血管正常化，使TME由免疫抑制转变为免疫激活状态的医药制品。
  4. 根据权利要求1所述的应用，其特征在于，所述戈氏梭菌为戈氏梭菌MW-DCG-LCv-26菌株或者戈氏梭菌驯化后获得的菌株；
    所述戈氏梭菌MW-DCG-LCv-26菌株保藏于澳大利亚国家计量研究院，菌株编号为V12/001486。
  5. 根据权利要求4所述的应用，其特征在于，所述戈氏梭菌驯化后获得的菌株包括MW-DCG-HNCv-18菌株或MW-DCG-CCv-17菌株；
    所述MW-DCG-HNCv-18菌株保藏于澳大利亚国家计量研究院，菌株保藏号V12/001485；
    所述MW-DCG-CCv-17菌株保藏于澳大利亚国家计量研究院，菌株保藏号V12/001487。
  6. 根据权利要求1～5任一项所述的应用，其特征在于，所述的戈氏梭菌为芽孢形式。
  7. 根据权利要求1～5任一项所述的应用，其特征在于，所述肿瘤血管生成抑制剂包括艾坦、舒尼替尼、帕唑帕尼、贝伐珠单抗、雷莫芦单抗、康柏西普、阿柏西普、索拉非尼和瑞戈非尼中的一种或两者以上的组合。
  8. 根据权利要求1～5任一项所述的应用，其特征在于，所述肿瘤为结肠癌、Lewis肺癌、鼻咽癌、非小细胞肺癌、纤维肉瘤或黑色素瘤。
  9. 一种治疗肿瘤的药物，其特征在于，所述药物中的有效成分包括戈氏梭菌和肿瘤血管生成抑制剂。
  10. 根据权利要求9所述的药物，其特征在于，所述的戈氏梭菌为戈氏梭菌MW-DCG-LCv-26菌株或者戈氏梭菌驯化后获得的菌株；
    所述戈氏梭菌MW-DCG-LCv-26菌株保藏于澳大利亚国家计量研究院，菌株编号为V12/001486。
  11. 根据权利要求10所述的药物，其特征在于，所述戈氏梭菌驯化后获得的菌株包括MW-DCG-HNCv-18菌株或MW-DCG-CCv-17菌株；
    所述MW-DCG-HNCv-18菌株保藏于澳大利亚国家计量研究院，菌株保藏号V12/001485；
    所述MW-DCG-CCv-17菌株保藏于澳大利亚国家计量研究院，菌株保藏号V12/001487。
  12. 根据权利要求10所述的药物，其特征在于，所述的戈氏梭菌为芽孢形式。
  13. 根据权利要求10所述的药物，其特征在于，所述肿瘤血管生成抑制剂包括艾坦、舒尼替尼、帕唑帕尼、贝伐珠单抗、雷莫芦单抗、康柏西普、阿柏西普、索拉非尼和瑞戈非尼中的一种或两者以上的组合。
  14. 根据权利要求10～13任一项所述的药物，其特征在于，所述药效成分戈氏梭菌与肿瘤血管生成抑制剂的给药顺序为先后给药或同时给药。
  15. 根据权利要求12所述的药物，其特征在于，每1×10 7CFU所述芽孢形式的戈氏梭菌联合艾坦的剂量是60mg/kg/d。
  16. 根据权利要求10～13任一项所述的药物，其特征在于，所述肿瘤为结肠癌、Lewis肺癌、鼻咽癌、非小细胞肺癌、纤维肉瘤或黑色素瘤。
  17. 根据权利要求10所述的药物，其特征在于，所述的戈氏梭菌为戈氏梭菌芽孢冻干粉。
  18. 根据权利要求17所述的药物，其特征在于，所述戈氏梭菌芽孢冻干粉以戈氏梭菌芽孢为活性成分，以1％蔗糖为辅料，经过-40℃4h；-35℃10min同时抽真空、-30℃10min、-25℃10min、-20℃26h、-15℃2h、-10℃10min、-5℃10min、0℃10min、10℃2h、15℃10min、20℃3h、27℃3h冻干程序制备而成。
  19. 根据权利要求18所述的药物，其特征在于，每支所述戈氏梭菌芽孢冻干粉中戈氏梭菌芽孢的个数为1×10 8CFU。
  20. 一种治疗肿瘤的方法，其特征在于，戈氏梭菌联合艾坦给药；
    所述的戈氏梭菌为芽孢形式，戈氏梭菌芽孢剂量为1×10 7cfu/瘤/次，瘤内给药，先给予艾坦3天，每天1次，再进行戈氏梭菌芽孢给药，隔1天给药1次， 共给药2次；
    所述艾坦灌胃给药，给药剂量为60mg/kg/d或120mg/kg/d或180mg/kg/d。
  21. 根据权利要求20所述的方法，其特征在于，所述肿瘤为结肠癌、Lewis肺癌、鼻咽癌、非小细胞肺癌、纤维肉瘤或黑色素瘤。
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