WO2019095508A1 - 一种用于增强t细胞免疫功能的组合物及其制备方法 - Google Patents

一种用于增强t细胞免疫功能的组合物及其制备方法 Download PDF

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WO2019095508A1
WO2019095508A1 PCT/CN2017/118590 CN2017118590W WO2019095508A1 WO 2019095508 A1 WO2019095508 A1 WO 2019095508A1 CN 2017118590 W CN2017118590 W CN 2017118590W WO 2019095508 A1 WO2019095508 A1 WO 2019095508A1
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bacteroides fragilis
cancer
bacteroides
inactivated
composition
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French (fr)
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曾谷城
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中山大学
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Priority to AU2017440014A priority Critical patent/AU2017440014A1/en
Priority to CA3082716A priority patent/CA3082716A1/en
Priority to EP17932406.6A priority patent/EP3712246A4/en
Priority to US16/764,413 priority patent/US20200353014A1/en
Priority to KR1020207014957A priority patent/KR20200071123A/ko
Priority to JP2020545408A priority patent/JP2021503504A/ja
Publication of WO2019095508A1 publication Critical patent/WO2019095508A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2200/00—Function of food ingredients
    • A23V2200/30—Foods, ingredients or supplements having a functional effect on health
    • A23V2200/324—Foods, ingredients or supplements having a functional effect on health having an effect on the immune system
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales

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  • the present invention belongs to the field of biomedical technology, and in particular relates to a composition for enhancing T cell immune function, a composition for preparing a prophylactic and/or therapeutic tumor, and a preparation method of the composition.
  • Malignant tumors have become the "first killer” threatening human health and life. Among the causes of human death, cases of death from malignant tumors have risen to the top. About 8.7 million people worldwide suffer from cancer every year, and about 6.9 million people die. In China alone, 1.05 million people die from advanced malignant tumors every year. Current methods for treating malignant tumors are very limited.
  • Bacteroides fragilis (B. fragilis) is an obligate anaerobic bacterium that is negative for Gram staining, rod-shaped, blunt and densely stained at both ends, and has a capsule, no spores, and no power. Enterotoxin-type and non-enteric enterotoxin-type. Bacteroides fragilis is a part of normal intestinal bacteria in humans and animals. It is mainly found in the colon. In addition, the respiratory tract and genitourinary tract can also grow. Numerous studies have shown that Bacteroides fragilis has good curative effect on prevention and treatment of acute and chronic enteritis, dysbacteriosis, upper respiratory tract infection and neurosis.
  • Another object of the invention is to provide a process for the preparation of said compositions.
  • the present invention provides an application of Bacteroides fragilis for enhancing the T cell immune function of the body.
  • the invention also provides the use of Bacteroides fragilis for the prevention and/or treatment of tumors.
  • the invention also provides the use of Bacteroides fragilis for the preparation of a medicament for enhancing the immune function of the body T cells.
  • the present invention also provides the use of Bacteroides fragilis for the preparation of a medicament for preventing and/or treating a tumor.
  • the present invention also provides a composition for enhancing the T cell immune function of the body, which comprises Bacteroides fragilis.
  • the Bacteroides fragilis is inactivated, attenuated, low infective or non-infectious, and contains a bacterial protein component of a natural structure, which is capable of effectively reducing T cell expression depleting molecules and enhancing T cell immunity.
  • the Bacteroides fragilis is one or more of the following: Bacteroides fragilis live cells; Bacteroides fragilis inactivated, genetically recombined, engineered or modified, attenuated, chemically treated, physically treated or inactivated; Bacteroides lysate; and/or Bacteroides fragilis liquid culture supernatant.
  • the Bacteroides fragilis is any one or more of dry heat, moist heat, filtration, organic solvent, chemical reagent, ultraviolet or infrared ray, fermentation, lyophilization, genetic recombination, genetic modification or modification. Inactivated.
  • the composition includes, but is not limited to, any one of a pharmaceutical composition, a food, a health supplement, or a food additive.
  • the invention also provides a preparation method of the composition for enhancing the T cell immune function of the body, the steps of which are as follows:
  • the 15% glycerol/physiological saline solution means that 15% by volume of glycerin is contained in physiological saline.
  • the present invention also provides a composition for preventing and/or treating a tumor comprising Bacteroides fragilis.
  • the tumor preferably includes, but is not limited to, breast cancer, colorectal cancer, ovarian cancer, pancreatic cancer, prostate cancer, bladder cancer, melanoma, lung cancer, stomach cancer, liver cancer, cholangiocarcinoma, glioma, head and neck squamous
  • breast cancer colorectal cancer
  • ovarian cancer pancreatic cancer
  • prostate cancer bladder cancer
  • melanoma lung cancer
  • stomach cancer liver cancer
  • cholangiocarcinoma glioma
  • head and neck squamous One or more of cell cancer, cervical cancer, renal cell carcinoma, thyroid cancer, acute myeloid leukemia, myeloma, esophageal cancer, lymphoma, and skin cancer.
  • the Bacteroides fragilis is inactivated, attenuated, low infective or non-infectious, and contains a bacterial protein component of a native structure.
  • the Bacteroides fragilis is any one or more of dry heat, moist heat, filtration, organic solvent, chemical reagent, ultraviolet or infrared ray, fermentation, lyophilization, genetic recombination, genetic modification or modification. Inactivated.
  • the composition includes, but is not limited to, any one of a pharmaceutical composition, a food, a health supplement, or a food additive.
  • the invention also provides a method for enhancing immune function of a body T cell, the method comprising the steps of:
  • the present invention also provides a method for preventing or treating a tumor, the method comprising the step of administering to a patient a composition comprising an inactivated, low-infectious or non-infectious Bacteroides fragilis, thereby reducing T cells It inhibits the expression of the molecule Tim-3 and enhances the immune function of T cells.
  • the invention also provides a preparation method of the composition for preventing and/or treating a tumor, the steps of which are as follows:
  • the experimental results show that the effect of Bacteroides fragilis on function enhancement, reduction or control of tumor growth can reach or exceed 40%, or even 40-90%, compared with saline or inactivated live intestinal control group.
  • the enhanced function of Bacteroides fragilis can reduce immune cells, especially T cell function inhibitory molecules or T cell depletion molecules including T-cell immunoglobulin and mucin-domain containing-3 (T cell immunoglobulin mucin molecule 3) :Tim-3 or Tim3), etc. but not limited to the expression of Tim-3, and the functionally enhanced Bacteroides fragilis reduces the expression of T cell depletion molecules such as Tim-3 compared with saline or inactivated live intestinal control group. Reached or exceeded 35 to 65%.
  • the enhanced function of Bacteroides fragilis can simultaneously reduce both CD4+ T cells and CD8+ T cells.
  • the most important anti-tumor immune cells express T cell depletion molecules including Tim-3 but not limited to Tim-3, thereby enhancing CD4+T.
  • Fig. 1 is a schematic diagram showing the experimental procedure for detecting and/or preventing the action of Bacteroides fragilis and inactivating Bacteroides fragilis on mouse melanoma.
  • Figure 2 shows the in situ detection of Bacteroides fragilis and inactivated Bacteroides fragilis on tumor growth inhibition in mice.
  • the dotted circle indicates the location and size of the tumor. The larger the diameter of the circle, the faster the tumor grows and the larger the tumor volume. It can be seen that the tumor volume of mice inactivated by Bacteroides fragilis is significantly smaller than that of the other two groups of mice.
  • Fig. 3 is a photograph showing the tumor detection of Bacteroides fragilis and inactivated Bacteroides fragilis on tumor growth in mice. As can be seen from the figure, the tumor volume of the mice inactivated by Bacteroides fragilis was significantly reduced compared to the saline or inactivated control group.
  • Figure 4 is a graph showing statistical analysis of tumor volume in mice after prevention and/or treatment of Bacteroides fragilis and inactivated Bacteroides fragilis.
  • the effect of inactivating Bacteroides fragilis inhibiting tumor growth volume was 40-40% higher than that of physiological saline or inactivated control group.
  • Fig. 5 is a flow cytometric analysis diagram of one mouse of each treatment group of Bacteroides fragilis and inactivated Bacteroides fragilis for preventing and/or treating the expression of the suppressor molecule Tim-3 in CD4+ T cells after melanoma.
  • the number in the upper right quadrant shows the percentage of total spleen CD4+ T cells in Tim-3+CD4+ T cells (T cells expressing both Tim-3 and CD4). From the flow cell analysis quadrant map, it can be seen that the inactivated Bacteroides fragilis has an effect of reducing the expression of Tim-3 molecules by 38 to 65% compared to the physiological saline or inactivated control group.
  • Fig. 6 is a statistical analysis diagram of the expression of the inhibitory molecule Tim-3 in CD4+ T cells after prevention and/or treatment of melanoma by Bacteroides fragilis and inactivated Bacteroides fragilis. It can be seen from the statistical graph that inactivation of Bacteroides fragilis significantly reduced the expression of Tim-3 molecules in CD4+ T cells compared to saline or inactivated controls. In the statistical analysis chart, ** indicates student t-test p ⁇ 0.01. p ⁇ 0.01 had statistically significant differences. There were 12 mice in each treatment group.
  • Figure 7 is a flow cytometric analysis of the expression of the inhibitory molecule Tim-3 in CD8+ T cells after prevention and/or treatment of melanoma by Bacteroides fragilis and inactivated Bacteroides fragilis.
  • the number in the upper right quadrant shows the percentage of total spleen CD8+ T cells in Tim-3+CD8+ T cells (T cells expressing both Tim-3 and CD8).
  • the inactivation of Bacteroides fragilis reduced the expression of Tim-3 molecules by 35 to 60% compared to the saline or inactivated control group.
  • Figure 8 is a graph showing the statistical analysis of the expression of the inhibitory molecule Tim-3 in CD8+ T cells after prevention and/or treatment of melanoma by Bacteroides fragilis and inactivated Bacteroides fragilis. It can be seen from the statistical graph that the inactivation of Bacteroides fragilis significantly reduced the expression of Tim-3 molecules in CD8+ T cells compared to the saline or inactivated control group. * in the statistical analysis chart indicates student t-test p ⁇ 0.05. p ⁇ 0.05 was statistically significant. There were 12 mice in each treatment group.
  • the present invention will be further specifically described below in conjunction with the embodiments, but is not limited thereto. It is to be noted that the inactivated Bacteroides fragilis or the pharmaceutical composition, food, health supplement and food containing the Bacteroides fragilis of the present invention for enhancing the T cell immune function of the body and treating and/or preventing the tumor in the present invention
  • the additives, after administration to a subject can be applied to the indications described above and exhibit the functions described above, all of which are tested within the scope of the invention, hereinafter, merely for illustration, Only a part of the description is described in the embodiment, however, it should not be construed as limiting the invention.
  • Bacteroides fragilis is inactivated, attenuated, low infective or non-infectious and is capable of reducing T cell depletion molecules expressed by CD4+ T cells and/or CD8+ T cells.
  • Bacteroides fragilis is preferably inactivated by any one or more of dry heat, moist heat, filtration, organic solvents, chemical agents, ultraviolet or infrared radiation, fermentation, lyophilization, genetic recombination, genetic modification or engineering.
  • the tumor/cancer includes, but is not limited to, a solid tumor/cancer.
  • the tumor/cancer can be breast cancer, colorectal cancer, ovarian cancer, pancreatic cancer, prostate cancer, bladder cancer, melanoma, lung cancer, gastric cancer, liver cancer, cholangiocarcinoma, glioma, Head or neck squamous cell carcinoma, cervical cancer, renal cell carcinoma, thyroid cancer, acute myeloid leukemia, myeloma, esophageal cancer, lymphoma, skin cancer, etc. or a combination of these tumors.
  • Step 1 Take a freeze-dried Bacteroides fragilis strain (purchased from the ATCC official website), add 200 ⁇ L of TSB medium, reconstitute, blood plate, and anaerobic tank gas control system after pumping Analytical culture in a biochemical incubator at 37 ° C for 48 hours (h);
  • Step 2 Pick up the monoclonal colonies into 10 ml TSB medium, anaerobic culture for 12 h at 37 ° C;
  • Step 3 Take 1 bottle of 500ml TSB medium, connect 1% (v/v) strain, 37 ° C, anaerobic culture for 48h;
  • Step 4 Take the bacteria solution and centrifuge at 6000 rpm for 10 min. Wash twice with physiological saline, and finally reconstitute the bacterial sludge with physiological saline for use and count the viable bacteria.
  • Example 2 Therapeutic effect of Bacteroides fragilis on mouse tumors (eg, melanoma)
  • FIG 1 is a schematic diagram showing the experimental procedure for the detection and/or prevention of tumors (melanoma) in mice by Bacteroides fragilis and inactivated Bacteroides fragilis.
  • the culture method of Bacteroides fragilis is the same as in Example 1.
  • Step 1 Take a freeze-preserved strain (purchased from the ATCC official website), add 200 ⁇ L of lyophilized preservation culture medium, reconstitute, absorb 20 ⁇ L, and draw a blood plate.
  • the anaerobic tank gas control system is pumped after biochemical culture. 37 ° C in the box, anaerobic culture for 48h;
  • Step 2 Pick up the monoclonal colonies into 10 ml TSB medium, anaerobic culture for 12 h at 37 ° C;
  • Step 3 Take 1 bottle of 500ml TSB medium, connect 1% (v/v) strain, 37 ° C, anaerobic culture for 48h;
  • Step 4 The bacteria solution was centrifuged, centrifuged with a centrifuge, and centrifuged at 6000 rpm for 10 minutes. Wash twice with physiological saline, and finally reconstitute the bacterial sludge with physiological saline for use and count the viable bacteria.
  • the inactivated bacterial solution was obtained by heating in a water bath at a temperature of 70 ° C for 30 minutes (min).
  • mice C57BL/6 mice were 36 in 3 to 4 weeks, and they were in good mental state and purchased from the Experimental Animal Center of Sun Yat-sen University. The mice were randomly divided into 3 groups, 12 in each group. The gender of each group was matched. The three groups were saline control group and Bacteroides fragilis gavage group (or short-lived group or non-inactivated group). Inactivate the Bacteroides fragilis gavage group (or simply: inactivated group).
  • mice were intragastrically administered with 1*10 9 CFU of Bacteroides fragilis, 1*10 9 CFU of Bacteroides fragilis and saline control, and were continuously intragastrically administered for 2 weeks.
  • mouse tumor (melanoma) B16 cells were grown to log phase, cells were digested with TE, neutralized in medium, cells were collected by centrifugation, and washed twice with DPBS to remove residual serum, and the cells were resuspended in DPBS.
  • 1*10 6 B16 cells were inoculated into the right flank of each mouse. The mice were continuously treated with intragastric administration. After 2 weeks, the tumor-bearing mice were sacrificed, and the subcutaneous tumor masses were dissected to measure the tumor size.
  • CD4+ T cells and CD8+ T cells are two key T cell subsets that are critical for anti-tumor, they are also isolated using anti-CD3, anti-CD4, anti-CD8 and anti-Tim-3 monoclonal fluorescent antibody markers.
  • the lymphocytes of the mouse spleen were then analyzed by flow cytometry for the percentage of expression of Tim-3 by CD4+ T cells and CD8+ T cells in the mouse spleen.
  • Tim-3 is a depletion molecule that inhibits the anti-tumor effect of T cells.
  • the higher expression of Tim-3 indicates that the body's T cell anti-tumor immune function is in a state of inhibition, low energy and/or depletion. Therefore, controlling and reducing the expression of Tim-3 on T cells is the key to enhance or enhance the anti-tumor immune function of human T cells.
  • the lower the expression of Tim-3 the stronger the anti-tumor immune function of T cells.
  • the results of Fig. 5, Fig. 6, Fig. 7 and Fig. 8 indicate that the inactivated Bacteroides fragilis can inhibit CD4+ T cells and/or CD8+ T cells more efficiently and significantly than physiological saline or inactivated Bacteroides fragilis.
  • Tim-3 (the effect of inhibiting the expression of Tim-3 is statistically significant) enhances T cell immune function and enhances the body's ability to resist tumor immunity. This result also further explains clearly why the inactivated Bacteroides fragilis has a stronger anti-tumor effect than the saline or Bacteroides fragilis live bacteria group.

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Abstract

提供了一种包括脆弱拟杆菌的组合物，该组合物用于增强机体T细胞免疫功能，或用于预防和/或治疗肿瘤。

Description

一种用于增强T细胞免疫功能的组合物及其制备方法 技术领域
本发明属于生物医疗技术领域，具体涉及一种用于增强T细胞免疫功能的组合物、一种用于制备预防和/或治疗肿瘤的组合物、以及所述组合物的制备方法。
背景技术
恶性肿瘤已成为威胁人类健康和生命的“第一杀手”，在人类死亡原因中，因患恶性肿瘤致死的病例已上升到首位。全世界每年大约有870万人患肿瘤，死亡约690万人，仅我国每年就有105万人死于晚期恶性肿瘤。目前治疗恶性肿瘤的方法十分有限。
脆弱拟杆菌(Bacteroides fragilis，简称B.fragilis)是一种革兰染色阴性、杆状、两端钝圆而浓染，有荚膜、无芽胞、无动力的专性厌氧细菌，其分为产肠毒素型和非产肠毒素型。脆弱拟杆菌作为人及动物肠道正常菌的一部分，主要存于结肠中，此外，呼吸道胃肠道及泌尿生殖道也可定植生长。众多研究表明，脆弱拟杆菌对防治急慢性肠炎、菌群失调、上呼吸道感染和神经官能症等具有较好疗效。
发明内容
本发明的一个目的是提供一种增强机体T细胞免疫功能以及预防和/或治疗肿瘤的组合物。
本发明的另一个目的是提供所述组合物的制备方法。
为了实现以上目的，本发明提供了脆弱拟杆菌用于增强机体T细胞免疫功能的应用。
本发明还提供了脆弱拟杆菌用于预防和/或治疗肿瘤的应用。
本发明还提供了脆弱拟杆菌用于制备增强机体T细胞免疫功能的药物的应用。
本发明还提供了脆弱拟杆菌用于制备预防和/或治疗肿瘤的药物的应用。
本发明还提供了一种用于增强机体T细胞免疫功能的组合物，其包括脆弱拟杆菌(Bacteroides fragilis)。
优选地，所述脆弱拟杆菌是灭活的、减毒的、低感染性或非感染性的，并且含有天然结构的细菌蛋白组分，能够有效降低T细胞表达耗竭分子从而增强T细胞免疫。所述脆弱拟杆菌是以下中的一种或多种：脆弱拟杆菌活菌体；经过灭活、基因重组、改造或修饰、减毒、化学处理、物理处理或灭活的脆弱拟杆菌；脆弱拟杆菌裂解物；和/或脆弱拟杆菌液体培养上清液。
优选地，所述脆弱拟杆菌是通过干热、湿热、过滤、有机溶剂、化学试剂、紫外或红外射线、发酵、冻干、基因重组、基因修饰或改造的方法中的任意一种或多种灭活的。
所述组合物包括但不限于是药物组合物、食品、保健品或食品添加剂中的任意一种。
本发明还提供了所述用于增强机体T细胞免疫功能的组合物的制备方法，其步骤如下：
(1)收集活脆弱拟杆菌的培养物；
(2)用15％甘油/生理盐水溶液洗涤所述脆弱拟杆菌；
(3)将所述脆弱拟杆菌重悬于所述15％甘油/生理盐水溶液中；
(4)通过70℃水浴加热30分钟所述脆弱拟杆菌，获得灭活菌体。
所述的15％甘油/生理盐水溶液是指生理盐水中含有15％(体积百分比)的甘油。
本发明还提供了一种用于预防和/或治疗肿瘤的组合物，其包含脆弱拟杆菌。
所述肿瘤优选包括但不限于乳腺癌、结直肠癌、卵巢癌、胰腺癌、前列腺癌、膀胱癌、黑色素瘤、肺癌、胃癌、肝癌、胆管癌、神经胶质瘤、头、颈部鳞状细胞癌、子宫颈癌、肾细胞癌、甲状腺癌、急性髓性白血病、骨髓瘤、食管癌、淋巴瘤、皮肤癌中的一种或多种。
优选地，所述脆弱拟杆菌是灭活的、减毒的、低感染性或非感染性的，并且含有天然结构的细菌蛋白组分。
优选地，所述脆弱拟杆菌是通过干热、湿热、过滤、有机溶剂、化学试剂、紫外或红外射线、发酵、冻干、基因重组、基因修饰或改造的方法中的任意一种或多种灭活的。
所述组合物包括但不限于是药物组合物、食品、保健品或食品添加剂中的任意一种。
本发明还提供了用于增强机体T细胞免疫功能的方法，所述方法包括步骤：
(1)提供包含灭活的或非感染性的脆弱拟杆菌的组合物；和
(2)给患者施用有效量的所述组合物。
本发明还提供了用于预防或治疗肿瘤的方法，所述方法包括给患者施用包含灭活的、低感染性的或非感染性的脆弱拟杆菌的组合物的步骤，由此减少T细胞中抑制分子Tim-3的表达并增强T细胞的免疫功能。
本发明还提供了所述用于预防和/或治疗肿瘤的组合物的制备方法，其步骤如下：
(1)收集活脆弱拟杆菌的培养物；
(2)用15％甘油/生理盐水溶液洗涤所述脆弱拟杆菌；
(3)将所述脆弱拟杆菌重悬于所述15％甘油/生理盐水溶液中；
(4)通过70℃水浴加热30分钟所述脆弱拟杆菌，获得灭活菌体。
实验结果表明，脆弱拟杆菌经功能增强，减少或控制肿瘤生长的效果对比生理盐水或不灭活肠道菌对照组，可以达到或超过40％，甚至40～90％。
脆弱拟杆菌经功能增强，可以减少免疫细胞特别是T细胞功能抑制分子或又称T细胞耗竭分子包括T-cell immunoglobulin and mucin-domain containing-3(T细胞免疫球蛋白黏蛋白分子3)(简称：Tim-3或Tim3)等但不仅限于Tim-3的表达，而且经功能增强的脆弱拟杆菌减少Tim-3等T细胞耗竭分子表达的效果对比生理盐水或不灭活肠道菌对照组可以达到或超过35～65％。
脆弱拟杆菌经功能增强，可以同时减少CD4+T细胞与CD8+T细胞这两类最为重要的抗肿瘤免疫细胞表达T细胞耗竭分子包括Tim-3但不限于Tim-3从而同时增强CD4+T细胞与CD8+T细胞的抗肿瘤免疫功能。
附图说明
图1为检测脆弱拟杆菌及灭活脆弱拟杆菌对小鼠黑色素瘤的治疗和/或预防作用实验流程示意图。
图2为脆弱拟杆菌及灭活脆弱拟杆菌对小鼠体内肿瘤生长抑制作用的小鼠原位检测，虚线圆圈标识的为肿瘤的位置与大小。圆圈的直径越大，表示肿瘤的生长速度更快并且肿瘤体积越大。图中可以看出灭活脆弱拟杆菌处理的小鼠的肿瘤体积显著小于其它两组小鼠的肿瘤。
图3为脆弱拟杆菌及灭活脆弱拟杆菌对小鼠体内肿瘤生长抑制作用的肿瘤检测照片。从图可以看出，相对比生理盐水或不灭活对照组，灭活脆弱拟杆菌处理的小鼠的肿瘤体积大幅度减小。
图4为脆弱拟杆菌及灭活脆弱拟杆菌预防和/或治疗后小鼠体内肿瘤体积对比统计分析图。相对比生理盐水或不灭活对照组，灭活脆弱拟杆菌抑制肿瘤生长体积的效果达到或超过40～90％。*表示student t-test p<0.05，**表示student t-test p<0.01。p<0.05或p<0.01都具有统计学显著差异意义。每种处理组有12只小鼠。
图5为脆弱拟杆菌及灭活脆弱拟杆菌预防和/或治疗黑色素瘤后CD4+T细胞中抑制分子Tim-3的表达情况的每种处理组各一只小鼠的流式细胞分析图。右上象限的数字显示的为Tim-3+CD4+T细胞(既表达Tim-3也表达CD4的T细胞)占脾脏总体CD4+T细胞的百分比情况。从流式细胞分析象限图可以看出，相对比生理盐水或不灭活对照组，灭活脆弱拟杆菌减少Tim-3分子表达的效果达到或超过38～65％。
图6为脆弱拟杆菌及灭活脆弱拟杆菌预防和/或治疗黑色素瘤后CD4+T细胞中抑制分子Tim-3的表达情况的统计分析图。从统计图可以看出，相对比生理盐水或不灭活对照组，灭活脆弱拟杆菌显著减少CD4+T细胞中Tim-3分子的表达。统计分析图中**表示student t-test p<0.01。p<0.01具有统计学显著差异意义。每种处理组有12只小鼠。
图7为脆弱拟杆菌及灭活脆弱拟杆菌预防和/或治疗黑色素瘤后CD8+T细胞中抑制分子Tim-3的表达情况的流式细胞分析图。右上象限的数字显示的为Tim-3+CD8+T细胞(既表达Tim-3也表达CD8的T细胞)占脾脏总体CD8+T细胞的百分比情况。从象限图可以看出，相对比生理盐水或不灭活对照组，灭活脆弱拟杆菌减少Tim-3分子表达的效果达到或超过35～60％。
图8为脆弱拟杆菌及灭活脆弱拟杆菌预防和/或治疗黑色素瘤后CD8+T细胞中抑制分子Tim-3的表达情况的统计分析图。从统计图可以看出，相对比生理盐水或不灭活对照组，灭活脆弱拟杆菌显著减少CD8+T细胞中Tim-3分子的表达。统计分析图中*表示student t-test p<0.05。p<0.05具有统计学显著差异意义。每种处理组有12只小鼠。
具体实施方式
以下结合实施例对本发明作进一步具体描述，但不局限于此。需要指出的是，由本发明中的用于增强机体T细胞免疫功能并治疗和/或预防肿瘤的灭活的脆弱拟杆菌或含有本发明的脆弱拟杆菌的药物组合物、食品、保健品和食品添加剂在施用于受试者后，都可以应用于上文所述的适应症并展现出上文所述的功能，在本发明范围内的所有剂型均已测试，下文中，仅仅是为说明，只在实施例中描述了其中一部分，然而不应将其理解为对本发明的限制。
所述脆弱拟杆菌是灭活的、减毒的、低感染性的或非感染性的，并能够降低CD4+T细胞和/或CD8+T细胞表达T细胞耗竭分子。脆弱拟杆菌优选通过干热、湿热、过滤、有机溶剂、化学试剂、紫外或红外射线、发酵、冻干、基因重组、基因修饰或改造的方法中的任意一种或多种灭活的。
所述肿瘤/癌症包括但不限于是实体瘤/癌症。在某些实施方案中，所述肿瘤/癌症可以为乳腺癌、结直肠癌、卵巢癌、胰腺癌、前列腺癌、膀胱癌、黑色素瘤、肺癌、胃癌、肝癌、胆管癌、神经胶质瘤、头、颈部鳞状细胞癌、子宫颈癌、肾细胞癌、甲状腺癌、急性髓性白血病、骨髓瘤、食管癌、淋巴瘤、皮肤癌等肿瘤或这些肿瘤的组合。
实施例1脆弱拟杆菌培养
培养方法
步骤1：，取一支冻干保存脆弱拟杆菌(Bacteroides fragilis)菌种(购自ATCC官网)，加入200μL TSB培养基，复溶，血平皿划线，厌氧罐气体控制系统抽气后在生化培养箱中37℃、厌氧培养48小时(h)；
步骤2：挑取单克隆菌落接入10ml TSB培养基，37℃、厌氧培养12h；
步骤3：取1瓶500ml TSB培养基，接入1％(v/v)菌种，37℃、厌氧培养48h；
步骤4：取菌液，6000rpm离心10min。用生理盐水洗涤2次，最后用生理盐水复溶菌泥备用并进行活菌计数。
实施例2脆弱拟杆菌对小鼠肿瘤(例如：黑色素瘤)的治疗作用实验
图1为检测脆弱拟杆菌及灭活脆弱拟杆菌对小鼠肿瘤(黑色素瘤)治疗和/或预防作用实验流程示意图。
1、培养方法
脆弱拟杆菌培养方法同实施例1。
2、样品准备
1)脆弱拟杆菌ZY-312活菌体的制备
步骤1：取一支冻干保存菌种(购自ATCC官网)，加入200μL冻干保存菌培养基，复溶，吸取20μL，血平皿划线，厌氧罐气体控制系统抽气后在生化培养箱中37℃、厌氧培养48h；
步骤2：挑取单克隆菌落接入10ml TSB培养基，37℃、厌氧培养12h；
步骤3：取1瓶500ml TSB培养基，接入1％(v/v)菌种，37℃、厌氧培养48h；
步骤4：取菌液离心，用离心机进行离心，离心条件6000rpm、10min。用生理盐水洗涤2次，最后用生理盐水复溶菌泥备用并进行活菌计数。
2)脆弱拟杆菌灭活菌体
在温度70℃水浴锅中加热30分钟(min)获得灭活菌液。
实验动物：C57BL/6小鼠3～4周36只，精神状态良好，购自中山大学实验动物中心。将小鼠随机分成3组，每组12只，每组小鼠的性别匹配，3组分别为生理盐水对照组、脆弱拟杆菌灌胃组(或简称：活菌组或未灭活组)、灭活脆弱拟杆菌灌胃组(或简称：灭活组)。
实验过程和结果：
如图1所示，对3组小鼠分别灌胃1*10 9CFU的脆弱拟杆菌、1*10 9CFU的灭活脆弱拟杆菌及生理盐水对照，连续灌胃2周，每天测定体重。随后待小鼠肿瘤(黑色素瘤)B16细胞生长到对数期，用TE消化细胞，培养基中和，离心收集细胞，并用DPBS洗涤两次，除去残留血清，用DPBS重悬细胞。细胞计数，将1*10 6个B16细胞接种到每只小鼠右腋皮下。并持续对小鼠分别进行灌胃治疗，2周后将荷瘤小鼠处死，剖取皮下瘤块，测量肿瘤大小。
鉴于CD4+T细胞与CD8+T细胞是机体抗肿瘤极为关键的两类T细胞亚群，同时利用anti-CD3、anti-CD4、anti-CD8及anti-Tim-3单克隆荧光抗体标记分离 自小鼠脾脏的淋巴细胞，然后利用流式细胞仪分析小鼠脾脏内CD4+T细胞和CD8+T细胞表达Tim-3的百分比例情况。
分析结果显示，脆弱拟杆菌及其灭活菌均对小鼠肿瘤的形成以及生长有明显的抑制作用(图2及图3，图2中箭头所示为肿瘤处)。但是，图2、图3及图4的实验结果表明，生理盐水对照组中小鼠肿瘤(黑色素瘤)随着时间肿块逐渐变大，灭活的脆弱拟杆菌组肿块明显小于对照盐水组及未灭活组(而且差异具有统计学意义)。这些结果(图2、图3及图4)表明脆弱拟杆菌经灭活后可以显著增强机体抗肿瘤效应，抑制肿瘤生长，对于防治肿瘤(例如：黑色素瘤)具有更好的效果。
Tim-3是一种抑制T细胞抗肿瘤效应功能的耗竭分子，越高的Tim-3的表达表明机体的T细胞抗肿瘤免疫功能处于被抑制、低能和/或者耗竭的状态。所以控制并且减少Tim-3在T细胞上的表达是提高或增强机体T细胞抗肿瘤免疫功能的关键，越低的Tim-3的表达说明T细胞抗肿瘤免疫功能越强。图5、图6、图7及图8的结果表明灭活的脆弱拟杆菌相比生理盐水或者不灭活的脆弱拟杆菌可以更加高效、显著抑制CD4+T细胞和/或CD8+T细胞中Tim-3的表达(抑制Tim-3表达的效果具有统计学意义)进而增强T细胞免疫功能并增强机体抗肿瘤的免疫能力。这一结果也更进一步清晰解释了为什么灭活脆弱拟杆菌相比生理盐水或者脆弱拟杆菌活菌组具有更强的抗肿瘤效果。
以上内容是结合本发明创造的优选实施方式对所提供技术方案所作的进一步详细说明，不能认定本发明创造具体实施只局限于上述这些说明，对于本发明创造所属技术领域的普通技术人员来说，在不脱离本发明创造构思的前提下，还可以做出若干简单推演或替换，都应当视为属于本发明创造的保护范围。

Claims (13)

  1. 一种用于增强机体T细胞免疫功能的组合物，其特征在于包括脆弱拟杆菌(Bacteroides fragilis)。
  2. 根据权利要求1所述的组合物，其特征在于，所述脆弱拟杆菌是以下中的一种或多种：脆弱拟杆菌活菌体；经过灭活、基因重组、改造或修饰、减毒、化学处理、物理处理或灭活的脆弱拟杆菌；脆弱拟杆菌裂解物；和/或脆弱拟杆菌液体培养上清液。
  3. 根据权利要求1所述的组合物，其特征在于，所述脆弱拟杆菌是通过干热、湿热、过滤、有机溶剂、化学试剂、紫外或红外射线、发酵、冻干、基因重组、基因修饰或改造的方法中的任意一种或多种灭活的。
  4. 根据权利要求1所述的组合物，其特征在于，所述组合物是药物组合物、食品、保健品或食品添加剂中的任意一种。
  5. 权利要求1所述组合物的制备方法，其特征在于步骤如下：
    (1)收集活脆弱拟杆菌的培养物；
    (2)用15％甘油/生理盐水溶液洗涤所述脆弱拟杆菌,所述15％甘油/生理盐水溶液是指生理盐水中含有体积百分比为15％的甘油；
    (3)将所述脆弱拟杆菌重悬于所述15％甘油/生理盐水溶液中；
    (4)通过70℃水浴加热30分钟所述脆弱拟杆菌，获得灭活菌体。
  6. 一种用于预防和/或治疗肿瘤的组合物，其特征在于包括脆弱拟杆菌。
  7. 根据权利要求6所述的组合物，其特征在于，所述脆弱拟杆菌是以下中的一种或多种：脆弱拟杆菌活菌体；经过灭活、基因重组、改造或修饰、减毒、化学处理、物理处理或灭活的脆弱拟杆菌；脆弱拟杆菌裂解物；和/或脆弱拟杆菌液体培养上清液。
  8. 根据权利要求6所述的组合物，其特征在于，所述脆弱拟杆菌是通过干热、湿热、过滤、有机溶剂、化学试剂、紫外或红外射线、发酵、冻干、基因重组、基因修饰或改造的方法中的任意一种或多种灭活的。
  9. 根据权利要求6所述的组合物，其特征在于，所述组合物是药物组合物、 食品、保健品或食品添加剂中的任意一种。
  10. 根据权利要求6所述的组合物，其特征在于，所述肿瘤为乳腺癌、结直肠癌、卵巢癌、胰腺癌、前列腺癌、膀胱癌、黑色素瘤、肺癌、胃癌、肝癌、胆管癌、神经胶质瘤、头、颈部鳞状细胞癌、子宫颈癌、肾细胞癌、甲状腺癌、急性髓性白血病、骨髓瘤、食管癌、淋巴瘤、皮肤癌中的一种或多种。
  11. 权利要求6所述组合物的制备方法，其特征在于步骤如下：
    (1)收集活脆弱拟杆菌的培养物；
    (2)用15％甘油/生理盐水溶液洗涤所述脆弱拟杆菌，所述15％甘油/生理盐水溶液是指生理盐水中含有体积百分比为15％的甘油；
    (3)将所述脆弱拟杆菌重悬于所述15％甘油/生理盐水溶液中；
    (4)通过70℃水浴加热30分钟所述脆弱拟杆菌，获得灭活菌体。
  12. 脆弱拟杆菌在制备用于增强机体T细胞免疫功能的组合物中的用途。
  13. 脆弱拟杆菌在制备用于预防或治疗肿瘤的组合物中的用途。
PCT/CN2017/118590 2017-11-17 2017-12-26 一种用于增强t细胞免疫功能的组合物及其制备方法 WO2019095508A1 (zh)

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