WO2023134210A1 - 脆弱拟杆菌荚膜多糖a与免疫检查点抑制剂的新应用 - Google Patents

脆弱拟杆菌荚膜多糖a与免疫检查点抑制剂的新应用 Download PDF

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WO2023134210A1
WO2023134210A1 PCT/CN2022/120110 CN2022120110W WO2023134210A1 WO 2023134210 A1 WO2023134210 A1 WO 2023134210A1 CN 2022120110 W CN2022120110 W CN 2022120110W WO 2023134210 A1 WO2023134210 A1 WO 2023134210A1
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administration
capsular polysaccharide
bacteroides fragilis
immune checkpoint
inhibitors
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PCT/CN2022/120110
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English (en)
French (fr)
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李平
郑晓蔓
刘洋洋
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广州知易生物科技有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

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  • the invention relates to the field of biomedicine, in particular to a new application of Bacteroides fragilis capsular polysaccharide A and immune checkpoint inhibitors.
  • Lymphoma generally refers to lymphoma. Lymphoma is a malignant tumor originating from the lymphatic hematopoietic system. According to the tumor cells, it is divided into two types: non-Hodgkin's lymphoma (NHL) and Hodgkin's lymphoma (HL). Pathological features In Hodgkin's lymphoma, the tumor tissue contains lymphocytes, eosinophils, plasma cells and specific Reed-Steinberg cells, and HL is divided into nodular lymphoid-rich Cellular and classical, the latter including lymphocyte predominant, nodular sclerosis, mixed cellular and lymphocyte depleted. The incidence of NHL is much higher than that of HL.
  • lymphocytes mainly lymphocytes, histiocytes or reticular cells with different degrees of differentiation.
  • lymphocytes mainly lymphocytes, histiocytes or reticular cells with different degrees of differentiation.
  • lymphocytes mainly lymphocytes, histiocytes or reticular cells with different degrees of differentiation.
  • lymphocytes According to the natural course of NHL, it can be classified as Three major clinical types, namely highly aggressive, aggressive and indolent lymphoma. According to the origin of different lymphocytes, it can be divided into B cell, T cell and NK cell lymphoma.
  • Lymphoma is highly heterogeneous, and the treatment effects vary greatly. Lymphomas of different pathological types and stages have great differences in terms of treatment intensity and prognosis.
  • the traditional treatment methods for lymphoma mainly include the following: radiotherapy, chemotherapy, bone marrow transplantation and surgery. At present, radiotherapy and chemotherapy are mostly used clinically as the main treatment. In recent years, with the progress of targeted therapy and immunotherapy, lymphoma cure has become a possibility. However, the high cost of treatment and many postoperative contraindications make the development of new Therapeutic drugs become an urgent need.
  • the main purpose of the present invention is to provide a new application of capsular polysaccharide A of Bacteroides fragilis and immune checkpoint inhibitors, mainly to prepare and prevent lymphatic Drugs for lymphoma have synergistic effects on lymphoma, providing a new solution for the prevention and treatment of lymphoma.
  • the present invention provides the application of capsular polysaccharide A of Bacteroides fragilis and immune checkpoint inhibitors in the preparation of drugs for preventing and treating lymphoma.
  • the deposit number of the Bacteroides fragilis is CGMCC No.10685.
  • the immune checkpoint inhibitor is selected from one or more of PD-1 inhibitors, PD-L1 inhibitors, PD-L2 inhibitors and CTLA-4 inhibitors.
  • the PD-1 inhibitor is selected from Nivolumab, Pembrolizumab, Cemiplimab, Toripalimab One or more of Toripalimab, Cindilimab, and Camrelizumab.
  • the weight average molecular weight of the capsular polysaccharide A is 70kDa-90kDa.
  • the medicine comprises one or more of the capsular polysaccharide A of B. fragilis and the immune checkpoint inhibitor, as well as pharmaceutically acceptable excipients.
  • the auxiliary materials include diluents, wetting agents, binders, disintegrants, lubricants, color and flavor regulators, solvents, solubilizers, co-solvents, emulsifiers, antioxidants, metal complexes
  • diluents wetting agents, binders, disintegrants, lubricants, color and flavor regulators, solvents, solubilizers, co-solvents, emulsifiers, antioxidants, metal complexes
  • solvents solubilizers, co-solvents
  • emulsifiers emulsifiers
  • antioxidants emulsifiers
  • metal complexes One or more of mixtures, inert gases, preservatives, local analgesics, pH regulators, and isotonic or isotonic regulators.
  • the dosage form of the drug includes pills, tablets, granules, capsules, powders, suspensions, oral liquids, tube feeding preparations or enemas.
  • the administration of the drug includes oral administration, enema administration or parenteral administration.
  • the administration cycle of the drug includes intermittent administration, periodic administration, continuous administration or long-term administration.
  • the present invention provides a drug for preventing and treating lymphoma, the drug comprising capsular polysaccharide A of Bacteroides fragilis and an immune checkpoint inhibitor.
  • the present invention has the following beneficial effects:
  • the present invention proves through a large number of experiments that the capsular polysaccharide A of Bacteroides fragilis is used in combination with immune checkpoint inhibitors, especially the capsular polysaccharide A of Bacteroides fragilis with the preservation number CGMCC No. 10685 is used in combination with PD-1 inhibitors , can be synergistically effective against lymphoma.
  • Fig. 1 is the colony characteristic figure of Bacteroides fragilis ZY-312 of the embodiment of the present invention 1;
  • Fig. 2 is the microscopic observation diagram after Gram staining of Bacteroides fragilis ZY-312 in Example 1 of the present invention
  • Fig. 3 is the capsular polysaccharide A nuclear magnetic resonance spectrometer analysis 1H spectrogram of embodiment 2 of the present invention
  • Fig. 4 is the capsular polysaccharide A nuclear magnetic resonance spectrometer analysis 13C spectrogram of embodiment 2 of the present invention
  • Fig. 5 is the COZY spectrogram analyzed by the capsular polysaccharide A NMR spectrometer of Example 2 of the present invention
  • Fig. 6 is the capsular polysaccharide A NMR spectrometer analysis HSQC spectrogram of embodiment 2 of the present invention
  • Fig. 7 is the HMBC spectrogram analyzed by the capsular polysaccharide A NMR spectrometer of Example 2 of the present invention.
  • Fig. 8 is the chemical structural formula of the structural unit of Bacteroides fragilis capsular polysaccharide A prepared in Example 2 of the present invention.
  • the raw materials and reagents used in the following examples are commercially available, all cells were purchased from ATCC; all cell culture materials and trypsin were purchased from Gibco; all experimental animals were purchased from Zhejiang Weitong Lihua Experimental Animal Technology Ltd; or may be prepared by known methods.
  • the experimental method that does not indicate specific conditions in the following examples, usually according to conventional conditions such as Sambrook et al., molecular cloning: the conditions described in the laboratory manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer suggested conditions.
  • the immune checkpoint is a protective molecule in the human immune system, which acts like a brake to prevent inflammatory damage caused by excessive activation of T cells.
  • Tumor cells take advantage of the characteristics of the human immune system to overexpress immune checkpoint molecules, inhibit the response of the human immune system, and escape human immune surveillance and killing, thereby promoting the growth of tumor cells. Inhibiting the expression of immune checkpoint molecules and their ligands can enhance the killing effect of T cells on tumors and achieve the purpose of anti-tumor.
  • the published immune checkpoints include CTLA-4, PD-1/PD-L1, LAG-3, TIM-3, VISTA, A2aR, etc.
  • Programmed cell death protein 1 is expressed on a variety of lymphocytes, especially on tumor-specific T cells. In the tumor microenvironment, it leads to the expansion of malignant tumor cells by interfering with protective immune responses. It has two ligands, programmed cell death ligand 1 and 2 (PD-L1, PD-L2), among which PD-L1 is expressed by tumor cells to evade the anti-tumor response to it by the immune system. Blocking the interaction between PD-1 and PD-L1 can maintain the response of T cells after T cells enter the tumor microenvironment and ensure the anti-tumor effect of T cells.
  • PD-1 programmed cell death ligand 1 and 2
  • Antibodies against PD-1/PD-L1 have Nivolumab, Pembrolizumab, JQ1, Atezolizumab, Avelumab and Simi Cemiplimab, etc. These mAbs are approved for the treatment of breast, lung, colorectal, cancer, bladder, pancreatic, prostate and diffuse large B-cell lymphoma (DLBCL). On December 24, 2018, the State Drug Administration approved the listing of Tyvyt (sintilimab). Dabur is used for the treatment of relapsed or refractory classical Hodgkin's lymphoma who have undergone at least second-line systemic chemotherapy, marking that my country's anti-tumor immunotherapy has entered a new era.
  • PD-1/PD-L1 immunosuppressants mainly kill tumor cells by activating the function of T lymphocytes.
  • Such drugs cannot directly prevent the development of tumors, and many clinical trials have found that PD-1 and PD -L1 antibody blockade leads to systemic immune disorders, and long-term treatment can also induce the occurrence of autoimmune diseases.
  • Immunotherapy may be affected by the gut microbiota, and existing studies have observed a correlation between the gut microbiota and gastrointestinal immunity.
  • the gut microbiota may also act as a modulator of gut immunity, and fecal microbiota transplantation and antibiotic administration may affect the efficacy and toxicity of immunotherapy through the microbiota.
  • Using precise microbiome modulation could make ICB therapy safer and more effective.
  • Bacteroides fragilis (B. fragilis) zwitterionic capsular polysaccharide (CPS) is the first recognized symbiotic factor that regulates the development of the host immune system and reverses morphological, cellular, and functional defects in germ-free animals. There is currently no documented use of zwitterionic capsular polysaccharides from Bacteroides fragilis and immunosuppressants in the synergistic treatment of lymphoma.
  • Bacteroides fragilis ZY-312 that the present invention adopts does not contain BFT gene, is non-toxigenic bacterial strain, and acute toxicity proves, and this bacterial strain is all nonpathogenic to normal mouse and nude mouse (Wang Y, Deng H, Li Z, Tan Y , Han Y, Wang X, Du Z, Liu Y, Yang R, Bai Y, Bi Y, Zhi F. Safety Evaluation of a Novel Strain of Bacteroides fragilis. Front Microbiol. 2017 Mar 17; 8:435.).
  • the present invention provides the application of capsular polysaccharide A of Bacteroides fragilis and immune checkpoint inhibitors in the preparation of drugs for preventing and treating lymphoma.
  • the deposit number of the Bacteroides fragilis is CGMCC No.10685.
  • the immune checkpoint inhibitor is selected from one or more of PD-1 inhibitors, PD-L1 inhibitors, PD-L2 inhibitors and CTLA-4 inhibitors.
  • the PD-1 inhibitors include but are not limited to one or more of the following types: Nivolumab, Pembrolizumab, Cemiplimab ), Toripalimab, Cindilimab, Camrelizumab and others that can bind to PD-1 and block the PD-1/PD-L1 signaling pathway , up-regulate T cell activation, substances that activate endogenous anti-tumor immune responses.
  • the capsular polysaccharide A has a weight average molecular weight of 70kDa-90kDa, preferably 80kDa-90kDa, wherein the portion with a weight average molecular weight distribution of 70kDa-100kDa accounts for 70wt-80wt% of the total.
  • the capsular polysaccharide A has a bound lipid content of less than 0.02% or does not contain lipid.
  • the drug comprises one or more of the capsular polysaccharide A of B. fragilis and the immune checkpoint inhibitor, as well as pharmaceutically acceptable excipients.
  • auxiliary materials are selected from, including but not limited to, one or more of the following types of auxiliary materials: diluents, wetting agents, binders, disintegrants, lubricants, color and flavor regulators, solvents , solubilizer, cosolvent, emulsifier, antioxidant, metal complexing agent, inert gas, preservative, local analgesic, pH regulator, isotonic or isotonic regulator.
  • diluent may be selected from, including but not limited to: starches, sugars, celluloses, and inorganic salts.
  • the wetting agent may be selected from, including but not limited to: water, ethanol.
  • the binder may be selected from, including but not limited to: starch slurry, dextrin, sugar, cellulose derivatives, gelatin, povidone, polyethylene glycol.
  • the disintegrating agent can be selected from, including but not limited to: starch, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, croscarmellose sodium, crospovidone, surfactant, foam Teng disintegrant.
  • the lubricant may be selected from, including but not limited to: talcum powder, calcium stearate, magnesium stearate, magnesium lauryl sulfate, micronized silica gel, polyethylene glycol.
  • the color and flavor regulator can be selected from, including but not limited to: pigments, spices, sweeteners, mucilage agents, and odorants.
  • the solvent may be selected from, including but not limited to: water, ethanol, glycerin, propylene glycol, polyethylene glycol, dimethyl sulfoxide, liquid paraffin, fatty oil, ethyl acetate.
  • the solubilizing agent can be selected from, including but not limited to: Tweens, mezes, polyoxyethylene fatty alcohol ethers, soaps, sulfates, and sulfonates.
  • the co-solvent can be selected from, including but not limited to: organic acids and their salts, amides and amines, inorganic salts, polyethylene glycol, povidone, and glycerin.
  • the emulsifier can be selected from, including but not limited to: Spans, Tweens, marzes, benzyls, glycerin fatty acid esters, higher fatty acid salts, sulfates, sulfonates, gum arabic, carbamide Tragacanth gum, gelatin, pectin, phospholipids, agar, sodium alginate, hydroxide, silica, bentonite.
  • the suspending agent can be selected from, including but not limited to: glycerin, syrup, gum arabic, tragacanth gum, agar, sodium alginate, cellulose derivatives, povidone, carbopol, polyvinyl alcohol, Gel.
  • the antioxidant may be selected from, including but not limited to: sulfites, pyrosulfites, bisulfites, ascorbic acid, gallic acid, and esters.
  • the metal complexing agent can be selected from, including but not limited to: disodium edetate, polycarboxylic acid compounds.
  • the inert gas can be selected from, including but not limited to: nitrogen, carbon dioxide.
  • the preservatives may be selected from, including but not limited to: parabens, organic acids and their salts, quaternary ammonium compounds, chlorhexidine acetate, alcohols, phenols, and volatile oils.
  • the local analgesic may be selected from, including but not limited to: benzyl alcohol, chlorobutanol, lidocaine, procaine.
  • the pH regulator can be selected from, including but not limited to: hydrochloric acid, sulfuric acid, phosphoric acid, citric acid, tartaric acid, acetic acid, sodium hydroxide, sodium bicarbonate, ethylenediamine, meglumine, phosphate, acetate , Citrate.
  • the isotonic or isotonic adjusting agent can be selected from, including but not limited to: glucose, sodium chloride, sodium citrate, sorbitol, xylitol.
  • the drug can be prepared into a suitable dosage form according to clinical needs, and the dosage form can be selected from, including but not limited to: pills, tablets, granules, capsules, powders, suspensions, oral liquids, tubes, etc. Feed preparations or enemas.
  • the drug can be administered according to clinical needs, using an appropriate route of administration, and the route of administration can be selected from, including but not limited to: oral administration, enema administration or parenteral administration.
  • the drug can be administered according to clinical needs, using a suitable dosing cycle, and the dosing cycle can be selected from, including but not limited to: intermittent dosing, periodic dosing, continuous dosing drug or long-term administration.
  • the medicine can be a human medicine or a veterinary medicine.
  • the present invention provides a drug for preventing and treating lymphoma, the drug comprising capsular polysaccharide A of Bacteroides fragilis and an immune checkpoint inhibitor.
  • the deposit number of the Bacteroides fragilis is CGMCC No.10685.
  • the immune checkpoint inhibitor is selected from one or more of PD-1 inhibitors, PD-L1 inhibitors, PD-L2 inhibitors and CTLA-4 inhibitors.
  • the PD-1 inhibitors include but are not limited to one or more of the following types: Nivolumab, Pembrolizumab, Cemiplimab ), Toripalimab, Cindilimab, Camrelizumab and others that can bind to PD-1 and block the PD-1/PD-L1 signaling pathway , up-regulate T cell activation, substances that activate endogenous anti-tumor immune responses.
  • the pharmaceutically effective dose of the capsular polysaccharide A is 5-15 mg/kg.
  • the preparation method of capsular polysaccharide A of Bacteroides fragilis comprises the following steps:
  • the bacteria liquid is centrifuged, the supernatant is discarded, and the bacteria are collected, which is the Bacteroides fragilis bacteria sludge; add purified water to the bacteria sludge to resuspend, and the addition ratio is 3 times the weight of the bacteria sludge- 10 times; adjust the pH of the bacterial suspension to 2.5-4.5; transfer the bacterial suspension to an extraction container, extract at 80°C-120°C for 0.5h-2.5h, cool, centrifuge and take the supernatant to obtain a crude capsular polysaccharide solution ;
  • the reflux liquid is chromatographed by anion exchange column, 20mM Tris-HCl (pH8.0, containing 0.2mol/L NaCl) gradient elution, the ion exchange column is preferably DEAE Sepharose Fast Flow, and the liquid collected by column chromatography is ultrafiltered Membrane ultrafiltration, adding purified water and repeating ultrafiltration until the conductivity is stable, collecting the reflux liquid and freeze-drying to obtain Bacteroides fragilis capsular polysaccharide A.
  • step (1) the centrifugation is 12000 g for 10 min.
  • the acid solution may be one or more of organic acid, inorganic acid and acid buffer.
  • the inorganic acid can be hydrochloric acid, sulfuric acid, phosphoric acid, etc.
  • the organic acid can be acetic acid, citric acid, etc.
  • the extraction temperature is 80°C-120°C.
  • step (1) the extraction time is 0.5h-2.5h;
  • step (3) 10KD ultrafiltration membrane is used for ultrafiltration and concentration.
  • Embodiment 1 the fermentation culture of Bacteroides fragilis
  • Colony characteristics Bacteroides fragilis ZY-312, after being cultured on a blood plate for 48 hours, is slightly convex, translucent, white, smooth, non-hemolytic, and the diameter of the colony is between 1mm and 3mm, see Figure 1.
  • Bacteroides fragilis ZY-312 was examined by Gram staining. It is a Gram-negative bacterium with a typical rod shape, blunt rounded ends and dense staining. The uncolored part in the middle of the bacteria is like a vacuole. figure 2.
  • the above bacterial liquid was taken and subjected to conventional heat inactivation treatment to obtain the inactivated bacterial liquid of Bacteroides fragilis ZY-312.
  • Test method Weigh 30 mg of the Bacteroides fragilis extract described in step (3), dissolve it in 0.5 mL of D 2 O, add 1 ⁇ l of acetone (1H, 2.22; 13C, 30.89) for calibration.
  • a 500MHz Bruker NMR spectrometer was used to analyze 1H spectrum (Figure 3), 13C spectrum ( Figure 4), COZY spectrum (Figure 5), HSQC spectrum ( Figure 6), and HMBC spectrum ( Figure 7).
  • GPC gel permeation chromatography
  • the prepared Bacteroides fragilis capsular polysaccharide A was named ZY-312-PSA.
  • Example 3 Application of capsular polysaccharide A combined with PD-1 antibody in the treatment of lymphoma
  • mice were selected in this case to construct a lymphoma model. Take well-cultured EL4 cells, adjust the cell concentration to 1 ⁇ 10 6 /ml, shave the hair on the back of the mouse with a shaver, and inoculate EL4 lymphoma cells on the subcutaneous right side of the back with an inoculation amount of 3 ⁇ 10 5 EL4 cells per
  • the mice were randomly divided into 6 groups, 10 mice in each group, specifically divided into model group, PD-1 antibody group, ZY-312-PSA group, low-dose ZY -312-PSA+PD-1 antibody group, medium-dose ZY-312-PSA+PD-1 antibody group and high-dose ZY-312-PSA+PD-1 antibody group, and the day of grouping was taken as day 0,
  • mice were euthanized, and mouse serum, tumor, spleen, feces, right cervical lymph and right axillary lymph were collected. All tumors were weighed and photographed. Tumors were divided into three parts, one was frozen for cytokine detection, one was fixed in formalin, and one was sent in vitro for flow analysis.
  • Tumor volume and antitumor efficacy starting from day 0, the tumor diameter was measured with a vernier caliper every 3 days.
  • TGI (%) [1-(Average tumor volume at the end of administration of a certain treatment group-Average tumor volume at the beginning of administration of this treatment group)/(Average tumor volume at the end of treatment of the same type control group- The average tumor volume at the beginning of treatment in the same type control group)] ⁇ 100%.
  • T cell subsets Flow cytometry analysis of the ratio of CD4+T cells and CD8+T cells in peripheral blood and spleen.
  • the growth inhibition rate of the test drug on the subcutaneous inoculation model of EL4 lymphoma cells was calculated.
  • TGI(%) [1-(Average tumor volume at the end of administration of a certain treatment group-Average tumor volume at the beginning of administration of this treatment group)/(Average tumor volume at the end of treatment of the same type control group Volume - the average tumor volume of the same type control group at the beginning of treatment)] ⁇ 100%;
  • each administration group can significantly inhibit tumor growth; when PD-1 antibody and B. fragilis capsular polysaccharide A are used in combination, there is a very significant difference. It can be seen that Bacteroides fragilis ZY-312 capsular polysaccharide A combined with PD-1 antibody can effectively inhibit tumor growth.
  • CD8+ T cells can kill target cells expressing antigens, and they are important effector cells in antiviral infection, acute allograft rejection and tumor cell killing.
  • CD4+ T cells are an important immune cell in the human immune system, mainly differentiated from helper T (Th) cells, can bind to the non-polypeptide region of MHC class II molecules, and participate in T cell antigen receptor (TCR) recognition of antigens signal transduction.
  • Th helper T
  • TCR T cell antigen receptor
  • CTL cytotoxic T lymphocytes
  • each administration group up-regulated the levels of CD4+ and CD8+ T cells in the peripheral blood of mice to varying degrees.
  • the combination group there was no significant PSA dose dependence.
  • each administration group up-regulated the levels of mouse spleen CD4+ and CD8+ T cells to varying degrees.
  • On the level of CD4+ T cells there was a significant difference between the middle and high doses of PSA combined with PD-1 antibody groups; on the level of CD8+ T cells, there were significant differences among the groups, and the combination of medium and high doses of PSA
  • the PD-1 antibody group had extremely significant differences. This shows that the combination of Bacteroides fragilis capsular polysaccharide A and PD-1 antibody can regulate the level of T cells in the peripheral blood and spleen of mice, and enhance the body's anti-tumor immune response.
  • Bacteroides fragilis especially the zwitterionic capsular polysaccharide A (PSA) of Bacteroides fragilis ZY-312 with the deposit number CGMCC No. 10685, combined with PD-1 antibody, can regulate the peripheral blood of mice. and the level of T cells in the spleen, enhance the body's anti-tumor immune response, inhibit the growth of EL4 lymphoid transplanted tumors in mice, and effectively prevent and treat lymphoma.
  • PSA zwitterionic capsular polysaccharide A

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Abstract

本发明涉及脆弱拟杆菌荚膜多糖A与免疫检查点抑制剂的新应用，主要涉及脆弱拟杆菌的荚膜多糖A和免疫检查点抑制剂在制备防治淋巴瘤的药物中的应用。本发明通过大量实验证明，脆弱拟杆菌的荚膜多糖A与免疫检查点抑制剂联合使用，能够对淋巴瘤协同显效。

Description

脆弱拟杆菌荚膜多糖A与免疫检查点抑制剂的新应用 技术领域
本发明涉及生物医药领域，具体涉及一种脆弱拟杆菌荚膜多糖A与免疫检查点抑制剂的新应用。
背景技术
淋巴癌，一般指的是淋巴瘤。淋巴瘤是起源于淋巴造血系统的恶性肿瘤，根据瘤细胞分为非霍奇金淋巴瘤(NHL)和霍奇金淋巴瘤(HL)两类。病理学特征在霍奇金淋巴瘤为瘤组织内含有淋巴细胞、嗜酸性粒细胞、浆细胞和特异性的里-斯(Reed-Steinberg)细胞，HL按照病理类型分为结节性富含淋巴细胞型和经典型，后者包括淋巴细胞为主型、结节硬化型、混合细胞型和淋巴细胞消减型。NHL发病率远高于HL,是具有很强异质性的一组独立疾病的总和，病理上主要是分化程度不同的淋巴细胞、组织细胞或网状细胞，根据NHL的自然病程，可以归为三大临床类型，即高度侵袭性、侵袭性和惰性淋巴瘤。根据不同的淋巴细胞起源，可以分为B细胞、T细胞和NK细胞淋巴瘤。
淋巴瘤具有高度异质性，治疗效果差别很大，不同病理类型和分期的淋巴瘤无论从治疗强度和预后上都存在很大差别。传统针对淋巴瘤的治疗方法主要有以下几种：放射治疗，化学药物治疗，骨髓移植和手术治疗。目前，临床上多采用放射化疗为主要治疗手段，近年来，随着靶向疗法和免疫疗法的进展，淋巴瘤治愈成为一种可能，然而高昂的治疗费用和较多的术后禁忌使得开发新的治疗药物成为一个迫切的需求。
发明内容
基于此，本发明的主要目的是提供一种脆弱拟杆菌的荚膜多糖A和免疫检 查点抑制剂的新应用，主要是用脆弱拟杆菌的荚膜多糖A和免疫检查点抑制剂制备防治淋巴瘤的药物，对淋巴瘤协同显效，为淋巴瘤防治提供新方案。
本发明的目的可以通过以下技术方案实现：
作为本发明的目的之一，本发明提供脆弱拟杆菌的荚膜多糖A和免疫检查点抑制剂在制备防治淋巴瘤的药物中的应用。
在其中一些实施例中，所述脆弱拟杆菌的保藏编号为CGMCC No.10685。
在其中一些实施例中，所述免疫检查点抑制剂选自PD-1抑制剂、PD-L1抑制剂、PD-L2抑制剂和CTLA-4抑制剂中的一种或多种。
在其中一些实施例中，所述PD-1抑制剂选自纳武利尤单抗(Nivolumab)、帕博利珠单抗(Pembrolizumab)、西米普利单抗(Cemiplimab)、特瑞普利单抗(Toripalimab)、信迪利单抗(Cindilimab)和卡瑞利珠单抗(Camrelizumab)中的一种或者多种。
在其中一些实施例中，所述荚膜多糖A的重均分子量为70kDa-90kDa。
在其中一些实施例中，所述药物包含所述脆弱拟杆菌的荚膜多糖A和所述免疫检查点抑制剂中的一种或多种，以及药学上可以接受的辅料。
在其中一些实施例中，所述辅料包含稀释剂、润湿剂、黏合剂、崩解剂、润滑剂、色香味调节剂、溶剂、增溶剂、助溶剂、乳化剂、抗氧剂、金属络合剂、惰性气体、防腐剂、局部止痛剂、pH调节剂以及等渗或等张调节剂中的一种或者多种。
在其中一些实施例中，所述药物的剂型包括丸剂、片剂、颗粒剂、胶囊、散剂、混悬剂、口服液、管饲制剂或者灌肠剂。
在其中一些实施例中，所述药物的给药方式包括口服给药、灌肠给药或者肠胃外给药。
在其中一些实施例中，所述药物的给药周期包括间歇性给药、周期性给药、持续性给药或者长期给药。
作为本发明的目的之二，本发明提供一种防治淋巴瘤的药物，所述药物包含脆弱拟杆菌的荚膜多糖A和免疫检查点抑制剂。
与传统技术相比，本发明具备如下有益效果：
本发明通过大量实验证明，脆弱拟杆菌的荚膜多糖A与免疫检查点抑制剂联合使用，特别是保藏编号为CGMCC No.10685的脆弱拟杆菌的荚膜多糖A与PD-1抑制剂联合使用，能够对淋巴瘤协同显效。
附图说明
图1为本发明实施例1的脆弱拟杆菌ZY-312的菌落特征图；
图2为本发明实施例1的脆弱拟杆菌ZY-312进行革兰氏染色后的显微镜观察图；
图3为本发明实施例2的荚膜多糖A核磁共振波谱仪分析1H谱图；
图4为本发明实施例2的荚膜多糖A核磁共振波谱仪分析13C谱图；
图5为本发明实施例2的荚膜多糖A核磁共振波谱仪分析COSY谱图；
图6为本发明实施例2的荚膜多糖A核磁共振波谱仪分析HSQC谱图；
图7为本发明实施例2的荚膜多糖A核磁共振波谱仪分析HMBC谱图；
图8为本发明实施例2制备得到的脆弱拟杆菌荚膜多糖A的结构单元的化学结构式。
具体实施方式
为了便于理解本发明，下面将对本发明进行更全面的描述。但是，本发明可以以许多不同的形式来实现，并不限于本文所描述的实施例。相反地，提供这些实施例的目的是使对本发明的公开内容的理解更加透彻全面。
除非另有定义，本文所使用的所有的技术和科学术语与属于本发明的技术领域的技术人员通常理解的含义相同。本文中在本发明的说明书中所使用的术语只是为了描述具体的实施例的目的，不是旨在于限制本发明。本文所使用的术语“和/或”包括一个或多个相关的所列项目的任意的和所有的组合。
除非另有说明，以下实施例中使用的原料和试剂均为市售商品，所有细胞购自ATCC；所有细胞培养材料及胰酶购自Gibco；所有实验动物购自浙江维通利华实验动物技术有限公司；或者可以通过已知方法制备。下列实施例中未注明具体条件的实验方法，通常按照常规条件如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。
免疫检查点本是人体免疫系统中起保护作用的分子，起类似刹车的作用，防止T细胞过度激活导致的炎症损伤等。而肿瘤细胞利用人体免疫系统这一特性，通过过度表达免疫检查点分子，抑制人体免疫系统反应，逃脱人体免疫监视与杀伤，从而促进肿瘤细胞的生长。抑制免疫检查点分子及其配体的表达能够增强T细胞对肿瘤的杀伤效应，达到抗肿瘤的目的。已被公布的免疫检查点有CTLA-4、PD-1/PD-L1、LAG-3、TIM-3、VISTA、A2aR等。
程序性细胞死亡蛋白1(PD-1)在多种淋巴细胞上表达，尤其在肿瘤特异性T细胞上高表达。在肿瘤微环境中，它通过干扰保护性免疫应答而导致恶性肿瘤细胞的扩张。它具有两个配体，即程序性细胞死亡配体1和2(PD-L1、PD-L2)，其中，PD-L1被肿瘤细胞表达，以逃逸免疫系统对它进行的抗肿瘤反应。阻断PD-1和PD-L1间的作用可以在T细胞进入肿瘤微环境后保持T细胞的应答，保证T细胞的抗肿瘤作用。针对PD-1/PD-L1的抗体已有纳武单抗(Nivolumab)、派姆单抗(Pembrolizumab)、JQ1、阿特珠单抗(Atezolizumab)、阿维鲁单抗 (Avelumab)和西米普利单抗(Cemiplimab)等。这些单抗被批准用于治疗乳腺癌、肺癌、大肠癌、癌症、膀胱癌、胰腺癌、前列腺癌和弥漫性大B细胞淋巴瘤(DLBCL)。2018年12月24日，国家药品监督管理局批准达伯舒(信迪利单抗)上市。达伯舒适用于至少经过二线系统化疗的复发或难治性经典型霍奇金淋巴瘤的治疗，标志着我国抗肿瘤免疫治疗进入新的时代。PD-1/PD-L1免疫抑制剂主要通过激活T淋巴细胞的功能，使其杀死肿瘤细胞，该类药物并不能直接阻止肿瘤的发展，且多项临床试验中均发现PD-1和PD-L1抗体阻断导致全身免疫紊乱，长期治疗还会诱导自身免疫疾病的发生。
免疫疗法可能会受到肠道微生物群的影响，已有的研究观察到肠道微生物群与胃肠道免疫之间的相互关系。肠道微生物群也可能充当肠道免疫的调节剂，粪便微生物群移植和抗生素给药可能会通过微生物群影响免疫疗法的疗效和毒性。使用精确的微生物调制可以使ICB治疗更安全、更有效。脆弱拟杆菌(Bacteroides fragilis，B.fragilis)两性离子荚膜多糖(capsular polysaccharide，CPS)是首个公认的调节宿主免疫系统的发育，逆转无菌动物的形态、细胞和功能缺陷的共生因子。目前没有关于利用脆弱拟杆菌的两性离子荚膜多糖和免疫抑制剂协同治疗淋巴癌的文献记载。
本发明采用的脆弱拟杆菌ZY-312不含BFT基因，是非产毒菌株，急性毒性证实，该菌株对正常小鼠和裸鼠均无致病性(Wang Y，Deng H，Li Z，Tan Y，Han Y，Wang X，Du Z，Liu Y，Yang R，Bai Y，Bi Y，Zhi F.Safety Evaluation of a Novel Strain of Bacteroides fragilis.Front Microbiol.2017 Mar 17；8:435.)。根据专利ZL201510459408.X和科技文献Xu W，Su P，Zheng L，Fan H，Wang Y，Liu Y，Lin Y，Zhi F.In vivo Imaging of a Novel Strain of Bacteroides fragilis via Metabolic Labeling.Front Microbiol.2018 Oct 1；9:2298.的报道，该菌株对胃酸、 胆盐有着较好的耐性，能够保证其在胃中的存活和有效定植。
作为本发明的一个方面，本发明提供脆弱拟杆菌的荚膜多糖A和免疫检查点抑制剂在制备防治淋巴瘤的药物中的应用。
在一个示例中，所述脆弱拟杆菌的保藏编号为CGMCC No.10685。
在其中一个示例中，所述免疫检查点抑制剂选自PD-1抑制剂、PD-L1抑制剂、PD-L2抑制剂和CTLA-4抑制剂中的一种或多种。进一步地，所述PD-1抑制剂包括但不限于如下种类中的一种或者多种：纳武利尤单抗(Nivolumab)、帕博利珠单抗(Pembrolizumab)、西米普利单抗(Cemiplimab)、特瑞普利单抗(Toripalimab)、信迪利单抗(Cindilimab)、卡瑞利珠单抗(Camrelizumab)及其他能够与PD-1结合，阻断PD-1/PD-L1信号通路，上调T细胞活化，激活内源性抗肿瘤免疫反应的物质。
在其中的一些实施例中，所述的荚膜多糖A的重均分子量为70kDa-90kDa，优选80kDa-90kDa，其中重均分子量分布于70kDa-100kDa的部分占总量的70wt-80wt％。
在其中的一些实施例中，所述的荚膜多糖A的结合脂质含量低于0.02％或不含脂质。
在其中一个示例中，所述药物包含所述脆弱拟杆菌的荚膜多糖A和所述免疫检查点抑制剂中的一种或多种，以及药学上可以接受的辅料。
可以理解的是，所述辅料选自，包括但不限于以下所示辅料种类的一种或者几种：稀释剂、润湿剂、黏合剂、崩解剂、润滑剂、色香味调节剂、溶剂、增溶剂、助溶剂、乳化剂、抗氧剂、金属络合剂、惰性气体、防腐剂、局部止痛剂、pH调节剂、等渗或等张调节剂。所述稀释剂可以选自，包括但不限于：淀粉类、糖类、纤维素类、无机盐类。所述润湿剂可以选自，包括但不限于： 水、乙醇。所述黏合剂可以选自，包括但不限于：淀粉浆、糊精、糖、纤维素衍生物、明胶、聚维酮、聚乙二醇。所述崩解剂可以选自，包括但不限于：淀粉、羧甲基淀粉钠、低取代羟丙基纤维素、交联羧甲基纤维素钠、交联聚维酮、表面活性剂、泡腾崩解剂。所述润滑剂可以选自，包括但不限于：滑石粉、硬脂酸钙、硬脂酸镁、十二烷基硫酸镁、微粉硅胶、聚乙二醇。所述色香味调节剂可以选自，包括但不限于：色素、香料、甜味剂、胶浆剂、矫臭剂。所述溶剂可以选自，包括但不限于：水、乙醇、甘油、丙二醇、聚乙二醇、二甲基亚砜、液体石蜡、脂肪油、乙酸乙酯。所述增溶剂可以选自，包括但不限于：吐温类、卖泽类、聚氧乙烯脂肪醇醚类、肥皂类、硫酸化物、磺酸化物。所述助溶剂可以选自，包括但不限于：有机酸及其盐类、酰胺及胺类化合物、无机盐、聚乙二醇、聚维酮、甘油。所述乳化剂可以选自，包括但不限于：司盘类、吐温类、卖泽类、苄泽类、甘油脂肪酸酯、高级脂肪酸盐、硫酸化物、磺酸化物、阿拉伯胶、西黄耆胶、明胶、果胶、磷脂、琼脂、海藻酸钠、氢氧化物、二氧化硅、皂土。所述助悬剂可以选自，包括但不限于：甘油、糖浆、阿拉伯胶、西黄耆胶、琼脂、海藻酸钠、纤维素衍生物、聚维酮、卡波普、聚乙烯醇、触变胶。所述抗氧剂可以选自，包括但不限于：亚硫酸盐、焦亚硫酸盐、亚硫酸氢盐、抗坏血酸、没食子酸、酯类。所述金属络合剂可以选自，包括但不限于：乙二胺四乙酸二钠、多羧酸化合物。所述惰性气体可以选自，包括但不限于：氮气、二氧化碳。所述防腐剂可以选自，包括但不限于：尼泊金类、有机酸及其盐、季铵类化合物、醋酸氯己定、醇类、酚类、挥发油。所述局部止痛剂可以选自，包括但不限于：苯甲醇、三氯叔丁醇、利多卡因、普鲁卡因。所述pH调节剂可以选自，包括但不限于：盐酸、硫酸、磷酸、枸橼酸、酒石酸、醋酸、氢氧化钠、碳酸氢钠、乙二胺、葡甲胺、磷酸盐、醋酸盐、枸橼酸盐。所述等 渗或等张调节剂可以选自，包括但不限于：葡萄糖、氯化钠、枸橼酸钠、山梨醇、木糖醇。
可以理解的是，所述药物可以根据临床的需求，制备成合适的剂型，剂型可以选自，包括但不限于：丸剂、片剂、颗粒剂、胶囊、散剂、混悬剂、口服液、管饲制剂或者灌肠剂。
可以理解的是，所述药物可以根据临床的需求，采用合适的给药途径进行给药，给药途径可以选自，包括但不限于：口服给药、灌肠给药或者肠胃外给药。
可以理解的是，所述药物可以根据临床的需求，采用合适的给药周期进行给药，给药的周期可以选自，包括但不限于：间歇性给药、周期性给药、持续性给药或者长期给药。
可以理解的是，所述药物可以是人药，也可以是兽药。
作为本发明的另一方面，本发明提供一种防治淋巴瘤的药物，所述药物包含脆弱拟杆菌的荚膜多糖A和免疫检查点抑制剂。
在其中一个示例中，所述脆弱拟杆菌的保藏编号为CGMCC No.10685。
在其中一个示例中，所述免疫检查点抑制剂选自PD-1抑制剂、PD-L1抑制剂、PD-L2抑制剂和CTLA-4抑制剂中的一种或多种。进一步地，所述PD-1抑制剂包括但不限于如下种类中的一种或者多种：纳武利尤单抗(Nivolumab)、帕博利珠单抗(Pembrolizumab)、西米普利单抗(Cemiplimab)、特瑞普利单抗(Toripalimab)、信迪利单抗(Cindilimab)、卡瑞利珠单抗(Camrelizumab)及其他能够与PD-1结合，阻断PD-1/PD-L1信号通路，上调T细胞活化，激活内源性抗肿瘤免疫反应的物质。
在其中一个示例中，所述药物中，所述荚膜多糖A药学有效剂量为 5-15mg/kg。
本发明提供的上述应用以及防治淋巴瘤的药物中，脆弱拟杆菌的荚膜多糖A的制备方法包括以下步骤：
(1)脆弱拟杆菌发酵培养后，菌液离心，弃上清，收集菌体，即为脆弱拟杆菌菌泥；往菌泥中加入纯化水重悬，添加比例为菌泥质量的3倍-10倍；调节细菌重悬液pH为2.5-4.5；细菌重悬液转移至提取容器中，80℃-120℃提取0.5h-2.5h，冷却，离心取上清，得粗提荚膜多糖溶液；
(2)粗提荚膜多糖溶液超滤除小分子杂质至电导率稳定，收集回流液；
(3)回流液利用阴离子交换柱层析，20mM Tris-HCl(pH8.0，含0.2mol/L NaCl)梯度洗脱，离子交换住优选为DEAE Sepharose Fast Flow，柱层析收集液经超滤膜超滤，加入纯化水反复超滤，至电导率稳定，收集回流液，冷冻干燥，得到脆弱拟杆菌荚膜多糖A。
在上述的脆弱拟杆菌的荚膜多糖A的制备方法中，步骤(1)中，所述离心为12000g离心10min。
在上述的脆弱拟杆菌的荚膜多糖A的制备方法中，步骤(1)中，所述酸溶液可以是有机酸、无机酸和酸性缓冲液中的一种或多种。其中，无机酸可以是盐酸、硫酸、磷酸等；有机酸可以是乙酸、柠檬酸等。
在上述的脆弱拟杆菌的荚膜多糖A的制备方法中，步骤(1)中，提取温度为80℃-120℃。
在上述的脆弱拟杆菌的荚膜多糖A的制备方法中，步骤(1)中，提取时间为0.5h-2.5h；
在上述的脆弱拟杆菌荚膜多糖A的制备方法中，步骤(3)中，采用10KD超滤膜超滤浓缩。
实施例1：脆弱拟杆菌的发酵培养
将脆弱拟杆菌ZY-312菌种划线接种于血平皿，厌氧培养48h。观察菌落形态特征、染色特性、大小、球杆状和分布情况等。
菌落特征：脆弱拟杆菌ZY-312在血平皿上培养48h后，呈现圆形微凸、半透明、白色、表面光滑、不溶血，菌落直径在1mm-3mm之间，参见图1。
显微镜下形态：脆弱拟杆菌ZY-312进行革兰氏染色镜检，为革兰阴性细菌，呈现典型的杆状，两端钝圆而浓染，菌体中间不着色部分形如空泡，参见图2。
选取单个菌落接种于植物源蛋白胨液体培养基中进行37℃，发酵培养8h，所得菌液离心沉淀，转速3000r/min，离心15min，去上清，收集沉淀物，即得脆弱拟杆菌ZY-312菌泥。
取上述菌液，常规热灭活处理，得脆弱拟杆菌ZY-312灭活菌液。
实施例2：脆弱拟杆菌荚膜多糖A的制备
一、采用实施例1制备的菌泥进行实验。
(1)取菌泥50g，加入300g纯化水使菌体重悬，用1mol/L盐酸溶液调节其pH至3.5，100℃提取1.5h，冷却至室温，12000g常温离心10min，取上清，得到粗糖溶液；
(2)粗糖溶液经10KD超滤膜超滤浓缩、除小分子杂质，至电导率稳定，收集回流液；
(3)回流液中加入等体积40mmol/L Tris-HCl(pH8.5)转盐；DEAE Sepharose Fast Flow离子交换柱层析(16mm×200mm)，流速20mL/min，20mmol/L Tris-HCl(pH8.5，含0.2mol/L NaCl)梯度洗脱25个柱体积，分段收集，100mL/瓶(组分)，SEC-HPLC跟踪监测，合并206nm吸收峰为单一、对称峰的组分，10KD超滤膜超滤，加入纯化水反复超滤，至电导率稳定，收集回流液，冻干，得到 脆弱拟杆菌提取物。
二、脆弱拟杆菌荚膜多糖A测试分析：
测试方法：称量30mg步骤(3)所述的脆弱拟杆菌提取物，溶于0.5mL D 2O，加入1μl丙酮(1H，2.22；13C，30.89)定标。采用500MHz Bruker核磁共振波谱仪分析1H谱(图3)、13C谱(图4)、COSY谱(图5)、HSQC谱(图6)、HMBC谱(图7)。
测试结果：确证步骤(3)收集的脆弱拟杆菌提取物为荚膜多糖A，结合脂质含量低于0.02％，蛋白残留低于1％，核酸残留低于0.05％。通过GPC(凝胶渗透色谱)分析，制得的荚膜多糖A重均分子量为80kDa-90kDa，Mw/Mn为1.0-1.2，化学结构参见附图8。
制得的脆弱拟杆菌荚膜多糖A命名为ZY-312-PSA。
实施例3：荚膜多糖A联合PD-1抗体在治疗淋巴瘤中的应用
一、实验设计
为了验证实施例2提供的荚膜多糖A联合PD-1抗体对淋巴瘤的治疗作用，本案例选用6周龄-8周龄C57BL/6J雌性小鼠来构建淋巴瘤模型。取培养状态良好的EL4细胞，调整细胞浓度为1×10 6/ml，剃毛器剃去小鼠背部毛发，在背部皮下右侧接种EL4淋巴瘤细胞，接种量为3×10 5EL4细胞每处，待肿瘤生长一周约100mm 3-150mm 3时，将小鼠随机分为6组，每组10只，具体分为模型组、PD-1抗体组、ZY-312-PSA组、低剂量ZY-312-PSA+PD-1抗体组、中剂量ZY-312-PSA+PD-1抗体组和高剂量ZY-312-PSA+PD-1抗体组，并以分组当日作为day 0，按表1进行给药，共给药21天。
在实验终点，所有小鼠安乐死，采集小鼠血清、肿瘤、脾脏、粪便、右侧颈部淋巴以及右侧腋窝淋巴。所有肿瘤称重和拍照。肿瘤分为三份，一份冻存 用于细胞因子检测，一份于福尔马林中固定，一份送于体外用于流式分析。
表1、实验动物分组及给药方案
Figure PCTCN2022120110-appb-000001
二、肿瘤测量和实验指标
肿瘤体积和抑瘤疗效：从day0开始，每3天用游标卡尺测量肿瘤直径。肿瘤体积的计算公式为：V＝0.5a×b 2，a和b分别表示肿瘤的长径和短径。
抑瘤疗效用TGI(％)评价，TGI(％)，反映肿瘤生长抑制率。TGI(％)的计算：TGI(％)＝[1-(某处理组给药结束时平均瘤体积－该处理组开始给药时平均瘤体积)/(同型对照组治疗结束时平均瘤体积－同型对照组开始治疗时平均瘤体积)]×100％。
T细胞亚群：流式细胞术分析外周血和脾脏内CD4+T细胞和CD8+T细胞的比例。
三、统计分析
基于实验结束时获得的数据进行统计学分析评估组间差异。两组间比较用T-test(one tailed)进行统计分析。使用GraphPad Prism软件进行所有数据分析，p<0.05认为有显著性差异。
四、实验结果
1、肿瘤体积和抑瘤疗效
基于分组给药后第21天肿瘤体积及瘤重，计算得出受试药物对EL4淋巴瘤细胞皮下接种模型的生长抑制率。
表2、受试物对小鼠EL4淋巴瘤模型的抑瘤药效评价
(基于分组给药后第21天肿瘤体积计算得出)
Figure PCTCN2022120110-appb-000002
注：
a.平均值±SEM；
b.肿瘤生长抑制评价指标根据公式TGI(％)＝[1-(某处理组给药结束时平均瘤体积－该处理组开始给药时平均瘤体积)/(同型对照组治疗结束时平均瘤体积－同型对照组开始治疗时平均瘤体积)]×100％；
c.根据肿瘤体积计算，两组间p值按照unpaired t-test(one-tailed)方法计算。
根据上表可知，与模型组相比，各给药组均可显著抑制肿瘤生长；PD-1抗体和脆弱拟杆菌荚膜多糖A联合用药时，具有极显著差异。可见，脆弱拟杆菌ZY-312荚膜多糖A联用PD-1抗体能够有效抑制肿瘤生长。
2、T细胞亚群
表3、各组小鼠外周血和脾CD4+、CD8+T细胞比例(mean±SD)
Figure PCTCN2022120110-appb-000003
注：与模型组比较，*表示差异显著p<0.05；**表示差异极显著p<0.01。
CD8+T细胞(TC或CTL细胞)能杀伤表达抗原的靶细胞，它在抗毒感染、急性同种异型移植物排斥和对肿瘤细胞的杀伤作用是重要的效应细胞。CD4+T细胞是人体免疫系统中的一种重要免疫细胞，主要由辅助T(Th)细胞分化而来，可与MHCⅡ类分子的非多肽区结合，参与T细胞抗原受体(TCR)识别抗原的信号转导。研究发现，在肿瘤免疫中，CD4+T细胞启动后可以通过多种机制激活CD8+T细胞，使其分化为细胞毒性T淋巴细胞(CTL)，同时维持并加强CTL的抗肿瘤反应。
如上表所示，与模型组相比，各给药组均不同程度地上调了小鼠外周血CD4+、CD8+T细胞的水平。其中，脆弱拟杆菌荚膜多糖A与PD-1抗体联用组具有显著性差异(p＜0.05或0.01)。联用组内，有不显著的PSA剂量依赖性。
与模型组相比，各给药组均不同程度地上调了小鼠脾脏CD4+、CD8+T细胞的水平。在CD4+T细胞水平上，中、高剂量PSA联用PD-1抗体组具有显著性差异；在CD8+T细胞水平上，各给药组均具有显著性差异，中、高剂量PSA联用PD-1抗体组具有极显著差异。这说明脆弱拟杆菌荚膜多糖A与PD-1抗体联合用药能够调节小鼠外周血和脾内T细胞水平，增强机体抗肿瘤免疫反应。
综上所述，脆弱拟杆菌，尤其是保藏编号为CGMCC No.10685的脆弱拟杆菌ZY-312的两性离子荚膜多糖A(PSA)，与PD-1抗体联用，能够调节小鼠外周血和脾内T细胞水平，增强机体抗肿瘤免疫反应，抑制小鼠EL4淋巴移植瘤的生长，有效防治淋巴瘤。
上所述实施例的各技术特征可以进行任意的组合，为使描述简洁，未对上述实施例中的各个技术特征所有可能的组合都进行描述，然而，只要这些技术特征的组合不存在矛盾，都应当认为是本说明书记载的范围。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。

Claims (10)

  1. 脆弱拟杆菌的荚膜多糖A和免疫检查点抑制剂在制备防治淋巴瘤的药物中的应用。
  2. 根据权利要求1所述的应用，其特征在于，所述脆弱拟杆菌的保藏编号为CGMCC No.10685。
  3. 根据权利要求1所述的应用，其特征在于，所述免疫检查点抑制剂选自PD-1抑制剂、PD-L1抑制剂、PD-L2抑制剂和CTLA-4抑制剂中的一种或多种。
  4. 根据权利要求3所述的应用，其特征在于，所述PD-1抑制剂选自纳武利尤单抗、帕博利珠单抗、西米普利单抗、特瑞普利单抗、信迪利单抗和卡瑞利珠单抗中的一种或者多种。
  5. 根据权利要求1所述的应用，其特征在于，所述荚膜多糖A的重均分子量为70kDa-90kDa。
  6. 根据权利要求1至5任一项所述的应用，其特征在于，所述药物包含所述脆弱拟杆菌的荚膜多糖A和所述免疫检查点抑制剂中的一种或多种，以及药学上可以接受的辅料。
  7. 根据权利要求6所述的应用，其特征在于，所述辅料包含稀释剂、润湿剂、黏合剂、崩解剂、润滑剂、色香味调节剂、溶剂、增溶剂、助溶剂、乳化剂、抗氧剂、金属络合剂、惰性气体、防腐剂、局部止痛剂、pH调节剂以及等渗或等张调节剂中的一种或者多种。
  8. 根据权利要求1至5和7任一项所述的应用，其特征在于，所述药物的剂型包括丸剂、片剂、颗粒剂、胶囊、散剂、混悬剂、口服液、管饲制剂或者灌肠剂。
  9. 根据权利要求1至5和7任一项所述的应用，其特征在于，所述药物的给药方式包括口服给药、灌肠给药或者肠胃外给药；
    或/和，所述药物的给药周期包括间歇性给药、周期性给药、持续性给药或者长期给药。
  10. 一种防治淋巴瘤的药物，特征在于，所述药物包含脆弱拟杆菌的荚膜多糖A和免疫检查点抑制剂。
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