WO2023134209A1 - 脆弱拟杆菌和免疫检查点抑制剂的新应用 - Google Patents

脆弱拟杆菌和免疫检查点抑制剂的新应用 Download PDF

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WO2023134209A1
WO2023134209A1 PCT/CN2022/120086 CN2022120086W WO2023134209A1 WO 2023134209 A1 WO2023134209 A1 WO 2023134209A1 CN 2022120086 W CN2022120086 W CN 2022120086W WO 2023134209 A1 WO2023134209 A1 WO 2023134209A1
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antibody
bacteroides fragilis
immune checkpoint
group
administration
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PCT/CN2022/120086
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French (fr)
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王晔
易晓敏
黄烁雅
王薇
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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
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention relates to the field of biomedicine, in particular to a new application of Bacteroides fragilis and immune checkpoint inhibitors.
  • Lymphoma is a malignant tumor originating from the lymphatic hematopoietic system, which can be divided into two types: non-Hodgkin's lymphoma (NHL) and Hodgkin's lymphoma (HL).
  • NHL non-Hodgkin's lymphoma
  • HL Hodgkin's lymphoma
  • the incidence of NHL is about 10 times that of HL.
  • the mortality rate of NHL is 20 times that of HL.
  • cHL nodular lymphoid-rich Cellular and classical
  • NHL lymphocyte-predominant, nodular sclerosis, mixed cellularity and lymphocyte depletion.
  • NHL is the sum of a group of independent diseases with strong heterogeneity. Pathologically, it mainly consists of lymphocytes, histiocytes or reticular cells with different degrees of differentiation.
  • NHL According to the natural course of NHL, it can be classified into three major clinical types, namely high Aggressive, aggressive and indolent lymphomas. According to the origin of different lymphocytes, it can be divided into B cell, T cell and NK cell lymphoma. For specific classification details, please refer to the 2016 edition of WHO lymphoma classification.
  • the lymphatic system includes the lymph nodes (lymph glands), spleen, thymus, and bone marrow. Lymphoma can affect not only the lymphatic system but also other organs throughout the body. Clinically, painless lymphadenopathy is the most typical, hepatosplenomegaly is common, and cachexia, fever and anemia are present in the late stage. Due to the difference in the location and extent of the lesion, the clinical manifestations are inconsistent.
  • the primary site can be in the lymph nodes or extranodal lymphatic tissues, such as tonsils, nasopharynx, gastrointestinal tract, spleen, bones, or skin. Primary changes of nodal lymphoid tissue are more common in NHL.
  • the traditional treatment methods for lymphoma are mainly combined radiotherapy and chemotherapy, and other treatment methods include hematopoietic stem cell transplantation, surgery, targeted therapy and immunotherapy.
  • the principle of treatment is stratified treatment based on staging and prognostic factors, and PET/CT is an important examination method for staging.
  • HL due to advances in chemotherapy and radiotherapy, most HL patients can achieve long-term survival or even be cured.
  • the first-line chemotherapy options include ABVD (doxorubicin + bleomycin + vinblastine + dacarbazine), CHOP ( cyclophosphamide + doxorubicin + vincristine + prednisone), CVP program (cyclophosphamide + vincristine + prednisone), EPOCH program (etoposide + vincristine + cyclophosphamide + a Rituximab, a targeted drug targeting CD20, is often used in the combined treatment of IB and above HL.
  • ABVD doxorubicin + bleomycin + vinblastine + dacarbazine
  • CHOP cyclophosphamide + doxorubicin + vincristine + prednisone
  • CVP program cyclophosphamide + vincristine + prednisone
  • EPOCH program etoposide + vincristine + cyclophosphamide + a Rituxima
  • auto-HSCT autologous hematopoietic stem cell transplantation
  • r/r cHL relapsed/refractory HL
  • auto-HSCT autologous hematopoietic stem cell transplantation
  • Diffuse large B-cell lymphoma is the most common type in NHL, and the treatment mode is comprehensive treatment including medical treatment and radiotherapy.
  • Medical treatment includes chemotherapy and biological targeted therapy.
  • the treatment strategy should be adjusted according to age, IPI score, and stage.
  • the commonly used treatment plan is R-CGOP, which is chemotherapy + local radiotherapy 30-36Gy.
  • Follicular lymphoma is the most common indolent lymphoma in Europe and the United States.
  • the standard first-line treatment is rituximab combined with chemotherapy.
  • Marginal zone lymphoma originates from B-cell lymphoma in the marginal zone and belongs to indolent lymphoma. According to the site and stage, choose a personalized treatment plan of local radiotherapy or surgery, chemotherapy and immunotherapy.
  • gastric mucosa-associated lymphoid tissue lymphoma is related to HP infection, and anti-HP treatment should be considered for positive cases.
  • Surgical treatment is mostly used for tissue biopsy or treatment of complications.
  • splenectomy may be considered to improve the blood picture, which is more conducive to future chemotherapy.
  • Surgical treatment may be considered for patients with gastrointestinal lymphoma who have complications such as gastric perforation, bleeding, or obstruction that cannot be relieved by medical treatment.
  • complications such as gastric perforation, bleeding, or obstruction that cannot be relieved by medical treatment.
  • microecological product for the treatment of lymphoma there is no microecological product for the treatment of lymphoma in the prior art.
  • the main purpose of the present invention is to provide a new application of Bacteroides fragilis and immune checkpoint inhibitors, mainly related to the preparation and prevention of lymphoma with the preservation number of Bacteroides fragilis and immune checkpoint inhibitors CGMCC No.10685
  • the drug has a synergistic effect on lymphoma, and provides a kind of microecological drug for the treatment of lymphoma.
  • Bacteroides fragilis and immune checkpoint inhibitors in the preparation of drugs for preventing and treating lymphoma, the preservation number of Bacteroides fragilis is CGMCC No.10685.
  • the Bacteroides fragilis is selected from one or more of the following species: live bacteria of Bacteroides fragilis, inactivated Bacteroides fragilis with complete or incomplete morphology, genetic recombination, transformation Or modified, attenuated, chemically or physically treated Bacteroides fragilis, Bacteroides fragilis lysate, Bacteroides fragilis liquid culture supernatant.
  • the immune checkpoint inhibitor is selected from PD-1 antibody, PD-L1 antibody, PD-L2 antibody, CTLA-4 antibody, LAG-3 antibody, TIM-3 antibody, VISTA antibody and A2aR One or more of the antibodies.
  • the immune checkpoint inhibitor is PD-1 antibody or/and PD-L1 antibody.
  • the PD-1 antibody is selected from Nivolumab, Pembrolizumab, Simiprimumab, Toripalimab, Sintilimab, and Carrelizumab One or more of Zhuzumab.
  • the PD-L1 antibody is selected from one or more of atezolizumab, avelumab and durvalumab.
  • the lymphoma is T cell lymphoma.
  • the medicine comprises one or more of the Bacteroides 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.
  • a medicine for preventing and treating lymphoma comprises Bacteroides fragilis and immune checkpoint inhibitors, and the preservation number of Bacteroides fragilis is CGMCC No.10685.
  • the Bacteroides fragilis is selected from one or more of the following species: live bacteria of Bacteroides fragilis, inactivated Bacteroides fragilis with complete or incomplete morphology, genetic recombination, transformation Or modified, attenuated, chemically or physically treated Bacteroides fragilis, Bacteroides fragilis lysate, Bacteroides fragilis liquid culture supernatant.
  • the immune checkpoint inhibitor is selected from PD-1 antibody, PD-L1 antibody, PD-L2 antibody, CTLA-4 antibody, LAG-3 antibody, TIM-3 antibody, VISTA antibody and A2aR One or more of the antibodies.
  • the immune checkpoint inhibitor is PD-1 antibody or/and PD-L1 antibody.
  • the PD-1 antibody is selected from Nivolumab, Pembrolizumab, Simiprimumab, Toripalimab, Sintilimab, and Carrelizumab One or more of Zhuzumab.
  • the PD-L1 antibody is selected from one or more of atezolizumab, avelumab and durvalumab.
  • the present invention has the following beneficial effects:
  • the present invention proves through a large number of experiments that the combined use of Bacteroides fragilis ZY-312 with the deposit number CGMCC No. 10685 and immune checkpoint inhibitors, especially PD-1 inhibitors, can have a synergistic effect on lymphoma.
  • Fig. 1 is the colony characteristic figure of Bacteroides fragilis ZY-312 of the embodiment of the present invention 1;
  • Fig. 2 is a microscope observation diagram of Bacteroides fragilis ZY-312 in Example 1 of the present invention after Gram staining.
  • the experimental methods used in the following examples, if no special instructions, are conventional methods; reagents, materials, etc. used in the following examples, if no special instructions, the raw materials and reagents used in the following examples are commercially available Commercial products, 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 Co., Ltd.; or can 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 advent of immunotherapy represented by immune checkpoint inhibitors (ICIs) has further improved the prognosis of relapsed and refractory patients.
  • the immune checkpoint inhibitor PD-1 monoclonal antibody has significantly improved the prognosis of patients with r/r cHL.
  • the objective response rate (ORR) of multiple PD1 monoclonal antibodies is above 70%-80%, the complete response (CR) is above 20%-30%, and the median 10-year progression-free survival rate (progression-free survival) -free survival, PFS) greater than 12 months.
  • ICIs Immune checkpoint inhibitors
  • PD-1 Programmed cell death receptor-1
  • PD-L1 Programmed cell death ligand 1
  • PD-1/PD-L1 inhibitors have great potential in the treatment of various tumors.
  • Effectively approved PD-1/PD-L1 inhibitors for Hodgkin's lymphoma in my country include Camrelizumab, Islelizumab, and Intilimab , Enpulimab (enpulimab).
  • Bifidobacterium is a genus that is positively correlated with anti-tumor immune responses, among which Bifidobacterium longum (B.longum) and Bifidobacterium breve (B.breve) It can indirectly regulate the expression of specific genes of dendritic cells, promote their maturation and secretion of IFN- ⁇ cytokines, thereby enhancing the infiltration of CTLs in tumor tissues and increasing their activity, and improving the anti-tumor immune response.
  • antibiotic treatment weakened the treatment effect of ICIs in patients with non-small cell lung cancer, renal cell carcinoma, and urothelial carcinoma.
  • the strain with the greatest impact on the curative effect is A. muciniphila, which can effectively improve the therapeutic effect of PD-1 inhibitors on tumor-bearing mice.
  • Bacteroides fragilis (B. fragilis) is Gram-negative, rod-shaped, blunt-rounded at both ends and densely stained, capsulated, non-spore-free, non-motive obligate anaerobic bacteria, divided into enterotoxigenic bacteria Enterotoxigenic type (ETBF) and non-enterotoxigenic type (NTBF) are part of the normal intestinal flora of humans and animals, and normally reside in the mucous membranes of the human respiratory tract, gastrointestinal tract, and genitourinary tract.
  • EBF enterotoxigenic type
  • NTBF non-enterotoxigenic type
  • 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.).
  • Bacteroides fragilis ZY-312 Bacteroides fragilis ZY-312
  • deposit number CGMCC No.10685 Bacteroides fragilis ZY-312 was isolated and obtained by the applicant unit of the present invention, and has been authorized for patent protection (patent number 201510459408.X). According to the provisions of the patent examination guidelines, the public can buy it from commercial channels or has been authorized without preservation, that is, No deposit certificate is required.
  • the present invention provides the application of Bacteroides fragilis and immune checkpoint inhibitors in the preparation of drugs for preventing and treating lymphoma.
  • the deposit number of Bacteroides fragilis is CGMCC No.10685.
  • Bacteroides fragilis and immune checkpoint inhibitors work together to reduce the levels of pro-inflammatory factors IL-6, IL-8, IL-2R, TNF- ⁇ , IL-21, increase the migration of CD8+ effector T cells, and reduce The recruitment of Treg cells reduces the weight of orthotopic tumors and increases the tumor suppressor rate.
  • Bacteroides fragilis is selected from one or more of the following species: live bacteria of Bacteroides fragilis, inactivated Bacteroides fragilis with complete or incomplete morphology, genetically recombined, modified or Bacteroides fragilis modified, attenuated, chemically or physically treated, lysate of Bacteroides fragilis, liquid culture supernatant of Bacteroides fragilis.
  • the immune checkpoint inhibitor is selected from PD-1 antibody, PD-L1 antibody, PD-L2 antibody, CTLA-4 antibody, LAG-3 antibody, TIM-3 antibody, VISTA antibody and A2aR antibody one or more of.
  • the immune checkpoint inhibitor is PD-1 antibody or/and PD-L1 antibody.
  • the PD-1 antibody is selected from the group consisting of nivolumab, pembrolizumab, simiprizumab, toripalimab, sintilimab and camrelizumab one or more of.
  • the PD-L1 antibody is selected from one or more of atezolizumab, avelumab and durvalumab.
  • the lymphoma is T cell lymphoma.
  • the drug comprises one or more of the Bacteroides 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 can 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 compound.
  • 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 may 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 medicament for preventing and treating lymphoma, the medicament comprises Bacteroides fragilis and immune checkpoint inhibitors, and the preservation number of Bacteroides fragilis is CGMCC No.10685.
  • Bacteroides fragilis is selected from one or more of the following species: live bacteria of Bacteroides fragilis, inactivated Bacteroides fragilis with complete or incomplete morphology, genetically recombined, modified or Modified, attenuated, chemically treated, physically treated Bacteroides fragilis, Bacteroides fragilis lysate, Bacteroides fragilis liquid culture supernatant.
  • the immune checkpoint inhibitor is selected from PD-1 antibody, PD-L1 antibody, PD-L2 antibody, CTLA-4 antibody, LAG-3 antibody, TIM-3 antibody, VISTA antibody and A2aR antibody one or more of.
  • the immune checkpoint inhibitor is PD-1 antibody or/and PD-L1 antibody.
  • the PD-1 antibody is selected from the group consisting of nivolumab, pembrolizumab, simiprizumab, toripalimab, sintilimab and camrelizumab one or more of.
  • the PD-L1 antibody is selected from one or more of atezolizumab, avelumab and durvalumab.
  • the lymphoma is T cell lymphoma.
  • Embodiment 1 the fermentation culture of Bacteroides fragilis
  • Bacteroides fragilis ZY-312 was cultured on a blood plate for 48 hours, and it appeared round, slightly convex, translucent, white, smooth, non-hemolytic, and the diameter of the colony was between 1-3 mm, 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.
  • Embodiment 2 Preparation of Bacteroides fragilis live bacteria liquid
  • 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 1-3mm.
  • Live Bacteroides fragilis solution use the McFarland turbidimetric tube to measure the bacterial count of the bacteria solution prepared in step (1), dilute it with normal saline to 10 7 CFU/ml and 10 9 CFU/ml, and save it for later use .
  • Embodiment 3 Preparation of the inactivated bacteria powder of Bacteroides fragilis
  • step (3) Centrifuge the inactivated bacteria liquid obtained in step (2) to collect the inactivated bacteria sludge.
  • step (3) Add excipients to the inactivated bacteria slime collected in step (3) to make the total weight consistent with the weight of the bacteria liquid before inactivation, stir to dissolve completely, and obtain the inactivated bacteria stock solution.
  • step (4) Vacuum freeze-dry the stock solution of inactivated bacteria obtained in step (4), pre-freeze at -40 ⁇ 2°C for 1-3 hours, then pre-freeze at -20 ⁇ 2°C for 0.5-1h, and finally freeze at -40 ⁇ 2°C Pre-freeze for 0.5-2 hours, under 0.25mbar vacuum degree, after one drying (-5 ⁇ 2°C and 0 ⁇ 2°C), analytical drying (35 ⁇ 2°C) to prepare inactivated bacteria powder, the number of bacteria in the powder reaches 1 ⁇ More than 10 11 Cell/g.
  • Example 4 Drug efficacy experiment of Bacteroides fragilis and PD-1 inhibitors in synergistic treatment of EL4 mouse T-cell lymphoma model
  • Tumor programmed cell death receptor-1 can inhibit the activation of T cells by binding to its ligand, so as to realize the immune escape of tumors.
  • PD-1 Tumor programmed cell death receptor-1
  • CD8+ effector T cells undertake the main tumor killing activity, while regulatory T cells accumulated in the microenvironment play a role in tumor immunosuppression.
  • FOXP3+CD4+CD25+Treg cells are considered to be the main obstacle in anti-tumor immunotherapy.
  • EL4 cells were cultured in DMEM/F12 medium containing 10% fetal bovine serum, the culture temperature was 37°C, the gas environment was 5% CO 2 /95% air, and the humidity was saturated humidity; Change the culture medium for color change, digest and pass with 0.25% trypsin. According to the growth of the cells, the cells in the logarithmic growth phase were prepared into a single cell suspension, and the cell concentration was adjusted to 1 ⁇ 10 7 /mL.
  • mice Seven-week-old male C57B6/L mice were used in the experiment, and the tumors were inoculated after the mice grew naturally for one week. Inoculate all mice except the normal control group with tumor cells, take one side of the mouse under the armpit, and inject 0.2ml of the counted cell suspension subcutaneously with a 6-gauge needle. Before aspirating the cell suspension, mix gently to ensure that each mouse The number of cells seeded was the same. Observe the tumor growth in mice.
  • mice showed signs of exhaustion such as emaciation, arched back, and listlessness, and palpable lumps at the inoculation site of the mice.
  • CD8+ effector T cells CD8+ effector T cells, regulatory T cells (Treg cells), tumor weight, tumor suppression rate, and cytokines IL-6, IL-8, IL-2R, TNF- ⁇ , IL-21, etc.
  • Tumor inhibition rate 100% (average tumor weight in model group-average tumor weight in administration group)/average tumor weight in model group.
  • the average tumor weight of the model group was significantly higher than that of the other groups (P ⁇ 0.01), slightly higher in the ZY-312 group than in the PD-1 group (P ⁇ 0.05); low, medium and high doses of Bacteroides fragilis ZY
  • the -312 combined with PD-1 inhibitor group was lower than that of the PD-1 inhibitor group (P ⁇ 0.05), and the tumor weight of the high-dose Bacteroides fragilis ZY-312 combined with PD-1 inhibitor group was the lowest;
  • Bacteroides ZY-312 inactivated bacteria combined with PD-1 inhibitor group was lower than that in PD-1 inhibitor group (P ⁇ 0.05), and the high-dose Bacteroides fragilis ZY-312 inactivated bacteria combined with PD-1 inhibitor group
  • the body weight was the lowest.
  • the low-dose ZY-312 inactivated bacteria combined with PD-1 inhibitor group was slightly lower than the PD-1 group, there was no statistical difference.
  • each concentration of Bacteroides fragilis ZY-312 and inactivated bacteria combined with PD-1 inhibitor group was higher than that of PD-1 inhibitor group alone (P ⁇ 0.05), and ZY-312 group was slightly lower than PD-1 inhibitor group. 1 group, but there was no statistical difference.
  • the model group was higher than the saline blank control group, the PD-1 inhibitor group was higher than the model group, and there were statistical differences (P ⁇ 0.01), and the ZY-312 group was higher than the model group (P ⁇ 0.05).
  • the model group was higher than the saline blank control group, the PD-1 inhibitor group was higher than the model group, and there were statistical differences (P ⁇ 0.05), and the ZY-312 group was higher than the model group, but there was no statistical difference. Differences; low, medium and high doses of Bacteroides fragilis ZY-312 combined with PD-1 inhibitor groups were all higher than those of PD-1 inhibitors group (P ⁇ 0.05), and high and high doses of Bacteroides fragilis ZY-312 combined with PD-1 inhibited CD8+ effector T cells accounted for the highest proportion in the dose group; low, medium, and high doses of B. The proportion of CD8+ effector T cells was the highest in the Bacteroides fragilis ZY-312 inactivated bacteria combined with PD-1 inhibitor group.
  • Treg cells Regulatory T cells
  • the model group was significantly higher than the saline blank control group (P ⁇ 0.001), the PD-1 inhibitor group was lower than the model group (P ⁇ 0.05), and there was no statistical difference between the ZY-312 group and the PD-1 inhibitor group Differences; low, medium and high doses of Bacteroides fragilis ZY-312 combined with PD-1 inhibitor groups were all lower than those of PD-1 inhibitors group (P ⁇ 0.05), and medium doses of Bacteroides fragilis ZY-312 combined with PD-1 inhibited
  • the proportion of Treg cells in the dose group was the lowest; the low, medium and high doses of B.
  • the proportion of Treg cells in the Bacillus ZY-312 inactivated bacteria combined with PD-1 inhibitor group was the lowest, but higher than that in the saline blank control group (P ⁇ 0.05).
  • the model group was significantly higher than the saline blank control group (P ⁇ 0.001), the PD-1 inhibitor group was lower than the model group (P ⁇ 0.05), and there was no statistical difference between the ZY-312 group and the PD-1 inhibitor group.
  • the low, medium and high doses of B. fragilis ZY-312 combined with PD-1 inhibitor group were all lower than those of the PD-1 inhibitor group (P ⁇ 0.05), and the low dose of B. fragilis ZY-312 combined with PD-1
  • the proportion of CD8+ effector T cells in the inhibitor group was the lowest; the low, medium and high doses of B. fragilis ZY-312 inactivated bacteria combined with PD-1 inhibitor group were lower than those in the PD-1 inhibitor group (P ⁇ 0.05).
  • IL-6, IL-8, IL-2R, TNF- ⁇ , IL-21, IL-10 and other cytokines in the EL4 lymphoma mouse model were detected by luminex technology.
  • pro-inflammatory factors such as IL-6, IL-8, IL-2R, TNF- ⁇ , and IL-21 in the model group were significantly increased.
  • PD-1 inhibitors can effectively regulate the above cytokines and down-regulate the levels of pro-inflammatory factors such as IL-6, IL-8, IL-2R, TNF- ⁇ , and IL-21. Similar effects were also observed in the ZY-312 group. Bacteroides fragilis ZY-312 can enhance the effect of PD-1 inhibitors.

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Abstract

本发明涉及脆弱拟杆菌与免疫检查点抑制剂的新应用。脆弱拟杆菌和免疫检查点抑制剂的组合在制备防治淋巴瘤的药物中的应用，所述脆弱拟杆菌的保藏编号为CGMCC No.10685。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，保藏编号为CGMCC No.10685的脆弱拟杆菌ZY-312与免疫检查点抑制剂联合使用，特别是与PD-1抑制剂联合使用，能够对淋巴瘤协同显效。

Description

脆弱拟杆菌和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与免疫检查点抑制剂的新应用。
背景技术
淋巴瘤(lymphoma)是起源于淋巴造血系统的恶性肿瘤，可分为非霍奇金淋巴瘤(NHL)和霍奇金淋巴瘤(HL)两类，其中NHL发病率约为HL的10倍，死亡率NHL是HL的20倍。霍奇金淋巴瘤的病理学特征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(cHL)，cHL是霍奇金淋巴瘤中最常见类型，约占90％，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。具体分类详情可参考2016版WHO淋巴瘤分类。
淋巴系统包括淋巴结(淋巴腺)、脾脏、胸腺和骨髓。淋巴瘤可以影响淋巴系统以外还会影响全身其他器官。临床以无痛性淋巴结肿大最为典型，肝脾常肿大，晚期有恶病质、发热及贫血。由于病变部位和范围的不同，临床表现不一致，原发部位可在淋巴结，也可在结外的淋巴组织，例如扁桃体、鼻咽部、胃肠道、脾、骨骼或皮肤等。结淋巴组织原发部变多见于NHL。
传统的针对淋巴瘤的治疗方法以放疗和化疗联合治疗为主，其他治疗手段 还包括造血干细胞移植、手术治疗、靶向治疗和免疫疗法。治疗原则为根据分期和预后因素进行分层治疗，对于分期PET/CT是重要的检查手段。对于HL，由于化疗及放疗的进步，多数HL患者可以获得长期生存甚至治愈，一线化疗方案可选择ABVD方案(阿霉素+博来霉素+长春花碱+氮烯咪胺)、CHOP方案(环磷酰胺+阿霉素+长春新碱+泼尼松)、CVP方案(环磷酰胺+长春新碱+泼尼松)、EPOCH方案(足叶乙甙+长春新碱+环磷酰胺+阿霉素+泼尼松)，以CD20为靶点的靶向药物利妥昔单抗常用于ⅠB及以上的HL的联合治疗。对于复发/难治性HL患者(r/r cHL)，可考虑自体造血干细胞移植(auto-HSCT)增加二次治愈的可能。由于NHL病理类型多，异质性强，需要根据不同病理亚型、分期、预后因素和治疗目的制定治疗方案。放化疗联合治疗为主要的治疗手段。以下介绍几种常见的NHL病理类型治疗方案。(1)弥漫大B细胞淋巴瘤为NHL中最常见的类型，治疗模式为包括内科治疗和放疗在内的综合治疗。内科治疗包括化疗和生物靶向治疗。治疗策略应根据年龄、IPI评分和分期等进行相应的调整，常用的治疗方案如R-CGOP方案即化疗+局部放疗30-36Gy。(2)滤泡性淋巴瘤为欧美地区最常见的惰性淋巴瘤，标准一线治疗方案为利妥昔单抗联合化疗，联合化疗方案可有多种选择。(3)边缘区淋巴瘤起源于边缘带区的B细胞淋瘤，属于惰性淋巴瘤。根据部位与分期选择局部放疗或手术、化疗和免疫治疗的个性化治疗方案。其中胃黏膜相关淋巴样组织淋巴瘤与HP感染相关，阳性者考虑抗HP治疗。手术治疗多用于组织活检或并发症处理，如合并脾功能亢进患者排除禁忌症可考虑行脾切除手术，以提高血象，更有利于以后的化疗。胃肠道淋巴瘤患者若合并胃穿孔、出血或梗阻等并发症，内科治疗无法缓解可考虑行外科治疗。然而，现有技术尚无用于治疗淋巴瘤的微生态产品。
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和免疫检查点抑制剂的新应用，主要涉及用保藏编号为CGMCC No.10685的脆弱拟杆菌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，为淋巴瘤的治疗提供一种微生态药物。
本发明的目的可以通过以下技术方案实现：
脆弱拟杆菌和免疫检查点抑制剂在制备防治淋巴瘤的药物中的应用，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，所述脆弱拟杆菌选自如下种类中的一种或者多种：脆弱拟杆菌的活菌，形态结构完整或者不完整的灭活的脆弱拟杆菌，经基因重组、改造或修饰、减毒、化学处理或者物理处理的脆弱拟杆菌，脆弱拟杆菌裂解物，脆弱拟杆菌液体培养上清液。
在其中一些实施例中，所述免疫检查点抑制剂选自PD-1抗体、PD-L1抗体、PD-L2抗体、CTLA-4抗体、LAG-3抗体、TIM-3抗体、VISTA抗体和A2aR抗体中的一种或多种。
在其中一些实施例中，所述免疫检查点抑制剂为PD-1抗体或/和PD-L1抗体。
在其中一些实施例中，所述PD-1抗体选自纳武利尤单抗、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。
在其中一些实施例中，所述PD-L1抗体选自阿特朱单抗、阿维鲁单抗和度伐鲁单抗中的一种或者多种。在其中一些实施例中，所述淋巴瘤为T细胞淋巴瘤。
在其中一些实施例中，所述药物包含所述脆弱拟杆菌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。
一种防治淋巴瘤的药物，所述药物包含脆弱拟杆菌和免疫检查点抑制剂，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，所述脆弱拟杆菌选自如下种类中的一种或者多种：脆弱拟杆菌的活菌，形态结构完整或者不完整的灭活的脆弱拟杆菌，经基因重组、改造或修饰、减毒、化学处理或者物理处理的脆弱拟杆菌，脆弱拟杆菌裂解物，脆弱拟杆菌液体培养上清液。
在其中一些实施例中，所述免疫检查点抑制剂选自PD-1抗体、PD-L1抗体、PD-L2抗体、CTLA-4抗体、LAG-3抗体、TIM-3抗体、VISTA抗体和A2aR抗体中的一种或多种。
在其中一些实施例中，所述免疫检查点抑制剂为PD-1抗体或/和PD-L1抗体。
在其中一些实施例中，所述PD-1抗体选自纳武利尤单抗、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。
在其中一些实施例中，所述PD-L1抗体选自阿特朱单抗、阿维鲁单抗和度伐鲁单抗中的一种或者多种。
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，保藏编号为CGMCC No.10685的脆弱拟杆菌ZY-312与免疫检查点抑制剂联合使用，特别是与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。
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。
下述实施例中所使用的实验方法如无特殊说明，均为常规方法；下述实施例中所用的试剂、材料等，如无特殊说明，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。
除非另外定义或由背景清楚指示，否则在本公开中的全部技术与科学术语具有如本公开所属领域的普通技术人员通常理解的相同含义。
近年来以免疫检查点抑制剂(ICIs)为代表的免疫治疗问世，进一步改善复发难治患者的预后，例如，免疫检查点抑制剂PD-1单抗明显改善了r/r cHL患者的预后，多个PD1单抗的客观缓解率(objective response rate，ORR)均在70％-80％以上，完全缓解(complete response，CR)20％-30％以上，中位10年无进展生存率(progression-free survival，PFS)大于12个月。
免疫检查点抑制剂(ICIs)在肿瘤治疗领域已展现卓越的疗效，程序性细胞死亡受体-1(PD-1)主要是存在于T细胞上的一种抑制性受体，与肿瘤细胞表达的程序性细胞死亡配体1(PD-L1)相互作用，抑制T细胞增殖、活化以及细胞因子的分泌，从而发生肿瘤免疫逃逸。越来越多的证据证明PD-1/PD-L1抑制剂在多种肿瘤治疗方面有巨大潜力。我国针对霍奇金淋巴瘤有效获批的PD-1/PD-L1抑制剂有：卡瑞利珠单抗(Camrelizumab)、替雷利珠单抗(islelizumab)、信迪利单抗(intilimab)、派安普利单抗(enpulimab)。
有研究表明，通过比对与筛选得到双歧杆菌属(Bifidobacterium)是与抗肿瘤免疫反应呈正相关的菌属，其中的长双歧杆菌(B.longum)和短双歧杆菌(B.breve)能够间接调节树突状细胞特定基因的表达，促进其成熟以及IFN-γ细胞因子分泌，进而增强CTLs在肿瘤组织浸润并增加其活性，提高抗肿瘤免疫反应。法国古斯塔夫·罗西研究所肿瘤研究中心的研究人员发现，抗生素治疗减弱非小细胞肺癌、肾细胞癌、尿路上皮癌患者的ICIs治疗效果，通过宏基因组测序比对与筛选发现对疗效影响最大的菌株是嗜黏蛋白-艾克曼菌 (A.muciniphila)，能够有效提高PD-1抑制剂对荷瘤小鼠的治疗效果。
脆弱拟杆菌(Bacteroides fragilis，B.fragilis)为革兰氏染色阴性、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，分为产肠毒素型(ETBF)和非产肠毒素型(NTBF)，是人及动物肠道正常菌群的一部分，正常寄居于人体呼吸道、胃肠道及泌尿生殖道粘膜等。申请人研究团队从2012年开始探索并从健康婴儿粪便中分离脆弱拟杆菌B.fragilis(菌株ZY-312)。但是目前没有利用该脆弱拟杆菌与免疫检查点抑制剂协同治疗淋巴瘤的报道。
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。
本发明在实施过程中所使用的微生物菌种已于2015年4月2日在中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)(北京市朝阳区北辰西路1号院3号)保藏。分类命名：脆弱拟杆菌ZY-312(bacteroides fragilis ZY-312)，保藏编号CGMCC No.10685。脆弱拟杆菌ZY-312由本发明申请单位自行分离获得，并且已经在授权专利保护(专利号201510459408.X)，按照专利审查指南的规定，公众能够从商业渠道买到或已经授权，不用保藏，即不用提供保藏证明。
作为本发明的主要目的，本发明提供脆弱拟杆菌和免疫检查点抑制剂在制备防治淋巴瘤的药物中的应用，所述脆弱拟杆菌的保藏编号为CGMCC  No.10685。
脆弱拟杆菌和免疫检查点抑制剂对淋巴瘤共同作用，降低促炎因子IL-6、IL-8、IL-2R、TNF-α、IL-21的水平，增加CD8+效应T细胞的迁移，减少Treg细胞的募集，降低原位肿瘤的重量，增加抑癌率。
在其中一个示例中，所述脆弱拟杆菌选自如下种类中的一种或者多种：脆弱拟杆菌的活菌，形态结构完整或者不完整的灭活的脆弱拟杆菌，经基因重组、改造或修饰、减毒、化学处理或者物理处理的脆弱拟杆菌，脆弱拟杆菌裂解物，脆弱拟杆菌液体培养上清液。
在其中一个示例中，所述免疫检查点抑制剂选自PD-1抗体、PD-L1抗体、PD-L2抗体、CTLA-4抗体、LAG-3抗体、TIM-3抗体、VISTA抗体和A2aR抗体中的一种或多种。
优选地，所述免疫检查点抑制剂为PD-1抗体或/和PD-L1抗体。
优选地，所述PD-1抗体选自纳武利尤单抗、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。
优选地，所述PD-L1抗体选自阿特朱单抗、阿维鲁单抗和度伐鲁单抗中的一种或者多种。
在其中一个示例中，所述淋巴瘤为T细胞淋巴瘤。
在其中一个示例中，所述药物包含所述脆弱拟杆菌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和免疫检查点抑制剂，所述脆弱拟杆菌的保藏编号为CGMCC No.10685。
在其中一个示例中，所述脆弱拟杆菌选自如下种类中的一种或者多种：脆弱拟杆菌的活菌，形态结构完整或者不完整的灭活的脆弱拟杆菌，经基因重组、改造或修饰、减毒、化学处理、物理处理的脆弱拟杆菌，脆弱拟杆菌裂解物，脆弱拟杆菌液体培养上清液。
在其中一个示例中，所述免疫检查点抑制剂选自PD-1抗体、PD-L1抗体、PD-L2抗体、CTLA-4抗体、LAG-3抗体、TIM-3抗体、VISTA抗体和A2aR抗体中的一种或多种。
优选地，所述免疫检查点抑制剂为PD-1抗体或/和PD-L1抗体。
优选地，所述PD-1抗体选自纳武利尤单抗、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。
优选地，所述PD-L1抗体选自阿特朱单抗、阿维鲁单抗和度伐鲁单抗中的一种或者多种。
在其中一个示例中，所述淋巴瘤为T细胞淋巴瘤。
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1-3mm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发酵培养8小时(温度为37℃)，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。
实施例2：脆弱拟杆菌活菌液的制备
(1)将菌种划线接种于血平皿，37℃、厌氧培养48h。
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1-3mm。
(2)增菌：从步骤(1)中选取单个菌落接种于TSB(胰蛋白胨大豆肉汤，含5％胎牛血清)中进行增菌培养，所得菌液保存备用。
(3)脆弱拟杆菌活菌液：将步骤(1)制备的菌液，用麦氏比浊管做菌数测 定，用生理盐水稀释至10 7CFU/ml和10 9CFU/ml，保存备用。
实施例3：脆弱拟杆菌的灭活菌粉的制备
(1)取实施例1制得的脆弱拟杆菌发酵液对发酵液进行离心处理，收集湿菌体，按菌体：生理盐水＝1:(10-30)(m:v)比例加入生理盐水对菌泥进行重悬洗涤，再次离心收集洗涤后的菌体。
(2)向步骤(1)所得菌体加入5wt％麦芽糊精+0.9wt％氯化钠混合成的赋形剂，按菌体：赋形剂＝1:(5-15)(m:m)比例进行添加，搅拌分散后在(70-100)±5℃热灭活(20-40)±5分钟，得灭活菌液。
(3)将步骤(2)所得的灭活菌液离心收集灭活菌泥。
(4)在步骤(3)收集的灭活菌泥中加入赋形剂使总重量与灭活前菌液重量一致，搅拌完全溶解，得灭活菌原液。
(5)将步骤(4)所得的灭活菌原液进行真空冷冻干燥，-40±2℃预冻1-3小时后，-20±2℃预冻0.5-1h，最后-40±2℃再预冻0.5-2h，0.25mbar真空度下经一次干燥(-5±2℃和0±2℃)、解析干燥(35±2℃)制备成灭活菌粉，菌粉的菌数达到1×10 11Cell/g以上。
实施例4：脆弱拟杆菌与PD-1抑制剂协同治疗EL4小鼠T细胞淋巴瘤系模型的药效实验
肿瘤局部微环境中浸润淋巴细胞对肿瘤的演进起着不容小觑的作用。肿瘤程序性细胞死亡受体-1(PD-1)可通过与其配体的结合抑制T细胞活化，从而实现肿瘤的免疫逃逸。肿瘤免疫反应中CD8+效应T细胞承担主要的肿瘤杀伤活性，而微环境中聚集的调节性T细胞起着肿瘤免疫抑制的作用。大量研究显示，不同类型的肿瘤患者外周血中的调节性T细胞数量远高于正常人群，FOXP3+CD4+CD25+Treg细胞被认为是抗肿瘤免疫治疗中的主要阻碍。
本实验观察了脆弱拟杆菌与PD-1抑制剂(BE0273，BioXcell)协同治疗EL4小鼠T细胞淋巴瘤系模型中CD8+效应T细胞、调节性T细胞(Treg细胞)、肿瘤重量、抑瘤率、小鼠生存率及相关细胞因子的变化情况。
一、EL4系T淋巴细胞瘤细胞培养
EL4细胞培养于含10％胎牛血清的DMEM/F12培养基中，培养温度为37℃，气体环境为5％CO 2/95％空气，湿度为饱和湿度；根据细胞生长的速度及培养液的颜色变化更换培养液，用0.25％的胰蛋白酶消化传代。根据细胞生长情况，将对数生长期的细胞制备成单细胞悬液，将细胞浓度调整至1×10 7/mL。
完成细胞系扩大培养后，在注射前计数保证每只小鼠量为5×10 5个EL4细胞。
二、小鼠肿瘤模型建立
(1)瘤体接种
实验用7周雄性C57B6/L小鼠，待小鼠自然生长1周后，进行瘤体接种。分别给除正常对照组外全部小鼠接种肿瘤细胞，取小鼠单侧腋下，6号针皮下注射0.2ml已计数细胞悬液，在吸取细胞悬液前，轻柔混合以确保每只小鼠接种的细胞数相同。观察小鼠肿瘤生长情况。
(2)建模成功标志
小鼠出现消瘦、弓背、精神萎靡等衰竭体征，小鼠接种部位可触及肿块。
三、小鼠体内实验
(1)实验分组
表1、分组和给药方案
Figure PCTCN2022120086-appb-000001
(2)瘤体接种7天后开始给药，并于3周给药时间结束后，统计各组小鼠生存时间，处死全部小鼠，无菌操作下剥离小鼠肿块及脾脏，剥离的每组肿块称重，分别研磨为细胞悬液，700目过滤网过滤组织残渣后，PBS洗涤2遍，离心计数，每组标本取两管细胞样本分别上机检测CD8+效应T细胞、Treg细胞水平及细胞因子的水平。
(3)观察指标
CD8+效应T细胞、调节性T细胞(Treg细胞)、肿瘤重量、抑瘤率及细胞因子IL-6、IL-8、IL-2R、TNF-α、IL-21等。
四、实验结果
(1)肿瘤重量及抑瘤率
表2、各组肿瘤重量及抑瘤率(
Figure PCTCN2022120086-appb-000002
n＝8)
组别 肿瘤重量(g) 抑瘤率(％)
模型组 8.04±2.5 -
PD-1抗体组 5.85±0.43* 36.18±0.077
ZY-312组 6.75±0.55 34.93±0.065
低剂量ZY-312+PD-1抗体 4.93±0.29** 68.82±0.074
中剂量ZY-312+PD-1抗体 5.00±0.21** 72.94±0.063
高剂量ZY-312+PD-1抗体 4.66±0.43** 74.16±0.109
低剂量ZY-312灭活菌+PD-1抗体 5.85±0.42* 65.86±0.091
中剂量ZY-312灭活菌+PD-1抗体 5.38±0.53* 69.17±0.086
高剂量ZY-312灭活菌+PD-1抗体 4.68±0.55** 71.80±0.061
注：与模型组相比，*表示P＜0.05，**表示P＜0.01，***表示P＜0.001
抑瘤率＝100％(模型组肿瘤平均重量-给药组肿瘤平均重量)/模型组肿瘤平均重量。
如表2所示，模型组的肿瘤平均重量显著高于其他组(P＜0.01)，ZY-312组稍高与PD-1组(P＜0.05)；低、中、高剂量脆弱拟杆菌ZY-312联合PD-1抑制剂组均低于PD-1抑制剂组(P＜0.05)，其中高剂量脆弱拟杆菌ZY-312联合PD-1抑制剂组瘤体重量最低；中、高剂量脆弱拟杆菌ZY-312灭活菌联合PD-1抑制剂组均低于PD-1抑制剂组(P＜0.05)，其中高剂量脆弱拟杆菌ZY-312灭活菌联合PD-1抑制剂组瘤体重量最低，低剂量ZY-312灭活菌联合PD-1抑制剂组虽稍低于PD-1组，但无统计学差异。
抑瘤率方面，各浓度脆弱拟杆菌ZY-312和灭活菌联合PD-1抑制剂组均高于单用PD-1抑制剂组(P＜0.05)，ZY-312组稍低于PD-1组，但无统计学差异。
(2)CD8+效应T细胞
使用流式细胞计数分别分析各组处理3周后脾脏和瘤体中CD8+效应T细胞占比，如表3所示。
在脾脏中，模型组高于生理盐水空白对照组，PD-1抑制剂组高于模型组，均有统计学差异(P＜0.01)，ZY-312组高于模型组(P＜0.05)，稍低于PD-1抑制剂组，但无统计学差异；低、中、高剂量脆弱拟杆菌ZY-312联合PD-1抑制剂组均高于PD-1抑制剂组(P＜0.05)，其中中剂量脆弱拟杆菌ZY-312联合PD-1抑制剂组CD8+效应T细胞占比最高；低、中、高剂量脆弱拟杆菌ZY-312灭活菌联合PD-1抑制剂组均高于PD-1抑制剂组(P＜0.05)，其中中剂量脆弱拟杆菌ZY-312灭活菌联合PD-1抑制剂组CD8+效应T细胞占比最高。
在瘤体中，模型组高于生理盐水空白对照组，PD-1抑制剂组高于模型组，均有统计学差异(P＜0.05)，ZY-312组高于模型组，但无统计学差异；低、中、高剂量脆弱拟杆菌ZY-312联合PD-1抑制剂组均高于PD-1抑制剂组(P＜0.05)，其高中剂量脆弱拟杆菌ZY-312联合PD-1抑制剂组CD8+效应T细胞占比最高；低、中、高剂量脆弱拟杆菌ZY-312灭活菌联合PD-1抑制剂组均高于PD-1抑制剂组(P＜0.05)，其高中剂量脆弱拟杆菌ZY-312灭活菌联合PD-1抑制剂组CD8+效应T细胞占比最高。
(3)调节性T细胞(Treg细胞)
使用流式细胞计数检测脾脏和瘤体中Treg细胞占比，如表3所示。
在脾脏中，模型组显著高于生理盐水空白对照组(P＜0.001)，PD-1抑制剂组低于模型组(P＜0.05)，ZY-312组与PD-1抑制剂组无统计学差异；低、中、高剂量脆弱拟杆菌ZY-312联合PD-1抑制剂组均低于PD-1抑制剂组(P＜0.05)，其中中剂量脆弱拟杆菌ZY-312联合PD-1抑制剂组Treg细胞占比最低；低、中、高剂量脆弱拟杆菌ZY-312灭活菌联合PD-1抑制剂组均低于PD-1抑制剂组(P＜0.05)，其中低剂量脆弱拟杆菌ZY-312灭活菌联合PD-1抑制剂组Treg细胞占比最低，但高于生理盐水空白对照组(P＜0.05)。
在瘤体中，模型组显著高于生理盐水空白对照组(P＜0.001)，PD-1抑制剂组低于模型组(P＜0.05)，ZY-312组与PD-1抑制剂组无统计学差异；低、中、高剂量脆弱拟杆菌ZY-312联合PD-1抑制剂组均低于PD-1抑制剂组(P＜0.05)，其中低剂量脆弱拟杆菌ZY-312联合PD-1抑制剂组CD8+效应T细胞占比最低；低、中、高剂量脆弱拟杆菌ZY-312灭活菌联合PD-1抑制剂组均低于PD-1抑制剂组(P＜0.05)。
表3、各组免疫细胞百分比(
Figure PCTCN2022120086-appb-000003
n＝8)
Figure PCTCN2022120086-appb-000004
注：与模型组相比，*表示P＜0.05，**表示P＜0.01，***表示P＜0.001。
(4)细胞因子
表4、各组细胞因子(
Figure PCTCN2022120086-appb-000005
n＝8)
Figure PCTCN2022120086-appb-000006
采用luminex技术检测EL4淋巴瘤小鼠模型中IL-6、IL-8、IL-2R、TNF-α、IL-21、IL-10等细胞因子的水平。如表4所示，相较于正常对照组，模型组中IL-6、IL-8、IL-2R、TNF-α、IL-21等促炎因子显著升高。PD-1抑制剂能够有效调节上述细胞因子，下调IL-6、IL-8、IL-2R、TNF-α、IL-21等促炎因子水平，在ZY-312组也观察到类似的效果，脆弱拟杆菌ZY-312能够加强PD-1抑制剂效果。
综上可知，本发明提供的脆弱拟杆菌与免疫检查点抑制剂联合使用，可以有效抑制淋巴瘤的生长，具有治疗淋巴瘤的显著效果。
以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. 脆弱拟杆菌和免疫检查点抑制剂在制备防治淋巴瘤的药物中的应用，所述脆弱拟杆菌的保藏编号为CGMCC No.10685。
  2. 根据权利要求1所述的应用，其特征在于，所述脆弱拟杆菌选自如下种类中的一种或者多种：脆弱拟杆菌的活菌，形态结构完整或者不完整的灭活的脆弱拟杆菌，经基因重组、改造或修饰、减毒、化学处理或者物理处理的脆弱拟杆菌，脆弱拟杆菌裂解物，脆弱拟杆菌液体培养上清液。
  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抗体、PD-L1抗体、PD-L2抗体、CTLA-4抗体、LAG-3抗体、TIM-3抗体、VISTA抗体和A2aR抗体中的一种或多种；
    优选地，所述免疫检查点抑制剂为PD-1抗体或/和PD-L1抗体；
    优选地，所述PD-1抗体选自纳武利尤单抗、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；
    优选地，所述PD-L1抗体选自阿特朱单抗、阿维鲁单抗和度伐鲁单抗中的一种或者多种。
  4. 根据权利要求1所述的应用，其特征在于，所述淋巴瘤为T细胞淋巴瘤。
  5. 根据权利要求1至4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。
  6. 根据权利要求5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。
  7. 根据权利要求1至4和6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；
    或/和，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。
  8. 一种防治淋巴瘤的药物，其特征在于，所述药物包含脆弱拟杆菌和免疫检查点抑制剂，所述脆弱拟杆菌的保藏编号为CGMCC No.10685。
  9. 根据权利要求8所述的防治淋巴瘤的药物，其特征在于，所述脆弱拟杆菌选自如下种类中的一种或者多种：脆弱拟杆菌的活菌，形态结构完整或者不完整的灭活的脆弱拟杆菌，经基因重组、改造或修饰、减毒、化学处理、物理处理的脆弱拟杆菌，脆弱拟杆菌裂解物，脆弱拟杆菌液体培养上清液。
  10. 根据权利要求8或者9所述的防治淋巴瘤的药物，其特征在于，所述免疫检查点抑制剂选自PD-1抗体、PD-L1抗体、PD-L2抗体、CTLA-4抗体、LAG-3抗体、TIM-3抗体、VISTA抗体和A2aR抗体中的一种或多种；
    优选地，所述免疫检查点抑制剂为PD-1抗体或/和PD-L1抗体；
    优选地，所述PD-1抗体选自纳武利尤单抗、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；
    优选地，所述PD-L1抗体选自阿特朱单抗、阿维鲁单抗和度伐鲁单抗中的一种或者多种。
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