[go: up one dir, main page]

WO2022222591A1 - 一种增强免疫检查点抑制剂治疗效应的副干酪乳杆菌株及其应用 - Google Patents

一种增强免疫检查点抑制剂治疗效应的副干酪乳杆菌株及其应用 Download PDF

Info

Publication number
WO2022222591A1
WO2022222591A1 PCT/CN2022/077453 CN2022077453W WO2022222591A1 WO 2022222591 A1 WO2022222591 A1 WO 2022222591A1 CN 2022077453 W CN2022077453 W CN 2022077453W WO 2022222591 A1 WO2022222591 A1 WO 2022222591A1
Authority
WO
WIPO (PCT)
Prior art keywords
tumor
strain
antibody
paracasei
therapeutic effect
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/CN2022/077453
Other languages
English (en)
French (fr)
Inventor
王立顺
韩冰
张世龙
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shanghai Yaodan Biotechnology Co Ltd
Original Assignee
Shanghai Yaodan Biotechnology Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Shanghai Yaodan Biotechnology Co Ltd filed Critical Shanghai Yaodan Biotechnology Co Ltd
Publication of WO2022222591A1 publication Critical patent/WO2022222591A1/zh
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • 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
    • 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
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • 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
    • A23V2400/00—Lactic or propionic acid bacteria
    • A23V2400/11—Lactobacillus
    • A23V2400/125—Casei
    • 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 belongs to the field of biotechnology, and in particular relates to a Lactobacillus paracasei strain for increasing the therapeutic effect of an immune checkpoint inhibitor and its application.
  • Immune checkpoint inhibitors are monoclonal antibodies targeting the corresponding immune checkpoints, blocking the inhibitory effect of tumor cells on immune cells through immune checkpoints, enabling immune cells to kill tumor cells. effect.
  • ICIs are monoclonal antibodies targeting the corresponding immune checkpoints, blocking the inhibitory effect of tumor cells on immune cells through immune checkpoints, enabling immune cells to kill tumor cells. effect.
  • PD-1 programmed death-1
  • PD-L1 programmed death ligand-1
  • CTLA-4 Cytotoxic T-lymphocyte associated protein 4
  • the MD Anderson Cancer Center study found that those patients with high diversity of gut microbial species had a high response rate to PD-1 antibody therapy, a significant increase in CD8 + T lymphocytes, and Clostridiales bacteria and Faecalibacterium in their guts. Faecalibaterium); similarly, tumor-bearing mice transplanted with microbiota that responded to PD-1 antibody treatment had a better therapeutic effect on PD-1 antibody.
  • the University of Chicago also found that in patients with metastatic melanoma, the intestinal commensal microbiome was associated with the efficacy of PD-1 antibodies, and Bifidobaterium was significantly enriched in the intestinal flora.
  • the purpose of the present invention is to provide a Lactobacillus paracasei strain, which can effectively enhance the therapeutic effect of immune checkpoint inhibitors.
  • the second object of the present invention is to provide the application of the Lactobacillus paracasei strain in preparing food or medicine for enhancing the therapeutic effect of immune checkpoint inhibitor.
  • the present invention provides a Lactobacillus paracasei strain, which is named Lactobacillus paracasei Shanghai 2020 Lacticaseibacillus paracasei strain Shanghai 2020 (hereinafter referred to as L.paracasei-sh2020), and the deposit number is CCTCC NO:M 2020474.
  • the present invention provides the application of the Lactobacillus paracasei strain in preparing food or medicine for enhancing the therapeutic effect of immune checkpoint inhibitor.
  • the immune checkpoint inhibitor includes PD-1 antibody, PD-L1 antibody and CTLA-4 antibody.
  • the strain of the present invention is preserved in the China Collection Center for Type Cultures (CCTCC), and the preservation address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei province, and the preservation date is September 9, 2020.
  • the strain of the present invention was originally named "Lactobacillus casei strain Shanghai 2020" (application number 202110434508.2 application date: 2021-04-22), and after uploading the measured whole genome sequencing to the NCBI database, NCBI performed a comparison , in view of the higher homology between the strain and Lactobacillus paracasei, in this application, the name of the strain is modified to "Lactobacillus paracasei Shanghai 2020 Lacticaseibacillus paracasei strain Shanghai 2020", and issued by the China Collection of Type Cultures (CCTCC) Corresponding amendments have also been made to the deposit certificate of .
  • the viable count of the bacterial powder of the L. paracasei-sh2020 strain of the present invention is 1.0 ⁇ 10 10 to 3.0 ⁇ 10 11 CFU/g.
  • the preparation method of the bacterial powder of described Lactobacillus paracasei strain L.paracasei-sh2020 comprises the following steps:
  • Lactobacillus paracasei strain L.paracasei-sh2020 fermented liquid is centrifuged, and the sediment is collected to obtain the Lactobacillus paracasei strain L.paracasei-sh2020 bacterial slurry;
  • L. paracasei strain L. paracasei-sh2020 bacterial slurry was vacuum freeze-dried to obtain the bacterial powder of L. paracasei-sh2020 strain.
  • the advantage of the present invention is that the L. paracasei-sh2020 strain provided by the present invention can improve the intestinal microecology of tumor model mice, promote the expression of CXCL10 chemokine by tumor cells, and recruit and increase the lethality of tumor cells in tissue.
  • the infiltration and activation of CD8 T lymphocytes can effectively promote the therapeutic effect of PD-1 antibody and significantly inhibit tumor growth.
  • PD-1 antibody has better antitumor effect in tumor-bearing mice transplanted with healthy donor gut flora.
  • FIG. 1 Significantly increased infiltration of effector immune cells in tumor tissues of mice transplanted with healthy donor gut microbiota.
  • Lactobacillus paracasei enhances the efficacy of PD-1 antibody dependent on CD8 + T cells.
  • mice In C57BL/6 mice, an antibiotic combination (ATB) consisting of vancomycin, neomycin, metronidazole, and ampicillin was first treated for 7 days to clear the mouse gut microbiota; then the guts of healthy donors were administered 20 ⁇ l of microflora was administered by gavage for 10 days, and 20 ⁇ l of intestinal microflora of tumor patients was used as a control; then, mice were inoculated with MC38 cells, and tumor volume was measured twice a week after tumor inoculation, and tumor growth curves were drawn; after 7 days, tumors were 150-200 mm 3 in size, treated with PD-1 antibody for 21 days; 28 days after tumor seeding, tumor size was assessed, mice were sacrificed, and intestinal, blood, spleen, colon and stool samples were taken from the mice.
  • ATB antibiotic combination
  • Figure 1 shows that PD-1 antibody has better anti-tumor effect in tumor-bearing mice transplanted with healthy donor intestinal flora.
  • A-C After antibiotics cleared the intestinal flora of mice, PD-1 antibody lost tumor cells MC38 inhibitory effect;
  • D Construction of humanized tumor-bearing mice with gut microbiota and treatment procedure with PD-1 antibody;
  • E-G PD-1 antibody is better in mice transplanted with healthy donor gut microbiota antitumor effect.
  • FMT flora transplantation
  • C tumor patient
  • H healthy donor.
  • ATB antibiotic cocktail.
  • lymphocytes CD4 + T Fig. 2A
  • CD8 + T Fig. 2B
  • ICOS expression in CD4 + T
  • Fig. 2C CD8 + T
  • Fig. 2D CD8 + T
  • INF- ⁇ INF- ⁇ that promote antitumor effects
  • FIG. 2E The expression of CD8 + T ( FIG. 2E ) was significantly increased in the tumor tissues of mice transplanted with healthy donor gut flora, while Treg (CD4 + CD25 + FoxP3 + ) cells, which inhibited the antitumor effect, were not significantly changed.
  • Lactobacillus in healthy donors was significantly higher than that in tumor patients, and was positively correlated with the therapeutic effect of anti-PD-1 antibodies.
  • mice with gut microbiota constructed from the gut microbiota of the above tumor patients and healthy donors were collected.
  • 16S was used to sequence the intestinal flora in stool samples, and analyze the expression of cytokines and the number and classification of immune cells in mice treated with PD-1 antibody, to explore the relationship between the intestinal flora and the therapeutic effect of PD-1 antibody. Correlation.
  • Fig. 3A taxonomic evolutionary tree
  • Fig. 3B LDA score
  • Fig. 3C genus-level analysis
  • Fig. 3C relative abundance comparison
  • Example 3 A new strain of L. paracasei L. paracasei-sh2020 from healthy donors enhances the anti-tumor effect of PD-1 antibody
  • mice were inoculated with MC38 cells, and tumor volumes were measured twice a week after tumor inoculation, and plotted Tumor growth curve; 7 days after tumor size 150-200mm 3 , PD-1 antibody treatment was given for 21 days; 28 days after tumor seeding, tumor size was assessed, mice were sacrificed and mouse intestinal, blood, spleen, colon and stool samples were taken .
  • ARB antibiotic combination
  • L. paracasei-sh2020 significantly enhanced the therapeutic effect of PD-1 antibody, promoted CD8 + T cell infiltration and induced CD8 + T cell-dependent antitumor immunity.
  • L. paracasei-sh2020 The Lactobacillus strain is a new strain, named L. paracasei-sh2020 (Fig. 6), and has been sent to the China Center for Type Culture Collection for preservation under the number CCTCC NO: M 2020474.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Microbiology (AREA)
  • Mycology (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Genetics & Genomics (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Biotechnology (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Epidemiology (AREA)
  • Biomedical Technology (AREA)
  • Virology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Nutrition Science (AREA)
  • Food Science & Technology (AREA)
  • Polymers & Plastics (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

提供了一种增强免疫检查点抑制剂治疗效应的副干酪乳杆菌株及其应用，所述菌株的保藏编号为CCTCC NO：M 2020474，该菌株能够募集和增加肿瘤细胞组织内杀伤性CD8 T淋巴细胞的浸润和活化，促进PD-1抗体的治疗效应。

Description

一种增强免疫检查点抑制剂治疗效应的副干酪乳杆菌株及其应用 技术领域
本发明属于生物技术领域，具体涉及一种用于增加免疫检查点抑制剂治疗效应的副干酪乳杆菌株及其应用。
背景技术
免疫检查点抑制剂(immune checkpoint inhibitor,ICI)，是针对相应免疫检查点的单抗类药物，阻断肿瘤细胞通过免疫检查点对免疫细胞的抑制作用，使免疫细胞能够发挥对肿瘤细胞的杀伤作用。目前有3类ICI批准上市，包括程序性死亡分子-1(programmed death-1,PD-1)单抗、程序性死亡配体-1(programmed death ligand-1,PD-L1)单抗、以及细胞毒性T淋巴细胞相关蛋白-4(cytotoxic T-lymphocyte associated protein 4,CTLA-4)单抗。ICI自问世以来用于治疗多种肿瘤，在20％-30％的患者中取得了肯定的疗效，部分晚期肿瘤患者经过ICI治疗后甚至完全缓解，能够长期生存，成为肿瘤免疫治疗历史上重要的里程碑，为恶性肿瘤患者带来新的希望。但是，大部分肿瘤患者ICI初始治疗无效，或在初始治疗后继发耐药，发现克服ICI耐药的方法，进一步提高ICI疗效，成为肿瘤学治疗领域的难点。
近几年的研究表明，患者肠道菌群是导致PD-1抗体等ICI疗效个体差异的原因之一。2020年12月，Science在线刊发了以色列特拉维夫大学医学中心研究发现：10名对PD-1抗体治疗无响应的患者移植了对PD-1抗体治疗有积极响应患者的肠道菌群后，有3名患者对PD-1抗体治疗出现积极响应，其中1名晚期患者达到完全缓解，这些患者肿瘤组织内CD8 +T淋巴细胞显著增加。这3名患者都接受了同一个供体的肠道菌群，而接受另1位供体肠道菌群的五名患者没有效应。2021年2月，《Science》发表了UPMC Hillman癌症中心和美国国家癌症研究所的研究成果：改变肠道菌群可以提高晚期黑色素瘤患者的免疫治疗效果。在这项研究中，研究人员对抗PD-1抗体无效的黑色素瘤患者进行了肠道菌群移植和PD-1抗体免疫治疗，在接受菌群移植和PD-1抗体联合治疗的15名晚期黑色素瘤患者中，有6名肿瘤减少或疾病稳定持续了超过一年。这些应答者肿瘤微环境中的免疫CD8T细胞激活增加，而非应答者的免疫抑制细胞增加。
近年来人们从肠道菌群中分离到了多个菌株，可以促进PD-1抗体的抗肿瘤效应。法国免疫学家研究了249名应用PD-1抗体的肿瘤患者，发现接受PD-1抗体治疗前或期间服用抗生素，其PD-1抗体治疗效果显著下降。移植对PD-1抗体有良好响应者肠道菌群的小鼠，对PD-1抗体的响应更好。PD-1治疗响应者肠道菌群中阿克曼菌(Akkermansia muciniphila)显著富集。对PD-1抗体响应较差的小鼠通过喂食阿克曼菌可以产生好的响应，其肿瘤微环境中效应淋巴细胞显著增加。MD Anderson癌症中心研究发现，那些肠道微生物种类多样性高的患者对PD-1抗体治疗响应率高，CD8 +T淋巴细胞显著增加，其肠道中梭菌目细菌(Clostridiales bacteria)和粪杆菌(Faecalibaterium)含量高；类似的，移植了响应PD-1抗体治疗患者菌群的荷瘤小鼠对PD-1抗体有更好的治疗效应。美国芝加哥大学也发现，在转移性黑色素瘤患者中，患者肠道共生微生物组与PD-1抗体的疗效相关，其肠道菌群中双歧杆菌(Bifidobaterium)等显著富集。紧接着，Nature报道了日本科学家团队鉴定出能够协同增强免疫检查点抑制剂作用的肠道菌群中11种细菌菌株“组合”(11-mix)。他们在小鼠试验中发现口服11-mix可以增加肿瘤微环境中IFN-γ +CD8T细胞，提高免疫检查点抑制剂(PD-1抗体、CTLA-4抗体)的抗肿瘤效果。
尽管临床前的动物实验和临床小样本的研究发现肠道菌群影响ICI的治疗效应，但是肠道菌群影响ICI的治疗效应的机制还很不清晰。不同供体肠道菌群对ICI效应的影响不同；同一个供体的菌群移植给不同受体后，对ICI治疗效应的影响也不相同；同时，来自法国、美国和日本的科学家分离获得的影响ICI治疗效应的肠道细菌也完全不同。这些差异意味着深入研究肠道菌群如何影响PD-1抗体等ICI制剂的治疗效应，发现肠道菌群中新的影响ICI治疗效应的菌种，解析其分子机制，对于有效地克服ICI耐药有重要意义。
发明内容
本发明的目的在于，提供一种副干酪乳杆菌株，所述菌株能够有效增强免疫检查点抑制剂治疗效应。
本发明的第二个目的在于，提供所述副干酪乳杆菌株在制备增强免疫检查点抑制剂治疗效应的食品或药物中的应用。
为了实现上述目的，本发明提供了一种副干酪乳杆菌株，所述菌株命名为 副干酪乳杆菌Shanghai 2020 Lacticaseibacillus paracasei strain Shanghai 2020(下简称L.paracasei-sh2020)，保藏编号为CCTCC NO：M 2020474。
为了实现上述第二个目的，本发明提供了所述副干酪乳杆菌株在制备增强免疫检查点抑制剂治疗效应的食品或药物中的应用。
作为一个优选方案，所述免疫检查点抑制剂包括PD-1抗体、PD-L1抗体和CTLA-4抗体。
本发明菌株保藏于中国典型培养物保藏中心(CCTCC)，保藏地址：湖北省武汉市武昌区八一路299号，保藏日期为2020年09月09日。本发明菌株原命名为“干酪乳杆菌Shanghai 2020 Lactobacillus casei strain Shanghai 2020”(申请号202110434508.2申请日：2021-04-22)，将所测得的全基因组测序上传至NCBI数据库后，NCBI进行比对，鉴于该菌株与副干酪乳杆菌的同源性更高，所以在本申请中该菌株命名修改为“副干酪乳杆菌Shanghai 2020Lacticaseibacillus paracasei strain Shanghai 2020”，并且中国典型培养物保藏中心(CCTCC)出具的保藏证明中也进行了相应修改(保藏证明中保藏号和保藏日期没有变化)。
本发明副干酪乳杆菌株L.paracasei-sh2020的菌粉的活菌数为1.0×10 10～3.0×10 11CFU/g。
所述副干酪乳杆菌株L.paracasei-sh2020的菌粉的制备方法包括如下步骤：
将所述副干酪乳杆菌株L.paracasei-sh2020接种在MRS培养液中在36～38℃下发酵12～36h，得到副干酪乳杆菌株L.paracasei-sh2020发酵液；
将上述副干酪乳杆菌株L.paracasei-sh2020发酵液离心，收集沉淀物，得到副干酪乳杆菌株L.paracasei-sh2020菌泥；
将上述副干酪乳杆菌株L.paracasei-sh2020菌泥进行真空冷冻干燥，得到副干酪乳杆菌株L.paracasei-sh2020的菌粉。
研究发现来自健康供体的肠道菌群可以增强抗PD-1抗体治疗肿瘤的效应，健康供体肠道菌群中乳酸菌显著高于肿瘤患者，并与抗PD-1抗体的治疗效应正相关；我们进一步从健康供体肠道菌群中分离获得了多株乳酸菌，并将该菌给予移植了肿瘤患者肠道菌群的荷瘤小鼠，发现副干酪乳杆菌可以显著提 高PD-1抗体治疗肿瘤的效应，经全基因组测序分析，发现该副干酪乳杆菌为新菌株，已送中国典型培养物保藏中心保藏。我们发现的影响抗PD-1抗体治疗效应的副干酪乳杆菌完全不同于国际上其他团队发现的阿克曼、粪杆菌、双歧杆菌等肠道菌。
本发明的优点在于，本发明提供的副干酪乳杆菌株L.paracasei-sh2020能够改善肿瘤模型小鼠的肠道微生态，促进肿瘤细胞表达CXCL10趋化因子，募集和增加肿瘤细胞组织内杀伤性CD8 T淋巴细胞的浸润和活化，高效促进PD-1抗体的治疗效应，显著抑制肿瘤的生长。
附图说明
图1.PD-1抗体在移植健康供体肠道菌群的荷瘤小鼠有更好的抗肿瘤效应。
图2.在移植健康供体肠道菌群的小鼠肿瘤组织中效应免疫细胞的浸润显著增多。
图3.健康供体肠道中乳酸菌增高，并与免疫杀伤细胞正相关。
图4.补充副干酪乳杆菌显著增强PD-1抗体的治疗效应。
图5.副干酪乳杆菌增强PD-1抗体疗效依赖CD8 +T细胞。
图6.基因组测序分析发现该菌株为副干酪乳杆菌新菌株。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白，以下结合附图及实施例，对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅仅用以解释本发明，并不用于限定本发明。
除特殊说明的之外，各实施例及试验例中所用的设备和试剂均可从商业途径得到。
实施例1.来自健康供体的肠道菌群增强PD-1抗体的抗肿瘤效应
在C57BL/6小鼠，先用万古霉素、新霉素、甲硝唑和氨苄青霉素组成的抗生素组合(ATB)处理7天，清除小鼠肠道菌群；然后给予健康供体的肠道菌群20μl灌胃10天，以肿瘤患者的肠道菌群20μl灌胃作为对照；接着，给小鼠接种MC38细胞，种瘤后每周两次测量肿瘤体积，绘制肿瘤生长曲线；7天后肿瘤大小150-200mm 3，给予PD-1抗体治疗21天；种瘤后28天，评估肿瘤大 小，处死小鼠并取小鼠肠道、血液、脾脏、结肠和粪便样本。
图1为PD-1抗体在移植健康供体肠道菌群的荷瘤小鼠有更好的抗肿瘤效应，(A-C)在抗生素清除小鼠肠道菌群后，PD-1抗体失去肿瘤细胞MC38抑制效应；(D)肠道菌群人源化荷瘤小鼠的构建及PD-1抗体治疗程序；(E-G)PD-1抗体在移植健康供体肠道菌群的小鼠有更好的抗肿瘤效应。FMT,菌群移植；C,肿瘤患者；H，健康供体。ATB，抗生素混合物。我们发现PD-1抗体在正常小鼠可以显著抑制肿瘤增长(图1A)，在应用在抗生素清除小鼠肠道菌群后，PD-1抗体失去对肿瘤细胞MC38的抑制效应(图1B-C)；(D)而利用肿瘤患者肠道菌群移植建立的肠道菌群人源化荷瘤小鼠中，应用PD-1抗体，其肿瘤增殖速度和体积显著高于健康供体肠道菌群构建的小鼠(图1D-F)，其生存期显著低于健康供体肠道菌群构建的小鼠(图1G)。
我们进一步发现促进抗肿瘤效应的淋巴细胞CD4 +T(图2A)、CD8 +T(图2B)、ICOS在CD4 +T(图2C)和CD8 +T的表达(图2D)、以及INF-γ在CD8 +T的表达(图2E)在移植健康供体肠道菌群的小鼠肿瘤组织中显著增加，而抑制抗肿瘤效应的Treg(CD4 +CD25 +FoxP3 +)细胞无明显变化。*P<0.05,**P<0.01,***P<0.001。这些结果提示来自健康供体的肠道菌群可以增强抗PD-1抗体治疗肿瘤的效应。
实施例2.肠道菌群与PD-1抗体的治疗效应的相关性
健康供体肠道菌群中乳酸菌显著高于肿瘤患者，并与抗PD-1抗体的治疗效应正相关。
收集上述肿瘤患者和健康供体肠道菌群构建的肠道菌群人源化小鼠的肠道、血液、脾脏、结肠和粪便样本。用16S测序粪便样本中肠道菌群，并分析其PD-1抗体的治疗后小鼠细胞因子的表达情况以及免疫细胞数量和分类，探索肠道菌群中与PD-1抗体的治疗效应的相关性。我们发现肠道菌群的分类进化树(图3A)、LDA评分(图3B)、属水平分析(图3C)、相对丰度比较(图3D)均提示健康供体的肠道中乳酸菌显著增高；并且免疫细胞与肠道菌群的相关性分析(图3E-I)提示肠道中乳酸菌的数量与免疫杀伤细胞正相关。
实施例3.来自健康供体的副干酪乳杆菌新菌株L.paracasei-sh2020增强PD-1抗体的抗肿瘤效应
利用MRS乳酸菌选择性培养基，对PD-1抗体有良好促进效应的健康供体的粪便样本进行分离培养，我们获得能够高效扩增的多株乳酸菌。接着，我们在C57BL/6小鼠，先用万古霉素、新霉素、甲硝唑和氨苄青霉素组成的抗生素组合(ATB)处理7天，清除小鼠肠道菌群；然后给予培养的L.paracasei-sh202020μl灌胃10天，以其他乳酸菌种以及健康供体的肠道菌群各20μl灌胃作为对照；接着，给小鼠接种MC38细胞，种瘤后每周两次测量肿瘤体积，绘制肿瘤生长曲线；7天后肿瘤大小150-200mm 3，给予PD-1抗体治疗21天；种瘤后28天，评估肿瘤大小，处死小鼠并取小鼠肠道、血液、脾脏、结肠和粪便样本。我们发现用抗生素清除肠道菌群后，制作肠道菌群人源化荷瘤小鼠，给予乳酸菌混合物显著增强PD-1抗体的治疗效应(图4A-D)；在肿瘤患者肠道菌群人源化荷瘤小鼠，补充副干酪乳杆菌株L.paracasei-sh2020具有最显著的增强PD-1抗体的治疗效应(图4E)；在抗生素清除肠道菌群的小鼠，给予副干酪乳杆菌株L.paracasei-sh2020同样显著地增强PD-1抗体的治疗效应(图4F)。接下来，使用流式细胞术检查对照和副干酪乳杆菌株L.paracasei-sh2020处理的肿瘤的免疫细胞浸润，结果显示L.paracasei-sh2020处理的肿瘤中CD8 +T细胞显著扩增，而其它免疫细胞未发现明显变化(图5A)。此外，在L.paracasei-sh2020处理后，IFNγ +CD8 +T细胞显著增加(图5B-C)。为了确定L.paracasei-sh2020增强PD-1抗体功效需要哪些T细胞亚群，使用针对CD8、CD4的体内中和抗体耗尽了CD4 +或CD8 +T细胞(图5D)。CD8 +T细胞的消耗完全消除了L.paracasei-sh2020的作用，表明CD8 +T细胞对于L.paracasei-sh2020增强PD-1抗体的抗肿瘤免疫是必不可少的。相反，CD4 +T细胞的消耗不影响副干酪乳杆菌株L.paracasei-sh2020联合PD-1抗体的抗肿瘤作用(图5E)。总的来说，L.paracasei-sh2020显著增强PD-1抗体的治疗效应，促进CD8 +T细胞浸润并诱导CD8 +T细胞依赖性抗肿瘤免疫。
进一步，对副干酪乳杆菌株L.paracasei-sh2020全基因测序，组装出来的基因组与NCBI数据中副干酪乳杆菌比对分析，发现其序列与已知菌株相似度约为77.56％，该副干酪乳杆菌株为新菌株，命名为L.paracasei-sh2020(图6)，已送中国典型培养物保藏中心保藏，编号CCTCC NO：M 2020474。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (3)

  1. 一种副干酪乳杆菌株，其特征在于，所述菌株命名为副干酪乳杆菌Shanghai 2020 Lacticaseibacillus paracasei strain Shanghai 2020，保藏编号为CCTCC NO：M 2020474。
  2. 权利要求1所述的副干酪乳杆菌株在制备增强免疫检查点抑制剂治疗效应的食品或药物中的应用。
  3. 根据权利要求2所述的副干酪乳杆菌株在制备增强免疫检查点抑制剂治疗效应的食品或药物中的应用，其特征在于，所述免疫检查点抑制剂包括PD-1抗体、PD-L1抗体和CTLA-4抗体。
PCT/CN2022/077453 2021-04-22 2022-02-23 一种增强免疫检查点抑制剂治疗效应的副干酪乳杆菌株及其应用 Ceased WO2022222591A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202110434508.2A CN112877268A (zh) 2021-04-22 2021-04-22 一种增强免疫检查点抑制剂治疗效应的干酪乳杆菌株及其应用
CN202110434508.2 2021-04-22

Publications (1)

Publication Number Publication Date
WO2022222591A1 true WO2022222591A1 (zh) 2022-10-27

Family

ID=76040091

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2022/077453 Ceased WO2022222591A1 (zh) 2021-04-22 2022-02-23 一种增强免疫检查点抑制剂治疗效应的副干酪乳杆菌株及其应用

Country Status (2)

Country Link
CN (2) CN112877268A (zh)
WO (1) WO2022222591A1 (zh)

Families Citing this family (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112877268A (zh) * 2021-04-22 2021-06-01 上海耀旦生物科技有限公司 一种增强免疫检查点抑制剂治疗效应的干酪乳杆菌株及其应用
CN114164148B (zh) * 2021-11-29 2022-11-25 天津科技大学 一株马乳酒样乳杆菌、菌剂及其应用
CN114657086A (zh) * 2021-12-27 2022-06-24 苏州善佰生物技术有限公司 一种双歧杆菌的制备方法及其在肿瘤治疗中的应用
CN114306616B (zh) * 2022-01-12 2023-04-28 广州知易生物科技有限公司 脆弱拟杆菌和免疫检查点抑制剂的新应用
CN115414390B (zh) * 2022-08-30 2024-08-23 复旦大学附属中山医院 具有改善肠道微生态性能和增强肿瘤免疫检查点抑制剂治疗效果的益生菌复合制剂及应用
CN115747097B (zh) * 2022-09-28 2025-02-11 复旦大学附属中山医院 一种Bifidobacterium bifidum菌株及其在肿瘤辅助治疗中的应用
CN117987297B (zh) * 2023-08-11 2024-06-14 合肥瀚微生物科技有限公司 一株肠道菌及其在抗肿瘤免疫治疗中的应用

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017129769A1 (en) * 2016-01-28 2017-08-03 INSERM (Institut National de la Santé et de la Recherche Médicale) Methods for enhancing the potency of the immune checkpoint inhibitors
WO2018222969A1 (en) * 2017-06-02 2018-12-06 Board Of Regents, The University Of Texas System Specific bacterial species and metabolite that improves immune checkpoint inhibitor therapy efficacy
CN109771445A (zh) * 2019-03-20 2019-05-21 青岛东海药业有限公司 酪酸梭菌在制备诱导抗肿瘤免疫及免疫检查点抑制剂增敏制剂中的应用
CN109966320A (zh) * 2019-04-26 2019-07-05 青岛东海药业有限公司 凝结芽孢杆菌在制备诱导抗肿瘤免疫及免疫检查点抑制剂增敏制剂中的应用
CN112334141A (zh) * 2018-06-14 2021-02-05 株式会社明治 用于促进免疫检查点抑制疗法的组合物
CN112877268A (zh) * 2021-04-22 2021-06-01 上海耀旦生物科技有限公司 一种增强免疫检查点抑制剂治疗效应的干酪乳杆菌株及其应用

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP7542946B2 (ja) * 2016-09-27 2024-09-02 ボード オブ リージェンツ， ザ ユニヴァーシティー オブ テキサス システム マイクロバイオームをモジュレートすることにより、免疫チェックポイント遮断療法を増強するための方法
CN110305821B (zh) * 2019-08-28 2019-12-17 鲁东大学 副干酪乳杆菌配合car-t细胞治疗应用
CN111560330B (zh) * 2020-05-12 2022-04-26 天津科技大学 一种具有免疫调节、抗炎和抗宫颈癌作用的干酪乳杆菌及应用

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017129769A1 (en) * 2016-01-28 2017-08-03 INSERM (Institut National de la Santé et de la Recherche Médicale) Methods for enhancing the potency of the immune checkpoint inhibitors
WO2018222969A1 (en) * 2017-06-02 2018-12-06 Board Of Regents, The University Of Texas System Specific bacterial species and metabolite that improves immune checkpoint inhibitor therapy efficacy
CN112334141A (zh) * 2018-06-14 2021-02-05 株式会社明治 用于促进免疫检查点抑制疗法的组合物
CN109771445A (zh) * 2019-03-20 2019-05-21 青岛东海药业有限公司 酪酸梭菌在制备诱导抗肿瘤免疫及免疫检查点抑制剂增敏制剂中的应用
CN109966320A (zh) * 2019-04-26 2019-07-05 青岛东海药业有限公司 凝结芽孢杆菌在制备诱导抗肿瘤免疫及免疫检查点抑制剂增敏制剂中的应用
CN112877268A (zh) * 2021-04-22 2021-06-01 上海耀旦生物科技有限公司 一种增强免疫检查点抑制剂治疗效应的干酪乳杆菌株及其应用

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
CHEN PING, LIN JIANGUANG, DAI YANGBIN, ZHAO AIYUE, DAI YIJUN, XU TIANWEN: "Progress in understanding the relationship between gut microbiota and immune checkpoint inhibitors ", CHINESE JOURNAL OF CLINICAL ONCOLOGY, TIANJIN SHI YIXUE ZAZHISHE, TIANJIN, CH, vol. 46, no. 24, 30 December 2019 (2019-12-30), CH , pages 1292 - 1296, XP055977952, ISSN: 1000-8179, DOI: 10.3969/j.issn.1000-8179.2019.24.239 *
JIN YUEPING, DONG HUI, XIA LILIANG, YANG YI, ZHU YONGQIANG, SHEN YAN, ZHENG HUAJUN, YAO CHENGCHENG, WANG YING, LU SHUN: "The Diversity of Gut Microbiome is Associated With Favorable Responses to Anti–Programmed Death 1 Immunotherapy in Chinese Patients With NSCLC", JOURNAL OF THORACIC ONCOLOGY, ELSEVIER INC., US, vol. 14, no. 8, 1 August 2019 (2019-08-01), US , pages 1378 - 1389, XP055809261, ISSN: 1556-0864, DOI: 10.1016/j.jtho.2019.04.007 *
VYARA MATSON ET AL.: "The commensal microbiome is associated with anti-PD-1 efficacy in metastatic melanoma patients", SCIENCE, vol. 359, no. 6371, 5 January 2018 (2018-01-05), XP055564226, DOI: 10.1126/science.aao3290 *

Also Published As

Publication number Publication date
CN114540229B (zh) 2024-10-01
CN112877268A (zh) 2021-06-01
CN114540229A (zh) 2022-05-27

Similar Documents

Publication Publication Date Title
WO2022222591A1 (zh) 一种增强免疫检查点抑制剂治疗效应的副干酪乳杆菌株及其应用
EP3795676A1 (en) Application of b. fragilis or akkermansia muciniphila in preparation of drug for preventing or treating tumor
US20220331385A1 (en) Anti-cancer oncolytic virus combination therapies and elite responder selection platforms
CN109966320B (zh) 凝结芽孢杆菌在制备诱导抗肿瘤免疫及免疫检查点抑制剂增敏制剂中的应用
CN105087488A (zh) 一种肿瘤抗原诱导的dc-cik细胞的制备方法及应用
CN109771445B (zh) 酪酸梭菌在制备诱导抗肿瘤免疫及免疫检查点抑制剂增敏制剂中的应用
CN116731894B (zh) 一种巨球形菌菌株及其应用
CN114917252A (zh) 毛螺菌科微生物菌株、预防和/或治疗肿瘤的药物及应用
CN116103205B (zh) 一种粘液玫瑰单胞菌、菌剂和胞外多糖及其制备方法和应用
CN114452382B (zh) 脆弱拟杆菌荚膜多糖a与pd-1及pd-l1抗体联合治疗呼吸系统肿瘤的应用
CN117018036B (zh) 巨球形菌属细菌在制备治疗癌症的药物中的应用
CN117987297B (zh) 一株肠道菌及其在抗肿瘤免疫治疗中的应用
CN114404455A (zh) 脆弱拟杆菌及其两性离子荚膜多糖在制备用于治疗呼吸系统肿瘤药物中的应用
CN115806893A (zh) 普通拟杆菌及其组合物在辅助癌症免疫治疗中的应用
EP4201416A1 (en) Use of bacterium in preparation of synergist of immune checkpoint inhibitor
Miyaguchi et al. Treatment with Lactobacillus retards the tumor growth of head and neck squamous cell carcinoma cells inoculated in mice
CN114344340A (zh) 脆弱拟杆菌与pd-1及pd-l1抗体联合用药治疗呼吸系统肿瘤中的应用
CN114796284A (zh) 微生物菌株、预防或治疗肿瘤的药物及应用
CN117089490B (zh) 青春双歧杆菌basj001及应用
Xu et al. Overcoming suppression of antitumor immune reactivity in tumor-bearing rats by treatment with bleomycin
CN106479973A (zh) 一种体外iak免疫细胞培养方法
CN115747097B (zh) 一种Bifidobacterium bifidum菌株及其在肿瘤辅助治疗中的应用
CN111991427A (zh) 肠道细菌在制备用于促进TCR γδ+T细胞增殖的药物中的应用
CN110205293A (zh) 一种加强型高效治疗肺癌的nk免疫细胞的制备方法及应用
Halle-Pannenko et al. Comparison of various BCG preparations in the EORTC—ICIG experimental screening for systemic immunity adjuvants applicable to cancer immunotherapy

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22790701

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 22790701

Country of ref document: EP

Kind code of ref document: A1

122 Ep: pct application non-entry in european phase

Ref document number: 22790701

Country of ref document: EP

Kind code of ref document: A1

32PN Ep: public notification in the ep bulletin as address of the adressee cannot be established

Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 112(1) EPC (EPO FORM 1205A DATED 04/04/2024)

122 Ep: pct application non-entry in european phase

Ref document number: 22790701

Country of ref document: EP

Kind code of ref document: A1