CN112877268A - 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 - Google Patents
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 Download PDFInfo
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Abstract
本发明提供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,所述菌株命名为干酪乳杆菌Shanghai 2020Lactobacillus casei strain Shanghai 2020(下简称L.casei‑sh2020),保藏编号为CCTCC NO:M 2020474。本发明提供的干酪乳杆菌L.casei‑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CD8T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。
Description
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(programmeddeath-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。
从生态学角度看,人体是一个微生态系统。正常成年人体内大约有1014个微生物,是人体自身细胞数量的10倍,与人体共生,形成人体微生态。人体肠道内的微生物最多,约有1.5kg,以菌群为主,因此肠道微生物往往被称为肠道菌群。肠道菌群对宿主免疫系统有极其重要的调控作用,肠道菌群是推动免疫系统发育成熟和维持免疫稳态的重要基础。宿主通过肠道免疫细胞严格控制肠道菌群,减少组织炎症和细菌移位,人体免疫系统也因此受到了肠道菌群的刺激和训练。
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CD8 T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(Akkermansiamuciniphila)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+CD8 T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。这些差异意味着发现肠道菌群中新的影响ICI治疗效应的菌种,有助于有效地克服ICI耐药。
发明内容
本发明的目的在于,提供一种用于增强免疫检查点抑制剂治疗效应的干酪乳杆菌株,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,本发明提供了一种增强免疫检查点抑制剂治疗效应的干酪乳杆菌株,所述菌株命名为干酪乳杆菌Shanghai 2020 Lactobacillus casei strainShanghai 2020(下简称L.casei-sh2020),保藏编号为CCTCC NO:M 2020474。
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。
本发明干酪乳杆菌株L.casei-sh2020的菌粉的活菌数为1.0×1010~3.0×1011CFU/g。
所述干酪乳杆菌株L.casei-sh2020的菌粉的制备方法包括如下步骤:
将所述干酪乳杆菌株L.casei-sh2020接种在MRS培养液中在36~38℃下发酵12~36h,得到干酪乳杆菌株L.casei-sh2020发酵液;
将上述干酪乳杆菌株L.casei-sh2020发酵液离心,收集沉淀物,得到干酪乳杆菌株L.casei-sh2020菌泥;
将上述干酪乳杆菌株L.casei-sh2020菌泥进行真空冷冻干燥,得到干酪乳杆菌株L.casei-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,发现干酪乳酸菌可以显著提高PD-1抗体治疗肿瘤的效应,经全基因组测序分析,发现该干酪乳酸菌为新菌株,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抗体治疗效应的干酪乳酸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,本发明提供的干酪乳杆菌L.casei-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.补充干酪乳酸菌显著增强PD-1抗体的治疗效应。
图5.基因组测序分析发现该菌株为干酪乳酸菌新菌株。
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150-200mm3,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.来自健康供体的干酪乳酸菌新菌株L.casei-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,我们鉴定出一个能够高校扩增的多种乳酸菌。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.casei-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150-200mm3,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,补充干酪乳酸菌L.casei具有最显著的增强PD-1抗体的治疗效应(图4E-F);在抗生素清除肠道菌群的小鼠,给予干酪乳酸菌L.casei同样显著地增强PD-1抗体的治疗效应(图4G)。进一步,对干酪乳酸菌L.casei全基因测序,组装出来的基因组与NCBI数据中干酪乳酸菌比对分析,发现其序列与已知菌株相似度约为77.56%,该干酪乳酸菌为新菌株,命名为L.casei-sh2020(图5),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 (2)
1.一种增强免疫检查点抑制剂治疗效应的干酪乳杆菌株,其特征在于,所述菌株命名为干酪乳杆菌Shanghai 2020Lactobacillus casei strain Shanghai 2020,保藏编号为CCTCC NO:M 2020474。
2.权利要求1所述的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。
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