CN117503802A - Ac菌在抗肿瘤免疫治疗中的应用 - Google Patents
Ac菌在抗肿瘤免疫治疗中的应用 Download PDFInfo
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Abstract
本发明涉及癌症的免疫治疗领域。具体的,本发明涉及肠道特定菌Anaerotruncus colihominis对抗肿瘤免疫的作用,并提供提高患者肿瘤免疫治疗响应率的方法。本发明首次提示Anaerotruncus colihominis能够用于预防或治疗肿瘤,丰富了本领域的选择,具有巨大的临床应用价值。
Description
技术领域
本发明涉及癌症的免疫治疗领域。具体的,本发明涉及肠道特定菌Anaerotruncus colihominis对抗肿瘤免疫的作用,并提供提高患者肿瘤免疫治疗响应率的方法。
背景技术
肠道菌群包含至少38万亿个细菌,对于维持体内平衡和健康至关重要。肠道菌群被认为是一种微生物人体器官,是人类基因组的100倍,参与免疫调节、代谢功能等。许多研究表明,包括癌症在内的许多疾病可能是由微生物群失调引起的。重要的是,微生物病原体在高百分比(约20%)的癌症中引起肿瘤发生。因此,研究这些微生物病原体在肿瘤发生过程中的作用是非常重要的。
Paul A. Lawson等人2004年对从人类粪便中分离出的两株未确定的革兰氏阳性、厌氧、不形成孢子的棒状细菌进行了表型和系统发育学研究。这些生物是过氧化氢酶阴性的,产生乙酸和丁酸作为代谢的最终产物,DNA G+C含量约为54 mol%。比较16S rRNA基因测序结果表明,这两个分离株亲缘关系密切,在柔嫩梭菌(Clostridium leptum)rRNA群中形成了一个迄今未知的亚谱系。根据系统发育和表型证据,该未知细菌应被分类为大肠杆菌厌氧菌属(Anaerotruncus colihominis gen. nov., sp. nov.),模式菌株为WAL 14565T=CCUG 45055T=CIP 107754T(Lawson, PA et al. Anaerotruncus colihominis gen. nov.,sp nov., from human faeces. International Journal of Systematic andEvolutionary Microbiology (2004), 54, 413–417)。
目前为止,NCBI上也仅公开一株Anaerotruncus colihominis DSM 17241(=WAL14565T=CCUG 45055T=CIP 107754T)。现有技术中也没有专门研究 Anaerotruncus colihominis的专利,没有明确的Anaerotruncus colihominis治疗肿瘤的启示。
发明内容
本发明提供一种肠道菌,能够通过增强CD8T细胞效应因子分泌能力,从而增加免疫检查点阻断疗法的疗效,达到改善肿瘤患者生存期的目的。
本发明是通过以下技术方案实现的:
本发明保护微生物菌株,或所述微生物菌株的培养物或其加工物在制备用于预防或治疗肿瘤的药物中的应用,所述微生物菌株属于Anaerotruncus colihominis种。
在本说明书中,术语“培养物”指在适合Anaerotruncus colihominis存活和/或生长条件下悬于培养基的Anaerotruncus colihominis群体。如本领域普通技术人员所清楚了解的,在一些方面,本文所用的这些术语涉及包含Anaerotruncus colihominis群体和该群体在其中悬浮的培养基的组合。在另一方面,本文所用的这些术语还涉及指将本发明的Anaerotruncus colihominis的培养完成后的培养上清液和培养成分。在本发明中,所述培养物包括但不限于:Anaerotruncus colihominis接种或移植于任意形态(液体或固体)的培养基中所得到的含菌溶液、培养上清或含菌培养基。
在本说明书中,术语“加工物”是指只要来自于培养物则不作特别限定,可以通过例如培养物的浓缩、糊化、喷雾干燥、冻结干燥、真空干燥、滚筒干燥、液化、稀释、粉碎等加工而得到。在这些加工中,可以适当使用公知的方法。
在本发明中,所述培养物中或所述加工物中,本发明的微生物菌株可以是活菌,也可以是死菌。
在本说明书中,术语“菌株”可以是由保藏的菌株直接培养获得,也可以是子代菌株(后代)或从原始菌株培养(亚克隆菌株)的菌株。
优选地,所述预防或治疗肿瘤包括但不限于如下至少一种表型:
(a)在体外促进CD8T细胞效应因子TNF-α和/或IFN-γ的分泌;
(b)在体内促进肠道固有层淋巴细胞中CD8T细胞的效应因子TNF-α和/或IFN-γ的分泌;
(c)在体内促进肿瘤浸润淋巴细胞中CD8T细胞的效应因子TNF-α和/或IFN-γ的分泌;
(d)减缓肿瘤生长曲线;
(e)减小肿瘤体积;
(f)减轻肿瘤重量。
优选地,所述微生物菌株的培养物为其代谢物,更优选为分子量小于3kDa的代谢物;当所述药物用于治疗肿瘤时可以单用或者与其他抗癌治疗联合施用而增强抗癌效果;
所述其他抗癌治疗包括抗PD-1治疗剂(优选为抗PD-1的抗体、CTLA-4抗体、PD-L1抗体和/或PD-L1抑制剂),或除了抗PD-1抗体以外的其他抗体药物(优选为化疗药物、光敏剂、光热剂、免疫治疗药物中的至少一种)。
根据本发明可使用Anaerotruncus colihominis种的微生物菌株治疗的肿瘤为实体瘤(优选为选自由胃癌、食管癌、结肠癌、直肠癌、肝癌、胰腺癌、乳腺癌、肾癌、纤维肉瘤、肺癌、胆管癌、黑色素瘤组成的组中的一种或多种,更优选为结肠癌或黑色素瘤)。
本发明还保护微生物菌株,或所述微生物菌株的培养物或其加工物在制备提高生物体(优选为哺乳动物,更优选为人)的免疫应答(优选为提高T细胞中CD8阳性T细胞的比率)的药物中的应用,所述微生物菌株属于Anaerotruncus colihominis种。
上述应用中,所述微生物菌株为能够在生理生化水平和/或分子生物学水平鉴定为Anaerotruncus colihominis的任一菌株。优选地,所述微生物菌株与选自包括A. colihominis CCUG 45055、A. colihominis CIP 107754、A. colihominis DSM 17241、A. colihominis JCM 15631、A. colihominis WAL 14565、A. colihominis VTT E-062942和A. colihominis CCTCC M 20232246的A. colihominis菌株组中的至少一种具有至少95%、96%、97%、98%、98.65%、99%、99.41%、99.5%、99.9%或100%一致性的16s rRNA基因序列、重要功能性基因序列、调控子序列、重要的分子标志物序列、分化性能更高的基因序列或全基因组序列。或者针对上述菌株中的至少一种,针对全基因组序列的任一段特定序列或任意多种特定或不特定序列,利用PCR扩增进而测序,若某微生物菌株的扩增子中至少一个具有至少95%、96%、97%、98%、98.65%、99%、99.41%、99.5%、99.9%或100%一致性,也认为是本发明的所述微生物菌株。
在本发明的一些实施方案中,所述微生物菌株选自包括A. colihominis CCUG45055、A. colihominis CIP 107754、A. colihominis DSM 17241、A. colihominis JCM15631、A. colihominis WAL 14565、A. colihominis VTT E-062942、和A. colihominisCCTCC M 20232246的A. colihominis菌株组中的至少一种。
上述应用中,所述A. colihominis CCTCC M 20232246菌株在中国典型培养物保藏中心保藏,地址为湖北省武汉市武昌区八一路武汉大学中国典型培养物保藏中心,保藏日期2023年11月16日。
上述应用中,优选所述药物还包括药学上可接受的辅料。
辅料在制剂中的分类方式有多种,可从来源、作用和用途、给药途径等进行分类。按来源可分为天然产物、半合成产物和全合成产物。按辅料在制剂中的作用和用途分类有65种,分别是pH调节剂、螯合剂、包合剂、包衣剂、保护剂、保湿剂、崩解剂、表面活性剂、病毒灭活剂、补剂、沉淀剂、成膜材料、调香剂、冻干用赋形剂、二氧化碳吸附剂、发泡剂、芳香剂、防腐剂、赋形剂、干燥剂、固化剂、缓冲剂、缓控释材料、胶黏剂、矫味剂、抗氧剂、抗氧增效剂、抗黏着剂、空气置换剂、冷凝剂、膏剂基材、凝胶材料、抛光剂、抛射剂、溶剂、柔软剂、乳化剂、软膏基质、软胶囊材料、润滑剂、润湿剂、渗透促进剂、渗透压调节剂、栓剂基质、甜味剂、填充剂、丸芯、稳定剂、吸附剂、吸收剂、稀释剂、消泡剂、絮凝剂、乙醇改性剂、硬膏基质、油墨、增稠剂、增溶剂、增塑剂、黏合剂、中药炮制辅料、助滤剂、助溶剂、助悬剂、着色剂。
所述在药学上可接受的辅料包括佐剂、稳定剂或保护剂、抑菌剂、赋形剂、助溶剂、矫味剂、稀释剂、缓冲剂中的至少一种。
佐剂:是与一种疫苗抗原结合以增强[如加强、加快、延长和(或)可能的定向]其特异性免疫反应和疫苗临床效果的一种或多种成分混合的物质。
稳定剂或保护剂:用于稳定或保护生物制品有效成分、防止其降解或失去活性的物质。
抑菌剂:用于抑制微生物生长、防止微生物污染的物质。赋形剂:用于冻干制品中使药品成型、起支架作用的物质。助溶剂:用于增加药品溶解性的物质。矫味剂:用于改善口服药品口感的物质。稀释剂、缓冲剂:用于溶解、稀释制品,调整制品酸碱度的溶剂,如注射用水、氯化钠注射液、磷酸盐缓冲生理氯化钠溶液(PBS)等。
例示性辅料包括但不限于:丁基化羟基甲苯(BHT)、碳酸钙、磷酸钙(一氢)、硬脂酸钙、交联羧甲纤维素、交联聚乙烯吡咯烷酮、柠檬酸、交联聚维酮、半胱氨酸、乙基纤维素、明胶、羟基丙基纤维素、羟基丙基甲基纤维素、乳糖、硬脂酸镁、麦芽糖醇、甘露糖醇、蛋氨酸、甲基纤维素、对羟基苯甲酸甲酯、微晶纤维素、聚乙二醇、聚维酮、预胶化淀粉、对羟基苯甲酸丙酯、视黄醇棕榈酸酯、虫胶、二氧化硅、羧基甲基纤维素钠、柠檬酸钠、乙醇酸淀粉钠、山梨糖醇、淀粉(玉米)、硬脂酸、硬脂酸、蔗糖、滑石、二氧化钛、维生素A、维生素E、维生素C及木糖醇。
所述药物可呈注射制剂的形式或口服制剂形式经制备。所述的注射制剂依据物态分类包括液体注射剂、注射用粉剂、注射用片剂;依据注射部分分类包括皮内注射剂、皮下注射剂、肌肉注射剂、静脉注射剂、脊椎腔注射剂;优选的所述注射制剂的溶剂包括注射用水或生理盐水。
用于口服使用的制剂包括含有与无毒的药学上可接受的赋形剂混合的活性成分的片剂。这些赋形剂可以是例如惰性稀释剂或填充剂(例如,蔗糖、山梨糖醇、糖、甘露糖醇、微晶纤维素、包括马铃薯淀粉的淀粉、碳酸钙、氯化钠、乳糖、磷酸钙、硫酸钙或磷酸钠);制粒剂及崩解剂(例如,包括微晶纤维素的纤维素衍生物、包括马铃薯淀粉的淀粉、交联羧甲纤维素钠、藻酸盐或藻酸);粘合剂(例如,蔗糖、葡萄糖、山梨糖醇、阿拉伯胶、藻酸、藻酸钠、明胶、淀粉、预胶化淀粉、微晶纤维素、硅酸镁铝、羧甲基纤维素钠、甲基纤维素、羟丙基甲基纤维素、乙基纤维素、聚乙烯吡咯烷酮或聚乙二醇);以及润滑剂、助流剂及抗粘剂(例如,硬脂酸镁、硬脂酸锌、硬脂酸、二氧化硅、氢化植物油或滑石)。用于口服使用的制剂也可呈可咀嚼片剂形式,或呈硬明胶胶囊形式,其中活性成分与惰性固体稀释剂(例如,马铃薯淀粉、乳糖、微晶纤维素、碳酸钙、磷酸钙或高岭土)混合,或呈软明胶胶囊形式,其中活性成分与水或油介质(例如,花生油、液体石蜡或橄榄油)混合。粉剂、颗粒剂及丸剂可以使用上文在片剂或胶囊下提到的成分以常规方式使用例如混合器、流化床设备或喷雾干燥设备来制备。
用于口服制剂的其他药学上可接受的赋形剂包括但不限于着色剂、调味剂、增塑剂、保湿剂及缓冲剂。用于口服使用的制剂也可呈可咀嚼片剂形式,或呈硬明胶胶囊形式,其中活性成分与惰性固体稀释剂(例如,马铃薯淀粉、乳糖、微晶纤维素、碳酸钙、磷酸钙或高岭土)混合,或呈软明胶胶囊形式,其中活性成分与水或油介质(例如,花生油、液体石蜡或橄榄油)混合。粉剂、颗粒剂及丸剂可以使用上文在片剂或胶囊下提到的成分以常规方式使用例如混合器、流化床设备或喷雾干燥设备来制备。
在一些实施方案中,施用包括经肌肉内、静脉内(例如,呈无菌溶液形式且在适用于静脉内使用的溶剂系统中)、皮内、动脉内、腹膜内、病变内、颅内、关节内、前列腺内、胸膜内、气管内、鼻内、玻璃体内、阴道内、直肠内、经表面、肿瘤内、经腹膜、皮下、结膜下、囊内、经粘膜、心包内、脐内、眼内、经口(例如,片剂、胶囊、囊片、囊形片或糖浆)、经表面(例如,呈乳膏、凝胶、洗剂或软膏形式)、经局部、通过吸入、通过注射或通过输注(例如,以乳膏或脂质组合物形式连续输注、直接地浸泡靶标细胞的局部灌注、置管、灌洗)施用本文所述的药物。
优选地,所述功能性菌剂或药物还包括其他与Anaerotruncus colihominis起到协同作用的药物。
本发明还保护一种微生物菌株,所述微生物菌株是以保藏号CCTCC M 20232246保藏的Anaerotruncus colihominis ibiome014菌株,所述Anaerotruncus colihominisibiome014菌株在中国典型培养物保藏中心保藏,地址为湖北省武汉市武昌区八一路武汉大学中国典型培养物保藏中心,保藏日期2023年11月16日。
本发明同时保护上述微生物菌株的培养物或其加工物。
本发明还保护一种药物,包含上述的微生物菌株,或与上述的微生物菌株16srRNA序列一致性达到至少95%、至少96%、至少97%、至少98%、至少98.65%、至少99%、至少99.41%、至少99.5%、至少99.9%或100%的菌株,或包含上述微生物菌株的培养物或其加工物,和药学上可接受的辅料,优选地,所述辅料包括佐剂、稳定剂或保护剂、抑菌剂、赋形剂、助溶剂、矫味剂、稀释剂、缓冲剂中的至少一种。
本发明还保护一种药物组合物,包含上述的菌株和联用药物,所述联用药物包括抗PD-1治疗剂(优选为抗PD-1的抗体、CTLA-4抗体、PD-L1抗体和/或PD-L1抑制剂),或除了抗PD-1抗体以外的其他抗体药物(优选为化疗药物、光敏剂、光热剂、免疫治疗药物中的至少一种)。
本发明所述的药物的剂型选自由片剂、颗粒剂、胶囊剂、悬液、冻干制剂灯组成的组。
本发明的有效剂量是,Anaerotruncus colihominis作为主要药物活性成分制成活菌制剂包含的总活菌数为106-1014CFU。
用药周期以能够实现效果为准,包括但不限于每日1-3次,每周3-7天服用等,还和具体制剂的作用浓度有关。
本发明还保护一种发酵剂/功能性菌剂/营养组合物,包含上述微生物菌株,或与上述的微生物菌株16s rRNA序列一致性达到至少95%、至少96%、至少97%、至少98%、至少98.65%、至少99%、至少99.41%、至少99.5%、至少99.9%或100%的菌株,或上述微生物菌株的培养物或其加工物。
发酵剂或功能性菌剂,包括上述微生物菌株制备得到的菌液、或进一步处理得到的粉剂、或颗粒剂;所述发酵剂中还可以进一步含有一种以上互不拮抗的微生物菌剂,选自克里斯滕森菌、副拟杆菌、嗜黏蛋白阿克曼氏菌、多形拟杆菌等中的一种以上制备得到复合菌剂。
有效菌剂浓度、活菌数为106-1014CFU。
发酵剂或功能性菌剂亦可用作功能性食品、营养品。
营养组合物,包括上述微生物菌株,或与上述的微生物菌株16s rRNA序列一致性达到至少95%、至少96%、至少97%、至少98%、至少98.65%、至少99%、至少99.41%、至少99.5%、至少99.9%或100%的菌株,或微生物菌株的培养物或加工物。优选地,所述营养组合物为食品、营养品、补充物、益生菌或共生菌。
所述食品,包括上述微生物菌株,或与上述的微生物菌株16s rRNA序列一致性达到至少95%、至少96%、至少97%、至少98%、至少98.65%、至少99%、至少99.41%、至少99.5%、至少99.9%或100%的菌株,或微生物菌株的培养物或加工物以及实现食品功能的辅助物质,呈现的形式包括但不限于“膳食补充剂”“发酵食品”等。
所述膳食补充剂包括上述微生物菌株的培养物或加工物,并进一步加入纤维素、维生素、矿物质等营养物质处理得到。
所述发酵食品包括乳制品、豆制品或者果蔬制品等。所述的乳制品是牛奶、酸奶油或干酪等。所述的豆制品是豆奶、豆豉或豆酱等。所述的果蔬制品是黄瓜、胡萝卜、甜菜、芹菜或圆白菜制品等。
所述益生菌是指活的微生物,当它以合适的量提供时,对于宿主有机体的健康会有益。
所述共生菌是指那些含有益生素和益生菌的混合物的食品。它们通常包含利于生长和/或代谢活性的益生组分,以及总的来说与诸如但不限于上述微生物菌株与低聚果糖或低聚半乳糖相组合的益生菌效果。
本发明还提供了一种提高生物体免疫应答的方法,包括对生物体施用上述的微生物菌株,或与上述的微生物菌株16s rRNA序列一致性达到至少95%、至少96%、至少97%、至少98%、至少98.65%、至少99%、至少99.41%、至少99.5%、至少99.9%或100%的菌株,或包含上述微生物菌株的培养物或其加工物,或上述的药物。
本发明同时提供了一种检查上述的微生物菌株是否在生物体中定植的方法,包括从生物体的粪便或肠道提取物中提取细菌16s rRNA,并以其为模板,使用具有如SEQ IDNo:2所示的序列的上游引物和具有如SEQ ID No:3所示的序列的下游引物进行PCR。
本发明的有益效果在于:
本发明首次提示Anaerotruncus colihominis能够用于预防或治疗肿瘤,丰富了本领域的选择,具有巨大的临床应用价值。具体来说:
1.本菌株Anaerotruncus colihominis从健康志愿者体内分离,易于在体外培养,能够在小鼠肠道中定殖。
2.在体外实验中,该菌能够增强CD8T细胞的效应因子IFN-γ分泌能力,提高CD8T细胞效应功能。
3.该菌提高CD8T细胞效应功能主要依赖其产生的小分子代谢物。
4.在小鼠体内实验中,通过检测定殖后小鼠肠道CD8T细胞效应因子分泌情况,发现该菌能促进小鼠LPL(固有淋巴细胞)细胞CD8T细胞分泌IFN-γ,结果与体外实验相吻合。
5.对SPF小鼠皮下荷瘤MC38,并灌胃Anaerotruncus colihominis,单独施用该菌即能发挥肿瘤抑制效果,同时该菌还能够联合PD-1抗体,发挥更优的肿瘤抑制功能。
6. 对SPF小鼠皮下荷瘤B16黑色素瘤,并灌胃Anaerotruncus colihominis,该菌能发挥肿瘤抑制功能,且能够极显著的增加LPL中的CD8 T细胞分泌肿瘤坏死因子-α(TNF-α)和γ干扰素(IFN-γ)。
7.对无菌小鼠皮下荷瘤MC38,并灌胃Anaerotruncus colihominis,该菌株能够联合PD-1抗体,发挥肿瘤抑制功能,表明Anaerotruncus colihominis单一菌株就能达到联合PD-1的功能。
8.该菌株具有协同肿瘤免疫治疗、提高免疫检查点阻断疗法疗效、提高肿瘤患者anti-PD-1疗法响应率、提高患者生存期的潜力。生物保藏
Anaerotruncus colihominis ibiome014,保藏日期2023年11月16日,保藏地点为中国典型培养物保藏中心,地址为湖北省武汉市武昌区八一路武汉大学中国典型培养物保藏中心,保藏号为CCTCC M 20232246。
附图说明
图1为实施例3采用培养基对照及其小分子或A. colihominis培养上清液及其小分子刺激CD8T细胞后,使用流式细胞术检测CD8T细胞效应因子IFN-γ的分泌情况;其中,****代表p<0.0001。
图2为实施例4中A. colihominis在SPF小鼠体内定殖实验结果。
图3为实施例5中A. colihominis定殖的肠道免疫流式示意图。
图4为实施例5中A. colihominis定殖对肠道免疫的影响结果图。
图5为实施例6中SPF小鼠MC38肿瘤注射后各组的肿瘤生长曲线图。
图6为实施例6中SPF小鼠MC38肿瘤注射后14天对小鼠肿瘤组织进行的剥离拍照图。
图7为图6的肿瘤称重统计结果。
图8为图6肿瘤的流式分析结果图。
图9为实施例7中灌胃A. colihominis对SPF小鼠皮下注射B16黑色素瘤的肿瘤生长曲线的影响。
图10为实施例7中灌胃A. colihominis的SPF小鼠皮下注射B16黑色素瘤17天后对小鼠肿瘤组织进行的剥离拍照图。
图11为图10的肿瘤称重统计结果。
图12为图10肿瘤浸润的CD8T细胞流式分析图。
图13为实施例8中A. colihominis及联合PD-1对无菌(GF)小鼠肿瘤生长曲线的影响。
图14实施例8中灌胃A. colihominis及联合PD-1的无菌(GF)小鼠MC38肿瘤注射后19天对小鼠肿瘤组织进行的剥离拍照图。
图15为图14的肿瘤称重统计结果。
实施方式
为更好理解本发明,下面结合实施例及附图对本发明作进一步描述,以下实施例仅是对本发明进行说明而非对其加以限定。
实施例1:菌库建立
从健康志愿者及PD-1阻断治疗响应患者粪便中分离培养单菌落:志愿者粪便样品用20%的甘油磷酸盐缓冲液保存,分别梯度稀释至10-5、10-6、10-7倍,将各浓度分别涂布于GAM肉汤(Solarbio LA4450)、BBE(拟胆汁七叶苷固体培养基,Solarbio LA7310),RCM(梭菌增菌培养基,青岛海博HB0316),MRS肉汤(青岛海博HB0384-1)、TSB(胰酪大豆胨液体培养基,青岛海博HB4114-19)、BHI(脑心浸出液培养基,OXOID,CM1136)、哥伦比亚血平板(比克曼生物)。挑单克隆至相应的液体培养基,通过16s rRNA通用引物(上游引物27F:AGAGTTTGATCCTGGCTCAG,下游引物1492R:TACGGCTACCTTGTTACGACTT)测序确定种属(扩增条件:预变性:95℃ 3min;[变性 95℃ 15s、退火 58℃ 15s、延伸 72℃ 30s] 35个循环;彻底延伸 72℃ 5min),用甘油于-80℃保存。将细菌利用37℃摇床培养至OD600=0.8,3000rpm离心10min,收集细菌培养上清液,-40℃冻存备用。共建立包含5个门、12个纲、15个目、30个科、1000株菌的菌库,包含了人肠道微生物的主要类群。
实施例2:IFN-γ+CD8T细胞体外筛选
IFN-γ是一种通常由NK细胞、NKT细胞、CD4以及CD8T细胞表达的细胞因子,具有广泛的刺激和调节免疫系统的能力。IFN-γ+ CD8T细胞在抗肿瘤免疫中也起着至关重要的作用,并被报道能够影响免疫检查点抑制剂(ICI)疗法的疗效。Honda实验室的研究发现,从健康人体内分离的11种肠道菌的混合物能够通过上调CD8T细胞分泌IFN-γ的能力,在小鼠实验中,达到显著抑制肿瘤生长的作用。因此,能够刺激CD8T细胞IFN-γ分泌的肠道菌,可作为筛选潜在的抗肿瘤功能菌的指标。
然而,先前的研究采用的是用抗生素富集的方法,11种肠道菌的混合物具有氨苄青霉素抗性,有在服用抗生素情况下大量增殖的可能,在安全性方面具有风险。此外,由于先前的研究尚未确定具体的菌株,并且混合物中的梭杆菌对肿瘤的影响仍有争议,因此,寻找具体的、单一的作用菌株,具有重要意义。
从6-8周的IFN-γ荧光报告小鼠(在IFN-γ蛋白转录序列后插入IRES序列和katushka荧光蛋白序列,使IFN-γ表达的同时细胞表达katushka荧光,构建方法可参考Villegas-Mendez, A. et al. Parasite-Specific CD4+ IFN-γ+ IL-10+ T CellsDistribute within Both Lymphoid and Nonlymphoid Compartments and AreControlled Systemically by Interleukin-27 and ICOS during Blood-Stage MalariaInfection. Infection and immunity84, 34-46, doi:10.1128/iai.01100-15 (2016).)脾脏中分离CD8T细胞:脾脏组织去除表面脂肪,加入磷酸盐缓冲溶液,研磨成细胞悬液,用钢网过滤残余组织。通过500g、4℃离心5min,将细胞收集于管底,加入2mL红细胞裂解液去除红细胞。剩余细胞通过CD8T细胞磁珠分选试剂盒(Biolegend 480035)分离CD8T细胞。
对细胞进行计算,取3×105 的CD8T细胞加入anti-CD3抗体(终浓度5μg/mL)、anti-CD28抗体(终浓度2μg/mL)(分别为Biolegend 100223、Biolegend 102112)体外活化CD8T细胞,按3×105/孔分于96孔板。向孔中分别加入实施例1冻存的细菌上清液各20μL进行刺激,培养(即刺激)48h。
收集上述经刺激后的细胞(约1×106/管),每孔加入60 µL含有特异性表面标记荧光抗体的混合液(FITC-CD3 Biolegend 100204 0.2µL/孔,APC-Cy7 CD8 Biolegend100714 0.2µg/孔),4℃避光放置15-20min。加满1×PBS 洗一遍(6000rpm×2min)。
用200~300µL 1×PBS 重悬细胞,上样浓度在3~5×106/mL。细胞悬液经200目尼龙网过滤去除杂质,转至Ep管中,加DAPI瞬染(200倍稀释,博士德AR1177)上机检测,用Flowjo软件分析。以IFN-γ为例,先以FSC-H与FSC-A为横纵轴圈出单细胞,在单细胞门中圈出DAPI阴性的活细胞,之后圈出CD3阳性、CD8阳性的CD8T细胞,检测CD8T细胞IFN-γ分泌情况。
分析检测结果,发现一株健康志愿者来源、GAM肉汤培养基分离、并能在GAM肉汤厌氧培养、长出灰白色大菌落的菌株,该菌株来源的代谢物能够显著增强CD8T细胞IFN-γ分泌情况,以及提高IFN-γ阳性的CD8T细胞中TNF-α阳性细胞的比例。
经实施例1的方法测序得到该菌株的16s rRNA基因序列(SEQ ID NO.1)提交到NCBI Basic Local Alignment Search Tool,进行菌株16s rRNA基因分析。比对结果表明,与其相似性最高的菌株是Anaerotruncus colihominis strain WAL 14565(相似度99.41%),因此该菌株属于Anaerotruncus colihominis种,将其命名为Anaerotruncus colihominis ibiome014,保藏至中国典型培养物保藏中心,保藏地址为:湖北省武汉市武昌区八一路珞珈山;保藏日期为:2023年11月16日;保藏编号为:CCTCC M 20232246。
实施例3:A. colihominis菌小分子代谢物增加体外培养的CD8 T细胞的IFN-γ分泌能力
收集A. colihominis ibiome014上清液和GAM肉汤培养基,分别用0.2µm的滤膜过滤除菌,用孔径<3kDa的分子筛超滤,除去上清中的蛋白组分待用。准备4组样品:(1)GAM肉汤培养基,(2)经分子筛超滤后的GAM肉汤培养基,(3)A. colihominis ibiome014上清液,(4)经分子筛超滤后的A. colihominis ibiome014上清液。提前一天用4µg/mL的CD3抗体60µL铺板96孔板。
按照实施例2的方法,仅在刺激培养时替换加入上述4组样品,分别检测4组样品存在情况下CD8T细胞IFN-γ分泌情况。
结果如图1所示,A. colihominis培养上清液具有促进CD8T细胞效应因子分泌的能力,且发挥功能的有效成分为细菌来源的小分子代谢物(<3kDa)。
实施例4:A. colihominis在SPF小鼠模型中的定殖效果检测
对购买自集萃药康C57/B6J小鼠喂ABX(1g/L氨苄青霉素、新霉素、甲硝唑,以及0.5g/L万古霉素盐酸盐)清菌2周(以ABX替换饮用水),随后分为两组:对照组灌胃PBS,定殖组灌胃5×106CFU/mL的菌液,各200μL,两天一次,一周后收集小鼠粪便,检测粪便中A. colihominis含量。用20%(w/v)的SDS裂菌,用酚氯仿抽提细菌DNA,用菌株特异性引物SEQID No:2和SEQ ID No:3(F:GACATCGGATGCATAGCC,R:CTCATTAGAGTGCTCTTGCGTA)进行PCR,以16s rRNA通用引物(qPCR-27F:AGAGTTTGATCMTGGCTCAG,qPCR-1492R:TACGGCTACCTTGTTACGACTT)为内参,检测菌株定殖效果。
结果如图2所示:与对照组相比,定殖组小鼠粪便中A. colihominis含量明显增高。表明灌胃后,小鼠肠道内A. colihominis水平增加,A. colihominis能在小鼠体内定殖。
实施例5:A. colihominis增加肠道固有层CD8 T细胞的IFN-γ分泌能力
6-8周的集萃药康C57/B6J小鼠提前用四联抗生素(ABX)清除肠道菌:每250mL水分别加入0.25g氨苄青霉素、新霉素、甲硝唑,以及0.125g万古霉素盐酸盐,饲喂2周。之后换为普通水饮用,将小鼠随机分为2组:每天实验组灌胃1×109CFU/mL A. colihominis菌液,对照组灌胃PBS,各200μL。定殖2周后,牺牲小鼠,用PBS冲洗肠道内容物并剖开肠管,肠子剪成1-2cm小段投入25mL预消化液中(RPMI 65mL,FBS 1.2mL,EDTA 120μL),37℃,220rpm摇20min。将所得的培养基通过200目钢网过滤,肠段加入预消化液供后续处理,过滤后的培养基离心可得肠上皮细胞及上皮内淋巴细胞。重复上述步骤2次,剩余组织加入含胶原酶和DNA酶的消化液(RPMI 10mL,DNASE I STOCK 20μL,Type II Collagenase 5mg (sigma),FBS 0.2mL),过滤后所得为肠道固有层淋巴细胞。对固有层淋巴细胞用特异性荧光抗体标记,流式上机检测IFN-γ分泌情况。
如图3所示,为流式细胞术结果的划门示意图:分离小鼠肠道上皮细胞后,先圈出活细胞,进一步圈出CD45+的淋巴细胞,之后圈出CD3+的T细胞,进一步区分CD4和CD8,最后检测CD8 IFN-γ分泌情况。图4为流式细胞术的结果统计图(即菌定殖对肠道免疫的影响),图中表明,与对照组相比,A. colihominis灌胃的实验组的大肠固有层内淋巴细胞中,CD8T细胞的效应因子TNF-α(图4左)和IFN-γ(图4右)分泌增多。
实施例6:A. colihominis对SPF小鼠的抗肿瘤效应
MC38,小鼠结肠癌细胞,又名MCA38。来源于长期暴露于致癌物DMH (1,2-二甲基肼二盐酸盐)而患有结肠腺癌的C57BL6小鼠结肠肿瘤组织,并可高水平表达人癌胚抗原(CEA)。结肠直肠癌(CRC)是全球癌症死亡的第二大原因。而啮齿动物结肠癌模型研究是新药物临床前评估的有效方法。MC38细胞对免疫调节抗体具有良好的应答特性,证实了肿瘤微环境可修饰免疫激活。因此,MC38被定位为功能强大的免疫肿瘤学模型,在药物研发中具有重要的用途,常用于抗肿瘤药物、特别是抗结肠癌肿瘤药物在体内的免疫功能研究。
6-8周的集萃药康C57/B6J小鼠提前用四联抗生素(ABX)清除肠道菌,每250mL水分别加入0.25g氨苄青霉素、新霉素、甲硝唑,以及0.125g万古霉素盐酸盐,饲喂2周。对小鼠进行随机分组,分别为空白对照组、A. colihominis菌组、PD-1抗体治疗组和A. colihominis菌联合PD-1抗体治疗组共4组,A. colihominis菌组和A. colihominis菌联合PD-1抗体治疗组每天灌胃200μL 1×109CFU/mL的A. colihominis菌液,空白对照组和PD-1抗体治疗组每天灌胃PBS,直到实验结束。定殖3天后对所有组小鼠剃毛,皮下荷瘤MC38,3.5*105/只。荷瘤后隔天测量肿瘤体积,采用0.5*长*宽*宽近似计算肿瘤体积。荷瘤后7天、9天、11天,PD-1抗体治疗组和A. colihominis菌联合PD-1抗体治疗组对小鼠腹腔注射anti-PD-1(BioXcell BE0146),注射剂量为100 μg/只。荷瘤后第14天牺牲小鼠,对小鼠肿瘤进行拍照称重。
如图5-7所示,A. colihominis菌能够联合PD-1抗体,达到抑制肿瘤生长的效果。具体来说,单独灌胃A. colihominis菌即可使肿瘤生长曲线明显变缓(图5)、体积明显变小(图6)、肿瘤重量明显减轻(图7),且效果优于PD-1;当灌胃A. colihominis菌的同时还注射PD-1抗体,效果更佳,生长曲线(图5)、体积(图6)、重量(图7)的效果均明显优于PD-1抗体治疗组。
此外,将需要消化的肿瘤组织块放入5 mL EP 管内,加入少量消化液(1 mg/mL胶原酶 IV,20 µg/mL DNase I);用手术剪刀快速剪碎,保证充分剪碎后,补充消化液到 4mL;将 EP 置于恒温摇床37℃,180 rpm,消化30 mins至1 h,期间观察消化情况;随后取出消化后的肿瘤组织消化液,上清通过200目网过滤至15 mL离心管,加满PBS,1500 rpm x 5min离心,收集沉淀,进行流式分析。结果如图8所示:A. colihominis菌定殖后,肿瘤浸润的CD8T细胞TNF-α(图8左)和IFN-γ(图8右)比例显著升高。
实施例7:A. colihominis对SPF小鼠皮下B16黑色素瘤的抗肿瘤效应
B16,小鼠黑色素瘤细胞,是来源于C57BL/6小鼠皮肤的自发性肿瘤,此瘤株主要发生肺转移。B16细胞在小鼠体内的生长及转移特性与人黑色素瘤非常相似,为理想的黑色素瘤动物模型,已广泛地用于研究黑色素瘤发生发展、转移和药物筛选等研究。
将集萃药康C57/B6J雄性鼠适应性饲养一周后,用四联抗生素(ABX)清除肠道菌,每250mL水分别加入0.25g氨苄青霉素、新霉素、甲硝唑,以及0.125g万古霉素盐酸盐,饲喂2周。停用四联抗生素(ABX)后四天对小鼠进行随机分组:灌胃PBS对照组和灌胃A. colihominis菌治疗组。对灌胃A. colihominis菌治疗组每天灌胃200μL 1×109CFU/mL的A. colihominis菌液,灌胃PBS对照组每只鼠每天灌胃200μL PBS,直到实验结束。灌胃5天后对所有组小鼠剃毛,皮下荷瘤B16,1*105/只。荷瘤第7天后开始测量肿瘤体积并记录,此后每隔天测量一次,采用0.5*长*宽*宽近似计算肿瘤体积。在荷瘤后17天牺牲小鼠,对小鼠肿瘤进行拍照。拍照后将小鼠的肿瘤制成单细胞悬液,用于流式染色分析(方法同实施例6)。结果如图9-12所示,A. colihominis菌定殖后,B16肿瘤生长趋势明显变缓(图9)、肿瘤体积明显变小(图10)、肿瘤重量明显减轻(图11),肿瘤浸润的CD8T细胞TNF-α、IFN-γ分泌显著增加(图12)。
总体实验表明A. colihominis菌不仅对小鼠结肠癌瘤MC38的生长有抑制作用,也可以对小鼠黑色素瘤B16的生长有显著抑制作用。
实施例8:A. colihominis及联合PD-1抗体增强无菌小鼠抗肿瘤效应
将集萃药康C57/B6J雄性GF鼠随机分为四组:空白对照组、A. colihominis菌组、PD-1抗体治疗组和A. colihominis菌联合PD-1抗体治疗组。对A. colihominis菌组和A. colihominis菌联合PD-1抗体治疗组灌胃A. colihominis菌一次,每只鼠200μL,5×108CFU/mL。定殖1周后对所有组小鼠剃毛,皮下荷瘤MC38,3.5*105/只。荷瘤后隔天测量肿瘤体积,采用0.5*长*宽*宽近似计算肿瘤体积。荷瘤后的7天、9天、11天、13天,对小鼠腹腔注射anti-PD-1,注射剂量为200 μg/只。在荷瘤后19天牺牲小鼠,对小鼠肿瘤进行拍照。结果如图13-15所示,单独灌胃A. colihominis菌即可使肿瘤生长曲线明显变缓(图13)、体积明显变小(图14)、肿瘤重量明显减轻(图15);当灌胃A. colihominis菌的同时还注射PD-1抗体,能够进一步抑制肿瘤生长的效果,生长曲线(图13)、体积(图14)、重量(图15)的效果均明显优于PD-1抗体治疗组,表明A. colihominis单一菌株就能达到联合PD-1的功能。
以上所述实施方式仅仅是对本发明的优选实施方式进行描述,并非对本发明的范围进行限定,在不脱离本发明设计精神的前提下,本领域普通技术人员对本发明的技术方案作出的各种变形和改进,均应落入本发明的权利要求书确定的保护范围内。
Claims (10)
1.微生物菌株、或所述微生物菌株的培养物或加工物在制备用于预防或治疗肿瘤的药物中的应用,其特征在于:所述微生物菌株属于Anaerotruncus colihominis种。
2.根据权利要求1所述的应用,其特征在于,所述预防或治疗肿瘤包括但不限于如下至少一种表型:
(a)在体外促进CD8T细胞效应因子TNF-α和/或IFN-γ的分泌;
(b)在体内促进肠道固有层淋巴细胞中CD8T细胞的效应因子TNF-α和/或IFN-γ的分泌;
(c)在体内促进肿瘤浸润淋巴细胞中CD8T细胞的效应因子TNF-α和/或IFN-γ的分泌;
(d)减缓肿瘤生长曲线;
(e)减小肿瘤体积;
(f)减轻肿瘤重量。
3.根据权利要求1或2所述的应用,其特征在于:所述微生物菌株的培养物为其代谢物,优选为分子量小于3kDa的代谢物;当所述药物用于治疗肿瘤时可以单用或者与其他抗癌治疗联合施用而增强抗癌效果;
所述其他抗癌治疗包括抗PD-1治疗剂(优选为抗PD-1的抗体、CTLA-4抗体、PD-L1抗体和/或PD-L1抑制剂),或除了抗PD-1抗体以外的其他抗体药物(优选为化疗药物、光敏剂、光热剂、免疫治疗药物中的至少一种)。
4.根据权利要求1或2所述的应用,其特征在于:所述肿瘤为实体瘤(优选为选自由胃癌、食管癌、结肠癌、直肠癌、肝癌、胰腺癌、乳腺癌、肾癌、纤维肉瘤、肺癌、胆管癌、黑色素瘤组成的组中的一种或多种,更优选为结肠癌或黑色素瘤)。
5. 微生物菌株、或所述微生物菌株的培养物或加工物在制备提高生物体(优选为哺乳动物,更优选为人)的免疫应答(优选为提高T细胞中CD8阳性T细胞的比率)的药物中的应用,其特征在于:所述微生物菌株属于Anaerotruncus colihominis种。
6.根据权利要求1或5所述的应用,其特征在于:所述药物还包括药学上可接受的辅料,所述辅料包括佐剂、稳定剂或保护剂、抑菌剂、赋形剂、助溶剂、矫味剂、稀释剂、缓冲剂中的至少一种。
7. 根据权利要求1或5所述的应用,其特征在于:所述微生物菌株是以保藏号CCTCC M20232246保藏的Anaerotruncus colihominis ibiome014菌株,所述Anaerotruncus colihominis ibiome014菌株在中国典型培养物保藏中心保藏,地址为湖北省武汉市武昌区八一路武汉大学中国典型培养物保藏中心,保藏日期2023年11月16日。
8. 一种微生物菌株或其培养物或加工物,其特征在于:所述微生物菌株是以保藏号CCTCC M 20232246保藏的Anaerotruncus colihominis ibiome014菌株,所述Anaerotruncus colihominis ibiome014菌株在中国典型培养物保藏中心保藏,地址为湖北省武汉市武昌区八一路武汉大学中国典型培养物保藏中心,保藏日期2023年11月16日。
9. 一种药物,其特征在于:包含Anaerotruncus colihominis种微生物菌株,或其培养物或加工物,和药学上可接受的辅料,优选地,所述辅料包括佐剂、稳定剂或保护剂、抑菌剂、赋形剂、助溶剂、矫味剂、稀释剂、缓冲剂中的至少一种。
10. 一种发酵剂/功能性菌剂/营养组合物,其特征在于:包含Anaerotruncus colihominis种微生物菌株,或其培养物或加工物;优选地,所述营养组合物为食品、营养品、补充物、益生菌或共生菌。
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