CN115569193B - 戈氏梭菌芽孢联合帕博利珠单抗的应用及治疗结肠癌药物和戈氏梭菌芽孢冻干粉制备方法 - Google Patents
戈氏梭菌芽孢联合帕博利珠单抗的应用及治疗结肠癌药物和戈氏梭菌芽孢冻干粉制备方法 Download PDFInfo
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Abstract
本发明涉及戈氏梭菌联合帕博利珠单抗在癌症治疗中的应用和戈氏梭菌芽孢冻干粉制备方法。本发明提出了戈氏梭菌芽孢联合帕博利珠单抗在制备治疗结肠癌医药制品中的应用。本发明首次发现戈氏梭菌芽孢联合帕博利珠单抗可以显著提高抗结肠癌的疗效,同时降低帕博利珠单抗的使用剂量,高效低毒。戈氏梭菌溶瘤可通过多种方式影响TME免疫原性,使TME从一个免疫抑制状态变成免疫激活状态,同时调节免疫抑制性TME,打破免疫耐受。在最佳组合条件下戈氏梭菌芽孢与帕博利珠单抗联合约有20%小鼠肿瘤组织彻底清除,将PD‑1抗体治疗肿瘤患者的受益范围扩大,甚至对PD‑1抗体治疗失败患者也有突出疗效。
Description
本申请是申请日为2021年10月09日、申请号为202111177854.3、发明名称为《戈氏梭菌芽孢联合PD-1抗体的应用》的分案申请。
技术领域
本发明属于生物医药技术领域,具体涉及戈氏梭菌芽孢联合帕博利珠单抗的应用及治疗结肠癌药物和戈氏梭菌芽孢冻干粉制备方法,该治疗结肠癌医药制品可显著抑制结肠癌生长,增强PD-1抗体抗肿瘤效果。
背景技术
肿瘤微环境(Tumor microenvironment,TME)是在肿瘤生长过程中,由肿瘤细胞、基质细胞(包括成纤维细胞、免疫、炎性细胞以及一些血管内皮细胞等)和细胞外基质以及浸润在其中的生物分子等共同构成局部稳态环境。TME为肿瘤发生、发展、侵袭等提供了必要物质基础,调控肿瘤转移、复发等多种生物学行为。同时TME增加肿瘤耐药性和耐辐射性,降低治疗效果。TME内的免疫调节在肿瘤发生发展中具有重要功能,其可通过多种机制,形成肿瘤局部免疫抑制。如何调控TME免疫治疗策略,重塑积极的免疫微环境是抗肿瘤治疗的重点和难点。
Pembrolizumab是一种PD-1抑制剂药物,PD-1是一种重要的免疫抑制分子,PD-1抗体临床表现出显著抗肿瘤效果,包括对晚期转移患者的持久治疗效果。一般认为,对免疫检查点阻断剂的敏感性取决于肿瘤新生抗原负荷以及肿瘤微环境(TME)中免疫细胞浸润程度和组成。不幸的是,多数常见癌症并未表现出大量的突变和免疫细胞浸润,因此肿瘤对免疫检查点抑制剂不敏感,响应率低,仅20%患者使用有效。因此,开发能使这些肿瘤对免疫治疗更敏感方法也是目前重点研究方向之一。
戈氏梭菌(Clostridium ghonii)为严格厌氧菌,具有趋肿瘤低氧特性,仅能定植在肿瘤乏氧区。戈氏梭菌溶瘤可募集大量免疫细胞向TME浸润,包括树突状细胞、中性粒细胞、巨噬细胞在内的先天免疫细胞及CD3+T、CD4+T、CD8+T等特异性免疫细胞,改变TME中免疫细胞浸润程度和组成。同时促进肿瘤中TNF-α、IFN-γ、IL-6等细胞因子和趋化因子表达增强。可见,戈氏梭菌溶瘤可以通过多种方式影响TME免疫原性,使TME从一个免疫抑制状态变成免疫激活状态,打破免疫耐受,使得戈氏梭菌与免疫检查点抑制剂联用高效抗肿瘤成为可能。
发明内容
本发明根据现有技术的不足,提供戈氏梭菌芽孢联合PD-1抗体的应用。
本发明技术方案如下:
本发明提供了戈氏梭菌芽孢联合帕博利珠单抗(Pembrolizumab)在制备治疗结肠癌医药制品中的应用,所述戈氏梭菌包括戈氏梭菌MW-DCG-LCv-26菌株或MW-DCG-HNCv-18菌株;
所述戈氏梭菌MW-DCG-LCv-26菌株保藏于澳大利亚国家计量研究院,菌株编号为V12/001486,所述MW-DCG-HNCv-18菌株保藏于澳大利亚国家计量研究院,菌株保藏号V12/001485;
所述帕博利珠单抗(Pembrolizumab)为PD-1抗体(Pembrolizumab)注射液。
优选的,所述治疗结肠癌医药制品中戈氏梭菌芽孢剂量为1×107cfu/次;所述PD-1抗体(Pembrolizumab)注射液的剂量为0.2mg/次。
本发明提供了一种治疗结肠癌的药物,药物中的药效成分包括戈氏梭菌芽孢和帕博利珠单抗(Pembrolizumab)。
根据本发明优选的,所述的戈氏梭菌为戈氏梭菌MW-DCG-LCv-26菌株或者戈氏梭菌驯化后获得的菌株;戈氏梭菌MW-DCG-LCv-26菌株保藏于澳大利亚国家计量研究院,菌株编号为V12/001486。其它优选的菌株为MW-DCG-HNCv-18菌株,该菌株保藏于澳大利亚国家计量研究院,菌株保藏号V12/001485;MW-DCG-CCv-17菌株,该菌株保藏于澳大利亚国家计量研究院,菌株保藏号V12/001487等。
根据本发明优选的,所述的戈氏梭菌为芽孢形式。
本发明优选的,所述戈氏梭菌的芽孢形式为冻干粉剂,其辅料为1%蔗糖;
本发明优选的,所述帕博利珠单抗(Pembrolizumab)为PD-1抗体(Pembrolizumab)注射液。
进一步优选的戈氏梭菌芽孢冻干粉剂冻干工艺为:冻结阶段-40℃4h;-35℃10min同时抽真空、-30℃10min、-25℃10min、-20℃26h、-15℃2h、-10℃10min、-5℃10min、0℃10min、10℃2h、15℃10min、20℃3h、27℃3h冻干制备。
本发明优选的药物组合是1×107CFU戈氏梭菌芽孢冻干粉联合浓度1mg/mL的帕博利珠单抗(Pembrolizumab)注射液0.2mg。
进一步优选的,所述帕博利珠单抗(Pembrolizumab)注射液的溶媒为质量百分比浓度0.9%的氯化钠注射液;戈氏梭菌芽孢冻干粉的溶媒为灭菌注射用水和质量百分比浓度0.9%氯化钠注射液。
进一步优选的,所述戈氏梭菌芽孢联合帕博利珠单抗(Pembrolizumab)使用顺序为先戈氏梭菌芽孢后帕博利珠单抗(Pembrolizumab)。
本发明提供了一种戈氏梭菌芽孢冻干粉的制备方法,所述戈氏梭菌芽孢冻干粉以戈氏梭菌芽孢为活性成分,以1%蔗糖为辅料,经冻干程序制备得到;
所述冻干程序包括-40℃4h;-35℃10min同时抽真空、-30℃10min、-25℃10min、-20℃26h、-15℃2h、-10℃10min、-5℃10min、0℃10min、10℃2h、15℃10min、20℃3h、27℃3h。
有益效果
本发明首次发现戈氏梭菌芽孢联合Pembrolizumab可以显著提高抗结肠癌的疗效,同时降低帕博利珠单抗(Pembrolizumab)的使用剂量,高效低毒。戈氏梭菌溶瘤可通过多种方式影响TME免疫原性,使TME从一个免疫抑制状态变成免疫激活状态,同时调节免疫抑制性TME,打破免疫耐受。在最佳组合条件下戈氏梭菌芽孢与帕博利珠单抗(Pembrolizumab)联合约有20%小鼠肿瘤组织彻底清除,将PD-1抗体治疗肿瘤患者的受益范围扩大,甚至对PD-1抗体治疗失败患者也有突出疗效。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例中所需要使用的附图作简单地介绍。
图1实施例1中戈氏梭菌芽孢治疗CT26.WT结肠癌荷瘤鼠模型各组肿瘤重量(a)、抑瘤率(b);数据表示为:*与Control组相比P<0.05;**与Control组相比P<0.01;
图2实施例2中戈氏梭菌芽孢联合Pembrolizumab治疗MC38结肠癌荷瘤鼠模型各组肿瘤重量(a)、肿瘤重量抑瘤率(b);数据表示为:*P<0.05;**P<0.01;
图3实施例2中戈氏梭菌芽孢联合Pembrolizumab治疗MC38结肠癌荷瘤鼠模型各组肿瘤体积变化;数据表示为:*与Control组相比P<0.05;**与Control组相比P<0.01;#与C.ghonii组相比,C.ghonii+Pembrolizumab组P<0.05;##与C.ghonii组相比,C.ghonii+Pembrolizumab组P<0.01;
图4实施例2中戈氏梭菌芽孢联合Pembrolizumab治疗MC38结肠癌荷瘤鼠模型实验终点肿瘤体积抑瘤率;数据表示为:*P<0.05;**P<0.01;
图5实施例2中戈氏梭菌芽孢联合Pembrolizumab治疗MC38结肠癌荷瘤鼠模型各批肿瘤体积抑瘤率;a图实验批次1,b图实验批次2,c图实验批次3;
图6实施例2中戈氏梭菌芽孢联合Pembrolizumab治疗MC38结肠癌荷瘤鼠模型各组肿瘤瘤体形态照片;
图7实施例3中戈氏梭菌芽孢联合Pembrolizumab不同给药顺序治疗MC38结肠癌荷瘤鼠模型各组肿瘤重量;
图8实施例3中戈氏梭菌芽孢联合Pembrolizumab不同给药顺序治疗MC38结肠癌荷瘤鼠模型各组肿瘤体积变化。
具体实施方式
下面将结合具体实施例对本发明做进一步的详细说明,所描述的实施例仅仅是本发明为了使公众便于理解所列举的一部分技术方案,这些实施例仅用于说明本发明而不用于限制本发明的保护范围,本发明的保护范围由权利要求限定。
实施例1戈氏梭菌芽孢对结肠癌荷瘤鼠模型治疗效果
材料:注射用戈氏梭菌芽孢冻干粉,菌株为MW-DCG-LCv-26菌株,该菌株保藏于澳大利亚国家计量研究院,菌株保藏号V12/001486,山东新创生物科技有限公司研制。注射用戈氏梭菌芽孢冻干粉以戈氏梭菌芽孢为活性成分,以1%蔗糖为辅料,经过-40℃4h;-35℃10min同时抽真空、-30℃10min、-25℃10min、-20℃26h、-15℃2h、-10℃10min、-5℃10min、0℃10min、10℃2h、15℃10min、20℃3h、27℃3h冻干程序制备,规格为1×108CFU/支;对照品冻干粉,批号:201803001F,山东新创生物科技有限公司研制,以1mL 1%蔗糖溶液经过上述冻干程序制备;0.9%氯化钠注射液,批号:1803122161,辰欣药业股份有限公司有售;灭菌注射用水,批号:1704242163,辰欣药业股份有限公司有售;CT26.WT结肠癌细胞,编号:3131C0001000800037,中国科学院上海生命科学研究院细胞资源中心有售;BALB/c雌性小鼠,动物合格证号:No.11401300088557(120只),No.11401300089721(60只),No.11401300089721(60只),北京华阜康生物科技股份有限公司有售。
方法:CT26.WT细胞复苏传代至所需细胞数量,制备浓度为7.5×106个/mL的接种用细胞悬液,细胞活率在90%以上用于建立BALB/c小鼠结肠癌皮下移植瘤模型,小鼠右前肢皮下接种细胞悬液0.2mL,10天左右选择肿瘤体积约0.30cm3的实验动物用于试验;成瘤合格的小鼠以抽签法随机分为4组:对照组(Control)、低剂量治疗组(L C.ghonii)、中剂量治疗组(M C.ghonii)、高剂量治疗组(H C.ghonii),每组8只;戈氏梭菌芽孢冻干粉先用0.1mL灭菌注射用水复溶后,再用质量百分比浓度0.9%氯化钠注射液配制浓度分别为5×107cfu/mL、1×108cfu/mL、2×108cfu/mL的混悬液,瘤内注射0.1mL,隔天给药1次,共给药5次,给药剂量分别为5×106cfu/次、1×107cfu/次、2×107cfu/次,对照组的对照品浓度与高剂量治疗组相同;开始给药后,每天观察动物行为、死亡或濒死等临床症状,所有存活动物于末次给药后第2天进行解剖,称量肿瘤重量,并以肿瘤重量计算抑瘤率,肿瘤重量抑瘤率(IRTW%)=(对照组平均瘤重-实验组平均瘤重)/对照组平均瘤重×100%。以上述实验方法,分别进行3批独立重复实验。
结果:实验期间,3批实验所有动物均未见濒死/死亡情况。实验结束,3批实验各组荷瘤鼠平均肿瘤重量及治疗组抑瘤率如表1、表2所示。
表1各组荷瘤鼠平均肿瘤重量(g)
实验批次 | Control组 | LC.ghonii组 | MC.ghonii组 | HC.ghonii组 |
1 | 2.85±0.59 | 1.98±0.76 | 1.84±0.67 | 2.45±0.34 |
2 | 4.15±0.51 | 2.00±0.61 | 2.23±0.78 | 2.50±0.71 |
3 | 2.69±0.52 | 1.73±0.56 | 1.72±0.42 | 1.93±0.39 |
表2治疗组肿瘤重量抑瘤率(%)
实验批次 | LC.ghonii组 | MC.ghonii组 | HC.ghonii组 |
1 | 30.40 | 35.58 | 14.12 |
2 | 51.90 | 46.17 | 39.79 |
3 | 35.46 | 35.97 | 28.06 |
与Control组相比,每批治疗组肿瘤重量均小于Control组,以M C.ghonii组抗肿瘤效果最好(图1中a)。各剂量治疗组均表现出对肿瘤生长的抑制作用,且实验批次1和3中MC.ghonii组肿瘤重量抑瘤率效果最好,实验批次2中L C.ghonii组肿瘤重量抑瘤率效果最好。根据3批次实验结果进行统计学分析发现,H C.ghonii组肿瘤重量抑瘤率与Control组相比具有显著性差异(p<0.05),L C.ghonii组和M C.ghonii组肿瘤重量抑瘤率与Control组相比具有极显著差异(p<0.01),图1中b所示。
综上所述,戈氏梭菌芽孢冻干粉对结肠癌生长具有抑制作用,且研究表明MC.ghonii组(1×107cfu/次)优于其它治疗组效果。因此,在接下来与Pembrolizumab组合用药中C.ghonii优选剂量1×107cfu/次。
实施例2戈氏梭菌芽孢联合Pembrolizumab对PD-1人源化转基因结肠癌荷瘤鼠模型治疗效果
材料:注射用戈氏梭菌芽孢冻干粉,山东新创生物科技有限公司研制。制备方法同实施例1中材料;Pembrolizumab注射液,批号:S001188,规格:100mg/4mL,美国默沙东公司有售;对照品冻干粉,批号201910002F,201803001F,山东新创生物科技有限公司研制,制备方法同实施例1中材料;0.9%氯化钠注射液,批号:1809282161,辰欣药业股份有限公司有售;灭菌注射用水,批号:1902212162,辰欣药业股份有限公司有售;MC38结肠癌细胞,货号T1917,Abm生物科技有限公司有售;C57BL/6PD-1人源化基因工程小鼠,动物合格证号:No.20170010002500(90只),No.312024300008757(35只),No.20170010001319(50只),上海南方模式生物科技股份有限公司有售。
方法:MC38细胞复苏传代至所需细胞数量,制备浓度为7.5×106个/mL的接种用细胞悬液,细胞活率在90%以上建立C57BL/6PD-1人源化基因工程小鼠结肠癌皮下移植瘤模型,小鼠右前肢皮下接种0.2mL细胞悬液,10天左右选择肿瘤体积大于0.15cm3的实验动物用于试验;采用随机原则将筛选出符合要求的动物以抽签法分为4组:对照组(Control)、戈氏梭菌芽孢治疗组(C.ghonii)、Pembrolizumab组和戈氏梭菌芽孢联合Pembrolizumab组(C.ghonii+Pembrolizumab),每组不少于5只;戈氏梭菌芽孢冻干粉先用0.1mL灭菌注射用水复溶后,再用质量百分比0.9%氯化钠注射液配制浓度1×108cfu/mL的混悬液,瘤内注射0.1mL,C.ghonii组和C.ghonii+Pembrolizumab组注射芽孢1×107cfu/次,Control组和Pembrolizumab组剂量为0cfu/次,隔1天给药1次,共给药6次;以0.9%氯化钠注射液将Pembrolizumab注射液稀释成终浓度为1mg/mL工作液,腹腔注射0.2mL,Pembrolizumab组和C.ghonii+Pembrolizumab组注射Pembrolizumab为0.2mg/次,Control组和C.ghonii组注射0.2mL 0.9%氯化钠注射液,每周给药2次,共4次。
优先给予戈氏梭菌芽孢再给予PD-1抗体,给药顺序如下:戈氏梭菌芽孢于第1天起,隔一天注射一次;Pembrolizumab于第3、6、9和13天分别进行注射。
观测及评价指标:开始给药后,每天观察动物行为、死亡或濒死情况;每隔1-2天观测肿瘤体积,并以实验末次测量的肿瘤体积计算抑瘤率;所有存活动物于末次给药第2天后进行解剖,称量肿瘤重量,并以肿瘤重量计算抑瘤率。
肿瘤重量抑瘤率(IRTW%)=(对照组平均瘤重-实验组平均瘤重)/对照组平均瘤重×100%;
肿瘤体积抑瘤率(IRTV%)=1-(实验组开始平均肿瘤体积-实验组结束平均肿瘤体积)/(对照组开始平均肿瘤体积-对照组结束平均肿瘤体积)×100%;
治愈率(%)=每组治愈的动物数/每组总实验动物数×100%(时间节点为试验结束)。
以上述实验方法,分别进行3批独立重复实验。
结果:实验期间,实验批次1中对照组1只小鼠死亡,其他各批各组未出现死亡或濒死情况。
1、各组荷瘤鼠平均肿瘤重量与抑瘤率
3批实验各组荷瘤鼠平均肿瘤重量如表3所示。各治疗组平均肿瘤重量均小于Control组,且C.ghonii+Pembrolizumab组肿瘤重量均小于任何一种药物单独治疗组,与Control组相比,极显著降低(P<0.01)。C.ghonii+Pembrolizumab组平均肿瘤重量也显著小于单独C.ghonii组和单独Pembrolizumab组(P<0.05,P<0.05)(图2中a)。
表3各组荷瘤鼠平均肿瘤重量(g)
根据3批次实验结果进行统计学分析发现各治疗组表现出对肿瘤生长的抑制作用,抑瘤率均在30%以上,抑瘤率表现为C.ghonii+Pembrolizumab组>Pembrolizumab组>C.ghonii组(图2中b)。Pembrolizumab给药相同剂量后,C.ghonii+Pembrolizumab组比单独Pembrolizumab组抑瘤率分别提高了约20%,20%,25%(表4)。
表4治疗组肿瘤重量抑瘤率(%)
实验批次 | C.ghonii组 | Pembrolizumab组 | C.ghonii+Pembrolizumab组 |
1 | 35.33 | 76.04 | 94.76 |
2 | 32.03 | 75.95 | 92.95 |
3 | 43.82 | 61.27 | 86.61 |
2、各组荷瘤鼠平均肿瘤体积与抑瘤率
实验开始前分组时各批次各组平均肿瘤体积均无显著性差异(P>0.05)。批次3实验中荷瘤鼠给药末期,与Control组相比,C.ghonii组、Pembrolizumab组和C.ghonii+Pembrolizumab肿瘤体积显著小于Control组(p=0.001,p=0.000053,p=0.000)。C.ghonii+Pembrolizumab组肿瘤体积明显小于C.ghonii组和Pembrolizumab组,且在第10天联合组与Control组平均肿瘤体积出现显著性差异,而此时C.ghonii+Pembrolizumab组肿瘤体积比Pembrolizumab组小近1倍(图3)。
以肿瘤体积计算抑瘤率,3批试验期间C.ghonii+Pembrolizumab组抑瘤率均大于其它各组抑瘤率(图4)。
3批次实验中给药后第7天,Pembrolizumab完成给药2次,Pembrolizumab给药剂量0.4mg后,C.ghonii+Pembrolizumab组抑瘤率分别为51.29%(大于50%),49.78%(约50%),59.33%(大于50%),而此时单独Pembrolizumab组抑瘤率分别为25.67%,7.68%,35.10%。单独Pembrolizumab组给药3次,Pembrolizumab给药剂量0.6mg后,其抑瘤率才达到C.ghonii+Pembrolizumab组给药2次Pembrolizumab(给药剂量0.4mg)时的抑瘤率,分别为69.31%,52.83%,53.08%(图5)。可见,C.ghonii+Pembrolizumab组显著提高了Pembrolizumab抗肿瘤效果,在获得同样治疗效果C.ghonii+Pembrolizumab组所需Pembrolizumab使用剂量降低50%,高效低毒。
3、治愈率
3批实验C.ghonii+Pembrolizumab组分别有约20%(1/6、1/5、1/6)的小鼠肿瘤完全消除(表5),至实验终点也未见肿瘤生长,而Pembrolizumab组和C.ghonii组均未出现肿瘤彻底消失情况,治愈率0%。这可能与戈氏梭菌芽孢在肿瘤乏氧区萌发,通过多种方式影响TME免疫原性,使TME从一个免疫抑制状态变成免疫激活状态,同时调节免疫抑制性TME,打破免疫耐受。联合Pembrolizumab后可将PD-1抗体治疗效果进一步放大,约20%小鼠被治愈。戈氏梭菌有望成为肿瘤病人免疫治疗优秀的“增敏剂”。各组肿瘤瘤体形态见图6。
表5治愈率(%)
实施例3戈氏梭菌芽孢和Pembrolizumab给药顺序对PD-1人源化转基因结肠癌荷瘤鼠模型治疗效果的影响
材料:注射用戈氏梭菌芽孢冻干粉,山东新创生物科技有限公司研制,制备方法同实施例1中材料;Pembrolizumab注射液,批号:S006648,规格:100mg/4mL,美国默沙东公司有售;对照品冻干粉,批号201910002F,山东新创生物科技有限公司研制,制备方法同实施例1中材料;0.9%氯化钠注射液,批号:J18070104,山东华鲁制药有限公司有售;灭菌注射用水,批号:1902212162,辰欣药业股份有限公司有售;MC38结肠癌细胞,货号T1917,Abm生物科技有限公司有售;C57BL/6PD-1人源化基因工程小鼠,动物合格证号:No.20170010002500(90只),上海南方模式生物科技股份有限公司有售。
方法:C57BL/6PD-1人源化基因工程小鼠MC38结肠癌皮下移植瘤模型建立方法同实施例2;以抽签法将建模成功的小鼠随机分为A对照组(Control)、B戈氏梭菌芽孢组(C.ghonii)、C Pembrolizumab组、D先Pembrolizumab后C.ghonii组、E先C.ghonii后Pembrolizumab组、F C.ghonii+Pembrolizumab同时组,每组8只;C.ghonii给药剂量分别为0cfu/次、1×107cfu/次、0cfu/次、1×107cfu/次、1×107cfu/次和1×107cfu/次,给药容量为0.1mL/次,隔1天给药1次;1mg/mLPembrolizumab注射液腹腔给药0.2mL,剂量为0.2mg/次,隔1天给药1次。第一阶段给药过程如表6所示。
表6第一阶段给药过程
第一阶段给药结束,观察6天后,第二阶段重复第一阶段的给药过程。
观测及评价指标:开始给药后,每天观察动物行为、死亡或濒死情况,每隔1-2天观测肿瘤体积,并以实验末次测量的肿瘤体积计算抑瘤率。所有存活动物于第二阶段末次给药后第8天进行解剖。称量肿瘤重量,并以肿瘤重量计算抑瘤率。
肿瘤重量抑瘤率(IRTW%)=(对照组平均瘤重-实验组平均瘤重)/对照组平均瘤重×100%;
治愈率(%)=每组治愈的动物数/每组总实验动物数×100%(时间节点为试验结束)。
结果:实验期间,Control组因肿瘤过大或破溃出现4只动物死亡,其它各组未出现动物死亡或濒死情况。
1、肿瘤重量与抑瘤率
与Control组相比,C.ghonii组、Pembrolizumab组、先Pembrolizumab后C.ghonii组、先C.ghonii后Pembrolizumab组、C.ghonii+Pembrolizumab组肿瘤重量均显著小于Control组(p=0.005,p=0.000,p=0.000,p=0.000,p=0.000)。各组肿瘤重量检测结果见表7,图7。
表7肿瘤重量
备注:与Control组相比,**p<0.01。
根据肿瘤重量计算抑瘤率,结果显示C.ghonii组、Pembrolizumab组、先Pembrolizumab后C.ghonii组、先C.ghonii后Pembrolizumab组、C.ghonii+Pembrolizumab组抑瘤率分别为37.39%、76.20%、87.26%、92.71%和92.19%,各治疗组均表现出对肿瘤生长的明显抑制作用,见表8。
表8抑瘤率
2、肿瘤体积与抑瘤率
治疗前,各组荷瘤鼠肿瘤体积均无显著差异(p>0.05)。给药末期,与Control组相比,C.ghonii组、Pembrolizumab组、先Pembrolizumab后C.ghonii组、先C.ghonii后Pembrolizumab组、C.ghonii+Pembrolizumab同时组肿瘤体积均显著小于Control组(p=0.002,p=0.000,p=0.000,p=0.000,p=0.000,图8)。抑瘤率表现为先C.ghonii后Pembrolizumab组>C.ghonii+Pembrolizumab同时组>先Pembrolizumab后C.ghonii组>Pembrolizumab组>Control组,见表9。
表9抑瘤率
3、治愈率
试验期间,各组的治愈率见表10。
表10治愈率
先C.ghonii后Pembrolizumab组小鼠肿瘤治愈率明显高于其他组,为先Pembrolizumab后C.ghonii组和C.ghonii+Pembrolizumab同时组治愈率的2倍。这可能是由于先瘤内给予戈氏梭菌芽孢有效而不加区分溶解肿瘤组织,破坏TME,同时细菌溶瘤后也会募集免疫细胞向TME的浸润,改变了TME中免疫细胞浸润程度和组成,包括细胞裂解产物会吸引大量的CD8+T细胞聚集,在溶瘤细菌之后应用PD-1抗体治疗,避免免疫治疗中的免疫抑制,发动T细胞对肿瘤细胞的大规模攻击,从而发挥“双重”抗癌功效。因此溶瘤细菌与免疫抑制剂联合使用时给药顺序对于肿瘤的治疗效果也十分关键。
综上,优先使用戈氏梭菌芽孢治疗再与免疫疗法联用可以增强抗肿瘤功效,降低免疫药物的使用剂量,高效低毒。
Claims (6)
1.戈氏梭菌芽孢联合帕博利珠单抗在制备治疗结肠癌医药制品中的应用,其特征在于,所述戈氏梭菌包括戈氏梭菌MW-DCG-LCv-26菌株;
所述戈氏梭菌MW-DCG-LCv-26菌株保藏于澳大利亚国家计量研究院,菌株编号为V12/001486;
所述帕博利珠单抗为PD-1抗体注射液。
2.根据权利要求1所述的应用,其特征在于,所述治疗结肠癌医药制品中戈氏梭菌芽孢剂量为1×107cfu/次;所述PD-1抗体注射液的剂量为0.2mg/次。
3.一种治疗结肠癌的药物,其特征在于,所述药物中的药效成分包括戈氏梭菌芽孢和帕博利珠单抗;所述的戈氏梭菌为戈氏梭菌MW-DCG-LCv-26菌株;
所述戈氏梭菌MW-DCG-LCv-26菌株保藏于澳大利亚国家计量研究院,菌株编号为V12/001486;
所述帕博利珠单抗为PD-1抗体注射液。
4.如权利要求3所述的药物,其特征在于,所述戈氏梭菌的芽孢形式为冻干粉剂,其辅料为1%蔗糖;
所述冻干粉剂的冻干程序为:冻结阶段-40℃ 4h;-35℃ 10min同时抽真空、-30℃10min、-25℃ 10min、-20℃ 26h、-15℃ 2h、-10℃ 10min、-5℃10 min、0℃ 10min、10℃2h、15℃ 10min、20℃ 3h、27℃ 3h冻干制备。
5.如权利要求3所述的药物,其特征在于,所述帕博利珠单抗溶液的溶媒为质量百分比浓度0.9%的氯化钠注射液;戈氏梭菌芽孢冻干粉的溶媒为灭菌注射用水和质量百分比浓度0.9%氯化钠注射液。
6.如权利要求3所述的药物,其特征在于,所述戈氏梭菌芽孢联合帕博利珠单抗的使用顺序为先戈氏梭菌芽孢后帕博利珠单抗。
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