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CN113186225B - Pd1/pdl1单抗致免疫性心肌炎模型及制备方法 - Google Patents

Pd1/pdl1单抗致免疫性心肌炎模型及制备方法 Download PDF

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CN113186225B
CN113186225B CN202110524213.4A CN202110524213A CN113186225B CN 113186225 B CN113186225 B CN 113186225B CN 202110524213 A CN202110524213 A CN 202110524213A CN 113186225 B CN113186225 B CN 113186225B
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monoclonal antibody
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pdl1
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王怡宁
曲婵娟
王健
李颜玉
周慷
曹剑
林路
李潇
金征宇
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Peking Union Medical College Hospital Chinese Academy of Medical Sciences
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Abstract

本发明提供一种PD1/PDL1单抗致免疫性心肌炎模型的制备方法，包括通过AAV9介导模型心肌组织高表达PDL1，再对心肌组织高表达PDL1的模型应用PD‑1/PD‑L1单抗造模。本发明还提供了该制备方法所制备的动物模型的用途。本发明制备的模型真实模拟应用PD‑1/PD‑L1单抗发生免疫性心肌炎的患者的发病机制和临床经过，模型更接近于临床患者病理生理状态，成模率高并可动态监测。

Description

PD1/PDL1单抗致免疫性心肌炎模型及制备方法
技术领域
本发明涉及生物医药领域，具体涉及PD1/PDL1单抗致免疫性心肌炎模型及制备方法。
背景技术
PD-1/PD-L1单抗是肿瘤免疫治疗史上的里程碑，为新近备受瞩目的免疫检查点抑制剂之一，是肺癌、黑色素瘤等许多恶性肿瘤的一、二线治疗药物，明显延长了患者总生存期和中位无进展生存期，且适应证仍在增加，但是其副作用也逐渐受到关注，其中心肌炎是最为致命的，因其干扰了心脏正常的电机械功能，且心肌再生能力差，发生免疫性损伤后通常修复不良，因此需要高度警惕肿瘤免疫治疗导致的免疫性心肌炎，规定如应用PD-1/PD-L1单抗后发生心肌炎，需要永久性停药，并立即给予大剂量激素及免疫抑制剂治疗，然而预后仍旧很差，一旦发生，死亡率为40％，当与CTLA-4单抗如伊匹木单抗联用时，死亡率可高达67％。据文献报道PD-1/PD-L1单抗单药应用免疫性心肌炎发病率约万分之六，与CTLA-4单抗联用则增至千分之三，但是随着PD-1/PD-L1单抗应用增多，关于免疫性心肌炎的报道逐渐增加，由于我国恶性肿瘤患者基数大，近年来应用PD-1/PD-L1单抗的患者比例迅速增加，因此发生心肌炎的患者数量多，其高致死性已成为我国肿瘤慢病管理的难题。
由于自然状态下患者应用PD-1/PD-L1单抗发生自身免疫性心肌炎概率低，要想研究其机制、早期预警标志物及新的治疗方式有赖于动物模型的成功建立。我们前期的动物试验结果也说明直接给予PD-1/PD-L1单抗造模成功率非常之低。Keynote024、042等多项研究显示肿瘤组织TPS(PDL1表达率)越高，应用PD-1/PD-L1单抗抗肿瘤效果越好。文献中提及心肌组织表达PDL1，是应用PD-1/PD-L1单抗导致免疫性心肌炎的原因之一；采用PDL1Ig基因重组腺病毒为载体，将PDL1Ig基因转入BN大鼠树突状细胞同时输注受体，以Lewis大鼠为受体，行同种肾移植，转染组为实验组，发现PDL1基因修饰的DC(树突状细胞)可显著延长移植肾存活时间。说明如果能够使物种心肌组织高表达PDL1，那么应用PD-1/PD-L1单抗发生免疫性心肌炎的概率也大大提高。
腺相关病毒是微小病毒科家族的成员之一。这一家族成员是一类微小、无被膜及具有二十面体结构的病毒。病毒颗粒的直径在20～26nm之间，含有大小在4.7～6kb之间的线状单链DNA基因组。它的复制和增殖依赖于辅助病毒的存在。作为一种基因导入系统，AAV病毒(腺相关病毒)载体具有安全性好，免疫原性低，能感染分裂细胞和非分裂细胞，能介导基因的长期稳定表达等优点。其中，AAV9是公认的高效特异性靶向心脏表达的病毒载体血清型。AAV9不仅会感染心肌细胞，也会感染心脏其他细胞,本团队拟用心肌细胞特异性启动子cTNT来启动目的基因表达。
目前PD-1/PD-L1单抗相关的自身免疫性心肌炎多为药物临床试验的安全性评价的报道，或临床个案报道，只有为数甚少的基础和临床研究，且基础研究中采用的动物模型为PD-1/PD-L1敲除小鼠模型或与放疗等联用所导致的小鼠心脏毒性动物模型，与真实世界PD-1/PD-L1单抗所致的免疫性心肌炎发病机制、病理及临床表现有所差别。
发明内容
本发明提供一种PD1/PDL1单抗致免疫性心肌炎模型的制备方法及制备的模型，真实模拟应用PD-1/PD-L1单抗发生免疫性心肌炎的患者的发病机制和临床经过，模型更接近于临床患者病理生理状态，成模率高并可通过心脏磁共振来动态监测心肌病变。
本发明的一个方面，提供一种PD1/PDL1单抗致免疫性心肌炎模型的制备方法，包括通过AAV9介导模型心肌组织高表达PDL1，再对心肌组织高表达PDL1的模型应用PD-1/PD-L1单抗造模。
以上所述的制备方法，其中，所述模型为动物模型。
以上所述的制备方法，其中，具体步骤包括：
(1)PDL1Ig基因重组腺病毒载体的构建；
(2)将步骤(1)中构建的载体静脉注射所述动物模型，使所述动物模型心肌组织高表达PDL1，再腹腔注射PD1或PDL1单抗造模。
以上所述的制备方法，其中，所述步骤(1)中构建的载体为CD274基因过表达的AAV9腺病毒载体。
以上所述的制备方法，其中，所述CD274基因的过表达通过心肌细胞特异性启动子cTNT启动。
以上所述的制备方法，其中，所述动物模型为小鼠。
以上所述的制备方法，其中，所述小鼠为雄性小鼠。
以上任一所述制备方法制备的模型在与自身免疫性心肌炎方向相关用途。
有益效果
本申请采用PDL1(CD274)过表达的AAV9病毒尾静脉注射小鼠，使心肌组织高表达PDL1，再腹腔注射PD1或PDL1单抗，真实模拟应用PD-1/PD-L1单抗发生免疫性心肌炎的患者的发病机制和临床经过，构建的免疫性心肌炎动物模型，具有成模率高和可动态监测的优点，可用于临床研究PD-1/PD-L1单抗所致的免疫性心肌炎的发病机制、早期预警标志物和治疗等。
附图说明
图1.本实施例步骤一中构建的包含目的基因的pHBAAV-TNT-3flag-P2A-EGFP(lsz)载体图谱；
图2本发明实施例中A组小鼠心肌细胞病理切片HE染色和CD3免疫组化：可见心肌细胞肌丝溶解，巨噬细胞增多，T细胞浸润，巨细胞存在；
图3本发明实施例中A组小鼠心肌细胞病理切片HE染色和CD3免疫组化：再示T细胞浸润；
图4本发明实施例中A组小鼠心脏磁共振T2加权像长T2信号；
图5本发明实施例中A组小鼠心脏磁共振T2mapping值增高。
具体实施方式
以下实施例用于说明本发明，但不用来限制本发明的范围。若未特别指明，实施例中所用的技术手段为本领域技术人员所熟知的常规手段。所述试剂和生物材料，如无特殊说明，均可从商业途径获得。
实施例1 PDL1Ig基因重组腺病毒载体的构建
一、载体构建的主要流程如下：
1.选择对应的载体，并进行设计目的片段PCR引物；
具体使用的引物序列如下：
Figure BDA0003065136380000042
2.选用合适的限制性内切酶进行酶切载体，琼脂糖凝胶回收得到纯化的线性化载体；
根据如下表中顺序依次加入每种试剂，轻轻吸打混匀，置于37℃水浴锅中反应1-2h；酶切结束之后进行琼脂糖凝胶电泳，回收目的片段；
载体酶切体系如下：
Figure BDA0003065136380000041
注：限制性内切酶使用的量及酶切的时间可根据酶活性进行调整；若是单酶切，则进行相应体系的调整。
3.根据设计的引物进行目的片段PCR，琼脂糖凝胶回收得到正确大小的目的片段；
4.将线性化载体和目的片段按照同源重组或者T4连接的方法进行连接；
5.转化感受态DH5a或者stbl3，菌液涂板，培养12-16h；
6.挑选单克隆行进菌落验证；
7.选择菌落验证正确的阳性克隆进行测序；
8.测序正确的克隆样品进行质粒抽提。
二、构建的载体及目的基因信息
本实施例步骤一中构建的包含目的基因的pHBAAV-TNT-3flag-P2A-EGFP(lsz)载体图谱如图1所示。目的基因序列如SEQ ID NO.1所示。
实施例2转染小鼠构建模型
2.1实验小鼠
本实施例选择生长状况良好、体质健康的小鼠作为实验对象，具有廉价易得、来源广泛且易于饲养的特点，为近交系C57/B6J小鼠，购买于华阜康生物科技股份有限公司，体重20-30g，标准化程度高，便于给药且采样容易，且优选为雄性小鼠，且更为健壮，且符合PD1单抗引起的免疫性心肌炎多发于男性的特点，在试验过程中可尽量避免由于自身体质不足而导致的试验误差。
2.2饲养条件
饲养环境温度为22-34℃，饲养环境湿度为40％-70％。实验小鼠为自由采食和饮水。
2.3造模过程
将SPF级C57/BL6雄性小鼠80只，适应性饲养1周，每天更换垫料，在此期间喂养基础饲料和纯净水。
将上个步骤的小鼠分为4组：
A组：30只，尾静脉注射PDL1过表达CD274基因的AAV9 100μl，3周后腹腔注射PD1单抗10mg/kg，每周一次，共3次。
B组：30只，尾静脉注射PDL1过表达表达CD274基因的AAV9对照组100μl，3周后腹腔注射PD1单抗10mg/kg，每周一次，共3次。
C组：30只，腹腔注射PD1单抗10mg/kg，每周一次，共3次；
D组：30只，腹腔注射小鼠IgG 10mg/kg，每周一次，共3次。继续喂养基础饲料和纯净水。
每周1次，行心脏磁共振、心电图后，断颈处死小鼠10只，取心脏行HE染色。
2.4结果
采用心脏病理判断，心脏病理为判断造模成功与否的金标准。
A组：注射1次后10只小鼠行心脏病理5只示心肌细胞肌丝溶解，T细胞浸润，呈弥漫性，生理情况下心肌不存在T细胞，小鼠无死亡，造模成功率50％；注射2次后10只小鼠行心脏病理8只示心肌细胞肌丝溶解，T细胞浸润，呈弥漫性，造模成功率80％；注射3次后10只小鼠行心脏病理均示心肌细胞肌丝溶解，T细胞浸润，呈弥漫性，造模成功率100％；
B组：注射1次后10只小鼠行心脏病理1只示心肌细胞肌丝溶解，T细胞浸润，呈弥漫性，生理情况下心肌不存在T细胞，小鼠无死亡，造模成功率10％；注射2次后10只小鼠行心脏病理4只示心肌细胞肌丝溶解，T细胞浸润，呈弥漫性，造模成功率40％；注射3次后10只小鼠有7只行心脏病理均示心肌细胞肌丝溶解，T细胞浸润，呈弥漫性，小鼠无死亡，造模成功率70％，程度较A组轻。
C组：注射1次后10只小鼠行心脏病理2只示心肌细胞肌丝溶解，T细胞浸润，呈弥漫性，生理情况下心肌不存在T细胞，小鼠无死亡，造模成功率20％；注射2次后10只小鼠行心脏病理5只示心肌细胞肌丝溶解，T细胞浸润，呈弥漫性，造模成功率50％；注射3次后10只小鼠有6只行心脏病理均示心肌细胞肌丝溶解，T细胞浸润，呈弥漫性，小鼠无死亡，造模成功率60％，程度较A组轻。
D组：注射1次后10只小鼠行心脏病理正常；注射2次后10只小鼠行心脏病理正常；注射3次后10只小鼠行心脏病理正常。
虽然，上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述，但在本发明基础上，可以对之作一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。
SEQUENCE LISTING
<110> 中国医学科学院北京协和医院
<120> PD1/PDL1单抗致免疫性心肌炎模型及制备方法
<130> P210065
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<170> PatentIn version 3.5
<210> 1
<211> 873
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<213> 人工序列（Synthetic sequences）
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agattccctg tagaacggga gctggacctg cttgcgttag tggtgtactg ggaaaaggaa 180
gatgagcaag tgattcagtt tgtggcagga gaggaggacc ttaagcctca gcacagcaac 240
ttcaggggga gagcctcgct gccaaaggac cagcttttga agggaaatgc tgcccttcag 300
atcacagacg tcaagctgca ggacgcaggc gtttactgct gcataatcag ctacggtggt 360
gcggactaca agcgaatcac gctgaaagtc aatgccccat accgcaaaat caaccagaga 420
atttccgtgg atccagccac ttctgagcat gaactaatat gtcaggccga gggttatcca 480
gaagctgagg taatctggac aaacagtgac caccaacccg tgagtgggaa gagaagtgtc 540
accacttccc ggacagaggg gatgcttctc aatgtgacca gcagtctgag ggtcaacgcc 600
acagcgaatg atgttttcta ctgtacgttt tggagatcac agccagggca aaaccacaca 660
gcggagctga tcatcccaga actgcctgca acacatcctc cacagaacag gactcactgg 720
gtgcttctgg gatccatcct gttgttcctc attgtagtgt ccacggtcct cctcttcttg 780
agaaaacaag tgagaatgct agatgtggag aaatgtggcg ttgaagatac aagctcaaaa 840
aaccgaaatg atacacaatt cgaggagacg taa 873
<210> 2
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<212> DNA
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ttaatacgac tcactatagg ctagcgccac catgaggata tttgc 45
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ccttgtagtc aagcttggtg ctagccgtct cctcgaattg tgtat 45

Claims (7)

1.一种PD1/PDL1单抗致免疫性心肌炎模型的制备方法，其特征在于，包括通过AAV9介导模型心肌组织高表达PDL1，再对心肌组织高表达PDL1的模型应用PD-1/PD-L1单抗造模。
2.如权利要求1所述的制备方法，其特征在于，所述模型为动物模型。
3.如权利要求2所述的制备方法，其特征在于，具体步骤包括：
(1)PDL1Ig基因重组腺病毒载体的构建；
(2)将步骤(1)中构建的载体静脉注射所述动物模型，使所述动物模型心肌组织高表达PDL1，再腹腔注射PD1或PDL1单抗造模。
4.如权利要求3所述的制备方法，其特征在于，所述步骤(1)中构建的载体为CD274基因过表达的AAV9腺病毒载体。
5.如权利要求4所述的制备方法，其特征在于，所述CD274基因的过表达通过心肌细胞特异性启动子cTNT启动。
6.如权利要求3所述的制备方法，其特征在于，所述动物模型为小鼠。
7.如权利要求6所述的制备方法，其特征在于，所述小鼠为雄性小鼠。
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