CN111777686A - 用于治疗卵巢癌的folr1-msln双靶向性car-t细胞、嵌合抗原受体及载体 - Google Patents
用于治疗卵巢癌的folr1-msln双靶向性car-t细胞、嵌合抗原受体及载体 Download PDFInfo
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- CN111777686A CN111777686A CN202010677570.XA CN202010677570A CN111777686A CN 111777686 A CN111777686 A CN 111777686A CN 202010677570 A CN202010677570 A CN 202010677570A CN 111777686 A CN111777686 A CN 111777686A
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Abstract
本发明提供一种嵌合抗原受体，包括依次连接的单链抗体区段、胞外铰链区、跨膜区和胞内免疫受体酪氨酸活化基序、IL‑12基因区；其中，单链抗体区段选自叶酸受体α(FOLR1)单链抗体、间皮素(MSLN)单链抗体或者叶酸受体α单链抗体‑间皮素单链抗体串联复合体中的一个。本发明还提供了包含上述嵌合抗原受体的质粒载体、慢病毒载体，以及三种CAR‑T细胞。基于本发明的FOLR1‑CAR、MSLN‑CAR和Tandem‑CAR三种CAR‑T均能明显分泌IL‑12并抑制肿瘤的生长，其中Tandem‑CAR效果最为明显。
Description
技术领域
本发明属于涉及免疫学和分子生物学领域，具体而言，涉及一种用于治疗卵巢癌的FOLR1-MSLN双靶向性CAR-T细胞、嵌合抗原受体及载体。
背景技术
卵巢癌的死亡率位于女性生殖***三大恶性肿瘤的首位，其中90％为上皮性卵巢癌(epithelial ovarian cancer,EOC)，由于其发病隐匿，70％的患者就诊时已为晚期并伴有远处转移。一线治疗后缓解的患者中仍有55％～75％在2年内复发，导致其五年生存率不到40％，因此探索新的治疗方法具有重要意义。
1989年Gross第一次提出“嵌合受体”的概念，即将T细胞受体的可变区用单链抗体替代，这样修饰的T细胞便具备了抗原靶向性功能。嵌合抗原受体T细胞(chimericantigenreceptor modified T cells,CAR-T)的设计由此诞生，CAR-T可定向识别肿瘤细胞并具有特异性的杀伤功能。CAR基因由胞外的单链可变区(single chain variablefragment,scFv)和胞内的活化基序组成，经过这样修饰的T细胞解决了肿瘤细胞MHC(MajorHistocompatibility Complex)I类分子丢失而产生的免疫逃逸问题，因而具有巨大的应用前景。经过近三十多年的发展，2011年开发出的第二代CAR-T在急性淋巴细胞白血病的治疗上取得了近90％的完全缓解率，开启了人类抗肿瘤历史上的新纪元，为恶性肿瘤患者带来了希望。
然而CAR-T治疗方法在实体瘤上却遭遇困境，主要面临的瓶颈有：1)实体瘤的异质性，单个靶点的CAR-T治疗后易复发(抗原逃逸)；2)实体瘤的免疫抑制微环境(缺氧、低pH、Treg细胞、吲哚胺2,3-双加氧酶)导致T细胞激活不足和/或T细胞抑制；3)T细胞的持久性不足，扩增不足；4)实体瘤具有包膜，T细胞难浸润、难归巢。
发明内容
针对以上现有技术中存在的问题，本发明首先经过前期生物信息学的筛选和临床样本的验证，选择了两种在EOC中高表达的肿瘤相关抗原(tumor-associated antigen,TAA)：叶酸受体α(folate receptor alpha,FOLR1)和间皮素(mesothelin,MSLN)，并进一步构建同时靶向FOLR1和MSLN的串联型CAR-T(Tandem CAR)，并在此基础上设计了同时靶向FOLR1与MSLN的串联型CAR-T(Tandem CAR-T)，从而使CAR-T细胞具有更广的杀伤覆盖率。同时，本发明通过IL-12的免疫激活作用(如激活细胞毒性T细胞，募集B细胞、NK细胞、巨噬细胞等)，进一步强化肿瘤微环境中T细胞的免疫应答，CAR-T细胞的存活和扩增能力将进一步提高，从而解决了CAR-T的浸润和激活问题。
本发明提供的技术方案如下：
一种嵌合抗原受体，包括依次连接的单链抗体区段、胞外铰链区(Hing area)、跨膜区和胞内免疫受体酪氨酸活化基序(ITAM)；其中，
所述单链抗体区段选自叶酸受体1单链抗体(FOLR1scFV)、间皮素单链抗体(MSLNscFV)或者叶酸受体1单链抗体(FOLR1scFV)-间皮素单链抗体(MSLN scFV)串联复合体中的一个。通过SNU119细胞移植瘤模型验证，活体成像结果显示与Mock-T组相比，FOLR1-CAR、MSLN-CAR和Tandem-CAR(以FOLR1scFV-MSLN scFV串联复合体为嵌合抗原受体制备的CAR)三种CAR-T均能明显抑制肿瘤的生长(P<0.0001)。
在根据本发明的一个实施方案中，所述胞内免疫受体酪氨酸活化基序(ITAM)由CD28肽段、4-1BB肽段和CD3ζ链肽段依次连接构成。由于包含了CD28、4-1BB两个共刺激分子，使CAR-T细胞的活化能力和持久性更强。
优选地，在CD3ζ链肽段之后还连接有IL-12肽段。CAR-T细胞表达IL-12并分泌高水平的细胞因子能够显著促进抗肿瘤效应。
在根据本发明的一个实施方案中，所述叶酸受体1单链抗体(FOLR1scFV)的氨基酸序列为SEQ ID NO:1；
所述间皮素单链抗体(MSLN scFV)的氨基酸序列为SEQ ID NO:2；
所述叶酸受体1单链抗体(FOLR1scFV)-间皮素单链抗体(MSLN scFV)串联复合体中叶酸受体1单链抗体(FOLR1scFV)和间皮素单链抗体(MSLN scFV)通过Llinker肽段连接，所述Linker肽段的氨基酸序列为SEQ ID NO:3。
本发明还提供了一种用于表达上述的嵌合抗原受体的质粒载体，包含
单链抗体区段的编码基因、胞内免疫受体酪氨酸活化基序的编码基因和慢病毒质粒；其中，所述单链抗体区段的编码基因选自叶酸受体1单链抗体(FOLR1scFV)的编码基因、间皮素单链抗体(MSLN scFV)的编码基因或叶酸受体1单链抗体(FOLR1scFV)-间皮素单链抗体(MSLN scFV)串联复合体的编码基因中的一种。所述慢病毒质粒优选为pCDH。
在根据本发明的一个实施方案中，所述胞内免疫受体酪氨酸活化基序的编码基因的核苷酸序列为SEQ ID NO4。
在根据本发明的一个实施方案中，所述叶酸受体1单链抗体(FOLR1scFV)的编码基因的核苷酸序列为SEQ ID NO:5；
所述间皮素单链抗体(MSLN scFV)的编码基因的核苷酸序列为SEQ ID NO:6。
在根据本发明的一个实施方案中，所述叶酸受体1单链抗体(FOLR1scFV)与间皮素单链抗体(MSLN scFV)串联复合体的编码基因的核苷酸序列为SEQ ID NO:7。优选地，所述联复合体的氨基酸序列为SEQ ID NO:8.
本发明还提供了一种慢病毒载体，所述慢病毒载体包含编码上述的嵌合抗原受体的编码基因或者由上述的质粒载体转染细胞后包装形成。
本发明进一步提供了一种用于治疗卵巢癌的CAR-T细胞，所述CAR-T细胞是通过上述的慢病毒载体感染CD3阳性T细胞得到。
本发明提供的技术方案具有以下有益效果：
1)成功构建了Tandem-CAR、FOLR1-CAR、MSLN-CAR的慢病毒载体，经测序验证正确，包装慢病毒并感染CD3细胞，建立了不同种类的CAR-T。
2)LDH实验表明，相比于对照组和两个单靶点CAR-T，Tandem-CAR细胞能明显杀伤FOLR1和MSLN同时高表达的SNU119细胞，而对FOLR1和MSLN低表达的A2780细胞杀伤率较低。
3)流式细胞检测IL-2、IL-12、TNF-α、IFN-γ水平，结果表明与SNU119细胞共培养后，Tandem-CAR释放细胞因子的阳性比例最高，三种CAR-T均成功表达了IL-12，而Mock-T细胞(未转染病毒的T细胞)几乎不表达IL-12。三种CAR-T表达IL-2、TNF-α、IFN-γ阳性细胞的比例均明显高于对照组(Mock-T)。
4)小鼠实验表明相比于Mock-T组，三种CAR-T均能有效抑制肿瘤的生长，MSLN-CAR的治疗作用体现为肿瘤先缩小后增长，FOLR1-CAR和Tandem-CAR治疗组肿瘤荧光强度均持续减小。Tandem-CAR的治疗效果最为明显，其中有1例小鼠的肿瘤发生消退。
附图说明
图1为单CAR和Tandem-CAR示意图；
图2为荧光显微镜下观察48h转染结果示意图，其中，放大倍率为×100倍；
图3为三种病毒感染293T细胞72h后流式细胞术测滴度示意图；
图4为免疫磁珠从PBMC中分选CD3示意图；
图5为F(ab')2检测不同CAR的表达率示意图；
图6为荧光显微镜下观察感染的CD3细胞示意图，其中放大倍率为×100；
图7为三种不同肿瘤细胞靶抗原的表达水平示意图；
图8为不同效靶比(E/T)下四种T细胞的杀伤率示意图，其中，*P<0.05，*P<0.01，***P<0.001，****P<0.0001；
图9为T细胞与SNU119肿瘤细胞共培养后四种细胞因子阳性细胞的比例示意图，其中，*P<0.05，**P<0.01，***P<0.001，****P<0.0001；
图10为SNU119卵巢癌细胞荷瘤后CAR-T的体内治疗效果比较示意图；
图11为活体成像荧光值比较示意图，其中，*P<0.05，**P<0.01，***P<0.001，****P<0.0001。
具体实施方式
以下实施例用于说明本申请，但不用来限制本申请的范围。
下面将更详细地描述本申请的具体实施例。提供这些实施例是为了能够更透彻地理解本申请，并且能够将本申请的范围完整的传达给本领域的技术人员。
如在通篇说明书及权利要求当中所提及的“包含”或“包括”为一开放式用语，故应解释成“包含但不限定于”。说明书后续描述为实施本申请的较佳实施方式，然所述描述乃以说明书的一般原则为目的，并非用以限定本申请的范围。本申请的保护范围当视所附权利要求所界定者为准。
如无特殊说明，本发明中所用到的试剂均可以商购途径购买得到。
细胞及试剂耗材
实验仪器
相关试剂配置
1)LB液体培养基：取NaCl、胰蛋白胨、酵母提取物各10g，加入1L的ddH2O，充分溶解至溶液澄亮透明，分装为100ml每锥形瓶，121℃高温高压30min，做好标记并密封于4℃。
2)LB固体培养基：于配置的100ml LB液体培养基中加入1％的Agar，灭菌完成后，置于55℃水浴锅中，待温度下降后加入0.1％Amp，充分摇匀迅速倒10个板子，凝固后封口胶封边，4℃保存。
3)人源化FOLR1-scFv(氨基酸序列为SEQ ID NO:1，核苷酸序列为SDQ ID NO:5)、MSLN-scFv(氨基酸序列为SEQ ID NO:2，核苷酸序列为SEQ ID NO:6)、Tandem-scFv序列(氨基酸序列为SEQ ID NO:8，核苷酸序列为SEQ ID NO:7)由北京擎科生物合成并测序验证，其中，叶酸受体1单链抗体(FOLR1scFV)和间皮素单链抗体(MSLN scFV)通过Llinker肽段连接，所述Linker肽段的氨基酸序列为SEQ ID NO:3；酪氨酸活化基序(ITAM)氨基酸序列SEQID NO:4。
实施例1慢病毒载体构建
一、质粒载体的构建和验证
1、重组质粒FOLR1-CAR、MSLN-CAR和Tandem-CAR的构建的构建流程：利用PCR搭桥的基因合成方法，人工合成CD8信号肽、单链抗体(FOLR1-scFv或MSLN-scFv或Tandem-scFv)、CD8a铰链区、CD28-41BB-CD3z CAR结构、IL-12基因序列，在其上游引入XbaI酶切位点，下游***NotI酶切位点，委托北京擎科生物公司合成，将其装入用XbaI和NotI双酶切的pCDH载体中(购自Addgene公司)，
PCDH。
2、大量抽提质粒pCDH
1)取少量冻存的甘油菌接种于含有Amp抗性的LB固体培养板上，置于37℃孵箱过夜培养。
2)次日下午挑菌，接种到150ml含有Amp的LB液体培养基中，置于37℃摇床，260rpm，摇菌18h。
3)第三日使用天根试剂盒大抽质粒，首先将洗脱缓冲液TB放入66℃水浴锅中预热。
4)分装菌液至干净的50ml的离心管中，做好标记，8000rpm离心3min收菌，弃上清，重复三次后用吸水纸吸干净管壁的液体。
5)移液器取10ml溶液P1(已加入RNase A)加入到离心管中,上下颠倒2次后迅速放置在涡旋仪上使菌块混匀。
6)取10ml的P2溶液加入到离心管中，上下颠倒10次，室温放置5min保证菌体充***解。
7)取10ml的P4溶液加入到离心管中，上下颠倒10次以混匀菌液，室温放置15min后可看到大量白色絮状沉淀。8,200rpm离心35min后可看到管底的白色沉淀，用移液枪将全部溶液加入到滤器CS1中进行过滤，取一干净的50ml离心管收集滤液。
8)取3ml ER溶液(去内毒素)加入到离心管中，上下翻转直至溶液出现均匀的黄色。
9)取9ml的异丙醇加入到离心管中，上下翻转溶液以混匀，每次取10ml液体加到吸附柱CP6中(平衡液已处理)。室温8,200rpm离心2min后弃废液，逐次将全部液体小心过柱，此时质粒DNA均吸附于CP6的柱中。
10)取10ml ED缓冲液加入到吸附柱CP6中，室温8,200rpm离心2min后弃废液，将吸附柱放回收集管并重复操作一次。
11)取10ml漂洗液PW(无水乙醇已加)加入到吸附柱CP6中，室温8,200rpm离心2min后弃废液，将吸附柱放回收集管并重复操作一次。
12)将吸附柱CP6放回收集管中并以8,200rpm离心5min，打开吸附柱CP6的盖子于室温中放5分钟，使吸附膜上的残余漂洗液能充分晾干。
13)取一干净的50ml离心管并将吸附柱CP6放入，吸取1ml之前预热好的洗脱缓冲液TB，小心的滴加在吸附膜的各个部位并放置6min，然后以8,200rpm的速度离心6min。将管中液体再次滴加到吸附膜上重复洗脱一次，最后将离心管中的液体转移至灭菌的1.5ml离心管，分装后-20℃长期保存。
3、酶切质粒骨架
1)采用如下体系37℃酶切2h
2)凝胶电泳：取100ml的1×TAE，加入2g琼脂糖粉，微波炉加热至琼脂糖完全融化，待温度下降至55℃，加入3ul的GoldView，倒胶后***梳子。待凝固后将凝胶放入电泳槽内，倒入1×TAE电泳缓冲液，第一个孔不加样，第二个孔加入3ul的DNA Marker，第三孔加入酶切产物和DNA loading Buffer的混合液，120V电泳30min，电泳结束后于凝胶扫描成像仪观察条带大小，判断切胶回收的片段位置。
4、回收质粒骨架片段
1)将凝胶置于紫外灯下，快速切取所需的目的片段于1.5ml的EP管中。
2)称取凝胶块的重量，按每100mg的凝胶块等于100μl溶液计算，取3倍体积的GDPBuffer加入到EP管中，于55℃水浴锅中溶解15min，每隔5min上下翻转3次以加速凝胶块的溶解。
3)将EP管以12,000rpm的速度离心1min使管壁的液滴收集到管底，将回收柱放入干净的1.5ml EP管中，把液体转移至柱子中，12,000rpm离心1min后弃滤液。
4)将柱子放回EP管中，取300μl GDP Buffer小心加入到柱子中并放置1min，12,000rpm离心1min后弃滤液。
5)将柱子放回EP管中，取600μl DW2Buffer(已加无水乙醇)加入到柱子中。12,000rpm离心1min后重复一次上述操作。
6)将滤液倒弃，把柱子放回EP管中以12,000rpm的速度离心3min后弃滤液。
7)取一干净的1.5ml EP管并将柱子放入其中，用移液枪取15μl Elution Buffer(已预热)小心滴入柱子的膜上所有位置，室温静置3min后以12,000rpm的速度离心1min，仍掉柱子并将管底的DNA于-20℃保存。
5、连接
将目的片段和载体按照4：1的比例混合加入T4DNA连接酶进行连接，4℃连接过夜。
6、感受态细胞的转化
1)从-80℃冰箱取出感受态细胞Stbl3于冰上化冻，加入连接混合液，用枪头轻轻混合后放在冰上30min静置。
2)42℃热激60秒：把EP管中的感受态放在42℃水浴锅中60秒钟，然后迅速将EP管放入冰上冷却2min，注意不要晃动EP管。
3)取600ul灭菌的LB培养基(不含抗生素)加入EP管中，用移液枪混匀后放入摇床(37℃，150rpm)振荡培养60min复苏菌体。
4)无菌条件下，将菌液加到含Amp的LB固体培养基平板上，用无菌的细菌L棒将细胞均匀涂开，等平板中的液体完全吸收后，倒置平板，37℃培养16h后挑菌。
7、小抽质粒、酶切、凝胶电泳、测序
1)从LB平板上挑取5个单克隆菌落，加入到5个标记好的15ml离心管中，每管加入2ml含有Amp的LB液体培养基，280rpm，37℃振荡培养14h。
2)将菌液加入1.5ml的EP管中，12,000rpm离心2min收菌后弃上清，管壁液体用吸水纸吸去。
3)取500ul P1溶液(RNase A已加)加入到含有菌体沉淀的EP管中，上下颠倒2次后将EP管放置在涡旋仪上涡旋，一定要使菌块混匀。
4)取500ul的P2溶液加入到离心管中，上下颠倒10次，保证菌体充***解。
5)取700ul的P3溶液加入到离心管中，上下颠倒10次以混匀菌液，室温放置3min后可看到大量白色絮状沉淀。1,400rpm离心15min，可看到管底的白色沉淀。
6)用移液枪分次将上清加入到CS过滤柱中进行过滤，14,000rpm离心2min。
7)将收集的滤液小心的加入到CP4吸附柱(已用平衡液活化)中，以14,000rpm的速度离心1min后弃废液，把CP4吸附柱重新放回收集管。
8)取500ul的PD(去蛋白液)加入到吸附柱CP4中，14,000rpm离心1min后弃废液。
9)取600ul的漂洗液PW(无水乙醇已加)加入到吸附柱CP4中，室温14,000rpm离心2min后弃废液，将吸附柱放回收集管并重复操作一次。
10)将吸附柱CP4放回收集管中并以14,000rpm离心5min，打开吸附柱CP4的盖子于室温中放3min，使吸附膜上的残余漂洗液能充分晾干。
11)取一干净的1.5ml离心管，将吸附柱CP4放入，取100ul的TB(洗脱缓冲液)缓慢的滴加在吸附膜的各个部位，静置3min后以14,000rpm的速度离心2min，管中所得便是质粒DNA溶液。
12)微量分光光度计测浓度。
13)酶切质粒，步骤同前。
14)凝胶电泳分析条带大小，初步判断后送测序验证。
二、慢病毒的包装和浓缩测滴度
1、大抽质粒，步骤同前，确保质粒浓度在1000ng/ul以上。
2、复苏293T，从液氮中取出293T细胞，迅速置于37℃水浴锅中解冻细胞，于无菌操作台中将细胞转移至10ml的离心管中(已灭菌)，取1ml 10％的DMEM完全培养基加入离心管，1000rpm低速离心5min，弃上清，1ml培养基重悬细胞，用移液枪轻轻吹打，将细胞接种于10cm培养皿中，标记好日期、种类和传代次数等，于37℃孵箱培养。
3、转染293T
1)转染前一天晚上计数细胞，取5×106铺一个10cm板子，保证次日早晨细胞密度为80％左右，细胞不可过密。
2)次日早晨转染前给细胞更换新的完全培养基，取2个1.5ml的EP管，分别标记为A、B。A管加入500ul减血清培养基(Opti-MEM)和21.7ulLip3000，充分混匀涡旋振荡2-3秒。B管加入500ul Opti-MEM后加入两个包装质粒psPAX2、pMD2G以及主质粒共16.5ug，B管加入P3000试剂48ul，充分混匀。
3)将B管液体加入到A管中混匀，室温孵育15min后将混合物加入到293T细胞培养基中，十字法轻轻混匀，48h后拍照查看荧光。
4、浓缩病毒
1)48h收病毒，1500rpm离心，去除细胞碎片，上清用0.22um的滤头过滤，按照1：3的比例加入Lenti-x Concentrator慢病毒浓缩液，4℃冰箱过夜，次日早上4℃、1500g离心45min。
2)所得白色沉淀即为病毒浓缩产物，弃上清，取100ul DMEM溶解病毒，分装10ul每管，浇淋液氮速冻后-80℃冰箱保存备用。
5、测病毒滴度
1)Day 0：将生长状态良好的293T细胞消化计数后按照每孔1×105个铺12孔板。
2)Day 1：按照梯度稀释法取病毒浓缩液0.0001ul、0.001ul、0.01ul、0.1ul、1ul、10ul加入到12孔板中，设置1个阴性对照不加病毒，加入Polybrene 8ug/ml，十字法混匀，放入37℃，5％的CO2孵箱中培养72h。
3)Day 4：荧光显微镜下观察，流式检测感染率，计算滴度。
图2为荧光显微镜下观察48h转染结果示意图，其中，放大倍率为×100倍；图3为三种病毒感染293T细胞72h后测滴度示意图；如图2和图3所示，48h转染率在百分之八十以上，三种慢病毒的包装、浓缩、测滴度结果为测得浓缩后FOLR1-CAR、MSLN-CAR、Tandem-CAR病毒的滴度分别为3.4×108、3.1×108、3.5×108TU/ml。
实施例2分离PBMC和和CD3
1、离PBMC
1)收取健康志愿者的外周血10ml，立即放入冰盒，带回实验室细胞间，PBS冲洗三次，加入PBS+2％FBS缓冲液10ml。
2)密度梯度离心法：加入Stemcell的Ficoll液15ml，1500rpm、19℃，离心30分钟，离心后可见管内分为4层：最上层是血浆，含部分血小板；第二层为薄薄的白膜层，主要是单个核细胞，还混杂有少量血小板；第三层为分离液层；第四层为粒细胞及红细胞，红细胞沉于管底，而粒细胞则紧贴在压积红细胞上呈一层很薄的白膜。
3)小心吸取白膜细胞，加5倍PBS液于所得的PBMC中，用毛细吸管轻轻吹打均匀，避免产小气泡，液柱高度不超过离心管的2/3。混匀后以1500r/min的速度离心10min，弃上清后重复洗涤2次。再以10ml含10％FBS的RPMI 1640培养基重悬后机器计数，细胞总数为1.38×107个。
2、分离PBMC中的CD3细胞
1)40um尼龙网过滤，以去除可能堵塞柱子的细胞团块。配buffer(PBScontaining2％FBS and 1mM EDTA)：配制100ml buffer＝0.5M EDTA 0.2ml+98ml PBS+2mlFBS,过滤除菌。
2)确定细胞数。Buffer重悬，调整细胞数为5×10 7cells/ml。
3)5ml灭菌流式管准备，加入细胞0.25-2ml到流式管。
4)加入Cocktail 50ul/ml到细胞中，充分混匀，室温孵育5分钟。
5)涡旋试剂Rapd Spheres 30秒，保证颗粒均匀。
6)加入Rapd Spheres比例为40ul/ml到细胞中，充分混匀。
7)加入Buffer，将液体总体积补充至2.5ml，将管子(不带盖子)放入磁铁中，室温孵育3分钟。
8)拿起磁块和流式管，直接将离心管中的液体倒入一干净的新离心管，里面就是分离的CD3，机器计数细胞。
9)流式鉴定CD3：调整细胞浓度为10 7/ml，每管各取100ul，设置空白对照组、单管染色组、全染色组，加入CD3-PE/cy7、CD8-APC/CY7、CD4-percp-cy5.5三种流式抗体，冰上避光孵育30min，其余细胞用含10％FBS、IL-2 100U/ml的RPMI 1640培养基重悬，调整细胞浓度至1×10 6/ml于24孔板中，每1ml细胞悬液中加入25ulImmunoCultTMHuman CD3/CD28T细胞激活剂，将培养板置于37℃、5％CO 2培养箱中激活培养48h。
从PMBC中分选CD3细胞，结果显示分选的CD3纯度高达99.6％(图4)，其中CD4和CD8的比例分别为74.4％和17.4％，
实施例3慢病毒感染CD3并测定感染结果
1、感染复数(multiplicity of infection，MOI)为10感染每孔CD3细胞，加入ploybrene 8ug/ml，进行感染。
2、感染72h后APC-F(ab')2染色，流式上机检测感染率。
3、实验结果
第4代FOLR1-CAR质粒、MSLN-CAR质粒和Tandem-CAR质粒，结构示意图见图1，构建的质粒经过酶切，核酸凝胶电泳分析符合目的基因片段大小。同时我们对三种质粒进行测序验证，序列正确，表明成功构建了包含两个共刺激分子CD28和4-1BB以及IL-12基因的三种慢病毒质粒。
以MOI＝10感染CD3细胞5天，流式结果显示CAR的表达分别为40％、36％、32％(图5)，荧光显微镜下可以看到感染的CD3细胞聚团并发绿色荧光(图6)。
实施例4不同CAR-T细胞在体内外对卵巢癌细胞的靶向杀伤作用
一、细胞系表面抗原的检测
(1)收集对数生长期的A2780、SKOV3、SNU119卵巢癌细胞，消化细胞，离心收细胞，弃上清，1ml冰冷染色Buffer重悬，洗两次。
(2)细胞计数，用细胞染色Buffer将细胞制成1x10 7/ml悬液。将100ul细胞悬液加入流式管中备用。
(3)加入流式抗体5ul，第1管：Blank，第2管：APC-MSLN，第3管：PE-FOLR1，第4管：双阳，4℃，30min，避光。
(4)加入5ml细胞染色Buffer重悬细胞，1000rpm离心细胞悬液5min，弃掉上清，洗两次，加入200ul细胞染色Buffer重悬细胞，40um筛网过滤，用流式细胞仪进行检测和分析。
利用流式分析A2780、SKOV3、SNU119三种人卵巢癌细胞系表面FOLR1和MSLN抗原的表达水平，结果表明，FOLR1的表达水平SNU119>SKOV3>A2780(图7)，MSLN表达水平也是SNU119>SKOV3>A2780(图7)，因此SNU119细胞可作为Tandem-CAR理想的靶细胞，因此接下来我们以SNU119细胞作为体内外杀伤的主要研究模型。
二、CAR-T细胞杀伤实验
1、LDH释放实验
1)实验原理：
正常情况下，细胞内含有丰富的乳酸脱氢酶(LDH)，当细胞膜完整时不能通过细胞膜，而当细胞受损或死亡时，细胞膜破裂，LDH释放到胞外，胞外的LDH在一定时间内较为稳定，所以细胞死亡的数量与上清中的LDH含量成正比，因此可以通过测定LDH的含量来计算CAR-T的杀伤效能。
2)靶细胞铺板，取对数生长期的A2780、SKOV3、SNU119细胞，消化计数，调整浓度为105/ml，取三块新的96孔板，每孔加入5000个肿瘤细胞，四周的空白孔加入100ul的PBS，防止中间细胞的水分蒸发，铺完肿瘤细胞后将96孔板放回孵箱培养过夜。
3)次日按比例加入三种CAR-T和一种Mock T细胞，留空白对照孔(TargetSpontaneous和Effector Spontaneous)和单纯培养基孔(Medium)，将96孔板放回孵箱培养18h，杀伤结束前45min，向最大裂解孔加入10ul的10×Lysis Buffer，培养45min，然后将96孔板以250g的速度离心5min，将每孔上清取50ul转移至一新的96孔板中，取出12ml检测缓冲液(Assay Buffer)，每孔加入50ul的Assay Buffer，室温孵育30min，注意全程避光，30min后每孔加入50ul的终止液。
4)酶标仪490nm检测吸光度值
5)靶细胞的裂解率计算公式如下：
杀伤率＝(Experimental-Target Spontaneous)/(Target Maximum-TargetSpontaneous)×100％。
实验结果：
用LDH细胞毒性检测试剂盒分析了四种效应细胞(Tandem-CAR、FOLR1-CAR、MSLN-CAR、Mock-T)和三种肿瘤细胞(SNU119、SKOV3、A2780)共培养18h后杀伤效能的比较，结果显示除了A2780组(E/T＝2:1)，Tandem-CAR的杀伤效能明显高于Mock-T组(P<0.0001)。Tandem-CAR的杀伤率也显著高于FOLR1-CAR和MSLN-CAR组(E/T＝8:1，P<0.0001)。并且Tandem-CAR的特异杀伤力是依赖于FOLR1和MSLN这两种抗原的表达高低，如图8所示，对于FOLR1和MSLN抗原高表达的SNU119(FHMH)肿瘤细胞，效应细胞的杀伤能力为：Tandem-CAR>FOLR1-CAR>MSLN-CAR>Mock-T，当E/T比为8：1时Tandem-CAR的最高杀伤效能达到79.96％。当靶细胞为SKOV3(FHML)时，Tandem-CAR和FOLR1-CAR的杀伤效能明显高于MSLN-CAR和Mock-T(P<0.0001)，MSLN-CAR和Mock-T的杀伤能力无统计学差异。而当靶细胞为A2780(FLML)时，Tandem-CAR的最高杀伤率仅为30.9％，MSLN-CAR和Mock-T的杀伤能力存在较小的统计学差异(E/T＝4:1，E/T＝8:1，P＝0.0110，P＝0.0102)，这表明Tandem-CAR能特异性的识别并杀伤肿瘤细胞。
三、流式检测细胞因子释放
(1)Day 0：靶细胞SNU119铺板，取一块96孔板，每孔加入细胞肿瘤细胞104个，培养过夜使细胞贴壁。
(2)Day 1：按照E/T为2：1的比例加入Mock-T、FOLR1-CAR、MSLN-CAR、Tandem-CAR细胞，每组设三个复孔，共培养14h后，加入离子霉素(Ionomycin)500ng/ml和PMA(佛波酯)50ng/ml刺激4h，最后加入高尔基体阻断剂，进行细胞因子染色。
(3)离心收细胞，50ul重悬细胞，每50ul中取出5ul混在一起得150ul混合液，从混合液中各取10ul染表面Marker CD3、CD4、CD8，剩余120ul离心弃上清，加入250ulFixation/Permeabilization Solution，离心后加入200ul的Wash Buffer重悬细胞。
(4)取30ul做Blank，剩下170ul分4管染单色(IL-2、IL-12、TNF-α、IFN-γ)。
(5)45ul的样品各加入50ul的混合抗体(CD3、CD4、CD8)，染色30min，Wash Buffer洗两次。
(6)250ul Fixation/Permeabilization Solution重悬细胞，固定通透15min后洗两次，加入55ul的细胞因子混合抗体，冰上孵育30min后Wash Buffer洗两次。
(7)上机检测。
实验结果：
IL-2、FN-γ、IL-12、TNF-α等细胞因子的分泌是CAR-T细胞发挥杀伤效能的重要标志，因此利用流式检测四种T细胞与SNU119靶细胞共培养后细胞因子的分泌水平可以有效判断不同T细胞的杀伤能力，如图9所示，Tandem-CAR的细胞因子分泌能力最强，三种CAR-T的细胞因子分泌水平明显高于Mock-T细胞组P<0.0001)，
并且由于Mock-T未感染病毒，因此IL-12的分泌水平极低，这表明我们构建的四代CAR-T成功感染并表达了IL-12。三组CAR-T间TNF-α阳性细胞的比例无统计学差异(P>0.05)，FOLR1-CAR和MSLN-CAR相比，四种细胞因子的分泌能力无统计学差异(P>0.05)。
四、小鼠移植瘤模型
(1)购买5周龄的重度免疫缺陷小鼠(B-NDG)24只，随机分为四组，每组6只，耳标进行标记。
(2)大量扩增表达荧光素酶的SNU119卵巢癌细胞(SNU119-Luc)，每只小鼠于大腿根部皮下接种5×106的SNU119-Luc细胞，预约10天后的活体成像仪，注意观察成瘤情况。
(3)10天后观察发现小鼠均已成瘤，活体成像拍照，根据荧光强度每组选取5只，四组小鼠分别经尾静脉给予1×107的Mock-T、FOLR1-CAR、MSLN-CAR、Tandem-CAR细胞，记为第0天，以后每7天进行一次活体成像拍照观察。
实验结果：
如图10、11所示，B-NDG小鼠荷瘤10天后分别给予Mock-T、FOLR1-CAR、MSLN-CAR、Tandem-CAR细胞进行治疗，结果表明与对照组(Mock-T)相比，FOLR1-CAR、MSLN-CAR、Tandem-CAR均能明显抑制肿瘤的生长(P<0.0001)，Mock-T组有两只小鼠分别在21天、28天死亡。MSLN-CAR组的肿瘤先缩小后增长，FOLR1-CAR和Tandem-CAR组的肿瘤持续缩小，Tandem-CAR组的抑瘤效果最为明显，显著小于MSLN-CAR组(P<0.0001)，并且有一只小鼠肿瘤发生消退。Tandem-CAR组与FOLR1-CAR组之间无统计学差异，FOLR1-CAR组的治疗效果要优于MSLN-CAR组(P＝0.0153)。
应注意的是，以上实例仅用于说明本发明的技术方案而非对其进行限制。尽管参照所给出的实例对本发明进行了详细说明，但是本领域的普通技术人员可根据需要对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的精神和范围。
序列表
<110> 中国人民解放军陆军军医大学第一附属医院
<120> 用于治疗卵巢癌的FOLR1-MSLN双靶向性CAR-T细胞、嵌合抗原受体及载体
<141> 2020-07-13
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 245
<212> PRT
<213> 人类(Human)
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Ser Ser Ile Ser Ser Ser Ser Ser Tyr Ile Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Glu Arg Tyr Asp Phe Trp Ser Gly Met Asp Val Trp Gly Lys
100 105 110
Gly Thr Thr Val Thr Val Ser Ser Gly Ser Thr Ser Gly Ser Gly Lys
115 120 125
Pro Gly Ser Gly Glu Gly Ser Gln Ser Ala Leu Thr Gln Pro Ala Ser
130 135 140
Val Ser Gly Ser Pro Gly Gln Ser Ile Thr Ile Ser Cys Thr Gly Thr
145 150 155 160
Ser Ser Asp Val Gly Ser Tyr Asn Leu Val Ser Trp Tyr Gln Gln His
165 170 175
Pro Gly Lys Ala Pro Lys Leu Met Ile Tyr Glu Gly Ser Lys Arg Pro
180 185 190
Ser Gly Val Ser Asn Arg Phe Ser Gly Ser Lys Ser Gly Asn Ala Ala
195 200 205
Ser Leu Thr Ile Ser Gly Leu Gln Ala Glu Asp Glu Ala Asp Tyr Tyr
210 215 220
Cys Gln Ser Tyr Asp Ser Ser Leu Ser Val Val Phe Gly Gly Gly Thr
225 230 235 240
Lys Leu Thr Val Leu
245
<210> 2
<211> 236
<212> PRT
<213> 人类(Human)
<400> 2
Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Arg Tyr Tyr
20 25 30
Leu Ser Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Thr Ala Ser Ile Leu Gln Asn Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Thr Tyr Thr Thr Pro Asp
85 90 95
Phe Gly Pro Gly Thr Lys Val Glu Ile Lys Gly Ser Thr Ser Gly Ser
100 105 110
Gly Lys Pro Gly Ser Gly Glu Gly Ser Gln Val Gln Leu Val Gln Ser
115 120 125
Gly Ala Glu Val Glu Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys
130 135 140
Ala Ser Gly Tyr Thr Phe Thr Asp Tyr Tyr Met His Trp Val Arg Gln
145 150 155 160
Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Trp Ile Asn Pro Asn Ser
165 170 175
Gly Gly Thr Asn Tyr Ala Gln Lys Phe Gln Gly Arg Val Thr Met Thr
180 185 190
Arg Asp Thr Ser Ile Ser Thr Ala Tyr Met Glu Leu Ser Arg Leu Arg
195 200 205
Ser Asp Asp Thr Ala Val Tyr Tyr Cys Ala Ser Gly Trp Asp Phe Asp
210 215 220
Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
225 230 235
<210> 3
<211> 20
<212> PRT
<213> 人工序列(Atificial sequence)
<400> 3
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
1 5 10 15
Gly Gly Gly Ser
20
<210> 4
<211> 112
<212> PRT
<213> 人类(human)
<400> 4
Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly
1 5 10 15
Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr
20 25 30
Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys
35 40 45
Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys
50 55 60
Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg
65 70 75 80
Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala
85 90 95
Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
100 105 110
<210> 5
<211> 735
<212> DNA
<213> 人类(human)
<400> 5
caggtgcagc tggtggagtc tgggggaggc ttggtacagc cagggcggtc cctgagactc 60
tcctgcacaa cttctggatt cacttttggt gattatgcta tgatctgggc ccgccaggct 120
ccagggaagg ggctggagtg ggtctcatcc attagtagta gtagtagtta catatactac 180
gcagactcag tgaagggccg attcaccatc tccagagaca acgccaagaa ctcactgtat 240
ctgcaaatga acagcctgag agccgaggac acggctgtgt attactgtgc gagagaacga 300
tacgattttt ggagtggaat ggacgtctgg ggcaaaggga ccacggtcac cgtgtcgagt 360
ggcagcacca gcggcagcgg caagcccggc agcggcgagg gcagccagtc tgccctgact 420
cagcctgcct ccgtgtctgg gtctcctgga cagtcgatca ccatctcctg cactggaacc 480
agcagtgatg ttgggagtta taaccttgtc tcctggtacc aacagcaccc aggcaaagcc 540
cccaaactca tgatttatga gggcagtaag cggccctcag gggtttctaa tcgcttctct 600
ggctccaagt ctggcaacgc ggcctccctg acaatctctg ggctccaggc tgaggacgag 660
gctgattatt actgccagtc ctatgacagc agcctgagtg tggtattcgg cggagggacc 720
aagctgaccg tccta 735
<210> 6
<211> 708
<212> DNA
<213> 人类(human)
<400> 6
gatatcgtga tgacgcaatc gccttcctcg ttgtccgcat ccgtgggaga cagggtgacc 60
attacttgca gagcgtccca gtccattcgg tactacctgt cgtggtacca gcagaagccg 120
gagaaagccc caaaactgct tatctatact gcctcgatcc tccaaaacgg cgtgccatca 180
agattcagcg gttcgggcag cgggaccgac tttaccctga ctatcagcag cctgcagccg 240
gaagatttcg ccacgtacta ctgcctgcaa acctacacca ccccggactt cggacctgga 300
accaaggtgg agatcaaggg cagcaccagc ggcagcggca agcccggcag cggcgagggc 360
agccaagtcc aactcgttca atcaggcgca gaagtcgaaa agcccggagc atcagtcaaa 420
gtctcttgca aggcttccgg ctacaccttc acggactact acatgcactg ggtgcgccag 480
gctccaggcc agggactgga gtggatggga tggatcaacc cgaattccgg gggaactaac 540
tacgcccaga agtttcaggg ccgggtgact atgactcgcg atacctcgat ctcgactgcg 600
tacatggagc tcagccgcct ccggtcggac gataccgccg tgtactattg tgcgtcggga 660
tgggacttcg actactgggg gcagggcact ctggtcactg tgtcaagc 708
<210> 7
<211> 1503
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
caggtgcagc tggtggagtc tgggggaggc ttggtacagc cagggcggtc cctgagactc 60
tcctgcacaa cttctggatt cacttttggt gattatgcta tgatctgggc ccgccaggct 120
ccagggaagg ggctggagtg ggtctcatcc attagtagta gtagtagtta catatactac 180
gcagactcag tgaagggccg attcaccatc tccagagaca acgccaagaa ctcactgtat 240
ctgcaaatga acagcctgag agccgaggac acggctgtgt attactgtgc gagagaacga 300
tacgattttt ggagtggaat ggacgtctgg ggcaaaggga ccacggtcac cgtgtcgagt 360
ggcagcacca gcggcagcgg caagcccggc agcggcgagg gcagccagtc tgccctgact 420
cagcctgcct ccgtgtctgg gtctcctgga cagtcgatca ccatctcctg cactggaacc 480
agcagtgatg ttgggagtta taaccttgtc tcctggtacc aacagcaccc aggcaaagcc 540
cccaaactca tgatttatga gggcagtaag cggccctcag gggtttctaa tcgcttctct 600
ggctccaagt ctggcaacgc ggcctccctg acaatctctg ggctccaggc tgaggacgag 660
gctgattatt actgccagtc ctatgacagc agcctgagtg tggtattcgg cggagggacc 720
aagctgaccg tcctaggcgg cggcggcagc ggcggcggcg gcagcggcgg cggcggcagc 780
ggcggcggcg gcagcgatat cgtgatgacg caatcgcctt cctcgttgtc cgcatccgtg 840
ggagacaggg tgaccattac ttgcagagcg tcccagtcca ttcggtacta cctgtcgtgg 900
taccagcaga agccggagaa agccccaaaa ctgcttatct atactgcctc gatcctccaa 960
aacggcgtgc catcaagatt cagcggttcg ggcagcggga ccgactttac cctgactatc 1020
agcagcctgc agccggaaga tttcgccacg tactactgcc tgcaaaccta caccaccccg 1080
gacttcggac ctggaaccaa ggtggagatc aagggcagca ccagcggcag cggcaagccc 1140
ggcagcggcg agggcagcca agtccaactc gttcaatcag gcgcagaagt cgaaaagccc 1200
ggagcatcag tcaaagtctc ttgcaaggct tccggctaca ccttcacgga ctactacatg 1260
cactgggtgc gccaggctcc aggccaggga ctggagtgga tgggatggat caacccgaat 1320
tccgggggaa ctaactacgc ccagaagttt cagggccggg tgactatgac tcgcgatacc 1380
tcgatctcga ctgcgtacat ggagctcagc cgcctccggt cggacgatac cgccgtgtac 1440
tattgtgcgt cgggatggga cttcgactac tgggggcagg gcactctggt cactgtgtca 1500
agc 1503
<210> 8
<211> 501
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 8
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Thr Thr Ser Gly Phe Thr Phe Gly Asp Tyr
20 25 30
Ala Met Ile Trp Ala Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ser Ile Ser Ser Ser Ser Ser Tyr Ile Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Glu Arg Tyr Asp Phe Trp Ser Gly Met Asp Val Trp Gly Lys
100 105 110
Gly Thr Thr Val Thr Val Ser Ser Gly Ser Thr Ser Gly Ser Gly Lys
115 120 125
Pro Gly Ser Gly Glu Gly Ser Gln Ser Ala Leu Thr Gln Pro Ala Ser
130 135 140
Val Ser Gly Ser Pro Gly Gln Ser Ile Thr Ile Ser Cys Thr Gly Thr
145 150 155 160
Ser Ser Asp Val Gly Ser Tyr Asn Leu Val Ser Trp Tyr Gln Gln His
165 170 175
Pro Gly Lys Ala Pro Lys Leu Met Ile Tyr Glu Gly Ser Lys Arg Pro
180 185 190
Ser Gly Val Ser Asn Arg Phe Ser Gly Ser Lys Ser Gly Asn Ala Ala
195 200 205
Ser Leu Thr Ile Ser Gly Leu Gln Ala Glu Asp Glu Ala Asp Tyr Tyr
210 215 220
Cys Gln Ser Tyr Asp Ser Ser Leu Ser Val Val Phe Gly Gly Gly Thr
225 230 235 240
Lys Leu Thr Val Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
245 250 255
Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser
260 265 270
Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys
275 280 285
Arg Ala Ser Gln Ser Ile Arg Tyr Tyr Leu Ser Trp Tyr Gln Gln Lys
290 295 300
Pro Glu Lys Ala Pro Lys Leu Leu Ile Tyr Thr Ala Ser Ile Leu Gln
305 310 315 320
Asn Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe
325 330 335
Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr
340 345 350
Cys Leu Gln Thr Tyr Thr Thr Pro Asp Phe Gly Pro Gly Thr Lys Val
355 360 365
Glu Ile Lys Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu
370 375 380
Gly Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Glu Lys Pro
385 390 395 400
Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr
405 410 415
Asp Tyr Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
420 425 430
Trp Met Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln
435 440 445
Lys Phe Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr
450 455 460
Ala Tyr Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr
465 470 475 480
Tyr Cys Ala Ser Gly Trp Asp Phe Asp Tyr Trp Gly Gln Gly Thr Leu
485 490 495
Val Thr Val Ser Ser
500
Claims (10)
1.一种嵌合抗原受体，其特征在于，包括依次连接的单链抗体区段、胞外铰链区(Hingarea)、跨膜区和胞内免疫受体酪氨酸活化基序(ITAM)；其中，
所述单链抗体区段选自叶酸受体1单链抗体(FOLR1 scFV)、间皮素单链抗体(MSLNscFV)或者叶酸受体1单链抗体(FOLR1 scFV)-间皮素单链抗体(MSLN scFV)串联复合体中的一个。
2.如权利要求1所述的嵌合抗原受体，其特征在于，所述胞内免疫受体酪氨酸活化基序(ITAM)由CD28肽段、4-1BB肽段和CD3ζ链肽段依次连接构成。
3.如权利要求2所述的嵌合抗原受体，其特征在于，在所述CD3ζ链肽段之后还连接有IL-12肽段。
4.如权利要求1所述的嵌合抗原抗体，其特征在于，所述叶酸受体1单链抗体(FOLR1scFV)的氨基酸序列为SEQ ID NO:1；
所述间皮素单链抗体(MSLN scFV)的氨基酸序列为SEQ ID NO:2；
所述叶酸受体1单链抗体(FOLR1 scFV)-间皮素单链抗体(MSLN scFV)串联复合体中叶酸受体1单链抗体(FOLR1 scFV)和间皮素单链抗体(MSLN scFV)通过Llinker肽段连接，所述Linker肽段的氨基酸序列为SEQ ID NO:3。
5.一种用于表达如权利要求1-4中任一项所述的嵌合抗原受体的质粒载体，其特征在于，包含：
单链抗体区段的编码基因、胞内免疫受体酪氨酸活化基序的编码基因和慢病毒质粒；其中，所述单链抗体区段的编码基因选自叶酸受体1单链抗体(FOLR1 scFV)的编码基因、间皮素单链抗体(MSLN scFV)的编码基因或叶酸受体1单链抗体(FOLR1 scFV)-间皮素单链抗体(MSLN scFV)串联复合体的编码基因中的一种。
6.如权利要求5所述的质粒载体，其特征在于，所述胞内免疫受体酪氨酸活化基序的氨基酸序列为SEQ ID NO4。
7.如权利要求5所述的质粒载体，其特征在于，所述叶酸受体1单链抗体(FOLR1 scFV)的编码基因的核苷酸序列为SEQ ID NO:5；
所述间皮素单链抗体(MSLN scFV)的编码基因的核苷酸序列为SEQ ID NO:6；
8.如权利要求5所述的质粒载体，其特征在于，所述叶酸受体1单链抗体(FOLR1 scFV)-间皮素单链抗体(MSLN scFV)串联复合体的编码基因的核苷酸序列为SEQ ID NO:7。
9.一种慢病毒载体，其特征在于，所述慢病毒载体包含编码如权利要求1-3中任一项所述的嵌合抗原受体的编码基因或者由如权利要求5-8所述的质粒载体转染细胞后包装形成。
10.一种用于治疗卵巢癌的CAR-T细胞，其特征在于，所述CAR-T细胞是通过将如权利要求8所述的慢病毒载体感染CD3阳性T细胞得到。
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115161345A (zh) * | 2022-06-14 | 2022-10-11 | 同宜医药（苏州）有限公司 | 高表达FRα的重组载体、重组细胞及其构建方法和用途 |
| CN115491358A (zh) * | 2021-06-17 | 2022-12-20 | 复星凯特生物科技有限公司 | 一种靶向b7-h3和folr1双打靶点car t的制备及应用 |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2018219278A1 (en) * | 2017-06-01 | 2018-12-06 | Innovative Cellular Therapeutics CO., LTD. | Chimeric antigen receptor cell preparation and uses thereof |
| CN109306014A (zh) * | 2017-07-27 | 2019-02-05 | 上海细胞治疗研究院 | 一种靶向间皮素的嵌合抗原受体修饰t细胞及其用途 |
| CN109796535A (zh) * | 2019-01-30 | 2019-05-24 | 重庆福美干细胞生物科技发展有限公司 | 靶向叶酸受体α的嵌合抗原受体及其在制备预防或治疗恶性肿瘤药物中的用途 |
-
2020
- 2020-07-13 CN CN202010677570.XA patent/CN111777686B/zh active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2018219278A1 (en) * | 2017-06-01 | 2018-12-06 | Innovative Cellular Therapeutics CO., LTD. | Chimeric antigen receptor cell preparation and uses thereof |
| CN109306014A (zh) * | 2017-07-27 | 2019-02-05 | 上海细胞治疗研究院 | 一种靶向间皮素的嵌合抗原受体修饰t细胞及其用途 |
| CN109796535A (zh) * | 2019-01-30 | 2019-05-24 | 重庆福美干细胞生物科技发展有限公司 | 靶向叶酸受体α的嵌合抗原受体及其在制备预防或治疗恶性肿瘤药物中的用途 |
Non-Patent Citations (2)
| Title |
|---|
| NP_000725.1: "T细胞表面糖蛋白CD3zeta链异构体2前体", 《NCBI》 * |
| 王泽凡 等: "CAR-T在实体瘤中的研究进展", 《中国肿瘤》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115491358A (zh) * | 2021-06-17 | 2022-12-20 | 复星凯特生物科技有限公司 | 一种靶向b7-h3和folr1双打靶点car t的制备及应用 |
| CN115161345A (zh) * | 2022-06-14 | 2022-10-11 | 同宜医药（苏州）有限公司 | 高表达FRα的重组载体、重组细胞及其构建方法和用途 |
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