CN117143825B - 一种msln嵌合抗原受体修饰的t细胞及其应用 - Google Patents
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Abstract
本发明提供一种MSLN嵌合抗原受体修饰的T细胞及其应用,属于遗传工程技术领域,所述MSLN嵌合抗原受体由以下模块依次串联得到:导引子、单链抗体scFv‑MSLN、CD8 Hinge区、CD28跨膜区、CD28‑4‑1BB共刺激区、CD3ζ胞内区、自剪切区T2A、自杀基因RQR8;所述单链抗体scFv‑MSLN的核苷酸序列如序列表中SEQ ID NO.3所示。本发明制备的MSLN嵌合抗原受体修饰的T细胞(CAR‑MSLN‑T)与靶细胞共培养后,IFN‑γ的释放量明显增多,对靶细胞的杀伤性明显提高;本发明制备的CAR‑MSLN‑T细胞对C57BL6小鼠卵巢癌肿瘤生长具有明显的抑制效果。
Description
技术领域
本发明涉及一种MSLN嵌合抗原受体修饰的T细胞及其应用,属于遗传工程技术领域。
背景技术
CAR-T(Chimeric antigen receptor T cell,嵌合抗原受体T细胞)疗法,是指通过基因修饰技术,将带有特异性抗原识别结构域及T细胞激活信号的遗传物质转入T细胞,使T细胞直接与肿瘤细胞表面的特异性抗原相结合而被激活,通过释放穿孔素、颗粒酶素B等直接杀伤肿瘤细胞,同时还通过释放细胞因子募集人体内源性免疫细胞杀伤肿瘤细胞,从而达到治疗肿瘤的目的。
CAR-T设计的关键在于靶抗原的选择,研究发现,间皮素(MSLN)是一种细胞表面糖蛋白,在大多数恶性胸膜间皮瘤、卵巢癌、胰腺癌和直肠癌中过表达,而正常组织表达很少。由于MSLN在正常组织和肿瘤组织之间差异表达,MSLN成为肿瘤治疗的潜在靶点之一。
目前CAR-T疗法虽然已日渐成熟,但是始终面临着肿瘤复发、抗药的问题,并且在实际的运用中受限于MSLN表达量等原因,治疗效果与预期有差距,因此,有必要对嵌合抗原受体进行进一步改进,提高嵌合抗原受体对肿瘤抗原的靶向结合能力。
现有技术中公开了靶向MSLN的CAR-T细胞的制备方法,具体如下:
CN114891815A提供一种编码CCR5、编码嵌合抗原受体、编码IL-12的DNA片段,整合入嵌合抗原受体T细胞,能够提高其浸润肿瘤的能力,减弱肿瘤微环境对CAR-T细胞的抑制作用。但该方法制备的细胞转染效率不高,其中靶向间皮素的CAR-T细胞(CARTmeso细胞)转染效率为44 .1%,CCR5和IL-12修饰改造的CAR-T细胞(CART5-12)的转染效率为47.7%。
CN16445549A提供一种负载紫杉醇的CAR-T外泌体在制备雾化吸入法原位治疗肺癌药物中的应用,该专利主要是利用CAR-T细胞的外泌体,并没有直接涉及到CAR-T细胞的效果。
CN116589597A制备一种分泌Mesothelin抗体的Meso-CAR-T细胞,属于二代CAR结构,包括抗MSLN的Scfv片段、CD8α的铰链区、CD8α跨膜结构域和一个共刺激信号4-1BB+CD3ζ,杀伤48h,在效靶比1:1时,杀伤效率最高达到45%。
CN114015720A中构建得到的MSLN-CAR-T细胞对实体肿瘤具有靶向杀伤性,但阳性细胞表达率和杀伤率有限,该发明中获得的MSLN-CAR-T细胞的CAR+CD8+表达率为12.51%,肿瘤杀伤实验中,当效靶比为1:1时,肿瘤杀伤率低于20%。
发明内容
针对现有技术存在的不足,本发明提供一种MSLN嵌合抗原受体修饰的T细胞及其应用,实现以下发明目的:
一种MSLN嵌合抗原受体修饰的T细胞,所述MSLN嵌合抗原受体由以下模块依次串联得到:导引子、单链抗体scFv-MSLN、CD8 Hinge区、CD28跨膜区、CD28-4-1BB共刺激区、CD3ζ胞内区、自剪切区T2A、自杀基因RQR8;所述单链抗体scFv-MSLN的核苷酸序列如序列表中SEQ ID NO.3所示。
所述导引子的核苷酸序列如序列表SEQ ID NO.2所示;所述CD8 Hinge区的核苷酸序列如序列表SEQ ID NO.4所示;所述CD28跨膜区的核苷酸序列如序列表SEQ ID NO.5所示;所述CD28-4-1BB共刺激区的核苷酸序列如序列表SEQ ID NO.6所示;所述CD3ζ胞内区的核苷酸序列如序列表SEQID NO.7所示;所述自剪切区T2A的核苷酸序列如序列表SEQ IDNO.8所示;所述自杀基因RQR8的核苷酸序列如序列表SEQID NO.9所示。
所述MSLN嵌合抗原受体修饰的T细胞在制备治疗抗肿瘤药物中的应用。
与现有技术相比,本发明取得以下有益效果:
(1)本发明对单链抗体scFv-MSLN的核苷酸序列进行了优化,制备的MSLN嵌合抗原受体修饰的T细胞(CAR-MSLN-T)与靶细胞共培养后,IFN-γ的释放量明显增多,对靶细胞的杀伤性明显提高。
CAR-MSLN-T与靶细胞KYSE-510共培养24h后,IFN-γ释放量达到9673pg/mL,对靶细胞的杀伤率为92.3%;CAR-MSLN-T与靶细胞SKOV3共培养24h后,IFN-γ释放量达到7911pg/mL pg/mL,对靶细胞的杀伤率89.1%。
(2)本发明制备的CAR-MSLN-T细胞对C57BL6小鼠卵巢癌肿瘤生长具有明显的抑制效果。
附图说明
图1为CAR-MSLN模块的结构示意图;
图2为流式细胞术检测T细胞活化指标CD69的表达率的流式图;
图3为慢病毒转染293T细胞48h后的荧光图;
图4含pLent-EF1α-CAR-MSLN的重组慢病毒感染活化T细胞后的流式图;
图5为含pLent-EF1α-CAR-MSLN-2的重组慢病毒感染活化T细胞后的流式图;
图6为含pLent-EF1α-空载质粒的重组慢病毒感染活化T细胞后的流式图;
图7为CAR-MSLN-T细胞、CAR-MSLN-2-T细胞、空载T细胞分别和靶细胞共培养24h后, IFN-γ释放量的柱状图;
图8为CAR-MSLN-T细胞、CAR-MSLN-2-T细胞、空载T细胞对靶细胞的体外杀伤率的柱状图;
图9为体内毒性实验中小鼠体重的变化图;
图10为CAR-T细胞对C57BL6小鼠乳腺癌肿瘤生长的抑制效率图。
具体实施方式
实施例1重组表达载体pLent-EF1α-CAR-MSLN的构建
CAR-MSLN模块示意见图1(完整核酸序列见附录SEQ ID NO.1)。
CAR-MSLN各模块序列:
(1)导引子Leader (SEQ ID NO.2)
(2)单链抗体scFv-MSLN(SEQ ID NO.3)
(3)CD8 Hinge区(SEQ ID NO.4)
(4)CD28跨膜区 (SEQ ID NO.5)
(5)CD28-4-1BB共刺激区(SEQ ID NO.6)
(6)CD3ζ胞内区(SEQ ID NO.7)
(7)自剪切区T2A(SEQ ID NO.8)
(8)自杀基因RQR8 (SEQ ID NO.9)
按照上述从(1)-(8)的顺序委托山东弘诺生物科技有限公司合成其整个表达框,插入pLent-EF1α载体(购自Vigene公司) BamHI-NotI位点,转化到E.coli(Top10),经测序正确后,使用OMEGA公司的质粒提取试剂盒提取质粒,获得重组表达载体,命名为pLent-EF1α-CAR-MSLN。本发明中,重组表达载体pLent-EF1α-CAR-MSLN的浓度为1.0µg/µL。
同时采用CN115947865A中公开的Anti-MSLN-scFV作为单链抗体(SEQ ID NO.10),其余模块均采用本发明的序列,构建包含CN115947865A中公开的Anti-MSLN-scFV序列的CAR结构,插入pLent-EF1α载体,得到重组表达载体,命名为pLent-EF1α-CAR-MSLN-2,其质粒浓度为1.0µg/µL。
提取Plent-EF1α-空载质粒:对慢病毒空载直接利用试剂盒提取,其质粒浓度调节为1.0µg/µL。
实施例2 pLent-EF1α-CAR-MSLN修饰T细胞的制备
1、T细胞的分离及活化
取75mL患者自体外周血,用TBD样本密度分离液(购自天津灏洋华科生物),分离外周血。具体方法如下:
(1)将外周血75mL与生理盐水按体积比为1:1进行稀释,得到稀释后的血液,取6个离心管,分别加入20mL的分离液,将25mL稀释后的血液缓慢地加在淋巴细胞分离液上,形成明显分层,室温水平离心900g/min,20min。此时离心管中自上而下形成4层;血清、由PBMC构成的白膜层、淋巴细胞分离液层和最下面的红细胞沉淀层。
(2)用移液枪小心吸取白膜层,全部吸出PBMC,加2倍量的生理盐水,洗涤细胞2次,每次混匀后离心400g/min,5min,低速离心有利于去除细胞悬液中留存的血小板和淋巴细胞分离液,离心后弃去上清。
(3)分离后的细胞用BD公司提供的CD8+分选试剂分选出CD8+T细胞,进行细胞计数,按1×106个细胞/mL,加入相应体积的KBM551细胞培养基(购自Corning,货号:88-551-CM)。放在37℃、5%CO2培养箱中培养24h,加入与细胞量相同的CD3CD28磁珠进行活化T细胞,活化24h后取出磁珠,得到活化T细胞,用流式细胞仪检测CD69的表达率,本发明中CD69的表达率为61.2%(见图2),本发明中的活化T细胞即为CD8+T细胞。
2、慢病毒包装
按照公司常用方法,重组载体pLent-EF1α-MSLN(pLent-EF1α- CAR-MSLN-2、Plent-EF1α-空载质粒)与慢病毒包装质粒pMDNA2G与psPAX2采用lipo3000转染试剂转染293T细胞。
48小时后,显微镜下观察293T细胞转染后的形态变化(见图3)。72h后将含有病毒的细胞培养上清收集到离心管中,3500rpm/min离心10min,去除细胞碎片,4.5μm滤器过滤后以70000g离心力,4℃离心2h,将沉淀用PBS重悬,分装后放入-80℃保存,同时测定病毒滴度。
本发明中含有pLent-EF1α-CAR-MSLN的病毒液的病毒滴度为1.13×108TU/mL,含pLent-EF1α-CAR-MSLN-2的病毒液的病毒滴度为1.04×108TU/mL,含有pLent-EF1α空载质粒的病毒液的病毒滴度为9.87×107TU/mL。
3、慢病毒感染活化的T细胞
从-80℃拿出上述制备的三种病毒液,解冻后加入含有终浓度为1500IU/mL IL-2的KBM551的培养基,将病毒滴度稀释到3×107TU/mL,得到稀释后的病毒液。用稀释后的病毒液100µL重悬1×106个活化T细胞,使病毒颗粒数与活化T细胞的数量比例为3:1,得到病毒和细胞悬液。将病毒和细胞悬液加入到6孔板中,每孔2mL,37℃,5%的CO2培养箱中培养48小时,收集细胞,400g、5min离心,弃上清,对细胞计数,按照1×106个细胞/mL密度接种。每隔3天倍比加液,加入含有终浓度为1500IU/mL IL-2的KBM551培养基,37℃,5%的CO2培养箱培养13天,使细胞扩增至足够的用量,得到含pLent-EF1α-CAR-MSLN的重组慢病毒感染后的T细胞,简称为CAR-MSLN-T细胞;含pLent-EF1α-CAR-MSLN-2的重组慢病毒感染后的T细胞,简称为CAR-MSLN-2-T细胞;含pLent-EF1α-空载质粒的重组慢病毒感染后的T细胞,简称为空载T细胞。
通过流式细胞术检测嵌合抗原受体表达。以活化T细胞作为阴性对照,如图4-6所示,本发明中含pLent-EF1α-CAR-MSLN的重组慢病毒对活化T细胞的感染率为62.2%,含pLent-EF1α-CAR-MSLN-2的重组慢病毒对活化T细胞的感染率为57.6%,含pLent-EF1α-空载质粒的重组慢病毒对活化T细胞的感染率为60.4%。
实施例3 CAR-MSLN-T细胞对KYSE-510、细胞、SKOV3细胞的杀伤活性
以高表达间皮素的人食管癌细胞系KYSE-510细胞、卵巢癌细胞SKOV3分别作为靶细胞,效应细胞为CAR-MSLN-T细胞、CAR-MSLN-2-T细胞、空载T细胞。
按E:T(效应细胞和靶细胞比)为1:1、5:1、10:1,96孔板中每孔加入100μL的1×104个靶细胞待细胞完全贴壁后,收集CAR-MSLN-T细胞、CAR-MSLN-2-T细胞和空载T细胞,分别调整细胞浓度为1×105/mL、5×105/mL、1×106/mL,每孔加入100μL效应细胞,37℃,5%的CO2条件下培养。各组均设3个复孔,取3个复孔的平均值。效应细胞和靶细胞共同培养24h后,收集细胞上清,用ELISA试剂盒检测IFN-γ的含量。同时将细胞中加入20μL稀释的CCK8(购自MCE公司),孵育5小时,酶标仪检测OD450的吸光值。
结果如表1、图7、图8所示,针对KYSE-510和SKOV3两种靶细胞,本发明的CAR-MSLN-T细胞相较于CAR-MSLN-2-T细胞对靶细胞的杀伤力更强,释放的IFN-γ更多。对于KYSE-510细胞,在效靶比10:1时IFN-γ释放量达到9673pg/mL,杀伤率为92.3%。对SKOV3细胞,在效靶比10:1时IFN-γ释放量达到7911pg/mL,杀伤率为89.1%。
表1 CAR-MSLN-T细胞体外杀伤活性研究的IFN-γ释放量和杀伤率
实施例4 CAR-T细胞在C57BL6小鼠体内毒性实验
购买6-8周的C57BL6小鼠(购自南京君科生物工程有限公司),分成5组,每组10只,验证本发明制备的CAR-T细胞对C57BL6小鼠体内毒性实验。分成5个实验组,具体如下:
a.实验一组,尾部静脉注射2×107个细胞/只本发明的CAR-MSLN-T细胞。
b.实验二组,尾部静脉注射2×107个细胞/只对照的CAR-MSLN-2-T细胞。
c.实验三组,尾部静脉注射2×107个细胞/只空载T细胞;
d.实验四组,尾部静脉注射2×107个细胞/只活化T细胞;
e.对照组,尾部静脉注射同等体积的生理盐水;
注射后每天观察小鼠的行为表现,每隔3天对小鼠称重一次,记录小鼠是否出现死亡。利用动物活体成像系统监测小鼠体内CAR-T细胞是否存在。45天后,解剖小鼠,对小鼠的主要组织如脑、心脏、肺、肝、结肠和肾脏进行病理观察。
实验过程中,小鼠没有异常的行为表现,也没有出现死亡。如表2及图9所示,小鼠的体重均明显增加且无差异,CAR-T细胞持续存在28天。解剖小鼠后对主要组织的病理观察,CAR-T小鼠并没有发现组织的病变。
表2 各实验组培养45天后小鼠体重增重变化
实施例5 CAR-T细胞对C57BL6小鼠卵巢癌肿瘤生长抑制作用
6-8周的雄性C57BL6小鼠(购自南京君科生物工程有限公司)于动物房饲养(室温23±2℃,湿度50%±10%),采用常规方法利用SKOV3肿瘤细胞对小鼠进行建模,待腋下出现米粒大小较硬的结节作为建模成功的标准。
C57BL6卵巢癌模型小鼠,肿瘤组织块在95mm3左右时,随机分成6组,每组10只,开始注射治疗实验。实验组分别为:
a.治疗一组,尾部静脉注射2×106个细胞/只本发明的CAR-MSLN-T细胞;
b.治疗二组,尾部静脉注射2×106个细胞/只CAR-MSLN-2-T细胞;
c.治疗三组,尾部静脉注射2×106个细胞/只空载T细胞;
d.治疗四组,尾部静脉注射2×106个细胞/只活化T细胞;
e.治疗五组,尾部静脉注射2×106个细胞/只CAR-MSLN-T细胞,24h后注射利妥昔单抗,每天一次;
f.对照组,尾部静脉注射同等体积的生理盐水;
间隔1周,对各组小鼠免疫1次,共2次。首次注射当天记为第0天,每三天测量一次不同小组各个小鼠皮下肿瘤大小,计算均值绘制肿瘤生长曲线图,5周后解剖小鼠,将肿瘤组织利用石蜡包埋,切片。
结果如表3和图10所示:本发明中的CAR-MSLN-T细胞对C57BL6小鼠卵巢癌肿瘤生长的抑制效果最佳,注射利妥昔单抗后CAR-MSLN-T细胞不再抑制肿瘤的生长,说明CAR-MSLN-T细胞的作用受到抑制。因此注射CAR-MSLN-T细胞后,如果发生副作用,可以注射利妥昔单抗抑制CAR-MSLN-T细胞。
表3 小鼠肿瘤体积的变化(mm3)
。
Claims (3)
1. 一种MSLN嵌合抗原受体修饰的T细胞,其特征在于:所述MSLN嵌合抗原受体由以下模块依次串联得到:导引子、单链抗体scFv-MSLN、CD8 Hinge区、CD28跨膜区、CD28-4-1BB共刺激区、CD3ζ胞内区、自剪切区T2A、自杀基因RQR8;
所述单链抗体scFv-MSLN的核苷酸序列如序列表SEQ ID NO.3所示;所述导引子的核苷酸序列如序列表SEQ ID NO.2所示;所述CD8 Hinge区的核苷酸序列如序列表SEQ ID NO.4所示;所述CD28跨膜区的核苷酸序列如序列表SEQ ID NO.5所示;所述CD28-4-1BB共刺激区的核苷酸序列如序列表SEQ ID NO.6所示;所述CD3ζ胞内区的核苷酸序列如序列表SEQ IDNO.7所示;所述自剪切区T2A的核苷酸序列如序列表SEQ ID NO.8所示;所述自杀基因RQR8的核苷酸序列如序列表SEQ ID NO.9所示。
2.根据权利要求1所述的一种MSLN嵌合抗原受体修饰的T细胞,其特征在于:所述MSLN嵌合抗原受体修饰的T细胞的制备方法为将MSLN嵌合抗原受体插入pLent-EF1α载体,得到重组表达载体,经慢病毒包装后,感染活化T细胞,得到MSLN嵌合抗原受体修饰的T细胞。
3.权利要求1所述MSLN嵌合抗原受体修饰的T细胞在制备治疗抗肿瘤药物中的应用,其特征在于:所述肿瘤为食管癌或卵巢癌。
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