CN116555187A - 一种Mucin1嵌合抗原受体修饰的T细胞及其应用 - Google Patents
一种Mucin1嵌合抗原受体修饰的T细胞及其应用 Download PDFInfo
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Abstract
本发明提供一种Mucin1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Mucin1嵌合抗原受体包括导引子、单链抗体scFv‑MUC1、CD8 Hinge区、CD8跨膜区、CD226‑CD28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‑MUC1的核苷酸序列如序列表中SEQ ID NO.3所示。本发明制备的CAR‑MUC1‑T细胞对MCF‑7细胞的杀伤率为86.2%,对T47D‑GL细胞的杀伤率为89.4%;本发明制备的CAR‑MUC1‑T细胞对C57BL6小鼠的肿瘤生长具有明显的抑制作用。
Description
技术领域
本发明涉及一种Mucin1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
目前,肿瘤免疫治疗CAR-T(嵌合抗原受体T细胞免疫疗法)是继手术、化疗、放疗和靶向疗法之后出现的一种新型治疗方法,被称为治疗癌症的“第五大疗法”,近年来取得了越来越多的令人鼓舞的研究成果。
CAR应用的关键在于确定一种在肿瘤细胞表面高表达,而在正常组织中低表达或无表达的相关抗原。并且实体瘤具有高度的异质性,不同患者、同一患者不同病灶、同一病灶不同肿瘤细胞之间均存在高度的差异。这种高度的异质性致使肿瘤靶向治疗缺乏理想的通用、广谱靶点,限制了CAR-T细胞治疗实体瘤的疗效。因而,寻找有效的CAR-T细胞治疗靶点已然成为CAR-T细胞治疗的重中之重。
Mucin 1是膜结合型粘蛋白家族成员,简称MUC1,对上皮细胞更新、分化和完整性维持,癌症发生和转移等方面都起着重要作用。在正常情况下,MUC1在乳腺、胰腺、胃肠道、呼吸道及泌尿生殖道的上皮细胞的近管腔面呈低水平表达,不能被机体免疫系统识别,其表达特点为极性表达、顶端分布、糖基化丰富。但MUC1在乳腺癌、卵巢癌、肺癌、前列腺癌、结肠癌、肝癌和胰腺癌等恶性肿瘤细胞中呈异常过表达,并失去极性,遍布于整个细胞表面,可通过调控多个信号通路调节细胞恶性转化,是促进肿瘤发生与发展的重要癌基因。因此,MUC1蛋白可作为CAR-T细胞抗肿瘤治疗的一个理想靶点。
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MUC1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。
目前提供靶向性抗肿瘤T细胞的方法多种多样, CN112940137A提供一种PD-1基因敲除的靶向MUC1的CAR-T细胞制备方法,但细胞的培养周期较长且工序繁琐,该方法只涉及在临床上对癌症患者疾病控制率和安全性评价的统计。CN113248619A提供一种双靶向嵌合抗原受体,以串联方式连接MSLN、MUC1单链抗体的重链VH和轻链VL、及连接MSLN、MUC1单链抗体的第二铰链区,无法判断单靶点MUC1修饰的CAR-T的具体杀伤率。CN107227299A提供一种Anti MUC1 CAR细胞及其制备方法,并以乳腺癌细胞系MCF-7和胰腺癌细胞系BxPC-3作为靶细胞,在效靶比10:1时对靶细胞杀伤率在40%左右,杀伤率较低。
发明内容
针对现有技术存在的不足,本发明提供一种Mucin1嵌合抗原受体修饰的T细胞及其应用,实现以下发明目的:提高对靶细胞的杀伤率,提高IFN-γ的释放量,对小鼠肿瘤生长具有明显抑制作用。
为解决上述问题,本发明采取以下技术方案:
一种Mucin1嵌合抗原受体修饰的T细胞,所述Mucin1嵌合抗原受体包括导引子、单链抗体scFv-MUC1、CD8 Hinge区、CD8跨膜区、CD226-CD28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-MUC1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中SEQ ID NO.2所示;所述CD8 Hinge区的核苷酸序列如序列表中SEQ ID NO.4所示;所述CD8跨膜区的核苷酸序列如序列表中SEQ IDNO.5所示;所述CD226-CD28共刺激区的核苷酸序列如序列表中SEQ ID NO.6所示;所述CD3ζ胞内区的核苷酸序列如序列表中SEQ ID NO.7所示;所述自剪切区T2A的核苷酸序列如序列表中SEQ ID NO.8所示;所述自杀基因RQR8的核苷酸序列如序列表中SEQ ID NO.9所示。
所述Mucin1嵌合抗原受体修饰的T细胞的制备方法为将含Mucin1嵌合抗原受体的重组表达载体进行慢病毒包装,得到重组慢病毒,然后感染活化T细胞,得到Mucin1嵌合抗原受体修饰的T细胞。
所述Mucin1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)本发明制备的CAR-MUC1-T细胞与靶细胞共培养后,IFN-γ的释放量明显增多。本发明CAR-MUC1-T细胞和MCF-7细胞按照效靶比为10:1共培养24h后,IFN-γ释放量为9581pg/mL;本发明CAR-MUC1-T细胞和T47D-GL细胞按照效靶比为10:1共培养24h后,IFN-γ释放量为7836 pg/mL。
(2)本发明制备的CAR-MUC1-T细胞对MCF-7细胞的杀伤率为86.2%(效靶比为10:1),对T47D-GL细胞的杀伤率为89.4%(效靶比为10:1)。
(3)本发明制备的CAR-MUC1-T细胞对C57BL6小鼠的肿瘤生长具有明显的抑制作用,且细胞活性受自杀基因系统的控制。
附图说明
图1为CAR-MUC1模块的连接示意图;
图2为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为含pLent-EF1α- CAR-MUC1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-MUC1-2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-MUC1-T细胞、CAR-MUC1-2-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-MUC1-T细胞、CAR-MUC1-2-T细胞、空载T细胞对靶细胞的体外杀伤率的柱状图;
图9为小鼠体内注射CAR-MUC1-T细胞、CAR-MUC1-2-T细胞、空载T细胞、活化T细胞后的体重变化图;
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-MUC1的构建
CAR-MUC1模块示意见图1(完整核酸序列见附录SEQ ID NO.1);
CAR-MUC1各模块序列:
(1)导引子Leader (SEQ ID NO.2)
(2)单链抗体scFv-MUC1(SEQ ID NO.3)
(3)CD8 Hinge区(SEQ ID NO.4)
(4)CD8跨膜区 (SEQ ID NO.5)
(5)CD226-CD28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)
分别按照上述从(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pLent-EF1α-CAR-MUC1浓度为1.1µg/µL。
同时采用CN106220736A中公开的Anti-MUC-1-scFV作为单链抗体(SEQ IDNO.10),替代上述SEQ ID NO.3,其余模块均采用上述本发明的序列,构建包含CN106220736A中公开的Anti-MUC-1-scFV序列的重组表达载体,命名为pLent-EF1α-CAR-MUC1-2,浓度为1.0µg/µL。
构建Plent-EF1α-空载质粒:浓度为1.0µg/µL。
实施例2 pLent-EF1α-CAR-MUC1修饰T细胞的制备
1. 活化T细胞的制备
取75mL患者自体外周血,用Ficoll-Paque淋巴细胞分离液分离外周血单个核细胞。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进行接种,加入含有终浓度为1500IU/mL IL-2的KBM551细胞培养基(购自Corning,货号:88-551-CM);然后加入与细胞相同数量的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,利用流式细胞仪检测CD69的表达率, CD69的表达率为73.2%,见图2。
2. 慢病毒包装
复苏293T细胞,培养3天,根据细胞密度进行传代,传1代后,细胞融合度达到80%时进行转染。六孔板中按6×105个细胞/孔进行接种,每孔加入2mLDMEM培养基(购自Gibco公司,货号11960-044), 为第二天的转染做准备。转染前将六孔板换成新鲜DMEM培养基,37℃温箱中培养1h。
转染试剂的准备:在5mL离心管中,分别配制A管与B管试剂(Tube A and Tube B);A管与B管的配制见表1。
表1
配好后,放置5min,然后将A管缓慢加入B管,混合均匀,室温放置20min,形成脂质体-DNA混合物;将混合物加入培养瓶中,轻微混匀,置于37℃,5%CO2培养箱中培养。
48小时后,显微镜下观察293T细胞转染后的形态变化(图3)。72小时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100μL的PBS重悬,分装后放入-80℃保存,得到病毒液,同时测定病毒滴度。本发明中含有pLent-EF1α-CAR-MUC1的病毒液的病毒滴度为2.18×108TU/mL,含pLent-EF1α-CAR-MUC1-2的病毒液的病毒滴度为1.95×108TU/mL,含有pLent-EF1α-空载质粒的病毒液的病毒滴度为2.26×108TU/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48h,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,加入含有终浓度为1500IU/mL IL-2的KBM551培养基,每隔3天倍比加液,37℃、5%的CO2培养箱中培养13天使细胞扩增至足够的用量,得到含pLent-EF1α-CAR-MUC1的重组慢病毒感染后的T细胞,简称为CAR-MUC1-T细胞;含pLent-EF1α-CAR-MUC1-2的重组慢病毒感染后的T细胞,简称为CAR-MUC1-2-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,本发明中含pLent-EF1α- CAR-MUC1的重组慢病毒对活化T细胞的感染率为56.9%,含pLent-EF1α- CAR-MUC1-2的重组慢病毒对活化T细胞的感染率为50.7%,含pLent-EF1α-空载质粒的重组慢病毒对活化T细胞的感染率为57.4%(见图4-6)。
实施例3 三种T细胞的体外IFN-γ释放实验
以乳腺癌细胞系MCF-7、T47D-GL分别作为靶细胞,效应细胞为CAR-MUC1-T细胞、CAR-MUC1-2-T细胞、空载T细胞, 效靶比分别为1:1、5:1、10:1,靶细胞数量为1×105/孔,根据不同效靶比对应效应细胞,每孔加入200μL含有10vol% FBS的DMEM培养基,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。
结果如表2、图7所示,以MCF-7和T47D-GL作为靶细胞,本发明的CAR-MUC1-T细胞相较于CAR-MUC1-2-T细胞来说,IFN-γ的释放量明显增多,杀伤力更强,并呈现效靶比梯度依赖性即效靶比越高细胞毒性作用越强。
表2 三种T细胞和靶细胞共同培养24h后IFN-γ释放量(pg/mL)
。
实施例4三种T细胞的体外杀伤实验
将MCF-7细胞和T47D-GL细胞作为靶细胞,CAR-MUC1-T细胞、CAR-MUC1-2-T细胞和空载T细胞作为效应细胞进行杀伤活性测定。按照效应细胞(1×105/孔)与靶细胞(1×104/孔)数量比例为10:1加入48孔培养板中,每孔加入200μL含有10vol% FBS的DMEM培养基,置于5% CO2、37℃培养箱培养,24h后每孔加入20µL CCK-8,继续孵育2h后,酶标仪检测450nm波长,读取OD值。
具体分组如下:
实验组A:CAR-MUC1-T细胞和MCF-7细胞共培养;
实验组B:CAR-MUC1-T细胞和T47D-GL细胞共培养;
实验组C:CAR-MUC1-2-T细胞和MCF-7细胞共培养;
实验组D:CAR-MUC1-2-T细胞和T47D-GL细胞共培养;
对照组E:空载T细胞和MCF-7细胞共培养;
对照组F:空载T细胞和T47D-GL细胞共培养;
空白组G:MCF-7细胞、T47D-GL细胞。
按以下公式计算细胞杀伤率:杀伤率(%)=[1-(空白组OD值-效应细胞组OD值)/空白组OD值]×100%。
结果表明(见图8),实验A-D,以及对照E-F的杀伤率依次为86.2%、89.4%、45.3%、46.1%和15.3%、16.8%, CAR-MUC1-T细胞对两种靶细胞的杀伤效率明显高于CAR-MUC1-2-T细胞,两者均高于对照组。因此,本发明制备的CAR-MUC1-T细胞,能够增强细胞的杀伤能力。
实施例5 CAR-T细胞的体内毒性实验
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CAR-T细胞的体内毒性实验。实验组分别为:
a.对照组,尾部静脉注射同等体积的生理盐水;
b.实验一组,尾部静脉注射2×107个细胞/只(活化T细胞);
c.实验二组,尾部静脉注射2×107个细胞/只(空载T细胞);
d.实验三组,尾部静脉注射2×107个细胞/只(CAR-MUC1-T细胞);
e.实验四组,尾部静脉注射2×107个细胞/只(CAR-MUC1-2-T细胞)。
注射后每天观察小鼠的行为表现,每周对小鼠称重一次,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,并未观察到小鼠有异常的行为表现,如图9所示,小鼠的体重增加没有明显差异,且血液中检测到CAR-T细胞持续存在35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3 各实验组培养45天后小鼠体重增重变化
。
实施例6 CAR-T细胞对C57BL6小鼠乳腺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%),收集对数期的MCF-7细胞,磷酸盐缓冲液(PBS)稀释至2×106个/mL。无菌条件下,小鼠左侧腋下接种0.2mL MCF-7细胞悬浮液,观察两周,待腋下出现米粒大小较硬的结节作为建模成功的标准。
C57BL6乳腺癌模型小鼠(游标卡尺量取皮下肿瘤组织块的大小为90-100mm3)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.对照组,尾部静脉注射同等体积的生理盐水;
b.治疗一组,尾部静脉注射2×106个细胞/只(活化T细胞);
c.治疗二组,尾部静脉注射2×106个细胞/只(空载T细胞);
d.治疗三组,尾部静脉注射2×106个细胞/只(CAR-MUC1-T细胞);
e.治疗四组,尾部静脉注射2×106个细胞/只(CAR-MUC1-2-T细胞);
f.治疗五组,尾部静脉注射2×106个细胞/只(CAR-MUC1-T细胞),24h后注射利妥昔单抗,每天一次。
每周免疫上述各组小鼠一次,连续免疫两周,以首次注射当天为0天记录,每三天通过游标卡尺测量各个治疗组小鼠皮下肿瘤组织块大小,用肿块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。
肿瘤体积增长结果如表4 所示。结果表明本发明制备的CAR-MUC1-T细胞对C57BL6小鼠肿瘤生长的抑制作用最好(图10),相较于CAR-MUC1-2-T细胞细胞,可以明显抑制肿瘤的增长,注射利妥昔单抗后CAR-T细胞对肿瘤基本不起作用,说明利妥昔单抗可以启动本发明中CAR-MUC1-T细胞的自杀系统RQR8,细胞活性受自杀基因系统的控制。
表4 治疗过程中小鼠肿瘤体积的统计(mm3)
。
Claims (4)
1.一种Mucin1嵌合抗原受体修饰的T细胞,其特征在于:所述Mucin1嵌合抗原受体包括导引子、单链抗体scFv-MUC1、CD8 Hinge区、CD8跨膜区、CD226-CD28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-MUC1的核苷酸序列如序列表中SEQ IDNO.3所示。
2.根据权利要求1所述的一种Mucin1嵌合抗原受体修饰的T细胞,其特征在于:所述导引子的核苷酸序列如序列表中SEQ ID NO.2所示;所述CD8 Hinge区的核苷酸序列如序列表中SEQ ID NO.4所示;所述CD8跨膜区的核苷酸序列如序列表中SEQ ID NO.5所示;所述CD226-CD28共刺激区的核苷酸序列如序列表中SEQ ID NO.6所示;所述CD3ζ胞内区的核苷酸序列如序列表中SEQ ID NO.7所示;所述自剪切区T2A的核苷酸序列如序列表中SEQ IDNO.8所示;所述自杀基因RQR8的核苷酸序列如序列表中SEQ ID NO.9所示。
3.根据权利要求1所述的一种Mucin1嵌合抗原受体修饰的T细胞,其特征在于:所述Mucin1嵌合抗原受体修饰的T细胞的制备方法为将含Mucin1嵌合抗原受体的重组表达载体进行慢病毒包装,得到重组慢病毒,然后感染活化T细胞,得到Mucin1嵌合抗原受体修饰的T细胞。
4.权利要求1所述的Mucin1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。
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CN117143825A (zh) * | 2023-11-01 | 2023-12-01 | 上海兴瑞一达生物科技有限公司 | 一种msln嵌合抗原受体修饰的t细胞及其应用 |
CN117143825B (zh) * | 2023-11-01 | 2024-01-23 | 上海兴瑞一达生物科技有限公司 | 一种msln嵌合抗原受体修饰的t细胞及其应用 |
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