CN116606884A - 一种制备car-t细胞的方法以及制得的car-t细胞及其应用 - Google Patents
一种制备car-t细胞的方法以及制得的car-t细胞及其应用 Download PDFInfo
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Abstract
本发明提供了一种制备CAR‑T细胞的方法以及制得的CAR‑T细胞及其应用,本发明提供的嵌合抗原受体能够特异识别HER2抗原,具有良好的靶向性,包含该嵌合抗原受体的CAR‑T细胞在体外表现出很好杀伤能力,并且其在体内实验中,同样显示出很好的抗原靶向性和肿瘤杀伤活性,因此,本发明制备的CAR‑T细胞能够用于肿瘤的靶向治疗。
Description
技术领域
本发明涉及CAR-T细胞技术领域,特别涉及一种制备CAR-T细胞的方法以及制得的CAR-T细胞及其应用。
背景技术
目前,细胞治疗领域因其高度精准化和个性化的优势,已成为未来人类医学发展的热门方向。细胞治疗是指应用人的自体、同种异体或异种( 非人体) 的细胞,经体外操作后回输( 或植入) 人体的治疗方法。其中嵌合抗原受体T 细胞免疫疗法( CAR-T细胞疗法)是近几年兴起的过继性细胞免疫治疗,主要通过将嵌合抗原受体的特异性与T 细胞的免疫作用相结合,再经过特异性识别对恶性肿瘤细胞进行杀伤。CAR-T 细胞疗法的传统领域主要是治疗血液恶性肿瘤,临床效果较好。
人表皮生长因子受体2(HER2)是一种受体酪氨酸蛋白激酶,其过表达对于肿瘤的发生、发展与转移起着关键性作用。自1998年FDA批准曲妥珠单抗以来,特异性单克隆抗体治疗改变了HER2阳性肿瘤的治疗,在化疗中加入曲妥珠单抗可降低死亡率延长生存期,并降低20%的死亡风险。然而,一些HER2阳性肿瘤患者用药后并没有取得良好效果或者渐渐产生耐药反应;因此,需要进一步研究新的方法来治疗HER2过表达的肿瘤患者。因此,本发明的目的旨在设计一种靶向HER2的CAR-T细胞制备方法以及制得的CAR-T细胞及其应用。
发明内容
针对现有技术所存在的技术问题,本发明提供了一种制备CAR-T细胞的方法以及制得的CAR-T细胞及其应用。本发明提供的嵌合抗原受体能够特异识别HER2抗原,具有良好的靶向性,能够用于肿瘤的靶向治疗。
本发明的目的之一在于提供一种靶向HER2的CAR-T表达载体,其特征在于,所述表达载体包含靶向HER2的CAR基因序列;
优选地,所述靶向HER2的CAR依次是由CD8α信号肽、靶向HER2的单链抗体、CD8a铰链区、CD8a跨膜结构域、CD28蛋白共刺激结构域和CD3ζ胞信号传导结构域组成;
进一步地,所述靶向HER2的CAR-T的氨基酸序列如SEQ ID NO.1所示,其编码的核苷酸序列如SEQ ID NO.2所示;
优选地,所述CD8α信号肽序列的氨基酸序列如SEQ ID NO.3所示,其编码的核苷酸序列如SEQ ID NO.4所示;
优选地,靶向HER2的单链抗体的氨基酸序列如SEQ ID NO.5所示,其编码的核苷酸序列如SEQ ID NO.6所示;
优选地,CD8a铰链区的氨基酸序列如SEQ ID NO.7所示,其编码的核苷酸序列如SEQ ID NO.8所示;
优选地,CD8a跨膜结构域的氨基酸序列如SEQ ID NO.9所示,其编码的核苷酸序列如SEQ ID NO.10所示;
优选地,CD28蛋白共刺激结构域的氨基酸序列如SEQ ID NO.11所示,其编码的核苷酸序列如SEQ ID NO.12所示;
优选地,CD3ζ胞信号传导结构域的氨基酸序列如SEQ ID NO.13所示,其编码的核苷酸序列如SEQ ID NO.14所示;
本发明的另一个目的之一在于提供一种靶向HER2的CAR-T表达载体的构建方法,以靶向HER2的嵌合抗原受体基因为目的基因,利用重组慢病毒包装系统,构建获得靶向HER2的CAR-T表达载体。
本发明的另一个目的之一在于提供一种CAR-T细胞,使用上述CAR-T表达载体转导T淋巴细胞获得。
本发明的另一个目的之一在于上述CAR-T表达载体或CAR-T细胞在制备抗肿瘤药物中的应用。
优选地,上述肿瘤为HER2过表达肿瘤。
优选地,上述肿瘤包括HER2阳性乳腺癌。
本发明的优点如下:本发明提供的嵌合抗原受体能够特异识别HER2抗原,具有良好的靶向性,包含该嵌合抗原受体的CAR-T细胞在体外表现出很好杀伤能力,并且其在体内实验中,同样显示出很好的抗原靶向性和肿瘤杀伤活性,因此,本发明制备的CAR-T细胞能够用于肿瘤的靶向治疗。
附图说明
图1. CAR-T细胞体外对肿瘤细胞特异性杀伤作用;
图2. CAR-T细胞体内对肿瘤细胞特异性杀伤作用。
具体实施方式
下面结合具体实施例对本发明作进一步的详细说明,以使本领域的技术人员更加清楚地理解本发明。
以下各实施例,仅用于说明本发明,并不用来限制本发明的范围。基于本发明中的具体实施例,本领域普通技术人员在没有做出创造性劳动的情况下,所获得的其他所有实施例,都属于本发明的保护范围。
在本发明实施例中,若无特殊说明,所有原料组分均为本领域技术人员熟知的市售产品;在本发明实施例中,若未具体指明,所用的技术手段均为本领域技术人员所熟知的常规手段。
1.1 CAR表达序列的设计
一种靶向HER2的CAR依次是由CD8α信号肽、靶向HER2的单链抗体、CD8a铰链区、CD8a跨膜结构域、CD28蛋白共刺激结构域和CD3ζ胞信号传导结构域组成,其氨基酸序列如SEQ ID NO.1所示,其编码的核苷酸序列如SEQ ID NO.2所示。
其中,CD8α信号肽序列的氨基酸序列如SEQ ID NO.3所示,其编码的核苷酸序列如SEQ ID NO.4所示;靶向HER2的单链抗体的氨基酸序列如SEQ ID NO.5所示,其编码的核苷酸序列如SEQ ID NO.6所示;CD8a铰链区的氨基酸序列如SEQ ID NO.7所示,其编码的核苷酸序列如SEQ ID NO.8所示;CD8a跨膜结构域的氨基酸序列如SEQ ID NO.9所示,其编码的核苷酸序列如SEQ ID NO.10所示;CD28蛋白共刺激结构域的氨基酸序列如SEQ ID NO.11所示,其编码的核苷酸序列如SEQ ID NO.12所示;CD3ζ胞信号传导结构域的氨基酸序列如SEQ ID NO.13所示,其编码的核苷酸序列如SEQ ID NO.14所示;
1.2 慢病毒的构建
将设计的CAR基因序列插入至慢病毒骨架质粒pCDH-EF1-MCS(购自Addgene)中的EcoRI和BamH1酶切位点之间,将重组慢病毒质粒于所设计基因序列两端的酶切位点使用限制性内切酶进行双酶切后,进行SDS-PAGE电泳,选择与设计酶切片段一致大小的片段区域进行胶回收,经测序鉴定获得序列正确的阳性克隆质粒;将阳性克隆质粒与DH5α感受态大肠杆菌混合,加入不含抗生素的LB液体培养基培养,制备成原始菌液并接种至100mL的Amp抗性的LB培养基中,37℃摇床200rpm过夜,第二天收获菌液进行质粒抽提,检测质粒纯度和浓度,-20℃备用。
1.3慢病毒制备
提前24h将293T细胞接种到6cm培养皿,保证转染时细胞融合率达到80%。次日观察293T细胞,确认细胞状态良好且融合度在80%-90%时,更换新鲜培养基,将上述重组慢病毒质粒与包装质粒(psPAX2和pMD2 .G)使用Thermofisher生产的Lipofectamine 3000试剂盒(Cat#L3000008)依照说明书混合共孵育后逐滴加入到293T细胞培养皿中,轻轻晃动培养皿充分混匀。将培养皿置于37℃、5%CO2培养箱,培养6~8h后进行换液处理。连续培养72小时后,收集培养皿中含有病毒的培养基上清,用0.45μm的滤膜过滤,滤液转至无菌离心管中,超高速离心浓缩获得慢病毒并分装于病毒管,置于-80℃保存。
2.1 CAR-T细胞的制备
使用T细胞纯化试剂盒(美天旎,操作按产品说明书进行)从人外周血中获得T细胞后,按照4×106个T细胞加入40μL活化磁珠(美天旎)比例混匀,并加入IL-2至200IU/mL进行活化。
取激活后的T细胞计数,用T细胞培养基调整细胞密度,以5×105/孔加入24孔板中,加入5mL病毒上清,放入培养箱感染培养。病毒感染24h各补加500μL新鲜培养基,病毒感染48h收集病毒转导后的T细胞,1500rpm 5min离心弃上清,加入新鲜培养基重悬混匀细胞悬液并转移至培养瓶扩大培养,获得CART-Her2细胞。
3.1 CAR-T细胞体外对肿瘤细胞特异性杀伤作用
将1×106个HER2阳性的MCF7肿瘤细胞加入细胞培养皿,待其贴壁后加入1×104个T细胞或CAR-T细胞。培养5天后,采用LDH检测试剂盒说明书分析细胞的杀伤率。结果如图1所示:T细胞对HER2阳性的MCF7基本无杀伤作用,CAR-T细胞对靶细胞MCF7均有显著杀伤效果。
3.2 CAR-T细胞的体内抗肿瘤活性
利用小鼠异种移植瘤模型观察了CAR-T细胞的体内抗肿瘤活性。在免疫缺陷的NSG小鼠乳房垫处皮下注射1×106个MCF7细胞,构建小鼠肿瘤模型,待肿瘤体积为100mm3,经尾静脉给予1×104个T细胞或者CAR-T细胞回输(每组5只小鼠)。T细胞回输后,每周进行2次肿瘤测量并计算肿瘤体积,根据肿瘤体积制作肿瘤生长曲线。由图2可知,与T细胞相比,CAR-T细胞均能显著抑制肿瘤生长。由此说明,构建的CAR-T细胞在体内具有良好的抗肿瘤活性。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明技术原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
上述实施例的作用在于说明本发明的实质性内容,但并不以此限定本发明的保护范围。本领域的普通技术人员应当理解,可以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实质和保护范围。
Claims (10)
1. 一种靶向HER2的CAR-T表达载体,其特征在于,所述表达载体包含靶向HER2的CAR基因序列;所述靶向HER2的CAR依次是由CD8α信号肽、靶向HER2的单链抗体、CD8a铰链区、CD8a跨膜结构域、CD28蛋白共刺激结构域和CD3ζ胞信号传导结构域组成;所述靶向HER2的CAR-T的氨基酸序列如SEQ ID NO.1所示,其编码的核苷酸序列如SEQ ID NO.2所示。
2. 如权利要求1所述的靶向HER2的CAR-T表达载体,其特征在于,所述CD8α信号肽的氨基酸序列如SEQ ID NO.3所示,其编码的核苷酸序列如SEQ ID NO.4所示。
3. 如权利要求1所述的靶向HER2的CAR-T表达载体,其特征在于,所述靶向HER2的单链抗体的氨基酸序列如SEQ ID NO.5所示,其编码的核苷酸序列如SEQ ID NO.6所示。
4. 如权利要求1所述的靶向HER2的CAR-T表达载体,其特征在于,所述CD8a铰链区的氨基酸序列如SEQ ID NO.7所示,其编码的核苷酸序列如SEQ ID NO.8所示。
5. 如权利要求1所述的靶向HER2的CAR-T表达载体,其特征在于,所述CD8a跨膜结构域的氨基酸序列如SEQ ID NO.9所示,其编码的核苷酸序列如SEQ ID NO.10所示。
6. 如权利要求1所述的靶向HER2的CAR-T表达载体,其特征在于,所述CD28蛋白共刺激结构域的氨基酸序列如SEQ ID NO.11所示,其编码的核苷酸序列如SEQ ID NO.12所示。
7. 如权利要求1所述的靶向HER2的CAR-T表达载体,其特征在于,所述CD3ζ胞信号传导结构域的氨基酸序列如SEQ ID NO.13所示,其编码的核苷酸序列如SEQ ID NO.14所示。
8.如权利要求1-7中任一项所述的靶向HER2的CAR-T表达载体,其特征在于,以靶向HER2的嵌合抗原受体基因为目的基因,利用重组慢病毒包装系统,构建获得靶向HER2的CAR-T表达载体。
9.一种CAR-T细胞,其特征在于,使用权利要求8所述的靶向HER2的CAR-T表达载体来转导T淋巴细胞获得。
10.根据权利要求1-7中任一项所述的靶向HER2的CAR-T表达载体或根据权利要求9所述的CAR-T细胞在制备抗肿瘤药物中的应用,其特征在于,所述肿瘤为HER2过表达肿瘤。
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Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20190010220A1 (en) * | 2014-08-28 | 2019-01-10 | Bioatla, Llc | Conditionally active chimeric antigen receptors for modified t-cells |
CN111533808A (zh) * | 2020-03-10 | 2020-08-14 | 南京医科大学 | 一种可自分泌TLR4 scFv且靶向cMet的嵌合抗原受体修饰的T细胞及其应用 |
CN112391414A (zh) * | 2020-11-17 | 2021-02-23 | 中国医学科学院基础医学研究所 | 一种靶向her2的car-t表达载体及其构建和应用 |
CN112522208A (zh) * | 2020-12-25 | 2021-03-19 | 赜誉(上海)生物科技有限公司 | 一种转基因肿瘤浸润淋巴细胞及其用途 |
US20220185893A1 (en) * | 2018-12-20 | 2022-06-16 | West China Hospital, Sichuan University | Genetically engineered dual-targeting chimeric antigen receptor and use thereof |
CN115666594A (zh) * | 2019-11-29 | 2023-01-31 | 苏州诺沃泰医药科技有限公司 | Cart细胞在制备治疗癌症的药物中的应用 |
CN116041546A (zh) * | 2022-12-26 | 2023-05-02 | 湖南光琇高新生命科技有限公司 | 嵌合抗原受体、分离的核酸、重组载体、car-t细胞及其应用 |
-
2023
- 2023-07-17 CN CN202310870795.0A patent/CN116606884B/zh active Active
Patent Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20190010220A1 (en) * | 2014-08-28 | 2019-01-10 | Bioatla, Llc | Conditionally active chimeric antigen receptors for modified t-cells |
US20220185893A1 (en) * | 2018-12-20 | 2022-06-16 | West China Hospital, Sichuan University | Genetically engineered dual-targeting chimeric antigen receptor and use thereof |
CN115666594A (zh) * | 2019-11-29 | 2023-01-31 | 苏州诺沃泰医药科技有限公司 | Cart细胞在制备治疗癌症的药物中的应用 |
CN111533808A (zh) * | 2020-03-10 | 2020-08-14 | 南京医科大学 | 一种可自分泌TLR4 scFv且靶向cMet的嵌合抗原受体修饰的T细胞及其应用 |
CN112391414A (zh) * | 2020-11-17 | 2021-02-23 | 中国医学科学院基础医学研究所 | 一种靶向her2的car-t表达载体及其构建和应用 |
CN112522208A (zh) * | 2020-12-25 | 2021-03-19 | 赜誉(上海)生物科技有限公司 | 一种转基因肿瘤浸润淋巴细胞及其用途 |
CN116041546A (zh) * | 2022-12-26 | 2023-05-02 | 湖南光琇高新生命科技有限公司 | 嵌合抗原受体、分离的核酸、重组载体、car-t细胞及其应用 |
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