CN107881185A - 一种单链tcr‑t载体及单链tcr‑t细胞生产工艺 - Google Patents
一种单链tcr‑t载体及单链tcr‑t细胞生产工艺 Download PDFInfo
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Abstract
一种单链TCR‑T载体及单链TCR‑T细胞生产工艺,该方案包括完整的HPV特异性TCR选取、TCR‑T载体构建、TCR‑T细胞工程以及人工改造TCR‑T细胞的活性检测。本发明采用了可识别主要组织相容性复合体(MHC)为HLA‑A2呈递HPV‑16 E6多肽的TCR序列,并将该序列转化为功能性的单链构象,在载体构建方面提出了全新的TCRα链和β链的稳定组装方式,在TCR‑T细胞工程方面使用稳定TCR‑T逆转录病毒生产细胞系。由该系列技术生产的HPVTCR‑T细胞能够被呈递HPV病毒多肽的受体细胞有效激活,将能够用于HPV引发的头颈癌、宫颈癌等疾病的治疗。
Description
技术领域
本发明属于生物技术领域,具体涉及TCR-T类免疫治疗技术及应用领域。
背景技术
TCR-T技术是通过人工改造自体T细胞使其表达肿瘤抗原特异性TCR的细胞治疗技术。TCR-T细胞治疗包括肿瘤特异性TCR的选取、TCR表达载体的构建、TCR-T改造后细胞回输、免疫进程监测等关键技术和治疗手段。与同类型的CAR-T(嵌合抗原受体T细胞)细胞治疗技术相比,TCR-T具有更广泛抗原选择空间,因此不仅能扩充其适用肿瘤范围,还有望减少脱靶效应[1]。然而,尽管TCR-T在临床前测试和小范围病人治疗中取得了一定成功[2,3],TCR-T的临床试验数据仍然匮乏。同时,由于TCR-T的研发仍在初级阶段,临床治疗品质的TCR-T的载体构建和细胞生产策略还有待改进,更重要的是,TCR-T的有效患者人群以及新型构造的TCR-T的临床治疗效果仍有待证实。
HPV病毒是一种人群感染率十分普遍的DNA病毒。HPV感染会造成粘膜上皮细胞增生,而高危型HPV感染会引发头颈癌、宫颈癌等病变。有报告指出,15-25%的头颈癌和几乎所有的宫颈癌都由HPV感染引起[4]。目前这类癌症的治疗方案较为单一,主要治疗手段包括放疗和顺柏类化疗药物。由于缺乏特征性突变,并没有针对HPV癌症的靶向药物。因此,研发HPV引起的头颈癌、宫颈癌的新型治疗方案有非常重要的临床意义。由于这类肿瘤有明确的HPV抗原表达谱,针对HPV抗原的主要TCR序列在过往的研究中已被阐明[5],同时HPV抗原并无在正常组织中的表达,因此HPV特异性的TCR-T细胞将能够安全且有效的治疗HPV引发的头颈癌、宫颈癌。
TCR-T的细胞治疗方案包括以下几个核心技术难点:1)TCR的选取;2)TCR-T载体的构建;3)TCR-T的制备和回输。在TCR-T载体构建方面,国际上并没有统一的标准。目前已在进行中的TCR-T临床试验采用完整TCRα链和β链的表达载体[2,3],但是该载体序列较长,在检测TCR是否成功导入T细胞时需使用特殊的多肽-MHC四聚体,会一定程度影响TCR-T的表达和质量监测。最后,现有的TCR-T技术在病人T细胞中直接导入外源TCR,但是未有效限制外源TCR与內源TCR的错配情况。错配将有可能产生非特异性TCR识别,一旦有少量细胞识别自身抗原并克隆增殖,则会造成大规模的自体免疫反应,在临床中会有极大的安全风险。
发明内容
本发明要解决的技术问题是提供一种单链TCR-T载体及单链TCR-T细胞生产工艺。
本发明目的之一是这样实现的:
一种单链TCR-T载体,其特征在于:
(1)含有,但不限于针对HLA-A2呈递的HPV-16E6多肽的单链TCR序列,包括细胞膜定向信号肽序列SEQ ID No.1、TCRα链的可变区Vα序列SEQ ID No.2以及TCRβ链的可变区Vβ序列SEQ ID No.4;
(2)含有,但不限于连接TCRVα以及Vβ的连接序列SEQ ID No.3;
(4)其中,Vα与Vβ形成的单链TCR删去了可变区中不识别抗原部分;
(4)含有,但不限于改造后可以形成二硫键的鼠源恒定区Cβ序列SEQ ID No.5以及恒定区Cα序列SEQ ID No.8;
(5)含有,但不限于用于同时表达Cβ和Cα的间隔多肽P2A序列SEQ ID No.6以及Cα部分细胞膜信号肽SEQ ID No.7。
其中,鼠源Cβ和Cα可以有效降低TCR-T与人体T细胞中的人源Cβ或者Cα错配,从而减少因错配产生的非HPV特异性TCR;
其中,改造后鼠源Cβ和Cα增加一处二硫键配对的半胱氨酸,从而进一步促进导入TCR-T的自配对,降低与人源TCR配对的可能性;
其中,鼠源Cβ和Cα含有细胞内信号转导区,在单链TCR识别抗原后可以激活T细胞下游分子通路;
该载体含有独立的Cα部分(SEQ ID No.8),用于支撑整个TCR结构,同时允许仅更换Vα-Vβ-Cβ部分获得识别其它抗原的TCR-T,从而增加该载体构建方案的可扩展性;
鼠源Cβ和Cα可用于免疫荧光染色方法检测TCR-T载体的表达强度。
本发明目的之二是这样实现的:
一种TCR-T细胞生产工艺,其特征在于,包括:
(1)使用单链TCR-T载体、病毒结构载体共转染逆转录病毒包装细胞系;
(2)筛选产生高滴度的单链TCR-T逆转录病毒的单细胞;
(3)扩增出稳定并持续生产单链TCR-T逆转录病毒的病毒生产细胞系。
鉴于传统TCR-T疗法的局限和不足,本发明对TCR-T的设计进行根本性创新。首先,传统TCR-T的TCR识别区被分散在两条肽链上,而本发明构建出单链TCR,直接连接TCRα链的可变区Vα序列以及TCRβ链的可变区Vβ序列,极大简化了克隆步骤并加强了表达效率。第二,本发明通过使用改造的鼠源的Cβ和Cα,并在这两条肽链上增加了一个二硫键配对位点,极大程度减少了外源导入TCR和內源TCR错配引起的非特异性识别。这一并允许使用通用方法,即检测鼠源Cβ和Cα来检测TCR-T的表达效果。在TCR-T的制备方面,本发明涵盖单链TCR-T逆转录病毒生产细胞系的生产工艺,能够稳定快速的制备临床应用级的TCR-T细胞。
SEQIDNo.1(HPV16-E6长链信号肽序列)
MALEHLLIILWMQLTWVS
SEQIDNo.2(HPV16-E6Vα氨基酸序列)
GQQLNQSPQSMFIQEGEDVSMNCTSSSIFNTWLWYKQDPGEGPVLLIALYKAGELTSNGRLTAQFGITRKDSFLNISASIPSDVGIYFCAGHPSSNSGYALNFGKGTSLLVTP
SEQ ID No.3(单链TCR连接序列)
GGGGSGGGGSGGGGSGGGGS
SEQ ID No.4(HPV16-E6Vβ氨基酸序列)
GAGVSQSPRYKVTKRGQDVALRCDPISGHVSLYWYRQALGQGPEFLTYFNYEAQQDKSGLPNDRFSAERPEGSISTLTIQRTEQRDSAMYRCASSSQTGARTDTQYFGPGTRLTVL
SEQIDNo.5(改造后增加二硫键的鼠源Cβ氨基酸序列)
EDLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVSTDPQAYKESNYSYCLSSRLRVCATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYQQGVLSATILYEILLGKATLYAVLVSTLVVMAMVKRKNS
SEQID No.6(P2A氨基酸序列)
GSGATNFSLLKQAGDVEENPGP
SEQIDNo.7(HPV16-E6短链信号肽序列)
MALPVTALLLPLALLLHAARP
SEQIDNo.8(改造后增加二硫键的鼠源Cα氨基酸序列)
DIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKTVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNACYPSSDVPCDATLTEKSFETDMNLNFQNLSVMGLRILLLKVAGFNLLMTLRLWSS
有益效果:本发明公开了一种新型的TCR-T的构建方案,该方案能有效确保特异性TCR的表达,并通过增强TCR的特异性提高TCR-T疗法的安全性,对HPV感染引发的难治性宫颈癌、头颈癌的治疗有重要意义。
附图说明
图1A为HPV16-E6单链TCR-T的结构图示;
图1B为HPV16-E6单链TCR-T的核酸序列连接图;
图1C为表达HPV6-E6单链TCR的逆转录病毒载体图谱;
图2A为HPV16-E6单链TCR-T在Jurkat细胞上表达的流式分析图谱;
图2B为TCR-T表达的Jurkat细胞在受到抗原呈递细胞激活之后CD69的表达的流式分析图谱;
图3A为单链TCR-T病毒批量化生产的流程图;
图3B单链TCR-T在人外周血T细胞中的表达流式分析图谱;
图3C表达HPV16-E6单链TCR-T的外周血T细胞被抗原呈递细胞加载HPV16-E6多肽后激活的IFN-γ表达流式分析图谱。
具体实施方式
实施例
步骤(1)
HPV-16E6单链TCR-T载体的设计及构建
HPV-16E6单链TCR-T的总结构如图1A所示,其中抗原识别部分Hu Vα以及Hu Vβ由四段重复的GGGS连接多肽构成。该单链部分连有鼠源TCR Cβ链,包含跨膜区以及突变后新产生的二硫键位点(红色-S-S-标注)。另外,该单链TCR含有鼠源TCR Cα链,包含跨膜区以及突变后产生的二硫键位点,其位置可以与鼠源Cβ链配对,使得鼠源Cα链与鼠源Cβ链有两个二硫键配对位点。载体设计上,为保持Hu Vα-Linker-Hu Vβ-Mu Cβ肽链与Mu Cα肽链表达量相当,用P2A序列连接这两段肽链。P2A序列可以在蛋白质翻译完成后在内质网中自降解,从而得到P2A序列前面及后面的两条肽链。
完整的单链TCR片段(图1B)的核苷酸序列在密码子优化后,由Genscript公司合成,再通过分子克隆方法克隆至MSCV逆转录病毒载体中,其步骤包括以下要点:
1)将小量MSCV-mPGK载体转化到DH5α感受态细菌中,在37摄氏度扩增。使用QIAgenMaxiprep试剂盒大量提取MSCV-mPGK载体;
2)使用NEBNcoI-HF/BamHI-HF两种限制性内切酶切MSCV-mPGK载体以及合成的HPV单链TCR片段,在37摄氏度进行1小时酶切反应;
3)用电泳胶方法分离酶切完成的MSCV-mPGK载体片段,并使用NEBT4DNAligase连接酶切完成的载体片段以及单链TCR片段,在14摄氏度连接16小时;
4)将连接产物转化至DH5α感受态细菌中,并在氨苄抗性的LB平板中培养转化完成的菌落。挑选3个转化成功的菌落进行测序鉴定。测序鉴定正确的质粒图谱如图1C所示。对测序鉴定正确的菌落进行扩增和大量提取质粒。
步骤(2)
单链TCR-T表达效率及功能测试
1)使用Lipofectamin2000转染试剂盒将1微克单链TCR质粒转染至BOSC病毒包装细胞系。48小时之后,收取病毒包装细胞系培养上清液中的TCR-T逆转录病毒。速冻于-80摄氏度可长期保存;
2)培养Jurkat细胞至1x106细胞/毫升。Jurkat细胞是人的一种T细胞系,有持续分裂的能力,并能够被常规TCR通路激活,提高CD69等激活分子标记的表达。取1毫升Jurkat细胞于24孔培养板中,加入1毫升TCR-T病毒,使用离心机在800g,37摄氏度下离心感染1.5小时。病毒感染完成后移除病毒液,更换为Jurkat细胞培养基;
3)继续培养感染后的TCR-Jurkat细胞。48小时之后,使用抗鼠源Cβ抗体对细胞染色,并使用流式细胞仪检测HPVTCR-T表达效率,其结果如图2A所示。经重复测试,该TCR-T表达效率稳定可达20%以上;
4)使用HPV-16E6多肽加载MHC表型为HLA-A2的T2细胞,在37摄氏度加载1小时。并将TCR-Jurkat细胞与之共培养。二十四小时后检测TCR-Jurkat细胞的激活情况:使用抗人CD69抗体对细胞进行染色,CD69表达量升高表示细胞被TCR信号通路激活,其结果如图2B所示。
步骤(3)
单链TCR-T病毒生产细胞系的生成和在人原代T细胞中的表达测试
1)用HPV16-E6单链TCR-T载体和包装载体共转染PG13包装细胞系;
2)挑选单克隆包装细胞,并在96孔板中测试病毒滴度;
3)筛选生产病毒滴度最高的单克隆包装细胞,并大量扩增与储存;
4)获取人外周血单核细胞,使用Gibco人CD3/CD28抗体磁珠激活T细胞,在24小时后使用单链TCR-T病毒感染细胞,感染方法同实施例2中Jurkat细胞的感染。在感染48小时后使用抗鼠源Cβ抗体对细胞染色,并使用流式细胞仪检测HPVTCR-T表达效率。其结果如图3B所示;
5)将感染完成的TCR-T细胞与加载HPV16-E6多肽的T2细胞系,进行T细胞激活。在TCR-T细胞激活后第6天和第17天的时候,取一定数量的细胞,使用PMA和Ionomycin刺激细胞,同时加入eBiosicencegolgiblock试剂,在37摄氏度培养4小时,用2%PFA固定,用0.5%Saponin穿孔细胞膜,最后使用IFN-γ抗体染色(图3C)。IFN-γ是T细胞肿瘤杀伤过程中的重要效应分子,其表达增高可以反映TCR-T细胞杀伤活性。
Claims (2)
1.一种单链TCR-T载体,其特征在于:
(1)含有,但不限于针对HLA-A2呈递的HPV-16E6多肽的单链TCR序列,包括细胞膜定向信号肽序列SEQ ID No.1、TCRα链的可变区Vα序列SEQ ID No.2以及TCRβ链的可变区Vβ序列SEQ ID No.4;
(2)含有,但不限于连接TCRVα以及Vβ的连接序列SEQ ID No.3;
(3)其中,Vα与Vβ形成的单链TCR删去了可变区中不识别抗原部分;
(4)含有,但不限于改造后可以形成二硫键的鼠源恒定区Cβ序列SEQ ID No.5以及恒定区Cα序列SEQ ID No.8;
(5)含有,但不限于用于同时表达Cβ和Cα的间隔多肽P2A序列SEQ ID No.6以及Cα部分细胞膜信号肽SEQ ID No.7。
2.一种TCR-T细胞生产工艺,其特征在于,包括:
(1)使用单链TCR-T载体、病毒结构载体共转染逆转录病毒包装细胞系;
(2)筛选产生高滴度的单链TCR-T逆转录病毒的单细胞;
(3)扩增出稳定并持续生产单链TCR-T逆转录病毒的病毒生产细胞系。
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