CN112940105B - HLA-A11限制性乙型肝炎病毒HBc141-151表位肽的T细胞受体及其应用 - Google Patents
HLA-A11限制性乙型肝炎病毒HBc141-151表位肽的T细胞受体及其应用 Download PDFInfo
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Abstract
本发明公开了HLA‑A11限制性乙型肝炎病毒HBc141‑151表位肽的T细胞受体及其应用。该T细胞受体包含α链和β链;α链包含三个互补决定区,氨基酸序列分别如SEQ ID No.2的第48‑53位、第71‑77位和第112‑121位所示;β链包含三个互补决定区,氨基酸序列分别如SEQ ID No.4的第46‑50位、第68‑73位和第111‑122位所示。实验证明,该T细胞受体不仅具有HBV多肽表位依赖性的活化和增殖能力,而且体内和体外均具有良好的杀伤靶细胞的活性,还能够有效清除体内HBV的慢性感染。本发明具有很重要的应用价值。
Description
技术领域
本发明属于生物医学领域,具体涉及HLA-A11限制性乙型肝炎病毒HBc141-151表位肽的 T细胞受体及其应用。
背景技术
肝细胞癌(Hepatocellular Carcinoma,HCC)简称肝癌,其临床表现隐匿,早期缺乏典型症状,尽管近年来肝癌的诊断和治疗有了一定的进展,其预后仍然不理想,五年生存率极低。究其原因,一方面是肝癌对目前化疗药物不敏感,大多数肝癌患者缺乏有效的治疗方法;另一方面,肝癌往往在疾病的晚期才被诊断,这就排除了局部消融这些原本可以改善病人病情的方法。目前,手术切除与肝移植仍然是治疗肝癌最有效的方法。但是肝癌发展到一定阶段,肿瘤细胞可能转移到其它器官(如肺部、骨骼、脑等),行肝移植前,现阶段的检查检测不到,术后因免疫抑制状态,潜伏在其它器官的微病灶可能导致肝癌复发,患者术后生存期不理想。因此,亟需找寻新的有效辅助治疗方案。
免疫细胞治疗技术是生物医药领域中发展最为迅速的板块。2013年,科学转化医学 (Science Translational Medicine)杂志预测,细胞治疗将成为“未来医学第三大支柱”。2017 年10月以来,美国FDA相继批准诺华与KITE公司的CAR-T药物上市,开创免疫细胞治疗药物的新时代,在临床治疗中展现出广阔的应用前景。在实体肿瘤免疫细胞治疗领域,理想的目标抗原是仅在肿瘤细胞表面表达的肿瘤特异性抗原。不幸的是肿瘤表达的大多数抗原不具备肿瘤特异,因此大多数的CAR-T、TCR-T都以肿瘤相关性抗原作为靶点,但这往往会导致“脱靶”的可能性。目前使用较多的一些肿瘤相关抗原,甲胎蛋白、NY-ESO、MAGE等蛋白都属于这一状态。
在诸多肝癌相关环境危险因素中,乙型肝炎病毒(Hepatitis B Virus,HBV)或丙型肝炎病毒(Hepatitis C Virus,HCV)感染与肝癌的发生直接相关。高达80%的肝癌归因于HBV 或HCV感染,在我国主要是HBV感染为主,90%肝癌患者HBsAg呈阳性。HBV感染人体后可刺激机体产生一系列体液免疫和细胞免疫反应,通常可以清除感染的病毒而痊愈;但如果机体免疫反应低下,不足以清除病毒,则病毒可持续存在,可发展成为慢性乙肝。慢性乙肝是肝硬化和肝癌发生的重要危险因素。慢性乙肝主要涉及免疫反应各个阶段的抗病毒免疫反应缺陷,尤其是HBV特异性CD8+T细胞的数量下降、功能降低,引起免疫耐受;同时伴随免疫介导的炎症性肝损伤。
目前,尚未发现能够治愈乙肝的药物。临床上针对慢性乙肝的治疗手段主要为抗病毒相关药物的治疗,包括干扰素类(IFNs)及核苷和核苷酸类药物等,通过免疫调节或干扰HBV 复制发挥抗病毒作用。然而,这些治疗手段不能彻底清除病毒,使得患者容易出现耐药、病毒变异以及病情反复等情况。
HBV特异性CD8+T细胞在控制病毒的复制、清除病毒和HBV感染的临床恢复上起着决定性作用。HBV特异性TCR基因修饰的T细胞(TCR-T)过继转移,已初步证明有非常好的抗病毒活性。在肝癌领域,HBV也可作为一种独特的肿瘤相关性抗原。虽然已知的 HBV-HCC肝癌细胞不表达完整的HBV抗原,但是在慢性乙型肝炎的自然史感染中,病毒往往将自身整合到人类基因组中,最终形成HCC细胞并携带这些HBV基因组。新加坡学者研究发现,尽管不表达完整HBV抗原,HBV-HCC肝癌细胞却带有短片段的HBV mRNAs,这些mRNAs可以编码能够被HBV特异性T细胞识别并活化的表位多肽,他们入组了2例肝移植患者肝癌复发和肺部转移瘤,根据肿瘤中HBV mRNA表达选择HBV特异性T细胞受体(TCRs),利用基因工程手段让自体T细胞表达这些TCRs,并过继转移到病人体内。这些TCR-T细胞不影响肝功能,在1例患者中,5/6肺转移瘤在1年内体积减小。该结果提示在肝癌病人,特别是肝移植患者治疗中,将肝癌细胞上HBV抗原表位当做肿瘤相关抗原进行T细胞过继免疫治疗有可能将HBV-HCC肝癌细胞清除掉。这种肝特异性标记意味着肿瘤外的不良反应在很大程度上是可以预测的,很少或不涉及其它器官。此外,也有其它团队临床研究通过体外诱导TCR重定向的T细胞的方法,将特异识别乙肝病毒HLA-A2限制性表位HBs183-191的TCR-T细胞用于治疗肝移植患者中的肝癌转移,证明了这种重定向TCR-T细胞具有靶向特定肝癌细胞的活性,展现出巨大的应用潜力和价值。
现有报道的治疗性T细胞的研究主要局限在HLA-A2人群的研究,而针对其它HLA人群的关注却明显不足。HLA-A3超家族(包含HLA-A11、HLA-A33、HLA-A68、HLA-A31 等)在中国人群HLA分型中占着最大比例(约52.7%)。HLA-A3超家族成员的显著特征之一是不同的成员具有共同的多肽结合特性,即偏好结合C末端带碱性氨基酸的多肽表位。因此,针对HLA-A3超家族限制性的人群进行肝癌的治疗研究将具有很好的大众性和通用性。其中,HLA-A11是A3超家族中分布最广的,统计分析也表明中国乙肝患者中HLA-A11基因频率最高。
HLA转基因动物模型在临床前试验和基础实验研究中发挥了重要作用。HLA-A11转基因小鼠之前已有报道,然而多方面的证据表明,HLA-A11分子偏好结合C末端碱性的表位多肽,与小鼠自身抗原提呈系统中抗原相关转运蛋白(TAP)的多肽结合偏好性不符,因而HLA-A11转基因小鼠在递呈HLA-A11限制性表位上存在明显缺陷。
将含人源TAP-LMP基因簇的BAC转基因小鼠(hTAP-LMP转基因小鼠)与HLA-A11 转基因小鼠杂交,得到优化型的HLA-A11/hTAP-LMP转基因小鼠。与HLA-A11转基因小鼠相比,HLA-A11/hTAP-LMP转基因小鼠具有更强的提呈HLA-A11限制性CTL表位的能力。
综上所述,免疫细胞技术治疗乙肝和肝癌进入到临床试验阶段并取得了进展,显示出广阔的应用前景。但是在中国人群为主的HLA-A11限制性肝癌治疗亟需进行深入的研究。
发明内容
本发明的目的是制备用于预防和/或治疗由HBV感染所致的疾病的药物。
本发明首先保护一种识别HLA-A11限制性乙型肝炎病毒HBc141-151表位肽的T细胞受体,可包含α链和β链。所述α链可包含三个互补决定区,氨基酸序列分别如SEQ ID No.2的第 48-53位、第71-77位和第112-121位所示;或这些序列的具有至多3个、2个或1个氨基酸改变的变体。所述β链可包含三个互补决定区,氨基酸序列分别如SEQ ID No.4的第46-50位、第68-73位和第111-122位所示;或这些序列的具有至多3个、2个或1个氨基酸改变的变体。
所述T细胞受体中,所述α链的可变区的氨基酸序列可如SEQ ID No.2的第22-112位所示;或这些序列的具有至多3个、2个或1个氨基酸改变的变体。所述β链的可变区的氨基酸序列可如SEQ ID No.4的第20-113位所示;或这些序列的具有至多3个、2个或1个氨基酸改变的变体。
所述α链的恒定区的氨基酸序列为SEQ ID No.2的第133-268位所示。
所述β链的恒定区的氨基酸序列为SEQ ID No.4的第133-305位所示。
所述T细胞受体中,所述α链的氨基酸序列可如SEQ ID No.2所示。所述β链的氨基酸序列可如SEQ ID No.4所示。
编码上述任一所述T细胞受体的核酸分子也属于本发明的保护范围。
编码上述任一所述T细胞受体的核酸分子可包含编码所述T细胞受体的α链的核酸分子和编码所述T细胞受体的β链的核酸分子。
编码所述T细胞受体的α链中三个互补决定区的核苷酸序列分别可如SEQ ID No.1的第 142-159位、第211-231位和第334-363位所示;或与这些序列具有99%以上、95%以上、90%以上、85%以上或者80%以上同一性且编码相同氨基酸残基的序列。
编码所述T细胞受体的β链中三个互补决定区的核苷酸序列分别可如SEQ ID No.3的第 136-150位、第202-219位和第331-366位所示;或与这些序列具有99%以上、95%以上、90%以上、85%以上或者80%以上同一性且编码相同氨基酸残基的序列。
编码所述α链的可变区的核苷酸序列可如SEQ ID No.1的第64-336位所示;或与这些序列具有99%以上、95%以上、90%以上、85%以上或者80%以上同一性且编码相同氨基酸残基的序列。
编码所述β链的可变区的核苷酸序列如SEQ ID No.3的第58-339位所示;或与这些序列具有99%以上、95%以上、90%以上、85%以上或者80%以上同一性且编码相同氨基酸残基的序列。
所述α链的恒定区的核苷酸序列为SEQ ID No.1的第397-807位所示。
所述β链的恒定区的核苷酸序列为SEQ ID No.3的第397-918位所示。
编码所述α链的核酸分子的核苷酸序列如SEQ ID No.1所示。
编码所述β链的核酸分子的核苷酸序列如SEQ ID No.3所示。
含有上述任一所述核酸分子的表达盒、载体或细胞也属于本发明的保护范围。
所述细胞可为Jurkat细胞或T细胞。所述T细胞具体可为人T细胞或小鼠T细胞。
所述载体可为逆转录病毒载体或慢病毒载体。
所述逆转录病毒载体具体可为向逆转录病毒载体MSCV-IRES-GFP的多克隆位点(如限制性内切酶XhoI和EcoRI)之间插入编码所述T细胞受体的α链的核酸分子和编码所述T 细胞受体的β链的核酸分子,得到的重组质粒。
所述逆转录病毒载体具体可为向逆转录病毒载体MSCV-IRES-GFP的多克隆位点(如限制性内切酶XhoI和EcoRI)之间插入DNA序列,得到的重组质粒;所述DNA序列为将编码所述α链的核酸分子和编码所述β链的核酸分子用连接肽(如T2A自剪切多肽)的编码序列连接而成。
在本发明的一个实施例中,所述逆转录病毒载体具体可为将逆转录病毒载体MSCV-IRES-GFP的限制性内切酶XhoI和EcoRI之间的DNA小片段替换为SEQ ID No.5所示的DNA分子,得到的重组质粒。SEQ ID No.5中,第1至804位为α链的完整编码基因,第805至867位为T2A自剪切多肽的编码基因,第868至1785位为β链的完整编码基因。
上述任一所述逆转录病毒载体MSCV-IRES-GFP是将MO载体的限制性内切酶XhoI和ClaI之间的DNA小片段替换为IRES核苷酸序列(Genbank:MG550106.1)和荧光标记蛋白 GFP核苷酸序列(Genbank:MH777595.1),得到的重组质粒。
所述慢病毒载体具体可为向慢病毒包装载体pCDH-MSCV-MCS-IRES-GFP(SystemBiosciences,编号:CD731B-1)的多克隆位点(如限制性内切酶EcoRI和BamHI)之间插入编码所述T细胞受体的α链的核酸分子和编码所述T细胞受体的β链的核酸分子,得到的重组质粒。
所述慢病毒载体具体可为向慢病毒包装载体pCDH-MSCV-MCS-IRES-GFP的多克隆位点(如限制性内切酶EcoRI和BamHI)之间插入DNA序列,得到的重组质粒;所述DNA 序列为将编码所述α链的核酸分子和编码所述β链的核酸分子用连接肽(如T2A自剪切多肽) 的编码序列连接而成。
在本发明的一个实施例中,所述慢病毒载体具体可为将慢病毒包装载体 pCDH-MSCV-MCS-IRES-GFP的限制性内切酶EcoRI和BamHI之间的DNA小片段替换为 SEQ ID No.5所示的DNA分子,得到的重组质粒。SEQ ID No.5中,第1至804位为α链的完整编码基因,第805至867位为T2A自剪切多肽的编码基因,第868至1785位为β链的完整编码基因。
具有上述任一所述T细胞受体的T细胞也属于本发明的保护范围。
含有上述任一所述T细胞受体的T细胞或“上述任一所述表达盒、载体或细胞”的药物组合物也属于本发明的保护范围。所述药物组合物可用于预防和/或治疗由HBV感染所致的疾病。
本发明还保护上述任一所述T细胞受体、或、上述任一所述核酸分子、或、上述任一所述载体或细胞、或、上述任一所述T细胞受体的T细胞,的应用,可为A1)-A4)中的至少一种:
A1)制备预防和/或治疗由HBV感染所致的疾病的药物;
A2)预防和/或治疗由HBV感染所致的疾病;
A3)体内或体外杀伤靶细胞;
A4)清除HBV的慢性感染。
上述应用中,所述靶细胞可为脾细胞或PBMC细胞。所述脾细胞具体可为负载多肽HBc141-151的脾细胞。所述PBMC细胞具体可为负载多肽HBc141-151的PBMC细胞。所述负载多肽HBc141-151的脾细胞具体可为小鼠(如HLA-A11转基因小鼠)负载多肽HBc141-151的脾细胞。所述负载多肽HBc141-151的PBMC细胞可为负载多肽HBc141-151的小鼠(如HLA-A11 转基因小鼠)PBMC或HLA-A11+PBMC细胞。HLA-A11+PBMC细胞是指HLA-A11阳性健康人的PBMC细胞。
本发明还要求保护一种预防和/或治疗由HBV感染所致疾病的方法,可包括如下步骤:以前文所述的T细胞受体、或、所述核酸分子、或、“所述表达盒、载体或细胞”、或、含有上述任一所述T细胞受体的T细胞来预防和/或治疗由HBV感染所致疾病。
上述任一所述由HBV感染所致的疾病可为慢性乙型肝炎或肝细胞癌。
本发明的发明人经过大量实验,分离并鉴定了一对HBV特异性的TCR序列,成功构建了这对TCR的转基因小鼠,并且在体外验证了TCR转基因阳性的CD8细胞(即TCR-T细胞)具有HBV多肽表位依赖性的活化和增殖能力;同时利用动物体内和体外杀伤靶细胞实验,证明了这对TCR具有很好的杀伤靶细胞(HLA-A11转基因小鼠负载多肽HBc141-151的脾细胞或PBMC细胞)的活性;体外验证Human TCR-T也具有特异性的杀伤靶细胞(负载多肽HBc141-151的HLA-A11限制性人PBMC细胞)的能力;此外,动物实验提示这对TCR序列可能是清除HBV感染细胞的有效方法之一。本发明提供的HLA-A11限制性HBc141-151表位肽的T细胞受体具有重要的应用价值。
附图说明
图1为筛选HLA-A11限制性HBV特异性TCR序列的流程示意图。
图2为表达TCR的Jurkat细胞的染色结果。
图3为TCR转基因小鼠的鉴定。
图4为TCR转基因小鼠的CD8+T细胞(即TCR-T细胞)具有HBV多肽依赖的活化增殖能力。
图5为TCR-T细胞具有体外杀伤靶细胞的功能。
图6为TCR-T细胞具有体内杀伤靶细胞的功能。
图7为TCR-T细胞可以有效的清除体内HBV的慢性感染。
图8为Human TCR-T细胞具有体外杀伤靶细胞的功能。
具体实施方式
以下的实施例便于更好地理解本发明,但并不限定本发明。
下述实施例中的实验方法,如无特殊说明,均为常规方法。
下述实施例中所用的试验材料,如无特殊说明,均为自常规生化试剂商店购买得到的。
以下实施例中的定量试验,均设置三次重复实验,结果取平均值。
HLA-A11/hTAP-LMP转基因小鼠、多肽HBc123-157、辅助多肽HBc128-140和多肽HBc141-151记载于如下文献中:Man Huang,Wei Zhang,Jie Guo,Xundong Wei,Krung Phiwpan,Jianhua Zhang,Xuyu Zhou.Improved Transgenic Mouse Model for Studying HLAClass I Antigen Presentation.Scientific Report.2016,doi:10.1038/srep33612.
下述实施例中,尾静脉高压注射法记载于如下文献中:Liu F,Song Y,Liu D.Hydrodynamics-based transfection in animals by systemic administration ofplasmid DNA.Gene Therapy.1999,doi:10.1038/sj.gt.3300947。
下述实施例中,pAAV/HBV1.2质粒记载于如下文献中:Huang,L.R.,Wu,H.L.,Chen,P.J.&Chen,D.S.An immunocompetent mouse model for the tolerance of humanchronic hepatitis B virus infection.Proc Natl Acad Sci USA 103.17862–17867,doi: 10.1073/pnas.0608578103(2006)
10×PCR buffer、dNTPmix和Taq DNA polymerase均为TAKARA公司的产品。C57BL/6小鼠和ICR小鼠均为北京华阜康生物科技股份有限公司的产品。B6D2F1小鼠为北京维通利华实验动物技术有限公司的产品。
实施例1、TCR-T细胞的获得及其应用
一、应用HLA-A11/hTAP-LMP转基因小鼠筛选获得A11限制性HBV特异性TCR序列
1、将100μL含100μg多肽HBc123-157和100μg辅助多肽HBc128-140的PBS缓冲液和100μLIFA 混合,乳化;然后皮下多点注射至HLA-A11/hTAP-LMP转基因小鼠。HLA-A11/hTAP-LMP转基因小鼠体内将诱导出针对HBc141-151表位的初次CTL免疫应答。
2、完成步骤1后第14天,采用尾静脉高压注射法向HLA-A11/hTAP-LMP转基因小鼠的尾静脉注射pAAV/HBV1.2质粒,每只小鼠注射10μg pAAV/HBV1.2质粒。
pAAV/HBV1.2质粒包含HBV病毒1.2个拷贝的基因组,采用尾静脉高压注射法可瞬时转染小鼠肝细胞并表达乙肝病毒抗原以及产生病毒颗粒,因而可用于在小鼠体内模拟HBV的早期感染。进行该步骤的目的是HBV的早期感染后,诱导针对HBc141-151的二次CTL免疫应答,产生大量HBc141-151抗原特异性的CD8+T细胞。
3、完成步骤2后第8天,处死HLA-A11/hTAP-LMP转基因小鼠并收集外周血单核细胞,进行Tetramer染色,之后流式分选出HLA-A11/HBc141-151Tetramer(NIH TetramerFacility)和 CD8双阳性的T细胞,使用口吸式显微注射针将抗原特异性CTL在显微镜下手动吸取单个细胞,即获得单个抗原特异性CTL细胞。
4、完成步骤3后,首先配制反转录体系Mix1(由0.5μL Random引物和4.5μLDEPC处理的 H2O组成)和反转录体系Mix2(由5μL 2×buffer和0.5μL RT酶组成);然后在显微镜下使用显微注射针将单个抗原特异性CTL细胞吹入反转录体系Mix1中,70℃冰浴5min,冰浴2min;之后加入反转录体系Mix2,25℃5min,42℃30min,85℃5min,合成单细胞cDNA。
Random引物为生工生物工程(上海)股份有限公司的产品。
2×buffer和RT酶为北京全式金生物技术有限公司的产品。
5、完成步骤4后,进行两轮TCR特异性兼并引物PCR。
第一轮PCR:TCRα-mix,V区23条,C区1条;TCRβ-mix,V区19条,C区1条。
第二轮PCR:TCRα-mix(in),V区23条,C区1条;TCRβ-mix(in),V区19条,C区1条。
引物的核苷酸序列具体见表1。
表1
第一轮PCR反应体系为25μL,具体见表2。反应程序为:95℃5min;95℃20s,56℃20s, 72℃45s,34个循环;72℃7min。
表2
第二轮PCR反应体系为25μL,具体见表3。反应程序为:95℃5min;95℃20s,56℃20s, 72℃45s,34个循环;72℃7min。
表3
TCR | |
第一轮产物 | 1μL(梯度稀释使用) |
10×PCR buffer | 2.5μL |
10mM dNTPmix | 0.5μL |
Primer Mix(5μM) | 0.5μL(终浓度为0.1μM) |
Taq DNA Polymerase | 0.15μL(0.75U) |
H<sub>2</sub>O | 20.35μL |
6、完成步骤5后,两轮PCR结束后得到配对的TCR-α和TCR-β可变区序列。切胶回收PCR 扩增产物,测序。将测序结果在IMGT网站(网址为: http://www.imgt.org/IMGT_vquest/vquest?livret=0&Option=mouseTcR)上进行分析,得到单个细胞的α链序列和β链序列。
共检测40个抗原特异性CTL。其中5个抗原特异性CTL可以获得α链序列和β链序列,即得到5对TCR受体序列。经过分析,这5个抗原特异性CTL的α链和β链完全相同。
α链可变区的编码基因如SEQ ID No.1的第64-336位所示;其中,SEQ ID No.1中,第 142-159位、第211-231位和第334-363位分别为三个CDR的编码基因。
α链可变区的氨基酸序列如SEQ ID No.2的第22-112位所示;其中,SEQ ID No.2中,第 48-53位、第71-77位和第112-121位分别为三个互补决定区。
β链可变区的编码基因如SEQ ID No.3的第58-339位所示;其中,SEQ ID No.3中,第 136-150位、第202-219位和第331-366位分别为三个CDR的编码基因。
β链可变区的氨基酸序列如SEQ ID No.4的第20-113位所示;其中,SEQ ID No.4中,第 46-50位、第68-73位和第111-122位分别为三个互补决定区。
这对α链序列和β链序列组成的TCR受体可能对HLA-A11限制性CTL表位HBc141-151具有高亲和力,并来自同一个T细胞克隆。
步骤3-6的实验流程图见图1。
二、表达TCR的Jurkat细胞的获得及鉴定
1、根据步骤一得到的α链可变区和β链可变区的序列,参考NCBI上小鼠基因组α链和α链的恒定区序列,获得并人工合成HLA-A11限制性乙型肝炎病毒HBc141-151特异性TCR受体α链和β链的完整编码基因。
α链的完整编码基因如SEQ ID No.1所示,编码SEQ ID No.2所示的α链。
β链的完整编码基因如SEQ ID No.3所示,编码SEQ ID No.4所示的β链。
2、将逆转录病毒载体MSCV-IRES-GFP的限制性内切酶XhoI和EcoRI之间的DNA小片段替换为SEQ ID No.5所示的DNA分子,其它序列均不变,得到重组质粒MSCV-TCR-IRES-GFP(即重组质粒MSCV-TCR-GFP)。SEQ ID No.5中,第1至804位为α链的完整编码基因,第805 至867位为T2A自剪切多肽的编码基因,第868至1785位为β链的完整编码基因。
逆转录病毒载体MSCV-IRES-GFP是将MO载体的限制性内切酶XhoI和ClaI之间的DNA 小片段替换为IRES核苷酸序列(Genbank:MG550106.1)和荧光标记蛋白GFP核苷酸序列(Genbank:MH777595.1),得到的重组质粒。
MO载体记载于如下文献:Tanyu Hu,Krung Phiwpan,Jitao Guo,et al.MicroRNA-142-3p Negatively Regulates Canonical Wnt Signaling Pathway.PLOS ONE.2016,DOI:10.1371/journal.pone.0158432.
3、培养Jurkat细胞至数量2×107以上,收集细胞,用不含抗生素的1640培养基清洗2遍,最后一遍洗后用不含抗生素1640培养基重悬至5×107/mL,将上述细胞分为400μL/份加至电转杯(BIO-RAD,货号:165-2088),同时加入40μg重组质粒MSCV-TCR-GFP,混匀,将电转杯放入电转仪(BIO-RAD,Gene Pulser XcellTM)中,电压250V,电容950μF电转,将重组质粒MSCV-TCR-GFP导入Jurkat细胞,得到表达TCR的Jurkat细胞。
按照上述方法,将重组质粒MSCV-TCR-GFP替换为逆转录病毒载体MSCV-NGFR-GFP,其它步骤均不变,得到表达NGFR的Jurkat细胞。
逆转录病毒载体MSCV-NGFR-GFP是将MO载体的限制性内切酶XhoI和EcoRI之间的DNA小片段替换为NGFR核苷酸序列(序列来源于addgene NGFR质粒,Plasmid#27489),再将限制性内切酶EcoRI和ClaI之间的DNA小片段替换为IRES核苷酸序列(Genbank:MG550106.1)和荧光标记蛋白GFP核苷酸序列(Genbank:MH777595.1),得到的重组质粒。
4、利用Tetramer(HLA-A11/HBc141-151)对步骤3得到的表达TCR的Jurkat细胞或表达NGFR 的Jurkat细胞进行染色。
染色结果见图2。结果表明,TCR对HLA-A11限制性表位具有较强的亲和力。
三、HBc141-151特异性TCR转基因小鼠(以下简称TCR转基因小鼠)的构建和鉴定
1、重组质粒phCD2-TCR-α和重组质粒p428-TCR-β的获得
(1)向phCD2质粒的限制性内切酶EcoRI的识别位点处插入SEQ ID No.1所示的TCR-α基因序列,其它序列均不变,得到重组质粒phCD2-TCR-α。
phCD2质粒记载于如下文献:Zhumabekov T,Corbella P,Tolaini M.KioussisD.Improved version of a human CD2 minigene based vector for T cell-specificexpression in transgenic mice.Journal of Immunological Methods.1995 Sep 11;185(1):133-40.
(2)向p428质粒的限制性内切酶SalI的识别位点处插入SEQ ID No.3所示的TCR-β基因序列,其它序列均不变,得到重组质粒p428-TCR-β。
p428质粒记载于如下文献:Sawada S,Scarborough JD,Killeen N,Littman DR.A lineage-specific transcriptional silencer regulates CD4 gene expressionduring T lymphocyte development.Cell.1994 Jun 17;77(6):917-29.
2、超排以及假孕母鼠的准备:
(1)超排
向3周龄的C57BL/6雌性小鼠腹腔注射PMSG(5U/只);48h以后,腹腔注射HCG(5U/只);注射HCG后立即与B6D2F1雄鼠1:1合笼,合笼18~24h后检查阴栓,有阴栓的小鼠提出待用;
(2)假孕母鼠的准备
在步骤(1)中C57BL/6雌性小鼠与B6D2F1雄鼠合笼的同时,将ICR雌鼠与结扎的ICR雄鼠以2:1的比例合笼,合笼18~24h后检查,有阴栓的小鼠(即假孕母鼠)提出待用。
3、取卵
将步骤2中(1)有阴栓的C57BL/6雌性小鼠脱颈处死,并解剖小鼠,用镊子夹住子宫上部,将输卵管分离至M2培养基中,在显微镜下划开输卵管的壶腹部,使卵流到培养液中。向培养液里加入1mg/mL的透明质酸酶,以去除受精卵周围的颗粒细胞。选择状态较好的受精卵,并转移至塑料皿(直径35mm)里由矿物油覆盖着的M2培养基液滴之中,在二氧化碳孵箱(37℃,5%二氧化碳,95%空气)中培养直到受精卵适合注射为止。
4、向受精卵原核注入DNA溶液
首先,将1mL注射器的活塞切两段长度为0.5cm的小柱,用砂轮切割一片宽度为0.5cm的盖玻片,75%(v/v)乙醇水溶液消毒。用凡士林把切下来的两段小柱按盖玻片的长度黏在载玻片上,利用1mL的注射器在两小柱中央滴两小滴M2溶液,同时在盖玻片上滴一滴,然后将盖玻片反转盖在两小柱上压紧,将注射器针头插入盖玻片的M2液滴里缓慢推入M2溶液,使围成的小室充满M2溶液。随后将完成的注射室放到显微操作仪的载物台上,用胚胎移植管将一组受精卵(约100枚)移到注射室中。将固定针和注射针装上并调整到视野中央,调整各自的X、Y和Z轴,使固定针、注射针和合子处于同一水平面上。推动注射针透过透明质带,进入原核,利用压力泵的持续压力(约150hPa)向原核注入DNA(浓度为3-5ng/μL的重组质粒 phCD2-TCR-α和浓度为3-5ng/μL的重组质粒p428-TCR-β按体积比1:1混合而成)至原核稍微膨大,然后将注射针迅速撤出。调节固定针为正压,使注射过的合子脱落,再调节为负压吸附另一个合子进行注射。合子注射完毕后,立即移回M16培养基中,至于37℃的培养箱培养 (约12h)。
5、受精卵移植
将假孕母鼠麻醉,手术取出卵巢连接输卵管并用脂肪镊固定,在显微镜下找到输卵管开口。移植管吸取经显微注射DNA后培养成活的受精卵,将移植管口插入输卵管口后轻轻将移植管内的液体吹入,将卵巢连同输卵管放回腹腔,缝合肌肉和皮肤。
6、TCR转基因小鼠的鉴定
取步骤5处理后假孕母鼠分娩的首建小鼠进行基因型鉴定,具体步骤如下:取首建小鼠的尾静脉血,使用ACK红细胞裂解液裂解红细胞,离心,收集白细胞。使用anti-mouseCD8a(Biolegend,克隆号:53-6.7)以及anti-mouse TCRVβ10(BDBioscience,克隆号:B21.5) 的抗体对获得的白细胞进行染色。
按照上述方法,将首建小鼠替换为野生型小鼠,其它步骤均不变,作为对照。
染色结果见图3。结果表明,TCR转基因小鼠的体内可以检测到CD8细胞高表达特异性 TCRVβ。
四、TCR转基因小鼠的CD8+T细胞(即TCR-T细胞)具有HBV多肽依赖的活化增殖能力
TCR转基因小鼠中特异性TCR转基因阳性T细胞即为TCR-T细胞。
1、取TCR转基因小鼠淋巴结以及脾脏细胞,计数并将细胞浓度稀释至3×107/mL。每3 ×107个细胞加入1μganti-CD4抗体(BioXcell,克隆号:GK1.5),4℃旋转孵育30min。
2、完成步骤1后,加入2%FBSDMEM培养基使体积达到15mL,4℃、2000rpm离心2min,去除上清,以洗去未结合的抗体。然后每3×107个细胞加入1mLGoatAnti-MouseIgG(QIAGEN, 310007)、1mL GoatAnti-RatIgG(QIAGEN,货号:310107)和磁珠重悬液(取磁珠,用含 0.5%(v/v)BSA和2mM EDTA的1×PBS缓冲液洗涤2次,然后用含0.5%(v/v)BSA和2mMEDTA 的1×PBS缓冲液重悬获得),4℃旋转孵育30min。
3、完成步骤2后,用磁铁吸附磁珠,除去CD4+T细胞和B细胞,上清液中剩余细胞大约有90%为CD8+T细胞,随后对得到的CD8+细胞进行CFSE标记,CFSE标记的基本过程为:取准备标记的细胞,根据需要加入CFSE稀释液或CFSE原液,充分混匀,37℃、5%CO2培养10min;之后加入10倍体积预热的1640完全培养基以停止标记,并用PBS重悬,得到CFSE标记的TCR-T细胞。
4、取HLA-A11/hTAP-LMP转基因小鼠的脾脏细胞,裂解红细胞,细胞计数,加入新鲜1640培养基将细胞稀释至1×107个/mL,然后向HLA-A11/hTAP-LMP转基因小鼠的脾脏细胞中加入多肽HBc141-151并使其在体系中的浓度为10μg/mL,37℃、5%CO2培养1h。
5、完成步骤4后,用1640培养基洗涤两次,细胞计数。
6、将步骤5得到的细胞和CFSE标记的TCR-T细胞1:1混合,37℃、5%CO2培养1d、2d或 3d。
7、完成步骤6后,进行流式检测,分析TCR-T细胞的增殖和活化情况。
将上述方法中的多肽HBc141-151替换为对照多肽,其它步骤均不变,作为对照。对照多肽具体为NP91(NP91为PR8流感病毒NP蛋白91-99肽段,由北京旭和源生物科技有限公司合成)。
将上述方法中的多肽HBc141-151替换为Anti-CD3抗体(克隆号145-2C11,Bioxcell),其它步骤均不变,作为阳性对照。
检测结果见图4(特异性多肽为多肽HBc141-151,Anti-CD3为Anti-CD3抗体)。结果表明, HBc141-151可以有效的活化TCR-T细胞,并且可以有效的刺激TCR-T细胞的增殖。由此可见, TCR-T细胞具有HBV多肽依赖的体外活化增殖能力,当把TCR-T细胞回输给HBV阳性的动物或者患者时,可以有效的体内激活和扩增回输的TCR-T细胞。
五、TCR-T细胞具有体外杀伤靶细胞的功能
为了体外验证TCR具有特异性的杀伤靶细胞的能力,进行体外杀伤实验。具体步骤如下:
1、TCR-T细胞的活化
(1)取TCR转基因小鼠淋巴结以及脾脏细胞,用结合CD4+T细胞以及B细胞的磁珠处理样品,去除CD4+T细胞以及B细胞,从而富集CD8+T细胞,细胞计数。
(2)取HLA-A11/hTAP-LMP转基因小鼠的脾脏细胞,裂解红细胞,细胞计数,加入新鲜1640培养基将细胞稀释至1×107个/mL,然后向HLA-A11/hTAP-LMP转基因小鼠的脾脏细胞中加入多肽HBc141-151并使其在体系中的浓度为10μg/mL,37℃、5%CO2培养1h。
(3)完成步骤(2)后,用1640培养基洗涤两次,细胞计数。
(4)将步骤(3)得到的细胞和TCR转基因小鼠的CD8+T细胞等数量混合,加入 400U/mLIL-2,37℃、5%CO2培养5d,得到体外活化的TCR-T细胞。
2、取HLA-A11/hTAP-LMP转基因小鼠的脾脏细胞,裂解红细胞,细胞计数,加入新鲜1640培养基将细胞稀释至1×107个/mL,然后向HLA-A11/hTAP-LMP转基因小鼠的脾脏细胞中加入多肽HBc141-151并使其在体系中的浓度为10μg/mL,37℃、5%CO2培养1h,之后1640培养基洗涤两次,细胞计数,得到负载多肽HBc141-151的靶细胞。
3、将步骤2得到的负载多肽HBc141-151的靶细胞加入96孔板中,1×104个/孔;随后按10:1、 5:1或1:1的比例加入体外活化的TCR-T细胞,37℃、5%CO2培养5h。
4、取96孔板,250g离心4min;然后将50μL上清转移至新的96孔板,每孔加入底物50μ L,室温、避光孵育30min;之后每孔加入50μL终止液,使用酶标仪(波长490nm)读取数值,即实验组数值。
5、杀伤实验本底数值检测
(1)按照上述步骤1-4,将步骤3替换为步骤3A),其它步骤均不变,得到T细胞自释放值。步骤3A)为:取96孔板,加入与实验组同等数量的体外活化的TCR-T细胞,37℃、5%CO2培养5h。
(2)按照上述步骤1-4,将步骤3替换为步骤3B),其它步骤均不变,得到靶细胞自释放值。步骤3B)为:取96孔板,加入与实验组同等数量的负载多肽HBc141-151的靶细胞,37℃、5%CO2培养5h。
(3)按照上述步骤1-4,将步骤3替换为步骤3C),其它步骤均不变,得到靶细胞最大释放值。步骤3C)为:取96孔板,加入与实验组同等数量的负载多肽HBc141-151的靶细胞和10μL 细胞裂解液,37℃、5%CO2培养5h。
6、计算杀伤活性
杀伤活性=(实验组数值-T细胞自释放值-靶细胞自释放值)/(靶细胞最大释放值-靶细胞自释放值)
将上述方法中的多肽HBc141-151替换为NP91,其它步骤均不变,作为对照。
检测结果见图5(E:T表示TCR-T细胞和靶细胞的比例)。结果表明,TCR-T细胞在体外可以有效的杀伤靶细胞。
六、TCR-T细胞具有体内杀伤靶细胞的功能
为了体内验证TCR具有特异性的杀伤靶细胞的能力,进行体内杀伤实验。将负载多肽 HBc141-151的HLA-A11/hTAP-LMP细胞用较低浓度CFSE(CFSElow)标记,未负载多肽的 HLA-A11/hTAP-LMP细胞作为对照用较高浓度CFSE(CFSEhigh)标记,将两种细胞等量混合后回输至C57/B6J小鼠体内,随后回输预先活化的TCR-T细胞,使用流式细胞仪检测C57/B6J 小鼠体内CFSElow标记的细胞群是否会被TCR-T细胞杀伤。具体步骤如下:
1、TCR-T细胞的活化
(1)取TCR转基因小鼠淋巴结以及脾脏细胞,用结合CD4+T细胞以及B细胞的磁珠处理样品,去除CD4+T细胞以及B细胞,从而富集CD8+T细胞,细胞计数。
(2)取HLA-A11/hTAP-LMP转基因小鼠的脾脏细胞,裂解红细胞,细胞计数,加入新鲜1640培养基将细胞稀释至1×107个/mL,然后向HLA-A11/hTAP-LMP转基因小鼠的脾脏细胞中加入多肽HBc141-151并使其在体系中的浓度为10μg/mL,37℃、5%CO2培养1h。
(3)完成步骤(2)后,用1640培养基洗涤两次,细胞计数。
(4)将步骤(3)得到的细胞和TCR转基因小鼠的CD8+T细胞等数量混合,加入400U/mL IL-2,37℃、5%CO2培养5d,得到体外活化的TCR-T细胞。
2、靶细胞的制备
(1)取HLA-A11/hTAP-LMP转基因小鼠的脾脏细胞,裂解红细胞,细胞计数,加入新鲜1640培养基将细胞稀释至1×107个/mL,然后向HLA-A11/hTAP-LMP转基因小鼠的脾脏细胞中加入多肽HBc141-151并使其在体系中的浓度为10μg/mL,37℃、5%CO2培养1h,之后 1640培养基洗涤两次,细胞计数。
(2)完成步骤(1)后,用PBS重悬细胞,得到浓度为5×107/mL的负载多肽HBc141-151的脾细胞重悬液。
(3)完成步骤(2)后,用较低浓度的CFSE(CFSElow)标记负载多肽HBc141-151的脾细胞(每mL负载多肽HBc141-151的脾细胞需加入1μL浓度为0.5mM的CFSE稀释液;进行CFSE标记时,CFSE的浓度为0.5μM),得到负载多肽HBc141-151且CFSElow标记的脾细胞。
(4)取HLA-A11/hTAP-LMP转基因小鼠的脾脏细胞,裂解红细胞,细胞计数;然后37℃、 5%CO2培养1h(培养基为1640培养基)。
(5)完成步骤(4)后,用1640培养基洗涤两次,细胞计数。
(6)完成步骤(5)后,用PBS缓冲液重悬,得到浓度为5×107/mL的细胞重悬液。
(7)完成步骤(6)后,用较高浓度的CFSE(CFSEhigh)标记未负载多肽的脾细胞(每mL未负载多肽的脾细胞需加入1μL浓度为5mM的CFSE原液;进行CFSE标记时,CFSE 的浓度为5μM),得到CFSEhigh标记的脾细胞。
3、TCR-T细胞具有体内杀伤靶细胞的功能
(1)将负载多肽HBc141-151且CFSElow标记的脾细胞和CFSEhigh标记的脾细胞等数量混合,得到混合细胞。
(2)取C57/B6J小鼠,每只C57/B6J小鼠回输(尾静脉输入)混合细胞2×107个。
(3)完成步骤(2)的2h后,每只C57/B6J小鼠回输(尾静脉输入)步骤1获得的体外活化的TCR-T细胞1×107个。
(4)完成步骤(3)的24h后,处死小鼠,收获外周血和脾细胞,裂解红细胞,离心,获得白细胞,制成单细胞悬液。流式细胞仪检测外周血以及脾脏中CFSElow与CFSEhigh的比例。通过CFSElow与CFSEhigh的比例定量分析TCR-T细胞的杀伤功能。
按照上述步骤(1)-(4),将步骤(3)替换为步骤K,其它步骤均不变,作为回输PBS对照。步骤K为:完成步骤(2)的2h后,每只C57/B6J小鼠回输与步骤(3)中体外活化的TCR-T细胞等体积的PBS缓冲液。
检测结果见图6。与回输PBS对照组相比,不论在外周血或是脾脏中,CFSElow标记的细胞群相较CFSEhigh的细胞群均有明显的下降。结果表明,TCR-T细胞在体内可以有效的杀伤靶细胞。
七、TCR-T细胞可以有效的清除体内HBV的慢性感染
1、小鼠慢性HBV感染模型的建立
小鼠慢性HBV感染模型的建立的方法记载于如下文献中:董小岩,尉迟捷,王刚,等. 高嗜肝性8型重组腺相关病毒体内转导法制备乙型肝炎病毒持续感染小鼠模型.病毒学报. 2010,26(6):425-431。具体步骤如下:
(1)向HLA-A11/hTAP-LMP转基因小鼠尾静脉注射rAAV/HBV1.3病毒(北京五加和分子医学研究所有限公司的产品),注射剂量为5×109vg/只。
完成步骤(1)后,观察自然情况下病毒梯度(乙肝病毒s抗原、乙肝病毒e抗原或病毒 DNA)变化曲线,并根据变化曲线预测TCR-T细胞的给药时间。
(2)完成步骤(1)后1个月,得到小鼠慢性HBV感染模型。小鼠慢性HBV感染模型中HBsAg和HBeAg的表达受HBV病毒DNA自身的启动子等元件调控,在肝脏和外周血中可以连续10周观察到持续表达的HBsAg和HBeAg。
将上述方法中的HLA-A11/hTAP-LMP转基因小鼠替换为C57BL/6小鼠,其它步骤均不变,获得慢性HBV感染的C57BL/6小鼠,作为阴性对照。
2、TCR-T细胞治疗HLA-A11/hTAP-LMP小鼠的慢性HBV感染
(1)取TCR转基因小鼠淋巴结以及脾脏细胞,用结合CD4+T细胞以及B细胞的磁珠处理样品,去除CD4+T细胞以及B细胞,从而富集CD8+T细胞,细胞计数。
(2)取HLA-A11/hTAP-LMP转基因小鼠的脾脏细胞,裂解红细胞,细胞计数,加入新鲜1640培养基将细胞稀释至1×107个/mL,然后向HLA-A11/hTAP-LMP转基因小鼠的脾脏细胞中加入多肽HBc141-151并使其在体系中的浓度为10μg/mL,37℃、5%CO2培养1h。
(3)完成步骤(2)后,用1640培养基洗涤两次,细胞计数,然后将细胞和TCR转基因小鼠的CD8+T细胞等数量混合,加入400U/mLIL-2,37℃、5%CO2培养5d,得到体外活化的TCR-T细胞。
(4)取HLA-A11/hTAP-LMP转基因小鼠慢性HBV感染模型,每只小鼠回输(尾静脉输入)体外活化的TCR-T细胞1×107个。
(5)完成步骤(4)后记为第0天,每隔两天检测血清中HBsAg的OD450nm值(HBsAg检测试剂盒,上海科华)和ALT浓度(ALT/GPT kit,全自动生化分析仪MEDSOUL AMS-18),从而判断体内HBV病毒的感染情况。共检测8天。
(6)完成步骤(4)后第7天,取HLA-A11/hTAP-LMP转基因小鼠慢性HBV感染模型,二次回输体外活化的TCR-T细胞1×107个。
(7)完成步骤(6)后2天,检测血清中HBsAg的OD450nm值和ALT浓度。
(8)将步骤(4)-(7)中的HLA-A11/hTAP-LMP转基因小鼠慢性HBV感染模型替换为C57BL/6小鼠慢性HBV感染模型,其它步骤均不变,作为对照。
血清中HBsAg的OD450nm值的检测结果见图7中A(WT 1#,2#,3#,4#均为C57BL/6小鼠慢性HBV感染模型,A11.hTAP 36#、A11.hTAP 37#、A11.hTAP 39#、A11.hTAP 40#和A11.hTAP 43#均为HLA-A11/hTAP-LMP转基因小鼠慢性HBV感染模型)。血清中ALT浓度检测结果见图7中B(WT 1#,2#,3#,4#均为C57BL/6小鼠慢性HBV感染模型,A11.hTAP 36#、A11. hTAP37#、A11.hTAP 39#、A11.hTAP 40#和A11.hTAP 43#均为HLA-A11/hTAP-LMP转基因小鼠慢性HBV感染模型)。结果表明,TCR-T细胞可以有效的清除HLA-A11/hTAP-LMP转基因小鼠慢性HBV感染模型体内的HBV病毒感染。
八、Human TCR-T细胞具有体外杀伤靶细胞的功能
为了体外验证Human TCR-T具有特异性的杀伤靶细胞的能力,进行体外杀伤实验。具体步骤如下:
1、慢病毒的包装与浓缩
(1)将慢病毒包装载体pCDH-MSCV-MCS-IRES-GFP(SystemBiosciences,编号:CD731B-1)的限制性内切酶EcoRI与BamHI之间的DNA小片段替换为TCRDNA片段(SEQ IDNo.5所示),得到pCDH-MSCV-TCR-GFP质粒。
(2)将293T细胞吹打重悬成单细胞,计数,然后用含10%(v/v)FBS的DMEM培养基调节,得到浓度为5×105个/mL的细胞悬浮液。取培养皿(规格为10cm),铺10mL细胞悬浮液,培养过夜。
(3)完成步骤(2)后,待293T细胞汇合度为75%时进行转染,转染前30min更换培养基为DMEM培养基。
(4)转染预混物准备
向500μLDMEM培养基中加入12μg pCDH-MSCV-TCR-GFP质粒、9μg psPAX2和6μgpMG2.D,涡旋充分混匀,得到质粒混合物。
psPAX2和pMG2.D均为北京天恩泽基因科技有限公司的产品。
向500μLDMEM培养基中加入27μgPEI,涡旋混匀,静置5min,得到PEI混合物。
(5)完成步骤(4)后,向500μL质粒混合物中加入500μLPEI混合物,涡旋充分混匀,室温孵育20min;然后将孵育完成的混合物轻柔沿培养皿侧壁加入293T细胞中,轻晃培养皿混匀,37℃培养箱培养。6-8h后将培养基更换为10mL含10%(v/v)FBS的DMEM培养基。
(6)完成步骤(5)后第48h,收集第一次病毒上清,补充新鲜DMEM培养基,病毒上清4℃保存。
(7)完成步骤(5)后第72h,收集第二次病毒上清。将第一次病毒上清和第二次病毒上清合并,800g、室温离心5min,收集上清。
(8)完成步骤(7)后,将所述上清使用0.45μmPES滤膜过滤除去细胞碎片,收集病毒上清。
(9)完成步骤(8)后,将所述病毒上清转移至超速离心管。4℃、70000g离心120min,小心弃上清,收集沉淀(白色病毒颗粒)。
(10)完成步骤(9)后,取所述沉淀,加入100倍浓缩体积的1640培养基重悬,4℃溶解过夜,得到浓缩慢病毒。将浓缩慢病毒分装保存于-80℃超低温冰箱,备用。
2、人外周淋巴细胞的分离
(1)抽取人外周静脉血5mL。
(2)完成步骤(1)后,取离心管(规格为50mL),加入5mL外周血和5mLPBS缓冲液,充分混匀。
(3)完成步骤(2)后,向离心管中加入5mL人外周血淋巴细胞分离液(天津市灏洋生物制品科技有限公司,货号LTS1077),使用一次性无菌滴管取稀释后外周血沿管壁小心叠加于分离液液面之上,注意保持清楚界面。
(4)完成步骤(3)后,将离心管置于离心机中,调节升速和降速为最低,800g离心20min。
(5)完成步骤(4)后,离心结束后管内分为四层,第一层为血浆和PBS,第二层为环状乳白色淋巴细胞层,第三层为透明分离液层,第四层为红细胞和粒细胞层。用移液器小心吸取第二层环状乳白色淋巴细胞层至无菌离心管(规格为50mL),向离心管中加入40mL PBS缓冲液,混匀细胞,800g离心5min,弃上清,之后使用1640培养基重悬细胞,计数备用。
3、人外周T淋巴细胞的活化
(1)取24孔板,每孔加入500μL anti-CD3抗体稀释液和500μL anti-CD28抗体稀释液,4℃包被过夜。
anti-CD3抗体稀释液:用PBS缓冲液稀释anti-CD3抗体(BioXcell,克隆号:OKT3)至浓度为3μg/mL获得。
anti-CD28抗体稀释液:用PBS缓冲液稀释anti-CD28抗体(BioXcell,克隆号:CD28.2) 至浓度为1μg/mL获得。
(2)完成步骤(1)后,取所述24孔板,移除液体,并用PBS缓冲液清洗一次。
(3)完成步骤(2)后,取所述24孔板,加入500μL人外周T淋巴细胞稀释液,37℃培养箱培养48h;之后400g离心5min,收集沉淀并使用1640培养基重悬,得到活化的人外周T淋巴细胞。活化的人外周T淋巴细胞用于感染慢病毒。
人外周T淋巴细胞稀释液:用1640培养基将步骤2获得的人外周T淋巴细胞稀释至4×106个/mL获得。
4、慢病毒感染人外周T淋巴细胞
(1)取24孔板,每孔加入5×105个活化的人外周T淋巴细胞和200μL浓缩慢病毒,然后用1640培养基补体积至500μL;最后加入polybrene和IL2,并使polybrene和IL2在体系中的浓度分别为8μg/mL和40U/mL。
(2)完成步骤(1)后,取24孔板,600g、32℃离心90min;然后将24孔板放入37℃培养箱感染24h。
(3)完成步骤(2)后,取所述24孔板,小心吸掉感染孔中350μL培养基,然后加入1640 培养基补足至体积2mL并吹打混匀,37℃培养箱继续培养48h。
(4)完成步骤(3)后,先取适量感染后T细胞进行流式检测感染效率;然后使用anti-mouse CD8a抗体(Biolegend,克隆号:53-6.7)和anti-mouse TCRVβ10抗体(BDBioscience,克隆号:B21.5)对感染后T细胞进行染色,4℃染色30min后进行上机检测,感染效率(TCRVβ 10阳性率)大于15%,阳性细胞即为成功转入TCR的Human TCR-T细胞,用于后续杀伤实验。
5、HumanTCR-T体外具有杀伤靶细胞的功能
(1)抽取HLA-A11阳性健康人外周静脉血5mL,然后按照步骤2的方法分离获得淋巴细胞,即HLA-A11+PBMC细胞。
(2)完成步骤(1)后,用1640培养基将HLA-A11+PBMC细胞稀释至浓度为1×107个/mL,然后加入多肽HBc141-151并使其在体系中的浓度为10μg/mL,37℃、5%CO2培养1h,之后1640 培养基洗涤两次,细胞计数,得到负载多肽HBc141-151的靶细胞。
(3)将步骤(2)得到的负载多肽HBc141-151的靶细胞加入96孔板中,2×104个/孔;随后按效靶比例为2:1或1:1加入Human TCR-T细胞,37℃、5%CO2培养5h。
(4)取96孔板,250g离心4min;然后将50μL上清转移至新的96孔板,每孔加入底物50 μL,室温、避光孵育30min;之后每孔加入50μL终止液,使用酶标仪(波长490nm)读取数值,即实验组数值。
(5)杀伤实验本底数值检测
①按照上述步骤(1)-(4),将步骤(3)替换为步骤3A),其它步骤均不变,得到T 细胞自释放值。步骤3A)为:取96孔板,加入与实验组同等数量的HumanTCR-T细胞,37℃、 5%CO2培养5h。
②按照上述步骤(1)-(4),将步骤(3)替换为步骤3B),其它步骤均不变,得到靶细胞自释放值。步骤3B)为:取96孔板,加入与实验组同等数量的负载多肽HBc141-151的靶细胞,37℃、5%CO2培养5h。
③按照上述步骤(1)-(4),将步骤(3)替换为步骤3C),其它步骤均不变,得到靶细胞最大释放值。步骤3C)为:取96孔板,加入与实验组同等数量的负载多肽HBc141-151的靶细胞和10μL细胞裂解液,37℃、5%CO2培养5h。
6、计算杀伤活性
杀伤活性=(实验组数值-T细胞自释放值-靶细胞自释放值)/(靶细胞最大释放值-靶细胞自释放值)
将上述方法中的多肽HBc141-151替换为NP91,其它步骤均不变,作为对照。
将上述方法中的HumanTCR-T替换为未转染TCR的Human T细胞,其它步骤均不变,作为对照。
检测结果见图8(A为Human TCR-T细胞的获得;B为Human TCR-T细胞体外杀伤靶细胞实验,其中E:T表示Human TCR-T细胞和靶细胞的比例,T+HBc141-151为Human T细胞杀伤孵育HBc141-151多肽的靶细胞,TCR-T+NP91为Human TCR-T杀伤孵育NP91多肽的对照细胞, TCR-T+HBc141-151为Human TCR-T细胞杀伤孵育HBc141-151多肽的靶细胞)。结果表明,Human TCR-T细胞在体外可以有效的杀伤靶细胞。
综上所述,本发明的发明人分离并鉴定了一对HBV特异性的TCR序列,成功构建了这对 TCR的转基因小鼠,并且在体外验证了TCR转基因阳性的CD8细胞(即TCR-T细胞)具有HBV 多肽表位依赖性的活化和增殖能力;利用动物体内和体外杀伤靶细胞实验,证明了这对TCR 具有很好的杀伤靶细胞的活性;体外验证Human TCR-T也具有特异性的杀伤HLA-A11+靶细胞的能力;此外,动物实验提示这对TCR序列可能是清除HBV感染细胞的有效方法之一。
<110> 中国科学院微生物研究所
<120> HLA-A11限制性乙型肝炎病毒HBc141-151表位肽的T细胞受体及其应用
<160> 5
<170> PatentIn version 3.5
<210> 1
<211> 807
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tctgcaacca tcctctatga gatcctactg gggaaggcca ccctatatgc tgtgctggtc 1740
agtggcctgg tgctgatggc tatggtcaag aaaaaaaatt cctga 1785
Claims (13)
1.一种识别HLA-A11限制性HBc141-151表位肽的T细胞受体,包含α链和β链;
所述α链包含三个互补决定区,氨基酸序列分别如SEQ ID No.2的第48-53位、第71-77位和第112-121位所示;
所述β链包含三个互补决定区,氨基酸序列分别如SEQ ID No.4的第46-50位、第68-73位和第111-122位所示。
2.如权利要求1所述T细胞受体,其特征在于:
所述α链的可变区的氨基酸序列如SEQ ID No.2的第22-112位所示;
所述β链的可变区的氨基酸序列如SEQ ID No.4的第20-113位所示。
3.如权利要求2所述T细胞受体,其特征在于:
所述α链的氨基酸序列如SEQ ID No.2所示;
所述β链的氨基酸序列如SEQ ID No.4所示。
4.编码权利要求1至3任一所述T细胞受体的核酸分子。
5.如权利要求4所述的核酸分子,其特征在于:编码所述T细胞受体的核酸分子包含编码所述T细胞受体的α链的核酸分子和编码所述T细胞受体的β链的核酸分子;
编码所述T细胞受体的α链中三个互补决定区的核苷酸序列分别如SEQ ID No.1的第142-159位、第211-231位和第334-363位所示;
编码所述T细胞受体的β链中三个互补决定区的核苷酸序列分别如SEQ ID No.3的第136-150位、第202-219位和第331-366位所示。
6.如权利要求5所述的核酸分子,其特征在于:
编码所述α链的可变区的核苷酸序列如SEQ ID No.1的第64-336位所示;
编码所述β链的可变区的核苷酸序列如SEQ ID No.3的第58-339位所示。
7.如权利要求6所述的核酸分子,其特征在于:
编码所述α链的核酸分子的核苷酸序列如SEQ ID No.1所示;
编码所述β链的核酸分子的核苷酸序列如SEQ ID No.3所示。
8.含有权利要求4至7任一所述核酸分子的表达盒、载体或细胞。
9.如权利要求8所述的载体,其特征在于:所述载体为逆转录病毒载体或慢病毒载体;
所述逆转录病毒载体为向逆转录病毒载体MSCV-IRES-GFP的多克隆位点之间插入编码所述T细胞受体的α链的核酸分子和编码所述T细胞受体的β链的核酸分子,得到的重组质粒;
所述慢病毒载体为向慢病毒包装载体pCDH-MSCV-MCS-IRES-GFP的多克隆位点之间插入编码所述T细胞受体的α链的核酸分子和编码所述T细胞受体的β链的核酸分子,得到的重组质粒。
10.如权利要求8所述的细胞,其特征在于:所述细胞为T细胞或Jurkat细胞。
11.具有权利要求1至3任一所述T细胞受体的T细胞。
12.含有权利要求8至10任一所述载体或细胞或含有权利要求11所述T细胞的药物组合物。
13.权利要求1至3任一所述T细胞受体或权利要求4至7任一所述的核酸分子或权利要求8至10任一所述载体或细胞或权利要求11所述T细胞受体的T细胞在制备用于预防和/或治疗HLA-A11人群中由HBV感染所致的疾病的药物中的应用。
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