CN114106144B - 识别hla-a*02/wt1靶点的tcr及其应用 - Google Patents
识别hla-a*02/wt1靶点的tcr及其应用 Download PDFInfo
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- CN114106144B CN114106144B CN202010875385.1A CN202010875385A CN114106144B CN 114106144 B CN114106144 B CN 114106144B CN 202010875385 A CN202010875385 A CN 202010875385A CN 114106144 B CN114106144 B CN 114106144B
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Abstract
本发明公开了一种特异性识别HLA‑A*02:01/WT1靶点的TCR及其应用。所述的TCR的α链可变区的CDR1、CDR2和CDR3分别包含如SEQ ID NO.1、SEQ ID NO.2和SEQ ID NO.3所示的序列，或者分别包含如SEQ ID NO.1、SEQ ID NO.2和SEQ ID NO.3所示序列的变体；所述变体与突变前的序列相比具有4个、3个或者2个以下突变，并且所述变体至少保留突变前序列的功能。本发明TCR与HLA‑A*02:01/RMFPNAPYL靶点的亲和力高，KD(M)值甚至可达1.7μM。且这些突变体转导的T细胞对靶细胞特异性激活。
Description
技术领域
本发明属于生物技术领域，具体涉及一种识别HLA-A*02/WT1靶点的TCR及其应用。
背景技术
WT1基因最初被认识于一种儿童肾肿瘤(Wilms’tumor)中而被命名为WT1(Cell,1990,60,509)。它位于人染色体11p13，是一个包含锌指结构域的转录因子，能结合到GC富集序列，是很多生长因子基因的转录活化因子或受体(J Med Invest,1999,46,130)。WT1基因与肿瘤的关系研究最初集中于急性白血病(Leukemia,1992,6,405)，随着研究的深入，人们发现除了在大部分血液瘤中高表达，在乳腺癌、肺癌、大肠癌、脑癌、头颈癌、甲状腺癌等一系列实体瘤中，也存在过表达的现象(Exp Cell Res,2001,264,74；Jpn J Clin Oncol,2010,40,377)。后来学者们还发现WT1基因在肿瘤中的功能方面展现出更多的是致癌功能，因此WT1产物成为一个有吸引力的肿瘤相关抗原(Int J Hematol,2001,73,177)。
鉴于WT1在肿瘤细胞中的广泛表达而在正常组织中的有限表达，WT1成为一个免疫治疗的潜在靶点。多个研究报道，WT1具有良好的免疫源性，能够诱导病人体内产生WT1-特异性Th细胞，释放出抗体。而在MDS或AML病人也有数据证明患者能产生针对WT1的细胞免疫应答(Expert Rev Vaccines,2005,4,503)。一些WT1相关的免疫源性表位随着以WT1为靶点的深入研究相继被报道，包括HLA I和HLA II两类的表位(Immunogenetics,2000,51,99；Blood,2000,95,2198)。随着WT1的免疫源性表位的发现，以WT1为靶点的免疫治疗也先后被开展。
近来，越来越多的实验证明过继T细胞疗法能在体内有效清除细胞细胞(NatImmunol,2001,2,957)，尤其是在Rosenberg团队开展第一个针对黑色素瘤的临床试验证明了TCR-T的可行性后(Science,2006,314,126)，截至2019年，在clinicaltrials.gov网站上就已经有超过84个TCR-T免疫治疗临床数据登记记录，预示着TCR-T在肿瘤免疫治疗中有着巨大潜能(Technol Cancer Res Treat,2019,18,1533033819831068)。WT1 TCR-T的安全性和在一些肿瘤中的潜在临床疗效在Tawara(Blood.2017,130,1985)和Greenberg团队开展的I期临床试验中得到证实,在结合骨髓移植之后的临床疗效尤为显著(Sci TranslMed.2013,5,174ra27；Nat Med.2019,25,1064)。
总之，针对WT1的肿瘤相关靶点，尤其是针对HLA-A*02:01/RMFPNAPYL靶点，TCR-T免疫细胞治疗方法对AML等疾病具有明显疗效，并且没有观察到On Target/Off-Tumor的毒副作用。然而，这些临床研究、试验所使用的TCR，都没有证据显示经过蛋白工程的改造和亲和力优化。故现有技术方案存在表达不稳定、亲和力未经优化的问题。
发明内容
本发明所要解决的技术问题是为克服现有技术中缺乏有效TCR的缺陷，提供一种识别HLA-A*02/WT1靶点的TCR及其应用。本发明TCR具有极高的亲和力，且KD值甚至可达1.7μM。
本发明的技术方案之一为：一种TCR，所述的TCR的α链可变区的CDR1、CDR2和CDR3分别包含如SEQ ID NO.1、SEQ ID NO.2和SEQ ID NO.3所示的序列，或者分别包含如SEQ IDNO.1、SEQ ID NO.2和SEQ ID NO.3所示序列的变体；
所述变体与突变前的序列相比具有4个、3个或者2个以下突变，并且所述变体至少保留突变前序列的功能。
较佳地，如SEQ ID NO.3所示序列的第1、2、4、6和7位中的一位或者多位被替换后获得SEQ ID NO.3所示序列的变体。
更佳地，如SEQ ID NO.3所示序列的第2位和第4位被替换；进一步更佳地，第1位或者第6位被替换；最更佳地，第7位也被替换。
其中，所述的第1位优选被替换为天冬酰胺、丙氨酸、色氨酸或者缬氨酸；所述的第2位优选被替换为脯氨酸、丙氨酸、谷氨酸、苯丙氨酸、缬氨酸或者苏氨酸；所述的第4位优选被替换为缬氨酸或者天冬氨酸；所述的第6位优选被替换为甲硫氨酸、精氨酸或者谷氨酰胺；所述的第7位优选被替换为异亮氨酸；
在本发明一具体实施方案中，如SEQ ID NO.3所示序列的变体的氨基酸序列如序列表中SEQ ID NO.4～18中任一所示。
在本发明一具体实施方案中，如SEQ ID NO.2所示序列的变体的氨基酸序列如序列表中SEQ ID NO.45所示。
本发明中所述TCR的β链可变区的CDR1、CDR2和CDR3优选分别包含如SEQ IDNO.19、SEQ ID NO.20和SEQ ID NO.21所示的序列，或者分别包含如SEQ ID NO.19、SEQ IDNO.20和SEQ ID NO.21所示序列的变体；所述变体与突变前的序列相比具有2个以下突变，并且所述变体至少保留突变前序列的功能。
较佳地，所述TCR的α链和/或β链还包含框架区；所述α链的框架区优选来源于种系TRAV39，所述β链的框架区优选来源于种系TRBV7-9。
更佳地，所述的述α链的FR1～4的氨基酸序列分别如SEQ ID NO.23～26或其变体所示，和/或，所述β链的FR1～4的氨基酸序列分别如SEQ ID NO.23～26或其变体所示；其中，所述α链的FR1变体的氨基酸序列优选如SEQ ID NO.38所示，所述α链的FR3变体的氨基酸序列优选如SEQ ID NO.39所示，所述α链的FR4变体的氨基酸序列优选如SEQ ID NO.40所示；所述β链的FR1变体的氨基酸序列优选如SEQ ID NO.41所示，所述β链的FR2变体的氨基酸序列优选如SEQ ID NO.42所示，所述β链的FR3变体的氨基酸序列优选如SEQ ID NO.43所示，所述β链的FR4变体的氨基酸序列优选如SEQ ID NO.44所示。
在本发明一具体实施方案中，所述的TCR为scTCR，所述scTCR中TCRα可变区与TCRβ可变区之间通过接头连接；所述接头的氨基酸序列优选如序列表中SEQ ID NO.22所示。
如上所述的TCR的TCRα链和/或TCRβ链较佳地还包括恒定区，所述TCRα链的恒定区优选来源于种系TRAC；和/或所述TCRβ链的恒定区优选来源于种系TRBC2。
更佳地，所述的TCR的TCRα链和/或TCRβ链还包括膜外区和跨膜区；进一步更佳地，所述的TCR的TCRα链和/或TCRβ链还包括胞内序列。所述的胞内序列可为本领域常规。
本发明的技术方案之二为：一种编码如上所述的TCR的核酸。
本发明的技术方案之三为：一种包含如上所述的核酸的载体，所述的载体优选慢病毒载体；所述的核酸在单个开放阅读框中，或者在两个不同的开放阅读框中分别编码TCRα链和TCRβ链。
本发明的技术方案之四为：一种含有如上所述的核酸或者如上所述的载体的细胞；较佳地，所述的细胞为T细胞或者干细胞，所述的T细胞优选CD8+T细胞。
本发明的技术方案之五为：一种提呈如上所述的TCR的分离的或非天然存在的细胞，所述的细胞优选T细胞。
本发明的技术方案之六为：一种药物组合物，其含有如上所述的TCR或者如上所述的细胞；较佳地，所述的药物组合物还包含药物上可接受的载体。
本发明的技术方案之七为：一种如上所述TCR、如上所述的细胞或者如上所述的药物组合物在制备防治WT1表达相关肿瘤的药物中的应用；较佳地，所述的肿瘤包括乳腺癌、肺癌、大肠癌、脑癌、甲状腺癌以及头颈癌。
在符合本领域常识的基础上，上述各优选条件，可任意组合，即得本发明各较佳实例。
本发明所用试剂和原料均市售可得。
本发明的积极进步效果在于：
本发明TCR与HLA-A*02:01/RMFPNAPYL靶点的亲和力高，KD(M)值甚至可达1.7μM。且这些突变体转导的T细胞对靶细胞特异性激活。
附图说明
图1为双阳性T细胞克隆的分选。
图2为单克隆T细胞体外特异性分析。
图3A和图3B为scTCR纯化阴离子交换层析和电泳图。
图4A和图4B为scTCR纯化分子筛层析和电泳图。
图5A和图5B为突变体A7B0阴离子交换层析和电泳图。
图6A和图6B为突变体A7B0分子筛层析和电泳图。
图7A和图7B为pMHC阴离子交换层析和电泳图。
图8A和图8B为pMHC分子筛层析和电泳图。
图9为A15B0亲和测试。
图10为慢病毒感染CD8+T细胞的感染效率。
图11为WT1特异性TCR功能检测T2细胞多肽负载的INF-γ释放。
图12为WT1特异性TCR的肿瘤细胞系LDH特异性杀伤。
具体实施方式
下面通过实施例的方式进一步说明本发明，但并不因此将本发明限制在所述的实施例范围之中。下列实施例中未注明具体条件的实验方法，按照常规方法和条件，或按照商品说明书选择。
MHC分子属于免疫球蛋白超家族成员，可以是I类或II类MHC分子。其对于抗原的呈递具有特异性，不同的个体有不同的MHC，能呈递一种蛋白抗原中不同的短肽到各自的抗原呈递细胞(APC)表面。人类的MHC通常称为HLA基因或HLA复合体。
T细胞受体(TCR)，是呈递在主要组织相容性复合体(MHC)上的特异性抗原的唯一受体。在免疫系统中，通过抗原特异性的TCR与pMHC复合物的结合引发T细胞与APC直接的物理接触，然后T细胞及APC两者的其他细胞膜表面分子就发生相互作用，这就引起了一系列后续的细胞信号传递和其他生理反应，从而使得不同抗原特异性的T细胞对其靶细胞发挥免疫效应。
TCR是T淋巴细胞识别抗原的功能单位，属免疫球蛋白超级家族，其编码链包括α、β、γ和δ四条链，TCR是由α链/β链或者γ链/δ链以异质二聚体形式存在的细胞膜表面的糖蛋白。外周血95％的TCR是由α和β两条多肽链构成的异源二聚体。但重组TCR还可以由一条单独的TCRβ链或者TCRα链组成，其已被证明能够结合抗原肽-MHC分子(WO 2005/113595)。
广义上讲，α和β各链包含可变区、连接区和恒定区，β链通常还在可变区和连接区之间含有短的多变区，但该多变区常被视作连接区的一部分。各可变区包含嵌合在框架结构(framework regions)中的3个CDR(互补决定区)，CDR1、CDR2和CDR3。CDR区决定了TCR与pMHC复合物的结合，其中CDR3由可变区和连接区重组而成，被称为超变区。TCR的α和β链一般看作各有两个"结构域"即可变域和恒定域，可变域由连接的可变区和连接区构成。TCR恒定域的序列可以在国际免疫遗传学信息系统(IMGT)的公开数据库中找到，如TCR分子α链的恒定域序列为TRAC(也可称为TRAC*01)，TCR分子β链的恒定域序列为TRBC1(也可称为TRBC1*01)或TRBC2(也可称为TRBC2*01)。此外，TCR的α和β链还包含跨膜区和胞质区，胞质区很短。
另外，α链由胚系基因中TRAV、TRAJ、TRAC重排组成；β链由胚系基因中的TRBV、TRBD、TRBJ、TRBC重排组成，不同V(D)J重拍后，由V-J(或V-D和D-J)连接时随机插入不同数量的核苷酸形成一个可变化的区域(N)成为互补决定区3(CDR3)，这种随机插入使TCRα链和β链序列呈现高度多样性；不同克隆T淋巴细胞的TCR重排时CDR3长度和碱基序列不同，这是TCR特异地识别抗原的区域，它决定了TCR的特异性。
另外：本发明中除明确说明的突变之外，还存在本领域公知的保守修饰或者保守置换或取代。所述的保守修饰或者所述的保守置换或取代是指具有类似特征(例如电荷、侧链大小、疏水性/亲水性、主链构象和刚性等)的其它氨基酸置换蛋白中的氨基酸，使得可频繁进行改变而不改变蛋白的生物学活性。本领域技术人员知晓，一般而言，多肽的非必需区域中的单个氨基酸置换基本上不改变生物学活性(参见例如Watson等(1987)MolecμlarBiology of the Gene,The Benjamin/Cummings Pub.Co.,第224页，(第4版))。另外，结构或功能类似的氨基酸的置换不大可能破环生物学活性。氨基酸常见的保守取代如下：
实施例1克隆特异性CD8+T细胞，获得异二聚体TCR序列(野生型的TCR的取得和鉴定)
T细胞克隆用到的方法、试剂和耗材，主要参考Curr.Protoc.Immunol.2002,7,1；PLoS One，2011,6,e27930；Onco Immunology 2016,5,e1175795及其引用文献。用EBV(EB病毒)转导的负载有WT1短肽(RMFPNAPYL)的B细胞(EBV-B)(J Vis Exp.2011，8,3321)(EBV病毒：ATCC产品号VR-1492)刺激HLA-A*02:01基因型健康志愿者的CD8+T细胞。其中单克隆T细胞培养方法，主要参考相关文献的工作(J Immunol Methods.2006,310,40；PLoSOne.2014,9,e110741)。用PE标记的短肽-HLA四聚体(MBL,产品号TS-M047-1)及APC标记的抗CD8抗体(Biolegend,产品号301014)分选双阳性T细胞。被RMFPNAPYL短肽刺激后的T细胞扩增至一定数量后进行分选。经多次刺激培养和分选后采用有限稀释进行单克隆培养。增殖后的单克隆T细胞用四聚体染色，经Tetramer,抗CD8抗体进行多次分选检测到的双阳性克隆细胞如图1所示。单克隆T细胞生长到1×105次方个以上取适量细胞用Tetramer和抗CD8抗体染色进行分选，阳性细胞(单克隆T细胞)加入RNA提取试剂盒Quick-RNATMMiniPrep(ZYMO research,产品号R1050)中的裂解液，吹匀裂解后-80℃保存备用。
为了从单克隆T细胞中克隆到TCR基因进一步开展体外活性研究，提取单克隆T细胞的mRNA进行逆转录cDNA，然后从中克隆出TCR基因并构建慢病毒载体。把实施例1中的分选出来的单克隆T细胞裂解液解冻，按照说明书用试剂盒Quick-RNATMMiniPrep(ZYMOresearch,产品号R1050)抽提T细胞的总RNA。最后用10μL Tricine-EDTA buffer(SMARTRACE cDNA扩增试剂盒)洗脱,RNA保存在-80℃备用。cDNA的合成采用clontech的SMARTRACE cDNA扩增试剂盒。按说明书经PCR把目标基因扩增出来后连接至pUC19载体(SMARTRACE cDNA扩增试剂盒附带)，转化大肠杆菌DH5α涂板，挑单克隆进行测序。测序结果在IMGT数据库进行比较分析，TCRα和β链Germline分别为TRAV39和TRBV7-9。
野生型TCR的α链全长：
野生型TCR的β链全长：
依据IMGT规则，野生型TCR的V区的特有关键序列CDR区氨基酸序列如下：
α链CDR1-TTSDR(SEQ ID NO.1)
α链CDR2-LLSNGAV(SEQ ID NO.2)
α链CDR3-GGGADGLT(SEQ ID NO.3)
β链CDR1-SEHNR(SEQ ID NO.19)
β链CDR2-FQNEAQ(SEQ ID NO.20)
β链CDR3-ASSLYGGGDTQY(SEQ ID NO.21)
α链FR1-ELKVEQNPLFLSMQEGKNYTIYCNYS(SEQ ID NO.23)
α链FR2-LYWYRQDPGKSLESLFV(SEQ ID NO.24)
α链FR3-KQEGRLMASLDTKARLSTLHITAAVHDLSATYFC(SEQ ID NO.25)
α链FR4-FGKGTHLIIQP(SEQ ID NO.26)
β链FR1-DTGVSQDPRHKITKRGQNVTFRCDPI(SEQ ID NO.27)
β链FR2-LYWYRQTLGQGPEFLTY(SEQ ID NO.28)
β链FR3-SRLLSDRFSAERPKGSFSTLEIQRTEQGDSAMYLC(SEQ ID NO.29)
β链FR4-FGPGTRLTVL(SEQ ID NO.30)。
加粗且下划线标注的分别为α链的FR1～FR4序列和β链的FR1～FR4序列。
上述α链或者β链的全长序列中：下划线标注部分为信号肽序列，无标记序列为Vα(α链的可变区)或者Vβ(β链的可变区)、加粗部分为Cα(α链的恒定区)或者Cβ(β链的恒定区)序列、斜体部分为胞外linker、斜体且加下划线部分为跨膜区和胞内序列。
将α链全长以及β链全长的跨膜区和胞内序列(斜体且下划线标注的部分)分别去除后获得氨基酸序列分别为A0和B0(其中“A”和“B”分别代表α链和β链，“0”代表未经突变)。
实施例2单克隆T细胞的体外特异性分析
为了分析实验例1中WT1四聚体阳性单克隆的体外活性，用ELISPOT试验分析单克隆T细胞与负载10-6M浓度特异性多肽或非特异性多肽的抗原递呈细胞共培养后INF-γ的释放情况。WT1单克隆T细胞对WT1多肽显示出最强烈的活性(图2)，对其他HLA-A*02结合多肽，如NY-ESO-1多肽或不含多肽培养基无明显的活性。
实施例3针对实施例1制备得到的野生型TCR进行scTCR结构域的稳定性改造
将实施例1中获得的Vα以及Vβ(α链全长以及β链全长的可变区序列分别利用Linker氨基酸序列连接起来获得野生型scTCR(以下简称scTCR-wt),这种结构非常不稳定，需要进行稳定性优化，才能用于亲和力优化(PNAS,1999,96,5651；Nat Biotechnol.2000,18,754；Front.Oncol.2015,4,1；WO2016124142)和包涵体复性(scTCR-wt的克隆、表达以及纯化过程按照本领域的常规方法)。
对scTCR-wt的β-turn结构，根据不同氨基酸侧链对β-turn不同位置的偏好性，引入有助于β-turn结构稳定性的突变。由于去除了Cα、Cβ结构域，Vα、Vβ结构域中有些位置的疏水性氨基酸暴露在表面，减少表面疏水特性的氨基酸突变，获得稳定的scTCR_X0序列。scTCR-wt和scTCR-X0直接合成基因，然后通过Nco I/Not I内切酶克隆到pET28a载体。
scTCR_X0氨基酸序列(粗体为CDR区，小写为linker)：
α链可变区
其中FR1～FR4的氨基酸序列分别如SEQ ID NO.38、SEQ ID NO.24、SEQ ID NO.39和SEQ ID NO.40所示。
CDR2的氨基酸序列如SEQ ID NO.45所示。
Linker:GSADDAKKDAAKKDGKS(SEQ ID NO.22)
β链可变区
其中：FR1～FR4的氨基酸序列分别如SEQ ID NO.41～44所示。
scTCR_X0碱基序列(粗体为CDR区，小写为linker)：
α链可变区：
Linker：
GGTAGCGCCGATGATGCAAAAAAGGATGCCGCAAAAAAGGACGGTAAAAGT(SEQ ID NO.34)
β链可变区：
scTCR-X0的包涵体表达、蛋白复性、纯化和亲和力测试，按照实施例5、实施例6和实施例8所述完成，结果如图3A、图3B以及图4A、图4B所示。
实施例4噬菌体展示
采用Nco I/Not I酶切位点，把工程改造后的RMF4 scTCR的基因插入噬菌体展示载体，作为噬菌体展示文库构建模板，对可变区CDR3α/CDR3β，设计不连续的突变引物建库，电转进入TG1感受态，用于后续2-3轮噬菌体筛选。噬菌体筛选的整体过程，除了传统的分子生物学手册之外，主要参考Nature Protocols,2007,2,3001和Nat.Biotech.2005,23,349两篇文章、Cardiff University药学系的一篇博士论文(Liddy S.2013,Molecularengineering of high affinity T-cell receptors for bispecific therapeutics)。
(a)噬菌体筛选
文库菌使用含有ampicillin和葡萄糖的2×YT培养基培养至OD600＝0.4～0.5，适量M13KO7辅助噬菌体(NEB,产品号N0315S)感染1h，更换含ampicillin和kanamycin的2×YT培养基，26℃包装过夜。细菌培养液高速离心，上清使用PEG/NaCl溶液沉淀，再次离心弃上清后1mL PBS重悬，获得噬菌体溶液。适当体积的上述噬菌体溶液经牛奶封闭后，依次与适量的生物素化标记的pMHC和链霉亲和素磁珠进行孵育，形成噬菌体-TCR-pMHC-磁珠复合体，然后使用0.1％PBST(v/v)和PBS分别洗涤磁珠3-5次。经胰酶消化释放的噬菌体感染对数生长期的TG1，涂板、30℃倒置培养过夜。重复以上流程进行第二、三轮筛选。
(b)噬菌体ELISA
参考Nature Protocols,2007,2,3001中所述的工作流程，第一天在96孔深孔板中包装phage 200μL，并使用链霉亲和素包被96孔ELISA板(1μg/孔)。第二天ELISA板依次加100μL pMHC(0.5μg/孔)、400μL 3％牛奶PBS溶液、100μL牛奶溶液封闭的噬菌体以及100μLM13-HRP抗体(义翘神州，产品号11973-MM05T-H,1:10000比例稀释)室温孵育1h，每次加样前弃板中溶液并0.1％PBST(v/v)洗涤3次。然后用TMB显色液(Sigma,产品号T4444)显色90秒，1M硫酸终止反应，酶标仪测定吸收值(OD450)。挑选OD450值大于0.20的单克隆，DNA测序分析获得相应的突变体。
实施例5基因克隆与包涵体表达
将实施例3中获得的突变位点如表1所示对应引入到TCR的A0当中，获得氨基酸序列A1～A16的可变区(“A”代表α链、“B”代表β链，带有不同的数字“A”表示含有不同突变的“α链”；另，在下述实施例中，由TCRα链和TCRβ链组成的异二聚体简称为AmB0；其中m为0～16的整数)。
发生于CDR3α和CDR3β上的突变位点如下表1(其中的加粗氨基酸即为突变氨基酸)：
表1突变位点展示
具体的表达过程如下：
TCRα-链(A0～A16)、β-链(B0)、scTCR、HLA-A*02:01和β2M的基因和RMFPNAPYL三者同时复性形成pMHC复合体；其中HLA-A*02:01的UniProt ID是为P01892、β2M的UniProt ID是P61769)采用Nco I/Not I酶切位点克隆到pET28a的载体(购自Novagen)中，分别转化E.coli BL21(DE3)(购自NEB公司)，挑取单克隆到LB培养基中，37℃振荡培养至OD600＝0.6～0.8，然后加入IPTG至终浓度为1mM，37℃继续培养3小时。6000rpm离心10min，收集菌体，放入-20℃保存。
实施例6包涵体纯化、复性和纯化
用裂解液(0.5％TritonX 100的PBS)重悬菌体，超声破碎后，12000rpm高速离心20分钟。弃上清，用裂解液重悬沉淀直至无肉眼可见的颗粒，再高速离心10分钟，重复以上操作2-3次，用6M盐酸胍溶液溶解沉淀，高速离心10分钟，收集上清，上清即为纯化后的包涵体，定量、分装，-80℃冻存。
将20mg TCRα链和15mgβ链(实施例5制备得到)分别稀释在5mL的6M盐酸胍溶液中。将TCRα链、TCRβ链依次缓慢加入到预冷的复性缓冲液(Science 1996,274,209；J.Mol.Biol.1999,285,1831；Protein Eng.2003,16,707)，4℃下持续搅拌、混合30分钟。然后将其加入透析袋中，放入10倍体积预冷的去离子水中，搅拌透析8-12小时。在预冷的透析外液(主要为了维持pH值，但是pH值不固定，围绕蛋白的pI值做调整，可以获得更佳的复性效果)、4℃透析4-8小时，重复2-3次。
将透析袋中的溶液倒出，高速离心10分钟去除沉淀和气泡，通过HiTrap Q HP(5mL)进行阴离子交换层析，0-2M NaCl,20mM Tris pH 8.1线性洗脱。收集洗脱峰，合并浓缩含有目标蛋白组分的洗脱峰，在非还原性SDS-PAGE电泳中，45kD附近有一条带，但是纯度还不满足要求，需进一步纯化。浓缩后的蛋白样品，用superdex 75 10/300进行分子筛层析。非还原性SDS-PAGE电泳检测，45kD附近有一高浓度条带，采用还原性SDS-PAGE电泳检测，有两条带，分别是α-链和β-链，纯度达90％左右。具体如图5A、图5B以及图6A、图6B所示(鉴于A0～A16的分子量相同，B1～B4的分子量相同，此处仅以A7B0为例显示其纯化结果)。
实施例7生物素化抗原肽-MHC(pMHC)制备
pMHC的复性和纯化，按照NIH Tetramer Core Facility的方法进行制备。现以HLA-A*02:01/RMFPNAPYL为实施代表例，按照在线protocols所述，将多肽溶液与β2M，HLA-A*02:01的包涵体溶液依次加入复性缓冲液(0.1M Tris-HCl，0.4M L-arginine，2mM EDTA；0.5mM氧化性谷胱甘肽和5mM还原性谷胱甘肽，0.2mM PMSF)，4℃搅拌过夜，第二天早上和晚上分别再加入同量的HLA-A*02:01的包涵体溶液，4℃搅拌1～3天。然后在10倍体积透析液(pH 8.1，20mM Tris-HCl)中透析3次。将透析后的蛋白样品使用HiTrap Q HP(5ml)进行阴离子交换层析，采用0～2M NaCl,20mM Tris pH 8.1溶液线性洗脱，收集洗脱峰，采用SDS-PAGE电泳分析，有较纯HLA-A*02:01和β2M两条带，而RMFPNAPYL分子量太小，胶图看不到条带。合并浓缩含有pMHC组分的洗脱峰，经凝胶过滤层析(Superdex 75 10/300)进一步纯化，然后用SDS-PAGE电泳检测，从电泳图上可知，得到纯度更好的pMHC复合体。用重组酶BirA(BPS Bioscience产品，产品号：70031)进行生物素化(Protein Expr.Purif.2012,82,162；J.Bacteriol.2012,194,1113)，具体实验方法，如反应体系等按照NIH Tetramer CoreFacility的方法进行制备、Gel Shift纯度鉴定。从Gel Shift电泳图来看，纯度符合要求。结果详见图7A、7B以及图8A和图8B。
实施例8亲和力测试
Octet是一种采用SPR技术检测亲和力的仪器，根据基于光纤生物传感器的生物膜层光学干涉技术，检测相互作用分子间的动力学参数，进行动力学和亲和力分析，计算出结合解离常数。在本实验中，我们采用SA传感器固定生物素化的pMHC，检测其与不同TCR的结合解离常数，计算出KD值。以A15B0作为实施代表例，测试HLA-A*02:01/RMFPNAPYL的亲和力，详见图9。以下表2是对RMF4的TCR突变体和HLA-A*02:01/RMFPNAPYL结合亲和力的一个整理总结。
需说明的是：如本领域人员所知，SPR技术是当前测定亲和力最常用、可靠的方法之一，但是涉及到蛋白定量、芯片新旧程度、仪器状态等，不同批次间的实验，会有一定的误差，误差值甚至可达3～5倍；而本发明为使用了同一蛋白定量、同一芯片以及同一仪器所进行的同批次实验，因此各数据之间可用于进行亲和力大小的比较，但具体数值并不构成对本发明保护范围的限制。
表2 WT1 TCR亲和力
实施例9慢病毒制备和感染CD8+T细胞
(a)WT1 TCR慢病毒包装。
采用第三代慢病毒包装系统(Invitrogen,pLenti6/V5 DirectionalTOPOTMCloning Kit，产品号K495510)包装含有编码所需TCR的基因的慢病毒。将质粒pLenti-WT1 TRA-2A-TRB-IRES-NGFR和pLenti-eGFP，分别与包装质粒pMDLg/pRRE(addgene，产品号k12251),pRSV-REV(addgene,产品号12253)和pMD2.G(addgene,产品号12259)按4:2:1:1的比例混匀，瞬时转染处于对数生长期的293T细胞(购自ATCC，产品号CRL-3216),转染试剂PEI-MAX(Polyscience,产品号23966-1)与质粒(40μg/15cm平皿)的比例为2:1(体积质量比)，具体操作步骤按照说明书进行。第3和第4天收集含有包装的慢病毒的培养基上清。为进一步浓缩慢病毒，把所收集到的培养基上清3000rpm离心15min，再经过0.45μM滤器(Merck Millipore)过滤，去除碎片。最后用50kD分子量截留的浓缩管(MerckMillipore)浓缩至1mL，分装后-80℃冻存。取假病毒样品进行病毒滴度测定，步骤参照p24ELISA(Clontech，产品号632200)试剂盒说明书。
(b)用含有WT1特异性T细胞受体基因的慢病毒转导原代CD8+T细胞
从健康志愿者的血液中，通过负分离法富集CD8+T细胞(抗体偶联磁珠购自Miltenyi Biotec)。计数细胞，于48孔板中每孔接种1×106个细胞，每孔含有0.5mL 100IU/mL IL-2(Peprotech，产品号AF-200-02)的RPMI 1640完全培养基(10％FBS)，与预洗涤的抗CD3/CD28抗体-包被小珠(life technologies，产品号11452D)共孵育过夜刺激。
刺激过夜后，按MOI＝10的比例加入已浓缩的表达WT1野生或GFP基因的慢病毒，32℃，900g离心感染1小时。感染完毕去除慢病毒感染液，以包含100IU/mL IL-2的RPMI 1640完全培养基重悬细胞，37℃/5％CO2条件下培养3天。此后每两天计数细胞一次，替换或加入含有100IU/mL IL-2的新鲜培养基，维持细胞在1×106-2×106-个细胞/毫升。转导第3天通过流式细胞术分析细胞感染效率(图10)。结果显示，WT1 TCR转导的T细胞双染WT1四聚体和抗CD8抗体，A0B0阳性率为20.887％，A12B0为21.0％；阳性对照1G4转导的T细胞(WT1 TCR)双染WT1四聚体和抗CD8抗体，阳性率为46.3％(APC-H代表的是CD8抗体染色，R-PE-H代表的是tetramer(即抗原肽识别的TCR)的染色)。
实施例10验证WT1特异性TCR功能-ELISPOT方案检测T2细胞多肽负载的INF-γ释放
本实验方案以IFN-γ的产量作为T细胞激活的标识，检测TCR-转导的T细胞对靶细胞特异性激活反应。
本试验的靶细胞是T2细胞和H226细胞，效应细胞是实施例49中经流式细胞术分析表达WT1 TCR的CD8+T细胞(细胞名称、HLA分型以及WT1抗原表达情况见表3)，并以同一志愿者的CD8+T细胞作为阴性对照效应细胞。在实施例9中，经抗CD3/CD28包被珠刺激，并用表达WT1 TCR基因的慢病毒转导的T细胞，在含有100IU/mL IL-2的10％FBS的RPMI 1640培养基扩增直至转导后9-12天，然后将这些细胞置于实验培养基(10％FBS的RPMI 1640)中，300g常温离心10分钟进行洗涤。然后将细胞以2×所需终浓度重悬在试验培养基中。同样方法处理阴性对照效应细胞。
表3所用细胞名称、HLA分型以及WT1抗原表达情况
| 细胞名称 | HLA分型 | WT1抗原 |
| NCI H226 | 24:02；01:01 | + |
| NCI H226-A0201 | 过表达A02:01；24:02；01:01 | + |
| NCI H226-A0203 | 过表达A02:03；24:02；01:01 | + |
| T2 | A02:01；A02:01 | - |
| CD8+T细胞 | A02:03；A11:01 | - |
按照生产商提供的说明书，包被PVDF ELISPOT 96孔板(Merck Millipore，产品号MSIPS4510)，4℃孵育过夜。种板前洗涤除去多余的捕捉抗体后，用含有10％FBS的PBS并在室温下封闭孔板2小时，去除液体。
依次将试验的诸组分加入ELISPOT孔板：①20000个靶细胞(T2细胞)/孔；②1350个WT1 TCR CD8+双阳性T细胞，或者CD8+阴性对照效应细胞；③20微升10-5摩尔/升的与TCR相应短肽(终浓度为10-6摩尔/升)。所有实验组一式两份。
温育孔板过夜(37℃/5％CO2)，第二天按照人IFN-γELISPOT PVDF-酶试剂盒(BD，产品号551849)使用说明进行检测，最终加入BCIP/NBT溶液显影5-15分钟。在此期间常规检测显影孔板的斑点，确定终止反应的最佳时间。双蒸水冲洗孔板以中止显影，室温下完全干燥孔板。
结果如图11所示，负载WT1短肽的T2细胞和表达A0201的H226细胞强烈刺激T细胞释放INF-γ形成明显多的斑点，而H226细胞、表达A0201不负载WT1短肽的H226细胞、表达A0203不负载多肽的H226细胞和负载非特异短肽(NY-ESO-1)的H226细胞都不能刺激T细胞释放INF-γ形成明显多的斑点。
实施例11验证WT1特异性TCR功能-肿瘤细胞系LDH特异性杀伤
本试验是51Cr释放细胞毒性试验的比色替代试验，定量测定细胞裂解后释放的乳酸脱氢酶(LDH)，实验方案参考文献(Eur.J Immunol.1993,23,3217)，具体检测方法按试剂盒说明书进行(Promega，G1780)。采用30分钟偶联的酶反应来检测释放在培养基中的LDH，在酶反应中LDH可使一种四唑盐(INT)转化为红色的甲臜(formazan)。生成的红色产物的量与裂解的细胞数成正比。可用标准的96孔读板仪收集490nm可见光吸光值数据。
Elispot检测结束之后，慢病毒感染T细胞第13天时进行。
①负载多肽：靶细胞(2×105/mL)与梯度稀释多肽(终浓度为10-8，10-7，10-6M)混匀，37度培养箱孵育4小时后离心，洗去未结合多肽，细胞用于下一步接种。
②接种细胞：肿瘤细胞1×104/孔；A12B0和A0B0为3×104/孔(效靶比1:3)。每孔培养体系200μL，细胞在接种时均置换成仅含5％FBS的RPMI1640培养基。将细胞置于37度5％CO2培养箱中培养24小时。(注：H226-A0201-WT1-1E-8表示H226-A0201细胞负载了10-8M的WT1多肽，H226-A0201-1E-7表示H226-A0201细胞负载了10-7M的多肽，H226-A0201-1E-6表示H226-A0201细胞负载了10-6M的多肽)。
③LDH反应显色：按杀伤实验试剂盒(Promega，G1780)生产商提供的说明书进行检测操作，于1小时内490nm光波下读板。
计算公式：细胞毒性百分比％，即LDH释放百分比％＝(实验组释放量-肿瘤细胞自释放量-TCRT细胞自释放量)/(肿瘤细胞最大释放量-肿瘤细胞自释放量)×100％。本图中纵坐标1为100％。计算时，各组LDH释放量数值均减去培养基本底光吸收值。
实验结果整理如图12所示，表达高亲和力TCR突变体A12B0的CD8+T细胞较野生型有更强烈的杀伤效果。
SEQUENCE LISTING
<110> 深圳普瑞金生物药业有限公司
<120> 识别HLA-A*02/WT1靶点的TCR及其应用
<130> P20012985C
<160> 45
<170> PatentIn version 3.5
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<212> PRT
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<223> α链 CDR1
<400> 1
Thr Thr Ser Asp Arg
1 5
<210> 2
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> α链 CDR2
<400> 2
Leu Leu Ser Asn Gly Ala Val
1 5
<210> 3
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> α链 CDR3
<400> 3
Gly Gly Gly Ala Asp Gly Leu Thr
1 5
<210> 4
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> CDR3 变体
<400> 4
Asn Pro Gly Val Asp Gly Leu Thr
1 5
<210> 5
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> CDR3 变体
<400> 5
Ala Ala Gly Asp Asp Gly Leu Thr
1 5
<210> 6
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> CDR3变体
<400> 6
Gly Phe Gly Val Asp Gly Leu Thr
1 5
<210> 7
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> CDR3变体
<400> 7
Gly Glu Gly Val Asp Gly Leu Thr
1 5
<210> 8
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> CDR3变体
<400> 8
Ser Pro Gly Asp Asp Gly Leu Thr
1 5
<210> 9
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> CDR3变体
<400> 9
Val Val Gly Val Asp Gly Leu Thr
1 5
<210> 10
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> AEGVDGLT
<400> 10
Ala Glu Gly Val Asp Gly Leu Thr
1 5
<210> 11
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> CDR3变体
<400> 11
Gly Val Gly Asp Asp Gly Leu Thr
1 5
<210> 12
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> CDR3变体
<400> 12
Gly Glu Gly Asp Asp Gly Leu Thr
1 5
<210> 13
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> CDR3变体
<400> 13
Gly Glu Gly Val Asp Met Leu Thr
1 5
<210> 14
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> CDR3变体
<400> 14
Gly Glu Gly Asp Asp Met Leu Thr
1 5
<210> 15
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> CDR3变体
<400> 15
Gly Thr Gly Asp Asp Arg Leu Thr
1 5
<210> 16
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> CDR3变体
<400> 16
Gly Thr Gly Asp Asp Gln Leu Thr
1 5
<210> 17
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> CDR3变体
<400> 17
Gly Thr Gly Asp Asp Met Ile Thr
1 5
<210> 18
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> CDR3变体
<400> 18
Gly Glu Gly Asp Asp Gln Leu Thr
1 5
<210> 19
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> β链 CDR1
<400> 19
Ser Glu His Asn Arg
1 5
<210> 20
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
<223> β链 CDR2
<400> 20
Phe Gln Asn Glu Ala Gln
1 5
<210> 21
<211> 12
<212> PRT
<213> Artificial Sequence
<220>
<223> β链 CDR3
<400> 21
Ala Ser Ser Leu Tyr Gly Gly Gly Asp Thr Gln Tyr
1 5 10
<210> 22
<211> 17
<212> PRT
<213> Artificial Sequence
<220>
<223> 接头
<400> 22
Gly Ser Ala Asp Asp Ala Lys Lys Asp Ala Ala Lys Lys Asp Gly Lys
1 5 10 15
Ser
<210> 23
<211> 26
<212> PRT
<213> Artificial Sequence
<220>
<223> α链 FR1
<400> 23
Glu Leu Lys Val Glu Gln Asn Pro Leu Phe Leu Ser Met Gln Glu Gly
1 5 10 15
Lys Asn Tyr Thr Ile Tyr Cys Asn Tyr Ser
20 25
<210> 24
<211> 17
<212> PRT
<213> Artificial Sequence
<220>
<223> α链 FR2
<400> 24
Leu Tyr Trp Tyr Arg Gln Asp Pro Gly Lys Ser Leu Glu Ser Leu Phe
1 5 10 15
Val
<210> 25
<211> 34
<212> PRT
<213> Artificial Sequence
<220>
<223> α链 FR3
<400> 25
Lys Gln Glu Gly Arg Leu Met Ala Ser Leu Asp Thr Lys Ala Arg Leu
1 5 10 15
Ser Thr Leu His Ile Thr Ala Ala Val His Asp Leu Ser Ala Thr Tyr
20 25 30
Phe Cys
<210> 26
<211> 11
<212> PRT
<213> Artificial Sequence
<220>
<223> α链 FR4
<400> 26
Phe Gly Lys Gly Thr His Leu Ile Ile Gln Pro
1 5 10
<210> 27
<211> 26
<212> PRT
<213> Artificial Sequence
<220>
<223> β链 FR1
<400> 27
Asp Thr Gly Val Ser Gln Asp Pro Arg His Lys Ile Thr Lys Arg Gly
1 5 10 15
Gln Asn Val Thr Phe Arg Cys Asp Pro Ile
20 25
<210> 28
<211> 17
<212> PRT
<213> Artificial Sequence
<220>
<223> β链 FR2
<400> 28
Leu Tyr Trp Tyr Arg Gln Thr Leu Gly Gln Gly Pro Glu Phe Leu Thr
1 5 10 15
Tyr
<210> 29
<211> 35
<212> PRT
<213> Artificial Sequence
<220>
<223> β链 FR3
<400> 29
Ser Arg Leu Leu Ser Asp Arg Phe Ser Ala Glu Arg Pro Lys Gly Ser
1 5 10 15
Phe Ser Thr Leu Glu Ile Gln Arg Thr Glu Gln Gly Asp Ser Ala Met
20 25 30
Tyr Leu Cys
35
<210> 30
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> β链 FR4
<400> 30
Phe Gly Pro Gly Thr Arg Leu Thr Val Leu
1 5 10
<210> 31
<211> 109
<212> PRT
<213> Artificial Sequence
<220>
<223> scTCR_X0 α链可变区
<400> 31
Met Ala Glu Leu Lys Val Glu Gln Asn Pro Thr Asn Leu Ser Val Pro
1 5 10 15
Glu Gly Ala Asn Val Thr Ile Tyr Cys Asn Tyr Ser Thr Thr Ser Asp
20 25 30
Arg Leu Tyr Trp Tyr Arg Gln Asp Pro Gly Lys Ser Leu Glu Ser Leu
35 40 45
Phe Val Leu Leu Ser Asn Gly Ala Thr Lys Gln Glu Gly Arg Leu Gln
50 55 60
Ala Ser Leu Asp Thr Lys Ala Arg Leu Ser Thr Leu His Ile Thr Ala
65 70 75 80
Ala Thr Pro Asp Leu Ser Ala Thr Tyr Phe Cys Gly Gly Gly Ala Asp
85 90 95
Gly Leu Thr Phe Gly Ala Gly Thr Asn Leu Thr Ile Gln
100 105
<210> 32
<211> 114
<212> PRT
<213> Artificial Sequence
<220>
<223> scTCR_X0 β链可变区
<400> 32
Asp Thr Gly Val Ser Gln Asp Pro Thr Asn Ile Thr Val Pro Arg Gly
1 5 10 15
Ala Asn Val Thr Phe Arg Cys Asp Pro Ile Ser Glu His Asn Arg Leu
20 25 30
Tyr Trp Tyr Arg Gln Asp Pro Gly Gln Gly Pro Glu Phe Leu Thr Tyr
35 40 45
Phe Gln Asn Glu Ala Gln Leu Glu Lys Ser Arg Leu Leu Ser Asp Arg
50 55 60
Phe Ser Ala Glu Arg Pro Lys Gly Ser Phe Ser Thr Leu Glu Ile Gln
65 70 75 80
Arg Val Glu Pro Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Ser Leu
85 90 95
Tyr Gly Gly Gly Asp Thr Gln Tyr Phe Gly Pro Gly Thr Gln Leu Thr
100 105 110
Val Asn
<210> 33
<211> 327
<212> DNA
<213> Artificial Sequence
<220>
<223> scTCR_X0 α链可变区
<400> 33
atggctgaac tgaaagttga acagaatccg accaatctga gcgtgccgga aggcgcaaat 60
gtgaccatct attgcaatta tagcaccacc agtgatcgcc tgtattggta tcgtcaggat 120
ccgggtaaaa gcctggaaag cctgtttgtt ctgctgagca atggcgccac caaacaggaa 180
ggccgcctgc aggccagcct ggataccaaa gcccgtctga gcaccctgca tattaccgca 240
gcaaccccgg atctgagtgc cacctatttt tgtggtggtg gtgcagatgg tctgaccttt 300
ggtgccggca ccaatctgac cattcag 327
<210> 34
<211> 51
<212> DNA
<213> Artificial Sequence
<220>
<223> 接头的碱基序列
<400> 34
ggtagcgccg atgatgcaaa aaaggatgcc gcaaaaaagg acggtaaaag t 51
<210> 35
<211> 342
<212> DNA
<213> Artificial Sequence
<220>
<223> scTCR_X0 β链可变区
<400> 35
gacaccggcg tgagtcagga tccgaccaat attaccgtgc cgcgcggtgc aaatgttacc 60
tttcgttgtg atccgattag tgaacataat cgcctgtatt ggtaccgcca ggatccgggc 120
cagggtccgg aatttctgac ctattttcag aatgaagccc agctggaaaa aagtcgtctg 180
ctgagtgatc gttttagcgc cgaacgtccg aaaggcagct ttagtaccct ggaaattcag 240
cgcgtggaac cgggcgatag cgccatgtat ctgtgcgcca gcagtctgta tggtggcggt 300
gacacccagt attttggccc gggcacccag ctgaccgtta at 342
<210> 36
<211> 267
<212> PRT
<213> Artificial Sequence
<220>
<223> 野生型TCR的α链全长
<400> 36
Met Lys Lys Leu Leu Ala Met Ile Leu Trp Leu Gln Leu Asp Arg Leu
1 5 10 15
Ser Gly Glu Leu Lys Val Glu Gln Asn Pro Leu Phe Leu Ser Met Gln
20 25 30
Glu Gly Lys Asn Tyr Thr Ile Tyr Cys Asn Tyr Ser Thr Thr Ser Asp
35 40 45
Arg Leu Tyr Trp Tyr Arg Gln Asp Pro Gly Lys Ser Leu Glu Ser Leu
50 55 60
Phe Val Leu Leu Ser Asn Gly Ala Val Lys Gln Glu Gly Arg Leu Met
65 70 75 80
Ala Ser Leu Asp Thr Lys Ala Arg Leu Ser Thr Leu His Ile Thr Ala
85 90 95
Ala Val His Asp Leu Ser Ala Thr Tyr Phe Cys Gly Gly Gly Ala Asp
100 105 110
Gly Leu Thr Phe Gly Lys Gly Thr His Leu Ile Ile Gln Pro Tyr Ile
115 120 125
Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser Lys Ser Ser
130 135 140
Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln Thr Asn Val
145 150 155 160
Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys Thr Val Leu
165 170 175
Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val Ala Trp Ser
180 185 190
Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn Ser Ile Ile
195 200 205
Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser Cys Asp Val Lys
210 215 220
Leu Val Glu Lys Ser Phe Glu Thr Asp Thr Asn Leu Asn Phe Gln Asn
225 230 235 240
Leu Ser Val Ile Gly Phe Arg Ile Leu Leu Leu Lys Val Ala Gly Phe
245 250 255
Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser
260 265
<210> 37
<211> 312
<212> PRT
<213> Artificial Sequence
<220>
<223> 野生型TCR的β链全长
<400> 37
Met Gly Thr Ser Leu Leu Cys Trp Met Ala Leu Cys Leu Leu Gly Ala
1 5 10 15
Asp His Ala Asp Thr Gly Val Ser Gln Asp Pro Arg His Lys Ile Thr
20 25 30
Lys Arg Gly Gln Asn Val Thr Phe Arg Cys Asp Pro Ile Ser Glu His
35 40 45
Asn Arg Leu Tyr Trp Tyr Arg Gln Thr Leu Gly Gln Gly Pro Glu Phe
50 55 60
Leu Thr Tyr Phe Gln Asn Glu Ala Gln Leu Glu Lys Ser Arg Leu Leu
65 70 75 80
Ser Asp Arg Phe Ser Ala Glu Arg Pro Lys Gly Ser Phe Ser Thr Leu
85 90 95
Glu Ile Gln Arg Thr Glu Gln Gly Asp Ser Ala Met Tyr Leu Cys Ala
100 105 110
Ser Ser Leu Tyr Gly Gly Gly Asp Thr Gln Tyr Phe Gly Pro Gly Thr
115 120 125
Arg Leu Thr Val Leu Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val
130 135 140
Ala Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala
145 150 155 160
Thr Leu Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu
165 170 175
Ser Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp
180 185 190
Pro Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys
195 200 205
Leu Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg
210 215 220
Asn His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp
225 230 235 240
Glu Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala
245 250 255
Glu Ala Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Glu Ser Tyr Gln
260 265 270
Gln Gly Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys
275 280 285
Ala Thr Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala Met
290 295 300
Val Lys Arg Lys Asp Ser Arg Gly
305 310
<210> 38
<211> 28
<212> PRT
<213> Artificial Sequence
<220>
<223> scTCR_X0 α链 FR1
<400> 38
Met Ala Glu Leu Lys Val Glu Gln Asn Pro Thr Asn Leu Ser Val Pro
1 5 10 15
Glu Gly Ala Asn Val Thr Ile Tyr Cys Asn Tyr Ser
20 25
<210> 39
<211> 34
<212> PRT
<213> Artificial Sequence
<220>
<223> scTCR_X0 α链 FR3
<400> 39
Lys Gln Glu Gly Arg Leu Gln Ala Ser Leu Asp Thr Lys Ala Arg Leu
1 5 10 15
Ser Thr Leu His Ile Thr Ala Ala Thr Pro Asp Leu Ser Ala Thr Tyr
20 25 30
Phe Cys
<210> 40
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> scTCR_X0 α链 FR4
<400> 40
Phe Gly Ala Gly Thr Asn Leu Thr Ile Gln
1 5 10
<210> 41
<211> 26
<212> PRT
<213> Artificial Sequence
<220>
<223> scTCR_X0 β链FR1
<400> 41
Asp Thr Gly Val Ser Gln Asp Pro Thr Asn Ile Thr Val Pro Arg Gly
1 5 10 15
Ala Asn Val Thr Phe Arg Cys Asp Pro Ile
20 25
<210> 42
<211> 17
<212> PRT
<213> Artificial Sequence
<220>
<223> scTCR_X0 β链FR2
<400> 42
Leu Tyr Trp Tyr Arg Gln Asp Pro Gly Gln Gly Pro Glu Phe Leu Thr
1 5 10 15
Tyr
<210> 43
<211> 38
<212> PRT
<213> Artificial Sequence
<220>
<223> scTCR_X0 β链 FR3
<400> 43
Leu Glu Lys Ser Arg Leu Leu Ser Asp Arg Phe Ser Ala Glu Arg Pro
1 5 10 15
Lys Gly Ser Phe Ser Thr Leu Glu Ile Gln Arg Val Glu Pro Gly Asp
20 25 30
Ser Ala Met Tyr Leu Cys
35
<210> 44
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> scTCR_X0 β链 FR4
<400> 44
Phe Gly Pro Gly Thr Gln Leu Thr Val Asn
1 5 10
<210> 45
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> CDR2变体
<400> 45
Leu Leu Ser Asn Gly Ala Thr
1 5
Claims (20)
1.一种TCR，其特征在于，所述的TCR的α链可变区的CDR1、CDR2和CDR3分别为如SEQ IDNO. 1、SEQ ID NO. 2和SEQ ID NO. 3所示的序列，或者CDR3为SEQ ID NO. 3所示序列的变体；所述如SEQ ID NO. 3所示序列的变体的氨基酸序列如序列表中SEQ ID NO. 5~7或9~18中任一所示；
所述TCR的β链可变区的CDR1、CDR2和CDR3分别为如SEQ ID NO. 19、SEQ ID NO. 20和SEQ ID NO. 21所示的序列。
2.如权利要求1所述的TCR，其特征在于，所述TCR的α链和/或β链还包含框架区。
3.如权利要求2所述的TCR，其特征在于，所述α链的框架区来源于种系TRAV39，所述β链的框架区来源于种系TRBV7-9。
4. 如权利要求2所述的TCR，其特征在于，所述α链的FR1~4的氨基酸序列分别如SEQ IDNO. 23~26或其变体所示，和/或，所述β链的FR1~4的氨基酸序列分别如SEQ ID NO. 23~26或其变体所示。
5. 如权利要求4所述的TCR，其特征在于，所述α链的FR1变体的氨基酸序列如SEQ IDNO. 38所示，所述α链的FR3变体的氨基酸序列如SEQ ID NO. 39所示，所述α链的FR4变体的氨基酸序列如SEQ ID NO. 40所示；所述β链的FR1变体的氨基酸序列如SEQ ID NO. 41所示，所述β链的FR2变体的氨基酸序列如SEQ ID NO. 42所示，所述β链的FR3变体的氨基酸序列如SEQ ID NO. 43所示，所述β链的FR4变体的氨基酸序列如SEQ ID NO. 44所示。
6.如权利要求1所述的TCR，其特征在于，所述的TCR为scTCR，所述scTCR中TCRα可变区与TCRβ可变区之间通过接头连接。
7. 如权利要求6所述的TCR，其特征在于，所述接头的氨基酸序列如序列表中SEQ IDNO. 22所示。
8.如权利要求1~7任一项所述的TCR，其特征在于，所述TCR的TCRα链和/或TCRβ链还包括恒定区。
9.如权利要求8所述的TCR，其特征在于，所述TCRα链的恒定区来源于种系TRAC；和/或所述TCRβ链的恒定区来源于种系TRBC2。
10.如权利要求1~7任一项所述的TCR，其特征在于，所述的TCR的TCRα链和/或TCRβ链还包括膜外区和跨膜区。
11.如权利要求1~7任一项所述的TCR，其特征在于，所述的TCR的TCRα链和/或TCRβ链还包括胞内序列。
12.一种编码权利要求1~11任一项所述的TCR的核酸。
13.一种包含如权利要求12所述的核酸的载体。
14. 如权利要求13所述的载体，其特征在于，所述的载体为慢病毒载体；所述的核酸在单个开放阅读框中，或者在两个不同的开放阅读框中分别编码TCR α链和TCR β链。
15.一种含有如权利要求12所述的核酸或者如权利要求13所述的载体的细胞。
16.如权利要求15所述的细胞，其特征在于，所述的细胞为T细胞或者干细胞。
17. 如权利要求16所述的细胞，其特征在于，所述的T细胞为CD8+ T细胞。
18.一种药物组合物，其特征在于，其含有如权利要求1~11任一项所述的TCR或者如权利要求15-17任一项所述的细胞。
19.如权利要求18所述的药物组合物，其特征在于，所述的药物组合物还包含药物上可接受的载体。
20.一种如权利要求1~11任一项所述TCR、如权利要求15-17任一项所述的细胞或者如权利要求18或19所述的药物组合物在制备防治WT1表达相关肿瘤的药物中的应用；所述的肿瘤为乳腺癌或肺癌。
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| CN105255834A (zh) * | 2010-09-20 | 2016-01-20 | 生物技术公司 | 抗原特异性t细胞受体和t细胞表位 |
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| CN110121336A (zh) * | 2017-01-05 | 2019-08-13 | 弗莱德哈钦森癌症研究中心 | 改善疫苗功效的系统和方法 |
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