CN116554328B - 一种靶向trbv12的单链抗体、car、car-nk细胞及应用 - Google Patents
一种靶向trbv12的单链抗体、car、car-nk细胞及应用 Download PDFInfo
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- CN116554328B CN116554328B CN202310528437.1A CN202310528437A CN116554328B CN 116554328 B CN116554328 B CN 116554328B CN 202310528437 A CN202310528437 A CN 202310528437A CN 116554328 B CN116554328 B CN 116554328B
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明提供一种靶向TRBV12的单链抗体、CAR、CAR‑NK细胞及应用，其中，靶向TRBV12的单链抗体包括重链可变区和轻链可变区，其中，重链可变区包括CDR‑H1、CDR‑H2和CDR‑H3组成的三个互补决定区，轻链可变区包括CDR‑L1、CDR‑L2和CDR‑L3组成的三个互补决定区；CDR‑H1的氨基酸序列如SEQ ID NO:2所示、CDR‑H2的氨基酸序列如SEQ ID NO:3所示、CDR‑H3的氨基酸序列如SEQ ID NO:4所示；CDR‑L1氨基酸序列如SEQ ID NO:5所示、CDR‑L2的氨基酸序列如SEQ ID NO:6所示、CDR‑L3的氨基酸序列如SEQ ID NO:7所示。
Description
技术领域
本发明涉及生物技术领域，具体涉及一种基于靶向TRBV12的单链抗体的CAR-NK细胞及其制备与应用。
背景技术
T细胞恶性肿瘤是一组广泛存在的异质性疾病，每一种类型都表现为不同发育阶段功能失调的T细胞克隆性增生，通常预后较差。T细胞恶性肿瘤包括T细胞急性淋巴细胞白血病(T-ALL)、皮肤和外周T细胞淋巴瘤(CTCL和PTCL)以及成人T细胞白血病(ATL)。T细胞恶性肿瘤进展很快，可以在短期内快速浸润到淋巴结、肝、脾、中枢神经系统等组织器官，目前缺乏有效的治疗方法。虽然免疫疗法通过使用单克隆抗体、检查点抑制剂、双特异性T细胞接合剂和嵌合抗原受体(CAR)T细胞在癌症的治疗中取得一定的疗效，但在T细胞疾病中仅观察到有限的反应。因此对于恶性T细胞肿瘤的治疗，是目前临床所面临的一大难题。
目前细胞疗法治疗T细胞恶性肿瘤主要面临两个方面的限制：第一，目前CAR-T细胞治疗在复发/难治性B细胞恶性肿瘤取得很好的疗效，以广谱性靶点CD19 CAR-T为典型代表，在血液瘤的治疗中取得了巨大成就。从临床数据来看患者对于正常B细胞的消耗具有良好的耐受性，被认为是可接受的副作用。然而，靶向广泛T细胞抗原虽然可以控制T细胞肿瘤，但伴随正常T细胞耗竭会导致严重的免疫功能破坏。目前少数研究以CD7或CD5作为T细胞恶性肿瘤的治疗靶点，由于CD7/CD5也表达于正常T细胞表面，而这种广泛靶向T细胞抗原引起了严重T细胞再生障碍和免疫功能损伤。因此不同于B细胞肿瘤，针对T细胞恶性肿瘤而言，寻求一种不依赖于广泛T细胞抗原而针对特定肿瘤细胞群的靶点，进而保留正常T细胞，避免严重免疫破坏成为治疗T细胞恶性肿瘤的一大难题。第二，随着CAR-T在临床应用越来越广泛，也显现出一定弊端，其中最主要的就是细胞因子释放综合征和神经毒性，这也极大的影响了CAR-T的治疗效果。此外自体细胞获取给临床应用CAR-T细胞带来了挑战，一方面一些病人由于疾病影响无法获得足够的高质量T细胞；另一方面制备细胞需要一定的时间，使得一些病人无法及时得到治疗。因此选取一种低免疫源性，实现异体回输的免疫细胞疗法，替代传统的CAR-T细胞成为亟待进一步的解决的关键科学问题。
αβT细胞受体(TCR)是一种跨膜异源二聚体，由α、β两条肽链组成，负责特异性识别与MHC(主要组织相容性复合体)结合的抗原肽，激活T细胞产生后续的免疫应答。TCR在正常T细胞和T细胞肿瘤上均有表达，TCRβ链(TRB)种系基因座由68个可变(V)基因片段，以及2个多样性(D)、13个连接(J)和2个恒定(C)基因片段组成。TCRβ链基于核苷酸序列相似性，可变基因(TRBV)片段被分为30个TRBV家族。在T细胞发育过程中，V、D、J基因片段经历重排，导致连续的VDJ转录本在每个T细胞表面表达1种独特的TCRβ链，占正常人外周血T细胞总数的1％至5％。而在T细胞恶性肿瘤中，T细胞发生克隆性恶性增生，仅表达一种TRBV，这为选择性清除恶性克隆性T细胞同时保留大部分正常T细胞提供了潜在机会。有研究利用动物模型证实，表达单个TRBV的T细胞缺失，不影响机体的正常免疫反应。因此本研究从T细胞恶性肿瘤的靶点选择问题出发，建立了抗TRBV12的嵌合抗原受体，可以选择性的杀死TRBV12(+)肿瘤细胞，而保留其他TRBV12(-)的正常T细胞。与针对广泛T细胞类抗原相比，避免了因治疗而导致的T细胞再生障碍，保留了机体正常的免疫功能。
NK细胞，即自然杀伤细胞，作为人体免疫系统的第一道防线，可以主动识别和杀伤入侵机体的病毒以及被病毒感染的细胞。与具有广泛克隆重排的TCRs库的T细胞相比，NK细胞激活或抑制信号由种系编码受体如NKG2D或KIR(Ig样受体)和CD94-NKG2异源二聚体介导，除了使用颗粒酶和穿孔素的直接细胞毒性外，触发的NK细胞还可以通过刺激炎症细胞因子的产生导致靶细胞的破坏。与T细胞相比，TCR的缺失大大降低了GvHD的风险，从而允许使用同种异体CAR-NK细胞进行回输。此外与CAR-T细胞相比，CAR-NK细胞诱导细胞因子释放综合征(CRS)可能性更低，具有更高安全性。其次与使用自体T细胞制备CAR-T相比，同种异体CAR-NK细胞不仅可以克服终产品恶性肿瘤细胞污染的风险，还可以降低自体产品制备所需的成本与时间，方便随时取用。
发明内容
基于上述背景存在的目前对T细胞恶性肿瘤尚无有效治疗手段的技术缺陷，本发明提供一种基于人源的靶向TRBV12的单链抗体，将该单链抗体作为抗原结合结构域构建嵌合抗原受体CAR，通过CAR-NK细胞表达，可以有效清除TRBV12(+)T细胞恶性肿瘤。
本发明的第一个目的，提供一种靶向TRBV12的单链抗体，包括重链可变区和轻链可变区，其中，重链可变区包括CDR-H1、CDR-H2和CDR-H3组成的三个互补决定区，轻链可变区包括CDR-L1、CDR-L2和CDR-L3组成的三个互补决定区；CDR-H1的氨基酸序列如SEQ IDNO:2所示、CDR-H2的氨基酸序列如SEQ ID NO:3所示、CDR-H3的氨基酸序列如SEQ ID NO:4所示；CDR-L1氨基酸序列如SEQ ID NO:5所示、CDR-L2的氨基酸序列如SEQ ID NO:6所示、CDR-L3的氨基酸序列如SEQ ID NO:7所示。
SEQ ID NO:2:NFRMH；
SEQ ID NO:3:YISSGSSTIYYADTMKG；
SEQ ID NO:4:RGEGAMDY；
SEQ ID NO:5:RAGSSVNYIY；
SEQ ID NO:6:YTSNLAP；
SEQ ID NO:7:QRFTSSPFT；
进一步地，所述抗体的氨基酸全长序列如SEQ ID NO:1所示。
SEQ ID NO:1：
DVQLVESGGGLVQPKGSRKLSCAASGFTFSNFRMHWVRRAPGKGLEMVAYISSGSSTIYYADTMKGRFTISRDNPKNTLFLQMTSLRSEDTAMYYCARRGEGAMDYWGQGTSVTVSSGGGGSGGGGSGGGGSENVLTQSPATMSASLGIKVTMSCRAGSSVNYIYWYQQKSDASPKLWIYYTSNLAPGVPTRFSGSGSGNSLSLTISSMEGEDAATYYCQQFTSSPFTFGQGTKLEIK。
本发明的第二个目的，提供一种分离的核酸，其编码上述述的单链抗体。
进一步地，其核苷酸序列如SEQ ID NO:8所示。
SEQ ID NO:8:
GACGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGCAGCCCAAGGGCAGCAGGAAGCTGAGCTGCGCCGCCAGCGGCTTCACCTTCAGCAACTTCAGGATGCACTGGGTGAGGAGGGCCCCCGGCAAGGGCCTGGAGATGGTGGCCTACATCAGCAGCGGCAGCAGCACCATCTACTACGCCGACACCATGAAGGGCAGGTTCACCATCAGCAGGGACAACCCCAAGAACACCCTGTTCCTGCAGATGACCAGCCTGAGGAGCGAGGACACCGCCATGTACTACTGCGCCAGGAGGGGCGAGGGCGCCATGGACTACTGGGGCCAGGGCACCAGCGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGAGAACGTGCTGACCCAGAGCCCCGCCACCATGAGCGCCAGCCTGGGCATCAAGGTGACCATGAGCTGCAGGGCCGGCAGCAGCGTGAACTACATCTACTGGTACCAGCAGAAGAGCGACGCCAGCCCCAAGCTGTGGATCTACTACACCAGCAACCTGGCCCCCGGCGTGCCCACCAGGTTCAGCGGCAGCGGCAGCGGCAACAGCCTGAGCCTGACCATCAGCAGCATGGAGGGCGAGGACGCCGCCACCTACTACTGCCAGAGGTTCACCAGCAGCCCCTTCACCTTCGGCCAGGGCACCAAGCTGGAGATCAAG。
本发明的第三个目的，提供一种包含上述分离的核酸的表达载体、或宿主细胞。
本发明的第四个目的，提供一种包含上述的单链抗体的嵌合抗原受体CAR，其特征在于：所述嵌合抗原受体CAR还包括跨膜结构域和共刺激信号传导区。
进一步地，所述跨膜结构域选自CD3ζ、CD8、CD28、NKG2D、2B4、DNAM1中的一种；
所述共刺激信号传导区选自CD3ζ、CD3γ、CD3δ、CD3ε、CD5、CD22、CD79a、CD79b、CD66d、CD2、CD4、CD5、CD28家族、DAP10、DAP12、NKp44、NKG2D、NKp46，NKp30、TNFR家族或SLAM受体家族中的一种或多种组合。
进一步地，所述CD28家族为CD28或ICOS；所述TNFR家族为4-1BB、OX40或CD27；SLAM受体家族为2B4。
本发明的第五个目的，提供一种表达上述的嵌合抗原受体CAR的CAR-NK细胞。
本发明的第六个目的，提供了一种CAR-NK细胞的制备方法，于包括以下步骤：
分离培养健康人的NK细胞；
利用PB转座子系统电转NK细胞；所述PB转座子系统为表达抗TRBV12的CAR；
检测电转后的NK细胞CAR的表达效率，并进一步扩大培养。
本发明的第七个目的，提供一种将上述单链抗体、嵌合抗原受体CAR、或CAR-NK细胞在制备抗TRBV12(+)T细胞恶性肿瘤或TRBV12(+)T细胞增值性疾病药品或产品中的应用。
本发明所达到的有益技术效果：本发明针对T细胞肿瘤的靶点，利用靶向TRBV12的抗体，构建突变体库，结合噬菌体展示技术，筛选出突变抗体，即靶向TRBV12的单链抗体。以该单链抗体作为嵌合抗原受体CAR的scFV部分。将嵌合抗原受体CAR整合到NK细胞中，即制备出CAR-NK细胞，并验证了其抗T细胞恶性肿瘤的疗效。不仅为TRBV12(+)T细胞肿瘤提供治疗手段，也以TRBV12作为切入点，继续开发出针对其他TRBV家族的CAR-NK细胞进一步为临床攻克T细胞恶性肿瘤打下了研究基础。其次，本发明还提供一种靶向TRBV12的CAR-NK细胞，所述CAR-NK细胞能够表达嵌合抗原受体CAR，其中，所述嵌合抗原受体CAR包含抗原结合结构域，跨膜结构域和共刺激信号传导区。所述的抗原结构域为靶向TRBV12的单链抗体，能够特异性的结合TRBV12(+)的T细胞，通过跨膜结构域和共刺激信号传导区激活该NK细胞；该CAR-NK细胞具有以下优势：1.靶向性强，具有清除恶性肿瘤细胞潜力，同时又能保留正常T细胞；2.副作用小，相较于CAR-T，CAR-NK既能降低细胞因子风暴等风险又能避免终产品恶性细胞污染的风险；3.通用性好，利用NK细胞，有效降低GvHD，并降低制备时间和成本；4.理念创新，选用靶向TRBV12的单链抗体作为CAR的靶向识别区，通过技术升级实现功能突破。
附图说明
图1为TRBV12-His抗体对表达TRBV12(+)细胞的亲和力；其中，A表示商业化的TRBV12-biolegend抗体和本申请TRBV12-His抗体分别识别2名供体的正常外周血中TRBV12(+)细胞的能力；B表示TRBV12-His抗体与商业化TRBV12-biolegend抗体对内源性表达TRBV12的Jurkat细胞的识别能力；C表示TRB V12-His抗体与商业化TRBV12-biolegend抗体分别对正常外周血中TRBV12(+)细胞的识别能力及分别内源性表达TRBV12的Jurkat细胞的识别能力的统计图表；
图2为表达抗TRBV12 CAR的PB转座子DNA载体；
图3为表达PiggyBac的转座酶DNA载体；
图4为CAR-NK细胞表达效率及NK细胞表型检测；其中，A表示CAR-NK细胞分选前及分选后CAR表达效率的检测；B表示CAR-NK细胞分选前及分选后CAR表达效率的统计分析；C表示分选后继续培养14天之后CAR-NK细胞表面NK表面分子的表达情况；D表示CAR-NK细胞表面NK表面分子表达统计图；
图5为(TRBV12-His)CAR-NK与(α-TRBV12)CAR-NK细胞对内源性表达TR BV12的Jurkat细胞的杀伤性检测；其中，A表示将CAR-NK细胞与靶细胞以不同效靶比共培养之后靶细胞的裂解情况，B和C表示上清中细胞因子TNF-α和IFN-γ的表达情况；
图6为(TRBV12-His)CAR-NK与(α-TRBV12)CAR-NK细胞对过表达TRBV12的CCRF细胞(CCRF-TRBV12)的杀伤性检测，A表示将CAR-NK细胞与靶细胞以不同效靶比共培养之后靶细胞的裂解情况，B和C表示上清中细胞因子TNF-α,IFN-γ的表达情况。
具体实施方式
下面结合具体实施例对本发明作进一步描述。以下实施例仅用于更加清楚地说明本发明的技术方案，而不能以此来限制本发明的保护范围。
下面结合附图和实施例对本发明专利进一步说明。
实施例1靶向TRBV12的人重链抗体筛选
靶向TRBV12的人重链抗体是基于如SEQ ID NO:9所示的抗体序列构建突变体库，再利用噬菌体展示技术，从突变抗体库中筛选的突变抗体序列。最终筛选出靶向TRBV12的单链抗体，氨基酸序列如SEQ ID NO:1所示。具体过程如下：
SEQ ID NO:9：DVQLVESGGGLVQPGGSRKLSCAASGFTFSNFGMHWVR QAPGKGLEWVAYISSGSSTIYYADTLKGRFTISRDNPKNTLFLQMTSLRSEDTAMYYCARRGEGAMDYWGQGTSVTVSSGGGGSGGGGSGGGGSENVLTQSPAIMSASLGEKVTMSCRASSSVNYIYWYQQKSDASPKLWIYYTSNLAPGVPTRFSGSGSGNSYSLTISSMEGEDAATYYCQQFTSSPFTFGQGTKLEIK。
①基于SEQ ID NO:9所示抗体序列，利用易错PCR技术，配制PCR反应体系，扩增出目的片段；将PCR产物进行2％琼脂糖凝胶电泳分析，并切下约500bp左右的目的条带，用胶回收柱回收目的片段扩增产物。③用SfiⅠ和NotⅠ双酶切pCANTAB5E载体和抗体基因。琼脂糖凝胶电泳回收上一步的酶切片段。按载体1.5ug，抗体0.5ug，T4连接酶2μL，10xbuffer 10μL，加水至100μL，在PCR仪上16℃连接过夜，将抗体基因连入酶切后的pCANTAB5E载体。④向100uL TG1感受态中加入5uL连接产物，放于冰上预冷，转入预冷后的电转杯中；调节电转仪电压2.5KV电击时间5ms，电击后，迅速加入0.9ml培养基，37℃振荡培养2小时；取10uL梯度稀释，涂布于SOBAG平板上，计算库容。⑤将1.5mL的抗体库接种到300mL培养基中，37℃振荡培养约1.5h，按5倍的体积加入辅助噬菌体(M13K07)，37℃振荡培养约1h；4000rpm 15℃离心15min，去除培养基；加入200mL培养基(100μg/mL Amp，50μg/mL Kan)重悬细菌，37℃培养2h；10000rpm离心20min去除沉淀；上清加入40mL PEG/NaCl沉噬菌体，冰浴过夜；10000rpm离心20min，去掉上清；用0.6mL培养基重悬噬菌体，4℃备用。获得的噬菌体经过梯度稀释，感染TG1菌，涂布SOBAG平板，通过菌落计数计算噬菌体库滴度。⑥利用His Bind Resin结合抗原蛋白，从噬菌体展示的抗体库中淘选抗体，最终成功筛选出靶向TRBV12的单链抗体，并通过后续功能实验表明该突变抗体具有比初始抗体更有的杀伤效果。
实施例2靶向TRBV12人重链抗体对于TRBV12(+)T细胞的亲和力验证
首先，将筛选到的单链抗体如SEQ ID NO:8所示的DNA序列，作为模板合成cDNA，并连接C端人IgG1 FC将该融合物克隆到PCDH-EF1α载体中。进一步将质粒助染进293T细胞并利用His柱纯化抗体(TRBV12-His)。
由于表达TRBV12的T细胞在人群中有少量占比，因此分离2名健康成人外周血T淋巴细胞，分别利用商业化TRBV12抗体(biolegend)和我们筛选的抗体(TRBV12-His)同时结合2名供体的正常T淋巴细胞。结果如图1中A和C所示这些抗体能识别的T细胞比例类似于商业化抗体(biolegend)。进一步的利用天然表达TRBV12的Jurkat细胞验证该抗体活性，结果仍与商业化TRBV12抗体(biolegend)检测效率一致图1中B和C所示，证明筛选的抗体可以很好的识别靶点TRBV12。
实施例3表达抗TRBV12的CAR结构的PB转座子系统的构建
将上述得到的单链抗体与CD8跨膜结构域如SEQ ID NO:10，CD28-CD3ζ共刺激域如SEQ ID NO:11构成靶向TRBV12的CAR结构，如SEQ ID NO:12。连接到载体质粒PUC的EcoRI和SalI酶切位点之间，命名为PUC-EF1α-#3，如图2所示。此外将转座酶部分连接到PUC载体的SFiI酶切位点之间，命名为PUC-CMV-PiggyBac，如图3所示。
SEQ ID NO:10
ACCACCACCCCCGCCCCCCGCCCCCCCACCCCCGCCCCCACCATCGCCAGCCAGCCCCTGAGCCTGCGCCCCGAGGCCTGCCGCCCCGCCGCCGGCGGCGCCGTGCACACCCGCGGCCTGGACTTCGCCTGCGACATCTACATCTGGGCCCCCCTGGCCGGCACCTGCGGCGTGCTGCTGCTGAGCCTGGTGATCACC。
SEQ ID NO:11
AAGCGCGGCCGCAAGAAGCTGCTGTACATCTTCAAGCAGCCCTTCATGCGCCCCGTGCAGACCACCCAGGAGGAGGACGGCTGCAGCTGCCGCTTCCCCGAGGAGGAGGAGGGCGGCTGCGAGCTGCGCGTGAAGTTCAGCCGCAGCGCCGACGCCCCCGCCTACCAGCAGGGCCAGAACCAGCTGTACAACGAGCTGAACCTGGGCCGCCGCGAGGAGTACGACGTGCTGGACAAGCGCCGCGGCCGCGACCCCGAGATGGGCGGCAAGCCCCAGCGCCGCAAGAACCCCCAGGAGGGCCTGTACAACGAGCTGCAGAAGGACAAGATGGCCGAGGCCTACAGCGAGATCGGCATGAAGGGCGAGCGCCGCCGCGGCAAGGGCCACGACGGCCTGTACCAGGGCCTGAGCACCGCCACCAAGGACACCTACGACGCCCTGCACATGCAGGCCCTGCCCCCCCGC。
SEQ ID NO:12
ATGGCCCTGCCCGTGACCGCCCTGCTGCTGCCCCTGGCCCTGCTGCTGCACGCCGCCCGCCCCGACGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGCAGCCCAAGGGCAGCAGGAAGCTGAGCTGCGCCGCCAGCGGCTTCACCTTCAGCAACTTCAGGATGCACTGGGTGAGGAGGGCCCCCGGCAAGGGCCTGGAGATGGTGGCCTACATCAGCAGCGGCAGCAGCACCATCTACTACGCCGACACCATGAAGGGCAGGTTCACCATCAGCAGGGACAACCCCAAGAACACCCTGTTCCTGCAGATGACCAGCCTGAGGAGCGAGGACACCGCCATGTACTACTGCGCCAGGAGGGGCGAGGGCGCCATGGACTACTGGGGCCAGGGCACCAGCGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGAGAACGTGCTGACCCAGAGCCCCGCCACCATGAGCGCCAGCCTGGGCATCAAGGTGACCATGAGCTGCAGGGCCGGCAGCAGCGTGAACTACATCTACTGGTACCAGCAGAAGAGCGACGCCAGCCCCAAGCTGTGGATCTACTACACCAGCAACCTGGCCCCCGGCGTGCCCACCAGGTTCAGCGGCAGCGGCAGCGGCAACAGCCTGAGCCTGACCATCAGCAGCATGGAGGGCGAGGACGCCGCCACCTACTACTGCCAGAGGTTCACCAGCAGCCCCTTCACCTTCGGCCAGGGCACCAAGCTGGAGATCAAGACCACCACCCCCGCCCCCCGCCCCCCCACCCCCGCCCCCACCATCGCCAGCCAGCCCCTGAGCCTGCGCCCCGAGGCCTGCCGCCCCGCCGCCGGCGGCGCCGTGCACACCCGCGGCCTGGACTTCGCCTGCGACATCTACATCTGGGCCCCCCTGGCCGGCACCTGCGGCGTGCTGCTGCTGAGCCTGGTGATCACCAAGCGCGGCCGCAAGAAGCTGCTGTACATCTTCAAGCAGCCCTTCATGCGCCCCGTGCAGACCACCCAGGAGGAGGACGGCTGCAGCTGCCGCTTCCCCGAGGAGGAGGAGGGCGGCTGCGAGCTGCGCGTGAAGTTCAGCCGCAGCGCCGACGCCCCCGCCTACCAGCAGGGCCAGAACCAGCTGTACAACGAGCTGAACCTGGGCCGCCGCGAGGAGTACGACGTGCTGGACAAGCGCCGCGGCCGCGACCCCGAGATGGGCGGCAAGCCCCAGCGCCGCAAGAACCCCCAGGAGGGCCTGTACAACGAGCTGCAGAAGGACAAGATGGCCGAGGCCTACAGCGAGATCGGCATGAAGGGCGAGCGCCGCCGCGGCAAGGGCCACGACGGCCTGTACCAGGGCCTGAGCACCGCCACCAAGGACACCTACGACGCCCTGCACATGCAGGCCCTGCCCCCCCGC。
实施例4靶向TRBV12的CAR-NK细胞的制备
1.NK的细胞培养
将新鲜血液于2000转/分钟离心10分钟，吸取上层淡黄色血浆于50ml离心管，56℃水浴30min灭活，然后400g离心10min去除沉淀，将上层血浆转移至新的50ml离心管于4℃冰箱备用，下层血细胞用生理盐水按照1:1稀释混匀,在新的50ml离心管中先加入15ml人外周血淋巴细胞分离液(TBD)，之后沿壁小心加入25ml稀释后的全血，不可破坏两种液体之间的分界面。750g速度离心25-30分。离心后，吸取中间的白细胞层，PBS清洗两次并计数。起始培养按照2×106+3ml NK细胞无血清培养液+5％自体血清+试剂A(ZY-NKZ-0104,IL-21natural killer cell amplificati-on system)比例进行放大培养，刺激培养6天后进行电转。
2.PB转座子系统电转NK细胞
按照A液：B液＝1:1比例配置电转液(Celetrix)，NK细胞计数1000万，加入100μl电转液重悬细胞，加入10μg PB转座子质粒和5μg PB转座酶质粒，Celetrix电转仪电压设置为cell line模式480V。电转结束后转入培养基中继续培养，2天后流式检测电转效率。
3.抗TRBV12的CAR-NK细胞效率检测
按上述流程电转结束后继续培养48小时流式检测感染效率，并进一步在培养14天后做磁珠分选，分选后流式细胞术检测感染效率。如图4中A和B所示，分选之后CAR的表达效率远高于未分选之前CAR的阳性率，且继续培养14天之后检测NK细胞表型发现NK占比约为80％，如图4中C和D所示。
实施例5CAR-NK对表达TRBV12靶细胞的杀伤性检测
1.CAR-NK对内源性表达TRBV12的靶细胞(Jurkat)的杀伤性检测
为了对比(TRBV12-His)CAR-NK细胞与(α-TRBV12)CAR-NK细胞清除天然表达TRBV12的靶细胞的能力，我们分别将两种CAR-NK细胞与Jurkat细胞以不同效靶比(0.2:1,0.4:1,0.8:1,1:1)共孵育，利用细胞裂解实验检测CAR-NK的直接杀伤作用，如图5中A所示；上清细胞因子IFN-γ和TNF-α检测间接杀伤作用如图5中B和C所示。结果均显示TRBV12CAR-NK比对照NK细胞表现出更强的杀伤性，且(TRBV12-His)CAR-NK对靶细胞的杀伤性优于(α-TRBV12)CAR-NK细胞。
2.CAR-NK对过表达TRBV12的靶细胞(CCRF-TRBV12)的杀伤性检测
由于CCRF细胞自身不表达TRBV12，我们首先利用慢病毒转导方式构建过表达TRBV12的靶细胞CCRF-TRBV12，其次以不同效靶比(0.2:1,0.4:1,0.8:1,1:1)进行共孵育，利用细胞裂解实验检测CAR-NK的直接杀伤作用上清细胞因子IFN-γ和TNF-α检测间接杀伤作用，如图6所示，其中，A表示将CAR-NK细胞与靶细胞以不同效靶比共培养之后靶细胞的裂解情况，B和C表示上清中细胞因子TNF-α,IFN-γ的表达情况。结果均显示CAR-NK比对照NK细胞表现出更强的杀伤性，且(TRBV12-His)CAR-NK对CCRF-TRBV12靶细胞的杀伤性优于(α-TRBV12)CAR-NK细胞。
以上已以较佳实施例公布了本发明，然其并非用以限制本发明，凡采取等同替换或等效变换的方案所获得的技术方案，均落在本发明的保护范围内。
Claims (11)
1.一种靶向TRBV12的单链抗体，其特征在于：包括重链可变区和轻链可变区，其中，重链可变区包括CDR-H1、CDR-H2和CDR-H3组成的三个互补决定区，轻链可变区包括CDR-L1、CDR-L2和CDR-L3组成的三个互补决定区；CDR-H1的氨基酸序列如SEQ ID NO:2所示、CDR-H2的氨基酸序列如SEQ ID NO:3所示、CDR-H3的氨基酸序列如SEQ ID NO:4所示；CDR-L1氨基酸序列如SEQ ID NO:5所示、CDR-L2的氨基酸序列如SEQ ID NO:6所示、CDR-L3的氨基酸序列如SEQ ID NO:7所示。
2.根据权利要求1所述的靶向TRBV12的单链抗体，其特征在于：所述抗体的氨基酸全长序列如SEQ ID NO:1所示。
3.一种分离的核酸，其编码权利要求1-2任一项所述的单链抗体。
4.根据权利要求3所述的核酸，其特征在于：其核苷酸序列如SEQ ID NO:8所示。
5.一种包含如权利要求3所述的分离的核酸的表达载体或宿主细胞。
6.一种包含权利要求1-2任一项所述的单链抗体的嵌合抗原受体CAR，其特征在于：所述嵌合抗原受体CAR还包括跨膜结构域和共刺激信号传导区。
7.根据权利要求6所述的嵌合抗原受体CAR，其特征在于：所述跨膜结构域选自CD3ζ、CD8、CD28、NKG2D、2B4、DNAM1中的一种；
或/和
所述共刺激信号传导区选自CD3ζ、CD3γ、CD3δ、CD3ε、CD5、CD22、CD79a、CD79b、CD66d、CD2、CD4、CD5、CD28家族、DAP10、DAP12、NKp44、NKG2D 、NKp46，NKp30、TNFR家族或SLAM受体家族中的一种或多种组合。
8.根据权利要求7所述的嵌合抗原受体CAR，其特征在于：所述CD28家族为CD28或ICOS；所述TNFR家族为4-1BB、OX40或CD27；SLAM受体家族为2B4。
9.一种表达权利要求6所述的嵌合抗原受体CAR的CAR-NK细胞。
10.权利要求9所述的CAR-NK细胞的制备方法，其特征在于包括以下步骤：
分离培养健康人的NK细胞；
利用PB转座子系统电转NK细胞；所述PB转座子系统为表达抗TRBV12的CAR；
检测电转后的NK细胞CAR的表达效率，并进一步扩大培养。
11.如权利要求1-2任意一项所述的单链抗体、权利要求6所述的嵌合抗原受体CAR、权利要求9所述的CAR-NK细胞在制备抗TRBV12(+)T细胞恶性肿瘤或TRBV12(+)T细胞增值性疾病药品或产品中的应用。
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