CN113817056B - 一种靶向cd70的单链抗体及其应用 - Google Patents

一种靶向cd70的单链抗体及其应用 Download PDF

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CN113817056B
CN113817056B CN202010559366.8A CN202010559366A CN113817056B CN 113817056 B CN113817056 B CN 113817056B CN 202010559366 A CN202010559366 A CN 202010559366A CN 113817056 B CN113817056 B CN 113817056B
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齐亚男
赵文旭
陈军
赵永春
徐艳敏
黄霞
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Chongqing Jingzhun Biological Industrial Technology Institute Co ltd
Chongqing Precision Biotech Co ltd
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Abstract

本发明属于免疫治疗技术领域，具体涉及一种靶向CD70的ScFv，一种可识别CD70的人源化单链抗体及其应用。所述靶向CD70的ScFv的轻链可变区的氨基酸序列如SEQ ID NO:8或其功能性变体所示，重链可变区的氨基酸序列如SEQ ID NO:7或其功能性变体所示。将所述靶向CD70的ScFv进行人源化改造后的ScFv以及包含所述人源化ScFv的载体、CAR和免疫细胞可以有效的清除表达CD70抗原的肿瘤靶细胞。

Description

一种靶向CD70的单链抗体及其应用
技术领域
本发明属于免疫治疗技术领域，具体涉及一种靶向CD70的ScFv，一种可识别CD70的人源化单链抗体及其应用。
背景技术
CD70是肿瘤坏死因子受体(TNFR)超家族的成员之一，是一种Ⅱ型跨膜蛋白，主要表达于活化的T细胞、B细胞、自然杀伤细胞以及树突状细胞中。CD70在肾癌、血源性恶性肿瘤(包括AML、NHL、HL等均高表达)、胸腺肿瘤、卵巢癌、成神经胶质细胞瘤、鼻咽癌等多种肿瘤组织中高表达。
嵌合抗原受体(chimeric antigen receptor，CAR)是模拟TCR功能的人工受体，包括一个肿瘤相关抗原结合区、一个胞外间隔区、一个跨膜区和一个胞内信号区。胞内信号域通常为CD3ζ链或FcRγ，或与一种或多种共刺激分子相连，如4-1BB(CD137)，CD28，ICOS(CD278)。CD70在多种肿瘤中均呈现高表达状态，仅在正常的活化的T细胞、B细胞、自然杀伤细胞以及树突状细胞中表达，设计靶向CD70的CAR其可应用肿瘤范围更广，安全性更高。
发明内容
本发明的目的之一在于提供一种分离的抗体或抗原结合片段，其特征在于，可以识别CD70。
为实现上述目的，本发明的技术方案为：
所述ScFv的轻链CDR区包含氨基酸序列如SEQ ID NO:1所示的CDR1、SEQ ID NO:2所示的CDR2和SEQ ID NO:3所示的CDR3。
进一步，所述ScFv的重链CDR区包含氨基酸序列如SEQ ID NO:4所示的CDR1、SEQID NO:5所示的CDR2和SEQ ID NO:6所示的CDR3。
进一步，所述靶向CD70的ScFv的轻链可变区的氨基酸序列如SEQ ID NO:8或其功能性变体所示，重链可变区的氨基酸序列如SEQ ID NO:7或其功能性变体所示。
本发明的目的之二在于提供一种可识别CD70的人源化单链抗体。
虽然人源化改造的方案已有多方报道，但是尤其是为了进行稳定性和亲和力优化，进行定点突变后，改造获得的人源化单链抗体需要经过大量验证才能确认其有效性；理论上“单链抗体”可以用于制备嵌合抗原受体，但实际并不是每一单链抗体都可以用于制备嵌合抗原受体。这需要发明人付出创造性的劳动，才能在众多的改造的单链抗体中找到具有预料不到的效果的产品。
改造单链抗体的方法，是通过检索人源框架库获得与鼠源框架相似度较高的框架序列，将鼠源CDR(可变区)植入人源框架的可变区形成人源化抗体，再通过多位或单位定点突变的方式对人源化FR区(恒定区)进行突变以恢复抗体的稳定性和抗原识别活性。方法的结果存在随机性，只有在配置得当的情况下，该人源化抗体结构才能保持稳定，其亲和力才有可能适合识别肿瘤表面的靶抗原进而激活CAR-T细胞。
为实现上述目的，本发明的技术方案为：
将所述的靶向CD70的ScFv进行人源化改造，所述人源化单链抗体的轻链可变区氨基酸序列如SEQ ID NO:9-12任一所示，所述人源化单链抗体的重链可变区氨基酸序列如SEQ ID NO:13-16任一所示。
进一步，所述的识别CD70的抗体或抗原结合片段的重链可变区和轻链可变区通过一段Linker连接，在有些实施例中，Linker氨基酸序列如SEQ ID NO:17所示；在有些实施例中，Linker氨基酸序列如SEQ ID NO:18或SEQ ID NO:31或SEQ ID NO:32或SEQ ID NO:33所示；在某些实施例中重链可变区和轻链可变区的连接方式是VH-linker-VL，在某些实施例中重链可变区和轻链可变区的连接方式是VL-linker-VH。
本发明的目的之三在于提供一种包含所述可识别CD70的的ScFv的CAR结构和可识别CD70的人源化单链抗体的CAR结构、表达载体和免疫细胞。
为实现上述目的，本发明的技术方案为：
所述包含所述可识别CD70的人源化单链抗体的CAR结构包含识别CD70抗原的抗原识别区，铰链区，跨膜区和胞内信号域。
进一步，识别CD70抗原的抗原识别区选自ScFv，其特征在于，轻链CDR区包含氨基酸序列如SEQ ID NO:1所示的CDR1、SEQ ID NO:2所示的CDR2和SEQ ID NO:3所示的CDR3，重链CDR区包含氨基酸序列如SEQ ID NO:4所示的CDR1、SEQ ID NO:5所示的CDR2和SEQ IDNO:6所示的CDR3。
进一步，识别CD70抗原的抗原识别区包含重链可变区如SEQ ID NO:7所示，轻链可变区如SEQ ID NO:8所示或重链可变区如SEQ ID NO:13所示，轻链可变区如SEQ ID NO:9所示或重链可变区如SEQ ID NO:14所示，轻链可变区如SEQ ID NO:10所示或重链可变区如SEQ ID NO:15所示，轻链可变区如SEQ ID NO:11所示或重链可变区如SEQ ID NO:16所示，轻链可变区如SEQ ID NO:12所示的ScFv。
优选的，所述识别CD70抗原的单链抗体包含重链可变区如SEQ ID NO:14所示，轻链可变区如SEQ ID NO:10所示或重链可变区如SEQ ID NO:16所示，轻链可变区如SEQ IDNO:12所示的氨基酸序列。
在某些实施例中，所述抗CD70抗原的嵌合抗原受体的铰链区可以来源于CD8、IgG4以及CD7等。
进一步，铰链区的氨基酸序列如SEQ ID NO:19或SEQ ID NO:20或SEQ ID NO:21，在有些实施例中铰链区还可能源自IgG4 hinge区；所述跨膜区来源于CD8分子或CD28分子跨膜区；所述胞内信号来源于CD28或CD137胞内信号区和CD3胞内序列。
进一步，所述跨膜区氨基酸序列如SEQ ID NO:26或SEQ ID NO:27所示。
进一步，所述胞内信号氨基酸序列如SEQ ID NO:28或SEQ ID NO:29所示。
进一步，所述CD3活化区氨基酸序列如SEQ ID NO:30所示。
发明人设计了4种不同的人源化ScFv和3种不同的CAR结构进行随机组合，通过ScFv亲和力、稳定性，CAR表达稳定性、CAR-T细胞有效性和安全性5方面评估，验证了发明人所述的识别CD70的抗体或抗原结合片段及改造的人源化单链抗体在不同的CAR结构中均能发挥功能，对靶向CD70的所有CAR组合均具有适用性。
优选的，所述嵌合抗原受体氨基酸序列为SEQ ID NO:22、SEQ ID NO:23、SEQ IDNO:24、SEQ ID NO:25。
进一步，包含所述的CAR结构的表达载体为慢病毒表达载体、逆转录病毒表达载体、腺病毒表达载体、腺相关病毒表达载体、DNA载体，RNA载体、质粒中的任一种。
进一步，包含CAR基因的表达载体启动子采用重复的5个HRE调控元件与弱启动的CMV mini Promoter联用构成5HRE-CMVmini启动子。
优选的，所述载体为慢病毒载体，其特征在于所述载体含有RRE元件和oPRE元件。
进一步，所述的载体，其特征在于，包含用于质粒复制的原核复制子pUCOri序列；用于目的菌株大量扩增的卡纳霉素或者氨苄青霉素抗性基因序列；用于增强真核细胞内的复制病毒复制子SV40Ori序列；用于慢病毒包装的慢病毒包装顺式元件；用于嵌合抗原受体基因的真核转录的启动子；以及用于组成集靶点识别、信号传递、信号启动于一体的CAR编码基因。
在某些实施方案中，载体包含左(5')逆转录病毒LTR、Psi(Ψ)包装信号、中心多嘌呤段/DNA瓣(cPPT/FLAP)、逆转录病毒导出元件、可操作地连接到编码本文所涵盖的CAR的多核苷酸的启动子和右(3')逆转录病毒LTR。
在某些实施方案中，CAR载体包含乙型肝炎病毒转录后调节元件(HPRE)或土拔鼠转录后调节元件(WPRE)；5'LTR的启动子经异源启动子置换，所述异源启动子是巨细胞病毒(CMV)启动子、劳斯肉瘤病毒(Rous Sarcoma Virus，RSV)启动子或猿猴病毒40(SV40)启动子。
所述的慢病毒载体进行T细胞转导时，CAR阳性表达率高，在病人细胞培养过程中很稳定，并且不会随着时间的推移会导致阳性率下降。于是，用所述慢病毒载体感染的细胞，这样的细胞具备杀伤靶细胞的功能。
本发明的目的之四在于提供一种药物组合物以及所述药物组合物在制备恶性肿瘤药物中的应用。
为实现上述目的，本发明的技术方案为：
所述药物组合物包含所述的可识别CD70的ScFv或所述的人源化单链抗体或所述的CAR结构或所述的免疫细胞。
在某些实施方案中，CAR表达细胞还可以表达另外的活性剂或与活性剂联合应用，这些活性剂可以是免疫检查点抑制剂，如PD1/PDL1、TIM3、CTLA4、LAG3以及TGFRβ抑制剂等；在其他实施方案中，这些活性剂可以是多肽或融合蛋白，如PD-1-CD137-CD3分子形成的融合蛋白；在其他实施方案中，这些活性剂可以是抗体药物，如单抗药物、双特异性抗体药物等；在其他实施方案中，这些活性剂可以是激酶抑制剂，如酪氨酸激酶抑制剂达沙替尼。
进一步，所述恶性肿瘤为表达CD70的恶性肿瘤，包括但不限于肾细胞癌、食管癌、间皮瘤、胃癌、腺囊癌、卵巢癌、子宫内膜癌、乳腺癌、头颈部鳞状细胞癌、胶质瘤、肺癌、骨肉瘤、甲状腺癌、黑色素瘤、胰腺癌、淋巴瘤、急性髓系白血病、或多发性骨髓瘤。
在某些实施方案中，所述细胞在PI3K通路抑制剂存在下经活化和刺激。
在某些实施方案中，通过包含所述细胞的组合物进行应用，施用所述组合物的途径包括：口服、静脉注射、腹膜输注、皮下注射以及肿瘤或器官局部给药。优选地，施用所述组合物的方法为：通过静脉内、腹膜内或皮下注射。
总的来说，本发明所述的靶向CD70的CAR(嵌合抗原受体)在免疫细胞中表达后，不仅可以维持靶向CD70的嵌合抗原受体(chimeric antigen receptor，CAR)在病人细胞培养过程中的阳性率并且能够加强CAR-T的增殖和杀伤肿瘤的能力，能够用于肿瘤的靶向治疗。
本发明的有益效果在于：
1)本发明提供的识别CD70的抗体或抗原结合片段可以高效的识别CD70抗原；
2)本发明提供的识别CD70的抗体或抗原结合片段可以识别CD70阳性的组织、细胞和血液样本。
3)本发明提供的识别CD70的抗体或抗原结合片段构建的抗CD70抗原的嵌合抗原受体在免疫细胞中表达后，可以有效的清除表达CD70抗原的肿瘤靶细胞，并且能够维持靶向CD70的嵌合抗原受体(chimeric antigen receptor，CAR)在病人细胞培养过程中的阳性率，能够用于肿瘤的靶向治疗。
附图说明
图1为CD70在组织中表达检测。
图2为CD70鼠源抗体和人源化设计。
图3为CD70抗体亲和力检测。
图4为CD70抗体识别细胞表面CD70能力验证。
图5为CD70抗体ScFv构建的CAR结构。
图6为靶向CD70 CAR表达验证。
图7为靶向CD70 CART体外有效性杀伤。
图8为靶向CD70 CART体内有效性杀伤。
具体实施方式
以下将参照附图，对本发明的优选实施例进行详细描述。优选实施例中未注明具体条件的实验方法，通常按照常规条件，例如分子克隆实验指南(第三版，J.萨姆布鲁克等著)中所述的条件，或按照制造厂商所建议的条件。所举实施例是为了更好地对本发明的内容进行说明，但并不是本发明的内容仅限于所举实施例。所以熟悉本领域的技术人员根据上述发明内容对实施方案进行非本质的改进和调整，仍属于本发明的保护范围。
实施例1：CD70在正常组织表达的检测
选用单重PCR(一块板扩增一个基因，内参基因一块板，目的基因一块板)。以Skov3细胞的cDNA作为阳性对照的模板；阴性对照为不加cDNA模板的反应混合液，以dH2O作为模板，本试验采用来自同一次逆转录合成的Skov3细胞cDNA扩增同一对引物下的GAPDH，作用是证明试验板和内参板之间的可比性。以外周血淋巴细胞作为对照组，按相对表达量经典计算方法2-ΔΔCt计算各个组织中CD70的相对表达量。结果如图1所示：以外周血淋巴细胞作对照，***、扁桃体中CD70表达稍高于对照样本，而小肠、肺、脾、胃、胸腺、***、气管略低于对照，其他组织样本则远低于对照样本。可见CD70在除淋巴细胞丰富的组织表达外，在其他正常组织几乎不表达。
实施例2：CD70鼠源抗体和人源化设计
鼠源抗体CDR结构如图2所示，重链可变区氨基酸序列如SEQ ID NO:7所示，轻链可变区氨基酸序列如SEQ ID NO:8所示。
经过分子对接模拟，对鼠源抗体骨架序列进行人源化修饰改造，获得2个人源化ScFv，在此基础上分别对这2个ScFv进行回复突变和免疫原性优化，以获得活性好和免疫原性更低的ScFv，共获得4个优化后的人源化ScFv。
设计人源化ScFv序列，其中轻链可变区氨基酸序列分别如SEQ ID NO:9，SEQ IDNO:10，SEQ ID NO:11，SEQ ID NO:12任一所示；重链可变区氨基酸序列分别如SEQ ID NO:13，SEQ ID NO:14，SEQ ID NO:15，SEQ ID NO:16任一所示。
实施例3：CD70抗体亲和力检测
使用HIS1K传感器固化靶向CD70 ScFv，分析hCD70-hFc与之相互作用的动力学。分析物浓度梯度：200nM，100nM，50nM，25nM具体实验步骤见仪器操作规程。结果如图3和表1所示，ScFv具有对CD70较好的亲和力，亲和力常数KD＝16.4nM和4.65nM。
表1 CD70抗体亲和力检测
Conc.(nM) KD(M) Kon(1/Ms) Kdis(1/s) Full R<sup>2</sup>
100 2.138E-08 73993.971 0.0016 0.9965
50 2.138E-08 73993.971 0.0016 0.9965
25 2.138E-08 73993.971 0.0016 0.9965
12.5 2.138E-08 73993.971 0.0016 0.9965
实施例4：CD70分子识别细胞表面CD70能力验证
靶向CD70的ScFv对CD70阳性细胞786-O，外源构建K562-CD70和CD70阴性细胞K562进行流式染色。结果如图4所示，靶向CD70的ScFv能够识别CD70阳性的786-O细胞和外源构建的K562-CD70细胞表面的CD70。因此，我们公开的包含SEQ ID NO:1-SEQ ID NO:6CDR区的鼠源和人源化抗CD70抗体或多肽片段能够作为CD70检测试剂使用。
实施例5：表达靶向人CD70抗原的嵌合抗原受体的慢病毒制备
(1)制备靶向人CD70抗原的嵌合抗原受体构建
合成包含抗人CD70抗原的单链抗体ScFv，hFc铰链区、跨膜区和胞内信号段的嵌合抗原受体序列。其中抗人CD70抗原的人源化单链抗体包含重链可变区如SEQ ID NO:7所示，轻链可变区如SEQ ID NO:8所示或重链可变区如SEQ ID NO:13所示，轻链可变区如SEQ IDNO:9所示或重链可变区如SEQ ID NO:14所示，轻链可变区如SEQ ID NO:10所示或重链可变区如SEQ ID NO:15所示，轻链可变区如SEQ ID NO:11所示或重链可变区如SEQ ID NO:16所示，轻链可变区如SEQ ID NO:12所示的氨基酸序列；跨膜区来源于CD8或CD28序列；胞内信号段来源于CD28或CD137及CD3序列。最终合成的人源化抗CD70嵌合抗原受体12组，鼠源对照1组。结构如图5所示。
酶切反应按说明书进行。酶切产物分离后回收，然后将上述目的片段和载体片段通过T4连接酶(购自Promega公司)进行连接，获得表达嵌合抗原受体的慢病毒载体，质粒抽提试剂盒(Invitrogen公司)抽提质粒，具体方法见说明书。
(2)慢病毒的包装
本实施例包装慢病毒采用磷酸钙法，具体步骤如下：用含10％FBS(w/v)的DMEM培养基培养293T细胞至较佳状态，将ddH2O、慢病毒载体和2.5mM CaCl2以一定比例加入15mL离心管中，混合均匀，并向混合液中逐滴加入2×HBS，混匀后室温静置15min，然后将混合液逐滴加入上述制备的293T细胞中，继续培养过夜后，用更换新鲜的含10％FBS(w/v)的DMEM培养基。细胞在培养48h、72h后分别收集细胞上清，纯化分装。
实施例6：CD70抗原的嵌合抗原受体修饰T细胞的制备
(1)慢病毒感染T细胞
1)人外周血单核细胞的分离
利用梯度离心法进行淋巴细胞分离；离心后，取第二层白色淋巴细胞层，生理盐水洗涤，加入含有10％FBS的RPMI 1640完全培养基培养，得人外周血单核细胞。
2)慢病毒载体感染T淋巴细胞
获得PBMC细胞经抗CD3、CD28单克隆抗体活化后进行慢病毒感染；感染后继续培养10天左右进行CAR-T细胞体内体外生物学特性验证。获得的嵌合抗原受体T细胞，以对应慢病毒载体命名。
3)靶向人CD70抗原的嵌合抗原受体(CAR)表达检测
在培养过程中对培养至第6天和9天的已感染病毒的T细胞进行CAR阳性率检测。检测方法为流式检测，抗体为：Protein-L-PE，Protein-L可识别抗体轻链，CAR抗原识别区的ScFv序列的轻链可被Protein-L识别，因此利用Protein-L可检测CAR阳性率。结果如图6所示不同病毒感染T细胞后CAR能够正常表达在T细胞表面。
实施例7：表达靶向CD70的嵌合抗原受体的T淋巴细胞抗肿瘤效果验证
以稳定表达萤火虫荧光素酶的CD70阳性786-O(简称为786-O-luc)、阴性细胞A549-Luc作为靶细胞。
使用健康供者外周血单核细胞制备的CART，采用8:1/4:1/2:1效靶比铺效应细胞。使用
Figure BDA0002545713580000101
Luciferase Assay System(Promega Cat.#E2520)试剂盒提供的标准方法检测杀伤效果，杀伤率用下列公式计算：
Figure BDA0002545713580000102
杀伤结果如图7所示，构建的CART均具有较好的体外杀伤效果。
实施例8：表达靶向CD70嵌合抗原受体T淋巴细胞在动物模型中的抗肿瘤效果验证
建立人CD70阳性肿瘤细胞系的小鼠移植瘤模型用于验证表达靶向CD70的嵌合抗原受体的T淋巴细胞在动物模型中的抗肿瘤效果。
体内验证使用小鼠为NOD.Cg-PrkdcscidII2rgtm1Sug/JicCrl，简称NOG小鼠，由日本实验动物研究所(CIEA)的Mamoru Ito培育而成，为国际上CAR-T体内相关成瘤实验最常见品系。体内验证使用的成瘤靶细胞为786-O细胞。小鼠成瘤后，分别尾静脉注射CAR-T细胞，未感染病毒的PBMC细胞。注射CAR-T细胞后每隔7天测量肿瘤体积，期间每天观察小鼠存活情况并记录。结果如图8所示：构建的CD70 CAR-T治疗的小鼠肿瘤体积显著减小，具有较好的体内杀伤活性。
最后说明的是，以上实施例仅用以说明本发明的技术方案而非限制，尽管参照较佳实施例对本发明进行了详细说明，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宗旨和范围，其均应涵盖在本发明的权利要求范围当中。
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<110> 重庆精准生物技术有限公司
重庆精准生物产业技术研究院有限公司
<120> 一种靶向CD70的单链抗体及其应用
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Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala
20 25 30
Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp
35 40 45
<210> 20
<211> 36
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 20
Ala Pro Pro Arg Ala Ser Ala Leu Pro Ala Pro Pro Thr Gly Ser Ala
1 5 10 15
Leu Pro Asp Pro Gln Thr Ala Ser Ala Leu Pro Asp Pro Pro Ala Ala
20 25 30
Ser Ala Leu Pro
35
<210> 21
<211> 45
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 21
Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr
1 5 10 15
Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Arg Pro Ala Ala
20 25 30
Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Asp
35 40 45
<210> 22
<211> 473
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 22
Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Gln Arg Ala Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Ser Ile His
20 25 30
Gly Thr His Leu Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro
35 40 45
Val Leu Val Ile Tyr Ala Ala Ser Asn Leu Glu Ser Gly Val Pro Ala
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile His
65 70 75 80
Pro Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Gln Gln Ser Ile
85 90 95
Glu Asp Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly
100 105 110
Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys
115 120 125
Gly Gln Val Gln Leu Val Gln Ser Gly Pro Glu Val Lys Lys Pro Gly
130 135 140
Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp
145 150 155 160
Tyr Tyr Met Asn Trp Val Arg Gln Met His Gly Lys Gly Leu Glu Trp
165 170 175
Met Gly Val Ile Asn Pro Tyr Asn Gly Gly Thr Asp Tyr Asn Gln Lys
180 185 190
Phe Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala
195 200 205
Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr
210 215 220
Cys Ala Arg Ser Val Tyr Asp Tyr Pro Phe Asp Tyr Trp Gly Gln Gly
225 230 235 240
Thr Leu Val Thr Val Ser Ser Lys Pro Thr Thr Thr Pro Ala Pro Arg
245 250 255
Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg
260 265 270
Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly
275 280 285
Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr
290 295 300
Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Val Lys
305 310 315 320
Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg
325 330 335
Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro
340 345 350
Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser
355 360 365
Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu
370 375 380
Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg
385 390 395 400
Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln
405 410 415
Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr
420 425 430
Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp
435 440 445
Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala
450 455 460
Leu His Met Gln Ala Leu Pro Pro Arg
465 470
<210> 23
<211> 462
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 23
Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Gln Arg Ala Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Ser Ile His
20 25 30
Gly Thr His Leu Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro
35 40 45
Val Leu Val Ile Tyr Ala Ala Ser Asn Leu Glu Ser Gly Val Pro Ala
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile His
65 70 75 80
Pro Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Gln Gln Ser Ile
85 90 95
Glu Asp Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly
100 105 110
Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys
115 120 125
Gly Gln Val Gln Leu Val Gln Ser Gly Pro Glu Val Lys Lys Pro Gly
130 135 140
Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp
145 150 155 160
Tyr Tyr Met Asn Trp Val Arg Gln Met His Gly Lys Gly Leu Glu Trp
165 170 175
Met Gly Val Ile Asn Pro Tyr Asn Gly Gly Thr Asp Tyr Asn Gln Lys
180 185 190
Phe Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala
195 200 205
Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr
210 215 220
Cys Ala Arg Ser Val Tyr Asp Tyr Pro Phe Asp Tyr Trp Gly Gln Gly
225 230 235 240
Thr Leu Val Thr Val Ser Ser Ala Pro Pro Arg Ala Ser Ala Leu Pro
245 250 255
Ala Pro Pro Thr Gly Ser Ala Leu Pro Asp Pro Gln Thr Ala Ser Ala
260 265 270
Leu Pro Asp Pro Pro Ala Ala Ser Ala Leu Pro Ile Tyr Ile Trp Ala
275 280 285
Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr
290 295 300
Leu Tyr Cys Val Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys
305 310 315 320
Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys
325 330 335
Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val
340 345 350
Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn
355 360 365
Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val
370 375 380
Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg
385 390 395 400
Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys
405 410 415
Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg
420 425 430
Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys
435 440 445
Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
450 455 460
<210> 24
<211> 473
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 24
Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Glu Arg Ala Thr Ile Asn Cys Arg Ala Ser Glu Ser Val Ser Ile His
20 25 30
Gly Thr His Leu Met His Trp Tyr Gln Gln Leu Pro Gly Thr Pro Pro
35 40 45
Lys Leu Leu Ile Tyr Ala Ala Ser Asn Leu Glu Ser Gly Val Pro Asp
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser
65 70 75 80
Pro Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Gln Gln Ser Ile
85 90 95
Glu Asp Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly
100 105 110
Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys
115 120 125
Gly Gln Val Gln Leu Val Gln Ser Gly Pro Ala Leu Val Lys Pro Gly
130 135 140
Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp
145 150 155 160
Tyr Tyr Met Asn Trp Val Arg Gln Ala His Gly Gln Gly Leu Glu Trp
165 170 175
Met Gly Val Ile Asn Pro Tyr Asn Gly Gly Thr Asp Tyr Asn Gln Lys
180 185 190
Phe Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala
195 200 205
Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr
210 215 220
Cys Ala Arg Ser Val Tyr Asp Tyr Pro Phe Asp Tyr Trp Gly Gln Gly
225 230 235 240
Thr Leu Val Thr Val Ser Ser Lys Pro Thr Thr Thr Pro Ala Pro Arg
245 250 255
Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg
260 265 270
Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly
275 280 285
Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr
290 295 300
Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Val Lys
305 310 315 320
Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg
325 330 335
Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro
340 345 350
Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser
355 360 365
Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu
370 375 380
Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg
385 390 395 400
Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln
405 410 415
Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr
420 425 430
Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp
435 440 445
Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala
450 455 460
Leu His Met Gln Ala Leu Pro Pro Arg
465 470
<210> 25
<211> 473
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 25
Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Gln Arg Ala Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Ser Ile His
20 25 30
Gly Thr His Leu Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro
35 40 45
Val Leu Val Ile Tyr Ala Ala Ser Asn Leu Glu Ser Gly Val Pro Ala
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile His
65 70 75 80
Pro Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Gln Gln Ser Ile
85 90 95
Glu Asp Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly
100 105 110
Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys
115 120 125
Gly Gln Val Gln Leu Val Gln Ser Gly Pro Glu Val Lys Lys Pro Gly
130 135 140
Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp
145 150 155 160
Tyr Tyr Met Asn Trp Val Arg Gln Met His Gly Lys Gly Leu Glu Trp
165 170 175
Met Gly Val Ile Asn Pro Tyr Asn Gly Gly Thr Asp Tyr Asn Gln Lys
180 185 190
Phe Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala
195 200 205
Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr
210 215 220
Cys Ala Arg Ser Val Tyr Asp Tyr Pro Phe Asp Tyr Trp Gly Gln Gly
225 230 235 240
Thr Leu Val Thr Val Ser Ser Lys Pro Thr Thr Thr Pro Ala Pro Arg
245 250 255
Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg
260 265 270
Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly
275 280 285
Leu Asp Phe Ala Cys Asp Phe Trp Val Leu Val Val Val Gly Gly Val
290 295 300
Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp
305 310 315 320
Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met
325 330 335
Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala
340 345 350
Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Val Lys Phe Ser Arg Ser
355 360 365
Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu
370 375 380
Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg
385 390 395 400
Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln
405 410 415
Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr
420 425 430
Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp
435 440 445
Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala
450 455 460
Leu His Met Gln Ala Leu Pro Pro Arg
465 470
<210> 26
<211> 24
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 26
Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu
1 5 10 15
Ser Leu Val Ile Thr Leu Tyr Cys
20
<210> 27
<211> 27
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 27
Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu
1 5 10 15
Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val
20 25
<210> 28
<211> 41
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 28
Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr
1 5 10 15
Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro
20 25 30
Pro Arg Asp Phe Ala Ala Tyr Arg Ser
35 40
<210> 29
<211> 44
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 29
Val Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe
1 5 10 15
Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg
20 25 30
Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg
35 40
<210> 30
<211> 111
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 30
Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln
1 5 10 15
Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp
20 25 30
Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro
35 40 45
Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp
50 55 60
Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg
65 70 75 80
Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr
85 90 95
Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
100 105 110
<210> 31
<211> 10
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 31
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
1 5 10
<210> 32
<211> 15
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 32
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
1 5 10 15
<210> 33
<211> 20
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 33
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
1 5 10 15
Gly Gly Gly Ser
20

Claims (1)

1. 一种包含靶向CD70的ScFv的CAR结构在制备恶性肿瘤药物中的应用，其特征在于，所述ScFv的轻链CDR区包含氨基酸序列如SEQ ID NO:1所示的CDR1、SEQ ID NO:2所示的CDR2和SEQ ID NO:3所示的CDR3；所述ScFv的重链CDR区包含氨基酸序列如SEQ ID NO:4所示的CDR1、SEQ ID NO:5所示的CDR2和SEQ ID NO:6所示的CDR3；所述恶性肿瘤为表达CD70的肾细胞癌。
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