CN110950965B - 抗人cd123的嵌合抗原受体及其应用 - Google Patents

抗人cd123的嵌合抗原受体及其应用 Download PDF

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CN110950965B
CN110950965B CN201811125906.0A CN201811125906A CN110950965B CN 110950965 B CN110950965 B CN 110950965B CN 201811125906 A CN201811125906 A CN 201811125906A CN 110950965 B CN110950965 B CN 110950965B
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张巍
单娟娟
徐艳敏
黄霞
赵文旭
陈军
赵永春
张茜真
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Chongqing Precision Biotech Co ltd
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Abstract

本发明属于基因工程领域，具体涉及一种抗人CD123的嵌合抗原受体及其应用。本发明的识别CD123抗原的多肽包含如SEQ ID NO:4‑7所示的氨基酸序列，其作为(chimeric antigen receptor，CAR)结构中抗原识别区均能诱导CAR‑T细胞活化。抗CD123抗原的嵌合抗原受体在免疫细胞中表达后，不仅可以有效的清除表达CD123抗原的肿瘤靶细胞，而对阴性抗原(不表达CD123)的肿瘤细胞没有毒性作用；并且能够维持靶向CD123的CAR在病人细胞培养过程中的阳性率，在靶抗原刺激后能够长时间的进行增殖，能够用于肿瘤的靶向治疗。

Description

抗人CD123的嵌合抗原受体及其应用
技术领域
本发明属于基因工程领域，具体涉及一种抗人CD123的嵌合抗原受体及其应用。
背景技术
急性髓性白血病(Acute myeloid leukemia，AML)是髓系造血干/祖细胞恶性疾病，以骨髓与外周血中原始和幼稚髓性细胞异常增生为主要特征多数病例病情急重，预后凶险，如不及时治疗常可危及生命；常规的诱导化疗虽然能够缓解AML但是最终并不能阻止AML的复发以及复发患者高的死亡率。然而AML的常规治疗已经50年未有改变，急需寻找新的改变。嵌合抗原受体T细胞技术在治疗表达CD19的急性淋巴细胞白血病(ALL)方面取得了优异的治疗效果，但是其靶点为CD19分子是否适用于AML需要进行探索。
CD123又称白介素3受体α链(IL-3Rα)高表达于白血病干细胞或白血病幼稚细胞，在正常造血干细胞不表达或者低表达，是白血病相关抗原也是急性髓性白血病的特异性抗原。CD123靶标的出现是AML治疗的新突破；由于CD123在AML高表达，理论上以CD123为靶标的免疫治疗具有更安全有效的治疗效果，国外也已经在进行以CD123为靶标的靶向药物的临床试验，但是功效均很有限并且仍有安全性问题的发生；因此需要寻找特异性更好更有效的靶向CD123的新疗法，目前虽然有一些靶点的CAR正在试验正在进行，但是用于治疗AML的CAR相对较少，在CAR性能的稳定性和安全性上仍需进一步的改造试验，如何选择更稳定适合的scFv(单链抗体)也是CAR研究中未能解决的关键问题，所以一种更安全有效的以CD123为靶点的CD123-CAR的研发制备是极有必要的。
嵌合抗原受体(chimeric antigen receptor)简称CAR，是模拟TCR功能的人工受体，由抗原识别域、铰链区和跨膜区及胞内信号域依次连接组成，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)。肿瘤细胞表面的抗原(受体)与嵌合抗原受体的抗体(配体)结合时，通过铰链区和跨膜区将信号传递至胞内，胞内信号域将信号转化为活化信号，激活效应细胞，效应细胞增殖、产生细胞因子从而杀伤肿瘤细胞。CAR-T治疗不同的肿瘤疾病针对的抗原不同，不同的抗原抗原结合位点不同，需要完全不同的scFv识别。又因为不同抗原大小不同，scFv亲和力不同，CAR结构中的scFv要正常发挥作用与铰链区密切相关，而哪种scFv最适合选择哪种铰链结构目前并没有研究，需要经过大量实验摸索。
单链抗体作为CAR结构中重要的组成部分，其选择对CAR-T疗效起到至关重要的作用，传统的鼠源抗体，因为鼠抗的异质性会引起人抗鼠抗体反应(Human anti-mouseantibody reaction，HAMA)，导致CAR-T在循环***中被很快清除，失去疗效。因此，治疗用鼠源单抗需要进行人源化修饰以提高抗体的人源化程度、减弱HAMA。但是抗体的改造通常会导致抗体失去原有抗原结合活性，因此需要对影响抗原抗体结合的关键残基进行反复修改，而后通过大量抗原抗体结合特异性、亲和力检测，从而筛选得到具有活性的抗体序列。
因此，改造适用于CAR-T治疗的scFv以及筛选适合改造的人源化scFv的CAR结构对于解决利用靶向CD123的CAR-T治疗AML是很有必要的。
发明内容
有鉴于此，本发明的目的之一在于提供一种抗CD123的嵌合抗原受体和应用，本发明的包含CD123-scFv的嵌合抗原受体能够更稳定的表达于T淋巴细胞，能更好的清除表达CD123抗原的肿瘤靶细胞，对阴性抗原(不表达CD123)的肿瘤细胞没有毒性作用。
为实现上述目的，本发明的技术方案为：
抗人CD123抗原的嵌合抗原受体，其特征在于，包含氨基酸序列如SEQ ID NO:4或SEQ ID NO:5或SEQ ID NO:6或SEQ ID NO:7所示的单链抗体构成的抗原识别区。
所述的嵌合抗原受体需要逾越两个技术障碍，一是寻找更稳定有效的识别CD123抗原的人源化单克隆抗体(scFv)，二是获得最佳的CAR的组合方式。
本发明所述识别人CD123抗原的单链抗体(scFv)是通过CDR移植和多位或单位FR区定点突变的方式进行改造获得人源化单链抗体。多位或单位FR区定点突变目的是恢复抗体的稳定性和抗原识别活性，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，方法的结果存在随机性。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。
发明人设计了7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，最终仅仅由本发明所保护的这几组，具有预料不到的技术效果。
本部分保护的CAR的组合方式，经过测试之后，可以起到稳定表达于病人来源的T淋巴细胞，并且具有更好的清除肿瘤细胞的能力，用于针对恶性血液病的过继细胞治疗。
本发明所保护的包含如SEQ ID NO:4或SEQ ID NO:5或SEQ ID NO:6或SEQ ID NO:7所示的单链抗体，具有预料不到的技术效果。比再次改造前表达更稳定，且特异性高，具有更好的清除肿瘤细胞的能力。
作为优选的方案，所述嵌合抗原受体还包含铰链区，跨膜区和胞内信号域；
作为优选的方案，所述铰链区的氨基酸序列如SEQ ID NO:8或SEQ ID NO:9或SEQID NO:10或SEQ ID NO:11所示。
作为优选的方案，所述跨膜区为CD8TM或CD28TM来源的氨基酸序列，所述CD8TM的氨基酸序列如SEQ ID NO:12，所述CD28TM的氨基酸序列如SEQ ID NO:13所示。
作为优选的方案，所述胞内信号域为CD28和/或CD137和CD3；所述CD28的氨基酸序列如SEQ ID NO:14，所述CD137的氨基酸序列如SEQ ID NO:15，所述CD3的氨基酸序列如SEQID NO:16。
上述胞内信号域由共刺激结构域和初级信号传导结构域连接组成，所述共刺激结构域为CD28和/或CD137，所述初级信号传导结构域为CD3。
进一步，所述嵌合抗原受体的氨基酸序列如SEQ ID NO.17或SEQ ID NO.18或SEQID NO.19或SEQ ID NO.20或SEQ ID NO.21或SEQ ID NO.22所示。
进一步，所述嵌合抗原受体的基因序列如SEQ ID NO.23或SEQ ID NO.24或SEQ IDNO.25或SEQ ID NO.26或SEQ ID NO.27或SEQ ID NO.28。
本发明的目的之二在于提供一种重组载体，其包含编码上述嵌合抗原受体的核苷酸序列。
作为优选的方案，上述重组载体为慢病毒载体或重组质粒。
表达所述抗人CD123抗原的嵌合抗原受体的慢病毒载体的制备方法，包括以下步骤：
1)合成抗人CD123抗原的嵌合抗原受体的基因序列；
2)构建表达抗体的慢病毒载体：设计引物，以所述抗体的基因序列为模板进行PCR扩增，得DNA片段；
酶切反应按说明书进行。酶切产物经琼脂糖凝胶电泳分离后用琼脂糖凝胶DNA片段回收试剂盒进行DNA片段回收，然后将目的片段和载体片段通过T4连接酶进行连接，获得表达所述抗体的慢病毒载体。
进一步，在步骤2)之后，包装并纯化所述慢病毒载体。
在所述的方法下得到所述的慢病毒载体，这样的慢病毒载体的阳性表达率高，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CAR阳性率下降。用所述慢病毒载体感染的T细胞，这样的T细胞具备杀伤靶细胞的功能。
本发明的目的还在于提供一种所述的重组载体感染的细胞。
优选的，所述细胞可选自：干细胞、T细胞、NK细胞、单核细胞或巨噬细胞。
本发明的目的还在于提供一种所述的细胞在用于制备治疗恶性血液病的药物中的应用。
进一步，所述恶性血液病的细胞或组织能够表达CD123。
进一步，所述恶性血液病为表达CD123的急性粒细胞白血病、急性淋巴细胞白血病以及B细胞慢性淋巴细胞增殖性疾病。
本发明的有益效果在于：
1)本发明提供的抗CD123人源化单链抗体人源化程度高，用于制备嵌合抗原受体(CAR)可以有效地降低CAR的免疫原性，增强CAR-T在体内的持续和安全性。
2)本发明提供的抗CD123人源化单链抗体制备的嵌合抗原受体识别抗人CD123抗原，能够更稳定的表达于T淋巴细胞，具有更好的清除肿瘤细胞的能力，不仅可以维持靶向CD123的嵌合抗原受体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，对抗原阴性的细胞无毒副作用，能够用于肿瘤的靶向治疗。
3)本发明提供的抗CD123人源化单链抗体制备的嵌合抗原受体能够稳定表达于T淋巴细胞尤其是病人来源的T淋巴细胞，可以用于制备治疗血液***肿瘤的药物中针对肿瘤的过继细胞治疗。
附图说明
图1为不同的CD123-CAR表达。
图2为不同的CD123-CAR杀伤效率。
图3为人源化抗CD123scFv亲和力检测。
图4为靶向人CD123抗原的嵌合抗原受体(CAR)的结构示意图。
图5为靶向人CD123抗原的嵌合抗原受体(CAR)表达检测。
图6为表达靶向CD123嵌合抗原受体的T细胞体外对CD123阳性和阴性肿瘤细胞的杀伤情况。
图7为表达靶向CD123嵌合抗原受体的T细胞体外特异性检测。
图8为表达靶向CD123嵌合抗原受体的T细胞在小鼠肿瘤模型中的治疗情况。
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，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：设计抗人CD123抗原的人源化单克隆抗体
(1)将鼠抗人CD123抗原的单克隆抗体轻、重链的6段FR区氨基酸序列经过IMGT/BLAST数据库经序列分析及比对，选择同源性最高人抗体序列作为改造模板。鼠源抗体scFv序列如下表1所示。
表1：鼠源抗体scFv序列
Figure BDA0001812411140000061
(2)经过分子对接模拟，对骨架序列进行人源化修饰改造，获得3个人源化scFv，氨基酸序列如SEQ ID NO.1、SEQ ID NO.2和SEQ ID NO.3所示。进行CAR构建，经验证人源化后的scFv构建的CAR虽然能够正常表达于T细胞表面但均不能正常工作，而鼠源未改造的scFv组合的CAR能够正常工作，识别并杀伤表达CD123的肿瘤细胞；结果如图1和图2所示。
(3)重新设计人源化改造策略：对人源化改造的序列进行PCR定点突变，进行定点突变后获得4个新的改造scFv，突变获得的scFv氨基酸序列如SEQ ID NO.4、SEQ ID NO.5和SEQ ID NO.6和SEQ ID NO.7所示。
实施例2、靶向CD123人源化单克隆抗体亲和力测定
优选的单克隆抗体scFv标记有His标签后克隆进质粒载体并转染HEK293细胞，纯化scFv-His，scFv-His和阴性对照用PBS稀释6个梯度，分别孵育CD123表达阳性的THP-1细胞，流式检测浓度梯度下THP-1CD123检测的阳性率，进行流式阳性率及MFI(平均荧光强度)统计，对不同scFv于CD123的亲和力进行分析。实验进行了三个独立重复，结果如图3所示，其中Kd(nM)越大亲和力越小。
实施例3、表达靶向人CD123抗原的嵌合抗原受体的慢病毒制备
(1)制备靶向人CD123抗原的嵌合抗原受体的基因序列
合成依次含前导肽(又称信号肽)、抗人CD123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，其结构如图4所示。前导肽的核苷酸序列为atgggatggagctgtatcatcctcttcctggtagcaacagctacaggcgtgcacagt；抗人CD123抗原的人源化单链抗体核苷酸序列如SEQ ID NO.18、SEQ ID NO.19、SEQ ID NO20、SEQ ID NO21、SEQ IDNO22、SEQ ID NO23和SEQ ID NO24；hFc铰链区的核苷酸序列为氨基酸序列如SEQ ID NO.8或SEQ ID NO9或SEQ ID NO10或SEQ ID NO11所对应的氨基酸序列；跨膜区为CD8TM或CD28TM来源的氨基酸序列；胞内信号段为CD28或CD137及CD3来源的核苷酸序列。
(2)构建表达嵌合抗原受体的慢病毒载体
根据装载ScFv和跨膜结构以及胞内信号的不同，将嵌合抗原受体表达载体分别命名为CAR12301、CAR12302、CAR12303、CAR12304、CAR12305、CAR12306、CAR12307、CAR12308，CAR12309、CAR12310、CAR12311，CAR12300慢病毒载体。其中CAR12300为鼠源对照。
酶切反应按说明书进行。酶切产物经琼脂糖凝胶电泳分离后用琼脂糖凝胶DNA片段回收试剂盒进行DNA片段回收，然后将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，结构如图4所示3种CAR结构与4种人源化抗体组合共计12种组合。质粒抽提试剂盒(Invitrogen公司)抽提质粒，具体方法见说明书。
实施例6、CD123抗原的嵌合抗原受体修饰T细胞的制备
(1)慢病毒的包装
本实施例包装慢病毒采用磷酸钙法，具体步骤见分子克隆实验指南(第三版，J.萨姆布鲁克等著)。
(2)慢病毒的纯化
收集病毒上清，离心过滤后转移至新的离心管；根据病毒上清量，利用终浓度为8.5％的PEG6000、终浓度为0.3M的NaCl进行病毒纯化，纯化后的病毒用200μL含10％FBS的DMEM培养基重悬，1.5mL EP管分装，-80℃保存备用。
(3)慢病毒滴度测定
病毒感染293T细胞，感染72h后利用QIAamp DNA Blood Mini Kit(购于Qiagen公司，货号511004)基因组抽提试剂盒抽提基因组。按试剂盒说明书操作。qRT-PCR测定病毒滴度用分析软件分析数据，根据标准曲线计算病毒滴度，结果用TU/mL表示。滴度如下表2所示，均大于1*108TU/ml。
表2慢病毒滴度
病毒名称 病毒滴度病毒滴度
CAR12301 1.00E+08
CAR12302 2.63E+08
CAR12303 1.64E+08
CAR12304 4.58E+08
CAR12305 3.86E+08
CAR12306 2.40E+08
CAR12309 1.40E+08
CAR12310 2.14E+08
CAR12311 2.00E+08
(4)慢病毒感染T细胞
1)人外周血单核细胞的分离
用加有抗凝剂的采血管采集外周血约60ml，加入羟乙基淀粉稀释后室温(18～25℃)自然沉降约30min，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2次，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
用完全培养基培养新制备的单个核细胞PBMC，抗CD3单克隆抗体活化后进行慢病毒感染；分别加入慢病毒载体，未感染的外周血淋巴细胞(PBMC)作为空白对照；24h后将培养基更换为含有500IU/mL重组人IL-2的RPMI 1640完全培养基，继续培养10-20天。感染CAR12301、CAR12302、CAR12303、CAR12304、CAR12305、CAR12306、CAR12307、CAR12308，CAR12309、CAR12310、CAR12311，CAR12300慢病毒载体获得的嵌合抗原受体T细胞以病毒名称命名即：CAR12301、CAR12302、CAR12303、CAR12304、CAR12305、CAR12306、CAR12307、CAR12308、CAR12309、CAR12310、CAR12311，CAR12300。
3)靶向人CD123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5天和11天的已感染病毒的T细胞进行CAR阳性率检测。收集细胞调整细胞密度为1×106个/ml，分别分装，利用流式细胞术检测Protein-L阳性率，检测结果即表示不同结构CAR在T淋巴细胞表达阳性率。结果如表3和图5所示不同病毒感染T细胞后CAR阳性率，其中CAR12303、CAR12311以及CAR12302感染效率较低，CAR12301随着培养时间的持续CAR表达率下降，其它结构CAR阳性率和稳定性均较好。
表3 CAR在T淋巴细胞表达阳性率
CAR C-171011(5d/11d) B-171017(5d/11d) C-171017 B-171115(4d/9d)
CAR12301 59.1/30.8 64.1/50.7 61.8 66.1/29.69
CAR12302 11.7/8.3 22.4/15 19.4 18.59/11.52
CAR12303 3.0/1.6
CAR12304 78.5/87.6 82.1/87 72.7 86.56/95.07
CAR12305 51.9/64.9 65.9/67.2 43.2 64.72/81.95
CAR12306 28.1/34.6 35.5/41.5 26.8 23.79/53.73
CAR12307 24.4
CAR12308 21.0
CAR12309 65.6/74.5 67.9/76.6 50.5
CAR12310 27.3/36.9 39.9/43.7 27.5
CAR12311 9.2/10.6
CAR12300 35.8/21.9 30.7 16.35/8.79
实施例7、表达靶向CD123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CD123阳性THP-1细胞(简称为Raji-luc)以及阴性细胞Raji作为靶细胞，按照1:1效靶比铺效应细胞。使用Steady-
Figure BDA0001812411140000091
Luciferase AssaySystem(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 BDA0001812411140000101
杀伤结果如图6所示，结果表明CAR12301、CAR12302，CAR12305、CAR12304，CAR12310、CAR12309，均具有较好的体外杀伤效果和特异性。其中CAR12301的CAR结构的氨基酸序列如SEQ ID NO:17所示，核苷酸序列如SEQ ID NO:23所示；CAR12302的CAR结构的氨基酸序列如SEQ ID NO:18所示，核苷酸序列如SEQ ID NO:24所示；CAR12304的CAR结构的氨基酸序列如SEQ ID NO:19所示，核苷酸序列如SEQ ID NO:25所示；CAR12305的CAR结构的氨基酸序列如SEQ ID NO:20所示，核苷酸序列如SEQ ID NO:26所示；CAR12309的CAR结构的氨基酸序列如SEQ ID NO:21所示，核苷酸序列如SEQ ID NO:27所示；CAR12310的CAR结构的氨基酸序列如SEQ ID NO:22所示，核苷酸序列如SEQ ID NO:28所示。
实施例8、靶向人CD123的亲和抗原受体T细胞特异性检测
以稳定表达萤火虫荧光素酶的CD123阴性细胞Raji作为靶细胞，按照1:1效靶比铺效应细胞。使用Steady-
Figure BDA0001812411140000102
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 BDA0001812411140000103
实验进行了4次独立重复，结果如图7所示，除CAR12304外，其余靶向CD123CAR-T均具有较好的特异性。
实施例9、表达靶向CD123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验证
建立人CD123阳性肿瘤细胞系的小鼠移植瘤模型用于验证表达靶向CD123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体内相关成瘤实验的最常见品系。体内验证使用的成瘤靶向细胞为前期体外验证使用的稳定表达萤火虫荧光素酶的CD123阳性细胞系THP-1。小鼠成瘤后，分别尾静脉注射CAR-T细胞，生理盐水组对照、未感染病毒的PBMC细胞。注射CAR T细胞后每隔7天通过PerkinElmer公司的IVS活体成像***拍照成像，显示肿瘤生长情况，荧光值越小存活小鼠越多治疗效果越好。期间每天观察小鼠存活情况并记录。结果如图8所示：CAR12301、CAR12304和CAR12310均具有较好的体内杀伤结果，优选的CAR12301和CAR12310体内杀伤结果最优。
本部分实验通过对鼠源单克隆抗体(表位肽不同)的单链抗体进行获得，并对单链抗体进行人源化改造和定点突变，将定点突变的人源化抗体用于制备嵌合抗原受体，并对嵌合抗原受体进行功能验证试验。通过本部分的实验得出结论：虽然人源化改造的方案已有多方报道，但是人源化改造的结果存在未知性，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。
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。
<110> 重庆精准生物技术有限公司
<120>抗人CD123的嵌合抗原受体及其应用
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Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gly
210 215 220
Asn Trp Asp Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser
225 230 235 240
<210>5
<211> 240
<212> PRT
<213> Artificial
<220>
<223> scFv-humanized 5
<400> 5
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Pro Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Lys Ser Ile Ser Lys Asp
20 25 30
Leu Ala Trp Tyr Gln Glu Lys Pro Gly Lys Thr Asn Lys Leu Leu Ile
35 40 45
Tyr Ser Gly Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Asn Lys Tyr Pro Tyr
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly Ser Thr Ser Gly
100 105 110
Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Gln Val Gln
115 120 125
Leu Val Gln Pro Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys
130 135 140
Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Trp Met Asn
145 150 155 160
Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile Gly Arg Ile
165 170 175
Asp Pro Tyr Asp Ser Glu Thr His Tyr Asn Gln Lys Phe Lys Asp Arg
180 185 190
Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr Met Glu Leu
195 200 205
Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gly
210 215 220
Asn Trp Asp Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser
225 230 235 240
<210>6
<211> 240
<212> PRT
<213> Artificial
<220>
<223> scFv-humanized 6
<400> 6
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Pro Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Lys Ser Ile Ser Lys Asp
20 25 30
Leu Ala Trp Tyr Gln Glu Lys Pro Gly Lys Thr Asn Lys Leu Leu Ile
35 40 45
Tyr Ser Gly Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Asn Lys Tyr Pro Tyr
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly Ser Thr Ser Gly
100 105 110
Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Gln Val Gln
115 120 125
Leu Val Gln Pro Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys
130 135 140
Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Trp Met Asn
145 150 155 160
Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Arg Ile
165 170 175
Asp Pro Tyr Asp Ser Glu Thr His Tyr Asn Gln Lys Phe Lys Gly Arg
180 185 190
Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr Met Glu Leu
195 200 205
Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gly
210 215 220
Asn Trp Asp Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser
225 230 235 240
<210>7
<211> 240
<212> PRT
<213> Artificial
<220>
<223> scFv-humanized 7
<400> 7
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Pro Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Lys Ser Ile Ser Lys Asp
20 25 30
Leu Ala Trp Tyr Gln Glu Lys Pro Gly Lys Thr Asn Lys Leu Leu Ile
35 40 45
Tyr Ser Gly Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Asn Lys Tyr Pro Tyr
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly Ser Thr Ser Gly
100 105 110
Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Gln Val Gln
115 120 125
Leu Val Gln Pro Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys
130 135 140
Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Trp Met Asn
145 150 155 160
Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Arg Ile
165 170 175
Asp Pro Tyr Asp Ser Glu Thr His Tyr Asn Gln Lys Phe Lys Asp Arg
180 185 190
Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr Met Glu Leu
195 200 205
Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gly
210 215 220
Asn Trp Asp Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser
225 230 235 240
<210>8
<211> 47
<212> PRT
<213>
<220>
<223> CD8h铰链
<400> 8
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 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>9
<211> 36
<212> PRT
<213>
<220>
<223> 7h铰链
<400> 9
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>10
<211> 45
<212> PRT
<213>
<220>
<223> 8h(dc)铰链
<400> 10
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>11
<211> 229
<212> PRT
<213>
<220>
<223> IgG4h铰链
<400> 11
Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe
1 5 10 15
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
20 25 30
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
35 40 45
Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val
50 55 60
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser
65 70 75 80
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu85 90 95
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser
100 105 110
Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
115 120 125
Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln
130 135 140
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
145 150 155 160
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
165 170 175
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu
180 185 190
Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser
195 200 205
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
210 215 220
Leu Ser Leu Gly Lys
225
<210> 12
<211> 24
<212> PRT
<213>
<220>
<223> CD8跨膜区
<400>12
Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu
5 10 15
Leu Ser Leu Val Ile Thr Leu Tyr Cys
20
<211> 13
<212> PRT
<213> 27
<220>
<223> CD28跨膜区
<400> 13
Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser
5 10 15
Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val
20 25
<210>14
<211> 41
<212> PRT
<213>
<220>
<223> CD28共刺激信号
<400> 14
Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met
5 10 15
Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr
20 25 30
Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser
35 40
<210> 15
<211> 44
<212> PRT
<213>
<220>
<223> CD137共刺激信号
<400> 15
Val Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro
5 10 15
Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser
20 25 30
Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg
35 40
<210>16
<211> 111
<212> PRT
<213>
<220>
<223> CD3共刺激信号
<400> 16
Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly
5 10 15
Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu
20 25 30
Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly
35 40 45
Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu
50 55 60
Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met
65 70 75
Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln
80 85 90
Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met
95 100 105
Gln Ala Leu Pro Pro Arg
110
<210>17
<211> 465
<212> PRT
<213> Artificial
<220>
<223> CAR12301
<400> 17
Asp Ile Gln Met Thr Gln Ser Pro Ser Tyr Leu Ala Ala Ser Pro Gly
1 5 10 15
Asp Thr Ile Thr Ile Asn Cys Arg Ala Ser Lys Ser Ile Ser Lys Asp
20 25 30
Leu Ala Trp Tyr Gln Glu Lys Pro Gly Lys Thr Asn Lys Leu Leu Ile
35 40 45
Tyr Ser Gly Ser Thr Leu Gln Ser Gly Ile Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Met Tyr Tyr Cys Gln Gln His Asn Lys Tyr Pro Tyr
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly Ser Thr Ser Gly
100 105 110
Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Gln Val Gln
115 120 125
Leu Val Gln Pro Gly Ala Glu Leu Val Arg Pro Gly Ala Ser Val Lys
130 135 140
Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Trp Met Asn
145 150 155 160
Trp Val Arg Gln Ala Pro Asp Gln Gly Leu Glu Trp Ile Gly Arg Ile
165 170 175
Asp Pro Tyr Asp Ser Glu Thr His Tyr Asn Gln Lys Phe Lys Asp Lys
180 185 190
Ala Ile Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr Met Glu Leu
195 200 205
Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gly
210 215 220
Asn Trp Asp Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser
225 230 235 240
Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr
245 250 255
Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala
260 265 270
Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Phe
275 280 285
Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu
290 295 300
Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu
305 310 315 320
Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr
325 330 335
Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr
340 345 350
Arg Ser Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln
355 360 365
Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu
370 375 380
Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly
385 390 395 400
Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln
405 410 415
Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu
420 425 430
Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr
435 440 445
Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro
450 455 460
Arg
465
<210>18
<211> 466
<212> PRT
<213> Artificial
<220>
<223> CAR12302
<400> 18
Asp Ile Gln Met Thr Gln Ser Pro Ser Tyr Leu Ala Ala Ser Pro Gly
1 5 10 15
Asp Thr Ile Thr Ile Asn Cys Arg Ala Ser Lys Ser Ile Ser Lys Asp
20 25 30
Leu Ala Trp Tyr Gln Glu Lys Pro Gly Lys Thr Asn Lys Leu Leu Ile
35 40 45
Tyr Ser Gly Ser Thr Leu Gln Ser Gly Ile Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Met Tyr Tyr Cys Gln Gln His Asn Lys Tyr Pro Tyr
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly Ser Thr Ser Gly
100 105 110
Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Gln Val Gln
115 120 125
Leu Val Gln Pro Gly Ala Glu Leu Val Arg Pro Gly Ala Ser Val Lys
130 135 140
Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Trp Met Asn
145 150 155 160
Trp Val Arg Gln Ala Pro Asp Gln Gly Leu Glu Trp Ile Gly Arg Ile
165 170 175
Asp Pro Tyr Asp Ser Glu Thr His Tyr Asn Gln Lys Phe Lys Asp Lys
180 185 190
Ala Ile Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr Met Glu Leu
195 200 205
Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gly
210 215 220
Asn Trp Asp Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser
225 230 235 240
Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr
245 250 255
Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala
260 265 270
Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile
275 280 285
Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser
290 295 300
Leu Val Ile Thr Leu Tyr Cys Val Lys Arg Gly Arg Lys Lys Leu Leu
305 310 315 320
Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu
325 330 335
Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys
340 345 350
Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln
355 360 365
Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu
370 375 380
Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly
385 390 395 400
Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu
405 410 415
Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly
420 425 430
Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser
435 440 445
Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro
450 455 460
Pro Arg
465
<210>19
<211> 465
<212> PRT
<213> Artificial
<220>
<223> CAR12304
<400> 19
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Pro Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Lys Ser Ile Ser Lys Asp
20 25 30
Leu Ala Trp Tyr Gln Glu Lys Pro Gly Lys Thr Asn Lys Leu Leu Ile
35 40 45
Tyr Ser Gly Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Asn Lys Tyr Pro Tyr
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly Ser Thr Ser Gly
100 105 110
Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Gln Val Gln
115 120 125
Leu Val Gln Pro Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys
130 135 140
Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Trp Met Asn
145 150 155 160
Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile Gly Arg Ile
165 170 175
Asp Pro Tyr Asp Ser Glu Thr His Tyr Asn Gln Lys Phe Lys Asp Arg
180 185 190
Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr Met Glu Leu
195 200 205
Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gly
210 215 220
Asn Trp Asp Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser
225 230 235 240
Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr
245 250 255
Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala
260 265 270
Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Phe
275 280 285
Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu
290 295 300
Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu
305 310 315 320
Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr
325 330 335
Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr
340 345 350
Arg Ser Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln
355 360 365
Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu
370 375 380
Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly
385 390 395 400
Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln
405 410 415
Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu
420 425 430
Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr
435 440 445
Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro
450 455 460
Arg
465
<210>20
<211> 466
<212> PRT
<213> Artificial
<220>
<223> CAR12305
<400> 20
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Pro Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Lys Ser Ile Ser Lys Asp
20 25 30
Leu Ala Trp Tyr Gln Glu Lys Pro Gly Lys Thr Asn Lys Leu Leu Ile
35 40 45
Tyr Ser Gly Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Asn Lys Tyr Pro Tyr
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly Ser Thr Ser Gly
100 105 110
Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Gln Val Gln
115 120 125
Leu Val Gln Pro Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys
130 135 140
Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Trp Met Asn
145 150 155 160
Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile Gly Arg Ile
165 170 175
Asp Pro Tyr Asp Ser Glu Thr His Tyr Asn Gln Lys Phe Lys Asp Arg
180 185 190
Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr Met Glu Leu
195 200 205
Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gly
210 215 220
Asn Trp Asp Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser
225 230 235 240
Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr
245 250 255
Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala
260 265 270
Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile
275 280 285
Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser
290 295 300
Leu Val Ile Thr Leu Tyr Cys Val Lys Arg Gly Arg Lys Lys Leu Leu
305 310 315 320
Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu
325 330 335
Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys
340 345 350
Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln
355 360 365
Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu
370 375 380
Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly
385 390 395 400
Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu
405 410 415
Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly
420 425 430
Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser
435 440 445
Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro
450 455 460
Pro Arg
465
<210>21
<211> 464
<212> PRT
<213> Artificial
<220>
<223> CAR12309
<400> 21
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Pro Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Lys Ser Ile Ser Lys Asp
20 25 30
Leu Ala Trp Tyr Gln Glu Lys Pro Gly Lys Thr Asn Lys Leu Leu Ile
35 40 45
Tyr Ser Gly Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Asn Lys Tyr Pro Tyr
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly Ser Thr Ser Gly
100 105 110
Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Gln Val Gln
115 120 125
Leu Val Gln Pro Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys
130 135 140
Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Trp Met Asn
145 150 155 160
Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Arg Ile
165 170 175
Asp Pro Tyr Asp Ser Glu Thr His Tyr Asn Gln Lys Phe Lys Asp Arg
180 185 190
Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr Met Glu Leu
195 200 205
Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gly
210 215 220
Asn Trp Asp Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser
225 230 235 240
Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr
245 250 255
Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala
260 265 270
Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Phe
275 280 285
Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu
290 295 300
Val Thr Val Ala Phe Ile Ile Trp Val Arg Ser Lys Arg Ser Arg Leu Leu
305 310 315 320
His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg
325 330 335
Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg
340 345 350
Ser Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly
355 360 365
Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr
370 375 380
Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys
385 390 395 400
Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys
405 410 415
Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg
420 425 430
Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala
435 440 445
Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
450 455 460
<210>22
<211> 466
<212> PRT
<213> Artificial
<220>
<223> CAR12310
<400> 22
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Pro Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Lys Ser Ile Ser Lys Asp
20 25 30
Leu Ala Trp Tyr Gln Glu Lys Pro Gly Lys Thr Asn Lys Leu Leu Ile
35 40 45
Tyr Ser Gly Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Asn Lys Tyr Pro Tyr
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly Ser Thr Ser Gly
100 105 110
Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Gln Val Gln
115 120 125
Leu Val Gln Pro Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys
130 135 140
Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Trp Met Asn
145 150 155 160
Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Arg Ile
165 170 175
Asp Pro Tyr Asp Ser Glu Thr His Tyr Asn Gln Lys Phe Lys Asp Arg
180 185 190
Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr Met Glu Leu
195 200 205
Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gly
210 215 220
Asn Trp Asp Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser
225 230 235 240
Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr
245 250 255
Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala
260 265 270
Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile
275 280 285
Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser
290 295 300
Leu Val Ile Thr Leu Tyr Cys Val Lys Arg Gly Arg Lys Lys Leu Leu
305 310 315 320
Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu
325 330 335
Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys
340 345 350
Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln
355 360 365
Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu
370 375 380
Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly
385 390 395 400
Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu
405 410 415
Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly
420 425 430
Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser
435 440 445
Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro
450 455 460
Pro Arg
465
<210>23
<211> 1473
<212> DNA
<213> Artificial
<220>
<223> CAR12301
<400> 23
gctagcatgg cactgccagt gaccgccctg ctgctgcctc tggccctgct gctgcacgca 60
gcaaggccag acatccagat gacacagagc ccatcctacc tggcagccag cccaggcgac 120
accatcacaa tcaactgccg cgcctctaag agcatctcca aggatctggc ctggtaccag 180
gagaagcctg gcaagaccaa taagctgctg atctattctg gcagcacact gcagagcggc 240
atcccatccc ggttctccgg atctggaagc ggaaccgact ttaccctgac aatcagctcc 300
ctgcagccag aggatttcgc catgtactat tgccagcagc acaacaagta cccctatacc 360
tttggcggcg gcacaaagct ggagatcaag ggatccacct ctggaagcgg caagcctgga 420
tctggagagg gaagcacaaa gggacaggtg cagctggtgc agcctggagc agagctggtg 480
aggccaggag cctccgtgaa ggtgtcttgt aaggccagcg gctacacctt cacatcctat 540
tggatgaact gggtgcggca ggcaccagac cagggactgg agtggatcgg cagaatcgac 600
ccttacgatt ctgagaccca ctataatcag aagtttaagg acaaggccat cctgacagcc 660
gataagtcca cctctacagc ctacatggag ctgtctagcc tgacctccga ggatacagcc 720
gtgtactatt gtgccagagg caattgggac gattattggg gccagggcac cacactgacc 780
gtgtcctctc tcgagaagcc caccacgacg ccagcgccgc gaccaccaac accggcgccc 840
accatcgcgt cgcagcccct gtccctgcgc ccagaggcgt gccggccagc ggcggggggc 900
gcagtgcaca cgagggggct ggacttcgcc tgcgactttt gggtgctggt ggtggttggt 960
ggagtcctgg cttgctatag cttgctagta acagtggcct ttattatttt ctgggtgagg 1020
agtaagagga gcaggctcct gcacagtgac tacatgaaca tgactccccg ccgccccggg 1080
cccacccgca agcattacca gccctatgcc ccaccacgcg acttcgcagc ctatcgctcc 1140
gtgaagttca gcaggagcgc agacgccccc gcgtaccagc agggccagaa ccagctctat 1200
aacgagctca atctaggacg aagagaggag tacgatgttt tggacaagag acgtggccgg 1260
gaccctgaga tggggggaaa gccgagaagg aagaaccctc aggaaggcct gtacaatgaa 1320
ctgcagaaag ataagatggc ggaggcctac agtgagattg ggatgaaagg cgagcgccgg 1380
aggggcaagg ggcacgatgg cctttaccag ggtctcagta cagccaccaa ggacacctac 1440
gacgcccttc acatgcaggc cctgccccct cgc 1473
<210>24
<211> 1473
<212> DNA
<213> Artificial
<220>
<223> CAR12302
<400> 24
gctagcatgg cactgccagt gaccgccctg ctgctgcctc tggccctgct gctgcacgca 60
gcaaggccag acatccagat gacacagagc ccatcctacc tggcagccag cccaggcgac 120
accatcacaa tcaactgccg cgcctctaag agcatctcca aggatctggc ctggtaccag 180
gagaagcctg gcaagaccaa taagctgctg atctattctg gcagcacact gcagagcggc 240
atcccatccc ggttctccgg atctggaagc ggaaccgact ttaccctgac aatcagctcc 300
ctgcagccag aggatttcgc catgtactat tgccagcagc acaacaagta cccctatacc 360
tttggcggcg gcacaaagct ggagatcaag ggatccacct ctggaagcgg caagcctgga 420
tctggagagg gaagcacaaa gggacaggtg cagctggtgc agcctggagc agagctggtg 480
aggccaggag cctccgtgaa ggtgtcttgt aaggccagcg gctacacctt cacatcctat 540
tggatgaact gggtgcggca ggcaccagac cagggactgg agtggatcgg cagaatcgac 600
ccttacgatt ctgagaccca ctataatcag aagtttaagg acaaggccat cctgacagcc 660
gataagtcca cctctacagc ctacatggag ctgtctagcc tgacctccga ggatacagcc 720
gtgtactatt gtgccagagg caattgggac gattattggg gccagggcac cacactgacc 780
gtgtcctctc tcgagaagcc caccacgacg ccagcgccgc gaccaccaac accggcgccc 840
accatcgcgt cgcagcccct gtccctgcgc ccagaggcgt gccggccagc ggcggggggc 900
gcagtgcaca cgagggggct ggacttcgcc tgcgacatct acatctgggc gcccttggcc 960
gggacttgtg gggtccttct cctgtcactg gttatcaccc tttactgcgt taaacggggc 1020
agaaagaaac tcctgtatat attcaaacaa ccatttatga gaccagtaca aactactcaa 1080
gaggaagatg gctgtagctg ccgatttcca gaagaagaag aaggaggatg tgaactgaga 1140
gtgaagttca gcaggagcgc agacgccccc gcgtaccagc agggccagaa ccagctctat 1200
aacgagctca atctaggacg aagagaggag tacgatgttt tggacaagag acgtggccgg 1260
gaccctgaga tggggggaaa gccgagaagg aagaaccctc aggaaggcct gtacaatgaa 1320
ctgcagaaag ataagatggc ggaggcctac agtgagattg ggatgaaagg cgagcgccgg 1380
aggggcaagg ggcacgatgg cctttaccag ggtctcagta cagccaccaa ggacacctac 1440
gacgcccttc acatgcaggc cctgccccct cgc 1473
<210>25
<211> 1473
<212> DNA
<213> Artificial
<220>
<223> CAR12304
<400> 25
gctagcatgg cactgccagt gaccgccctg ctgctgcctc tggccctgct gctgcacgca 60
gcaaggccag acatccagat gacacagagc ccaagctccc tgtctgccag cccaggcgac 120
agggtgacca tcacatgcag agcctccaag tctatcagca aggatctggc ctggtaccag 180
gagaagcctg gcaagaccaa caagctgctg atctattccg gctctacact gcagtctgga 240
gtgccaagcc gcttcagcgg atccggatct ggaaccgact ttaccctgac aatctctagc 300
ctgcagccag aggatttcgc cacatactat tgccagcagc acaataagta cccctatacc 360
tttggcggcg gcacaaagct ggagatcaag ggaagcacct ccggatctgg caagcctgga 420
tccggagagg gctctacaaa gggacaggtg cagctggtgc agcctggagc agaggtgaag 480
aagccaggag ccagcgtgaa ggtgtcctgt aaggcctctg gctacacctt cacaagctat 540
tggatgaact gggtgcggca ggcaccagga cagggactgg agtggatcgg cagaatcgac 600
ccttacgatt ccgagaccca ctataatcag aagtttaagg accgggtgac catcacagcc 660
gataagagca cctccacagc ctacatggag ctgtcctctc tgaggtccga ggataccgcc 720
gtgtactatt gtgccagagg caactgggac gattattggg gccagggcac cacactgacc 780
gtgagctccc tcgagaagcc caccacgacg ccagcgccgc gaccaccaac accggcgccc 840
accatcgcgt cgcagcccct gtccctgcgc ccagaggcgt gccggccagc ggcggggggc 900
gcagtgcaca cgagggggct ggacttcgcc tgcgactttt gggtgctggt ggtggttggt 960
ggagtcctgg cttgctatag cttgctagta acagtggcct ttattatttt ctgggtgagg 1020
agtaagagga gcaggctcct gcacagtgac tacatgaaca tgactccccg ccgccccggg 1080
cccacccgca agcattacca gccctatgcc ccaccacgcg acttcgcagc ctatcgctcc 1140
gtgaagttca gcaggagcgc agacgccccc gcgtaccagc agggccagaa ccagctctat 1200
aacgagctca atctaggacg aagagaggag tacgatgttt tggacaagag acgtggccgg 1260
gaccctgaga tggggggaaa gccgagaagg aagaaccctc aggaaggcct gtacaatgaa 1320
ctgcagaaag ataagatggc ggaggcctac agtgagattg ggatgaaagg cgagcgccgg 1380
aggggcaagg ggcacgatgg cctttaccag ggtctcagta cagccaccaa ggacacctac 1440
gacgcccttc acatgcaggc cctgccccct cgc 1473
<210>26
<211> 1473
<212> DNA
<213> Artificial
<220>
<223> CAR12305
<400> 26
gctagcatgg cactgccagt gaccgccctg ctgctgcctc tggccctgct gctgcacgca 60
gcaaggccag acatccagat gacacagagc ccaagctccc tgtctgccag cccaggcgac 120
agggtgacca tcacatgcag agcctccaag tctatcagca aggatctggc ctggtaccag 180
gagaagcctg gcaagaccaa caagctgctg atctattccg gctctacact gcagtctgga 240
gtgccaagcc gcttcagcgg atccggatct ggaaccgact ttaccctgac aatctctagc 300
ctgcagccag aggatttcgc cacatactat tgccagcagc acaataagta cccctatacc 360
tttggcggcg gcacaaagct ggagatcaag ggaagcacct ccggatctgg caagcctgga 420
tccggagagg gctctacaaa gggacaggtg cagctggtgc agcctggagc agaggtgaag 480
aagccaggag ccagcgtgaa ggtgtcctgt aaggcctctg gctacacctt cacaagctat 540
tggatgaact gggtgcggca ggcaccagga cagggactgg agtggatcgg cagaatcgac 600
ccttacgatt ccgagaccca ctataatcag aagtttaagg accgggtgac catcacagcc 660
gataagagca cctccacagc ctacatggag ctgtcctctc tgaggtccga ggataccgcc 720
gtgtactatt gtgccagagg caactgggac gattattggg gccagggcac cacactgacc 780
gtgagctccc tcgagaagcc caccacgacg ccagcgccgc gaccaccaac accggcgccc 840
accatcgcgt cgcagcccct gtccctgcgc ccagaggcgt gccggccagc ggcggggggc 900
gcagtgcaca cgagggggct ggacttcgcc tgcgacatct acatctgggc gcccttggcc 960
gggacttgtg gggtccttct cctgtcactg gttatcaccc tttactgcgt taaacggggc 1020
agaaagaaac tcctgtatat attcaaacaa ccatttatga gaccagtaca aactactcaa 1080
gaggaagatg gctgtagctg ccgatttcca gaagaagaag aaggaggatg tgaactgaga 1140
gtgaagttca gcaggagcgc agacgccccc gcgtaccagc agggccagaa ccagctctat 1200
aacgagctca atctaggacg aagagaggag tacgatgttt tggacaagag acgtggccgg 1260
gaccctgaga tggggggaaa gccgagaagg aagaaccctc aggaaggcct gtacaatgaa 1320
ctgcagaaag ataagatggc ggaggcctac agtgagattg ggatgaaagg cgagcgccgg 1380
aggggcaagg ggcacgatgg cctttaccag ggtctcagta cagccaccaa ggacacctac 1440
gacgcccttc acatgcaggc cctgccccct cgc 1473
<210>27
<211> 1473
<212> DNA
<213> Artificial
<220>
<223> CAR12309
<400> 27
gctagcatgg cactgccagt gaccgccctg ctgctgcctc tggccctgct gctgcacgca 60
gcaaggccag acatccagat gacacagagc ccaagctccc tgtctgccag cccaggcgac 120
agggtgacca tcacatgcag agcctccaag tctatcagca aggatctggc ctggtaccag 180
gagaagcctg gcaagaccaa caagctgctg atctattccg gctctacact gcagtctgga 240
gtgccaagcc gcttcagcgg atccggatct ggaaccgact ttaccctgac aatctctagc 300
ctgcagccag aggatttcgc cacatactat tgccagcagc acaataagta cccctatacc 360
tttggcggcg gcacaaagct ggagatcaag ggaagcacct ccggatctgg caagcctgga 420
tccggagagg gctctacaaa gggacaggtg cagctggtgc agcctggagc agaggtgaag 480
aagccaggag ccagcgtgaa ggtgtcctgt aaggcctctg gctacacctt cacaagctat 540
tggatgaact gggtgcggca ggcaccagga cagggactgg agtggatggg cagaatcgac 600
ccttacgatt ccgagaccca ctataatcag aagtttaagg accgggtgac catcacagcc 660
gataagagca cctccacagc ctacatggag ctgtcctctc tgaggtccga ggataccgcc 720
gtgtactatt gtgccagagg caactgggac gattattggg gccagggcac cacactgacc 780
gtgagctccc tcgagaagcc caccacgacg ccagcgccgc gaccaccaac accggcgccc 840
accatcgcgt cgcagcccct gtccctgcgc ccagaggcgt gccggccagc ggcggggggc 900
gcagtgcaca cgagggggct ggacttcgcc tgcgactttt gggtgctggt ggtggttggt 960
ggagtcctgg cttgctatag cttgctagta acagtggcct ttattatttt ctgggtgagg 1020
agtaagagga gcaggctcct gcacagtgac tacatgaaca tgactccccg ccgccccggg 1080
cccacccgca agcattacca gccctatgcc ccaccacgcg acttcgcagc ctatcgctcc 1140
gtgaagttca gcaggagcgc agacgccccc gcgtaccagc agggccagaa ccagctctat 1200
aacgagctca atctaggacg aagagaggag tacgatgttt tggacaagag acgtggccgg 1260
gaccctgaga tggggggaaa gccgagaagg aagaaccctc aggaaggcct gtacaatgaa 1320
ctgcagaaag ataagatggc ggaggcctac agtgagattg ggatgaaagg cgagcgccgg 1380
aggggcaagg ggcacgatgg cctttaccag ggtctcagta cagccaccaa ggacacctac 1440
gacgcccttc acatgcaggc cctgccccct cgc 1473
<210>28
<211> 1473
<212> DNA
<213> Artificial
<220>
<223> CAR12310
<400> 28
gctagcatgg cactgccagt gaccgccctg ctgctgcctc tggccctgct gctgcacgca 60
gcaaggccag acatccagat gacacagagc ccaagctccc tgtctgccag cccaggcgac 120
agggtgacca tcacatgcag agcctccaag tctatcagca aggatctggc ctggtaccag 180
gagaagcctg gcaagaccaa caagctgctg atctattccg gctctacact gcagtctgga 240
gtgccaagcc gcttcagcgg atccggatct ggaaccgact ttaccctgac aatctctagc 300
ctgcagccag aggatttcgc cacatactat tgccagcagc acaataagta cccctatacc 360
tttggcggcg gcacaaagct ggagatcaag ggaagcacct ccggatctgg caagcctgga 420
tccggagagg gctctacaaa gggacaggtg cagctggtgc agcctggagc agaggtgaag 480
aagccaggag ccagcgtgaa ggtgtcctgt aaggcctctg gctacacctt cacaagctat 540
tggatgaact gggtgcggca ggcaccagga cagggactgg agtggatggg cagaatcgac 600
ccttacgatt ccgagaccca ctataatcag aagtttaagg accgggtgac catcacagcc 660
gataagagca cctccacagc ctacatggag ctgtcctctc tgaggtccga ggataccgcc 720
gtgtactatt gtgccagagg caactgggac gattattggg gccagggcac cacactgacc 780
gtgagctccc tcgagaagcc caccacgacg ccagcgccgc gaccaccaac accggcgccc 840
accatcgcgt cgcagcccct gtccctgcgc ccagaggcgt gccggccagc ggcggggggc 900
gcagtgcaca cgagggggct ggacttcgcc tgcgacatct acatctgggc gcccttggcc 960
gggacttgtg gggtccttct cctgtcactg gttatcaccc tttactgcgt taaacggggc 1020
agaaagaaac tcctgtatat attcaaacaa ccatttatga gaccagtaca aactactcaa 1080
gaggaagatg gctgtagctg ccgatttcca gaagaagaag aaggaggatg tgaactgaga 1140
gtgaagttca gcaggagcgc agacgccccc gcgtaccagc agggccagaa ccagctctat 1200
aacgagctca atctaggacg aagagaggag tacgatgttt tggacaagag acgtggccgg 1260
gaccctgaga tggggggaaa gccgagaagg aagaaccctc aggaaggcct gtacaatgaa 1320
ctgcagaaag ataagatggc ggaggcctac agtgagattg ggatgaaagg cgagcgccgg 1380
aggggcaagg ggcacgatgg cctttaccag ggtctcagta cagccaccaa ggacacctac 1440
gacgcccttc acatgcaggc cctgccccct cgc 1473

Claims (11)

1.抗人CD123抗原的嵌合抗原受体，其特征在于，包含氨基酸序列如SEQ ID NO:4或SEQID NO:7所示的单链抗体构成的抗原识别区；所述嵌合抗原受体还包含铰链区，跨膜区和胞内信号域；所述跨膜区为CD8TM或CD28TM来源的氨基酸序列；所述胞内信号域为CD28和/或CD137和CD3；所述抗原识别域、所述铰链区和所述跨膜区及所述胞内信号域依次连接组成。
2.根据权利要求1所述的嵌合抗原受体，其特征在于，所述CD8TM来源的氨基酸序列如SEQ ID NO:12所示，所述CD28TM来源的氨基酸序列如SEQ ID NO:13所示。
3.根据权利要求2所述的嵌合抗原受体，其特征在于，所述铰链区的氨基酸序列如SEQID NO:8或SEQ ID NO:9或SEQ ID NO:10或SEQ ID NO:11所示。
4.根据权利要求2所述的嵌合抗原受体，其特征在于，所述CD28的氨基酸序列如SEQ IDNO:14，所述CD137的氨基酸序列如SEQ ID NO:15，所述CD3的氨基酸序列如SEQ ID NO:16。
5.根据权利要求1-4任一项所述的嵌合抗原受体，其特征在于，所述嵌合抗原受体的氨基酸序列如SEQ ID NO.17或SEQ ID NO.18或SEQ ID NO.21或SEQ ID NO.22所示。
6.重组载体，其特征在于，所述重组载体包含编码权利要求1-5任一项所述的嵌合抗原受体的核苷酸序列。
7.根据权利要求6所述载体，其特征在于，所述载体可以选自慢病毒表达载体、逆转录病毒表达载体、腺病毒表达载体。
8.权利要求6或7所述的重组载体感染的细胞。
9.权利要求8所述的细胞在用于制备治疗恶性血液病的药物中的应用。
10.根据权利要求9所述的应用，其特征在于，所述恶性血液病的细胞或组织能够表达CD123。
11.根据权利要求10所述的应用，其特征在于，所述恶性血液病为表达CD123的急性粒细胞白血病、急性淋巴细胞白血病以及B细胞慢性淋巴细胞增殖性疾病。
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