CN113999319A - 表达pd-l1单链抗体的靶向gpc3的嵌合抗原受体、t细胞及制备方法和应用 - Google Patents

表达pd-l1单链抗体的靶向gpc3的嵌合抗原受体、t细胞及制备方法和应用 Download PDF

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CN113999319A
CN113999319A CN202111290859.7A CN202111290859A CN113999319A CN 113999319 A CN113999319 A CN 113999319A CN 202111290859 A CN202111290859 A CN 202111290859A CN 113999319 A CN113999319 A CN 113999319A
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万晓春
曹国政
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Abstract

本发明公开了一种表达PD‑L1单链抗体的靶向GPC3的嵌合抗原受体、嵌合抗原受体T细胞及制备方法和应用，属于生物医药技术领域。本发明构建表达PD‑L1单链抗体同时靶向GPC3的嵌合抗原受体T细胞，这种设计使得PD‑L1单链抗体能在局部肿瘤位点结合肿瘤细胞表达的PD‑L1，阻断PD‑1/PD‑L1信号，减弱T细胞耗竭，增强CAR‑T细胞在体内外的抗肿瘤活性，从而提高靶向GPC3的嵌合抗原受体T细胞对肝细胞癌的治疗效果。

Description

表达PD-L1单链抗体的靶向GPC3的嵌合抗原受体、T细胞及制 备方法和应用
技术领域
本发明属于生物医药技术领域，具体涉及一种表达PD-L1单链抗体的靶向GPC3的嵌合抗原受体、嵌合抗原受体T细胞及制备方法和应用。
背景技术
肝细胞癌是原发性肝癌的主要类型，是世界范围内第三大致死性癌症。索拉菲尼作为治疗肝细胞癌的一线治疗药物只能将总生存期延长2-3个月。其它肝细胞癌的治疗方法比如手术、化疗和抗体治疗都不能取得令人满意的治疗效果。
GPC-3即磷脂酰肌醇聚糖-3，在70％以上的肝细胞癌患者中过表达，是CAR-T治疗肝细胞癌的常用靶点。以GPC-3为靶点的CAR-T疗法在一期临床试验中展现出对复发或难治的肝细胞癌患者的安全和有效性(Chimeric Antigen Receptor-Glypican-3 T-CellTherapy for Advanced Hepatocellular Carcinoma:Results of Phase I Trials 2020，10.1158/1078-0432.CCR-19-3259)。然而CAR-T治疗实体瘤面临肿瘤内部免疫抑制微环境的限制，肝细胞癌具有强烈的免疫抑制性微环境，肝细胞癌高表达PD-L1，PD-L1与T细胞表面的PD-1相互作用抑制了T细胞的效应功能，阻碍了嵌合抗原受体T细胞在肝细胞癌中的治疗效果。PD-L1被发现表达在人原发性肝癌手术标本中，PD-1/PD-L1通路在肝细胞癌免疫抑制微环境中发挥重要作用，能导致T细胞耗竭(1.Obeid,J.M.,Kunk,P.R.,Zaydfudim,V.M.,Bullock,T.N.,Slingluff,C.L.Jr.,and Rahma,O.E.(2018).Immunotherapy forhepatocellular carcinoma patients:is it ready for prime time？CancerImmunol.Immunother.67,161–174.doi:10.1007/s00262-017-2082-z；2.Thorn,M.,Guha,P.,Cunetta,M.,Espat,N.J.,Miller,G.,Junghans,R.P.,et al.(2016).Tumor-associated GM-CSF overexpression induces immunoinhibitory molecules via STAT3in myeloid-suppressor cells infiltrating liver metastases.Cancer GeneTher.23,188–198.doi:10.1038/cgt.2016.19)。通过单克隆抗体对PD-1/PD-L1免疫检查点通路的阻断虽然在不同癌症患者中产生了强大的抗肿瘤效果，然而系统性的服用单克隆抗体会产生免疫相关的副反应。
目前，以GPC3为靶点的嵌合抗原受体T细胞疗法在临床一期试验中展现了安全和有效性，然而肝细胞癌是免疫抑制性癌症，高表达PD-L1分子，免疫检查点分子PD-1/PD-L1的相互作用导致T细胞耗竭，抑制T细胞的效应功能，因此单靶点的CAR-T细胞疗法在治疗肝细胞癌中并不能取得良好的疗效。
发明内容
为了克服上述现有技术的缺点，本发明的目的在于提供一种表达PD-L1单链抗体的靶向GPC3的嵌合抗原受体、嵌合抗原受体T细胞及制备方法和应用。
为了达到上述目的，本发明采用以下技术方案予以实现：
本发明的内容之一在于提供一种表达PD-L1单链抗体的靶向GPC3的嵌合抗原受体，所述表达PD-L1单链抗体的靶向GPC3的嵌合抗原受体包括如SEQ ID NO:1所示的氨基酸序列。
优选地，所述表达PD-L1单链抗体的靶向GPC3的嵌合抗原受体的编码基因包括如SEQ ID NO:2所示的核苷酸序列。
优选地，所述表达PD-L1单链抗体的靶向GPC3的嵌合抗原受体包括从氨基端到羧基端依次为靶向GPC3的单链抗体、胞外铰链区、跨膜区、共刺激结构区、CD3ζ信号结构区、P2A、IGHV3-2信号肽、靶向PD-L1的单链抗体、连接子G4S、标签蛋白的氨基酸序列。
优选地，所述靶向GPC3的单链抗体的氨基酸序列包括如SEQ ID NO:3所示的氨基酸序列。
进一步优选地，所述靶向GPC3的单链抗体的编码基因包括如SEQ ID NO:4所示的核苷酸序列。
更进一步优选地，所述靶向CD276的单链抗体的编码基因包括如SEQ ID NO:4所示的核苷酸序列。
进一步优选地，所述胞外铰链区为CD8α铰链区。
更进一步优选地，所述CD8α铰链区的氨基酸序列包括如SEQ ID NO:5所示的氨基酸序列。
更进一步优选地，所述CD8α铰链区的编码基因包括如SEQ ID NO:6所示的核苷酸序列。
当然，胞外铰链区还可以使用CD28铰链区、IgG1铰链区或IgG4铰链区。
进一步优选地，所述跨膜区为CD8α跨膜区。
更进一步优选地，所述CD8α跨膜区的氨基酸序列包括如SEQ ID NO:7所示的氨基酸序列。
更进一步优选地，所述CD8α跨膜区的编码基因包括如SEQ ID NO:8所示的核苷酸序列。
当然，跨膜区还可以使用CD4跨膜区、CD28跨膜区、CD3ζ跨膜区或ICOS的跨膜区。
更进一步优选地，所述4-1BB共刺激结构区的氨基酸序列如SEQ ID NO:9所示，编码该4-1BB共刺激结构区的核苷酸序列如SEQ ID NO:10所示。
更进一步优选地，所述CD3ζ信号结构区的氨基酸序列如SEQ ID NO:11所示，编码该CD3ζ信号结构区的核苷酸序列如SEQ ID NO:12所示。
优选地，所述共刺激结构区还可以使用CD28、OX40、ICOS或CD27的胞内区。
进一步优选地，所述P2A氨基酸序列如SEQ ID NO:13所示；编码P2A的的核苷酸序列如SEQ ID NO:14所示。
进一步优选地，所述IGHV3-2信号肽的氨基酸序列包括如SEQ ID NO:15所述的氨基酸序列。所述IGHV3-2信号肽的编码基因包括如SEQ ID NO:16所述的核苷酸序列。
进一步优选地，所述靶向PD-L1的单链抗体的氨基酸序列如SEQ ID NO:17所述的氨基酸序列。所述靶向PD-L1的单链抗体的编码基因如SEQ ID NO:18所述的核苷酸序列。
进一步优选地，所述连接子G4S的氨基酸序列如SEQ ID NO:19所述的氨基酸序列。所述连接子G4S的编码基因如SEQ ID NO:20所述的核苷酸序列。
优选地，所述标签蛋白为6×His蛋白。
进一步优选地，所述6×His蛋白的氨基酸序列如SEQ ID NO:21所述的氨基酸序列。所述6×His蛋白的编码基因如SEQ ID NO:22所述的核苷酸序列。
本发明的内容之二在于提供一种靶向GPC3的嵌合抗原受体T细胞，包括上述的表达PD-L1单链抗体的靶向GPC3的嵌合抗原受体；该表达PD-L1单链抗体的靶向GPC3的嵌合抗原受体的氨基酸序列包括如SEQ ID NO:1所示的氨基酸序列。
进一步地，表达PD-L1单链抗体的靶向GPC3的嵌合抗原受体CAR-CD276-28BBζ的编码基因包括如SEQ ID NO:2所示的核苷酸序列。
本发明的内容之三在于提供一种靶向GPC3的嵌合抗原受体T细胞的制备方法，包括以下步骤：
1)将表达PD-L1单链抗体的靶向GPC3的嵌合抗原受体CAR-CD276-28BBζ的编码基因插入到pWPXLd载体中，得到pWPXLd-GPC3CAR.T-PD-L1scFv重组质粒；
其中，表达PD-L1单链抗体的靶向GPC3的嵌合抗原受体CAR-CD276-28BBζ的编码基因的核苷酸序列如SEQ ID NO:2所示；
2)将所述pWPXLd-GPC3CAR.T-PD-L1scFv重组质粒与包膜质粒、包装质粒共转染宿主细胞，得到重组慢病毒；
3)将所述重组慢病毒转染CD3阳性T淋巴细胞，经分离获得靶向GPC3的嵌合抗原受体T细胞。
进一步地，所述包膜质粒为PMD2G，所述包装质粒为psPAX2，所述宿主细胞为HEK293T细胞。
进一步地，步骤3)中，所述CD3阳性T淋巴细胞是从人外周血单个核细胞中分离获得。
更进一步地，所述人源外周血单个核细胞来源于自体静脉血、自体骨髓、脐带血和胎盘血等。
本发明的内容之四在于提供一种重组病毒载体，所述重组病毒载体包括表达PD-L1单链抗体的靶向GPC3的嵌合抗原受体GPC3CAR.T-PD-L1scFv的编码基因。该编码基因的核苷酸序列如SEQ ID NO:2或SEQ ID NO:23所示；
如SEQ ID NO:23所示的核苷酸序列与如SEQ ID NO:2所示的核苷酸序列相比，多了CD8α信号肽的编码基因。所述信号肽的编码基因可以较好地指导所述嵌合抗原受体表达到细胞表面。
优选地，所述表达PD-L1单链抗体的靶向GPC3的嵌合抗原受体GPC3CAR.T-PD-L1scFv的编码基因，包括从5’端到3’端依次连接的CD8α信号肽(CD8αSP)的编码基因、靶向GPC3的单链抗体(GPC3-scFv)的编码基因、CD8α胞外铰链区(CD8αhinge)的编码基因、CD8α跨膜区(CD8αTM)的编码基因、4-1BB共刺激结构区的编码基因、CD3ζ信号结构区的编码基因、P2A的编码基因、IGHV3-2信号肽(IGHV3-2 SP)的编码基因、靶向PD-L1的单链抗体(CD276-scFv)的编码基因、连接子G4S的编码基因、标签蛋白6×His的编码基因；其中：
优选地，GPC3CAR.T-PD-L1scFv的氨基酸序列如SEQ ID NO:24所示；
优选地，CD8α信号肽的编码基因的核苷酸序列如SEQ ID NO:25所示；
优选地，CD8α信号肽的氨基酸序列如SEQ ID NO:26所示。
优选地，所述病毒载体为pWPXLd慢病毒载体。
本发明的内容之五在于提供上述的表达PD-L1单链抗体的靶向GPC3的嵌合抗原受体、靶向GPC3的嵌合抗原受体T细胞或重组病毒载体在制备治疗肝癌的药物中的应用。
本发明的内容之六在于提供一种治疗肝细胞癌的药物，该药物包括上述的表达PD-L1单链抗体的靶向GPC3的嵌合抗原受体、靶向GPC3的嵌合抗原受体T细胞或重组病毒载体。
当然，所述靶向GPC3的嵌合抗原受体T细胞也适用于其它细胞表面高表达GPC3的肿瘤细胞。
与现有技术相比，本发明具有以下有益效果：
本发明公开的表达PD-L1单链抗体的靶向GPC3的嵌合抗原受体T细胞，通过PD-L1单链抗体与肿瘤细胞表面的PD-L1结合，从而解除PD-1/PD-L1相互作用对T细胞的抑制作用，增强嵌合抗原受体T细胞对肝细胞癌治疗效果。
本发明构建表达PD-L1单链抗体同时靶向GPC3的嵌合抗原受体T细胞，这种设计使得PD-L1单链抗体能在局部肿瘤位点结合肿瘤细胞表达的PD-L1，阻断PD-1/PD-L1信号，减弱T细胞耗竭，增强CAR-T细胞在体内外的抗肿瘤活性，从而提高靶向GPC3的嵌合抗原受体T细胞对肝细胞癌的治疗效果。
附图说明
图1为GPC3CAR.T-PD-L1scFv基因结构图；
图2为表达PD-L1单链抗体的靶向GPC3的嵌合抗原受体在T细胞表面表达结果图；其中，(a)中UTD非转导病毒的T细胞；(b)中GPC3CAR.T-PD-L1scFv为转导病毒的T细胞；
图3为表达PD-L1单链抗体的靶向GPC3的嵌合抗原受体T细胞对肝细胞癌细胞系Huh7的杀伤结果图。
具体实施方式
为了使本技术领域的人员更好地理解本发明方案，下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分的实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都应当属于本发明保护的范围。
需要说明的是，本发明的说明书和权利要求书及上述附图中的术语“第一”、“第二”等是用于区别类似的对象，而不必用于描述特定的顺序或先后次序。应该理解这样使用的数据在适当情况下可以互换，以便这里描述的本发明的实施例能够以除了在这里图示或描述的那些以外的顺序实施。此外，术语“包括”和“具有”以及他们的任何变形，意图在于覆盖不排他的包含，例如，包含了一系列步骤或单元的过程、方法、系统、产品或设备不必限于清楚地列出的那些步骤或单元，而是可包括没有清楚地列出的或对于这些过程、方法、产品或设备固有的其它步骤或单元。
下面结合附图对本发明做进一步详细描述：
一、一种表达PD-L1单链抗体的靶向GPC3的嵌合抗原受体T细胞的制备方法，包括以下步骤:
(1)制备表达PD-L1单链抗体的靶向GPC3的嵌合抗原受体GPC3CAR.T-PD-L1scFv的基因序列
所述嵌合抗原受体GPC3CAR.T-PD-L1scFv的结构包括CD8α信号肽的编码基因、靶向GPC3单链抗体的编码基因、CD8α铰链区的编码基因、CD8α跨膜区的编码基因、4-1BB共刺激结构区的编码基因和CD3ζ信号结构区的编码基因、IGHV3-2信号肽的编码基因、靶向PD-L1单链抗体的编码基因、连接子G4S的编码基因、标签蛋白6×His的编码基因。
所述CD8α信号肽的编码基因如SEQ ID NO:25所示的核苷酸序列、所述靶向GPC3单链抗体的编码基因包括如SEQ ID NO:4所示的核苷酸序列、所述CD8α胞外铰链区的编码基因包括如SEQ ID NO:6所示的核苷酸序列、所述CD8α跨膜区的编码基因包括如SEQ ID NO:8所示的核苷酸序列、所述4-1BB共刺激结构区的编码基因包括如SEQ ID NO:10所示的核苷酸序列、所述CD3ζ信号结构区的编码基因包括如SEQ ID NO:12所示的核苷酸序列、所述P2A的编码基因如SEQ ID NO:14所述的核苷酸序列、所述IGHV3-2信号肽的编码基因包括如SEQID NO:16所述的核苷酸序列、所述靶向PD-L1的单链抗体的编码基因如SEQ ID NO:18所述的核苷酸序列、所述连接子G4S的编码基因如SEQ ID NO:20所述的核苷酸序列、所述6×His蛋白的编码基因如SEQ ID NO:22所述的核苷酸序列。
所述嵌合抗原受体结构由江苏金唯智生物技术有限公司进行基因合成，得到嵌合抗原受体GPC3CAR.T-PD-L1scFv，如图1所示为GPC3CAR.T-PD-L1scFv基因结构示意图。所述GPC3CAR.T-PD-L1scFv的编码基因包括如SEQ ID NO:23所示的核苷酸序列。
(2)构建pWPXLd-GPC3CAR.T-PD-L1scFv重组质粒
将合成的GPC3CAR.T-PD-L1scFv的编码基因插入到pWPXLd载体的BamH1和EcoR1酶切位点之间，然后转入大肠杆菌感受态细胞DH5α，进行阳性克隆PCR鉴定和测序鉴定。经验证正确的质粒标记为pWPXLd-GPC3CAR.T-PD-L1scFv重组质粒用于后续实验。
(3)重组慢病毒构建
将pWPXLd-GPC3CAR.T-PD-L1scFv重组质粒、包装质粒psPAX2、包膜质粒pMD2G使用lipofectamine3000转染试剂共转染入培养好的HEK293T细胞。第48h收集含病毒的上清，首先2000rpm室温离心5分钟，取上层上清，然后经0.45μm滤膜过滤，得到的重组慢病毒上清用于T细胞感染。
(4)嵌合抗原受体T细胞的制备
a)PBMC(外周血单个核细胞)的分离
PBMC来源于健康志愿者的自体静脉血。
抽取所述健康志愿者血液，使用Ficoll收集外周血单个核细胞，离心分离后取中间层细胞；经PBS洗涤计数后得到PBMC。
b)免疫磁珠法分离抗原特异性T淋巴细胞
取上述PBMC，加入KBM581培养基，配成细胞悬液；按磁珠与细胞的比例为1:1，加入CD3/CD28免疫磁珠，室温下于摇床孵育；采用磁铁对孵育磁珠的细胞进行筛选；去除未吸附的细胞悬液后，加入上述KBM581培养基重悬磁珠-细胞混合物得到CD3阳性T淋巴细胞，继续培养24小时后用于慢病毒感染。
c)病毒转染法制备抗原特异性T淋巴细胞
取上述经过免疫磁珠分离法得到的CD3阳性T淋巴细胞，加入与CD3阳性细胞数相应的所述重组慢病毒进行培养。
培养的第3天，收集合适数量的感染慢病毒的T细胞，流式细胞术分析表面CAR的表达，使用生物素标记的anti-human-fab一抗进行染色，然后使用链霉亲和素偶联的APC二抗进行流式分析，结果如图2所示，(a)UTD为非转导病毒的T细胞，(b)GPC3CAR.T-PD-L1scFv为转导病毒的T细胞。GPC3CAR.T-PD-L1scFv约有14％的表面表达，表明嵌合抗原受体T细胞制备成功。继续培养48小时，收集嵌合抗原受体T细胞用于杀伤实验分析或保存在细胞冻存液中，放置于程序降温盒中-80℃保存24小时，然后转移至液氮灌长期保存。
二、体外功能测试
本发明经过体外功能测试，表达PD-L1单链抗体的靶向GPC3的嵌合抗原受体T细胞对GPC3阳性肝癌细胞系有杀伤活性。具体方法和结果如下：
(1)使用RTCA系统分析表达PD-L1单链抗体的靶向GPC3的嵌合抗原受体T细胞(GPC3CAR.T-PD-L1scFv)对GPC3阳性肿瘤细胞系杀伤。
首先用50ul DMEM培养基进行RTCA单板的平衡，然后收集培养好的靶细胞Huh7，细胞计数，在每个孔中加入含有5000个Huh7细胞的50ul细胞悬液，37℃培养箱中放置15分钟，然后放置在RTCA电阻系统中，18小时后，收集GPC3CAR.T-PD-L1scFv细胞，细胞计数，按不同的效应细胞与靶细胞的比例(E:T)20:1，10:1，5:1把相应细胞数目的嵌合抗原受体T细胞悬液100ul加入到Huh7细胞中，设置对照组(control，加培养基组)，UTD组(加未转导病毒的T细胞)，CAR-T组(加GPC3CAR.T-PD-L1scFv细胞)，每组设置3个复孔，放入RTCA电阻系统中，24小时后进行杀伤活性分析。
(2)结果分析
使用指定时间点的细胞指数(cell index)进行UTD或CAR-T杀伤率分析。
所述UTD杀伤率计算公式为：
UTD杀伤率＝(对照组细胞指数值-UTD组细胞指数值)/对照组细胞指数值×100
所述CAR-T杀伤率计算公式为：
CAR-T杀伤率＝(对照组细胞指数值-CAR-T组细胞指数值)/对照组细胞指数值×100
结果如图3所示显示，UTD为非转导病毒的T细胞，GPC3CAR.T-PD-L1scFv为转导病毒的T细胞。****代表P<0.0001,*代表P<0.05，ns代表无显著差异。CAR-T与UTD组相比，对Huh7细胞有更强的杀伤活性，表明构建的表达PD-L1单链抗体的靶向GPC3的嵌合抗原受体T细胞对表达GPC3的阳性肿瘤细胞系有较强的杀伤活性，预示着所述表达PD-L1单链抗体的靶向GPC3的嵌合抗原受体T细胞在肝细胞癌的治疗中有较大的应用前景。
以上内容仅为说明本发明的技术思想，不能以此限定本发明的保护范围，凡是按照本发明提出的技术思想，在技术方案基础上所做的任何改动，均落入本发明权利要求书的保护范围之内。
序列表
<110> 中国科学院深圳先进技术研究院
<120> 表达PD-L1单链抗体的靶向GPC3的嵌合抗原受体、T细胞及制备方法和应用
<141> 2021-10-18
<160> 26
<170> SIPOSequenceListing 1.0
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Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser
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Asn Ala Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser
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Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro
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Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Asn
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Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105 110
Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
115 120 125
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
130 135 140
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
145 150 155 160
Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
165 170 175
Gly Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe
180 185 190
Lys Gly Arg Val Thr Leu Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr
195 200 205
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
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Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr
225 230 235 240
Val Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro
245 250 255
Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro
260 265 270
Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp
275 280 285
Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu
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Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu
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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
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Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys
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Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu
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Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly
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Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu
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Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly
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Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser
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Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro
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Pro Arg Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu
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Glu Asn Pro Gly Pro Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val
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Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala
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His His His
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gatgtggtta tgacccaaag ccccctgagc ctgcctgtga ctcctgggga gcctgctagc 60
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tacctgcaga agcctgggca gagccctcag ctgctgatct acaaggtgag caatagattt 180
tctggggtgc ctgacagatt ttctggctct ggctctggca cagacttcac cctgaagatc 240
agcagagtgg aggctgagga tgtgggggtg tactactgct ctcagaacac ccatgtgccc 300
cccacctttg gccaaggcac aaagctggag atcaagagag gtggcggtgg ctcgggcggt 360
ggtgggtcgg gtggcggcgg atctcaagtg cagctggtgc agtctggggc tgaggtgaag 420
aagcctgggg cctctgtgaa ggtgagctgc aaggcctctg gctacacctt cacagactat 480
gagatgcact gggtcagaca agcccctggc caaggcctag aatggatggg ggccctggac 540
cccaagactg gggacacagc ctactctcag aagttcaagg gcagagtgac cctgacagct 600
gatgagagca caagcacagc ctacatggag ctgagcagcc tgagatctga ggacacagct 660
gtgtactact gcactagatt ctacagctac acctactggg gccaaggcac cctggtgaca 720
gtgagcagca ccacgacgcc agcgccgcga ccaccaacac cggcgcccac catcgcgtcg 780
cagcccctgt ccctgcgccc agaggcgtgc cggccagcgg cggggggcgc agtgcacacg 840
agggggctgg acttcgcctg tgatatctac atctgggcgc ccttggccgg gacttgtggg 900
gtccttctcc tgtcactggt tatcaccctt tactgcaaac ggggcagaaa gaaactcctg 960
tatatattca aacaaccatt tatgagacca gtacaaacta ctcaagagga agatggctgt 1020
agctgccgat ttccagaaga agaagaagga ggatgtgaac tgagagtgaa gttcagcagg 1080
agcgcagacg cccccgcgta caagcagggc cagaaccagc tctataacga gctcaatcta 1140
ggacgaagag aggagtacga tgttttggac aagagacgtg gccgggaccc tgagatgggg 1200
ggaaagccga gaaggaagaa ccctcaggaa ggcctgtaca atgaactgca gaaagataag 1260
atggcggagg cctacagtga gattgggatg aaaggcgagc gccggagggg caaggggcac 1320
gatggccttt accagggtct cagtacagcc accaaggaca cctacgacgc ccttcacatg 1380
caggccctgc cccctcgcgc aacaaacttc tctctgctga aacaagccgg agatgtcgaa 1440
gagaatcctg gaccgatgga gtttgggctg agctggcttt ttcttgtggc tattttaaaa 1500
ggtgtccagt gtgaggtcca gctggtggag tcaggagggg gactggtaca accaggcggc 1560
tcactgagac tgagctgcgc cgccagcggc ttcaccttca gcgacagctg gattcactgg 1620
gtcaggcagg ctcccggcaa gggcctggag tgggtggcat ggatcagccc ctacggcggc 1680
agcacctact acgccgacag cgtgaagggc agattcacca tcagcgccga caccagcaag 1740
aacaccgcct acctgcagat gaacagcctg agagccgagg acaccgccgt gtattactgc 1800
gcccggagac actggcccgg cggcttcgac tactggggtc agggcacctt agtgaccgta 1860
tcgagcggtg gcggtggctc gggcggtggt gggtcgggtg gcggcggatc tgatatccag 1920
atgacacaga gcccctcgag cctgagcgcc agcgtgggcg acagagtgac catcacctgc 1980
agagccagcc aggacgtgag caccgccgtg gcctggtacc agcagaaacc cggaaaggcc 2040
cccaagctgc tgatttatag cgccagcttc ctgtactccg gagtgcccag tagattcagt 2100
gggagcggca gcggcaccga tttcacgctg acgattagca gcctgcagcc cgaggacttc 2160
gccacctact actgccagca gtacctgtac caccccgcaa cctttggcca aggtacaaag 2220
gtggagatca agggtggcgg tggctcgcat catcaccatc accattga 2268
<210> 3
<211> 243
<212> PRT
<213> Artificial Sequence
<400> 3
Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly
1 5 10 15
Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser
20 25 30
Asn Ala Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser
35 40 45
Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Asn
85 90 95
Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105 110
Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
115 120 125
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
130 135 140
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
145 150 155 160
Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
165 170 175
Gly Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe
180 185 190
Lys Gly Arg Val Thr Leu Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr
195 200 205
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
210 215 220
Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr
225 230 235 240
Val Ser Ser
<210> 4
<211> 729
<212> DNA
<213> Artificial Sequence
<400> 4
gatgtggtta tgacccaaag ccccctgagc ctgcctgtga ctcctgggga gcctgctagc 60
atcagctgca gaagctctca gagcctggtg cacagcaatg ccaacaccta cctgcactgg 120
tacctgcaga agcctgggca gagccctcag ctgctgatct acaaggtgag caatagattt 180
tctggggtgc ctgacagatt ttctggctct ggctctggca cagacttcac cctgaagatc 240
agcagagtgg aggctgagga tgtgggggtg tactactgct ctcagaacac ccatgtgccc 300
cccacctttg gccaaggcac aaagctggag atcaagagag gtggcggtgg ctcgggcggt 360
ggtgggtcgg gtggcggcgg atctcaagtg cagctggtgc agtctggggc tgaggtgaag 420
aagcctgggg cctctgtgaa ggtgagctgc aaggcctctg gctacacctt cacagactat 480
gagatgcact gggtcagaca agcccctggc caaggcctag aatggatggg ggccctggac 540
cccaagactg gggacacagc ctactctcag aagttcaagg gcagagtgac cctgacagct 600
gatgagagca caagcacagc ctacatggag ctgagcagcc tgagatctga ggacacagct 660
gtgtactact gcactagatt ctacagctac acctactggg gccaaggcac cctggtgaca 720
gtgagcagc 729
<210> 5
<211> 45
<212> PRT
<213> Artificial Sequence
<400> 5
Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala
1 5 10 15
Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly
20 25 30
Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp
35 40 45
<210> 6
<211> 135
<212> DNA
<213> Artificial Sequence
<400> 6
accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60
tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120
gacttcgcct gtgat 135
<210> 7
<211> 24
<212> PRT
<213> Artificial Sequence
<400> 7
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> 8
<211> 72
<212> DNA
<213> Artificial Sequence
<400> 8
atctacatct gggcgccctt ggccgggact tgtggggtcc ttctcctgtc actggttatc 60
accctttact gc 72
<210> 9
<211> 42
<212> PRT
<213> Artificial Sequence
<400> 9
Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met
1 5 10 15
Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe
20 25 30
Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu
35 40
<210> 10
<211> 126
<212> DNA
<213> Artificial Sequence
<400> 10
aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 60
actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 120
gaactg 126
<210> 11
<211> 112
<212> PRT
<213> Artificial Sequence
<400> 11
Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly
1 5 10 15
Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr
20 25 30
Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys
35 40 45
Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys
50 55 60
Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg
65 70 75 80
Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala
85 90 95
Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
100 105 110
<210> 12
<211> 336
<212> DNA
<213> Artificial Sequence
<400> 12
agagtgaagt tcagcaggag cgcagacgcc cccgcgtaca agcagggcca gaaccagctc 60
tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 120
cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 180
gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 240
cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 300
tacgacgccc ttcacatgca ggccctgccc cctcgc 336
<210> 13
<211> 19
<212> PRT
<213> Artificial Sequence
<400> 13
Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn
1 5 10 15
Pro Gly Pro
<210> 14
<211> 57
<212> DNA
<213> Artificial Sequence
<400> 14
gcaacaaact tctctctgct gaaacaagcc ggagatgtcg aagagaatcc tggaccg 57
<210> 15
<211> 19
<212> PRT
<213> Artificial Sequence
<400> 15
Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly
1 5 10 15
Val Gln Cys
<210> 16
<211> 57
<212> DNA
<213> Artificial Sequence
<400> 16
atggagtttg ggctgagctg gctttttctt gtggctattt taaaaggtgt ccagtgt 57
<210> 17
<211> 240
<212> PRT
<213> Artificial Sequence
<400> 17
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ser
20 25 30
Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Trp Ile Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Arg His Trp Pro Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
115 120 125
Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
130 135 140
Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln
145 150 155 160
Asp Val Ser Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala
165 170 175
Pro Lys Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro
180 185 190
Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
195 200 205
Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr
210 215 220
Leu Tyr His Pro Ala Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
225 230 235 240
<210> 18
<211> 720
<212> DNA
<213> Artificial Sequence
<400> 18
gaggtccagc tggtggagtc aggaggggga ctggtacaac caggcggctc actgagactg 60
agctgcgccg ccagcggctt caccttcagc gacagctgga ttcactgggt caggcaggct 120
cccggcaagg gcctggagtg ggtggcatgg atcagcccct acggcggcag cacctactac 180
gccgacagcg tgaagggcag attcaccatc agcgccgaca ccagcaagaa caccgcctac 240
ctgcagatga acagcctgag agccgaggac accgccgtgt attactgcgc ccggagacac 300
tggcccggcg gcttcgacta ctggggtcag ggcaccttag tgaccgtatc gagcggtggc 360
ggtggctcgg gcggtggtgg gtcgggtggc ggcggatctg atatccagat gacacagagc 420
ccctcgagcc tgagcgccag cgtgggcgac agagtgacca tcacctgcag agccagccag 480
gacgtgagca ccgccgtggc ctggtaccag cagaaacccg gaaaggcccc caagctgctg 540
atttatagcg ccagcttcct gtactccgga gtgcccagta gattcagtgg gagcggcagc 600
ggcaccgatt tcacgctgac gattagcagc ctgcagcccg aggacttcgc cacctactac 660
tgccagcagt acctgtacca ccccgcaacc tttggccaag gtacaaaggt ggagatcaag 720
<210> 19
<211> 5
<212> PRT
<213> Artificial Sequence
<400> 19
Gly Gly Gly Gly Ser
1 5
<210> 20
<211> 15
<212> DNA
<213> Artificial Sequence
<400> 20
ggtggcggtg gctcg 15
<210> 21
<211> 6
<212> PRT
<213> Artificial Sequence
<400> 21
His His His His His His
1 5
<210> 22
<211> 18
<212> DNA
<213> Artificial Sequence
<400> 22
catcatcacc atcaccat 18
<210> 23
<211> 2331
<212> DNA
<213> Artificial Sequence
<400> 23
atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60
ccggatgtgg ttatgaccca aagccccctg agcctgcctg tgactcctgg ggagcctgct 120
agcatcagct gcagaagctc tcagagcctg gtgcacagca atgccaacac ctacctgcac 180
tggtacctgc agaagcctgg gcagagccct cagctgctga tctacaaggt gagcaataga 240
ttttctgggg tgcctgacag attttctggc tctggctctg gcacagactt caccctgaag 300
atcagcagag tggaggctga ggatgtgggg gtgtactact gctctcagaa cacccatgtg 360
ccccccacct ttggccaagg cacaaagctg gagatcaaga gaggtggcgg tggctcgggc 420
ggtggtgggt cgggtggcgg cggatctcaa gtgcagctgg tgcagtctgg ggctgaggtg 480
aagaagcctg gggcctctgt gaaggtgagc tgcaaggcct ctggctacac cttcacagac 540
tatgagatgc actgggtcag acaagcccct ggccaaggcc tagaatggat gggggccctg 600
gaccccaaga ctggggacac agcctactct cagaagttca agggcagagt gaccctgaca 660
gctgatgaga gcacaagcac agcctacatg gagctgagca gcctgagatc tgaggacaca 720
gctgtgtact actgcactag attctacagc tacacctact ggggccaagg caccctggtg 780
acagtgagca gcaccacgac gccagcgccg cgaccaccaa caccggcgcc caccatcgcg 840
tcgcagcccc tgtccctgcg cccagaggcg tgccggccag cggcgggggg cgcagtgcac 900
acgagggggc tggacttcgc ctgtgatatc tacatctggg cgcccttggc cgggacttgt 960
ggggtccttc tcctgtcact ggttatcacc ctttactgca aacggggcag aaagaaactc 1020
ctgtatatat tcaaacaacc atttatgaga ccagtacaaa ctactcaaga ggaagatggc 1080
tgtagctgcc gatttccaga agaagaagaa ggaggatgtg aactgagagt gaagttcagc 1140
aggagcgcag acgcccccgc gtacaagcag ggccagaacc agctctataa cgagctcaat 1200
ctaggacgaa gagaggagta cgatgttttg gacaagagac gtggccggga ccctgagatg 1260
gggggaaagc cgagaaggaa gaaccctcag gaaggcctgt acaatgaact gcagaaagat 1320
aagatggcgg aggcctacag tgagattggg atgaaaggcg agcgccggag gggcaagggg 1380
cacgatggcc tttaccaggg tctcagtaca gccaccaagg acacctacga cgcccttcac 1440
atgcaggccc tgccccctcg cgcaacaaac ttctctctgc tgaaacaagc cggagatgtc 1500
gaagagaatc ctggaccgat ggagtttggg ctgagctggc tttttcttgt ggctatttta 1560
aaaggtgtcc agtgtgaggt ccagctggtg gagtcaggag ggggactggt acaaccaggc 1620
ggctcactga gactgagctg cgccgccagc ggcttcacct tcagcgacag ctggattcac 1680
tgggtcaggc aggctcccgg caagggcctg gagtgggtgg catggatcag cccctacggc 1740
ggcagcacct actacgccga cagcgtgaag ggcagattca ccatcagcgc cgacaccagc 1800
aagaacaccg cctacctgca gatgaacagc ctgagagccg aggacaccgc cgtgtattac 1860
tgcgcccgga gacactggcc cggcggcttc gactactggg gtcagggcac cttagtgacc 1920
gtatcgagcg gtggcggtgg ctcgggcggt ggtgggtcgg gtggcggcgg atctgatatc 1980
cagatgacac agagcccctc gagcctgagc gccagcgtgg gcgacagagt gaccatcacc 2040
tgcagagcca gccaggacgt gagcaccgcc gtggcctggt accagcagaa acccggaaag 2100
gcccccaagc tgctgattta tagcgccagc ttcctgtact ccggagtgcc cagtagattc 2160
agtgggagcg gcagcggcac cgatttcacg ctgacgatta gcagcctgca gcccgaggac 2220
ttcgccacct actactgcca gcagtacctg taccaccccg caacctttgg ccaaggtaca 2280
aaggtggaga tcaagggtgg cggtggctcg catcatcacc atcaccattg a 2331
<210> 24
<211> 776
<212> PRT
<213> Artificial Sequence
<400> 24
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu
20 25 30
Pro Val Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln
35 40 45
Ser Leu Val His Ser Asn Ala Asn Thr Tyr Leu His Trp Tyr Leu Gln
50 55 60
Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg
65 70 75 80
Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
85 90 95
Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr
100 105 110
Tyr Cys Ser Gln Asn Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr
115 120 125
Lys Leu Glu Ile Lys Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
130 135 140
Gly Gly Gly Gly Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
145 150 155 160
Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr
165 170 175
Thr Phe Thr Asp Tyr Glu Met His Trp Val Arg Gln Ala Pro Gly Gln
180 185 190
Gly Leu Glu Trp Met Gly Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala
195 200 205
Tyr Ser Gln Lys Phe Lys Gly Arg Val Thr Leu Thr Ala Asp Glu Ser
210 215 220
Thr Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
225 230 235 240
Ala Val Tyr Tyr Cys Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln
245 250 255
Gly Thr Leu Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro
260 265 270
Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro
275 280 285
Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu
290 295 300
Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys
305 310 315 320
Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly
325 330 335
Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val
340 345 350
Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu
355 360 365
Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp
370 375 380
Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn
385 390 395 400
Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg
405 410 415
Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly
420 425 430
Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu
435 440 445
Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu
450 455 460
Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His
465 470 475 480
Met Gln Ala Leu Pro Pro Arg Ala Thr Asn Phe Ser Leu Leu Lys Gln
485 490 495
Ala Gly Asp Val Glu Glu Asn Pro Gly Pro Met Glu Phe Gly Leu Ser
500 505 510
Trp Leu Phe Leu Val Ala Ile Leu Lys Gly Val Gln Cys Glu Val Gln
515 520 525
Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg
530 535 540
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ser Trp Ile His
545 550 555 560
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Trp Ile
565 570 575
Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg
580 585 590
Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met
595 600 605
Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Arg
610 615 620
His Trp Pro Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr
625 630 635 640
Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
645 650 655
Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
660 665 670
Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser
675 680 685
Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
690 695 700
Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe
705 710 715 720
Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu
725 730 735
Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Leu Tyr His
740 745 750
Pro Ala Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Gly Gly
755 760 765
Gly Ser His His His His His His
770 775
<210> 25
<211> 63
<212> DNA
<213> Artificial Sequence
<400> 25
atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60
ccg 63
<210> 26
<211> 21
<212> PRT
<213> Artificial Sequence
<400> 26
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro
20

Claims (16)

1.一种表达PD-L1单链抗体的靶向GPC3的嵌合抗原受体，其特征在于，该靶向GPC3的嵌合抗原受体的氨基酸序列如SEQ ID NO:1所示。
2.根据权利要求1所述的表达PD-L1单链抗体的靶向GPC3的嵌合抗原受体，其特征在于，编码所述表达PD-L1单链抗体的靶向GPC3的嵌合抗原受体的核苷酸序列如SEQ ID NO:2所示。
3.根据权利要求1所述的表达PD-L1单链抗体的靶向GPC3的嵌合抗原受体，其特征在于，该表达PD-L1单链抗体的靶向GPC3的嵌合抗原受体包括从氨基端到羧基端依次连接的靶向GPC3的单链抗体、胞外铰链区、跨膜区、4-1BB共刺激结构区、CD3ζ信号结构区、P2A、IGHV3-2信号肽、靶向PD-L1的单链抗体、连接子G4S、标签蛋白的氨基酸序列。
4.根据权利要求3所述的表达PD-L1单链抗体的靶向GPC3的嵌合抗原受体，其特征在于，所述靶向GPC3的单链抗体的氨基酸序列如SEQ ID NO:3所示，编码该靶向GPC3的单链抗体的核苷酸序列如SEQ ID NO:4所示。
5.根据权利要求3所述的表达PD-L1单链抗体的靶向GPC3的嵌合抗原受体，其特征在于，所述胞外铰链区为CD8α铰链区，其氨基酸序列如SEQ ID NO:5所示，编码该CD8α铰链区的核苷酸序列如SEQ ID NO:6所示；所述跨膜区为CD8α跨膜区，其氨基酸序列如SEQ ID NO:7所示，编码该CD8α跨膜区的核苷酸序列如SEQ ID NO:8所示。
6.根据权利要求3所述的表达PD-L1单链抗体的靶向GPC3的嵌合抗原受体，其特征在于，所述4-1BB共刺激结构区的氨基酸序列包括如SEQ ID NO:9所示的氨基酸序列；
所述4-1BB共刺激结构区的编码基因包括如SEQ ID NO:10所示的核苷酸序列；
所述CD3ζ信号结构区的氨基酸序列包括如SEQ ID NO:11所示的氨基酸序列；
CD3ζ信号结构区的编码基因包括如SEQ ID NO:12所示的核苷酸序列。
7.根据权利要求3所述的表达PD-L1单链抗体的靶向GPC3的嵌合抗原受体，其特征在于，所述P2A氨基酸序列如SEQ ID NO:13所示；编码P2A的核苷酸序列如SEQ ID NO:14所示；所述IGHV3-2信号肽的氨基酸序列如SEQ ID NO:15所示；编码所述IGHV3-2信号肽的核苷酸序列如SEQ ID NO:16所示。
8.根据权利要求3所述的表达PD-L1单链抗体的靶向GPC3的嵌合抗原受体，其特征在于，所述靶向PD-L1的单链抗体的氨基酸序列如SEQ ID NO:17所示；编码所述靶向PD-L1的单链抗体的核苷酸序列如SEQ ID NO:18所示。
9.根据权利要求3所述的表达PD-L1单链抗体的靶向GPC3的嵌合抗原受体，其特征在于，所述连接子G4S的氨基酸序列如SEQ ID NO:19所示；编码连接子G4S的核苷酸序列如SEQID NO:20所示；所述标签蛋白为6×His蛋白，所述6×His蛋白的氨基酸序列如SEQ ID NO:21所示；编码6×His蛋白的核苷酸序列如SEQ ID NO:22所示。
10.一种靶向GPC3的嵌合抗原受体T细胞，其特征在于，包括权利要求1～9中任意一项所述的表达PD-L1单链抗体的靶向GPC3的嵌合抗原受体。
11.一种靶向GPC3的嵌合抗原受体T细胞的制备方法，其特征在于，包括以下步骤：
1)将表达PD-L1单链抗体的靶向GPC3的嵌合抗原受体CAR-CD276-28BBζ的编码基因插入到pWPXLd载体中，得到pWPXLd-GPC3CAR.T-PD-L1scFv重组质粒；
其中，表达PD-L1单链抗体的靶向GPC3的嵌合抗原受体CAR-CD276-28BBζ的编码基因的核苷酸序列如SEQ ID NO:2所示；
2)将所述pWPXLd-GPC3CAR.T-PD-L1scFv重组质粒与包膜质粒、包装质粒共转染宿主细胞，得到重组慢病毒；
3)将所述重组慢病毒转染CD3阳性T淋巴细胞，经分离获得靶向GPC3的嵌合抗原受体T细胞。
12.根据权利要求11所述的靶向GPC3的嵌合抗原受体T细胞的制备方法，其特征在于，步骤2)中，所述包膜质粒为PMD2G，所述包装质粒为psPAX2，所述宿主细胞为HEK293T细胞；步骤3)中，CD3阳性T淋巴细胞是从人源外周血单个核细胞中分离获得。
13.一种重组病毒载体，其特征在于，包含表达PD-L1单链抗体的靶向GPC3的嵌合抗原受体GPC3CAR.T-PD-L1scFv的编码基因，该编码基因的核苷酸序列如SEQ ID NO:2或SEQ IDNO:23所示。
14.根据权利要求13所述的重组病毒载体，其特征在于，所述表达PD-L1单链抗体的靶向GPC3的嵌合抗原受体GPC3CAR.T-PD-L1scFv的编码基因，自5’端到3’端依次由CD8α信号肽的编码基因、靶向GPC3的单链抗体的编码基因、CD8α胞外铰链区的编码基因、CD8α跨膜区的编码基因、4-1BB共刺激结构区的编码基因、CD3ζ信号结构区的编码基因、P2A的编码基因、IGHV3-2信号肽的编码基因、靶向PD-L1的单链抗体的编码基因、连接子G4S的编码基因和标签蛋白6×His的编码基因串联相连；其中：
GPC3CAR.T-PD-L1scFv的氨基酸序列如SEQ ID NO:24所示；
CD8α信号肽的编码基因的核苷酸序列如SEQ ID NO:25所示；
CD8α信号肽的氨基酸序列如SEQ ID NO:26所示。
15.权利要求1～9中任意一项所述的表达PD-L1单链抗体的靶向GPC3的嵌合抗原受体、权利要求10所述的靶向GPC3的嵌合抗原受体T细胞或权利要求13所述的重组病毒载体在制备治疗肝癌的药物中的应用。
16.一种治疗肝细胞癌的药物，其特征在于，该药物包括权利要求1～9中任意一项所述的表达PD-L1单链抗体的靶向GPC3的嵌合抗原受体、权利要求10所述的靶向GPC3的嵌合抗原受体T细胞或权利要求13所述的重组病毒载体。
CN202111290859.7A 2021-11-02 2021-11-02 表达pd-l1单链抗体的靶向gpc3的嵌合抗原受体、t细胞及制备方法和应用 Pending CN113999319A (zh)

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CN111944850A (zh) * 2020-08-28 2020-11-17 澳门大学 表达抗cd22嵌合抗原受体和pd-l1阻断蛋白的细胞的制备方法、表达载体及应用
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CN113286615A (zh) * 2018-11-08 2021-08-20 同生运营公司 用于治疗癌症的微生物和免疫调节剂的组合疗法
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