CN108164600B - 一种抗gpc3抗体及其制备方法和用途 - Google Patents
一种抗gpc3抗体及其制备方法和用途 Download PDFInfo
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Abstract
本发明涉及生物技术领域，特别是涉及一种抗GPC3抗体及其制备方法和用途。本发明提供一种抗GPC3抗体，所述抗GPC3抗体的重链可变区的CDR包括氨基酸序列如SEQ ID No.1所示的CDR‑H1、氨基酸序列如SEQ ID No.2所示的CDR‑H2和氨基酸序列如SEQ ID No.3所示的CDR‑H3；所述抗GPC3抗体的轻链可变区的CDR包括氨基酸序列如SEQ ID No.4所示的CDR‑L1、氨基酸序列如SEQ ID No.5所示的CDR‑L2和氨基酸序列如SEQ ID No.6所示的CDR‑L3。本发明发明人利用噬菌体展示技术对GPC3单链抗体进行了亲和力成熟筛选，从而获得了高亲和力的对GPC3的单链抗体。
Description
技术领域
本发明涉及生物技术领域，特别是涉及一种抗GPC3抗体及其制备方法和用途。
背景技术
Glypican3(GPC3)是一种70kd膜蛋白，属于Glypicans家族，高度且集中表达于胚胎发育期并且呈现组织特异性，表达之后会被furin酶切割产生N端40kd可溶性部分以及30kd部分通过GPI分子锚定于细胞膜的C端。基因组学和功能研究表明，GPC3对于维持Wnt通路、Hedgehogs通路的激活中有重要作用，例如GPC3偶联的硫酸乙酰肝素分子可以增强Wnts与其受体的结合从而对于维持Wnt通路有重要作用。GPC3表达于脑、消化道、膀胱，性腺和皮肤并高度表达于肝细胞癌表面；肝癌发生中Wnt通路起到了重要的作用，例如20％肝细胞癌β-Catenin通路突变以及Frizzled-7受体过度表达，因此GPC3在部分肝细胞癌发生过程中可能起到了促进作用。
GPC3高度表达于肝细胞癌HCC表面，并少量表达于其他的组织(不包括正常肝组织)，因此是一个很好的免疫肿瘤靶点。肝癌是世界第五大肿瘤，其中肝细胞癌(Hepatocellular Carcinoma，HCC)占肝癌总数75％；其中中国是肝癌发生大国，根据WHO数据统计，中国肝癌年病人数为29万，死亡人数28万，1年生存率不足5％。FDA批准用于肝细胞癌的药物只有Sorafenib，PFS延长不足2个月，因此急需新型的治疗方式。
嵌合抗原受体T细胞疗法是新兴的肿瘤免疫治疗方法，通过基因工程改造患者的T细胞，使得T细胞表达嵌合抗原受体；经过修饰的嵌合抗原受体T细胞可以特异性识别表达癌细胞抗原的肿瘤细胞，从而激活T细胞对肿瘤细胞的杀伤。
发明内容
鉴于以上所述现有技术的缺点，本发明的目的在于提供一种抗GPC3抗体及其制备方法和用途，用于解决现有技术中的问题。
为实现上述目的及其他相关目的，本发明一方面提供一种抗GPC3抗体，所述抗GPC3抗体包括重链可变区和轻链可变区，所述抗GPC3抗体具有如下技术特征中的一个或多个：
<1>重链可变区的互补决定区包括氨基酸序列如SEQ ID No.1所示的CDR-H1；
<2>重链可变区的互补决定区包括氨基酸序列如SEQ ID No.2所示的CDR-H2；
<3>重链可变区的互补决定区包括氨基酸序列如SEQ ID No.3所示的CDR-H3；
<4>轻链可变区的互补决定区包括氨基酸序列如SEQ ID No.4所示的CDR-L1；
<5>轻链可变区的互补决定区包括氨基酸序列如SEQ ID No.5所示的CDR-L2；
<6>轻链可变区的互补决定区包括氨基酸序列如SEQ ID No.6所示的CDR-L3。
GYTFTXYEMH (SEQ ID No.1)
所述SEQ ID No.1中，X为D或E。
ALDPKTGDTAYSQKFKG (SEQ ID No.2)
TRFYSYTY (SEQ ID No.3)
RSSQSLVXSNXNXYLX (SEQ ID No.4)
所述SEQ ID No.4中，自N端至C端的第一个X氨基酸残基选自H或R；
所述SEQ ID No.4中，自N端至C端的第二个X氨基酸残基选自A、R、H、G或W；
所述SEQ ID No.4中，自N端至C端的第三个X氨基酸残基选自T或V；
所述SEQ ID No.4中，自N端至C端的第四个X氨基酸残基选自H或Q。
KVSNRFS (SEQ ID No.5)
XQNTHVPXX (SEQ ID No.6)
所述SEQ ID No.6中，自N端至C端的第一个X氨基酸残基选自S、L或V；
所述SEQ ID No.6中，自N端至C端的第二个X氨基酸残基选自P或Y；
所述SEQ ID No.6中，自N端至C端的第三个X氨基酸残基选自T或V。
CDR(互补决定区，complementarity determining region)通常指抗体中在空间结构上可以与抗原决定簇形成互补的区域。抗体中的可变性通常并不均匀地分布在整个抗体的可变区中，单克隆抗体的重链可变区和轻链可变区通常均具有3个超变区(hypervariable region，HVR)，这些区域在空间结构上通常可以与抗原决定簇形成互补，所以超变区也被称为互补决定区(complementarity determining region，CDR)，即重链可变区通常包括三个互补决定区，即HCDR1、HCDR2和HCDR3，轻链可变区通常包括三个互补决定区，即LCDR1、LCDR2和LCDR3。
在本发明某些实施方式中，所述抗GPC3抗体的重链可变区的互补决定区包括氨基酸序列如SEQ ID No.1所示的CDR-H1、氨基酸序列如SEQ ID No.2所示的CDR-H2和氨基酸序列如SEQ ID No.3所示的CDR-H3。
在本发明某些实施方式中，所述抗GPC3抗体的轻链可变区的互补决定区包括氨基酸序列如SEQ ID No.4其中之一所示的CDR-L1、氨基酸序列如SEQ ID No.5所示的CDR-L2和氨基酸序列如SEQ ID No.6所示的CDR-L3。
在本发明某些实施方式中，重链可变区的互补决定区包括氨基酸序列如SEQ IDNo.1所示的CDR-H1、氨基酸序列如SEQ ID No.2所示的CDR-H2和氨基酸序列如SEQ ID No.3所示的CDR-H3，轻链可变区的互补决定区包括氨基酸序列如SEQ ID No.4所示的CDR-L1、氨基酸序列如SEQ ID No.5所示的CDR-L2和氨基酸序列如SEQ ID No.6所示的CDR-L3。
在本发明某些实施方式中，所述抗GPC3抗体为单克隆抗体。单克隆抗体通常指一个抗体的群体，该群体中所包括的抗体是基本相同的(除少数可能存在的天然发生的突变外)。单克隆抗体通常针对抗原上特定的决定簇。
在本发明某些实施方式中，所述抗GPC3抗体为单链抗体(single chain Fv,scFv)。单链抗体通常可以为包含抗体的VH(重链可变区)和VL(轻链可变区)的多肽链。通常来说，单链抗体还可以包括连接肽(linker)，连接肽通常位于VH和VL之间，以使scFv形成能与抗原结合的期望结构。例如，所述抗GPC3抗体可以包括VH和VL，VH和VL之间可以设有连接肽，所述单链抗GPC3抗体自N段至C端可以依次包括VH、连接肽和VL，所述抗GPC3单链抗体自N段至C端也可以依次包括VL、连接肽和VH。所述连接肽可以是本领域中各种适用于形成scFv的连接肽，例如，所述连接肽可以是G4S3linker，所述G4S3linker的选择或设计可以参考文献Michel Sadelain etc，Science Translational Medicine，2013；Carl H.June etc,Science Translational Medicine,2015。
在本发明某些实施方式中，所述抗GPC3抗体衍生自GPC3特异性的单克隆抗体GC33(具体信息可参见WO 2009/122667A1)。
在本发明某些实施方式中，所述抗GPC3抗体的重链可变区的氨基酸序列包括：
a)如SEQ ID No.80-108其中之一所示的氨基酸序列；或
b)与SEQ ID No.80-108其中之一所示的氨基酸序列具有80％以上同源性、且具有a)所限定的氨基酸序列功能的氨基酸序列。
具体的，所述b)中的氨基酸序列具体指：如SEQ ID No.80-108其中之一所示的氨基酸序列经过取代、缺失或者添加一个或多个(具体可以是1-50、1-30个、1-20个、1-10个、1-5个、或1-3个)氨基酸而得到的，或者在N-末端和/或C-末端添加一个或多个(具体可以是1-50个、1-30个、1-20个、1-10个、1-5个、或1-3个)氨基酸而得到的，且具有如SEQ IDNo.80-108其中之一所示的氨基酸序列功能的氨基酸序列。所述b)中的氨基酸序列可与SEQID No.80-108其中之一具有80％、85％、90％、93％、95％、97％、或99％以上的同源性。
在本发明某些实施方式中，所述抗GPC3抗体的轻链可变区的氨基酸序列包括：
c)如SEQ ID No.51-79其中之一所示的氨基酸序列；或
d)与SEQ ID No.51-79其中之一所示的氨基酸序列具有80％以上同源性、且具有c)所限定的氨基酸序列功能的氨基酸序列。
具体的，所述d)中的氨基酸序列具体指：如SEQ ID No.51-79其中之一所示的氨基酸序列经过取代、缺失或者添加一个或多个(具体可以是1-50、1-30个、1-20个、1-10个、1-5个、或1-3个)氨基酸而得到的，或者在N-末端和/或C-末端添加一个或多个(具体可以是1-50个、1-30个、1-20个、1-10个、1-5个、或1-3个)氨基酸而得到的，且具有如SEQ ID No.51-79其中之一所示的氨基酸序列功能的氨基酸序列。所述b)中的氨基酸序列可与SEQ IDNo.51-79其中之一具有80％、85％、90％、93％、95％、97％、或99％以上的同源性。
在本发明某些实施方式中，所述抗GPC3抗体的氨基酸序列如SEQ ID No.22-50。
本发明另一方面提供一种分离的多核苷酸，编码所述抗GPC3抗体的重链可变区和/或轻链可变区或全长氨基酸。
本发明另一方面提供一种构建体，含有所述分离的多核苷酸。
在本发明某些实施方式中，所述构建体由所述分离的多核苷酸插入到表达载体的多克隆位点构建而成。本发明中的表达载体通常指本领域熟知的各种市售表达载体，例如可以是细菌质粒、噬菌体、酵母质粒、植物细胞病毒、哺乳动物细胞病毒如腺病毒、逆转录病毒或其他载体。
在本发明某些实施方式中，所述表达载体选自GV401，吉凯基因。
本发明另一方面提供一种抗体的表达系统，所述表达系统含有所述构建体或基因组中整合有外源的所述多核苷酸。任何适用于表达载体进行表达的细胞都可以作为宿主细胞，例如，所述宿主细胞可以是原核细胞，如细菌细胞；或是低等真核细胞，如酵母细胞；或是高等真核细胞，如哺乳动物细胞。
在本发明某些实施方式中，宿主细胞选自T细胞、NK细胞中的一种或多种的组合。
本发明另一方面提供所述抗GPC3抗体的制备方法，包括如下步骤：在适合表达所述抗GPC3抗体的条件下，培养所述的抗GPC3抗体的表达系统，从而表达出所述的抗GPC3抗体，纯化分离出所述的抗GPC3抗体。
本发明中所用的宿主细胞均为现有技术，可通过商业途径直接获取，培养中所用的培养基亦为各种常规培养基，本领域技术人员可根据经验选择适用的培养基，在适于宿主细胞生长的条件下进行培养。当宿主细胞生长到适当的细胞密度后，用合适的方法(如温度转换或化学诱导)诱导选择的启动子，将细胞再培养一段时间。在上面的方法中的重组多肽可在细胞内、或在细胞膜上表达、或分泌到细胞外。如果需要，可利用其物理的、化学的和其它特性通过各种分离方法分离和纯化重组的蛋白。这些方法是本领域技术人员所熟知的。这些方法的例子包括但并不限于：常规的复性处理、用蛋白沉淀剂处理(盐析方法)、离心、渗透破菌、超处理、超离心、分子筛层析(凝胶过滤)、吸附层析、离子交换层析、高效液相层析(HPLC)和其它各种液相层析技术及这些方法的结合。
本发明另一方面提供所述抗GPC3抗体在制备或筛选治疗药物中的用途、或制备诊断药物中的用途。
所述治疗药物可以是以GPC3抗原为作用靶标，结合或作用于所述GPC3抗原，从而治疗和/或预防适应症的药物。
在本发明某些实施方式中，所述治疗药物可以是肿瘤治疗药物。所述肿瘤治疗药物可以是以肿瘤细胞表面功能性表达的GPC3抗原为靶标，结合或作用于GPC3抗原，从而治疗和/或预防肿瘤的药物。所述肿瘤可以是与GPC3相关的肝细胞癌(HepatocellularCarcinoma，HCC)、卵巢癌、膀胱癌、前列腺癌、结直肠癌等。所述与GPC3相关的肝细胞癌可以是具有GPC3高表达表现的肝细胞癌。
在本发明某些实施方式中，所述治疗药物为嵌合抗原受体(CAR，chimericantigen receptor)细胞治疗药物。
所述嵌合抗原受体细胞治疗药物通常包括嵌合抗原受体细胞，所述嵌合抗原受体细胞可以是嵌合抗原受体T细胞、嵌合抗原受体NK细胞等，所述嵌合抗原受体T细胞通常包括T淋巴细胞，其还包括嵌合抗原受体。所述嵌合抗原受体NK细胞通常包括NK细胞，其还包括嵌合抗原受体。所述嵌合抗原受体包括跨膜域、胞内域和胞外域。在本发明某些实施方式中，所述胞外域包括所述抗GPC3抗体，即所述嵌合抗原受体细胞可以在细胞表面表达所述抗GPC3抗体，从而可以引导该细胞对表达GPC3抗原的细胞(例如，肿瘤细胞)进行作用的药物。所述对表达GPC3抗原的细胞进行作用可以是杀死表达GPC3抗原的细胞等。
所以诊断药物具体指针对作用靶标GPC3抗原，以GPC3抗原作为生物标志物进行诊断的试剂。
本发明另一方面提供一种分离的多肽，所述多肽包括跨膜域、胞内域和胞外域，所述胞外域包括所述抗GPC3抗体。
在本发明某些实施方式中，所述多肽为嵌合抗原受体。
在本发明某些实施方式中，所述跨膜域可以包括CD8α(NM_001145873)、CD28(NM_006139)、DAP10(NM_014266)等蛋白分子的跨膜结构域。
在本发明某些实施方式中，所述胞内域可以包括共刺激结构域和/或信号结构域，例如，所述胞内域可以包括4-1BB(NM_001561)、CD28(NM_006139)、OX40(NM_003327)、ICOS(NM_012092)、CD3zeta(NM_198053)、DAP10(NM_014266)等蛋白分子的信号转导结构域。
在本发明某些实施方式中，所述多肽自N端至C端依次包括所述抗GPC3单链抗体、跨膜域、胞内域。在本发明一些具体实施方式中，所述多肽自N端至C端依次包括所述抗GPC3单链抗体、CD8α跨膜区、4-1BB共刺激结构域、CD3zeta信号结构域。在本发明一具体实施方式中，所述多肽自N端至C端依次包括所述抗GPC3单链抗体、CD28跨膜区、CD28共刺激结构域、CD3zeta信号结构域。在本发明另一具体实施方式中，所述多肽自N端至C端依次包括所述抗GPC3单链抗体、CD8α跨膜区、OX40共刺激结构域、CD3zeta信号结构域。在本发明另一具体实施方式中，所述多肽自N端至C端依次包括所述抗GPC3单链抗体、CD8α跨膜区、ICOS共刺激结构域、CD3zeta信号结构域。在本发明另一具体实施方式中，所述多肽自N端至C端依次包括所述抗GPC3单链抗体、CD8α跨膜区、4-1BB共刺激结构域、CD28共刺激结构域、CD3zeta。在本发明另一具体实施方式中，所述多肽自N端至C端依次包括所述抗GPC3单链抗体、CD28跨膜区、CD28共刺激结构域、OX40共刺激结构域、CD3zeta信号结构域。
本发明另一方面一种T淋巴细胞，所述T淋巴细胞含膜结合的所述多肽。
在本发明某些实施方式中，所述多肽为嵌合抗原受体。
所述T淋巴细胞通常可以表达所述多肽，其通常可以结合于GPC3抗原，更具体可以通过包含所述抗GPC3抗体的胞外域结合于GPC3抗原，当所述多肽结合于所述GPC3抗原时，所述T淋巴细胞通常可以活化和/或刺激从而得以增殖。在本发明某些实施方式中，所述胞外域包括所述抗GPC3抗体，即所述嵌合抗原受体T细胞可以在T淋巴细胞表面表达所述抗GPC3抗体，从而可以引导T淋巴细胞对表达GPC3抗原的细胞(例如，肿瘤细胞)进行作用的，所述作用可以是杀死表达GPC3抗原的细胞等。
本发明另一方面一种NK细胞，所述NK细胞含膜结合的所述多肽。
在本发明某些实施方式中，所述多肽为嵌合抗原受体。
所述NK细胞通常可以表达所述多肽，其通常可以结合于GPC3抗原，更具体可以通过包含所述抗GPC3抗体的胞外域结合于GPC3抗原，当所述多肽结合于所述抗原时，所述NK细胞通常可以活化和/或刺激从而得以增殖。在本发明某些实施方式中，所述胞外域包括所述抗GPC3抗体，即所述嵌合抗原受体NK细胞可以在NK细胞表面表达所述抗GPC3抗体，从而可以引导NK细胞对表达GPC3抗原的细胞(例如，肿瘤细胞)进行作用的，所述作用可以是杀死表达GPC3抗原的细胞等。
本发明另一方面提供一种诊断试剂盒，包含诊断有效剂量的所述抗GPC3抗体或其免疫偶联物。有效量通常指能够提供诊断效益的量。
所以诊断试剂盒通常可以针对作用靶标GPC3抗原，以GPC3抗原作为生物标志物进行诊断。所述诊断试剂盒还可以包括抗GPC3抗体的标记物，所述抗GPC3抗体的标记物通常可以用于标记抗GPC3抗体，可选用的标记物的种类包括但不限于荧光标记物、放射性标记物、酶标标记物、化学发光性标记物等中的一种或多种的组合。根据试剂盒的检测原理，所述试剂盒通常还可包含检测所需的一种或多种试剂。此外，所述试剂盒中还可根据需要包括：容器、对照物(阴性或阳性对照)、缓冲剂、助剂等，本领域技术人员可根据具体情况对其进行选择。
本发明发明人利用噬菌体展示技术对GPC3单链抗体进行了亲和力优化，从而获得了高亲和力的抗GPC3的单链抗体，这些优化的单链抗体针对GPC3抗原的亲合力或结合力得到很大提高。此外，发明人进一步提供了基于这些高亲合力单链抗体的嵌合抗原受体，例如表达抗GPC3嵌合抗原受体的T细胞，并进一步验证了这些嵌合抗原受体可以提高对表达GPC3抗原的肿瘤细胞的识别，从而提高针对肿瘤细胞的杀伤能力。
具体实施方式
以下通过特定的具体实例说明本发明的实施方式，本领域技术人员可由本说明书所揭露的内容轻易地了解本发明的其他优点与功效。本发明还可以通过另外不同的具体实施方式加以实施或应用，本说明书中的各项细节也可以基于不同观点与应用，在没有背离本发明的精神下进行各种修饰或改变。
在进一步描述本发明具体实施方式之前，应理解，本发明的保护范围不局限于下述特定的具体实施方案；还应当理解，本发明实施例中使用的术语是为了描述特定的具体实施方案，而不是为了限制本发明的保护范围；在本发明说明书和权利要求书中，除非文中另外明确指出，单数形式“一个”、“一”和“这个”包括复数形式。
当实施例给出数值范围时，应理解，除非本发明另有说明，每个数值范围的两个端点以及两个端点之间任何一个数值均可选用。除非另外定义，本发明中使用的所有技术和科学术语与本技术领域技术人员通常理解的意义相同。除实施例中使用的具体方法、设备、材料外，根据本技术领域的技术人员对现有技术的掌握及本发明的记载，还可以使用与本发明实施例中所述的方法、设备、材料相似或等同的现有技术的任何方法、设备和材料来实现本发明。
除非另外说明，本发明中所公开的实验方法、检测方法、制备方法均采用本技术领域常规的分子生物学、生物化学、染色质结构和分析、分析化学、细胞培养、重组DNA技术及相关领域的常规技术。这些技术在现有文献中已有完善说明，具体可参见Sambrook等MOLECULAR CLONING：A LABORATORY MANUAL，Second edition，Cold Spring HarborLaboratory Press，1989and Third edition，2001；Ausubel等，CURRENT PROTOCOLS INMOLECULAR BIOLOGY，John Wiley&Sons，New York，1987and periodic updates；theseries METHODS IN ENZYMOLOGY，Academic Press，San Diego；Wolffe，CHROMATINSTRUCTURE AND FUNCTION，Third edition，Academic Press，San Diego，1998；METHODS INENZYMOLOGY，Vol.304，Chromatin(P.M.Wassarman and A.P.Wolffe，eds.)，AcademicPress，San Diego，1999；和METHODS IN MOLECULAR BIOLOGY，Vol.119，ChromatinProtocols(P.B.Becker，ed.)Humana Press，Totowa，1999等。
实施例1
scFv GC33的重链(H)及轻链(L)的CDR1、CDR2、CDR3突变库的构建：
以pCAN-scFv GC33质粒为模板，模板序列为：
>GC33scFv VL to VH
GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGATCTAGTCAGAGCCTTGTACACAGTAATGCCAACACCTATTTACATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATAAAGTTTCCAACCGATTTTCTGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCTCTCAAAATACACATGTTCCTCCTACGTTTGGCCAGGGGACCAAGCTGGAGATCAAAGGTGGAGGCGGTTCAGGCGGAGGTGGCTCTGGCGGTGGCGGATCGCAGGTGCAGCTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGATACACCTTCACCGACTATGAAATGCACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGCTCTTGATCCTAAAACTGGTGATACTGCCTACAGTCAGAAGTTCAAGGGCAGAGTCACGCTGACCGCGGACGAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTACAAGATTCTACTCCTATACTTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA(SEQ ID No.7)
利用随机引物PCR引入突变，引物如表1所示。获得的scFv GC33的重链(H)及轻链(L)的CDR1、CDR2、CDR3突变库PCR产物分别命名为H1、H2、H3、L1、L2和L3。用Sfi I和Not I对PCR产物及pCANTAB 5E(GE，27-9400-1)酶切回收后，经T4DNA连接酶16℃连接过夜。连接产物电转至TG1，2YT重悬并于37℃复苏1h后，取菌液梯度稀释进行平板计数，得到各个突变库库容至少108，其余菌液全部涂布2YT(GA)平板。从上述突变库中分别随机挑取20个单克隆送测序，多样性均100％。
表1 scFv GC33的重链(H)及轻链(L)的CDR1、CDR2、CDR 3突变库的引物
实施例2
噬菌体抗体库的淘选：
加入20nM GPC3-His-biotin抗原与噬菌体抗体库室温孵育2h，再将混合物转入链霉亲和素磁珠中室温共孵育15min。PBST-PBS洗去未结合的噬菌体，再加入胰酶37℃作用30min，从而洗脱下结合的噬菌体。将胰酶消化洗脱下的噬菌体感染4ml对数期的TG1菌体，37℃静置30min，取部分菌液梯度稀释进行平板计数，其余菌液全部涂布2YT(GA)平板，用于下一轮的包装。包装后的噬菌体可用于下一轮的淘选，共进行四轮淘选富集，每轮淘选10倍稀释梯度降低抗原浓度，并逐轮增加PBST-PBS洗涤次数(四轮淘选的GPC3-his-biotin抗原浓度分别为20nM、2nM、0.2nM和0.02nM，PBST-PBS洗涤次数分别为7，10，15，20次)。
实施例3
高亲和力scFv的筛选和鉴定：
经过四轮的淘选后，随机挑取单克隆，IPTG诱导后取上清进行ELISA，ELISA初步筛选后，挑取阳性信号至少2倍大于阴性信号的克隆送测序，分析测序结果，提取出富集较多的CDR区域对应的克隆。
实施例4
GC33突变体Koff测定：
将不同克隆的TG1菌株，用IPTG过夜诱导后，利用TSE buffer(Tris，Surose，ETDA)抽取菌体外周质；将GPC3-His-biotin固定于AR2G sensor(ForteBio)上，将外周质上清作为流动相，测定Kon时间为300s，将外周质流动相撤去，测定Koff时间为600s；芯片再生用Glycine溶液pH＝1.5至完全解离，各克隆的检测结果如表2所示，表2中，kdis表示解离速率，Full R^2表示拟合系数，Response表示偏振光位移度。
表2克隆对GPC3抗原Koff测定
| Clone ID | kdis(1/s) | Full R^2 | Response |
| GB5004-8F3 | 7.97E-04 | 0.991826 | 0.727 |
| GB5004-4A1 | 7.98E-04 | 0.991694 | 0.6625 |
| GB5004-8G4 | 6.44E-04 | 0.999337 | 0.59 |
| GB5004-4G2 | 8.87E-04 | 0.991722 | 0.7199 |
| GB5004-3A12 | 9.85E-04 | 0.979548 | 0.9313 |
| GB5004-3C1 | 8.51E-04 | 0.989459 | 0.4164 |
| GB5004-7A3 | 3.90E-04 | 0.996443 | 0.6619 |
| GB5004-8D5 | 7.16E-04 | 0.998904 | 0.6497 |
| GB5004-4B11 | 5.57E-04 | 0.993161 | 0.4011 |
| GB5004-12A9 | 4.49E-04 | 0.999141 | 0.5317 |
| GB5004-9B10 | 4.30E-04 | 0.998512 | 0.5699 |
| GB5004-7A4 | 5.44E-04 | 0.999239 | 0.6986 |
| GB5004-4A3 | 7.20E-04 | 0.995297 | 0.4872 |
| GB5004-10G3 | 3.24E-04 | 0.99576 | 0.6106 |
| GB5004-5F12 | 2.94E-04 | 0.998303 | 0.5212 |
| GB5004-4D4 | 1.06E-03 | 0.99641 | 0.5522 |
| GB5004-7A10 | 9.22E-04 | 0.99773 | 0.3635 |
| GB5004-2A6 | 8.87E-04 | 0.998637 | 0.4138 |
| GB5004-4H8 | 8.39E-04 | 0.995207 | 0.4748 |
| GB5004-12E9 | 6.79E-04 | 0.98264 | 0.6926 |
| GB5004-5B12 | 5.63E-04 | 0.997784 | 0.4492 |
| GB5004-4F5 | 9.97E-04 | 0.99251 | 0.6784 |
| GB5004-9D9 | 6.14E-04 | 0.99804 | 0.3975 |
| GB5004-7A9 | 5.12E-04 | 0.997541 | 0.39 |
| GB5004-9B11 | 4.42E-04 | 0.995355 | 0.2149 |
| GB5004-9D2 | 8.63E-04 | 0.998489 | 0.4125 |
| GB5004-7C6 | 2.89E-04 | 0.997121 | 0.5056 |
| GB5004-11E1 | 3.12E-04 | 0.995633 | 0.5564 |
| Negative Ctrl | <1.0E-07 | 0 | -0.0168 |
| GB5004-WT | 5.32E-04 | 0.998416 | 0.315 |
| GB5004-WT | 5.00E-04 | 0.998323 | 0.3646 |
| GB5004-WT | 5.02E-04 | 0.998504 | 0.3608 |
| GB5004-WT | 4.97E-04 | 0.99936 | 0.3479 |
| GB5004-WT | 5.16E-04 | 0.998538 | 0.3471 |
| GB5004-WT | 4.97E-04 | 0.99878 | 0.3439 |
其中，各克隆号所对应的scFv的序列信息如下：
>GB5004-WT
DVVMTQSPLSLPVTPGEPASISCRSSQSLVHSNANTYLHWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVPPTFGQGTKLEIK(SEQ ID No.51)
QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYEMHWVRQAPGQGLEWMGALDPKTGDTAYSQKFKGRVTLTADESTSTAYMELSSLRSEDTAVYYCTRFYSYTYWGQGTLVTVSS(SEQ ID No.80)
>4A3
DVVMTQSPLSLPVTPGEPASISCRSSQSLVHSNANTYLHWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVPPTFGQGTKLEIK(SEQ ID No.52)
QVQLVQSGAEVKKPGASVKVSCKASGYTFEDYEMHWVRQAPGQGLEWMGALDPKTGDTAYSQKFKGRVTLTADESTSTAYMELSSLRSEDTAVYYCTRFYSYTYWGQGTLVTVSS(SEQ ID No.81)
>2A6
DVVMTQSPLSLPVTPGEPASISCRSSQSLVHSNANTYLHWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVPPTFGQGTKLEIK(SEQ ID No.53)QVQLVQSGAEVKKPGASVKVSCKASGYTFRDYEMHWVRQAPGQGLEWMGALDPKTGDTAYSQKFKGRVTLTADESTSTAYMELSSLRSEDTAVYYCTRFYSYTYWGQGTLVTVSS(SEQ ID No.82)
>3C1
DVVMTQSPLSLPVTPGEPASISCRSSQSLVHSNANTYLHWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVPPTFGQGTKLEIK(SEQ ID No.54)
QVQLVQSGAEVKKPGASVKVSCKASGYTFSDYEMHWVRQAPGQGLEWMGALDPKTGDTAYSQKFKGRVTLTADESTSTAYMELSSLRSEDTAVYYCTRFYSYTYWGQGTLVTVSS(SEQ ID No.83)
>4B11
DVVMTQSPLSLPVTPGEPASISCRSSQSLVHSNANTYLHWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVPPTFGQGTKLEIK(SEQ ID No.55)
QVQLVQSGAEVKKPGASVKVSCKASGYEFTDYEMHWVRQAPGQGLEWMGALDPKTGDTAYSQKFKGRVTLTADESTSTAYMELSSLRSEDTAVYYCTRFYSYTYWGQGTLVTVSS(SEQ ID No.84)
>4D4
DVVMTQSPLSLPVTPGEPASISCRSSQSLVHSNANTYLHWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVPPTFGQGTKLEIK(SEQ ID No.56)
QVQLVQSGAEVKKPGASVKVSCKASGYHFTAYEMHWVRQAPGQGLEWMGALDPKTGDTAYSQKFKGRVTLTADESTSTAYMELSSLRSEDTAVYYCTRFYSYTYWGQGTLVTVSS(SEQ ID No.85)
>3A12
DVVMTQSPLSLPVTPGEPASISCRSSQSLVHSNANTYLHWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVPPTFGQGTKLEIK(SEQ ID No.57)
QVQLVQSGAEVKKPGASVKVSCKASGYRFTDYEMHWVRQAPGQGLEWMGALDPKTGDTAYSQKFKGRVTLTADESTSTAYMELSSLRSEDTAVYYCTRFYSYTYWGQGTLVTVSS(SEQ ID No.86)
>4A1
DVVMTQSPLSLPVTPGEPASISCRSSQSLVHSNANTYLHWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVPPTFGQGTKLEIK(SEQ ID No.58)
QVQLVQSGAEVKKPGASVKVSCKASGYRFTEYEMHWVRQAPGQGLEWMGALDPKTGDTAYSQKFKGRVTLTADESTSTAYMELSSLRSEDTAVYYCTRFYSYTYWGQGTLVTVSS(SEQ ID No.87)
>5F12
DVVMTQSPLSLPVTPGEPASISCRSSQSLVHSNANTYLHWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVPPTFGQGTKLEIK(SEQ ID No.59)
QVQLVQSGAEVKKPGASVKVSCKASGYTFTEYEMHWVRQAPGQGLEWMGALDPKTGDTAYSQKFKGRVTLTADESTSTAYMELSSLRSEDTAVYYCTRFYSYTYWGQGTLVTVSS(SEQ ID No.88)
>5B12
DVVMTQSPLSLPVTPGEPASISCRSSQSLVHSNANTYLHWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVPPTFGQGTKLEIK(SEQ ID No.60)
QVQLVQSGAEVKKPGASVKVSCKASGYVFTDYEMHWVRQAPGQGLEWMGALDPKTGDTAYSQKFKGRVTLTADESTSTAYMELSSLRSEDTAVYYCTRFYSYTYWGQGTLVTVSS(SEQ ID No.89)
>4G2
DVVMTQSPLSLPVTPGEPASISCRSSQSLVHSNANTYLHWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVPPTFGQGTKLEIK(SEQ ID No.61)
QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYEMHWVRQAPGQGLEWMGALDPKTGDTAYSQKFKGRVTLTADESTSTAYMELSSLRSEDTAVYYCTRFYSYAYWGQGTLVTVSS(SEQ ID No.90)
>4F5
DVVMTQSPLSLPVTPGEPASISCRSSQSLVHSNANTYLHWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVPPTFGQGTKLEIK(SEQ ID No.62)
QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYEMHWVRQAPGQGLEWMGALDPKTGDTAYSQKFKGRVTLTADESTSTAYMELSSLRSEDTAVYYCTRFYSYRYWGQGTLVTVSS(SEQ ID No.91)
>9D2
DVVMTQSPLSLPVTPGEPASISCRSSQSLRHSNANTYLQWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVPPTFGQGTKLEIK(SEQ ID No.63)
QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYEMHWVRQAPGQGLEWMGALDPKTGDTAYSQKFKGRVTLTADESTSTAYMELSSLRSEDTAVYYCTRFYSYTYWGQGTLVTVSS(SEQ ID No.92)
>8D5
DVVMTQSPLSLPVTPGEPASISCRSSQSLRHSNKNTYLQWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVPPTFGQGTKLEIK(SEQ ID No.64)
QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYEMHWVRQAPGQGLEWMGALDPKTGDTAYSQKFKGRVTLTADESTSTAYMELSSLRSEDTAVYYCTRFYSYTYWGQGTLVTVSS(SEQ ID No.93)
>8G4
DVVMTQSPLSLPVTPGEPASISCRSSQSLRHSNRNTYLQWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVPPTFGQGTKLEIK(SEQ ID No.65)
QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYEMHWVRQAPGQGLEWMGALDPKTGDTAYSQKFKGRVTLTADESTSTAYMELSSLRSEDTAVYYCTRFYSYTYWGQGTLVTVSS(SEQ ID No.94)
>12E9
DVVMTQSPLSLPVTPGEPASISCRSSQSLRHSNGNQYLQWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVPPTFGQGTKLEIK(SEQ ID No.66)
QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYEMHWVRQAPGQGLEWMGALDPKTGDTAYSQKFKGRVTLTADESTSTAYMELSSLRSEDTAVYYCTRFYSYTYWGQGTLVTVSS(SEQ ID No.95)
>12A9
DVVMTQSPLSLPVTPGEPASISCRSSQSLRHSRANTYLQWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVPPTFGQGTKLEIK(SEQ ID No.67)
QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYEMHWVRQAPGQGLEWMGALDPKTGDTAYSQKFKGRVTLTADESTSTAYMELSSLRSEDTAVYYCTRFYSYTYWGQGTLVTVSS(SEQ ID No.96)
>7A4
DVVMTQSPLSLPVTPGEPASISCRSSQSLVHSNRNTYLQWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVPPTFGQGTKLEIK(SEQ ID No.68)
QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYEMHWVRQAPGQGLEWMGALDPKTGDTAYSQKFKGRVTLTADESTSTAYMELSSLRSEDTAVYYCTRFYSYTYWGQGTLVTVSS(SEQ ID No.97)
>7A9
DVVMTQSPLSLPVTPGEPASISCRSSQSLVHSNHNTYLQWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVPPTFGQGTKLEIK(SEQ ID No.69)
QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYEMHWVRQAPGQGLEWMGALDPKTGDTAYSQKFKGRVTLTADESTSTAYMELSSLRSEDTAVYYCTRFYSYTYWGQGTLVTVSS(SEQ ID No.98)
>11E1
DVVMTQSPLSLPVTPGEPASISCRSSQSLVHSNANTYLHWYLQKPGQSPQLLIYKVSNRFPGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVPPTFGQGTKLEIK(SEQ ID No.70)
QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYEMHWVRQAPGQGLEWMGALDPKTGDTAYSQKFKGRVTLTADESTSTAYMELSSLRSEDTAVYYCTRFYSYTYWGQGTLVTVSS(SEQ ID No.99)
>9D9
DVVMTQSPLSLPVTPGEPASISCRSSQSLVHSNQNTYLQWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVPPTFGQGTKLEIK(SEQ ID No.71)
QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYEMHWVRQAPGQGLEWMGALDPKTGDTAYSQKFKGRVTLTADESTSTAYMELSSLRSEDTAVYYCTRFYSYTYWGQGTLVTVSS(SEQ ID No.100)
>9B10
DVVMTQSPLSLPVTPGEPASISCRSSQSLVHSNGNVYLQWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVPPTFGQGTKLEIK(SEQ ID No.72)
QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYEMHWVRQAPGQGLEWMGALDPKTGDTAYSQKFKGRVTLTADESTSTAYMELSSLRSEDTAVYYCTRFYSYTYWGQGTLVTVSS(SEQ ID No.101)
>9B11
DVVMTQSPLSLPVTPGEPASISCRSSQSLVHSNWNTYLQWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVPPTFGQGTKLEIK(SEQ ID No.73)
QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYEMHWVRQAPGQGLEWMGALDPKTGDTAYSQKFKGRVTLTADESTSTAYMELSSLRSEDTAVYYCTRFYSYTYWGQGTLVTVSS(SEQ ID No.102)
>7A10
DVVMTQSPLSLPVTPGEPASISCRSSQSLVHSNANTYLQWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVPPTFGQGTKLEIK(SEQ ID No.74)
QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYEMHWVRQAPGQGLEWMGALDPKTGDTAYSQKFKGRVTLTADESTSTAYMELSSLRSEDTAVYYCTRFYSYTYWGQGTLVTVSS(SEQ ID No.103)
>8F3
DVVMTQSPLSLPVTPGEPASISCRSSQSLVHSNANTYLHWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQNTHVPPTFGQGTKLEIK(SEQ ID No.75)
QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYEMHWVRQAPGQGLEWMGALDPKTGDTAYSQKFKGRVTLTADESTSTAYMELSSLRSEDTAVYYCTRFYSYTYWGQGTLVTVSS(SEQ ID No.104)
>7C6
DVVMTQSPLSLPVTPGEPASISCRSSQSLVHSNANTYLHWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCLQNTHVPYTFGQGTKLEIK(SEQ ID No.76)
QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYEMHWVRQAPGQGLEWMGALDPKTGDTAYSQKFKGRVTLTADESTSTAYMELSSLRSEDTAVYYCTRFYSYTYWGQGTLVTVSS(SEQ ID No.105)
>7A3
DVVMTQSPLSLPVTPGEPASISCRSSQSLVHSNANTYLHWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCLQNTHVPPVFGQGTKLEIK(SEQ ID No.77)
QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYEMHWVRQAPGQGLEWMGALDPKTGDTAYSQKFKGRVTLTADESTSTAYMELSSLRSEDTAVYYCTRFYSYTYWGQGTLVTVSS(SEQ ID No.106)
>10G3
DVVMTQSPLSLPVTPGEPASISCRSSQSLVHSNANTYLHWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCVQNTHVPPVFGQGTKLEIK(SEQ ID No.78)
QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYEMHWVRQAPGQGLEWMGALDPKTGDTAYSQKFKGRVTLTADESTSTAYMELSSLRSEDTAVYYCTRFYSYTYWGQGTLVTVSS(SEQ ID No.107)
>4H8
DVVMTQSPLSLPVTPGEPASISCRSSQSLVHSNANTYLHWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVPPTFGQGTKLEIK(SEQ ID No.79)
QVQLVQSGAEVKKPGASVKVSCKASGYSFTDYEMHWVRQAPGQGLEWMGALDPKTGDTAYSQKFKGRVTLTADESTSTAYMELSSLRSEDTAVYYCTRFYSYTYWGQGTLVTVSS(SEQ ID No.108)
实施例5
将高亲和力的scFv改造为CAR：
参照上述各克隆号所对应的scFv序列，按照CDR突变的氨基酸对GC33-BBz嵌合抗原受体进行突变，具体为将scFv序列与CD8α-4-1BB-CD3zeta通过标准生物分子生物方法进行PCR连接，得到突变后的GC33-Mutant-BBz系列嵌合抗原受体，GC33 Mutant-BBz序列通过标准分子生物学方法插入CV401质粒(吉凯基因)。
实施例6
GC33 Mutant-BBz体外验证：
将各scFv序列构建获得的GC33 Mutant-BBz(构建于CV401质粒，吉凯基因)通过与表达VSVg(Envelop)、gag/pol以及rev(Packaging)包装质粒共同感染293T细胞，经过48-72小时，收集培养基上清，上清中包含GC33 Mutant-BBz慢病毒(Lentivirus)，经过感染人类原代PBMC；感染后的T细胞在体外扩增和培养，通过细胞因子释放和杀伤实验评估GC33mutation能否增强T细胞杀伤肿瘤细胞的功能，具体结果如下：
细胞因子释放实验：
将人类PBMC用CD3和CD28抗体(OKT3克隆和15E8克隆，Miltenyi Biotec)刺激激活24小时候，将各scFv序列构建获得的GC33 Mutant-BBz包装成慢病毒(Lentivirus)后感染上述PBMC，感染表达抗CD19嵌合抗原受体，PBMC继续用完全培养基(TexMACS GMP+10％FBS+200IU/mlhIL2)培养8-10天后，收集T细胞，并将T细胞和HepG2细胞(表达GPC3)按照比例1：1混合培养于2％血清的RPMI1640培养基16小时，将混合培养上清利用BD Cytometric beadarray kit测定细胞因子的释放。实验表明，感染获得的PBMC(各scFv序列构建获得的GC33Mutant-BBz包装成慢病毒感染获得，表达GC33 Mutant-BBz嵌合抗原受体)与表达GC33 WT-BBz相比均具有很强的与HepG2细胞结合的能力并能够释放细胞因子。
肿瘤杀伤实验：
将上述制备获得的T细胞(PBMC继续培养8-10天后，收集T细胞)和HepG2细胞按照一定的E：T比例，例如30：1，10：1，3：1，1：1比例混合培养于2％血清的RPMI1640培养基中培养4小时，将培养上清与LDH底物(CytoTox96 Non-Radioactive Cytotoxicity Assay Kit,Promega)1：1体积混合，室温孵育30分钟后读取490nm光吸收。实验结果表明，感染获得的PBMC(各scFv序列构建获得的GC33 Mutant-BBz包装成慢病毒感染获得)与GC33 WT-BBz相比均具有很强的肿瘤杀伤能力。
综上所述，本发明有效克服了现有技术中的种种缺点而具高度产业利用价值。
上述实施例仅例示性说明本发明的原理及其功效，而非用于限制本发明。任何熟悉此技术的人士皆可在不违背本发明的精神及范畴下，对上述实施例进行修饰或改变。因此，举凡所属技术领域中具有通常知识者在未脱离本发明所揭示的精神与技术思想下所完成的一切等效修饰或改变，仍应由本发明的权利要求所涵盖。
SEQUENCE LISTING
<110> 上海吉凯基因化学技术有限公司
<120> 一种抗GPC3抗体及其制备方法和用途
<130> PCN
<160> 108
<170> PatentIn version 3.3
<210> 1
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> CDR-H1
<220>
<221> misc_feature
<222> (6)..(6)
<223> Xaa can be any naturally occurring amino acid
<400> 1
Gly Tyr Thr Phe Thr Xaa Tyr Glu Met His
1 5 10
<210> 2
<211> 17
<212> PRT
<213> Artificial Sequence
<220>
<223> CDR-H2
<400> 2
Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe Lys
1 5 10 15
Gly
<210> 3
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> CDR-H3
<400> 3
Thr Arg Phe Tyr Ser Tyr Thr Tyr
1 5
<210> 4
<211> 16
<212> PRT
<213> Artificial Sequence
<220>
<223> CDR-L1
<220>
<221> misc_feature
<222> (8)..(8)
<223> Xaa can be any naturally occurring amino acid
<220>
<221> misc_feature
<222> (11)..(11)
<223> Xaa can be any naturally occurring amino acid
<220>
<221> misc_feature
<222> (13)..(13)
<223> Xaa can be any naturally occurring amino acid
<220>
<221> misc_feature
<222> (16)..(16)
<223> Xaa can be any naturally occurring amino acid
<400> 4
Arg Ser Ser Gln Ser Leu Val Xaa Ser Asn Xaa Asn Xaa Tyr Leu Xaa
1 5 10 15
<210> 5
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> CDR-L2
<400> 5
Lys Val Ser Asn Arg Phe Ser
1 5
<210> 6
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> CDR-L3
<220>
<221> misc_feature
<222> (1)..(1)
<223> Xaa can be any naturally occurring amino acid
<220>
<221> misc_feature
<222> (8)..(9)
<223> Xaa can be any naturally occurring amino acid
<400> 6
Xaa Gln Asn Thr His Val Pro Xaa Xaa
1 5
<210> 7
<211> 726
<212> DNA
<213> Artificial Sequence
<220>
<223> 模板序列
<400> 7
gatgttgtga tgactcagtc tccactctcc ctgcccgtca cccctggaga gccggcctcc 60
atctcctgca gatctagtca gagccttgta cacagtaatg ccaacaccta tttacattgg 120
tacctgcaga agccagggca gtctccacag ctcctgatct ataaagtttc caaccgattt 180
tctggggtcc ctgacaggtt cagtggcagt ggatcaggca cagattttac actgaaaatc 240
agcagagtgg aggctgagga tgttggggtt tattactgct ctcaaaatac acatgttcct 300
cctacgtttg gccaggggac caagctggag atcaaaggtg gaggcggttc aggcggaggt 360
ggctctggcg gtggcggatc gcaggtgcag ctggtgcagt ctggagctga ggtgaagaag 420
cctggggcct cagtgaaggt ctcctgcaag gcttctggat acaccttcac cgactatgaa 480
atgcactggg tgcgacaggc ccctggacaa gggcttgagt ggatgggagc tcttgatcct 540
aaaactggtg atactgccta cagtcagaag ttcaagggca gagtcacgct gaccgcggac 600
gaatccacga gcacagccta catggagctg agcagcctga gatctgagga cacggccgtg 660
tattactgta caagattcta ctcctatact tactggggcc agggaaccct ggtcaccgtc 720
tcctca 726
<210> 8
<211> 61
<212> DNA
<213> Artificial Sequence
<220>
<223> 引物
<400> 8
tctcctgcaa ggcttctgga tacaccttca ccgactatga aatgcactgg gtgcgacagg 60
c 61
<210> 9
<211> 76
<212> DNA
<213> Artificial Sequence
<220>
<223> 引物
<400> 9
aagggcttga gtggatggga gctcttgatc ctaaaactgg tgatactgcc tacagtcaga 60
agttcaaggg cagagt 76
<210> 10
<211> 64
<212> DNA
<213> Artificial Sequence
<220>
<223> 引物
<400> 10
acacggccgt gtattactgt acaagattct actcctatac ttactggggc cagggaaccc 60
tggt 64
<210> 11
<211> 70
<212> DNA
<213> Artificial Sequence
<220>
<223> 引物
<400> 11
gcagatctag tcagagcctt gtacacagta atgccaacac ctatttacat tggtacctgc 60
agaagccagg 70
<210> 12
<211> 58
<212> DNA
<213> Artificial Sequence
<220>
<223> 引物
<400> 12
ctccacagct cctgatctat aaagtttcca accgattttc tggggtccct gacaggtt 58
<210> 13
<211> 67
<212> DNA
<213> Artificial Sequence
<220>
<223> 引物
<400> 13
atgttggggt ttattactgc tctcaaaata cacatgttcc tcctacgttt ggccagggga 60
ccaagct 67
<210> 14
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> 引物
<400> 14
ccctcatagt tagcgtaacg 20
<210> 15
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> 引物
<400> 15
agcggataac aatttcacac agga 24
<210> 16
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> 引物
<400> 16
tccagaagcc ttgcaggaga cct 23
<210> 17
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> 引物
<400> 17
tcccatccac tcaagccctt 20
<210> 18
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> 引物
<400> 18
acagtaatac acggccgtgt cctcagatct 30
<210> 19
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> 引物
<400> 19
aaggctctga ctagatctgc ag 22
<210> 20
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> 引物
<400> 20
atagatcagg agctgtggag 20
<210> 21
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> 引物
<400> 21
gcagtaataa accccaacat 20
<210> 22
<211> 242
<212> PRT
<213> Artificial Sequence
<220>
<223> ScFv氨基酸序列
<400> 22
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
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln
115 120 125
Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser
130 135 140
Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr Glu
145 150 155 160
Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly
165 170 175
Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe Lys
180 185 190
Gly Arg Val Thr Leu Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr Met
195 200 205
Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Thr
210 215 220
Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr Val
225 230 235 240
Ser Ser
<210> 23
<211> 242
<212> PRT
<213> Artificial Sequence
<220>
<223> ScFv氨基酸序列
<400> 23
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
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln
115 120 125
Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser
130 135 140
Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Glu Asp Tyr Glu
145 150 155 160
Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly
165 170 175
Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe Lys
180 185 190
Gly Arg Val Thr Leu Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr Met
195 200 205
Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Thr
210 215 220
Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr Val
225 230 235 240
Ser Ser
<210> 24
<211> 242
<212> PRT
<213> Artificial Sequence
<220>
<223> ScFv氨基酸序列
<400> 24
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
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln
115 120 125
Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser
130 135 140
Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Arg Asp Tyr Glu
145 150 155 160
Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly
165 170 175
Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe Lys
180 185 190
Gly Arg Val Thr Leu Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr Met
195 200 205
Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Thr
210 215 220
Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr Val
225 230 235 240
Ser Ser
<210> 25
<211> 242
<212> PRT
<213> Artificial Sequence
<220>
<223> ScFv氨基酸序列
<400> 25
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
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln
115 120 125
Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser
130 135 140
Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Asp Tyr Glu
145 150 155 160
Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly
165 170 175
Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe Lys
180 185 190
Gly Arg Val Thr Leu Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr Met
195 200 205
Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Thr
210 215 220
Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr Val
225 230 235 240
Ser Ser
<210> 26
<211> 242
<212> PRT
<213> Artificial Sequence
<220>
<223> ScFv氨基酸序列
<400> 26
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
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln
115 120 125
Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser
130 135 140
Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Glu Phe Thr Asp Tyr Glu
145 150 155 160
Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly
165 170 175
Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe Lys
180 185 190
Gly Arg Val Thr Leu Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr Met
195 200 205
Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Thr
210 215 220
Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr Val
225 230 235 240
Ser Ser
<210> 27
<211> 242
<212> PRT
<213> Artificial Sequence
<220>
<223> ScFv氨基酸序列
<400> 27
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
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln
115 120 125
Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser
130 135 140
Val Lys Val Ser Cys Lys Ala Ser Gly Tyr His Phe Thr Ala Tyr Glu
145 150 155 160
Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly
165 170 175
Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe Lys
180 185 190
Gly Arg Val Thr Leu Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr Met
195 200 205
Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Thr
210 215 220
Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr Val
225 230 235 240
Ser Ser
<210> 28
<211> 242
<212> PRT
<213> Artificial Sequence
<220>
<223> ScFv氨基酸序列
<400> 28
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
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln
115 120 125
Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser
130 135 140
Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Arg Phe Thr Asp Tyr Glu
145 150 155 160
Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly
165 170 175
Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe Lys
180 185 190
Gly Arg Val Thr Leu Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr Met
195 200 205
Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Thr
210 215 220
Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr Val
225 230 235 240
Ser Ser
<210> 29
<211> 242
<212> PRT
<213> Artificial Sequence
<220>
<223> ScFv氨基酸序列
<400> 29
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
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln
115 120 125
Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser
130 135 140
Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Arg Phe Thr Glu Tyr Glu
145 150 155 160
Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly
165 170 175
Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe Lys
180 185 190
Gly Arg Val Thr Leu Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr Met
195 200 205
Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Thr
210 215 220
Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr Val
225 230 235 240
Ser Ser
<210> 30
<211> 242
<212> PRT
<213> Artificial Sequence
<220>
<223> ScFv氨基酸序列
<400> 30
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
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln
115 120 125
Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser
130 135 140
Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Glu Tyr Glu
145 150 155 160
Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly
165 170 175
Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe Lys
180 185 190
Gly Arg Val Thr Leu Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr Met
195 200 205
Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Thr
210 215 220
Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr Val
225 230 235 240
Ser Ser
<210> 31
<211> 242
<212> PRT
<213> Artificial Sequence
<220>
<223> ScFv氨基酸序列
<400> 31
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
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln
115 120 125
Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser
130 135 140
Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Val Phe Thr Asp Tyr Glu
145 150 155 160
Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly
165 170 175
Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe Lys
180 185 190
Gly Arg Val Thr Leu Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr Met
195 200 205
Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Thr
210 215 220
Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr Val
225 230 235 240
Ser Ser
<210> 32
<211> 242
<212> PRT
<213> Artificial Sequence
<220>
<223> ScFv氨基酸序列
<400> 32
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
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln
115 120 125
Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser
130 135 140
Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr Glu
145 150 155 160
Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly
165 170 175
Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe Lys
180 185 190
Gly Arg Val Thr Leu Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr Met
195 200 205
Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Thr
210 215 220
Arg Phe Tyr Ser Tyr Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val
225 230 235 240
Ser Ser
<210> 33
<211> 242
<212> PRT
<213> Artificial Sequence
<220>
<223> ScFv氨基酸序列
<400> 33
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
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln
115 120 125
Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser
130 135 140
Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr Glu
145 150 155 160
Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly
165 170 175
Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe Lys
180 185 190
Gly Arg Val Thr Leu Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr Met
195 200 205
Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Thr
210 215 220
Arg Phe Tyr Ser Tyr Arg Tyr Trp Gly Gln Gly Thr Leu Val Thr Val
225 230 235 240
Ser Ser
<210> 34
<211> 242
<212> PRT
<213> Artificial Sequence
<220>
<223> ScFv氨基酸序列
<400> 34
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 Arg His Ser
20 25 30
Asn Ala Asn Thr Tyr Leu Gln 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
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln
115 120 125
Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser
130 135 140
Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr Glu
145 150 155 160
Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly
165 170 175
Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe Lys
180 185 190
Gly Arg Val Thr Leu Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr Met
195 200 205
Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Thr
210 215 220
Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr Val
225 230 235 240
Ser Ser
<210> 35
<211> 242
<212> PRT
<213> Artificial Sequence
<220>
<223> ScFv氨基酸序列
<400> 35
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 Arg His Ser
20 25 30
Asn Lys Asn Thr Tyr Leu Gln 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
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln
115 120 125
Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser
130 135 140
Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr Glu
145 150 155 160
Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly
165 170 175
Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe Lys
180 185 190
Gly Arg Val Thr Leu Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr Met
195 200 205
Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Thr
210 215 220
Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr Val
225 230 235 240
Ser Ser
<210> 36
<211> 242
<212> PRT
<213> Artificial Sequence
<220>
<223> ScFv氨基酸序列
<400> 36
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 Arg His Ser
20 25 30
Asn Arg Asn Thr Tyr Leu Gln 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
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln
115 120 125
Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser
130 135 140
Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr Glu
145 150 155 160
Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly
165 170 175
Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe Lys
180 185 190
Gly Arg Val Thr Leu Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr Met
195 200 205
Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Thr
210 215 220
Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr Val
225 230 235 240
Ser Ser
<210> 37
<211> 242
<212> PRT
<213> Artificial Sequence
<220>
<223> ScFv氨基酸序列
<400> 37
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 Arg His Ser
20 25 30
Asn Gly Asn Gln Tyr Leu Gln 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
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln
115 120 125
Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser
130 135 140
Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr Glu
145 150 155 160
Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly
165 170 175
Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe Lys
180 185 190
Gly Arg Val Thr Leu Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr Met
195 200 205
Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Thr
210 215 220
Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr Val
225 230 235 240
Ser Ser
<210> 38
<211> 242
<212> PRT
<213> Artificial Sequence
<220>
<223> ScFv氨基酸序列
<400> 38
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 Arg His Ser
20 25 30
Arg Ala Asn Thr Tyr Leu Gln 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
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln
115 120 125
Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser
130 135 140
Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr Glu
145 150 155 160
Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly
165 170 175
Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe Lys
180 185 190
Gly Arg Val Thr Leu Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr Met
195 200 205
Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Thr
210 215 220
Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr Val
225 230 235 240
Ser Ser
<210> 39
<211> 242
<212> PRT
<213> Artificial Sequence
<220>
<223> ScFv氨基酸序列
<400> 39
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 Arg Asn Thr Tyr Leu Gln 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
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln
115 120 125
Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser
130 135 140
Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr Glu
145 150 155 160
Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly
165 170 175
Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe Lys
180 185 190
Gly Arg Val Thr Leu Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr Met
195 200 205
Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Thr
210 215 220
Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr Val
225 230 235 240
Ser Ser
<210> 40
<211> 242
<212> PRT
<213> Artificial Sequence
<220>
<223> ScFv氨基酸序列
<400> 40
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 His Asn Thr Tyr Leu Gln 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
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln
115 120 125
Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser
130 135 140
Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr Glu
145 150 155 160
Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly
165 170 175
Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe Lys
180 185 190
Gly Arg Val Thr Leu Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr Met
195 200 205
Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Thr
210 215 220
Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr Val
225 230 235 240
Ser Ser
<210> 41
<211> 242
<212> PRT
<213> Artificial Sequence
<220>
<223> ScFv氨基酸序列
<400> 41
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 Pro 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
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln
115 120 125
Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser
130 135 140
Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr Glu
145 150 155 160
Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly
165 170 175
Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe Lys
180 185 190
Gly Arg Val Thr Leu Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr Met
195 200 205
Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Thr
210 215 220
Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr Val
225 230 235 240
Ser Ser
<210> 42
<211> 242
<212> PRT
<213> Artificial Sequence
<220>
<223> ScFv氨基酸序列
<400> 42
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 Gln Asn Thr Tyr Leu Gln 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
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln
115 120 125
Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser
130 135 140
Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr Glu
145 150 155 160
Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly
165 170 175
Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe Lys
180 185 190
Gly Arg Val Thr Leu Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr Met
195 200 205
Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Thr
210 215 220
Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr Val
225 230 235 240
Ser Ser
<210> 43
<211> 242
<212> PRT
<213> Artificial Sequence
<220>
<223> ScFv氨基酸序列
<400> 43
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 Gly Asn Val Tyr Leu Gln 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
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln
115 120 125
Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser
130 135 140
Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr Glu
145 150 155 160
Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly
165 170 175
Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe Lys
180 185 190
Gly Arg Val Thr Leu Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr Met
195 200 205
Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Thr
210 215 220
Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr Val
225 230 235 240
Ser Ser
<210> 44
<211> 242
<212> PRT
<213> Artificial Sequence
<220>
<223> ScFv氨基酸序列
<400> 44
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 Trp Asn Thr Tyr Leu Gln 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
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln
115 120 125
Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser
130 135 140
Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr Glu
145 150 155 160
Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly
165 170 175
Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe Lys
180 185 190
Gly Arg Val Thr Leu Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr Met
195 200 205
Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Thr
210 215 220
Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr Val
225 230 235 240
Ser Ser
<210> 45
<211> 242
<212> PRT
<213> Artificial Sequence
<220>
<223> ScFv氨基酸序列
<400> 45
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 Gln 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
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln
115 120 125
Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser
130 135 140
Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr Glu
145 150 155 160
Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly
165 170 175
Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe Lys
180 185 190
Gly Arg Val Thr Leu Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr Met
195 200 205
Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Thr
210 215 220
Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr Val
225 230 235 240
Ser Ser
<210> 46
<211> 242
<212> PRT
<213> Artificial Sequence
<220>
<223> ScFv氨基酸序列
<400> 46
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 Gly Gln Asn
85 90 95
Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105 110
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln
115 120 125
Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser
130 135 140
Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr Glu
145 150 155 160
Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly
165 170 175
Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe Lys
180 185 190
Gly Arg Val Thr Leu Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr Met
195 200 205
Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Thr
210 215 220
Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr Val
225 230 235 240
Ser Ser
<210> 47
<211> 242
<212> PRT
<213> Artificial Sequence
<220>
<223> ScFv氨基酸序列
<400> 47
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 Leu Gln Asn
85 90 95
Thr His Val Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105 110
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln
115 120 125
Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser
130 135 140
Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr Glu
145 150 155 160
Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly
165 170 175
Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe Lys
180 185 190
Gly Arg Val Thr Leu Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr Met
195 200 205
Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Thr
210 215 220
Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr Val
225 230 235 240
Ser Ser
<210> 48
<211> 242
<212> PRT
<213> Artificial Sequence
<220>
<223> ScFv氨基酸序列
<400> 48
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 Leu Gln Asn
85 90 95
Thr His Val Pro Pro Val Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105 110
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln
115 120 125
Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser
130 135 140
Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr Glu
145 150 155 160
Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly
165 170 175
Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe Lys
180 185 190
Gly Arg Val Thr Leu Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr Met
195 200 205
Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Thr
210 215 220
Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr Val
225 230 235 240
Ser Ser
<210> 49
<211> 242
<212> PRT
<213> Artificial Sequence
<220>
<223> ScFv氨基酸序列
<400> 49
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 Val Gln Asn
85 90 95
Thr His Val Pro Pro Val Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105 110
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln
115 120 125
Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser
130 135 140
Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr Glu
145 150 155 160
Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly
165 170 175
Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe Lys
180 185 190
Gly Arg Val Thr Leu Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr Met
195 200 205
Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Thr
210 215 220
Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr Val
225 230 235 240
Ser Ser
<210> 50
<211> 242
<212> PRT
<213> Artificial Sequence
<220>
<223> ScFv氨基酸序列
<400> 50
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
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln
115 120 125
Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser
130 135 140
Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asp Tyr Glu
145 150 155 160
Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly
165 170 175
Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe Lys
180 185 190
Gly Arg Val Thr Leu Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr Met
195 200 205
Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Thr
210 215 220
Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr Val
225 230 235 240
Ser Ser
<210> 51
<211> 112
<212> PRT
<213> Artificial Sequence
<220>
<223> 轻链可变区
<400> 51
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
<210> 52
<211> 112
<212> PRT
<213> Artificial Sequence
<220>
<223> 轻链可变区
<400> 52
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
<210> 53
<211> 112
<212> PRT
<213> Artificial Sequence
<220>
<223> 轻链可变区
<400> 53
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
<210> 54
<211> 112
<212> PRT
<213> Artificial Sequence
<220>
<223> 轻链可变区
<400> 54
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
<210> 55
<211> 112
<212> PRT
<213> Artificial Sequence
<220>
<223> 轻链可变区
<400> 55
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
<210> 56
<211> 112
<212> PRT
<213> Artificial Sequence
<220>
<223> 轻链可变区
<400> 56
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
<210> 57
<211> 112
<212> PRT
<213> Artificial Sequence
<220>
<223> 轻链可变区
<400> 57
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
<210> 58
<211> 112
<212> PRT
<213> Artificial Sequence
<220>
<223> 轻链可变区
<400> 58
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
<210> 59
<211> 112
<212> PRT
<213> Artificial Sequence
<220>
<223> 轻链可变区
<400> 59
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
<210> 60
<211> 112
<212> PRT
<213> Artificial Sequence
<220>
<223> 轻链可变区
<400> 60
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
<210> 61
<211> 112
<212> PRT
<213> Artificial Sequence
<220>
<223> 轻链可变区
<400> 61
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
<210> 62
<211> 112
<212> PRT
<213> Artificial Sequence
<220>
<223> 轻链可变区
<400> 62
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
<210> 63
<211> 112
<212> PRT
<213> Artificial Sequence
<220>
<223> 轻链可变区
<400> 63
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 Arg His Ser
20 25 30
Asn Ala Asn Thr Tyr Leu Gln 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
<210> 64
<211> 112
<212> PRT
<213> Artificial Sequence
<220>
<223> 轻链可变区
<400> 64
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 Arg His Ser
20 25 30
Asn Lys Asn Thr Tyr Leu Gln 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
<210> 65
<211> 112
<212> PRT
<213> Artificial Sequence
<220>
<223> 轻链可变区
<400> 65
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 Arg His Ser
20 25 30
Asn Arg Asn Thr Tyr Leu Gln 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
<210> 66
<211> 112
<212> PRT
<213> Artificial Sequence
<220>
<223> 轻链可变区
<400> 66
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 Arg His Ser
20 25 30
Asn Gly Asn Gln Tyr Leu Gln 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
<210> 67
<211> 112
<212> PRT
<213> Artificial Sequence
<220>
<223> 轻链可变区
<400> 67
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 Arg His Ser
20 25 30
Arg Ala Asn Thr Tyr Leu Gln 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
<210> 68
<211> 112
<212> PRT
<213> Artificial Sequence
<220>
<223> 轻链可变区
<400> 68
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 Arg Asn Thr Tyr Leu Gln 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
<210> 69
<211> 112
<212> PRT
<213> Artificial Sequence
<220>
<223> 轻链可变区
<400> 69
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 His Asn Thr Tyr Leu Gln 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
<210> 70
<211> 112
<212> PRT
<213> Artificial Sequence
<220>
<223> 轻链可变区
<400> 70
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 Pro 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
<210> 71
<211> 112
<212> PRT
<213> Artificial Sequence
<220>
<223> 轻链可变区
<400> 71
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 Gln Asn Thr Tyr Leu Gln 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
<210> 72
<211> 112
<212> PRT
<213> Artificial Sequence
<220>
<223> 轻链可变区
<400> 72
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 Gly Asn Val Tyr Leu Gln 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
<210> 73
<211> 112
<212> PRT
<213> Artificial Sequence
<220>
<223> 轻链可变区
<400> 73
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 Trp Asn Thr Tyr Leu Gln 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
<210> 74
<211> 112
<212> PRT
<213> Artificial Sequence
<220>
<223> 轻链可变区
<400> 74
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 Gln 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
<210> 75
<211> 112
<212> PRT
<213> Artificial Sequence
<220>
<223> 轻链可变区
<400> 75
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 Gly Gln Asn
85 90 95
Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105 110
<210> 76
<211> 112
<212> PRT
<213> Artificial Sequence
<220>
<223> 轻链可变区
<400> 76
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 Leu Gln Asn
85 90 95
Thr His Val Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105 110
<210> 77
<211> 112
<212> PRT
<213> Artificial Sequence
<220>
<223> 轻链可变区
<400> 77
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 Leu Gln Asn
85 90 95
Thr His Val Pro Pro Val Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105 110
<210> 78
<211> 112
<212> PRT
<213> Artificial Sequence
<220>
<223> 轻链可变区
<400> 78
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 Val Gln Asn
85 90 95
Thr His Val Pro Pro Val Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105 110
<210> 79
<211> 112
<212> PRT
<213> Artificial Sequence
<220>
<223> 轻链可变区
<400> 79
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
<210> 80
<211> 115
<212> PRT
<213> Artificial Sequence
<220>
<223> 重链可变区
<400> 80
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe
50 55 60
Lys Gly Arg Val Thr Leu Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr
100 105 110
Val Ser Ser
115
<210> 81
<211> 115
<212> PRT
<213> Artificial Sequence
<220>
<223> 重链可变区
<400> 81
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Glu Asp Tyr
20 25 30
Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe
50 55 60
Lys Gly Arg Val Thr Leu Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr
100 105 110
Val Ser Ser
115
<210> 82
<211> 115
<212> PRT
<213> Artificial Sequence
<220>
<223> 重链可变区
<400> 82
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Arg Asp Tyr
20 25 30
Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe
50 55 60
Lys Gly Arg Val Thr Leu Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr
100 105 110
Val Ser Ser
115
<210> 83
<211> 115
<212> PRT
<213> Artificial Sequence
<220>
<223> 重链可变区
<400> 83
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Asp Tyr
20 25 30
Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe
50 55 60
Lys Gly Arg Val Thr Leu Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr
100 105 110
Val Ser Ser
115
<210> 84
<211> 115
<212> PRT
<213> Artificial Sequence
<220>
<223> 重链可变区
<400> 84
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Glu Phe Thr Asp Tyr
20 25 30
Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe
50 55 60
Lys Gly Arg Val Thr Leu Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr
100 105 110
Val Ser Ser
115
<210> 85
<211> 115
<212> PRT
<213> Artificial Sequence
<220>
<223> 重链可变区
<400> 85
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr His Phe Thr Ala Tyr
20 25 30
Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe
50 55 60
Lys Gly Arg Val Thr Leu Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr
100 105 110
Val Ser Ser
115
<210> 86
<211> 115
<212> PRT
<213> Artificial Sequence
<220>
<223> 重链可变区
<400> 86
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Arg Phe Thr Asp Tyr
20 25 30
Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe
50 55 60
Lys Gly Arg Val Thr Leu Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr
100 105 110
Val Ser Ser
115
<210> 87
<211> 115
<212> PRT
<213> Artificial Sequence
<220>
<223> 重链可变区
<400> 87
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Arg Phe Thr Glu Tyr
20 25 30
Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe
50 55 60
Lys Gly Arg Val Thr Leu Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr
100 105 110
Val Ser Ser
115
<210> 88
<211> 115
<212> PRT
<213> Artificial Sequence
<220>
<223> 重链可变区
<400> 88
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Glu Tyr
20 25 30
Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe
50 55 60
Lys Gly Arg Val Thr Leu Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr
100 105 110
Val Ser Ser
115
<210> 89
<211> 115
<212> PRT
<213> Artificial Sequence
<220>
<223> 重链可变区
<400> 89
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Val Phe Thr Asp Tyr
20 25 30
Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe
50 55 60
Lys Gly Arg Val Thr Leu Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr
100 105 110
Val Ser Ser
115
<210> 90
<211> 115
<212> PRT
<213> Artificial Sequence
<220>
<223> 重链可变区
<400> 90
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe
50 55 60
Lys Gly Arg Val Thr Leu Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Thr Arg Phe Tyr Ser Tyr Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr
100 105 110
Val Ser Ser
115
<210> 91
<211> 115
<212> PRT
<213> Artificial Sequence
<220>
<223> 重链可变区
<400> 91
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe
50 55 60
Lys Gly Arg Val Thr Leu Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Thr Arg Phe Tyr Ser Tyr Arg Tyr Trp Gly Gln Gly Thr Leu Val Thr
100 105 110
Val Ser Ser
115
<210> 92
<211> 115
<212> PRT
<213> Artificial Sequence
<220>
<223> 重链可变区
<400> 92
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe
50 55 60
Lys Gly Arg Val Thr Leu Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr
100 105 110
Val Ser Ser
115
<210> 93
<211> 115
<212> PRT
<213> Artificial Sequence
<220>
<223> 重链可变区
<400> 93
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe
50 55 60
Lys Gly Arg Val Thr Leu Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr
100 105 110
Val Ser Ser
115
<210> 94
<211> 115
<212> PRT
<213> Artificial Sequence
<220>
<223> 重链可变区
<400> 94
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe
50 55 60
Lys Gly Arg Val Thr Leu Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr
100 105 110
Val Ser Ser
115
<210> 95
<211> 115
<212> PRT
<213> Artificial Sequence
<220>
<223> 重链可变区
<400> 95
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe
50 55 60
Lys Gly Arg Val Thr Leu Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr
100 105 110
Val Ser Ser
115
<210> 96
<211> 115
<212> PRT
<213> Artificial Sequence
<220>
<223> 重链可变区
<400> 96
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe
50 55 60
Lys Gly Arg Val Thr Leu Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr
100 105 110
Val Ser Ser
115
<210> 97
<211> 115
<212> PRT
<213> Artificial Sequence
<220>
<223> 重链可变区
<400> 97
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe
50 55 60
Lys Gly Arg Val Thr Leu Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr
100 105 110
Val Ser Ser
115
<210> 98
<211> 115
<212> PRT
<213> Artificial Sequence
<220>
<223> 重链可变区
<400> 98
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe
50 55 60
Lys Gly Arg Val Thr Leu Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr
100 105 110
Val Ser Ser
115
<210> 99
<211> 115
<212> PRT
<213> Artificial Sequence
<220>
<223> 重链可变区
<400> 99
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe
50 55 60
Lys Gly Arg Val Thr Leu Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr
100 105 110
Val Ser Ser
115
<210> 100
<211> 115
<212> PRT
<213> Artificial Sequence
<220>
<223> 重链可变区
<400> 100
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe
50 55 60
Lys Gly Arg Val Thr Leu Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr
100 105 110
Val Ser Ser
115
<210> 101
<211> 115
<212> PRT
<213> Artificial Sequence
<220>
<223> 重链可变区
<400> 101
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe
50 55 60
Lys Gly Arg Val Thr Leu Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr
100 105 110
Val Ser Ser
115
<210> 102
<211> 115
<212> PRT
<213> Artificial Sequence
<220>
<223> 重链可变区
<400> 102
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe
50 55 60
Lys Gly Arg Val Thr Leu Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr
100 105 110
Val Ser Ser
115
<210> 103
<211> 115
<212> PRT
<213> Artificial Sequence
<220>
<223> 重链可变区
<400> 103
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe
50 55 60
Lys Gly Arg Val Thr Leu Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr
100 105 110
Val Ser Ser
115
<210> 104
<211> 115
<212> PRT
<213> Artificial Sequence
<220>
<223> 重链可变区
<400> 104
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe
50 55 60
Lys Gly Arg Val Thr Leu Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr
100 105 110
Val Ser Ser
115
<210> 105
<211> 115
<212> PRT
<213> Artificial Sequence
<220>
<223> 重链可变区
<400> 105
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe
50 55 60
Lys Gly Arg Val Thr Leu Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr
100 105 110
Val Ser Ser
115
<210> 106
<211> 115
<212> PRT
<213> Artificial Sequence
<220>
<223> 重链可变区
<400> 106
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe
50 55 60
Lys Gly Arg Val Thr Leu Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr
100 105 110
Val Ser Ser
115
<210> 107
<211> 115
<212> PRT
<213> Artificial Sequence
<220>
<223> 重链可变区
<400> 107
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe
50 55 60
Lys Gly Arg Val Thr Leu Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr
100 105 110
Val Ser Ser
115
<210> 108
<211> 115
<212> PRT
<213> Artificial Sequence
<220>
<223> 重链可变区
<400> 108
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asp Tyr
20 25 30
Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe
50 55 60
Lys Gly Arg Val Thr Leu Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr
100 105 110
Val Ser Ser
115
Claims (11)
1.一种抗GPC3抗体，所述抗GPC3抗体包括重链可变区和轻链可变区，所述抗GPC3抗体具有如下技术特征：
<1> 重链可变区的CDR包括氨基酸序列如SEQ ID No.1所示的CDR-H1；
<2> 重链可变区的CDR包括氨基酸序列如SEQ ID No.2所示的CDR-H2；
<3> 重链可变区的CDR包括氨基酸序列如SEQ ID No.3所示的CDR-H3；
<4> 轻链可变区的CDR包括氨基酸序列如SEQ ID No.4所示的CDR-L1；
<5> 轻链可变区的CDR包括氨基酸序列如SEQ ID No.5所示的CDR-L2；
<6> 轻链可变区的CDR包括氨基酸序列如SEQ ID No.6所示的CDR-L3；
所述SEQ ID No.1中，X为D；所述SEQ ID No.4中，自N端至C端的第一个X氨基酸残基选自H，自N端至C端的第二个X氨基酸残基为A，自N端至C端的第三个X氨基酸残基选自T，自N端至C端的第四个X氨基酸残基选自H；所述SEQ ID No.6中，自N端至C端的第一个X氨基酸残基为L，自N端至C端的第二个X氨基酸残基为Y，自N端至C端的第三个X氨基酸残基为T。
2.如权利要求1所述的一种抗GPC3抗体，其特征在于，还包含如下特征中的一种或多种：
所述抗GPC3抗体为单克隆抗体；
和/或，所述抗GPC3抗体为单链抗体；
和/或，所述抗GPC3抗体的重链可变区的氨基酸序列包括：
a）如SEQ ID No. 105所示的氨基酸序列；或
b）与SEQ ID No. 105所示的氨基酸序列具有80%以上同源性、且具有a）所限定的氨基酸序列功能的氨基酸序列；
和/或，所述抗GPC3抗体的轻链可变区的氨基酸序列包括：
c）如SEQ ID No. 76所示的氨基酸序列；或
d）与SEQ ID No. 76所示的氨基酸序列具有80%以上同源性、且具有c）所限定的氨基酸序列功能的氨基酸序列。
3.一种分离的多核苷酸，编码如权利要求1-2任一权利要求所述的抗GPC3抗体的重链可变区和/或轻链可变区或全长氨基酸。
4.一种构建体，含有如权利要求3所述的分离的多核苷酸。
5.一种抗体的表达系统，所述表达系统含有如权利要求4所述的构建体或基因组中整合有外源的如权利要求3所述的多核苷酸。
6.如权利要求1-2任一权利要求所述的抗GPC3抗体的制备方法，包括如下步骤：在适合表达所述抗GPC3抗体的条件下，培养如权利要求5所述的抗体的表达系统，从而表达出所述的抗GPC3抗体，纯化分离出所述的抗GPC3抗体。
7.如权利要求1-2任一权利要求所述的抗GPC3抗体在制备或筛选肝癌治疗的CAR-T药物中的用途、或制备肝癌诊断药物中的用途。
8.一种分离的多肽，所述多肽包括跨膜域、胞内域和胞外域，所述胞外域包括如权利要求1-2任一权利要求所述的抗GPC3抗体。
9.如权利要求8所述的多肽，其特征在于，所述多肽为嵌合抗原受体；
和/或，所述跨膜域包括CD8α、CD28、DAP10；
和/或，所述胞内域包括4-1BB、CD28、OX40、ICOS、CD3zeta、DAP10；
和/或，所述多肽自N端至C端依次包括所述抗GPC3抗体、跨膜域、胞内域。
10.一种细胞，所述细胞含膜结合的如权利要求8-9任一权利要求所述的多肽，所述细胞为T淋巴细胞和/或NK细胞；
和/或，所述多肽为嵌合抗原受体；
和/或，当所述多肽结合于GPC3抗原时，所述T淋巴细胞和/或NK细胞可以活化和/或刺激从而得以增殖；
和/或，所述T淋巴细胞和/或NK细胞表面表达所述抗GPC3抗体。
11.一种诊断试剂盒，包含诊断有效剂量的如权利要求1-2任一权利要求所述的抗GPC3抗体或其免疫偶联物。
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