CN110291108A - 新型tnfr激动剂及其用途 - Google Patents

新型tnfr激动剂及其用途 Download PDF

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CN110291108A
CN110291108A CN201780086444.3A CN201780086444A CN110291108A CN 110291108 A CN110291108 A CN 110291108A CN 201780086444 A CN201780086444 A CN 201780086444A CN 110291108 A CN110291108 A CN 110291108A
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S·布莱因
F·卢梭
R·利西拉
J·拜克
J·麦考因
C·斯图茨
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Ichnos Sciences SA
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Abstract

本发明涉及一类新的TNFR激动剂，其包含与同一TNFR的两个不同部分结合的多个结合部分。本发明还涉及通过施用根据本发明的TNFR激动剂来激活患者免疫系统组分的方法，以及这些材料用于进一步治疗和其他目的的用途。

Description

新型TNFR激动剂及其用途
技术领域
本发明涉及一类新的肿瘤坏死因子受体超家族(TNFR)激动剂，其包含与TNFR的至少两个不同部分结合的多个结合部分。本发明还涉及通过施用根据本发明的TNFR激动剂来激活患者免疫系统组分的方法，以及这些材料用于治疗和其他目的的用途。
背景技术
免疫疗法已经成为抗癌疗法开发创新的一个主要焦点，因为当成功的患者具有持久的抗肿瘤免疫反应时，不仅可以根除原发性肿瘤，还可以根除转移性病变，并可以导致建立保护性抗肿瘤记忆免疫反应。研究人员已特别关注并且取得了巨大的成功，这些疗法抵消了检查点抑制剂，例如CTLA-4和PD-1，它们通过抗体介导的这些受体的拮抗作用去除了体内对抗肿瘤T细胞反应的抑制。然而，越来越清楚的是，去除一种或多种检查点抑制剂的作用不足以促进大多数患者的肿瘤消退。产生强大的治疗性免疫应答不仅需要去除抑制途径，还需要激活刺激途径。
在肿瘤内存在检查点抑制剂，抑制T细胞功能以抑制抗肿瘤免疫应答。检查点抑制剂，例如CTLA-4和PD-1，减弱T细胞增殖和细胞因子的产生。CD8T细胞应答还需要T细胞受体激活加共刺激，其可通过结合肿瘤坏死因子受体家族成员提供，所述成员包括OX40(CD134)和4-1BB(CD137)。OX40特别令人感兴趣，因为用活化(激动剂)抗OX40单克隆抗体处理可增强T细胞分化和细胞溶解功能，从而增强针对多种肿瘤的抗肿瘤免疫力。当用作单一药剂时，这些药物可以在转移性疾病患者中诱导有效的临床和免疫应答。然而，这些药物中的每一种仅对一部分患者有益，突出了对通过免疫系统的更多途径/组分起作用的更有效的组合治疗策略的迫切需求。
肿瘤坏死因子(TNF)/肿瘤坏死因子受体(TNFR)超家族的成员主要参与维持免疫系统的体内平衡。免疫系统的生物学功能包括炎症和宿主防御中的有益和保护作用以及器官发生中的关键作用。
TNFR超家族的成员列于下表1中。
表1
OX40(CD134；TN FRSF4)是TNFR超家族的成员，最初被表征为一种受体，其为在伴刀豆球蛋白A的刺激后主要由来自胸腺和淋巴结的大鼠CD4T细胞表达。随后的研究表明，在小鼠和人类中，OX40在抗原特异性引发期间由CD4和CD8T细胞表达，并且在TCR/CD3交联后诱导OX40表达，并且通过包括IL-1、IL-2和TNF-α的炎性细胞因子的存在诱导OX40表达。抗原接触后OX40的表达对于CD4和CD8T细胞(24-72小时)而言都是短暂的，据报道CD8T细胞的OX40表达持续时间短于CD4T细胞。在没有激活信号的情况下，已经显示相对较少的成熟T细胞亚群在生物学相关水平表达OX40。然而，已经在小鼠和人中描述了滤泡辅助CD4T细胞(Tfh)对OX40的组成型表达。在生发中心，已经显示Tfh细胞的CD4+/CXCR5+/CCR7-亚群具有最高水平的OX40表达，并且被认为是抗体产生的重要调节剂。在小鼠中，OX40也在FoxP3+调节性T细胞(Treg细胞)上组成型表达，与表达是可诱导的人Treg细胞相反。相反，抗原特异性激活可以通过分化的CD4和CD8T细胞的多个亚群诱导OX40表达。在鼠模型系统(OT-II)中，Th1和Th17细胞都能够响应肽激活而类似地强烈诱导OX40。在人类中，相当大比例的肿瘤浸润性CD4T细胞表达OX40，可能是由于肿瘤抗原的识别，并且OX40+CD4T细胞的频率可能是患者预后的结果。类似地，已经显示活化的外周CD8T细胞在小鼠和人中表达OX40。
使用其天然配体(OX40L)或激动剂抗体在CD8和常规(非调节)CD4T细胞上连接OX40可促进其存活和扩增。这方面的证据来自使用OX40和OX40L缺陷小鼠的研究，这些小鼠在最近的几篇综述中有详细讨论。这些研究表明，OX40-或OX40L-敲除小鼠具有减少的CD4和CD8T细胞的扩增，结合抗原攻击后的缺陷记忆反应，表明内源性OX40表达在调节T细胞扩增中的重要性。此外，用激动剂抗OX40单克隆抗体(mAb)处理并使用TCR刺激在野生型动物中诱导扩增、分化并增加CD4和CD8T细胞的存活。同样，CD8或CD4T细胞的消耗消除了抗OX40单克隆抗体在几种肿瘤模型中诱导肿瘤消退的能力。一项研究表明，抗OX40给药足以克服CD8T细胞对自身抗原的耐受性并恢复其细胞毒活性，突出了OX40激动剂的治疗潜力。这对于患有癌症的患者尤其重要，因为T细胞对肿瘤的耐受性是治疗方式的主要障碍。
另一组已经证明，抗OX40治疗后增强的CD8T细胞功能是通过诱导效应T细胞上的CD40L表达介导的，从而促进DC成熟，因为CD40-/-小鼠具有显著更少的CD11c+树突细胞迁移到引流淋巴中遵循抗OX40单克隆抗体的节点。事实上，与具有60％存活率的野生型小鼠相比，用抗OX40mAb处理的CD40-/-小鼠全部死于它们的肿瘤，表明OX40刺激后CD40表达的重要性。总的来说，这些数据表明外源性操纵OX40信号可以促进停滞的T细胞反应。一些研究人员进行了研究，以确定OX40促进T细胞存活的机制。已经证明，在激活后，OX40缺陷的CD4T细胞不能维持抗凋亡蛋白Bc1-xL和Bc1-2的表达。此外，通过Bc1-xL或Bc1-2的逆转录病毒转导拯救了活化的CD4T细胞的存活。在OX40共刺激后，Bcl-xL的持续表达对于肿瘤反应性CD8T细胞的存活也是必需的。随后的研究表明，T细胞中的OX40信号传导诱导了存活蛋白的表达，并且这需要随时间调节和维持T细胞分裂。通过OX40信号传导持续激活PI3K和PKB维持存活蛋白表达。然而，为了抑制T细胞凋亡，存活蛋白表达不会取代OX40信号传导后对Bcl-xL和Bcl-2的需求。通过OX40信号传导后激活IκB激酶和NF-κB1介导存活蛋白和Bcl-2家族成员的增强表达。其他研究者已经表明，在抗原特异性CD4T细胞中OX40信号传导后需要TRAF2，因为CD4T细胞中显性失活TRAF2的表达抑制了它们的扩增、存活和细胞因子产生。TRAF2的功能之一似乎是通过OX40预防T细胞共刺激后的CTLA-4表达，因为用抗原和抗OX40单克隆抗体引发T细胞时的CTLA-4阻断部分恢复了表达显性负性TRAF2蛋白的小鼠的缺陷扩增。在配体与其他TNFR家族成员(例如CD27和GITR)结合后，是否在T细胞中激活相同的TRAF衔接子和NF-κB途径仍然是未知的。
由T细胞共刺激受体(包括TNFR家族成员，如OX40和CD27，以及免疫球蛋白超家族成员，如CD28和B7家族)激活的信号传导途径的相似性和差异，已在其他地方进行了广泛的综述。共刺激受体超家族激活多种途径导致细胞生长和效应功能增强，并提高存活率。目前，许多研究人员正在测试这些受体对各种临床应用和免疫疗法的调节作用。临床前研究表明，用OX40激动剂(包括抗OX40单克隆抗体和OX40L-Fc融合蛋白)治疗肿瘤宿主导致几种临床前模型中的肿瘤消退。最近的研究已经研究了这些激动剂起作用的机制。除了促进效应T细胞扩增，由于OX40在Treg细胞上组成型表达，OX40激动剂具有直接调节Treg细胞的能力。关于这些激动剂是否促进或减弱Treg细胞反应的报道相互矛盾。一些人观察到抗OX40单克隆抗体在体内阻断了Treg细胞的抑制功能，而其他人观察到Treg细胞扩增。这些研究表明，抗OX40可以在两个方向上推动Treg细胞，这取决于刺激和细胞因子环境的背景。实际上，OX40共刺激途径在调节免疫中的重要性通过在具有组成型表达OX40L的小鼠中存在自身免疫样疾病来举例说明。还显示OX40信号传导通过Treg细胞抑制IL-10的产生和抑制Treg细胞的功能。支持这些数据，在肿瘤植入前施用抗OX40单克隆抗体使Treg细胞通过抑制IL-10产生和消除Treg细胞介导的CD8T细胞应答抑制而功能失活。最近的一篇报道观察到，表达活化FcγR的细胞是从肿瘤中选择性消除Treg细胞所必需的，而在抗OX40治疗后第5天，引流淋巴结中的Treg细胞没有变化。其他研究证实，即使在抗OX40治疗后的较晚时间点，引流淋巴结中Treg细胞的频率也没有变化，因此这种作用可能局限于肿瘤。事实上，这种效应可能是短暂的，因为另一份报告显示，在第7天，使用相同的CT26结肠癌模型，对照处理的小鼠和抗OX40处理的小鼠之间的肿瘤中的Treg细胞频率没有差异。该研究还特别表明，抗OX40治疗的免疫学效果可根据所检查的肿瘤模型而变化。因此，必须谨慎对OX40激动剂的精确机制进行概括。其他研究报道，抗OX40单克隆抗体在体外和体内降低Treg细胞的抑制活性。抗OX40是否通过Treg细胞抑制、缺失或两者起作用，用这些激动剂治疗应该减少Treg细胞介导的抑制作用，从而促进维持长期抗肿瘤免疫应答所必需的抗肿瘤CD8T细胞应答。多种机制可能对OX40激动剂的抗肿瘤活性很重要。
鉴于人体免疫系统的复杂性和可塑性以及处理癌症患者的免疫系统的进一步复杂性，其被肿瘤细胞有目的地破坏以逃避引发治愈性免疫应答，组合疗法调节不同的免疫系统受体/在临床前和患者中越来越多地提出和验证细胞群。例如，该领域的工作人员已经表明，PD-1拮抗性抗体和OX40激动性抗体的测序对于同时给药是至关重要的，导致OX40激动剂的作用被否定(Shrimali等，Cancer Immunol Res；5(9)；1-12)和Messenhiemer等，Clin Cancer Res.2017年10月15日；23(20)：6165-6177)。
这种复杂性还意味着随着免疫肿瘤学领域的进一步发展以及对使用免疫调节剂引发治疗性免疫应答的最佳方法的理解的增加，生成药理活性物质对于尽可能广泛的相关靶标的范围至关重要。TNFR可能是最重要的免疫肿瘤学目标类别，并且迄今为止难以产生药理学活性激动剂。
发明内容
本发明涉及TNFR激动剂，其包含与TNFR的至少两个不同部分结合的结合部分。
本发明人惊奇地发现，与不包含在相同激动剂中的结合部分的效果以及与TNFR的天然配体和其他先前抑制的TNFR相比，包含与TNFR的至少两个不同部分或表位结合的结合部的激动剂显示出更好的激动水平。
根据本发明，TNFR选自表1中所示的组或TNFR超家族的任何其他成员。
优选地，TNFR参与T细胞应答的共刺激。
优选地，TNFR选自包括CD27、4-1BB(CD137)、OX40(CD134)、HVEM、CD30和GITR的组，并且最优选是OX40。
根据本发明，术语“TNF受体的两个不同部分”是指可以同时被每个结合部中的一个结合的TNFR的两个部分，这意味着它们可以同时结合在相同的TNFR上或在两种相同的TNFR之间通过同时结合这些来桥接。
特别地，本发明涉及来自基于蛋白质的靶特异性结合分子的结合部，例如抗体、DARPin、Fynomer、Affimer、可变淋巴细胞受体、anticalin、nanofitin、可变新抗原受体(VNAR)，但不限于这些。
特别地，TNFR包含取自或衍生自抗体的结合部分，例如Fab、Fab′、Fab′-SH、Fd、Fv、dAb、F(ab′)2、scFv、Fcab、双特异性单链Fv二聚体、双抗体、三抗体。在优选的实施方案中，激动剂包含取自或衍生自Fab、ScFv和dAb的结合部分。
根据本发明的另一个方面，包含激动剂的结合部分是不同类型的，优选的实施方案是在同一激动剂中组合Fab和scFv或Fab和dAb结合部分。
已知方法将Fab结合部分转化为其他类型的结合部分，例如scFv、dAb、scFab，并且类似地将这些结合部分可互换地转化成Fab。
特别地，结合部分可以遗传融合至包含相同或不同抗体Fc或其部分的支架。根据本发明的这个方面，第一全长抗体如IgG可以形成本发明激动剂的基础，并且第二组结合部分可以移植到根据本发明的起始抗体上。
或者，结合部分可以遗传融合至除了衍生自免疫球蛋白Fc的支架之外的支架，例如基于Fynomer中使用的Fyn的SH3结构域的那些结合部分和基于Affimer中使用的人蛋白酶抑制剂Stefin A的那些结合部分。
根据本发明，结合TNFR的不同部分的结合部分分别位于TNFR激动剂内包含的支架的C和N末端。
根据本发明的另一个方面，结合部分位于C或N末端并连接。
优选地，与TNFR的相同部分结合的结合部分位于激动剂的相同末端。根据本发明，TNFR的第一部分的结合部分设置在C或N末端，TNFR的第二部分的结合部分设置在相对的末端。本发明人发现，靶TNFR的相同部分的结合部分应优先置于激动剂的相同末端。
根据本发明的另一个方面，结合部分可以是基于核苷酸的，例如适体。
优选地，激动剂包含多于两个的结合部分。
更优选地，激动剂包含四个或更多个结合部分。
优选地，激动剂包含至少两个结合部分，其结合TNFR的相同部分/表位。
最优选地，激动剂包含至少两组两个相同的结合部分。本发明人已经发现，包含TNFR的每个部分/表位的两个结合部分并且位于激动剂任一端的TNFR激动剂显示出始终如一的高水平的激动作用。
特别地，本发明人已发现包含结合部分的激动剂结合相同TNFR的不同富含半胱氨酸的结构域(CRD)，这意味着它们包含来自TNFR的不同富含半胱氨酸的结构域(CRD)的膜近端和膜远端结合部分。
优选地，激动剂结合膜近端和膜远端表位。
根据本发明的另一方面，涉及OX40受体(OX40)激动剂，其包含与OX40的两个不同部分/表位结合的多个OX40结合部分。
根据本发明，OX40激动剂结合OX40的富含半胱氨酸的结构域(CRD)1和CRD 3中的表位。或者，OX40激动剂结合CRD1和CRD4。
根据本发明的另一方面，OX40结合部分选自选自下组的序列：SEQ ID NO：2、3、12、13、14、15、16、17、18、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49，或具有与其至少50％、60％、65％、70％、75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的氨基酸序列的分离多肽。本发明还涉及包含说明书和序列表中包含的任何其他OX40结合部分的构建体。
根据本发明的一个优选实施方案，OX40激动剂由SEQ ID NO：45和16编码或为与其具有至少50％、60％、65％、70％、75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列的分离的多肽。
本发明还涉及通过施用根据本发明的OX40激动剂来激活患者免疫系统组分的方法。
本发明还涉及根据本发明的OX40激动剂作为药物的用途。
本发明还涉及根据本发明的OX40激动剂作为药物的用途，所述药物用于治疗癌症、免疫病症或通过激活患者免疫系统而表征或激动的其他疾病。
本发明还涉及治疗患有癌症的患者的方法，包括给患者施用有效量的OX40激动剂。
本发明还涉及治疗患有癌症的患者的方法，包括给患者施用有效量的OX40激动剂和一种或多种其他药剂，例如小分子或生物药物，以进一步调节患者的免疫系统。此类药剂的实例包括抗PD-1抗体和抗肿瘤小分子，例如多激酶抑制剂。
此外，本发明涉及将根据本发明的OX40激动剂和另一种药物共同施用于患者，其中所述其他药物具有协同或累加效应。
根据本发明的另一方面，涉及CD40受体(CD40)激动剂，其包含多个CD40结合部分。
优选地，激动剂包含多于两个的结合部分。
更优选地，激动剂包含四个结合部分
优选地，激动剂包含至少两个相同的结合部分。
优选地，激动剂包含至少两组两个相同的结合部分。
或者，激动剂包含与相同表位结合的四个结合部分。
本发明还涉及通过施用根据本发明的CD40激动剂激活患者免疫系统组分的方法。
根据本发明的CD40激动剂作为药物的用途。
根据本发明的另一个方面，TNFR激动剂包含两种单克隆抗体，其识别并结合相同TNFR的两个不同部分，并且可以共同施用于需要其的患者。
此外，本发明涉及根据本发明的TNFR激动剂和另一种药物共同给予患者，其中所述其他药物具有协同或累加效应。
非详尽列举的药物列表包括T细胞重定向多特异性抗体、检查点抑制剂、免疫调节剂。
本发明还涉及这些材料用于进一步治疗和其他用途的用途。
除非另外定义，否则结合本发明使用的科学和技术术语应具有本领域普通技术人员通常理解的含义。此外，除非上下文另有要求，否则单数术语应包括复数，复数术语应包括单数。通常，与本文所述的细胞和组织培养、分子生物学和蛋白质和寡核苷酸或多核苷酸化学和杂交相关的术语和技术是本领域公知和常用的那些。标准技术用于重组DNA，寡核苷酸合成和组织培养和转化(例如，电穿孔、脂质转染)。酶促反应和纯化技术根据制造商的说明书进行或如本领域通常实现的或如本文所述进行。前述技术和程序通常根据本领域熟知的常规方法进行，并且如在本说明书通篇引用和讨论的各种一般和更具体的参考文献中所述。参见例如Sambrook等，Molecular Cloning：A Laboratory Manual(第2版，Cold SpringHarbor Laboratory Press，Cold Spring Harbor，N.Y.(1989))。与本文所述的分析化学、合成有机化学以及药物和药物化学相关的术语和实验室程序和技术是本领域公知和常用的那些。标准技术用于化学合成、化学分析、药物制备、配制和递送以及患者的治疗。
已知基本抗体结构单元包含四聚体。每个四聚体由两对相同的多肽链组成，每对具有一个“轻”链(约25kDa)和一个“重”链(约50-70kDa)。每条链的氨基末端部分包括主要负责抗原识别的约100至110或更多氨基酸的可变区。每条链的羧基末端部分限定了主要负责效应子功能的恒定区。通常，从人获得的抗体分子涉及IgG、IgM、IgA、IgE和IgD中的任何类别，它们通过分子中存在的重链的性质彼此不同。某些类也具有亚类(也称为同种型)，例如IgG1、IgG2等。此外，在人类中，轻链可以是κ链或λ链。
如本文所用，术语“单克隆抗体”(MAb)或“单克隆抗体组合物”是指抗体分子群，其仅含有一种分子种类的抗体分子，其由独特的轻链基因产物和独特的重链基因产物组成。特别地，单克隆抗体的互补决定区(CDR)在群体的所有分子中是相同的。MAb含有能够与抗原的特定表位发生免疫反应的抗原结合位点，其特征在于对其具有独特的结合亲和力。
术语“抗原结合位点”或“结合部分”是指参与抗原结合的免疫球蛋白分子部分。抗原结合位点由重(“H”)链和轻(“L”)链的N-末端可变(“V”)区的氨基酸残基形成。重链和轻链的V区内的三个高度发散的区段(称为“高变区”)介于称为“框架区”或“FR”的更保守的侧翼区段之间。因此，术语“FR”是指天然存在于免疫球蛋白中的高变区之间并与其相邻的氨基酸序列。在抗体分子中，轻链的三个高变区和重链的三个高变区在三维空间中相对于彼此设置以形成抗原结合表面。抗原结合表面与结合抗原的三维表面互补，并且每条重链和轻链的三个高变区被称为“互补决定区”或“CDR”。每个结构域的氨基酸分配符合KabatSequences of Proteins of Immunological Interest(National Institutes ofHealth，Bethesda，Md.(1987和1991))或Chothia&Lesk J.Mol.Biol.196：901-917(1987)，Chothia等，Nature 342：878-883(1989)的限定。
本发明的融合蛋白的单结构域抗体(sdAb)片段部分在本文中可互换地称为靶向多肽。
如本文所用，术语“表位”包括能够与免疫球蛋白或其片段特异性结合的任何蛋白质决定簇，或T细胞受体。术语“表位”包括能够与免疫球蛋白或T细胞受体特异性结合的任何蛋白质决定簇。表位决定簇通常由分子的化学活性表面基团组成，例如氨基酸或糖侧链，并且通常具有特定的三维结构特征以及特定的电荷特征。当解离常数≤1mM时，例如在一些实施方案中≤1μM，如≤100nM，≤10nM或≤1nM，认为抗体特异性结合抗原。
如本文所用，术语“免疫结合”和“免疫结合特性”是指在免疫球蛋白分子和免疫球蛋白特异性的抗原之间发生的类型的非共价相互作用。免疫结合相互作用的强度或亲和力可以用相互作用的解离常数(Kd)表示，其中较小的Kd表示较大的亲和力。可以使用本领域熟知的方法定量所选多肽的免疫结合特性。一种这样的方法需要测量抗原结合位点/抗原复合物形成和解离的速率，其中所述速率取决于复合物配偶体的浓度、相互作用的亲和力、以及同样影响两个方向上的速率的几何参数。因此，“结合速率常数”(kon)和“解离速率常数”(koff)都可以通过计算浓度和实际的缔合和解离速率来确定。(参见Nature 361：186-87(1993))。koff/kon的比率使得能够消除与亲和力无关的所有参数，并且等于解离常数Kd。(一般参见Davies等(1990)，Annual Rev Biochem 59：439-473)。当平衡结合常数(Kd)≤1mM时，在一些实施方案中，≤1μM，≤100nM，＜10nM，或≤100pM至约1pM，通过诸如放射性配体结合、表面等离子体共振(SPR)、流式细胞术结合测定或本领域技术人员已知的类似测定的测定法测定，认为本发明的抗体特异性结合抗原。
本文提及的术语“分离的蛋白质”是指cDNA、重组RNA或合成来源的蛋白质或其某些组合，根据其来源或衍生来源，“分离的蛋白质”(1)与在自然界中发现的蛋白质无关，(2)不含来自相同来源的其他蛋白质，例如，不含海洋蛋白质，(3)由来自不同物种的细胞表达，或(4)不存在于自然界中。
术语“多肽”在本文中用作通用术语，指多肽序列的天然蛋白质、片段或类似物。因此，天然蛋白质片段和类似物是多肽属的种类。
如本文所用，术语“天然存在的”应用于物体是指物体可以在自然界中找到的事实。例如，存在于生物体(包括病毒)中的多肽或多核苷酸序列是天然存在的，所述多肽或多核苷酸序列可以从自然界中的来源分离并且在实验室中没有被人有意地修饰。
术语“序列同一性”是指两个多核苷酸或氨基酸序列在比较窗口上是相同的(即，在核苷酸-核苷酸或残基-残基的基础上)。术语“序列同一性百分比”通过比较比较窗口上的两个最佳比对序列，确定相同核酸碱基(例如A，T，C，G，U或I)的位置数或残基发生在两个序列中以产生匹配位置的数量，将匹配位置的数量除以比较窗口中的位置总数(即窗口大小)，并将结果乘以100以得到序列同一性的百分比。本文所用的术语“基本同一性”表示多核苷酸或氨基酸序列的特征，其中多核苷酸或氨基酸包含具有至少85％序列同一性的序列，例如，至少90％至95％的序列同一性，通常在至少18个核苷酸(6个氨基酸)位置的比较窗口上，通常在至少24-48个核苷酸(8-16个氨基酸)位置的窗口上，与参考序列相比，通常具有至少99％的序列同一性，其中序列同一性的百分比通过比较参考序列与序列来计算，所述序列可包括在比较窗口上总共20％或更少参考序列的缺失或添加。参考序列可以是较大序列的子集。
如本文所用，二十种常规氨基酸及其缩写遵循常规用法。参见Immunology-ASynthesis(第2版，E.S.Golub和D.R.Gren著，Sinauer Associates，Sunderland7Mass.(1991))。二十种常规氨基酸的立体异构体(例如D-氨基酸)，非天然氨基酸如α-，α-二取代氨基酸，N-烷基氨基酸，乳酸和其他非常规氨基酸也可以是本公开的多肽的合适的组分。非常规氨基酸的实例包括：4-羟基脯氨酸，γ-羧基谷氨酸，ε-N，N，N-三甲基赖氨酸，ε-N-乙酰基赖氨酸，O-磷酸丝氨酸，N-乙酰丝氨酸，N-甲酰基甲硫氨酸，3-甲基组氨酸，5-羟基赖氨酸，σ-N-甲基精氨酸和其他类似的氨基酸和亚氨基酸(例如，4-羟基脯氨酸)。在本文使用的多肽符号中，根据标准用法和惯例，左手方向是氨基末端方向，右手方向是羧基末端方向。
类似地，除非另有说明，否则单链多核苷酸序列的左手末端是5′末端，双链多核苷酸序列的左手方向称为5′方向。新生RNA转录物的5′至3′添加方向称为与RNA具有相同序列的DNA链上的转录方向序列区域，其在RNA转录物的5′末端至5′末端被称为作为“上游序列”，DNA链上具有与RNA相同序列并且在RNA转录物的3′至3′末端的序列区域被称为“下游序列”。
当应用于多肽时，术语“基本同一性”是指两个肽序列在最佳比对时，例如通过程序GAP或BESTFIT使用默认空位权重，共享至少80％的序列同一性，例如，至少90％序列同一性，至少95％的序列同一性，或至少99％的序列同一性。
在一些实施方案中，不相同的残基位置因保守氨基酸取代而不同。
保守氨基酸取代是指具有相似侧链的残基的可互换性。例如，具有脂肪族侧链的一组氨基酸是甘氨酸、丙氨酸，缬氨酸，亮氨酸和异亮氨酸。具有脂肪族-羟基侧链的一组氨基酸是丝氨酸和苏氨酸；具有含酰胺侧链的一组氨基酸是天冬酰胺和谷氨酰胺；具有芳香族侧链的一组氨基酸是苯丙氨酸、酪氨酸和色氨酸；具有碱性侧链的一组氨基酸是赖氨酸、精氨酸和组氨酸；具有含硫侧链的一组氨基酸是半胱氨酸和甲硫氨酸。合适的保守氨基酸取代基是：缬氨酸-亮氨酸-异亮氨酸、苯丙氨酸-酪氨酸、赖氨酸-精氨酸、丙氨酸缬氨酸、谷氨酸-天冬氨酸和天冬酰胺-谷氨酰胺。
如本文所讨论的，预期抗体或免疫球蛋白分子的氨基酸序列的微小变化被本公开涵盖，条件是氨基酸序列的变化保持至少75％，例如，至少80％、90％、95％或99％。特别是，考虑了保守氨基酸替代。保守替代是在与其侧链相关的氨基酸家族中发生的那些。遗传编码的氨基酸通常分为以下家族：(1)酸性氨基酸是天冬氨酸、谷氨酸；(2)碱性氨基酸有赖氨酸、精氨酸、组氨酸；(3)非极性氨基酸是丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、蛋氨酸、色氨酸；和(4)不带电荷的极性氨基酸是甘氨酸、天冬酰胺、谷氨酰胺、半胱氨酸、丝氨酸、苏氨酸、酪氨酸。亲水性氨基酸包括精氨酸、天冬酰胺、天冬氨酸、谷氨酰胺、谷氨酸、组氨酸、赖氨酸、丝氨酸和苏氨酸。疏水性氨基酸包括丙氨酸、半胱氨酸、异亮氨酸、亮氨酸、甲硫氨酸、苯丙氨酸、脯氨酸、色氨酸、酪氨酸和缬氨酸。其他氨基酸家族包括(i)丝氨酸和苏氨酸，它们是脂肪族-羟基家族；(ii)天冬酰胺和谷氨酰胺，它们是含有酰胺的家族；(iii)丙氨酸、缬氨酸、亮氨酸和异亮氨酸，它们是脂肪族；(iv)苯丙氨酸、色氨酸和酪氨酸，它们是芳香族。例如，可以合理地预期用异亮氨酸或缬氨酸替换亮氨酸，用谷氨酸替换天冬氨酸，用丝氨酸替换苏氨酸，或用结构相关氨基酸替换氨基酸的类似替换，将不会对所得分子的结合或性质的主要影响，特别是如果替换不涉及框架位点内的氨基酸。通过测定多肽衍生物的比活性，可以容易地确定氨基酸变化是否导致功能性肽。本文详细描述了测定方法。本领域普通技术人员可以容易地制备抗体或免疫球蛋白分子的片段或类似物。合适的片段或类似物的氨基和羧基末端出现在功能域的边界附近。可以通过将核苷酸和/或氨基酸序列数据与公共或专有序列数据库进行比较来鉴定结构域和功能域。在一些实施方案中，计算机化比较方法用于鉴定在已知结构和/或功能的其他蛋白质中发生的序列基序或预测的蛋白质构象结构域。鉴定折叠成已知三维结构的蛋白质序列的方法是已知的。Bowie等，Science 253：164(1991)。因此，前述实施例证明，本领域技术人员可以识别可用于定义根据本发明的结构域和功能域的序列基序和结构构象。
合适的氨基酸取代是：(1)降低对蛋白水解的易感性，(2)降低对氧化的易感性，(3)改变对形成蛋白质复合物的结合亲和力，(4)改变结合亲和力，和(4)赋予或修饰这些类似物的其他物理化学或功能特性。类似物可包括除天然存在的肽序列之外的序列的各种突变蛋白。例如，可以在天然存在的序列中进行单个或多个氨基酸取代(例如，保守氨基酸取代)(例如，在形成分子间接触的结构域外的多肽部分中)。保守氨基酸取代不应该显著改变亲本序列的结构特征(例如，取代氨基酸不应该破坏在亲本序列中发生的螺旋，或破坏表征亲本序列的其他类型的二级结构)。蛋白质识别的多肽二级和三级结构的实例描述于Proteins，Structures and Molecular Principles(Creighton著，W.H.Freeman andCompany，New York(1984))；Introduction to Protein Structure(C.Branden和J.Tooze著，Garland Publishing，New York，N.Y.(1991))；和Thornton等，Nature354：105(1991)。
如本文所用的术语“多肽片段”是指具有氨基末端和/或羧基末端缺失的多肽，但其中剩余的氨基酸序列与得出其的天然序列中的相应位置相同，例如，来自全长cDNA序列。片段通常长至少5、6、8或10个氨基酸，例如，至少14个氨基酸长，至少20个氨基酸长，至少50个氨基酸长，或至少70个氨基酸长。本文所用的术语“类似物”是指在合适的结合条件下由至少25个氨基酸的片段组成的多肽，其与推定的氨基酸序列的一部分具有基本同一性并且具有与CD47的特异性结合。通常，多肽类似物包含相对于天然存在的序列的保守氨基酸取代(或添加或缺失)。类似物通常至少20个氨基酸长，例如，至少50个氨基酸长或更长，并且通常可以与全长天然存在的多肽一样长。
肽类似物通常在制药工业中用作具有与模板肽类似性质的非肽药物。这些类型的非肽化合物被称为“肽模拟物”或“多肽模拟物”。Fauchere，J.Adv.Drug Res.15：29(1986)，Veber和Freidinger TINS p.392(1985)；和Evans等，J.Med.Chem.30：1229(1987)。这些化合物通常借助于计算机化的分子模型来开发。结构上与治疗上有用的肽类似的肽模拟物可用于产生等效的治疗或预防效果。通常，肽模拟物在结构上类似于范例多肽(即，具有生物化学性质或药理学活性的多肽)，例如人抗体，但具有一个或多个肽键，任选地被选自下组的连接通过本领域熟知的方法取代：--CH2NH--、--CH2S--、--CH2-CH2--、--CH＝CH--(顺式和反式)、--COCH2--、CH(OH)CH2--和-CH2SO--。用相同类型的D-氨基酸(例如，D-赖氨酸代替L-赖氨酸)系统取代共有序列的一个或多个氨基酸可用于产生更稳定的肽。另外，可以通过本领域已知的方法产生包含共有序列或基本相同的共有序列变异的受约束的肽(Rizo和Gierasch Ann.Rev.Biochem.61：387(1992))；例如，通过添加能够形成环化肽的分子内二硫键的内部半胱氨酸残基。
术语“药剂”在本文中用于表示化学化合物、化学化合物的混合物、生物大分子和/或由生物材料制成的提取物。
如本文所用，术语“标记”或“标记的”是指掺入可检测标记，例如通过掺入放射性标记的氨基酸或附着于生物素基部分的多肽，其可以通过标记的抗生物素蛋白(例如包含荧光标记物的链霉抗生物素蛋白或者可通过光学或量热法检测荧光标记物或酶活性)检测。在某些情况下，标签或标记物也可以是治疗性的。标记多肽和糖蛋白的各种方法是本领域已知的并且可以使用。多肽标记的实例包括但不限于以下：放射性同位素或放射性核素(例如，3H、14C、15N、35S、90Y、99Tc、111In、125I，131I)、荧光标记(例如，FITC、罗丹明、镧系元素荧光粉)、酶标记(例如，辣根过氧化物酶、β-半乳糖苷酶、荧光素酶、碱性磷酸酶)、化学发光、生物素基团、由二级报道分子识别的预定多肽表位(例如，亮氨酸拉链对序列、二抗的结合位点、金属结合域、表位标签)。在一些实施方案中，标记通过各种长度的间隔臂连接以减少潜在的空间位阻。如本文所用的术语“药剂或药物”是指当适当地施用于患者时能够诱导所需治疗效果的化合物或组合物。
术语“抗肿瘤剂”在本文中用于指具有抑制人类肿瘤发展或进展的功能特性的药剂，所述肿瘤特别是恶性(癌性)病变，例如癌、肉瘤、淋巴瘤或白血病。转移的抑制通常是抗肿瘤剂的特性。
如本文所用，术语“治疗”、“处理”、“医治”等是指减少和/或改善与其相关的病症和/或症状。“减轻”和/或“减缓”是指减少、抑制、减弱、弱化、阻止和/或稳定疾病(例如癌症)的发展或进展。应当理解，尽管不排除，但治疗病症或疾病不需要完全消除与其相关的紊乱、病症或症状。
本文中的其他化学术语根据本领域的常规用法使用，例如The McGraw-HillDictionary of Chemical Terms(Parker，S.著，McGraw-Hill，San Francisco(1985))。
如本文所用，“基本上纯的”是指目标物质是存在的主要物质(即，以摩尔计，它比组合物中的任何其他个体物种更充裕)，并且在一些实施方案中，基本上纯化的部分是组合物。其中目标物质包含至少约50％(以摩尔计)存在的所有大分子物质。
通常，基本上纯的组合物将占组合物中存在的所有大分子物质的大于约80％，例如，大于约85％、90％、95％和99％。在一些实施方案中，将目标物质纯化至基本均匀性(通过常规检测方法不能在组合物中检测到污染物质)，其中组合物基本上由单一大分子物质组成。在本公开中，“包含”、“含”、“含有”，“具有”等可以具有美国和/或欧洲专利法中赋予它们的含义，并且可以表示“包括”、“包括有”等；术语“基本上由......组成”或“基本上由......构成”同样具有美国专利法中赋予的含义，并且这些术语是开放式的，允许存在多于所述的那些物质，只要其具有基本或新颖的特征即可。所引用的内容不会因所述内容的存在而改变，但不包括现有技术实施例。
“有效量”是指相对于未治疗的患者改善疾病症状所需的量。用于实施本发明治疗疾病的活性化合物的有效量根据给药方式、受试者的年龄、体重和一般健康状况而变化。最终，主治医师或兽医将决定适当的量和剂量方案。该量称为“有效”量。
“受试者”是指哺乳动物，包括但不限于人或非人哺乳动物，例如牛、马、犬、啮齿动物、绵羊、灵长类动物、骆驼科动物或猫科动物。如本文所用，术语“施用”是指将治疗剂转移、递送、引入或运输至需要用这种药剂治疗的受试者的任何模式。这些模式包括但不限于口服、局部、静脉内、腹膜内、肌肉内、皮内、鼻内和皮下给药。
附图说明
图1：固定浓度(200nM)下2H6 scFv-Fc(上线)和2H6M108L scFv-Fc(下线)与CM5芯片上捕获的人OX40R结合的SPR传感图，配体密度为600RU，25℃。
图2：该图显示了来自4个独立MLR实验的标准化3H-胸苷掺入的结果，用平均值±SD表示。每个数据点是各个同种异体组合的一式三份的平均值。虚线表示同种异体反应的水平(无抗体)。所有组合没有显著差异(ns)。
图3：PBMC在有或没有抗体的SEB存在下孵育7天；在第5天收获上清液。该图显示来自5个独立实验的每孔CD4CD25+的标准化绝对计数的平均值±SD(A)和标准化的IL-2浓度(B)。每个数据点是三次重复值的平均值，并代表独立的PBMC供体。虚线表示PBMC仅与SEB(无抗体)孵育的条件水平，ns，不显著；*，p＜0.05；***，p＜0.001，使用单尾非参数Mann-Whitney检验获得。
图4：PBMC在有或没有抗体的PHA存在下孵育5天。该图显示了来自3次独立实验的归一化3H-胸苷掺入的结果，用平均值±SD表示。每个数据点是三次重复值的平均值，并代表独立的PBMC供体。虚线表示PBMC仅与PHA(无抗体)一起孵育的条件水平，ns表示不显著。
图5：Tetra-1和Tetra-8的SDS-PAGE分析。在蛋白A纯化后获得的Tetra-1和Tetra-8的非还原条件下的考马斯蓝染色SDS-PAGE凝胶的照片。(MW)分子量标记如图所示。
图6：Tetra-1和Tetra-8的分析尺寸排阻色谱。图6A和6B是一系列图，描绘了来自Tetra-1(图6A)和Tetra-8(图6B)的尺寸排阻色谱(SEC)柱的洗脱曲线。根据其保留时间计算每个峰的峰面积百分比(％)，其表示样品色谱图中总“可检测”峰的百分比(取100％)，并在Tetra-1表中显示(图6C)和Tetra-8(图6D)。
图7：Tetra-8的阳离子交换纯化。图7A显示了描绘来自阳离子交换HiTrapSP HP柱的Tetra-8(虚线)的洗脱曲线的图。用于蛋白质分离的乙酸钠梯度用黑线表示。图7B是在从Tetra-8的阳离子交换纯化色谱法收集的不同级分的非还原条件下的考马斯蓝染色SDS-PAGE凝胶的照片。
图8：通过差示扫描量热法评估Tetra-1和Tetra-8的热稳定性。图8A和8B分别是使用差示扫描量热法(DSC)表示Tetra-1和Tetra-8的热稳定性测量的图。数据表示为过量摩尔热容量(缩写为Cp[kcal/mol/℃]；Y轴)对温度(℃；X轴)。显示了对应于scFv、Fab、CH2和CH3结构域的解折叠事件。
图9：OX40的细胞外结构域的结构。条带表示人OX40的细胞外结构域(RCSB：2HEV)。富含半胱氨酸的结构域(CRD)可选地使用灰色或黑色突出显示。二硫键由球体描绘。
图10：人、食蟹猴和大鼠OX40细胞外结构域的比对。用T-coffee制备的人(SEQ IDNO：1)、食蟹猴(SEQ ID NO：122)(缩写的cyno)和大鼠OX40(SEO ID NO：121)的细胞外结构域的多序列比对。CRD由白色或黑色框表示。二硫键配对用箭头表示。在物种之间严格保守的残留物用黑色阴影表示，具有70％保守性的残留物用灰色阴影表示。
图11：将各种抗体的剂量反应在重组人OX40受体上孵育，然后用与辣根过氧化物酶特异性偶联的抗人Fab片段检测。该图显示了每种处理的非线性S形回归结合曲线(450nM的吸光度)。测试以下处理：Tetra-8(○)，7H11_v8 IgG1(□)，Tetra-22每个数据点是重复值的平均值±SD。
图12：将各种抗体的剂量反应在JURKAT-NFkB-OX40细胞上孵育，然后用与藻红蛋白特异性偶联的抗人Fc片段进行检测。该图显示了每种处理的非线性S形回归结合曲线(几何平均强度)。测试以下处理：Tetra-8(○)，7H11_v8 IgG1(□)，2H6 IgG1(△)，对照IgG
图13：将各种抗体的剂量反应在各种受体(肿瘤坏死因子受体家族成员)上孵育，然后用链霉抗生物素蛋白与辣根过氧化物酶偶联检测。对所有受体测试相同的处理：Tetra-8(○)，7H11 IgG1(□)，2H6 IgG1(△)，对照IgG各自的商业阳性对照(×)。该图显示了每种处理的非线性S形回归结合曲线(450nM的吸光度)。每个数据点是重复值的平均值，除了以单纯形式执行的控制曲线。
图14：将抗体的剂量反应在重组食蟹猴OX40上孵育，然后用与辣根过氧化物酶特异性偶联的抗人Fab片段检测。该图显示了每种条件下的非线性S形回归结合曲线(450nM的吸光度)。测试以下处理：Tetra-8(○)，7H11 IgG1(□)，Tetra-22(Δ)。每个数据点是重复值的平均值±SD。
图15：将抗体在人和食蟹猴PBMC上孵育，然后用与藻红蛋白特异性偶联的抗人Fc片段进行检测。该图表示人或Rhesus CD4+T细胞上的每种抗体的多个直方图(荧光的几何平均值)的叠加。
图16：将JURKAT-NFkB-OX40细胞转移至OKT3预包被(5μg/mL；过夜)或常规发光板。随后，将抗体或对照的剂量反应在JURKAT-NFkB-OX40细胞上孵育。孵育5小时后，将荧光素酶底物加入孔中，并使用酶标仪读取发光(读取带-终点；积分时间-1分钟；发射-孔；光学位置-顶部；增加135；读取高度-1.00mm)。该图显示了每种条件的非线性S形回归结合曲线(发光)。测试以下处理：Tetra-8(○)，7H11 IgG1 LALA(□)，2H6 IgG1 LALA(△)，对照IgGOX40L(×)。每个数据点是重复值的平均值±SD。
图17：将树突细胞(DC)分化6天，然后与新鲜分离的CD4+T细胞共培养。将抗体的剂量-反应或对照在这些细胞上孵育，然后在孵育6天后，加入氚化胸苷进行18至20小时的孵育。用以下方法计算增殖指数：在每个样品(对每个CD4+T细胞供体特异)中减去在自体条件下诱导的胸苷掺入背景(仅CD4+T细胞)，然后将该结果除以在同种异体条件下诱导的胸苷掺入。图表显示了每种治疗的增殖指数。每个数据点是每个DC-CD4+T细胞组合获得的一式三份重复值的平均值。N＝36种组合。线Y＝1表示归一化的同种异体反应。
图18：从过滤器中分离PBMC，并在抗体或对照存在下与葡萄球菌肠毒素B超抗原(SEB)一起温育。温育5天后，收获上清液并在Luminex上定量IL-2释放。用以下方法计算标准化的IL-2释放：对于每个样品(对于每个PBMC供体特异性)减去在未刺激的细胞(不含SEB的PBMC)中诱导的IL-2定量，然后将该结果除以IL-2。在SEB刺激的细胞中诱导释放(未处理)。填充的粗线表示响应阈值。该图显示了每种处理的标准化IL-2释放。每个数据点是每个PBMC供体获得的一式三份重复值的平均值。N＝17PBMC供体。线Y＝1表示仅归一化的SEB诱导响应。
图19：从过滤器中分离PBMC，并在80和10nM的抗体或对照存在下与葡萄球菌肠毒素B超抗原(SEB)一起温育。温育5天后，收获上清液并在Luminex上定量IL-2释放。用以下方法计算标准化的IL-2释放：对于每个样品(对于每个PBMC供体特异性)减去在未刺激的细胞(不含SEB的PBMC)中诱导的IL-2定量，然后将该结果除以IL-2。在SEB刺激的细胞中诱导释放(未处理)。填充的粗线表示响应阈值。该图显示了每种处理的标准化IL-2释放。每个数据点是每个PBMC供体获得的一式三份重复值的平均值。线Y＝1表示标准化的仅SEB诱导响应。
图20：基于具有不同化合价和结构的7H11和2H6结合单元的分子的示意图。
图21：当在C末端融合为Fab或scFv形式时，7H11和2H6与OX40结合的分析。在其铰链区附近蛋白水解切割的四价分子的表面等离子体共振(SPR)测量(Fc-2H6 Fab/2H6 Fab，Fc-2H6 Fab/7H11 scFv，FC-7H11 Fab/7H11 Fab和Fc-7H11 Fab/2H6 scFv，如图所示)嵌合OX40分子chiOX40R-Fc HHRH(图21A)或chiOX40R-Fc RRHH(图21B)。数据表示为响应单元的数量(缩写为RU；Y轴)对时间(X轴)。图21C显示了分析中使用的激动剂的示意图。
图22：当在C末端融合时7H11 Fab和2H6 scFv的0X40共接合的测定。通过SPR对Fc-7H11 Fab/2H6 scFv片段与嵌合OX40分子chiOX40R-Fc HHRH的共同接合测量(图22A)或固定在CHIP上的chiOX40R-Fc RRHH(图22B)和顺序注射的人OX40(HHHH)，chiOX40R-Fc(HHRH)和chiOX40R-Fc(RRHH)。数据表示为响应单元的数量(缩写为RU；Y轴)对时间(X轴)。图22C显示了分析中使用的激动剂的示意图。
图23：当在C末端融合时，7H11 scFv和2H6 Fab的0X40共接合的测定。通过SPR对Fc-2H6 Fab/7H11 scFv片段与嵌合OX40分子chiOX40R-Fc HHRH的共同接合测量(图23A)或固定在CHIP上的chiOX40R-Fc RRHH(图23B)和顺序注射的人OX40(HHHH)、chiOX40R-Fc(HHRH)和chiOX40R-Fc(RRHH)。数据表示为响应单元的数量(缩写为RU；Y轴)对时间(X轴)。图23C显示了分析中使用的激动剂的示意图。
图24：从过滤器分离PBMC，并在80和10nM的抗体或对照存在下与葡萄球菌肠毒素B超抗原(SEB)一起温育。温育5天后，收获上清液并在Luminex上定量IL-2释放。用以下方法计算标准化的IL-2释放：对于每个样品(对于每个PBMC供体特异)减去在未刺激的细胞(不含SEB的PBMC)中诱导的IL-2定量，然后将该结果除以IL-2。在SEB刺激的细胞中诱导释放(未处理)。填充的粗线表示响应阈值。该图显示了每种处理的标准化IL-2释放。每个数据点是每个PBMC供体获得的一式三份重复值的平均值。线Y＝1表示标准化的仅SEB诱导响应。
图25：从过滤器中分离PBMC，并在80和10nM的抗体或对照存在下与葡萄球菌肠毒素B超抗原(SEB)一起温育。温育5天后，收获上清液并在Luminex上定量IL-2释放。用以下方法计算标准化的IL-2释放：对于每个样品(对于每个PBMC供体特异)减去在未刺激的细胞(不含SEB的PBMC)中诱导的IL-2定量，然后将该结果除以IL-2。在SEB刺激的细胞中诱导释放(未处理)。填充的粗线表示响应阈值。该图显示了每种处理的标准化IL-2释放。每个数据点是每个PBMC供体获得的一式三份重复值的平均值。线Y＝1表示标准化的仅SEB诱导响应。
图26：分析凝胶过滤色谱图的叠加。叠加单独的Tetra-8、单独的hOX40和1∶4比例的抗体-hOX40复合物的色谱图。表示预期分子量的箭头对应于校准运行的峰，并且是铁蛋白(440kDa)、醛缩酶(158kDa)和碳酸酐酶(29kDa)。注意Tetra-8和反向Tetra-8(用箭头表示)-Tetra-8之间的差异具有在V0的肩部，第二个峰值与反转Tetra-8相比转移到更高的分子量。
图27：Tetra-8-hOX40晶体状晶格。显示了大的二维晶格结构的一种可能性。每个TETRA-8的两个hOX40用于构建理论上无限大的结构。
图28：用Tetra-8处理后，OX40-GFP在Jurkat OX40-GFP细胞系上的时间流逝。将表达Jurkat的OX40 eGFP细胞在预先涂有纤连蛋白(PBS中的1μg/cm2)的Fluorodish(WPI)细胞培养皿(20000个细胞/cm2)上于37℃和5％CO2温育过夜。然后将Tetra-8以80nM终浓度加入细胞培养基中不同的时间间隔(范围为2.5至27.5分钟)，并使用配备有共焦模块LSM 800的Zeiss倒置显微镜Z1以63x放大率对细胞成像。
图29：由Tetra-8和其他OX40靶向分子诱导的OX40簇的共聚焦图像。用靶向OX40(Tetra-8、1A7、OX40L和Tetra-14)的各种分子处理Jurkat OX40-GFP细胞5、10或20分钟，在20nM(A)或80nM(B)下使用。
图30：各种抗OX40分子诱导的Jurkat-OX40 GFP细胞系OX40聚类的定量分析。如实施例中所述，使用峰度(Kurtosis)方法分析由Tetra-8和其他OX40靶向分子在JurkatOX40-GFP细胞上诱导的OX40簇的共聚焦图像。
图31：DC活化测定。从PBMC(来自过滤器的3个供体和来自全血的一个供体)分离树突细胞(DC)并分化6天，然后在抗体或对照存在下再培养2天。孵育后，收获细胞并用抗-CDlc-APC、抗-CD80-PE、抗-CD86-PerCP-eF710(对于第1组)或抗-CDlc-APC、抗-CD83-FITC、抗-HLA-DR-PerCP5.5染色(对于第2组)。该图显示了与未处理的DC相比，CD83和CD86标记的过表达细胞的百分比，其也组成性地表达这些标记中的一些。每个数据点是一个DC供体的值。N＝4供体。
图32：在解冻和使用的NFkB-Luc2P/U2OS细胞上孵育抗体或对照的剂量反应。孵育4小时后，将荧光素酶底物加入到孔中，并使用酶标仪读取发光(读取带-终点；积分时间-1分钟；发射-孔；光学位置-顶部；增加135；读取高度-1.00mm)。该图显示了每种条件的非线性S形回归结合曲线(发光)。测试以下处理：Selicrelumab IgG(○)，ADC-1013 IgG1(□)，3h56 IgG1 LALA(Δ)，Selicrelumab_3h56(●)，ADC-1013_3h56(■)，CD40L(×)。每个数据点是重复值的平均值±SD。
具体实施方式
实施例1：
小鼠抗人OX40抗体的产生和筛选
为了产生重组人OX40-his蛋白，通过PCR扩增人TNFRSF4的细胞外区域(SEQ IDNO：1中所示的氨基酸1-214)，添加3′GSG-6xHis接头和限制性位点用于克隆。随后将PCR产物克隆到上述修饰的pcDNA3.1(-)质粒中。该重组质粒允许在哺乳动物细胞中表达人OX40-his蛋白，并分泌到由人TNFRSF4的天然信号肽驱动的细胞培养基中。对于蛋白质生产，使用jetPEITM转染试剂(Polyplus-transfection S.A.，斯特拉斯堡，法国；经销商：Brunschwig，巴塞尔，瑞士)将重组载体转染到悬浮适应的HEK 293细胞(ATCC号CRL 1573)中。转染后5天收集细胞培养上清液，并使用在FPLC系统(GE Healthcare Europe GmbH，格拉特布鲁格，瑞士)上操作的Ni2+-NTA琼脂糖柱(HiTrap Ni2+-NTA琼脂糖柱；GE HealthcareEurope GmbH，格拉特布鲁格，瑞士)纯化。
通过SDS-PAGE判断，发现重组人OX40-Fc和OX40-his蛋白纯度为95％，并且在使用前进一步缓冲交换成磷酸盐缓冲盐水(PBS)。
为了产生重组人OX40L-Fc蛋白，人TNFSF4的cDNA购自imaGenes(克隆名称：IOH46203，柏林，德国)并且人TNFSF4配体的细胞外部分(氨基酸51-183)(根据UniprotQ6FGS4序列编号)用侧翼限制性位点扩增，随后克隆到基于来自Invitrogen(InvitrogenAG，巴塞尔，瑞士，Cat.No.V795-20)的pcDNA3.1(-)质粒的改良哺乳动物表达载体中，含有人IgG1的人Fc区(EU位置223-451)，描述于美国专利5924939中的具有Ig供体受体片段(第一内含子)的人CMV启动子，OriP序列(Koons等，2001，J.Virol.75(22)：10582-92)，SV40增强子，和如Kim等人所述(2003，Biotechnol Prog.19(5)，p.1620-2)的与胃泌素终止子融合的SV40 polyA。该重组质粒允许在哺乳动物细胞中表达人TNFSF4细胞外结构域-Fc融合蛋白，并分泌到由VJ2C前导肽驱动的细胞培养基中。对于重组蛋白质生产，使用阳离子聚合物将上述重组载体转染到悬浮适应的HEK293细胞(ATCC号CRL 1573)中。5天后收集细胞培养上清液，并使用CaptivATMprimAB亲和珠(Repligen，Waltham，马萨诸塞州，USA)分批进一步纯化，并在使用前进一步缓冲液交换至磷酸盐缓冲盐水(PBS)。
为了产生重组猕猴OX40-Fc蛋白，产生对应于猕猴OX40的细胞外部分(NCBI序列XP_001090870.1的氨基酸29-214)的合成基因(GeneArt，ThermoFisher Scientific，Waltham，马萨诸塞州)，其具有限制性位点。随后基于来自Invitrogen(InvitrogenAG，巴塞尔，瑞士，Cat.No.V795-20)的pcDNA3.1(-)质粒克隆到经修饰的哺乳动物表达载体中，含有人IgG1的人Fc区(EU位置223-451)，描述于美国专利5924939的具有Ig供体受体片段(第一内含子)的人CMV启动子，OriP序列(Koons等，2001，J Virol.75(22)：10582-92)，SV40增强子，以及如Kim等(2003，Biotechnol Prog.19(5)，p.1620-2)所述的与胃泌素终止子融合的SV40 polyA。该重组质粒允许在哺乳动物细胞中表达猕猴OX40细胞外结构域-Fc融合蛋白，并分泌到由VJ2C前导肽驱动的细胞培养基中。对于重组蛋白质生产，使用阳离子聚合物将上述重组载体转染到悬浮适应的HEK293细胞(ATCC号CRL 1573)中。5天后收集细胞培养上清液，并使用CaptivATMprimAB亲和珠(Repligen，Waltham，马萨诸塞州，USA)分批进一步纯化，并在使用前进一步缓冲液交换至磷酸盐缓冲盐水(PBS)。为了产生重组人OX40-Fc蛋白，人TNFRSF4的cDNA购自imaGenes(克隆号：RZPDB737H0329D；柏林，德国)。该cDNA用作模板以PCR扩增人TNFRSF4细胞外结构域的DNA编码区(如SEQ ID NO：1所示的氨基酸1-214)。在单独的PCR反应中，扩增人IgG1的Fc区(EU位置223-451)。然后使用具有侧翼引物的重叠延伸PCR将两个所得产物融合，添加限制性位点用于随后基于来自Invitrogen(Invitrogen AG，巴塞尔，瑞士，Cat No.V795-20)的pcDNA3.1(-)质粒，包含美国专利号5924939中描述的具有Ig供体受体片段(第一内含子)的人CMV启动子，OriP序列(Koons等，2001，J Virol.75(22)：10582-92)，SV40增强子，以及如Kim等(2003，Biotechnol Prog.19(5)，p.1620-2)所述的与胃泌素终止子融合的SV40polyA。该重组质粒允许在哺乳动物细胞中表达人TNFRSF4细胞外结构域-Fc融合蛋白，并分泌到由人TNFRSF4蛋白的天然信号肽驱动的细胞培养基中。对于重组蛋白质生产，使用jetPEITM转染试剂(Polyplus-transfection S.A.，Strasbourg，法国；经销商：Brunschwig，巴塞尔，瑞士)将上述重组载体转染到悬浮适应的HEK 293细胞(ATCC号CRL 1573)中。5天后收集细胞培养上清液，并使用在FPLC系统(GE Healthcare Europe GmbH，格拉特布鲁格，瑞士)上操作的蛋白A亲和纯化柱(HiTrapProtein A sepharose column；GE Healthcare Europe GmbH，格拉特布鲁格，瑞士)进一步纯化。
将溶解在PBS中的重组人OX40-Fc蛋白与等体积的Stimune佐剂(Phonics，瑞士，ref：7925000)混合，制备乳剂。将乳液转移至0.5mL胰岛素注射器(BD Pharmingen，Allschwil，瑞士)，并将BALB/c动物(Harlan，荷兰)在后足垫、尾巴基部和颈部皮下免疫50μg乳化蛋白质。两周后用相同量的抗原和相同的注射途径重复免疫。
使用包被有重组人OX40-his蛋白的平板通过直接ELISA评估免疫小鼠血清中循环抗人OX40抗体的存在。将不同小鼠血清的连续稀释液(从1∶100至1∶109)加入板中，并使用山羊抗小鼠H+L全分子-HRP检测结合的抗体(Sigma-Aldrich Chemie GmbH，Buchs，瑞士)。
在处死前3天显示最佳抗人OX40IgG血清滴度的动物中进行用50μg无佐剂抗原的最终皮下加强。
将动物安乐死并收集腹股沟、腋窝、臂、膝后和坐骨神经淋巴结以通过用DNAse(Roche Diagnostics(Schweiz)AG，Rotkreuz，瑞士)中的两个25G针干扰淋巴结结构来制备单细胞悬液。胶原酶(Roche Diagnostics(Schweiz)AG，Rotkreuz，瑞士)溶液。将单细胞悬浮液与骨髓瘤细胞系X63AG8.653(小鼠BALB/c骨髓瘤细胞系；ATCC登录号：CRL 1580；JImmunol 1979,123：1548-1550)以7∶1的比例与聚乙二醇1500(Roche Diagnostics(Schweiz)AG，Rotkreuz，瑞士)融合。将融合细胞接种于含有在DMEM-10培养基(InvitrogenAG，巴塞尔，瑞士)中的小鼠巨噬细胞的96孔平底板中，所述培养基补充有10％胎牛血清(FBS，PAA Laboratories，Pasching，奥地利)，2mM L-谷氨酰胺，100U/ml(Biochrom AG，德国)青霉素，100μg/ml的链霉素(BiochromAG，德国)，10mMHEPES(InvitrogenAG，巴塞尔，瑞士)，50μMβ-巯基乙醇(Sigma-Aldrich Chemie GmbH，Buchs，瑞士)，HAT(Sigma-Aldrich Chemie GmbH，Buchs，瑞士)和1％生长因子(Hybridokine，Interchim/Uptima，Montlugon，法国)。
通过ELISA筛选来自融合体的大约800个孔中存在识别人OX40的小鼠IgG。扩增阳性孔并进行两轮亚克隆。收集细胞并克隆重链和轻链并测序。
实施例2：
克隆和测序来自杂交瘤细胞的抗OX40抗体的VH和VL链
对于每个阳性选择的杂交瘤，制备总RNA，逆转录成cDNA，并通过PCR分别扩增VH和VL基因。将这些PCR产物连接到拯救载体(rescue-vector)(pDrive载体；QIAGEN AG，Hombrechtikon，瑞士；Cat.No.231124)，允许单个PCR产物的DNA测序和确定所选杂交瘤的单克隆或多克隆性。该载体允许在含有IPTG和X-gal的LB-琼脂平板上进行蓝/白选择(没有插入物的菌落是蓝色的，因为LacZa-肽降解了X-gal)。制备来自阳性(白色)细菌克隆的重组质粒，并使用对载体骨架特异的标准DNA测序引物(M13rev，M13fwd，T7或SP6)测序。最后将DNA序列亚克隆到表达载体中，用于在哺乳动物细胞中重组表达目的抗体。
RNA分离
使用来自QIAGEN的RNeasy Mini试剂盒(QIAGEN AG，Hombrechtikon，瑞士；Cat.No.74106)根据制造商的方案从2-10×106个细胞中分离总RNA。使用NanoDrop ND-1000分光光度计(WITEC AG，Littau，瑞士)定量样品。
一步法RT-PCR
将上述总RNA制备物进一步逆转录成cDNA，并使用两种不同的简并引物混合物通过PCR扩增VH和VL片段，每种引物允许回收所有不同的小鼠免疫球蛋白重链可变片段和可变重链连接区或所有小鼠免疫球蛋白轻链κ可变片段和可变轻链κ结区的恢复。用于逆转录和扩增的引物通过Microsynth(Balgach，瑞士)合成，并进行HPLC纯化(表1-4)。逆转录和PCR扩增均使用QIAGEN一步法RT-PCR试剂盒(QIAGEN AG，Hombrechtikon，瑞士；Cat.No.210212)。由于该技术使用特异性引物，然后将每个mRNA样品一式两份处理，允许VH或VL片段的单独逆转录和扩增。将溶解于无RNase水中的2μg总RNA至终体积30μl与10μlQIAGEN一步法RT-PCR缓冲液的5x储备液、2μl浓度为10mM的dNTPs混合物、3μl浓度为10μM引物混合物和2μl的QIAGEN一步法RT-PCR酶混合物混合。然后将最终混合物置于PCR管中，并使用以下设置在PCR-混合器(BioRad iCycler version 4.006，Bio-rad LaboratoriesAG，Reinach，瑞士)中循环：
50℃进行30分钟
95℃进行15分钟
40循环：94℃进行30秒
55℃进行30秒
72℃进行1分钟
72℃进行10分钟
保持在4℃
pDrive克隆
将PCR产物加到2％琼脂糖凝胶上。DNA电泳后，从琼脂糖凝胶中切下目的片段(约450bp)，并使用Macherey-Nagel NucloSpin Extract II试剂盒250(Macherey-Nagel，Oensingen，瑞士；Cat.No.740609.250)。对于DNA测序，将提取的PCR产物克隆到上述的拯救载体中(pDrive载体，QIAGEN AG，Hombrechtikon，瑞士；Cat.No.231124)。将其转化到大肠杆菌TOP10菌株(Invitrogen AG，巴塞尔，瑞士；Cat.No.C404006)。
微量提取
将阳性菌落在37℃(振荡250RPM)下在1.5ml Luria Bertani(LB)培养基中培养过夜，所述培养基补充有接种于Macherey-Nagel Square-well Block板(Macherey-Nagel，Oensingen，瑞士；Cat.No.740488.24)的10μg/ml氨苄青霉素。第二天，使用NucleoSpinMulti-8质粒试剂盒(Macherey-Nagel，Oensingen，瑞士；Cat.No.740620.5)。
测序
将样品送至DNA测序服务公司Fasteris(Plan-les-Ouates，瑞士)进行DNA测序。使用标准引物：M13rev，M13fwd，T7，SP6(表5)。
序列分析
使用克隆管理器9专业版(Scientific&Educational Software，NC，USA)和BioEdit Sequence Alignment Editor(Hall，T.A.1999.BioEdit：a user-friendlybiological sequence alignment editor and analysis program for Windows 95/98/NT.Nucl.Acids.Symp.Ser.41：95-98)分析DNA序列。
重组嵌合抗体表达载体的克隆
为了在哺乳动物细胞中重组表达，使用基于组装的PCR方法将分离的鼠VH和VL片段格式化为嵌合免疫球蛋白。这些嵌合抗体由其中鼠重链可变结构域与人IgG1重链恒定结构域(γ1、铰链、γ2和γ3区域)融合的重链和其中鼠轻链可变结构域与人κ恒定域(CK)融合的轻链组成。随后将PCR组装的鼠可变部分和人恒定部分克隆至基于来自实施例1中提及的Invitrogen的经修饰的pcDNA3.1(-)载体克隆到经修饰的哺乳动物表达载体中，不同之处在于使用人免疫球蛋白轻链κ前导肽驱动蛋白质分泌。对于免疫球蛋白候选物的蛋白质产生，将等量的重链和轻链载体DNA共转染到悬浮适应的HEK-293(ATCC号：CRL-1573)中。5天后收集细胞培养上清液，并使用在FPLC系统(GE Healthcare Europe GmbH，格拉特布鲁格，瑞士)上操作的蛋白A亲和纯化柱(HiTrap Protein A琼脂糖柱；GE HealthcareEurope GmbH，格拉特布鲁格，瑞士)纯化。
表2：引物混合物VH-back(SEQ ID NO：50-68)
表3：引物混合物VH-FOR(SEQ ID NO：69-72)
表4：引物混合物VL-BACK(SEQ ID NO：73-92)
表5：引物混合物VL-FOR(SEQ ID NO：93-96)
表6：测序引物(SEQ ID NO：97-100)
实施例3：
抗人OX40抗体的生物学特征
OX40特异性抗体检测ELISA
通过直接ELISA测定抗体滴度、特异性和杂交瘤和重组抗体候选物的产生。简言之，将96孔微量滴定板(Costar USA，经销商VWR AG，Nyon，瑞士)用100μl在PBS中的2μg/ml的重组人OX40-his包被(参见实施例1，用于产生OX40-his蛋白质)。将平板在4℃温育过夜，然后在室温(RT)下用PBS 2％BSA(Bovine Serum Albumine，PAA Laboratories，Pasching，奥地利)封闭1小时。除去封闭溶液，加入杂交瘤上清液或纯化的抗体。将板在室温下孵育30分钟，然后用PBS 0.01％Tween-20(Sigma-Aldrich Chemie GmbH，Buchs，瑞士)和辣根过氧化物酶(HRP)标记的山羊抗小鼠H+L-洗涤9次。以1∶1000的稀释度加入检测抗体(Sigma-Aldrich Chemie GmbH，Buchs，瑞士)。为了检测具有人Fc的重组嵌合抗体(参见实施例2)，使用1∶1000稀释的HRP标记的兔抗人IgG抗体(Sigma-Aldrich Chemie GmbH，Buchs，瑞士)作为检测抗体。将板在室温下孵育30分钟，用PBS 0.01％的Tween-20洗涤9次，并将TMB底物(Bio-rad Laboratories AG，Reinach，瑞士)加入板中，6分钟后通过加入H2SO4终止反应。然后通过酶标仪(Biotek，USA；经销商：WITTEC AG，Littau，瑞士)在450nm处读取吸光度。在阳性克隆中，选择杂交瘤7H11和2H6，获得其可变结构域的编码DNA序列，并如实施例2中所述制备小鼠-人IgG1嵌合体。
实施例4：小鼠7H11抗体的人源化和优化
本文描述了人源化抗人OX40小鼠抗体7H11，其包括选择人受体框架、回复突变和基本上保留和/或改善人CDR-移植受体框架的结合和性质同时去除潜在的翻译后修饰的突变。小鼠7H11抗体具有SEQ ID NO：2所示的可变重链结构域序列和SEQ ID NO：3所示的可变轻链结构域序列。
方法
抗体的重组生产
由GENEART AG(Regensburg，德国)以scFv形式合成不同VH和VL结构域的编码DNA序列(cDNA)，从而允许单个DNA序列包含两个可变结构域。通过PCR从该scFv构建体中回收单个可变域cDNA，并使用PCR组装技术进一步在它们各自的恒定域cDNA序列的上游组装。最后，将完整的重链和轻链cDNA连接到独立的载体中，所述载体基于携带CMV启动子和牛生长激素多腺苷酸化信号的修饰的pcDNA3.1载体(Invitrogen，CA，USA)。通过使用BamHI和BsiWI限制酶位点在κ轻链恒定域cDNA前连接感兴趣的轻链可变域cDNA，轻链特异性载体允许表达κ同种型轻链；而重链特异性载体经过工程改造，允许使用BamHI和SalI限制酶位点在编码IGHG1 CH1、IGHG1铰链区、IGHG1CH2和IGHG1 CH3恒定区的cDNA序列前连接感兴趣的重链可变区cDNA。在重链和轻链表达载体中，分泌由含有BamHI位点的小鼠VJ2C前导肽驱动。BsiWI限制酶位点位于κ恒定域中；而SaIl限制酶位点存在于IGHG1 CH1结构域中。
通过使用聚乙烯亚胺(PEI，Sigma，Buchs，瑞士)将等量的重链和轻链载体共转染到悬浮适应的HEK293-EBNA1细胞(目录号：CRL-10852)中来瞬时产生抗体。通常，用含有50μg编码重链的表达载体和50μg编码轻链的表达载体的DNA-PEI混合物转染100ml悬浮液，其密度为0.8-1.2百万个细胞/ml。当将编码抗体基因的重组表达载体导入宿主细胞时，通过进一步培养细胞4至5天以产生抗体，以允许分泌到培养基中(EX-CELL 293，HEK293-无血清培养基)；Sigma，Buchs，瑞士)，补充0.1％的pluronic acid，4mM谷氨酰胺和0.25μg/ml遗传霉素)。
使用重组蛋白-A流线型培养基(GE Healthcare Europe GmbH，格拉特布鲁格，瑞士)从无细胞上清液中纯化人源化抗体，并在测定前将其缓冲交换到磷酸盐缓冲盐水中。
通过FACS对HPB-ALL细胞进行亲和力测量
HPB-ALL细胞(DSMZ，Braunschweig，德国，Cat.No：ACC483)用作人OX40阳性细胞系用于FACS染色。将HPB-ALL维持在补充有10％FCS和100U/ml青霉素和100μg/ml链霉素的RPMI 1640中。将FACS缓冲液中的4x10e5HPB-ALL细胞(补充有1％BSA和0.1％叠氮化钠的PBS)在冰上孵育45分钟，其中感兴趣的抗OX40抗体保持在10μg/ml的浓度。无关的人IgG1用作同种型对照；将细胞与1/200稀释的抗人Fc-PE(EBioscience，维也纳，奥地利)在冰上孵育45分钟。然后再次洗涤细胞并重悬于200μl FACS缓冲液中。在FACSCalibur仪器(BDBiosciences，Allschwil，瑞士)上测量每个样品的相对平均荧光。
SPR的亲和力测量
SPR分析用于测量抗OX40抗体的结合动力学的结合和解离速率常数。在室温下在BIAcore 2000(BIAcore-GE Healthcare Europe GmbH，格拉特布鲁格，瑞士)上测量结合动力学，并用BiaEvaluation软件(v4.1，GE Healthcare Europe GmbH)分析。
在CM5传感器芯片上进行测量(Biacore 2000，GE Healthcare Europe GmbH，Cat.No：BR-1000-14)单独与蛋白A偶联(Sigma，Buchs，瑞士，Cat.No：P7837)使用商业胺偶联试剂盒(GE Healthcare Europe GmbH，Cat.No：BR-1000-50)。捕获200-600RU的人源化抗体。OX40-his的稀释系列以10μl/min的流速在HBS-EP缓冲液(GE Healthcare EuropeGmbH，Cat.No：BR1001-88)。在每次结合事件后，用10μl pH1.5的甘氨酸缓冲液再生表面。使用具有局部Rmax的1∶1Langmuir模型处理实验数据。解离时间约为7分钟。测量一式两份或一式三份进行，并包括零浓度样品用于参考。Chi2和残差值均用于评估实验数据和个体结合模型之间的拟合质量。
通过差示扫描量热法评估热稳定性
使用差示扫描量热法(DSC)测量人源化抗体的热稳定性。熔解特征的单克隆抗体是其同种型的特征(Garber和Demarest(2007)，BBRC 355：751-7)，然而，即使在全长IgG的背景下，FAB片段的中点解链温度也可以容易地鉴定。FAB部分的这种中点熔化用于监测人源化候选物的单克隆稳定性。
在VP-DSC差示扫描微量热计(Malvern Instruments Ltd，Malvem，UK)上进行量热测量。细胞体积为0.128ml，加热速率为200℃/h，过压保持在65p.s.i.所有抗体在PBS(pH7.4)中以1mg/ml的浓度使用。通过与含有相同缓冲液的一式两份样品进行比较来估计抗体的摩尔热容量，其中已经省略了抗体。使用标准程序分析部分摩尔热容和熔化曲线。在使用软件Origin v7.0中的非双态模型进一步分析之前，将热谱图进行基线校正并浓度标准化。
结果
重构变量区域的设计
使用同源匹配来选择小鼠7H11抗体的CDR的人受体框架。数据库(例如来自人和小鼠免疫球蛋白基因座的种系可变基因数据库，IMGT数据库(国际ImMunoGeneTicsLefranc MP等，Nucleic Acids Res，27(1)：209-12(1999)；Ruiz M等，Nucleic Acids Res，28(1)：219-21(2000)；Lefranc MP，Nucleic Acids Res，29(1)：207-9(20010；Lefranc MP，Nucleic Acids Res，31(1)：307-10(2003)；Lefranc MP等，Dev CompImmunol，29(3)：185-203(2005)；Kaas Q等，Briefings in Functional Genomics&Proteomics，6(4)：253-64(2007))或VBASE2(Retter I.等，2005，Nucleic Acids Res.，33，Database issue D671-D674)，或Kabat数据库(Johnson G.等，2000，Nucleic Acids Res.，28，p214-218))或出版物(例如，Kabat等，Sequences of Proteins of ImmunologicalInterest，1992))可用于鉴定鼠重链和轻链V区所属的人亚家族和确定最合适的人类生殖框架以用作小鼠CDR的受体分子。在这些亚家族中选择用作受体的重链和轻链可变序列(VH和VL)可以基于序列同源性和/或CDR1和CDR2区域的结构匹配，以帮助保持移植后的六个CDR的适当相对呈现。
例如，使用IMGT数据库表明7H11重链可变结构域框架与人重链可变结构域亚家族1的成员之间具有良好的同源性。对于种系序列观察到CDR和框架序列的最高同源性和同一性：IGHV1-3*01(SEQ.ID NO：4)，IGHV1-2*02(SEQ ID NO：5)和IGHV1-46*01(SEQ ID NO：6)，所有这些序列在整个序列直至CDR3时具有高于68％的序列同一性。IGHV1-8*01(SEQ IDNO：7)具有较低的序列同一性(66.3％)。
使用相同的方法，7H11轻链可变结构域序列显示出与人轻链可变结构域κ亚家族3和4的成员的良好同源性。对于种系序列观察到CDR和框架序列的最高同源性和同一性：IGKV4-1*01(SEQ ID NO：8)(81.2％同源性)，IGKV3D-7*01(SEQ ID NO：9)(67.3％同源性)，IGKV3D-15*01(SEQ ID NO：10)(67.3％同源性)和IGKV3-20*01(SEQ ID NO：11)(65.3％同源性)。
从上述IMGT搜索中鉴定出与人受体框架最佳匹配的JH和JK区段序列。
作为人源化过程的起点，选择上述四个可变重链和轻链结构域作为小鼠7H11CDR的受体。制备第一组16种人γ1同种型的人源化抗体。评估这些第一种人源化候选物在HEK293E细胞中的瞬时表达并通过流式细胞术与HB-ALL细胞结合(表7)。
表7：第一人源化7H11抗体候选物(IgG1)的表征。HPB-ALL细胞系上抗OX40抗体的FACS染色。MFI值对应于使用10μg/ml抗体候选物通过流式细胞术测量的抗体中点荧光。以每升培养物的mg报告瞬时表达产量。指出了原始的人种系框架。
最佳人源化候选物是抗体VH1/VL1、VH2/VL1和VH3/VL1。这些抗体显示FACS染色水平接近于亲本小鼠抗体所观察到的水平，其表达产率高于候选物的其余部分。
通过SPR进一步测定三种候选物的亲和力等级(表8)。令人惊讶的是，发现人源化VH2/VL1 IgG1抗体与嵌合7H11抗体相比具有更高的亲和力(即更低的KD)。此外，表达产量、对HBP-ALL细胞的表观亲和力以及Fab稳定性与其他两种变体相当。
表8：最佳第一移植人源化抗体的表征。显示了通过DSC测量的亲和常数和通过DSC测量的Fab中点变性温度。基于其良好的结合、表达和Fab稳定性，通过称为反向诱变的过程选择VH2/VL1抗体用于进一步的亲和力改善，其中将来自小鼠抗体序列的氨基酸引入人源化抗体序列中。认为通过该方法可以进一步改善亲和力，而不管VH2/VL1抗体具有比其亲本小鼠抗体更好的亲和力。
移植人体框架的回复突变
回复突变的过程需要从小鼠抗体中鉴定和选择需要保留的关键框架残基，以保持或改善亲和力，同时使人源化抗体中潜在的免疫原性最小化。为了鉴定可能影响最多CDR构象和/或互变域填充的残基，使用以自动模式设置的结构同源性建模服务器SWISS-MODEL(Arnold K等，(2006)Bioinformatics 22(2)：195-201；http：//swissmodel.expasy.org)计算VH2/VL1对可变结构域的3D模型。模型分析允许基于它们对CDR区和/或重链-轻链可变区填充的推定影响来选择位置子集。该位置子集选自在可变重链中发现的26个可能的回复突变，并且由位置组成：37、58、60、61、85、89和91(Kabat编号)(表9)。
表9：选择用于人源化VH2/VL1候选物和小鼠7H11抗体之间的回复突变的位置的细节。
使用标准PCR诱变和上述方法，在VH2/VL1抗体序列的背景下制备基于这些单一回复突变的其他人源化候选物。然后通过DSC测定人源化抗体候选物的SPR和Fab热稳定性的结合亲和力。表10中显示了产率、结合亲和力和热解折叠的Fab中点。在测试的七种抗体中，N58K回复突变显著提高了亲和力，同时保持了良好的Fab热稳定性和表达。因此，选择人源化VH2-N58K/VL1抗体用于进一步优化。
表10：人源化VH2/VL1回复突变抗体的表征。
去除潜在的异构化位点。
7H11 VH2 N58K的序列分析突出显示在位置54和55(Kabat编号)的7H11CDRH2中存在推定的天冬氨酸异构化位点(DG)。为了消除该异构化位点，进行定点诱变以通过带负电或中性极性氨基酸如谷氨酸、丝氨酸和苏氨酸替换7H11天冬氨酸54残基以及通过丙氨酸替换7H11甘氨酸55残基。使用PCR装配技术，在基于携带CMV启动子和牛生长激素多腺苷酸化信号的经修饰的pcDNA3.1载体(Invitrogen，CA，USA)的载体中连接之前，将突变D54E、D54S、D54T和G55A引入7H11 VH的cDNA中。
使用这些方法，几种载体产生编码人源化7H11 VH D54E(人源化7H11-VH2 N58K-D54E)、7H11 VH D54S(人源化7H11-VH2 N58K-D54S)、7H11 VH D54T(人源化7H11-VH2N58K-D54T)和7H11-VH G55A(人源化7H11-VH2N58K-G55A)。在HEK293-EBNA1细胞中用7H11轻链共转染人源化7H11 VH的亲本序列和变体。然后在转染后4天收集细胞上清液，用蛋白A进一步纯化。与亲本抗体相比，测试的突变在哺乳动物细胞中没有改变7H11表达(表10)。
为了确定这些突变是否可以改变抗体热稳定性，进行差示扫描荧光测定。首先将PBS中的抗体与浓度为250μg/ml的10X浓度的SYPRO橙(Thermo Fisher Scientific，Ecublens，瑞士)溶液混合，最终体积为20μl。为了记录蛋白质去折叠，然后在Rotor-Gene Q2plex HRM(QIAGEN，Hilden，德国)中将样品暴露于温度的逐渐升高，热解折叠之后存在SYPRO橙染料，其荧光在极性环境中被淬灭，但在暴露于疏水环境(例如展开时蛋白质μl的疏水核心)时强烈发出荧光信号。对于7H11的亲本和突变形式，记录的荧光信号是相似的，表明在人源化7H11VH中引入的突变不改变抗体热稳定性(表11)。
最后，如前所述，应用表面等离子体共振分析来控制抗体变体亲和力。表11中的结果显示7H11中引入的突变不改变抗体亲和力。
表11：异构化位点去除后人源化7H11变体的总结
实施例5：小鼠2H6抗体的人源化和优化
小鼠单克隆2H6的人源化
抗人OX40小鼠抗体2H6的人源化包括选择人受体框架和突变，其基本上保留人CDR-移植受体框架的结合特性，同时本发明描述了去除潜在的翻译后修饰。
选择用于移植2H6CDR的人受体框架以赋予人源化形式的2H6最大表达和/或稳定性。选择用作受体的人重链和轻链可变序列(VH和VL)可以基于具有良好生物物理特性的种系(如Ewert S等，(2003)J.Mol.Biol，325，531-553)和/或在天然抗体库中发现的配对(如Glanville J等，(1999)Proc Natl Acad Sci U S A，106(48)：20216-21；DeKosky BJ等，(2015)Nat Med，21(1)：86-91中所述)。本领域已知的用于良好配对和/或稳定性的框架序列是人IGHV3-23*01(SED ID NO：33)和IGKV1-16*01(SED ID NO：34)，其用作2H6人源化的受体框架。
制备人γ一同种型的第一人源化抗体。抗体包括人-小鼠杂交重链可变结构域和人-小鼠杂合轻链可变结构域。杂合重链可变结构域基于人重链可变结构域IGHV3-23*01，其中种系CDRH1和H2分别替换为2H6CDRH1和CDRH2。使用同源性搜索从IMGT数据库鉴定与人受体框架最佳匹配的JH区段序列。得到的具有人IGHV3-23*01框架区、2H6小鼠CDR并与人受体最佳匹配的JH的人-鼠杂交重链可变序列在本发明中称为具有SEO ID NO：31的重链可变区VH1。
类似地，用于该第一人源化抗体候选物的人-鼠杂交轻链可变结构域具有人IGKV1-16*01框架区、2H6小鼠CDR并与人受体最佳匹配的JK，并且在本发明中称为具有SEOID NO：32的轻链可变结构域VL1。包含VH1和VL1的第一种人源化抗体在本发明中缩写为2H6VH1/VL1抗体。
人源化2H6 scFv-Fc的生产
由GENEART AG(Regensburg，德国)以scFv形式合成VH1和VL1的编码DNA序列(cDNA)，从而允许单个DNA序列包含两个可变域(SEQ.ID NO：35)。将scFv cDNA连接到基于前面描述的修饰的pcDNA3.1载体(Invitrogen，CA，USA)的载体中。将scFv-Fc特异性载体工程化，以允许使用BamHI和KpnI限制酶位点在编码人IGHG1铰链区、IGHG1 CH2和IGHG1 CH3恒定区的cDNA序列前连接感兴趣的scFv cDNA。分泌由含有BamHI位点的小鼠VJ2C前导肽驱动。在含有KpnI位点的scFv的C末端部分引入人工甘氨酸-苏氨酸接头。
如前所述，通过将scFv-Fc载体转染到悬浮适应的HEK293-EBNA1细胞(目录号：CRL-10852)中瞬时产生scFv-Fc。然后，使用重组蛋白-A流线型培养基(GEHealthcare Europe GmbH，格拉特布鲁格，瑞士)从无细胞上清液中纯化scFv-Fc，并在测定前将其缓冲交换到磷酸盐缓冲盐水中。通过如下所述的表面等离振子共振测量与人和食蟹猴OX40的结合。包含VH1和VL1的2H6人源化scFv在本文中缩写为2H6scFv1。
通过表面等离子体共振(SPR)，人源化2H6 scFv1-Fc对人和食蟹猴OX40受体细胞外结构域的动力学结合亲和力常数
使用重组组氨酸标记的人OX40受体细胞外结构域和在CM5芯片上捕获的重组Fc融合的食蟹猴OX40受体细胞外结构域和2H6 scFv1-Fc和小鼠嵌合2H6 scFv-Fc作为分析物测量动力学结合亲和力常数(KD)。在室温下在BIAcore T200(GE Healthcare-BIAcore，Uppsala，Sweden)上进行测量，并用Biacore T200评估软件分析。通过注射35μl的1∶1的N-羟基磺基琥珀酰亚胺(NHS)/1-乙基-3-[3-二甲基氨基丙基]碳二亚胺盐酸盐(EDC)溶液(v/v；5μl/min流速；在流路1、2、3和4上)来活化CM5研究级传感器芯片(GE Healthcare EuropeGmbH，格拉特布鲁格，瑞士；BR100530)。
将食蟹猴OX40R-Fc在醋酸盐缓冲液pH 4.0(GE，BR-1003-49)中稀释至终浓度25nM，随后通过在流路2上注射10μl(10μl/min)(大约相当于600个响应单位(RU))，固定在先前活化的CM5传感器芯片上。将人OX40R-His在乙酸盐缓冲液pH 4.0(GE，BR-1003-49)中稀释至终浓度25nM，随后通过在流路4上注射45μl(10μl/min)(大约相当于400个响应单位(RU))，固定在先前活化的CM5传感器芯片上。然后通过注入35μl的乙醇胺溶液(5μl/ml)使OX40R-CM5传感器芯片失活。最后，进行两次注射10μl甘氨酸溶液(GE，ref.BR-1003-54；10mM；pH 1.5)以释放未交联的(人和食蟹猴)OX40R分子。
以30μl/min的流速在4个流动路径(流路1和3用作参考)上以不同浓度(0.78nM至0.2μM)注射2H6 scFv-Fc。在每次结合事件后，利用注射pH 1.5的甘氨酸缓冲液再生表面30秒(10μl/min)。
测量(传感图：fc2-fc1和fc4-fc3)最适合具有质量转移的2∶1二价分析物模型。解离时间至少为300-600秒。Chi2值表示每个点的实验数据和参考数据之间的平方差的总和；而残差图表示拟合中每个点的实验数据和参考数据之间的差异。Chi2和残差值均用于评估实验数据和个体结合模型之间的拟合质量。
表12中显示的结果表明，与亲本小鼠2H6 scFv相比，人源化2H6 scFv1对人和食蟹猴OX40具有相似的亲和力。
表12：人源化2H6-scFv通过SPR的表征
从JH中除去2H6 Met
2H6 scFv的序列分析突出显示在2H6 VH JH区域中存在推定的氧化位点(甲硫氨酸108)。为了消除这种潜在的氧化位点，进行定点诱变以用亮氨酸氨基酸取代2H6 VH甲硫氨酸108残基。使用PCR组装技术，在基于如前所述的修饰的pcDNA3.1载体的载体中连接之前，将VH突变M108L(kabbat编号)引入2H6 scFv的cDNA中。使用该方法，产生编码人源化2H6scFv M108L(缩写为2H6 scFv2)的载体。如前所述，在HEK293-EBNA1细胞中转染2H6 scFv-Fc的亲本和突变形式。然后在转染后4天收集细胞上清液，用蛋白A进一步纯化。与亲本抗体相比，测试的突变在哺乳动物细胞中不改变2H6 scFv-Fc表达(表13)。为了确定M108L突变是否可以改变scFv热稳定性，如前所述进行差示扫描荧光测定。对于2H6的亲本和突变形式，记录的荧光信号是相似的，表明M108L突变不改变scFv热稳定性(表12)。最后，如前所述，应用表面等离子体共振分析来控制2H6 scFv2变体的结合特性。图1中的结果显示2H6scFv中引入的突变未改变其结合。
表13：M108L突变对人源化2H6-scFv的影响的表征
实施例7：四价抗人OX40抗体的工程化和生产
第一四价分子
使用的四价形式是scFv通过(Gly4Thr)接头在重链的C-末端连接的完整IgG。为了产生具有与2H6 scFv融合的7H11 IgG1的四价抗体，对人源化7H11-VH2 N58K(SEQ ID NO：21)、VL1(SEQ ID NO：16)和2H6 scFv1的编码DNA序列(cDNA)在基于前面描述的修饰的pcDNA3.1载体(Invitrogen，CA，USA)的载体中进行消化和连接之前进行PCR扩增。使用BamHI和BsiWI限制酶位点工程化轻链特异性载体以允许在编码人κ恒定结构域的cDNA序列之前连接感兴趣的VL cDNA。使用BamHI和SalI限制酶位点工程化重链特异性载体以允许在编码IGHG1铰链区的cDNA、具有降低Fc-FcγRs相互作用的L234A/L235A双突变(LALA，Eu编号，Hezareh M等，(2001)J Virol，75：12161-8)的修饰的IGHG1 CH2结构域和在其C-末端部分具有(Gly4Thr)接头的修饰的IGHG1CH3恒定结构域之前连接感兴趣的VH cDNA。然后，使用KpnI和NotI限制酶位点，在IGHG1 CH3恒定结构域和重链特异性载体的(Gly4Thr)接头之后连接感兴趣的scFv cDNA。在重链和轻链表达载体中，分泌由含有BamHI位点的小鼠VJ2C前导肽驱动。BsiWI限制酶位点位于κ恒定域中；而Sail限制酶位点存在于IGHG1 CH1结构域中。甘氨酸-苏氨酸接头含有KpnI位点，而NotI位点存在于编码重链的经修饰的pcDNA3.1载体中的牛生长激素多腺苷酸化信号之前。
如前所述，通过将等量的7H11VL1轻链和Tetra-1重链载体共转染到悬浮适应的HEK293-EBNA1细胞中，瞬时产生这种四价抗体(缩写为Tetra-1)。使用重组蛋白-A流线型培养基(GE Healthcare Europe GmbH，格拉特布鲁格，瑞士)从无细胞上清液中纯化四价抗体，并在测定前将其缓冲交换到磷酸盐缓冲盐水中。
优化的7H11x2H6四价抗体：
在上述四价分子中，scFv在IgG的C末端融合。因此，抗体的最后C末端残基是天然存在于JK区域的赖氨酸。为了避免循环中四价分子的C-末端赖氨酸剪切可能对抗体的生物学产生影响，使用定点指导用亮氨酸残基取代2H6 scFv的C端赖氨酸。使用PCR装配技术，在编码上述四价抗体的载体中，在连接前将VL突变K107L(Kabbat编号)引入2H6 scFv的cDNA中。如前所述，在HEK293-EBNA1细胞中转染亲本和突变形式的四价抗体。然后在转染后4天收集细胞上清液，用蛋白A进一步纯化。与亲本抗体相比，测试的突变不改变哺乳动物细胞中的四价抗体表达(表14)。为了确定K107L突变是否可以改变C-末端scFv热稳定性，如前所述进行差示扫描荧光测定。对于2H6 scFv的亲本和突变形式，记录的荧光信号是相似的，表明K107L突变不改变scFv热稳定性(表13)。
表14：K107L突变对人源化Tetra-1抗体的影响的表征
为了最终确定四价抗体的优化，通过如前所述的定点诱变，在编码四价分子重链(缩写为Tetra-6)的载体中引入7H11突变D54E，2H6 VH突变M108L和VL突变K107L。如前所示通过共转染7H11VL1轻链和Tetra-6重链载体产生Tetra-6，并使用重组蛋白-A流线型培养基从无细胞上清液中纯化。
实施例8：四价抗人OX40抗体的体外表征
实施例8.1 Tetra-1和Tetra-6在混合淋巴细胞反应(MLR)测定中显示增殖作用
使用ficoll密度梯度从健康供体的柠檬酸化全血中分离PBMC。使用单核细胞分离试剂盒(Miltenyi)从PBMC分离单核细胞，并用50ng/mL(R&D)的GM-CSF和20ng/mL(R&D)的rhLL-4培养7天以将它们分化成树突细胞(DC)。。使用CD1c APC(eBioScience)通过流式细胞术验证树突细胞的表型。在第7天，使用EasySep试剂盒(StemCell Technologies)从PBMC分离CD4T细胞(来自同种异体供体)。将CD4T细胞(40000细胞/孔)和DC(8000细胞/孔)与抗体在80nM下在96孔圆底板中在完全培养基中共培养6天，一式三份。在第13天，加入3H-胸苷(Perkin Elmer，每孔0.5μl)。脉冲后20小时，收获细胞并在Wallac β计数器上定量掺入放射物。使用以下公式确定归一化刺激指数(SI)：
(样品-仅Resp)/(Allo-仅Resp)
“样品”对应于共培养DC+CD4T细胞+测试抗体的条件的计数。“仅Resp”对应于仅添加应答细胞(CD4T细胞)的条件下的计数。“Allo”对应于共同孵育DC(刺激细胞)和同种异体CD4T细胞(应答细胞)的条件。使用Graphpad Prism 7软件分析数据；用Mann-Whitney检验(非参数检验)或Wilcoxon匹配对检验进行统计学分析。P＜0.05被认为具有统计学意义。
OX40L-Fc是有效的激动分子，其可以有效地接合和交联T细胞表面上的OX40(Müller FEBS J.2008年5月；275(9)：2296-304)。与此特征一致，OX40L-Fc能够增加混合淋巴细胞反应。在该测定中，Tetra-1和Tetra-6均将同种异体反应增强至与OX40L-Fc相似的水平(这三种分子之间的差异无统计学意义；图1)。比较在同一实验中测试的Tetra-1和Tetra-6的SI的Wilcoxon匹配对测试显示这两种四价分子类似地增强了增殖(数据未显示)。因此，这些结果强调了用Tetra-1和Tetra-6靶向OX40提供了相关的免疫刺激潜力。
实施例8.2 Tetra-1和Tetra-6在葡萄球菌肠毒素B刺激试验中诱导强免疫刺激作用
使用ficoll密度梯度分离从La Chaux-de-Fonds输血中心获得的血液过滤器收获外周血单核细胞(PMBC)。PBMC(105)一式三份地分布在96孔圆底板中。将最终浓度为50或100ng/mL(次优浓度)的葡萄球菌肠毒素B(SEB)和最终浓度为80nM的抗体加入孔中。将板在37℃下在CO2培养箱中孵育7天。在第5天使用ProcartaPlex试剂盒(eBiosciences)用Luminex测量培养上清液中IL-2的产生。在第7天，收获细胞并用抗人CD4ECD和抗人CD25Pacific Blue(eBioSciences)标记。将染色的细胞重悬于100μL的FACS缓冲液中，并通过CytoFLEX S(Beckman)上的流式细胞术分析。使用CytExpert(Beckman)分析流式细胞术数据。门控策略包括对活细胞(基于FSC和SSC图)细胞，CD4阳性细胞和随后CD4+CD25+进行门控。使用以下公式计算该感兴趣子集内每孔的细胞总数：
(用于重悬细胞的体积*门中的事件数)/获得的样品体积
将每个数据点标准化为PBMC仅与SEB孵育的条件(无抗体)。使用Graphpad Prism7软件分析数据，并使用Mann-Whitney检验(非参数检验)进行统计分析。P＜0.05被认为具有统计学意义。
在该测定中，Tetra-1能够显著增加CD4+CD25+细胞的数量(与仅SEB相比，91％的受试者达到1.2倍诱导；任意定义阈值)，而OX40L-Fc和Tetra-6在较小程度上这样(分别为67％和88％)。CD25的表达定义了活化的T细胞。表达CD25的CD4T细胞的增加可归因于T细胞活化的增加和/或活化T细胞增殖的增加。类似地，与仅SEB相比，添加Tetra-1、Tetra-6和OX40L-Fc也显著增强IL-2产生。IL-2产生也表明T细胞活化并且与T细胞增殖直接相关。在该读数和实验的这个阶段(第5天)，三个分子之间没有统计学差异。
总体而言，在这种超抗原介导的PBMC刺激中靶向OX40显著改善T细胞应答，如通过增强的细胞因子产生(IL-2)或增强的T细胞活化(CD25)所显示的。
实施例8.3 Tetra-1在PHA刺激测定中显示出强烈的免疫刺激作用
以与SEB测定相同的方式制备PBMC。PBMC(105)一式三份地分布在96孔圆底板中。加入终浓度为2或1μg/mL的PHA和终浓度为80nM的抗体。将板在37℃下在CO2培养箱中孵育7天。在测定开始后6天，用每孔0.5μCi的3H-胸苷(Perkin Elmer)脉冲细胞。脉冲后20小时，收获细胞并在Wallac β计数器上定量掺入放射物。使用以下公式确定刺激指数：
样品/仅PHA
“样品”对应于条件PBMC+PHA+测试抗体的计数。“仅PHA”对应于仅用PHA培养PBMC的条件计数(无抗体)。将每个数据点标准化为PBMC仅与PHA(无抗体)孵育的条件。使用Graphpad Prism 7软件分析数据。用Mann-Whitney检验(非参数检验)进行统计学分析。P＜0.05被认为具有统计学意义。
在该测定中，Tetra-1和Tetra-6响应于次优的PHA浓度而增加细胞增殖。在Tetra-1、Tetra-6和OX40L-FC之间未检测到显著差异。
实施例9：二硫键稳定的Tetra-8分子的产生
为了进一步增强Tetra-6分子，通过在VH和VL之间引入二硫键来改造携带VH突变M108L和VL突变K107L的2H6 scFv以增加其稳定性。结构域(ReiterY等，Nat Biotechnol.，14(10)：1239-45，1996年10月)。使用PCR组装技术，在编码新的四价分子重链(缩写为Tetra-8)的载体中连接之前，将VH G44C和VLQ100C突变引入突变的2H6 scFv的cDNA中。然后通过如前所述共转染7H11VL1轻链(SEQ ID NO：16)和Tetra-8重链(SEO ID NO：45)载体产生Tetra-8。蛋白A纯化后，通过非还原SDS-PAGE(图5)和SEC-HPLC(图6)分析分子。两种分析方法均显示四价分子的二硫键工程诱导共价多聚体的形成。为了将多聚体与单体分离，进行另外的阳离子交换纯化步骤。简而言之，首先使用50mM乙酸钠pH 5.5缓冲液平衡HiTrap SP HP柱(GE Healthcare Europe GmbH，格拉特布鲁格，瑞士)。然后，加载蛋白质样品，并使用50mM乙酸钠+1M NaCl pH 5.5的梯度从5％至20％和100％分离分子(图7)。然后合并含有单体形式的Tetra-8的级分，并进行与PBS的缓冲液交换(Gibco，ThermoFischerscientific，MA，USA)。最后，通过差示扫描量热法(DSC)评估单体Tetra-8的热稳定性，并与Tetra-1比较(图8)。测量Tetra-8中突变的2H6 scFv结构域的热稳定性(3℃)的明显增加，表明二硫键工程有效地增强2H6 scFv解链温度。
Tetra-8抗体的生物物理表征
通过Biacore测定2H6 scFv结合物在Tetra-8中的亲和力。为了进行精确测量，用FabALACTICA蛋白酶(Genovis AB，Lund，Sweden)消化Tetra-8分子以除去7H11 Fab。简言之，在每μg抗体加入1个FabALACTICA单位之前，首先将Tetra-8分子在150nM磷酸钠pH7.0中进行缓冲液交换。将抗体/蛋白酶混合物在37℃温育过夜。然后，使用CaptureSelectTMFcXL亲和基质树脂(ThermoFischer scientific，MA，USA)进一步纯化该材料，以从捕获Fc-2H6scFv片段的同时从混合物中除去7H11 Fab和蛋白酶。用PBS洗涤树脂，然后用0.1M甘氨酸pH3.0洗脱特异性Fc-2H6片段，最后配制在PBS pH7.4中。使用固定在CM5芯片上的抗人IgGFc，通过捕获600RU的Fc-2H6片段并通过注射稀释系列的OX40-CRD-Avi-his(SEQ.ID NO：159)以流速测量动力学。在HBS-EP缓冲液中30μl/min(GE Healthcare Europe GmbH，Cat.No：BR1001-88)。在每次结合事件后，用10μl的MgCl2(3M)缓冲液再生表面。使用具有局部Rmax的1∶1Langmuir模型处理实验数据。解离时间约为7分钟。在Tetra-8分子的C末端融合的2H6 scFv(VH/M108L-VL/K107L-二硫化物工程化)的测量亲和力为60nM，与人源化2H6scFv1-Fc相比仅表现出2倍的损失(表6)并且表明2H6结合臂是有功能的。
抗OX40抗体
为了评估Tetra-8激动剂活性，产生了几种已知的激动剂抗OX40。11D4 lgG2(US2012/0225086 A1)，9B12 IgG1(OX40mab24，US 2016/0137740 A1)，106-222IgG1(US 2016/0068604A1)，1A7 IgG1(WO 2015/153513 A1)和pabl949(US2016/0347847 A1)从它们各自的专利申请中检索重链和轻链序列，并通过Geneart AG(ThermoFischer scientific，MA，USA)作为cDNA进行基因合成。然后将重链和轻链序列连接在独立的载体中，该载体基于先前描述的修饰的pcDNA3.1载体。如前所述(表15)，在HEK293-EBNA1细胞中共转染编码每种抗体各自重链和轻链的载体。然后在转染后4天收集细胞上清液，用蛋白A进一步纯化。Tetra-hzG3V9、Tetra-hz1D10v1、Hexa-hzG3V9和Hexa-hz1D10v1(WO2017/123673A2)的结构域抗体(dAb)序列也是基因合成的。在将突变的IgG1 Fc序列(LALA)框内克隆到修饰的pcDNA3.1载体中之前，由GeneartAG进行。然后将编码各个分子的载体单独转染到HEK293-EBNA1细胞中，并在蛋白A纯化之前收集上清液。最后，将7H11-VH2 N58K-D54E克隆到人IgG1或IgG1 LALA序列的框架中，并与7H11VL1组合以产生7H11IgG1或IgG1LALA(表x)。将2H6scFv1克隆到IgG1-Fc LALA结构域的框架中以产生2H6 scFv-FcLALA。将2H6 VH1克隆到人IgG1或IgG1 LALA序列的框架中。用2H6LC共转染这些序列以产生2H6 IgG1和IgG1 LALA(表15)。
表15：用于抗体产生的重链和轻链的组合。
用于抗体表位作图(epitope mapping)的嵌合OX40
OX40分子是TNFR超家族的成员，其特征在于在其细胞外部分中存在四个被定义为富含半胱氨酸结构域(CRD)的结构域(图9)。为了确定拮抗剂抗体靶向的结构域，必须设计和表达OX40嵌合体以用作表位作图的工具。从Uniprot数据库检索人、大鼠和食蟹猴OX40的细胞外结构域的序列(分别为SEQ ID NO：1、121、122)，通过Geneart合成的基因，然后克隆为Fc-融合蛋白(分别为SEQ ID NO：123、124、125)。这些构建体在HEK293-EBNA1细胞中表达并使用蛋白A纯化。然后，在ELISA和Biacore中测试人和大鼠OX40-Fc以确定抗体交叉反应性。对于ELISA，用PBS中的2μg/ml的100μl重组人或大鼠OX40-Fc包被96孔微量滴定板(Costar USA，经销商VWR AG，Nyon，瑞士)。将平板在4℃温育过夜，然后在室温(RT)下用PBS2％BSA(Bovine Serum Albumine，PAA Laboratories，Pasching，奥地利)封闭1小时。除去封闭溶液，并将纯化的抗体以10μg/ml加入PBS 2％BSA中。将板在室温下孵育1小时，然后用PBS0.01％Tween-20(Sigma-Aldrich Chemie GmbH，Buchs，瑞士)洗涤5次。为了检测具有人Fab的重组抗体，使用在PBS 2％BSA中以1∶2000稀释的过氧化物酶缀合的山羊抗人IgG，Fab片段特异性(Jackson，I mmunoResearch，109-035-006)作为检测抗体。将板在室温下孵育1小时，用PBS 0.01％Tween-20洗涤5次，并将TMB底物(Bio-rad Laboratories AG，Reinach，瑞士)加入板中，5分钟后通过加入H2SO4终止反应。然后通过酶标仪(Biotek，USA；经销商：WITTEC AG，Littau，瑞士)在450nm处读取吸光度。Biacore实验用Hexa-hzG3V9和Hexa-hz1D10v1特异性地进行，它们是dAb-Fc融合蛋白。简而言之，通过将5μlTetra-hz1D10v1(5μg/mL)以10μl/min的流速注入流路2将其固定在先前活化的CM5传感器芯片上，其对应于大约206RU。类似地，将15μlTetra-hzG3V9(5μg/mL)以10μl/min的流速注入流路4，其大约相当于251RU。人和食蟹猴OX40-Fc分子的结合通过在400nM浓度和30uL/min的流速下连续注射这4种流路(流路1和3作为参考)来确定240秒。使用3M MgCl2以30uL/min进行两次注射之间的再生60秒。使用这两种方法，评估人/大鼠OX40结合(表16)。
表16：抗OX40抗体的交叉反应性的表征
这些实验清楚地证明大多数这些抗体(1A7除外)缺乏与大鼠OX40的交叉反应性，表明该蛋白质可用于产生具有人OX40序列的嵌合体用于表位作图目的。
表位映射
用T-coffee(Notredame C.等，J Mol Biol，302(205-217)2000)进行人(SEQ IDNO：1)、食蟹猴(SEQ ID NO：122)和大鼠(SEQ ID NO：121)OX40细胞外结构域的序列比对(图10)。基于OX40序列中的二硫键模式鉴定CRD。通过如下混合CRD建立人/大鼠OX40嵌合体的理论序列：人CRD1、CRD2、CRD3和大鼠CRD4(HHHR)(SEOID NO：126)；人CRD1、CRD2，大鼠CRD3，人CRD4(HHRH)(SEO ID NO：127)；人CRD1、CRD2，大鼠CRD3、CRD4(HHRR)(SEOID NO：128)；人CRD1，大鼠CRD2、CRD3、CRD4(HRRR)(SEO ID NO：129)；大鼠CRD1、CRD2，人CRD3、CRD4(RRHH)(SEO ID NO：130)；大鼠CRD1，人CRD2，大鼠CRD3、CRD4(RHRR)(SEO ID NO：131)。然后由Geneart AG基因合成设计的序列，并将获得的cDNA克隆到人IgG1-Fc区的框架中，形成修饰的pcDNA3.1载体。然后如前所述产生和纯化蛋白质。然后将这些分子用于先前描述的ELISA和Biacore实验中以确定这些抗体靶向的OX40结构域。结果总结在表17中。
表17：抗OX40抗体结构域特异性的表征
使用OX40嵌合体，我们发现7H11和11D4主要与OX40 CRD1结合。9B12和106-222主要与OX40 CRD2结合。2H6和pAB1949主要与CRD3结合。hzG3V9和ID10v1 dAb主要与CRD4结合。当1A7与大鼠OX40交叉反应时，该抗体的表位未被表征。
实施例10：Tetra-8的体外生物学表征
10.1 Tetra-8特异性结合OX40
按照上文实施例3中详述的方法，通过直接ELISA评估Tetra-8对可溶性OX40的结合活性。简而言之，在96孔微量滴定板中以不同浓度(范围从10到0.00017μg/ml)测试Tetra-8，用在PBS中以2μg/ml稀释的重组人OX40 His蛋白预涂覆过夜(参见实施例1，用于生成OX40-his蛋白质)。为了单独测试Tetra-8的两个结合单位，在同一测定中包括7H11_v8IgG1和Tetra-22分子。如后所述，Tetra-22是对照分子，其由不相关的IgG1 LALA组成，其中2H6ScFv已融合至C-末端。图11的结果显示Tetra-8、7H11_v8 IgG1和Tetra-22分子识别具有相同结合特征的重组人OX40蛋白。
使用GloResponseTM NFkB luc2/OX40-Jurkat细胞系(Promega)通过流式细胞术评估Tetra-8在膜结合的OX40上的结合。简而言之，收获细胞，计数并以100000个细胞/孔接种在96孔圆底板中。将板以350g离心3分钟，将细胞重悬于50μl含有各种浓度(100至0.00056g/ml)Tetra-8、7H11_v8 IgG1或2H6 IgG1抗体的FACS缓冲液(PBS+10％versene+2％FBS)中。将染色的细胞在4℃温育20分钟，用FACS缓冲液以350g洗涤3分钟，并重悬于100μl用FACS缓冲液稀释的抗人IgG PE二抗(Thermofischer)中。然后将细胞洗涤两次，并重悬于200μl FACS缓冲液中，并在FACSCalibur仪器(BD Biosciences，Allschwil，瑞士)上获得样品。基于FSC对SSC的大小门控细胞，并使用FlowJo软件分析PE-几何平均荧光强度。如图12中所示，Tetra-8、7H11_v8 IgG1和2H6 IgG1抗体识别在转染的Jurkat细胞上表达的膜结合的OX40。根据制造商的说明书(Thermofischer)，还用AF647染料直接标记3种分子，随后评估其与表达不同水平的OX40的其他细胞的结合。表18总结了所有测试分子和细胞系的KD值。
表18
为了进一步证明Tetra-8与OX40的选择性结合，对其他TNFR成员进行了直接ELISA。按照与先前描述的相同的方案进行实验。在该测定中，针对重组BAFF、CD40、DR3、DR6、GITR和TWEAK分子(R&D)测试Tetra-8的连续稀释(范围从10至0.0001μg/ml)。这些分子均在PBS中以2μg/ml包被在4℃下过夜。图13的结果显示Tetra-8选择性结合OX40分子，并且不识别TNFR家族的其他成员，包括那些在其氨基酸序列中显示高达40％同一性的成员。
10.2 Tetra-8通过其2H6部分与食蟹猴OX40结合
为了评估Tetra-8与食蟹猴0X40的交叉反应性，按照前述方案进行直接ELISA。在该测定中，Tetra-8和利妥昔单抗-2H6(其是相当于Tetra-22的对照分子，其中无关的IgG1LALA部分来自利妥昔单抗)与重组食蟹猴OX40蛋白结合，但不结合7H11_v8 IgG1(图14)。这些数据表明Tetra-8通过其2H6部分识别食蟹猴OX40。结合活性对人和食蟹猴OX40和EC50值的结果总结在表19中。
表19
通过流式细胞术进一步证明了Tetra-8与食蟹猴OX40的交叉反应性。在该方法中，将来自商业来源(Silabe)的食蟹猴PBMC在cRPMI中稀释，并在5μg/mlPHA存在下于37℃在CO2培养箱中以1×106个细胞/ml接种在T-25烧瓶中。使用如前所述分离的人PBMC进行相同的实验。两天后，收获活化的细胞并用抗人CD4 APC(Thermofischer)标记。随后用Tetra-8、7H11_v8 IgG1或2H6 IgG1抗体染色，然后用抗人IgG PE(Thermofischer)检测。洗涤染色的细胞并重悬于100μl的FACS缓冲液中，并使用CytExpert(Beckman)通过流式细胞术分析。门控策略包括门控活细胞(基于FSC和SSC图)和CD4阳性细胞。图15的结果显示Tetra-8、2H6IgG1而非7HII_v8 IgG1与CD4阳性细胞上表达的膜结合的食蟹猴OX40结合。这些数据证实Tetra-8的食蟹猴交叉反应活性是由其2H6部分介导的。
10.3 Tetra-8以不依赖FcγR的方式诱导OX40-NFkB荧光素酶报告细胞系的显著活化
为了评估Tetra-8激活OX40-信号传导的潜力，使用表达OX40的GloResponseTMNFkBluc2/OX40-Jurkat细胞系，按照制造商的说明书(Promega)进行荧光素酶测定。简言之，收获Jurkat NFkB，计数并以2×106细胞/ml重悬于完全RPMI培养基(RPMI 1640+10％FBS+1％NEAA+1％NaPyr+潮霉素500μg/ml+G418800μg/ml)。将50μl细胞分布在96孔发光板中，并在37℃，5％CO2下与25μl Tetra-8、7H11 IgG1 LALA或2H6 IgG1 LALA一起温育，在测定培养基中连续稀释(RPMI 1640+％FBS)。平行地，通过在96孔发光板上固定5μg/ml抗CD3抗体(OKT3克隆)，在施加TCR刺激的条件下测试这些分子。5小时后，向孔中加入75μl Bio-Glo溶液，并在发光酶标仪(Synergy HT2-分光光度计，Biotek)中读取所有板的信号。荧光素酶测定在没有任何交联条件下进行，即不存在二抗或表达FcγR的细胞。如图16所示，Tetra-8在两种不同的实验环境中(有或没有TCR刺激)诱导OX40NFkB荧光素酶报告基因Jurkat细胞系的剂量依赖性激活，并促进比其单独结合单位更高的OX40信号传导(7H11 IgG1LALA和2H6IgGl LALA)。重要的是，Tetra-8在ADCC测定中进一步测试，并且没有像预期的那样显示出显著的活性(数据未显示)。这些数据显示了Tetra-8与其他单克隆OX40激动剂抗体之间的重要差异，其被描述为通过Fc R介导的交联显示其活性。
10.4 Tetra-8在MLR测定中增加T细胞同种异体活性
MLR测定通常用于评估靶向共刺激分子(例如OX40)的免疫调节剂的潜力，以增强同种异体T细胞应答(Keli L.Hippen等，Blood 2008)。为了评估Tetra-8增强T细胞应答的潜力，按照实施例3中描述的方案进行同种异体MLR测定。在该测定中，在6种不同浓度(范围从160至0.001nM)下测试Tetra-8，并在80和10nM下测试OX40L。图17中描绘的结果显示Tetra-8在MLR测定中强烈且显著增强(2至3倍)同种异体反应性T细胞增殖。这种效应是剂量依赖性的，甚至比表20中总结的OX40L更有效。
表20
10.5 Tetra-8在葡萄球菌肠毒素B刺激试验中诱导强烈的免疫刺激作用
SEB刺激测定也已广泛用于评估靶向OX40和TNFR家族的其他成员的免疫调节剂增强T细胞应答的潜力。该测定，其方案描述于实施例8.2，用于评估Tetra-8的功能活性。测试Tetra-8的各种浓度范围为80至0.01nM。如图18所示，人PBMC与Tetra-8的温育导致响应SEB抗原的T细胞的增殖活性显著增加。还与在80和10nM下使用的其他单克隆抗OX40激动剂相比，测试了Tetra-8。图19的结果表明，与同种型对照相比，在相同SEB测定中测试的7H11_v8IgG1、2H6 IgG1和其他抗OX40单克隆激动性抗体增强SEB诱导的T细胞增殖。然而，与所有测试的单特异性二价抗OX40分子相比，Tetra-8显示出显著更高水平的激动作用。总体而言，SEB和MLR测定的结果显示Tetra-8增强T细胞应答并显示出比其他激动性抗OX40分子更高的效力。与描述基本上通过FcγR介导的交联起作用的单克隆激动性抗OX40抗体相反，Tetra-8的活性是FcγR非依赖性的，如前所示。此外，在SEB测定中测试9B12导致IL-2水平增加但活化的CD4+T细胞(CD4+CD25+)数量减少。相反，Tetra-8促进该T细胞亚群的扩增，同时诱导高水平的IL-2(数据未显示)。Tetra-8的这种强烈的FcyR非依赖性激动活性可能与更高的化合价和/或其结构有关。在实施例12和14中进一步探讨了该假设。
实施例11：替代Tetra-8架构的生成
分子设计和表达
为了监测FcγR参与对Tetra-8生物活性的影响，如前所述克隆并产生具有wtIgG1 Fc(Tetra-13)的Tetra-8的修饰形式。然后，为了确定Tetra-8结构对其激动剂性质的影响，产生了几种具有不同结合物(Binder)组合、取向和化合价的构建体(图20)。
Tetra-14分子是四价抗体，其中7H11-VH2 N58K-D54E和7H11VL1序列被格式化为用二硫键工程化的scFv(Geneart AG合成的基因)并进一步在修饰的pcDNA3.1载体、在2H6IgG1 LALA重链序列的3′中克隆，如先前对Tetra-8所描述的。将2H6VL克隆到经修饰的pcDNA3.1载体中的人κ恒定区(2H6-LC)的框架中。通过共转染HEK293-EBNA1细胞中编码Tetra-14HC和2H6LC的载体产生Tetra-14(表21)。然后收集细胞上清液，并使用如前所述的蛋白A亲和纯化柱纯化分子。然后，进行另外的阳离子交换纯化步骤以除去共价多聚体污染物，如前面对Tetra-8所述的。因此，该分子由Tetra-8中使用的相同的结合物(Binder)组成，但是反向取向。
还设计了具有相同4种结合物的四价分子，并由Geneart AG合成基因。Tetra-15抗体由2H6结合物组成，而Tetra-16由7H11结合物组成。在该形式中，用二硫键工程化的2H6和7H11 scFv序列分别与2H6和7H11 IgG1 LALA重链序列融合。通过分别用编码2H6LC和7H11VL1的载体转染编码Tetra-15和Tetra-16重链的载体来产生Tetra-15和Tetra-16。如前所述，使用蛋白A色谱法纯化蛋白质，并通过阳离子交换进一步抛光(polished)。因此，Tetra-15和Tetra-16具有类似于Tetra-8分子的形式，同时具有4个相关的结合臂。此外，Tetra-17和Tetra-18抗体被设计为具有4个相同的结合臂。在Tetra-17和18形式中，Fab融合在IgG1重链的C-末端。Tetra-17重链序列由携带LALA突变的7H11-VH2N58K-D54E IgG1重链组成，其中7H11-VH2 N58K-D54E-IgG1 CH1序列通过短的Glv4Thr(G4T)接头与IgG1CH3结构域融合。类似地，Tetra-18重链由与编码2H6 Fab的序列连接的2H6IgG1 LALA序列组成。使用前面描述的相同方法，在HEK293-EBNA1细胞中分别用编码7H11VL1和2H6LC的载体共转染编码Tetra-17和Tetra-18重链的载体以产生Tetra-17和Tetra-18抗体(表21)。
设计其他四价抗体以将三种相同的结合物与一种不相关的结合物组合，并由Geneart AG合成基因。Tetra-19抗体是三种7H11 Fab与一种2H6 scFv的组合。在这种形式中，需要重链异二聚化。因此，BEAT技术(Skegro D.等，J Biol Chem.，292(23)：9745-59，2017年6月)用于产生和纯化Fc异二聚体。构建了两种不同的重链并克隆到两种不同的载体中。第一条重链(Tetra-19HC1)从N末端到C末端包含7H11-VH2N58K-D54E、IgG1 CH1、IgG1铰链、含有LALA突变的IgG1CH2、IgG3 CH3 BEAT(A)、G4T接头以及用二硫键改造的2H6 scFv的结构域序列。第二链(Tetra-19 HC2)由N末端至C末端制备7H11-VH2 N58K-D54E、IgG1 CH1、IgG1铰链、含有LALA突变的IgG1 CH2、IgG1 CH3 BEAT(B)、G4T接头、7H11-VH2 N58K-D54E和IgG1 CH1的结构域序列。使用与先前描述的相同的方案(表21)，在HEK293-EBNA1细胞中以编码7H11 VL1轻链的载体以等摩尔比转染编码这两条不同重链的载体。BEAT技术诱导含有链的BEAT(A)和BEAT(B)的优先异二聚化。然而，仍然可以产生一些同型二聚体杂质。然而，BEAT技术被设计用于重链不对称蛋白A结合，其允许从表达细胞的上清液中存在的同型二聚体污染物中有效纯化异二聚体。简而言之，将澄清的转染细胞上清液上样到在pH 6的0.2M柠檬酸盐/磷酸盐缓冲液中预平衡的HiTrapTMMabSelect SuReTM蛋白A柱上，并在纯化仪色谱系统(均来自GE Healthcare Europe GmbH)上操作。流速为1ml/分钟。运行缓冲液是0.2M柠檬酸盐/磷酸盐缓冲液，pH 6。洗涤缓冲液是0.2M柠檬酸盐/磷酸盐缓冲液，pH 5。使用pH 4.1的20mM乙酸钠缓冲液进行异二聚体洗脱。洗脱后，在280nm处读取吸光度；合并含有异二聚体Tetra-19的相关级分，并用0.1体积的1MTrisHCl(pH 8)中和。然后进行另外的阳离子交换纯化步骤以除去共价多聚体。使用相同的方案产生并纯化Tetra-20抗体，其是三种2H6 Fab与一种7H11 scFv的组合。Tetra-20HC1包含从N到C-末端的2H6VH、IgG1 CH1、IgG1铰链、含有LALA突变的IgG1 CH2，IgG3 CH3 BEAT(A)、G4T接头和用二硫键工程化的7H11 scFv的结构域序列。Tetra-20 HC2由N至C末端产生2H6 VH、IgG1CH1、IgG1铰链、含有LALA突变的IgG1 CH2、IgG1 CH3 BEAT(B)、G4T接头、2H6VH和IgG1 CH1的结构域序列。将这两条链与2H6轻链共表达以产生Tetra-20(表21)，其使用差异蛋白A和阳离子交换层析纯化。
设计Tetra-21抗体含有四个抗体结合结构域，其中一个2H6 scFv和一个7H11 Fab融合在Fc区的N末端，一个2H6 scFv和一个7H11 Fab融合在相同Fc区的C末端。如前所述，通过共表达Tetra-19 HC1与Tetra-21 HC2和7H11 VL1轻链(表21)产生该异二聚体。构建Tetra-21 HC2，从N到C末端含有：与含有LALA突变的IgG1 CH2连接的2H6 scFv，接着是IgG1CH3 BEAT(B)、G4T接头、7H11-VH2 N58K-D54E和IgG1 CH1。该链由Geneart AG合成并克隆到修饰的pcDNA3.1载体中。然后使用差异蛋白A色谱法纯化Tetra-21，然后进行另外的阳离子交换纯化步骤，如前所述。
设计Tetra-22以将曲妥珠单抗Fab与融合在Fc区的C末端的2H6 scFv组合作为单独的2H6 scFv激动剂活性的对照。构建Tetra-22 HC以包含从N末端到C末端的曲妥珠单抗VH、IgG1 CH1、IgG1铰链区、含有LALA突变的IgG1 CH2、IgGl CH3、G4T接头和二硫化键工程化的2H6 scFv。该重链是由Geneart AG以及曲妥珠单抗VL(Tetra-22LC)合成的基因，然后将两条链克隆到经修饰的pcDNA3.1载体中。然后如前面对Tetra-8所述制备和纯化Tetra-22(表21)。类似于2H6 scFv与曲妥珠单抗IgG1 LALA的C-末端融合，利妥昔单抗用作无关的结合物，并且使用Tetra-8和Tetra-22结构作为模板产生四价分子利妥昔单抗-2H6。
最后，产生Tri-8分子以产生具有两个7H11 Fab和在C末端融合一个2H6scFv的三价分子。该异二聚体通过将Tetra-19 HC1与Tri-8 HC2和7H11VL1轻链组合而制成(表21)。Tri-8 HC2包括N末端至C末端的7H11-VH2 N58K-D54E、IgG1 CH1、含有LALA突变的IgG1CH2，然后是IgG1 CH3 BEAT(B)。该链由Geneart AG基因合成，并克隆到修饰的pcDNA3.1载体中。然后如前所述对Tetra-19、20和21产生和纯化Tri-8。
表21：用于产生四价和三价抗体的重链和轻链的组合。
表征具有替代Tetra-8结构的抗体
然后使用前面描述的方法通过Biacore进一步表征分子。使用FabALACTICA消化蛋白质，并使用CaptureSelectTM FcXL纯化蛋白水解后获得的Fc融合的C末端结合物。然后将获得的材料用于研究Biacore的C末端结合物效力。
使用与测量Tetra-8中OX40的2H6 scFv亲和力相同的方法测量在Tetra-14分子的C末端融合的7H11 scFv对OX40的亲和力。测定19nM的亲和力(表22)，表明在C-末端融合的Tetra-14结合臂是有功能的，尽管测量的7H11的亲和力降低了2倍。
表22：当在N或C末端融合时7H11和2H6亲和力的表征。
Tetra-17、Tetra-18、Tetra-19和Tetra-20也被消化，由Biacore使用OX40嵌合体进行研究。将chiOX40R HHRH-Fc和chiOX40R RRHH-Fc分别通过将它们注射到流路2和4固定在先前活化的CM5传感器芯片(3000RU)上，以达到两个分子的3000RU。然后，将纯化的Tetra-17、Tetra-18、Tetra-19和Tetra-20的消化产物用作分析物并注入4个流路径(流路1和3用作参考)，浓度为200nM，流速为30uL/min，持续240秒。然后使用HBS-EP缓冲液进行第二次注射3分钟，然后进行5分钟的解离。使用甘氨酸pH 1.5缓冲液进行注射之间的再生1分钟(图21和表23)。7H11结合物(Binder)是OX40 CRD1的特异性，因此它只能与chiOX40RHHRH-Fc结合，而不与chiOX40R RRHH-Fc结合(表23)。在这些情况下，我们观察到7H11与chiOX40R HHRH-Fc在C末端融合为Fab时的结合比scFv形式好4倍(分别为450响应单位(RU)与100RU的Fc-7H11Fab/2H6 scFv与Fc-7H11 scFv/2H6 fab的比较)，其表示7H11无论其格式如何都能正常工作。我们还确定，在二价形式中，7H11 Fab与OX40 CRD1的结合比单价形式好2倍(分别为870 RU与450 RU的Fc-7H11Fab/7H11 Fab与Fc-7H11 Fab/2H6 scFv的比较)，表明在C末端融合的两个Fab都是功能性的并且可能与两个OX40分子共同结合。2H6结合物(Binder)是OX40 CRD3的特异性，因此它只能与chiOX40R RRHH-Fc结合，而不与chiOX40R HHRH-Fc结合。我们还观察到，作为Fab在C末端融合的2H6的chiOX40R RRHH-Fc的结合优于scFv形式(Fc-7H11Fab/2H6 scFv与Fc-7H11 scFv/2H6 fab的比较)，证实当格式化为Fab或scFv时2H6是功能性的。我们还观察到，在二价形式中，2H6 Fab与OX40 CRD3的结合优于单价形式(Fc-2H6 Fab/2H6 Fab与Fc-7H11scFv/2H6 Fab的比较)，表明融合的2H6结合单位在C-末端都是功能性的并且可能与两个OX40分子共同结合。
表23：当在具有不同化合价和/或形式的C-末端融合时7H11和2H6结合的表征。
为了清楚地证明共结合事件，我们稍微修改了之前描述的Biacore设置，仅使用Fc-7H11 Fab/2H6-scFv或Fc-7H11 scFv/2H6 Fab作为第一注射的分析物并注射缓冲液、人OX40-Fc，chiOX40R HHRH-Fc(7H11特异性)和chiOX40RRRHH-Fc(2H6特异性)嵌合体在400nM下持续3分钟，然后在第二注射步骤中解离5分钟。使用甘氨酸pH 1.5缓冲液进行注射之间的再生1分钟(图22和23)。使用这些方法可以监测共同结合事件。有效地，当Fc-7H11 Fab/2H6 scFv部分通过7H11与OX40 CRD1的结合在CHIP上捕获时，仍然可以与人OX40-Fc相互作用，但也与chiOX40R RRHH-Fc(2H6特异性)相互作用(图22a)。类似地，通过2H6与OX40 CRD3结合而在CHIP上捕获的Fc-7H11 Fab/2H6 scFv部分可以与人OX40和chiOX40R OX40 HHRH-Fc(7H11特异性)相互作用(图22b)。用Fc-7H11 scFv/2H6 Fab获得了类似的结果(图23a和23b)。总之，这些数据表明，在C末端融合的7H11或2H6结合单位是功能性的，无论它们的Fab或scFv形式如何，并且它们可以同时共同结合两个不同的OX40单元。
实施例13：tetra-8的激动活性与其结构和化合价有关
与单克隆二价7H11_v8 IgG1或2H6 IgG1相比，展示四种结合单位的Tetra-8显示出更高的激动活性，如实施例10中所示。为了评估Tetra-8结构在其生物学功能中的贡献，在SEB测定中生成并测试了显示不同结构和效价的几种Tetra-8变体。这些分子列于图20中。如图24所示，由来自2个不同克隆的4个结合部分组成的Tetra-8引发比由i)来自一个克隆(7H11或2H6)的2个结合部分ii)衍生自2个不同克隆的3个结合部分(Tri-8)，iii)由4个相似结合部分(Tetra-15和Tetra-16)组成的四价分子组成的分子更高的激动活性。在相同的测定中测试了具有与Tetra-8不同的结构的另外两种四价抗OX40变体分子：Tetra-21和Tetra-14。这两个分子由与Tetra-8(衍生自7H11和2H6克隆)相同的OX40结合部分组成，但具有不同的取向。如图24所示，与SEB测定中的Tetra-8抗体相比，两种四价分子都显示出弱的激动势。此外，Tetra-8诱导的IL-2水平高于7H11和2H6(7H11IgG1 LALA+Tetra-22)的组合，如表24所示，其表明在同一分子中存在四个OX40结合单位是是推动Tetra-8活动的关键。总之，图24的结果表明Tetra-8的强烈激动特性与其四倍体及其结构有关。
表24
实施例13靶向OX40结构域的四价抗体
Tetra-8激动剂抗体分别通过其7h11和2H6结合单位与OX40 CRD1和CRD3结合。Tetra-8四价结构，包含于与IgG1 LALA重链的C-末端部分融合的二硫键工程化的scFv，似乎对其激动剂活性是最佳的。此外，用与Tetra-8共享相似结合单位但具有不同结构的抗体获得的数据也表明，N末端Fab部分必须靶向膜远端OX40结构域，而C末端scFv应与膜近端OX40结构域相互作用。使用2H6、7H11、9B12、11D4、1A7、106-222、pab199和hzG3V9 OX40结合单位来确定以四价形式组合的其他OX40结合物是否可以激动OX40。使用Tetra-8特异性形式作为模板组装抗OX40抗体序列(即由VH、IgG1 CH1、IgG1-铰链、IgG1 CH2LALA、IgG1 CH3、接头、二硫化物改造的scFv或来自N末端至C末端的dAb组成)。在N-末端，选择7H11、11D4、1A7、9B12、106-222和2H6的VH，而在C-末端，选择9B12、2H6、pab1949和hzG3v9dAb的scFv。设计结合物(Binder)组合以探索四价分子的不同OX40表位结合(表25)。然后，编码所设计的重链1A7_2H6_8、106-222_2H6_8、7H11_1949_8、11D4_1949、177_1949、9312_1949、106-222_1949、7H11_9B12_8、7HII_hzG3v9_8、HD4_hzG3v9、106-222_hzG3v9、9312_hzG3v9和2H6_hzG3v9_8的cDNA由GeneArt基因合成，然后在修饰中克隆pCDNA3.1载体，如前所述。为了产生这些四价抗体，在HEK293-EBNA1细胞中用它们各自的轻链(表26)共转染重链，并在蛋白A纯化之前收集上清液。然后，使用另外的阳离子交换纯化步骤除去与四价分子形成的共价多聚体，所述四价分子具有在C-末端融合的二硫化物改造的scFv。
表25：基于其表位的4价抗体形式的OX40结合物的组合。
表26：用于产生四价抗体的重链和轻链的组合
表征pab1949融合在N或C末端时的亲和力。
使用与测量Tetra-8中OX40的2H6 scFv亲和力相同的方法测量在C末端融合的pab1949 scFv的亲和力。测定460nM的亲和力(表27)。为了确定pab1949IgG1对OX40的亲和力，在CM5芯片上捕获约600RU的该抗体，所述CM5芯片中先前固定了抗人IgG Fc。然后注入稀释系列的hsOX40_CRD_Avi_His(SED ID NO：159)。在该形式中，测量了304nM的pab1949的亲和力。
表27：pab1949作为在C末端融合的IgG1或scFv的亲和力。
实施例14：OX40的多个表位的结合增加了其激动潜力
在Tetra-8和Tetra-16之间或在Tetra-8和Tetra-15之间观察到的功能差异强烈表明由靶向OX40的不同表位的多个结合单元组成的分子显示出比多价单特异性分子更高的激动潜力。为了验证该假设，产生了由识别不同OX40表位的结合单元组成的分子，并在SEB测定中进行测试。如图25所示，Tetra-8、7H11_1949_8和11D4_1949分子是由对OX40的结构域1和3特异的结合单元组成的四价分子，表现出比Tetra-15或Tetra-16四价单特异性抗体更高的激动作用。类似地，靶向OX40的结构域1和4的四价7H11_hzG3v9_8和HD4_hzG3v9比其二价对应物引发更强的激动活性。这些数据显示Tetra-8通过OX40的膜远端和近端结构域的多价和双表位靶向显示出有效的OX40激动活性。此外，与单个分子相比，7H11 IgG1LALA+Tetra-22的组合诱导更高的IL-2水平，但没有重现Tetra-8诱导的IL-2水平，如表24所示。这表明由四价Tetra-8抗体介导的OX40的双表位靶向引发比两个二价分子的组合介导的双表位靶向更高的激动活性。
实施例15 TETRA-8+hOX40的多聚化与体外活性相关
为了确定Tetra-8+hOX40复合物的化学计量，在连接到系统的Superdex 20010/300GL增加柱(均为GE Healthcare Europe GmbH，格拉特布鲁格，瑞士)上进行分析性凝胶过滤色谱。运行缓冲液是PBS pH 7.4(Gibco，Thermo Fisher Scientific，Reinach，瑞士)，流速为0.5ml/min。注射的样品体积不超过柱体积的2％，一般为0.3-0.5ml。在第一轮中，注射2500pmol(1份)抗体。在第二轮中，注射5000pmol(2份)hOX40(SEQID NO：160)。对于第三次运行，将2500pmol抗体(1份)与10000pmol hOX40(4份)混合，并在注射前在室温下温育10分钟。在使用高分子量和低分子量校准试剂盒(28-4038-41，28-4038-42，GE Healthcare)之前进行校准运行。单独的Tetra-8，单独的hOX40和1∶4比例的Tetra-8/hOX40的色谱图显示在图26中。考虑到16kDa配体的结合，不是观察复合物的峰值以最小的方式向单个峰形状的早期洗脱体积移动，而是在比预期更高的分子量下观察到许多新的峰。发现组装的峰早于440kDa校准标记物洗脱，表明形成了由多于一种抗体组成的复合物。过量的约5000pmol的未结合的hOX40(来自曲线下面积)和在复合物混合物中加入10000pmol的事实表明每个Tetra-8结合了两个hOX40分子。考虑到Tetra-8的理论分子量为198kDa，hOX40的理论分子量为16kDa，可以推断第二个峰包含2-3个与hOX40复合的Tetra-8分子，第一个峰和它的肩包含更高阶的多聚体(由两种以上抗体和多种hOX40组成的复合物)。我们假设，由于其双互补性，Tetra-8可以与hOX40多聚化以形成大的类晶体晶格(图27)。每个Tetra-8一个hOX40足以在理论上产生无限大的晶格，尽管如上所述，每个抗体似乎结合了两个受体。在同一实验中测试了许多对照分子，以使多聚化与体外活性相关联。仅含有7H11结构域的对照分子在约440kDa处显示出峰，表明由hOX40结合诱导的非特异性二聚体的形成(图26，为简单起见，未单独显示对照抗体的峰)。没有观察到更高级的多聚体。与该结果一致，没有观察到这些分子的体外活性。Tri-8在～440kDa处显示出峰，表明二聚体形成，但是没有观察到显著量的高级多聚体。与该结果一致，Tri-8未显示体外活性。Tetra-14显示出两个峰，第一个可能包含更高级的多聚体和第二个峰，在440kDa标记之前洗脱，其可能包含二聚体或与多个hOX40分子复合的单个抗体。Tetra-14在体外没有显示出活性，我们假设这是与Tetra-8相比其较低的多聚化倾向的结果。Tetra-8在440kDa标记处或之前未显示峰，但仅显示高级多聚体的峰。此外，Tetra-8/hOX40显示在空隙体积(V0)中洗脱的肩部，观察不能制成Tetra-14。7H11_1949_8在体外具有最高活性，同时在分析凝胶过滤中显示出最高的多聚化程度，与测试的任何其他分子相比，大多数蛋白质在V0中洗脱。7H11_v8 IgG1也包括在实验中并且在～440kDa处显示出峰值，这可能是非特异性二聚体形成的结果。正如预期的那样，对于该分子，没有观察到更高级多聚化的峰。总之，我们提出表位和抗体结构的组合决定了多聚化的倾向，并且更高级的多聚化与体外活性相关。
实施例16：Tetra-8诱导细胞表面上的局部OX40聚集
16.1产生表达人OX40-eGFP的稳定Jurkat细胞系
使用电穿孔(转染系统)用pT1-hsOX40-eGFP_fusion_IRES_Puromycin质粒(GSY935a)转染Jurkat E6.1细胞(ECACC 88042803)，hsOX40-eGFP是融合蛋白，其中eGFP融合在hsOX40的C末端部分。在第二天在含有嘌呤霉素(RPMI 1640，谷氨酰胺+10％FBS+0.25ug/mL嘌呤霉素)的生长培养基中进行有限稀释。在37℃和5％CO2温育2周后，使用EasyCyte流式细胞仪分析单个库的eGFP表达，并选择19个库。五天后，使用FACS分析这些库的hsOX40表达，并保留具有不同表达水平的hsOX40-eGFP融合蛋白的7个同源库。将它们扩增并在含有10％DMSO的90％FBS中冷冻。OX40-eGFP融合蛋白在细胞表面上的表达允许在细胞活化时直接观察OX40聚集。
16.2 Tetra-8诱导Jurkat OX40-eGFP细胞系上膜结合OX40的聚集
16.2.1用Tetra-8处理的OX40-GFP Jurkat细胞上的Time lapse共聚焦显微镜检查。
至于TNFRSF的其他成员，下游信号的激活依赖于OX40多聚化。Tetra-8以不依赖于FcγR的方式触发OX40信号传导的事实强烈暗示抗体对OX40多聚化的直接影响。为了使Tetra-8触发膜结合OX40聚集的能力相互作用，对表达用Tetra-8预孵育的Jurkat表达OX40eGFP细胞进行Time lapse共聚焦显微镜实验。简言之，在室温下将Fluorodish(WPI)细胞培养皿用1mL纤连蛋白(在PBS中的1μg/cm2)预涂覆45分钟。然后用PBS洗涤培养皿2次，并将3mL RPMI中的细胞悬浮液和嘌呤霉素(20000细胞/cm2)倒入培养皿中。将细胞在37℃和5％CO2下孵育过夜。将培养皿放置在显微镜下，将焦点设置在典型的细胞上，并且每30秒重复拍摄一次照片。在1.5分钟时，将含有Tetra-8的溶液以80nM终浓度加入培养基中。使用配备有共焦模块LSM 800的Zeiss倒置显微镜Z1以63x放大率对细胞成像。如图28所示，在用Tetra-8处理Jurkat OX40-eGFP细胞后，OX40-GFP的荧光模式从质膜的均匀染色开始超时到质膜上的离散斑块中的清晰聚集。
16.2.2测试Tetra-8诱导的OX40聚类与其他OX40特异性分子的比较
如前面部分所示，Tetra-8比许多其他单克隆OX40激动剂显示出更有效的FcγR非依赖性激动活性。使用荧光共聚焦显微镜在OX40聚类实验中进一步研究了这些差异。在这些实验中，与Tetra-14、1AM7和OX40L分子相比，测试了Tetra-8在诱导膜结合的OX40的局部聚集中的作用。简而言之，按照前一段中描述的方案，在Jurkat-OX40eGFP细胞上以20和80nM的两种浓度测试这些分子。基于先前共聚焦实验的结果，选择时间点5、10和20min来监测测试的化合物对OX40聚类的影响。图29的结果显示，使用20或80nM的Tetra-8，在37℃温育5分钟后，OX40-GFP的荧光模式已经显著受到影响。相比之下，即使在20分钟后，所测试的其他分子的效果也不太明显：1A7似乎没有任何定性效果，而OX40L和Tetra-14诱导非常微弱的OX40-GFP浓度。为了更准确地评估这些差异，开发了一种定量方法。第一步是基于通过共聚焦显微镜获得的OX40-GFP荧光的3维堆叠来绘制细胞膜的荧光强度。该荧光强度显示在θ-z坐标系中，其中θ是细胞膜相对于细胞中心的任意点的角度，z是盖玻片的高度。作为第二步，从该荧光图中提取单个数值：表面的峰度。这个参数常用于表面测量，是因为它是衡量平均线上下的尖峰分布的指标(对于尖刺表面，R_ku＞3；对于凹凸不平的表面，R_ku＜3；完全随机的表面有峰度3)。测量每个样品细胞的峰度值，得到平均峰度值，每个细胞实验条件的平均值的标准误差(即药物类型、药物浓度、注射后的时间)。来自均值的标准误差由σ_x＝σ/√n定义，其中σ是总体的标准偏差，n是观测数量；n通常等于每个实验条件下4个细胞样品。如图30所示，来自OX40聚类的定量分析的结果显示，与其他处理(1A7、OX40L和Tetra-14)相比，用Tetra-8处理Jurkat-OX40-GFP细胞后，峰度值随时间增加。
实施例17靶向CD40的不同结构域的四价抗体
OX40是TNFR超家族的成员，其包含TNFR1、TNFR2、BAFFR、BCMA、TACI、GITR、CD27、4-1BB、CD40、DR3、HVEM、LTβR、RANK、Fn14、FAS、TRAILR1和TRAILR2。这些受体的特征在于其细胞外部分中的共同结构域，其是富含半胱氨酸的结构域(CRD)。我们的数据显示，激活OX40的四价抗体的作用机制似乎依赖于两种不同OX40 CRD的共同作用。为了确定这种作用机制是否可以在TNFR超家族中有更广泛的应用，我们已经产生抗CD40四价分子作为概念的证据。从文献检索中鉴定抗CD40抗体，并从专利申请或数据库检索中检索它们各自的序列。2C10(WO 2017/040932 A1)、ADC-1013(US 2014/0348836 A1)、CD40.1(US 2016/0376371A1)、selicrelumab(US 8,388,971B2)、替奈昔单抗(RCSB，5DMI)和3h56-5(选择US2017/0015754 A1)抗CD40抗体作为其在CD40上的表位是已知的，并且因为还报道了它们的拮抗剂或激动剂活性。据报道，2C10、CD40.1、ADC-1013和替奈昔单抗靶向CD40CRD1。selicrelumab与CD40的CRD1和2结合，而3h56-5与CRD3相互作用。2C10和3h56-5被描述为拮抗剂，而其他抗体是激动剂。大多数这些抗体靶向CD40膜远端结构域，除了3h56-5dAb，其结合该受体的膜近端结构域。因此，通过将3h56-5dAb序列与2C10、selicrelumab、CD40.1、ADC-1013和替奈昔单抗的重链的C末端融合，产生抗CD40四价分子。这些抗体的VH、VL和dAb序列由Geneart AG合成。用于这些四价抗体的形式类似于前面描述的四价抗体。将VH cDNA序列克隆到修饰的pcDNA3.1载体中，在人IgG1-LALA骨架的框架中，然后是短的G4T接头序列和3h56-5dAb序列。将VL cDNA序列克隆到经修饰的pcDNA3.1载体中的κ或λ恒定结构域的框架中。此外，将selicrelumab和ADC-1013VH序列分别克隆到人IgG1 LALA或人IgG1骨架的框架中，同时将3H56-5序列克隆到含有LALA突变的人IgG1 Fc片段的框架中。通过共转染HEK293-EBNA1细胞中的重链和轻链(表28)或单个重链产生四价、selicrelumab IgGl LALA和3h56 IgG1 LALA分子。在蛋白A纯化之前收集上清液。
表28：用于抗体产生的重链和轻链的组合
实施例18：CD40的双表位靶向导致增加的激动活性
通过双表位靶向增强OX40激动的方法进一步扩展至CD40，其是TNFR超家族的另一成员，其显示与OX40的结构同源性。为此，产生了组合衍生自抗CD40单克隆抗体的结合单位的各种分子，如表28中所列，并在DC成熟测定中进行测试。在该测定中，如前所述分离人PBMC，并使用单核细胞纯化试剂盒按照制造商的说明书(干细胞)纯化单核细胞。为了产生DC，将纯化的单核细胞在GM-CSF以50ng/mL(R&D)和持续6天的rhIL-4以20ng/mL(R&D)存在下在37℃，5％CO2下培养6天。使用CD1c APC(Thermofischer)通过流式细胞术验证树突细胞的表型。然后在抗CD40抗体或对照的存在下培养细胞。两天后，收获DC并用抗-CDlc-APC、抗-CD80-PE、抗-CD86-PerCP-eF710抗-CD83-FITC、抗-HLA-DR-PerCP5.5(Thermofischer)染色。用100μl FACS缓冲液洗涤细胞，并在Cytoflex上获得。为了评估测试的抗CD40抗体在DC上上调CD83和CD86的潜力，使用CytExpert进行表达CD83和CD86的细胞百分比的分析。如图31所示，用单克隆单特异性或双表位抗CD40抗体处理单核细胞衍生的DC导致DC成熟标记CD83和CD86的上调。同样，CD40和HLA-DR也被上调(数据未显示)。虽然大多数测试的抗体诱导与可溶性CD40L(Selicrelumab IgG1 LALA、ADC-1013_3h56、2C10_3h56和CD40.1_3h56)相等的激动作用，但两种双表位抗CD40抗体显示甚至更高的激动活性(Selicrelumab_3h56和替奈昔单抗_3h56)。
根据制造商的说明书(Promega)，使用CD40-生物测定试剂盒进一步评估先前在DC成熟测定中测试的抗CD40分子的激动潜力。在该测定中，将NFkB-Luc2P/U2O以3×105个细胞/ml重悬于完全RPMI培养基(RPMI1640，10％FBS)中，并将100ml该细胞悬浮液分配在96个发光板中。然后将板在37℃，5％CO2下孵育过夜。第二天，将所有测试的抗CD40抗体在测定缓冲液(RPMI1640+1％FBS)中连续稀释，并将75ml该制剂加入细胞中。在37℃，5％CO2下孵育5小时后，向孔中加入75μL Bio-Glo溶液，并在酶标仪中获得平板。使用以下设置测量发光：读取带-端点。图32的结果显示抗CD40抗体显示出不同水平的CD40依赖性荧光素酶活性。在该测定中，如所预期的，报道的抗CD40拮抗抗体3h56 IgG1 LALA不激活CD40信号。然而，其结合单元与源自Selicrelumab(Selicrelumab_3h56)或ADC-1013(ADC-1013_3h56)的部分的组合分别导致与单独的Selicrelumab或ADC-1013相比增加的活性。
总之，图31和32的结果显示，如用OX40所观察到的，使用四价双表位抗体靶向CD40的方法与其单特异性对应物相比促进了增强的激动活性。
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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 Ser Tyr
20 25 30
Asp Ile Asn Trp Val Arg Gln Ala Thr Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Trp Met Asn Pro Asn Ser Gly Asn Thr Gly Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Thr Met Thr Arg Asn Thr Ser Ile 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
Ala Arg
<210> 8
<211> 101
<212> PRT
<213> 人工序列
<220>
<223> IGKV4-1*01
<400> 8
Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Tyr Ser
20 25 30
Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val
50 55 60
Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln
85 90 95
Tyr Tyr Ser Thr Pro
100
<210> 9
<211> 96
<212> PRT
<213> 人工序列
<220>
<223> IGKV3D-7*01
<400> 9
Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser
20 25 30
Tyr Leu Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu
35 40 45
Ile Tyr Gly Ala Ser Thr Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser
50 55 60
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln
65 70 75 80
Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Asp Tyr Asn Leu Pro
85 90 95
<210> 10
<211> 95
<212> PRT
<213> 人工序列
<220>
<223> IGKV3D-15*01
<400> 10
Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Asn
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45
Tyr Gly Ala Ser Thr Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser
65 70 75 80
Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Asn Asn Trp Pro
85 90 95
<210> 11
<211> 96
<212> PRT
<213> 人工序列
<220>
<223> IGKV3-20*01
<400> 11
Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser
20 25 30
Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu
35 40 45
Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser
50 55 60
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu
65 70 75 80
Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro
85 90 95
<210> 12
<211> 446
<212> PRT
<213> 人工序列
<220>
<223> 人源化7H11-VH1
<400> 12
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 Arg Tyr
20 25 30
Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Trp Ile Tyr Pro Gly Asp Gly Ser Thr Lys Tyr Ser Gln Lys Phe
50 55 60
Gln Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala 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
Ala Arg Glu Tyr Gly Asn Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala
115 120 125
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu
130 135 140
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
145 150 155 160
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
165 170 175
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
180 185 190
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr
195 200 205
Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr
210 215 220
Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
225 230 235 240
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
245 250 255
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
260 265 270
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
275 280 285
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
290 295 300
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
305 310 315 320
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
325 330 335
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
340 345 350
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
355 360 365
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
370 375 380
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
385 390 395 400
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
405 410 415
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
420 425 430
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 445
<210> 13
<211> 446
<212> PRT
<213> 人工序列
<220>
<223> 人源化7H11-VH2
<400> 13
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 Arg Tyr
20 25 30
Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Trp Ile Tyr Pro Gly Asp Gly Ser Thr Asn Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Glu Tyr Gly Asn Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala
115 120 125
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu
130 135 140
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
145 150 155 160
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
165 170 175
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
180 185 190
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr
195 200 205
Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr
210 215 220
Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
225 230 235 240
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
245 250 255
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
260 265 270
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
275 280 285
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
290 295 300
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
305 310 315 320
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
325 330 335
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
340 345 350
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
355 360 365
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
370 375 380
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
385 390 395 400
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
405 410 415
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
420 425 430
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 445
<210> 14
<211> 446
<212> PRT
<213> 人工序列
<220>
<223> 人源化7H11-VH3
<400> 14
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 Arg Tyr
20 25 30
Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Ile Ile Tyr Pro Gly Asp Gly Ser Thr Ser Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Glu Tyr Gly Asn Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala
115 120 125
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu
130 135 140
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
145 150 155 160
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
165 170 175
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
180 185 190
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr
195 200 205
Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr
210 215 220
Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
225 230 235 240
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
245 250 255
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
260 265 270
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
275 280 285
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
290 295 300
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
305 310 315 320
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
325 330 335
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
340 345 350
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
355 360 365
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
370 375 380
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
385 390 395 400
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
405 410 415
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
420 425 430
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 445
<210> 15
<211> 446
<212> PRT
<213> 人工序列
<220>
<223> 人源化7H11-VH4
<400> 15
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 Arg Tyr
20 25 30
Tyr Ile Asn Trp Val Arg Gln Ala Thr Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Trp Ile Tyr Pro Gly Asp Gly Ser Thr Gly Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Thr Met Thr Arg Asn Thr Ser Ile 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
Ala Arg Glu Tyr Gly Asn Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala
115 120 125
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu
130 135 140
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
145 150 155 160
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
165 170 175
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
180 185 190
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr
195 200 205
Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr
210 215 220
Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
225 230 235 240
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
245 250 255
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
260 265 270
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
275 280 285
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
290 295 300
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
305 310 315 320
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
325 330 335
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
340 345 350
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
355 360 365
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
370 375 380
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
385 390 395 400
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
405 410 415
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
420 425 430
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 445
<210> 16
<211> 220
<212> PRT
<213> 人工序列
<220>
<223> 人源化7H11-VL1
<400> 16
Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser
20 25 30
Gly Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Pro Pro Lys Leu Leu Ile Tyr Gly Ala Ser Thr Arg Glu Ser Gly Val
50 55 60
Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn
85 90 95
Asp His Ser Tyr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile
100 105 110
Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp
115 120 125
Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn
130 135 140
Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu
145 150 155 160
Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp
165 170 175
Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr
180 185 190
Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser
195 200 205
Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
210 215 220
<210> 17
<211> 220
<212> PRT
<213> 人工序列
<220>
<223> 人源化7H11-VL2
<400> 17
Glu Ile Val Met Thr Gln Ser Pro Pro Thr Leu Ser Leu Ser Pro Gly
1 5 10 15
Glu Arg Val Thr Leu Ser Cys Arg Ala Ser Gln Ser Leu Leu Asn Ser
20 25 30
Gly Asn Gln Lys Asn Tyr Leu Ser Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Ala Pro Arg Leu Leu Ile Tyr Gly Ala Ser Thr Arg Ala Thr Gly Ile
50 55 60
Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Asn
85 90 95
Asp His Ser Tyr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile
100 105 110
Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp
115 120 125
Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn
130 135 140
Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu
145 150 155 160
Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp
165 170 175
Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr
180 185 190
Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser
195 200 205
Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
210 215 220
<210> 18
<211> 220
<212> PRT
<213> 人工序列
<220>
<223> 人源化7H11-VL3
<400> 18
Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Leu Leu Asn Ser
20 25 30
Gly Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Ala Pro Arg Leu Leu Ile Tyr Gly Ala Ser Thr Arg Ala Thr Gly Ile
50 55 60
Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Leu Gln Ser Glu Asp Phe Ala Val Tyr Tyr Cys Gln Asn
85 90 95
Asp His Ser Tyr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile
100 105 110
Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp
115 120 125
Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn
130 135 140
Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu
145 150 155 160
Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp
165 170 175
Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr
180 185 190
Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser
195 200 205
Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
210 215 220
<210> 19
<211> 220
<212> PRT
<213> 人工序列
<220>
<223> 人源化7H11-VL4
<400> 19
Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Leu Leu Asn Ser
20 25 30
Gly Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Ala Pro Arg Leu Leu Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile
50 55 60
Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Ser Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Asn
85 90 95
Asp His Ser Tyr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile
100 105 110
Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp
115 120 125
Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn
130 135 140
Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu
145 150 155 160
Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp
165 170 175
Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr
180 185 190
Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser
195 200 205
Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
210 215 220
<210> 20
<211> 446
<212> PRT
<213> 人工序列
<220>
<223> 人源化7H11-VH2 V37M
<400> 20
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 Arg Tyr
20 25 30
Tyr Met His Trp Met Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Trp Ile Tyr Pro Gly Asp Gly Ser Thr Asn Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Glu Tyr Gly Asn Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala
115 120 125
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu
130 135 140
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
145 150 155 160
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
165 170 175
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
180 185 190
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr
195 200 205
Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr
210 215 220
Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
225 230 235 240
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
245 250 255
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
260 265 270
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
275 280 285
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
290 295 300
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
305 310 315 320
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
325 330 335
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
340 345 350
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
355 360 365
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
370 375 380
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
385 390 395 400
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
405 410 415
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
420 425 430
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 445
<210> 21
<211> 446
<212> PRT
<213> 人工序列
<220>
<223> 人源化7H11-VH2 N58K
<400> 21
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 Arg Tyr
20 25 30
Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Trp Ile Tyr Pro Gly Asp Gly Ser Thr Lys Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Glu Tyr Gly Asn Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala
115 120 125
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu
130 135 140
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
145 150 155 160
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
165 170 175
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
180 185 190
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr
195 200 205
Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr
210 215 220
Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
225 230 235 240
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
245 250 255
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
260 265 270
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
275 280 285
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
290 295 300
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
305 310 315 320
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
325 330 335
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
340 345 350
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
355 360 365
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
370 375 380
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
385 390 395 400
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
405 410 415
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
420 425 430
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 445
<210> 22
<211> 446
<212> PRT
<213> 人工序列
<220>
<223> 人源化7H11-VH2 A60N
<400> 22
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 Arg Tyr
20 25 30
Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Trp Ile Tyr Pro Gly Asp Gly Ser Thr Asn Tyr Asn Gln Lys Phe
50 55 60
Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Glu Tyr Gly Asn Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala
115 120 125
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu
130 135 140
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
145 150 155 160
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
165 170 175
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
180 185 190
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr
195 200 205
Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr
210 215 220
Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
225 230 235 240
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
245 250 255
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
260 265 270
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
275 280 285
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
290 295 300
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
305 310 315 320
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
325 330 335
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
340 345 350
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
355 360 365
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
370 375 380
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
385 390 395 400
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
405 410 415
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
420 425 430
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 445
<210> 23
<211> 446
<212> PRT
<213> 人工序列
<220>
<223> 人源化7H11-VH2 Q61E
<400> 23
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 Arg Tyr
20 25 30
Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Trp Ile Tyr Pro Gly Asp Gly Ser Thr Asn Tyr Ala Glu Lys Phe
50 55 60
Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Glu Tyr Gly Asn Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala
115 120 125
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu
130 135 140
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
145 150 155 160
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
165 170 175
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
180 185 190
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr
195 200 205
Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr
210 215 220
Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
225 230 235 240
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
245 250 255
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
260 265 270
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
275 280 285
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
290 295 300
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
305 310 315 320
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
325 330 335
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
340 345 350
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
355 360 365
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
370 375 380
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
385 390 395 400
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
405 410 415
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
420 425 430
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 445
<210> 24
<211> 446
<212> PRT
<213> 人工序列
<220>
<223> 人源化7H11-VH2 D85E
<400> 24
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 Arg Tyr
20 25 30
Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Trp Ile Tyr Pro Gly Asp Gly Ser Thr Asn Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Arg Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Glu Tyr Gly Asn Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala
115 120 125
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu
130 135 140
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
145 150 155 160
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
165 170 175
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
180 185 190
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr
195 200 205
Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr
210 215 220
Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
225 230 235 240
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
245 250 255
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
260 265 270
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
275 280 285
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
290 295 300
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
305 310 315 320
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
325 330 335
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
340 345 350
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
355 360 365
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
370 375 380
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
385 390 395 400
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
405 410 415
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
420 425 430
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 445
<210> 25
<211> 446
<212> PRT
<213> 人工序列
<220>
<223> 人源化7H11-VH2 V89I
<400> 25
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 Arg Tyr
20 25 30
Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Trp Ile Tyr Pro Gly Asp Gly Ser Thr Asn Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Ile Tyr Tyr Cys
85 90 95
Ala Arg Glu Tyr Gly Asn Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala
115 120 125
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu
130 135 140
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
145 150 155 160
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
165 170 175
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
180 185 190
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr
195 200 205
Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr
210 215 220
Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
225 230 235 240
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
245 250 255
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
260 265 270
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
275 280 285
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
290 295 300
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
305 310 315 320
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
325 330 335
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
340 345 350
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
355 360 365
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
370 375 380
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
385 390 395 400
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
405 410 415
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
420 425 430
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 445
<210> 26
<211> 446
<212> PRT
<213> 人工序列
<220>
<223> 人源化7H11-VH2 Y91F
<400> 26
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 Arg Tyr
20 25 30
Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Trp Ile Tyr Pro Gly Asp Gly Ser Thr Asn Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Phe Cys
85 90 95
Ala Arg Glu Tyr Gly Asn Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala
115 120 125
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu
130 135 140
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
145 150 155 160
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
165 170 175
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
180 185 190
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr
195 200 205
Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr
210 215 220
Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
225 230 235 240
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
245 250 255
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
260 265 270
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
275 280 285
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
290 295 300
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
305 310 315 320
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
325 330 335
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
340 345 350
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
355 360 365
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
370 375 380
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
385 390 395 400
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
405 410 415
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
420 425 430
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 445
<210> 27
<211> 446
<212> PRT
<213> 人工序列
<220>
<223> 人源化7H11-VH2 N58K-D54E
<400> 27
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 Arg Tyr
20 25 30
Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Trp Ile Tyr Pro Gly Glu Gly Ser Thr Lys Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Glu Tyr Gly Asn Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala
115 120 125
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu
130 135 140
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
145 150 155 160
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
165 170 175
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
180 185 190
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr
195 200 205
Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr
210 215 220
Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
225 230 235 240
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
245 250 255
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
260 265 270
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
275 280 285
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
290 295 300
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
305 310 315 320
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
325 330 335
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
340 345 350
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
355 360 365
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
370 375 380
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
385 390 395 400
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
405 410 415
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
420 425 430
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 445
<210> 28
<211> 446
<212> PRT
<213> 人工序列
<220>
<223> 人源化7H11-VH2 N58K-D54S
<400> 28
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 Arg Tyr
20 25 30
Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Trp Ile Tyr Pro Gly Ser Gly Ser Thr Lys Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Glu Tyr Gly Asn Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala
115 120 125
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu
130 135 140
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
145 150 155 160
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
165 170 175
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
180 185 190
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr
195 200 205
Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr
210 215 220
Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
225 230 235 240
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
245 250 255
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
260 265 270
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
275 280 285
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
290 295 300
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
305 310 315 320
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
325 330 335
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
340 345 350
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
355 360 365
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
370 375 380
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
385 390 395 400
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
405 410 415
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
420 425 430
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 445
<210> 29
<211> 446
<212> PRT
<213> 人工序列
<220>
<223> 人源化7H11-VH2 N58K-D54T
<400> 29
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 Arg Tyr
20 25 30
Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Trp Ile Tyr Pro Gly Thr Gly Ser Thr Lys Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Glu Tyr Gly Asn Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala
115 120 125
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu
130 135 140
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
145 150 155 160
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
165 170 175
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
180 185 190
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr
195 200 205
Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr
210 215 220
Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
225 230 235 240
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
245 250 255
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
260 265 270
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
275 280 285
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
290 295 300
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
305 310 315 320
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
325 330 335
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
340 345 350
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
355 360 365
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
370 375 380
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
385 390 395 400
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
405 410 415
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
420 425 430
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 445
<210> 30
<211> 446
<212> PRT
<213> 人工序列
<220>
<223> 人源化7H11-VH2 N58K-G55A
<400> 30
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 Arg Tyr
20 25 30
Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Trp Ile Tyr Pro Gly Asp Ala Ser Thr Lys Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Glu Tyr Gly Asn Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala
115 120 125
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu
130 135 140
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
145 150 155 160
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
165 170 175
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
180 185 190
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr
195 200 205
Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr
210 215 220
Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
225 230 235 240
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
245 250 255
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
260 265 270
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
275 280 285
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
290 295 300
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
305 310 315 320
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
325 330 335
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
340 345 350
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
355 360 365
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
370 375 380
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
385 390 395 400
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
405 410 415
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
420 425 430
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 445
<210> 31
<211> 117
<212> PRT
<213> 人工序列
<220>
<223> 小鼠2H6 VH
<400> 31
Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln
1 5 10 15
Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr Ser Asp
20 25 30
Tyr Ala Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp
35 40 45
Met Gly Tyr Ile Ser Tyr Ser Gly Ser Thr Ala Tyr Asn Pro Ser Leu
50 55 60
Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe
65 70 75 80
Leu Gln Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys
85 90 95
Ala Arg Gly Pro Tyr Asn Tyr Leu Ala Tyr Trp Gly Gln Gly Thr Leu
100 105 110
Val Thr Val Ser Ser
115
<210> 32
<211> 111
<212> PRT
<213> 人工序列
<220>
<223> 小鼠2H6 VL
<400> 32
Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Asn Tyr
20 25 30
Gly Ile Ser Phe Met Asn Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro
35 40 45
Lys Leu Leu Ile Tyr Ala Ala Ser Asn Gln Lys Ser Gly Val Pro Ala
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Ser Leu Asn Ile His
65 70 75 80
Pro Met Glu Glu Asp Asp Thr Ala Met Tyr Phe Cys Gln Gln Ser Lys
85 90 95
Glu Val Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile Lys
100 105 110
<210> 33
<211> 98
<212> PRT
<213> 人工序列
<220>
<223> IGHV3-23*01
<400> 33
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Lys
<210> 34
<211> 95
<212> PRT
<213> 人工序列
<220>
<223> IGKV1-16*01
<400> 34
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Asn Tyr
20 25 30
Leu Ala Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro
85 90 95
<210> 35
<211> 475
<212> PRT
<213> 人工序列
<220>
<223> 小鼠2H6 scFv-Fc
<400> 35
Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln
1 5 10 15
Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr Ser Asp
20 25 30
Tyr Ala Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp
35 40 45
Met Gly Tyr Ile Ser Tyr Ser Gly Ser Thr Ala Tyr Asn Pro Ser Leu
50 55 60
Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe
65 70 75 80
Leu Gln Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys
85 90 95
Ala Arg Gly Pro Tyr Asn Tyr Leu Ala Tyr Trp Gly Gln Gly Thr Leu
100 105 110
Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
115 120 125
Gly Gly Gly Ser Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala
130 135 140
Val Ser Leu Gly Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser
145 150 155 160
Val Asp Asn Tyr Gly Ile Ser Phe Met Asn Trp Phe Gln Gln Lys Pro
165 170 175
Gly Gln Pro Pro Lys Leu Leu Ile Tyr Ala Ala Ser Asn Gln Lys Ser
180 185 190
Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Ser
195 200 205
Leu Asn Ile His Pro Met Glu Glu Asp Asp Thr Ala Met Tyr Phe Cys
210 215 220
Gln Gln Ser Lys Glu Val Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu
225 230 235 240
Glu Ile Lys Gly Gly Gly Gly Thr Asp Lys Thr His Thr Cys Pro Pro
245 250 255
Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro
260 265 270
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
275 280 285
Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn
290 295 300
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
305 310 315 320
Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val
325 330 335
Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
340 345 350
Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
355 360 365
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp
370 375 380
Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
385 390 395 400
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
405 410 415
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
420 425 430
Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
435 440 445
Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
450 455 460
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
465 470 475
<210> 36
<211> 475
<212> PRT
<213> 人工序列
<220>
<223> 2H6 scFv1-Fc
<400> 36
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Tyr Ser Ile Thr Ser Asp
20 25 30
Tyr Ala Trp Asn Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
35 40 45
Met Gly Tyr Ile Ser Tyr Ser Gly Ser Thr Ala Tyr Asn Pro Ser Leu
50 55 60
Lys Ser Arg Ile Thr Ile Ser Arg Asp Thr Ser Lys Asn Thr Phe Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gly Pro Tyr Asn Tyr Leu Ala Tyr Trp Gly Gln Gly Thr Met
100 105 110
Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
115 120 125
Gly Gly Gly Ser Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser
130 135 140
Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Ser
145 150 155 160
Val Asp Asn Tyr Gly Ile Ser Phe Met Asn Trp Phe Gln Gln Lys Pro
165 170 175
Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Asn Gln Lys Ser
180 185 190
Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
195 200 205
Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Phe Cys
210 215 220
Gln Gln Ser Lys Glu Val Pro Leu Thr Phe Gly Gln Gly Thr Lys Val
225 230 235 240
Glu Ile Lys Gly Gly Gly Gly Thr Asp Lys Thr His Thr Cys Pro Pro
245 250 255
Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro
260 265 270
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
275 280 285
Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn
290 295 300
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
305 310 315 320
Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val
325 330 335
Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
340 345 350
Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
355 360 365
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp
370 375 380
Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
385 390 395 400
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
405 410 415
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
420 425 430
Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
435 440 445
Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
450 455 460
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
465 470 475
<210> 37
<211> 475
<212> PRT
<213> 人工序列
<220>
<223> 2H6 scFv2-Fc
<400> 37
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Tyr Ser Ile Thr Ser Asp
20 25 30
Tyr Ala Trp Asn Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
35 40 45
Met Gly Tyr Ile Ser Tyr Ser Gly Ser Thr Ala Tyr Asn Pro Ser Leu
50 55 60
Lys Ser Arg Ile Thr Ile Ser Arg Asp Thr Ser Lys Asn Thr Phe Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gly Pro Tyr Asn Tyr Leu Ala Tyr Trp Gly Gln Gly Thr Leu
100 105 110
Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
115 120 125
Gly Gly Gly Ser Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser
130 135 140
Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Ser
145 150 155 160
Val Asp Asn Tyr Gly Ile Ser Phe Met Asn Trp Phe Gln Gln Lys Pro
165 170 175
Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Asn Gln Lys Ser
180 185 190
Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
195 200 205
Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Phe Cys
210 215 220
Gln Gln Ser Lys Glu Val Pro Leu Thr Phe Gly Gln Gly Thr Lys Val
225 230 235 240
Glu Ile Lys Gly Gly Gly Gly Thr Asp Lys Thr His Thr Cys Pro Pro
245 250 255
Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro
260 265 270
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
275 280 285
Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn
290 295 300
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
305 310 315 320
Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val
325 330 335
Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
340 345 350
Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
355 360 365
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp
370 375 380
Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
385 390 395 400
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
405 410 415
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
420 425 430
Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
435 440 445
Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
450 455 460
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
465 470 475
<210> 38
<211> 693
<212> PRT
<213> 人工序列
<220>
<223> Tetra-1 HC
<400> 38
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 Arg Tyr
20 25 30
Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Trp Ile Tyr Pro Gly Asp Gly Ser Thr Lys Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Glu Tyr Gly Asn Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala
115 120 125
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu
130 135 140
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
145 150 155 160
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
165 170 175
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
180 185 190
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr
195 200 205
Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr
210 215 220
Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe
225 230 235 240
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
245 250 255
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
260 265 270
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
275 280 285
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
290 295 300
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
305 310 315 320
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
325 330 335
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
340 345 350
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
355 360 365
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
370 375 380
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
385 390 395 400
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
405 410 415
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
420 425 430
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly Gly
435 440 445
Gly Thr Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro
450 455 460
Gly Gly Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Tyr Ser Ile Thr
465 470 475 480
Ser Asp Tyr Ala Trp Asn Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu
485 490 495
Glu Trp Met Gly Tyr Ile Ser Tyr Ser Gly Ser Thr Ala Tyr Asn Pro
500 505 510
Ser Leu Lys Ser Arg Ile Thr Ile Ser Arg Asp Thr Ser Lys Asn Thr
515 520 525
Phe Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
530 535 540
Tyr Cys Ala Arg Gly Pro Tyr Asn Tyr Leu Ala Tyr Trp Gly Gln Gly
545 550 555 560
Thr Met Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
565 570 575
Ser Gly Gly Gly Gly Ser Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser
580 585 590
Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser
595 600 605
Glu Ser Val Asp Asn Tyr Gly Ile Ser Phe Met Asn Trp Phe Gln Gln
610 615 620
Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Asn Gln
625 630 635 640
Lys Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
645 650 655
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr
660 665 670
Phe Cys Gln Gln Ser Lys Glu Val Pro Leu Thr Phe Gly Gln Gly Thr
675 680 685
Lys Val Glu Ile Lys
690
<210> 39
<211> 693
<212> PRT
<213> 人工序列
<220>
<223> Tetra-6 HC
<400> 39
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 Arg Tyr
20 25 30
Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Trp Ile Tyr Pro Gly Glu Gly Ser Thr Lys Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Glu Tyr Gly Asn Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala
115 120 125
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu
130 135 140
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
145 150 155 160
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
165 170 175
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
180 185 190
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr
195 200 205
Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr
210 215 220
Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe
225 230 235 240
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
245 250 255
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
260 265 270
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
275 280 285
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
290 295 300
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
305 310 315 320
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
325 330 335
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
340 345 350
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
355 360 365
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
370 375 380
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
385 390 395 400
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
405 410 415
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
420 425 430
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly Gly
435 440 445
Gly Thr Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro
450 455 460
Gly Gly Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Tyr Ser Ile Thr
465 470 475 480
Ser Asp Tyr Ala Trp Asn Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu
485 490 495
Glu Trp Met Gly Tyr Ile Ser Tyr Ser Gly Ser Thr Ala Tyr Asn Pro
500 505 510
Ser Leu Lys Ser Arg Ile Thr Ile Ser Arg Asp Thr Ser Lys Asn Thr
515 520 525
Phe Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
530 535 540
Tyr Cys Ala Arg Gly Pro Tyr Asn Tyr Leu Ala Tyr Trp Gly Gln Gly
545 550 555 560
Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
565 570 575
Ser Gly Gly Gly Gly Ser Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser
580 585 590
Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser
595 600 605
Glu Ser Val Asp Asn Tyr Gly Ile Ser Phe Met Asn Trp Phe Gln Gln
610 615 620
Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Asn Gln
625 630 635 640
Lys Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
645 650 655
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr
660 665 670
Phe Cys Gln Gln Ser Lys Glu Val Pro Leu Thr Phe Gly Gln Gly Thr
675 680 685
Lys Val Glu Ile Leu
690
<210> 40
<211> 693
<212> PRT
<213> 人工序列
<220>
<223> Tetra-2 HC
<400> 40
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 Arg Tyr
20 25 30
Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Trp Ile Tyr Pro Gly Asp Gly Ser Thr Lys Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Glu Tyr Gly Asn Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala
115 120 125
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu
130 135 140
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
145 150 155 160
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
165 170 175
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
180 185 190
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr
195 200 205
Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr
210 215 220
Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
225 230 235 240
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
245 250 255
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
260 265 270
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
275 280 285
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
290 295 300
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
305 310 315 320
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
325 330 335
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
340 345 350
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
355 360 365
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
370 375 380
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
385 390 395 400
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
405 410 415
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
420 425 430
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly Gly
435 440 445
Gly Thr Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro
450 455 460
Gly Gly Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Tyr Ser Ile Thr
465 470 475 480
Ser Asp Tyr Ala Trp Asn Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu
485 490 495
Glu Trp Met Gly Tyr Ile Ser Tyr Ser Gly Ser Thr Ala Tyr Asn Pro
500 505 510
Ser Leu Lys Ser Arg Ile Thr Ile Ser Arg Asp Thr Ser Lys Asn Thr
515 520 525
Phe Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
530 535 540
Tyr Cys Ala Arg Gly Pro Tyr Asn Tyr Leu Ala Tyr Trp Gly Gln Gly
545 550 555 560
Thr Met Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
565 570 575
Ser Gly Gly Gly Gly Ser Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser
580 585 590
Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser
595 600 605
Glu Ser Val Asp Asn Tyr Gly Ile Ser Phe Met Asn Trp Phe Gln Gln
610 615 620
Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Asn Gln
625 630 635 640
Lys Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
645 650 655
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr
660 665 670
Phe Cys Gln Gln Ser Lys Glu Val Pro Leu Thr Phe Gly Gln Gly Thr
675 680 685
Lys Val Glu Ile Lys
690
<210> 41
<211> 695
<212> PRT
<213> 人工序列
<220>
<223> Tetra-3 HC
<400> 41
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Tyr Ser Ile Thr Ser Asp
20 25 30
Tyr Ala Trp Asn Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
35 40 45
Met Gly Tyr Ile Ser Tyr Ser Gly Ser Thr Ala Tyr Asn Pro Ser Leu
50 55 60
Lys Ser Arg Ile Thr Ile Ser Arg Asp Thr Ser Lys Asn Thr Phe Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gly Pro Tyr Asn Tyr Leu Ala Tyr Trp Gly Gln Gly Thr Met
100 105 110
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
115 120 125
Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys
130 135 140
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser
145 150 155 160
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
165 170 175
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser
180 185 190
Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn
195 200 205
Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His
210 215 220
Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val
225 230 235 240
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
245 250 255
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
260 265 270
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
275 280 285
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
290 295 300
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
305 310 315 320
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
325 330 335
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
340 345 350
Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
355 360 365
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
370 375 380
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
385 390 395 400
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
405 410 415
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
420 425 430
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly
435 440 445
Gly Gly Thr Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys
450 455 460
Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe
465 470 475 480
Thr Arg Tyr Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu
485 490 495
Glu Trp Met Gly Trp Ile Tyr Pro Gly Asp Gly Ser Thr Lys Tyr Ala
500 505 510
Gln Lys Phe Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser
515 520 525
Thr Ala Tyr Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val
530 535 540
Tyr Tyr Cys Ala Arg Glu Tyr Gly Asn Tyr Asp Tyr Trp Gly Gln Gly
545 550 555 560
Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
565 570 575
Ser Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser Pro Asp Ser
580 585 590
Leu Ala Val Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser
595 600 605
Gln Ser Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu Ala Trp Tyr
610 615 620
Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Gly Ala Ser
625 630 635 640
Thr Arg Glu Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly
645 650 655
Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala
660 665 670
Val Tyr Tyr Cys Gln Asn Asp His Ser Tyr Pro Tyr Thr Phe Gly Gln
675 680 685
Gly Thr Lys Leu Glu Ile Lys
690 695
<210> 42
<211> 694
<212> PRT
<213> 人工序列
<220>
<223> Tetra-4 HC
<400> 42
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 Arg Tyr
20 25 30
Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Trp Ile Tyr Pro Gly Asp Gly Ser Thr Lys Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Glu Tyr Gly Asn Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala
115 120 125
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu
130 135 140
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
145 150 155 160
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
165 170 175
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
180 185 190
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr
195 200 205
Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr
210 215 220
Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe
225 230 235 240
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
245 250 255
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
260 265 270
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
275 280 285
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
290 295 300
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
305 310 315 320
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
325 330 335
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
340 345 350
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
355 360 365
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
370 375 380
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
385 390 395 400
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
405 410 415
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
420 425 430
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly Gly
435 440 445
Gly Thr Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
450 455 460
Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr
465 470 475 480
Arg Tyr Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
485 490 495
Trp Met Gly Trp Ile Tyr Pro Gly Asp Gly Ser Thr Lys Tyr Ala Gln
500 505 510
Lys Phe Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr
515 520 525
Ala Tyr Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr
530 535 540
Tyr Cys Ala Arg Glu Tyr Gly Asn Tyr Asp Tyr Trp Gly Gln Gly Thr
545 550 555 560
Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
565 570 575
Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu
580 585 590
Ala Val Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln
595 600 605
Ser Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln
610 615 620
Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Gly Ala Ser Thr
625 630 635 640
Arg Glu Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr
645 650 655
Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val
660 665 670
Tyr Tyr Cys Gln Asn Asp His Ser Tyr Pro Tyr Thr Phe Gly Gln Gly
675 680 685
Thr Lys Leu Glu Ile Lys
690
<210> 43
<211> 694
<212> PRT
<213> 人工序列
<220>
<223> Tetra-5 HC
<400> 43
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Tyr Ser Ile Thr Ser Asp
20 25 30
Tyr Ala Trp Asn Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
35 40 45
Met Gly Tyr Ile Ser Tyr Ser Gly Ser Thr Ala Tyr Asn Pro Ser Leu
50 55 60
Lys Ser Arg Ile Thr Ile Ser Arg Asp Thr Ser Lys Asn Thr Phe Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gly Pro Tyr Asn Tyr Leu Ala Tyr Trp Gly Gln Gly Thr Met
100 105 110
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
115 120 125
Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys
130 135 140
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser
145 150 155 160
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
165 170 175
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser
180 185 190
Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn
195 200 205
Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His
210 215 220
Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val
225 230 235 240
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
245 250 255
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
260 265 270
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
275 280 285
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
290 295 300
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
305 310 315 320
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
325 330 335
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
340 345 350
Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
355 360 365
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
370 375 380
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
385 390 395 400
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
405 410 415
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
420 425 430
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly
435 440 445
Gly Gly Thr Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln
450 455 460
Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Tyr Ser Ile
465 470 475 480
Thr Ser Asp Tyr Ala Trp Asn Trp Ile Arg Gln Ala Pro Gly Lys Gly
485 490 495
Leu Glu Trp Met Gly Tyr Ile Ser Tyr Ser Gly Ser Thr Ala Tyr Asn
500 505 510
Pro Ser Leu Lys Ser Arg Ile Thr Ile Ser Arg Asp Thr Ser Lys Asn
515 520 525
Thr Phe Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
530 535 540
Tyr Tyr Cys Ala Arg Gly Pro Tyr Asn Tyr Leu Ala Tyr Trp Gly Gln
545 550 555 560
Gly Thr Met Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly
565 570 575
Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Leu Thr Gln Ser Pro Ser
580 585 590
Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala
595 600 605
Ser Glu Ser Val Asp Asn Tyr Gly Ile Ser Phe Met Asn Trp Phe Gln
610 615 620
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Asn
625 630 635 640
Gln Lys Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr
645 650 655
Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr
660 665 670
Tyr Phe Cys Gln Gln Ser Lys Glu Val Pro Leu Thr Phe Gly Gln Gly
675 680 685
Thr Lys Val Glu Ile Lys
690
<210> 44
<211> 693
<212> PRT
<213> 人工序列
<220>
<223> Tetra-7 HC
<400> 44
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 Arg Tyr
20 25 30
Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Trp Ile Tyr Pro Gly Asp Gly Ser Thr Lys Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Glu Tyr Gly Asn Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala
115 120 125
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu
130 135 140
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
145 150 155 160
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
165 170 175
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
180 185 190
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr
195 200 205
Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr
210 215 220
Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe
225 230 235 240
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
245 250 255
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
260 265 270
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
275 280 285
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
290 295 300
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
305 310 315 320
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
325 330 335
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
340 345 350
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
355 360 365
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
370 375 380
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
385 390 395 400
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
405 410 415
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
420 425 430
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly Gly
435 440 445
Gly Thr Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro
450 455 460
Gly Gly Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Tyr Ser Ile Thr
465 470 475 480
Ser Asp Tyr Ala Trp Asn Trp Ile Arg Gln Ala Pro Gly Lys Cys Leu
485 490 495
Glu Trp Met Gly Tyr Ile Ser Tyr Ser Gly Ser Thr Ala Tyr Asn Pro
500 505 510
Ser Leu Lys Ser Arg Ile Thr Ile Ser Arg Asp Thr Ser Lys Asn Thr
515 520 525
Phe Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
530 535 540
Tyr Cys Ala Arg Gly Pro Tyr Asn Tyr Leu Ala Tyr Trp Gly Gln Gly
545 550 555 560
Thr Met Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
565 570 575
Ser Gly Gly Gly Gly Ser Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser
580 585 590
Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser
595 600 605
Glu Ser Val Asp Asn Tyr Gly Ile Ser Phe Met Asn Trp Phe Gln Gln
610 615 620
Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Asn Gln
625 630 635 640
Lys Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
645 650 655
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr
660 665 670
Phe Cys Gln Gln Ser Lys Glu Val Pro Leu Thr Phe Gly Cys Gly Thr
675 680 685
Lys Val Glu Ile Lys
690
<210> 45
<211> 693
<212> PRT
<213> 人工序列
<220>
<223> Tetra-8 HC
<400> 45
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 Arg Tyr
20 25 30
Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Trp Ile Tyr Pro Gly Glu Gly Ser Thr Lys Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Glu Tyr Gly Asn Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala
115 120 125
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu
130 135 140
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
145 150 155 160
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
165 170 175
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
180 185 190
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr
195 200 205
Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr
210 215 220
Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe
225 230 235 240
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
245 250 255
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
260 265 270
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
275 280 285
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
290 295 300
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
305 310 315 320
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
325 330 335
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
340 345 350
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
355 360 365
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
370 375 380
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
385 390 395 400
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
405 410 415
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
420 425 430
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly Gly
435 440 445
Gly Thr Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro
450 455 460
Gly Gly Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Tyr Ser Ile Thr
465 470 475 480
Ser Asp Tyr Ala Trp Asn Trp Ile Arg Gln Ala Pro Gly Lys Cys Leu
485 490 495
Glu Trp Met Gly Tyr Ile Ser Tyr Ser Gly Ser Thr Ala Tyr Asn Pro
500 505 510
Ser Leu Lys Ser Arg Ile Thr Ile Ser Arg Asp Thr Ser Lys Asn Thr
515 520 525
Phe Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
530 535 540
Tyr Cys Ala Arg Gly Pro Tyr Asn Tyr Leu Ala Tyr Trp Gly Gln Gly
545 550 555 560
Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
565 570 575
Ser Gly Gly Gly Gly Ser Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser
580 585 590
Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser
595 600 605
Glu Ser Val Asp Asn Tyr Gly Ile Ser Phe Met Asn Trp Phe Gln Gln
610 615 620
Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Asn Gln
625 630 635 640
Lys Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
645 650 655
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr
660 665 670
Phe Cys Gln Gln Ser Lys Glu Val Pro Leu Thr Phe Gly Cys Gly Thr
675 680 685
Lys Val Glu Ile Leu
690
<210> 46
<211> 693
<212> PRT
<213> 人工序列
<220>
<223> Tetra-9 HC
<400> 46
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 Arg Tyr
20 25 30
Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Trp Ile Tyr Pro Gly Glu Gly Ser Thr Lys Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Glu Tyr Gly Asn Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala
115 120 125
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu
130 135 140
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
145 150 155 160
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
165 170 175
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
180 185 190
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr
195 200 205
Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr
210 215 220
Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe
225 230 235 240
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
245 250 255
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
260 265 270
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
275 280 285
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
290 295 300
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
305 310 315 320
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
325 330 335
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
340 345 350
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
355 360 365
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
370 375 380
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
385 390 395 400
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
405 410 415
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
420 425 430
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly Gly
435 440 445
Gly Thr Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro
450 455 460
Gly Gly Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Tyr Ser Ile Thr
465 470 475 480
Ser Asp Tyr Ala Trp Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
485 490 495
Glu Trp Val Ser Tyr Ile Ser Tyr Ser Gly Ser Thr Ala Tyr Asn Pro
500 505 510
Ser Leu Lys Ser Arg Phe Thr Ile Ser Arg Asp Thr Ser Lys Asn Thr
515 520 525
Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
530 535 540
Tyr Cys Ala Arg Gly Pro Tyr Asn Tyr Leu Ala Tyr Trp Gly Gln Gly
545 550 555 560
Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
565 570 575
Ser Gly Gly Gly Gly Ser Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser
580 585 590
Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser
595 600 605
Glu Ser Val Asp Asn Tyr Gly Ile Ser Phe Met Asn Trp Phe Gln Gln
610 615 620
Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Asn Gln
625 630 635 640
Lys Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
645 650 655
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr
660 665 670
Phe Cys Gln Gln Ser Lys Glu Val Pro Leu Thr Phe Gly Gln Gly Thr
675 680 685
Lys Val Glu Ile Leu
690
<210> 47
<211> 693
<212> PRT
<213> 人工序列
<220>
<223> Tetra-10 HC
<400> 47
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 Arg Tyr
20 25 30
Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Trp Ile Tyr Pro Gly Glu Gly Ser Thr Lys Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Glu Tyr Gly Asn Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala
115 120 125
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu
130 135 140
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
145 150 155 160
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
165 170 175
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
180 185 190
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr
195 200 205
Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr
210 215 220
Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe
225 230 235 240
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
245 250 255
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
260 265 270
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
275 280 285
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
290 295 300
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
305 310 315 320
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
325 330 335
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
340 345 350
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
355 360 365
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
370 375 380
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
385 390 395 400
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
405 410 415
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
420 425 430
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly Gly
435 440 445
Gly Thr Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro
450 455 460
Gly Gly Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Tyr Ser Ile Thr
465 470 475 480
Ser Asp Tyr Ala Trp Asn Trp Val Arg Gln Ala Pro Gly Lys Cys Leu
485 490 495
Glu Trp Val Ser Tyr Ile Ser Tyr Ser Gly Ser Thr Ala Tyr Asn Pro
500 505 510
Ser Leu Lys Ser Arg Phe Thr Ile Ser Arg Asp Thr Ser Lys Asn Thr
515 520 525
Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
530 535 540
Tyr Cys Ala Arg Gly Pro Tyr Asn Tyr Leu Ala Tyr Trp Gly Gln Gly
545 550 555 560
Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
565 570 575
Ser Gly Gly Gly Gly Ser Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser
580 585 590
Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser
595 600 605
Glu Ser Val Asp Asn Tyr Gly Ile Ser Phe Met Asn Trp Phe Gln Gln
610 615 620
Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Asn Gln
625 630 635 640
Lys Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
645 650 655
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr
660 665 670
Phe Cys Gln Gln Ser Lys Glu Val Pro Leu Thr Phe Gly Cys Gly Thr
675 680 685
Lys Val Glu Ile Leu
690
<210> 48
<211> 693
<212> PRT
<213> 人工序列
<220>
<223> Tetra-11 HC
<400> 48
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 Arg Tyr
20 25 30
Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Trp Ile Tyr Pro Gly Glu Gly Ser Thr Lys Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Glu Tyr Gly Asn Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala
115 120 125
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu
130 135 140
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
145 150 155 160
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
165 170 175
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
180 185 190
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr
195 200 205
Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr
210 215 220
Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe
225 230 235 240
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
245 250 255
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
260 265 270
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
275 280 285
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
290 295 300
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
305 310 315 320
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
325 330 335
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
340 345 350
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
355 360 365
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
370 375 380
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
385 390 395 400
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
405 410 415
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
420 425 430
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly Gly
435 440 445
Gly Thr Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro
450 455 460
Gly Gly Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Tyr Ser Ile Thr
465 470 475 480
Ser Asp Tyr Ala Trp Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
485 490 495
Glu Trp Val Gly Tyr Ile Ser Tyr Ser Gly Ser Thr Ala Tyr Asn Pro
500 505 510
Ser Leu Lys Ser Arg Phe Thr Ile Ser Arg Asp Thr Ser Lys Asn Thr
515 520 525
Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
530 535 540
Tyr Cys Ala Arg Gly Pro Tyr Asn Tyr Leu Ala Tyr Trp Gly Gln Gly
545 550 555 560
Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
565 570 575
Ser Gly Gly Gly Gly Ser Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser
580 585 590
Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser
595 600 605
Glu Ser Val Asp Asn Tyr Gly Ile Ser Phe Met Asn Trp Phe Gln Gln
610 615 620
Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Asn Gln
625 630 635 640
Lys Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
645 650 655
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr
660 665 670
Phe Cys Gln Gln Ser Lys Glu Val Pro Leu Thr Phe Gly Gln Gly Thr
675 680 685
Lys Val Glu Ile Leu
690
<210> 49
<211> 693
<212> PRT
<213> 人工序列
<220>
<223> Tetra-12 HC
<400> 49
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 Arg Tyr
20 25 30
Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Trp Ile Tyr Pro Gly Glu Gly Ser Thr Lys Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Glu Tyr Gly Asn Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala
115 120 125
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu
130 135 140
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
145 150 155 160
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
165 170 175
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
180 185 190
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr
195 200 205
Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr
210 215 220
Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe
225 230 235 240
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
245 250 255
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
260 265 270
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
275 280 285
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
290 295 300
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
305 310 315 320
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
325 330 335
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
340 345 350
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
355 360 365
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
370 375 380
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
385 390 395 400
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
405 410 415
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
420 425 430
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly Gly
435 440 445
Gly Thr Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro
450 455 460
Gly Gly Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Tyr Ser Ile Thr
465 470 475 480
Ser Asp Tyr Ala Trp Asn Trp Val Arg Gln Ala Pro Gly Lys Cys Leu
485 490 495
Glu Trp Val Gly Tyr Ile Ser Tyr Ser Gly Ser Thr Ala Tyr Asn Pro
500 505 510
Ser Leu Lys Ser Arg Phe Thr Ile Ser Arg Asp Thr Ser Lys Asn Thr
515 520 525
Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
530 535 540
Tyr Cys Ala Arg Gly Pro Tyr Asn Tyr Leu Ala Tyr Trp Gly Gln Gly
545 550 555 560
Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
565 570 575
Ser Gly Gly Gly Gly Ser Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser
580 585 590
Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser
595 600 605
Glu Ser Val Asp Asn Tyr Gly Ile Ser Phe Met Asn Trp Phe Gln Gln
610 615 620
Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Asn Gln
625 630 635 640
Lys Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
645 650 655
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr
660 665 670
Phe Cys Gln Gln Ser Lys Glu Val Pro Leu Thr Phe Gly Cys Gly Thr
675 680 685
Lys Val Glu Ile Leu
690
<210> 50
<211> 41
<212> DNA
<213> 人工序列
<220>
<223> 引物混合物VH - BACK_1
<400> 50
gtgatcgcca tggcgtcgac cgakgtrmag cttcaggagt c 41
<210> 51
<211> 41
<212> DNA
<213> 人工序列
<220>
<223> 引物混合物VH - BACK_2
<400> 51
gtgatcgcca tggcgtcgac cgaggtbcag ctbcagcagt c 41
<210> 52
<211> 41
<212> DNA
<213> 人工序列
<220>
<223> 引物混合物VH - BACK_3
<400> 52
gtgatcgcca tggcgtcgac ccaggtgcag ctgaagsart c 41
<210> 53
<211> 41
<212> DNA
<213> 人工序列
<220>
<223> 引物混合物VH - BACK_4
<400> 53
gtgatcgcca tggcgtcgac cgaggtccar ctgcaacart c 41
<210> 54
<211> 41
<212> DNA
<213> 人工序列
<220>
<223> 引物混合物VH - BACK_5
<400> 54
gtgatcgcca tggcgtcgac ccaggtycag ctbcagcart c 41
<210> 55
<211> 41
<212> DNA
<213> 人工序列
<220>
<223> 引物混合物VH - BACK_6
<400> 55
gtgatcgcca tggcgtcgac ccaggtycar ctgcagcart c 41
<210> 56
<211> 41
<212> DNA
<213> 人工序列
<220>
<223> 引物混合物VH - BACK_7
<400> 56
gtgatcgcca tggcgtcgac ccaggtccac gtgaagcart c 41
<210> 57
<211> 41
<212> DNA
<213> 人工序列
<220>
<223> 引物混合物VH - BACK_8
<400> 57
gtgatcgcca tggcgtcgac cgaggtgaas stggtggart c 41
<210> 58
<211> 41
<212> DNA
<213> 人工序列
<220>
<223> 引物混合物VH - BACK_9
<400> 58
gtgatcgcca tggcgtcgac cgavgtgawg stggtggagt c 41
<210> 59
<211> 41
<212> DNA
<213> 人工序列
<220>
<223> 引物混合物VH - BACK_10
<400> 59
gtgatcgcca tggcgtcgac cgaggtgcag stggtggart c 41
<210> 60
<211> 41
<212> DNA
<213> 人工序列
<220>
<223> 引物混合物VH - BACK_11
<400> 60
gtgatcgcca tggcgtcgac cgakgtgcam ctggtggart c 41
<210> 61
<211> 41
<212> DNA
<213> 人工序列
<220>
<223> 引物混合物VH - BACK_12
<400> 61
gtgatcgcca tggcgtcgac cgaggtgaag ctgatggart c 41
<210> 62
<211> 41
<212> DNA
<213> 人工序列
<220>
<223> 引物混合物VH - BACK_13
<400> 62
gtgatcgcca tggcgtcgac cgaggtgcar cttgttgart c 41
<210> 63
<211> 41
<212> DNA
<213> 人工序列
<220>
<223> 引物混合物VH - BACK_14
<400> 63
gtgatcgcca tggcgtcgac cgargtraag cttctcgart c 41
<210> 64
<211> 41
<212> DNA
<213> 人工序列
<220>
<223> 引物混合物VH - BACK_15
<400> 64
gtgatcgcca tggcgtcgac cgaagtgaar sttgaggart c 41
<210> 65
<211> 41
<212> DNA
<213> 人工序列
<220>
<223> 引物混合物VH - BACK_16
<400> 65
gtgatcgcca tggcgtcgac ccaggttact ctraaasart c 41
<210> 66
<211> 41
<212> DNA
<213> 人工序列
<220>
<223> 引物混合物VH - BACK_17
<400> 66
gtgatcgcca tggcgtcgac ccaggtccaa ctvcagcarc c 41
<210> 67
<211> 41
<212> DNA
<213> 人工序列
<220>
<223> 引物混合物VH - BACK_18
<400> 67
gtgatcgcca tggcgtcgac cgatgtgaac ttggaasart c 41
<210> 68
<211> 41
<212> DNA
<213> 人工序列
<220>
<223> 引物混合物VH - BACK_19
<400> 68
gtgatcgcca tggcgtcgac cgaggtgaag gtcatcgart c 41
<210> 69
<211> 38
<212> DNA
<213> 人工序列
<220>
<223> 引物混合物VH - FOR_1
<400> 69
cctccaccac tcgagcccga ggaaacggtg accgtggt 38
<210> 70
<211> 38
<212> DNA
<213> 人工序列
<220>
<223> 引物混合物VH - FOR_2
<400> 70
cctccaccac tcgagcccga ggagactgtg agagtggt 38
<210> 71
<211> 38
<212> DNA
<213> 人工序列
<220>
<223> 引物混合物VH - FOR_3
<400> 71
cctccaccac tcgagcccgc agagacagtg accagagt 38
<210> 72
<211> 38
<212> DNA
<213> 人工序列
<220>
<223> 引物混合物VH - FOR_4
<400> 72
cctccaccac tcgagcccga ggagacggtg actgaggt 38
<210> 73
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> 引物混合物VL - BACK_1
<400> 73
ggcggtggcg ctagcgayat ccagctgact cagcc 35
<210> 74
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> 引物混合物VL - BACK_2
<400> 74
ggcggtggcg ctagccaaat tgttctcacc cagtc 35
<210> 75
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> 引物混合物VL - BACK_3
<400> 75
ggcggtggcg ctagcgayat tgtgmtmact cagtc 35
<210> 76
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> 引物混合物VL - BACK_4
<400> 76
ggcggtggcg ctagcgayat tgtgytraca cagtc 35
<210> 77
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> 引物混合物VL - BACK_5
<400> 77
ggcggtggcg ctagcgayat tgtratgacm cagtc 35
<210> 78
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> 引物混合物VL - BACK_6
<400> 78
ggcggtggcg ctagcgayat tmagatramc cagtc 35
<210> 79
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> 引物混合物VL - BACK_7
<400> 79
ggcggtggcg ctagcgayat tcagatgayd cagtc 35
<210> 80
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> 引物混合物VL - BACK_8
<400> 80
ggcggtggcg ctagcgayat ycagatgaca cagac 35
<210> 81
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> 引物混合物VL - BACK_9
<400> 81
ggcggtggcg ctagcgayat tgttctcawc cagtc 35
<210> 82
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> 引物混合物VL - BACK_10
<400> 82
ggcggtggcg ctagcgayat tgwgctsacc caatc 35
<210> 83
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> 引物混合物VL - BACK_11
<400> 83
ggcggtggcg ctagcgayat tstratgacc cartc 35
<210> 84
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> 引物混合物VL - BACK_12
<400> 84
ggcggtggcg ctagcgayrt tktgatgacc carac 35
<210> 85
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> 引物混合物VL - BACK_13
<400> 85
ggcggtggcg ctagcgayat tgtgatgacb cagkc 35
<210> 86
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> 引物混合物VL - BACK_14
<400> 86
ggcggtggcg ctagcgayat tgtgataacy cagga 35
<210> 87
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> 引物混合物VL - BACK_15
<400> 87
ggcggtggcg ctagcgayat tgtgatgacc cagwt 35
<210> 88
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> 引物混合物VL - BACK_16
<400> 88
ggcggtggcg ctagcgayat tgtgatgaca caacc 35
<210> 89
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> 引物混合物VL - BACK_17
<400> 89
ggcggtggcg ctagcgayat tttgctgact cagtc 35
<210> 90
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> 引物混合物VL - BACK_18
<400> 90
ggcggtggcg ctagcgaaac aactgtgacc cagtc 35
<210> 91
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> 引物混合物VL - BACK_19
<400> 91
ggcggtggcg ctagcgaaaa tgtkctsacc cagtc 35
<210> 92
<211> 38
<212> DNA
<213> 人工序列
<220>
<223> 引物混合物VL - BACK_20
<400> 92
ggcggtggcg ctagccaggc tgttgtgact caggaatc 38
<210> 93
<211> 36
<212> DNA
<213> 人工序列
<220>
<223> 引物混合物VL - FOR_1
<400> 93
atgctgacgc ggccgcacgt ttkatttcca gcttgg 36
<210> 94
<211> 36
<212> DNA
<213> 人工序列
<220>
<223> 引物混合物VL - FOR_2
<400> 94
atgctgacgc ggccgcacgt tttatttcca actttg 36
<210> 95
<211> 36
<212> DNA
<213> 人工序列
<220>
<223> 引物混合物VL - FOR_3
<400> 95
atgctgacgc ggccgcacgt ttcagctcca gcttgg 36
<210> 96
<211> 36
<212> DNA
<213> 人工序列
<220>
<223> 引物混合物VL - FOR_4
<400> 96
atgctgacgc ggccgcacct aggacagtca gtttgg 36
<210> 97
<211> 17
<212> DNA
<213> 人工序列
<220>
<223> M13-Fwd
<400> 97
gtaaaacgac ggccagt 17
<210> 98
<211> 16
<212> DNA
<213> 人工序列
<220>
<223> M13-Rev
<400> 98
aacagctatg accatg 16
<210> 99
<211> 19
<212> DNA
<213> 人工序列
<220>
<223> T7
<400> 99
taatacgact cactatagg 19
<210> 100
<211> 19
<212> DNA
<213> 人工序列
<220>
<223> SP6
<400> 100
gatttaggtg acactatag 19
<210> 101
<211> 444
<212> PRT
<213> 人工序列
<220>
<223> 11D4 IgG2 HC
<400> 101
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Tyr Ile Ser Ser Ser Ser Ser Thr Ile Asp Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Asp Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Glu Ser Gly Trp Tyr Leu Phe Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
115 120 125
Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly
130 135 140
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
145 150 155 160
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
165 170 175
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
180 185 190
Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser
195 200 205
Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys Cys Val Glu Cys
210 215 220
Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe
225 230 235 240
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
245 250 255
Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe
260 265 270
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
275 280 285
Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr
290 295 300
Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
305 310 315 320
Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr
325 330 335
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg
340 345 350
Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
355 360 365
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
370 375 380
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser
385 390 395 400
Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
405 410 415
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
420 425 430
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440
<210> 102
<211> 214
<212> PRT
<213> 人工序列
<220>
<223> 11D4 LC
<400> 102
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile
35 40 45
Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Pro
85 90 95
Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys
210
<210> 103
<211> 451
<212> PRT
<213> 人工序列
<220>
<223> 9B12 IgG1 HC
<400> 103
Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln
1 5 10 15
Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Ser Gly
20 25 30
Tyr Trp Asn Trp Ile Arg Lys His Pro Gly Lys Gly Leu Glu Tyr Ile
35 40 45
Gly Tyr Ile Ser Tyr Asn Gly Ile Thr Tyr His Asn Pro Ser Leu Lys
50 55 60
Ser Arg Ile Thr Ile Asn Arg Asp Thr Ser Lys Asn Gln Tyr Ser Leu
65 70 75 80
Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Arg Tyr Lys Tyr Asp Tyr Asp Gly Gly His Ala Met Asp Tyr Trp Gly
100 105 110
Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
115 120 125
Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala
130 135 140
Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val
145 150 155 160
Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
165 170 175
Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
180 185 190
Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His
195 200 205
Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys
210 215 220
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
225 230 235 240
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
245 250 255
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
260 265 270
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
275 280 285
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
290 295 300
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
305 310 315 320
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
325 330 335
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
340 345 350
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser
355 360 365
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
370 375 380
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
385 390 395 400
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
405 410 415
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
420 425 430
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
435 440 445
Pro Gly Lys
450
<210> 104
<211> 214
<212> PRT
<213> 人工序列
<220>
<223> 9B12 LC
<400> 104
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Tyr Thr Ser Lys Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Ser Ala Leu Pro Trp
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys
210
<210> 105
<211> 452
<212> PRT
<213> 人工序列
<220>
<223> 106-222 IgG1 HC
<400> 105
Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu 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
Ser Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Lys Trp Met
35 40 45
Gly Trp Ile Asn Thr Glu Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe
50 55 60
Lys Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr
65 70 75 80
Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Asn Pro Tyr Tyr Asp Tyr Val Ser Tyr Tyr Ala Met Asp Tyr Trp
100 105 110
Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
115 120 125
Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr
130 135 140
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
145 150 155 160
Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
165 170 175
Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
180 185 190
Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn
195 200 205
His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser
210 215 220
Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu
225 230 235 240
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
245 250 255
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
260 265 270
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
275 280 285
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
290 295 300
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
305 310 315 320
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
325 330 335
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
340 345 350
Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val
355 360 365
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
370 375 380
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
385 390 395 400
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
405 410 415
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
420 425 430
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
435 440 445
Ser Pro Gly Lys
450
<210> 106
<211> 214
<212> PRT
<213> 人工序列
<220>
<223> 106-222 LC
<400> 106
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Thr Ala
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ser Ala Ser Tyr Leu Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro Arg
85 90 95
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys
210
<210> 107
<211> 447
<212> PRT
<213> 人工序列
<220>
<223> 1A7 IgG1 HC
<400> 107
Glu 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 Ser
20 25 30
Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Asp Met Tyr Pro Asp Asn Gly Asp Ser Ser Tyr Asn Gln Lys Phe
50 55 60
Arg Glu Arg Val Thr Ile Thr Arg Asp Thr Ser Thr Ser Thr Ala Tyr
65 70 75 80
Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Val Leu Ala Pro Arg Trp Tyr Phe Ser Val Trp Gly Gln Gly Thr Leu
100 105 110
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
115 120 125
Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys
130 135 140
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser
145 150 155 160
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
165 170 175
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser
180 185 190
Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn
195 200 205
Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His
210 215 220
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val
225 230 235 240
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
245 250 255
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
260 265 270
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
275 280 285
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
290 295 300
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
305 310 315 320
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
325 330 335
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
340 345 350
Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
355 360 365
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
370 375 380
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
385 390 395 400
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
405 410 415
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
420 425 430
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 445
<210> 108
<211> 214
<212> PRT
<213> 人工序列
<220>
<223> 1A7 LC
<400> 108
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Tyr Thr Ser Arg Leu Arg Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly His Thr Leu Pro Pro
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys
210
<210> 109
<211> 451
<212> PRT
<213> 人工序列
<220>
<223> pab1949 IgG1 HC
<400> 109
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Gly Ser
20 25 30
Ala Met His Trp Val Arg Gln Ala Ser Gly Lys Cys Leu Glu Trp Val
35 40 45
Gly Arg Ile Arg Ser Lys Ala Asn Ser Tyr Ala Thr Ala Tyr Ala Ala
50 55 60
Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr
65 70 75 80
Ala Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr
85 90 95
Tyr Cys Thr Ser Gly Ile Tyr Asp Ser Ser Gly Tyr Asp Tyr Trp Gly
100 105 110
Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
115 120 125
Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala
130 135 140
Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val
145 150 155 160
Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
165 170 175
Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
180 185 190
Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His
195 200 205
Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys
210 215 220
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
225 230 235 240
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
245 250 255
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
260 265 270
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
275 280 285
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
290 295 300
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
305 310 315 320
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
325 330 335
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
340 345 350
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser
355 360 365
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
370 375 380
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
385 390 395 400
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
405 410 415
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
420 425 430
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
435 440 445
Pro Gly Lys
450
<210> 110
<211> 219
<212> PRT
<213> 人工序列
<220>
<223> pab1949 LC
<400> 110
Asp Ile 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 Leu His Ser
20 25 30
Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser
35 40 45
Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala 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 Met Gln Ala
85 90 95
Leu Gln Thr Pro Leu Thr Phe Gly Cys Gly Thr Lys Val Glu Ile Lys
100 105 110
Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
115 120 125
Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
130 135 140
Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
145 150 155 160
Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
165 170 175
Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
180 185 190
Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
195 200 205
Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
210 215
<210> 111
<211> 468
<212> PRT
<213> 人工序列
<220>
<223> Tetra-hz1D10v1 HC
<400> 111
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Glu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ala
20 25 30
Phe Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ser Ile Ser Asn Arg Gly Leu Lys Thr Ala Tyr Ala Glu Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ser Arg Asp Val Asp Gly Asp Phe Arg Gly Gln Gly Thr Leu Val Thr
100 105 110
Val Lys Pro Gly Gly Ser Gly Gly Ser Glu Val Gln Leu Leu Glu Ser
115 120 125
Gly Gly Gly Glu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala
130 135 140
Ala Ser Gly Phe Thr Phe Ser Asp Ala Phe Met Tyr Trp Val Arg Gln
145 150 155 160
Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Ser Ile Ser Asn Arg Gly
165 170 175
Leu Lys Thr Ala Tyr Ala Glu Ser Val Lys Gly Arg Phe Thr Ile Ser
180 185 190
Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Ser Ser Leu Arg
195 200 205
Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ser Arg Asp Val Asp Gly Asp
210 215 220
Phe Arg Gly Gln Gly Thr Leu Val Thr Val Lys Pro Gly Gly Gly Gly
225 230 235 240
Thr Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala
245 250 255
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
260 265 270
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
275 280 285
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
290 295 300
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
305 310 315 320
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
325 330 335
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
340 345 350
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
355 360 365
Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val
370 375 380
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
385 390 395 400
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
405 410 415
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
420 425 430
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
435 440 445
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
450 455 460
Ser Pro Gly Lys
465
<210> 112
<211> 470
<212> PRT
<213> 人工序列
<220>
<223> Tetra-hzG3V9 HC
<400> 112
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Glu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ala
20 25 30
Phe Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Glu Arg Glu Trp Val
35 40 45
Ser Ser Ile Ser Asn Arg Gly Leu Lys Thr Ala Tyr Ala Glu Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Val Glu Gly Asp Trp Asn Leu Gly Pro Arg Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Lys Pro Gly Gly Ser Gly Gly Ser Glu Val Gln Leu Leu Glu
115 120 125
Ser Gly Gly Gly Glu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys
130 135 140
Ala Ala Ser Gly Phe Thr Phe Ser Asp Ala Phe Met Tyr Trp Val Arg
145 150 155 160
Gln Ala Pro Gly Lys Glu Arg Glu Trp Val Ser Ser Ile Ser Asn Arg
165 170 175
Gly Leu Lys Thr Ala Tyr Ala Glu Ser Val Lys Gly Arg Phe Thr Ile
180 185 190
Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Ser Ser Leu
195 200 205
Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Val Glu Gly Asp Trp Asn
210 215 220
Leu Gly Pro Arg Gly Gln Gly Thr Leu Val Thr Val Lys Pro Gly Gly
225 230 235 240
Gly Gly Thr Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
245 250 255
Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
260 265 270
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
275 280 285
Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
290 295 300
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
305 310 315 320
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
325 330 335
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
340 345 350
Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
355 360 365
Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn
370 375 380
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
385 390 395 400
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
405 410 415
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
420 425 430
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
435 440 445
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
450 455 460
Ser Leu Ser Pro Gly Lys
465 470
<210> 113
<211> 594
<212> PRT
<213> 人工序列
<220>
<223> Hexa-hz1D10v1 HC
<400> 113
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Glu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ala
20 25 30
Phe Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ser Ile Ser Asn Arg Gly Leu Lys Thr Ala Tyr Ala Glu Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ser Arg Asp Val Asp Gly Asp Phe Arg Gly Gln Gly Thr Leu Val Thr
100 105 110
Val Lys Pro Gly Gly Ser Gly Gly Ser Glu Val Gln Leu Leu Glu Ser
115 120 125
Gly Gly Gly Glu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala
130 135 140
Ala Ser Gly Phe Thr Phe Ser Asp Ala Phe Met Tyr Trp Val Arg Gln
145 150 155 160
Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Ser Ile Ser Asn Arg Gly
165 170 175
Leu Lys Thr Ala Tyr Ala Glu Ser Val Lys Gly Arg Phe Thr Ile Ser
180 185 190
Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Ser Ser Leu Arg
195 200 205
Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ser Arg Asp Val Asp Gly Asp
210 215 220
Phe Arg Gly Gln Gly Thr Leu Val Thr Val Lys Pro Gly Gly Ser Gly
225 230 235 240
Gly Ser Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Glu Val Gln Pro
245 250 255
Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser
260 265 270
Asp Ala Phe Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
275 280 285
Trp Val Ser Ser Ile Ser Asn Arg Gly Leu Lys Thr Ala Tyr Ala Glu
290 295 300
Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr
305 310 315 320
Leu Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
325 330 335
Tyr Cys Ser Arg Asp Val Asp Gly Asp Phe Arg Gly Gln Gly Thr Leu
340 345 350
Val Thr Val Lys Pro Gly Gly Gly Gly Thr Gly Gly Gly Gly Thr Asp
355 360 365
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly
370 375 380
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
385 390 395 400
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
405 410 415
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
420 425 430
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
435 440 445
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
450 455 460
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
465 470 475 480
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
485 490 495
Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu
500 505 510
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
515 520 525
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
530 535 540
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
545 550 555 560
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
565 570 575
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
580 585 590
Gly Lys
<210> 114
<211> 597
<212> PRT
<213> 人工序列
<220>
<223> Hexa-hzG3V9 HC
<400> 114
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Glu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ala
20 25 30
Phe Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Glu Arg Glu Trp Val
35 40 45
Ser Ser Ile Ser Asn Arg Gly Leu Lys Thr Ala Tyr Ala Glu Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Val Glu Gly Asp Trp Asn Leu Gly Pro Arg Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Lys Pro Gly Gly Ser Gly Gly Ser Glu Val Gln Leu Leu Glu
115 120 125
Ser Gly Gly Gly Glu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys
130 135 140
Ala Ala Ser Gly Phe Thr Phe Ser Asp Ala Phe Met Tyr Trp Val Arg
145 150 155 160
Gln Ala Pro Gly Lys Glu Arg Glu Trp Val Ser Ser Ile Ser Asn Arg
165 170 175
Gly Leu Lys Thr Ala Tyr Ala Glu Ser Val Lys Gly Arg Phe Thr Ile
180 185 190
Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Ser Ser Leu
195 200 205
Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Val Glu Gly Asp Trp Asn
210 215 220
Leu Gly Pro Arg Gly Gln Gly Thr Leu Val Thr Val Lys Pro Gly Gly
225 230 235 240
Ser Gly Gly Ser Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Glu Val
245 250 255
Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
260 265 270
Phe Ser Asp Ala Phe Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Glu
275 280 285
Arg Glu Trp Val Ser Ser Ile Ser Asn Arg Gly Leu Lys Thr Ala Tyr
290 295 300
Ala Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys
305 310 315 320
Asn Thr Leu Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala
325 330 335
Val Tyr Tyr Cys Val Glu Gly Asp Trp Asn Leu Gly Pro Arg Gly Gln
340 345 350
Gly Thr Leu Val Thr Val Lys Pro Gly Gly Gly Gly Thr Gly Gly Gly
355 360 365
Gly Thr Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala
370 375 380
Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
385 390 395 400
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
405 410 415
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
420 425 430
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
435 440 445
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
450 455 460
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
465 470 475 480
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
485 490 495
Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
500 505 510
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
515 520 525
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
530 535 540
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
545 550 555 560
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
565 570 575
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
580 585 590
Leu Ser Pro Gly Lys
595
<210> 115
<211> 447
<212> PRT
<213> 人工序列
<220>
<223> 2H6 IgG1 HC
<400> 115
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Tyr Ser Ile Thr Ser Asp
20 25 30
Tyr Ala Trp Asn Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
35 40 45
Met Gly Tyr Ile Ser Tyr Ser Gly Ser Thr Ala Tyr Asn Pro Ser Leu
50 55 60
Lys Ser Arg Ile Thr Ile Ser Arg Asp Thr Ser Lys Asn Thr Phe Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gly Pro Tyr Asn Tyr Leu Ala Tyr Trp Gly Gln Gly Thr Met
100 105 110
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
115 120 125
Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys
130 135 140
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser
145 150 155 160
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
165 170 175
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser
180 185 190
Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn
195 200 205
Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His
210 215 220
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val
225 230 235 240
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
245 250 255
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
260 265 270
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
275 280 285
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
290 295 300
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
305 310 315 320
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
325 330 335
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
340 345 350
Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
355 360 365
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
370 375 380
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
385 390 395 400
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
405 410 415
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
420 425 430
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 445
<210> 116
<211> 218
<212> PRT
<213> 人工序列
<220>
<223> 2H6 LC
<400> 116
Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Asp Asn Tyr
20 25 30
Gly Ile Ser Phe Met Asn Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro
35 40 45
Lys Leu Leu Ile Tyr Ala Ala Ser Asn Gln Lys Ser Gly Val Pro Ser
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
65 70 75 80
Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Ser Lys
85 90 95
Glu Val Pro Leu Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg
100 105 110
Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
115 120 125
Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
130 135 140
Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
145 150 155 160
Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
165 170 175
Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
180 185 190
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
195 200 205
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
210 215
<210> 117
<211> 447
<212> PRT
<213> 人工序列
<220>
<223> 2H6 IgG1 LALA HC
<400> 117
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Tyr Ser Ile Thr Ser Asp
20 25 30
Tyr Ala Trp Asn Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
35 40 45
Met Gly Tyr Ile Ser Tyr Ser Gly Ser Thr Ala Tyr Asn Pro Ser Leu
50 55 60
Lys Ser Arg Ile Thr Ile Ser Arg Asp Thr Ser Lys Asn Thr Phe Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gly Pro Tyr Asn Tyr Leu Ala Tyr Trp Gly Gln Gly Thr Met
100 105 110
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
115 120 125
Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys
130 135 140
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser
145 150 155 160
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
165 170 175
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser
180 185 190
Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn
195 200 205
Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His
210 215 220
Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val
225 230 235 240
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
245 250 255
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
260 265 270
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
275 280 285
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
290 295 300
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
305 310 315 320
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
325 330 335
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
340 345 350
Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
355 360 365
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
370 375 380
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
385 390 395 400
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
405 410 415
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
420 425 430
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 445
<210> 118
<211> 475
<212> PRT
<213> 人工序列
<220>
<223> 2H6 scFv-Fc LALA HC
<400> 118
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Tyr Ser Ile Thr Ser Asp
20 25 30
Tyr Ala Trp Asn Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
35 40 45
Met Gly Tyr Ile Ser Tyr Ser Gly Ser Thr Ala Tyr Asn Pro Ser Leu
50 55 60
Lys Ser Arg Ile Thr Ile Ser Arg Asp Thr Ser Lys Asn Thr Phe Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gly Pro Tyr Asn Tyr Leu Ala Tyr Trp Gly Gln Gly Thr Met
100 105 110
Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
115 120 125
Gly Gly Gly Ser Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser
130 135 140
Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Ser
145 150 155 160
Val Asp Asn Tyr Gly Ile Ser Phe Met Asn Trp Phe Gln Gln Lys Pro
165 170 175
Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Asn Gln Lys Ser
180 185 190
Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
195 200 205
Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Phe Cys
210 215 220
Gln Gln Ser Lys Glu Val Pro Leu Thr Phe Gly Gln Gly Thr Lys Val
225 230 235 240
Glu Ile Lys Gly Gly Gly Gly Thr Asp Lys Thr His Thr Cys Pro Pro
245 250 255
Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro
260 265 270
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
275 280 285
Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn
290 295 300
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
305 310 315 320
Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val
325 330 335
Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
340 345 350
Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
355 360 365
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp
370 375 380
Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
385 390 395 400
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
405 410 415
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
420 425 430
Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
435 440 445
Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
450 455 460
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
465 470 475
<210> 119
<211> 446
<212> PRT
<213> 人工序列
<220>
<223> 7H11_v8 IgG1 HC
<400> 119
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 Arg Tyr
20 25 30
Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Trp Ile Tyr Pro Gly Glu Gly Ser Thr Lys Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Glu Tyr Gly Asn Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala
115 120 125
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu
130 135 140
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
145 150 155 160
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
165 170 175
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
180 185 190
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr
195 200 205
Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr
210 215 220
Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
225 230 235 240
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
245 250 255
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
260 265 270
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
275 280 285
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
290 295 300
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
305 310 315 320
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
325 330 335
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
340 345 350
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
355 360 365
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
370 375 380
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
385 390 395 400
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
405 410 415
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
420 425 430
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 445
<210> 120
<211> 446
<212> PRT
<213> 人工序列
<220>
<223> 7H11 IgG1 LALA HC
<400> 120
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 Arg Tyr
20 25 30
Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Trp Ile Tyr Pro Gly Asp Gly Ser Thr Lys Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Glu Tyr Gly Asn Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala
115 120 125
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu
130 135 140
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
145 150 155 160
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
165 170 175
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
180 185 190
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr
195 200 205
Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr
210 215 220
Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe
225 230 235 240
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
245 250 255
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
260 265 270
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
275 280 285
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
290 295 300
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
305 310 315 320
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
325 330 335
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
340 345 350
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
355 360 365
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
370 375 380
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
385 390 395 400
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
405 410 415
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
420 425 430
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 445
<210> 121
<211> 187
<212> PRT
<213> 褐家鼠
<400> 121
Leu Asn Cys Val Lys Asp Thr Tyr Pro Ser Gly His Lys Cys Cys Arg
1 5 10 15
Glu Cys Gln Pro Gly His Gly Met Val Ser Arg Cys Asp His Thr Arg
20 25 30
Asp Thr Val Cys His Pro Cys Glu Pro Gly Phe Tyr Asn Glu Ala Val
35 40 45
Asn Tyr Asp Thr Cys Lys Gln Cys Thr Gln Cys Asn His Arg Ser Gly
50 55 60
Ser Glu Leu Lys Gln Asn Cys Thr Pro Thr Glu Asp Thr Val Cys Gln
65 70 75 80
Cys Arg Pro Gly Thr Gln Pro Arg Gln Asp Ser Ser His Lys Leu Gly
85 90 95
Val Asp Cys Val Pro Cys Pro Pro Gly His Phe Ser Pro Gly Ser Asn
100 105 110
Gln Ala Cys Lys Pro Trp Thr Asn Cys Thr Leu Ser Gly Lys Gln Ile
115 120 125
Arg His Pro Ala Ser Asn Ser Leu Asp Thr Val Cys Glu Asp Arg Ser
130 135 140
Leu Leu Ala Thr Leu Leu Trp Glu Thr Gln Arg Thr Thr Phe Arg Pro
145 150 155 160
Thr Thr Val Pro Ser Thr Thr Val Trp Pro Arg Thr Ser Gln Leu Pro
165 170 175
Ser Thr Pro Thr Leu Val Ala Pro Glu Gly Pro
180 185
<210> 122
<211> 186
<212> PRT
<213> 食蟹猴
<400> 122
Leu His Cys Val Gly Asp Thr Tyr Pro Ser Asn Asp Arg Cys Cys Gln
1 5 10 15
Glu Cys Arg Pro Gly Asn Gly Met Val Ser Arg Cys Asn Arg Ser Gln
20 25 30
Asn Thr Val Cys Arg Pro Cys Gly Pro Gly Phe Tyr Asn Asp Val Val
35 40 45
Ser Ala Lys Pro Cys Lys Ala Cys Thr Trp Cys Asn Leu Arg Ser Gly
50 55 60
Ser Glu Arg Lys Gln Pro Cys Thr Ala Thr Gln Asp Thr Val Cys Arg
65 70 75 80
Cys Arg Ala Gly Thr Gln Pro Leu Asp Ser Tyr Lys Pro Gly Val Asp
85 90 95
Cys Ala Pro Cys Pro Pro Gly His Phe Ser Pro Gly Asp Asn Gln Ala
100 105 110
Cys Lys Pro Trp Thr Asn Cys Thr Leu Ala Gly Lys His Thr Leu Gln
115 120 125
Pro Ala Ser Asn Ser Ser Asp Ala Ile Cys Glu Asp Arg Asp Pro Pro
130 135 140
Pro Thr Gln Pro Gln Glu Thr Gln Gly Pro Pro Ala Arg Pro Thr Thr
145 150 155 160
Val Gln Pro Thr Glu Ala Trp Pro Arg Thr Ser Gln Arg Pro Ser Thr
165 170 175
Arg Pro Val Glu Val Pro Arg Gly Pro Ala
180 185
<210> 123
<211> 424
<212> PRT
<213> 人工序列
<220>
<223> hsOX40(ECD)-Fc-His
<400> 123
Leu His Cys Val Gly Asp Thr Tyr Pro Ser Asn Asp Arg Cys Cys His
1 5 10 15
Glu Cys Arg Pro Gly Asn Gly Met Val Ser Arg Cys Ser Arg Ser Gln
20 25 30
Asn Thr Val Cys Arg Pro Cys Gly Pro Gly Phe Tyr Asn Asp Val Val
35 40 45
Ser Ser Lys Pro Cys Lys Pro Cys Thr Trp Cys Asn Leu Arg Ser Gly
50 55 60
Ser Glu Arg Lys Gln Leu Cys Thr Ala Thr Gln Asp Thr Val Cys Arg
65 70 75 80
Cys Arg Ala Gly Thr Gln Pro Leu Asp Ser Tyr Lys Pro Gly Val Asp
85 90 95
Cys Ala Pro Cys Pro Pro Gly His Phe Ser Pro Gly Asp Asn Gln Ala
100 105 110
Cys Lys Pro Trp Thr Asn Cys Thr Leu Ala Gly Lys His Thr Leu Gln
115 120 125
Pro Ala Ser Asn Ser Ser Asp Ala Ile Cys Glu Asp Arg Asp Pro Pro
130 135 140
Ala Thr Gln Pro Gln Glu Thr Gln Gly Pro Pro Ala Arg Pro Ile Thr
145 150 155 160
Val Gln Pro Thr Glu Ala Trp Pro Arg Thr Ser Gln Gly Pro Ser Thr
165 170 175
Arg Pro Val Glu Val Pro Gly Gly Arg Ala Gly Ser Gly Gly Gly Thr
180 185 190
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
195 200 205
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
210 215 220
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
225 230 235 240
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
245 250 255
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
260 265 270
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
275 280 285
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
290 295 300
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
305 310 315 320
Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys
325 330 335
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
340 345 350
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
355 360 365
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
370 375 380
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
385 390 395 400
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
405 410 415
Ser Ala His His His His His His
420
<210> 124
<211> 416
<212> PRT
<213> 人工序列
<220>
<223> cynoOX40R(ECD)-Fc
<400> 124
Leu His Cys Val Gly Asp Thr Tyr Pro Ser Asn Asp Arg Cys Cys Gln
1 5 10 15
Glu Cys Arg Pro Gly Asn Gly Met Val Ser Arg Cys Asn Arg Ser Gln
20 25 30
Asn Thr Val Cys Arg Pro Cys Gly Pro Gly Phe Tyr Asn Asp Val Val
35 40 45
Ser Ala Lys Pro Cys Lys Ala Cys Thr Trp Cys Asn Leu Arg Ser Gly
50 55 60
Ser Glu Arg Lys Gln Pro Cys Thr Ala Thr Gln Asp Thr Val Cys Arg
65 70 75 80
Cys Arg Ala Gly Thr Gln Pro Leu Asp Ser Tyr Lys Pro Gly Val Asp
85 90 95
Cys Ala Pro Cys Pro Pro Gly His Phe Ser Pro Gly Asp Asn Gln Ala
100 105 110
Cys Lys Pro Trp Thr Asn Cys Thr Leu Ala Gly Lys His Thr Leu Gln
115 120 125
Pro Ala Ser Asn Ser Ser Asp Ala Ile Cys Glu Asp Arg Asp Pro Pro
130 135 140
Pro Thr Gln Pro Gln Glu Thr Gln Gly Pro Pro Ala Arg Pro Thr Thr
145 150 155 160
Val Gln Pro Thr Glu Ala Trp Pro Arg Thr Ser Gln Arg Pro Ser Thr
165 170 175
Arg Pro Val Glu Val Pro Arg Gly Pro Ala Gln Ala Ser Gly Gly Thr
180 185 190
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
195 200 205
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
210 215 220
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
225 230 235 240
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
245 250 255
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
260 265 270
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
275 280 285
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
290 295 300
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
305 310 315 320
Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys
325 330 335
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
340 345 350
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
355 360 365
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
370 375 380
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
385 390 395 400
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
405 410 415
<210> 125
<211> 421
<212> PRT
<213> 人工序列
<220>
<223> rnOX40(ECD)-Fc
<400> 125
Val Thr Val Lys Leu Asn Cys Val Lys Asp Thr Tyr Pro Ser Gly His
1 5 10 15
Lys Cys Cys Arg Glu Cys Gln Pro Gly His Gly Met Val Ser Arg Cys
20 25 30
Asp His Thr Arg Asp Thr Val Cys His Pro Cys Glu Pro Gly Phe Tyr
35 40 45
Asn Glu Ala Val Asn Tyr Asp Thr Cys Lys Gln Cys Thr Gln Cys Asn
50 55 60
His Arg Ser Gly Ser Glu Leu Lys Gln Asn Cys Thr Pro Thr Glu Asp
65 70 75 80
Thr Val Cys Gln Cys Arg Pro Gly Thr Gln Pro Arg Gln Asp Ser Ser
85 90 95
His Lys Leu Gly Val Asp Cys Val Pro Cys Pro Pro Gly His Phe Ser
100 105 110
Pro Gly Ser Asn Gln Ala Cys Lys Pro Trp Thr Asn Cys Thr Leu Ser
115 120 125
Gly Lys Gln Ile Arg His Pro Ala Ser Asn Ser Leu Asp Thr Val Cys
130 135 140
Glu Asp Arg Ser Leu Leu Ala Thr Leu Leu Trp Glu Thr Gln Arg Thr
145 150 155 160
Thr Phe Arg Pro Thr Thr Val Pro Ser Thr Thr Val Trp Pro Arg Thr
165 170 175
Ser Gln Leu Pro Ser Thr Pro Thr Leu Val Ala Pro Glu Gly Pro Gln
180 185 190
Ala Ser Gly Gly Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
195 200 205
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
210 215 220
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
225 230 235 240
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
245 250 255
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
260 265 270
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
275 280 285
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
290 295 300
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
305 310 315 320
Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln
325 330 335
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
340 345 350
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
355 360 365
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
370 375 380
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
385 390 395 400
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
405 410 415
Leu Ser Pro Gly Lys
420
<210> 126
<211> 415
<212> PRT
<213> 人工序列
<220>
<223> chiOX40R(ECD)-HHHR-Fc
<400> 126
Leu His Cys Val Gly Asp Thr Tyr Pro Ser Asn Asp Arg Cys Cys His
1 5 10 15
Glu Cys Arg Pro Gly Asn Gly Met Val Ser Arg Cys Ser Arg Ser Gln
20 25 30
Asn Thr Val Cys Arg Pro Cys Gly Pro Gly Phe Tyr Asn Asp Val Val
35 40 45
Ser Ser Lys Pro Cys Lys Pro Cys Thr Trp Cys Asn Leu Arg Ser Gly
50 55 60
Ser Glu Arg Lys Gln Leu Cys Thr Ala Thr Gln Asp Thr Val Cys Arg
65 70 75 80
Cys Arg Ala Gly Thr Gln Pro Leu Asp Ser Tyr Lys Pro Gly Val Asp
85 90 95
Cys Ala Pro Cys Pro Pro Gly His Phe Ser Pro Gly Ser Asn Gln Ala
100 105 110
Cys Lys Pro Trp Thr Asn Cys Thr Leu Ser Gly Lys Gln Ile Arg His
115 120 125
Pro Ala Ser Asn Ser Leu Asp Thr Val Cys Glu Asp Arg Ser Leu Leu
130 135 140
Ala Thr Leu Leu Trp Glu Thr Gln Arg Thr Thr Phe Arg Pro Thr Thr
145 150 155 160
Val Pro Ser Thr Thr Val Trp Pro Arg Thr Ser Gln Leu Pro Ser Thr
165 170 175
Pro Thr Leu Val Ala Pro Glu Gly Pro Gly Gly Gly Gly Gly Thr His
180 185 190
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val
195 200 205
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
210 215 220
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
225 230 235 240
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
245 250 255
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
260 265 270
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
275 280 285
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
290 295 300
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
305 310 315 320
Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
325 330 335
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
340 345 350
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
355 360 365
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
370 375 380
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
385 390 395 400
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
405 410 415
<210> 127
<211> 418
<212> PRT
<213> 人工序列
<220>
<223> chiOX40R(ECD)-HHRH-Fc
<400> 127
Leu His Cys Val Gly Asp Thr Tyr Pro Ser Asn Asp Arg Cys Cys His
1 5 10 15
Glu Cys Arg Pro Gly Asn Gly Met Val Ser Arg Cys Ser Arg Ser Gln
20 25 30
Asn Thr Val Cys Arg Pro Cys Gly Pro Gly Phe Tyr Asn Asp Val Val
35 40 45
Ser Ser Lys Pro Cys Lys Pro Cys Thr Trp Cys Asn Leu Arg Ser Gly
50 55 60
Ser Glu Arg Lys Gln Leu Cys Thr Ala Thr Gln Asp Thr Val Cys Arg
65 70 75 80
Cys Arg Pro Gly Thr Gln Pro Arg Gln Asp Ser Ser His Lys Leu Gly
85 90 95
Val Asp Cys Val Pro Cys Pro Pro Gly His Phe Ser Pro Gly Asp Asn
100 105 110
Gln Ala Cys Lys Pro Trp Thr Asn Cys Thr Leu Ala Gly Lys His Thr
115 120 125
Leu Gln Pro Ala Ser Asn Ser Ser Asp Ala Ile Cys Glu Asp Arg Asp
130 135 140
Pro Pro Ala Thr Gln Pro Gln Glu Thr Gln Gly Pro Pro Ala Arg Pro
145 150 155 160
Ile Thr Val Gln Pro Thr Glu Ala Trp Pro Arg Thr Ser Gln Gly Pro
165 170 175
Ser Thr Arg Pro Val Glu Val Pro Gly Gly Arg Ala Gly Gly Gly Gly
180 185 190
Gly Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly
195 200 205
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
210 215 220
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
225 230 235 240
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
245 250 255
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
260 265 270
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
275 280 285
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
290 295 300
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
305 310 315 320
Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu
325 330 335
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
340 345 350
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
355 360 365
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
370 375 380
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
385 390 395 400
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
405 410 415
Gly Lys
<210> 128
<211> 428
<212> PRT
<213> 人工序列
<220>
<223> chiOX40R(ECD)-HHRR-Fc
<400> 128
Leu His Cys Val Gly Asp Thr Tyr Pro Ser Asn Asp Arg Cys Cys His
1 5 10 15
Glu Cys Arg Pro Gly Asn Gly Met Val Ser Arg Cys Ser Arg Ser Gln
20 25 30
Asn Thr Val Cys Arg Pro Cys Gly Pro Gly Phe Tyr Asn Asp Val Val
35 40 45
Ser Ser Lys Pro Cys Lys Pro Cys Thr Trp Cys Asn Leu Arg Ser Gly
50 55 60
Ser Glu Arg Lys Gln Leu Cys Thr Ala Thr Gln Asp Thr Val Cys Gln
65 70 75 80
Cys Arg Pro Gly Thr Gln Pro Arg Gln Asp Ser Ser His Lys Leu Gly
85 90 95
Val Asp Cys Val Pro Cys Pro Pro Gly His Phe Ser Pro Gly Ser Asn
100 105 110
Gln Ala Cys Lys Pro Trp Thr Asn Cys Thr Leu Ser Gly Lys Gln Ile
115 120 125
Arg His Pro Ala Ser Asn Ser Leu Asp Thr Val Cys Glu Asp Arg Ser
130 135 140
Leu Leu Ala Thr Leu Leu Trp Glu Thr Gln Arg Thr Thr Phe Arg Pro
145 150 155 160
Thr Thr Val Pro Ser Thr Thr Val Trp Pro Arg Thr Ser Gln Leu Pro
165 170 175
Ser Thr Pro Thr Leu Val Ala Pro Glu Gly Pro Ala Ala Ala Leu Glu
180 185 190
Val Leu Phe Gln Gly Pro Leu Gly Ser Gly Ser Thr His Thr Cys Pro
195 200 205
Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe
210 215 220
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
225 230 235 240
Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe
245 250 255
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
260 265 270
Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
275 280 285
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
290 295 300
Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
305 310 315 320
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg
325 330 335
Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
340 345 350
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
355 360 365
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
370 375 380
Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
385 390 395 400
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
405 410 415
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
420 425
<210> 129
<211> 428
<212> PRT
<213> 人工序列
<220>
<223> chiOX40R(ECD)-HRRR-Fc
<400> 129
Leu His Cys Val Gly Asp Thr Tyr Pro Ser Asn Asp Arg Cys Cys His
1 5 10 15
Glu Cys Arg Pro Gly Asn Gly Met Val Ser Arg Cys Ser Arg Ser Gln
20 25 30
Asn Thr Val Cys His Pro Cys Glu Pro Gly Phe Tyr Asn Glu Ala Val
35 40 45
Asn Tyr Asp Thr Cys Lys Gln Cys Thr Gln Cys Asn His Arg Ser Gly
50 55 60
Ser Glu Leu Lys Gln Asn Cys Thr Pro Thr Glu Asp Thr Val Cys Gln
65 70 75 80
Cys Arg Pro Gly Thr Gln Pro Arg Gln Asp Ser Ser His Lys Leu Gly
85 90 95
Val Asp Cys Val Pro Cys Pro Pro Gly His Phe Ser Pro Gly Ser Asn
100 105 110
Gln Ala Cys Lys Pro Trp Thr Asn Cys Thr Leu Ser Gly Lys Gln Ile
115 120 125
Arg His Pro Ala Ser Asn Ser Leu Asp Thr Val Cys Glu Asp Arg Ser
130 135 140
Leu Leu Ala Thr Leu Leu Trp Glu Thr Gln Arg Thr Thr Phe Arg Pro
145 150 155 160
Thr Thr Val Pro Ser Thr Thr Val Trp Pro Arg Thr Ser Gln Leu Pro
165 170 175
Ser Thr Pro Thr Leu Val Ala Pro Glu Gly Pro Ala Ala Ala Leu Glu
180 185 190
Val Leu Phe Gln Gly Pro Leu Gly Ser Gly Ser Thr His Thr Cys Pro
195 200 205
Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe
210 215 220
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
225 230 235 240
Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe
245 250 255
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
260 265 270
Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
275 280 285
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
290 295 300
Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
305 310 315 320
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg
325 330 335
Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
340 345 350
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
355 360 365
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
370 375 380
Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
385 390 395 400
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
405 410 415
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
420 425
<210> 130
<211> 430
<212> PRT
<213> 人工序列
<220>
<223> chiOX40R(ECD)-RRHH-Fc
<400> 130
Val Thr Val Lys Leu Asn Cys Val Lys Asp Thr Tyr Pro Ser Gly His
1 5 10 15
Lys Cys Cys Arg Glu Cys Gln Pro Gly His Gly Met Val Ser Arg Cys
20 25 30
Asp His Thr Arg Asp Thr Val Cys His Pro Cys Glu Pro Gly Phe Tyr
35 40 45
Asn Glu Ala Val Asn Tyr Asp Thr Cys Lys Gln Cys Thr Gln Cys Asn
50 55 60
His Arg Ser Gly Ser Glu Leu Lys Gln Asn Cys Thr Pro Thr Glu Asp
65 70 75 80
Thr Val Cys Arg Cys Arg Ala Gly Thr Gln Pro Leu Asp Ser Tyr Lys
85 90 95
Pro Gly Val Asp Cys Ala Pro Cys Pro Pro Gly His Phe Ser Pro Gly
100 105 110
Asp Asn Gln Ala Cys Lys Pro Trp Thr Asn Cys Thr Leu Ala Gly Lys
115 120 125
His Thr Leu Gln Pro Ala Ser Asn Ser Ser Asp Ala Ile Cys Glu Asp
130 135 140
Arg Asp Pro Pro Ala Thr Gln Pro Gln Glu Thr Gln Gly Pro Pro Ala
145 150 155 160
Arg Pro Ile Thr Val Gln Pro Thr Glu Ala Trp Pro Arg Thr Ser Gln
165 170 175
Gly Pro Ser Thr Arg Pro Val Glu Val Pro Gly Gly Arg Ala Ala Ala
180 185 190
Leu Glu Val Leu Phe Gln Gly Pro Leu Gly Ser Gly Ser Thr His Thr
195 200 205
Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
210 215 220
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
225 230 235 240
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
245 250 255
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
260 265 270
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
275 280 285
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
290 295 300
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
305 310 315 320
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
325 330 335
Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
340 345 350
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
355 360 365
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
370 375 380
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
385 390 395 400
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
405 410 415
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
420 425 430
<210> 131
<211> 432
<212> PRT
<213> 人工序列
<220>
<223> chiOX40R(ECD)-RHRR-Fc
<400> 131
Val Thr Val Lys Leu Asn Cys Val Lys Asp Thr Tyr Pro Ser Gly His
1 5 10 15
Lys Cys Cys Arg Glu Cys Gln Pro Gly His Gly Met Val Ser Arg Cys
20 25 30
Asp His Thr Arg Asp Thr Val Cys Arg Pro Cys Gly Pro Gly Phe Tyr
35 40 45
Asn Asp Val Val Ser Ser Lys Pro Cys Lys Pro Cys Thr Trp Cys Asn
50 55 60
Leu Arg Ser Gly Ser Glu Arg Lys Gln Leu Cys Thr Ala Thr Gln Asp
65 70 75 80
Thr Val Cys Gln Cys Arg Pro Gly Thr Gln Pro Arg Gln Asp Ser Ser
85 90 95
His Lys Leu Gly Val Asp Cys Val Pro Cys Pro Pro Gly His Phe Ser
100 105 110
Pro Gly Ser Asn Gln Ala Cys Lys Pro Trp Thr Asn Cys Thr Leu Ser
115 120 125
Gly Lys Gln Ile Arg His Pro Ala Ser Asn Ser Leu Asp Thr Val Cys
130 135 140
Glu Asp Arg Ser Leu Leu Ala Thr Leu Leu Trp Glu Thr Gln Arg Thr
145 150 155 160
Thr Phe Arg Pro Thr Thr Val Pro Ser Thr Thr Val Trp Pro Arg Thr
165 170 175
Ser Gln Leu Pro Ser Thr Pro Thr Leu Val Ala Pro Glu Gly Pro Ala
180 185 190
Ala Ala Leu Glu Val Leu Phe Gln Gly Pro Leu Gly Ser Gly Ser Thr
195 200 205
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
210 215 220
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
225 230 235 240
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
245 250 255
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
260 265 270
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
275 280 285
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
290 295 300
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
305 310 315 320
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
325 330 335
Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys
340 345 350
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
355 360 365
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
370 375 380
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
385 390 395 400
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
405 410 415
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
420 425 430
<210> 132
<211> 693
<212> PRT
<213> 人工序列
<220>
<223> Tetra-13 HC
<400> 132
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 Arg Tyr
20 25 30
Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Trp Ile Tyr Pro Gly Glu Gly Ser Thr Lys Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Glu Tyr Gly Asn Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala
115 120 125
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu
130 135 140
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
145 150 155 160
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
165 170 175
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
180 185 190
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr
195 200 205
Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr
210 215 220
Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
225 230 235 240
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
245 250 255
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
260 265 270
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
275 280 285
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
290 295 300
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
305 310 315 320
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
325 330 335
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
340 345 350
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
355 360 365
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
370 375 380
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
385 390 395 400
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
405 410 415
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
420 425 430
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly Gly
435 440 445
Gly Thr Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro
450 455 460
Gly Gly Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Tyr Ser Ile Thr
465 470 475 480
Ser Asp Tyr Ala Trp Asn Trp Ile Arg Gln Ala Pro Gly Lys Cys Leu
485 490 495
Glu Trp Met Gly Tyr Ile Ser Tyr Ser Gly Ser Thr Ala Tyr Asn Pro
500 505 510
Ser Leu Lys Ser Arg Ile Thr Ile Ser Arg Asp Thr Ser Lys Asn Thr
515 520 525
Phe Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
530 535 540
Tyr Cys Ala Arg Gly Pro Tyr Asn Tyr Leu Ala Tyr Trp Gly Gln Gly
545 550 555 560
Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
565 570 575
Ser Gly Gly Gly Gly Ser Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser
580 585 590
Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser
595 600 605
Glu Ser Val Asp Asn Tyr Gly Ile Ser Phe Met Asn Trp Phe Gln Gln
610 615 620
Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Asn Gln
625 630 635 640
Lys Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
645 650 655
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr
660 665 670
Phe Cys Gln Gln Ser Lys Glu Val Pro Leu Thr Phe Gly Cys Gly Thr
675 680 685
Lys Val Glu Ile Leu
690
<210> 133
<211> 695
<212> PRT
<213> 人工序列
<220>
<223> Tetra-14 HC
<400> 133
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Tyr Ser Ile Thr Ser Asp
20 25 30
Tyr Ala Trp Asn Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
35 40 45
Met Gly Tyr Ile Ser Tyr Ser Gly Ser Thr Ala Tyr Asn Pro Ser Leu
50 55 60
Lys Ser Arg Ile Thr Ile Ser Arg Asp Thr Ser Lys Asn Thr Phe Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gly Pro Tyr Asn Tyr Leu Ala Tyr Trp Gly Gln Gly Thr Leu
100 105 110
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
115 120 125
Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys
130 135 140
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser
145 150 155 160
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
165 170 175
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser
180 185 190
Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn
195 200 205
Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His
210 215 220
Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val
225 230 235 240
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
245 250 255
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
260 265 270
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
275 280 285
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
290 295 300
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
305 310 315 320
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
325 330 335
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
340 345 350
Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
355 360 365
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
370 375 380
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
385 390 395 400
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
405 410 415
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
420 425 430
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly
435 440 445
Gly Gly Thr Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys
450 455 460
Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe
465 470 475 480
Thr Arg Tyr Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Cys Leu
485 490 495
Glu Trp Met Gly Trp Ile Tyr Pro Gly Glu Gly Ser Thr Lys Tyr Ala
500 505 510
Gln Lys Phe Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser
515 520 525
Thr Ala Tyr Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val
530 535 540
Tyr Tyr Cys Ala Arg Glu Tyr Gly Asn Tyr Asp Tyr Trp Gly Gln Gly
545 550 555 560
Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
565 570 575
Ser Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser Pro Asp Ser
580 585 590
Leu Ala Val Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser
595 600 605
Gln Ser Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu Ala Trp Tyr
610 615 620
Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Gly Ala Ser
625 630 635 640
Thr Arg Glu Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly
645 650 655
Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala
660 665 670
Val Tyr Tyr Cys Gln Asn Asp His Ser Tyr Pro Tyr Thr Phe Gly Cys
675 680 685
Gly Thr Lys Leu Glu Ile Leu
690 695
<210> 134
<211> 694
<212> PRT
<213> 人工序列
<220>
<223> Tetra-15 HC
<400> 134
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Tyr Ser Ile Thr Ser Asp
20 25 30
Tyr Ala Trp Asn Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
35 40 45
Met Gly Tyr Ile Ser Tyr Ser Gly Ser Thr Ala Tyr Asn Pro Ser Leu
50 55 60
Lys Ser Arg Ile Thr Ile Ser Arg Asp Thr Ser Lys Asn Thr Phe Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gly Pro Tyr Asn Tyr Leu Ala Tyr Trp Gly Gln Gly Thr Leu
100 105 110
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
115 120 125
Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys
130 135 140
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser
145 150 155 160
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
165 170 175
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser
180 185 190
Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn
195 200 205
Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His
210 215 220
Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val
225 230 235 240
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
245 250 255
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
260 265 270
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
275 280 285
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
290 295 300
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
305 310 315 320
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
325 330 335
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
340 345 350
Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
355 360 365
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
370 375 380
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
385 390 395 400
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
405 410 415
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
420 425 430
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly
435 440 445
Gly Gly Thr Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln
450 455 460
Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Tyr Ser Ile
465 470 475 480
Thr Ser Asp Tyr Ala Trp Asn Trp Ile Arg Gln Ala Pro Gly Lys Cys
485 490 495
Leu Glu Trp Met Gly Tyr Ile Ser Tyr Ser Gly Ser Thr Ala Tyr Asn
500 505 510
Pro Ser Leu Lys Ser Arg Ile Thr Ile Ser Arg Asp Thr Ser Lys Asn
515 520 525
Thr Phe Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
530 535 540
Tyr Tyr Cys Ala Arg Gly Pro Tyr Asn Tyr Leu Ala Tyr Trp Gly Gln
545 550 555 560
Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly
565 570 575
Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Leu Thr Gln Ser Pro Ser
580 585 590
Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala
595 600 605
Ser Glu Ser Val Asp Asn Tyr Gly Ile Ser Phe Met Asn Trp Phe Gln
610 615 620
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Asn
625 630 635 640
Gln Lys Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr
645 650 655
Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr
660 665 670
Tyr Phe Cys Gln Gln Ser Lys Glu Val Pro Leu Thr Phe Gly Cys Gly
675 680 685
Thr Lys Val Glu Ile Leu
690
<210> 135
<211> 694
<212> PRT
<213> 人工序列
<220>
<223> Tetra-16 HC
<400> 135
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 Arg Tyr
20 25 30
Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Trp Ile Tyr Pro Gly Glu Gly Ser Thr Lys Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Glu Tyr Gly Asn Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala
115 120 125
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu
130 135 140
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
145 150 155 160
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
165 170 175
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
180 185 190
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr
195 200 205
Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr
210 215 220
Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe
225 230 235 240
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
245 250 255
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
260 265 270
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
275 280 285
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
290 295 300
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
305 310 315 320
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
325 330 335
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
340 345 350
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
355 360 365
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
370 375 380
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
385 390 395 400
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
405 410 415
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
420 425 430
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly Gly
435 440 445
Gly Thr Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
450 455 460
Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr
465 470 475 480
Arg Tyr Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Cys Leu Glu
485 490 495
Trp Met Gly Trp Ile Tyr Pro Gly Glu Gly Ser Thr Lys Tyr Ala Gln
500 505 510
Lys Phe Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr
515 520 525
Ala Tyr Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr
530 535 540
Tyr Cys Ala Arg Glu Tyr Gly Asn Tyr Asp Tyr Trp Gly Gln Gly Thr
545 550 555 560
Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
565 570 575
Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu
580 585 590
Ala Val Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln
595 600 605
Ser Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln
610 615 620
Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Gly Ala Ser Thr
625 630 635 640
Arg Glu Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr
645 650 655
Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val
660 665 670
Tyr Tyr Cys Gln Asn Asp His Ser Tyr Pro Tyr Thr Phe Gly Cys Gly
675 680 685
Thr Lys Leu Glu Ile Leu
690
<210> 136
<211> 669
<212> PRT
<213> 人工序列
<220>
<223> Tetra-17 HC
<400> 136
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 Arg Tyr
20 25 30
Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Trp Ile Tyr Pro Gly Glu Gly Ser Thr Lys Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Glu Tyr Gly Asn Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala
115 120 125
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu
130 135 140
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
145 150 155 160
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
165 170 175
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
180 185 190
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr
195 200 205
Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr
210 215 220
Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe
225 230 235 240
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
245 250 255
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
260 265 270
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
275 280 285
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
290 295 300
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
305 310 315 320
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
325 330 335
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
340 345 350
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
355 360 365
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
370 375 380
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
385 390 395 400
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
405 410 415
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
420 425 430
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly Gly
435 440 445
Gly Thr Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
450 455 460
Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr
465 470 475 480
Arg Tyr Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
485 490 495
Trp Met Gly Trp Ile Tyr Pro Gly Glu Gly Ser Thr Lys Tyr Ala Gln
500 505 510
Lys Phe Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr
515 520 525
Ala Tyr Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr
530 535 540
Tyr Cys Ala Arg Glu Tyr Gly Asn Tyr Asp Tyr Trp Gly Gln Gly Thr
545 550 555 560
Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
565 570 575
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
580 585 590
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
595 600 605
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
610 615 620
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
625 630 635 640
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser
645 650 655
Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys
660 665
<210> 137
<211> 671
<212> PRT
<213> 人工序列
<220>
<223> Tetra-18 HC
<400> 137
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Tyr Ser Ile Thr Ser Asp
20 25 30
Tyr Ala Trp Asn Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
35 40 45
Met Gly Tyr Ile Ser Tyr Ser Gly Ser Thr Ala Tyr Asn Pro Ser Leu
50 55 60
Lys Ser Arg Ile Thr Ile Ser Arg Asp Thr Ser Lys Asn Thr Phe Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gly Pro Tyr Asn Tyr Leu Ala Tyr Trp Gly Gln Gly Thr Leu
100 105 110
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
115 120 125
Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys
130 135 140
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser
145 150 155 160
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
165 170 175
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser
180 185 190
Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn
195 200 205
Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His
210 215 220
Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val
225 230 235 240
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
245 250 255
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
260 265 270
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
275 280 285
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
290 295 300
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
305 310 315 320
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
325 330 335
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
340 345 350
Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
355 360 365
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
370 375 380
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
385 390 395 400
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
405 410 415
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
420 425 430
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly
435 440 445
Gly Gly Thr Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln
450 455 460
Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Tyr Ser Ile
465 470 475 480
Thr Ser Asp Tyr Ala Trp Asn Trp Ile Arg Gln Ala Pro Gly Lys Gly
485 490 495
Leu Glu Trp Met Gly Tyr Ile Ser Tyr Ser Gly Ser Thr Ala Tyr Asn
500 505 510
Pro Ser Leu Lys Ser Arg Ile Thr Ile Ser Arg Asp Thr Ser Lys Asn
515 520 525
Thr Phe Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
530 535 540
Tyr Tyr Cys Ala Arg Gly Pro Tyr Asn Tyr Leu Ala Tyr Trp Gly Gln
545 550 555 560
Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
565 570 575
Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
580 585 590
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
595 600 605
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
610 615 620
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
625 630 635 640
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
645 650 655
Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys
660 665 670
<210> 138
<211> 693
<212> PRT
<213> 人工序列
<220>
<223> Tetra-19 HC1
<400> 138
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 Arg Tyr
20 25 30
Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Trp Ile Tyr Pro Gly Glu Gly Ser Thr Lys Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Glu Tyr Gly Asn Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala
115 120 125
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu
130 135 140
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
145 150 155 160
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
165 170 175
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
180 185 190
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr
195 200 205
Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr
210 215 220
Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe
225 230 235 240
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
245 250 255
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
260 265 270
Gln Phe Lys Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
275 280 285
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Phe Arg Val Val Ser Val
290 295 300
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
305 310 315 320
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
325 330 335
Lys Thr Lys Gly Gln Pro Arg Glu Pro Ala Val Tyr Thr Leu Pro Pro
340 345 350
Ser Arg Glu Glu Met Thr Lys Asn Gln Val Lys Leu Val Cys Leu Val
355 360 365
Thr Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Ser Gly
370 375 380
Gln Pro Glu Asn Asn Tyr Tyr Thr Thr Pro Pro Met Leu Asp Ser Asp
385 390 395 400
Gly Ser Phe Ser Leu Val Ser Trp Leu Asn Val Asp Lys Ser Arg Trp
405 410 415
Gln Gln Gly Asn Ile Phe Ser Cys Ser Val Met His Glu Ala Leu His
420 425 430
Asn Arg Phe Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly Gly
435 440 445
Gly Thr Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro
450 455 460
Gly Gly Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Tyr Ser Ile Thr
465 470 475 480
Ser Asp Tyr Ala Trp Asn Trp Ile Arg Gln Ala Pro Gly Lys Cys Leu
485 490 495
Glu Trp Met Gly Tyr Ile Ser Tyr Ser Gly Ser Thr Ala Tyr Asn Pro
500 505 510
Ser Leu Lys Ser Arg Ile Thr Ile Ser Arg Asp Thr Ser Lys Asn Thr
515 520 525
Phe Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
530 535 540
Tyr Cys Ala Arg Gly Pro Tyr Asn Tyr Leu Ala Tyr Trp Gly Gln Gly
545 550 555 560
Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
565 570 575
Ser Gly Gly Gly Gly Ser Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser
580 585 590
Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser
595 600 605
Glu Ser Val Asp Asn Tyr Gly Ile Ser Phe Met Asn Trp Phe Gln Gln
610 615 620
Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Asn Gln
625 630 635 640
Lys Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
645 650 655
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr
660 665 670
Phe Cys Gln Gln Ser Lys Glu Val Pro Leu Thr Phe Gly Cys Gly Thr
675 680 685
Lys Val Glu Ile Leu
690
<210> 139
<211> 669
<212> PRT
<213> 人工序列
<220>
<223> Tetra-19 HC2
<400> 139
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 Arg Tyr
20 25 30
Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Trp Ile Tyr Pro Gly Glu Gly Ser Thr Lys Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Glu Tyr Gly Asn Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala
115 120 125
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu
130 135 140
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
145 150 155 160
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
165 170 175
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
180 185 190
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr
195 200 205
Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr
210 215 220
Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe
225 230 235 240
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
245 250 255
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
260 265 270
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
275 280 285
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
290 295 300
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
305 310 315 320
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
325 330 335
Lys Ala Lys Gly Gln Pro Arg Glu Pro Glu Val Ala Thr Phe Pro Pro
340 345 350
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Thr Leu Val Cys Leu Val
355 360 365
Thr Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
370 375 380
Gln Pro Glu Asn Asn Tyr Lys Thr Asp Pro Pro Leu Leu Glu Ser Gln
385 390 395 400
Gly Ser Phe Ala Leu Ser Ser Arg Leu Arg Val Asp Lys Ser Arg Trp
405 410 415
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
420 425 430
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly Gly
435 440 445
Gly Thr Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
450 455 460
Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr
465 470 475 480
Arg Tyr Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
485 490 495
Trp Met Gly Trp Ile Tyr Pro Gly Glu Gly Ser Thr Lys Tyr Ala Gln
500 505 510
Lys Phe Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr
515 520 525
Ala Tyr Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr
530 535 540
Tyr Cys Ala Arg Glu Tyr Gly Asn Tyr Asp Tyr Trp Gly Gln Gly Thr
545 550 555 560
Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
565 570 575
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
580 585 590
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
595 600 605
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
610 615 620
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
625 630 635 640
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser
645 650 655
Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys
660 665
<210> 140
<211> 695
<212> PRT
<213> 人工序列
<220>
<223> Tetra-20 HC1
<400> 140
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Tyr Ser Ile Thr Ser Asp
20 25 30
Tyr Ala Trp Asn Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
35 40 45
Met Gly Tyr Ile Ser Tyr Ser Gly Ser Thr Ala Tyr Asn Pro Ser Leu
50 55 60
Lys Ser Arg Ile Thr Ile Ser Arg Asp Thr Ser Lys Asn Thr Phe Tyr
65 70 75 80
Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gly Pro Tyr Asn Tyr Leu Ala Tyr Trp Gly Gln Gly Thr Leu
100 105 110
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
115 120 125
Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys
130 135 140
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser
145 150 155 160
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
165 170 175
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser
180 185 190
Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn
195 200 205
Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His
210 215 220
Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val
225 230 235 240
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
245 250 255
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
260 265 270
Val Gln Phe Lys Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
275 280 285
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Phe Arg Val Val Ser
290 295 300
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
305 310 315 320
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
325 330 335
Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Ala Val Tyr Thr Leu Pro
340 345 350
Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Lys Leu Val Cys Leu
355 360 365
Val Thr Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Ser
370 375 380
Gly Gln Pro Glu Asn Asn Tyr Tyr Thr Thr Pro Pro Met Leu Asp Ser
385 390 395 400
Asp Gly Ser Phe Ser Leu Val Ser Trp Leu Asn Val Asp Lys Ser Arg
405 410 415
Trp Gln Gln Gly Asn Ile Phe Ser Cys Ser Val Met His Glu Ala Leu
420 425 430
His Asn Arg Phe Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly
435 440 445
Gly Gly Thr Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys
450 455 460
Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe
465 470 475 480
Thr Arg Tyr Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Cys Leu
485 490 495
Glu Trp Met Gly Trp Ile Tyr Pro Gly Glu Gly Ser Thr Lys Tyr Ala
500 505 510
Gln Lys Phe Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser
515 520 525
Thr Ala Tyr Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val
530 535 540
Tyr Tyr Cys Ala Arg Glu Tyr Gly Asn Tyr Asp Tyr Trp Gly Gln Gly
545 550 555 560
Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
565 570 575
Ser Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser Pro Asp Ser
580 585 590
Leu Ala Val Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser
595 600 605
Gln Ser Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu Ala Trp Tyr
610 615 620
Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Gly Ala Ser
625 630 635 640
Thr Arg Glu Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly
645 650 655
Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala
660 665 670
Val Tyr Tyr Cys Gln Asn Asp His Ser Tyr Pro Tyr Thr Phe Gly Cys
675 680 685
Gly Thr Lys Leu Glu Ile Leu
690 695
<210> 141
<211> 671
<212> PRT
<213> 人工序列
<220>
<223> Tetra-20 HC2
<400> 141
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Tyr Ser Ile Thr Ser Asp
20 25 30
Tyr Ala Trp Asn Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
35 40 45
Met Gly Tyr Ile Ser Tyr Ser Gly Ser Thr Ala Tyr Asn Pro Ser Leu
50 55 60
Lys Ser Arg Ile Thr Ile Ser Arg Asp Thr Ser Lys Asn Thr Phe Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gly Pro Tyr Asn Tyr Leu Ala Tyr Trp Gly Gln Gly Thr Leu
100 105 110
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
115 120 125
Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys
130 135 140
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser
145 150 155 160
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
165 170 175
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser
180 185 190
Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn
195 200 205
Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His
210 215 220
Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val
225 230 235 240
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
245 250 255
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
260 265 270
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
275 280 285
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
290 295 300
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
305 310 315 320
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
325 330 335
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Glu Val Ala Thr Phe Pro
340 345 350
Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Thr Leu Val Cys Leu
355 360 365
Val Thr Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
370 375 380
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Asp Pro Pro Leu Leu Glu Ser
385 390 395 400
Asp Gly Ser Phe Ala Leu Ser Ser Arg Leu Arg Val Asp Lys Ser Arg
405 410 415
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
420 425 430
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly
435 440 445
Gly Gly Thr Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln
450 455 460
Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Tyr Ser Ile
465 470 475 480
Thr Ser Asp Tyr Ala Trp Asn Trp Ile Arg Gln Ala Pro Gly Lys Gly
485 490 495
Leu Glu Trp Met Gly Tyr Ile Ser Tyr Ser Gly Ser Thr Ala Tyr Asn
500 505 510
Pro Ser Leu Lys Ser Arg Ile Thr Ile Ser Arg Asp Thr Ser Lys Asn
515 520 525
Thr Phe Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
530 535 540
Tyr Tyr Cys Ala Arg Gly Pro Tyr Asn Tyr Leu Ala Tyr Trp Gly Gln
545 550 555 560
Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
565 570 575
Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
580 585 590
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
595 600 605
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
610 615 620
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
625 630 635 640
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
645 650 655
Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys
660 665 670
<210> 142
<211> 700
<212> PRT
<213> 人工序列
<220>
<223> Tetra-21 HC2
<400> 142
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Tyr Ser Ile Thr Ser Asp
20 25 30
Tyr Ala Trp Asn Trp Ile Arg Gln Ala Pro Gly Lys Cys Leu Glu Trp
35 40 45
Met Gly Tyr Ile Ser Tyr Ser Gly Ser Thr Ala Tyr Asn Pro Ser Leu
50 55 60
Lys Ser Arg Ile Thr Ile Ser Arg Asp Thr Ser Lys Asn Thr Phe Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gly Pro Tyr Asn Tyr Leu Ala Tyr Trp Gly Gln Gly Thr Leu
100 105 110
Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
115 120 125
Gly Gly Gly Ser Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser
130 135 140
Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Ser
145 150 155 160
Val Asp Asn Tyr Gly Ile Ser Phe Met Asn Trp Phe Gln Gln Lys Pro
165 170 175
Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Asn Gln Lys Ser
180 185 190
Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
195 200 205
Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Phe Cys
210 215 220
Gln Gln Ser Lys Glu Val Pro Leu Thr Phe Gly Cys Gly Thr Lys Val
225 230 235 240
Glu Ile Lys Arg Thr Gly Gly Gly Gly Thr Asp Lys Thr His Thr Cys
245 250 255
Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu
260 265 270
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
275 280 285
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys
290 295 300
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
305 310 315 320
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
325 330 335
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
340 345 350
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
355 360 365
Ala Lys Gly Gln Pro Arg Glu Pro Glu Val Ala Thr Phe Pro Pro Ser
370 375 380
Arg Asp Glu Leu Thr Lys Asn Gln Val Thr Leu Val Cys Leu Val Thr
385 390 395 400
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
405 410 415
Pro Glu Asn Asn Tyr Lys Thr Asp Pro Pro Leu Leu Glu Ser Gln Gly
420 425 430
Ser Phe Ala Leu Ser Ser Arg Leu Arg Val Asp Lys Ser Arg Trp Gln
435 440 445
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
450 455 460
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly Gly Gly
465 470 475 480
Thr Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly
485 490 495
Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Arg
500 505 510
Tyr Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp
515 520 525
Met Gly Trp Ile Tyr Pro Gly Glu Gly Ser Thr Lys Tyr Ala Gln Lys
530 535 540
Phe Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala
545 550 555 560
Tyr Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr
565 570 575
Cys Ala Arg Glu Tyr Gly Asn Tyr Asp Tyr Trp Gly Gln Gly Thr Leu
580 585 590
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
595 600 605
Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys
610 615 620
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser
625 630 635 640
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
645 650 655
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser
660 665 670
Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn
675 680 685
Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys
690 695 700
<210> 143
<211> 684
<212> PRT
<213> 人工序列
<220>
<223> Tetra-22 HC
<400> 143
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr
20 25 30
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
115 120 125
Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
130 135 140
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
145 150 155 160
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
165 170 175
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
180 185 190
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
195 200 205
Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
210 215 220
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly
225 230 235 240
Pro Ser Val Phe Leu Phe Pro Glu Val Thr Cys Val Val Val Asp Val
245 250 255
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
260 265 270
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
275 280 285
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
290 295 300
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
305 310 315 320
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
325 330 335
Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
340 345 350
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
355 360 365
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
370 375 380
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
385 390 395 400
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
405 410 415
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
420 425 430
Leu Ser Pro Gly Gly Gly Gly Gly Thr Glu Val Gln Leu Val Glu Ser
435 440 445
Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala
450 455 460
Val Ser Gly Tyr Ser Ile Thr Ser Asp Tyr Ala Trp Asn Trp Ile Arg
465 470 475 480
Gln Ala Pro Gly Lys Cys Leu Glu Trp Met Gly Tyr Ile Ser Tyr Ser
485 490 495
Gly Ser Thr Ala Tyr Asn Pro Ser Leu Lys Ser Arg Ile Thr Ile Ser
500 505 510
Arg Asp Thr Ser Lys Asn Thr Phe Tyr Leu Gln Met Asn Ser Leu Arg
515 520 525
Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gly Pro Tyr Asn Tyr
530 535 540
Leu Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly
545 550 555 560
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln
565 570 575
Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val
580 585 590
Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Asp Asn Tyr Gly Ile Ser
595 600 605
Phe Met Asn Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu
610 615 620
Ile Tyr Ala Ala Ser Asn Gln Lys Ser Gly Val Pro Ser Arg Phe Ser
625 630 635 640
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln
645 650 655
Pro Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Ser Lys Glu Val Pro
660 665 670
Leu Thr Phe Gly Cys Gly Thr Lys Val Glu Ile Leu
675 680
<210> 144
<211> 214
<212> PRT
<213> 人工序列
<220>
<223> Tetra-22 LC
<400> 144
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys
210
<210> 145
<211> 446
<212> PRT
<213> 人工序列
<220>
<223> Tri-8 HC2
<400> 145
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 Arg Tyr
20 25 30
Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Trp Ile Tyr Pro Gly Glu Gly Ser Thr Lys Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Glu Tyr Gly Asn Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala
115 120 125
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu
130 135 140
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
145 150 155 160
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
165 170 175
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
180 185 190
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr
195 200 205
Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr
210 215 220
Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe
225 230 235 240
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
245 250 255
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
260 265 270
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
275 280 285
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
290 295 300
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
305 310 315 320
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
325 330 335
Lys Ala Lys Gly Gln Pro Arg Glu Pro Glu Val Ala Thr Phe Pro Pro
340 345 350
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Thr Leu Val Cys Leu Val
355 360 365
Thr Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
370 375 380
Gln Pro Glu Asn Asn Tyr Lys Thr Asp Pro Pro Leu Leu Glu Ser Gln
385 390 395 400
Gly Ser Phe Ala Leu Ser Ser Arg Leu Arg Val Asp Lys Ser Arg Trp
405 410 415
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
420 425 430
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 445
<210> 146
<211> 694
<212> PRT
<213> 人工序列
<220>
<223> 1A7_2H6_8 HC
<400> 146
Glu 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 Ser
20 25 30
Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Asp Met Tyr Pro Asp Asn Gly Asp Ser Ser Tyr Asn Gln Lys Phe
50 55 60
Arg Glu Arg Val Thr Ile Thr Arg Asp Thr Ser Thr Ser Thr Ala Tyr
65 70 75 80
Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Val Leu Ala Pro Arg Trp Tyr Phe Ser Val Trp Gly Gln Gly Thr Leu
100 105 110
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
115 120 125
Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys
130 135 140
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser
145 150 155 160
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
165 170 175
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser
180 185 190
Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn
195 200 205
Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His
210 215 220
Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val
225 230 235 240
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
245 250 255
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
260 265 270
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
275 280 285
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
290 295 300
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
305 310 315 320
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
325 330 335
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
340 345 350
Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
355 360 365
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
370 375 380
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
385 390 395 400
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
405 410 415
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
420 425 430
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly
435 440 445
Gly Gly Thr Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln
450 455 460
Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Tyr Ser Ile
465 470 475 480
Thr Ser Asp Tyr Ala Trp Asn Trp Ile Arg Gln Ala Pro Gly Lys Cys
485 490 495
Leu Glu Trp Met Gly Tyr Ile Ser Tyr Ser Gly Ser Thr Ala Tyr Asn
500 505 510
Pro Ser Leu Lys Ser Arg Ile Thr Ile Ser Arg Asp Thr Ser Lys Asn
515 520 525
Thr Phe Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
530 535 540
Tyr Tyr Cys Ala Arg Gly Pro Tyr Asn Tyr Leu Ala Tyr Trp Gly Gln
545 550 555 560
Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly
565 570 575
Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Leu Thr Gln Ser Pro Ser
580 585 590
Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala
595 600 605
Ser Glu Ser Val Asp Asn Tyr Gly Ile Ser Phe Met Asn Trp Phe Gln
610 615 620
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Asn
625 630 635 640
Gln Lys Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr
645 650 655
Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr
660 665 670
Tyr Phe Cys Gln Gln Ser Lys Glu Val Pro Leu Thr Phe Gly Cys Gly
675 680 685
Thr Lys Val Glu Ile Leu
690
<210> 147
<211> 699
<212> PRT
<213> 人工序列
<220>
<223> 106-222_2H6_8 HC
<400> 147
Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu 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
Ser Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Lys Trp Met
35 40 45
Gly Trp Ile Asn Thr Glu Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe
50 55 60
Lys Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr
65 70 75 80
Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Asn Pro Tyr Tyr Asp Tyr Val Ser Tyr Tyr Ala Met Asp Tyr Trp
100 105 110
Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
115 120 125
Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr
130 135 140
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
145 150 155 160
Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
165 170 175
Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
180 185 190
Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn
195 200 205
His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser
210 215 220
Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala
225 230 235 240
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
245 250 255
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
260 265 270
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
275 280 285
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
290 295 300
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
305 310 315 320
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
325 330 335
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
340 345 350
Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val
355 360 365
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
370 375 380
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
385 390 395 400
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
405 410 415
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
420 425 430
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
435 440 445
Ser Pro Gly Gly Gly Gly Gly Thr Glu Val Gln Leu Val Glu Ser Gly
450 455 460
Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Val
465 470 475 480
Ser Gly Tyr Ser Ile Thr Ser Asp Tyr Ala Trp Asn Trp Ile Arg Gln
485 490 495
Ala Pro Gly Lys Cys Leu Glu Trp Met Gly Tyr Ile Ser Tyr Ser Gly
500 505 510
Ser Thr Ala Tyr Asn Pro Ser Leu Lys Ser Arg Ile Thr Ile Ser Arg
515 520 525
Asp Thr Ser Lys Asn Thr Phe Tyr Leu Gln Met Asn Ser Leu Arg Ala
530 535 540
Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gly Pro Tyr Asn Tyr Leu
545 550 555 560
Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly
565 570 575
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Leu
580 585 590
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr
595 600 605
Ile Thr Cys Arg Ala Ser Glu Ser Val Asp Asn Tyr Gly Ile Ser Phe
610 615 620
Met Asn Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
625 630 635 640
Tyr Ala Ala Ser Asn Gln Lys Ser Gly Val Pro Ser Arg Phe Ser Gly
645 650 655
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
660 665 670
Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Ser Lys Glu Val Pro Leu
675 680 685
Thr Phe Gly Cys Gly Thr Lys Val Glu Ile Leu
690 695
<210> 148
<211> 699
<212> PRT
<213> 人工序列
<220>
<223> 7H11_1949_8 HC
<400> 148
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 Arg Tyr
20 25 30
Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Trp Ile Tyr Pro Gly Glu Gly Ser Thr Lys Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Glu Tyr Gly Asn Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala
115 120 125
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu
130 135 140
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
145 150 155 160
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
165 170 175
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
180 185 190
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr
195 200 205
Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr
210 215 220
Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe
225 230 235 240
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
245 250 255
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
260 265 270
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
275 280 285
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
290 295 300
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
305 310 315 320
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
325 330 335
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
340 345 350
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
355 360 365
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
370 375 380
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
385 390 395 400
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
405 410 415
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
420 425 430
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly Gly
435 440 445
Gly Thr Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro
450 455 460
Gly Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser
465 470 475 480
Gly Ser Ala Met His Trp Val Arg Gln Ala Ser Gly Lys Cys Leu Glu
485 490 495
Trp Val Gly Arg Ile Arg Ser Lys Ala Asn Ser Tyr Ala Thr Ala Tyr
500 505 510
Ala Ala Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys
515 520 525
Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala
530 535 540
Val Tyr Tyr Cys Thr Ser Gly Ile Tyr Asp Ser Ser Gly Tyr Asp Tyr
545 550 555 560
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser
565 570 575
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr Asp Ile Val Met Thr
580 585 590
Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly Glu Pro Ala Ser Ile
595 600 605
Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser Asn Gly Tyr Asn Tyr
610 615 620
Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile
625 630 635 640
Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro Asp Arg Phe Ser Gly
645 650 655
Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala
660 665 670
Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala Leu Gln Thr Pro Leu
675 680 685
Thr Phe Gly Cys Gly Thr Lys Val Glu Ile Lys
690 695
<210> 149
<211> 701
<212> PRT
<213> 人工序列
<220>
<223> 11D4_1949 HC
<400> 149
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Tyr Ile Ser Ser Ser Ser Ser Thr Ile Asp Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Asp Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Glu Ser Gly Trp Tyr Leu Phe Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
115 120 125
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
130 135 140
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
145 150 155 160
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
165 170 175
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
180 185 190
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser
195 200 205
Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr
210 215 220
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser
225 230 235 240
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
245 250 255
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
260 265 270
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
275 280 285
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
290 295 300
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
305 310 315 320
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
325 330 335
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
340 345 350
Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys
355 360 365
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
370 375 380
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
385 390 395 400
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
405 410 415
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
420 425 430
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly
435 440 445
Gly Gly Gly Thr Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val
450 455 460
Gln Pro Gly Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr
465 470 475 480
Phe Ser Gly Ser Ala Met His Trp Val Arg Gln Ala Ser Gly Lys Cys
485 490 495
Leu Glu Trp Val Gly Arg Ile Arg Ser Lys Ala Asn Ser Tyr Ala Thr
500 505 510
Ala Tyr Ala Ala Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp
515 520 525
Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp
530 535 540
Thr Ala Val Tyr Tyr Cys Thr Ser Gly Ile Tyr Asp Ser Ser Gly Tyr
545 550 555 560
Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly
565 570 575
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr Asp Ile Val
580 585 590
Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly Glu Pro Ala
595 600 605
Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser Asn Gly Tyr
610 615 620
Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Gln Leu
625 630 635 640
Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro Asp Arg Phe
645 650 655
Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val
660 665 670
Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala Leu Gln Thr
675 680 685
Pro Leu Thr Phe Gly Cys Gly Thr Lys Val Glu Ile Lys
690 695 700
<210> 150
<211> 720
<212> PRT
<213> 人工序列
<220>
<223> 1A7_1949 HC
<400> 150
Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro
1 5 10 15
Gly Ser Thr Gly Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys
20 25 30
Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr
35 40 45
Phe Thr Asp Ser Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Gln Gly
50 55 60
Leu Glu Trp Ile Gly Asp Met Tyr Pro Asp Asn Gly Asp Ser Ser Tyr
65 70 75 80
Asn Gln Lys Phe Arg Glu Arg Val Thr Ile Thr Arg Asp Thr Ser Thr
85 90 95
Ser Thr Ala Tyr Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala
100 105 110
Val Tyr Tyr Cys Val Leu Ala Pro Arg Trp Tyr Phe Ser Val Trp Gly
115 120 125
Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
130 135 140
Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala
145 150 155 160
Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val
165 170 175
Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
180 185 190
Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
195 200 205
Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His
210 215 220
Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys
225 230 235 240
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly
245 250 255
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
260 265 270
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
275 280 285
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
290 295 300
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
305 310 315 320
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
325 330 335
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
340 345 350
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
355 360 365
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser
370 375 380
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
385 390 395 400
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
405 410 415
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
420 425 430
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
435 440 445
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
450 455 460
Pro Gly Gly Gly Gly Gly Thr Glu Val Gln Leu Val Glu Ser Gly Gly
465 470 475 480
Gly Leu Val Gln Pro Gly Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser
485 490 495
Gly Phe Thr Phe Ser Gly Ser Ala Met His Trp Val Arg Gln Ala Ser
500 505 510
Gly Lys Cys Leu Glu Trp Val Gly Arg Ile Arg Ser Lys Ala Asn Ser
515 520 525
Tyr Ala Thr Ala Tyr Ala Ala Ser Val Lys Gly Arg Phe Thr Ile Ser
530 535 540
Arg Asp Asp Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu Lys
545 550 555 560
Thr Glu Asp Thr Ala Val Tyr Tyr Cys Thr Ser Gly Ile Tyr Asp Ser
565 570 575
Ser Gly Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
580 585 590
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr
595 600 605
Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly
610 615 620
Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser
625 630 635 640
Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser
645 650 655
Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro
660 665 670
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
675 680 685
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala
690 695 700
Leu Gln Thr Pro Leu Thr Phe Gly Cys Gly Thr Lys Val Glu Ile Lys
705 710 715 720
<210> 151
<211> 704
<212> PRT
<213> 人工序列
<220>
<223> 9B12_1949 HC
<400> 151
Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln
1 5 10 15
Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Ser Gly
20 25 30
Tyr Trp Asn Trp Ile Arg Lys His Pro Gly Lys Gly Leu Glu Tyr Ile
35 40 45
Gly Tyr Ile Ser Tyr Asn Gly Ile Thr Tyr His Asn Pro Ser Leu Lys
50 55 60
Ser Arg Ile Thr Ile Asn Arg Asp Thr Ser Lys Asn Gln Tyr Ser Leu
65 70 75 80
Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Arg Tyr Lys Tyr Asp Tyr Asp Gly Gly His Ala Met Asp Tyr Trp Gly
100 105 110
Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
115 120 125
Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala
130 135 140
Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val
145 150 155 160
Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
165 170 175
Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
180 185 190
Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His
195 200 205
Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys
210 215 220
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly
225 230 235 240
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
245 250 255
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
260 265 270
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
275 280 285
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
290 295 300
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
305 310 315 320
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
325 330 335
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
340 345 350
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser
355 360 365
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
370 375 380
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
385 390 395 400
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
405 410 415
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
420 425 430
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
435 440 445
Pro Gly Gly Gly Gly Gly Thr Glu Val Gln Leu Val Glu Ser Gly Gly
450 455 460
Gly Leu Val Gln Pro Gly Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser
465 470 475 480
Gly Phe Thr Phe Ser Gly Ser Ala Met His Trp Val Arg Gln Ala Ser
485 490 495
Gly Lys Cys Leu Glu Trp Val Gly Arg Ile Arg Ser Lys Ala Asn Ser
500 505 510
Tyr Ala Thr Ala Tyr Ala Ala Ser Val Lys Gly Arg Phe Thr Ile Ser
515 520 525
Arg Asp Asp Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu Lys
530 535 540
Thr Glu Asp Thr Ala Val Tyr Tyr Cys Thr Ser Gly Ile Tyr Asp Ser
545 550 555 560
Ser Gly Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
565 570 575
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr
580 585 590
Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly
595 600 605
Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser
610 615 620
Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser
625 630 635 640
Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro
645 650 655
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
660 665 670
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala
675 680 685
Leu Gln Thr Pro Leu Thr Phe Gly Cys Gly Thr Lys Val Glu Ile Lys
690 695 700
<210> 152
<211> 705
<212> PRT
<213> 人工序列
<220>
<223> 106-222_1949 HC
<400> 152
Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu 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
Ser Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Lys Trp Met
35 40 45
Gly Trp Ile Asn Thr Glu Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe
50 55 60
Lys Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr
65 70 75 80
Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Asn Pro Tyr Tyr Asp Tyr Val Ser Tyr Tyr Ala Met Asp Tyr Trp
100 105 110
Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
115 120 125
Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr
130 135 140
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
145 150 155 160
Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
165 170 175
Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
180 185 190
Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn
195 200 205
His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser
210 215 220
Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala
225 230 235 240
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
245 250 255
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
260 265 270
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
275 280 285
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
290 295 300
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
305 310 315 320
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
325 330 335
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
340 345 350
Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val
355 360 365
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
370 375 380
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
385 390 395 400
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
405 410 415
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
420 425 430
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
435 440 445
Ser Pro Gly Gly Gly Gly Gly Thr Glu Val Gln Leu Val Glu Ser Gly
450 455 460
Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Lys Leu Ser Cys Ala Ala
465 470 475 480
Ser Gly Phe Thr Phe Ser Gly Ser Ala Met His Trp Val Arg Gln Ala
485 490 495
Ser Gly Lys Cys Leu Glu Trp Val Gly Arg Ile Arg Ser Lys Ala Asn
500 505 510
Ser Tyr Ala Thr Ala Tyr Ala Ala Ser Val Lys Gly Arg Phe Thr Ile
515 520 525
Ser Arg Asp Asp Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu
530 535 540
Lys Thr Glu Asp Thr Ala Val Tyr Tyr Cys Thr Ser Gly Ile Tyr Asp
545 550 555 560
Ser Ser Gly Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
565 570 575
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
580 585 590
Thr Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro
595 600 605
Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His
610 615 620
Ser Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln
625 630 635 640
Ser Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val
645 650 655
Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys
660 665 670
Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln
675 680 685
Ala Leu Gln Thr Pro Leu Thr Phe Gly Cys Gly Thr Lys Val Glu Ile
690 695 700
Lys
705
<210> 153
<211> 693
<212> PRT
<213> 人工序列
<220>
<223> 7H11_9B12_8 HC
<400> 153
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 Arg Tyr
20 25 30
Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Trp Ile Tyr Pro Gly Glu Gly Ser Thr Lys Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Glu Tyr Gly Asn Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala
115 120 125
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu
130 135 140
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
145 150 155 160
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
165 170 175
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
180 185 190
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr
195 200 205
Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr
210 215 220
Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe
225 230 235 240
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
245 250 255
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
260 265 270
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
275 280 285
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
290 295 300
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
305 310 315 320
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
325 330 335
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
340 345 350
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
355 360 365
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
370 375 380
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
385 390 395 400
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
405 410 415
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
420 425 430
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly Gly
435 440 445
Gly Thr Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro
450 455 460
Ser Gln Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser
465 470 475 480
Ser Gly Tyr Trp Asn Trp Ile Arg Lys His Pro Gly Lys Cys Leu Glu
485 490 495
Tyr Ile Gly Tyr Ile Ser Tyr Asn Gly Ile Thr Tyr His Asn Pro Ser
500 505 510
Leu Lys Ser Arg Ile Thr Ile Asn Arg Asp Thr Ser Lys Asn Gln Tyr
515 520 525
Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val Tyr Tyr
530 535 540
Cys Ala Arg Tyr Lys Tyr Asp Tyr Asp Gly Gly His Ala Met Asp Tyr
545 550 555 560
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser
565 570 575
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln
580 585 590
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr
595 600 605
Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr Leu Asn Trp Tyr Gln Gln
610 615 620
Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Tyr Thr Ser Lys Leu
625 630 635 640
His Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
645 650 655
Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr
660 665 670
Tyr Cys Gln Gln Gly Ser Ala Leu Pro Trp Thr Phe Gly Cys Gly Thr
675 680 685
Lys Val Glu Ile Lys
690
<210> 154
<211> 566
<212> PRT
<213> 人工序列
<220>
<223> 7H11_hzG3v9_8 HC
<400> 154
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 Arg Tyr
20 25 30
Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Trp Ile Tyr Pro Gly Glu Gly Ser Thr Lys Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Glu Tyr Gly Asn Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala
115 120 125
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu
130 135 140
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
145 150 155 160
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
165 170 175
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
180 185 190
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr
195 200 205
Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr
210 215 220
Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe
225 230 235 240
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
245 250 255
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
260 265 270
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
275 280 285
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
290 295 300
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
305 310 315 320
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
325 330 335
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
340 345 350
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
355 360 365
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
370 375 380
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
385 390 395 400
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
405 410 415
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
420 425 430
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly Gly
435 440 445
Gly Thr Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Glu Val Gln Pro
450 455 460
Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser
465 470 475 480
Asp Ala Phe Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Glu Arg Glu
485 490 495
Trp Val Ser Ser Ile Ser Asn Arg Gly Leu Lys Thr Ala Tyr Ala Glu
500 505 510
Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr
515 520 525
Leu Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
530 535 540
Tyr Cys Val Glu Gly Asp Trp Asn Leu Gly Pro Arg Gly Gln Gly Thr
545 550 555 560
Leu Val Thr Val Lys Pro
565
<210> 155
<211> 568
<212> PRT
<213> 人工序列
<220>
<223> 11D4_hzG3v9 HC
<400> 155
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Tyr Ile Ser Ser Ser Ser Ser Thr Ile Asp Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Asp Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Glu Ser Gly Trp Tyr Leu Phe Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
115 120 125
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
130 135 140
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
145 150 155 160
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
165 170 175
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
180 185 190
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser
195 200 205
Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr
210 215 220
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser
225 230 235 240
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
245 250 255
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
260 265 270
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
275 280 285
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
290 295 300
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
305 310 315 320
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
325 330 335
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
340 345 350
Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys
355 360 365
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
370 375 380
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
385 390 395 400
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
405 410 415
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
420 425 430
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly
435 440 445
Gly Gly Gly Thr Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Glu Val
450 455 460
Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
465 470 475 480
Phe Ser Asp Ala Phe Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Glu
485 490 495
Arg Glu Trp Val Ser Ser Ile Ser Asn Arg Gly Leu Lys Thr Ala Tyr
500 505 510
Ala Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys
515 520 525
Asn Thr Leu Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala
530 535 540
Val Tyr Tyr Cys Val Glu Gly Asp Trp Asn Leu Gly Pro Arg Gly Gln
545 550 555 560
Gly Thr Leu Val Thr Val Lys Pro
565
<210> 156
<211> 572
<212> PRT
<213> 人工序列
<220>
<223> 106-222_hzG3v9 HC
<400> 156
Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu 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
Ser Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Lys Trp Met
35 40 45
Gly Trp Ile Asn Thr Glu Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe
50 55 60
Lys Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr
65 70 75 80
Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Asn Pro Tyr Tyr Asp Tyr Val Ser Tyr Tyr Ala Met Asp Tyr Trp
100 105 110
Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
115 120 125
Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr
130 135 140
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
145 150 155 160
Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
165 170 175
Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
180 185 190
Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn
195 200 205
His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser
210 215 220
Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala
225 230 235 240
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
245 250 255
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
260 265 270
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
275 280 285
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
290 295 300
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
305 310 315 320
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
325 330 335
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
340 345 350
Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val
355 360 365
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
370 375 380
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
385 390 395 400
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
405 410 415
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
420 425 430
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
435 440 445
Ser Pro Gly Gly Gly Gly Gly Thr Glu Val Gln Leu Leu Glu Ser Gly
450 455 460
Gly Gly Glu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala
465 470 475 480
Ser Gly Phe Thr Phe Ser Asp Ala Phe Met Tyr Trp Val Arg Gln Ala
485 490 495
Pro Gly Lys Glu Arg Glu Trp Val Ser Ser Ile Ser Asn Arg Gly Leu
500 505 510
Lys Thr Ala Tyr Ala Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg
515 520 525
Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Ser Ser Leu Arg Ala
530 535 540
Glu Asp Thr Ala Val Tyr Tyr Cys Val Glu Gly Asp Trp Asn Leu Gly
545 550 555 560
Pro Arg Gly Gln Gly Thr Leu Val Thr Val Lys Pro
565 570
<210> 157
<211> 571
<212> PRT
<213> 人工序列
<220>
<223> 9B12_hzG3v9 HC
<400> 157
Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln
1 5 10 15
Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Ser Gly
20 25 30
Tyr Trp Asn Trp Ile Arg Lys His Pro Gly Lys Gly Leu Glu Tyr Ile
35 40 45
Gly Tyr Ile Ser Tyr Asn Gly Ile Thr Tyr His Asn Pro Ser Leu Lys
50 55 60
Ser Arg Ile Thr Ile Asn Arg Asp Thr Ser Lys Asn Gln Tyr Ser Leu
65 70 75 80
Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Arg Tyr Lys Tyr Asp Tyr Asp Gly Gly His Ala Met Asp Tyr Trp Gly
100 105 110
Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
115 120 125
Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala
130 135 140
Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val
145 150 155 160
Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
165 170 175
Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
180 185 190
Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His
195 200 205
Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys
210 215 220
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly
225 230 235 240
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
245 250 255
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
260 265 270
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
275 280 285
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
290 295 300
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
305 310 315 320
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
325 330 335
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
340 345 350
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser
355 360 365
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
370 375 380
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
385 390 395 400
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
405 410 415
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
420 425 430
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
435 440 445
Pro Gly Gly Gly Gly Gly Thr Glu Val Gln Leu Leu Glu Ser Gly Gly
450 455 460
Gly Glu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser
465 470 475 480
Gly Phe Thr Phe Ser Asp Ala Phe Met Tyr Trp Val Arg Gln Ala Pro
485 490 495
Gly Lys Glu Arg Glu Trp Val Ser Ser Ile Ser Asn Arg Gly Leu Lys
500 505 510
Thr Ala Tyr Ala Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp
515 520 525
Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu
530 535 540
Asp Thr Ala Val Tyr Tyr Cys Val Glu Gly Asp Trp Asn Leu Gly Pro
545 550 555 560
Arg Gly Gln Gly Thr Leu Val Thr Val Lys Pro
565 570
<210> 158
<211> 567
<212> PRT
<213> 人工序列
<220>
<223> 2H6_hzG3v9_8 HC
<400> 158
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Tyr Ser Ile Thr Ser Asp
20 25 30
Tyr Ala Trp Asn Trp Ile Arg Gln Ala Pro Gly Lys Cys Leu Glu Trp
35 40 45
Met Gly Tyr Ile Ser Tyr Ser Gly Ser Thr Ala Tyr Asn Pro Ser Leu
50 55 60
Lys Ser Arg Ile Thr Ile Ser Arg Asp Thr Ser Lys Asn Thr Phe Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gly Pro Tyr Asn Tyr Leu Ala Tyr Trp Gly Gln Gly Thr Leu
100 105 110
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
115 120 125
Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys
130 135 140
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser
145 150 155 160
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
165 170 175
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser
180 185 190
Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn
195 200 205
Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His
210 215 220
Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val
225 230 235 240
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
245 250 255
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
260 265 270
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
275 280 285
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
290 295 300
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
305 310 315 320
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
325 330 335
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
340 345 350
Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
355 360 365
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
370 375 380
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
385 390 395 400
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
405 410 415
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
420 425 430
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly
435 440 445
Gly Gly Thr Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Glu Val Gln
450 455 460
Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
465 470 475 480
Ser Asp Ala Phe Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Glu Arg
485 490 495
Glu Trp Val Ser Ser Ile Ser Asn Arg Gly Leu Lys Thr Ala Tyr Ala
500 505 510
Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn
515 520 525
Thr Leu Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val
530 535 540
Tyr Tyr Cys Val Glu Gly Asp Trp Asn Leu Gly Pro Arg Gly Gln Gly
545 550 555 560
Thr Leu Val Thr Val Lys Pro
565
<210> 159
<211> 163
<212> PRT
<213> 人工序列
<220>
<223> hsOX40-CRD(29-168)-Avi-his
<400> 159
Leu His Cys Val Gly Asp Thr Tyr Pro Ser Asn Asp Arg Cys Cys His
1 5 10 15
Glu Cys Arg Pro Gly Asn Gly Met Val Ser Arg Cys Ser Arg Ser Gln
20 25 30
Asn Thr Val Cys Arg Pro Cys Gly Pro Gly Phe Tyr Asn Asp Val Val
35 40 45
Ser Ser Lys Pro Cys Lys Pro Cys Thr Trp Cys Asn Leu Arg Ser Gly
50 55 60
Ser Glu Arg Lys Gln Leu Cys Thr Ala Thr Gln Asp Thr Val Cys Arg
65 70 75 80
Cys Arg Ala Gly Thr Gln Pro Leu Asp Ser Tyr Lys Pro Gly Val Asp
85 90 95
Cys Ala Pro Cys Pro Pro Gly His Phe Ser Pro Gly Asp Asn Gln Ala
100 105 110
Cys Lys Pro Trp Thr Asn Cys Thr Leu Ala Gly Lys His Thr Leu Gln
115 120 125
Pro Ala Ser Asn Ser Ser Asp Ala Ile Cys Glu Asp Arg Asp Gly Leu
130 135 140
Asn Asp Ile Phe Glu Ala Gln Lys Ile Glu Trp His Glu His His His
145 150 155 160
His His His
<210> 160
<211> 148
<212> PRT
<213> 人工序列
<220>
<223> hsOX40-CRD(29-168)-His
<400> 160
Leu His Cys Val Gly Asp Thr Tyr Pro Ser Asn Asp Arg Cys Cys His
1 5 10 15
Glu Cys Arg Pro Gly Asn Gly Met Val Ser Arg Cys Ser Arg Ser Gln
20 25 30
Asn Thr Val Cys Arg Pro Cys Gly Pro Gly Phe Tyr Asn Asp Val Val
35 40 45
Ser Ser Lys Pro Cys Lys Pro Cys Thr Trp Cys Asn Leu Arg Ser Gly
50 55 60
Ser Glu Arg Lys Gln Leu Cys Thr Ala Thr Gln Asp Thr Val Cys Arg
65 70 75 80
Cys Arg Ala Gly Thr Gln Pro Leu Asp Ser Tyr Lys Pro Gly Val Asp
85 90 95
Cys Ala Pro Cys Pro Pro Gly His Phe Ser Pro Gly Asp Asn Gln Ala
100 105 110
Cys Lys Pro Trp Thr Asn Cys Thr Leu Ala Gly Lys His Thr Leu Gln
115 120 125
Pro Ala Ser Asn Ser Ser Asp Ala Ile Cys Glu Asp Ser Ala His His
130 135 140
His His His His
145
<210> 161
<211> 497
<212> PRT
<213> 人工序列
<220>
<223> hsOX40-eGFP
<400> 161
Leu His Cys Val Gly Asp Thr Tyr Pro Ser Asn Asp Arg Cys Cys His
1 5 10 15
Glu Cys Arg Pro Gly Asn Gly Met Val Ser Arg Cys Ser Arg Ser Gln
20 25 30
Asn Thr Val Cys Arg Pro Cys Gly Pro Gly Phe Tyr Asn Asp Val Val
35 40 45
Ser Ser Lys Pro Cys Lys Pro Cys Thr Trp Cys Asn Leu Arg Ser Gly
50 55 60
Ser Glu Arg Lys Gln Leu Cys Thr Ala Thr Gln Asp Thr Val Cys Arg
65 70 75 80
Cys Arg Ala Gly Thr Gln Pro Leu Asp Ser Tyr Lys Pro Gly Val Asp
85 90 95
Cys Ala Pro Cys Pro Pro Gly His Phe Ser Pro Gly Asp Asn Gln Ala
100 105 110
Cys Lys Pro Trp Thr Asn Cys Thr Leu Ala Gly Lys His Thr Leu Gln
115 120 125
Pro Ala Ser Asn Ser Ser Asp Ala Ile Cys Glu Asp Arg Asp Pro Pro
130 135 140
Ala Thr Gln Pro Gln Glu Thr Gln Gly Pro Pro Ala Arg Pro Ile Thr
145 150 155 160
Val Gln Pro Thr Glu Ala Trp Pro Arg Thr Ser Gln Gly Pro Ser Thr
165 170 175
Arg Pro Val Glu Val Pro Gly Gly Arg Ala Val Ala Ala Ile Leu Gly
180 185 190
Leu Gly Leu Val Leu Gly Leu Leu Gly Pro Leu Ala Ile Leu Leu Ala
195 200 205
Leu Tyr Leu Leu Arg Arg Asp Gln Arg Leu Pro Pro Asp Ala His Lys
210 215 220
Pro Pro Gly Gly Gly Ser Phe Arg Thr Pro Ile Gln Glu Glu Gln Ala
225 230 235 240
Asp Ala His Ser Thr Leu Ala Lys Ile Gly Gly Ser Ala Thr Thr Ser
245 250 255
Ala Thr Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro
260 265 270
Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val
275 280 285
Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys
290 295 300
Phe Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val
305 310 315 320
Thr Thr Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His
325 330 335
Met Lys Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val
340 345 350
Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg
355 360 365
Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu
370 375 380
Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu
385 390 395 400
Glu Tyr Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln
405 410 415
Lys Asn Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp
420 425 430
Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly
435 440 445
Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser
450 455 460
Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu
465 470 475 480
Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr
485 490 495
Lys
<210> 162
<211> 561
<212> PRT
<213> 人工序列
<220>
<223> 2C10_3h56 HC
<400> 162
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 Asn Tyr
20 25 30
Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Ile
35 40 45
Gly Tyr Ile Asn Pro Ser Asn Asp Tyr Thr Lys Tyr Asn Gln Lys Phe
50 55 60
Lys Asp Arg Ala Thr Leu Thr Ala Asp Lys Ser Ala Asn 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
Ala Arg Gln Gly Phe Pro Tyr Trp Gly Gln Gly Thr Leu Val Thr Val
100 105 110
Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser
115 120 125
Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys
130 135 140
Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu
145 150 155 160
Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu
165 170 175
Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr
180 185 190
Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val
195 200 205
Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro
210 215 220
Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe
225 230 235 240
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
245 250 255
Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe
260 265 270
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
275 280 285
Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
290 295 300
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
305 310 315 320
Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
325 330 335
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg
340 345 350
Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
355 360 365
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
370 375 380
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
385 390 395 400
Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
405 410 415
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
420 425 430
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Thr Glu Val Gln
435 440 445
Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg
450 455 460
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Asp Tyr Glu Met Trp
465 470 475 480
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Arg Val Ser Ala Ile
485 490 495
Asn Pro Gln Gly Thr Arg Thr Tyr Tyr Ala Asp Ser Val Met Gly Arg
500 505 510
Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met
515 520 525
Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys Leu
530 535 540
Pro Phe Thr Phe Asp Asp Trp Gly Gln Gly Thr Leu Val Thr Val Ser
545 550 555 560
Ser
<210> 163
<211> 566
<212> PRT
<213> 人工序列
<220>
<223> ADC-1013_3h56 HC
<400> 163
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr
20 25 30
Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu
35 40 45
Ser Tyr Ile Ser Gly Gly Ser Ser Tyr Ile Phe Tyr Ala Asp Ser Val
50 55 60
Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Glu Asn Ala Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Ile Leu Arg Gly Gly Ser Gly Met Asp Leu Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
115 120 125
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu
130 135 140
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
145 150 155 160
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
165 170 175
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
180 185 190
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro
195 200 205
Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys
210 215 220
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro
225 230 235 240
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
245 250 255
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
260 265 270
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
275 280 285
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
290 295 300
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
305 310 315 320
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
325 330 335
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
340 345 350
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr
355 360 365
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
370 375 380
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
385 390 395 400
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
405 410 415
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
420 425 430
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
435 440 445
Gly Thr Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro
450 455 460
Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg
465 470 475 480
Asp Tyr Glu Met Trp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
485 490 495
Arg Val Ser Ala Ile Asn Pro Gln Gly Thr Arg Thr Tyr Tyr Ala Asp
500 505 510
Ser Val Met Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
515 520 525
Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
530 535 540
Tyr Cys Ala Lys Leu Pro Phe Thr Phe Asp Asp Trp Gly Gln Gly Thr
545 550 555 560
Leu Val Thr Val Ser Ser
565
<210> 164
<211> 566
<212> PRT
<213> 人工序列
<220>
<223> CD40.1_3h56 HC
<400> 164
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 Gly Tyr
20 25 30
Asn Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Asn Ile Asp Pro Tyr Tyr Gly Asn Thr Asn Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Leu Gly Leu Gln Leu Tyr Ala Leu Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
115 120 125
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu
130 135 140
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
145 150 155 160
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
165 170 175
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
180 185 190
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro
195 200 205
Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys
210 215 220
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro
225 230 235 240
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
245 250 255
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
260 265 270
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
275 280 285
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
290 295 300
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
305 310 315 320
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
325 330 335
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
340 345 350
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr
355 360 365
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
370 375 380
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
385 390 395 400
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
405 410 415
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
420 425 430
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
435 440 445
Gly Thr Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro
450 455 460
Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg
465 470 475 480
Asp Tyr Glu Met Trp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
485 490 495
Arg Val Ser Ala Ile Asn Pro Gln Gly Thr Arg Thr Tyr Tyr Ala Asp
500 505 510
Ser Val Met Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
515 520 525
Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
530 535 540
Tyr Cys Ala Lys Leu Pro Phe Thr Phe Asp Asp Trp Gly Gln Gly Thr
545 550 555 560
Leu Val Thr Val Ser Ser
565
<210> 165
<211> 573
<212> PRT
<213> 人工序列
<220>
<223> selicrelumab_3h56 HC
<400> 165
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 Gly Tyr
20 25 30
Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Trp Ile Asn Pro Asp Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Asn Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Asp Gln Pro Leu Gly Tyr Cys Thr Asn Gly Val Cys Ser Tyr
100 105 110
Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser
115 120 125
Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr
130 135 140
Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro
145 150 155 160
Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
165 170 175
His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser
180 185 190
Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile
195 200 205
Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val
210 215 220
Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
225 230 235 240
Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
245 250 255
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
260 265 270
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
275 280 285
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
290 295 300
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
305 310 315 320
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
325 330 335
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
340 345 350
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
355 360 365
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
370 375 380
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
385 390 395 400
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
405 410 415
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
420 425 430
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
435 440 445
Ser Leu Ser Leu Ser Pro Gly Gly Thr Glu Val Gln Leu Leu Glu Ser
450 455 460
Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala
465 470 475 480
Ala Ser Gly Phe Thr Phe Arg Asp Tyr Glu Met Trp Trp Val Arg Gln
485 490 495
Ala Pro Gly Lys Gly Leu Glu Arg Val Ser Ala Ile Asn Pro Gln Gly
500 505 510
Thr Arg Thr Tyr Tyr Ala Asp Ser Val Met Gly Arg Phe Thr Ile Ser
515 520 525
Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg
530 535 540
Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys Leu Pro Phe Thr Phe
545 550 555 560
Asp Asp Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
565 570
<210> 166
<211> 569
<212> PRT
<213> 人工序列
<220>
<223> 替奈昔单抗_3h56 HC
<400> 166
Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu
1 5 10 15
Thr Val Arg Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Thr Thr
20 25 30
Gly Met Gln Trp Val Gln Glu Met Pro Gly Lys Gly Leu Lys Trp Ile
35 40 45
Gly Trp Ile Asn Thr His Ser Gly Val Pro Lys Tyr Val Glu Asp Phe
50 55 60
Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Asn Thr Ala Tyr
65 70 75 80
Leu Gln Ile Ser Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys
85 90 95
Val Arg Ser Gly Asn Gly Asn Tyr Asp Leu Ala Tyr Phe Ala Tyr Trp
100 105 110
Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
115 120 125
Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr
130 135 140
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
145 150 155 160
Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
165 170 175
Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
180 185 190
Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn
195 200 205
His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser
210 215 220
Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala
225 230 235 240
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
245 250 255
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
260 265 270
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
275 280 285
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
290 295 300
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
305 310 315 320
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
325 330 335
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
340 345 350
Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val
355 360 365
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
370 375 380
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
385 390 395 400
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
405 410 415
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
420 425 430
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
435 440 445
Ser Pro Gly Gly Thr Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu
450 455 460
Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
465 470 475 480
Thr Phe Arg Asp Tyr Glu Met Trp Trp Val Arg Gln Ala Pro Gly Lys
485 490 495
Gly Leu Glu Arg Val Ser Ala Ile Asn Pro Gln Gly Thr Arg Thr Tyr
500 505 510
Tyr Ala Asp Ser Val Met Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser
515 520 525
Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
530 535 540
Ala Val Tyr Tyr Cys Ala Lys Leu Pro Phe Thr Phe Asp Asp Trp Gly
545 550 555 560
Gln Gly Thr Leu Val Thr Val Ser Ser
565
<210> 167
<211> 213
<212> PRT
<213> 人工序列
<220>
<223> 2C10 LC
<400> 167
Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser Ser Val Ser Tyr Met
20 25 30
His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Arg Trp Ile Tyr
35 40 45
Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser
50 55 60
Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu
65 70 75 80
Asp Phe Ala Val Tyr Tyr Cys His Gln Leu Ser Ser Asp Pro Phe Thr
85 90 95
Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro
100 105 110
Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr
115 120 125
Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys
130 135 140
Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu
145 150 155 160
Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser
165 170 175
Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala
180 185 190
Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe
195 200 205
Asn Arg Gly Glu Cys
210
<210> 168
<211> 217
<212> PRT
<213> 人工序列
<220>
<223> ADC-1013 LC
<400> 168
Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln
1 5 10 15
Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly
20 25 30
Tyr Asn Val Tyr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu
35 40 45
Leu Ile Tyr Gly Asn Ile Asn Arg Pro Ser Gly Val Pro Asp Arg Phe
50 55 60
Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu
65 70 75 80
Arg Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Lys Ser
85 90 95
Ile Ser Gly Leu Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly
100 105 110
Gln Pro Lys Ala Asn Pro Thr Val Thr Leu Phe Pro Pro Ser Ser Glu
115 120 125
Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe
130 135 140
Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Gly Ser Pro Val
145 150 155 160
Lys Ala Gly Val Glu Thr Thr Lys Pro Ser Lys Gln Ser Asn Asn Lys
165 170 175
Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser
180 185 190
His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu
195 200 205
Lys Thr Val Ala Pro Thr Glu Cys Ser
210 215
<210> 169
<211> 219
<212> PRT
<213> 人工序列
<220>
<223> CD40.1 LC
<400> 169
Asp Ile Val Met Thr Gln Thr 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 Leu Thr Asn Arg Phe Phe 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 Ser
85 90 95
Ile His Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105 110
Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
115 120 125
Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
130 135 140
Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
145 150 155 160
Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
165 170 175
Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
180 185 190
Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
195 200 205
Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
210 215
<210> 170
<211> 214
<212> PRT
<213> 人工序列
<220>
<223> Selicrelumab LC
<400> 170
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Tyr Ser Trp
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Asn Leu Leu Ile
35 40 45
Tyr Thr Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Asn Ile Phe Pro Leu
85 90 95
Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys
210
<210> 171
<211> 214
<212> PRT
<213> 人工序列
<220>
<223> 替奈昔单抗 LC
<400> 171
Asp Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Val Thr Pro Gly
1 5 10 15
Asp Arg Val Ser Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Asp Tyr
20 25 30
Leu His Trp Tyr Gln Gln Lys Ser His Glu Ser Pro Arg Leu Leu Ile
35 40 45
Lys Tyr Ala Ser His Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Ser Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Pro
65 70 75 80
Glu Asp Val Gly Ile Tyr Tyr Cys Gln His Gly His Ser Phe Pro Trp
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys
210
<210> 172
<211> 455
<212> PRT
<213> 人工序列
<220>
<223> selicrelumab IgG1 LALA HC
<400> 172
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 Gly Tyr
20 25 30
Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Trp Ile Asn Pro Asp Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Asn Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Asp Gln Pro Leu Gly Tyr Cys Thr Asn Gly Val Cys Ser Tyr
100 105 110
Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser
115 120 125
Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr
130 135 140
Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro
145 150 155 160
Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
165 170 175
His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser
180 185 190
Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile
195 200 205
Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val
210 215 220
Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
225 230 235 240
Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
245 250 255
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
260 265 270
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
275 280 285
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
290 295 300
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
305 310 315 320
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
325 330 335
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
340 345 350
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
355 360 365
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
370 375 380
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
385 390 395 400
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
405 410 415
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
420 425 430
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
435 440 445
Ser Leu Ser Leu Ser Pro Gly
450 455
<210> 173
<211> 448
<212> PRT
<213> 人工序列
<220>
<223> ADC-1013 IgG1 HC
<400> 173
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr
20 25 30
Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu
35 40 45
Ser Tyr Ile Ser Gly Gly Ser Ser Tyr Ile Phe Tyr Ala Asp Ser Val
50 55 60
Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Glu Asn Ala Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Ile Leu Arg Gly Gly Ser Gly Met Asp Leu Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
115 120 125
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu
130 135 140
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
145 150 155 160
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
165 170 175
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
180 185 190
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro
195 200 205
Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys
210 215 220
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro
225 230 235 240
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
245 250 255
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
260 265 270
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
275 280 285
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
290 295 300
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
305 310 315 320
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
325 330 335
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
340 345 350
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr
355 360 365
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
370 375 380
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
385 390 395 400
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
405 410 415
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
420 425 430
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
435 440 445
<210> 174
<211> 348
<212> PRT
<213> 人工序列
<220>
<223> 3h-56 IgG1 LALA HC
<400> 174
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Asp Tyr
20 25 30
Glu Met Trp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Arg Val
35 40 45
Ser Ala Ile Asn Pro Gln Gly Thr Arg Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Met Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Lys Leu Pro Phe Thr Phe Asp Asp Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Ser Ser Gly Gly Gly Gly Thr Asp Lys Thr His Thr Cys Pro
115 120 125
Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe
130 135 140
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
145 150 155 160
Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe
165 170 175
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
180 185 190
Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
195 200 205
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
210 215 220
Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
225 230 235 240
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg
245 250 255
Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
260 265 270
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
275 280 285
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
290 295 300
Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
305 310 315 320
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
325 330 335
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
340 345

Claims (15)

1.一种TNFR激动剂，包含对所述TNFR的至少两个不同部分特异的结合部分。
2.根据权利要求1所述的TNFR激动剂，其中所述TNFR参与T细胞应答的共刺激。
3.根据权利要求1或2所述的TNFR激动剂，其中所述TNFR选自由CD27、4-1BB(CD137)、OX40(CD134)、HVEM、CD30和GITR所组成的组。
4.根据权利要求1至3中任一项所述的TNFR激动剂，其中所述结合部分能够同时结合所述TNFR。
5.根据权利要求1至4中任一项所述的TNFR激动剂，包含与被所述激动剂结合的所述TNFR的每个部分结合的至少两个结合部分。
6.根据权利要求1至5中任一项所述的TNFR激动剂，其中所述结合部分选自包含抗体、DARPin、Fynomer、Affimer、可变淋巴细胞受体、anticalin、nanofitin、可变新抗原受体(VNAR)，及其衍生物例如Fab、Fab′、Fab′-SH、Fd、Fv、dAb、F(ab′)2、scFv、Fcab、双特异性单链Fv二聚体、双抗体、三抗体的组。
7.根据权利要求5或6所述的TNFR激动剂，其中与所述TNFR的相同部分结合的所述至少两个结合部分位于所述激动剂的相同肽末端。
8.根据权利要求1至7中任一项所述的TNFR激动剂，其中所述结合部分结合所述TNFR的不同的富含半胱氨酸的结构域(CRD)。
9.根据权利要求1至8中任一项所述的TNFR激动剂，其激动OX40并结合OX40的CRD1和CRD3或CRD1和CRD4中的表位。
10.根据权利要求9所述的TNFR激动剂，其中至少一个OX40结合部分选自包含以下的组：SEQ ID NO：2、3、12、13、14、15、16、17、18、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49，或具有与其至少50％、60％、65％、70％、75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的氨基酸序列的分离多肽。
11.一种OX40激动剂，由SEQ ID NO：45和16编码或是具有与其至少50％、60％、65％、70％、75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的氨基酸序列的分离的多肽。
12.根据权利要求1至11中任一项所述的激动剂作为药物的用途。
13.根据权利要求1至11中任一项所述的激动剂激活人免疫系统的组成部分的用途。
14.根据权利要求1至11中任一项所述的OX40激动剂作为药物的用途。
15.根据权利要求12至14中任一项所述的用途，与另一种药物组合。
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