CN111647077B - 新型冠状病毒(sars-cov-2)刺突蛋白结合分子及其应用 - Google Patents

新型冠状病毒(sars-cov-2)刺突蛋白结合分子及其应用 Download PDF

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CN111647077B
CN111647077B CN202010491549.0A CN202010491549A CN111647077B CN 111647077 B CN111647077 B CN 111647077B CN 202010491549 A CN202010491549 A CN 202010491549A CN 111647077 B CN111647077 B CN 111647077B
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张军方
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Shenzhen Yinnuosai Biology Technology Co ltd
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Abstract

本发明涉及医药生物技术领域，具体公开一种新型冠状病毒(SARS‑COV‑2)刺突蛋白结合分子及其应用。所述结合分子能特异性结合SARS‑COV‑2的刺突蛋白且包含至少一个免疫球蛋白单一可变结构域。本发明提供的SARS‑COV‑2‑Spike蛋白结合分子能够特异性的结合SARS‑COV‑2‑Spike蛋白，并有效阻断SARS‑COV‑2‑Spike蛋白与人体细胞ACE2受体的结合，进而阻断SARS‑COV‑2对细胞的感染过程，抑制SARS‑COV‑2的传染和扩增。

Description

新型冠状病毒(SARS-COV-2)刺突蛋白结合分子及其应用
技术领域
本发明涉及医药生物技术领域，尤其涉及一种新型冠状病毒(SARS-COV-2)刺突蛋白结合分子及其应用。
背景技术
新型冠状病毒肺炎(COVID-19)在全球累计感染超过400多万人，且感染人数依然在快速增加，对COVID-19目前临床上缺乏特异有效的治疗手段。虽然我国疫情已经得到全面控制，但国外疫情却暴发出来，并且还在迅速增长。此外，越来越多的研究显示，新型冠状病毒(SARS-COV-2)感染可能存在慢性携带状态；部分出院复阳病人也提示病毒可能会长期存在人体。目前尚不清楚长期携带存在的机制、时间等关键因素，未来防止SARS-COV-2卷土重来至关重要。受COVID-19的影响，我国及世界各国由此而产生的经济损失、社会负担及其它负面影响难以计量。
目前针对COVID-19尚无特效药物，亟需快速研制有效的药物。国内外众多研发机构都在针对COVID-19的治疗策略研究上分秒必争。虽然已发掘的瑞德西韦、法匹拉韦等广谱小分子抗病毒药物对COVID-19具有一定疗效，但由于针对SARS-COV-2并无特异性，治疗效果受限，难以成为COVID-19的特效药。
发明内容
针对现有抗病毒药物对新型冠状病毒新型冠状病毒无特异性，治疗效果受限，难以成为SARS-COV-2的特效药的问题，本发明提供一种新型冠状病毒(SARS-COV-2)刺突蛋白结合分子及其应用。
为达到上述发明目的，本发明实施例采用了如下的技术方案：
一种新型冠状病毒(SARS-COV-2)刺突蛋白结合分子，能特异性结合SARS-COV-2刺突蛋白且包含至少一个免疫球蛋白单一可变结构域，所述免疫球蛋白单一可变结构域中的CDR1、CDR2和CDR3选自如下组合中的任意一组：
1)SEQ ID NO:1所示的CDR1，SEQ ID NO:2所示的CDR2和SEQ ID NO:3所示的CDR3；
2)SEQ ID NO:4所示的CDR1，SEQ ID NO:5所示的CDR2和SEQ ID NO:6所示的CDR3；
3)SEQ ID NO:7所示的CDR1，SEQ ID NO:8所示的CDR2和SEQ ID NO:9所示的CDR3；
4)SEQ ID NO:10所示的CDR1，SEQ ID NO:11所示的CDR2和SEQ ID NO:12所示的CDR3；
5)SEQ ID NO:13所示的CDR1，SEQ ID NO:14所示的CDR2和SEQ ID NO:15所示的CDR3；
6)SEQ ID NO:16所示的CDR1，SEQ ID NO:17所示的CDR2和SEQ ID NO:18所示的CDR3；
7)SEQ ID NO:19所示的CDR1，SEQ ID NO:20所示的CDR2和SEQ ID NO:21所示的CDR3；
8)SEQ ID NO:22所示的CDR1，SEQ ID NO:23所示的CDR2和SEQ ID NO:24所示的CDR3；
9)SEQ ID NO:25所示的CDR1，SEQ ID NO:26所示的CDR2和SEQ ID NO:27所示的CDR3；
10)SEQ ID NO:28所示的CDR1，SEQ ID NO:29所示的CDR2和SEQ ID NO:30所示的CDR3；
11)SEQ ID NO:31所示的CDR1，SEQ ID NO:32所示的CDR2和SEQ ID NO:33所示的CDR3；
12)SEQ ID NO:34所示的CDR1，SEQ ID NO:35所示的CDR2和SEQ ID NO:36所示的CDR3；
13)SEQ ID NO:37所示的CDR1，SEQ ID NO:38所示的CDR2和SEQ ID NO:39所示的CDR3；
14)SEQ ID NO:40所示的CDR1，SEQ ID NO:41所示的CDR2和SEQ ID NO:42所示的CDR3；
15)SEQ ID NO:43所示的CDR1，SEQ ID NO:44所示的CDR2和SEQ ID NO:45所示的CDR3；
16)SEQ ID NO:46所示的CDR1，SEQ ID NO:47所示的CDR2和SEQ ID NO:48所示的CDR3；
17)SEQ ID NO:49所示的CDR1，SEQ ID NO:50所示的CDR2和SEQ ID NO:51所示的CDR3；
18)SEQ ID NO:52所示的CDR1，SEQ ID NO:53所示的CDR2和SEQ ID NO:54所示的CDR3；
19)SEQ ID NO:55所示的CDR1，SEQ ID NO:56所示的CDR2和SEQ ID NO:57所示的CDR3；
20)SEQ ID NO:58所示的CDR1，SEQ ID NO:59所示的CDR2和SEQ ID NO:60所示的CDR3；
21)SEQ ID NO:61所示的CDR1，SEQ ID NO:62所示的CDR2和SEQ ID NO:63所示的CDR3；
22)SEQ ID NO:64所示的CDR1，SEQ ID NO:65所示的CDR2和SEQ ID NO:66所示的CDR3；
23)SEQ ID NO:67所示的CDR1，SEQ ID NO:68所示的CDR2和SEQ ID NO:69所示的CDR3；
24)SEQ ID NO:70所示的CDR1，SEQ ID NO:71所示的CDR2和SEQ ID NO:72所示的CDR3；
25)SEQ ID NO:73所示的CDR1，SEQ ID NO:74所示的CDR2和SEQ ID NO:75所示的CDR3；
26)SEQ ID NO:76所示的CDR1，SEQ ID NO:77所示的CDR2和SEQ ID NO:78所示的CDR3；
27)SEQ ID NO:79所示的CDR1，SEQ ID NO:80所示的CDR2和SEQ ID NO:81所示的CDR3。
相对于现有技术，本发明提供的SARS-COV-2刺突蛋白(SARS-COV-2-Spike蛋白)结合分子能够特异性的结合SARS-COV-2-Spike蛋白，并有效阻断SARS-COV-2-Spike蛋白与人体细胞ACE2受体的结合，进而阻断SARS-COV-2对细胞的感染过程，抑制SARS-COV-2的传染和扩增。且本发明提供的SARS-COV-2-Spike蛋白结合分子还具有与SARS-COV-2-Spike蛋白结合的特异性好，生物活性和稳定性高以及无毒副作用的特点。
优选的，所述免疫球蛋白单一可变域为单域抗体。
优选的，所述单域抗体包含与SEQIDNO:82-108中任一序列具有至少80％的序列相同性的氨基酸序列。
优选的，所述单域抗体包含与SEQIDNO:82-108中任一序列具有至少90％的序列相同性的氨基酸序列。
优选的，所述单域抗体包含与SEQ ID NO:82-108中任一序列具有至少99％的序列相同性的氨基酸序列。
所述单域抗体的氨基酸序列与SEQIDNO:82-108中任一项比包含一或多个氨基酸取代，优选保守氨基酸取代。
优选的，所述单域抗体包含SEQ ID NO:82-108中任意一种氨基酸序列。
优选的，所述的SARS-COV-2刺突蛋白结合分子还包含免疫球蛋白Fc区。
在本发明的SARS-COV-2刺突蛋白结合分子中包含免疫球蛋白Fc区可以使所述结合分子形成二聚体，同时进一步延长所述分子的体内半衰期。可用于本发明的Fc区可以来自不同亚型的免疫球蛋白，例如，IgG(IgG1、IgG2、IgG3或IgG4亚型)、IgA1、IgA2、IgD、IgE或IgM。
优选的，所述免疫球蛋白Fc区是人免疫球蛋白Fc区。
优选的，所述免疫球蛋白Fc区是人IgG1的Fc区。
优选的，所述免疫球蛋白Fc区的氨基酸序列为SEQIDNO:109。
优选的，包含SEQ ID NO:110-136中的至少一种氨基酸序列。
与上述Fc区融合后的结合分子，稳定性和生物活性进一步提高，并进一步降低了其与SARS-COV-2刺突蛋白结合的KD值。
优选的，所述SARS-COV-2刺突蛋白结合分子具有下述特征中的至少一种：
a、与SARS-COV-2刺突蛋白结合的KD值小于1×10-8M；
b、阻断SARS-COV-2与人体细胞受体ACE2的结合；
c、抑制SARS-COV-2的传染和扩增。
本发明还提供了编码所述SARS-COV-2刺突蛋白结合分子的核酸分子，所述核酸分子为RNA、DNA或cDNA，其可以通过人工合成的方式获得，也可从适合的天然来源加以分离获得。
本发明还提供了包含所述核酸分子及其表达调控原件的表达载体。该表达载体通常包含至少一种本发明提供的核酸分子，其可操作地连接至一个或多个适合的表达调控元件(启动子、增强子、终止子、整合因子、选择标记物、前导序列、报告基因等)。针对在特定宿主细胞中的表达对所述元件及其序列进行选择为本领域技术人员的常识。
本发明还提供了包含所述核酸分子并进行表达的宿主细胞。所述的宿主细胞为用于表达异源蛋白的细胞，包括细菌细胞、真菌细胞或哺乳动物细胞。
本发明还提供了获得所述的SARS-COV-2刺突蛋白结合分子的方法，包括：
a、在允许所述SARS-COV-2刺突蛋白结合分子表达的条件下培养上述宿主细胞；
b、从步骤a的培养物中收集由所述宿主细胞表达的SARS-COV-2刺突蛋白结合分子。
将特定的核酸分子重组到表达载体上并通过转化或转染方法进入宿主细胞中表达、选择标记物、诱导蛋白表达的方法、培养条件等在本领域中是已知的。同时蛋白结合分子的分离及纯化技术为本领域技术人员所公知。
本发明的SARS-COV-2刺突蛋白结合分子也可以通过本领域已知的其它产生蛋白质的方法获得，例如化学合成。
本发明还提供了一种免疫缀合物，包含与治疗性部分缀合的上述任一项所述的SARS-COV-2刺突蛋白结合分子。
本发明还提供了一种药物组合物，包含上述任一项所述的SARS-COV-2刺突蛋白结合分子和/或所述的免疫缀合物，以及药学上可接受的载体。
本发明所述的“药学上可接受的载体”包括生理学相容的任何溶剂、分散介质、包衣、抗细菌剂和抗真菌剂、等渗剂和吸收延迟剂等。该载体适合于静脉内、肌内、皮下、肠胃外、脊柱或表皮施用(如通过注射或输注)。根据施用途径，可将活性化合物即结合分子、免疫缀合物包裹于一种材料中，以保护该化合物免受可使该化合物失活的酸和其他天然条件的作用，为本领域技术人员公知。
本发明所述的药物组合物根据需要还可以包括其它佐剂和辅料等。
本发明还提供了所述药物组合物在制备治疗或预防新型冠状病毒病肺炎药物中的应用。
本发明还提供了一种用于检测SARS-COV-2的试剂盒，包含上述任一项所述的SARS-COV-2刺突蛋白结合分子。
本发明还提供了所述用于检测SARS-COV-2的试剂盒的使用方法，在上述任一项所述的SARS-COV-2刺突蛋白结合分子与SARS-COV-2刺突蛋白之间能够形成复合物的条件下，使检测样品和对照样品接触上述任一项所述的SARS-COV-2刺突蛋白结合分子，检测复合物的形成；通过所述检测样品与对照样品之间复合物形成的差异判断样品中SARS-COV-2的存在。
附图说明
图1是本发明实施例1中提取的总RNA的琼脂糖凝胶电泳图，其中，M：DNA marker2000，泳道1：总RNA；
图2是本发明实施例1中巢式PCR扩增单域抗体基因的Step1-PCR扩增产物的琼脂糖凝胶电泳图，其中，M：DNA marker 2000，泳道1：扩增产物；
图3是本发明实施例1中巢式PCR扩增单域抗体基因的Step2-PCR扩增产物的琼脂糖凝胶电泳图，其中，DNA marker 2000，泳道1和2：扩增产物；
图4是本发明实施例1中目的单域抗体基因和载体pHEN1的SfiI和Not1双酶切产物的琼脂糖凝胶电泳图，其中，DNA marker 2000，泳道1：pHEN1；泳道2：sfil/notI酶切后的pHEN1；泳道3：sfil/notI酶切后的单域抗体基因；
图5是本发明实施例1中测算文库***率的菌落PCR扩增产物的琼脂糖凝胶电泳图，其中，M：DNA marker 2000；泳道1-48：挑取的48个菌落；
图6是本发明实施例2中随天数变化治疗组与对照组恒河猴的病毒载量变化图。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白，以下结合实施例，对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅用以解释本发明，并不用于限定本发明。
为了更好的说明本发明实施例提供的，下面通过实施例做进一步的举例说明。
定义
除非另有指示或定义，否则所有所用术语均具有本领域中的通常含义，该含义将为本领域技术人员所了解。此外，除非另有说明，否则未具体详述的所有方法、步骤、技术及操作均可以且已经以本身已知的方式进行，该方式将为本领域技术人员所了解。
除非另有说明，否则可互换使用的术语“抗体”或“免疫球蛋白”在本文中无论是指重链抗体还是指常规4链抗体，均用作一般术语以包括全长抗体、其单个的链以及其所有部分、结构域或片段(包括但不限于抗原结合结构域或片段)。此外，本文所用的术语“序列”(例如在“免疫球蛋白序列”、“抗体序列”、“单一可变结构域序列”、“单域抗体序列”或“蛋白序列”等的术语中)一般应理解为既包括相关氨基酸序列，又包括编码所述序列的核酸序列或核苷酸序列，除非本文需要更限定的解释。
如本文所用的术语“免疫球蛋白可变结构域”是指基本上由本领域及下文中分别称为“框架区1”或“FR1”、“框架区2”或“FR2”、“框架区3”或“FR3”、及“框架区4”或“FR4”的四个“框架区”组成的免疫球蛋白结构域，其中所述框架区由本领域及下文中分别称为“互补决定区1”或“CDR1”、“互补决定区2”或“CDR2”、及“互补决定区3”或“CDR3”的三个“互补决定区”或“CDR”间隔开。因此，免疫球蛋白可变结构域的一般结构或序列可如下表示为：FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4。免疫球蛋白可变结构域因具有抗原结合位点而赋予抗体对抗原的特异性。
传统IgG抗体分子一般由轻链和重链组成，轻链包含1个可变区(VL)和1个恒定区(CL)，重链包含1个可变区(VH)和3个恒定区(CH1，CH2，CH3)。单域抗体(Single domainantibody，sdAb)，是指缺失抗体轻链而只有重链可变区的一类抗体，因其分子量小，也被称为纳米抗体(Nanobody)。单域抗体特异性结合表位而无需其他抗原结合结构域。单域抗体为由单一免疫球蛋白结构域形成的小型稳定及高效的抗原识别单元。
如本领域中对于单域抗体中的各CDR中的氨基酸残基的总数可能不同。
单域抗体中的氨基酸残基的总数将通常在110至120范围内，常常介于112与115之间。然而应注意较小及较长序列也可适于本发明所述的目的。
单域抗体及含有其的多肽的其他结构特性及功能性质可总结如下：
单域抗体(其已经天然“设计”以在不存在轻链可变结构域且不与轻链可变结构域相互作用的情况下与抗原功能性结合)可用作单一且相对较小的功能性抗原结合结构单元、结构域或多肽。此区分单域抗体与常规4链抗体的VH及VL结构域，这些VH及VL结构域自身通常不适于作为单一抗原结合蛋白或免疫球蛋白单一可变结构域进行实际应用，但需要以某种形式或另一形式组合以提供功能性抗原结合单元(如以诸如Fab片段等常规抗体片段的形式；或以由与VL结构域共价连接的VH结构域组成的scFv的形式)。
由于上述这些独特性质，使用单域抗体或作为较大多肽的一部分提供许多优于使用常规VH及VL结构域、scFv或常规抗体片段(例如Fab-或F(ab’)2-片段)的显著优势，例如，单域抗体仅需要单一结构域以高亲和力及高选择性结合抗原，从而使得既不需要存在两个单独结构域，也不需要确保该两个结构域以适当空间构象及构型存在(例如scFv一般需要使用经特别设计的接头)；单域抗体可自单一基因表达且不需要翻译后折叠或修饰；单域抗体可容易地改造成多价及多特异性格式；单域抗体高度可溶且无聚集趋势；单域抗体对热、pH、蛋白酶及其他变性剂或条件高度稳定，且因此可在制备、储存或运输中不使用冷冻设备，从而达成节约成本、时间及环境；单域抗体易于制备且相对廉价，甚至在生产所需的规模上亦如此；单域抗体与常规4链抗体及其抗原结合片段相比相对较小(大约15kDa或大小为常规IgG的1/10)，因此相比于常规4链抗体及其抗原结合片段，显示较高的组织渗透性且可以较高剂量给药；单域抗体可显示所谓腔结合性质(尤其由于与常规VH结构域相比其延长的CDR3环)，从而可到达常规4链抗体及其抗原结合片段不可到达的靶及表位。
获得结合特定抗原或表位的单域抗体的方法，先前已公开于以下文献中：R.vanderLindenetal.,JournalofImmunologicalMethods,240(2000)185–195；Lietal.,JBiolChem.,287(2012)13713–13721；Deffaretal.,AfricanJournalofBiotechnologyVol.8(12),pp.2645-2652,17June,2009和WO94/04678。
此外，本领域技术人员还将了解，有可能将一个或多个上述CDR“移植”于其他“支架”(包括但不限于人支架或非免疫球蛋白支架)上。适于所述CDR移植的支架及技术在本领域中是已知的。
一般而言，术语“特异性”是指特定抗原结合分子或抗原结合蛋白(例如本发明的免疫球蛋白单一可变结构域)分子可结合的不同类型抗原或表位的数目。可基于抗原结合分子的亲和力和/或亲合力确定其特异性。由抗原与抗原结合蛋白的解离平衡常数(KD)所表示的亲和力，是表位与抗原结合蛋白上抗原结合位点之间结合强度的量度：KD值越小，表位与抗原结合分子之间的结合强度越强(或者，亲和力也可表示为缔合常数(KA)，其为1/KD)。如本领域技术人员将了解，取决于具体感兴趣的抗原，可以以已知方式测定亲和力。亲合力为抗原结合分子(例如免疫球蛋白、抗体、免疫球蛋白单一可变结构域或含有其的多肽)与相关抗原之间结合强度的量度。亲合力与以下两者有关：与其抗原结合分子上的抗原结合位点之间的亲和力，以及存在于抗原结合分子上的相关结合位点的数目。
发明所用术语“SARS-COV-2刺突蛋白结合分子(SARS-COV-2-Spike蛋白结合分子)”意指任何能够特异性结合SARS-COV-2刺突蛋白的分子。SARS-COV-2刺突蛋白结合分子可以包括针对SARS-COV-2刺突蛋白的如本发明定义的单域抗体或其缀合物。SARS-COV-2刺突蛋白结合分子还涵盖所谓的“SMIP”(“小模块免疫药物”)，或者免疫球蛋白超家族抗体(IgSF)或CDR移植分子。
本发明的“SARS-COV-2刺突蛋白结合分子”可以包含至少一个结合SARS-COV-2刺突蛋白的免疫球蛋白单一可变结构域如单域抗体。在一些实施方案中，本发明的“SARS-COV-2刺突蛋白结合分子”可以包含两个结合SARS-COV-2刺突蛋白的免疫球蛋白单一可变结构域如单域抗体。含有一个以上的免疫球蛋白单一可变结构域的SARS-COV-2刺突蛋白结合分子亦称为“格式化的”SARS-COV-2刺突蛋白结合分子。格式化的SARS-COV-2刺突蛋白结合分子除结合SARS-COV-2刺突蛋白的免疫球蛋白单一可变结构域外也可包含接头和/或具有效应器功能的部分，例如半衰期延长部分(如结合血清白蛋白的免疫球蛋白单一可变结构域)、和/或融合配偶体(如血清白蛋白)和/或缀合的聚合物(如PEG)和/或Fc区。本发明的“SARS-COV-2刺突蛋白结合分子”还涵盖双特异性抗体，其含有结合不同抗原的免疫球蛋白单一可变结构域。
通常，本发明的SARS-COV-2刺突蛋白结合分子将以如于Biacore或Kin ExA测定中测量的优选10-8至10-12摩尔/升(M)、更优选10-9至10-11摩尔/升、甚至更优选10-10至10-12、甚至更优选10-11至10-12或更低的解离常数(KD)。任何大于10-4M的KD值一般都视为指示非特异性结合。抗原结合蛋白对抗原或表位的特异性结合可以以已知的任何适合方式来测定，包括例如本文所述的表面等离子体共振术(SPR)测定、和/或竞争性结合测定(例如酶免疫测定(EIA)及夹心式竞争性测定。
氨基酸残基将根据如本领域中公知且达成一致的标准三字母或一字母氨基酸编码加以表示。所述保守氨基酸取代在本领域中是公知的，例如保守氨基酸取代优选是以下组(1)-(5)内的一个氨基酸被同一组内的另一氨基酸残基所取代：(1)较小脂族非极性或弱极性残基：Ala、Ser、Thr、Pro及Gly；(2)极性带负电残基及其(不带电)酰胺：Asp、Asn、Glu及Gln；(3)极性带正电残基：His、Arg及Lys；(4)较大脂族非极性残基：Met、Leu、Ile、Val及Cys；及(5)芳族残基：Phe、Tyr及Trp。特别优选的保守氨基酸取代如下：Ala被Gly或Ser取代；Arg被Lys取代；Asn被Gln或His取代；Asp被Glu取代；Cys被Ser取代；Gln被Asn取代；Glu被Asp取代；Gly被Ala或Pro取代；His被Asn或Gln取代；Ile被Leu或Val取代；Leu被Ile或Val取代；Lys被Arg、Gln或Glu取代；Met被Leu、Tyr或Ile取代；Phe被Met、Leu或Tyr取代；Ser被Thr取代；Thr被Ser取代；Trp被Tyr取代；Tyr被Trp或Phe取代；Val被Ile或Leu取代。
两个多肽序列之间的“序列相同性”指示序列之间相同氨基酸的百分比。用于评价氨基酸或核苷酸之间的序列相同性程度的方法是本领域技术人员已知的。例如，氨基酸序列相同性通常使用序列分析软件来测量。例如，可使用NCBI数据库的BLAST程序来确定相同性。对于序列相同性的确定，可以参见例如：SequenceAnalysisinMolecularBiology,vonHeinje,G.,AcademicPress,1987和SequenceAnalysisPrimer,Gribskov,M.andDevereux,J.,eds.,MStocktonPress,NewYork,1991。
相比于其天然生物来源和/或获得该多肽或核酸分子的反应介质或培养基，当其已与至少一种在该来源或介质(培养基)中通常与之相关的其他组分(例如另一蛋白/多肽、另一核酸、另一生物组分或大分子或至少一种污染物、杂质或微量组分)分离时，多肽或核酸分子视为“基本上分离的”。特别地，多肽或核酸分子在其已纯化至少2倍、特别是至少10倍、更特别是至少100倍且多达1000倍或1000倍以上时被视为“基本上分离的”。经适合的技术(例如适合色谱技术，如聚丙烯酰胺凝胶电泳)确定，“基本上分离的”多肽或核酸分子优选基本上为均质的。
实施例1
筛选针对SARS-COV-2-Spike蛋白的单域抗体
1.1文库的构建
1.1.1免疫
用新型冠状病毒的Spike-RBD蛋白免疫羊驼，并分别在第1、2、4、6周免疫，共免疫4次，每次免疫剂量为200ug。
1.1.2提取总RNA
取第6周免疫后的羊驼外周血50ml，分离淋巴细胞，用Trizol提取淋巴细胞的总RNA，采用紫外分光光度计检测提取的RNA的结果为：OD260/280＝1.99，OD260/230＝1.43，说明提取的RNA没有明显降解，纯度较好；总RNA浓度为809.3ng/μL。用提取的总RNA进行琼脂糖凝胶电泳，结果如图1所示，可以看到28S和18S两条条带。
1.1.3RNA反转录
RNA反转录体系如下：
Step 1：
Figure BDA0002521253740000131
混匀后，65℃保温5min，迅速冰浴；
Step 2
Figure BDA0002521253740000132
混匀后，进行反转录得到cDNA，反转录条件为：42℃，30min；50℃，15min；70℃，15min。
1.1.4单域抗体(VHH)基因扩增
采用巢式PCR扩增VHH基因，方法如下：
Step1
Figure BDA0002521253740000133
Figure BDA0002521253740000141
混匀后，进行PCR反应，反应条件：98℃10s，50℃30s，72℃1min，共20个循环。扩增引物的序列为：Primer For-1:5′-GTCCTGGCTGCTCTTCTACAAGG-3′(SEQIDNO:138)；PrimerRev-1:5′-GGTACGTGCTGTTGAACTGTTCC-3′(SEQIDNO:139)。
PCR产物经DNA纯化试剂盒纯化浓缩后，进行琼脂糖凝胶电泳，得到的琼脂糖凝胶电泳图如图2所示，采用DNA产物凝胶回收试剂盒回收750bp条带，紫外分光光度计定量；
Step 2
Figure BDA0002521253740000142
混匀后，进行PCR反应，反应条件：98℃10s，55℃30s，72℃30s，共20个循环。扩增引物的序列为：Primer For-2:5′-CTAGTGCGGCCGCTGGAGACGGTGACCTGGGT-3′(SEQIDNO:140)；Primer Rev-2:5′-GATGTGCAGCTGCAGGAGTCTGGRGGAGG-3′(SEQIDNO:141)。
得到的PCR产物进行琼脂糖凝胶电泳，琼脂糖凝胶电泳图如图3所示，采用DNA产物凝胶回收试剂盒回收，紫外分光光度计定量。最终得到约500bp的目的基因(VHH)200μL，浓度为400ng/μL。
1.1.5文库转化
将得到的目的基因和载体pHEN1采用SfiI和Not1进行双酶切，将酶切产物进行琼脂糖凝胶电泳，结果如图4所示，将酶切后的目的基因和pHEN1采用T4DNA连接酶连接后，转化至大肠杆菌电转感受态细胞TG1中，构建针对SARS-COV-2-Spike蛋白的单域抗体基因文库，命名为J2-Lib。共转化15次，混合后均匀涂布于6块Ф150mm的培养皿(含氨苄青霉素的LB固体培养基)中。
分别取0.1μL，0.01μL，0.001μL和0.0001μL混合后的转化液均匀涂布于Ф90mm的培养皿(含氨苄青霉素的LB固体培养基)，用于文库库容量的计算(以菌落数为30-300的平板为准计数)，如表1所示，计算库容量为1.395×108cfu。
表1
Figure BDA0002521253740000151
在上述用于计算库容量的培养皿中随机挑选48个菌落，进行菌落PCR，并将PCR产物进行琼脂糖凝胶电泳，测算文库的目的基因***率，琼脂糖凝胶电泳如图5所示，说明文库***率为100％，文库实际库容量为1.395×108cfu。
菌落PCR体系如下：
Figure BDA0002521253740000152
菌落PCR反应条件为：98℃10s，50℃30s，72℃1min，共30个循环。
1.1.6文库救援
从上述J2-Lib基因文库中取10-100倍库容量的活细胞进行接种培养，培养至对数期后采用M13K07噬菌体进行救援，救援培养后，离心收集噬菌体，采用PEG-NaCl纯化噬菌体，即得噬菌体展示文库，命名为J2-PDL，滴度为3.4×1013cfu/mL。可直接用于后续特异性噬菌体的亲和筛选。
1.2针对SARS-COV-2-Spike蛋白单域抗体的筛选
用Spike-RBD蛋白(刺突蛋白受体结合区蛋白)3μg/孔包被平板，4℃放置过夜；用1wt％脱脂奶粉室温封闭2h，加入100μl噬菌体(8×1011tfu，来自1.1.6所构建的噬菌体展示文库J2-PDL)，在室温下作用1h。之后用PBST(PBS中含有0.05vt％吐温20)洗脱5遍，以洗掉不结合的噬菌体；用三乙基胺(100mM)将与Spike-RBD蛋白特异性结合的噬菌体解离下，并感染处于对数期生长的大肠杆菌TG1，产生并纯化噬菌体用于下一轮的筛选。相同筛选过程重复3-4轮。由此，阳性的克隆被富集，达到了利用噬菌体展示文库筛取抗体库中Spike-RBD蛋白特异抗体的目的。并将获得的阳性的噬菌体进行测序，获得抗体基因序列。
将获得的抗体基因序列分别构建在pcDNA3.4载体上，用HEK-293细胞表达抗体，用proteinA介质纯化收集培养基上清中的抗体。纯化后的抗体与包被Spike-RBD的板孵育进行ELISA测定。获得可特异性结合Spike-RBD蛋白的抗体。
根据序列比对软件VectorNTI分析获得的抗体序列。把CDR1、CDR2、CDR3序列均相同的克隆视为同一抗体株，而CDR序列不同的克隆视为不同抗体株。共获得27个不同的单域抗体株，单域抗体序列如SEQIDNO:82-108，分别携带SEQ ID NO:1-81中的27组CDR1-3序列，具体如表2所示：
表2
单域抗体 CDR1 CDR2 CDR3
COV2-114-1 SEQ ID NO:1 SEQIDNO:2 SEQIDNO:3
COV2-187-2 SEQ ID NO:4 SEQIDNO:5 SEQIDNO:6
COV2-360-3 SEQIDNO:7 SEQIDNO:8 SEQIDNO:9
COV2-143-4 SEQIDNO:10 SEQIDNO:11 SEQIDNO:12
COV2-13-5 SEQIDNO:13 SEQIDNO:14 SEQIDNO:15
COV2-356-6 SEQIDNO:16 SEQIDNO:17 SEQIDNO:18
COV2-115-7 SEQIDNO:19 SEQIDNO:20 SEQIDNO:21
COV2-22-8 SEQIDNO:22 SEQIDNO:23 SEQIDNO:24
COV2-116-9 SEQIDNO:25 SEQIDNO:26 SEQIDNO:27
COV2-122-10 SEQIDNO:28 SEQIDNO:29 SEQIDNO:30
COV2-171-11 SEQIDNO:31 SEQIDNO:32 SEQIDNO:33
COV2-136-12 SEQIDNO:34 SEQIDNO:35 SEQIDNO:36
COV2-237-13 SEQIDNO:37 SEQIDNO:38 SEQIDNO:39
COV2-353-14 SEQIDNO:40 SEQIDNO:41 SEQIDNO:42
COV2-121-15 SEQIDNO:43 SEQIDNO:44 SEQIDNO:45
COV2-324-16 SEQIDNO:46 SEQIDNO:47 SEQIDNO:48
COV2-120-17 SEQIDNO:49 SEQIDNO:50 SEQIDNO:51
COV2-153-18 SEQIDNO:52 SEQIDNO:53 SEQIDNO:54
COV2-39-19 SEQIDNO:55 SEQIDNO:56 SEQIDNO:57
COV2-124-20 SEQIDNO:58 SEQIDNO:59 SEQIDNO:60
COV2-18-21 SEQIDNO:61 SEQIDNO:62 SEQIDNO:63
COV2-119-22 SEQIDNO:64 SEQIDNO:65 SEQIDNO:66
COV2-151-23 SEQIDNO:67 SEQIDNO:68 SEQIDNO:69
COV2-214-24 SEQIDNO:70 SEQIDNO:71 SEQIDNO:72
COV2-331-25 SEQIDNO:73 SEQIDNO:74 SEQIDNO:75
COV2-32-26 SEQIDNO:76 SEQIDNO:77 SEQIDNO:78
COV2-336-27 SEQIDNO:79 SEQIDNO:80 SEQIDNO:81
。
1.3针对SARS-COV-2-Spike蛋白的单域抗体的评价鉴定
1.3.1单域抗体在宿主菌大肠杆菌中表达、纯化
将获得的27个不同CDR1-3的单域抗体的基因编码序列分别重组至表达载体PET32b(Novagen，产品号：69016-3)中，并将测序鉴定正确的重组质粒分别转化到表达型宿主菌BL1(DE3)(天根生化科技，产品号：CB105-02)中，将其涂布在含有100μg/mL的氨苄青霉素的LB平板上，37℃过夜。挑选单菌落接种、培养过夜，第二天将过夜菌种转接扩增，37℃摇床培养至OD值达到0.5-1时，加入0.5mM IPTG诱导，28℃摇床培养过夜。第二天，离心收集菌体，并将收集的菌体破碎获得抗体粗提液。然后纯化27株单域抗体蛋白，使其纯度达到90％以上。
1.3.2竞争ELISA考察SARS-COV-2-Spike蛋白单域抗体对Spike-RBD蛋白与受体ACE2结合的阻断效果
先通过HEK293细胞(pCDNA4，Invitrogen，Cat V86220)表达获得Spike-RBD蛋白与ACE2蛋白。再利用Thermo公司的Biotinlytion试剂盒，得到生物素化的ACE2蛋白。
用Spike-RBD蛋白0.5μg/孔，4℃过夜包被平板，之后每孔加入100ng的1.3.1纯化所得的单域抗体以及5μg生物素化的ACE2蛋白，并设置对照组，对照组1的孔中不加入单域抗体，对照组2的孔中不加入生物素化的ACE2蛋白，室温下反应2h。之后加入SA-HRP(购自Sigma公司)，室温反应1小时后加入显色液，450nm波长读取吸收值。当样品OD值比对照OD值<0.8时，则认为单域抗体有阻断效果。
结果如表3所示，出现27个不同的单域抗体株表现出对Spike蛋白/ACE2蛋白相互作用的阻断效应。
表3
Figure BDA0002521253740000191
Figure BDA0002521253740000201
在生物安全级别为P3的实验室，通过用病毒感染VERO细胞模型，将纯化得到的27株单域抗体分别加入到培养体系中，具体操作为：将104/孔VERO细胞加到96孔板，24小时后，用PBS洗细胞2次，将27株单域抗体分别与病毒混合加入96孔板，抗体初始浓度为100μg/mL，再分别2倍稀释10个梯度，5个复孔，37度孵育2小时，第5天检测VERO细胞是否发生病变。如果单域抗体处理后细胞不发生病变说明单域抗体具有中和病毒阻断病毒感染VERO细胞的效果。
检测结果显示，经上述27株单域抗体处理后VERO细胞均未发生病变，而只加入病毒未加入单域抗体的VERO细胞大部分病变和死亡，说明所获得的27株单域抗体全部能够阻断病毒感染细胞的过程，显示其为有效的中和抗体。
实施例2
1.1制备SARS-COV-2-Spike蛋白单域抗体的Fc融合蛋白
根据蛋白数据库Uniprot上人免疫球蛋白(IgG1)的恒定区氨基酸序列，得到人IgG1-Fc区氨基酸序列(SEQIDNO:109)。通过逆转录PCR，从人PBMC总RNA中获得编码人IgG1-Fc的核酸片段(核酸序列如SEQIDNO:137)，再通过overlappingPCR得到SARS-COV-2-Spike蛋白单域抗体与Fc的融合蛋白的编码核酸片段，并重组至载体pCDNA4(Invitrogen，CatV86220)。
将构建成功的含有SARS-COV-2-Spike蛋白单域抗体与Fc的融合蛋白的核酸片段的pCDNA4质粒转染HEK293细胞进行表达。具体是将重组表达质粒用Freestyle293培养基稀释并加入转化所需PEI(Polyethylenimine)溶液，将质粒/PEI混合物分别加入HEK293细胞悬液中，放置在37℃，10％的CO2，100rpm的摇床中培养；补加50μg/L IGF-1。4h后补加EX293培养基、2mM谷氨酰胺和50μg/L IGF-1，120rpm摇培。24h后加3.8mM VPA。培养5天后，收集表达培养上清液，通过Protein A亲和层析法，纯化得到SARS-COV-2-Spike蛋白单域抗体与Fc的融合蛋白。
获得的27个SARS-COV-2-Spike蛋白单域抗体与Fc的融合蛋白的序列如SEQIDNO:110-136。
1.2鉴定SARS-COV-2-Spike蛋白单域抗体与Fc的融合蛋白的功能
通过SPR法鉴定SARS-COV-2-Spike蛋白单域抗体与Fc融合蛋白对SARS-COV-2-Spike蛋白的结合能力。具体操作是：将获得的27株SARS-COV-2-Spike蛋白单域抗体与Fc的融合蛋白针对spike-RBD的结合动力学通过表面等离振子共振(SRP)方法，使用BIAcoreX100仪器测量，将spike-RBD蛋白直接包被于CM5生物传感器芯片上以获得大约1000应答单位(response units，RU)。对于动力学测量，将SARS-COV-2-Spike蛋白单域抗体与Fc的融合蛋白用HBS-EP+1×缓冲液(GE，cat#BR-1006-69)三倍连续稀释(1.62nm至1000nm)，在25℃进样120s，解离时间为30min，加入10mM甘氨酸-HCl(pH2.0)再生120s。使用简单一对一Languir结合模型(BIAcore Evaluation Software version 3.2)计算出融合蛋白与SARS-COV-2-Spike蛋白的结合速率(kon)、解离速率(koff)和平衡解离常数(kD)(以比率koff/kon计算)。计算结果如表4所示。
表4
Figure BDA0002521253740000211
Figure BDA0002521253740000221
由表4可知，SARS-COV-2-Spike蛋白单域抗体与Fc融合蛋白对SARS-COV-2-Spike蛋白的结合速率较高的，解离速率较低，平衡解离常数KD小于1.05E-9，说明融合蛋白能更快速的结合SARS-COV-2-Spike蛋白并很难解离下来，进一步说明SARS-COV-2-Spike蛋白单域抗体与Fc融合蛋白作为一个阻断型抗体，具有极佳的阻断效果。
1.3通过竞争ELISA法鉴定SARS-COV-2-Spike蛋白单域抗体与Fc融合蛋白对Spike蛋白/ACE2的相互作用的阻断能力
利用HEK293细胞表达获得ACE2蛋白。利用Thermo公司的Biotinlytion试剂盒，得到生物素化的蛋白ACE2-Biotin。
用Spike-RBD蛋白0.5μg/孔4℃过夜包被平板，之后每孔加入获得的27株SARS-COV-2-Spike蛋白单域抗体与Fc融合蛋白200ng和ACE2-Biotin5ug，对照组1中不加入融合蛋白，对照组2中不加入ACE2-Biotin，室温下反应2h。洗涤之后加入SA-HRP(购自Sigma公司)，室温反应1小时，洗涤之后加入显色液，450nm波长读取吸收值。结果如表5所示：
表5
Figure BDA0002521253740000231
Figure BDA0002521253740000241
结果显示，SARS-COV-2-Spike蛋白单域抗体与Fc融合蛋白能有效阻断Spike蛋白与ACE2之间的相互作用。
1.4分析SARS-COV-2-Spike蛋白单域抗体与Fc融合蛋白对Spike蛋白结合的特异性
利用人HEK293细胞通过瞬时转染，获得带有目前已知的7种冠状病毒(SARS-COV-2、HCoV-229E、HCoV-OC43、HCoV-NL63、HCoV-HKU1、SARS-CoV、MERS-CoV)Spike蛋白全长基因的质粒(pCDNA4,Invitrogen，Cat V86220)，并于膜上瞬时表达Spike蛋白。该质粒使得Spike蛋白C端融合EGFP蛋白，从而可以通过绿色荧光强度来考察膜上Spike蛋白的表达水平。将构建好的细胞重悬于0.5％的PBS-BSA Buffer中，加入SARS-COV-2-Spike蛋白单域抗体与Fc融合蛋白，同时设置阴性对照，冰上孵育20min。洗涤后加入eBioscience二抗anti-hIg-PE，冰上20min。洗涤后将细胞重悬于500μl的0.5％PBS-BSABuffer中，流式细胞仪进行检测。结果显示，27株SARS-COV-2-Spike蛋白单域抗体-Fc融合蛋白只特异性结合SARS-COV-2-Spike蛋白，而不与其他冠状病毒的Spike蛋白结合。
1.5SARS-COV-2-Spike蛋白单域抗体与Fc融合蛋白阻断SARS-COV-2感染细胞
将提前给药(SARS-COV-2-Spike蛋白单域抗体与Fc融合蛋白100μg/恒河猴)的15只健康恒河猴和15只未提前给药的健康恒河猴与感染SARS-COV-2的2只恒河猴在同一环境中喂养10天。10天后观察发现提前给药的15只健康恒河猴均未出现异常，病毒核酸检测均为阴性；而15只未提前给药的健康恒河猴有12只出现感染症状，经病毒核酸检测均呈阳性。
上述试验结果说明本发明提供的SARS-COV-2-Spike蛋白单域抗体能够完全阻断新冠病毒感染，具有优良的预防作用。
将上述感染SARS-COV-2病毒并出现症状的12只恒河猴中的6只(治疗组)利用本发明提供的SARS-COV-2-Spike蛋白单域抗体与Fc融合蛋白(SARS-COV-2-Spike蛋白单域抗体与Fc融合蛋白100μg/恒河猴)进行给药治疗，另外6只(对照组)不进行给药治疗。在治疗后的6天内，每天进行一次病毒载量的检测。经检测，治疗组的6只恒河猴每毫升血液中新冠病毒的平均载量相比对照组显著降低，如图6所示。其中新冠病毒的载量的检测过程为：分别采取给药治疗的恒河猴(治疗组)和未进行给药治疗的恒河猴(对照组)的血液，提取血液中的病毒的核酸进行检测，检测过程为：采用RNA提取试剂盒(Qiagen)按照说明书操作提取SARS-COV-2的RNA，将获得的RNA溶解在50μL洗脱buffer并作为模板进行RT-PCR扩增。以RT-PCR产物为模板，用引物RBD-qF1(5′-CAATGGTTTAACAGGCACAGG-3′，SEQIDNO:142)和RBD-qR1(5′-CTCAAGTGTCTGTGGATCACG-3′，SEQIDNO:143)扩增病毒S区基因。采用HiScriptR II OneStep qRT-PCR SYBRRGreen Kit(Vazyme Biotech Co.,Ltd)试剂盒，根据试剂盒说明书进行操作，设置PCR扩增条件为；50℃3min，95℃10s，60℃30s，40个循环，所用PCR扩增仪为ABI定量PCR仪。
上述体内实验结果表明，本发明SARS-COV-2-Spike蛋白单域抗体与Fc融合蛋白在对感染SARS-COV-2的恒河猴体内表现出显著的抑制SARS-COV-2感染细胞并扩增的效果。
1.6SARS-COV-2-Spike蛋白单域抗体与Fc融合蛋白稳定性研究
使用500mM碳酸氢铵作为碱性破坏试剂，37℃处理48h。选择1％双氧水作为氧化剂，室温处理10h。
使用竞争ELISA法检测处理前后的上述27种SARS-COV-2-Spike蛋白单域抗体与Fc融合蛋白的生物学活性变化。48h碱处理后的竞争ELISA活性相对碱处理0小时平均为107％；10h氧化后的竞争ELISA活性相对氧化0h平均为111％。
说明本发明提供的27种SARS-COV-2-Spike蛋白单域抗体与Fc融合蛋白具有较高的稳定性。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内所作的任何修改、等同替换或改进等，均应包含在本发明的保护范围之内。
SEQUENCE LISTING
<110> 深圳市因诺赛生物科技有限公司
<120> 新型冠状病毒（SARS-COV-2）刺突蛋白结合分子及其应用
<130> 2020
<160> 143
<170> PatentIn version 3.5
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Ile Thr Lys His Gly Gly Thr Thr
1 5
<210> 9
<211> 20
<212> PRT
<213> CDR3
<400> 9
Ala Ala Asp His Arg Ala Leu Arg Phe Gly Cys Ala Val Val Pro Gly
1 5 10 15
Pro Tyr Asp Tyr
20
<210> 10
<211> 8
<212> PRT
<213> CDR1
<400> 10
Gly Leu Thr Leu Asn Asn Asn Thr
1 5
<210> 11
<211> 8
<212> PRT
<213> CDR2
<400> 11
Ile Ser Pro Ser Gly Ile Val Thr
1 5
<210> 12
<211> 16
<212> PRT
<213> CDR3
<400> 12
Ala Val Leu Gln Trp Asn Gly Ala Gly Leu Pro Ser Pro Leu Gly Ser
1 5 10 15
<210> 13
<211> 8
<212> PRT
<213> CDR1
<400> 13
Gly Phe Thr Leu Gly Asn Tyr Glu
1 5
<210> 14
<211> 8
<212> PRT
<213> CDR2
<400> 14
Ile Ser Arg Ser Gly Val Ile Phe
1 5
<210> 15
<211> 22
<212> PRT
<213> CDR3
<400> 15
Ala Ala Arg Phe Arg Ala Gly Leu Val Gln Ser Leu Cys Gln Ser Thr
1 5 10 15
Pro Glu Phe Tyr Gln Asp
20
<210> 16
<211> 8
<212> PRT
<213> CDR1
<400> 16
Gly Phe Thr Leu Gly Asn Tyr Ala
1 5
<210> 17
<211> 8
<212> PRT
<213> CDR2
<400> 17
Ile Ser Leu Ser Gly Asp Ile Ile
1 5
<210> 18
<211> 22
<212> PRT
<213> CDR3
<400> 18
Ala Ala Pro Phe Pro Ser Leu Leu Tyr Cys Ser Leu Cys Pro Ser Asn
1 5 10 15
Pro Glu Phe Cys Arg His
20
<210> 19
<211> 8
<212> PRT
<213> CDR1
<400> 19
Gly Phe Ser Leu Asp Gly Tyr Ala
1 5
<210> 20
<211> 8
<212> PRT
<213> CDR2
<400> 20
Ile Gly Arg Leu Gly Thr Thr Thr
1 5
<210> 21
<211> 18
<212> PRT
<213> CDR3
<400> 21
Ala Ala Lys Arg Ser Thr Trp Phe Gly Cys Ser Thr Leu Asp Asp Tyr
1 5 10 15
Asp Tyr
<210> 22
<211> 8
<212> PRT
<213> CDR1
<400> 22
Gly Phe Thr Leu Asp Arg Tyr Ala
1 5
<210> 23
<211> 7
<212> PRT
<213> CDR2
<400> 23
Ile Ser Ser Gly Gly Thr Thr
1 5
<210> 24
<211> 19
<212> PRT
<213> CDR3
<400> 24
Ala Ala Gly Thr Gln Leu Arg Tyr Gly Cys Ser Arg Gln Ala Arg Arg
1 5 10 15
Tyr Asp Tyr
<210> 25
<211> 8
<212> PRT
<213> CDR1
<400> 25
Gly Phe Thr Leu Asp Tyr Tyr Ala
1 5
<210> 26
<211> 8
<212> PRT
<213> CDR2
<400> 26
Ile Ser Ser Ser Gly Thr Ser Thr
1 5
<210> 27
<211> 19
<212> PRT
<213> CDR3
<400> 27
Ala Ala Leu Gln Gly Ser Tyr Tyr Tyr Thr Ser Leu His Gln Thr Glu
1 5 10 15
Tyr Asp Tyr
<210> 28
<211> 8
<212> PRT
<213> CDR1
<400> 28
Gly Ala Thr Ser Asp Ile Tyr Ala
1 5
<210> 29
<211> 8
<212> PRT
<213> CDR2
<400> 29
Ile Ser Pro Ser Gly Gly Ser Thr
1 5
<210> 30
<211> 19
<212> PRT
<213> CDR3
<400> 30
Ala Ala Ala Ser Pro Ser Trp His Tyr Cys Ser Gly Tyr Glu His Glu
1 5 10 15
Tyr Asp Asp
<210> 31
<211> 8
<212> PRT
<213> CDR1
<400> 31
Gly Phe Thr Leu Asp Tyr Tyr Ala
1 5
<210> 32
<211> 8
<212> PRT
<213> CDR2
<400> 32
Ile Ser Pro Ser Gly Thr Ser Thr
1 5
<210> 33
<211> 19
<212> PRT
<213> CDR3
<400> 33
Ala Ala Ala Asn Pro Ser Tyr Tyr Tyr Cys Ser Gly Tyr Pro His Glu
1 5 10 15
Tyr Asp Asp
<210> 34
<211> 8
<212> PRT
<213> CDR1
<400> 34
Gly Phe Thr Phe His Tyr Tyr Ala
1 5
<210> 35
<211> 8
<212> PRT
<213> CDR2
<400> 35
Ile Ser Ser Gly Gly Gly Arg Thr
1 5
<210> 36
<211> 19
<212> PRT
<213> CDR3
<400> 36
Ala Ala Leu Gln Gly Ser Leu His Tyr Cys Ser Gly Leu Val Gly Glu
1 5 10 15
Tyr Glu Tyr
<210> 37
<211> 8
<212> PRT
<213> CDR1
<400> 37
Phe Lys Ser Tyr His Ile Ala Trp
1 5
<210> 38
<211> 8
<212> PRT
<213> CDR2
<400> 38
Ser Leu Thr Gly Ala Ser Ile Thr
1 5
<210> 39
<211> 20
<212> PRT
<213> CDR3
<400> 39
Ala Ala Arg Asp Tyr Ala Ser Ser Leu Tyr Cys Ser Arg Asn Glu Ala
1 5 10 15
Met Tyr Ala Tyr
20
<210> 40
<211> 8
<212> PRT
<213> CDR1
<400> 40
Gly Phe Arg Leu Gly Gly Tyr Ala
1 5
<210> 41
<211> 8
<212> PRT
<213> CDR2
<400> 41
Ile Ser Gly Ser Gly Ile Ile Ser
1 5
<210> 42
<211> 24
<212> PRT
<213> CDR3
<400> 42
Ala Ala Pro Ser Gly Ser Ser Tyr Gly Ser Arg Arg Leu Pro Cys Gly
1 5 10 15
Ile Asp Ser Gln Asp Tyr Gln Thr
20
<210> 43
<211> 8
<212> PRT
<213> CDR1
<400> 43
Phe Asp Asp Phe Ala Ile Gly Trp
1 5
<210> 44
<211> 8
<212> PRT
<213> CDR2
<400> 44
Ser Ala Arg Gly Ile Val Asn Thr
1 5
<210> 45
<211> 20
<212> PRT
<213> CDR3
<400> 45
Ala Ala Leu Val Gly Leu Asn Asp Arg Ser Leu Pro His Glu Ala Glu
1 5 10 15
Glu Tyr Asp Tyr
20
<210> 46
<211> 8
<212> PRT
<213> CDR1
<400> 46
Gly Phe Thr Phe Asp Asp Tyr Ser
1 5
<210> 47
<211> 8
<212> PRT
<213> CDR2
<400> 47
Phe Ser Ser Leu Asp Gly Ser Thr
1 5
<210> 48
<211> 18
<212> PRT
<213> CDR3
<400> 48
Ala Ala Thr Arg Gly Ser Ser Trp Tyr Trp Tyr Arg Pro Ser Glu Tyr
1 5 10 15
Asp Tyr
<210> 49
<211> 8
<212> PRT
<213> CDR1
<400> 49
Gly His Thr Gly Ser Thr Tyr Ile
1 5
<210> 50
<211> 8
<212> PRT
<213> CDR2
<400> 50
Ile Ser Arg Ser Gly Ile Leu Arg
1 5
<210> 51
<211> 19
<212> PRT
<213> CDR3
<400> 51
Ala Ala Arg Asp Ser Leu Thr Gly Val Ile Gly Asn Thr Pro Gly Glu
1 5 10 15
Tyr Asp Tyr
<210> 52
<211> 8
<212> PRT
<213> CDR1
<400> 52
Gly Arg Thr Val Ser Ser Tyr Val
1 5
<210> 53
<211> 8
<212> PRT
<213> CDR2
<400> 53
Ile Ser Arg Ser Gly Ile Ile Thr
1 5
<210> 54
<211> 17
<212> PRT
<213> CDR3
<400> 54
Ala Ala Ala Gln Thr Gln Val Leu Pro Thr Glu Asp Arg Ser Tyr Asp
1 5 10 15
Tyr
<210> 55
<211> 8
<212> PRT
<213> CDR1
<400> 55
Gly Arg Thr Phe Thr Arg Tyr Pro
1 5
<210> 56
<211> 8
<212> PRT
<213> CDR2
<400> 56
Ile Ser Gly Ser Gly Ile Ile Thr
1 5
<210> 57
<211> 19
<212> PRT
<213> CDR3
<400> 57
Ala Val Asp Asn Ala Pro Thr Ser Ser Leu Pro Leu Thr Pro Gly Glu
1 5 10 15
Tyr Asp Tyr
<210> 58
<211> 8
<212> PRT
<213> CDR1
<400> 58
Gly Arg Thr Phe Ser Ser Tyr Ala
1 5
<210> 59
<211> 7
<212> PRT
<213> CDR2
<400> 59
Ile Ser Ser Gly Gly Ile Thr
1 5
<210> 60
<211> 18
<212> PRT
<213> CDR3
<400> 60
Ala Ala Asn Arg Gly Arg Phe Thr Gly Trp Arg Ser Leu Thr Asp Tyr
1 5 10 15
Thr Tyr
<210> 61
<211> 8
<212> PRT
<213> CDR1
<400> 61
Gly Arg Thr Phe Ser Thr Tyr Ala
1 5
<210> 62
<211> 8
<212> PRT
<213> CDR2
<400> 62
Ile Ser Pro Ser Arg Ile Ile Thr
1 5
<210> 63
<211> 19
<212> PRT
<213> CDR3
<400> 63
Ala Ala Asp Ser Ser Tyr Gly Gly Arg Leu Gly Arg Arg Ile Asp Asp
1 5 10 15
Tyr Glu Tyr
<210> 64
<211> 8
<212> PRT
<213> CDR1
<400> 64
Gly Ser Gly Val Ser Thr Gly Ser
1 5
<210> 65
<211> 7
<212> PRT
<213> CDR2
<400> 65
Tyr Thr Ser Val Gly Ser Ile
1 5
<210> 66
<211> 10
<212> PRT
<213> CDR3
<400> 66
Ala Phe Phe Val Pro Phe Gly Asp Thr Tyr
1 5 10
<210> 67
<211> 8
<212> PRT
<213> CDR1
<400> 67
Ala Ser Gly Arg Thr Ser Ser Thr
1 5
<210> 68
<211> 8
<212> PRT
<213> CDR2
<400> 68
Ile Thr Trp Ser Gly Gly Ser Thr
1 5
<210> 69
<211> 17
<212> PRT
<213> CDR3
<400> 69
Ala Ala Gly Leu Ser Pro Leu Met Leu Thr Ala Ser Arg Arg Tyr Glu
1 5 10 15
Tyr
<210> 70
<211> 8
<212> PRT
<213> CDR1
<400> 70
Gly Arg Thr Val Ser Met Tyr Ala
1 5
<210> 71
<211> 8
<212> PRT
<213> CDR2
<400> 71
Ile Ser Arg Ser Gly Arg Ser Thr
1 5
<210> 72
<211> 20
<212> PRT
<213> CDR3
<400> 72
Ala Ala Arg Asp Gly Gly Leu His Tyr Ala Leu Tyr Val Glu Ala Ser
1 5 10 15
His Tyr Asn Tyr
20
<210> 73
<211> 8
<212> PRT
<213> CDR1
<400> 73
Gly Phe Thr Phe Ser Ser Tyr Ala
1 5
<210> 74
<211> 8
<212> PRT
<213> CDR2
<400> 74
Leu Asp Val Thr Gly Arg Ser Thr
1 5
<210> 75
<211> 20
<212> PRT
<213> CDR3
<400> 75
Ala Ala Val Tyr Thr Pro Phe Arg Phe Ala Phe Lys Thr Asp Pro Asp
1 5 10 15
Arg Gln Asp His
20
<210> 76
<211> 8
<212> PRT
<213> CDR1
<400> 76
Gly Arg Thr Phe Gly Thr Tyr Arg
1 5
<210> 77
<211> 8
<212> PRT
<213> CDR2
<400> 77
Ile Phe Trp Ser Gly Gly Gly Ala
1 5
<210> 78
<211> 12
<212> PRT
<213> CDR3
<400> 78
Ala Ala Arg Ala Pro Thr Thr Gln Glu Tyr Asp Tyr
1 5 10
<210> 79
<211> 8
<212> PRT
<213> CDR1
<400> 79
Gly Arg Thr Leu Ser Thr Tyr Glu
1 5
<210> 80
<211> 8
<212> PRT
<213> CDR2
<400> 80
Val Thr Pro Thr Gly Phe Gly Thr
1 5
<210> 81
<211> 16
<212> PRT
<213> CDR3
<400> 81
Ala Gly Ser Gly Lys Gly Leu Gly Ser Leu Gln Arg Ser Pro Asp Asp
1 5 10 15
<210> 82
<211> 127
<212> PRT
<213> VHH1
<400> 82
Gln Leu Gln Leu Val Glu Ser Gly Gly Asp Leu Val Gln Pro Gly Glu
1 5 10 15
Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Leu Ser Leu Glu His Tyr
20 25 30
Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ser Cys Ile Ser Asp Ser Gly Ala Thr Arg Ser Tyr Ala Asp Ser Val
50 55 60
Arg Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Ala Val Tyr
65 70 75 80
Leu Glu Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ser Ala Thr Pro Gly Ala Phe Arg Thr Cys Ala Phe Arg Asp Phe Ser
100 105 110
His Leu Ala Asn Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 83
<211> 126
<212> PRT
<213> VHH2
<400> 83
Gln Leu Gln Leu Val Glu Ser Gly Gly Asp Leu Val Gln Pro Gly Glu
1 5 10 15
Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Leu Ser Leu Glu His Tyr
20 25 30
Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ser Cys Ile Asp Ser Ser Gly Gly Ser Thr Ser Tyr Ala Asp Ser Val
50 55 60
Arg Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Ala Val Tyr
65 70 75 80
Leu Glu Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Leu Gln Gly Ser Tyr Tyr Tyr Thr Ser Leu His Gln Thr Glu
100 105 110
Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 84
<211> 127
<212> PRT
<213> VHH3
<400> 84
Gln Leu Gln Leu Val Glu Ser Gly Gly Asp Leu Val Gln Pro Gly Glu
1 5 10 15
Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Phe Thr Leu Asp Ser Tyr
20 25 30
Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ser Cys Ile Thr Lys His Gly Gly Thr Thr Ser Tyr Ala Asp Ser Val
50 55 60
Arg Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Ala Val Tyr
65 70 75 80
Leu Glu Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Asp His Arg Ala Leu Arg Phe Gly Cys Ala Val Val Pro Gly
100 105 110
Pro Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 85
<211> 123
<212> PRT
<213> VHH4
<400> 85
Gln Leu Gln Leu Val Glu Ser Gly Gly Asp Leu Val Gln Pro Gly Glu
1 5 10 15
Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Leu Thr Leu Asn Asn Asn
20 25 30
Thr Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ser Cys Ile Ser Pro Ser Gly Ile Val Thr Ser Tyr Ala Asp Ser Val
50 55 60
Arg Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Ala Val Tyr
65 70 75 80
Leu Glu Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Val Leu Gln Trp Asn Gly Ala Gly Leu Pro Ser Pro Leu Gly Ser
100 105 110
Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 86
<211> 129
<212> PRT
<213> VHH5
<400> 86
Gln Leu Gln Leu Val Glu Ser Gly Gly Asp Leu Val Gln Pro Gly Glu
1 5 10 15
Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Phe Thr Leu Gly Asn Tyr
20 25 30
Glu Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ser Cys Ile Ser Arg Ser Gly Val Ile Phe Ser Tyr Ala Asp Ser Val
50 55 60
Arg Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Ala Val Tyr
65 70 75 80
Leu Glu Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Arg Phe Arg Ala Gly Leu Val Gln Ser Leu Cys Gln Ser Thr
100 105 110
Pro Glu Phe Tyr Gln Asp Trp Gly Gln Gly Thr Gln Val Thr Val Ser
115 120 125
Ser
<210> 87
<211> 129
<212> PRT
<213> VHH6
<400> 87
Gln Leu Gln Leu Val Glu Ser Gly Gly Asp Leu Val Gln Pro Gly Glu
1 5 10 15
Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Phe Thr Leu Gly Asn Tyr
20 25 30
Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ser Cys Ile Ser Leu Ser Gly Asp Ile Ile Ser Tyr Ala Asp Ser Val
50 55 60
Arg Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Ala Val Tyr
65 70 75 80
Leu Glu Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Pro Phe Pro Ser Leu Leu Tyr Cys Ser Leu Cys Pro Ser Asn
100 105 110
Pro Glu Phe Cys Arg His Trp Gly Gln Gly Thr Gln Val Thr Val Ser
115 120 125
Ser
<210> 88
<211> 125
<212> PRT
<213> VHH7
<400> 88
Gln Leu Gln Leu Val Glu Ser Gly Gly Asp Leu Val Gln Pro Gly Glu
1 5 10 15
Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Phe Ser Leu Asp Gly Tyr
20 25 30
Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ser Cys Ile Gly Arg Leu Gly Thr Thr Thr Ser Tyr Ala Asp Ser Val
50 55 60
Arg Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Ala Val Tyr
65 70 75 80
Leu Glu Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Lys Arg Ser Thr Trp Phe Gly Cys Ser Thr Leu Asp Asp Tyr
100 105 110
Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 89
<211> 125
<212> PRT
<213> VHH8
<400> 89
Gln Leu Gln Leu Val Glu Ser Gly Gly Asp Leu Val Gln Pro Gly Glu
1 5 10 15
Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Phe Thr Leu Asp Arg Tyr
20 25 30
Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ser Cys Ile Ser Ser Gly Gly Thr Thr Ser Tyr Ala Asp Ser Val Arg
50 55 60
Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Ala Val Tyr Leu
65 70 75 80
Glu Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Ala Gly Thr Gln Leu Arg Tyr Gly Cys Ser Arg Gln Ala Arg Arg Tyr
100 105 110
Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 90
<211> 126
<212> PRT
<213> VHH9
<400> 90
Gln Leu Gln Leu Val Glu Ser Gly Gly Asp Leu Val Gln Pro Gly Glu
1 5 10 15
Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Phe Thr Leu Asp Tyr Tyr
20 25 30
Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ser Cys Ile Ser Ser Ser Gly Thr Ser Thr Ser Tyr Ala Asp Ser Val
50 55 60
Arg Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Ala Val Tyr
65 70 75 80
Leu Glu Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Leu Gln Gly Ser Tyr Tyr Tyr Thr Ser Leu His Gln Thr Glu
100 105 110
Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 91
<211> 126
<212> PRT
<213> VHH10
<400> 91
Gln Leu Gln Leu Val Glu Ser Gly Gly Asp Leu Val Gln Pro Gly Glu
1 5 10 15
Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Ala Thr Ser Asp Ile Tyr
20 25 30
Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ser Cys Ile Ser Pro Ser Gly Gly Ser Thr Ser Tyr Ala Asp Ser Val
50 55 60
Arg Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Ala Val Tyr
65 70 75 80
Leu Glu Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Ala Ser Pro Ser Trp His Tyr Cys Ser Gly Tyr Glu His Glu
100 105 110
Tyr Asp Asp Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 92
<211> 126
<212> PRT
<213> VHH11
<400> 92
Gln Leu Gln Leu Val Glu Ser Gly Gly Asp Leu Val Gln Pro Gly Glu
1 5 10 15
Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Phe Thr Leu Asp Tyr Tyr
20 25 30
Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ser Cys Ile Ser Pro Ser Gly Thr Ser Thr Ser Tyr Ala Asp Ser Val
50 55 60
Arg Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Ala Val Tyr
65 70 75 80
Leu Glu Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Ala Asn Pro Ser Tyr Tyr Tyr Cys Ser Gly Tyr Pro His Glu
100 105 110
Tyr Asp Asp Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 93
<211> 126
<212> PRT
<213> VHH12
<400> 93
Gln Leu Gln Leu Val Glu Ser Gly Gly Asp Leu Val Gln Pro Gly Glu
1 5 10 15
Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Phe Thr Phe His Tyr Tyr
20 25 30
Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ser Cys Ile Ser Ser Gly Gly Gly Arg Thr Ser Tyr Ala Asp Ser Val
50 55 60
Arg Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Ala Val Tyr
65 70 75 80
Leu Glu Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Leu Gln Gly Ser Leu His Tyr Cys Ser Gly Leu Val Gly Glu
100 105 110
Tyr Glu Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 94
<211> 127
<212> PRT
<213> VHH13
<400> 94
Gln Leu Gln Leu Val Glu Ser Gly Gly Asp Leu Val Gln Pro Gly Glu
1 5 10 15
Ser Leu Arg Leu Ser Cys Val Ala Ser Phe Lys Ser Tyr His Ile Ala
20 25 30
Trp Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ser Cys Ser Leu Thr Gly Ala Ser Ile Thr Ser Tyr Ala Asp Ser Val
50 55 60
Arg Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Ala Val Tyr
65 70 75 80
Leu Glu Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Arg Asp Tyr Ala Ser Ser Leu Tyr Cys Ser Arg Asn Glu Ala
100 105 110
Met Tyr Ala Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 95
<211> 131
<212> PRT
<213> VHH14
<400> 95
Gln Leu Gln Leu Val Glu Ser Gly Gly Asp Leu Val Gln Pro Gly Glu
1 5 10 15
Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Phe Arg Leu Gly Gly Tyr
20 25 30
Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ser Cys Ile Ser Gly Ser Gly Ile Ile Ser Ser Tyr Ala Asp Ser Val
50 55 60
Arg Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Ala Val Tyr
65 70 75 80
Leu Glu Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Pro Ser Gly Ser Ser Tyr Gly Ser Arg Arg Leu Pro Cys Gly
100 105 110
Ile Asp Ser Gln Asp Tyr Gln Thr Trp Gly Gln Gly Thr Gln Val Thr
115 120 125
Val Ser Ser
130
<210> 96
<211> 127
<212> PRT
<213> VHH15
<400> 96
Gln Leu Gln Leu Val Glu Ser Gly Gly Asp Leu Val Gln Pro Gly Glu
1 5 10 15
Ser Leu Arg Leu Ser Cys Val Ala Ser Phe Asp Asp Phe Ala Ile Gly
20 25 30
Trp Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ser Cys Ser Ala Arg Gly Ile Val Asn Thr Ser Tyr Ala Asp Ser Val
50 55 60
Arg Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Ala Val Tyr
65 70 75 80
Leu Glu Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Leu Val Gly Leu Asn Asp Arg Ser Leu Pro His Glu Ala Glu
100 105 110
Glu Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 97
<211> 125
<212> PRT
<213> VHH16
<400> 97
Gln Leu Gln Leu Val Glu Ser Gly Gly Asp Leu Val Gln Pro Gly Glu
1 5 10 15
Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Asp Asp Tyr
20 25 30
Ser Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ser Cys Phe Ser Ser Leu Asp Gly Ser Thr Ser Tyr Ala Asp Ser Val
50 55 60
Arg Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Ala Val Tyr
65 70 75 80
Leu Glu Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Thr Arg Gly Ser Ser Trp Tyr Trp Tyr Arg Pro Ser Glu Tyr
100 105 110
Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 98
<211> 126
<212> PRT
<213> VHH17
<400> 98
Gln Leu Gln Leu Val Glu Ser Gly Gly Asp Leu Val Gln Pro Gly Glu
1 5 10 15
Ser Leu Arg Leu Ser Cys Val Ala Ser Gly His Thr Gly Ser Thr Tyr
20 25 30
Ile Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ser Cys Ile Ser Arg Ser Gly Ile Leu Arg Ser Tyr Ala Asp Ser Val
50 55 60
Arg Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Ala Val Tyr
65 70 75 80
Leu Glu Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Arg Asp Ser Leu Thr Gly Val Ile Gly Asn Thr Pro Gly Glu
100 105 110
Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 99
<211> 124
<212> PRT
<213> VHH18
<400> 99
Gln Leu Gln Leu Val Glu Ser Gly Gly Asp Leu Val Gln Pro Gly Glu
1 5 10 15
Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Arg Thr Val Ser Ser Tyr
20 25 30
Val Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ser Cys Ile Ser Arg Ser Gly Ile Ile Thr Ser Tyr Ala Asp Ser Val
50 55 60
Arg Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Ala Val Tyr
65 70 75 80
Leu Glu Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Ala Gln Thr Gln Val Leu Pro Thr Glu Asp Arg Ser Tyr Asp
100 105 110
Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 100
<211> 126
<212> PRT
<213> VHH19
<400> 100
Gln Leu Gln Leu Val Glu Ser Gly Gly Asp Leu Val Gln Pro Gly Glu
1 5 10 15
Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Arg Thr Phe Thr Arg Tyr
20 25 30
Pro Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ser Cys Ile Ser Gly Ser Gly Ile Ile Thr Ser Tyr Ala Asp Ser Val
50 55 60
Arg Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Ala Val Tyr
65 70 75 80
Leu Glu Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Val Asp Asn Ala Pro Thr Ser Ser Leu Pro Leu Thr Pro Gly Glu
100 105 110
Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 101
<211> 124
<212> PRT
<213> VHH20
<400> 101
Gln Leu Gln Leu Val Glu Ser Gly Gly Asp Leu Val Gln Pro Gly Glu
1 5 10 15
Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Arg Thr Phe Ser Ser Tyr
20 25 30
Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ser Cys Ile Ser Ser Gly Gly Ile Thr Ser Tyr Ala Asp Ser Val Arg
50 55 60
Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Ala Val Tyr Leu
65 70 75 80
Glu Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Ala Asn Arg Gly Arg Phe Thr Gly Trp Arg Ser Leu Thr Asp Tyr Thr
100 105 110
Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 102
<211> 126
<212> PRT
<213> VHH21
<400> 102
Gln Leu Gln Leu Val Glu Ser Gly Gly Asp Leu Val Gln Pro Gly Glu
1 5 10 15
Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Arg Thr Phe Ser Thr Tyr
20 25 30
Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ser Cys Ile Ser Pro Ser Arg Ile Ile Thr Ser Tyr Ala Asp Ser Val
50 55 60
Arg Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Ala Val Tyr
65 70 75 80
Leu Glu Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Asp Ser Ser Tyr Gly Gly Arg Leu Gly Arg Arg Ile Asp Asp
100 105 110
Tyr Glu Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 103
<211> 116
<212> PRT
<213> VHH22
<400> 103
Gln Leu Gln Leu Val Glu Ser Gly Gly Asp Leu Val Gln Pro Gly Glu
1 5 10 15
Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Ser Gly Val Ser Thr Gly
20 25 30
Ser Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ser Cys Tyr Thr Ser Val Gly Ser Ile Ser Tyr Ala Asp Ser Val Arg
50 55 60
Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Ala Val Tyr Leu
65 70 75 80
Glu Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Phe Phe Val Pro Phe Gly Asp Thr Tyr Trp Gly Gln Gly Thr Gln Val
100 105 110
Thr Val Ser Ser
115
<210> 104
<211> 124
<212> PRT
<213> VHH23
<400> 104
Gln Leu Gln Leu Val Glu Ser Gly Gly Asp Leu Val Gln Pro Gly Glu
1 5 10 15
Ser Leu Arg Leu Ser Cys Val Ala Ser Ala Ser Gly Arg Thr Ser Ser
20 25 30
Thr Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ser Cys Ile Thr Trp Ser Gly Gly Ser Thr Ser Tyr Ala Asp Ser Val
50 55 60
Arg Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Ala Val Tyr
65 70 75 80
Leu Glu Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Gly Leu Ser Pro Leu Met Leu Thr Ala Ser Arg Arg Tyr Glu
100 105 110
Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 105
<211> 127
<212> PRT
<213> VHH24
<400> 105
Gln Leu Gln Leu Val Glu Ser Gly Gly Asp Leu Val Gln Pro Gly Glu
1 5 10 15
Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Arg Thr Val Ser Met Tyr
20 25 30
Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ser Cys Ile Ser Arg Ser Gly Arg Ser Thr Ser Tyr Ala Asp Ser Val
50 55 60
Arg Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Ala Val Tyr
65 70 75 80
Leu Glu Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Arg Asp Gly Gly Leu His Tyr Ala Leu Tyr Val Glu Ala Ser
100 105 110
His Tyr Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 106
<211> 127
<212> PRT
<213> VHH25
<400> 106
Gln Leu Gln Leu Val Glu Ser Gly Gly Asp Leu Val Gln Pro Gly Glu
1 5 10 15
Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ser Cys Leu Asp Val Thr Gly Arg Ser Thr Ser Tyr Ala Asp Ser Val
50 55 60
Arg Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Ala Val Tyr
65 70 75 80
Leu Glu Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Val Tyr Thr Pro Phe Arg Phe Ala Phe Lys Thr Asp Pro Asp
100 105 110
Arg Gln Asp His Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 107
<211> 119
<212> PRT
<213> VHH26
<400> 107
Gln Leu Gln Leu Val Glu Ser Gly Gly Asp Leu Val Gln Pro Gly Glu
1 5 10 15
Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Arg Thr Phe Gly Thr Tyr
20 25 30
Arg Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ser Cys Ile Phe Trp Ser Gly Gly Gly Ala Ser Tyr Ala Asp Ser Val
50 55 60
Arg Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Ala Val Tyr
65 70 75 80
Leu Glu Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Arg Ala Pro Thr Thr Gln Glu Tyr Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Gln Val Thr Val Ser Ser
115
<210> 108
<211> 123
<212> PRT
<213> VHH27
<400> 108
Gln Leu Gln Leu Val Glu Ser Gly Gly Asp Leu Val Gln Pro Gly Glu
1 5 10 15
Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Arg Thr Leu Ser Thr Tyr
20 25 30
Glu Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ser Cys Val Thr Pro Thr Gly Phe Gly Thr Ser Tyr Ala Asp Ser Val
50 55 60
Arg Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Ala Val Tyr
65 70 75 80
Leu Glu Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Gly Ser Gly Lys Gly Leu Gly Ser Leu Gln Arg Ser Pro Asp Asp
100 105 110
Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 109
<211> 232
<212> PRT
<213> Fc
<400> 109
Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
1 5 10 15
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
20 25 30
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
35 40 45
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
50 55 60
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
65 70 75 80
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
85 90 95
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
100 105 110
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
115 120 125
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
130 135 140
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
145 150 155 160
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
165 170 175
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
180 185 190
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
195 200 205
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
210 215 220
Ser Leu Ser Leu Ser Pro Gly Lys
225 230
<210> 110
<211> 359
<212> PRT
<213> VHH1-Fc
<400> 110
Gln Leu Gln Leu Val Glu Ser Gly Gly Asp Leu Val Gln Pro Gly Glu
1 5 10 15
Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Leu Ser Leu Glu His Tyr
20 25 30
Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ser Cys Ile Ser Asp Ser Gly Ala Thr Arg Ser Tyr Ala Asp Ser Val
50 55 60
Arg Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Ala Val Tyr
65 70 75 80
Leu Glu Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ser Ala Thr Pro Gly Ala Phe Arg Thr Cys Ala Phe Arg Asp Phe Ser
100 105 110
His Leu Ala Asn Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Glu
115 120 125
Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
130 135 140
Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
145 150 155 160
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
165 170 175
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
180 185 190
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
195 200 205
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
210 215 220
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
225 230 235 240
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
245 250 255
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys
260 265 270
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
275 280 285
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
290 295 300
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
305 310 315 320
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
325 330 335
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
340 345 350
Leu Ser Leu Ser Pro Gly Lys
355
<210> 111
<211> 358
<212> PRT
<213> VHH2-Fc
<400> 111
Gln Leu Gln Leu Val Glu Ser Gly Gly Asp Leu Val Gln Pro Gly Glu
1 5 10 15
Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Leu Ser Leu Glu His Tyr
20 25 30
Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ser Cys Ile Asp Ser Ser Gly Gly Ser Thr Ser Tyr Ala Asp Ser Val
50 55 60
Arg Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Ala Val Tyr
65 70 75 80
Leu Glu Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Leu Gln Gly Ser Tyr Tyr Tyr Thr Ser Leu His Gln Thr Glu
100 105 110
Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Glu Pro
115 120 125
Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
130 135 140
Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
145 150 155 160
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
165 170 175
Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
180 185 190
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
195 200 205
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
210 215 220
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
225 230 235 240
Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
245 250 255
Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn
260 265 270
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
275 280 285
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
290 295 300
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
305 310 315 320
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
325 330 335
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
340 345 350
Ser Leu Ser Pro Gly Lys
355
<210> 112
<211> 359
<212> PRT
<213> VHH3-Fc
<400> 112
Gln Leu Gln Leu Val Glu Ser Gly Gly Asp Leu Val Gln Pro Gly Glu
1 5 10 15
Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Phe Thr Leu Asp Ser Tyr
20 25 30
Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ser Cys Ile Thr Lys His Gly Gly Thr Thr Ser Tyr Ala Asp Ser Val
50 55 60
Arg Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Ala Val Tyr
65 70 75 80
Leu Glu Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Asp His Arg Ala Leu Arg Phe Gly Cys Ala Val Val Pro Gly
100 105 110
Pro Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Glu
115 120 125
Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
130 135 140
Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
145 150 155 160
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
165 170 175
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
180 185 190
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
195 200 205
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
210 215 220
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
225 230 235 240
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
245 250 255
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys
260 265 270
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
275 280 285
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
290 295 300
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
305 310 315 320
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
325 330 335
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
340 345 350
Leu Ser Leu Ser Pro Gly Lys
355
<210> 113
<211> 355
<212> PRT
<213> VHH4-Fc
<400> 113
Gln Leu Gln Leu Val Glu Ser Gly Gly Asp Leu Val Gln Pro Gly Glu
1 5 10 15
Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Leu Thr Leu Asn Asn Asn
20 25 30
Thr Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ser Cys Ile Ser Pro Ser Gly Ile Val Thr Ser Tyr Ala Asp Ser Val
50 55 60
Arg Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Ala Val Tyr
65 70 75 80
Leu Glu Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Val Leu Gln Trp Asn Gly Ala Gly Leu Pro Ser Pro Leu Gly Ser
100 105 110
Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Glu Pro Lys Ser Ser
115 120 125
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
130 135 140
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
145 150 155 160
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
165 170 175
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
180 185 190
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
195 200 205
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
210 215 220
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
225 230 235 240
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
245 250 255
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser
260 265 270
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
275 280 285
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
290 295 300
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
305 310 315 320
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
325 330 335
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
340 345 350
Pro Gly Lys
355
<210> 114
<211> 361
<212> PRT
<213> VHH5-Fc
<400> 114
Gln Leu Gln Leu Val Glu Ser Gly Gly Asp Leu Val Gln Pro Gly Glu
1 5 10 15
Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Phe Thr Leu Gly Asn Tyr
20 25 30
Glu Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ser Cys Ile Ser Arg Ser Gly Val Ile Phe Ser Tyr Ala Asp Ser Val
50 55 60
Arg Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Ala Val Tyr
65 70 75 80
Leu Glu Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Arg Phe Arg Ala Gly Leu Val Gln Ser Leu Cys Gln Ser Thr
100 105 110
Pro Glu Phe Tyr Gln Asp Trp Gly Gln Gly Thr Gln Val Thr Val Ser
115 120 125
Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro
130 135 140
Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
145 150 155 160
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
165 170 175
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
180 185 190
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
195 200 205
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
210 215 220
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
225 230 235 240
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
245 250 255
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu
260 265 270
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
275 280 285
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
290 295 300
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
305 310 315 320
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
325 330 335
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
340 345 350
Lys Ser Leu Ser Leu Ser Pro Gly Lys
355 360
<210> 115
<211> 361
<212> PRT
<213> VHH6-Fc
<400> 115
Gln Leu Gln Leu Val Glu Ser Gly Gly Asp Leu Val Gln Pro Gly Glu
1 5 10 15
Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Phe Thr Leu Gly Asn Tyr
20 25 30
Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ser Cys Ile Ser Leu Ser Gly Asp Ile Ile Ser Tyr Ala Asp Ser Val
50 55 60
Arg Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Ala Val Tyr
65 70 75 80
Leu Glu Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Pro Phe Pro Ser Leu Leu Tyr Cys Ser Leu Cys Pro Ser Asn
100 105 110
Pro Glu Phe Cys Arg His Trp Gly Gln Gly Thr Gln Val Thr Val Ser
115 120 125
Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro
130 135 140
Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
145 150 155 160
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
165 170 175
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
180 185 190
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
195 200 205
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
210 215 220
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
225 230 235 240
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
245 250 255
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu
260 265 270
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
275 280 285
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
290 295 300
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
305 310 315 320
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
325 330 335
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
340 345 350
Lys Ser Leu Ser Leu Ser Pro Gly Lys
355 360
<210> 116
<211> 357
<212> PRT
<213> VHH7-Fc
<400> 116
Gln Leu Gln Leu Val Glu Ser Gly Gly Asp Leu Val Gln Pro Gly Glu
1 5 10 15
Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Phe Ser Leu Asp Gly Tyr
20 25 30
Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ser Cys Ile Gly Arg Leu Gly Thr Thr Thr Ser Tyr Ala Asp Ser Val
50 55 60
Arg Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Ala Val Tyr
65 70 75 80
Leu Glu Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Lys Arg Ser Thr Trp Phe Gly Cys Ser Thr Leu Asp Asp Tyr
100 105 110
Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Glu Pro Lys
115 120 125
Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
130 135 140
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
145 150 155 160
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
165 170 175
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
180 185 190
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
195 200 205
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
210 215 220
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
225 230 235 240
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
245 250 255
Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
260 265 270
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
275 280 285
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
290 295 300
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
305 310 315 320
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
325 330 335
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
340 345 350
Leu Ser Pro Gly Lys
355
<210> 117
<211> 357
<212> PRT
<213> VHH8-Fc
<400> 117
Gln Leu Gln Leu Val Glu Ser Gly Gly Asp Leu Val Gln Pro Gly Glu
1 5 10 15
Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Phe Thr Leu Asp Arg Tyr
20 25 30
Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ser Cys Ile Ser Ser Gly Gly Thr Thr Ser Tyr Ala Asp Ser Val Arg
50 55 60
Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Ala Val Tyr Leu
65 70 75 80
Glu Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Ala Gly Thr Gln Leu Arg Tyr Gly Cys Ser Arg Gln Ala Arg Arg Tyr
100 105 110
Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Glu Pro Lys
115 120 125
Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
130 135 140
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
145 150 155 160
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
165 170 175
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
180 185 190
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
195 200 205
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
210 215 220
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
225 230 235 240
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
245 250 255
Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
260 265 270
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
275 280 285
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
290 295 300
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
305 310 315 320
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
325 330 335
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
340 345 350
Leu Ser Pro Gly Lys
355
<210> 118
<211> 358
<212> PRT
<213> VHH9-Fc
<400> 118
Gln Leu Gln Leu Val Glu Ser Gly Gly Asp Leu Val Gln Pro Gly Glu
1 5 10 15
Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Phe Thr Leu Asp Tyr Tyr
20 25 30
Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ser Cys Ile Ser Ser Ser Gly Thr Ser Thr Ser Tyr Ala Asp Ser Val
50 55 60
Arg Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Ala Val Tyr
65 70 75 80
Leu Glu Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Leu Gln Gly Ser Tyr Tyr Tyr Thr Ser Leu His Gln Thr Glu
100 105 110
Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Glu Pro
115 120 125
Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
130 135 140
Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
145 150 155 160
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
165 170 175
Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
180 185 190
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
195 200 205
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
210 215 220
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
225 230 235 240
Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
245 250 255
Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn
260 265 270
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
275 280 285
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
290 295 300
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
305 310 315 320
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
325 330 335
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
340 345 350
Ser Leu Ser Pro Gly Lys
355
<210> 119
<211> 358
<212> PRT
<213> VHH10-Fc
<400> 119
Gln Leu Gln Leu Val Glu Ser Gly Gly Asp Leu Val Gln Pro Gly Glu
1 5 10 15
Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Ala Thr Ser Asp Ile Tyr
20 25 30
Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ser Cys Ile Ser Pro Ser Gly Gly Ser Thr Ser Tyr Ala Asp Ser Val
50 55 60
Arg Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Ala Val Tyr
65 70 75 80
Leu Glu Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Ala Ser Pro Ser Trp His Tyr Cys Ser Gly Tyr Glu His Glu
100 105 110
Tyr Asp Asp Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Glu Pro
115 120 125
Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
130 135 140
Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
145 150 155 160
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
165 170 175
Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
180 185 190
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
195 200 205
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
210 215 220
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
225 230 235 240
Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
245 250 255
Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn
260 265 270
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
275 280 285
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
290 295 300
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
305 310 315 320
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
325 330 335
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
340 345 350
Ser Leu Ser Pro Gly Lys
355
<210> 120
<211> 358
<212> PRT
<213> VHH11-Fc
<400> 120
Gln Leu Gln Leu Val Glu Ser Gly Gly Asp Leu Val Gln Pro Gly Glu
1 5 10 15
Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Phe Thr Leu Asp Tyr Tyr
20 25 30
Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ser Cys Ile Ser Pro Ser Gly Thr Ser Thr Ser Tyr Ala Asp Ser Val
50 55 60
Arg Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Ala Val Tyr
65 70 75 80
Leu Glu Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Ala Asn Pro Ser Tyr Tyr Tyr Cys Ser Gly Tyr Pro His Glu
100 105 110
Tyr Asp Asp Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Glu Pro
115 120 125
Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
130 135 140
Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
145 150 155 160
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
165 170 175
Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
180 185 190
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
195 200 205
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
210 215 220
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
225 230 235 240
Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
245 250 255
Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn
260 265 270
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
275 280 285
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
290 295 300
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
305 310 315 320
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
325 330 335
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
340 345 350
Ser Leu Ser Pro Gly Lys
355
<210> 121
<211> 358
<212> PRT
<213> VHH12-Fc
<400> 121
Gln Leu Gln Leu Val Glu Ser Gly Gly Asp Leu Val Gln Pro Gly Glu
1 5 10 15
Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Phe Thr Phe His Tyr Tyr
20 25 30
Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ser Cys Ile Ser Ser Gly Gly Gly Arg Thr Ser Tyr Ala Asp Ser Val
50 55 60
Arg Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Ala Val Tyr
65 70 75 80
Leu Glu Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Leu Gln Gly Ser Leu His Tyr Cys Ser Gly Leu Val Gly Glu
100 105 110
Tyr Glu Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Glu Pro
115 120 125
Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
130 135 140
Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
145 150 155 160
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
165 170 175
Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
180 185 190
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
195 200 205
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
210 215 220
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
225 230 235 240
Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
245 250 255
Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn
260 265 270
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
275 280 285
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
290 295 300
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
305 310 315 320
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
325 330 335
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
340 345 350
Ser Leu Ser Pro Gly Lys
355
<210> 122
<211> 359
<212> PRT
<213> VHH13-Fc
<400> 122
Gln Leu Gln Leu Val Glu Ser Gly Gly Asp Leu Val Gln Pro Gly Glu
1 5 10 15
Ser Leu Arg Leu Ser Cys Val Ala Ser Phe Lys Ser Tyr His Ile Ala
20 25 30
Trp Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ser Cys Ser Leu Thr Gly Ala Ser Ile Thr Ser Tyr Ala Asp Ser Val
50 55 60
Arg Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Ala Val Tyr
65 70 75 80
Leu Glu Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Arg Asp Tyr Ala Ser Ser Leu Tyr Cys Ser Arg Asn Glu Ala
100 105 110
Met Tyr Ala Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Glu
115 120 125
Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
130 135 140
Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
145 150 155 160
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
165 170 175
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
180 185 190
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
195 200 205
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
210 215 220
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
225 230 235 240
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
245 250 255
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys
260 265 270
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
275 280 285
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
290 295 300
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
305 310 315 320
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
325 330 335
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
340 345 350
Leu Ser Leu Ser Pro Gly Lys
355
<210> 123
<211> 363
<212> PRT
<213> VHH14-Fc
<400> 123
Gln Leu Gln Leu Val Glu Ser Gly Gly Asp Leu Val Gln Pro Gly Glu
1 5 10 15
Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Phe Arg Leu Gly Gly Tyr
20 25 30
Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ser Cys Ile Ser Gly Ser Gly Ile Ile Ser Ser Tyr Ala Asp Ser Val
50 55 60
Arg Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Ala Val Tyr
65 70 75 80
Leu Glu Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Pro Ser Gly Ser Ser Tyr Gly Ser Arg Arg Leu Pro Cys Gly
100 105 110
Ile Asp Ser Gln Asp Tyr Gln Thr Trp Gly Gln Gly Thr Gln Val Thr
115 120 125
Val Ser Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro
130 135 140
Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro
145 150 155 160
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
165 170 175
Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn
180 185 190
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
195 200 205
Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val
210 215 220
Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
225 230 235 240
Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
245 250 255
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp
260 265 270
Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
275 280 285
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
290 295 300
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
305 310 315 320
Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
325 330 335
Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
340 345 350
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
355 360
<210> 124
<211> 359
<212> PRT
<213> VHH15-Fc
<400> 124
Gln Leu Gln Leu Val Glu Ser Gly Gly Asp Leu Val Gln Pro Gly Glu
1 5 10 15
Ser Leu Arg Leu Ser Cys Val Ala Ser Phe Asp Asp Phe Ala Ile Gly
20 25 30
Trp Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ser Cys Ser Ala Arg Gly Ile Val Asn Thr Ser Tyr Ala Asp Ser Val
50 55 60
Arg Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Ala Val Tyr
65 70 75 80
Leu Glu Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Leu Val Gly Leu Asn Asp Arg Ser Leu Pro His Glu Ala Glu
100 105 110
Glu Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Glu
115 120 125
Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
130 135 140
Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
145 150 155 160
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
165 170 175
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
180 185 190
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
195 200 205
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
210 215 220
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
225 230 235 240
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
245 250 255
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys
260 265 270
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
275 280 285
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
290 295 300
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
305 310 315 320
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
325 330 335
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
340 345 350
Leu Ser Leu Ser Pro Gly Lys
355
<210> 125
<211> 357
<212> PRT
<213> VHH16-Fc
<400> 125
Gln Leu Gln Leu Val Glu Ser Gly Gly Asp Leu Val Gln Pro Gly Glu
1 5 10 15
Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Asp Asp Tyr
20 25 30
Ser Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ser Cys Phe Ser Ser Leu Asp Gly Ser Thr Ser Tyr Ala Asp Ser Val
50 55 60
Arg Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Ala Val Tyr
65 70 75 80
Leu Glu Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Thr Arg Gly Ser Ser Trp Tyr Trp Tyr Arg Pro Ser Glu Tyr
100 105 110
Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Glu Pro Lys
115 120 125
Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
130 135 140
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
145 150 155 160
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
165 170 175
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
180 185 190
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
195 200 205
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
210 215 220
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
225 230 235 240
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
245 250 255
Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
260 265 270
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
275 280 285
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
290 295 300
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
305 310 315 320
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
325 330 335
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
340 345 350
Leu Ser Pro Gly Lys
355
<210> 126
<211> 358
<212> PRT
<213> VHH17-Fc
<400> 126
Gln Leu Gln Leu Val Glu Ser Gly Gly Asp Leu Val Gln Pro Gly Glu
1 5 10 15
Ser Leu Arg Leu Ser Cys Val Ala Ser Gly His Thr Gly Ser Thr Tyr
20 25 30
Ile Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ser Cys Ile Ser Arg Ser Gly Ile Leu Arg Ser Tyr Ala Asp Ser Val
50 55 60
Arg Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Ala Val Tyr
65 70 75 80
Leu Glu Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Arg Asp Ser Leu Thr Gly Val Ile Gly Asn Thr Pro Gly Glu
100 105 110
Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Glu Pro
115 120 125
Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
130 135 140
Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
145 150 155 160
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
165 170 175
Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
180 185 190
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
195 200 205
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
210 215 220
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
225 230 235 240
Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
245 250 255
Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn
260 265 270
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
275 280 285
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
290 295 300
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
305 310 315 320
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
325 330 335
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
340 345 350
Ser Leu Ser Pro Gly Lys
355
<210> 127
<211> 356
<212> PRT
<213> VHH18-Fc
<400> 127
Gln Leu Gln Leu Val Glu Ser Gly Gly Asp Leu Val Gln Pro Gly Glu
1 5 10 15
Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Arg Thr Val Ser Ser Tyr
20 25 30
Val Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ser Cys Ile Ser Arg Ser Gly Ile Ile Thr Ser Tyr Ala Asp Ser Val
50 55 60
Arg Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Ala Val Tyr
65 70 75 80
Leu Glu Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Ala Gln Thr Gln Val Leu Pro Thr Glu Asp Arg Ser Tyr Asp
100 105 110
Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Glu Pro Lys Ser
115 120 125
Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu
130 135 140
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
145 150 155 160
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
165 170 175
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
180 185 190
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
195 200 205
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
210 215 220
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
225 230 235 240
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
245 250 255
Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val
260 265 270
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
275 280 285
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
290 295 300
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
305 310 315 320
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
325 330 335
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
340 345 350
Ser Pro Gly Lys
355
<210> 128
<211> 358
<212> PRT
<213> VHH19-Fc
<400> 128
Gln Leu Gln Leu Val Glu Ser Gly Gly Asp Leu Val Gln Pro Gly Glu
1 5 10 15
Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Arg Thr Phe Thr Arg Tyr
20 25 30
Pro Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ser Cys Ile Ser Gly Ser Gly Ile Ile Thr Ser Tyr Ala Asp Ser Val
50 55 60
Arg Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Ala Val Tyr
65 70 75 80
Leu Glu Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Val Asp Asn Ala Pro Thr Ser Ser Leu Pro Leu Thr Pro Gly Glu
100 105 110
Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Glu Pro
115 120 125
Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
130 135 140
Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
145 150 155 160
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
165 170 175
Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
180 185 190
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
195 200 205
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
210 215 220
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
225 230 235 240
Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
245 250 255
Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn
260 265 270
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
275 280 285
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
290 295 300
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
305 310 315 320
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
325 330 335
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
340 345 350
Ser Leu Ser Pro Gly Lys
355
<210> 129
<211> 356
<212> PRT
<213> VHH20-Fc
<400> 129
Gln Leu Gln Leu Val Glu Ser Gly Gly Asp Leu Val Gln Pro Gly Glu
1 5 10 15
Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Arg Thr Phe Ser Ser Tyr
20 25 30
Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ser Cys Ile Ser Ser Gly Gly Ile Thr Ser Tyr Ala Asp Ser Val Arg
50 55 60
Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Ala Val Tyr Leu
65 70 75 80
Glu Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Ala Asn Arg Gly Arg Phe Thr Gly Trp Arg Ser Leu Thr Asp Tyr Thr
100 105 110
Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Glu Pro Lys Ser
115 120 125
Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu
130 135 140
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
145 150 155 160
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
165 170 175
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
180 185 190
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
195 200 205
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
210 215 220
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
225 230 235 240
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
245 250 255
Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val
260 265 270
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
275 280 285
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
290 295 300
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
305 310 315 320
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
325 330 335
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
340 345 350
Ser Pro Gly Lys
355
<210> 130
<211> 358
<212> PRT
<213> VHH21-Fc
<400> 130
Gln Leu Gln Leu Val Glu Ser Gly Gly Asp Leu Val Gln Pro Gly Glu
1 5 10 15
Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Arg Thr Phe Ser Thr Tyr
20 25 30
Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ser Cys Ile Ser Pro Ser Arg Ile Ile Thr Ser Tyr Ala Asp Ser Val
50 55 60
Arg Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Ala Val Tyr
65 70 75 80
Leu Glu Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Asp Ser Ser Tyr Gly Gly Arg Leu Gly Arg Arg Ile Asp Asp
100 105 110
Tyr Glu Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Glu Pro
115 120 125
Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
130 135 140
Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
145 150 155 160
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
165 170 175
Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
180 185 190
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
195 200 205
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
210 215 220
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
225 230 235 240
Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
245 250 255
Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn
260 265 270
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
275 280 285
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
290 295 300
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
305 310 315 320
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
325 330 335
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
340 345 350
Ser Leu Ser Pro Gly Lys
355
<210> 131
<211> 348
<212> PRT
<213> VHH22-Fc
<400> 131
Gln Leu Gln Leu Val Glu Ser Gly Gly Asp Leu Val Gln Pro Gly Glu
1 5 10 15
Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Ser Gly Val Ser Thr Gly
20 25 30
Ser Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ser Cys Tyr Thr Ser Val Gly Ser Ile Ser Tyr Ala Asp Ser Val Arg
50 55 60
Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Ala Val Tyr Leu
65 70 75 80
Glu Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Phe Phe Val Pro Phe Gly Asp Thr Tyr Trp Gly Gln Gly Thr Gln Val
100 105 110
Thr Val Ser Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro
115 120 125
Pro Cys Pro Ala Pro Glu Leu Leu 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
<210> 132
<211> 356
<212> PRT
<213> VHH23-Fc
<400> 132
Gln Leu Gln Leu Val Glu Ser Gly Gly Asp Leu Val Gln Pro Gly Glu
1 5 10 15
Ser Leu Arg Leu Ser Cys Val Ala Ser Ala Ser Gly Arg Thr Ser Ser
20 25 30
Thr Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ser Cys Ile Thr Trp Ser Gly Gly Ser Thr Ser Tyr Ala Asp Ser Val
50 55 60
Arg Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Ala Val Tyr
65 70 75 80
Leu Glu Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Gly Leu Ser Pro Leu Met Leu Thr Ala Ser Arg Arg Tyr Glu
100 105 110
Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Glu Pro Lys Ser
115 120 125
Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu
130 135 140
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
145 150 155 160
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
165 170 175
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
180 185 190
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
195 200 205
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
210 215 220
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
225 230 235 240
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
245 250 255
Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val
260 265 270
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
275 280 285
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
290 295 300
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
305 310 315 320
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
325 330 335
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
340 345 350
Ser Pro Gly Lys
355
<210> 133
<211> 359
<212> PRT
<213> VHH24-Fc
<400> 133
Gln Leu Gln Leu Val Glu Ser Gly Gly Asp Leu Val Gln Pro Gly Glu
1 5 10 15
Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Arg Thr Val Ser Met Tyr
20 25 30
Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ser Cys Ile Ser Arg Ser Gly Arg Ser Thr Ser Tyr Ala Asp Ser Val
50 55 60
Arg Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Ala Val Tyr
65 70 75 80
Leu Glu Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Arg Asp Gly Gly Leu His Tyr Ala Leu Tyr Val Glu Ala Ser
100 105 110
His Tyr Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Glu
115 120 125
Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
130 135 140
Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
145 150 155 160
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
165 170 175
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
180 185 190
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
195 200 205
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
210 215 220
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
225 230 235 240
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
245 250 255
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys
260 265 270
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
275 280 285
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
290 295 300
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
305 310 315 320
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
325 330 335
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
340 345 350
Leu Ser Leu Ser Pro Gly Lys
355
<210> 134
<211> 359
<212> PRT
<213> VHH25-Fc
<400> 134
Gln Leu Gln Leu Val Glu Ser Gly Gly Asp Leu Val Gln Pro Gly Glu
1 5 10 15
Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ser Cys Leu Asp Val Thr Gly Arg Ser Thr Ser Tyr Ala Asp Ser Val
50 55 60
Arg Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Ala Val Tyr
65 70 75 80
Leu Glu Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Val Tyr Thr Pro Phe Arg Phe Ala Phe Lys Thr Asp Pro Asp
100 105 110
Arg Gln Asp His Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Glu
115 120 125
Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
130 135 140
Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
145 150 155 160
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
165 170 175
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
180 185 190
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
195 200 205
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
210 215 220
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
225 230 235 240
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
245 250 255
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys
260 265 270
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
275 280 285
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
290 295 300
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
305 310 315 320
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
325 330 335
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
340 345 350
Leu Ser Leu Ser Pro Gly Lys
355
<210> 135
<211> 351
<212> PRT
<213> VHH26-Fc
<400> 135
Gln Leu Gln Leu Val Glu Ser Gly Gly Asp Leu Val Gln Pro Gly Glu
1 5 10 15
Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Arg Thr Phe Gly Thr Tyr
20 25 30
Arg Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ser Cys Ile Phe Trp Ser Gly Gly Gly Ala Ser Tyr Ala Asp Ser Val
50 55 60
Arg Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Ala Val Tyr
65 70 75 80
Leu Glu Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Arg Ala Pro Thr Thr Gln Glu Tyr Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Gln Val Thr Val Ser Ser Glu Pro Lys Ser Ser Asp Lys Thr His
115 120 125
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val
130 135 140
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
145 150 155 160
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
165 170 175
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
180 185 190
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
195 200 205
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
210 215 220
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
225 230 235 240
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
245 250 255
Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
260 265 270
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
275 280 285
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
290 295 300
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
305 310 315 320
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
325 330 335
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
340 345 350
<210> 136
<211> 355
<212> PRT
<213> VHH27-Fc
<400> 136
Gln Leu Gln Leu Val Glu Ser Gly Gly Asp Leu Val Gln Pro Gly Glu
1 5 10 15
Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Arg Thr Leu Ser Thr Tyr
20 25 30
Glu Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ser Cys Val Thr Pro Thr Gly Phe Gly Thr Ser Tyr Ala Asp Ser Val
50 55 60
Arg Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Ala Val Tyr
65 70 75 80
Leu Glu Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Gly Ser Gly Lys Gly Leu Gly Ser Leu Gln Arg Ser Pro Asp Asp
100 105 110
Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Glu Pro Lys Ser Ser
115 120 125
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
130 135 140
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
145 150 155 160
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
165 170 175
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
180 185 190
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
195 200 205
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
210 215 220
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
225 230 235 240
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
245 250 255
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser
260 265 270
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
275 280 285
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
290 295 300
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
305 310 315 320
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
325 330 335
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
340 345 350
Pro Gly Lys
355
<210> 137
<211> 705
<212> DNA
<213> Fc基因序列
<400> 137
cgtacggagc ccaaatcttg tgacaaaact cacacatgcc caccgtgccc agcacctgaa 60
ctcctggggg gaccgtcagt cttcctcttc cccccaaaac ccaaggacac cctcatgatc 120
tcccggaccc ctgaggtcac atgcgtggtg gtggacgtga gccacgaaga ccctgaggtc 180
aagttcaact ggtacgtgga cggcgtggag gtgcataatg ccaagacaaa gccgcgggag 240
gagcagtaca acagcacgta ccgtgtggtc agcgtcctca ccgtcctgca ccaggactgg 300
ctgaatggca aggagtacaa gtgcaaggtc tccaacaaag ccctcccagc ccccatcgag 360
aaaaccatct ccaaagccaa agggcagccc cgagaaccac aggtgtacac cctgccccca 420
tcccgggatg agctgaccaa gaaccaggtc agcctgacct gcctggtcaa aggcttctat 480
cccagcgaca tcgccgtgga gtgggagagc aatgggcagc cggagaacaa ctacaagacc 540
acgcctcccg tgctggactc cgacggctcc ttcttcctct acagcaagct caccgtggac 600
aagagcaggt ggcagcaggg gaacgtcttc tcatgctccg tgatgcatga ggctctgcac 660
aaccactaca cgcagaagag cctctccctg tctccgggta aatga 705
<210> 138
<211> 23
<212> DNA
<213> Primer For-1
<400> 138
gtcctggctg ctcttctaca agg 23
<210> 139
<211> 23
<212> DNA
<213> Primer Rev-1
<400> 139
ggtacgtgct gttgaactgt tcc 23
<210> 140
<211> 32
<212> DNA
<213> Primer For-2
<400> 140
ctagtgcggc cgctggagac ggtgacctgg gt 32
<210> 141
<211> 29
<212> DNA
<213> Primer Rev-2
<400> 141
gatgtgcagc tgcaggagtc tggrggagg 29
<210> 142
<211> 21
<212> DNA
<213> RBD-qF1
<400> 142
caatggttta acaggcacag g 21
<210> 143
<211> 21
<212> DNA
<213> RBD-qR1
<400> 143
ctcaagtgtc tgtggatcac g 21

Claims (20)

1.一种SARS-COV-2刺突蛋白结合分子，其特征在于：能特异性结合SARS-COV-2刺突蛋白的免疫球蛋白单一可变结构域，所述免疫球蛋白单一可变域为单域抗体；所述免疫球蛋白单一可变结构域中的CDR1、CDR2和CDR3选自如下组合中的任意一组：
1）SEQ ID NO:1所示的CDR1，SEQ ID NO:2所示的CDR2和SEQ ID NO:3所示的CDR3；
2）SEQ ID NO:4所示的CDR1，SEQ ID NO:5所示的CDR2和SEQ ID NO:6所示的CDR3；
3）SEQ ID NO:7所示的CDR1，SEQ ID NO:8所示的CDR2和SEQ ID NO:9所示的CDR3；
4）SEQ ID NO:10所示的CDR1，SEQ ID NO:11所示的CDR2和SEQ ID NO:12所示的CDR3；
5）SEQ ID NO:13所示的CDR1，SEQ ID NO:14所示的CDR2和SEQ ID NO:15所示的CDR3；
6）SEQ ID NO:16所示的CDR1，SEQ ID NO:17所示的CDR2和SEQ ID NO:18所示的CDR3；
7）SEQ ID NO:19所示的CDR1，SEQ ID NO:20所示的CDR2和SEQ ID NO:21所示的CDR3；
8）SEQ ID NO:22所示的CDR1，SEQ ID NO:23所示的CDR2和SEQ ID NO:24所示的CDR3；
9）SEQ ID NO:25所示的CDR1，SEQ ID NO:26所示的CDR2和SEQ ID NO:27所示的CDR3；
10）SEQ ID NO:28所示的CDR1，SEQ ID NO:29所示的CDR2和SEQ ID NO:30所示的CDR3；
11）SEQ ID NO:31所示的CDR1，SEQ ID NO:32所示的CDR2和SEQ ID NO:33所示的CDR3；
12）SEQ ID NO:34所示的CDR1，SEQ ID NO:35所示的CDR2和SEQ ID NO:36所示的CDR3；
13）SEQ ID NO:37所示的CDR1，SEQ ID NO:38所示的CDR2和SEQ ID NO:39所示的CDR3；
14）SEQ ID NO:40所示的CDR1，SEQ ID NO:41所示的CDR2和SEQ ID NO:42所示的CDR3；
15）SEQ ID NO:43所示的CDR1，SEQ ID NO:44所示的CDR2和SEQ ID NO:45所示的CDR3；
16）SEQ ID NO:46所示的CDR1，SEQ ID NO:47所示的CDR2和SEQ ID NO:48所示的CDR3；
17）SEQ ID NO:49所示的CDR1，SEQ ID NO:50所示的CDR2和SEQ ID NO:51所示的CDR3；
18）SEQ ID NO:52所示的CDR1，SEQ ID NO:53所示的CDR2和SEQ ID NO:54所示的CDR3；
19）SEQ ID NO:55所示的CDR1，SEQ ID NO:56所示的CDR2和SEQ ID NO:57所示的CDR3；
20）SEQ ID NO:58所示的CDR1，SEQ ID NO:59所示的CDR2和SEQ ID NO:60所示的CDR3；
21）SEQ ID NO:61所示的CDR1，SEQ ID NO:62所示的CDR2和SEQ ID NO:63所示的CDR3；
22）SEQ ID NO:64所示的CDR1，SEQ ID NO:65所示的CDR2和SEQ ID NO:66所示的CDR3；
23）SEQ ID NO:67所示的CDR1，SEQ ID NO:68所示的CDR2和SEQ ID NO:69所示的CDR3；
24）SEQ ID NO:70所示的CDR1，SEQ ID NO:71所示的CDR2和SEQ ID NO:72所示的CDR3；
25）SEQ ID NO:73所示的CDR1，SEQ ID NO:74所示的CDR2和SEQ ID NO:75所示的CDR3；
26）SEQ ID NO:76所示的CDR1，SEQ ID NO:77所示的CDR2和SEQ ID NO:78所示的CDR3；
27）SEQ ID NO:79所示的CDR1，SEQ ID NO:80所示的CDR2和SEQ ID NO:81所示的CDR3。
2.如权利要求1所述的SARS-COV-2刺突蛋白结合分子，其特征在于：所述单域抗体包含与SEQIDNO:82-108中任一序列具有至少80％的序列相同性的氨基酸序列。
3.如权利要求1所述的SARS-COV-2刺突蛋白结合分子，其特征在于：所述单域抗体包含与SEQIDNO:82-108中任一序列具有至少90％的序列相同性的氨基酸序列。
4.如权利要求1所述的SARS-COV-2刺突蛋白结合分子，其特征在于：所述单域抗体包含与SEQIDNO:82-108中任一序列具有至少99％的序列相同性的氨基酸序列。
5.如权利要求1所述的SARS-COV-2刺突蛋白结合分子，其特征在于：所述单域抗体包含SEQ ID NO:82-108中的任意一种氨基酸序列。
6.如权利要求1-5任一项所述的SARS-COV-2刺突蛋白结合分子，其特征在于：还包含免疫球蛋白Fc区。
7.如权利要求6所述的SARS-COV-2刺突蛋白结合分子，其特征在于：所述免疫球蛋白Fc区是人免疫球蛋白Fc区。
8.如权利要求7所述的SARS-COV-2刺突蛋白结合分子，其特征在于：所述免疫球蛋白Fc区是人IgG1的Fc区。
9.如权利要求8所述的SARS-COV-2刺突蛋白结合分子，其特征在于：所述免疫球蛋白Fc区的氨基酸序列为SEQIDNO:109。
10.如权利要求9所述的SARS-COV-2刺突蛋白结合分子，其特征在于：包含SEQIDNO:110-136中的至少一种氨基酸序列。
11.如权利要求1-5、7-10中任一项所述的SARS-COV-2刺突蛋白结合分子，其特征在于：具有下述特征中的至少一种：
a、与SARS-COV-2刺突蛋白结合的KD值小于1×10-8M；
b、阻断SARS-COV-2与人体细胞受体ACE2的结合；
c、抑制SARS-COV-2的传染和扩增。
12.如权利要求6所述的SARS-COV-2刺突蛋白结合分子，其特征在于：具有下述特征中的至少一种：
a、与SARS-COV-2刺突蛋白结合的KD值小于1×10-8M；
b、阻断SARS-COV-2刺突蛋白与人体细胞受体ACE2的结合；
c、抑制SARS-COV-2传染和扩增。
13.编码权利要求1-12任一项所述的SARS-COV-2刺突蛋白结合分子的核酸分子。
14.包含权利要求13所述的核酸分子及其表达调控原件的表达载体。
15.包含权利要求13所述的核酸分子并进行表达的宿主细胞。
16.获得权利要求1-12中任一项所述的SARS-COV-2刺突蛋白结合分子的方法，其特征在于，包括：
a、在允许所述SARS-COV-2刺突蛋白结合分子表达的条件下培养权利要求15所述的宿主细胞；
b、从步骤a的培养物中收集由所述宿主细胞表达的SARS-COV-2刺突蛋白结合分子。
17.一种免疫缀合物，其特征在于，包含与治疗性部分缀合的权利要求1-12任一项所述的SARS-COV-2刺突蛋白结合分子。
18.一种药物组合物，其特征在于，包含权利要求1-12任一项所述的SARS-COV-2刺突蛋白结合分子和/或权利要求17所述的免疫缀合物，以及药学上可接受的载体。
19.权利要求18所述的药物组合物在制备治疗或预防新型冠状病毒病肺炎药物中的应用。
20.一种用于检测SARS-COV-2的试剂盒，其特征在于，包含权利要求1-12任一项所述的SARS-COV-2刺突蛋白结合分子。
CN202010491549.0A 2020-06-02 2020-06-02 新型冠状病毒(sars-cov-2)刺突蛋白结合分子及其应用 Active CN111647077B (zh)

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PCT/CN2021/079568 WO2021244089A1 (zh) 2020-06-02 2021-03-08 新型冠状病毒（sars-cov-2）刺突蛋白结合分子及其应用
US18/000,515 US20230331822A1 (en) 2020-06-02 2021-03-08 SARS-COV-2 spike protein binding molecule and application thereof
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JP2022574538A JP2023527927A (ja) 2020-06-02 2021-03-08 新型コロナウイルス（ｓａｒｓ－ｃｏｖ－２）スパイクタンパク質結合分子及びその使用

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