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仅用以解释本发明，并不用于限定本发明。

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。

如本文所用的术语“免疫球蛋白单一可变结构域”是指基本上由本领域所称的“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所称的“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，免疫球蛋白单一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。免疫球蛋白单一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。单域抗体为由免疫球蛋白单一可变结构域形成的小型稳定及高效的抗原识别单元。

本领域公知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。

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.vanderLindenet al.,Journal of Immunological Methods,240(2000)185–195；Li etal.,JBiol Chem.,287(2012)13713–13721；Deffar et al.,African Journal ofBiotechn ology Vol.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“SM IP”(“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KinExA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：Sequence Analysis in Molecular Biology,vonHeinje,G.,Academic Press,1987和Sequence Analysis Primer,Gribskov,M.andDevereux,J.,eds.,M Stockton Press,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300ug。

1.1.2提取总RNA

取第6周免疫后的羊驼的外周血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＝1.97，OD260/230＝2.14，说明提取的RNA没有明显降解，纯度较好；总RNA浓度为937.5ng/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：

混匀后，65℃保温5min，迅速冰浴；

Step 2

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

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′(SEQ ID NO:108)；PrimerRev-1:5′-GGTACGTGCTGTTGAACTGTTCC-3′(SEQ ID NO:109)。

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，作为Step 2的DNA模板；

Step 2

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′(SEQ ID NO:110)；Primer Rev-2:5′-GATGTGCAGCTGCAGGAGTCTGGRGGAGG-3′(SEQ ID NO:111)。

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458ng/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，将酶切后的目的基因和pHEN1片段采用T4 DNA连接酶连接后，转化至大肠杆菌电穿孔感受态细胞TG1中，构建针对SARS-COV-2-Spike蛋白的单域抗体基因文库，命名为S2-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1.425×10⁹cfu。

表1

在上述用于计算库容量的培养皿中随机挑选8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插入率，琼脂糖凝胶电泳如图4所示，说明文库插入率为100％，文库实际库容量为1.425×10⁹cfu。

菌落PCR体系如下：

菌落PCR反应条件为：98℃10s，50℃30s，72℃1min，共31个循环。

1.1.6文库救援

从上述S2-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S2-PDL，滴度为3.5×10¹³cfu/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S2-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Vector NTI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21个不同的可特异性结合Spike-RBD蛋白的单域抗体株，单域抗体序列如SEQ ID NO:64-84，分别携带SEQID NO:1-63中的21组CDR1-3序列，具体如表2所示。

表2

21个不同的单域抗体株与包被Spike-RBD的板孵育进行ELISA测定，测定的单域抗体与Spike-RBD反应后的复孔中的OD450的值如表3所示。

表3

其中，空白为不加抗体的复孔中的OD450值。

由表3中的数据可知单域抗体与Spike-RBD蛋白进行了结合反应。

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21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21株单域抗体蛋白，使其纯度达到90％以上。

1.3.2竞争ELISA考察21株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，CatV86220)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4所示，21株不同的单域抗体株均表现出对Spike蛋白/ACE2蛋白相互作用的阻断效应。

表4

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21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21株单域抗体分别与病毒混合加入96孔板，抗体初始浓度为10μg/mL，再分别2倍稀释10个梯度，5个复孔，37℃孵育2小时，第5天检测VERO细胞是否感染病毒(若细胞不发生病变说明单域抗体具有中和病毒并阻断病毒感染VERO细胞的过程)。

检测结果如表5所示，21株单域抗体在5μg/ml以上的浓度下均可有效阻断病毒感染细胞的过程，且部分抗体在低于0.02μg/ml的浓度下依然可以有效阻断病毒感染细胞的过程。根据表5中得到的IC50(μg/ml)数据说明所获得的21株单域抗体能够阻断病毒感染细胞的过程，为有效的中和抗体。

表5

注：“+”表示可以阻断病毒感染细胞，“-”表示不能阻断病毒感染细胞。

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(SEQ ID NO:85)。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SEQ ID NO:107)，再通过overlapping PCR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，Cat V86220)。

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/LIGF-1。4h后补加EX293培养基、2mM谷氨酰胺和50μg/LIGF-1，120rpm摇培。24h后加3.8mMVPA。培养5天后，收集表达培养上清液，通过ProteinA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21个SARS-COV-2-Spike蛋白单域抗体与Fc的融合蛋白的序列如SEQ IDNO:86-106。

1.2鉴定SARS-COV-2-Spike蛋白单域抗体与Fc的融合蛋白(SEQ ID NO:86-106)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21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(1.37nm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6所示。

表6

由表6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1.58E-10，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21株SARS-COV-2-Spike蛋白单域抗体与Fc融合蛋白200ng和ACE2-Biotin 5ug，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7所示。

表7

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，21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感染恒河猴

将感染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，每天进行一次呼吸道病毒载量的检测。经检测，治疗组的6只恒河猴呼吸道中新冠病毒的平均载量相比对照组显著降低，如图5所示。继续对治疗组的6只恒河猴进行观察，每隔一周对其症状和呼吸道病毒载量进行检测。持续观察2周后，检测不出其呼吸道中的病毒载量，也未出现相应的发病症状。持续观察3个月，治疗组的6只恒河猴体内的病毒均无复发的情况，说明上述SARS-COV-2-Spike蛋白单域抗体与Fc融合蛋白可在体内发挥长效作用，且可以避免感染新型冠状病毒的活体治愈后出现复阳。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)的咽拭子，提取咽拭子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。用引物RBD-qF1(5′-CAATGGTTTAACAGGCACAGG-3′，SEQ ID NO:112)和RBD-qR1(5′-CTCAAGTGTCTGTGGATCACG-3′，SEQ ID NO:113)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长效抑制SARS-COV-2感染细胞并扩增的效果，且经过治疗后的恒河猴的复阳率为0。

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> 113

<170> PatentIn version 3.5

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<213> VHH-6

<400> 69

Gln Leu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Asp

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly His Thr Phe Ser Arg Phe

20 25 30

Gly Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Gly Arg Asn Asp Gly Gly Phe Pro Tyr Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Gly Lys Thr Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Ala Ala Thr Ser Pro Glu Ser Gly Arg Tyr Asp Tyr Arg Gly Gln Gly

100 105 110

Thr Gln Val Thr Val Ser Ser

115

<210> 70

<211> 129

<212> PRT

<213> VHH-7

<400> 70

Gln Leu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Asp

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Thr Ser Arg Thr Phe Ser Val Gly Glu

20 25 30

Val Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Gly Ile Arg Trp Gly Ser Glu Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Gly Lys Thr Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Ala Thr Glu Lys Val Tyr Ser Ala Tyr Tyr Tyr Tyr Ser Arg

100 105 110

Asp Arg Leu Tyr Asp Tyr Arg Gly Gln Gly Thr Gln Val Thr Val Ser

115 120 125

Ser

<210> 71

<211> 128

<212> PRT

<213> VHH-8

<400> 71

Gln Leu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Asp

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Phe Thr Phe Asp Asp Tyr

20 25 30

Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Gly Ile Ser Ser Arg Asp Gly Arg Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Gly Lys Thr Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Ala Asn His Leu Thr Tyr Tyr Ser Gly Ser Tyr Tyr Phe Pro

100 105 110

Tyr Glu Tyr His Tyr Arg Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210> 72

<211> 129

<212> PRT

<213> VHH-9

<400> 72

Gln Leu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Asp

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Thr Ser Arg Cys Thr Phe Asn Trp Asp

20 25 30

Gly Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Gly Ile Ser Trp Ser Gly Gln Glu Pro Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Gly Lys Thr Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Ala Ala Gln Tyr Thr Gly Ala Ser Tyr Ser Ile Leu Arg Asp

100 105 110

Gln Val Gly Tyr Asp Tyr Arg Gly Gln Gly Thr Gln Val Thr Val Ser

115 120 125

Ser

<210> 73

<211> 123

<212> PRT

<213> VHH-10

<400> 73

Gln Leu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Asp

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Thr Ser Thr Arg Thr Phe Ser Asn Tyr

20 25 30

Asn Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Gly Ile Ser Arg Ala Gly Ala Glu Ile Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Gly Lys Thr Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Ala Ala Arg Val Met Gly Val Ser Pro Ala Ile Tyr Asn Tyr

100 105 110

Arg Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210> 74

<211> 126

<212> PRT

<213> SP11

<400> 74

Gln Leu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Asp

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly His Thr Phe Ser Ser Tyr

20 25 30

Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Gly Ile Asp Trp Ser Gly Asp Glu Ala Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Gly Lys Thr Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Ala Asp Arg Gly Leu Ser Tyr Tyr Tyr Asp Arg Thr Gln Glu

100 105 110

Tyr Gly Ile Arg Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210> 75

<211> 124

<212> PRT

<213> VHH-12

<400> 75

Gln Leu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Asp

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Arg Thr Leu Arg Ser Phe

20 25 30

Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Gly Ile Ser Trp Ser Gly Asp Asp Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Gly Lys Thr Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Ala Gly Gly His Thr Ile Asn His Asp Ile Ile Ser Pro Asp

100 105 110

Tyr Arg Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210> 76

<211> 126

<212> PRT

<213> VHH-13

<400> 76

Gln Leu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Asp

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Leu Thr Gly Ser Thr Phe

20 25 30

Val Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Gly Ile Arg Trp Thr Tyr Gly Arg Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Gly Lys Thr Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Ala Arg Glu Gly Tyr Arg Gly Ser Ala Tyr Gln Thr Ser Asp

100 105 110

Phe Asp Tyr Arg Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210> 77

<211> 119

<212> PRT

<213> VHH-14

<400> 77

Gln Leu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Asp

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Thr Ser Ala Ser Gly Arg Ser Thr His

20 25 30

Arg Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Gly Ile Met Pro Gly Gly Gly Phe Pro Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Gly Lys Thr Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Ala Arg Ser Glu Thr Gly Glu Tyr Asp Asn Arg Gly Gln Gly

100 105 110

Thr Gln Val Thr Val Ser Ser

115

<210> 78

<211> 133

<212> PRT

<213> VHH-15

<400> 78

Gln Leu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Asp

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Phe Thr Leu Asp Tyr Tyr

20 25 30

Arg Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Gly Ile Ser Ser Ser Asp Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Gly Lys Thr Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Thr Gly Pro Leu Thr Tyr Tyr Ser Gly Arg Tyr Tyr Leu Gly

100 105 110

Gly Cys Phe Asp Thr Tyr Ala Met Asp Phe Arg Gly Gln Gly Thr Gln

115 120 125

Val Thr Val Ser Ser

130

<210> 79

<211> 125

<212> PRT

<213> VHH-16

<400> 79

Gln Leu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Asp

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Arg Thr Phe Ser Ser Tyr

20 25 30

Thr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Gly Ile Ser Arg Ser Gly Gly Gly Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Gly Lys Thr Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Val Ser Arg Arg Val Leu Leu Thr Asp Thr Pro Arg Asp Tyr

100 105 110

Asp Arg Arg Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210> 80

<211> 128

<212> PRT

<213> VHH-17

<400> 80

Gln Leu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Asp

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Asn Thr Leu Asp Tyr Tyr

20 25 30

Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Gly Ile Lys Ser Ser Asp Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Gly Lys Thr Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Ile Gly Pro Leu Thr Tyr Tyr Ser Gly Arg Tyr Tyr Phe Pro

100 105 110

Tyr Asp Tyr Asp Tyr Arg Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210> 81

<211> 128

<212> PRT

<213> VHH-18

<400> 81

Gln Leu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Asp

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Arg Thr Ser Ser Arg Tyr

20 25 30

Val Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Gly Ile Thr Ser Thr Gly Phe Gly Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Gly Lys Thr Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Ala Asp Thr Ser Leu Leu Thr Arg Arg Pro Ser Asp Ser Ala

100 105 110

Ser Pro Tyr Asp Tyr Arg Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210> 82

<211> 120

<212> PRT

<213> VHH-19

<400> 82

Gln Leu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Asp

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Ser Ile Ala Ser Ile Thr

20 25 30

Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Gly Ser Asn Ser Arg Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Gly Lys Thr Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Tyr Val Asn Thr Leu Arg Ala Glu Met Pro Glu Ala Tyr Arg Gly Gln

100 105 110

Gly Thr Gln Val Thr Val Ser Ser

115 120

<210> 83

<211> 128

<212> PRT

<213> VHH-20

<400> 83

Gln Leu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Asp

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Asp Thr Phe Asn Asn Tyr

20 25 30

Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Gly Ile Ala Arg Ser Gly Arg Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Gly Lys Thr Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Ala Asp Pro Ala Pro Leu Trp Phe Ala Trp Thr Ser Asp Met

100 105 110

Ala Lys Tyr Lys Phe Arg Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210> 84

<211> 127

<212> PRT

<213> VHH-21

<400> 84

Gln Leu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Asp

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Arg Ile Pro Ser Trp Asn

20 25 30

Ile Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Gly Ile Arg Trp Thr Ser Gly Thr Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Gly Lys Thr Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Gly Trp Arg Arg Gly Gly Val Gln Gln Ile Ser Thr Gly Ser His

100 105 110

Asp Tyr Asp Pro Arg Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210> 85

<211> 232

<212> PRT

<213> Fc

<400> 85

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> 86

<211> 355

<212> PRT

<213> VHH-1-Fc

<400> 86

Gln Leu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Asp

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Arg Ala Val Thr Ile Tyr

20 25 30

His Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Gly Ile Ser Pro Ser Arg Val Ile Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Gly Lys Thr Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Thr Arg Gly Ser Phe Asp Arg Asn Arg Val Asn Tyr Asp Tyr

100 105 110

Arg 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> 87

<211> 358

<212> PRT

<213> VHH-2-Fc

<400> 87

Gln Leu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Asp

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Arg Thr Phe Ser Ser Tyr

20 25 30

Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Gly Ile Ser Trp Thr Gly Asp Thr Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Gly Lys Thr Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Ala Asp Arg Gly Leu Ser Tyr Tyr Tyr Tyr Arg Thr Gln Asp

100 105 110

Tyr Asp Tyr Arg 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> 88

<211> 363

<212> PRT

<213> VHH-3-Fc

<400> 88

Gln Leu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Asp

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Phe Thr Leu Asp Tyr Tyr

20 25 30

Thr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Gly Thr Ser Ser Ala Asp Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Gly Lys Thr Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Ala Ala Pro Gly Ala Tyr Tyr Ser Gly Thr Tyr Tyr Arg Ala

100 105 110

Cys His Ser Ala Gly Met Glu Tyr Arg 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> 89

<211> 360

<212> PRT

<213> VHH-4-Fc

<400> 89

Gln Leu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Asp

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Phe Thr Phe Asp Asp Tyr

20 25 30

Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Gly Ile Gly Ser Arg Asp Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Gly Lys Thr Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Ala Pro Asp Ser Val Ser Tyr Tyr Gly Trp Ser Leu Asn Pro

100 105 110

Tyr Glu Tyr Asp Tyr Arg Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

130 135 140

Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

145 150 155 160

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

165 170 175

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

180 185 190

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

195 200 205

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

210 215 220

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

225 230 235 240

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

245 250 255

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr

260 265 270

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

275 280 285

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

290 295 300

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

305 310 315 320

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

325 330 335

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

340 345 350

Ser Leu Ser Leu Ser Pro Gly Lys

355 360

<210> 90

<211> 351

<212> PRT

<213> VHH-5-Fc

<400> 90

Gln Leu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Asp

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Gly Thr Phe Ser Arg Ser

20 25 30

Gly Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Gly Arg Ala Asp Val Gly Phe Pro Tyr Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Gly Lys Thr Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Ala Ala Thr Ser Leu Gln Ser Gly Lys Tyr Asp Tyr Arg 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> 91

<211> 351

<212> PRT

<213> VHH-6-Fc

<400> 91

Gln Leu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Asp

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly His Thr Phe Ser Arg Phe

20 25 30

Gly Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Gly Arg Asn Asp Gly Gly Phe Pro Tyr Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Gly Lys Thr Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Ala Ala Thr Ser Pro Glu Ser Gly Arg Tyr Asp Tyr Arg 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> 92

<211> 361

<212> PRT

<213> VHH-7-Fc

<400> 92

Gln Leu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Asp

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Thr Ser Arg Thr Phe Ser Val Gly Glu

20 25 30

Val Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Gly Ile Arg Trp Gly Ser Glu Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Gly Lys Thr Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Ala Thr Glu Lys Val Tyr Ser Ala Tyr Tyr Tyr Tyr Ser Arg

100 105 110

Asp Arg Leu Tyr Asp Tyr Arg 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> 93

<211> 360

<212> PRT

<213> VHH-8-Fc

<400> 93

Gln Leu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Asp

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Phe Thr Phe Asp Asp Tyr

20 25 30

Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Gly Ile Ser Ser Arg Asp Gly Arg Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Gly Lys Thr Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Ala Asn His Leu Thr Tyr Tyr Ser Gly Ser Tyr Tyr Phe Pro

100 105 110

Tyr Glu Tyr His Tyr Arg Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

130 135 140

Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

145 150 155 160

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

165 170 175

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

180 185 190

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

195 200 205

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

210 215 220

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

225 230 235 240

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

245 250 255

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr

260 265 270

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

275 280 285

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

290 295 300

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

305 310 315 320

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

325 330 335

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

340 345 350

Ser Leu Ser Leu Ser Pro Gly Lys

355 360

<210> 94

<211> 361

<212> PRT

<213> VHH-9-Fc

<400> 94

Gln Leu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Asp

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Thr Ser Arg Cys Thr Phe Asn Trp Asp

20 25 30

Gly Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Gly Ile Ser Trp Ser Gly Gln Glu Pro Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Gly Lys Thr Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Ala Ala Gln Tyr Thr Gly Ala Ser Tyr Ser Ile Leu Arg Asp

100 105 110

Gln Val Gly Tyr Asp Tyr Arg 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> 95

<211> 355

<212> PRT

<213> VHH-10-Fc

<400> 95

Gln Leu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Asp

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Thr Ser Thr Arg Thr Phe Ser Asn Tyr

20 25 30

Asn Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Gly Ile Ser Arg Ala Gly Ala Glu Ile Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Gly Lys Thr Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Ala Ala Arg Val Met Gly Val Ser Pro Ala Ile Tyr Asn Tyr

100 105 110

Arg 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> 96

<211> 358

<212> PRT

<213> VHH-11-Fc

<400> 96

Gln Leu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Asp

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly His Thr Phe Ser Ser Tyr

20 25 30

Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Gly Ile Asp Trp Ser Gly Asp Glu Ala Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Gly Lys Thr Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Ala Asp Arg Gly Leu Ser Tyr Tyr Tyr Asp Arg Thr Gln Glu

100 105 110

Tyr Gly Ile Arg 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> 97

<211> 356

<212> PRT

<213> VHH-12-Fc

<400> 97

Gln Leu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Asp

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Arg Thr Leu Arg Ser Phe

20 25 30

Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Gly Ile Ser Trp Ser Gly Asp Asp Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Gly Lys Thr Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Ala Gly Gly His Thr Ile Asn His Asp Ile Ile Ser Pro Asp

100 105 110

Tyr Arg 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> 98

<211> 358

<212> PRT

<213> VHH-13-Fc

<400> 98

Gln Leu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Asp

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Leu Thr Gly Ser Thr Phe

20 25 30

Val Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Gly Ile Arg Trp Thr Tyr Gly Arg Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Gly Lys Thr Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Ala Arg Glu Gly Tyr Arg Gly Ser Ala Tyr Gln Thr Ser Asp

100 105 110

Phe Asp Tyr Arg 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> 99

<211> 351

<212> PRT

<213> VHH-14-Fc

<400> 99

Gln Leu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Asp

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Thr Ser Ala Ser Gly Arg Ser Thr His

20 25 30

Arg Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Gly Ile Met Pro Gly Gly Gly Phe Pro Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Gly Lys Thr Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Ala Arg Ser Glu Thr Gly Glu Tyr Asp Asn Arg 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> 100

<211> 365

<212> PRT

<213> VHH-15-Fc

<400> 100

Gln Leu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Asp

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Phe Thr Leu Asp Tyr Tyr

20 25 30

Arg Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Gly Ile Ser Ser Ser Asp Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Gly Lys Thr Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Thr Gly Pro Leu Thr Tyr Tyr Ser Gly Arg Tyr Tyr Leu Gly

100 105 110

Gly Cys Phe Asp Thr Tyr Ala Met Asp Phe Arg Gly Gln Gly Thr Gln

115 120 125

Val Thr Val Ser Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys

130 135 140

Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu

145 150 155 160

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

165 170 175

Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys

180 185 190

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

195 200 205

Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu

210 215 220

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

225 230 235 240

Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys

245 250 255

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

260 265 270

Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

275 280 285

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

290 295 300

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

305 310 315 320

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

325 330 335

Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

340 345 350

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

355 360 365

<210> 101

<211> 357

<212> PRT

<213> VHH-16-Fc

<400> 101

Gln Leu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Asp

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Arg Thr Phe Ser Ser Tyr

20 25 30

Thr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Gly Ile Ser Arg Ser Gly Gly Gly Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Gly Lys Thr Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Val Ser Arg Arg Val Leu Leu Thr Asp Thr Pro Arg Asp Tyr

100 105 110

Asp Arg Arg 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> 102

<211> 360

<212> PRT

<213> VHH-17-Fc

<400> 102

Gln Leu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Asp

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Asn Thr Leu Asp Tyr Tyr

20 25 30

Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Gly Ile Lys Ser Ser Asp Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Gly Lys Thr Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Ile Gly Pro Leu Thr Tyr Tyr Ser Gly Arg Tyr Tyr Phe Pro

100 105 110

Tyr Asp Tyr Asp Tyr Arg Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

130 135 140

Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

145 150 155 160

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

165 170 175

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

180 185 190

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

195 200 205

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

210 215 220

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

225 230 235 240

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

245 250 255

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr

260 265 270

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

275 280 285

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

290 295 300

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

305 310 315 320

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

325 330 335

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

340 345 350

Ser Leu Ser Leu Ser Pro Gly Lys

355 360

<210> 103

<211> 360

<212> PRT

<213> VHH-18-Fc

<400> 103

Gln Leu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Asp

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Arg Thr Ser Ser Arg Tyr

20 25 30

Val Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Gly Ile Thr Ser Thr Gly Phe Gly Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Gly Lys Thr Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Ala Asp Thr Ser Leu Leu Thr Arg Arg Pro Ser Asp Ser Ala

100 105 110

Ser Pro Tyr Asp Tyr Arg Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

130 135 140

Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

145 150 155 160

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

165 170 175

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

180 185 190

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

195 200 205

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

210 215 220

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

225 230 235 240

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

245 250 255

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr

260 265 270

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

275 280 285

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

290 295 300

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

305 310 315 320

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

325 330 335

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

340 345 350

Ser Leu Ser Leu Ser Pro Gly Lys

355 360

<210> 104

<211> 352

<212> PRT

<213> VHH-19-Fc

<400> 104

Gln Leu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Asp

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Ser Ile Ala Ser Ile Thr

20 25 30

Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Gly Ser Asn Ser Arg Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Gly Lys Thr Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Tyr Val Asn Thr Leu Arg Ala Glu Met Pro Glu Ala Tyr Arg Gly Gln

100 105 110

Gly Thr Gln Val Thr Val Ser Ser Glu Pro Lys Ser Ser Asp Lys Thr

115 120 125

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

130 135 140

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

145 150 155 160

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

165 170 175

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

180 185 190

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

195 200 205

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

210 215 220

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

225 230 235 240

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

245 250 255

Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys

260 265 270

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

275 280 285

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

290 295 300

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

305 310 315 320

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

325 330 335

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

340 345 350

<210> 105

<211> 360

<212> PRT

<213> VHH-20-Fc

<400> 105

Gln Leu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Asp

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Asp Thr Phe Asn Asn Tyr

20 25 30

Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Gly Ile Ala Arg Ser Gly Arg Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Gly Lys Thr Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Ala Asp Pro Ala Pro Leu Trp Phe Ala Trp Thr Ser Asp Met

100 105 110

Ala Lys Tyr Lys Phe Arg Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

130 135 140

Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

145 150 155 160

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

165 170 175

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

180 185 190

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

195 200 205

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

210 215 220

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

225 230 235 240

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

245 250 255

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr

260 265 270

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

275 280 285

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

290 295 300

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

305 310 315 320

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

325 330 335

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

340 345 350

Ser Leu Ser Leu Ser Pro Gly Lys

355 360

<210> 106

<211> 359

<212> PRT

<213> VHH-21-Fc

<400> 106

Gln Leu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Asp

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Arg Ile Pro Ser Trp Asn

20 25 30

Ile Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Gly Ile Arg Trp Thr Ser Gly Thr Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Gly Lys Thr Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Gly Trp Arg Arg Gly Gly Val Gln Gln Ile Ser Thr Gly Ser His

100 105 110

Asp Tyr Asp Pro Arg 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> 107

<211> 705

<212> DNA

<213> Fc DNA 序列

<400> 107

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> 108

<211> 23

<212> DNA

<213> Primer For-1

<400> 108

gtcctggctg ctcttctaca agg 23

<210> 109

<211> 23

<212> DNA

<213> Primer Rev-1

<400> 109

ggtacgtgct gttgaactgt tcc 23

<210> 110

<211> 32

<212> DNA

<213> Primer For-2

<400> 110

ctagtgcggc cgctggagac ggtgacctgg gt 32

<210> 111

<211> 29

<212> DNA

<213> Primer Rev-2

<400> 111

gatgtgcagc tgcaggagtc tggrggagg 29

<210> 112

<211> 21

<212> DNA

<213> RBD-qF1

<400> 112

caatggttta acaggcacag g 21

<210> 113

<211> 21

<212> DNA

<213> RBD-qR1

<400> 113

ctcaagtgtc tgtggatcac g 21