CN114349865A - 一种pd-1/lag-3四价双特异性抗体、其制备方法和用途 - Google Patents

一种pd-1/lag-3四价双特异性抗体、其制备方法和用途 Download PDF

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CN114349865A
CN114349865A CN202111641121.0A CN202111641121A CN114349865A CN 114349865 A CN114349865 A CN 114349865A CN 202111641121 A CN202111641121 A CN 202111641121A CN 114349865 A CN114349865 A CN 114349865A
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赵杰
黄浩旻
朱祯平
朱云霞
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Sunshine Guojian Pharmaceutical Shanghai Co Ltd
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Abstract

本发明描述了一种基于共同轻链的结构对称的四价双特异性抗体及其构建方法。本发明制备的四价双特异性抗体具有与单抗相似甚至更优的生物学活性和理化性质，可以用于治疗各种炎性疾病、癌症和其它疾病。

Description

一种PD-1/LAG-3四价双特异性抗体、其制备方法和用途
本申请为202180003485.8的分案申请。
技术领域
本发明涉及抗体领域，更具体地，本发明涉及一类四价双特异性抗体、其制备方法和用途。
背景技术
双特异性抗体是指能同时特异性结合两种抗原或两种表位的抗体分子。根据对称性，双特异性抗体可以分为结构对称的和不对称的分子。根据结合位点的多少，双特异性抗体可以分为二价、三价、四价和多价分子。
ScFv由重链可变区(VH)和轻链可变区(VL)组成，其中VH和VL通过柔性肽接头连接在一起。在ScFv中，结构域的顺序可以是VH-接头-VL或VL-接头-VH。已经报道了各种接头用于连接VH和VL，例如短丙氨酸接头，富含甘氨酸-丝氨酸的接头，采用螺旋构象的接头，以及衍生自各种免疫球蛋白和非免疫球蛋白分子的接头(Ahmad Z A,Yeap S K,Ali AM,etal.scFv antibody:principles and clinical application[J].Clinical anddevelopmental immunology,2012,2012:980250.)。ScFv通常代表一个抗体的最小结合位点。可以通过接头连接两个ScFv来构建双特异性抗体。如此构建成的双抗分子是二价的，每种抗原具有一个结合位点，分子量通常在50-60kDa左右。两个串联的ScFv片段会独立折叠并形成各自的抗原结合位点。双特异的串联scFv形式已广泛应用于癌症免疫疗法中，用于将T细胞重新靶向肿瘤细胞或肿瘤微环境中的肿瘤相关细胞。这种形式构成了双特异性T细胞衔接子(bispecific T-cell engager，BiTE)的分子基础，第一个被批准上市的BiTE分子是Blinatumomab(Huehls AM,Coupet T A,Sentman C L.Bispecific T-cell engagersfor cancer immunotherapy[J].Immunology and cell biology,2015,93(3):290-296.)。
Diabody(Db)是由两条链组成的二价分子，每条链包含一个VH和VL结构域，来自相同或不同的抗体。在Diabody中，两个可变结构域通过短接头连接，通常是5个氨基酸残基，例如GGGGS。因为接头显著短于允许链内装配所需的长度，这将导致两条ScFv链以头对尾方向二聚化，从而产生分子量与串联ScFv相当的包装紧密的Diabody分子(Wu C.Diabodies:molecular engineering and therapeutic applications[J].Drug News Perspect,2009,22(8):453.)。Diabody的两条不同的链在细胞内表达时会出现可变结构域的错误配对。此外，由于两条链之间没有非共价键相连，Diabody并不稳定(Kipriyanov S M,Moldenhauer G,Braunagel M,et al.Effect of domain order on the activity ofbacterially produced bispecific single-chain Fv antibodies[J].Journal ofmolecular biology,2003,330(1):99-111.)。
Diabody可以与人类Fc融合产生更类似免疫球蛋白的分子，称为Di-diabody(LuD,Zhang H,Koo H,et al.A fully human recombinant IgG-like bispecific antibodyto both the epidermal growth factor receptor and the insulin-like growthfactor receptor for enhanced antitumor activity[J].Journal of BiologicalChemistry,2005,280(20):19665-19672.)。两个串联的ScFv也可以与人类Fc融合产生更类似免疫球蛋白的分子，称为TaFv-Fc(Chen Z,Xie W,Acheampong D O,et al.A human IgG-like bispecific antibody co-targeting epidermal growth factor receptor andthe vascular endothelial growth factor receptor 2for enhanced antitumoractivity[J].Cancer biology&therapy,2016,17(2):139-150.)。
除以上描述的Di-diabody和TaFv-Fc之外，结构对称的四价双特异性抗体还有(不限于)以下几种类型。
在天然IgG的重链或轻链的N末端或C末端通过多肽接头连接ScFv，如此可以形成IgG-ScFv形式的双特异性抗体(Coloma M J,Morrison S L.Design and production ofnovel tetravalent bispecific antibodies[J].Nature biotechnology,1997,15(2):159.)。
在天然抗体的轻链和重链的N末端通过多肽接头分别再接入另一个抗体的VL和VH，如此可以形成DVD-Ig形式的双特异性抗体(Wu C,Ying H,Grinnell C,etal.Simultaneous targeting of multiple disease mediators by a dual-variable-domain immunoglobulin[J].Nature biotechnology,2007,25(11):1290.)。
CrossMAb是一种抗体Fab臂的功能区互换的技术，是由罗氏开发的技术平台。该技术解决了同源轻重链正确装配的问题，进一步提高了装配的成功率。基于CrossMAb的多种形式的双特异性抗体的形式已被公开(Klein C,Schaefer W,Regula J T.The use ofCrossMAb technology for the generation of bi-and multispecific antibodies[C]//MAbs.Taylor&Francis,2016,8(6):1010-1020.)。
在天然抗体重链的N末端通过多肽接头连接另一个抗体的轻链，然后VH+CH1作为独立的短链与连接到N末端的轻链配对，如此可以形成FIT-IgG形式的双特异性抗体(US10266608B2)。
IgG-TCR通过用T细胞受体(T cell receptors，TCR)的α和β链的恒定区分别替换重链的CH1和轻链的CL来提高相关的重链和轻链的配对效率(Wu X,Sereno AJ,Huang F,etal.Protein design of IgG/TCR chimeras for the co-expression of Fab-likemoieties within bispecific antibodies[C]//MAbs.Taylor&Francis,2015,7(2):364-376.)。WuXiBody在IgG-TCR的基础上进行了改进，在α和β链的恒定区之间增加了一个工程化二硫键，进一步提高了相关的重链和轻链配对的效率，以及双特异性抗体分子的稳定性(WO 2019/057122A1)。
最近还报道了一种构建四价双特异性抗体的方法。该方法基于晶体结构对Fab的重链和轻链之间的界面进行人工设计，并通过实验方法筛选出一种正交的(orthogonal)Fab界面，该正交的Fab的重链和轻链能够特异配对而不会与野生型的重链和轻链错配(Lewis S M,Wu X,Pustilnik A,et al.Generation of bispecific IgG antibodies bystructure-based design of an orthogonal Fab interface[J].Naturebiotechnology,2014,32(2):191.；Wu X,Sereno AJ,Huang F,et al.Fab-basedbispecific antibody formats with robust biophysical properties and biologicalactivity[C]//MAbs.Taylor&Francis,2015,7(3):470-482.)。
另外，结构不对称的双特异性抗体还可能采用以下几种结构(不限于)。
杵臼结构(knob-in-hole，KIH)的主要功能是促使双特异性抗体的两条不同重链异二聚化。其结构特点为：组成双特异性抗体的一条重链的CH3区发生突变形成一个突起的“杵”的结构，另一条重链的CH3区发生突变形成一个凹陷的“臼”的结构，杵臼结构设计有利于两种异源抗体重链的正确装配(Merchant AM,Zhu Z,Yuan J Q,et al.An efficientroute to human bispecific IgG[J].Nature biotechnology,1998,16(7):677.)。DuetMab通过使用KIH技术实现2条不同重链的异二聚化，并通过用工程化二硫键替换CH1-CL界面之间的天然二硫键来提高相关的重链和轻链的配对效率(Mazor Y,Oganesyan V,Yang C,et al.Improving target cell specificity using a novel monovalentbispecific IgG design[C]//MAbs.Taylor&Francis,2015,7(2):377-389.)。
另外一种增强不同重链异二聚化的方法为静电转向突变(ElectrostaticSteering Mutations)技术，该方法基于具有静电相互作用的带电残基。该方法选择改变了CH3界面中的电荷极性，使得静电匹配的Fc结构域因为有利的电荷吸引作用而利于异二聚体形成，而不利的电荷排斥作用则抑制同源二聚化(US 10011858B2；Gunasekaran K,Pentony M,Shen M,et al.Enhancing antibody Fc heterodimer formation throughelectrostatic steering effects applications to bispecific molecules andmonovalent IgG[J].Journal of Biological Chemistry,2010,285(25):19637-19646.)。
共同轻链(Common Light Chain)经常被用于双特异性抗体的构建。共同轻链能够减少重链和轻链错配引起的副产物的产生，增加双特异性抗体的产率(Brinkmann U,Kontermann RE.The making of bispecific antibodies[C]//MAbs.Taylor&Francis,2017,9(2):182-212.)。通常情况下，共同轻链需要通过实验方法筛选出来，一般可以通过杂交瘤或噬菌体展示技术获得。通常情况下，需要对抗A抗体和抗B抗体的抗体库进行高通量筛选，这要求两个抗体库的库容量不能太小(Sampei Z,Igawa T,Soeda T,etal.Identification and multidimensional optimization of an asymmetricbispecific IgG antibody mimicking the function of factor VIII cofactoractivity[J].PloS one,2013,8(2):e57479.；US 9657102B2)。
双特异性抗体正在逐步成为一类新的治疗性抗体，可以用于治疗各种炎性疾病、癌症和其它疾病。虽然最近报道了大量新的双特异性抗体的构造形式，然而，生产双特异性抗体的主要技术难点在于获得正确配对的分子。上述双特异性抗体的形式均存在错配的问题，因此会产生一种或多种错配导致的副产物或者聚集体，从而影响目的双特异性抗体的产率、纯度和理化稳定性，进而影响双特异性抗体在体内的安全性和有效性。
发明内容
本发明描述了一种基于共同轻链的结构对称的四价双特异性抗体及其构建方法。通常情况下，双特异性抗体的重链和轻链之间会出现错配，重链和重链之间也会出现错配，正确装配的效率越高错配的程度就越低。在此，本发明用共同轻链解决了重链和轻链之间的错配问题，并将一个抗体的重链可变区VH-B与CH1连接，然后通过肽接头与另一个抗体的重链可变区VH-A连接，再与重链恒定区CH1-CH2-CH3连接，构成一条长重链。长重链和共同轻链的基因在同一细胞内表达时，每条长重链会与两条共同轻链配对，长重链与共同轻链之间不会出现错配，因为与每条长重链配对的两条轻链是相同的；长重链之间会进行同源二聚化而不是异源二聚化，因此长重链之间也不会有错配问题。本发明的四价双特异性抗体不需要进行Fc修饰，制备方法简便，具有与单抗相似甚至更优的生物学活性和理化性质。
进一步的，本申请的发明人在对上述四价双特异性抗体长期研究的过程中意外地发现：抗人PD-1抗体mAb1-25-Hu(609)主要通过重链结合PD-1分子，而对轻链依赖较小。因此，可以将609的重链可变区/重链与其他靶点的抗体的重链可变区/重链和轻链可变区/轻链(作为共同轻链)组合用于构建结合PD-1以及其他靶点的双特异性抗体，可应用于包括但不限于本发明的四价双特异性抗体结构或本领域已知的其他形式的包含共同轻链的双特异性抗体结构。
因此，本发明的第一个目的在于提供一种四价双特异性抗体。
本发明的第二个目的在于提供一种编码所述四价双特异性抗体的分离的核苷酸。
本发明的第三个目的在于提供一种包含所述核苷酸的表达载体。
本发明的第四个目的在于提供一种包含所述表达载体的宿主细胞。
本发明的第五个目的在于提供一种所述的四价双特异性抗体的制备方法。
本发明的第六个目的在于提供一种包含所述的四价双特异性抗体的药物组合物。
本发明的第七个目的在于提供所述的四价双特异性抗体或所述的药物组合物在制备治疗癌症、炎性疾病、自体免疫性疾病和其它病症的药物中的用途。
本发明的第八个目的在于提供一种四价双特异性抗体的构建方法。
本发明的第九个目的在于提供一种包含如SEQ ID NO：83所示的氨基酸序列的重链可变区的双特异性抗体。
本发明的第十个目的在于提供包含如SEQ ID NO：83所示的氨基酸序列的重链可变区用于构建双特异性抗体的用途。
为了达到上述目的，本发明提供了以下技术方案：
本发明的第一个方面提供了一种四价双特异性抗体，包含：
两条多肽链，所述多肽链从N末端到C末端包含VH-B-CH1-肽接头-VH-A-CH1-CH2-CH3，其中，所述VH-A为第一抗体的重链可变区，VH-B为第二抗体的重链可变区，所述CH1为重链恒定区的第一结构域，所述CH2为重链恒定区的第二结构域，所述CH3为重链恒定区的第三结构域，所述第一抗体特异性结合第一抗原，所述第二抗体特异性结合第二抗原；
四条共同轻链，所述共同轻链从N末端到C末端包含VL-CL，其中，所述VL为轻链可变区，所述CL为轻链恒定区，所述多肽链的VH-A-CH1和所述多肽链的VH-B-CH1分别与所述共同轻链的VL-CL配对，所述VH-A和所述VL形成第一抗原结合位点，所述VH-B与所述VL形成第二抗原结合位点。
本发明的双特异性抗体的结构如图1所示。
根据本发明，所述共同轻链通过以下方法筛选获得：
将第一抗体和第二抗体的重链和轻链分别交换获得第一抗体的重链与第二抗体的轻链组合以及第二抗体的重链与第一抗体的轻链组合的杂合抗体，如果：
(a)第一抗体的重链与第二抗体的轻链组合成的杂合抗体能够特异性结合第一抗原，则选择第二抗体的轻链作为共同轻链；
(b)第二抗体的重链与第一抗体的轻链组合成的杂合抗体能够特异性结合第二抗原，则选择第一抗体的轻链作为共同轻链；
(c)第一抗体的重链与第二抗体的轻链组合成的杂合抗体不能特异性结合第一抗原，且第二抗体的重链与第一抗体的轻链组合成的杂合抗体不能特异性结合第二抗原，则将第一抗体的轻链进行回复突变使得第二抗体的重链与第一抗体的突变轻链组合的杂合抗体能够特异性结合第二抗原，然后选择第一抗体的突变轻链作为共同轻链；或
(d)第一抗体的重链与第二抗体的轻链组合成的杂合抗体不能特异性结合第一抗原，且第二抗体的重链与第一抗体的轻链组合成的杂合抗体不能特异性结合第二抗原，则将第二抗体的轻链进行回复突变使得第一抗体的重链与第二抗体的突变轻链组合的杂合抗体能够特异性结合第一抗原，然后选择第二抗体的突变轻链作为共同轻链。
其中，对于上述(a)和(b)的情形，当杂合抗体能够特异性结合抗原时，作为优选的实施方式，同样可以选择将轻链进行回复突变从而进一步提高杂合抗体与抗原结合的亲和力。
因此，进一步的，所述共同轻链通过以下方法筛选获得：
将第一抗体和第二抗体的重链和轻链分别交换获得第一抗体的重链与第二抗体的轻链组合以及第二抗体的重链与第一抗体的轻链组合的杂合抗体，如果：
(a’)第一抗体的重链与第二抗体的轻链组合成的杂合抗体能够特异性结合第一抗原，则将第二抗体的轻链进行回复突变使得第一抗体的重链与第二抗体的突变轻链组合的杂合抗体结合第一抗原的亲和力优于第一抗体的重链与第二抗体的轻链组合的杂合抗体结合第一抗原的亲和力，然后选择第二抗体的突变轻链作为共同轻链；
(b’)第二抗体的重链与第一抗体的轻链组合成的杂合抗体能够特异性结合第二抗原，则将第一抗体的轻链进行回复突变使得第二抗体的重链与第一抗体的突变轻链组合的杂合抗体结合第二抗原的亲和力优于第二抗体的重链与第一抗体的轻链组合的杂合抗体结合第二抗原的亲和力，然后选择第一抗体的突变轻链作为共同轻链。
根据本发明，所述肽接头为人工连接子。优选的，所述人工连接子选自由以下组成的组：G、GS、SG、GGS、GSG、SGG、GGG、GGGS、SGGG、GGGGS、GGGGSGS、GGGGSGGS、GGGGSGGGGS、GGGGSGGGGSGGGGS(SEQ ID NO：150)、AKTTPKLEEGEFSEAR、AKTTPKLEEGEFSEARV、AKTTPKLGG、SAKTTPKLGG、SAKTTP、RADAAP、RADAAPTVS、RADAAAAGGPGS、SAKTTPKLEEGEFSEARV、ADAAP、ADAAPTVSIFPP、TVAAP、TVAAPSVFIFPP、QPKAAP、QPKAAPSVTLFPP、AKTTPP、AKTTPPSVTPLAP、AKTTAPSVYPLAP、ASTKGP、ASTKGPSVFPLAP、GENKVEYAPALMALS、GPAKELTPLKEAKVS、GHEAAAVMQVQYPAS等。更优选的，所述人工连接子为GGGGSGGGGSGGGGS(SEQ ID NO：150)。
根据本发明，所述多肽链中靠近N末端的CH1结构域和CH1-CH2-CH3可以来自相同或不同亚型的重链恒定区，包括由IgG1、IgG2、IgG3、IgG4组成的组的重链恒定区，优选来自IgG1或IgG4的重链恒定区，优选的，所述IgG4包含S228P(根据EU编码)突变。
根据本发明，所述CL为κ轻链恒定区或λ轻链恒定区。
根据本发明的优选实施例，所述第一抗原和所述第二抗原选自由以下组成的组：VEGF/PD-1、PD-1/VEGF、TGF-β/PD-1、PD-1/TGF-β、HER2/CD47、CD47/HER2、HER2/CD137、CD137/HER2、PD-1/CD137、CD137/PD-1、PD-1/CD40、CD40/PD-1、PD-1/EGFR、EGFR/PD-1、PD-1/HER2、HER2/PD-1、PD-1/CTLA-4、CTLA-4/PD-1、PD-1/LAG-3、LAG-3/PD-1。
根据本发明的优选实施例，所述VH-A或所述VH-B具有如SEQ ID NO：83所示的序列。
根据本发明的优选实施例，所述VH-A、所述VH-B和所述VL的序列选自由以下组成的组：SEQ ID NO：1、11、15；SEQ ID NO：11、1、15；SEQ ID NO：20、11、15；SEQ ID NO：11、20、15；SEQ ID NO：29、41、42；SEQ ID NO：41、29、42；SEQ ID NO：31、41、42；SEQ ID NO：41、31、42；SEQ ID NO：53、61、54；SEQ ID NO：61、53、54；SEQ ID NO：53、77、54；SEQ ID NO：77、53、54；SEQ ID NO：83、91、92；SEQ ID NO：91、83、92；SEQ ID NO：83、105、106；SEQ ID NO：105、83、106；SEQ ID NO：83、113、114；SEQ ID NO：113、83、114；SEQ ID NO：83、117、118；SEQ IDNO：117、83、118；SEQ ID NO：83、119、120；SEQ ID NO：119、83、120；SEQ ID NO：83、121、122；SEQ ID NO：121、83、122。
根据本发明的优选实施例，所述多肽链和所述共同轻链的序列选自由以下组成的组：SEQ ID NO：16、13；SEQ ID NO：18、13；SEQ ID NO：21、13；SEQ ID NO：45、43；SEQ ID NO：47、43；SEQ ID NO：49、43；SEQ ID NO：51、43；SEQ ID NO：65、63；SEQ ID NO：67、63；SEQ IDNO：79、63；SEQ ID NO：81、63；SEQ ID NO：95、93；SEQ ID NO：97、93；SEQ ID NO：109、107；SEQ ID NO：111、107；SEQ ID NO：131、123；SEQ ID NO：133、125；SEQ ID NO：135、127；SEQID NO：137、127；SEQ ID NO：139、127；SEQ ID NO：141、127；SEQ ID NO：143、129；SEQ IDNO：145、129。
本发明的第二个方面提供了一种分离的核苷酸，所述的核苷酸编码所述的四价双特异性抗体。
本发明的第三个方面提供了一种表达载体，所述的表达载体含有所述的核苷酸。
根据本发明的优选实施例，所述的核苷酸编码所述多肽链和所述共同轻链，所述核苷酸具有选自由以下组成的组的序列：SEQ ID NO：17、14；SEQ ID NO：19、14；SEQ ID NO：22、14；SEQ ID NO：46、44；SEQ ID NO：48、44；SEQ ID NO：50、44；SEQ ID NO：52、44；SEQ IDNO：66、64；SEQ ID NO：68、64；SEQ ID NO：80、64；SEQ ID NO：82、64；SEQ ID NO：96、94；SEQID NO：98、94；SEQ ID NO：110、108；SEQ ID NO：112、108；SEQ ID NO：132、124；SEQ ID NO：134、126；SEQ ID NO：136、128；SEQ ID NO：138、128；SEQ ID NO：140、128；SEQ ID NO：142、128；SEQ ID NO：144、130；SEQ ID NO：146、130。
本发明的第四个方面提供了一种宿主细胞，所述的宿主细胞含有所述的表达载体。
本发明的第五个方面提供了所述的四价双特异性抗体的制备方法，所述方法包含以下步骤：
(a)在表达条件下，培养所述的宿主细胞，从而表达所述的四价双特异性抗体；
(b)分离并纯化(a)所述的四价双特异性抗体。
本发明的第六个方面提供了一种药物组合物，所述药物组合物含有如上所述的四价双特异性抗体和药学上可接受的载体。
本发明的第七个方面提供了所述的四价双特异性抗体或所述的药物组合物在制备治疗癌症和炎性疾病、自体免疫性疾病和其它病症的药物中的用途。本发明还提供了治疗癌症和炎性疾病、自体免疫性疾病和其它病症的方法，包括向有需要的受试者施用所述的四价双特异性抗体或所述的药物组合物。所述癌症包括但不限于：黑素瘤(例如，转移性恶性黑素瘤)、肾癌(例如，透明细胞癌)、前列腺癌(例如，激素难治性前列腺腺癌)、胰腺癌、乳腺癌、结肠癌、肺癌(例如，非小细胞肺癌)、食道癌、头颈鳞状细胞癌、肝癌、卵巢癌、宫颈癌、甲状腺癌、成胶质细胞瘤、神经胶质瘤、白血病、淋巴瘤及其它赘生性恶性疾病。所述炎性疾病、自体免疫性疾病和其它病症包括但不限于：眼科、纤维化、哮喘、类风湿性关节炎、系统性红斑狼疮、多发性硬化、银屑病、特应性皮炎等。其中，所述受试者包括但不限于人。
本发明的第八个方面提供了一种四价双特异性抗体的构建方法，包含以下步骤：
(a)将第二抗体的重链可变区VH-B与重链恒定区的第一结构域CH1连接，所述第二抗体特异性结合第二抗原；
(b)将(a)通过肽接头与第一抗体的重链可变区VH-A连接，所述第一抗体特异性结合第一抗原；
(c)将(b)与重链恒定区CH1-CH2-CH3连接，形成多肽链；
(d)将(c)与共同轻链VL-CL分别构建入表达载体中组合表达，从而获得目的四价双特异性抗体；
其中，所述CH1为重链恒定区的第一结构域，所述CH2为重链恒定区的第二结构域，所述CH3为重链恒定区的第三结构域，所述VL为轻链可变区，所述CL为轻链恒定区，所述VH-A-CH1和所述VH-B-CH1分别与所述VL-CL配对，所述VH-A与所述VL形成第一抗原结合位点，所述VH-B与所述VL形成第二抗原结合位点。
本发明的第九个方面提供了一种双特异性抗体，其中，包含至少两个不同的重链可变区以及至少两条共同轻链，所述共同轻链包含相同的轻链可变区，所述重链可变区与所述轻链可变区形成抗原结合位点，其中，所述重链可变区包含如SEQ ID NO：83所示的氨基酸序列。
本发明的第十个方面提供了包含如SEQ ID NO：83所示的氨基酸序列的重链可变区用于构建双特异性抗体的用途，其中，所述双特异性抗体包含至少两个不同的重链可变区以及至少两条共同轻链，所述共同轻链包含相同的轻链可变区，所述重链可变区与所述轻链可变区形成抗原结合位点。
本发明中，术语“抗体(Antibody，缩写Ab)”和“免疫球蛋白G(Immunoglobulin G，缩写IgG)”是有相同结构特征的约150000道尔顿的异四聚糖蛋白，其由两条相同的轻链(LC)和两条相同的重链(HC)组成。每条轻链通过一个共价二硫键与重链相连，而不同免疫球蛋白同种型(isotype)的重链间的二硫键数目不同。每条重链和轻链也有规则间隔的链内二硫键。每条重链的一端有可变区(VH)，其后是恒定区，重链恒定区由三个结构域CH1、CH2、以及CH3构成。每条轻链的一端有可变区(VL)，另一端有恒定区，轻链恒定区包括一个结构域CL；轻链的恒定区与重链恒定区的CH1结构域配对，轻链的可变区与重链的可变区配对。恒定区不直接参与抗体与抗原的结合，但是它们表现出不同的效应功能，例如参与抗体依赖的细胞介导的细胞毒性作用(ADCC，antibody-dependent cell-mediatedcytotoxicity)等。重链恒定区包括IgG1、IgG2、IgG3、IgG4亚型；轻链恒定区包括κ(Kappa)或λ(Lambda)。抗体的重链和轻链通过重链的CH1结构域和轻链的CL结构域之间的二硫键共价连接在一起，抗体的两条重链通过铰链区之间形成的多肽间二硫键共价连接在一起。
本发明中，术语“双特异性抗体(双抗)”是指能同时特异性结合两种抗原(靶点)或两种表位的抗体分子。
本发明中，术语“单克隆抗体(单抗)”指从一类基本均一的群体获得的抗体，即该群体中包含的单个抗体是相同的，除少数可能存在的天然发生的突变外。单克隆抗体高特异性地针对单个抗原位点。而且，与常规多克隆抗体制剂(通常是具有针对不同抗原决定簇的不同抗体的混合物)不同，各单克隆抗体是针对抗原上的单个决定簇。除了它们的特异性外，单克隆抗体的好处还在于它们可以通过杂交瘤培养来合成，不会被其它免疫球蛋白污染。修饰语“单克隆”表示了抗体的特性，是从基本均一的抗体群中获得的，这不应被解释成需要用任何特殊方法来生产抗体。
本发明中，术语“Fab”和“Fc”是指木瓜蛋白酶可将抗体裂解为两个完全相同的Fab段和一个Fc段。Fab段由抗体的重链的VH和CH1以及轻链的VL和CL结构域组成。Fc段即可结晶片段(fragment crystallizable，Fc)，由抗体的CH2和CH3结构域组成。Fc段无抗原结合活性，是抗体与效应分子或细胞相互作用的部位。
本发明中，术语“可变”表示抗体中可变区的某些部分在序列上有所不同，它形成各种特定抗体对其特定抗原的结合和特异性。然而，可变性并不均匀地分布在整个抗体可变区中。它集中于重链可变区和轻链可变区中称为互补决定区(complementarity-determining region，CDR)或超变区中的三个片段中。可变区中较保守的部分称为框架区(frame region，FR)。天然重链和轻链的可变区中各自包含四个FR区，它们大致上呈β-折叠构型，由形成连接环的三个CDR相连，在某些情况下可形成部分β折叠结构。每条链中的CDR通过FR区紧密地靠在一起并与另一链的CDR一起形成了抗体的抗原结合部位(参见Kabat等，NIH Publ.No.91-3242，卷I，647-669页(1991))。
本发明中，术语“鼠源抗体”是指来源于大鼠或小鼠的抗体，优选小鼠。
本发明中，术语“人源化抗体”是指其CDR来源于非人物种(优选小鼠)抗体，抗体分子中残余的部分(包括框架区和恒定区)来源于人抗体。此外，框架区残基可被改变以维持结合亲和性。
本发明中，术语“特异性结合”和“结合”是指两分子间的非随机的结合反应，如抗体和其所针对的抗原之间的反应。通常，抗体以小于大约10-7M，例如小于大约10-8M、10-9M、10-10M、10-11M或更小的平衡解离常数(KD)结合该抗原。本发明中，术语“KD”是指特定抗体-抗原相互作用的平衡解离常数，其用于描述抗体与抗原之间的结合亲和力。平衡解离常数越小，抗体-抗原结合越紧密，抗体与抗原之间的亲和力越高。例如，使用表面等离子体共振术(Surface Plasmon Resonance，缩写SPR)在BIACORE仪中测定抗体与抗原的结合亲和力或使用ELISA测定抗体与抗原结合的相对亲和力。
本发明中，术语“价”是指抗体分子中存在指定数量的抗原结合位点。优选的，本发明的双特异性抗体具有四个抗原结合位点，是四价的。本发明中，抗原结合位点包含重链可变区(VH)和轻链可变区(VL)。
本发明中，术语“表位”是指与抗体特异性结合的多肽决定簇。本发明的表位是抗原中被抗体结合的区域。
本发明中，术语“肽接头”是指具有氨基酸序列的肽。本发明的肽接头是天然连接子或人工连接子。优选的，本发明的肽接头是人工连接子。本发明的多肽接头可以选自G、GS、SG、GGS、GSG、SGG、GGG、GGGS、SGGG、GGGGS、GGGGSGS、GGGGSGGS、GGGGSGGGGS、GGGGSGGGGSGGGGS(SEQ ID NO：150)、AKTTPKLEEGEFSEAR、AKTTPKLEEGEFSEARV、AKTTPKLGG、SAKTTPKLGG、SAKTTP、RADAAP、RADAAPTVS、RADAAAAGGPGS、SAKTTPKLEEGEFSEARV、ADAAP、ADAAPTVSIFPP、TVAAP、TVAAPSVFIFPP、QPKAAP、QPKAAPSVTLFPP、AKTTPP、AKTTPPSVTPLAP、AKTTAPSVYPLAP、ASTKGP、ASTKGPSVFPLAP、GENKVEYAPALMALS、GPAKELTPLKEAKVS和GHEAAAVMQVQYPAS等。接头还可以是在体内可断裂的肽接头、蛋白酶(如MMP)敏感性接头、可以通过还原而断裂的基于二硫键的接头等，参见先前所描述的(Fusion ProteinTechnologies for Biopharmaceuticals：Applications andChallenges，由StefanR.Schmidt编辑)，或本领域已知的任何可断裂的接头。这些可断裂的接头可以用于在体内释放分子顶部的Fab，以便提高组织渗透和分布，增强与靶标的结合，减少潜在副作用，以及调节2个不同的Fab区的体内功能和半衰期。最优选的，本发明的人工连接子为GGGGSGGGGSGGGGS(SEQ ID NO：150)。
本发明中，术语“共同轻链”是指包含相同的轻链可变区和轻链恒定区的轻链，其能够与结合第一抗原的第一抗体的重链配对，形成特异性结合第一抗原的结合位点，也能够与结合第二抗原的第二抗体的重链配对，形成特异性结合第二抗原的结合位点。进一步的，共同轻链的轻链可变区与第一抗体的重链可变区形成第一抗原结合位点，共同轻链的轻链可变区与第二抗体的重链可变区形成第二抗原结合位点。
本发明中，术语“表达载体”可以为pTT5，pSECtag系列，pCGS3系列，pcDNA系列载体等，以及其它用于哺乳动物表达系统的载体等，表达载体中包括连接有合适的转录和翻译调节序列的融合DNA序列。
本发明中，术语“宿主细胞”是指适用于表达上述表达载体的细胞，可以是真核细胞，如哺乳动物或昆虫宿主细胞培养系统均可用于本发明的融合蛋白的表达，CHO(中国仓鼠卵巢，Chinese Hamster Ovary)，HEK293，COS，BHK以及上述细胞的衍生细胞均可适用于本发明。
本发明中，术语“药物组合物”是指本发明的双特异性四价抗体可以和药学上可以接受的载体一起组成药物制剂组合物从而更稳定地发挥疗效，这些制剂可以保证本发明公开的双特异性四价抗体的氨基酸核心序列的构象完整性，同时还保护蛋白质的多官能团防止其降解(包括但不限于凝聚、脱氨或氧化)。
本发明描述了一种基于共同轻链的结构对称的四价双特异性抗体及其构建方法。本发明制备的双特异性抗体具有与单抗相似甚至更优的生物学活性和理化性质，可以用于治疗各种炎性疾病、癌症和其它疾病。
附图说明
图1为本发明的双特异性抗体的结构示意图，其中，VH-A表示第一抗体的重链可变区，VH-B表示第二抗体的重链可变区，VL表示共同轻链的轻链可变区，CH1、CH2和CH3是重链恒定区的三个结构域，CL是共同轻链的轻链恒定区，两条重链之间的线段表示二硫键，重链和轻链之间的线段也表示二硫键，靠近多肽链N末端的CH1和VH-A之间的线段表示人工设计的连接子，靠近多肽链C末端的CH1和CH2之间的线段表示抗体天然的连接子和铰链区(如果重链是人IgG4亚型，铰链区会含有S228P点突变)。
图2A和图2B为601和20-Hu及其杂合抗体的ELISA结果。
图3A和图3B为20-Fab-601-IgG4-V94L和601-Fab-20-IgG4-V94L的ELISA结果。
图4A和图4B为20-Fab-601-IgG4-V94L的HPLC-SEC图谱。
图5A和图5B为20-Fab-601-IgG4-V94L的HPLC-IEC图谱。
图6A～图6D为20-Fab-601-IgG4-V94L的CE-SDS图谱。
图7A和图7B为20-Fab-601-IgG4-V94L的DSC图谱。
图8为测定20-Fab-0313-IgG4-V94L对VEGF生物活性的中和能力的结果。
图9A和图9B为评估20-Fab-0313-IgG4-V94L增强MLR的功能活性的结果。
图10为评估20-Fab-0313-IgG4-V94L同时结合PD-1和VEGF165的能力。
图11为20-Fab-0313-IgG4-V94L的药代动力学性质。
图12为抗PD-1和VEGF双特异性抗体在小鼠体内的抗肿瘤生长曲线。
图13A和图13B为1D11-Hu和14-Hu及其杂合抗体的ELISA结果。
图14为mAb127和14-Hu及其杂合抗体的ELISA结果。
图15A和图15B为14-Fab-1D11-IgG4、1D11-Fab-14-IgG4、14-Fab-127-IgG4和127-Fab-14-IgG4的ELISA结果。
图16A和图16B为评估14-Fab-127-IgG4增强MLR的功能活性的结果。
图17为评估14-Fab-127-IgG4同时结合TGF-β1和PD-1的能力。
图18为抗PD-1和TGF-beta双特异性抗体在小鼠体内的抗肿瘤生长曲线。
图19A和图19B为19H6-Hu和Anti-CD47B-Hu及其杂合抗体的ELISA结果。
图20A和图20B为CD47B-Fab-19H6-IgG1和19H6-Fab-CD47B-IgG1的ELISA结果。
图21A和图21B为19H6-Hu和94-Hu及其杂合抗体的ELISA结果。
图22A和图22B为94-Fab-19H6-IgG1-LALA和19H6-Fab-94-IgG1-LALA的ELISA结果。
图23A和图23B为609和Anti-CD137-Hu及其杂合抗体的ELISA结果。
图24A和图24B为609-Fab-137-IgG4和137-Fab-609-IgG4的ELISA结果。
图25A和图25B为609和Anti-CD40-Hu及其杂合抗体的ELISA结果。
图26A和图26B为609-Fab-40-IgG4和40-Fab-609-IgG4的ELISA结果。
图27为609-HC+Cetuximab-LC、609-HC+Bevacizumab-LC、609-HC+Trastuzumab-LC、609-HC+Pertuzumab-LC、609-HC+Ipilimumab-LC和609-HC+5E7-Hu-LC的ELISA结果。
图28为609-HC+Cetuximab-LC、609-HC+Bevacizumab-LC、609-HC+Trastuzumab-LC、609-HC+Pertuzumab-LC、609-HC+Ipilimumab-LC和609-HC+5E7-Hu-LC阻断PD-1/PD-L1相互作用的能力结果。
图29为609-HC+Cetuximab-LC、609-HC+Bevacizumab-LC、609-HC+Trastuzumab-LC、609-HC+Pertuzumab-LC、609-HC+Ipilimumab-LC和609-HC+5E7-Hu-LC增强混合淋巴细胞反应的能力结果。
图30为流式细胞法测定609、609-HC+Cetuximab-LC、609-HC+Bevacizumab-LC、609-HC+Pertuzumab-LC、609-HC+Ipilimumab-LC和609-HC+5E7-Hu-LC对细胞表面PD-1的结合能力。
图31为609轻链可变区的丙氨酸扫描结果。
图32A和图32B为609-Fab-Cetuximab-IgG4的ELISA结果。
图33A和图33B为609-Fab-Pertuzumab-IgG4的ELISA结果。
图34A和图34B为609-Fab-Ipilimumab-IgG1、Ipilimumab-Fab-609-IgG1、609-Fab-Ipilimumab-IgG4和Ipilimumab-Fab-609-IgG4的ELISA结果。
图35为抗PD-1和CTLA-4双特异性抗体的功能活性测定结果。
图36为抗PD-1和CTLA-4双特异性抗体的ADCC活性测定结果。
图37为抗PD-1和CTLA-4双特异性抗体在大鼠体内的药代动力学。
图38为抗PD-1和CTLA-4双特异性抗体在小鼠体内的抗肿瘤生长曲线。
图39A和图39B为609-Fab-5E7-IgG4和5E7-Fab-609-IgG4的ELISA结果。
图40为测定PD-1和LAG-3双特异性抗体同时结合两种抗原的能力。
图41为抗PD-1和LAG-3双特异性抗体的功能活性测定。
图42为抗PD-1和LAG-3双特异性抗体在小鼠体内的抗肿瘤生长曲线。
图43A～图43E为杂合抗体的特异性检验。
图44A～图44H为609-Fab-Cetuximab-IgG4、609-Fab-Pertuzumab-IgG4、609-Fab-Ipilimumab-IgG1、Ipilimumab-Fab-609-IgG1、609-Fab-Ipilimumab-IgG4、Ipilimumab-Fab-609-IgG4、609-Fab-5E7-IgG4和5E7-Fab-609-IgG4的HPLC-SEC图谱。
图45A和45B为609-Fab-Ipilimumab-IgG1和609-Fab-5E7-IgG4的HPLC-IEC图谱。
图46A～图46D为609-Fab-Ipilimumab-IgG1和609-Fab-5E7-IgG4的NR-CE-SDS和R-CE-SDS的图谱。
具体实施方式
以下实施例中使用的表达纯化抗体方法说明如下：将外源基因构建到pcDNA3.4(购自Thermo Fisher Scientific)表达载体中，利用PEI(Polyethylenimine)转染法将表达载体的组合转入HEK293F细胞(购自Thermo Fisher Scientific)中以表达抗体，然后用ProteinA亲和层析纯化抗体。
以下实施例中使用的ELISA检测方法说明如下：将重组蛋白分别包被酶标板，用含有1％牛血清白蛋白的PBST(PBST为含0.05％Tween-20的磷酸盐缓冲液)封闭酶标板。将待测抗体进行梯度稀释，然后转移到上述包被重组蛋白的酶标板中，室温孵育半小时后洗板；加入适当稀释的HRP(Horseradish Peroxidase)标记的羊抗人抗体(Fc或Fab-specific，购自Sigma)，室温孵育半小时后洗板；每孔加入100μl以TMB(3,3′,5,5′-Tetramethylbenzidine)为底物的显色液，室温孵育1～5min；加50μl终止液(2M H2SO4)终止反应；酶标仪(SpectraMax 190)读取OD450，用GraphPad Prism6进行作图和数据分析，并计算EC50。
以下实施例中使用的评估增强混合淋巴细胞反应(Mixed Lymphocyte Reaction，MLR)的能力方法说明如下：用Histopaque(购自Sigma)从人血液中分离出外周血单个核细胞(Peripheral Blood Mononuclear Cell，缩写PBMC)，然后将PBMC中的单核细胞通过贴壁法分离出来，然后用IL-4(25ng/ml)联合GM-CSF(25ng/ml)诱导单核细胞分化成诱导的树突状细胞。七天之后，消化收集上述诱导的树突状细胞。用上述方法从另外供体的血液中分离出PBMC，然后用MACS磁铁和CD4MicroBeads(购自Miltenyi biotec)从中分离CD4+T细胞。将诱导的树突状细胞(104/孔)和分离出的CD4+T细胞(105/孔)按比例混匀后接种到96孔板中，每孔150μl；数小时后，在上述96孔板中加入50μl梯度稀释的抗体；将96孔板置于37℃细胞培养箱中孵育3天。上述实验过程中使用AIM-V培养基(Thermo Fisher Scientific)培养细胞。然后按照标准操作流程检测IL-2和IFN-γ的分泌。IL-2和IFN-γ的检测使用标准的双抗夹心法(相关检测用的配对抗体购自BD Biosciences)。用酶标仪(SpectraMax 190)读取OD450，用GraphPad Prism6进行作图并计算EC50。
以下实施例中使用的理化性质检测方法说明如下：
HPLC-SEC
抗体是高分子量蛋白质，具有高度复杂的二级和三级结构。由于翻译后修饰、聚集和降解等变化，抗体在生物化学和生物物理特性方面是异质的。当通过分离技术分析双特异性抗体时，通常会观察到变体、聚集体和降解片段，它们的存在可能会损害安全性和有效性。在生产和存储抗体的过程中容易出现聚集体、降解片段和不完整组装的分子。本发明使用高效液相色谱-尺寸排阻色谱(High-performance liquid chromatography–sizeexclusion chromatography，HPLC-SEC)检测样品中上述杂质的含量。聚集体的分子量要大于单体，因此相应峰的保留时间较短；降解片段或不完整组装分子的分子量要小于单体，因此相应峰的保留时间较长。HPLC-SEC所用色谱仪为Dionex Ultimate 3000；流动相配制方法如下：取适量20mM磷酸二氢钠母液，用20mM磷酸氢二钠调节PH至6.8±0.1；进样量：20μg；色谱柱为TSK G3000SWXL，规格为7.8×300mm 5μm；流速0.5ml/min，洗脱时间30min；柱温25℃，样品室温度10℃；检测波长214nm。
HPLC-IEC
许多翻译后修饰(例如N糖基化、C末端赖氨酸残基修饰、N末端谷氨酰胺或谷氨酸环化、天冬酰胺脱酰胺化、天冬氨酸异构化和氨基酸残基氧化等)会直接或间接地引起抗体表面电荷的改变，导致电荷异质性的产生。基于所带电荷可对电荷变体进行分离和分析，常用的分析方法有阳离子交换色谱法(cation exchange chromatography，CEX)和阴离子交换色谱法(anionexchange chromatography，AEX)。当通过基于色谱法的方法分析时，酸性种类(acidic species)和碱性种类(basic species)基于它们相对于主峰(main peak)的保留时间来定义。酸性种类是早于CEX的主峰或晚于AEX的主峰洗脱出来的变体，而碱性种类是晚于CEX的主峰或早于AEX的主峰洗脱出来的变体。酸性种类和碱性种类所对应的峰分别称作酸性峰和碱性峰。在生产和存储抗体的过程中容易产生电荷变体。在此使用高效液相色谱-离子交换色谱(High-performance liquid chromatography-ionexchangechromatography，HPLC-IEC)分析样品的电荷异质性。HPLC-IEC所用色谱仪为DionexUltimate 3000；流动相A：20mM PB pH6.3，流动相B：20mM PB+200mM NaCl pH6.3，两种流动相混合的比例按照预先设置的程序随时间而改变，流速1.0ml/min；色谱柱：ThermoPropacTM WCX-10；柱温30℃，样品室温度10℃；进样量：20μg；检测波长：214nm。
CE-SDS
本发明使用CE-SDS(Capillary Electrophoresis-Sodium Dodecyl Sulfate)分析样品中降解片段或不完整组装的分子的含量。CE分为非还原和还原两种类型，用于前者的样品在变性时不需要用还原剂DTT将分子内的二硫键破坏，而用于后者的样品在变性时需要用还原剂DTT将分子内的二硫键破坏。非还原和还原CE-SDS分别记作NR-CE-SDS和R-CE-SDS。所用毛细管电泳仪为ProteomeLabTM PA800plus(Beckman Coulter)，配备UV 214nm检测器，毛细管型号为Bare Fused-Silica Capillary，规格30.7cm×50μm，有效长度20.5cm；其它相关试剂购自Beckman Coulter。仪器关键参数设置如下：毛细管和样品室温度为20±2℃，分离电压为15kV。
DSC
差示扫描量热法(Differential Scanning Calorimeter，DSC)主要通过在可控的升温或降温过程检测生物分子中的热量变化来反映样品的热稳定性。通过加热，蛋白样品的去折叠会吸收热量，而消除样品池温差所需的补充能量则通过设备记录下来，这些热量变化会在图谱上形成一个峰形，其中蛋白质样品发生去折叠时所对应的峰顶温度作为熔融温度Tm。Tm是蛋白热稳定性的一个重要指示，Tm越高，蛋白的稳定性越好。
本发明中涉及的序列信息总结在表1中。
表1、本发明的抗体的序列信息
Figure BDA0003443428400000141
Figure BDA0003443428400000151
Figure BDA0003443428400000161
Figure BDA0003443428400000171
Figure BDA0003443428400000181
以下实施例、实验例是对本发明进行进一步的说明，不应理解为对本发明的限制。实施例不包括对传统方法的详细描述，如那些用于构建载体和质粒的方法，将编码蛋白的基因插入到这样的载体和质粒的方法或将质粒引入宿主细胞的方法。这样的方法对于本领域中具有普通技术的人员是众所周知的，并且在许多出版物中都有所描述，包括Sambrook,J.,Fritsch,E.F.and Maniais，T.(1989)Molecular Cloning：A Laboratory Manual，2ndedition，Cold spring Harbor Laboratory Press。
实施例1构建抗PD-1和VEGF的双特异性抗体
实施例1.1序列
从公开的文献资料(Magdelaine-Beuzelin C,Kaas Q,Wehbi V,etal.Structure–function relationships of the variable domains of monoclonalantibodies approved for cancer treatment[J].Critical reviews in oncology/hematology,2007,64(3):210-225.)中获得Bevacizumab(后文用601替代)抗体的重链可变区和轻链可变区序列(SEQ ID NO：1和2)。由上海生工生物工程有限公司合成编码上述可变区的DNA。601的重链可变区(601-VH)和轻链可变区(601-VL)分别与人IgG1重链恒定区(SEQID NO：147)和人Kappa轻链恒定区(SEQ ID NO：149)相连，构建成全长的601抗体的重链和轻链基因，分别命名为601-HC和601-LC。
根据WO2018/137576A1中实施例1-5所述，依据筛选结果，最终挑取20号鼠源抗人PD-1单抗作为先导抗体，并获得其重链可变区核苷酸序列和轻链可变区核苷酸序列，并翻译成氨基酸序列(SEQ ID NO：3和4)。
对20号抗体的重链可变区和轻链可变区氨基酸序列进行分析，依据Kabat规则分别确定20号抗体重链和轻链的抗原互补决定区和框架区。20号抗体重链CDR的氨基酸序列为H-CDR1：NYDMS(SEQ ID NO：5)、H-CDR2：TISGGGGYTYYSDSVKG(SEQ ID NO：6)和H-CDR3：PYGHYGFEY(SEQ ID NO：7)，轻链CDR的氨基酸序列为L-CDR1：SASQGISNFLS(SEQ ID NO：8)、L-CDR2：YTSSLHS(SEQ ID NO：9)和L-CDR3：QQYSNLPWT(SEQ ID NO：10)。
在https://www.ncbi.nlm.nih.gov/igblast/，将鼠源20号抗体的重链可变区与人IgG胚系序列进行同源性比较，选择IGHV3-21*01为重链CDR移植模板，将鼠源的20号抗体的重链CDR移植入IGHV3-21*01骨架区，并在H-CDR3之后加入WGQGTLVTVSS(SEQ ID NO：151)作为第四个框架区，获得CDR移植重链可变区序列。同样地，将鼠源20号抗体的轻链可变区与人IgG胚系序列同源性比较，选择IGKV1-39*01为轻链CDR移植模板，将鼠源20号抗体的轻链CDR移植入IGKV1-39*01的骨架区，并在L-CDR3之后加入FGQGTKVEIK(SEQ ID NO：152)作为第四个框架区，获得CDR移植轻链可变区序列。在CDR移植可变区的基础上，对一些框架区的氨基酸位点进行回复突变(回复突变就是将人源框架区的某些氨基酸突变成鼠源框架区同一位置的氨基酸，回复突变的位点一般对维持抗体的结构和/或亲和力是至关重要的)。在进行回复突变时，将氨基酸序列进行Kabat编码，位点的位置由Kabat码指示。
优选的，对于CDR移植重链可变区，根据Kabat编码，将第28位的T回复突变为鼠源的V，将第44位的G回复为R，第94位的R回复为S。对于CDR移植轻链可变区，将第43位的A回复为T，第44位的P回复为V，第71位的F回复为Y。
上述带有回复突变位点的重链可变区和轻链可变区分别定义为人源化的重链可变区和轻链可变区(SEQ ID NO：11和12)。由上海生工生物工程有限公司合成编码上述人源化的重链和轻链可变区的DNA。将合成的人源化重链可变区与人IgG4(铰链区含有S228P点突变)恒定区(SEQ ID NO：148)相连，获得全长的人源化重链基因，命名为20-Hu-HC；将人源化轻链可变区与人Kappa链恒定区(SEQ ID NO：149)相连，获得全长的人源化轻链基因，命名为20-Hu-LC。
上述抗体的重链和轻链基因分别构建到pcDNA3.4表达载体中，利用PEI转染法将所得重链和轻链表达载体一起转入HEK293F细胞中以表达抗体。HEK293F细胞在Free Style293Expression Medium中培养5天后收取细胞上清，利用Protein A亲和层析法纯化抗体。20-Hu-HC与20-Hu-LC组合后获得的抗体命名为20-Hu。
实施例1.2共同轻链的选择
用BLAST对20-Hu轻链可变区与601轻链可变区的氨基酸序列进行对比分析，结果显示，两者之间完全相同的氨基酸占比89％(Identities)，性质相似的氨基酸占比94％(Positives)。
将601-HC和601-LC的基因序列分别构建到pcDNA3.4表达载体中。将20-Hu-HC、20-Hu-LC、601-HC和601-LC的表达载体按照下述方式进行组合：20-Hu-HC+20-Hu-LC、601-HC+601-LC、20-Hu-HC+601-LC和601-HC+20-Hu-LC，表达纯化抗体，所得的抗体分别命名为20-Hu、601、20-Hu-HC+601-LC和601-HC+20-Hu-LC。
ELISA检测方法如下：自制带有6*His标签的人PD-1的胞外区蛋白(PD-1的胞外区来源如WO2018/137576A1中所述)，将这种重组蛋白记作PD1-His。自制带有6*His标签的人VEGF165(序列来自NCBI，Accession：AAM03108)，将这种重组蛋白记作VEGF165-His。用PD1-His和VEGF165-His分别包被酶标板，包被浓度分别为20ng/孔和10ng/孔。
如图2A所示，20-Hu和20-Hu-HC+601-LC能够有效结合PD1-His，EC50分别是0.2062nM和0.9747nM；而601和601-HC+20-Hu-LC不能有效结合PD1-His，无法准确计算出EC50。如图2B所示，601和601-HC+20-Hu-LC能够有效结合VEGF165-His，EC50为0.4681nM和8.217nM，而20-Hu和20-Hu-HC+601-LC不能有效结合VEGF165-His。
与20-Hu相比，20-Hu-HC+601-LC对PD1-His的相对亲和力明显下降；与601相比，601-HC+20-Hu-LC对VEGF165-His的相对亲和力也明显下降。在此尝试用回复突变的方式，使20-Hu-HC+601-LC对PD1-His的相对亲和力得到增强。分析发现，601轻链可变区和20-Hu轻链可变区之间有12个氨基酸残基存在差异，其中28、32、34、46、50、71、93和94位上的氨基酸残基(按照Kabat规则编码)对维持抗体的亲和力可能是至关重要的。在此，通过定点诱变，将601-LC上述位置的氨基酸残基分别突变成20-Hu-LC对应位置的氨基酸残基，这些带有点突变的601-LC分别记作601-LC-D28G、601-LC-Y32F、601-LC-N34S、601-LC-V46L、601-LC-F50Y、601-LC-F71Y、601-LC-T93N和601-LC-V94L。
将上述轻链的基因序列分别构建到pcDNA3.4表达载体中。将20-Hu-HC与上述带有点突变的601-LC的表达载体分别进行组合，表达纯化抗体，所得的抗体分别命名为20-Hu-HC+601-LC-D28G、20-Hu-HC+601-LC-Y32F、20-Hu-HC+601-LC-N34S、20-Hu-HC+601-LC-V46L、20-Hu-HC+601-LC-F50Y、20-Hu-HC+601-LC-F71Y、20-Hu-HC+601-LC-T93N、20-Hu-HC+601-LC-V94L。用上述实施例中描述的ELISA评估上述抗体结合PD-1的相对亲和力，20-Hu-HC+601-LC作为参照。ELISA结果显示，上述突变体和20-Hu-HC+601-LC的EC50分别为0.4849nM、0.4561nM、0.1751nM、0.5333nM、0.5255nM、1.0345nM、0.4859nM、0.3079nM和0.6251nM；与20-Hu-HC+601-LC相比，20-Hu-HC+601-LC-N34S和20-Hu-HC+601-LC-V94L对PD-1的相对亲和力明显更高；其它突变抗体的相对亲和力与20-Hu-HC+601-LC基本相同甚至更低。
将601-HC与上述带有点突变的601-LC的表达载体分别进行组合，表达纯化抗体，所得的抗体分别命名为601-HC+601-LC-D28G、601-HC+601-LC-Y32F、601-HC+601-LC-N34S、601-HC+601-LC-V46L、601-HC+601-LC-F50Y、601-HC+601-LC-F71Y、601-HC+601-LC-T93N、601-HC+601-LC-V94L。用上述实施例中描述的ELISA评估上述抗体结合VEGF165的相对亲和力，601作为参照。ELISA结果显示，上述突变体和601的EC50分别为0.1328nM、0.1254nM、0.2081nM、0.3256nM、0.1400nM、0.1481nM、0.1259nM、0.1243nM和0.1291nM；与601相比，601-HC+601-LC-N34S和601-HC+601-LC-V46L对VEGF165的相对亲和力明显降低；其它突变抗体的相对亲和力与601基本相同。
综上所述，V94L突变能够增强20-Hu-HC+601-LC对PD-1的相对亲和力，同时又不降低601对VEGF165的相对亲和力。在此选择601-LC-V94L(SEQ ID NO：13和14)作为共同轻链构建双特异性抗体。
实施例1.3双特异性抗体的构建
将20-Hu的重链可变区与人IgG4的CH1结构域相连，然后再通过人工连接子(在此使用的连接子是三个串联的GGGGS)连接601的重链可变区，最后再连接人IgG4的重链恒定区(CH1+CH2+CH3，铰链区含有S228P突变)，通过此程序构建成的含有两个重链可变区和两个CH1结构域的长重链基因命名为20-Fab-601-IgG4(SEQ ID NO：16和17)。相似地，将601的重链可变区与人IgG4的CH1结构域相连，然后再通过人工连接子(在此使用的连接子是三个串联的GGGGS)连接20-Hu的重链可变区，最后再连接人IgG4的重链恒定区(CH1+CH2+CH3，铰链区含有S228P突变)，通过此程序构建成的含有两个重链可变区和两个CH1结构域的长重链基因命名为601-Fab-20-IgG4(SEQ ID NO：18和19)。
将上述序列分别构建到pcDNA3.4表达载体中，将20-Fab-601-IgG4和601-Fab-20-IgG4表达载体分别与601-LC-V94L表达载体组合，表达纯化抗体，所得的抗体分别命名为20-Fab-601-IgG4-V94L和601-Fab-20-IgG4-V94L。
实施例1.4ELISA测定相对亲和力
如图3A所示，20-Hu-HC+601-LC-V94L、20-Fab-601-IgG4-V94L和601-Fab-20-IgG4-V94L能够有效结合PD1-His，EC50分别是0.3314nM、0.4768nM和1.772nM。如图3B所示，601-HC+601-LC-V94L、20-Fab-601-IgG4-V94L和601-Fab-20-IgG4-V94L能够有效结合VEGF165-His，EC50为0.01872nM、0.05859nM和0.03886nM。20-Fab-601-IgG4-V94L和601-Fab-20-IgG4-V94L既能够结合PD-1又能结合VEGF，这说明它们是双特异性抗体。
实施例1.5Biacore测定亲和力
在此通过Biacore 8K(GE healthcare)检测上述抗体与PD-1或VEGF之间的亲和力。在Biacore 8K上，使用偶联有ProteinA/G的芯片分别捕获各种抗体，再将重组蛋白PD1-His或VEGF165-His进样，得到结合-解离曲线，用6M盐酸胍再生缓冲液洗脱后重复下一个循环；利用Biacore 8K Evaluation Software对数据进行分析。结果如表2所示。
表2-1.对PD-1的结合和解离动力学参数以及平衡解离常数
Figure BDA0003443428400000221
表2-2.对VEGF的结合和解离动力学参数以及平衡解离常数
Figure BDA0003443428400000222
表2-1显示，20-Hu-HC+601-LC-V94L和20-Fab-601-IgG4-V94L对PD-1的平衡解离常数(KD)十分相近，KD分别是1.59E-09和8.85E-10。表2-2显示，601-HC+601-LC-V94L和20-Fab-601-IgG4-V94L对VEGF165-His的结合常数(Kon)和解离常数(Koff)十分相近，平衡解离常数(KD)也基本相当，KD分别是7.46E-12和9.26E-12。平衡解离常数(KD)与亲和力高低成反比。
实施例1.6物理化学性质的表征
实施例1.6.1 HPLC-SEC
图4A表示单抗601-HC+601-LC-V94L的HPLC-SEC图谱，其中存在2个明显的峰，分别是Peak1和Peak2，占比分别为0.7％和99.3％。其中Peak1的保留时间短于主峰Peak2，说明Peak1可能是聚集体产生的；图中没有出现可能代表降解片段或不完整组装分子的峰。图4B表示20-Fab-601-IgG4-V94L的HPLC-SEC图谱，其中存在两个明显的峰，分别是Peak1和Peak2，占比分别为0.7％和99.3％。其中Peak1的保留时间短于主峰Peak2，说明Peak1可能是聚集体产生的；图中没有出现可能代表降解片段或不完整组装分子的峰。
实施例1.6.2HPLC-IEC
图5A和5B表示601-HC+601-LC-V94L和20-Fab-601-IgG4-V94L的HPLC-IEC图谱，它们的主峰分别占比79.31％和80.64％，该结果表明20-Fab-601-IgG4-V94L的电荷异质性与601-HC+601-LC-V94L相当。
实施例1.6.3 CE-SDS
图6A和图6B分别表示601-HC+601-LC-V94L的NR-CE-SDS和R-CE-SDS的图谱，NR-CE-SDS图谱中主峰Peak9占比97.90％；R-CE-SDS图谱中两个主峰Peak6(对应轻链)和Peak12(对应重链)分别占比30.92％和65.27％，两者峰面积之比为1:2.1。图6C和图6D分别表示20-Fab-601-IgG4-V94L的NR-CE-SDS和R-CE-SDS的图谱，NR-CE-SDS图谱中主峰Peak13占比96.74％；R-CE-SDS图谱中两个主峰Peak3(对应轻链)和Peak12(对应重链)分别占比38.42％和59.74％，两者峰面积之比为2:3.1。NR-CE-SDS中，601-HC+601-LC-V94L和20-Fab-601-IgG4-V94L的主峰占比十分相近；R-CE-SDS中，601-HC+601-LC-V94L和20-Fab-601-IgG4-V94L的轻链和重链的峰面积之比均符合预期。
实施例1.6.4 DSC
图7A和7B分别表示601-HC+601-LC-V94L和20-Fab-601-IgG4-V94L的DSC图谱。其中601-HC+601-LC-V94L的TmOnset和Tm分别为66.46℃和75.37℃，20-Fab-601-IgG4-V94L的TmOnset和Tm分别为65.92℃和74.28℃，该结果表明20-Fab-601-IgG4-V94L的热稳定性与601-HC+601-LC-V94L非常相近。
实施例1.7改进型双特异性抗体的构建
实施例1.7.1双特异性抗体的构建
在美国专利申请US20020032315A1中，相关发明人利用噬菌体展示法对Bevacizumab的重链可变区和轻链可变区进行改造，获得了亲和力和中和活性更高的重链可变区氨基酸序列Y0313-1(US20020032315A1中的SEQ ID NO：114，即本发明中的SEQ IDNO：20)。
在此用Y0313-1置换了20-Fab-601-IgG4-V94L中的601(Bevacizumab)重链可变区。方法如下：将20-Hu的重链可变区与人IgG4的CH1结构域相连，然后再通过人工连接子(在此使用的连接子是三个串联的GGGGS)连接Y0313-1的重链可变区，最后再连接人IgG4的重链恒定区(CH1+CH2+CH3，铰链区含有S228P突变)，通过此程序构建成的含有两个重链可变区和两个CH1结构域的长重链基因命名为20-Fab-0313-IgG4(SEQ ID NO：21和22)。
将上述序列构建到pcDNA3.4表达载体中，将20-Fab-0313-IgG4与601-LC-V94L表达载体组合，表达纯化抗体，所得的抗体分别命名为20-Fab-0313-IgG4-V94L。
实施例1.7.2测定对VEGF生物活性的中和能力
人脐静脉内皮细胞(Human Umbilical Vein Endothelial Cells，HUVEC)购自澳赛尔斯(AllCells)生物技术有限公司。用脐静脉内皮细胞完全培养基(购自AllCells，货号和规格：H-004/500ml)对HUVEC进行培养和传代。待HUVEC生长至对数生长期时，用胰酶消化使细胞脱落，离心后用完全培养基重悬细胞，然后按照8000细胞/孔的密度接种于96孔细胞培养板中；24小时后，将96孔板中的完全培养基置换为基础培养基(购自AllCells，货号和规格：H-004B/500ml)，150μl/孔；在含有400ng/ml重组VEGF165(购自Acrobiosystems，货号：VE5-H4210)的基础培养中对抗体进行梯度稀释，然后将VEGF165和抗体的混合溶液加入96孔板中，50μl/孔；在37℃、5％CO2细胞培养箱中继续孵育3天；3天后每孔加入20μl CCK-8(Dojindo)溶液，在培养箱中继续孵育4小时；用酶标仪读取OD450；GraphPad Prism6进行数据分析，作图并计算IC50。
如图8所示，601、20-Fab-601-IgG4-V94L、601-Fab-20-IgG4-V94L和20-Fab-0313-IgG4-V94L均能有效抑制VEGF165诱导的HUVEC细胞增殖，它们的IC50分别为4.422nM、9.039nM、3.84nM和1.632nM。上述结果显示，20-Fab-0313-IgG4-V94L中和VEGF165生物活性的能力最强。
实施例1.7.3评估增强MLR的功能活性
如图9A和图9B所示，20-Humanized(即20-Hu)、20-Hu-HC+601-LC-V94L和20-Fab-0313-IgG4-V94L均能有效刺激MLR分泌IL-2和IFN-γ，刺激MLR分泌IL-2的EC50分别为0.2571nM、0.3703nM和0.7554nM，刺激MLR分泌IFN-γ的EC50分别为0.1426nM、0.247nM和1.036nM。
实施例1.7.4测定抗PD-1和VEGF双特异性抗体同时结合两种抗原的能力
用VEGF165(购自Acrobiosystems，货号：VE5-H4210)包被微孔板。用含1％牛血清白蛋白的PBST将待测抗体进行梯度稀释，然后转移到上述微孔板中，室温孵育半小时左右；PBST洗板三遍；将生物素化的人PD-1胞外段重组蛋白(购自Sino Biological，货号：10377-H08H-B)稀释到200ng/ml，转移到微孔板中，室温孵育半小时左右；PBST洗板三遍；将Streptavidin-HRP(购自BD Biosciences，货号：554066)稀释1000倍，转移到微孔板中，室温孵育半小时左右；PBST洗板三遍；加入TMB显色液(100μl/孔)，室温孵育1～5min；加终止液(50μl/孔)，终止显色反应；用酶标仪读取OD450；用GraphPad Prism6进行数据分析和作图，并计算EC50。
如图10所示，20-Fab-0313-IgG4-V94L在结合VEGF165之后仍然能够有效地继续结合人PD-1，EC50为0.3293nM。601和20-Hu-HC+601-LC-V94L都不能同时结合PD-1和VEGF165。
实施例1.7.5测定抗PD-1和VEGF双特异性抗体在大鼠体内的药代动力学
采用SD(Sprague-Dawley)大鼠(购自浙江维通利华实验动物技术有限公司)进行双特异性抗体的药代动力学研究。每组四只大鼠，体重200g左右，每只通过静脉注射(Intravenous Injection，I.V.)剂量为1mg的抗体；分别在给药后的特定时间眼眶取血，血液自然凝固后离心取血清。
血清中目的抗体浓度的测量方法如下：用双特异性抗体对应的两种相关抗原(VEGF165购自Acrobiosystems，货号：VE5-H4210；带有6*His标签的人PD-1的胞外区重组蛋白的来源如实施例1.2中所述)分别包被酶标板，然后用含有1％牛血清白蛋白的PBST封闭酶标板。将适当稀释的大鼠血清分别转移到上述包被两种相关抗原的酶标板中；经过1小时的室温孵育之后洗板，然后加入HRP标记的羊抗人(Fc-Specific)抗体(购自Sigma；该抗体经过种属交叉吸附处理，不识别大鼠抗体)；经过半小时的室温孵育之后洗板，每孔加入100μl以TMB为底物的显色液，室温孵育1～5min；加50μl终止液(2M H2SO4)终止反应；酶标仪读取OD450，并用标准曲线将OD450换算成抗体血清浓度；用GraphPad Prism6进行数据分析和作图；用Phoenix软件计算抗体药物在大鼠体内的半衰期。
如图11所示，计算出20-Fab-0313-IgG4-V94L的半衰期为16.9天(以PD-1为抗原的检测结果)和17.3天(以VEGF165为抗原的检测结果)。上述结果表明，20-Fab-0313-IgG4-V94L具有良好的药代动力学性质。
实施例1.7.6抗PD-1和VEGF双特异性抗体在小鼠体内的抗肿瘤作用
利用人外周血单个核细胞(PBMC)在NSG小鼠体内重建人源免疫系统，并在此小鼠上建立人肺癌NCI-H460皮下移植瘤模型。该小鼠模型同时存在表达人PD-1蛋白的T细胞，以及表达人VEGF的人类肿瘤细胞，因此可以用来评价抗PD-1和VEGF双特异性抗体的体内抗肿瘤活性。具体实施步骤如下：收集体外培养的人非小细胞肺癌NCI-H460细胞(
Figure BDA0003443428400000251
HTB-177TM)，将细胞悬液浓度调整为1×108/ml，与Matrigel(购自BD Biosciences，货号：356234)以等体积混合。体外复苏购买的PBMC(购自Allcells，货号：PB005-C)，用PBS重悬PBMC细胞，将PBMC悬液浓度调整为1×107/ml。将混合好的肿瘤细胞悬液和PBMC悬液以等体积混合。在无菌条件下，接种200μl细胞混合悬液于M-NSG小鼠(购自上海南方模式生物研究中心)右侧上背部皮下。当天将接种混合细胞的小鼠按体重随机分组，每组10只小鼠。各组小鼠药物处理情况如下：对照组，仅注射生理盐水；Avastin组，注射10mg/kg的抗VEGF阳性对照抗体Avastin(由罗氏制药生产)；Opdivo组，注射10mg/kg的抗PD-1阳性对照抗体Opdivo(由百时美施贵宝生产)；20-Fab-0313-IgG4-V94L组，注射16mg/kg的20-Fab-0313-IgG4-V94L。考虑到双特异性抗体和单克隆抗体的分子量存在差异，本实验中药物剂量按照等物质的量进行设定。随后，按照上述设计好的方案给药，每周给药2次，共给药10次，每周测定肿瘤体积2次。最终，测定的各组肿瘤随时间的生长曲线如图12所示。
结果显示，在第31天实验结束时，Avastin、Opdivo和20-Fab-0313-IgG4-V94L各组的抑瘤率分别为84.5％、35.8％和96.6％(抑瘤率＝(对照组平均体积－实验组平均体积)/对照组平均体积×100％)。与Avastin和Opdivo相比，20-Fab-0313-IgG4-V94L能够更加有效地抑制肿瘤生长。
实施例2构建抗PD-1和TGF-Beta的双特异性抗体
实施例2.1序列
US5571714A公开了一系列抗TGF-β(Transforming growth factor beta)单克隆抗体，其中鼠源单抗1D11.16(后续简称1D11)能够有效结合TGF-β1和-β2。相关发明人已将1D11对应的杂交瘤存储到美国典型培养物保藏中心(
Figure BDA0003443428400000261
HB-9849TM)。从US20180244763A1获得鼠源1D11序列。
对1D11号抗体的重链可变区和轻链可变区氨基酸序列进行分析，依据Kabat规则分别确定1D11号抗体重链和轻链的抗原互补决定区和框架区。1D11号抗体重链CDR的氨基酸序列为H-CDR1：TYWMN(SEQ ID NO：23)、H-CDR2：QIFPASGSTNYNEMFEG(SEQ ID NO：24)和H-CDR3：GDGNYALDAMDY(SEQ ID NO：25)，轻链CDR的氨基酸序列为L-CDR1：RASESVDSYGNSFMH(SEQ ID NO：26)、L-CDR2：LASNLES(SEQ ID NO：27)和L-CDR3：QQNNEDPLT(SEQ ID NO：28)。
在https://www.ncbi.nlm.nih.gov/igblast/，将鼠源1D11号抗体的重链可变区与人IgG胚系序列进行同源性比较，选择IGHV1-46*01为重链CDR移植模板，将鼠源的1D11号抗体的重链CDR移植入IGHV1-46*01骨架区，并在H-CDR3之后加入WGQGTLVTVSS(SEQ ID NO：151)作为第四个框架区，获得CDR移植重链可变区序列。同样地，将鼠源1D11号抗体的轻链可变区与人IgG胚系序列同源性比较，选择IGKV7-3*01为轻链CDR移植模板，将鼠源1D11号抗体的轻链CDR移植入IGKV7-3*01的骨架区，并在L-CDR3之后加入FGGGTKVELK(SEQ ID NO：153)作为第四个框架区，获得CDR移植轻链可变区序列。在CDR移植可变区的基础上，对一些框架区的氨基酸位点进行回复突变。在进行回复突变时，将氨基酸序列进行Kabat编码，位点的位置由Kabat码指示。
优选的，对于CDR移植重链可变区，将第28位的T回复为鼠源的I，第30位的T回复为I，第48位的M回复为I，第67位的V回复为A，第69位的M回复为L，第71位的R回复为V，第78位的V回复为A。对于CDR移植轻链可变区，将第81位的N回复为D。
上述人源化的重链可变区和轻链可变区(SEQ ID NO：29和30)由上海生工生物工程有限公司合成编码上述可变区的DNA。将合成的人源化重链可变区与人IgG4(铰链区含有S228P点突变)重链恒定区(SEQ ID NO：148)相连，获得全长的人源化重链基因，命名为1D11-Hu-HC；将人源化轻链可变区与人Kappa轻链恒定区(SEQ ID NO：149)相连，获得全长的人源化轻链基因，命名为1D11-Hu-LC。
上述抗体的重链和轻链基因分别构建到pcDNA3.4表达载体中，表达纯化抗体。1D11-Hu-HC与1D11-Hu-LC组合后获得的抗体定义为1D11-Hu。
US20100136021A1也公开了一系列抗TGF-β抗体，其中单抗mAb12.7(后续简称mAb127)能够有效结合并中和TGF-β1、TGF-β2和TGF-β3。从US20100136021A1中获得mAb127的重链可变区和轻链可变区序列(SEQ ID NO：31和32)。
由上海生工生物工程有限公司合成编码上述可变区的DNA。将合成的重链可变区与人IgG4(铰链区含有S228P点突变)重链恒定区(SEQ ID NO：148)相连，获得全长的重链基因，命名为mAb127-HC；将轻链可变区与人Kappa轻链恒定区(SEQ ID NO：149)相连，获得全长的轻链基因，命名为mAb127-LC。
根据WO2018/137576A1中实施例1-5所述，挑取14号鼠源单抗作为先导抗体并获得其重链可变区核苷酸序列和轻链可变区核苷酸序列，并翻译成氨基酸序列(SEQ ID NO：33和34)。
对14号抗体的重链可变区和轻链可变区氨基酸序列进行分析，依据Kabat规则分别确定14号抗体重链和轻链的抗原互补决定区和框架区。14号抗体重链CDR的氨基酸序列为H-CDR1：GYTMN(SEQ ID NO：35)、H-CDR2：LINPYNGDTSYNQKFKG(SEQ ID NO：36)和H-CDR3：WRYTMDY(SEQ ID NO：37)，轻链CDR的氨基酸序列为L-CDR1：RASESVDNYGNSFMN(SEQ ID NO：38)、L-CDR2：FASNLES(SEQ ID NO：39)和L-CDR3：QQNNEAPPT(SEQ ID NO：40)。
在https://www.ncbi.nlm.nih.gov/igblast/，将鼠源14号抗体的重链可变区与人IgG胚系序列进行同源性比较，选择IGHV1-46*01为重链CDR移植模板，将鼠源的14号抗体的重链CDR移植入IGHV1-46*01骨架区，并在H-CDR3之后加入WGQGTLVTVSS(SEQ ID NO：151)作为第四个框架区，获得CDR移植重链可变区序列。同样地，将鼠源14号抗体的轻链可变区与人IgG胚系序列进行同源性比较，选择IGKV7-3*01为轻链CDR移植模板，将鼠源14号抗体的轻链CDR移植入IGKV7-3*01的骨架区，并在L-CDR3之后加入FGQGTKVEIK(SEQ ID NO：152)作为第四个框架区，获得CDR移植轻链可变区序列。在CDR移植可变区的基础上，对一些框架区的氨基酸位点进行回复突变。在进行回复突变时，将氨基酸序列进行Kabat编码，位点的位置由Kabat码指示。
优选的，对于CDR移植重链可变区，将第28位(Kabat编码)的T回复为鼠源的S，第48位的M回复为I，第67位的V回复为A，第69位的M回复为V，第71位的R回复为V，第73位的T回复为K，第78位的V回复为A。对于CDR移植轻链可变区，将第46位的L回复为P，第68位的G回复为R，第81位的N回复为D。
上述带有回复突变位点的重链和轻链可变区分别定义为人源化的重链可变区和轻链可变区(SEQ ID NO：41和42)。由上海生工生物工程有限公司合成编码上述人源化的重链和轻链可变区的DNA。将合成的人源化重链可变区与人IgG4(铰链区含有S228P点突变)重链恒定区(SEQ ID NO：148)相连，获得全长的人源化重链基因，命名为14-Hu-HC；将人源化轻链可变区与人Kappa轻链恒定区(SEQ ID NO：149)相连，获得全长的人源化轻链基因，命名为14-Hu-LC。
上述抗体的重链和轻链基因分别构建到pcDNA3.4表达载体中，表达纯化抗体。14-Hu-HC与14-Hu-LC组合后获得的抗体定义为14-Hu。
实施例2.2共同轻链的选择
用BLAST对14-Hu轻链可变区与1D11-Hu轻链可变区的氨基酸序列进行对比分析，结果显示，两者之间完全相同的氨基酸占比92％(Identities)，性质相似的氨基酸占比94％(Positives)。
将14-Hu-HC、14-Hu-LC、1D11-Hu-HC和1D11-Hu-LC的表达载体按照下述方式进行组合：14-Hu-HC+14-Hu-LC、1D11-Hu-HC+1D11-Hu-LC、14-Hu-HC+1D11-Hu-LC和1D11-Hu-HC+14-Hu-LC，表达纯化抗体，所得的抗体分别命名为14-Hu、1D11-Hu、14-Hu-HC+1D11-Hu-LC和1D11-Hu-HC+14-Hu-LC。
ELISA检测方法如下：自制带有6*His标签的人PD-1的胞外区蛋白(PD-1胞外区来源如WO2018/137576A1中所述)，将这种重组蛋白记作PD1-His并用其包被酶标板(20ng/孔)；用TGF-β1(购自Sino Biological)包被酶标板(5ng/孔)。
如图13A所示，14-Hu能够有效结合PD1-His，EC50是0.3924nM；而1D11-Hu、14-Hu-HC+1D11-Hu-LC和1D11-Hu-HC+14-Hu-LC不能有效结合PD1-His。如图13B所示，1D11-Hu能够有效结合TGF-β1，EC50为0.06624nM，1D11-Hu-HC+14-Hu-LC也能结合TGF-β1，EC50为0.5255nM，而14-Hu和14-Hu-HC+1D11-Hu-LC不能结合TGF-β1。在此选择14-Hu-LC(SEQ IDNO：43和44)作为共同轻链构建双特异性抗体。
用BLAST对14-Hu轻链可变区与mAb127轻链可变区的氨基酸序列进行对比分析，结果显示，两者之间完全相同的氨基酸占比75％(Identities)，性质相似的氨基酸占比83％(Positives)。
将14-Hu-LC、mAb127-HC和mAb127-LC的表达载体按照下述方式进行组合：mAb127-HC+mAb127-LC和mAb127-HC+14-Hu-LC，表达纯化抗体，所得的抗体分别命名为mAb127和mAb127-HC+14-Hu-LC。
用上述实施例中描述的ELISA检测1D11-Hu、1D11-Hu-HC+14-Hu-LC、mAb127和mAb127-HC+14-Hu-LC对TGF-β1的相对亲和力，用GraphPad Prism6进行作图和数据分析，并计算EC50。
如图14所示，1D11-Hu、mAb127和mAb127-HC+14-Hu-LC均能够有效结合TGF-β1，EC50分别是0.1338nM、0.04136nM和0.07105nM。与1D11-Hu相比，mAb127和mAb127-HC+14-Hu-LC有更低的EC50和更高的平台，因此两者对TGF-β1具有更高的亲和力。在此选择14-Hu-LC(SEQ ID NO：43和44)作为共同轻链构建双特异性抗体。
实施例2.3双特异性抗体的构建
将14-Hu的重链可变区与人IgG4的CH1结构域相连，然后再通过人工连接子(在此使用的连接子是三个串联的GGGGS)连接1D11的重链可变区，最后再连接人IgG4的重链恒定区(CH1+CH2+CH3，铰链区含有S228P突变)，通过此程序构建成的含有两个重链可变区和两个CH1结构域的长重链基因命名为14-Fab-1D11-IgG4(SEQ ID NO：45和46)。相似地，将1D11的重链可变区与人IgG4的CH1结构域相连，然后再通过人工连接子(在此使用的连接子是三个串联的GGGGS)连接14-Hu的重链可变区，最后再连接人IgG4的重链恒定区(CH1+CH2+CH3，铰链区含有S228P突变)，通过此程序构建成的含有两个重链可变区和两个CH1结构域的长重链基因命名为1D11-Fab-14-IgG4(SEQ ID NO：47和48)。
按照上述实施例中描述的方法，将14-Hu的重链可变区与人IgG4的CH1结构域相连，然后再通过人工连接子(在此使用的连接子是三个串联的GGGGS)连接mAb127的重链可变区，最后再连接人IgG4的重链恒定区(CH1+CH2+CH3，铰链区含有S228P突变)，通过此程序构建成的含有两个重链可变区和两个CH1结构域的长重链基因命名为14-Fab-127-IgG4(SEQ ID NO：49和50)。相似地，将mAb127的重链可变区与人IgG4的CH1结构域相连，然后再通过人工连接子(在此使用的连接子是三个串联的GGGGS)连接14-Hu的重链可变区，最后再连接人IgG4的重链恒定区(CH1+CH2+CH3，铰链区含有S228P突变)，通过此程序构建成的含有两个重链可变区和两个CH1结构域的长重链基因命名为127-Fab-14-IgG4(SEQ ID NO：51和52)。
将上述序列分别构建到pcDNA3.4表达载体中，将14-Fab-1D11-IgG4、1D11-Fab-14-IgG4、14-Fab-127-IgG4和127-Fab-14-IgG4表达载体分别与14-Hu-LC表达载体组合，表达纯化抗体，所得的抗体分别命名为14-Fab-1D11-IgG4、1D11-Fab-14-IgG4、14-Fab-127-IgG4和127-Fab-14-IgG4。
实施例2.4 ELISA测定相对亲和力
如图15A所示，14-Hu、14-Fab-1D11-IgG4、1D11-Fab-14-IgG4、14-Fab-127-IgG4和127-Fab-14-IgG4均能够结合PD1-His，EC50分别是0.4321nM、0.4367nM、1.996nM、0.3873nM和3.955nM；与14-Fab-1D11-IgG4和14-Fab-127-IgG4相比，1D11-Fab-14-IgG4和127-Fab-14-IgG4对PD1-His的相对亲和力更弱，这可能是由空间位阻造成的。如图15B所示，1D11-Hu-HC+14-Hu-LC、mAb127-HC+14-Hu-LC、14-Fab-1D11-IgG4、1D11-Fab-14-IgG4、14-Fab-127-IgG4和127-Fab-14-IgG4均能结合TGF-β1，EC50分别是1.267nM、0.0803nM、0.6985nM、0.3628nM、0.1525nM和0.1083nM。与1D11-Hu-HC+14-Hu-LC、14-Fab-1D11-IgG4和1D11-Fab-14-IgG4相比，mAb127-HC+14-Hu-LC、14-Fab-127-IgG4和127-Fab-14-IgG4对TGF-β1的相对亲和力更强。
实施例2.5评估增强MLR的功能活性
如图16A和16B所示，14-Hu和14-Fab-127-IgG4均能有效刺激MLR分泌IL-2和IFN-γ，刺激MLR分泌IL-2的EC50分别为0.1008nM和0.3185nM，刺激MLR分泌IFN-γ的EC50分别为0.04716nM和0.5871nM。另外，实验结果显示，在较高浓度下，与14-Hu相比，14-Fab-127-IgG4能够刺激MLR分泌更多IL-2和IFN-γ。
实施例2.6测定抗PD-1和TGF-Beta双特异性抗体同时结合两种抗原的能力
用TGF-β1(购自Sino Biological，货号：10804-HNAC)包被微孔板。用含1％牛血清白蛋白的PBST将待测抗体进行梯度稀释，然后转移到上述微孔板中，室温孵育半小时左右。后续实验步骤与实施例1.7.4相同。
如图17所示，14-Fab-127-IgG4在结合TGF-β1之后仍然能够有效地继续结合人PD-1，EC50为0.2784nM。14-Hu和mAb127-HC+14-Hu-LC都不能同时结合PD-1和TGF-β1。
实施例2.7抗PD-1和TGF-beta双特异性抗体在小鼠体内的抗肿瘤作用
人PD-1转基因小鼠(种系背景为C57BL/6)和MC38小鼠结直肠癌细胞购自上海南方模式生物研究中心，该转基因小鼠中用人源PD-1基因的胞外段替换了小鼠的同源部分。本发明的双特异性抗体能够识别该转基因小鼠中的PD-1分子，也可以结合小鼠内源的TGF-beta。具体实施步骤如下：将MC38在体外培养，培养基为含有10％血清的DMEM(血清和培养基购自Gibco)；将培养的MC38细胞接种于人PD-1转基因小鼠中，每只小鼠皮下接种2×106个细胞；待接种的肿瘤细胞生长至体积接近100mm3时，将小鼠随机分组，每组8只小鼠。各组小鼠药物处理情况如下：对照组，仅注射生理盐水；Keytruda组(两个剂量组)，注射2mg/kg或10mg/kg的抗PD-1阳性对照抗体Keytruda(由默沙东公司生产)；14-Fab-127-IgG4组(两个剂量组)，注射3.2mg/kg或16mg/kg的14-Fab-127-IgG4。考虑到双特异性抗体和单克隆抗体的分子量存在差异，本实验中药物剂量按照等物质的量进行设定。随后，按照上述设计好的方案给药，每周给药2次，共给药6次，每周测定肿瘤体积2次。最终，测定的各组肿瘤随时间的生长曲线如图18所示。
结果显示，在第25天实验结束时，Keytruda(两个剂量组)和14-Fab-127-IgG4(两个剂量组)各组的抑瘤率分别为79.2％(2mg/kg)、77.7％(10mg/kg)、85.1％(3.2mg/kg)和100％(16mg/kg)(抑瘤率＝(对照组平均体积－实验组平均体积)/对照组平均体积×100％)。与Keytruda相比，14-Fab-127-IgG4能够更加有效地抑制肿瘤生长，并且14-Fab-127-IgG4在高剂量时能够导致所有小鼠的肿瘤完全消退。
实施例3构建抗HER-2和CD47的双特异性抗体
实施例3.1序列
19H6-Hu是人源化的抗人HER2单抗，其重链可变区和轻链可变区序列来自于WO2020/025013A1，人源化的重链可变区和轻链可变区分别命名为19H6-Hu-VH和19H6-Hu-VL(SEQ ID NO：53和54)。
由上海生工生物工程有限公司合成编码上述人源化的重链可变区和轻链可变区的DNA。将合成的人源化重链可变区与人IgG1重链恒定区(SEQ ID NO：147)相连，获得全长的人源化重链基因，命名为19H6-Hu-HC；将人源化轻链可变区与人Kappa链恒定区(SEQ IDNO：149)相连，获得全长的人源化轻链基因，命名为19H6-Hu-LC。将19H6-Hu-HC和19H6-Hu-LC基因分别构建到pcDNA3.4表达载体中，表达纯化抗体，所得抗体命名为19H6-Hu。
MABL-2(后文中称为Anti-CD47B)是抗人CD47的鼠源单抗，其重链可变区和轻链可变区氨基酸序列来自于US20030108546A中的SEQ ID NO：12和SEQ ID NO：10。
对Anti-CD47B抗体的重链可变区和轻链可变区氨基酸序列进行分析，依据Kabat规则分别确定Anti-CD47B抗体重链和轻链的抗原互补决定区和框架区。Anti-CD47B抗体重链CDR的氨基酸序列为H-CDR1：NHVIH(SEQ ID NO：55)、H-CDR2：YIYPYNDGTKYNEKFKD(SEQ IDNO：56)和H-CDR3：GGYYTYDD(SEQ ID NO：57)，轻链CDR的氨基酸序列为L-CDR1：RSSQSLVHSNGKTYLH(SEQ ID NO：58)、L-CDR2：KVSNRFS(SEQ ID NO：59)和L-CDR3：SQSTHVPYT(SEQ ID NO：60)。
在https://www.ncbi.nlm.nih.gov/igblast/，将鼠源Anti-CD47B抗体的重链可变区与人IgG胚系序列进行同源性比较，选择IGHV1-46*01为重链CDR移植模板，将鼠源的Anti-CD47B抗体的重链CDR移植入IGHV1-46*01骨架区，并在H-CDR3之后加入WGQGTLVTVSS(SEQ ID NO：151)作为第四个框架区，获得CDR移植重链可变区序列。同样地，将鼠源Anti-CD47B抗体的轻链可变区与人IgG胚系序列同源性比较，选择IGKV2-30*01为轻链CDR移植模板，将鼠源Anti-CD47B抗体的轻链CDR移植入IGKV2-30*01的骨架区，并在L-CDR3之后加入FGQGTKVEIK(SEQ ID NO：152)作为第四个框架区，获得CDR移植轻链可变区序列。在CDR移植可变区的基础上，对一些框架区的氨基酸位点进行回复突变。在进行突变时，将氨基酸序列进行Kabat编码，位点的位置由Kabat码指示。
优选的，对于CDR移植重链可变区，根据Kabat编码，将第30位的T突变为A，将第69位的M突变为L，将第71位的R突变为S，将第73位的T突变为K。对于CDR移植轻链可变区，将第36位的F突变为Y，第46位的R突变为L。
上述带有突变位点的重链可变区和轻链可变区分别定义为人源化的重链可变区和轻链可变区，分别命名为Anti-CD47B-Hu-VH和Anti-CD47B-Hu-VL(SEQ ID NO：61和62)。
由上海生工生物工程有限公司合成编码上述人源化的重链可变区和轻链可变区的DNA。将合成的人源化重链可变区与人IgG1重链恒定区(SEQ ID NO：147)相连，获得全长的人源化重链基因，命名为Anti-CD47B-Hu-HC；将人源化轻链可变区与人Kappa链恒定区(SEQ ID NO：149)相连，获得全长的人源化轻链基因，命名为Anti-CD47B-Hu-LC。将Anti-CD47B-Hu-HC和Anti-CD47B-Hu-LC基因分别构建到pcDNA3.4表达载体中，表达纯化抗体，所得抗体命名为Anti-CD47B-Hu。
实施例3.2共同轻链的选择
用BLAST对19H6-Hu轻链可变区与Anti-CD47B-Hu轻链可变区的氨基酸序列进行对比分析，结果显示，两者之间完全相同的氨基酸占比96％(Identities)，性质相似的氨基酸占比99％(Positives)。
将19H6-Hu和Anti-CD47B-Hu的重链和轻链基因按照下述方式进行组合：19H6-Hu-HC+Anti-CD47B-Hu-LC和Anti-CD47B-Hu-HC+19H6-Hu-LC，表达纯化抗体，所得的抗体分别命名为19H6-Hu-HC+Anti-CD47B-Hu-LC和Anti-CD47B-Hu-HC+19H6-Hu-LC。
ELISA检测方法如下：带有多聚组氨酸标签的人Her-2胞外段的重组蛋白购自ACROBiosystems(货号：HE2-H5225)，带多聚组氨酸标签的人CD47胞外段的重组蛋白购自Sino Biological(货号：12283-H08H)，将这两种重组蛋白名称分别记作HER2-ECD-His和CD47-ECD-His。用HER2-ECD-His和CD47-ECD-His分别包被酶标板，包被浓度均为10ng/孔。
如图19A所示，19H6-Hu和19H6-Hu-HC+Anti-CD47B-Hu-LC均能够有效结合HER2-ECD-His，EC50分别是0.07701nM和0.1388nM；而Anti-CD47B-Hu和Anti-CD47B-Hu-HC+19H6-Hu-LC不能有效结合HER2-ECD-His。如图19B所示，Anti-CD47B-Hu和Anti-CD47B-Hu-HC+19H6-Hu-LC均能有效结合CD47-ECD-His，EC50分别是0.04276nM和0.0541nM，而19H6-Hu和19H6-Hu-HC+Anti-CD47B-Hu-LC不能有效结合CD47-ECD-His。在此选择19H6-Hu-LC(SEQ IDNO：63和64)作为共同轻链构建双特异性抗体。
实施例3.3双特异性抗体的构建
将Anti-CD47B-Hu的重链可变区与人IgG4的CH1结构域相连，然后再通过人工连接子(在此使用的连接子是三个串联的GGGGS)连接19H6-Hu的重链可变区，最后再连接人IgG1的重链恒定区(CH1+CH2+CH3)，通过此程序构建成的含有两个重链可变区和两个CH1结构域的长重链命名为CD47B-Fab-19H6-IgG1(SEQ ID NO：65和66)。相似地，将19H6-Hu的重链可变区与人IgG4的CH1结构域相连，然后再通过人工连接子(在此使用的连接子是三个串联的GGGGS)连接Anti-CD47B-Hu的重链可变区，最后再连接人IgG1的重链恒定区(CH1+CH2+CH3)，通过此程序构建成的含有两个重链可变区和两个CH1结构域的长重链命名为19H6-Fab-CD47B-IgG1(SEQ ID NO：67和68)。
将上述序列分别构建到pcDNA3.4表达载体中，将CD47B-Fab-19H6-IgG1和19H6-Fab-CD47B-IgG1表达载体分别与19H6-Hu-LC表达载体组合，表达纯化抗体，所得的抗体分别命名为CD47B-Fab-19H6-IgG1和19H6-Fab-CD47B-IgG1(为简明起见，此处只取重链的名字作为抗体的名称)。
实施例3.4ELISA测定相对亲和力
如图20A所示，19H6-Hu、CD47B-Fab-19H6-IgG1和19H6-Fab-CD47B-IgG1均能有效结合HER2-ECD-His，EC50分别是0.1262nM、0.1057nM和0.1543nM。如图20B所示，Anti-CD47B-Hu-HC+19H6-Hu-LC、CD47B-Fab-19H6-IgG1和19H6-Fab-CD47B-IgG1均能有效结合CD47-ECD-His，EC50分别是0.06166nM、0.07817nM和0.1945nM。上述结果显示，CD47B-Fab-19H6-IgG1和19H6-Fab-CD47B-IgG1既能够结合HER-2又能结合CD47，这说明它们是双特异性抗体。
实施例4构建抗HER-2和CD137的双特异性抗体
实施例4.1序列
19H6-Hu是人源化的抗人HER2单抗，其来源如实施例3.1所述。
94号抗体是本公司实验室内部制备的鼠源抗人CD137抗体，制备方法参照WO2018/137576A1中实施例1-5所述，不同之处在于使用人CD137重组蛋白免疫小鼠和使用人CD137重组蛋白对杂交瘤细胞进行ELISA筛选，其重链可变区氨基酸序列和轻链可变区氨基酸序列分别如SEQ ID NO：69和70所示。
对94号抗体的重链可变区和轻链可变区氨基酸序列进行分析，依据Kabat规则分别确定94号抗体重链和轻链的抗原互补决定区和框架区。94号抗体重链CDR的氨基酸序列为H-CDR1：SYDIS(SEQ ID NO：71)、H-CDR2：VIWTGGGTNYNSAFMS(SEQ ID NO：72)和H-CDR3：MDY(SEQ ID NO：73)，轻链CDR的氨基酸序列为L-CDR1：RSSQSLLHSNGNTYLH(SEQ ID NO：74)、L-CDR2：KVSNRFS(SEQ ID NO：75)和L-CDR3：SQSTHVPWT(SEQ ID NO：76)。
在https://www.ncbi.nlm.nih.gov/igblast/，将鼠源94号抗体的重链可变区与人IgG胚系序列进行同源性比较，选择IGHV4-59*01为重链CDR移植模板，将鼠源的94号抗体的重链CDR移植入IGHV4-59*01骨架区，并在H-CDR3之后加入WGQGTLVTVSS(SEQ ID NO：151)作为第四个框架区，获得CDR移植重链可变区序列。同样地，将鼠源94号抗体的轻链可变区与人IgG胚系序列同源性比较，选择IGKV2-30*01为轻链CDR移植模板，将鼠源Anti-CD47B抗体的轻链CDR移植入IGKV2-30*01的骨架区，并在L-CDR3之后加入FGQGTKVEIK(SEQ ID NO：152)作为第四个框架区，获得CDR移植轻链可变区序列。在CDR移植可变区的基础上，对一些框架区的氨基酸位点进行回复突变。在进行突变时，将氨基酸序列进行Kabat编码，位点的位置由Kabat码指示。
优选的，对于CDR移植重链可变区，根据Kabat编码，将第27位的G突变为F，将第29位的I突变为L，将第48位的I突变为L，将第71位的R突变为S，将第71位的V突变为K，将第73位的T突变为N，将第76位的N突变为S，将第78位的F突变为V，将第93位的A突变为V。对于CDR移植轻链可变区，将第36位的F突变为Y，第46位的R突变为L，将第48位的I突变为F，将第87位的Y突变为F。
上述带有突变位点的重链可变区和轻链可变区分别定义为人源化的重链可变区和轻链可变区，分别命名为94-Hu-VH和94-Hu-VL(SEQ ID NO：77和78)。
由上海生工生物工程有限公司合成编码上述人源化的重链可变区和轻链可变区的DNA。将合成的人源化重链可变区与人IgG1重链恒定区(SEQ ID NO：147)相连，获得全长的人源化重链基因，命名为94-Hu-HC；将人源化轻链可变区与人Kappa链恒定区(SEQ IDNO：149)相连，获得全长的人源化轻链基因，命名为94-Hu-LC。将94-Hu-HC和94-Hu-LC基因分别构建到pcDNA3.4表达载体中，表达纯化抗体，所得抗体命名为94-Hu。
实施例4.2共同轻链的选择
用BLAST对19H6-Hu轻链可变区与94-Hu轻链可变区的氨基酸序列进行对比分析，结果显示，两者之间完全相同的氨基酸占比97％(Identities)，性质相似的氨基酸占比99％(Positives)。
将19H6-Hu和94-Hu的重链和轻链基因按照下述方式进行组合：19H6-Hu-HC+94-Hu-LC和94-Hu-HC+19H6-Hu-LC，表达纯化抗体，所得的抗体分别命名为19H6-Hu-HC+94-Hu-LC和94-Hu-HC+19H6-Hu-LC。
ELISA检测方法如下：带有多聚组氨酸标签的人Her-2胞外段的重组蛋白购自ACROBiosystems(货号：HE2-H5225)，带Fc标签的人CD137胞外段的重组蛋白购自SinoBiological(货号：10041-H02H)，将这两种重组蛋白名称分别记作HER2-ECD-His和CD137-ECD-Fc。用HER2-ECD-His和CD137-ECD-Fc分别包被酶标板，包被浓度均为10ng/孔。
如图21A所示，19H6-Hu和19H6-Hu-HC+94-Hu-LC均能够有效结合HER2-ECD-His，EC50分别是0.1222nM和0.1391nM；而94-Hu和94-Hu-HC+19H6-Hu-LC不能有效结合HER2-ECD-His。如图21B所示，94-Hu和94-Hu-HC+19H6-Hu-LC均能有效结合CD137-ECD-Fc，EC50分别是0.3913nM和0.634nM，而19H6-Hu和19H6-Hu-HC+94-Hu-LC不能有效结合CD137-ECD-Fc。在此选择19H6-Hu-LC(SEQ ID NO：63和64)作为共同轻链构建双特异性抗体(实际上也可以选择94-Hu-LC作为共同轻链构建双特异性抗体)。
实施例4.3双特异性抗体的构建
将94-Hu的重链可变区与人IgG4的CH1结构域相连，然后再通过人工连接子(在此使用的连接子是三个串联的GGGGS)连接19H6-Hu的重链可变区，最后再连接人IgG1的重链恒定区(CH1+CH2+CH3)，通过此程序构建成的含有两个重链可变区和两个CH1结构域的长重链命名为94-Fab-19H6-IgG1。相似地，将19H6-Hu的重链可变区与人IgG4的CH1结构域相连，然后再通过人工连接子(在此使用的连接子是三个串联的GGGGS)连接94-Hu的重链可变区，最后再连接人IgG1的重链恒定区(CH1+CH2+CH3)，通过此程序构建成的含有两个重链可变区和两个CH1结构域的长重链命名为19H6-Fab-94-IgG1。
为降低上述双抗分子的Fc段与FcγRs(Fc gamma receptors)之间的相互作用，将它们Fc段的第234的亮氨酸和235位的亮氨酸都突变为丙氨酸，将该突变标记为LALA(参考文献：Wang X,Mathieu M,Brezski R J.IgG Fc engineering to modulate antibodyeffector functions[J].Protein&cell,2018,9(1):63-73.)。突变后的上述双抗分子的名称分别为94-Fab-19H6-IgG1-LALA(SEQ ID NO：79和80)和19H6-Fab-94-IgG1-LALA(SEQ IDNO：81和82)。在此，LALA突变的目的主要是降低潜在的体内毒性(参考文献：Ho S K,Xu Z,Thakur A,et al.Epitope and Fc-mediated Crosslinking,but not High Affinity,AreCritical for Antitumor Activity of CD137Agonist Antibody with Reduced LiverToxicity[J].Molecular Cancer Therapeutics,2020.pp.1040–1051.)。
将上述序列分别构建到pcDNA3.4表达载体中，将94-Fab-19H6-IgG1-LALA和19H6-Fab-94-IgG1-LALA表达载体分别与19H6-Hu-LC表达载体组合，表达纯化抗体，所得的抗体分别命名为94-Fab-19H6-IgG1-LALA和19H6-Fab-94-IgG1-LALA(为简明起见，此处只取重链的名字作为抗体的名称)。
实施例4.4 ELISA测定相对亲和力
如图22A所示，19H6-Hu、94-Fab-19H6-IgG1-LALA和19H6-Fab-94-IgG1-LALA均能有效结合HER2-ECD-His，EC50分别是0.1933nM、0.1579nM和0.1201nM。如图22B所示，94-Hu-HC+19H6-Hu-LC和94-Fab-19H6-IgG1-LALA均能有效结合CD137-ECD-Fc，EC50分别是0.634nM和0.2411nM；19H6-Fab-94-IgG1-LALA对CD137-ECD-Fc的结合较弱，EC50为和27.56nM。上述结果显示，94-Fab-19H6-IgG1-LALA和19H6-Fab-94-IgG1-LALA既能够结合HER-2又能结合CD137，这说明它们是双特异性抗体。
实施例5构建抗PD-1和CD137的双特异性抗体
实施例5.1序列
mAb1-25-Hu(后文中简称为609)是人源化的抗人PD-1单抗，其重链可变区和轻链可变区序列来自于WO2018/137576A1，人源化的重链可变区和轻链可变区(SEQ ID NO：83和84)分别与人IgG4(S228P)重链恒定区(SEQ ID NO：148)和Kappa轻链恒定区(SEQ ID NO：149)相连，最终获得完整的人源化mAb1-25-Hu单抗(609)的重链和轻链基因。
4B4-1-1(后文中称为Anti-CD137)是抗人CD137的鼠源单抗，其重链可变区和轻链可变区氨基酸序列来自于US5928893中的SEQ ID NO：10和SEQ ID NO：11。
对Anti-CD137抗体的重链可变区和轻链可变区氨基酸序列进行分析，依据Kabat规则分别确定Anti-CD137抗体重链和轻链的抗原互补决定区和框架区。Anti-CD137抗体重链CDR的氨基酸序列为H-CDR1：SYWMH(SEQ ID NO：85)、H-CDR2：EINPGNGHTNYNEKFKS(SEQ IDNO：86)和H-CDR3：SFTTARGFAY(SEQ ID NO：87)，轻链CDR的氨基酸序列为L-CDR1：RASQTISDYLH(SEQ ID NO：88)、L-CDR2：YASQSIS(SEQ ID NO：89)和L-CDR3：QDGHSFPPT(SEQID NO：90)。
在https://www.ncbi.nlm.nih.gov/igblast/，将鼠源Anti-CD137抗体的重链可变区与人IgG胚系序列进行同源性比较，选择IGHV1-46*01为重链CDR移植模板，将鼠源的Anti-CD137抗体的重链CDR移植入IGHV1-46*01骨架区，并在H-CDR3之后加入WGQGTLVTVSS(SEQ ID NO：151)作为第四个框架区，获得CDR移植重链可变区序列。同样地，将鼠源Anti-CD137抗体的轻链可变区与人IgG胚系序列同源性比较，选择IGKV6-21*02为轻链CDR移植模板，将鼠源Anti-CD137抗体的轻链CDR移植入IGKV6-21*02的骨架区，并在L-CDR3之后加入FGQGTKVEIK(SEQ ID NO：152)作为第四个框架区，获得CDR移植轻链可变区序列。在CDR移植可变区的基础上，对一些框架区的氨基酸位点进行回复突变。在进行突变时，将氨基酸序列进行Kabat编码，位点的位置由Kabat码指示。
优选的，对于CDR移植重链可变区，根据Kabat编码，将第30位的T突变为S，将第69位的M突变为L，将第71位的R突变为V，将第73位的T突变为K。对于CDR移植轻链可变区，将第4位的L突变为M，将第58位的V突变为I，第69位的T突变为S。
上述带有突变位点的重链可变区和轻链可变区分别定义为人源化的重链可变区和轻链可变区，分别命名为Anti-CD137-Hu-VH和Anti-CD137-Hu-VL(SEQ ID NO：91和92)。
由上海生工生物工程有限公司合成编码上述人源化的重链可变区和轻链可变区的DNA。将合成的人源化重链可变区与人IgG4(S228P)重链恒定区(SEQ ID NO：148)相连，获得全长的人源化重链基因，命名为Anti-CD137-Hu-HC；将人源化轻链可变区与人Kappa链恒定区(SEQ ID NO：149)相连，获得全长的人源化轻链基因，命名为Anti-CD137-Hu-LC。将Anti-CD137-Hu-HC和Anti-CD137-Hu-LC基因分别构建到pcDNA3.4表达载体中，表达纯化抗体，所得抗体命名为Anti-CD137-Hu。
实施例5.2共同轻链的选择
用BLAST对609轻链可变区与Anti-CD137-Hu轻链可变区的氨基酸序列进行对比分析，结果显示，两者之间完全相同的氨基酸占比73％(Identities)，性质相似的氨基酸占比88％(Positives)。
将609和Anti-CD137-Hu的重链和轻链基因按照下述方式进行组合：609-HC+Anti-CD137-Hu-LC和Anti-CD137-Hu-HC+609-LC，表达纯化抗体，所得的抗体分别命名为609-HC+Anti-CD137-Hu-LC和Anti-CD137-Hu-HC+609-LC。
ELISA检测方法如下：带有多聚组氨酸标签的人PD-1胞外段的重组蛋白购自SinoBiological(货号：10377-H08H)，带Fc标签的人CD137胞外段的重组蛋白购自SinoBiological(货号：10041-H02H)，将这两种重组蛋白名称分别记作PD1-ECD-His和CD137-ECD-Fc。用PD1-ECD-His和CD137-ECD-Fc分别包被酶标板，包被浓度均为10ng/孔。
如图23A所示，609和609-HC+Anti-CD137-Hu-LC均能够有效结合PD1-ECD-His，EC50分别是0.1358nM和0.2067nM；而Anti-CD137-Hu和Anti-CD137-Hu-HC+609-LC不能有效结合PD1-ECD-His。如图23B所示，Anti-CD137-Hu能有效结合CD137-ECD-Fc，EC50分别为0.461nM，而609、609-HC+Anti-CD137-Hu-LC和Anti-CD137-Hu-HC+609-LC不能有效结合CD137-ECD-Fc。在此选择Anti-CD137-Hu-LC(SEQ ID NO：93和94)作为共同轻链构建双特异性抗体。
实施例5.3双特异性抗体的构建
将609的重链可变区与人IgG4的CH1结构域相连，然后再通过人工连接子(在此使用的连接子是三个串联的GGGGS)连接Anti-CD137-Hu的重链可变区，最后再连接人IgG4的重链恒定区(CH1+CH2+CH3，铰链区含有S228P突变)，通过此程序构建成的含有两个重链可变区和两个CH1结构域的长重链命名为609-Fab-137-IgG4(SEQ ID NO：95和96)。相似地，将Anti-CD137-Hu的重链可变区与人IgG4的CH1结构域相连，然后再通过人工连接子(在此使用的连接子是三个串联的GGGGS)连接609的重链可变区，最后再连接人IgG4的重链恒定区(CH1+CH2+CH3，铰链区含有S228P突变)，通过此程序构建成的含有两个重链可变区和两个CH1结构域的长重链命名为137-Fab-609-IgG4(SEQ ID NO：97和98)。
将上述序列分别构建到pcDNA3.4表达载体中，将609-Fab-137-IgG4和137-Fab-609-IgG4表达载体分别与Anti-CD137-Hu-LC表达载体组合，表达纯化抗体，所得的抗体分别命名为609-Fab-137-IgG4和137-Fab-609-IgG4(为简明起见，此处只取重链的名字作为抗体的名称)。
实施例5.4ELISA测定相对亲和力
如图24A所示，609-HC+Anti-CD137-Hu-LC、609-Fab-137-IgG4和137-Fab-609-IgG4均能有效结合PD1-ECD-His，EC50分别是0.2067nM、0.2293nM和1.415nM。如图24B所示，Anti-CD137-Hu、609-Fab-137-IgG4和137-Fab-609-IgG4均能有效结合CD137-ECD-Fc，EC50分别是0.461nM、0.3572nM和0.2424nM。上述结果显示，609-Fab-137-IgG4和137-Fab-609-IgG4既能够结合PD-1又能结合CD137，这表明它们是双特异性抗体。
实施例6构建抗PD-1和CD40的双特异性抗体
实施例6.1序列
609是人源化的抗人PD-1单抗，其来源如实施例5.1所述。
MAb2.220(后文中称为Anti-CD40)是抗人CD40的鼠源单抗，其重链可变区和轻链可变区氨基酸序列来自于US6312693中的SEQ ID NO：2和SEQ ID NO：1。
对Anti-CD40抗体的重链可变区和轻链可变区氨基酸序列进行分析，依据Kabat规则分别确定Anti-CD40抗体重链和轻链的抗原互补决定区和框架区。Anti-CD40抗体重链CDR的氨基酸序列为H-CDR1：TTGMQ(SEQ ID NO：99)、H-CDR2：WINTHSGVPKYVEDFKG(SEQ IDNO：100)和H-CDR3：SGNGNYDLAYFAY(SEQ ID NO：101)，轻链CDR的氨基酸序列为L-CDR1：RASQSISDYLH(SEQ ID NO：102)、L-CDR2：YASHSIS(SEQ ID NO：103)和L-CDR3：QHGHSFPWT(SEQ ID NO：104)。
在https://www.ncbi.nlm.nih.gov/igblast/，将鼠源Anti-CD40抗体的重链可变区与人IgG胚系序列进行同源性比较，选择IGHV7-4-1*02为重链CDR移植模板，将鼠源的Anti-CD40抗体的重链CDR移植入IGHV7-4-1*02骨架区，并在H-CDR3之后加入WGQGTLVTVSS(SEQ ID NO：151)作为第四个框架区，获得CDR移植重链可变区序列。同样地，将鼠源Anti-CD40抗体的轻链可变区与人IgG胚系序列同源性比较，选择IGKV3-11*01为轻链CDR移植模板，将鼠源Anti-CD40抗体的轻链CDR移植入IGKV3-11*01的骨架区，并在L-CDR3之后加入FGQGTKVEIK(SEQ ID NO：152)作为第四个框架区，获得CDR移植轻链可变区序列。在CDR移植可变区的基础上，对一些框架区的氨基酸位点进行回复突变。在进行突变时，将氨基酸序列进行Kabat编码，位点的位置由Kabat码指示。
优选的，对于CDR移植重链可变区，根据Kabat编码，将第2位的V突变为I，将第28位的T突变为A，将第39位的Q突变为E，将第48位的M突变为I，将第76位的S突变为N，将第93位的A突变为V。对于CDR移植轻链可变区，将第43位的A突变为S，将第49位的Y突变为K，将第69位的T突变为S。
上述带有突变位点的重链可变区和轻链可变区分别定义为人源化的重链可变区和轻链可变区，分别命名为Anti-CD40-Hu-VH和Anti-CD40-Hu-VL(SEQ ID NO：105和106)。
由上海生工生物工程有限公司合成编码上述人源化的重链可变区和轻链可变区的DNA。将合成的人源化重链可变区与人IgG4(S228P)重链恒定区(SEQ ID NO：148)相连，获得全长的人源化重链基因，命名为Anti-CD40-Hu-HC；将人源化轻链可变区与人Kappa链恒定区(SEQ ID NO：149)相连，获得全长的人源化轻链基因，命名为Anti-CD40-Hu-LC。将Anti-CD40-Hu-HC和Anti-CD40-Hu-LC基因分别构建到pcDNA3.4表达载体中，表达纯化抗体，所得抗体命名为Anti-CD40-Hu。
实施例6.2共同轻链的选择
用BLAST对609轻链可变区与Anti-CD40-Hu轻链可变区的氨基酸序列进行对比分析，结果显示，两者之间完全相同的氨基酸占比90％(Identities)，性质相似的氨基酸占比96％(Positives)。
将609和Anti-CD40-Hu的重链和轻链基因按照下述方式进行组合：609-HC+Anti-CD40-Hu-LC和Anti-CD40-Hu-HC+609-LC，表达纯化抗体，所得的抗体分别命名为609-HC+Anti-CD40-Hu-LC和Anti-CD40-Hu-HC+609-LC。
ELISA检测方法如下：带有多聚组氨酸标签的人PD-1胞外段的重组蛋白购自SinoBiological(货号：10377-H08H)，带多聚组氨酸标签的人CD40胞外段的重组蛋白购自SinoBiological(货号：10774-H08H)，将这两种重组蛋白名称分别记作PD1-ECD-His和CD40-ECD-His。用PD1-ECD-His和CD40-ECD-His分别包被酶标板，包被浓度均为10ng/孔。
如图25A所示，609和609-HC+Anti-CD40-Hu-LC均能够有效结合PD1-ECD-His，EC50分别是0.1263nM和0.1387nM；而Anti-CD40-Hu和Anti-CD40-Hu-HC+609-LC不能有效结合PD1-ECD-His。如图25B所示，Anti-CD40-Hu能有效结合CD40-ECD-His，EC50是0.1104nM，而609、609-HC+Anti-CD40-Hu-LC和Anti-CD40-Hu-HC+609-LC均不能有效结合CD40-ECD-His。在此选择Anti-CD40-Hu-LC(SEQ ID NO：107和108)作为共同轻链构建双特异性抗体。
实施例6.3双特异性抗体的构建
将609的重链可变区与人IgG4的CH1结构域相连，然后再通过人工连接子(在此使用的连接子是三个串联的GGGGS)连接Anti-CD40-Hu的重链可变区，最后再连接人IgG4的重链恒定区(CH1+CH2+CH3，铰链区含有S228P突变)，通过此程序构建成的含有两个重链可变区和两个CH1结构域的长重链命名为609-Fab-40-IgG4(SEQ ID NO：109和110)。相似地，将Anti-CD40-Hu的重链可变区与人IgG4的CH1结构域相连，然后再通过人工连接子(在此使用的连接子是三个串联的GGGGS)连接609的重链可变区，最后再连接人IgG4的重链恒定区(CH1+CH2+CH3，铰链区含有S228P突变)，通过此程序构建成的含有两个重链可变区和两个CH1结构域的长重链命名为40-Fab-609-IgG4(SEQ ID NO：111和112)。
将上述序列分别构建到pcDNA3.4表达载体中，将609-Fab-40-IgG4和40-Fab-609-IgG4表达载体分别与Anti-CD40-Hu-LC表达载体组合，表达纯化抗体，所得的抗体分别命名为609-Fab-40-IgG4和40-Fab-609-IgG4(为简明起见，此处只取重链的名字作为抗体的名称)。
实施例6.4 ELISA测定相对亲和力
如图26A所示，609-HC+Anti-CD40-Hu-LC、609-Fab-40-IgG4和40-Fab-609-IgG4均能有效结合PD1-ECD-His，EC50分别是0.1387nM、0.1723nM和1.017nM。如图26B所示，Anti-CD40-Hu、609-Fab-40-IgG4和40-Fab-609-IgG4均能有效结合CD40-ECD-His，EC50分别是0.1104nM、0.1047nM和0.09556nM。上述结果显示，609-Fab-40-IgG4和40-Fab-609-IgG4既能够结合PD-1又能结合CD40，这表明它们是双特异性抗体。
实施例7构建抗PD-1和其它靶点的双特异性抗体
实施例7.1序列
609是人源化的抗人PD-1单抗，其来源如实施例5.1所述。
从公开的文献资料(Magdelaine-Beuzelin C,Kaas Q,Wehbi V,etal.Structure–function relationships of the variable domains of monoclonalantibodies approved for cancer treatment[J].Critical reviews in oncology/hematology,2007,64(3):210-225.)中获得Cetuximab、Bevacizumab、Trastuzumab、Pertuzumab等抗体的重链可变区和轻链可变区氨基酸序列(SEQ ID NO：1-2、113-118)。10D1(后文中称为Ipilimumab)是抗人CTLA-4单抗，其重链可变区和轻链可变区氨基酸序列来自于US20020086014A1中的SEQ ID NO：17和SEQ ID NO：7(即本发明中的SEQ ID NO：119和120)。
由上海生工生物工程有限公司合成编码上述重链可变区和轻链可变区的DNA。重链可变区和轻链可变区编码序列分别与人IgG1重链恒定区(SEQ ID NO：147)和人Kappa轻链恒定区(SEQ ID NO：149)相连，构建成全长的抗体重链和轻链基因。上述抗体的重链基因分别命名为Cetuximab-HC、Bevacizumab-HC、Trastuzumab-HC、Pertuzumab-HC和Ipilimumab-HC，上述抗体的轻链基因分别命名为Cetuximab-LC、Bevacizumab-LC、Trastuzumab-LC、Pertuzumab-LC和Ipilimumab-LC。将上述重链和轻链基因分别构建到pcDNA3.4表达载体中，将名称对应的重链和轻链基因进行组合，表达纯化抗体，所得抗体分别命名为Cetuximab-IgG1、Bevacizumab-IgG1、Trastuzumab-IgG1、Pertuzumab-IgG1和Ipilimumab-IgG1。
5E7-Hu是人源化的抗人LAG-3抗体，其重链可变区和轻链可变区序列来自于PCT/CN2020/076023中的SEQ ID NO：26和SEQ ID NO：28(即本发明中的SEQ ID NO：121和122)，由上海生工生物工程有限公司合成编码上述人源化的重链可变区和轻链可变区的DNA。将合成的人源化重链可变区与人IgG4(S228P)重链恒定区(SEQ ID NO：148)相连，获得全长的人源化重链基因，命名为5E7-Hu-HC；将人源化轻链可变区与人Kappa链恒定区(SEQ ID NO：149)相连，获得全长的人源化轻链基因，命名为5E7-Hu-LC。将5E7-Hu-HC和5E7-Hu-LC基因分别构建到pcDNA3.4表达载体中，表达纯化抗体，所得抗体重新命名为5E7-Hu。
实施例7.2共同轻链的选择
实施例7.2.1检测杂合抗体与抗原结合的亲和力
将609的重链和上述抗体的轻链基因分别组合，表达纯化抗体，所得的抗体分别命名为609-HC+Cetuximab-LC、609-HC+Bevacizumab-LC、609-HC+Trastuzumab-LC、609-HC+Pertuzumab-LC、609-HC+Ipilimumab-LC和609-HC+5E7-Hu-LC。
ELISA检测方法如下：带有多聚组氨酸标签的人PD-1胞外段的重组蛋白购自SinoBiological(货号：10377-H08H)，将这种重组蛋白名称记作PD1-ECD-His。用PD1-ECD-His包被酶标板，包被浓度均为10ng/孔。如图27所示，Opdivo(购自Bristol-Myers Squibb)、609、609-HC+Cetuximab-LC、609-HC+Bevacizumab-LC、609-HC+Pertuzumab-LC、609-HC+Ipilimumab-LC和609-HC+5E7-Hu-LC均能够有效结合PD1-ECD-His，EC50分别是0.2887nM、0.1100nM、0.2424nM、0.1530nM、0.2244nM、0.1547nM和0.1709nM；而609-HC+Trastuzumab-LC结合PD1-ECD-His的相对亲和力较弱，EC50为0.7219nM。因此，在此可以选择Cetuximab-LC(SEQ ID NO：123和124)、Bevacizumab-LC、Pertuzumab-LC(SEQ ID NO：125和126)、Ipilimumab-LC(SEQ ID NO：127和128)和5E7-Hu-LC(SEQ ID NO：129和130)作为共同轻链构建相应的双特异性抗体。其中，同型对照抗体为不结合PD-1的人IgG4抗体。
实施例7.2.2检测杂合抗体阻断PD-1/PD-L1相互作用的能力
抗体阻断PD-1/PD-L1相互作用的能力的检测方法如下：自制带有人Fc标签的人PD-1和PD-L1的胞外区融合蛋白(制备方法如WO2018/137576A1中所述)，将这两种重组蛋白分别记作PD1-ECD-hFc和PD-L1-ECD-hFc。用无水DMSO将Biotin化标记试剂Biotin N-hydroxysuccinimide ester(购自Sigma，货号/规格：H1759-100MG)配制成100mM的母液；根据PD-L1-ECD-hFc的分子量和浓度计算相应的物质的量浓度；取适当体积的PD-L1-ECD-hFc融合蛋白，计算物质的量之后，分别与Biotin N-hydroxysuccinimide ester按照1:20的比例混匀，室温标记1小时；透析之后用紫外分光光度法测定蛋白浓度。用包被缓冲液将人PD-1-ECD-hFc稀释到2μg/ml，用排枪加到96孔ELISA酶标板中，100μl/孔，室温孵育4h；用PBST清洗1次，用含1％BSA的PBST封闭，200μl/孔，室温孵育2h；弃封闭液，拍干，于4℃备用。在96孔板中将Biotin化的PD-L1-ECD-hFc用PBST+1％BSA(含有1％牛血清白蛋白的PBST溶液)稀释至500ng/ml；用上述稀释好的Biotin化融合蛋白分别梯度稀释抗PD-1抗体；将上述稀释好的抗体和Biotin化融合蛋白的混合溶液转移到上述用人PD-1-ECD-hFc包被好的ELISA板中，室温孵育1小时；PBST洗板3次；加入用PBST+1％BSA以1:1000稀释的Streptavidin-HRP(购自BD Biosciences)，室温孵育45min；PBST洗板3次；加显色液(TMB底物溶液)，100μl/孔，室温孵育1～5min；加终止液终止显色反应，50μl/孔；用酶标仪读取OD450值；用GraphPad Prism6进行数据整理分析和作图，计算IC50。
如图28所示，Opdivo、609、609-HC+Cetuximab-LC、609-HC+Bevacizumab-LC、609-HC+Trastuzumab-LC、609-HC+Pertuzumab-LC、609-HC+Ipilimumab-LC和609-HC+5E7-Hu-LC均能有效阻断PD-1和PD-L1之间的相互作用，IC50分别为0.05729nM、0.1309nM、0.1199nM、0.1191nM、0.1162nM、0.09876nM、0.1052nM和0.1312nM。其中，同型对照抗体为不结合PD-1的人IgG4抗体。
实施例7.2.3测定杂合抗体增强混合淋巴细胞反应的能力
然后测定上述抗体增强混合淋巴细胞反应的能力。如图29所示，609、609-HC+Cetuximab-LC、609-HC+Bevacizumab-LC、609-HC+Pertuzumab-LC、609-HC+Ipilimumab-LC和609-HC+5E7-Hu-LC均能有效刺激混合淋巴细胞反应分泌IL-2，EC50分别为0.08623nM、0.2510nM、0.1211nM、0.5171nM、0.2040nM和0.09101nM。其中，609-HC+Trastuzumab-LC不能有效刺激混合淋巴细胞反应分泌IL-2。同型对照抗体为不结合PD-1的人IgG4抗体。
实施例7.2.4流式细胞法测定杂合抗体对细胞表面PD-1的结合能力
使用流式细胞法测定杂合抗体对细胞表面PD-1的结合能力。表达PD-1的TF-1细胞的建立过程如下：将全长的人PD-1基因(序列来自UniProt，Entry：Q15116)构建到慢病毒表达载体pLVX-Puro(购自Clontech)中。用Lipofectamine 3000(购自Thermo FisherScientific，货号：L3000001)将慢病毒包装载体和装载目的基因的pLVX-Puro转染入HEK293FT细胞中(购自Thermo Fisher Scientific，货号：R70007)，细胞培养箱中孵育48小时之后收集细胞培养上清，用0.45微米滤膜过滤除去细胞残骸。用上述含有病毒颗粒的上清液感染TF-1细胞(购自ATCC，货号CRL-2003TM)，48小时之后用Puromycin处理细胞，筛选出稳定表达目的基因的细胞群体。稳定表达PD-1的TF-1细胞株命名为TF1-PD1。
流式细胞法检测抗体对细胞结合的方法如下：将TF1-PD1细胞接种到圆底96孔板中(每孔20万个细胞)；离心后吸掉上清，加入梯度稀释的抗体，室温孵育半小时左右；细胞用PBS洗涤2遍；离心后吸掉上清，每孔加入以适当稀释的Anti-Human IgG(Fc specific)-FITC抗体(购自Sigma，货号：F9512)，室温孵育半小时左右；细胞用PBS洗涤2遍；吸掉上清后加入Fix Buffer I(购自BD Biosciences)以固定细胞，室温孵育5min；细胞用PBS洗涤2遍，最终用200μl PBS重悬细胞；在流式细胞仪上检测FITC通道的荧光强度；用流式细胞仪自带软件处理实验数据并导出到Excel；用GraphPad Prism6进行数据分析和作图，计算EC50。
如图30所示，609、609-HC+Cetuximab-LC、609-HC+Bevacizumab-LC、609-HC+Pertuzumab-LC、609-HC+Ipilimumab-LC和609-HC+5E7-Hu-LC均能有效结合细胞表面的PD-1，EC50分别为0.3761nM、0.577nM、0.5193nM、0.4302nM、0.4773nM和0.3864nM。其中，609-HC+Trastuzumab-LC对TF1-PD1的结合作用明显弱于其它杂合抗体。同型对照抗体为不结合PD-1的人IgG4抗体。
实施例7.2.5丙氨酸扫描研究609轻链可变区CDR在609结合PD-1中的作用
上述实验结果显示，609的重链与许多其它靶点抗体的轻链组合之后产生的杂合抗体仍然能够有效结合PD-1分子，并且具有阻断PD-1/PD-L1相互作用、刺激混合淋巴细胞反应和结合细胞表面PD-1的能力。在此，用丙氨酸扫描(Alanine Scanning)研究609轻链可变区CDR在609结合PD-1中的作用。方法如下：将609轻链CDR中的氨基酸分别突变为丙氨酸(CDR中原有的丙氨酸不做改变)，然后609重链分别与这些轻链突变体组合后，按照上述实施例中的方法表达并纯化抗体，然后按照上述实施例中的ELISA方法，测定上述抗体对PD-1的相对亲和力。如表3中所示，609-HC+609-LC-R24A中的R24A表示第24位的精氨酸突变为丙氨酸，突变氨基酸的位置由Kabat编码指示，其余以此类推。
表3、609轻链可变区的丙氨酸扫描结果
抗体名称 EC50(nM)
Opdivo 0.3054
609 0.0980
609-HC+609-LC-R24A 0.0967
609-HC+609-LC-S26A 0.1094
609-HC+609-LC-Q27A 0.1105
609-HC+609-LC-S28A 0.1059
609-HC+609-LC-I29A 0.0969
609-HC+609-LC-S30A 0.1068
609-HC+609-LC-N31A 0.1148
609-HC+609-LC-F32A 0.1547
609-HC+609-LC-L33A 0.1060
609-HC+609-LC-H34A 0.1112
609-HC+609-LC-Y50A 0.1272
609-HC+609-LC-S52A 0.1074
609-HC+609-LC-Q53A 0.1103
609-HC+609-LC-S54A 0.1237
609-HC+609-LC-I55A 0.1140
609-HC+609-LC-S56A 0.1240
609-HC+609-LC-Q89A 0.1322
609-HC+609-LC-Q90A 0.1457
609-HC+609-LC-S91A 0.1334
609-HC+609-LC-N92A 0.0971
609-HC+609-LC-S93A 0.1106
609-HC+609-LC-W94A 0.1045
609-HC+609-LC-P95A 0.1317
609-HC+609-LC-H96A 0.1266
609-HC+609-LC-T97A 0.1143
如图31和表3所示，丙氨酸扫描结果显示，轻链CDR氨基酸分别突变为丙氨酸后均没有显著影响抗体对PD-1的结合，这说明609主要通过重链结合PD-1分子，而对轻链依赖较小。
实施例7.3抗PD-1和EGFR的双特异性抗体的构建
将609的重链可变区与人IgG4的CH1结构域相连，然后再通过人工连接子(在此使用的连接子是三个串联的GGGGS)连接Cetuximab-IgG1的重链可变区，最后再连接人IgG4的重链恒定区(CH1+CH2+CH3，铰链区含有S228P突变)，通过此程序构建成的含有两个重链可变区和两个CH1结构域的长重链命名为609-Fab-Cetuximab-IgG4(SEQ ID NO：131和132)。
将上述序列分别构建到pcDNA3.4表达载体中，将609-Fab-Cetuximab-IgG4与Cetuximab-LC表达载体组合，表达纯化抗体，所得的抗体命名为609-Fab-Cetuximab-IgG4(为简明起见，此处只取重链的名字作为抗体的名称)。
ELISA检测方法如下：带有多聚组氨酸标签的人PD-1胞外段的重组蛋白购自SinoBiological(货号：10377-H08H)，带多聚组氨酸标签的人EGFR胞外段的重组蛋白购自SinoBiological(货号：10001-H08H)，将这两种重组蛋白名称分别记作PD1-ECD-His和EGFR-ECD-His。用PD1-ECD-His和EGFR-ECD-His分别包被酶标板，包被浓度均分别为10ng/孔和20ng/孔。
如图32A所示，609-HC+Cetuximab-LC和609-Fab-Cetuximab-IgG4均能有效结合PD1-ECD-His，EC50分别是0.7172nM和0.2616nM。如图32B所示，Cetuximab-IgG1和609-Fab-Cetuximab-IgG4均能有效结合EGFR-ECD-His，EC50分别是0.07609nM和0.09327nM。上述结果显示，609-Fab-Cetuximab-IgG4既能够结合PD-1又能结合EGFR，这说明它是双特异性抗体。
实施例7.4抗PD-1和HER-2的双特异性抗体的构建
将609的重链可变区与人IgG4的CH1结构域相连，然后再通过人工连接子(在此使用的连接子是三个串联的GGGGS)连接Pertuzumab-IgG1的重链可变区，最后再连接人IgG4的重链恒定区(CH1+CH2+CH3，铰链区含有S228P突变)，通过此程序构建成的含有两个重链可变区和两个CH1结构域的长重链命名为609-Fab-Pertuzumab-IgG4(SEQ ID NO：133和134)。
将上述序列分别构建到pcDNA3.4表达载体中，将609-Fab-Pertuzumab-IgG4与Pertuzumab-LC表达载体组合，表达纯化抗体，所得的抗体命名为609-Fab-Pertuzumab-IgG4(为简明起见，此处只取重链的名字作为抗体的名称)。
ELISA检测方法如下：带有多聚组氨酸标签的人PD-1胞外段的重组蛋白购自SinoBiological(货号：10377-H08H)，带有多聚组氨酸标签的人Her-2胞外段的重组蛋白购自ACROBiosystems(货号：HE2-H5225)，将这两种重组蛋白名称分别记作PD1-ECD-His和HER2-ECD-His。用PD1-ECD-His和HER2-ECD-His分别包被酶标板，包被浓度均为10ng/孔。
如图33A所示，609-HC+Pertuzumab-LC和609-Fab-Pertuzumab-IgG4均能有效结合PD1-ECD-His，EC50分别是0.1422nM和0.1196nM。如图33B所示，Pertuzumab-IgG1和609-Fab-Pertuzumab-IgG4均能有效结合HER2-ECD-His，EC50分别是0.5352nM和2.616nM。上述结果显示，609-Fab-Pertuzumab-IgG4既能够结合PD-1又能结合HER-2，这说明它是双特异性抗体。
实施例7.5抗PD-1和CTLA-4的双特异性抗体的构建
实施例7.5.1双特异性抗体的构建
将609的重链可变区与人IgG4的CH1结构域相连，然后再通过人工连接子(在此使用的连接子是三个串联的GGGGS)连接Ipilimumab-IgG1的重链可变区，最后再连接人IgG1的重链恒定区(CH1+CH2+CH3)，通过此程序构建成的含有两个重链可变区和两个CH1结构域的长重链基因命名为609-Fab-Ipilimumab-IgG1(SEQ ID NO：135和136)。相似地，将Ipilimumab-IgG1的重链可变区与人IgG4的CH1结构域相连，然后再通过人工连接子(在此使用的连接子是三个串联的GGGGS)连接609的重链可变区，最后再连接人IgG1的重链恒定区(CH1+CH2+CH3)，通过此程序构建成的含有两个重链可变区和两个CH1结构域的长重链基因命名为Ipilimumab-Fab-609-IgG1(SEQ ID NO：137和138)。
将609的重链可变区与人IgG4的CH1结构域相连，然后再通过人工连接子(在此使用的连接子是三个串联的GGGGS)连接Ipilimumab-IgG1的重链可变区，最后再连接人IgG4的重链恒定区(CH1+CH2+CH3，铰链区含有S228P突变)，通过此程序构建成的含有两个重链可变区和两个CH1结构域的长重链命名为609-Fab-Ipilimumab-IgG4(SEQ ID NO：139和140)。相似地，将Ipilimumab-IgG1的重链可变区与人IgG4的CH1结构域相连，然后再通过人工连接子(在此使用的连接子是三个串联的GGGGS)连接609的重链可变区，最后再连接人IgG4的重链恒定区(CH1+CH2+CH3，铰链区含有S228P突变)，通过此程序构建成的含有两个重链可变区和两个CH1结构域的长重链命名为Ipilimumab-Fab-609-IgG4(SEQ ID NO：141和142)。
将上述序列分别构建到pcDNA3.4表达载体中，将609-Fab-Ipilimumab-IgG1、Ipilimumab-Fab-609-IgG1、609-Fab-Ipilimumab-IgG4和Ipilimumab-Fab-609-IgG4分别与Ipilimumab-LC表达载体组合，表达纯化抗体，所得的抗体分别命名为609-Fab-Ipilimumab-IgG1、Ipilimumab-Fab-609-IgG1、609-Fab-Ipilimumab-IgG4和Ipilimumab-Fab-609-IgG4(为简明起见，此处只取重链的名字作为抗体的名称)。
ELISA检测方法如下：带有多聚组氨酸标签的人PD-1胞外段的重组蛋白购自SinoBiological(货号：10377-H08H)，带有人Fc标签的人CTLA-4胞外段的重组蛋白购自SinoBiological(货号：11159-H31H5)，将这两种重组蛋白名称分别记作PD1-ECD-His和CTLA4-ECD-Fc。用PD1-ECD-His和CTLA4-ECD-Fc分别包被酶标板，包被浓度均为10ng/孔。
如图34A所示，609-HC+Ipilimumab-LC、609-Fab-Ipilimumab-IgG1、Ipilimumab-Fab-609-IgG1、609-Fab-Ipilimumab-IgG4和Ipilimumab-Fab-609-IgG4均能有效结合PD1-ECD-His，EC50分别是0.2337nM、0.1734nM、0.7954nM、0.2078nM和0.9643nM。如图34B所示，Ipilimumab-IgG1、609-Fab-Ipilimumab-IgG1、Ipilimumab-Fab-609-IgG1、609-Fab-Ipilimumab-IgG4和Ipilimumab-Fab-609-IgG4均能有效结合CTLA4-ECD-Fc，EC50分别是0.8354nM、2.123nM、0.3376nM、2.626nM和0.392nM。上述结果显示，609-Fab-Ipilimumab-IgG1、Ipilimumab-Fab-609-IgG1、609-Fab-Ipilimumab-IgG4和Ipilimumab-Fab-609-IgG4既能够结合PD-1又能结合CTLA-4，这说明它们是双特异性抗体。
实施例7.5.2测定抗PD-1和CTLA-4双特异性抗体的功能活性
在RPMI1640中加入以下添加剂制成RPMI 1640完全培养基：10％胎牛血清；1％MEMNon-Essential Amino Acids Solution；1％Sodium Pyruvate；1％HEPES；1‰2-Mercaptoethanol；1％Penicillin-Streptomycin；1％GlutaMAX(上述添加剂购自ThermoFisher Scientific)。用上述RPMI 1640完全培养基将新鲜分离的PBMC(购自Allcells，货号：PB005-C)洗涤并重悬，加入一定量的超抗原金黄色葡萄球菌肠毒素B(staphylococcalenterotoxin B，SEB)。该超抗原为内部制备(SEB氨基酸序列来自https://www.uniprot.org/uniprot/P01552)，使用大肠杆菌生产。将PBMC细胞悬液接种到圆底96孔细胞培养板中，每孔150μl悬液和20万个细胞；在上述96孔板中加入50μl梯度稀释的抗体；将96孔板置于37℃细胞培养箱中孵育4天。从96孔板中取适量细胞培养上清。然后按照标准操作流程检测IL-2分泌。用双抗体夹心法(sandwich ELISA)检测上清中的IL-2(相关检测用的配对抗体购自BD Biosciences)。用酶标仪(SpectraMax 190)读取OD450，用GraphPadPrism6进行作图并计算EC50。
表4、抗PD-1和CTLA-4双特异性抗体的功能活性参数
Figure BDA0003443428400000461
如图35和表4所示，609、609-HC+Ipilimumab-LC和Ipilimumab-IgG1具有相当的EC50和Top(高平台)，表明这三个抗体具有相近的功能活性。由高到低，抗PD-1和CTLA-4双特异性抗体的功能活性排序如下：609-Fab-Ipilimumab-IgG1>Ipilimumab-Fab-609-IgG1>609-Fab-Ipilimumab-IgG4>Ipilimumab-Fa b-609-IgG4。609-Fab-Ipilimumab-IgG1和Ipilimumab-Fab-609-IgG1的功能活性明显优于单克隆抗体609和Ipilimumab-IgG1，以及杂合抗体609-HC+Ipilimumab-LC，而与609-HC+Ipilimumab-LC和Ipilimumab-IgG1联合应用(609-HC+Ipilimumab-LC/Ipilimumab-IgG1表示两种抗体以物质的量之比1：1联合应用)的效果近似。
实施例7.5.3测定抗PD-1和CTLA-4双特异性抗体的ADCC活性
抗体依赖细胞介导的细胞毒作用(antibody-dependent cell-mediatedcytotoxicity，ADCC)是人IgG抗体的普遍功能，ADCC强弱与抗体亚型相关。IgG1亚型的抗PD-1和CTLA-4双特异性抗体可能会对表达PD-1的细胞产生潜在细胞毒性。在此，用新鲜分离的PBMC(购自Allcells，货号：PB005-C)作为效应细胞，表达PD-1的TF-1细胞作为靶细胞，测定ADCC。
如图36所示，IgG4同型对照抗体(为不结合PD-1和CTLA-4的单抗)没有体现出明显的ADCC。Ipilimumab-IgG1也没有体现出明显的ADCC作用，因为TF1-PD1细胞不表达CTLA-4。609具有较弱的ADCC(重链恒定区为IgG4亚型，靶细胞裂解最高10％左右)，609-IgG1具有较强的ADCC(将609的重链恒定区替换为IgG1亚型，靶细胞裂解最高50％左右)，因为两者的重链恒定区分别为IgG4和IgG1，而IgG1通常具有比IgG4更强的ADCC活性。609-Fab-Ipilimumab-IgG1的ADCC与609相似，Ipilimumab-Fab-609-IgG1的ADCC与609-IgG1相似。609-Fab-Ipilimumab-IgG1的ADCC明显弱于Ipilimumab-Fab-609-IgG1，可能与双特异性抗体的空间排序方式有关联。
实施例7.5.4抗PD-1和CTLA-4双特异性抗体在大鼠体内的药代动力学
采用实施例1.7.5中描述的方法测定抗PD-1和CTLA-4双特异性抗体在大鼠体内的药代动力学。不同之处在于，用抗PD-1和CTLA-4双特异性抗体对应的两种相关抗原(PD1-ECD-His和CTLA4-ECD-Fc的来源如实施例7.5.1中所述)分别包被酶标板。
如图37所示，计算出609-Fab-Ipilimumab-IgG1的半衰期为15.2天(以PD-1为抗原的检测结果)和14.6天(以CTLA-4为抗原的检测结果)。上述结果表明，609-Fab-Ipilimumab-IgG1具有良好的药代动力学性质。
实施例7.5.5抗PD-1和CTLA-4双特异性抗体在小鼠体内的抗肿瘤作用
人PD-1/CTLA-4双转基因小鼠(种系背景为C57BL/6)购自北京百奥赛图科技股份有限公司，MC38小鼠结直肠癌细胞购自广州吉妮欧生物科技有限公司。PD-1/CTLA-4双转基因小鼠中用人源PD-1及CTLA-4基因的胞外段替换了小鼠的同源部分，因此本发明的双特异性抗体能够识别该转基因小鼠中的PD-1和CTLA-4。具体实施步骤如下：将MC38在体外培养，培养基为含有10％血清的DMEM(血清和培养基购自Gibco)；将培养的MC38细胞接种于人PD-1转基因小鼠中，每只小鼠皮下接种2×106个细胞；待接种的肿瘤细胞生长至体积接近100mm3时，将小鼠随机分组，每组8只小鼠。各组小鼠药物处理情况如下：对照组，仅注射生理盐水；609组，注射10mg/kg的抗PD-1抗体609；Yervoy组，注射10mg/kg的抗CTLA-4阳性对照抗体Yervoy(由百时美施贵宝生产)；609+Yervoy组，注射10mg/kg的609和10mg/kg的Yervoy；609-Fab-Ipilimumab-IgG1组，注射16mg/kg的609-Fab-Ipilimumab-IgG1。考虑到双特异性抗体和单克隆抗体的分子量存在差异，本实验中药物剂量按照等物质的量进行设定。随后，按照上述设计好的方案给药，每周给药2次，共给药4次，每周测定肿瘤体积2次。最终，测定的各组肿瘤随时间的生长曲线如图38所示。
结果显示，在第14天实验结束时，609、Yervoy、609+Yervoy和609-Fab-Ipilimumab-IgG1各组的抑瘤率分别为48.6％、79.1％、85.9％和92.2％(抑瘤率＝(对照组平均体积－实验组平均体积)/对照组平均体积×100％)。与609和Yervoy单药相比，609-Fab-Ipilimumab-IgG1能够更加有效地抑制肿瘤生长。609-Fab-Ipilimumab-IgG1与609和Yervoy联合应用的治疗效果相当。
实施例7.6抗PD-1和LAG-3的双特异性抗体的构建
实施例7.6.1双特异性抗体的构建
将609的重链可变区与人IgG4的CH1结构域相连，然后再通过人工连接子(在此使用的连接子是三个串联的GGGGS)连接5E7-Hu的重链可变区，最后再连接人IgG4的重链恒定区(CH1+CH2+CH3，铰链区含有S228P突变)，通过此程序构建成的含有两个重链可变区和两个CH1结构域的长重链命名为609-Fab-5E7-IgG4(SEQ ID NO：143和144)。相似地，将5E7-Hu的重链可变区与人IgG4的CH1结构域相连，然后再通过人工连接子(在此使用的连接子是三个串联的GGGGS)连接609的重链可变区，最后再连接人IgG4的重链恒定区(CH1+CH2+CH3，铰链区含有S228P突变)，通过此程序构建成的含有两个重链可变区和两个CH1结构域的长重链命名为5E7-Fab-609-IgG4(SEQ ID NO：145和146)。
将上述序列分别构建到pcDNA3.4表达载体中，将609-Fab-5E7-IgG4和5E7-Fab-609-IgG4分别与5E7-Hu-LC表达载体组合，利用上述实施例中描述的方法表达并纯化抗体，所得的抗体分别命名为609-Fab-5E7-IgG4和5E7-Fab-609-IgG4(为简明起见，此处只取重链的名字作为抗体的名称)。
ELISA检测方法如下：带有多聚组氨酸标签的人PD-1胞外段的重组蛋白购自SinoBiological(货号：10377-H08H)，带有多聚组氨酸标签的人LAG-3胞外段的重组蛋白购自Sino Biological(货号：16498-H08H)，将这两种重组蛋白名称分别记作PD1-ECD-His和LAG3-ECD-His。用PD1-ECD-His和LAG3-ECD-His分别包被酶标板，包被浓度均为10ng/孔。
如图39A所示，609-HC+5E7-Hu-LC、609-Fab-5E7-IgG4和5E7-Fab-609-IgG4均能有效结合PD1-ECD-His，EC50分别是0.1523nM、0.161nM和0.8138nM。如图39B所示，5E7-Hu、609-Fab-5E7-IgG4和5E7-Fab-609-IgG4均能有效结合LAG3-ECD-His，EC50分别是0.1472nM、0.2082nM和0.1529nM。上述结果显示，609-Fab-5E7-IgG4和5E7-Fab-609-IgG4既能够结合PD-1又能结合LAG-3，这说明它们是双特异性抗体。
实施例7.6.2测定抗PD-1和LAG-3双特异性抗体同时结合两种抗原的能力
用LAG3-ECD-His包被微孔板。用含1％牛血清白蛋白的PBST将待测抗体进行梯度稀释，然后转移到上述微孔板中，室温孵育半小时左右。后续实验步骤与实施例1.7.4相同。
如图40所示，609-Fab-5E7-IgG4在结合LAG-3之后仍然能够有效地继续结合人PD-1，EC50为0.5294nM。5E7-Hu和609-HC+5E7-Hu-LC都不能同时结合PD-1和LAG-3。
实施例7.6.3测定抗PD-1和LAG-3双特异性抗体的功能活性
在此，按照实施例7.5.2中所述的方法测定抗PD-1和LAG-3双特异性抗体的功能活性。
表5、抗PD-1和LAG-3双特异性抗体的功能活性参数
Figure BDA0003443428400000491
如图41和表5所示，5E7-Hu仅能微弱刺激IL-2的分泌。609和609-HC+5E7-Hu-LC具有相当的EC50和Top(高平台)，表明这二者具有相近的功能活性。在浓度大于1nM时，609-Fab-5E7-IgG4刺激IL-2分泌的能力明显高于单克隆抗体5E7-Hu和609，以及杂合抗体609-HC+5E7-Hu-LC，而与5E7-Hu和609-HC+5E7-Hu-LC联合应用(5E7-Hu/609-HC+5E7-Hu-LC表示两种抗体以物质的量之比1：1联合应用)的效果近似。
实施例7.6.4抗PD-1和LAG-3双特异性抗体在小鼠体内的抗肿瘤作用
人PD-1/LAG-3双转基因小鼠(种系背景为C57BL/6)购自北京百奥赛图科技股份有限公司，MC38小鼠结直肠癌细胞购自广州吉妮欧生物科技有限公司。PD-1/LAG-3双转基因小鼠中用人源PD-1及LAG-3基因的胞外段替换了小鼠的同源部分，因此本发明的双特异性抗体能够识别该转基因小鼠中的PD-1和LAG-3。具体实施步骤如下：将MC38在体外培养，培养基为含有10％血清的DMEM(血清和培养基购自Gibco)；将培养的MC38细胞接种于人PD-1转基因小鼠中，每只小鼠皮下接种2×106个细胞；待接种的肿瘤细胞生长至体积接近100mm3时，将小鼠随机分组，每组8只小鼠。各组小鼠药物处理情况如下：对照组，仅注射生理盐水；609组，注射20mg/kg的抗PD-1抗体609；5E7-Hu组，注射20mg/kg的抗LAG-3抗体5E7-Hu；609+5E7-Hu组，注射20mg/kg的609和20mg/kg的5E7-Hu；609-Fab-5E7-IgG4组，注射32mg/kg的609-Fab-5E7-IgG4。考虑到双特异性抗体和单克隆抗体的分子量存在差异，本实验中药物剂量按照等物质的量进行设定。随后，按照上述设计好的方案给药，每周给药2次，共给药4次，每周测定肿瘤体积2次。最终，测定的各组肿瘤随时间的生长曲线如图42所示。
结果显示，在第14天实验结束时，609、5E7-Hu、609+5E7-Hu和609-Fab-5E7-IgG4各组的抑瘤率分别为70.8％、13.1％、71.5％和82.8％(抑瘤率＝(对照组平均体积－实验组平均体积)/对照组平均体积×100％)。与609和5E7-Hu单药相比，609-Fab-5E7-IgG4能够更加有效地抑制肿瘤生长。609和5E7-Hu联合没有比609单药的药效更优，因此可以推测，609-Fab-5E7-IgG4作为抗PD-1和LAG-3的双特异性抗体展现出协同效应。
实施例7.7检验杂合抗体的特异性
ELISA检测方法如下：用上述实施例中提到的相关抗原(EGFR-ECD-His、VEGF165-His、HER2-ECD-His、LAG3-ECD-His和CTLA4-ECD-Fc)分别包被酶标板，实验条件如前所述，然后检测609重链与其它靶点抗体轻链的杂合抗体能否识别这些靶点。Cetuximab-IgG1、601、Trastuzumab-IgG1、5E7-Hu和Ipilimumab-IgG1等抗体的来源如上述实施例中所述(其中Trastuzumab-IgG1的可变区来源如实施例7.1中所述，恒定区与Ipilimumab-IgG1相同，制备方法与其它抗体相同)，在此分别作为结合各种抗原的阳性对照抗体。
图43A～图43E显示，609重链与其它靶点抗体轻链的杂合抗体不能识别其它靶点，这表明这些杂合抗体具有较好的特异性。
实施例7.8 HPLC-SEC
图44A表示609-Fab-Cetuximab-IgG4的HPLC-SEC图谱，主峰占比99.13％。图44B表示609-Fab-Pertuzumab-IgG4的HPLC-SEC图谱，主峰占比99.2％。图44C表示609-Fab-Ipilimumab-IgG1的HPLC-SEC图谱，主峰占比99.3％。图44D表示Ipilimumab-Fab-609-IgG1的HPLC-SEC图谱，主峰占比99.2％。图44E表示609-Fab-Ipilimumab-IgG4的HPLC-SEC图谱，主峰占比99.3％。图44F表示Ipilimumab-Fab-609-IgG4的HPLC-SEC图谱，主峰占比99.1％。图44G表示609-Fab-5E7-IgG4的HPLC-SEC图谱，主峰占比99.2％。图44H表示5E7-Fab-609-IgG4的HPLC-SEC图谱，主峰占比99.0％。
实施例7.9 HPLC-IEC
图45A表示609-Fab-Ipilimumab-IgG1的HPLC-IEC图谱，主峰占比83.52％。该结果表明，609-Fab-Ipilimumab-IgG1电荷异质性良好。
图45B表示609-Fab-5E7-IgG4的HPLC-IEC图谱，主峰占比85.43％。该结果表明，609-Fab-5E7-IgG4电荷异质性良好。
实施例7.10 CE-SDS
图46A和图46B分别表示609-Fab-Ipilimumab-IgG1的NR-CE-SDS和R-CE-SDS的图谱，NR-CE-SDS图谱中主峰Peak13占比97.02％；R-CE-SDS图谱中两个主峰Peak2(对应轻链)和Peak12(对应长重链)分别占比39.14％和59.13％，两者峰面积之比为2:3.0。R-CE-SDS中，609-Fab-Ipilimumab-IgG1的轻链和长重链的峰面积之比符合预期。
图46C和图46D分别表示609-Fab-5E7-IgG4的NR-CE-SDS和R-CE-SDS的图谱，NR-CE-SDS图谱中主峰Peak11占比94.73％；R-CE-SDS图谱中两个主峰Peak7(对应轻链)和Peak16(对应长重链)分别占比38.32％和59.58％，两者峰面积之比为2:3.1。R-CE-SDS中，609-Fab-5E7-IgG4的轻链和长重链的峰面积之比符合预期。
序列表
<110> 三生国健药业（上海）股份有限公司
<120> 一种PD-1/LAG-3四价双特异性抗体、其制备方法和用途
<130> SH3502-21P450594CN-DIV
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<210> 7
<211> 9
<212> PRT
<213> Mus musculus
<400> 7
Pro Tyr Gly His Tyr Gly Phe Glu Tyr
1 5
<210> 8
<211> 11
<212> PRT
<213> Mus musculus
<400> 8
Ser Ala Ser Gln Gly Ile Ser Asn Phe Leu Ser
1 5 10
<210> 9
<211> 7
<212> PRT
<213> Mus musculus
<400> 9
Tyr Thr Ser Ser Leu His Ser
1 5
<210> 10
<211> 9
<212> PRT
<213> Mus musculus
<400> 10
Gln Gln Tyr Ser Asn Leu Pro Trp Thr
1 5
<210> 11
<211> 118
<212> PRT
<213> Homo
<400> 11
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Val Phe Ser Asn Tyr
20 25 30
Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Arg Leu Glu Trp Val
35 40 45
Ala Thr Ile Ser Gly Gly Gly Gly Tyr Thr Tyr Tyr Ser Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ser Pro Tyr Gly His Tyr Gly Phe Glu Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser
115
<210> 12
<211> 107
<212> PRT
<213> Homo
<400> 12
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Gln Gly Ile Ser Asn Phe
20 25 30
Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Thr Val Lys Leu Leu Ile
35 40 45
Tyr Tyr Thr Ser Ser Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Tyr Thr Phe Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Asn Leu Pro Trp
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105
<210> 13
<211> 214
<212> PRT
<213> Homo
<400> 13
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Gln Asp Ile Ser Asn Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Val Leu Ile
35 40 45
Tyr Phe Thr Ser Ser Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Thr Leu Pro Trp
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys
210
<210> 14
<211> 642
<212> DNA
<213> Homo
<400> 14
gacatccaga tgacccagtc ccccagcagc ctgagcgcca gcgtgggcga cagggtgacc 60
atcacctgct ccgcctccca ggacatctcc aactacctga actggtacca gcagaagccc 120
ggcaaggccc ccaaggtgct gatctacttc acctcctccc tgcactccgg cgtgcccagc 180
aggttctccg gcagcggctc cggcaccgac ttcaccctga ccatctccag cctgcagccc 240
gaggacttcg ccacctacta ctgccagcag tactccaccc tgccctggac cttcggccag 300
ggcaccaagg tggagatcaa gaggaccgtg gccgccccct ccgtgttcat cttccccccc 360
tccgacgagc agctgaagag cggcaccgcc agcgtggtgt gcctgctgaa caacttctac 420
cccagggagg ccaaggtgca gtggaaggtg gacaacgccc tgcagagcgg caactcccag 480
gagagcgtga ccgagcagga ctccaaggac tccacctaca gcctgagctc caccctgacc 540
ctgtccaagg ccgactacga gaagcacaag gtgtacgcct gcgaggtgac ccaccagggc 600
ctgtcctccc ccgtgaccaa gtccttcaac aggggcgagt gc 642
<210> 15
<211> 107
<212> PRT
<213> Homo
<400> 15
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Gln Asp Ile Ser Asn Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Val Leu Ile
35 40 45
Tyr Phe Thr Ser Ser Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Thr Leu Pro Trp
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105
<210> 16
<211> 681
<212> PRT
<213> Homo
<400> 16
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Val Phe Ser Asn Tyr
20 25 30
Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Arg Leu Glu Trp Val
35 40 45
Ala Thr Ile Ser Gly Gly Gly Gly Tyr Thr Tyr Tyr Ser Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ser Pro Tyr Gly His Tyr Gly Phe Glu Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
115 120 125
Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly
130 135 140
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
145 150 155 160
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
165 170 175
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
180 185 190
Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser
195 200 205
Asn Thr Lys Val Asp Lys Arg Val Gly Gly Gly Gly Ser Gly Gly Gly
210 215 220
Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly
225 230 235 240
Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser
245 250 255
Gly Tyr Thr Phe Thr Asn Tyr Gly Met Asn Trp Val Arg Gln Ala Pro
260 265 270
Gly Lys Gly Leu Glu Trp Val Gly Trp Ile Asn Thr Tyr Thr Gly Glu
275 280 285
Pro Thr Tyr Ala Ala Asp Phe Lys Arg Arg Phe Thr Phe Ser Leu Asp
290 295 300
Thr Ser Lys Ser Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu
305 310 315 320
Asp Thr Ala Val Tyr Tyr Cys Ala Lys Tyr Pro His Tyr Tyr Gly Ser
325 330 335
Ser His Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val
340 345 350
Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys
355 360 365
Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys
370 375 380
Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu
385 390 395 400
Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu
405 410 415
Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr
420 425 430
Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val
435 440 445
Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro
450 455 460
Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
465 470 475 480
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
485 490 495
Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr
500 505 510
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
515 520 525
Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
530 535 540
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
545 550 555 560
Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
565 570 575
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met
580 585 590
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
595 600 605
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
610 615 620
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
625 630 635 640
Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val
645 650 655
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
660 665 670
Lys Ser Leu Ser Leu Ser Leu Gly Lys
675 680
<210> 17
<211> 2043
<212> DNA
<213> Homo
<400> 17
gaggtccagc tggtggagag cggaggagga ctggtgcagc ccggcggctc tctgaggctg 60
agctgtgccg ctagcggctt cgtcttctcc aactacgaca tgagctgggt gaggcaagcc 120
cccggcaaga ggctggagtg ggtggctaca atcagcggcg gcggaggcta cacatactac 180
tccgactccg tgaagggaag gttcactatc tctagggaca acgccaagaa ctctctgtat 240
ctgcagatga actctctgag agccgaggat acagctgtgt actactgtgc ctccccatac 300
ggccactacg gcttcgagta ctggggccaa ggcactctgg tcactgtgag cagcgcaagt 360
accaagggac ctagtgtttt ccctcttgca ccttgctcca ggtcaacatc agagtccaca 420
gctgctcttg gatgtctcgt taaggactac ttcccagagc cagttaccgt atcctggaac 480
tccggagctt tgacaagcgg cgttcataca ttcccagctg tgttgcagag ttctgggttg 540
tacagtttga gctcagtggt gaccgtgcct tcatcttctt tgggcactaa gacctacacc 600
tgcaacgtgg atcacaagcc aagcaacacc aaggtggata agagggtggg tggaggcggt 660
tcaggcggag gtggcagcgg aggtggcggg agtgaggtgc agctggtgga gtctggcggc 720
ggactggtgc agcccggcgg cagcctgcgc ctgtcctgcg ctgcctccgg ctacaccttc 780
accaactacg gcatgaactg ggtgaggcag gcccccggaa agggcctgga gtgggtgggc 840
tggatcaaca cctacaccgg cgagcccacc tacgccgctg actttaagcg caggtttacc 900
ttctccctgg acacctccaa gtccaccgcc tacctgcaga tgaacagcct gagggccgag 960
gacaccgccg tgtactactg cgccaagtac ccccactact atggctccag ccactggtac 1020
tttgacgtgt ggggccaggg caccctggtc acagtgtcta gtgcctccac caagggccca 1080
tcggtcttcc ccctggcacc ctgctccagg agcacctctg agtccacagc ggccctgggc 1140
tgcctggtca aggactactt ccccgaaccg gtgacggtgt cgtggaactc aggcgccctg 1200
accagcggcg tgcacacctt cccggctgtc ctacagtcct caggactcta ctccctcagc 1260
agcgtggtga ccgtgccctc cagcagcttg ggcaccaaga catatacctg taatgtggat 1320
cacaagcctt ccaatacaaa agtggacaag agagttgagt ccaagtacgg cccaccatgt 1380
cctccatgtc cagcccctga atttttgggc gggccttctg tctttctgtt tcctcctaaa 1440
cctaaagata ccctgatgat cagccgcaca cccgaagtca cttgtgtggt cgtggatgtg 1500
tctcaggaag atcccgaagt gcagtttaac tggtatgtcg atggcgtgga agtgcataat 1560
gccaaaacta agccccgcga agaacagttc aacagcactt atcgggtcgt gtctgtgctc 1620
acagtcctcc atcaggattg gctgaatggg aaagaatata agtgcaaggt gagcaataag 1680
ggcctcccca gcagcatcga gaagactatt agcaaagcca aagggcagcc acgggaaccc 1740
caggtgtaca ctctgccccc ctctcaggag gagatgacta aaaatcaggt ctctctgact 1800
tgtctggtga aagggtttta tcccagcgac attgccgtgg agtgggagtc taatggccag 1860
cccgagaata attataagac aactcccccc gtcctggact ctgacggcag ctttttcctg 1920
tattctcggc tgacagtgga caaaagtcgc tggcaggagg gcaatgtctt tagttgcagt 1980
gtcatgcatg aggccctgca caatcactat acacagaaaa gcctgtctct gagtctgggc 2040
aaa 2043
<210> 18
<211> 681
<212> PRT
<213> Homo
<400> 18
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Asn Tyr
20 25 30
Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Ala Asp Phe
50 55 60
Lys Arg Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser Thr Ala Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Lys Tyr Pro His Tyr Tyr Gly Ser Ser His Trp Tyr Phe Asp Val
100 105 110
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly
115 120 125
Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser
130 135 140
Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
145 150 155 160
Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe
165 170 175
Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
180 185 190
Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val
195 200 205
Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Gly Gly Gly
210 215 220
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu
225 230 235 240
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu
245 250 255
Ser Cys Ala Ala Ser Gly Phe Val Phe Ser Asn Tyr Asp Met Ser Trp
260 265 270
Val Arg Gln Ala Pro Gly Lys Arg Leu Glu Trp Val Ala Thr Ile Ser
275 280 285
Gly Gly Gly Gly Tyr Thr Tyr Tyr Ser Asp Ser Val Lys Gly Arg Phe
290 295 300
Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gln Met Asn
305 310 315 320
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ser Pro Tyr
325 330 335
Gly His Tyr Gly Phe Glu Tyr Trp Gly Gln Gly Thr Leu Val Thr Val
340 345 350
Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys
355 360 365
Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys
370 375 380
Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu
385 390 395 400
Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu
405 410 415
Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr
420 425 430
Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val
435 440 445
Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro
450 455 460
Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
465 470 475 480
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
485 490 495
Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr
500 505 510
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
515 520 525
Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
530 535 540
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
545 550 555 560
Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
565 570 575
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met
580 585 590
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
595 600 605
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
610 615 620
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
625 630 635 640
Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val
645 650 655
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
660 665 670
Lys Ser Leu Ser Leu Ser Leu Gly Lys
675 680
<210> 19
<211> 2043
<212> DNA
<213> Homo
<400> 19
gaggtgcagc tggtggagtc tggcggcgga ctggtgcagc ccggcggcag cctgcgcctg 60
tcctgcgctg cctccggcta caccttcacc aactacggca tgaactgggt gaggcaggcc 120
cccggaaagg gcctggagtg ggtgggctgg atcaacacct acaccggcga gcccacctac 180
gccgctgact ttaagcgcag gtttaccttc tccctggaca cctccaagtc caccgcctac 240
ctgcagatga acagcctgag ggccgaggac accgccgtgt actactgcgc caagtacccc 300
cactactatg gctccagcca ctggtacttt gacgtgtggg gccagggcac cctggtgact 360
gtgagcagcg caagtaccaa gggacctagt gttttccctc ttgcaccttg ctccaggtca 420
acatcagagt ccacagctgc tcttggatgt ctcgttaagg actacttccc agagccagtt 480
accgtatcct ggaactccgg agctttgaca agcggcgttc atacattccc agctgtgttg 540
cagagttctg ggttgtacag tttgagctca gtggtgaccg tgccttcatc ttctttgggc 600
actaagacct acacctgcaa cgtggatcac aagccaagca acaccaaggt ggataagagg 660
gtgggtggag gcggttcagg cggaggtggc agcggaggtg gcgggagtga ggtccagctg 720
gtggagagcg gaggaggact ggtgcagccc ggcggctctc tgaggctgag ctgtgccgct 780
agcggcttcg tcttctccaa ctacgacatg agctgggtga ggcaagcccc cggcaagagg 840
ctggagtggg tggctacaat cagcggcggc ggaggctaca catactactc cgactccgtg 900
aagggaaggt tcactatctc tagggacaac gccaagaact ctctgtatct gcagatgaac 960
tctctgagag ccgaggatac agctgtgtac tactgtgcct ccccatacgg ccactacggc 1020
ttcgagtact ggggccaagg cactctggtc acagtgtcta gtgcctccac caagggccca 1080
tcggtcttcc ccctggcacc ctgctccagg agcacctctg agtccacagc ggccctgggc 1140
tgcctggtca aggactactt ccccgaaccg gtgacggtgt cgtggaactc aggcgccctg 1200
accagcggcg tgcacacctt cccggctgtc ctacagtcct caggactcta ctccctcagc 1260
agcgtggtga ccgtgccctc cagcagcttg ggcaccaaga catatacctg taatgtggat 1320
cacaagcctt ccaatacaaa agtggacaag agagttgagt ccaagtacgg cccaccatgt 1380
cctccatgtc cagcccctga atttttgggc gggccttctg tctttctgtt tcctcctaaa 1440
cctaaagata ccctgatgat cagccgcaca cccgaagtca cttgtgtggt cgtggatgtg 1500
tctcaggaag atcccgaagt gcagtttaac tggtatgtcg atggcgtgga agtgcataat 1560
gccaaaacta agccccgcga agaacagttc aacagcactt atcgggtcgt gtctgtgctc 1620
acagtcctcc atcaggattg gctgaatggg aaagaatata agtgcaaggt gagcaataag 1680
ggcctcccca gcagcatcga gaagactatt agcaaagcca aagggcagcc acgggaaccc 1740
caggtgtaca ctctgccccc ctctcaggag gagatgacta aaaatcaggt ctctctgact 1800
tgtctggtga aagggtttta tcccagcgac attgccgtgg agtgggagtc taatggccag 1860
cccgagaata attataagac aactcccccc gtcctggact ctgacggcag ctttttcctg 1920
tattctcggc tgacagtgga caaaagtcgc tggcaggagg gcaatgtctt tagttgcagt 1980
gtcatgcatg aggccctgca caatcactat acacagaaaa gcctgtctct gagtctgggc 2040
aaa 2043
<210> 20
<211> 123
<212> PRT
<213> Homo
<400> 20
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Asp Phe Thr His Tyr
20 25 30
Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Ala Asp Phe
50 55 60
Lys Arg Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser Thr Ala Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Lys Tyr Pro Tyr Tyr Tyr Gly Thr Ser His Trp Tyr Phe Asp Val
100 105 110
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 21
<211> 681
<212> PRT
<213> Homo
<400> 21
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Val Phe Ser Asn Tyr
20 25 30
Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Arg Leu Glu Trp Val
35 40 45
Ala Thr Ile Ser Gly Gly Gly Gly Tyr Thr Tyr Tyr Ser Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ser Pro Tyr Gly His Tyr Gly Phe Glu Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
115 120 125
Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly
130 135 140
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
145 150 155 160
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
165 170 175
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
180 185 190
Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser
195 200 205
Asn Thr Lys Val Asp Lys Arg Val Gly Gly Gly Gly Ser Gly Gly Gly
210 215 220
Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly
225 230 235 240
Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser
245 250 255
Gly Tyr Asp Phe Thr His Tyr Gly Met Asn Trp Val Arg Gln Ala Pro
260 265 270
Gly Lys Gly Leu Glu Trp Val Gly Trp Ile Asn Thr Tyr Thr Gly Glu
275 280 285
Pro Thr Tyr Ala Ala Asp Phe Lys Arg Arg Phe Thr Phe Ser Leu Asp
290 295 300
Thr Ser Lys Ser Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu
305 310 315 320
Asp Thr Ala Val Tyr Tyr Cys Ala Lys Tyr Pro Tyr Tyr Tyr Gly Thr
325 330 335
Ser His Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val
340 345 350
Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys
355 360 365
Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys
370 375 380
Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu
385 390 395 400
Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu
405 410 415
Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr
420 425 430
Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val
435 440 445
Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro
450 455 460
Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
465 470 475 480
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
485 490 495
Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr
500 505 510
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
515 520 525
Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
530 535 540
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
545 550 555 560
Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
565 570 575
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met
580 585 590
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
595 600 605
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
610 615 620
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
625 630 635 640
Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val
645 650 655
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
660 665 670
Lys Ser Leu Ser Leu Ser Leu Gly Lys
675 680
<210> 22
<211> 2043
<212> DNA
<213> Homo
<400> 22
gaggtccagc tggtggagag cggaggagga ctggtgcagc ccggcggctc tctgaggctg 60
agctgtgccg ctagcggctt cgtcttctcc aactacgaca tgagctgggt gaggcaagcc 120
cccggcaaga ggctggagtg ggtggctaca atcagcggcg gcggaggcta cacatactac 180
tccgactccg tgaagggaag gttcactatc tctagggaca acgccaagaa ctctctgtat 240
ctgcagatga actctctgag agccgaggat acagctgtgt actactgtgc ctccccatac 300
ggccactacg gcttcgagta ctggggccaa ggcactctgg tcactgtgag cagcgcaagt 360
accaagggac ctagtgtttt ccctcttgca ccttgctcca ggtcaacatc agagtccaca 420
gctgctcttg gatgtctcgt taaggactac ttcccagagc cagttaccgt atcctggaac 480
tccggagctt tgacaagcgg cgttcataca ttcccagctg tgttgcagag ttctgggttg 540
tacagtttga gctcagtggt gaccgtgcct tcatcttctt tgggcactaa gacctacacc 600
tgcaacgtgg atcacaagcc aagcaacacc aaggtggata agagggtggg tggaggcggt 660
tcaggcggag gtggcagcgg aggtggcggg agtgaggtgc agctggtgga gtctggagga 720
ggcctggtgc agcctggcgg ctctctgaga ctgtcttgcg ctgctagtgg atatgatttt 780
acacattatg gcatgaactg ggtgagacag gctccaggca agggcctgga atgggtgggc 840
tggattaata cctatacagg cgaacctacc tacgccgctg attttaagag aagattcacc 900
ttttctctgg atacatctaa gagcacagct tacctgcaga tgaacagcct gcgggccgag 960
gacaccgccg tgtactactg cgccaagtac ccctactact acggcacctc ccactggtac 1020
ttcgacgtgt ggggccaggg caccctggtg accgtgtcct ccgcctccac caagggccca 1080
tcggtcttcc ccctggcacc ctgctccagg agcacctctg agtccacagc ggccctgggc 1140
tgcctggtca aggactactt ccccgaaccg gtgacggtgt cgtggaactc aggcgccctg 1200
accagcggcg tgcacacctt cccggctgtc ctacagtcct caggactcta ctccctcagc 1260
agcgtggtga ccgtgccctc cagcagcttg ggcaccaaga catatacctg taatgtggat 1320
cacaagcctt ccaatacaaa agtggacaag agagttgagt ccaagtacgg cccaccatgt 1380
cctccatgtc cagcccctga atttttgggc gggccttctg tctttctgtt tcctcctaaa 1440
cctaaagata ccctgatgat cagccgcaca cccgaagtca cttgtgtggt cgtggatgtg 1500
tctcaggaag atcccgaagt gcagtttaac tggtatgtcg atggcgtgga agtgcataat 1560
gccaaaacta agccccgcga agaacagttc aacagcactt atcgggtcgt gtctgtgctc 1620
acagtcctcc atcaggattg gctgaatggg aaagaatata agtgcaaggt gagcaataag 1680
ggcctcccca gcagcatcga gaagactatt agcaaagcca aagggcagcc acgggaaccc 1740
caggtgtaca ctctgccccc ctctcaggag gagatgacta aaaatcaggt ctctctgact 1800
tgtctggtga aagggtttta tcccagcgac attgccgtgg agtgggagtc taatggccag 1860
cccgagaata attataagac aactcccccc gtcctggact ctgacggcag ctttttcctg 1920
tattctcggc tgacagtgga caaaagtcgc tggcaggagg gcaatgtctt tagttgcagt 1980
gtcatgcatg aggccctgca caatcactat acacagaaaa gcctgtctct gagtctgggc 2040
aaa 2043
<210> 23
<211> 5
<212> PRT
<213> Mus musculus
<400> 23
Thr Tyr Trp Met Asn
1 5
<210> 24
<211> 17
<212> PRT
<213> Mus musculus
<400> 24
Gln Ile Phe Pro Ala Ser Gly Ser Thr Asn Tyr Asn Glu Met Phe Glu
1 5 10 15
Gly
<210> 25
<211> 12
<212> PRT
<213> Mus musculus
<400> 25
Gly Asp Gly Asn Tyr Ala Leu Asp Ala Met Asp Tyr
1 5 10
<210> 26
<211> 15
<212> PRT
<213> Mus musculus
<400> 26
Arg Ala Ser Glu Ser Val Asp Ser Tyr Gly Asn Ser Phe Met His
1 5 10 15
<210> 27
<211> 7
<212> PRT
<213> Mus musculus
<400> 27
Leu Ala Ser Asn Leu Glu Ser
1 5
<210> 28
<211> 9
<212> PRT
<213> Mus musculus
<400> 28
Gln Gln Asn Asn Glu Asp Pro Leu Thr
1 5
<210> 29
<211> 121
<212> PRT
<213> Homo
<400> 29
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ile Phe Ile Thr Tyr
20 25 30
Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Gln Ile Phe Pro Ala Ser Gly Ser Thr Asn Tyr Asn Glu Met Phe
50 55 60
Glu Gly Arg Ala Thr Leu Thr Val Asp Thr Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gly Asp Gly Asn Tyr Ala Leu Asp Ala Met Asp Tyr Trp Gly
100 105 110
Gln Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 30
<211> 111
<212> PRT
<213> Homo
<400> 30
Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Pro Gly
1 5 10 15
Gln Arg Ala Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Asp Ser Tyr
20 25 30
Gly Asn Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro
35 40 45
Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn
65 70 75 80
Pro Val Glu Ala Asp Asp Thr Ala Asn Tyr Tyr Cys Gln Gln Asn Asn
85 90 95
Glu Asp Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Leu Lys
100 105 110
<210> 31
<211> 121
<212> PRT
<213> Homo
<400> 31
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Glu
20 25 30
Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Gln Ile Phe Pro Ala Leu Gly Ser Thr Asn Tyr Asn Glu Met Tyr
50 55 60
Glu Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gly Ile Gly Asn Tyr Ala Leu Asp Ala Met Asp Tyr Trp Gly
100 105 110
Gln Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 32
<211> 111
<212> PRT
<213> Homo
<400> 32
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Asp Phe Tyr
20 25 30
Gly Asn Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
35 40 45
Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ser
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
65 70 75 80
Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Asn Ile
85 90 95
Glu Asp Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105 110
<210> 33
<211> 116
<212> PRT
<213> Mus musculus
<400> 33
Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Met Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr
20 25 30
Thr Met Asn Trp Val Lys Gln Ser His Gly Lys Thr Leu Glu Trp Ile
35 40 45
Gly Leu Ile Asn Pro Tyr Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Lys Ala Thr Val Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Leu Asn Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Trp Arg Tyr Thr Met Asp Tyr Trp Gly Gln Gly Thr Ser Val
100 105 110
Thr Val Ser Ser
115
<210> 34
<211> 111
<212> PRT
<213> Mus musculus
<400> 34
Asn Ile Ala Leu Thr Gln Ser Pro Ala Ser Val Ala Val Ser Leu Gly
1 5 10 15
Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Asn Tyr
20 25 30
Gly Asn Ser Phe Met Asn Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro
35 40 45
Lys Pro Leu Ile Tyr Phe Ala Ser Asn Leu Glu Ser Gly Val Pro Ala
50 55 60
Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Asp
65 70 75 80
Pro Val Glu Ala Asp Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Asn Asn
85 90 95
Glu Ala Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105 110
<210> 35
<211> 5
<212> PRT
<213> Mus musculus
<400> 35
Gly Tyr Thr Met Asn
1 5
<210> 36
<211> 17
<212> PRT
<213> Mus musculus
<400> 36
Leu Ile Asn Pro Tyr Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe Lys
1 5 10 15
Gly
<210> 37
<211> 7
<212> PRT
<213> Mus musculus
<400> 37
Trp Arg Tyr Thr Met Asp Tyr
1 5
<210> 38
<211> 15
<212> PRT
<213> Mus musculus
<400> 38
Arg Ala Ser Glu Ser Val Asp Asn Tyr Gly Asn Ser Phe Met Asn
1 5 10 15
<210> 39
<211> 7
<212> PRT
<213> Mus musculus
<400> 39
Phe Ala Ser Asn Leu Glu Ser
1 5
<210> 40
<211> 9
<212> PRT
<213> Mus musculus
<400> 40
Gln Gln Asn Asn Glu Ala Pro Pro Thr
1 5
<210> 41
<211> 116
<212> PRT
<213> Homo
<400> 41
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr
20 25 30
Thr Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Leu Ile Asn Pro Tyr Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Arg Ala Thr Val Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Trp Arg Tyr Thr Met Asp Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Ser Ser
115
<210> 42
<211> 111
<212> PRT
<213> Homo
<400> 42
Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Pro Gly
1 5 10 15
Gln Arg Ala Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Asp Asn Tyr
20 25 30
Gly Asn Ser Phe Met Asn Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro
35 40 45
Lys Pro Leu Ile Tyr Phe Ala Ser Asn Leu Glu Ser Gly Val Pro Ala
50 55 60
Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Asn
65 70 75 80
Pro Val Glu Ala Asp Asp Thr Ala Asn Tyr Tyr Cys Gln Gln Asn Asn
85 90 95
Glu Ala Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105 110
<210> 43
<211> 218
<212> PRT
<213> Homo
<400> 43
Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Pro Gly
1 5 10 15
Gln Arg Ala Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Asp Asn Tyr
20 25 30
Gly Asn Ser Phe Met Asn Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro
35 40 45
Lys Pro Leu Ile Tyr Phe Ala Ser Asn Leu Glu Ser Gly Val Pro Ala
50 55 60
Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Asn
65 70 75 80
Pro Val Glu Ala Asp Asp Thr Ala Asn Tyr Tyr Cys Gln Gln Asn Asn
85 90 95
Glu Ala Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg
100 105 110
Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
115 120 125
Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
130 135 140
Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
145 150 155 160
Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
165 170 175
Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
180 185 190
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
195 200 205
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
210 215
<210> 44
<211> 654
<212> DNA
<213> Homo
<400> 44
gatatcgtgc tgactcagag cccagcctct ctggctgtga gccccggcca gagagccaca 60
atcacttgta gggccagcga gagcgtggac aactacggca actccttcat gaattggtac 120
cagcagaagc ccggccagcc tccaaagcct ctgatctact tcgcctccaa tctggaaagc 180
ggcgtgccag ctaggtttag cggctccggc tctaggacag acttcactct gactatcaac 240
ccagtggagg ccgatgacac agccaactac tactgccagc agaacaacga ggcccctcca 300
actttcggcc aaggcactaa ggtcgagatc aagagaaccg tcgccgctcc cagcgtcttc 360
atcttccccc ccagcgatga gcagctgaag agcggaaccg ccagcgtggt gtgcctgctg 420
aacaacttct accccaggga ggccaaggtg caatggaagg tggacaacgc cctacagagc 480
ggcaactccc aggagagcgt gaccgagcag gacagcaagg atagcaccta cagcctgagc 540
agcaccctca ccctgagcaa ggccgactac gagaagcaca aggtgtacgc ctgcgaggtg 600
acccatcagg gcctgagcag ccctgtgacc aagagcttca acaggggcga gtgc 654
<210> 45
<211> 677
<212> PRT
<213> Homo
<400> 45
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr
20 25 30
Thr Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Leu Ile Asn Pro Tyr Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Arg Ala Thr Val Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Trp Arg Tyr Thr Met Asp Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala
115 120 125
Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu
130 135 140
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
145 150 155 160
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
165 170 175
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
180 185 190
Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr
195 200 205
Lys Val Asp Lys Arg Val Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
210 215 220
Gly Gly Gly Gly Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
225 230 235 240
Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr
245 250 255
Ile Phe Ile Thr Tyr Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln
260 265 270
Gly Leu Glu Trp Ile Gly Gln Ile Phe Pro Ala Ser Gly Ser Thr Asn
275 280 285
Tyr Asn Glu Met Phe Glu Gly Arg Ala Thr Leu Thr Val Asp Thr Ser
290 295 300
Thr Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
305 310 315 320
Ala Val Tyr Tyr Cys Ala Arg Gly Asp Gly Asn Tyr Ala Leu Asp Ala
325 330 335
Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser
340 345 350
Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr
355 360 365
Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro
370 375 380
Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
385 390 395 400
His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser
405 410 415
Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr
420 425 430
Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val
435 440 445
Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe
450 455 460
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
465 470 475 480
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
485 490 495
Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val
500 505 510
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser
515 520 525
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
530 535 540
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser
545 550 555 560
Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
565 570 575
Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln
580 585 590
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
595 600 605
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
610 615 620
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu
625 630 635 640
Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser
645 650 655
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
660 665 670
Leu Ser Leu Gly Lys
675
<210> 46
<211> 2031
<212> DNA
<213> Homo
<400> 46
caagtgcagc tggtccagag cggcgctgag gtgaagaagc ccggcgccag cgtgaaagtc 60
agctgcaaag ctagcggcta ctccttcact ggctacacta tgaactgggt gaggcaagcc 120
cccggccaag gcctcgagtg gatcggactg atcaacccat acaacggcga cacaagctac 180
aaccagaagt tcaagggaag ggccacagtg actgtggaca agtccacaag cactgcctac 240
atggaactga gctctctgag gagcgaggat acagccgtgt actactgcgc taggtggaga 300
tacacaatgg actattgggg ccaaggcact ctggtcactg tgagcagcgc aagtaccaag 360
ggacctagtg ttttccctct tgcaccttgc tccaggtcaa catcagagtc cacagctgct 420
cttggatgtc tcgttaagga ctacttccca gagccagtta ccgtatcctg gaactccgga 480
gctttgacaa gcggcgttca tacattccca gctgtgttgc agagttctgg gttgtacagt 540
ttgagctcag tggtgaccgt gccttcatct tctttgggca ctaagaccta cacctgcaac 600
gtggatcaca agccaagcaa caccaaggtg gataagaggg tgggtggagg cggttcaggc 660
ggaggtggca gcggaggtgg cgggagtcag gtgcagctgg tgcagtctgg agctgaggtg 720
aagaagcctg gcgcttctgt gaaggtgtct tgtaaggctt ctggatatat ctttattacc 780
tattggatga attgggtgag acaggctcct ggccagggcc tggagtggat cggacagatt 840
tttccagctt ctggctccac aaattataat gagatgtttg agggcagagc tacactgaca 900
gtggatacat ctacatctac cgcctacatg gaactgtctt ctctgagatc tgaggataca 960
gctgtgtact attgtgctag aggcgatggc aattatgctc tggatgctat ggattattgg 1020
ggccagggaa cactggtgac cgtgtcttct gcctccacca agggcccatc ggtcttcccc 1080
ctggcaccct gctccaggag cacctctgag tccacagcgg ccctgggctg cctggtcaag 1140
gactacttcc ccgaaccggt gacggtgtcg tggaactcag gcgccctgac cagcggcgtg 1200
cacaccttcc cggctgtcct acagtcctca ggactctact ccctcagcag cgtggtgacc 1260
gtgccctcca gcagcttggg caccaagaca tatacctgta atgtggatca caagccttcc 1320
aatacaaaag tggacaagag agttgagtcc aagtacggcc caccatgtcc tccatgtcca 1380
gcccctgaat ttttgggcgg gccttctgtc tttctgtttc ctcctaaacc taaagatacc 1440
ctgatgatca gccgcacacc cgaagtcact tgtgtggtcg tggatgtgtc tcaggaagat 1500
cccgaagtgc agtttaactg gtatgtcgat ggcgtggaag tgcataatgc caaaactaag 1560
ccccgcgaag aacagttcaa cagcacttat cgggtcgtgt ctgtgctcac agtcctccat 1620
caggattggc tgaatgggaa agaatataag tgcaaggtga gcaataaggg cctccccagc 1680
agcatcgaga agactattag caaagccaaa gggcagccac gggaacccca ggtgtacact 1740
ctgcccccct ctcaggagga gatgactaaa aatcaggtct ctctgacttg tctggtgaaa 1800
gggttttatc ccagcgacat tgccgtggag tgggagtcta atggccagcc cgagaataat 1860
tataagacaa ctccccccgt cctggactct gacggcagct ttttcctgta ttctcggctg 1920
acagtggaca aaagtcgctg gcaggagggc aatgtcttta gttgcagtgt catgcatgag 1980
gccctgcaca atcactatac acagaaaagc ctgtctctga gtctgggcaa a 2031
<210> 47
<211> 677
<212> PRT
<213> Homo
<400> 47
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ile Phe Ile Thr Tyr
20 25 30
Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Gln Ile Phe Pro Ala Ser Gly Ser Thr Asn Tyr Asn Glu Met Phe
50 55 60
Glu Gly Arg Ala Thr Leu Thr Val Asp Thr Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gly Asp Gly Asn Tyr Ala Leu Asp Ala Met Asp Tyr Trp Gly
100 105 110
Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
115 120 125
Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala
130 135 140
Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val
145 150 155 160
Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
165 170 175
Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
180 185 190
Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His
195 200 205
Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Gly Gly Gly Gly Ser
210 215 220
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Val Gln
225 230 235 240
Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys
245 250 255
Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr Thr Met Asn Trp Val Arg
260 265 270
Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile Gly Leu Ile Asn Pro Tyr
275 280 285
Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe Lys Gly Arg Ala Thr Val
290 295 300
Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu
305 310 315 320
Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Trp Arg Tyr Thr
325 330 335
Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser
340 345 350
Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr
355 360 365
Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro
370 375 380
Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
385 390 395 400
His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser
405 410 415
Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr
420 425 430
Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val
435 440 445
Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe
450 455 460
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
465 470 475 480
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
485 490 495
Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val
500 505 510
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser
515 520 525
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
530 535 540
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser
545 550 555 560
Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
565 570 575
Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln
580 585 590
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
595 600 605
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
610 615 620
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu
625 630 635 640
Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser
645 650 655
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
660 665 670
Leu Ser Leu Gly Lys
675
<210> 48
<211> 2031
<212> DNA
<213> Homo
<400> 48
caggtgcagc tggtgcagtc tggagctgag gtgaagaagc ctggcgcttc tgtgaaggtg 60
tcttgtaagg cttctggata tatctttatt acctattgga tgaattgggt gagacaggct 120
cctggccagg gcctggagtg gatcggacag atttttccag cttctggctc cacaaattat 180
aatgagatgt ttgagggcag agctacactg acagtggata catctacatc taccgcctat 240
atggagctgt cttctctgag atctgaagat accgctgtgt attattgtgc tagaggagat 300
ggcaactatg ctctggatgc catggattac tggggccagg gcaccctggt gactgtgagc 360
agcgcaagta ccaagggacc tagtgttttc cctcttgcac cttgctccag gtcaacatca 420
gagtccacag ctgctcttgg atgtctcgtt aaggactact tcccagagcc agttaccgta 480
tcctggaact ccggagcttt gacaagcggc gttcatacat tcccagctgt gttgcagagt 540
tctgggttgt acagtttgag ctcagtggtg accgtgcctt catcttcttt gggcactaag 600
acctacacct gcaacgtgga tcacaagcca agcaacacca aggtggataa gagggtgggt 660
ggaggcggtt caggcggagg tggcagcgga ggtggcggga gtcaagtgca gctggtccag 720
agcggcgctg aggtgaagaa gcccggcgcc agcgtgaaag tcagctgcaa agctagcggc 780
tactccttca ctggctacac tatgaactgg gtgaggcaag cccccggcca aggcctcgag 840
tggatcggac tgatcaaccc atacaacggc gacacaagct acaaccagaa gttcaaggga 900
agggccacag tgactgtgga caagtccaca agcactgcct acatggaact gagctctctg 960
aggagcgagg atacagccgt gtactactgc gctaggtgga gatacacaat ggactattgg 1020
ggccaaggca ctctggtcac agtgtctagt gcctccacca agggcccatc ggtcttcccc 1080
ctggcaccct gctccaggag cacctctgag tccacagcgg ccctgggctg cctggtcaag 1140
gactacttcc ccgaaccggt gacggtgtcg tggaactcag gcgccctgac cagcggcgtg 1200
cacaccttcc cggctgtcct acagtcctca ggactctact ccctcagcag cgtggtgacc 1260
gtgccctcca gcagcttggg caccaagaca tatacctgta atgtggatca caagccttcc 1320
aatacaaaag tggacaagag agttgagtcc aagtacggcc caccatgtcc tccatgtcca 1380
gcccctgaat ttttgggcgg gccttctgtc tttctgtttc ctcctaaacc taaagatacc 1440
ctgatgatca gccgcacacc cgaagtcact tgtgtggtcg tggatgtgtc tcaggaagat 1500
cccgaagtgc agtttaactg gtatgtcgat ggcgtggaag tgcataatgc caaaactaag 1560
ccccgcgaag aacagttcaa cagcacttat cgggtcgtgt ctgtgctcac agtcctccat 1620
caggattggc tgaatgggaa agaatataag tgcaaggtga gcaataaggg cctccccagc 1680
agcatcgaga agactattag caaagccaaa gggcagccac gggaacccca ggtgtacact 1740
ctgcccccct ctcaggagga gatgactaaa aatcaggtct ctctgacttg tctggtgaaa 1800
gggttttatc ccagcgacat tgccgtggag tgggagtcta atggccagcc cgagaataat 1860
tataagacaa ctccccccgt cctggactct gacggcagct ttttcctgta ttctcggctg 1920
acagtggaca aaagtcgctg gcaggagggc aatgtcttta gttgcagtgt catgcatgag 1980
gccctgcaca atcactatac acagaaaagc ctgtctctga gtctgggcaa a 2031
<210> 49
<211> 677
<212> PRT
<213> Homo
<400> 49
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr
20 25 30
Thr Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Leu Ile Asn Pro Tyr Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Arg Ala Thr Val Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Trp Arg Tyr Thr Met Asp Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala
115 120 125
Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu
130 135 140
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
145 150 155 160
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
165 170 175
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
180 185 190
Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr
195 200 205
Lys Val Asp Lys Arg Val Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
210 215 220
Gly Gly Gly Gly Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
225 230 235 240
Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr
245 250 255
Thr Phe Thr Ser Glu Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln
260 265 270
Gly Leu Glu Trp Met Gly Gln Ile Phe Pro Ala Leu Gly Ser Thr Asn
275 280 285
Tyr Asn Glu Met Tyr Glu Gly Arg Val Thr Met Thr Thr Asp Thr Ser
290 295 300
Thr Ser Thr Ala Tyr Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr
305 310 315 320
Ala Val Tyr Tyr Cys Ala Arg Gly Ile Gly Asn Tyr Ala Leu Asp Ala
325 330 335
Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser
340 345 350
Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr
355 360 365
Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro
370 375 380
Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
385 390 395 400
His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser
405 410 415
Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr
420 425 430
Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val
435 440 445
Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe
450 455 460
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
465 470 475 480
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
485 490 495
Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val
500 505 510
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser
515 520 525
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
530 535 540
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser
545 550 555 560
Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
565 570 575
Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln
580 585 590
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
595 600 605
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
610 615 620
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu
625 630 635 640
Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser
645 650 655
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
660 665 670
Leu Ser Leu Gly Lys
675
<210> 50
<211> 2031
<212> DNA
<213> Homo
<400> 50
caagtgcagc tggtccagag cggcgctgag gtgaagaagc ccggcgccag cgtgaaagtc 60
agctgcaaag ctagcggcta ctccttcact ggctacacta tgaactgggt gaggcaagcc 120
cccggccaag gcctcgagtg gatcggactg atcaacccat acaacggcga cacaagctac 180
aaccagaagt tcaagggaag ggccacagtg actgtggaca agtccacaag cactgcctac 240
atggaactga gctctctgag gagcgaggat acagccgtgt actactgcgc taggtggaga 300
tacacaatgg actattgggg ccaaggcact ctggtcactg tgagcagcgc aagtaccaag 360
ggacctagtg ttttccctct tgcaccttgc tccaggtcaa catcagagtc cacagctgct 420
cttggatgtc tcgttaagga ctacttccca gagccagtta ccgtatcctg gaactccgga 480
gctttgacaa gcggcgttca tacattccca gctgtgttgc agagttctgg gttgtacagt 540
ttgagctcag tggtgaccgt gccttcatct tctttgggca ctaagaccta cacctgcaac 600
gtggatcaca agccaagcaa caccaaggtg gataagaggg tgggtggagg cggttcaggc 660
ggaggtggca gcggaggtgg cgggagtcaa gtgcagctgg tgcagagcgg cgctgaggtc 720
aaaaagcccg gcgcctccgt gaaggtgagc tgtaaggcca gcggctacac attcactagc 780
gagtggatga actgggtgag acaagccccc ggccaaggac tggaatggat gggccagatc 840
ttcccagctc tgggctccac taactacaac gagatgtacg agggaagggt cactatgact 900
acagacacta gcactagcac tgcctacatg gaactgaggt ctctgagaag cgacgataca 960
gccgtgtact actgcgccag aggcatcggc aactatgctc tggatgccat ggactactgg 1020
ggccaaggca ctctcgtgac tgtgagctcc gcctccacca agggcccatc ggtcttcccc 1080
ctggcaccct gctccaggag cacctctgag tccacagcgg ccctgggctg cctggtcaag 1140
gactacttcc ccgaaccggt gacggtgtcg tggaactcag gcgccctgac cagcggcgtg 1200
cacaccttcc cggctgtcct acagtcctca ggactctact ccctcagcag cgtggtgacc 1260
gtgccctcca gcagcttggg caccaagaca tatacctgta atgtggatca caagccttcc 1320
aatacaaaag tggacaagag agttgagtcc aagtacggcc caccatgtcc tccatgtcca 1380
gcccctgaat ttttgggcgg gccttctgtc tttctgtttc ctcctaaacc taaagatacc 1440
ctgatgatca gccgcacacc cgaagtcact tgtgtggtcg tggatgtgtc tcaggaagat 1500
cccgaagtgc agtttaactg gtatgtcgat ggcgtggaag tgcataatgc caaaactaag 1560
ccccgcgaag aacagttcaa cagcacttat cgggtcgtgt ctgtgctcac agtcctccat 1620
caggattggc tgaatgggaa agaatataag tgcaaggtga gcaataaggg cctccccagc 1680
agcatcgaga agactattag caaagccaaa gggcagccac gggaacccca ggtgtacact 1740
ctgcccccct ctcaggagga gatgactaaa aatcaggtct ctctgacttg tctggtgaaa 1800
gggttttatc ccagcgacat tgccgtggag tgggagtcta atggccagcc cgagaataat 1860
tataagacaa ctccccccgt cctggactct gacggcagct ttttcctgta ttctcggctg 1920
acagtggaca aaagtcgctg gcaggagggc aatgtcttta gttgcagtgt catgcatgag 1980
gccctgcaca atcactatac acagaaaagc ctgtctctga gtctgggcaa a 2031
<210> 51
<211> 677
<212> PRT
<213> Homo
<400> 51
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Glu
20 25 30
Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Gln Ile Phe Pro Ala Leu Gly Ser Thr Asn Tyr Asn Glu Met Tyr
50 55 60
Glu Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gly Ile Gly Asn Tyr Ala Leu Asp Ala Met Asp Tyr Trp Gly
100 105 110
Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
115 120 125
Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala
130 135 140
Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val
145 150 155 160
Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
165 170 175
Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
180 185 190
Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His
195 200 205
Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Gly Gly Gly Gly Ser
210 215 220
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Val Gln
225 230 235 240
Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys
245 250 255
Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr Thr Met Asn Trp Val Arg
260 265 270
Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile Gly Leu Ile Asn Pro Tyr
275 280 285
Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe Lys Gly Arg Ala Thr Val
290 295 300
Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu
305 310 315 320
Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Trp Arg Tyr Thr
325 330 335
Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser
340 345 350
Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr
355 360 365
Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro
370 375 380
Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
385 390 395 400
His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser
405 410 415
Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr
420 425 430
Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val
435 440 445
Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe
450 455 460
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
465 470 475 480
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
485 490 495
Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val
500 505 510
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser
515 520 525
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
530 535 540
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser
545 550 555 560
Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
565 570 575
Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln
580 585 590
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
595 600 605
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
610 615 620
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu
625 630 635 640
Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser
645 650 655
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
660 665 670
Leu Ser Leu Gly Lys
675
<210> 52
<211> 2031
<212> DNA
<213> Homo
<400> 52
caagtgcagc tggtgcagag cggcgctgag gtcaaaaagc ccggcgcctc cgtgaaggtg 60
agctgtaagg ccagcggcta cacattcact agcgagtgga tgaactgggt gagacaagcc 120
cccggccaag gactggaatg gatgggccag atcttcccag ctctgggctc cactaactac 180
aacgagatgt acgagggaag ggtcactatg actacagaca ctagcactag cactgcctac 240
atggaactga ggtctctgag aagcgacgat acagccgtgt actactgcgc cagaggcatc 300
ggcaactatg ctctggatgc catggactac tggggccaag gcactctcgt gactgtgagc 360
tccgcaagta ccaagggacc tagtgttttc cctcttgcac cttgctccag gtcaacatca 420
gagtccacag ctgctcttgg atgtctcgtt aaggactact tcccagagcc agttaccgta 480
tcctggaact ccggagcttt gacaagcggc gttcatacat tcccagctgt gttgcagagt 540
tctgggttgt acagtttgag ctcagtggtg accgtgcctt catcttcttt gggcactaag 600
acctacacct gcaacgtgga tcacaagcca agcaacacca aggtggataa gagggtgggt 660
ggaggcggtt caggcggagg tggcagcgga ggtggcggga gtcaagtgca gctggtccag 720
agcggcgctg aggtgaagaa gcccggcgcc agcgtgaaag tcagctgcaa agctagcggc 780
tactccttca ctggctacac tatgaactgg gtgaggcaag cccccggcca aggcctcgag 840
tggatcggac tgatcaaccc atacaacggc gacacaagct acaaccagaa gttcaaggga 900
agggccacag tgactgtgga caagtccaca agcactgcct acatggaact gagctctctg 960
aggagcgagg atacagccgt gtactactgc gctaggtgga gatacacaat ggactattgg 1020
ggccaaggca ctctggtcac agtgtctagt gcctccacca agggcccatc ggtcttcccc 1080
ctggcaccct gctccaggag cacctctgag tccacagcgg ccctgggctg cctggtcaag 1140
gactacttcc ccgaaccggt gacggtgtcg tggaactcag gcgccctgac cagcggcgtg 1200
cacaccttcc cggctgtcct acagtcctca ggactctact ccctcagcag cgtggtgacc 1260
gtgccctcca gcagcttggg caccaagaca tatacctgta atgtggatca caagccttcc 1320
aatacaaaag tggacaagag agttgagtcc aagtacggcc caccatgtcc tccatgtcca 1380
gcccctgaat ttttgggcgg gccttctgtc tttctgtttc ctcctaaacc taaagatacc 1440
ctgatgatca gccgcacacc cgaagtcact tgtgtggtcg tggatgtgtc tcaggaagat 1500
cccgaagtgc agtttaactg gtatgtcgat ggcgtggaag tgcataatgc caaaactaag 1560
ccccgcgaag aacagttcaa cagcacttat cgggtcgtgt ctgtgctcac agtcctccat 1620
caggattggc tgaatgggaa agaatataag tgcaaggtga gcaataaggg cctccccagc 1680
agcatcgaga agactattag caaagccaaa gggcagccac gggaacccca ggtgtacact 1740
ctgcccccct ctcaggagga gatgactaaa aatcaggtct ctctgacttg tctggtgaaa 1800
gggttttatc ccagcgacat tgccgtggag tgggagtcta atggccagcc cgagaataat 1860
tataagacaa ctccccccgt cctggactct gacggcagct ttttcctgta ttctcggctg 1920
acagtggaca aaagtcgctg gcaggagggc aatgtcttta gttgcagtgt catgcatgag 1980
gccctgcaca atcactatac acagaaaagc ctgtctctga gtctgggcaa a 2031
<210> 53
<211> 117
<212> PRT
<213> Homo
<400> 53
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Ala Ile His Trp Val Arg Gln Ala Pro Gly Gln Ser Leu Glu Trp Ile
35 40 45
Gly Val Phe Ser Ile Tyr Tyr Glu Asn Ile Asn Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Arg Ala Thr Met Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Arg Asp Gly Gly Thr Ile Asn Tyr Trp Gly Gln Gly Thr Leu
100 105 110
Val Thr Val Ser Ser
115
<210> 54
<211> 112
<212> PRT
<213> Homo
<400> 54
Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly
1 5 10 15
Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser
20 25 30
Asn Gly Asn Thr Tyr Leu His Trp Tyr Gln Gln Arg Pro Gly Gln Ser
35 40 45
Pro Arg Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Phe Cys Ser Gln Ser
85 90 95
Thr His Ile Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105 110
<210> 55
<211> 5
<212> PRT
<213> Mus musculus
<400> 55
Asn His Val Ile His
1 5
<210> 56
<211> 17
<212> PRT
<213> Mus musculus
<400> 56
Tyr Ile Tyr Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Glu Lys Phe Lys
1 5 10 15
Asp
<210> 57
<211> 8
<212> PRT
<213> Mus musculus
<400> 57
Gly Gly Tyr Tyr Thr Tyr Asp Asp
1 5
<210> 58
<211> 16
<212> PRT
<213> Mus musculus
<400> 58
Arg Ser Ser Gln Ser Leu Val His Ser Asn Gly Lys Thr Tyr Leu His
1 5 10 15
<210> 59
<211> 7
<212> PRT
<213> Mus musculus
<400> 59
Lys Val Ser Asn Arg Phe Ser
1 5
<210> 60
<211> 9
<212> PRT
<213> Mus musculus
<400> 60
Ser Gln Ser Thr His Val Pro Tyr Thr
1 5
<210> 61
<211> 117
<212> PRT
<213> Homo
<400> 61
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ala Asn His
20 25 30
Val Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Tyr Ile Tyr Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Glu Lys Phe
50 55 60
Lys Asp Arg Val Thr Leu Thr Ser Asp Lys Ser Thr Ser Thr Val Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gly Gly Tyr Tyr Thr Tyr Asp Asp Trp Gly Gln Gly Thr Leu
100 105 110
Val Thr Val Ser Ser
115
<210> 62
<211> 112
<212> PRT
<213> Homo
<400> 62
Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly
1 5 10 15
Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser
20 25 30
Asn Gly Lys Thr Tyr Leu His Trp Tyr Gln Gln Arg Pro Gly Gln Ser
35 40 45
Pro Arg Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Ser
85 90 95
Thr His Val Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105 110
<210> 63
<211> 219
<212> PRT
<213> Homo
<400> 63
Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly
1 5 10 15
Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser
20 25 30
Asn Gly Asn Thr Tyr Leu His Trp Tyr Gln Gln Arg Pro Gly Gln Ser
35 40 45
Pro Arg Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Phe Cys Ser Gln Ser
85 90 95
Thr His Ile Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105 110
Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
115 120 125
Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
130 135 140
Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
145 150 155 160
Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
165 170 175
Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
180 185 190
Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
195 200 205
Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
210 215
<210> 64
<211> 657
<212> DNA
<213> Homo
<400> 64
gacgtggtga tgacccagag ccctttatct ttacccgtta cactgggaca gcccgccagc 60
atcagctgtc gtagcagcca gtctttagtg cacagcaacg gcaacaccta tttacactgg 120
taccagcaga gacccggcca gagccccaga ctgctgatct acaaggtgag caatcgtttc 180
tccggcgtgc ccgacagatt cagcggcagc ggaagcggca ccgacttcac tttaaagatc 240
agcagagtgg aggccgagga cgtgggcgtg tacttctgca gccagagcac ccacatccct 300
tggaccttcg gccaaggtac caaggtggag atcaagagaa ccgtcgccgc tcccagcgtc 360
ttcatcttcc cccccagcga tgagcagctg aagagcggaa ccgccagcgt ggtgtgcctg 420
ctgaacaact tctaccccag ggaggccaag gtgcaatgga aggtggacaa cgccctacag 480
agcggcaact cccaggagag cgtgaccgag caggacagca aggatagcac ctacagcctg 540
agcagcaccc tcaccctgag caaggccgac tacgagaagc acaaggtgta cgcctgcgag 600
gtgacccatc agggcctgag cagccctgtg accaagagct tcaacagggg cgagtgc 657
<210> 65
<211> 677
<212> PRT
<213> Homo
<400> 65
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ala Asn His
20 25 30
Val Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Tyr Ile Tyr Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Glu Lys Phe
50 55 60
Lys Asp Arg Val Thr Leu Thr Ser Asp Lys Ser Thr Ser Thr Val Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gly Gly Tyr Tyr Thr Tyr Asp Asp Trp Gly Gln Gly Thr Leu
100 105 110
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
115 120 125
Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys
130 135 140
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser
145 150 155 160
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
165 170 175
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser
180 185 190
Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn
195 200 205
Thr Lys Val Asp Lys Arg Val Gly Gly Gly Gly Ser Gly Gly Gly Gly
210 215 220
Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu
225 230 235 240
Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Gly Ser Gly
245 250 255
Tyr Thr Phe Thr Asp Tyr Ala Ile His Trp Val Arg Gln Ala Pro Gly
260 265 270
Gln Ser Leu Glu Trp Ile Gly Val Phe Ser Ile Tyr Tyr Glu Asn Ile
275 280 285
Asn Tyr Asn Gln Lys Phe Lys Gly Arg Ala Thr Met Thr Val Asp Lys
290 295 300
Ser Thr Ser Thr Ala Tyr Met Glu Leu Arg Ser Leu Arg Ser Asp Asp
305 310 315 320
Thr Ala Val Tyr Tyr Cys Ala Arg Arg Asp Gly Gly Thr Ile Asn Tyr
325 330 335
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly
340 345 350
Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly
355 360 365
Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
370 375 380
Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe
385 390 395 400
Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
405 410 415
Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val
420 425 430
Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys
435 440 445
Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
450 455 460
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
465 470 475 480
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
485 490 495
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
500 505 510
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
515 520 525
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
530 535 540
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
545 550 555 560
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
565 570 575
Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln
580 585 590
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
595 600 605
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
610 615 620
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
625 630 635 640
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
645 650 655
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
660 665 670
Leu Ser Pro Gly Lys
675
<210> 66
<211> 2031
<212> DNA
<213> Homo
<400> 66
caggtgcagc tggtgcagtc cggcgccgag gtgaagaagc ccggcgcctc cgtgaaagtg 60
agctgcaagg catccggcta caccttcgcc aaccacgtga tccactgggt gcggcaggcc 120
cccggccagg gcctggagtg gatgggctac atctatcctt ataatgatgg cacaaagtat 180
aatgagaagt ttaaggatag ggtaacactg acatctgata agtctacatc tacagtgtac 240
atggagctgt cctctctgag gtccgaggac accgccgtgt actactgcgc cagaggcggc 300
tactacacct acgacgactg gggccagggc accctggtga ccgtgtcctc cgcaagtacc 360
aagggaccta gtgttttccc tcttgcacct tgctccaggt caacatcaga gtccacagct 420
gctcttggat gtctcgttaa ggactacttc ccagagccag ttaccgtatc ctggaactcc 480
ggagctttga caagcggcgt tcatacattc ccagctgtgt tgcagagttc tgggttgtac 540
agtttgagct cagtggtgac cgtgccttca tcttctttgg gcactaagac ctacacctgc 600
aacgtggatc acaagccaag caacaccaag gtggataaga gggtgggtgg aggcggttca 660
ggcggaggtg gcagcggagg tggcgggagt caagttcagc tggtgcagag cggagctgag 720
gtgaagaagc ccggcgccag cgtgaaggtg agctgtaagg gcagcggcta caccttcacc 780
gactacgcca tccactgggt gagacaagct cccggtcagt ctttagaatg gatcggcgtg 840
ttcagcatct actacgagaa catcaactat aaccagaagt tcaagggtcg tgccaccatg 900
accgtggaca agagcaccag caccgcctac atggagctga ggtctttaag gagcgacgac 960
accgccgtgt actactgcgc tcgtagggac ggcggcacca tcaactactg gggccaaggt 1020
actttagtga cagtgagcag cgcgagcacc aagggacctt ccgtgtttcc cctcgccccc 1080
agctccaaaa gcaccagcgg cggaacagct gctctcggct gtctcgtcaa ggattacttc 1140
cccgagcccg tgaccgtgag ctggaacagc ggagccctga caagcggcgt ccacaccttc 1200
cctgctgtcc tacagtcctc cggactgtac agcctgagca gcgtggtgac agtccctagc 1260
agctccctgg gcacccagac atatatttgc aacgtgaatc acaagcccag caacaccaag 1320
gtcgataaga aggtggagcc taagtcctgc gacaagaccc acacatgtcc cccctgtccc 1380
gctcctgaac tgctgggagg cccttccgtg ttcctgttcc cccctaagcc caaggacacc 1440
ctgatgattt ccaggacacc cgaggtgacc tgtgtggtgg tggacgtcag ccacgaggac 1500
cccgaggtga aattcaactg gtacgtcgat ggcgtggagg tgcacaacgc taagaccaag 1560
cccagggagg agcagtacaa ttccacctac agggtggtgt ccgtgctgac cgtcctccat 1620
caggactggc tgaacggcaa agagtataag tgcaaggtga gcaacaaggc cctccctgct 1680
cccatcgaga agaccatcag caaagccaag ggccagccca gggaacctca agtctatacc 1740
ctgcctccca gcagggagga gatgaccaag aaccaagtga gcctcacatg cctcgtcaag 1800
ggcttctatc cttccgatat tgccgtcgag tgggagtcca acggacagcc cgagaacaac 1860
tacaagacaa caccccccgt gctcgattcc gatggcagct tcttcctgta ctccaagctg 1920
accgtggaca agtccagatg gcaacaaggc aacgtcttca gttgcagcgt catgcatgag 1980
gccctccaca accactacac ccagaaaagc ctgtctctga gtcctggcaa a 2031
<210> 67
<211> 677
<212> PRT
<213> Homo
<400> 67
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Ala Ile His Trp Val Arg Gln Ala Pro Gly Gln Ser Leu Glu Trp Ile
35 40 45
Gly Val Phe Ser Ile Tyr Tyr Glu Asn Ile Asn Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Arg Ala Thr Met Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Arg Asp Gly Gly Thr Ile Asn Tyr Trp Gly Gln Gly Thr Leu
100 105 110
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
115 120 125
Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys
130 135 140
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser
145 150 155 160
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
165 170 175
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser
180 185 190
Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn
195 200 205
Thr Lys Val Asp Lys Arg Val Gly Gly Gly Gly Ser Gly Gly Gly Gly
210 215 220
Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu
225 230 235 240
Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly
245 250 255
Tyr Thr Phe Ala Asn His Val Ile His Trp Val Arg Gln Ala Pro Gly
260 265 270
Gln Gly Leu Glu Trp Met Gly Tyr Ile Tyr Pro Tyr Asn Asp Gly Thr
275 280 285
Lys Tyr Asn Glu Lys Phe Lys Asp Arg Val Thr Leu Thr Ser Asp Lys
290 295 300
Ser Thr Ser Thr Val Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
305 310 315 320
Thr Ala Val Tyr Tyr Cys Ala Arg Gly Gly Tyr Tyr Thr Tyr Asp Asp
325 330 335
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly
340 345 350
Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly
355 360 365
Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
370 375 380
Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe
385 390 395 400
Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
405 410 415
Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val
420 425 430
Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys
435 440 445
Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
450 455 460
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
465 470 475 480
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
485 490 495
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
500 505 510
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
515 520 525
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
530 535 540
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
545 550 555 560
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
565 570 575
Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln
580 585 590
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
595 600 605
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
610 615 620
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
625 630 635 640
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
645 650 655
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
660 665 670
Leu Ser Pro Gly Lys
675
<210> 68
<211> 2031
<212> DNA
<213> Homo
<400> 68
caagttcagc tggtgcagag cggagctgag gtgaagaagc ccggcgccag cgtgaaggtg 60
agctgtaagg gcagcggcta caccttcacc gactacgcca tccactgggt gagacaagct 120
cccggtcagt ctttagaatg gatcggcgtg ttcagcatct actacgagaa catcaactat 180
aaccagaagt tcaagggtcg tgccaccatg accgtggaca agagcaccag caccgcctac 240
atggagctga ggtctttaag gagcgacgac accgccgtgt actactgcgc tcgtagggac 300
ggcggcacca tcaactactg gggccaaggt actttagtga cagtgagcag cgcaagtacc 360
aagggaccta gtgttttccc tcttgcacct tgctccaggt caacatcaga gtccacagct 420
gctcttggat gtctcgttaa ggactacttc ccagagccag ttaccgtatc ctggaactcc 480
ggagctttga caagcggcgt tcatacattc ccagctgtgt tgcagagttc tgggttgtac 540
agtttgagct cagtggtgac cgtgccttca tcttctttgg gcactaagac ctacacctgc 600
aacgtggatc acaagccaag caacaccaag gtggataaga gggtgggtgg aggcggttca 660
ggcggaggtg gcagcggagg tggcgggagt caggtgcagc tggtgcagtc cggcgccgag 720
gtgaagaagc ccggcgcctc cgtgaaagtg agctgcaagg catccggcta caccttcgcc 780
aaccacgtga tccactgggt gcggcaggcc cccggccagg gcctggagtg gatgggctac 840
atctatcctt ataatgatgg cacaaagtat aatgagaagt ttaaggatag ggtaacactg 900
acatctgata agtctacatc tacagtgtac atggagctgt cctctctgag gtccgaggac 960
accgccgtgt actactgcgc cagaggcggc tactacacct acgacgactg gggccagggc 1020
accctggtga ccgtgtcctc cgcgagcacc aagggacctt ccgtgtttcc cctcgccccc 1080
agctccaaaa gcaccagcgg cggaacagct gctctcggct gtctcgtcaa ggattacttc 1140
cccgagcccg tgaccgtgag ctggaacagc ggagccctga caagcggcgt ccacaccttc 1200
cctgctgtcc tacagtcctc cggactgtac agcctgagca gcgtggtgac agtccctagc 1260
agctccctgg gcacccagac atatatttgc aacgtgaatc acaagcccag caacaccaag 1320
gtcgataaga aggtggagcc taagtcctgc gacaagaccc acacatgtcc cccctgtccc 1380
gctcctgaac tgctgggagg cccttccgtg ttcctgttcc cccctaagcc caaggacacc 1440
ctgatgattt ccaggacacc cgaggtgacc tgtgtggtgg tggacgtcag ccacgaggac 1500
cccgaggtga aattcaactg gtacgtcgat ggcgtggagg tgcacaacgc taagaccaag 1560
cccagggagg agcagtacaa ttccacctac agggtggtgt ccgtgctgac cgtcctccat 1620
caggactggc tgaacggcaa agagtataag tgcaaggtga gcaacaaggc cctccctgct 1680
cccatcgaga agaccatcag caaagccaag ggccagccca gggaacctca agtctatacc 1740
ctgcctccca gcagggagga gatgaccaag aaccaagtga gcctcacatg cctcgtcaag 1800
ggcttctatc cttccgatat tgccgtcgag tgggagtcca acggacagcc cgagaacaac 1860
tacaagacaa caccccccgt gctcgattcc gatggcagct tcttcctgta ctccaagctg 1920
accgtggaca agtccagatg gcaacaaggc aacgtcttca gttgcagcgt catgcatgag 1980
gccctccaca accactacac ccagaaaagc ctgtctctga gtcctggcaa a 2031
<210> 69
<211> 111
<212> PRT
<213> Mus musculus
<400> 69
Gln Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln
1 5 10 15
Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Ser Tyr
20 25 30
Asp Ile Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu
35 40 45
Gly Val Ile Trp Thr Gly Gly Gly Thr Asn Tyr Asn Ser Ala Phe Met
50 55 60
Ser Arg Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Leu
65 70 75 80
Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Lys Tyr Tyr Cys Val
85 90 95
Arg Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser
100 105 110
<210> 70
<211> 112
<212> PRT
<213> Mus musculus
<400> 70
Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly
1 5 10 15
Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser
20 25 30
Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser
35 40 45
Pro Lys Leu Leu Phe Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser
85 90 95
Thr His Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105 110
<210> 71
<211> 5
<212> PRT
<213> Mus musculus
<400> 71
Ser Tyr Asp Ile Ser
1 5
<210> 72
<211> 16
<212> PRT
<213> Mus musculus
<400> 72
Val Ile Trp Thr Gly Gly Gly Thr Asn Tyr Asn Ser Ala Phe Met Ser
1 5 10 15
<210> 73
<211> 3
<212> PRT
<213> Mus musculus
<400> 73
Met Asp Tyr
1
<210> 74
<211> 16
<212> PRT
<213> Mus musculus
<400> 74
Arg Ser Ser Gln Ser Leu Leu His Ser Asn Gly Asn Thr Tyr Leu His
1 5 10 15
<210> 75
<211> 7
<212> PRT
<213> Mus musculus
<400> 75
Lys Val Ser Asn Arg Phe Ser
1 5
<210> 76
<211> 9
<212> PRT
<213> Mus musculus
<400> 76
Ser Gln Ser Thr His Val Pro Trp Thr
1 5
<210> 77
<211> 111
<212> PRT
<213> Homo
<400> 77
Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu
1 5 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Ser Tyr
20 25 30
Asp Ile Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu
35 40 45
Gly Val Ile Trp Thr Gly Gly Gly Thr Asn Tyr Asn Ser Ala Phe Met
50 55 60
Ser Arg Leu Thr Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Ser Leu
65 70 75 80
Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Val
85 90 95
Arg Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
100 105 110
<210> 78
<211> 112
<212> PRT
<213> Homo
<400> 78
Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly
1 5 10 15
Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser
20 25 30
Asn Gly Asn Thr Tyr Leu His Trp Tyr Gln Gln Arg Pro Gly Gln Ser
35 40 45
Pro Arg Leu Leu Phe Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Phe Cys Ser Gln Ser
85 90 95
Thr His Val Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105 110
<210> 79
<211> 671
<212> PRT
<213> Homo
<400> 79
Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu
1 5 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Ser Tyr
20 25 30
Asp Ile Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu
35 40 45
Gly Val Ile Trp Thr Gly Gly Gly Thr Asn Tyr Asn Ser Ala Phe Met
50 55 60
Ser Arg Leu Thr Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Ser Leu
65 70 75 80
Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Val
85 90 95
Arg Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala
100 105 110
Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser
115 120 125
Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe
130 135 140
Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly
145 150 155 160
Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu
165 170 175
Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr
180 185 190
Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg
195 200 205
Val Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
210 215 220
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
225 230 235 240
Ser Val Lys Val Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp Tyr
245 250 255
Ala Ile His Trp Val Arg Gln Ala Pro Gly Gln Ser Leu Glu Trp Ile
260 265 270
Gly Val Phe Ser Ile Tyr Tyr Glu Asn Ile Asn Tyr Asn Gln Lys Phe
275 280 285
Lys Gly Arg Ala Thr Met Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr
290 295 300
Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
305 310 315 320
Ala Arg Arg Asp Gly Gly Thr Ile Asn Tyr Trp Gly Gln Gly Thr Leu
325 330 335
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
340 345 350
Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys
355 360 365
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser
370 375 380
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
385 390 395 400
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser
405 410 415
Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn
420 425 430
Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His
435 440 445
Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val
450 455 460
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
465 470 475 480
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
485 490 495
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
500 505 510
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
515 520 525
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
530 535 540
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
545 550 555 560
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
565 570 575
Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
580 585 590
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
595 600 605
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
610 615 620
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
625 630 635 640
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
645 650 655
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
660 665 670
<210> 80
<211> 2013
<212> DNA
<213> Homo
<400> 80
caggtgcagc tgcaggaatc tggcccaggc ctggtgaagc cttctgagac actgtctttg 60
acctgtacag tgtctggctt ttctctgtct tcttatgata tttcctggat tagacagcct 120
cctggcaagg gcctggagtg gctgggagtg atttggacag gaggcggaac aaattataac 180
tctgctttta tgtctaggct gaccatctct aaggataata gtaaatctca ggtgtctctg 240
aagctgtctt ctgtgaccgc tgctgataca gccgtgtact attgtgttag aatggattat 300
tggggccagg gcacactggt gaccgtgtct tctgcaagta ccaagggacc tagtgttttc 360
cctcttgcac cttgctccag gtcaacatca gagtccacag ctgctcttgg atgtctcgtt 420
aaggactact tcccagagcc agttaccgta tcctggaact ccggagcttt gacaagcggc 480
gttcatacat tcccagctgt gttgcagagt tctgggttgt acagtttgag ctcagtggtg 540
accgtgcctt catcttcttt gggcactaag acctacacct gcaacgtgga tcacaagcca 600
agcaacacca aggtggataa gagggtgggt ggaggcggtt caggcggagg tggcagcgga 660
ggtggcggga gtcaagttca gctggtgcag agcggagctg aggtgaagaa gcccggcgcc 720
agcgtgaagg tgagctgtaa gggcagcggc tacaccttca ccgactacgc catccactgg 780
gtgagacaag ctcccggtca gtctttagaa tggatcggcg tgttcagcat ctactacgag 840
aacatcaact ataaccagaa gttcaagggt cgtgccacca tgaccgtgga caagagcacc 900
agcaccgcct acatggagct gaggtcttta aggagcgacg acaccgccgt gtactactgc 960
gctcgtaggg acggcggcac catcaactac tggggccaag gtactttagt gacagtgagc 1020
agcgcgagca ccaagggacc ttccgtgttt cccctcgccc ccagctccaa aagcaccagc 1080
ggcggaacag ctgctctcgg ctgtctcgtc aaggattact tccccgagcc cgtgaccgtg 1140
agctggaaca gcggagccct gacaagcggc gtccacacct tccctgctgt cctacagtcc 1200
tccggactgt acagcctgag cagcgtggtg acagtcccta gcagctccct gggcacccag 1260
acatatattt gcaacgtgaa tcacaagccc agcaacacca aggtcgataa gaaggtggag 1320
cctaagtcct gcgacaagac ccacacatgt cccccctgtc ccgctcctga agctgctgga 1380
ggcccttccg tgttcctgtt cccccctaag cccaaggaca ccctgatgat ttccaggaca 1440
cccgaggtga cctgtgtggt ggtggacgtc agccacgagg accccgaggt gaaattcaac 1500
tggtacgtcg atggcgtgga ggtgcacaac gctaagacca agcccaggga ggagcagtac 1560
aattccacct acagggtggt gtccgtgctg accgtcctcc atcaggactg gctgaacggc 1620
aaagagtata agtgcaaggt gagcaacaag gccctccctg ctcccatcga gaagaccatc 1680
agcaaagcca agggccagcc cagggaacct caagtctata ccctgcctcc cagcagggag 1740
gagatgacca agaaccaagt gagcctcaca tgcctcgtca agggcttcta tccttccgat 1800
attgccgtcg agtgggagtc caacggacag cccgagaaca actacaagac aacacccccc 1860
gtgctcgatt ccgatggcag cttcttcctg tactccaagc tgaccgtgga caagtccaga 1920
tggcaacaag gcaacgtctt cagttgcagc gtcatgcatg aggccctcca caaccactac 1980
acccagaaga gcctctccct gagccctgga aag 2013
<210> 81
<211> 671
<212> PRT
<213> Homo
<400> 81
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Ala Ile His Trp Val Arg Gln Ala Pro Gly Gln Ser Leu Glu Trp Ile
35 40 45
Gly Val Phe Ser Ile Tyr Tyr Glu Asn Ile Asn Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Arg Ala Thr Met Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Arg Asp Gly Gly Thr Ile Asn Tyr Trp Gly Gln Gly Thr Leu
100 105 110
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
115 120 125
Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys
130 135 140
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser
145 150 155 160
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
165 170 175
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser
180 185 190
Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn
195 200 205
Thr Lys Val Asp Lys Arg Val Gly Gly Gly Gly Ser Gly Gly Gly Gly
210 215 220
Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly
225 230 235 240
Leu Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly
245 250 255
Phe Ser Leu Ser Ser Tyr Asp Ile Ser Trp Ile Arg Gln Pro Pro Gly
260 265 270
Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Thr Gly Gly Gly Thr Asn
275 280 285
Tyr Asn Ser Ala Phe Met Ser Arg Leu Thr Ile Ser Lys Asp Asn Ser
290 295 300
Lys Ser Gln Val Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr
305 310 315 320
Ala Val Tyr Tyr Cys Val Arg Met Asp Tyr Trp Gly Gln Gly Thr Leu
325 330 335
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
340 345 350
Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys
355 360 365
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser
370 375 380
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
385 390 395 400
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser
405 410 415
Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn
420 425 430
Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His
435 440 445
Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val
450 455 460
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
465 470 475 480
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
485 490 495
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
500 505 510
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
515 520 525
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
530 535 540
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
545 550 555 560
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
565 570 575
Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
580 585 590
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
595 600 605
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
610 615 620
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
625 630 635 640
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
645 650 655
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
660 665 670
<210> 82
<211> 2013
<212> DNA
<213> Homo
<400> 82
caagttcagc tggtgcagag cggagctgag gtgaagaagc ccggcgccag cgtgaaggtg 60
agctgtaagg gcagcggcta caccttcacc gactacgcca tccactgggt gagacaagct 120
cccggtcagt ctttagaatg gatcggcgtg ttcagcatct actacgagaa catcaactat 180
aaccagaagt tcaagggtcg tgccaccatg accgtggaca agagcaccag caccgcctac 240
atggagctga ggtctttaag gagcgacgac accgccgtgt actactgcgc tcgtagggac 300
ggcggcacca tcaactactg gggccaaggt actttagtga cagtgagcag cgcaagtacc 360
aagggaccta gtgttttccc tcttgcacct tgctccaggt caacatcaga gtccacagct 420
gctcttggat gtctcgttaa ggactacttc ccagagccag ttaccgtatc ctggaactcc 480
ggagctttga caagcggcgt tcatacattc ccagctgtgt tgcagagttc tgggttgtac 540
agtttgagct cagtggtgac cgtgccttca tcttctttgg gcactaagac ctacacctgc 600
aacgtggatc acaagccaag caacaccaag gtggataaga gggtgggtgg aggcggttca 660
ggcggaggtg gcagcggagg tggcgggagt caggtgcagc tgcaggaatc tggcccaggc 720
ctggtgaagc cttctgagac actgtctttg acctgtacag tgtctggctt ttctctgtct 780
tcttatgata tttcctggat tagacagcct cctggcaagg gcctggagtg gctgggagtg 840
atttggacag gaggcggaac aaattataac tctgctttta tgtctaggct gaccatctct 900
aaggataata gtaaatctca ggtgtctctg aagctgtctt ctgtgaccgc tgctgataca 960
gccgtgtact attgtgttag aatggattat tggggccagg gcacactggt gaccgtgtct 1020
tctgcgagca ccaagggacc ttccgtgttt cccctcgccc ccagctccaa aagcaccagc 1080
ggcggaacag ctgctctcgg ctgtctcgtc aaggattact tccccgagcc cgtgaccgtg 1140
agctggaaca gcggagccct gacaagcggc gtccacacct tccctgctgt cctacagtcc 1200
tccggactgt acagcctgag cagcgtggtg acagtcccta gcagctccct gggcacccag 1260
acatatattt gcaacgtgaa tcacaagccc agcaacacca aggtcgataa gaaggtggag 1320
cctaagtcct gcgacaagac ccacacatgt cccccctgtc ccgctcctga agctgctgga 1380
ggcccttccg tgttcctgtt cccccctaag cccaaggaca ccctgatgat ttccaggaca 1440
cccgaggtga cctgtgtggt ggtggacgtc agccacgagg accccgaggt gaaattcaac 1500
tggtacgtcg atggcgtgga ggtgcacaac gctaagacca agcccaggga ggagcagtac 1560
aattccacct acagggtggt gtccgtgctg accgtcctcc atcaggactg gctgaacggc 1620
aaagagtata agtgcaaggt gagcaacaag gccctccctg ctcccatcga gaagaccatc 1680
agcaaagcca agggccagcc cagggaacct caagtctata ccctgcctcc cagcagggag 1740
gagatgacca agaaccaagt gagcctcaca tgcctcgtca agggcttcta tccttccgat 1800
attgccgtcg agtgggagtc caacggacag cccgagaaca actacaagac aacacccccc 1860
gtgctcgatt ccgatggcag cttcttcctg tactccaagc tgaccgtgga caagtccaga 1920
tggcaacaag gcaacgtctt cagttgcagc gtcatgcatg aggccctcca caaccactac 1980
acccagaaga gcctctccct gagccctgga aag 2013
<210> 83
<211> 117
<212> PRT
<213> Homo
<400> 83
Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Ser Ser Tyr
20 25 30
Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Arg Leu Glu Trp Val
35 40 45
Ala Thr Ile Ser Gly Gly Gly Arg Tyr Thr Tyr Tyr Pro Asp Thr Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser His Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys
85 90 95
Ala Ser Pro Tyr Gly Gly Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu
100 105 110
Val Thr Val Ser Ser
115
<210> 84
<211> 107
<212> PRT
<213> Homo
<400> 84
Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Asn Phe
20 25 30
Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45
Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ala Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro
65 70 75 80
Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Ser Asn Ser Trp Pro His
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105
<210> 85
<211> 5
<212> PRT
<213> Mus musculus
<400> 85
Ser Tyr Trp Met His
1 5
<210> 86
<211> 17
<212> PRT
<213> Mus musculus
<400> 86
Glu Ile Asn Pro Gly Asn Gly His Thr Asn Tyr Asn Glu Lys Phe Lys
1 5 10 15
Ser
<210> 87
<211> 10
<212> PRT
<213> Mus musculus
<400> 87
Ser Phe Thr Thr Ala Arg Gly Phe Ala Tyr
1 5 10
<210> 88
<211> 11
<212> PRT
<213> Mus musculus
<400> 88
Arg Ala Ser Gln Thr Ile Ser Asp Tyr Leu His
1 5 10
<210> 89
<211> 7
<212> PRT
<213> Mus musculus
<400> 89
Tyr Ala Ser Gln Ser Ile Ser
1 5
<210> 90
<211> 9
<212> PRT
<213> Mus musculus
<400> 90
Gln Asp Gly His Ser Phe Pro Pro Thr
1 5
<210> 91
<211> 119
<212> PRT
<213> Homo
<400> 91
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Ser Tyr
20 25 30
Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Glu Ile Asn Pro Gly Asn Gly His Thr Asn Tyr Asn Glu Lys Phe
50 55 60
Lys Ser Arg Val Thr Leu Thr Val Asp Lys Ser Thr Ser Thr Val Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Ser Phe Thr Thr Ala Arg Gly Phe Ala Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ser
115
<210> 92
<211> 107
<212> PRT
<213> Homo
<400> 92
Glu Ile Val Met Thr Gln Ser Pro Asp Phe Gln Ser Val Thr Pro Lys
1 5 10 15
Glu Lys Val Thr Ile Thr Cys Arg Ala Ser Gln Thr Ile Ser Asp Tyr
20 25 30
Leu His Trp Tyr Gln Gln Lys Pro Asp Gln Ser Pro Lys Leu Leu Ile
35 40 45
Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Ser Asp Phe Thr Leu Thr Ile Asn Ser Leu Glu Ala
65 70 75 80
Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Asp Gly His Ser Phe Pro Pro
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105
<210> 93
<211> 214
<212> PRT
<213> Homo
<400> 93
Glu Ile Val Met Thr Gln Ser Pro Asp Phe Gln Ser Val Thr Pro Lys
1 5 10 15
Glu Lys Val Thr Ile Thr Cys Arg Ala Ser Gln Thr Ile Ser Asp Tyr
20 25 30
Leu His Trp Tyr Gln Gln Lys Pro Asp Gln Ser Pro Lys Leu Leu Ile
35 40 45
Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Ser Asp Phe Thr Leu Thr Ile Asn Ser Leu Glu Ala
65 70 75 80
Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Asp Gly His Ser Phe Pro Pro
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys
210
<210> 94
<211> 642
<212> DNA
<213> Homo
<400> 94
gagatcgtga tgacccagtc tcctgatttt cagtctgtga cccctaagga gaaggtgaca 60
atcacctgta gagcttctca gaccatctct gattatctgc attggtatca gcagaagcct 120
gatcagtctc ctaagctgct gatcaagtat gcttctcagt ctatctctgg cattccttct 180
aggttttccg gctctggctc tggctctgat tttacactga ccatcaattc tctggaggct 240
gaggatgctg ccacctatta ttgtcaggac ggccactctt ttcctcctac ctttggccag 300
ggaaccaagg tggagatcaa gagaaccgtc gccgctccca gcgtcttcat cttccccccc 360
agcgatgagc agctgaagag cggaaccgcc agcgtggtgt gcctgctgaa caacttctac 420
cccagggagg ccaaggtgca atggaaggtg gacaacgccc tacagagcgg caactcccag 480
gagagcgtga ccgagcagga cagcaaggat agcacctaca gcctgagcag caccctcacc 540
ctgagcaagg ccgactacga gaagcacaag gtgtacgcct gcgaggtgac ccatcagggc 600
ctgagcagcc ctgtgaccaa gagcttcaac aggggcgagt gc 642
<210> 95
<211> 676
<212> PRT
<213> Homo
<400> 95
Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Ser Ser Tyr
20 25 30
Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Arg Leu Glu Trp Val
35 40 45
Ala Thr Ile Ser Gly Gly Gly Arg Tyr Thr Tyr Tyr Pro Asp Thr Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser His Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys
85 90 95
Ala Ser Pro Tyr Gly Gly Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu
100 105 110
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
115 120 125
Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys
130 135 140
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser
145 150 155 160
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
165 170 175
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser
180 185 190
Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn
195 200 205
Thr Lys Val Asp Lys Arg Val Gly Gly Gly Gly Ser Gly Gly Gly Gly
210 215 220
Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu
225 230 235 240
Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly
245 250 255
Tyr Thr Phe Ser Ser Tyr Trp Met His Trp Val Arg Gln Ala Pro Gly
260 265 270
Gln Gly Leu Glu Trp Met Gly Glu Ile Asn Pro Gly Asn Gly His Thr
275 280 285
Asn Tyr Asn Glu Lys Phe Lys Ser Arg Val Thr Leu Thr Val Asp Lys
290 295 300
Ser Thr Ser Thr Val Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
305 310 315 320
Thr Ala Val Tyr Tyr Cys Ala Arg Ser Phe Thr Thr Ala Arg Gly Phe
325 330 335
Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr
340 345 350
Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser
355 360 365
Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu
370 375 380
Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His
385 390 395 400
Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
405 410 415
Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys
420 425 430
Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu
435 440 445
Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu
450 455 460
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
465 470 475 480
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
485 490 495
Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu
500 505 510
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr
515 520 525
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
530 535 540
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser
545 550 555 560
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
565 570 575
Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val
580 585 590
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
595 600 605
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
610 615 620
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr
625 630 635 640
Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val
645 650 655
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
660 665 670
Ser Leu Gly Lys
675
<210> 96
<211> 2028
<212> DNA
<213> Homo
<400> 96
gaggtcaagc tggtggaaag cggcggcggc ctggtgcagc ctggaggatc cctgcggctg 60
agctgcgctg cctccggctt cgctttcagc tcctatgaca tgtcctgggt gaggcaggcc 120
cctggaaaga ggctggagtg ggtggctacc atctccggag gcggaaggta cacctactac 180
cccgacacag tgaagggaag gttcaccatc agccgggata acgccaaaaa cagccactat 240
ctccagatga actccctgag ggccgaagat acagccgtgt atttctgtgc ctccccctac 300
ggaggctatt ttgacgtgtg gggacagggc accctggtga ccgtctcctc cgcaagtacc 360
aagggaccta gtgttttccc tcttgcacct tgctccaggt caacatcaga gtccacagct 420
gctcttggat gtctcgttaa ggactacttc ccagagccag ttaccgtatc ctggaactcc 480
ggagctttga caagcggcgt tcatacattc ccagctgtgt tgcagagttc tgggttgtac 540
agtttgagct cagtggtgac cgtgccttca tcttctttgg gcactaagac ctacacctgc 600
aacgtggatc acaagccaag caacaccaag gtggataaga gggtgggtgg aggcggttca 660
ggcggaggtg gcagcggagg tggcgggagt caggtgcagc tggtgcagag cggcgctgaa 720
gtgaagaagc caggcgcttc tgtaaaggtg tcttgtaagg cttctggcta tacattttct 780
tcttattgga tgcactgggt gagacaggct cctggccagg gactggagtg gatgggtgag 840
atcaatcctg gcaatggcca tactaactac aacgagaagt tcaagtccag ggtgaccctg 900
accgtggaca agtccacctc caccgtgtac atggagctga gctccctgag gtccgaggac 960
accgccgtgt actactgcgc ccggagcttc actaccgccc gcggcttcgc ctattggggc 1020
cagggcaccc tggtgaccgt gagctccgcc tccaccaagg gcccatcggt cttccccctg 1080
gcaccctgct ccaggagcac ctctgagtcc acagcggccc tgggctgcct ggtcaaggac 1140
tacttccccg aaccggtgac ggtgtcgtgg aactcaggcg ccctgaccag cggcgtgcac 1200
accttcccgg ctgtcctaca gtcctcagga ctctactccc tcagcagcgt ggtgaccgtg 1260
ccctccagca gcttgggcac caagacatat acctgtaatg tggatcacaa gccttccaat 1320
acaaaagtgg acaagagagt tgagtccaag tacggcccac catgtcctcc atgtccagcc 1380
cctgaatttt tgggcgggcc ttctgtcttt ctgtttcctc ctaaacctaa agataccctg 1440
atgatcagcc gcacacccga agtcacttgt gtggtcgtgg atgtgtctca ggaagatccc 1500
gaagtgcagt ttaactggta tgtcgatggc gtggaagtgc ataatgccaa aactaagccc 1560
cgcgaagaac agttcaacag cacttatcgg gtcgtgtctg tgctcacagt cctccatcag 1620
gattggctga atgggaaaga atataagtgc aaggtgagca ataagggcct ccccagcagc 1680
atcgagaaga ctattagcaa agccaaaggg cagccacggg aaccccaggt gtacactctg 1740
cccccctctc aggaggagat gactaaaaat caggtctctc tgacttgtct ggtgaaaggg 1800
ttttatccca gcgacattgc cgtggagtgg gagtctaatg gccagcccga gaataattat 1860
aagacaactc cccccgtcct ggactctgac ggcagctttt tcctgtattc tcggctgaca 1920
gtggacaaaa gtcgctggca ggagggcaat gtctttagtt gcagtgtcat gcatgaggcc 1980
ctgcacaatc actatacaca gaaaagcctg tctctgagtc tgggcaaa 2028
<210> 97
<211> 676
<212> PRT
<213> Homo
<400> 97
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Ser Tyr
20 25 30
Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Glu Ile Asn Pro Gly Asn Gly His Thr Asn Tyr Asn Glu Lys Phe
50 55 60
Lys Ser Arg Val Thr Leu Thr Val Asp Lys Ser Thr Ser Thr Val Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Ser Phe Thr Thr Ala Arg Gly Phe Ala Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
115 120 125
Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu
130 135 140
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
145 150 155 160
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
165 170 175
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
180 185 190
Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro
195 200 205
Ser Asn Thr Lys Val Asp Lys Arg Val Gly Gly Gly Gly Ser Gly Gly
210 215 220
Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Lys Leu Val Glu Ser Gly
225 230 235 240
Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala
245 250 255
Ser Gly Phe Ala Phe Ser Ser Tyr Asp Met Ser Trp Val Arg Gln Ala
260 265 270
Pro Gly Lys Arg Leu Glu Trp Val Ala Thr Ile Ser Gly Gly Gly Arg
275 280 285
Tyr Thr Tyr Tyr Pro Asp Thr Val Lys Gly Arg Phe Thr Ile Ser Arg
290 295 300
Asp Asn Ala Lys Asn Ser His Tyr Leu Gln Met Asn Ser Leu Arg Ala
305 310 315 320
Glu Asp Thr Ala Val Tyr Phe Cys Ala Ser Pro Tyr Gly Gly Tyr Phe
325 330 335
Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr
340 345 350
Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser
355 360 365
Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu
370 375 380
Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His
385 390 395 400
Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
405 410 415
Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys
420 425 430
Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu
435 440 445
Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu
450 455 460
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
465 470 475 480
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
485 490 495
Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu
500 505 510
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr
515 520 525
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
530 535 540
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser
545 550 555 560
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
565 570 575
Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val
580 585 590
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
595 600 605
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
610 615 620
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr
625 630 635 640
Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val
645 650 655
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
660 665 670
Ser Leu Gly Lys
675
<210> 98
<211> 2028
<212> DNA
<213> Homo
<400> 98
caggtgcagc tggtgcagag cggcgctgaa gtgaagaagc caggcgcttc tgtaaaggtg 60
tcttgtaagg cttctggcta tacattttct tcttattgga tgcactgggt gagacaggct 120
cctggccagg gactggagtg gatgggtgag atcaatcctg gcaatggcca tactaactac 180
aacgagaagt tcaagtccag ggtgaccctg accgtggaca agtccacctc caccgtgtac 240
atggagctga gctccctgag gtccgaggac accgccgtgt actactgcgc ccggagcttc 300
actaccgccc gcggcttcgc ctattggggc cagggcaccc tggtgaccgt gagctccgca 360
agtaccaagg gacctagtgt tttccctctt gcaccttgct ccaggtcaac atcagagtcc 420
acagctgctc ttggatgtct cgttaaggac tacttcccag agccagttac cgtatcctgg 480
aactccggag ctttgacaag cggcgttcat acattcccag ctgtgttgca gagttctggg 540
ttgtacagtt tgagctcagt ggtgaccgtg ccttcatctt ctttgggcac taagacctac 600
acctgcaacg tggatcacaa gccaagcaac accaaggtgg ataagagggt gggtggaggc 660
ggttcaggcg gaggtggcag cggaggtggc gggagtgagg tcaagctggt ggaaagcggc 720
ggcggcctgg tgcagcctgg aggatccctg cggctgagct gcgctgcctc cggcttcgct 780
ttcagctcct atgacatgtc ctgggtgagg caggcccctg gaaagaggct ggagtgggtg 840
gctaccatct ccggaggcgg aaggtacacc tactaccccg acacagtgaa gggaaggttc 900
accatcagcc gggataacgc caaaaacagc cactatctcc agatgaactc cctgagggcc 960
gaagatacag ccgtgtattt ctgtgcctcc ccctacggag gctattttga cgtgtgggga 1020
cagggcaccc tggtgaccgt ctcctccgcc tccaccaagg gcccatcggt cttccccctg 1080
gcaccctgct ccaggagcac ctctgagtcc acagcggccc tgggctgcct ggtcaaggac 1140
tacttccccg aaccggtgac ggtgtcgtgg aactcaggcg ccctgaccag cggcgtgcac 1200
accttcccgg ctgtcctaca gtcctcagga ctctactccc tcagcagcgt ggtgaccgtg 1260
ccctccagca gcttgggcac caagacatat acctgtaatg tggatcacaa gccttccaat 1320
acaaaagtgg acaagagagt tgagtccaag tacggcccac catgtcctcc atgtccagcc 1380
cctgaatttt tgggcgggcc ttctgtcttt ctgtttcctc ctaaacctaa agataccctg 1440
atgatcagcc gcacacccga agtcacttgt gtggtcgtgg atgtgtctca ggaagatccc 1500
gaagtgcagt ttaactggta tgtcgatggc gtggaagtgc ataatgccaa aactaagccc 1560
cgcgaagaac agttcaacag cacttatcgg gtcgtgtctg tgctcacagt cctccatcag 1620
gattggctga atgggaaaga atataagtgc aaggtgagca ataagggcct ccccagcagc 1680
atcgagaaga ctattagcaa agccaaaggg cagccacggg aaccccaggt gtacactctg 1740
cccccctctc aggaggagat gactaaaaat caggtctctc tgacttgtct ggtgaaaggg 1800
ttttatccca gcgacattgc cgtggagtgg gagtctaatg gccagcccga gaataattat 1860
aagacaactc cccccgtcct ggactctgac ggcagctttt tcctgtattc tcggctgaca 1920
gtggacaaaa gtcgctggca ggagggcaat gtctttagtt gcagtgtcat gcatgaggcc 1980
ctgcacaatc actatacaca gaaaagcctg tctctgagtc tgggcaaa 2028
<210> 99
<211> 5
<212> PRT
<213> Mus musculus
<400> 99
Thr Thr Gly Met Gln
1 5
<210> 100
<211> 17
<212> PRT
<213> Mus musculus
<400> 100
Trp Ile Asn Thr His Ser Gly Val Pro Lys Tyr Val Glu Asp Phe Lys
1 5 10 15
Gly
<210> 101
<211> 13
<212> PRT
<213> Mus musculus
<400> 101
Ser Gly Asn Gly Asn Tyr Asp Leu Ala Tyr Phe Ala Tyr
1 5 10
<210> 102
<211> 11
<212> PRT
<213> Mus musculus
<400> 102
Arg Ala Ser Gln Ser Ile Ser Asp Tyr Leu His
1 5 10
<210> 103
<211> 7
<212> PRT
<213> Mus musculus
<400> 103
Tyr Ala Ser His Ser Ile Ser
1 5
<210> 104
<211> 9
<212> PRT
<213> Mus musculus
<400> 104
Gln His Gly His Ser Phe Pro Trp Thr
1 5
<210> 105
<211> 122
<212> PRT
<213> Homo
<400> 105
Gln Ile Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Thr Thr
20 25 30
Gly Met Gln Trp Val Arg Glu Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Trp Ile Asn Thr His Ser Gly Val Pro Lys Tyr Val Glu Asp Phe
50 55 60
Lys Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Asn Thr Ala Tyr
65 70 75 80
Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Val Arg Ser Gly Asn Gly Asn Tyr Asp Leu Ala Tyr Phe Ala Tyr Trp
100 105 110
Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 106
<211> 107
<212> PRT
<213> Homo
<400> 106
Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Asp Tyr
20 25 30
Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Arg Leu Leu Ile
35 40 45
Lys Tyr Ala Ser His Ser Ile Ser Gly Ile Pro Ala Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Ser Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro
65 70 75 80
Glu Asp Phe Ala Val Tyr Tyr Cys Gln His Gly His Ser Phe Pro Trp
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105
<210> 107
<211> 214
<212> PRT
<213> Homo
<400> 107
Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Asp Tyr
20 25 30
Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Arg Leu Leu Ile
35 40 45
Lys Tyr Ala Ser His Ser Ile Ser Gly Ile Pro Ala Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Ser Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro
65 70 75 80
Glu Asp Phe Ala Val Tyr Tyr Cys Gln His Gly His Ser Phe Pro Trp
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys
210
<210> 108
<211> 642
<212> DNA
<213> Homo
<400> 108
gaaatcgtgc tgacacagtc tcctgctaca ctgtctctga gtcctggaga aagagctaca 60
ctgtcttgta gagcttccca gtctatctcc gattatctgc actggtacca gcagaagcct 120
ggccagtctc ctagactgct gatcaagtat gcttctcatt ctatttctgg catcccagct 180
aggtttagtg gatctggctc tggaagcgac tttacactga ccatttcttc tctggagcct 240
gaggattttg ctgtgtatta ctgtcagcac ggccattctt ttccttggac ctttggccag 300
ggcacaaagg tggagatcaa gagaaccgtc gccgctccca gcgtcttcat cttccccccc 360
agcgatgagc agctgaagag cggaaccgcc agcgtggtgt gcctgctgaa caacttctac 420
cccagggagg ccaaggtgca atggaaggtg gacaacgccc tacagagcgg caactcccag 480
gagagcgtga ccgagcagga cagcaaggat agcacctaca gcctgagcag caccctcacc 540
ctgagcaagg ccgactacga gaagcacaag gtgtacgcct gcgaggtgac ccatcagggc 600
ctgagcagcc ctgtgaccaa gagcttcaac aggggcgagt gc 642
<210> 109
<211> 679
<212> PRT
<213> Homo
<400> 109
Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Ser Ser Tyr
20 25 30
Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Arg Leu Glu Trp Val
35 40 45
Ala Thr Ile Ser Gly Gly Gly Arg Tyr Thr Tyr Tyr Pro Asp Thr Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser His Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys
85 90 95
Ala Ser Pro Tyr Gly Gly Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu
100 105 110
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
115 120 125
Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys
130 135 140
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser
145 150 155 160
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
165 170 175
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser
180 185 190
Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn
195 200 205
Thr Lys Val Asp Lys Arg Val Gly Gly Gly Gly Ser Gly Gly Gly Gly
210 215 220
Ser Gly Gly Gly Gly Ser Gln Ile Gln Leu Val Gln Ser Gly Ser Glu
225 230 235 240
Leu Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly
245 250 255
Tyr Ala Phe Thr Thr Thr Gly Met Gln Trp Val Arg Glu Ala Pro Gly
260 265 270
Gln Gly Leu Glu Trp Ile Gly Trp Ile Asn Thr His Ser Gly Val Pro
275 280 285
Lys Tyr Val Glu Asp Phe Lys Gly Arg Phe Val Phe Ser Leu Asp Thr
290 295 300
Ser Val Asn Thr Ala Tyr Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp
305 310 315 320
Thr Ala Val Tyr Tyr Cys Val Arg Ser Gly Asn Gly Asn Tyr Asp Leu
325 330 335
Ala Tyr Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
340 345 350
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg
355 360 365
Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
370 375 380
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
385 390 395 400
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
405 410 415
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr
420 425 430
Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys
435 440 445
Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro
450 455 460
Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
465 470 475 480
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
485 490 495
Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp
500 505 510
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe
515 520 525
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
530 535 540
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu
545 550 555 560
Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
565 570 575
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys
580 585 590
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
595 600 605
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
610 615 620
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
625 630 635 640
Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser
645 650 655
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
660 665 670
Leu Ser Leu Ser Leu Gly Lys
675
<210> 110
<211> 2037
<212> DNA
<213> Homo
<400> 110
gaggtcaagc tggtggaaag cggcggcggc ctggtgcagc ctggaggatc cctgcggctg 60
agctgcgctg cctccggctt cgctttcagc tcctatgaca tgtcctgggt gaggcaggcc 120
cctggaaaga ggctggagtg ggtggctacc atctccggag gcggaaggta cacctactac 180
cccgacacag tgaagggaag gttcaccatc agccgggata acgccaaaaa cagccactat 240
ctccagatga actccctgag ggccgaagat acagccgtgt atttctgtgc ctccccctac 300
ggaggctatt ttgacgtgtg gggacagggc accctggtga ccgtctcctc cgcaagtacc 360
aagggaccta gtgttttccc tcttgcacct tgctccaggt caacatcaga gtccacagct 420
gctcttggat gtctcgttaa ggactacttc ccagagccag ttaccgtatc ctggaactcc 480
ggagctttga caagcggcgt tcatacattc ccagctgtgt tgcagagttc tgggttgtac 540
agtttgagct cagtggtgac cgtgccttca tcttctttgg gcactaagac ctacacctgc 600
aacgtggatc acaagccaag caacaccaag gtggataaga gggtgggtgg aggcggttca 660
ggcggaggtg gcagcggagg tggcgggagt cagatccagc tggtgcagtc cggctccgag 720
ctgaagaagc ccggcgcctc cgtcaaggtg tcctgcaagg ccagtggcta cgcctttacc 780
accaccggca tgcagtgggt gagggaggcc ccaggccagg gcctggagtg gatcggctgg 840
atcaacacac attctggagt gcctaagtat gtggaagact ttaagggcag attcgtgttt 900
tctctggata cctctgtgaa taccgcttac ctgcagattt cttctctgaa ggctgaggac 960
acagctgtgt attactgtgt gagatctgga aatggaaact atgatctggc ttattttgcc 1020
tattggggcc agggcacact ggtgaccgtg tcttctgcct ccaccaaggg cccatcggtc 1080
ttccccctgg caccctgctc caggagcacc tctgagtcca cagcggccct gggctgcctg 1140
gtcaaggact acttccccga accggtgacg gtgtcgtgga actcaggcgc cctgaccagc 1200
ggcgtgcaca ccttcccggc tgtcctacag tcctcaggac tctactccct cagcagcgtg 1260
gtgaccgtgc cctccagcag cttgggcacc aagacatata cctgtaatgt ggatcacaag 1320
ccttccaata caaaagtgga caagagagtt gagtccaagt acggcccacc atgtcctcca 1380
tgtccagccc ctgaattttt gggcgggcct tctgtctttc tgtttcctcc taaacctaaa 1440
gataccctga tgatcagccg cacacccgaa gtcacttgtg tggtcgtgga tgtgtctcag 1500
gaagatcccg aagtgcagtt taactggtat gtcgatggcg tggaagtgca taatgccaaa 1560
actaagcccc gcgaagaaca gttcaacagc acttatcggg tcgtgtctgt gctcacagtc 1620
ctccatcagg attggctgaa tgggaaagaa tataagtgca aggtgagcaa taagggcctc 1680
cccagcagca tcgagaagac tattagcaaa gccaaagggc agccacggga accccaggtg 1740
tacactctgc ccccctctca ggaggagatg actaaaaatc aggtctctct gacttgtctg 1800
gtgaaagggt tttatcccag cgacattgcc gtggagtggg agtctaatgg ccagcccgag 1860
aataattata agacaactcc ccccgtcctg gactctgacg gcagcttttt cctgtattct 1920
cggctgacag tggacaaaag tcgctggcag gagggcaatg tctttagttg cagtgtcatg 1980
catgaggccc tgcacaatca ctatacacag aaaagcctgt ctctgagtct gggcaaa 2037
<210> 111
<211> 679
<212> PRT
<213> Homo
<400> 111
Gln Ile Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Thr Thr
20 25 30
Gly Met Gln Trp Val Arg Glu Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Trp Ile Asn Thr His Ser Gly Val Pro Lys Tyr Val Glu Asp Phe
50 55 60
Lys Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Asn Thr Ala Tyr
65 70 75 80
Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Val Arg Ser Gly Asn Gly Asn Tyr Asp Leu Ala Tyr Phe Ala Tyr Trp
100 105 110
Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
115 120 125
Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr
130 135 140
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
145 150 155 160
Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
165 170 175
Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
180 185 190
Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp
195 200 205
His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Gly Gly Gly Gly
210 215 220
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Lys Leu Val
225 230 235 240
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser
245 250 255
Cys Ala Ala Ser Gly Phe Ala Phe Ser Ser Tyr Asp Met Ser Trp Val
260 265 270
Arg Gln Ala Pro Gly Lys Arg Leu Glu Trp Val Ala Thr Ile Ser Gly
275 280 285
Gly Gly Arg Tyr Thr Tyr Tyr Pro Asp Thr Val Lys Gly Arg Phe Thr
290 295 300
Ile Ser Arg Asp Asn Ala Lys Asn Ser His Tyr Leu Gln Met Asn Ser
305 310 315 320
Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys Ala Ser Pro Tyr Gly
325 330 335
Gly Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
340 345 350
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg
355 360 365
Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
370 375 380
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
385 390 395 400
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
405 410 415
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr
420 425 430
Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys
435 440 445
Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro
450 455 460
Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
465 470 475 480
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
485 490 495
Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp
500 505 510
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe
515 520 525
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
530 535 540
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu
545 550 555 560
Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
565 570 575
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys
580 585 590
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
595 600 605
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
610 615 620
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
625 630 635 640
Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser
645 650 655
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
660 665 670
Leu Ser Leu Ser Leu Gly Lys
675
<210> 112
<211> 2037
<212> DNA
<213> Homo
<400> 112
cagatccagc tggtgcagtc cggctccgag ctgaagaagc ccggcgcctc cgtcaaggtg 60
tcctgcaagg ccagtggcta cgcctttacc accaccggca tgcagtgggt gagggaggcc 120
ccaggccagg gcctggagtg gatcggctgg atcaacacac attctggagt gcctaagtat 180
gtggaagact ttaagggcag attcgtgttt tctctggata cctctgtgaa taccgcttac 240
ctgcagattt cttctctgaa ggctgaggac acagctgtgt attactgtgt gagatctgga 300
aatggaaact atgatctggc ttattttgcc tattggggcc agggcacact ggtgaccgtg 360
tcttctgcaa gtaccaaggg acctagtgtt ttccctcttg caccttgctc caggtcaaca 420
tcagagtcca cagctgctct tggatgtctc gttaaggact acttcccaga gccagttacc 480
gtatcctgga actccggagc tttgacaagc ggcgttcata cattcccagc tgtgttgcag 540
agttctgggt tgtacagttt gagctcagtg gtgaccgtgc cttcatcttc tttgggcact 600
aagacctaca cctgcaacgt ggatcacaag ccaagcaaca ccaaggtgga taagagggtg 660
ggtggaggcg gttcaggcgg aggtggcagc ggaggtggcg ggagtgaggt caagctggtg 720
gaaagcggcg gcggcctggt gcagcctgga ggatccctgc ggctgagctg cgctgcctcc 780
ggcttcgctt tcagctccta tgacatgtcc tgggtgaggc aggcccctgg aaagaggctg 840
gagtgggtgg ctaccatctc cggaggcgga aggtacacct actaccccga cacagtgaag 900
ggaaggttca ccatcagccg ggataacgcc aaaaacagcc actatctcca gatgaactcc 960
ctgagggccg aagatacagc cgtgtatttc tgtgcctccc cctacggagg ctattttgac 1020
gtgtggggac agggcaccct ggtgaccgtc tcctccgcct ccaccaaggg cccatcggtc 1080
ttccccctgg caccctgctc caggagcacc tctgagtcca cagcggccct gggctgcctg 1140
gtcaaggact acttccccga accggtgacg gtgtcgtgga actcaggcgc cctgaccagc 1200
ggcgtgcaca ccttcccggc tgtcctacag tcctcaggac tctactccct cagcagcgtg 1260
gtgaccgtgc cctccagcag cttgggcacc aagacatata cctgtaatgt ggatcacaag 1320
ccttccaata caaaagtgga caagagagtt gagtccaagt acggcccacc atgtcctcca 1380
tgtccagccc ctgaattttt gggcgggcct tctgtctttc tgtttcctcc taaacctaaa 1440
gataccctga tgatcagccg cacacccgaa gtcacttgtg tggtcgtgga tgtgtctcag 1500
gaagatcccg aagtgcagtt taactggtat gtcgatggcg tggaagtgca taatgccaaa 1560
actaagcccc gcgaagaaca gttcaacagc acttatcggg tcgtgtctgt gctcacagtc 1620
ctccatcagg attggctgaa tgggaaagaa tataagtgca aggtgagcaa taagggcctc 1680
cccagcagca tcgagaagac tattagcaaa gccaaagggc agccacggga accccaggtg 1740
tacactctgc ccccctctca ggaggagatg actaaaaatc aggtctctct gacttgtctg 1800
gtgaaagggt tttatcccag cgacattgcc gtggagtggg agtctaatgg ccagcccgag 1860
aataattata agacaactcc ccccgtcctg gactctgacg gcagcttttt cctgtattct 1920
cggctgacag tggacaaaag tcgctggcag gagggcaatg tctttagttg cagtgtcatg 1980
catgaggccc tgcacaatca ctatacacag aaaagcctgt ctctgagtct gggcaaa 2037
<210> 113
<211> 119
<212> PRT
<213> Homo
<400> 113
Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln
1 5 10 15
Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asn Tyr
20 25 30
Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu
35 40 45
Gly Val Ile Trp Ser Gly Gly Asn Thr Asp Tyr Asn Thr Pro Phe Thr
50 55 60
Ser Arg Leu Ser Ile Asn Lys Asp Asn Ser Lys Ser Gln Val Phe Phe
65 70 75 80
Lys Met Asn Ser Leu Gln Ser Asn Asp Thr Ala Ile Tyr Tyr Cys Ala
85 90 95
Arg Ala Leu Thr Tyr Tyr Asp Tyr Glu Phe Ala Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ala
115
<210> 114
<211> 107
<212> PRT
<213> Homo
<400> 114
Asp Ile Leu Leu Thr Gln Ser Pro Val Ile Leu Ser Val Ser Pro Gly
1 5 10 15
Glu Arg Val Ser Phe Ser Cys Arg Ala Ser Gln Ser Ile Gly Thr Asn
20 25 30
Ile His Trp Tyr Gln Gln Arg Thr Asn Gly Ser Pro Arg Leu Leu Ile
35 40 45
Lys Tyr Ala Ser Glu Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Ser
65 70 75 80
Glu Asp Ile Ala Asp Tyr Tyr Cys Gln Gln Asn Asn Asn Trp Pro Thr
85 90 95
Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys
100 105
<210> 115
<211> 120
<212> PRT
<213> Homo
<400> 115
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr
20 25 30
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 116
<211> 107
<212> PRT
<213> Homo
<400> 116
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105
<210> 117
<211> 119
<212> PRT
<213> Homo
<400> 117
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Tyr
20 25 30
Thr Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Asp Val Asn Pro Asn Ser Gly Gly Ser Ile Tyr Asn Gln Arg Phe
50 55 60
Lys Gly Arg Phe Thr Leu Ser Val Asp Arg Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Asn Leu Gly Pro Ser Phe Tyr Phe Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ser
115
<210> 118
<211> 107
<212> PRT
<213> Homo
<400> 118
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Ile Gly
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Ile Tyr Pro Tyr
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105
<210> 119
<211> 118
<212> PRT
<213> Homo
<400> 119
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Thr Phe Ile Ser Tyr Asp Gly Asn Asn Lys Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Ile Tyr Tyr Cys
85 90 95
Ala Arg Thr Gly Trp Leu Gly Pro Phe Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser
115
<210> 120
<211> 108
<212> PRT
<213> Homo
<400> 120
Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Gly Ser Ser
20 25 30
Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu
35 40 45
Ile Tyr Gly Ala Phe Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser
50 55 60
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu
65 70 75 80
Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro
85 90 95
Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105
<210> 121
<211> 116
<212> PRT
<213> Homo
<400> 121
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Asp
20 25 30
Tyr Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Arg Leu Glu Trp Val
35 40 45
Ala Ser Ile Ser His Gly Gly Asp Tyr Ile Tyr Tyr Ala Asp Asn Leu
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys
85 90 95
Ser Arg Asp Arg Arg Ser Ile Asp Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Ser Ser
115
<210> 122
<211> 107
<212> PRT
<213> Homo
<400> 122
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr
20 25 30
Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Thr Ile Lys Leu Leu Ile
35 40 45
Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Tyr Thr Phe Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Lys Thr Leu Pro Tyr
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105
<210> 123
<211> 214
<212> PRT
<213> Homo
<400> 123
Asp Ile Leu Leu Thr Gln Ser Pro Val Ile Leu Ser Val Ser Pro Gly
1 5 10 15
Glu Arg Val Ser Phe Ser Cys Arg Ala Ser Gln Ser Ile Gly Thr Asn
20 25 30
Ile His Trp Tyr Gln Gln Arg Thr Asn Gly Ser Pro Arg Leu Leu Ile
35 40 45
Lys Tyr Ala Ser Glu Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Ser
65 70 75 80
Glu Asp Ile Ala Asp Tyr Tyr Cys Gln Gln Asn Asn Asn Trp Pro Thr
85 90 95
Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys
210
<210> 124
<211> 642
<212> DNA
<213> Homo
<400> 124
gacatcctgc tgacccagtc ccccgtgatc ctgtccgtgt cccctggcga gagggtgtcc 60
ttctcctgca gggcctccca gtccatcggc accaacatcc actggtacca gcagaggacc 120
aacggctccc ccaggctgct gatcaagtac gcctccgaga gcatcagcgg catcccctcc 180
aggttttccg gctccggctc cggaaccgac ttcaccctgt ccatcaactc cgtggagtcc 240
gaggacatcg ccgactacta ctgccagcag aacaacaact ggcccaccac ctttggcgcc 300
ggcaccaagc tggagctgaa gaggaccgtg gccgccccct ccgtgttcat ttttccccct 360
agcgacgagc agctgaagag cggcaccgct agcgtggtgt gcctgctgaa caacttctac 420
cccagggagg ccaaggtgca gtggaaagtg gacaacgccc tgcagagcgg caactcccag 480
gagtccgtga ccgagcagga cagcaaggac agcacctact ccctgtcctc caccctgacc 540
ctgtccaagg ccgactacga gaagcacaag gtgtacgcct gtgaggtgac acaccagggc 600
ctgtcctccc ccgtgacaaa gtccttcaac aggggcgagt gc 642
<210> 125
<211> 214
<212> PRT
<213> Homo
<400> 125
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Ile Gly
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Ile Tyr Pro Tyr
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys
210
<210> 126
<211> 642
<212> DNA
<213> Homo
<400> 126
gacatccaga tgacccagtc tccttcttct ctgtctgctt ctgtgggcga tagagtgacc 60
atcacctgta aggcttctca ggatgtgtct atcggcgtgg cctggtatca gcagaagcct 120
ggaaaggctc ctaagctgct gatctactct gcttcttaca gatatactgg cgtgccttct 180
agattttctg gctctggctc tggcacagat tttacactga ccatctcttc tctgcagcct 240
gaggacttcg ctacctatta ctgtcagcag tactacatct atccttatac ctttggccag 300
ggcaccaagg tggagatcaa gagaaccgtc gccgctccca gcgtcttcat cttccccccc 360
agcgatgagc agctgaagag cggaaccgcc agcgtggtgt gcctgctgaa caacttctac 420
cccagggagg ccaaggtgca atggaaggtg gacaacgccc tacagagcgg caactcccag 480
gagagcgtga ccgagcagga cagcaaggat agcacctaca gcctgagcag caccctcacc 540
ctgagcaagg ccgactacga gaagcacaag gtgtacgcct gcgaggtgac ccatcagggc 600
ctgagcagcc ctgtgaccaa gagcttcaac aggggcgagt gc 642
<210> 127
<211> 215
<212> PRT
<213> Homo
<400> 127
Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Gly Ser Ser
20 25 30
Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu
35 40 45
Ile Tyr Gly Ala Phe Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser
50 55 60
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu
65 70 75 80
Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro
85 90 95
Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala
100 105 110
Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser
115 120 125
Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu
130 135 140
Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser
145 150 155 160
Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu
165 170 175
Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val
180 185 190
Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys
195 200 205
Ser Phe Asn Arg Gly Glu Cys
210 215
<210> 128
<211> 645
<212> DNA
<213> Homo
<400> 128
gagatcgtgc tgactcagag ccccggcact ctgtctctga gccccggcga aagggccaca 60
ctgagctgta gggctagcca gagcgtgggc agcagctatc tggcttggta ccagcagaag 120
cccggccaag cccctagact gctgatctac ggcgccttct ctagggctac tggcatccca 180
gataggttta gcggcagcgg atccggcaca gacttcactc tgacaatctc taggctggag 240
ccagaagact tcgccgtgta ctattgccag cagtacggca gcagcccatg gactttcggc 300
caaggcacaa aggtcgagat caagagaacc gtcgccgctc ccagcgtctt catcttcccc 360
cccagcgatg agcagctgaa gagcggaacc gccagcgtgg tgtgcctgct gaacaacttc 420
taccccaggg aggccaaggt gcaatggaag gtggacaacg ccctacagag cggcaactcc 480
caggagagcg tgaccgagca ggacagcaag gatagcacct acagcctgag cagcaccctc 540
accctgagca aggccgacta cgagaagcac aaggtgtacg cctgcgaggt gacccatcag 600
ggcctgagca gccctgtgac caagagcttc aacaggggcg agtgc 645
<210> 129
<211> 214
<212> PRT
<213> Homo
<400> 129
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr
20 25 30
Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Thr Ile Lys Leu Leu Ile
35 40 45
Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Tyr Thr Phe Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Lys Thr Leu Pro Tyr
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys
210
<210> 130
<211> 642
<212> DNA
<213> Homo
<400> 130
gacatccaga tgacccagtc cccttcctcc ctgtccgctt ccgtgggcga cagggtgacc 60
atcacctgca gggccagcca ggacatctcc aactacctgt cctggtacca gcagaagccc 120
ggcaagacca tcaagctgct gatctactac acctccaggc tgcacagcgg cgtgcctagc 180
aggttctccg gttctggctc cggcaccgac tacaccttca ccatcagctc cctgcagccc 240
gaggacatcg ccacctactt ctgccagcag ggcaagaccc tgccctacac cttcggccag 300
ggcaccaagg tggagatcaa gcgcactgtg gctgccccca gtgttttcat atttcccccc 360
agtgatgagc aactgaagtc cggcacagcc tctgttgtat gtctgctgaa taatttttat 420
ccacgggagg ccaaggtgca gtggaaggtg gacaatgccc tgcagtctgg gaactctcaa 480
gagagtgtga cagagcagga cagtaaagac agcacctata gcctcagcag caccctgacc 540
ctgtctaaag ccgactatga aaaacacaaa gtgtatgcct gcgaagtgac ccatcagggg 600
ctcagctctc ccgttaccaa gagctttaac cgaggcgaat gt 642
<210> 131
<211> 676
<212> PRT
<213> Homo
<400> 131
Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Ser Ser Tyr
20 25 30
Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Arg Leu Glu Trp Val
35 40 45
Ala Thr Ile Ser Gly Gly Gly Arg Tyr Thr Tyr Tyr Pro Asp Thr Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser His Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys
85 90 95
Ala Ser Pro Tyr Gly Gly Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu
100 105 110
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
115 120 125
Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys
130 135 140
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser
145 150 155 160
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
165 170 175
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser
180 185 190
Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn
195 200 205
Thr Lys Val Asp Lys Arg Val Gly Gly Gly Gly Ser Gly Gly Gly Gly
210 215 220
Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Lys Gln Ser Gly Pro Gly
225 230 235 240
Leu Val Gln Pro Ser Gln Ser Leu Ser Ile Thr Cys Thr Val Ser Gly
245 250 255
Phe Ser Leu Thr Asn Tyr Gly Val His Trp Val Arg Gln Ser Pro Gly
260 265 270
Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Ser Gly Gly Asn Thr Asp
275 280 285
Tyr Asn Thr Pro Phe Thr Ser Arg Leu Ser Ile Asn Lys Asp Asn Ser
290 295 300
Lys Ser Gln Val Phe Phe Lys Met Asn Ser Leu Gln Ser Asn Asp Thr
305 310 315 320
Ala Ile Tyr Tyr Cys Ala Arg Ala Leu Thr Tyr Tyr Asp Tyr Glu Phe
325 330 335
Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala Ala Ser Thr
340 345 350
Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser
355 360 365
Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu
370 375 380
Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His
385 390 395 400
Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
405 410 415
Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys
420 425 430
Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu
435 440 445
Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu
450 455 460
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
465 470 475 480
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
485 490 495
Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu
500 505 510
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr
515 520 525
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
530 535 540
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser
545 550 555 560
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
565 570 575
Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val
580 585 590
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
595 600 605
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
610 615 620
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr
625 630 635 640
Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val
645 650 655
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
660 665 670
Ser Leu Gly Lys
675
<210> 132
<211> 2028
<212> DNA
<213> Homo
<400> 132
gaggtcaagc tggtggaaag cggcggcggc ctggtgcagc ctggaggatc cctgcggctg 60
agctgcgctg cctccggctt cgctttcagc tcctatgaca tgtcctgggt gaggcaggcc 120
cctggaaaga ggctggagtg ggtggctacc atctccggag gcggaaggta cacctactac 180
cccgacacag tgaagggaag gttcaccatc agccgggata acgccaaaaa cagccactat 240
ctccagatga actccctgag ggccgaagat acagccgtgt atttctgtgc ctccccctac 300
ggaggctatt ttgacgtgtg gggacagggc accctggtga ccgtctcctc cgcaagtacc 360
aagggaccta gtgttttccc tcttgcacct tgctccaggt caacatcaga gtccacagct 420
gctcttggat gtctcgttaa ggactacttc ccagagccag ttaccgtatc ctggaactcc 480
ggagctttga caagcggcgt tcatacattc ccagctgtgt tgcagagttc tgggttgtac 540
agtttgagct cagtggtgac cgtgccttca tcttctttgg gcactaagac ctacacctgc 600
aacgtggatc acaagccaag caacaccaag gtggataaga gggtgggtgg aggcggttca 660
ggcggaggtg gcagcggagg tggcgggagt caggtgcagc tgaagcagtc cggacctggc 720
ctggtgcagc cttcccagtc cctgtccatc acctgcaccg tgtccggctt ttccctgacc 780
aactacggcg tgcactgggt gaggcagtcc cctggcaagg gcctggaatg gctgggcgtg 840
atctggtccg gcggcaacac cgactacaac acccccttca cctcccggct gtccatcaac 900
aaggacaaca gcaagtccca ggtgttcttc aagatgaact ccctgcagag caacgacacc 960
gccatctact actgcgccag agccctgacc tattacgact acgagttcgc ctactggggc 1020
cagggcacac tggtgaccgt gtccgccgcc tccaccaagg gcccatcggt cttccccctg 1080
gcaccctgct ccaggagcac ctctgagtcc acagcggccc tgggctgcct ggtcaaggac 1140
tacttccccg aaccggtgac ggtgtcgtgg aactcaggcg ccctgaccag cggcgtgcac 1200
accttcccgg ctgtcctaca gtcctcagga ctctactccc tcagcagcgt ggtgaccgtg 1260
ccctccagca gcttgggcac caagacatat acctgtaatg tggatcacaa gccttccaat 1320
acaaaagtgg acaagagagt tgagtccaag tacggcccac catgtcctcc atgtccagcc 1380
cctgaatttt tgggcgggcc ttctgtcttt ctgtttcctc ctaaacctaa agataccctg 1440
atgatcagcc gcacacccga agtcacttgt gtggtcgtgg atgtgtctca ggaagatccc 1500
gaagtgcagt ttaactggta tgtcgatggc gtggaagtgc ataatgccaa aactaagccc 1560
cgcgaagaac agttcaacag cacttatcgg gtcgtgtctg tgctcacagt cctccatcag 1620
gattggctga atgggaaaga atataagtgc aaggtgagca ataagggcct ccccagcagc 1680
atcgagaaga ctattagcaa agccaaaggg cagccacggg aaccccaggt gtacactctg 1740
cccccctctc aggaggagat gactaaaaat caggtctctc tgacttgtct ggtgaaaggg 1800
ttttatccca gcgacattgc cgtggagtgg gagtctaatg gccagcccga gaataattat 1860
aagacaactc cccccgtcct ggactctgac ggcagctttt tcctgtattc tcggctgaca 1920
gtggacaaaa gtcgctggca ggagggcaat gtctttagtt gcagtgtcat gcatgaggcc 1980
ctgcacaatc actatacaca gaaaagcctg tctctgagtc tgggcaaa 2028
<210> 133
<211> 676
<212> PRT
<213> Homo
<400> 133
Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Ser Ser Tyr
20 25 30
Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Arg Leu Glu Trp Val
35 40 45
Ala Thr Ile Ser Gly Gly Gly Arg Tyr Thr Tyr Tyr Pro Asp Thr Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser His Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys
85 90 95
Ala Ser Pro Tyr Gly Gly Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu
100 105 110
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
115 120 125
Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys
130 135 140
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser
145 150 155 160
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
165 170 175
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser
180 185 190
Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn
195 200 205
Thr Lys Val Asp Lys Arg Val Gly Gly Gly Gly Ser Gly Gly Gly Gly
210 215 220
Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly
225 230 235 240
Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
245 250 255
Phe Thr Phe Thr Asp Tyr Thr Met Asp Trp Val Arg Gln Ala Pro Gly
260 265 270
Lys Gly Leu Glu Trp Val Ala Asp Val Asn Pro Asn Ser Gly Gly Ser
275 280 285
Ile Tyr Asn Gln Arg Phe Lys Gly Arg Phe Thr Leu Ser Val Asp Arg
290 295 300
Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
305 310 315 320
Thr Ala Val Tyr Tyr Cys Ala Arg Asn Leu Gly Pro Ser Phe Tyr Phe
325 330 335
Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr
340 345 350
Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser
355 360 365
Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu
370 375 380
Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His
385 390 395 400
Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
405 410 415
Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys
420 425 430
Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu
435 440 445
Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu
450 455 460
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
465 470 475 480
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
485 490 495
Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu
500 505 510
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr
515 520 525
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
530 535 540
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser
545 550 555 560
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
565 570 575
Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val
580 585 590
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
595 600 605
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
610 615 620
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr
625 630 635 640
Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val
645 650 655
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
660 665 670
Ser Leu Gly Lys
675
<210> 134
<211> 2028
<212> DNA
<213> Homo
<400> 134
gaggtcaagc tggtggaaag cggcggcggc ctggtgcagc ctggaggatc cctgcggctg 60
agctgcgctg cctccggctt cgctttcagc tcctatgaca tgtcctgggt gaggcaggcc 120
cctggaaaga ggctggagtg ggtggctacc atctccggag gcggaaggta cacctactac 180
cccgacacag tgaagggaag gttcaccatc agccgggata acgccaaaaa cagccactat 240
ctccagatga actccctgag ggccgaagat acagccgtgt atttctgtgc ctccccctac 300
ggaggctatt ttgacgtgtg gggacagggc accctggtga ccgtctcctc cgcaagtacc 360
aagggaccta gtgttttccc tcttgcacct tgctccaggt caacatcaga gtccacagct 420
gctcttggat gtctcgttaa ggactacttc ccagagccag ttaccgtatc ctggaactcc 480
ggagctttga caagcggcgt tcatacattc ccagctgtgt tgcagagttc tgggttgtac 540
agtttgagct cagtggtgac cgtgccttca tcttctttgg gcactaagac ctacacctgc 600
aacgtggatc acaagccaag caacaccaag gtggataaga gggtgggtgg aggcggttca 660
ggcggaggtg gcagcggagg tggcgggagt gaagtgcagc tggtggagtc tggaggcgga 720
ctggtgcagc ctggaggctc tctgagactg tcttgtgctg cttctggctt tacctttacc 780
gattatacca tggattgggt gagacaggcc cctggaaagg gactggagtg ggtggctgat 840
gtgaacccta actctggagg atctatctat aatcagaggt ttaagggcag attcaccctg 900
tctgtggata gatctaagaa tacactgtat ctgcagatga actctctgag agctgaagat 960
acagctgtgt attactgcgc tagaaatctg ggcccttcct tttactttga ttactggggc 1020
cagggaacac tggtgaccgt gtcttctgcc tccaccaagg gcccatcggt cttccccctg 1080
gcaccctgct ccaggagcac ctctgagtcc acagcggccc tgggctgcct ggtcaaggac 1140
tacttccccg aaccggtgac ggtgtcgtgg aactcaggcg ccctgaccag cggcgtgcac 1200
accttcccgg ctgtcctaca gtcctcagga ctctactccc tcagcagcgt ggtgaccgtg 1260
ccctccagca gcttgggcac caagacatat acctgtaatg tggatcacaa gccttccaat 1320
acaaaagtgg acaagagagt tgagtccaag tacggcccac catgtcctcc atgtccagcc 1380
cctgaatttt tgggcgggcc ttctgtcttt ctgtttcctc ctaaacctaa agataccctg 1440
atgatcagcc gcacacccga agtcacttgt gtggtcgtgg atgtgtctca ggaagatccc 1500
gaagtgcagt ttaactggta tgtcgatggc gtggaagtgc ataatgccaa aactaagccc 1560
cgcgaagaac agttcaacag cacttatcgg gtcgtgtctg tgctcacagt cctccatcag 1620
gattggctga atgggaaaga atataagtgc aaggtgagca ataagggcct ccccagcagc 1680
atcgagaaga ctattagcaa agccaaaggg cagccacggg aaccccaggt gtacactctg 1740
cccccctctc aggaggagat gactaaaaat caggtctctc tgacttgtct ggtgaaaggg 1800
ttttatccca gcgacattgc cgtggagtgg gagtctaatg gccagcccga gaataattat 1860
aagacaactc cccccgtcct ggactctgac ggcagctttt tcctgtattc tcggctgaca 1920
gtggacaaaa gtcgctggca ggagggcaat gtctttagtt gcagtgtcat gcatgaggcc 1980
ctgcacaatc actatacaca gaaaagcctg tctctgagtc tgggcaaa 2028
<210> 135
<211> 678
<212> PRT
<213> Homo
<400> 135
Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Ser Ser Tyr
20 25 30
Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Arg Leu Glu Trp Val
35 40 45
Ala Thr Ile Ser Gly Gly Gly Arg Tyr Thr Tyr Tyr Pro Asp Thr Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser His Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys
85 90 95
Ala Ser Pro Tyr Gly Gly Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu
100 105 110
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
115 120 125
Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys
130 135 140
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser
145 150 155 160
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
165 170 175
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser
180 185 190
Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn
195 200 205
Thr Lys Val Asp Lys Arg Val Gly Gly Gly Gly Ser Gly Gly Gly Gly
210 215 220
Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Val Glu Ser Gly Gly Gly
225 230 235 240
Val Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
245 250 255
Phe Thr Phe Ser Ser Tyr Thr Met His Trp Val Arg Gln Ala Pro Gly
260 265 270
Lys Gly Leu Glu Trp Val Thr Phe Ile Ser Tyr Asp Gly Asn Asn Lys
275 280 285
Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
290 295 300
Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
305 310 315 320
Thr Ala Ile Tyr Tyr Cys Ala Arg Thr Gly Trp Leu Gly Pro Phe Asp
325 330 335
Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys
340 345 350
Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly
355 360 365
Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
370 375 380
Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr
385 390 395 400
Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
405 410 415
Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn
420 425 430
Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro
435 440 445
Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
450 455 460
Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
465 470 475 480
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
485 490 495
Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
500 505 510
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
515 520 525
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
530 535 540
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
545 550 555 560
Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
565 570 575
Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn
580 585 590
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
595 600 605
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
610 615 620
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
625 630 635 640
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
645 650 655
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
660 665 670
Ser Leu Ser Pro Gly Lys
675
<210> 136
<211> 2034
<212> DNA
<213> Homo
<400> 136
gaggtcaagc tggtggaaag cggcggcggc ctggtgcagc ctggaggatc cctgcggctg 60
agctgcgctg cctccggctt cgctttcagc tcctatgaca tgtcctgggt gaggcaggcc 120
cctggaaaga ggctggagtg ggtggctacc atctccggag gcggaaggta cacctactac 180
cccgacacag tgaagggaag gttcaccatc agccgggata acgccaaaaa cagccactat 240
ctccagatga actccctgag ggccgaagat acagccgtgt atttctgtgc ctccccctac 300
ggaggctatt ttgacgtgtg gggacagggc accctggtga ccgtctcctc cgcaagtacc 360
aagggaccta gtgttttccc tcttgcacct tgctccaggt caacatcaga gtccacagct 420
gctcttggat gtctcgttaa ggactacttc ccagagccag ttaccgtatc ctggaactcc 480
ggagctttga caagcggcgt tcatacattc ccagctgtgt tgcagagttc tgggttgtac 540
agtttgagct cagtggtgac cgtgccttca tcttctttgg gcactaagac ctacacctgc 600
aacgtggatc acaagccaag caacaccaag gtggataaga gggtgggtgg aggcggttca 660
ggcggaggtg gcagcggagg tggcgggagt caagtgcagc tggtcgaaag cggcggagga 720
gtcgtccagc ccggcagatc tctgagactg agctgtgctg cctccggctt cacattcagc 780
tcctacacta tgcactgggt gaggcaagcc cccggcaagg gactggagtg ggtgactttc 840
atcagctacg acggcaacaa caagtactac gccgacagcg tgaagggaag gttcactatc 900
tctagggaca acagcaagaa cactctgtat ctgcagatga actctctgag ggccgaggat 960
actgccatct actactgcgc taggactggc tggctgggcc ctttcgatta ctggggccaa 1020
ggcactctgg tcactgtgag cagcgcgagc accaagggac cttccgtgtt tcccctcgcc 1080
cccagctcca aaagcaccag cggcggaaca gctgctctcg gctgtctcgt caaggattac 1140
ttccccgagc ccgtgaccgt gagctggaac agcggagccc tgacaagcgg cgtccacacc 1200
ttccctgctg tcctacagtc ctccggactg tacagcctga gcagcgtggt gacagtccct 1260
agcagctccc tgggcaccca gacatatatt tgcaacgtga atcacaagcc cagcaacacc 1320
aaggtcgata agaaggtgga gcctaagtcc tgcgacaaga cccacacatg tcccccctgt 1380
cccgctcctg aactgctggg aggcccttcc gtgttcctgt tcccccctaa gcccaaggac 1440
accctgatga tttccaggac acccgaggtg acctgtgtgg tggtggacgt cagccacgag 1500
gaccccgagg tgaaattcaa ctggtacgtc gatggcgtgg aggtgcacaa cgctaagacc 1560
aagcccaggg aggagcagta caattccacc tacagggtgg tgtccgtgct gaccgtcctc 1620
catcaggact ggctgaacgg caaagagtat aagtgcaagg tgagcaacaa ggccctccct 1680
gctcccatcg agaagaccat cagcaaagcc aagggccagc ccagggaacc tcaagtctat 1740
accctgcctc ccagcaggga ggagatgacc aagaaccaag tgagcctcac atgcctcgtc 1800
aagggcttct atccttccga tattgccgtc gagtgggagt ccaacggaca gcccgagaac 1860
aactacaaga caacaccccc cgtgctcgat tccgatggca gcttcttcct gtactccaag 1920
ctgaccgtgg acaagtccag atggcaacaa ggcaacgtct tcagttgcag cgtcatgcat 1980
gaggccctcc acaaccacta cacccagaaa agcctgtctc tgagtcctgg caaa 2034
<210> 137
<211> 678
<212> PRT
<213> Homo
<400> 137
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Thr Phe Ile Ser Tyr Asp Gly Asn Asn Lys Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Ile Tyr Tyr Cys
85 90 95
Ala Arg Thr Gly Trp Leu Gly Pro Phe Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
115 120 125
Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly
130 135 140
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
145 150 155 160
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
165 170 175
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
180 185 190
Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser
195 200 205
Asn Thr Lys Val Asp Lys Arg Val Gly Gly Gly Gly Ser Gly Gly Gly
210 215 220
Gly Ser Gly Gly Gly Gly Ser Glu Val Lys Leu Val Glu Ser Gly Gly
225 230 235 240
Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser
245 250 255
Gly Phe Ala Phe Ser Ser Tyr Asp Met Ser Trp Val Arg Gln Ala Pro
260 265 270
Gly Lys Arg Leu Glu Trp Val Ala Thr Ile Ser Gly Gly Gly Arg Tyr
275 280 285
Thr Tyr Tyr Pro Asp Thr Val Lys Gly Arg Phe Thr Ile Ser Arg Asp
290 295 300
Asn Ala Lys Asn Ser His Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu
305 310 315 320
Asp Thr Ala Val Tyr Phe Cys Ala Ser Pro Tyr Gly Gly Tyr Phe Asp
325 330 335
Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys
340 345 350
Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly
355 360 365
Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
370 375 380
Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr
385 390 395 400
Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
405 410 415
Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn
420 425 430
Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro
435 440 445
Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
450 455 460
Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
465 470 475 480
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
485 490 495
Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
500 505 510
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
515 520 525
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
530 535 540
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
545 550 555 560
Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
565 570 575
Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn
580 585 590
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
595 600 605
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
610 615 620
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
625 630 635 640
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
645 650 655
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
660 665 670
Ser Leu Ser Pro Gly Lys
675
<210> 138
<211> 2034
<212> DNA
<213> Homo
<400> 138
caagtgcagc tggtcgaaag cggcggagga gtcgtccagc ccggcagatc tctgagactg 60
agctgtgctg cctccggctt cacattcagc tcctacacta tgcactgggt gaggcaagcc 120
cccggcaagg gactggagtg ggtgactttc atcagctacg acggcaacaa caagtactac 180
gccgacagcg tgaagggaag gttcactatc tctagggaca acagcaagaa cactctgtat 240
ctgcagatga actctctgag ggccgaggat actgccatct actactgcgc taggactggc 300
tggctgggcc ctttcgatta ctggggccaa ggcactctgg tcactgtgag cagcgcaagt 360
accaagggac ctagtgtttt ccctcttgca ccttgctcca ggtcaacatc agagtccaca 420
gctgctcttg gatgtctcgt taaggactac ttcccagagc cagttaccgt atcctggaac 480
tccggagctt tgacaagcgg cgttcataca ttcccagctg tgttgcagag ttctgggttg 540
tacagtttga gctcagtggt gaccgtgcct tcatcttctt tgggcactaa gacctacacc 600
tgcaacgtgg atcacaagcc aagcaacacc aaggtggata agagggtggg tggaggcggt 660
tcaggcggag gtggcagcgg aggtggcggg agtgaggtca agctggtgga aagcggcggc 720
ggcctggtgc agcctggagg atccctgcgg ctgagctgcg ctgcctccgg cttcgctttc 780
agctcctatg acatgtcctg ggtgaggcag gcccctggaa agaggctgga gtgggtggct 840
accatctccg gaggcggaag gtacacctac taccccgaca cagtgaaggg aaggttcacc 900
atcagccggg ataacgccaa aaacagccac tatctccaga tgaactccct gagggccgaa 960
gatacagccg tgtatttctg tgcctccccc tacggaggct attttgacgt gtggggacag 1020
ggcaccctgg tgaccgtctc ctccgcgagc accaagggac cttccgtgtt tcccctcgcc 1080
cccagctcca aaagcaccag cggcggaaca gctgctctcg gctgtctcgt caaggattac 1140
ttccccgagc ccgtgaccgt gagctggaac agcggagccc tgacaagcgg cgtccacacc 1200
ttccctgctg tcctacagtc ctccggactg tacagcctga gcagcgtggt gacagtccct 1260
agcagctccc tgggcaccca gacatatatt tgcaacgtga atcacaagcc cagcaacacc 1320
aaggtcgata agaaggtgga gcctaagtcc tgcgacaaga cccacacatg tcccccctgt 1380
cccgctcctg aactgctggg aggcccttcc gtgttcctgt tcccccctaa gcccaaggac 1440
accctgatga tttccaggac acccgaggtg acctgtgtgg tggtggacgt cagccacgag 1500
gaccccgagg tgaaattcaa ctggtacgtc gatggcgtgg aggtgcacaa cgctaagacc 1560
aagcccaggg aggagcagta caattccacc tacagggtgg tgtccgtgct gaccgtcctc 1620
catcaggact ggctgaacgg caaagagtat aagtgcaagg tgagcaacaa ggccctccct 1680
gctcccatcg agaagaccat cagcaaagcc aagggccagc ccagggaacc tcaagtctat 1740
accctgcctc ccagcaggga ggagatgacc aagaaccaag tgagcctcac atgcctcgtc 1800
aagggcttct atccttccga tattgccgtc gagtgggagt ccaacggaca gcccgagaac 1860
aactacaaga caacaccccc cgtgctcgat tccgatggca gcttcttcct gtactccaag 1920
ctgaccgtgg acaagtccag atggcaacaa ggcaacgtct tcagttgcag cgtcatgcat 1980
gaggccctcc acaaccacta cacccagaaa agcctgtctc tgagtcctgg caaa 2034
<210> 139
<211> 675
<212> PRT
<213> Homo
<400> 139
Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Ser Ser Tyr
20 25 30
Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Arg Leu Glu Trp Val
35 40 45
Ala Thr Ile Ser Gly Gly Gly Arg Tyr Thr Tyr Tyr Pro Asp Thr Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser His Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys
85 90 95
Ala Ser Pro Tyr Gly Gly Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu
100 105 110
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
115 120 125
Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys
130 135 140
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser
145 150 155 160
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
165 170 175
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser
180 185 190
Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn
195 200 205
Thr Lys Val Asp Lys Arg Val Gly Gly Gly Gly Ser Gly Gly Gly Gly
210 215 220
Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Val Glu Ser Gly Gly Gly
225 230 235 240
Val Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
245 250 255
Phe Thr Phe Ser Ser Tyr Thr Met His Trp Val Arg Gln Ala Pro Gly
260 265 270
Lys Gly Leu Glu Trp Val Thr Phe Ile Ser Tyr Asp Gly Asn Asn Lys
275 280 285
Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
290 295 300
Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
305 310 315 320
Thr Ala Ile Tyr Tyr Cys Ala Arg Thr Gly Trp Leu Gly Pro Phe Asp
325 330 335
Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys
340 345 350
Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu
355 360 365
Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
370 375 380
Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr
385 390 395 400
Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
405 410 415
Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn
420 425 430
Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser
435 440 445
Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly
450 455 460
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
465 470 475 480
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln
485 490 495
Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val
500 505 510
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr
515 520 525
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
530 535 540
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile
545 550 555 560
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
565 570 575
Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser
580 585 590
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
595 600 605
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
610 615 620
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val
625 630 635 640
Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met
645 650 655
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
660 665 670
Leu Gly Lys
675
<210> 140
<211> 2025
<212> DNA
<213> Homo
<400> 140
gaggtcaagc tggtggaaag cggcggcggc ctggtgcagc ctggaggatc cctgcggctg 60
agctgcgctg cctccggctt cgctttcagc tcctatgaca tgtcctgggt gaggcaggcc 120
cctggaaaga ggctggagtg ggtggctacc atctccggag gcggaaggta cacctactac 180
cccgacacag tgaagggaag gttcaccatc agccgggata acgccaaaaa cagccactat 240
ctccagatga actccctgag ggccgaagat acagccgtgt atttctgtgc ctccccctac 300
ggaggctatt ttgacgtgtg gggacagggc accctggtga ccgtctcctc cgcaagtacc 360
aagggaccta gtgttttccc tcttgcacct tgctccaggt caacatcaga gtccacagct 420
gctcttggat gtctcgttaa ggactacttc ccagagccag ttaccgtatc ctggaactcc 480
ggagctttga caagcggcgt tcatacattc ccagctgtgt tgcagagttc tgggttgtac 540
agtttgagct cagtggtgac cgtgccttca tcttctttgg gcactaagac ctacacctgc 600
aacgtggatc acaagccaag caacaccaag gtggataaga gggtgggtgg aggcggttca 660
ggcggaggtg gcagcggagg tggcgggagt caagtgcagc tggtcgaaag cggcggagga 720
gtcgtccagc ccggcagatc tctgagactg agctgtgctg cctccggctt cacattcagc 780
tcctacacta tgcactgggt gaggcaagcc cccggcaagg gactggagtg ggtgactttc 840
atcagctacg acggcaacaa caagtactac gccgacagcg tgaagggaag gttcactatc 900
tctagggaca acagcaagaa cactctgtat ctgcagatga actctctgag ggccgaggat 960
actgccatct actactgcgc taggactggc tggctgggcc ctttcgatta ctggggccaa 1020
ggcactctgg tcactgtgag cagcgcctcc accaagggcc catcggtctt ccccctggca 1080
ccctgctcca ggagcacctc tgagtccaca gcggccctgg gctgcctggt caaggactac 1140
ttccccgaac cggtgacggt gtcgtggaac tcaggcgccc tgaccagcgg cgtgcacacc 1200
ttcccggctg tcctacagtc ctcaggactc tactccctca gcagcgtggt gaccgtgccc 1260
tccagcagct tgggcaccaa gacatatacc tgtaatgtgg atcacaagcc ttccaataca 1320
aaagtggaca agagagttga gtccaagtac ggcccaccat gtcctccatg tccagcccct 1380
gaatttttgg gcgggccttc tgtctttctg tttcctccta aacctaaaga taccctgatg 1440
atcagccgca cacccgaagt cacttgtgtg gtcgtggatg tgtctcagga agatcccgaa 1500
gtgcagttta actggtatgt cgatggcgtg gaagtgcata atgccaaaac taagccccgc 1560
gaagaacagt tcaacagcac ttatcgggtc gtgtctgtgc tcacagtcct ccatcaggat 1620
tggctgaatg ggaaagaata taagtgcaag gtgagcaata agggcctccc cagcagcatc 1680
gagaagacta ttagcaaagc caaagggcag ccacgggaac cccaggtgta cactctgccc 1740
ccctctcagg aggagatgac taaaaatcag gtctctctga cttgtctggt gaaagggttt 1800
tatcccagcg acattgccgt ggagtgggag tctaatggcc agcccgagaa taattataag 1860
acaactcccc ccgtcctgga ctctgacggc agctttttcc tgtattctcg gctgacagtg 1920
gacaaaagtc gctggcagga gggcaatgtc tttagttgca gtgtcatgca tgaggccctg 1980
cacaatcact atacacagaa aagcctgtct ctgagtctgg gcaaa 2025
<210> 141
<211> 675
<212> PRT
<213> Homo
<400> 141
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Thr Phe Ile Ser Tyr Asp Gly Asn Asn Lys Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Ile Tyr Tyr Cys
85 90 95
Ala Arg Thr Gly Trp Leu Gly Pro Phe Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
115 120 125
Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly
130 135 140
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
145 150 155 160
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
165 170 175
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
180 185 190
Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser
195 200 205
Asn Thr Lys Val Asp Lys Arg Val Gly Gly Gly Gly Ser Gly Gly Gly
210 215 220
Gly Ser Gly Gly Gly Gly Ser Glu Val Lys Leu Val Glu Ser Gly Gly
225 230 235 240
Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser
245 250 255
Gly Phe Ala Phe Ser Ser Tyr Asp Met Ser Trp Val Arg Gln Ala Pro
260 265 270
Gly Lys Arg Leu Glu Trp Val Ala Thr Ile Ser Gly Gly Gly Arg Tyr
275 280 285
Thr Tyr Tyr Pro Asp Thr Val Lys Gly Arg Phe Thr Ile Ser Arg Asp
290 295 300
Asn Ala Lys Asn Ser His Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu
305 310 315 320
Asp Thr Ala Val Tyr Phe Cys Ala Ser Pro Tyr Gly Gly Tyr Phe Asp
325 330 335
Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys
340 345 350
Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu
355 360 365
Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
370 375 380
Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr
385 390 395 400
Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
405 410 415
Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn
420 425 430
Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser
435 440 445
Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly
450 455 460
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
465 470 475 480
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln
485 490 495
Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val
500 505 510
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr
515 520 525
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
530 535 540
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile
545 550 555 560
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
565 570 575
Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser
580 585 590
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
595 600 605
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
610 615 620
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val
625 630 635 640
Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met
645 650 655
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
660 665 670
Leu Gly Lys
675
<210> 142
<211> 2025
<212> DNA
<213> Homo
<400> 142
caagtgcagc tggtcgaaag cggcggagga gtcgtccagc ccggcagatc tctgagactg 60
agctgtgctg cctccggctt cacattcagc tcctacacta tgcactgggt gaggcaagcc 120
cccggcaagg gactggagtg ggtgactttc atcagctacg acggcaacaa caagtactac 180
gccgacagcg tgaagggaag gttcactatc tctagggaca acagcaagaa cactctgtat 240
ctgcagatga actctctgag ggccgaggat actgccatct actactgcgc taggactggc 300
tggctgggcc ctttcgatta ctggggccaa ggcactctgg tcactgtgag cagcgcaagt 360
accaagggac ctagtgtttt ccctcttgca ccttgctcca ggtcaacatc agagtccaca 420
gctgctcttg gatgtctcgt taaggactac ttcccagagc cagttaccgt atcctggaac 480
tccggagctt tgacaagcgg cgttcataca ttcccagctg tgttgcagag ttctgggttg 540
tacagtttga gctcagtggt gaccgtgcct tcatcttctt tgggcactaa gacctacacc 600
tgcaacgtgg atcacaagcc aagcaacacc aaggtggata agagggtggg tggaggcggt 660
tcaggcggag gtggcagcgg aggtggcggg agtgaggtca agctggtgga aagcggcggc 720
ggcctggtgc agcctggagg atccctgcgg ctgagctgcg ctgcctccgg cttcgctttc 780
agctcctatg acatgtcctg ggtgaggcag gcccctggaa agaggctgga gtgggtggct 840
accatctccg gaggcggaag gtacacctac taccccgaca cagtgaaggg aaggttcacc 900
atcagccggg ataacgccaa aaacagccac tatctccaga tgaactccct gagggccgaa 960
gatacagccg tgtatttctg tgcctccccc tacggaggct attttgacgt gtggggacag 1020
ggcaccctgg tgaccgtctc ctccgcctcc accaagggcc catcggtctt ccccctggca 1080
ccctgctcca ggagcacctc tgagtccaca gcggccctgg gctgcctggt caaggactac 1140
ttccccgaac cggtgacggt gtcgtggaac tcaggcgccc tgaccagcgg cgtgcacacc 1200
ttcccggctg tcctacagtc ctcaggactc tactccctca gcagcgtggt gaccgtgccc 1260
tccagcagct tgggcaccaa gacatatacc tgtaatgtgg atcacaagcc ttccaataca 1320
aaagtggaca agagagttga gtccaagtac ggcccaccat gtcctccatg tccagcccct 1380
gaatttttgg gcgggccttc tgtctttctg tttcctccta aacctaaaga taccctgatg 1440
atcagccgca cacccgaagt cacttgtgtg gtcgtggatg tgtctcagga agatcccgaa 1500
gtgcagttta actggtatgt cgatggcgtg gaagtgcata atgccaaaac taagccccgc 1560
gaagaacagt tcaacagcac ttatcgggtc gtgtctgtgc tcacagtcct ccatcaggat 1620
tggctgaatg ggaaagaata taagtgcaag gtgagcaata agggcctccc cagcagcatc 1680
gagaagacta ttagcaaagc caaagggcag ccacgggaac cccaggtgta cactctgccc 1740
ccctctcagg aggagatgac taaaaatcag gtctctctga cttgtctggt gaaagggttt 1800
tatcccagcg acattgccgt ggagtgggag tctaatggcc agcccgagaa taattataag 1860
acaactcccc ccgtcctgga ctctgacggc agctttttcc tgtattctcg gctgacagtg 1920
gacaaaagtc gctggcagga gggcaatgtc tttagttgca gtgtcatgca tgaggccctg 1980
cacaatcact atacacagaa aagcctgtct ctgagtctgg gcaaa 2025
<210> 143
<211> 673
<212> PRT
<213> Homo
<400> 143
Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Ser Ser Tyr
20 25 30
Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Arg Leu Glu Trp Val
35 40 45
Ala Thr Ile Ser Gly Gly Gly Arg Tyr Thr Tyr Tyr Pro Asp Thr Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser His Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys
85 90 95
Ala Ser Pro Tyr Gly Gly Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu
100 105 110
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
115 120 125
Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys
130 135 140
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser
145 150 155 160
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
165 170 175
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser
180 185 190
Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn
195 200 205
Thr Lys Val Asp Lys Arg Val Gly Gly Gly Gly Ser Gly Gly Gly Gly
210 215 220
Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly
225 230 235 240
Leu Val Lys Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
245 250 255
Phe Thr Phe Ser Asp Asp Tyr Met Ala Trp Phe Arg Gln Ala Pro Gly
260 265 270
Lys Arg Leu Glu Trp Val Ala Ser Ile Ser His Gly Gly Asp Tyr Ile
275 280 285
Tyr Tyr Ala Asp Asn Leu Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
290 295 300
Ala Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
305 310 315 320
Thr Ala Val Tyr Phe Cys Ser Arg Asp Arg Arg Ser Ile Asp Tyr Trp
325 330 335
Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
340 345 350
Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr
355 360 365
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
370 375 380
Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
385 390 395 400
Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
405 410 415
Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp
420 425 430
His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr
435 440 445
Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro
450 455 460
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
465 470 475 480
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp
485 490 495
Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
500 505 510
Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val
515 520 525
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
530 535 540
Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys
545 550 555 560
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
565 570 575
Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr
580 585 590
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
595 600 605
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
610 615 620
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys
625 630 635 640
Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu
645 650 655
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly
660 665 670
Lys
<210> 144
<211> 2019
<212> DNA
<213> Homo
<400> 144
gaggtcaagc tggtggaaag cggcggcggc ctggtgcagc ctggaggatc cctgcggctg 60
agctgcgctg cctccggctt cgctttcagc tcctatgaca tgtcctgggt gaggcaggcc 120
cctggaaaga ggctggagtg ggtggctacc atctccggag gcggaaggta cacctactac 180
cccgacacag tgaagggaag gttcaccatc agccgggata acgccaaaaa cagccactat 240
ctccagatga actccctgag ggccgaagat acagccgtgt atttctgtgc ctccccctac 300
ggaggctatt ttgacgtgtg gggacagggc accctggtga ccgtctcctc cgcaagtacc 360
aagggaccta gtgttttccc tcttgcacct tgctccaggt caacatcaga gtccacagct 420
gctcttggat gtctcgttaa ggactacttc ccagagccag ttaccgtatc ctggaactcc 480
ggagctttga caagcggcgt tcatacattc ccagctgtgt tgcagagttc tgggttgtac 540
agtttgagct cagtggtgac cgtgccttca tcttctttgg gcactaagac ctacacctgc 600
aacgtggatc acaagccaag caacaccaag gtggataaga gggtgggtgg aggcggttca 660
ggcggaggtg gcagcggagg tggcgggagt gaagtgcagc tggtggagtc cggaggcgga 720
ctggtgaagc ctggaggctc cctgaggctg tcctgtgccg cttccggctt caccttctcc 780
gacgactaca tggcctggtt caggcaggcc cctggaaaga ggctggagtg ggtggcttcc 840
atctcccacg gcggcgacta catctactac gccgacaacc tgaagggcag gttcaccatc 900
tccagggaca acgccaagaa ctccctgtac ctgcagatga actccctgag ggccgaggac 960
accgccgtgt acttctgctc cagggacagg aggtccatcg actattgggg ccagggcacc 1020
ctggtgacag tgtcctccgc ctccaccaag ggcccatcgg tcttccccct ggcaccctgc 1080
tccaggagca cctctgagtc cacagcggcc ctgggctgcc tggtcaagga ctacttcccc 1140
gaaccggtga cggtgtcgtg gaactcaggc gccctgacca gcggcgtgca caccttcccg 1200
gctgtcctac agtcctcagg actctactcc ctcagcagcg tggtgaccgt gccctccagc 1260
agcttgggca ccaagacata tacctgtaat gtggatcaca agccttccaa tacaaaagtg 1320
gacaagagag ttgagtccaa gtacggccca ccatgtcctc catgtccagc ccctgaattt 1380
ttgggcgggc cttctgtctt tctgtttcct cctaaaccta aagataccct gatgatcagc 1440
cgcacacccg aagtcacttg tgtggtcgtg gatgtgtctc aggaagatcc cgaagtgcag 1500
tttaactggt atgtcgatgg cgtggaagtg cataatgcca aaactaagcc ccgcgaagaa 1560
cagttcaaca gcacttatcg ggtcgtgtct gtgctcacag tcctccatca ggattggctg 1620
aatgggaaag aatataagtg caaggtgagc aataagggcc tccccagcag catcgagaag 1680
actattagca aagccaaagg gcagccacgg gaaccccagg tgtacactct gcccccctct 1740
caggaggaga tgactaaaaa tcaggtctct ctgacttgtc tggtgaaagg gttttatccc 1800
agcgacattg ccgtggagtg ggagtctaat ggccagcccg agaataatta taagacaact 1860
ccccccgtcc tggactctga cggcagcttt ttcctgtatt ctcggctgac agtggacaaa 1920
agtcgctggc aggagggcaa tgtctttagt tgcagtgtca tgcatgaggc cctgcacaat 1980
cactatacac agaaaagcct gtctctgagt ctgggcaaa 2019
<210> 145
<211> 673
<212> PRT
<213> Homo
<400> 145
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Asp
20 25 30
Tyr Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Arg Leu Glu Trp Val
35 40 45
Ala Ser Ile Ser His Gly Gly Asp Tyr Ile Tyr Tyr Ala Asp Asn Leu
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys
85 90 95
Ser Arg Asp Arg Arg Ser Ile Asp Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala
115 120 125
Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu
130 135 140
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
145 150 155 160
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
165 170 175
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
180 185 190
Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr
195 200 205
Lys Val Asp Lys Arg Val Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
210 215 220
Gly Gly Gly Gly Ser Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu
225 230 235 240
Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
245 250 255
Ala Phe Ser Ser Tyr Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys
260 265 270
Arg Leu Glu Trp Val Ala Thr Ile Ser Gly Gly Gly Arg Tyr Thr Tyr
275 280 285
Tyr Pro Asp Thr Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala
290 295 300
Lys Asn Ser His Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
305 310 315 320
Ala Val Tyr Phe Cys Ala Ser Pro Tyr Gly Gly Tyr Phe Asp Val Trp
325 330 335
Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
340 345 350
Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr
355 360 365
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
370 375 380
Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
385 390 395 400
Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
405 410 415
Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp
420 425 430
His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr
435 440 445
Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro
450 455 460
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
465 470 475 480
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp
485 490 495
Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
500 505 510
Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val
515 520 525
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
530 535 540
Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys
545 550 555 560
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
565 570 575
Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr
580 585 590
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
595 600 605
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
610 615 620
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys
625 630 635 640
Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu
645 650 655
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly
660 665 670
Lys
<210> 146
<211> 2019
<212> DNA
<213> Homo
<400> 146
gaagtgcagc tggtggagtc cggaggcgga ctggtgaagc ctggaggctc cctgaggctg 60
tcctgtgccg cttccggctt caccttctcc gacgactaca tggcctggtt caggcaggcc 120
cctggaaaga ggctggagtg ggtggcttcc atctcccacg gcggcgacta catctactac 180
gccgacaacc tgaagggcag gttcaccatc tccagggaca acgccaagaa ctccctgtac 240
ctgcagatga actccctgag ggccgaggac accgccgtgt acttctgctc cagggacagg 300
aggtccatcg actattgggg ccagggcacc ctggtgacag tgtcctccgc aagtaccaag 360
ggacctagtg ttttccctct tgcaccttgc tccaggtcaa catcagagtc cacagctgct 420
cttggatgtc tcgttaagga ctacttccca gagccagtta ccgtatcctg gaactccgga 480
gctttgacaa gcggcgttca tacattccca gctgtgttgc agagttctgg gttgtacagt 540
ttgagctcag tggtgaccgt gccttcatct tctttgggca ctaagaccta cacctgcaac 600
gtggatcaca agccaagcaa caccaaggtg gataagaggg tgggtggagg cggttcaggc 660
ggaggtggca gcggaggtgg cgggagtgag gtcaagctgg tggaaagcgg cggcggcctg 720
gtgcagcctg gaggatccct gcggctgagc tgcgctgcct ccggcttcgc tttcagctcc 780
tatgacatgt cctgggtgag gcaggcccct ggaaagaggc tggagtgggt ggctaccatc 840
tccggaggcg gaaggtacac ctactacccc gacacagtga agggaaggtt caccatcagc 900
cgggataacg ccaaaaacag ccactatctc cagatgaact ccctgagggc cgaagataca 960
gccgtgtatt tctgtgcctc cccctacgga ggctattttg acgtgtgggg acagggcacc 1020
ctggtgaccg tctcctccgc ctccaccaag ggcccatcgg tcttccccct ggcaccctgc 1080
tccaggagca cctctgagtc cacagcggcc ctgggctgcc tggtcaagga ctacttcccc 1140
gaaccggtga cggtgtcgtg gaactcaggc gccctgacca gcggcgtgca caccttcccg 1200
gctgtcctac agtcctcagg actctactcc ctcagcagcg tggtgaccgt gccctccagc 1260
agcttgggca ccaagacata tacctgtaat gtggatcaca agccttccaa tacaaaagtg 1320
gacaagagag ttgagtccaa gtacggccca ccatgtcctc catgtccagc ccctgaattt 1380
ttgggcgggc cttctgtctt tctgtttcct cctaaaccta aagataccct gatgatcagc 1440
cgcacacccg aagtcacttg tgtggtcgtg gatgtgtctc aggaagatcc cgaagtgcag 1500
tttaactggt atgtcgatgg cgtggaagtg cataatgcca aaactaagcc ccgcgaagaa 1560
cagttcaaca gcacttatcg ggtcgtgtct gtgctcacag tcctccatca ggattggctg 1620
aatgggaaag aatataagtg caaggtgagc aataagggcc tccccagcag catcgagaag 1680
actattagca aagccaaagg gcagccacgg gaaccccagg tgtacactct gcccccctct 1740
caggaggaga tgactaaaaa tcaggtctct ctgacttgtc tggtgaaagg gttttatccc 1800
agcgacattg ccgtggagtg ggagtctaat ggccagcccg agaataatta taagacaact 1860
ccccccgtcc tggactctga cggcagcttt ttcctgtatt ctcggctgac agtggacaaa 1920
agtcgctggc aggagggcaa tgtctttagt tgcagtgtca tgcatgaggc cctgcacaat 1980
cactatacac agaaaagcct gtctctgagt ctgggcaaa 2019
<210> 147
<211> 330
<212> PRT
<213> Homo
<400> 147
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
1 5 10 15
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
65 70 75 80
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95
Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
100 105 110
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
115 120 125
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
130 135 140
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
145 150 155 160
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
165 170 175
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
180 185 190
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
195 200 205
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
210 215 220
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu
225 230 235 240
Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
245 250 255
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
260 265 270
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
275 280 285
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
290 295 300
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
305 310 315 320
Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
325 330
<210> 148
<211> 327
<212> PRT
<213> Homo
<400> 148
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg
1 5 10 15
Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr
65 70 75 80
Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95
Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro
100 105 110
Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
115 120 125
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
130 135 140
Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp
145 150 155 160
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe
165 170 175
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
180 185 190
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu
195 200 205
Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
210 215 220
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys
225 230 235 240
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
245 250 255
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
260 265 270
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
275 280 285
Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser
290 295 300
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
305 310 315 320
Leu Ser Leu Ser Leu Gly Lys
325
<210> 149
<211> 107
<212> PRT
<213> Homo
<400> 149
Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
1 5 10 15
Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
20 25 30
Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
35 40 45
Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
50 55 60
Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
65 70 75 80
Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
85 90 95
Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
100 105
<210> 150
<211> 15
<212> PRT
<213> Homo
<400> 150
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
1 5 10 15
<210> 151
<211> 11
<212> PRT
<213> Homo
<400> 151
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
1 5 10
<210> 152
<211> 10
<212> PRT
<213> Homo
<400> 152
Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
1 5 10
<210> 153
<211> 10
<212> PRT
<213> Homo
<400> 153
Phe Gly Gly Gly Thr Lys Val Glu Leu Lys
1 5 10

Claims (9)

1.一种PD-1/LAG-3四价双特异性抗体，其特征在于，包含两条多肽链和四条共同轻链，所述的多肽链的氨基酸序列如SEQ ID NO.143所示，所述的共同轻链的氨基酸序列如SEQID NO.129所示。
2.一种分离的核苷酸，其特征在于，编码权利要求1所述的PD-1/LAG-3四价双特异性抗体。
3.根据权利要求2所述的分离的核苷酸，其特征在于，编码所述的多肽链的核苷酸的序列如SEQ ID NO.144所示，编码所述的共同轻链的核苷酸的序列如SEQ ID NO.130所示。
4.一种表达载体，其特征在于，含有权利要求2或3所述的分离的核苷酸。
5.一种宿主细胞，其特征在于，含有权利要求2或3所述的分离的核苷酸或权利要求4所述的表达载体。
6.一种药物组合物，其特征在于，含有权利要求1所述的PD-1/LAG-3四价双特异性抗体。
7.权利要求1所述的PD-1/LAG-3四价双特异性抗体或权利要求6所述的药物组合物在制备治疗癌症、炎性疾病、自体免疫性疾病的药物中的用途。
8.根据权利要求7所述的用途，其特征在于，其中所述的PD-1/LAG-3四价双特异性抗体或含有其的药物组合物用于治疗癌症。
9.权利要求1所述的PD-1/LAG-3四价双特异性抗体的制备方法，其特征在于，所述方法包含以下步骤：
(a)构建如权利要求4所述的表达载体，并转化宿主细胞；
(b)在表达条件下，培养步骤(a)所述的宿主细胞，表达所述的PD-1/LAG-3四价双特异性抗体；
(c)分离并纯化(b)所述的PD-1/LAG-3四价双特异性抗体。
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