CN114989305A - 新型双特异性抗体及其用途 - Google Patents

新型双特异性抗体及其用途 Download PDF

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CN114989305A
CN114989305A CN202210655873.0A CN202210655873A CN114989305A CN 114989305 A CN114989305 A CN 114989305A CN 202210655873 A CN202210655873 A CN 202210655873A CN 114989305 A CN114989305 A CN 114989305A
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刘志刚
刘玉兰
郝小勃
郭晶晶
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Beijing Wisdomab Biotechnology Co ltd
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Abstract

本申请提供了双特异性人IgG1抗体，其包含两种具有相同铰链区的Fc片段，所述铰链区的氨基酸序列为SEQ ID NO:1或SEQ ID NO:28并替换了天然人IgG1抗体恒定区的第216‑230位序列，抗体恒定区氨基酸位置按照EU numbering确定。此外，本申请还提供了所述双特异性抗体的用途。

Description

新型双特异性抗体及其用途
技术领域
本申请大体涉及抗体药物领域，具体而言，本申请提供了新型双特异性抗体及其医学和生物学用途。
背景技术
双特异性抗体(bispecific antibody,BsAb)是一类人工抗体，其包含两个不同的抗原结合位点。双特异性抗体在生物医药领域，尤其是肿瘤免疫治疗方面应用广泛。
双特异性抗体从作用机制上可分为双重信号阻断型和介导细胞功能型。通常，介导细胞功能型双特异性抗体指介导T细胞杀伤的抗CD3双特异性抗体。1985年，利用T细胞杀死肿瘤细胞的概念就已被提出(Stearz at al.Nature 1985,314:628-631)。通常认为有效激活T细胞需要双重信号，第一信号来自抗原呈递细胞上MHC-抗原复合物与T细胞受体TCR-CD3的结合，第二信号为T细胞与抗原呈递细胞表达的共刺激分子相互作用后产生的非抗原特异性共刺激信号。由于多数癌细胞表面MHC的表达下调甚至缺失，从而使癌细胞逃逸免疫杀伤。靶向CD3的双特异性抗体则能够分别结合T细胞表面CD3分子和癌细胞表面抗原，从而拉近细胞毒性T细胞(cytotoxic T cell，Tc或CTL)与癌细胞的距离，引导T细胞直接杀伤癌细胞，而不再依赖于T细胞的双重激活信号。双特异性抗体可以是三功能抗体，即一条臂靶向肿瘤细胞上的抗原，另一条臂靶向T细胞的CD3分子，Fc段结合Fc受体。这种抗体使得T细胞、肿瘤细胞和结合抗体Fc结构域的效应细胞形成复合体(Muller and Kontermann,BioDrugs 2010；24:89-98)。
CD3分子有δ、ε、γ、ζ共4个亚基，分子量分别为18.9kDa、23.1kDa、20.5kDa、18.7kDa，各包括171个、207个、182个和164个氨基酸残基。四种亚基组成的6条多肽链与T细胞受体(T cell receptor,TCR)紧密结合，形成包含8条多肽链的TCR-CD3复合物，该复合物传导T细胞激活信号，稳定TCR结构。CD3的胞内部分含有免疫受体酪氨酸激活基序(immunoreceptor tyrosine-based activation motif,ITAM)，TCR识别和结合MHC分子呈递的抗原肽，使得T细胞内的酪氨酸蛋白激酶p561ck磷酸化CD3分子中ITAM的酪氨酸残基，之后募集含有SH2(Scr homology 2)结构域的酪氨酸蛋白激酶(如ZAP-70)。ITAM磷酸化和结合ZAP-70是T细胞激活早期信号传导过程的重要生化反应之一。因此，CD3分子有传导TCR识别抗原产生的激活信号的功能。
由于早期在免疫原性、结构稳定性以及抗体质量控制等方面的不足，限制了双特异性抗体的进一步发展。近年来，上游基因工程抗体和下游生产工艺技术的改进，克服了传统双特异性抗体的缺陷，从而推动了多类新型双特异性抗体进入临床开发阶段。为了解决将两个不同的半抗体进行正确装配的问题，设计开发了多种结构的双特异性抗体。
一类双特异性抗体不含Fc区。这类结构抗体的优点是分子量小，可以在原核细胞中表达，不需要考虑正确装配的问题；缺点是由于没有抗体Fc段，不能介导相应的生物学功能，而且半衰期短，因而临床应用受到一定限制。目前已有报道的此类双特异性抗体包括BiTE、DART、TrandAbs、bi-Nanobody等。德国Micromet公司开发的BiTE(bispecific T-cellengager)系列产品是将抗CD3 scfv与不同抗肿瘤细胞表面抗原scfv通过肽段进行连接获得的，可同时结合CD3+T细胞和肿瘤细胞。此类抗体克服了稳定性差、表达量低、溶解性低等生产问题，其中Blinatumomab已成功上市。
另一类双特异性抗体保留抗体Fc结构域。此类抗体形成IgG样结构，具有Fc介导的生物学功能。目前已有报道的此类双特异性抗体包括Triomabs、kih IgG、Cross-mab、orthoFab IgG、DVD IgG、IgG scFv、scFv2-Fc等。这些包含Fc段的双特异性抗体具有体内半衰期长、能够介导ADCC和CDC等优点。
双特异性抗体(例如靶向CD3和肿瘤表面抗原)将在肿瘤治疗中有良好的应用前景。
发明内容
第一方面，本申请提供双特异性人IgG1抗体，其包含两种具有相同铰链区的Fc片段，所述铰链区的氨基酸序列为SEQ ID NO:1或SEQ ID NO:28并替换了天然人IgG1抗体恒定区的第216-230位序列，抗体恒定区氨基酸位置按照EU numbering确定。
在一些实施方案中，两种Fc片段包含能够确保重链异聚化的突变。在一些实施方案中，一个Fc片段包含点突变S354C和T366W，另一个Fc片段包含点突变Y349C、T366S、L368A和Y407V。
在一些实施方案中，两种Fc片段的N端分别融合识别不同抗原表位的单链抗体(scfv)。
在一些实施方案中，一种Fc片段的N端融合单链抗体(scfv)，另一种Fc片段的N端融合Fab片段，单链抗体与Fab片段识别不同的抗原表位。
在一些实施方案中，两种Fc片段的N端分别融合识别不同抗原表位的Fab片段。在一些实施方案中，识别不同抗原表位的Fab片段包含相同的轻链。
在一些实施方案中，一种Fab片段包含天然的人IgG1抗体恒定区CH1片段，另一种Fab片段包含突变的人IgG1抗体恒定区CH1片段，突变的人IgG1抗体恒定区CH1片段包含点突变G137E、N203D和R214T中的任意1个、2个或3个，其中CH1片段的氨基酸序列为抗体恒定区第118-215位的氨基酸序列。在一些实施方案中，天然的人IgG1抗体恒定区CH1片段的氨基酸序列为SEQ ID NO:2。在一些实施方案中，突变的人IgG1抗体恒定区CH1片段的氨基酸序列为SEQ ID NO:30。
在一些实施方案中，两种Fc片段的CH2片段中均包含点突变L234F、L235E和P331S，其中CH2片段的氨基酸序列为抗体恒定区第231-340位的氨基酸序列。在一些实施方案中，两种Fc片段的CH2片段的氨基酸序列均为SEQ ID NO:31。
在一些实施方案中，双特异性人IgG1抗体识别人免疫细胞表面抗原和肿瘤细胞表面抗原。
在一些实施方案中，人免疫细胞表面抗原为CD3E。
在一些实施方案中，所述肿瘤表面抗原选自HER1、HER2、HER3、EpCAM、CEA、PSMA、CD19、CD20、CD22、CD38和BCMA。
第二方面，本申请提供了包含第一方面所述的双特异性人IgG1抗体的药物组合物。
在一些实施方案中，药物组合物还包含药学可接受的载体、赋形剂、稀释剂等。
第三方面，本申请提供了第一方面所述的双特异性人IgG1抗体，或者第二方面所述的药物组合物在制备用于预防或治疗肿瘤的药物中的用途。
第四方面，本申请提供了预防或治疗肿瘤的方法，包括向有需要的个体给予第一方面所述的双特异性人IgG1抗体或者第二方面所述的药物组合物。
在第三或第四方面的一些实施方案中，肿瘤表达双特异性人IgG1抗体所针对的肿瘤表面抗原。
在第三或第四方面的一些实施方案中，肿瘤选自乳腺癌、胃癌、结直肠癌、前列腺癌、胰腺癌、白血病、多发性骨髓瘤以及恶性淋巴瘤。
附图说明
图1a-图1d为本申请设计的四种不同结构双特异性抗体的结构示意图。
图2显示了采用ELISA方法检测各种双特异性抗体同时结合CD3E和HER2两种抗原的结果。
图3显示了利用流式细胞术分析各种双特异性抗体与MDA-MB-453人乳腺癌细胞表面HER2结合的结果。
图4显示了利用流式细胞术分析各种双特异性抗体与PBMC表面CD3结合的结果。
图5a-5g显示了不同双特异性抗体对肿瘤细胞的杀伤作用结果，其中图5a显示了双特异性抗体对HER2阳性细胞的杀伤结果；图5b显示了双特异性抗体对HER2阴性细胞的杀伤结果；图5c-图5e显示了双特异性抗体的不同铰链区结构对杀伤活性的影响；图5f和图5g为双特异性抗体的不同抗原结合部设计对杀伤活性的影响。
图6a-6c显示了不同双特异性抗体介导HER2阳性靶细胞(MDA-MB-453人乳腺癌细胞)对T细胞的激活，其中图6a和图6b显示了不同双特异性抗体激活T细胞表达早期活化标志分子CD69的结果；图6c显示了不同双特异性抗体诱导T细胞产生细胞因子IL-2的结果。
序列说明
SEQ ID NO:1为本申请的示例性双特异性抗体包含的铰链区的氨基酸序列，其为天然的人IgG2抗体的铰链区。
SEQ ID NO:2为天然的人IgG1抗体恒定区CH1片段的氨基酸序列。
SEQ ID NO:3为本申请的示例性抗人CD3E单克隆抗体的HCDR1的氨基酸序列。
SEQ ID NO:4为本申请的示例性抗人CD3E单克隆抗体的HCDR2的氨基酸序列。
SEQ ID NO:5为本申请的示例性抗人CD3E单克隆抗体的HCDR3的氨基酸序列。
SEQ ID NO:6为本申请的示例性抗人CD3E单克隆抗体和抗HER2单克隆抗体的LCDR1的氨基酸序列。
SEQ ID NO:7为本申请的示例性抗人CD3E单克隆抗体和抗HER2单克隆抗体的LCDR2的氨基酸序列。
SEQ ID NO:8为本申请的示例性抗人CD3E单克隆抗体和抗HER2单克隆抗体的LCDR3的氨基酸序列。
SEQ ID NO:9为本申请的示例性抗HER2单克隆抗体的HCDR1的氨基酸序列。
SEQ ID NO:10为本申请的示例性抗HER2单克隆抗体的HCDR2的氨基酸序列。
SEQ ID NO:11为本申请的示例性抗HER2单克隆抗体的HCDR3的氨基酸序列。
SEQ ID NO:12为本申请的示例性抗人CD3E单克隆抗体的重链可变区的氨基酸序列。
SEQ ID NO:13为本申请的示例性抗人CD3E单克隆抗体和抗HER2单克隆抗体的轻链可变区的氨基酸序列。
SEQ ID NO:14为本申请的示例性抗HER2单克隆抗体的重链可变区的氨基酸序列。
SEQ ID NO:15为本申请的示例性双特异性抗体的含抗HER2 scfv的臂(抗HER2scfv-Fc融合蛋白)的氨基酸序列。
SEQ ID NO:16为本申请的示例性双特异性抗体的含抗人CD3Escfv的臂(抗人CD3Escfv-Fc融合蛋白)的氨基酸序列。
SEQ ID NO:17为本申请的示例性双特异性抗体的含抗HER2 Fab的臂的重链部分的氨基酸序列。
SEQ ID NO:18为本申请的示例性双特异性抗体的含抗HER2 Fab或含抗人CD3EFab的臂的轻链部分的氨基酸序列。
SEQ ID NO:19为本申请的示例性双特异性抗体的含抗人CD3EFab的臂的重链部分的氨基酸序列。
SEQ ID NO:20为本申请的示例性双特异性抗体的含抗HER2 Fab的臂的重链部分的氨基酸序列。
SEQ ID NO:21为本申请的示例性双特异性抗体的含抗HER2 scfv的臂(抗HER2scfv-Fc融合蛋白)的氨基酸序列。
SEQ ID NO:22为本申请的示例性双特异性抗体的含抗人CD3Escfv的臂(抗人CD3Escfv-Fc融合蛋白)的氨基酸序列。
SEQ ID NO:23为本申请的示例性双特异性抗体的含抗HER2 Fab的臂的重链部分的氨基酸序列。
SEQ ID NO:24为本申请的示例性双特异性抗体的含抗人CD3EFab的臂的重链部分的氨基酸序列。
SEQ ID NO:25为本申请的示例性双特异性抗体的含抗HER2 Fab的臂的重链部分的氨基酸序列。
SEQ ID NO:26为本申请的示例性双特异性抗体的含抗HER2 Fab的臂的重链部分的氨基酸序列。
SEQ ID NO:27为本申请的示例性双特异性抗体的含抗人CD3EFab的臂的重链部分的氨基酸序列。
SEQ ID NO:28为本申请的示例性双特异性抗体包含的铰链区的氨基酸序列，其为天然的人IgG2抗体的铰链区的变体。
SEQ ID NO:29为本申请的示例性双特异性抗体包含的铰链区的氨基酸序列，其为天然的人IgG1抗体的铰链区。
SEQ ID NO:30为本申请的示例性双特异性抗体包含的Fab片段中的CH1片段的氨基酸序列。
SEQ ID NO:31为本申请的示例性双特异性抗体包含的Fc片段中的CH2片段的氨基酸序列。
SEQ ID NO:32为人CD3E胞外区(CD3E)的氨基酸序列。
SEQ ID NO:33为人CD3D胞外区(CD3D)的氨基酸序列。
SEQ ID NO:34为猴CD3E胞外区(mfCD3E)的氨基酸序列。
SEQ ID NO:35为猴CD3D胞外区(mfCD3D)的氨基酸序列。
SEQ ID NO:36为小鼠CD3E胞外区(mCD3E)的氨基酸序列。
SEQ ID NO:37为小鼠CD3D胞外区(mCD3D)的氨基酸序列。
SEQ ID NO:38为HER2胞外区D1D2D3部分(HER2)的氨基酸序列。
SEQ ID NO:39为His标签的氨基酸序列。
SEQ ID NO:40为鼠抗体IgG2a的Fc片段(mFc)的氨基酸序列。
SEQ ID NO:41为人IgG1抗体的Fc片段变体(FcK)的氨基酸序列。
SEQ ID NO:42为人IgG1抗体的Fc片段变体(FcH)的氨基酸序列。
SEQ ID NO:43为天然的人IgG1抗体重链恒定区的氨基酸序列。
SEQ ID NO:44为人IgG1抗体重链恒定区的变体(IgG1Hn)的氨基酸序列。
SEQ ID NO:45为人IgG1抗体重链恒定区的变体(IgG1Kn)的氨基酸序列。
SEQ ID NO:46为人IgG1抗体重链恒定区的变体(IgG1Hn-m3)的氨基酸序列。
SEQ ID NO:47为人IgG1抗体重链恒定区的变体(IgG1kn-m3)的氨基酸序列。
SEQ ID NO:48为人IgG1抗体重链恒定区的变体(IgG1H3n-m3)的氨基酸序列。
SEQ ID NO:49为人IgG1抗体重链恒定区的变体(IgG1kn1-m3)的氨基酸序列。
SEQ ID NO:50为人IgG1抗体重链恒定区的变体(IgG1H3n1-m3)的氨基酸序列。
SEQ ID NO:51为人IgG1抗体κ亚型轻链恒定区的氨基酸序列。
发明详述
定义
除非另外指明，本申请中所用的术语具有本领域技术人员通常所理解的含义。
在本文描述抗体结构时，涉及氨基酸位置编号的描述参照人IgG1抗体的EUnumbering定义，这是本领域技术人员公知且容易查询到的。此外，在本文结合EUnumbering位置描述突变时，是指相对于天然抗体序列产生的突变。
本文所用术语“Fc片段”、“Fc结构域”、“Fc部分”或类似的术语是指抗体重链恒定区的一部分，包括铰链区(hinge)、恒定区的CH2片段和CH3片段。参照人IgG1抗体的EUnumbering定义，Fc片段是抗体恒定区中第216-447位的氨基酸序列。
本文所用术语“Fab(fragment antigen binding)片段”、“Fab部分”或类似的术语是指完整的抗体用木瓜蛋白酶处理后产生的能够与抗原结合的抗体片段，包括完整的轻链(VL-CL)、重链可变区和CH1片段(VH-CH1)。
本文所用术语“单链抗体(scfv,single chain fragment variable)”是指一般利用基因工程技术构建的单链结构的抗体，包含重链可变区(VH)和轻链可变区(VL)的一条多肽链。在重链可变区和轻链可变区之间通常会设计一段柔性的连接肽(linker)以便重链可变区和轻链可变区可以折叠成为能够结合抗原的正确构象。
本文所用术语“双特异性抗体”是具有结合两种不同抗原能力的抗体，其可以由两个Fc片段以及分别与其融合的两个抗原结合部分组成，每个抗原结合部分可以为Fab片段的形式或单链抗体的形式。
本文所用术语“双特异性人IgG1抗体”是指基于人IgG1抗体的双特异性抗体，并且除了本文说明的改变结构之外，其具备人IgG1抗体的基本特征和功能。
在本文的某些实施方案中，将双特异性抗体描述为具有两个“臂”，例如，在图1a-图1d所示的四种结构中，以中间为界，可以将双特异性抗体分为两个臂。双特异性抗体的臂可以由Fc片段和抗原结合部分(Fab片段或单链抗体)组成。对于由Fc片段和Fab片段组成的臂，其结构类似于通常的抗体，含有完整的重链和轻链。
本领域技术人员公知，互补决定区(CDR，通常有CDR1、CDR2及CDR3)是可变区中对抗体的亲和力和特异性影响最大的区域。VH或VL的CDR序列有两种常见的定义方式，即kabat定义和Chothia定义。(参阅例如Kabat,“Sequences of Proteins of ImmunologicalInterest”，National Institutes of Health,Bethesda，Md.(1991)；A1-Lazikani etal.，J.Mol.Biol.273:927-948(1997)；以及Martin et al.，Proc.Natl.Acad.Sci.USA86:9268-9272(1989))。对于给定抗体的可变区序列，可以根据Kabat定义或者Chothia定义来确定VH和VL序列中CDR区序列。在本申请的实施方案中，利用Kabat定义CDR序列。
对于给定抗体的可变区序列，可以通过多种方式分析可变区序列中CDR区序列，例如可以利用在线软件Abysis确定(http://www.abysis.org/)。
如本文所用术语“特异性结合”，是指两个分子之间的非随机结合反应，例如抗体至抗原表位的结合。
第一方面，双特异性人IgG1抗体，其包含两种具有相同铰链区的Fc片段，所述铰链区的氨基酸序列为SEQ ID NO:1或SEQ ID NO:28并替换了天然人IgG1抗体恒定区的第216-230位序列，抗体恒定区氨基酸位置按照EU numbering确定。
SEQ ID NO:1为天然的人IgG2抗体的铰链区，SEQ ID NO:28为天然的人IgG2抗体的铰链区的变体。本申请的发明人通过研究意想不到地发现，在双特异性人IgG1抗体中引入人IgG2抗体的铰链区能够改进双特异性抗体的一些功能和性质。
当构建保留抗体Fc结构域的双特异性抗体时，可以从以下两个角度优化双特异性抗体的结构：一是重链异聚化，二是轻链和重链的正确装配。在一些实施方案中，两种Fc片段包含能够确保重链异聚化的突变。KIH技术(knob-in-hole，KIH)是解决重链异聚化的一种策略。通常，KIH技术是指通过改造CH3区的氨基酸序列，形成有利于异种半抗体相互配对的结构，可以在构成双特异性抗体的同时又尽可能地保持正常抗体的结构。在一些实施方案中，所利用的KIH技术包括，使一个Fc片段包含点突变S354C和T366W，另一个Fc片段包含点突变Y349C、T366S、L368A和Y407V。关于KIH技术的指导，例如可参见“An efficientroute to human bispecific IgG”,A.Margaret Merchant et al.,NatureBiotechnology,Volume16,1998，通过引用的方式将该文献全文并入本文。
在一些实施方案中，两种Fc片段的N端分别融合识别不同抗原表位的单链抗体(scfv)。
在一些实施方案中，一种Fc片段的N端融合单链抗体(scfv)，另一种Fc片段的N端融合Fab片段，所述单链抗体与所述Fab片段识别不同的抗原表位。
在一些实施方案中，两种Fc片段的N端分别融合识别不同抗原表位的Fab片段。由此形成的双特异性抗体的结构接近于天然抗体，也是优选的一种实施方案。
在一些实施方案中，识别不同抗原表位的Fab片段包含相同的轻链。该实施方案有利于轻链和重链的正确装配，也是优选的一种实施方案。
在一些实施方案中，一种Fab片段包含天然的人IgG1抗体恒定区CH1片段，另一种Fab片段包含突变的人IgG1抗体恒定区CH1片段，突变的人IgG1抗体恒定区CH1片段包含点突变G137E、N203D和R214T中的任意1个、2个或3个，其中CH1片段的氨基酸序列为抗体恒定区第118-215位的氨基酸序列。在一些实施方案中，天然的人IgG1抗体恒定区CH1片段的氨基酸序列为SEQ ID NO:2。在一些实施方案中，突变的人IgG1抗体恒定区CH1片段的氨基酸序列为SEQ ID NO:30。
不被任何理论所束缚，在一种Fab片段中引入上述突变能在不改变抗体恒定区结构/功能以及免疫原性的前提下，改变含有该突变的重链的电荷特性(等电点，pI)，进而有利于双特异性抗体的后期纯化。137、203和214这三个位点位于CH1结构域的亲水区(结构域的表面)，对其进行突变不会改变CH1的构象，所涉及的突变是将碱性氨基酸改为中性氨基酸(如R214T)，或者将中性氨基酸改为酸性氨基酸(如G137E、N203D)，这样的突变能导致含有突变的这条重链的等电点(pI)下降，低于未突变的另一条重链的等电点，这有利于后期纯化过程中利用等电点不同将目的双特异性抗体与例如KIH技术可能产生的同聚体进行有效的分离。而且，上述突变也天然存在于其它亚型的抗体(如IgG2、IgG4)，预期不会导致明显的免疫原性问题。
在一些实施方案中，两种Fc片段的CH2片段中均包含点突变L234F、L235E和P331S，其中CH2片段的氨基酸序列为抗体恒定区第231-340位的氨基酸序列。在一些实施方案中，两种Fc片段的CH2片段的氨基酸序列均为SEQ ID NO:31。
在CH2片段中引入上述突变能够降低抗体Fc段介导的抗体依赖性细胞毒作用(ADCC)，从而可能减少双特异性抗体在体内导致的副作用。关于上述突变的指导，例如可参见“The binding affinity of human IgG for its high affinity Fc receptor isdetermined by multiple amino acids in the CH2domain and is modulated by thehinge region”,Stephen M.Canfield et al.,J.Exp.Med.Volume 173,1991，通过引用的方式将该文献全文并入本文。
在一些实施方案中，双特异性人IgG1抗体识别人免疫细胞表面抗原和肿瘤细胞表面抗原。
在一些实施方案中，人免疫细胞表面抗原为CD3E。
抗CD3E抗体能结合T细胞表面TCR受体复合物中的CD3E亚基，能够提供T细胞激活的第一信号(类似于抗原呈递细胞上的MHC-肽复合物结合到TCR)，有利于T细胞的激活。而且包含针对CD3E的的抗原结合部的双特异性抗体，可以实现T细胞在肿瘤细胞周边的富集，提高T细胞对肿瘤细胞的杀伤效率。
在一些实施方案中，所述肿瘤表面抗原选自HER1、HER2、HER3、EpCAM、CEA、PSMA、CD19、CD20、CD22、CD38和BCMA。
HER2基因位于人17q21染色体，编码分子量185kD的跨膜蛋白，该蛋白有酪氨酸激酶活性，正常状态下以无活性形式存在，参与调节细胞正常分化，通常只在婴儿期表达，成人只在少数组织中低水平表达。HER2基因在正常细胞中是双拷贝基因，通过基因突变激活，其扩增导致转录上调，蛋白表达增加。HER2是人表皮生长因子受体(epidermal growthfactor receptor,EGFR)家族的第二个成员，属于I型受体酪氨酸激酶家族，在许多正常和异常表皮细胞的生长、分化和代谢过程中起重要的调节作用，多种肿瘤的发生、发展和疾病状态都与HER2相关。该家族共有4种受体，分别命名为HER1、HER2、HER3和HER4，这些受体相互作用形成同源或异源二聚体，尤以HER2异二聚体为主，在细胞的信号传导过程中发挥重要作用。HER2激活抑制肿瘤细胞凋亡，促进肿瘤细胞增殖；上调VEGF/VPF，加速肿瘤血管生成，促进肿瘤细胞转移，破坏组织抗侵袭功能(Artufel MV,Valero AC,Llado R R,etal.Clin Transl Oncol,2005,7.(11):504-511)。HER2蛋白过表达对诱导细胞分化、增殖和转化以及促进肿瘤转移、侵袭和粘附具有重要作用(Hynes N E,Stem D F.BiochemBiophys AcTa,1994,1198(2-3):165-184.)。由于在20％的乳腺癌病例中过表达，而且与不良预后相关，因此HER2在乳腺癌中的作用格外受到关注(Reese et al.,Stem Cells 1997；15:1-8；Andrechek et al.,Proc Natl Acad Sci USA2000；97:3444-3449.Slamon etal.,Science 1987；235:177-182)。
CEA(癌胚抗原,carcino-embryonic antigen)是大肠癌组织产生的一种糖蛋白，作为抗原可引起患者的免疫反应。其广泛存在于内胚叶起源的消化系统癌，也存在于正常胚胎的消化管组织中，在正常人血清中也可有微量存在。癌胚抗原是一个广谱性肿瘤标志物，它能向人们反映出多种肿瘤的存在，对大肠癌、乳腺癌和肺癌的疗效判断、病情发展、监测和预后估计是一个较好的肿瘤标志物。
PSMA是110kDa的II型穿膜蛋白，基因定位于染色体断臂11q上，表达于正常前列腺上皮和前列腺肿瘤细胞中，其中在肿瘤细胞中的表达量上调，有研究认为PSMA参与了前列腺癌发生过程中细胞迁移的调控。在前列腺癌中PSMA的表达和肿瘤的级别及是否存在激素抵抗呈正相关，PSMA的强表达意味着更高的复发率。现认为PSMA是前列腺癌参考依据之一。
CD19是表达于B淋巴细胞及滤泡树突状细胞的表面蛋白，属于免疫球蛋白(Ig)超家族成员，位于16号染色体短臂上(16p11.2)，编码556个氨基酸的Ⅰ型跨膜糖蛋白，分子量95KD。CD19只在正常和恶性B细胞中表达，几乎不在其他组织中表达；其次，CD19在B细胞恶性转化过程中不丢失，难治/复发性病例仍然有效；再者，CD19在造血干细胞和pro-B细胞中不表达，治疗停止后，B细胞可以得到有效地补充。因而近年来靶向CD19的各种白血病治疗策略取得较好的临床效果，包括CD3+CD19双特异性抗体和靶向CD19的CAR-T等。
CD20抗原是一种B细胞分化抗原，仅表达于前B细胞和成熟B细胞表面，它在95％以上的B细胞性淋巴瘤中表达，而在造血干细胞、血浆细胞和其他正常组织中不表达。靶向CD20的多个单抗(如Rituxan)已经在临床上成功的用于多种淋巴瘤的治疗。
CD38是一个定位于膜上的糖蛋白，催化环腺苷二磷酸核糖(cADPR，cyclic ADP-ribose)的合成和降解。CD38是一个45kDa的单链跨膜糖蛋白，整体结构分为N末端短的胞质尾，单次跨膜域和C端长的胞外区。成年人中，CD38在大多数自然杀伤细胞、T细胞、B细胞，单核细胞/巨噬细胞。血小板和红细胞上也有一定程度的表达。靶向CD38的单抗(daratumumab)已经批准用于多发性骨髓瘤的治疗。
BCMA(B细胞成熟抗原，B-cell maturation antigen)是由185个氨基酸残基组成的III型跨膜蛋白。它属于TNF受体家族成员，与其配体B细胞激活因子BAFF或增殖诱导配体APRIL结合可刺激B细胞增生。BCMA正常表达于成熟的B细胞和浆细胞，在多发性骨髓瘤(MM)中也有广泛的表达，是多发性骨髓瘤的一个非常理想的免疫治疗靶点。
第二方面，本申请提供了包含第一方面所述的双特异性人IgG1抗体的药物组合物。
在一些实施方案中，药物组合物还包含药学可接受的载体、赋形剂、稀释剂等。
在一些实施方案中，药物组合物用于治疗肿瘤，例如表达双特异性人IgG1抗体所针对的肿瘤表面抗原的肿瘤。
在一些实施方案中，药物组合物还可包含润滑剂，如滑石粉、硬脂酸镁和矿物油；润湿剂；乳化剂；悬浮剂；防腐剂，如苯甲酸、山梨酸和丙酸钙；增甜剂和/或调味剂等。
在一些实施方案中，可将本申请中的药物组合物配制为片剂、丸剂、粉剂、锭剂、酏剂、悬液、乳剂、溶液、糖浆、栓剂或胶囊等形式。
在一些实施方案中，可以利用任何生理上可接受的给药方式递送本申请的药物组合物，这些给药方式包括但不限于：口服给药、肠胃外给药、经鼻给药、直肠给药、腹膜内给药、血管内注射、皮下给药、经皮给药、吸入给药等。
在一些实施方案中，可以通过混合具有所需纯度的试剂与视情况的药学上可接受的载体、赋形剂等，以冻干制剂或水溶液的形式配制用于治疗用途的药物组合物用于存储。
第三方面，本申请提供了第一方面所述的双特异性人IgG1抗体，或者第二方面所述的药物组合物在制备用于预防或治疗肿瘤的药物中的用途。
第四方面，本申请提供了预防或治疗肿瘤的方法，包括向有需要的个体给予第一方面所述的双特异性人IgG1抗体或者第二方面所述的药物组合物。
在第三或第四方面的一些实施方案中，肿瘤表达双特异性人IgG1抗体所针对的肿瘤表面抗原。
在第三或第四方面的一些实施方案中，肿瘤选自乳腺癌、胃癌、结直肠癌、前列腺癌、胰腺癌、白血病、多发性骨髓瘤以及恶性淋巴瘤。
应当理解，以上详细描述仅为了使本领域技术人员更清楚地了解本申请的内容，而并非意图在任何方面加以限制。本领域技术人员能够对所述实施方案进行各种改动和变化。
以下实施例仅用于说明而非限制本申请范围的目的。
实施例
实施例1单克隆抗体的制备和确认
作为展示本申请的双特异性人IgG1抗体的一个实例，制备了靶向于人CD3E和HER2的双特异性人IgG1抗体。
作为制备该双特异性人IgG1抗体的基础，本申请的发明人首先制备了针对人CD3E和HER2的两种单克隆抗体，该过程需要利用多种不同的重组蛋白，包括重组人CD3E的胞外区(CD3E，SEQ ID NO:32)、人CD3D-胞外区(CD3D，SEQ ID NO:33)、猴CD3E胞外区(mfCD3E，SEQ ID NO:34)、猴CD3D胞外区(mfCD3D，SEQ ID NO:35)、小鼠CD3E胞外区(mCD3E，SEQ IDNO:36)、小鼠CD3D胞外区(mCD3D，SEQ ID NO:37)、重组HER2胞外区D1D2D3部分(HER2，SEQID NO:38)。在自然环境中，CD3E与CD3D形成异源二聚体，因此为了制备有天然构象的CD3E抗原，我们将CD3E和CD3D同时表达，并利用FcK+FcH异源二聚体方法将CD3D和CD3E形成异源二聚体，用作本研究中的抗原。这些重组蛋白都有大量的翻译后修饰(如：糖基化或二硫键等)，因而利用哺乳动物细胞表达系统将更有利于保持重组蛋白的结构和功能。此外，为了方便纯化，非抗体类的重组蛋白在C端添加了His标签(SEQ ID NO:39)或者鼠抗体IgG2a的Fc段(mFc，SEQ ID NO:40)。重组抗体制备时，抗体重链恒定区可以来自人IgG1抗体(SEQ IDNO:43)或者为IgG1恒定区的各种突变体，如：IgG1Hn(SEQ ID NO:44)、IgG1Kn(SEQ ID NO:45)、IgG1Hn-m3(SEQ ID NO:46)、IgG1kn-m3(SEQ ID NO:47)、IgG1H3n-m3(SEQ ID NO:48)、IgG1kn1-m3(SEQ ID NO:49)、IgG1H3n1-m3(SEQ ID NO:50)，轻链恒定区是kappa亚型(SEQID NO:51)。
根据Uniprot数据库的各种目的重组蛋白的氨基酸序列，设计并合成上述各种重组蛋白的基因(包含His-tag、mFc或者Fc编码基因)。利用常规的分子生物学技术将合成的各种重组蛋白基因克隆至合适的真核表达载体(如invitrogen公司的pcDNA3.1等)，然后利用脂质体(如invitrogen公司的293fectin等)或其它转染试剂(如PEI等)将制备的重组蛋白表达质粒转染入HEK293细胞(如invitrogen公司的HEK293F)，在无血清悬浮培养条件下培养3-5天。然后通过离心等方式收获培养上清。
His标签融合表达的重组蛋白利用金属螯合亲和层析柱(如GE公司的HisTrap FF等)对培养上清中的重组蛋白进行一步纯化。而mFc融合表达的重组蛋白和重组抗体用ProteinA/G亲和层析柱(如GE公司的Mabselect SURE等)进行一步纯化。然后利用脱盐柱(如GE公司的Hitrap desaulting等)将重组蛋白保存缓冲液置换为PBS(pH7.0)或者其它合适的缓冲液。必要时，可以对抗体样品进行过滤除菌，然后分装保存于-20℃。
利用重组抗体技术得到靶向于肿瘤抗原HER2的单克隆抗体(指定为C6G9+L1A7)和靶向于人CD3E的单克隆抗体(指定为H10B7+L1A7)。C6G9+L1A7的重链可变区序列为SEQ IDNO:14，轻链可变区序列为SEQ ID NO:13。H10B7+L1A7的重链可变区序列为SEQ ID NO:12，轻链可变区序列为SEQ ID NO:13。C6G9+L1A7和H10B7+L1A7具有相同的轻链。此外，C6G9+L1A7和H10B7+L1A7各自的抗体功能和性质经过实验确认。
实施例2不同结构双特异性抗体的设计和制备
基于实施例1制备和验证的两种单克隆抗体，设计了一系列针对人CD3E和HER2的双特异性抗体。
由于双特异性抗体的每个臂可以包含Fab片段或scfv，因此根据不同的组合方式设计了四种结构的双特异性抗体(参见图1a-图1d)。
此外，为了促进形成异源二聚体，使用了基于KIH(Knob-Into-Hole)技术的人IgG1抗体的Fc片段突变体(FcK，SEQ ID NO:41或者FcH，SEQ ID NO:42)，即，将针对人CD3E(源自H10B7+L1A7)的抗原结合区融合在含有Knob突变的Fc(FcK)的N端，将针对HER2(源自C6G9+L1A7)的抗原结合区融合在含有Hole突变的Fc(FcH)的N端。
按照与实施例1类似的重组抗体技术，制备了表1中所示的双特异性抗体(BsAb)和作为对照的单克隆抗体(MAb)。
表1.不同结构的人CD3E+HER2双特异性抗体和对照单克隆抗体的结构
Figure BDA0003687652280000161
Figure BDA0003687652280000171
Figure BDA0003687652280000181
实施例3各种双特异性抗体的亲和力分析
用GE的Biacore X100 plus进行抗体亲和力测定。胺基偶联试剂(Amine couplingkit)、人抗体捕获试剂(human antibody capture kit)、His捕获试剂(his capture kit)以及CM5芯片和pH7.4的10×HBS-EP等相关试剂和耗材均购自GE Healthcare公司。
采用捕获法测定不同双特异性抗体的亲和力。在测定实施例2中制备的单克隆抗体或者双特异性抗体对CD3E的亲和力时，将抗His的抗体偶联至CM5芯片表面，将带有His标签的CD3E抗原(CD3E-FcK-His/CD3D-FcH)捕获到CM5芯片表面作为固定相，采用单循环的方法将各种抗CD3E抗体蛋白设置一系列的浓度梯度流经固定相表面，测定各抗体蛋白的亲和力。
在测定实施例2中制备的单克隆抗体或者双特异性抗体对HER2抗原的亲和力时，将抗Fc的抗体偶联至CM5芯片表面，稀释各种抗HER2抗体蛋白至合适浓度，分别捕获到CM5芯片表面作为固定相，采用单循环的方法将重组的HER2-His设置一系列的浓度梯度流经固定相表面，测定不同抗体蛋白的亲和力。
本实施例中使用的人CD3E和HER2抗原的制备见实施例1的描述。
如表2和表3的结果所示，各种双特异性抗体均能够分别有效结合CD3E和HER2两种抗原。
表2.对照单克隆抗体和双特异性抗体结合CD3E的亲和力常数
Figure BDA0003687652280000182
Figure BDA0003687652280000191
表3.对照单克隆抗体和双特异性抗体结合HER2的亲和力常数
抗体 Ka Kd KD
MAb2 1.194E+5 6.676E+-4 5.313E-9
BsAb1 9.659E+4 6.846E+-4 7.088E-9
BsAb2 7.427E+4 9.622E+-4 1.296E-8
BsAb3 1.32E+5 9.474E+-4 7.175E-9
BsAb4 8.231E+4 7.118E+-4 8.648E-9
BsAb5 6.666E+-4 7.172E+-4 1.076E-8
BsAb6 8.252E+4 6.984E+-4 8.463E-9
BsAb7 9.533E+4 6.473E+-4 6.79E-9
BsAb8 5.236E+4 5.071E-4 9.686E-9
实施例4各种双特异性抗体同时识别CD3E和HER2两种抗原的能力鉴定
利用常规ELISA方法检测实施例2中制备的双特异性抗体对CD3E和HER2两种抗原的同时结合，ELISA流程如下：首先用CD3E-FcK/CD3D-FcH抗原包被ELISA板，4℃冰箱过夜；然后用含有1％BSA的封闭液37℃封闭1小时；洗涤后加入双特异性抗体37℃孵育1小时；洗涤后加HER2-His抗原，37℃孵育1小时；洗涤后加入HRP-抗His IgG，37℃孵育1小时；洗涤后加入HRP底物液进行显色分析。结果如图2所示：构建的各种双特异性抗体均能够同时识别CD3E和HER2两种抗原。
实施例5各种双特异性抗体识别细胞表面的CD3E和HER2的能力鉴定
通过流式细胞术分析实施例2中制备的双特异性抗体与MDA-MB-453人乳腺癌细胞上表达的人HER2或人PBMC上表达的人CD3的结合能力。MDA-MB-453细胞购自中国医学科学院基础医学研究所基础医学细胞中心，并培养于RPMI1640培养基中。PBMC利用Ficoll密度梯度离心法从志愿者全血中分离，并培养于RPMI1640培养基中。
采集正常志愿者的血液(各50mL)，其中所采集的血液由发明人及其同事作为志愿者提供，所有志愿者均已签署知情同意书。志愿者的纳入标准为：
1.年龄大于18周岁；
2.无HIV、HBV感染；
3.血常规检测正常；
4.非孕妇或哺乳期妇女。
收集培养的细胞，并使用台盼蓝染色评估细胞活力。然后将活细胞在含有0.1％BSA的PBS缓冲液中调整至3×106个细胞/ml，向圆底96孔板中每孔加入90μl的细胞悬浮液。向含细胞的孔中加入10μl的双特异性抗体或相应的两种单克隆抗体(MAb1和MAb2)以获17.8nM(MDA-MB-453结合研究)或者53.3nM(PBMC结合研究)的终浓度。于4℃下孵育30分钟后，将细胞离心(5分钟，350g)，利用150μl/孔含BSA的PBS染色缓冲液洗涤，重悬浮并与100μl/孔荧光染料缀合的羊抗人IgG抗体于4℃下孵育额外的30分钟。然后通过加入150μl/孔PBS染色缓冲液并在350g下离心5分钟来洗涤细胞。利用150μl/孔PBS染色缓冲液进行第二个洗涤步骤。将样品重悬浮在100μl/孔PBS染色缓冲液中，使用流式细胞仪(BDBiosciences)获得并分析所述样品。结果如图3和图4中所示：实施例2中制备的双特异性抗体均能够分别识别MDA-MB-453人乳腺癌细胞上表达的人HER2和PBMC上表达的人CD3。
实施例6各种双特异性抗体介导T细胞对HER2阳性肿瘤细胞的杀伤
收集MDA-MB-453人乳腺癌细胞(HER2阳性细胞)或MDA-MB-468人乳腺癌细胞(HER2阴性细胞，购自中国医学科学院基础医学研究所)，计数，并使用台盼蓝评估细胞活力。调整细胞密度为2×105/ml，每孔100μl接种于96孔细胞培养板，然后加入起始浓度为3.3nM，10倍梯度稀释的实施例2中制备的CD3+HER2双特异性抗体。为了方便比较，将不同的CD3+HER2双特异性抗体或者对照单克隆抗体(MAb1和MAb2)调整至相同的摩尔浓度。最后向孔中加入人PBMC细胞(效应物)以获得5:1的最终E:T比例。同时设置单独靶细胞(MDA-MB-453人乳腺癌细胞或MDA-MB-468人乳腺癌细胞)对照、单独PBMC细胞(效应细胞)对照、单独培养基做空白对照。孵育20小时后，取上清，参照CytoTox
Figure BDA0003687652280000213
非放射性细胞毒性检测试剂盒说明书(CytoTox
Figure BDA0003687652280000211
Non-Radioactive Cytotoxicity Assay，promega，G1780)检测和分析双特异性抗体介导的T细胞对靶细胞MDA-MB-453人乳腺癌细胞的杀伤率。检测结果如图5a-图5g和表4所示：本申请构建的各种双特异性抗体(BsAb)均能有效介导T细胞对HER2阳性靶细胞的杀伤，不能介导T细胞对HER2阴性靶细胞的杀伤，双特异性抗体中抗原结合部的形式或铰链区的序列的不同选择带来的双特异性抗体的活性存在一定差异。当采用SEQ ID NO:1或者SEQ ID NO:28所示的铰链区时，双特异性抗体能够介导T细胞对肿瘤细胞实现更强的杀伤能力。
表4.不同双特异性抗体介导T细胞对靶细胞MDA-MB-453杀伤的EC50
Figure BDA0003687652280000212
实施例7各种双特异性抗体介导的T细胞激活
收集表达HER2的MDA-MB-453人乳腺癌细胞，计数并使用台盼蓝评估细胞活力。调整MDA-MB-453人乳腺癌细胞的细胞密度为2×105/ml，每孔100μl接种于96孔细胞培养板，然后加入系列稀释的实施例2制备的各种CD3+HER2双特异性抗体。为了方便比较，将不同的CD3+HER2双特异性抗体或者对照单克隆抗体(MAb1和MAb2)调整至相同的摩尔浓度。最后向孔中加入人PBMC(效应物)以获得5:1的最终E:T比例。同时设置单独靶细胞(MDA-MB-453人乳腺癌细胞)对照。孵育20小时后，通过离心(5分钟，350g)沉淀细胞并利用150μl/孔PBS缓冲液洗涤两次。加入抗人CD3和人CD69的抗体(eBioscience，11-0037-42，12-0699-42)，于4℃下避光孵育30分钟。然后利用150μl/孔PBS缓冲液洗涤细胞两次，重悬浮于100μl/孔PBS缓冲液中，并使用流式细胞仪(BD Biosciences C6)对细胞样品进行分析并比较不同样品处理后CD3阳性细胞群中激活标志物CD69的表达差异。或者在孵育20小时后，取培养上请，利用ELISA分析上清中IL2水平(人IL-2ELISA试剂，DAKEWE，DKW12-1020-096)。结果如图6a-图6c所示：本申请构建的多种双特异性抗体均能够实现HER2阳性靶细胞对T细胞的特异性激活。
虽然，上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述，但在本发明基础上，可以对之作一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。
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<110> 北京智仁美博生物科技有限公司
<120> 新型双特异性抗体及其用途
<130> D22C11725CN
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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 Asn 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 Ser Ser Ile Tyr Asn Glu 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> 15
<211> 472
<212> PRT
<213> 人工序列(Artificial Sequence)
<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 Arg Ala Ser Gln Asn Ile Asn Asn Trp
20 25 30
Leu Thr Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ala Ala Ser Ser Arg Gln 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 Ala Lys Leu Pro Val
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser
100 105 110
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu
115 120 125
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys
130 135 140
Ala Ala Ser Gly Phe Thr Phe Asn Asp Tyr Thr Met Asp Trp Val Arg
145 150 155 160
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Asp Val Asn Pro Asn
165 170 175
Ser Gly Ser Ser Ile Tyr Asn Glu Arg Phe Lys Gly Arg Phe Thr Leu
180 185 190
Ser Val Asp Arg Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu
195 200 205
Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asn Leu Gly Pro
210 215 220
Ser Phe Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
225 230 235 240
Ser Ala Ser Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala
245 250 255
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
260 265 270
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
275 280 285
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
290 295 300
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
305 310 315 320
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
325 330 335
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
340 345 350
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
355 360 365
Arg Glu Pro Gln Val Cys Thr Leu Pro Pro Ser Arg Glu Glu Met Thr
370 375 380
Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser
385 390 395 400
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
405 410 415
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val
420 425 430
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
435 440 445
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
450 455 460
Ser Leu Ser Leu Ser Pro Gly Lys
465 470
<210> 16
<211> 474
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 16
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 Lys Phe Ser Gly Tyr
20 25 30
Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Val Ile Trp Phe Asp Gly Ser Arg Lys Tyr Tyr Val Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gln Met Gly Tyr Trp His Phe Gly Leu Trp Gly Arg Gly Thr
100 105 110
Leu Val Thr Val Ser Ser Gly Glu Gly Thr Ser Thr Gly Ser Gly Gly
115 120 125
Ser Gly Gly Ser Gly Gly Ala Asp Asp Ile Gln Met Thr Gln Ser Pro
130 135 140
Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg
145 150 155 160
Ala Ser Gln Asn Ile Asn Asn Trp Leu Thr Trp Tyr Gln Gln Lys Pro
165 170 175
Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Ser Arg Gln Thr
180 185 190
Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
195 200 205
Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys
210 215 220
Gln Gln Tyr Ala Lys Leu Pro Val Thr Phe Gly Gln Gly Thr Lys Val
225 230 235 240
Glu Ile Lys Ala Ser Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys
245 250 255
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
260 265 270
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
275 280 285
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
290 295 300
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
305 310 315 320
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
325 330 335
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
340 345 350
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
355 360 365
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg Glu Glu
370 375 380
Met Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr
385 390 395 400
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
405 410 415
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
420 425 430
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
435 440 445
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
450 455 460
Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
465 470
<210> 17
<211> 446
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 17
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 Asn 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 Ser Ser Ile Tyr Asn Glu 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 Ala Ser Thr Lys Gly Pro Ser Val Phe
115 120 125
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu
130 135 140
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
145 150 155 160
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
165 170 175
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
180 185 190
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro
195 200 205
Ser Asn Thr Lys Val Asp Lys Arg Val Glu Arg Lys Cys Cys Val Glu
210 215 220
Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
225 230 235 240
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
245 250 255
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
260 265 270
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
275 280 285
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
290 295 300
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
305 310 315 320
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
325 330 335
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro
340 345 350
Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val
355 360 365
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
370 375 380
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
385 390 395 400
Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
405 410 415
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
420 425 430
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 445
<210> 18
<211> 214
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 18
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 Asn Ile Asn Asn Trp
20 25 30
Leu Thr Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ala Ala Ser Ser Arg Gln 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 Ala Lys Leu Pro Val
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> 19
<211> 445
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 19
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 Lys Phe Ser Gly Tyr
20 25 30
Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Val Ile Trp Phe Asp Gly Ser Arg Lys Tyr Tyr Val Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gln Met Gly Tyr Trp His Phe Gly Leu Trp Gly Arg Gly Thr
100 105 110
Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
115 120 125
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
130 135 140
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
145 150 155 160
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
165 170 175
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
180 185 190
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser
195 200 205
Asn Thr Lys Val Asp Lys Arg Val Glu Arg Lys Cys Cys Val Glu Cys
210 215 220
Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu
225 230 235 240
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
245 250 255
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys
260 265 270
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
275 280 285
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
290 295 300
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
305 310 315 320
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
325 330 335
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys
340 345 350
Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys
355 360 365
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
370 375 380
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
385 390 395 400
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
405 410 415
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
420 425 430
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 445
<210> 20
<211> 446
<212> PRT
<213> 人工序列(Artificial Sequence)
<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 Phe Thr Phe Asn 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 Ser Ser Ile Tyr Asn Glu 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 Ala Ser Thr Lys Gly Pro Ser Val Phe
115 120 125
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Glu Gly Thr Ala Ala Leu
130 135 140
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
145 150 155 160
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
165 170 175
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
180 185 190
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asp His Lys Pro
195 200 205
Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys Cys Val Glu
210 215 220
Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
225 230 235 240
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
245 250 255
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
260 265 270
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
275 280 285
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
290 295 300
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
305 310 315 320
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
325 330 335
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro
340 345 350
Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val
355 360 365
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
370 375 380
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
385 390 395 400
Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
405 410 415
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
420 425 430
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 445
<210> 21
<211> 472
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 21
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 Asn Ile Asn Asn Trp
20 25 30
Leu Thr Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ala Ala Ser Ser Arg Gln 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 Ala Lys Leu Pro Val
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser
100 105 110
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu
115 120 125
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys
130 135 140
Ala Ala Ser Gly Phe Thr Phe Asn Asp Tyr Thr Met Asp Trp Val Arg
145 150 155 160
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Asp Val Asn Pro Asn
165 170 175
Ser Gly Ser Ser Ile Tyr Asn Glu Arg Phe Lys Gly Arg Phe Thr Leu
180 185 190
Ser Val Asp Arg Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu
195 200 205
Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asn Leu Gly Pro
210 215 220
Ser Phe Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
225 230 235 240
Ser Ala Ser Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala
245 250 255
Pro Glu Phe Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
260 265 270
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
275 280 285
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
290 295 300
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
305 310 315 320
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
325 330 335
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
340 345 350
Leu Pro Ala Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
355 360 365
Arg Glu Pro Gln Val Cys Thr Leu Pro Pro Ser Arg Glu Glu Met Thr
370 375 380
Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser
385 390 395 400
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
405 410 415
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val
420 425 430
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
435 440 445
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
450 455 460
Ser Leu Ser Leu Ser Pro Gly Lys
465 470
<210> 22
<211> 474
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 22
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 Lys Phe Ser Gly Tyr
20 25 30
Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Val Ile Trp Phe Asp Gly Ser Arg Lys Tyr Tyr Val Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gln Met Gly Tyr Trp His Phe Gly Leu Trp Gly Arg Gly Thr
100 105 110
Leu Val Thr Val Ser Ser Gly Glu Gly Thr Ser Thr Gly Ser Gly Gly
115 120 125
Ser Gly Gly Ser Gly Gly Ala Asp Asp Ile Gln Met Thr Gln Ser Pro
130 135 140
Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg
145 150 155 160
Ala Ser Gln Asn Ile Asn Asn Trp Leu Thr Trp Tyr Gln Gln Lys Pro
165 170 175
Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Ser Arg Gln Thr
180 185 190
Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
195 200 205
Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys
210 215 220
Gln Gln Tyr Ala Lys Leu Pro Val Thr Phe Gly Gln Gly Thr Lys Val
225 230 235 240
Glu Ile Lys Ala Ser Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys
245 250 255
Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
260 265 270
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
275 280 285
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
290 295 300
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
305 310 315 320
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
325 330 335
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
340 345 350
Lys Ala Leu Pro Ala Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
355 360 365
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg Glu Glu
370 375 380
Met Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr
385 390 395 400
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
405 410 415
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
420 425 430
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
435 440 445
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
450 455 460
Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
465 470
<210> 23
<211> 446
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 23
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 Asn 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 Ser Ser Ile Tyr Asn Glu 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 Ala Ser Thr Lys Gly Pro Ser Val Phe
115 120 125
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu
130 135 140
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
145 150 155 160
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
165 170 175
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
180 185 190
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro
195 200 205
Ser Asn Thr Lys Val Asp Lys Arg Val Glu Arg Lys Cys Cys Val Glu
210 215 220
Cys Pro Pro Cys Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser Val Phe
225 230 235 240
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
245 250 255
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
260 265 270
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
275 280 285
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
290 295 300
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
305 310 315 320
Lys Val Ser Asn Lys Ala Leu Pro Ala Ser Ile Glu Lys Thr Ile Ser
325 330 335
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro
340 345 350
Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val
355 360 365
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
370 375 380
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
385 390 395 400
Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
405 410 415
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
420 425 430
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 445
<210> 24
<211> 445
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 24
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 Lys Phe Ser Gly Tyr
20 25 30
Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Val Ile Trp Phe Asp Gly Ser Arg Lys Tyr Tyr Val Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gln Met Gly Tyr Trp His Phe Gly Leu Trp Gly Arg Gly Thr
100 105 110
Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
115 120 125
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
130 135 140
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
145 150 155 160
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
165 170 175
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
180 185 190
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser
195 200 205
Asn Thr Lys Val Asp Lys Arg Val Glu Arg Lys Cys Cys Val Glu Cys
210 215 220
Pro Pro Cys Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser Val Phe Leu
225 230 235 240
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
245 250 255
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys
260 265 270
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
275 280 285
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
290 295 300
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
305 310 315 320
Val Ser Asn Lys Ala Leu Pro Ala Ser Ile Glu Lys Thr Ile Ser Lys
325 330 335
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys
340 345 350
Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys
355 360 365
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
370 375 380
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
385 390 395 400
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
405 410 415
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
420 425 430
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 445
<210> 25
<211> 446
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 25
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 Asn 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 Ser Ser Ile Tyr Asn Glu 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 Ala Ser Thr Lys Gly Pro Ser Val Phe
115 120 125
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Glu Gly Thr Ala Ala Leu
130 135 140
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
145 150 155 160
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
165 170 175
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
180 185 190
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asp His Lys Pro
195 200 205
Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys Cys Val Glu
210 215 220
Cys Pro Pro Cys Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser Val Phe
225 230 235 240
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
245 250 255
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
260 265 270
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
275 280 285
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
290 295 300
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
305 310 315 320
Lys Val Ser Asn Lys Ala Leu Pro Ala Ser Ile Glu Lys Thr Ile Ser
325 330 335
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro
340 345 350
Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val
355 360 365
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
370 375 380
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
385 390 395 400
Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
405 410 415
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
420 425 430
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 445
<210> 26
<211> 446
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 26
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 Asn 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 Ser Ser Ile Tyr Asn Glu 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 Ala Ser Thr Lys Gly Pro Ser Val Phe
115 120 125
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Glu Gly Thr Ala Ala Leu
130 135 140
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
145 150 155 160
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
165 170 175
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
180 185 190
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asp His Lys Pro
195 200 205
Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Ser Cys Val Glu
210 215 220
Cys Pro Pro Cys Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser Val Phe
225 230 235 240
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
245 250 255
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
260 265 270
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
275 280 285
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
290 295 300
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
305 310 315 320
Lys Val Ser Asn Lys Ala Leu Pro Ala Ser Ile Glu Lys Thr Ile Ser
325 330 335
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro
340 345 350
Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val
355 360 365
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
370 375 380
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
385 390 395 400
Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
405 410 415
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
420 425 430
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 445
<210> 27
<211> 445
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 27
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 Lys Phe Ser Gly Tyr
20 25 30
Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Val Ile Trp Phe Asp Gly Ser Arg Lys Tyr Tyr Val Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gln Met Gly Tyr Trp His Phe Gly Leu Trp Gly Arg Gly Thr
100 105 110
Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
115 120 125
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
130 135 140
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
145 150 155 160
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
165 170 175
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
180 185 190
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser
195 200 205
Asn Thr Lys Val Asp Lys Arg Val Glu Arg Lys Ser Cys Val Glu Cys
210 215 220
Pro Pro Cys Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser Val Phe Leu
225 230 235 240
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
245 250 255
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys
260 265 270
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
275 280 285
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
290 295 300
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
305 310 315 320
Val Ser Asn Lys Ala Leu Pro Ala Ser Ile Glu Lys Thr Ile Ser Lys
325 330 335
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys
340 345 350
Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys
355 360 365
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
370 375 380
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
385 390 395 400
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
405 410 415
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
420 425 430
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 445
<210> 28
<211> 12
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 28
Glu Arg Lys Ser Cys Val Glu Cys Pro Pro Cys Pro
1 5 10
<210> 29
<211> 15
<212> PRT
<213> 智人（Homo sapiens）
<400> 29
Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro
1 5 10 15
<210> 30
<211> 98
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 30
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
1 5 10 15
Ser Thr Ser Glu 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 Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95
Thr Val
<210> 31
<211> 110
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 31
Ala Pro Glu Phe Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
1 5 10 15
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
20 25 30
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
35 40 45
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
50 55 60
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
65 70 75 80
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
85 90 95
Ala Leu Pro Ala Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys
100 105 110
<210> 32
<211> 104
<212> PRT
<213> 智人（Homo sapiens）
<400> 32
Asp Gly Asn Glu Glu Met Gly Gly Ile Thr Gln Thr Pro Tyr Lys Val
1 5 10 15
Ser Ile Ser Gly Thr Thr Val Ile Leu Thr Cys Pro Gln Tyr Pro Gly
20 25 30
Ser Glu Ile Leu Trp Gln His Asn Asp Lys Asn Ile Gly Gly Asp Glu
35 40 45
Asp Asp Lys Asn Ile Gly Ser Asp Glu Asp His Leu Ser Leu Lys Glu
50 55 60
Phe Ser Glu Leu Glu Gln Ser Gly Tyr Tyr Val Cys Tyr Pro Arg Gly
65 70 75 80
Ser Lys Pro Glu Asp Ala Asn Phe Tyr Leu Tyr Leu Arg Ala Arg Val
85 90 95
Cys Glu Asn Cys Met Glu Met Asp
100
<210> 33
<211> 84
<212> PRT
<213> 智人（Homo sapiens）
<400> 33
Phe Lys Ile Pro Ile Glu Glu Leu Glu Asp Arg Val Phe Val Asn Cys
1 5 10 15
Asn Thr Ser Ile Thr Trp Val Glu Gly Thr Val Gly Thr Leu Leu Ser
20 25 30
Asp Ile Thr Arg Leu Asp Leu Gly Lys Arg Ile Leu Asp Pro Arg Gly
35 40 45
Ile Tyr Arg Cys Asn Gly Thr Asp Ile Tyr Lys Asp Lys Glu Ser Thr
50 55 60
Val Gln Val His Tyr Arg Met Cys Gln Ser Cys Val Glu Leu Asp Pro
65 70 75 80
Ala Thr Val Ala
<210> 34
<211> 96
<212> PRT
<213> 食蟹猴（Macaca fascicularis）
<400> 34
Gln Asp Gly Asn Glu Glu Met Gly Ser Ile Thr Gln Thr Pro Tyr Gln
1 5 10 15
Val Ser Ile Ser Gly Thr Thr Val Ile Leu Thr Cys Ser Gln His Leu
20 25 30
Gly Ser Glu Ala Gln Trp Gln His Asn Gly Lys Asn Lys Glu Asp Ser
35 40 45
Gly Asp Arg Leu Phe Leu Pro Glu Phe Ser Glu Met Glu Gln Ser Gly
50 55 60
Tyr Tyr Val Cys Tyr Pro Arg Gly Ser Asn Pro Glu Asp Ala Ser His
65 70 75 80
His Leu Tyr Leu Lys Ala Arg Val Cys Glu Asn Cys Met Glu Met Asp
85 90 95
<210> 35
<211> 84
<212> PRT
<213> 食蟹猴（Macaca fascicularis）
<400> 35
Phe Lys Ile Pro Val Glu Glu Leu Glu Asp Arg Val Phe Val Lys Cys
1 5 10 15
Asn Thr Ser Val Thr Trp Val Glu Gly Thr Val Gly Thr Leu Leu Thr
20 25 30
Asn Asn Thr Arg Leu Asp Leu Gly Lys Arg Ile Leu Asp Pro Arg Gly
35 40 45
Ile Tyr Arg Cys Asn Gly Thr Asp Ile Tyr Lys Asp Lys Glu Ser Ala
50 55 60
Val Gln Val His Tyr Arg Met Cys Gln Asn Cys Val Glu Leu Asp Pro
65 70 75 80
Ala Thr Leu Ala
<210> 36
<211> 87
<212> PRT
<213> 小鼠（Mus musculus）
<400> 36
Asp Asp Ala Glu Asn Ile Glu Tyr Lys Val Ser Ile Ser Gly Thr Ser
1 5 10 15
Val Glu Leu Thr Cys Pro Leu Asp Ser Asp Glu Asn Leu Lys Trp Glu
20 25 30
Lys Asn Gly Gln Glu Leu Pro Gln Lys His Asp Lys His Leu Val Leu
35 40 45
Gln Asp Phe Ser Glu Val Glu Asp Ser Gly Tyr Tyr Val Cys Tyr Thr
50 55 60
Pro Ala Ser Asn Lys Asn Thr Tyr Leu Tyr Leu Lys Ala Arg Val Cys
65 70 75 80
Glu Tyr Cys Val Glu Val Asp
85
<210> 37
<211> 84
<212> PRT
<213> 小鼠（Mus musculus）
<400> 37
Phe Lys Ile Gln Val Thr Glu Tyr Glu Asp Lys Val Phe Val Thr Cys
1 5 10 15
Asn Thr Ser Val Met His Leu Asp Gly Thr Val Glu Gly Trp Phe Ala
20 25 30
Lys Asn Lys Thr Leu Asn Leu Gly Lys Gly Val Leu Asp Pro Arg Gly
35 40 45
Ile Tyr Leu Cys Asn Gly Thr Glu Gln Leu Ala Lys Val Val Ser Ser
50 55 60
Val Gln Val His Tyr Arg Met Cys Gln Asn Cys Val Glu Leu Asp Ser
65 70 75 80
Gly Thr Met Ala
<210> 38
<211> 488
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 38
Thr Gln Val Cys Thr Gly Thr Asp Met Lys Leu Arg Leu Pro Ala Ser
1 5 10 15
Pro Glu Thr His Leu Asp Met Leu Arg His Leu Tyr Gln Gly Cys Gln
20 25 30
Val Val Gln Gly Asn Leu Glu Leu Thr Tyr Leu Pro Thr Asn Ala Ser
35 40 45
Leu Ser Phe Leu Gln Asp Ile Gln Glu Val Gln Gly Tyr Val Leu Ile
50 55 60
Ala His Asn Gln Val Arg Gln Val Pro Leu Gln Arg Leu Arg Ile Val
65 70 75 80
Arg Gly Thr Gln Leu Phe Glu Asp Asn Tyr Ala Leu Ala Val Leu Asp
85 90 95
Asn Gly Asp Pro Leu Asn Asn Thr Thr Pro Val Thr Gly Ala Ser Pro
100 105 110
Gly Gly Leu Arg Glu Leu Gln Leu Arg Ser Leu Thr Glu Ile Leu Lys
115 120 125
Gly Gly Val Leu Ile Gln Arg Asn Pro Gln Leu Cys Tyr Gln Asp Thr
130 135 140
Ile Leu Trp Lys Asp Ile Phe His Lys Asn Asn Gln Leu Ala Leu Thr
145 150 155 160
Leu Ile Asp Thr Asn Arg Ser Arg Ala Cys His Pro Cys Ser Pro Met
165 170 175
Cys Lys Gly Ser Arg Cys Trp Gly Glu Ser Ser Glu Asp Cys Gln Ser
180 185 190
Leu Thr Arg Thr Val Cys Ala Gly Gly Cys Ala Arg Cys Lys Gly Pro
195 200 205
Leu Pro Thr Asp Cys Cys His Glu Gln Cys Ala Ala Gly Cys Thr Gly
210 215 220
Pro Lys His Ser Asp Cys Leu Ala Cys Leu His Phe Asn His Ser Gly
225 230 235 240
Ile Cys Glu Leu His Cys Pro Ala Leu Val Thr Tyr Asn Thr Asp Thr
245 250 255
Phe Glu Ser Met Pro Asn Pro Glu Gly Arg Tyr Thr Phe Gly Ala Ser
260 265 270
Cys Val Thr Ala Cys Pro Tyr Asn Tyr Leu Ser Thr Asp Val Gly Ser
275 280 285
Cys Thr Leu Val Cys Pro Leu His Asn Gln Glu Val Thr Ala Glu Asp
290 295 300
Gly Thr Gln Arg Cys Glu Lys Cys Ser Lys Pro Cys Ala Arg Val Cys
305 310 315 320
Tyr Gly Leu Gly Met Glu His Leu Arg Glu Val Arg Ala Val Thr Ser
325 330 335
Ala Asn Ile Gln Glu Phe Ala Gly Cys Lys Lys Ile Phe Gly Ser Leu
340 345 350
Ala Phe Leu Pro Glu Ser Phe Asp Gly Asp Pro Ala Ser Asn Thr Ala
355 360 365
Pro Leu Gln Pro Glu Gln Leu Gln Val Phe Glu Thr Leu Glu Glu Ile
370 375 380
Thr Gly Tyr Leu Tyr Ile Ser Ala Trp Pro Asp Ser Leu Pro Asp Leu
385 390 395 400
Ser Val Phe Gln Asn Leu Gln Val Ile Arg Gly Arg Ile Leu His Asn
405 410 415
Gly Ala Tyr Ser Leu Thr Leu Gln Gly Leu Gly Ile Ser Trp Leu Gly
420 425 430
Leu Arg Ser Leu Arg Glu Leu Gly Ser Gly Leu Ala Leu Ile His His
435 440 445
Asn Thr His Leu Cys Phe Val His Thr Val Pro Trp Asp Gln Leu Phe
450 455 460
Arg Asn Pro His Gln Ala Leu Leu His Thr Ala Asn Arg Pro Glu Asp
465 470 475 480
Glu Cys Val Gly Glu Gly Leu Ala
485
<210> 39
<211> 6
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 39
His His His His His His
1 5
<210> 40
<211> 232
<212> PRT
<213> 小鼠（Mus musculus）
<400> 40
Pro Arg Gly Pro Thr Ile Lys Pro Cys Pro Pro Cys Lys Cys Pro Ala
1 5 10 15
Pro Asn Leu Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Ile
20 25 30
Lys Asp Val Leu Met Ile Ser Leu Ser Pro Ile Val Thr Cys Val Val
35 40 45
Val Asp Val Ser Glu Asp Asp Pro Asp Val Gln Ile Ser Trp Phe Val
50 55 60
Asn Asn Val Glu Val His Thr Ala Gln Thr Gln Thr His Arg Glu Asp
65 70 75 80
Tyr Asn Ser Thr Leu Arg Val Val Ser Ala Leu Pro Ile Gln His Gln
85 90 95
Asp Trp Met Ser Gly Lys Glu Phe Lys Cys Lys Val Asn Asn Lys Asp
100 105 110
Leu Pro Ala Pro Ile Glu Arg Thr Ile Ser Lys Pro Lys Gly Ser Val
115 120 125
Arg Ala Pro Gln Val Tyr Val Leu Pro Pro Pro Glu Glu Glu Met Thr
130 135 140
Lys Lys Gln Val Thr Leu Thr Cys Met Val Thr Asp Phe Met Pro Glu
145 150 155 160
Asp Ile Tyr Val Glu Trp Thr Asn Asn Gly Lys Thr Glu Leu Asn Tyr
165 170 175
Lys Asn Thr Glu Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe Met Tyr
180 185 190
Ser Lys Leu Arg Val Glu Lys Lys Asn Trp Val Glu Arg Asn Ser Tyr
195 200 205
Ser Cys Ser Val Val His Glu Gly Leu His Asn His His Thr Thr Lys
210 215 220
Ser Phe Ser Arg Thr Pro Gly Lys
225 230
<210> 41
<211> 232
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 41
Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
1 5 10 15
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
20 25 30
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
35 40 45
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
50 55 60
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
65 70 75 80
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
85 90 95
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
100 105 110
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
115 120 125
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg Glu Glu Met Thr
130 135 140
Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser
145 150 155 160
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
165 170 175
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
180 185 190
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
195 200 205
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
210 215 220
Ser Leu Ser Leu Ser Pro Gly Lys
225 230
<210> 42
<211> 232
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 42
Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
1 5 10 15
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
20 25 30
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
35 40 45
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
50 55 60
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
65 70 75 80
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
85 90 95
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
100 105 110
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
115 120 125
Arg Glu Pro Gln Val Cys Thr Leu Pro Pro Ser Arg Glu Glu Met Thr
130 135 140
Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser
145 150 155 160
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
165 170 175
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val
180 185 190
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
195 200 205
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
210 215 220
Ser Leu Ser Leu Ser Pro Gly Lys
225 230
<210> 43
<211> 330
<212> PRT
<213> 智人（Homo sapiens）
<400> 43
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
Arg 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> 44
<211> 327
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 44
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
Arg Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro
100 105 110
Glu Leu 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 His Glu Asp Pro Glu Val Lys 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 Tyr
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 Ala Leu
195 200 205
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
210 215 220
Glu Pro Gln Val Cys Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys
225 230 235 240
Asn Gln Val Ser Leu Ser Cys Ala 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 Val Ser
275 280 285
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 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 Pro Gly Lys
325
<210> 45
<211> 327
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 45
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
Arg Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro
100 105 110
Glu Leu 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 His Glu Asp Pro Glu Val Lys 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 Tyr
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 Ala Leu
195 200 205
Pro Ala Pro 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 Cys Arg Glu Glu Met Thr Lys
225 230 235 240
Asn Gln Val Ser Leu Trp 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
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 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 Pro Gly Lys
325
<210> 46
<211> 327
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 46
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
Arg Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro
100 105 110
Glu Phe Glu 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 His Glu Asp Pro Glu Val Lys 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 Tyr
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 Ala Leu
195 200 205
Pro Ala Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
210 215 220
Glu Pro Gln Val Cys Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys
225 230 235 240
Asn Gln Val Ser Leu Ser Cys Ala 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 Val Ser
275 280 285
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 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 Pro Gly Lys
325
<210> 47
<211> 327
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 47
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
Arg Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro
100 105 110
Glu Phe Glu 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 His Glu Asp Pro Glu Val Lys 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 Tyr
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 Ala Leu
195 200 205
Pro Ala 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 Cys Arg Glu Glu Met Thr Lys
225 230 235 240
Asn Gln Val Ser Leu Trp 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
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 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 Pro Gly Lys
325
<210> 48
<211> 327
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 48
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
1 5 10 15
Ser Thr Ser Glu 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 Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95
Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro
100 105 110
Glu Phe Glu 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 His Glu Asp Pro Glu Val Lys 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 Tyr
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 Ala Leu
195 200 205
Pro Ala Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
210 215 220
Glu Pro Gln Val Cys Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys
225 230 235 240
Asn Gln Val Ser Leu Ser Cys Ala 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 Val Ser
275 280 285
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 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 Pro Gly Lys
325
<210> 49
<211> 327
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 49
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
Arg Val Glu Arg Lys Ser Cys Val Glu Cys Pro Pro Cys Pro Ala Pro
100 105 110
Glu Phe Glu 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 His Glu Asp Pro Glu Val Lys 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 Tyr
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 Ala Leu
195 200 205
Pro Ala 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 Cys Arg Glu Glu Met Thr Lys
225 230 235 240
Asn Gln Val Ser Leu Trp 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
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 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 Pro Gly Lys
325
<210> 50
<211> 327
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 50
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
1 5 10 15
Ser Thr Ser Glu 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 Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95
Thr Val Glu Arg Lys Ser Cys Val Glu Cys Pro Pro Cys Pro Ala Pro
100 105 110
Glu Phe Glu 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 His Glu Asp Pro Glu Val Lys 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 Tyr
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 Ala Leu
195 200 205
Pro Ala Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
210 215 220
Glu Pro Gln Val Cys Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys
225 230 235 240
Asn Gln Val Ser Leu Ser Cys Ala 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 Val Ser
275 280 285
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 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 Pro Gly Lys
325
<210> 51
<211> 107
<212> PRT
<213> 智人（Homo sapiens）
<400> 51
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

Claims (10)

1.双特异性人IgG1抗体，其包含两种具有相同铰链区的Fc片段，所述铰链区的氨基酸序列为SEQ ID NO:1或SEQ ID NO:28并替换了天然人IgG1抗体恒定区的第216-230位序列；以及
所述两种Fc片段的CH2片段中均包含点突变L234F、L235E和P331S，其中CH2片段的氨基酸序列为抗体恒定区第231-340位的氨基酸序列；
其中所述抗体恒定区氨基酸位置按照EU numbering确定。
2.如权利要求1所述的双特异性人IgG1抗体，其中所述两种Fc片段包含能够确保重链异聚化的突变；优选地，一个Fc片段包含点突变S354C和T366W，另一个Fc片段包含点突变Y349C、T366S、L368A和Y407V。
3.如权利要求1或2所述的双特异性人IgG1抗体，其中所述两种Fc片段的N端分别融合识别不同抗原表位的单链抗体(scfv)。
4.如权利要求1或2所述的双特异性人IgG1抗体，其中一种Fc片段的N端融合单链抗体(scfv)，另一种Fc片段的N端融合Fab片段，所述单链抗体与所述Fab片段识别不同的抗原表位。
5.如权利要求1或2所述的双特异性人IgG1抗体，其中所述两种Fc片段的N端分别融合识别不同抗原表位的Fab片段；
优选地，识别不同抗原表位的Fab片段包含相同的轻链。
6.如权利要求5所述的双特异性人IgG1抗体，其中一种Fab片段包含天然的人IgG1抗体恒定区CH1片段，另一种Fab片段包含突变的人IgG1抗体恒定区CH1片段，所述突变的人IgG1抗体恒定区CH1片段包含点突变G137E、N203D和R214T中的任意1个、2个或3个，其中CH1片段的氨基酸序列为抗体恒定区第118-215位的氨基酸序列；
优选地，所述天然的人IgG1抗体恒定区CH1片段的氨基酸序列为SEQ ID NO:2，和/或所述突变的人IgG1抗体恒定区CH1片段的氨基酸序列为SEQ ID NO:30。
7.如权利要求1-6中任一项所述的双特异性人IgG1抗体，其中所述两种Fc片段的CH2片段的氨基酸序列均为SEQ ID NO:31。
8.如权利要求1-7中任一项所述的双特异性人IgG1抗体，其识别人免疫细胞表面抗原和肿瘤细胞表面抗原；
优选地，所述人免疫细胞表面抗原为CD3E。
9.药物组合物，其包含权利要求1-8中任一项所述的双特异性人IgG1抗体。
10.权利要求1-8中任一项所述的双特异性人IgG1抗体或者权利要求9所述的药物组合物在制备用于预防或治疗肿瘤的药物中的用途。
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