CN113135996A - 一种双特异抗体及其应用 - Google Patents

一种双特异抗体及其应用 Download PDF

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CN113135996A
CN113135996A CN202011427863.9A CN202011427863A CN113135996A CN 113135996 A CN113135996 A CN 113135996A CN 202011427863 A CN202011427863 A CN 202011427863A CN 113135996 A CN113135996 A CN 113135996A
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屈向东
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Qiyu Biotechnology Shanghai Co Ltd
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Abstract

本发明提供一种双特异抗体及其应用，本发明的双特异性抗体与HER2蛋白的亲和力高，本发明的双特异性抗体对肿瘤细胞的增殖抑制作用强。

Description

一种双特异抗体及其应用
技术领域
本发明涉及一种双特异抗体，本发明还涉及这种双特异性抗体的应用，属于生物医药领域。
背景技术
双特异性抗体(BsAb)又称双功能抗体，可同时识别和结合两种不同的抗原和表位，并阻断两种不同的信号通路以发挥其作用。BsAb与普通抗体相比增加了一个特异性抗原结合位点，在治疗方面表现出了以下优势：
介导免疫细胞对肿瘤的杀伤：双特异性抗体的一个重要作用机制是介导免疫细胞杀伤，双特异性抗体有两条抗原结合臂，其中一条与靶抗原结合，另一条与效应细胞上的标记抗原结合，后者可以激活效应细胞，使其靶向杀伤肿瘤细胞。目前已经批准上市的2个双特异性抗体产品都属于这个类别，TrionPharma公司开发的catumaxomab能够靶向肿瘤表面抗原EpCAM和T细胞表面受体CD3，而Micromet公司和Amgen公司开发的Blinatumomab可以同时结合CD19和CD3。两者都是通过激活并召集杀伤性T细胞，从而达到治疗肿瘤的目的；
双靶点信号阻断，发挥独特的或重叠的功能，有效防止耐药：同时结合双靶点，阻断双信号通路是双特异性抗体的另一个重要作用机制。受体络氨酸激酶(receptortyrosine kinase，RTKs)是最大的一类酶联受体，在细胞增殖过程中发挥重要的调节作用，如Her家族等。RTKs在肿瘤细胞表面异常高表达，导致肿瘤细胞恶性增生，因此也是肿瘤治疗的重要靶点。针对RTKs的单靶点单克隆抗体已在肿瘤治疗中得到广泛应用，但是，肿瘤细胞可以通过转换信号通路或通过HER家族成员自身或不同成员之间的同源或异源二聚体激活细胞内信号进行免疫逃逸。因此采用双特异性抗体药物同时阻断两个或多个RTKs或其配体，可以减少肿瘤细胞逃逸，提高治疗效果；
具备更强特异性、靶向性和降低脱靶毒性：利用双特异性抗体两个抗原结合臂可以结合不同抗原的特点，两个抗原结合臂分别结合癌细胞表面2种抗原，可以有效增强抗体对癌细胞的结合特异性和靶向性，降低脱靶等副作用；
有效降低治疗成本：以BiTE为例，与传统抗体相比在组织渗透率、杀伤肿瘤细胞效率、脱靶率和临床适应症等指标方面都具有较强的竞争力，临床优势显著。特别在使用剂量方面，由于其治疗效果可以达到普通抗体的100-1000倍，使用剂量最低可将为原来的1/2000，显著降低药物治疗成本。相对于组合疗法，双特异性抗体的成本也远远低于两个单药联合治疗。
目前，全球有三款双特异性抗体药物获得上市批准：
2009年，欧盟批准了第一个治疗性双特异性抗体—TrionPharma公司的Catumaxomab(靶向CD3和EpCAM)，用于治疗癌性腹水。2014年，FDA快速批准Amgen公司基于
Figure BDA0002825622600000011
技术开发的双特异性抗体药物Blinatumomab(靶向CD3和CD19)用于治疗急性B淋巴细胞白血病，它也是第一个获批的靶点为CD19的药物。Amgen一系列的血液恶性肿瘤和实体瘤的临床开发中就拥有十几种
Figure BDA0002825622600000021
分子。其中靶向BCMA/CD3的AMG420已获得FDA授权快速审批通道地位。在实体肿瘤的治疗中，靶向前列腺特异膜抗原(PSMA)的
Figure BDA0002825622600000022
分子AMG212(Pasotuxizumab)的临床试验结果也展露锋芒。2017年11月，FDA快速批准了罗氏的双特异性抗体Emicizumab(靶向凝血因子X和因子IXa)，用于治疗血友病。2018年12月在中国获批上市，是首个在国内获批的双特异性抗体。
1.IL15/IL15Ra
IL-15是一种长14–15kDa的细胞因子，对于NK细胞、NKT细胞和memoryCD8+T细胞功能非常重要。IL-15在体内含量很少，但通过与其受体IL-15Rα结合产生具有极高生物效价的复合物IL-15超激动剂(IL-15SA)后一起被转导转运至靶细胞。IL-15SA强烈激活响应IL-15的细胞，特别是NK细胞，从而促进抗肿瘤和抗病毒功能。
研究人员在1994年首次将IL-15鉴定为T淋巴细胞生长因子，它与IL-2具有大约19％的同源序列，许多生物学特性也非常类似。IL-15三维结构与IL-2类似，由四个“上-下-下-下”螺旋束组成，IL-4，IL-7和IL-9等其他细胞因子也包含这种构象。IL-15与其他细胞因子作用方式不同，IL-15受体α表达于产生IL-15的细胞(例如巨噬细胞和树突状细胞)，与IL-15形成IL-15SA将信号递送至表达IL-15Rβ(也称为IL-2Rβ)和共同γ链(与IL-2，IL-4，IL-7，IL-9和IL-21共享)的NK、NKT和memory CD8+T细胞，很有可能正是由于这一独特的呈递方式赋予了IL-15介导其独特功能的能力。小鼠IL-15与人IL-15具有70％的氨基酸序列同源性，人IL-15和小鼠IL-15也具有相似的反式表达模式，信号通路以及生物学活性。IL-15在许多细胞类型和组织中表达，包括单核细胞、巨噬细胞、DC、角质形成细胞、成纤维细胞、肌细胞和神经细胞。IL-15作为多效细胞因子在先天性和适应性免疫中起重要作用。
反式表达的IL-15/IL-15Rα信号通过响应细胞上表达的β和γ链，诱导JAK1和JAK3的募集和激活，活化的JAK1和JAK3进一步磷酸化STAT3和STAT5。STAT3和STAT5磷酸化后形成同源二聚体，易位至细胞核并促进靶基因的转录。IL-15信号传导刺激一系列下游反应，诱导细胞生长、减少凋亡以及增强免疫细胞的激活和转移。在没有高亲和力IL-15Rα的情况下，IL-15也可以单独与中等亲和力(Ka＝1.109/M)的β、γ受体复合物(IL-15Rβγ)结合，诱导其他酪氨酸激酶(如Lck、Fyn、Lyn、Syk)磷酸化激活，并与PI3K、MAPK途径发生作用。有研究表明，代谢检查点激酶mTOR也可以被高浓度的IL-15激活，这与NK细胞的增殖和激活增强有关：选择性敲除mTOR会导致骨髓NK细胞成熟受阻。IL-15促进NK细胞增殖的能力部分是由于IL-15介导的有氧糖酵解作用引起的，在没有IL-15的情况下NK细胞的基础代谢很低，但通过提高IL-15浓度可以明显增强这一生理活动。
由于IL-15具有与IL-2类似的免疫学特性：诱导T细胞的增殖和存活，促进NK细胞的增殖和分化，并诱导细胞毒性T淋巴细胞的产生。但不同于IL-2，IL-15对Treg细胞没有明显作用，并且不会在小鼠或非人类灵长类动物(NHP)中引起毛细血管渗漏综合征，因此相较于IL-2，IL-15是肿瘤免疫疗法的更优选择。猕猴IL-15(rIL-15)是第一种用于体内实验的IL-15形式，研究人员认为rIL-15可以优先结合细胞表面IL-15Rα。异二聚体IL-15/IL-15Rα是从细胞上裂解下来的IL-15的天然形式，可以独立于细胞刺激相互作用的应答，目前诺华公司正在实体瘤中开展这种形式的分子(NIZ985)的临床试验。Cytune开发的RLI是一种融合蛋白，由与IL-15Rα的Sushi结构域相连的IL-15组成，可以作为可溶性的IL-15激动剂。IL-15/IL-15Rα-Fc复合物通过将市售的IL-15Rα-Fc嵌合融合蛋白与rIL-15混合产生的，已在临床前研究中广泛使用。IL-15与IL-15Rα有很高亲和力，KD＝30-100pM。
本专利发明了一种全新的双特异靶向细胞因子抗体形式：细胞因子IL15与IL15Ra之间有超高的亲和力(KD约30-100pM)，本发明用IL15及IL15Ra分别替代其中一个抗体结构中的CL及CH1结构域，解决双特异抗体轻链错配问题。通过FC异源二聚体形式解决重链错配。将有Fab的FC端进行突变，使其不能结合Protein A。纯化通过结合FC端的Protein A亲和层析及结合CH1结构域CH1-XL亲和层析的2步纯化出双特异靶向抗体。
2.Her2背景
受体酪氨酸激酶的ErbB家族的成员是细胞生长，分化和存活的重要介导物。该受体家族包括四个独特成员，包括表皮生长因子受体(EGFR或ErbB1)，HER2(ErbB2)，HER3(ErbB3)和HER4(ErbB4或tyro2)。HER2是一种跨膜的表面结合的受体酪氨酸激酶，而且正常情况下牵涉导致细胞生长和分化的信号转导途径。
Her2过度表达可能导致细胞正常功能紊乱，并且通常与肿瘤的发生和发展密切相关。Her2的同源或异源聚合可导致受体酪氨酸残基磷酸化，并且引发许多信号途径并引起细胞增殖和肿瘤发生。作为预后和预测的生物标志物，Her2基因的扩增或过度表达在约15-30％的乳腺癌和10-30％的胃癌/食道癌中发生。在其它肿瘤诸如卵巢、子宫内膜、膀胱、肺、结肠和头颈肿瘤中也可观察到Her2过度表达。
曲妥珠单抗(Trastuzumab)识别Her2胞外结构域IV近膜表位。具体为Her2胞外结构域IV部分的C端处由3个环(557-561、570-573和593-603)组成的表位。因为表位可以靠近其二聚化伴侣的结合结构域或直接与之相互作用，所以曲妥珠单抗与表位的结合可以诱导抑制二聚化过程的空间位阻。另外，曲妥珠单抗的结合还可以保护Her2受体的细胞外结构域免受蛋白酶的攻击而水解。
目前将曲妥珠单抗用作治疗乳腺癌的一线药物，有效治疗Her2过度表达的转移性乳腺癌，并且单药一线治疗的客观反映率为30-50％；但是在治疗Her2表达较低的转移性乳腺癌方面效果不理想，并且抗体最初在1年内有效的许多患者中发展出耐药性。Her2连同该家族的其它成员(Her1、Her3和Her4)可以形成配体依赖性或配体非依赖性异源二聚体，从而激活下游途径，然后导致肿瘤细胞增殖，而曲妥珠单抗无法抑制异源二聚体形成，所以这可能是耐药性发展的原因之一。
帕妥珠单抗(Pertuzumab)是一种人源化单克隆抗体，其特异性设计成阻止HER2受体与细胞表面上的其它HER受体(EGFR/HER1，HER3和HER4)配对(二聚化)，认为该过程在肿瘤生长和存活中发挥作用。帕妥珠单抗对晚期前列腺癌、非小细胞肺癌、卵巢癌和乳腺癌有一定治疗效果，但其治疗效果还取决于Her2表达水平。帕妥珠单抗识别Her2细胞外结构域II异源二聚化的关键位点，由此识别的表位位于II亚区中心的区段245-311，关键残基为H245、V286、S288、L295、H296和K311。其中，L295、H296是介导Her2和Her3异源二聚化的关键位点，并且L295A/H296A双重突变可完全阻断Her2/Her3的异源二聚化(Franklin，M.C.等人，Insights into ErbB signaling from the structure ofthe ErbB2-pertuzumabcomplex.Cancer Cell，2004.5(4):第317-28页)。因此，帕妥珠单抗可用于有效抑制Her2/Her3异源二聚体的形成，但对EGFR/Her2异源二聚体的形成并未显示出明显的抑制作用。
发明内容
本发明的目的在于提供一种特异性结合HER2的双特异性抗体及其应用。
本发明采用了如下技术方案：
一种全新的双特异抗体分子包括但不限于这种形式：
{[TAA]1，[TAA]2，[IL15/IL15Ra]，[FC异源二聚体]}，
每种形式同时包含:
[TAA]1：结合一个抗原的抗体、抗体或多肽片段；
[TAA]2：结合另一个抗原或同一抗原不同表位的抗体、抗体或多肽片段；
[IL15/IL15Ra]：IL15及其能结合IL15Ra的突变，截短及各种衍生物；
IL15Ra及其能结合IL15的突变，截短及各种衍生物；
这两者形成的复合物；
[FC异源二聚体]：FC形成的异源二聚体。
作为优选，本发明的双特异抗体分子，还具有这样的特征：包含IL15/IL15Ra复合物，IL15/IL15Ra复合物包括但不限于1)IL15及其能结合IL15Ra的突变，截短及各种衍生物；2)IL15Ra及其能结合IL15的突变，截短及各种衍生物；
包括但不限于下表所示突变，计数方式根据SEQ ID No.1所示IL15第一个氨基酸开始算为第1位；SEQ ID No.3所示IL15Ra第一个氨基酸开始算为第1位：
组合 IL15 IL15Ra
1 wt D96
2 wt D96/P97
3 wt D96/P97/A98
4 E87C D96/C97
5 E87C D96/P97/C98
6 E87C D96/C97/A98
7 V49C S40C
8 L52C S40C
9 E89C K34C
10 Q48C G38C
11 E53C L42C
12 C425 A37C
13 L45C G38C
14 L45C A37C
同时包括但不限于下表所示IL15相关各种突变，计数方式根据SEQ ID No.1所示IL15第一个氨基酸开始算为第1位：
组合 IL15突变
1 N1D
2 ND4
3 D8N
4 D30N
5 D61N
6 E64Q
7 N65D
8 Q108E
9 N1D/D61N
10 N1D/E64Q
11 N4D/D61N
12 N4D/E64Q
13 D8N/D61N
14 D8N/E64Q
15 D61N/F64Q
16 E64Q/Q108E
17 N1D/N4D/D8N
18 D61N/E64Q/N65D
19 N1D/D61N/E64Q/Q108E
20 N4D/D61N/E64Q/Q108E
作为优选，本发明的双特异抗体分子，还具有这样的特征：与IL15或IL15Ra融合的是针对抗原的抗体可变区VL1或VH1，包括但不限于以下突变，根据EU计数：
Figure BDA0002825622600000061
其中，VL1与VH1之间可有一对或多对二硫键。
作为优选，本发明的双特异抗体分子，还具有这样的特征：包含抗体的FC段，是FC异源二聚体形式，FC异源二聚体包含但不限于以下突变的组合，根据EU计数，
Figure BDA0002825622600000071
作为优选，本发明的双特异抗体分子，还具有这样的特征：其中，人FC片段包括但不限于Human IgG1 FC、Human IgG2 FC、Human IgG3 FC、Human IgG4 FC及其变异体，其中一条链能结合proteinA；则另一条链可选择不结合proteinA的突变体，包括但不限于突变H435R或H435R/Y436F，根据EU计数。
作为优选，本发明的双特异抗体分子，还具有这样的特征：其中结合抗原的各个抗体或多肽片段是单价或多价分子，TAA结合的抗原或其特异突变包括但不限于CD3、CD20、CD19、CD30、CD33、CD38、CD40、CD52、slamf7、GD2、CD24、CD47、CD133、CD239、CD276、PD-1等表面抗原，CEA、Epcam、Trop2、TAG72、MUC1、MUC16、mesothelin、folr1、CLDN18.2、PDL1、EGFR、EGFR VIII、C-MET、HER2、FGFR2、FGFR3、PSMA、PSCA、EphA2、ADAM17、17-A1、NKG2D ligands、MCSP、LGR5、SSEA3、SLC34A2、BCMA、GPNMB等靶点的抗体，形式包括Fab、ScFv、VHH或多肽片段等。
作为优选，本发明的双特异抗体分子，还具有这样的特征：其中各个抗体是嵌合、人源化、全人源或单链抗体。
作为优选，本发明的双特异抗体分子，还具有这样的特征：连接序列为免疫原性低的氨基酸序列。
作为优选，本发明的双特异抗体分子，还具有这样的特征：当由4条链组成时，包括但不限于如下形式：
Figure BDA0002825622600000081
其中：
链1：结合抗原的抗体可变区VL1或VH1融合在IL15或IL15Ra的N端或C端；
链2：IL15或IL15Ra的N端或C端融合了结合抗原的抗体可变区VL1或VH1,在其C端融合可形成异源二聚体的FC；
链3：结合另一个抗原的抗体可变区VH2融合CH1片段,在其C端融合可形成异源二聚体的FC，组成抗体重链；
链4：结合另一个抗原的抗体可变区VL2的C端融合CL片段，组成抗体轻链，
当由3条链组成时，包括但不限于以下形式：
Figure BDA0002825622600000082
其中：
链1：结合抗原的抗体可变区VL1或VH1融合在IL15或IL15Ra的N端或C端；
链2：IL15或IL15Ra的N端或C端融合了结合抗原的抗体可变区VL1或VH1,在其C端融合可形成异源二聚体的FC；
链3：结合另一个靶点的亲和多肽，包括SCFV，VHH及其人源化形式、多肽或蛋白质，在其C端融合可形成异源二聚体的FC。
作为优选，本发明的双特异抗体分子，还具有这样的特征：与IL15或IL15Ra融合的蛋白链1及蛋白链2的组合形式包括但不限于以下组合：
Figure BDA0002825622600000091
作为优选，本发明的双特异抗体分子，还具有这样的特征：还具有如下形式：
Figure BDA0002825622600000092
其中：
链1：结合抗原的抗体可变区VL1融合在IL15的N端；
链2：IL15Ra的N端融合了结合抗原的抗体可变区VH1，C端融合FC异源二聚体；
链3：结合另一个抗原的抗体可变区VH2融合CH1片段,C端融合FC异源二聚体，组成抗体重链；
链4：结合另一个抗原的抗体可变区VL2的C端融合CL片段，组成抗体轻链。
作为优选，本发明的双特异抗体分子，还具有这样的特征：其中，IL15及IL15Ra复合物包括：1)IL15及其能结合IL15Ra的突变，截短及各种衍生物；2)IL15Ra及其能结合IL15的突变，截短及各种衍生物；包括但不限于权利要求3所述突变以及本实施例中所述序列SEQ ID No.1、SEQ ID No.2、SEQ ID No.3、SEQ ID No.4、SEQ ID No.5、SEQ ID No.6、SEQID No.7、SEQ ID No.8、SEQ ID No.9。
作为优选，本发明的双特异抗体分子，还具有这样的特征：人FC片段,是FC异源二聚体形式，FC异源二聚体包含但不限于权利要求3所述突变以及序列SEQ ID No.94、SEQ IDNo.95。
作为优选，本发明的双特异抗体分子，还具有这样的特征：人FC片段，包括但不限于Human IgG1 FC、Human IgG2 FC、Human IgG3 FC、Human IgG4 FC及其变异体。其中一条链能结合proteinA；另一条链可选择不结合proteinA，包括但不限于突变H435R或H435R/Y436F，根据EU计数。
作为优选，本发明的双特异抗体分子，还具有这样的特征：结合抗原的TAA1和/或TAA2抗体包括但不限于CD3、CD20、CD19、CD38、CD40、CD30、CD33、CD52、slamf7、GD2、CD24、CD47、CD133、CD239、CD276、PD-1等表面抗原，CEA、Epcam、Trop2、TAG72、MUC1、MUC16、mesothelin、folr1、CLDN18.2、PDL1、EGFR、EGFR VIII、C-MET、HER2、FGFR2、FGFR3、PSMA、PSCA、EphA2、ADAM17、17-A1、NKG2D ligands、MCSP、LGR5、SSEA3、SLC34A2、BCMA、GPNMB等靶点的抗体，包含但不限于序列SEQ ID No.18、SEQ ID No.19、SEQ ID No.20、SEQ ID No.21、SEQ ID No.22、SEQ ID No.23、SEQ ID No.24、SEQ ID No.25、SEQ ID No.26、SEQ IDNo.27、SEQ ID No.28、SEQ ID No.29、SEQ ID No.30、SEQ ID No.31、SEQ ID No.32、SEQ IDNo.33、SEQ ID No.34、SEQ ID No.35、SEQ ID No.36、SEQ ID No.37、SEQ ID No.38、SEQ IDNo.39、SEQ ID No.40、SEQ ID No.41、SEQ ID No.42、SEQ ID No.43、SEQ ID No.44、SEQ IDNo.45、SEQ ID No.46、SEQ ID No.47、SEQ ID No.48、SEQ ID No.49、SEQ ID No.50、SEQ IDNo.51、SEQ ID No.52、SEQ ID No.53、SEQ ID No.54、SEQ ID No.55、SEQ ID No.56、SEQ IDNo.57、SEQ ID No.58、SEQ ID No.59、SEQ ID No.60、SEQ ID No.61、SEQ ID No.62、SEQ IDNo.63、SEQ ID No.64、SEQ ID No.65、SEQ ID No.66、SEQ ID No.67、SEQ ID No.68、SEQ IDNo.69、SEQ ID No.70、SEQ ID No.71、SEQ ID No.72、SEQ ID No.73、SEQ ID No.74、SEQ IDNo.75、SEQ ID No.76、SEQ ID No.77、SEQ ID No.78、SEQ ID No.79、SEQ ID No.80、SEQ IDNo.81、SEQ ID No.82、SEQ ID No.83、SEQ ID No.84、SEQ ID No.85、SEQ ID No.86、SEQ IDNo.87、SEQ ID No.88、SEQ ID No.89、SEQ ID No.90、SEQ ID No.91、SEQ ID No.92、SEQ IDNo.93。
作为优选，本发明的双特异抗体分子，还具有这样的特征：其中，TAA为Her2/Her3；FC为IgG1异源二聚体；链1和链2的IL15及IL15Ra复合物融合蛋白有二硫键形成，包含但不限于本实施例中所述序列SEQ ID No.10、SEQ ID No.11、SEQ ID No.12、SEQ ID No.13。
作为优选，本发明的双特异抗体分子，还具有这样的特征：其中，TAA为CD38/CS1；FC为IgG1异源二聚体；链1和链2的IL15及IL15Ra复合物融合蛋白有二硫键形成，包含但不限于本实施例中所述序列SEQ ID No.14、SEQ ID No.15、SEQ ID No.16、SEQ ID No.17。
本发明还提供上述任意一项所述的双特异抗体分子在制备癌症、感染和免疫调节疾病的药物中的应用。
本发明还提供上述任意一项所述的双特异抗体分子，在制备抑制肿瘤生长的药物中的应用。
进一步，本发明的应用，其中，所述癌症或肿瘤包括：骨髓癌、淋巴癌、白血病、小细胞肺癌、非小细胞肺癌、黑素瘤、基底细胞皮肤癌、鳞状细胞皮肤癌、隆突性皮肤纤维肉瘤、梅克尔细胞癌、成胶质细胞瘤、胶质瘤、肉瘤、间皮瘤，和骨髓增生异常综合症；
或者包括来自如下部位的癌症或肿瘤：结直肠、乳腺、卵巢、胰腺、胃、前列腺、肾、宫颈、甲状腺、子宫内膜、子宫、膀胱、神经内分泌、头部颈部、肝、鼻咽、睾丸。
本发明提供的特异性结合HER2的双特异性抗体，其包含对HER2胞外域II特异性的第一抗原结合位点和对HER2胞外域IV特异性的第二抗原结合位点，其中该双特异性抗体对HER2胞外域II和IV二者均是单价的。
发明的有益效果
本发明的双特异性抗体与HER2蛋白的亲和力高，本发明的双特异性抗体对肿瘤细胞的增殖抑制作用强。
附图说明
图1是双特异抗体的结构形式一。
图2是双特异抗体的结构形式二。
图3是双特异抗体的结构形式三。
图4是双特异抗体的结构形式四。
图5是双特异抗体的结构形式五。
图6是双特异抗体的结构形式六。
图7是双特异抗体的结构形式七。
图8是双特异抗体的结构形式八。
图9是SDS-PAGE电泳分析QP34563457蛋白还原与非还原条带。
图10是HPLC-SEC分析QP34563457蛋白纯度。
图11是QP34563457蛋白的肽图分析，图11a显示QP34563457蛋白对于TrastuzumabVL-IL15链36％的序列覆盖率，图11b显示QP34563457蛋白对于Trastuzumab VH-IL15Ra-FC链56％的序列覆盖率，图11c显示QP34563457蛋白对于pertuzumab VL-CL链64％的序列覆盖率，图11d显示QP34563457蛋白对于pertuzumab VH-CH1-FC链56％的序列覆盖率。
图12是ELISA检测QP34563457等分子结合Her2-FC融合蛋白。
图13是ELISA检测QP34563457等分子结合Her2M2-FC融合蛋白。
图14是ELISA检测QP34623463等分子结合CD38蛋白。
图15是ELISA检测QP34623463等分子结合CS1蛋白。
图16是FACS检测QP34563457等分子结合SK-BR-3细胞。
图17是FACS检测QP34563457等分子结合BT-474细胞。
图18是FACS检测QP34563457等分子结合SK-OV-3细胞。
图19是QP34563457等分子对人乳腺癌细胞SK-BR-3的增殖抑制曲线。
图20是QP34563457等分子对人乳腺癌细胞BT-474的增殖抑制曲线。
图21是QP34563457等分子对人卵巢癌细胞SK-OV-3的增殖抑制曲线。
图22是QP34563457等介导的PBMC杀伤人乳腺癌细胞SK-BR-3细胞。
图23是QP34563457等介导的PBMC杀伤人乳腺癌细胞BT-474细胞。
图24是QP34563457等介导的PBMC杀伤人卵巢癌细胞SK-OV-3细胞。
图25是检测QP34563457等分子对Mo7e细胞增殖实验。
图26是SK-OV-3体内药效模型肿瘤生长曲线。
具体实施方式
以下实施方式中未注明具体条件的实验方法，通常是按照常规条件，或按照原料或商品制造厂商所建议的条件和方法进行。如分子克隆，实验室手册，冷泉港实验室，当代分子生物学方法，细胞生物学等等。未注明具体来源的试剂，为市场购买的常规试剂。
实施例1：融合蛋白的克隆和表达
蛋白序列如下：
anti Her2 VL-IL15 SEQ ID NO:10
DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGGGGSGGGGSGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISCESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS
anti Her2 VH-IL15Ra-FC(Knob)SEQ ID NO:11
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTCSLTECVLNKATNVAHWTTPSLKCIRGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
anti Her3 VL-CLSEQ ID NO:12
DIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
anti Her3 VH-CH1-CH2-CH3(Hole)SEQ ID NO:13
EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRYTQKSLSLSPGK
anti CS1 VL-IL15 SEQ ID NO:14
DIQMTQSPSSLSASVGDRVTITCKASQDVGIAVAWYQQKPGKVPKLLIYWASTRHTGVPDRFSGSGSGTDFTLTISSLQPEDVATYYCQQYSSYPYTFGQGTKVEIKGGGGSGGGGSGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISCESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS
anti CS1 VH-IL15Ra-FC(Knob)SEQ ID NO:15
EVQLVESGGGLVQPGGSLRLSCAASGFDFSRYWMSWVRQAPGKGLEWIGEINPDSSTINYAPSLKDKFIISRDNAKNSLYLQMNSLRAEDTAVYYCARPDGNYWYFDVWGQGTLVTVSSGGGGSGGGGSGGGGSITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTCSLTECVLNKATNVAHWTTPSLKCIRGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKanti CD38 VL-CL SEQ ID NO:16
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
anti CD38 VH-CH1-CH2-CH3(Hole)SEQ ID NO:17
EVQLLESGGGLVQPGGSLRLSCAVSGFTFNSFAMSWVRQAPGKGLEWVSAISGSGGGTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYFCAKDKILWFGEPVFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRYTQKSLSLSPGK
克隆构建方法：
克隆设计如表1所示：
如表1所示，分别构建载体编码anti Her2 VL(Tmab)-IL15(SEQ ID NO:10)、antiHer2 VH(Tmab)-IL15Ra-FC(Knob)(SEQ ID NO:11)、anti CS1 VL-IL15(SEQ ID NO:14)、anti CS1 VH-IL15Ra-FC(Knob)(SEQ ID NO:15)。质粒含有DHFR作为筛选标记,可以用于稳定株筛选；构建载体编码抗肿瘤特异性抗原抗体的轻链序列(序列)及抗肿瘤特异性抗原抗体的重链序列(其中Fc含有Hole突变即(T366S,L368A,Y407V)anti Her2 VL(Pmab)-CL(SEQID NO:12)、anti Her2 VH(Pmab)-CH1-CH2-CH3(Hole)(SEQ ID NO:13)、anti CD38 VL-CL(SEQ ID NO:16)、anti CD38VH-CH1-CH2-CH3(Hole)(SEQ ID NO:17)，质粒含有GS作为筛选标记,可以用于稳定株筛选。蛋白分子的结构示意图如图1所示。在其它的实施方式中，蛋白分子可以为其它结构形式，分别如图2至图8所示。
表1：克隆设计构建
Figure BDA0002825622600000141
如表2所示，设计构建对照抗体Trastuzumab，Pertuzumab，Elotuzumab，Daratumumab。
表2：对照蛋白序列及编号
Figure BDA0002825622600000151
瞬转表达目标分子
接种ExpiCHO-S细胞到FortiCHO培养基中(Gibco，A1148301)额外加入8mMGlutaMax，37℃，120rpm，8％CO2培养。转染前一天，将ExpiCHO-S细胞密度调整为3*10E6/mL，置于摇床中，37℃，120rpm，8％CO2培养；转染当天，取样，计数，将细胞密度稀释为6*10E6/ml，每瓶40ml，置于125ml的摇瓶中；质粒QD3208，QD3209各20ug与4.8mL Opti MEM混合，加入120ul Polyplus-FectoPRO转染试剂，将DNA和转染试剂混合均匀后，置于室温10min，将混合物缓慢置于细胞中，混合均匀后，置于摇床培养。培养过程中，分别在第1，4，6，8天每瓶补加2mL Feed PFF05(OPM，F81279-001)及1m的30％葡萄糖溶液。转染第一天，将温度降为32℃，CO2浓度降为5％。13天收样，8000rpm离心20min，取上清，待纯化。
实施例2：融合蛋白的纯化
Protein A亲和层析纯化
用平衡液过柱，至少3CV，实际体积20ml，确保最终仪器中流出的溶液pH和电导与平衡液一致，流速1ml/min；将离心后培养液上清过柱，上样40ml，流速0.33ml/min；用平衡液过柱，至少3CV，实际体积20ml，确保最终仪器中流出的溶液pH和电导与平衡液一致，流速0.33ml/min；用洗脱液过柱，UV280上升至15mAU时开始收集洗脱峰(PAC-EP)，UV280下降至15mAU时停止收集，流速1ml/min。样品收集完成后，用pH调节液将PAC-EP调至中性。
CH1-XL亲和层析
将Protein A处理后样品8000rpm×15min离心，取上清；用平衡液过柱，至少3CV，实际体积20ml，确保最终仪器中流出的溶液pH和电导与平衡液一致，流速1ml/min；将离心后上清经上样环过柱，流速0.33ml/min；用平衡液过柱，至少3CV，实际体积20ml，确保最终仪器中流出的溶液pH和电导与平衡液一致，流速0.33ml/min；用洗脱液过柱，UV280上升至10mAU时开始收集洗脱峰(PAC-EP)，UV280下降至10mAU时停止收集，流速1ml/min。样品收集完成后，用pH调节液将CH1-EP调至中性。
PNGase F酶切：
取39.5μl样品，加入1.98μl 10％SDS和1.58μl 1M DTT，混合均匀后100℃煮沸10分钟；待样品冷却，加入4.8μl 10％NP-40和1.0μl PNGase F，混匀后4℃水浴过夜；将过夜后样品75℃加热10分钟热失活，样品进行SDS-PAGE电泳及SEC，检测蛋白纯度。
SDS-PAGE电泳分析:
酶切前和酶切后样品各取5μg，用1×PBS将体积定容至48μl，再加入12μl5×Protein Loading Dye，95℃加热10分钟。在电泳槽中倒入pH为8.3的Tris-HCl缓冲液(含0.1％SDS)，用移液枪向样品槽中加入样品，每个样品槽加样60μl。将电压设置为140V，时间设置为100分钟即可开始电泳。电泳结束后取出凝胶，浸泡在染色液中30分钟，再用脱色液脱色，直至蛋白质区条带清晰。图9是SDS-PAGE电泳分析QP34563457蛋白还原与非还原条带。图10是HPLC-SEC分析QP34563457蛋白纯度。
实施例3：蛋白QP34563457肽图分析
本实施例利用LC-MS来鉴定QP34563457蛋白的氨基酸序列。
QP34563457蛋白变性还原后，加入胰蛋白酶酶解后用LC-MS/MS分析，(LC仪器Agilent 1290 Infinity II，柱子Agilent Peptide Plus column。MS仪器Agilent 6545Q-TOF)，将得到的数据采用Peaks进行搜库，Peaks搜库结果经严格卡值过滤后得到可信肽段，实现了对于QP34563457蛋白Trastuzumab VL-IL15链36％的序列覆盖率(图3a，灰色为鉴定到的氨基酸序列)，Trastuzumab VH-IL15Ra-FC链56％的序列覆盖率(图3b，灰色为鉴定到的氨基酸序列)，pertuzumab VL-CL链64％的序列覆盖率(图3c，灰色为鉴定到的氨基酸序列)，pertuzumab VH-CH1-FC链56％的序列覆盖率(图3d，灰色为鉴定到的氨基酸序列)。
结果如图11a、图11b、图11c和图11d所示：图11a:Trastuzumab VL-IL15链肽图分析；图11b：Trastuzumab VH-IL15Ra-FC链肽图分析；图11c:pertuzumab VL-CL链肽图分析；图11d:pertuzumab VH-CH1-FC链肽图分析。
结果：通过LC-MS进行肽图分析，分别找到了QP34563457分子中各个片段，包括，Trastuzumab VL、IL15、Trastuzumab VH、IL15Ra、FC、pertuzumab VL、CL、pertuzumab VH、CH1、FC等。
实施例4：ELISA检测QP34563457结合Her2及其突变体活性
只结合帕托珠单抗Pertuzumab的Her2变体蛋白的设计.Matthew C.Franklin在Cancer cell上发表了帕托珠单抗Fab与Her2胞外结构的复合物结构。该团队同时用丙氨酸扫描的方法研究了Her2哪些关键氨基酸会影响与帕托珠单抗Fab的结合。将只结合帕托珠单抗Pertuzumab而不结合曲妥珠单抗trastuzumab的Her2变体蛋白命名为Her2M2(蛋白编号QP3732)。Her2wt(蛋白编号QP3731)及Her2M2(蛋白编号QP3732)的氨基酸序列分别如下：
QP3731:(Her2ECD-FC)
MEFGLSWLFLVAILKGVQCTQVCTGTDMKLRLPASPETHLDMLRHLYQGCQVVQGNLELTYLPTNASLSFLQDIQEVQGYVLIAHNQVRQVPLQRLRIVRGTQLFEDNYALAVLDNGDPLNNTTPVTGASPGGLRELQLRSLTEILKGGVLIQRNPQLCYQDTILWKDIFHKNNQLALTLIDTNRSRACHPCSPMCKGSRCWGESSEDCQSLTRTVCAGGCARCKGPLPTDCCHEQCAAGCTGPKHSDCLACLHFNHSGICELHCPALVTYNTDTFESMPNPEGRYTFGASCVTACPYNYLSTDVGSCTLVCPLHNQEVTAEDGTQRCEKCSKPCARVCYGLGMEHLREVRAVTSANIQEFAGCKKIFGSLAFLPESFDGDPASNTAPLQPEQLQVFETLEEITGYLYISAWPDSLPDLSVFQNLQVIRGRILHNGAYSLTLQGLGISWLGLRSLRELGSGLALIHHNTHLCFVHTVPWDQLFRNPHQALLHTANRPEDECVGEGLACHQLCARGHCWGPGPTQCVNCSQFLRGQECVEECRVLQGLPREYVNARHCLPCHPECQPQNGSVTCFGPEADQCVACAHYKDPPFCVARCPSGVKPDLSYMPIWKFPDEEGACQPCPINCTHSCVDLDDKGCPAEQRASPLTEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK*
QP3732(Her2M2-FC):
MEFGLSWLFLVAILKGVQCTQVCTGTDMKLRLPASPETHLDMLRHLYQGCQVVQGNLELTYLPTNASLSFLQDIQEVQGYVLIAHNQVRQVPLQRLRIVRGTQLFEDNYALAVLDNGDPLNNTTPVTGASPGGLRELQLRSLTEILKGGVLIQRNPQLCYQDTILWKDIFHKNNQLALTLIDTNRSRACHPCSPMCKGSRCWGESSEDCQSLTRTVCAGGCARCKGPLPTDCCHEQCAAGCTGPKHSDCLACLHFNHSGICELHCPALVTYNTDTFESMPNPEGRYTFGASCVTACPYNYLSTDVGSCTLVCPLHNQEVTAEDGTQRCEKCSKPCARVCYGLGMEHLREVRAVTSANIQEFAGCKKIFGSLAFLPESFDGDPASNTAPLQPEQLQVFETLEEITGYLYISAWPDSLPDLSVFQNLQVIRGRILHNGAYSLTLQGLGISWLGLRSLRELGSGLALIHHNTHLCFVHTVPWDQLFRNPHQALLHTANRPEDECVGEGLACHQLCARGHCWGPGPTQCVNCSQFLRGQECVEECRVLQGLPREYVNARHCLPCHPECQPQNGSVTCFGPAADQCVACAHYKDPAFCVARCPSGVKPDLSYMPIWKFPDEEGACQPCPINCTHSCVDLDDKGCPAEQRASPLTEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK*
检测试剂：milk(BD,232100)，PBS(生工，B548117-0500)；HRP-anti human IgG(H+L)(jackson，109-035-088)；TMB(洛阳佰奥通实验材料中心，C060201)；Elisa板(costa,9018)1×PBS缓冲液：称取NaCl 8.00g、KCl 0.20g、Na2HPO4·12H20 2.9g、KH2PO4 0.2g至800mL ddH2O中溶解，充分溶解后定容至1L，调节pH至7.4，高温灭菌备用。或购买商品化10×、20×的PBS溶液稀释至1×PBS缓冲液使用。封闭液：称取5g的milk至PBS中,封闭液需现配现用。终止液(1mol/LH2SO4)：取109mL 98％的浓H2SO4缓慢滴加至2000mL ddH2O中。TMB37℃显色10min,置于摇床中(120rpm)，100μl/well；
实验步骤：Her2及其变体蛋白QP3731及QP3732包板，1ug/ml，4度过夜；PBS洗3遍。按200μL/孔加入封闭液5％milk，37度孵育1h。封闭结束后，PBS洗3遍，孵育样品，按照20ug/ml 5倍稀释，共稀释7个梯度最后一孔稀释100倍,100μl/well充分混匀后，孵育1h,PBST洗3遍；加酶标抗体：孵育HRP-anti human Fab抗体，按照1:5000稀释比例,100μl/孔，充分混匀后，孵育1h，PBST洗6遍。加底物显色液：按100μL/孔用量加底物显色液TMB，置于摇床中，200rpm,35℃避光显色10min。
终止：显色完成后，按100μL/孔用量快速加入终止液终止反应。检测：在酶标仪上，测其A450nm的OD值，使用graphpad prism软件分析结果。结果如图12，图13所示。
实施例5：ELISA检测QP34623463结合CD38及CS1活性
检测试剂：milk(BD,232100)，PBS(生工，B548117-0500)；HRP-anti human IgG(H+L)(jackson，109-035-088)；TMB(洛阳佰奥通实验材料中心，C060201)；Elisa板(costa,9018)1×PBS缓冲液：称取NaCl 8.00g、KCl 0.20g、Na2HPO4·12H20 2.9g、KH2PO4 0.2g至800mL ddH2O中溶解，充分溶解后定容至1L，调节pH至7.4，高温灭菌备用。或购买商品化10×、20×的PBS溶液稀释至1×PBS缓冲液使用。封闭液：称取5g的milk至PBS中,封闭液需现配现用。终止液(1mol/LH2SO4)：取109mL 98％的浓H2SO4缓慢滴加至2000mL ddH2O中。TMB37℃显色10min,置于摇床中(120rpm)，100μl/well；
实验步骤：CD38-FC和CS1-FC融合蛋白分别包板，1ug/ml，4度过夜；PBS洗3遍。按200μL/孔加入封闭液5％milk，37度孵育1h。封闭结束后，PBS洗3遍，孵育样品，按照20ug/ml5倍稀释，共稀释7个梯度最后一孔稀释100倍，100μl/well充分混匀后，孵育1h,PBST洗3遍；加酶标抗体：孵育HRP-anti human Fab抗体，按照1:5000稀释比例，100μl/孔，充分混匀后，孵育1h，PBST洗6遍。加底物显色液：按100μL/孔用量加底物显色液TMB，置于摇床中，200rpm,35℃避光显色10min。终止：显色完成后，按100μL/孔用量快速加入终止液终止反应。检测：在酶标仪上，测其A450nm的OD值，使用graphpad prism软件分析结果。结果如图14和图15所示。
实施例6：FACS检测QP34563457结合Her2高表达细胞
实验目的：FACS检测QP34563457结合Her2高表达细胞
实验材料：
BT474细胞(人乳腺癌细胞株)，购于中国医学科学院基础医学研究所细胞资源中心。
SK-BR-3细胞(人乳腺癌细胞株)，购于中国医学科学院基础医学研究所细胞资源中心。SK-OV-3细胞(人卵巢癌细胞株)，购于中国医学科学院基础医学研究所细胞资源中心。
实验方法：BT474细胞使用含有10％FBS，0.11g/L丙酮酸钠和2.5g/L葡萄糖的RPMI1640(Gibco)培养基，SK-BR-3细胞使用含有10％FBS的DMEM培养基(Gibco)，SK-OV-3细胞使用含有10％FBS的McCoy′s 5a培养基(Gibco)。于37℃5％CO2培养箱中培养，胰酶消化收集细胞，按100000个细胞每孔接种于96孔板中，2％FBS/PBS冰上封闭1h，孵育不同浓度蛋白QP34563457及对照蛋白，冰上1h，PBS洗3遍，孵育PE-anti human FC(1：200)稀释，PBS洗3遍，200微升PBS重悬，FACS读取平均荧光值，使用graphpad prism软件分析结果。结果如图16、图17和图18所示。
实施例7：Her2双特异抗体介导的增殖抑制
实验目的：确定Her2双特异抗体对肿瘤细胞的增殖抑制作用
实验材料：
BT474细胞(人乳腺癌细胞株)，购于中国医学科学院基础医学研究所细胞资源中心。SK-BR-3细胞(人乳腺癌细胞株)，购于中国医学科学院基础医学研究所细胞资源中心。SK-OV-3细胞(人卵巢癌细胞株)，购于中国医学科学院基础医学研究所细胞资源中心。
实验试剂：细胞增殖及毒性检测试剂盒(CCK-8)，购自美仑生物，货号MA0218；人IgG，购自Sigma，货号I4506；其他抗体来自内部制备。
实验方法：BT474细胞使用含有10％FBS，0.11g/L丙酮酸钠和2.5g/L葡萄糖的RPMI1640(Gibco)培养基，SK-BR-3细胞使用含有10％FBS的DMEM培养基(Gibco)，SK-OV-3细胞使用含有10％FBS的McCoy′s 5a培养基(Gibco)。于37℃，5％CO2培养箱中培养，胰酶消化收集细胞，离心后用含有1％FBS的培养基重悬，按10000个细胞，50μl每孔接种于96孔板中，37℃贴壁培养3小时；将各个待测抗体用培养基按3倍梯度稀释后，50μl每孔与细胞悬液混合均匀，37℃，5％CO2培养箱中培养3天；将CCK-8试剂按10μl每孔加入待测的96孔板中，37℃，5％CO2培养箱中孵育2小时；将96孔板取出，于酶标仪中检测450nm波长的吸光值，计算细胞活力值，与样品浓度对数作图，使用graphpad prism软件分析结果。通过四参数拟合出细胞增殖抑制曲线。结果如图19、图20和图21所示。
实施例8：HER2双特异抗体介导的ADCC作用
实验目的：确定HER2双特异抗体的ADCC作用
实验材料：PBMC购买自上海赛笠生物科技有限公司，BT474细胞(人乳腺癌细胞株)，购于中国医学科学院基础医学研究所细胞资源中心。SK-BR-3细胞(人乳腺癌细胞株)，购于中国医学科学院基础医学研究所细胞资源中心。SK-OV-3细胞(人卵巢癌细胞株)，购于中国医学科学院基础医学研究所细胞资源中心，Cytotox96 non-radioactivecytotoxicity assay检测试剂盒购自Promega(G1780)。
实验步骤：复苏PBMC，收集细胞待第二天备用。准备靶细胞:SK-BR-3，BT-474，SK-OV-3用胰酶消化，1000rpm 5min。PBS清洗2次后，铺96孔板，20000个每孔，50μl每孔，37℃5％CO2孵育2h。准备抗体:用培养基(RPMI1640含10％low-IgG FBS)1:4梯度稀释抗体(80ug/ml—0.000512ug/ml,0ug/ml)10个浓度。加入上面稀释各浓度的抗体50μl每孔，37℃孵育15min。准备PBMC:将第一天复苏的PBMC离心，用培养基(RPMI1640含10％low-IgG FBS)重悬，计数。按PBMC:Target cell＝30:1，加入上面的孔板中50μl每孔，37度孵育4小时。LDH检测:对于最大释放组和体积矫正组，提前45min加入10μl裂解液,继续放置培养箱培养。培养4h后，吸取50μl上清至酶标板中,按照Cytotox96 non-radioactive cytotoxicityassay检测试剂盒(Promega，G1780)说明书，加入50μl reagent。室温，避光反应30min后，加入50μl stop solution.490nm吸光值读数(加入stop solution后1h内完成读数)。计算：杀伤百分比＝(样品释放-靶细胞自发-效应细胞自发释放)/(靶细胞最大释放-靶细胞自发释放)*100。将自发释放(对应于在没有抗体的情况下与效应细胞一起温育的靶细胞)定义为0％细胞毒性，并将最大释放(用1％Triton X-100裂解的靶细胞)定义为100％细胞毒性。结果如图22、图23和图24所示。
实施例9：Mo7e细胞增殖实验
实验试剂：Mo7e细胞(人巨细胞白血病细胞株)，购于中国医学科学院基础医学研究所细胞资源中心,细胞增殖及毒性检测试剂盒(CCK-8)，购自美仑生物，货号MA0218；重组人GM-CSF，购自perprotech，货号300-03；人IgG，购自Sigma，货号I4506；其他抗体来自内部制备。
实验方法：Mo7e细胞使用含有10％FBS，2mM L-谷氨酰胺和8ng/ml GM-CSF的RPMI1640培养基于37℃，5％CO2培养箱中培养；收集Mo7e细胞，800rpm离心5分钟倒掉上清，细胞用不含有GM-CSF的RPMI1640培养基洗涤2遍；用无GM-CSF的RPMI1640培养基重悬细胞并计数，将细胞按2×104个，80μl每孔接种于96孔板中，37℃，5％CO2培养箱中培养1小时；将各个待测抗体用培养基按4倍梯度稀释后，20μl每孔与细胞悬液混合均匀，37℃，5％CO2培养箱中培养3天；将CCK-8试剂按10μl每孔加入待测的96孔板中，37℃，5％CO2培养箱中孵育4小时；将96孔板取出，于酶标仪中检测450nm波长的吸光值。实验结果如图25所示
实施例10：动物药效实验
在高表达HER2的人卵巢癌细胞SK-OV-3模型中评价QP34563457对肿瘤生长的抑制作用。
SK-OV-3细胞培养在含10％胎牛血清的McCoy’s 5A+10％FBS培养液中。收集指数生长期的SK-OV-3细胞，PBS重悬至适合浓度接种。每只BALB/C-nude小鼠于右侧背部皮下接种1×106SK-OV-3细胞，定期观察肿瘤生长情况，待肿瘤生长至平均体积约50mm3时根据肿瘤大小和小鼠体重随机分组给药，第一次给药当天定义为D0。给药方案如下：
表3：给药方案
Figure BDA0002825622600000211
给药体积为10ul/g体重。
每次给药观察、记录小鼠体重和肿瘤体积，用游标卡尺测量肿瘤长短径，并按公式肿瘤体积(mm3)＝0.5×(a×b2)计算、记录肿瘤生长情况，绘制肿瘤生长曲线。最后一次给药后观察6天，结束实验，并记录实验数据。
结果显示，Tastuzumab、Pertuzumab、Trastuzumab+Pertuzumab及QP34563457均能明显抑制SK-OV-3肿瘤生长，6次给药后肿瘤发生消退(TGItv＞100％)。结果如图26和表4所示。
表4：SK-OV-3体内药效数据及分析
Figure BDA0002825622600000221
Sequence：
SEQ ID NO:1 IL15
NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS
SEQ ID NO:2 IL-15(L52C)
NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISCESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS
SEQ ID NO:3 IL15Ra
ECDITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTT
SEQ ID NO:4 IL15Ra Sushi 65">sp|Q13261|31-95
ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR"
SEQ ID NO:5 IL15Ra Sushi 73
ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQR
SEQ ID NO:6 IL15Ra Sushi 77
ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPP
SEQ ID NO:7 IL15Ra Sushi 86
ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVT
SEQ ID NO:8 IL15Ra Sushi 102
ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAAS
SEQ ID NO:9 IL15Ra-sushi(S40C)
ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTCSLTECVLNKATNVAHWTTPSLKCIR
SEQ ID NO:10anti Her2 VL-IL15
DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGGGGSGGGGSGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISCESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS
SEQ ID NO:11 anti Her2 VH-IL15Ra-FC(Knob)
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTCSLTECVLNKATNVAHWTTPSLKCIRGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID NO:12 anti Her3 VL-CL
DIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
SEQ ID NO:13anti Her3 VH-CH1-CH2-CH3(Hole)
EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRYTQKSLSLSPGK
SEQ ID NO:14anti CS1 VL-IL15
DIQMTQSPSSLSASVGDRVTITCKASQDVGIAVAWYQQKPGKVPKLLIYWASTRHTGVPDRFSGSGSGTDFTLTISSLQPEDVATYYCQQYSSYPYTFGQGTKVEIKGGGGSGGGGSGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISCESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS
SEQ ID NO:15anti CS1 VH-IL15Ra-FC(Knob)
EVQLVESGGGLVQPGGSLRLSCAASGFDFSRYWMSWVRQAPGKGLEWIGEINPDSSTINYAPSLKDKFIISRDNAKNSLYLQMNSLRAEDTAVYYCARPDGNYWYFDVWGQGTLVTVSSGGGGSGGGGSGGGGSITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTCSLTECVLNKATNVAHWTTPSLKCIRGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID NO:16anti CD38 VL-CL
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
SEQ ID NO:17 anti CD38 VH-CH1-CH2-CH3(Hole)
EVQLLESGGGLVQPGGSLRLSCAVSGFTFNSFAMSWVRQAPGKGLEWVSAISGSGGGTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYFCAKDKILWFGEPVFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRYTQKSLSLSPGK
SEQ ID NO:18 VH
QVQLQQSGPELEKPGASVKISCKASGYSFTGYTMNWVKQSHGKSLEWIGLITPYNGASSYNQKFRGKATLTVDKSSSTAYMDLLSLTSEDSAVYFCARGGYDGRGFDYWGSGTPVTVSS
SEQ ID NO:19 VL
DIELTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPGRFSGSGSGNSYSLTISSVEAEDDATYYCQQWSKHPLTFGSGTKVEIK
SEQ ID NO:20 VH
EVQLLESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDMGWGSGWRPYYYYGMDVWGQGTTVTVSS
SEQ ID NO:21 VL
ELQMTQSPSSLSASVGDRVTITCRTSQSISSYLNWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQPEDSATYYCQQSYDIPYTFGQGTKLEIK
SEQ ID NO:22 VH
QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSA
SEQ ID NO:23 VL
DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELK
SEQ ID NO:24 VH
XVQLQESGPGLVRPSQTLSLTCTVSGYSITSDHAWSWVRQPPGRGLEWIGYISYSGITTYNPSLKSRVTMLRDTSKNQFSLRLSSVTAADTAVYYCARSLARTTAMDYWGQGSLVTVSS
SEQ ID NO:25 VL
DIQMTQSPSSLSASVGDRVTITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGNTLPYTFGQGTKVEIK
SEQ ID NO:26 VH
EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSS
SEQ ID NO:27 VL
DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIK
SEQ ID NO:28 VH
EVQLLQSGPELEKPGASVMISCKASGSSFTGYNMNWVRQNIGKSLEWIGAIDPYYGGTSYNQKFKGRATLTVDKSSSTAYMHLKSLTSEDSAVYYCVSGMEYWGQGTSVTVSS
SEQ ID NO:29 VL
EIVMTQSPATLSVSPGERATLSCRSSQSLVHRNGNTYLHWYLQKPGQSPKLLIHKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPPLTFGAGTKLELK
SEQ ID NO:30 VH
QVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYRMHWVRQAPGQGLEWIGYINPSTGYTEYNQKFKDKATITADESTNTAYMELSSLRSEDTAVYYCARGGGVFDYWGQGTLVTVSS
SEQ ID NO:31 VL
DIQMTQSPSTLSASVGDRVTITCSASSSISYMHWYQQKPGKAPKLLIYTTSNLASGVPARFSGSGSGTEFTLTISSLQPDDFATYYCHQRSTYPLTFGQGTKVEVK
SEQ ID NO:32 VH
QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSS
SEQ ID NO:33 VL
EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIK
SEQ ID NO:34 VH
QVQLVQSGAEVKKPGASVKVSCKASGYTFTRYGISWVRQAPGQGLEWMGWISTYSGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARRQLYFDYWGQGTLVTVSS
SEQ ID NO:35 VL
EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWFQQKPGQAPRPLIYDASTRATGVPARFSGSGSGTDFTLTISSLQSEDFAVYYCQQYDNWPLTFGGGTKVEIK
SEQ ID NO:36 VH
EVQLVESGGGLVQPGGSLRLSCAASGFDFSRYWMSWVRQAPGKGLEWIGEINPDSSTINYAPSLKDKFIISRDNAKNSLYLQMNSLRAEDTAVYYCARPDGNYWYFDVWGQGTLVTVSS
SEQ ID NO:37 VL
DIQMTQSPSSLSASVGDRVTITCKASQDVGIAVAWYQQKPGKVPKLLIYWASTRHTGVPDRFSGSGSGTDFTLTISSLQPEDVATYYCQQYSSYPYTFGQGTKVEIK
SEQ ID NO:38 VH
EVQLVESGGDLVQPGRSLRLSCAASGFIFSNYGMSWVRQAPGKGLEWVATISSASTYSYYPDSVKGRFTISRDNAKNSLYLQMNSLRVEDTALYYCGRHSDGNFAFGYWGQGTLVTVSS
SEQ ID NO:39 VL
DVLMTQSPLSLPVTPGEPASISCRSSRNIVHINGDTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSLLPWTFGQGTKVEIK
SEQ ID NO:40 VH
QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSS
SEQ ID NO:41 VL
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIK
SEQ ID NO:42 VH
QVQLVQSGAEVKKPGSSVKVSCKASGYAFSYSWINWVRQAPGQGLEWMGRIFPGDGDTDYNGKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARNVFDGYWLVYWGQGTLVTVSS
SEQ ID NO:43 VL
DIVMTQTPLSLPVTPGEPASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLVSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCAQNLELPYTFGGGTKVEIK
SEQ ID NO:44 VH
EVQLLESGGGLVQPGGSLRLSCAVSGFTFNSFAMSWVRQAPGKGLEWVSAISGSGGGTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYFCAKDKILWFGEPVFDYWGQGTLVTVSS
SEQ ID NO:45 VL
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPTFGQGTKVEIK
SEQ ID NO:46 VH
QVQLQESGPGLVKPSQTLSLTCTVSGGSISSGDYYWSWIRQPPGKGLEWIGYIYYSGSTDYNPSLKSRVTMSVDTSKNQFSLKVNSVTAADTAVYYCARVSIFGVGTFDYWGQGTLVTVSS
SEQ ID NO:47 VL
EIVMTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCHQYGSTPLTFGGGTKAEIK
SEQ ID NO:48 VH
EVQLVQSGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARVTDAFDIWGQGTMVTVSS
SEQ ID NO:49 VL
DIQMTQSPSSVSASIGDRVTITCRASQGIDNWLGWYQQKPGKAPKLLIYDASNLDTGVPSRFSGSGSGTYFTLTISSLQAEDFAVYFCQQAKAFPPTFGGGTKVDIK
SEQ ID NO:50 VH
EVQLVESGGGLVKPGGSLKLSCAASGFKFSRYAMSWVRQAPGKRLEWVATISSGGSYIYYPDSVKGRFTISRDNVKNTLYLQMSSLRSEDTAMYYCARRDYDLDYFDSWGQGTLVTVSS
SEQ ID NO:51 VL
DIQMTQSPSSLSASVGDRVTITCKASRDIRSYLTWYQQKPGKAPKTLIYYATSLADGVPSRFSGSGSGQDYSLTISSLESDDTATYYCLQHGESPFTLGSGTKLEIK
SEQ ID NO:52 VH
EVQLVESGGGLVQPGRSLRLSCAASGFTFNDYAMHWVRQAPGKGLEWVSTISWNSGSIGYADSVKGRFTISRDNAKKSLYLQMNSLRAEDTALYYCAKDIQYGNYYYGMDVWGQGTTVTVSS
SEQ ID NO:53 VL
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPITFGQGTRLEIK
SEQ ID NO:54 VH
QIQLQQSGPEVVKPGASVKISCKASGYTFTDYYITWVKQKPGQGLEWIGWIYPGSGNTKYNEKFKGKATLTVDTSSSTAFMQLSSLTSEDTAVYFCANYGNYWFAYWGQGTQVTVSA
SEQ ID NO:55 VL
DIVLTQSPASLAVSLGQRATISCKASQSVDFDGDSYMNWYQQKPGQPPKVLIYAASNLESGIPARFSGSGSGTDFTLNIHPVEEEDAATYYCQQSNEDPWTFGGGTKLEIK
SEQ ID NO:56 VH
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS
SEQ ID NO:57 VL
DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK
SEQ ID NO:58 VH
QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYTMHWVRQAPGKGLEWVTFISYDGNNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARTGWLGPFDYWGQGTLVTVSS
SEQ ID NO:59 VL
EIVLTQSPGTLSLSPGERATLSCRASQSVGSSYLAWYQQKPGQAPRLLIYGAFSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIK
SEQ ID NO:60 VH
EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSS
SEQ ID NO:61 VL
DIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIK
SEQ ID NO:62 VH
QVQLQQSGAEVKKPGSSVKVSCKASGYTFTNYYIYWVRQAPGQGLEWIGGINPTSGGSNFNEKFKTRVTITVDESTNTAYMELSSLRSEDTAFYFCARQGLWFDSDGRGFDFWGQGSTVTVSS
SEQ ID NO:63 VL
DIQMTQSPSSLSASVGDRVTITCRSSQNIVHSNGNTYLDWYQQTPGKAPKLLIYKVSNRFSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCFQYSHVPWTFGQGTKLQITRE
SEQ ID NO:64 VH
QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGDYYWTWIRQSPGKGLEWIGHIYYSGNTNYNPSLKSRLTISIDTSKTQFSLKLSSVTAADTAIYYCVRDRVTGAFDIWGQGTMVTVSS
SEQ ID NO:65 VL
DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYFCQHFDHLPLAFGGGTKVEIK
SEQ ID NO:66 VH
QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSA
SEQ ID NO:67 VL
QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIK
SEQ ID NO:68 VH
QVQLVQSGAEVKKPGASVKVSCKASGFNIKDTYIHWVRQAPGQRLEWMGRIDPANGYTKYDPKFQGRVTITADTSASTAYMELSSLRSEDTAVYYCAREGYYGNYGVYAMDYWGQGTLVTVSS
SEQ ID NO:69 VL
DIQMTQSPSSLSASVGDRVTITCKTSQDINKYMAWYQQTPGKAPRLLIHYTSALQPGIPSRFSGSGSGRDYTFTISSLQPEDIATYYCLQYDNLWTFGQGTKVEIK
SEQ ID NO:70 VH
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS
SEQ ID NO:71 VL
DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK
SEQ ID NO:72 VH
XVQLQESGPGLVRPSQTLSLTCTVSGYSITSDHAWSWVRQPPGRGLEWIGYISYSGITTYNPSLKSRVTMLRDTSKNQFSLRLSSVTAADTAVYYCARSLARTTAMDYWGQGSLVTVSS
SEQ ID NO:73 VL
DIQMTQSPSSLSASVGDRVTITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGNTLPYTFGQGTKVEIK
SEQ ID NO:74 VH
EVQLVQSGAEVKKPGSSVKVSCKASGYTITDSNIHWVRQAPGQSLEWIGYIYPYNGGTDYNQKFKNRATLTVDNPTNTAYMELSSLRSEDTAFYYCVNGNPWLAYWGQGTLVTVSS
SEQ ID NO:75 VL
DIQLTQSPSTLSASVGDRVTITCRASESLDNYGIRFLTWFQQKPGKAPKLLMYAASNQGSGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQTKEVPWSFGQGTKVEVK
SEQ ID NO:76 VH
QVQLKESGPGLVAPSQSLSITCTVSGFSLTDYGVRWIRQPPGKGLEWLGVIWGGGSTYYNSALKSRLSISKDNSKSQVFLKMNSLQTDDTAMYYCAKEKRRGYYYAMDYWGQGTSVTVSS
SEQ ID NO:77 VL
ENVLTQSPAIMSASPGEKVTMTCRASSSVSSSYLHWYQQKSGASPKLWIYSTSNLASGVPARFSGSGSGTSYSLTISSVEAEDAATYYCQQYSGYPLTFGGGTKLEIK
SEQ ID NO:78 VH
QVQLQESGPGLVRPSQTLSLTCTVSGFTFTDFYMNWVRQPPGRGLEWIGFIRDKAKGYTTEYNPSVKGRVTMLVDTSKNQFSLRLSSVTAADTAVYYCAREGHTAAPFDYWGQGSLVTVSS
SEQ ID NO:79 VL
DIQMTQSPSSLSASVGDRVTITCKASQNIDKYLNWYQQKPGKAPKLLIYNTNNLQTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCLQHISRPRTFGQGTKVEIK
SEQ ID NO:80 VH
QLQLQESGPGLVKPSETLSLTCTVSGGSINSSSYYWGWLRQSPGKGLEWIGSFFYTGSTYYNPSLRSRLTISVDTSKNQFSLMLSSVTAADTAVYYCARQSTYYYGSGNYYGWFDRWDQGTLVTVSS
SEQ ID NO:81 VL
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPAFGQGTKVEIK
SEQ ID NO:82 VH
QVQLQQSGAELVKPGASVKLSCKASGYTFTSYDINWVRQRPEQGLEWIGWIFPGDGSTKYNEKFKGKATLTTDKSSSTAYMQLSRLTSEDSAVYFCAREDYYDNSYYFDYWGQGTTLTVSS
SEQ ID NO:83 VL
DIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFGGGTKLEIK
SEQ ID NO:84 VH
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYIMMWVRQAPGKGLEWVSSIYPSGGITFYADTVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARIKLGTVTTVDYWGQGTLVTVSS
SEQ ID NO:85 VL
QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTRVFGTGTKVTVL
SEQ ID NO:86 VH
EVQLVESGGGLEQPGGSLRLSCAGSGFTFRDYAMTWVRQAPGKGLEWVSSISGSGGNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDRLSITIRPRYYGLDVWGQGTTVTVSS
SEQ ID NO:87 VL
DIVMTQSPLSLPVTPGEPASISCRSSQSLLYSIGYNYLDWYLQKSGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGFYYCMQALQTPYTFGQGTKLEIK
SEQ ID NO:88 VH
EVQLVESGGGLVQPGRSLRLSCAASRFTFDDYAMHWVRQAPGKGLEWVSGISWNSGRIGYADSVKGRFTISRDNAENSLFLQMNGLRAEDTALYYCAKGRDSFDIWGQGTMVTVSS
SEQ ID NO:89 VL
DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYGASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFASYYCQQANSFPYTFGQGTKLEIK
SEQ ID NO:90 VH
EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWVGAIYPGNGDTSYNQKFKGRFTISVDKSKNTLYLQMNSLRAEDTAVYYCARVVYYSNSYWYFDVWGQGTLVTVSS
SEQ ID NO:91 VL
DIQMTQSPSSLSASVGDRVTITCRASSSVSYMHWYQQKPGKAPKPLIYAPSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSFNPPTFGQGTKVEIK
SEQ ID NO:92 VH
EVQLLQSGPELEKPGASVMISCKASGSSFTGYNMNWVRQNIGKSLEWIGAIDPYYGGTSYNQKFKGRATLTVDKSSSTAYMHLKSLTSEDSAVYYCVSGMEYWGQGTSVTVSS
SEQ ID NO:93 VL
EIVMTQSPATLSVSPGERATLSCRSSQSLVHRNGNTYLHWYLQKPGQSPKLLIHKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPPLTFGAGTKLELK
FC-Knob(hIgG1)SEQ ID NO:94
EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
FC-Hole(hIgG1)SEQ ID NO:95
EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
Figure BDA0002825622600000321
Figure BDA0002825622600000331
Figure BDA0002825622600000341
Figure BDA0002825622600000351
Figure BDA0002825622600000361
Figure BDA0002825622600000371
Figure BDA0002825622600000381
Figure BDA0002825622600000391
Figure BDA0002825622600000401
Figure BDA0002825622600000411
Figure BDA0002825622600000421
Figure BDA0002825622600000431
Figure BDA0002825622600000441
Figure BDA0002825622600000451
Figure BDA0002825622600000461
Figure BDA0002825622600000471
Figure BDA0002825622600000481
Figure BDA0002825622600000491
Figure BDA0002825622600000501
Figure BDA0002825622600000511
Figure BDA0002825622600000521
Figure BDA0002825622600000531
Figure BDA0002825622600000541
Figure BDA0002825622600000551
Figure BDA0002825622600000561
Figure BDA0002825622600000571
Figure BDA0002825622600000581
Figure BDA0002825622600000591
Figure BDA0002825622600000601
Figure BDA0002825622600000611
Figure BDA0002825622600000621
Figure BDA0002825622600000631
Figure BDA0002825622600000641
Figure BDA0002825622600000651
Figure BDA0002825622600000661
Figure BDA0002825622600000671
Figure BDA0002825622600000681
Figure BDA0002825622600000691
Figure BDA0002825622600000701
Figure BDA0002825622600000711
Figure BDA0002825622600000721
Figure BDA0002825622600000731
Figure BDA0002825622600000741
Figure BDA0002825622600000751
Figure BDA0002825622600000761
Figure BDA0002825622600000771
Figure BDA0002825622600000781
Figure BDA0002825622600000791
Figure BDA0002825622600000801
Figure BDA0002825622600000811
Figure BDA0002825622600000821
Figure BDA0002825622600000831
Figure BDA0002825622600000841
Figure BDA0002825622600000851
序列表
<110> 屈向东
<120> 一种双特异抗体及其应用
<150> 201911253241.6
<151> 2019-12-09
<160> 95
<170> SIPOSequenceListing 1.0
<210> 1
<211> 114
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile
1 5 10 15
Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His
20 25 30
Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln
35 40 45
Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu
50 55 60
Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val
65 70 75 80
Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile
85 90 95
Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn
100 105 110
Thr Ser
<210> 2
<211> 114
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile
1 5 10 15
Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His
20 25 30
Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln
35 40 45
Val Ile Ser Cys Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu
50 55 60
Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val
65 70 75 80
Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile
85 90 95
Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn
100 105 110
Thr Ser
<210> 3
<211> 178
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 3
Glu Cys Asp Ile Thr Cys Pro Pro Pro Met Ser Val Glu His Ala Asp
1 5 10 15
Ile Trp Val Lys Ser Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys
20 25 30
Asn Ser Gly Phe Lys Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys
35 40 45
Val Leu Asn Lys Ala Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu
50 55 60
Lys Cys Ile Arg Asp Pro Ala Leu Val His Gln Arg Pro Ala Pro Pro
65 70 75 80
Ser Thr Val Thr Thr Ala Gly Val Thr Pro Gln Pro Glu Ser Leu Ser
85 90 95
Pro Ser Gly Lys Glu Pro Ala Ala Ser Ser Pro Ser Ser Asn Asn Thr
100 105 110
Ala Ala Thr Thr Ala Ala Ile Val Pro Gly Ser Gln Leu Met Pro Ser
115 120 125
Lys Ser Pro Ser Thr Gly Thr Thr Glu Ile Ser Ser His Glu Ser Ser
130 135 140
His Gly Thr Pro Ser Gln Thr Thr Ala Lys Asn Trp Glu Leu Thr Ala
145 150 155 160
Ser Ala Ser His Gln Pro Pro Gly Val Tyr Pro Gln Gly His Ser Asp
165 170 175
Thr Thr
<210> 4
<211> 65
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 4
Ile Thr Cys Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp Val
1 5 10 15
Lys Ser Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly
20 25 30
Phe Lys Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val Leu Asn
35 40 45
Lys Ala Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile
50 55 60
Arg
65
<210> 5
<211> 73
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 5
Ile Thr Cys Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp Val
1 5 10 15
Lys Ser Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly
20 25 30
Phe Lys Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val Leu Asn
35 40 45
Lys Ala Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile
50 55 60
Arg Asp Pro Ala Leu Val His Gln Arg
65 70
<210> 6
<211> 77
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 6
Ile Thr Cys Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp Val
1 5 10 15
Lys Ser Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly
20 25 30
Phe Lys Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val Leu Asn
35 40 45
Lys Ala Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile
50 55 60
Arg Asp Pro Ala Leu Val His Gln Arg Pro Ala Pro Pro
65 70 75
<210> 7
<211> 86
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 7
Ile Thr Cys Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp Val
1 5 10 15
Lys Ser Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly
20 25 30
Phe Lys Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val Leu Asn
35 40 45
Lys Ala Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile
50 55 60
Arg Asp Pro Ala Leu Val His Gln Arg Pro Ala Pro Pro Ser Thr Val
65 70 75 80
Thr Thr Ala Gly Val Thr
85
<210> 8
<211> 102
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 8
Ile Thr Cys Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp Val
1 5 10 15
Lys Ser Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly
20 25 30
Phe Lys Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val Leu Asn
35 40 45
Lys Ala Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile
50 55 60
Arg Asp Pro Ala Leu Val His Gln Arg Pro Ala Pro Pro Ser Thr Val
65 70 75 80
Thr Thr Ala Gly Val Thr Pro Gln Pro Glu Ser Leu Ser Pro Ser Gly
85 90 95
Lys Glu Pro Ala Ala Ser
100
<210> 9
<211> 65
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 9
Ile Thr Cys Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp Val
1 5 10 15
Lys Ser Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly
20 25 30
Phe Lys Arg Lys Ala Gly Thr Cys Ser Leu Thr Glu Cys Val Leu Asn
35 40 45
Lys Ala Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile
50 55 60
Arg
65
<210> 10
<211> 236
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 10
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 Gly Gly Gly Gly Ser
100 105 110
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asn Trp Val Asn Val Ile
115 120 125
Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile Gln Ser Met His Ile Asp
130 135 140
Ala Thr Leu Tyr Thr Glu Ser Asp Val His Pro Ser Cys Lys Val Thr
145 150 155 160
Ala Met Lys Cys Phe Leu Leu Glu Leu Gln Val Ile Ser Cys Glu Ser
165 170 175
Gly Asp Ala Ser Ile His Asp Thr Val Glu Asn Leu Ile Ile Leu Ala
180 185 190
Asn Asn Ser Leu Ser Ser Asn Gly Asn Val Thr Glu Ser Gly Cys Lys
195 200 205
Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile Lys Glu Phe Leu Gln Ser
210 215 220
Phe Val His Ile Val Gln Met Phe Ile Asn Thr Ser
225 230 235
<210> 11
<211> 437
<212> PRT
<213> 人工序列(Artificial Sequence)
<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 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 Gly Gly Gly Gly Ser Gly Gly Gly
115 120 125
Gly Ser Gly Gly Gly Gly Ser Ile Thr Cys Pro Pro Pro Met Ser Val
130 135 140
Glu His Ala Asp Ile Trp Val Lys Ser Tyr Ser Leu Tyr Ser Arg Glu
145 150 155 160
Arg Tyr Ile Cys Asn Ser Gly Phe Lys Arg Lys Ala Gly Thr Cys Ser
165 170 175
Leu Thr Glu Cys Val Leu Asn Lys Ala Thr Asn Val Ala His Trp Thr
180 185 190
Thr Pro Ser Leu Lys Cys Ile Arg Gly Gly Gly Gly Ser Glu Pro Lys
195 200 205
Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
210 215 220
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
225 230 235 240
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
245 250 255
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
260 265 270
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
275 280 285
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
290 295 300
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
305 310 315 320
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
325 330 335
Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
340 345 350
Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
355 360 365
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
370 375 380
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
385 390 395 400
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
405 410 415
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
420 425 430
Leu Ser Pro Gly Lys
435
<210> 12
<211> 214
<212> PRT
<213> 人工序列(Artificial Sequence)
<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 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> 13
<211> 449
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 13
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 Ala Ser Thr Lys Gly Pro Ser Val Phe
115 120 125
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu
130 135 140
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
145 150 155 160
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
165 170 175
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
180 185 190
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro
195 200 205
Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys
210 215 220
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro
225 230 235 240
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
245 250 255
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
260 265 270
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
275 280 285
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
290 295 300
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
305 310 315 320
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
325 330 335
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
340 345 350
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser
355 360 365
Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
370 375 380
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
385 390 395 400
Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys
405 410 415
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
420 425 430
Ala Leu His Asn Arg Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
435 440 445
Lys
<210> 14
<211> 236
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 14
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 Gly Ile Ala
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile
35 40 45
Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp 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 Val Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Ser Tyr Pro Tyr
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 Asn Trp Val Asn Val Ile
115 120 125
Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile Gln Ser Met His Ile Asp
130 135 140
Ala Thr Leu Tyr Thr Glu Ser Asp Val His Pro Ser Cys Lys Val Thr
145 150 155 160
Ala Met Lys Cys Phe Leu Leu Glu Leu Gln Val Ile Ser Cys Glu Ser
165 170 175
Gly Asp Ala Ser Ile His Asp Thr Val Glu Asn Leu Ile Ile Leu Ala
180 185 190
Asn Asn Ser Leu Ser Ser Asn Gly Asn Val Thr Glu Ser Gly Cys Lys
195 200 205
Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile Lys Glu Phe Leu Gln Ser
210 215 220
Phe Val His Ile Val Gln Met Phe Ile Asn Thr Ser
225 230 235
<210> 15
<211> 436
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 15
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 Asp Phe Ser Arg Tyr
20 25 30
Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile
35 40 45
Gly Glu Ile Asn Pro Asp Ser Ser Thr Ile Asn Tyr Ala Pro Ser Leu
50 55 60
Lys Asp Lys Phe Ile 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 Arg Pro Asp Gly Asn Tyr Trp Tyr Phe Asp Val Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
115 120 125
Ser Gly Gly Gly Gly Ser Ile Thr Cys Pro Pro Pro Met Ser Val Glu
130 135 140
His Ala Asp Ile Trp Val Lys Ser Tyr Ser Leu Tyr Ser Arg Glu Arg
145 150 155 160
Tyr Ile Cys Asn Ser Gly Phe Lys Arg Lys Ala Gly Thr Cys Ser Leu
165 170 175
Thr Glu Cys Val Leu Asn Lys Ala Thr Asn Val Ala His Trp Thr Thr
180 185 190
Pro Ser Leu Lys Cys Ile Arg Gly Gly Gly Gly Ser Glu Pro Lys Ser
195 200 205
Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu
210 215 220
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
225 230 235 240
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
245 250 255
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
260 265 270
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
275 280 285
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
290 295 300
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
305 310 315 320
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
325 330 335
Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val
340 345 350
Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
355 360 365
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
370 375 380
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
385 390 395 400
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
405 410 415
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
420 425 430
Ser Pro Gly Lys
435
<210> 16
<211> 214
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 16
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 Val Ser Ser Tyr
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45
Tyr Asp Ala Ser Asn Arg Ala Thr 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 Tyr Cys Gln Gln Arg Ser Asn Trp 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> 17
<211> 452
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 17
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Thr Phe Asn Ser Phe
20 25 30
Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ala Ile Ser Gly Ser Gly Gly Gly Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys
85 90 95
Ala Lys Asp Lys Ile Leu Trp Phe Gly Glu Pro Val Phe Asp Tyr Trp
100 105 110
Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
115 120 125
Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr
130 135 140
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
145 150 155 160
Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
165 170 175
Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
180 185 190
Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn
195 200 205
His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser
210 215 220
Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu
225 230 235 240
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
245 250 255
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
260 265 270
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
275 280 285
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
290 295 300
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
305 310 315 320
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
325 330 335
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
340 345 350
Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val
355 360 365
Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
370 375 380
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
385 390 395 400
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr
405 410 415
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
420 425 430
Met His Glu Ala Leu His Asn Arg Tyr Thr Gln Lys Ser Leu Ser Leu
435 440 445
Ser Pro Gly Lys
450
<210> 18
<211> 119
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 18
Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Glu Lys Pro Gly Ala
1 5 10 15
Ser Val 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 Ser Leu Glu Trp Ile
35 40 45
Gly Leu Ile Thr Pro Tyr Asn Gly Ala Ser Ser Tyr Asn Gln Lys Phe
50 55 60
Arg Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Asp Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys
85 90 95
Ala Arg Gly Gly Tyr Asp Gly Arg Gly Phe Asp Tyr Trp Gly Ser Gly
100 105 110
Thr Pro Val Thr Val Ser Ser
115
<210> 19
<211> 106
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 19
Asp Ile Glu Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly
1 5 10 15
Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met
20 25 30
His Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr
35 40 45
Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Gly Arg Phe Ser Gly Ser
50 55 60
Gly Ser Gly Asn Ser Tyr Ser Leu Thr Ile Ser Ser Val Glu Ala Glu
65 70 75 80
Asp Asp Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Lys His Pro Leu Thr
85 90 95
Phe Gly Ser Gly Thr Lys Val Glu Ile Lys
100 105
<210> 20
<211> 127
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 20
Glu Val Gln Leu Leu 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
Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Val Ile Ser Tyr Asp Gly Ser 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 Val Tyr Tyr Cys
85 90 95
Ala Lys Asp Met Gly Trp Gly Ser Gly Trp Arg Pro Tyr Tyr Tyr Tyr
100 105 110
Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser
115 120 125
<210> 21
<211> 107
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 21
Glu Leu 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 Thr Ser Gln Ser Ile Ser Ser Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile
35 40 45
Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val Pro Asp 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 Ser Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Asp Ile Pro Tyr
85 90 95
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 22
<211> 119
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 22
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> 23
<211> 107
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 23
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> 24
<211> 119
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 24
Xaa Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln
1 5 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp
20 25 30
His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp
35 40 45
Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu
50 55 60
Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gln Phe Ser
65 70 75 80
Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Gln Gly
100 105 110
Ser Leu Val Thr Val Ser Ser
115
<210> 25
<211> 107
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 25
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 Ser Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Tyr
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105
<210> 26
<211> 118
<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 Ser Asp Ser
20 25 30
Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Trp Ile Ser Pro Tyr Gly Gly Ser Thr Tyr 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
Ala Arg Arg His Trp Pro Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser
115
<210> 27
<211> 107
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 27
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 Ser Thr Ala
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ser Ala Ser Phe Leu Tyr 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 Leu Tyr His Pro Ala
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105
<210> 28
<211> 113
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 28
Glu Val Gln Leu Leu Gln Ser Gly Pro Glu Leu Glu Lys Pro Gly Ala
1 5 10 15
Ser Val Met Ile Ser Cys Lys Ala Ser Gly Ser Ser Phe Thr Gly Tyr
20 25 30
Asn Met Asn Trp Val Arg Gln Asn Ile Gly Lys Ser Leu Glu Trp Ile
35 40 45
Gly Ala Ile Asp Pro Tyr Tyr Gly Gly Thr Ser Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Arg Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met His Leu Lys Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Val Ser Gly Met Glu Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser
100 105 110
Ser
<210> 29
<211> 113
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 29
Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ser Ser Gln Ser Leu Val His Arg
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 Ile His 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 Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu
100 105 110
Lys
<210> 30
<211> 116
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 30
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30
Arg Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Tyr Ile Asn Pro Ser Thr Gly Tyr Thr Glu Tyr Asn Gln Lys Phe
50 55 60
Lys Asp Lys Ala Thr Ile Thr Ala Asp Glu Ser Thr Asn Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gly Gly Gly Val Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Ser Ser
115
<210> 31
<211> 106
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 31
Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Ile Ser Tyr Met
20 25 30
His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr
35 40 45
Thr Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser
50 55 60
Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp
65 70 75 80
Asp Phe Ala Thr Tyr Tyr Cys His Gln Arg Ser Thr Tyr Pro Leu Thr
85 90 95
Phe Gly Gln Gly Thr Lys Val Glu Val Lys
100 105
<210> 32
<211> 120
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 32
Gln Val Gln Leu Val Gln Ser Gly Val Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr
20 25 30
Tyr Met Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Gly Ile Asn Pro Ser Asn Gly Gly Thr Asn Phe Asn Glu Lys Phe
50 55 60
Lys Asn Arg Val Thr Leu Thr Thr Asp Ser Ser Thr Thr Thr Ala Tyr
65 70 75 80
Met Glu Leu Lys Ser Leu Gln Phe Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Arg Asp Tyr Arg Phe Asp Met Gly Phe Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Thr Val Thr Val Ser Ser
115 120
<210> 33
<211> 111
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 33
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 Lys Gly Val Ser Thr Ser
20 25 30
Gly Tyr Ser Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro
35 40 45
Arg Leu Leu Ile Tyr Leu Ala Ser Tyr 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 Ser
65 70 75 80
Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln His Ser Arg
85 90 95
Asp Leu Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105 110
<210> 34
<211> 116
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 34
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Arg Tyr
20 25 30
Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Trp Ile Ser Thr Tyr Ser Gly Asn Thr Asn Tyr Ala Gln Lys Leu
50 55 60
Gln 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 Arg Gln Leu Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Ser Ser
115
<210> 35
<211> 107
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 35
Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Asn
20 25 30
Leu Ala Trp Phe Gln Gln Lys Pro Gly Gln Ala Pro Arg Pro Leu Ile
35 40 45
Tyr Asp Ala Ser Thr Arg Ala Thr Gly Val Pro Ala Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser
65 70 75 80
Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Asp Asn Trp Pro Leu
85 90 95
Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105
<210> 36
<211> 119
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 36
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asp Phe Ser Arg Tyr
20 25 30
Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile
35 40 45
Gly Glu Ile Asn Pro Asp Ser Ser Thr Ile Asn Tyr Ala Pro Ser Leu
50 55 60
Lys Asp Lys Phe Ile 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 Arg Pro Asp Gly Asn Tyr Trp Tyr Phe Asp Val Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ser
115
<210> 37
<211> 107
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 37
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 Gly Ile Ala
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile
35 40 45
Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp 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 Val Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Ser Tyr Pro Tyr
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105
<210> 38
<211> 119
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 38
Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Phe Ser Asn Tyr
20 25 30
Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Thr Ile Ser Ser Ala Ser Thr Tyr Ser Tyr Tyr Pro 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 Val Glu Asp Thr Ala Leu Tyr Tyr Cys
85 90 95
Gly Arg His Ser Asp Gly Asn Phe Ala Phe Gly Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ser
115
<210> 39
<211> 112
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 39
Asp Val Leu Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly
1 5 10 15
Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Arg Asn Ile Val His Ile
20 25 30
Asn Gly Asp Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser
35 40 45
Pro Gln 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 Phe Gln Gly
85 90 95
Ser Leu Leu Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105 110
<210> 40
<211> 113
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 40
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Asp Cys Lys Ala Ser Gly Ile Thr Phe Ser Asn Ser
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 Tyr Asp Gly Ser Lys Arg Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Thr Asn Asp Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
100 105 110
Ser
<210> 41
<211> 107
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 41
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 Val Ser Ser Tyr
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45
Tyr Asp Ala Ser Asn Arg Ala Thr 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 Tyr Cys Gln Gln Ser Ser Asn Trp Pro Arg
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105
<210> 42
<211> 119
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 42
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Tyr Ser
20 25 30
Trp Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe
50 55 60
Lys Gly Arg Val Thr Ile Thr Ala 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 Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ser
115
<210> 43
<211> 112
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 43
Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly
1 5 10 15
Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser
20 25 30
Asn Gly Ile Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Ser
35 40 45
Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn Leu Val 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 Ala Gln Asn
85 90 95
Leu Glu Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105 110
<210> 44
<211> 122
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 44
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Thr Phe Asn Ser Phe
20 25 30
Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ala Ile Ser Gly Ser Gly Gly Gly Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys
85 90 95
Ala Lys Asp Lys Ile Leu Trp Phe Gly Glu Pro Val Phe Asp Tyr Trp
100 105 110
Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 45
<211> 107
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 45
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 Val Ser Ser Tyr
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45
Tyr Asp Ala Ser Asn Arg Ala Thr 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 Tyr Cys Gln Gln Arg Ser Asn Trp Pro Pro
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105
<210> 46
<211> 121
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 46
Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln
1 5 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly
20 25 30
Asp Tyr Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu
35 40 45
Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Asp Tyr Asn Pro Ser
50 55 60
Leu Lys Ser Arg Val Thr Met Ser Val Asp Thr Ser Lys Asn Gln Phe
65 70 75 80
Ser Leu Lys Val Asn Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr
85 90 95
Cys Ala Arg Val Ser Ile Phe Gly Val Gly Thr Phe Asp Tyr Trp Gly
100 105 110
Gln Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 47
<211> 107
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 47
Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45
Tyr Asp Ala Ser Asn Arg Ala Thr 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 Tyr Cys His Gln Tyr Gly Ser Thr Pro Leu
85 90 95
Thr Phe Gly Gly Gly Thr Lys Ala Glu Ile Lys
100 105
<210> 48
<211> 116
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 48
Glu Val Gln Leu Val Gln 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 Ser Tyr
20 25 30
Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ser Ile Ser Ser Ser Ser Ser Tyr Ile Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Val Thr Asp Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val
100 105 110
Thr Val Ser Ser
115
<210> 49
<211> 107
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 49
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Ile Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Asp Asn Trp
20 25 30
Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Asp Ala Ser Asn Leu Asp Thr Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Tyr Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala
65 70 75 80
Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Ala Lys Ala Phe Pro Pro
85 90 95
Thr Phe Gly Gly Gly Thr Lys Val Asp Ile Lys
100 105
<210> 50
<211> 119
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 50
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly
1 5 10 15
Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Lys Phe Ser Arg Tyr
20 25 30
Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Arg Leu Glu Trp Val
35 40 45
Ala Thr Ile Ser Ser Gly Gly Ser Tyr Ile Tyr Tyr Pro Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Val Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys
85 90 95
Ala Arg Arg Asp Tyr Asp Leu Asp Tyr Phe Asp Ser Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ser
115
<210> 51
<211> 107
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 51
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 Arg Asp Ile Arg Ser Tyr
20 25 30
Leu Thr Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Thr Leu Ile
35 40 45
Tyr Tyr Ala Thr Ser Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Gln Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Ser
65 70 75 80
Asp Asp Thr Ala Thr Tyr Tyr Cys Leu Gln His Gly Glu Ser Pro Phe
85 90 95
Thr Leu Gly Ser Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 52
<211> 122
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 52
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Asp Tyr
20 25 30
Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Thr Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Lys Ser Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys
85 90 95
Ala Lys Asp Ile Gln Tyr Gly Asn Tyr Tyr Tyr Gly Met Asp Val Trp
100 105 110
Gly Gln Gly Thr Thr Val Thr Val Ser Ser
115 120
<210> 53
<211> 107
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 53
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 Val Ser Ser Tyr
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45
Tyr Asp Ala Ser Asn Arg Ala Thr 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 Tyr Cys Gln Gln Arg Ser Asn Trp Pro Ile
85 90 95
Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys
100 105
<210> 54
<211> 117
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 54
Gln Ile Gln Leu Gln Gln Ser Gly Pro Glu Val Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Tyr Ile Thr Trp Val Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Trp Ile Tyr Pro Gly Ser Gly Asn Thr Lys Tyr Asn Glu Lys Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Val Asp Thr Ser Ser Ser Thr Ala Phe
65 70 75 80
Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Phe Cys
85 90 95
Ala Asn Tyr Gly Asn Tyr Trp Phe Ala Tyr Trp Gly Gln Gly Thr Gln
100 105 110
Val Thr Val Ser Ala
115
<210> 55
<211> 111
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 55
Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Gln Arg Ala Thr Ile Ser Cys Lys Ala Ser Gln Ser Val Asp Phe Asp
20 25 30
Gly Asp Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro
35 40 45
Lys Val Leu Ile Tyr Ala Ala Ser Asn Leu Glu Ser Gly Ile Pro Ala
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His
65 70 75 80
Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Ser Asn
85 90 95
Glu Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105 110
<210> 56
<211> 120
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 56
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> 57
<211> 107
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 57
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> 58
<211> 118
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 58
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> 59
<211> 108
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 59
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> 60
<211> 119
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 60
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> 61
<211> 107
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 61
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> 62
<211> 123
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 62
Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr
20 25 30
Tyr Ile Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Gly Ile Asn Pro Thr Ser Gly Gly Ser Asn Phe Asn Glu Lys Phe
50 55 60
Lys Thr Arg Val Thr Ile Thr Val Asp Glu Ser Thr Asn Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Phe Tyr Phe Cys
85 90 95
Ala Arg Gln Gly Leu Trp Phe Asp Ser Asp Gly Arg Gly Phe Asp Phe
100 105 110
Trp Gly Gln Gly Ser Thr Val Thr Val Ser Ser
115 120
<210> 63
<211> 114
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 63
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 Ser Ser Gln Asn Ile Val His Ser
20 25 30
Asn Gly Asn Thr Tyr Leu Asp Trp Tyr Gln Gln Thr Pro Gly Lys Ala
35 40 45
Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro
50 55 60
Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile
65 70 75 80
Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Phe Gln Tyr
85 90 95
Ser His Val Pro Trp Thr Phe Gly Gln Gly Thr Lys Leu Gln Ile Thr
100 105 110
Arg Glu
<210> 64
<211> 119
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 64
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 Gly Ser Val Ser Ser Gly
20 25 30
Asp Tyr Tyr Trp Thr Trp Ile Arg Gln Ser Pro Gly Lys Gly Leu Glu
35 40 45
Trp Ile Gly His Ile Tyr Tyr Ser Gly Asn Thr Asn Tyr Asn Pro Ser
50 55 60
Leu Lys Ser Arg Leu Thr Ile Ser Ile Asp Thr Ser Lys Thr Gln Phe
65 70 75 80
Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Ile Tyr Tyr
85 90 95
Cys Val Arg Asp Arg Val Thr Gly Ala Phe Asp Ile Trp Gly Gln Gly
100 105 110
Thr Met Val Thr Val Ser Ser
115
<210> 65
<211> 107
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 65
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 Gln Ala Ser Gln Asp Ile Ser Asn Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Asp Ala Ser Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Ile Ala Thr Tyr Phe Cys Gln His Phe Asp His Leu Pro Leu
85 90 95
Ala Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105
<210> 66
<211> 121
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 66
Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30
Asn Met His Trp Val Lys Gln Thr Pro Gly Arg Gly Leu Glu Trp Ile
35 40 45
Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Ser Thr Tyr Tyr Gly Gly Asp Trp Tyr Phe Asn Val Trp Gly
100 105 110
Ala Gly Thr Thr Val Thr Val Ser Ala
115 120
<210> 67
<211> 106
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 67
Gln Ile Val Leu Ser Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro Gly
1 5 10 15
Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Ile
20 25 30
His Trp Phe Gln Gln Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr
35 40 45
Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Val Arg Phe Ser Gly Ser
50 55 60
Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu Ala Glu
65 70 75 80
Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Thr Ser Asn Pro Pro Thr
85 90 95
Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 68
<211> 123
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 68
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 Phe Asn Ile Lys Asp Thr
20 25 30
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met
35 40 45
Gly Arg Ile Asp Pro Ala Asn Gly Tyr Thr Lys Tyr Asp Pro Lys Phe
50 55 60
Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Ala Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Glu Gly Tyr Tyr Gly Asn Tyr Gly Val Tyr Ala Met Asp Tyr
100 105 110
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 69
<211> 106
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 69
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 Thr Ser Gln Asp Ile Asn Lys Tyr
20 25 30
Met Ala Trp Tyr Gln Gln Thr Pro Gly Lys Ala Pro Arg Leu Leu Ile
35 40 45
His Tyr Thr Ser Ala Leu Gln Pro Gly Ile Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Arg Asp Tyr Thr Phe Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Asn Leu Trp Thr
85 90 95
Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105
<210> 70
<211> 120
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 70
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> 71
<211> 107
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 71
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> 72
<211> 119
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 72
Xaa Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln
1 5 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp
20 25 30
His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp
35 40 45
Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu
50 55 60
Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gln Phe Ser
65 70 75 80
Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Gln Gly
100 105 110
Ser Leu Val Thr Val Ser Ser
115
<210> 73
<211> 107
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 73
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 Ser Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Tyr
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105
<210> 74
<211> 116
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 74
Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Ile Thr Asp Ser
20 25 30
Asn Ile His Trp Val Arg Gln Ala Pro Gly Gln Ser Leu Glu Trp Ile
35 40 45
Gly Tyr Ile Tyr Pro Tyr Asn Gly Gly Thr Asp Tyr Asn Gln Lys Phe
50 55 60
Lys Asn Arg Ala Thr Leu Thr Val Asp Asn Pro Thr Asn Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Phe Tyr Tyr Cys
85 90 95
Val Asn Gly Asn Pro Trp Leu Ala Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Ser Ser
115
<210> 75
<211> 111
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 75
Asp Ile Gln Leu Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Ser Leu Asp Asn Tyr
20 25 30
Gly Ile Arg Phe Leu Thr Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro
35 40 45
Lys Leu Leu Met Tyr Ala Ala Ser Asn Gln Gly Ser Gly Val Pro Ser
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser
65 70 75 80
Ser Leu Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Thr Lys
85 90 95
Glu Val Pro Trp Ser Phe Gly Gln Gly Thr Lys Val Glu Val Lys
100 105 110
<210> 76
<211> 120
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 76
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 Thr Asp Tyr
20 25 30
Gly Val Arg Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu
35 40 45
Gly Val Ile Trp Gly Gly Gly Ser Thr Tyr Tyr Asn Ser Ala Leu Lys
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 Met Tyr Tyr Cys Ala
85 90 95
Lys Glu Lys Arg Arg Gly Tyr Tyr Tyr Ala Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Ser Val Thr Val Ser Ser
115 120
<210> 77
<211> 108
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 77
Glu Asn Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly
1 5 10 15
Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Ser Ser
20 25 30
Tyr Leu His Trp Tyr Gln Gln Lys Ser Gly Ala Ser Pro Lys Leu Trp
35 40 45
Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser
50 55 60
Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Val Glu
65 70 75 80
Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Gly Tyr Pro
85 90 95
Leu Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 78
<211> 121
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 78
Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln
1 5 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Thr Phe Thr Asp Phe
20 25 30
Tyr Met Asn Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp Ile
35 40 45
Gly Phe Ile Arg Asp Lys Ala Lys Gly Tyr Thr Thr Glu Tyr Asn Pro
50 55 60
Ser Val Lys Gly Arg Val Thr Met Leu Val Asp Thr Ser Lys Asn Gln
65 70 75 80
Phe Ser Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr
85 90 95
Tyr Cys Ala Arg Glu Gly His Thr Ala Ala Pro Phe Asp Tyr Trp Gly
100 105 110
Gln Gly Ser Leu Val Thr Val Ser Ser
115 120
<210> 79
<211> 107
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 79
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 Asn Ile Asp Lys Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Asn Thr Asn Asn Leu Gln Thr Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln His Ile Ser Arg Pro Arg
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105
<210> 80
<211> 127
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 80
Gln Leu 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 Gly Ser Ile Asn Ser Ser
20 25 30
Ser Tyr Tyr Trp Gly Trp Leu Arg Gln Ser Pro Gly Lys Gly Leu Glu
35 40 45
Trp Ile Gly Ser Phe Phe Tyr Thr Gly Ser Thr Tyr Tyr Asn Pro Ser
50 55 60
Leu Arg Ser Arg Leu Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe
65 70 75 80
Ser Leu Met Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr
85 90 95
Cys Ala Arg Gln Ser Thr Tyr Tyr Tyr Gly Ser Gly Asn Tyr Tyr Gly
100 105 110
Trp Phe Asp Arg Trp Asp Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 81
<211> 107
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 81
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 Val Ser Ser Tyr
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45
Tyr Asp Ala Ser Asn Arg Ala Thr 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 Tyr Cys Gln Gln Arg Ser Asn Trp Pro Pro
85 90 95
Ala Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105
<210> 82
<211> 121
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 82
Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30
Asp Ile Asn Trp Val Arg Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile
35 40 45
Gly Trp Ile Phe Pro Gly Asp Gly Ser Thr Lys Tyr Asn Glu Lys Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Gln Leu Ser Arg Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys
85 90 95
Ala Arg Glu Asp Tyr Tyr Asp Asn Ser Tyr Tyr Phe Asp Tyr Trp Gly
100 105 110
Gln Gly Thr Thr Leu Thr Val Ser Ser
115 120
<210> 83
<211> 107
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 83
Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly
1 5 10 15
Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val 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 Ser Leu Thr Ile Ser Asn Leu Glu Gln
65 70 75 80
Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Trp
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 84
<211> 120
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 84
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Ile Met Met Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ser Ile Tyr Pro Ser Gly Gly Ile Thr Phe Tyr Ala Asp Thr 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 Ile Lys Leu Gly Thr Val Thr Thr Val Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 85
<211> 110
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 85
Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln
1 5 10 15
Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr
20 25 30
Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu
35 40 45
Met Ile Tyr Asp Val Ser Asn Arg Pro Ser Gly Val Ser Asn Arg Phe
50 55 60
Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu
65 70 75 80
Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Ser
85 90 95
Ser Thr Arg Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu
100 105 110
<210> 86
<211> 125
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 86
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Glu Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Gly Ser Gly Phe Thr Phe Arg Asp Tyr
20 25 30
Ala Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ser Ile Ser Gly Ser Gly Gly Asn Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Lys Asp Arg Leu Ser Ile Thr Ile Arg Pro Arg Tyr Tyr Gly Leu
100 105 110
Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser
115 120 125
<210> 87
<211> 112
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 87
Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly
1 5 10 15
Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu Tyr Ser
20 25 30
Ile Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Ser Gly Gln Ser
35 40 45
Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Val Gly Phe Tyr Tyr Cys Met Gln Ala
85 90 95
Leu Gln Thr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105 110
<210> 88
<211> 116
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 88
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Arg Phe Thr Phe Asp Asp Tyr
20 25 30
Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Gly Ile Ser Trp Asn Ser Gly Arg Ile Gly Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Glu Asn Ser Leu Phe
65 70 75 80
Leu Gln Met Asn Gly Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys
85 90 95
Ala Lys Gly Arg Asp Ser Phe Asp Ile Trp Gly Gln Gly Thr Met Val
100 105 110
Thr Val Ser Ser
115
<210> 89
<211> 107
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 89
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Gly Ala Ser Ser Leu Glu 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 Ser Tyr Tyr Cys Gln Gln Ala Asn Ser Phe Pro Tyr
85 90 95
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 90
<211> 122
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 90
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 Ser Tyr
20 25 30
Asn Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Arg Phe Thr Ile Ser Val Asp Lys 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 Val Val Tyr Tyr Ser Asn Ser Tyr Trp Tyr Phe Asp Val Trp
100 105 110
Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 91
<211> 106
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 91
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 Ser Ser Val Ser Tyr Met
20 25 30
His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Pro Leu Ile Tyr
35 40 45
Ala Pro Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser
50 55 60
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu
65 70 75 80
Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Phe Asn Pro Pro Thr
85 90 95
Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105
<210> 92
<211> 113
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 92
Glu Val Gln Leu Leu Gln Ser Gly Pro Glu Leu Glu Lys Pro Gly Ala
1 5 10 15
Ser Val Met Ile Ser Cys Lys Ala Ser Gly Ser Ser Phe Thr Gly Tyr
20 25 30
Asn Met Asn Trp Val Arg Gln Asn Ile Gly Lys Ser Leu Glu Trp Ile
35 40 45
Gly Ala Ile Asp Pro Tyr Tyr Gly Gly Thr Ser Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Arg Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met His Leu Lys Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Val Ser Gly Met Glu Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser
100 105 110
Ser
<210> 93
<211> 113
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 93
Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ser Ser Gln Ser Leu Val His Arg
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 Ile His 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 Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu
100 105 110
Lys
<210> 94
<211> 231
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 94
Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
1 5 10 15
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
20 25 30
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
35 40 45
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
50 55 60
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
65 70 75 80
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
85 90 95
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
100 105 110
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
115 120 125
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
130 135 140
Lys Asn Gln Val Ser Leu 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
225 230
<210> 95
<211> 231
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 95
Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
1 5 10 15
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
20 25 30
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
35 40 45
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
50 55 60
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
65 70 75 80
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
85 90 95
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
100 105 110
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
115 120 125
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
130 135 140
Lys Asn Gln Val Ser Leu 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
225 230

Claims (20)

1.一种双特异抗体分子，包括但不限于这种形式：
{[TAA]1，[TAA]2，[IL15/IL15Ra]，[FC异源二聚体]}，
每种形式同时包含：
[TAA]1：结合一个抗原的抗体、抗体或多肽片段；
[TAA]2：结合另一个抗原或同一抗原不同表位的抗体、抗体或多肽片段；
[IL15/IL15Ra]：IL15及其能结合IL15Ra的突变，截短及各种衍生物；
IL15Ra及其能结合IL15的突变，截短及各种衍生物；
这两者形成的复合物；
[FC异源二聚体]：FC形成的异源二聚体。
2.如权利要求1中所述的双特异抗体分子，其特征在于：
包含IL15/IL15Ra复合物，IL15/IL15Ra复合物包括但不限于1)IL15及其能结合IL15Ra的突变，截短及各种衍生物；2)IL15Ra及其能结合IL15的突变，截短及各种衍生物；
包括但不限于下表所示突变，计数方式根据SEQ ID No.1所示IL15第一个氨基酸开始算为第1位；SEQ ID No.3所示IL15Ra第一个氨基酸开始算为第1位：
组合 IL15 IL15Ra 1 wt D96 2 wt D96/P97 3 wt D96/P97/A98 4 E87C D96/C97 5 E87C D96/P97/C98 6 E87C D96/C97/A98 7 V49C S40C 8 L52C S40C 9 E89C K34C 10 Q48C G38C 11 E53C L42C 12 C42S A37C 13 L45C G38C 14 L45C A37C
同时包括但不限于下表所示IL15相关各种突变，计数方式根据SEQ ID No.1所示IL15第一个氨基酸开始算为第1位：
组合 IL15突变 1 N1D 2 N4D 3 D8N 4 D30N 5 D61N 6 E64Q 7 N65D 8 Q108E 9 N1D/D61N 10 N1D/E64Q 11 N4D/D61N 12 N4D/E64Q 13 D8N/D61N 14 D8N/E64Q 15 D61N/E64Q 16 E64Q/Q108E 17 N1D/N4D/D8N 18 D61N/E64Q/N65D 19 N1D/D61N/E64Q/Q108E 20 N4D/D61N/E64Q/Q108E
3.如权利要求1中所述的双特异抗体分子，其特征在于：
与IL15或IL15Ra融合的是针对抗原的抗体可变区VL1或VH1，包括但不限于以下突变，根据EU计数：
Figure FDA0002825622590000021
其中，VL1与VH1之间可有一对或多对二硫键。
4.权利要求1中所述的双特异抗体分子，其特征在于：
包含抗体的FC段，是FC异源二聚体形式，FC异源二聚体包含但不限于以下突变的组合，根据EU计数，
Figure FDA0002825622590000031
5.如权利要求1所述的双特异抗体分子，其特征在于：
其中，人FC片段包括但不限于Human IgG1 FC、Human IgG2 FC、Human IgG3 FC、HumanIgG4 FC及其变异体，其中一条链能结合proteinA；则另一条链可选择不结合proteinA的突变体，包括但不限于突变H435R或H435R/Y436F，根据EU计数。
6.如权利要求1所述的双特异抗体分子，其特征在于：
其中结合抗原的各个抗体或多肽片段是单价或多价分子，TAA结合的抗原或其特异突变包括但不限于CD3、CD20、CD19、CD30、CD33、CD38、CD40、CD52、slamf7、GD2、CD24、CD47、CD133、CD239、CD276、PD-1等表面抗原，CEA、Epcam、Trop2、TAG72、MUC1、MUC16、mesothelin、folr1、CLDN18.2、PDL1、EGFR、EGFR VIII、C-MET、HER2、FGFR2、FGFR3、PSMA、PSCA、EphA2、ADAM17、17-A1、NKG2D ligands、MCSP、LGR5、SSEA3、SLC34A2、BCMA、GPNMB等靶点的抗体，形式包括Fab、ScFv、VHH或多肽片段等。
7.如权利要求1-6中任一项所述的的双特异抗体分子，其特征在于：
其中各个抗体是嵌合、人源化、全人源或单链抗体。
8.如权利要求1-7中任一项所述的的双特异抗体分子，其特征在于：
连接序列为免疫原性低的氨基酸序列。
9.如权利要求1-8所述的双特异抗体分子，其特征在于：
当由4条链组成时，包括但不限于如下形式：
Figure FDA0002825622590000041
其中：
链1：结合抗原的抗体可变区VL1或VH1融合在IL15或IL15Ra的N端或C端；
链2：IL15或IL15Ra的N端或C端融合了结合抗原的抗体可变区VL1或VH1，在其C端融合可形成异源二聚体的FC；
链3：结合另一个抗原的抗体可变区VH2融合CH1片段，在其C端融合可形成异源二聚体的FC，组成抗体重链；
链4：结合另一个抗原的抗体可变区VL2的C端融合CL片段，组成抗体轻链，
当由3条链组成时，包括但不限于以下形式：
Figure FDA0002825622590000051
其中：
链1：结合抗原的抗体可变区VL1或VH1融合在IL15或IL15Ra的N端或C端；
链2：IL15或IL15Ra的N端或C端融合了结合抗原的抗体可变区VL1或VH1，在其C端融合可形成异源二聚体的FC；
链3：结合另一个靶点的亲和多肽，包括SCFV，VHH及其人源化形式、多肽或蛋白质，在其C端融合可形成异源二聚体的FC。
10.如权利要求9的双特异抗体分子，其特征在于：
与IL15或IL15Ra融合的蛋白链1及蛋白链2的组合形式包括但不限于以下组合：
Figure FDA0002825622590000061
11.如权利要求1-8所述的双特异抗体分子，其特征在于：
还具有如下形式：
Figure FDA0002825622590000062
其中:
链1：结合抗原的抗体可变区VL1融合在IL15的N端；
链2：IL15Ra的N端融合了结合抗原的抗体可变区VH1，C端融合FC异源二聚体；
链3：结合另一个抗原的抗体可变区VH2融合CH1片段，C端融合FC异源二聚体，组成抗体重链；
链4：结合另一个抗原的抗体可变区VL2的C端融合CL片段，组成抗体轻链。
12.如权利要求9-11所述的双特异抗体分子，其特征在于：
其中，IL15及IL15Ra复合物包括：1)IL15及其能结合IL15Ra的突变，截短及各种衍生物；2)IL15Ra及其能结合IL15的突变，截短及各种衍生物；包括但不限于权利要求3所述突变以及本实施例中所述序列SEQ ID No.1、SEQ ID No.2、SEQ ID No.3、SEQ ID No.4、SEQID No.5、SEQ ID No.6、SEQ ID No.7、SEQ ID No.8、SEQ ID No.9。
13.如权利要求9-11所述的双特异抗体分子，其特征在于：
人FC片段，是FC异源二聚体形式，FC异源二聚体包含但不限于权利要求3所述突变以及序列SEQ ID No.94、SEQ ID No.95。
14.如权利要求9-11所述的双特异抗体分子，其特征在于：
人FC片段，包括但不限于Human IgG1 FC、Human IgG2 FC、Human IgG3 FC、Human IgG4FC及其变异体。其中一条链能结合proteinA；另一条链可选择不结合proteinA，包括但不限于突变H435R或H435R/Y436F，根据EU计数。
15.如权利要求1-8所述的双特异抗体分子，其特征在于：
结合抗原的TAA1和/或TAA2抗体，包括但不限于CD3、CD20、CD19、CD38、CD40、CD30、CD33、CD52、slamf7、GD2、CD24、CD47、CD133、CD239、CD276、PD-1等表面抗原，CEA、Epcam、Trop2、TAG72、MUC1、MUC16、mesothelin、folr1、CLDN18.2、PDL1、EGFR、EGFRVIII、C-MET、HER2、FGFR2、FGFR3、PSMA、PSCA、EphA2、ADAM17、17-A1、NKG2D ligands、MCSP、LGR5、SSEA3、SLC34A2、BCMA、GPNMB等靶点的抗体，包含但不限于序列SEQ ID No.18、SEQ ID No.19、SEQID No.20、SEQ ID No.21、SEQ ID No.22、SEQ ID No.23、SEQ ID No.24、SEQ ID No.25、SEQID No.26、SEQ ID No.27、SEQ ID No.28、SEQ ID No.29、SEQ ID No.30、SEQ ID No.31、SEQID No.32、SEQ ID No.33、SEQ ID No.34、SEQ ID No.35、SEQ ID No.36、SEQ ID No.37、SEQID No.38、SEQ ID No.39、SEQ ID No.40、SEQ ID No.41、SEQ ID No.42、SEQ ID No.43、SEQID No.44、SEQ ID No.45、SEQ ID No.46、SEQ ID No.47、SEQ ID No.48、SEQ ID No.49、SEQID No.50、SEQ ID No.51、SEQ ID No.52、SEQ ID No.53、SEQ ID No.54、SEQ ID No.55、SEQID No.56、SEQ ID No.57、SEQ ID No.58、SEQ ID No.59、SEQ ID No.60、SEQ ID No.61、SEQID No.62、SEQ ID No.63、SEQ ID No.64、SEQ ID No.65、SEQ ID No.66、SEQ ID No.67、SEQID No.68、SEQ ID No.69、SEQ ID No.70、SEQ ID No.71、SEQ ID No.72、SEQ ID No.73、SEQID No.74、SEQ ID No.75、SEQ ID No.76、SEQ ID No.77、SEQ ID No.78、SEQ ID No.79、SEQID No.80、SEQ ID No.81、SEQ ID No.82、SEQ ID No.83、SEQ ID No.84、SEQ ID No.85、SEQID No.86、SEQ ID No.87、SEQ ID No.88、SEQ ID No.89、SEQ ID No.90、SEQ ID No.91、SEQID No.92、SEQ ID No.93。
16.如权利要求9-11所述权利要求1-8所述的双特异抗体分子，其特征在于：
其中，TAA为Her2/Her3；FC为IgG1异源二聚体；链1和链2的IL15及IL15Ra复合物融合蛋白有二硫键形成，包含但不限于本实施例中所述序列SEQ ID No.10、SEQ ID No.11、SEQ IDNo.12、SEQ ID No.13。
17.如权利要求9-11所述的双特异抗体分子，其特征在于：
其中，TAA为CD38/CS1；FC为IgG1异源二聚体；链1和链2的IL15及IL15Ra复合物融合蛋白有二硫键形成，包含但不限于本实施例中所述序列SEQ ID No.14、SEQ ID No.15、SEQ IDNo.16、SEQ ID No.17。
18.如权利要求1-17中任意一项所述的双特异抗体分子在制备癌症、感染和免疫调节疾病的药物中的应用。
19.如权利要求1-17中任意一项所述的双特异抗体分子，在制备抑制肿瘤生长的药物中的应用。
20.如权利要求18或19所述的应用，其中，所述癌症或肿瘤包括：骨髓癌、淋巴癌、白血病、小细胞肺癌、非小细胞肺癌、黑素瘤、基底细胞皮肤癌、鳞状细胞皮肤癌、隆突性皮肤纤维肉瘤、梅克尔细胞癌、成胶质细胞瘤、胶质瘤、肉瘤、间皮瘤，和骨髓增生异常综合症；
或者包括来自如下部位的癌症或肿瘤：结直肠、乳腺、卵巢、胰腺、胃、前列腺、肾、宫颈、甲状腺、子宫内膜、子宫、膀胱、神经内分泌、头部颈部、肝、鼻咽、睾丸。
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Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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WO2023020315A1 (zh) * 2021-08-19 2023-02-23 南京吉盛澳玛生物医药有限公司 靶向pd-l1/pd-1的抗体及其应用
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Family Cites Families (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ES2774337T3 (es) 2008-01-07 2020-07-20 Amgen Inc Método para fabricación de moléculas heterodímeras Fc de anticuerpos utilizando efectos de conducción electrostática
EP2537933A1 (en) * 2011-06-24 2012-12-26 Institut National de la Santé et de la Recherche Médicale (INSERM) An IL-15 and IL-15Ralpha sushi domain based immunocytokines
US20150183877A1 (en) * 2012-07-18 2015-07-02 Eli Lilly And Company Multi-Specific IgG-(Fab)2 Constructs Containing T-Cell Receptor Constant Domains
CN105189562B (zh) * 2014-01-08 2019-08-16 上海恒瑞医药有限公司 Il-15异源二聚体蛋白及其用途
US10905743B2 (en) * 2014-12-19 2021-02-02 Jiangsu Hengrui Medicine Co., Ltd. Interleukin 15 protein complex
KR102463844B1 (ko) * 2016-05-27 2022-11-08 알토 바이오사이언스 코포레이션 Cd3 결합 도메인을 가지는 다량체 il-15 기반 분자의 구성 및 특성규명
AU2018334886A1 (en) * 2017-09-22 2020-04-09 WuXi Biologics Ireland Limited Novel bispecific polypeptide complexes
US11168138B2 (en) * 2018-03-26 2021-11-09 Altor Bioscience, Llc Anti-PDL1, IL-15 and TGF-beta receptor combination molecules
CA3097741A1 (en) * 2018-04-18 2019-10-24 Xencor, Inc. Tim-3 targeted heterodimeric fusion proteins containing il-15/il-15ra fc-fusion proteins and tim-3 antigen binding domains
CN113195534A (zh) * 2018-04-18 2021-07-30 Xencor股份有限公司 包含il-15/il-15ra fc融合蛋白和lag-3抗原结合结构域的靶向lag-3的异源二聚体融合蛋白
JP2021521784A (ja) * 2018-04-18 2021-08-30 ゼンコア インコーポレイテッド ＩＬ−１５／ＩＬ−１５ＲａＦｃ融合タンパク質とＰＤ−１抗原結合ドメインを含むＰＤ−１標的化ヘテロダイマー融合タンパク質およびそれらの使用
CN110437339B (zh) * 2018-05-04 2021-08-13 免疫靶向有限公司 一种以白介素15为活性成分的融合蛋白型药物前体
EP3988574A4 (en) * 2019-02-03 2022-10-12 Shanghai Bao Pharmaceuticals Co., Ltd. AGAINST HER2 BI-SPECIFIC ANTIBODIES AND THEIR USE
JP2022530934A (ja) * 2019-02-22 2022-07-05 武▲漢▼友芝友生物制▲薬▼有限公司 改変されたＦｃ断片、それを含む抗体、及びそれらの使用
CN113135996A (zh) * 2019-12-09 2021-07-20 启愈生物技术(上海)有限公司 一种双特异抗体及其应用

Cited By (14)

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