CN112142842A - 抗PD-L1纳米抗体及其Fc融合蛋白和应用 - Google Patents

抗PD-L1纳米抗体及其Fc融合蛋白和应用 Download PDF

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CN112142842A
CN112142842A CN202010574992.4A CN202010574992A CN112142842A CN 112142842 A CN112142842 A CN 112142842A CN 202010574992 A CN202010574992 A CN 202010574992A CN 112142842 A CN112142842 A CN 112142842A
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屈向东
潘琴
金后聪
都业杰
郑翰
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Qiyu Biotechnology Shanghai Co Ltd
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Abstract

本发明提供一种抗PD‑L1纳米抗体及其Fc融合蛋白和应用，本发明的抗PD‑L1纳米抗体及其Fc融合蛋白特异性强，亲和力高，对人的免疫原性弱。并且稳定性高，具有显著的抗肿瘤效果。

Description

抗PD-L1纳米抗体及其Fc融合蛋白和应用
技术领域
本发明涉及一种抗PD-L1纳米抗体，本发明还涉及一种抗PD-L1纳米抗体的Fc融合蛋白，本发明还涉及抗PD-L1纳米抗体的应用，本发明还涉及抗PD-L1纳米抗体的Fc融合蛋白的应用，属于生物医药领域。
背景技术
在经典免疫监视理论中，免疫系统可以识别肿瘤抗原并将其消除。如果免疫系统能够完全消除肿瘤细胞，那么免疫清除可以稳定进行。如果肿瘤细胞通过突变逃避免疫系统的清除，免疫系统将会进行重新平衡(rebalance)。在这一过程中，肿瘤细胞的免疫原性逐渐降低。肿瘤细胞的增殖能力在免疫系统的压力下变弱，使得肿瘤细胞的侦测变得更为困难。
致癌基因的激活导致肿瘤细胞改变自身及肿瘤微环境，使得免疫系统和肿瘤细胞间的平衡被打破。当免疫系统和肿瘤细胞进入逃逸阶段，肿瘤细胞的恶性程度会提高，而肿瘤细胞丢失MHC分子使其避免被免疫细胞识别和消除。肿瘤微环境也可以通过释放免疫抑制因子抑制免疫系统，如IL-10，TGF-β等。肿瘤细胞表面也会高表达免疫抑制蛋白(如程序化死亡配体-1，PD-L1)，当效应T细胞与肿瘤细胞结合时，PD-L1 与PD-1相互作用并诱导T细胞发生凋亡，这是肿瘤对免疫系统产生耐受的主要原因之一，肿瘤迅速生长，发生转移。如果人为激活宿主的免疫系统并将其重定向到肿瘤细胞，从理论上来讲肿瘤组织就能够被清除，而免疫治疗的理论已经在临床治疗中得到广泛证明。
免疫疗法可分为两类：特异性治疗和非特异性治疗。在类别中特定疗法，包括以下治疗策略：肿瘤疫苗通过注射肿瘤激活免疫细胞对患者的抗原。肿瘤疫苗包括：灭活的肿瘤细胞疫苗，肿瘤抗原疫苗，肿瘤DNA 疫苗，树突状细胞(DC)疫苗和细菌疫苗。特异性ACT免疫疗法主要包括三种治疗方法，
肿瘤浸润淋巴细胞(TIL)：淋巴细胞从肿瘤组织中分离并在体外培养。TIL可以分泌具有特异性抗肿瘤的IL-2能力。
T细胞受体(TCR)治疗：T细胞识别肿瘤抗原通过其单链抗体片段 (scFv)，并将单链抗体片段TCR通过病毒载体克隆到正常T细胞中。因此，正常T细胞变为特异性肿瘤杀伤T细胞。
CAR-T：T细胞通过基因修饰获得具有肿瘤特异性受体T细胞。与常规T细胞识别机制不同，CAR-T细胞识别肿瘤抗原不受到MHC分子的限制。因此，CAR-T细胞能够通过增加共刺激信号分子克服肿瘤的免疫逃逸机制，增强T细胞对肿瘤细胞的杀伤能能力。
在非特异性ACT免疫疗法中，有两种主要应用治疗方法：淋巴因子激活的杀手(lymphokine activated killer,LAK)细胞治疗和细胞因子诱导的杀手(cytokineinduced killer,CIK)细胞治疗。
LAK细胞治疗：LAK细胞一方面利用IL-2刺激外周血淋巴细胞中的免疫细胞，包括NK细胞和T细胞等，另一方面通过过表达FAS配体，增强对靶细胞的识别能力，并通过释放穿孔素和颗粒酶细胞杀死肿瘤细胞。
CIK细胞治疗：CIK细胞来源于病人或健康人的外周血淋巴细胞(PBL)，在ex vivo条件下受到anti-CD3抗体、IFN-γ和IL-2刺激下扩增。CIK细胞主要通过FasL和穿孔素发挥抗肿瘤的作用。
免疫检查点是人体免疫系统中的保护性分子，在正常机体中防止由于T细胞过度活化引起的炎性损伤。肿瘤细胞能够利用这一特性，过度表达免疫检查点分子，抑制机体的免疫反应，逃避人体免疫系统的监视和杀伤，从而促进肿瘤细胞的生长。免疫检查点抑制剂治疗可以通过抑制肿瘤微环境中的免疫检查点活性，重新激活T细胞对肿瘤的免疫应答反应，实现抗肿瘤的效果。T细胞的完全活化受“双信号”系统调节：第一信号来自其自身TCR(T细胞受体)与抗原的MHC的特异性结合，即 T细胞识别抗原；第二信号来自共刺激分子，该信号参与由抗原呈递细胞(APC)表达的共刺激分子与T细胞表面上的相应受体或配体(例如CD28)的相互作用。例如CD28-B7是正向共刺激信号，而负向共刺激分子主要是CTLA4-B7途径和PD-1/PD-L1途径。在肿瘤细胞侵入后，这种抑制途径被肿瘤细胞利于抑制T细胞活化，从而逃避免疫系统的清除作用。
PD-1(CD279)最早于1992年被报道，人PD-1编码基因PDCD1位于2q37.3，全长2097bp，由6个外显子组成，翻译产物为288个氨基酸组成的PD-1前体蛋白，剪切前20个氨基酸组成的信号肽后得到成熟蛋白质。PD-1包括胞外免疫球蛋白可变区IgV结构域，疏水跨膜结构域和胞内结构域，胞内尾部结构域N端ITIM基序包含2个磷酸化位点，C端则是一个ITSM基序。PD-1是膜蛋白，属于CD28免疫球蛋白超家族，主要表达在激活后的T细胞表面，此外还在胸腺的CD4-CD8-T细胞、活化的NK细胞和单核细胞有低丰度表达。PD-1有2个配体，分别是B7蛋白家族的PD-L1(CD274,B7-H1)和PD-L2(CD273,B7-DC)，PD-L1和PD-L2 氨基酸序列有40％相同。两者区别主要在于表达模式不同，PD-L1组成性的低表达于APCs、非造血细胞(如血管内皮细胞、胰岛细胞)和免疫豁免部位(如胎盘、睾丸和眼睛)，炎性细胞因子如I型和II型干扰素、 TNF-α和VEGF等均可以诱导PD-L1的表达。PD-L2则只在被激活的巨噬细胞和树突细胞中有表达。PD-1与PD-L1在激活的T细胞结合后，PD-1 的ITSM基序发生酪氨酸磷酸化，进而导致下游蛋白激酶Syk和PI3K的去磷酸化，抑制下游AKT、ERK等通路的活化，最终抑制T细胞活化所需基因及细胞因子的转录和翻译，发挥负向调控T细胞活性的作用。
在肿瘤细胞中，肿瘤细胞及肿瘤微环境通过上调PD-L1表达并与肿瘤特异的CD8+T细胞表面的PD-1结合，负调控T细胞活性，抑制免疫反应。肿瘤细胞可以通过以下4种途径上调PD-L1表达：1.编码PD-L1 的基因扩增(9p24.1)；2.EGFR、MAPK、PI3K-Akt信号通路激活，HIF-1 转录因子等可以从转录水平上调PD-L1的表达；3.EB病毒的诱导(EB 病毒阳性的胃癌和鼻咽癌表现为PD-L1高表达)；4.表观遗传学的调控。在肿瘤微环境中，interferon-γ等炎症因子的刺激同样可以诱导PD-L1 和PD-L2的表达。炎症因子可以诱导肿瘤微环境中其他细胞，包括巨噬细胞、树突状细胞和基质细胞表达PD-L1和PD-L2，而能够识别肿瘤抗原的肿瘤浸润性T细胞能够分泌interferon-γ，进而诱导PD-L1表达上调，这一过程被称为“适应性免疫抵抗”，肿瘤细胞通过这一机制可以实现自我保护。有越来越多的证据表明肿瘤利用PD-1依赖的免疫抑制免疫逃避。在各种实体瘤和血液系统恶性肿瘤种均已经发现PD-L1和 PD-L2的高表达。此外，PD-Ls的表达与肿瘤细胞的不良预后之间具有很强相关性，证明了包括食道癌、胃癌、肾癌、卵巢癌、膀胱癌、胰腺癌和黑色素瘤等。
目前FDA已经批准上市的PD-1治疗性单抗有Nivolumab(Opdivo， 2014年9月)，Pembrolizumab(Keytruda，2014年12月)和Cemiplimab (Libtayo，2018年9月)，已上市的PD-L1治疗性单抗有Atezolizumab (Tecentriq，2014年9月)，avelumab(Bavencio，2016年5月)以及Duravulumab(Imfinzi，2017年5月)，已批准的适应症见下表所示
Figure BDA0002551001190000051
此外还有Pidilizumab，AMP-224，AMP-514，PDR001等PD-1单抗和 BMS-936559，CK-301等PD-L1单抗处于研发和临床试验中。
然而现有的单抗，亲和力并未达到理想状态，并且由于体积较大，因而免疫原性较强。
发明内容
本发明的目的在于提供一种抗PD-L1纳米抗体及其Fc融合蛋白和应用，以解决上述问题。
本发明提供一种抗PD-L1纳米抗体，其特征在于：至少包含一个VHH 片段，在所述VHH片段中，包含CDR1、CDR2和CDR3三个氨基酸片段,CDR1、CDR2、CDR3分别选自以下序列：
1)SEQ ID No.44至SEQ ID No.60所示的CDR1；
2)SEQ ID No.61至SEQ ID No.82所示的CDR2；
3)SEQ ID No.83至SEQ ID No.99所示的CDR3。
进一步，本发明的抗PD-L1纳米抗体，其特征在于：其序列如： SEQIDNo.1至SEQNo.41所示。
本发明还提供一种抗PD-L1纳米抗体的Fc融合蛋白，其特征在于：包含如权利要求1或2所述的抗PD-L1纳米抗体以及Fc段，所述Fc段的序列如SEQ ID No.42所示。
进一步，本发明的抗PD-L1纳米抗体，其特征在于：其序列中，除 CDR1、CDR2和CDR3以外，有80％的氨基酸序列与SEQIDNo.1至SEQNo.41 所示的序列相同。
本发明还提供一种抗PD-L1纳米抗体在制备阻断PD-L1和PD-1结合试剂中的应用。
本发明还提供一种抗PD-L1纳米抗体的Fc融合蛋白在制备阻断 PD-L1和PD-1结合试剂中的应用。
进一步，本发明的抗PD-L1纳米抗体其特征在于：其用量为20ug/ml 至0.000128ug/ml。
进一步，本发明的抗PD-L1纳米抗体人源化改造后，其特征在于：其序列如SEQ IDNo.100至SEQ ID No.105所示。
本发明还提供一种人源化抗PD-L1纳米抗体的Fc融合蛋白，其特征在于：包含如权利要求8所述的抗PD-L1纳米抗体以及Fc段，所述Fc 段的序列如SEQ ID No.42所示。
上述任一项所述的纳米抗体或者其Fc融合蛋白，在制备治疗癌症、感染或免疫调节疾病的药物中的应用。
上述任一项所述的纳米抗体，在制备抑制肿瘤生长的药物中的应用。
上述任一项所述的纳米抗体或者其Fc融合蛋白的应用中：癌症或肿瘤选自下组织或部位：结直肠、乳腺、卵巢、胰腺、胃、食管、前列腺、肾、宫颈、骨髓癌、淋巴癌、白血病、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、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，或者骨髓增生异常综合症。
发明的有益效果
本发明的纳米抗体及其Fc融合蛋白特异性强，亲和力高，对人的免疫原性弱，具有显著的抗肿瘤效果。
附图说明
图1A是PD-L1纳米抗体编号QP1120、QP1122、QP1123、QP1124、QP1125、 QP1126、QP1127、QP1128、QP1129、QP1130和QP1139的human Fc融合蛋白对PD-L1蛋白的结合曲线；
图1B是PD-L1纳米抗体编号QP1140、QP1141、QP1142、QP1143、QP1144、 QP1145、QP1146、QP1147、QP1148、QP1149、和QP1150的human Fc融合蛋白对PD-L1蛋白的结合曲线；
图1C是PD-L1纳米抗体编号QP1151、QP1152、QP1153、QP1154、QP1155、 QP1156、QP1157、QP1158、QP1159、QP1160、和QP1161的human Fc融合蛋白对PD-L1蛋白的结合曲线；
图1D是PD-L1纳米抗体编号QP1162、QP1163、QP1164、QP1165、QP1166、 QP1168和QP1169的human Fc融合蛋白对PD-L1蛋白的结合曲线；
图2A是PD-L1纳米抗体编号QP1120、QP1122、QP1123、QP1124、QP1125、QP1126、QP1127、QP1128、QP1129和QP1130的human Fc融合蛋白对生物素化小鼠PD-L1蛋白结合曲线；
图2B是PD-L1纳米抗体编号QP1122、QP1142、QP1143、QP1144、QP1145、 QP1146、QP1147、QP1148、QP1149、QP1150和QP1151的human Fc融合蛋白对生物素化小鼠PD-L1蛋白结合曲线；
图2C是PD-L1纳米抗体编号QP1126、QP1152、QP1153、QP1154、QP1155、 QP1156、QP1157、QP1158、QP1159、QP1160、QP1161的human Fc融合蛋白对生物素化小鼠PD-L1蛋白结合曲线；
图2D是QP1162、QP1163、QP1164、QP1165、QP1166、QP1168、QP1169、 QP1139、QP1140、和QP1141、的human Fc融合蛋白对生物素化小鼠PD-L1 蛋白结合曲线；
图3A是PD-L1纳米抗体编号QP1120、QP1162、QP1163、QP1164、QP1165、 QP1166、QP1168、QP1169、QP1139、QP1140和QP1141对PD-1/PD-L1相互作用阻断曲线；
图3B PD-L1纳米抗体：编号QP1122、QP1142、QP1143、QP1144、QP1145、 QP1146、QP1147、QP1148、QP1149、QP1150和QP1151的human Fc融合蛋白对PD-1/PD-L1相互作用阻断曲线；
图3C PD-L1纳米抗体编号QP1126、QP1152、QP1153、QP1154、QP1155、QP1156、QP1157、QP1158、QP1159、QP1160、QP1161的human Fc融合蛋白对PD-1/PD-L1相互作用阻断曲线；
图4A是PD-L1纳米抗体编号QP1120、QP1162、QP1163、QP1164和QP1165 的human Fc融合蛋白对PD-1/PD-L1相互作用阻断曲线；
图4B是PD-L1纳米抗体编号QP1166、QP1168、QP1169、QP1122、QP1126 的human Fc融合蛋白对PD-1/PD-L1相互作用阻断曲线；
图4C是PD-L1纳米抗体编号QP1139、QP1140、QP1141、QP1142、QP1143 的human Fc融合蛋白对PD-1/PD-L1相互作用阻断曲线；
图4D是PD-L1纳米抗体编号QP1144、QP1145、QP1146、QP1147和QP1148 的human Fc融合蛋白：对PD-1/PD-L1相互作用阻断曲线；
图4E是PD-L1纳米抗体编号QP1149、QP1150、QP1151、QP1152和QP1153 的human Fc融合蛋白对PD-1/PD-L1相互作用阻断曲线；
图4F是PD-L1纳米抗体编号QP1149、QP1155、QP1156、QP1157和QP1158 的human Fc融合蛋白对PD-1/PD-L1相互作用阻断曲线；
图4G是PD-L1纳米抗体编号QP1159、QP1160和QP1161的human Fc融合蛋白对PD-1/PD-L1相互作用阻断曲线；
图5A是PD-L1纳米抗体编号QP1120、QP1162、QP1163、QP1164、QP1165 和QP1166的human Fc融合蛋白：对人非小细胞肺癌细胞HCC827结合曲线；
图5B是PD-L1纳米抗体编号QP1168、QP1169、QP1122、QP1126、QP1139 和QP1141的human Fc融合蛋白：对人非小细胞肺癌细胞HCC827结合曲线；
图5C是PD-L1纳米抗体编号QP1142、QP1149、QP1151、QP1156、QP1157、 QP1158的human Fc融合蛋白对人非小细胞肺癌细胞HCC827结合曲线；
图6是人源化PD-L1纳米抗体编号QP1162、QP320、QP321、QP322、QP1166、 QP323、QP324、QP325的human Fc融合蛋白对生物素化人PDL1蛋白结合曲线；
图7是人源化PD-L1纳米抗体编号QP1162、QP320、QP321、QP322、QP1166、 QP323、QP324、QP325的human Fc融合蛋白对PD-1/PD-L1相互作用阻断曲线；
图8是抗CLDN18.2/抗PD-L1双特异抗体分子的结构示意图；
图9是抗CLDN18.2/抗PD-L1双特异抗体PD-L1功能活性鉴定(混合淋巴细胞反应MLR中QP3711461对T细胞激活后产生的细胞因子IFNγ的浓度依赖结果。
图10是抗CLDN18.2/抗PD-L1双特异抗体PD-L1功能活性鉴定(混合淋巴细胞反应MLR中QP3711461对T细胞激活后产生的细胞因子IL-2的浓度依赖结果。
图11是抗CLDN18.2/抗PD-L1双特异抗体在免疫靶点人源化转基因小鼠 C57BL/6-hPDL1模型MC38-hPDL1中的肿瘤生长曲线结果。
具体实施方式
以下进一步详细描述本发明的技术方案。
实施例1PD-L1纳米抗体Fc融合蛋白的制备方法：
1.针对PD-L1的纳米抗体的筛选
1.1文库构建
a)经镍柱纯化得到免疫骆驼的PD-L1-his融合蛋白，序列如 SEQ ID NO.43所示。选1只新疆双峰驼进行皮下多点注射免疫，四次免疫后采集50mL外周血分离PBMC；
b)用TRIzol试剂提取总RNA。电泳鉴定RNA纯度足够后，使用Super
Figure BDA0002551001190000111
IIIFirst-Strand Synthesis System for RT-PCR转录8ug RNA，2轮巢式PCR后用DNA产物纯化试剂盒回收、纯化得到目的核酸片段。
c)将噬菌体载体pComb3XSS与目的片段分别用sfiI进行酶切，50℃过夜酶切，然后割胶回收目的片段。连接摩尔比例为 Vector∶VHH＝1∶3。
d)电转化至TG1大肠杆菌后立即加入1mL SOC培养基复苏， 37℃，180rpm复苏45min，然后离心，加入5mL SOC重悬，取10 μL测定库容量，其余涂布于200mm的平板上共8块。第二天10-5 共有104个克隆，因此库容量为5.04×109(104*5*100*105)。随机从滴度平板上挑取48个克隆进行鉴定，结果表明插入率100％，且大小正确。
1.2针对PD-L1的纳米抗体淘选
a)经过3轮淘选后，获得的与PD-L1结合的噬菌体克隆铺在一块96孔板中，并加入150ul 2×TY-Amp-0.1％蔗糖培养基，37℃培养3h；
b)每孔加入30ul 2×TY-Amp-5mM IPTG(IPTG最终浓度为 1mM)，诱导可溶性抗体片段表达，30℃培养过夜；
c)ELISA板包被PD-L1-his蛋白2ng/ul，50ul/孔，4℃过夜；
d)1×PBS洗板1次，加入200ul 2％Milk-PBS封闭ELISA板， 37℃孵育1h。
e)过夜培养的菌液，4000g离心10mins，转移上清至新的96 孔板中。
f)1×PBST洗板2次，加入25ul 2％Milk-PBS封闭液，再加入25ul培养上清，混匀。25℃孵育1h。
g)1×PBST洗板3次，加入100ul anti-Fab-HRP antibody(1∶5000in 2％Milk-PBS)，25℃孵育1h。
h)1×PBST洗板4次，加入TMB 100ul/孔，室温避光显色10min，加入2M H2SO450ul/孔，终止反应，上机于450nm读值；
i)样品孔读值高于对照孔读值2倍以上认为是阳性克隆孔，将阳性孔菌液扩大培养并提取质粒测序；将CDR1、2、3均相同的序列视为同一个克隆，从而获得41个独特的纳米抗体序列，序列如SEQ ID NO：1-41所示。
为了方便筛选进行，将41个克隆转化为C端为人IgG1的PD-L1-FC 融合蛋白。重新构建的质粒在HEK293细胞中进行表达，通过protein A 亲和层析纯化，除QP1121-FC外，一共获得40个候选PD-L1-FC融合蛋白，序列为SEQ ID NO.1-41所示的纳米抗体后端连接SEQID NO.42的 Fc段所组成。相应的纳米抗体的Fc融合蛋白的编号在相应的纳米抗体后加Fc后缀形成。
2.PD-L1纳米抗体Fc融合蛋白对PD-L1蛋白结合曲线
a)包被PDL1-mFc融合蛋白，1ug/ml，100ul/孔，4℃过夜；
b)1×PBS洗板3次，加入3％BSA封闭，250ul/孔，室温孵育1h；
c)1×PBST洗板3次，1×PBS洗板3次，加入5倍梯度稀释(10ug/ml 至0.000128ug/ml)的待测抗体，室温孵育1h；
d)1×PBST洗板3次，1×PBS洗板3次，加入HRP-anti-hFc(1∶2500)， 50ul/孔，室温孵育1h；
e)1×PBST洗板3次，1×PBS洗板3次，加入TM B 100uI/孔，室温避光显色10min，加入2M H2SO4 50ul/孔，终止反应，上机于450nm 读值。
结果如图1A至图1D，和表1至表4所示。
表1：与图1A中实验结果对应的各抗体的EC50值
EC50
QP1120-FC 0.0008491
QP1122-FC 0.001204
QP1123-FC 0.001286
QP1124-FC 0.001256
QP1125-FC 0.001066
QP1126-FC 0.001212
QP1127-FC 0.4651
QP1128-FC 0.00333
QP1129-FC 0.003955
QP1130-FC 0.005762
QP1139-FC 0.001327
3280A 0.002289
表2与图1B中实验结果对应的各抗体的EC50值
EC50
QP1140-FC 0.001131
QP1141-FC 0.001456
QP1142-FC 0.001027
QP1143-FC 0.001669
QP1144-FC 0.001197
QP1145-FC 0.001776
QP1146-FC 0.001938
QP1147-FC 0.001695
QP1148-FC 0.00116
QP1149-FC 0.001819
QP1150-FC 0.001391
3280A 0.00246
表3与图1C中实验结果对应的各抗体的EC50值
EC50
QP1151-FC 0.001037
QP1152-FC 0.001559
QP1153-FC 0.001419
QP1154-FC 0.00112
QP1155-FC 0.001707
QP1156-FC 0.001241
QP1157-FC 0.001216
QP1158-FC 0.001674
QP1159-FC 0.0009905
QP1160-FC 0.0007973
QP1161-FC 0.001055
3280A 0.002356
表4与图1D中实验结果对应的各抗体的EC50值
EC50
QP1162-FC 0.0007951
QP1163-FC 0.0007316
QP1164-FC 0.0007242
QP1165-FC 0.0008409
QP1166-FC 0.0008137
QP1168-FC 0.0008302
QP1169-FC 0.0007612
3280A 0.002705
3.PD-L1纳米抗体human Fc融合蛋白对生物素化小鼠PD-L1蛋白结合曲线
a)小鼠PDL1由HEK293表达获得。使用Thermo公司Biotinlytion试剂盒得到生物素化蛋白mPDL1-Biotin；
b)包被Strepavidin，4ug/ml，50ul/孔，4℃过夜；
c)1×PBS洗板3次，加入3％BSA封闭，250ul/孔，室温孵育1h；
d)1×PBST洗板3次，1×PBS洗板3次，加入mPDL1-Biotin，1ug/ml， 50ul/孔，室温孵育1h；
e)1×PBST洗板3次，1×PBS洗板3次，加入5倍梯度稀释10ug/ml 至0.000128ug/ml的待测抗体，室温孵育1h；
f)1×PBST洗板3次，1×PBS洗板3次，加入HRP-anti-hFc(1∶2500)， 50ul/孔，室温孵育1h；
g)1×PBST洗板6次，1×PBS洗板3次，加入TMB 100ul/孔，室温避光显色10min，加入2M H2SO4 50ul/孔，终止反应，上机于450nm 读值。
结果如图2A至图2D所示。
表5是与图2A中实验结果对应的各抗体的EC50值
EC50
QP1120-FC N/A
QP1122-FC ～37681
QP1123-FC 0.01144
QP1124-FC ～185556
QP1125-FC 16.29
QP1126-FC N/A
QP1127-FC 26.98
QP1128-FC 94261
QP1129-FC 0.07072
QP1130-FC ～126139
3280A 0.01098
表6是图2B中实验结果对应的各抗体的EC50值
EC50
QP1122-FC N/A
QP1142-FC N/A
QP1143-FC ～26132
QP1144-FC ～95487
QP1145-FC ～257223
QP1146-FC ～61912
QP1147-FC ～63480
QP1148-FC ～69726
QP1149-FC ～36588
QP1150-FC N/A
QP1151-FC ～44027
3280A 0.008206
表7是图2C中实验结果对应的各抗体的EC50值
EC50
QP1126-FC ～6.932e-008
QP1152-FC ～118144
QP1153-FC 7.139
QP1154-FC 72.92
QP1155-FC ～55064
QP1156-FC 0.09957
QP1157-FC 0.1189
QP1158-FC N/A
QP1159-FC 3.502
QP1160-FC ～47090
QP1161-FC 0.06164
3280A 0.01322
表8是图2D中实验结果对应的各抗体的EC50值
EC50
QP1162-FC 0.04891
QP1163-FC 0.007373
QP1164-FC 0.01443
QP1165-FC 0.01341
QP1166-FC 0.01765
QP1168-FC 0.04416
QP1169-FC 0.1067
QP1139-FC 0.02131
QP1140-FC 0.08768
QP1141-FC ～44849
3280A 0.00701
4.PD-L1纳米抗体human Fc融合蛋白对PD-1/PD-L1相互作用阻断曲线采用竞争性ELISA进行检测
a)包被PD1-hFc融合蛋白，1ug/ml，100ul/孔，4℃过夜；
b)1×PBS洗板3次，加入3％BSA封闭，250ul/孔，室温孵育1h；
c)1×PBST洗板3次，1×PBS洗板3次，加入PDL1-mFc，2ug/ml， 50ul/孔，同时加入等体积5倍梯度稀释(20ug/ml至 0.000256ug/ml)待测抗体，室温孵育1h；
d)1×PBST洗板3次，1×PBS洗板3次，加入HRP-anti-mFc(1∶2500)， 50ul/孔，室温孵育1h；
e)1×PBST洗板3次，1×PBS洗板3次，加入TMB 100ul/孔，室温避光显色10min，加入2M H2SO4 50ul/孔，终止反应，上机于450nm 读值。
结果如图3A至图3C所示。
表9是与图3A中实验结果对应的各抗体的IC50值
IC50
QP1120-FC 0.3191
QP1162-FC ～1.037
QP1163-FC 0.9799
QP1164-FC 0.3334
QP1165-FC 0.3223
QP1166-FC 0.6531
QP1168-FC 0.3524
QP1169-FC ～0.5939
QP1139-FC 0.3279
QP1140-FC 0.6766
QP1141-FC 0.3986
3280A ～1.01
表10是与图3B中实验结果对应的各抗体的IC50值
IC50
QP1122-FC 0.6305
QP1142-FC 0.2909
QP1143-FC ～0.572
QP1144-FC 0.3105
QP1145-FC 0.967
QP1146-FC 0.3395
QP1147-FC ～0.711
QP1148-FC 0.3541
QP1149-FC 0.442
QP1150-FC 0.4183
QP1151-FC 0.3254
3280A ～1.006
表11是与图3C中实验结果对应的各抗体的IC50值
IC50
QP1126-FC 0.3245
QP1152-FC 0.3295
QP1153-FC 0.4048
QF1154-FC 0.2776
QP1155-FC 1.074
QP1156-FC 0.3237
QP1157-FC 0.3289
QP1158-FC 0.247
QP1159-FC 0.2817
QP1160-FC 0.3077
QP1161-FC 0.3263
3280A ～1.07
5.PD-L1纳米抗体human Fc融合蛋白对PD-1/PD-L1相互作用阻断曲线，采用竞争性ELISA，biotin检测
a)使用thermo公司Biotinlytion试剂盒得到生物素化蛋白 PDL1-Biotin；
b)包被PD1-hFc融合蛋白，1ug/ml，100ul/孔，4℃过夜；
c)1×PBS洗板3次，加入3％BSA封闭，250ul/孔，室温孵育1h；
d)1×PBST洗板3次，1×PBS洗板3次，加入PDL1-Biotin，2ug/ml， 50ul/孔，同时加入等体积3倍梯度稀释(100nM至0.0457nM，对应质量浓度为15ug/ml至0.00686ug/ml)待测抗体，室温孵育1h；
e)1×PBST洗板3次，1×PBS洗板3次，加入HRP-Strepavidin(1∶ 5000)，50ul/孔，室温孵育1h；
f)1×PBST洗板6次，1×PBS洗板3次，加入TMB 100ul/孔，室温避光显色10min，加入2M H2SO4 50ul/孔，终止反应，上机于450nm 读值。
结果如图4A至图4G所示。
表12是与图4A中实验结果对应的各抗体的IC50值
IC50
QP1120-FC 4.903
QP1162-FC 4.754
QP1163-FC 14.18
QP1164-FC 11.46
QP1165-FC 13.66
3280A 4.914
表13是与图4B中实验结果对应的各抗体的IC50值
IC50
QP1166-FC 12.75
QP1168-FC 13.45
QP1169-FC 4.551
QP1122-FC 4.085
QP1126-FC 4.291
3280A 4.273
表14是与图4C中实验结果对应的各抗体的IC50值
IC50
QP1139-FC ～11.74
QP1140-FC 14.58
QP1141-FC 13.91
QP1142-FC 13.49
QP1143-FC 14.01
3280A 5.44
表15是与图4D中实验结果对应的各抗体的IC50值
IC50
QP1144-FC 13.3
QP1145-FC 21.99
QP1146-FC 15.24
QP1147-FC 14.39
QP1148-FC 14.15
3280A 4.557
表16是与图4E中实验结果对应的各抗体的IC50值
IC50
QP1149-FC 13.33
QP1150-FC 13.99
QP1151-FC 14.03
QP1152-FC 13.55
QP1153-FC 13.19
3280A 4.721
表17是与图4F中实验结果对应的各抗体的IC50值
IC50
QP1149-FC 14.14
QP1155-FC ～11.71
QP1156-FC ～11.88
QP1157-FC 13.46
QP1158-FC 4.652
3280A 4.881
表18是与图4G中实验结果对应的各抗体的IC50值
IC50
QP1159-FC ～11.66
QP1160-FC 11.94
QP1161-FC 11.94
3280A 4.189
6.PD-L1纳米抗体human Fc融合蛋白对人非小细胞肺癌细胞HCC827 结合曲线
a)人非小细胞肺癌细胞HCC827天然高表达PD-L1。准备对数期生长 HCC827细胞(汇合度80％)，调整浓度并铺板种于costar 96孔板中，1E5细胞/孔，1×PBS洗板一次，加入3％BSA，250ul/孔，37℃孵育1h；
b)加入4倍梯度稀释(33.33nM至0.008nM，对应质量浓度为5ug/mL 至0.001ug/mL)待测抗体，50ul/孔，冰上孵育1h；
c)1×PBS洗板2次，加入PE-anti hFc(1∶200)，50ul/孔，冰上孵育1h；
d)1×PBS洗板3次，上机读数。
结果如图5A至图5C所示。
表19是与图5A中实验结果对应的各抗体的EC50值
EC50
QP1120-FC 0.2912
QP1162-FC 0.2302
QP1163-FC 0.1946
QP1164-FC 0.1926
QP1165-FC 0.2325
QP1166-FC 0.161
3280A 0.3181
表20是与图5B中实验结果对应的各抗体的EC50值
EC50
QP1168-FC 0.1927
QP1169-FC 0.1898
QP1122-FC 0.1719
QP1126-FC 0.1714
QP1139-FC 0.2182
QP1141-FC 0.2044
3280A 0.3181
表20是与图5C中实验结果对应的各抗体的EC50值
EC50
QP1142-FC 0.203
QP1149-FC 0.248
QP1151-FC 0.2359
QP1156-FC 0.1605
QP1157-FC 0.161
QP1158-FC 0.1788
3280A 0.3181
实施例2抗PD-L1纳米抗体的人源化
通过比对IMGT人类抗体重轻链可变区种系基因数据库和MOE软件，分别挑选与QP1162(SEQ ID No.35)、QP1166(SEQ ID No.39)同源性高的重轻链可变区种系基因作为模板，将鼠源抗体的CDR分别移植到相应的人源模板中，形成次序为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4的可变区序列。再选择一些重要的氨基酸残基做回复突变组合。其中氨基酸残基由Kabat编号系统确定并注释。
设计引物PCR搭建各人源化抗体VH基因片段，再与带信号肽及恒定区基因(FC)片段的表达载体pQD进行同源重组，构建抗体全长表达载体VH-FC-pQD。
利用在线软件DNAWorks(v3.2.2)(http：//helixweb.nih.gov/dnaworks/) 设计多条引物合成VH/VK含重组所需基因片段：5’-30bp信号肽 +VH+30bp FC-3’。按照TaKaRa公司Primer STAR GXL DNA聚合酶操作说明书，用上面设计的多条引物，分两步PCR扩增得到VH/VK含重组所需基因片段。带信号肽及恒定区基因(FC)片段的表达载体pQD的构建及酶切，利用限制性内切酶，如BsmBI，识别序列与酶切位点不同的特性设计构建带信号肽及恒定区基因(FC)片段的表达载体pQD。BsmBI酶切载体，切胶回收备用。重组构建表达载体VH-FC-pQD。VH含重组所需基因片段与BsmBI酶切回收表达载体pQD(带信号肽及恒定区基因(FC)片段) 按3∶1比例分别加入DH5H感受态细胞中，0℃冰浴30min，42℃热击 90s，加入5倍体积LB介质，37℃孵育45min，涂布LB-Amp平板，37℃培养过夜，挑取单克隆送测序得到各目的克隆。
各克隆人源化设计轻重链可变区序列及蛋白表达编号如下所示，此表中抗体在其C端融合人IgG1-FC恒定区：
表21：QP1162和QP1166人源化设计
Figure BDA0002551001190000231
同时设计克隆表达人源化前嵌合抗体及对照抗体如下表所示：
表22：人源化前嵌合抗体及对照抗体
Figure BDA0002551001190000232
2.抗PD-L1纳米抗体人源化蛋白表达
293E细胞培养密度维持在0.2-3×106/ml之间，维护阶段培养基 (GIBCOFreestyle 293expression medium)进行培养，转染前一天将待转染细胞离心换液，调整细胞密度为0.5-0.8×106/ml。转染当天，293E细胞密度为1-1.5×106/ml。准备质粒和转染试剂PEI，需转染质粒量为100ug/100ml细胞，使用PEI和质粒的质量比为2∶1。将质粒和PEI进行混匀，静置15min，不宜超过20min。将质粒和PEI混合物缓慢加入293E 的细胞中，放入8％CO2，120rpm，37℃的摇床中培养，转染第五天，水平离心机4700rpm离心20min收集细胞上清。
3.抗PD-L1纳米抗体人源化蛋白纯化
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0.33 ml/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。
4.人源化抗PD-L1纳米抗体活性鉴定(Binding-ELISA)
包被抗体QP1162/QP320/QP321/QP322/QP1166/QP323/QP324/ QP325 0.75ug/ml，QP11801181 1.5ug/ml 50ul/孔，4℃过夜。PBS 3times。封闭：3％BSA 250ul/孔，RT 1h。分别孵育2ug/ml biotin-PDL1-FC 1∶4稀释至不同浓度，室温孵育1h。PBST洗3次，PBS洗3次。孵育二抗： HRP-strepavidin(1∶5000)50ul/孔，PBST洗6次，PBS洗3次。显色：TMB 100ul/孔，显色10min。2M H2SO4 50ul/孔终止。结果如图6和表23所示。
表23：人源化抗PD-L1纳米抗体活性鉴定(Binding-ELISA)结果
QP1162 1162-V1 1162-V2 1162-V3 QP1166 1166-V1 1166-V2 1166-V3
conc.(ug/ml) QP1162 QP320 QP321 QP322 QP1166 QP323 QP324 QP325 QP11801181
2.0000 3.4366 0.2297 0.2251 3.0042 3.4143 2.8564 0.4130 3.4216 3.4911
0.5000 3.4482 0.1419 0.1378 2.7861 3.2805 1.8773 0.2012 3.2090 3.4778
0.1667 3.2597 0.0957 0.0836 2.7811 3.1343 0.7893 0.1285 3.1059 3.7196
0.0556 2.8355 0.0843 0.0756 2.7008 3.2327 0.3141 0.0882 2.7828 3.2350
0.0185 2.5016 0.0792 0.0960 2.0715 2.5660 0.1810 0.1070 2.1246 2.5747
0.0062 1.5396 0.1076 0.1201 1.2735 1.5464 0.1663 0.1502 1.3189 1.9496
0.0021 0.5232 0.1140 0.1561 0.4107 0.5605 0.2577 0.1414 0.5978 0.8702
0.0007 0.1214 0.1248 0.1562 0.1756 0.2169 0.1810 0.1917 0.1817 0.1986
5.人源化抗PD-L1纳米抗体活性鉴定(Blocking-ELISA)
包被蛋白QP1138(PD1-FC)2ug/ml 50ul/孔，4℃过夜。PBS洗3次。封闭：3％BSA250ul/孔，室温孵育1h。分别配制2ug/ml biotin-PDL1-FC 和不同浓度QP1120 15ug/ml，QP11801181 30ug/ml，1∶3稀释，等体积混匀，室温孵育1h。PBST洗3次，PBS洗3次。孵育二抗：HRP-strepavidin(1∶5000)50ul/孔，PBST洗6次，PBS洗3次。显色：TMB 100ul/孔，显色10min。2M H2SO4 50ul/孔终止。结果如图7和表24所示。
表24：人源化抗PD-L1纳米抗体活性鉴定(Blocking-ELISA)
Figure BDA0002551001190000251
7.人源化抗PD-L1纳米抗体SPR鉴定亲和力
表面等离子体共振(SPR)检测亲和力
通过Biacore T200(GE)测定待检分子与蛋白人PD-L1及cynoPD-L1 的亲和力
抗原信息如下：
表25：蛋白编号
蛋白编号 蛋白描述 货号
QPP09.1 PD-L1 Protein，Human，Recombinant(His Tag) SinoBiologic，10084-H08H
QPP10.1 PD-L1 Protein，Cynomolgus，Recombinant(His Tag) SinoBiologic，90251-C08H
表26：SPR亲和力结果
Figure BDA0002551001190000261
实施例3人源化抗PD-L1纳米抗体活性鉴定
构建设计抗CLDN18.2/抗PD-L1双特异抗体分子QP3711461，形式如图8所示。
Figure BDA0002551001190000262
1.PD-L1功能体外活性鉴定(混合淋巴细胞反应MLR)
准备DC(donor1)细胞：复苏PBMC，用EasySepTMHuman Monocyte Isolation Kit(Stemcell 19359)分离单核细胞monocytes，加入 rhGM-CSF(1000U/ml)和rhIL4(500U/ml)，37℃培养细胞6天诱导为iDC；每2-3天半换液，同时补充rhGM-CSF(1000U/ml)和rhIL4(500U/ml)；收集细胞300xg离心5min，用加入rhGM-CSF(1000U/ml)和rhIL4(500U/ml) 的培养基重悬，同时加入LPS(1μg/ml)，37℃继续培养细胞1天诱导为成熟DC；收集细胞，计数备用。
准备T(donor2)细胞：复苏PBMC，用EasySepTMHuman CD4+T Cell Isolation Kit(Stemcell 17952)分离CD4+Tcell。
准备抗体：用培养基1∶5梯度稀释抗体(初始浓度10ug/ml)6个浓度。将DC细胞：T细胞为1∶10的比例混合，加入不同浓度的抗体，混合培养，第2天检测培养上清中IL2的表达，第5天检测培养上清中 IFNg的表达。
在混合淋巴细胞反应实验中，QP3711461对T细胞激活后产生的细胞因子IFNγ及IL-2的浓度有明显的抗体浓度依赖。证明QP3711461中 PD-L1抗体的生物学功能。如图9和图10所示。
2.PD-L1功能体内活性鉴定
利用小鼠结肠癌细胞MC38-hPDL1在转基因小鼠C57BL/6-hPDL1皮下模型，评估抗PD-L1纳米抗体的体内药效。
实验方法：取对数生长期小鼠结肠癌细胞MC38-hPDL1细胞(该细胞敲除小鼠的PDL1，表达人的PDL1)，去除培养液并用PBS洗两次后接种于C57BL/6-hPDL1小鼠右侧胁腹部皮下，接种量：5×105/100μL/只。观察接种后小鼠并监测肿瘤的生长，接种后第8天，平均肿瘤体积达到92.9mm3时，根据肿瘤体积随机分成4组，每组9只。分组当天定义为 D0天，并于D0天开始给药。
实验结果：如图11和表25所示。
表25：肿瘤体积
分组 第27天肿瘤体积 第27天TGI％ P值(t test)
溶媒对照组(PBS) -- --
QP1461371-4mpk 477.00 72.00％ 0.0095**
QP1461371-10mpk 279.97 86.14％ 0.0018**
QP1461371-25mpk 293.96 85.14％ 0.0037**
待测分子为抗CLDN18.2/抗PD-L1双特异抗体QP3711461，给药剂量分别为4mpk、10mpk、25mpk，BIW×3，i.p.给药。给药后第27天PBS 组(阴性对照组)平均肿瘤体积达到1445.20mm3，QP3711461(4mpk) 组平均肿瘤体积477.00mm3，TGI＝72.00％，QP3711461(10mpk)组平均肿瘤体积279.97mm3，TGI＝86.14％，QP3711461(25mpk)组平均肿瘤体积293.96mm3，TGI＝85.14％；各剂量给药组与PBS组的肿瘤体积均有统计学意义极显著差异(t检验，p<0.01)。
该实验说明在免疫靶点人源化转基因小鼠的MC38-hPDL1模型中，本发明的抗CLDN18.2/抗PD-L1双特异抗体中的抗PD-L1分子表现出优越的抗肿瘤能力。
本发明所提供的抗PD-L1纳米抗体编号及对应序列如下所示。
>SEQ ID No.1QP1120
QVQLVESGGGSVQSGGSLRLSCAASGFTYGTYAMSWFRQAPGKEREGVACIDIYGRTSYTDPVKGRFTISQDNAKNTLYLQMNSLKPEDTAMYYCAARDFGYCTASWVHEGFSRYWGQGTQVTVSS
>SEQ ID No.2QP1121
QVQLVESGGGSVHAGGSLRLSCVRVSMYTYTGTCMAWFRQAPGKEREGVAGLWTGDGVTYYADSVKGRFTISQDDAKNTLYLQMDSLKPEDTAMYYCASNGMCGQYWALEDEYKYWGQGTQVTVSS
>SEQ ID No.3QP1122
QVQLVESGGGSVQAGGSLTLSCAVSGNTYSTNVMGWFRQAPGKEREGVAAILAGGRNTYYADSVKGRFTISQDNAKNTVYLQMNSLKPEDTAMYYCAADTRAAFWNIGPLNSDQYNIWGQGTQVTVSS
>SEQ ID No.4QP1123
QVQLVESGGGSVQSGGSLRLSCAASGYTVRHYCMGWFRQTPGKEREGVASIDTFGIPKYADSVKGRFTISQDNAKNTLNLQMDSLKPEDTAMYFCAGRSYTNCRDGPPSASHYSHWGQGTQVTVSS
>SEQ ID No.5QP1124
QVQLVESGGGLVQPGGSLRLSCTAPGFTSNTCAMGWYRQAAGMQREWVSSISKYGITTYANSVKGRFTISKDKAEDTVYLQMNNLKPEDTAMYVCKTFSCRNRGGAYLADA WGQGTQVTVSS
>SEQ ID No.6QP1125
QVQLVESGGGLVQPGGSLRLSCTAPGFTDKTCAMAWYRQVAGIEREWVSSISTLGTTNYASSVKGRFTISKDNAKDTVYLQMNNLKPEDTAMYVCKTFSCRNRGGSYLPDTW GQGTQVTVSS
>SEQ ID No.7 QP1126
QVQLVESGGGSVQAGGSLTLSCAVSGNTYSTKYMSWFRQAPGKEREGVAAILAGGRNTYYADSVKGRFTISQDNAKNTVYLRMNSLKPEDTAMYYCAADTRAALWYIGPLNSDQYNTWGQGTQVTVSS
>SEQ ID No.8 QP1127
QVQLVESGGGSVQSGGSLTLSCAISGYAYATYSMAWFRQAHGKEREGVAAINSDGHTTYVDSVKGRFTISRDNTNKNSYTLTLTMNNLNPEDTAMYYCAATSQLGFWAQKLWEAIRDGTWSPSTTDFGFWGRGTQVTVSS
>SEQ ID No.9 QP1128
QVQLVESGGGSVQSGGSLRLSCAASGYTASAYYMAWFRQNSRKQREGVAAINRDGDTKYADSVKGRFTISRDDAKNTLYLQMNSLKPEDTAVYYCAASDWSRLYKIYWLDDNYYVRWGQGTQVTVSS
>SEQ ID No.10 QP1129
QVQLVESGGGSVQSGGSLRLSCAASGYSSSRYSVGWFRQAPGKEREGVAGQTPRGTTTYADSVKDRFTISRDNAKNTVYLQMNSLKPEDTAMYYCAAGQALLWASLRQTSYQFWGQGTQVTVSS
>SEQ ID No.11 QP1130
QVQLVESGGGLVQPGGSLRLSCAASGFTFSTSTMMWVRRAPGKGLEWVSGIHNDGGPIAYADSVKGRFTISRDNAKNTLYLQMTSLKSEDTALYYCARGWYFSGDYVPMTQG TQVTVSS
>SEQ ID No.12 QP1139
QVQLVESGGGSVQAGGSLTLSCAVSGNTYGTNAMGWFRQAPGKEREGVAAILGGGRNTYYADSVKGRFTISQDNAKNTVYLQMNSLKPEDTAMYYCAADTRAAFWNIGPLNSDQYNIWGQGTQVTVSS
>SEQ ID No.13 QP1140
QVQLVESGGGSVQAGGSLTLSCVVSGNTYSTKYMGWFRQAPGKEREGVAAILAGGRNTYYADSVKGRFTISQDNAKNTVYLQMNSLKPEDTAMYYCAADTRAAFWNIGPLNSDQYNIWGQGTQVTVSS
>SEQ ID No.14 QP1141
QVQLVESGGGSVQAGGSLTLSCAVSGNTYSTNAMGWFRQAPGKEREGVAAILGGGRNTYYADSVKGRFTISQDNAKNTVYLQMNSLKPEDTAMYYCAADTRAAFWNIGPLNSDQYNIWGQGTQVTVSS
>SEQ ID No.15 QP1142
QVQLVESGGGSVQAGGSLTLSCAVSGNTYSTNIMGWFRQAPGKEREGVAAILAGGRNTYYADSVKGRFTISQDNAKNTVYLQMNSLKPEDTAMYYCAADTRAAFWNIGPLNSDQYNIWGQGTQVTVSS
>SEQ ID No.16 QP1143
QVQLVESGGGSVQAGGSLTLSCAVSGNTYSTKYMGWFRQAPGKEREGVAAILAGGRDTYYADSVKGRFTISQDNAKNTVYLQMNSLKPEDTAMYYCAADTRAAFWYIGPLNSDQYNIWGQGTQVTVSS
>SEQ ID No.17 QP1144
QVQLVESGGGSVQAGGSLTLSCAVSGNTYSTKYMGYFRQAPGKEREGVAAILAGGRNTNYADSVKGRFTISQDNAKNTVYLQMNSLKPEDTAMYYCAADTRAAFWYIGPLNSDQYNIWGQGTQVTVSS
>SEQ ID No.18 QP1145
QVQLVESGGGSVQAGGSLTLSCAVSGNTYSTKYMAWFRQAPGKEREGVAAILAGGRNTSYADSVKGRFTISQDNAKNTVYLQTNSLKPEDTAMYYCAADTRAAFWYIGPLNSHQYNIWGQGTQVTVSS
>SEQ ID No.19 QP1146
QVQLVESGGGSVQAGGSLTLSCAVSGNTYSTNYMGWFRQAPGKEREGVAAILVGGRNTYYADSVKGRFTISQDNAKNLVYLQMNSLKPEDTAMYYCAADTRAAFWNIGPLNSDQYNIWGQGTQVTVSS
>SEQ ID No.20 QP1147
QVQLVESGGGSVQAGGSLTLSCAVSGNTYSTKYMGWFRQAPGKEREGVAAILAGGRNTYYADSVKGRFTISQDNAKNTVYLQMNSLKPEDTAMYYCAADTRAAFWYIGPLNSDQYNIWGQGTQVTVSS
>SEQ ID No.21 QP1148
QVQLVESGGGSVQAGGSLTLSCAVSGNTYSTNYMGWFRQAPGKEREGVAAILAGGRNTAYADSVKGRFTISQDNAKNTVYLQMNSLKPEDTAMYYCAADTRAAFWNIGPLNSDQYNIWGQGTQVTVSS
>SEQ ID No.22 QP1149
QVQLVESGGGSVQAGGSLTLSCAVSGNTYSTNYMGWFRQAPGKEREGVAAILAGGRNTYYADSVKGRFTISQDNAKDTVYLQMNSLKPEDTAMYYCAADTRAAFWNIGPLNSDQYNIWGQGTQVTVSS
>SEQ ID No.23 QP1150
QVQLVESGGGSVQAGGSLTLSCAVSGNTYSTKYMGWFRQAPGKEREGVAAILAGGRNTHYADSVKGRFAISQDNAKNTVYLQMNSLKPEDTAMYYCAADTRAAFWNIGPLNSDQYNIWGQGTQVTVSS
>SEQ ID No.24 QP1151
QVQLVESGGGSVQAGGSLTLSCAVSGNTYSTNYMGWFRQAPGKEREGVAAILTGGRNTYYADSVKGRFTISQDNAKNTVYLQMNSLKPEDTAMYYCAADTRAAFWNIGPLNSDQYNIWGQGTQVTVSS
>SEQ ID No.25 QP1152
QVQLVESGGGSVQAGGSLTLSCAISGNTYSTKYMGWFRQAPGKEREGVAAILAGGRNTYYADSVKGRFTISQDNAKNTVYLQMHSLKPEDTAMYYCAADTRAAFWYIGPLNSDQYNLWGQGTQVTVSS
>SEQ ID No.26 QP1153
QVQLVESGGGSVQAGGSLTLSCAVSGNTYSTKYMGWFRQAPGKEREGVAAILAGGRNTDYADSVKGRFTISQDNAKNTVYLQMNSLKPEDTAMYYCAADTRAAFWSIGPLNSDQYNIWGQGTQVTVSS
>SEQ ID No.27 QP1154
QVQLVESGGGSVQAGGSLTLSCAVSGNTYSTNYMAWFRQAPGKEREGVAAIRAGGRNTDYADSVKGRFTISQDNAKNTVYLQMNSLKPEDTAMYYCAADTRAAFWYIGPLNSDQYNIWGQGTQVTVSS
>SEQ ID No.28 QP1155
QVQLVESGGGSVQAGGSLTLSCEVSGSTYSTNYMGWFRQAPGKEREGVAAILAGGRNTDYADSVKGRFTISQDNAKNTVYLQMNSLKPEDTAMYYCAADTRAAFWYIGPLNSDQYNIWGQGTQVTVSS
>SEQ ID No.29 QP1156
QVQLVESGGGSVQAGGSLTLSCAVSGNTYSTKYMGWFRQAPGKEREGVAAILAGGRNTYYADSVKGRFTISQDSAKNTVYLQMNSLKPEDTAMYYCAADTRAAFWYIGPLNSDQYNSWGQGTQVTVSS
>SEQ ID No.30 QP1157
QVQLVESGGGSVQAGGSLTLSCAVSGNTYSTNYMGWFRQAPGKEREGVAAILVGGRNTYYADPVKGRFTISQDNAKNLVYLQMNSLKPEDTAMYYCAADTRAAFWNIGPLNSDQYNIWGQGTQVTVSS
>SEQ ID No.31 QP1158
QVQLVESGGGSVQAGGSLTLSCAVSGNTYSTKYMGWFRQAPGKEREGVAAILAGGRNTYYADSVKGRFTISQDNAKNTVYLRMNSLKPEDTAMYYCAADTRAALWYIGPLNSDQYNTWGQGTQVTVSS
>SEQ ID No.32 QP1159
QVQLVESGGGSVQAGGSLTLSCAVSGNTYSTKYMGWFRQAPGKEREGVAAILAGGRNTSYADSVKGRFTISQDNAKNTVYLQMNSLKPEDTAMYYCAADTRAAFWYIGPLNSDQYNIWGQGTQVTVSS
>SEQ ID No.33 QP1160
QVQLVESGGGSVQAGGSLTLSCAISGNTYSTKYMGWFRQAPGKEREGVAAILAGGRNTYYADSVKGRFTISQDNAKNTVYLQMNSLKPEDTAMYYCAADARAAFWYIGPLNSDQYNIWGQGTQVTVSS
>SEQ ID No.34 QP1161
QVQLVESGGGSVQAGGSLTLSCAVSGNTYSTNYMAWFRQAPGKEREGVAAIRVGGRNTDYADSVKGRFTISQDNAKNTVYLQMNSLKPEDTAMYYCAADTRAAFWYIGPLNSDQYNIWGQGTQVTVSS
>SEQ ID No.35 QP1162
QVQLVESGGGSVQSGGSLRLSCAASGFTYGTYAMSWFRQAPGKEREGVACIDIYGRASYTDPVKGRFTISQDNAKNTLYLQMNSLKPEDTAMYYCAARDFGYCTASWVHEGFSRYWGQGTQVTVSS
>SEQ ID No.36 QP1163
QVQLVESGGGSVQSGGSLRLSCAASGFTYGTYAMSWFRQAPGKEREGVACIDIYGRTSYTDPVKGRFTISQDNAKNTLYLQMNSLKPEDTAMYYCAARDFGYCTASWVHAGFSRYWGQGTQVTVSS
>SEQ ID No.37 QP1164
QVQLVESGGGSVQSGGSLRLSCAASGFTYGTYAMSWFRQTPGKEREGVACIDIYGRTSYTDPVKGRFTISQDNAKNTLYLQMNSLKPEDTAMYYCAARDFGYCTASWVHEGFSRYWGQGTQVTVSS
>SEQ ID No.38 QP1165
QVQLVESGGGSVQSGGSLRLSCAASGFTYGAYAMSWFRQAPGKEREGVACIDIYGRTSYTDPVKGRFTISQDNAKNTLYLQMNSLKPEDTAMYYCAARDFGYCTASWVHEGFSRYWGQGTQVTVSS
>SEQ ID No.39 QP1166
QVQLVESGGDSVQPGGSLRLSCAASGFTYGTYAMSWFRQAPGKEREGVACIDIYGRTSYTDPVKGRFTISQDNAKNTLYLQMNSLKPEDTAMYYCAARDFGYCTASWVHEGFSRYWGQGTQVTVSS
>SEQ ID No.40 QP1168
QVQLVESGGGSVQSGGSLRLSCAASGFTYETYAMSWFRQAPGKEREGVACIDIYGRTSYTDPVKGRFTISQDNAKNTLYLQMNSLKPEDTAMYYCAARDFGYCTASWVHEGFSRYWGQGTQVTVSS
>SEQ ID No.41 QP1169
QVQLVESGGGSVQSGGSLRLSCAASGFTYGTYAMSWFRQAPGKEREGVACIDIYGRTSYTDPVKGRFTISQDNAKNTLYLQMNSLKPGDTAMYYCAARDFGYCTASWVHEGFSRYWGQGTQVTVSS
>SEQ ID No.42 Human IgG1：
EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA PIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K
>SEQ ID No.43 PD-L1-his
MRIFAVFIFMTYWHLLNAFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWE MEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRC MISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDH QVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAH PPNEREQKLISEEDLHHHHHH
>SEQ ID No.44 QP1120-CDR1
TYAMS
>SEQ ID No.45 QP1121-CDR1
GTCMA
>SEQ ID No.46 QP1122-CDR1
TNVMG
>SEQ ID No.47 QP1123-CDR1
HYCMG
>SEQ ID No.48 QP1124-CDR1
TCAMG
>SEQ ID No.49 QP1125-CDR1
TCAMA
>SEQ ID No.50 QP1126-CDR1
TKYMS
>SEQ ID No.51 QP1127-CDR1
TYSMA
>SEQ ID No.52 QP1128-CDR1
AYYMA
>SEQ ID No.53 QP1129-CDR1
RYSVG
>SEQ ID No.54 QP1130-CDR1
TSTMM
>SEQ ID No.55 QP1139-CDR1
TNAMG
>SEQ ID No.56 QP1140-CDR1
TKYMG
>SEQ ID No.57 QP1142-CDR1
TNIMG
>SEQ ID No.58 QP1145-CDR1
TKYMA
>SEQ ID No.59 QP1154-CDR1
TNYMA
>SEQ ID No.60 QP1165-CDR1
AYAMS
>SEQ ID No.61 QP1120-CDR2
CIDIYGRTSYTDPVKG
>SEQ ID No.62 QP1121-CDR2
GLWTGDGVTYYADSVKG
>SEQ ID No.63 QP1122-CDR2
AILAGGRNTYYADSVKG
>SEQ ID No.64 QP1123-CDR2
SIDTFGIPKYADSVKG
>SEQ ID No.65 QP1124-CDR2
SISKYGITTYANSVKG
>SEQ ID No.66 QP1125-CDR2
SISTLGTTNYASSVKG
>SEQ ID No.67 QP1127-CDR2
AINSDGHTTYVDSVKG
>SEQ ID No.68 QP1128-CDR2
AINRDGDTKYADSVKG
>SEQ ID No.69 QP1129-CDR2
GQTPRGTTTYADSVKD
>SEQ ID No.70 QP1130-CDR2
GIHNDGGPIAYADSVKG
>SEQ ID No.71 QP1139-CDR2
AILGGGRNTYYADSVKG
>SEQ ID No.72 QP1143-CDR2
AILAGGRDTYYADSVKG
>SEQ ID No.73 QP1144-CDR2
AILAGGRNTNYADSVKG
>SEQ ID No.74 QP1145-CDR2
AILAGGRNTSYADSVKG
>SEQ ID No.75 QP1146-CDR2
AILVGGRNTYYADSVKG
>SEQ ID No.76 QP1148-CDR2
AILAGGRNTAYADSVKG
>SEQ ID No.77 QP1150-CDR2
AILAGGRNTHYADSVKG
>SEQ ID No.78 QP1151-CDR2
AILTGGRNTYYADSVKG
>SEQ ID No.79 QP1153-CDR2
AILAGGRNTDYADSVKG
>SEQ ID No.80 QP1157-CDR2
AILVGGRNTYYADPVKG
>SEQ ID No.81 QP1161-CDR2
AIRVGGRNTDYADSVKG
>SEQ ID No.82 QP1162-CDR2
CIDIYGRASYTDPVKG
>SEQ ID No.83 QP1120-CDR3
ARDFGYCTASWVHEGFSRY
>SEQ ID No.84 QP1121-CDR3
SNGMCGQYWALEDEYKY
>SEQ ID No.85 QP1122-CDR3
ADTRAAFWNIGPLNSDQYNI
>SEQ ID No.86 QP1123-CDR3
GRSYTNCRDGPPSASHYSH
>SEQ ID No.87 QP1124-CDR3
KTFSCRNRGGAYLADA
>SEQ ID No.88 QP1126-CDR3
ADTRAALWYIGPLNSDQYNT
>SEQ ID No.89 QP1127-CDR3
ATSQLGFWAQKLWEAIRDGTWSPSTTDFGF
>SEQ ID No.90 QP1128-CDR3
ASDWSRLYKIYWLDDNYYVR
>SEQ ID No.91 QP1129-CDR3
AGQALLWASLRQTSYQF
>SEQ ID No.92 QP1130-CDR3
GWYFSGDYVP
>SEQ ID No.93 QP1143-CDR3
ADTRAAFWYIGPLNSDQYNI
>SEQ ID No.94 QP1145-CDR3
ADTRAAFWYIGPLNSHQYNI
>SEQ ID No.95 QP1152-CDR3
ADTRAAFWYIGPLNSDQYNL
>SEQ ID No.96 QP1153-CDR3
ADTRAAFWSIGPLNSDQYNI
>SEQ ID No.97 QP1156-CDR3
ADTRAAFWYIGPLNSDQYNS
>SEQ ID No.98 QP1160-CDR3
ADARAAFWYIGPLNSDQYNI
>SEQ ID No.99 QP1163-CDR3
ARDFGYCTASWVHAGFSRY
>SEQ ID No.100 QP320
EVQLLESGGGLVQPGGSLRLSCAASGFTYGTYAMSWVRQAPGKGLEWVSCIDIYGR ASYTDPVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAARDFGYCTASWVHEGFS RYWGQGTLVTVSS
>SEQ ID No.101 QP321
EVQLLESGGGLVQPGGSLRLSCAASGFTYGTYAMSWVRQAPGKGLEWVACIDIYGR ASYTDPVKGRFTISRDNSKNTLYLQMNSLKAEDTAVYYCAARDFGYCTASWVHEGFS RYWGQGTLVTVSS
>SEQ ID No.102 QP322
EVQLLESGGGLVQPGGSLRLSCAASGFTYGTYAMSWFRQAPGKGREGVACIDIYGRA SYTDPVKGRFTISQDNSKNTLYLQMNSLKAEDTAVYYCAARDFGYCTASWVHEGFSR YWGQGTLVTVSS
>SEQ ID No.103 QP323
EVQLLESGGGLVQPGGSLRLSCAASGFTYGTYAMSWVRQAPGKGLEWVSCIDIYGR TSYTDPVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAARDFGYCTASWVHEGFS RYWGQGTLVTVSS
>SEQ ID No.104 QP324
EVQLLESGGGLVQPGGSLRLSCAASGFTYGTYAMSWVRQAPGKGLEWVACIDIYGR TSYTDPVKGRFTISQDNSKNTLYLQMNSLRAEDTAVYYCAARDFGYCTASWVHEGFS RYWGQGTLVTVSS
>SEQ ID No.105 QP325
EVQLLESGGGLVQPGGSLRLSCAASGFTYGTYAMSWFRQAPGKGLEGVACIDIYGRT SYTDPVKGRFTISQDNSKNTLYLQMNSLRAEDTAVYYCAARDFGYCTASWVHEGFSR YWGQGTLVTVSS
>SEQ ID No.106 QP1180
EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYG GSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTC PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ PREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
>SEQ ID No.107 QP1181
DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGV PSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKRTVAAPSVFIF PPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSL SSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
>SEQ ID No.108 QP1461
DIVMTQSPDSLAVSLGERATINCKSSQSLLNSGNQKNYLAWYQQKPGQPPKLLIYGA STRESGVPDRFTGSGSGTDFTLTISSLQAEDVAVYYCQNDHSYPFTFGQGTKLEIKRTV AAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
>SEQ ID No.109 QP371
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYIMHWVRQAPGQGLEWMGYINPYN DGTKYNEKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARLGFTTRNAMDYWG QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGG SGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTYGTYAMSWFRQAPGKG REGVACIDIYGRASYTDPVKGRFTISQDNSKNTLYLQMNSLKAEDTAVYYCAARDFGYCTASWVHEGFSRYWGQGTLVTVSS
Figure BDA0002551001190000461
Figure BDA0002551001190000471
Figure BDA0002551001190000481
Figure BDA0002551001190000491
Figure BDA0002551001190000501
Figure BDA0002551001190000511
Figure BDA0002551001190000521
Figure BDA0002551001190000531
Figure BDA0002551001190000541
Figure BDA0002551001190000551
Figure BDA0002551001190000561
Figure BDA0002551001190000571
Figure BDA0002551001190000581
Figure BDA0002551001190000591
Figure BDA0002551001190000601
Figure BDA0002551001190000611
Figure BDA0002551001190000621
Figure BDA0002551001190000631
Figure BDA0002551001190000641
Figure BDA0002551001190000651
Figure BDA0002551001190000661
Figure BDA0002551001190000671
Figure BDA0002551001190000681
Figure BDA0002551001190000691
Figure BDA0002551001190000701
Figure BDA0002551001190000711
Figure BDA0002551001190000721
Figure BDA0002551001190000731
Figure BDA0002551001190000741
Figure BDA0002551001190000751
Figure BDA0002551001190000761
Figure BDA0002551001190000771
Figure BDA0002551001190000781
Figure BDA0002551001190000791
Figure BDA0002551001190000801
Figure BDA0002551001190000811
Figure BDA0002551001190000821
Figure BDA0002551001190000831
Figure BDA0002551001190000841
Figure BDA0002551001190000851
Figure BDA0002551001190000861
Figure BDA0002551001190000871
序列表
<110> 启愈生物技术(上海)有限公司
<120> 抗PD-L1纳米抗体及其Fc融合蛋白和应用
<160> 109
<170> SIPOSequenceListing 1.0
<210> 1
<211> 126
<212> PRT
<213> artificial
<400> 1
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ser Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Tyr Gly Thr Tyr
20 25 30
Ala Met Ser Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Cys Ile Asp Ile Tyr Gly Arg Thr Ser Tyr Thr Asp Pro Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn Thr Leu Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala
85 90 95
Ala Arg Asp Phe Gly Tyr Cys Thr Ala Ser Trp Val His Glu Gly Phe
100 105 110
Ser Arg Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 2
<211> 126
<212> PRT
<213> artificial
<400> 2
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val His Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Val Arg Val Ser Met Tyr Thr Tyr Thr Gly
20 25 30
Thr Cys Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly
35 40 45
Val Ala Gly Leu Trp Thr Gly Asp Gly Val Thr Tyr Tyr Ala Asp Ser
50 55 60
Val Lys Gly Arg Phe Thr Ile Ser Gln Asp Asp Ala Lys Asn Thr Leu
65 70 75 80
Tyr Leu Gln Met Asp Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr
85 90 95
Cys Ala Ser Asn Gly Met Cys Gly Gln Tyr Trp Ala Leu Glu Asp Glu
100 105 110
Tyr Lys Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 3
<211> 128
<212> PRT
<213> artificial
<400> 3
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Thr Leu Ser Cys Ala Val Ser Gly Asn Thr Tyr Ser Thr Asn
20 25 30
Val Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Ala Ile Leu Ala Gly Gly Arg Asn Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys
85 90 95
Ala Ala Asp Thr Arg Ala Ala Phe Trp Asn Ile Gly Pro Leu Asn Ser
100 105 110
Asp Gln Tyr Asn Ile Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 4
<211> 126
<212> PRT
<213> artificial
<400> 4
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ser Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Val Arg His Tyr
20 25 30
Cys Met Gly Trp Phe Arg Gln Thr Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Ser Ile Asp Thr Phe Gly Ile Pro Lys Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn Thr Leu Asn Leu
65 70 75 80
Gln Met Asp Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Phe Cys Ala
85 90 95
Gly Arg Ser Tyr Thr Asn Cys Arg Asp Gly Pro Pro Ser Ala Ser His
100 105 110
Tyr Ser His Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 5
<211> 122
<212> PRT
<213> artificial
<400> 5
Gln 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 Thr Ala Pro Gly Phe Thr Ser Asn Thr Cys
20 25 30
Ala Met Gly Trp Tyr Arg Gln Ala Ala Gly Met Gln Arg Glu Trp Val
35 40 45
Ser Ser Ile Ser Lys Tyr Gly Ile Thr Thr Tyr Ala Asn Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Lys Asp Lys Ala Glu Asp Thr Val Tyr Leu
65 70 75 80
Gln Met Asn Asn Leu Lys Pro Glu Asp Thr Ala Met Tyr Val Cys Lys
85 90 95
Thr Phe Ser Cys Arg Asn Arg Gly Gly Ala Tyr Leu Ala Asp Ala Trp
100 105 110
Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 6
<211> 122
<212> PRT
<213> artificial
<400> 6
Gln 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 Thr Ala Pro Gly Phe Thr Asp Lys Thr Cys
20 25 30
Ala Met Ala Trp Tyr Arg Gln Val Ala Gly Ile Glu Arg Glu Trp Val
35 40 45
Ser Ser Ile Ser Thr Leu Gly Thr Thr Asn Tyr Ala Ser Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Lys Asp Asn Ala Lys Asp Thr Val Tyr Leu
65 70 75 80
Gln Met Asn Asn Leu Lys Pro Glu Asp Thr Ala Met Tyr Val Cys Lys
85 90 95
Thr Phe Ser Cys Arg Asn Arg Gly Gly Ser Tyr Leu Pro Asp Thr Trp
100 105 110
Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 7
<211> 128
<212> PRT
<213> artificial
<400> 7
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Thr Leu Ser Cys Ala Val Ser Gly Asn Thr Tyr Ser Thr Lys
20 25 30
Tyr Met Ser Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Ala Ile Leu Ala Gly Gly Arg Asn Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Arg Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys
85 90 95
Ala Ala Asp Thr Arg Ala Ala Leu Trp Tyr Ile Gly Pro Leu Asn Ser
100 105 110
Asp Gln Tyr Asn Thr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 8
<211> 140
<212> PRT
<213> artificial
<400> 8
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ser Gly Gly
1 5 10 15
Ser Leu Thr Leu Ser Cys Ala Ile Ser Gly Tyr Ala Tyr Ala Thr Tyr
20 25 30
Ser Met Ala Trp Phe Arg Gln Ala His Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Ala Ile Asn Ser Asp Gly His Thr Thr Tyr Val Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Thr Asn Lys Asn Ser Tyr Thr
65 70 75 80
Leu Thr Leu Thr Met Asn Asn Leu Asn Pro Glu Asp Thr Ala Met Tyr
85 90 95
Tyr Cys Ala Ala Thr Ser Gln Leu Gly Phe Trp Ala Gln Lys Leu Trp
100 105 110
Glu Ala Ile Arg Asp Gly Thr Trp Ser Pro Ser Thr Thr Asp Phe Gly
115 120 125
Phe Trp Gly Arg Gly Thr Gln Val Thr Val Ser Ser
130 135 140
<210> 9
<211> 127
<212> PRT
<213> artificial
<400> 9
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ser Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Ala Ser Ala Tyr
20 25 30
Tyr Met Ala Trp Phe Arg Gln Asn Ser Arg Lys Gln Arg Glu Gly Val
35 40 45
Ala Ala Ile Asn Arg Asp Gly Asp Thr Lys Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asp Ala Lys Asn Thr Leu Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Ala Ser Asp Trp Ser Arg Leu Tyr Lys Ile Tyr Trp Leu Asp Asp Asn
100 105 110
Tyr Tyr Val Arg Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 10
<211> 124
<212> PRT
<213> artificial
<400> 10
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ser Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ser Ser Ser Arg Tyr
20 25 30
Ser Val Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Gly Gln Thr Pro Arg Gly Thr Thr Thr Tyr Ala Asp Ser Val Lys
50 55 60
Asp Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala
85 90 95
Ala Gly Gln Ala Leu Leu Trp Ala Ser Leu Arg Gln Thr Ser Tyr Gln
100 105 110
Phe Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 11
<211> 119
<212> PRT
<213> artificial
<400> 11
Gln 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 Thr Ser
20 25 30
Thr Met Met Trp Val Arg Arg Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Gly Ile His Asn Asp Gly Gly Pro Ile Ala Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Thr Ser Leu Lys Ser Glu Asp Thr Ala Leu Tyr Tyr Cys
85 90 95
Ala Arg Gly Trp Tyr Phe Ser Gly Asp Tyr Val Pro Met Thr Gln Gly
100 105 110
Thr Gln Val Thr Val Ser Ser
115
<210> 12
<211> 128
<212> PRT
<213> artificial
<400> 12
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Thr Leu Ser Cys Ala Val Ser Gly Asn Thr Tyr Gly Thr Asn
20 25 30
Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Ala Ile Leu Gly Gly Gly Arg Asn Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys
85 90 95
Ala Ala Asp Thr Arg Ala Ala Phe Trp Asn Ile Gly Pro Leu Asn Ser
100 105 110
Asp Gln Tyr Asn Ile Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 13
<211> 128
<212> PRT
<213> artificial
<400> 13
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Thr Leu Ser Cys Val Val Ser Gly Asn Thr Tyr Ser Thr Lys
20 25 30
Tyr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Ala Ile Leu Ala Gly Gly Arg Asn Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys
85 90 95
Ala Ala Asp Thr Arg Ala Ala Phe Trp Asn Ile Gly Pro Leu Asn Ser
100 105 110
Asp Gln Tyr Asn Ile Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 14
<211> 128
<212> PRT
<213> artificial
<400> 14
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Thr Leu Ser Cys Ala Val Ser Gly Asn Thr Tyr Ser Thr Asn
20 25 30
Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Ala Ile Leu Gly Gly Gly Arg Asn Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys
85 90 95
Ala Ala Asp Thr Arg Ala Ala Phe Trp Asn Ile Gly Pro Leu Asn Ser
100 105 110
Asp Gln Tyr Asn Ile Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 15
<211> 128
<212> PRT
<213> artificial
<400> 15
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Thr Leu Ser Cys Ala Val Ser Gly Asn Thr Tyr Ser Thr Asn
20 25 30
Ile Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Ala Ile Leu Ala Gly Gly Arg Asn Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys
85 90 95
Ala Ala Asp Thr Arg Ala Ala Phe Trp Asn Ile Gly Pro Leu Asn Ser
100 105 110
Asp Gln Tyr Asn Ile Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 16
<211> 128
<212> PRT
<213> artificial
<400> 16
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Thr Leu Ser Cys Ala Val Ser Gly Asn Thr Tyr Ser Thr Lys
20 25 30
Tyr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Ala Ile Leu Ala Gly Gly Arg Asp Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys
85 90 95
Ala Ala Asp Thr Arg Ala Ala Phe Trp Tyr Ile Gly Pro Leu Asn Ser
100 105 110
Asp Gln Tyr Asn Ile Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 17
<211> 128
<212> PRT
<213> artificial
<400> 17
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Thr Leu Ser Cys Ala Val Ser Gly Asn Thr Tyr Ser Thr Lys
20 25 30
Tyr Met Gly Tyr Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Ala Ile Leu Ala Gly Gly Arg Asn Thr Asn Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys
85 90 95
Ala Ala Asp Thr Arg Ala Ala Phe Trp Tyr Ile Gly Pro Leu Asn Ser
100 105 110
Asp Gln Tyr Asn Ile Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 18
<211> 128
<212> PRT
<213> artificial
<400> 18
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Thr Leu Ser Cys Ala Val Ser Gly Asn Thr Tyr Ser Thr Lys
20 25 30
Tyr Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Ala Ile Leu Ala Gly Gly Arg Asn Thr Ser Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Thr Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys
85 90 95
Ala Ala Asp Thr Arg Ala Ala Phe Trp Tyr Ile Gly Pro Leu Asn Ser
100 105 110
His Gln Tyr Asn Ile Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 19
<211> 128
<212> PRT
<213> artificial
<400> 19
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Thr Leu Ser Cys Ala Val Ser Gly Asn Thr Tyr Ser Thr Asn
20 25 30
Tyr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Ala Ile Leu Val Gly Gly Arg Asn Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn Leu Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys
85 90 95
Ala Ala Asp Thr Arg Ala Ala Phe Trp Asn Ile Gly Pro Leu Asn Ser
100 105 110
Asp Gln Tyr Asn Ile Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 20
<211> 128
<212> PRT
<213> artificial
<400> 20
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Thr Leu Ser Cys Ala Val Ser Gly Asn Thr Tyr Ser Thr Lys
20 25 30
Tyr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Ala Ile Leu Ala Gly Gly Arg Asn Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys
85 90 95
Ala Ala Asp Thr Arg Ala Ala Phe Trp Tyr Ile Gly Pro Leu Asn Ser
100 105 110
Asp Gln Tyr Asn Ile Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 21
<211> 128
<212> PRT
<213> artificial
<400> 21
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Thr Leu Ser Cys Ala Val Ser Gly Asn Thr Tyr Ser Thr Asn
20 25 30
Tyr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Ala Ile Leu Ala Gly Gly Arg Asn Thr Ala Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys
85 90 95
Ala Ala Asp Thr Arg Ala Ala Phe Trp Asn Ile Gly Pro Leu Asn Ser
100 105 110
Asp Gln Tyr Asn Ile Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 22
<211> 128
<212> PRT
<213> artificial
<400> 22
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Thr Leu Ser Cys Ala Val Ser Gly Asn Thr Tyr Ser Thr Asn
20 25 30
Tyr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Ala Ile Leu Ala Gly Gly Arg Asn Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asp Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys
85 90 95
Ala Ala Asp Thr Arg Ala Ala Phe Trp Asn Ile Gly Pro Leu Asn Ser
100 105 110
Asp Gln Tyr Asn Ile Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 23
<211> 128
<212> PRT
<213> artificial
<400> 23
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Thr Leu Ser Cys Ala Val Ser Gly Asn Thr Tyr Ser Thr Lys
20 25 30
Tyr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Ala Ile Leu Ala Gly Gly Arg Asn Thr His Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Ala Ile Ser Gln Asp Asn Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys
85 90 95
Ala Ala Asp Thr Arg Ala Ala Phe Trp Asn Ile Gly Pro Leu Asn Ser
100 105 110
Asp Gln Tyr Asn Ile Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 24
<211> 128
<212> PRT
<213> artificial
<400> 24
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Thr Leu Ser Cys Ala Val Ser Gly Asn Thr Tyr Ser Thr Asn
20 25 30
Tyr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Ala Ile Leu Thr Gly Gly Arg Asn Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys
85 90 95
Ala Ala Asp Thr Arg Ala Ala Phe Trp Asn Ile Gly Pro Leu Asn Ser
100 105 110
Asp Gln Tyr Asn Ile Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 25
<211> 128
<212> PRT
<213> artificial
<400> 25
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Thr Leu Ser Cys Ala Ile Ser Gly Asn Thr Tyr Ser Thr Lys
20 25 30
Tyr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Ala Ile Leu Ala Gly Gly Arg Asn Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met His Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys
85 90 95
Ala Ala Asp Thr Arg Ala Ala Phe Trp Tyr Ile Gly Pro Leu Asn Ser
100 105 110
Asp Gln Tyr Asn Leu Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 26
<211> 128
<212> PRT
<213> artificial
<400> 26
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Thr Leu Ser Cys Ala Val Ser Gly Asn Thr Tyr Ser Thr Lys
20 25 30
Tyr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Ala Ile Leu Ala Gly Gly Arg Asn Thr Asp Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys
85 90 95
Ala Ala Asp Thr Arg Ala Ala Phe Trp Ser Ile Gly Pro Leu Asn Ser
100 105 110
Asp Gln Tyr Asn Ile Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 27
<211> 128
<212> PRT
<213> artificial
<400> 27
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Thr Leu Ser Cys Ala Val Ser Gly Asn Thr Tyr Ser Thr Asn
20 25 30
Tyr Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Ala Ile Arg Ala Gly Gly Arg Asn Thr Asp Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys
85 90 95
Ala Ala Asp Thr Arg Ala Ala Phe Trp Tyr Ile Gly Pro Leu Asn Ser
100 105 110
Asp Gln Tyr Asn Ile Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 28
<211> 128
<212> PRT
<213> artificial
<400> 28
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Thr Leu Ser Cys Glu Val Ser Gly Ser Thr Tyr Ser Thr Asn
20 25 30
Tyr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Ala Ile Leu Ala Gly Gly Arg Asn Thr Asp Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys
85 90 95
Ala Ala Asp Thr Arg Ala Ala Phe Trp Tyr Ile Gly Pro Leu Asn Ser
100 105 110
Asp Gln Tyr Asn Ile Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 29
<211> 128
<212> PRT
<213> artificial
<400> 29
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Thr Leu Ser Cys Ala Val Ser Gly Asn Thr Tyr Ser Thr Lys
20 25 30
Tyr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Ala Ile Leu Ala Gly Gly Arg Asn Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Gln Asp Ser Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys
85 90 95
Ala Ala Asp Thr Arg Ala Ala Phe Trp Tyr Ile Gly Pro Leu Asn Ser
100 105 110
Asp Gln Tyr Asn Ser Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 30
<211> 128
<212> PRT
<213> artificial
<400> 30
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Thr Leu Ser Cys Ala Val Ser Gly Asn Thr Tyr Ser Thr Asn
20 25 30
Tyr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Ala Ile Leu Val Gly Gly Arg Asn Thr Tyr Tyr Ala Asp Pro Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn Leu Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys
85 90 95
Ala Ala Asp Thr Arg Ala Ala Phe Trp Asn Ile Gly Pro Leu Asn Ser
100 105 110
Asp Gln Tyr Asn Ile Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 31
<211> 128
<212> PRT
<213> artificial
<400> 31
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Thr Leu Ser Cys Ala Val Ser Gly Asn Thr Tyr Ser Thr Lys
20 25 30
Tyr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Ala Ile Leu Ala Gly Gly Arg Asn Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Arg Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys
85 90 95
Ala Ala Asp Thr Arg Ala Ala Leu Trp Tyr Ile Gly Pro Leu Asn Ser
100 105 110
Asp Gln Tyr Asn Thr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 32
<211> 128
<212> PRT
<213> artificial
<400> 32
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Thr Leu Ser Cys Ala Val Ser Gly Asn Thr Tyr Ser Thr Lys
20 25 30
Tyr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Ala Ile Leu Ala Gly Gly Arg Asn Thr Ser Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys
85 90 95
Ala Ala Asp Thr Arg Ala Ala Phe Trp Tyr Ile Gly Pro Leu Asn Ser
100 105 110
Asp Gln Tyr Asn Ile Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 33
<211> 128
<212> PRT
<213> artificial
<400> 33
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Thr Leu Ser Cys Ala Ile Ser Gly Asn Thr Tyr Ser Thr Lys
20 25 30
Tyr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Ala Ile Leu Ala Gly Gly Arg Asn Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys
85 90 95
Ala Ala Asp Ala Arg Ala Ala Phe Trp Tyr Ile Gly Pro Leu Asn Ser
100 105 110
Asp Gln Tyr Asn Ile Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 34
<211> 128
<212> PRT
<213> artificial
<400> 34
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Thr Leu Ser Cys Ala Val Ser Gly Asn Thr Tyr Ser Thr Asn
20 25 30
Tyr Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Ala Ile Arg Val Gly Gly Arg Asn Thr Asp Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys
85 90 95
Ala Ala Asp Thr Arg Ala Ala Phe Trp Tyr Ile Gly Pro Leu Asn Ser
100 105 110
Asp Gln Tyr Asn Ile Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 35
<211> 126
<212> PRT
<213> artificial
<400> 35
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ser Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Tyr Gly Thr Tyr
20 25 30
Ala Met Ser Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Cys Ile Asp Ile Tyr Gly Arg Ala Ser Tyr Thr Asp Pro Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn Thr Leu Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala
85 90 95
Ala Arg Asp Phe Gly Tyr Cys Thr Ala Ser Trp Val His Glu Gly Phe
100 105 110
Ser Arg Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 36
<211> 126
<212> PRT
<213> artificial
<400> 36
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ser Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Tyr Gly Thr Tyr
20 25 30
Ala Met Ser Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Cys Ile Asp Ile Tyr Gly Arg Thr Ser Tyr Thr Asp Pro Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn Thr Leu Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala
85 90 95
Ala Arg Asp Phe Gly Tyr Cys Thr Ala Ser Trp Val His Ala Gly Phe
100 105 110
Ser Arg Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 37
<211> 126
<212> PRT
<213> artificial
<400> 37
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ser Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Tyr Gly Thr Tyr
20 25 30
Ala Met Ser Trp Phe Arg Gln Thr Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Cys Ile Asp Ile Tyr Gly Arg Thr Ser Tyr Thr Asp Pro Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn Thr Leu Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala
85 90 95
Ala Arg Asp Phe Gly Tyr Cys Thr Ala Ser Trp Val His Glu Gly Phe
100 105 110
Ser Arg Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 38
<211> 126
<212> PRT
<213> artificial
<400> 38
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ser Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Tyr Gly Ala Tyr
20 25 30
Ala Met Ser Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Cys Ile Asp Ile Tyr Gly Arg Thr Ser Tyr Thr Asp Pro Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn Thr Leu Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala
85 90 95
Ala Arg Asp Phe Gly Tyr Cys Thr Ala Ser Trp Val His Glu Gly Phe
100 105 110
Ser Arg Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 39
<211> 126
<212> PRT
<213> artificial
<400> 39
Gln Val Gln Leu Val Glu Ser Gly Gly Asp Ser Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Tyr Gly Thr Tyr
20 25 30
Ala Met Ser Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Cys Ile Asp Ile Tyr Gly Arg Thr Ser Tyr Thr Asp Pro Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn Thr Leu Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala
85 90 95
Ala Arg Asp Phe Gly Tyr Cys Thr Ala Ser Trp Val His Glu Gly Phe
100 105 110
Ser Arg Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 40
<211> 126
<212> PRT
<213> artificial
<400> 40
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ser Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Tyr Glu Thr Tyr
20 25 30
Ala Met Ser Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Cys Ile Asp Ile Tyr Gly Arg Thr Ser Tyr Thr Asp Pro Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn Thr Leu Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala
85 90 95
Ala Arg Asp Phe Gly Tyr Cys Thr Ala Ser Trp Val His Glu Gly Phe
100 105 110
Ser Arg Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 41
<211> 126
<212> PRT
<213> artificial
<400> 41
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ser Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Tyr Gly Thr Tyr
20 25 30
Ala Met Ser Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Cys Ile Asp Ile Tyr Gly Arg Thr Ser Tyr Thr Asp Pro Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn Thr Leu Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Lys Pro Gly Asp Thr Ala Met Tyr Tyr Cys Ala
85 90 95
Ala Arg Asp Phe Gly Tyr Cys Thr Ala Ser Trp Val His Glu Gly Phe
100 105 110
Ser Arg Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 42
<211> 232
<212> PRT
<213> human
<400> 42
Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
1 5 10 15
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
20 25 30
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
35 40 45
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
50 55 60
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
65 70 75 80
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
85 90 95
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
100 105 110
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
115 120 125
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
130 135 140
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
145 150 155 160
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
165 170 175
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
180 185 190
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
195 200 205
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
210 215 220
Ser Leu Ser Leu Ser Pro Gly Lys
225 230
<210> 43
<211> 254
<212> PRT
<213> artificial
<400> 43
Met Arg Ile Phe Ala Val Phe Ile Phe Met Thr Tyr Trp His Leu Leu
1 5 10 15
Asn Ala Phe Thr Val Thr Val Pro Lys Asp Leu Tyr Val Val Glu Tyr
20 25 30
Gly Ser Asn Met Thr Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu
35 40 45
Asp Leu Ala Ala Leu Ile Val Tyr Trp Glu Met Glu Asp Lys Asn Ile
50 55 60
Ile Gln Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser
65 70 75 80
Tyr Arg Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn
85 90 95
Ala Ala Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr
100 105 110
Arg Cys Met Ile Ser Tyr Gly Gly Ala Asp Tyr Lys Arg Ile Thr Val
115 120 125
Lys Val Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val
130 135 140
Asp Pro Val Thr Ser Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr
145 150 155 160
Pro Lys Ala Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser
165 170 175
Gly Lys Thr Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn
180 185 190
Val Thr Ser Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr
195 200 205
Cys Thr Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu
210 215 220
Val Ile Pro Glu Leu Pro Leu Ala His Pro Pro Asn Glu Arg Glu Gln
225 230 235 240
Lys Leu Ile Ser Glu Glu Asp Leu His His His His His His
245 250
<210> 44
<211> 5
<212> PRT
<213> artificial
<400> 44
Thr Tyr Ala Met Ser
1 5
<210> 45
<211> 5
<212> PRT
<213> artificial
<400> 45
Gly Thr Cys Met Ala
1 5
<210> 46
<211> 5
<212> PRT
<213> artificial
<400> 46
Thr Asn Val Met Gly
1 5
<210> 47
<211> 5
<212> PRT
<213> artificial
<400> 47
His Tyr Cys Met Gly
1 5
<210> 48
<211> 5
<212> PRT
<213> artificial
<400> 48
Thr Cys Ala Met Gly
1 5
<210> 49
<211> 5
<212> PRT
<213> artificial
<400> 49
Thr Cys Ala Met Ala
1 5
<210> 50
<211> 5
<212> PRT
<213> artificial
<400> 50
Thr Lys Tyr Met Ser
1 5
<210> 51
<211> 5
<212> PRT
<213> artificial
<400> 51
Thr Tyr Ser Met Ala
1 5
<210> 52
<211> 5
<212> PRT
<213> artificial
<400> 52
Ala Tyr Tyr Met Ala
1 5
<210> 53
<211> 5
<212> PRT
<213> artificial
<400> 53
Arg Tyr Ser Val Gly
1 5
<210> 54
<211> 5
<212> PRT
<213> artificial
<400> 54
Thr Ser Thr Met Met
1 5
<210> 55
<211> 5
<212> PRT
<213> artificial
<400> 55
Thr Asn Ala Met Gly
1 5
<210> 56
<211> 5
<212> PRT
<213> artificial
<400> 56
Thr Lys Tyr Met Gly
1 5
<210> 57
<211> 5
<212> PRT
<213> artificial
<400> 57
Thr Asn Ile Met Gly
1 5
<210> 58
<211> 5
<212> PRT
<213> artificial
<400> 58
Thr Lys Tyr Met Ala
1 5
<210> 59
<211> 5
<212> PRT
<213> artificial
<400> 59
Thr Asn Tyr Met Ala
1 5
<210> 60
<211> 5
<212> PRT
<213> artificial
<400> 60
Ala Tyr Ala Met Ser
1 5
<210> 61
<211> 16
<212> PRT
<213> artificial
<400> 61
Cys Ile Asp Ile Tyr Gly Arg Thr Ser Tyr Thr Asp Pro Val Lys Gly
1 5 10 15
<210> 62
<211> 17
<212> PRT
<213> artificial
<400> 62
Gly Leu Trp Thr Gly Asp Gly Val Thr Tyr Tyr Ala Asp Ser Val Lys
1 5 10 15
Gly
<210> 63
<211> 17
<212> PRT
<213> artificial
<400> 63
Ala Ile Leu Ala Gly Gly Arg Asn Thr Tyr Tyr Ala Asp Ser Val Lys
1 5 10 15
Gly
<210> 64
<211> 16
<212> PRT
<213> artificial
<400> 64
Ser Ile Asp Thr Phe Gly Ile Pro Lys Tyr Ala Asp Ser Val Lys Gly
1 5 10 15
<210> 65
<211> 16
<212> PRT
<213> artificial
<400> 65
Ser Ile Ser Lys Tyr Gly Ile Thr Thr Tyr Ala Asn Ser Val Lys Gly
1 5 10 15
<210> 66
<211> 16
<212> PRT
<213> artificial
<400> 66
Ser Ile Ser Thr Leu Gly Thr Thr Asn Tyr Ala Ser Ser Val Lys Gly
1 5 10 15
<210> 67
<211> 16
<212> PRT
<213> artificial
<400> 67
Ala Ile Asn Ser Asp Gly His Thr Thr Tyr Val Asp Ser Val Lys Gly
1 5 10 15
<210> 68
<211> 16
<212> PRT
<213> artificial
<400> 68
Ala Ile Asn Arg Asp Gly Asp Thr Lys Tyr Ala Asp Ser Val Lys Gly
1 5 10 15
<210> 69
<211> 16
<212> PRT
<213> artificial
<400> 69
Gly Gln Thr Pro Arg Gly Thr Thr Thr Tyr Ala Asp Ser Val Lys Asp
1 5 10 15
<210> 70
<211> 17
<212> PRT
<213> artificial
<400> 70
Gly Ile His Asn Asp Gly Gly Pro Ile Ala Tyr Ala Asp Ser Val Lys
1 5 10 15
Gly
<210> 71
<211> 17
<212> PRT
<213> artificial
<400> 71
Ala Ile Leu Gly Gly Gly Arg Asn Thr Tyr Tyr Ala Asp Ser Val Lys
1 5 10 15
Gly
<210> 72
<211> 17
<212> PRT
<213> artificial
<400> 72
Ala Ile Leu Ala Gly Gly Arg Asp Thr Tyr Tyr Ala Asp Ser Val Lys
1 5 10 15
Gly
<210> 73
<211> 17
<212> PRT
<213> artificial
<400> 73
Ala Ile Leu Ala Gly Gly Arg Asn Thr Asn Tyr Ala Asp Ser Val Lys
1 5 10 15
Gly
<210> 74
<211> 17
<212> PRT
<213> artificial
<400> 74
Ala Ile Leu Ala Gly Gly Arg Asn Thr Ser Tyr Ala Asp Ser Val Lys
1 5 10 15
Gly
<210> 75
<211> 17
<212> PRT
<213> artificial
<400> 75
Ala Ile Leu Val Gly Gly Arg Asn Thr Tyr Tyr Ala Asp Ser Val Lys
1 5 10 15
Gly
<210> 76
<211> 17
<212> PRT
<213> artificial
<400> 76
Ala Ile Leu Ala Gly Gly Arg Asn Thr Ala Tyr Ala Asp Ser Val Lys
1 5 10 15
Gly
<210> 77
<211> 17
<212> PRT
<213> artificial
<400> 77
Ala Ile Leu Ala Gly Gly Arg Asn Thr His Tyr Ala Asp Ser Val Lys
1 5 10 15
Gly
<210> 78
<211> 17
<212> PRT
<213> artificial
<400> 78
Ala Ile Leu Thr Gly Gly Arg Asn Thr Tyr Tyr Ala Asp Ser Val Lys
1 5 10 15
Gly
<210> 79
<211> 17
<212> PRT
<213> artificial
<400> 79
Ala Ile Leu Ala Gly Gly Arg Asn Thr Asp Tyr Ala Asp Ser Val Lys
1 5 10 15
Gly
<210> 80
<211> 17
<212> PRT
<213> artificial
<400> 80
Ala Ile Leu Val Gly Gly Arg Asn Thr Tyr Tyr Ala Asp Pro Val Lys
1 5 10 15
Gly
<210> 81
<211> 17
<212> PRT
<213> artificial
<400> 81
Ala Ile Arg Val Gly Gly Arg Asn Thr Asp Tyr Ala Asp Ser Val Lys
1 5 10 15
Gly
<210> 82
<211> 16
<212> PRT
<213> artificial
<400> 82
Cys Ile Asp Ile Tyr Gly Arg Ala Ser Tyr Thr Asp Pro Val Lys Gly
1 5 10 15
<210> 83
<211> 19
<212> PRT
<213> atificial
<400> 83
Ala Arg Asp Phe Gly Tyr Cys Thr Ala Ser Trp Val His Glu Gly Phe
1 5 10 15
Ser Arg Tyr
<210> 84
<211> 17
<212> PRT
<213> artificial
<400> 84
Ser Asn Gly Met Cys Gly Gln Tyr Trp Ala Leu Glu Asp Glu Tyr Lys
1 5 10 15
Tyr
<210> 85
<211> 20
<212> PRT
<213> artificial
<400> 85
Ala Asp Thr Arg Ala Ala Phe Trp Asn Ile Gly Pro Leu Asn Ser Asp
1 5 10 15
Gln Tyr Asn Ile
20
<210> 86
<211> 19
<212> PRT
<213> artificial
<400> 86
Gly Arg Ser Tyr Thr Asn Cys Arg Asp Gly Pro Pro Ser Ala Ser His
1 5 10 15
Tyr Ser His
<210> 87
<211> 16
<212> PRT
<213> artificial
<400> 87
Lys Thr Phe Ser Cys Arg Asn Arg Gly Gly Ala Tyr Leu Ala Asp Ala
1 5 10 15
<210> 88
<211> 20
<212> PRT
<213> artificial
<400> 88
Ala Asp Thr Arg Ala Ala Leu Trp Tyr Ile Gly Pro Leu Asn Ser Asp
1 5 10 15
Gln Tyr Asn Thr
20
<210> 89
<211> 30
<212> PRT
<213> artificial
<400> 89
Ala Thr Ser Gln Leu Gly Phe Trp Ala Gln Lys Leu Trp Glu Ala Ile
1 5 10 15
Arg Asp Gly Thr Trp Ser Pro Ser Thr Thr Asp Phe Gly Phe
20 25 30
<210> 90
<211> 20
<212> PRT
<213> artificial
<400> 90
Ala Ser Asp Trp Ser Arg Leu Tyr Lys Ile Tyr Trp Leu Asp Asp Asn
1 5 10 15
Tyr Tyr Val Arg
20
<210> 91
<211> 17
<212> PRT
<213> artificial
<400> 91
Ala Gly Gln Ala Leu Leu Trp Ala Ser Leu Arg Gln Thr Ser Tyr Gln
1 5 10 15
Phe
<210> 92
<211> 10
<212> PRT
<213> artificial
<400> 92
Gly Trp Tyr Phe Ser Gly Asp Tyr Val Pro
1 5 10
<210> 93
<211> 20
<212> PRT
<213> artificial
<400> 93
Ala Asp Thr Arg Ala Ala Phe Trp Tyr Ile Gly Pro Leu Asn Ser Asp
1 5 10 15
Gln Tyr Asn Ile
20
<210> 94
<211> 20
<212> PRT
<213> artificial
<400> 94
Ala Asp Thr Arg Ala Ala Phe Trp Tyr Ile Gly Pro Leu Asn Ser His
1 5 10 15
Gln Tyr Asn Ile
20
<210> 95
<211> 20
<212> PRT
<213> artificial
<400> 95
Ala Asp Thr Arg Ala Ala Phe Trp Tyr Ile Gly Pro Leu Asn Ser Asp
1 5 10 15
Gln Tyr Asn Leu
20
<210> 96
<211> 20
<212> PRT
<213> artificial
<400> 96
Ala Asp Thr Arg Ala Ala Phe Trp Ser Ile Gly Pro Leu Asn Ser Asp
1 5 10 15
Gln Tyr Asn Ile
20
<210> 97
<211> 20
<212> PRT
<213> artificial
<400> 97
Ala Asp Thr Arg Ala Ala Phe Trp Tyr Ile Gly Pro Leu Asn Ser Asp
1 5 10 15
Gln Tyr Asn Ser
20
<210> 98
<211> 20
<212> PRT
<213> artificial
<400> 98
Ala Asp Ala Arg Ala Ala Phe Trp Tyr Ile Gly Pro Leu Asn Ser Asp
1 5 10 15
Gln Tyr Asn Ile
20
<210> 99
<211> 19
<212> PRT
<213> artificial
<400> 99
Ala Arg Asp Phe Gly Tyr Cys Thr Ala Ser Trp Val His Ala Gly Phe
1 5 10 15
Ser Arg Tyr
<210> 100
<211> 126
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 100
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 Tyr Gly Thr Tyr
20 25 30
Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Cys Ile Asp Ile Tyr Gly Arg Ala Ser Tyr Thr Asp Pro Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Ala Arg Asp Phe Gly Tyr Cys Thr Ala Ser Trp Val His Glu Gly Phe
100 105 110
Ser Arg Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 101
<211> 126
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 101
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 Tyr Gly Thr Tyr
20 25 30
Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Cys Ile Asp Ile Tyr Gly Arg Ala Ser Tyr Thr Asp Pro Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Ala Arg Asp Phe Gly Tyr Cys Thr Ala Ser Trp Val His Glu Gly Phe
100 105 110
Ser Arg Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 102
<211> 126
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 102
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 Tyr Gly Thr Tyr
20 25 30
Ala Met Ser Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Gly Val
35 40 45
Ala Cys Ile Asp Ile Tyr Gly Arg Ala Ser Tyr Thr Asp Pro Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Gln Asp Asn Ser Lys Asn Thr Leu Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Ala Arg Asp Phe Gly Tyr Cys Thr Ala Ser Trp Val His Glu Gly Phe
100 105 110
Ser Arg Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 103
<211> 126
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 103
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 Tyr Gly Thr Tyr
20 25 30
Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Cys Ile Asp Ile Tyr Gly Arg Thr Ser Tyr Thr Asp Pro Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Ala Arg Asp Phe Gly Tyr Cys Thr Ala Ser Trp Val His Glu Gly Phe
100 105 110
Ser Arg Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 104
<211> 126
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 104
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 Tyr Gly Thr Tyr
20 25 30
Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Cys Ile Asp Ile Tyr Gly Arg Thr Ser Tyr Thr Asp Pro Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Gln Asp Asn Ser Lys Asn Thr Leu Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Ala Arg Asp Phe Gly Tyr Cys Thr Ala Ser Trp Val His Glu Gly Phe
100 105 110
Ser Arg Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 105
<211> 126
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 105
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 Tyr Gly Thr Tyr
20 25 30
Ala Met Ser Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Gly Val
35 40 45
Ala Cys Ile Asp Ile Tyr Gly Arg Thr Ser Tyr Thr Asp Pro Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Gln Asp Asn Ser Lys Asn Thr Leu Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Ala Arg Asp Phe Gly Tyr Cys Thr Ala Ser Trp Val His Glu Gly Phe
100 105 110
Ser Arg Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 106
<211> 448
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 106
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 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
115 120 125
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
130 135 140
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
145 150 155 160
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
165 170 175
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
180 185 190
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser
195 200 205
Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr
210 215 220
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
225 230 235 240
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
245 250 255
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
260 265 270
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
275 280 285
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val
290 295 300
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
305 310 315 320
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
325 330 335
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
340 345 350
Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys
355 360 365
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
370 375 380
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
385 390 395 400
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
405 410 415
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
420 425 430
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 445
<210> 107
<211> 214
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 107
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 Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys
210
<210> 108
<211> 220
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 108
Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser
20 25 30
Gly Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Pro Pro Lys Leu Leu Ile Tyr Gly Ala Ser Thr Arg Glu Ser Gly Val
50 55 60
Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn
85 90 95
Asp His Ser Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile
100 105 110
Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp
115 120 125
Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn
130 135 140
Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu
145 150 155 160
Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp
165 170 175
Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr
180 185 190
Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser
195 200 205
Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
210 215 220
<210> 109
<211> 595
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 109
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30
Ile Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Glu Lys Phe
50 55 60
Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Leu Gly Phe Thr Thr Arg Asn Ala Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
115 120 125
Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
130 135 140
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
145 150 155 160
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
165 170 175
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
180 185 190
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
195 200 205
Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
210 215 220
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly
225 230 235 240
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
245 250 255
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
260 265 270
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
275 280 285
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
290 295 300
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
305 310 315 320
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
325 330 335
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
340 345 350
Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu
355 360 365
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
370 375 380
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
385 390 395 400
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
405 410 415
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
420 425 430
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
435 440 445
Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
450 455 460
Gly Gly Gly Gly Ser Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu
465 470 475 480
Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
485 490 495
Thr Tyr Gly Thr Tyr Ala Met Ser Trp Phe Arg Gln Ala Pro Gly Lys
500 505 510
Gly Arg Glu Gly Val Ala Cys Ile Asp Ile Tyr Gly Arg Ala Ser Tyr
515 520 525
Thr Asp Pro Val Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ser Lys
530 535 540
Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Lys Ala Glu Asp Thr Ala
545 550 555 560
Val Tyr Tyr Cys Ala Ala Arg Asp Phe Gly Tyr Cys Thr Ala Ser Trp
565 570 575
Val His Glu Gly Phe Ser Arg Tyr Trp Gly Gln Gly Thr Leu Val Thr
580 585 590
Val Ser Ser
595

Claims (12)

1.一种抗PD-L1纳米抗体，其特征在于：
至少包含一个VHH片段，在所述VHH片段中，包含CDR1、CDR2和CDR3三个氨基酸片段,CDR1、CDR2、CDR3分别选自以下序列：
1)SEQ ID No.44至SEQ ID No.60所示的CDR1；
2)SEQ ID No.61至SEQ ID No.82所示的CDR2；
3)SEQ ID No.83至SEQ ID No.99所示的CDR3。
2.如权利要求1所述的抗PD-L1纳米抗体，其特征在于：
其序列如：SEQ ID No.1至SEQNo.41所示。
3.一种抗PD-L1纳米抗体的Fc融合蛋白，其特征在于：包含如权利要求1或2所述的抗PD-L1纳米抗体以及Fc段，所述Fc段的序列如SEQ ID No.42所示。
4.如权利要求1所述的抗PD-L1纳米抗体，其特征在于：其序列中，除CDR1、CDR2和CDR3以外，有80％的氨基酸序列与SEQ ID No.1至SEQNo.41所示的序列相同。
5.抗PD-L1纳米抗体在制备阻断PD-L1和PD-1结合试剂中的应用。
6.抗PD-L1纳米抗体的Fc融合蛋白在制备阻断PD-L1和PD-1结合试剂中的应用。
7.如权利要求5所述的抗PD-L1纳米抗体其特征在于:其用量为20ug/ml至0.000128ug/ml。
8.如权利要求1所述的抗PD-L1纳米抗体人源化改造后，其特征在于：其序列如SEQ IDNo.100至SEQ ID No.105所示。
9.一种人源化抗PD-L1纳米抗体的Fc融合蛋白，其特征在于：包含如权利要求8所述的抗PD-L1纳米抗体以及Fc段，所述Fc段的序列如SEQ ID No.42所示。
10.如权利要求1-4或权利要求9中任一项所述的纳米抗体或者其Fc融合蛋白，在制备治疗癌症、感染或免疫调节疾病的药物中的应用。
11.如权利要求1-4或权利要求9中任一项所述的纳米抗体，在制备抑制肿瘤生长的药物中的应用。
12.如权利要求10所述的应用，其特征在于：
所述癌症或肿瘤选自下组织或部位：结直肠、乳腺、卵巢、胰腺、胃、食管、前列腺、肾、宫颈、骨髓癌、淋巴癌、白血病、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、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，或者骨髓增生异常综合症。
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