CN113683697A - 抗b7-h3抗体、其制备方法及用途 - Google Patents

抗b7-h3抗体、其制备方法及用途 Download PDF

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CN113683697A
CN113683697A CN202110996629.6A CN202110996629A CN113683697A CN 113683697 A CN113683697 A CN 113683697A CN 202110996629 A CN202110996629 A CN 202110996629A CN 113683697 A CN113683697 A CN 113683697A
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CN113683697B (zh
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魏海涛
褚敏
尚玉栓
李亚男
王平
董国良
李秀兰
罗雪琴
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Shanghai Xiangyao Biotechnology Co ltd
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Abstract

本发明公开了抗B7‑H3抗体、其制备方法及用途，所述抗体包含重链可变区和/或轻链可变区：所述重链可变区，其包含由HCDR1(SEQ ID NO:1)、HCDR2(SEQ ID NO:2)和HCDR3(SEQ ID NO:3)组成的重链互补决定区；所述轻链可变区，其包含由LCDR1(SEQ ID NO:4)、LCDR2(WAS)和LCDR3(SEQ ID NO:5)组成的轻链互补决定区。所述抗B7‑H3抗体具有较强的结合活性、物种交叉反应性和ADCC活性，适应于后续癌症等相关疾病的治疗和产品开发。

Description

抗B7-H3抗体、其制备方法及用途
技术领域
本发明涉及抗体技术领域，具体涉及抗B7-H3抗体、其制备方法及用途。
背景技术
免疫检查点(immune checkpoints)是指免疫系统中存在的一些抑制性信号通路，机体在正常抗肿瘤免疫应答情况下，共刺激信号和共抑制信号保持平衡，通过调节自身免疫反应的强度来维持免疫耐受。机体在受到肿瘤侵袭时，通常会阻断免疫检查点信号通路从而抑制自身免疫，给肿瘤细胞的生长和逃逸提供机会。随着对肿瘤微环境和肿瘤免疫逃逸机制的研究，发现负性B7家族分子异常表达于多种肿瘤组织及肿瘤浸润免疫细胞中，是肿瘤微环境的重要组成部分，并参与肿瘤免疫逃逸。目前已有CTLA-4、PD-1以及PD-L1(Bowyer,Prithviraj et al.2016,Pento 2017,Fujita,Uchida et al.2019)等抗免疫检查点的单克隆抗体应用于肿瘤治疗并取得了显著疗效。这些成果使阻断免疫检查点的治疗策略备受关注。
B7-H3又名CD276，最早由Andrei I.Chapoval等人于2001年从人树突状细胞的cDNA文库中克隆获得，在氨基酸水平与B7免疫球蛋白超家族存在20–27％的同源性，属于免疫球蛋白超家族B7成员(Chapoval,Ni et al.2001)。B7-H3蛋白属于I型跨膜蛋白，包含一个信号肽，一个C端的免疫球蛋白恒定区(IgC)和N端的可变区(IgV)、一个跨膜区和一个胞内区(Vigdorovich,Ramagopal et al.2013)。B7-H3蛋白有两种变异剪接体，变体1胞外段由IgV-IgC-IgV-IgC 4个免疫球蛋白结构域组成，称为4Ig-B7-H3，变体2胞外段由IgV-IgC2个免疫球蛋白结构域组成，称为2Ig-B7-H3。在人体中有两种不同的变体形式存在，其主要形式为4Ig-B7-H3，而在小鼠中只含有2Ig-B7-H3形式(Y-H,Y-J et al.2007)。
临床上已经报道，B7-H3在健康组织中低表达，但在大量的恶性肿瘤中高表达。研究表明，B7-H3可在食管癌、黑色素瘤、结直肠癌、腺癌、卵巢癌、非小细胞肺癌、肾癌、胃癌、膀胱癌、多形性胶质母细胞瘤以及骨肉瘤等诸多癌症中高表达(Tang,Zhao et al.2019,Tang,Liu et al.2020)。B7-H3不仅表达于肿瘤细胞上，在肿瘤新生血管内皮细胞上同样高表达，是一个非常广谱的肿瘤标志性抗原。B7-H3蛋白高表达可促进癌症进展并与患者的不良预后有关，因此，抑制B7-H3蛋白的表达对治疗癌症具有潜在作用。
目前，还没有靶向B7-H3的抗体药物上市，因此，有必要进一步开发具有更高活性、更高亲和力和治疗效果的B7-H3抗体，以进行相关疾病的治疗研究和应用。
发明内容
本发明旨在至少解决现有技术中存在的技术问题之一。为此，本发明提出一种抗B7-H3抗体，能够高特异性结合B7-H3。
本发明还提出与上述抗体相关的重组蛋白、药物组合物、多核苷酸、重组质粒和分离细胞。
本发明还提出上述抗体的制备方法。
本发明还提出上述抗体在制备抗癌药物中的应用。
本发明还提出上述抗体在制备抗体检测试剂盒中的应用。
根据本发明的第一方面实施方式的抗体，所述抗体包含重链可变区和/或轻链可变区：所述重链可变区，其包含：由SEQ ID NO:1所示的氨基酸序列组成的重链互补决定区HCDR1，由SEQ ID NO:2所示的氨基酸序列组成的重链互补决定区HCDR2，由SEQ ID NO:3所示的氨基酸序列组成的重链互补决定区HCDR3；所述轻链可变区，其包含：由SEQ ID NO:4所示的氨基酸序列组成的轻链互补决定区LCDR1，由WAS氨基酸序列组成的轻链互补决定区LCDR2，由SEQ ID NO:5所示的氨基酸序列组成的轻链互补决定区LCDR3。
根据本发明实施方式的抗体，至少具有如下有益效果：本发明的抗体具有(1)结合活性好，与B7-H3蛋白较强的结合能力；(2)物种交叉反应性，与猴B7-H3具有交叉反应性，利于开展验证实验和便于后续有治疗用途的产品开发；(3)具有ADCC活性。
在本发明中，抗体依赖的细胞介导的细胞毒作用(ADCC，antibody-dependentcell-mediated cytotoxicity)是一种细胞介导的免疫防御机制，是免疫细胞消灭已结合特异性抗体的病原靶细胞的作用方式。现如今，ADCC作用机制被用来检测、评定抗体或靶细胞的功效。
根据本发明的一些实施方式，所述为鼠源抗体、嵌合抗体或人源抗体。
根据本发明的一些实施方式，所述重链可变区选自如SEQ ID NO:6、SEQ ID NO:18、SEQ ID NO:19、SEQ ID NO:20、SEQ ID NO:21或SEQ ID NO:22所示的氨基酸序列；所述轻链可变区由选自如SEQ ID NO:7、SEQ ID NO:23、SEQ ID NO:24、SEQ ID NO:25、SEQ IDNO:26或SEQ ID NO:27所示的氨基酸序列。
根据本发明的一些实施方式，所述抗体包含重链恒定区和/或轻链恒定区：所述重链恒定区包含如SEQ ID NO:10所示的氨基酸序列；所述轻链恒定区包含如SEQ ID NO:12所示的氨基酸序列。
根据本发明的一些实施方式，所述抗体包含重链和/或轻链：所述重链选自如SEQID NO:14、SEQ ID NO:46和SEQ ID NO:47所示的氨基酸序列；所述轻链选自如SEQ ID NO:15、SEQ ID NO:48和SEQ ID NO:49所示的氨基酸序列。
根据本发明的一些实施方式，所述抗体包含以下i至v中任一种性质：
i、所述抗体能够特异性结合B7-H3；
ii、所述抗体具有抗体依赖的细胞介导的细胞毒作用；
iii、所述抗体特异性地结合人4Ig-B7-H3并与猴4Ig-B7-H3交叉反应；
iv、所述抗体具有减少的糖基化或无糖基化或被低岩藻糖基化。
在本发明中，上述抗体序列具有最佳的结合活性、反应特异性、物种交叉反应性和ADCC活性。通过实施例实验可以证明本发明的抗体与B7-H3蛋白结合力强；具有物种交叉反应这一特点，有助于抗体在猴子体内进行毒性分析，有利于在相关动物中开展验证试验，进而有利于后续的治疗用途的应用开发；有较强的ADCC活性，更适合抗体药物的后续开发。
在本发明中，与未经修饰的Fc区相比，所述Fc区的氨基酸位点被修饰以增强FcγR结合。在一些实施方式中，所述Fc区具有更强的FcγRIII结合的能力，从而发挥更强的抗体介导的细胞毒(ADCC)作用。
根据本发明第二方面的实施方式的重组蛋白、药物组合物、多核苷酸、载体或分离细胞：所述重组蛋白包含上述抗体；所述药物组合物包含上述抗体或上述重组蛋白；所述多核苷酸包含编码上述抗体或重组蛋白的核苷酸序列；所述载体包含上述多核苷酸；所述分离细胞产生上述抗体。
根据本发明的一些实施方式，所述重组蛋白还包含协助表达和/或纯化的标签序列。
根据本发明的一些实施方式，所述重组蛋白为双抗，即还包括能够与其他靶蛋白结合的抗体。进一步地，所述双抗还包括特异性结合B7-H3不同表位的抗体。
根据本发明的一些实施方式，所述药物组合物还包括上述双抗。
根据本发明的一些实施方式，所述药物组合物还包括含有上述抗体的ADC药物。进一步地，所述ADC药物还包括连接臂和毒性分子。
根据本发明地一些实施方式，所述药物组合物还包括药学上可用的辅料。
根据本发明第三方面的实施方式的制备方法，包括以下步骤：培养上述分离细胞，从培养物中回收所述抗体。
在本发明中，具体的制备方法为：将编码上述抗B7-H3抗体的重链可变区序列克隆到含有IgG1重链恒定区氨基酸序列的重组质粒1中，轻链可变区序列克隆到含有Kappa轻链恒定区氨基酸序列的重组质粒2中，将重组质粒1和重组质粒2同时转染细胞并进行培养，从培养物中回收所述抗B7-H3抗体。
根据本发明第四方面的实施方式的应用，所述抗体可应用于抗癌药物和/或抗体检测试剂盒的制备中。
在本发明中，所述抗癌药物主要用于预防或治疗癌症，其中所述癌症是表达B7-H3蛋白的乳腺癌、子宫内膜癌、卵巢癌、肺癌、胃癌、前列腺癌、肾癌、肝癌、胰腺癌、结肠直肠癌、食道癌、膀胱癌、子宫颈癌、血液癌、淋巴瘤或恶性黑色素瘤。
在本发明中，抗癌药物还包含与靶向其它靶点的抗体，如：CD3、BCMA、CD38等构建成双特异性抗体，开发成各种具有调节肿瘤细胞作用的方法。
在本发明中，本发明的抗体还可以通过偶联其它类型的分子，如：毒素、核酸分子等，通过该抗体特异地将偶联的分子带到肿瘤细胞体内，从而调节肿瘤细胞的作用。
在本发明中，可对所述的B7-H3特异性抗体或抗原结合片段进行标记以用于所述方法或本领域技术人员已知的其它方法。例如，本发明所述的抗体或其抗原结合片段可用放射标记物、荧光标记物、表位标签、生物素、发色团标记物、ECL标记物、酶、钌、111In-DOTA、111In-二乙烯三胺五乙酸(DTPA)、辣根过氧化物酶、碱性磷酸酶和β-半乳糖苷酶，或者聚组氨酸或本领域已知的类似此类标记物进行标记。
在本发明中，该抗体还可以开发成通过免疫手段，对组织细胞表面B7-H3表达量进行检测的方法。例如，该抗体还可用于检测生物样品诸如血液或血清中B7-H3的存在、用于定量分析生物样品诸如血液或血清中B7-H3的量、用于诊断B7-H3表达型癌症、用于确定治疗患有癌症的受治疗者的方法、或用于监测受治疗者中B7-H3表达型癌症的进展等。
在本发明中，术语“抗体”具体包括狭义抗体，还包括“嵌合”抗体以及该抗体片段，其中部分重链和/轻链与衍生自特定物种或属于特定抗体类型或亚类抗体的对应序列相同或同源，而该链的剩余部分与衍生自另一物种或属于另一抗体类型或亚类抗体的对应序列相同或同源，只要其特异性结合靶抗原和/或显示所需的生物活性(美国专利号4,816,567，和Morrison等，Proc.Natl.Acad.Sci.USA 81:6851-6855(1984))。其中，“狭义抗体”是指由抗原进入机体刺激B细胞分化增殖为浆细胞而合并分泌的一类能与相应抗原发生特异性结合并产生免疫效应的含有糖基的球蛋白。1964年世界卫生组织召开会议，将具有抗体活性及化学结构与抗体相似的球蛋白统称为免疫球蛋白。现代免疫学认为，抗体与免疫球蛋白是等同概念，只是抗体侧重于其生物学活性的描述，而免疫球蛋白侧重强调其化学结构。
免疫球蛋白的基本结构包含四条肽链：两条重链(Heavy chain，H链)和两条轻链(Light chain，L链)，轻链与重链由二硫键连接形成一个对称四肽链分子，成为免疫球蛋白分子单体，单体是构成所有免疫球蛋白的基本结构。每条重链和轻链分为氨基端(N端)和羧基端(C端)。通过对H链或L链的氨基酸序列比较分析发现：其N端序列变化很大，称此区为可变区(Variable region，V区)；C端氨基酸相对稳定，变化很小，称此区为恒定区(Constantregion，C区)。可变区可分为高可变区(Hypervariable region，HVR)和骨架区(Frameworkregion，FR)。重链可变区和轻链可变区的分别有3个HVR，从N端往C端，分别称为重链或轻链的HVR1、HVR2和HVR3。高可变区为抗体与抗原的结合位置，称为互补决定区(Complementarity-determining region，CDR)，因此，重链或轻链的HVR1、HVR2和HVR3又称CDR1、CDR2和CDR3。
在本发明中，术语“双抗”，即“双特异性”抗体，指某一抗体，一般是单克隆抗体，具有至少两个不同抗原性表位的结合特性。在一个实施方式中，该表位来自相同抗原。在另一个实施方式中，该表位来自两个不同抗原。制备双特异性抗体的方法为本领域已知。例如，双特异性抗体可通过共同表达两种免疫球蛋白重/轻链对来重组生产。参见例如，Milstein等，Nature305:537-39(1983)。或者，可利用化学连接制备双特异性抗体。参见例如，Brennan等，Science229:81(1985)。双特异性抗体包括双特异性抗体片段(例如，Hollinger等，Proc.Natl.Acad.Sci.U.S.A.90:6444-48(1993)，Gruber等，J.Immunol.152:5368(1994))。
本发明中，术语“人源化抗体”指含有来自非人(例如鼠)抗体以及人抗体序列的抗体形式。该抗体是含有衍生自非人免疫球蛋白最小序列的嵌合抗体。通常，所述人源化抗体包含几乎所有的至少一个且通常两个可变区，其中全部或基本上全部的高变环对应于非人免疫球蛋白的高变环，全部或基本上全部的FR区是人免疫球蛋白序列的FR区。人源化抗体还任选包含至少一部分免疫球蛋白恒定区(Fc)，一般是人免疫球蛋白的Fc。参见例如，Cabilly美国专利号4,816,567；Queen等(1989)Proc.Natl.Acad.Sci.USA86:10029-10033；和Antibody Engineering:A Practical Approach(《抗体工程：实践方法》)(牛津大学出版社(OxfordUniversityPress)1996)。
本发明所用氨基酸三字母代码和单字母代码如J.Biol.Chem,243,P3558(1968)中所述。
本发明的附加方面和优点将在下面的描述中部分给出，部分将从下面的描述中变得明显，或通过本发明的实践了解到。
附图说明
图1示出了本发明实施例3中嵌合抗体27B4与MDA-MB-231的结合力；
图2示出了本发明实施例3中嵌合抗体27B4的ADCC活性；
图3示出了本发明实施例4中不同27B4人源化scFv抗体与B7-H3抗原的结合力；
图4示出了本发明实施例4中不同Whole IgG形式27B4人源化抗体B7-H3抗原的结合力；
图5示出了本发明实施例4中不同Whole IgG形式27B4人源化抗体ADCC活性。
图6示出了本发明实施例4中27B4人源化抗体h27B4-H4-L3(27B4Z02分子)结合B7-H32Ig与4Ig蛋白的活性。
图7示出了本发明实施例4中27B4人源化抗体h27B4-H4-L3(27B4Z02分子)结合人和猴B7-H3蛋白的活性。
具体实施方式
为详细说明本发明的技术内容、所实现目的及效果，以下结合实施方式并配合附图予以说明。
实施例1.B7-H3抗原、表达载体及稳转细胞株的制备
1-1.B7-H3抗原及检测用蛋白的制备
筛选及检测用人4Ig-B7-H3抗原为商品化产品(Acro，货号B7B-H52E7)，序列如下：
Figure BDA0003234280210000071
筛选及检测用人2Ig-B7-H3抗原为商品化产品(Acro，货号B73-H52E2)，序列如下：
Figure BDA0003234280210000072
筛选及检测用食猕猴4Ig-B7-H3抗原为商品化产品(Acro，货号B73-C52Ha)，序列如下：
Figure BDA0003234280210000073
筛选及检测用鼠2Ig-B7-H3抗原为商品化产品(Acro，货号B73-M52H4)，序列如下：
Figure BDA0003234280210000081
注释：划横线部分为B7-H3胞外区；斜体部分为linker；黑色加粗部分为His-tag标签。
1-2.人4Ig-B7-H3表达载体的制备
从NCBI数据库中获取编码人B7-H3变体1(4Ig-B7-H3)的CDS区，由生工生物工程(上海)股份有限公司常规合成全长基因序列，并通过酶切位点XbaI与NotI构建到慢病毒过表达载体pCDH-EF1-T2A-copGFP中，经测序验证序列正确，即成功构建质粒pCDH-EF1-h4Ig-B7-H3-T2A-copGFP。在液体LB培养基中培养菌种pCDH-EF1-h4Ig-B7-H3-T2A-copGFP，并使用天根生化科技(北京)有限公司无内毒素质粒大提试剂盒(DP117)，按照说明书常规步骤提取质粒DNA。
人B7-H3变体1(4Ig-B7-H3)的CDS区碱基序列：
atgctgcgtcggcggggcagccctggcatgggtgtgcatgtgggtgcagccctgggagcactgtggttctgcctcacaggagccctggaggtccaggtccctgaagacccagtggtggcactggtgggcaccgatgccaccctgtgctgctccttctcccctgagcctggcttcagcctggcacagctcaacctcatctggcagctgacagataccaaacagctggtgcacagctttgctgagggccaggaccagggcagcgcctatgccaaccgcacggccctcttcccggacctgctggcacagggcaacgcatccctgaggctgcagcgcgtgcgtgtggcggacgagggcagcttcacctgcttcgtgagcatccgggatttcggcagcgctgccgtcagcctgcaggtggccgctccctactcgaagcccagcatgaccctggagcccaacaaggacctgcggccaggggacacggtgaccatcacgtgctccagctaccagggctaccctgaggctgaggtgttctggcaggatgggcagggtgtgcccctgactggcaacgtgaccacgtcgcagatggccaacgagcagggcttgtttgatgtgcacagcatcctgcgggtggtgctgggtgcaaatggcacctacagctgcctggtgcgcaaccccgtgctgcagcaggatgcgcacagctctgtcaccatcacaccccagagaagccccacaggagccgtggaggtccaggtccctgaggacccggtggtggccctagtgggcaccgatgccaccctgcgctgctccttctcccccgagcctggcttcagcctggcacagctcaacctcatctggcagctgacagacaccaaacagctggtgcacagtttcaccgaaggccgggaccagggcagcgcctatgccaaccgcacggccctcttcccggacctgctggcacaaggcaatgcatccctgaggctgcagcgcgtgcgtgtggcggacgagggcagcttcacctgcttcgtgagcatccgggatttcggcagcgctgccgtcagcctgcaggtggccgctccctactcgaagcccagcatgaccctggagcccaacaaggacctgcggccaggggacacggtgaccatcacgtgctccagctaccggggctaccctgaggctgaggtgttctggcaggatgggcagggtgtgcccctgactggcaacgtgaccacgtcgcagatggccaacgagcagggcttgtttgatgtgcacagcgtcctgcgggtggt gctgggtgcgaatggcacctacagctgcctggtgcgcaaccccgtgctgcagcaggatgcgcacggctctgtcaccatcacagggcagcctatgacattccccccagaggccctgtgggtgaccgtggggctgtctgtctgtctcattgcactgctggtggccctggctttcgtgtgctggagaaagatcaaacagagctgtgaggaggagaatgcaggagctgaggaccaggatggggagggagaaggctccaagacagccctgcagcctctgaaacactctgacagcaaagaagatgatggacaagaaatagcc；
人B7-H3变体1(4Ig-B7-H3)的CDS区氨基酸序列：
MLRRRGSPGMGVHVGAALGALWFCLTGALEVQVPEDPVVALVGTDATLCCSFSPEPGFSLAQLNLIWQLTDTKQLVHSFAEGQDQGSAYANRTALFPDLLAQGNASLRLQRVRVADEGSFTCFVSIRDFGSAAVSLQVAAPYSKPSMTLEPNKDLRPGDTVTITCSSYQGYPEAEVFWQDGQGVPLTGNVTTSQMANEQGLFDVHSILRVVLGANGTYSCLVRNPVLQQDAHSSVTITPQRSPTGAVEVQVPEDPVVALVGTDATLRCSFSPEPGFSLAQLNLIWQLTDTKQLVHSFTEGRDQGSAYANRTALFPDLLAQGNASLRLQRVRVADEGSFTCFVSIRDFGSAAVSLQVAAPYSKPSMTLEPNKDLRPGDTVTITCSSYRGYPEAEVFWQDGQGVPLTGNVTTSQMANEQGLFDVHSVLRVVLGANGTYSCLVRNPVLQQDAHGSVTITGQPMTFPPEALWVTVGLSVCLIALLVALAFVCWRKIKQSCEEENAGAEDQDGEGEGSKTALQPLKHSDSKEDDGQEIA。
1-3.表达人4Ig-B7-H3抗原的293稳转细胞株制备
选取生长状态良好的对数增长期的HEK293细胞(来源于ATCC)，在一个10cm培养皿中接种8E6数量的细胞，DMEM中添加10％胎牛血清，37℃、5％CO2培养箱内培养，当细胞融合率达到70％～80％左右时转染。使用转染试剂PEI共转染三质粒psPAX2、pMD2.G和pCDH-EF1-h4Ig-B7-H3-T2A-copGFP。分别收集转染后48小时与72小时的病毒液，0.45μM针头滤器过滤病毒，按照MOI为10感染目的细胞CHO-S(来源于ATCC)，RPMI-1640加10％胎牛血清培养。病毒感染48小时后换液。构建的细胞株通过流式检测人4Ig-B7-H3表达阳性率，测试阳性率在95％以上，293-h4Ig-B7-H3-copGFP稳转细胞株构建成功。
实施例2.单克隆抗体的制备和抗体的筛选
2-1.免疫接种
选择4只6周龄SPF级BALB/C品系的雌性健康小鼠(购自上海吉辉实验动物饲养有限公司)，免疫抗原为His标签的人重组4Ig-B7-H3抗原(Acro，货号B7B-H52E7)，抗原与等体积佐剂乳化。首次(第0天)免疫使用弗氏完全佐剂(Sigma，货号F5881)乳化抗原，于小鼠颈背部皮下进行多点注射，每只小鼠免疫50μg抗原。第14天、28天、42天及56天用弗氏不完全佐剂(Sigma，货号F5602)乳化抗原进行免疫，每只小鼠免疫25μg抗原。于第20天、34天、48天及62天对小鼠进行眼眶采血，按照实施例2-4的方法通过ELISA检测小鼠免疫血清效价。免疫前血清作为阴性对照，将免疫血清按1:100稀释后，再3倍梯度稀释11个浓度，以3倍阴性对照OD值对应的血清稀释度作为其效价。挑选效价最高且两次加强免疫后血清效价趋于平稳的小鼠，在细胞融合前3天采用25μg重组人4Ig-B7-H3抗原腹腔注射进行一次加强免疫，此次抗原无需佐剂乳化，缓冲液为PBS。
2-2.杂交瘤制备及筛选鉴定
收集生长状态良好的对数增长期的SP2/0骨髓瘤细胞(购自中科院细胞库)与脾细胞按照1：1的比例进行电融合，96孔细胞培养板每孔接种15000个细胞，HAT完全培养基37℃5％CO2培养箱中培养，第4天换成HT完全培养基继续培养。当杂交瘤细胞培养到第7天时，按照实施例2-4的方法通过ELISA法检测细胞上清与人4Ig-B7-H3重组抗原的结合力。对ELISA检测阳性的克隆，按照实施例2-5的方法进行流式细胞仪(Flow Cytometry，FACS)检测，检测上清与过稳转细胞293-h4Ig-B7-H3-copGFP的结合力。采用有限稀释法对阳性杂交瘤细胞进行单克隆化，然后进行ELISA检测及FACS检测，FACS方法同时检测了样品与过稳转细胞293-h4Ig-B7-H3-copGFP及内源表达细胞MDA-MB-231的结合情况，挑选阳性单克隆孔重复进行1次单克隆化及阳性克隆筛选鉴定。
2-3.单克隆抗体的制备
将杂交瘤细胞以2.5×105/mL接种至250mL细胞培养瓶中，培养体积为50mL，待细胞活率降至30％时，离心收获含有抗体的上清液，并使用0.45μM滤器过滤，Protein G介质纯化小鼠单克隆抗体并通过透析方法将抗体置换至PBS pH7.2缓冲液中。通过Nanodrop测定吸光度来确定抗体浓度和纯度，并通过十二烷基硫酸钠凝胶电泳和考马斯染色来检查纯度。
2-4.ELISA方法确定单克隆抗体与不同种属B7-H3及其变体的交叉反应性
将商品化的重组B7-H3抗原(人4Ig-B7-H3抗原，Acro，货号B7B-H52E7或人2Ig-B7-H3，Acro，货号B73-H52E2或食猕猴4Ig-B7-H3，Acro，货号B73-C52Ha或鼠2Ig-B7-H3，Acro，货号B73-M52H4)用PBS稀释至1μg/mL，25μL/孔包被384孔酶标板，4℃孵育过夜。弃去包被液并加入80μg/孔封闭液(含2％脱脂奶粉的PBS)室温封闭2小时。弃去封闭液，PBST(1‰Tween-20)洗板3次。系列稀释单克隆抗体，25μL/孔加入酶标板，室温孵育1小时。PBST洗板3次后，加入HRP标记的羊抗鼠IgG(Jackson，货号115-035-071)，室温孵育1小时。弃去二抗，PBST洗板7次后加入底物TMB显色12min。最后每孔加入25μL终止液(2M HCl)终止反应，用酶标仪读取450nm处吸光度值。
作为结果，发现了1株杂交瘤细胞27B4生产的抗体与人4Ig-B7-H3抗原及人2Ig-B7-H3抗原结合活性最高，且具有物种交叉反应性，能同时结合食猕猴4Ig-B7-H3。该交叉反应活性有利于抗体应用于动物实验中。
2-5.FACS方法确定单克隆抗体与癌细胞系的结合活性
通过FACS的方法，检验实施例2-4中获得的单克隆抗体是否结合过表达4Ig-B7-H3的稳转细胞293-h4Ig-B7-H3-copGFP及高表达4Ig-B7-H3的癌细胞MDA-MB-231(ATCC)。每孔加入5×105个293-h4Ig-B7-H3-copGFP细胞或MDA-MB-231细胞于96孔V型微孔板中，1500r/min离心1min，弃上清。稀释单克隆抗体到20μg/mL，每孔50μL重悬细胞，冰上孵育30min。然后每孔150μL PBS，1500r/min离心1min，弃上清，重复洗板4次。加入APC标记的羊抗鼠IgG(Jackson，货号115-605-164，PBS 1:800稀释)，每孔50μL，冰上孵育30min。用PBS洗板4次后每孔加入150μL PBS重悬细胞，用CytoFLEX进行检测(Beckman)。结果证实，单克隆抗体27B4结合稳转细胞293-h4Ig-B7-H3-copGFP及内源高表达细胞MDA-MB-231。
2-6.杂交瘤测序
收集对数增长期的27B4杂交瘤细胞，用RNAiso Plus(TAKARA，货号9109)提取总RNA，5’RACE法(SMARTer RACE5’/3’Kit，Clontech，货号634859)反转录获得cDNA。以cDNA为模板，Adaptor为上游引物，CL和CH1分别为抗体轻重链的下游引物(参考IMGT恒定区CL和CH1设计下游引物，http://www.imgt.org/genedb/)，使用ExTaq PCR(94℃，3min；94℃，30s，55℃，30s，72℃，45s，32cycle；72℃，5min)扩增抗体轻重链的可变区序列，PCR产物经天根通用型DNA纯化回收试剂盒回收纯化后，以摩尔比3:1分别与pMD18-T载体(Takara，货号6011)连接。连接产物转化到TG-1大肠杆菌感受态细胞进行测序。用VBASE2(http://www.vbase2.org/vbscAb.php)分析测序结果，获得抗体轻重链可变区序列。以下将该筛选出来的抗体记为27B4。
其中，27B4重链可变区的氨基酸序列包括重链互补决定区HCDR1(SEQ ID NO：1)、HCDR2(SEQ ID NO：2)和HCDR3(SEQ ID NO：3)；轻链可变区包括轻链互补决定区LCDR1(SEQID NO：4)、LCDR2(WAS)和LCDR3(SEQ ID NO：5)。
27B4重链可变区27B4 VH的氨基酸序列：SEQ ID NO：6。
27B4轻链可变区27B4 VL的氨基酸序列：SEQ ID NO：7。
表达27B4重链可变区27B4 VH的碱基序列：SEQ ID NO：8。
表达27B4轻链可变区27B4 VL的碱基序列：SEQ ID NO：9。
实施例3.嵌合抗体27B4的制备及活性检测
3-1.重链表达载体pTT5-hIgG1及轻链表达载体pTT5-HL的构建
从NCBI获取抗体的γ重链恒定区氨基酸序列hIgG1(AWK57454.1)，并引进突变L235V,F243L,R292P,Y300L和P396L以增强ADCC效应(antibody-dependent cell-mediatedcytotoxicity，抗体依赖的细胞介导的细胞毒作用)，并在序列N端依次加入Kozak序列(5'-GCCACCATGG-3')、信号肽序列和多克隆位点区序列，进行真核密码子优化获得碱基序列，其中克隆位点区包含酶切位点KpnI(5'-GGTACC-3')及MfeI(5'-CAATTG-3’)，在生工生物工程(上海)股份有限公司常规合成基因序列构建到pTT5载体(Biovector)的多克隆位点中，获得含抗体重链恒定区序列的真核重链表达载体pTT5-hIgG1。
优化的hIgG1氨基端序列：SEQ ID NO：10。
优化的hIgG1碱基序列：SEQ ID NO：11。
从NCBI获取抗体的κ轻链恒定区氨基酸序列HL(CAR58102.1)，并在序列N端依次加入Kozak序列(5'-GCCACCATGG-3')、信号肽序列和多克隆位点区序列，进行真核表达密码子优化获得碱基序列，其中克隆位点区包含酶切位点KpnI(5'-GGTACC-3')及MfeI(5'-CAATTG-3’)，在生工生物工程(上海)股份有限公司常规合成基因序列构建到pTT5载体的多克隆位点中，获得含抗体轻链恒定区序列的真核重链表达载体pTT5-HL。
κ轻链恒定区氨基酸序列：SEQ ID NO：12。
κ轻链恒定区碱基序列：SEQ ID NO：13。
Kozak序列是位于真核生物mRNA 5’端帽子结构后面的一段核酸序列，它可以与翻译起始因子结合而介导含有5’帽子结构的mRNA翻译起始。
3-2.27B4嵌合表达载体的构建
将27B4重链和轻链可变区序列在生工生物工程(上海)股份有限公司常规合成，并通过KpnI及MfeI分别克隆进入pTT5-hIgG1及pTT5-HL载体质粒，得到重链表达质粒pTT5-27B4-VH-hIgG1及轻链表达质粒pTT5-27B4-VL-HL。
嵌合抗体xw.M0883B9.27B4重链氨基酸序列：SEQ ID NO：14。
嵌合抗体xw.M0883B9.27B4轻链氨基酸序列：SEQ ID NO：15。
嵌合抗体xw.M0883B9.27B4重链碱基序列：SEQ ID NO：16。
嵌合抗体xw.M0883B9.27B4轻链碱基序列：SEQ ID NO：17。
3-3.嵌合抗体27B4(xw.M0883B9.27B4抗体)的表达纯化
收集生长状态良好的对数增长期的293F细胞接种至250mL细胞培养瓶中并在50mL培养基中培养，PEI共转染轻链表达质粒pTT5-27B4-VL-HL与重链表达质粒pTT5-27B4-VH-hIgG1各25μg。收集转染后培养第7天的细胞上清，离心并使用0.45μM过滤器过滤，ProteinA介质纯化抗体并通过透析方法将抗体置换至PBS pH7.2缓冲液中。通过Nanodrop测定吸光度来确定抗体浓度和纯度，并通过十二烷基硫酸钠凝胶电泳和考马斯染色来检查纯度。
3-4.嵌合抗体结合活性检测
通过FACS的方法，检验实施例3-3中获得的嵌合抗体xw.M0883B9.27B4与B7-H3抗原的结合活性。阳性参照抗体为MGA-271(NCT02475213)，每孔加入5×105个MDA-MB-231于96孔V型微孔板中，1500r/min离心1min，弃上清。稀释xw.M0883B9.27B4抗体，浓度为100、50、25、12.5、3.125、0.78、0.19、0.0488、0.0122、0.003、0.0007和0.00038μg/mL，每孔50μL，冰上孵育30min。然后每孔150μL PBS，1500r/min离心1min，弃上清，重复洗板4次。加入APC标记的羊抗鼠IgG(Jackson，货号115-605-164，PBS 1:800稀释)，每孔50μL，冰上孵育30min。用PBS洗板4次后每孔加入150μL PBS重悬细胞，用CytoFLEX进行检测(Beckman)。经GraphPad 8.0.2分析，xw.M0883B9.27B4的EC50为3.2μg/mL，MGA-271的EC50为23.8μg/mL，xw.M0883B9.27B4有更高的结合活性，结果如图1所示。因此，说明xw.M0883B9.27B4与B7-H3抗原具有极好的结合活性。
3-5.xw.M0883B9.27B4抗体的ADCC活性
根据常规方法分离健康捐赠者的人外周血单核细胞(PBMC，peripheral bloodmononuclear cell)，并将其悬于含1％FBS的RPMI 1640(称为ADCC培养基)中，使用40μm细胞滤网(BD Biosciences,Ltd.)过滤细胞并计数，调整细胞密度为2x107/mL，作为效应细胞。收集生长状态良好的对数增长期的MDA-MB-231细胞，使用ADCC培养基洗细胞一遍，40μm细胞滤网过滤，细胞计数并调整细胞密度为2x105/mL，作为靶细胞。96孔板中每孔加入靶细胞50μL，即每孔细胞数为10000个。稀释xw.M0883B9.27B4抗体及阳性对照抗体MGA-271，最高浓度为180nM/mL，依次十倍稀释，共8个浓度，即180、18、1.8、0.18、0.018、0.0018、0.00018和0.000018nM/mL，每孔加入50μL，与靶细胞混匀，37℃，5％CO2培养箱培养0.5小时。然后每孔加入效应细胞50μL，即每孔细胞500000个，效靶比为50:1，混匀，37℃，5％CO2培养箱培养4小时。使用试剂
Figure BDA0003234280210000141
Non-Radioactive(Promega，货号G1780)按照说明书常规方法检测细胞毒性。根据如下公式进行计算，xw.M0883B9.27B4抗体具有较对照抗体更强的ADCC活性，结果见图2。
Figure BDA0003234280210000142
备注：实验组＝实验孔-培养基背景对照孔；
靶细胞自发＝靶细胞自发LDH释放孔-培养基背景对照孔；
效应细胞自发＝效应细胞自发LDH释放孔-培养基背景对照孔；
靶细胞最大LDH释放＝靶细胞最大LDH释放孔-体积校正孔。
实施例4.小鼠抗人抗体27B4的人源化
4-1.27B4抗体的人源化设计方案
根据通常称作的CDR移植方法对鼠抗人27B4单克隆抗体进行人源化。简言之，使用IMGT/V-QUEST工具(http://www.imgt.org/IMGT_vquest/input)分析27B4抗体VH和VK碱基序列，确定抗体轻链和重链的CDR区序列。使用IgBlast tool工具(https://www.ncbi.nlm.nih.gov/igblast/)分析27B4抗体的氨基酸序列，获得27B4抗体最接近的人种系VH和VK序列。将27B4抗体的CDR移植到选定的VH和VK人种系序列的框架区中，该序列即为人源化的抗体序列。对27B4抗体VH的人种系VH进行分析，选择5个不同的序列，将27B4抗体VH的CDR移植到这5个序列的框架区中，得到5条重链序列可变区27B4-H1、27B4-H2、27B4-H3、27B4-H4和27B4-H5。以相同的方法对轻链进行CDR移植，得到5条轻链可变区序列27B4-L1、27B4-L2、27B4-L3、27B4-L4和27B4-L5。
27B4-H1、27B4-H2、27B4-H3、27B4-H4和27B4-H5的氨基酸序列分别记作SEQ IDNO：18、SEQ ID NO：19、SEQ ID NO：20、SEQ ID NO：21和SEQ ID NO：22。
27B4-L1、27B4-L2、27B4-L3、27B4-L4和27B4-L5的氨基酸序列分别记作SEQ IDNO：23、SEQ ID NO：24、SEQ ID NO：25、SEQ ID NO：26和SEQ ID NO：27。
27B4-H1、27B4-H2、27B4-H3、27B4-H4和27B4-H5的碱基序列分别记作SEQ ID NO：28、SEQ ID NO：29、SEQ ID NO：30、SEQ ID NO：31和SEQ ID NO：32。
27B4-L1、27B4-L2、27B4-L3、27B4-L4和27B4-L5的碱基序列见序列表SEQ ID NO：33、SEQ ID NO：34、SEQ ID NO：35、SEQ ID NO：36和SEQ ID NO：37。
4-2.27B4人源化scFv的构建
在重链可变区序列5’端添加碱基ttactcgcggcccagccggccatggcc，在重链可变区序列3’端添加碱基acagtctcgagtggtggtgg，得到对应的重链可变区序列27B4-H1-F、27B4-H2-F、27B4-H3-F、27B4-H4-F和27B4-H5-F。设计linker序列。在轻链可变区序列5’端添加碱基gtggcggtggcggtgctagc，在轻链可变区序列3’端添加碱基gcggccgcaggcgcggaacaaaaac，得到对应的轻链可变区序列27B4-L1-F、27B4-L2-F、27B4-L3-F、27B4-L4-F和27B4-L5-F。
在生工生物工程(上海)股份有限公司常规合成序列，分别选择一条重链可变区序列、linker(碱基序列信息为：ggtggtggcggttctggtggtggtggtagcggtggcggtggtagtggcggtggcggtgctagc)及一条轻链可变区序列与经过NcoI及NotI酶切处理的噬菌体展示载体pXY，使用重组酶(
Figure BDA0003234280210000151
HiFi DNA Assembly Master Mix，NEB，货号E2621S)，按照说明书常规方法进行重组。将通过27B4-H1-F、linker及27B4-L1-F组合得到的人源化scFv 27B4抗体命名为“scFv-27B4-H1-L1”，将通过27B4-H1-F、linker及27B4-L2-F组合得到的人源化scFv 27B4抗体命名为“scFv-27B4-H1-L2”，将通过27B4-H1-F、linker及27B4-L3-F组合得到的人源化scFv 27B4抗体命名为“scFv-27B4-H1-L3”，将通过27B4-H1-F、linker及27B4-L4-F组合得到的人源化scFv 27B4抗体命名为“scFv-27B4-H1-L4”，将通过27B4-H1-F、linker及27B4-L5-F组合得到的人源化scFv 27B4抗体命名为“scFv-27B4-H1-L5”，将通过27B4-H2-F、linker及27B4-L1-F组合得到的人源化scFv 27B4抗体命名为“scFv-27B4-H2-L1”，将通过27B4-H2-F、linker及27B4-L2-F组合得到的人源化scFv 27B4抗体命名为“scFv-27B4-H2-L2”，将通过27B4-H2-F、linker及27B4-L3-F组合得到的人源化scFv 27B4抗体命名为“scFv-27B4-H2-L3”，将通过27B4-H2-F、linker及27B4-L4-F组合得到的人源化scFv 27B4抗体命名为“scFv-27B4-H2-L4”，将通过27B4-H2-F、linker及27B4-L5-F组合得到的人源化scFv 27B4抗体命名为“scFv-27B4-H2-L5”，将通过27B4-H3-F、linker及27B4-L1-F组合得到的人源化scFv 27B4抗体命名为“scFv-27B4-H3-L1”，将通过27B4-H3-F、linker及27B4-L2-F组合得到的人源化scFv 27B4抗体命名为“scFv-27B4-H3-L2”，将通过27B4-H3-F、linker及27B4-L3-F组合得到的人源化scFv 27B4抗体命名为“scFv-27B4-H3-L3”，将通过27B4-H3-F、linker及27B4-L4-F组合得到的人源化scFv 27B4抗体命名为“scFv-27B4-H3-L4”，将通过27B4-H3-F、linker及27B4-L5-F组合得到的人源化scFv 27B4抗体命名为“scFv-27B4-H3-L5”，将通过27B4-H4-F、linker及27B4-L1-F组合得到的人源化scFv 27B4抗体命名为“scFv-27B4-H4-L1”，将通过27B4-H4-F、linker及27B4-L2-F组合得到的人源化scFv 27B4抗体命名为“scFv-27B4-H4-L2”，将通过27B4-H4-F、linker及27B4-L3-F组合得到的人源化scFv 27B4抗体命名为“scFv-27B4-H4-L3”，将通过27B4-H4-F、linker及27B4-L4-F组合得到的人源化scFv 27B4抗体命名为“scFv-27B4-H4-L4”，将通过27B4-H4-F、linker及27B4-L5-F组合得到的人源化scFv 27B4抗体命名为“scFv-27B4-H4-L5”，将通过27B4-H5-F、linker及27B4-L1-F组合得到的人源化scFv 27B4抗体命名为“scFv-27B4-H5-L1”，将通过27B4-H5-F、linker及27B4-L2-F组合得到的人源化scFv 27B4抗体命名为“scFv-27B4-H5-L2”，将通过27B4-H5-F、linker及27B4-L3-F组合得到的人源化scFv 27B4抗体命名为“scFv-27B4-H5-L3”，将通过27B4-H5-F、linker及27B4-L4-F组合得到的人源化scFv 27B4抗体命名为“scFv-27B4-H5-L4”，将通过27B4-H5-F、linker及27B4-L5-F组合得到的人源化scFv 27B4抗体命名为“scFv-27B4-H5-L5”。
4-3.27B4人源化scFv表达
本实施例对实施例4-2中构建的25个27B4人源化序列的scFv单链抗体进行表达纯化。将TG1表达上清200mL高速离心去除杂质，使用20mL平衡衡缓冲液(50mM Na2HPO4，0.3MNaCl，pH＝8.0)预处理镍柱。将离心后的样品上柱，依次用25mL含有10mM、20mM咪唑的洗涤缓冲液洗涤柱子，最后用5mL含有250mM咪唑的洗涤缓冲液洗涤柱子，收集洗脱液。通过透析方法将抗体置换至PBS pH7.2缓冲液中。通过Nanodrop测定吸光度来确定抗体浓度和纯度，并通过十二烷基硫酸钠凝胶电泳和考马斯染色来检查纯度。
4-4.27B4人源化scFv的结合活性检测
按照实施例2-5的方法进行流式细胞仪(FACS)检测，检测27B4人源化scFv与稳转细胞293-h4Ig-B7-H3-copGFP及内源编表达细胞MDA-MB-231的结合力。作为结果，25个27B4人源化scFv与细胞均有不同程度的结合，其中scFv-27B4-H2-L2、scFv-27B4-H2-L3、scFv-27B4-H4-L2及scFv-27B4-H4-L3结合力与细胞表面的B7-H3抗原的结合力与27B4结合力相当或更高，结果见图3。
scFv-27B4-H2-L2、scFv-27B4-H2-L3、scFv-27B4-H4-L2及scFv-27B4-H4-L3的氨基酸序列分别为SEQ ID NO：38、SEQ ID NO：39、SEQ ID NO：40、SEQ ID NO：41。
scFv-27B4-H2-L2、scFv-27B4-H2-L3、scFv-27B4-H4-L2及scFv-27B4-H4-L3的碱基序列分别为SEQ ID NO：42、SEQ ID NO：43、SEQ ID NO：44、SEQ ID NO：45。
4-5.Whole IgG形式27B4人源化抗体的制备
对实施例4-4中获得的scFv-27B4-H2-L2、scFv-27B4-H2-L3、scFv-27B4-H4-L2及scFv-27B4-H4-L3进行Whole IgG形式27B4人源化抗体的制备。将重链人源化序列27B4-H2及27B4-H4在生工生物工程(上海)股份有限公司常规合成，并通过KpnI及MfeI分别克隆进入pTT5-hIgG1中，将轻链人源化序列27B4-L2及27B4-L3在生工生物工程(上海)股份有限公司常规合成，并通过KpnI及MfeI分别克隆进入pTT5-HL载体质粒，得到重链表达质粒pTT5-27B4-H2-hIgG1、pTT5-27B4-H4-hIgG1及轻链表达质粒pTT5-27B4-L2-HL、pTT5-27B4-L3-HL。将生长状态良好的对数增长期的293F细胞接种至250mL细胞培养瓶中并在50mL培养基中培养，PEI共转染轻重链表达质粒各25μg。收集转染后培养第7天的细胞上清，离心并使用0.45μM滤器过滤，Protein A介质纯化抗体并通过透析方法将抗体置换至PBS pH7.2缓冲液中。通过Nanodrop测定吸光度来确定抗体浓度和纯度，并通过十二烷基硫酸钠凝胶电泳和考马斯染色来检查纯度。将通过pTT5-27B4-H2-hIgG1和pTT5-27B4-L2-HL的组合得到的抗体命名为“h27B4-H2-L2”，将通过pTT5-27B4-H2-hIgG1和pTT5-27B4-L3-HL的组合得到的抗体命名为“h27B4-H2-L3”，将通过pTT5-27B4-H4-hIgG1和pTT5-27B4-L2-HL的组合得到的抗体命名为“h27B4-H4-L2”，将通过pTT5-27B4-H4-hIgG1和pTT5-27B4-L3-HL的组合得到的抗体命名为“h27B4-H4-L3”。
h27B4-H2、h27B4-H4的重链氨基酸序列：SEQ ID NO：46、SEQ ID NO：47。
h27B4-L2、h27B4-L3的轻链氨基酸序列：SEQ ID NO：48、SEQ ID NO：49。
h27B4-H2、h27B4-H4的重链碱基序列：SEQ ID NO：50、SEQ ID NO：51。
h27B4-L2、h27B4-L3的轻链碱基序列：SEQ ID NO：52、SEQ ID NO：53。
4-6.Whole IgG形式27B4人源化抗体结合活性检测
按照实施例2-5的方法进行流式细胞仪(FACS)检测，检测whole IgG形式27B4人源抗体与稳转细胞293-h4Ig-B7-H3-copGFP及内源表达细胞MDA-MB-231的结合力。作为结果，4个Whole IgG形式27B4人源化抗体体h27B4-H2-L2、h27B4-H4-L3、h27B4-H2-L3和h27B4-H4-L2(在图中依次标识为zw.M0883B9.27B4Z01、zw.M0883B9.27B4Z02、zw.M0883B9.27B4Z03、zw.M0883B9.27B4Z04)与细胞表面的B7-H3抗原均有较强结合，与人源化之前的27B4嵌合抗体(图中标识为：xw.M0883B9.27B4)结合力相当，比MGA271结合力强，结果见图4。
4-7.Whole IgG形式27B4人源化抗体ADCC活性检测
按照实施例3-5的方法检测whole IgG形式27B4人源抗体的ADCC活性。作为结果，4个Whole IgG形式27B4人源化抗体h27B4-H2-L2、h27B4-H4-L3、h27B4-H2-L3和h27B4-H4-L2(在图中依次标识为zw.M0883B9.27B4Z01、zw.M0883B9.27B4Z02、zw.M0883B9.27B4Z03、zw.M0883B9.27B4Z04)均有ADCC活性，与人源化之前的27B4抗体ADCC活性相当，结果见图5。
4-8.Whole IgG形式27B4人源化抗体的交叉结合实验
根据ADCC活性确定zw.M0883B9.27B4Z02号分子(h27B4-H4-L3,简称Z02号分子)的活性最高，按照实施例2-4的方法检测Z02号分子的交叉反应性。通过对Z02分子进一步的结合EC50和特种交叉结合实验证明，该分子能同时识别人4Ig-B7-H3抗原及人2Ig-B7-H3抗原，且具有同猴4Ig-B7-H3蛋白结合，该交叉反应活性有利于抗体应用于动物实验中。结果见图6和图7。
由上述实施例的实验结果可知，本发明制备得到的抗B7-H3抗体均具有较高的B7-H3抗原结合活性和ADCC活性，能够应用于癌症治疗等领域。
以上所述仅为本发明的实施例，并非因此限制本发明的专利范围，凡是利用本发明说明书及附图内容所作的等同变换，或直接或间接运用在相关的技术领域，均同理包括在本发明的专利保护范围内。
序列表
<110> 上海祥耀生物科技有限责任公司
<120> 抗B7-H3抗体、其制备方法及用途
<160> 53
<170> SIPOSequenceListing 1.0
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Ile Tyr Pro Gly Asp Gly Gly Ser
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Ala Arg Gly Gly Asn Tyr Ile Ser Phe Pro Met Tyr Tyr
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Trp Met Gln Trp Ile Gln Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
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Gly Ala Ile Tyr Pro Gly Asp Gly Gly Ser Arg Tyr Thr Gln Arg Phe
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caggttcagc tccagcagtc tggggctgaa ctggcaagac ctggggcttc agtgaagttg 60
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actcagaggt tcaagggcaa ggccacaatg actgcagatc catcttccag cacagtctac 240
atgcaactca ccagcttggc atctgaggac tctgcggtct atttctgtgc aagaggtggt 300
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gggcaatctc ctaaactact gatttactgg gcatccaccc ggcacactgg agtccctgat 180
cgcttcacag gcagtggatc tgggacagat tatactctca ccatcagcag tgtgcaggct 240
gaagacctgg cactttatta ctgtcagcaa cattatgaca ctccgtacac gttcggaggg 300
gggaccaagc tggagattca a 321
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Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
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Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
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Pro Ala Pro Glu Leu Val Gly Gly Pro Ser Val Phe Leu Leu Pro Pro
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Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
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His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
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gcctccacaa aggggccttc tgtgtttcca ctggccccct cctctaagag caccagcggt 60
gggacagccg ccctgggatg cctggtgaag gactatttcc ctgagcccgt gaccgtgtcc 120
tggaattccg gcgccctgac ttccggcgtg cacaccttcc ccgccgtgct gcagagctct 180
ggcctgtact ctctgtcctc cgtggtgacc gtgccctctt cctctctggg cacccagacc 240
tacatctgca atgtgaacca caagccttcc aacaccaagg tggataaacg ggtggagccc 300
aagtcctgcg acaagaccca tacctgcccc ccctgccccg cccctgaact ggtgggagga 360
ccctccgtgt tcctgttgcc ccccaagccc aaggacactc tgatgatctc ccgcaccccc 420
gaggtcacct gcgtggtcgt ggacgtctct cacgaagacc ccgaggtgaa attcaactgg 480
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agcaccctca gagtggtgtc cgtgctcacc gtgctgcacc aggactggct caacggcaag 600
gaatacaagt gcaaggtgtc caacaaggcc ctccccgccc ccattgaaaa gaccatctcc 660
aaagccaaag gccagcccag agaaccccag gtctacaccc tccccccctc cagagaagaa 720
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cagcagggca acgtgttctc ctgctccgtg atgcacgagg ccctgcacaa ccattacacc 960
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tccaaggact ccacctactc cctctcctcc accctgaccc tctccaaagc cgattacgag 240
aaacacaaag tgtacgcctg cgaggtgaca caccagggcc tgtccagccc cgtcaccaaa 300
agcttcaaca gaggcgagtg c 321
<210> 14
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Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
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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 Arg Val Glu Pro Lys Ser Cys Asp
210 215 220
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Val Gly Gly
225 230 235 240
Pro Ser Val Phe Leu Leu 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 Pro Glu Glu Gln Tyr Asn Ser Thr Leu 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 Glu Glu Met 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 Leu 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 Lys
450
<210> 15
<211> 214
<212> PRT
<213> Human
<400> 15
Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Val Gly
1 5 10 15
Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Arg Gly Val Ser Thr Ala
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile
35 40 45
Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Thr Gly
50 55 60
Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Val Gln Ala
65 70 75 80
Glu Asp Leu Ala Leu Tyr Tyr Cys Gln Gln His Tyr Asp Thr Pro Tyr
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Gln 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> 16
<211> 1350
<212> DNA
<213> Human
<400> 16
caggttcagc tccagcagtc tggggctgaa ctggcaagac ctggggcttc agtgaagttg 60
tcctgcaagg cttctggcta cacctttact acctactgga tgcagtggat acaacagagg 120
cctggacagg gtctggaatg gattggggct atttatcctg gagatggtgg ttctcggtac 180
actcagaggt tcaagggcaa ggccacaatg actgcagatc catcttccag cacagtctac 240
atgcaactca ccagcttggc atctgaggac tctgcggtct atttctgtgc aagaggtggt 300
aactacataa gttttcctat gtattactgg ggtcaaggaa cctcagtcac cgtctcccca 360
gcctccacaa aggggccttc tgtgtttcca ctggccccct cctctaagag caccagcggt 420
gggacagccg ccctgggatg cctggtgaag gactatttcc ctgagcccgt gaccgtgtcc 480
tggaattccg gcgccctgac ttccggcgtg cacaccttcc ccgccgtgct gcagagctct 540
ggcctgtact ctctgtcctc cgtggtgacc gtgccctctt cctctctggg cacccagacc 600
tacatctgca atgtgaacca caagccttcc aacaccaagg tggataaacg ggtggagccc 660
aagtcctgcg acaagaccca tacctgcccc ccctgccccg cccctgaact ggtgggagga 720
ccctccgtgt tcctgttgcc ccccaagccc aaggacactc tgatgatctc ccgcaccccc 780
gaggtcacct gcgtggtcgt ggacgtctct cacgaagacc ccgaggtgaa attcaactgg 840
tacgtggacg gcgtcgaggt gcacaacgcc aagaccaagc cccccgagga acagtataac 900
agcaccctca gagtggtgtc cgtgctcacc gtgctgcacc aggactggct caacggcaag 960
gaatacaagt gcaaggtgtc caacaaggcc ctccccgccc ccattgaaaa gaccatctcc 1020
aaagccaaag gccagcccag agaaccccag gtctacaccc tccccccctc cagagaagaa 1080
atgaccaaga accaggtgag cctgacctgc ctcgtcaaag ggttctaccc ctccgacatc 1140
gccgtggagt gggagagcaa cggccagccc gagaacaact acaaaaccac ccccctcgtg 1200
ctcgactccg acggatcctt cttcctgtac agcaaactga ccgtggacaa atcccgctgg 1260
cagcagggca acgtgttctc ctgctccgtg atgcacgagg ccctgcacaa ccattacacc 1320
cagaagtccc tgtccctgtc ccccggcaag 1350
<210> 17
<211> 642
<212> DNA
<213> Human
<400> 17
gacattgtga tgacccagtc tcacaaattc atgtccacat cagtaggaga cagggtcagc 60
atcacctgta aggccagtcg gggtgtgagt actgctgttg cctggtatca acaaaaacca 120
gggcaatctc ctaaactact gatttactgg gcatccaccc ggcacactgg agtccctgat 180
cgcttcacag gcagtggatc tgggacagat tatactctca ccatcagcag tgtgcaggct 240
gaagacctgg cactttatta ctgtcagcaa cattatgaca ctccgtacac gttcggaggg 300
gggaccaagc tggagattca aagaaccgtg gccgccccct ccgtgtttat cttccccccc 360
tctgacgagc agctcaagtc cggcaccgcc tccgtcgtct gcctcctgaa caatttctac 420
ccccgcgaag ctaaagtcca gtggaaggtg gataacgccc tgcagtccgg caactcccag 480
gagagcgtca ccgagcagga ctccaaggac tccacctact ccctctcctc caccctgacc 540
ctctccaaag ccgattacga gaaacacaaa gtgtacgcct gcgaggtgac acaccagggc 600
ctgtccagcc ccgtcaccaa aagcttcaac agaggcgagt gc 642
<210> 18
<211> 120
<212> PRT
<213> Human
<400> 18
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 Thr Tyr
20 25 30
Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Ile Ile Tyr Pro Gly Asp Gly Gly Ser Ser Tyr Ala Gln Lys Phe
50 55 60
Gln 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 Gly Gly Asn Tyr Ile Ser Phe Pro Met Tyr Tyr Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 19
<211> 120
<212> PRT
<213> Human
<400> 19
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 Thr Tyr
20 25 30
Trp Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Gly Ile Tyr Pro Gly Asp Gly Gly Ser Asn Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gly Gly Asn Tyr Ile Ser Phe Pro Met Tyr Tyr Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 20
<211> 120
<212> PRT
<213> Human
<400> 20
Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Tyr
20 25 30
Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Trp Ile Tyr Pro Gly Asp Gly Gly Ser Thr Tyr Ala Gln Gly Phe
50 55 60
Thr Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr
65 70 75 80
Leu Gln Ile Cys Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gly Gly Asn Tyr Ile Ser Phe Pro Met Tyr Tyr Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 21
<211> 120
<212> PRT
<213> Human
<400> 21
Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu
1 5 10 15
Ser Leu Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Tyr
20 25 30
Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met
35 40 45
Gly Ile Ile Tyr Pro Gly Asp Gly Gly Ser Arg Tyr Ser Pro Ser Phe
50 55 60
Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr
65 70 75 80
Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys
85 90 95
Ala Arg Gly Gly Asn Tyr Ile Ser Phe Pro Met Tyr Tyr Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 22
<211> 120
<212> PRT
<213> Human
<400> 22
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Tyr
20 25 30
Trp Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Val Ile Tyr Pro Gly Asp Gly Gly Ser Tyr Tyr Thr Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gly Gly Asn Tyr Ile Ser Phe Pro Met Tyr Tyr Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 23
<211> 107
<212> PRT
<213> Human
<400> 23
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Met Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Arg Gly Val Ser Thr Ala
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Trp Ala Ser Ser Leu Gln 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 His Tyr Asp Thr Pro Tyr
85 90 95
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 24
<211> 107
<212> PRT
<213> Human
<400> 24
Val Ile Trp Met Thr Gln Ser Pro Ser Leu Leu Ser Ala Ser Thr Gly
1 5 10 15
Asp Arg Val Thr Ile Ser Cys Lys Ala Ser Arg Gly Val Ser Thr Ala
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Glu Leu Leu Ile
35 40 45
Tyr Trp Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Cys Leu Gln Ser
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Asp Thr Pro Tyr
85 90 95
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 25
<211> 107
<212> PRT
<213> Human
<400> 25
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 Lys Ala Ser Arg Gly Val Ser Thr Ala
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45
Tyr Trp Ala Ser Thr Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser
65 70 75 80
Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln His Tyr Asp Thr Pro Tyr
85 90 95
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 26
<211> 107
<212> PRT
<213> Human
<400> 26
Glu Ile Val Leu Thr Gln Ser Pro Asp Phe Gln Ser Val Thr Pro Lys
1 5 10 15
Glu Lys Val Thr Ile Thr Cys Lys Ala Ser Arg Gly Val Ser Thr Ala
20 25 30
Leu His Trp Tyr Gln Gln Lys Pro Asp Gln Ser Pro Lys Leu Leu Ile
35 40 45
Lys Trp Ala Ser Gln Ser Phe Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser Leu Glu Ala
65 70 75 80
Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln His Tyr Asp Thr Pro Tyr
85 90 95
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 27
<211> 107
<212> PRT
<213> Human
<400> 27
Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly
1 5 10 15
Gln Pro Ala Ser Ile Ser Cys Lys Ala Ser Arg Gly Val Ser Thr Ala
20 25 30
Leu Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Pro Pro Gln Leu Leu Ile
35 40 45
Tyr Trp Ala Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala
65 70 75 80
Glu Asp Val Gly Val Tyr Tyr Cys Gln Gln His Tyr Asp Thr Pro Tyr
85 90 95
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 28
<211> 360
<212> DNA
<213> Human
<400> 28
caggtgcagc tggtgcagag cggcgcggaa gtgaagaagc ctggtgcgag cgtgaaagtg 60
agctgcaaag cgagcggcta tacctttacc acctattgga tgcattgggt gcgccaggcg 120
ccgggccagg gcctggaatg gatgggcatt atttatccgg gcgatggcgg cagcagctat 180
gcgcagaaat ttcagggccg cgtgaccatg acccgcgata ccagcaccag caccgtgtat 240
atggaactga gcagcctgcg cagcgaagat accgcggtgt attattgcgc gcgcggcggc 300
aactatatta gctttccgat gtattattgg ggccaaggca ccctggtgac agtctcgagt 360
<210> 29
<211> 360
<212> DNA
<213> Human
<400> 29
caggtgcagc tggtgcagag cggcgcggaa gtgaagaagc ctggtagcag cgtgaaagtg 60
agctgcaaag cgagcggcta tacctttacc acctattgga ttagctgggt gcgccaggcg 120
ccgggccagg gcctggaatg gatgggcggc atttatccgg gcgatggcgg cagcaactat 180
gcgcagaaat ttcagggccg cgtgaccatt accgcggatg aaagcaccag caccgcgtat 240
atggaactga gcagcctgcg cagcgaagat accgcggtgt attattgcgc gcgcggcggc 300
aactatatta gctttccgat gtattattgg ggccaaggca ccctggtgac agtctcgagt 360
<210> 30
<211> 360
<212> DNA
<213> Human
<400> 30
caggtgcagc tggtgcagag cggcagcgaa ctgaagaagc ctggtgcgag cgtgaaagtg 60
agctgcaaag cgagcggcta tacctttacc acctattgga tgaactgggt gcgccaggcg 120
ccgggccagg gcctggaatg gatgggctgg atttatccgg gcgatggcgg cagcacctat 180
gcgcagggct ttaccggccg ctttgtgttt agcctggata ccagcgtgag caccgcgtat 240
ctgcagattt gcagcctgaa agcggaagat accgcggtgt attattgcgc gcgcggcggc 300
aactatatta gctttccgat gtattattgg ggccaaggca ccctggtgac agtctcgagt 360
<210> 31
<211> 360
<212> DNA
<213> Human
<400> 31
gaagtgcagc tggtgcagag cggcgcggaa gtgaagaagc caggagaaag cctgaagata 60
tcttgcaaag cgagcggcta tacctttacc acctattgga ttggctgggt gcgccagatg 120
ccgggcaaag gcctggaatg gatgggcatt atttatccgg gcgatggcgg cagccgctat 180
agcccgagct ttcagggcca ggtgaccatt agcgcggata agtcaatcag caccgcgtat 240
ctgcagtgga gcagcctgaa agcgagcgat accgccatgt attattgcgc gcgcggcggc 300
aactatatta gctttccgat gtattattgg ggccaaggca ccctggtgac agtctcgagt 360
<210> 32
<211> 360
<212> DNA
<213> Human
<400> 32
caagtacaac tagtcgagtc gggtggcgga gtggtccaac ctggaagaag cctgcgcctg 60
agctgcaaag cgagcggcta tacctttacc acctattgga tgcattgggt gcgccaggcg 120
ccgggcaaag gcctggaatg ggtggcggtg atttatccgg gcgatggcgg cagctattat 180
accgatagcg tgaaaggccg ctttaccatt agccgcgata acagcaagaa tactctgtat 240
ctgcagatga acagcctgcg cgcggaagat accgcggtgt attattgcgc gcgcggcggc 300
aactatatta gctttccgat gtattattgg ggccaaggca ccctggtgac agtctcgagt 360
<210> 33
<211> 321
<212> DNA
<213> Human
<400> 33
gatattcaga tgacccagag cccgagcagc ctgagcgcga gcatgggcga tcgcgtgacc 60
attacctgca aagcgagccg cggcgtgagc accgcgctga actggtatca gcagaaaccg 120
ggcaaagcgc cgaaactgct gatttattgg gcgtctagtc tccaatcggg tgtgccgagc 180
cgctttagcg gcagcggcag cggcaccgat ttcactctca ccatttcgtc tctccaacca 240
gaggatttcg ctacttatta ttgccagcag cattatgata cgccctacac ctttggccag 300
ggcaccaaac tggaaattaa a 321
<210> 34
<211> 321
<212> DNA
<213> Human
<400> 34
gtcatttgga tgacccagag cccgagcctg ctgagcgcga gcaccggcga tcgcgttaca 60
atctcatgca aagcgagccg cggcgtgagc accgcgctgg cgtggtatca gcagaaaccg 120
ggcaaagcgc cggaactgct gatttattgg gcgtctaccc tgcagagcgg cgtgccgagc 180
cgctttagcg gcagcggcag cggcaccgat ttcactctga cgatatcgtg tctccaaagc 240
gaagatttcg caacgtatta ttgccagcag cattatgata cgccctacac ctttggccag 300
ggcaccaaac tggaaattaa a 321
<210> 35
<211> 321
<212> DNA
<213> Human
<400> 35
gaaattgtaa tgactcaatc tcccgcaacg ctatcggtta gccctggcga acgcgcgacg 60
ttgagttgca aagcgagccg cggcgtgagc accgcgctgg cgtggtatca gcagaaaccg 120
ggccaggcgc cgcgcctgct gatttattgg gcgtctaccc gcgcgaccgg cattccggcg 180
cgctttagcg gcagcggcag cggcaccgaa tttaccctga ccattagcag cctgcagagc 240
gaagatttcg ctgtctatta ttgccagcag cattatgata cgccctacac ctttggccag 300
ggcaccaaac tggaaattaa a 321
<210> 36
<211> 321
<212> DNA
<213> Human
<400> 36
gaaattgtgc tgacccagag cccggatttc caatcggtga cgccaaagga gaaggttaca 60
attacctgca aagcgagccg cggcgtgagc accgcgctgc attggtatca gcagaaaccg 120
gatcagagcc cgaaactgct gattaaatgg gcgtctcaga gctttagcgg cgtgccgagc 180
cgctttagcg gcagcggcag cggcaccgat ttcacgctaa ccattaacag cctggaagcg 240
gaagatgcgg cgacctatta ttgccagcag cattatgata cgccctacac ctttggccag 300
ggcaccaaac tggaaattaa a 321
<210> 37
<211> 321
<212> DNA
<213> Human
<400> 37
gatattgtga tgacccagac accgctgtcg ctgagcgtga cgcccggtca acccgcatca 60
attagctgca aagcgagccg cggcgtgagc accgcgctgt attggtatct gcagaaaccc 120
gggcaaccac cgcagctgct gatttattgg gcgtctaaca gattctctgg agttcccgat 180
cggttctctg ggagtggaag cggcaccgat ttcactctaa agatatcgcg cgtggaagcg 240
gaagatgtgg gcgtgtatta ttgccagcag cattatgata cgccctacac ctttggccag 300
ggcaccaaac tggaaattaa a 321
<210> 38
<211> 248
<212> PRT
<213> Human
<400> 38
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 Thr Tyr
20 25 30
Trp Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Gly Ile Tyr Pro Gly Asp Gly Gly Ser Asn Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gly Gly Asn Tyr Ile Ser Phe Pro Met Tyr 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 Gly Gly Gly Gly Ala Ser Val Ile Trp
130 135 140
Met Thr Gln Ser Pro Ser Leu Leu Ser Ala Ser Thr Gly Asp Arg Val
145 150 155 160
Thr Ile Ser Cys Lys Ala Ser Arg Gly Val Ser Thr Ala Leu Ala Trp
165 170 175
Tyr Gln Gln Lys Pro Gly Lys Ala Pro Glu Leu Leu Ile Tyr Trp Ala
180 185 190
Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser
195 200 205
Gly Thr Asp Phe Thr Leu Thr Ile Ser Cys Leu Gln Ser Glu Asp Phe
210 215 220
Ala Thr Tyr Tyr Cys Gln Gln His Tyr Asp Thr Pro Tyr Thr Phe Gly
225 230 235 240
Gln Gly Thr Lys Leu Glu Ile Lys
245
<210> 39
<211> 248
<212> PRT
<213> Human
<400> 39
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 Thr Tyr
20 25 30
Trp Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Gly Ile Tyr Pro Gly Asp Gly Gly Ser Asn Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gly Gly Asn Tyr Ile Ser Phe Pro Met Tyr 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 Gly Gly Gly Gly Ala Ser Glu Ile Val
130 135 140
Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly Glu Arg Ala
145 150 155 160
Thr Leu Ser Cys Lys Ala Ser Arg Gly Val Ser Thr Ala Leu Ala Trp
165 170 175
Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr Trp Ala
180 185 190
Ser Thr Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser
195 200 205
Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser Glu Asp Phe
210 215 220
Ala Val Tyr Tyr Cys Gln Gln His Tyr Asp Thr Pro Tyr Thr Phe Gly
225 230 235 240
Gln Gly Thr Lys Leu Glu Ile Lys
245
<210> 40
<211> 248
<212> PRT
<213> Human
<400> 40
Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu
1 5 10 15
Ser Leu Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Tyr
20 25 30
Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met
35 40 45
Gly Ile Ile Tyr Pro Gly Asp Gly Gly Ser Arg Tyr Ser Pro Ser Phe
50 55 60
Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr
65 70 75 80
Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys
85 90 95
Ala Arg Gly Gly Asn Tyr Ile Ser Phe Pro Met Tyr 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 Gly Gly Gly Gly Ala Ser Val Ile Trp
130 135 140
Met Thr Gln Ser Pro Ser Leu Leu Ser Ala Ser Thr Gly Asp Arg Val
145 150 155 160
Thr Ile Ser Cys Lys Ala Ser Arg Gly Val Ser Thr Ala Leu Ala Trp
165 170 175
Tyr Gln Gln Lys Pro Gly Lys Ala Pro Glu Leu Leu Ile Tyr Trp Ala
180 185 190
Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser
195 200 205
Gly Thr Asp Phe Thr Leu Thr Ile Ser Cys Leu Gln Ser Glu Asp Phe
210 215 220
Ala Thr Tyr Tyr Cys Gln Gln His Tyr Asp Thr Pro Tyr Thr Phe Gly
225 230 235 240
Gln Gly Thr Lys Leu Glu Ile Lys
245
<210> 41
<211> 248
<212> PRT
<213> Human
<400> 41
Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu
1 5 10 15
Ser Leu Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Tyr
20 25 30
Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met
35 40 45
Gly Ile Ile Tyr Pro Gly Asp Gly Gly Ser Arg Tyr Ser Pro Ser Phe
50 55 60
Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr
65 70 75 80
Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys
85 90 95
Ala Arg Gly Gly Asn Tyr Ile Ser Phe Pro Met Tyr 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 Gly Gly Gly Gly Ala Ser Glu Ile Val
130 135 140
Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly Glu Arg Ala
145 150 155 160
Thr Leu Ser Cys Lys Ala Ser Arg Gly Val Ser Thr Ala Leu Ala Trp
165 170 175
Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr Trp Ala
180 185 190
Ser Thr Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser
195 200 205
Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser Glu Asp Phe
210 215 220
Ala Val Tyr Tyr Cys Gln Gln His Tyr Asp Thr Pro Tyr Thr Phe Gly
225 230 235 240
Gln Gly Thr Lys Leu Glu Ile Lys
245
<210> 42
<211> 744
<212> DNA
<213> Human
<400> 42
caggtgcagc tggtgcagag cggcgcggaa gtgaagaagc ctggtagcag cgtgaaagtg 60
agctgcaaag cgagcggcta tacctttacc acctattgga ttagctgggt gcgccaggcg 120
ccgggccagg gcctggaatg gatgggcggc atttatccgg gcgatggcgg cagcaactat 180
gcgcagaaat ttcagggccg cgtgaccatt accgcggatg aaagcaccag caccgcgtat 240
atggaactga gcagcctgcg cagcgaagat accgcggtgt attattgcgc gcgcggcggc 300
aactatatta gctttccgat gtattattgg ggccaaggca ccctggtgac agtctcgagt 360
ggtggtggcg gttctggtgg tggtggtagc ggtggcggtg gtagtggcgg tggcggtgct 420
agcgtcattt ggatgaccca gagcccgagc ctgctgagcg cgagcaccgg cgatcgcgtt 480
acaatctcat gcaaagcgag ccgcggcgtg agcaccgcgc tggcgtggta tcagcagaaa 540
ccgggcaaag cgccggaact gctgatttat tgggcgtcta ccctgcagag cggcgtgccg 600
agccgcttta gcggcagcgg cagcggcacc gatttcactc tgacgatatc gtgtctccaa 660
agcgaagatt tcgcaacgta ttattgccag cagcattatg atacgcccta cacctttggc 720
cagggcacca aactggaaat taaa 744
<210> 43
<211> 744
<212> DNA
<213> Human
<400> 43
caggtgcagc tggtgcagag cggcgcggaa gtgaagaagc ctggtagcag cgtgaaagtg 60
agctgcaaag cgagcggcta tacctttacc acctattgga ttagctgggt gcgccaggcg 120
ccgggccagg gcctggaatg gatgggcggc atttatccgg gcgatggcgg cagcaactat 180
gcgcagaaat ttcagggccg cgtgaccatt accgcggatg aaagcaccag caccgcgtat 240
atggaactga gcagcctgcg cagcgaagat accgcggtgt attattgcgc gcgcggcggc 300
aactatatta gctttccgat gtattattgg ggccaaggca ccctggtgac agtctcgagt 360
ggtggtggcg gttctggtgg tggtggtagc ggtggcggtg gtagtggcgg tggcggtgct 420
agcgaaattg taatgactca atctcccgca acgctatcgg ttagccctgg cgaacgcgcg 480
acgttgagtt gcaaagcgag ccgcggcgtg agcaccgcgc tggcgtggta tcagcagaaa 540
ccgggccagg cgccgcgcct gctgatttat tgggcgtcta cccgcgcgac cggcattccg 600
gcgcgcttta gcggcagcgg cagcggcacc gaatttaccc tgaccattag cagcctgcag 660
agcgaagatt tcgctgtcta ttattgccag cagcattatg atacgcccta cacctttggc 720
cagggcacca aactggaaat taaa 744
<210> 44
<211> 744
<212> DNA
<213> Human
<400> 44
gaagtgcagc tggtgcagag cggcgcggaa gtgaagaagc caggagaaag cctgaagata 60
tcttgcaaag cgagcggcta tacctttacc acctattgga ttggctgggt gcgccagatg 120
ccgggcaaag gcctggaatg gatgggcatt atttatccgg gcgatggcgg cagccgctat 180
agcccgagct ttcagggcca ggtgaccatt agcgcggata agtcaatcag caccgcgtat 240
ctgcagtgga gcagcctgaa agcgagcgat accgccatgt attattgcgc gcgcggcggc 300
aactatatta gctttccgat gtattattgg ggccaaggca ccctggtgac agtctcgagt 360
ggtggtggcg gttctggtgg tggtggtagc ggtggcggtg gtagtggcgg tggcggtgct 420
agcgtgattt ggatgaccca gagcccgagc ctgctgagcg cgagcaccgg cgatcgcgtt 480
acaatctcat gcaaagcgag ccgcggcgtg agcaccgcgc tggcgtggta tcagcagaaa 540
ccgggcaaag cgccggaact gctgatttat tgggcgtcta ccctgcagag cggcgtgccg 600
agccgcttta gcggcagcgg cagcggcacc gatttcactc tgacgatatc gtgtctccaa 660
agcgaagatt tcgcaacgta ttattgccag cagcattatg atacgcccta cacctttggc 720
cagggcacca aactggaaat taaa 744
<210> 45
<211> 744
<212> DNA
<213> Human
<400> 45
gaagtgcagc tggtgcagag cggcgcggaa gtgaagaagc caggagaaag cctgaagata 60
tcttgcaaag cgagcggcta tacctttacc acctattgga ttggctgggt gcgccagatg 120
ccgggcaaag gcctggaatg gatgggcatt atttatccgg gcgatggcgg cagccgctat 180
agcccgagct ttcagggcca ggtgaccatt agcgcggata agtcaatcag caccgcgtat 240
ctgcagtgga gcagcctgaa agcgagcgat accgccatgt attattgcgc gcgcggcggc 300
aactatatta gctttccgat gtattattgg ggccaaggca ccctggtgac agtctcgagt 360
ggtggtggcg gttctggtgg tggtggtagc ggtggcggtg gtagtggcgg tggcggtgct 420
agcgaaattg taatgactca atctcccgca acgctatcgg ttagccctgg cgaacgcgcg 480
acgttgagtt gcaaagcgag ccgcggcgtg agcaccgcgc tggcgtggta tcagcagaaa 540
ccgggccagg cgccgcgcct gctgatttat tgggcgtcta cccgcgcgac cggcattccg 600
gcgcgcttta gcggcagcgg cagcggcacc gaatttaccc tgaccattag cagcctgcag 660
agcgaagatt tcgctgtcta ttattgccag cagcattatg atacgcccta cacctttggc 720
cagggcacca aactggaaat taaa 744
<210> 46
<211> 450
<212> PRT
<213> Human
<400> 46
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 Thr Tyr
20 25 30
Trp Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Gly Ile Tyr Pro Gly Asp Gly Gly Ser Asn Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gly Gly Asn Tyr Ile Ser Phe Pro Met Tyr 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 Arg Val Glu Pro Lys Ser Cys Asp
210 215 220
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Val Gly Gly
225 230 235 240
Pro Ser Val Phe Leu Leu 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 Pro Glu Glu Gln Tyr Asn Ser Thr Leu 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 Glu Glu Met 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 Leu 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 Lys
450
<210> 47
<211> 450
<212> PRT
<213> Human
<400> 47
Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu
1 5 10 15
Ser Leu Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Tyr
20 25 30
Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met
35 40 45
Gly Ile Ile Tyr Pro Gly Asp Gly Gly Ser Arg Tyr Ser Pro Ser Phe
50 55 60
Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr
65 70 75 80
Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys
85 90 95
Ala Arg Gly Gly Asn Tyr Ile Ser Phe Pro Met Tyr 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 Arg Val Glu Pro Lys Ser Cys Asp
210 215 220
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Val Gly Gly
225 230 235 240
Pro Ser Val Phe Leu Leu 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 Pro Glu Glu Gln Tyr Asn Ser Thr Leu 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 Glu Glu Met 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 Leu 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 Lys
450
<210> 48
<211> 214
<212> PRT
<213> Human
<400> 48
Val Ile Trp Met Thr Gln Ser Pro Ser Leu Leu Ser Ala Ser Thr Gly
1 5 10 15
Asp Arg Val Thr Ile Ser Cys Lys Ala Ser Arg Gly Val Ser Thr Ala
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Glu Leu Leu Ile
35 40 45
Tyr Trp Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Cys Leu Gln Ser
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Asp Thr Pro Tyr
85 90 95
Thr Phe Gly Gln Gly Thr Lys Leu 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> 49
<211> 214
<212> PRT
<213> Human
<400> 49
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 Lys Ala Ser Arg Gly Val Ser Thr Ala
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45
Tyr Trp Ala Ser Thr Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser
65 70 75 80
Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln His Tyr Asp Thr Pro Tyr
85 90 95
Thr Phe Gly Gln Gly Thr Lys Leu 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> 50
<211> 1350
<212> DNA
<213> Human
<400> 50
caggtgcagc tggtgcagag cggcgcggaa gtgaagaagc ctggtagcag cgtgaaagtg 60
agctgcaaag cgagcggcta tacctttacc acctattgga ttagctgggt gcgccaggcg 120
ccgggccagg gcctggaatg gatgggcggc atttatccgg gcgatggcgg cagcaactat 180
gcgcagaaat ttcagggccg cgtgaccatt accgcggatg aaagcaccag caccgcgtat 240
atggaactga gcagcctgcg cagcgaagat accgcggtgt attattgcgc gcgcggcggc 300
aactatatta gctttccgat gtattattgg ggccaaggca ccctggtgac agtctcgagt 360
gcctccacaa aggggccttc tgtgtttcca ctggccccct cctctaagag caccagcggt 420
gggacagccg ccctgggatg cctggtgaag gactatttcc ctgagcccgt gaccgtgtcc 480
tggaattccg gcgccctgac ttccggcgtg cacaccttcc ccgccgtgct gcagagctct 540
ggcctgtact ctctgtcctc cgtggtgacc gtgccctctt cctctctggg cacccagacc 600
tacatctgca atgtgaacca caagccttcc aacaccaagg tggataaacg ggtggagccc 660
aagtcctgcg acaagaccca tacctgcccc ccctgccccg cccctgaact ggtgggagga 720
ccctccgtgt tcctgttgcc ccccaagccc aaggacactc tgatgatctc ccgcaccccc 780
gaggtcacct gcgtggtcgt ggacgtctct cacgaagacc ccgaggtgaa attcaactgg 840
tacgtggacg gcgtcgaggt gcacaacgcc aagaccaagc cccccgagga acagtataac 900
agcaccctca gagtggtgtc cgtgctcacc gtgctgcacc aggactggct caacggcaag 960
gaatacaagt gcaaggtgtc caacaaggcc ctccccgccc ccattgaaaa gaccatctcc 1020
aaagccaaag gccagcccag agaaccccag gtctacaccc tccccccctc cagagaagaa 1080
atgaccaaga accaggtgag cctgacctgc ctcgtcaaag ggttctaccc ctccgacatc 1140
gccgtggagt gggagagcaa cggccagccc gagaacaact acaaaaccac ccccctcgtg 1200
ctcgactccg acggatcctt cttcctgtac agcaaactga ccgtggacaa atcccgctgg 1260
cagcagggca acgtgttctc ctgctccgtg atgcacgagg ccctgcacaa ccattacacc 1320
cagaagtccc tgtccctgtc ccccggcaag 1350
<210> 51
<211> 1350
<212> DNA
<213> Human
<400> 51
gaagtgcagc tggtgcagag cggcgcggaa gtgaagaagc caggagaaag cctgaagata 60
tcttgcaaag cgagcggcta tacctttacc acctattgga ttggctgggt gcgccagatg 120
ccgggcaaag gcctggaatg gatgggcatt atttatccgg gcgatggcgg cagccgctat 180
agcccgagct ttcagggcca ggtgaccatt agcgcggata agtcaatcag caccgcgtat 240
ctgcagtgga gcagcctgaa agcgagcgat accgccatgt attattgcgc gcgcggcggc 300
aactatatta gctttccgat gtattattgg ggccaaggca ccctggtgac agtctcgagt 360
gcctccacaa aggggccttc tgtgtttcca ctggccccct cctctaagag caccagcggt 420
gggacagccg ccctgggatg cctggtgaag gactatttcc ctgagcccgt gaccgtgtcc 480
tggaattccg gcgccctgac ttccggcgtg cacaccttcc ccgccgtgct gcagagctct 540
ggcctgtact ctctgtcctc cgtggtgacc gtgccctctt cctctctggg cacccagacc 600
tacatctgca atgtgaacca caagccttcc aacaccaagg tggataaacg ggtggagccc 660
aagtcctgcg acaagaccca tacctgcccc ccctgccccg cccctgaact ggtgggagga 720
ccctccgtgt tcctgttgcc ccccaagccc aaggacactc tgatgatctc ccgcaccccc 780
gaggtcacct gcgtggtcgt ggacgtctct cacgaagacc ccgaggtgaa attcaactgg 840
tacgtggacg gcgtcgaggt gcacaacgcc aagaccaagc cccccgagga acagtataac 900
agcaccctca gagtggtgtc cgtgctcacc gtgctgcacc aggactggct caacggcaag 960
gaatacaagt gcaaggtgtc caacaaggcc ctccccgccc ccattgaaaa gaccatctcc 1020
aaagccaaag gccagcccag agaaccccag gtctacaccc tccccccctc cagagaagaa 1080
atgaccaaga accaggtgag cctgacctgc ctcgtcaaag ggttctaccc ctccgacatc 1140
gccgtggagt gggagagcaa cggccagccc gagaacaact acaaaaccac ccccctcgtg 1200
ctcgactccg acggatcctt cttcctgtac agcaaactga ccgtggacaa atcccgctgg 1260
cagcagggca acgtgttctc ctgctccgtg atgcacgagg ccctgcacaa ccattacacc 1320
cagaagtccc tgtccctgtc ccccggcaag 1350
<210> 52
<211> 642
<212> DNA
<213> Human
<400> 52
gtcatttgga tgacccagag cccgagcctg ctgagcgcga gcaccggcga tcgcgttaca 60
atctcatgca aagcgagccg cggcgtgagc accgcgctgg cgtggtatca gcagaaaccg 120
ggcaaagcgc cggaactgct gatttattgg gcgtctaccc tgcagagcgg cgtgccgagc 180
cgctttagcg gcagcggcag cggcaccgat ttcactctga cgatatcgtg tctccaaagc 240
gaagatttcg caacgtatta ttgccagcag cattatgata cgccctacac ctttggccag 300
ggcaccaagc tggaaatcaa aagaaccgtg gccgccccct ccgtgtttat cttccccccc 360
tctgacgagc agctcaagtc cggcaccgcc tccgtcgtct gcctcctgaa caatttctac 420
ccccgcgaag ctaaagtcca gtggaaggtg gataacgccc tgcagtccgg caactcccag 480
gagagcgtca ccgagcagga ctccaaggac tccacctact ccctctcctc caccctgacc 540
ctctccaaag ccgattacga gaaacacaaa gtgtacgcct gcgaggtgac acaccagggc 600
ctgtccagcc ccgtcaccaa aagcttcaac agaggcgagt gc 642
<210> 53
<211> 642
<212> DNA
<213> Human
<400> 53
gaaattgtaa tgactcaatc tcccgcaacg ctatcggtta gccctggcga acgcgcgacg 60
ttgagttgca aagcgagccg cggcgtgagc accgcgctgg cgtggtatca gcagaaaccg 120
ggccaggcgc cgcgcctgct gatttattgg gcgtctaccc gcgcgaccgg cattccggcg 180
cgctttagcg gcagcggcag cggcaccgaa tttaccctga ccattagcag cctgcagagc 240
gaagatttcg ctgtctatta ttgccagcag cattatgata cgccctacac ctttggccag 300
ggcaccaaac tggaaattaa aagaaccgtg gccgccccct ccgtgtttat cttccccccc 360
tctgacgagc agctcaagtc cggcaccgcc tccgtcgtct gcctcctgaa caatttctac 420
ccccgcgaag ctaaagtcca gtggaaggtg gataacgccc tgcagtccgg caactcccag 480
gagagcgtca ccgagcagga ctccaaggac tccacctact ccctctcctc caccctgacc 540
ctctccaaag ccgattacga gaaacacaaa gtgtacgcct gcgaggtgac acaccagggc 600
ctgtccagcc ccgtcaccaa aagcttcaac agaggcgagt gc 642

Claims (14)

1.一种抗体，其特征在于，所述抗体包含重链可变区和/或轻链可变区：
所述重链可变区，其包含：
由SEQ ID NO:1所示的氨基酸序列组成的重链互补决定区HCDR1，
由SEQ ID NO:2所示的氨基酸序列组成的重链互补决定区HCDR2，
由SEQ ID NO:3所示的氨基酸序列组成的重链互补决定区HCDR3；
所述轻链可变区，其包含：
由SEQ ID NO:4所示的氨基酸序列组成的轻链互补决定区LCDR1，
由WAS氨基酸序列组成的轻链互补决定区LCDR2，
由SEQ ID NO:5所示的氨基酸序列组成的轻链互补决定区LCDR3。
2.根据权利要求1所述的抗体，其特征在于，所述抗体为鼠源抗体、嵌合抗体或人源抗体。
3.根据权利要求1或2所述的抗体，其特征在于，所述重链可变区选自如SEQ ID NO:6、SEQ ID NO:18、SEQ ID NO:19、SEQ ID NO:20、SEQ ID NO:21或SEQ ID NO:22所示的氨基酸序列；所述轻链可变区由选自如SEQ ID NO:7、SEQ ID NO:23、SEQ ID NO:24、SEQ ID NO:25、SEQ ID NO:26或SEQ ID NO:27所示的氨基酸序列。
4.根据权利要求1或2所述的抗体，其特征在于，所述抗体包含重链恒定区和/或轻链恒定区：所述重链恒定区包含如SEQ ID NO:10所示的氨基酸序列；所述轻链恒定区包含如SEQID NO:12所示的氨基酸序列。
5.根据权利要求1或2所述的抗体，其特征在于，所述抗体包含重链和/或轻链：
所述重链选自如SEQ ID NO:14、SEQ ID NO:46和SEQ ID NO:47所示的氨基酸序列；
所述轻链选自如SEQ ID NO:15、SEQ ID NO:48和SEQ ID NO:49所示的氨基酸序列。
6.根据权利要求1或2所述的抗体，其特征在于，所述包含以下i至iv中任一种性质：
i、所述抗体能够特异性结合B7-H3；
ii、所述抗体具有抗体依赖的细胞介导的细胞毒作用；
iii、所述抗体特异性地结合人4Ig-B7-H3并与猴4Ig-B7-H3交叉反应；
iv、所述抗体具有减少的糖基化或无糖基化或被低岩藻糖基化。
7.一种重组蛋白，其特征在于，所述重组蛋白包含如权利要求1或2所述的抗体。
8.一种药物组合物，其特征在于，所述药物组合物包含如权利要求1或2所述的抗体或如权利要求7所述的重组蛋白。
9.一种多核苷酸，其特征在于，所述多核苷酸包含编码如权利要求1或2所述抗体或如权利要求7所述的重组蛋白的核苷酸序列。
10.一种载体，其特征在于，所述载体包含如权利要求9所述的多核苷酸。
11.一种分离细胞，其特征在于，所述细胞产生如权利要求1或2所述的抗体或如权利要求7所述的重组蛋白。
12.一种抗体的制备方法，其特征在于，包括以下步骤：培养权利要求11所述的分离细胞，从培养物中回收所述抗体。
13.权利要求1或2的抗体或权利要求7所述的重组蛋白在制备治疗癌症的药物或药物组合物中的应用。
14.权利要求1或2的抗体或权利要求7所述的重组蛋白在制备抗体检测试剂盒中的应用。
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