CN107987169B - 一种以ROBO1为靶点的双特异性抗体scFv及其制备和应用 - Google Patents
一种以ROBO1为靶点的双特异性抗体scFv及其制备和应用 Download PDFInfo
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Abstract
本发明公开了一种双特异性抗体scFv，其包含特异性结合肿瘤细胞表面抗原分子的抗原结合结构域(简称R‑scFv)和特异性结合免疫细胞(例如T细胞、NKT细胞和/或CIK细胞)的抗原结合结构域(简称I‑scFv)，所述I‑scFv与R‑scFv直接连接或通过连接肽连接。本发明提供份以ROBO1为靶点的双特异性抗体scFv，其能够同时结合T细胞表面的CD3分子和肿瘤细胞表面ROBO1分子，从而拉近肿瘤细胞和T细胞间的距离，能够快速激活静止的T细胞，快速有效的杀伤肿瘤细胞。将其用于抗肿瘤(高表达ROBO1分子)的药物的开发具有广阔的应用前景。
Description
技术领域
本发明涉及生物技术领域，具体涉及一种以ROBO1为靶点的双特异性抗体scFv及其制备和应用。
背景技术
双特异性抗体（bispecific monoclonal antibody，BsAb）能够双重识别肿瘤靶细胞和免疫效应细胞，因此兼有抗体特异性和介导效应细胞的细胞毒作用，经过合适设计的双特异性抗体能够结合和聚集效应细胞于肿瘤部位，激活效应细胞的活性，诱导肿瘤细胞的溶解。自然状态下不存在，只能通过人工方法制备。
双特异性抗体可通过以下途径得到：
①杂交瘤法：杂交瘤细胞是由B细胞和骨髓瘤细胞融合形成的可以产生特异性单克隆抗体的永生化细胞系。两种杂交瘤细胞进一步杂交形成的杂交瘤细胞可以在同一细胞内产生两种不同的轻重链。在同一个细胞内两种轻重链可以随机组装，产生双特异性分子和一系列无功能的单特异性分子。其组装成双特异性分子的概率仅有1/10。因此，要得到单纯的双特异性分子，需要精细的纯化步骤。
②化学交联法：化学交联法是将两种抗体或抗体片段进行交联生产双特异性抗体。最初使用氧化重组方法进行化学交联，现在主要使用异型或同型双功能交联剂。异型双功能交联剂对两种不同的活性集团进行交联。例如氨基和巯基基团。但使用异型双功能交联剂的过程中，会导致一个抗体分子或抗体片段拥有大量的氨基或羧基基团，进而导致教练抗体的不均一性。同型双功能交联剂可以避免这种状况。
以上两种方法中，虽然杂交瘤法生产的双特异性抗体来源可靠，但是由于轻链、重链随机配对组装可产生多种抗体分子形式，使得双特异性抗体的生产和纯化都变得非常困难。而化学交联法交联生产的双特异性抗体成分较均一，但是费时费力且产量很低。基因工程抗体技术的发展为双特异性抗体的研制奠定了基础。
发明内容
CD3分子由4个亚基组成：δ、ε、γ、ζ，其分子质量分别为18.9kDa、23.1kDa、20.5kDa、18.7kDa，其长度分别由171、207、182、164个氨基酸残基组成。它们一起组成6条肽链，常与T细胞受体(T cell receptor，TCR)紧密结合形成含有8条肽链的TCR-CD3复合体，结构示意图见图1所示。此复合体具有T细胞活化信号转导，稳定TCR结构的功能。CD3胞质段含有免疫受体酪氨酸活化基序(immunoreceptor tyrosine-based activationmotif,ITAM)，TCR识别并结合由MHC(major histo-compatibility complex)分子提呈的抗原肽，导致CD3的ITAM的保守序列的酪氨酸残基被T细胞内的酪氨酸蛋白激酶p56lck磷酸化，然后可募集其他含有SH2(Scr homology 2)结构域的酪氨酸蛋白激酶(如ZAP-70等)。ITAM的磷酸化、以及与ZAP-70的结合是T细胞活化信号传导过程早期阶段的重要生化反应之一。因此，CD3分子的功能是转导TCR识别抗原所产生的活化信号。
Robo是一次跨膜的受体蛋白,在哺乳动物中，已经克隆到4个Robo基因。从物种进化的角度看，Robo1,2,3的胞外部分非常的保守，从果蝇到人类都是由5个Ig样功能区和3个Fibronectin III型重复序列组成。Robos有很短的跨膜区域和一个较长的胞内区；按照序列的保守性，胞内区被划分为4个更小的区域，分别命名为：CC0，CC1，CC2，CC3。Robo4的结构与其它三个家族成员有很大的不同，它的胞外只有2个Ig样功能区和3个Fibronectin III型重复序列；胞内也只有CC0和CC2两个区域。Robos胞外的IgG domains被认为是与配体Slit结合所必需的，较长的胞内区域则和一些重要的信号分子相互作用，参与Slit/Robo下游的信号转导，从而完成刺激信号由细胞外部到内部骨架的传递。目前，已完成slit2与Robo相互作用区域蛋白质的机构解析，发现slit2的第二个结构域D2与Robo1的Ig1结合，进而启动信号传导。目前，针对高表达ROBO1分子的肿瘤细胞或慢性疾病并没有一种有效的治疗方法或药物。
基于此，本发明利用基因工程方法提供一种以ROBO1为靶点的双体异性抗体scFv及其制备和应用。其可作为高表达ROBO1分子的肿瘤或慢性疾病药物的开发。
本发明第一方面提供一种双特异性抗体scFv，其包含特异性结合肿瘤细胞表面抗原分子的抗原结合结构域（简称R-scFv）和特异性结合免疫细胞（例如T细胞、NKT细胞和/或CIK细胞）的抗原结合结构域（简称I-scFv），所述I-scFv与R-scFv直接连接或通过连接肽连接。
优选地，所述R-scFv能够特异性识别肿瘤特异性抗原ROBO1。
示例性地，所述R-scFv能够特异性识别肿瘤特异性抗原ROBO1的Ig1、Ig2、Ig3、Ig4、Ig5、FN1、FN2和FN3结构域中的一种或多种。
示例性地，所述I-scFv能够特异性结合T细胞、NKT细胞和/或CIK细胞等免疫细胞表面的相关抗原分子。例如：CD3、T细胞受体（TCR）、CD28、CD16、NKG2D、Ox40、4-1BB、CD2、CD5、CD95等。
在本发明的一个具体实施例中，所述R-scFv能够特异性识别肿瘤特异性抗原ROBO1的FN3结构域；优选地，所述R-scFv为slit2D2分子。
在本发明的一个具体实施例中，所述I-scFv能够特异性结合免疫细胞表面抗原CD3。
示例性地，所述scFv包括Anti ROBO1 VH结构域、Anti ROBO1 VL结构域、Anti CD3VH结构域和Anti CD3 VL结构域。
示例性地，所述R-scFv包括Anti ROBO1 VH结构域和Anti ROBO1 VL结构域。
示例性地，所述I-scFv包括Anti CD3 VH结构域和Anti CD3 VL结构域。
在本发明的一个具体实施例中，所述scFv的结构为：
Anti ROBO1 VL-Anti ROBO1 VH-Anti CD3 VH-Anti CD3 VL。
在本发明的一个具体实施例中，所述scFv的结构为：
Anti ROBO1 VH-Anti ROBO1 VL-Anti CD3 VH-Anti CD3 VL。
在本发明的一个具体实施例中，所述scFv的结构为：
Anti ROBO1 VL-Anti ROBO1 VH-Anti CD3 VL-Anti CD3 VH。
在本发明的一个具体实施例中，所述scFv的结构为：
Anti ROBO1 VH-Anti ROBO1 VL-Anti CD3 VL-Anti CD3 VH。
在本发明的一个具体实施例中，所述Anti CD3 VH的氨基酸序列如SEQ ID NO：3所示或其同源序列。
在本发明的一个具体实施例中，所述Anti CD3VL的氨基酸序列如SEQ ID NO：4所示或其同源序列。
在本发明的一个具体实施例中，所述Anti ROBO1 VH的氨基酸序列如SEQ ID NO：2所示或其同源序列。
在本发明的一个具体实施例中，所述Anti ROBO1 VL的氨基酸序列如SEQ ID NO：1所示或其同源序列。
在本发明的一个具体实施例中，所述Anti CD3 VH、Anti CD3 VL、Anti ROBO1 VH、Anti ROBO1 VL之间直接连接。
在本发明的一个优选地实施例中，所述Anti CD3 VH、Anti CD3 VL、Anti ROBO1VH、Anti ROBO1 VL之间通过连接肽连接。
示例性地，所述连接肽的序列为：如SEQ ID NO：5所示的序列、如SEQ ID NO：6所示的序列、如SEQ ID NO：7所示的序列中的一种或多种。
在本发明的一个具体实施例中，所述scFv的氨基酸序列如SEQ ID NO：8所示的序列或其同源序列。
在本发明的一个具体实施例中，所述scFv的氨基酸序列如SEQ ID NO：9所示的序列或其同源序列。
在本发明的一个具体实施例中，所述scFv的氨基酸序列如SEQ ID NO：10所示的序列或其同源序列。
在本发明的一个具体实施例中，所述scFv的氨基酸序列如SEQ ID NO：11所示的序列或其同源序列。
在本发明的一个具体实施例中，所述scFv的核苷酸序列如SEQ ID NO：12所示的序列或其简并序列。
本发明第二方面提供一种核酸分子，其编码上述双特异性抗体scFv。
本发明第三方面提供一种上述双特异性抗体scFv的制备方法，所述方法包括：
将上述双特异性抗体scFv的基因克隆到表达载体上；以及任选地，将表达载体转入宿主细胞中表达。
示例性地，所述表达载体选自质粒、细菌和病毒中的一种或多种，优选地，所述表达载体为pCDNA3.4载体。
在本发明提供的一个具体实施方式中，上述双特异性抗体scFv的制备方法具体包括：
(1) 将带有His标签的双特异性抗体scFv的基因克隆到pCDNA3.4表达载体上；
(2) 将表达载体转染到宿主细胞中，培养并取上清。优选地，所述宿主细胞为EXPICHO细胞。
在本发明提供的一个具体实施方式中，通过镍柱从表达上清中捕获带His标签的蛋白，通过透析纯化双特异性抗体scFv。
本发明第四方面提供一种双体异性抗体，其包括上述双特异性抗体scFv。
本发明第五方面提供一种药物组合物，其包含选自：本发明上述的双特异性抗体scFv、核酸分子和双体异性抗体的一项或多项。
在本发明的一个实施例中，所述药物组合物还包括药学上可接受的辅料。
在本发明的一个实施例中，所述药物组合物中，所述辅料选自：缓冲液、碳水化合物、抗氧化剂、螯合剂和防腐剂中的一种或多种。
本发明第六方面提供一种本发明上述的双特异性抗体scFv、核酸分子、双体异性抗体和药物组合物中的一项或多项在制备治疗和/或预防癌症或其他慢性病的药物中的应用。
在本发明的一个实施例中，所述的应用中，所述的癌症为高表达Robo1的肿瘤及相关疾病，此处所述的高表达指肿瘤细胞中Robo1的表达水平高于正常细胞中其表达水平。
在本发明的一个实施例中，所述癌症为肝癌、乳腺癌、结肠癌、胰腺癌、***癌、神经胶质瘤、肺癌或其他。
在本发明的一个优选实施例中，所述癌症为肝癌。
本发明至少具有以下优势之一：
本发明提供一种以ROBO1为靶点的双特异性抗体scFv，其能够同时结合T细胞表面的CD3分子和肿瘤细胞表面ROBO1分子，从而拉近肿瘤细胞和T细胞间的距离，能够有效地激活静止的T细胞，快速杀伤肿瘤细胞。将其用于抗肿瘤（高表达ROBO1分子）药物的开发具有广阔的应用前景。
附图说明
图1所示为本发明实施例提供的RCD3分子示意图。
图2所示为本发明实施例提供的利用凝胶电泳检测收集的RCD3蛋白的实验结果图。
图3a-b所示为本发明实施例提供的MHCC97-H肿瘤细胞表达的ROBO1分子的流式检测结果图，其中，图3a为未加抗体的对照组；图3b为加入抗体的实验组。
图4所示为本发明实施例提供的利用IncuCyte Zoom检测RCD3药物对肿瘤细胞的杀伤结果图，其中，■表示A组；○表示B组；▲表示C组；X表示D组；▽表示E组。
图5所示为本发明实施例提供的T细胞表面CD25分子表达检测结果图，其中图A、B为对照组；图C、D为RCD3实验组。
具体实施方式
除非另有定义，本发明中使用的所有技术和科学术语具有与本发明所述技术领域的普通技术人员通常理解的相同含义。
本发明中，术语“抗体”指的是与抗原特异性结合的免疫球蛋白分子。抗体可为源于自然源或源于重组源的完整的免疫球蛋白，并可为完整免疫球蛋白的免疫反应部分。抗体通常为免疫球蛋白分子的四聚物。本发明中的抗体可以以多种形式存在，包括例如，多克隆抗体、单克隆抗体、Fv、Fab和F(ab)2，以及单链抗体和人源化抗体等(Harlow等，1999,In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, NY；Harlow等，1989, In: Antibodies: A Laboratory Manual,Cold SpringHarbor, New York；Houston等，1988, Proc. Natl. Acad. Sci. USA 85:5879-5883；Bird等，1988, Science 242: 423-426)。
术语“抗体片段”指的是完整抗体的一部分，并指的是完整抗体的抗原决定可变区。抗体片段的例子包括但不限于Fab、Fab'、F(ab')2和Fv片段，由抗体片段形成的线性抗体、scFv抗体和多特异性抗体。
术语“编码”指的是多核苷酸诸如基因、cDNA或mRNA中核苷酸的特异性序列用作模板合成在生物学过程中的其他多聚体和大分子的固有性质，所述多聚体和大分子具有核苷酸(即，rRNA、tRNA和mRNA)的限定序列或氨基酸的限定序列中的任一个和由其产生的生物学性质。因此，如果相应于那个基因的mRNA的转录和翻译在细胞或其他生物学***中产生蛋白质，则基因编码蛋白质。核苷酸序列等同mRNA序列并通常提供在序列表中的编码链，和用作转录基因或cDNA的模板的非编码链两者，都可被称为编码那个基因或cDNA的蛋白质或其他产物。
除非另有规定，“编码氨基酸序列的核苷酸序列”包括为彼此简并版本并编码相同的氨基酸序列的所有核苷酸序列。编码蛋白质和RNA的核苷酸序列可包括内含子。
术语“特异性结合”指识别特异性抗原但基本上不识别或结合样本中的其他分子。
术语“载体”为物质组合物，其包括分离的核酸，并且其可用于传递分离的核酸至细胞内部。很多载体在本领域中是已知的，包括但不限于线性多核苷酸、与离子或两性分子化合物相关的多核苷酸、质粒和病毒。因此，术语“载体”包括自主复制的质粒或病毒。该术语也应被解释为包括便于将核酸转移入细胞的非质粒和非病毒化合物，诸如例如聚赖氨酸化合物、脂质体等等。
术语“癌症”被定义为以畸变细胞的快速和失控生长为特征的疾病。癌症细胞可局部蔓延或通过血流和淋巴***蔓延至身体的其他部分。各种癌症的例子包括但不限于乳腺癌、***癌、卵巢癌、子***、皮肤癌、胰腺癌、结肠直肠癌、肾癌、肝癌、脑癌、淋巴瘤、白血病、肺癌等等。
下面将结合本发明实施例，对本发明的技术方案进行清楚、完整地描述，显然，所描述的实施例仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
以下实施例以能够特异性结合ROBO1分子和CD3分子的双特异性抗体scFv（简称RCD3）为例进行说明。
实施例1 双特异性抗体RCD3的制备
材料：
细胞株ExpiCHO-S™ cells(Gibco Catalog No. A29127)；
转染试剂盒ExpiFectamine CHO Transfection Kit (Gibco Catalog No.A29129)；
OptiPRO SFM (Gibco Catalog No. 12309-050)；
培养基ExpiCHO Expression Medium(Gibco Catalog No. A29100-01)；
培养条件：37℃，8% CO2培养箱，摇床培养；
pCDNA3.4质粒载体；
1.1 RCD3蛋白表达
RCD3分子设计形式如图1所示。以SEQ ID NO：8所示氨基酸序列，SEQ ID NO：12所示基因序列为例的RCD3进行说明。
通过全基因合成得到SEQ ID NO：12所示的基因片段，在合成时，加入His标签，便于RCD3蛋白的纯化。以SEQ ID NO：12所示的基因序列为模板，通过PCR扩增得到PCR产物，用胶回收试剂盒纯化上述产物，利用T-A克隆原理，将其克隆至pCDNA3.4载体上，之后转化至TOP10菌株，氨苄青霉素筛选并挑取阳性克隆，通过测序确认载体构建成功。用无内毒素DNA提取试剂盒提取质粒DNA，用于转染ExpiCHO-S™细胞使用。
培养ExpiCHO-S™细胞，当密度达到2.5x106细胞/毫升时转染，转染14天后收集上清。高速离心ExpiCHO-S™培养液取上清，过滤，用于后续纯化。
1.2 RCD3蛋白纯化
材料：
Binding Buffer：
20mM磷酸盐；0.5M NaCl；20mM咪唑；pH 7.4。
Elution Buffer：
20mM磷酸盐；0.5M NaCl；500mM咪唑；pH 7.4。
实验流程：
启动AKTA设备，将His柱与AKTA连接。
使用3-5个柱体积的去离子水清洗His柱。
使用5个柱体积的Binding Buffer平衡His柱。
取出过滤的细胞上清液，依次流过His柱。
使用Binding Buffer清洗杂蛋白，直至UV280处吸光值趋近于0。
使用Elution Buffer洗脱目的蛋白，当UV280>400的时候开始收集洗脱液。
4℃，利用PBS透析收集到的蛋白洗脱液，透析后的蛋白RCD3保存于-80℃。
利用凝胶电泳检测收集的RCD3蛋白，其结果如图2所示。
由图2可知，利用本实施例提供的制备方法成功表达制备并纯化获得RCD3目的蛋白，将其用于后续试验。
实施例2 肿瘤细胞ROBO1表达分析鉴定
本实施例选择高表达ROBO1抗原的肝癌细胞MHCC97-H细胞作为靶细胞。为了确认MHCC97-H细胞为ROBO1分子高表达，进行流式染色，具体方法如下：
取5*105MHCC97-H细胞用于染色。将MHCC97-H细胞与一抗共孵育45min，取50ul共孵育物置于冰上，流式染色buffer洗脱一次；将MHCC97-H细胞与二抗共孵育30min；再利用流式染色buffer洗脱一次，之后重悬于120ul FACS试剂中，流式细胞仪器检测分析。其实验结果如图3所示。
由图3可知，MHCC97-H细胞高表达ROBO1分子。
实施例3 肿瘤杀伤实验检测药物活性
利用IncuCyte Zoom长时间动态细胞成像分析***实时观察细胞的状态。将肿瘤细胞MHCC97-H和免疫细胞PBMC加入孔板放在该分析***下观察，记录免疫细胞的整个杀伤过程。利用NucView 488 Caspase-3检测杀伤效果。
NucView 488 Caspase-3是一种死细胞染料，当肿瘤细胞被杀伤后，该染料可结合在DNA上，激光激发后呈现绿色，绿色荧光信号可以被IncuCyte Zoom设备捕捉记录。通过分析不同实验孔绿色荧光强度信号，可判断各实验组杀伤效果的强弱。
试剂与材料：
细胞：MHCC97-H，PBMC；
药物：RCD3；
培养基：DMEM（10%FBS，5%双抗）(Gibco)，T细胞培养基(Gibco)；
试剂准备：NucView 488 Caspase-3底物（Biotium，#30067）；
设备：IncuCyte Zoom长时间动态细胞成像分析***；
实验流程：
靶细胞MHCC97-H按100μl/4×103/孔加入96孔板，37℃培养过夜（18-24小时）。
以100K cells/well的加入量将PBMC细胞加入到96孔板中；
RCD3药物分别按不同浓度加入实验孔，将已加入细胞的96孔板置于IncuCyteZoom设备中，打开绿光通道观察，设备放入37℃、体积分数5%CO2培养箱中培养过夜。
实验分组如下：
| 组别 | 药物 | 免疫细胞 | 肿瘤细胞 |
| A | / | PBMC(100K cells/well) | MHCC97-H(10K cells/well) |
| B | RCD3(0.25ug/ml) | / | MHCC97-H(10K cells/well) |
| C | RCD3(0.25ug/ml) | PBMC(100K cells/well) | MHCC97-H(10K cells/well) |
| D | RCD3(2.5ug/ml) | / | MHCC97-H(10K cells/well) |
| E | RCD3(2.5ug/ml) | PBMC(100K cells/well) | MHCC97-H(10K cells/well) |
杀伤活性计算公式：
利用IncuCyte Zoom设备自带的荧光分析软件绘制荧光强度曲线图（肿瘤细胞杀伤效果图）分析上述各个组别中肿瘤细胞的杀伤状况。其实验结果如图4所示。从图4中可以看出，与对照组（A/B/D）相比，RCD3药物在2.5ug/ml和0.25ug/ml浓度下均可有效激活T细胞，启动杀伤肿瘤MHCC97-H细胞。
实施例4 T细胞活性分析
CD25分子是IL-2R的α链，单独构成低亲和力受体，其不能传导信号，但对形成高亲和力受体有重要意义。IL-2R的另外两条链是β、γ链。三条连构成的三聚体受体具有高亲和力，βγ链构成的中等亲和力受体负责传递IL-2信号。T细胞被激活后，CD25分子表达会显著上调，为了进一步确认T细胞是否被激活，利用CD25抗体对杀伤实验结束后的T细胞进行染色，染色方法为常规流式染色（如实施例2所述），其实验结果如图5所示。
图5中，其中纵坐标SSC-H表示侧向散射信号、横坐标FSH-H表示前向散射信号，APC信号表示CD25分子表达，信号越强，CD25分子表达越高。用SSC-H和FSH-H两个值圈定用于流式分析的活细胞，用APC和FSH-H两个值分析表达CD25分子细胞的比例。如A和C图。A/B图用于分析对照组T细胞，C/D图用于分析RCD3实验组T细胞。与对照组相比(B图)，实验组RCD3给药后，CD25分子表达明显升高，说明T细胞被活化，证明RCD3分子是通过激活T细胞而杀伤肿瘤细胞。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换等，均应包含在本发明的保护范围之内。
序列表
<110> 阿思科力（苏州）生物科技有限公司
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Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly
1 5 10
<210> 8
<211> 489
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> RCD3-1
<400> 8
Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly
1 5 10 15
Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Asn Phe
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile
35 40 45
Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Ser Leu Thr Ile Ser Lys Leu Glu Gln
65 70 75 80
Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Leu
85 90 95
Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Gly Gly Gly Gly Ser
100 105 110
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Gln Gln
115 120 125
Ser Gly Pro Glu Leu Val Lys Pro Gly Ala Ser Val Lys Ile Ser Cys
130 135 140
Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr Tyr Met Asn Trp Val Lys
145 150 155 160
Leu Ser His Gly Lys Ser Leu Glu Trp Ile Gly Asp Ile Val Pro Asn
165 170 175
Asn Gly Asp Thr Thr Tyr Asn Gln Asn Phe Arg Gly Lys Ala Thr Leu
180 185 190
Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr Met Glu Leu Arg Ser Leu
195 200 205
Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Phe Ser Asn Tyr
210 215 220
Val Tyr Pro Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser
225 230 235 240
Ser Gly Gly Gly Gly Ser Asp Ile Lys Leu Gln Gln Ser Gly Ala Glu
245 250 255
Leu Ala Arg Pro Gly Ala Ser Val Lys Met Ser Cys Lys Thr Ser Gly
260 265 270
Tyr Thr Phe Thr Arg Tyr Thr Met His Trp Val Lys Gln Arg Pro Gly
275 280 285
Gln Gly Leu Glu Trp Ile Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr
290 295 300
Asn Tyr Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys
305 310 315 320
Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp
325 330 335
Ser Ala Val Tyr Tyr Cys Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu
340 345 350
Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Val Glu Gly
355 360 365
Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Val Asp Asp
370 375 380
Ile Gln Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly Glu
385 390 395 400
Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met Asn
405 410 415
Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp
420 425 430
Thr Ser Lys Val Ala Ser Gly Val Pro Tyr Arg Phe Ser Gly Ser Gly
435 440 445
Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu Asp
450 455 460
Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr Phe
465 470 475 480
Gly Ala Gly Thr Lys Leu Glu Leu Lys
485
<210> 9
<211> 489
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> RCD3-2
<400> 9
Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Tyr Met Asn Trp Val Lys Leu Ser His Gly Lys Ser Leu Glu Trp Ile
35 40 45
Gly Asp Ile Val Pro Asn Asn Gly Asp Thr Thr Tyr Asn Gln Asn Phe
50 55 60
Arg Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Phe Ser Asn Tyr Val Tyr Pro Phe Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Thr Leu Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
115 120 125
Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Thr Thr Ser Ser
130 135 140
Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser
145 150 155 160
Gln Asp Ile Ser Asn Phe Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly
165 170 175
Thr Val Lys Leu Leu Ile Tyr Tyr Thr Ser Arg Leu His Ser Gly Val
180 185 190
Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Ser Leu Thr
195 200 205
Ile Ser Lys Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln
210 215 220
Gly Asn Thr Leu Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu
225 230 235 240
Lys Gly Gly Gly Gly Ser Asp Ile Lys Leu Gln Gln Ser Gly Ala Glu
245 250 255
Leu Ala Arg Pro Gly Ala Ser Val Lys Met Ser Cys Lys Thr Ser Gly
260 265 270
Tyr Thr Phe Thr Arg Tyr Thr Met His Trp Val Lys Gln Arg Pro Gly
275 280 285
Gln Gly Leu Glu Trp Ile Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr
290 295 300
Asn Tyr Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys
305 310 315 320
Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp
325 330 335
Ser Ala Val Tyr Tyr Cys Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu
340 345 350
Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Val Glu Gly
355 360 365
Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Val Asp Asp
370 375 380
Ile Gln Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly Glu
385 390 395 400
Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met Asn
405 410 415
Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp
420 425 430
Thr Ser Lys Val Ala Ser Gly Val Pro Tyr Arg Phe Ser Gly Ser Gly
435 440 445
Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu Asp
450 455 460
Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr Phe
465 470 475 480
Gly Ala Gly Thr Lys Leu Glu Leu Lys
485
<210> 10
<211> 489
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> RCD3-3
<400> 10
Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly
1 5 10 15
Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Asn Phe
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile
35 40 45
Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Ser Leu Thr Ile Ser Lys Leu Glu Gln
65 70 75 80
Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Leu
85 90 95
Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Gly Gly Gly Gly Ser
100 105 110
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Gln Gln
115 120 125
Ser Gly Pro Glu Leu Val Lys Pro Gly Ala Ser Val Lys Ile Ser Cys
130 135 140
Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr Tyr Met Asn Trp Val Lys
145 150 155 160
Leu Ser His Gly Lys Ser Leu Glu Trp Ile Gly Asp Ile Val Pro Asn
165 170 175
Asn Gly Asp Thr Thr Tyr Asn Gln Asn Phe Arg Gly Lys Ala Thr Leu
180 185 190
Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr Met Glu Leu Arg Ser Leu
195 200 205
Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Phe Ser Asn Tyr
210 215 220
Val Tyr Pro Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser
225 230 235 240
Ser Gly Gly Gly Gly Ser Val Asp Asp Ile Gln Leu Thr Gln Ser Pro
245 250 255
Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg
260 265 270
Ala Ser Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser Gly
275 280 285
Thr Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Val Ala Ser Gly
290 295 300
Val Pro Tyr Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu
305 310 315 320
Thr Ile Ser Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln
325 330 335
Gln Trp Ser Ser Asn Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu
340 345 350
Leu Lys Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly
355 360 365
Asp Ile Lys Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala
370 375 380
Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Arg Tyr
385 390 395 400
Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
405 410 415
Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe
420 425 430
Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr
435 440 445
Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
450 455 460
Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly
465 470 475 480
Thr Thr Leu Thr Val Ser Ser Val Glu
485
<210> 11
<211> 489
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> RCD3-4
<400> 11
Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Tyr Met Asn Trp Val Lys Leu Ser His Gly Lys Ser Leu Glu Trp Ile
35 40 45
Gly Asp Ile Val Pro Asn Asn Gly Asp Thr Thr Tyr Asn Gln Asn Phe
50 55 60
Arg Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Phe Ser Asn Tyr Val Tyr Pro Phe Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Thr Leu Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
115 120 125
Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Thr Thr Ser Ser
130 135 140
Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser
145 150 155 160
Gln Asp Ile Ser Asn Phe Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly
165 170 175
Thr Val Lys Leu Leu Ile Tyr Tyr Thr Ser Arg Leu His Ser Gly Val
180 185 190
Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Ser Leu Thr
195 200 205
Ile Ser Lys Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln
210 215 220
Gly Asn Thr Leu Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu
225 230 235 240
Lys Gly Gly Gly Gly Ser Val Asp Asp Ile Gln Leu Thr Gln Ser Pro
245 250 255
Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg
260 265 270
Ala Ser Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser Gly
275 280 285
Thr Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Val Ala Ser Gly
290 295 300
Val Pro Tyr Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu
305 310 315 320
Thr Ile Ser Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln
325 330 335
Gln Trp Ser Ser Asn Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu
340 345 350
Leu Lys Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly
355 360 365
Asp Ile Lys Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala
370 375 380
Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Arg Tyr
385 390 395 400
Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
405 410 415
Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe
420 425 430
Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr
435 440 445
Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
450 455 460
Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly
465 470 475 480
Thr Thr Leu Thr Val Ser Ser Val Glu
485
<210> 12
<211> 1506
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> RCD3-1nt
<400> 12
atgggcggcg gcggcagcga catccagatg acacagacca ccagcagcct gagcgccagc 60
ctgggcgaca gagtgaccat ctcctgtcgg gcctcccagg atatctccaa tttcctgaat 120
tggtaccagc agaagcccga cggcaccgtg aagctgctga tctactacac atcccggctg 180
cactccggcg tgcccagcag attcagcggc agcggcagcg gcaccgattt ctccctgaca 240
atctccaagc tggagcagga ggatatcgcc acatacttct gccagcaggg caatacactg 300
ccactgacct tcggcgccgg cacaaagctg gagctgaagg gcggcggcgg cagcggcggc 360
ggcggcagcg gcggcggcgg cagcgaggtg cagctgcagc agagcggccc agagctggtg 420
aagcctggcg ccagcgtgaa gatcagctgc aaggccagcg gctacacatt tacagattac 480
tacatgaact gggtgaagct gagccacggc aagtccctgg agtggatcgg cgacatcgtg 540
cctaataacg gcgacaccac atacaatcag aatttcagag gcaaggccac actgacagtg 600
gacaagagca gcagcacagc ctacatggag ctgcggagcc tgacaagcga ggacagcgcc 660
gtgtactact gcgcccggtt ctccaattac gtgtacccat tcgattactg gggccagggc 720
accaccctga ccgtgagcag cggcggcggc ggcagcgaca tcaagctgca gcagagcggc 780
gccgagctgg ccagacctgg cgccagcgtg aagatgtcct gcaagacaag cggctacaca 840
tttacaaggt acaccatgca ctgggtgaag cagaggcctg gccagggcct ggagtggatc 900
ggctacatca acccttcccg cggctacacc aactacaacc agaagttcaa ggacaaggcc 960
acactgacca cagataagag ctcctccaca gcctacatgc agctgagcag cctgaccagc 1020
gaggactccg ccgtgtacta ctgtgccagg tactacgatg atcactactg cctggattac 1080
tggggccagg gcaccaccct gacagtgagc tccgtggagg gcggcagcgg cggcagcggc 1140
ggcagcggcg gcagcggcgg cgtggacgac atccagctga cccagtcacc agccatcatg 1200
tccgcctcac ctggcgagaa ggtgacaatg acctgtaggg cctcctcctc cgtgtcctac 1260
atgaactggt accagcagaa gtccggcacc tcaccaaagc gctggatcta cgatacctcc 1320
aaggtggcct ccggcgtgcc ataccgcttc tccggctccg gctccggcac ctcctactcc 1380
ctgaccatct cctccatgga ggccgaggat gccgccacct actactgtca gcagtggtcc 1440
tccaacccac tgaccttcgg cgccggcacc aagctggagc tgaagcacca ccaccaccac 1500
cactga 1506
Claims (9)
1.双特异性抗体scFv，其特征在于，其包含特异性结合肿瘤细胞表面抗原分子的抗原结合结构域（简称R-scFv）和特异性结合免疫细胞的抗原结合结构域（简称I-scFv），所述I-scFv与R-scFv直接连接或通过连接肽连接；所述R-scFv能够特异性结合肿瘤特异性抗原ROBO1的FN3结构域；所述I-scFv能够特应性结合免疫细胞表面抗原CD3；
所述R-scFv包括Anti ROBO1 VH结构域和Anti ROBO1 VL结构域；所述Anti ROBO1 VH的氨基酸序列如SEQ ID NO：2所示的序列；和所述Anti ROBO1 VL的氨基酸序列如SEQ IDNO：1所示的序列；
所述I-scFv包括Anti CD3 VH结构域和Anti CD3 VL结构域；
所述双特异性抗体scFv的结构为：Anti ROBO1 VL-Anti ROBO1 VH-Anti CD3 VH-AntiCD3 VL；所述双特异性抗体scFv的氨基酸序列如SEQ ID NO：8所示的序列。
2.如权利要求1所述的双特异性抗体scFv，其特征在于，所述免疫细胞选自：T细胞、NKT细胞和CIK细胞。
3.编码双特异性抗体scFv的核苷酸序列，其特征在于，所述scFv的核苷酸序列如SEQID NO：12所示的序列或其简并序列。
4.权利要求1或2所述双特异性抗体scFv的制备方法，其包括：将双特异性抗体scFv的基因克隆到表达载体上；以及将表达载体转入宿主细胞中表达。
5.如权利要求4所述的制备方法，其特征在于，所述表达载体选自质粒、细菌和病毒中的一种或多种。
6.如权利要求4所述的制备方法，其特征在于，所述表达载体为pCDNA3.4载体。
7.一种药物组合物，其特征在于，其包含选自：权利要求1或2所述的双特异性抗体scFv，或权利要求4-6任一项所制备的双特异性抗体scFv。
8.如权利要求7所述的药物组合物，其还包括药学上可接受的辅料。
9.一种权利要求1或2所述的双特异性抗体scFv、权利要求4-6任一项所制备的双特异性抗体scFv在制备治疗和/或预防癌症的药物中的应用；所述的癌症为高表达Robo1的肿瘤及相关疾病。
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| CN105907719B (zh) * | 2016-04-18 | 2019-10-18 | 阿思科力(苏州)生物科技有限公司 | Anti ROBO1 CAR-T细胞及其制备和应用 |
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