CN108264562B - 一种结合cd3和t细胞正共刺激分子的双功能分子及其应用 - Google Patents
一种结合cd3和t细胞正共刺激分子的双功能分子及其应用 Download PDFInfo
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Abstract
本发明属于生物医药技术领域，具体涉及一种结合CD3和T细胞正共刺激分子的双功能分子及其应用。本发明将能够结合并激活T细胞表面CD3分子的第一功能域和能够结合并激活T细胞正共刺激分子的第二功能域融合于同一蛋白肽链形成双功能分子，采用真核细胞表达系统生产，表达产物结构单一，纯化工艺简便，蛋白产量高，制备工艺及产品稳定；与抗CD3和抗T细胞正共刺激分子全长抗体联用相比，本发明所述双功能分子对T细胞的体外扩增效果更优，蛋白用量更少，且使用简便，可通过溶液形式直接添加，无需优化两种全长抗体的相对比例。
Description
技术领域
本发明属于生物医药技术领域，具体涉及一种结合CD3和T细胞正共刺激分子的双功能分子及其应用。
背景技术
T淋巴细胞(T lymphocyte)来源于胸腺(Thymus)，故称T细胞。成熟T细胞存在于外周免疫器官的胸腺依赖区，在适应性细胞免疫应答中占据核心地位，同时在胸腺依赖性抗原诱导的体液免疫应答中亦发挥重要的辅助作用。根据功能的不同，T细胞可分为细胞毒性T细胞(Cytotoxic T lymphocyte,CTL)、辅助T细胞(Helper T cell,Th)和调节性T细胞(Regulatory T cell，Treg)。其中CTL表达CD8，是适应性细胞免疫的主要效应细胞，其主要功能是特异性识别靶细胞表面的内源性抗原肽/MHC I类分子复合物，自身活化后可分泌穿孔素(Peforin)、颗粒酶(Granzyme)、颗粒溶素(Granulysin)等物质直接杀伤靶细胞(肿瘤细胞或寄生病原体感染的细胞)，也可通过Fas/FasL信号途径诱导靶细胞凋亡；而Th均表达CD4，通过分泌不同种类的细胞因子以及与其它细胞之间直接的相互作用，调节CTL的细胞活性，间接参与细胞免疫；此外，Treg可通过直接接触抑制靶细胞活化以及分泌IL-10、TGFβ等细胞因子对细胞免疫应答进行负调控，在免疫耐受、自身免疫病、感染性疾病及肿瘤等多种疾病中发挥重要的作用。
CD8阳性T细胞的完全活化与高效扩增是其有效杀伤靶细胞的基础，依赖于双信号传递途径的作用：其中抗原递呈细胞(Antigen presenting cell，APC)表面的MHC I/内源性抗原肽复合物特异性识别T细胞表达的TCR/CD3复合物，导致CD3与共受体CD8的胞质段相互作用，激活与胞质段尾部相连的蛋白质酪氨酸激酶，使CD3胞质区免疫受体酪氨酸激酶激活模体(Immunoreceptor tyrosine-based activation motif，ITAM)中的酪氨酸磷酸化，启动信号传导分子级联反应，激活转录因子，使得T细胞初步活化，这是T细胞活化的第一信号；同时，T细胞表面的共刺激分子(Co-stimulatory molecule)可与APC细胞表面相应的共刺激分子配体相互作用，产生T细胞活化的第二信号(共刺激信号)，其中正共刺激分子产生的第二信号可导致T细胞的完全活化，而负共刺激分子产生的第二信号主要是下调和终止T细胞的活化。
目前针对T细胞活化的第一信号途径，已见报道通过基因工程设计并构建了一系列的抗CD3单克隆全长抗体(Beverley PC等人,Eur J Immunol,11:329-334,1981；Lanzavecchia A等人,Eur J Immunol,17:105-111,1987；Yannelli JR等人,J ImmunolMethods,130:91-100,1990)。已有的实验数据表明，该类单克隆抗体能够特异性识别T细胞表面的CD3分子，产生T细胞活化的第一信号。然而，仅有第一信号传递途径非但不能有效激活T细胞，反而会导致T细胞失能甚至产生激活诱导的T细胞死亡(Activation inducedcell death，AICD)。
发明内容
为了克服现有技术中所存在的问题，本发明的目的在于提供一种同时结合CD3和T细胞正共刺激分子的双功能分子及其应用。
为了实现上述目的以及其他相关目的，本发明采用如下技术方案：
本发明的第一方面，提供一种双功能分子，其结构中包括能够结合并激活T细胞表面CD3分子的第一功能域和能够结合并激活T细胞正共刺激分子的第二功能域。
优选地，所述双功能分子能够同时结合并激活T细胞表面CD3分子和T细胞正共刺激分子，从而产生T细胞活化所需的第一信号和第二信号。
优选地，所述第一功能域为抗CD3的抗体，所述第二功能域为抗T细胞正共刺激分子的抗体。
优选地，所述抗体为小分子抗体。
优选地，所述抗体选自Fab抗体、Fv抗体或单链抗体(scFv)。
优选地，所述第一功能域和所述第二功能域通过连接片段连接。所述连接片段的氨基酸数量可为≥2个。
优选地，所述连接片段选自以G4S为单位的连接片段或免疫球蛋白IgD的铰链区片段。
所述G4S具体为GGGGS。所述以G4S为单位的连接片段包括一个或多个G4S单位。例如，可以包括是一个、二个、三个或四个以上的G4S单位。本发明的一些实施例中，列举了一单体形式的双功能分子中，第一功能域和第二功能域之间通过以G4S为单位的连接片段连接，所述连接片段含有三个G4S单位，所述连接片段的氨基酸序列如SEQ ID NO.1所示。
所述免疫球蛋白IgD的铰链区片段可以为免疫球蛋白IgD的铰链Ala90-Val170。本发明的一些实施例中，列举了一二聚体形式的双功能分子中，第一功能域和第二功能域之间通过免疫球蛋白IgD的铰链区片段连接，所述免疫球蛋白IgD的铰链区片段为免疫球蛋白IgD的铰链Ala90-Val170，所述免疫球蛋白IgD的铰链区片段的氨基酸序列如SEQ ID NO.3所示。所述连接片段可通过二硫键相互连接形成二聚体。
优选地，所述第一功能域的C末端与所述第二结构域的N末端连接。
优选地，所述第一功能域为抗CD3的单链抗体，所述第二功能域为抗T细胞正共刺激分子的单链抗体，所述单链抗体包括重链可变区和轻链可变区。
优选地，所述抗CD3的单链抗体的重链可变区的氨基酸序列如SEQ ID NO.37所示。所述抗CD3的单链抗体的轻链可变区的氨基酸序列如SEQ ID NO.38所示。
优选地，所述抗T细胞正共刺激分子的单链抗体可以是抗4-1BB的单链抗体、抗ICOS的单链抗体、抗OX40的单链抗体、抗GITR的单链抗体、抗CD40L的单链抗体或抗CD27的单链抗体之任一。
优选地，所述抗4-1BB的单链抗体的重链可变区的氨基酸序列如SEQ ID NO.40所示。所述抗4-1BB的单链抗体的轻链可变区的氨基酸序列如SEQ ID NO.41所示。
优选地，所述抗ICOS的单链抗体的重链可变区的氨基酸序列如SEQ ID NO.43所示。所述抗ICOS的单链抗体的轻链可变区的氨基酸序列如SEQ ID NO.44所示。
优选地，所述抗OX40的单链抗体的重链可变区的氨基酸序列如SEQ ID NO.46所示。所述抗OX40的单链抗体的轻链可变区的氨基酸序列如SEQ ID NO.47所示。
优选地，所述抗GITR的单链抗体的重链可变区的氨基酸序列如SEQ ID NO.49所示。所述抗GITR的单链抗体的轻链可变区的氨基酸序列如SEQ ID NO.50所示。
优选地，所述抗CD40L的单链抗体的重链可变区的氨基酸序列如SEQ ID NO.52所示。所述抗CD40L的单链抗体的轻链可变区的氨基酸序列如SEQ ID NO.53所示。
优选地，所述抗CD27的单链抗体的重链可变区的氨基酸序列如SEQ ID NO.55所示。所述抗CD27的单链抗体的轻链可变区的氨基酸序列如SEQ ID NO.56所示。
本发明一些实施例中，列举了所述抗CD3的单链抗体的氨基酸序列如SEQ IDNO.36所示。所述抗4-1BB的单链抗体的氨基酸序列如SEQ ID NO.39所示。所述抗ICOS的单链抗体的氨基酸序列如SEQ ID NO.42所示。所述抗OX40的单链抗体的氨基酸序列如SEQ IDNO.45所示。所述抗GITR的单链抗体的氨基酸序列如SEQ ID NO.48所示。所述抗CD40L的单链抗体的氨基酸序列如SEQ ID NO.51所示。所述抗CD27的单链抗体的氨基酸序列如SEQ IDNO.54所示。
本发明一些实施例中，还列举了单体形式的双功能分子的氨基酸序列如SEQ IDNO.12、SEQ ID NO.16、SEQ ID NO.20、SEQ ID NO.24、SEQ ID NO.28或SEQ ID NO.32之任一所示。二聚体形式的双功能分子的氨基酸序列如SEQ ID NO.14、SEQ ID NO.18、SEQ IDNO.22、SEQ ID NO.26、SEQ ID NO.30或SEQ ID NO.34之任一所示。
本发明的第二方面，提供一种多核苷酸，其编码前述双功能分子。
本发明的第三方面，提供一种表达载体，其含有前述多核苷酸。
本发明的第四方面，提供一种宿主细胞，其被前述表达载体所转化。
本发明的第五方面，提供一种制备前述双功能分子的方法，包括：构建含有双功能分子基因序列的表达载体，然后将含双功能分子基因序列的表达载体转化至宿主细胞中诱导表达，从表达产物中分离获得所述的双功能分子。
本发明的较佳案例中，所述表达载体采用pcDNA3.1。所述宿主细胞采用中国仓鼠卵巢细胞(Chinese hamster ovary ce1l，CHO)。
本发明的第六方面，提供前述双功能分子用于制备T细胞体外扩增剂的用途。
本发明的第七方面，提供一种T细胞体外扩增剂，含有前述双功能分子。
本发明的第八方面，公开了一种体外扩增T细胞的方法，包括将前述双功能分子作用于T细胞。所述方法可以是非治疗目的的。
与现有技术相比，本发明具有如下有益效果：
(1)本发明所述的双功能分子将能够结合并激活T细胞表面CD3分子的第一功能域和能够结合并激活T细胞正共刺激分子的第二功能域融合于同一蛋白肽链，采用真核细胞表达系统生产，表达产物结构单一，纯化工艺简便，蛋白产量高，制备工艺及产品稳定；而抗CD3单克隆全长抗体与抗T细胞正共刺激分子单克隆全长抗体如果联合使用，两个抗体需分别表达纯化，制备工艺更复杂，工作量和生产成本显著增加。
(2)本发明所述的双功能分子为单一蛋白，相对于抗CD3全长抗体与抗T细胞正共刺激分子全长抗体联合使用，对T细胞的体外活化与扩增效果更优，蛋白用量更少，且使用简便，可通过溶液形式直接添加，无需优化两种全长抗体的相对比例。
附图说明
图1：A.单体形式抗CD3/抗T细胞正共刺激分子双特异性抗体的结构图；B.二聚体形式抗CD3/抗T细胞正共刺激分子双特异性抗体的结构图。
图2：A.纯化的CD3-4-1BB BsAb_M SDS-PAGE分析图，泳道1：分子量蛋白Marker；泳道2：还原性CD3-4-1BB BsAb_M；泳道3：非还原性CD3-4-1BB BsAb_M；B.纯化的CD3-4-1BBBsAb_D SDS-PAGE分析图，泳道1：分子量蛋白Marker；泳道2：还原性CD3-4-1BB BsAb_D；泳道3：非还原性CD3-4-1BB BsAb_D。
图3A：CD3-4-1BB BsAb_M的ELISA鉴定结果，图中的曲线分别代表三种检测结果：■包被1μg/ml重组抗原CD3-hFc，●包被1μg/ml重组抗原4-1BB-hFc；▲不包被任何抗原的测定结果。
图3B：CD3-4-1BB BsAb_D的ELISA鉴定结果，图中的曲线分别代表三种检测结果：■包被1μg/ml重组抗原CD3-hFc；●包被1μg/ml重组抗原4-1BB-hFc；▲不包被任何抗原的测定结果。
图4：CIK细胞扩增倍数曲线，以外周血PBMC为实验细胞，分别添加CD3-4-1BBBsAb_M、CD3-4-1BB BsAb_D或抗CD3/抗CD28单克隆全长抗体联合使用(Anti-CD3/Anti-CD28)，总计培养30天，以每次计数的细胞数量除以第1天的细胞数量，计数比较细胞扩增倍数，其中对照组：5ug/ml Anti-CD3和5ug/ml Anti-CD28包被细胞培养板；实验组1：溶液状态下添加10ng/ml CD3-4-1BB BsAb_M；实验组2：溶液状态下添加10ng/ml CD3-4-1BBBsAb_D。
图5：基于流式细胞分析方法测定CIK细胞CD8+/CD4+比例，取图4所述扩增30天后的细胞，分别测定CD8+阳性与CD4+阳性的细胞比例；其中，A：对照组；B：实验组1；C：实验组2。
图6：A.纯化的CD3-ICOS BsAb_M SDS-PAGE分析图，泳道1：分子量蛋白Marker；泳道2：还原性CD3-ICOS BsAb_M；泳道3：非还原性CD3-ICOS BsAb_M；B.纯化的CD3-ICOSBsAb_D SDS-PAGE分析图，泳道1：分子量蛋白Marker；泳道2：还原性CD3-ICOS BsAb_D；泳道3：非还原性CD3-ICOS BsAb_D。
图7A：CD3-ICOS BsAb_M的ELISA鉴定结果，图中的曲线分别代表三种检测结果：■包被1μg/ml重组抗原CD3-hFc，●包被1μg/ml重组抗原ICOS-hFc；▲不包被任何抗原的测定结果。
图7B：CD3-ICOS BsAb_D的ELISA鉴定结果，图中的曲线分别代表三种检测结果：■包被1μg/ml重组抗原CD3-hFc；●包被1μg/ml重组抗原ICOS-hFc；▲不包被任何抗原的测定结果。
图8：CIK细胞扩增倍数曲线，以外周血PBMC为实验细胞，分别添加CD3-ICOS BsAb_M、CD3-ICOS BsAb_D或抗CD3/抗CD28单克隆全长抗体联合使用(Anti-CD3/Anti-CD28)，总计培养14天，以每次计数的细胞数量除以第1天的细胞数量，计数比较细胞扩增倍数，其中对照组：5ug/ml Anti-CD3和5ug/ml Anti-CD28包被细胞培养板；实验组1：溶液状态下添加10ng/ml CD3-ICOS BsAb_M；实验组2：溶液状态下添加10ng/ml CD3-ICOS BsAb_D。
图9：基于流式细胞分析方法测定CD3+CD56+CIK细胞比例，取图8所述扩增14天后的细胞，分别测定CD3+CD56+双阳性的细胞比例；其中，A：对照组；B：实验组1；C：实验组2。
图10：A.纯化的CD3-OX40 BsAb_M SDS-PAGE分析图，泳道1：分子量蛋白Marker；泳道2：还原性CD3-OX40 BsAb_M；泳道3：非还原性CD3-OX40 BsAb_M；B.纯化的CD3-OX40BsAb_D SDS-PAGE分析图，泳道1：分子量蛋白Marker；泳道2：还原性CD3-OX40 BsAb_D；泳道3：非还原性CD3-OX40 BsAb_D。
图11A：CD3-OX40 BsAb_M的ELISA鉴定结果，图中的曲线分别代表三种检测结果：■包被1μg/ml重组抗原CD3-hFc，●包被1μg/ml重组抗原OX40-hFc；▲不包被任何抗原的测定结果。
图11B：CD3-OX40 BsAb_D的ELISA鉴定结果，图中的曲线分别代表三种检测结果：■包被1μg/ml重组抗原CD3-hFc；●包被1μg/ml重组抗原OX40-hFc；▲不包被任何抗原的测定结果。
图12：CIK细胞扩增倍数曲线，以外周血PBMC为实验细胞，分别添加CD3-OX40BsAb_M、CD3-OX40 BsAb_D或抗CD3/抗CD28单克隆全长抗体联合使用(Anti-CD3/Anti-CD28)，总计培养30天，以每次计数的细胞数量除以第1天的细胞数量，计数比较细胞扩增倍数，其中对照组：5ug/ml Anti-CD3和5ug/ml Anti-CD28包被细胞培养板；实验组1：溶液状态下添加10ng/ml CD3-OX40 BsAb_M；实验组2：溶液状态下添加10ng/ml CD3-OX40 BsAb_D。
图13：扩增后的CIK细胞对肿瘤细胞的杀伤活性检测，分别取图12所述扩增14天和30天后的CIK细胞作为杀伤效应细胞，以Raji淋巴瘤细胞作为靶细胞，分别检测CIK细胞对Raji细胞的杀伤效率，效应细胞：靶细胞(E:T比)＝1：1，杀伤时间：3h。
图14：A.纯化的CD3-GITR BsAb_M SDS-PAGE分析图，泳道1：分子量蛋白Marker；泳道2：还原性CD3-GITR BsAb_M；泳道3：非还原性CD3-GITR BsAb_M；B.纯化的CD3-GITRBsAb_D SDS-PAGE分析图，泳道1：分子量蛋白Marker；泳道2：还原性CD3-GITR BsAb_D；泳道3：非还原性CD3-GITR BsAb_D。
图15A：CD3-GITR BsAb_M的ELISA鉴定结果，图中的曲线分别代表三种检测结果：■包被1μg/ml重组抗原CD3-hFc，●包被1μg/ml重组抗原GITR-hFc；▲不包被任何抗原的测定结果。
图15B：CD3-GITR BsAb_D的ELISA鉴定结果，图中的曲线分别代表三种检测结果：■包被1μg/ml重组抗原CD3-hFc；●包被1μg/ml重组抗原GITR-hFc；▲不包被任何抗原的测定结果。
图16：CIK细胞扩增倍数曲线，以外周血PBMC为实验细胞，分别添加CD3-GITRBsAb_M、CD3-GITR BsAb_D或抗CD3/抗CD28单克隆全长抗体联合使用(Anti-CD3/Anti-CD28)，总计培养14天，以每次计数的细胞数量除以第1天的细胞数量，计数比较细胞扩增倍数。其中，对照组：5ug/ml Anti-CD3和5ug/ml Anti-CD28包板；实验组1：溶液状态下添加10ng/ml CD3-GITR BsAb_M；实验组2：溶液状态下添加10ng/ml CD3-GITR BsAb_D。
图17：A.纯化的CD3-CD40L BsAb_M SDS-PAGE分析图，泳道1：分子量蛋白Marker；泳道2：还原性CD3-CD40L BsAb_M；泳道3：非还原性CD3-CD40L BsAb_M；B.纯化的CD3-CD40LBsAb_D SDS-PAGE分析图，泳道1：分子量蛋白Marker；泳道2：还原性CD3-CD40L BsAb_D；泳道3：非还原性CD3-CD40L BsAb_D。
图18A：CD3-CD40L BsAb_M的ELISA鉴定结果，图中的曲线分别代表三种检测结果：■包被1μg/ml重组抗原CD3-hFc，●包被1μg/ml重组抗原CD40L-hFc；▲不包被任何抗原的测定结果。
图18B：CD3-CD40L BsAb_D的ELISA鉴定结果，图中的曲线分别代表三种检测结果：■包被1μg/ml重组抗原CD3-hFc；●包被1μg/ml重组抗原CD40L-hFc；▲不包被任何抗原的测定结果。
图19：CIK细胞扩增倍数曲线，以外周血PBMC为实验细胞，分别添加CD3-CD40LBsAb_M、CD3-CD40L BsAb_D或抗CD3/抗CD28单克隆全长抗体联合使用(Anti-CD3/Anti-CD28)，总计培养14天，以每次计数的细胞数量除以第1天的细胞数量，计数比较细胞扩增倍数。其中，对照组：5ug/ml Anti-CD3和5ug/ml Anti-CD28包板；实验组1：溶液状态下添加10ng/ml CD3-CD40L BsAb_M；实验组2：溶液状态下添加10ng/ml CD3-CD40L BsAb_D。
图20：A.纯化的CD3-CD27 BsAb_M SDS-PAGE分析图，泳道1：分子量蛋白Marker；泳道2：还原性CD3-CD27 BsAb_M；泳道3：非还原性CD3-CD27 BsAb_M；B.纯化的CD3-CD27BsAb_D SDS-PAGE分析图，泳道1：分子量蛋白Marker；泳道2：还原性CD3-CD27 BsAb_D；泳道3：非还原性CD3-CD27 BsAb_D。
图21A：CD3-CD27 BsAb_M的ELISA鉴定结果，图中的曲线分别代表三种检测结果：■包被1μg/ml重组抗原CD3-hFc，●包被1μg/ml重组抗原CD27-hFc；▲不包被任何抗原的测定结果。
图21B：CD3-CD27 BsAb_D的ELISA鉴定结果，图中的曲线分别代表三种检测结果：■包被1μg/ml重组抗原CD3-hFc；●包被1μg/ml重组抗原CD27-hFc；▲不包被任何抗原的测定结果。
图22：CIK细胞扩增倍数曲线，以外周血PBMC为实验细胞，分别添加CD3-CD27BsAb_M、CD3-CD27 BsAb_D或抗CD3/抗CD28单克隆全长抗体联合使用(Anti-CD3/Anti-CD28)，总计培养30天，以每次计数的细胞数量除以第1天的细胞数量，计数比较细胞扩增倍数。其中，对照组：5ug/ml Anti-CD3和5ug/ml Anti-CD28包板；实验组1：溶液状态下添加10ng/ml CD3-CD27 BsAb_M；实验组2：溶液状态下添加10ng/ml CD3-CD27 BsAb_D。
具体实施方式
一、术语和缩略语：
BsAb：双特异性抗体(Bi-specific Antibody)
Fab：抗原结合片段(Fragment of antigen binding)
Fv：可变区片段(Variable fragment)
scFv：单链可变区片段(Single-chain variable fragment)，又称为单链抗体
VH：重链可变区(Heavy chain variable region)
VL：轻链可变区(Light chain variable region)
Linker：连接片段
Extracellular domain：胞外区
CD3-4-1BB BsAb_M:单体形式的抗CD3/抗4-1BB双特异性抗体
CD3-4-1BB BsAb_D:二聚体形式的抗CD3/抗4-1BB双特异性抗体
CD3-ICOS BsAb_M:单体形式的抗CD3/抗ICOS双特异性抗体
CD3-ICOS BsAb_D:二聚体形式的抗CD3/抗ICOS双特异性抗体
CD3-OX40 BsAb_M:单体形式的抗CD3/抗OX40双特异性抗体
CD3-OX40 BsAb_D:二聚体形式的抗CD3/抗OX40双特异性抗体
CD3-GITR BsAb_M:单体形式的抗CD3/抗GITR双特异性抗体
CD3-GITR BsAb_D:二聚体形式的抗CD3/抗GITR双特异性抗体
CD3-CD40L BsAb_M:单体形式的抗CD3/抗CD40L双特异性抗体
CD3-CD40L BsAb_D:二聚体形式的抗CD3/抗CD40L双特异性抗体
CD3-CD27 BsAb_M:单体形式的抗CD3/抗CD27双特异性抗体
CD3-CD27 BsAb_D:二聚体形式的抗CD3/抗CD27双特异性抗体
二、双功能分子
本发明所述的双功能分子，其结构中包括能够结合并激活T细胞表面CD3分子的第一功能域和能够结合并激活T细胞正共刺激分子的第二功能域。
进一步地，所述双功能分子能够同时结合并激活T细胞表面CD3分子和T细胞正共刺激分子，从而产生T细胞活化所需的第一信号和第二信号。所述T细胞正共刺激分子包括但不限于人类CD28、4-1BB、ICOS、OX40、GITR、CD40L或CD27。
本发明对于第一功能域和第二功能域并无特殊限制，只要能够同时结合并激活T细胞表面CD3分子和T细胞正共刺激分子，从而产生T细胞活化所需的第一信号和第二信号即可。例如，所述第一功能域可以是抗CD3的抗体，所述第二功能域可以是抗T细胞正共刺激分子的抗体。所述抗体可以是任意形式。但无论是何种形式的抗体，其抗原结合部位均含有重链可变区和轻链可变区。所述抗体优选地可以是小分子抗体。所述小分子抗体是分子量较小的抗体片段，其抗原结合部位包括重链可变区和轻链可变区。所述小分子抗体的分子量虽小但保持了亲本单抗的亲和力，具有亲本单抗一样的特异性。所述小分子抗体的种类主要包括Fab抗体、Fv抗体、单链抗体(scFv)等。Fab抗体由完整的轻链(可变区VL和恒定区CL)和重链Fd段(可变区VH和第一恒定区CH1)通过二硫键连接形成。Fv抗体仅由轻链和重链的可变区通过非共价键连接，是抗体分子保留完整抗原结合部位的最小功能片段。单链抗体(scFv)是重链可变区和轻链可变区通过连接片段连接而成的单一蛋白肽链分子。
所述第一功能域和所述第二功能域通过连接片段连接。本发明对于连接顺序没有特殊要求，只要不限制本发明的目的即可。例如，所述第一功能域的C末端可以与所述第二结构域的N末端连接。所述连接片段的氨基酸数量优选≥2个。本发明对于连接片段也没有特殊的限制，只要是不限制本发明的目的即可。
进一步地，所述连接片段选自以G4S为单位的连接片段或免疫球蛋白IgD的铰链区片段。
所述G4S具体为GGGGS。所述以G4S为单位的连接片段包括一个或多个G4S单位。例如，可以包括是一个、二个、三个或四个以上的G4S单位。本发明的一些实施例中，列举了一单体形式的双功能分子中，第一功能域和第二功能域之间通过以G4S为单位的连接片段连接，所述连接片段含有三个G4S单位，所述连接片段的氨基酸序列如SEQ ID NO.1所示。
所述免疫球蛋白IgD的铰链区片段可以为免疫球蛋白IgD的铰链Ala90-Val170。本发明的一些实施例中，列举了一二聚体形式的双功能分子中，第一功能域和第二功能域之间通过免疫球蛋白IgD的铰链区片段连接，所述免疫球蛋白IgD的铰链区片段为免疫球蛋白IgD的铰链Ala90-Val170，所述免疫球蛋白IgD的铰链区片段的氨基酸序列如SEQ ID NO.3所示。所述连接片段可通过二硫键相互连接形成二聚体。
在本发明的较佳实施例中，所述双功能分子的结构示意图如图1所示，为双特异性抗体。所述双功能分子可以是单体形式也可以是二聚体形式。本发明的单体形式的双功能分子的结构示意图如图1中A所示，所述双功能分子的结构中含有一个与CD3抗原结合的第一功能域和一个与任一T细胞正共刺激分子抗原结合的第二功能域，所述第一功能域为与CD3抗原结合的单链抗体(scFv)，所述第二功能域为与T细胞正共刺激分子胞外区(Extracellular domain)结合的单链抗体(scFv)。本发明的二聚体形式的双功能分子的结构示意图如图1中B所示，所述双功能分子的结构中含有两个与CD3抗原结合的第一功能域和两个与任一T细胞正共刺激分子抗原结合的第二功能域，所述第一功能域为与CD3抗原结合的单链抗体(scFv)，所述第二功能域为与T细胞正共刺激分子胞外区(Extracellulardomain)结合的单链抗体(scFv)。本发明的二聚体形式的双功能分子的抗原结合效价是单体形式的二倍以上，体外扩增T细胞的效果更优。
所述T细胞正共刺激分子可以是CD28、4-1BB、ICOS、OX40、GITR、CD40L或CD27等。
T细胞正共刺激分子人类CD28胞外区的氨基酸序列如SEQ ID NO.5所示，具体为：
NKILVKQSPMLVAYDNAVNLSCKYSYNLFSREFRASLHKGLDSAVEVCVVYGNYSQQLQVYSKTGFNCDGKLGNESVTFYLQNLYVNQTDIYFCKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP。
T细胞正共刺激分子人类4-1BB胞外区的氨基酸序列如SEQ ID NO.6所示，具体为：
LQDPCSNCPAGTFCDNNRNQICSPCPPNSFSSAGGQRTCDICRQCKGVFRTRKECSSTSNAECDCTPGFHCLGAGCSMCEQDCKQGQELTKKGCKDCCFGTFNDQKRGICRPWTNCSLDGKSVLVNGTKERDVVCGPSPADLSPGASSVTPPAPAREPGHSPQ。
T细胞正共刺激分子人类ICOS胞外区的氨基酸序列如SEQ ID NO.7所示，具体为：
EINGSANYEMFIFHNGGVQILCKYPDIVQQFKMQLLKGGQILCDLTKTKGSGNTVSIKSLKFCHSQLSNNSVSFFLYNLDHSHANYYFCNLSIFDPPPFKVTLTGGYLHIYESQLCCQLK。
T细胞正共刺激分子人类OX40胞外区的氨基酸序列如SEQ ID NO.8所示，具体为：
LHCVGDTYPSNDRCCHECRPGNGMVSRCSRSQNTVCRPCGPGFYNDVVSSKPCKPCTWCNLRSGSERKQLCTATQDTVCRCRAGTQPLDSYKPGVDCAPCPPGHFSPGDNQACKPWTNCTLAGKHTLQPASNSSDAICEDRDPPATQPQETQGPPARPITVQPTEAWPRTSQGPSTRPVEVPGGRA。
T细胞正共刺激分子人类GITR胞外区的氨基酸序列如SEQ ID NO.9所示，具体为：
QRPTGGPGCGPGRLLLGTGTDARCCRVHTTRCCRDYPGEECCSEWDCMCVQPEFHCGDPCCTTCRHHPCPPGQGVQSQGKFSFGFQCIDCASGTFSGGHEGHCKPWTDCTQFGFLTVFPGNKTHNAVCVPGSPPAEP。
T细胞正共刺激分子人类CD40L胞外区的氨基酸序列如SEQ ID NO.10所示，具体为：
HRRLDKIEDERNLHEDFVFMKTIQRCNTGERSLSLLNCEEIKSQFEGFVKDIMLNKEETKKENSFEMQKGDQNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL。
T细胞正共刺激分子人类CD27胞外区的氨基酸序列如SEQ ID NO.11所示，具体为：
ATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIR。
具体地，所述第一功能域为抗CD3的单链抗体。所述抗CD3的单链抗体包括重链可变区和轻链可变区。所述抗CD3的单链抗体的重链可变区的氨基酸序列如SEQ ID NO.37所示。所述抗CD3的单链抗体的轻链可变区的氨基酸序列如SEQ ID NO.38所示。进一步地，所述抗CD3的单链抗体的氨基酸序列如SEQ ID NO.36所示。
所述第二功能域为抗T细胞正共刺激分子的单链抗体。所述抗T细胞正共刺激分子的单链抗体包括重链可变区和轻链可变区。
所述抗T细胞正共刺激分子的单链抗体可以是抗4-1BB的单链抗体、抗ICOS的单链抗体、抗OX40的单链抗体、抗GITR的单链抗体、抗CD40L的单链抗体或抗CD27的单链抗体之任一。
所述抗4-1BB的单链抗体的重链可变区的氨基酸序列如SEQ ID NO.40所示。所述抗4-1BB的单链抗体的轻链可变区的氨基酸序列如SEQ ID NO.41所示。所述抗4-1BB的单链抗体的氨基酸序列如SEQ ID NO.39所示。
所述抗ICOS的单链抗体的重链可变区的氨基酸序列如SEQ ID NO.43所示。所述抗ICOS的单链抗体的轻链可变区的氨基酸序列如SEQ ID NO.44所示。所述抗ICOS的单链抗体的氨基酸序列如SEQ ID NO.42所示。
所述抗OX40的单链抗体的重链可变区的氨基酸序列如SEQ ID NO.46所示。所述抗OX40的单链抗体的轻链可变区的氨基酸序列如SEQ ID NO.47所示。所述抗OX40的单链抗体的氨基酸序列如SEQ ID NO.45所示。
所述抗GITR的单链抗体的重链可变区的氨基酸序列如SEQ ID NO.49所示。所述抗GITR的单链抗体的轻链可变区的氨基酸序列如SEQ ID NO.50所示。所述抗GITR的单链抗体的氨基酸序列如SEQ ID NO.48所示。
所述抗CD40L的单链抗体的重链可变区的氨基酸序列如SEQ ID NO.52所示。所述抗CD40L的单链抗体的轻链可变区的氨基酸序列如SEQ ID NO.53所示。所述抗CD40L的单链抗体的氨基酸序列如SEQ ID NO.51所示。
所述抗CD27的单链抗体的重链可变区的氨基酸序列如SEQ ID NO.55所示。所述抗CD27的单链抗体的轻链可变区的氨基酸序列如SEQ ID NO.56所示。所述抗CD27的单链抗体的氨基酸序列如SEQ ID NO.54所示。
在本案的较佳案例中，单体形式的双功能分子的氨基酸序列如SEQ ID NO.12、SEQID NO.16、SEQ ID NO.20、SEQ ID NO.24、SEQ ID NO.28或SEQ ID NO.32之任一所示。二聚体形式的双功能分子的氨基酸序列如SEQ ID NO.14、SEQ ID NO.18、SEQ ID NO.22、SEQ IDNO.26、SEQ ID NO.30或SEQ ID NO.34之任一所示。但不限于本发明较佳案例中所列举的具体形式。
三、编码双功能分子的多核苷酸
本发明的编码所述双功能分子的多核苷酸，可以是DNA形式或RNA形式。DNA形式包括cDNA、基因组DNA或人工合成的DNA。DNA可以是单链的或是双链的。
本发明的编码所述双功能分子的多核苷酸，可以通过本领域技术人员熟知的任何适当的技术制备。所述技术见于本领域的一般描述，如《分子克隆实验指南》(J.萨姆布鲁克等，科学出版社，1995)。包括但不限于重组DNA技术、化学合成等方法；例如采用重叠延伸PCR法。
在本发明的较佳实施例中，编码所述抗CD3的单链抗体的重链可变区的核苷酸序列如SEQ ID NO.58所示。编码所述抗CD3的单链抗体的轻链可变区的核苷酸序列如SEQ IDNO.59所示。编码所述抗CD3的单链抗体的核苷酸序列如SEQ ID NO.57所示。
编码所述抗4-1BB的单链抗体的重链可变区的核苷酸序列如SEQ ID NO.61所示。编码所述抗4-1BB的单链抗体的轻链可变区的核苷酸序列如SEQ ID NO.62所示。编码所述抗4-1BB的单链抗体的核苷酸序列如SEQ ID NO.60所示。
编码所述抗ICOS的单链抗体的重链可变区的核苷酸序列如SEQ ID NO.64所示。编码所述抗ICOS的单链抗体的轻链可变区的核苷酸序列如SEQ ID NO.65所示。编码所述抗ICOS的单链抗体的核苷酸序列如SEQ ID NO.63所示。
编码所述抗OX40的单链抗体的重链可变区的核苷酸序列如SEQ ID NO.67所示。编码所述抗OX40的单链抗体的轻链可变区的核苷酸序列如SEQ ID NO.68所示。编码所述抗OX40单链抗体的核苷酸序列如SEQ ID NO.66所示。
编码所述抗GITR的单链抗体的重链可变区的核苷酸序列如SEQ ID NO.70所示。编码所述抗GITR的单链抗体的轻链可变区的核苷酸序列如SEQ ID NO.71所示。编码所述抗GITR单链抗体的核苷酸序列如SEQ ID NO.69所示。
编码所述抗CD40L的单链抗体的重链可变区的核苷酸序列如SEQ ID NO.73所示。编码所述抗CD40L的单链抗体的轻链可变区的核苷酸序列如SEQ ID NO.74所示。编码所述抗CD40L单链抗体的核苷酸序列如SEQ ID NO.72所示。
编码所述抗CD27的单链抗体的重链可变区的核苷酸序列如SEQ ID NO.76所示。编码所述抗CD27的单链抗体的轻链可变区的核苷酸序列如SEQ ID NO.77所示。编码所述抗CD27单链抗体的核苷酸序列如SEQ ID NO.75所示。
编码氨基酸序列如SEQ ID NO.1所示的连接片段的核苷酸序列如SEQ ID NO.2所示。
编码氨基酸序列如SEQ ID NO.3所示的连接片段的核苷酸序列如SEQ ID NO.4所示。
进一步地，编码单体形式的双功能分子的核苷酸序列如SEQ ID NO.13、SEQ IDNO.17、SEQ ID NO.21、SEQ ID NO.25、SEQ ID NO.29或SEQ ID NO.33之任一所示。编码二聚体形式的双功能分子的核苷酸序列如如SEQ ID NO.15、SEQ ID NO.19、SEQ ID NO.23、SEQID NO.27、SEQ ID NO.31或SEQ ID NO.35之任一所示。
四、表达载体
本发明的所述表达载体含有编码所述双功能分子的多核苷酸。本领域的技术人员熟知的方法能用于构建所述表达载体。这些方法包括重组DNA技术、DNA合成技术等。可将编码所述融合蛋白的DNA有效连接到载体中的多克隆位点上，以指导mRNA合成进而表达蛋白，或者用于同源重组。本发明的较佳案例中，所述表达载体采用pcDNA3.1。所述宿主细胞采用中国仓鼠卵巢细胞(Chinese hamster ovary ce1l，CHO)。
五、制备双功能分子的方法
本发明的制备前述双功能分子的方法，包括：构建含有双功能分子基因序列的表达载体，然后将含双功能分子基因序列的表达载体转化至宿主细胞中诱导表达，从表达产物中分离获得所述的双功能分子。本发明的较佳案例中，所述表达载体采用pcDNA3.1。所述宿主细胞采用中国仓鼠卵巢细胞(Chinese hamster ovary ce1l，CHO)。
六、双功能分子的用途
本发明的双功能分子可用于制备T细胞体外扩增剂。本发明较佳实施例中，通过试验证明单体和二聚体形式的双功能分子均具有与CD3和正共刺激分子重组抗原的体外结合活性，可应用于T细胞体外活化与扩增，其中二聚体较单体具有更好的效果。
七、体外扩增T细胞的方法
本发明的体外扩增T细胞的方法，包括将前述双功能分子作用于T细胞。所述方法可以是非治疗目的的。本发明较佳实施例中，通过试验证明单体和二聚体形式的双功能分子均具有与CD3和正共刺激分子重组抗原的体外结合活性，可应用于T细胞体外活化与扩增，其中二聚体较单体具有更好的效果。
本发明针对抗CD3和抗T细胞正共刺激分子全长抗体联合应用的不足之处，通过基因工程和抗体工程的方法构建能够同时识别并激活CD3和任何一个T细胞正共刺激分子的双功能分子，该双功能分子不仅具有上述双抗体联合使用的特性，同时在制备工艺和实际应用方面具有明显的优势，以溶液形式添加时能够达到甚至优于两个抗体联合添加或包被培养板的效果，大大提高了体外活化与扩增T细胞的功效，增加了使用的方便性。
在进一步描述本发明具体实施方式之前，应理解，本发明的保护范围不局限于下述特定的具体实施方案；还应当理解，本发明实施例中使用的术语是为了描述特定的具体实施方案，而不是为了限制本发明的保护范围。下列实施例中未注明具体条件的试验方法，通常按照常规条件，或者按照各制造商所建议的条件。
当实施例给出数值范围时，应理解，除非本发明另有说明，每个数值范围的两个端点以及两个端点之间任何一个数值均可选用。除非另外定义，本发明中使用的所有技术和科学术语与本技术领域技术人员通常理解的意义相同。除实施例中使用的具体方法、设备、材料外，根据本技术领域的技术人员对现有技术的掌握及本发明的记载，还可以使用与本发明实施例中所述的方法、设备、材料相似或等同的现有技术的任何方法、设备和材料来实现本发明。
除非另外说明，本发明中所公开的实验方法、检测方法、制备方法均采用本技术领域常规的分子生物学、生物化学、染色质结构和分析、分析化学、细胞培养、重组DNA技术及相关领域的常规技术。这些技术在现有文献中已有完善说明，具体可参见Sambrook等MOLECULAR CLONING：A LABORATORY MANUAL，Second edition，Cold Spring HarborLaboratory Press，1989 and Third edition，2001；Ausubel等，CURRENT PROTOCOLS INMOLECULAR BIOLOGY，John Wiley&Sons，New York，1987 and periodic updates；theseries METHODS IN ENZYMOLOGY，Academic Press，San Diego；Wolffe，CHROMATINSTRUCTURE AND FUNCTION，Third edition，Academic Press，San Diego，1998；METHODS INENZYMOLOGY，Vol.304，Chromatin(P.M.Wassarman and A.P.Wolffe，eds.)，AcademicPress，San Diego，1999；和METHODS IN MOLECULAR BIOLOGY，Vol.119，ChromatinProtocols(P.B.Becker，ed.)Humana Press，Totowa，1999等。
实施例1 CD3-4-1BB BsAb_M和CD3-4-1BB BsAb_D真核表达载体的构建
在本发明中，以T细胞表面人类CD3蛋白和T细胞正共刺激分子4-1BB蛋白为靶点的双特异性抗体被命名为CD3-4-1BB BsAb。
一、CD3-4-1BB BsAb_M和CD3-4-1BB BsAb_D构建方案设计
单体形式的CD3-4-1BB BsAb_M具体构建方案为：抗CD3 scFv和抗4-1BB scFv序列之间通过(GGGGS)3 Linker相连。
二聚体形式的CD3-4-1BB BsAb_D具体构建方案为：抗CD3 scFv和抗4-1BB scFv序列之间通过IgD铰链区作为Linker相连。
为使双特异性抗体在哺乳细胞中进行表达，针对抗CD3 scFv，抗4-1BB scFv及连接片段(Linker)的序列均进行了哺乳系统表达的密码子优化。
具体地，抗CD3 scFv的重链可变区的核苷酸序列如SEQ ID NO.58所示，具体为：
GACATCAAGCTGCAGCAGAGCGGCGCCGAGCTGGCCCGCCCCGGCGCCAGCGTGAAGATGAGCTGCAAGACCAGCGGCTACACCTTCACCCGCTACACCATGCACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGGCTACATCAACCCCAGCCGCGGCTACACCAACTACAACCAGAAGTTCAAGGACAAGGCCACCCTGACCACCGACAAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCCGCTACTACGACGACCACTACTGCCTGGACTACTGGGGCCAGGGCACCACCCTGACCGTGAGCAGC。
抗CD3 scFv的轻链可变区的核苷酸序列如SEQ ID NO.59所示，具体为：
GACATCCAGCTGACCCAGAGCCCCGCCATCATGAGCGCCAGCCCCGGCGAGAAGGTGACCATGACCTGCCGCGCCAGCAGCAGCGTGAGCTACATGAACTGGTACCAGCAGAAGAGCGGCACCAGCCCCAAGCGCTGGATCTACGACACCAGCAAGGTGGCCAGCGGCGTGCCCTACCGCTTCAGCGGCAGCGGCAGCGGCACCAGCTACAGCCTGACCATCAGCAGCATGGAGGCCGAGGACGCCGCCACCTACTACTGCCAGCAGTGGAGCAGCAACCCCCTGACCTTCGGCGCCGGCACCAAGCTGGAGCTGAAG。
抗CD3 scFv的核苷酸序列如SEQ ID NO.57所示，具体为：
GACATCAAGCTGCAGCAGAGCGGCGCCGAGCTGGCCCGCCCCGGCGCCAGCGTGAAGATGAGCTGCAAGACCAGCGGCTACACCTTCACCCGCTACACCATGCACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGGCTACATCAACCCCAGCCGCGGCTACACCAACTACAACCAGAAGTTCAAGGACAAGGCCACCCTGACCACCGACAAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCCGCTACTACGACGACCACTACTGCCTGGACTACTGGGGCCAGGGCACCACCCTGACCGTGAGCAGCGTGGAGGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCGTGGACGACATCCAGCTGACCCAGAGCCCCGCCATCATGAGCGCCAGCCCCGGCGAGAAGGTGACCATGACCTGCCGCGCCAGCAGCAGCGTGAGCTACATGAACTGGTACCAGCAGAAGAGCGGCACCAGCCCCAAGCGCTGGATCTACGACACCAGCAAGGTGGCCAGCGGCGTGCCCTACCGCTTCAGCGGCAGCGGCAGCGGCACCAGCTACAGCCTGACCATCAGCAGCATGGAGGCCGAGGACGCCGCCACCTACTACTGCCAGCAGTGGAGCAGCAACCCCCTGACCTTCGGCGCCGGCACCAAGCTGGAGCTGAAG。
抗4-1BB scFv的重链可变区的核苷酸序列如SEQ ID NO.61所示，具体为：
CAGGTGCAGCTGCAGCAGTGGGGCGCCGGCCTGCTGAAGCCCAGCGAGACCCTGAGCCTGACCTGCGCCGTGTACGGCGGCAGCTTCAGCGGCTACTACTGGAGCTGGATCCGCCAGAGCCCCGAGAAGGGCCTGGAGTGGATCGGCGAGATCAACCACGGCGGCTACGTGACCTACAACCCCAGCCTGGAGAGCCGCGTGACCATCAGCGTGGACACCAGCAAGAACCAGTTCAGCCTGAAGCTGAGCAGCGTGACCGCCGCCGACACCGCCGTGTACTACTGCGCCCGCGACTACGGCCCCGGCAACTACGACTGGTACTTCGACCTGTGGGGCCGCGGCACCCTGGTGACCGTGAGCAGC。
抗4-1BB scFv的轻链可变区的核苷酸序列如SEQ ID NO.62所示，具体为：
GAGATCGTGCTGACCCAGAGCCCCGCCACCCTGAGCCTGAGCCCCGGCGAGCGCGCCACCCTGAGCTGCCGCGCCAGCCAGAGCGTGAGCAGCTACCTGGCCTGGTACCAGCAGAAGCCCGGCCAGGCCCCCCGCCTGCTGATCTACGACGCCAGCAACCGCGCCACCGGCATCCCCGCCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGGAGCCCGAGGACTTCGCCGTGTACTACTGCCAGCAGCGCAGCAACTGGCCCCCCGCCCTGACCTTCTGCGGCGGCACCAAGGTGGAGATCAAGCGC。
抗4-1BB scFv的核苷酸序列如SEQ ID NO.60所示，具体为：
CAGGTGCAGCTGCAGCAGTGGGGCGCCGGCCTGCTGAAGCCCAGCGAGACCCTGAGCCTGACCTGCGCCGTGTACGGCGGCAGCTTCAGCGGCTACTACTGGAGCTGGATCCGCCAGAGCCCCGAGAAGGGCCTGGAGTGGATCGGCGAGATCAACCACGGCGGCTACGTGACCTACAACCCCAGCCTGGAGAGCCGCGTGACCATCAGCGTGGACACCAGCAAGAACCAGTTCAGCCTGAAGCTGAGCAGCGTGACCGCCGCCGACACCGCCGTGTACTACTGCGCCCGCGACTACGGCCCCGGCAACTACGACTGGTACTTCGACCTGTGGGGCCGCGGCACCCTGGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGAGATCGTGCTGACCCAGAGCCCCGCCACCCTGAGCCTGAGCCCCGGCGAGCGCGCCACCCTGAGCTGCCGCGCCAGCCAGAGCGTGAGCAGCTACCTGGCCTGGTACCAGCAGAAGCCCGGCCAGGCCCCCCGCCTGCTGATCTACGACGCCAGCAACCGCGCCACCGGCATCCCCGCCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGGAGCCCGAGGACTTCGCCGTGTACTACTGCCAGCAGCGCAGCAACTGGCCCCCCGCCCTGACCTTCTGCGGCGGCACCAAGGTGGAGATCAAGCGC。
单体形式的CD3-4-1BB BsAb_M连接片段的核苷酸序列如SEQ ID NO.2所示，具体为：GGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGC。
二聚体形式的CD3-4-1BB BsAb_D连接片段的核苷酸序列如SEQ ID NO.4所示，具体为：
GCCAGCAAGAGCAAGAAGGAGATCTTCCGCTGGCCCGAGAGCCCCAAGGCCCAGGCCAGCAGCGTGCCCACCGCCCAGCCCCAGGCCGAGGGCAGCCTGGCCAAGGCCACCACCGCCCCCGCCACCACCCGCAACACCGGCCGCGGCGGCGAGGAGAAGAAGAAGGAGAAGGAGAAGGAGGAGCAGGAGGAGCGCGAGACCAAGACCCCCGAGTGCCCCAGCCACACCCAGCCCCTGGGCGTG。
为使双特异性抗体在CHO-S细胞中表达并成功分泌到培养基中，选择了抗体分泌型表达的信号肽用于此实施例。
该分泌表达信号肽的氨基酸序列如SEQ ID NO.78所示，具体为：
MTRLTVLALLAGLLASSRA。
该分泌表达信号肽的核苷酸序列如SEQ ID NO.79所示，具体为：
ATGACCCGGCTGACCGTGCTGGCCCTGCTGGCCGGCCTGCTGGCCTCCTCCAGGGCC。
二、CD3-4-1BB BsAb_M和CD3-4-1BB BsAb_D真核表达载体构建
本发明双特异性抗体的构建与表达，选用哺乳细胞蛋白瞬时表达载体pcDNA3.1(购自上海英骏生物科技有限公司)。为构建单体和二聚体形式的双特异性抗体，分别设计了如表1所示引物，所有引物由苏州金唯智生物科技有限公司合成，扩增所需基因模板由苏州鸿讯科技有限公司合成。
针对CD3-4-1BB BsAb_M的克隆构建，首先使用引物pcDNA3.1-Sig-F和Sig-R扩增出信号肽片段，然后分别利用引物Sig-CD3-F和CD3-R、CD3-(GGGGS)3-4-1BB-F和pcDNA3.1-4-1BB-R扩增出抗CD3 scFv、(GGGGS)3 Linker、抗4-1BB scFv的基因序列；针对CD3-4-1BBBsAb_D的克隆构建，同样首先使用引物pcDNA3.1-Sig-F和Sig-R扩增出信号肽片段，然后分别利用引物Sig-CD3-F和CD3-R、CD3-IgD-F和IgD-R、IgD-4-1BB-F和pcDNA3.1-4-1BB-R扩增出抗CD3 scFv、IgD铰链区、抗4-1BB scFv的基因序列。扩增完毕后，利用
PCR一步定向克隆试剂盒(购自吴江近岸蛋白质科技有限公司)分别拼接单体和二聚体形式双特异性抗体全长基因序列并无缝克隆至经EcoRI和HindIII线性化处理的pcDNA3.1表达载体上。目的载体转化大肠杆菌DH5α，利用菌落PCR进行阳性克隆鉴定，鉴定为阳性的重组子(重组质粒)进行测序鉴定。随后将测序正确的重组子(重组质粒)安排质粒中抽，用于CHO-S细胞的转染。
经测序获知，单体形式的CD3-4-1BB BsAb_M和二聚体形式的CD3-4-1BB BsAb_D的全长基因序列正确，均与预期相符。
具体地，单体形式的CD3-4-1BB BsAb_M的核苷酸序列如SEQ ID NO.13所示，具体为：GACATCAAGCTGCAGCAGAGCGGCGCCGAGCTGGCCCGCCCCGGCGCCAGCGTGAAGATGAGCTGCAAGACCAGCGGCTACACCTTCACCCGCTACACCATGCACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGGCTACATCAACCCCAGCCGCGGCTACACCAACTACAACCAGAAGTTCAAGGACAAGGCCACCCTGACCACCGACAAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCCGCTACTACGACGACCACTACTGCCTGGACTACTGGGGCCAGGGCACCACCCTGACCGTGAGCAGCGTGGAGGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCGTGGACGACATCCAGCTGACCCAGAGCCCCGCCATCATGAGCGCCAGCCCCGGCGAGAAGGTGACCATGACCTGCCGCGCCAGCAGCAGCGTGAGCTACATGAACTGGTACCAGCAGAAGAGCGGCACCAGCCCCAAGCGCTGGATCTACGACACCAGCAAGGTGGCCAGCGGCGTGCCCTACCGCTTCAGCGGCAGCGGCAGCGGCACCAGCTACAGCCTGACCATCAGCAGCATGGAGGCCGAGGACGCCGCCACCTACTACTGCCAGCAGTGGAGCAGCAACCCCCTGACCTTCGGCGCCGGCACCAAGCTGGAGCTGAAGGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCCAGGTGCAGCTGCAGCAGTGGGGCGCCGGCCTGCTGAAGCCCAGCGAGACCCTGAGCCTGACCTGCGCCGTGTACGGCGGCAGCTTCAGCGGCTACTACTGGAGCTGGATCCGCCAGAGCCCCGAGAAGGGCCTGGAGTGGATCGGCGAGATCAACCACGGCGGCTACGTGACCTACAACCCCAGCCTGGAGAGCCGCGTGACCATCAGCGTGGACACCAGCAAGAACCAGTTCAGCCTGAAGCTGAGCAGCGTGACCGCCGCCGACACCGCCGTGTACTACTGCGCCCGCGACTACGGCCCCGGCAACTACGACTGGTACTTCGACCTGTGGGGCCGCGGCACCCTGGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGAGATCGTGCTGACCCAGAGCCCCGCCACCCTGAGCCTGAGCCCCGGCGAGCGCGCCACCCTGAGCTGCCGCGCCAGCCAGAGCGTGAGCAGCTACCTGGCCTGGTACCAGCAGAAGCCCGGCCAGGCCCCCCGCCTGCTGATCTACGACGCCAGCAACCGCGCCACCGGCATCCCCGCCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGGAGCCCGAGGACTTCGCCGTGTACTACTGCCAGCAGCGCAGCAACTGGCCCCCCGCCCTGACCTTCTGCGGCGGCACCAAGGTGGAGATCAAGCGC。
具体地，二聚体形式的CD3-4-1BB BsAb_D的核苷酸序列如SEQ ID NO.15所示，具体为：
GACATCAAGCTGCAGCAGAGCGGCGCCGAGCTGGCCCGCCCCGGCGCCAGCGTGAAGATGAGCTGCAAGACCAGCGGCTACACCTTCACCCGCTACACCATGCACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGGCTACATCAACCCCAGCCGCGGCTACACCAACTACAACCAGAAGTTCAAGGACAAGGCCACCCTGACCACCGACAAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCCGCTACTACGACGACCACTACTGCCTGGACTACTGGGGCCAGGGCACCACCCTGACCGTGAGCAGCGTGGAGGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCGTGGACGACATCCAGCTGACCCAGAGCCCCGCCATCATGAGCGCCAGCCCCGGCGAGAAGGTGACCATGACCTGCCGCGCCAGCAGCAGCGTGAGCTACATGAACTGGTACCAGCAGAAGAGCGGCACCAGCCCCAAGCGCTGGATCTACGACACCAGCAAGGTGGCCAGCGGCGTGCCCTACCGCTTCAGCGGCAGCGGCAGCGGCACCAGCTACAGCCTGACCATCAGCAGCATGGAGGCCGAGGACGCCGCCACCTACTACTGCCAGCAGTGGAGCAGCAACCCCCTGACCTTCGGCGCCGGCACCAAGCTGGAGCTGAAGGCCAGCAAGAGCAAGAAGGAGATCTTCCGCTGGCCCGAGAGCCCCAAGGCCCAGGCCAGCAGCGTGCCCACCGCCCAGCCCCAGGCCGAGGGCAGCCTGGCCAAGGCCACCACCGCCCCCGCCACCACCCGCAACACCGGCCGCGGCGGCGAGGAGAAGAAGAAGGAGAAGGAGAAGGAGGAGCAGGAGGAGCGCGAGACCAAGACCCCCGAGTGCCCCAGCCACACCCAGCCCCTGGGCGTGCAGGTGCAGCTGCAGCAGTGGGGCGCCGGCCTGCTGAAGCCCAGCGAGACCCTGAGCCTGACCTGCGCCGTGTACGGCGGCAGCTTCAGCGGCTACTACTGGAGCTGGATCCGCCAGAGCCCCGAGAAGGGCCTGGAGTGGATCGGCGAGATCAACCACGGCGGCTACGTGACCTACAACCCCAGCCTGGAGAGCCGCGTGACCATCAGCGTGGACACCAGCAAGAACCAGTTCAGCCTGAAGCTGAGCAGCGTGACCGCCGCCGACACCGCCGTGTACTACTGCGCCCGCGACTACGGCCCCGGCAACTACGACTGGTACTTCGACCTGTGGGGCCGCGGCACCCTGGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGAGATCGTGCTGACCCAGAGCCCCGCCACCCTGAGCCTGAGCCCCGGCGAGCGCGCCACCCTGAGCTGCCGCGCCAGCCAGAGCGTGAGCAGCTACCTGGCCTGGTACCAGCAGAAGCCCGGCCAGGCCCCCCGCCTGCTGATCTACGACGCCAGCAACCGCGCCACCGGCATCCCCGCCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGGAGCCCGAGGACTTCGCCGTGTACTACTGCCAGCAGCGCAGCAACTGGCCCCCCGCCCTGACCTTCTGCGGCGGCACCAAGGTGGAGATCAAGCGC。
表1.CD3-4-1BB双特异性抗体基因克隆中使用的引物
实施例2：CD3-4-1BB BsAb_M和CD3-4-1BB BsAb_D的表达与纯化
一、CD3-4-1BB BsAb_M和CD3-4-1BB BsAb_D的表达
1.1.CHO-S细胞(购自Thermo Fisher Scientific公司)转染前1天传代密度为0.5～0.6×106/ml；
1.2.转染当天进行细胞密度统计，当密度为1～1.4×106/ml、活力>90％时，可用于质粒转染；
1.3.转染复合物配制：每个项目(CD3-4-1BB BsAb_M和CD3-4-1BB BsAb_D)需准备两个离心管/培养瓶，以20ml为例，分别放置，取实施例1中所制备重组质粒：
管①中加入600μl PBS，20μg重组质粒，混匀；
管②中加入600μl PBS，20ul FreeStyleTM MAX Transfection Reagent(购自Thermo Fisher Scientific公司)，混匀；
1.4.将稀释后的转染试剂，加入至稀释后的重组质粒中，混合均匀，配制成转染复合物；
1.5.转染复合物静置15～20min后，单滴匀速加入细胞培养物中；
1.6.于37℃，CO2浓度8％，摇床转速130rpm条件下进行转染后细胞培养，5天后收集培养上清进行目的蛋白表达检测。
二、CD3-4-1BB BsAb_M和CD3-4-1BB BsAb_D的纯化
2.1样品预处理
取上述转染后细胞培养上清20ml，加入缓冲液20mM PB，200mM NaCl调节pH至7.5；
2.2 Protein L亲和层析柱纯化
蛋白纯化层析柱：Protein L亲和层析柱(购自GE Healthcare公司，柱体积1.0ml)
缓冲液A(Buffer A)：PBS，pH7.4
缓冲液B(Buffer B)：0.1M Glycine,pH3.0
缓冲液C(Buffer C)：0.1M Glycine,pH2.7
纯化过程：采用AKTA explorer 100型蛋白纯化系统(购自GE Healthcare公司)，用Buffer A预处理Protein L亲和层析柱，取培养上清上样，收集流出液。上样完毕后，用至少1.5ml Buffer A平衡层析柱，平衡后分别用Buffer B和Buffer C洗脱，收集目的蛋白洗脱液(洗脱液的收集管需要预先加入1％的1M Tris,pH8.0来中和洗脱液pH值，Tris终浓度约为10mM)，最后浓缩透析至缓冲液PBS中。
最终纯化的CD3-4-1BB BsAb_M和CD3-4-1BB BsAb_D重组蛋白经SDS-PAGE分析，还原和非还原条件下电泳图如图2所示。从图中可以看出，经Protein L亲和层析柱纯化后，CD3-4-1BB BsAb_M和CD3-4-1BB BsAb_D重组蛋白的纯度均>95％；其中CD3-4-1BB BsAb_M重组蛋白的理论分子量为53.7kDa，还原和非还原条件下该蛋白均呈现单一电泳条带，分子量与单体一致，因此该双特异性抗体为单体形式(图2A)；CD3-4-1BB BsAb_D重组蛋白的理论分子量为61.5kDa，还原条件下该蛋白电泳条带所呈现分子量与单体一致，非还原条件下电泳条带所呈现分子量与二聚体一致(图2B)，说明两个蛋白分子可通过二硫键相互连接，因此该双特异性抗体为二聚体形式。
此外，纯化的重组蛋白样品经N/C末端序列分析，结果表明所表达的重组蛋白样品均读框无误，与理论N/C末端氨基酸序列一致，质谱分析进一步确认CD3-4-1BB BsAb_M为单体形式，CD3-4-1BB BsAb_D为二聚体形式。
因此，可得知，单体形式的CD3-4-1BB BsAb_M的氨基酸序列如SEQ ID NO.12所示，具体为：
DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKGGGGSGGGGSGGGGSQVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQSPEKGLEWIGEINHGGYVTYNPSLESRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDYGPGNYDWYFDLWGRGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPALTFCGGTKVEIKR。
二聚体形式的CD3-4-1BB BsAb_D的氨基酸序列如SEQ ID NO.14所示，具体为：
DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKASKSKKEIFRWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEKEKEEQEERETKTPECPSHTQPLGVQVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQSPEKGLEWIGEINHGGYVTYNPSLESRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDYGPGNYDWYFDLWGRGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPALTFCGGTKVEIKR。
抗CD3 scFv的氨基酸序列如SEQ ID NO.36所示，具体为：
DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELK。
抗CD3 scFv的重链可变区的氨基酸序列如SEQ ID NO.37所示，具体为：
DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSS。
抗CD3 scFv的轻链可变区的氨基酸序列如SEQ ID NO.38所示，具体为：
DIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELK。
抗4-1BB scFv的氨基酸序列如SEQ ID NO.39所示，具体为：
QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQSPEKGLEWIGEINHGGYVTYNPSLESRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDYGPGNYDWYFDLWGRGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPALTFCGGTKVEIKR。
抗4-1BB scFv的重链可变区的氨基酸序列如SEQ ID NO.40所示，具体为：
QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQSPEKGLEWIGEINHGGYVTYNPSLESRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDYGPGNYDWYFDLWGRGTLVTVSS。
抗4-1BB scFv的轻链可变区的氨基酸序列如SEQ ID NO.41所示，具体为：
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPALTFCGGTKVEIKR。
单体形式的CD3-4-1BB BsAb_D中连接片段的氨基酸序列如SEQ ID NO.1所示，具体为：
GGGGSGGGGSGGGGS。
二聚体形式的CD3-4-1BB BsAb_D中连接片段的氨基酸序列如SEQ ID NO.3所示，具体为：
ASKSKKEIFRWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEKEKEEQEERETKTPECPSHTQPLGV。
实施例3：ELISA检测CD3-4-1BB BsAb_M和CD3-4-1BB BsAb_D的抗原结合活性
ELISA操作步骤：
1.重组抗原包被：人类CD3-hFc与人类4-1BB-hFc融合蛋白(购自吴江近岸蛋白质科技有限公司)分别包被96孔板，抗原浓度为1μg/ml，包被体积为100μl/孔，包被条件为37℃1小时或4℃过夜，包被缓冲液(PBS)的配方为：3.58g Na2HPO4，0.24g NaH2PO4，0.2g KCl，8.2g NaCl，950ml H2O，用1mol/L HCl或1mol/L NaOH调pH至7.4，补水至1L；
2.封闭：PBS洗板4次后，加入封闭液PBSA(PBS+2％BSA(V/W))，200μl/孔，37℃封闭1小时；
3.加样：PBS洗板4次后，分别加入纯化的双特异性抗体样品，100μl/孔，37℃孵育1小时，样品梯度配制方法：以10μg/ml纯化的CD3-4-1BB BsAb_M或CD3-4-1BB BsAb_D作为起始浓度，进行倍比稀释6个梯度，每个梯度设置2个复孔；
4.显色：PBST(PBS+0.05％Tween-20(V/V))洗板4次后，用封闭液PBSA按1/5000稀释HRP标记的显色抗体(购自Abcam公司)，按100μl/孔加入，37℃孵育1小时。PBS洗板4次后，添加显色液TMB(购自KPL公司)，100μl/孔，室温避光显色5～10分钟；
5.终止反应与结果测定：添加终止液(1M HCl)，100μl/孔，在酶标仪上450nm波长下读取吸光值。
ELISA结果如图3A和图3B所示：图3A说明CD3-4-1BB BsAb_M与重组抗原CD3-hFc和4-1BB-hFc均具有体外结合活性，其中4-1BB结合活性较CD3结合活性更高；图3B说明CD3-4-1BB BsAb_D与重组抗原CD3-hFc和4-1BB-hFc同样具有体外结合活性，其中4-1BB结合活性更高。
实施例4：CD3-4-1BB双特异抗体介导的CIK(Cytokine induced killer)细胞增殖
以人外周血单个核细胞(Peripheral blood mononuclear cell，PBMC)为实验材料，用本发明所制备的上述单体形式的双特异抗体CD3-4-1BB BsAb_M、二聚体形式的双特异抗体CD3-4-1BB BsAb_D、以及抗CD3/抗CD28单克隆全长抗体联用(Anti-CD3/Anti-CD28)分别作用于同一供体来源的人血PBMC，细胞培养后进行计数，比较扩增倍数。
1.PBMC的分离：取抗凝血，加入等体积的医用生理盐水，沿离心管壁缓慢加入与血液等体积的淋巴细胞分离液(购自GE Healthcare公司)，保持液面分层明显，2000rpm离心20min，吸取中间白雾状的细胞层于新的离心管中，加入2倍以上体积的PBS缓冲液洗涤，1100rpm离心10min，重复洗涤一次，用少量预冷的X-vivo 15无血清培养基(购自Lonza公司)重悬，细胞计数待用；
2.CIK细胞培养与扩增：将PBMC用CIK基础培养基(90％X-vivo15+10％FBS)重悬，调整细胞密度为1×106/ml，分别设计以下3个实验组：对照组(Anti-CD3 5ug/ml和Anti-CD285ug/ml包被细胞培养板)；实验组1(溶液状态下添加双特异性抗体CD3-4-1BB BsAb_M10ng/ml)；实验组2(溶液状态下添加双特异性抗体CD3-4-1BB BsAb_D 10ng/ml)。此外，3组实验细胞同时添加细胞因子IFN-γ(200ng/ml，购自吴江近岸蛋白质科技有限公司)和IL-1β(2ng/ml，购自吴江近岸蛋白质科技有限公司)，置于培养箱，在饱和湿度、37℃、5.0％CO2的条件下进行培养。培养过夜后，添加500U/ml的IL-2(购自吴江近岸蛋白质科技有限公司)继续培养，每2-3天计数并用添加500U/ml IL-2的CIK基础培养基按1×106/ml的密度进行细胞传代。照此方法培养30天，最终统计细胞的扩增倍数，绘制生长曲线；
检测结果如图4所示，单体和二聚体形式的CD3-4-1BB双特异性抗体单一使用对CIK细胞的增殖效果均优于抗CD3/抗CD28单克隆全长抗体联合使用：培养18天后，Anti-CD3/Anti-CD28联用出现大量细胞死亡，细胞扩增倍数显著下降；而添加单体形式的CD3-4-1BB BsAb_M或二聚体形式的CD3-4-1BB BsAb_D均未出现细胞死亡，只是细胞扩增速度相对放缓。因此本发明制备的两种形式的CD3-4-1BB双特异性抗体均可有效扩增和延长CIK细胞的生存期，其中二聚体形式效果更好。
实施例5：CD3-4-1BB双特异抗体介导的CIK细胞增殖后表型检测CD8+/CD4+阳性细胞的流式检测
取实施例4所述培养30天后的3组实验细胞，分别进行Anti-CD4-FITC和Anti-CD8-PE(均购自Ebiosciense公司)抗体双重染色，流式细胞仪检测CD8+和CD4+阳性细胞数目，统计各自比例。
流式检测步骤：
1.取对照组细胞4份，其他2组(实验组1、实验组2)各取细胞1份，每份细胞数目1×106；
2. 1000rpm离心5min，弃上清，用200ul 2％BSA/PBS重悬细胞，离心清洗2次；
3.对照组的4份细胞分别添加5ul PBS、Anti-CD4-FITC、Anti-CD8-PE以及Anti-CD4-FITC和Anti-CD8-PE同时添加，其余2份细胞只同时添加Anti-CD4-FITC和Anti-CD8-PE，4℃孵育1h；
4.所有组处理的细胞均用PBS清洗两次，最后用100ul PBS重悬，流式细胞仪检测。
结果如图5所示：CD3-4-1BB BsAb_D介导CIK细胞增殖30天后，CD8+阳性细胞比例达到88.17％，CD3-4-1BB BsAb_M介导CIK细胞增殖30天后，CD8+阳性细胞比例为78.02％，均明显优于Anti-CD3/Anti-CD28联合(CD8+阳性细胞比例：包被使用48.47％)，说明两种形式的CD3-4-1BB双特异抗体比抗CD3/抗CD28单克隆全长抗体联合使用更有利于CD8+阳性细胞的生长扩增，其中二聚体较单体具有更好的效果。
实施例6：CD3-ICOS BsAb_M和CD3-ICOS BsAb_D真核表达载体的构建
在本发明中，以T细胞表面人类CD3蛋白和T细胞正共刺激分子ICOS蛋白为靶点的双特异性抗体被命名为CD3-ICOS BsAb。
一、CD3-ICOS BsAb_M和CD3-ICOS BsAb_D构建方案设计
单体形式的CD3-ICOS BsAb_M具体构建方案为：抗CD3 scFv和抗ICOS scFv序列之间通过(GGGGS)3 Linker相连。
二聚体形式的CD3-ICOS BsAb_D具体构建方案为：抗CD3 scFv和抗ICOS scFv序列之间通过IgD铰链区作为Linker相连。
为使双特异性抗体在哺乳细胞中进行表达，针对抗CD3 scFv，抗ICOS scFv及连接片段(Linker)序列均进行了哺乳系统表达的密码子优化。
具体地，抗CD3 scFv的重链可变区的核苷酸序列如SEQ ID NO.58所示。
具体地，抗CD3 scFv的轻链可变区的核苷酸序列如SEQ ID NO.59所示。
具体地，抗CD3 scFv的核苷酸序列如SEQ ID NO.57所示。
具体地，抗ICOS scFv的重链可变区的核苷酸序列如SEQ ID NO.64所示，具体为：
CAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCGCCAGCGTGAAGGTGAGCTGCAAGGCCAGCGGCTACACCTTCACCGGCTACTACATGCACTGGGTGCGCCAGGCCCCCGGCCAGGGCCTGGAGTGGATGGGCTGGATCAACCCCCACAGCGGCGGCACCAACTACGCCCAGAAGTTCCAGGGCCGCGTGACCATGACCCGCGACACCAGCATCAGCACCGCCTACATGGAGCTGAGCCGCCTGCGCAGCGACGACACCGCCGTGTACTACTGCGCCCGCACCTACTACTACGACAGCAGCGGCTACTACCACGACGCCTTCGACATCTGGGGCCAGGGCACCATGGTGACCGTGAGCAGC。
具体地，抗ICOS scFv的轻链可变区的核苷酸序列如SEQ ID NO.65所示，具体为：
GACATCCAGATGACCCAGAGCCCCAGCAGCGTGAGCGCCAGCGTGGGCGACCGCGTGACCATCACCTGCCGCGCCAGCCAGGGCATCAGCCGCCTGCTGGCCTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGTGGCCAGCAGCCTGCAGAGCGGCGTGCCCAGCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGGCCAACAGCTTCCCCTGGACCTTCGGCCAGGGCACCAAGGTGGAGATCAAG。
具体地，抗ICOS scFv的核苷酸序列如SEQ ID NO.63所示，具体为：
CAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCGCCAGCGTGAAGGTGAGCTGCAAGGCCAGCGGCTACACCTTCACCGGCTACTACATGCACTGGGTGCGCCAGGCCCCCGGCCAGGGCCTGGAGTGGATGGGCTGGATCAACCCCCACAGCGGCGGCACCAACTACGCCCAGAAGTTCCAGGGCCGCGTGACCATGACCCGCGACACCAGCATCAGCACCGCCTACATGGAGCTGAGCCGCCTGCGCAGCGACGACACCGCCGTGTACTACTGCGCCCGCACCTACTACTACGACAGCAGCGGCTACTACCACGACGCCTTCGACATCTGGGGCCAGGGCACCATGGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGACATCCAGATGACCCAGAGCCCCAGCAGCGTGAGCGCCAGCGTGGGCGACCGCGTGACCATCACCTGCCGCGCCAGCCAGGGCATCAGCCGCCTGCTGGCCTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGTGGCCAGCAGCCTGCAGAGCGGCGTGCCCAGCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGGCCAACAGCTTCCCCTGGACCTTCGGCCAGGGCACCAAGGTGGAGATCAAG。
单体形式的CD3-ICOS BsAb_M连接片段的核苷酸序列如SEQ ID NO.2所示。
二聚体形式的CD3-ICOS BsAb_D连接片段的核苷酸序列如SEQ ID NO.4所示。
为使双特异性抗体在CHO-S细胞中表达并成功分泌到培养基中，选择了抗体分泌型表达的信号肽用于此实施例。
该分泌表达信号肽的氨基酸序列如SEQ ID NO.78所示。
该分泌表达信号肽的核苷酸序列如SEQ ID NO.79所示。
二、CD3-ICOS BsAb_M和CD3-ICOS BsAb_D真核表达载体构建
本发明双特异性抗体的构建与表达，选用哺乳细胞蛋白瞬时表达载体pcDNA3.1(购自上海英骏生物科技有限公司)。为构建单体和二聚体形式的双特异性抗体，分别设计了如表2所示引物，所有引物由苏州金唯智生物科技有限公司合成，扩增所需基因模板由苏州鸿讯科技有限公司合成。
针对CD3-ICOS BsAb_M的克隆构建，首先使用引物pcDNA3.1-Sig-F和Sig-R扩增出信号肽片段，然后分别利用引物Sig-CD3-F和CD3-R、CD3-(GGGGS)3-ICOS-F和pcDNA3.1-ICOS-R扩增出抗CD3 scFv、(GGGGS)3 Linker、抗ICOS scFv的基因序列；针对CD3-ICOSBsAb_D的克隆构建，同样首先使用引物pcDNA3.1-Sig-F和Sig-R扩增出信号肽片段，然后分别利用引物Sig-CD3-F和CD3-R、CD3-IgD-F和IgD-R、IgD-ICOS-F和pcDNA3.1-ICOS-R扩增出抗CD3 scFv、IgD铰链区、抗ICOS scFv的基因序列。扩增完毕后，利用
PCR一步定向克隆试剂盒(购自吴江近岸蛋白质科技有限公司)分别拼接单体和二聚体形式双特异性抗体全长基因序列并无缝克隆至经EcoRI和HindIII线性化处理的pcDNA3.1表达载体上。目的载体转化大肠杆菌DH5α，利用菌落PCR进行阳性克隆鉴定，鉴定为阳性的重组子(重组质粒)进行测序鉴定。随后将测序正确的重组子(重组质粒)安排质粒中抽，用于CHO-S细胞的转染。
经测序获知，单体形式的CD3-ICOS BsAb_M和二聚体形式的CD3-ICOS BsAb_D的全长基因序列正确，均与预期相符。
具体地，单体形式的CD3-ICOS BsAb_M的核苷酸序列如SEQ ID NO.17所示，具体为：
GACATCAAGCTGCAGCAGAGCGGCGCCGAGCTGGCCCGCCCCGGCGCCAGCGTGAAGATGAGCTGCAAGACCAGCGGCTACACCTTCACCCGCTACACCATGCACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGGCTACATCAACCCCAGCCGCGGCTACACCAACTACAACCAGAAGTTCAAGGACAAGGCCACCCTGACCACCGACAAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCCGCTACTACGACGACCACTACTGCCTGGACTACTGGGGCCAGGGCACCACCCTGACCGTGAGCAGCGTGGAGGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCGTGGACGACATCCAGCTGACCCAGAGCCCCGCCATCATGAGCGCCAGCCCCGGCGAGAAGGTGACCATGACCTGCCGCGCCAGCAGCAGCGTGAGCTACATGAACTGGTACCAGCAGAAGAGCGGCACCAGCCCCAAGCGCTGGATCTACGACACCAGCAAGGTGGCCAGCGGCGTGCCCTACCGCTTCAGCGGCAGCGGCAGCGGCACCAGCTACAGCCTGACCATCAGCAGCATGGAGGCCGAGGACGCCGCCACCTACTACTGCCAGCAGTGGAGCAGCAACCCCCTGACCTTCGGCGCCGGCACCAAGCTGGAGCTGAAGGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCCAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCGCCAGCGTGAAGGTGAGCTGCAAGGCCAGCGGCTACACCTTCACCGGCTACTACATGCACTGGGTGCGCCAGGCCCCCGGCCAGGGCCTGGAGTGGATGGGCTGGATCAACCCCCACAGCGGCGGCACCAACTACGCCCAGAAGTTCCAGGGCCGCGTGACCATGACCCGCGACACCAGCATCAGCACCGCCTACATGGAGCTGAGCCGCCTGCGCAGCGACGACACCGCCGTGTACTACTGCGCCCGCACCTACTACTACGACAGCAGCGGCTACTACCACGACGCCTTCGACATCTGGGGCCAGGGCACCATGGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGACATCCAGATGACCCAGAGCCCCAGCAGCGTGAGCGCCAGCGTGGGCGACCGCGTGACCATCACCTGCCGCGCCAGCCAGGGCATCAGCCGCCTGCTGGCCTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGTGGCCAGCAGCCTGCAGAGCGGCGTGCCCAGCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGGCCAACAGCTTCCCCTGGACCTTCGGCCAGGGCACCAAGGTGGAGATCAAG。
具体地，二聚体形式的CD3-ICOS BsAb_D的核苷酸序列如SEQ ID NO.19所示，具体为：
GACATCAAGCTGCAGCAGAGCGGCGCCGAGCTGGCCCGCCCCGGCGCCAGCGTGAAGATGAGCTGCAAGACCAGCGGCTACACCTTCACCCGCTACACCATGCACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGGCTACATCAACCCCAGCCGCGGCTACACCAACTACAACCAGAAGTTCAAGGACAAGGCCACCCTGACCACCGACAAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCCGCTACTACGACGACCACTACTGCCTGGACTACTGGGGCCAGGGCACCACCCTGACCGTGAGCAGCGTGGAGGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCGTGGACGACATCCAGCTGACCCAGAGCCCCGCCATCATGAGCGCCAGCCCCGGCGAGAAGGTGACCATGACCTGCCGCGCCAGCAGCAGCGTGAGCTACATGAACTGGTACCAGCAGAAGAGCGGCACCAGCCCCAAGCGCTGGATCTACGACACCAGCAAGGTGGCCAGCGGCGTGCCCTACCGCTTCAGCGGCAGCGGCAGCGGCACCAGCTACAGCCTGACCATCAGCAGCATGGAGGCCGAGGACGCCGCCACCTACTACTGCCAGCAGTGGAGCAGCAACCCCCTGACCTTCGGCGCCGGCACCAAGCTGGAGCTGAAGGCCAGCAAGAGCAAGAAGGAGATCTTCCGCTGGCCCGAGAGCCCCAAGGCCCAGGCCAGCAGCGTGCCCACCGCCCAGCCCCAGGCCGAGGGCAGCCTGGCCAAGGCCACCACCGCCCCCGCCACCACCCGCAACACCGGCCGCGGCGGCGAGGAGAAGAAGAAGGAGAAGGAGAAGGAGGAGCAGGAGGAGCGCGAGACCAAGACCCCCGAGTGCCCCAGCCACACCCAGCCCCTGGGCGTGCAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCGCCAGCGTGAAGGTGAGCTGCAAGGCCAGCGGCTACACCTTCACCGGCTACTACATGCACTGGGTGCGCCAGGCCCCCGGCCAGGGCCTGGAGTGGATGGGCTGGATCAACCCCCACAGCGGCGGCACCAACTACGCCCAGAAGTTCCAGGGCCGCGTGACCATGACCCGCGACACCAGCATCAGCACCGCCTACATGGAGCTGAGCCGCCTGCGCAGCGACGACACCGCCGTGTACTACTGCGCCCGCACCTACTACTACGACAGCAGCGGCTACTACCACGACGCCTTCGACATCTGGGGCCAGGGCACCATGGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGACATCCAGATGACCCAGAGCCCCAGCAGCGTGAGCGCCAGCGTGGGCGACCGCGTGACCATCACCTGCCGCGCCAGCCAGGGCATCAGCCGCCTGCTGGCCTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGTGGCCAGCAGCCTGCAGAGCGGCGTGCCCAGCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGGCCAACAGCTTCCCCTGGACCTTCGGCCAGGGCACCAAGGTGGAGATCAAG。
表2.CD3-ICOS双特异性抗体基因克隆中使用的引物
实施例7：CD3-ICOS BsAb_M和CD3-ICOS BsAb_D的表达与纯化
一、CD3-ICOS BsAb_M和CD3-ICOS BsAb_D的表达
1.1.CHO-S细胞(购自Thermo Fisher Scientific公司)转染前1天传代密度为0.5～0.6×106/ml；
1.2.转染当天进行细胞密度统计，当密度为1～1.4×106/ml、活力>90％时，可用于质粒转染；
1.3.转染复合物配制：每个项目(CD3-ICOS BsAb_M和CD3-ICOS BsAb_D)需准备两个离心管/培养瓶，以20ml为例，分别放置，取实施例6中所制备重组质粒：
管①中加入600μl PBS，20μg重组质粒，混匀；
管②中加入600μl PBS，20ul FreeStyleTM MAX Transfection Reagent(购自Thermo Fisher Scientific公司)，混匀；
1.4.将稀释后的转染试剂，加入至稀释后的重组质粒中，混合均匀，配制成转染复合物；
1.5.转染复合物静置15～20min后，单滴匀速加入细胞培养物中；
1.6.于37℃，CO2浓度8％，摇床转速130rpm条件下进行转染后细胞培养，5天后收集培养上清进行目的蛋白表达检测
二、CD3-ICOS BsAb_M和CD3-ICOS BsAb_D的纯化
2.1样品预处理
取上述转染后细胞培养上清20ml，加入缓冲液20mM PB，200mM NaCl调节pH至7.5；
2.2 Protein L亲和层析柱纯化
蛋白纯化层析柱：Protein L亲和层析柱(购自GE Healthcare公司，柱体积1.0ml)
缓冲液A(Buffer A)：PBS，pH7.4
缓冲液B(Buffer B)：0.1M Glycine,pH3.0
缓冲液C(Buffer C)：0.1M Glycine,pH2.7
纯化过程：采用AKTA explorer 100型蛋白纯化系统(购自GE Healthcare公司)，用Buffer A预处理Protein L亲和层析柱，取培养上清上样，收集流出液。上样完毕后，用至少1.5ml Buffer A平衡层析柱，平衡后分别用Buffer B和Buffer C洗脱，收集目的蛋白洗脱液(洗脱液的收集管需要预先加入1％的1M Tris,pH8.0来中和洗脱液pH值，Tris终浓度约为10mM)，最后浓缩透析至缓冲液PBS中。
最终纯化的CD3-ICOS BsAb_M和CD3-ICOS BsAb_D重组蛋白经SDS-PAGE分析，还原和非还原条件下电泳图如图6所示。从图中可以看出，经Protein L亲和层析柱纯化后，CD3-ICOS BsAb_M和CD3-ICOS BsAb_D重组蛋白的纯度均>95％；其中CD3-ICOS BsAb_M重组蛋白的理论分子量为53.8kDa，还原和非还原条件下该蛋白均呈现单一电泳条带，分子量与单体一致，因此该双特异性抗体为单体形式(图6A)；CD3-ICOS BsAb_D重组蛋白的理论分子量为61.7kDa，还原条件下该蛋白电泳条带所呈现分子量与单体一致，非还原条件下电泳条带所呈现分子量与二聚体一致(图6B)，说明两个蛋白分子可通过二硫键相互连接，因此该双特异性抗体为二聚体形式。
此外，纯化的重组蛋白样品经N/C末端序列分析，结果表明所表达的重组蛋白样品均读框无误，与理论N/C末端氨基酸序列一致，质谱分析进一步确认CD3-ICOS BsAb_M为单体形式，CD3-ICOS BsAb_D为二聚体形式。
因此，可得知，单体形式的CD3-ICOS BsAb_M的氨基酸序列如SEQ ID NO.16所示，具体为：
DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPHSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARTYYYDSSGYYHDAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQGISRLLAWYQQKPGKAPKLLIYVASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPWTFGQGTKVEIK。
二聚体形式的CD3-ICOS BsAb_D的氨基酸序列如SEQ ID NO.18所示，具体为：
DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKASKSKKEIFRWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEKEKEEQEERETKTPECPSHTQPLGVQVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPHSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARTYYYDSSGYYHDAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQGISRLLAWYQQKPGKAPKLLIYVASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPWTFGQGTKVEIK。
抗CD3 scFv的氨基酸序列如SEQ ID NO.36所示。
抗CD3 scFv的重链可变区的氨基酸序列如SEQ ID NO.37所示。
抗CD3 scFv的轻链可变区的氨基酸序列如SEQ ID NO.38所示。
抗ICOS scFv的氨基酸序列如SEQ ID NO.42所示，具体为：
QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPHSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARTYYYDSSGYYHDAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQGISRLLAWYQQKPGKAPKLLIYVASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPWTFGQGTKVEIK。
抗ICOS scFv的重链可变区的氨基酸序列如SEQ ID NO.43所示，具体为：
QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPHSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARTYYYDSSGYYHDAFDIWGQGTMVTVSS。
抗ICOS scFv的轻链可变区的氨基酸序列如SEQ ID NO.44所示，具体为：
DIQMTQSPSSVSASVGDRVTITCRASQGISRLLAWYQQKPGKAPKLLIYVASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPWTFGQGTKVEIK。
单体形式的CD3-ICOS BsAb_M中连接片段的氨基酸序列如SEQ ID NO.1所示。
二聚体形式的CD3-ICOS BsAb_D中连接片段的氨基酸序列如SEQ ID NO.3所示。
实施例8：ELISA检测CD3-ICOS BsAb_M和CD3-ICOS BsAb_D的抗原结合活性
ELISA操作步骤：
1.重组抗原包被：人类CD3-hFc与人类ICOS-hFc融合蛋白(购自吴江近岸蛋白质科技有限公司)分别包被96孔板，抗原浓度为1μg/ml，包被体积为100μl/孔，包被条件为37℃1小时或4℃过夜，包被缓冲液(PBS)的配方为：3.58g Na2HPO4，0.24g NaH2PO4，0.2g KCl，8.2g NaCl，950ml H2O，用1mol/L HCl或1mol/L NaOH调pH至7.4，补水至1L；
2.封闭：PBS洗板4次后，加入封闭液PBSA(PBS+2％BSA(V/W))，200μl/孔。37℃封闭1小时；
3.加样：PBS洗板4次后，分别加入纯化的双特异性抗体样品，100μl/孔，37℃孵育1小时，样品梯度配制方法：以10μg/ml纯化的CD3-ICOS BsAb_M或CD3-ICOS BsAb_D作为起始浓度，进行倍比稀释6个梯度，每个梯度设置2个复孔；
4.显色：PBST(PBS+0.05％Tween-20(V/V))洗板4次后，用封闭液PBSA按1/5000稀释HRP标记的显色抗体(购自Abcam公司)，按100μl/孔加入，37℃孵育1小时。PBS洗板4次后，添加显色液TMB(购自KPL公司)，100μl/孔，室温避光显色5～10分钟；
5.终止反应与结果测定：添加终止液(1M HCl)，100μl/孔，在酶标仪上450nm波长下读取吸光值。
ELISA结果如图7A和图7B所示：图7A说明CD3-ICOS BsAb_M与重组抗原CD3-hFc和ICOS-hFc均具有体外结合活性，其中ICOS结合活性较CD3结合活性更高；图7B说明CD3-ICOSBsAb_D与重组抗原CD3-hFc和ICOS-hFc同样具有体外结合活性，其中ICOS结合活性更高。
实施例9：CD3-ICOS双特异抗体介导的CIK细胞增殖
以人外周血单个核细胞(Peripheral blood mononuclear cell，PBMC)为实验材料，用本发明所制备的上述单体形式的双特异抗体CD3-ICOS BsAb_M、二聚体形式的双特异抗体CD3-ICOS BsAb_D、以及抗CD3/抗CD28单克隆全长抗体联合使用(Anti-CD3/Anti-CD28)分别作用于同一供体来源的人血PBMC，细胞培养后进行计数，比较扩增倍数。
1.PBMC的分离：取抗凝血，加入等体积的医用生理盐水，沿离心管壁缓慢加入与血液等体积的淋巴细胞分离液(购自GE Healthcare公司)，保持液面分层明显，2000rpm离心20min，吸取中间白雾状的细胞层于新的离心管中，加入2倍以上体积的PBS缓冲液洗涤，1100rpm离心10min，重复洗涤一次，用少量预冷的X-vivo 15无血清培养基(购自Lonza公司)重悬，细胞计数待用；
2.CIK细胞培养与扩增：将PBMC用CIK基础培养基(90％X-vivo15+10％FBS)重悬，调整细胞密度为1×106/ml，分别设计以下3个实验组：对照组(Anti-CD3 5ug/ml和Anti-CD28 5ug/ml包被细胞培养板)；实验组1(溶液状态下添加双特异性抗体CD3-ICOS BsAb_M10ng/ml)；实验组2(溶液状态下添加双特异性抗体CD3-ICOS BsAb_D 10ng/ml)。此外，3组实验细胞同时添加细胞因子IFN-γ(200ng/ml，购自吴江近岸蛋白质科技有限公司)和IL-1β(2ng/ml，购自吴江近岸蛋白质科技有限公司)，置于培养箱，在饱和湿度、37℃、5.0％CO2的条件下进行培养。培养过夜后，添加500U/ml的IL-2(购自吴江近岸蛋白质科技有限公司)继续培养，每2-3天计数并用添加500U/ml IL-2的CIK基础培养基按1×106/ml的密度进行细胞传代。照此方法培养14天，最终统计细胞的扩增倍数，绘制生长曲线；
检测结果如表3和图8所示，单体和二聚体形式的CD3-ICOS双特异性抗体单一使用对CIK细胞的增殖效果均不同程度地优于抗CD3/抗CD28单克隆全长抗体联合使用，且蛋白用量更少(10ng/ml vs 5ug/ml)：其中二聚体形式的CD3-ICOS BsAb_D可以介导CIK细胞两周扩增352倍，效果最优(实验组2)；单体形式的CD3-ICOS BsAb_M可以介导CIK细胞两周扩增298倍，效果次之(实验组1)；Anti CD3/Anti CD28联用可以介导CIK细胞两周扩增224倍，效果最弱(对照组)。
表3 CIK细胞扩增倍数
| 实验组名 | 对照组 | 实验组1 | 实验组2 |
| 14天扩增倍数 | 224 | 298 | 352 |
实施例10：CD3-ICOS双特异抗体介导的CIK细胞增殖后表型检测CD3+/CD56+双阳性CIK细胞的流式检测
取实施例9所述培养14天后的3组实验细胞，分别进行Anti-CD3-FITC和Anti-CD56-PE(均购自Ebiosciense公司)抗体双重染色，流式细胞仪检测CD3+CD56+双阳性细胞比例。流式检测步骤：
1.1取对照组细胞4份，其他2组(实验组1、实验组2)各取细胞1份，每份细胞数目1×106；
1.2 1000rpm离心5min，弃上清，用200ul 2％BSA/PBS重悬细胞，离心清洗2次；
1.3对照组的4份细胞分别添加5ul PBS、Anti-CD3-FITC、Anti-CD56-PE以及Anti-CD3-FITC和Anti-CD56-PE同时添加，其余2份细胞只同时添加Anti-CD3-FITC和Anti-CD56-PE，4℃孵育1h；
1.4所有组处理的细胞均用PBS清洗两次，最后用100ul PBS重悬，流式细胞仪检测。
结果如图9所示：其中Anti-CD3/Anti-CD28联用介导CIK细胞增殖14天后，CD3+CD56+双阳性的细胞比例为12.9％(图9A)；单体形式的CD3-ICOS BsAb_M介导CIK细胞增殖14天后，CD3+CD56+双阳性的细胞比例为24.18％(图9B)；二聚体形式的CD3-ICOS BsAb_D介导CIK细胞增殖14天后，CD3+CD56+双阳性的细胞比例为39.71％(图9C)，说明单体和二聚体形式CD3-ICOS双特异抗体均可用于替代抗CD3/抗CD28两种全长抗体联合使用，且介导的CIK细胞在增殖后具有更高的CD3+CD56+双阳性比例，其中二聚体较单体具有更好的效果。
实施例11：CD3-OX40 BsAb_M和CD3-OX40 BsAb_D真核表达载体的构建
在本发明中，以T细胞表面人类CD3蛋白和T细胞正共刺激分子OX40蛋白为靶点的双特异性抗体被命名为CD3-OX40 BsAb。
一、CD3-OX40 BsAb_M和CD3-OX40 BsAb_D构建方案设计
单体形式的CD3-OX40 BsAb_M具体构建方案为：抗CD3 scFv和抗OX40 scFv序列之间通过(GGGGS)3 Linker相连。
二聚体形式的CD3-OX40 BsAb_D具体构建方案为：抗CD3 scFv和抗OX40 scFv序列之间通过IgD铰链区作为Linker相连。
为使双特异性抗体在哺乳细胞中进行表达，针对抗CD3 scFv，抗OX40 scFv及连接片段(Linker)序列均进行了哺乳系统表达的密码子优化。
具体地，抗CD3 scFv的重链可变区的核苷酸序列如SEQ ID NO.58所示。
具体地，抗CD3 scFv的轻链可变区的核苷酸序列如SEQ ID NO.59所示。
具体地，抗CD3 scFv的核苷酸序列如SEQ ID NO.57所示。
具体地，抗OX40 scFv的重链可变区的核苷酸序列如SEQ ID NO.67所示，具体为：CAGCTGGTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCTGCGCCTGAGCTGCGCCGCCAGCGGCTTCACCTTCAGCAGCTACAGCATGAACTGGGTGCGCCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGAGCTACATCAGCAGCAGCAGCAGCACCATCTACTACGCCGACAGCGTGAAGGGCCGCTTCACCATCAGCCGCGACAACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGCGCGACGAGGACACCGCCGTGTACTACTGCGCCCGCGGCGTGTACCACAACGGCTGGAGCTTCTTCGACTACTGGGGCCAGGGCACCCTGCTGACCGTGAGCAGC。
具体地，抗OX40 scFv的轻链可变区的核苷酸序列如SEQ ID NO.68所示，具体为：GACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCAACCGCGTGACCATCACCTGCCGCGCCAGCCAGGACATCAGCAGCTGGCTGGCCTGGTACCAGCAGAAGCCCGAGAAGGCCCCCAAGAGCCTGATCTACGCCGCCAGCAGCCTGCAGAGCGGCGTGCCCAGCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGTACAACAGCTACCCCCTGACCTTCGGCCAGGGCACCCGCCTGGAGATCAAGCGC。
具体地，抗OX40 scFv的核苷酸序列如SEQ ID NO.66所示，具体为：
CAGCTGGTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCTGCGCCTGAGCTGCGCCGCCAGCGGCTTCACCTTCAGCAGCTACAGCATGAACTGGGTGCGCCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGAGCTACATCAGCAGCAGCAGCAGCACCATCTACTACGCCGACAGCGTGAAGGGCCGCTTCACCATCAGCCGCGACAACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGCGCGACGAGGACACCGCCGTGTACTACTGCGCCCGCGGCGTGTACCACAACGGCTGGAGCTTCTTCGACTACTGGGGCCAGGGCACCCTGCTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCAACCGCGTGACCATCACCTGCCGCGCCAGCCAGGACATCAGCAGCTGGCTGGCCTGGTACCAGCAGAAGCCCGAGAAGGCCCCCAAGAGCCTGATCTACGCCGCCAGCAGCCTGCAGAGCGGCGTGCCCAGCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGTACAACAGCTACCCCCTGACCTTCGGCCAGGGCACCCGCCTGGAGATCAAGCGC。
单体形式的CD3-OX40 BsAb_M连接片段的核苷酸序列如SEQ ID NO.2所示。
二聚体形式的CD3-OX40 BsAb_D连接片段的核苷酸序列如SEQ ID NO.4所示。
为使双特异性抗体在CHO-S细胞中表达并成功分泌到培养基中，选择了抗体分泌型表达的信号肽用于此实施例。
该分泌表达信号肽的氨基酸序列如SEQ ID NO.78所示。
该分泌表达信号肽的核苷酸序列如SEQ ID NO.79所示。
二、CD3-OX40 BsAb_M和CD3-OX40 BsAb_D真核表达载体构建
本发明双特异性抗体的构建与表达，选用哺乳细胞蛋白瞬时表达载体pcDNA3.1(购自上海英骏生物科技有限公司)。为构建单体和二聚体形式的双特异性抗体，分别设计了如表4所示引物，所有引物由苏州金唯智生物科技有限公司合成，扩增所需基因模板由苏州鸿讯科技有限公司合成。
针对CD3-OX40 BsAb_M的克隆构建，首先使用引物pcDNA3.1-Sig-F和Sig-R扩增出信号肽片段，然后分别利用引物Sig-CD3-F和CD3-R、CD3-(GGGGS)3-OX40-F和pcDNA3.1-OX40-R扩增出抗CD3 scFv、(GGGGS)3 Linker、抗OX40 scFv的基因序列；针对CD3-OX40BsAb_D的克隆构建，同样首先使用引物pcDNA3.1-Sig-F和Sig-R扩增出信号肽片段，然后分别利用引物Sig-CD3-F和CD3-R、CD3-IgD-F和IgD-R、IgD-OX40-F和pcDNA3.1-OX40-R扩增出抗CD3 scFv、IgD铰链区、抗OX40 scFv的基因序列。扩增完毕后，利用
PCR一步定向克隆试剂盒(购自吴江近岸蛋白质科技有限公司)分别拼接单体和二聚体形式双特异性抗体全长基因序列并无缝克隆至经EcoRI和HindIII线性化处理的pcDNA3.1表达载体上。目的载体转化大肠杆菌DH5α，利用菌落PCR进行阳性克隆鉴定，鉴定为阳性的重组子(重组质粒)进行测序鉴定。随后将测序正确的重组子(重组质粒)安排质粒中抽，用于CHO-S细胞的转染。
经测序获知，单体形式的CD3-OX40 BsAb_M和二聚体形式的CD3-OX40 BsAb_D的全长基因序列正确，均与预期相符。
具体地，单体形式的CD3-OX40 BsAb_M的核苷酸序列如SEQ ID NO.21所示，具体为：
GACATCAAGCTGCAGCAGAGCGGCGCCGAGCTGGCCCGCCCCGGCGCCAGCGTGAAGATGAGCTGCAAGACCAGCGGCTACACCTTCACCCGCTACACCATGCACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGGCTACATCAACCCCAGCCGCGGCTACACCAACTACAACCAGAAGTTCAAGGACAAGGCCACCCTGACCACCGACAAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCCGCTACTACGACGACCACTACTGCCTGGACTACTGGGGCCAGGGCACCACCCTGACCGTGAGCAGCGTGGAGGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCGTGGACGACATCCAGCTGACCCAGAGCCCCGCCATCATGAGCGCCAGCCCCGGCGAGAAGGTGACCATGACCTGCCGCGCCAGCAGCAGCGTGAGCTACATGAACTGGTACCAGCAGAAGAGCGGCACCAGCCCCAAGCGCTGGATCTACGACACCAGCAAGGTGGCCAGCGGCGTGCCCTACCGCTTCAGCGGCAGCGGCAGCGGCACCAGCTACAGCCTGACCATCAGCAGCATGGAGGCCGAGGACGCCGCCACCTACTACTGCCAGCAGTGGAGCAGCAACCCCCTGACCTTCGGCGCCGGCACCAAGCTGGAGCTGAAGGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCTGCGCCTGAGCTGCGCCGCCAGCGGCTTCACCTTCAGCAGCTACAGCATGAACTGGGTGCGCCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGAGCTACATCAGCAGCAGCAGCAGCACCATCTACTACGCCGACAGCGTGAAGGGCCGCTTCACCATCAGCCGCGACAACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGCGCGACGAGGACACCGCCGTGTACTACTGCGCCCGCGGCGTGTACCACAACGGCTGGAGCTTCTTCGACTACTGGGGCCAGGGCACCCTGCTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCAACCGCGTGACCATCACCTGCCGCGCCAGCCAGGACATCAGCAGCTGGCTGGCCTGGTACCAGCAGAAGCCCGAGAAGGCCCCCAAGAGCCTGATCTACGCCGCCAGCAGCCTGCAGAGCGGCGTGCCCAGCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGTACAACAGCTACCCCCTGACCTTCGGCCAGGGCACCCGCCTGGAGATCAAGCGC。
具体地，二聚体形式的CD3-OX40 BsAb_D的核苷酸序列如SEQ ID NO.23所示，具体为：
GACATCAAGCTGCAGCAGAGCGGCGCCGAGCTGGCCCGCCCCGGCGCCAGCGTGAAGATGAGCTGCAAGACCAGCGGCTACACCTTCACCCGCTACACCATGCACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGGCTACATCAACCCCAGCCGCGGCTACACCAACTACAACCAGAAGTTCAAGGACAAGGCCACCCTGACCACCGACAAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCCGCTACTACGACGACCACTACTGCCTGGACTACTGGGGCCAGGGCACCACCCTGACCGTGAGCAGCGTGGAGGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCGTGGACGACATCCAGCTGACCCAGAGCCCCGCCATCATGAGCGCCAGCCCCGGCGAGAAGGTGACCATGACCTGCCGCGCCAGCAGCAGCGTGAGCTACATGAACTGGTACCAGCAGAAGAGCGGCACCAGCCCCAAGCGCTGGATCTACGACACCAGCAAGGTGGCCAGCGGCGTGCCCTACCGCTTCAGCGGCAGCGGCAGCGGCACCAGCTACAGCCTGACCATCAGCAGCATGGAGGCCGAGGACGCCGCCACCTACTACTGCCAGCAGTGGAGCAGCAACCCCCTGACCTTCGGCGCCGGCACCAAGCTGGAGCTGAAGGCCAGCAAGAGCAAGAAGGAGATCTTCCGCTGGCCCGAGAGCCCCAAGGCCCAGGCCAGCAGCGTGCCCACCGCCCAGCCCCAGGCCGAGGGCAGCCTGGCCAAGGCCACCACCGCCCCCGCCACCACCCGCAACACCGGCCGCGGCGGCGAGGAGAAGAAGAAGGAGAAGGAGAAGGAGGAGCAGGAGGAGCGCGAGACCAAGACCCCCGAGTGCCCCAGCCACACCCAGCCCCTGGGCGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCTGCGCCTGAGCTGCGCCGCCAGCGGCTTCACCTTCAGCAGCTACAGCATGAACTGGGTGCGCCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGAGCTACATCAGCAGCAGCAGCAGCACCATCTACTACGCCGACAGCGTGAAGGGCCGCTTCACCATCAGCCGCGACAACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGCGCGACGAGGACACCGCCGTGTACTACTGCGCCCGCGGCGTGTACCACAACGGCTGGAGCTTCTTCGACTACTGGGGCCAGGGCACCCTGCTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCAACCGCGTGACCATCACCTGCCGCGCCAGCCAGGACATCAGCAGCTGGCTGGCCTGGTACCAGCAGAAGCCCGAGAAGGCCCCCAAGAGCCTGATCTACGCCGCCAGCAGCCTGCAGAGCGGCGTGCCCAGCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGTACAACAGCTACCCCCTGACCTTCGGCCAGGGCACCCGCCTGGAGATCAAGCGC。
表4.CD3-OX40双特异性抗体基因克隆中使用的引物
实施例12：CD3-OX40 BsAb_M和CD3-OX40 BsAb_D的表达与纯化
一、CD3-OX40 BsAb_M和CD3-OX40 BsAb_D的表达
1.1.CHO-S细胞(购自Thermo Fisher Scientific公司)转染前1天传代密度为0.5～0.6×106/ml；
1.2.转染当天进行细胞密度统计，当密度为1～1.4×106/ml、活力>90％时，可用于质粒转染；
1.3.转染复合物配制：每个项目(CD3-OX40 BsAb_M和CD3-OX40 BsAb_D)需准备两个离心管/培养瓶，以20ml为例，分别放置，取实施例11中所制备重组质粒：
管①中加入600μl PBS，20μg重组质粒，混匀；
管②中加入600μl PBS，20ul FreeStyleTM MAX Transfection Reagent(购自Thermo Fisher Scientific公司)，混匀；
1.4.将稀释后的转染试剂，加入至稀释后的重组质粒中，混合均匀，配制成转染复合物；
1.5.转染复合物静置15～20min后，单滴匀速加入细胞培养物中；
1.6.于37℃，CO2浓度8％，摇床转速130rpm条件下进行转染后细胞培养，5天后收集培养上清进行目的蛋白表达检测。
二、CD3-OX40 BsAb_M和CD3-OX40 BsAb_D的纯化
2.1样品预处理
取上述转染后细胞培养上清20ml，加入缓冲液20mM PB，200mM NaCl调节pH至7.5；
2.2 Protein L亲和层析柱纯化
蛋白纯化层析柱：Protein L亲和层析柱(购自GE Healthcare公司，柱体积1.0ml)
缓冲液A(Buffer A)：PBS，pH7.4
缓冲液B(Buffer B)：0.1M Glycine,pH3.0
缓冲液C(Buffer C)：0.1M Glycine,pH2.7
纯化过程：采用AKTA explorer 100型蛋白纯化系统(购自GE Healthcare公司)，用Buffer A预处理Protein L亲和层析柱，取培养上清上样，收集流出液。上样完毕后，用至少1.5ml Buffer A平衡层析柱，平衡后分别用Buffer B和Buffer C洗脱，收集目的蛋白洗脱液(洗脱液的收集管需要预先加入1％的1M Tris,pH8.0来中和洗脱液pH值，Tris终浓度约为10mM)，最后浓缩透析至缓冲液PBS中。
最终纯化的CD3-OX40 BsAb_M和CD3-OX40 BsAb_D重组蛋白经SDS-PAGE分析，还原和非还原条件下电泳图如图10所示。从图中可以看出，经Protein L亲和层析柱纯化后，CD3-OX40 BsAb_M和CD3-OX40 BsAb_D重组蛋白的纯度均>95％；其中CD3-OX40 BsAb_M重组蛋白的理论分子量为53.2kDa，还原和非还原条件下该蛋白均呈现单一电泳条带，分子量与单体一致，因此该双特异性抗体为单体形式(图10A)；CD3-OX40 BsAb_D重组蛋白的理论分子量为61.1kDa，还原条件下该蛋白电泳条带所呈现分子量与单体一致，非还原条件下电泳条带所呈现分子量与二聚体一致(图10B)，说明两个蛋白分子可通过二硫键相互连接，因此该双特异性抗体为二聚体形式。
此外，纯化的重组蛋白样品经N/C末端序列分析，结果表明所表达的重组蛋白样品均读框无误，与理论N/C末端氨基酸序列一致，质谱分析进一步确认CD3-OX40 BsAb_M为单体形式，CD3-OX40 BsAb_D为二聚体形式。
因此，可得知，单体形式的CD3-OX40 BsAb_M的氨基酸序列如SEQ ID NO.20所示，具体为：
DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKGGGGSGGGGSGGGGSQLVESGGGLVQPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSYISSSSSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCARGVYHNGWSFFDYWGQGTLLTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGNRVTITCRASQDISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPLTFGQGTRLEIKR。
二聚体形式的CD3-OX40 BsAb_D的氨基酸序列如SEQ ID NO.22所示，具体为：
DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKASKSKKEIFRWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEKEKEEQEERETKTPECPSHTQPLGVQLVESGGGLVQPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSYISSSSSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCARGVYHNGWSFFDYWGQGTLLTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGNRVTITCRASQDISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPLTFGQGTRLEIKR。
抗CD3 scFv的氨基酸序列如SEQ ID NO.36所示。
抗CD3 scFv的重链可变区的氨基酸序列如SEQ ID NO.37所示。
抗CD3 scFv的轻链可变区的氨基酸序列如SEQ ID NO.38所示。
抗OX40 scFv的氨基酸序列如SEQ ID NO:45所示，具体为：
QLVESGGGLVQPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSYISSSSSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCARGVYHNGWSFFDYWGQGTLLTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGNRVTITCRASQDISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPLTFGQGTRLEIKR。
抗OX40 scFv的重链可变区的氨基酸序列如SEQ ID NO:46所示，具体为：
QLVESGGGLVQPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSYISSSSSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCARGVYHNGWSFFDYWGQGTLLTVSS。
抗OX40 scFv的轻链可变区的氨基酸序列如SEQ ID NO:47所示，具体为：
DIQMTQSPSSLSASVGNRVTITCRASQDISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPLTFGQGTRLEIKR。
单体形式的CD3-OX40 BsAb_M中连接片段的氨基酸序列如SEQ ID NO.1所示。
二聚体形式的CD3-OX40 BsAb_D中连接片段的氨基酸序列如SEQ ID NO.3所示。
实施例13：ELISA检测CD3-OX40 BsAb_M和CD3-OX40 BsAb_D的抗原结合活性
ELISA操作步骤：
1.重组抗原包被：人类CD3-hFc与人类OX40-hFc融合蛋白(购自吴江近岸蛋白质科技有限公司)分别包被96孔板，抗原浓度为1μg/ml，包被体积为100μl/孔，包被条件为37℃1小时或4℃过夜，包被缓冲液(PBS)的配方为：3.58g Na2HPO4，0.24g NaH2PO4，0.2g KCl，8.2g NaCl，950ml H2O，用1mol/L HCl或1mol/L NaOH调pH至7.4，补水至1L；
2.封闭：PBS洗板4次后，加入封闭液PBSA(PBS+2％BSA(V/W))，200μl/孔。37℃封闭1小时；
3.加样：PBS洗板4次后，分别加入纯化的双特异性抗体样品，100μl/孔，37℃孵育1小时，样品梯度配制方法：以10μg/ml纯化的CD3-OX40 BsAb_M或CD3-OX40 BsAb_D作为起始浓度，进行倍比稀释6个梯度，每个梯度设置2个复孔；
4.显色：PBST(PBS+0.05％Tween-20(V/V))洗板4次后，用封闭液PBSA按1/5000稀释HRP标记的显色抗体(购自Abcam公司)，按100μl/孔加入，37℃孵育1小时。PBS洗板4次后，添加显色液TMB(购自KPL公司)，100μl/孔，室温避光显色5～10分钟；
5.终止反应与结果测定：添加终止液(1M HCl)，100μl/孔，在酶标仪上450nm波长下读取吸光值。
ELISA结果如图11A和图11B所示：图11A说明CD3-OX40 BsAb_M与重组抗原CD3-hFc和OX40-hFc均具有体外结合活性，其中OX40结合活性较CD3结合活性更高；图11B说明CD3-OX40 BsAb_D与重组抗原CD3-hFc和OX40-hFc同样具有体外结合活性，其中OX40结合活性更高。
实施例14：CD3-OX40双特异抗体介导的CIK细胞增殖
以人外周血单个核细胞(Peripheral blood mononuclear cell，PBMC)为实验材料，用本发明所制备的上述单体形式的双特异抗体CD3-OX40 BsAb_M、二聚体形式的双特异抗体CD3-OX40 BsAb_D、以及抗CD3/抗CD28单克隆全长抗体联用(Anti-CD3/Anti-CD28)分别作用于同一供体来源的人血PBMC，细胞培养后进行计数，比较扩增倍数。
1.PBMC的分离：取抗凝血，加入等体积的医用生理盐水，沿离心管壁缓慢加入与血液等体积的淋巴细胞分离液(购自GE Healthcare公司)，保持液面分层明显，2000rpm离心20min，吸取中间白雾状的细胞层于新的离心管中，加入2倍以上体积的PBS缓冲液洗涤，1100rpm离心10min，重复洗涤一次，用少量预冷的X-vivo 15无血清培养基(购自Lonza公司)重悬，细胞计数待用；
2.CIK细胞培养与扩增：将PBMC用CIK基础培养基(90％X-vivo15+10％FBS)重悬，调整细胞密度为1×106/ml，分别设计以下3个实验组：对照组(Anti-CD3 5ug/ml和Anti-CD28 5ug/ml包被细胞培养板，全长抗体均购自吴江近岸蛋白质科技有限公司)；实验组1(溶液状态下添加双特异性抗体CD3-OX40 BsAb_M 10ng/ml)；实验组2(溶液状态下添加双特异性抗体CD3-OX40 BsAb_D 10ng/ml)。此外，3组实验细胞同时添加细胞因子IFN-γ(200ng/ml，购自吴江近岸蛋白质科技有限公司)和IL-1β(2ng/ml，购自吴江近岸蛋白质科技有限公司)，置于培养箱，在饱和湿度、37℃、5.0％CO2的条件下进行培养。培养过夜后，添加500U/ml的IL-2(购自吴江近岸蛋白质科技有限公司)继续培养，每2-3天计数并用添加500U/ml IL-2的CIK基础培养基按1×106/ml的密度进行细胞传代。照此方法培养30天，最终统计细胞的扩增倍数，绘制生长曲线。
检测结果如图12所示，单体和二聚体形式的CD3-OX40双特异性抗体单一使用对CIK细胞的增殖效果均优于抗CD3/抗CD28单克隆全长抗体联合使用：培养18天后，Anti-CD3/Anti-CD28联用出现大量细胞死亡，细胞扩增倍数显著下降；而添加单体形式的CD3-OX40 BsAb_M或二聚体形式的CD3-OX40 BsAb_D均未出现细胞死亡，只是细胞扩增速度相对放缓，因此本发明制备的两种形式的CD3-OX40双特异性抗体均可有效扩增和延长CIK细胞的生存期，其中二聚体较单体效果稍好。
实施例15：添加CD3-OX40双特异抗体的CIK细胞对肿瘤细胞杀伤活性检测
分别取实施例14所述培养14天或30天后的3组实验细胞作为杀伤效应细胞，以CCL-86 Raji淋巴瘤细胞(购自ATCC)作为靶细胞，将两种细胞混合培养，检测CIK细胞对Raji细胞的杀伤活性。
CIK细胞对Raji细胞的杀伤效率检测：
在96孔板中进行细胞杀伤实验，反应体积为100uL，设置以下实验组别：组1(5ug/ml Anti-CD3/Anti-CD28联用培养14天后的CIK细胞)、组2(10ng/ml CD3-OX40 BsAb_M培养14天后的CIK细胞)、组3(10ng/ml CD3-OX40 BsAb_D培养14天后的CIK细胞)、组4(5ug/mlAnti-CD3/Anti-CD28联用培养30天后的CIK细胞)、组5(10ng/ml CD3-OX40 BsAb_M培养30天后的CIK细胞)、组6(10ng/ml CD3-OX40 BsAb_D培养30天后的CIK细胞)。分别取上述6组CIK细胞1×105个，加入Raji细胞1×105个(CIK效应细胞：Raji靶细胞(E:T比)为1：1)，37℃共培养3h后，每孔添加10ul的CCK8,37℃继续反应2-3h，随后用酶标仪测OD450值，按照以下公式计算细胞杀伤效率，每组实验重复检测3次；同时以未添加任何抗体进行培养的CIK细胞对Raji细胞的杀伤效率作为空白对照。
结果如图13显示，培养14天后，添加CD3-OX40双特异抗体的CIK细胞对Raji细胞的杀伤效率均不同程度地优于抗CD3/抗CD28单克隆全长抗体联合使用：其中添加二聚体形式的CD3-OX40 BsAb_D，杀伤效率约为32％，效果最优(组3)；添加单体形式的CD3-OX40 BsAb_M，杀伤效率约为25％，效果次之(组2)；Anti CD3/Anti CD28联用，杀伤效率约为22％，效果最弱(组1)。培养30天后，添加CD3-OX40双特异抗体的CIK细胞杀伤活性进一步提高：其中添加二聚体形式的CD3-OX40 BsAb_D，杀伤效率约为40％(组6)；添加单体形式的CD3-OX40BsAb_M，杀伤效率约为35％(组5)；与之相比，Anti CD3/Anti CD28联用培养的CIK细胞杀伤Raji肿瘤细胞的能力已大幅下降，杀伤效率仅约为10％(组4)。
其中，杀伤效率的计算公式为：
实施例16：CD3-GITR BsAb_M和CD3-GITR BsAb_D真核表达载体的构建
在本发明中，以T细胞表面人类CD3蛋白和T细胞正共刺激分子GITR蛋白为靶点的双特异性抗体被命名为CD3-GITR BsAb。
一、CD3-GITR BsAb_M和CD3-GITR BsAb_D构建方案设计
单体形式的CD3-GITR BsAb_M具体构建方案为：抗CD3 scFv和抗GITR scFv序列之间通过(GGGGS)3 Linker相连。
二聚体形式的CD3-GITR BsAb_D具体构建方案为：抗CD3 scFv和抗GITR scFv序列之间通过IgD铰链区作为Linker相连。
为使双特异性抗体在哺乳细胞中进行表达，针对抗CD3 scFv，抗GITR scFv及连接片段(Linker)序列均进行了哺乳系统表达的密码子优化。
具体地，抗CD3 scFv的重链可变区的核苷酸序列如SEQ ID NO.58所示。
具体地，抗CD3 scFv的轻链可变区的核苷酸序列如SEQ ID NO.59所示。
具体地，抗CD3 scFv的核苷酸序列如SEQ ID NO.57所示。
具体地，抗GITR scFv的重链可变区的核苷酸序列如SEQ ID NO.70所示，具体为：
CAGGTGACCCTGAAGGAGAGCGGCCCCGGCATCCTGAAGCCCAGCCAGACCCTGAGCCTGACCTGCAGCTTCAGCGGCTTCAGCCTGAGCACCAGCGGCATGGGCGTGGGCTGGATCCGCCAGCCCAGCGGCAAGGGCCTGGAGTGGCTGGCCCACATCTGGTGGGACGACGACAAGTACTACAACCCCAGCCTGAAGAGCCAGCTGACCATCAGCAAGGACACCAGCCGCAACCAGGTGTTCCTGAAGATCACCAGCGTGGACACCGCCGACGCCGCCACCTACTACTGCGCCCGCACCCGCCGCTACTTCCCCTTCGCCTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGC。
具体地，抗GITR scFv的轻链可变区的核苷酸序列如SEQ ID NO.71所示，具体为：
GACATCGTGATGACCCAGAGCCAGAAGTTCATGAGCACCAGCGTGGGCGACCGCGTGAGCGTGACCTGCAAGGCCAGCCAGAACGTGGGCACCAACGTGGCCTGGTACCAGCAGAAGCCCGGCCAGAGCCCCAAGGCCCTGATCTACAGCGCCAGCTACCGCTACAGCGGCGTGCCCGACCGCTTCACCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAACAACGTGCACAGCGAGGACCTGGCCGAGTACTTCTGCCAGCAGTACAACACCGACCCCCTGACCTTCGGCGCCGGCACCAAGCTGGAGATCAAG。
具体地，抗GITR scFv的核苷酸序列如SEQ ID NO.69所示，具体为：
CAGGTGACCCTGAAGGAGAGCGGCCCCGGCATCCTGAAGCCCAGCCAGACCCTGAGCCTGACCTGCAGCTTCAGCGGCTTCAGCCTGAGCACCAGCGGCATGGGCGTGGGCTGGATCCGCCAGCCCAGCGGCAAGGGCCTGGAGTGGCTGGCCCACATCTGGTGGGACGACGACAAGTACTACAACCCCAGCCTGAAGAGCCAGCTGACCATCAGCAAGGACACCAGCCGCAACCAGGTGTTCCTGAAGATCACCAGCGTGGACACCGCCGACGCCGCCACCTACTACTGCGCCCGCACCCGCCGCTACTTCCCCTTCGCCTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGACATCGTGATGACCCAGAGCCAGAAGTTCATGAGCACCAGCGTGGGCGACCGCGTGAGCGTGACCTGCAAGGCCAGCCAGAACGTGGGCACCAACGTGGCCTGGTACCAGCAGAAGCCCGGCCAGAGCCCCAAGGCCCTGATCTACAGCGCCAGCTACCGCTACAGCGGCGTGCCCGACCGCTTCACCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAACAACGTGCACAGCGAGGACCTGGCCGAGTACTTCTGCCAGCAGTACAACACCGACCCCCTGACCTTCGGCGCCGGCACCAAGCTGGAGATCAAG。
单体形式的CD3-GITR BsAb_M连接片段的核苷酸序列如SEQ ID NO.2。
二聚体形式的CD3-GITR BsAb_D连接片段的核苷酸序列如SEQ ID NO.4。
为使双特异性抗体在CHO-S细胞中表达并成功分泌到培养基中，选择了抗体分泌型表达的信号肽用于此实施例。
该分泌表达信号肽的氨基酸序列如SEQ ID NO.78所示。
该分泌表达信号肽的核苷酸序列如SEQ ID NO.79所示。
二、CD3-GITR BsAb_M和CD3-GITR BsAb_D真核表达载体构建
本发明双特异性抗体的构建与表达，选用哺乳细胞蛋白瞬时表达载体pcDNA3.1(购自上海英骏生物科技有限公司)。为构建单体和二聚体形式的双特异性抗体，分别设计了如表5所示引物，所有引物由苏州金唯智生物科技有限公司合成，扩增所需基因模板由苏州鸿讯科技有限公司合成。
针对CD3-GITR BsAb_M的克隆构建，首先使用引物pcDNA3.1-Sig-F和Sig-R扩增出信号肽片段，然后分别利用引物Sig-CD3-F和CD3-R、CD3-(GGGGS)3-GITR-F和pcDNA3.1-GITR-R扩增出抗CD3 scFv、(GGGGS)3 Linker、抗GITR scFv的基因序列；针对CD3-GITRBsAb_D的克隆构建，同样首先使用引物pcDNA3.1-Sig-F和Sig-R扩增出信号肽片段，然后分别利用引物Sig-CD3-F和CD3-R、CD3-IgD-F和IgD-R、IgD-GITR-F和pcDNA3.1-GITR-R扩增出抗CD3 scFv、IgD铰链区、抗GITR scFv的基因序列。扩增完毕后，利用
PCR一步定向克隆试剂盒(购自吴江近岸蛋白质科技有限公司)分别拼接单体和二聚体形式双特异性抗体全长基因序列并无缝克隆至经EcoRI和HindIII线性化处理的pcDNA3.1表达载体上。目的载体转化大肠杆菌DH5α，利用菌落PCR进行阳性克隆鉴定，鉴定为阳性的重组子(重组质粒)进行测序鉴定。随后将测序正确的重组子(重组质粒)安排质粒中抽，用于CHO-S细胞的转染。
经测序获知，单体形式的CD3-GITR BsAb_M和二聚体形式的CD3-GITR BsAb_D的全长基因序列正确，均与预期相符。
具体地，单体形式的CD3-GITR BsAb_M的核苷酸序列如SEQ ID NO.25所示，具体为：GACATCAAGCTGCAGCAGAGCGGCGCCGAGCTGGCCCGCCCCGGCGCCAGCGTGAAGATGAGCTGCAAGACCAGCGGCTACACCTTCACCCGCTACACCATGCACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGGCTACATCAACCCCAGCCGCGGCTACACCAACTACAACCAGAAGTTCAAGGACAAGGCCACCCTGACCACCGACAAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCCGCTACTACGACGACCACTACTGCCTGGACTACTGGGGCCAGGGCACCACCCTGACCGTGAGCAGCGTGGAGGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCGTGGACGACATCCAGCTGACCCAGAGCCCCGCCATCATGAGCGCCAGCCCCGGCGAGAAGGTGACCATGACCTGCCGCGCCAGCAGCAGCGTGAGCTACATGAACTGGTACCAGCAGAAGAGCGGCACCAGCCCCAAGCGCTGGATCTACGACACCAGCAAGGTGGCCAGCGGCGTGCCCTACCGCTTCAGCGGCAGCGGCAGCGGCACCAGCTACAGCCTGACCATCAGCAGCATGGAGGCCGAGGACGCCGCCACCTACTACTGCCAGCAGTGGAGCAGCAACCCCCTGACCTTCGGCGCCGGCACCAAGCTGGAGCTGAAGGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCCAGGTGACCCTGAAGGAGAGCGGCCCCGGCATCCTGAAGCCCAGCCAGACCCTGAGCCTGACCTGCAGCTTCAGCGGCTTCAGCCTGAGCACCAGCGGCATGGGCGTGGGCTGGATCCGCCAGCCCAGCGGCAAGGGCCTGGAGTGGCTGGCCCACATCTGGTGGGACGACGACAAGTACTACAACCCCAGCCTGAAGAGCCAGCTGACCATCAGCAAGGACACCAGCCGCAACCAGGTGTTCCTGAAGATCACCAGCGTGGACACCGCCGACGCCGCCACCTACTACTGCGCCCGCACCCGCCGCTACTTCCCCTTCGCCTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGACATCGTGATGACCCAGAGCCAGAAGTTCATGAGCACCAGCGTGGGCGACCGCGTGAGCGTGACCTGCAAGGCCAGCCAGAACGTGGGCACCAACGTGGCCTGGTACCAGCAGAAGCCCGGCCAGAGCCCCAAGGCCCTGATCTACAGCGCCAGCTACCGCTACAGCGGCGTGCCCGACCGCTTCACCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAACAACGTGCACAGCGAGGACCTGGCCGAGTACTTCTGCCAGCAGTACAACACCGACCCCCTGACCTTCGGCGCCGGCACCAAGCTGGAGATCAAG。
具体地，二聚体形式的CD3-GITR BsAb_D的核苷酸序列如SEQ ID NO.27所示，具体为：
GACATCAAGCTGCAGCAGAGCGGCGCCGAGCTGGCCCGCCCCGGCGCCAGCGTGAAGATGAGCTGCAAGACCAGCGGCTACACCTTCACCCGCTACACCATGCACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGGCTACATCAACCCCAGCCGCGGCTACACCAACTACAACCAGAAGTTCAAGGACAAGGCCACCCTGACCACCGACAAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCCGCTACTACGACGACCACTACTGCCTGGACTACTGGGGCCAGGGCACCACCCTGACCGTGAGCAGCGTGGAGGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCGTGGACGACATCCAGCTGACCCAGAGCCCCGCCATCATGAGCGCCAGCCCCGGCGAGAAGGTGACCATGACCTGCCGCGCCAGCAGCAGCGTGAGCTACATGAACTGGTACCAGCAGAAGAGCGGCACCAGCCCCAAGCGCTGGATCTACGACACCAGCAAGGTGGCCAGCGGCGTGCCCTACCGCTTCAGCGGCAGCGGCAGCGGCACCAGCTACAGCCTGACCATCAGCAGCATGGAGGCCGAGGACGCCGCCACCTACTACTGCCAGCAGTGGAGCAGCAACCCCCTGACCTTCGGCGCCGGCACCAAGCTGGAGCTGAAGGCCAGCAAGAGCAAGAAGGAGATCTTCCGCTGGCCCGAGAGCCCCAAGGCCCAGGCCAGCAGCGTGCCCACCGCCCAGCCCCAGGCCGAGGGCAGCCTGGCCAAGGCCACCACCGCCCCCGCCACCACCCGCAACACCGGCCGCGGCGGCGAGGAGAAGAAGAAGGAGAAGGAGAAGGAGGAGCAGGAGGAGCGCGAGACCAAGACCCCCGAGTGCCCCAGCCACACCCAGCCCCTGGGCGTGCAGGTGACCCTGAAGGAGAGCGGCCCCGGCATCCTGAAGCCCAGCCAGACCCTGAGCCTGACCTGCAGCTTCAGCGGCTTCAGCCTGAGCACCAGCGGCATGGGCGTGGGCTGGATCCGCCAGCCCAGCGGCAAGGGCCTGGAGTGGCTGGCCCACATCTGGTGGGACGACGACAAGTACTACAACCCCAGCCTGAAGAGCCAGCTGACCATCAGCAAGGACACCAGCCGCAACCAGGTGTTCCTGAAGATCACCAGCGTGGACACCGCCGACGCCGCCACCTACTACTGCGCCCGCACCCGCCGCTACTTCCCCTTCGCCTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGACATCGTGATGACCCAGAGCCAGAAGTTCATGAGCACCAGCGTGGGCGACCGCGTGAGCGTGACCTGCAAGGCCAGCCAGAACGTGGGCACCAACGTGGCCTGGTACCAGCAGAAGCCCGGCCAGAGCCCCAAGGCCCTGATCTACAGCGCCAGCTACCGCTACAGCGGCGTGCCCGACCGCTTCACCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAACAACGTGCACAGCGAGGACCTGGCCGAGTACTTCTGCCAGCAGTACAACACCGACCCCCTGACCTTCGGCGCCGGCACCAAGCTGGAGATCAAG。
表5.CD3-GITR双特异性抗体基因克隆中使用的引物
实施例17：CD3-GITR BsAb_M和CD3-GITR BsAb_D的表达与纯化
一、CD3-GITR BsAb_M和CD3-GITR BsAb_D的表达
1.1.CHO-S细胞(购自Thermo Fisher Scientific公司)转染前1天传代密度为0.5～0.6×106/ml；
1.2.转染当天进行细胞密度统计，当密度为1～1.4×106/ml、活力>90％时，可用于质粒转染；
1.3.转染复合物配制：每个项目(CD3-GITR BsAb_M和CD3-GITR BsAb_D)需准备两个离心管/培养瓶，以20ml为例，分别放置，取实施例16中所制备重组质粒：
管①中加入600μl PBS，20μg重组质粒，混匀；
管②中加入600μl PBS，20ul FreeStyleTM MAX Transfection Reagent(购自Thermo Fisher Scientific公司)，混匀；
1.4.将稀释后的转染试剂，加入至稀释后的重组质粒中，混合均匀，配制成转染复合物；
1.5.转染复合物静置15～20min后，单滴匀速加入细胞培养物中；
1.6.于37℃，CO2浓度8％，摇床转速130rpm条件下进行转染后细胞培养，5天后收集培养上清进行目的蛋白表达检测。
二、CD3-GITR BsAb_M和CD3-GITR BsAb_D的纯化
2.1样品预处理
取上述转染后细胞培养上清20ml，加入缓冲液20mM PB，200mM NaCl调节pH至7.5；
2.2 Protein L亲和层析柱纯化
蛋白纯化层析柱：Protein L亲和层析柱(购自GE Healthcare公司，柱体积1.0ml)
缓冲液A(Buffer A)：PBS，pH7.4
缓冲液B(Buffer B)：0.1M Glycine,pH3.0
缓冲液C(Buffer C)：0.1M Glycine,pH2.7
纯化过程：采用AKTA explorer 100型蛋白纯化系统(购自GE Healthcare公司)，用Buffer A预处理Protein L亲和层析柱，取培养上清上样，收集流出液。上样完毕后，用至少1.5ml Buffer A平衡层析柱，平衡后分别用Buffer B和Buffer C洗脱，收集目的蛋白洗脱液(洗脱液的收集管需要预先加入1％的1M Tris,pH8.0来中和洗脱液pH值，Tris终浓度约为10mM)，最后浓缩透析至缓冲液PBS中。
最终纯化的CD3-GITR BsAb_M和CD3-GITR BsAb_D重组蛋白经SDS-PAGE分析，还原和非还原条件下电泳图如图14所示。从图中可以看出，经Protein L亲和层析柱纯化后，CD3-GITR BsAb_M和CD3-GITR BsAb_D重组蛋白的纯度均>95％；其中CD3-GITR BsAb_M重组蛋白的理论分子量为53.2kDa，还原和非还原条件下该蛋白均呈现单一电泳条带，分子量与单体一致，因此该双特异性抗体为单体形式(图14A)；CD3-GITR BsAb_D重组蛋白的理论分子量为61.1kDa，还原条件下该蛋白电泳条带所呈现分子量与单体一致，非还原条件下电泳条带所呈现分子量与二聚体一致(图14B)，说明两个蛋白分子可通过二硫键相互连接，因此该双特异性抗体为二聚体形式。
此外，纯化的重组蛋白样品经N/C末端序列分析，结果表明所表达的重组蛋白样品均读框无误，与理论N/C末端氨基酸序列一致，质谱分析进一步确认CD3-GITR BsAb_M为单体形式，CD3-GITR BsAb_D为二聚体形式。
因此，可得知，单体形式的CD3-GITR BsAb_M的氨基酸序列如SEQ ID NO.24所示，具体为：
DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKGGGGSGGGGSGGGGSQVTLKESGPGILKPSQTLSLTCSFSGFSLSTSGMGVGWIRQPSGKGLEWLAHIWWDDDKYYNPSLKSQLTISKDTSRNQVFLKITSVDTADAATYYCARTRRYFPFAYWGQGTLVTVSSGGGGSGGGGSGGGGSDIVMTQSQKFMSTSVGDRVSVTCKASQNVGTNVAWYQQKPGQSPKALIYSASYRYSGVPDRFTGSGSGTDFTLTINNVHSEDLAEYFCQQYNTDPLTFGAGTKLEIK。
二聚体形式的CD3-GITR BsAb_D的氨基酸序列如SEQ ID NO.26所示，具体为：
DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKASKSKKEIFRWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEKEKEEQEERETKTPECPSHTQPLGVQVTLKESGPGILKPSQTLSLTCSFSGFSLSTSGMGVGWIRQPSGKGLEWLAHIWWDDDKYYNPSLKSQLTISKDTSRNQVFLKITSVDTADAATYYCARTRRYFPFAYWGQGTLVTVSSGGGGSGGGGSGGGGSDIVMTQSQKFMSTSVGDRVSVTCKASQNVGTNVAWYQQKPGQSPKALIYSASYRYSGVPDRFTGSGSGTDFTLTINNVHSEDLAEYFCQQYNTDPLTFGAGTKLEIK。
抗CD3 scFv的氨基酸序列如SEQ ID NO.36所示。
抗CD3 scFv的重链可变区的氨基酸序列如SEQ ID NO.37所示。
抗CD3 scFv的轻链可变区的氨基酸序列如SEQ ID NO.38所示。
抗GITR scFv的氨基酸序列如SEQ ID NO.48所示，具体为：
QVTLKESGPGILKPSQTLSLTCSFSGFSLSTSGMGVGWIRQPSGKGLEWLAHIWWDDDKYYNPSLKSQLTISKDTSRNQVFLKITSVDTADAATYYCARTRRYFPFAYWGQGTLVTVSSGGGGSGGGGSGGGGSDIVMTQSQKFMSTSVGDRVSVTCKASQNVGTNVAWYQQKPGQSPKALIYSASYRYSGVPDRFTGSGSGTDFTLTINNVHSEDLAEYFCQQYNTDPLTFGAGTKLEIK。
抗GITR scFv的重链可变区的氨基酸序列如SEQ ID NO.49所示，具体为：
QVTLKESGPGILKPSQTLSLTCSFSGFSLSTSGMGVGWIRQPSGKGLEWLAHIWWDDDKYYNPSLKSQLTISKDTSRNQVFLKITSVDTADAATYYCARTRRYFPFAYWGQGTLVTVSS。
抗GITR scFv的轻链可变区的氨基酸序列如SEQ ID NO.50所示，具体为：
DIVMTQSQKFMSTSVGDRVSVTCKASQNVGTNVAWYQQKPGQSPKALIYSASYRYSGVPDRFTGSGSGTDFTLTINNVHSEDLAEYFCQQYNTDPLTFGAGTKLEIK。
单体形式的CD3-GITR BsAb_M中连接片段的氨基酸序列如SEQ ID NO.1所示。
二聚体形式的CD3-GITR BsAb_D中连接片段的氨基酸序列如SEQ ID NO.3所示。
实施例18：ELISA检测CD3-GITR BsAb_M和CD3-GITR BsAb_D的抗原结合活性
ELISA操作步骤：
1.重组抗原包被：人类CD3-hFc与人类GITR-hFc融合蛋白(购自吴江近岸蛋白质科技有限公司)分别包被96孔板，抗原浓度为1μg/ml，包被体积为100μl/孔，包被条件为37℃1小时或4℃过夜，包被缓冲液(PBS)的配方为：3.58g Na2HPO4，0.24g NaH2PO4，0.2g KCl，8.2g NaCl，950ml H2O，用1mol/L HCl或1mol/L NaOH调pH至7.4，补水至1L；
2.封闭：PBS洗板4次后，加入封闭液PBSA(PBS+2％BSA(V/W))，200μl/孔。37℃封闭1小时；
3.加样：PBS洗板4次后，分别加入纯化的双特异性抗体样品，100μl/孔，37℃孵育1小时，样品梯度配制方法：以10μg/ml纯化的CD3-GITR BsAb_M或CD3-GITR BsAb_D作为起始浓度，进行倍比稀释6个梯度，每个梯度设置2个复孔；
4.显色：PBST(PBS+0.05％Tween-20(V/V))洗板4次后，用封闭液PBSA按1/5000稀释HRP标记的显色抗体(购自Abcam公司)，按100μl/孔加入，37℃孵育1小时。PBS洗板4次后，添加显色液TMB(购自KPL公司)，100μl/孔，室温避光显色5～10分钟；
5.终止反应与结果测定：添加终止液(1M HCl)，100μl/孔，在酶标仪上450nm波长下读取吸光值。
ELISA结果如图15A和图15B所示：图15A说明CD3-GITR BsAb_M与重组抗原CD3-hFc和GITR-hFc均具有体外结合活性，其中GITR结合活性较CD3结合活性更高；图15B说明CD3-GITR BsAb_D与重组抗原CD3-hFc和GITR-hFc同样具有体外结合活性，其中GITR结合活性更高。
实施例19：CD3-GITR双特异抗体介导的CIK细胞增殖
以人外周血单个核细胞(Peripheral blood mononuclear cell，PBMC)为实验材料，用本发明所制备的上述单体形式的双特异抗体CD3-GITR BsAb_M、二聚体形式的双特异抗体CD3-GITR BsAb_D、以及抗CD3/抗CD28单克隆全长抗体联用(Anti-CD3/Anti-CD28)分别作用于同一供体来源的人血PBMC，细胞培养后进行计数，比较扩增倍数。
1.PBMC的分离：取抗凝血，加入等体积的医用生理盐水，沿离心管壁缓慢加入与血液等体积的淋巴细胞分离液(购自GE Healthcare公司)，保持液面分层明显，2000rpm离心20min，吸取中间白雾状的细胞层于新的离心管中，加入2倍以上体积的PBS缓冲液洗涤，1100rpm离心10min，重复洗涤一次，用少量预冷的X-vivo 15无血清培养基(购自Lonza公司)重悬，细胞计数待用；
2.CIK细胞培养与扩增：将PBMC用CIK基础培养基(90％X-vivo15+10％FBS)重悬，调整细胞密度为1×106/ml，分别设计以下3个实验组：对照组(Anti-CD3 5ug/ml和Anti-CD28 5ug/ml包被细胞培养板，全长抗体均购自吴江近岸蛋白质科技有限公司)；实验组1(溶液状态下添加双特异性抗体CD3-GITR BsAb_M 10ng/ml)；实验组2(溶液状态下添加双特异性抗体CD3-GITR BsAb_D 10ng/ml)。此外，3组实验细胞同时添加细胞因子IFN-γ(200ng/ml，购自吴江近岸蛋白质科技有限公司)和IL-1β(2ng/ml，购自吴江近岸蛋白质科技有限公司)，置于培养箱，在饱和湿度、37℃、5.0％CO2的条件下进行培养。培养过夜后，添加500U/ml的IL-2(购自吴江近岸蛋白质科技有限公司)继续培养，每2-3天计数并用添加500U/ml IL-2的CIK基础培养基按1×106/ml的密度进行细胞传代。照此方法培养14天，最终统计细胞的扩增倍数，绘制生长曲线；
检测结果如表6和图16所示，单体和二聚体形式的CD3-GITR双特异性抗体单一使用对CIK细胞的增殖效果均稍优于抗CD3/抗CD28单克隆全长抗体联合使用，且蛋白用量更少(10ng/ml vs 5ug/ml)：其中二聚体形式的CD3-GITR BsAb_D可以介导CIK细胞两周扩增287倍，效果最优(实验组2)；单体形式的CD3-GITR BsAb_M可以介导CIK细胞两周扩增248倍，效果次之(实验组1)；Anti CD3/Anti CD28联用可以介导CIK细胞两周扩增224倍，效果最弱(对照组)。
表6 CIK细胞扩增倍数
| 实验组名 | 对照组 | 实验组1 | 实验组2 |
| 14天扩增倍数 | 224 | 248 | 287 |
实施例20：CD3-CD40L BsAb_M和CD3-CD40L BsAb_D真核表达载体的构建
在本发明中，以T细胞表面人类CD3蛋白和T细胞正共刺激分子CD40L蛋白为靶点的双特异性抗体被命名为CD3-CD40L BsAb。
一、CD3-CD40L BsAb_M和CD3-CD40L BsAb_D构建方案设计
单体形式的CD3-CD40L BsAb_M具体构建方案为：抗CD3 scFv和抗CD40L scFv序列之间通过(GGGGS)3 Linker相连。
二聚体形式的CD3-CD40L BsAb_D具体构建方案为：抗CD3 scFv和抗CD40L scFv序列之间通过IgD铰链区作为Linker相连。
为使双特异性抗体在哺乳细胞中进行表达，针对抗CD3 scFv，抗CD40L scFv及连接片段(Linker)序列均进行了哺乳系统表达的密码子优化。
具体地，抗CD3 scFv的重链可变区的核苷酸序列如SEQ ID NO.58所示。
具体地，抗CD3 scFv的轻链可变区的核苷酸序列如SEQ ID NO.59所示。
具体地，抗CD3 scFv的核苷酸序列如SEQ ID NO.57所示。
具体地，抗CD40L scFv的重链可变区的核苷酸序列如SEQ ID NO.73所示，具体为：
GAGGTGCAGCTGCTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCTGCGCCTGAGCTGCGCCGCCAGCGGCTTCACCTTCAGCAGCTACGCCATGAGCTGGGTGCGCCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGAGCGCCATCAGCGGCAGCGGCGGCAGCACCTACTACGCCGACAGCGTGAAGGGCCGCTTCACCATCAGCCGCGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGCGCCGAGGACACCGCCGTGTACTACTGCGCCAAGAGCTACGGCGCCTTCGACTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGC。
具体地，抗CD40L scFv的轻链可变区的核苷酸序列如SEQ ID NO.74所示，具体为：
GACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCGACCGCGTGACCATCACCTGCCGCGCCAGCCAGAGCATCAGCAGCTACCTGAACTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGCCGCCAGCAGCCTGCAGAGCGGCGTGCCCAGCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGAGCTACAGCACCCCCAACACCTTCGGCCAGGGCACCAAGGTGGAGATCAAGCGC。
具体地，抗CD40L scFv的核苷酸序列如SEQ ID NO.72所示，具体为：
GAGGTGCAGCTGCTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCTGCGCCTGAGCTGCGCCGCCAGCGGCTTCACCTTCAGCAGCTACGCCATGAGCTGGGTGCGCCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGAGCGCCATCAGCGGCAGCGGCGGCAGCACCTACTACGCCGACAGCGTGAAGGGCCGCTTCACCATCAGCCGCGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGCGCCGAGGACACCGCCGTGTACTACTGCGCCAAGAGCTACGGCGCCTTCGACTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCGACCGCGTGACCATCACCTGCCGCGCCAGCCAGAGCATCAGCAGCTACCTGAACTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGCCGCCAGCAGCCTGCAGAGCGGCGTGCCCAGCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGAGCTACAGCACCCCCAACACCTTCGGCCAGGGCACCAAGGTGGAGATCAAGCGC。
单体形式的CD3-CD40L BsAb_M连接片段的核苷酸序列如SEQ ID NO.2。
二聚体形式的CD3-CD40L BsAb_D连接片段的核苷酸序列如SEQ ID NO.4。
为使双特异性抗体在CHO-S细胞中表达并成功分泌到培养基中，选择了抗体分泌型表达的信号肽用于此实施例。
该分泌表达信号肽的氨基酸序列如SEQ ID NO.78所示。
该分泌表达信号肽的核苷酸序列如SEQ ID NO.79所示。
二、CD3-CD40L BsAb_M和CD3-CD40L BsAb_D真核表达载体构建
本发明双特异性抗体的构建与表达，选用哺乳细胞蛋白瞬时表达载体pcDNA3.1(购自上海英骏生物科技有限公司)。为构建单体和二聚体形式的双特异性抗体，分别设计了如表7所示引物，所有引物由苏州金唯智生物科技有限公司合成，扩增所需基因模板由苏州鸿讯科技有限公司合成。
针对CD3-CD40L BsAb_M的克隆构建，首先使用引物pcDNA3.1-Sig-F和Sig-R扩增出信号肽片段，然后分别利用引物Sig-CD3-F和CD3-R、CD3-(GGGGS)3-CD40L-F和pcDNA3.1-CD40L-R扩增出抗CD3 scFv、(GGGGS)3 Linker、抗CD40L scFv的基因序列；针对CD3-CD40LBsAb_D的克隆构建，同样首先使用引物pcDNA3.1-Sig-F和Sig-R扩增出信号肽片段，然后分别利用引物Sig-CD3-F和CD3-R、CD3-IgD-F和IgD-R、IgD-CD40L-F和pcDNA3.1-CD40L-R扩增出抗CD3 scFv、IgD铰链区、抗CD40L scFv的基因序列。扩增完毕后，利用
PCR一步定向克隆试剂盒(购自吴江近岸蛋白质科技有限公司)分别拼接单体和二聚体形式双特异性抗体全长基因序列并无缝克隆至经EcoRI和HindIII线性化处理的pcDNA3.1表达载体上。目的载体转化大肠杆菌DH5α，利用菌落PCR进行阳性克隆鉴定，鉴定为阳性的重组子(重组质粒)进行测序鉴定。随后将测序正确的重组子(重组质粒)安排质粒中抽，用于CHO-S细胞的转染。
经测序获知，单体形式的CD3-CD40L BsAb_M和二聚体形式的CD3-CD40L BsAb_D的全长基因序列正确，均与预期相符。
具体地，单体形式的CD3-CD40L BsAb_M的核苷酸序列如SEQ ID NO.29所示，具体为：
GACATCAAGCTGCAGCAGAGCGGCGCCGAGCTGGCCCGCCCCGGCGCCAGCGTGAAGATGAGCTGCAAGACCAGCGGCTACACCTTCACCCGCTACACCATGCACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGGCTACATCAACCCCAGCCGCGGCTACACCAACTACAACCAGAAGTTCAAGGACAAGGCCACCCTGACCACCGACAAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCCGCTACTACGACGACCACTACTGCCTGGACTACTGGGGCCAGGGCACCACCCTGACCGTGAGCAGCGTGGAGGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCGTGGACGACATCCAGCTGACCCAGAGCCCCGCCATCATGAGCGCCAGCCCCGGCGAGAAGGTGACCATGACCTGCCGCGCCAGCAGCAGCGTGAGCTACATGAACTGGTACCAGCAGAAGAGCGGCACCAGCCCCAAGCGCTGGATCTACGACACCAGCAAGGTGGCCAGCGGCGTGCCCTACCGCTTCAGCGGCAGCGGCAGCGGCACCAGCTACAGCCTGACCATCAGCAGCATGGAGGCCGAGGACGCCGCCACCTACTACTGCCAGCAGTGGAGCAGCAACCCCCTGACCTTCGGCGCCGGCACCAAGCTGGAGCTGAAGGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGAGGTGCAGCTGCTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCTGCGCCTGAGCTGCGCCGCCAGCGGCTTCACCTTCAGCAGCTACGCCATGAGCTGGGTGCGCCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGAGCGCCATCAGCGGCAGCGGCGGCAGCACCTACTACGCCGACAGCGTGAAGGGCCGCTTCACCATCAGCCGCGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGCGCCGAGGACACCGCCGTGTACTACTGCGCCAAGAGCTACGGCGCCTTCGACTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCGACCGCGTGACCATCACCTGCCGCGCCAGCCAGAGCATCAGCAGCTACCTGAACTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGCCGCCAGCAGCCTGCAGAGCGGCGTGCCCAGCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGAGCTACAGCACCCCCAACACCTTCGGCCAGGGCACCAAGGTGGAGATCAAGCGC。
具体地，二聚体形式的CD3-CD40L BsAb_D的核苷酸序列如SEQ ID NO.31所示，具体为：
GACATCAAGCTGCAGCAGAGCGGCGCCGAGCTGGCCCGCCCCGGCGCCAGCGTGAAGATGAGCTGCAAGACCAGCGGCTACACCTTCACCCGCTACACCATGCACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGGCTACATCAACCCCAGCCGCGGCTACACCAACTACAACCAGAAGTTCAAGGACAAGGCCACCCTGACCACCGACAAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCCGCTACTACGACGACCACTACTGCCTGGACTACTGGGGCCAGGGCACCACCCTGACCGTGAGCAGCGTGGAGGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCGTGGACGACATCCAGCTGACCCAGAGCCCCGCCATCATGAGCGCCAGCCCCGGCGAGAAGGTGACCATGACCTGCCGCGCCAGCAGCAGCGTGAGCTACATGAACTGGTACCAGCAGAAGAGCGGCACCAGCCCCAAGCGCTGGATCTACGACACCAGCAAGGTGGCCAGCGGCGTGCCCTACCGCTTCAGCGGCAGCGGCAGCGGCACCAGCTACAGCCTGACCATCAGCAGCATGGAGGCCGAGGACGCCGCCACCTACTACTGCCAGCAGTGGAGCAGCAACCCCCTGACCTTCGGCGCCGGCACCAAGCTGGAGCTGAAGGCCAGCAAGAGCAAGAAGGAGATCTTCCGCTGGCCCGAGAGCCCCAAGGCCCAGGCCAGCAGCGTGCCCACCGCCCAGCCCCAGGCCGAGGGCAGCCTGGCCAAGGCCACCACCGCCCCCGCCACCACCCGCAACACCGGCCGCGGCGGCGAGGAGAAGAAGAAGGAGAAGGAGAAGGAGGAGCAGGAGGAGCGCGAGACCAAGACCCCCGAGTGCCCCAGCCACACCCAGCCCCTGGGCGTGGAGGTGCAGCTGCTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCTGCGCCTGAGCTGCGCCGCCAGCGGCTTCACCTTCAGCAGCTACGCCATGAGCTGGGTGCGCCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGAGCGCCATCAGCGGCAGCGGCGGCAGCACCTACTACGCCGACAGCGTGAAGGGCCGCTTCACCATCAGCCGCGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGCGCCGAGGACACCGCCGTGTACTACTGCGCCAAGAGCTACGGCGCCTTCGACTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCGACCGCGTGACCATCACCTGCCGCGCCAGCCAGAGCATCAGCAGCTACCTGAACTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGCCGCCAGCAGCCTGCAGAGCGGCGTGCCCAGCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGAGCTACAGCACCCCCAACACCTTCGGCCAGGGCACCAAGGTGGAGATCAAGCGC。
表7.CD3-CD40L双特异性抗体基因克隆中使用的引物
实施例21：CD3-CD40L BsAb_M和CD3-CD40L BsAb_D的表达与纯化
一、CD3-CD40L BsAb_M和CD3-CD40L BsAb_D的表达
1.1.CHO-S细胞(购自Thermo Fisher Scientific公司)转染前1天传代密度为0.5～0.6×106/ml；
1.2.转染当天进行细胞密度统计，当密度为1～1.4×106/ml、活力>90％时，可用于质粒转染；
1.3.转染复合物配制：每个项目(CD3-CD40L BsAb_M和CD3-CD40L BsAb_D)需准备两个离心管/培养瓶，以20ml为例，分别放置，取实施例20中所制备重组质粒：
管①中加入600μl PBS，20μg重组质粒，混匀；
管②中加入600μl PBS，20ul FreeStyleTM MAX Transfection Reagent(购自Thermo Fisher Scientific公司)，混匀；
1.4.将稀释后的转染试剂，加入至稀释后的重组质粒中，混合均匀，配制成转染复合物；
1.5.转染复合物静置15～20min后，单滴匀速加入细胞培养物中；
1.6.于37℃，CO2浓度8％，摇床转速130rpm条件下进行转染后细胞培养，5天后收集培养上清进行目的蛋白表达检测。
二、CD3-CD40L BsAb_M和CD3-CD40L BsAb_D的纯化
2.1样品预处理
取上述转染后细胞培养上清20ml，加入缓冲液20mM PB，200mM NaCl调节pH至7.5；
2.2 Protein L亲和层析柱纯化
蛋白纯化层析柱：Protein L亲和层析柱(购自GE Healthcare公司，柱体积1.0ml)
缓冲液A(Buffer A)：PBS，pH7.4
缓冲液B(Buffer B)：0.1M Glycine,pH3.0
缓冲液C(Buffer C)：0.1M Glycine,pH2.7
纯化过程：采用AKTA explorer 100型蛋白纯化系统(购自GE Healthcare公司)，用Buffer A预处理Protein L亲和层析柱，取培养上清上样，收集流出液。上样完毕后，用至少1.5ml Buffer A平衡层析柱，平衡后分别用Buffer B和Buffer C洗脱，收集目的蛋白洗脱液(洗脱液的收集管需要预先加入1％的1M Tris,pH8.0来中和洗脱液pH值，Tris终浓度约为10mM)，最后浓缩透析至缓冲液PBS中。
最终纯化的CD3-CD40L BsAb_M和CD3-CD40L BsAb_D重组蛋白经SDS-PAGE分析，还原和非还原条件下电泳图如图17所示。从图中可以看出，经Protein L亲和层析柱纯化后，CD3-CD40L BsAb_M和CD3-CD40L BsAb_D重组蛋白的纯度均>95％；其中CD3-CD40L BsAb_M重组蛋白的理论分子量为53.2kDa，还原和非还原条件下该蛋白均呈现单一电泳条带，分子量与单体一致，因此该双特异性抗体为单体形式(图17A)；CD3-CD40L BsAb_D重组蛋白的理论分子量为61.2kDa，还原条件下该蛋白电泳条带所呈现分子量与单体一致，非还原条件下电泳条带所呈现分子量与二聚体一致(图17B)，说明两个蛋白分子可通过二硫键相互连接，因此该双特异性抗体为二聚体形式。
此外，纯化的重组蛋白样品经N/C末端序列分析，结果表明所表达的重组蛋白样品均读框无误，与理论N/C末端氨基酸序列一致，质谱分析进一步确认CD3-CD40L BsAb_M为单体形式，CD3-CD40L BsAb_D为二聚体形式。
因此，可得知，单体形式的CD3-CD40L BsAb_M的氨基酸序列如SEQ ID NO.28所示，具体为：
DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSYGAFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPNTFGQGTKVEIKR。
二聚体形式的CD3-CD40L BsAb_D的氨基酸序列如SEQ ID NO.30所示，具体为：
DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKASKSKKEIFRWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEKEKEEQEERETKTPECPSHTQPLGVEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSYGAFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPNTFGQGTKVEIKR。
抗CD3 scFv的氨基酸序列如SEQ ID NO.36所示。
抗CD3 scFv的重链可变区的氨基酸序列如SEQ ID NO.37所示。
抗CD3 scFv的轻链可变区的氨基酸序列如SEQ ID NO.38所示。
抗CD40L scFv的氨基酸序列如SEQ ID NO.51所示，具体为：
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSYGAFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPNTFGQGTKVEIKR。
抗CD40L scFv的重链可变区的氨基酸序列如SEQ ID NO.52所示，具体为：
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSYGAFDYWGQGTLVTVSS。
抗CD40L scFv的重链可变区的氨基酸序列如SEQ ID NO.53所示，具体为：
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPNTFGQGTKVEIKR。
单体形式的CD3-CD40L BsAb_M中连接片段的氨基酸序列如SEQ ID NO.1所示。
二聚体形式的CD3-CD40L BsAb_D中连接片段的氨基酸序列如SEQ ID NO.3所示。
实施例22：ELISA检测CD3-CD40L BsAb_M和CD3-CD40L BsAb_D的抗原结合活性
ELISA操作步骤：
1.重组抗原包被：人类CD3-hFc与人类CD40L-hFc融合蛋白(购自吴江近岸蛋白质科技有限公司)分别包被96孔板，抗原浓度为1μg/ml，包被体积为100μl/孔，包被条件为37℃1小时或4℃过夜，包被缓冲液(PBS)的配方为：3.58g Na2HPO4，0.24g NaH2PO4，0.2g KCl，8.2g NaCl，950ml H2O，用1mol/L HCl或1mol/L NaOH调pH至7.4，补水至1L；
2.封闭：PBS洗板4次后，加入封闭液PBSA(PBS+2％BSA(V/W))，200μl/孔。37℃封闭1小时；
3.加样：PBS洗板4次后，分别加入纯化的双特异性抗体样品，100μl/孔，37℃孵育1小时，样品梯度配制方法：以10μg/ml纯化的CD3-CD40L BsAb_M或CD3-CD40L BsAb_D作为起始浓度，进行倍比稀释6个梯度，每个梯度设置2个复孔；
4.显色：PBST(PBS+0.05％Tween-20(V/V))洗板4次后，用封闭液PBSA按1/5000稀释HRP标记的显色抗体(购自Abcam公司)，按100μl/孔加入，37℃孵育1小时。PBS洗板4次后，添加显色液TMB(购自KPL公司)，100μl/孔，室温避光显色5～10分钟；
5.终止反应与结果测定：添加终止液(1M HCl)，100μl/孔，在酶标仪上450nm波长下读取吸光值。
ELISA结果如图18A和图18B所示：图18A说明CD3-CD40L BsAb_M与重组抗原CD3-hFc和CD40L-hFc均具有体外结合活性，其中CD40L结合活性较CD3结合活性更高；图18B说明CD3-CD40L BsAb_D与重组抗原CD3-hFc和CD40L-hFc同样具有体外结合活性，其中CD40L结合活性更高。
实施例23：CD3-CD40L双特异抗体介导的CIK细胞增殖
以人外周血单个核细胞(Peripheral blood mononuclear cell，PBMC)为实验材料，用本发明所制备的上述单体形式的双特异抗体CD3-CD40L BsAb_M、二聚体形式的双特异抗体CD3-CD40L BsAb_D、以及抗CD3/抗CD28单克隆全长抗体联用(Anti-CD3/Anti-CD28)分别作用于同一供体来源的人血PBMC，细胞培养后进行计数，比较扩增倍数。
1.PBMC的分离：取抗凝血，加入等体积的医用生理盐水，沿离心管壁缓慢加入与血液等体积的淋巴细胞分离液(购自GE Healthcare公司)，保持液面分层明显，2000rpm离心20min，吸取中间白雾状的细胞层于新的离心管中，加入2倍以上体积的PBS缓冲液洗涤，1100rpm离心10min，重复洗涤一次，用少量预冷的X-vivo 15无血清培养基(购自Lonza公司)重悬，细胞计数待用；
2.CIK细胞培养与扩增：将PBMC用CIK基础培养基(90％X-vivo15+10％FBS)重悬，调整细胞密度为1×106/ml，分别设计以下3个实验组：对照组(Anti-CD3 5ug/ml和Anti-CD28 5ug/ml包被细胞培养板，全长抗体均购自吴江近岸蛋白质科技有限公司)；实验组1(溶液状态下添加双特异性抗体CD3-CD40L BsAb_M 10ng/ml)；实验组2(溶液状态下添加双特异性抗体CD3-CD40L BsAb_D 10ng/ml)。此外，3组实验细胞同时添加细胞因子IFN-γ(200ng/ml，购自吴江近岸蛋白质科技有限公司)和IL-1β(2ng/ml，购自吴江近岸蛋白质科技有限公司)，置于培养箱，在饱和湿度、37℃、5.0％CO2的条件下进行培养。培养过夜后，添加500U/ml的IL-2(购自吴江近岸蛋白质科技有限公司)继续培养，每2-3天计数并用添加500U/ml IL-2的CIK基础培养基按1×106/ml的密度进行细胞传代。照此方法培养14天，最终统计细胞的扩增倍数，绘制生长曲线；
检测结果如表8和图19所示，单体和二聚体形式的CD3-CD40L双特异性抗体单一使用对CIK细胞的增殖效果均显著地优于抗CD3/抗CD28单克隆全长抗体联合使用，且蛋白用量更少(10ng/ml vs 5ug/ml)：其中二聚体形式的CD3-CD40L BsAb_D可以介导CIK细胞两周扩增367倍，效果最优(实验组2)；单体形式的CD3-CD40L BsAb_M可以介导CIK细胞两周扩增301倍，效果次之(实验组1)；Anti CD3/Anti CD28联用可以介导CIK细胞两周扩增224倍，效果最弱(对照组)。
表8 CIK细胞扩增倍数
| 实验组名 | 对照组1 | 实验组1 | 实验组2 |
| 14天扩增倍数 | 224 | 301 | 367 |
实施例24：CD3-CD27 BsAb_M和CD3-CD27 BsAb_D真核表达载体的构建
在本发明中，以T细胞表面人类CD3蛋白和T细胞正共刺激分子CD27蛋白为靶点的双特异性抗体被命名为CD3-CD27 BsAb。
一、CD3-CD27 BsAb_M和CD3-CD27 BsAb_D构建方案设计
单体形式的CD3-CD27 BsAb_M具体构建方案为：抗CD3 scFv和抗CD27 scFv序列之间通过(GGGGS)3 Linker相连。
二聚体形式的CD3-CD27 BsAb_D具体构建方案为：抗CD3 scFv和抗CD27 scFv序列之间通过IgD铰链区作为Linker相连。
为使双特异性抗体在哺乳细胞中进行表达，针对抗CD3 scFv，抗CD27 scFv及IgD铰链区序列均进行了哺乳系统表达的密码子优化。
具体地，抗CD3 scFv的重链可变区的核苷酸序列如SEQ ID NO.58。
具体地，抗CD3 scFv的轻链可变区的核苷酸序列如SEQ ID NO.59。
具体地，抗CD3 scFv的核苷酸序列如SEQ ID NO.57所示。
具体地，抗CD27 scFv的重链可变区的核苷酸序列如SEQ ID NO.76所示，具体为：
CAGGTGCAGCTGGTGGAGAGCGGCGGCGGCGTGGTGCAGCCCGGCCGCAGCCTGCGCCTGAGCTGCGCCGCCAGCGGCTTCACCTTCAGCAGCTACGACATGCACTGGGTGCGCCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGGCCGTGATCTGGTACGACGGCAGCAACAAGTACTACGCCGACAGCGTGAAGGGCCGCTTCACCATCAGCCGCGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGCGCCGAGGACACCGCCGTGTACTACTGCGCCCGCGGCAGCGGCAACTGGGGCTTCTTCGACTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGC。
具体地，抗CD27 scFv的轻链可变区的核苷酸序列如SEQ ID NO.77所示，具体为：
GACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCGACCGCGTGACCATCACCTGCCGCGCCAGCCAGGGCATCAGCCGCTGGCTGGCCTGGTACCAGCAGAAGCCCGAGAAGGCCCCCAAGAGCCTGATCTACGCCGCCAGCAGCCTGCAGAGCGGCGTGCCCAGCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGTACAACACCTACCCCCGCACCTTCGGCCAGGGCACCAAGGTGGAGATCAAG。
具体地，抗CD27 scFv的核苷酸序列如SEQ ID NO.75所示，具体为：
CAGGTGCAGCTGGTGGAGAGCGGCGGCGGCGTGGTGCAGCCCGGCCGCAGCCTGCGCCTGAGCTGCGCCGCCAGCGGCTTCACCTTCAGCAGCTACGACATGCACTGGGTGCGCCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGGCCGTGATCTGGTACGACGGCAGCAACAAGTACTACGCCGACAGCGTGAAGGGCCGCTTCACCATCAGCCGCGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGCGCCGAGGACACCGCCGTGTACTACTGCGCCCGCGGCAGCGGCAACTGGGGCTTCTTCGACTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCGACCGCGTGACCATCACCTGCCGCGCCAGCCAGGGCATCAGCCGCTGGCTGGCCTGGTACCAGCAGAAGCCCGAGAAGGCCCCCAAGAGCCTGATCTACGCCGCCAGCAGCCTGCAGAGCGGCGTGCCCAGCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGTACAACACCTACCCCCGCACCTTCGGCCAGGGCACCAAGGTGGAGATCAAG。
单体形式的CD3-CD27 BsAb_M连接片段的核苷酸序列如SEQ ID NO.2所示。
二聚体形式的CD3-CD27 BsAb_D连接片段的核苷酸序列如SEQ ID NO.4所示。
为使双特异性抗体在CHO-S细胞中表达并成功分泌到培养基中，选择了抗体分泌型表达的信号肽用于此实施例。
该分泌表达信号肽的氨基酸序列如SEQ ID NO.78所示。
该分泌表达信号肽的核苷酸序列如SEQ ID NO.79所示。
二、CD3-CD27 BsAb_M和CD3-CD27 BsAb_D真核表达载体构建
本发明双特异性抗体的构建与表达，选用哺乳细胞蛋白瞬时表达载体pcDNA3.1(购自上海英骏生物科技有限公司)。为构建单体和二聚体形式的双特异性抗体，分别设计了如表9所示引物，所有引物由苏州金唯智生物科技有限公司合成，扩增所需基因模板由苏州鸿讯科技有限公司合成。
针对CD3-CD27 BsAb_M的克隆构建，首先使用引物pcDNA3.1-Sig-F和Sig-R扩增出信号肽片段，然后分别利用引物Sig-CD3-F和CD3-R、CD3-(GGGGS)3-CD27-F和pcDNA3.1-CD27-R扩增出抗CD3 scFv、(GGGGS)3 Linker、抗CD27 scFv的基因序列；针对CD3-CD27BsAb_D的克隆构建，同样首先使用引物pcDNA3.1-Sig-F和Sig-R扩增出信号肽片段，然后分别利用引物Sig-CD3-F和CD3-R、CD3-IgD-F和IgD-R、IgD-CD27-F和pcDNA3.1-CD27-R扩增出抗CD3 scFv、IgD铰链区、抗CD27 scFv的基因序列。扩增完毕后，利用
PCR一步定向克隆试剂盒(购自吴江近岸蛋白质科技有限公司)分别拼接单体和二聚体形式双特异性抗体全长基因序列并无缝克隆至经EcoRI和HindIII线性化处理的pcDNA3.1表达载体上。目的载体转化大肠杆菌DH5α，利用菌落PCR进行阳性克隆鉴定，鉴定为阳性的重组子(重组质粒)进行测序鉴定。随后将测序正确的重组子(重组质粒)安排质粒中抽，用于CHO-S细胞的转染。
经测序获知，单体形式的CD3-CD27 BsAb_M和二聚体形式的CD3-CD27 BsAb_D的全长基因序列正确，均与预期相符。
具体地，单体形式的CD3-CD27 BsAb_M的核苷酸序列如SEQ ID NO.33所示，具体为：
GACATCAAGCTGCAGCAGAGCGGCGCCGAGCTGGCCCGCCCCGGCGCCAGCGTGAAGATGAGCTGCAAGACCAGCGGCTACACCTTCACCCGCTACACCATGCACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGGCTACATCAACCCCAGCCGCGGCTACACCAACTACAACCAGAAGTTCAAGGACAAGGCCACCCTGACCACCGACAAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCCGCTACTACGACGACCACTACTGCCTGGACTACTGGGGCCAGGGCACCACCCTGACCGTGAGCAGCGTGGAGGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCGTGGACGACATCCAGCTGACCCAGAGCCCCGCCATCATGAGCGCCAGCCCCGGCGAGAAGGTGACCATGACCTGCCGCGCCAGCAGCAGCGTGAGCTACATGAACTGGTACCAGCAGAAGAGCGGCACCAGCCCCAAGCGCTGGATCTACGACACCAGCAAGGTGGCCAGCGGCGTGCCCTACCGCTTCAGCGGCAGCGGCAGCGGCACCAGCTACAGCCTGACCATCAGCAGCATGGAGGCCGAGGACGCCGCCACCTACTACTGCCAGCAGTGGAGCAGCAACCCCCTGACCTTCGGCGCCGGCACCAAGCTGGAGCTGAAGGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCCAGGTGCAGCTGGTGGAGAGCGGCGGCGGCGTGGTGCAGCCCGGCCGCAGCCTGCGCCTGAGCTGCGCCGCCAGCGGCTTCACCTTCAGCAGCTACGACATGCACTGGGTGCGCCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGGCCGTGATCTGGTACGACGGCAGCAACAAGTACTACGCCGACAGCGTGAAGGGCCGCTTCACCATCAGCCGCGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGCGCCGAGGACACCGCCGTGTACTACTGCGCCCGCGGCAGCGGCAACTGGGGCTTCTTCGACTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCGACCGCGTGACCATCACCTGCCGCGCCAGCCAGGGCATCAGCCGCTGGCTGGCCTGGTACCAGCAGAAGCCCGAGAAGGCCCCCAAGAGCCTGATCTACGCCGCCAGCAGCCTGCAGAGCGGCGTGCCCAGCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGTACAACACCTACCCCCGCACCTTCGGCCAGGGCACCAAGGTGGAGATCAAG。
具体地，二聚体形式的CD3-CD27 BsAb_D的核苷酸序列如SEQ ID NO.35所示，具体为：
GACATCAAGCTGCAGCAGAGCGGCGCCGAGCTGGCCCGCCCCGGCGCCAGCGTGAAGATGAGCTGCAAGACCAGCGGCTACACCTTCACCCGCTACACCATGCACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGGCTACATCAACCCCAGCCGCGGCTACACCAACTACAACCAGAAGTTCAAGGACAAGGCCACCCTGACCACCGACAAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCCGCTACTACGACGACCACTACTGCCTGGACTACTGGGGCCAGGGCACCACCCTGACCGTGAGCAGCGTGGAGGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCGTGGACGACATCCAGCTGACCCAGAGCCCCGCCATCATGAGCGCCAGCCCCGGCGAGAAGGTGACCATGACCTGCCGCGCCAGCAGCAGCGTGAGCTACATGAACTGGTACCAGCAGAAGAGCGGCACCAGCCCCAAGCGCTGGATCTACGACACCAGCAAGGTGGCCAGCGGCGTGCCCTACCGCTTCAGCGGCAGCGGCAGCGGCACCAGCTACAGCCTGACCATCAGCAGCATGGAGGCCGAGGACGCCGCCACCTACTACTGCCAGCAGTGGAGCAGCAACCCCCTGACCTTCGGCGCCGGCACCAAGCTGGAGCTGAAGGCCAGCAAGAGCAAGAAGGAGATCTTCCGCTGGCCCGAGAGCCCCAAGGCCCAGGCCAGCAGCGTGCCCACCGCCCAGCCCCAGGCCGAGGGCAGCCTGGCCAAGGCCACCACCGCCCCCGCCACCACCCGCAACACCGGCCGCGGCGGCGAGGAGAAGAAGAAGGAGAAGGAGAAGGAGGAGCAGGAGGAGCGCGAGACCAAGACCCCCGAGTGCCCCAGCCACACCCAGCCCCTGGGCGTGCAGGTGCAGCTGGTGGAGAGCGGCGGCGGCGTGGTGCAGCCCGGCCGCAGCCTGCGCCTGAGCTGCGCCGCCAGCGGCTTCACCTTCAGCAGCTACGACATGCACTGGGTGCGCCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGGCCGTGATCTGGTACGACGGCAGCAACAAGTACTACGCCGACAGCGTGAAGGGCCGCTTCACCATCAGCCGCGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGCGCCGAGGACACCGCCGTGTACTACTGCGCCCGCGGCAGCGGCAACTGGGGCTTCTTCGACTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCGACCGCGTGACCATCACCTGCCGCGCCAGCCAGGGCATCAGCCGCTGGCTGGCCTGGTACCAGCAGAAGCCCGAGAAGGCCCCCAAGAGCCTGATCTACGCCGCCAGCAGCCTGCAGAGCGGCGTGCCCAGCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGTACAACACCTACCCCCGCACCTTCGGCCAGGGCACCAAGGTGGAGATCAAG。
表9.CD3-CD27双特异性抗体基因克隆中使用的引物
实施例25：CD3-CD27 BsAb_M和CD3-CD27 BsAb_D的表达与纯化
一、CD3-CD27 BsAb_M和CD3-CD27 BsAb_D的表达
1.1.CHO-S细胞(购自Thermo Fisher Scientific公司)转染前1天传代密度为0.5～0.6×106/ml；
1.2.转染当天进行细胞密度统计，当密度为1～1.4×106/ml、活力>90％时，可用于质粒转染；
1.3.转染复合物配制：每个项目(CD3-CD27 BsAb_M和CD3-CD27 BsAb_D)需准备两个离心管/培养瓶，以20ml为例，分别放置，取实施例24中所制备重组质粒：
管①中加入600μl PBS，20μg重组质粒，混匀；
管②中加入600μl PBS，20ul FreeStyleTM MAX Transfection Reagent(购自Thermo Fisher Scientific公司)，混匀；
1.4.将稀释后的转染试剂，加入至稀释后的重组质粒中，混合均匀，配制成转染复合物；
1.5.转染复合物静置15～20min后，单滴匀速加入细胞培养物中；
1.6.于37℃，CO2浓度8％，摇床转速130rpm条件下进行转染后细胞培养，5天后收集培养上清进行目的蛋白表达检测。
二、CD3-CD27 BsAb_M和CD3-CD27 BsAb_D的纯化
2.1样品预处理
取上述转染后细胞培养上清20ml，加入缓冲液20mM PB，200mM NaCl调节pH至7.5；
2.2 Protein L亲和层析柱纯化
蛋白纯化层析柱：Protein L亲和层析柱(购自GE Healthcare公司，柱体积1.0ml)
缓冲液A(Buffer A)：PBS，pH7.4
缓冲液B(Buffer B)：0.1M Glycine,pH3.0
缓冲液C(Buffer C)：0.1M Glycine,pH2.7
纯化过程：采用AKTA explorer 100型蛋白纯化系统(购自GE Healthcare公司)，用Buffer A预处理Protein L亲和层析柱，取培养上清上样，收集流出液。上样完毕后，用至少1.5ml Buffer A平衡层析柱，平衡后分别用Buffer B和Buffer C洗脱，收集目的蛋白洗脱液(洗脱液的收集管需要预先加入1％的1M Tris,pH8.0来中和洗脱液pH值，Tris终浓度约为10mM)，最后浓缩透析至缓冲液PBS中。
最终纯化的CD3-CD27 BsAb_M和CD3-CD27 BsAb_D重组蛋白经SDS-PAGE分析，还原和非还原条件下电泳图如图20所示。从图中可以看出，经Protein L亲和层析柱纯化后，CD3-CD27 BsAb_M和CD3-CD27 BsAb_D重组蛋白的纯度均>95％；其中CD3-CD27 BsAb_M重组蛋白的理论分子量为53.2kDa，还原和非还原条件下该蛋白均呈现单一电泳条带，分子量与单体一致，因此该双特异性抗体为单体形式(图20A)；CD3-CD27 BsAb_D重组蛋白的理论分子量为61.1kDa，还原条件下该蛋白电泳条带所呈现分子量与单体一致，非还原条件下电泳条带所呈现分子量与二聚体一致(图20B)，说明两个蛋白分子可通过二硫键相互连接，因此该双特异性抗体为二聚体形式。
此外，纯化的重组蛋白样品经N/C末端序列分析，结果表明所表达的重组蛋白样品均读框无误，与理论N/C末端氨基酸序列一致，质谱分析进一步确认CD3-CD27 BsAb_M为单体形式，CD3-CD27 BsAb_D为二聚体形式。
因此，可得知，单体形式的CD3-CD27 BsAb_M的氨基酸序列如SEQ ID NO.32所示，具体为：
DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKGGGGSGGGGSGGGGSQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYDMHWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGSGNWGFFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQGISRWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNTYPRTFGQGTKVEIK。
二聚体形式的CD3-CD27 BsAb_D的氨基酸序列如SEQ ID NO.34所示，具体为：
DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKASKSKKEIFRWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEKEKEEQEERETKTPECPSHTQPLGVQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYDMHWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGSGNWGFFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQGISRWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNTYPRTFGQGTKVEIK。
抗CD3 scFv的氨基酸序列如SEQ ID NO.36所示。
抗CD3 scFv的重链可变区的氨基酸序列如SEQ ID NO.37所示。
抗CD3 scFv的轻链可变区的氨基酸序列如SEQ ID NO.38所示。
抗CD27 scFv的氨基酸序列如SEQ ID NO.54所示，具体为：
QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYDMHWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGSGNWGFFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQGISRWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNTYPRTFGQGTKVEIK。
抗CD27 scFv的重链可变区的氨基酸序列如SEQ ID NO.55所示，具体为：
QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYDMHWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGSGNWGFFDYWGQGTLVTVSS。
抗CD27 scFv的轻链可变区的氨基酸序列如SEQ ID NO.56所示，具体为：
DIQMTQSPSSLSASVGDRVTITCRASQGISRWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNTYPRTFGQGTKVEIK。
单体形式的CD3-CD27 BsAb_M中连接片段的氨基酸序列如SEQ ID NO.1所示。
二聚体形式的CD3-CD27 BsAb_D中连接片段的氨基酸序列如SEQ ID NO.3所示。
实施例26：ELISA检测CD3-CD27 BsAb_M和CD3-CD27 BsAb_D的抗原结合活性
ELISA操作步骤：
1.重组抗原包被：人类CD3-hFc与人类CD27-hFc融合蛋白(购自吴江近岸蛋白质科技有限公司)分别包被96孔板，抗原浓度为1μg/ml，包被体积为100μl/孔，包被条件为37℃1小时或4℃过夜，包被缓冲液(PBS)的配方为：3.58g Na2HPO4，0.24g NaH2PO4，0.2g KCl，8.2g NaCl，950ml H2O，用1mol/L HCl或1mol/L NaOH调pH至7.4，补水至1L；
2.封闭：PBS洗板4次后，加入封闭液PBSA(PBS+2％BSA(V/W))，200μl/孔。37℃封闭1小时；
3.加样：PBS洗板4次后，分别加入纯化的双特异性抗体样品，100μl/孔，37℃孵育1小时，样品梯度配制方法：以10μg/ml纯化的CD3-CD27 BsAb_M或CD3-CD27 BsAb_D作为起始浓度，进行倍比稀释6个梯度，每个梯度设置2个复孔；
4.显色：PBST(PBS+0.05％Tween-20(V/V))洗板4次后，用封闭液PBSA按1/5000稀释HRP标记的显色抗体(购自Abcam公司)，按100μl/孔加入，37℃孵育1小时。PBS洗板4次后，添加显色液TMB(购自KPL公司)，100μl/孔，室温避光显色5～10分钟；
5.终止反应与结果测定：添加终止液(1M HCl)，100μl/孔，在酶标仪上450nm波长下读取吸光值。
ELISA结果如图21A和图21B所示：图21A说明CD3-CD27 BsAb_M与重组抗原CD3-hFc和CD27-hFc均具有体外结合活性，其中CD27结合活性较CD3结合活性更高；图21B说明CD3-CD27 BsAb_D与重组抗原CD3-hFc和CD27-hFc同样具有体外结合活性，其中CD27结合活性更高。
实施例27：CD3-CD27双特异抗体介导的CIK细胞增殖
以人外周血单个核细胞(Peripheral blood mononuclear cell，PBMC)为实验材料，用本发明所制备的上述单体形式的双特异抗体CD3-CD27 BsAb_M、二聚体形式的双特异抗体CD3-CD27 BsAb_D、以及抗CD3/抗CD28单克隆全长抗体联用(Anti-CD3/Anti-CD28)分别作用于同一供体来源的人血PBMC，细胞培养后进行计数，比较扩增倍数。
1.PBMC的分离：取抗凝血，加入等体积的医用生理盐水，沿离心管壁缓慢加入与血液等体积的淋巴细胞分离液(购自GE Healthcare公司)，保持液面分层明显，2000rpm离心20min，吸取中间白雾状的细胞层于新的离心管中，加入2倍以上体积的PBS缓冲液洗涤，1100rpm离心10min，重复洗涤一次，用少量预冷的X-vivo 15无血清培养基(购自Lonza公司)重悬，细胞计数待用；
2.CIK细胞培养与扩增：将PBMC用CIK基础培养基(90％X-vivo15+10％FBS)重悬，调整细胞密度为1×106/ml，分别设计以下3个实验组：对照组(Anti-CD3 5ug/ml和Anti-CD28 5ug/ml包被细胞培养板，全长抗体均购自吴江近岸蛋白质科技有限公司)；实验组1(溶液状态下添加双特异性抗体CD3-CD27 BsAb_M 10ng/ml)；实验组2(溶液状态下添加双特异性抗体CD3-CD27 BsAb_D 10ng/ml)。此外，三组实验细胞同时添加细胞因子IFN-γ(200ng/ml，购自吴江近岸蛋白质科技有限公司)和IL-1β(2ng/ml，购自吴江近岸蛋白质科技有限公司)，置于培养箱，在饱和湿度、37℃、5.0％CO2的条件下进行培养。培养过夜后，添加500U/ml的IL-2(购自吴江近岸蛋白质科技有限公司)继续培养，每2-3天计数并用添加500U/ml IL-2的CIK基础培养基按1×106/ml的密度进行细胞传代。照此方法培养30天，最终统计细胞的扩增倍数，绘制生长曲线；
检测结果如图22所示，单体和二聚体形式的CD3-CD27双特异性抗体单一使用对CIK细胞的增殖效果均优于抗CD3/抗CD28单克隆全长抗体联合使用：培养18天后，Anti-CD3/Anti-CD28联用出现大量细胞死亡，细胞扩增倍数显著下降；而添加单体形式的CD3-CD27 BsAb_M或二聚体形式的CD3-CD27 BsAb_D均未出现细胞死亡，细胞扩增速度未见明显下降。因此本发明制备的两种形式的CD3-CD27双特异性抗体均可有效扩增和延长CIK细胞的生存期，其中二聚体形式的效果更好。
以上所述，仅为本发明的较佳实施例，并非对本发明任何形式上和实质上的限制，应当指出，对于本技术领域的普通技术人员，在不脱离本发明方法的前提下，还将可以做出若干改进和补充，这些改进和补充也应视为本发明的保护范围。凡熟悉本专业的技术人员，在不脱离本发明的精神和范围的情况下，当可利用以上所揭示的技术内容而做出的些许更动、修饰与演变的等同变化，均为本发明的等效实施例；同时，凡依据本发明的实质技术对上述实施例所作的任何等同变化的更动、修饰与演变，均仍属于本发明的技术方案的范围内。
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<213> Artificial
<220>
<223> T细胞正共刺激分子人类CD27胞外区的氨基酸序列
<400> 11
Ala Thr Pro Ala Pro Lys Ser Cys Pro Glu Arg His Tyr Trp Ala Gln
1 5 10 15
Gly Lys Leu Cys Cys Gln Met Cys Glu Pro Gly Thr Phe Leu Val Lys
20 25 30
Asp Cys Asp Gln His Arg Lys Ala Ala Gln Cys Asp Pro Cys Ile Pro
35 40 45
Gly Val Ser Phe Ser Pro Asp His His Thr Arg Pro His Cys Glu Ser
50 55 60
Cys Arg His Cys Asn Ser Gly Leu Leu Val Arg Asn Cys Thr Ile Thr
65 70 75 80
Ala Asn Ala Glu Cys Ala Cys Arg Asn Gly Trp Gln Cys Arg Asp Lys
85 90 95
Glu Cys Thr Glu Cys Asp Pro Leu Pro Asn Pro Ser Leu Thr Ala Arg
100 105 110
Ser Ser Gln Ala Leu Ser Pro His Pro Gln Pro Thr His Leu Pro Tyr
115 120 125
Val Ser Glu Met Leu Glu Ala Arg Thr Ala Gly His Met Gln Thr Leu
130 135 140
Ala Asp Phe Arg Gln Leu Pro Ala Arg Thr Leu Ser Thr His Trp Pro
145 150 155 160
Pro Gln Arg Ser Leu Cys Ser Ser Asp Phe Ile Arg
165 170
<210> 12
<211> 504
<212> PRT
<213> Artificial
<220>
<223> 单体形式的CD3-4-1BB BsAb_M的氨基酸序列
<400> 12
Asp Ile Lys Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala
1 5 10 15
Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Arg Tyr
20 25 30
Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe
50 55 60
Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Thr Leu Thr Val Ser Ser Val Glu Gly Gly Ser Gly Gly Ser Gly
115 120 125
Gly Ser Gly Gly Ser Gly Gly Val Asp Asp Ile Gln Leu Thr Gln Ser
130 135 140
Pro Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys
145 150 155 160
Arg Ala Ser Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser
165 170 175
Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Val Ala Ser
180 185 190
Gly Val Pro Tyr Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser
195 200 205
Leu Thr Ile Ser Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys
210 215 220
Gln Gln Trp Ser Ser Asn Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu
225 230 235 240
Glu Leu Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
245 250 255
Gly Ser Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro
260 265 270
Ser Glu Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser
275 280 285
Gly Tyr Tyr Trp Ser Trp Ile Arg Gln Ser Pro Glu Lys Gly Leu Glu
290 295 300
Trp Ile Gly Glu Ile Asn His Gly Gly Tyr Val Thr Tyr Asn Pro Ser
305 310 315 320
Leu Glu Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe
325 330 335
Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr
340 345 350
Cys Ala Arg Asp Tyr Gly Pro Gly Asn Tyr Asp Trp Tyr Phe Asp Leu
355 360 365
Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser
370 375 380
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Leu Thr Gln
385 390 395 400
Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser
405 410 415
Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr Leu Ala Trp Tyr Gln Gln
420 425 430
Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr Asp Ala Ser Asn Arg
435 440 445
Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
450 455 460
Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr
465 470 475 480
Tyr Cys Gln Gln Arg Ser Asn Trp Pro Pro Ala Leu Thr Phe Cys Gly
485 490 495
Gly Thr Lys Val Glu Ile Lys Arg
500
<210> 13
<211> 1512
<212> DNA
<213> Artificial
<220>
<223> 单体形式的CD3-4-1BB BsAb_M的核苷酸序列
<400> 13
gacatcaagc tgcagcagag cggcgccgag ctggcccgcc ccggcgccag cgtgaagatg 60
agctgcaaga ccagcggcta caccttcacc cgctacacca tgcactgggt gaagcagcgc 120
cccggccagg gcctggagtg gatcggctac atcaacccca gccgcggcta caccaactac 180
aaccagaagt tcaaggacaa ggccaccctg accaccgaca agagcagcag caccgcctac 240
atgcagctga gcagcctgac cagcgaggac agcgccgtgt actactgcgc ccgctactac 300
gacgaccact actgcctgga ctactggggc cagggcacca ccctgaccgt gagcagcgtg 360
gagggcggca gcggcggcag cggcggcagc ggcggcagcg gcggcgtgga cgacatccag 420
ctgacccaga gccccgccat catgagcgcc agccccggcg agaaggtgac catgacctgc 480
cgcgccagca gcagcgtgag ctacatgaac tggtaccagc agaagagcgg caccagcccc 540
aagcgctgga tctacgacac cagcaaggtg gccagcggcg tgccctaccg cttcagcggc 600
agcggcagcg gcaccagcta cagcctgacc atcagcagca tggaggccga ggacgccgcc 660
acctactact gccagcagtg gagcagcaac cccctgacct tcggcgccgg caccaagctg 720
gagctgaagg gcggcggcgg cagcggcggc ggcggcagcg gcggcggcgg cagccaggtg 780
cagctgcagc agtggggcgc cggcctgctg aagcccagcg agaccctgag cctgacctgc 840
gccgtgtacg gcggcagctt cagcggctac tactggagct ggatccgcca gagccccgag 900
aagggcctgg agtggatcgg cgagatcaac cacggcggct acgtgaccta caaccccagc 960
ctggagagcc gcgtgaccat cagcgtggac accagcaaga accagttcag cctgaagctg 1020
agcagcgtga ccgccgccga caccgccgtg tactactgcg cccgcgacta cggccccggc 1080
aactacgact ggtacttcga cctgtggggc cgcggcaccc tggtgaccgt gagcagcggc 1140
ggcggcggca gcggcggcgg cggcagcggc ggcggcggca gcgagatcgt gctgacccag 1200
agccccgcca ccctgagcct gagccccggc gagcgcgcca ccctgagctg ccgcgccagc 1260
cagagcgtga gcagctacct ggcctggtac cagcagaagc ccggccaggc cccccgcctg 1320
ctgatctacg acgccagcaa ccgcgccacc ggcatccccg cccgcttcag cggcagcggc 1380
agcggcaccg acttcaccct gaccatcagc agcctggagc ccgaggactt cgccgtgtac 1440
tactgccagc agcgcagcaa ctggcccccc gccctgacct tctgcggcgg caccaaggtg 1500
gagatcaagc gc 1512
<210> 14
<211> 570
<212> PRT
<213> Artificial
<220>
<223> 二聚体形式的CD3-4-1BB BsAb_D的氨基酸序列
<400> 14
Asp Ile Lys Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala
1 5 10 15
Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Arg Tyr
20 25 30
Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe
50 55 60
Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Thr Leu Thr Val Ser Ser Val Glu Gly Gly Ser Gly Gly Ser Gly
115 120 125
Gly Ser Gly Gly Ser Gly Gly Val Asp Asp Ile Gln Leu Thr Gln Ser
130 135 140
Pro Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys
145 150 155 160
Arg Ala Ser Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser
165 170 175
Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Val Ala Ser
180 185 190
Gly Val Pro Tyr Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser
195 200 205
Leu Thr Ile Ser Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys
210 215 220
Gln Gln Trp Ser Ser Asn Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu
225 230 235 240
Glu Leu Lys Ala Ser Lys Ser Lys Lys Glu Ile Phe Arg Trp Pro Glu
245 250 255
Ser Pro Lys Ala Gln Ala Ser Ser Val Pro Thr Ala Gln Pro Gln Ala
260 265 270
Glu Gly Ser Leu Ala Lys Ala Thr Thr Ala Pro Ala Thr Thr Arg Asn
275 280 285
Thr Gly Arg Gly Gly Glu Glu Lys Lys Lys Glu Lys Glu Lys Glu Glu
290 295 300
Gln Glu Glu Arg Glu Thr Lys Thr Pro Glu Cys Pro Ser His Thr Gln
305 310 315 320
Pro Leu Gly Val Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu
325 330 335
Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser
340 345 350
Phe Ser Gly Tyr Tyr Trp Ser Trp Ile Arg Gln Ser Pro Glu Lys Gly
355 360 365
Leu Glu Trp Ile Gly Glu Ile Asn His Gly Gly Tyr Val Thr Tyr Asn
370 375 380
Pro Ser Leu Glu Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn
385 390 395 400
Gln Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val
405 410 415
Tyr Tyr Cys Ala Arg Asp Tyr Gly Pro Gly Asn Tyr Asp Trp Tyr Phe
420 425 430
Asp Leu Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly
435 440 445
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Leu
450 455 460
Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr
465 470 475 480
Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr Leu Ala Trp Tyr
485 490 495
Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr Asp Ala Ser
500 505 510
Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly
515 520 525
Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu Asp Phe Ala
530 535 540
Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Pro Ala Leu Thr Phe
545 550 555 560
Cys Gly Gly Thr Lys Val Glu Ile Lys Arg
565 570
<210> 15
<211> 1710
<212> DNA
<213> Artificial
<220>
<223> 二聚体形式的CD3-4-1BB BsAb_D的核苷酸序列
<400> 15
gacatcaagc tgcagcagag cggcgccgag ctggcccgcc ccggcgccag cgtgaagatg 60
agctgcaaga ccagcggcta caccttcacc cgctacacca tgcactgggt gaagcagcgc 120
cccggccagg gcctggagtg gatcggctac atcaacccca gccgcggcta caccaactac 180
aaccagaagt tcaaggacaa ggccaccctg accaccgaca agagcagcag caccgcctac 240
atgcagctga gcagcctgac cagcgaggac agcgccgtgt actactgcgc ccgctactac 300
gacgaccact actgcctgga ctactggggc cagggcacca ccctgaccgt gagcagcgtg 360
gagggcggca gcggcggcag cggcggcagc ggcggcagcg gcggcgtgga cgacatccag 420
ctgacccaga gccccgccat catgagcgcc agccccggcg agaaggtgac catgacctgc 480
cgcgccagca gcagcgtgag ctacatgaac tggtaccagc agaagagcgg caccagcccc 540
aagcgctgga tctacgacac cagcaaggtg gccagcggcg tgccctaccg cttcagcggc 600
agcggcagcg gcaccagcta cagcctgacc atcagcagca tggaggccga ggacgccgcc 660
acctactact gccagcagtg gagcagcaac cccctgacct tcggcgccgg caccaagctg 720
gagctgaagg ccagcaagag caagaaggag atcttccgct ggcccgagag ccccaaggcc 780
caggccagca gcgtgcccac cgcccagccc caggccgagg gcagcctggc caaggccacc 840
accgcccccg ccaccacccg caacaccggc cgcggcggcg aggagaagaa gaaggagaag 900
gagaaggagg agcaggagga gcgcgagacc aagacccccg agtgccccag ccacacccag 960
cccctgggcg tgcaggtgca gctgcagcag tggggcgccg gcctgctgaa gcccagcgag 1020
accctgagcc tgacctgcgc cgtgtacggc ggcagcttca gcggctacta ctggagctgg 1080
atccgccaga gccccgagaa gggcctggag tggatcggcg agatcaacca cggcggctac 1140
gtgacctaca accccagcct ggagagccgc gtgaccatca gcgtggacac cagcaagaac 1200
cagttcagcc tgaagctgag cagcgtgacc gccgccgaca ccgccgtgta ctactgcgcc 1260
cgcgactacg gccccggcaa ctacgactgg tacttcgacc tgtggggccg cggcaccctg 1320
gtgaccgtga gcagcggcgg cggcggcagc ggcggcggcg gcagcggcgg cggcggcagc 1380
gagatcgtgc tgacccagag ccccgccacc ctgagcctga gccccggcga gcgcgccacc 1440
ctgagctgcc gcgccagcca gagcgtgagc agctacctgg cctggtacca gcagaagccc 1500
ggccaggccc cccgcctgct gatctacgac gccagcaacc gcgccaccgg catccccgcc 1560
cgcttcagcg gcagcggcag cggcaccgac ttcaccctga ccatcagcag cctggagccc 1620
gaggacttcg ccgtgtacta ctgccagcag cgcagcaact ggccccccgc cctgaccttc 1680
tgcggcggca ccaaggtgga gatcaagcgc 1710
<210> 16
<211> 505
<212> PRT
<213> Artificial
<220>
<223> 单体形式的CD3-ICOS BsAb_M的氨基酸序列
<400> 16
Asp Ile Lys Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala
1 5 10 15
Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Arg Tyr
20 25 30
Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe
50 55 60
Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Thr Leu Thr Val Ser Ser Val Glu Gly Gly Ser Gly Gly Ser Gly
115 120 125
Gly Ser Gly Gly Ser Gly Gly Val Asp Asp Ile Gln Leu Thr Gln Ser
130 135 140
Pro Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys
145 150 155 160
Arg Ala Ser Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser
165 170 175
Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Val Ala Ser
180 185 190
Gly Val Pro Tyr Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser
195 200 205
Leu Thr Ile Ser Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys
210 215 220
Gln Gln Trp Ser Ser Asn Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu
225 230 235 240
Glu Leu Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
245 250 255
Gly Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
260 265 270
Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr
275 280 285
Gly Tyr Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
290 295 300
Trp Met Gly Trp Ile Asn Pro His Ser Gly Gly Thr Asn Tyr Ala Gln
305 310 315 320
Lys Phe Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr
325 330 335
Ala Tyr Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr
340 345 350
Tyr Cys Ala Arg Thr Tyr Tyr Tyr Asp Ser Ser Gly Tyr Tyr His Asp
355 360 365
Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Gly
370 375 380
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile
385 390 395 400
Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly Asp Arg
405 410 415
Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Arg Leu Leu Ala
420 425 430
Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Val
435 440 445
Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly
450 455 460
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp
465 470 475 480
Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Asn Ser Phe Pro Trp Thr Phe
485 490 495
Gly Gln Gly Thr Lys Val Glu Ile Lys
500 505
<210> 17
<211> 1515
<212> DNA
<213> Artificial
<220>
<223> 单体形式的CD3-ICOS BsAb_M的核苷酸序列
<400> 17
gacatcaagc tgcagcagag cggcgccgag ctggcccgcc ccggcgccag cgtgaagatg 60
agctgcaaga ccagcggcta caccttcacc cgctacacca tgcactgggt gaagcagcgc 120
cccggccagg gcctggagtg gatcggctac atcaacccca gccgcggcta caccaactac 180
aaccagaagt tcaaggacaa ggccaccctg accaccgaca agagcagcag caccgcctac 240
atgcagctga gcagcctgac cagcgaggac agcgccgtgt actactgcgc ccgctactac 300
gacgaccact actgcctgga ctactggggc cagggcacca ccctgaccgt gagcagcgtg 360
gagggcggca gcggcggcag cggcggcagc ggcggcagcg gcggcgtgga cgacatccag 420
ctgacccaga gccccgccat catgagcgcc agccccggcg agaaggtgac catgacctgc 480
cgcgccagca gcagcgtgag ctacatgaac tggtaccagc agaagagcgg caccagcccc 540
aagcgctgga tctacgacac cagcaaggtg gccagcggcg tgccctaccg cttcagcggc 600
agcggcagcg gcaccagcta cagcctgacc atcagcagca tggaggccga ggacgccgcc 660
acctactact gccagcagtg gagcagcaac cccctgacct tcggcgccgg caccaagctg 720
gagctgaagg gcggcggcgg cagcggcggc ggcggcagcg gcggcggcgg cagccaggtg 780
cagctggtgc agagcggcgc cgaggtgaag aagcccggcg ccagcgtgaa ggtgagctgc 840
aaggccagcg gctacacctt caccggctac tacatgcact gggtgcgcca ggcccccggc 900
cagggcctgg agtggatggg ctggatcaac ccccacagcg gcggcaccaa ctacgcccag 960
aagttccagg gccgcgtgac catgacccgc gacaccagca tcagcaccgc ctacatggag 1020
ctgagccgcc tgcgcagcga cgacaccgcc gtgtactact gcgcccgcac ctactactac 1080
gacagcagcg gctactacca cgacgccttc gacatctggg gccagggcac catggtgacc 1140
gtgagcagcg gcggcggcgg cagcggcggc ggcggcagcg gcggcggcgg cagcgacatc 1200
cagatgaccc agagccccag cagcgtgagc gccagcgtgg gcgaccgcgt gaccatcacc 1260
tgccgcgcca gccagggcat cagccgcctg ctggcctggt accagcagaa gcccggcaag 1320
gcccccaagc tgctgatcta cgtggccagc agcctgcaga gcggcgtgcc cagccgcttc 1380
agcggcagcg gcagcggcac cgacttcacc ctgaccatca gcagcctgca gcccgaggac 1440
ttcgccacct actactgcca gcaggccaac agcttcccct ggaccttcgg ccagggcacc 1500
aaggtggaga tcaag 1515
<210> 18
<211> 571
<212> PRT
<213> Artificial
<220>
<223> 二聚体形式的CD3-ICOS BsAb_D的氨基酸序列
<400> 18
Asp Ile Lys Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala
1 5 10 15
Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Arg Tyr
20 25 30
Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe
50 55 60
Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Thr Leu Thr Val Ser Ser Val Glu Gly Gly Ser Gly Gly Ser Gly
115 120 125
Gly Ser Gly Gly Ser Gly Gly Val Asp Asp Ile Gln Leu Thr Gln Ser
130 135 140
Pro Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys
145 150 155 160
Arg Ala Ser Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser
165 170 175
Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Val Ala Ser
180 185 190
Gly Val Pro Tyr Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser
195 200 205
Leu Thr Ile Ser Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys
210 215 220
Gln Gln Trp Ser Ser Asn Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu
225 230 235 240
Glu Leu Lys Ala Ser Lys Ser Lys Lys Glu Ile Phe Arg Trp Pro Glu
245 250 255
Ser Pro Lys Ala Gln Ala Ser Ser Val Pro Thr Ala Gln Pro Gln Ala
260 265 270
Glu Gly Ser Leu Ala Lys Ala Thr Thr Ala Pro Ala Thr Thr Arg Asn
275 280 285
Thr Gly Arg Gly Gly Glu Glu Lys Lys Lys Glu Lys Glu Lys Glu Glu
290 295 300
Gln Glu Glu Arg Glu Thr Lys Thr Pro Glu Cys Pro Ser His Thr Gln
305 310 315 320
Pro Leu Gly Val Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys
325 330 335
Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr
340 345 350
Phe Thr Gly Tyr Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly
355 360 365
Leu Glu Trp Met Gly Trp Ile Asn Pro His Ser Gly Gly Thr Asn Tyr
370 375 380
Ala Gln Lys Phe Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile
385 390 395 400
Ser Thr Ala Tyr Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala
405 410 415
Val Tyr Tyr Cys Ala Arg Thr Tyr Tyr Tyr Asp Ser Ser Gly Tyr Tyr
420 425 430
His Asp Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser
435 440 445
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
450 455 460
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly
465 470 475 480
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Arg Leu
485 490 495
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
500 505 510
Tyr Val Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
515 520 525
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
530 535 540
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Asn Ser Phe Pro Trp
545 550 555 560
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
565 570
<210> 19
<211> 1713
<212> DNA
<213> Artificial
<220>
<223> 二聚体形式的CD3-ICOS BsAb_D的核苷酸序列
<400> 19
gacatcaagc tgcagcagag cggcgccgag ctggcccgcc ccggcgccag cgtgaagatg 60
agctgcaaga ccagcggcta caccttcacc cgctacacca tgcactgggt gaagcagcgc 120
cccggccagg gcctggagtg gatcggctac atcaacccca gccgcggcta caccaactac 180
aaccagaagt tcaaggacaa ggccaccctg accaccgaca agagcagcag caccgcctac 240
atgcagctga gcagcctgac cagcgaggac agcgccgtgt actactgcgc ccgctactac 300
gacgaccact actgcctgga ctactggggc cagggcacca ccctgaccgt gagcagcgtg 360
gagggcggca gcggcggcag cggcggcagc ggcggcagcg gcggcgtgga cgacatccag 420
ctgacccaga gccccgccat catgagcgcc agccccggcg agaaggtgac catgacctgc 480
cgcgccagca gcagcgtgag ctacatgaac tggtaccagc agaagagcgg caccagcccc 540
aagcgctgga tctacgacac cagcaaggtg gccagcggcg tgccctaccg cttcagcggc 600
agcggcagcg gcaccagcta cagcctgacc atcagcagca tggaggccga ggacgccgcc 660
acctactact gccagcagtg gagcagcaac cccctgacct tcggcgccgg caccaagctg 720
gagctgaagg ccagcaagag caagaaggag atcttccgct ggcccgagag ccccaaggcc 780
caggccagca gcgtgcccac cgcccagccc caggccgagg gcagcctggc caaggccacc 840
accgcccccg ccaccacccg caacaccggc cgcggcggcg aggagaagaa gaaggagaag 900
gagaaggagg agcaggagga gcgcgagacc aagacccccg agtgccccag ccacacccag 960
cccctgggcg tgcaggtgca gctggtgcag agcggcgccg aggtgaagaa gcccggcgcc 1020
agcgtgaagg tgagctgcaa ggccagcggc tacaccttca ccggctacta catgcactgg 1080
gtgcgccagg cccccggcca gggcctggag tggatgggct ggatcaaccc ccacagcggc 1140
ggcaccaact acgcccagaa gttccagggc cgcgtgacca tgacccgcga caccagcatc 1200
agcaccgcct acatggagct gagccgcctg cgcagcgacg acaccgccgt gtactactgc 1260
gcccgcacct actactacga cagcagcggc tactaccacg acgccttcga catctggggc 1320
cagggcacca tggtgaccgt gagcagcggc ggcggcggca gcggcggcgg cggcagcggc 1380
ggcggcggca gcgacatcca gatgacccag agccccagca gcgtgagcgc cagcgtgggc 1440
gaccgcgtga ccatcacctg ccgcgccagc cagggcatca gccgcctgct ggcctggtac 1500
cagcagaagc ccggcaaggc ccccaagctg ctgatctacg tggccagcag cctgcagagc 1560
ggcgtgccca gccgcttcag cggcagcggc agcggcaccg acttcaccct gaccatcagc 1620
agcctgcagc ccgaggactt cgccacctac tactgccagc aggccaacag cttcccctgg 1680
accttcggcc agggcaccaa ggtggagatc aag 1713
<210> 20
<211> 500
<212> PRT
<213> Artificial
<220>
<223> 单体形式的CD3-OX40 BsAb_M的氨基酸序列
<400> 20
Asp Ile Lys Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala
1 5 10 15
Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Arg Tyr
20 25 30
Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe
50 55 60
Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Thr Leu Thr Val Ser Ser Val Glu Gly Gly Ser Gly Gly Ser Gly
115 120 125
Gly Ser Gly Gly Ser Gly Gly Val Asp Asp Ile Gln Leu Thr Gln Ser
130 135 140
Pro Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys
145 150 155 160
Arg Ala Ser Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser
165 170 175
Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Val Ala Ser
180 185 190
Gly Val Pro Tyr Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser
195 200 205
Leu Thr Ile Ser Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys
210 215 220
Gln Gln Trp Ser Ser Asn Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu
225 230 235 240
Glu Leu Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
245 250 255
Gly Ser Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
260 265 270
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
275 280 285
Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
290 295 300
Ser Tyr Ile Ser Ser Ser Ser Ser Thr Ile Tyr Tyr Ala Asp Ser Val
305 310 315 320
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr
325 330 335
Leu Gln Met Asn Ser Leu Arg Asp Glu Asp Thr Ala Val Tyr Tyr Cys
340 345 350
Ala Arg Gly Val Tyr His Asn Gly Trp Ser Phe Phe Asp Tyr Trp Gly
355 360 365
Gln Gly Thr Leu Leu Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly
370 375 380
Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro
385 390 395 400
Ser Ser Leu Ser Ala Ser Val Gly Asn Arg Val Thr Ile Thr Cys Arg
405 410 415
Ala Ser Gln Asp Ile Ser Ser Trp Leu Ala Trp Tyr Gln Gln Lys Pro
420 425 430
Glu Lys Ala Pro Lys Ser Leu Ile Tyr Ala Ala Ser Ser Leu Gln Ser
435 440 445
Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
450 455 460
Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys
465 470 475 480
Gln Gln Tyr Asn Ser Tyr Pro Leu Thr Phe Gly Gln Gly Thr Arg Leu
485 490 495
Glu Ile Lys Arg
500
<210> 21
<211> 1500
<212> DNA
<213> Artificial
<220>
<223> 单体形式的CD3-OX40 BsAb_M的核苷酸序列
<400> 21
gacatcaagc tgcagcagag cggcgccgag ctggcccgcc ccggcgccag cgtgaagatg 60
agctgcaaga ccagcggcta caccttcacc cgctacacca tgcactgggt gaagcagcgc 120
cccggccagg gcctggagtg gatcggctac atcaacccca gccgcggcta caccaactac 180
aaccagaagt tcaaggacaa ggccaccctg accaccgaca agagcagcag caccgcctac 240
atgcagctga gcagcctgac cagcgaggac agcgccgtgt actactgcgc ccgctactac 300
gacgaccact actgcctgga ctactggggc cagggcacca ccctgaccgt gagcagcgtg 360
gagggcggca gcggcggcag cggcggcagc ggcggcagcg gcggcgtgga cgacatccag 420
ctgacccaga gccccgccat catgagcgcc agccccggcg agaaggtgac catgacctgc 480
cgcgccagca gcagcgtgag ctacatgaac tggtaccagc agaagagcgg caccagcccc 540
aagcgctgga tctacgacac cagcaaggtg gccagcggcg tgccctaccg cttcagcggc 600
agcggcagcg gcaccagcta cagcctgacc atcagcagca tggaggccga ggacgccgcc 660
acctactact gccagcagtg gagcagcaac cccctgacct tcggcgccgg caccaagctg 720
gagctgaagg gcggcggcgg cagcggcggc ggcggcagcg gcggcggcgg cagccagctg 780
gtggagagcg gcggcggcct ggtgcagccc ggcggcagcc tgcgcctgag ctgcgccgcc 840
agcggcttca ccttcagcag ctacagcatg aactgggtgc gccaggcccc cggcaagggc 900
ctggagtggg tgagctacat cagcagcagc agcagcacca tctactacgc cgacagcgtg 960
aagggccgct tcaccatcag ccgcgacaac gccaagaaca gcctgtacct gcagatgaac 1020
agcctgcgcg acgaggacac cgccgtgtac tactgcgccc gcggcgtgta ccacaacggc 1080
tggagcttct tcgactactg gggccagggc accctgctga ccgtgagcag cggcggcggc 1140
ggcagcggcg gcggcggcag cggcggcggc ggcagcgaca tccagatgac ccagagcccc 1200
agcagcctga gcgccagcgt gggcaaccgc gtgaccatca cctgccgcgc cagccaggac 1260
atcagcagct ggctggcctg gtaccagcag aagcccgaga aggcccccaa gagcctgatc 1320
tacgccgcca gcagcctgca gagcggcgtg cccagccgct tcagcggcag cggcagcggc 1380
accgacttca ccctgaccat cagcagcctg cagcccgagg acttcgccac ctactactgc 1440
cagcagtaca acagctaccc cctgaccttc ggccagggca cccgcctgga gatcaagcgc 1500
<210> 22
<211> 566
<212> PRT
<213> Artificial
<220>
<223> 二聚体形式的CD3-OX40 BsAb_D的氨基酸序列
<400> 22
Asp Ile Lys Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala
1 5 10 15
Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Arg Tyr
20 25 30
Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe
50 55 60
Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Thr Leu Thr Val Ser Ser Val Glu Gly Gly Ser Gly Gly Ser Gly
115 120 125
Gly Ser Gly Gly Ser Gly Gly Val Asp Asp Ile Gln Leu Thr Gln Ser
130 135 140
Pro Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys
145 150 155 160
Arg Ala Ser Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser
165 170 175
Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Val Ala Ser
180 185 190
Gly Val Pro Tyr Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser
195 200 205
Leu Thr Ile Ser Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys
210 215 220
Gln Gln Trp Ser Ser Asn Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu
225 230 235 240
Glu Leu Lys Ala Ser Lys Ser Lys Lys Glu Ile Phe Arg Trp Pro Glu
245 250 255
Ser Pro Lys Ala Gln Ala Ser Ser Val Pro Thr Ala Gln Pro Gln Ala
260 265 270
Glu Gly Ser Leu Ala Lys Ala Thr Thr Ala Pro Ala Thr Thr Arg Asn
275 280 285
Thr Gly Arg Gly Gly Glu Glu Lys Lys Lys Glu Lys Glu Lys Glu Glu
290 295 300
Gln Glu Glu Arg Glu Thr Lys Thr Pro Glu Cys Pro Ser His Thr Gln
305 310 315 320
Pro Leu Gly Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro
325 330 335
Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser
340 345 350
Ser Tyr Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
355 360 365
Trp Val Ser Tyr Ile Ser Ser Ser Ser Ser Thr Ile Tyr Tyr Ala Asp
370 375 380
Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser
385 390 395 400
Leu Tyr Leu Gln Met Asn Ser Leu Arg Asp Glu Asp Thr Ala Val Tyr
405 410 415
Tyr Cys Ala Arg Gly Val Tyr His Asn Gly Trp Ser Phe Phe Asp Tyr
420 425 430
Trp Gly Gln Gly Thr Leu Leu Thr Val Ser Ser Gly Gly Gly Gly Ser
435 440 445
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln
450 455 460
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asn Arg Val Thr Ile Thr
465 470 475 480
Cys Arg Ala Ser Gln Asp Ile Ser Ser Trp Leu Ala Trp Tyr Gln Gln
485 490 495
Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile Tyr Ala Ala Ser Ser Leu
500 505 510
Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
515 520 525
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr
530 535 540
Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Leu Thr Phe Gly Gln Gly Thr
545 550 555 560
Arg Leu Glu Ile Lys Arg
565
<210> 23
<211> 1698
<212> DNA
<213> Artificial
<220>
<223> 二聚体形式的CD3-OX40 BsAb_D的核苷酸序列
<400> 23
gacatcaagc tgcagcagag cggcgccgag ctggcccgcc ccggcgccag cgtgaagatg 60
agctgcaaga ccagcggcta caccttcacc cgctacacca tgcactgggt gaagcagcgc 120
cccggccagg gcctggagtg gatcggctac atcaacccca gccgcggcta caccaactac 180
aaccagaagt tcaaggacaa ggccaccctg accaccgaca agagcagcag caccgcctac 240
atgcagctga gcagcctgac cagcgaggac agcgccgtgt actactgcgc ccgctactac 300
gacgaccact actgcctgga ctactggggc cagggcacca ccctgaccgt gagcagcgtg 360
gagggcggca gcggcggcag cggcggcagc ggcggcagcg gcggcgtgga cgacatccag 420
ctgacccaga gccccgccat catgagcgcc agccccggcg agaaggtgac catgacctgc 480
cgcgccagca gcagcgtgag ctacatgaac tggtaccagc agaagagcgg caccagcccc 540
aagcgctgga tctacgacac cagcaaggtg gccagcggcg tgccctaccg cttcagcggc 600
agcggcagcg gcaccagcta cagcctgacc atcagcagca tggaggccga ggacgccgcc 660
acctactact gccagcagtg gagcagcaac cccctgacct tcggcgccgg caccaagctg 720
gagctgaagg ccagcaagag caagaaggag atcttccgct ggcccgagag ccccaaggcc 780
caggccagca gcgtgcccac cgcccagccc caggccgagg gcagcctggc caaggccacc 840
accgcccccg ccaccacccg caacaccggc cgcggcggcg aggagaagaa gaaggagaag 900
gagaaggagg agcaggagga gcgcgagacc aagacccccg agtgccccag ccacacccag 960
cccctgggcg tgcagctggt ggagagcggc ggcggcctgg tgcagcccgg cggcagcctg 1020
cgcctgagct gcgccgccag cggcttcacc ttcagcagct acagcatgaa ctgggtgcgc 1080
caggcccccg gcaagggcct ggagtgggtg agctacatca gcagcagcag cagcaccatc 1140
tactacgccg acagcgtgaa gggccgcttc accatcagcc gcgacaacgc caagaacagc 1200
ctgtacctgc agatgaacag cctgcgcgac gaggacaccg ccgtgtacta ctgcgcccgc 1260
ggcgtgtacc acaacggctg gagcttcttc gactactggg gccagggcac cctgctgacc 1320
gtgagcagcg gcggcggcgg cagcggcggc ggcggcagcg gcggcggcgg cagcgacatc 1380
cagatgaccc agagccccag cagcctgagc gccagcgtgg gcaaccgcgt gaccatcacc 1440
tgccgcgcca gccaggacat cagcagctgg ctggcctggt accagcagaa gcccgagaag 1500
gcccccaaga gcctgatcta cgccgccagc agcctgcaga gcggcgtgcc cagccgcttc 1560
agcggcagcg gcagcggcac cgacttcacc ctgaccatca gcagcctgca gcccgaggac 1620
ttcgccacct actactgcca gcagtacaac agctaccccc tgaccttcgg ccagggcacc 1680
cgcctggaga tcaagcgc 1698
<210> 24
<211> 499
<212> PRT
<213> Artificial
<220>
<223> 单体形式的CD3-GITR BsAb_M的氨基酸序列
<400> 24
Asp Ile Lys Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala
1 5 10 15
Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Arg Tyr
20 25 30
Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe
50 55 60
Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Thr Leu Thr Val Ser Ser Val Glu Gly Gly Ser Gly Gly Ser Gly
115 120 125
Gly Ser Gly Gly Ser Gly Gly Val Asp Asp Ile Gln Leu Thr Gln Ser
130 135 140
Pro Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys
145 150 155 160
Arg Ala Ser Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser
165 170 175
Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Val Ala Ser
180 185 190
Gly Val Pro Tyr Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser
195 200 205
Leu Thr Ile Ser Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys
210 215 220
Gln Gln Trp Ser Ser Asn Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu
225 230 235 240
Glu Leu Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
245 250 255
Gly Ser Gln Val Thr Leu Lys Glu Ser Gly Pro Gly Ile Leu Lys Pro
260 265 270
Ser Gln Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu Ser
275 280 285
Thr Ser Gly Met Gly Val Gly Trp Ile Arg Gln Pro Ser Gly Lys Gly
290 295 300
Leu Glu Trp Leu Ala His Ile Trp Trp Asp Asp Asp Lys Tyr Tyr Asn
305 310 315 320
Pro Ser Leu Lys Ser Gln Leu Thr Ile Ser Lys Asp Thr Ser Arg Asn
325 330 335
Gln Val Phe Leu Lys Ile Thr Ser Val Asp Thr Ala Asp Ala Ala Thr
340 345 350
Tyr Tyr Cys Ala Arg Thr Arg Arg Tyr Phe Pro Phe Ala Tyr Trp Gly
355 360 365
Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly
370 375 380
Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser Gln
385 390 395 400
Lys Phe Met Ser Thr Ser Val Gly Asp Arg Val Ser Val Thr Cys Lys
405 410 415
Ala Ser Gln Asn Val Gly Thr Asn Val Ala Trp Tyr Gln Gln Lys Pro
420 425 430
Gly Gln Ser Pro Lys Ala Leu Ile Tyr Ser Ala Ser Tyr Arg Tyr Ser
435 440 445
Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr
450 455 460
Leu Thr Ile Asn Asn Val His Ser Glu Asp Leu Ala Glu Tyr Phe Cys
465 470 475 480
Gln Gln Tyr Asn Thr Asp Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu
485 490 495
Glu Ile Lys
<210> 25
<211> 1497
<212> DNA
<213> Artificial
<220>
<223> 单体形式的CD3-GITR BsAb_M的核苷酸序列
<400> 25
gacatcaagc tgcagcagag cggcgccgag ctggcccgcc ccggcgccag cgtgaagatg 60
agctgcaaga ccagcggcta caccttcacc cgctacacca tgcactgggt gaagcagcgc 120
cccggccagg gcctggagtg gatcggctac atcaacccca gccgcggcta caccaactac 180
aaccagaagt tcaaggacaa ggccaccctg accaccgaca agagcagcag caccgcctac 240
atgcagctga gcagcctgac cagcgaggac agcgccgtgt actactgcgc ccgctactac 300
gacgaccact actgcctgga ctactggggc cagggcacca ccctgaccgt gagcagcgtg 360
gagggcggca gcggcggcag cggcggcagc ggcggcagcg gcggcgtgga cgacatccag 420
ctgacccaga gccccgccat catgagcgcc agccccggcg agaaggtgac catgacctgc 480
cgcgccagca gcagcgtgag ctacatgaac tggtaccagc agaagagcgg caccagcccc 540
aagcgctgga tctacgacac cagcaaggtg gccagcggcg tgccctaccg cttcagcggc 600
agcggcagcg gcaccagcta cagcctgacc atcagcagca tggaggccga ggacgccgcc 660
acctactact gccagcagtg gagcagcaac cccctgacct tcggcgccgg caccaagctg 720
gagctgaagg gcggcggcgg cagcggcggc ggcggcagcg gcggcggcgg cagccaggtg 780
accctgaagg agagcggccc cggcatcctg aagcccagcc agaccctgag cctgacctgc 840
agcttcagcg gcttcagcct gagcaccagc ggcatgggcg tgggctggat ccgccagccc 900
agcggcaagg gcctggagtg gctggcccac atctggtggg acgacgacaa gtactacaac 960
cccagcctga agagccagct gaccatcagc aaggacacca gccgcaacca ggtgttcctg 1020
aagatcacca gcgtggacac cgccgacgcc gccacctact actgcgcccg cacccgccgc 1080
tacttcccct tcgcctactg gggccagggc accctggtga ccgtgagcag cggcggcggc 1140
ggcagcggcg gcggcggcag cggcggcggc ggcagcgaca tcgtgatgac ccagagccag 1200
aagttcatga gcaccagcgt gggcgaccgc gtgagcgtga cctgcaaggc cagccagaac 1260
gtgggcacca acgtggcctg gtaccagcag aagcccggcc agagccccaa ggccctgatc 1320
tacagcgcca gctaccgcta cagcggcgtg cccgaccgct tcaccggcag cggcagcggc 1380
accgacttca ccctgaccat caacaacgtg cacagcgagg acctggccga gtacttctgc 1440
cagcagtaca acaccgaccc cctgaccttc ggcgccggca ccaagctgga gatcaag 1497
<210> 26
<211> 565
<212> PRT
<213> Artificial
<220>
<223> 二聚体形式的CD3-GITR BsAb_D的氨基酸序列
<400> 26
Asp Ile Lys Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala
1 5 10 15
Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Arg Tyr
20 25 30
Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe
50 55 60
Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Thr Leu Thr Val Ser Ser Val Glu Gly Gly Ser Gly Gly Ser Gly
115 120 125
Gly Ser Gly Gly Ser Gly Gly Val Asp Asp Ile Gln Leu Thr Gln Ser
130 135 140
Pro Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys
145 150 155 160
Arg Ala Ser Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser
165 170 175
Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Val Ala Ser
180 185 190
Gly Val Pro Tyr Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser
195 200 205
Leu Thr Ile Ser Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys
210 215 220
Gln Gln Trp Ser Ser Asn Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu
225 230 235 240
Glu Leu Lys Ala Ser Lys Ser Lys Lys Glu Ile Phe Arg Trp Pro Glu
245 250 255
Ser Pro Lys Ala Gln Ala Ser Ser Val Pro Thr Ala Gln Pro Gln Ala
260 265 270
Glu Gly Ser Leu Ala Lys Ala Thr Thr Ala Pro Ala Thr Thr Arg Asn
275 280 285
Thr Gly Arg Gly Gly Glu Glu Lys Lys Lys Glu Lys Glu Lys Glu Glu
290 295 300
Gln Glu Glu Arg Glu Thr Lys Thr Pro Glu Cys Pro Ser His Thr Gln
305 310 315 320
Pro Leu Gly Val Gln Val Thr Leu Lys Glu Ser Gly Pro Gly Ile Leu
325 330 335
Lys Pro Ser Gln Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser
340 345 350
Leu Ser Thr Ser Gly Met Gly Val Gly Trp Ile Arg Gln Pro Ser Gly
355 360 365
Lys Gly Leu Glu Trp Leu Ala His Ile Trp Trp Asp Asp Asp Lys Tyr
370 375 380
Tyr Asn Pro Ser Leu Lys Ser Gln Leu Thr Ile Ser Lys Asp Thr Ser
385 390 395 400
Arg Asn Gln Val Phe Leu Lys Ile Thr Ser Val Asp Thr Ala Asp Ala
405 410 415
Ala Thr Tyr Tyr Cys Ala Arg Thr Arg Arg Tyr Phe Pro Phe Ala Tyr
420 425 430
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser
435 440 445
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln
450 455 460
Ser Gln Lys Phe Met Ser Thr Ser Val Gly Asp Arg Val Ser Val Thr
465 470 475 480
Cys Lys Ala Ser Gln Asn Val Gly Thr Asn Val Ala Trp Tyr Gln Gln
485 490 495
Lys Pro Gly Gln Ser Pro Lys Ala Leu Ile Tyr Ser Ala Ser Tyr Arg
500 505 510
Tyr Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp
515 520 525
Phe Thr Leu Thr Ile Asn Asn Val His Ser Glu Asp Leu Ala Glu Tyr
530 535 540
Phe Cys Gln Gln Tyr Asn Thr Asp Pro Leu Thr Phe Gly Ala Gly Thr
545 550 555 560
Lys Leu Glu Ile Lys
565
<210> 27
<211> 1695
<212> DNA
<213> Artificial
<220>
<223> 二聚体形式的CD3-GITR BsAb_D的核苷酸序列
<400> 27
gacatcaagc tgcagcagag cggcgccgag ctggcccgcc ccggcgccag cgtgaagatg 60
agctgcaaga ccagcggcta caccttcacc cgctacacca tgcactgggt gaagcagcgc 120
cccggccagg gcctggagtg gatcggctac atcaacccca gccgcggcta caccaactac 180
aaccagaagt tcaaggacaa ggccaccctg accaccgaca agagcagcag caccgcctac 240
atgcagctga gcagcctgac cagcgaggac agcgccgtgt actactgcgc ccgctactac 300
gacgaccact actgcctgga ctactggggc cagggcacca ccctgaccgt gagcagcgtg 360
gagggcggca gcggcggcag cggcggcagc ggcggcagcg gcggcgtgga cgacatccag 420
ctgacccaga gccccgccat catgagcgcc agccccggcg agaaggtgac catgacctgc 480
cgcgccagca gcagcgtgag ctacatgaac tggtaccagc agaagagcgg caccagcccc 540
aagcgctgga tctacgacac cagcaaggtg gccagcggcg tgccctaccg cttcagcggc 600
agcggcagcg gcaccagcta cagcctgacc atcagcagca tggaggccga ggacgccgcc 660
acctactact gccagcagtg gagcagcaac cccctgacct tcggcgccgg caccaagctg 720
gagctgaagg ccagcaagag caagaaggag atcttccgct ggcccgagag ccccaaggcc 780
caggccagca gcgtgcccac cgcccagccc caggccgagg gcagcctggc caaggccacc 840
accgcccccg ccaccacccg caacaccggc cgcggcggcg aggagaagaa gaaggagaag 900
gagaaggagg agcaggagga gcgcgagacc aagacccccg agtgccccag ccacacccag 960
cccctgggcg tgcaggtgac cctgaaggag agcggccccg gcatcctgaa gcccagccag 1020
accctgagcc tgacctgcag cttcagcggc ttcagcctga gcaccagcgg catgggcgtg 1080
ggctggatcc gccagcccag cggcaagggc ctggagtggc tggcccacat ctggtgggac 1140
gacgacaagt actacaaccc cagcctgaag agccagctga ccatcagcaa ggacaccagc 1200
cgcaaccagg tgttcctgaa gatcaccagc gtggacaccg ccgacgccgc cacctactac 1260
tgcgcccgca cccgccgcta cttccccttc gcctactggg gccagggcac cctggtgacc 1320
gtgagcagcg gcggcggcgg cagcggcggc ggcggcagcg gcggcggcgg cagcgacatc 1380
gtgatgaccc agagccagaa gttcatgagc accagcgtgg gcgaccgcgt gagcgtgacc 1440
tgcaaggcca gccagaacgt gggcaccaac gtggcctggt accagcagaa gcccggccag 1500
agccccaagg ccctgatcta cagcgccagc taccgctaca gcggcgtgcc cgaccgcttc 1560
accggcagcg gcagcggcac cgacttcacc ctgaccatca acaacgtgca cagcgaggac 1620
ctggccgagt acttctgcca gcagtacaac accgaccccc tgaccttcgg cgccggcacc 1680
aagctggaga tcaag 1695
<210> 28
<211> 497
<212> PRT
<213> Artificial
<220>
<223> 单体形式的CD3-CD40L BsAb_M的氨基酸序列
<400> 28
Asp Ile Lys Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala
1 5 10 15
Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Arg Tyr
20 25 30
Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe
50 55 60
Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Thr Leu Thr Val Ser Ser Val Glu Gly Gly Ser Gly Gly Ser Gly
115 120 125
Gly Ser Gly Gly Ser Gly Gly Val Asp Asp Ile Gln Leu Thr Gln Ser
130 135 140
Pro Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys
145 150 155 160
Arg Ala Ser Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser
165 170 175
Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Val Ala Ser
180 185 190
Gly Val Pro Tyr Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser
195 200 205
Leu Thr Ile Ser Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys
210 215 220
Gln Gln Trp Ser Ser Asn Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu
225 230 235 240
Glu Leu Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
245 250 255
Gly Ser Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro
260 265 270
Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser
275 280 285
Ser Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
290 295 300
Trp Val Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp
305 310 315 320
Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
325 330 335
Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
340 345 350
Tyr Cys Ala Lys Ser Tyr Gly Ala Phe Asp Tyr Trp Gly Gln Gly Thr
355 360 365
Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
370 375 380
Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
385 390 395 400
Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln
405 410 415
Ser Ile Ser Ser Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala
420 425 430
Pro Lys Leu Leu Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro
435 440 445
Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
450 455 460
Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser
465 470 475 480
Tyr Ser Thr Pro Asn Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
485 490 495
Arg
<210> 29
<211> 1491
<212> DNA
<213> Artificial
<220>
<223> 单体形式的CD3-CD40L BsAb_M的核苷酸序列
<400> 29
gacatcaagc tgcagcagag cggcgccgag ctggcccgcc ccggcgccag cgtgaagatg 60
agctgcaaga ccagcggcta caccttcacc cgctacacca tgcactgggt gaagcagcgc 120
cccggccagg gcctggagtg gatcggctac atcaacccca gccgcggcta caccaactac 180
aaccagaagt tcaaggacaa ggccaccctg accaccgaca agagcagcag caccgcctac 240
atgcagctga gcagcctgac cagcgaggac agcgccgtgt actactgcgc ccgctactac 300
gacgaccact actgcctgga ctactggggc cagggcacca ccctgaccgt gagcagcgtg 360
gagggcggca gcggcggcag cggcggcagc ggcggcagcg gcggcgtgga cgacatccag 420
ctgacccaga gccccgccat catgagcgcc agccccggcg agaaggtgac catgacctgc 480
cgcgccagca gcagcgtgag ctacatgaac tggtaccagc agaagagcgg caccagcccc 540
aagcgctgga tctacgacac cagcaaggtg gccagcggcg tgccctaccg cttcagcggc 600
agcggcagcg gcaccagcta cagcctgacc atcagcagca tggaggccga ggacgccgcc 660
acctactact gccagcagtg gagcagcaac cccctgacct tcggcgccgg caccaagctg 720
gagctgaagg gcggcggcgg cagcggcggc ggcggcagcg gcggcggcgg cagcgaggtg 780
cagctgctgg agagcggcgg cggcctggtg cagcccggcg gcagcctgcg cctgagctgc 840
gccgccagcg gcttcacctt cagcagctac gccatgagct gggtgcgcca ggcccccggc 900
aagggcctgg agtgggtgag cgccatcagc ggcagcggcg gcagcaccta ctacgccgac 960
agcgtgaagg gccgcttcac catcagccgc gacaacagca agaacaccct gtacctgcag 1020
atgaacagcc tgcgcgccga ggacaccgcc gtgtactact gcgccaagag ctacggcgcc 1080
ttcgactact ggggccaggg caccctggtg accgtgagca gcggcggcgg cggcagcggc 1140
ggcggcggca gcggcggcgg cggcagcgac atccagatga cccagagccc cagcagcctg 1200
agcgccagcg tgggcgaccg cgtgaccatc acctgccgcg ccagccagag catcagcagc 1260
tacctgaact ggtaccagca gaagcccggc aaggccccca agctgctgat ctacgccgcc 1320
agcagcctgc agagcggcgt gcccagccgc ttcagcggca gcggcagcgg caccgacttc 1380
accctgacca tcagcagcct gcagcccgag gacttcgcca cctactactg ccagcagagc 1440
tacagcaccc ccaacacctt cggccagggc accaaggtgg agatcaagcg c 1491
<210> 30
<211> 563
<212> PRT
<213> Artificial
<220>
<223> 二聚体形式的CD3-CD40L BsAb_D的氨基酸序列
<400> 30
Asp Ile Lys Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala
1 5 10 15
Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Arg Tyr
20 25 30
Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe
50 55 60
Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Thr Leu Thr Val Ser Ser Val Glu Gly Gly Ser Gly Gly Ser Gly
115 120 125
Gly Ser Gly Gly Ser Gly Gly Val Asp Asp Ile Gln Leu Thr Gln Ser
130 135 140
Pro Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys
145 150 155 160
Arg Ala Ser Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser
165 170 175
Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Val Ala Ser
180 185 190
Gly Val Pro Tyr Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser
195 200 205
Leu Thr Ile Ser Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys
210 215 220
Gln Gln Trp Ser Ser Asn Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu
225 230 235 240
Glu Leu Lys Ala Ser Lys Ser Lys Lys Glu Ile Phe Arg Trp Pro Glu
245 250 255
Ser Pro Lys Ala Gln Ala Ser Ser Val Pro Thr Ala Gln Pro Gln Ala
260 265 270
Glu Gly Ser Leu Ala Lys Ala Thr Thr Ala Pro Ala Thr Thr Arg Asn
275 280 285
Thr Gly Arg Gly Gly Glu Glu Lys Lys Lys Glu Lys Glu Lys Glu Glu
290 295 300
Gln Glu Glu Arg Glu Thr Lys Thr Pro Glu Cys Pro Ser His Thr Gln
305 310 315 320
Pro Leu Gly Val Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val
325 330 335
Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
340 345 350
Phe Ser Ser Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly
355 360 365
Leu Glu Trp Val Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr
370 375 380
Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys
385 390 395 400
Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
405 410 415
Val Tyr Tyr Cys Ala Lys Ser Tyr Gly Ala Phe Asp Tyr Trp Gly Gln
420 425 430
Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly
435 440 445
Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser
450 455 460
Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala
465 470 475 480
Ser Gln Ser Ile Ser Ser Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly
485 490 495
Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly
500 505 510
Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
515 520 525
Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln
530 535 540
Gln Ser Tyr Ser Thr Pro Asn Thr Phe Gly Gln Gly Thr Lys Val Glu
545 550 555 560
Ile Lys Arg
<210> 31
<211> 1689
<212> DNA
<213> Artificial
<220>
<223> 二聚体形式的CD3-CD40L BsAb_D的核苷酸序列
<400> 31
gacatcaagc tgcagcagag cggcgccgag ctggcccgcc ccggcgccag cgtgaagatg 60
agctgcaaga ccagcggcta caccttcacc cgctacacca tgcactgggt gaagcagcgc 120
cccggccagg gcctggagtg gatcggctac atcaacccca gccgcggcta caccaactac 180
aaccagaagt tcaaggacaa ggccaccctg accaccgaca agagcagcag caccgcctac 240
atgcagctga gcagcctgac cagcgaggac agcgccgtgt actactgcgc ccgctactac 300
gacgaccact actgcctgga ctactggggc cagggcacca ccctgaccgt gagcagcgtg 360
gagggcggca gcggcggcag cggcggcagc ggcggcagcg gcggcgtgga cgacatccag 420
ctgacccaga gccccgccat catgagcgcc agccccggcg agaaggtgac catgacctgc 480
cgcgccagca gcagcgtgag ctacatgaac tggtaccagc agaagagcgg caccagcccc 540
aagcgctgga tctacgacac cagcaaggtg gccagcggcg tgccctaccg cttcagcggc 600
agcggcagcg gcaccagcta cagcctgacc atcagcagca tggaggccga ggacgccgcc 660
acctactact gccagcagtg gagcagcaac cccctgacct tcggcgccgg caccaagctg 720
gagctgaagg ccagcaagag caagaaggag atcttccgct ggcccgagag ccccaaggcc 780
caggccagca gcgtgcccac cgcccagccc caggccgagg gcagcctggc caaggccacc 840
accgcccccg ccaccacccg caacaccggc cgcggcggcg aggagaagaa gaaggagaag 900
gagaaggagg agcaggagga gcgcgagacc aagacccccg agtgccccag ccacacccag 960
cccctgggcg tggaggtgca gctgctggag agcggcggcg gcctggtgca gcccggcggc 1020
agcctgcgcc tgagctgcgc cgccagcggc ttcaccttca gcagctacgc catgagctgg 1080
gtgcgccagg cccccggcaa gggcctggag tgggtgagcg ccatcagcgg cagcggcggc 1140
agcacctact acgccgacag cgtgaagggc cgcttcacca tcagccgcga caacagcaag 1200
aacaccctgt acctgcagat gaacagcctg cgcgccgagg acaccgccgt gtactactgc 1260
gccaagagct acggcgcctt cgactactgg ggccagggca ccctggtgac cgtgagcagc 1320
ggcggcggcg gcagcggcgg cggcggcagc ggcggcggcg gcagcgacat ccagatgacc 1380
cagagcccca gcagcctgag cgccagcgtg ggcgaccgcg tgaccatcac ctgccgcgcc 1440
agccagagca tcagcagcta cctgaactgg taccagcaga agcccggcaa ggcccccaag 1500
ctgctgatct acgccgccag cagcctgcag agcggcgtgc ccagccgctt cagcggcagc 1560
ggcagcggca ccgacttcac cctgaccatc agcagcctgc agcccgagga cttcgccacc 1620
tactactgcc agcagagcta cagcaccccc aacaccttcg gccagggcac caaggtggag 1680
atcaagcgc 1689
<210> 32
<211> 499
<212> PRT
<213> Artificial
<220>
<223> 单体形式的CD3-CD27 BsAb_M的氨基酸序列
<400> 32
Asp Ile Lys Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala
1 5 10 15
Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Arg Tyr
20 25 30
Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe
50 55 60
Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Thr Leu Thr Val Ser Ser Val Glu Gly Gly Ser Gly Gly Ser Gly
115 120 125
Gly Ser Gly Gly Ser Gly Gly Val Asp Asp Ile Gln Leu Thr Gln Ser
130 135 140
Pro Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys
145 150 155 160
Arg Ala Ser Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser
165 170 175
Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Val Ala Ser
180 185 190
Gly Val Pro Tyr Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser
195 200 205
Leu Thr Ile Ser Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys
210 215 220
Gln Gln Trp Ser Ser Asn Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu
225 230 235 240
Glu Leu Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
245 250 255
Gly Ser Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro
260 265 270
Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser
275 280 285
Ser Tyr Asp Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
290 295 300
Trp Val Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp
305 310 315 320
Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
325 330 335
Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
340 345 350
Tyr Cys Ala Arg Gly Ser Gly Asn Trp Gly Phe Phe Asp Tyr Trp Gly
355 360 365
Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly
370 375 380
Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro
385 390 395 400
Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg
405 410 415
Ala Ser Gln Gly Ile Ser Arg Trp Leu Ala Trp Tyr Gln Gln Lys Pro
420 425 430
Glu Lys Ala Pro Lys Ser Leu Ile Tyr Ala Ala Ser Ser Leu Gln Ser
435 440 445
Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
450 455 460
Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys
465 470 475 480
Gln Gln Tyr Asn Thr Tyr Pro Arg Thr Phe Gly Gln Gly Thr Lys Val
485 490 495
Glu Ile Lys
<210> 33
<211> 1497
<212> DNA
<213> Artificial
<220>
<223> 单体形式的CD3-CD27 BsAb_M的核苷酸序列
<400> 33
gacatcaagc tgcagcagag cggcgccgag ctggcccgcc ccggcgccag cgtgaagatg 60
agctgcaaga ccagcggcta caccttcacc cgctacacca tgcactgggt gaagcagcgc 120
cccggccagg gcctggagtg gatcggctac atcaacccca gccgcggcta caccaactac 180
aaccagaagt tcaaggacaa ggccaccctg accaccgaca agagcagcag caccgcctac 240
atgcagctga gcagcctgac cagcgaggac agcgccgtgt actactgcgc ccgctactac 300
gacgaccact actgcctgga ctactggggc cagggcacca ccctgaccgt gagcagcgtg 360
gagggcggca gcggcggcag cggcggcagc ggcggcagcg gcggcgtgga cgacatccag 420
ctgacccaga gccccgccat catgagcgcc agccccggcg agaaggtgac catgacctgc 480
cgcgccagca gcagcgtgag ctacatgaac tggtaccagc agaagagcgg caccagcccc 540
aagcgctgga tctacgacac cagcaaggtg gccagcggcg tgccctaccg cttcagcggc 600
agcggcagcg gcaccagcta cagcctgacc atcagcagca tggaggccga ggacgccgcc 660
acctactact gccagcagtg gagcagcaac cccctgacct tcggcgccgg caccaagctg 720
gagctgaagg gcggcggcgg cagcggcggc ggcggcagcg gcggcggcgg cagccaggtg 780
cagctggtgg agagcggcgg cggcgtggtg cagcccggcc gcagcctgcg cctgagctgc 840
gccgccagcg gcttcacctt cagcagctac gacatgcact gggtgcgcca ggcccccggc 900
aagggcctgg agtgggtggc cgtgatctgg tacgacggca gcaacaagta ctacgccgac 960
agcgtgaagg gccgcttcac catcagccgc gacaacagca agaacaccct gtacctgcag 1020
atgaacagcc tgcgcgccga ggacaccgcc gtgtactact gcgcccgcgg cagcggcaac 1080
tggggcttct tcgactactg gggccagggc accctggtga ccgtgagcag cggcggcggc 1140
ggcagcggcg gcggcggcag cggcggcggc ggcagcgaca tccagatgac ccagagcccc 1200
agcagcctga gcgccagcgt gggcgaccgc gtgaccatca cctgccgcgc cagccagggc 1260
atcagccgct ggctggcctg gtaccagcag aagcccgaga aggcccccaa gagcctgatc 1320
tacgccgcca gcagcctgca gagcggcgtg cccagccgct tcagcggcag cggcagcggc 1380
accgacttca ccctgaccat cagcagcctg cagcccgagg acttcgccac ctactactgc 1440
cagcagtaca acacctaccc ccgcaccttc ggccagggca ccaaggtgga gatcaag 1497
<210> 34
<211> 565
<212> PRT
<213> Artificial
<220>
<223> 二聚体形式的CD3-CD27 BsAb_D的氨基酸序列
<400> 34
Asp Ile Lys Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala
1 5 10 15
Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Arg Tyr
20 25 30
Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe
50 55 60
Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Thr Leu Thr Val Ser Ser Val Glu Gly Gly Ser Gly Gly Ser Gly
115 120 125
Gly Ser Gly Gly Ser Gly Gly Val Asp Asp Ile Gln Leu Thr Gln Ser
130 135 140
Pro Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys
145 150 155 160
Arg Ala Ser Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser
165 170 175
Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Val Ala Ser
180 185 190
Gly Val Pro Tyr Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser
195 200 205
Leu Thr Ile Ser Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys
210 215 220
Gln Gln Trp Ser Ser Asn Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu
225 230 235 240
Glu Leu Lys Ala Ser Lys Ser Lys Lys Glu Ile Phe Arg Trp Pro Glu
245 250 255
Ser Pro Lys Ala Gln Ala Ser Ser Val Pro Thr Ala Gln Pro Gln Ala
260 265 270
Glu Gly Ser Leu Ala Lys Ala Thr Thr Ala Pro Ala Thr Thr Arg Asn
275 280 285
Thr Gly Arg Gly Gly Glu Glu Lys Lys Lys Glu Lys Glu Lys Glu Glu
290 295 300
Gln Glu Glu Arg Glu Thr Lys Thr Pro Glu Cys Pro Ser His Thr Gln
305 310 315 320
Pro Leu Gly Val Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val
325 330 335
Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
340 345 350
Phe Ser Ser Tyr Asp Met His Trp Val Arg Gln Ala Pro Gly Lys Gly
355 360 365
Leu Glu Trp Val Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr
370 375 380
Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys
385 390 395 400
Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
405 410 415
Val Tyr Tyr Cys Ala Arg Gly Ser Gly Asn Trp Gly Phe Phe Asp Tyr
420 425 430
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser
435 440 445
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln
450 455 460
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr
465 470 475 480
Cys Arg Ala Ser Gln Gly Ile Ser Arg Trp Leu Ala Trp Tyr Gln Gln
485 490 495
Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile Tyr Ala Ala Ser Ser Leu
500 505 510
Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
515 520 525
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr
530 535 540
Tyr Cys Gln Gln Tyr Asn Thr Tyr Pro Arg Thr Phe Gly Gln Gly Thr
545 550 555 560
Lys Val Glu Ile Lys
565
<210> 35
<211> 1695
<212> DNA
<213> Artificial
<220>
<223> 二聚体形式的CD3-CD27 BsAb_D的核苷酸酸序列
<400> 35
gacatcaagc tgcagcagag cggcgccgag ctggcccgcc ccggcgccag cgtgaagatg 60
agctgcaaga ccagcggcta caccttcacc cgctacacca tgcactgggt gaagcagcgc 120
cccggccagg gcctggagtg gatcggctac atcaacccca gccgcggcta caccaactac 180
aaccagaagt tcaaggacaa ggccaccctg accaccgaca agagcagcag caccgcctac 240
atgcagctga gcagcctgac cagcgaggac agcgccgtgt actactgcgc ccgctactac 300
gacgaccact actgcctgga ctactggggc cagggcacca ccctgaccgt gagcagcgtg 360
gagggcggca gcggcggcag cggcggcagc ggcggcagcg gcggcgtgga cgacatccag 420
ctgacccaga gccccgccat catgagcgcc agccccggcg agaaggtgac catgacctgc 480
cgcgccagca gcagcgtgag ctacatgaac tggtaccagc agaagagcgg caccagcccc 540
aagcgctgga tctacgacac cagcaaggtg gccagcggcg tgccctaccg cttcagcggc 600
agcggcagcg gcaccagcta cagcctgacc atcagcagca tggaggccga ggacgccgcc 660
acctactact gccagcagtg gagcagcaac cccctgacct tcggcgccgg caccaagctg 720
gagctgaagg ccagcaagag caagaaggag atcttccgct ggcccgagag ccccaaggcc 780
caggccagca gcgtgcccac cgcccagccc caggccgagg gcagcctggc caaggccacc 840
accgcccccg ccaccacccg caacaccggc cgcggcggcg aggagaagaa gaaggagaag 900
gagaaggagg agcaggagga gcgcgagacc aagacccccg agtgccccag ccacacccag 960
cccctgggcg tgcaggtgca gctggtggag agcggcggcg gcgtggtgca gcccggccgc 1020
agcctgcgcc tgagctgcgc cgccagcggc ttcaccttca gcagctacga catgcactgg 1080
gtgcgccagg cccccggcaa gggcctggag tgggtggccg tgatctggta cgacggcagc 1140
aacaagtact acgccgacag cgtgaagggc cgcttcacca tcagccgcga caacagcaag 1200
aacaccctgt acctgcagat gaacagcctg cgcgccgagg acaccgccgt gtactactgc 1260
gcccgcggca gcggcaactg gggcttcttc gactactggg gccagggcac cctggtgacc 1320
gtgagcagcg gcggcggcgg cagcggcggc ggcggcagcg gcggcggcgg cagcgacatc 1380
cagatgaccc agagccccag cagcctgagc gccagcgtgg gcgaccgcgt gaccatcacc 1440
tgccgcgcca gccagggcat cagccgctgg ctggcctggt accagcagaa gcccgagaag 1500
gcccccaaga gcctgatcta cgccgccagc agcctgcaga gcggcgtgcc cagccgcttc 1560
agcggcagcg gcagcggcac cgacttcacc ctgaccatca gcagcctgca gcccgaggac 1620
ttcgccacct actactgcca gcagtacaac acctaccccc gcaccttcgg ccagggcacc 1680
aaggtggaga tcaag 1695
<210> 36
<211> 243
<212> PRT
<213> Artificial
<220>
<223> 抗CD3 scFv的氨基酸序列
<400> 36
Asp Ile Lys Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala
1 5 10 15
Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Arg Tyr
20 25 30
Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe
50 55 60
Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Thr Leu Thr Val Ser Ser Val Glu Gly Gly Ser Gly Gly Ser Gly
115 120 125
Gly Ser Gly Gly Ser Gly Gly Val Asp Asp Ile Gln Leu Thr Gln Ser
130 135 140
Pro Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys
145 150 155 160
Arg Ala Ser Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser
165 170 175
Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Val Ala Ser
180 185 190
Gly Val Pro Tyr Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser
195 200 205
Leu Thr Ile Ser Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys
210 215 220
Gln Gln Trp Ser Ser Asn Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu
225 230 235 240
Glu Leu Lys
<210> 37
<211> 119
<212> PRT
<213> Artificial
<220>
<223> 抗CD3 scFv的重链可变区的氨基酸序列
<400> 37
Asp Ile Lys Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala
1 5 10 15
Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Arg Tyr
20 25 30
Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe
50 55 60
Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Thr Leu Thr Val Ser Ser
115
<210> 38
<211> 106
<212> PRT
<213> Artificial
<220>
<223> 抗CD3 scFv的轻链可变区的氨基酸序列
<400> 38
Asp Ile Gln Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly
1 5 10 15
Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met
20 25 30
Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr
35 40 45
Asp Thr Ser Lys Val Ala Ser Gly Val Pro Tyr Arg Phe Ser Gly Ser
50 55 60
Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu
65 70 75 80
Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr
85 90 95
Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys
100 105
<210> 39
<211> 246
<212> PRT
<213> Artificial
<220>
<223> 抗4-1BB scFv的氨基酸序列
<400> 39
Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu
1 5 10 15
Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr
20 25 30
Tyr Trp Ser Trp Ile Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Ile
35 40 45
Gly Glu Ile Asn His Gly Gly Tyr Val Thr Tyr Asn Pro Ser Leu Glu
50 55 60
Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu
65 70 75 80
Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Arg Asp Tyr Gly Pro Gly Asn Tyr Asp Trp Tyr Phe Asp Leu Trp Gly
100 105 110
Arg Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly
115 120 125
Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Leu Thr Gln Ser Pro
130 135 140
Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg
145 150 155 160
Ala Ser Gln Ser Val Ser Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro
165 170 175
Gly Gln Ala Pro Arg Leu Leu Ile Tyr Asp Ala Ser Asn Arg Ala Thr
180 185 190
Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
195 200 205
Leu Thr Ile Ser Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys
210 215 220
Gln Gln Arg Ser Asn Trp Pro Pro Ala Leu Thr Phe Cys Gly Gly Thr
225 230 235 240
Lys Val Glu Ile Lys Arg
245
<210> 40
<211> 121
<212> PRT
<213> Artificial
<220>
<223> 抗4-1BB scFv的重链可变区的氨基酸序列
<400> 40
Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu
1 5 10 15
Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr
20 25 30
Tyr Trp Ser Trp Ile Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Ile
35 40 45
Gly Glu Ile Asn His Gly Gly Tyr Val Thr Tyr Asn Pro Ser Leu Glu
50 55 60
Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu
65 70 75 80
Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Arg Asp Tyr Gly Pro Gly Asn Tyr Asp Trp Tyr Phe Asp Leu Trp Gly
100 105 110
Arg Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 41
<211> 110
<212> PRT
<213> Artificial
<220>
<223> 抗4-1BB scFv的轻链可变区的氨基酸序列
<400> 41
Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45
Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro
65 70 75 80
Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Pro
85 90 95
Ala Leu Thr Phe Cys Gly Gly Thr Lys Val Glu Ile Lys Arg
100 105 110
<210> 42
<211> 247
<212> PRT
<213> Artificial
<220>
<223> 抗ICOS scFv的氨基酸序列
<400> 42
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 Gly Tyr
20 25 30
Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Trp Ile Asn Pro His Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Thr Tyr Tyr Tyr Asp Ser Ser Gly Tyr Tyr His Asp Ala Phe
100 105 110
Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Gly Gly Gly
115 120 125
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met
130 135 140
Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly Asp Arg Val Thr
145 150 155 160
Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Arg Leu Leu Ala Trp Tyr
165 170 175
Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Val Ala Ser
180 185 190
Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly
195 200 205
Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala
210 215 220
Thr Tyr Tyr Cys Gln Gln Ala Asn Ser Phe Pro Trp Thr Phe Gly Gln
225 230 235 240
Gly Thr Lys Val Glu Ile Lys
245
<210> 43
<211> 125
<212> PRT
<213> Artificial
<220>
<223> 抗ICOS scFv的重链可变区的氨基酸序列
<400> 43
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 Gly Tyr
20 25 30
Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Trp Ile Asn Pro His Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Thr Tyr Tyr Tyr Asp Ser Ser Gly Tyr Tyr His Asp Ala Phe
100 105 110
Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser
115 120 125
<210> 44
<211> 107
<212> PRT
<213> Artificial
<220>
<223> 抗ICOS scFv的轻链可变区的氨基酸序列
<400> 44
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Arg Leu
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Val 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 Ala Asn Ser Phe Pro Trp
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105
<210> 45
<211> 242
<212> PRT
<213> Artificial
<220>
<223> 抗OX40 scFv的氨基酸序列
<400> 45
Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu
1 5 10 15
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Ser Met
20 25 30
Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Tyr
35 40 45
Ile Ser Ser Ser Ser Ser Thr Ile Tyr Tyr Ala Asp Ser Val Lys Gly
50 55 60
Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gln
65 70 75 80
Met Asn Ser Leu Arg Asp Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg
85 90 95
Gly Val Tyr His Asn Gly Trp Ser Phe Phe Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Leu 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 Ser Pro Ser Ser
130 135 140
Leu Ser Ala Ser Val Gly Asn Arg Val Thr Ile Thr Cys Arg Ala Ser
145 150 155 160
Gln Asp Ile Ser Ser Trp Leu Ala Trp Tyr Gln Gln Lys Pro Glu Lys
165 170 175
Ala Pro Lys Ser Leu Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val
180 185 190
Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
195 200 205
Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
210 215 220
Tyr Asn Ser Tyr Pro Leu Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile
225 230 235 240
Lys Arg
<210> 46
<211> 119
<212> PRT
<213> Artificial
<220>
<223> 抗OX40 scFv的重链可变区的氨基酸序列
<400> 46
Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu
1 5 10 15
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Ser Met
20 25 30
Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Tyr
35 40 45
Ile Ser Ser Ser Ser Ser Thr Ile Tyr Tyr Ala Asp Ser Val Lys Gly
50 55 60
Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gln
65 70 75 80
Met Asn Ser Leu Arg Asp Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg
85 90 95
Gly Val Tyr His Asn Gly Trp Ser Phe Phe Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Leu Thr Val Ser Ser
115
<210> 47
<211> 108
<212> PRT
<213> Artificial
<220>
<223> 抗OX40 scFv的轻链可变区的氨基酸序列
<400> 47
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asn Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Ser Trp
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile
35 40 45
Tyr Ala 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 Tyr Asn Ser Tyr Pro Leu
85 90 95
Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys Arg
100 105
<210> 48
<211> 241
<212> PRT
<213> Artificial
<220>
<223> 抗GITR scFv的氨基酸序列
<400> 48
Gln Val Thr Leu Lys Glu Ser Gly Pro Gly Ile Leu Lys Pro Ser Gln
1 5 10 15
Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu Ser Thr Ser
20 25 30
Gly Met Gly Val Gly Trp Ile Arg Gln Pro Ser Gly Lys Gly Leu Glu
35 40 45
Trp Leu Ala His Ile Trp Trp Asp Asp Asp Lys Tyr Tyr Asn Pro Ser
50 55 60
Leu Lys Ser Gln Leu Thr Ile Ser Lys Asp Thr Ser Arg Asn Gln Val
65 70 75 80
Phe Leu Lys Ile Thr Ser Val Asp Thr Ala Asp Ala Ala Thr Tyr Tyr
85 90 95
Cys Ala Arg Thr Arg Arg Tyr Phe Pro Phe Ala Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
115 120 125
Ser Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser Gln Lys Phe
130 135 140
Met Ser Thr Ser Val Gly Asp Arg Val Ser Val Thr Cys Lys Ala Ser
145 150 155 160
Gln Asn Val Gly Thr Asn Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln
165 170 175
Ser Pro Lys Ala Leu Ile Tyr Ser Ala Ser Tyr Arg Tyr Ser Gly Val
180 185 190
Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
195 200 205
Ile Asn Asn Val His Ser Glu Asp Leu Ala Glu Tyr Phe Cys Gln Gln
210 215 220
Tyr Asn Thr Asp Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile
225 230 235 240
Lys
<210> 49
<211> 119
<212> PRT
<213> Artificial
<220>
<223> 抗GITR scFv的重链可变区的氨基酸序列
<400> 49
Gln Val Thr Leu Lys Glu Ser Gly Pro Gly Ile Leu Lys Pro Ser Gln
1 5 10 15
Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu Ser Thr Ser
20 25 30
Gly Met Gly Val Gly Trp Ile Arg Gln Pro Ser Gly Lys Gly Leu Glu
35 40 45
Trp Leu Ala His Ile Trp Trp Asp Asp Asp Lys Tyr Tyr Asn Pro Ser
50 55 60
Leu Lys Ser Gln Leu Thr Ile Ser Lys Asp Thr Ser Arg Asn Gln Val
65 70 75 80
Phe Leu Lys Ile Thr Ser Val Asp Thr Ala Asp Ala Ala Thr Tyr Tyr
85 90 95
Cys Ala Arg Thr Arg Arg Tyr Phe Pro Phe Ala Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ser
115
<210> 50
<211> 107
<212> PRT
<213> Artificial
<220>
<223> 抗GITR scFv的轻链可变区的氨基酸序列
<400> 50
Asp Ile Val Met Thr Gln Ser Gln Lys Phe Met Ser Thr Ser Val Gly
1 5 10 15
Asp Arg Val Ser Val Thr Cys Lys Ala Ser Gln Asn Val Gly Thr Asn
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Ala Leu Ile
35 40 45
Tyr Ser Ala Ser Tyr Arg Tyr Ser Gly Val Pro Asp Arg Phe Thr Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Asn Val His Ser
65 70 75 80
Glu Asp Leu Ala Glu Tyr Phe Cys Gln Gln Tyr Asn Thr Asp Pro Leu
85 90 95
Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 51
<211> 239
<212> PRT
<213> Artificial
<220>
<223> 抗CD40L scFv的氨基酸序列
<400> 51
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Lys Ser Tyr Gly Ala Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
115 120 125
Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala
130 135 140
Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile
145 150 155 160
Ser Ser Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys
165 170 175
Leu Leu Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg
180 185 190
Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
195 200 205
Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser
210 215 220
Thr Pro Asn Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg
225 230 235
<210> 52
<211> 116
<212> PRT
<213> Artificial
<220>
<223> 抗CD40L scFv的重链可变区的氨基酸序列
<400> 52
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Lys Ser Tyr Gly Ala Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Ser Ser
115
<210> 53
<211> 108
<212> PRT
<213> Artificial
<220>
<223> 抗CD40L scFv的轻链可变区的氨基酸序列
<400> 53
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 Ser Ile Ser Ser Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ala 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 Ser Tyr Ser Thr Pro Asn
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg
100 105
<210> 54
<211> 241
<212> PRT
<213> Artificial
<220>
<223> 抗CD27 scFv的氨基酸序列
<400> 54
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Asp Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gly Ser Gly Asn Trp Gly Phe Phe Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
115 120 125
Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
130 135 140
Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser
145 150 155 160
Gln Gly Ile Ser Arg Trp Leu Ala Trp Tyr Gln Gln Lys Pro Glu Lys
165 170 175
Ala Pro Lys Ser Leu Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val
180 185 190
Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
195 200 205
Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
210 215 220
Tyr Asn Thr Tyr Pro Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
225 230 235 240
Lys
<210> 55
<211> 119
<212> PRT
<213> Artificial
<220>
<223> 抗CD27 scFv的重链可变区的氨基酸序列
<400> 55
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Asp Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gly Ser Gly Asn Trp Gly Phe Phe Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ser
115
<210> 56
<211> 107
<212> PRT
<213> Artificial
<220>
<223> 抗CD27 scFv的轻链可变区的氨基酸序列
<400> 56
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 Gly Ile Ser Arg Trp
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile
35 40 45
Tyr Ala 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 Tyr Asn Thr Tyr Pro Arg
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105
<210> 57
<211> 729
<212> DNA
<213> Artificial
<220>
<223> 抗CD3 scFv的核苷酸序列
<400> 57
gacatcaagc tgcagcagag cggcgccgag ctggcccgcc ccggcgccag cgtgaagatg 60
agctgcaaga ccagcggcta caccttcacc cgctacacca tgcactgggt gaagcagcgc 120
cccggccagg gcctggagtg gatcggctac atcaacccca gccgcggcta caccaactac 180
aaccagaagt tcaaggacaa ggccaccctg accaccgaca agagcagcag caccgcctac 240
atgcagctga gcagcctgac cagcgaggac agcgccgtgt actactgcgc ccgctactac 300
gacgaccact actgcctgga ctactggggc cagggcacca ccctgaccgt gagcagcgtg 360
gagggcggca gcggcggcag cggcggcagc ggcggcagcg gcggcgtgga cgacatccag 420
ctgacccaga gccccgccat catgagcgcc agccccggcg agaaggtgac catgacctgc 480
cgcgccagca gcagcgtgag ctacatgaac tggtaccagc agaagagcgg caccagcccc 540
aagcgctgga tctacgacac cagcaaggtg gccagcggcg tgccctaccg cttcagcggc 600
agcggcagcg gcaccagcta cagcctgacc atcagcagca tggaggccga ggacgccgcc 660
acctactact gccagcagtg gagcagcaac cccctgacct tcggcgccgg caccaagctg 720
gagctgaag 729
<210> 58
<211> 357
<212> DNA
<213> Artificial
<220>
<223> 抗CD3 scFv的重链可变区的核苷酸序列
<400> 58
gacatcaagc tgcagcagag cggcgccgag ctggcccgcc ccggcgccag cgtgaagatg 60
agctgcaaga ccagcggcta caccttcacc cgctacacca tgcactgggt gaagcagcgc 120
cccggccagg gcctggagtg gatcggctac atcaacccca gccgcggcta caccaactac 180
aaccagaagt tcaaggacaa ggccaccctg accaccgaca agagcagcag caccgcctac 240
atgcagctga gcagcctgac cagcgaggac agcgccgtgt actactgcgc ccgctactac 300
gacgaccact actgcctgga ctactggggc cagggcacca ccctgaccgt gagcagc 357
<210> 59
<211> 318
<212> DNA
<213> Artificial
<220>
<223> 抗CD3 scFv的轻链可变区的核苷酸序列
<400> 59
gacatccagc tgacccagag ccccgccatc atgagcgcca gccccggcga gaaggtgacc 60
atgacctgcc gcgccagcag cagcgtgagc tacatgaact ggtaccagca gaagagcggc 120
accagcccca agcgctggat ctacgacacc agcaaggtgg ccagcggcgt gccctaccgc 180
ttcagcggca gcggcagcgg caccagctac agcctgacca tcagcagcat ggaggccgag 240
gacgccgcca cctactactg ccagcagtgg agcagcaacc ccctgacctt cggcgccggc 300
accaagctgg agctgaag 318
<210> 60
<211> 738
<212> DNA
<213> Artificial
<220>
<223> 抗4-1BB scFv的核苷酸序列
<400> 60
caggtgcagc tgcagcagtg gggcgccggc ctgctgaagc ccagcgagac cctgagcctg 60
acctgcgccg tgtacggcgg cagcttcagc ggctactact ggagctggat ccgccagagc 120
cccgagaagg gcctggagtg gatcggcgag atcaaccacg gcggctacgt gacctacaac 180
cccagcctgg agagccgcgt gaccatcagc gtggacacca gcaagaacca gttcagcctg 240
aagctgagca gcgtgaccgc cgccgacacc gccgtgtact actgcgcccg cgactacggc 300
cccggcaact acgactggta cttcgacctg tggggccgcg gcaccctggt gaccgtgagc 360
agcggcggcg gcggcagcgg cggcggcggc agcggcggcg gcggcagcga gatcgtgctg 420
acccagagcc ccgccaccct gagcctgagc cccggcgagc gcgccaccct gagctgccgc 480
gccagccaga gcgtgagcag ctacctggcc tggtaccagc agaagcccgg ccaggccccc 540
cgcctgctga tctacgacgc cagcaaccgc gccaccggca tccccgcccg cttcagcggc 600
agcggcagcg gcaccgactt caccctgacc atcagcagcc tggagcccga ggacttcgcc 660
gtgtactact gccagcagcg cagcaactgg ccccccgccc tgaccttctg cggcggcacc 720
aaggtggaga tcaagcgc 738
<210> 61
<211> 363
<212> DNA
<213> Artificial
<220>
<223> 抗4-1BB scFv的重链可变区的核苷酸序列
<400> 61
caggtgcagc tgcagcagtg gggcgccggc ctgctgaagc ccagcgagac cctgagcctg 60
acctgcgccg tgtacggcgg cagcttcagc ggctactact ggagctggat ccgccagagc 120
cccgagaagg gcctggagtg gatcggcgag atcaaccacg gcggctacgt gacctacaac 180
cccagcctgg agagccgcgt gaccatcagc gtggacacca gcaagaacca gttcagcctg 240
aagctgagca gcgtgaccgc cgccgacacc gccgtgtact actgcgcccg cgactacggc 300
cccggcaact acgactggta cttcgacctg tggggccgcg gcaccctggt gaccgtgagc 360
agc 363
<210> 62
<211> 330
<212> DNA
<213> Artificial
<220>
<223> 抗4-1BB scFv的轻链可变区的核苷酸序列
<400> 62
gagatcgtgc tgacccagag ccccgccacc ctgagcctga gccccggcga gcgcgccacc 60
ctgagctgcc gcgccagcca gagcgtgagc agctacctgg cctggtacca gcagaagccc 120
ggccaggccc cccgcctgct gatctacgac gccagcaacc gcgccaccgg catccccgcc 180
cgcttcagcg gcagcggcag cggcaccgac ttcaccctga ccatcagcag cctggagccc 240
gaggacttcg ccgtgtacta ctgccagcag cgcagcaact ggccccccgc cctgaccttc 300
tgcggcggca ccaaggtgga gatcaagcgc 330
<210> 63
<211> 741
<212> DNA
<213> Artificial
<220>
<223> 抗ICOS scFv的核苷酸序列
<400> 63
caggtgcagc tggtgcagag cggcgccgag gtgaagaagc ccggcgccag cgtgaaggtg 60
agctgcaagg ccagcggcta caccttcacc ggctactaca tgcactgggt gcgccaggcc 120
cccggccagg gcctggagtg gatgggctgg atcaaccccc acagcggcgg caccaactac 180
gcccagaagt tccagggccg cgtgaccatg acccgcgaca ccagcatcag caccgcctac 240
atggagctga gccgcctgcg cagcgacgac accgccgtgt actactgcgc ccgcacctac 300
tactacgaca gcagcggcta ctaccacgac gccttcgaca tctggggcca gggcaccatg 360
gtgaccgtga gcagcggcgg cggcggcagc ggcggcggcg gcagcggcgg cggcggcagc 420
gacatccaga tgacccagag ccccagcagc gtgagcgcca gcgtgggcga ccgcgtgacc 480
atcacctgcc gcgccagcca gggcatcagc cgcctgctgg cctggtacca gcagaagccc 540
ggcaaggccc ccaagctgct gatctacgtg gccagcagcc tgcagagcgg cgtgcccagc 600
cgcttcagcg gcagcggcag cggcaccgac ttcaccctga ccatcagcag cctgcagccc 660
gaggacttcg ccacctacta ctgccagcag gccaacagct tcccctggac cttcggccag 720
ggcaccaagg tggagatcaa g 741
<210> 64
<211> 375
<212> DNA
<213> Artificial
<220>
<223> 抗ICOS scFv的重链可变区的核苷酸序列
<400> 64
caggtgcagc tggtgcagag cggcgccgag gtgaagaagc ccggcgccag cgtgaaggtg 60
agctgcaagg ccagcggcta caccttcacc ggctactaca tgcactgggt gcgccaggcc 120
cccggccagg gcctggagtg gatgggctgg atcaaccccc acagcggcgg caccaactac 180
gcccagaagt tccagggccg cgtgaccatg acccgcgaca ccagcatcag caccgcctac 240
atggagctga gccgcctgcg cagcgacgac accgccgtgt actactgcgc ccgcacctac 300
tactacgaca gcagcggcta ctaccacgac gccttcgaca tctggggcca gggcaccatg 360
gtgaccgtga gcagc 375
<210> 65
<211> 321
<212> DNA
<213> Artificial
<220>
<223> 抗ICOS scFv的轻链可变区的核苷酸序列
<400> 65
gacatccaga tgacccagag ccccagcagc gtgagcgcca gcgtgggcga ccgcgtgacc 60
atcacctgcc gcgccagcca gggcatcagc cgcctgctgg cctggtacca gcagaagccc 120
ggcaaggccc ccaagctgct gatctacgtg gccagcagcc tgcagagcgg cgtgcccagc 180
cgcttcagcg gcagcggcag cggcaccgac ttcaccctga ccatcagcag cctgcagccc 240
gaggacttcg ccacctacta ctgccagcag gccaacagct tcccctggac cttcggccag 300
ggcaccaagg tggagatcaa g 321
<210> 66
<211> 726
<212> DNA
<213> Artificial
<220>
<223> 抗OX40 scFv的核苷酸序列
<400> 66
cagctggtgg agagcggcgg cggcctggtg cagcccggcg gcagcctgcg cctgagctgc 60
gccgccagcg gcttcacctt cagcagctac agcatgaact gggtgcgcca ggcccccggc 120
aagggcctgg agtgggtgag ctacatcagc agcagcagca gcaccatcta ctacgccgac 180
agcgtgaagg gccgcttcac catcagccgc gacaacgcca agaacagcct gtacctgcag 240
atgaacagcc tgcgcgacga ggacaccgcc gtgtactact gcgcccgcgg cgtgtaccac 300
aacggctgga gcttcttcga ctactggggc cagggcaccc tgctgaccgt gagcagcggc 360
ggcggcggca gcggcggcgg cggcagcggc ggcggcggca gcgacatcca gatgacccag 420
agccccagca gcctgagcgc cagcgtgggc aaccgcgtga ccatcacctg ccgcgccagc 480
caggacatca gcagctggct ggcctggtac cagcagaagc ccgagaaggc ccccaagagc 540
ctgatctacg ccgccagcag cctgcagagc ggcgtgccca gccgcttcag cggcagcggc 600
agcggcaccg acttcaccct gaccatcagc agcctgcagc ccgaggactt cgccacctac 660
tactgccagc agtacaacag ctaccccctg accttcggcc agggcacccg cctggagatc 720
aagcgc 726
<210> 67
<211> 357
<212> DNA
<213> Artificial
<220>
<223> 抗OX40 scFv的重链可变区的核苷酸序列
<400> 67
cagctggtgg agagcggcgg cggcctggtg cagcccggcg gcagcctgcg cctgagctgc 60
gccgccagcg gcttcacctt cagcagctac agcatgaact gggtgcgcca ggcccccggc 120
aagggcctgg agtgggtgag ctacatcagc agcagcagca gcaccatcta ctacgccgac 180
agcgtgaagg gccgcttcac catcagccgc gacaacgcca agaacagcct gtacctgcag 240
atgaacagcc tgcgcgacga ggacaccgcc gtgtactact gcgcccgcgg cgtgtaccac 300
aacggctgga gcttcttcga ctactggggc cagggcaccc tgctgaccgt gagcagc 357
<210> 68
<211> 324
<212> DNA
<213> Artificial
<220>
<223> 抗OX40 scFv的轻链可变区的核苷酸序列
<400> 68
gacatccaga tgacccagag ccccagcagc ctgagcgcca gcgtgggcaa ccgcgtgacc 60
atcacctgcc gcgccagcca ggacatcagc agctggctgg cctggtacca gcagaagccc 120
gagaaggccc ccaagagcct gatctacgcc gccagcagcc tgcagagcgg cgtgcccagc 180
cgcttcagcg gcagcggcag cggcaccgac ttcaccctga ccatcagcag cctgcagccc 240
gaggacttcg ccacctacta ctgccagcag tacaacagct accccctgac cttcggccag 300
ggcacccgcc tggagatcaa gcgc 324
<210> 69
<211> 723
<212> DNA
<213> Artificial
<220>
<223> 抗GITR scFv的核苷酸酸序列
<400> 69
caggtgaccc tgaaggagag cggccccggc atcctgaagc ccagccagac cctgagcctg 60
acctgcagct tcagcggctt cagcctgagc accagcggca tgggcgtggg ctggatccgc 120
cagcccagcg gcaagggcct ggagtggctg gcccacatct ggtgggacga cgacaagtac 180
tacaacccca gcctgaagag ccagctgacc atcagcaagg acaccagccg caaccaggtg 240
ttcctgaaga tcaccagcgt ggacaccgcc gacgccgcca cctactactg cgcccgcacc 300
cgccgctact tccccttcgc ctactggggc cagggcaccc tggtgaccgt gagcagcggc 360
ggcggcggca gcggcggcgg cggcagcggc ggcggcggca gcgacatcgt gatgacccag 420
agccagaagt tcatgagcac cagcgtgggc gaccgcgtga gcgtgacctg caaggccagc 480
cagaacgtgg gcaccaacgt ggcctggtac cagcagaagc ccggccagag ccccaaggcc 540
ctgatctaca gcgccagcta ccgctacagc ggcgtgcccg accgcttcac cggcagcggc 600
agcggcaccg acttcaccct gaccatcaac aacgtgcaca gcgaggacct ggccgagtac 660
ttctgccagc agtacaacac cgaccccctg accttcggcg ccggcaccaa gctggagatc 720
aag 723
<210> 70
<211> 357
<212> DNA
<213> Artificial
<220>
<223> 抗GITR scFv的重链可变区的核苷酸序列
<400> 70
caggtgaccc tgaaggagag cggccccggc atcctgaagc ccagccagac cctgagcctg 60
acctgcagct tcagcggctt cagcctgagc accagcggca tgggcgtggg ctggatccgc 120
cagcccagcg gcaagggcct ggagtggctg gcccacatct ggtgggacga cgacaagtac 180
tacaacccca gcctgaagag ccagctgacc atcagcaagg acaccagccg caaccaggtg 240
ttcctgaaga tcaccagcgt ggacaccgcc gacgccgcca cctactactg cgcccgcacc 300
cgccgctact tccccttcgc ctactggggc cagggcaccc tggtgaccgt gagcagc 357
<210> 71
<211> 321
<212> DNA
<213> Artificial
<220>
<223> 抗GITR scFv的轻链可变区的核苷酸序列
<400> 71
gacatcgtga tgacccagag ccagaagttc atgagcacca gcgtgggcga ccgcgtgagc 60
gtgacctgca aggccagcca gaacgtgggc accaacgtgg cctggtacca gcagaagccc 120
ggccagagcc ccaaggccct gatctacagc gccagctacc gctacagcgg cgtgcccgac 180
cgcttcaccg gcagcggcag cggcaccgac ttcaccctga ccatcaacaa cgtgcacagc 240
gaggacctgg ccgagtactt ctgccagcag tacaacaccg accccctgac cttcggcgcc 300
ggcaccaagc tggagatcaa g 321
<210> 72
<211> 717
<212> DNA
<213> Artificial
<220>
<223> 抗CD40L scFv的核苷酸序列
<400> 72
gaggtgcagc tgctggagag cggcggcggc ctggtgcagc ccggcggcag cctgcgcctg 60
agctgcgccg ccagcggctt caccttcagc agctacgcca tgagctgggt gcgccaggcc 120
cccggcaagg gcctggagtg ggtgagcgcc atcagcggca gcggcggcag cacctactac 180
gccgacagcg tgaagggccg cttcaccatc agccgcgaca acagcaagaa caccctgtac 240
ctgcagatga acagcctgcg cgccgaggac accgccgtgt actactgcgc caagagctac 300
ggcgccttcg actactgggg ccagggcacc ctggtgaccg tgagcagcgg cggcggcggc 360
agcggcggcg gcggcagcgg cggcggcggc agcgacatcc agatgaccca gagccccagc 420
agcctgagcg ccagcgtggg cgaccgcgtg accatcacct gccgcgccag ccagagcatc 480
agcagctacc tgaactggta ccagcagaag cccggcaagg cccccaagct gctgatctac 540
gccgccagca gcctgcagag cggcgtgccc agccgcttca gcggcagcgg cagcggcacc 600
gacttcaccc tgaccatcag cagcctgcag cccgaggact tcgccaccta ctactgccag 660
cagagctaca gcacccccaa caccttcggc cagggcacca aggtggagat caagcgc 717
<210> 73
<211> 348
<212> DNA
<213> Artificial
<220>
<223> 抗CD40L scFv的重链可变区的核苷酸序列
<400> 73
gaggtgcagc tgctggagag cggcggcggc ctggtgcagc ccggcggcag cctgcgcctg 60
agctgcgccg ccagcggctt caccttcagc agctacgcca tgagctgggt gcgccaggcc 120
cccggcaagg gcctggagtg ggtgagcgcc atcagcggca gcggcggcag cacctactac 180
gccgacagcg tgaagggccg cttcaccatc agccgcgaca acagcaagaa caccctgtac 240
ctgcagatga acagcctgcg cgccgaggac accgccgtgt actactgcgc caagagctac 300
ggcgccttcg actactgggg ccagggcacc ctggtgaccg tgagcagc 348
<210> 74
<211> 324
<212> DNA
<213> Artificial
<220>
<223> 抗CD40L scFv的轻链可变区的核苷酸序列
<400> 74
gacatccaga tgacccagag ccccagcagc ctgagcgcca gcgtgggcga ccgcgtgacc 60
atcacctgcc gcgccagcca gagcatcagc agctacctga actggtacca gcagaagccc 120
ggcaaggccc ccaagctgct gatctacgcc gccagcagcc tgcagagcgg cgtgcccagc 180
cgcttcagcg gcagcggcag cggcaccgac ttcaccctga ccatcagcag cctgcagccc 240
gaggacttcg ccacctacta ctgccagcag agctacagca cccccaacac cttcggccag 300
ggcaccaagg tggagatcaa gcgc 324
<210> 75
<211> 723
<212> DNA
<213> Artificial
<220>
<223> 抗CD27 scFv的核苷酸序列
<400> 75
caggtgcagc tggtggagag cggcggcggc gtggtgcagc ccggccgcag cctgcgcctg 60
agctgcgccg ccagcggctt caccttcagc agctacgaca tgcactgggt gcgccaggcc 120
cccggcaagg gcctggagtg ggtggccgtg atctggtacg acggcagcaa caagtactac 180
gccgacagcg tgaagggccg cttcaccatc agccgcgaca acagcaagaa caccctgtac 240
ctgcagatga acagcctgcg cgccgaggac accgccgtgt actactgcgc ccgcggcagc 300
ggcaactggg gcttcttcga ctactggggc cagggcaccc tggtgaccgt gagcagcggc 360
ggcggcggca gcggcggcgg cggcagcggc ggcggcggca gcgacatcca gatgacccag 420
agccccagca gcctgagcgc cagcgtgggc gaccgcgtga ccatcacctg ccgcgccagc 480
cagggcatca gccgctggct ggcctggtac cagcagaagc ccgagaaggc ccccaagagc 540
ctgatctacg ccgccagcag cctgcagagc ggcgtgccca gccgcttcag cggcagcggc 600
agcggcaccg acttcaccct gaccatcagc agcctgcagc ccgaggactt cgccacctac 660
tactgccagc agtacaacac ctacccccgc accttcggcc agggcaccaa ggtggagatc 720
aag 723
<210> 76
<211> 357
<212> DNA
<213> Artificial
<220>
<223> 抗CD27 scFv的重链可变区的核苷酸序列
<400> 76
caggtgcagc tggtggagag cggcggcggc gtggtgcagc ccggccgcag cctgcgcctg 60
agctgcgccg ccagcggctt caccttcagc agctacgaca tgcactgggt gcgccaggcc 120
cccggcaagg gcctggagtg ggtggccgtg atctggtacg acggcagcaa caagtactac 180
gccgacagcg tgaagggccg cttcaccatc agccgcgaca acagcaagaa caccctgtac 240
ctgcagatga acagcctgcg cgccgaggac accgccgtgt actactgcgc ccgcggcagc 300
ggcaactggg gcttcttcga ctactggggc cagggcaccc tggtgaccgt gagcagc 357
<210> 77
<211> 321
<212> DNA
<213> Artificial
<220>
<223> 抗CD27 scFv的轻链可变区的核苷酸序列
<400> 77
gacatccaga tgacccagag ccccagcagc ctgagcgcca gcgtgggcga ccgcgtgacc 60
atcacctgcc gcgccagcca gggcatcagc cgctggctgg cctggtacca gcagaagccc 120
gagaaggccc ccaagagcct gatctacgcc gccagcagcc tgcagagcgg cgtgcccagc 180
cgcttcagcg gcagcggcag cggcaccgac ttcaccctga ccatcagcag cctgcagccc 240
gaggacttcg ccacctacta ctgccagcag tacaacacct acccccgcac cttcggccag 300
ggcaccaagg tggagatcaa g 321
<210> 78
<211> 19
<212> PRT
<213> Artificial
<220>
<223> 分泌表达信号肽的氨基酸序列
<400> 78
Met Thr Arg Leu Thr Val Leu Ala Leu Leu Ala Gly Leu Leu Ala Ser
1 5 10 15
Ser Arg Ala
<210> 79
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 分泌表达信号肽的核苷酸序列
<400> 79
atgacccggc tgaccgtgct ggccctgctg gccggcctgc tggcctcctc cagggcc 57
<210> 80
<211> 59
<212> DNA
<213> Artificial
<220>
<223> pcDNA3.1-Sig-F
<400> 80
gtgctggata tctgcagaat tcgccgccac catgacccgg ctgaccgtgc tggccctgc 59
<210> 81
<211> 49
<212> DNA
<213> Artificial
<220>
<223> Sig-R
<400> 81
ggccctggag gaggccagca ggccggccag cagggccagc acggtcagc 49
<210> 82
<211> 41
<212> DNA
<213> Artificial
<220>
<223> Sig-CD3-F
<400> 82
gctggcctcc tccagggccg acatcaagct gcagcagagc g 41
<210> 83
<211> 20
<212> DNA
<213> Artificial
<220>
<223> CD3-R
<400> 83
cttcagctcc agcttggtgc 20
<210> 84
<211> 85
<212> DNA
<213> Artificial
<220>
<223> CD3-(GGGGS)3-4-1BB-F
<400> 84
gcaccaagct ggagctgaag ggcggcggcg gcagcggcgg cggcggcagc ggcggcggcg 60
gcagccaggt gcagctgcag cagtg 85
<210> 85
<211> 51
<212> DNA
<213> Artificial
<220>
<223> pcDNA3.1-4-1BB-R
<400> 85
ctgatcagcg gtttaaactt aagctttcag cgcttgatct ccaccttggt g 51
<210> 86
<211> 41
<212> DNA
<213> Artificial
<220>
<223> CD3-IgD-F
<400> 86
gcaccaagct ggagctgaag gccagcaaga gcaagaagga g 41
<210> 87
<211> 21
<212> DNA
<213> Artificial
<220>
<223> IgD-R
<400> 87
cacgcccagg ggctgggtgt g 21
<210> 88
<211> 42
<212> DNA
<213> Artificial
<220>
<223> IgD-4-1BB-F
<400> 88
cacacccagc ccctgggcgt gcaggtgcag ctgcagcagt gg 42
<210> 89
<211> 86
<212> DNA
<213> Artificial
<220>
<223> CD3-(GGGGS)3-ICOS-F
<400> 89
gcaccaagct ggagctgaag ggcggcggcg gcagcggcgg cggcggcagc ggcggcggcg 60
gcagccaggt gcagctggtg cagagc 86
<210> 90
<211> 50
<212> DNA
<213> Artificial
<220>
<223> pcDNA3.1-ICOS-R
<400> 90
ctgatcagcg gtttaaactt aagctttcac ttgatctcca ccttggtgcc 50
<210> 91
<211> 42
<212> DNA
<213> Artificial
<220>
<223> IgD-ICOS-F
<400> 91
cacacccagc ccctgggcgt gcaggtgcag ctggtgcaga gc 42
<210> 92
<211> 85
<212> DNA
<213> Artificial
<220>
<223> CD3-(GGGGS)3-OX40-F
<400> 92
gcaccaagct ggagctgaag ggcggcggcg gcagcggcgg cggcggcagc ggcggcggcg 60
gcagccagct ggtggagagc ggcgg 85
<210> 93
<211> 52
<212> DNA
<213> Artificial
<220>
<223> pcDNA3.1-OX40-R
<400> 93
ctgatcagcg gtttaaactt aagctttcag cgcttgatct ccaggcgggt gc 52
<210> 94
<211> 45
<212> DNA
<213> Artificial
<220>
<223> IgD-OX40-F
<400> 94
gccacaccca gcccctgggc gtgcagctgg tggagagcgg cggcg 45
<210> 95
<211> 85
<212> DNA
<213> Artificial
<220>
<223> CD3-(GGGGS)3-GITR-F
<400> 95
gcaccaagct ggagctgaag ggcggcggcg gcagcggcgg cggcggcagc ggcggcggcg 60
gcagccaggt gaccctgaag gagag 85
<210> 96
<211> 52
<212> DNA
<213> Artificial
<220>
<223> pcDNA3.1-GITR-R
<400> 96
ctgatcagcg gtttaaactt aagctttcac ttgatctcca gcttggtgcc gg 52
<210> 97
<211> 43
<212> DNA
<213> Artificial
<220>
<223> IgD-GITR-F
<400> 97
gccacaccca gcccctgggc gtgcaggtga ccctgaagga gag 43
<210> 98
<211> 87
<212> DNA
<213> Artificial
<220>
<223> CD3-(GGGGS)3-CD40L-F
<400> 98
ggcaccaagc tggagctgaa gggcggcggc ggcagcggcg gcggcggcag cggcggcggc 60
ggcagcgagg tgcagctgct ggagagc 87
<210> 99
<211> 51
<212> DNA
<213> Artificial
<220>
<223> pcDNA3.1-CD40L-R
<400> 99
ctgatcagcg gtttaaactt aagctttcag cgcttgatct ccaccttggt g 51
<210> 100
<211> 43
<212> DNA
<213> Artificial
<220>
<223> IgD-CD40L-F
<400> 100
gccacaccca gcccctgggc gtggaggtgc agctgctgga gag 43
<210> 101
<211> 86
<212> DNA
<213> Artificial
<220>
<223> CD3-(GGGGS)3-CD27-F
<400> 101
gcaccaagct ggagctgaag ggcggcggcg gcagcggcgg cggcggcagc ggcggcggcg 60
gcagccaggt gcagctggtg gagagc 86
<210> 102
<211> 51
<212> DNA
<213> Artificial
<220>
<223> pcDNA3.1-CD27-R
<400> 102
ctgatcagcg gtttaaactt aagctttcac ttgatctcca ccttggtgcc c 51
<210> 103
<211> 43
<212> DNA
<213> Artificial
<220>
<223> IgD-CD27-F
<400> 103
gccacaccca gcccctgggc gtgcaggtgc agctggtgga gag 43
Claims (11)
1.一种双功能分子，其结构中包括能够结合并激活T细胞表面CD3分子的第一功能域和能够结合并激活T细胞正共刺激分子的第二功能域，所述第一功能域为抗CD3的单链抗体，所述第二功能域为选自抗4-1BB的单链抗体、抗 ICOS的单链抗体、抗OX40的单链抗体、抗GITR的单链抗体、抗CD40L的单链抗体或抗CD27的单链抗体之任一的抗T细胞正共刺激分子的单链抗体，所述单链抗体包括重链可变区和轻链可变区；
其中所述第一功能域和所述第二功能域通过连接片段连接，所述连接片段为SEQ IDNO. 3所示的免疫球蛋白IgD的铰链区片段。
2.根据权利要求1所述的双功能分子，其特征在于，所述双功能分子能够同时结合并激活T细胞表面CD3分子和T细胞正共刺激分子，从而产生T细胞活化所需的第一信号和第二信号。
3.根据权利要求1所述的双功能分子，其特征在于，所述抗CD3的单链抗体的重链可变区的氨基酸序列如SEQ ID NO.37所示；所述抗CD3的单链抗体的轻链可变区的氨基酸序列如SEQ ID NO.38所示；所述抗4-1BB的单链抗体的重链可变区的氨基酸序列如SEQ IDNO.40所示；所述抗4-1BB的单链抗体的轻链可变区的氨基酸序列如SEQ ID NO.41所示；所述抗ICOS 的单链抗体的重链可变区的氨基酸序列如SEQ ID NO.43所示；所述抗ICOS的单链抗体的轻链可变区的氨基酸序列如SEQ ID NO.44所示；所述抗OX40的单链抗体的重链可变区的氨基酸序列如SEQ ID NO.46所示；所述抗OX40的单链抗体的轻链可变区的氨基酸序列如SEQ ID NO.47所示；所述抗GITR的单链抗体的重链可变区的氨基酸序列如SEQ IDNO.49所示；所述抗GITR的单链抗体的轻链可变区的氨基酸序列如SEQ ID NO.50所示；所述抗CD40L的单链抗体的重链可变区的氨基酸序列如SEQ ID NO.52所示；所述抗CD40L的单链抗体的轻链可变区的氨基酸序列如SEQ ID NO.53所示；所述抗CD27的单链抗体的重链可变区的氨基酸序列如SEQ ID NO.55所示；所述抗CD27的单链抗体的轻链可变区的氨基酸序列如SEQ ID NO.56所示。
4.根据权利要求3所述的双功能分子，其特征在于，所述抗CD3的单链抗体的氨基酸序列如SEQ ID NO.36所示；所述抗4-1BB的单链抗体的氨基酸序列如SEQ ID NO.39所示；所述抗ICOS的单链抗体的氨基酸序列如SEQ ID NO.42所示；所述抗OX40的单链抗体的氨基酸序列如SEQ ID NO.45所示；所述抗GITR的单链抗体的氨基酸序列如SEQ ID NO.48所示；所述抗CD40L的单链抗体的氨基酸序列如SEQ ID NO.51所示；所述抗CD27的单链抗体的氨基酸序列如SEQ ID NO.54所示。
5.根据权利要求1所述的双功能分子，其特征在于，所述双功能分子的氨基酸序列如SEQ ID NO.14、SEQ ID NO.18、SEQ ID NO.22、SEQ ID NO.26、SEQ ID NO.30或SEQ IDNO.34之任一所示。
6.一种多核苷酸，其编码如权利要求1～5任一项所述双功能分子。
7.一种表达载体，其含有如权利要求6所述的多核苷酸。
8.一种宿主细胞，其被如权利要求7所述的表达载体所转化。
9.如权利要求1～5任一项所述双功能分子的制备方法，包括：构建含有双功能分子基因序列的表达载体，然后将含双功能分子基因序列的表达载体转化至宿主细胞中诱导表达，从表达产物中分离获得所述的双功能分子。
10.如权利要求1～5任一项所述双功能分子用于制备T细胞体外扩增剂的用途。
11.一种体外扩增T细胞的方法，包括将如权利要求1～5任一项所述双功能分子作用于T细胞。
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