CN108264560B - 一种结合cd3和cd28的双功能分子及其应用 - Google Patents
一种结合cd3和cd28的双功能分子及其应用 Download PDFInfo
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- CN108264560B CN108264560B CN201611260781.3A CN201611260781A CN108264560B CN 108264560 B CN108264560 B CN 108264560B CN 201611260781 A CN201611260781 A CN 201611260781A CN 108264560 B CN108264560 B CN 108264560B
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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CD28的双功能分子及其应用。本发明所述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细胞表面CD28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CD28全长抗体联用相比，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CD28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(例如CD28、4-1BB、CD40L、CTLA-4和PD1等)与APC细胞表面的共刺激分子配体(例如CD80、CD86、4-1BBL、CD40、PD-L1和PD-L2等)相互作用，产生T细胞活化的第二信号(共刺激信号)：其中CD28、4-1BB和CD40L等属于正共刺激分子，其产生的第二信号可导致T细胞的完全活化；而CTLA-4和PD1等属于负共刺激分子，其作用主要是下调和终止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)。为了克服CD3单克隆全长抗体的这一缺点，人们设计并构建了抗CD28的激活型单克隆全长抗体(US Patent 20100168400A1)，通过与抗CD3全长抗体联合使用，可以为T细胞提供完整的双信号活化途径。然而，两种单克隆全长抗体联合使用的方式在具体应用上仍存在一些不足，如明显增加了重组抗体表达与纯化的工作量与生产成本，实际应用于T细胞体外活化扩增时必须优化两种全长抗体的相对比例。此外，两个全长抗体联合使用时，为促进受体活化，需要添加较高浓度的抗体溶液或将抗体包被到培养板或微球上以增强其对受体的活化效果。
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CD28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细胞表面CD28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CD28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CD28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:17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.19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CD28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.6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.7所示。所述抗CD28的单链抗体的重链可变区的氨基酸序列如SEQ ID NO.9所示。所述抗CD28的单链抗体的轻链可变区的氨基酸序列如SEQ ID NO.10所示。
本发明一些实施例中，列举了抗CD3的单链抗体的氨基酸序列如SEQ ID NO.5所示。抗CD28的单链抗体的氨基酸序列如SEQ ID NO.8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.1所示。二聚体形式的双功能分子的氨基酸序列如SEQ ID NO.3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，包括步骤：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细胞表面CD28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；而抗CD3单克隆全长抗体与抗CD28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CD28全长抗体联合使用，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。
附图说明
图1：A.单体形式抗CD3/抗CD28双特异性抗体(CD3-CD28BsAb_M)的结构图；B.二聚体形式抗CD3/抗CD28双特异性抗体(CD3-CD28BsAb_D)的结构图。
图2：最终纯化的CD3-CD28BsAb_M和CD3-CD28BsAb_D重组蛋白经SDS-PAGE分析，还原和非还原条件下电泳图，A.纯化的CD3-CD28BsAb_M SDS-PAGE分析图，泳道1：分子量蛋白Marker；泳道2：还原性CD3-CD28BsAb_M；泳道3：非还原性CD3-CD28BsAb_M；B.纯化的CD3-CD28BsAb_D SDS-PAGE分析图，泳道1：分子量蛋白Marker；泳道2：还原性CD3-CD28BsAb_D；泳道3：非还原性CD3-CD28BsAb_D。
图3A：CD3-CD28BsAb_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CD28-hFc；▲不包被任何抗原的测定结果。
图3B：CD3-CD28BsAb_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CD28-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-CD28BsAb_M、CD3-CD28BsAb_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1：5ug/ml Anti-CD3和5ug/ml Anti-CD28包板；对照组2：溶液状态下添加100ng/mlAnti-CD3和100ng/ml Anti-CD28；实验组1：溶液状态下添加10ng/ml CD3-CD28BsAb_M；实验组2：溶液状态下添加10ng/ml CD3-CD28BsAb_D。
图5：基于流式细胞分析方法测定CD3+CD56+CIK细胞比例，取图4所述扩增后细胞，分别测定CD3+CD56+双阳性CIK细胞比例；其中，A：对照组1；B：对照组2；C：实验组1；D：实验组2。
图6：基于流式细胞分析方法测定CIK细胞CD8+/CD4+比例。取图4所述扩增后细胞，分别测定CD8+阳性与CD4+阳性的细胞比例。其中，A：对照组1；B：对照组2；C：实验组1；D：实验组2。
具体实施方式
一、术语和缩略语：
BsAb：双特异性抗体(Bi-specific Antibody)
scFv:单链可变区片段(Single-chain variable fragment)，又称为单链抗体
Fab：抗原结合片段(Fragement of antigen binding)
Fv：可变区片段(Variable fragment)
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
CD3-CD28BsAb_M:单体形式的抗CD3/抗CD28双特异性抗体
CD3-CD28BsAb_D:二聚体形式的抗CD3/抗CD28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细胞表面CD28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CD28分子，从而产生T细胞活化所需的第一信号和第二信号。
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CD28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CD28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.17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.19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1A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CD28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CD28抗原结合的单链抗体(scFv)。本发明的二聚体形式的双功能分子的结构示意图如图1B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CD28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CD28抗原结合的单链抗体(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。
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.6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.7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.5所示。所述第二功能域为抗CD28的单链抗体。所述抗CD28的单链抗体包括重链可变区和轻链可变区。所述抗CD28的单链抗体的重链可变区的氨基酸序列如SEQ ID NO.9所示。所述抗CD28的单链抗体的轻链可变区的氨基酸序列如SEQ ID NO.10所示。所述抗CD28的单链抗体的氨基酸序列如SEQ ID NO.8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.1所示。二聚体形式的双功能分子的氨基酸序列如SEQ ID NO.3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.12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 ID NO.13所示。
编码所述抗CD3的单链抗体的核苷酸序列如SEQ ID NO.11所示。
编码所述抗CD28的单链抗体的重链可变区的核苷酸序列如SEQ ID NO.15所示。
编码所述抗CD28的单链抗体的轻链可变区的核苷酸序列如SEQ ID NO.16所示。
编码所述抗CD28的单链抗体的核苷酸序列如SEQ ID NO.14所示。
编码氨基酸序列如SEQ ID NO:17所示的连接片段的核苷酸序列如SEQ ID NO.18所示。
编码氨基酸序列如SEQ ID NO:19所示的连接片段的核苷酸序列如SEQ ID NO.20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.2所示。编码二聚体形式的双功能分子的核苷酸序列如SEQ ID NO.4。
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，以人外周血单个核细胞(Peripheral blood mononuclear cell，PBMC)为实验材料，用本发明所制备的双功能分子、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。结果，所述双功能分子能够很好地介导CIK(Cytokine induced killer)细胞增殖，且使用本发明的双功能分子对于CIK细胞的增殖效果优于抗CD3/抗CD28单克隆全长抗体联合使用，且蛋白用量更少。
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，以人外周血单个核细胞(Peripheral bloodmononuclear cell，PBMC)为实验材料，用本发明所制备的双功能分子、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。结果，所述双功能分子能够很好地介导CIK(Cytokineinduced killer)细胞增殖，且使用本发明的双功能分子对于CIK细胞的增殖效果优于抗CD3/抗CD28单克隆全长抗体联合使用，且蛋白用量更少。
本发明针对抗CD3和抗CD28单克隆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CD28的双功能分子，该双功能分子不仅具有上述抗CD3和抗CD28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，1989and Third edition，2001；Ausubel等，CURRENT PROTOCOLS INMOLECULAR BIOLOGY，John Wiley&Sons，New York，1987and 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1CD3-CD28BsAb_M和CD3-CD28BsAb_D真核表达载体的构建
在本发明中，以T细胞表面人类CD3和CD28蛋白为靶点的双特异性抗体被命名为CD3-CD28BsAb。
一、CD3-CD28BsAb_M和CD3-CD28BsAb_D构建方案设计
单体形式的CD3-CD28BsAb_M具体构建方案为：抗CD3scFv和抗CD28scFv序列之间通过(GGGGS)3Linker相连。
二聚体形式的CD3-CD28BsAb_D具体构建方案为：抗CD3scFv和抗CD28scFv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CD3scFv，抗CD28scFv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CD3scFv的重链可变区的核苷酸序列如SEQ ID NO.12所示，具体为：
GACATCAAGCTGCAGCAGAGCGGCGCCGAGCTGGCCCGCCCCGGCGCCAGCGTGAAGATGAGCTGCAAGACCAGCGGCTACACCTTCACCCGCTACACCATGCACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGGCTACATCAACCCCAGCCGCGGCTACACCAACTACAACCAGAAGTTCAAGGACAAGGCCACCCTGACCACCGACAAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCCGCTACTACGACGACCACTACTGCCTGGACTACTGGGGCCAGGGCACCACCCTGACCGTGAGCAGC。
抗CD3scFv的轻链可变区的核苷酸序列如SEQ ID NO.13所示，具体为：
GACATCCAGCTGACCCAGAGCCCCGCCATCATGAGCGCCAGCCCCGGCGAGAAGGTGACCATGACCTGCCGCGCCAGCAGCAGCGTGAGCTACATGAACTGGTACCAGCAGAAGAGCGGCACCAGCCCCAAGCGCTGGATCTACGACACCAGCAAGGTGGCCAGCGGCGTGCCCTACCGCTTCAGCGGCAGCGGCAGCGGCACCAGCTACAGCCTGACCATCAGCAGCATGGAGGCCGAGGACGCCGCCACCTACTACTGCCAGCAGTGGAGCAGCAACCCCCTGACCTTCGGCGCCGGCACCAAGCTGGAGCTGAAG。
抗CD3scFv的核苷酸序列如SEQ ID NO.11所示，具体为：
GACATCAAGCTGCAGCAGAGCGGCGCCGAGCTGGCCCGCCCCGGCGCCAGCGTGAAGATGAGCTGCAAGACCAGCGGCTACACCTTCACCCGCTACACCATGCACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGGCTACATCAACCCCAGCCGCGGCTACACCAACTACAACCAGAAGTTCAAGGACAAGGCCACCCTGACCACCGACAAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCCGCTACTACGACGACCACTACTGCCTGGACTACTGGGGCCAGGGCACCACCCTGACCGTGAGCAGCGTGGAGGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCGTGGACGACATCCAGCTGACCCAGAGCCCCGCCATCATGAGCGCCAGCCCCGGCGAGAAGGTGACCATGACCTGCCGCGCCAGCAGCAGCGTGAGCTACATGAACTGGTACCAGCAGAAGAGCGGCACCAGCCCCAAGCGCTGGATCTACGACACCAGCAAGGTGGCCAGCGGCGTGCCCTACCGCTTCAGCGGCAGCGGCAGCGGCACCAGCTACAGCCTGACCATCAGCAGCATGGAGGCCGAGGACGCCGCCACCTACTACTGCCAGCAGTGGAGCAGCAACCCCCTGACCTTCGGCGCCGGCACCAAGCTGGAGCTGAAG。
抗CD28scFv的重链可变区的核苷酸序列如SEQ ID NO.15所示，具体为：
CAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCGCCAGCGTGAAGGTGAGCTGCAAGGCCAGCGGCTACACCTTCACCAGCTACTACATCCACTGGGTGCGCCAGGCCCCCGGCCAGGGCCTGGAGTGGATCGGCTGCATCTACCCCGGCAACGTGAACACCAACTACAACGAGAAGTTCAAGGACCGCGCCACCCTGACCGTGGACACCAGCATCAGCACCGCCTACATGGAGCTGAGCCGCCTGCGCAGCGACGACACCGCCGTGTACTTCTGCACCCGCAGCCACTACGGCCTGGACTGGAACTTCGACGTGTGGGGCCAGGGCACCACCGTGACCGTGAGCAGC。
抗CD28scFv的轻链可变区的核苷酸序列如SEQ ID NO.16所示，具体为：
GACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCGACCGCGTGACCATCACCTGCCACGCCAGCCAGAACATCTACGTGTGGCTGAACTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACAAGGCCAGCAACCTGCACACCGGCGTGCCCAGCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGGGCCAGACCTACCCCTACACCTTCGGCGGCGGCACCAAGGTGGAGATCAAGCGC。
抗CD28scFv的核苷酸序列如SEQ ID NO.14所示，具体为：
CAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCGCCAGCGTGAAGGTGAGCTGCAAGGCCAGCGGCTACACCTTCACCAGCTACTACATCCACTGGGTGCGCCAGGCCCCCGGCCAGGGCCTGGAGTGGATCGGCTGCATCTACCCCGGCAACGTGAACACCAACTACAACGAGAAGTTCAAGGACCGCGCCACCCTGACCGTGGACACCAGCATCAGCACCGCCTACATGGAGCTGAGCCGCCTGCGCAGCGACGACACCGCCGTGTACTTCTGCACCCGCAGCCACTACGGCCTGGACTGGAACTTCGACGTGTGGGGCCAGGGCACCACCGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCGACCGCGTGACCATCACCTGCCACGCCAGCCAGAACATCTACGTGTGGCTGAACTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACAAGGCCAGCAACCTGCACACCGGCGTGCCCAGCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGGGCCAGACCTACCCCTACACCTTCGGCGGCGGCACCAAGGTGGAGATCAAGCGC。
单体形式的CD3-CD28BsAb_M连接片段的核苷酸序列如SEQ ID NO.18所示，具体为：
GGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGC。
二聚体形式的CD3-CD28BsAb_D连接片段的核苷酸序列如SEQ ID NO.20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.21所示，具体为：
MTRLTVLALLAGLLASSRA。
该分泌表达信号肽的核苷酸序列如SEQ ID NO.22所示，具体为：
ATGACCCGGCTGACCGTGCTGGCCCTGCTGGCCGGCCTGCTGGCCTCCTCCAGGGCC。
二、CD3-CD28BsAb_M和CD3-CD28BsAb_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-CD28BsAb_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-CD28-F和pcDNA3.1-CD28-R扩增出抗CD3scFv、(GGGGS)3Linker、抗CD28scFv的基因序列；针对CD3-CD28BsAb_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-CD28-F和pcDNA3.1-CD28-R扩增出抗CD3scFv、IgD铰链区、抗CD28scFv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-CD28BsAb_M和二聚体形式的CD3-CD28BsAb_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-CD28BsAb_M的核苷酸序列如SEQ ID NO.2所示，具体为：
GACATCAAGCTGCAGCAGAGCGGCGCCGAGCTGGCCCGCCCCGGCGCCAGCGTGAAGATGAGCTGCAAGACCAGCGGCTACACCTTCACCCGCTACACCATGCACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGGCTACATCAACCCCAGCCGCGGCTACACCAACTACAACCAGAAGTTCAAGGACAAGGCCACCCTGACCACCGACAAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCCGCTACTACGACGACCACTACTGCCTGGACTACTGGGGCCAGGGCACCACCCTGACCGTGAGCAGCGTGGAGGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCGTGGACGACATCCAGCTGACCCAGAGCCCCGCCATCATGAGCGCCAGCCCCGGCGAGAAGGTGACCATGACCTGCCGCGCCAGCAGCAGCGTGAGCTACATGAACTGGTACCAGCAGAAGAGCGGCACCAGCCCCAAGCGCTGGATCTACGACACCAGCAAGGTGGCCAGCGGCGTGCCCTACCGCTTCAGCGGCAGCGGCAGCGGCACCAGCTACAGCCTGACCATCAGCAGCATGGAGGCCGAGGACGCCGCCACCTACTACTGCCAGCAGTGGAGCAGCAACCCCCTGACCTTCGGCGCCGGCACCAAGCTGGAGCTGAAGGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCCAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCGCCAGCGTGAAGGTGAGCTGCAAGGCCAGCGGCTACACCTTCACCAGCTACTACATCCACTGGGTGCGCCAGGCCCCCGGCCAGGGCCTGGAGTGGATCGGCTGCATCTACCCCGGCAACGTGAACACCAACTACAACGAGAAGTTCAAGGACCGCGCCACCCTGACCGTGGACACCAGCATCAGCACCGCCTACATGGAGCTGAGCCGCCTGCGCAGCGACGACACCGCCGTGTACTTCTGCACCCGCAGCCACTACGGCCTGGACTGGAACTTCGACGTGTGGGGCCAGGGCACCACCGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCGACCGCGTGACCATCACCTGCCACGCCAGCCAGAACATCTACGTGTGGCTGAACTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACAAGGCCAGCAACCTGCACACCGGCGTGCCCAGCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGGGCCAGACCTACCCCTACACCTTCGGCGGCGGCACCAAGGTGGAGATCAAGCGC。
二聚体形式的CD3-CD28BsAb_D的核苷酸序列如SEQ ID NO.4所示，具体为：
GACATCAAGCTGCAGCAGAGCGGCGCCGAGCTGGCCCGCCCCGGCGCCAGCGTGAAGATGAGCTGCAAGACCAGCGGCTACACCTTCACCCGCTACACCATGCACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGGCTACATCAACCCCAGCCGCGGCTACACCAACTACAACCAGAAGTTCAAGGACAAGGCCACCCTGACCACCGACAAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCCGCTACTACGACGACCACTACTGCCTGGACTACTGGGGCCAGGGCACCACCCTGACCGTGAGCAGCGTGGAGGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCGTGGACGACATCCAGCTGACCCAGAGCCCCGCCATCATGAGCGCCAGCCCCGGCGAGAAGGTGACCATGACCTGCCGCGCCAGCAGCAGCGTGAGCTACATGAACTGGTACCAGCAGAAGAGCGGCACCAGCCCCAAGCGCTGGATCTACGACACCAGCAAGGTGGCCAGCGGCGTGCCCTACCGCTTCAGCGGCAGCGGCAGCGGCACCAGCTACAGCCTGACCATCAGCAGCATGGAGGCCGAGGACGCCGCCACCTACTACTGCCAGCAGTGGAGCAGCAACCCCCTGACCTTCGGCGCCGGCACCAAGCTGGAGCTGAAGGCCAGCAAGAGCAAGAAGGAGATCTTCCGCTGGCCCGAGAGCCCCAAGGCCCAGGCCAGCAGCGTGCCCACCGCCCAGCCCCAGGCCGAGGGCAGCCTGGCCAAGGCCACCACCGCCCCCGCCACCACCCGCAACACCGGCCGCGGCGGCGAGGAGAAGAAGAAGGAGAAGGAGAAGGAGGAGCAGGAGGAGCGCGAGACCAAGACCCCCGAGTGCCCCAGCCACACCCAGCCCCTGGGCGTGCAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCGCCAGCGTGAAGGTGAGCTGCAAGGCCAGCGGCTACACCTTCACCAGCTACTACATCCACTGGGTGCGCCAGGCCCCCGGCCAGGGCCTGGAGTGGATCGGCTGCATCTACCCCGGCAACGTGAACACCAACTACAACGAGAAGTTCAAGGACCGCGCCACCCTGACCGTGGACACCAGCATCAGCACCGCCTACATGGAGCTGAGCCGCCTGCGCAGCGACGACACCGCCGTGTACTTCTGCACCCGCAGCCACTACGGCCTGGACTGGAACTTCGACGTGTGGGGCCAGGGCACCACCGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCGACCGCGTGACCATCACCTGCCACGCCAGCCAGAACATCTACGTGTGGCTGAACTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACAAGGCCAGCAACCTGCACACCGGCGTGCCCAGCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGGGCCAGACCTACCCCTACACCTTCGGCGGCGGCACCAAGGTGGAGATCAAGCGC。
表1.双特异性抗体基因克隆中使用的引物
实施例2：CD3-CD28BsAb_M和CD3-CD28BsAb_D的表达与纯化
一、CD3-CD28BsAb_M和CD3-CD28BsAb_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-CD28BsAb_M和CD3-CD28BsAb_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-CD28BsAb_M和CD3-CD28BsAb_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；
2.2Protein 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BufferA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1.5mlBuffer 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-CD28BsAb_M和CD3-CD28BsAb_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-CD28BsAb_M和CD3-CD28BsAb_D重组蛋白的纯度均>95％；其中CD3-CD28BsAb_M重组蛋白的理论分子量为54.4kDa，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-CD28BsAb_D重组蛋白的理论分子量为62.2kDa，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-CD28BsAb_M为单体形式，CD3-CD28BsAb_D为二聚体形式。
因此，可得知，单体形式的CD3-CD28BsAb_M的氨基酸序列如SEQ ID NO.1所示，具体为：
DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGLEWIGCIYPGNVNTNYNEKFKDRATLTVDTSISTAYMELSRLRSDDTAVYFCTRSHYGLDWNFDVWGQGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCHASQNIYVWLNWYQQKPGKAPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGQTYPYTFGGGTKVEIKR。
二聚体形式的CD3-CD28BsAb_D的氨基酸序列如SEQ ID NO.3所示，具体为：
DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKASKSKKEIFRWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEKEKEEQEERETKTPECPSHTQPLGVQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGLEWIGCIYPGNVNTNYNEKFKDRATLTVDTSISTAYMELSRLRSDDTAVYFCTRSHYGLDWNFDVWGQGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCHASQNIYVWLNWYQQKPGKAPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGQTYPYTFGGGTKVEIKR。
抗CD3scFv的氨基酸序列如SEQ ID NO.5所示，具体为：
DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELK。
抗CD3scFv的重链可变区的氨基酸序列如SEQ ID NO.6所示，具体为：
DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSS。
抗CD3scFv的轻链可变区的氨基酸序列如SEQ ID NO.7所示，具体为：
DIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELK。
抗CD28scFv的氨基酸序列如SEQ ID NO.8所示，具体为：
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGLEWIGCIYPGNVNTNYNEKFKDRATLTVDTSISTAYMELSRLRSDDTAVYFCTRSHYGLDWNFDVWGQGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCHASQNIYVWLNWYQQKPGKAPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGQTYPYTFGGGTKVEIKR。
抗CD28scFv的重链可变区的氨基酸序列如SEQ ID NO.9所示，具体为：
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGLEWIGCIYPGNVNTNYNEKFKDRATLTVDTSISTAYMELSRLRSDDTAVYFCTRSHYGLDWNFDVWGQGTTVTVSS。
抗CD28scFv的轻链可变区的氨基酸序列如SEQ ID NO.10所示，具体为：
DIQMTQSPSSLSASVGDRVTITCHASQNIYVWLNWYQQKPGKAPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGQTYPYTFGGGTKVEIKR。
单体形式的CD3-CD28BsAb_M中连接片段的氨基酸序列如SEQ ID NO.17所示，具体为：GGGGSGGGGSGGGGS。
二聚体形式的CD3-CD28BsAb_D中连接片段的氨基酸序列如SEQ ID NO.19所示，具体为：
ASKSKKEIFRWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEKEKEEQEERETKTPECPSHTQPLGV。
实施例3：ELISA检测CD3-CD28BsAb_M和CD3-CD28BsAb_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CD28-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-CD28BsAb_M或CD3-CD28BsAb_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-CD28BsAb_M与重组抗原CD3-hFc和CD28-hFc均具有体外结合活性，其中CD28结合活性较CD3结合活性更高；图3B说明CD3-CD28BsAb_D与重组抗原CD3-hFc和CD28-hFc同样具有体外结合活性，其中CD28结合活性更高。
实施例4：CD3-CD28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-CD28BsAb_M、二聚体形式的双特异抗体CD3-CD28BsAb_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，分别设计以下实验组：对照组1(Anti-CD3 5ug/ml和Anti-CD285ug/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)；对照组2(溶液状态下添加全长抗体Anti-CD3 100ng/ml和Anti-CD28 100ng/ml)；实验组1(溶液状态下添加双特异性抗体CD3-CD28BsAb_M 10ng/ml)；实验组2(溶液状态下添加双特异性抗体CD3-CD28BsAb_D 10ng/ml)。此外，四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2和图4所示，单体和二聚体形式的CD3-CD28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 100ng/ml)，其中二聚体形式的CD3-CD28BsAb_D可以介导CIK细胞两周扩增373倍，效果最优(实验组2)；单体形式的CD3-CD28BsAb_M可以介导CIK细胞两周扩增278倍，效果次之(实验组1)。
表2CIK细胞扩增倍数
| 实验组名 | 对照组1 | 对照组2 | 实验组1 | 实验组2 |
| 14天扩增倍数 | 224 | 196 | 278 | 373 |
实施例5：CD3-CD28双特异抗体介导的CIK细胞增殖后表型检测
1.CD3+CD56+双阳性CIK细胞的流式检测
取实施例4所述培养14天后的4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1细胞4份，其他3组(对照组2、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1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3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5所示：其中CD3-CD28BsAb_M介导CIK细胞增殖2周后，CD3+CD56+双阳性的细胞比例为13.23％，CD3-CD28BsAb_D介导CIK细胞增殖2周后，CD3+CD56+双阳性的细胞比例为13.92％，与Anti-CD3/Anti-CD28联用(CD3+CD56+双阳性比例：包被使用为12.90％；溶液添加为11.40％)的效果无明显差别，说明单体和二聚体形式CD3-CD28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。
2.CD8+/CD4+阳性细胞的流式检测
取实施例4所述培养14天后的4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：
2.1取对照组1细胞4份，其他3组(对照组2、实验组1、实验组2)各取细胞1份，每份细胞数目1×106；
2.2 1000rpm离心5min，弃上清，用200ul 2％BSA/PBS重悬细胞，离心清洗2次；
2.3对照组1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3份细胞只同时添加Anti-CD4-FITC和Anti-CD8-PE，4℃孵育1h；
2.4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6所示：CD3-CD28BsAb_M介导CIK细胞增殖2周后，CD8+阳性细胞比例为67.60％，CD3-CD28BsAb_D介导CIK细胞增殖2周后，CD8+阳性细胞比例为78.65％，均明显优于Anti-CD3/Anti-CD28联用(CD8+阳性比例：包被使用为48.95％；溶液添加为48.47％)，说明CD3-CD28双特异抗体比抗CD3/抗CD28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。
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。
SEQUENCE LISTING
<110> 上海欣百诺生物科技有限公司
<120> 一种结合CD3和CD28的双功能分子及其应用
<130> 163904
<160> 31
<170> PatentIn version 3.3
<210> 1
<211> 501
<212> PRT
<213> Artificial
<220>
<223> CD3-CD28 BsAb_M的氨基酸序列
<400> 1
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
Ser Tyr Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
290 295 300
Trp Ile Gly Cys Ile Tyr Pro Gly Asn Val Asn Thr Asn Tyr Asn Glu
305 310 315 320
Lys Phe Lys Asp Arg Ala Thr Leu Thr Val 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
Phe Cys Thr Arg Ser His Tyr Gly Leu Asp Trp Asn Phe Asp Val Trp
355 360 365
Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly
370 375 380
Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser
385 390 395 400
Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys
405 410 415
His Ala Ser Gln Asn Ile Tyr Val Trp Leu Asn Trp Tyr Gln Gln Lys
420 425 430
Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Lys Ala Ser Asn Leu His
435 440 445
Thr Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe
450 455 460
Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr
465 470 475 480
Cys Gln Gln Gly Gln Thr Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys
485 490 495
Val Glu Ile Lys Arg
500
<210> 2
<211> 1503
<212> DNA
<213> Artificial
<220>
<223> CD3-CD28 BsAb_M的核苷酸序列
<400> 2
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 caccagctac tacatccact gggtgcgcca ggcccccggc 900
cagggcctgg agtggatcgg ctgcatctac cccggcaacg tgaacaccaa ctacaacgag 960
aagttcaagg accgcgccac cctgaccgtg gacaccagca tcagcaccgc ctacatggag 1020
ctgagccgcc tgcgcagcga cgacaccgcc gtgtacttct gcacccgcag ccactacggc 1080
ctggactgga acttcgacgt gtggggccag ggcaccaccg tgaccgtgag cagcggcggc 1140
ggcggcagcg gcggcggcgg cagcggcggc ggcggcagcg acatccagat gacccagagc 1200
cccagcagcc tgagcgccag cgtgggcgac cgcgtgacca tcacctgcca cgccagccag 1260
aacatctacg tgtggctgaa ctggtaccag cagaagcccg gcaaggcccc caagctgctg 1320
atctacaagg ccagcaacct gcacaccggc gtgcccagcc gcttcagcgg cagcggcagc 1380
ggcaccgact tcaccctgac catcagcagc ctgcagcccg aggacttcgc cacctactac 1440
tgccagcagg gccagaccta cccctacacc ttcggcggcg gcaccaaggt ggagatcaag 1500
cgc 1503
<210> 3
<211> 567
<212> PRT
<213> Artificial
<220>
<223> CD3-CD28 BSAb_D的氨基酸序列
<400> 3
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 Ser Tyr Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly
355 360 365
Leu Glu Trp Ile Gly Cys Ile Tyr Pro Gly Asn Val Asn Thr Asn Tyr
370 375 380
Asn Glu Lys Phe Lys Asp Arg Ala Thr Leu Thr Val 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 Phe Cys Thr Arg Ser His Tyr Gly Leu Asp Trp Asn Phe Asp
420 425 430
Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly
435 440 445
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr
450 455 460
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile
465 470 475 480
Thr Cys His Ala Ser Gln Asn Ile Tyr Val Trp Leu Asn Trp Tyr Gln
485 490 495
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Lys Ala Ser Asn
500 505 510
Leu His Thr Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr
515 520 525
Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr
530 535 540
Tyr Tyr Cys Gln Gln Gly Gln Thr Tyr Pro Tyr Thr Phe Gly Gly Gly
545 550 555 560
Thr Lys Val Glu Ile Lys Arg
565
<210> 4
<211> 1701
<212> DNA
<213> Artificial
<220>
<223> CD3-CD28 BsAb_D的核苷酸序列
<400> 4
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 ccagctacta catccactgg 1080
gtgcgccagg cccccggcca gggcctggag tggatcggct gcatctaccc cggcaacgtg 1140
aacaccaact acaacgagaa gttcaaggac cgcgccaccc tgaccgtgga caccagcatc 1200
agcaccgcct acatggagct gagccgcctg cgcagcgacg acaccgccgt gtacttctgc 1260
acccgcagcc actacggcct ggactggaac ttcgacgtgt ggggccaggg caccaccgtg 1320
accgtgagca gcggcggcgg cggcagcggc ggcggcggca gcggcggcgg cggcagcgac 1380
atccagatga cccagagccc cagcagcctg agcgccagcg tgggcgaccg cgtgaccatc 1440
acctgccacg ccagccagaa catctacgtg tggctgaact ggtaccagca gaagcccggc 1500
aaggccccca agctgctgat ctacaaggcc agcaacctgc acaccggcgt gcccagccgc 1560
ttcagcggca gcggcagcgg caccgacttc accctgacca tcagcagcct gcagcccgag 1620
gacttcgcca cctactactg ccagcagggc cagacctacc cctacacctt cggcggcggc 1680
accaaggtgg agatcaagcg c 1701
<210> 5
<211> 243
<212> PRT
<213> Artificial
<220>
<223> 抗CD3 scFv的氨基酸序列
<400> 5
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> 6
<211> 119
<212> PRT
<213> Artificial
<220>
<223> 抗CD3 scFv的重链可变区的氨基酸序列
<400> 6
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> 7
<211> 106
<212> PRT
<213> Artificial
<220>
<223> 抗CD3 scFv的轻链可变区的氨基酸序列
<400> 7
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> 8
<211> 243
<212> PRT
<213> Artificial
<220>
<223> 抗CD28 scFv的氨基酸序列
<400> 8
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Cys Ile Tyr Pro Gly Asn Val Asn Thr Asn Tyr Asn Glu Lys Phe
50 55 60
Lys Asp Arg Ala Thr Leu Thr Val 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 Phe Cys
85 90 95
Thr Arg Ser His Tyr Gly Leu Asp Trp Asn Phe Asp Val Trp Gly Gln
100 105 110
Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly
115 120 125
Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser
130 135 140
Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys His Ala
145 150 155 160
Ser Gln Asn Ile Tyr Val Trp Leu Asn Trp Tyr Gln Gln Lys Pro Gly
165 170 175
Lys Ala Pro Lys Leu Leu Ile Tyr Lys Ala Ser Asn Leu His Thr Gly
180 185 190
Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
195 200 205
Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln
210 215 220
Gln Gly Gln Thr Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu
225 230 235 240
Ile Lys Arg
<210> 9
<211> 120
<212> PRT
<213> Artificial
<220>
<223> 抗CD28 scFv的重链可变区的氨基酸序列
<400> 9
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Cys Ile Tyr Pro Gly Asn Val Asn Thr Asn Tyr Asn Glu Lys Phe
50 55 60
Lys Asp Arg Ala Thr Leu Thr Val 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 Phe Cys
85 90 95
Thr Arg Ser His Tyr Gly Leu Asp Trp Asn Phe Asp Val Trp Gly Gln
100 105 110
Gly Thr Thr Val Thr Val Ser Ser
115 120
<210> 10
<211> 108
<212> PRT
<213> Artificial
<220>
<223> 抗CD28 scFv的轻链可变区的氨基酸序列
<400> 10
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys His Ala Ser Gln Asn Ile Tyr Val Trp
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Lys Ala Ser Asn Leu His Thr Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Gln Thr Tyr Pro Tyr
85 90 95
Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg
100 105
<210> 11
<211> 729
<212> DNA
<213> Artificial
<220>
<223> 抗CD3 scFv的核苷酸序列
<400> 11
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> 12
<211> 357
<212> DNA
<213> Artificial
<220>
<223> 抗CD3 scFv的重链可变区的核苷酸序列
<400> 12
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> 13
<211> 318
<212> DNA
<213> Artificial
<220>
<223> 抗CD3 scFv的轻链可变区的核苷酸序列
<400> 13
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> 14
<211> 729
<212> DNA
<213> Artificial
<220>
<223> 抗CD28 scFv的核苷酸序列
<400> 14
caggtgcagc tggtgcagag cggcgccgag gtgaagaagc ccggcgccag cgtgaaggtg 60
agctgcaagg ccagcggcta caccttcacc agctactaca tccactgggt gcgccaggcc 120
cccggccagg gcctggagtg gatcggctgc atctaccccg gcaacgtgaa caccaactac 180
aacgagaagt tcaaggaccg cgccaccctg accgtggaca ccagcatcag caccgcctac 240
atggagctga gccgcctgcg cagcgacgac accgccgtgt acttctgcac ccgcagccac 300
tacggcctgg actggaactt cgacgtgtgg ggccagggca ccaccgtgac cgtgagcagc 360
ggcggcggcg gcagcggcgg cggcggcagc ggcggcggcg gcagcgacat ccagatgacc 420
cagagcccca gcagcctgag cgccagcgtg ggcgaccgcg tgaccatcac ctgccacgcc 480
agccagaaca tctacgtgtg gctgaactgg taccagcaga agcccggcaa ggcccccaag 540
ctgctgatct acaaggccag caacctgcac accggcgtgc ccagccgctt cagcggcagc 600
ggcagcggca ccgacttcac cctgaccatc agcagcctgc agcccgagga cttcgccacc 660
tactactgcc agcagggcca gacctacccc tacaccttcg gcggcggcac caaggtggag 720
atcaagcgc 729
<210> 15
<211> 360
<212> DNA
<213> Artificial
<220>
<223> 抗CD28 scFv的重链可变区的核苷酸序列
<400> 15
caggtgcagc tggtgcagag cggcgccgag gtgaagaagc ccggcgccag cgtgaaggtg 60
agctgcaagg ccagcggcta caccttcacc agctactaca tccactgggt gcgccaggcc 120
cccggccagg gcctggagtg gatcggctgc atctaccccg gcaacgtgaa caccaactac 180
aacgagaagt tcaaggaccg cgccaccctg accgtggaca ccagcatcag caccgcctac 240
atggagctga gccgcctgcg cagcgacgac accgccgtgt acttctgcac ccgcagccac 300
tacggcctgg actggaactt cgacgtgtgg ggccagggca ccaccgtgac cgtgagcagc 360
<210> 16
<211> 324
<212> DNA
<213> Artificial
<220>
<223> 抗CD28 scFv的轻链可变区的核苷酸序列
<400> 16
gacatccaga tgacccagag ccccagcagc ctgagcgcca gcgtgggcga ccgcgtgacc 60
atcacctgcc acgccagcca gaacatctac gtgtggctga actggtacca gcagaagccc 120
ggcaaggccc ccaagctgct gatctacaag gccagcaacc tgcacaccgg cgtgcccagc 180
cgcttcagcg gcagcggcag cggcaccgac ttcaccctga ccatcagcag cctgcagccc 240
gaggacttcg ccacctacta ctgccagcag ggccagacct acccctacac cttcggcggc 300
ggcaccaagg tggagatcaa gcgc 324
<210> 17
<211> 15
<212> PRT
<213> Artificial
<220>
<223> CD3-CD28 BsAb_M中连接片段的氨基酸序列
<400> 17
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
1 5 10 15
<210> 18
<211> 45
<212> DNA
<213> Artificial
<220>
<223> CD3-CD28 BsAb_M连接片段的核苷酸序列
<400> 18
ggcggcggcg gcagcggcgg cggcggcagc ggcggcggcg gcagc 45
<210> 19
<211> 81
<212> PRT
<213> Artificial
<220>
<223> CD3-CD28 BsAb_D中连接片段的氨基酸序列
<400> 19
Ala Ser Lys Ser Lys Lys Glu Ile Phe Arg Trp Pro Glu Ser Pro Lys
1 5 10 15
Ala Gln Ala Ser Ser Val Pro Thr Ala Gln Pro Gln Ala Glu Gly Ser
20 25 30
Leu Ala Lys Ala Thr Thr Ala Pro Ala Thr Thr Arg Asn Thr Gly Arg
35 40 45
Gly Gly Glu Glu Lys Lys Lys Glu Lys Glu Lys Glu Glu Gln Glu Glu
50 55 60
Arg Glu Thr Lys Thr Pro Glu Cys Pro Ser His Thr Gln Pro Leu Gly
65 70 75 80
Val
<210> 20
<211> 243
<212> DNA
<213> Artificial
<220>
<223> CD3-CD28 BsAb_D连接片段的核苷酸序列
<400> 20
gccagcaaga gcaagaagga gatcttccgc tggcccgaga gccccaaggc ccaggccagc 60
agcgtgccca ccgcccagcc ccaggccgag ggcagcctgg ccaaggccac caccgccccc 120
gccaccaccc gcaacaccgg ccgcggcggc gaggagaaga agaaggagaa ggagaaggag 180
gagcaggagg agcgcgagac caagaccccc gagtgcccca gccacaccca gcccctgggc 240
gtg 243
<210> 21
<211> 19
<212> PRT
<213> Artificial
<220>
<223> 分泌表达信号肽的氨基酸序列
<400> 21
Met Thr Arg Leu Thr Val Leu Ala Leu Leu Ala Gly Leu Leu Ala Ser
1 5 10 15
Ser Arg Ala
<210> 22
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 分泌表达信号肽的核苷酸序列
<400> 22
atgacccggc tgaccgtgct ggccctgctg gccggcctgc tggcctcctc cagggcc 57
<210> 23
<211> 59
<212> DNA
<213> Artificial
<220>
<223> pcDNA3.1-Sig-F
<400> 23
gtgctggata tctgcagaat tcgccgccac catgacccgg ctgaccgtgc tggccctgc 59
<210> 24
<211> 49
<212> DNA
<213> Artificial
<220>
<223> Sig-R
<400> 24
ggccctggag gaggccagca ggccggccag cagggccagc acggtcagc 49
<210> 25
<211> 41
<212> DNA
<213> Artificial
<220>
<223> Sig-CD3-F
<400> 25
gctggcctcc tccagggccg acatcaagct gcagcagagc g 41
<210> 26
<211> 20
<212> DNA
<213> Artificial
<220>
<223> CD3-R
<400> 26
cttcagctcc agcttggtgc 20
<210> 27
<211> 86
<212> DNA
<213> Artificial
<220>
<223> CD3-(GGGGS)3-CD28-F
<400> 27
gcaccaagct ggagctgaag ggcggcggcg gcagcggcgg cggcggcagc ggcggcggcg 60
gcagccaggt gcagctggtg cagagc 86
<210> 28
<211> 51
<212> DNA
<213> Artificial
<220>
<223> pcDNA3.1-CD28-R
<400> 28
ctgatcagcg gtttaaactt aagctttcag cgcttgatct ccaccttggt g 51
<210> 29
<211> 41
<212> DNA
<213> Artificial
<220>
<223> CD3-IgD-F
<400> 29
gcaccaagct ggagctgaag gccagcaaga gcaagaagga g 41
<210> 30
<211> 21
<212> DNA
<213> Artificial
<220>
<223> IgD-R
<400> 30
cacgcccagg ggctgggtgt g 21
<210> 31
<211> 42
<212> DNA
<213> Artificial
<220>
<223> IgD-CD28-F
<400> 31
cacacccagc ccctgggcgt gcaggtgcag ctggtgcaga gc 42
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细胞表面CD28分子的第二功能域，所述第一功能域为抗CD3的单链抗体，所述第二功能域为抗CD28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，所述连接片段为如SEQ IDNO.19所示的免疫球蛋白IgD。
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CD28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.6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.7所示；所述抗CD28的单链抗体的重链可变区的氨基酸序列如SEQ ID NO.9所示；所述抗CD28的单链抗体的轻链可变区的氨基酸序列如SEQ ID NO.10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.5所示；所述抗CD28的单链抗体的氨基酸序列如SEQ ID NO.8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.3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，包括步骤：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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| US16/474,554 US20230242876A1 (en) | 2016-12-30 | 2017-08-09 | Bifunctional molecule and use thereof |
| EP17886498.9A EP3575319A4 (en) | 2016-12-30 | 2017-08-09 | BIFUNCTIONAL MOLECULE AND USE OF IT |
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| WO2021260064A1 (en) * | 2020-06-25 | 2021-12-30 | F. Hoffmann-La Roche Ag | Anti-cd3/anti-cd28 bispecific antigen binding molecules |
| WO2022077108A1 (en) * | 2020-10-15 | 2022-04-21 | Sunnybook Research Institute | Bispecific constructs for expanding t cells and related methods |
| CN117467009A (zh) * | 2022-07-28 | 2024-01-30 | 四川思柏沃生物技术有限公司 | 抗cd28人源化单域抗体 |
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