CN112250767A - 一种结合Strep-Tag II标签的抗体及其应用 - Google Patents
一种结合Strep-Tag II标签的抗体及其应用 Download PDFInfo
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Abstract
本发明公开了本发明涉及一种特异性结合Strep标签II的抗体，进一步公开了本发明抗体的氨基酸序列、克隆或表达载体、宿主细胞、以及用于表达或分离抗体的方法，还公开了包含本发明抗体的组合物。本发明还公开了使用本发明抗体检测嵌合抗原受体细胞或工程化细胞受体细胞的方法。
Description
技术领域
本发明涉及一种结合Strep-Tag II标签的抗体，并公开了该抗体的氨基酸序列、克隆或表达载体、宿主细胞、生产抗体的方法以及使用该抗体检测融合多肽的方法。
背景技术
随着肿瘤免疫治疗的发展，CAR-T（Chimeric antigen receptor-T cell，嵌合抗原受体T细胞）和TCR-T（T cell receptor-T cell，工程化T细胞受体T细胞）等细胞免疫治疗策略受到极大关注。CAR-T或TCR-T在体内的扩增水平是指征其疗效的一个重要指标，因此，研发快速准确检测细胞的试剂或方法，将有助于有效监测其在体内外的扩增效率。
目前对于CAR-T或TCR-T细胞进行检测的方法有两种：使用定量实时聚合酶联反应（Quantitative real time PCR，qPCR）检测T细胞中的CAR或TCR的基因表达；以及检测T细胞中的CAR蛋白或TCR蛋白表达。其中，对于检测T细胞中的CAR蛋白表达，常用特异性结合CAR蛋白中scFv（Single-chain variable fragment）片段的抗体对T细胞进行流式细胞检测，或用结合Fab片段的抗体或protein L等检测CAR蛋白中的scFv片段。对于检测T细胞中的TCR蛋白的表达，常用特异针对某TCR的HLA和多肽的复合物对T细胞进行流式检测。但这些方法存在明显的局限性：qPCR检测基因拷贝数方法不能准确反映表达CAR的T细胞，因为对于整合了CAR基因但未正常表达CAR蛋白的细胞，使用qPCR检测方法会造成检测的假阳性；而对于CAR蛋白阳性的T细胞的检测方法，Fab抗体的检测背景值较高，容易造成假阳性；protein L只适用于检测带有κ轻链的scFv的CAR，而针对CAR蛋白中scFv片段的抗体，以及针对TCR的HLA和多肽复合物，则需要针对不同的CAR蛋白或TCR筛选出不同的抗体或复合物，并建立相关检测方法，耗时长，效率低，成本高。如果能开发出一种准确、灵敏且通用的抗体检测CAR-T，将大大提高基因转导的细胞治疗药物的检测效率，降低检测成本。
发明内容
本发明公开了一种特异性结合Strep-Tag II标签的分离的抗体，其包括：重链可变区（以下缩写为VH）和轻链可变区（以下缩写为VL），其中所述VH包括：包含SEQ ID NO:1的氨基酸序列的VH-CDR1，包含SEQ ID NO:2的氨基酸序列的VH-CDR2和包含SEQ ID NO:3或SEQ ID NO:4的氨基酸序列的VH-CDR3；所述VL包括：包含SEQ ID NO:5或SEQ ID NO:6的氨基酸序列的VL-CDR1，包含SEQ ID NO:7或SEQ ID NO:8的氨基酸序列的VL-CDR2和包含SEQID NO:9或SEQ ID NO:10的氨基酸序列的VL-CDR3。
在一些实施方案中，一种特异性结合Strep-Tag II标签的分离的抗体，其包括：重链可变区和轻链可变区，其中所述重链可变区包括：由SEQ ID NO:1所示的氨基酸序列组成的VH-CDR1，由SEQ ID NO:2所示的氨基酸序列组成的VH-CDR2和由SEQ ID NO:3或SEQ IDNO:4所示的氨基酸序列组成的VH-CDR3；所述轻链可变区包括：由SEQ ID NO:5或SEQ IDNO:6的氨基酸序列所示的VL-CDR1，由SEQ ID NO:7 或SEQ ID NO:8的氨基酸序列所示的VL-CDR2和由SEQ ID NO:9 或SEQ ID NO:10的氨基酸序列所示的VL-CDR3。
在一些实施方案中，所述VH包括：包含SEQ ID NO:1的氨基酸序列的VH-CDR1，包含SEQ ID NO:2的氨基酸序列的VH-CDR2和包含SEQ ID NO:3的氨基酸序列的VH-CDR3。
在一些实施方案中，所述VH包括：包含SEQ ID NO:1的氨基酸序列的VH-CDR1，包含SEQ ID NO:2的氨基酸序列的VH-CDR2和包含SEQ ID NO:4的氨基酸序列的VH-CDR3。
在一些实施方案中，所述抗体的VL包括：包含SEQ ID NO:5的氨基酸序列的VL-CDR1，包含SEQ ID NO:7的氨基酸序列的VL-CDR2和包含SEQ ID NO:9的氨基酸序列的VL-CDR3。
在一些实施方案中，所述抗体的VL包括：包含SEQ ID NO:6的氨基酸序列的VL-CDR1，包含SEQ ID NO:8的氨基酸序列的VL-CDR2和包含SEQ ID NO:10的氨基酸序列的VL-CDR3。
在一些实施方案中，所述抗体包括VH和VL，其中所述VH包括：包含SEQ ID NO:1的氨基酸序列的VH-CDR1，包含SEQ ID NO:2的氨基酸序列的VH-CDR2和包含SEQ ID NO:3的氨基酸序列的VH-CDR3；并且所述VL包括：包含SEQ ID NO:5的氨基酸序列的VL-CDR1，包含SEQID NO:7的氨基酸序列的VL-CDR2和包含SEQ ID NO:9的氨基酸序列的VL-CDR3。
在一些实施方案中，所述抗体包括VH和VL，其中所述VH包括：包含SEQ ID NO:1的氨基酸序列的VH-CDR1，包含SEQ ID NO:2的氨基酸序列的VH-CDR2和包含SEQ ID NO:4的氨基酸序列的VH-CDR3；并且所述VL包括：包含SEQ ID NO:6的氨基酸序列的VL-CDR1，包含SEQID NO:8的氨基酸序列的VL-CDR2和包含SEQ ID NO:10的氨基酸序列的VL-CDR3。
在一些实施方案中，所述抗体包括VH和VL，
所述VH包括：由SEQ ID NO:1所示的氨基酸序列组成的VH-CDR1，由SEQ ID NO:2所示的氨基酸序列组成的VH-CDR2和由SEQ ID NO:3所示的氨基酸序列组成的VH-CDR3；和所述VL包括：由SEQ ID NO:5所示的氨基酸序列组成的VL-CDR1，由SEQ ID NO:7所示的氨基酸序列组成的VL-CDR2和由SEQ ID NO:9所示的氨基酸序列组成的VL-CDR3；或
所述VH包括：由SEQ ID NO:1所示的氨基酸序列组成的VH-CDR1，由SEQ ID NO:2所示的氨基酸序列组成的VH-CDR2和由SEQ ID NO:4所示的氨基酸序列组成的VH-CDR3；和所述VL包括：由SEQ ID NO:6所示的氨基酸序列组成的VL-CDR1，由SEQ ID NO:8所示的氨基酸序列组成的VL-CDR2和由SEQ ID NO:10所示的氨基酸序列组成的VL-CDR3。
在本发明中，所述VH和所述VL中各CDR由Kabat编号系统确定。
在一些实施方案中，所述抗体的VH包含SEQ ID NO:23或24的氨基酸序列，或包含与SEQ ID NO:23或24具有至少85%同一性的氨基酸序列。在一些具体实施方案中，前述抗体的VH包含与SEQ ID NO:23或24具有至少85%以上、90%以上、95%以上或99%以上同一性的氨基酸序列。
在一些实施方案中，所述抗体的VL包含SEQ ID NO:25或26的氨基酸序列，或包含与SEQ ID NO:25或26具有至少85%同一性的氨基酸序列。在一些具体实施方案中，前述抗体的VL包含与SEQ ID NO:25或26具有至少85%以上、90%以上、95%以上或99%以上同一性的氨基酸序列。
在一些实施方案中，所述抗体的VH或VL进一步包括一个以上的框架区（以下缩写为FWR）。
在一些实施方案中，所述抗体的VH包括一个、两个、三个或四个FWR，每个FWR包含一个氨基酸序列，所述氨基酸序列选自由SEQ ID NO:11、12、13、14、15和16，与其具有至少90％、至少95%、至少99%同一性的氨基酸序列，与SEQ ID NO:11，12，13，14，15和16中任一个的氨基酸序列相比具有一个以上的氨基酸置换、插入或缺失的氨基酸序列组成的组中的氨基酸序列。在一些具体实施方案中，前述抗体的VH包括四个FWR，其中FWR1包含SEQ ID NO:11，FWR2包含SEQ ID NO:12或SEQ ID NO:13，FWR3包含SEQ ID NO:14或SEQ ID NO:15，FWR4包含SEQ ID NO:16。
在一些实施方案中，所述抗体VL包括一个、两个、三个或四个FWR，每个FWR包含一个氨基酸序列，所述氨基酸序列选自由SEQ ID NO:17、18、19、20、21和22，与其至少90％、至少95%、至少99%同一性的氨基酸序列，与SEQ ID NO: 17，18，19，20，21和22中任一个的氨基酸序列相比具有一个以上氨基酸置换、插入或缺失的氨基酸序列组成的组中的氨基酸序列。在一些具体实施方案中，前述抗体的VL包括四个FWR，其中FWR1包含SEQ ID NO:17或SEQID NO:18，FWR2包含SEQ ID NO:19，FWR3包含SEQ ID NO:20或SEQ ID NO:21，FWR4包含SEQID NO:22。
在一些实施方案中，所述抗体包括：a) 包含SEQ ID NO:23的氨基酸序列的VH和包含SEQ ID NO:25的氨基酸序列的VL；或b) 包含SEQ ID NO:24的氨基酸序列的VH和包含SEQID NO:26的氨基酸序列的VL。
在一些实施方案中，所述抗体的重链包含SEQ ID NO:27或29的氨基酸序列，或包含与SEQ ID NO:27或29具有至少85%、至少90%、至少95%或至少99%同一性的氨基酸序列。
在一些实施方案中，所述抗体的轻链包含SEQ ID NO:28或30的氨基酸序列，或包含与SEQ ID NO:28或30具有至少85%、至少90%、至少95%或至少99%同一性的氨基酸序列。
在一些实施方案中，前述抗体进一步包括重链恒定区和轻链恒定区的至少一个。所述重链恒定区选自小鼠IgG1、小鼠IgG2a、小鼠IgG2b或小鼠IgG3，所述轻链恒定区选自κ链或者λ链。优选所述重链恒定区为小鼠IgG1，且所述轻链恒定区为小鼠抗体的κ链。
在一些实施方案中，前述抗体是全长抗体、Fab、Fab’、F(ab’)2、Fv、scFv、单克隆抗体、双特异性抗体或多特异性抗体。
所述示例性抗体的序列如表1和序列表所示。
表 1示例性抗体氨基酸序列
所述示例性抗体的CDR序列和FWR序列如表2和表3所示。
表 2 示例性抗体的CDR序列
表3示例性抗体的FWR序列
在另一方面，本发明公开了一种分离的核酸分子，该核酸分子编码如前所述的抗体。
本发明公开了一种克隆载体或表达载体，该表达载体包含如前所述的核酸分子。
本发明公开了一种宿主细胞，该宿主细胞包含一个以上如前所述的表达载体。
在另一方面中，本发明公开了一种用于生产如前所述的抗体的方法，包括培养如前所述的宿主细胞，并且分离所述抗体。
在又一方面中，本发明公开了一种组合物或试剂盒，包含如前所述的抗体，以及一种以上药学可接受的赋形剂、稀释剂或载体。
本发明公开了一种偶联物，包含连接至可检测标签的如前所述的抗体。示例性的可检测标签可包括荧光标签（例如，荧光素、罗丹明、丹西尔、藻红蛋白或德州红），酶底物标签（例如，辣根过氧化物酶、碱性磷酸酶、糖化酶、溶菌酶、糖氧化酶或β-D-半乳糖苷酶），放射性同位素（例如，123I、124I、125I、131I、35S、3H、111In、112In、14C、64Cu、67Cu、86Y、88Y、90Y、177Lu、211At、186Re、188Re、153Sm、212Bi、32P、其它镧系元素），发光标记，发色基团，地高辛素，生物素/亲和素，用于检测的DNA分子或金等。
本发明还公开了一种组合物或试剂盒，包含如前所述的偶联物和一种以上药学可接受的赋形剂、稀释剂或载体。
本发明还公开了如前所述的抗体在制备用于在样品中检测含有Strep-Tag II标签的融合多肽的试剂中的用途。
本发明还公开了一种检测样品中含有Strep-Tag II标签的融合多肽的方法，包括(i)在允许抗体和多肽发生相互作用的条件下，体外使样品接触本发明公开的分离的抗体，和(ii)检测所述抗体和样品之间的复合物的形成。其中检测所述复合物的方法包括但不限于流式、蛋白质印迹、免疫组化、免疫荧光中的至少一种。
本发明还公开了一种检测样品中含有Strep-Tag II标签的CAR-T细胞的方法，包括(i)在允许抗体和CAR-T细胞上的Strep-Tag II发生相互作用的条件下，体外使样品接触本发明公开的分离的抗体，和(ii)检测所述CAR-T细胞的数量。其中检测方法包括但不限于流式、蛋白质印迹、免疫组化、免疫荧光中的至少一种。
本发明还公开了一种纯化样品中含有Strep-Tag II标签的融合多肽的方法，包括(i)在允许所述抗体和多肽发生相互作用的条件下，体外使样品接触本发明公开的的分离的抗体，和(ii)纯化抗体和样品之间的复合物。其中纯化方法包括但不限于亲和层析、免疫沉淀等。
在一些实施方案中，所述样品是包括血液、尿液、唾液、淋巴液、脑脊液、骨髓、组织器官、细胞中至少一种的生物样品。
在一些实施方案中，所述样品是嵌合抗原受体细胞或工程化细胞受体细胞。
发明的有益效果
本发明抗体的亲和力更强，可更特异地结合CAR-T或TCR-T中的Strep-Tag II标签，有效监测CAR-T或TCR-T在体内外的扩增效率。
附图说明
图1示出小鼠经过四次免疫后血清的效价曲线。
图2示出抗体8F8D1的聚丙烯酰胺凝胶电泳图谱。泳道M为分子量标准（300KD以内）；泳道1为抗体8F8D1的重链（约55KD）和轻链（约24 KD）。
图3示出抗体8F8D1和8A882的流式检测图。
图4示出抗体8F8D1和8A882的亲和力检测图。
图5示出使用抗体8F8D1监测小鼠体内CAR-T细胞的代谢曲线图。
具体实施方式
下面通过具体实施方式及实验数据对本发明作进一步的说明。除非另有限定，本文中所使用的所有技术和科学术语具有与本发明所属技术领域的普通技术人员通常理解相同的含义。
本文所用的术语“抗原”是指引起免疫应答的分子，该免疫应答可涉及抗体产生，或特异性免疫活性细胞的活化。本领域技术人员均可理解任何大分子包括所有的蛋白质或肽，可用作抗原。例如本发明中的免疫抗原可以为Strep-Tag II，其氨基酸序列为NWSHPQFEK，亦可为偶联血蓝素（Keyhole Limpet Hemocyanin，KLH）的Strep-Tag II（NWSHPQFEK-KLH）。抗原可被产生、合成或可源自生物学样品，这种生物学样品包括但不限于组织样品、肿瘤样品、细胞或生物学流体。本文使用的术语“检测抗原”是指用于测定抗体效价的抗原，例如偶联鸡卵白蛋白（Ovalbumin，OVA）的Strep-Tag II（NWSHPQFEK-OVA），但本领域技术人员均可理解检测抗原并不局限于NWSHPQFEK-OVA，包含NWSHPQFEK或者其部分的抗原也可作为检测抗原使用，因此该具体检测抗原的使用并不应认为是对抗体制备方法的限制。
本文使用的术语“肽”和“多肽”是指由肽键共价连接的氨基酸残基组成的化合物。如本文使用的“融合多肽”指的是连接了Strep-Tag II标签的多肽，例如在嵌合抗原受体的胞外结构域scFv中重链可变区或轻链可变区的N端或C端连接Strep-Tag II标签。多肽包括天然肽、重组肽、合成肽或其组合。
本文所用的术语“抗体”指与抗原特异性结合的免疫球蛋白分子。抗体可为源于天然来源或源于重组来源的完整的免疫球蛋白，并可为完整免疫球蛋白的免疫反应部分。本发明中的抗体可以以多种形式存在，包括多克隆、单克隆、单特异性的、多特异性的、非特异性的、人源化的、单链的、嵌合的、合成的、重组的、杂合的、突变的、以及嫁接的抗体；本发明中的抗体形式还包括全长抗体、抗体片段，例如Fab、Fab’、F(ab’)2、Fv、scFv、di-scFv、tri-scFv、Fd和其它保留抗原结合功能的抗体片段；还可以是二聚体结构Diabody或三聚体结构Triabody。通常情况下，所述片段应当包括抗原结合片段，抗原结合片段通常包括抗体轻链可变区（VL）和抗体重链可变区（VH），VH和VL区可以进一步细分成：称为互补决定区（CDR）的高变区，以及穿插分布的称为框架区（FWR）的更保守区域。本发明公开的抗体和抗原结合片段的CDR由Kabat编号所定义或识别。在一个实施方案中，每个VH和VL一般包括从氨基端到羧基端按以下顺序排列的3个CDR和4个FWR：FWR1、CDR1、FWR2、CDR2、FWR3、CDR3、FWR4。
全长抗体是指包含至少两条重链（H）和两条轻链（L）并通过二硫键相互连接的蛋白质。每条重链由重链可变区（VH）和重链恒定区（CH）组成。重链恒定区由三个结构域CH1、CH2和CH3组成。每条轻链由轻链可变区（VL）和轻链恒定区组成。轻链恒定区由一个结构域CL组成。全长的抗体包括重链恒定区和轻链恒定区。本发明具体实施方案中所述重链恒定区可以选自小鼠来源的IgG1、IgG2a、IgG2b或IgG3，所述轻链恒定区选自κ链或者λ链。
在本发明的一些实施方案中，抗体包括：包含SEQ ID NO:1的VH-CDR1氨基酸序列，包含SEQ ID NO:2的VH-CDR2氨基酸序列和包含SEQ ID NO:3的VH-CDR3氨基酸序列的重链可变区；和包含SEQ ID NO:5的VL-CDR1氨基酸序列，包含SEQ ID NO:7的VL-CDR2氨基酸序列和包含SEQ ID NO:9的VL-CDR3氨基酸序列的轻链可变区；或者，包含SEQ ID NO:1的VH-CDR1氨基酸序列，包含SEQ ID NO:2的VH-CDR2氨基酸序列和包含SEQ ID NO:4的VH-CDR3氨基酸序列的重链可变区；和包含SEQ ID NO:6的VL-CDR1氨基酸序列，包含SEQ ID NO:8的VL-CDR2氨基酸序列和包含SEQ ID NO:10的VL-CDR3氨基酸序列的轻链可变区。
本文使用的术语“嵌合抗原受体”或“CAR”是指被工程化以在免疫效应细胞上表达并能特异性地结合抗原的人工受体。“嵌合抗原受体细胞”是指细胞上表达能特异性地结合抗原的人工受体的免疫细胞，其中免疫细胞包括淋巴细胞、树突状细胞、单核/巨噬细胞、粒细胞、肥大细胞等。CAR可以被用于使用过继细胞转移的疗法。CAR可以包括胞内活化结构域、跨膜结构域和包括肿瘤相关抗原结合区的胞外结构域。本发明所用的术语“工程化细胞受体”或“TCR”，又称工程化T细胞受体，是通过体外基因工程化改造TCR的肽链构成的异元二聚体，使之更有效地识别由MHC递呈的肿瘤胞内抗原多肽，进而杀伤和治疗肿瘤。本发明中的融合多肽包括融合了Strep-Tag II标签的CAR分子或TCR分子。
本文使用的“分离的”是指从自然状态改变或移出。例如，天然存在于活动物中的核酸或肽不是“分离的”，但是部分或完全与它的自然状态的共存物质分开的相同的核酸或肽是“分离的”。分离的核酸或蛋白质可以以基本上纯化的形式存在，或可以存在于非自然环境，例如，宿主细胞中。“分离的”抗体是已鉴定并与其天然环境的组分分开和/或从其回收的抗体。在一些实施方案中，将抗体纯化至大于95％或99％纯度，如通过电泳（例如SDS-PAGE、等电聚焦IEF、毛细管电泳）或色谱法（例如离子交换或反相HPLC）所测定。
本文使用的“表达载体”是指包括重组多核苷酸的载体，所述重组多核苷酸包括可操作地连接至待表达的核苷酸序列的表达控制序列。本文使用的“克隆载体”是指在宿主细胞中具有自主复制能力的DNA分子例如质粒、粘粒或噬菌体。
宿主细胞可以是含有克隆载体或表达载体的任何原核细胞或真核细胞，包括已经进行基因工程改造以在宿主细胞的染色体或基因组中含有克隆化基因的那些原核细胞或真核细胞。合适的哺乳动物宿主细胞包括骨髓瘤细胞，例如SP2/0细胞和NS0细胞以及中国仓鼠卵巢(CHO)细胞、杂交瘤细胞系及其它可用于表达抗体的哺乳动物宿主细胞。具有经修饰的免疫系统的特殊转基因动物也可用于制备抗体。
本文使用的“同一性”是指在两种核酸分子或多肽之间的序列一致性。可以通过比较为比较目的而对齐的每个序列中位置来确定同一性。当比较的序列中的位置被相同碱基占据时，那么在该位置处分子是相同的。可使用各种比对算法和/或程序来计算两个序列之间的同一性，包括可获得为GCG序列分析包（University of Wisconsin，Madison，Wis.）的一部分，并且可以以例如默认设置使用的FASTA或BLAST。例如，本领域技术人员可以预期到与在本文中描述的特定多肽具有至少70％、85％、90％、95％、98％或99％的同一性的这些序列能够展现出基本上相同的功能。
在本文中陈述数值极限或范围的情况下，包括端点。此外，具体包括在数值极限或范围内的所有值和子范围。
下述实施例中的实验方法，如无特殊说明，均为常规方法。
实施例：
实施例1 抗原、小鼠免疫及杂交瘤制备
1. 抗原
免疫抗原是偶联血蓝素（Keyhole Limpet Hemocyanin，KLH）的Strep-Tag II（NWSHPQFEK-KLH）；检测抗原是偶联鸡卵白蛋白（Ovalbumin，OVA）的Strep-Tag II（NWSHPQFEK-OVA），均购自杭州中肽生化有限公司。
免疫
将免疫抗原蛋白（偶联KLH的Strep-Tag II（NWSHPQFEK-KLH）多肽）溶解于生理盐水中，浓度为0.5 mg/mL。将100μL的上述抗原多肽溶液和等体积的弗氏完全佐剂（Sigma；Cat#：1001646446）混匀，经过腹部皮下多点注射免疫Balb/c小鼠（雌性，6-8周，购自北京维通利华实验动物技术有限公司）的方式使其产生免疫应答，之后将上述抗原蛋白用生理盐水稀释至浓度为0.25 mg/mL，分别在第15天、第29天、第43天将100ul稀释后的抗原蛋白和弗氏不完全佐剂（Sigma；Cat#: 1002036152）按体积比1: 1混合，经过腹部皮下多点注射免疫Balb/c小鼠进行加强免疫。每次每只小鼠注射200 μL体积，共计抗原免疫4次。在第22天、36天和50天分别进行断尾采血测定血清效价。小鼠血清效价通过使用NWSHPQFEK-OVA多肽进行酶联免疫吸附测定（ELISA）检测，方法如下：在4℃下，将1 μg/mL偶联OVA的Strep-Tag II（NWSHPQFEK-OVA）添加到96孔板（购自康宁公司）中过夜，清洗酶标板并用含0.05% Tween-20的磷酸盐缓冲液（PBST）洗涤5次。室温下，用1%牛血清白蛋白将平板封闭2小时，并用PBST清洗5次。后将小鼠血清从1:1000进行4倍梯度稀释至1:102400加入酶标板，室温孵育1小时后用PBST洗涤5次。加入100 μL/孔的标记HRP的羊抗鼠二抗（Invitrogen，Cat#：31430），并在洗涤后室温孵育1小时。然后在平板中加入50 μL/孔的3,3',5,5'-四甲基联苯胺（TMB），在室温下孵育15分钟，然后停止反应并读取结果。
根据免疫小鼠血清效价的检测结果显示了5只小鼠的效价结果（图1），编号#4小鼠的效价相较于其它小鼠偏高。挑选#4小鼠进行杂交瘤融合，融合前3天，以50 μg免疫抗原腹腔注射小鼠进行冲击。
融合
通过聚乙二醇融合法，提取#4小鼠的脾脏，研碎后用杜氏磷酸缓冲液（DPBS）重悬，以提取小鼠脾脏B细胞单细胞，500 g离心5分钟，用DPBS重悬沉淀后，500 g再离心5分钟洗涤细胞，将脾细胞与小鼠骨髓瘤细胞系sp2/0（购自北欧生物科技(北京)有限公司）按照3:1（细胞数量比）的比例混合，加入1 mL聚乙二醇（罗氏；Cat#：10783641001），将融合后的细胞用HAT培养基（Gibco；Cat#：21060-017）重悬，铺在96孔细胞培养板，在二氧化碳培养箱中37℃培养7天后用ELISA和流式细胞术进行杂交瘤的初筛。筛选出来的杂交瘤细胞在二氧化碳培养箱中37℃培养过夜后，将融合细胞以有限稀释法接种到96孔板中。
实施例2 阳性杂交瘤细胞的筛选
通过ELISA试验和流式细胞术检测杂交瘤培养上清液中抗Strep-Tag II抗体的存在，最终筛到既可以无限增殖又可以分泌抗体的杂交瘤细胞。具体如下：
1. ELISA筛选
在4℃下，将1 μg/mL偶联OVA的Strep-Tag II（NWSHPQFEK-OVA）添加到96孔板（购自康宁公司）中过夜，然后丢弃上清并用含0.05% Tween-20的磷酸盐缓冲液（PBST）洗涤5次。室温下，用1%牛血清白蛋白将平板封闭2小时，并用PBST清洗5次。加入100 μL/孔杂交瘤上清液，室温孵育1小时，然后用PBST洗涤5次。加入100 μL/孔的标记HRP的羊抗鼠二抗（Invitrogen，Cat#：31430），并在洗涤后室温孵育1小时。然后在平板中加入50 μL/孔的3,3',5,5'-四甲基联苯胺（TMB），在室温下孵育15分钟，然后停止反应并读取结果。ELISA筛选挑取阳性杂交瘤，96孔培养板中转到24孔培养板扩大培养，5天后对24孔培养板孔中的上清进行复筛，复筛采用流式细胞术方法（FACS）分析。
流式细胞术筛选
编码靶向CD19的CAR慢病毒转移质粒的构建和慢病毒制备：1）将编码Strep-Tag II和靶向CD19的scFv的嵌合基因通过基因合成的方法合成（北京博迈德基因技术有限公司）。2）以已有的靶向CD19的CAR质粒为模板（靶向CD19的CAR核苷酸序列参见专利CN 105177031 B中SEQ ID NO: 13），利用PCR克隆出CAR分子中含有CD8α铰链区、CD8α跨膜区、4-1BB（对应于NP_001552.2）的胞内区、以及CD3ζ（对应于NP_000725.1）胞内区的核酸片段。3）以步骤1）获得的嵌合基因和步骤2）获得的核酸片段为模板，通过PCR克隆出编码Strep-Tag II和靶向CD19 CAR的完整核酸片段。4）通过限制性酶切和连接的方法，将步骤3）获得的完整核酸片段插入慢病毒载体pLenti6.3/V5（Thermo Fisher，Waltham，MA，USA），获得载带Strep-TagII和靶向CD19 CAR基因的慢病毒转移质粒。5）将慢病毒包装质粒pLP/VSVG, pLP1/MDK,pLP2/RSK（Thermo Fisher，Waltham，MA，USA）与步骤4）获得的转移质粒，用Lipofectamine3000（Thermo Fisher，Waltham，MA，USA）转染至HEK293T细胞，48小时后收集培养基，300 g离心去除细胞碎片后，用超速离心机25000 rpm离心3小时。将沉淀用1 mL生理盐水溶解，即为所需的慢病毒载体。
制备包含Strep-Tag II标签的CAR-T细胞：使用CD3/CD28 Dynabeads（ThermoFisher）从健康志愿者的外周血单个核细胞（妙通（上海）生物科技有限公司，中国）中分离T细胞，将分离得到的T细胞（此时的T细胞与CD3/CD28 Dynabeads连在一起）-含IL-2（500IU/ml）的新鲜X-VIVO 15培养体系中培养48小时后，用上述慢病毒载体感染T细胞。病毒感染细胞24小时后，细胞离心换液，并于上述培养体系中继续培养。细胞培养4天后，收集培养体系中的所有细胞，并用磁力架去除培养体系中的Dynabeads，T细胞离心并计数，用流式细胞仪（NovoCyte 2060R，ACEA Biosciences，San Diego，CA，USA）检测CAR-T细胞含量。
收集上述包含Strep-Tag II标签的CAR-T细胞，用DPBS清洗细胞并计数，将细胞分装到EP管中，每管约3×105个细胞。离心取上清后加入100 μL/孔杂交瘤上清液，在室温孵育15分钟。洗涤2次后每管加入5 μL标记PE标记的大鼠抗小鼠IgG（BD，Cat#：550083），在室温孵育15分钟，然后用DPBS洗涤细胞2次后进行流式细胞术FACS方法分析。
阳性亚克隆
通过有限稀释法亚克隆阳性杂交瘤克隆至96孔板，并通过FACS筛选方法（同上述步骤）鉴定和挑选阳性单克隆。将得到的单克隆细胞提取RNA，反转录为DNA后，用抗体重链和轻链基因特异结合的引物进行PCR，将PCR产物进行测序。
实施例3 测序
挑选阳性单克隆，采用TRIZOL法提取总RNA。RT-PCR产生cDNA，随后重链和轻链分别用PCR扩增（RT-PCR试剂盒购自全式金，Cat#：AE311-03，PCR使用的是GXL高保真DNA聚合酶，购自TaKaRa，Cat#：R050A，具体操作见产品说明书）。然后将PCR产物用清洁试剂盒（AXYGEN，Cat#：155。具体操作见产品说明书）清洁后进行测序，测序交予北京睿博兴科有限公司。轻链可变区和重链可变区的氨基酸序列参见表1和表2，以及序列表。筛选的抗体克隆号分别是8A882和8F8D1。
实施例4 腹水制备和抗体纯化
1. 腹水制备
订购5只6~8周的Balb/c小鼠，饲养一周后注射1 mL石蜡（购自江西益普生药业），次周每只小鼠注射1×106的单克隆杂交瘤细胞。待一周后小鼠观察到有明显的腹胀，每隔48小时对小鼠穿刺抽液。每只小鼠收集3~4 mL腹水，共收集到20 mL腹水。
抗体纯化
采用亲和纯化法纯化抗体，用Binding Buffer（pH7.0）平衡重力柱，2-5倍柱体积冲洗。将过滤的腹水过柱（ProteinA/G柱，Thermo Fisher订购，Cat#：89930）；用5-10倍柱体积的Binding Buffer冲洗柱子，再用pH 3.5的0.1M甘氨酸洗脱目的蛋白，后用pH 9.0的Tris-HCl调节至中性。最后用BCA试剂盒（Thermo；Cat#：23227，具体操作见产品说明书）测定抗体浓度。通过SDS-PAGE鉴定抗体纯度，图2显示抗体有较高的纯度。
实施例5 抗体的验证和亲和力的检测
1. 流式细胞术验证
将纯化出的高纯度抗体进行FACS鉴定。收集实施例2.2中制备的含有Strep-Tag II标签的CAR-T细胞（本公司制备），用FACS缓冲液清洗细胞并计数。将细胞分装到EP管中，每管约3×105个细胞。离心去上清后加入终浓度为1 μg/mL的8A882抗体或8F8D1抗体或阳性对照抗体，在室温孵育15分钟。洗涤2次后每管加入5 μL PE标记的大鼠抗小鼠IgG，在室温孵育15分钟。然后洗涤细胞2次后进行FACS分析。实验结果显示，在检测细胞（CAR-T）相同量的情况下，使用本发明的8F8D1和8A882抗体处理后，含有Strep-Tag II标签的CAR-T细胞的表达率分别为48.07%和36.41%，远远高于阳性对照抗体处理的27.63%的表达率。上述结果证明本发明抗体可以特异性识别Strep-Tag II短肽且相比商用的阳性对照抗体，本发明的抗体对Strep-Tag II短肽的亲和力更高（图3）。阴性对照为未加入Strep-Tag II标签抗体而仅加入阴性对照IgG，阳性对照为商用抗Strep-Tag II标签抗体（Abcam；Cat#：ab184224）。
抗体荧光标记
将本发明抗体分别用FITC和PE两种不同荧光标记，目的为了多位点检测分析。抗体荧光标记由北京四正柏生物科技有限公司完成。
荧光标记抗体亲和力检测
本发明抗体亲和力检测采用动态平衡测定法（饱和浓度法）。少量抗原存在的情况下，将抗体进行梯度稀释，检测抗原抗体复合物的浓度，当抗原抗体复合物的浓度占总抗原浓度的一半时，抗体对应的浓度值（EC50）即抗体相对于抗原的KD值。
收集含有Strep-Tag II标签的CAR-T细胞，用DPBS清洗细胞并计数。分装到数个EP管中，每管约3×105个细胞。离心去上清后加入连续梯度稀释10个浓度的8A882或8F8D1以及阳性对照抗体（商用的Strep-Tag II抗体，Abcam；Cat#：ab184224），在室温孵育15分钟。用DPBS洗涤细胞2次后再加入PE标记的大鼠抗小鼠IgG，室温孵育15分钟，洗涤细胞2次后进行FACS分析以及GraphPad Prism 软件进行EC50值的计算。通过软件分析得到8A882和8F8D1以及阳性对照抗体的EC50值分别是0.4nM、0.2nM、0.55nM（图4）。与阳性对照抗体相比，本发明的两株抗体具有高度亲和力。
实施例6 利用抗体检测荷瘤小鼠外周血中的CAR-T细胞
6-8周龄NCG小鼠（江苏集萃药康生物科技有限公司，中国）共6只，每只小鼠从尾静脉注射1.0×106个Nalm-6-LAE细胞（ATCC，USA），5天后对小鼠进行荧光素酶活体成像（LuminaII小动物活体成像系统，PerkinElmer，USA）分析，确定小鼠白血病模型制作成功后，每只小鼠从尾静脉注射CD19 CAR-T细胞（参照实施例2.2中方法由本公司制备，2×106个细胞/只），小鼠于CAR-T细胞注射后第2、4、8、12、21、28天，分别从眼眶取100 μL外周血进行CAR-T检测。具体操作为，将外周血分为2等份，每份50 μL，每份加入3 μLPE标记的8F8D1抗体，混匀后室温避光孵育20分钟；向样品中加入溶血剂，室温避光孵育15分钟，用DPBS洗涤两次后，再分别用100 μL DPBS重悬，用FACS分析CAR-T比例，并计算每100 μL外周血中CAR-T细胞数，绘制CAR-T在每只小鼠体内的代谢曲线（图5）。
数据显示，使用PE标记的8F8D1抗体可以有效检测小鼠体内CAR-T细胞的分布情况。
序列表
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Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Gln Arg Ala Thr Ile Ser Cys Lys Ala Ser Gln Ser Val Asp Tyr Asp
20 25 30
Gly Asp Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro
35 40 45
Lys Leu Leu Ile Tyr Ala Ala Ser Asn Leu Gln Ser Gly Ile Pro Ala
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His
65 70 75 80
Pro Val Glu Glu Glu Asp Thr Ala Thr Tyr Tyr Cys Gln Gln Ile Thr
85 90 95
Glu Asp Pro Trp Thr Phe Gly Gly Arg Thr Lys Leu Glu Ile Lys Arg
100 105 110
Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln
115 120 125
Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr
130 135 140
Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln
145 150 155 160
Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr
165 170 175
Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg
180 185 190
His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro
195 200 205
Ile Val Lys Ser Phe Asn Arg Asn Glu Cys
210 215
<210> 29
<211> 444
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 重链
<400> 29
Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
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Tyr Met Lys Trp Val Lys Gln Ser His Gly Lys Arg Leu Glu Trp Ile
35 40 45
Gly Asp Ile Asn Pro Asn Asn Gly Asp Thr Phe Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Tyr Tyr Gly Arg Ser Ser Pro Trp Phe Pro Tyr Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ala Ala Lys Thr Thr Pro Pro Ser Val
115 120 125
Tyr Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr
130 135 140
Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr
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Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val
165 170 175
Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser
180 185 190
Ser Pro Arg Pro Ser Glu Thr Val Thr Cys Asn Val Ala His Pro Ala
195 200 205
Ser Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys
210 215 220
Lys Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe
225 230 235 240
Pro Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val
245 250 255
Thr Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe
260 265 270
Ser Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln Pro
275 280 285
Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro
290 295 300
Ile Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val
305 310 315 320
Asn Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr
325 330 335
Lys Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys
340 345 350
Glu Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp
355 360 365
Phe Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro
370 375 380
Ala Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asn Thr Asn Gly Ser
385 390 395 400
Tyr Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala
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Gly Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His
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His Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys
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<210> 30
<211> 218
<212> PRT
<213> 人工序列(Artificial Sequence)
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Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Gly Val Ser Leu Gly
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35 40 45
Lys Leu Leu Ile Tyr Ala Ala Ser Asn Leu Glu Ser Gly Ile Pro Ala
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Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His
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Glu Asp Pro Trp Thr Phe Gly Gly Arg Thr Lys Leu Glu Ile Lys Arg
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Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln
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Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr
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Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln
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Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr
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Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg
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Claims (24)
1.一种特异性结合Strep-Tag II标签的分离的抗体，其包括：重链可变区和轻链可变区，其中所述重链可变区包括：由SEQ ID NO:1所示的氨基酸序列组成的VH-CDR1，由SEQ IDNO:2所示的氨基酸序列组成的VH-CDR2和由SEQ ID NO:3或SEQ ID NO:4所示的氨基酸序列组成的VH-CDR3；所述轻链可变区包括：由SEQ ID NO:5或SEQ ID NO:6的氨基酸序列所示的VL-CDR1，由SEQ ID NO:7 或SEQ ID NO:8的氨基酸序列所示的VL-CDR2和由SEQ ID NO:9或SEQ ID NO:10的氨基酸序列所示的VL-CDR3。
2. 如权利要求1所述抗体，其中所述抗体包括：重链可变区和轻链可变区，
所述重链可变区包括：由SEQ ID NO:1所示的氨基酸序列组成的VH-CDR1，由SEQ IDNO:2所示的氨基酸序列组成的VH-CDR2和由SEQ ID NO:3所示的氨基酸序列组成的VH-CDR3；和所述轻链可变区包括：由SEQ ID NO:5所示的氨基酸序列组成的VL-CDR1，由SEQ IDNO:7所示的氨基酸序列组成的VL-CDR2和由SEQ ID NO:9所示的氨基酸序列组成的VL-CDR3；或
所述重链可变区包括：由SEQ ID NO:1所示的氨基酸序列组成的VH-CDR1，由SEQ IDNO:2所示的氨基酸序列组成的VH-CDR2和由SEQ ID NO:4所示的氨基酸序列组成的VH-CDR3；和所述轻链可变区包括：由SEQ ID NO:6所示的氨基酸序列组成的VL-CDR1，由SEQ IDNO:8所示的氨基酸序列组成的VL-CDR2和由SEQ ID NO:10所示的氨基酸序列组成的VL-CDR3。
3.如权利要求1所述的抗体，其中所述重链可变区包含SEQ ID NO:23或24的氨基酸序列，或包含与SEQ ID NO:23或24至少85%、至少90%、至少95%或至少99%同一性的氨基酸序列。
4.如权利要求1所述的抗体，其中所述轻链可变区包含SEQ ID NO:25或26的氨基酸序列，或包含与SEQ ID NO:25或26至少85%、至少90%、至少95%或至少99%同一性的氨基酸序列。
5.如权利要求1所述的抗体，其中所述重链可变区包括一个以上的框架区，所述框架区包含一个氨基酸序列，所述氨基酸序列选自由SEQ ID NO:11、12、13、14、15和16，与其至少90％同一性的氨基酸序列，与SEQ ID NO:11、12、13、14、15和16中任一个的氨基酸序列相比具有一个以上氨基酸置换、插入或缺失的氨基酸序列组成的组中的氨基酸序列。
6.如权利要求1所述的抗体，其中所述轻链可变区包括一个以上的框架区，所述框架区包含一个氨基酸序列，所述氨基酸序列选自由SEQ ID NO:17、18、19、20、21和22，与其至少90％同一性的氨基酸序列，与SEQ ID NO: 17、18、19、20、21和22中任一个的氨基酸序列相比具有一个以上氨基酸置换、插入或缺失的氨基酸序列组成的组中的氨基酸序列。
7.如权利要求1所述的抗体，其中所述抗体包括：重链可变区和轻链可变区，
所述重链可变区包含SEQ ID NO:23的氨基酸序列和所述轻链可变区包含SEQ ID NO:25的氨基酸序列；或
所述重链可变区包含SEQ ID NO:24的氨基酸序列和所述轻链可变区包含SEQ ID NO:26的氨基酸序列。
8.如权利要求1所述的抗体，其中所述重链包含SEQ ID NO:27或29的氨基酸序列，或包含与SEQ ID NO:27或29具有至少85%同一性的氨基酸序列。
9.如权利要求1所述的抗体，其中所述轻链包含SEQ ID NO:28或30的氨基酸序列，或包含与SEQ ID NO:28或30具有至少85%同一性的氨基酸序列。
10.如权利要求1所述的抗体，其中所述抗体包括重链和轻链，
所述重链包含SEQ ID NO:27的氨基酸序列，所述轻链包含SEQ ID NO:28的氨基酸序列；或
所述重链包含SEQ ID NO:29的氨基酸序列，所述轻链包含SEQ ID NO:30的氨基酸序列。
11.如权利要求1-10中任一项所述的抗体，其中所述抗体进一步包括重链恒定区和轻链恒定区的至少一个。
12.一种编码如权利要求1-11任一项的抗体的分离的核酸。
13.一种包括如权利要求12所述的核酸的克隆载体或表达载体。
14.一种包括如权利要求12所述的核酸的宿主细胞。
15.一种产生抗体的方法，包括在适于基因表达的条件下培养如权利要求14所述的宿主细胞，并且分离所述抗体。
16.一种抗体偶联物，包含连接至荧光标记的如权利要求1-11任一项的抗体，其中，所述荧光标记选自荧光标签诸如荧光素、罗丹明、丹西尔、藻红蛋白或德州红；酶底物标签诸如辣根过氧化物酶、碱性磷酸酶、糖化酶、溶菌酶、糖氧化酶或β-D-半乳糖苷酶；放射性同位素诸如123I、124I、125I、131I、35S、3H、111In、112In、14C、64Cu、67Cu、86Y、88Y、90Y、177Lu、211At、186Re、188Re、153Sm、212Bi、32P、其它镧系元素；发光标记；发色基团；地高辛素；生物素或亲和素；用于检测的DNA分子或金。
17.一种组合物或试剂盒，其包含如权利要求1至11中任一项所述的抗体或如权利要求16所述的抗体偶联物，以及一种以上药学可接受的赋形剂、稀释剂或载体。
18.如权利要求1至11中任一项所述的抗体在制备用于在样品中检测含有Strep-TagII标签的融合多肽的试剂中的用途。
19.如权利要求18所述的用途，其中所述试剂用于流式、蛋白质印迹、免疫组化、免疫荧光中的至少一种。
20.一种检测样品中含有Strep-Tag II标签的融合多肽的方法，包括(i)在允许抗体和多肽发生相互作用的条件下，体外使样品接触如权利要求1至11任一项的分离的抗体，和(ii)检测抗体和样品之间的复合物的形成。
21.如权利要求20所述的方法，其中检测所述复合物的方法包括流式、蛋白质印迹、免疫组化、免疫荧光中的至少一种。
22.一种纯化样品中含有Strep-Tag II标签的融合多肽的方法，包括(i)在允许抗体和多肽发生相互作用的条件下，体外使样品接触如权利要求1至11任一项的分离的抗体，和(ii)纯化抗体和样品之间的复合物。
23.如权利要求18所述的用途或权利要求20或22所述的方法，其中所述样品是包括血液、尿液、唾液、淋巴液、脑脊液、骨髓、组织器官、细胞中至少一种的生物样品。
24.如权利要求18所述的用途或权利要求20或22所述的方法，其中所述样品是嵌合抗原受体细胞或工程化细胞受体细胞。
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| EP21902604.4A EP4261228A1 (en) | 2020-12-08 | 2021-12-07 | Antibody specifically binding to strep-tag ii tag and use thereof |
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| WO2015067768A1 (en) * | 2013-11-07 | 2015-05-14 | Institut Pasteur | High-affinity monoclonal anti-strep-tag antibody |
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| CN111065409A (zh) * | 2017-09-06 | 2020-04-24 | 弗雷德哈钦森癌症研究中心 | Strep-tag特异性结合蛋白及其用途 |
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| WO2015067768A1 (en) * | 2013-11-07 | 2015-05-14 | Institut Pasteur | High-affinity monoclonal anti-strep-tag antibody |
| CN111051349A (zh) * | 2017-09-06 | 2020-04-21 | 弗雷德哈钦森癌症研究中心 | Strep-tag特异性嵌合受体及其用途 |
| CN111065409A (zh) * | 2017-09-06 | 2020-04-24 | 弗雷德哈钦森癌症研究中心 | Strep-tag特异性结合蛋白及其用途 |
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