Site-Specific Fluorescent Labeling of Antibodies and Diabodies Using SpyTag/SpyCatcher System for In Vivo Optical Imaging
Site-Specific Fluorescent Labeling of Antibodies and Diabodies Using SpyTag/SpyCatcher System for In Vivo Optical Imaging
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DOI：
10.1007/s11307-018-1222-y
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发表时间：
2019-02-01
影响因子：
3.1
通讯作者：
Geyer, C. Ronald
中科院分区：
文献类型：
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作者：
Alam, Md. Kausar;El-Sayed, Ayman;Geyer, C. Ronald
目的构建以抗体为基础的分子靶向光学成像探针需要用荧光团标记抗体。最常见的方法是将荧光团非特异性地结合到抗体上，从而产生定义不明确的异质成像探针，这些探针通常具有次优的体内行为。我们使用SpyCatcher/SpyTag蛋白连接酶系统测试了一种新的策略来位点特异性标记基于抗体的成像探针。程序我们使用SpyCatcher/SpyTag蛋白连接酶系统来位点特异性标记尼妥珠单抗、抗EGFR抗体和抗HER 3双抗体。为了防止标记干扰抗原结合，我们分别在抗体和双抗体的C-末端引入SpyTag和SpyCatcher。C-末端抗体-SpyTag和双抗体-SpyCatcher融合体的表达和结合性质与抗体和双抗体相似，表明SpyTag和SpyCatcher融合体在该位置耐受良好。抗体和双抗体的位点特异性标记分两步进行。首先，我们用IRDye 800 CW-Maleimide标记SpyCatcher，用IRDye 800 CW-NHS标记SpyTag。其次，我们将IRDye 800 CW-SpyCatcher和IRDye 800 CW-SpyTag分别与抗体或双抗体缀合。我们使用生物层干涉法和流式细胞术证实了IRDye 800 CW标记的成像探针的亲和力和特异性。我们分析了在体内的IRDye 800 CW标记的尼妥珠单抗和抗HER 3双抗体在荷瘤裸鼠表达EGFR和HER3.ResultsExpression和C-末端抗体SpyTag和双抗体SpyCatcher融合的结合特性是类似的抗体和双抗体，表明SpyTag和SpyCatcher融合在这个位置耐受良好。我们使用生物层干涉法和流式细胞术证实了IRDye 800 CW标记的成像探针的亲和力和特异性。我们分析了IRDye 800 CW标记的尼妥珠单抗和抗HER 3双抗体在携带分别表达EGFR和HER 3的异种移植物的裸鼠中的体内生物分布和肿瘤蓄积。位点特异性IRDye 800 CW标记的成像探针结合到其固定的目标，细胞表达这些目标，并选择性地积累在xenografts.ConclusionsThese结果突出了使用模块化SpyTag/SpyCatcher蛋白连接酶系统的易用性和实用性的位点特异性的荧光标记的蛋白质为基础的成像探针。以这种方式标记的成像探针将可用于光学成像应用，例如图像引导手术，并且对于其他成像模态具有广泛的应用。
PurposeConstruction of antibody-based, molecular-targeted optical imaging probes requires the labeling of an antibody with a fluorophore. The most common method for doing this involves non-specifically conjugating a fluorophore to an antibody, resulting in poorly defined, heterogeneous imaging probes that often have suboptimal in vivo behavior. We tested a new strategy to site-specific label antibody-based imaging probes using the SpyCatcher/SpyTag protein ligase system.ProceduresWe used the SpyCatcher/SpyTag protein ligase system to site specifically label nimotuzumab, an anti-EGFR antibody and an anti-HER3 diabody. To prevent the labeling from interfering with antigen binding, we introduced the SpyTag and SpyCatcher at the C-terminus of the antibody and diabody, respectively. Expression and binding properties of the C-terminal antibody-SpyTag and diabody-SpyCatcher fusions were similar to the antibody and diabody, indicating that the SpyTag and SpyCatcher fusions were well tolerated at this position. Site-specific labeling of the antibody and diabody was performed in two steps. First, we labeled the SpyCatcher with IRDye800CW-Maleimide and the SpyTag with IRDye800CW-NHS. Second, we conjugated the IRDye800CW-SpyCatcher and the IRDye800CW-SpyTag to the antibody or diabody, respectively. We confirmed the affinity and specificity of the IRDye800CW-labeled imaging probes using biolayer interferometry and flow cytometry. We analyzed the in vivo biodistribution and tumor accumulation of the IRDye800CW-labeled nimotuzumab and anti-HER3 diabody in nude mice bearing xenografts that express EGFR and HER3, respectively.ResultsExpression and binding properties of the C-terminal antibody-SpyTag and diabody-SpyCatcher fusions were similar to the antibody and diabody, indicating that the SpyTag and SpyCatcher fusions were well tolerated at this position. We confirmed the affinity and specificity of the IRDye800CW-labeled imaging probes using biolayer interferometry and flow cytometry. We analyzed the in vivo biodistribution and tumor accumulation of the IRDye800CW-labeled nimotuzumab and anti-HER3 diabody in nude mice bearing xenografts that express EGFR and HER3, respectively. Site-specifically IRDye800CW-labeled imaging probes bound to their immobilized targets, cells expressing these targets, and selectively accumulated in xenografts.ConclusionsThese results highlight the ease and utility of using the modular SpyTag/SpyCatcher protein ligase system for site-specific fluorescent labeling of protein-based imaging probes. Imaging probes labeled in this manner will be useful for optical imaging applications such as image-guided surgery and have broad application for other imaging modalities.