Self-assembled "dock and lock" system for linking payloads to targeting proteins
Self-assembled "dock and lock" system for linking payloads to targeting proteins
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DOI：
10.1021/bc060037u
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发表时间：
2006-07-19
影响因子：
4.7
通讯作者：
Backer, Joseph M.
中科院分区：
文献类型：
--
作者：
Backer, Marina V.;Patel, Vimal;Backer, Joseph M.
治疗或诊断有效负载与靶向蛋白质的随机缀合产生功能异质的产物。将有效负载与接头结合，该接头与工程化到靶向蛋白中的肽标签结合，提供了一种替代策略。为了进入临床开发，适配器/对接标签系统应包含人源化成分并且在循环中稳定。我们在此描述了一种基于人 RNase I 突变片段的接头/对接标签系统，该系统自发地相互结合并在互补的半胱氨酸残基之间形成二硫键缀合物。这种自组装的“对接和锁定”系统利用了先前描述的融合C标签，即带有R4C氨基酸取代的人RNase I的1-15个氨基酸片段，以及新设计的接头蛋白（Ad-C），即带有V118C取代的人RNase I的21-127个氨基酸片段。两种截然不同的 C 标签重组蛋白，人血管内皮生长因子 (VEGF) 和炭疽致死因子 (LFn) 的 254 个氨基酸长的 N 末端片段，在 Ad-C 分别与 N 末端或 C 末端 C 标签自发缀合后保留了功能活性。 Ad-C 用聚乙二醇化磷脂修饰并插入载药脂质体 (Doxil) 的脂膜中，保留了结合 C 标签蛋白的能力，产生装饰有功能活性蛋白的靶向脂质体。为了进一步优化系统，我们设计了一个在第 88 位带有额外半胱氨酸残基的接头，用于位点特异性修饰，将其与 C 标记的 VEGF 缀合，并用近红外荧光染料 Cy5.5 标记，产生用于体内分子成像的独特功能活性探针。我们期望这种自组装的“对接和锁定”系统将为将功能活性蛋白用于生物医学目的提供新的机会。
Random conjugation of therapeutic or diagnostic payloads to targeting proteins generates functionally heterogeneous products. Conjugation of payloads to an adapter that binds to a peptide tag engineered into a targeting protein provides an alternative strategy. To progress into clinical development, an adapter/docking tag system should include humanized components and be stable in circulation. We describe here an adapter/docking tag system based on mutated fragments of human RNase I that spontaneously bind to each other and form a conjugate with a disulfide bond between complimentary cysteine residues. This self-assembled "dock and lock" system utilizes the previously described fusion C-tag, a 1-15 aa fragment of human RNase I with the R4C amino acid substitution, and a newly engineered adapter protein (Ad-C), a 21-127-aa fragment of human RNase I with the V118C substitution. Two vastly different C-tagged recombinant proteins, human vascular endothelial growth factor ( VEGF) and a 254-aa long N-terminal fragment of anthrax lethal factor (LFn), retain functional activities after spontaneous conjugation of Ad-C to N-terminal or C-terminal C-tag, respectively. Ad-C modified with pegylated phospolipid and inserted into the lipid membrane of drug-loaded liposomes (Doxil) retained the ability to conjugate C-tagged proteins, yielding targeted liposomes decorated with functionally active proteins. To further optimize the system, we engineered an adapter with an additional cysteine residue at position 88 for site-specific modification, conjugated it to C-tagged VEGF, and labeled with a near-infrared fluorescent dye Cy5.5, yielding a unique functionally active probe for in vivo molecular imaging. We expect that this self-assembled "dock and lock" system will provide new opportunities for using functionally active proteins for biomedical purposes.