A novel assay for monitoring internalization of nanocarrier coupled antibodies
A novel assay for monitoring internalization of nanocarrier coupled antibodies
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DOI：
10.1186/1471-2172-7-24
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发表时间：
2006-10-02
期刊：
影响因子：
3
通讯作者：
D Marks, James
中科院分区：
文献类型：
--
作者：
Nielsen, Ulrik B.;Kirpotin, Dmitri B.;D Marks, James
背景：肿瘤选择性抗体或抗体片段的发现是向抗原过度表达的癌症递送治疗剂的一种有前景的方法。因此，开发用于有效药物递送的目标和功能特异性抗体的鉴定方法非常重要。在这里，我们描述了一种高度选择性和灵敏的方法，用于表征与脂质体缀合的多价展示抗体或配体进入肿瘤细胞的内化潜力。该测定需要微量的组氨酸标记配体，并依赖于这些抗体与含有金属离子螯合脂质的荧光脂质体的非共价偶联。将细胞与抗体缀合的脂质体一起孵育后，轻轻去除表面结合的脂质体，并以高通量方式基于荧光对剩余的内化脂质体进行定量。我们将这种方法称为“螯合配体内化测定”，或 CLIA。 结果：使用针对 ErbB-2 和 EGF 受体的不同抗体证明了该测定的特异性。抗体摄取与具有一系列受体表达的肿瘤细胞系中的受体表达水平相关。此外，含有阿霉素的 Ni-NTA 脂质体用于筛选抗体赋予靶标特异性细胞毒性的能力。使用抗 ErbB2 单链 Fv (scFv) (F5) 抗体，可以赋予 ErbB2 过表达细胞细胞毒性；然而，聚（乙二醇）连接的脂质（DSPE-PEG-NTA-Ni）对于有效装载药物并减少测定过程中的非特异性药物渗漏是必要的。结论：我们在此描述的 CLIA 方法代表了一种快速、灵敏且稳健的测定方法，用于识别和表征与脂质体纳米载体缀合时能够实现高药物递送效率的肿瘤特异性抗体。
Background: Discovery of tumor-selective antibodies or antibody fragments is a promising approach for delivering therapeutic agents to antigen over-expressing cancers. Therefore it is important to develop methods for the identification of target- and function specific antibodies for effective drug delivery. Here we describe a highly selective and sensitive method for characterizing the internalizing potential of multivalently displayed antibodies or ligands conjugated to liposomes into tumor cells. The assay requires minute amounts of histidine-tagged ligand and relies on the non-covalent coupling of these antibodies to fluorescent liposomes containing a metal ion-chelating lipid. Following incubation of cells with antibody-conjugated liposomes, surface bound liposomes are gently removed and the remaining internalized liposomes are quantitated based on fluorescence in a high throughput manner. We have termed this methodology "Chelated Ligand Internalization Assay", or CLIA.Results: The specificity of the assay was demonstrated with different antibodies to the ErbB-2 and EGF receptors. Antibody-uptake correlated with receptor expression levels in tumor cell lines with a range of receptor expression. Furthermore, Ni-NTA liposomes containing doxorubicin were used to screen for the ability of antibodies to confer target-specific cytotoxicity. Using an anti-ErbB2 single chain Fv (scFv) (F5) antibody, cytotoxicity could be conferred to ErbB2-overexpressing cells; however, a poly(ethylene glycol)-linked lipid (DSPE-PEG-NTA-Ni) was necessary to allow for efficient loading of the drug and to reduce nonspecific drug leakage during the course of the assay.Conclusion: The CLIA method we describe here represents a rapid, sensitive and robust assay for the identification and characterization of tumor-specific antibodies capable of high drug-delivery efficiency when conjugated to liposomal nanocarriers.