Impact of single-chain fv antibody fragment affinity on nanoparticle targeting of epidermal growth factor receptor-expressing tumor cells
Impact of single-chain fv antibody fragment affinity on nanoparticle targeting of epidermal growth factor receptor-expressing tumor cells
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DOI：
10.1016/j.jmb.2007.05.011
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发表时间：
2007-08-24
影响因子：
5.6
通讯作者：
Marks, James D.
中科院分区：
文献类型：
--
作者：
Zhou, Yu;Daryl, C. Drummond;Marks, James D.
为了确定单链Fv （scFv）亲和力对肿瘤靶向纳米颗粒结合、摄取和细胞毒性的重要性，利用分子进化和酵母展示提高了表皮生长因子受体（EGFR） scFv抗体C10的亲和力。通过容易出错的PCR建立了一个包含scFv突变体的文库，并在酵母表面显示，并通过荧光激活细胞分选选择了高亲和力的克隆。与C10 scFv （K-D=264 nM）相比，10个突变scFv对表达egfr的A431肿瘤细胞的亲和力（K-D=15-88 nM）提高了3-18倍。通过突变组合，产生了高亲和度的scFv， K-D范围在0.9 ~ 10 nM之间。与亲本C10 scFv相比，亲和性最高的scFv亲和性提高了280倍。利用280倍亲和范围的EGFR scFv制备免疫脂质体纳米颗粒（ILs），并对其与表达EGFR的肿瘤细胞的结合和摄取进行量化。当scFv浓度大于148 scFv/IL时，scFv的亲和力对IL的结合和被肿瘤细胞摄取或细胞毒性没有影响。在较低的scFv密度下，由非常低亲和力的C10 scFv构建的il的摄取和结合较少。结果表明，抗体片段密度对纳米颗粒摄取的重要性，并表明工程超高亲和scFv可能不需要优化纳米颗粒靶向。(c) 2007 Elsevier Ltd.版权所有。
To determine the importance of single-chain Fv (scFv) affinity on binding, uptake, and cytotoxicity of tumor-targeting nanoparticles, the affinity of the epidermal growth factor receptor (EGFR) scFv antibody C10 was increased using molecular evolution and yeast display. A library containing scFv mutants was created by error-prone PCR, displayed on the surface of yeast, and higher affinity clones selected by fluorescence activated cell sorting. Ten mutant scFv were identified that had a 3-18-fold improvement in affinity (K-D=15-88 nM) for EGFR-expressing A431 tumor cells compared to C10 scFv (K-D=264 nM). By combining mutations, higher affinity scFv were generated with K-D ranging from 0.9 nM to 10 nM. The highest affinity scFv had a 280-fold higher affinity compared to that of the parental C10 scFv. Immunoliposome nanoparticles (ILs) were prepared using EGFR scFv with a 280-fold range of affinities, and their binding and uptake into EGFR-expressing tumor cells was quantified. At scFv densities greater than 148 scFv/IL, there was no effect of scFv affinity on IL binding and uptake into tumor cells, or on cytotoxicity. At lower scFv densities, there was less uptake and binding for ILs constructed from the very low affinity C10 scFv. The results show the importance of antibody fragment density on nanoparticle uptake, and suggest that engineering ultrahigh affinity scFv may be unnecessary for optimal nanoparticle targeting. (c) 2007 Elsevier Ltd. All rights reserved.