Zhou Yu, Drummond Daryl C, Zou Hao, Hayes Mark E, Adams Gregory P, Kirpotin Dmitri B, Marks James D
Department of Anesthesia and Pharmaceutical Chemistry, University of California, San Francisco Rm 3C-38, San Francisco General Hospital, 1001 Potrero Ave, San Francisco, CA 94110, USA.
J Mol Biol. 2007 Aug 24;371(4):934-47. doi: 10.1016/j.jmb.2007.05.011. Epub 2007 May 10.
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 (KD=15-88 nM) for EGFR-expressing A431 tumor cells compared to C10 scFv (KD=264 nM). By combining mutations, higher affinity scFv were generated with KD 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.
为了确定单链抗体片段（scFv）亲和力对肿瘤靶向纳米颗粒的结合、摄取及细胞毒性的重要性，利用分子进化和酵母展示技术提高了表皮生长因子受体（EGFR）单链抗体片段C10的亲和力。通过易错PCR构建了一个包含scFv突变体的文库，将其展示在酵母表面，并用荧光激活细胞分选技术筛选出亲和力更高的克隆。与C10单链抗体片段（解离常数KD = 264 nM）相比，鉴定出10个对表达EGFR的A431肿瘤细胞亲和力提高了3至18倍（KD = 15至88 nM）的突变单链抗体片段。通过组合突变，产生了解离常数在0.9 nM至10 nM之间的更高亲和力的单链抗体片段。与亲本C10单链抗体片段相比，亲和力最高的单链抗体片段的亲和力高280倍。使用亲和力范围达280倍的EGFR单链抗体片段制备了免疫脂质体纳米颗粒（IL），并对其与表达EGFR的肿瘤细胞的结合和摄取进行了定量分析。当单链抗体片段密度大于148个单链抗体片段/免疫脂质体时，单链抗体片段亲和力对免疫脂质体与肿瘤细胞的结合、摄取及细胞毒性均无影响。在较低的单链抗体片段密度下，由亲和力极低的C10单链抗体片段构建的免疫脂质体的摄取和结合较少。结果表明抗体片段密度对纳米颗粒摄取的重要性，并提示为实现最佳的纳米颗粒靶向，设计超高亲和力的单链抗体片段可能并无必要。
