Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan.
Laboratory of Biopharmaceutical Research (Pharmaceutical Proteomics), National Institute of Biomedical Innovation, Osaka, Japan.
Biomaterials. 2013 May;34(16):4191-4201. doi: 10.1016/j.biomaterials.2013.02.046. Epub 2013 Mar 6.
A major drawback of adenovirus (Ad) vectors is their nonspecific transduction into various types of cells or tissue after in vivo application, which might lead to unexpected toxicity and tissue damage. To overcome this problem, we developed a fiber-mutant Ad vector displaying a monobody specific for epidermal growth factor receptor (EGFR) or vascular endothelial growth factor receptor 2 (VEGFR2) in the C-terminus of the knobless fiber protein derived from T4 phage fibritin. A monobody, which is a single domain antibody mimic based on the tenth human fibronectin type III domain scaffold with a structure similar to the variable domains of antibodies, would be suitable as a targeting molecule for display on the Ad capsid proteins because of its highly stable structure even under reducing conditions and low molecular weight (approximately 10 kDa). Surface plasmon resonance (SPR) analysis revealed that the monobody-displaying Ad vector specifically bound to the targeted molecules, leading to significant increases in cellular binding and transduction efficiencies in the targeted cells. Transduction with the monobody-displaying Ad vectors was significantly inhibited in the presence of the Fc-chimera protein of EGFR and VEGFR2. This monobody-displaying Ad vector would be a crucial resource for targeted gene therapy.
腺病毒（Ad）载体的一个主要缺点是，在体内应用后，它们会非特异性地转导到各种类型的细胞或组织中，这可能导致意外的毒性和组织损伤。为了克服这个问题，我们开发了一种纤维突变型 Ad 载体，在来自 T4 噬菌体纤维蛋白的无结纤维蛋白的 C 末端展示了针对表皮生长因子受体（EGFR）或血管内皮生长因子受体 2（VEGFR2）的单域抗体。单域抗体是一种基于第十个人类纤维连接蛋白 III 结构域支架的单域抗体模拟物，具有与抗体可变区相似的结构，由于其高度稳定的结构（即使在还原条件下）和低分子量（约 10 kDa），非常适合作为展示在 Ad 衣壳蛋白上的靶向分子。表面等离子体共振（SPR）分析表明，展示单域抗体的 Ad 载体特异性地与靶向分子结合，导致靶向细胞的细胞结合和转导效率显著增加。在存在 EGFR 和 VEGFR2 的 Fc 嵌合蛋白的情况下，展示单域抗体的 Ad 载体的转导被显著抑制。这种展示单域抗体的 Ad 载体将成为靶向基因治疗的重要资源。
