Kawato Tatsuya, Mizohata Eiichi, Shimizu Yohei, Meshizuka Tomohiro, Yamamoto Tomohiro, Takasu Noriaki, Matsuoka Masahiro, Matsumura Hiroyoshi, Kodama Tatsuhiko, Kanai Motomu, Doi Hirofumi, Inoue Tsuyoshi, Sugiyama Akira
Division of Applied Chemistry, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan; Graduate School of Pharmaceutical Sciences, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan; Research Center for Advanced Science and Technology, The University of Tokyo, Tokyo 153-8904, Japan; and Radioisotope Center, The University of Tokyo, 2-11-16 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan.
Division of Applied Chemistry, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan; Graduate School of Pharmaceutical Sciences, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan; Research Center for Advanced Science and Technology, The University of Tokyo, Tokyo 153-8904, Japan; and Radioisotope Center, The University of Tokyo, 2-11-16 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan
J Biochem. 2015 Jun;157(6):467-75. doi: 10.1093/jb/mvv004. Epub 2015 Feb 2.
For a multistep pre-targeting method using antibodies, a streptavidin mutant with low immunogenicity, termed low immunogenic streptavidin mutant No. 314 (LISA-314), was produced previously as a drug delivery tool. However, endogenous biotins (BTNs) with high affinity (Kd < 10(-10) M) for the binding pocket of LISA-314 prevents access of exogenous BTN-labelled anticancer drugs. In this study, we improve the binding pocket of LISA-314 to abolish its affinity for endogenous BTN species, therefore ensuring that the newly designed LISA-314 binds only artificial BTN analogue. The replacement of three amino acid residues was performed in two steps to develop a mutant termed V212, which selectively binds to 6-(5-((3aS,4S,6aR)-2-iminohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)hexanoic acid (iminobiotin long tail, IMNtail). Surface plasmon resonance results showed that V212 has a Kd value of 5.9 × 10(-7) M towards IMNtail, but no binding affinity for endogenous BTN species. This V212/IMNtail system will be useful as a novel delivery tool for anticancer therapy.
对于一种使用抗体的多步预靶向方法，先前制备了一种免疫原性低的链霉亲和素突变体，称为低免疫原性链霉亲和素突变体314（LISA-314），作为一种药物递送工具。然而，对内源性生物素（BTNs）具有高亲和力（Kd < 10(-10) M）的LISA-314结合口袋会阻止外源性BTN标记的抗癌药物进入。在本研究中，我们改进了LISA-314的结合口袋，以消除其对内源性BTN种类的亲和力，从而确保新设计的LISA-314仅结合人工BTN类似物。通过两步替换三个氨基酸残基来开发一种称为V212的突变体，其选择性地结合6-(5-((3aS,4S,6aR)-2-亚氨基六氢-1H-噻吩并[3,4-d]咪唑-4-基)戊酰胺基)己酸（亚氨基生物素长尾，IMNtail）。表面等离子体共振结果表明，V212对IMNtail的Kd值为5.9 × 10(-7) M，但对内源性BTN种类没有结合亲和力。这种V212/IMNtail系统将作为一种新型的抗癌治疗递送工具。
