Department of Medicine, University of California at Los Angeles, California 90024, United States.
Bioconjug Chem. 2011 Aug 17;22(8):1611-8. doi: 10.1021/bc200174x. Epub 2011 Jul 6.
The genetic engineering, host cell expression, purity, identity, and in vivo brain drug targeting properties are described for a new IgG-fusion protein, designated the cTfRMAb-AV fusion protein. Avidin (AV) is fused to the carboxyl terminus of the heavy chain of the genetically engineered chimeric monoclonal antibody (mAb) against the mouse transferrin receptor (TfR). The TfRMAb binds the endogenous TfR on the blood-brain barrier (BBB), which triggers transport into brain from blood. The cTfRMAb-AV fusion protein is produced in stably transfected Chinese hamster ovary cells, which are grown in serum free medium under conditions of biotin starvation. Following affinity purification, the purity and identity of the cTfRMAb-AV fusion protein were verified by electrophoresis and Western blotting. The affinity of the cTfRMAb for the murine TfR is high, K(I) = 4.6 ± 0.5 nM, despite fusion of avidin to the antibody heavy chain. The model peptide radiopharmaceutical used in this study is the Aβ(1-40) amyloid peptide of Alzheimer's disease (AD), which in a brain-penetrating form could be used to image the amyloid plaque in brain in AD. The BBB transport and brain uptake of the [(125)I]-Aβ(1-40) peptide was measured in mice injected intravenously (IV) with the peptide either free or conjugated to the cTfRMAb-AV fusion protein. The brain uptake of the free Aβ(1-40) peptide was very low, 0.1% of injected dose (ID)/gram brain following i.v. injection, and is comparable to the brain uptake of a brain blood volume marker. However, the brain uptake of the Aβ(1-40) peptide was high, 2.1 ± 0.2% ID/gram brain, following attachment of the biotinylated peptide to the cTfRMAb-AV fusion protein. Capillary depletion analysis showed the peptide penetrated the brain parenchyma from blood. The cTfRMAb-AV fusion protein is a new drug delivery system that can target to mouse brain monobiotinylated peptide or antisense radiopharmaceuticals.
一种新的 IgG-融合蛋白，命名为 cTfRMAb-AV 融合蛋白，其基因工程、宿主细胞表达、纯度、身份和体内脑内药物靶向特性进行了描述。亲和素 (AV) 融合到针对小鼠转铁蛋白受体 (TfR) 的基因工程嵌合单克隆抗体 (mAb) 的重链羧基末端。TfRMAb 结合血脑屏障 (BBB) 上的内源性 TfR，触发从血液向大脑的转运。cTfRMAb-AV 融合蛋白在稳定转染的中国仓鼠卵巢细胞中产生，这些细胞在生物素饥饿的条件下在无血清培养基中生长。经过亲和纯化后，通过电泳和 Western 印迹验证了 cTfRMAb-AV 融合蛋白的纯度和身份。尽管抗体的重链与亲和素融合，但 cTfRMAb 对鼠 TfR 的亲和力很高，K(I) = 4.6 ± 0.5 nM。本研究中使用的模型肽放射性药物是阿尔茨海默病 (AD) 的 Aβ(1-40) 淀粉样肽，这种穿透性脑的形式可用于成像 AD 脑中的淀粉样斑块。在静脉内 (IV) 注射肽游离或与 cTfRMAb-AV 融合蛋白缀合的情况下，在注射肽的小鼠中测量了 [(125)I]-Aβ(1-40) 肽的 BBB 转运和脑摄取。静脉注射后，游离 Aβ(1-40) 肽的脑摄取非常低，为 0.1% 注入剂量 (ID)/克脑，与脑血容量标记物的脑摄取相当。然而，当将生物素化肽连接到 cTfRMAb-AV 融合蛋白上时，Aβ(1-40) 肽的脑摄取量很高，为 2.1 ± 0.2% ID/克脑。毛细血管耗竭分析表明肽从血液穿透脑实质。cTfRMAb-AV 融合蛋白是一种新的药物输送系统，可靶向小鼠脑单生物素化肽或反义放射性药物。
