Monoclonal Antibody-Glial-Derived Neurotrophic Factor Fusion Protein Penetrates the Blood-Brain Barrier in the Mouse
Monoclonal Antibody-Glial-Derived Neurotrophic Factor Fusion Protein Penetrates the Blood-Brain Barrier in the Mouse
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DOI：
10.1124/dmd.109.031534
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发表时间：
2010-04-01
影响因子：
3.9
通讯作者：
Pardridge, William M.
中科院分区：
文献类型：
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作者：
Zhou, Qing-Hui;Boado, Ruben J.;Pardridge, William M.
胶质源性神经营养因子（GDNF）是一种有效的神经保护剂，用于多种脑部疾病，包括帕金森病。然而，GDNF药物开发是困难的，因为GDNF不能穿过血脑屏障（BBB）。为了使GDNF在小鼠模型中的未来药物开发成为可能，将神经营养因子重新工程化为IgG融合蛋白，以使其在静脉内施用后能够穿透BBB。将134个氨基酸的GDNF融合到针对小鼠转铁蛋白受体（TfR）的嵌合单克隆抗体（MAb）的重链，称为cTfRMAb。这种抗体经历受体介导的跨血脑屏障转运，并作为分子特洛伊木马将GDNF运送到小鼠脑中。在稳定转染的中国仓鼠卵巢细胞中表达cTfRMAb-GDNF融合蛋白，亲和纯化后，用小鼠IgG和GDNF蛋白免疫印迹法鉴定其生物学特性。cTfRMAb-GDNF融合蛋白是双功能的，并且以高亲和力结合GDNF受体α 1（ED 50 = 1.7 +/- 0.2 nM）和小鼠TfR（ED 50 = 3.2 +/- 0.3 nM）。cTfRMAb-GDNF融合蛋白被脑迅速摄取，并且在静脉内注射1-mg/kg剂量的融合蛋白后60分钟，脑摄取为3.1 +/- 0.2%注射剂量/g脑。脑毛细血管耗竭分析显示，大部分融合蛋白通过血脑屏障转运并渗透到脑实质中。脑摄取结果表明，通过静脉内施用cTfRMAb-GDNF融合蛋白，有可能在小鼠脑中实现GDNF的治疗性升高。
Glial-derived neurotrophic factor (GDNF) is a potent neuroprotective agent for multiple brain disorders, including Parkinson's disease. However, GDNF drug development is difficult because GDNF does not cross the blood-brain barrier (BBB). To enable future drug development of GDNF in mouse models, the neurotrophin was re-engineered as an IgG fusion protein to enable penetration through the BBB after intravenous administration. The 134-amino acid GDNF was fused to the heavy chain of a chimeric monoclonal antibody (MAb) against the mouse transferrin receptor (TfR) designated the cTfRMAb. This antibody undergoes receptor-mediated transport across the BBB and acts as a molecular Trojan horse to ferry the GDNF into mouse brain. The cTfRMAb-GDNF fusion protein was expressed by stably transfected Chinese hamster ovary cells, affinity-purified, and the biochemical identity was confirmed by mouse IgG and GDNF Western blotting. The cTfRMAb-GDNF fusion protein was bifunctional and bound with high affinity to both the GDNF receptor alpha 1, ED50 = 1.7 +/- 0.2 nM, and the mouse TfR, ED50 = 3.2 +/- 0.3 nM. The cTfRMAb-GDNF fusion protein was rapidly taken up by brain, and the brain uptake was 3.1 +/- 0.2% injected dose/g brain at 60 min after intravenous injection of a 1-mg/kg dose of the fusion protein. Brain capillary depletion analysis showed the majority of the fusion protein was transcytosed across the BBB with penetration into brain parenchyma. The brain uptake results indicate it is possible to achieve therapeutic elevations of GDNF in mouse brain with intravenous administration of the cTfRMAb-GDNF fusion protein.