Pharmacokinetics and brain uptake in the rhesus monkey of a fusion protein of arylsulfatase a and a monoclonal antibody against the human insulin receptor.
Pharmacokinetics and brain uptake in the rhesus monkey of a fusion protein of arylsulfatase a and a monoclonal antibody against the human insulin receptor.
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DOI：
10.1002/bit.24795
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发表时间：
2013-05
影响因子：
3.8
通讯作者：
Pardridge, William M.
中科院分区：
文献类型：
--
作者：
Boado, Ruben J.;Lu, Jeff Zhiqiang;Hui, Eric K. -W.;Sumbria, Rachita K.;Pardridge, William M.
异染性脑白质营养不良（MLD）是由编码溶酶体硫酸酯酶（芳基硫酸酯酶A（阿萨））的基因突变引起的脑的溶酶体储存病症。不可能用重组阿萨治疗MLD的大脑，因为这种酶不能穿过血脑屏障（BBB）。在本研究中，设计并表达BBB穿透IgG-ASA融合蛋白，其中阿萨单体融合至针对人胰岛素受体（HIR）的工程化单克隆抗体（MAb）的每个重链的羧基末端。HIRMAb通过内源性BBB胰岛素受体上的受体介导的转运穿过BBB，并作为分子特洛伊木马将阿萨从血液运送到脑中。HIRMAb-ASA在无血清培养基中生长的稳定转染的中国仓鼠卵巢细胞中表达，并通过蛋白A亲和层析纯化。融合蛋白保留了与HIR的高亲和力结合，EC 50 = 0.34 ± 0.11 nM，并保留了高阿萨酶活性，20 ± 1单位/mg。HIRMAb-ASA融合蛋白被内吞并分流至MLD成纤维细胞中的溶酶体区室。用Bolton-Hunter试剂放射性标记融合蛋白，静脉内给药后，[125 I]-HIRMAb-ASA迅速穿透恒河猴的脑。灵长类动物脑的胶片和乳剂放射自显影显示融合蛋白在整个猴脑中的全局分布。这些研究描述了一种新的生物实体，其设计用于在非侵入性静脉输注IgG-ASA融合蛋白后治疗患有MLD的人脑。
Metachromatic leukodystrophy (MLD) is a lysosomal storage disorder of the brain caused by mutations in the gene encoding the lysosomal sulfatase, arylsulfatase A (ASA). It is not possible to treat the brain in MLD with recombinant ASA, because the enzyme does not cross the blood-brain barrier (BBB). In the present investigation, a BBB-penetrating IgG-ASA fusion protein is engineered and expressed, where the ASA monomer is fused to the carboxyl terminus of each heavy chain of an engineered monoclonal antibody (MAb) against the human insulin receptor (HIR). The HIRMAb crosses the BBB via receptor-mediated transport on the endogenous BBB insulin receptor, and acts as a molecular Trojan horse to ferry the ASA into brain from blood. The HIRMAb-ASA is expressed in stably transfected Chinese hamster ovary cells grown in serum free medium, and purified by protein A affinity chromatography. The fusion protein retains high affinity binding to the HIR, EC50 = 0.34 ± 0.11 nM, and retains high ASA enzyme activity, 20 ± 1 units/mg. The HIRMAb-ASA fusion protein is endocytosed and triaged to the lysosomal compartment in MLD fibroblasts. The fusion protein was radio-labeled with the Bolton-Hunter reagent, and the [125I]-HIRMAb-ASA rapidly penetrates the brain in the Rhesus monkey following intravenous administration. Film and emulsion autoradiography of primate brain shows global distribution of the fusion protein throughout the monkey brain. These studies describe a new biological entity that is designed to treat the brain of humans with MLD following non-invasive, intravenous infusion of an IgG-ASA fusion protein.
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