Insulin Receptor Antibody-α-N-Acetylglucosaminidase Fusion Protein Penetrates the Primate Blood-Brain Barrier and Reduces Glycosoaminoglycans in Sanfilippo Type B Fibroblasts
Insulin Receptor Antibody-α-N-Acetylglucosaminidase Fusion Protein Penetrates the Primate Blood-Brain Barrier and Reduces Glycosoaminoglycans in Sanfilippo Type B Fibroblasts
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DOI：
10.1021/acs.molpharmaceut.6b00037
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发表时间：
2016-04-01
影响因子：
4.9
通讯作者：
Pardridge, William M.
中科院分区：
文献类型：
--
作者：
Boado, Ruben J.;Lu, Jeff Zhiqiang;Pardridge, William M.
IIIB型粘多糖沉积症（MPSIIIB）由编码溶酶体酶α-N-乙酰氨基葡萄糖苷酶（NAGLU）的基因突变引起。MPSIIIB表现为严重的中枢神经系统疾病，但静脉内NAGLU酶替代疗法尚未开发，因为NAGLU酶不能穿过血脑屏障（BBB）。通过将NAGLU再工程化为IgG-酶融合蛋白来产生酶的BBB穿透形式，其中IgG结构域是针对人胰岛素受体（HIR）的单克隆抗体（mAb）。HIRMAb通过内源性胰岛素受体上的转运穿过BBB，并作为分子特洛伊木马从血液中运送融合的NAGLU穿过BBB。NAGLU通过延伸的31个氨基酸的接头与HIRMAb的每条重链的羧基末端融合，融合蛋白被命名为HIRMAb-LL-NAGLU。该融合蛋白保持与HIR的高亲和力结合，并且在摩尔基础上具有与重组人NAGLU相等的酶活性。用融合蛋白处理MPSIIIB成纤维细胞使细胞内NAGLU酶活性正常化并减少硫酸根掺入细胞内糖氨基聚糖。如共聚焦显微镜所示，融合蛋白靶向细胞的溶酶体区室。融合蛋白用[I-125]-Bolton-Hunter试剂放射性标记，并静脉注射到成年恒河猴中。融合蛋白被所有主要外周器官从血浆中迅速清除。融合蛋白的高脑摄取; 1%注射剂量/脑，使得脑NAGLU酶活性能够以lmg/kg的治疗剂量正常化。HIRMAb-LL-NAGLU融合蛋白是MPSIIIB脑的新治疗方法;可通过无创静脉输注给药。
Mucopolysaccharidosis Type IIIB (MPSIIIB) is caused by mutations in the gene encoding the lysosomal enzyme, alpha-N-acetylglucosaminidase (NAGLU). MPSIIIB presents with severe disease of the central nervous system, but intravenous NAGLU enzyme replacement therapy has not been developed because the NAGLU enzyme does not cross the blood brain barrier (BBB). A BBB-penetrating form of the enzyme was produced by re-engineering NAGLU as an IgG-enzyme fusion protein, where the IgG domain is a monoclonal antibody (mAb) against the human insulin receptor (HIR). The HIRMAb traverses the BBB via transport on the endogenous insulin receptor and acts as a molecular Trojan horse to ferry the fused NAGLU across the BBB from blood. The NAGLU was fused to the carboxyl terminus of each heavy chain of the HIRMAb via an extended 31-amino acid linker, and the fusion protein is designated HIRMAb-LL-NAGLU. The fusion protein retains high affinity binding to the HIR, and on a molar basis has an enzyme activity equal to that of recombinant human NAGLU. Treatment of MPSIIIB fibroblasts with the fusion protein normalizes intracellular NAGLU enzyme activity and reduces sulfate incorporation into intracellular glycosoaminoglycan. The fusion protein is targeted to the lysosomal compartment of the cells as shown by confocal microscopy. The fusion protein was radiolabeled with the [I-125]-Bolton-Hunter reagent and injected intravenously in the adult Rhesus monkey. The fusion protein was rapidly cleared from plasma by all major peripheral organs. The high brain uptake of the fusion protein; 1% injected dose/brain, enables normalization of brain NAGLU enzyme activity with a therapeutic dose of 1 mg/kg. The HIRMAb-LL-NAGLU fusion protein is a new treatment of the brain in MPSIIIB; which can be administered by noninvasive intravenous infusion.