Genetic engineering, expression, and activity of a fusion protein of a human neurotrophin and a molecular Trojan horse for delivery across the human blood-brain barrier
Genetic engineering, expression, and activity of a fusion protein of a human neurotrophin and a molecular Trojan horse for delivery across the human blood-brain barrier
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DOI：
10.1002/bit.21369
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发表时间：
2007-08-15
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.;Zhang, Yufeng;Pardridge, William M.
神经营养因子，例如脑源性神经营养因子 (BDNF)，不会穿过血脑屏障 (BBB)。某些针对人胰岛素受体 (HIR) 的单克隆抗体 (MAb) 确实通过受体介导的转运穿过 BBB，并且可以充当分子特洛伊木马，将附着的药物运送穿过 BBB。产生了一种基因工程融合蛋白，其中人 BDNF 的氨基末端与嵌合 HIRMAb 重链的羧基末端融合。 HIRMAb-BDNF 融合蛋白与人 IgG 和 BDNF 抗体的反应相同。融合蛋白的双功能被保留，因为融合蛋白对HIR的亲和力与嵌合HIRMAb的亲和力相同，并且融合蛋白对trkB受体的亲和力与BDNF的亲和力相同。在人类神经细胞的神经保护测定中，该融合蛋白与 BDNF 具有同等效力。在成年恒河猴中检查了融合蛋白的药代动力学 (PK)。融合蛋白在血液中的平均停留时间(MRT)比BDNF的MRT长>100倍。静脉注射融合蛋白后，灵长类动物大脑中产生了治疗水平的 BDNF。融合蛋白串联载体经过工程设计，允许分离在无血清培养基中高水平产生融合蛋白的 CHO 细胞系。神经营养素，如 BDNF，可以重新配制，使这些分子能够穿过人类血脑屏障，这种融合蛋白代表了一类新的人类神经治疗药物。
Neurotrophins, such as brain derived neurotrophic factor (BDNF), do not cross the blood-brain barrier (BBB). Certain monoclonal antibodies (MAb) to the human insulin receptor (HIR) do cross the BBB via receptor-mediated transport, and can act as a molecular Trojan horse to ferry across the BBB an attached drug. A genetically engineered fusion protein was produced whereby the amino terminus of human BDNF is fused to the carboxyl terminus of the heavy chain of a chimeric HIRMAb. The HIRMAb-BDNF fusion protein reacted equally with antibodies to human IgG and BDNF. The bi- functionality of the fusion protein was retained as the affinity of the fusion protein for the HIR was identical to that of the chimeric HIRMAb, and the affinity of the fusion protein for the trkB receptor was identical to that of BDNF. The fusion protein was equipotent with BDNF in a neuroprotection assay in human neural cells. The pharmacokinetics (PK) of the fusion protein was examined in the adult Rhesus monkey. The mean residence time (MRT) of the fusion protein in blood was >1 00-fold longer than the MRT of BDNF. Therapeutic levels of BDNF were produced in primate brain following the intravenous administration of the fusion protein. A fusion protein tandem vector was engineered that allowed for isolation of a CHO cell line that produced the fusion protein at high levels in serum free medium. Neurotrophins, such as BDNF, can be re-formulated to enable these molecules to cross the human BBB, and such fusion proteins represent a new class of human neurotherapeutics.