Recombinant LRP fragments production for Alzheimer?s disease treatment

用于阿尔茨海默病治疗的重组 LRP 片段生产

• 批准号: 7536964
• 负责人: RASHID DEANE
• 金额: $ 12.06万
• 依托单位: SOCRATECH, LLC
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-08-01 至 2010-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7536964
• 关键词: Advanced Glycosylation End Products; Affect; Affinity; Affinity Chromatography; Age; Alzheimer' s Disease; Alzheimer' s disease model; Amino Acids; Amyloid; Antibodies; Binding; Blood; Blood - brain barrier anatomy; Blood Vessels; Blood flow; Brain; Cell Line; Cerebrovascular Circulation; Cerebrum; Characteristics; Chinese Hamster; Chinese Hamster Ovary Cell; Classification; Complementary DNA; Conditioned Culture Media; Culture Media; Cultured Cells; Disease; Disease Progression; Endothelial Cells; Enzyme-Linked Immunosorbent Assay; Extracellular Domain; Facility Construction Funding Category; Factor VIII; Figs - dietary; Gangliosides; Gelsolin; Generations; Glutathione S-Transferase; Goals; Hamsters; Human; Immune; In Vitro; Inflammation; Kidney; LDL-Receptor Related Protein 1; Lead; Learning; Ligand Binding Domain; Ligands; Lipoprotein Receptor; Marketing; Mass Spectrum Analysis; Memory; Modification; Mus; N-terminal; Ovary; Patient Care; Peptide Leader Sequences; Peptides; Pharmaceutical Preparations; Phase; Plasma; Process; Production; Protein Isoforms; Proteins; Public Health; Purpose; Recombinant Proteins; Recombinants; Serum Proteins; Side; Suspension Culture; Symptoms; System; Therapeutic; Toxin; United States Food and Drug Administration; Validation; Western Blotting; activated Protein C; amyloid peptide; base; cost; disorder prevention; expression vector; flasks; improved; in vivo; kidney cell; prevent; receptor; response; validation studies; vector

DESCRIPTION (provided by applicant): Alzheimer's disease (AD) is associated with abnormal accumulation of amyloid-2 peptides (A2) in brain due mainly to faulty clearance. Rapid clearance from brain across the blood-brain barrier (BBB) is regulated by the low density lipoprotein receptor-related protein 1 (LRP1). LRP consists of four ligand binding domains of which domains II (LRP-II) and IV (LRP-IV) bind A240 and A242 in vitro with high affinity. Recently, we have shown that human plasma sLRP, soluble extracellular domain of LRP, is a key endogenous `brain A2 sinker'. We have also showed that in a mouse AD model (Tg 2576) treated with low levels of recombinant LRP-IV brain A2 levels are reduced, and cerebral blood flow (CBF) responses to brain activation and learning and memory are improved. Unlike other Ab `sinker' agents LRP-II and LRP-IV have the highest affinity for binding directly different A2 isoforms, and do not enter brain. Therefore, this therapeutic approach may not lead to potential neuro-inflammation as seen in immune based therapies. In our earlier studies we used purified LRP-IV produced by stably transfected Baby Hamster Kidney (BHK) cells expressing LRP-IV with a tag. For therapeutic purposes we need to produce the pure recombinant proteins, without extraneous amino acids, and to use a cell line that is acceptable to the FDA. To achieve this we propose to use Chinese hamster ovary (CHO) cells and culture the cells in a medium free of serum and proteins. The goals of Phase I are to generate an expression system for pure recombinant LRP-II and LRP-IV production, and to demonstrate that they preferentially bind A2 in vitro. Currently, there are no cures for AD, a devastating disease that affects about 4.5 million people in the US, and cost about $100 billion in patient care. The major unmet need in the AD therapeutic market is for disease modification and disease prevention drugs. Our disease modifying approach, using recombinant LRP-II or LRP-IV, which do not enter brain, may be effective in curing and preventing AD. PUBLIC HEALTH RELEVANCE Alzheimer's disease (AD) is associated with abnormal accumulation of brain toxins called amyloid-2 peptides (A2). This is due mainly to faulty clearance from brain directly into blood. Rapid clearance from brain is regulated mainly by a transporter called low density lipoprotein receptor-related protein 1 (LRP1) on the brain blood vessels. A2 directly attaches to LRP1 resulting in its clearance from brain into circulating blood. LRP consists of four regions that may bind molecules, two of which, region II (LRP-II) and IV (LRP-IV) strongly bind A2. It was shown that mice (AD model) injected with LRP-IV had lower brain A2, and this was associated with improved functional brain blood flow and learning and memory. Currently, there are no cures for AD, a devastating disease that affects about 4.5 million people in the US, and cost about $100 billion in patient care. Current drugs treat symptoms of the disease and these are of limited use in slowing the disease progression. The major unmet need in the AD therapeutic market is for disease modification and disease prevention drugs. Our disease modifying approach, using LRP-II or LRP-IV, may be effective in treating and preventing AD. This proposal will evaluate the production process of LRP-II and LRP-IV and their potential as new therapies for AD.

描述（由申请人提供）：阿尔茨海默氏病（AD）与淀粉样蛋白2肽（A2）在大脑中的异常积累有关，这主要是由于清除错误造成的。穿过血脑屏障 (BBB) 从大脑快速清除是由低密度脂蛋白受体相关蛋白 1 (LRP1) 调节的。 LRP 由四个配体结合结构域组成，其中结构域 II (LRP-II) 和 IV (LRP-IV) 在体外以高亲和力结合 A240 和 A242。最近，我们发现人血浆 sLRP（LRP 的可溶性胞外结构域）是一个关键的内源性“大脑 A2 沉降片”。我们还表明，在用低水平重组 LRP-IV 治疗的小鼠 AD 模型 (Tg 2576) 中，大脑 A2 水平降低，脑血流 (CBF) 对大脑激活以及学习和记忆的反应得到改善。与其他 Ab“沉降”剂不同，LRP-II 和 LRP-IV 具有直接结合不同 A2 同工型的最高亲和力，并且不进入大脑。因此，这种治疗方法可能不会导致免疫疗法中出现的潜在神经炎症。在我们早期的研究中，我们使用了稳定转染的幼仓鼠肾 (BHK) 细胞产生的纯化 LRP-IV，这些细胞表达带有标签的 LRP-IV。出于治疗目的，我们需要生产不含外来氨基酸的纯重组蛋白，并使用 FDA 可接受的细胞系。为了实现这一目标，我们建议使用中国仓鼠卵巢 (CHO) 细胞并在不含血清和蛋白质的培养基中培养细胞。第一阶段的目标是生成用于纯重组 LRP-II 和 LRP-IV 生产的表达系统，并证明它们在体外优先结合 A2。目前，AD 尚无治愈方法，这种毁灭性疾病影响着美国约 450 万人，患者护理费用约为 1000 亿美元。 AD 治疗市场中未满足的主要需求是疾病缓解和疾病预防药物。我们的疾病缓解方法使用不进入大脑的重组LRP-II或LRP-IV，可能有效治疗和预防AD。公共健康相关性 阿尔茨海默病 (AD) 与称为淀粉样蛋白 2 肽 (A2) 的脑毒素异常积累有关。这主要是由于从大脑直接进入血液的错误清除造成的。从大脑中的快速清除主要由脑血管上称为低密度脂蛋白受体相关蛋白 1 (LRP1) 的转运蛋白调节。 A2 直接附着在 LRP1 上，导致其从大脑清除到循环血液中。 LRP 由四个可结合分子的区域组成，其中两个区域 II (LRP-II) 和 IV (LRP-IV) 与 A2 强烈结合。结果表明，注射 LRP-IV 的小鼠（AD 模型）的大脑 A2 较低，这与功能性脑血流以及学习和记忆的改善有关。目前，AD 尚无治愈方法，这种毁灭性疾病影响着美国约 450 万人，患者护理费用约为 1000 亿美元。目前的药物治疗该疾病的症状，但在减缓疾病进展方面作用有限。 AD 治疗市场中未满足的主要需求是疾病缓解和疾病预防药物。我们使用 LRP-II 或 LRP-IV 的疾病缓解方法可能有效治疗和预防 AD。该提案将评估 LRP-II 和 LRP-IV 的生产工艺及其作为 AD 新疗法的潜力。