Recombinant LRP fragments production for Alzheimer?s disease treatment

描述（由申请人提供）：阿尔茨海默病（AD）与淀粉样蛋白-2肽（A2）在脑中的异常积累有关，主要是由于错误的清除。低密度脂蛋白受体相关蛋白1（LRP 1）可调节通过血脑屏障（BBB）从脑中的快速清除。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，我们已经表明，人血浆sLRP，可溶性细胞外结构域的LRP，是一个关键的内源性“脑A2下沉”。我们还表明，在用低水平的重组LRP-IV处理的小鼠AD模型（Tg 2576）中，脑A2水平降低，并且脑血流（CBF）对脑激活的反应以及学习和记忆得到改善。与其它Ab“下沉”剂不同，LRP-II和LRP-IV具有最高的直接结合不同A2同种型的亲和力，并且不进入脑。因此，这种治疗方法可能不会导致潜在的神经炎症，如在基于免疫的治疗中所见。在我们早期的研究中，我们使用了由稳定转染的婴儿肾脏（BHK）细胞产生的纯化的LRP-IV，所述细胞表达具有标签的LRP-IV。出于治疗目的，我们需要生产不含外源氨基酸的纯重组蛋白，并使用FDA可接受的细胞系。为了实现这一点，我们建议使用中国仓鼠卵巢（CHO）细胞，并在无血清和蛋白质的培养基中培养细胞。I期的目标是产生用于纯重组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是一种毁灭性的疾病，影响着美国约450万人，花费约1000亿美元用于患者护理。AD治疗市场中未满足的主要需求是疾病修饰和疾病预防药物。我们的疾病修饰方法，使用不进入大脑的重组LRP-II或LRP-IV，可能有效地治疗和预防AD。阿尔茨海默病（AD）与称为淀粉样蛋白-2肽（A2）的脑毒素的异常积累有关。这主要是由于从大脑直接进入血液的错误清除。从脑中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（LRP 1）的转运蛋白调节。A2直接附着于LRP 1，导致其从大脑清除到循环血液中。LRP由可以结合分子的四个区域组成，其中两个区域II（LRP-II）和IV（LRP-IV）强烈结合A2。结果表明，注射LRP-IV的小鼠（AD模型）具有较低的脑A2，并且这与改善的功能性脑血流和学习和记忆有关。目前，AD是一种毁灭性的疾病，影响着美国约450万人，花费约1000亿美元用于患者护理。目前的药物治疗疾病的症状，这些药物在减缓疾病进展方面的作用有限。AD治疗市场中未满足的主要需求是疾病修饰和疾病预防药物。我们的疾病修饰方法，使用LRP-II或LRP-IV，可能有效地治疗和预防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。

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.