Monoclonal Antibody Drug Development for Alzheimer?s Disease

描述（申请人提供）：阿尔茨海默病（AD）的痴呆是由Ab淀粉样蛋白在大脑中沉积多年引起的。一旦淀粉样斑块在大脑中形成，在没有斑块分解治疗的情况下，它是永久性的。最有效的斑块聚散治疗形式是针对AD的Ab淀粉样肽的单克隆抗体（MAb）， AD的被动免疫治疗目前正在临床试验中进行测试。然而，抗单克隆抗体不能穿过血脑屏障（BBB）。因此，必须给予非常高剂量的单抗来降低AD转基因小鼠脑中的斑块，而这些高剂量会导致脑微出血。目前的研究将通过基因工程设计一种新型的抗单克隆抗体，使其能够通过受体介导的血脑和脑血方向的运输穿过血脑屏障。PI先前已经通过基因工程设计了一种针对小鼠转铁蛋白受体（TfR）的嵌合单抗，该受体通过受体介导的转运在小鼠血脑屏障TfR上穿过血脑屏障，并在血脑屏障Fc受体（FcR）上穿过血脑屏障从脑到血方向。此外，PI通过基因工程表达了针对Ab肽氨基末端的单链Fv抗体（ScFv）。本研究将产生一种新的融合蛋白，将抗抗体ScFv融合到针对小鼠TfR的嵌合单抗的羧基端，这种新的融合蛋白被命名为TfRMAb-A2ScFv双特异性抗体（BSA）。在新BSA的基因工程之后，该蛋白将在COS细胞中短暂表达，并且BSA的双功能将通过小鼠TfR结合试验和Ab结合试验来证明。然后在中国仓鼠卵巢（CHO）细胞中永久表达BSA，随后进行选择，稀释克隆，亲和和阳离子交换层析纯化。纯化后的牛血清白蛋白将用于治疗APPswe/PSEN1（dE9）双转基因小鼠3个月。对照小鼠将接受生理盐水或常规的高剂量抗-单抗治疗，该抗-单抗不会穿过血脑屏障。用普鲁士蓝组织化学方法评估小鼠的神经行为、血浆Ab水平、脑Ab斑块含量和脑微出血。这项研究将提供必要的临床前药理学，以支持使用基因工程融合抗体通过受体介导穿过血脑屏障并结合和分解AD的Ab淀粉样斑块治疗人类AD的IND申请。公共卫生相关性：阿尔茨海默病（AD）痴呆是由Ab淀粉样蛋白在大脑中沉积多年引起的，一旦淀粉样蛋白斑块在大脑中形成，在没有斑块分解治疗的情况下是永久性的。最有效的斑块聚散治疗形式是针对AD的Ab淀粉样肽的单克隆抗体（MAb）， AD的被动免疫治疗目前正在临床试验中进行测试；然而，抗单克隆抗体不能穿过血脑屏障（BBB）。本研究将在AD转基因小鼠中测试一种新的基因工程融合抗体的功效，该抗体既可以通过受体介导穿过血脑屏障，又可以结合并分解AD的Ab淀粉样斑块。

DESCRIPTION (provided by applicant): The dementia of Alzheimer's disease (AD) is caused by the deposition of Ab amyloid in brain over many years. Once the amyloid plaque forms in brain, it is permanent in the absence of plaque disaggregation therapy. The most potent form of plaque diasaggregation therapy is a monoclonal antibody (MAb) against the Ab amyloid peptide of AD, and passive immune therapy of AD is currently being tested in clinical trials. However, the anti-Ab MAb does not cross the blood-brain barrier (BBB). Consequently, very high doses of MAb must be administered to lower plaque in brain of AD transgenic mice, and these high doses causes brain microhemorrhage. The present research will genetically engineer a new form of anti-Ab MAb that is enabled to cross the BBB via receptor-mediated transport in both the blood-to-brain and brain-to-blood directions. The PI has previously genetically engineered a chimeric MAb against the mouse transferrin receptor (TfR) that crosses the BBB in the blood-to-brain direction via receptor-mediated transport on the mouse BBB TfR, and also crosses the BBB in the brain-to-blood direction on the BBB Fc receptor (FcR). In addition, the PI has genetically engineered, and expressed a single chain Fv (ScFv) antibody against the amino terminal portion of the Ab peptide. The present research will produce a new fusion protein, wherein the anti-Ab ScFv is fused to the carboxyl terminus of the chimeric MAb against the mouse TfR, and this new fusion protein is designated the TfRMAb-A2ScFv bi-specific antibody (BSA). Following the genetic engineering of the new BSA, the protein will be transiently expressed in COS cells, and the bi-functionality of the BSA will be demonstrated with a mouse TfR binding assay and an Ab binding assay. The BSA will then be permanently expressed in Chinese hamster ovary (CHO) cells, followed by selection, and dilutional cloning, and purification with affinity and cation exchange chromatography. The purified BSA will then be used to treat APPswe/PSEN1(dE9) double transgenic mice over 3 month period. Control mice will be treated with either saline or with the conventional high dose anti-Ab MAb that does not cross the BBB. The mice will be evaluated for neurobehavior, plasma Ab levels, brain Ab plaque content, and brain micro-hemorrhage using Prussian blue histochemistry. This research will provide the necessary pre-clinical pharmacology to support an IND filing for the treatment of humans with AD using genetically engineered fusion antibodies that both cross the BBB via receptor-mediation and bind and disaggregate Ab amyloid plaque of AD. PUBLIC HEALTH RELEVANCE: The dementia of Alzheimer's disease (AD) is caused by the deposition of Ab amyloid in brain over many years, and once the amyloid plaque forms in brain, it is permanent in the absence of plaque disaggregation therapy. The most potent form of plaque diasaggregation therapy is a monoclonal antibody (MAb) against the Ab amyloid peptide of AD, and passive immune therapy of AD is currently being tested in clinical trials; however, the anti-Ab MAb does not cross the blood-brain barrier (BBB). The present research will test in AD transgenic mice the efficacy of a new genetically engineered fusion antibody that both crosses the BBB via receptor-mediation and binds and disaggregates Ab amyloid plaque of AD.