Monoclonal Antibody Drug Development for Alzheimer?s Disease

阿尔茨海默病单克隆抗体药物开发

• 批准号: 7870352
• 负责人: William M Pardridge
• 金额: $ 46.39万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-08-15 至 2012-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7870352
• 关键词: A Mouse; Affinity; Affinity Chromatography; Age-Months; Alzheimer' s Disease; Alzheimer' s disease model; Amino Acids; Amyloid; Animals; Antibodies; Binding; Binding Sites; Biochemical; Biological Assay; Bioreactors; Blood; Blood - brain barrier anatomy; Blood capillaries; Brain; Brain hemorrhage; Breeding; COS Cells; Cations; Cerebrum; Chimeric Proteins; Chinese Hamster; Chinese Hamster Ovary Cell; Chromatography; Clinical Pharmacology; Clinical Trials; Cloning; Complementary DNA; DNA Sequence; Data; Dementia; Deposition; Disease; Dose; Drug Delivery Systems; Drug Kinetics; Electroporation; Engineering; Fc Receptor; Filtration; G-substrate; Genes; Genetic Engineering; Genotype; Goals; Harvest; Hemorrhage; Histocytochemistry; Human; Hybridomas; IgG1; Immune; Immunoglobulin G; Immunoglobulin Variable Region; Immunoradiometric Assays; Immunotherapy; Inbred BALB C Mice; Injection of therapeutic agent; Insulin Receptor; Label; Laboratories; Light; Measurement; Mediating; Mediation; Medicine; Methods; Monoclonal Antibodies; Mus; Ovary; Peptides; Peripheral; Peritoneal; Pharmaceutical Preparations; Plasma; Plasmids; Proteins; Prussian blue; Publishing; RNA Splicing; Radio; Rattus; Reaction; Research; Rodent; Saline; Senile Plaques; Series; Serum-Free Culture Media; Staging; Structure; Testing; Therapeutic; Transfection; Transferrin Receptor; Transgenic Mice; Validation; Western Blotting; Work; amyloid peptide; animal efficacy; antibiotic G 418; capillary; drug development; drug discovery; human INSR protein; in vivo; interest; lipofection; molecular trojan horse; mouse model; nervous system disorder; neurobehavior; novel therapeutics; pre-clinical; public health relevance; receptor; receptor binding; uptake; variable region gene

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.

描述(申请人提供)：阿尔茨海默病(AD)的痴呆症是由于多年来抗淀粉样蛋白在大脑中沉积而引起的。淀粉样斑块一旦在脑内形成，在没有斑块解聚治疗的情况下是永久性的。斑块解聚治疗最有效的形式是抗AD的抗淀粉样多肽的单抗(MAb)，目前AD的被动免疫治疗正在进行临床试验。然而，抗单抗不能通过血脑屏障。因此，为了降低AD转基因小鼠脑内的斑块，必须给予极高剂量的单抗，而这些高剂量会导致脑微出血。目前的研究将从基因上设计一种新形式的抗单抗，这种单抗能够通过受体介导的血到脑和脑到血两个方向的运输穿过血脑屏障。PI此前曾通过基因工程设计出一种针对小鼠转铁蛋白受体(TFR)的嵌合单抗，通过小鼠BBB TFR上的受体介导的转运在血到脑方向穿过BBB，并在BBB Fc受体(FCR)上在脑到血方向上穿过BBB。此外，PI还进行了基因工程，并表达了针对抗体氨基端部分的单链抗体(ScFv)。本研究将产生一种新的融合蛋白，其中抗AbScFv与抗小鼠TfR嵌合单抗的羧基末端融合，这种新的融合蛋白被命名为TfRMAb-A2ScFv双特异性抗体(BSA)。在对新的BSA进行基因工程后，该蛋白将在COS细胞中瞬时表达，并将通过小鼠TFR结合试验和抗体结合试验证明BSA的双功能。然后将BSA在中国仓鼠卵巢(CHO)细胞中永久表达，然后进行筛选、稀释克隆，并用亲和层析和阳离子交换层析进行纯化。然后将纯化的BSA用于治疗APPswe/PSEN1(DE9)双转基因小鼠，为期3个月。对照组小鼠将接受生理盐水或常规大剂量不跨越血脑屏障的抗-Ab单抗治疗。将使用普鲁士蓝组织化学方法对小鼠的神经行为、血浆抗体水平、脑抗体斑块含量和脑微出血进行评估。这项研究将提供必要的临床前药理学，以支持IND申请使用基因工程融合抗体治疗人类AD，这些融合抗体既通过受体中介穿过血脑屏障，又结合和解聚AD的抗淀粉样蛋白斑块。公共卫生相关性：阿尔茨海默病(AD)的痴呆是由于多年来抗淀粉样蛋白在大脑中沉积而引起的，一旦淀粉样斑块在大脑中形成，在没有斑块解聚治疗的情况下，它是永久性的。斑块解聚治疗最有效的形式是抗AD的抗淀粉样多肽的单抗(MAb)，目前AD的被动免疫治疗正在进行临床试验；然而，抗AbMAb不能跨越血脑屏障(BBB)。本研究将在AD转基因小鼠身上测试一种新的基因工程融合抗体的有效性，这种抗体既可以通过受体介导跨越血脑屏障，又可以结合和解聚AD的抗淀粉样蛋白斑块。