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: 7674768
• 负责人: William M Pardridge
• 金额: $ 31.45万
• 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 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7674768
• 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.

产品描述（申请人提供）：阿尔茨海默病（AD）痴呆是由于Ab淀粉样蛋白在脑内多年沉积所致。一旦淀粉样斑块在脑中形成，在没有斑块解聚治疗的情况下，它是永久性的。斑块解聚疗法的最有效形式是针对AD的Ab淀粉样肽的单克隆抗体（MAb），并且AD的被动免疫疗法目前正在临床试验中测试。然而，抗Ab MAb不穿过血脑屏障（BBB）。因此，必须给予非常高剂量的MAb才能降低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形式的抗Ab MAb，使其能够通过受体介导的血-脑和脑-血两个方向的转运穿过BBB。PI先前已对针对小鼠转铁蛋白受体（TfR）的嵌合MAb进行了基因工程改造，该嵌合MAb通过小鼠BBB TfR上的受体介导的转运以血液至脑的方向穿过BBB，并且还通过BBB Fc受体（FcR）以脑至血液的方向穿过BBB。此外，PI已进行基因工程改造，并表达针对Ab肽氨基末端部分的单链Fv（ScFv）抗体。本研究将抗鼠TfR单克隆抗体的羧基端与抗鼠TfR单克隆抗体的羧基端融合，得到一种新的融合蛋白，命名为TfRMAb-A2 ScFv双特异性抗体（BSA）。在新BSA的基因工程之后，蛋白质将在COS细胞中瞬时表达，并且BSA的双功能性将用小鼠TfR结合测定和Ab结合测定来证明。然后，BSA将在中国仓鼠卵巢（CHO）细胞中永久表达，然后进行选择、稀释克隆，并采用亲和和阳离子交换色谱法进行纯化。然后将纯化的BSA用于治疗APPswe/PSEN 1（dE 9）双转基因小鼠3个月。对照小鼠将用盐水或用不穿过BBB的常规高剂量抗Ab MAb处理。将使用普鲁士蓝组织化学评价小鼠的神经行为、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IND申请，用于使用基因工程融合抗体治疗AD患者，这些抗体通过受体介导穿过BBB并结合和解聚AD的Ab淀粉样斑块。公共卫生相关性：阿尔茨海默病（Alzheimer's disease，AD）的痴呆是由于A β淀粉样蛋白（A β amyloid，A β）在脑内沉积多年而引起的，一旦A β淀粉样蛋白斑块在脑内形成，如果不进行斑块解聚治疗，A β淀粉样蛋白斑块是永久性的。斑块解聚疗法的最有效形式是针对AD的Ab淀粉样肽的单克隆抗体（MAb），并且AD的被动免疫疗法目前正在临床试验中测试;然而，抗Ab MAb不穿过血脑屏障（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，该抗体通过受体介导穿过BBB并结合和解聚AD的Ab淀粉样斑块。