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: 7867615
• 负责人: William M Pardridge
• 金额: $ 15.4万
• 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/7867615
• 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; Public Health; 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; abstracting; 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; receptor; receptor binding; uptake; variable region gene

ABSTRACT The dementia of Alzheimer's disease (AD) is caused by the deposition of A¿ 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 A¿ amyloid peptide of AD, and passive immune therapy of AD is currently being tested in clinical trials. However, the anti- A¿ 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-A¿ 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 A¿ peptide. The present research will produce a new fusion protein, wherein the anti-A¿ ScFv is fused to the carboxyl terminus of the chimeric MAb against the mouse TfR, and this new fusion protein is designated the TfRMAb-A¿ScFv 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 A¿ 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-A¿ MAb that does not cross the BBB. The mice will be evaluated for neurobehavior, plasma A¿ levels, brain A¿ 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 A¿ amyloid plaque of AD. RELEVANCE TO PUBLIC HEALTH The dementia of Alzheimer's disease (AD) is caused by the deposition of A¿ 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 A¿ amyloid peptide of AD, and passive immune therapy of AD is currently being tested in clinical trials; however, the anti- A¿ 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 A¿ amyloid plaque of AD.

抽象的 阿尔茨海默氏病（AD）的痴呆是由大脑中A淀粉样蛋白的沉积引起的 多年。一旦在大脑中形成淀粉样菌斑，它是永久的，在没有斑块的情况下 分解疗法。斑块二氧化糖疗法的最潜在形式是单克隆 抗体（mAb）针对AD的A淀粉样蛋白肽，并且AD的被动免疫疗法是 在临床试验中进行了测试。但是，抗ambAb不会越过血脑屏障（BBB）。 因此，必须将非常高剂量的mAb施用到AD大脑的较低斑块 转基因小鼠，这些高剂量会导致脑部微毛发。本研究将 从遗传学上设计一种新形式的抗a笔mab，可以通过受体越过BBB 介导的血液到脑和血液方向的转运。 PI以前有 基因设计的嵌合mab针对横穿小鼠转移蛋白受体（TFR） BBB通过受体介导的小鼠BBB TFR的传输在血液到脑方向，也是 在BBB FC受体（FCR）上沿脑对血液方向越过BBB。此外，PI具有 基因设计，并表达针对氨基终端的单链FV（SCFV）抗体 肽的一部分。本研究将产生一种新的融合蛋白，其中抗a。 SCFV融合到嵌合mab的羧基末端与鼠标TFR融合，这是新的 融合蛋白被指定为TFRMAB-A¿SCFV双特异性抗体（BSA）。遵循通用 新BSA的工程，该蛋白质将在COS细胞中瞬时表达，并且 BSA的功能将使用鼠标TFR结合测定和A binding证明 测定。然后，BSA将在中国仓鼠卵巢（CHO）细胞中永久表达 通过选择，稀释克隆以及与亲和力和阳离子交换的纯化 色谱法。然后，纯化的BSA将用于治疗Appswe/psen1（DE9）双转基因 小鼠超过3个月。对照小鼠将用盐水或常规高 剂量抗a笔mab不会越过BBB。将评估小鼠的神经行为， 血浆A级，大脑A斑块含量和使用普鲁士蓝的大脑微毛 组织化学。这项研究将提供必要的临床前药理学来支持IND 使用一般设计的融合抗体对人类进行AD的治疗 通过受体介导并结合并分解AD的淀粉样菌斑，越过BBB。与公共卫生有关 阿尔茨海默氏病（AD）的痴呆是由A淀粉样蛋白在 大脑多年，一旦大脑中的淀粉样菌斑形成，它是永久的 缺乏斑块分解疗法。斑块的最潜在形式 DIASACGREGATION疗法是针对A淀粉样胡椒的单克隆抗体（MAB） AD和AD的被动免疫疗法目前正在临床试验中进行测试； 但是，抗ambAb不会越过血脑屏障（BBB）。现在 研究将在AD转基因小鼠中测试新基因工程的效率 融合抗体均通过受体介入和结合以及结合以及 AD的淀粉样菌斑进行分解。