Targeting RAGE/Abeta interaction with single chain monoclonal antibodies

靶向 RAGE/Abeta 与单链单克隆抗体的相互作用

基本信息

批准号：
8307318
负责人：
Estelle Leclerc
金额：
$ 5.88万
依托单位：
NORTH DAKOTA STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-08-01 至 2013-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8307318
关键词：
Advanced Glycosylation End Products Affinity Alzheimer&apos s Disease Amyloid Amyloid beta-Protein Amyloid beta-Protein Precursor Animal Model Antibodies Apoptosis Binding Blood - brain barrier anatomy Brain Brain Diseases Cell Death Cell Line Cell Surface Receptors Cell model Cell surface Cells Cessation of life Cloning Complex Digestion Enzyme-Linked Immunosorbent Assay Fab Immunoglobulins Fc Receptor Flow Cytometry Goals Immunoglobulin G In Vitro Inflammation Lead Libraries Ligands Light MAPK3 gene Measures Mediating Monoclonal Antibodies Neurons Patients Pattern recognition receptor Peptides Phage Display Play Production Property Protocols documentation Recombinants Role S100 Proteins Signal Transduction Testing expression cloning member neuron apoptosis neuron loss neurotoxicity receptor receptor for advanced glycation endproducts vector

项目摘要

DESCRIPTION (provided by applicant): The Receptor for Advanced Glycation Endproducts is a pattern recognition receptor that can be activated by advanced glycation endproducts, members of the S100 protein family or amyloid b (Ab) peptides. Ab oligomers are generated by proteolytic digestion of the amyloid precursor protein (APP) and are currently believed to be the most toxic forms of Ab. We recently showed that Ab oligomers interact preferentially with the V domain of RAGE whereas other forms of Ab (fibrils, aggregates) interact with other domains of the receptor. We showed that engagement of RAGE by Ab oligomers lead to neuronal apoptosis. Blocking the interaction of RAGE by Ab oligomers could lead to significant reduction of neuronal death in Alzheimer's patients. We propose to use single chain monoclonal antibody (light chain) to block RAGE/ Ab interaction. Single chain domain antibodies have been shown to cross the blood brain barrier and are thus promising agents to target and block brain receptors. We have recently generated a Fab fragment antibody phage display library targeting RAGE. Ten selected Fabs reacting with RAGE presented truncated and thus non- functional heavy chains. We could sub-cloned the light chain of truncated Fab B2 into a His tag containing vector and purified it to homogeneity. We showed that the recombinant light chain could bind to RAGE and displace RAGE ligand S100B in vitro. We propose here to further evaluate these single domain light chain antibodies by measuring their binding affinities to RAGE in vitro (ELISA) and on the surface of cells. We will then test the ability of these antibodies to block cell death triggered by the engagement of RAGE by distinct b amyloid oligomeric forms.

描述（由申请人提供）：高级糖基化终产物受体是一种模式识别受体，可以被高级糖基化终产物、S100 蛋白家族成员或淀粉样蛋白 b (Ab) 肽激活。抗体寡聚物是通过淀粉样前体蛋白 (APP) 的蛋白水解消化产生的，目前被认为是毒性最强的抗体形式。我们最近表明，Ab 寡聚物优先与 RAGE 的 V 结构域相互作用，而其他形式的 Ab（原纤维、聚集体）与受体的其他结构域相互作用。我们发现 Ab 寡聚体与 RAGE 的结合会导致神经元凋亡。 Ab 寡聚体阻断 RAGE 的相互作用可能会显着减少阿尔茨海默病患者的神经元死亡。我们建议使用单链单克隆抗体（轻链）来阻断 RAGE/ Ab 相互作用。单链域抗体已被证明可以穿过血脑屏障，因此是有前途的靶向和阻断脑受体的药物。我们最近生成了一个针对 RAGE 的 Fab 片段抗体噬菌体展示库。与 RAGE 反应的十个选定的 Fab 呈现出截短的、因此无功能的重链。我们可以将截短的 Fab B2 的轻链亚克隆到含有 His 标签的载体中，并将其纯化至均质。我们证明重组轻链可以与 RAGE 结合并在体外取代 RAGE 配体 S100B。我们在此建议通过测量它们与 RAGE 的体外 (ELISA) 和细胞表面的结合亲和力来进一步评估这些单域轻链抗体。然后，我们将测试这些抗体阻止由不同的 b 淀粉样蛋白寡聚形式与 RAGE 结合而引发的细胞死亡的能力。