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

• 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
• 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' 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

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(申请人提供)：高级糖基化终末产物受体是一种模式识别受体，可被晚期糖基化终末产物、S100蛋白家族成员或淀粉样蛋白b(Ab)多肽激活。AB寡聚体是由淀粉样前体蛋白(APP)的蛋白分解产生的，目前被认为是最有毒的AB形式。我们最近发现，抗体寡聚体优先与RAGE的V结构域相互作用，而其他形式的抗体(纤维、聚集体)则与受体的其他结构域相互作用。我们发现，抗体寡聚体与RAGE的结合可导致神经细胞凋亡。用抗体寡聚体阻断RAGE的相互作用可以显著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抗(轻链)来阻断RAGE/Ab的相互作用。单链结构域抗体已被证明可以穿越血脑屏障，因此有望成为靶向和阻断大脑受体的药物。我们最近建立了针对RAGE的Fab片段噬菌体展示文库。与RAGE反应的10个选定的Fabs呈现截断的因此不起作用的重链。我们可以将截短的Fab B2轻链亚克隆到含有His标签的载体中，并将其纯化为均一。体外实验表明，重组轻链能够与RAGE结合并取代RAGE配体S100B。我们建议通过测量它们与RAGE的体外结合亲和力(ELISA法)和细胞表面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细胞死亡的能力，这些细胞死亡是由不同的b淀粉样寡聚体形式的RAGE引起的。