Targeting RAGE/Abeta interaction with single chain monoclonal antibodies

描述（由申请人提供）：晚期糖基化终产物受体是一种模式识别受体，可被晚期糖基化终产物、S100蛋白家族成员或淀粉样蛋白b （Ab）肽激活。Ab低聚物是由淀粉样前体蛋白（APP）的蛋白水解消化产生的，目前被认为是Ab最具毒性的形式。我们最近发现，Ab低聚物优先与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寡聚物参与RAGE可导致神经元凋亡。通过Ab寡聚物阻断RAGE的相互作用可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克隆抗体（轻链）阻断RAGE/ Ab相互作用。单链结构域抗体已被证明可以穿过血脑屏障，因此是靶向和阻断脑受体的有希望的药物。我们最近建立了一个靶向RAGE的Fab片段抗体噬菌体展示文库。与RAGE反应的10个晶圆片呈现截断的非功能重链。我们将截断的Fab B2轻链亚克隆到含有His标签的载体上，并将其纯化至均匀性。结果表明，重组轻链在体外可以与RAGE结合并取代RAGE配体S100B。我们建议通过测定这些单域轻链抗体在体外（ELISA）和细胞表面对RAGE的结合亲和力来进一步评价它们。然后，我们将测试这些抗体阻断由不同b淀粉样蛋白寡聚物形式的RAGE参与引发的细胞死亡的能力。

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. PUBLIC HEALTH RELEVANCE: The activation of the Receptor for Advanced Glycation Endproducts by amyloid b peptides plays an important role in Alzheimer's disease (AD). Single domain monoclonal antibodies cross the blood brain barrier and are promising agents to treat AD. We propose to use single domain light chain monoclonal antibodies to block RAGE mediated cell death in AD.