Targeting RAGE/Abeta interaction with single chain monoclonal antibodies

描述（由申请方提供）：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寡聚体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Ab毒性最大的形式。我们最近发现，抗体寡聚体相互作用优先与V域的的ESTA，而其他形式的抗体（原纤维，聚集体）与其他领域的受体相互作用。我们发现，通过Ab寡聚体的接合导致神经元凋亡。阻断Ab寡聚体与Alzheimer的相互作用可能会导致阿尔茨海默病患者神经元死亡的显着减少。我们建议使用单链单克隆抗体（轻链）来阻断抗体/抗体的相互作用。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前景的试剂。我们最近已经建立了一个Fab段抗体噬菌体展示库靶向的ESTA。十个选择的与Fab反应的Fab呈现截短的且因此无功能的重链。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，并将其纯化至均一。结果表明，重组轻链在体外可与人IgG 1结合并置换IgG 1配体S100 B。我们建议在这里进一步评估这些单域轻链抗体，通过测量它们的结合亲和力，在体外（ELISA）和细胞表面上的抗体。然后，我们将测试这些抗体阻断由不同的B淀粉样蛋白寡聚体形式与β-淀粉样蛋白的结合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.