Rational Design of Single-Domain Antibodies Targeting the Central Nervous System Neurite Outgrowth Inhibitor Nogo-A

Vaidehi Roy Chowdhury, Alexander Röntgen, Matthew Greenig, Yanira Méndez Gómez, Sebastian P. Spiegel, Magdalena Nowinska, Aubin Ramon, Pietro Sormanni, Andrew Chan, Michele Vendruscolo
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Abstract

The oligodendrocyte-derived membrane protein Nogo-A is one of the most potent inhibitors of neurite growth and regeneration in the adult mammalian central nervous system (CNS). It has been recently shown that the administration of an antibody targeting Nogo-A promoted functional and histopathological recovery in animal models of multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), spinal cord injury (SCI) and stroke. Based on these results, this study aims to develop rationally designed nanobodies to target Nogo-A for diagnostic or therapeutic purposes, as these antibody variants offer therapeutic opportunities for their small size and CNS penetrance. In the first step of our approach, we carried out computational and functional analyses of Nogo-A to identify targetable epitopes. We then rationally designed epitope-specific CDR3 loops and grafted them onto a pre-optimised human VHH scaffold to create a panel of nanobodies. These designed nanobodies were then screened in terms of their thermostability, solubility and binding affinity towards the target antigen to select the best candidate. In this way, we identified a nanobody that binds to an epitope within the ectodomain of human Nogo-A. These results indicate that the rational design method used in this study may facilitate the initial stages of nanobody development for Nogo-A detection and inhibition for CNS therapeutic applications.

Abstract Image

靶向中枢神经突生长抑制剂Nogo-A的单域抗体的合理设计
少突胶质细胞衍生的膜蛋白Nogo-A是成年哺乳动物中枢神经系统(CNS)中神经突生长和再生的最有效抑制剂之一。最近有研究表明,在多发性硬化症(MS)、肌萎缩侧索硬化症(ALS)、脊髓损伤(SCI)和中风的动物模型中,靶向Nogo-A的抗体可促进功能和组织病理学恢复。基于这些结果,本研究旨在开发合理设计的靶向Nogo-A的纳米体,以用于诊断或治疗目的,因为这些抗体变体具有小尺寸和CNS外显性,为治疗提供了机会。在我们方法的第一步,我们对Nogo-A进行了计算和功能分析,以确定可靶向的表位。然后,我们合理设计表位特异性CDR3环,并将其移植到预优化的人类VHH支架上,以创建纳米体面板。然后根据其热稳定性、溶解度和对目标抗原的结合亲和力对这些设计的纳米体进行筛选,以选择最佳候选物。通过这种方法,我们发现了一个纳米体,它与人类Nogo-A的外域内的一个表位结合。这些结果表明,本研究中采用的合理设计方法可以促进纳米体开发的初始阶段,用于检测和抑制中枢神经系统的治疗应用。
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CiteScore
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