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

<jats:title>ABSTRACT</jats:title><jats:p>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 V<jats:sub>H</jats:sub>H 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.</jats:p>