Rational Design of Nanobodies Targeting LINGO‐1

ABSTRACT The leucine‐rich repeat and immunoglobulin‐like domain‐containing Nogo receptor‐interacting protein 1 (LINGO‐1) is a transmembrane protein expressed in neurons and oligodendrocytes. Since LINGO‐1 is involved in the regulation of axonal outgrowth and neuronal survival as well as oligodendrocyte differentiation and axon myelination, it has been investigated as a potential drug target for demyelinating diseases such as multiple sclerosis. In this study, we implement a pipeline to generate and test single‐domain antibodies (nanobodies) targeting the functionally relevant immunoglobulin‐like (Ig‐like) domain of LINGO‐1 using an in‐silico design method. First, we designed complementarity‐determining region 3 (CDR3) loops and grafted them onto a suitable nanobody scaffold. We then recombinantly expressed the designed nanobodies in bacteria followed by purification using liquid chromatography. Next, after characterising the purity and structural integrity of the produced nanobodies, we validated their binding to LINGO‐1 using an enzyme‐linked immunosorbent assay (ELISA). Overall, the strategy that we employed in this study provides a framework for the generation of nanobodies against challenging targets based on in‐silico design methods followed by testing to identify suitable candidates.