Novel engineered nanobodies specific for N-terminal region of alpha-synuclein recognize Lewy-body pathology and inhibit in-vitro seeded aggregation and toxicity

Nanobodies (Nbs), the single-domain antigen-binding fragments of dromedary heavy-chain antibodies (HCAb), are excellent candidates as therapeutic and diagnostic tools in synucleinopathies because of their small size, solubility and stability. Here, we constructed an immune nanobody library specific to the monomeric form of alpha-synuclein (alpha-syn). Phage display screening of the library allowed the identification of a nanobody, Nb alpha-syn01, specific for alpha-syn. Unlike previously developed nanobodies, Nb alpha-syn01 recognized the N-terminal region which is critical for in vitro and in vivo aggregation and contains many point mutations involved in early PD cases. The affinity of the monovalent Nb alpha-syn01 and the engineered bivalent format BivNb alpha-syn01 measured by isothermal titration calorimetry revealed unexpected results where Nb alpha-syn01 and its bivalent format recognized preferentially alpha-syn fibrils compared to the monomeric form. Nb alpha-syn01 and BivNb alpha-syn01 were also able to inhibit alpha-syn-seeded aggregation in vitro and reduced alpha-syn-seeded aggregation and toxicity in cells showing their potential to reduce alpha-syn pathology. Moreover, both nanobody formats were able to recognize Lewy-body pathology in human post-mortem brain tissue from PD and DLB cases. Additionally, we present evidence through structural docking that Nb alpha-syn01 binds the N-terminal region of the alpha-syn aggregated form. Overall, these results highlight the potential of Nb alpha-syn01 and BivNb alpha-syn01 in developing into a diagnostic or a therapeutic tool for PD and related disorders.