Single domain antibodies for diagnosis and treatment of synucleinopathies

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Abstract Immunotherapies targeting aggregates of amyloid-β (Aβ), tau and α-synuclein (αsyn) are the most common experimental approach for diseases characterized by such depositions. Most of these potential therapies are whole antibodies. Antibody fragments have certain advantages over antibodies but their diagnostic and therapeutic potential has not been well explored. Five anti-αsyn IgG antibodies and one αsyn vaccine are in Phase 1-2 clinical trials, and one Aβ antibody recently showed clinical benefit in a Phase 3 trial, which strongly supports this therapeutic approach. Regarding antibody fragments, several anti-αsyn single chain variable fragments (scFvs) are effective in culture and in vivo, and two anti-αsyn single domain antibodies (sdAbs) have been characterized in a binding assay but their therapeutic efficacy or diagnostic imaging potential have not been reported. SdAbs have several advantages over scFvs, including higher affinity, binding to cryptic epitopes and simpler engineering. They are also more suitable for gene therapy because of uncomplicated expression and predictable protein folding, given their single domain, resulting in high solubility. We have developed 58 anti-αsyn sdAb clones with unique binding regions from a screen of phage display libraries generated from B-cells of a llama immunized with αsyn. Three aims are proposed to clarify their potential to detect and clear αsyn in vivo. Aim 1 will determine the in vivo imaging potential of sdAbs targeting αsyn. Aim 2 will clarify αsyn clearance potential and mechanism of action of sdAbs targeting αsyn, and Aim 3 will explore the feasibility of sdAb gene therapy targeting αsyn. Preliminary findings indicate that the sdAbs: 1) recognize various forms of αsyn with high affinity; 2) clear αsyn and prevent its toxicity in culture models; 3) clear αsyn from brain interstitial fluid in αsyn mice; 4) bind to αsyn in the brain of αsyn mice after intravenous injection with in vivo brain signal that correlates well with αsyn brain burden; 5) clear αsyn from the brain after intravenous injection of sdAb with improved clearance by its PROTAC derivative, and; 6) clear αsyn from the brain after intravenous sdAb gene therapy. It is hypothesized that the small size of the sdAbs will provide diagnostic and therapeutic benefits over whole antibodies, primarily because of greater access into the brain and to the target, proper folding for gene therapy, and to some extent due to their binding to novel epitopes that the larger antibodies cannot access. Overall, the proposed studies are likely to aid in clarifying αsyn pathogenesis, decipher the mechanism of action of the sdAbs, and identify promising diagnostic and therapeutic sdAbs for clinical trials on synucleinopathies such as Lewy Body Dementia, Parkinson's disease, Alzheimer's disease with Lewy Bodies, and Multiple System Atrophy.