Development of an antibody reformatting strategy for use in targeted protein degradation therapies for neurodegenerative diseases

Neurodegenerative diseases (NDDs) such as Parkinson’s disease (PD) and amyotrophic lateral sclerosis (ALS) are a leading cause of global mortality and place a great financial and emotional burden on healthcare systems and support networks. Misfolded insoluble aggregates and toxic soluble proteins are central hallmarks of these NDDs. α-Synuclein aggregation drives Parkinson’s disease pathology and is a suitable target for selective protein clearance. Biological proteolysis targeting chimeras (bioPROTACs) aim to eliminate disease-causing intracellular proteins using host cell ubiquitination and degradation pathways. Here, I describe a bioPROTAC comprising the E3 ubiquitin ligase domain of CHIP (carboxy terminus of Hsc70-interacting protein) fused to NbSyn87, a nanobody specific for α-synuclein. Successful target degradation was achieved using the CHIP-NbSyn87 bioPROTAC. In contrast, CHIP-based bioPROTACs targeting SOD1, a ubiquitously expressed protein prone to misfold and aggregate in ALS, failed to degrade its target. This work highlighted key parameters for consideration during BioPROTAC design including target half-life and solubility, recognition domain binding affinity, molecular chaperone activity, and interdomain linker optimisation. A strategy for rational repurposing of conformation-specific antibodies into soluble, functional intrabodies was also developed. Candidate antibodies were reformatted into scFvs and their solubility and specificity tested. A panel of misfolding-specific SOD1 intrabodies and conformation-specific α-synuclein intrabodies are described. Almost any antibody can be reformatted as a highly soluble (>70 %) and intracellularly stable intrabody using the described approach, based on the discovery of a strong negative correlation between net charge and intrabody solubility in cell models.