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The aggregation of alpha-synuclein (αSyn) into oligomers and fibrils is central to the disease progression of Parkinson’s disease and related pathologies, where αSyn aggregates spread between neurons and cause neurodegeneration. To this date, there is no treatment available that could interfere with the aggregation of αSyn to potentially stop the disease progression. Among the major limitations in the development of therapeutics against αSyn aggregation are the low extracellular concentration of αSyn, the low selectivity of therapeutics for the pathologically relevant αSyn species, and the lacking detailed knowledge about the actual pathological αSyn species.

In this thesis, different engineered antibodies and αSyn mutants were investigated with the aim to identify better strategies of antibody-based treatment of αSyn aggregation.

In Paper I, we engineered multivalent antibodies based on the αSyn aggregate-specific antibody SynO2 to enhance the antibody’s binding strength to a wide range of soluble αSyn aggregates. We could show that the higher valency increased the binding strength to αSyn aggregates up to 20-fold.

In Paper II, we aimed to improve the design of the antibody RmAb158-scFv8D3 to enhance its TfR-mediated brain uptake. By drastically reducing the linker length between the therapeutic antibody and its TfR-targeting scFv8D3, we showed a two-fold enhanced transcytosis across an in vitro BBB model.

In Paper III, we fused a negatively charged peptide to the αSyn aggregate-specific antibodies SynO2 and 9E4 to test whether those fusion antibodies had the potential to bind with higher avidity to αSyn aggregates. Our results showed lower binding strengths compared with the parental antibodies.

In Paper IV, we designed αSyn mutants with a stabilized beta-hairpin conformation to produce stable, small αSyn oligomers closely resembling native, pathological αSyn oligomers. We showed that two of the mutants formed exclusively pentameric and hexameric oligomers under conditions that promoted fibrillation of wild-type αSyn.

In conclusion, this thesis shows that increasing the valency of an antibody is a possible strategy to enhance its binding strength to αSyn aggregates. However, to effectively target pathologically relevant αSyn species, a more selective targeting approach may be required, possibly through a conformational epitope exclusive to αSyn oligomers.