Logo: to the web site of Uppsala University

Soluble aggregates are reported to be the most neurotoxic species of alpha-Synuclein (alpha Syn) in Parkinson's disease (PD) and hence are a promising target for diagnosis and treatment of PD. However, the predominantly intracellular location of alpha Syn limits its accessibility, especially for antibody-based molecules and prompts the need for exceptionally strong soluble alpha Syn aggregate binders to enhance their sensitivity and efficacy for targeting the extracellular alpha Syn pool. In this study, we have created the multivalent antibodies TetraSynO2 and HexaSynO2, derived from the alpha Syn oligomer-specific antibody SynO2, to increase avidity binding to soluble alpha Syn aggregate species through more binding sites in close proximity. The multivalency was achieved through recombinant fusion of single-chain variable fragments of SynO2 to the antibodies' original N-termini. Our ELISA results indicated a 20-fold increased binding strength of the multivalent formats to alpha Syn aggregates, while binding to alpha Syn monomers and unspecific binding to amyloid beta protofibrils remained low. Kinetic analysis using LigandTracer revealed that only 80% of SynO2 bound bivalently to soluble aSyn aggregates, whereas the proportion of TetraSynO2 and HexaSynO2 binding bi- or multivalently to soluble alpha Syn aggregates was increased to similar to 95% and 100%, respectively. The overall improved binding strength of TetraSynO2 and HexaSynO2 implies great potential for immunotherapeutic and diagnostic applications with targets of limited accessibility, like extra-cellular alpha Syn aggregates. The ability of the multivalent antibodies to bind a wider range of alpha Syn aggregate species, which are not targetable by conventional bivalent antibodies, thus could allow for an earlier and more effective intervention in the progression of PD.

 Transferrin Receptor (TfR)-mediated transcytosis across the blood-brain barrier (BBB) enables the uptake of bispecific therapeutic antibodies into the brain. At therapeutically relevant concentrations, bivalent binding to TfR appears to reduce the transcytosis efficiency by receptor crosslinking. In this study, we aimed to improve BBB transcytosis of symmetric antibodies through minimizing their ability to cause TfR crosslinking. We created variants of the previously published RmAb158-scFv8D3, where the linker length between RmAb158 and the mTfR-targeting scFv8D3 was adjusted. We investigated the effect of the linker length on the antibodies’ binding kinetics to mTfR using ELISA and LigandTracer assays, and their ability to transcytose across BBB endothelial cells (In-Cell BBB-Trans assay). We show that even a direct fusion without a linker does not alter the antibodies’ apparent affinities to mTfR indicating their valency is unlikely affected by the linker length. However, the shortest linker variants demonstrated BBB transcytosis levels comparable to that of the monovalent control at a high antibody concentration and showed an almost two-fold higher level of BBB transcytosis compared to the longer linker variants at the high concentration. Our new RmAb158-scFv8D3 short-linker variants are examples of symmetric, therapeutic antibodies with improved TfR-binding characteristics to facilitate more efficient brain uptake. We hypothesize that bivalent binding to TfR as such does not negatively affect BBB transcytosis in vitro, but a very short distance between TfR-targeting domains lowers the probability of receptor crosslinking. This study provides valuable insights into antibody-TfR interaction kinetics, contributing to future development of TfR-targeting antibody-based treatments for brain diseases.

The aggregation of α-Synuclein (αSyn) is strongly linked to neuronal death in Parkinson’s disease and other synucleinopathies. The spreading of aggregated αSyn between neurons is at least partly dependent on electrostatic interactions between positively charged stretches on αSyn fibrils and the negatively charged heparan sulphate proteoglycans on the cell surface. To date there is still no therapeutic option available that could halt the progression of Parkinson’s disease and one of the major limitations is likely the relatively low proportion of αSyn aggregates accessible to drugs in the extracellular space. Here, we investigated whether a negatively charged peptide tail fused to the αSyn aggregate-specific antibodies SynO2 and 9E4 could enhance the antibodies’ avidity to αSyn aggregates in order to improve their potential therapeutic effect through inhibiting cell-to-cell spreading and enhancing the clearance of extracellular aggregates. We performed ELISAs to test the avidity to αSyn aggregates of both monovalent and bivalent antibody formats with and without the peptide tail. Our results show that the addition of the negatively charged peptide tail decreased the binding strength of both antibodies to αSyn aggregates at physiological salt conditions, which can likely be explained by intermolecular repulsions between the tail and the negatively charged C-terminus of αSyn. Additionally, the tail might interact with the paratopes of the SynO2 antibody abolishing its binding to αSyn aggregates. Conclusively, our peptide tail did not fulfil the required characteristics to improve the antibodies’ binding to αSyn aggregates. Fine-tuning the design of the peptide tail to avoid its interaction with the antibodies’ CDR and to better mimic relevant characteristics of heparan sulphates for αSyn aggregate binding may help overcome the limitations observed in this study.