| 1. |
- Petersen, Inga, et al.
(författare)
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A charged tail on anti-α-Synuclein antibodies does not enhance their affinity to α-Synuclein fibrils
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Ingår i: PLOS ONE. - 1932-6203.
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 2. |
- Petersen, Inga, et al.
(författare)
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A shorter linker in the bispecific antibody RmAb158-scFv8D3 improves TfR-mediated Blood-Brain Barrier transcytosis in vitro
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Ingår i: Scientific Reports. - 2045-2322.
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- 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.
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| 3. |
- Rofo, Fadi, et al.
(författare)
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Blood-brain barrier penetrating neprilysin degrades monomeric amyloid-beta in a mouse model of Alzheimer’s disease
- 2022
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Ingår i: Alzheimer's Research & Therapy. - : BioMed Central (BMC). - 1758-9193. ; 14:1
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Tidskriftsartikel (refereegranskat)abstract
- BackgroundAggregation of the amyloid-β (Aβ) peptide in the brain is one of the key pathological events in Alzheimer’s disease (AD). Reducing Aβ levels in the brain by enhancing its degradation is one possible strategy to develop new therapies for AD. Neprilysin (NEP) is a membrane-bound metallopeptidase and one of the major Aβ-degrading enzymes. The secreted soluble form of NEP (sNEP) has been previously suggested as a potential protein-therapy degrading Aβ in AD. However, similar to other large molecules, peripherally administered sNEP is unable to reach the brain due to the presence of the blood–brain barrier (BBB).MethodsTo provide transcytosis across the BBB, we recombinantly fused the TfR binding moiety (scFv8D3) to either sNEP or a previously described variant of NEP (muNEP) suggested to have higher degradation efficiency of Aβ compared to other NEP substrates, but not per se to degrade Aβ more efficiently. To provide long blood half-life, an Fc-based antibody fragment (scFc) was added to the designs, forming sNEP-scFc-scFv8D3 and muNEP-scFc-scFv8D3. The ability of the mentioned recombinant proteins to degrade Aβ was first evaluated in vitro using synthetic Aβ peptides followed by sandwich ELISA. For the in vivo studies, a single injection of 125-iodine-labelled sNEP-scFc-scFv8D3 and muNEP-scFc-scFv8D3 was intravenously administered to a tg-ArcSwe mouse model of AD, using scFc-scFv8D3 protein that lacks NEP as a negative control. Different ELISA setups were applied to quantify Aβ concentration of different conformations, both in brain tissues and blood samples.ResultsWhen tested in vitro, sNEP-scFc-scFv8D3 retained sNEP enzymatic activity in degrading Aβ and both constructs efficiently degraded arctic Aβ. When intravenously injected, sNEP-scFc-scFv8D3 demonstrated 20 times higher brain uptake compared to sNEP. Both scFv8D3-fused NEP proteins significantly reduced aggregated Aβ levels in the blood of tg-ArcSwe mice, a transgenic mouse model of AD, following a single intravenous injection. In the brain, monomeric and oligomeric Aβ were significantly reduced. Both scFv8D3-fused NEP proteins displayed a fast clearance from the brain.ConclusionA one-time injection of a BBB-penetrating NEP shows the potential to reduce, the likely most toxic, Aβ oligomers in the brain in addition to monomers. Also, Aβ aggregates in the blood were reduced.
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