Blood-brain barrier penetrating neprilysin degrades monomeric amyloid-beta in a mouse model of Alzheimer's disease

Background: Aggregation of the amyloid-beta (A beta) peptide in the brain is one of the key pathological events in Alzheimer's disease (AD). Reducing A beta 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 A beta-degrading enzymes. The secreted soluble form of NEP (sNEP) has been previously suggested as a potential protein-therapy degrading A beta 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). Methods: To 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 A beta compared to other NEP substrates, but not per se to degrade A beta 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 A beta was first evaluated in vitro using synthetic A beta 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 A beta concentration of different conformations, both in brain tissues and blood samples. Results: When tested in vitro, sNEP-scFc-scFv8D3 retained sNEP enzymatic activity in degrading A beta and both constructs efficiently degraded arctic A beta. When intravenously injected, sNEP-scFc-scFv8D3 demonstrated 20 times higher brain uptake compared to sNEP. Both scFv8D3-fused NEP proteins significantly reduced aggregated A beta 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 A beta were significantly reduced. Both scFv8D3-fused NEP proteins displayed a fast clearance from the brain. Conclusion: A one-time injection of a BBB-penetrating NEP shows the potential to reduce, the likely most toxic, A beta oligomers in the brain in addition to monomers. Also, A beta aggregates in the blood were reduced.