A human scFv antibody that targets and neutralizes high molecular weight pathogenic amyloid‐β oligomers

Abstract

Brain accumulation of soluble oligomers of the amyloid-β peptide (AβOs) is increasingly considered a key early event in the pathogenesis of Alzheimer's disease (AD). A variety of AβO species have been identified, both in vitro and in vivo, ranging from dimers to 24mers and higher order oligomers. However, there is no consensus in the literature regarding which AβO species are most germane to AD pathogenesis. Antibodies capable of specifically recognizing defined subpopulations of AβOs would be a valuable asset in the identification, isolation, and characterization of AD-relevant AβO species. Here, we report the characterization of a human single chain antibody fragment (scFv) denoted NUsc1, one of a number of scFvs we have identified that stringently distinguish AβOs from both monomeric and fibrillar Aβ. NUsc1 readily detected AβOs previously bound to dendrites in cultured hippocampal neurons. In addition, NUsc1 blocked AβO binding and reduced AβO-induced neuronal oxidative stress and tau hyperphosphorylation in cultured neurons. NUsc1 further distinguished brain extracts from AD-transgenic mice from wild type (WT) mice, and detected endogenous AβOs in fixed AD brain tissue and AD brain extracts. Biochemical analyses indicated that NUsc1 targets a subpopulation of AβOs with apparent molecular mass greater than 50 kDa. Results indicate that NUsc1 targets a particular AβO species relevant to AD pathogenesis, and suggest that NUsc1 may constitute an effective tool for AD diagnostics and therapeutics.

A novel crosslinking protocol stabilizes amyloid β oligomers capable of inducing Alzheimer's‐associated pathologies

“…Considering NUsc1 targets a subset of NU2-reactive AbOs that are > 50 kDa (Velasco et al 2012;Sebollela et al 2017), this small, but statistically significant, EC50 difference may indicate that DFDNB is stabilizing this NUsc1reactive subset, especially considering the enrichment of AbOs within 50-300 kDa in this preparation. Additional testing is needed to validate this hypothesis.…”

“…Ab antibodies mAbs NU2 and NU4 were obtained by vaccination of mice with synthetic AbOs, as previously (Lambert et al 2007). NUsc1 was prepared free of phage from the Tomlinson I + J human scFv library, as previously (Sebollela et al 2017). NU2, NU4, and NUsc1 will be provided upon reasonable request.…”

“…Next, we investigated if DFDNB crosslinking disrupts the AD-associated pathobiological activity of AbOs. First, we tested for any DFDNB-induced changes in immunoreactivity to various AbO-selective antibodies shown immunoprotective in culture: NUsc1 (Sebollela et al 2017), NU2, and ACU-193. Utilizing dose-response ELISA assays, we find that DFDNB (fivefold) does not significantly alter AbO immunoreactivity ( Figure S4).…”

Free-floating adult human brain-derived slice cultures as a model to study the neuronal impact of Alzheimer’s disease-associated Aβ oligomers

“…Reactivity was revealed using DAB. ELISA was performed as in Sebollela et al (2017). Briefly, 96-well plates were coated overnight with 100 μL monoclonal anti-pan Aβ antibody (6E10, Covance) at 1.0 μg/mL in PBS at 4°C, washed and blocked with 200 μL of 2% NFD milk/PBS.…”

Fluorescence Detection and Dissociation of Amyloid‐β Species for the Treatment of Alzheimer's Disease

“…Schematic representation of the peptide-conjugated Au nanorods used for AD treatment. Klein and coworkers reported NUsc1, [148] one of the human single-chain antibody fragments, capable of differentiating monomeric from fibrillar Aβ, preventing Aβ binding, and reducing neuronal oxidative stress [149] and tau hyperphosphorylation. B) The peptide-conjugated Au nanorod with high NIR absorbance used for AD diagnosis and treatment.…”