Characterization of Novel Amyloid-β Peptide (Aβ) Binding Ligands

AD is a neurodegenerative disorder leading to brain atrophy and severe dementia. Thirtyfive million people are affected worldwide and the number is rapidly increasing. The cause of AD is the Aβ peptide, which aggregates in the brain of affected people via an unknown, poorly understood mechanism. Such self-association events give rise to a multitude of different species. The most toxic species are believed to be the soluble oligomers. Currently, there is neither a method available to diagnose Alzheimer’s disease $\textit{ante mortem}$ nor is there a causal therapeutic available. The main aim of this work was to characterize Aβ ligands that can potentially be used as a therapeutic or a tracer for diagnosis in AD. A single-chain variable fragment (scFv), scFv-IC16 constructed from an anti-Aβ antibody and two variants derived from scFv-IC16 were characterized and compared with respect totheir binding affinities to different N-terminal Aβ fragments and their binding specificities to different Aβ conformers. The binding epitope of scFv-IC16 and the variants was shown to be Aβ2-8. The amino acid residue Ala2 of the Aβ peptides was shown to play a crucial role in the binding of scFv-IC16 to Aβ. Furthermore, the influence of scFv-IC16 on the fibrilization process of Aβ was monitored using a ThT assay. scFv-IC16 inhibited the formation of ThT positive fibrils in a concentration dependent manner. Three novel D-peptides were characterized according to their binding to different Aβ species. In ELISA experiments these peptides were shown to bind preferentially to Aβfibrils, but also detected Aβ oligomers moderately and Aβ monomers weakly. ThT assays of the new peptides showed a strong concentration dependent inhibition of the formation of ThT positive fibrils. DLS measurements showed the formation of larger particles if Aβ was incubated in the presence of the novel D-peptides. Furthermore, the D-peptides enhanced the viability of cells if they were co-incubated with Aβ

Characterization of a Single-Chain Variable Fragment Recognizing a Linear Epitope of Aβ: A Biotechnical Tool for Studies on Alzheimer's Disease?

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder with devastating effects. Currently, therapeutic options are limited to symptomatic treatment. For more than a decade, research focused on immunotherapy for the causal treatment of AD. However, clinical trials with active immunization using Aβ encountered severe complications, for example meningoencephalitis. Consequently, attention focused on passive immunization using antibodies. As an alternative to large immunoglobulins (IgGs), Aβ binding single-chain variable fragments (scFvs) were used for diagnostic and therapeutic research approaches. scFvs can be expressed in E. coli and may provide improved pharmacokinetic properties like increased blood-brain barrier permeability or reduced side-effects in vivo. In this study, we constructed an scFv from an Aβ binding IgG, designated IC16, which binds the N-terminal region of Aβ (Aβ(1-8)). scFv-IC16 was expressed in E. coli, purified and characterized with respect to its interaction with different Aβ species and its influence on Aβ fibril formation. We were able to show that scFv-IC16 strongly influenced the aggregation behavior of Aβ and could be applied as an Aβ detection probe for plaque staining in the brains of transgenic AD model mice. The results indicate potential for therapy and diagnosis of AD

Characterization of a single-chain variable fragment recognizing a linear epitope of aβ: a biotechnical tool for studies on Alzheimer's disease?

Alzheimer's disease (AD) is a progressive neurodegenerative disorder with devastating effects. Currently, therapeutic options are limited to symptomatic treatment. For more than a decade, research focused on immunotherapy for the causal treatment of AD. However, clinical trials with active immunization using Aβ encountered severe complications, for example meningoencephalitis. Consequently, attention focused on passive immunization using antibodies. As an alternative to large immunoglobulins (IgGs), Aβ binding single-chain variable fragments (scFvs) were used for diagnostic and therapeutic research approaches. scFvs can be expressed in E. coli and may provide improved pharmacokinetic properties like increased blood-brain barrier permeability or reduced side-effects in vivo. In this study, we constructed an scFv from an Aβ binding IgG, designated IC16, which binds the N-terminal region of Aβ (Aβ(1-8)). scFv-IC16 was expressed in E. coli, purified and characterized with respect to its interaction with different Aβ species and its influence on Aβ fibril formation. We were able to show that scFv-IC16 strongly influenced the aggregation behavior of Aβ and could be applied as an Aβ detection probe for plaque staining in the brains of transgenic AD model mice. The results indicate potential for therapy and diagnosis of AD

Rate constants and dissociation constants of the interactions of scFv-IC16 and IC16 with N-terminal Aβ fragments.

<p>Data were fitted globally. scFv-IC16 data were fitted according to the Langmuir 1∶1 binding model, whereas IC16 data were fitted using a bivalent fit. The association rate constants (k_on), the dissociation rate constants (k_off) and the equilibrium dissociation constants (K_D) are given below. Both, scFv-IC16 and IC16, did not show any detectable binding towards Aβ(8-15). However, they showed binding to all N-terminal peptides, except scFv-IC16 did not bind to Aβ(pE3-8). The lowest K_D value was observed for the interaction of scFv-IC16 and IC16 to Aβ(2-8).</p

In vitro staining of brain sliced from transgenic 2576 AD mice using scFv-IC16, 6E10-Aβ-antibody and DAPI.

<p>(A). Pretreated deep frozen horizontal brain cryosections (20 µm thickness) from 9 months old Tg2576 mice were fixed in 4% paraformaldehyde, treated with 70%formic acid and either incubated with scFv-IC16 in a concentration of 400 nM or 6E10 in a concentration of 33 nM. As scFv-IC16 is fused with a His tag, the respective sections were incubated with Penta-His Mouse antibody. Detection was performed using goat anti mouse-Alexa488 using a fluorescent microscope (Leica LMD6000. Excitation Range: UV; blue; green. Excitation Filter: Bp 420/30; Bp 495/15; Bp 570/20) with camera (Leica DFC 310 FX). Images were processed with LAS software (Leica Application Suite, V.4.0.0) and ImageJ (1.45 s). Scale bar: 50 µm. B: In vitro staining of brain sliced from transgenic 2576 AD mice using scFv-IC16 and a respective wildtype control mouse. Scale bar:100 µm. C: Co-staining of brain sliced from transgenic 2576 AD mice using scFv-IC16 and anti Aβ(1-42) 1-11-3. As secondary antibodies, a mix of goat anti mouse-Alexa488 and goat anti rabbit-Alexa-568 was used. Scale bar: 50 µm.</p

SDS-PAGE analysis of the purification of scFv-IC16 via Ni-NTA and Aβ1-16 GB1 NHS sepharose affinity chromatography.

<p>1 µl of the lysate and 15 µl of the purified fractions were applied to 16% polyacrylamide gels <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059820#pone.0059820-Laemmli1" target="_blank">[56]</a>. Lane 1: cell lysate, lane 2: eluate after affinity chromatography via Ni NTA–Agarose, lane 3: eluate after Aβ1-16-GB1-NHS-Sepharose affinity chromatography. Target protein is more than 99% pure after final purification step.</p

Inhibition of ThT positive Aβ fibril formation in presence of different scFv-IC16 concentrations.

<p>(A). ScFv-IC16 was added in concentrations of: 7.5, 3.75, 1.5 and 0 µM (positive control) to 7.5 µM Aβ(1-42) samples. Data were recorded every 30 min during 22 hours of incubation at room temperature. Depicted is the absolute fluorescence value after 10 h of incubation. Upon addition of ThT, fluorescence was measured at 490 nm in relative units (mean +/− standard deviations of results, each measurement was repeated five times). All values are corrected by background fluorescence of ThT in PBS. ScFv-IC16 only control (7.5 µM) does not show any formation of ThT positive fibrils. A statistical significant difference between the fluorescence values of Aβ only and Aβ-scFv-IC16 co incubations was calculated by student’s T-test, as indicated (**: p<0.01; ***: p<0.001). Analysis of an ELISA experiment to quantify the binding specificity of scFv-IC16 to different Aβ1-42 conformers and Aβ1-10 peptides (B). 250 ng Aβ1-42 (100% N-biotinylated monomers, 100% C-terminally biotinylated monomers, 10% N-biotinylated oligomers and fibrils) and 60 ng Aβ peptides 1–10 (100% C-terminally biotinylated Aβ1-10, 100% N-terminally biotinylated Aβ1-10) were immobilized on streptavidin coated 96 well plates. 6E10 was used to monitor the immobilized amount of Aβ conformers. Similar absorption values of 6E10 to the different Aβ conformers except for 100% N-terminally biotinylated monomers, indicate similar molar amounts of immobilized Aβ regarding monomeric Aβ for monomers, oligomers and fibrils. Background absorption was subtracted from all samples. A highly significant difference in the relative fluorescence value between N-biotinylated monomers and C-biotinylated monomers, as well as between C-bio Aβ1-10 and N-bio Aβ1-10 was calculated by Student’s t-test (***: p<0.001). N-bio = N-terminally biotinylated; C-bio = C-terminally biotinylated. Western Blot of Aβ monomers and low-n oligomers immunoprecipitated by scFv-IC16 from conditioned medium (CM) of CHO or 7PA2 cells (C). The latter were grown either in medium with or without FCS. Monomeric Aβ is stabilized by the presence of FCS, whereas low-n oligomeric Aβ is eriched in CM-FCS. No Aβ was precititated from the supernatants of CHO cells. Monomeric Aβ (CM 7Pa2+ FCS) and low-n oligomeric Aβ (CM 7PA2− FCS) were equally bound by scFv-IC16. Detection antibody: 4G8.</p

Comparison of the CDR sequences of the light chains of antibody fragments.

<p>Comparison of the CDR sequences of the light chains of antibody fragments.</p

Comparison of the CDR sequences of the heavy chains of different antibody fragments.

<p>Comparison of the CDR sequences of the heavy chains of different antibody fragments.</p

Sequence alignment of the V_H (a) and V_L (b) region of IC16 and PFA1.

<p>Residues belonging to the CDRs are marked in red. Labeling was done according to the sequence of the heavy and light chain used for the scFv-IC16 construct, each starting with residue 1. Residues that interact with Aβ(1-8) are marked in bold and those residues that form hydrogen bonds or salt-bridges with Aβ(1-8) are additionally underlined. D1 of the IC16 light chain is marked in bold and blue, because it forms repulsive interactions with D1 of Aβ.</p