The Mode of Action of an Anti-Oligomeric Amyloid β-Protein Antibody Affects its Protective Efficacy

Abstract

The process of developing antibody drugs for Alzheimer's disease therapy has been both long and difficult; however, recent advances suggest that antibodies against neurotoxic Αβ42 can suppress the progression of AD, especially on its early stage. Here, we obtained and characterized a novel anti-oligomeric Aβ42 aggregate scFv antibody, HT7, which could induce the significant disaggregation of Aβ42 aggregates through the release of stable and non-cytotoxic hexameric complexes that were composed of three scFv HT7s and one Aβ42 trimer, the latter being found to serve as the assembled subunit within larger Aβ42 aggregates in addition to existing freely between the cells. The docking model of the scFv HT7-Aβ42 complex revealed that only the N-terminal peptide of the Aβ42 molecule was bound into the groove between the VH and VL domains of scFv HT7. Thus, it was suggested that the hydrophobic interaction between the C-terminal peptides of Aβ42 molecules maintained the stability of the Aβ42 trimers or the Aβ42 trimer subunits. The saturation of Aβ42 trimer subunits by scFv HT7 and the subsequent dissociation of the scFv HT7-saturated Aβ42 trimer subunits from larger Aβ42 aggregates constituted the primary mechanisms underlying the high efficacy of scFv HT7. Our findings revealed that it was not sufficient for an anti-oligomeric Aβ42 antibody to exhibit high specificity and high affinity to the oligomeric Aβ42 aggregates in order to promote Aβ42 aggregate clearance and neutralize their cytotoxic effects. Here, for the first time, we proposed a "post-saturation dissociation" mechanism of Aβ42 oligomeric subunits for effective anti-Aβ42 antibodies.

Different Extracellular β-Amyloid (1-42) Aggregates Differentially Impair Neural Cell Adhesion and Neurite Outgrowth through Differential Induction of Scaffold Palladin

“…In the group using Aβ42-containing DMEM medium, SH-SY5Y or HT22 cells were seeded in 96-well plates at a density of approximately 8 × 10 4 cells per well and cultured with 10% DMEM medium at 37 °C with 5% CO 2 from 4–6 h to enable attachment to the bottom of wells. Subsequently, the culture medium was aspirated and fresh 10% DMEM medium containing SAβ42 or its aggregates (final concentration: 0.02–2.0 μM) with or without anti-oligomeric Aβ42 single-chain variable fragment (scFv) HT7 (or HT6) antibody (final concentration: 2.0 μM) [ 26 , 27 ] was added to each well. Cells were cultured at 37 °C with 5% CO 2 for 12 h. The culture medium was collected, and the adhering cells were washed twice using 10 mM PBS (pH 7.2–7.4) and the washes were collected.…”

Conformational Essentials Responsible for Neurotoxicity of Aβ42 Aggregates Revealed by Antibodies against Oligomeric Aβ42

“…These anti-oligomeric Aβ scFvs display high selectivity for toxic Aβ42O species, neutralize their neurotoxicity in vivo or in vitro and reduce the toxicity of preformed oligomeric Aβ42 toward target cells. In addition, some anti-oligomeric Aβ scFvs have been reported to have relatively significant permeability in in vitro blood−brain barrier models [ 71 , 72 , 73 , 74 ].…”

“…A comparative analysis of the similar binding models of our three scFvs (MO6, HT6, and HT7) to Aβ42O [ 72 , 73 , 74 ] revealed that the closer the scFv-bound site is to the N-terminus of Aβ42O ( Figure 4 ), the larger the size of the corresponding scFv-bound Aβ42O target ( Table 1 ) and vice versa. The relationship between the site on the Aβ42O unit targeted by a scFv antibody and the size of the Aβ42O target has implications for antibody function.…”

“…For example, a scFv antibody with a binding site close to the N-terminus of Aβ42 would presumably have a relatively high ability to neutralize the toxicity of Aβ42O, based on the results that the N-terminally integrated structures of Aβ42O appear to directly result in the neurotoxicity of an Aβ42O unit. This has been demonstrated by the efficacies of the three scFvs (MO6, HT6, and HT7) in inhibiting Aβ42O toxicity by 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide (MTT) assay [ 72 , 73 , 74 ]. The potency of these three scFv antibodies correlate with an aggregate set of in vitro activities, such as recognizing Aβ42 oligomers and fibrils in a consistent manner.…”

“… Overview of the conformations of three scFv antibodies (MO6, HT6, and HT7) with certain homology (linkers are not shown) [ 72 , 73 , 74 ].VH or VL: heavy or light chain variable domain; C or N: C- or N-terminus. …”