Dual Efficacy of a Catalytic Anti-Oligomeric Aß42 scFv Antibody in Clearing Aß42 Aggregates and Reducing Aß Burden in the Brains of Alzheimer's Disease Mice.

One of the primary pathological mechanisms underlying Alzheimer's disease (AD) is the deposition of amyloid ß-protein (Aß42) aggregates in the brain. In this study, a catalytic anti-oligomeric Aß42 scFv antibody, HS72, was identified by screening a human antibody library, its ability to degrade Aß42 aggregates was defined, and its role in the reduction of Aß burden in the AD mouse brain was evaluated. HS72 specifically targeted Aß42 aggregates with an approximately 14-68 kDa range. Based on molecular docking simulations, HS72 likely catalyzed the hydrolytic cleavage of the His13-His14 bond of Aß42 chains in an Aß42 aggregate unit, releasing N/C-terminal fragments and Aß42 monomers. Degradation of Aß42 aggregates by HS72 triggered a considerable disassembly or breakdown of the Aß42 aggregates and greatly reduced their neurotoxicity. Aß deposit/plaque load in the hippocampus of AD mice was reduced by approximately 27% after 7 days (once daily) of intravenous HS72 administration, while brain neural cells were greatly restored and their morphology was drastically improved. The above efficacies of HS72 were all greater than those of HT7, a simple anti-oligomeric Aß42 scFv antibody. Although a catalytic anti-oligomeric Aß42 antibody may have a slightly lower affinity for Aß42 aggregates than a simple anti-oligomeric Aß42 antibody, the former may display a stronger overall efficacy (dual efficacy of induction and catalysis) than the latter (induction alone) in clearing Aß42 aggregates and improving histopathological changes in AD brain. Our findings on the catalytic antibody HS72 indicate the possibility of functional evolution of anti-oligomeric Aß42 antibodies and provide novel insights into the immunotherapy of AD.

Extracellular Amyloid ß-protein (1-42) Oligomers Anchor Brain Cells and Make them inert as an Unconventional Integrin-Coupled Ligand.

This study aimed to investigate the effect of extracellular Aß42 on neural cell migration, and the possible molecular mechanisms. Extracellular Aß42 monomers did not negatively affect the motility of neural cells; however, they could promote cell migration from toxic extracellular Aß42 oligomers. Contrastingly, extracellular Aß42 aggregates, especially Aß42 oligomers, significantly decreased neural cell migration while reducing their survival. Further, their soluble and deposited states showed different effects in causing the neural cells to become inert (incapable of moving). These findings were consistent with that of binding of Aß42 oligomers to the plasma membrane or integrin receptors of the inert cells. By combining the protection of cell migratory capability by anti-oligomeric Aß42 scFv antibody with the information obtained from our docking model of the Aß42 trimer and integrin molecule, our findings suggest that extracellular Aß42 aggregates disrupt the function of integrins mainly through the RHDS motif of Aß42 chain, which eventually causes neural cells to become inert. Thus, we propose an "anchor" opinion, where Aß42 aggregates in the ECM serve as the adverse "anchors" in the brain for anchoring neurons and for making neural cells inert, which causes their dysfunction. The neural cells with damaged motility could be restored or repaired if these anchoring effects of extracellular Aß42 aggregates on the neural cells were severed or reduced, even if the "anchors" themselves were not completely eliminated. Medicines targeting soluble and deposited anchors of Aß42 aggregates could be developed into effective treatments for Alzheimer disease.

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

Extracellular amyloid ß-protein (1-42) (Aß42) aggregates have been recognized as toxic agents for neural cells in vivo and in vitro. The aim of this study was to investigate the cytotoxic effects of extracellular Aß42 aggregates in soluble (or suspended, SAß42) and deposited (or attached, DAß42) forms on cell adhesion/re-adhesion, neurite outgrowth, and intracellular scaffold palladin using the neural cell lines SH-SY5Y and HT22, and to elucidate the potential relevance of these effects. The effect of extracellular Aß42 on neural cell adhesion was directly associated with their neurotrophic or neurotoxic activity, with SAß42 aggregates reducing cell adhesion and associated live cell de-adherence more than DAß42 aggregates, while causing higher mortality. The reduction in cell adhesion due to extracellular Aß42 aggregates was accompanied by the impairment of neurite outgrowth, both in length and number, and similarly, SAß42 aggregates impaired the extension of neurites more severely than DAß42 aggregates. Further, the disparate changes of intracellular palladin induced by SAß42 and DAß42 aggregates, respectively, might underlie their aforementioned effects on target cells. Further, the use of anti-oligomeric Aß42 scFv antibodies revealed that extracellular Aß42 aggregates, especially large DAß42 aggregates, had some independent detrimental effects, including physical barrier effects on neural cell adhesion and neuritogenesis in addition to their neurotoxicity, which might be caused by the rigid C-terminal clusters formed between adjacent Aß42 chains in Aß42 aggregates. Our findings, concerning how scaffold palladin responds to extracellular Aß42 aggregates, and is closely connected with declines in cell adhesion and neurite outgrowth, provide new insights into the cytotoxicity of extracellular Aß42 aggregates in Alzheimer disease.

Conformational Essentials Responsible for Neurotoxicity of Aß42 Aggregates Revealed by Antibodies against Oligomeric Aß42.

Soluble aggregation of amyloid ß-peptide 1-42 (Aß42) and deposition of Aß42 aggregates are the initial pathological hallmarks of Alzheimer's disease (AD). The bipolar nature of Aß42 molecule results in its ability to assemble into distinct oligomers and higher aggregates, which may drive some of the phenotypic heterogeneity observed in AD. Agents targeting Aß42 or its aggregates, such as anti-Aß42 antibodies, can inhibit the aggregation of Aß42 and toxicity of Aß42 aggregates to neural cells to a certain extent. However, the epitope specificity of an antibody affects its binding affinity for different Aß42 species. Different antibodies target different sites on Aß42 and thus elicit different neuroprotective or cytoprotective effects. In the present review, we summarize significant information reflected by anti-Aß42 antibodies in different immunotherapies and propose an overview of the structure (conformation)-toxicity relationship of Aß42 aggregates. This review aimed to provide a reference for the directional design of antibodies against the most pathogenic conformation of Aß42 aggregates.

Novel antibody against oligomeric amyloid-ß: Insight into factors for effectively reducing the aggregation and cytotoxicity of amyloid-ß aggregates.

Amyloid-beta 42 (Aß42) aggregates represent a prominent histopathological feature in Alzheimer's disease (AD); thus, immunotherapy against oligomeric Aß42 aggregates is considered to be a potentially safe and specific therapeutic strategy. In this study, we identified an anti-oligomeric Aß42 aggregate single-chain variable fragment (scFv) antibody, HT6, that is capable of efficiently binding to medium-sized Aß42 aggregates (mainly 18-45 kDa) in vitro with an equilibrium dissociation constant (KD) of 3.0 × 10-6 M, whether they were derived from Aß42 monomer, larger Aß42 oligomers, or even fibrils. This ability allowed scFv HT6 to induce the gradual disassembly of large Aß42 aggregates into small Aß42 oligomers while simultaneously effectively inhibiting the further development of Aß42 aggregates. Moreover, the scFv HT6-targeted conformational region on Aß42 aggregates was found to be more local and relatively close to the N-terminus of Aß42; thus, scFv HT6 significantly delayed or even prevented the aggregation of Aß42 protofibrils, while significantly reducing the cytotoxicity of Aß42 oligomers. Overall, this study demonstrate that even though the decrease in the cytotoxicity of Aß42 aggregates might be closely related to the reduction in Aß42 aggregates and vice versa, the reduction in Aß42 aggregates might not necessarily be accompanied by or followed by the reduction or even elimination of the cytotoxicity of Aß42 aggregates. This insight enriches the diversity of anti-oligomeric Aß42 antibodies, further providing a new understanding into the relationship between their binding pattern to Aß42 aggregates and the efficacy against their formation, offering a therapeutic strategy to delay the progression of AD.

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

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 Αß42 can suppress the progression of AD, especially on its early stage. Here, we obtained and characterized a novel anti-oligomeric Aß42 aggregate scFv antibody, HT7, which could induce the significant disaggregation of Aß42 aggregates through the release of stable and non-cytotoxic hexameric complexes that were composed of three scFv HT7s and one Aß42 trimer, the latter being found to serve as the assembled subunit within larger Aß42 aggregates in addition to existing freely between the cells. The docking model of the scFv HT7-Aß42 complex revealed that only the N-terminal peptide of the Aß42 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 Aß42 molecules maintained the stability of the Aß42 trimers or the Aß42 trimer subunits. The saturation of Aß42 trimer subunits by scFv HT7 and the subsequent dissociation of the scFv HT7-saturated Aß42 trimer subunits from larger Aß42 aggregates constituted the primary mechanisms underlying the high efficacy of scFv HT7. Our findings revealed that it was not sufficient for an anti-oligomeric Aß42 antibody to exhibit high specificity and high affinity to the oligomeric Aß42 aggregates in order to promote Aß42 aggregate clearance and neutralize their cytotoxic effects. Here, for the first time, we proposed a "post-saturation dissociation" mechanism of Aß42 oligomeric subunits for effective anti-Aß42 antibodies.