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.

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.

Functional Characteristics and Molecular Mechanism of a New scFv Antibody Against Aß42 Oligomers and Immature Protofibrils.

Amyloid ß peptide (Aß42) is a major determinant of Alzheimer's disease (AD). In this study, we studied a novel single-chain variable fragment (scFv), AS, generated from an antibody library of AD patients, which recognized and bound specifically to medium-size amyloid ß peptide (Aß42) oligomers and immature protofibrils (25-55 kDa) and, more importantly, reduced their level by blocking their formation or inducing their disassembly. Consequently, scFv AS ameliorated or prevented their cytotoxicity and protected SH-SY5Y cells and primary cultured neurons in vitro from their damage in a concentration-dependent manner. Comparison of its cytotoxicity-inhibiting and cytotoxicity-neutralizing activities indicated that scFv AS displayed its protective effect on target cells mainly due to its cytotoxicity-inhibitory activity though it could also neutralize the cytotoxicity. We also found that scFv AS could efficiently cross the in vitro BBB model with a delivery efficiency of over 70% after a 60-min post-administration. The scFv AS was a monovalent antibody with an affinity constant (KD) of 5.5 × 10(-6) M and a binding threshold of 6.25 × 10(-4) µM for Aß42 oligomers. The molecular docking simulations of Aß42 to scFv AS revealed that scFv AS tends to approached Aß42 oligomers and immature protofibrils mainly by their hydrophobic interaction and then drew Aß42 molecule into the gap between VL and VH domains of scFv AS by hydrophilic interaction between scFv AS and the N-terminal region (residues 1-15) of Aß42 and the hydrophobic interactions between scFv AS and the middle region (residues 20-33) of Aß42. The combination of scFv AS with Aß42 was realized likely through an induced-fit process.