An anti-Aβ (amyloid β) single-chain variable fragment prevents amyloid fibril formation and cytotoxicity by withdrawing Aβ oligomers from the amyloid pathway

Abstract

Aβ (amyloid β) immunotherapy has been revealed as a possible tool in Alzheimer's disease treatment. In contrast with complete antibodies, the administration of scFvs (single-chain variable fragments) produces neither meningoencephalitis nor cerebral haemorrhage. In the present study, the recombinant expression of scFv-h3D6, a derivative of an antibody specific for Aβ oligomers, is presented, as well as the subsequent proof of its capability to recover the toxicity induced by the Aβ1-42 peptide in the SH-SY5Y neuroblastoma cell line. To gain insight into the conformational changes underlying the prevention of Aβ toxicity by this antibody fragment, the conformational landscape of scFv-h3D6 upon temperature perturbation is also described. Heating the native state does not lead to any extent of unfolding, but rather directly to a β-rich intermediate state which initiates an aggregation pathway. This aggregation pathway is not an amyloid fibril pathway, as is that followed by the Aβ peptide, but rather a worm-like fibril pathway which, noticeably, turns out to be non-toxic. On the other hand, this pathway is thermodynamically and kinetically favoured when the scFv-h3D6 and Aβ1-42 oligomers form a complex in native conditions, explaining how the scFv-h3D6 withdraws Aβ1-42 oligomers from the amyloid pathway. To our knowledge, this is the first description of a conformational mechanism by which a scFv prevents Aβ-oligomer cytotoxicity.

Elongation of the C-terminal domain of an anti-amyloid β single-chain variable fragment increases its thermodynamic stability and decreases its aggregation tendency

“…In agreement with previous results, 13 the FTIR spectrum of scFv-h3D6 decomposes in 64% native β-sheet component, nm. The plateau at ~351 nm spans the 4-6 M urea region and is indicative of the occurrence of an intermediate state.…”

“…1), but a second ellipticity minimum at 230 nm and a positive shoulder at 237 nm were found. 13 These anomalies are contributions from the aromatic or cystinyl side-chains within the far-UV. 18 The minimum at 230 nm was also reported for an IgG1-Fc 19 and described for some V L domains, 20,21 the latter attributed to the interaction of the aromatic residues with the conserved Trp35.…”

“…7 Therefore, the use of single-chain variable fragments (scFv) has been proposed as a hopeful therapeutic strategy. [8][9][10][11][12][13] We previously described the recombinant expression and aggregation pathway of scFv-h3D6, a single chain variable fragment derived from mAb-h3D6, [14][15][16] which inhibits amyloid fibril formation and cytotoxicity of the Aβ 1-42 -peptide. 13 Addition of scFv-h3D6 completely precluded the toxic effect of Aβ-oligomers 22% loops/turns component, 11% β-turns components and 3% of a low-frequency component (see later).…”

Loss of deep cerebellar nuclei neurons in the 3xTg-AD mice and protection by an anti-amyloid β antibody fragment

“…16 Additionally, we recently demonstrated the benefits of scFv-h3D6 in five month-old female 3xTg-AD animals, which corresponds to early stages of the disease. 15 The 3xTg-AD mouse brain develops molecular and histological alterations characteristic of AD, following a regional and temporal involvement homologous to humans.…”

“…14 We recently used this model to show that immunotherapy with an anti-Aβ antibody fragment is a potential tool for the treatment of AD. 15 Specifically, we constructed a single-chain variable fragment (scFv), scFv-h3D6, 16 from the humanized monoclonal antibody AAB-001 (mAb-h3D6, bapineuzumab). 17 An advantage of the use of such fragments compared with the use of complete antibodies is that Fc-mediated activation of microglia cannot occur.…”