Structural differences of amyloid-β fibrils revealed by antibodies from phage display

Abstract

BackgroundBeside neurofibrillary tangles, amyloid plaques are the major histological hallmarks of Alzheimer’s disease (AD) being composed of aggregated fibrils of β-amyloid (Aβ). During the underlying fibrillogenic pathway, starting from a surplus of soluble Aβ and leading to mature fibrils, multiple conformations of this peptide appear, including oligomers of various shapes and sizes. To further investigate the fibrillization of β-amyloid and to have tools at hand to monitor the distribution of aggregates in the brain or even act as disease modulators, it is essential to develop highly sensitive antibodies that can discriminate between diverse aggregates of Aβ.ResultsHere we report the generation and characterization of a variety of amyloid-β specific human and human-like antibodies. Distinct fractions of monomers and oligomers of various sizes were separated by size exclusion chromatography (SEC) from Aβ42 peptides. These antigens were used for the generation of two Aβ42 specific immune scFv phage display libraries from macaque (Macaca fascicularis). Screening of these libraries as well as two naïve human phage display libraries resulted in multiple unique binders specific for amyloid-β. Three of the obtained antibodies target the N-terminal part of Aβ42 although with varying epitopes, while another scFv binds to the α-helical central region of the peptide. The affinities of the antibodies to various Aβ42 aggregates as well as their ability to interfere with fibril formation and disaggregation of preformed fibrils were determined. Most significantly, one of the scFv is fibril-specific and can discriminate between two different fibril forms resulting from variations in the acidity of the milieu during fibrillogenesis.ConclusionWe demonstrated that the approach of animal immunization and subsequent phage display based antibody selection is applicable to generate highly specific anti β-amyloid scFvs that are capable of accurately discriminating between minute conformational differences.

Phage display-derived human antibodies in clinical development and therapy

“…The optimal selection strategy depends on many parameters, including the target antigen, the optimal antigen immobilization strategy, the quality of the library (particularly the frequency of antigen specific antibodies therein) and the binding and washing conditions. The tight control of the in vitro conditions allows the pre-design of antibody properties during the selection including epitope specificity, 73,74 and even conformation specificity 75,76 and interspecies cross-reactivity. 77 Screening for monoclonal antibodies can be performed either by antibody phage using enyzme-linked immunosorbent assay (phage ELISA) or by soluble expression of antibody fragments in E. coli and immunoassays, including ELISA or flow cytometry.…”

Early Disruption of Synaptic Function, Impairment of Plasticity, and Decreased of Cortical Circuit Connectivity in an Alzheimer’S Mouse Model of Amyloid Deposition

“…Similarly, another vulnerable cortical population in terms of APP/A overloading is the subiculum neurons of the temporal hippocampus, whose functional changes remain to be investigated. Because the 6E10 antibody for A does not distinguish APP and A 33,34 , we cannot attribute these observed synaptic and circuit connectivity changes to APP or to intracellular A production. Yet, our IHC staining revealed that minimum microglia activation at P21, and there was no extracellular amyloid deposition or dense core fibrillar A plaques in extracellular space.…”

Designing Human Antibodies by Phage Display

“…Since macaque VH and VL sequences have an extremely high homology to their human counterparts [34], they are a more obvious choice for the development of therapeutic antibodies than rodents. They have successfully been used to generate antibodies against the most dangerous toxins, like ricin [35], anthrax lethal factor [36], or various botulinum toxins [37,38] but also against fungi [39], encephalitis viruses [40], or human proteins [41]. The last example also demonstrates the very high specificity achievable from phage display by design, since the discovered antibody is able to discriminate between very subtle conformation differences of Alzheimer peptide aggregates.…”