Abstract
Antibodies that preferentially and specifically target pathological oligomeric protein and peptide assemblies, as opposed to their monomeric and amyloid counterparts, provide therapeutic and diagnostic opportunities for protein misfolding diseases. Unfortunately, the molecular properties associated with oligomer-specific antibodies are not well understood, and this limits targeted design and development. We present here a generic method that enables the design and optimisation of oligomer-specific antibodies. The method takes a two-step approach where discrimination between oligomers and fibrils is first accomplished through identification of cryptic epitopes exclusively buried within the structure of the fibrillar form. The second step discriminates between monomers and oligomers based on differences in avidity. We show here that a simple divalent mode of interaction, as within e.g. the IgG isotype, can increase the binding strength of the antibody up to 1500 times compared to its monovalent counterpart. We expose how the ability to bind oligomers is affected by the monovalent affinity and the turnover rate of the binding and, importantly, also how oligomer specificity is only valid within a specific concentration range. We provide an example of the method by creating and characterising a spectrum of different monoclonal antibodies against both the Aβ peptide and α-synuclein that are associated with Alzheimer's and Parkinson's diseases, respectively. The approach is however generic, does not require identification of oligomer-specific architectures, and is, in essence, applicable to all polypeptides that form oligomeric and fibrillar assemblies.
Highlights
The pathological self-assembly of proteins and peptides into amyloid fibrils is the defining characteristic of a group of more than twenty human diseases, including Alzheimer’s disease (AD) and Parkinson’s disease (PD) [1]
Amyloid fibrils are invariably present in the affected individuals, many studies have shown that soluble oligomeric assemblies, which can either precede amyloid formation or represent a stand-alone entity formed in parallel with the fibrils, exert the most potent detrimental physiological effects [2,3,4,5,6,7,8,9,10,11]
Using a dot blot technique – in which an equal amount of fibrils and monomers from the Ab1–42 are applied to the membrane – we concluded that the region spanning residue 3–10 in a nice manner can be used to differentiate between the fibrillar and oligomeric forms
Summary
The pathological self-assembly of proteins and peptides into amyloid fibrils is the defining characteristic of a group of more than twenty human diseases, including Alzheimer’s disease (AD) and Parkinson’s disease (PD) [1]. Amyloid fibrils are invariably present in the affected individuals, many studies have shown that soluble oligomeric assemblies, which can either precede amyloid formation or represent a stand-alone entity formed in parallel with the fibrils, exert the most potent detrimental physiological effects [2,3,4,5,6,7,8,9,10,11] These oligomers are transient species and frequently only constitute a very minor fraction as compared to the amyloid and the non-aggregated native and precursor forms of the specific protein or peptide. There is, an urgent need for a method that can be used to consistently and reliably design oligomer-specific antibodies
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