Antibody Fragments as Tools for Elucidating Structure-Toxicity Relationships and for Diagnostic/Therapeutic Targeting of Neurotoxic Amyloid Oligomers.

The accumulation of amyloid protein aggregates in tissues is the basis for the onset of diseases known as amyloidoses. Intriguingly, many amyloidoses impact the central nervous system (CNS) and usually are devastating diseases. It is increasingly apparent that neurotoxic soluble oligomers formed by amyloidogenic proteins are the primary molecular drivers of these diseases, making them lucrative diagnostic and therapeutic targets. One promising diagnostic/therapeutic strategy has been the development of antibody fragments against amyloid oligomers. Antibody fragments, such as fragment antigen-binding (Fab), scFv (single chain variable fragments), and VHH (heavy chain variable domain or single-domain antibodies) are an alternative to full-length IgGs as diagnostics and therapeutics for a variety of diseases, mainly because of their increased tissue penetration (lower MW compared to IgG), decreased inflammatory potential (lack of Fc domain), and facile production (low structural complexity). Furthermore, through the use of in vitro-based ligand selection, it has been possible to identify antibody fragments presenting marked conformational selectivity. In this review, we summarize significant reports on antibody fragments selective for oligomers associated with prevalent CNS amyloidoses. We discuss promising results obtained using antibody fragments as both diagnostic and therapeutic agents against these diseases. In addition, the use of antibody fragments, particularly scFv and VHH, in the isolation of unique oligomeric assemblies is discussed as a strategy to unravel conformational moieties responsible for neurotoxicity. We envision that advances in this field may lead to the development of novel oligomer-selective antibody fragments with superior selectivity and, hopefully, good clinical outcomes.

The toxic nature of murine amylin and the immune responsivity of pancreatic islet to conformational antibody in mice.

The human amylin is a pancreatic peptide hormone found in hyperhormonemic state along with insulin in subclinical diabetes. Amylin has been associated with the pathology of type 2 diabetes, particularly due to its ability to assembly into toxic oligomers and amyloid specimens. On the other hand, some variants such as murine amylin has been described as non-amyloidogenic, either in vitro or in vivo. Recent data have demonstrated the amyloid propensity of murine amylin and the therapeutic analogue pramlintide, suggesting a universality for amylin amyloidosis. Here, we report the amyloidogenesis of murine amylin, which showed lower responsivity to the fluorescent probe thioflavin T compared to human amylin, but presented highly organized fibrilar amyloid material. The aggregation of murine amylin also resulted in the formation of cytotoxic specimens, as evaluated in vitro in INS-1 cells. The aggregation product from murine amylin was responsive to a specific antibody raised against amyloid oligomers, the A11 oligomer antibody. Pancreatic islets of wild-type Swiss male mice have also shown responsivity for the anti-oligomer, indicating the natural abundance of such specimen in rodents. These data provide for the first time evidences for the toxic nature of oligomeric assemblies of murine amylin and its existence in wild-type, non-transgenic mice.