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

Abstract

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.

Keywords: NUsc1; amyloid; antibody fragments; neurotoxicity; oligomer; single chain.

Figure 1

Overview of the structure of antibody fragments. (A) General schematic of domain framework and (B) ribbon diagrams of full-length IgG and fragment molecules. Structures were obtained from the Protein Data Bank (http://www.rcsb.org/pdb/). CH, CL, VH, and VL stand for constant heavy, constant light, variable heavy, and variable light domains, respectively. Heavy or light chains are depicted in dark blue or cyan, respectively. Complementarity-determining region (CDR) segments are highlighted in red. PDB codes: 1IGT (full length IgG), 5VH3 (Fab), 4NKO (scFv), and 3R0M (VHH) [58]. Fab: fragment antigen-binding; scFv: single chain variable fragments; VH/VHH: heavy chain variable domain fragment.

Figure 2

The scFv antibody NUsc1 is highly selective to high molecular weight Aβ oligomers (AβO). (A) NUsc1 shows high selectivity for Aβ oligomers over monomers and fibrils as determined via ELISA. The anti-pan Aβ IgG 6E10 is shown for comparison. Adapted with permission from (Velasco et al., ACS Chem. Neurosci. 2012 [64]). Copyright (2020) American Chemical Society. (B) Within a synthetic AβO population, NUsc1 selectively targets a high molecular weight subset, showing little binding to a lower molecular weight subset that is readily bound by the anti-AβO IgG NU1. Reactivity of both antibodies to AβO fractions separated by size-exclusion chromatography under non-denaturing conditions was determined by dot immunoblotting. Reprinted with permission from (Sebollela et al., Journal of Neurochem. 2017 [65]). Copyright (2020) John Wiley and Sons.