ABSTRACT
Anti-amyloid immunotherapy has been proposed as an appropriate therapeutic approach for Alzheimer's disease (AD). Significant efforts have been made towards the generation and assessment of antibody-based reagents capable of preventing and clearing amyloid aggregates as well as preventing their synaptotoxic effects. In this study, we selected a novel set of human anti-amyloid-beta peptide 1-42 (Abeta1-42) recombinant monoclonal antibodies in a single chain fragment variable (scFv) and a single-domain (VH) format. We demonstrated that these antibody fragments recognize in a specific manner amyloid-beta deposits in APP/Tg mouse brains, inhibit toxicity of oligomeric Abeta1-42 in neuroblastoma cell cultures in a concentration-dependent manner and reduced amyloid deposits in APP/Tg2576 mice after intracranial administration. These antibody fragments recognize epitopes in the middle/C-terminus region of Abeta, which makes them strong therapeutic candidates due to the fact that most of the Abeta species found in the brains of AD patients display extensive N-terminus truncations/modifications.
Subject(s)
Amyloid beta-Peptides/administration & dosage , Amyloid beta-Peptides/immunology , Antibodies, Monoclonal/chemistry , Bacteriophage M13/immunology , Epitopes/immunology , Immunoglobulin Heavy Chains/chemistry , Peptide Fragments/administration & dosage , Peptide Fragments/immunology , Peptide Library , Single-Chain Antibodies/chemistry , Amino Acid Sequence , Amyloid beta-Peptides/genetics , Animals , Antibodies, Monoclonal/genetics , Antibodies, Monoclonal/metabolism , Bacteriophage M13/chemistry , Bacteriophage M13/genetics , Binding Sites, Antibody/genetics , Cell Line, Tumor , Epitopes/genetics , Epitopes/metabolism , Humans , Immunoglobulin Heavy Chains/genetics , Immunoglobulin Heavy Chains/metabolism , Mice , Mice, Transgenic , Molecular Sequence Data , Peptide Fragments/genetics , Protein Structure, Tertiary/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Single-Chain Antibodies/biosynthesis , Single-Chain Antibodies/geneticsABSTRACT
N-truncated and N-modified forms of amyloid beta (Abeta) peptide are found in diffused and dense core plaques in Alzheimer's disease (AD) and Down's syndrome patients as well as transgenic mouse models of AD. Although the pathological significance of these shortened forms Abeta is not completely understood, previous studies have demonstrated that these peptides are significantly more resistant to degradation, aggregate more rapidly in vitro and exhibit similar or, in some cases, increased toxicity in hippocampal neuronal cultures compared to the full length peptides. In the present study we further investigated the mechanisms of toxicity of one of the most abundant N-truncated/modified Abeta peptide bearing amino-terminal pyroglutamate at position 3 (AbetaN3(pE)). We demonstrated that AbetaN3(pE) oligomers induce phosphatidyl serine externalization and membrane damage in SH-SY5Y cells. Also, we produced AbetaN3(pE)-specific polyclonal antibodies in rabbit and identified an immunodominant epitope recognized by anti-AbetaN3(pE) antibodies. Our results are important for developing new immunotherapeutic compounds specifically targeting AbetaN3(pE) aggregates since the most commonly used immunogens in the majority of vaccines for AD have been shown to induce antibodies that recognize the N-terminal immunodominant epitope (EFRH) of the full length Abeta, which is absent in N-amino truncated peptides.