Epitomic analysis of the specificity of conformation-dependent, anti-Aß amyloid monoclonal antibodies

Abstract

Antibodies against Aß amyloid are indispensable research tools and potential therapeutics for Alzheimer’s Disease, but display unusual properties, such as specificity for aggregated forms of the peptide, ability to distinguish several polymorphic aggregate structures and ability to recognize generic aggregation-related epitopes formed by unrelated amyloid sequences. Understanding the mechanisms underlying these unusual properties of anti-amyloid antibodies and the structures of their corresponding epitopes is crucial for the understanding why they display different therapeutic activities and for the development of more effective therapeutic agents. Here we employed a novel “epitomic” approach (1) to map the fine structure of the epitopes of 28 monoclonal antibodies against amyloid-beta using immunoselection of random sequences from a phage display, deep sequencing and pattern analysis to define the critical sequence elements recognized by the antibodies. Although most of the antibodies map to linear epitopes in the amino terminal 1-14 residues of Aß, the antibodies display differences in the target sequence residues that are critical for binding and in their individual preferences for non-target residues, indicating that the antibodies bind to alternative conformations of the sequence by different mechanisms. Epitomic analysis identifies more non-overlapping sequence Aß segments than peptide array approaches that may constitute the conformational epitopes that underly the aggregation specificity of antibodies. Aggregation specific antibodies also recognize sequences that display a significantly higher predicted propensity for forming amyloid than antibodies that recognize Aß monomer, indicating that the ability of the random sequences to aggregate into amyloid structures is a critical element of their binding mechanism.

Age-Related Oxidative Redox and Metabolic Changes Precede Intraneuronal Amyloid-β Accumulation and Plaque Deposition in a Transgenic Alzheimer’s Disease Mouse Model

“…To profile the development of A␤ aggregates across the lifespan of 3xTg AD-model mice, we performed immunostaining of brain sections using 6E10, an antibody that recognizes exposed residues 5-7 of the human A␤ peptide [51], and mOC78 antibodies that recognize a conformational epitope at positions 8-11 and 24-26 [52] of aggregates of oligomeric mouse and human A␤ peptide and other amyloids [7]. We examined the staining of large aggregates and plaques (>80 m 2 ) in CA1 of the hippocampus, a region that is known to be affected early in AD [53].…”

Inefficient quality control of ribosome stalling during APP synthesis generates CAT-tailed species that precipitate hallmarks of Alzheimer’s disease

“…Immunostaining with anti-Pum antibody showed that stalled NPCs were localized to the perinuclear ER compartment where some of them colocalized with the conformation-specific mOC78 antibody (Fig. 2e), which recognizes a discontinuous epitope consisting of residues 8-12 (SGYEV) and 24-26 (VGS) in misfolded or aggregated Aβ [55,56], suggesting that stalled NPCs adopted abnormal conformation and represented aberrant APP.C99 species. Thus, the translation of APP.C99 is stalled at two sites: one at or near the stop codon, another at an internal site ~ 30 AA upstream of the stop codon based on the size of the lower band.…”

Screening of sperm antigen epitopes by phage display technique and its preliminary clinical application

“…have been obtained successfully by using specific antibodies to screen phage display library combined with gene sequencing. These studies demonstrated that the obtained epitopes/mimotopes could be linear or conformational [11,18,20], and that these epitopes/ mimotopes were feasible for serological diagnosis and vaccine [10], and were more specific and sensitive [21,22]. However, there were few reports on the use of the phage display technique in the field of infertility.…”