An “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. They display several unusual properties, such as specificity for aggregated forms of the peptide, the ability to distinguish polymorphic aggregate structures, and the ability to recognize generic aggregation-related epitopes formed by unrelated amyloid sequences. Understanding the mechanisms underlying these unusual properties and the structures of their corresponding epitopes is crucial for the understanding why antibodies display different therapeutic activities and for the development of more effective therapeutic agents. Here we employed a novel “epitomic” approach to map the fine structure of the epitopes of 28 monoclonal antibodies against amyloid-beta using immunoselection of random sequences from a phage display library, deep sequencing, and pattern analysis to define the critical sequence elements recognized by the antibodies. Although most of the antibodies map to major linear epitopes in the amino terminal 1 to 14 residues of Aß, the antibodies display differences in the target sequence residues that are critical for binding and in their individual preferences for nontarget residues, indicating that the antibodies bind to alternative conformations of the sequence by different mechanisms. Epitomic analysis also identifies discontinuous, nonoverlapping sequence Aß segments that may constitute the conformational epitopes that underlie the aggregation specificity of antibodies. Aggregation-specific antibodies recognize sequences that display a significantly higher predicted propensity for forming amyloid than antibodies that recognize the monomer, indicating that the ability of random sequences to aggregate into amyloid is a critical element of their binding mechanism. Antibodies against Aß amyloid are indispensable research tools and potential therapeutics for Alzheimer’s disease. They display several unusual properties, such as specificity for aggregated forms of the peptide, the ability to distinguish polymorphic aggregate structures, and the ability to recognize generic aggregation-related epitopes formed by unrelated amyloid sequences. Understanding the mechanisms underlying these unusual properties and the structures of their corresponding epitopes is crucial for the understanding why antibodies display different therapeutic activities and for the development of more effective therapeutic agents. Here we employed a novel “epitomic” approach to map the fine structure of the epitopes of 28 monoclonal antibodies against amyloid-beta using immunoselection of random sequences from a phage display library, deep sequencing, and pattern analysis to define the critical sequence elements recognized by the antibodies. Although most of the antibodies map to major linear epitopes in the amino terminal 1 to 14 residues of Aß, the antibodies display differences in the target sequence residues that are critical for binding and in their individual preferences for nontarget residues, indicating that the antibodies bind to alternative conformations of the sequence by different mechanisms. Epitomic analysis also identifies discontinuous, nonoverlapping sequence Aß segments that may constitute the conformational epitopes that underlie the aggregation specificity of antibodies. Aggregation-specific antibodies recognize sequences that display a significantly higher predicted propensity for forming amyloid than antibodies that recognize the monomer, indicating that the ability of random sequences to aggregate into amyloid is a critical element of their binding mechanism. Amyloids are intermolecularly hydrogen-bonded ß-sheet aggregates that have a regularly repeating lattice structure (1Eisenberg D.S. Sawaya M.R. Structural studies of amyloid proteins at the molecular level.Annu. Rev. Biochem. 2017; 86: 69-95Crossref PubMed Scopus (282) Google Scholar, 2Glabe C.G. Structural classification of toxic amyloid oligomers.J. Biol. Chem. 2008; 283: 29639-29643Abstract Full Text Full Text PDF PubMed Scopus (650) Google Scholar). Unlike natively folded proteins that adopt a single or limited range of structures or states, amyloids can adopt a large number of different ß-sheet aggregate structures that vary in the parallel versus antiparallel strand arrangement, the segments of the sequence that form intermolecularly hydrogen-bonded ß-sheets, and the locations where the sheets fold and how the sheets stack together. Many proteins are able to form amyloids upon unfolding or misfolding and are frequently associated with neurodegenerative diseases such as Alzheimer’s disease (AD), Parkinson’s disease, amyotrophic lateral sclerosis, and prion diseases (3Dobson C.M. Protein misfolding, evolution and disease.Trends Biochem. Sci. 1999; 24: 329-332Abstract Full Text Full Text PDF PubMed Scopus (1698) Google Scholar). AD contains two canonical amyloids: Aß amyloid, derived from APP and tau amyloid, and in approximately 30% of AD, cortical Lewy bodies are also present containing α-synuclein amyloid. Monoclonal antibodies against Aß are a leading class of therapeutic for AD. Many of these antibodies have been evaluated in clinical trials, but so far none have demonstrated consistent therapeutic activity in slowing the progression of AD and none have been approved by the US Food and Drug Administration (reviewed in (4Panza F. Lozupone M. Logroscino critical of for Rev. PubMed Scopus Google Scholar, and in to PubMed Scopus Google monoclonal antibodies against are also for research in the of amyloids in AD recognize epitopes that are or folded of the peptide, into the of in the of the disease C.G. antibodies target diseases of Biochem. Sci. Full Text Full Text PDF PubMed Scopus Google Scholar). Many of the antibodies against Aß amyloid bind to aggregated or forms of the and bind to the or the amyloid C.G. structure of amyloid of PubMed Scopus Google Scholar, F. M. C.G. antibodies recognize a generic to amyloid and that is in PubMed Scopus Google Scholar, monoclonal antibodies distinguish different or of PubMed Scopus Google Scholar, Monoclonal antibodies against distinguish aggregation in and in disease Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of these aggregation antibodies recognize aggregates formed from unrelated indicating that amyloids display generic epitopes as a of their aggregated Understanding the mechanisms underlying the unusual of antibodies and the structures of their corresponding epitopes is crucial for the understanding of the to amyloid and for the development of effective therapeutic agents. have that distinguish two of amyloid against Aß of the to that but or C.G. structure of amyloid of PubMed Scopus Google Scholar). we that against that is for and but or F. M. C.G. antibodies recognize a generic to amyloid and that is in PubMed Scopus Google Scholar). and recognize and from several α-synuclein and amyloid indicating that of the antibodies in the recognize generic amyloid aggregates that the also against are structures, but the of as indicating that these have C.G. in disease Biol. Chem. 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Sawaya M.R. Structural studies of amyloid proteins at the molecular level.Annu. Rev. Biochem. 2017; 86: 69-95Crossref PubMed Scopus (282) Google Scholar, 2Glabe C.G. Structural classification of toxic amyloid oligomers.J. Biol. Chem. 2008; 283: 29639-29643Abstract Full Text Full Text PDF PubMed Scopus (650) Google Scholar, C.M. Protein misfolding, evolution and disease.Trends Biochem. Sci. 1999; 24: 329-332Abstract Full Text Full Text PDF PubMed Scopus (1698) Google Scholar, F. Lozupone M. Logroscino critical of for Rev. PubMed Scopus Google Scholar, and in to PubMed Scopus Google Scholar, C.G. antibodies target diseases of Biochem. Sci. Full Text Full Text PDF PubMed Scopus Google Scholar, C.G. structure of amyloid of PubMed Scopus Google Scholar, F. M. C.G. antibodies recognize a generic to amyloid and that is in PubMed Scopus Google Scholar, monoclonal antibodies distinguish different or of PubMed Scopus Google Scholar, Monoclonal antibodies against distinguish aggregation in and in disease Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, C.G. in disease Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, F. C.G. structure of a amyloid Sci. PubMed Scopus Google Scholar, F. are a and of amyloid Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, C.G. proteins and with amyloid PubMed Scopus Google or C.G. structure of amyloid of PubMed Scopus Google Scholar, in Sci. PubMed Scopus Google Scholar, using Biol. PubMed Scopus Google Scholar, a for the and of of aggregation in PubMed Scopus Google Scholar, M. M. F. the sequence of amyloid structure using PubMed Scopus Google Scholar, the proteins of forming Sci. 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