Nature-inspired design and evolution of anti-amyloid antibodies

Mark C Julian,Lilia A Rabia,Alec A Desai,Ammar Arsiwala,Julia E Gerson,Henry L Paulson,Ravi S Kane,Peter M Tessier

doi:10.1074/jbc.ra118.004731

Abstract

Antibodies that recognize amyloidogenic aggregates with high conformational and sequence specificity are important for detecting and potentially treating a wide range of neurodegenerative disorders, including Alzheimer's and Parkinson's diseases. However, these types of antibodies are challenging to generate because of the large size, hydrophobicity, and heterogeneity of protein aggregates. To address this challenge, we developed a method for generating antibodies specific for amyloid aggregates. First, we grafted amyloidogenic peptide segments from the target polypeptide [Alzheimer's amyloid-β (Aβ) peptide] into the complementarity-determining regions (CDRs) of a stable antibody scaffold. Next, we diversified the grafted and neighboring CDR sites using focused mutagenesis to sample each WT or grafted residue, as well as one to five of the most commonly occurring amino acids at each site in human antibodies. Finally, we displayed these antibody libraries on the surface of yeast cells and selected antibodies that strongly recognize Aβ-amyloid fibrils and only weakly recognize soluble Aβ. We found that this approach enables the generation of monovalent and bivalent antibodies with nanomolar affinity for Aβ fibrils. These antibodies display high conformational and sequence specificity as well as low levels of nonspecific binding and recognize a conformational epitope at the extreme N terminus of human Aβ. We expect that this systematic approach will be useful for generating antibodies with conformational and sequence specificity against a wide range of peptide and protein aggregates associated with neurodegenerative disorders.

Highlights

Antibodies that recognize amyloidogenic aggregates with high conformational and sequence specificity are important for detecting and potentially treating a wide range of neurodegenerative disorders, including Alzheimer’s and Parkinson’s diseases
Some conformational antibodies against amyloidogenic aggregates have been generated using in vivo or in vitro methods [11, 13,14,15], it remains extremely challenging to reliably identify monoclonal antibodies with high levels of conformational and sequence specificity for diverse types of amyloidogenic aggregates
Given that the scFv–Fc antibodies only differ in heavy chain CDR3 (HCDR3) sequence, these findings demonstrate that the selected mutations in AF1 HCDR3 promote specific binding to A␤ fibrils without significantly promoting nonspecific interactions

Summary

The abbreviations used are

A␤, amyloid ␤; CDR, complementarity-determining region; scFv, single-chain variable fragment; VH, variable domain of heavy-chain; Fc, fragment crystallizable; HCDR3, heavy chain CDR3; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; IAPP, islet amyloid polypeptide; RCF, relative centrifugal force; APP, amyloid precursor protein; HFIP, hexafluoro-2-propanol; Ni-NTA, nickel-nitrilotriacetic acid; DMEM, Dulbecco’s modified Eagle’s medium; KLH, keyhole limpet hemocyanin; ssDNA, single-stranded DNA; RFU, relative fluorescence units. Given that it is not possible to sample all combinations of mutations using 20 amino acids even in a single antibody CDR (e.g. Ͼ1019 possible variants for a CDR with 15 residues), our method involved sampling the WT residue at each targeted CDR site as well as one to five mutations that are most common in human antibodies and which are compatible with degenerate codons This approach enables testing all possible single, double, and higher-order combinations of CDR mutations in a single library by restricting the diversity at each CDR site. We previously used this library design approach for affinity maturation of single-domain (VHH) antibodies and found that it was highly effective for isolating antibody variants with significant improvements in affinity and specificity [17]. Design and selection methods that lead to the isolation of antiamyloid antibodies with high affinity and specificity

Results

Discussion

Experimental procedures