Structure-based design of conformation- and sequence-specific antibodies against amyloid β

Abstract

Conformation-specific antibodies that recognize aggregated proteins associated with several conformational disorders (e.g., Parkinson and prion diseases) are invaluable for diagnostic and therapeutic applications. However, no systematic strategy exists for generating conformation-specific antibodies that target linear sequence epitopes within misfolded proteins. Here we report a strategy for designing conformation-and sequence-specific antibodies against misfolded proteins that is inspired by the molecular interactions governing protein aggregation. We find that grafting small amyloidogenic peptides (6-10 residues) from the Aβ42 peptide associated with Alzheimer's disease into the complementarity determining regions of a domain (V H ) antibody generates antibody variants that recognize Aβ soluble oligomers and amyloid fibrils with nanomolar affinity. We refer to these antibodies as gammabodies for grafted amyloid-motif antibodies. Gammabodies displaying the central amyloidogenic Aβ motif ( 18 VFFA 21 ) are reactive with Aβ fibrils, whereas those displaying the amyloidogenic C terminus ( 34 LMVGGVVIA 42 ) are reactive with Aβ fibrils and oligomers (and weakly reactive with Aβ monomers). Importantly, we find that the grafted motifs target the corresponding peptide segments within misfolded Aβ conformers. Aβ gammabodies fail to cross-react with other amyloidogenic proteins and scrambling their grafted sequences eliminates antibody reactivity. Finally, gammabodies that recognize Aβ soluble oligomers and fibrils also neutralize the toxicity of each Aβ conformer. We expect that our antibody design strategy is not limited to Aβ and can be used to readily generate gammabodies against other toxic misfolded proteins.misfolding | beta-amyloid | protein engineering A hallmark of protein misfolding disorders is that polypeptides of unrelated sequence fold into similar oligomeric and fibrillar assemblies that are cytotoxic (1). The structures of these enigmatic conformers have captured the imagination of many investigators who have sought to explain the molecular basis of proteotoxicity in conformational disorders such as Alzheimer's disease. Because misfolded proteins are typically refractory to structural methods such as X-ray crystallography and solution NMR, few high-resolution structures of full-length misfolded proteins have been reported (ref. 2 and references therein). The structures of oligomeric intermediates have proven especially difficult to characterize because these conformers are labile, transient, and, in many cases, heterogeneous.Given the complexity of high-resolution structural analysis of misfolded proteins, alternative biochemical approaches are critical for understanding structure-function relationships of aggregated proteins. A breakthrough in this area has been the development of conformation-specific antibodies that selectively recognize uniquely folded conformers of amyloidogenic proteins (3-13). Indeed, multiple conformation-specific antibodies have been reported that recognize structural features ...

Rational design of potent domain antibody inhibitors of amyloid fibril assembly

“…S1) (9). Importantly, the charged Aβ gammabodies bind with much higher affinity to Aβ fibrils (IC 50 (4,9), and they fail to bind to soluble or aggregated conformers of other amyloidogenic polypeptides (IAPP and α-Synuclein; Fig. S1).…”

“…We used antibodies specific for prefibrillar oligomers (A11) and fibrillar conformers (OC) to monitor the aggregation of Aβ42 via immunoblotting ( Fig. 2A), as we reported previously (4,10). In the absence of gammabody inhibitors, Aβ forms prefibrillar oligomers (recognized by the A11 antibody) after 1 d; these oligomers convert into fibrillar conformers (recognized by the OC antibody) on the second day and persist for an additional 4 d (longer times not evaluated).…”

“…To evaluate our hypotheses related to the design of domain antibody inhibitors of amyloid assembly, we first sought to optimize the biophysical properties of a V H antibody scaffold for CDR grafting (4,8,9). We find that gammabodies presenting hydrophobic Aβ peptide segments (e.g., Aβ residues 15-24 and 33-42) within their third CDR (CDR3) readily aggregate when heated, stick to size-exclusion columns, and express at relatively low levels (5-7 mg/L) (4,9). However, inserting a triad of negatively charged residues (Asp-GluAsp) at each edge of the hydrophobic CDR3 loops results in gammabodies that fail to aggregate when heated, elute as monomeric peaks from size-exclusion columns, and express at relatively high levels (15-20 mg/L; Fig.…”

Design and Optimization of Anti-amyloid Domain Antibodies Specific for β-Amyloid and Islet Amyloid Polypeptide

“…To address this challenge, we are investigating the potential of designing conformational antibody fragments by mimicking the natural process of amyloid formation (38). Our approach is to graft amyloidogenic peptide segments from polypeptides such as the Alzheimer A␤ 2 peptide into the complementarity-determining regions (CDRs) of single-domain (V H ) antibodies.…”

Kinetic Rate Determination via Electrophoresis along a Varying Cross-Section Microchannel

“…U sed in a wide-range of immunoassays 1,2 and therapeutics, 3,4 over a million antibodies are estimated to be in production. Nevertheless, for immunoreagents, false positives (nonspecific binding), false negatives (low specificity), and reproducibility (lot-to-lot, vendor variability) remain problematic.…”