Structural Basis of C-terminal β-Amyloid Peptide Binding by the Antibody Ponezumab for the Treatment of Alzheimer's Disease

https://doi.org/10.1016/j.jmb.2011.11.047Get rights and content

Abstract

Alzheimer's disease, the most common cause of dementia in the elderly and characterized by the deposition and accumulation of plaques, is composed in part of β-amyloid (Aβ) peptides, loss of neurons, and the accumulation of neurofibrillary tangles. Here, we describe ponezumab, a humanized monoclonal antibody, and show how it binds specifically to the carboxyl (C)-terminus of Aβ40. Ponezumab can label Aβ that is deposited in brain parenchyma found in sections from Alzheimer's disease casualties and in transgenic mouse models that overexpress Aβ. Importantly, ponezumab does not label full-length, non-cleaved amyloid precursor protein on the cell surface. The C-terminal epitope of the soluble Aβ present in the circulation appears to be available for ponezumab binding because systemic administration of ponezumab greatly elevates plasma Aβ40 levels in a dose-dependent fashion after administration to a mouse model that overexpress human Aβ. Administration of ponezumab to transgenic mice also led to a dose-dependent reduction in hippocampal amyloid load. To further explore the nature of ponezumab binding to Aβ40, we determined the X-ray crystal structure of ponezumab in complex with Aβ40 and found that the Aβ40 carboxyl moiety makes extensive contacts with ponezumab. Furthermore, the structure–function analysis supported this critical requirement for carboxy group of AβV40 in the Aβ–ponezumab interaction. These findings provide novel structural insights into the in vivo conformation of the C-terminus of Aβ40 and the brain Aβ-lowering efficacy that we observed following administration of ponezumab in transgenic mouse models.

Graphical Abstract

Highlights

► Aβ40 C-terminus has been a challenging epitope to target. ► Ponezumab binds specifically to Aβ40 over other Aβ species and intact amyloid precursor protein. ► Ponezumab binds Aβ40 in the blood and in brain plaques. ► The crystal structure of the Aβ40–Fab complex reveals the Aβ40 C-terminal domain.

Introduction

The amyloid hypothesis of Alzheimer's disease (AD) pathogenesis posits that β-amyloid (Aβ)-driven synaptic failure and senile plaque formation in the cortex, limbic system, and basal ganglia lead to tau protein hyper-phosphorylation and neurofibrillary tangle formation that result in neuronal death and dementia.1, 2, 3 The Aβ peptides that are derived from sequential proteolytic processing of amyloid precursor protein (APP) accumulate in brain regions critical for learning and memory and are thought to play a pivotal role in the pathogenesis of AD. Therefore, inhibition of de novo Aβ synthesis and/or enhanced clearance has become a major target for many therapeutic interventions, including immunotherapy.4

Passive immunotherapy involves the use of antibodies directed against Aβ, and there are currently three proposed mechanisms by which Aβ can be cleared from the central nervous system (CNS). Two of these mechanisms—microglial activation and catalytic dissolution—require antibody entrance into the CNS, while the third mechanism, dubbed the “peripheral sink” hypothesis, relies on antibody activity against Aβ found in the blood to reduce CNS levels.5, 6 The peripheral sink hypothesis emerges from findings showing active Aβ transport across the blood–brain barrier from the CNS to the periphery via the low-density lipoprotein receptor-1 and from the periphery to the CNS via the receptor for advanced glycation end products.7, 8 This hypothesis proposes that anti-Aβ antibodies remain in the periphery, where they bind to and sequester free Aβ in the blood, thus altering the systemic dynamics of Aβ transport and promoting the net efflux of Aβ from the CNS.5, 9, 10 Different therapeutic antibodies target one or more mechanisms via epitope and the antibody Fc (fragment crystallizable) effector function selection.

Passive immunotherapy for AD holds promise, although some clinical Aβ-targeting antibodies have been shown to increase the occurrence of microhemorrhages secondary to cerebral amyloid angiopathy, a neurological disorder that is characterized by inflammation and the deposition of Aβ in the central vasculature.11, 12 Likewise, transient cerebral vasogenic edema has occurred more frequently in AD patients who received higher doses of bapineuzumab, an antibody targeting the N-terminal region of Aβ.13 Passive immunization with antibodies devoid of the Fc–receptor activity on effector cells, such as macrophages and microglia, has been suggested as an approach for decreasing the risk of a dangerous inflammatory response while diminishing central amyloid deposition.14

Section snippets

Ponezumab rationale as an immunotherapy for AD

Ponezumab is a humanized monoclonal antibody that binds to the C-terminus of Aβ1-40, the most abundant Aβ peptide. Another antibody, which binds to the C-terminus of Aβ1-40, mouse monoclonal antibody 2H6, has been shown to have activity in animal models of AD.15, 16 The ponezumab scaffold is human IgG2 wherein the Fc region contains two mutations (so-called IgG2Δa)17 that eliminate effector function—thus, it is immunologically inert. Consequently, ponezumab is designed to act through the sink

Relevance of Aβ40 structure in complex with ponezumab

Amyloid-containing plaques are the hallmark of AD pathology, and the hydrophobic C-terminal domain of Aβ peptides is believed to be essential for its amyloidogenic properties.22 Our results demonstrate that ponezumab binds specifically to the Aβ40 C-terminal domain, displaying avid and selective binding at Aβ residues 30–40, forming extensive contacts with the carboxylic acid moiety presented at the C-terminal residue AβV40, with the ponezumab–Fab Hv dominating the interaction. Several mouse

ELISA epitope mapping

For the epitope determination by peptide mapping ELISA, 10 nM biotinylated microscale peptide libraries of 15 or 10 amino acids spanning the length of Aβ1-42 peptide starting from Aβ20 (JPT Peptide Technologies GmbH) were incubated for 1 h in 96-well microtiter plates (Nunc MaxiSorp, Denmark), which were precoated with streptavidin (Pierce/Thermo Scientific) at 6 μg/mL in phosphate-buffered saline (PBS) buffer, pH 7.4. Plates were washed and then incubated with 5 μg/mL ponezumab for 1 h in PBS,

Acknowledgements

We thank the staff at Advanced Light Source sector 5 for assistance with X-ray diffraction data collection. We thank Kane and Finkel for early draft preparation assistance.

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    Competing financial interests: All authors work for Pfizer, Inc.

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