Structural basis of Alzheimer β-amyloid peptide recognition by engineered lipocalin proteins with aggregation-blocking activity

Abstract

We describe the structural analysis of two Anticalin® proteins that tightly bind Aβ 40, a peptide involved in the pathophysiology of Alzheimer’s disease. These anticalins, US7 and H1GA, were engineered on the basis of the human lipocalin 2, thus yielding compact single-domain binding proteins as an alternative to antibodies. Albeit selected under different conditions and mutually deviating in 13 amino acid positions within the binding pocket (of 17 mutated residues in total), both crystallised anticalins recognize the same epitope in the middle of the β-amyloid peptide. In the two complexes with the Aβ 40 peptide, its central part comprising residues LysP16 to LysP28 shows well defined electron density whereas the flanking regions appear structurally disordered. The compact zigzag-bend conformation which is seen in both structures may indicate a role during conversion of the soluble monomeric form into pathogenic Aβ state(s) and, thus, explain the aggregation-inhibiting effect of the anticalins. In contrast to solanezumab, which targets the same Aβ region in a different conformation, the anticalin H1GA does not show cross-reactivity with sequence-related human plasma proteins. Consequently, anticalins offer promising reagents to prevent oligomerization of Aβ peptides to neurotoxic species in vivo and their small size may enable new routes for brain delivery.

β-amyloid monomer scavenging by an anticalin protein prevents neuronal hyperactivity

“…Anticalins exhibit several bene cial properties compared with mAbs, in particular small size, high speci city and strong a nity for Aβ 22 , including a lack of interaction with plasma proteins 23 , as well as low immunogenic potential 20 . This may be in contrast to the use with the humanized IgG1 Lecanemab that caused clear side effects in a signi cant fraction of treated Alzheimer patients, including microhemorrhages and macrohemorrhages 9 .…”

“…2, E-G). While this result may be expected based on the binding properties of the Aβ-anticalin 23 , it is still remarkable as [AβS26C] 2 mimics the action of human Aβ dimers 18 . Thus, the experiment does not answer whether binding to monomers or dimers is necessary for the hyperactivityreducing action of the Aβ-anticalin but warrants further investigation of the interaction of the anticalin with different forms of Aβ.…”

“…We have previously demonstrated that the Aβ-anticalin binds Aβ monomers with high a nity 22,23 . To determine the precise Aβ-binding behavior of the Aβ-anticalin, we performed size exclusion chromatography (SEC) of the Aβ-anticalin alone, and after mixture with freshly prepared Aβ monomers.…”

“…Anticalins have already entered clinical trials for different indications 21 . Several Aβ-binding anticalins with high a nities and speci cities for the monomeric peptide target have recently been described 22 , and their mode of tight complex formation with the central Aβ epitope (Lys P16 to Lys P28 ) -which is common to both Aβ 40 and Aβ 42 peptide species -was elucidated by X-ray crystallography 23 .…”

β-amyloid monomer scavenging by an anticalin protein prevents neuronal hyperactivity in mouse models of Alzheimer’s Disease

“…These properties make them potent Aβ scavengers and the ideal tool to study the acute effects of Aβ removal in vivo. Several Aβ-binding anticalins with high affinities and specificities for the monomeric Aβ peptide target have recently been described 23 , and their mode of tight complex formation with the central Aβ epitope (Lys P16 to Lys P28 )—which is common to both Aβ 40 and Aβ 42 peptide species—was elucidated by X-ray crystallography 24 .…”