Structure of Crenezumab Complex with Aβ Shows Loss of β-Hairpin

Abstract

Accumulation of amyloid-β (Aβ) peptides and amyloid plaque deposition in brain is postulated as a cause of Alzheimer's disease (AD). The precise pathological species of Aβ remains elusive although evidence suggests soluble oligomers may be primarily responsible for neurotoxicity. Crenezumab is a humanized anti-Aβ monoclonal IgG4 that binds multiple forms of Aβ, with higher affinity for aggregated forms, and that blocks Aβ aggregation, and promotes disaggregation. To understand the structural basis for this binding profile and activity, we determined the crystal structure of crenezumab in complex with Aβ. The structure reveals a sequential epitope and conformational requirements for epitope recognition, which include a subtle but critical element that is likely the basis for crenezumab's versatile binding profile. We find interactions consistent with high affinity for multiple forms of Aβ, particularly oligomers. Of note, crenezumab also sequesters the hydrophobic core of Aβ and breaks an essential salt-bridge characteristic of the β-hairpin conformation, eliminating features characteristic of the basic organization in Aβ oligomers and fibrils, and explains crenezumab's inhibition of aggregation and promotion of disaggregation. These insights highlight crenezumab's unique mechanism of action, particularly regarding Aβ oligomers, and provide a strong rationale for the evaluation of crenezumab as a potential AD therapy.

Figure 1. Crystal structure of CreneFab/Aβ.

(A) The overall view of Aβ_11–25 binding to the antibody. CreneFab is shown in surface rendering. Blue, heavy chain; Yellow: light chain. Aβ peptide is shown in ribbon and sticks. Carbon atoms of Aβ peptide are in magenta; other atoms are colored by atom-type. The N- and C-termini of Aβ peptide are disordered in structures as indicated by dots. The Aβ residues are labeled. Green mesh shows the 2Fo-Fc electron density map (contoured at 1xRMSD) corresponding to the Aβ peptide. (B) In side-by-side stereo view, shows an overlap of the CDR region from the Fab alone structure (green) with the Fab/Aβ complex (blue: heavy chain, Yellow: light chain), denoted H1, H2, H3, L1, L2, L3. (C–F) Close-up views of the binding site. Details are described in the results. Color scheme is same as in (A).

Figure 2. NMR HSQC spectrum of Aβ_1–42 and CreneFab mapped the epitope.

Comparison of the ¹H/¹⁵N correlated NMR spectra of ¹³C/¹⁵N isotopically labeled Aβ_1–42 (R-Peptide, Georgia), in the absence (A) and presence (B) of CreneFab. The figures show the region of the resonances belonging to the backbone amide groups. (A) ¹⁵N HSQC spectrum of a 40 μM Aβ_1–42 solution. The peaks are labeled according to the assignments published elsewhere. (B) The same spectrum of Aβ_1–42 in the presence of a 1:1 ratio of labeled Aβ_1–42 to unlabeled CreneFab. The red circles show where peaks were broadened such that their intensity was below the noise level.

Figure 3. CreneFab alanine mutations impact on affinity.

(A) A bar graph showing the fold decrease in affinity of individual CreneFab alanine mutants compared to the wild-type determined with SPR kinetic measurements using BIAcore. Mutations at 32^HC, 95^HC or 101^HC abolished binding. N/D: not detectable. (B) Residues subject to this study are mapped on the crystal structure. The antibody is illustrated in surface presentation. The residues are colored by their impact on binding affinity.

Figure 4. Negative staining immunogold electron microscopy of crenezumab binding to Aβ fibrils.

TEM grids were either incubated with a preparation of Aβ fibrils (A–C) or were left empty as a control for nonspecific antibody adsorption to the grid surface (D). Primary labeling was either with crenezumab (A,B and D) or with an anti-gD control antibody (C). Detection was with a secondary biotinylated antibody followed by streptavidin conjugated with 10 nm gold particles (A–D). Arrows point to 10 nm gold particles specifically bound to Aβ fibrils (A) or to a low molecular weight (LMW) Aβ aggregated species (B). Representative images were taken at 20000× and scale bars are 500 nm. (E) Labeling densities were calculated as the average number and standard deviation (STD) of gold particles per 1 um² for N = 20 random areas analyzed: A (152.6+/−54.5), B (20.1+/−5.9), C (0.25+/−0.7) and D (0.25+/−0.4).

Figure 5. A comparison of crenezumab and solanezumab.

Crenezumab/Aβ structure is superimposed onto solanezumab/Aβ structure (4XXD). The color scheme for crenezumab and bound Aβ peptide is same as in Fig. 1A. Solanezumab Fab is shown in black ribbons, and the solanezumab-bound Aβ peptide is in green. The side chains of Glu22^Aβ, Asp23^Aβ, and resides at position 33^HC and 52a^HC of the antibodies are shown in sticks and labeled. The dotted lines indicate hydrogen bond interactions. Black dotted lines are of solanezumab, red dotted lines are of crenezumab. Red spheres indicate amino acid residues different between crenezumab and solanezumab. A more complete view of difference between crenezumab and solanezumab is shown in Figure S2.