Molecular basis for passive immunotherapy of Alzheimer's disease

Abstract

Amyloid aggregates of the amyloid-beta (Abeta) peptide are implicated in the pathology of Alzheimer's disease. Anti-Abeta monoclonal antibodies (mAbs) have been shown to reduce amyloid plaques in vitro and in animal studies. Consequently, passive immunization is being considered for treating Alzheimer's, and anti-Abeta mAbs are now in phase II trials. We report the isolation of two mAbs (PFA1 and PFA2) that recognize Abeta monomers, protofibrils, and fibrils and the structures of their antigen binding fragments (Fabs) in complex with the Abeta(1-8) peptide DAEFRHDS. The immunodominant EFRHD sequence forms salt bridges, hydrogen bonds, and hydrophobic contacts, including interactions with a striking WWDDD motif of the antigen binding fragments. We also show that a similar sequence (AKFRHD) derived from the human protein GRIP1 is able to cross-react with both PFA1 and PFA2 and, when cocrystallized with PFA1, binds in an identical conformation to Abeta(1-8). Because such cross-reactivity has implications for potential side effects of immunotherapy, our structures provide a template for designing derivative mAbs that target Abeta with improved specificity and higher affinity.

Fig. 1.

SPR reveals Aβ peptide binding to PFA1 via the EFRHD epitope. (a) Kinetic analysis of Aβ(1–40) (WT) monomer at 0, 1.23, 3.70, 11.1, 33.3, 100, and 300 nM, binding to PFA1 Fab immobilized at densities of 2,720 response units (RU) (shown) and 1,280 RU (data not shown). Duplicate binding responses for each monomer concentration are overlaid with the global fit of a simple 1:1 interaction model (smooth lines), which yielded k_a = (1.431 ± 0.003) × 10⁴ M⁻¹ s⁻¹, k_d = (5.58 ± 0.01) × 10⁻⁴ s⁻¹, and K_d = 39.0 ± 0.1 nM. (b) WT and Ala-substituted mutants of Aβ(1–40) monomers were sequentially flowed over PFA1 IgG captured on anti-IgG flow cell surfaces (SPR). Significant RU peaks show good peptide binding, whereas the absence of a peak shows no binding. Results from PFA2 were essentially identical. The D7A mutation limits but does not completely eliminate binding. The numbering scheme is Aβ(1–40)-specific.

Fig. 2.

PFA1 and PFA2 bind to the Aβ(1–8) peptide. (a) Stereoview of a simulated-annealing omit map contoured at 3σ shows the electron density for the free DAEFRHDS peptide bound to the CDR of PFA1. (b) Stereoview of the overlay of the peptides and CDRs highlights the similarity in binding. PFA1-pep is shown in blue, PFA2-pep is in green. Residues are numbered by the Kabat scheme.

Fig. 3.

Electrostatics of binding. The electrostatic potential surface of PFA1 with bound peptide. Blue represents positive charge, red indicates negative charge, and the apolar surface is shown in white. The Aβ(1–8) peptide is drawn with carbon (yellow), nitrogen (blue), and oxygen (red). Although the Arg 5 residue sits in a pocket of strong negative charge, the Glu 3 residue has no correspondingly positive region around it. This position is susceptible to substitution and cross-reaction.