One must know oneself and one's enemy to prevail in battle, according to the legendary militarist Sun Tzu. The first shot in the war against amyloid was fired long ago, but researchers are still getting to know which species of Aβ pose a threat and are fashioning their antibody weaponry accordingly.
Take a recent crystal structure of solanezumab bound to a fragment of Aβ. Published in the April 16 issue of Scientific Reports, it showed that in the antibody’s clutches, Aβ exhibited a unique configuration that was part helix, part extended coil. The study's authors, Luke Miles and collaborators at St. Vincent’s Institute for Medical Research in Fitzroy, Australia, noted that the amino acids in solanezumab that interacted with Aβ had nearly the same sequence as those in crenezumab. This suggested that the two antibodies bind Aβ in the same way, despite reports that the antibodies bind different forms of Aβ. A patent application on Biogen’s BIIB037/aducanumab antibody disclosed that it binds a linear stretch of Aβ. More broadly, researchers believe that the precise spatial details of how Aβ antibodies in clinical development engage their antigen may correlate with which ones succeed in therapeutic trials, both with regard to amyloid reduction and minimal collateral activity such as vasogenic edema. Binding experiments and a slowly growing portfolio of crystal structures are beginning to build this knowledge base.
Aβ Antibodies—What’s in That Pocket?
Each of the clinical anti-Aβ antibodies target specific parts of the infamous peptide. For example, solanezumab and crenezumab bind Aβ’s midsection, while bapineuzumab, gantenerumab, and aducanumab recognize the N-terminal region. In turn, the antibodies have been reported to preferentially recognize different Aβ complexes. Solanezumab has a penchant for soluble monomers, while crenezumab recognizes monomers, oligomers, and aggregates. The N-terminal antibodies recognize primarily aggregates, though some are reported to recognize soluble forms as well. Researchers speculate that these patterns reflect the exposure of Aβ’s N-terminus to solution in monomeric, aggregated, and plaque-deposited forms (Zago et al., 2012). In contrast, Aβ’s midsection is masked when it forms aggregates but exposed in monomers. The antibodies further differ by their affinity for their respective targets.
Five antibodies bind different epitopes and conformations of Aβ.
In 2012, Miles started to investigate how therapeutic antibodies engage Aβ. “We believe that developing structure-activity relationships for the immunotherapies will be informative in understanding why one antibody works and another does not,” Miles wrote to Alzforum. His group first reported that bapineuzumab bound the N-terminus of Aβ in a helical form, offering a possible explanation for why the antibody also recognizes aggregates (see Feb 2013 news).
To address the structural relationship between solanezumab and Aβ, first author Gabriella Crespi and colleagues crystallized the Fab portion of solanezumab (containing the Aβ binding region) with Aβ12-28—the portion of the peptide reportedly recognized by the antibody. X-ray analysis resolved Aβ residues 16-26 complexed with the antibody fragment. In this structure, the antibody made extensive contacts with the side chains of Aβ residues lysine 16, phenylalanine 19, phenylalanine 20, and aspartate 23, as well as with the Aβ peptide backbone. The researchers found the two phenylalanine side chains wedged deep within the antibody; their dive into the antibody’s folds consumed nearly half of the 960A2 surface area with which the antibody and Aβ interacted.
In the antibody binding pocket, residues 16 to18 assumed an extended coil configuration, lying flat over the solanezumab surface. Phenylalanine residues 19-20 projected down into the pocket. Residues 21-26 formed a helix held together by hydrogen bonds between Aβ residues. The researchers described the structure as an intermediate between a previously reported crystallographic β-sheet and a helical structure reported in NMR studies. They designated residues 16-20 (KLVFF) as the core epitope required for antibody specificity, and the helical region as affinity enhancer. They predicted that the core epitope would become masked in the context of Aβ oligomers, which could explain why solanezumab binds only monomers.
In a separate argument, Miles and colleagues claim that solanezumab recognizes hundreds of other proteins, a subset of which share the KLVFF motif. Researchers at Eli Lilly dispute this (see comment by DeMattos and colleagues, below, and Siemers et al., 2014).
While it is unclear what this hybrid Aβ structure represents, Colin Masters of the University of Melbourne, Australia, speculated that it could be an intermediate between monomeric and oligomeric forms. Miles agreed, writing, “The structure suggests to me that the conformation solanezumab recognizes is on a pathway to oligomerization.”
Next, Crespi and colleagues compared the binding pockets of a recombinant Fab fragment of solanezumab with that of crenezumab. The sequences are identical bar two residues—serine 33 and phenylalanine 36 in solanezumab are tyrosine and glycine, respectively, in crenezumab. Using homology modelling, the researchers determined that these changes would not affect the shape of the Aβ-antibody complex, and predicted that crenezumab mimics solanezumab folding and binds the same Aβ conformation. That said, exchange of the serine in solanezumab to tyrosine in crenezumab would remove a hydrogen bond between crenezumab and Aβ, while the second amino acid difference (glycine instead of phenylalanine) would lessen the hydrophobic interaction between the crenezumab and Aβ phenylalanines 19 and 20. Crespi suggested that these differences could explain why solanezumab binds Aβ with picomolar affinity, while crenezumab has a weaker, nanomolar affinity.
Unlike solanezumab, crenezumab is reported to bind aggregated forms of Aβ (Adolfsson et al., 2012). Miles suggested that perhaps solanezumab does as well, but this binding to aggregates may have gone unnoticed amid its high affinity for monomers. Ron Black, who headed Pfizer’s Phase 3 bapineuzumab trial, offered the same hypothesis.
How do the N-terminal antibodies compare? Researchers led by Guriqbal Basi, then at Elan, published a crystal structure showing 3D6, the murine parent of bapineuzumab, binds Aβ1-7 as an α-helix (see Jan 2009 conference news; Feinberg et al., 2014). Later, Miles solved a crystal structure that shows bapineuzumab binding a unique helix at the Aβ N-terminus, though he does not know which form of Aβ assumes this configuration (see Crespi et al., 2014).
In marked contrast, Biogen's patent application discloses that BIIB037/aducanumab binds residues 2 to 9 of Aβ in an extended coil conformation. Glutamate 3, phenylalanine 4, arginine 5, and histidine 6 all make contact with the antibody, with the phenylalanine and histidine being buried deep in the binding pocket. Epitope mapping identified amino acids 3-6 as the primary epitope for aducanumab.
Other monoclonal antibodies targeting Aβ at the N-terminus, for example 12A11 and others, also bind this linear Aβ structure and have sequence similarities with aducanumab in the binding region (see image at right and Basi et al., 2010). As for gantenerumab, it recognizes a linear conformation in Aβ as well, but its epitope comprises residues 1-11. Moreover, this antigen is bound by antibody in reverse orientation compared with the crystal structure of Aβ as reported for 12A11 (Feinberg et al., 2014). Furthermore, the complementarity determining regions of gantenerumab are different from those of aducanumab and the 12A11 family (see Bohrmann et al., 2012).
Epitope May Make a Difference in the Clinic
Are those just arcane details? Or does the atom-by-atom way each antibody binds Aβ influence the outcome of therapeutic trials? Some similarities in past trials hint as much, scientists say. Both the mid-region binding solanezumab and crenezumab had overall negative results, but sub-group and post-hoc analyses hinted at a benefit in mild AD (see Aug 2012 news; Oct 2012 news; July 2014 conference news). Both produced low rates of amyloid-related imaging abnormalities with brain edema (ARIA-E).
These similarities could arise from binding specificity and the extent of inflammation triggered, Masters suggested. Because solanezumab ignores plaques deposited in the brain, it largely avoids arousing the microglia-triggered inflammation associated with plaque clearance and ARIA-E. Crenezumab binds aggregated Aβ in addition to monomer. This would suggest that it triggers more ARIA-E, but whereas solanezumab is an IgG1 antibody, crenezumab’s IgG4 subclass may provide an explanation for the lower ARIA-E rate observed with crenezumab (see Jul 2012 news).
Trial results from N-terminal antibodies also share some commonalities. Bapineuzumab, gantenerumab, and aducanumab all reduced amyloid burden and provoked ARIA-E, in keeping with the idea that mobilizing plaque-bound Aβ activates microglia. But there were stark differences too. High ARIA-E sans cognitive benefit sunk bapineuzumab in 2012, and a Phase 3 trial of gantenerumab ended because the drug failed a futility analysis for lack of a cognitive benefit (see Dec 2014 news). In contrast, interim data from an ongoing Phase 1b trial showed aducanumab nearly eliminated brain amyloid with hints of a cognitive benefit.
Where do the differences come from? Masters speculated that the extended conformations recognized by aducanumab and gantenerumab could represent fibrillar Aβ species rather than the helical structure recognized by bapineuzumab. Furthermore, aducanumab was derived from a natural Aβ-specific antibody from an older person. This suggests this antibody recognizes an Aβ conformation that truly exists in the human brain and has clinical relevance, Masters said. Bapineuzumab and gantenerumab were raised against synthetic and phage-displayed peptides, respectively, and then humanized.
Even with a growing collection of antibody-Aβ structures in hand, it remains unclear whether targeting monomeric, oligomeric, or fibrillar forms of Aβ is the right strategy for treating AD, and which molecular interactions will prove to work best.—Jessica Shugart
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- Phase 3 Solanezumab Trials "Fail"—Is There a Silver Lining?
- The Solanezumab Benefit: Oh, So Small, But Probably Real
- Crenezumab Disappoints in Phase 2, Researchers Remain Hopeful
- A Close Look at Passive Immunotherapy Newbie, Crenezumab
- End of the RoAD for Gantenerumab? Roche Declares Prodromal Alzheimer’s Trial Futile
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- Crespi GA, Ascher DB, Parker MW, Miles LA. Crystallization and preliminary X-ray diffraction analysis of the Fab portion of the Alzheimer's disease immunotherapy candidate bapineuzumab complexed with amyloid-β. Acta Crystallogr F Struct Biol Commun. 2014 Mar;70(Pt 3):374-7. Epub 2014 Feb 20 PubMed.
- Basi GS, Feinberg H, Oshidari F, Anderson J, Barbour R, Baker J, Comery TA, Diep L, Gill D, Johnson-Wood K, Goel A, Grantcharova K, Lee M, Li J, Partridge A, Griswold-Prenner I, Piot N, Walker D, Widom A, Pangalos MN, Seubert P, Jacobsen JS, Schenk D, Weis WI. Structural correlates of antibodies associated with acute reversal of amyloid beta-related behavioral deficits in a mouse model of Alzheimer disease. J Biol Chem. 2010 Jan 29;285(5):3417-27. PubMed.
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No Available Further Reading
- Crespi GA, Hermans SJ, Parker MW, Miles LA. Molecular basis for mid-region amyloid-β capture by leading Alzheimer's disease immunotherapies. Sci Rep. 2015 Apr 16;5:9649. PubMed.