A slew of anti-Aβ antibodies have been developed for clinical trials, in hopes that they will spur the immune system to remove amyloid-β from the brain. But might they also shift the dynamic of Aβ aggregation? According to a paper published today in Nature Structural and Molecular Biology, four monoclonal antibodies against different epitopes of the peptide interfere with dramatically different aspects of this process. Employing in vitro kinetic and biochemical approaches, researchers led by Sara Linse and Oskar Hansson, both at Lund University in Sweden, reported that while solanezumab disrupts the initial formation of fibrils, both bapineuzumab and gantenerumab slow their elongation. Aducanumab stemmed the formation of oligomers, a secondary nucleation process fueled by interactions between monomers and fibrils. It may work by coating the surface of fibrils, effectively removing them as a source of oligomerization. Only these four antibodies were tested. Biogen has filed for FDA approval for aducanumab for the treatment of Alzheimer’s disease.
- In vitro at least, investigational Aβ antibodies interfere with different phases of Aβ fibrillization.
- Solanezumab inhibits primary nucleation; bapineuzumab and gantenerumab slow elongation; aducanumab thwarts secondary nucleation of oligomers.
- Aducanumab may completely coat fibril surfaces, while other antibodies leave gaps.
“This paper really makes the point that these monoclonal antibodies are not doing the same thing—not by a long shot,” said Eric Siemers of Siemers Integration, LLC. While working at Lilly, Siemers oversaw clinical development of solanezumab. While it is generally accepted that oligomers are the most toxic of Aβ species, only results of clinical trials can tell whether antibodies that reduce them also prove to be the most efficacious, Siemers added.
Indeed, while plaques packed with Aβ fibrils are the obvious pathological hallmark of AD, studies have increasingly pointed to those ephemeral Aβ species—low-molecular-weight oligomers—as inflicting the most damage to neurons (Benilova et al., 2012; Reiss et al., 2018). Oligomers are in constant flux, complicating efforts to detect them or to gauge the effectiveness of monoclonal antibodies at vanquishing them.
Oligomers do not primarily form when monomers bump into one another; rather, scientists believe most are born of secondary nucleation (Dec 2009 news). This happens when monomers interact with the surface of Aβ fibrils, an encounter that not only makes monomers more likely to meet, but also goads them into oligomerizing.
Previously, Linse and colleagues had developed an in vitro assay system to study Aβ aggregation kinetics (May 2013 news). They found that each phase of the Aβ aggregation process—including the primary nucleation of monomers into fibrils, the elongation of fibrils, and the secondary nucleation of monomers into oligomers at the fibril surface—is marked by a unique kinetic signature, with its own rate constant (Cohen et al., 2012; Arosio et al., 2015; Dear et al., 2020).
Do monoclonal antibodies perturb different parts of this process? And do any of them throw a wrench into secondary nucleation? Linse and Hansson set out to test this. They asked various companies for samples of their antibodies. Biogen offered a mouse version of aducanumab, and also mouse versions of solanezumab, bapineuzumab, and gantenerumab that Biogen had been studying. Hansson told Alzforum that he tried hard to get other antibodies, including BAN2401, as well, but to no avail.
Of the four antibodies Linse and Hansson were able to obtain, each binds a different spot on the Aβ peptide, which ultimately dictates which species of Aβ it prefers (May 2015 news). Solanezumab has a strong penchant for Aβ monomers, because monomers display its epitope—residues 16-26—while in aggregates this epitope is buried. Gantenerumab binds aggregated forms of Aβ, including fibrils. Although its epitope, residues 3-11 and 18-27, is hidden within most fibrils, it may be exposed at their ends. Bapineuzumab and aducanumab latch on to amino acids 1-5 and 3-7, respectively, part of the peptide’s freewheeling N-terminal region, which has little secondary structure. While both epitopes are exposed in all species of Aβ, both antibodies bind tightest to aggregated forms of the peptide.
To test how the antibodies would shift the dynamics of Aβ fibrillization, the researchers added increasing concentrations of each to a solution of Aβ monomers, then turned up the temperature from zero to 37 degrees to initiate the aggregation process. In some reactions, the researchers added preformed aggregate seeds to bypass the primary nucleation step and focus on elongation and secondary nucleation. Using thioflavin T fluorescence to track aggregation over time, the researchers compared how each antibody tweaked the kinetics of each phase of aggregation.
In a nutshell, they found that solanezumab primarily disrupted primary nucleation, while gantenerumab and bapineuzumab nipped elongation of fibrils in the bud. Aducanumab inhibited secondary nucleation (see model below). At the lowest concentration used, 0.25 nM, aducanumab cut secondary nucleation by nearly half; at 100 nM, threefold. The scientists obtained similar results when they carried out the fibrillization in cerebrospinal fluid to mimic a bit more closely what might happen in the brain.
Assembly Line. In a solution assay, solanezumab (m266) preferentially blocked primary nucleation; bapineuzumab (3D6) and gantenerumab curtailed fibril elongation; aducanumab reduced secondary nucleation. [Courtesy of Linse et al., Nature Structural and Molecular Biology, 2020.]
Did disrupting secondary nucleation translate into a meaningful drop in Aβ oligomerization? To find out, the researchers attempted to measure oligomers biochemically—no small task. They used size-exclusion chromatography (SEC) followed by matrix-assisted laser desorption ionization (MALDI) mass spectrometry to detect free oligomers at the halfway point in their aggregation assay. With this method, they found that aducanumab reduced the concentration of these oligomers by more than 80 percent compared to an isotype control antibody. Bapineuzumab took oligomers down by about a third, while gantenerumab had no effect. In the presence of solanezumab, which latches onto monomers, aggregation never took off in the first place in this in vitro test, which means the researchers were unable to measure any secondary nucleation effects. This might be different in the brain of a person who has already accumulated plaques.
How did the antibodies’ interactions with Aβ relate to their mechanisms of action? To investigate, the researchers examined how each binds monomers and fibrils using a technique called microfluidic diffusional sizing. It quantifies interactions between macromolecules in real time within a flowing solution, rather than trying to capture inherently dynamic species with biochemical techniques (Arosio et al., 2016). Oligomers were too fickle to measure in this assay.
In agreement with previous studies, the scientists found that aducanumab bound fibrils four orders of magnitude more tightly than it did monomers. Notably, the diffusion data revealed one aducanumab molecule for every 4.5 Aβ42 monomers. This stoichiometry jibes with the size of the antigen-binding region of aducanumab, which spans the length of about five monomer rungs within a fibril. Together, the data paint a picture of aducanumab coating the entire surface of an Aβ fibril, explaining its rebuff of monomers from the fibril surface.
Both gantenerumab and bapineuzumab also preferred fibrils over monomers in this assay, but the stoichiometry indicates that these antibodies were spaced farther apart. Gantenerumab bound once every 44 monomers, while bapineuzumab bound once every 40. This is consistent with kinetics suggesting that these antibodies slowed fibril elongation more than secondary nucleation. Finally, solanezumab had a high affinity for monomers and did not bind fibrils at all, consistent with its inhibition of primary nucleation.
Finally, the researchers compared the kinetic effects of aducanumab with that of Brichos—a molecular chaperone that powerfully blocks secondary nucleation (Cohen et al., 2015). Though weaker than Brichos, aducanumab changed the kinetics of the aggregation in the same way, while the other antibodies did not (see image below).
Predictive Pentagrams? Brichos (gray), aducanumab (green), solanezumab (purple), bapineuzumab (blue), and gantenerumab (red) shift nucleation (pKn), elongation (pK+), and secondary nucleation (pK2) kinetic constants, as well as the binding constants for monomers (pKDm) and fibrils (pKDf). Colored slices depict the effect size, from zero at the center to maximal at the pentagram tips. Aducanumab works similarly to Brichos, affecting secondary nucleation and fibril binding. [Courtesy of Linse et al., Nature Structural and Molecular Biology, 2020.]
Overall, the findings suggest that while all four monoclonals block Aβ aggregation at the macroscopic level, they do so by meddling with different parts of the process. The finding that aducanumab derails oligomer secondary nucleation bodes well for potentially dampening amyloid toxicity, Linse said. However, she also noted that aducanumab was a relatively weak blocker of secondary nucleation compared to Brichos. Some of the authors co-founded Wren Therapeutics, which is now using kinetic assays to screen for small molecules that more powerfully and specifically derail secondary nucleation of Aβ, unlike Brichos.
This work did not study a host of other antibodies, previously or currently in clinical development, that might perturb Aβ aggregation, including crenezumab, donanemab, and BAN2401, which was developed specifically to target the most toxic protofibrils (Apr 2011 conference news).
“These findings suggest that aducanumab … should, as an early intervention or prophylaxis, result in cognitive benefit if the amyloid hypothesis holds water,” commented Luke Miles of St. Vincent’s Institute of Medical Research in Fitzroy, Australia. “If realized, these benefits might well be enhanced in combination with a specific inhibitor of primary oligomer nucleation, such as solanezumab,” he added.—Jessica Shugart
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No Available Further Reading
- Linse S, Scheidt T, Bernfur K, Vendruscolo M, Dobson CM, Cohen SI, Sileikis E, Lundqvist M, Qian F, O'Malley T, Bussiere T, Weinreb PH, Xu CK, Meisl G, Devenish SR, Knowles TP, Hansson O. Kinetic fingerprints differentiate the mechanisms of action of anti-Aβ antibodies. Nat Struct Mol Biol. 2020 Sep 28; PubMed.