The 16th International AD/PD meeting was also the first hybrid version, drawing some 3,360 participants online, or in person in Barcelona, Spain. Scientists outlined Roche’s new Phase 3 secondary prevention trial of gantenerumab for the first time. Attendees also heard updates on Aduhelm, lecanemab, and α-synuclein immunotherapy. On the basic science front, tau emerged as an instigator of interferon responses, while presentations on postmortem single-nuclei data showed how collectives of cell subtypes, and their cross talk, wax and wane together with age and with AD pathology.
Gantenerumab Prevention Trial in Sporadic Alzheimer's Begins
Many researchers believe anti-amyloid antibodies may offer the most benefit in a preventative paradigm, before plaques have triggered extensive downstream damage. Now Roche will test this idea in a Phase 3 secondary prevention trial of its anti-amyloid antibody gantenerumab. At the 16th International Conference on Alzheimer’s and Parkinson’s Diseases, held March 15-20 in Barcelona, Spain, and virtually, Roche’s Szofia Bullain offered an overview. Dubbed Skyline, the trial will enroll 1,200 cognitively healthy, amyloid-positive participants in 17 countries. Researchers will look for delayed decline on a sensitive cognitive composite after four years on drug or placebo. “This trial will tell us if going quite early with this molecule can delay or avert symptoms,” Rachelle Doody at Roche told Alzforum.
Others hailed the news. “I am very excited that with the launch of Skyline, a third promising anti-amyloid immunotherapeutic is being tested in the presymptomatic (preclinical) sporadic AD population,” Paul Aisen at the University of Southern California, San Diego, wrote to Alzforum. He collaborates on the trial.
Jeffrey Cummings at the University of Nevada, Las Vegas, noted broader implications. “Initiation of the Skyline trial suggests that industry sponsors will continue to investigate treatment of Alzheimer’s disease despite the reimbursement uncertainties created by the Coverage with Evidence Development (CED) requirement proposed by the Centers for Medicare and Medicaid Services,” he wrote (full comments below).
So far, most trials of anti-amyloid antibodies have enrolled people with early symptomatic AD. Secondary prevention studies are harder to run, because it is difficult to recruit participants and detect changes in people who have no overt symptoms of the disease. To design Skyline, Roche scientists teamed up with researchers at Massachusetts General Hospital in Boston, the Banner Alzheimer’s Institute’s Alzheimer’s Prevention Initiative in Phoenix, and the University of Southern California Alzheimer’s Therapeutic Research Institute in San Diego. These groups have designed previous secondary prevention trials in sporadic AD, including A4, AHEAD 3-45, and API’s Generation program, and were able to offer advice on how to recruit and screen presymptomatic candidates and choose outcome measures.
Skyline will enroll people from 60 to 80 years of age who are amyloid positive by either PET scan or cerebrospinal fluid Aβ. Sites have the option to prescreen potential participants by plasma p-tau181 and plasma ApoE protein isoform levels to identify those likely to be amyloid-negative, cutting down on screen failures. Bullain expects this step to raise the amyloid positivity at screening from 15 to 30 percent. Audience members at AD/PD suggested recruits should be PET positive for tangles as well as amyloid, to ensure they are likely to progress to symptoms within the time frame of the trial.
Roche aims to recruit a diverse population. Since 2019, an external diversity council has advised Roche on outreach to underrepresented groups. Strategies can include providing practical resources, such as transport to medical appointments, Doody noted.
Half the participants will receive placebo and half gantenerumab, but dosing will be more frequent than in the Phase 3 GRADUATE studies, in which participants received two subcutaneous injections of 510 mg per month. Because some people find it easier to take medication on a weekly schedule, Roche has been testing a weekly dosing regimen in its ongoing Graduation program of newly recruited people with prodromal to mild AD.
In Skyline, participants will have the option to take 510 mg biweekly or 255 mg weekly. In either case, injections can be administered at home by the participants themselves or a family member, lessening travel time and the overall burden of trial participation. Most amyloid immunotherapies are administered by intravenous infusion in the clinic, but that is starting to change, with other programs testing subcutaneous administration as well. “We view subcutaneous administration as a major public health benefit,” Doody said.
If participants on placebo develop symptoms during the trial, they will be switched to active therapy, effectively making the trial an open-label extension for people in the prodromal phase of the disease. This assumes gantenerumab demonstrates efficacy in the Phase 3 prodromal trials reading out later this year. Patients already on drug who develop symptoms will undergo a mock titration to maintain the study blind up to that point, Bullain said.
Pierre Tariot at Banner praised the gantenerumab flexible dosing schedule, and other innovative aspects of the trial design. “I believe these features will appeal to a large number of people around the globe,” he noted (full comment below).
The primary outcome measure will be change on the PACC5 cognitive composite, which detects subtle memory deficits in preclinical AD (Jun 2014 news). As a secondary outcome, researchers will assess the time it takes participants to progress to cognitive impairment, defined as a CDR greater than zero. Doody considers both measures important. The PACC5 may be more sensitive because it assesses change along a continuum, while progression to symptomatic disease is more clinically meaningful. The Food and Drug Administration has said it would consider slowing of cognitive decline alone as the basis for a drug approval, though the agency did not specify particular tests that would qualify (Nov 2018 news).
Other secondary outcomes will include functional measures such as the Activities of Daily Living and the Cognitive Function Instrument. For biomarkers, all participants will undergo volumetric MRI and donate blood, which will be assessed for Aβ42, Aβ40, p-tau181, and the neuronal injury marker NfL. A subset of participants will undergo lumbar puncture and amyloid and tau PET.
Cummings welcomed the inclusion of blood biomarkers, which he noted would help researchers learn which of them changes first as a person develops AD. “[That] is encouraging as a means of identifying individuals who would benefit from prevention therapy,” Cummings wrote.
Gantenerumab was previously tested in the Dominantly Inherited Alzheimer Network secondary prevention study, where, in addition to mopping up plaques, it dropped CSF p-tau181 and total tau and slowed the rise in CSF NfL (Jun 2021 news). “That increased our confidence that there is a biologically relevant outcome from lowering amyloid,” Doody noted.
Gantenerumab continues to be evaluated in an open-label extension of that study, and has also been chosen for DIAN’s forthcoming primary prevention study in familial AD mutation carriers who do not yet have amyloid (Dec 2021 press release). The only previous AD primary prevention trial was API’s Colombian study, which is evaluating Roche and Genentech’s crenezumab in presenilin mutation carriers (Aug 2019 news). That trial is expected to read out this year, as are gantenerumab’s GRADUATE studies.
Doody stressed that Roche is committed to the AD field and increasing its investment every year. “That needs to be understood, so people don’t hang their hopes on the outcome of one study,” she said.
Meanwhile, several other anti-amyloid antibodies are in secondary prevention studies, including lecanemab in AHEAD3-45 and donanemab in TRAILBLAZER 3. Tariot noted that each of these trials is important, as the antibodies have different mechanisms of action and the trials test different treatment regimens. “Together, these trials represent a huge investment, for which I give thanks: We should not put all of our eggs in one basket,” Tariot said. “The stakes are enormous. ‘Merely’ delaying the onset of AD symptoms would cut the prevalence of the symptomatic phase of the disease in half.”—Madolyn Bowman Rogers
In First for the Field, α-Synuclein PET. Only for Multiple System Atrophy
For the first time, scientists have detected α-synuclein aggregates lurking in the brains of the living. This thanks to 18F-ACI-12589, a new tracer developed by AC Immune in Lausanne, Switzerland. Presented at AD/PD 2022, held March 15-20 in Barcelona, Spain, and online, the first PET scans using the tracer showed uptake in the cerebellar white matter of people with multiple system atrophy (MSA). 18F-ACI-12589 bound specifically to α-synuclein fibrils while shunning the other amyloids that often accompany Lewy bodies—namely amyloid plaques and tau tangles. Alas, PET signals were undetectable in the brains of people with other synucleinopathies, including Parkinson’s disease and dementia with Lewy bodies, despite selective binding of the tracer to postmortem brain samples of people who had died with those disorders.
Oskar Hansson of Lund University in Sweden led clinical studies of the tracer and unveiled the first glimpse of the PET scans at the meeting. He thinks the findings bode well for detection of α-synuclein pathology during life in people with MSA, and will aid in diagnosis as well as monitoring of treatment effects in clinical trials.
Why the tracer fell short in people with other synucleinopathies remains unclear. Hansson and others suspect it is because they have far fewer deposits, and tracer binding may have been too weak to detect them. Differences in α-synuclein fibril conformation, or in where the aggregates are in the brain, could also contribute, Hansson said.
“If verified in larger samples, this would represent a landmark moment, similar to first reports of imaging amyloid with PIB and tau with FTP (at the time T807),” wrote Gil Rabinovici of the University of California, San Francisco, in a comment to Alzforum. “Overall, while preliminary, these are exciting data, and I am cautiously optimistic that the field may have its first foothold into synuclein imaging.”
The road to synuclein imaging has been long and strewn with obstacles. Compared to amyloid plaques and neurofibrillary tangles, α-synuclein fibrils exist in low concentrations in the human brain. This, combined with its intracellular location and myriad structural conformations, makes α-synuclein a tough target for PET tracers. Multiple contenders have been developed over more than a decade of trying. All of them failed, most due to subpar binding affinity, off-target binding to aggregates of Aβ or tau, or both (for review, see Korat et al., 2021; Alzghool et al., 2022).
To meet the challenge, the Michael J. Fox Foundation had gathered consortia of scientists over the years (Eberling et al., 2013; Merchant et al., 2019). According to MJFF’s Jamie Eberling, the foundation is currently funding 10 different groups, including AC Immune. In 2016, it even offered a $2 million prize to the first group to develop an α-synuclein tracer. While AC Immune appears to be the top contender, Eberling said more data is needed to support their tracer's specificity. She noted that other groups are edging close to testing theirs in the clinic. “I would love to award that prize,” Eberling said.
At AD/PD 2022, AC Immune’s Francesca Capotosti presented preclinical data for ACI-12589. The tracer emerged from the company’s Morphomer platform, a library of some 15,000 small molecules designed to cross the blood-brain barrier, enter cells, and bind with high specificity to pathological forms of different amyloidogenic target proteins. Capotosti said more than 2,000 compounds from this library were designed and selected as candidates for α-synuclein tracers. She emphasized that the researchers used α-synuclein aggregates derived from brain samples of people with PD, then further validated using aggregates from other synucleinopathies.
The use of brain-derived α-synuclein to select potential tracers sets AC Immune’s approach apart from other groups, most of which rely upon synthetic α-synuclein fibrils, at least in initial screening steps. Fibrils generated in vitro are structurally distinct from brain-derived fibrils, and Capotosti told Alzforum that AC Immune has avoided using them for this reason. “I think our approach to screen on brain-derived material has been one of key elements that has allowed the discovery of AC-12589,” she wrote.
Previously, the researchers used this approach to develop a tau tracer, PI-2620, which binds 4R forms of the protein that get tangled up in progressive supranuclear palsy and other primary tauopathies (Jul 2020 news). Efforts are underway to develop a TDP-43 tracer as well, and at AD/PD, AC Immune’s Tamara Seredenina reported that lead candidates detect this pathology in brain tissue from people with frontotemporal lobar degeneration who had FTLD-TDP Type A, B, or C pathology.
Autoradiography. In brain sections from people with familial PD or MSA, 3H-ACI-12589 bound α-synuclein aggregates (top panels) with minimal non-specific binding (middle). The distribution matched the immunohistochemistry of phosphorylated α-synuclein (bottom). [Courtesy of AC Immune.] [Courtesy of AC Immune.]
ACI-12589 was the top compound to emerge as a potential α-synuclein tracer. A tritiated version bound Lewy body inclusions and Lewy body neurites in affected brain regions in people who had died with a synucleinopathy, be it familial PD, idiopathic PD, PDD, DLB, or MSA. Its distribution in these brain sections matched immunohistochemistry of phosphorylated α-synuclein, suggesting the tracer was binding its intended target. Capotosti reported similar findings when the tracer was radiolabeled with fluorine-18.
The tracer did not bind to brain sections or homogenates from people without α-synuclein pathology. It also ignored tau tangles or Aβ-plaque-ridden brain sections from people with AD, suggesting specificity for α-synuclein, Capotosti said.
Finally, Capotosti reported that the tracer only weakly bound monoamine oxidase B. This enzyme co-localizes with Lewy bodies and has ensnared other tracers in off-target binding, including some tau tracers.
Would ACI-12589 reach its target in the brain of a living person? Ruben Smith of Lund University presented results from a clinical study that monitored uptake in 25 participants via PET scan. Eight were healthy controls, seven had PD, two DLB, and eight MSA. MSA is broadly divided into cerebellar and parkinsonian subtypes, and this study included six people with MSA-c and two with MSA-p. Initially, Smith monitored uptake of the tracer continuously over 90 minutes. He found a favorable kinetic profile, with the tracer sticking around in the brain long enough to decipher its retention, and then washing away effectively thereafter.
A signal leapt out of the cerebellum in all people with MSA, Smith reported. In agreement with the distribution of α-synuclein pathology in this disorder, Smith saw tracer retention in the cerebellar white matter and a stalk-shaped structure called peduncles, but not in cerebellar gray matter. Though people with either MSA subtype evinced binding in these regions, it was greater, on average, in those with MSA-c. Tracer uptake in cerebellar white matter completely distinguished people with MSA from controls and from those with other synucleinopathies, none of whom had significant uptake in this region.
Smith spotted some unspecific binding in the pons (see image below). This signal was less visible in MSA due to atrophy in the region and to masking by the stronger signal in the cerebellum. It is more easily seen in PET scans overlaid on MRI scans, noted Hansson. He and Smith noted that this unspecific binding was relatively weak, and did not detract from the specific signal emanating from the cerebellar white matter.
Finally, α-Synuclein PET? 18F-ACI-12589 bound cerebellar white matter and cerebellar peduncles (white arrows) in people with MSA (bottom) but not in healthy controls (top). The green blob in controls reflects unspecific binding in the pons, which is masked by the stronger specific signal in MSA cases. [Courtesy of Ruben Smith, Lund University.]
Alas, in people with PD, PDD, or DLB, there was nothing much to see. Smith did spot some uptake in the basal ganglia of a person with PD as well as in a person with MSA-p, but some controls also had this signal, suggesting it was non-specific. Similarly, three MSA-p patients had strong tracer binding in the globus pallidus, but so did one control. In all, the only disease-specific signals arose from the cerebellar white matter and peduncles in people with MSA.
Quantity or Quality? Researchers at AD/PD were excited that a tracer appears to work in MSA, but their enthusiasm was tempered by the lack of a signal in people with the more common synucleinopathies. “Why do you think it didn’t work in PD?” asked session co-chair Tamara Shiner of Tel Aviv University. Smith was unsure, but said that given the small number of people scanned so far, including only seven with PD, it might be too soon to close the book on this tracer’s performance in other synucleinopathies.
Among the main α-synucleinopathies, MSA progresses faster and comes with more Lewy bodies than do the others, suggesting the tracer’s affinity may simply be too low to pick up all deposits.
Conformation of α-synuclein aggregates could also play a role, Hansson and Smith believe, as the fibrils are known to twist into distinctive shapes in each synucleinopathy (Mar 2020 conference news; Tarutani et al., 2018). Curiously, though, the tracer bound well to α-synuclein in brain sections from people with these different disorders, suggesting it was capable of latching onto multiple forms. Reaching a target in the brain of a living person is a far higher bar for a tracer than sticking to it in a brain section.
Robert Mach of the University of Pennsylvania, Philadelphia, heads a massive multi-institutional effort, called the Center without Walls for Imaging Proteinopathies with PET, to develop tracers for α-synuclein and 4R-tau. He favors the idea that differences in structure best explain why the tracer bound in MSA and not in other synucleinopathies. He noted that a wealth of data, from binding of fluorescent probes and tracers in his own work to antibody binding experiments and cryo-EM, support the idea that structural differences in α-synuclein influence the binding of ligands. As to why the tracer bound to multiple forms of α-synuclein in tissue sections, but not in vivo, Mach said that tissue sections are subjected to biochemical procedures that might expose binding sites that are normally cloaked in the human brain.
Chet Mathis of the University of Pittsburgh is a co-developer of PiB and has been searching for an α-synuclein tracer for years. He called the findings a definite advance for the field, noting that while past α-synuclein tracer candidates had bound to α-synuclein in the brains of people with MSA, this is the first one that does so specifically, without off-target binding to Aβ or tau. However, he considers the lack of binding in people with PD or DLB a disappointment that likely boils down to the tracer’s low affinity.
Capotosti reported that the tracer 's dissociation constant was between 8 to 30 nM for α-synuclein aggregates in tissue slices and brain homogenates from different α-synucleinopathies. This seemed shockingly weak to Mathis, who said most researchers agree the sweet spot for tracer affinity is 1 nM or lower. Oddly, the tracer’s affinity for brain-derived α-synuclein in vitro was weaker for MSA than it was for PD. Capotosti reported a Kd of 17 nM for tracer binding to α-synuclein in a frontal cortex section of a person with PD, compared to 30 nM in a cerebellum section from a person with MSA. Similarly, the tracer bound with Kd values of 8 nM and 22 nM in brain homogenates from people with PD and MSA, respectively.
“The surprising thing is that it does work in MSA despite this low affinity,” Mathis said. For whatever reason—be it abundance, location, or conformation—MSA α-synuclein appears to be more forgiving than PD α-synuclein in terms of the tracer affinity required to detect it, Mathis added.
Like Mathis, Mach was also surprised that a tracer with such a low affinity could work. On the one hand, those findings are highly encouraging, he said, because they mean that tracer candidates need not eclipse a 1 nM affinity before being put to the test in clinical studies. On the other hand, he said that the results beg a major question: “What are the properties of the ligand that enabled it to succeed where others have failed?”
Hansson told Alzforum he plans to submit a detailed manuscript soon. Capotosti said efforts are underway to test and optimize 18F-ACI-12589 in people with MSA and other synucleinopathies, and to try other promising compounds from their Morphomer library that may work for PD.
Progress on other α-synuclein tracers was reported at AD/PD, as well. Felix Schmidt of MODAG, a biotech company based in in Wendelsheim, Germany, and funded in part by MJFF, reported specific binding of their compound, MODAG-005, in tissue sections from people with PD and MSA. This tracer bound to brainstem and midbrain sections in people with PD, as well as cerebellar white matter in people with MSA. In mice, C11-MODAG-005 entered the brain and labeled recombinant α-synuclein fibrils that had been previously injected.
Ruiqing Ni, of the University of Zurich also reported promising data from α-synuclein tracer candidates in animal models. Specifically, Ni reported that one was taken up in the striata of M83 mice, a model for PD, but not in models with amyloid or tangle accumulation.—Jessica Shugart
Using Lecanemab Trial Data to Determine Maintenance Dose
With several anti-amyloid antibodies now proven to clear plaque from the human brain, researchers are turning their attention to fine-tuning immunotherapy for Alzheimer’s disease. At the 16th International Conference on Alzheimer’s and Parkinson’s Diseases held March 15-20 in Barcelona, Spain, speakers showed how analyses from the lecanemab program are helping them model antibody efficacy and figure out which maintenance dose might keep plaques from coming back. Technical improvements in measuring plasma biomarkers are enabling some of these advances. “The science has converged,” Michael Irizarry of Eisai said during a panel discussion, noting advancements in both biomarkers and therapies. He expects upcoming trials to provide even more information on dosing, safety, and efficacy.
Immunotherapy can banish plaque, but what happens next? The lecanemab Phase 2 trials offer some clues. At the high dose of 10 mg/kg biweekly, four in five participants were deemed amyloid-negative after 18 months (Jul 2018 conference news), and their stored plasma samples subsequently revealed that their Aβ42/40 rose and p-tau181 fell in tandem with plaque removal (Nov 2021 conference news).
At AD/PD, Eric McDade of Washington University in St. Louis added new data from this analysis. He tied plasma marker changes to clinical outcome. McDade found that larger changes in Aβ42/40 and p-tau181 correlated with slower slippage on the CDR-SB at the group level. For Aβ and p-tau181 the correlations were 0.57 and 0.47, respectively. The data hint that plasma markers could help monitor treatment response, McDade noted.
Between the end of Phase 2 and the start of the open-label extension study, there was a roughly two-year treatment gap, hence these data offer a glimpse of the long-term effects of amyloid removal. Previous reports had noted sustained improvement on biomarkers (Aug 2021 conference news) and in Barcelona, McDade put specific numbers to this. He reported that following 18 months of treatment at 10 mg/kg biweekly, plaque load and plasma p-tau181 both crept back up by about a quarter during this gap, while the plasma Aβ42/40 shifted halfway back to where it had started. These data suggest that staying on lecanemab might be necessary to maintain benefits, McDade said. This is quite different from the approach being taken by Eli Lilly with donanemab, where treatment is stopped once plaques are gone. Lilly scientists predict that it would take three to four years for amyloid to creep back into positive territory, and as long as 14 years to return to baseline levels (Nov 2021 conference news).
Feeding Phase 2 and gap period lecanemab data into a model of what such long-term treatment might look like led to a prediction that a maintenance dose of 10 mg/kg quarterly would prevent plaque growth, but that plasma markers would gradually worsen. A dose of 10 mg/kg monthly, however, would keep plasma markers flat, as well. McDade believes this prediction could change if the initial treatment more aggressively drove markers further into the normal range with higher dosage or longer duration.
Walking the Line. Modeling data predicts that without maintenance dosing (black), all AD biomarkers will rise after treatment stops at 18 months. A quarterly maintenance dose (blue) would keep plaque down (left) but not plasma p-tau181 (middle) or plasma Aβ42/40 (right), while a monthly dose (turquoise) would keep all biomarkers flat. Continued biweekly dosing (pink) would drive all lower still. [Courtesy of Eric McDade.]
Why is maintenance dosing required once plaque is gone? At AD/PD, Eisai’s Antonio Cabal suggested that protofibrils and oligomers that remain in interstitial fluid after plaque removal might be behind the worsening of plasma markers during the gap. These small soluble species are believed to be the most toxic forms of Aβ. If only there were good estimates of their concentration, doctors might be able to adjust dosing to mop them up, too, and prevent further damage to synapses and neurons, Cabal said.
Alas, such ISF markers do not exist. Meanwhile, McDade believes plasma biomarkers will be a sensitive tool for tracking drug effects. “These peripheral measures become critical when we think about monitoring chronic therapy and reaching maximal effect,” McDade said.
Irizarry described how Eisai is testing these ideas. Each participant in the Phase 2 OLE starts below the threshold for amyloid positivity, and will go on a maintenance dose of 10 mg/kg either monthly or quarterly. Researchers will track their changes in amyloid PET, plasma Aβ42/40, and plasma p-tau181 to find the minimum dose that maintains treatment gains.
This type of long-term treatment could become onerous if it requires monthly trips to a clinic for intravenous infusions for years or decades. To establish a more convenient route of administration, Eisai will use the open-label extension of its ongoing Clarity Phase 3 trial to explore subcutaneous injection. As each participant reaches the end of the blinded study period, they can switch from monthly IV to weekly subcutaneous, which can be done at home. All major anti-amyloid antibody companies are currently testing subcutaneous dosing.
Some safety benefits might be had this way, as well, Irizarry said. Plaque clearance correlates with the average plasma concentration of lecanemab, while the brain edema known as ARIA-E correlates with lecanamab’s maximum concentration. Because antibody makes its way into the plasma more slowly following a subcutaneous bolus, and it reaches a lower maximal concentration, Irizarry hopes this route of administration may produce less ARIA, perhaps half that seen with IV dosing.
Predicting Antibody Effects
How might the dynamics of plaque clearance and soluble species change when other antibodies are used? Each antibody currently in trials has its own distinct characteristics, recognizing different species of Aβ with varying affinities. In Barcelona, Cabal presented a quantitative systems pharmacology (QSP) model of Aβ aggregation kinetics that takes into account these affinities and the effect of age and APOE genotype on slowing clearance. Cabal then plugged different antibodies into this model, assuming 1.5 years of treatment followed by 2.5 years of follow-up. The idea was to link each antibody's distinct binding characteristics to their clinical effects.
The model showed that solanezumab and crenezumab, which bind Aβ monomers and oligomers, respectively, would halt plaque growth but not reduce existing plaque. Bapineuzumab, gantenerumab, and lecanemab, which bind fibrils, would remove plaque, with lecanemab having the strongest effect according to the Eisai model and gantenerumab the next. For protofibrils, the model forecast lecanemab to mop up the most, with crenezumab next, and the other antibodies doing little. Cabal said these predictions match observed pharmacologic and pharmacodynamic data for each antibody.
Cabal did not model donanemab or aducanumab treatment. Donanemab, which recognizes a pyroglutamated form of Aβ, cleared plaque rapidly in Phase 2, in many cases banishing deposits by six months (Mar 2021 conference news).
The QSP model also offered insight into ARIA-E. Researchers believe this condition arises when antibodies bind to Aβ at blood vessels, disrupting their walls and allowing fluid to leak into the brain. But do fibrils or protofibrils do this? Cabal modeled for both, and found that fibril binding produced the best fit to actual data. Fibrillar amyloid in the perivascular space, aka cerebral amyloid angiopathy, present at the start of treatment may explain differing rates of ARIA, Cabal concluded.
While researchers now have enough data to model what happens during immunotherapy, they still know little about what the AD brain looks like once plaque is gone. How does its biochemistry change? How do microglia respond? Autopsy data from immunotherapy trial participants will be crucial for determining this. “These are fascinating open questions,” Irizarry said.—Madolyn Bowman Rogers
Scientists Re-Analyze Aduhelm Data, Try to Parse Who Benefits
Last June’s accelerated FDA approval of Aduhelm based on amyloid plaque removal left unresolved whether the antibody truly shores up cognitive function. At the 16th International Conference on Alzheimer’s and Parkinson’s Diseases, held March 15-20 online and in Barcelona, Spain, speakers picked at this question with new analyses of the old Phase 3 data, and some argued that it does. Biogen presented an updated meta-analysis of anti-amyloid immunotherapies, reporting that plaque removal does slow cognitive decline across multiple trials and molecules. Other speakers sussed out patient characteristics that could help predict who will benefit from immunotherapy, showing analyses from the aducanumab and donanemab programs that hinted at possible effects of age and baseline p-tau181. Meanwhile, Philip Scheltens of VU University, Amsterdam, offered a sobering assessment of how heterogeneity in the AD patient population muddies clinical endpoints and may mask drug effects.
Despite their longstanding difficulties in demonstrating a meaningful benefit, pharma scientists remain focused on anti-amyloid therapy. They note its strong effects on multiple biomarkers of amyloid, tau, and neuroinflammation. “Biomarkers show changes in the underlying pathological state,” said Mark Mintun of Eli Lilly, which makes donanemab, during a panel discussion. “At Lilly, we have a lot of confidence that this is the right track.”
Aduhelm Controversy Continues
Nonetheless, the first antibody to gain approval in the U.S. is still treading a rocky road. After Biogen’s mixed Phase 3 results got a thumbs-down from the FDA's advisory committee, the agency granted accelerated approval based on plaque removal being “reasonably likely” to translate into a clinical benefit (Nov 2020 news; Jun 2021 news). The Phase 3 efficacy data on which this decision rests remained unpublished for three years after these trials were stopped, and nearly a year after aducanumab was approved.
Now the data are published, in the March 16 Journal of Prevention of Alzheimer’s Disease, an 8-year-old specialty journal. According to press reports, in 2021 Biogen withdrew the paper from consideration by JAMA (Axios news). Aducanumab's Phase 1 data had appeared in Nature (Sevigny et al., 2016).
JPAD ran three accompanying editorials. Ron Petersen, Mayo Clinic Rochester, Minnesota, cautioned that the results of the EMERGE trial must be interpreted in the context of the negative results of ENGAGE, meaning the impact of plaque removal at this stage of disease may be minimal. Zaven Khachaturian of the Campaign to Prevent Alzheimer’s Disease offered no specific opinion on the aducanumab data, but drew a historic analogy with the flawed, first-approved acetylcholinesterase inhibitor, Tacrine. He wrote, “Even if in the long run [aducanumab] fails the promise of becoming a treatment of choice, the FDA’s conditional approval has already created an encouraging environment for further investments in R&D of drugs in this class.” Lon Schneider, University of Southern California, Los Angeles, reminded the journal's readers of the flaws in the two trial's data analysis and presentation after the futility analysis ended the trials early, calling the JPAD paper's claim of a clinically meaningful impact "so wrong on many levels."
Like Schneider, other commentators were unimpressed. Madhav Thambisetty at the National Institute on Aging, Bethesda, Maryland, noted that the paper skirted key questions such as whether the faster placebo decline in the positive EMERGE study could have driven the apparent treatment effect. Rob Howard at University College London was quoted as saying, “The peer review has apparently been so gentle and unrevealing that they might as well not have bothered” (Endpoints news).
At AD/PD, some presentations attempted to calm the roiling waters with fresh looks at the Phase 3 data. Biogen's Kumar Kandadi Muralidharan presented an exposure-response analysis of the ENGAGE and EMERGE trials that linked reduction in amyloid PET SUVR to slowed decline on the CDR-SB. Only about a third of participants had PET scans. Biogen used data on how their drug exposure related to their plaque reduction to impute SUVR values for the rest, i.e., the majority of the cohort. With these imputed numbers, Biogen created an exposure-response model that rendered a statistically significant effect of amyloid removal on CDR-SB score. The effect predicted by this model was the same for both ENGAGE and EMERGE participants, as well as for people whose disease progressed at different rates. Overall, the model posits that 18 months of aducanumab titrated to 10 mg/kg would slash a person's plaque load by 75 percent and slow his or her slide on the CDR-SB by 0.38 points on average.
Sam Dickson of Pentara Corporation, which sells biostatistics services to drug sponsors, took a different tack. He combined the data from all endpoints into a single univariate scale and, based on that, claimed that even in the negative ENGAGE trial, this new endpoint favored aducanumab in both dose groups.
Ultimately, new efficacy data will be required to convince the field at large. On March 30, Biogen announced that it has submitted the final protocol for Envision, its required confirmatory trial, to the FDA for approval and expects to begin screening patients in May and to complete the trial in four years. Biogen will enroll around 1,500 participants, and pledged at least 18 percent of those from the U.S. will be black or Latino.
Also at AD/PD, Changyu Shen of Biogen argued that data from other anti-amyloid immunotherapy trials strengthen the case for cognitive benefits with this treatment approach. He revisited an oft-cited meta-analysis by Maria Glymour and colleagues at the University of California, San Francisco, which reported no significant correlation between amyloid lowering and MMSE scores. This meta-analysis combined 14 trials of eight purported amyloid-lowering drugs, including early immunotherapies, γ-secretase and BACE inhibitors, and bexarotene (Ackley et al., 2021).
Shen repeated this analysis, using a publicly available web interface Glymour and colleagues had provided to allow their findings to be updated with new data. Shen added two trials not previously included, the aducanumab Phase 1 and donanemab Phase 2 studies. He also corrected what he said were data inconsistencies in the original meta-analysis. Shen's updated meta-analysis found a statistically significant effect of amyloid removal on the three endpoints examined: CDR-SB, MMSE, and ADAS-Cog. Shen reported larger effect sizes than did Ackley et al. For example, the effect of a 0.1 SUVR reduction in amyloid rose from 0.06 points on the CDR-SB to 0.09. Restricting the meta-analysis to only immunotherapy trials further strengthened the effect size and statistical significance, Shen noted.
These findings broadly align with a recent review of immunotherapy trials by Eric Karran at AbbVie in Cambridge, Massachusetts, and Bart De Strooper at the UK Dementia Research Institute, London. They concluded that plaque needs to be lowered to 20 centiloids or less to produce a noticeable cognitive benefit, with a lag time of several months between amyloid removal and clinical effect. Therefore, trials such as the Phase 3 aducanumab program, where most people became amyloid-negative only at the end of the study, might not demonstrate a convincing clinical benefit (Karran and De Strooper, 2022).
Who Gets the Most Out of Adu?
Even if removing plaque eventually restores cognition, researchers agree that the effects will vary significantly because people’s rates of progression and even the confluence of genetic, environmental, and lifestyle factors leading to AD differ so much from one person to the next. At AD/PD, Scheltens provided a glimpse at how noisy Alzheimer's progression data can be. VU scientists analyzed cognitive change over 18 months in amyloid-positive, cognitively impaired participants in the ADNI observational cohort. They saw a broad range of outcomes. For example, on the CDR-SB, 95 percent of people had anything from a drop of 0.35 to a gain of the same amount over this time period. Most recent immunotherapy trials posted drug effect sizes within this range, meaning what signal there is could easily be lost in this noise, Scheltens noted.
Would selecting patients based on risk factors like an APOE4 allele or tau pathology tighten this variability? Alarmingly, it would not, Scheltens reported. The VU study found even more clinical variability in E4 carriers than noncarriers, and in people with higher concentrations of CSF total tau than in people with less CSF total tau. Variability was higher in younger than older people, and higher in women than men. Running longer studies to try to see an effect might not work either, as clinical variability increased over time (Jutten et al., 2021).
Instead, Scheltens suggested enrolling more people, using more sensitive outcome measures such as ADCOMS, and, importantly, comparing individual patient trajectories rather than group differences. For such trajectories, acquiring some run-in data before people start on drug can further help changes pop out, Scheltens noted. Some trials, such as the DIAN-TU platform, are already doing this.
Other talks at AD/PD offered tantalizing if inconclusive hints of factors that could influence whether a given patient responds. Oliver Peters of Charité University Hospital, Berlin, examined data from the 15 people who participated in the ENGAGE trial at his site, took aducanumab, and had CSF data. He noticed that the dose a person was on correlated with his or her change in CSF Aβ42/40 but not p-tau181. To parse this, Peters separated the eight participants who had a large drop in p-tau181 on aducanumab from the seven who did not. The main difference between these two groups was a higher baseline p-tau181 in the responders, though both groups started above the threshold for abnormal p-tau181.
Peters believes that within this abnormal range, low baseline p-tau181 may mark patients who have atypical AD and are therefore less likely to respond to immunotherapy. Although both groups had similar cognitive scores at baseline, and both declined while on aducanumab, those whose CSF p-tau181 dropped on drug declined less than the p-tau181 “non-responders” on the MMSE, ADAS-Cog, CDR-SB, and Activities of Daily Living scales.
“Aducanumab showed best results in typical AD patients who tested amyloid- and tangle-positive,” Peters wrote to Alzforum. He thinks this lends support to the use of tau PET as a trial inclusion criteria, as Lilly is doing with its donanemab studies.
Meanwhile, Oskar Hansson of Lund University, Sweden, spotted a relationship between p-tau181 change and an unexpected factor linked to treatment response. He previously reported that a dip in this marker in the Phase 3 aducanumab trials correlated with plaque clearance and a slowing of cognitive decline at the group level (Nov 2021 conference news). At AD/PD, Hansson added subgroup analyses, finding that a person's sex, APOE genotype, disease stage, or standard AD medications had no bearing here. Age did, however. In people older than 75, p-tau181 dropped more dramatically with plaque clearance than it did in younger people. Hansson was at a loss to explain this. “I would love to get ideas from anyone here on why this might be the case,” he told the audience.
Analysis of a different therapeutic antibody program, Lilly’s donanemab, implicated drug exposure as the main factor determining treatment response. Lilly’s Ivelina Gueorguieva presented a pharmacokinetic analysis based on serum samples from 304 participants in donanemab Phase 1 or 2 trials; 177 took drug, 127 placebo. Participants had amyloid PET scans at baseline, six, 12, and 18 months. Only one factor affected their amyloid removal: People who maintained an average serum concentration of at least 4.4 μg/mL donanemab during the trial cleared plaque rapidly, while those with less donanemab cleared it more slowly. About 80 percent of the cohort were in the former category. What determined this threshold effect? Gueorguieva does not know. People with a higher body weight, or more anti-donanemab antibodies in their blood, cleared donanemab from their bodies fastest, but neither factor had much impact on plaque removal, she said.
While scientists are slicing and dicing old trial data to squeeze out some additional learning, all eyes are on new trial results. Gene Kinney of Prothena noted that upcoming trial readouts for gantenerumab, lecanemab, and donanemab will give better information on patient selection, biomarker response, and the sensitivity of clinical endpoints. “I think we’ll have a rich dataset on which to learn more about every one of those components,” Kinney said in Barcelona.
For its part, Prothena announced on March 28 that it has begun a Phase 1 trial of a new anti-amyloid antibody, PRX012. It binds Aβ oligomers, plaques, and pyroglutamated forms with high affinity and can be injected under the skin.—Madolyn Bowman Rogers
Sevigny J, Chiao P, Bussière T, Weinreb PH, Williams L, Maier M, Dunstan R, Salloway S, Chen T, Ling Y, O'Gorman J, Qian F, Arastu M, Li M, Chollate S, Brennan MS, Quintero-Monzon O, Scannevin RH, Arnold HM, Engber T, Rhodes K, Ferrero J, Hang Y, Mikulskis A, Grimm J, Hock C, Nitsch RM, Sandrock A.
The antibody aducanumab reduces Aβ plaques in Alzheimer's disease.
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PubMed.
Known as first responders to viral infection, Type I interferons may also play a part—for better or worse—in neurodegenerative disease. At AD/PD 2022, held March 15-20 online and in Barcelona, Spain, researchers reported that not only viruses, but also aggregates of tau can set the interferon cascade in motion. Possibly by damaging mitochondria enough to let their DNA escape, tau aggregates triggered a cytosolic DNA sensor called cyclic GMP-AMP synthase. This, in turn, set off the interferon response, leading to destruction of synapses. Deleting or blocking cGAS spared synapses and memory in mouse models of tauopathy, despite having no effect on tau tangles. Scientists in Barcelona also reported that interferon signaling proteins were elevated in the cerebrospinal fluid of people who tested positive for amyloid plaques and/or tau tangles. The findings cast interferon as a bad actor responding to both pathologies, and may point to therapeutic targets that prevent the neuronal damage caused by this inflammatory response.
Innate immune mechanisms that have evolved to thwart invading microbes are increasingly recognized as players in neurodegenerative disease. Type I interferons are typically activated by cellular sensors that detect viral RNA or DNA, but recent studies suggest that in the face of Aβ or tau pathology, some microglia turn on a similar interferon cascade (Tan et al., 2018; Dec 2020 news; and Mar 2022 conference news). If not viruses, then what could be setting this off? Curiously, one study found that misplaced or damaged endogenous DNA—such as that found mingling within Aβ plaques—can do so, leading to dead synapses and faster cognitive decline (Roy et al., 2020).
How might tau pathology rev this interferon cascade? Using RNA-Seq, Sadaf Amin, a postdoc in Li Gan’s lab at Weill Cornell Medical College in New York, found that multiple interferon-stimulated genes ramped up in the brains of P301S tauopathy mice, and that cGAS could be the upstream trigger. This sensor detects foreign viral DNA as well as endogenous mitochondrial and nuclear DNA that have leaked into the cytosol. Upon detecting these misplaced nucleic acids, cGAS activates STING, which leads to recruitment and phosphorylation of TBK-1.This kinase phosphorylates the transcription factor IRF3, which then dimerizes and heads into the nucleus, where it sets off the transcription of type I interferon genes (for review, Hopfner and Hornung, 2020).
To find the source of the interferon in the mice, Amin first turned to the most likely suspects, the brain's immune cells. In Barcelona, Amin reported that when they treated cultured microglia with synthetic tau fibrils, levels of p-TBK1, IFN-β, and interferon-stimulated genes all increased, suggesting the cGAS-interferon pathway had been switched on. But how? The scientists spotted tau fibrils congregating in some mitochondria, and found that depleting cells of mitochondrial DNA dampened the interferon response. Knocking out cGAS almost completely abolished the response as well. Together, the findings suggested that tau fibrils may trigger leakage of mitochondrial DNA, which trips off cGAS and the interferon cascade.
How might tau aggregates leak mitochondrial DNA into the cytosol? This is an open question, but Amin spotted tau aggregates within the organelles. A recent study from Gan’s lab found that tau interacts with multiple mitochondrial proteins in neurons, suggesting that the microtubule-binding protein may have a proclivity for this organelle (Jan 2022 news). Another recent study found tau aggregates cavorting with a mitochondrial protein within synapses, ultimately leading the organelles to break in two (Mar 2022 conference news). It is also possible that the phenomenon extends beyond tau, since others have watched Aβ damage mitochondria, and TDP-43 was recently reported to enter these little factories as well, triggering the release of mtDNA and activating the cGAS–STING pathway (Yu et al., 2020).
Cooking with cGAS
To investigate the role of cGAS in tauopathy in vivo, the researchers generated cGAS homo- and heterozygous knockouts on a non-transgenic or P301S background. Using single-nuclei RNA sequencing, Amin found upregulation of multiple interferon-stimulated genes in microglia from P301S mice compared to controls. Knocking out one or two copies of cGAS suppressed this. Furthermore, ditching cGAS prevented synaptic loss, synaptic dysfunction, and spatial memory deficits in the tauopathy mice, although it did not lighten their load of neurofibrillary tangles.
Finally, a brain-permeable cGAS inhibitor, called TDI-8570, bestowed similar benefits. Added to mouse kibble for three months, it reduced synapse loss in P301S mice and sharpened their recognition of new objects. The inhibitor had no effect on the memory of non-transgenic mice.
Amin found evidence that the cGAS-STING pathway was cranked up in humans, as well. People with AD had more phospho-TBK1 in their brains than did controls. The findings mesh with previous transcriptomic studies that have identified subsets of microglia with ongoing interferon responses in the AD brain (Dec 2020 news).
Mapping Inflammation to PET Signals
While Amin’s study focused on tau, a jumble of different aggregated proteins typically haunt the brains of people with AD, and each may invoke its own brand of inflammation. Which pathways are triggered by plaques and tangles, and how do they relate to progression of disease? At AD/PD, Bruna Bellaver, from the lab of TharickPascoal at the University of Pittsburgh in Philadelphia, presented findings from a study that dissected the inflammatory pathways associated with plaques and tangles in members of the TRIAD cohort at McGill University in Montreal.
Bellaver and colleagues measured CSF levels of 92 inflammatory proteins among 35 cognitively normal and 25 cognitively impaired participants. They plugged these 92 proteins into a so-called STRING database, which integrates data from multiple studies to group proteins based on their involvement in similar signaling pathways. STRING identified six clusters within the CSF marker dataset, and each could be scored based on the CSF levels of each member protein. Comparing the scores to each participant’s PET scans, Bellaver found that two clusters associated with both plaques and tangles, while one cluster associated only with plaques and another only with tangles. Interestingly, none of the clusters correlated with cognitive status.
Essentially, Bellaver found that clusters relating to JAK-STAT signaling and glial cell neurotrophic receptor binding correlated with Aβ plaques as well as tau tangles. JAK-STAT signaling is triggered when interferon binds to its receptor, suggesting that the interferon response is associated with both amyloid and tau accumulation. The cluster that correlated only with plaques also reflects interferon responses, this time stimulation of interferon-gamma, a type II interferon. Lastly, a TNF-α signaling cluster only correlated with tangles. In all, the findings reveal distinct inflammatory signatures of Aβ plaques and tau tangles in the human brain, with substantial overlap between the two. Notably, Type I interferon signaling emerged as a commonality between the two signatures.
The data can’t predict whether the different inflammatory pathways were cause or consequence of each type of pathology. They do, however, suggest that inflammation is an early part of the disease process and heats up well before cognitive symptoms surface, Pascoal said. He noted that numerous other genetic, health, and environmental factors also contribute to inflammatory pathways active in the brain. Teasing apart the inflammatory signatures invoked by these different contributors will help scientists understand which ones to target, and when.—Jessica Shugart
Yu CH, Davidson S, Harapas CR, Hilton JB, Mlodzianoski MJ, Laohamonthonkul P, Louis C, Low RR, Moecking J, De Nardo D, Balka KR, Calleja DJ, Moghaddas F, Ni E, McLean CA, Samson AL, Tyebji S, Tonkin CJ, Bye CR, Turner BJ, Pepin G, Gantier MP, Rogers KL, McArthur K, Crouch PJ, Masters SL.
TDP-43 Triggers Mitochondrial DNA Release via mPTP to Activate cGAS/STING in ALS.
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PubMed.
Further Reading
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Eavesdropping on Cell-to-Cell Conversations in Aging, Alzheimer's
Hundreds, if not thousands, of transcripts and proteins get up- or downregulated in Alzheimer’s disease, but how this broadly affects the brain remains a mystery. Scientists are trying to solve it by simultaneously isolating millions of single cells or single nuclei from brain samples and scrutinizing patterns that correlate with disease traits. Such studies illuminate the cellular state of the brain, but not how it got there.
At this year’s AD/PD meeting, held March 15-20 in Barcelona, Spain, Philip de Jager, Columbia University, New York, described an unbiased look at how communities of cells change during age and disease. It turns out some cell types wax and wane in unison, and some of their shifts correlate with amyloid plaques, neurofibrillary tangles, and cognitive decline.
Also working to figure out what governs en masse changes, Ricardo D’Oliveira Albanus, a postdoctoral fellow in the lab of Oscar Harari at Washington University, St. Louis, analyzed ligand-receptor pairs to infer thousands of cell-cell interactions. Albanus predicted that the number of liaisons between cell types in the brain changes as AD worsens, with most of those changes involving microglia. Many of the proteins involved have cropped up in genome-wide association studies. He linked some of these proteins to signaling networks that correlated with pathology, but more correlated with resilience. “This could help us find new drug targets that slow or prevent AD,” said Albanus.
Together, the two approaches exemplify the power of merging single-cell and interactome analyses in scientists' quest to understand the cellular phases of AD and related diseases.
Cells of a Feather
Unlike birds, it turns out that cells in the AD brain not only flock with their own kind, but with other cells as well. That’s what de Jager, collaborating with Vilas Menon at Columbia and Naomi Habib, Hebrew University of Jerusalem, discovered when they looked at how the amounts of different cells change over time. The scientists counted cells in prefrontal cortex tissue taken postmortem from volunteers in the Religious Orders Study and the Memory and Aging Project at Rush University, Chicago (see image below). Some of the data was recently posted on bioRxiv (Cain et al., 2020).
Cell Tracking. Single-nuclei RNA-Seq of 24 ROSMAP samples (top) yielded high-resolution transcriptome data that could be used to identify proportions of brain cells in a larger sample of 640 individuals (bottom). [Courtesy of Cain et al., bioRxiv 2022.]
First, the scientists ran a pilot study of 24 ROSMAP participants, from whom they obtained more than 170,000 single-nuclei transcriptomes total. They reflect all the eight major cell subtypes of the brain, though the proportions were not the same in each person. Indeed, the relative frequency of certain cell types seemed highly coordinated. For example, when the number of microglia was high in a given person, so was that of oligodendrocytes, and vice versa. “We could see that there was structure in the data,” said de Jager (see image below).
Two main groups of cells seemed to fluctuate in unison. One comprised microglia, oligodendrocytes, pericytes, and endothelial cells; the other included astrocytes, oligodendrocyte precursor cells, and inhibitory neurons. Curiously, excitatory neuron counts did not track with any other cell type.
Fascimile? Single nuclei RNA-Seq data from 24 samples (left) and bulk RNA analysis of 640 samples (right) show that the frequencies of some cell subtypes in the prefrontal cortex are highly coordinated. [Courtesy Cain et al., bioRXiv 2022.]
Because two dozen samples harbor little statistical power, the scientists next turned to bulk RNA-Seq data from 640 people. Menon devised a method called CelMod, aka Cellular Landscapes Modeling by Deconvolution. It infers cell type and subtype from snRNA-Seq data. CelMod first matches snRNA-Seq and bulk RNA-Seq from the same person, and then uses that benchmark to infer cell types and subtypes for other samples where only bulk data is available. The scientists found cells behaved similarly in this larger data set, with the two main groups still changing in unison, although they were now able to identify 37 different cell subtypes (see image above).
Do these cellular undulations correlate with AD pathology? On this question, two things stood out. First, cell type compositions that correlated with cognitive decline and neurofibrillary tangle pathology were very similar to each other, but distinct from cells that correlated with amyloid plaques. Second, while some cell numbers bloomed with these pathology traits, others, often of subsets of the same cell type, shrank. For example, one subset of astrocytes expanded in the presence of tau pathology or cognitive impairment, whereas another subset of astrocytes waned. Neither subset correlated with plaques. When plaques were present, a subset of inhibitory neurons swelled in number, whereas subsets of endothelial cells and excitatory neurons dwindled. In this analysis, none of the cells that correlated with plaques seemed to be affected by tangles or cognition.
The scientists took their work a step further, obtaining snRNA-Seq data from 424 ROSMAP participants. This yielded 1.7 million transcriptomes, identifying 92 subsets of cells. With this higher resolution, the scientists have begun to home in on cells that change most as pathology advances. Again, some changed more with tangles than with plaques. One subset of excitatory neurons grew when tangles did, whereas another shrank as plaque burden increased. The most dramatic change was in a subcluster of microglia, dubbed M13. Numbers of these cells grew dramatically as both tangle and plaque burden rose, hinting at a connection. A subset of astrocytes, A10, also swelled with both pathologies.
What does this mean for AD progression? De Jager said it’s too early to tell. Meanwhile the scientists are beginning to add detail to the picture. Using mediation analysis, they predict that M13 may be bad news because as these microglia respond to Aβ they exacerbate tau pathology. Likewise, A10 astrocytes seem to exacerbate the effect of tau on cognitive decline. The researchers are now looking at how an individual’s cell profile might predict whether they had been on a path to disease before they died, or whether they might have been more resilient.
Cell-Cell Cross Talk Resilience emerged as an important cellular state in Albanus’s analysis. He also used snRNA-Seq data, this time from parietal cortex tissue donated by 67 people who had either been in the DIAN study of autosomal-dominant AD, or had been treated at the Knight Alzheimer’s Disease Research Center at WashU. Logan Brase in Hariri’s lab had previously used this data to uncover transcriptome differences between ADAD and late-onset AD (see Aug 2021 conference news).
Albanus wanted to know how the different subtypes of cells communicate with each other, and how that changes with disease. For this, he turned on CellPhoneDB. This is not the latest smart phone, but an algorithm that predicts cellular cross talk based on expression of ligands and receptors in transcriptome data (Efremova et al., 2020). It enabled Albanus to evaluate more than a million potential interactions between thousands of ligand pairs expressed by hundreds of cell subtypes. From control transcriptomes, he found 31,000l ligand/receptor pairs that drive interactions between different cells. From this he could predict patterns of cellular cross talk. For example, oligodendrocyte precursor cells shared more ligand/receptor pairs with endothelial cells than with any other cell type, and astrocytes had more lines of communication with microglia than with neurons or oligodendrocytes.
These patterns were different in Alzheimer's samples, i.e., both the types and number of interactions between cells had changed with disease. Interestingly, endothelial cells doubled their repertoire, going from fewer than 100 ligand/pair interactions with oligodendrocytes to almost 200. Their interactions between other cells increased similarly. Researchers hearing the talk were intrigued by this response and asked if it might be due to the number of cell adhesion molecules endothelial cells produce. Albanus said this is possible, but hard to study because the dataset contains so few of these cells. He agreed that it would be interesting to run a similar analysis of other tissues to see if this endothelial response is specific to the brain.
More statistically powerful data came from analysis of other cells. Interactions between microglia and excitatory neurons changed the most of all cell types, with the former bumping up interactions, and the latter tamping them down, by about 30 percent. Curiously, people with AD who carried a TREM2 mutation were an exception. Their microglial interactions were unchanged from controls. This may not be surprising, Albanus agreed, since TREM2 is a cell-surface signaling receptor on microglia, and pathogenic AD variants cause a loss of function, i.e., dampen microglial responses.
Alzheimer's Chatter. Ligand/receptor cross talk (left) involving AD-related genes buzzes more often between microglia and other cells, including neurons. In AD cases, those interactions occur even more often (right). [Courtesy of Ricardo Albanus, Washington University.]
Which of the thousands of ligand/pair interactions might be important in AD? To address this, Albanus looked to 800 genes that have popped up in AD genome-wide association and functional studies, and found that much of the overall cross talk involved AD-linked genes, for example TREM2/semaphorin and APP/CD74. These interactions were enriched in microglia, were mostly between microglia and either excitatory or inhibitory neurons, and occurred more often in tissue samples from AD brain.
Given that such interactions are sprouting in AD, Albanus next wanted to know what effect they might be having. CellPhoneDB offers little help in this regard, so Albanus instead used the CytoTalk bioinformatics algorithm, which reconstructs signaling networks downstream of ligand-receptor interactions (Hu et al., 2021). Albanus has only just begun to figure out what these networks do. For example, CytoTalk linked the TREM2/semaphorin cross-talk pair to a massive spiderweb of signaling networks inside both microglia and neurons. Intriguingly, known AD genes cropped up mostly in the microglial networks (see image below). Albanus found very similar patterns when he applied the same analysis to datasets from Marco Colonna’s lab at WashU (Zhou et al., 2020).
To better understand how these networks are linked to disease, Albanus has focused in on small subnetworks, many of which are enriched in AD-related genes, but not all of which are directly connected to the ligand/pair interactions identified by CellPhoneDB. One such subnetwork ties TREM2/semaphorin directly to ApoE and HLA immune pathways that regulate microglial responses to amyloid and other pathological changes in the brain. He found similar subnetworks in prefrontal cortex microglia when he analyzes snRNA-Seq data from Nancy Ip’s lab at Hong Kong University of Science and Technology (Lau et al., 2020). Albanus has identified 360 potential subnetworks with CytoTalk.
Cross Talk Networks. CytoTalk links ligand/receptor pairs identified by CellPhoneDB (center) to large protein-protein interaction networks in cells, including microglia (left) and excitatory neurons (right). AD-linked genes (circled red) crop up mostly in microglia, and cluster in sub-networks. [Image courtesy Ricardo Albanus, WashU.]
To get a handle on what these subnetworks might be doing in AD, he correlated their expression with Braak stage. Some, for example those involving APP, and presenilins, positively correlated and so likely increase risk for AD, Albanus said. But many others, including the TREM2 subnetwork, negatively correlated, suggesting they reflect cellular resilience. Intriguingly, only some of these subnetworks mediate ligand/pair crosstalk among cells, and most of them associated with resilience.
“This is why we are so excited about this cross talk,” Albanus told Alzforum. “Proteins that are tuned to those network signals are mostly intracellular and not easy targets to regulate therapeutically. But if we can find a shortcut where we can directly modulate the network by changing the cross-talk interaction on the cell surface, that could help us find new drug targets for AD,” he said.—Tom Fagan
UB-312 Synuclein Vaccine Safe in Controls. Next Up: Parkinson's.
Current treatments for Parkinson’s only address the disease's symptoms, and much like Alzheimer's scientists are pursuing that disease's central pathogenic proteins, Parkinson's scientists believe that targeting α-synuclein aggregates will slow or stop the movement disorder from getting worse. Following in the footsteps of α-synuclein antibodies, such as Roche/Prothena’s prasinezumab, scientists are testing active vaccines in the clinic. At the 16th International Conference on Alzheimer’s and Parkinson’s Diseases held online and in Barcelona, Spain, Hui Jing Yu of Vaxxinity, Dallas, reported that in a Phase 1 trial in healthy volunteers, UB-312 spurred production of C-terminal synuclein antibodies without causing serious side effects. The company is now enrolling people with Parkinson’s for a second trial. UB-312 is the second α-synuclein vaccine in the clinic; ACI-7104, developed by AFFiRiS and then licensed to AC Immune, also appeared safe in Phase 1.
Nipping at their heels are a pack of other α-synuclein immunotherapy approaches. In Barcelona, Robin Barbour of Prothena, San Francisco, showed how a vaccine carrying two C-terminal α-synuclein fragments jolted mice into making more antibodies than either snippet alone did. Those antibodies labeled Lewy bodies in brain tissue from PD cases. Sahar Roshanbin from Uppsala University, Sweden, showed how a hybrid α-synuclein/transferrin receptor antibody slipped into the mouse brain better than did the synuclein antibody alone. Chasing a different target, Jun Sung Lee of Neuramedy, a small biotech in Seoul, South Korea, reported that tomaralimab, an antibody against toll-like receptor 2, lowered α-synuclein aggregate load, neuroinflammation, and motor dysfunction in a mouse model of synucleinopathy.
The need for taking many shots on goal is pressing. “When you don’t have any disease-slowing therapies to keep people out of the late stages of Parkinson’s where dementia may develop, you want to try everything,” Mark Cookson, National Institute on Aging, Baltimore, told Alzforum.
Active Synuclein Immunotherapy
For one, the α-synuclein vaccine field is stirring. From 2012 to 2017, AFFiRiS’ PD01A/PD03A, based on an eight-amino acid fragment from the C-terminus, showed some safety and capacity to provoke an antibody response in Phase 1 trials in PD and multiple system atrophy (MSA, Volc et al., 2020; Meissner et al., 2020). The program stalled until July 2021, when AC Immune acquired rights and said it would start Phase 2 in a formulation called ACI-7104. According to the company website, this trial is to measure immunogenicity, imaging and fluid biomarkers, and progression of motor and non-motor symptoms in people with early sporadic PD, but no trials are listed on clinicaltrials.gov.
Meanwhile, UB-312 is catching up. Vaxxinity's vaccine links a 10-amino-acid fragment from α-synuclein's C-terminus to a small peptide that activates T-helper cells. In guinea pigs, a 12-amino-acid predecessor of UB-312 had generated antibodies against toxic α-synuclein fibrils and oligomers, but not monomers. In postmortem analysis, these antibodies bound synuclein inclusions within the substantia nigra and basal ganglia of people who had had PD, dementia with Lewy bodies, or MSA (Nimmo et al., 2020).
As for UB-312, when injected into 10-week-old Line 61 transgenic mice that express human α-synuclein, fewer α-synuclein oligomers formed in the cortex, hippocampus, and striatum. Vaccinated mice did better at crossing a beam and hanging onto a wire (Nimmo et al., 2022).
At AD/PD, Yu shared safety and immunogenicity data from 50 healthy adults in a Phase 1 trial conducted in the Netherlands. Participants had gotten intramuscular jabs at weeks 1, 5, and 13, following one of seven dosing schedules: either three doses each of 40, 100, 300, 1,000, or 2,000 μg, or 40 μg initially followed by either 300 or 1,000 μg for the last two doses. Six people received each vaccine regimen, eight a placebo. Volunteers donated CSF at baseline and week 21, and blood at baseline, one week after each jab, and every four weeks from week 1 to 21. All were monitored for adverse events for another 23 weeks.
Doses at or below 300 μg prompted no serious adverse events, but did cause mild headache, cold-like complaints, and pain or redness at the injection site. The dose-ranging stopped at the 1,000 and 2,000 μg doses, after one volunteer developed moderate flu-like symptoms after the second 1,000 μg injection.
UB-312 prompted a dose-dependent rise in α-synuclein antibodies in the blood and CSF, with the largest response coming after three 300 μg doses (see image below). The antibody concentration in the CSF reached only 0.2 percent of that in the blood, indicating that a small fraction had crossed the blood-brain barrier. Yu considers this good news, as it is on par with the reported serum-to-CSF ratios of 0.1 to 0.2 percent for monoclonal antibodies. [Editor's note: This data was published on April 15, 2022, in the journal Movement Disorders.]
Immune Response. Healthy adults who received three injections of 300 μg (red) or 100 μg (blue) UB-312 made more C-terminal α-synuclein antibodies in the blood (left) and CSF (right) than volunteers who got lower doses (yellow and green) or placebo (black). [Courtesy of Vaxxinity.]
Still in Phase 1, the researchers are now testing an additional 20 people ages 40 to 85 with early to mid-stage sporadic Parkinson's. Yu expects enrollment to wrap up by the end of April. These participants, too, will get three doses of either 100 or 300 μg UB-312 over the same 13-week period. Seven will receive each dose of active vaccine; six, placebo. Endpoints include change in the Unified Parkinson Disease Rating Scale and the Montreal Cognitive Assessment, and blood and CSF will be collected and monitored for anti-α-synuclein antibodies, as well as changes in total and free α-synuclein.
Unlike for AD, there are no established CSF or serum biomarkers for PD. Instead, the researchers will measure misfolded pathogenic synuclein before and after vaccination using the Protein Modification Cyclic Amplification technique. PMCA can detect minuscule amounts of α-synuclein oligomers in CSF from people with PD and other synucleinopathies (Dec 2016 news; Kang et al., 2019). “This method might help to diagnose these neurological disorders and provide a way to assess target engagement of PD treatments,” Yu said, adding that topline results could come by the end of this year.
Stronger in Tandem?
Both PD01A/PD03A and UB-312 contain two antigens—one against synuclein and one to stimulate T-cells. Can packing on more boost the immune response? Prothena has bumped the antigen count to three. At AD/PD, Barbour said the strategy is to deploy vaccines with a T-cell-stimulating epitope and two α-synuclein fragments separated by a dendritic endopeptidase cleavage site. Prothena is doing this for its dual Aβ-tau vaccine, as well (Aug 2021 conference news). Michael Agadjanyan, Institute for Molecular Medicine, Huntington Beach, California, told Alzforum he is unaware of other vaccines that include such a cleavage site between antigenic fragments and wondered why this is needed. “Dendritic cells chop up antigens into small fragments to present to T-cells regardless of an endopeptidase cleavage site,” he told Alzforum
Still, the trick might work. Barbour has tested six different vaccines using two C-terminal and one mid-domain fragment. One vaccine has both C-terminal fragments, two have the mid-domain tethered to either of the C-terminal antigens, and three comprise single peptides. In wild-type mice, the tandem vaccines evoked seven to 10 times higher antibody titers than the single-antigen varieties, even when the latter were given at twice the dose. Tandem vaccine antisera more robustly labeled Lewy bodies in Parkinson's brain tissue, and prevented cultured neurons from taking up recombinant, fluorescent α-synuclein aggregates. Tandem C-terminal vaccine antisera bound Lewy bodies and prevented synuclein uptake by cells better than did antisera from the C- and mid-domain fragment vaccines. Barbour said the dual C-terminal vaccine has been selected for clinical testing, though she did not say when.
Why stop at three antigens? Agadjanyan and colleagues are pushing the envelope by creating vaccines with 13 to 15 epitopes, including 12 antigenic peptides from infectious pathogens that stimulate T-cells. “This could induce a robust immune response in almost everyone, since each person has their own set of MHC class II molecules on their antigen-presenting cells that could recognize some, but not all, of the antigens,” he said. The scientists have developed a DNA-based, multi-fragment, α-synuclein vaccine (Kim et al., 2022; Davtyan et al., 2017). It contains a plasmid carrying genes for the 12 T-cell epitopes and three different α-synuclein fragments spanning the entire C-terminus. Once translated, the peptides run as one long string with no cleavage sites in between.
Much like the Prothena tandem vaccine, this DNA-based jab induced higher antibody titers in mice than vaccines encoding each α-synuclein fragment alone. Antibodies created by the multi-fragment version also bound Lewy bodies in postmortem brain tissue. The vaccine reduced the amount of total α-synuclein deposits in the brains of transgenic mice, slowed neuron loss, reduced micro- and astrogliosis, and improved motor function better than any single-fragment vaccines. Agadjanyan hopes to develop a protein-based multi-fragment vaccine as well.
Targeting Synuclein Using Antibodies
In parallel, researchers are testing therapeutic antibodies, as well. Such biologic drugs against α-synuclein have been in clinical development for more than a decade and seem safe, though it's still unclear if they work. Roche/Prothena’s prasinezumab is the furthest along in Phase 2 trials (see Mar 2013 conference news; Mar 2015 conference news). Just as vaccines against synuclein’s C-terminus seem promising, antibodies that recognize this portion of the peptide, such as prasinezumab and Lundbeck’s LuAF82422, have fared well in early trials. In contrast, the only antibody tested so far against the N-terminus, Biogen’s cinpanemab, posted negative results in Phase 2 (Apr 2021 conference news).
Whichever candidate antibody will succeed, more of it needs to get into the brain. In Barcelona, Roshanbin of Uppsala University described a tweaked version of the SynO2, which binds oligomeric and fibrillar α-synuclein species that are believed to be most toxic (Vaikath et al., 2015; Ingelsson, 2016).
Initially, Roshanbin aimed to develop an antibody-based PET ligand for α-synuclein. To drive SynO2 uptake into the brain, she tacked on an antibody fragment that binds the transferrin receptor. TfR transports transferrin across cells that line the blood-brain barrier, and scientists have piggybacked other cargoes onto the receptor to get them in (Dec 2021 conference news; Mar 2021 conference news; May 2020 news). Lo and behold, a radiolabeled version of the SynO2-TfR bispecific antibody reached levels in the brain 50 times higher than did SynO2, and, in PET scans, it lit up extracellular α-synuclein deposits in transgenic mice (see image below; Roshanbin et al., 2022).
Antibody PET. MicroPET scans of mouse brains show that more SynO2-TfR (left) than SynO2 (right) was able to cross the blood-brain barrier. [Courtesy of Sahar Roshanbin, Uppsala University.]
Roshanbin wondered if more uptake would mean a better therapeutic effect. She injected Line 61 mice with SynO2-TfR or SynO2 on days 1, 3, and 4, then sampled the brain on day 5. Compared to SynO2, the bispecific antibody lowered total α-synuclein and insoluble oligomer levels but increased the amount of soluble oligomers, hinting that the antibody was destabilizing or preventing synuclein aggregates.
Since microglia mop up antibody-antigen complexes in the brain, the scientists looked for signs of microglial activity. In mice injected with either antibody, brain levels of soluble TREM2 rose, as did the number of Iba1-positive cells, both signs of microglial activation. This was only a five-day experiment, hence the phosphorylated α-synuclein deposits did not budge. Roshanbin is planning longer experiments to track changes in pathology and see if the microglia better phagocytose α-synuclein when mobilized.
Last but not least, Neuramedy’s Lee described a different way to attack α-synuclein deposits. This company is developing tomaralimab, an antibody to toll-like receptor 2. In the brain, TLR2 ushers α-synuclein aggregates inside neurons, while it triggers neuroinflammation in microglia (Apr 2020 conference news; Oct 2020 news; Kim et al., 2021). A small molecule inhibitor of TLR2 signaling, NPT520-34, reduced α-synuclein aggregates and improved motor symptoms in Line 61 mice. In a Phase 1 trial for PD, it caused no serious adverse events in healthy adults.
Tomaralimab, aka OPN-305, was developed by Opsona Therapeutics, Ireland. It is the only TLR2 antibody that was temporarily in clinical development, between 2012 and 2019, with three Phase 1 studies for cancer and kidney transplant recipients (see clinicaltrials.gov). Neuramedy bought the rights to this antibody in 2019.
At AD/PD, Lee reported that tomaralimab reduced expression of the inflammatory cytokines interleukin-1β (IL-1β), IL-6, and tumor necrosis factor-α (TNF-α) in cultured microglia, and reduced the propagation of α-synuclein between cells.
To see if these effects held true in vivo, the researchers turned to a mouse model of synucleinopathy (Nov 2012 news). They injected preformed synuclein fibrils into 10-week-old wild-type mice to seed pathology and trigger neurodegeneration. Beginning a week later, they infused tomaralimab or placebo once weekly for 18 weeks, then assessed synuclein pathology and neuroinflammation. The antibody entered the brain, reaching CSF levels 0.1 percent of that in plasma. Phosphorylated α-synuclein aggregates, Iba1-positive microglia, and GFAP-positive astrocytes were less in treated mice than controls, Lee reported at AD/PD. Antibody treatment soothed inflammation, as well, with mice having fewer IL-1β-, IL-6-, and TNF-α-positive cells. Line 61 transgenic mice given the same tomaralimab regimen showed similar results.
Furthermore, the antibody improved muscle strength, motor activity, and coordination of the fibril-injected mice. Treated animals held onto a wire for longer, scurried farther in an open field, and spent more time walking a balance beam than controls. Lee said these results seem promising, but not whether Neuramedy will evaluate tomaralimab in PD.
Both Cookson and Martin Ingelsson,who is now at the University of Toronto, consider anti-synuclein vaccines and antibodies equally valid approaches. Ingelsson, who works with Roshanbin at Uppsala U, pointed out a wrinkle unique to vaccines against α-synuclein. “While AD seems to only engage the brain, PD affects the whole nervous system and includes peripheral protein deposits, so researchers need to ensure that α-synuclein species in vaccines will not potentiate peripheral and, secondarily, central neuropathy” he told Alzforum.
Cookson thinks the biggest challenge may be that the body tolerates only mild swings in α-synuclein level. “Three copies of the normal SNCA allele cause an early onset, aggressive form of Parkinson’s with a lot of cortical involvement, while lowering synuclein brain levels too much also causes cognitive impairment,” Cookson told Alzforum (Nov 2003 news). While vaccines are easier to administer than monthly antibody infusions, Cookson likes antibodies because they target only toxic α-synuclein species while leaving the normal protein alone, which lessens the concern about driving synuclein levels down too much.—Chelsea Weidman Burke
Roshanbin S, Xiong M, Hultqvist G, Söderberg L, Zachrisson O, Meier S, Ekmark-Lewén S, Bergström J, Ingelsson M, Sehlin D, Syvänen S.
In vivo imaging of alpha-synuclein with antibody-based PET.
Neuropharmacology. 2022 May 1;208:108985. Epub 2022 Feb 8
PubMed.
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