03 May 2023

Tau pathology is proving a tough nut to crack, with most antibodies directed against it posting negative results in clinical trials thus far. At the recent International Conference on Alzheimer’s and Parkinson’s Diseases in Gothenburg, Sweden, researchers discussed how they might hone antibodies and vaccination strategies to become effective.

Acknowledging that the bulk of tau stays inside cells, some speakers touted single-domain antibodies, small fragments that can more easily enter neurons. Others introduced vaccine strategies designed to provoke a stronger immune response or hit multiple types of fibril. For example, the vaccine PRX123 elicits antibodies against both Aβ and tau. Likewise, scientists at a pan-amyloid company debuted a peptide-antibody fusion, or “peptibody,” that binds Aβ, tau, and α-synuclein, three pathologies that often co-occur in Alzheimer’s disease.

These strategies are still preclinical, so it remains to be seen how well they perform in people. Nonetheless, the presentations showed a field branching out as it learns from early failure.

One Domain Curbs Tau. Tauopathy flies (solid red) die much faster than do wild-types (solid black), but treatment with anti-tau single-domain antibodies sdAbs (dotted red) extends (left) or restores (right) their lifespan. [Courtesy of Einar Sigurdsson.]

Get Small, Sneak In
Initially, most tau antibodies were intended to stop the spread of toxic aggregates from cell to cell. However, more recent research suggests that replication of tau seeds within neurons does more to propagate tangles than extracellular spreading (Nov 2021 news). In addition, aggregated tau may pass between cells via exosomes or nanotubes that shield it from antibodies (e.g., Leroux et al., 2022). These findings have heightened interest in targeting intracellular tau.

In Gothenburg, Einar Sigurdsson of New York University School of Medicine stressed the importance of this, noting that intracellular tau is 10,000 to 100,000 times more abundant than extracellular species (Han et al., 2017; Congdon et al., 2022). While antibodies can be taken up by cells to reach intracellular tau, cells do that mostly through receptor-mediated endocytosis. Antibody fragments, on the other hand, can be engulfed by bulk endocytosis and thus may enter cells more readily, Sigurdsson said.

To shrink antibodies, one option is to use single-domain versions. sdAbs come from mammals such as camels or llamas, whose antibodies contain a single variable heavy domain (VHH), unlike the double heavy and light chains of conventional antibodies. Also called a nanobody or llamabody, an isolated VHH domain weighs in at 13 kDa, compared to the 150 kDa of its big brother IgG. sdAbs have high affinity and specificity, Sigurdsson said. They are stable, enter the brain well and—importantly—can find epitopes that larger antibodies cannot reach.

To make tau-specific sdAbs, Sigurdsson’s group immunized llamas with the longest isoform of recombinant human tau, then boosted with paired helical filaments of tau from postmortem human brain. After screening a library of the resulting antibodies, the scientists selected a candidate, 2B8, that bound tangles in human brain. They injected fluorescently labeled 2B8 into tauopathy mice as a probe, finding that the signal correlated strongly with tau protein as seen by Western blot, at r=0.94. The probe ended up in endosomes and lysosomes, showing that it was internalized by neurons and positioned to influence tau degradation (Congdon et al., 2022).

But does this sdAb clean up aggregated tau? When expressed in tauopathy flies that make mutant R406W human tau, 2B8 greatly extended lifespan. A similar tau sdAb, 1D9, restored their lifespan to wild-type levels (image above).

Pursuing the same strategy to make sdAbs against aggregated α-synuclein, Sigurdsson's group found a candidate that halved the amount of α-synuclein in the brain of a synucleinopathy mouse. When the scientists conjugated this sdAb to thalidomide to bind E3 ubiquitin ligase and trigger degradation of the complex, the treatment nearly eliminated aggregated α-synuclein. Sigurdsson believes sdAbs have potential as both therapeutics and PET ligands, and has a paper coming out on the latter later this month.

Intriguingly, a recent paper found that engaging the E3 ubiquitin ligase TRIM21 is essential to enabling antibodies to clear tau seeds. In the March 31 Science, researchers led by William McEwan at the U.K. Dementia Research Institute, Cambridge, reported that a tau-clearing antibody became ineffective in tauopathy mice if they lacked TRIM21. Cytosolic TRIM21 binds to full-size antibodies via their Fc domain, stimulating proteasomal degradation of the tau-antibody complex (Mukadam et al., 2023).

McEwan’s finding bolsters the idea that antibodies operate inside neurons, rather than extracellularly, to eliminate aggregated tau. It also implies that antibody fragments lacking the Fc domain might be less potent at abolishing tau, unless researchers activate the E3 ubiquitin ligase as Sigurdsson did.

Research on anti-tau sdAbs is also happening in the lab of Luc Buée of the University of Lille, France. At AD/PD, Buée likened these small fragments to Legos that allow scientists to build whatever tools they wish. His group searched a humanized library of VHH fragments to find sdAbs that recognize the hexapeptide sequence in tau’s microtubule-binding region (MTBR) that controls formation of paired helical filaments (von Bergen et al., 2000). 

One candidate the scientists found was able to enter cells and bind tau, but did not stop aggregates. They then tweaked this sdAb to make a new version dubbed VHH Z70. It bound with micromolar affinity to the hexapeptide sequence and prevented tau aggregates in yeast. In THY-tau30 tauopathy mice, it halved tau deposits, as judged by AT8 staining. Buée believes that VHH Z70 blocks the growth of fibrils by preventing recruitment of tau into PHFs, but does not break apart existing strands (Danis et al., 2022).

One Treatment to Clear Them All?
In many neurodegenerative diseases, multiple types of aggregated protein build up in the brain, and scientists are seeking ways to remove these mixed deposits. In Gothenburg, Suganya Selvarajah of Attralus Inc., San Francisco, detailed her company’s strategy for homing in on such pathologies.

Instead of an antibody, Attralus generated a peptide sequence that selectively binds to heparan sulfate proteoglycans found on amyloid fibrils. Dubbed P5R, this peptide has a random shape in solution, but when it comes in contact with the dense, negatively charged surface of fibrils, it coils into a helix, attaching itself to the fibril via multiple side chains. Because this binding does not depend on the fibril's amino acid sequence, P5R can recognize multiple types of amyloid, including Aβ, tau, and α-synuclein, Selvarajah told the audience.

To build a therapeutic, aka peptibody, the researchers linked the peptide to Fc antibody fragments. The idea is to opsonize fibrils and tempt microglia to eat them. The hybrid molecule, AT-04, consists of two linked Fc fragments, each attached to a P5R peptide. It weighs 59 kDa. AT-04 binds Aβ with 0.2 nM affinity, similar to aducanumab’s binding strength, and orders of magnitude higher than that of VHH Z70. Unlike aducanumab, however, AT-04 also recognizes tau fibrils with 0.2 nM affinity, and α-synuclein fibrils at 0.8 nM. Because these latter two are intraneuronal, peptibodies would have to enter cells to access them.

To test AT-04 in vivo, the researchers infused 50 mg/kg into a vein of 5XFAD mice three times over the course of 10 days. The peptibody bound plaques in mouse brain, although Selvarajah did not say if it promoted clearance. AT-04 also recognized plaques in postmortem AD brain tissue.

The researchers made another peptibody, AT-07, for which they fused Fc domains to VNAR domains, variable heavy chain fragments made in sharks. Because the VNAR domain chosen for AT-07 recognizes the transferrin receptor expressed by blood vessels in the brain, this construct enters the brain better than does AT-04.

Scientists for some years have exploited the ability of transferrin receptors to shuttle large molecules past the blood-brain barrier (Hultqvist et al., 2017; Mar 2021 conference news; Jan 2023 news). About 1.4 percent of AT-04 passes the blood-brain barrier, slightly above the 0.5 to 1 percent uptake of conventional antibodies, while AT-07 uptake is 10-fold higher, with 15 percent of it reaching the parenchyma. At 80 kDa, AT-07 is larger than AT-04. It also binds Aβ fibrils with 0.2 nM affinity, and recognizes plaques in AD brain.

An audience member asked how the peptibodies will choose between amyloid, tau, and α-synuclein if all three types of aggregate are present in brain. “That’s the million-dollar question. We don’t know yet,” Selvarajah said, referring to ongoing in-vivo studies. Attralus peptibodies for peripheral amyloidoses are in Phase 1 (AT-02; AT-03).

Rallying the Body’s Own Defense
For long-term treatment, coaxing the body to produce its own antibodies would be far cheaper than therapeutic antibodies. Several groups are working on tau vaccines, including Axon Neuroscience, AC Immune, and an academic collaboration that develops the recombinant protein vaccine AV1980R/A (Jun 2021 news; Dec 2022 conference news).

In Gothenburg, Justin Boyd of Vaxxinity, Merritt Island, Florida, described his company's strategy. To make a tau vaccine, or “vaxxine” as they call it, company scientists link a short synthetic peptide that mimics a portion of tau to another that activates CD4+ T-helper cells. The latter helps stimulate an immune reaction and boost antibody production. The company has one against α-synuclein in Phase 2 (UB-312; Apr 2022 conference news).

For tau, 100+ peptides were screened to find those that induced the best immune response. Two candidates emerged: vaxxine A, which mimics part of tau’s N-terminus, and F, from the MTBR. Both produced antibodies that bound full-length tau monomers as well as tau fibrils from Alzheimer’s brain sections.

Differences between the two became apparent in cellular assays. Antibodies generated by vaxxine F better inhibited tau aggregation in the tau biosensor cell line developed by Marc Diamond of University of Texas, Southwestern Medical Center, Dallas, than did those generated by A (Oct 2014 news; Apr 2023 conference news). Compared head-to-head, polyclonal fractions of F antibodies slashed aggregation by 80 percent; A, 20 percent. For comparison, Genentech’s anti-tau monoclonal antibody semorinemab suppressed aggregation by 60 percent in this assay. On the other hand, A outdid F at blocking tau seed uptake by neuroblastoma cells. It diminished uptake of tau fibrils by three-quarters, compared to about 60 percent for F. In this assay, semorinemab was similar to A, at 80 percent.

How do the vaxxines perform in vivo? Boyd reported that vaccination with A slashed tau accumulation in the cortex of P301L mice almost to wild-type levels. Lysates from the treated P301L brain contained half as many tau seeds as untreated.

Rather than pick one of the two to take forward, company scientists decided to combine them. In cell cultures, A and F together better blocked tau uptake than did A alone, and this version, dubbed VXX-301, is now being evaluated in P301L mice. The company also intends to test a multivalent vaxxine against both tau and Aβ.

Likewise, Chad Swanson of Prothena introduced PRX123, a vaccine that produces antibodies against both Aβ and tau. In a talk devoted mostly to Prothena's anti-Aβ antibody PRX012 (Part 12 of this series), Swanson mentioned that Prothena will submit an Investigational New Drug application for PRX123 this year, but offered no details about the molecule. Given how easily vaccines can target multiple epitopes, there will likely be many more such efforts.—Madolyn Bowman Rogers

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References

News Citations

1. Doubling of Tau Seeds, Not Spread, Sets Pace of Tauopathy in Alzheimer's 3 Nov 2021
2. Shuttle Unloads More Gantenerumab Into the Brain 25 Mar 2021
3. Ferried Into Brain, TREM2 Antibody Stirs Microglia 20 Jan 2023
4. Phase 2 Data of AADvac1 in Alzheimer’s Disease Published 16 Jun 2021
5. Two New Stabs at Vaccinating People Against Pathologic Tau 10 Dec 2022
6. UB-312 Synuclein Vaccine Safe in Controls. Next Up: Parkinson's. 9 Apr 2022
7. Cellular Biosensor Detects Tau Seeds Long Before They Sprout Pathology 9 Oct 2014
8. Tau Chimeras Do Make Fibrils—and a Chaperone Rips Them Apart 8 Apr 2023
9. Next Goals for Immunotherapy: Make It Safer, Less of a Hassle 21 Apr 2023

Therapeutics Citations

1. Aduhelm
2. UB-312
3. Semorinemab
4. PRX012

Research Models Citations

1. 5xFAD (B6SJL)
2. Tau P301L

Paper Citations

1. Leroux E, Perbet R, Caillierez R, Richetin K, Lieger S, Espourteille J, Bouillet T, Bégard S, Danis C, Loyens A, Toni N, Déglon N, Deramecourt V, Schraen-Maschke S, Buée L, Colin M. Extracellular vesicles: Major actors of heterogeneity in tau spreading among human tauopathies. Mol Ther. 2022 Feb 2;30(2):782-797. Epub 2021 Sep 24 PubMed.
2. Han P, Serrano G, Beach TG, Caselli RJ, Yin J, Zhuang N, Shi J. A Quantitative Analysis of Brain Soluble Tau and the Tau Secretion Factor. J Neuropathol Exp Neurol. 2017 Jan 1;76(1):44-51. PubMed.
3. Congdon EE, Jiang Y, Sigurdsson EM. Targeting tau only extracellularly is likely to be less efficacious than targeting it both intra- and extracellularly. Semin Cell Dev Biol. 2022 Jun;126:125-137. Epub 2021 Dec 9 PubMed.
4. Congdon EE, Pan R, Jiang Y, Sandusky-Beltran LA, Dodge A, Lin Y, Liu M, Kuo MH, Kong XP, Sigurdsson EM. Single domain antibodies targeting pathological tau protein: Influence of four IgG subclasses on efficacy and toxicity. EBioMedicine. 2022 Oct;84:104249. Epub 2022 Sep 10 PubMed.
5. Mukadam AS, Miller LV, Smith AE, Vaysburd M, Sakya SA, Sanford S, Keeling S, Tuck BJ, Katsinelos T, Green C, Skov L, Kaalund SS, Foss S, Mayes K, O'Connell K, Wing M, Knox C, Banbury J, Avezov E, Rowe JB, Goedert M, Andersen JT, James LC, McEwan WA. Cytosolic antibody receptor TRIM21 is required for effective tau immunotherapy in mouse models. Science. 2023 Mar 31;379(6639):1336-1341. Epub 2023 Mar 30 PubMed.
6. von Bergen M, Friedhoff P, Biernat J, Heberle J, Mandelkow EM, Mandelkow E. Assembly of tau protein into Alzheimer paired helical filaments depends on a local sequence motif ((306)VQIVYK(311)) forming beta structure. Proc Natl Acad Sci U S A. 2000 May 9;97(10):5129-34. PubMed.
7. Danis C, Dupré E, Zejneli O, Caillierez R, Arrial A, Bégard S, Mortelecque J, Eddarkaoui S, Loyens A, Cantrelle FX, Hanoulle X, Rain JC, Colin M, Buée L, Landrieu I. Inhibition of Tau seeding by targeting Tau nucleation core within neurons with a single domain antibody fragment. Mol Ther. 2022 Apr 6;30(4):1484-1499. Epub 2022 Jan 7 PubMed.
8. Hultqvist G, Syvänen S, Fang XT, Lannfelt L, Sehlin D. Bivalent Brain Shuttle Increases Antibody Uptake by Monovalent Binding to the Transferrin Receptor. Theranostics. 2017;7(2):308-318. PubMed.

External Citations

1. THY-tau30
2. AT-02
3. AT-03

Further Reading

News

1. Paper Alert: Tau Antisense Oligonucleotide BIIB080 Hits Its Target 26 Apr 2023
2. First Hit on Aggregated Tau: Antisense Oligonucleotide Lowers Tangles 7 Apr 2023
3. TAURIEL Phase 2 Data Published 17 Jun 2022
4. More Tau Antibodies Bid Adieu; Semorinemab Keeps Foot in Door 23 Nov 2021
5. First Cognitive Signal that Tau Immunotherapy Works? 2 Sep 2021
6. Antisense Therapy Stifles CSF Tau in Mild Alzheimer’s Disease 6 Aug 2021
7. Biogen Shelves Gosuranemab After Negative Alzheimer’s Trial 18 Jun 2021
8. Phase 2 Data of AADvac1 in Alzheimer’s Disease Published 16 Jun 2021
9. Anti-Tau Antibody Data Leave Best Isotype Unclear 14 May 2021
10. N-Terminal Tau Antibodies Fade, Mid-Domain Ones Push to the Fore 27 Mar 2021
11. Aiming at the Tangle’s Heart? DIAN-TU Trial to Torpedo Tau’s Core 18 Mar 2021