Abstract Body

Tauopathies exhibit diverse pathological mechanisms but share intracellular Tau deposits. On a therapeutic point of view, there is a debate on targeting Tau inside or outside cells. Extracellular Tau, linked to the prion-like tau propagation hypothesis, is a potential target for Alzheimer's disease. Conversely, intracellular Tau is the target for tauopathies overall, including rare forms like frontotemporal lobar degeneration and progressive supranuclear palsy.

Early clinical strategies often overlook specifics of rare tauopathies differing from Alzheimer's patterns. While antisense oligonucleotides show promise, alternative approaches are crucial. Our results indicate that the Z70 Tau-specific intrabody can inhibit Tau self-association and limit seeding (Danis C. et al., Mol Ther 2022).

Leveraging our library of anti-Tau nanobodies with proven ability to block Tau aggregation within cells, we aim to develop them into therapeutics. In addition to Z70, we selected two other anti-tau nanobodies interfering with tau aggregation.

Intrahippocampal injections of AAV vectors encoding anti-tau VHHs (secreted minibody or intrabody) were done in both wild-type and Thy-tau transgenic mice. After eight months, wild-type mice did not show any deleterious effect. For tau mice, our analysis has focussed on crucial factors: anti-aggregation and pharmacological properties of these new biologics, efficacy in preserving cognitive/motor functions in preclinical models, engagement with Tau targets to reduce pathophysiological tauopathy signs, and safety of treatment.

In conclusion, both intra- and extra-cellular tau targeting might be an innovative approach in the treatment of tauopathies.

Abstract Body

Alzheimer’s disease (AD) drug development has entered a new era, bringing disease-modifying therapies to patients. With the initial success of anti-amyloid plaque therapies, a critical next step towards preventing AD progression is the targeting of key additional pathologies, such as tau. The progressive accumulation of the microtubule-associated protein tau in the brain is a characteristic hallmark of AD and other tauopathies, and tau aggregation is the final common pathway towards neurodegeneration in AD. By reducing intracellular tau aggregation, it may be possible to block further neuronal dysfunction and death. Inhibition of the O-GlcNAcase (OGA) enzyme is proposed to delay the progression of tau-related diseases by slowing the accumulation of hyper-phosphorylated, insoluble tau filaments. We will present the discovery and our innovative approach to clinical development of LY3372689, a low dose, central nervous system (CNS) penetrant OGA inhibitor currently under investigation in a phase 2 clinical trial in patients with early symptomatic AD.

Abstract Body

Objectives: The presence of β-amyloid (Aβ) plaques and neurofibrillary tangles of tau are two main neuropathological changes in the brain of Alzheimer disease (AD). Lowered efficiency of degradation pathways, such as Ubiquitin Proteasome System (UPS), further exacerbates the accumulation of aberrant Aβ, tau, α-synuclein and other proteins. Proteolysis-Targeting Chimeras (PROTACs) are hetero-bifunctional molecules that can bring the E3 ligase into the vicinity of protein of interest, leading to protein ubiquitination, followed by proteasomal degradation. Since 2016, PROTACs have been applied to resolve AD-related pathologies, progressing from being based on peptides to fully synthetic small molecules. These PROTACs were constructed to target tau, α-synuclein, or GSK-3β and most of their effects have only been proven in cell models. In this study, we aim to perform intensive research to promote the development of small-molecule PROTACs for AD treatment.

Methods: PROTACs can be developed to target aberrant tau, α-synuclein, intracellular Aβ and other proteins involved in AD pathology. Here we put tau as an example. Firstly, we are developing novel tau-binding ligands and link them to ligands binding to E3 ligase, for instance, VHL, CRBN or CHIP. Then protein binding affinities and tau-reducing effects are evaluated in vitro. Structure optimization is planned for the hit molecules and the most potent PROTACs with least off-target effects will move on to in vivo study: pharmacokinetics and evaluation of treatment effects in tau transgenic mice model.

Results: We perform the development of small-molecule PROTACs according to the methods listed above. We have chosen some published small ligands that can bind to tau, VHL or CRBN and use them to design PROTACs.

Conclusions: The development of small-molecule PROTACs will bring breakthrough medicines for AD and other neurodegenerative diseases.

Aims

HMTM is an oral treatment targeting pathological tau aggregation. It also has secondary tau-independent activity which increases acetylcholine levels in hippocampus. Key topline results are summarised.

Methods

LUCIDITY is a Phase 3 trial in MCI-AD and mild to moderate AD in an amyloid-PET positive population. An initial 12-month randomised double-blind period compared HMTM at 16 (target dose) and 8 mg/day with methylthioninium chloride (MTC) 4 mg twice weekly as a blinded control. After 12 months, all participants received 16 mg/day HMTM in a modified delayed-start design. Change on ADAS-cog₁₁ and ADCS-ADL₂₃ over 12 months were coprimary clinical outcomes and change in plasma neurofilament light chain (NfL) concentration was the prespecified blood biomarker outcome to measure progression of neurodegeneration. Various subgroup analyses were pre-specified including split by diagnosis.

Results

Participant numbers at randomisation, 12 months and 24 months were: HMTM 16 mg/day (252/191/162), 8 mg/day (80/57/39) and control (266/207/170). HMTM 16 mg/day produced a statistically significant 93% reduction in change in NfL as randomised (p=0.0278) compared to control. Overall cognitive decline did not differ between arms but was significantly less than closely matched real-world populations over 12 and 24 months. Relative to baseline, MCI-AD subjects receiving 16 mg/day showed statistically significant cognitive improvement sustained over 18 months and no decline at 24 months. Control MCI-AD subjects had transient symptomatic benefit at 6 months, but thereafter declined significantly. Control subjects with AD also experienced initial symptomatic exposure-dependent cognitive benefit. The safety profile was excellent with no ARIA.

Conclusions

HMTM combines slowing of neurodegeneration and symptomatic benefit. It is an accessible, safe, oral treatment for MCI-AD to mild/ moderate AD which could be delivered with minimal patient and physician burden worldwide.

Aims

Development of next generation of treatments and diagnostics for protein aggregation diseases.

Methods

Preclinical studies in various models.

Results

Immunotherapies for synucleinopathies and tauopathies are the most common disease-modifying approach in clinical trials for these diseases. The majority are whole antibodies and a few are vaccines. Single domain antibodies (sdAbs) have several advantages over whole antibodies and have shown promise in preclinical studies targeting α-synuclein (αsyn) or tau. Their advantages include greater brain entry and ability to bind to cryptic epitopes that whole antibodies cannot reach. They are also ideally suited for gene therapy because of their single domain, which facilitates proper folding within the cell. We have generated numerous sdAbs from phage display libraries from llamas immunized with αsyn or tau proteins. Following in vitro screening, several have shown efficacy in fly and mouse models by transgenetic, gene therapy and PROTAC approaches, supporting their further development. We recently reported on the initial characterization of the anti-tau sdAbs, including the influence of four Fc subclasses on efficacy and toxicity (Congdon EE et al EBioMedicine 2022). We have also explored their diagnostic potential. Small molecule imaging probes for tau are in use but αsyn probes have been more difficult to develop. These target β-sheets and are therefore only selective for their desired target whereas sdAbs can be specific and thereby more accurately reflect tau or αsyn burden. We recently reported their excellent in vivo diagnostic potential in mouse models (Jiang Y et al Sci Adv 2023), and are advancing those studies.

Conclusions

Single domain antibodies have several advantages over whole antibodies. Recent findings from my group and others support their development as the next generation of treatments and imaging agents for synucleinopathies and tauopathies.

Aims

Pathological tau aggregates in neurodegenerative tauopathies, including Alzheimer’s disease (AD)­­, are ~10,000 times more abundant in intracellular vs. extracellular. However, current tau immunotherapies are ineffective at clearing intracellular tau aggregates, or improving cognitive function in clinical trials. Aims of this study: (i) can toxic tau conformation–specific MAB-2–loaded micelles (TTCM2-ms) specifically detect disease-relevant tau aggregates in tauopathies ; (ii) can TTCM2-ms effectively neutralize the seeding activity of AD brain-derived tau oligomers (AD-BDTOs); (iii) do TTCM2-ms effectively and rapidly reach the brain following intranasal delivery; and (iv) can TTCM2-ms eliminate intracellular, synaptic, and seed-competent tau aggregates and ameliorate cognitive decline in tauopathy mice

Methods

We use intranasal route for delivery, thus relying on the nose-to-brain direct anatomic pathway, which is considered a viable, non-invasive, safe, and effective drug-delivery approach for administering of TTCM2-ms a in aged hTau mice ( 15 months). Investigated treatment effects on Cognition ,tau pathology and mechanism involved in the antibody-mediated clearance of tau aggregates.

Results

TTCM2-ms potently inhibit tau-seeding activity, an essential mechanism underlying tauopathy progression, and intranasally delivered TTCM2-ms can efficiently reach the brain in tauopathy mice, targeting various regions and intracellular compartments. Moreover, a single intranasal dose of TTCM2-ms effectively cleared pathological tau, increased levels of synaptic proteins, and improved cognitive functions in aged tauopathy mice. Mechanistic studies indicate that TTCM2-ms clears intracellular, synaptic, and seed-competent tau aggregates via tripartite motif-containing 21 (TRIM21), an intracellular antibody receptor and E3-ubiquitin ligase essential for TTCM2-ms–mediated clearance of tau pathology.

Conclusions

Our findings suggest intranasal TTCM2-ms rapidly enter the brain of tauopathy mice, and engage TRIM21 to reduce intracellular and synaptic tau pathology, leading to enhanced cognitive function. As the conformational epitopes recognized by TTCM2-ms might be structurally conserved and critical for tau-seeding activity.

Aims

Previous work showed a central role of pathological Tau in synapse dysfunction and consequently synapse degeneration. We have shown that Tau translocates to the pre-synapse and binds to synaptic vesicles via Synaptogyrin-3 (SYNGR3). Moreover, we found that lowering Synaptogyrin-3 levels in Tau-mutant mice rescues Tau-induced synapse degeneration and memory loss.

The aim of this project is to study whether Synaptogyrin-3 is sufficient to rescue Tau-induced synapse loss in human Alzheimer’s model.

Methods

To study if lowering SYNGR3 also rescues neuronal decline in human models of disease, we will use a xenotransplantation model were human induced neurons grow in a mouse host that produces Aβ. This drives Tau pathology in human neurons, causing them to undergo neurodegeneration. We will knockout SYNGR3 in the human neurons and ask if loss of SYNGR3 protects them against synapse- and neuro-degeneration.

Results

We have designed Synaptogyrin-3 knockout neurons which integrate as single neurons into the cortex of the mouse. Moreover, we have engineered a novel tool to study human synapses after xenotransplantation and shown that they form mature and functional synapses. However, when the cells are exposed to Aβ pathology, synapses become dystrophic and synapse loss is observed around plaques. Currently, we are studying the effects of Tau-induced pathology and Synaptogyirin-3 in this model.

Conclusions

This study highlights the possibility to study human-induced synapse loss in an in vivo model of disease. Moreover, it aims to rescue synapse loss in a therapeutic manner by using lowering Synaptogyrin-3 levels. This research recognizes the close correlation between cognitive impairment, Tau dysfunction and synaptic decline and aims to find a therapeutic candidate.

Aims

Regulatory T cells (Tregs) are a subset of T cells that play a neuroprotective role by suppressing inflammation. Treg immunomodulatory mechanisms are compromised in Alzheimer’s disease (AD) individuals, shifting the immune system toward pro-inflammatory status. We investigated the safety and feasibility of low-dose Interleukin-2 (IL-2) immunotherapy on restoring Tregs and modifying inflammation in an AD clinical setting.

Methods

Eight AD dementia (MMSE:12-25) individuals were enrolled in a phase 1, open-label, feasibility study of IL-2 treatment. The presence of brain amyloid pathology was confirmed in all participants. Enrolled individuals received monthly five-day-courses of subcutaneous IL-2 for four months and were followed for an additional two-month post-treatment. Safety measures were monitored throughout the trial. Treg immunophenotype and function were assayed serially at screening, on day 1 (before IL-2 treatment) and day 8 (three days after the fifth dose) of each treatment cycle during therapy, and then on days 120 and 168.

Results

Low-dose IL-2 immunotherapy was safe and well-tolerated. The percentage of CD4⁺CD25^highFoxP3⁺ Tregs increased after each IL-2 treatment cycle (p<0.01) and returned to baseline before the next cycle. Treg suppressive function also progressively increased throughout the four-month IL-2 treatment phase (p<0.01). IL-2 treatment down-regulated inflammatory cytokine transcript expressions in circulating monocytes and modulated plasma pro-inflammatory analytes (IL-15, CCL2, CCL4, CCL11 and FLT3LG; p<0.01). Clinical improvement was observed on the Mini-Mental State Exam and the Clinical Dementia Rating Sum of Boxes.

Conclusions

IL-2 immunotherapy restored Tregs and ameliorated systemic pro-inflammatory mediators in AD individuals. The results of this open-label feasibility study warrant conducting a placebo-controlled clinical trial to further evaluate the safety and efficacy of low-dose IL-2 as a potential treatment for AD.