Abstract Body

Title: In Vitro Amplification of Pathogenic Tau in a Strain-Dependent Manner

Presenter: Virginia MY Lee, University of Pennsylvania, USA

Abstract: The microtubule-associated protein tau (tau) forms hyperphosphorylated aggregates in tauopathy patient brains that can be pathologically and biochemically defined as distinct tau strains. Recent study shows that these tau strains exhibit strain-specific biological activities or pathogenicities in the tau spreading models. Currently, the specific pathogenicity of human-derived tau strains cannot be fully recapitulated by synthetic tau preformed fibrils (pffs), generated from the aggregation of recombinant tau protein. Understanding disease-relevant tau pathology necessitates the use of human brain-derived tau seeds to model tauopathy and the continued search for tau variants that can consistently and accurately mimic the pathogenicity of human-derived tau seeds. The in vitro seeding reaction based on their ability to template pathological conformations may serve as a faithful approach for the amplification of tau strains. However, little is known about the fidelity and efficacy of the amplification. Here, we used biochemically enriched brain-derived tau seeds from Alzheimer’s disease (AD), corticobasal degeneration (CBD) and progressive supranuclear palsy (PSP) patients’ brain with a modified seeding protocol to template the recruitment of recombinant 2N4R (T40) tau in vitro. We quantitatively interrogated the fidelity and efficacy of the amplification reactions in their pathogenicity using recently developed sporadic tau spreading models. Our data suggest that different tau strains can be faithfully amplified in vitro from tau isolated from different tauopathy brains, and that the amplified tau variants retain their strain-dependent pathogenic characteristics

Aims

Alzheimer’s disease (AD) is characterized by age-of-onset-related heterogeneity in initial patterns of cognitive deficits with non-memory-predominant presentations more likely with earlier onset. We sought to determine if this heterogeneity is related to differences in concomitant pathologies (Lewy Bodies, TDP-43 inclusions, vascular) or distributions of neurofibrillary tangle pathology across limbic and cortical regions.

Methods

Baseline neuropsychological testing from 121 cases with autopsy-confirmed, severe AD pathology (Braak V-VI) was examined. Estimated age of onset ranged from 50 to 87. Testing occurred on average 4.2±2.7 years after onset and 6.4±3.1 years before death. Memory, Language, Executive, and Visuospatial domain scores were generated. Concomitant Lewy body, TDP-43, and vascular pathology was staged, and the ratio of Midfrontal Cortex/Hippocampal CA1 tangles (“MF/HP tangle ratio”) was calculated as a proxy for tangle distribution.

Results

The MF/HP tangle ratio decreased with increasing age of onset (p = 1.5x10^-5) consistent with a more neocortical tangle distribution with earlier onset. TDP-43 (p=.02) and vascular (p=0.003), but not Lewy body pathology, increased with age of onset, as did Executive (p=.02) and Visuospatial (p=2.2x10^-4), but not Memory or Language scores. Mediation analyses showed that the relationship between age of onset and Executive scores was mediated by the tangle ratio (p=0.008), but not Lewy body, TDP-43, or vascular pathology. The relationship with Visuospatial scores was not mediated by any of the 4 pathology measures.

Conclusions

Age-of-onset-related variability in initial cognitive deficit profiles is mediated by age-related differences in the distribution of tangle pathology in AD and not by concomitant Lewy body, TDP-43, or vascular pathology.

Aims

Neuronal hyperexcitability linked to an increase in glutamate signalling is a peculiar trait of the early stages of Alzheimer's disease (AD) and tauopathies, however, a progressive reduction in glutamate release follows in advanced stages. We recently reported that in the early phases of the neurodegenerative process, soluble, non-aggregated Tau accumulates in the nucleus and modulates the expression of disease-relevant genes directly involved in glutamatergic transmission, thus establishing a link between Tau instability and altered neurotransmission.

Methods

We investigated, at cellular level, Tau aggregation mechanism in the nuclear compartment, and we evaluated the impact on disease-related gene expression.

Moreover, we evaluated the glutamatergic synapse function in PFC of DA patients by GSEA.

Results

Here we report that while the nuclear translocation of Tau in cultured cells is not impaired by its own aggregation, the nuclear amyloid inclusions of aggregated Tau abolish Tau-dependent increased expression of the glutamate transporter. Remarkably, we observed that in the prefrontal cortex (PFC) of AD patient brain, the glutamate transporter is upregulated at early stages and is downregulated at late stages.

Conclusions

The Gene Set Enrichment Analysis indicates that the modulation of Tau-dependent gene expression along the disease progression can be extended to all protein pathways of the glutamatergic synapse. Together, this evidence links the altered glutamatergic function in the PFC during AD progression to the newly discovered function of nuclear Tau.

Abstract Body

Tau has become an unavoidable therapeutic target in Alzheimer's disease and progressive supranuclear palsy. Decreasing the expression of tau is the most commonly used strategy. In addition to anti-sense oligonucleotides, anti-tau immunotherapy is one of the most advanced therapeutic approaches. However, targeting tau remains a challenge. Tau is primarily an intracellular protein. In such paradigm, immunological tools must reach the cytoplasm of the cells to be targeted. However, tau protein is also secreted, and its pro-aggregative species are believed to participate in tau pathology spreading by a prion-like mechanism. In this hypothesis, immunological tools should block extracellular pathological species. From a technological point of view, immunological tools have largely evolved and there is now a very wide panel: monoclonal antibody (immunoglobulin, IgG), Fab fragment, single-chain variable fragment (Fv), VHH single domain antibody (or nanobody), ... Each of them has advantage.

In the laboratory, we work with IgGs that recognise different tau regions or pathological post-translational modifications (phosphorylation or acetylation). We have also developed numerous anti-tau VHHs that can be used as recombinant protein or by viral vectorisation. We use many experimental models of tauopathy (FRET seeding assay, murine models (seeding/spreading)...) for preclinical studies.

In this work, we will discuss the advantages and disadvantages of using a total anti-tau immunoglobulin versus pathological anti-tau. We will also see the opportunity to use VHH-based gene therapy for certain tauopathies considered as orphan disease (FTLD-MAPT).

Altogether, we show that Tau is a promising therapeutic target, but targeted epitope is crucial to avoid side-effects.

Aims

Pinteon Therapeutics has generated a humanized monoclonal antibody, PNT001, that recognizes cis-pT231 tau with high affinity and selectivity and may reduce the spread of tau pathology and augment tau clearance in tauopathies. We performed a Phase 1, randomized, double-blind, placebo-controlled, single-ascending-dose trial to evaluate the safety, tolerability, immunogenicity, and pharmacokinetics of intravenous PNT001 in healthy volunteers.

Methods

The study was a single-dose, double-blind, placebo-controlled dose cohort escalation design with PNT001 at doses of 33mg, 100mg, 300 mg, 900 mg, 2700 mg, delivered as a single 30-minute intravenous infusion. Each dose cohort contained 6 active and 2 placebo subjects. Safety, tolerability, PK and biomarker data were collected over 16 weeks. CSF was collected at day 3 and day 28 to assess PNT001 CSF and biomarker concentrations. Dose escalation was determined by an external, independent data safety monitoring board based on review of adverse event data.

Results

Three non-serious adverse Grade 1 events were determined to be related to study drug/placebo and all resolved without sequelae. No maximum tolerated dose was identified and there were no premature discontinuations, dose reductions, or dose interruptions due to adverse events. Well-tolerated doses provided CSF concentrations of PNT001 several fold in excess of the K_d for PNT001-antigen binding.

Conclusions

PNT001 was well tolerated in human volunteers at doses that provide a potentially therapeutic CSF drug concentration. These results support further multiple ascending dose studies in patients with tauopathy.

Aims

Semorinemab is a humanized anti-tau IgG4 monoclonal antibody, designed to intercept the cell-to-cell propagation of pathological tau. The safety and efficacy of semorinemab in patients with prodromal-to-mild Alzheimer’s disease over 18-months was assessed in the Tauriel Study (NCT03289143), a Phase 2 multi-center, randomized, double-blind, placebo-controlled, parallel-group clinical trial. Pharmacokinetic (PK) and pharmacodynamic (PD) analyses were performed on blood and cerebrospinal fluid (CSF) samples from this study.

Methods

CSF was collected from a subset of patients at baseline and after 49 and 73 weeks of treatment with semorinemab or placebo. Drug concentrations were measured using an enzyme-linked immunosorbent assay in CSF. Protein biomarker concentrations were analyzed in plasma or CSF using multiple assays including the Elecsys® total tau (tTau) and phospho-tau 181P (pTau).

Results

Preliminary longitudinal CSF data were available for 71 patients and indicate semorinemab CSF exposure was approximately dose-proportional. In patients treated with placebo, tTau and pTau increased slightly over 73 weeks (1.2%, 2.4%). However, in patients treated with semorinemab, CSF tau levels trended towards decline (1500 mg tTau −15.6% and pTau −19.0%, respectively; 4500 mg −4.4%, −10.4%; 8100 mg −2.7%, −13.7%), with no consistent dose-response relationship. Similarly, there was no consistent PK/PD relationship with CSF tTau or pTau. Pending results from additional biomarker assays will be presented.

Conclusions

CSF PK indicate that the expected dose-dependent concentrations of semorinemab in the central nervous system were achieved in Tauriel. Trends towards lower CSF tTau and pTau levels were seen with semorinemab treatment, but no clear PK/PD relationship was observed.

Aims

Cell-to-cell transmission of pathogenic tau species has been proposed as the principal mechanism for the propagation of tau pathology across brain regions in Alzheimer’s disease (AD). In order to develop targeted therapeutics that may delay pathological progression in AD by impacting this prion-like mechanism, an unbiased epitope strategy was used to define critical regions of tau involved in neuronal uptake and to assess antibodies targeting regions throughout tau in multiple in vitro and in vivo assays.

Methods

Antibody binding profiles were assessed by surface plasmon resonance, Western blotting, ELISA, and immunohistochemistry. Inhibition of tau internalization and neurotoxicity was tested in B103 cells and mouse primary cortical neurons, respectively. Seeding and passive administration studies were performed in tau transgenic mice.

Results

A large antibody panel was created, consisting of antibodies with high affinity to tau irrespective of aggregation state. In vitro assessment of this panel identified antibodies targeting the MTBR region of tau as more effectively blocking tau internalization and toxicity than antibodies binding other regions of tau. Upon further examination, one of these antibodies, mPRX005 (the murine precursor of PRX005), demonstrated significant inhibition of tau seeding in vitro and in vivo, and slowed pathological progression in a tau transgenic mouse model.

Conclusions

Antibodies spanning the full-length tau molecule were tested for neutralization activity in a variety of in vitro models, with activity confirmed in vivo. MTBR-binding antibody mPRX005 displayed consistent potency across a range of neutralization and efficacy assays, supporting further investigation as a clinical candidate.

Aims

Progressive supranuclear palsy (PSP) is a relentlessly progressive tauopathy for which there are no approved therapies. Bepranemab (UCB0107) is a recombinant, humanized, full-length IgG4 monoclonal antibody that targets a central tau epitope. Our objective is to evaluate bepranemab versus placebo in people living with PSP.

Methods

This double-blind, placebo-controlled, Phase III study (PSP001; auTAUnomy) will assess the efficacy, safety, and tolerability of intravenously administered bepranemab. Patients ≥40 years old with possible/probable PSP-Richardson’s syndrome (MDS-PSP diagnostic criteria) will be recruited from study centers in Asia, Australia, Europe, and North America. Inclusion criteria include patients able to walk ≥5 steps with/without minimal assistance; exclusion criteria include patients with any clinically significant neurological disorder, other than PSP. Patients (N=384) will be randomized 1:1 to receive bepranemab or placebo, every 4 weeks up to Week 100. Primary endpoint: Combined Assessment of Function and Survival (CAFS), based on change from baseline in the signs and symptoms of PSP (measured by the PSP rating scale [PSPRS]) and time to death up to Week 104. Secondary endpoints include change from baseline in patients’ ability to attend to activities of daily living (ADL), measured by the Schwab and England ADL Scale, and safety and tolerability.

Results

Enrolment will begin in mid-2021.

Conclusions

This is the first study in PSP to utilize a 2-year study design with a joint assessment of PSPRS and survival as the primary endpoint. This design will enable robust assessment of the efficacy and safety of bepranemab in people living with PSP.