Immunotherapy targeting the C-terminal domain of TDP-43 decreases neuropathology and confers neuroprotection in mouse models of ALS/FTD.

Effective therapies are urgently needed to safely target TDP-43 pathology as it is closely associated with the onset and development of devastating diseases such as frontotemporal lobar degeneration with TDP-43 pathology (FTLD-TDP) and amyotrophic lateral sclerosis (ALS). In addition, TDP-43 pathology is present as a co-pathology in other neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. Our approach is to develop a TDP-43-specific immunotherapy that exploits Fc gamma-mediated removal mechanisms to limit neuronal damage while maintaining physiological TDP-43 function. Thus, using both in vitro mechanistic studies in conjunction with the rNLS8 and CamKIIa inoculation mouse models of TDP-43 proteinopathy, we identified the key targeting domain in TDP-43 to accomplish these therapeutic objectives. Targeting the C-terminal domain of TDP-43 but not the RNA recognition motifs (RRM) reduces TDP-43 pathology and avoids neuronal loss in vivo. We demonstrate that this rescue is dependent on Fc receptor-mediated immune complex uptake by microglia. Furthermore, monoclonal antibody (mAb) treatment enhances phagocytic capacity of ALS patient-derived microglia, providing a mechanism to restore the compromised phagocytic function in ALS and FTD patients. Importantly, these beneficial effects are achieved while preserving physiological TDP-43 activity. Our findings demonstrate that a mAb targeting the C-terminal domain of TDP-43 limits pathology and neurotoxicity, enabling clearance of misfolded TDP-43 through microglia engagement, and supporting the clinical strategy to target TDP-43 by immunotherapy. SIGNIFICANCE STATEMENT: TDP-43 pathology is associated with various devastating neurodegenerative disorders with high unmet medical needs such as frontotemporal dementia (FTD), amyotrophic lateral sclerosis (ALS) and Alzheimer's disease. Thus, safely and effectively targeting pathological TDP-43 represents a key paradigm for biotechnical research as currently there is little in clinical development. After years of research, we have determined that targeting the C-terminal domain of TDP-43 rescues multiple patho-mechanisms involved in disease progression in two animal models of FTD/ALS. In parallel, importantly, our studies establish that this approach does not alter the physiological functions of this ubiquitously expressed and indispensable protein. Together, our findings substantially contribute to the understanding of TDP-43 pathobiology and support the prioritization for clinical testing of immunotherapy approaches targeting TDP-43.

Structure-activity relationship around PI-2620 highlights the importance of the nitrogen atom position in the tricyclic core.

Tau aggregates represent a critical pathology in Alzheimer's disease (AD) and other forms of dementia. The extent of Tau neurofibrillary tangles across defined brain regions corresponds well to the observed level of cognitive decline in AD. Compound 1 (PI-2620) was recently identified as a promising Tau positron emission tomography tracer for AD and non-AD tauopathies. To evaluate the impact of the N-atom position with respect to Tau- and off-target binding, tricyclic core analogs of PI-2620 with nitrogen atoms at different positions were prepared. Affinity to aggregated Tau was evaluated using human AD brain homogenates, and their off-target binding was evaluated in a monoamine oxidase A (MAO-A) competition assay. The novel tricyclic core derivatives all displayed inferior Tau binding or MAO-A off-target selectivity, indicating PI-2620 to be the optimal design for high affinity binding to Tau and high MAO-A selectivity.

Discovery and preclinical characterization of [18F]PI-2620, a next-generation tau PET tracer for the assessment of tau pathology in Alzheimer's disease and other tauopathies.

PURPOSE: Tau deposition is a key pathological feature of Alzheimer's disease (AD) and other neurodegenerative disorders. The spreading of tau neurofibrillary tangles across defined brain regions corresponds to the observed level of cognitive decline in AD. Positron-emission tomography (PET) has proved to be an important tool for the detection of amyloid-beta (Aß) aggregates in the brain, and is currently being explored for detection of pathological misfolded tau in AD and other non-AD tauopathies. Several PET tracers targeting tau deposits have been discovered and tested in humans. Limitations have been reported, especially regarding their selectivity. METHODS: In our screening campaign we identified pyrrolo[2,3-b:4,5-c']dipyridine core structures with high affinity for aggregated tau. Further characterization showed that compounds containing this moiety had significantly reduced monoamine oxidase A (MAO-A) binding compared to pyrido[4,3-b]indole derivatives such as AV-1451. RESULTS: Here we present preclinical data of all ten fluoropyridine regioisomers attached to the pyrrolo[2,3-b:4,5-c']dipyridine scaffold, revealing compounds 4 and 7 with superior properties. The lead candidate [18F]PI-2620 (compound 7) displayed high affinity for tau deposits in AD brain homogenate competition assays. Specific binding to pathological misfolded tau was further demonstrated by autoradiography on AD brain sections (Braak I-VI), Pick's disease and progressive supranuclear palsy (PSP) pathology, whereas no specific tracer binding was detected on brain slices from non-demented donors. In addition to its high affinity binding to tau aggregates, the compound showed excellent selectivity with no off-target binding to Aß or MAO-A/B. Good brain uptake and fast washout were observed in healthy mice and non-human primates. CONCLUSIONS: Therefore, [18F]PI-2620 was selected for clinical validation.

Discovery and characterization of novel indole and 7-azaindole derivatives as inhibitors of ß-amyloid-42 aggregation for the treatment of Alzheimer's disease.

The aggregation of amyloid-ß peptides into cytotoxic oligomeric and fibrillary aggregates is believed to be one of the major pathological events in Alzheimer disease. Here we report the design and synthesis of a novel series of indole and 7-azaindole derivatives containing, nitrile, piperidine and N-methyl-piperidine substituents at the 3-position to prevent the pathological self-assembly of amyloid-ß. We have further demonstrated that substitution of the azaindole and indole derivatives at the 3 positions is required to obtain compounds with improved physicochemical properties to allow brain penetration.

Synthesis and structure-activity relationship of 2,6-disubstituted pyridine derivatives as inhibitors of ß-amyloid-42 aggregation.

It is assumed that amyloid-ß aggregation is a crucial event in the pathogenesis of Alzheimer's disease. Novel 2,6-disubstituted pyridine derivatives were designed to interact with the ß-sheet conformation of Aß via donor-acceptor-donor hydrogen bond formation. A series of pyridine derivatives were synthesized and tested regarding their potential to inhibit the aggregation of Aß. The 2,6-diaminopyridine moiety was identified as a key component to inhibit Aß aggregation. Overall, compounds having three 2,6-disubstituted pyridine units separated by at least one C2- or C3-linker displayed the most potent inhibition of Aß aggregation.

Human genome-guided identification of memory-modulating drugs.

In the last decade there has been an exponential increase in knowledge about the genetic basis of complex human traits, including neuropsychiatric disorders. It is not clear, however, to what extent this knowledge can be used as a starting point for drug identification, one of the central hopes of the human genome project. The aim of the present study was to identify memory-modulating compounds through the use of human genetic information. We performed a multinational collaborative study, which included assessment of aversive memory--a trait central to posttraumatic stress disorder--and a gene-set analysis in healthy individuals. We identified 20 potential drug target genes in two genomewide-corrected gene sets: the neuroactive ligand-receptor interaction and the long-term depression gene set. In a subsequent double-blind, placebo-controlled study in healthy volunteers, we aimed at providing a proof of concept for the genome-guided identification of memory modulating compounds. Pharmacological intervention at the neuroactive ligand-receptor interaction gene set led to significant reduction of aversive memory. The findings demonstrate that genome information, along with appropriate data mining methodology, can be used as a starting point for the identification of memory-modulating compounds.

TLR4- and TRIF-dependent stimulation of B lymphocytes by peptide liposomes enables T cell-independent isotype switch in mice.

Immunoglobulin class switching from IgM to IgG in response to peptides is generally T cell-dependent and vaccination in T cell-deficient individuals is inefficient. We show that a vaccine consisting of a dense array of peptides on liposomes induced peptide-specific IgG responses totally independent of T-cell help. Independency was confirmed in mice lacking T cells and in mice deficient for MHC class II, CD40L, and CD28. The IgG titers were high, long-lived, and comparable with titers obtained in wild-type animals, and the antibody response was associated with germinal center formation, expression of activation-induced cytidine deaminase, and affinity maturation. The T cell-independent (TI) IgG response was strictly dependent on ligation of TLR4 receptors on B cells, and concomitant TLR4 and cognate B-cell receptor stimulation was required on a single-cell level. Surprisingly, the IgG class switch was mediated by TIR-domain-containing adapter inducing interferon-ß (TRIF), but not by MyD88. This study demonstrates that peptides can induce TI isotype switching when antigen and TLR ligand are assembled and appropriately presented directly to B lymphocytes. A TI vaccine could enable efficient prophylactic and therapeutic vaccination of patients with T-cell deficiencies and find application in diseases where induction of T-cell responses contraindicates vaccination, for example, in Alzheimer disease.

An effector-reduced anti-ß-amyloid (Aß) antibody with unique aß binding properties promotes neuroprotection and glial engulfment of Aß.

Passive immunization against ß-amyloid (Aß) has become an increasingly desirable strategy as a therapeutic treatment for Alzheimer's disease (AD). However, traditional passive immunization approaches carry the risk of Fcγ receptor-mediated overactivation of microglial cells, which may contribute to an inappropriate proinflammatory response leading to vasogenic edema and cerebral microhemorrhage. Here, we describe the generation of a humanized anti-Aß monoclonal antibody of an IgG4 isotype, known as MABT5102A (MABT). An IgG4 subclass was selected to reduce the risk of Fcγ receptor-mediated overactivation of microglia. MABT bound with high affinity to multiple forms of Aß, protected against Aß1-42 oligomer-induced cytotoxicity, and increased uptake of neurotoxic Aß oligomers by microglia. Furthermore, MABT-mediated amyloid plaque removal was demonstrated using in vivo live imaging in hAPP((V717I))/PS1 transgenic mice. When compared with a human IgG1 wild-type subclass, containing the same antigen-binding variable domains and with equal binding to Aß, MABT showed reduced activation of stress-activated p38MAPK (p38 mitogen-activated protein kinase) in microglia and induced less release of the proinflammatory cytokine TNFα. We propose that a humanized IgG4 anti-Aß antibody that takes advantage of a unique Aß binding profile, while also possessing reduced effector function, may provide a safer therapeutic alternative for passive immunotherapy for AD. Data from a phase I clinical trial testing MABT is consistent with this hypothesis, showing no signs of vasogenic edema, even in ApoE4 carriers.

Discovery and structure activity relationship of small molecule inhibitors of toxic ß-amyloid-42 fibril formation.

Increasing evidence implicates Aß peptides self-assembly and fibril formation as crucial events in the pathogenesis of Alzheimer disease. Thus, inhibiting Aß aggregation, among others, has emerged as a potential therapeutic intervention for this disorder. Herein, we employed 3-aminopyrazole as a key fragment in our design of non-dye compounds capable of interacting with Aß42 via a donor-acceptor-donor hydrogen bond pattern complementary to that of the ß-sheet conformation of Aß42. The initial design of the compounds was based on connecting two 3-aminopyrazole moieties via a linker to identify suitable scaffold molecules. Additional aryl substitutions on the two 3-aminopyrazole moieties were also explored to enhance π-π stacking/hydrophobic interactions with amino acids of Aß42. The efficacy of these compounds on inhibiting Aß fibril formation and toxicity in vitro was assessed using a combination of biophysical techniques and viability assays. Using structure activity relationship data from the in vitro assays, we identified compounds capable of preventing pathological self-assembly of Aß42 leading to decreased cell toxicity.

Improved antibody pharmacokinetics by disruption of contiguous positive surface potential and charge reduction using alternate human framework.

Optimal pharmacokinetic (PK) properties of therapeutic monoclonal antibodies (mAbs) are essential to achieve the desired pharmacological benefits in patients. To accomplish this, we followed an approach comprising structure-based mAb charge engineering in conjunction with the use of relevant preclinical models to screen and select humanized candidates with PK suitable for clinical development. Murine mAb targeting TDP-43, ACI-5891, was humanized on a framework (VH1-3/VK2-30) selected based on the highest sequence homology. Since the initial humanized mAb (ACI-5891.1) presented a fast clearance in non-human primates (NHPs), reiteration of humanization on a less basic human framework (VH1-69-2/VK2-28) while retaining high sequence homology was performed. The resulting humanized variant, ACI-5891.9, presented a six-fold reduction in clearance in NHPs resulting in a significant increase in half-life. The observed reduced clearance of ACI-5891.9 was attributed not only to the overall reduction in isoelectric point (pI) by 2 units, but importantly to a more even surface potential. These data confirm the importance and contribution of surface charges to mAb disposition in vivo. Consistent low clearance of ACI-5891.9 in Tg32 mice, a human FcRn transgenic mouse model, further confirmed its utility for early assessment and prediction of human PK. These data demonstrate that mAb surface charge is an important parameter for consideration during the selection and screening of humanized candidates in addition to maintaining the other key physiochemical and target binding characteristics.

Modeling Alzheimer's disease related phenotypes in the Ts65Dn mouse: impact of age on Aß, Tau, pTau, NfL, and behavior.

Introduction: People with DS are highly predisposed to Alzheimer's disease (AD) and demonstrate very similar clinical and pathological features. Ts65Dn mice are widely used and serve as the best-characterized animal model of DS. Methods: We undertook studies to characterize age-related changes for AD-relevant markers linked to Aß, Tau, and phospho-Tau, axonal structure, inflammation, and behavior. Results: We found age related changes in both Ts65Dn and 2N mice. Relative to 2N mice, Ts65Dn mice showed consistent increases in Aß40, insoluble phospho-Tau, and neurofilament light protein. These changes were correlated with deficits in learning and memory. Discussion: These data have implications for planning future experiments aimed at preventing disease-related phenotypes and biomarkers. Interventions should be planned to address specific manifestations using treatments and treatment durations adequate to engage targets to prevent the emergence of phenotypes.

The α-synuclein PET tracer [18F] ACI-12589 distinguishes multiple system atrophy from other neurodegenerative diseases.

A positron emission tomography (PET) tracer detecting α-synuclein pathology will improve the diagnosis, and ultimately the treatment of α-synuclein-related diseases. Here we show that the PET ligand, [18F]ACI-12589, displays good in vitro affinity and specificity for pathological α-synuclein in tissues from patients with different α-synuclein-related disorders including Parkinson's disease (PD) and Multiple-System Atrophy (MSA) using autoradiography and radiobinding techniques. In the initial clinical evaluation we include 23 participants with α-synuclein related disorders, 11 with other neurodegenerative disorders and eight controls. In vivo [18F]ACI-12589 demonstrates clear binding in the cerebellar white matter and middle cerebellar peduncles of MSA patients, regions known to be highly affected by α-synuclein pathology, but shows limited binding in PD. The binding statistically separates MSA patients from healthy controls and subjects with other neurodegenerative disorders, including other synucleinopathies. Our results indicate that α-synuclein pathology in MSA can be identified using [18F]ACI-12589 PET imaging, potentially improving the diagnostic work-up of MSA and allowing for detection of drug target engagement in vivo of novel α-synuclein targeting therapies.

Targeting amyotrophic lateral sclerosis by neutralizing seeding-competent TDP-43 in CSF.

In amyotrophic lateral sclerosis, a disease driven by abnormal transactive response DNA-binding protein of 43 kDa aggregation, CSF may contain pathological species of transactive response DNA-binding protein of 43 kDa contributing to the propagation of pathology and neuronal toxicity. These species, released in part by degenerating neurons, would act as a template for the aggregation of physiological protein contributing to the spread of pathology in the brain and spinal cord. In this study, a robust seed amplification assay was established to assess the presence of seeding-competent transactive response DNA-binding protein of 43 kDa species in CSF of apparently sporadic amyotrophic lateral sclerosis patients. These samples resulted in a significant acceleration of substrate aggregation differentiating the kinetics from healthy controls. In parallel, a second assay was developed to determine the level of target engagement that would be necessary to neutralize such species in human CSF by a therapeutic monoclonal antibody targeting transactive response DNA-binding protein of 43 kDa. For this, evaluation of the pharmacokinetic/pharmacodynamic effect for the monoclonal antibody, ACI-5891.9, in vivo and in vitro confirmed that a CSF concentration of ≍1100 ng/mL would be sufficient for sustained target saturation. Using this concentration in the seed amplification assay, ACI-5891.9 was able to neutralize the transactive response DNA-binding protein of 43 kDa pathogenic seeds derived from amyotrophic lateral sclerosis patient CSF. This translational work adds to the evidence of transmission of transactive response DNA-binding protein of 43 kDa pathology via CSF that could contribute to the non-contiguous pattern of clinical manifestations observed in amyotrophic lateral sclerosis and demonstrates the ability of a therapeutic monoclonal antibody to neutralize the toxic, extracellular seeding-competent transactive response DNA-binding protein of 43 kDa species in the CSF of apparently sporadic amyotrophic lateral sclerosis patients.

Abeta42 neurotoxicity is mediated by ongoing nucleated polymerization process rather than by discrete Abeta42 species.

The identification of toxic Aß species and/or the process of their formation is crucial for understanding the mechanism(s) of Aß neurotoxicity in Alzheimer disease and also for the development of effective diagnostic and therapeutic interventions. To elucidate the structural basis of Aß toxicity, we developed different procedures to isolate Aß species of defined size and morphology distribution, and we investigated their toxicity in different cell lines and primary neurons. We observed that crude Aß42 preparations, containing a monomeric and heterogeneous mixture of Aß42 oligomers, were more toxic than purified monomeric, protofibrillar fractions, or fibrils. The toxicity of protofibrils was directly linked to their interactions with monomeric Aß42 and strongly dependent on their ability to convert into amyloid fibrils. Subfractionation of protofibrils diminished their fibrillization and toxicity, whereas reintroduction of monomeric Aß42 into purified protofibril fractions restored amyloid formation and enhanced their toxicity. Selective removal of monomeric Aß42 from these preparations, using insulin-degrading enzyme, reversed the toxicity of Aß42 protofibrils. Together, our findings demonstrate that Aß42 toxicity is not linked to specific prefibrillar aggregate(s) but rather to the ability of these species to grow and undergo fibril formation, which depends on the presence of monomeric Aß42. These findings contribute significantly to the understanding of amyloid formation and toxicity in Alzheimer disease, provide novel insight into mechanisms of Aß protofibril toxicity, and important implications for designing anti-amyloid therapies.

Sequence-independent control of peptide conformation in liposomal vaccines for targeting protein misfolding diseases.

Synthetic peptide immunogens that mimic the conformation of a target epitope of pathological relevance offer the possibility to precisely control the immune response specificity. Here, we performed conformational analyses using a panel of peptides in order to investigate the key parameters controlling their conformation upon integration into liposomal bilayers. These revealed that the peptide lipidation pattern, the lipid anchor chain length, and the liposome surface charge all significantly alter peptide conformation. Peptide aggregation could also be modulated post-liposome assembly by the addition of distinct small molecule ß-sheet breakers. Immunization of both mice and monkeys with a model liposomal vaccine containing ß-sheet aggregated lipopeptide (Palm1-15) induced polyclonal IgG antibodies that specifically recognized ß-sheet multimers over monomer or non-pathological native protein. The rational design of liposome-bound peptide immunogens with defined conformation opens up the possibility to generate vaccines against a range of protein misfolding diseases, such as Alzheimer disease.

Beneficial Effect of ACI-24 Vaccination on Aß Plaque Pathology and Microglial Phenotypes in an Amyloidosis Mouse Model.

Amyloid-ß (Aß) deposition is an initiating factor in Alzheimer's disease (AD). Microglia are the brain immune cells that surround and phagocytose Aß plaques, but their phagocytic capacity declines in AD. This is in agreement with studies that associate AD risk loci with genes regulating the phagocytic function of immune cells. Immunotherapies are currently pursued as strategies against AD and there are increased efforts to understand the role of the immune system in ameliorating AD pathology. Here, we evaluated the effect of the Aß targeting ACI-24 vaccine in reducing AD pathology in an amyloidosis mouse model. ACI-24 vaccination elicited a robust and sustained antibody response in APPPS1 mice with an accompanying reduction of Aß plaque load, Aß plaque-associated ApoE and dystrophic neurites as compared to non-vaccinated controls. Furthermore, an increased number of NLRP3-positive plaque-associated microglia was observed following ACI-24 vaccination. In contrast to this local microglial activation at Aß plaques, we observed a more ramified morphology of Aß plaque-distant microglia compared to non-vaccinated controls. Accordingly, bulk transcriptomic analysis revealed a trend towards the reduced expression of several disease-associated microglia (DAM) signatures that is in line with the reduced Aß plaque load triggered by ACI-24 vaccination. Our study demonstrates that administration of the Aß targeting vaccine ACI-24 reduces AD pathology, suggesting its use as a safe and cost-effective AD therapeutic intervention.

Safety, Tolerability, and Immunogenicity of the ACI-24 Vaccine in Adults With Down Syndrome: A Phase 1b Randomized Clinical Trial.

Importance: Individuals with Down syndrome (DS) are at high risk of developing Alzheimer disease due to an increased dose of the amyloid precursor protein gene, APP, which leads to increased levels of full-length APP and its products, including amyloid-ß (Aß). The liposome-based antiamyloid ACI-24 vaccine is intended to treat neurological disorders caused by misfolded Aß pathological protein. However, the safety, tolerability, and immunogenicity of the ACI-24 vaccine among adults with DS have not been fully examined. Objective: To assess the safety and tolerability of the ACI-24 vaccine among adults with DS as well as its ability to induce immunogenicity measured by anti-Aß immunoglobulin G titers. Design, Setting, and Participants: This multicenter double-blind placebo-controlled dose-escalation phase 1b randomized clinical trial was conducted at 3 US academic medical centers with affiliated Down syndrome clinics between March 30, 2016, and June 29, 2020. A total of 20 adults with DS were screened; of those, 16 adults were eligible to participate. Eligibility criteria included men or women aged 25 to 45 years with cytogenetic diagnosis of either trisomy 21 or complete unbalanced translocation of chromosome 21. Between April 27, 2016, and July 2, 2018, participants were randomized 3:1 into 2 dose-level cohorts (8 participants per cohort, with 6 participants receiving the ACI-24 vaccine and 2 receiving placebo) in a 96-week study. Participants received 48 weeks of treatment followed by an additional 48 weeks of safety follow-up. Interventions: Participants were randomized to receive 7 subcutaneous injections of ACI-24, 300 µg or 1000 µg, or placebo. Main Outcomes and Measures: Primary outcomes were measures of safety and tolerability as well as antibody titers. Results: Among 16 enrolled participants, the mean (SD) age was 32.6 (4.4) years; 9 participants were women, and 7 were men. All participants were White, and 1 participant had Hispanic or Latino ethnicity. Treatment adherence was 100%. There were no cases of meningoencephalitis, death, or other serious adverse events (AEs) and no withdrawals as a result of AEs. Most treatment-emergent AEs were of mild intensity (110 of 132 events [83.3%]) and unrelated or unlikely to be related to the ACI-24 vaccine (113 of 132 events [85.6%]). No amyloid-related imaging abnormalities with edema or cerebral microhemorrhage and no evidence of central nervous system inflammation were observed on magnetic resonance imaging scans. Increases in anti-Aß immunoglobulin G titers were observed in 4 of 12 participants (33.3%) receiving ACI-24 (2 receiving 300 µg and 2 receiving 1000 µg) compared with 0 participants receiving placebo. In addition, a greater increase was observed in plasma Aß1-40 and Aß1-42 levels among individuals receiving ACI-24. Conclusions and Relevance: In this study, the ACI-24 vaccine was safe and well tolerated in adults with DS. Evidence of immunogenicity along with pharmacodynamic and target engagement were observed, and anti-Aß antibody titers were not associated with any adverse findings. These results support progression to clinical trials using an optimized formulation of the ACI-24 vaccine among individuals with DS. Trial Registration: ClinicalTrials.gov Identifier: NCT02738450.

PI-2620 Lead Optimization Highlights the Importance of Off-Target Assays to Develop a PET Tracer for the Detection of Pathological Aggregated Tau in Alzheimer's Disease and Other Tauopathies.

The first candidate PI-2014 was tested in healthy controls and subjects with Alzheimer's disease (AD). As PI-2014 displayed off-target binding to monoamine oxidase A (MAO-A), a new lead with improved binding to Tau and decreased MAO-A binding was required. For compound optimization, Tau binding assays based on both human AD brain homogenate and Tau-paired helical filaments were employed. Furthermore, two MAO-A screening assays based on (1) human-recombinant MAO-A and (2) displacement of 2-fluoro-ethyl-harmine from mouse brain homogenate were employed. Removing the N-methyl group from the tricyclic core resulted in compounds displaying improved Tau binding. For the final round of optimization, the cyclic amine substituents were replaced by pyridine derivatives. PI-2620 (2-(2-fluoropyridin-4-yl)-9H-pyrrolo[2,3-b:4,5-c']dipyridine) emerged as a best candidate displaying high Tau binding, low MAO-A binding, high brain uptake, and fast and complete brain washout. Furthermore, PI-2620 showed Tau binding on brain sections from corticobasal degeneration, progressive supranuclear palsy, and Pick's disease.

Antibody semorinemab reduces tau pathology in a transgenic mouse model and engages tau in patients with Alzheimer's disease.

Tau has become an attractive alternative target for passive immunotherapy efforts for Alzheimer's disease (AD). The anatomical distribution and extent of tau pathology correlate with disease course and severity better than other disease markers to date. We describe here the generation, preclinical characterization, and phase 1 clinical characterization of semorinemab, a humanized anti-tau monoclonal antibody with an immunoglobulin G4 (igG4) isotype backbone. Semorinemab binds all six human tau isoforms and protects neurons against tau oligomer neurotoxicity in cocultures of neurons and microglia. In addition, when administered intraperitoneally once weekly for 13 weeks, murine versions of semorinemab reduced the accumulation of tau pathology in a transgenic mouse model of tauopathy, independent of antibody effector function status. Semorinemab also showed clear evidence of target engagement in vivo, with increases in systemic tau concentrations observed in tau transgenic mice, nonhuman primates, and humans. Higher concentrations of systemic tau were observed after dosing in AD participants compared to healthy control participants. No concerning safety signals were observed in the phase 1 clinical trial at single doses up to 16,800 mg and multiple doses totaling 33,600 mg in a month.

Quenched substrates for live-cell labeling of SNAP-tagged fusion proteins with improved fluorescent background.

Recent developments in fluorescence microscopy raise the demands for bright and photostable fluorescent tags for specific and background free labeling in living cells. Aside from fluorescent proteins and other tagging methods, labeling of SNAP-tagged proteins has become available thereby increasing the pool of potentially applicable fluorescent dyes for specific labeling of proteins. Here, we report on novel conjugates of benzylguanine (BG) which are quenched in their fluorescence and become highly fluorescent upon labeling of the SNAP-tag, the commercial variant of the human O(6)-alkylguanosyltransferase (hAGT). We identified four conjugates showing a strong increase, i.e., >10-fold, in fluorescence intensity upon labeling of SNAP-tag in vitro. Moreover, we screened a subset of nine BG-dye conjugates in living Escherichia coli and found them all suited for labeling of the SNAP-tag. Here, quenched BG-dye conjugates yield a higher specificity due to reduced contribution from excess conjugate to the fluorescence signal. We further extended the application of these conjugates by labeling a SNAP-tag fusion of the Tar chemoreceptor in live E. coli cells and the eukaryotic transcription factor STAT5b in NIH 3T3 mouse fibroblast cells. Aside from the labeling efficiency and specificity in living cells, we discuss possible mechanisms that might be responsible for the changes in fluorescence emission upon labeling of the SNAP-tag, as well as problems we encountered with nonspecific labeling with certain conjugates in eukaryotic cells.