INTRODUCTION:Semorinemab, an anti-tau monoclonal antibody, was assessed in two Phase II trials for Alzheimer's disease (AD). Plasma and cerebrospinal fluid (CSF) biomarkers provided insights into the drug's potential mechanism of action. METHODS:Qualified assays were used to measure biomarkers of tau, amyloidosis, glial activity, neuroinflammation, synaptic function, and neurodegeneration from participant samples in Tauriel (NCT03289143) and Lauriet (NCT03828747) Phase II trials. RESULTS:Plasma phosphorylated Tau 181 (pTau181) and CSF chitinase-3-like protein 1 (YKL-40) increased following semorinemab treatment in both studies. In Lauriet, increasing plasma glial fibrillary protein (GFAP) concentrations stabilized with semorinemab, while this was not observed in Tauriel. Other AD pathophysiology biomarkers showed no consistent response to semorinemab. DISCUSSION:Increases in CSF YKL-40 suggest that semorinemab may stimulate microglia activation in the presence of AD-associated Tau pathology, but not in healthy controls. Stabilization of plasma GFAP in Lauriet indicates a possible impact on reactive gliosis in mild-to-moderate AD. TRIAL REGISTRATION:Tauriel ClinicalTrials.gov Identifier: NCT03289143. Lauriet ClinicalTrials.gov Identifier: NCT03828747. Phase 1 ClinicalTrials.gov Identifier: NCT02820896. HIGHLIGHTS:AD pathophysiology biomarkers were measured to assess the mechanism of action. Semorinemab increased CSF YKL-40 in participants with AD but not in healthy controls. Semorinemab possibly stabilized plasma GFAP in the Lauriet trial. Semorinemab treatment may activate microglia and moderate reactive gliosis.

INTRODUCTION:Growing evidence suggests a role for neuroinflammation in Alzheimer's disease (AD). We investigated complement pathway activity in AD patient cerebrospinal fluid (CSF) and evaluated its modulation by the anti-tau antibody semorinemab. METHODS:Immunoassays were applied to measure CSF complement proteins C4, factor B (FB), C3 and their cleavage fragments C4a, C3a, and factor Bb (Bb) in AD patients and a separate cognitively unimpaired (CU) cohort. RESULTS:All measured CSF complement proteins were increased in AD versus CU subjects, with C4a displaying the most robust increase. Finally, semorinemab did not have a significant pharmacodynamic effect on CSF complement proteins. DISCUSSION:Elevated levels of CSF C4a, C4, C3a, C3, Bb, and FB are consistent with complement activation in AD brains. Despite showing a reduction in CSF soluble tau species, semorinemab did not impact complement protein levels or activity. Further studies are needed to determine the value of complement proteins as neuroinflammation biomarkers in AD. HIGHLIGHTS:Cerebrospinal fluid (CSF) complement proteins C4a, C3a, Bb, C4, C3, and factor B levels were increased in Alzheimer's disease (AD) patients compared to a separate cognitively unimpaired (CU) cohort. Baseline CSF complement protein levels were correlated with neuro-axonal degeneration and glial activation biomarkers in AD patients. The investigational anti-tau antibody semorinemab did not impact CSF complement protein levels or activity relative to the placebo arm.

The complement classical pathway (CP) is a key mediator of synapse loss and neurodegeneration in mouse models of Alzheimer′s (AD) and other neurodegenerative diseases. We analyzed human brain proteomics and found consistent elevations of all CP proteins, but not other complement pathways, in AD patient brains. We performed human genetics analysis that identified a rare variant in the C1S gene within the Finnish population that is associated with AD and we found that a common AD-associated C1S variant correlates with increased C1S protein levels. A targeted assay detected elevated C1S activation in AD patient CSF. Given this specific implication of the CP in AD, we next evaluated the therapeutic approach of targeting the CP in the brain using antisense oligonucleotides (ASOs). To identify promising CP targets for knockdown using ASOs we first tested for rescue of synapse loss in an AD mouse model using heterozygous and homozygous complement knockout mice and examined the relative brain expression levels of different CP genes. Based on these experiments we prioritized C1r, C1s and C4 as promising targets for therapeutic knockdown using ASOs. We then screened for ASOs for each target, evaluating in vitro and in vivo knockdown and toxicity, and identified optimal ASOs targeting C1r, C1s and C4. Experiments with AD model mice demonstrated significant rescue of synapse loss following treatment with C1r, C1s or C4 ASOs. Overall, our findings provide proof of concept for using nucleic acid-based medicine to target the CP in AD and demonstrate the translational potential of this approach. ### Competing Interest Statement The authors have declared no competing interest.

INTRODUCTION:Triggering receptor expressed on myeloid cells 2 (TREM2) agonists are being clinically evaluated as disease-modifying therapeutics for Alzheimer's disease. Clinically translatable pharmacodynamic (PD) biomarkers are needed to confirm drug activity and select the appropriate therapeutic dose in clinical trials. METHODS:We conducted multi-omic analyses on paired non-human primate brain and cerebrospinal fluid (CSF), and stimulation of human induced pluripotent stem cell-derived microglia cultures after TREM2 agonist treatment, followed by validation of candidate fluid PD biomarkers using immunoassays. We immunostained microglia to characterize proliferation and clustering. RESULTS:We report CSF soluble TREM2 (sTREM2) and CSF chitinase-3-like protein 1 (CHI3L1/YKL-40) as PD biomarkers for the TREM2 agonist hPara.09. The respective reduction of sTREM2 and elevation of CHI3L1 in brain and CSF after TREM2 agonist treatment correlated with transient microglia proliferation and clustering. DISCUSSION:CSF CHI3L1 and sTREM2 reflect microglial TREM2 agonism and can be used as clinical PD biomarkers to monitor TREM2 activity in the brain. HIGHLIGHTS:CSF soluble triggering receptor expressed on myeloid cells 2 (sTREM2) reflects brain target engagement for a novel TREM2 agonist, hPara.09. CSF chitinase-3-like protein 1 reflects microglial TREM2 agonism. Both can be used as clinical fluid biomarkers to monitor TREM2 activity in brain.

Background and ObjectivesAccumulation of tau pathology in Alzheimer disease (AD) correlates with cognitive decline. Anti-tau immunotherapies were proposed as potential interventions in AD. While antibodies targeting N-terminal tau failed to demonstrate clinical efficacy in prodromal-to-mild AD, their utility at other disease stages was not evaluated in prior studies. Lauriet is a phase 2 study of an anti-tau monoclonal antibody, semorinemab, in patients with mild-to-moderate AD.MethodsThe phase 2 Lauriet study included a randomized, placebo-controlled, double-blind period, during which participants with mild-to-moderate AD received 4,500 mg of IV semorinemab or placebo every 4 weeks for 48 or 60 weeks. Participants who chose to continue in the subsequent optional open-label extension received 4,500 mg of semorinemab every 4 weeks for up to 96 weeks. Coprimary efficacy endpoints were change from baseline to week 49 or 61 on the 11-item version of the Alzheimer's Disease Assessment Scale-Cognitive Subscale (ADAS-Cog11) and the Alzheimer's Disease Cooperative Study-Activities of Daily Living (ADCS-ADL) scale. Secondary efficacy endpoints included change from baseline on the Mini-Mental State Examination (MMSE) and Clinical Dementia Rating-Sum of Boxes (CDR-SB). Safety, pharmacokinetics, and pharmacodynamic effects were also evaluated.ResultsBetween December 3, 2018, and February 27, 2020, 624 individuals were screened, 272 participants were randomized, and 238 were included in the modified intent-to-treat population (received >= 1 dose(s) of study medication and underwent baseline and >= 1 postbaseline assessment(s)). Baseline characteristics were well balanced. At week 49, the semorinemab arm demonstrated a 42.2% reduction (-2.89 points, 95% CI -4.56 to -1.21, p = 0.0008) in decline on the ADAS-Cog11 (coprimary endpoint) relative to the placebo arm. However, no treatment effects were observed on the ADCS-ADL scale (coprimary endpoint; absolute difference between the 2 treatment arms in the ADCS-ADL score change from baseline of -0.83 points, 95% CI -3.39 to 1.72, p = 0.52) or on the MMSE or CDR-SB (secondary endpoints). Semorinemab was safe and well tolerated.DiscussionBased on the results of the prespecified coprimary endpoints, this study was negative. While semorinemab had a significant effect on cognition measured by the ADAS-Cog11, this effect did not extend to improved functional or global outcomes. These results may warrant further exploration of semorinemab or other anti-tau therapies in mild-to-moderate AD.Classification of EvidenceThis study provides Class I evidence that semorinemab does not slow functional decline in patients with mild-to-moderate AD.Trial Registration InformationThe Lauriet study is registered on ClinicalTrials.gov, NCT03828747, and EudraCT 2018-003398-87.

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.

The Tauriel Study (NCT03289143) was a Phase 2 randomized, double‐blind, placebo‐controlled, parallel‐group clinical trial that assessed the safety and efficacy of semorinemab in patients with prodromal‐to‐mild Alzheimer’s disease. We examined the concordance of fluid tau biomarkers with Tau PET for the identification of patients with high tau burden at enrollment.

Complement pathway overactivation can lead to neuronal damage in various neurological diseases. Although Alzheimer's disease (AD) is characterized by β-amyloid plaques and tau tangles, previous work examining complement has largely focused on amyloidosis models. We find that glial cells show increased expression of classical complement components and the central component C3 in mouse models of amyloidosis (PS2APP) and more extensively tauopathy (TauP301S). Blocking complement function by deleting C3 rescues plaque-associated synapse loss in PS2APP mice and ameliorates neuron loss and brain atrophy in TauP301S mice, improving neurophysiological and behavioral measurements. In addition, C3 protein is elevated in AD patient brains, including at synapses, and levels and processing of C3 are increased in AD patient CSF and correlate with tau. These results demonstrate that complement activation contributes to neurodegeneration caused by tau pathology and suggest that blocking C3 function might be protective in AD and other tauopathies.

Alzheimer's disease biomarker CSF Aβ1-42 is susceptible to numerous factors that compromise measurement accuracy. We evaluated assay stability, Aβ peptide recoveries, and Aβ peptide stabilities over a 2-year period to inform CSF handling and analysis protocols for clinical trials. A UP-RPLC-MS/MS assay was developed at Pharmaceutical Product Development with the Alzheimer's Association's Global Consortium for Biomarker Standardization to quantitate CSF Aβ1-42, 1-40, 1-38, 1-37, 1-34 using stable isotope-labeled peptides. CSF was collected from young normal controls and aged patients with dementia. CSF was denatured with 6M guanidine hydrochloride (GuHCl) at different stages of the collection and aliquoting procedure (after collection (“Collection”), before sub-aliquoting (“Aliquot”), or before solid phase extraction (“Analysis”)) and then stored at -80oC. In addition, a 1 mL aliquot was prepared in LoBind tubes at the time of collection, stored at -80oC, and GuHCl was added before extraction (“LoBind”). Measurements were made at baseline, and then every 3 months for 2 years. Longitudinal stabilities were assessed according to CLSI standards, with an acceptability cutoff of +/-10% of baseline. Earlier denaturation increased baseline CSF Aβ recovery relative to later denaturation (Mean (SD) CSF Aβ1-42collection=1061(476) pg/mL, CSF Aβ1-42Aliquot=996(414) pg/mL; vs. CSF Aβ1-42Analysis=822(385) pg/mL, CSF Aβ1-42LoBind=899(376) pg/mL (p<0.0002)). The Aβ1-42/Aβ1-40 ratio reduced recovery variability due to collection condition, and improved discrimination between control and dementia groups. The assay was stable for 2 years, but CSF Aβ peptide measurements and the Aβ1-42/Aβ1-40 ratio were not longitudinally stable for the study period. CSF Aβ1-42 was stable for 15months (“Collection”,“Aliquot”) or 18months (“Analysis”). The Aβ1-42/Aβ1-40 ratio was stable for 8 months (“Collection”,“Aliquot”) and 11months (“Analysis”). When measured with a UP-RPLC-MS/MS assay, earlier denaturation improved CSF Aβ peptide recovery. CSF Aβ1-42, Aβ1-40, Aβ1-38, Aβ1-34, and the Aβ1-42/Aβ1-40 ratio were not stable for the study period when samples were stored frozen. The Aβ1-42/Aβ1-40 ratio was useful for normalizing adsorption-related artifacts at baseline and for separating Dx groups, but was more sensitive to longitudinal instability. We recommend that UP-RPLC-MS/MS analysis of CSF Aβ peptides from frozen samples occur within the cutoffs specified or close to the time of collection.

We investigated the effect of crenezumab, a humanized anti-amyloid-beta (Aβ) immunoglobulin (Ig)G4 monoclonal antibody, on biomarkers of amyloid pathology, neurodegeneration, and disease progression in patients with mild-to-moderate Alzheimer’s disease (AD).

Amyloid-β 1–42 (Aβ1–42) peptide is a well-established cerebrospinal fluid (CSF) biomarker for Alzheimer’s disease (AD). Reduced levels of Aβ1–42 are indicative of AD, but significant variation in the absolute concentrations of this analyte has been described for both healthy and diseased populations. Preanalytical factors such as storage tube type are reported to impact Aβ recovery and quantification accuracy. Using complementary immunological and mass spectrometry-based approaches, we identified and characterized preanalytical factors that influence measured concentrations of CSF Aβ peptides in stored samples.

Post-translational modifications of Tau protein are of central importance in Alzheimer's disease (AD) pathogenesis. In addition to the well-established role of hyperphosphorylation in AD, emerging evidence indicates the involvement of proteolytic cleavages in Tau pathology development. In this study we explored the disease-related changes in the composition of Tau protein species, both full length and fragments, in cortical tissue from AD subjects. Cryosections of fresh-frozen fusiform gyrus cortex from AD subjects and cognitively normal, age matched control subjects were characterized using immunoprecipitation, ELISA, immunoblotting and transcriptomic analyses. Additionally, fresh-frozen frontal cortical tissues from control subjects were used to identify proteolytic cleavage sites using proteomic methods. Full-length Tau isoforms showed changes in protein expression pattern with high correlation to disease severity. Specifically, the longest Tau isoform, Tau441 (2N4R), is selectively enriched in AD patients versus controls, with the highest enrichment in Braak stages V and VI. No difference in Tau spliced forms were observed at the RNA level, indicating the enrichment of specific isoforms occurs post-transcriptionally. One possible mechanism is differential fragmentation of Tau isoforms via proteolytic cleavage. We observed that the Tau fragmentation pattern is common and stereotypical across all patient samples, regardless of disease status, with fragments representing ∼25% of total Tau signal. The extent of Tau fragmentation, however, is inversely correlated with the extent of tauopathy and Tau441 levels. Further analyses of the fragments suggest at least one common cleavage site for all isoforms, generating a N terminal-mid domain fragment and a C-terminal fragment. Our data suggest a proteolytic mechanism that shifts the Tau protein landscape toward the enrichment of longer isoforms during Alzheimer's disease progression. The fragments we identified are likely precursors of CSF Tau fragments, and therefore have implications for interpretation of current mid-domain Tau biomarkers for AD diagnosis and progression. Further understanding of the mechanisms of Tau fragmentation would provide insight into the biological significance of CSF Tau measurements beyond the most commonly used mid-domain assays.

Alzheimer’s disease (AD) is the leading cause of dementia affecting greater than 26 million people worldwide. Although cerebrospinal fluid (CSF) levels of Aβ42, tau, and p-tau181 are well established as diagnostic biomarkers of AD, there is a need for additional CSF biomarkers of neuronal function that continue to change during disease progression and could be used as pharmacodynamic measures in clinical trials. Multiple proteomic discovery experiments have reported a range of CSF biomarkers that differ between AD and control subjects. These potential biomarkers represent multiple aspects of the disease pathology. The performance of these markers has not been compared with each other, and their performance has not been evaluated longitudinally.

This study was conducted to determine the pharmacokinetics (PK) and pharmacodynamics (PD) of two novel inhibitors of β-site amyloid precursor protein (APP)–cleaving enzyme (BACE1), GNE-629 [(4S,4a′S,10a′S)-2-amino-8′-(2-fluoropyridin-3-yl)-1-methyl-3′,4′,4a′,10a′-tetrahydro-1′H-spiro[imidazole-4,10′-pyrano[4,3-b]chromen]-5(1H)-one] and GNE-892 [(R)-2-amino-1,3′,3′-trimethyl-7′-(pyrimidin-5-yl)-3′,4′-dihydro-2′H-spiro[imidazole-4,1′-naphthalen]-5(1H)-one], and to develop a PK-PD model to predict in vivo effects based solely on in vitro activity and PK. GNE-629 and GNE-892 concentrations and PD biomarkers including amyloid β (Aβ) in the plasma and cerebrospinal fluid (CSF), and secreted APPβ (sAPPβ) and secreted APPα (sAPPα) in the CSF were measured after a single oral administration of GNE-629 (100 mg/kg) or GNE-892 (30 or 100 mg/kg) in cynomolgus monkeys. A mechanistic PK-PD model was developed to simultaneously characterize the plasma Aβ and CSF Aβ, sAPPα, and sAPPβ using GNE-629 in vivo data. This model was used to predict the in vivo effects of GNE-892 after adjustments based on differences in in vitro cellular activity and PK. The PK-PD model estimated GNE-629 CSF and free plasma IC50 of 0.0033 μM and 0.065 μM, respectively. These differences in CSF and free plasma IC50 suggest that different mechanisms are involved in Aβ formation in these two compartments. The predicted in vivo effects for GNE-892 using the PK-PD model were consistent with the observed data. In conclusion, a PK-PD model was developed to mechanistically describe the effects of BACE1 inhibition on Aβ, sAPPβ, and sAPPα in the CSF, and Aβ in the plasma. This model can be used to prospectively predict in vivo effects of new BACE1 inhibitors using just their in vitro activity and PK data.

Alzheimer's disease is the leading cause of dementia affecting greater than 26 million people worldwide. Currently there are no effective treatments. Although CSF levels of A b, tau, and p-tau are well established as diagnostic biomarkers of AD, in the setting of clinical trials, additional CSF biomarkers are needed that change gradually as the disease progresses. Multiple proteomic discovery experiments have identified potential CSF biomarkers which reflect multiple aspects of the disease-pathology. We developed a targeted-proteomic MRM assay for evaluation of biomarker candidates in CSF, and piloted the assay using longitudinal CSF samples collected from controls (n=15), MCI (n=5), and AD (n=46) patients. A panel of 30 candidate CSF biomarkers was generated based on previously reported findings. Candidate peptides were selected based on detection in previous discovery experiments. Peptide performance was evaluated in pilot studies. At least one peptide was selected for each protein with a total of 40 peptides, including 5 plasma markers. Longitudinal CSF samples (2–3 samples per subject, spanning 1–2 years) were concentrated, denatured, reduced and alkylated and digested overnight. A stable-isotope labeled peptide standard for each peptide was used for quantitation. The ratio of the endogenous to heavy peptide was analyzed by nanoLC-MS operating in scheduled MRM mode. A b 42, tau and p-tau were also quantitated in CSF using the INNO-BIA Alzbio3 assay (Innogenetics). We evaluated each biomarker for diagnostic sensitivity, longitudinal consistency, and correlation with CSF A b42 and tau. Of the 40 peptides, the intra-assay CV for the ratio of light to heavy peptide was <10% for 32 peptides and the inter-assay CV was <20% for 27 peptides with 8 of the peptides being under LOD (5 of which corresponded to plasma peptides). 19 of 28 quantitatable CSF proteins were significantly different based on diagnostic group. Chitinase-3-like protein was the most significantly different between AD and control (p<0.0001). 28 of 30 CSF markers demonstrated < 20% CV in longitudinal samples. Interestingly, robust correlations were observed within some subgroups of proteins and these subgroups may reflect proteins with similar function or mechanism of release into CSF. Multiplexed MRM assays promise to increase the successful development of clinical biomarkers.

CSF Aβ42, tau, and phosphorylated tau are well-established biomarkers of Alzheimer's disease (AD), but significant inter-laboratory differences in the absolute levels of these analytes have been observed (Alz. Dem. 7:386). Understanding the pre-analytical variables that affect these biomarkers is essential for their use in clinical trials. In the current study, CSF from patients with mild to moderate AD that were Amyloid PET positive was collected in 15mL polypropylene tubes and immediately frozen. CSF was later thawed, aliquoted into Axygen MAXYMum Recovery polypropylene tubes, and re-frozen. The INNO-BIA AlzBio3 assay (Innogenetics) and the Human Aβ42 Assay (Meso Scale Discovery) were used to measure analytes in CSF. Using the AlzBio3 assay we compared results from 0.1mL aliquots and 0.5mL aliquots from the same patients. A b 42 recovery was significantly higher in the 0.5mL aliquots (median increase of 43%, range: 14–168%, p<0.0001, n=82 subjects). Tau and p-tau(181) levels were not different between 0.1 and 0.5 mL aliquots. The addition of Tween-20 has been proposed as a method to reduce binding of A b 42 to tubes (AAPS 14:510). Adding 0.2% Tween-20 resulted in a median Aβ42 increase of 112% (range 63%-192%) in 0.1mL aliquots and 39% (range 33%-51%) in 0.5mL aliquots. Although Tween-20 reduced the discrepancy in Aβ42 measurements between 0.1 and 0.5 mL aliquots, it did not eliminate the differences, and the percentage increase in recovery varied significantly across patients. Tau and p-tau(181) levels were unaffected by Tween-20 addition. The effects of aliquot size and Tween-20 on Aβ42 recovery were also observed when A b 42 was measured using a different ELISA kit (Meso Scale Discovery). Recent reports have highlighted the differences in performance of various polypropylene tubes (J Alz. Dis. 31:13). We measured A b 42 recovery in 0.1mL vs. 0.5mL aliquots stored in other “low-retention” polypropylene tubes. Most tubes performed similarly to the Axygen tubes, but we observed no loss of A b 42 in 0.1mL aliquots stored in 1.5mL Eppendorf LoBind tubes. As expected, Tween-20 had minimal effect on recovery from the LoBind tubes. Aliquot size and tube type can impact A b 42 recovery as measured by existing ELISAs and the magnitude of these effects varies significantly across subjects.

Centromere-associated protein-E (CENP-E) is a kinetochore-associated mitotic kinesin that is thought to function as the key receptor responsible for mitotic checkpoint signal transduction after interaction with spindle microtubules. We have identified GSK923295, an allosteric inhibitor of CENP-E kinesin motor ATPase activity, and mapped the inhibitor binding site to a region similar to that bound by loop-5 inhibitors of the kinesin KSP/Eg5. Unlike these KSP inhibitors, which block release of ADP and destabilize motor-microtubule interaction, GSK923295 inhibited release of inorganic phosphate and stabilized CENP-E motor domain interaction with microtubules. Inhibition of CENP-E motor activity in cultured cells and tumor xenografts caused failure of metaphase chromosome alignment and induced mitotic arrest, indicating that tight binding of CENP-E to microtubules is insufficient to satisfy the mitotic checkpoint. Consistent with genetic studies in mice suggesting that decreased CENP-E function can have a tumor-suppressive effect, inhibition of CENP-E induced tumor cell apoptosis and tumor regression.