Systemic immune-checkpoint blockade with anti-PD1 antibodies does not alter cerebral amyloid-ß burden in several amyloid transgenic mouse models.

Chronic inflammation represents a central component in the pathogenesis of Alzheimer's disease (AD). Recent work suggests that breaking immune tolerance by Programmed cell Death-1 (PD1) checkpoint inhibition produces an IFN-γ-dependent systemic immune response, with infiltration of the brain by peripheral myeloid cells and neuropathological as well as functional improvements even in mice with advanced amyloid pathology (Baruch et al., (): Nature Medicine, 22:135-137). Immune checkpoint inhibition was therefore suggested as potential treatment for neurodegenerative disorders when activation of the immune system is appropriate. Because a xenogeneic rat antibody (mAb) was used in the study, whether the effect was specific to PD1 target engagement was uncertain. In the present study we examined whether PD1 immunotherapy can lower amyloid-ß pathology in a range of different amyloid transgenic models performed at three pharmaceutical companies with the exact same anti-PD1 isotype and two mouse chimeric variants. Although PD1 immunotherapy stimulated systemic activation of the peripheral immune system, monocyte-derived macrophage infiltration into the brain was not detected, and progression of brain amyloid pathology was not altered. Similar negative results of the effect of PD1 immunotherapy on amyloid brain pathology were obtained in two additional models in two separate institutions. These results show that inhibition of PD1 checkpoint signaling by itself is not sufficient to reduce amyloid pathology and that additional factors might have contributed to previously published results (Baruch et al., (): Nature Medicine, 22:135-137). Until such factors are elucidated, animal model data do not support further evaluation of PD1 checkpoint inhibition as a therapeutic modality for Alzheimer's disease.

Exacerbation of cerebral amyloid angiopathy-associated microhemorrhage in amyloid precursor protein transgenic mice by immunotherapy is dependent on antibody recognition of deposited forms of amyloid beta.

Passive immunization with an antibody directed against the N terminus of amyloid beta (Abeta) has recently been reported to exacerbate cerebral amyloid angiopathy (CAA)-related microhemorrhage in a transgenic animal model. Although the mechanism responsible for the deleterious interaction is unclear, a direct binding event may be required. We characterized the binding properties of several monoclonal anti-Abeta antibodies to deposited Abeta in brain parenchyma and CAA. Biochemical analyses demonstrated that the 3D6 and 10D5, two N-terminally directed antibodies, bound with high affinity to deposited forms of Abeta, whereas 266, a central domain antibody, lacked affinity for deposited Abeta. To determine whether 266 or 3D6 would exacerbate CAA-associated microhemorrhage, we treated aged PDAPP mice with either antibody for 6 weeks. We observed an increase in both the incidence and severity of CAA-associated microhemorrhage when PDAPP transgenic mice were treated with the N-terminally directed 3D6 antibody, whereas mice treated with 266 were unaffected. These results may have important implications for future immune-based therapeutic strategies for Alzheimer's disease.

A digital enzyme-linked immunosorbent assay for ultrasensitive measurement of amyloid-ß 1-42 peptide in human plasma with utility for studies of Alzheimer's disease therapeutics.

BACKGROUND: Amyloid-ß 1-42 peptide (Aß1-42) is associated with plaque formation in the brain of patients with Alzheimer's disease (AD). Pharmacodynamic studies of AD therapeutics that lower the concentrations of Aß1-42 in peripheral blood require highly sensitive assays for its measurement. A digital enzyme-linked immunosorbent assay (ELISA) using single molecule array (Simoa) technology has been developed that provides improved sensitivity compared with conventional ELISA methods using the same antibody reagents. METHODS: A sensitive digital ELISA for measurement of Aß1-42 using antibodies 3D6 and 21F12 was developed. Assay performance was evaluated by repeated testing of pooled human plasma and buffer diluent quality control samples to determine relative accuracy, intra- and inter-assay precision, limit of detection (LOD), lower limit of quantification (LLOQ), dilutional linearity, and spike recovery. The optimized assay was used to quantify Aß1-42 in clinical samples from patients treated with the ß-site amyloid precursor protein cleaving enzyme 1 inhibitor LY2886721. RESULTS: The prototype assay measured Aß1-42 with an LOD of 0.3 pg/ml and an LLOQ of 2.8 pg/ml in plasma, calibrated using an Aß1-42 peptide standard from Fujirebio. Assay precision was acceptable with intra- and inter-assay coefficients of variation both being ≤10%. Dilutional linearity was demonstrated in sample diluent and immunodepleted human plasma. Analyte spike recovery ranged from 51% to 93% with a mean of 80%. This assay was able to quantify Aß1-42 in all of the 84 clinical samples tested. A rapid reduction in levels of Aß1-42 was detected within 1 h after drug treatment, and a dose-dependent decrease of Aß1-42 levels was also observed over the time course of sample collection. CONCLUSIONS: This digital ELISA has potential utility in clinical applications for quantification of Aß1-42 in plasma where high sensitivity and precision are required.

ß-site amyloid precursor protein-cleaving enzyme 1(BACE1) inhibitor treatment induces Aß5-X peptides through alternative amyloid precursor protein cleavage.

INTRODUCTION: The ß-secretase enzyme, ß-site amyloid precursor protein-cleaving enzyme 1 (BACE1), cleaves amyloid precursor protein (APP) in the first step in ß-amyloid (Aß) peptide production. Thus, BACE1 is a key target for candidate disease-modifying treatment of Alzheimer's disease. In a previous exploratory Aß biomarker study, we found that BACE1 inhibitor treatment resulted in decreased levels of Aß1-34 together with increased Aß5-40, suggesting that these Aß species may be novel pharmacodynamic biomarkers in clinical trials. We have now examined whether the same holds true in humans. METHODS: In an investigator-blind, placebo-controlled and randomized study, healthy subjects (n =18) were randomly assigned to receive a single dose of 30 mg of LY2811376 (n =6), 90 mg of LY2811376 (n =6), or placebo (n =6). We used hybrid immunoaffinity-mass spectrometry (HI-MS) and enzyme-linked immunosorbent assays to monitor a variety of Aß peptides. RESULTS: Here, we demonstrate dose-dependent changes in cerebrospinal fluid (CSF) Aß1-34, Aß5-40 and Aß5-X after treatment with the BACE1-inhibitor LY2811376. Aß5-40 and Aß5-X increased dose-dependently, as reflected by two independent methods, while Aß1-34 dose-dependently decreased. CONCLUSION: Using HI-MS for the first time in a study where subjects have been treated with a BACE inhibitor, we confirm that CSF Aß1-34 may be useful in clinical trials on BACE1 inhibitors to monitor target engagement. Since it is less hydrophobic than longer Aß species, it is less susceptible to preanalytical confounding factors and may thus be a more stable marker. By independent measurement techniques, we also show that BACE1 inhibition in humans is associated with APP-processing into N-terminally truncated Aß peptides via a BACE1-independent pathway. TRIAL REGISTRATION: ClinicalTrials.gov NCT00838084. Registered: First received: January 23, 2009, Last updated: July 14, 2009, Last verified: July 2009.

Validation of assays for measurement of amyloid-ß peptides in cerebrospinal fluid and plasma specimens from patients with Alzheimer's disease treated with solanezumab.

The aim of this study was to validate new assays for measurement of amyloid-ß (Aß) peptides in cerebrospinal fluid (CSF) and plasma specimens in clinical studies of solanezumab according to current regulatory recommendations. Four assays based on the INNOTEST® ß-AMYLOID(1-42) and prototype INNOTEST ß-AMYLOID(1-40) kits were developed and validated. To render these assays 'solanezumab-tolerant', excess drug was added to calibrators, quality control, and test samples via a 2-fold dilution with kit diluent. Validation parameters were evaluated by repeated testing of human CSF and EDTA-plasma pools containing solanezumab. Calibration curve correlation coefficients for the four assays were ≥0.9985. Intra- and inter-assay coefficients of variation for Aß1-40 and Aß1-42 were ≤13 and ≤15%, respectively for both matrices. Dilutional linearity, within and between assays, was demonstrated for both analytes in CSF and plasma at clinically relevant dilution factors. This dilution regimen was successfully applied during Phase 3 clinical sample analysis. Aß1-40 and Aß1-42 were stable in CSF and plasma containing solanezumab at 2-8°C and room temperature for up to 8 h and during 5 additional freeze-thaw cycles from ≤-20 and ≤-70°C. Results of parallel tests on stored clinical samples using INNOTEST methods and proprietary ELISA methods were closely correlated (r2 > 0.9), although bias in reported concentrations was observed between assays. In conclusion, the modified INNOTEST assays provided (relatively) accurate and precise quantification of Aß1-40 and Aß1-42 in CSF and plasma containing solanezumab according to established consensus validation criteria. The clinical experience with these assays post validation has shown them to be robust and reliable.

A plaque-specific antibody clears existing ß-amyloid plaques in Alzheimer's disease mice.

Aß Immunotherapy is a promising therapeutic approach for Alzheimer's disease. Preclinical studies demonstrate that plaque prevention is possible; however, the more relevant therapeutic removal of existing plaque has proven elusive. Monoclonal antibodies in development target both soluble and insoluble Aß peptide. We hypothesized that antibody specificity for deposited plaque was critical for plaque removal since soluble Aß peptide would block recognition of deposited forms. We developed a plaque-specific antibody that targets a modified Aß peptide (Aß(p3-42)), which showed robust clearance of pre-existing plaque without causing microhemorrhage. Interestingly, a comparator N-terminal Aß antibody 3D6, which binds both soluble and insoluble Aß(1-42), lacked efficacy for lowering existing plaque but manifested a significant microhemorrhage liability. Mechanistic studies suggested that the lack of efficacy for 3D6 was attributed to poor target engagement in plaques. These studies have profound implications for the development of therapeutic Aß antibodies for Alzheimer's disease.

ABCG1 influences the brain cholesterol biosynthetic pathway but does not affect amyloid precursor protein or apolipoprotein E metabolism in vivo.

Cholesterol homeostasis is of emerging therapeutic importance for Alzheimer's disease (AD). Agonists of liver-X-receptors (LXRs) stimulate several genes that regulate cholesterol homeostasis, and synthetic LXR agonists decrease neuropathological and cognitive phenotypes in AD mouse models. The cholesterol transporter ABCG1 is LXR-responsive and highly expressed in brain. In vitro, conflicting reports exist as to whether ABCG1 promotes or impedes Abeta production. To clarify the in vivo roles of ABCG1 in Abeta metabolism and brain cholesterol homeostasis, we assessed neuropathological and cognitive outcome measures in PDAPP mice expressing excess transgenic ABCG1. A 6-fold increase in ABCG1 levels did not alter Abeta, amyloid, apolipoprotein E levels, cholesterol efflux, or cognitive performance in PDAPP mice. Furthermore, endogenous murine Abeta levels were unchanged in both ABCG1-overexpressing or ABCG1-deficient mice. These data argue against a direct role for ABCG1 in AD. However, excess ABCG1 is associated with decreased levels of sterol precursors and increased levels of SREBP-2 and HMG-CoA-reductase mRNA, whereas deficiency of ABCG1 leads to the opposite effects. Although functions for ABCG1 in cholesterol efflux and Abeta metabolism have been proposed based on results with cellular model systems, the in vivo role of this enigmatic transporter may be largely one of regulating the sterol biosynthetic pathway.

Activation of caspase-3 alone is insufficient for apoptotic morphological changes in human neuroblastoma cells.

Activated caspase-3 is considered an important enzyme in the cell death pathway. To study the specific role of caspase-3 activation in neuronal cells, we generated a stable tetracycline-regulated SK-N-MC neuroblastoma cell line, which expressed a highly efficient self-activating chimeric caspase-3, consisting of the caspase-1 prodomain fused to the caspase-3 catalytic domain. Under expression-inducing conditions, we observed a time-dependent increase of processed caspase-3 by immunostaining for the active form of the enzyme, intracellular caspase-3 enzyme activity, as well as poly(ADP-ribose) polymerase (PARP) cleavage. Induced expression of the caspase fusion protein showed predominantly caspase-3 activity without any apoptotic morphological changes. In contrast, staurosporine treatment of the same cells resulted in activation of multiple caspases and profound apoptotic morphology. Our work provides evidence that auto-activation of caspase-3 can be efficiently achieved with a longer prodomain and that neuronal cell apoptosis may require another caspase or activation of multiple caspase enzymes.