John Tzaferis

A Plaque-Specific Antibody Clears Existing β-amyloid Plaques in Alzheimer's Disease Mice

Aβ Immunotherapy is a promising therapeutic approach for Alzheimers 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 Aβ peptide. We hypothesized that antibody specificity for deposited plaque was critical for plaque removal since soluble Aβ peptide would block recognition of deposited forms. We developed a plaque-specific antibody that targets a modified Aβ peptide (Aβ(p3-42)), which showed robust clearance of pre-existing plaque without causing microhemorrhage. Interestingly, a comparator N-terminal Aβ antibody 3D6, which binds both soluble and insoluble Aβ(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 Aβ antibodies for Alzheimers disease.

TREM2-mediated early microglial response limits diffusion and toxicity of amyloid plaques

Wang et al. report that TREM2 protects mice from Alzheimers disease by enabling resident microglia to insulate and alter Aβ plaque structure, thereby limiting neuritic damage.

Expression of Metabotropic Glutamate Receptors in Rat Meningeal and Brain Microvasculature and Choroid Plexus

This study investigated the distribution of metabotropic glutamate receptors (mGluRs) in meningeal and parenchymal microvasculature and in choroid plexus by means of Western blot analysis and immunohistochemistry. Western blot analysis demonstrated mGluR expression in both rat and human leptomeningeal tissues. In the rat, mGluR expression was developmentally regulated, with only mGluR2/3 showing expression at the embryonic day 19 developmental stage. In contrast, mGluR1α, mGluR2/3, mGluR4a, and mGluR7 were expressed in leptomeninges from adult rats. Immunohistochemical analyses showed intense mGluR1α immunoreactivity in the pia mater and blood vessels in the subarachnoid space and in the arachnoid layer of the meninges. mGluR2/3, mGluR4a, mGluR5, and mGluR7 were also expressed in meningeal microvasculature. In addition, the parenchymal microvasculature and choroid plexus were strongly immunoreactive for mGluR1α, mGluR2/3, mGluR4a, mGluR5, and mGluR7. We used antibodies specific for phenotypic markers of microvascular and glial cells to characterize the cell type(s) immunopositive for mGluRs. Comparison of staining with anti–von Willebrand factor antibody and anti‐mGluR antibodies revealed that mGluR immunoreactivity was present in cells that surrounded the luminal surface labeled by the endothelial cell marker. In these cells, smooth muscle actin and mGluR immunoreactivity overlapped, suggesting that, in addition to endothelial cells, pericytes within the microvasculature also express mGluRs. Furthermore, expression of mGluR1α was also observed in pure pericyte cultures isolated from bovine retina. These data suggest that glutamate by means of activation of mGluRs may have a broad sphere of physiological influence in the brain which in addition to modulating synaptic transmission may also have a role in determining microvascular function and dysfunction. J. Comp. Neurol. 461:317–332, 2003.

COMBINATION THERAPY WITH A PLAQUE-SPECIFIC ABETA ANTIBODY AND A BACE INHIBITOR RESULTS IN DRAMATIC PLAQUE REDUCTION IN A DOSE-DEPENDENT MANNER IN AGED PDAPP TRANSGENIC MICE

lowed. Results:High antibody responses were found in both species. In the rabbit, 277.16 118.4 mg/ml plasmaweremeasured after 5 immunizations with no significant differences between the dose groups. Antibody levels declined slightly to 246.66 109.5 mg/ml after a resting period of two months. Low levels of IFNg and IL-17 secreting splenocytes were found in the ELISPOTassays from rabbit splenocytes. Rhesusmonkeys reachedmean antibody levels of 114.2 6 41.67 mg/ml plasma after five immunizations. The isotype profile was high on IgG4 antibodies, indicative of a Th2 immune response. After three immunizations, no IL-17 or IFNg producing cells were found in ELISPOT assays from PBMCs. Conclusions: DNA Ab42 immunization leads to high antibody titers in large mammals, and is likely to produce high antibody levels and a safe (Th2 biased) immune response in humans as well. supported by NIA/NIH P30AG12300-21, Zale Foundation, Rudman Foundation, AWARE, Presbyterian Village North, Freiberger, and Denker Family Funds

A COMPETITIVE EX-VIVO SCREENING ASSAY TO DETERMINE ABETA ANTIBODY MEDIATED PHAGOCYTOSIS AND PLAQUE SPECIFICITY

being the most productive source of recruitment. However, thus far the respondents are predominantly Caucasian, college-educated participants. Other targeted recruitment strategies are needed to increase ethnic minority representation. The registry provides a platform for cohort studies and dementia risk analysis. Further risk assessment including genotyping and cognitive screen will be included in the next phase of the project to further characterize risk profiles in preclinical and symptomatic AD.

CHARACTERIZATION OF AMINO TERMINAL TRUNCATIONS OF PLAQUE-ASSOCIATED Aβ AFTER COMBINATION BACE INHIBITOR AND ANTIBODY THERAPY IN AGED PDAPP MICE

TRUNCATIONS OF PLAQUE-ASSOCIATED Ab AFTER COMBINATION BACE INHIBITOR AND ANTIBODY THERAPY IN AGED PDAPP MICE Margaret Racke, John Tzaferis, Justin Hole, Feng Liu, Cynthia DeLong, Patrick May, Michael Hutton, Christer Nordstedt, Ronald DeMattos, Eli Lilly and Company, Indianapolis, Indiana, United States; Eli Lilly and Company, Indianapolis, Indiana, United States; Eli Lilly and Company, Indianapolis, Indiana, United States; Eli Lilly and Company, Ltd., Windlesham, United Kingdom; 5 Eli Lilly and Company, Indianapolis, Indiana, United States. Contact e-mail: racke_margaret@ lilly.com

Standardization of beta-amyloid reference peptides

Samantha Budd Haeberlein, Gvido Cebers, Kina H€oglund, Hugh Salter, Susanna Eketj€all, Anna Bogstedt, Tina Olsson, Robert Alexander, Michael Poole, AstraZeneca R&D, Cambridge, Massachusetts, United States; Translational Science Centre, Solna, Sweden. Contact e-mail: samantha.budd@azneuro.com Background: A growing body of pathological, biomarker, genetic and mechanistic data suggests that amyloid accumulation as a result of changes in production, processing and/or clearance of brain Ab levels plays a key role in the pathogenesis of Alzheimer’s disease (AD). G enetic mutations in APP have been linked causally to earlyonset AD, and two mutations in APP (K670N/M671L the Swedish mutation, and the A673T variant) have been associated with changes in Beta-site amyloid precursor protein cleaving enzyme1 (BACE1) activity and confer early onset AD and reduced risk for AD respectively. BACE1 is the first step in the processing of APP to Ab peptides, and its inhibition is an attractive target for therapeutic intervention to stop the production of A b.Methods:We report here the pharmacological profile of a potent and selective, orally active, brain permeable BACE1 inhibitor AZD3293. Results: The potency of AZD3293 in cellular models on secretion of Ab40 has been studied in SHSY5Y/APP cells (human neuronal cells over expressing human APPwt), N2A cells (mouse neuronal cells), primary mouse neurons and primary guinea pig neurons, using ELISA technology. Mice treated with AZD3293 as a single administration, or repeated administrations twice daily during 7 days, demonstrated a statistically significant doseand time-dependent reduction of the levels of Ab40, Ab42 and sAPPb in plasma and brain. Guinea pigs treatedwithAZD3293 as a single administration demonstrated a statistically significant doseand time-dependent reduction of the levels of Ab40, Ab42 and sAPPb in plasma, CSF and brain. In vitro potency inmouse and guinea pig primary cortical neuronal cells was strongly correlated to potency in mouse mouse and guinea pig in vivo potency.Conclusions: In conclusion, AZD3293 is a potent and selective, orally active, brain permeable BACE1 inhibitor with a promising preclinical profile for treatment of AD.