Margaret M. Racke

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 β

Passive immunization with an antibody directed against the N terminus of amyloid β (Aβ) 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-Aβ antibodies to deposited Aβ 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 Aβ, whereas 266, a central domain antibody, lacked affinity for deposited Aβ. 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 Alzheimers disease.

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.

Identification, characterization, and comparison of amino-terminally truncated Aβ42 peptides in Alzheimer's disease brain tissue and in plasma from Alzheimer's patients receiving solanezumab immunotherapy treatment

observed in any Aß-degrading protease knockout mouse studied to date. Remarkably, this increase was selective for Aß42, resulting in elevated Aß42/ Aß40 ratios comparable to those induced by presenilin mutations that cause early-onset AD. Cultured neurons from CatD-KO mice also showed selective defects in the catabolism of Aß42, but not Aß40. Furthermore, relative to age-matched controls, the brains of CatD-KO mice showed pronounced increases in the levels of tau and phosphorylated forms thereof, while showing no increases in the levels of several other cytosolic proteins. Conclusions: Our findings demonstrate that, in vivo, CatD deficiency leads to large and highly selective increases in the two protein species most strongly linked to the pathogenesis of AD_Aß42 and tau. Combined with human molecular genetic data on the CatD gene (CTSD) to be presented at this meeting (Burgess et al.), our findings suggest that CatD plays a protective role in the pathogenesis of AD by mediating the catabolism of Aß42 and tau.

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

Characterization of amino-terminally truncated Abeta-42 peptides in plasma from Alzheimer's patients receiving solanezumab immunotherapy treatment

also assessed the specificity of E2012 for different gamma-secretase substrates. Results: E2012 was shown to decrease Ab(x-40) and Ab(x-42) and to increase Ab(1-38) without changing total Ab(1-x) levels. MALDITOF analysis also revealed that E2012 reduced Ab40 and Ab42 and increased shorter Ab peptides, such as Ab37 and Ab38. E2012 did not induce the accumulation of APP-CTFs, suggesting that E2012 modulates, but does not inhibit, the cleavage of APP-CTF by gamma-secretase. Production of Notch intracellular domain (NICD) was not inhibited by E2012. Conclusions: We have profiled E2012, a compound from a new chemical class of GSMs, and found that it preferentially reduced gamma-secretase mediated Ab40 and Ab42 production over Notch cleavage via a different mechanism from that of classical GSIs. These results provide the basis for the development of E2012 as a new therapeutic agent for Alzheimer’s disease.

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.

OPTIMIZATION OF THE PHARMACOKINETIC (PK) AND PHARMACODYNAMIC (PD) PROPERTIES OF AN ANTI-ABETA ANTIBODY M266 FAB FRAGMENT VARIANT BY SITE-SPECIFIC PEGYLATION

neuronal culture assay with an EC50of approximately 250 pM. Following acute oral treatment with 0, 0.3, 1.0, or 3.0 mg/kg LY3202626, dose-dependent reductions in Ab, sAPPb, and C99 were observed in cortex and hippocampus of PDAPPmice. In beagle dog, acute oral dosing of 1.5 mg/kg LY3202626 resulted in lowering of plasma and CSF Ab 1-x by approximately 80% at the nadir; the CSF Ab 1-x was still reduced by approximately 75% at 24 hours post-dosing. Exposure of LY3202626 in plasma and CSF correlated significantly with pharmacodynamic effects upon Ab in both PDAPP mice and beagle dogs. Conclusions:LY3202626 is a potent and selective inhibitor of the BACE1 and BACE2 enzymes. The robust in vivo effects of LY3202626 are consistent with its in vitro potency and exposure in target compartments. These data support further clinical development of LY3202626 for the treatment of 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

Effects of solanezumab versus placebo administration on biomarkers in people with mild-to-moderate Alzheimer's disease: Results from two phase III clinical trials

ratiowas 4:1, consistent with the ratio in the general population; that for avagacestat was 1:1.75. NMSC incidence rates among avagacestat-treated patients were higher than those from the claims database. Conclusions: This is the first clinical report characterizing NMSC in patients treated with a GSI. The NMSCs observed were readily identified and easily managed without recurrence. Increased NMSC incidence (especially SCC) and inversion of BCC:SCC ratio suggest that Notch inhibition oncogenically effects SCC. Further evaluation is warranted to understand potential oncogenic or suppressor effects of similar agents on skin cancer subtypes. The algorithms utilized in this study may help accurately identify and manage skin malignancies in studies using agents that affect epidermal differentiation.

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.