Aβ immunization in old mouse lemur primates induces cerebral microhemorrhages and accelerates age-associated iron deposits in the choroid plexus

Oral Sessions: O4-06: Therapeutics/Therapeutic Strategies: Immunisation—Mechanisms of Action and Alternative Approaches

constituent of peripheral amyloidoses has been shown to result in a broad amyloid clearance, targeting a minor brain plaque constituent leading to b-amyloid clearance has not been demonstrated. Methods: We showed that murine Ab accumulates in multiple human amyloid precursor protein transgenic mouse lines, representing w2-8% of the total Ab in the brain. We also determined that murine Ab co-localizes and interacts with the human Ab by co-immunolabeling of and co-immunoprecipitation from brain tissue. Given that endogenous murine Ab is a minor b-amyloid plaque component in these mouse models, we undertook an anti-murine-Ab passive immunization by intraperitoneal injection for eight weeks in Tg2576 mice to determine whether this immunotherapy reduces brain plaque pathology while improving performance in behavioral assays in these mice. Results: This immunization resulted in the clearance of more than one-half of the b-amyloid plaque pathology - including both the endogenous murine Ab and the human Ab components - following two months of treatment. The immunized mice additionally showed improvements in two behavioral assays, odor habituation and nesting behavior. Given the interactions we showed between the two Ab species, our results argue that anti-murineAb immunization clears human Ab with murine Ab as a complex while improving behavioral deficits which suggests that this immunization strategy may effectively clear oligomeric Ab. Conclusions: We conclude that passive anti-murine-Ab immunization clears Ab plaque pathology - including the major human Ab component - and decreases behavioral deficits, arguing that targeting minor, endogenous brain plaque constituents can be beneficial, broadening the range of plaque-associated targets for AD therapeutics. O4-06-05

Ab IMMUNIZATION IN OLD MOUSE LEMUR PRIMATES INDUCES CEREBRAL MICROHEMORRHAGES AND ACCELERATES AGE-ASSOCIATED IRON DEPOSITS IN THE CHOROID PLEXUS

Olene Dorieux1, Nelly Joseph-Mathurin2, Stephanie Trouche3, Audrey Kraska2, Mathieu Santin2, Allal Boutajangout4, JeanMichel Verdier5, Einar M. Sigurdsson4, Nadine Mestre-Frances3, Marc Dhenain1, 1CNRS, Brunoy, France; 2URA CEA-CNRS 2210 MIRCen, Fontenay-aux-Roses, France; 3INSERM U710- EPHE, Montpellier, France; 4 New York University School of Medicine, New York, New York, United States; 5EPHE, Montpellier, France. Background: Anti-amyloid immunotherapy reduces Ab plaques and prevents cognitive decline in transgenic mouse models of Alzheimer’s disease (AD) (Asuni et al, 2006). Nevertheless, in humans, a strategy based on Ab142 peptide induced encephalomyelitis and possibly microhemorrhages (Orgogozo et al, 2003; Ferrer et al, 2004). These outcomes were not expected from studies in rodents. Mouse lemur, as a primate model may be more predictive of human side effects. A small proportion of old animals develop spontaneously Ab plaques (Mestre-Frances et al, 2000). Thus this primate models prodromal AD stages, and we used it to evaluate side effects of immunotherapies. Methods: Animals were treated with K6Ab1-30 (n ¼ 4; 5.860.2 years) or Ab1-42 (n ¼ 4; 5.960.2 years) immunogens in alum adjuvant. They were followed-up for 9 months with biochemistry (anti-Ab40 antibodies and Ab40 levels in the plasma), as well as T2-weighted (T2w) and T2-weighted (T2w) MRI (7T PharmaScan-Bruker; resolutions¼(234x234x234)mm3. Histological analyses was performed to evaluate amyloidosis, neuroinflammation, and iron deposits/microhemorrhages. Age-associated occurrences of hypointense signals were evaluated in twenty other naive animals (1.5 to 4.9 years). Results: In this particular study, the animals responded mainly to the Ab1-42 immunogen, which differs from our prior study with this Ab derivative (Trouche et al, 2009). This treatment induced an immune response and increased Ab levels in plasma. No severe neuroinflammation were observed (either on MRI or histology). Compared to K6Ab1-30 vaccine, Ab1-42 immunogen increased microhemorrhages (Mann Whitney test U¼1, P<0.05) and the size of hypointense signal corresponding to iron deposits in the choroid plexus (CP) (F(2,5)¼ 4.627; P<0.05). The study in naive lemurs showed that iron accumulates in the CP with normal aging (r¼0.60; P<0.001). Hence, immunotherapy with Ab1-42 immunogen accelerated this age-associated phenomenon.

Conclusions: Ab-immunization can lead to side effects such as microhemorrhages in a primate model of normal aging or prodromal stage of AD with minimal extracellular Ab deposition. Also, iron accumulation in the CP is a potential side effect of Ab-immunization. This should be taken into account in future evaluations of clinical trials with AD patients. O4-06-06

NOVEL MONOCLONAL ANTIBODY FOR TREATING ALZHEIMER’S DISEASE

Ronald Ellis1, Sarit Samira2, Nurit Rachamim2, Idan Rakover3, Beka Solomon3, 1NasVax Ltd, Jerusalem, Israel; 2NasVax Ltd, Ness Ziona, Israel; 3Tel Aviv University, Tel Aviv, Israel. Background: Amyloid precursor protein (APP) is a major target for therapeutic development. The presence of the complex of APP with the protease b-secretase (BACE) in early endosomes suggests that interference in APPBACE interaction at the cell surface may affect Ab production. Our novel approach for inhibiting Aß production employs a monoclonal antibody (MAb) that binds to the BACE cleavage site on APP on the cell surface and internalizes to cells within two minutes, thereby blocking the first step by BACE in the proteolytic processing of APP to Ab and other peptides. This abstract describes the in vitro and in vivo activities of this MAb (BBS ¼ Blocking b-site) as an immunotherapeutic. Methods: Affinity of BBS MAb to its target sequence in APP was quantified by Biacore analysis. This MAb has been evaluated in transgenic AD mouse models of overproduction of APP. These mice display spontaneous, progressive accumulation of Ab in brain, eventually resulting in amyloid plaques and accompanied by cognitive deficits. Animals were dosed with BBS MAb or with an isotype control MAb. Treated mice were evaluated in two behavioral tests vs. nontransgenic controls, then sacrificed. Soluble and insoluble brain fractions were derived and analyzed for the levels of different forms of Ab and for safety-related markers. Results: BBS MAb binds to APP with very high affinity. Transgenic AD mice treated with BBS MAb compared to control MAb showed significant reductions in some forms of Ab including plaque, significantly improved performance in two behavioral tests to the same level as that of non-transgenic mice, and reduced levels of astrocytosis indicating an improved safety profile. Conclusions: BBS MAb shows an excellent efficacy and safety profile in mouse AD models by means of a novel and highly specific mechanism. The rapid cellular internalization of the MAb after binding to APP may be beneficial for avoiding microglia and complement activation and resultant deleterious effects that have been reported for anti-Ab Abs that bind to amyloid plaques. This approach also avoids the potential non-specific effects of small-molecule BACE inhibitors toward non-AD pathways. The BBS MAb is a strong candidate for clinical development. Object recognition test - Week 6 of MAb treatment * P<0.01 % with the new object

P626

% tim e

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% frequency

80 60 40 20 0 PBS / normal mice

Control MAb / AD mice

BBS3 MAb / AD mice

Object recognition test Mice were placed individually for 10 minutes of habituation in a Plexiglas open field box with a translucent floor dimly illuminated by a lamp placed above the box. During the habituation stage, Object A (black can) was placed in the open field box. On the third day, the recognition test was performed as follows: Object B (transparent bottle) was placed in the field of view. The % time and % frequency by which the mice explored the familiar Object A and the novel Object B were recorded for 5 minutes. The % time and % frequency for each treatment group are plotted as mean and standard deviation per group. *erformance for BBS MAb vs. Control MAb in AD mice; P<0.01