Poster Presentations P3P426

Genetics, KULeuven, Leuven, Belgium. Contact e-mail: kasia.piorkowska@

acimmune.com

Background: Alzheimer’s vaccine ACI-24 is a liposome-based vaccine

with tetra-palmitoylated amyloid beta 1-15 peptide. Recently, we demon-

strated the immunogenicity and therapeutic efficacy of ACI-24 in double

transgenic APPxPS1mice (Muhs et al., PNAS, 104, 9810-9815, 2007).

The data presented here focus on the safety profile of ACI-24 by analyz-

ing inflammation and hemorrhages in an Alzheimer’s disease (AD)

mouse model. Methods: 27-28 month old double transgenic APPxPS-1

mice were injected intraperitoneally with 5 doses of ACI-24 or empty li-

posomes, at 2-week intervals. The presence of activated microglia (MHC-

II and CD45), astrogliosis (GFAP), and penetration into the brain of pe-

ripheral peripheral T and B-cells (CD4 and CD45) cells were analyzed by

immunohistochemistry. Micro-hemorrhages were analyzed by Perl’s Iron

staining and local secretion of pro-inflammatory cytokines by ELISA.

Results: ACI-24 vaccine induced anti-amyloid beta antibody titers in

the APPxPS1 transgenic mice with high specificity for oligomeric Amy-

loid beta species. These antibodies were of IgG2b and IgG3 isotypes, in-

dicating a preferential Th2 vaccine response. The brain tissues showed no

evidence of microglia activation nor astrogliosis and no increase in the

levels of the pro-inflammatory cytokines IL-1beta, IL-6, TNF-alpha, or

IFN-gamma. The histological staining with Perl’s Iron revealed that the

number of sections per mouse containing large hemorrhages was signif-

icantly lower in mice treated with ACI-24 compared to the vehicle-treated

controls. Additionally, ACI-24 immunization did not induce infiltration of

peripheral monocytes (MHC-II) nor peripheral T- and B-cells (CD4 or

CD45) into the brains of the treated transgenic mice. Conclusions: Our

results show that immunization of old transgenic mice with ACI-24 in-

duces oligo-specific anti-Amyloid beta antibodies and decreases the num-

ber of large micro-hemorrhages without inducing micro-hemorrhages.

Furthermore, ACI-24 immunization does not cause cellular brain inflam-

mation or enhance the release of pro-inflammatory cytokines, nor does it

result in the penetration of peripheral monocytes into the brain. These re-

sults, together with the preferentially Th2 associated antibody isotype

profile, indicate a low risk of encephalitis and thus demonstrates a positive

safety profile for ACI-24 in a relevant AD animal model.

P3-282 INVESTIGATION OF LOW MOLECULAR

WEIGHT D-ENANTIOMERIC PEPTIDES

FOR DIAGNOSIS AND THERAPY OF

ALZHEIMER’S DISEASE

Dirk Bartnik1, Susanne A. Funke1, Yeliz Cinar1, Oleksandr Brener2,

Luitgard Nagel-Steger2, Katja Wiesehan1, Dieter Willbold1,2, 1For-

schungszentrum Julich, Julich, Germany; 2Heinrich-Heine-UniversitatDusseldorf, Dusseldorf, Germany. Contact e-mail: d.bartnik@fz-juelich.de

Background: Alzheimer’s Disease (AD) is a progressive neurodegenera-

tive disorder, affecting 20 million people world- wide. Recently there is

no cure, only palliative therapies are available and the diagnostic opportu-

nities are limited. Methods: Actually we are characterising our peptides on

their binding properties to Ab1-40, Ab1-42 and different Ab species

(monomers, oligomers, protofibrils, fibrils and plaques) in more detail us-

ing dynamic light scattering, size exclusion chromatography, PepSpot anal-

ysis, density gradient centrifugation, surface plasmon resonance,

fluorescence and electron microscopy. Results: Using mirror image phage

display, we identified two Ab binding D-enantiomeric peptides with sub-

micromolar binding affinity [1,2,3]. D-enantiomeric peptides are known

to be less immunogenic and much more resistant to proteolysis in compar-

ison to their respective L-enantiomers. Conclusions: Fluorescence labelled

derivatives of the peptide D1 stain specifically Ab plaques and leptomenin-

geal vessels containing Ab from human Alzheimer brains - other amyloid-

oses were not stained [4]. Peptide D3 shows interesting therapeutic

properties [5]. The peptide exerts strong influence on Ab aggregation, dis-

aggregation and cytotoxicity in vitro. In vivo, it reduces amyloid plaque

load and cerebral damage of transgenic mouse models. D3 treated mice

show enhanced cognitive performance in comparison to non treated

controls.

P3-283 SELECTION OF AN ANTI-ABETA ANTIBODY THAT

BINDS VARIOUS FORMS OF ABETA AND BLOCKS

TOXICITY BOTH IN VITRO AND IN VIVO

Ryan J. Watts1, Mark Chen1, Jasvinder Atwal1, Joan M. Greve1,

Yongjian Wu1, Deborah L. Mortensen1, Yvan Varisco2, Oskar Aldolfsson2,

Maria Pihlgren2, Andrea Pfeifer2, Andreas Muhs2, 1Genentech, South San

Francisco, CA, USA; 2AC Immune SA, Lausanne, Switzerland.Contact e-mail: rwatts@gene.com

Background: After a decade of research on immunological approaches to

treating Alzheimer’s disease (AD), much has been learned about selection

criteria for antibodies targeting b-amyloid (Abeta). Here we describe the

preclinical properties of an anti-Abeta MAb, MABT5102A, which has

been selected for testing as a disease modifying therapeutic in patients

with AD. Methods: Monoclonal antibodies (MAbs) were generated by

immunization of mice with pegylated Abeta peptide integrated into lipo-

somes. Murine anti-Abeta MAbs were further characterized using both in

vitro and in vivo methods to evaluate Abeta binding and toxicity. An anti-

Abeta MAb was then selected for humanization and affinity maturation

and further evaluated in vitro and in PKPD models. Results: A parental

murine MAb was selected that bound to multiple forms of Abeta. Treat-

ment with this MAb showed an increase in cognitive memory capacity in

an animal model of AD (hAPP-Tg). Long-term dosing studies with this

MAb in an aged AD mouse model resulted in a reduction in plaque

load and number. This murine MAb was affinity matured and humanized

to give rise to MABT5102A. In vitro, MABT5102A bound equally well

and with high affinity to monomer-, oligomer- and fiber-enriched prepara-

tions of Abeta1-42 peptide. Furthermore, binding of MABT5102A in-

hibited self-association and aggregation of Abeta peptides into

protofibrillar conformations, and it disaggregated pre-formed Abeta1-42

protofibrils. MABT5102A also blocked Abeta oligomer-induced toxicity

on primary neurons. In vivo PKPD and safety studies with MABT5102A

supported initiation of phase I clinical trials. Conclusions: A humanized

monoclonal antibody, MABT5102A, was selected based on various desir-

able in vitro and in vivo properties and is currently in a phase I clinical

study enrolling mild to moderate AD patients.

P3-284 DRUG DESIGN APPROACH FOR THE

DEVELOPMENT OF NOVEL NONPEPTIDE

CASPASE-3 INHIBITORS FOR THE TREATMENT

OF ALZHEIMER’S DISEASE

Simant Sharma, Ram Kishore Agrawal, Dr. Hari Singh Gour University,

Sagar (M.P.), India, Sagar, India. Contact e-mail: simpharma49@gmail.

com

Background: Alzheimer’s disease (AD), one form of dementia, is a pro-

gressive, degenerative brain disease. About 10 percent of all people over

70 have significant memory problems and about half of those are due to

AD. In early onset AD, symptoms first appear before age 60. However, it

tends to progress rapidly. Every seven seconds globally someone is diag-

nosed with dementia. In case of AD loss of hippocampal and cortical neu-

rons leads to impairment of memory and cognitive ability. Neuronal loss

due to unwanted apoptosis, correlates with neuronal dysfunction in Alz-

heimer’s disease (AD), which involves caspases, a group of cysteine pro-

teases that cleave their substrates after aspartic acid residues and are the

key executioners of apoptosis. Among the identified caspases, caspase 3

is of particular interest, since it appears to be very important in the pro-

gression of AD (Alzheimer’s disease). Apoptosis of human primary neu-

rons results in an increased production of amyloid b peptide, suggesting

that apoptosis-related activation of proteases is implicated in the metabo-

lism of amyloid precursor protein (APP). Further studies showed that cas-

pase-3 can cleave APP directly. Furthermore, caspase inhibitors eliminate

the apoptosis-mediated increase in amyloid b peptide. Novel medicines

are typically developed using a trial and error approach which is costly

and time-consuming. The application of quantitative structure activity re-

lationship methodologies to this problem has the potential to greatly de-

crease the time and effort required to improve current medicines in

Poster Presentations P3 P427

terms of their efficacy or to discover new ones. Methods: In pursuit of

better caspase-3 inhibitors, a quantitative structure-activity relationship

(QSAR) analysis was performed on a series of caspase-3 inhibitors using

molecular modeling software WIN CAChe 6.1 and statistical software

STATISTICA. Results: The present study results in partition coefficient

(log P), conformational energy (CME), and lowest unoccupied molecular

orbital energy (ELUMO) as optimum physico-chemical properties which

determine the caspase-3 inhibitory activity. Conclusions: On the basis

of above study a new series of caspase-3 inhibitors has been designed

with greater caspase-3 inhibitory activity. After synthesizing these com-

pounds, it can be hypothesized that biological evaluation of these com-

pounds would prove to be effective in addressing neurodegenerative

disorders.

P3-285 ANTIBODY IMMUNE RESPONSE IN

CYNOMOLGUS MONKEYS FOLLOWING

TREATMENT WITH THE ACTIVE Aß

IMMUNOTHERAPY CAD106

Georges Imbert1, Severine Marrony1, Peter Ulrich1, Paul Goldsmith2,1Novartis Pharma AG, Basel, Switzerland; 2Novartis Horsham Research

Centre, Horsham, United Kingdom. Contact e-mail: georges.imbert@

novartis.com

Background: CAD106 is an immunotherapeutic vaccine comprising the

Aß1-6 peptide coupled to the Qß virus-like particle. Treatment with

CAD106 induces production of IgG antibodies both against Aß1-6 and

Qß. These antibodies, and not CAD106 itself, are expected to play a pri-

mary role in the reduction of Ab deposits in the brain of Alzheimer’s dis-

ease patients. CAD106 is therefore a compound which is very different

from either small chemical entities or monoclonal antibodies, both from

its structure and its mechanism of action. As a consequence, primary phar-

macodynamic effect of CAD106 treatment is determined by measurement

of Ab and Qb IgGs. Methods: CAD106 has been administered to cyno-

molgus monkeys in the frame of three separate studies. Serum from these

animals was collected at different timepoints during the course of these

studies. Ab and Qb IgG titers were determined using sandwich ELISA as-

says, specifically developed and validated to quantify these IgG titers.

These assays are based on the binding of Ab and Qb antibodies to immo-

bilized Ab or Qb peptides and detection with anti-human-IgG-HRP. Re-

sults: CAD106 was administered 4 to 8 times in individual animals,

with doses ranging from 150 to 1000 ug. In each study, an increase in an-

tibody titers Ab and Qb IgG titers was observed when the dose was in-

creased. For Ab IgG titers, the titers increased by 1.2 to 2.0-fold under

such circumstances. When CAD106 injections were stopped, antibody ti-

ters levels diminished rapidly. Conclusions: Treatment of cynomolgus

monkey with CAD106 resulted in induction of Ab and Qb IgG titers in

all animals studied. A dose-response relationship was observed in several

independent studies.

P3-286 BIOPHYSICAL CHARACTERIZATION OF SCYLLO-

INOSITOL-Ab INTERACTIONS

JoAnne McLaurin1, James Shaw1, Noble Nemieboka2,

Edward Koellhoeffer3, Hideyo Inouye3, Daniel Kirschner3, Austin Yang2,1University of Toronto, Toronto, ON, Canada; 2University of Maryland,Baltimore, MD, USA; 3Boston College, Chestnut Hill, MA, USA.

Contact e-mail: j.mclaurin@utoronto.ca

Background: scyllo-Inositol, ELND005, is a small molecule that is pres-

ently in phase II clinical trials for AD. Although our preclinical data dem-

onstrated the efficacy in mouse models of AD, the direct binding of

scyllo-inositol with Ab is not well understood. Our previous in vitro

data suggest that scyllo-inositol inhibits Ab42 fibrillogenesis and stabi-

lizes a small Ab conformer but not Ab40. To further understand this in-

teraction, we examined the physical characteristics of this conformer using

several biophysical approaches. Methods: Atomic force microscopy, neu-

tral loss screening method using a triple quadrupole mass spectrometer

and X-ray diffraction were utilized. Results: By in situ atomic force mi-

croscopy we confirmed earlier electron microscopy data that Ab42 fibres

are not formed in the presence of scyllo-inositol, but rather form a mixture

of conformer sizes. By collision-induced dissociation mass spectrometry,

which incorporates a capillary ultra high pressure HPLC system coupled

to a triple quadrupole mass spectrometer, we were able to measure the

binding stoichiometry of the Ab42-scyllo-inositol complex. We also dem-

onstrated that scyllo-inositol preferentially binds to Ab42 rather than Ab40

under physiological conditions. Comparison of x-ray diffraction patterns

from Ab in the presence and absence of scyllo-inositol demonstrated an

absence of the intersheet reflection for Ab42 in the presence of scyllo-ino-

sitol whilst no changes could be detected for Ab40. Conclusions: Alto-

gether, our results suggest that scyllo-inositol binds to Ab42 in a 2:1

ratio thereby inhibiting aggregation through interaction between the two

C-terminal residues and the neighbouring ones that stabilizes the b-sheet

formation of Ab42.

P3-287 CARVEDILOL REDUCES Ab OLIGOMERIZATION

AND IMPROVES SPATIAL MEMORY IN MOUSE

MODELS OF ALZHEIMER’S DISEASE

Jun Wang1, Wei Zhao1, Lap Ho1, XJ Qian1, Isabel Arrieta-Cruz1,

Kenjiro Ono2, David Teplow2, Giulio Maria Pasinetti1, 1Mount Sinai School

of Medicine, New York, NY, USA; 2University of California, Los Angeles,

CA, USA. Contact e-mail: jun.wang@mssm.edu

Background: Abnormal oligomerization of myloid beta monomer to olig-

omers is a key pathological feature associated with AD cognitive deterio-

ration and presently Ab oligomerization is a major target for therapeuticl

interventions In AD. Carvedilol, a non-selective alpha beta blocker bears

the common structural feature that can inhibit beta-amyloid fibril forma-

tion. Methods: In this study, we used in vitro Photo-Induced Cross-linking

of Unmodified Proteins (PICUP) methodology (PICUP assay), circular di-

chroism and electron microscopy analysis to confirm the effect of carvedi-

lol on amyloid aggregation. We also tested the in vivo efficacy of

carvedilol in two animal models of alzheimer’s disease, namely, the

Tg2576 mouse model and TgCRND8 mouse model. Results: We found

that carvedilol treatment significantly improved spatial memory function

in both animal models by Morris Water Maze test and novel object recog-

nition test. This improvement is associated with reduced amyloid oligomer

content in the brain, as well as total amyloid content and amyloid plaque

burden in the brain. Moreover, we also found that the behavior improve-

ment is associated with improved neuroplasticity in the frontal cortical neu-

ron of the mice treated with carvedilol. Conclusions: Collectively, our

preclinical studies demonstrated that carvedilol could significantly inhibit Ab

oligomerization and improve cognitive function in two different models of Alz-

heimer’s disease.Our study may provide the appropriate impetus for clinical tri-

als in vulnerable human subjects, such as MCI patients.

P3-288 ANTIBODIES IN THE DIMER FRACTION OF IVIG

HAVE THE CAPACITY TO BIND BETA AMYLOID

Norman R. Relkin, Paul Szabo, Matthew Rotondi, Diana Mujalli,

Weill Cornell Medical College, New York City, NY, USA.Contact e-mail: nrelkin@med.cornell.edu

Background: Intravenous immunoglobulin (IVIG) is a human polyclonal

antibody (IgG) preparation currently being tested as a potential agent for

Alzheimer’s disease (AD) immunotherapy. IVIG contains antibodies

against the linear sequence of beta amyloid (Ab) as well as conforma-

tion-selective antibodies against Ab aggregates. A subset of the antibodies

in IVIG exist as dimers composed of monomeric IgGs reversibly bound to

anti-idiotypic antibodies. These dimers are known to exert important im-

munomodulatory effects but their possible relevance to AD anti-amyloid

immunotherapy has not been previously studied. The objective of this

study was to determine whether antibody dimers in IVIG possess signifi-

cant Ab binding capacity. Methods: IVIg was subjected to Size Exclusion

Chromatography to produce dimeric (D-IgG) and monomeric (M-IgG)

fractions that were confirmed to be >95% pure. The Ab-binding activity

of these fractions was measured by ELISAs on plates bearing synthetic