Field of the invention

The present invention relates to a combination of an Αβ antibody and a BACE1 inhibitor. Background of the invention

The primary pathology of Alzheimer's Disease (AD) is a wide-spread amyloidosis in the field of the central nervous system which is caused by a progressive deposition of amyloid-β- peptides (Αβ). Αβ is derived from the β-amyloid precursor protein through proteolytic processing by BACE1 (β-secretase) and γ-secretase. Abeta (Αβ) comprises a family of peptides which differ in length at their C-terminus, Αβ40 is the predominant species but a minor peptide of 42 amino acid length, Αβ42, is considered to be the most pathogenic peptide because of its high propensity to form toxic aggregates. The transgenic mouse model hAPP-TG which was used in this study carries a mutation in its APP transgene, V717I, which increases the formation of Αβ42 relative to Αβ40 and thus causes an aggressive amyloidosis which starts around 9-10 months of age Tanghe et al 2010) ¹ . The transgene is under the control of a mouse Thyl promoter which directs is expression preferentially to neuronal cells (Andra et al 1996, Vidal et al 1990) ² .

Because natural animal models for AD amyloidosis are lacking, APP-transgenic mice have become the preferred model to assess the in vivo efficacy of amyloid treatments (Games et al 2006, Higgins & Jacobsen 2003) ³ . While the BACE1 inhibitor blocks the de novo production of Αβ and thus reduces the formation of amyloid aggregates, the antibody binds to existing amyloid and enhances its clearance. Combining both therapeutic strategies causes an amyloid lowering effect exceeding that of mono -treatment with either compound alone. A combination of the specific Αβ antibody (Cmpd 1) and the specific BACE1 inhibitor 5-Cyano-pyridine-2-carboxylic acid [3-((R)-2- amino-5,5-difluoro-4-methyl-5,6-dihydro-4H-[l,3]oxazin-4-yl) -4-fluoro-phenyl]-amide, or a pharmaceutically acceptable salt thereof lead to a surprisingly high amyloid lowering effect (Cmpd 2).

5-Cyano-pyridine-2-carboxylic acid [3-((R)-2-amino-5,5-difluoro-4-methyl-5,6-dihydro- 4H-[l,3]oxazin-4-yl)-4-fluoro-phenyl]-amide, or a pharmaceutically acceptable salt thereof (Cmpd 2) has been described in WO 2011/069934 ⁴ and the Αβ antibody (Cmpd 1) has been described in WO 03/070760 ⁵ .

Alzheimer's Disease is the sixth leading cause of death in the United States and is a fast growing disease. Despite considerable progress of AD research in recent years and evolving paradigm shifts in both pathophysiological concepts as well as in diagnostic criteria fundamental challenges have not yet been reso lved (Hampel et al. 2011) ⁶ . It is an object of the present invention to provide a composition suitable for control, treatment and/or prevention of Alzheimer's Disease. WO 2007068412 ⁷ related to monoclonal antibodies used in methods and compositions for the therapeutic and diagnostic use in the treatment of diseases and disorders which are caused by or associated with amyloid or amyloid-like proteins including amyloidosis, a group of disorders and abnormalities associated with amyloid protein such as Alzheimer's disease. WO 2006014944 ⁸ relates to β secretase inhibitors. WO 03070760 ⁹ relates to antibody molecules capable of specifically recognizing two regions of the R-A4 peptide. WO 2011069934 ¹⁰ relates to 2-Amino-5,5-difluoro-5,6-dihydro-4H-[l,3]oxazin-4-yl)- phenyl]-amide derivatives having BACE1 and/or BACE2 inhibitory activity. Chow et al. ¹¹ describes a combination therapy for the treatment of Alzheimer's Disease. Wang et al. ¹² describes a mouse model of Alzheimer's Disease. Guo et a/. ¹³ describes the role of Αβ in the treatment of glaucoma. The present invention provides a combination of an Αβ antibody and a BACE1 inhibitor, pharmaceutical products based on that combination in accordance with the invention and their the use thereof in the control, treatment and/or prevention of illnesses such as Alzheimer's disease.

Definitions As stated above, compound 1 (Cmpd 1) shall herein refer to the Αβ antibody.

The terms "Abeta antibody" and "an antibody that binds to Abeta" refer to an antibody that is capable of binding Αβ peptide with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent in targeting Αβ peptide. Alternatively, the term "anti- Αβ antibody" can be used. The term "antibody" encompasses the various forms of antibody structures including but not being limited to whole antibodies and antibody fragments. The antibody according to the invention is preferably a humanized antibody, chimeric antibody, or further genetically engineered antibody as long as the characteristic properties according to the invention are retained. The terms "monoclonal antibody" or "monoclonal antibody composition" as used herein refer to a preparation of antibody molecules of a single amino acid composition. The term "mono-glycosylated Abeta antibody" relates to an antibody molecule comprising an N-glycosylation at position 52 of Seq. Id. No. 1 in one (VH)-region of an individual antibody molecule. The term "double-glycosylation Abeta antibody" defines an antibody molecule which is N-glycosylated at position 52 of Seq. Id. No. 1 on both variable regions of the heavy chain" (figure 1). Antibody molecules which lack a N-glycosylation on both heavy chain (VH)-domains are named "non-glycosylated antibodies". The mono-glycosylated antibody, the double- glycosylated antibody and the non-glycosylated antibody may comprise the identical amino acid sequences or different amino acid sequences. The mono-glycosylated antibody and the double- glycosylated antibody are herein referred to as "glycosylated antibody isoforms". A purified antibody molecule characterized in that at least one antigen binding site comprises a glycosylation in the variable region of the heavy chain (VH) is a mono-glycosylated antibody which is free of or to a very low extent associated with an isoform selected from a double- glycosylated antibody and a nonglycosylated antibody, i.e. a "purified mono-glycosylated antibody". A double-glycosylated antibody in context of this invention is free of or to a very low extent associated with an isoform selected from a mono-glycosylated antibody and a nonglycosylated antibody, i.e. a "purified double-glycosylated antibody".

Compound 2 (Cmpd 2) shall herein refer to 5-Cyano-pyridine-2-carboxylic acid [3-((R)-2- amino-5,5-difluoro-4-methyl-5,6-dihydro-4H-[l,3]oxazin-4-yl) -4-fluoro-phenyl]-amide of the following structure

or a pharmaceutically acceptable salt thereof The terms "excipient" or "pharmaceutically acceptable excipient" refers to surfactans, disintegrants, fillers, binders, glidants, lubricants and there like. The term "surfactant" refers to excipients that lower the surface tension of a liquid, the interfacial tension between two liquids, or that between a liquid and a solid. The term "disintegrant" refers to excipients that expand and dissolve when wet causing the tablet to break apart in the body and release the active ingredient for absorption. The term "filler" refers to excipients that fill out the size of a tablet by increasing the bulk volume. Fillers make it possible for the final product to have the proper volume for patient handling. The term "binder" refers to excipients that hold the ingredients in a tablet together. Binders ensure that tablets and granules can be formed with required mechanical strength, and give volume to low active dose tablets. The term "glidant" refers to excipients that enhance product flow by reducing interparticulate friction. The term "lubricant" refers to excipients that prevent ingredients from clumping together and from sticking to the tablet punches or capsule filling machine. Lubricants also ensure that tablet formation and ejection can occur with low fraction between active ingredient and wall.

The term "pharmaceutically acceptable salt" refers to any conventional salt or base addition salt that retains the biological effectiveness and properties of the compound and which is formed from a suitable non-toxic organic or inorganic acid or organic or inorganic base. An example is the hydrochloride salt. As used herein, the term "therapeutically effective" means an amount of drug, or composition, which is effective for producing a desired therapeutic effect upon administration to a patient, for example, to control, treat and/or prevent Alzheimer's Disease.

Detailed description of the invention

The present invention relates to a combination of an Αβ antibody and a BACEl inhibitor.

The combination of the Αβ antibody and the BACEl inhibitor 5-Cyano-pyridine-2- carboxylic acid [3-((R)-2-amino-5,5-difluoro-4-methyl-5,6-dihydro-4H-[l,3]ox azin-4-yl)-4- fluoro-phenyl]-amide, or a pharmaceutically acceptable salt thereof can be used in the therapeutic and/or prophylactic treatment of diseases and disorders characterized by elevated β- amyloid levels and/or β-amyloid oligomers and/or β-amyloid plaques and further deposits, particularly Alzheimer's disease.

All specific embodiments can be combined.

The invention relates to a composition comprising an Αβ antibody and a BACEl inhibitor. A specific embodiment relates to a composition comprising i) gantenerumab, and ii) 5-Cyano-pyridine-2-carboxylic acid [3-((R)-2-amino-5,5-difluoro-4-methyl-5,6- dihydro-4H-[l,3]oxazin-4-yl)-4-fluoro-phenyl]-amide, or a pharmaceutically acceptable salt thereof.

A specific embodiment relates to a composition consisting of i) gantenerumab, and ii) 5-Cyano-pyridine-2-carboxylic acid [3-((R)-2-amino-5,5-difluoro-4-methyl-5,6- dihydro-4H-[ 1 ,3]oxazin-4-yl)-4-fluoro-phenyl]-amide hydrochloride.

A specific embodiment relates to a composition comprising i) an Αβ antibody, wherein the heavy chain thereof comprises a VH domain which comprises:

• a CDR1 comprising the amino acid sequence of Seq. Id. No. 3,

• a CDR2 comprising the amino acid sequence of Seq. Id. No. 4,

• a CDR3 sequence comprising the amino acid sequence of Seq. Id. No. 5; and ii) a BACEl inhibitor which is 5-Cyano-pyridine-2-carboxylic acid [3-((R)-2-amino-5,5- difluoro-4-methyl-5,6-dihydro-4H-[l,3]oxazin-4-yl)-4-fluoro- phenyl]-amide, or a pharmaceutically acceptable salt thereof.

A specific embodiment relates to a composition comprising i) an Αβ antibody, wherein the light chain thereof comprises a VL domain which comprises:

• a CDR1 comprising the amino acid sequence of Seq. Id. No. 6,

• a CDR2 comprising the amino acid sequence of Seq. Id. No. 7,

• a CDR3 sequence comprising the amino acid sequence of Seq. Id. No. 8; and ii) a BACEl inhibitor which is 5-Cyano-pyridine-2-carboxylic acid [3-((R)-2-amino-5,5- difluoro-4-methyl-5,6-dihydro-4H-[l,3]oxazin-4-yl)-4-fluoro- phenyl]-amide, or a pharmaceutically acceptable salt thereof.

A specific embodiment relates to a composition comprising i) an Αβ antibody, wherein the VH domain thereof comprises the amino acid sequence of Seq. Id. No. 1 and the VL domain thereof comprises the amino acid sequence of Seq. Id. No. 2; and ii) a BACEl inhibitor which is 5-Cyano-pyridine-2-carboxylic acid [3-((R)-2-amino-5,5- difluoro-4-methyl-5,6-dihydro-4H-[l,3]oxazin-4-yl)-4-fluoro- phenyl]-amide, or a pharmaceutically acceptable salt thereof.

A specific embodiment relates to a composition comprising i) an Αβ antibody, wherein the heavy chain thereof comprises the amino acid sequence of Seq. Id. No. 9; and ii) a BACEl inhibitor which is 5-Cyano-pyridine-2-carboxylic acid [3-((R)-2-amino-5,5- difluoro-4-methyl-5,6-dihydro-4H-[l,3]oxazin-4-yl)-4-fluoro- phenyl]-amide, or a pharmaceutically acceptable salt thereof.

A specific embodiment relates to a composition comprising i) an Αβ antibody, wherein the light chain thereof comprises the amino acid sequence of Seq. Id. No. 10; and ii) a BACEl inhibitor which is 5-Cyano-pyridine-2-carboxylic acid [3-((R)-2-amino-5,5- difluoro-4-methyl-5,6-dihydro-4H-[l,3]oxazin-4-yl)-4-fluoro- phenyl]-amide, or a pharmaceutically acceptable salt thereof. A specific embodiment relates to a composition comprising i) an monoclonal Αβ antibody, which is a mixture of mono-glycosylated Αβ antibodies and double-glycosylated Αβ antibodies, wherein the mono-glycosylated Αβ antibody comprises a glycosylated asparagine (Asn) at position 52 of Seq. Id. No. 1 in the VH domain of one antibody binding site and wherein the double-glycosylated antibody comprises a glycosylated asparagine (Asn) at position 52 of Seq. Id. No. 1 in the VH domain of both antibody binding sites and whereby said mixture comprises less than 5% of an antibody being non-glycosylated at position 52 of Seq. Id. No. 1 in the VH domain; and ii) a BACEl inhibitor which is 5-Cyano-pyridine-2-carboxylic acid [3-((R)-2-amino-5,5- difluoro-4-methyl-5,6-dihydro-4H-[l,3]oxazin-4-yl)-4-fluoro- phenyl]-amide, or a pharmaceutically acceptable salt thereof.

A specific embodiment relates to a composition as described herein, wherein the a BACEl inhibitor is 5-Cyano-pyridine-2-carboxylic acid [3-((R)-2-amino-5,5-difluoro-4-methyl-5,6- dihydro-4H-[ 1 ,3]oxazin-4-yl)-4-fluoro-phenyl]-amide hydrochloride. A specific embodiment relates to a composition as described herein, wherein the Αβ antibody and 5-Cyano-pyridine-2-carboxylic acid [3-((R)-2-amino-5,5-difluoro-4-methyl-5,6- dihydro-4H-[l,3]oxazin-4-yl)-4-fluoro-phenyl]-amide, or a pharmaceutically acceptable salt thereof are administered separately, simultaneously, in a fixed combination or sequentially in any order. A specific embodiment relates to a composition as described herein, wherein the Αβ antibody and 5-Cyano-pyridine-2-carboxylic acid [3-((R)-2-amino-5,5-difluoro-4-methyl-5,6- dihydro-4H-[l,3]oxazin-4-yl)-4-fluoro-phenyl]-amide, or a pharmaceutically acceptable salt thereof are administered separately.

A specific embodiment relates to a composition as described herein, wherein the Αβ antibody and 5-Cyano-pyridine-2-carboxylic acid [3-((R)-2-amino-5,5-difluoro-4-methyl-5,6- dihydro-4H-[l,3]oxazin-4-yl)-4-fluoro-phenyl]-amide, or a pharmaceutically acceptable salt thereof are administered simultaneously.

A specific embodiment relates to a composition as described herein, wherein the Αβ antibody and 5-Cyano-pyridine-2-carboxylic acid [3-((R)-2-amino-5,5-difluoro-4-methyl-5,6- dihydro-4H-[l,3]oxazin-4-yl)-4-fluoro-phenyl]-amide, or a pharmaceutically acceptable salt thereof are administered in a fixed combination.

A specific embodiment relates to a composition as described herein, wherein the Αβ antibody and 5-Cyano-pyridine-2-carboxylic acid [3-((R)-2-amino-5,5-difluoro-4-methyl-5,6- dihydro-4H-[l,3]oxazin-4-yl)-4-fluoro-phenyl]-amide, or a pharmaceutically acceptable salt thereof are administered sequentially in any order.

A specific embodiment relates to a pharmaceutical product comprising a composition as described herein and one or more pharmaceutically acceptable excipients. A specific embodiment relates to a pharmaceutical product comprising a composition as described herein which is in the form suitable for oral administration.

A specific embodiment relates to a pharmaceutical product comprising a composition as described herein which is in the form suitable for intravenous administration.

A specific embodiment relates to a pharmaceutical product comprising a composition as described herein which is in the form suitable for subcutaneous administration

A specific embodiment relates to a use of a composition as described herein for the manufacture of a medicament for the treatment and/or prevention of Alzheimer's Disease.

A specific embodiment relates to a use of a composition as described herein for the manufacture of a medicament for the treatment of Alzheimer's Disease. A specific embodiment relates to a use of a composition as described herein for the manufacture of a medicament for the prevention of Alzheimer's Disease.

A specific embodiment relates to a composition as described herein for the use in the treatment and/or prevention of Alzheimer's Disease.

A specific embodiment relates to a composition as described herein for the use in the control of Alzheimer's Disease.

A specific embodiment relates to a composition as described herein for the use in the treatment of Alzheimer's Disease.

A specific embodiment relates to a composition as described herein for the use in the prevention of Alzheimer's Disease. A specific embodiment of the invention relates to a method of treatment and/or prevention of Alzheimer's Disease by administering a composition as described herein.

A specific embodiment of the invention relates to a kit comprising a preparation as described herein and instructions for the separate, simultaneous, fixed-dose or sequential in any order administration of the preparations to a patient in need thereof. The efficacy of BACEl inhibitor Cmpd 2 and Αβ antibody (Cmpd 1) as mono -treatments was confirmed in the tgAPP mouse model of CNS amyloidosis. A significantly enhanced treatment effect of combination-therapy was demonstrated. The different read-outs which we employed to assess brain amyloidosis reveal remarkable differences in efficacy for each treatment modality, pointing to their different mechanisms of action which are inhibition of de novo production vs enhanced clearance.

BACEl inhibition decreases total brain Αβ40 and Αβ42 to similar extends and with a clear dose-response as measured by specific immune-assays. The antibody by itself reduces Αβ42 but has little effect on Αβ40 in this specific mouse model. Since the Αβ antibody recognizes an epitope in the N-terminus of Αβ this differential effect does not reflect a preference for Αβ42 per se but is likely due its different conformation or aggregation state as compared to Ab40. the Αβ antibody prefers Αβ aggregates to monomers and it is thus conceivable that Αβ42'8 higher propensity for aggregate formation favors its binding to the Αβ antibody and thus enhances its clearance. BACEl inhibition, however, reduces both Αβ species to similar extends as clearly shown by their similar reduction in the CSF. This results in a comparably reduced de novo production for both peptides over the entire treatment period and thus reduces their accumulation as amyloid. The effect of combination treatment on total brain Αβ is significant for Αβ42. For Αβ40 there is a trend towards enhanced efficacy in the combination arm but because of the weak potency of the Αβ antibody towards Αβ40 in this specific mouse model its reduction is mostly driven by BACEl inhibition. The benefit of combination treatment is more pronounced when using the lower dose of Cmpd 2.

Measurement of amyloid load, either by areas covered by plaques or plaque number, gives a different picture for the relative efficacies of both treatment modalities. Here the the Αβ antibody y alone exceeds the BACEl inhibitor in efficacy, which is most pronounced in the effect on plaque number. Combining both treatments causes a significantly enhanced efficacy, both for reduction of plaque load and plaque number.

In summary, combining BACEl inhibitor Cmpd 2 and Αβ antibody Cmpd 1 significantly enhances the in vivo amyloid in a transgenic mouse model for CNS amyloidosis. This enhanced efficacy is partly due to their different mechanisms of action which, when combined, reduce the de novo production on Αβ and thus its accumulation and enhance the clearance of existing amyloid.

Experimental part

1. In vivo procedures

105 mice of an average age of 13.5 months were randomized to 7 groups of 15 animals each. Group N Strain Treatment

None, sacrificed at the age of 13.5 months >

1 15 hAPP-Tg

defines amyloidosis at study start

Vehicle (qd, po), sacrificed at the age of 17.5

2 15 hAPP-Tg

months > defines amyloidosis at study end

Cmpd 2 (qd, po, 30 mg/kg), sacrifice at the age of

3 15 hAPP-Tg

17.5 months

Cmpd 2 (qd, po, 90 mg/kg), sacrifice at the age of

4 15 hAPP-Tg

17.5 months

Vehicle (qd, po), Cmpd 1 (weekly 20 mg/kg, i.v. in

5 15 hAPP-Tg

tail vein), sacrifice at the age of 17.5 months

Combination Cmpd 2 (qd, po, 30 mg/kg) plus

6 15 hAPP-Tg Cmpd 1 (weekly 20 mg/kg, i.v. in tail vein),

sacrifice at the age of 17,5 months

Combination Cmpd 2 Bll (qd, po, 90 mg/kg) plus

7 15 hAPP-Tg Cmpd 1 (weekly 20 mg/kg, i.v. in tail vein),

sacrifice at the age of 17.5 months

In addition, all animals of groups 2 to 7 were treated with an mouse CD4 antibody (0.5mg, i.v.) one day before the group-specific treatments started. The purpose was to deplete the CH4- positive T-cells and thus prevent the formation of an immune response against the human Cmpd 1 antibody. The efficacy and validity of this procedure has been shown previously in chronic mono-therapies with Cmpd 1 alone (Bohrmann et al 2012) ¹⁴ .

2. Assays

2.1. Αβ-peptide measurements

Preparations of brain lysates: Frozen left brain halves (cerebellum removed) were homogenized in four volumes of 9M Urea/ 50mM Tris (vol/wet weight) using a MagNa Lyzer (Roche Applied Science) for 20" at 4000rpm in Green Bead Tubes (Roche Applied Science). The homogenates were incubated on ice for 2 hours, and then centrifuged for 20 minutes at 20'000g at 4°C. The supernatants were diluted from 1 :200 to 1 : 100'000 in AlphaLlSA assay buffer depending on Αβ content and assay sensitivity. Αβ was determined by a AlphaLlSA assays. AlphaLlSA: Brain extracts was diluted in AlphaLlSA Assay Buffer (25mM Hepes pH 7.4,

0.5% Triton X-100, 0.1% Casein, lmg/ml Dextran 500, pH 7.0). In a 96well half-area plate (PerkinElmer), 20ul of these dilutions were mixed with 5ul of biotinylated Αβ antibody 6E10 (Covance) an Αβ-C-terminal end- specific antibody BAP-24 or BAP- 15 conjugated to AlphaLlSA Acceptor Beads (Perkin Elmer). The specificities of BAP-24 and BAP- 15 have been described (Brockhaus et al 1998). The mix was incubated for lh at RT, then 20ul AlphaLISA Donor Beads (PerkinElmer) were added and the complex was allowed to form for another 30' before the plate was read on an EnVision MultiLabel Reader (PerkinElmer). For the quantification of Αβ40 or Αβ42 from brain extracts, the corresponding peptide standards (Anaspec) were diluted in AlphaLISA buffer containing 45mM Urea.

2.2 Determination of amyloid-β plaque deposits by immunohistochemistry and quantitative morphometry

Fresh frozen brains from animals were sagitally sectioned with a cryostat and processed for further analysis by immunofluorescence and quantitative morphometry as described previously (Bohrmann et al 2012) ¹⁴ . In this study, for quantifications 5 sections per animal were used. Quantification of plaque number in APP mice was obtained from five sagittal sections per mouse within the hippocampal formation, cortex and subiculum using every 10 ^th section cut at a nominal thickness of 10 μιη. After staining of sections with gantenerumab at 2 μg/mL for 1 hour at room temperature and detection by affinity-purified goat human IgG (H + L) conjugated to Alexa555 at 20 μg/mL for 1 hour at room temperature (#A21433, Molecular Probes, Eugene, OR, U.S.A.) fluorescence images were obtained from entire brain sections scanned with a Metafer4 slide scanner (MetaSystems, Altlussheim, Germany). Quantitative image analysis was done by a customized rule set developed in-house for automated detection of stained amyloid plaques after interactive selection of ROI in most affected brain regions using the Definiens XD 2.0 software package (Definiens Ltd., Munich, Germany). Calculations were made with common spreadsheet software (Microsoft Excel, Redmond, WA, USA). Statistical evaluation was done using a two -tailed Student's t-test and One-way ANOVA with Bonferroni's Multiple Comparison test (GraphPad Prism).

Results 1. Progressive amyloidosis in vehicle-treated mice

Based on previous studies it is known that this transgenic mouse strain starts to deposit Αβ- amyloid around 8-10 months of age and at 13.5 months already carries an extensive CNS amyloidosis (Tanghe et al 2010) ¹ . Thus, this study is designed to study treatment effect on preexisting amyloid and treatment efficacy to inhibit further amyloid deposition. In Fig. l solubilized Αβ40 and Αβ42 levels from brain extracts are shown for mice at 13.5 months of age (at study start), and for the vehicle-treated group 4 months later (at study end). Αβ42 increases approx. 7fold from a mean of 6ng per mg brain wet weight to a mean of 41ng while Αβ40 increases from 0.8ng to 5ng. The mean Αβ42 content thus exceeds that for Αβ40 by 8 fold, reflecting the increased Αβ42 production due to the V717I London mutation in the transgenic mice. The total amounts for the individual animals vary considerably; the extremes for Αβ42 are 31 and 47ng/mg brain. However, such variability of brain Αβ is commonly seen in APP-transgenic mice.

In Fig.2 representative brain sections stained for human Αβ are shown for an animal at study begin (a) and for a vehicle treated animal at study end (b). Typical dense plaques and diffuse plaques of various sizes are clearly shown and it is obvious that 13.5m old animals have already a significant amyloid load, thus confirming an established amyloidosis for this age group at study start. Quantitative data for amyloid plaque deposition are reported below.

2. Mono-treatments with Cmpd 1 or Cmpd 2 reduce Αβ deposition in brain

The treatment was started in an animal cohort that was on average 13.5 months old and presented with advanced amyloidosis, a condition closer to the 'therapeutic' treatment in a clinical trial. Cmpd 2 was applied in two doses, a medium dose of 30mg/kg and a high dose of 90mg/kg, once daily per os. Cmpd 1 was applied once weekly at 20mg/kg, iv via tail vein injection. Its efficacy at this dose and treatment schedule has been established before in a different APP-transgenic mouse model (Bohrmann et al 2012) ¹⁴ For Αβ42 a clear and significant reduction is observed for all 3 treatment arms; a decrease of 28% and 58% for the two Cmpd 2 treatment arms and 39% for the Cmpd 1 treatment (Fig.3). The variability is likely due to differences in brain amyloid level in the animals at study start (see Fig. l). For Αβ40, a significant reduction is noted for the Cmpd 2 treatment arms (reductions by 45% and 69%), however, the antibody treatment by itself did not result in a significant Αβ40 reduction. The reasons for the discrepancy between Αβ42 and Αβ40 are the binding properties of Cmpd 1. This antibody has a much higher affinity for aggregated Αβ over soluble, monomeric Αβ (Bohrmann et al 2012) ^M . It is presumed that in this mouse model Αβ40 is largely in a non- aggregated state and thus less accessible for binding by antibody Cmpd 1.

3. Combination-treatment of Cmpd 1 and Cmpd 2 reduces brain Αβ-deposition below levels obtained with mono-treatments

3. a) Effect of treatments on total brain Αβ

Fig.4 compares the brain Αβ level in the mono -treatment arms (same as in Fig.3) to the two combination arms. Cmpd 1 treatment in addition to Cmpd 2 leads to a substantial enhancement of the amyloid activity. For Αβ42 the amyloid reduction more than doubles for the lower dose arm, i.e. Cmpd 2 at 30mg/kg decreases Αβ42 by 28%, the addition of Cmpd 1 treatment causes a reduction of 66%. The difference is highly significant (p < 0.0001). For the higher dose treatment arms the corresponding number are 58% for Cmpd 2 alone and 79% after addition of Cmpd 1, the combined effect is less, but still reaches statistical significance (p < 0.001). The less-than-additive combo effect at the higher doseis probably due to the very high dose of Cmpd 2, which already approaches maximal inhibition. Nevertheless, for Αβ42 there is a clear benefit for combination treatment.

There is a trend towards lower Αβ40 values in the combination arms compared to the corresponding Cmpd 2 mono -treatments. The numbers for Αβ40 reduction of mono- vs. combination-treatments are 45% vs 57% and 69% vs 74%.

We then asked if treatment (i) slows the build-up of amyloid, (ii) stabilizes amyloidosis at the level of study start or (iii) even reduces any existing amyloid. The results are shown for total brain Αβ40 and Αβ42 for the Cmpd 2 high-dose arm alone or plus Cmpd 1 treatment (Fig.5). For Αβ40 there is a trend towards increased amyloidosis. For Αβ42 there is a clear, significant increase in the group treated with 90mg/kg Cmpd 2 alone whereas the combination treatment stabilizes the amyloidosis at the starting level.

3.b) Effect of treatments on plaque load and number

Plaque load and amyloid-β plaque numbers were measured by quantitative morphometry. The plaque load, i.e., area covered by amyloid deposits, was significantly reduced by all treatment arms (Fig.6). The reduction by treatment with Cmpd 1 alone was much stronger than what observed previously for its effect on total brain Αβ (Fig.3 and 4) and equaled or exceeded the reduction observed with Cmpd 2 alone. This is in accordance with the known preference for aggregated Αβ as it is in plaques. Combination treatments essentially prevented any increase of amyloid load over the entire treatment period. The findings for plaque load are mirrored in an additional read-out for the treatment effects on plaque number (Fig.7). All treatments significantly reduced total plaque number compared to the vehicle group. Cmpd 1 as mono -treatment was highly efficacious in this read-out and for the cortex sections its effect alone was comparable to the reduction seen in the combination arms, i.e., the reduction was almost exclusively antibody-driven. In contrast, in cortex and subiculum the reduction in the combination arms exceeded the mono -treatments. In cortex the plaque number in the Cmpd 1 alone and in the combination arms are even lower than in the baseline group.

In a more detailed analysis of the plaque sizes we obtained evidence that combination treatment is able to actually decrease the amyloid plaque numbers significantly in most size groups with a trend towards reduction below baseline levels (Figure 8).

In Fig.9 some brain sections of the various treatment paradigms are shown which visually demonstrate the impressive reduction of amyloid load and plaque number especially for the combination arms.

Brief description of the figures Fig 1 : Αβ40 and Αβ42 from brain extracts at 13.5m of age (start) and 17.5m (vehicle), expressed as ng per mg brain wet weight. Each point represents a single animal; the line shows the group median.

Fig.2: Representative brain sections stained for Αβ deposits showing amyloid plaque load in the hippocampal formation and dorsal cortical regions in an animal at study start (a) and in a vehicle-treated animal at study end (b).

Fig.3: Treatment with Cmpd 2 (BI30; BI90) and Cmpd 1 (mAb). Shown are the group means and standard deviations. Bars with or 'ns' indicate significant or non-significant group differences. Fig.4: Treatment with Cmpd 2at two dose levels (BI30 = 30mg/kg; BI90= 90mg/kg) and

Cmpd 1 (mAb) alone or in combination with Cmpd 2 (BI30+mAb; BI90+mAb). Shown are the group means and standard deviations at end of treatment (17.5m). Bars with or 'ns' indicate significant or non-significant group differences.

Fig.5: Αβ40 and 42 level at study start (13.5m) and study end (17.5m) in the groups receiving vehicle treatment, Cmpd 2 at 90mg/kg (BI90) or Cmpd 2 plus Cmpd 1 (BI90+mAb). Shown are the group means and standard deviations at end of treatment (17.5m). Bars with or 'ns' indicate significant or non-significant group differences.

Fig 6: Effect on total immuno detected amyloid plaques after treatment with Cmpd 2 at two dose levels (30 and 90mg/kg) and Cmpd 1 (20 mg/kg) or in combination. Data are shown as immunostained area in indicated brain regions obtained from 5 brain sections per animal with boxes resembling 2 quantiles (50% of data), mean values (line), median (+) and whiskers spanning minimum and maximum values. Significance levels given: * p<0.05, ** p<o0.01, *** pO.001

Fig 7: Effect on total amyloid plaque numbers after treatment with Cmpd 2 at two dose levels (30 and 90mg/kg) and Cmpd 1 (20 mg/kg) or in combination. Data are shown as total detectable plaque numbers with boxes resembling 2 quantiles (50% of data), mean values (line), median (+) and whiskers spanning minimum and maximum values. Significance levels given: * p<0.05, ** p<o0.01, *** pO.001

Fig 8: Effect on amyloid plaque size distribution after treatment with Cmpd 2 at two dose levels (30 and 90mg/kg) and Cmpd 1 (20 mg/kg) or in combination. Plaque sizes are shown in colors at surface classes indicated.

Fig 9: Representative brain sections stained for Αβ deposits showing amyloid plaque deposition in the hippocampal formation and dorsal cortical regions in animals treated with Cmpd 2 at 30 mg/kg (a), Cmpd 2 at 90 mg/kg (b), Cmpd 1 at 20 mg/kg (c), Cmpd 2 (30 mg/kg) plus Cmpd 1 (d) and Cmpd 2 (90 mg/kg) plus Cmpd 1 (e).

Exemplified Sequences:

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² Andra K, Abramowski D, Duke M, Probst A, Wiederhold KH, et al. 1996. Expression of APP in transgenic mice: a comparison of neuron-specific promoters. Neurobiology of aging 17: 183- 90, and

Vidal M, Morris R, Grosveld F, Spanopoulou E. 1990. Tissue-specific control elements of the Thy-1 gene. The EMBO journal 9: 833-40

³ Games D, Buttini M, Kobayashi D, Schenk D, Seubert P. 2006. Mice as models: transgenic approaches and Alzheimer's disease. J Alzheimers Dis 9: 133-49, and

Higgins GA, Jacobsen H. 2003. Transgenic mouse models of Alzheimer's disease: phenotype and application. Behav Pharmacol 14: 419-38.

⁴ WO 2011/069934 ⁶ Harald Hampel, David Prvulovic, Stefan Teipel, Frank Jessen, Christian Luckhaus, Lutz Frolich, Matthias W. Riepe, Richard Dodel, Thomas Leyhe, Lars Bertram, Wolfgang Hoffmann, Frank Faltraco, for the German Task Force on Alzheimer's Disease (GTF-AD), Progress in Neurobiology, Volume 95, Issue 4, December 2011, Pages 718-728

⁷ WO 2007068412

⁸ WO 2006014944

⁹ WO 03070760

¹⁰ WO 2011069934

¹¹ Chow et al., Alzheimers Dementia, vol.7,p. e6(2011) and Science Translational Medicine, vo 1.2,p .1 - 11 (2010)

¹² Wang et al., Journal of Neuroscience,vol.31,pp.4124-4136(2011)

¹³ Guo et al., Proceedings of the National Academy of Sciences, vol.104,pp.13444-13449(2007)

¹⁴ Bohrmann B, Baumann K, Benz J, Gerber F, Huber W, et al. 2012. Gantenerumab: a novel human anti-Abeta antibody demonstrates sustained cerebral amyloid-beta binding and elicits cell-mediated removal of human amyloid-beta. Journal of Alzheimer's disease : JAD 28: 49-69.