WO2004018997A2

WO2004018997A2 - Methods and compositions for modulating amyloid beta

Info

Publication number: WO2004018997A2
Application number: PCT/US2003/026173
Authority: WO
Inventors: Maria Kounnas; Aaron Patrick; Gonul Velicelebi; Steven Wagner
Original assignee: Neurogenetics, Inc.
Priority date: 2002-08-20
Filing date: 2003-08-20
Publication date: 2004-03-04
Also published as: WO2004018997A3; AU2003259965A8; AU2003259965A1

Abstract

Provided herein are methods and compositions for detecting, assessing and modulating β-amyloid peptide (Aβ) levels and/or processing of amyloid precursor protein. Methods for screening and/or identifying agents that modulate processing of APP or the levels of β-amyloid peptides, and methods for assessing presenilin activity and for modulating lipoprotein receptor-related protein (LRP), are also provided.

Description

METHODS AMD COMPOSITIONS FOR MODULATING AMYLOD3 BETA

RELATED APPLICATIONS

Benefit of priority under §119(e) is claimed to U.S. Provisional Application Serial No. 60/405,417, filed August 20, 2002, entitled "Methods of Modulating and Identifying Agents that Modulate Processing of Amyloid Precursor Protein" and U.S. Provisional Application Serial No. 60/411,974, filed September 18, 2002, entitled "Methods of Modulating and Identifying Agents that Modulate Processing of Amyloid Precursor Protein." The subject matter and contents, including sequence listings, of each of these provisional applications is incorporated by reference herein in its entirety. FD3LD OF THE INVENTION

The field of invention relates to methods and compositions for detecting, assessing and modulating _/3-amyloid peptide (Aβ) levels and processing of amyloid precursor protein. BACKGROUND

Alzheimer's disease (AD) is a progressive neurodegenerative disorder that is the predominant cause of dementia in people over 65 years of age. It is estimated to affect 4 million Americans. Clinical symptoms of the disease begin with subtle short term memory problems. As the disease progresses, difficulty with memory, language and orientation worsen to the point of interfering with the ability of the person to function independently. Other symptoms, which are variable, include myoclonus and seizures. Duration of AD from the first symptoms of memory loss until death is 10 years on average.

The AD brain is characterized by two distinct pathologies; 1) neurofibrillary tangles (NFT), comprised mostly of tau and 2) amyloid plaques, comprised primarily of highly hydrophobic amyloid precursor protein peptides called Aβ peptides. The characteristic Alzheimer's NFTs contain abnormal filaments bundled together in neurons and occupying much of the perinuclear cytoplasm. These filaments contain the microtubule-associated protein tau in a hyperphosphorylated form. "Ghost" NFTs are also observed in AD brains, which presumably mark the location of dead neurons. Ajβ aggregates into antiparallel filaments in a j3-pleated sheet structure resulting in the birefringent nature of the AD amyloid. Other neuropathological features include granulovascular changes, neuronal loss, gliosis and the variable presence of Lewy bodies. Although Aβ is the major component of AD amyloid, other proteins have also been found associated with amyloid plaques, e.g., alpha- 1-anti-chymotrypsin (Abraham et al. (1988) Cell 52:487-501), cathepsin D (Cataldo (1990) et al. Brain Res. 573:181- 192), non-amyloid component protein (Ueda et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90:11282-11286), apolipoprotein E (apoE) (Namba et al. (1991) Brain Res. 541:163- 166; isniewski and Frangione (1992) Neurosci. Lett. 735:235-238; Strittmatter et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90:1977-1981), apolipoprotein J (Choi-Mura (1992) et al. Acta Neuropathol. 83:260-264; McGeer (1992) et al. Brain Res. 579:337-341), heat shock protein 70 (Hamos et al. (1991) Neurology 47:345-350), complement components (McGeer and Rogers (1992) Neurology 43:447-449), alpha2-macroglobin (Strauss et al. (1992) Lab. Invest. 65:223-230), interleukin-6 (Strauss et al. (1992) Lab. Invest. 66:223- 230), proteoglycans (Snow et al. (1987) Lab. Invest. 58:454-458), and serum amyloid P (Coria et al. (1988) Lab. Invest. 58:454-458).

Plaques are often surrounded by asirocytes and activated microglial cells expressing immune-related proteins, such as the MHC class II glycoproteins HLA-DR, HLA-DP and HLA-DQ, as well as MHC class I glycoproteins, interleukin-2 (IL-2) receptors and IL-1. Also surrounding many plaques are dystrophic neurites, which are nerve endings containing abnormal filamentous structures.Currently, there is no cure or effective treatment for AD and the few approved drugs including Aricept, Exelon, Cognex and Reminyl are palliative at best. Effective treatments are needed. Therefore, among the objects herein, it is an object to provide methods for modulating and for identifying agents for modulating the processing of amyloid precursor protein (APP) and the levels of Aβ peptides. It is also an object to provide methods for identifying candidate agents for the treatment of AD and other neurodegenerative disorders characterized by altered levels of Aβ peptides and/or amyloidosis. SUMMARY

Provided herein are methods for assessing presenilin activity, comprising contacting a sample containing a presenilin and/or fragment(s) thereof with a lipoprotein receptor-related protein (LRP) and/or fragment(s) thereof; and assessing the processing and/or cleavage of the LRP or fragment(s) thereof. Also provided herein are methods for identifying an agent that modulates presenilin activity, comprising contacting a sample containing a presenilin, and/or fragment(s) thereof, and a lipoprotein receptor-related protein (LRP), and/or fragment(s) thereof with a test agent; and identifying an agent that alters the processing and/or cleavage of LRP and/or fragment(s) thereof. The processing and/or cleavage of LRP and/or fragment(s) thereof can be assessed by determining the presence, absence and/or level of one or more fragments of LRP and/or the composition of LRP. hi one embodiment, the step of identifying comprises comparing the cleavage and/or processing of LRP and/or fragment(s) thereof in a test sample that has been contacted with the test agent and a control sample that has not been contacted with the test agent and identifying an agent as an agent that alters the processing and/or cleavage of LRP and/or fragment(s) thereof if the processing and/or cleavage of LRP and/or fragment(s) thereof differs in the test and control samples. The control sample can be the test sample in the absence of test agent. In certain embodiments, the processing or cleavage of LRP and/or fragment(s) thereof is assessed by determining the presence or absence and/or level of an LRP fragment that has a molecular weight of about 20 kD.

The LRP fragment that has a molecular weight of about 20 kD can contain an amino acid sequence that is contained within a transmembrane region of LRP. In another embodiment, the LRP fragment that has a molecular weight of about 20 kD can bind with an antibody generated against a C-terminal amino acid sequence of an LRP. The C- terminal amino acid sequence of LRP can be a sequence of about the C-terminal 13 amino acids of an LRP. hi another embodiment, the LRP fragment that has a molecular weight of about 20 kD comprises an amino acid sequence located within the sequence of amino acids from about amino acid 4420 to about amino acid 4544 of SEQ ID NO: 10. The LRP fragment that has a molecular weight of about 20 kD can be present when an LRP is not cleaved by a presenilin-dependent activity; or can be in the presence of an inhibitor of a presenilin-dependent activity. In a particular embodiment, the inhibitor is DAPT.

In particular embodiments of the methods provided herein, the processing and/or cleavage of LRP or fragment(s) thereof can be assessed by determining the presence or absence and/or level of an LRP C-terminal fragment (CTF). The processing and/or cleavage of LRP and/or fragment(s) thereof is assessed by determining the presence or absence and/or level of a fragment of LRP that binds to an antibody. The antibody can bind to an epitope in the C-terminal about 13 amino acids of an LRP, and can be a polyclonal antibody. The sample can comprise a composition selected from the group consisting of a cell, a tissue, an organism, a cell or tissue lysate, a cell or tissue extract, a body fluid, a cell membrane or composition containing cell membranes, and a cell-free extract or other cell-free sample. In particular embodiments, the cell can contain presenilin, LRP and/or fragment(s) of presenilin and/or LRP. The cell can be either eukaryotic, mammalian, rodent or a human cell. Also provided herein are methods for identifying a candidate agent for treatment or prophylaxis of a disease associated with an altered presenilin, comprising contacting a sample that contains an altered presenilin and/or fragment(s) thereof and a lipoprotein receptor-related protein (LRP) and/or fragment(s) thereof with a test agent, wherein the altered presenilin and/or fragment(s) thereof is associated with an altered cleavage and/or processing of LRP and/or fragment(s) thereof; and identifying a candidate agent that restores LRP cleavage and/or processing to substantially that which occurs in the presence of a presenilin and/or fragment(s) thereof that is not associated with an altered cleavage and/or processing of LRP and/or fragment(s) thereof. The presenilin and/or fragment(s) thereof can comprise a mutation, and can be altered relative to a wild-type presenilin, wherein the wild-type is a predominant allele. The wild-type presenilin can be one that occurs in an organism that exhibits normal presenilin-dependent LRP processing patterns. The disease can be an amyloidosis-associated disease; a neurodegenerative disease, such as Alzheimer's Disease. The mutation can be linked to familial Alzheimer's disease. In one embodiment of the methods provided herein, LRP cleavage and/or processing is assessed by determining the presence or absence and/or level of one or more fragments of LRP and/or the composition of LRP. The step of identifying can comprise comparing the cleavage and/or processing of LRP and/or fragment(s) thereof in a test sample that has been contacted with the test agent and a control sample that has not been contacted with the test agent and identifying an agent as a candidate agent that restores LRP cleavage and/or processing if the cleavage and/or processing of LRP and/or fragment(s) thereof differs in the test and control samples; or is substantially similar; wherein the positive control sample contains LRP and/or fragment(s) thereof and a presenilin and/or fragment(s) thereof that is not associated with an altered processing of LRP. The presenilin and/or fragment(s) thereof in the positive control sample can be a wild-type presenilin. The cleavage or processing of LRP and/or fragment(s) thereof is assessed by determining the presence or absence and/or level of an LRP fragment that has a molecular weight of about 20 kD. The LRP fragment that has a molecular weight of about 20 kD can contain an amino acid sequence that is contained within a transmembrane region of LRP; or can bind with an antibody generated against a C- terminal amino acid sequence of an LRP. The C-terminal amino acid sequence of LRP is a sequence of about the C-terminal 13 amino acids of an LRP. The LRP fragment that has a molecular weight of about 20 kD can comprise an amino acid sequence located within the sequence of amino acids from about amino acid 4420 to about amino acid 4544 of SEQ ID NO: 10; can be one that is present when an LRP is not cleaved by a presenilin-dependent activity; or can occur in the presence of an inhibitor of a presenilin- dependent activity. In one embodiment, the inhibitor is DAPT. The LRP processing can be assessed by determining the presence or absence and/or level of an LRP C-terminal fragment (CTF); or by determining the presence or absence and/or level of a fragment of LRP that binds to an antibody. The antibody can bind to an epitope in the C-terminal about 13 amino acids of an LRP, and can be a polyclonal antibody. The sample can comprise a composition selected from the group consisting of a cell, a tissue, an organism, a cell or tissue lysate, a cell or tissue extract, a body fluid, a cell membrane or composition containing cell membranes, and a cell-free extract or other cell-free sample. In one embodiment, the sample can comprise a cell that contains the presenilin, LRP and/or fragment(s) of presenilin and/or LRP. The cell can be eukaryotic, mammalian, rodent or a human cell.

Also provided herein are methods for modulating LRP, comprising altering the structure, function and/or activity of a presenilin, and/or fragment(s) thereof, in a sample comprising LRP, and/or fragment(s) thereof, and a presenilin, and/or fragment(s) thereof, whereby the LRP is modulated. In another embodiment, provided herein are methods for modulating LRP, comprising contacting a sample comprising an LRP, and/or fragment(s) thereof, and presenilin, and/or fragment(s) thereof, with an agent that modulates the presenilin and/or fragment(s) thereof or a presenilin-dependent activity, whereby LRP is modulated. In these methods the cleavage, processing, structure, function and/or activity of LRP can be modulated. The method can further comprise selecting a sample for modulation of LRP. The sample can comprise a composition selected from the group consisting of a cell, tissue, organism, cell or tissue lysate, cell or tissue extract, a cell membrane, a membrane preparation from a cell and a cell-free sample. Also provide herein are methods for identifying an agent that modulates A/342 levels, comprising comparing the levels of bound antibody and/or fragment(s) thereof in a test sample contacted with the test agent and a control sample not contacted with the test agent; and identifying an agent as an agent that modulates A/342 levels if the levels of bound antibody differ in the test and control samples; wherein the sample comprises APP or portion(s) thereof; and the antibody and/or fragment(s) thereof comprises a sequence of amino acids selected from the group consisting of amino acids 1-50, 1-60, 1-70, 1-80, 1-90, 1-91, 1-92, 1-93, 1-94 and 1-95 of SEQ ID NO: 12 and any amino acid sequences containing modifications of these amino acid sequences that retain the antigen-binding properties of an antibody comprising one or both of the amino acid sequences set forth as amino acids 1-95 of SEQ ID NO: 12 and amino acids 1-97 of SEQ ID NO: 14. In another embodiment, provided herein are methods for identifying an agent that modulates A/342 levels, comprising comparing the levels of bound antibody and/or fragment(s) thereof in a test sample contacted with the test agent and a control sample not contacted with the test agent; and identifying an agent as an agent that modulates A/342 levels if the levels of bound antibody differ in the test and control samples; wherein the sample comprises APP or portion(s) thereof; and the antibody and/or fragment(s) thereof comprises a sequence of amino acids selected from the group consisting of amino acids 1-50, 1-60, 1-70, 1-80, 1-90, 1-91, 1-92, 1-93, 1-94, 1-95, 1-96 and 1-97 of SEQ ID NO: 14 and any amino acid sequences containing modifications of these amino acid sequences that retain the antigen-binding properties of an antibody comprising one or both of the amino acid sequences set forth as amino acids 1-95 of SEQ ID NO: 12 and amino acids 1-97 of SEQ ID NO: 14. In these methods, the antibody comprising one or both of the amino acid sequences set forth as amino acids 1-95 of SEQ ID NO: 12 and amino acids 1-97 of SEQ ID NO: 14 can be an IgG. The antibody and/or fragment(s) thereof can bind A/342 without substantially binding other Aβ forms, such as A/340. The antibody and/or fragment(s) thereof can have at least about 100-fold, 200- fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1000-fold or more specificity or selectivity for A/342 relative to other forms of A/3, such as A/340. In addition, the antibody and/or fragment(s) thereof can have an affinity constant for binding to A,842 of at least about 10⁵ 1/mol, 2 x 10⁵ 1 mol, 3 x 10⁵ 1/mol, 4 x 10⁵ 1/mol, 5 x 10⁵1/mol, 6 x 10⁵ 1/mol, 7 x 10⁵1/mol, 8 x 10⁵1/mol, 9 x 10⁵1/mol, 10⁶1/mol, 2 x 10⁶ 1/mol, 3 x 10 1/mol or 4 x 10 1/mol or more. In one embodiment, the agent identified as an agent that modulates A/342 levels can reduce A/342 levels. The concentration of test agent can be less than or equal to about 35 μM, 30 μM, 25 μM, 20 μM, 15 μM or 10 μM. The step of identifying an agent as an agent that modulates A/342 levels can comprise identifying an agent that reduces A/342 levels with and ICso of about 25 μM or less or about 20 μM or less.

Also provided herein are methods for identifying an agent that modulates A/3 levels, comprising assessing a test agent that modulates A/342 levels to determine if it modulates the level of one or more other Aβ peptides; and identifying an agent that modulates A/342 levels to a greater extent than it modulates the level of one or more other A/3 peptides. The step of identifying can comprise identifying an agent that modulates A/342 levels without substantially altering the level of one or more other Aβ peptides, such as A/340. The step of identifying can comprise identifying an agent that modulates A/342 levels to a greater extent than it modulates the level of A/340. In one embodiment, the test agent reduces A/342 levels. In another embodiment, the test agent increases A/342 levels.

Further provided herein are methods for identifying an agent that modulates A/3 levels, comprising assessing a test agent that modulates A/342 levels to determine if it modulates the level of one or more other Aβ peptides; and identifying an agent that modulates A/342 levels and A/339 levels. The test agent can reduce A/342 levels or can increase A/342 levels. The step of identifying can comprise identifying an agent that increases A/339 or that reduces A/339, h another embodiment, the step of identifying can comprise identifying an agent that modulates A/342 levels and A/339 levels to a greater extent than it modulates A/340 levels. The step of identifying can comprise identifying an agent that modulates A/342 levels and A/339 without substantially altering the level of A/340. In one embodiment, the step of assessing a test agent can comprise comparing the levels of one or more A/3 peptides other than A/342 in a test sample that has been contacted with the test agent and in a control sample that has not been contacted with the test agent; and the step of identifying comprises identifying an agent as an agent that modulates A/342 levels to a greater extent than it modulates the level of one or more other A/3 peptides if the difference in the levels of one or more Aβ peptides other than A/342 in the test and control samples is less than the difference in the A/342 levels of the test and control samples. In another embodiment, the step of assessing a test agent can comprise comparing the levels of one or more A/3 peptides other than A/342 in a test sample that has been contacted with the test agent and in a control sample that has not been contacted with the test agent; and the step of identifying comprises identifying an agent as an agent that modulates A/342 levels to a greater extent than it modulates the level of one or more other A/3 peptides if the levels of one or more A/3 peptides other than A/342 in the test and control samples are substantially unchanged. In another embodiment, the step of assessing a test agent can comprise comparing the levels of A/339 in a test sample that has been contacted with the test agent and in a control sample that has not been contacted with the test agent; and the step of identifying comprises identifying an agent as an agent that modulates A/342 levels and A/339 levels if A/339 levels in the test and control samples differ. Likewise, the step of identifying can comprise identifying an agent as an agent that modulates A/342 levels and A/339 levels to a greater extent than it modulates the level of A 340 if the difference in the levels of A/340 in a test sample that has been contacted with the test agent and a control sample that has not been contacted with a test agent is less than the difference in the A/342 and A/339 levels of the test and control samples. The step of identifying can also comprise identifying an agent as an agent that modulates Aj842 levels and A/339 levels to a greater extent than it modulates the level A/340 if the levels of A/340 in test sample that has been contacted with test agent and a control sample that has not been contacted with test agent are substantially unchanged. The methods can further comprise a step of identifying the test agent as an agent that modulates A/342 levels; wherein the step of identifying the test agent as an agent that modulates A/342 levels is performed prior to or simultaneously with the step of assessing the test agent; and if the test agent is identified as an agent that modulates A/342 levels simultaneously with the step of assessing the test agent, then the step of assessing includes determining if the test agent modulates the level of A/342. The step of identifying the test agent as an agent that modulates Aj842 levels can comprise comparing the levels of A/342 in a test sample that has been contacted with the test agent and in a control sample that has not been contacted with the test agent; and identifying a test agent as an agent that modulates A/342 levels if the levels of A/342 in the test and control samples differ. The levels of A/342 in the samples are assessed in a method comprising an immunoassay wherein an antibody and/or fragment(s) thereof that bind A/342 without substantially binding other Aβ forms is used. The antibody and/or fragment(s) thereof can be at least about 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1000-fold or more specificity or selectivity for A/342 relative to other forms of Aβ, such as A/340. The antibody and/or fragment(s) thereof bind A/342 without substantially binding A/340. In one embodiment, the test agent can reduce A/342 levels. In the step of identifying the test agent as an agent that modulates A/342 levels, the concentration of test agent can be less than or equal to about 35 μM, 30 μM, 25 μM, 20 μM, 15 μM or 10 μM. The step of identifying the test agent as an agent that modulates A/342 levels can comprise identifying an agent that reduces A/342 levels with and IC50 of about 25 μM or less or about 20 μM or less. The sample can comprise APP and/or portion(s) thereof. In other embodiments, the sample can comprise a composition selected from the group consisting of a cell, a tissue, an organism, a cell or tissue lysate, a cell or tissue extract, a body fluid, a cell membrane or composition containing cell membranes, and a cell-free extract or other cell-free sample. The sample can comprise a cell. The Aβ can be a cellular and/or extracellular A/3.

Also provided herein are methods for modulating A/3 levels of a sample, comprising altering the A/3 peptide-producing cleavage of APP, the processing of APP, the processing of A/3 and/or the levels of A/3 such that the level of A/342 is modulated to a greater extent than the level of one or more other Aβ peptides is modulated. Also provided are methods for modulating A/3 levels of a sample, comprising contacting a sample comprising APP and/or a portion(s) thereof with an agent that modulates the level of A/342 to a greater extent than the level of one or more other Aβ peptides. The level of A/342 can be modulated without substantially altering the level of one or more other A/3 peptides. The level of A/342 can be modulated to a greater extent than the level of A/340. The level of A/342 is modulated without substantially altering the level of A/340. The level of A/342 can be reduced or increased. The level of A/342 and the level of A/339 can be modulated to a greater extent than the level of one or more other A/3 peptides, such as A/340. The level of A/342 and the level of A/339 can be modulated without substantially altering the level of one or more other A/3 peptides, such as A/340. In particular embodiments, the level of A/342 is reduced; the level of A/339 is increased; or the level of A/342 is increased. The concentration of the agent can be less than or equal to about 35 μM, 30 μM, 25 μM, 20 μM, 15 μM or 10 μM. The sample can comprise APP and/or portion(s) thereof. The A/3 can be a cellular and/or extracellular A/3. Also provided herein are methods for identifying an agent that modulates Aβ levels, comprising assessing a test agent that alters the cleavage of APP that produces one or more Aβ peptides, the processing of APP, the processing of A/3 and/or the level of one or more Aβ peptides to determine if it effects one or more presenilin-dependent activities other than the presenilin-dependent processing of APP or portion(s) thereof; and identifying an agent that modulates Aβ levels without substantially altering one or more presenilin-dependent activities other than the presenilin-dependent processing of APP.

In the methods provided herein, the test agent can modulate A/342 levels, such as to a greater extent than it modulates the levels of other Aβ peptides; without substantially altering the level of one or more other Aβ peptides; to a greater extent than it modulates the levels of A/340; or without substantially altering the level of A/340. In another embodiment, the test agent can modulate A/342 and A/339 levels, such as to a greater extent than it modulates the levels of other A/3 peptides; without substantially altering the level of one or more other Aβ peptides; to a greater extent than it modulates the levels of A/340; or without substantially altering the level of A/340. In these methods, the step of assessing a test agent can comprise comparing one or more presenilin-dependent activities other than the presenilin-dependent processing of APP and/or portion(s) thereof in a test sample that has been contacted with the test agent and in a control sample that has not been contacted with the test agent; and the step of identifying comprises identifying an agent that modulates A/3 levels without substantially altering one or more presenilin-dependent activities other than the presenilin-dependent processing of APP if the one or more presenilin-dependent activities other than the presenilin-dependent processing of APP is (are) substantially unchanged in the test and control samples. The presenilin-dependent activity other than presenilin-dependent processing of APP can be the cleavage and/or processing of a substrate, and/or portion(s) thereof, other than APP. The test agent can reduce or increase A/342 levels.

Also provided herein are methods for identifying an agent that modulates Aβ levels, comprising assessing a test agent that modulates the cleavage of APP that produces one or more A/3 peptides, the processing of APP, the processing of Aβ and/or the level of one or more A/3 peptides to determine if it effects the cleavage and/or processing of a presenilin substrate and/or portion(s) thereof other than APP or other than the presenilin-dependent processing of APP or portion(s) thereof; and identifying an agent that modulates A/3 levels without substantially altering the cleavage and/or processing of the presenilin substrate and/or portion(s) thereof that is other than APP. The step of assessing the test agent can comprise comparing (a) the cleavage and/or processing of a presenilin substrate, and/or portion(s) thereof, other than APP, and/or (b) the levels of a fragment(s) of the presenilin substrate and/or portion(s) thereof in a test sample that has been contacted with the test agent and in a control sample that has not been contacted with the test agent; and the step of identifying comprises identifying an agent if the cleavage and/or processing of the presenilin substrate and/or portion(s) thereof and/or the levels of a fragment(s) of the presenilin substrate and/or portion(s) thereof in the test and control samples do not substantially differ. The step of assessing can comprise comparing (a) the cleavage and/or processing of a presenilin substrate, and/or portion(s) thereof, other than APP and/or (b) the levels of a fragment(s) of the presenilin substrate and/or portion(s) thereof in a test sample that has been contacted with the test agent and a positive control sample; and the step of identifying comprises identifying an agent if the cleavage and/or processing of a presenilin substrate, and/or portion(s) thereof, and/or the levels of fragment(s) of the presenilin substrate and/or portion(s) thereof in the test and positive control samples substantially differ; wherein the positive control sample is one that has been contacted with an modulator of presenilin and/or presenilin-dependent activity. The modulator of presenilin and/or presenilin- dependent activity is an inhibitor of presenilin and/or presenilin-dependent activity. The inhibitor can be DAPT. The level of a substrate fragment in the test sample can be less than about 40%, 35%, 30% or 20% of the level of the fragment in the positive control sample. The concentration of test agent is less than or equal to about 35 μM, 30 μM, 25 μM, 20 μM, 15 μM or 10 μM. The sample comprises a presenilin substrate and/or portion(s) thereof; and/or presenilin and/or portion(s) thereof. The method can further comprise a step of identifying the test agent as an agent that modulates Aβ levels; wherein the step of identifying the test agent as an agent that modulates Aj8 levels is performed prior to or simultaneously with the step of assessing the test agent; and if the test agent is identified as an agent that modulates A/3 levels simultaneously with the step of assessing the test agent, then the step of assessing includes determining if the test agent modulates Aβ levels. The step of identifying the test agent as an agent that modulates the cleavage of APP and/or portion(s) thereof that produces one or more A/3 peptides, the processing of APP, the processing of AjS and/or the level of one or more Aβ peptides can comprise comparing the Aβ peptide-producing cleavage of APP, or portion(s) thereof, APP processing, Aβ processing and/or A/3 levels in a test sample containing APP and/or portion(s) thereof that has been contacted with the test agent and in a control sample that has not been contacted with the test agent; and identifying an agent as an agent that modulates A/3 levels if the Aβ peptide-producing cleavage of APP, or portion(s) thereof, APP processing, A/3 processing and/or A/3 levels in the test and control samples differ. The presenilin substrate and/or portion(s) thereof can be selected from the group consisting of LRP, Notch, E-cadherin, Erb-B4, and portions of LRP, Notch, E-cadherin and Erb-B4. The step of assessing the test agent can comprise comparing the levels of an intracellular carboxyl domain fragment of Notch, E-cadherin and/or Erb-B4 in test and control samples; and the step of identifying comprises identifying an agent if the cleavage and/or processing of Notch, E-cadherin and/or Erb- B4 (and/or portion(s) thereof) and/or the levels of an intracellular carboxyl domain fragment of Notch, E-cadherin and/or Erb-B4 in the test and control samples do not substantially differ. The sample can comprise a composition selected from the group consisting of a cell, a tissue, an organism, a cell or tissue lysate, a cell or tissue extract, a body fluid, a cell membrane or composition containing cell membranes, and a cell-free extract or other cell-free sample.

Also provided herein are methods for identifying an agent that modulates A/3 levels, comprising assessing a test agent that modulates the cleavage of APP that produces one or more A/3 peptides, the processing of APP, the processing of Aβ and/or the level of one or more Aβ peptides to determine if it effects the cleavage and/or processing of LRP and/or portion(s) thereof; and identifying an agent that modulates A/3 levels without substantially altering the cleavage and/or processing of LRP and/or portion(s) thereof. The test agent can modulate A/342 levels; modulate A/342 levels to a greater extent than it modulates the levels of other Aβ peptides; modulate A/342 levels without substantially altering the level of one or more other Aβ peptides; modulate A/342 levels to a greater extent than it modulates the levels of A/340; modulate A/342 levels without substantially altering the level of A/340; modulate A/342 and A/339 levels; modulate A/342 and A/339 levels to a greater extent than it modulates the levels of other Aβ peptides; modulate A/342 and A/339 levels without substantially altering the level of one or more other Aβ peptides; modulate A/342 and A/339 levels to a greater extent than it modulates the levels of A/340; modulate A/342 and A/339 levels without substantially altering the level of A/340; reduces A/342 levels; increases A/339 levels. The step of assessing the test agent can comprise comparing (a) the cleavage and/or processing of LRP, and/or portion(s) thereof, and/or (b) the levels of a fragment(s) of LRP and/or portion(s) thereof in a test sample that has been contacted with the test agent and in a control sample that has not been contacted with the test agent; and the step of identifying comprises identifying an agent if the cleavage and/or processing of LRP and/or portion(s) thereof and/or the levels of a fragment(s) of LRP and/or portion(s) thereof in the test and control samples do not substantially differ. The step of assessing can comprise comparing (a) the cleavage and/or processing of LRP, and/or portion(s) thereof, and/or (b) the levels of a fragment(s) of LRP and/or portion(s) thereof in a test sample that has been contacted with the test agent and a positive control sample; and the step of identifying comprises identifying an agent if the cleavage and/or processing of LRP, and/or portion(s) thereof, and/or the levels of fragment(s) of LRP and/or portion(s) thereof in the test and positive control samples substantially differ; wherein the positive control sample is one that has been contacted with an modulator of presenilin and/or presenilin-dependent activity. In one embodiment, the modulator of presenilin and/or presenilin-dependent activity can be an inhibitor of presenilin and/or presenilin- dependent activity. The level of an LRP fragment in the test sample can be less than about 40%, 35%, 30% or 20% of the level of the fragment in the positive control sample. The concentration of test agent can be less than or equal to about 35 μM, 30 μM, 25 μM, 20 μM, 15 μM or 10 μM. The cleavage and/or processing of LRP, and/or portion(s) thereof, and/or the levels of a fragment(s) of LRP and/or portion(s) thereof can be assessed by determining the presence, absence and/or level of one or more fragments of LRP and/or the composition of LRP. The cleavage and/or processing of LRP, and/or portion(s) thereof, and/or the levels of a fragment(s) of LRP and/or portion(s) thereof can be assessed by determining the presence, absence and/or level of an LRP fragment that has a molecular weight of about 20 kD. The LRP fragment that has a molecular weight of about 20 kD can contain an amino acid sequence that is contained within a transmembrane region of LRP; can bind with an antibody generated against a C-terminal amino acid sequence of an LRP, wherein the C-terminal amino acid sequence of LRP is a sequence of about the C-terminal 13 amino acids of an LRP; can comprise an amino acid sequence located within the sequence of amino acids from about amino acid 4420 to about amino acid 4544 of SEQ ID NO: 10; is one that is present when an LRP is not cleaved by a presenilin-dependent activity; is one that occurs in the presence of an inhibitor of a presenilin-dependent activity. The cleavage and/or processing of LRP, and/or portion(s) thereof, and/or the levels of a fragment(s) of LRP and/or portion(s) thereof can be assessed by determining the presence, absence and/or level of an LRP C- terminal fragment (CTF); can be assessed by determining the presence or absence and/or level of a fragment of LRP that binds to an antibody. The sample can comprise LRP and/or portion(s) thereof; or presenilin and/or portion(s) thereof. The method can further comprise a step of identifying the test agent as an agent that modulates Aβ levels; wherein the step of identifying the test agent as an agent that modulates A/3 levels is performed prior to or simultaneously with the step of assessing the test agent; and if the test agent is identified as an agent that modulates Aβ levels simultaneously with the step of assessing the test agent, then the step of assessing includes determining if the test agent modulates Aβ levels. The step of identifying the test agent as an agent that modulates the cleavage of APP and/or portion(s) thereof that produces one or more A/3 peptides, the processing of APP, the processing of A/3 and/or the level of one or more Aβ peptides can further comprise: comparing the A/3 peptide-producing cleavage of APP, or portion(s) thereof, APP processing, A/3 processing and/or A/3 levels in a test sample containing APP and/or portion(s) thereof that has been contacted with the test agent and in a control sample that has not been contacted with the test agent; and identifying an agent as an agent that modulates A/3 levels if the A/3 peptide-producing cleavage of APP, or portion(s) thereof, APP processing, A/3 processing and/or Aβ levels in the test and control samples differ. The sample comprises a composition selected from the group consisting of a cell, a tissue, an organism, a cell or tissue lysate, a cell or tissue extract, a body fluid, a cell membrane or composition containing cell membranes, and a cell-free extract or other cell-free sample. The step of identifying the test agent as an agent that modulates A/3 levels can comprise identifying an agent that reduces A/342 levels in test samples contacted with the test agent by greater than or equal to about 50% compared to the levels of A/342 in a control sample that has not been contacted with the agent. The concentration of the identified agent can be less than or equal to about 35 μM, 30 μM, 25 μM, 20 μM, 15 μM or 10 μM. In one embodiment, the step of identifying an agent that modulates A/3 levels without substantially altering the cleavage and/or processing of LRP and/or portion(s) thereof can comprise identifying an agent if the level of an -20 kD fragment of LRP in a test sample is less than about 20% of the level of the fragment in a positive control sample that has been contacted with an inhibitor of presenilin and/or presenilin-dependent activity. In one embodiment, the A/3 levels are extracellular levels and the LRP fragment levels are cellular levels.

Further provided herein are methods of modulating the A/3 levels of a sample, comprising modulating the cleavage of APP that produces one or more Aβ peptides, the processing of APP, the processing of Aβ and/or the level of one or more A/3 peptides without substantially altering one or more presenilin-dependent activities other than the presenilin-dependent processing of APP. In one embodiment, the modulating can be effected by contacting the sample with an agent that modulates the cleavage of APP that produces one or more A/3 peptides, the processing of APP, the processing of A/3 and/or the level of one or more A/3 peptides without substantially altering one or more presenilin-dependent activities other than the presenilin-dependent processing of APP. Also provided are methods of modulating the A/3 levels of a sample, comprising modulating the cleavage of APP that produces one or more A/3 peptides, the processing of APP, the processing of A/3 and/or the level of one or more A/3 peptides without substantially altering the cleavage and/or processing of a presenilin substrate and/or portion(s) thereof that is other than APP. In one embodiment, the modulating can be effected by contacting the sample with an agent that modulates the cleavage of APP that produces one or more Aβ peptides, the processing of APP, the processing of Aβ and/or the level of one or more A/3 peptides without substantially altering the cleavage and/or processing of a presenilin substrate and/or portion(s) thereof that is other than APP . Further provided are methods of modulating the A/3 levels of a sample, comprising modulating the cleavage of APP that produces one or more Aβ peptides, the processing of APP, the processing of Aβ and/or the level of one or more A/3 peptides without substantially altering the cleavage and/or processing of LRP and/or portion(s) thereof, wherein modulating can be effected by contacting the sample with an agent that modulates the cleavage of APP that produces one or more A/3 peptides, the processing of APP, the processing of Aβ and/or the level of one or more A/3 peptides without substantially altering the cleavage and/or processing of LRP and/or portion(s) thereof. In the above methods, the levels of A/342 can be modulated: to a greater extent than the levels of other A/3 peptides; without substantially altering the level of one or more other A/3 peptides; to a greater extent than the levels of A/340; without substantially altering the level of A/340; and the like. In other embodiments, the levels of A/342 and A/339 can be modulated, such as: to a greater extent than the levels of other A/3 peptides; without substantially altering the level of one or more other A/3 peptides; to a greater extent than the levels of A/340; without substantially altering the level of A/340. In other embodiments, the level of A/342 can be reduced or increased. Likewise, the level of A/339 can be increased or reduced. The sample can comprise presenilin and/or portion(s) thereof; APP and/or portion(s) thereof; a presenilin substrate and/or portion(s) thereof; and the like. The sample can comprise one or more of LRP, Notch, E-cadherin, TrkB, APLP2, hlreloi, Erb-B4, portion(s) of LRP, portion(s) of Notch, portion(s) of E-cadherin, portion(s) of TrkB, portion(s) of APLP2, portion(s) of hlrelo; and portion(s) of Erb-B4. The sample can comprise a composition selected from the group consisting of a cell, a tissue, an organism, a cell or tissue lysate, a cell or tissue extract, a body fluid, a cell membrane or composition containing cell membranes, and a cell-free extract or other cell-free sample. In one embodiment, the sample comprises a cell, such as a eukaryotic, a mammalian, a rodent or a human cell. The A/3 can be a cellular and/or extracellular AjS. In particular embodiments of these methods, the A/342 levels of the sample can be reduced by greater than or equal to about 50%. The presenilin substrate and/or portion(s) thereof can be selected from the group consisting of Notch, E-cadherin, Erb-B4, and portions of Notch, E-cadherin and Erb-B4. In one embodiment, the levels of an intracellular carboxyl domain fragment of Notch, E-cadherin and/or Erb-B4 are substantially unchanged. In another, the level or absence of an -20 kD fragment of LRP is substantially unchanged. In other embodiments, the LRP fragment that has a molecular weight of about 20 kD: can contain an amino acid sequence that is contained within a transmembrane region of LRP; or can bind with an antibody generated against a C-terminal amino acid sequence of an LRP, wherein the C-terminal amino acid sequence of LRP is a sequence of about the C-terminal 13 amino acids of an LRP. In other embodiments, the LRP fragment that has a molecular weight of about 20 kD: can comprise an amino acid sequence located within the sequence of amino acids from about amino acid 4420 to about amino acid 4544 of SEQ ID NO: 10; can be one that is present when an LRP is not cleaved by a presenilin-dependent activity; can be one that occurs in the presence of an inhibitor of a presenilin-dependent activity. The inhibitor can be DAPT. In these methods, the level or absence of an LRP-CTF can be substantially unchanged. The concentration of agent can be less than or equal to about 35 μM, 30 μM, 25 μM, 20 μM, 15 μM or 10 μM. In a particular embodiment, the agent reduces A/342 levels with an IC50 of about 25 μM or less or about 20 μM or less.

Also provided herein are methods for treating or preventing a disease or disorder, comprising administering to a subject an agent that modulates the A/3 peptide-producing cleavage of APP, the processing of APP, the processing of A/3 and/or the levels of A/3 such that the level of A/342 is modulated to a greater extent than the level of one or more other A/3 peptides is modulated. The level of A/342 can be modulated: without substantially altering the level of one or more other A/3 peptides; to a greater extent than the level of AjS40; without substantially altering the level of A/340. In one embodiment, the level of A/342 is reduced. In other embodiments, the level of A/342 and the level of A/339 can be modulated to a greater extent than the level of one or more other A/3 peptides; to a greater extent than the level of A/340; without substantially altering the level of one or more other A/3 peptides; without substantially altering the level of A/340. The level of A/342 can be reduced or increased.

Also provided herein are methods for treating or preventing a disease or disorder, comprising administering to a subject an agent that modulates the A/3 peptide-producing cleavage of APP, the processing of APP, the processing of A/3 and/or the levels of A/3 without substantially altering one or more presenilin-dependent activities other than the presenilin-dependent processing of APP. Also provided are methods for treating or preventing a disease or disorder, comprising administering to a subject an agent that modulates the A 3 peptide-producing cleavage of APP, the processing of APP, the processing of A/3 and/or the levels of Aβ without substantially altering the cleavage and/or processing of a presenilin substrate and/or portion(s) thereof that is other than APP.

Also provided herein are methods for treating or preventing a disease or disorder, comprising administering to a subject an agent that modulates the A/3 peptide-producing cleavage of APP, the processing of APP, the processing of Aβ and/or the levels of A/3 without substantially altering the cleavage and/or processing of LRP and/or portion(s) thereof. The level of A/342 can be modulated: to a greater extent than the levels of other AjS peptides; to a greater extent than the levels of other A/3 peptides; without substantially altering the level of one or more other A/3 peptides; to a greater extent than the level of A/340; without substantially altering the level of A/340. In one embodiment, the level of A/342 is reduced. In other embodiments, the level of A/342 and the level of A/339 can be modulated: to a greater extent than the level of one or more other AjS peptides; to a greater extent than the level of A/340; without substantially altering the level of one or more other A/3 peptides; without substantially altering the level of A/340. The level of A/342 can be reduced or increased. The presenilin substrate and/or portion(s) thereof can be selected from the group consisting of Notch, E-cadherin, Erb- B4, and portions of Notch, E-cadherin and Erb-B4.

With respect to any of the methods provided herein for treating a disease or disorder, the disease or disorder can be one characterized by altered A/3 production, catabolism, processing and/or levels. The disease or disorder can be one associated with amyloidosis, can be a neurodegenerative disease, and in a particular embodiment, is Alzheimer's disease.

Also provided are systems for use in assessing presenilin activity, comprising a source of presenilin activity; a source of LRP protein; and a reagent for determining LRP protein composition. The reagent for determining LRP protein composition: can bind to LRP protein or a fragment of an LRP protein; can be an antibody or portion of an antibody that binds to LRP; can bind to a C-terminal portion of LRP; can bind to an -20 kD fragment of LRP. The LRP fragment that has a molecular weight of about 20 kD; can contain an amino acid sequence that is contained within a transmembrane region of LRP; can bind with an antibody generated against a C-terminal amino acid sequence of an LRP. The C-terminal amino acid sequence of LRP can be a sequence of about the C- terminal 13 amino acids of an LRP. In other embodiments, the LRP fragment that has a molecular weight of about 20 kD: can comprise an amino acid sequence located within the sequence of amino acids from about amino acid 4420 to about amino acid 4544 of SEQ ID NO: 10; can be one that is present when an LRP is not cleaved by a presenilin- dependent activity; can be one that occurs in the presence of an inhibitor of a presenilin- dependent activity. The inhibitor can be DAPT. The source of a presenilin activity can be selected from the group consisting of a cell comprising a presenilin, an extract of a cell comprising a presenilin and medium comprising a presenilin.

Also provided herein are antibodies or fragments thereof comprising the sequence of amino acids 1-95 as set forth in SEQ ID NO: 12 and/or the sequence of amino acids 1- 97 as set forth in SEQ ID NO: 14. The antibody or fragment thereof can comprise a light chain variable region that comprises the sequence of amino acids 1-95 as set forth in SEQ ID NO: 12. In another embodiment, the antibody or fragment thereof can comprise a light chain variable region that comprises the sequence of amino acids selected from the group consisting of 1-50, 1-60, 1-70, 1-80, 1-90, 1-91, 1-92, 1-93, and 1-94 of SEQ ID NO: 12. The antibody or fragment thereof can comprise a heavy chain variable region that comprises the sequence of amino acids 1-97 as set forth in SEQ ID NO: 14. The antibody or fragment thereof can comprise a heavy chain variable region that comprises the sequence of amino acids selected from the group consisting of 1-50, 1-60, 1-70, 1-80, 1-90, 1-91, 1-92, 1-93, 1-94, 1-95, and 1-96 of SEQ ID NO: 14. The antibodies or fragments thereof can further comprise one or more joining regions. In one embodiment, at least one joining region comprises the sequence of amino acids 96- 107 as set forth in SEQ ID NO: 12. The antibody or fragment thereof can further comprise one or more constant regions, wherein at least one constant region is a mouse constant region. The mouse constant region can comprise the sequence of amino acids selected from the group consisting of SEQ ID NOs: 63 and 65. In another embodiment, the at least one constant region is a human constant region. The human constant region can comprise the sequence of amino acids as set forth in SEQ ID NO: 81. The at least one joining region can comprise the sequence of amino acids 98-118 as set forth in SEQ ID NO: 14. In this embodiment, the antibody or fragment thereof can further comprise one or more constant regions. The at least one constant region can be a mouse constant region. The mouse constant region can comprise the sequence of amino acids selected from the group consisting of SEQ ID NOs: 69 and 71. The at least one constant region can be a human constant region. The human constant region can comprise the sequence of amino acids selected from the group consisting of SEQ ID NOs: 83, 85 and 87. The at least one joining region can comprise a mouse joining region. The mouse joining region can comprise the sequence of amino acids selected from the group consisting of SEQ ID NOs: 46, 48, 50, 52, 54, 55, 57, 59, 61 and 67. The antibody or fragment thereof can further comprise one or more constant regions, wherein at least one constant region is a mouse constant region. The mouse constant region can comprise the sequence of amino acids selected from the group consisting of SEQ ID NOs: 63, 65, 69 and 71. The at least one constant region can be a human constant region. The human constant region can comprise the sequence of amino acids selected from the group consisting of SEQ ID NOs: 81, 83, 85 and 87. In another embodiment, the at least one joining region can comprise a human joining region. The human joining region can comprise the sequence of amino acids selected from the group consisting of SEQ ID NOs: 73, 75, 77, 79, 89 and 91. The antibody or fragment thereof can further comprise one or more constant regions, wherein at least one constant region is a mouse constant region. The mouse constant region can comprise the sequence of amino acids selected from the group consisting of SEQ ID NOs: 63, 65, 69 and 71. The at least one constant region can be a human constant region. The human constant region can comprise the sequence of amino acids selected from the group consisting of SEQ ID NOs: 81, 83, 85 and 87. Also provide herein is an antibody or fragment thereof encoded by the sequence of nucleic acids as set forth in SEQ ID NO: 11 and/or the sequence of nucleic acids as set forth in SEQ ID NO: 13. The antibody or fragment thereof can comprise: a light chain variable region encoded by the sequence of nucleic acids as set forth in SEQ ID NO: 11; or a heavy chain variable region encoded by the sequence of nucleic acids as set forth in SEQ ID NO: 13.

Also provided herein is an antibody or fragment thereof comprising the sequence of amino acids as set forth in SEQ ID NO: 97 and/or the sequence of amino acids as set forth in SEQ ID NO: 98. In embodiment, the antibody reacts with A/342 with an affinity of at least about 4 x 10 1/mol. In another embodiment, the antibody reacts with A/342 with an affinity of at least about 10 1/mol, or 10 1/mol or 10 1/mol. The antibody or fragment thereof can comprise at least a portion of the antigen-binding region of the antibody, wherein the portion binds to the same antigenic determinant as the antibody with an affinity of at least about 1%, 5%, 10%, 50%, 70%, 80% or 100% of the entire antibody. Further provided is an antibody or fragment thereof comprising the sequence of amino acids 1-100 as set forth in SEQ ID NO: 16 and/or the sequence of amino acids 1-98 as set forth in SEQ ID NO: 18. The antibody or fragment thereof can comprise a light chain variable region that comprises the sequence of amino acids 1-100 as set forth in SEQ ID NO: 16. The antibody or fragment thereof can comprise a light chain variable region that comprises the sequence of amino acids selected from the group consisting of 1-50, 1-60, 1-70, 1-80, 1-90, 1-95, 1-96, 1-97, 1-98, and 1-99 of SEQ ID NO: 16. The antibody or fragment thereof can comprise a heavy chain variable region that comprises the sequence of amino acids 1-98 as set forth in SEQ ID NO: 18. The antibody or fragment thereof can comprise a heavy chain variable region that comprises the sequence of amino acids selected from the group consisting of 1-50, 1-60, 1-70, 1-80, 1-90, 1-91, 1-92, 1-93, and 1-94, 1-95, 1-96, and 1-97 of SEQ ID NO: 18. The antibody or fragment thereof can further comprise one or more joining regions, wherein at least one joining region comprises the sequence of amino acids 101-112 as set forth in SEQ ID NO: 16. The antibody or fragment thereof can further comprise one or more constant regions. At least one constant region can be a mouse constant region. The mouse constant region can comprise the sequence of amino acids selected from the group consisting of SEQ ID NOs: 63 and 65. In another embodiment, at least one constant region is a human constant region. The human constant region can comprise the sequence of amino acids as set forth in SEQ ID NO: 81. In another embodiment, at least one joining region can comprise the sequence of amino acids 99-114 as set forth in SEQ ID NO: 14. The antibody or fragment thereof can further comprise one or more constant regions, wherein at least one constant region is a mouse constant region. The mouse constant region can comprise the sequence of amino acids selected from the group consisting of SEQ ID NOs: 69 and 71. In another embodiment, at least one constant region can be a human constant region. The human constant region can comprise the sequence of amino acids selected from the group consisting of SEQ ID NOs: 83, 85 and 87. In one embodiment, at least one joining region comprises a mouse joining region. The mouse joining region can comprise the sequence of amino acids selected from the group consisting of SEQ ID NOs: 46, 48, 50, 52, 54, 55, 57, 59, 61 and 67. The antibody or fragment thereof can further comprise one or more constant regions. In another embodiment, at least one constant region can be a mouse constant region. The mouse constant region can comprise the sequence of amino acids selected from the group consisting of SEQ ID NOs: 63, 65, 69 and 71. In another embodiment, at least one constant region is a human constant region. The human constant region can comprise the sequence of amino acids selected from the group consisting of SEQ ID NOs: 81, 83, 85 and 87. At least one joining region can comprise a human joining region. The human joining region can comprise the sequence of amino acids selected from the group consisting of SEQ ID NOs: 73, 75, 77, 79, 89 and 91. The antibody or fragment thereof can further comprise one or more constant regions, wherein at least one constant region is a mouse constant region or a human constant region. The mouse constant region can comprise the sequence of amino acids selected from the group consisting of SEQ ID NOs: 63, 65, 69 and 71. The human constant region can comprise the sequence of amino acids selected from the group consisting of SEQ ID NOs: 81, 83, 85 and 87.

Also provided herein is an antibody or fragment thereof encoded by the sequence of nucleic acids as set forth in SEQ ID NO: 15 and/or the sequence of nucleic acids as set forth in SEQ ID NO: 17. The antibody or fragment thereof can comprise a light chain variable region encoded by the sequence of nucleic acids as set forth in SEQ ID NO: 15 and/or a heavy chain variable region encoded by the sequence of nucleic acids as set forth in SEQ ED NO: 17. Also provided is an antibody or fragment thereof comprising the sequence of amino acids as set forth in SEQ ID NO: 99 and/or the sequence of amino acids as set forth in SEQ ID NO: 100.

Also provided herein is a protein or fragment thereof comprising the sequence of amino acids 1-95 as set forth in SEQ ID NO: 12 and/or the sequence of amino acids 1-97 as set forth in SEQ ID NO: 14. Further provided herein is a protein or fragment thereof comprising the sequence of amino acids as set forth in SEQ ID NO: 97 and/or the sequence of amino acids as set forth in SEQ ID NO: 98. Also provided is a protein or fragment thereof comprising the sequence of amino acids 1-100 as set forth in SEQ ID NO: 16 and/or the sequence of amino acids 1-98 as set forth in SEQ ID NO: 18. Further provided is a protein or fragment thereof comprising the sequence of amino acids as set forth in SEQ ID NO: 99 and/or the sequence of amino acids as set forth in SEQ ID NO: 100. Also provided herein is an isolated nucleic acid molecule that encoding these proteins. Also provided are isolated nucleic acid molecules that encode the antibodies provided herein.

Also provided herein are assays for determining the A/342 content of a sample, comprising contacting an antibody or fragment thereof provided herein with the sample under conditions whereby the antibody forms complexes with AjS; and determining if the antibody or fragment thereof binds to a molecule in the sample. The Aβ can be A|842. The assay can be an enzyme-linked immunosorbant assay (ELISA). The antibody can be a capture antibody. The binding of the antibody or fragment thereof to a molecule in the sample can be determined by contacting the complex with a second antibody or fragment thereof, such as, for example an antibody or fragment thereof provided herein that contains the sequence of amino acids 1-100 of SEQ ID NO: 16 and/or the sequence of amino acids 1-98 of SEQ ID NO: 18.

Also provided herein is a kit containing a reagent for assessing cleavage of APP that produces one or more A/3 peptides, APP processing, A/3 processing and/or Aβ levels and a reagent for assessing cleavage and/or processing of a presenilin substrate. In one embodiment, the presenilin substrate is LRP and/or portion(s) thereof. The reagent for assessing A/3 levels can be, for example, an antibody and/or fragment(s) thereof that specifically react with A/342, such as any of the A/342 specific antibodies provided herein. A reagent for assessing A/3 levels can include an antibody and/or fragment(s) thereof that reacts with two or more or most Aβ peptides, such as antibodies provided herein that contains the sequence of amino acids 1 - 100 of SEQ ID NO : 16 and/or the sequence of amino acids 1-98 of SEQ ID NO: 18. The reagent for assessing cleavage and/or processing of LRP can be an antibody and/or fragment(s) thereof that recognizes a fragment of LRP. The antibody can be one that prepared against the carboxyl-terminal 13 amino acid peptide of LRP (C-GRGPEDEIGDPLA). The LRP fragment can be one that is generated by a presenilin-dependent activity or a fragment that occurs in the absence of such activity. The fragment can have a molecular weight of about 20 kD. Also provided is a method for identifying a candidate agent for the treatment or prophylaxis of a disease that includes steps of (a) contacting a sample that contains an altered test protein, and/or portion(s) thereof, and APP, and/or portion(s) thereof, with a test agent, wherein the altered protein is associated with altered A/342 production, catabolism, processing and/or A/342 levels; and (b) identifying a candidate agent that restores A/3 production, catabolism, processing and/or A/3 levels to substantially that which occurs in the presence of a test protein and/or portion(s) thereof that is not associated with altered A/342 production, catabolism, processing and/or A/342 levels without substantially altering the level of one or more other A/3 peptides. The method can be one wherein the candidate agent restores A/3 production, catabolism, processing and/or A/3 levels to substantially that which occurs in the presence of a test protein and/or portion(s) thereof that is not associated with altered A/342 production, catabolism, processing and/or A/342 levels without substantially altering the level of A/340. The method can be one wherein the candidate agent reduces the level of A/342 and/or increases A/339 levels. In one embodiment, the step of identifying a candidate agent comprises comparing AjS production, catabolism, processing and/or A/3 levels in a test sample that has been contacted with test agent and a control sample that has not been contacted with test agent and identifying an agent if A/3 production, catabolism, processing and/or A/3 levels in the test sample is such that A/342 levels differ in the test and control samples and the level of one or more other A/3 peptides is substantially unchanged in the test and control samples. The level of A/340 can be substantially unchanged in the test and control samples. The level of A/342 can be reduced in the test sample relative to the control sample. The level of A/339 can be increased. In another embodiment, the step of identifying comprises comparing A/3 production, catabolism, processing and/or A/3 levels in a test sample that has been contacted with the test agent and a positive control sample and identifying an agent as a candidate agent Aβ production, catabolism, processing and/or A/3 levels if A/3 production, catabolism, processing and/or AjS levels in the test and control samples is substantially similar; wherein the positive control sample contains test protein and/or portion(s) thereof that is not associated with altered A/342 production, catabolism, processing and/or A/342 levels. Another method provided herein for identifying a candidate agent for the treatment or prophylaxis of a disease includes steps of contacting a sample that contains an altered test protein, and/or portion(s) thereof, and APP, and/or portion(s) thereof, with a test agent, wherein the altered protein is associated with altered A/3 production, catabolism, processing and/or A/3 levels; and identifying a candidate agent that restores A/3 production, catabolism, processing and/or A/3 levels to substantially that which occurs in the presence of a test protein and/or portion(s) thereof that is not associated with altered A/3 production, catabolism, processing and/or Aβ levels without substantially altering (a) one or more presenilin-dependent activities other than the presenilin-dependent processing of APP, (b) the cleavage and/or processing of a presenilin substrate and/or portion(s) thereof that is other than APP and/or (c) the cleavage and/or processing of LRP and/or portion(s) thereof. For example, the candidate agent can restore A/3 production, catabolism, processing and/or A/3 levels to substantially that which occurs in the presence of a test protein and/or portion(s) thereof that is not associated with altered A/3 production, catabolism, processing and/or AjS levels without substantially altering the cleavage and/or processing of Notch, E-cadherin, Erb-B4 and/or portion(s) thereof. The candidate agent can reduce the level of A/342 and/or increase Aj839 levels. In one embodiment, the step of identifying a candidate agent comprises comparing A/3 production, catabolism, processing and/or A/3 levels in a test sample that has been contacted with test agent and a control sample that has not been contacted with test agent and identifying a candidate agent if A/3 production, catabolism, processing and/or A/3 levels in the test sample is such that A/342 levels differ in the test and control samples and one or more of the following is substantially similar in the test and control samples: (a) one or more presenilin-dependent activities other than the presenilin- dependent processing of APP, (b) the cleavage and/or processing of a presenilin substrate and/or portion(s) thereof that is other than APP and/or (c) the cleavage and/or processing of LRP and/or portion(s) thereof. In a particular embodiment, the step of identifying comprises identifying a candidate agent that restores A/3 production, catabolism, processing and/or Aβ levels to substantially that which occurs in the presence of a test protein and/or portion(s) thereof that is not associated with altered AjS production, catabolism, processing and/or A/3 levels without substantially altering the cleavage and/or processing of LRP. The altered protein in these methods can be one that is associated with altered A_/342 production, catabolism, processing and/or A/342 levels; and the method can include identifying a candidate agent that restores A/3 production, catabolism, processing and/or A/342 levels.

In any of the methods provided herein for identifying a candidate agent for the treatment or prophylaxis of a disease, the altered test protein and/or portion(s) thereof can contain a mutation and/or can be altered relative to a wild-type protein, such as a wild- type protein encoded by a predominant allele or that occurs in an organism that exhibits normal A/342 production, catabolism, processing and/or A 342 levels. The mutation can be linked to familial Alzheimer's disease. In particular embodiments, the test protein is an APP or a presenilin. If the test protein is an APP, the APP, and/or portion(s) thereof, that is not an altered test protein does not have to be included in the sample. An altered APP or presenilin can be one that is linked to Alzheimer's disease.

The disease can be, for example, an amyloidosis-associated disease, a neurodegenerative disease, and, in particular, Alzheimer's Disease. For any of the methods, the sample can, for example, comprise a cell or organism. The cell can be, for example, a eukaryotic cell, including a mammalian cell, such as, for example, a rodent or human cell. An organism may be, for example, a non-human transgenic animal.

Also provided are polypeptides comprising a sequence of amino acids that is selectively reactive with AjS 42 and preferentially binds to low molecular weight forms of A/342. The polypeptide can comprise at least one complementarity-determining region (CDR) selected from the group consisting of CDR-Ll, CDR-L2, CDR-L3, CDR-Hl, CDR-H2 or CDR-H3 of antibody A387. In one embodiment, the polypeptide comprises CDR-Ll, CDR-L2, CDR-L3, CDR-Hl, CDR-H2 and CDR-H3 of antibody A387. In another embodiment, the polypeptide comprises at least one CDR selected from the group consisting of amino acids 24-34 of SEQ ID NO: 12, amino acids 50-56 of SEQ ID NO: 12, amino acids 89-97 of SEQ ID NO: 12, amino acids 26-35 of SEQ ID NO: 14, amino acids 31-35 of SEQ ID NO: 14, amino acids 26-31 of SEQ ID NO: 14, amino acids 50-65 of SEQ IDNO:14, amino acids 50-58 of SEQ ID NO: 14, and amino acids 98-107 of SEQ ID NO: 14. The polypeptide can comprise at least a portion of a variable domain of the light chain or the heavy chain of an A/3 antibody. In one embodiment, the variable domain is selected from the group consisting of the light chain variable domain of A387, the heavy chain variable domain of A387, a polypeptide with at least 85% identity to the light chain variable domain of A387; a polypeptide with at least 85% identity to the heavy chain variable domain of A387. The polypeptide can further comprise a scaffold. In one embodiment, the scaffold is a polypeptide scaffold. In one embodiment, the scaffold is a human polypeptide scaffold. In one embodiment, the scaffold is an antibody scaffold. The antibody scaffold can be selected from the group consisting of an Fv fragment scaffold, an Fab fragment scaffold, and a single-chain (scFv) fragment. The polypeptide can further comprise a detectable moiety. The polypeptide can further comprise a clearance domain. The clearance domain can be a ligand for an Fc receptor.

Also provided is a polypeptide, comprising at least one complementarity- determining region (CDR) selected from the group consisting of CDR-Ll, CDR-L2, CDR-L3, CDR-Hl, CDR-H2 or CDR-H3 of antibody A387. In one embodiment, the polypeptide comprises CDR-Ll, CDR-L2, CDR-L3, CDR-Hl, CDR-H2 and CDR-H3 of antibody A387. In one embodiment, the polypeptide comprises at least one CDR selected from the group consisting of amino acids 24-34 of SEQ ID NO: 12, amino acids 50-56 of SEQ ID NO: 12, amino acids 89-97 of SEQ ID NO: 12, amino acids 26-35 of SEQ ID NO: 14, amino acids 31-35 of SEQ ID NO: 14, amino acids 26-31 of SEQ ID NO:14, amino acids 50-65 of SEQ ID NO:14, amino acids 50-58 of SEQ ID NO: 14, and amino acids 98-107 of SEQ ID NO: 14. The polypeptide can also be a chimeric polypeptide. The polypeptide can be an antibody.

The polypeptide can further comprising a clearance domain. The clearance domain can be a ligand for an Fc receptor. The polypeptide can further comprise a detectable moiety. The polypeptide can further comprising a scaffold. In one embodiment, the scaffold comprises a solid support. In another embodiment, the scaffold is a polypeptide scaffold. The scaffold can be a human polypeptide scaffold. The scaffold can be an antibody scaffold. The antibody scaffold can be selected from the group consisting of an Fv fragment scaffold, an Fab fragment scaffold, and a single-chain (scFv) fragment.

The polypeptide can comprise an amino acids 1-95 of SEQ ID NO: 12, or a fragment thereof and/or comprises amino acids 1-97 of SEQ ID NO: 14, or a fragment thereof. Such polypeptides can further comprise one or more joining regions. In one embodiment, the joining region comprises amino acids 96-107 of SEQ ID NO: 12 or amino acids 98-118 of SEQ ID NO: 14. In one embodiment, the joining region comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 46, 48, 50, 52, 54, 55, 57, 59, 61, 67, 73, 75, 77, 79, 89 and 91. The polypeptide can further comprising one or more constant regions. The constant region can be a mouse constant region. In one embodiment, the constant region comprises an amino acid sequence selected from the group consisting of SEQ ID NOS:63, 65, 69 and 71. The constant region can also be a human constant region. In one embodiment, the constant region comprises an amino acid sequence selected from the group consisting of SEQ ID NOS:81, 83, 85 and 87. The polypeptide can comprise the amino acid sequence of SEQ ID NO:97 and/or SEQ ID NO:98. The polypeptide can be specifically reactive with at least one AjS. In one embodiment, A/3 is A/342. In one embodiment, the polypeptide binds A/342 without substantially binding other Aβpeptides.

Also provided is a polypeptide comprising at least one complementarity- determining region (CDR) selected from the group consisting of CDR-Ll, CDR-L2, CDR-L3, CDR-Hl, CDR-H2 or CDR-H3 of antibody B436. In one embodiment, the polypeptide comprisesCDR-Ll, CDR-L2, CDR-L3, CDR-Hl, CDR-H2 and CDR-H3 of antibody B436. In one embodiment, the polypeptide comprises at least one CDR selected from the group consisting of amino acids 24-39 of SEQ ID NO: 16, amino acids 55-61 of SEQ ID NO:16, amino acids 94-102 of SEQ ID NO:16, amino acids 26-35 of SEQ ID NO:18, amino acids 31-35 ofSEQ ID NO:18, amino acids 26-31 ofSEQ ID

NO: 18, amino acids 50-66 of SEQ ID NO: 18, amino acids 50-59 of SEQ ID NO: 18, and amino acids 99-103 of SEQ ID NO:18. The polypeptide can a chimeric polypeptide. The polypeptide can be an antibody.

The polypeptide can further comprise a scaffold. In one embodiment, the scaffold comprises a solid support. In one embodiment, the scaffold is a polypeptide scaffold. The scaffold can be a human polypeptide scaffold. The scaffold can be an antibody scaffold. The antibody scaffold can be selected from the group consisting of an Fv fragment scaffold, an Fab fragment scaffold, and a single-chain (scFv) fragment. The polypeptide can be specifically reactive with at least one A/3 peptide. The polypeptide can further comprise a clearance domain. The clearance domain can be a ligand for an Fc receptor. The polypeptide can further comprise a detectable moiety.

The polypeptide can comprise amino acids 1-100 of SEQ ID NO: 16, or a fragment thereof and/or comprises amino acids 1-98 of SEQ ID NO: 18, or a fragment thereof. Such polypeptides can further comprise one or more joining regions. In one embodiment, the j oining region can comprise amino acids 101 - 112 of SEQ ID NO : 16 or amino acids 99-114 of SEQ ID NO: 18. In one embodiment, the joining region comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 46, 48, 50, 52, 54, 55, 57, 59, 61, 67, 73, 75, 77, 79, 89 and 91. The polypeptide can further comprising one or more constant regions. In one embodiment, the constant region is a mouse constant region. The mouse constant region can comprise an amino acid sequence selected from the group consisting of SEQ ID NOS:63, 65, 69 and 71. In one embodiment, the constant region is a human constant region. The human constant region can comprise an amino acid sequence selected from the group consisting of SEQ ID NOS:81, 83, 85 and 87. The polypeptide can comprise the amino acid sequence of SEQ ID NO:99 and/or SEQ ID NO: 100.

Also provided are nucleic acid molecules encoding polypeptides provided herein. In one embodiment, the nucleic acid molecule encodes a polypeptide comprising a sequence of amino acids that is selectively reactive with Aβ 42 and preferentially binds to low molecular weight forms of A/342. In one embodiment, the nucleic acid molecule encodes a polypeptide comprising at least one complementarity-determining region (CDR) selected from the group consisting of CDR-Ll, CDR-L2, CDR-L3, CDR-Hl, CDR-H2 or CDR-H3 of antibody A387. Also provided are nucleic acid molecules encoding a polypeptide comprising at least one complementarity-determining region (CDR) selected from the group consisting of CDR-Ll , CDR-L2, CDR-L3, CDR-Hl , CDR-H2 or CDR-H3 of antibody B436. Also provided are kits comprising the polypeptides described herein.

Further provided are methods for assessing the presence or amount of Aβ in a sample, comprising contacting a polypeptide provided herein with the sample under conditions whereby a complex is formed between the polypeptide and A/3, and assessing the presence or amount of the complex in the sample, and thereby determining the presence or amount of Aβ in the sample. The sample can be selected from the group consisting of a cell extract, extracellular medium, plasma, cerebrospinal fluid and brain. The presence or amount of the complex can be assessed using an enzyme-linked immunosorbent assay (ELISA).

Also provided are methods comprising administering to a subject a polypeptide provided herein. In one embodiment, the polypeptide comprises at least one complementarity-determining region (CDR) selected from the group consisting of CDR- Ll, CDR-L2, CDR-L3, CDR-Hl, CDR-H2 or CDR-H3 of antibody A387. In one embodiment, the polypeptide is selectively reactive with A/342 and preferentially binds to low molecular weight forms of A/342. h one embodiment, the polypeptide comprises at least one complementarity-determining region (CDR) selected from the group consisting of CDR-Ll, CDR-L2, CDR-L3, CDR-Hl, CDR-H2 or CDR-H3 of antibody B436. In one embodiment, the subject has, or is at risk of developing, a disease associated with accumulation of Aβ. The disease can be Alzheimer's disease.

Also provided are methods of binding A 3 comprising administering to a subject a polypeptide provided herein to bind A/3. In one embodiment, the polypeptide comprises at least one complementarity-determining region (CDR) selected from the group consisting of CDR-Ll, CDR-L2, CDR-L3, CDR-Hl, CDR-H2 or CDR-H3 of antibody A387 wherein the polypeptide is specifically reactive with at least one A/3 peptide. h one embodiment, the polypeptide is selectively reactive with A/342 and preferentially binds to low molecular weight forms of A/342. In one embodiment, the polypeptide comprises at least one complementarity-determining region (CDR) selected from the group consisting of CDR-Ll, CDR-L2, CDR-L3, CDR-Hl, CDR-H2 or CDR-H3 of antibody B436, wherein the polypeptide is specifically reactive with at least one A/3 peptide. h one embodiment, the subject has, or is at risk of developing, a disease associated with accumulation of A/3. The disease can be Alzheimer's disease.

Also provided are methods of reducing A/3 level in an subject, comprising administering to the subject an effective amount of a polypeptide provided herein to reduce the level of at least one Aβ peptide. In one embodiment, the polypeptide comprises at least one complementarity-determining region (CDR) selected from the group consisting of CDR-Ll, CDR-L2, CDR-L3, CDR-Hl, CDR-H2 or CDR-H3 of antibody A387 wherein the polypeptide is specifically reactive with at least one A/3 peptide. In one embodiment, the polypeptide is selectively reactive with A/342 and preferentially binds to low molecular weight forms of A/342. In one embodiment, the polypeptide comprises at least one complementarity-determining region (CDR) selected from the group consisting of CDR-Ll, CDR-L2, CDR-L3, CDR-Hl, CDR-H2 or CDR- H3 of antibody B436, wherein the polypeptide is specifically reactive with at least one Aβ peptide. In one embodiment, the subject has, or is at risk of developing, a disease associated with accumulation of A/3. The disease can be Alzheimer's disease. In one embodiment, the level of at least one Aβ peptide in blood or plasma is reduced. In one embodiment, the level at least one Aβpeptide in brain is reduced.

Also provided are methods for identifying an agent that modulates Aβ levels, comprising comparing the levels of bound Aβ binding protein in a test sample contacted with the test agent and a control sample not contacted with the test agent; and identifying an agent as an agent that modulates A/3 levels if the levels of bound A/3 binding protein differ in the test and control samples; wherein the sample comprises APP or portion(s) thereof. The A/3 binding protein comprises a polypeptide provided herein, hi one embodiment, the polypeptide comprises at least one complementarity-determining region (CDR) selected from the group consisting of CDR-Ll, CDR-L2, CDR-L3, CDR- Hl, CDR-H2 or CDR-H3 of antibody A387 wherein the polypeptide is specifically reactive with at least one Aβ peptide. In one embodiment, the polypeptide is selectively reactive with A/342 and preferentially binds to low molecular weight forms of A 342. In one embodiment, the polypeptide comprises at least one complementarity-determining region (CDR) selected from the group consisting of CDR-Ll , CDR-L2, CDR-L3 , CDR- Hl, CDR-H2 or CDR-H3 of antibody B436, wherein the polypeptide is specifically reactive with at least one Aβ peptide.

Also provided are methods for identifying an agent that modulates A/342 levels, comprising, comparing the levels of bound Aβ binding protein in a test sample contacted with the test agent and a control sample not conlacted^'with the test agent; and identifying an agent as an agent that modulates A/342 levels if the levels of bound Aβ binding protein differ in the test and control samples; wheremthe sample comprises APP or portion(s) thereof; and the Aβ binding protein comprises a sequence of amino acids selected from the group consisting of amino acids 1-50, 1-60, 1-70, 1-80, 1-90, 1-91, 1-92, 1-93, 1-94, 1-95, 1-96 and 1-97 of SEQ ID NO: 12 and 1-50, 1-60, 1-70, 1-80, 1-90, 1-91, 1-92, 1- 93, 1-94, 1-95, 1-96 and 1-97 of SEQ ID NO: 14 and any amino acid sequences containing modifications of these amino acid sequences that retain the Aj3 binding properties of an antibody comprising one or both of the amino acid sequences set forth as amino acids 1-95 of SEQ ID NO: 12 and amino acids 1-97 of SEQ ID NO: 14. Further provided herein are methods in the treatment or prophylaxis of disease involving or characterized by Aβ and/or specific A/3 forms. In one embodiment, the method includes a step of administering a polypeptide provided herein to a subject having such a disease or disorder or predisposed to such a disease or disorder. In one embodiment, the disease is Alzheimer's disease. In one embodiment, A/342 levels are modulated. In one embodiment, the polypeptide is an A/3 binding protein or A/3 antibody. In one embodiment, the polypeptide comprising a sequence of amino acids that is selectively reactive with A/342 and preferentially binds to low molecular weight forms of A/342. DETAILED DESCRIPTION A. DEFINITIONS

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which this invention belongs. All patents, applications, published applications and other publications and sequences from GenBank and other data bases referred to herein are incorporated by reference in their entirety. Where reference is made to a URL or other such identifier or address, it is understood that such identifiers can change and particular information on the internet can come and go, but equivalent information can be found by searching the internet. Reference thereto evidences the availability and public dissemination of such information. As used herein, "Alzheimer's disease" or "AD" refers to a group of visible, detectable or otherwise measurable properties characteristic of AD. Exemplary properties include, but are not limited to, dementia, aphasia (language problems), apraxia (complex movement problems), agnosia (problems in identifying objects), progressive memory impairment, disordered cognitive function, altered behavior, including paranoia, delusions and loss of social appropriateness, progressive decline in language function, slowing of motor functions such as gait and coordination in later stages of AD, amyloid- containing plaques which are foci of extracellular amyloid-/3 (A/3) protein deposition with dystrophic neurites and associated axonal and dendritic injury and microglia expressing surface antigens associated with activation (e.g., CD45 and HLA-DR), diffuse ("preamyloid") plaques and neuronal cytoplasmic inclusions such as neurofibrillary tangles containing hyperphosphorylated tau protein or Lewy bodies (containing - synuclein). Standardized clinical criteria for the diagnosis of AD have been established by NINCDS/ADRDA (National Institute of Neurological and Communicative Disorders and Stroke/Alzheimer's Disease and Related Disorders Association) (McKhann et al. (1984) Neurology 34:939-944). The clinical manifestations of AD as set forth in these criteria are included within the definition of AD. For example, dementia may be established by clinical exam and documented by any of several neuropsychological tests, including the Mini Mental State Exam (MMSE) (Folstein and McHugh (1975) J. Psychiatr. Res. 72:196-198; Cockrell and Folstein (1988) Psychopharm. Bull. 24:689- 692), the Blessed Test (Blessed et al. (1968) Br. J. Psychiatry 774:797-811), and the Alzheimer's Disease Assessment Scale-Cognitive (ADAS-COG) Test (Rosen et al. (1984) Am. J. Psychiatry 747:1356-1364; Weyer et al. (1997) Int. Psychogeriatr. 9:123- 138; and Ihl et al. (2000) Neuropsychobiol. 4:102-107).

As used herein, "amyloidosis" refers to a condition characterized by the presence of amyloid. Amyloid refers to a group of diverse but specific protein deposits observed in a number of different diseases. An example of an amyloid deposit is the /3-amyloid plaque that is a defining pathological hallmark of Alzheimer's disease. The major protein component of the jS-amyloid plaque is the A/3 peptide which is derived from processing of amyloid precursor protein (APP). Amyloid deposits, though diverse in their occurrence, can share some common morphologic properties. Many stain with specific dyes (e.g., Congo red), and have a characteristic red-green birefringent appearance in polarized light after staining. Some share ultrastructural features and common x-ray diffraction and infrared spectra. Amyloidosis can be classified clinically as primary, secondary, familial and/or isolated. Primary amyloid appears de novo without any preceding disorder. Secondary amyloid is that form which appears as a complication of a previously existing disorder. Familial amyloid is a genetically inherited form found in particular geographic populations. Isolated forms of amyloid are those that tend to involve a single organ system.

An amyloidosis-associated disease is a disease involving accumulation of amyloid. Such diseases include, but are not limited to, AD, Down's syndrome, familial amyloid polyneuropathy, familial amyloid cardiomyopathy (Danish type), isolated cardiac amyloid, amyloid angiopathy, systemic senile amyloidosis, idiopathic (primary) amyloidosis, reactive (secondary) amyloidosis, familial amyloidosis of Finnish type, and hereditary cerebral hemorrhage with amyloidosis-Dutch type (HCHWA-D) and Icelandic type.

As used herein, "amyloid precursor protein" or "APP" refers to a protein containing several characteristic domains, including a heparin-binding site, zinc- and copper-binding domains, a trophic domain containing an amino acid sequence (RERMS) that promotes fibroblast growth and a protease inhibitor domain for the matrix metalloprotease gelatinase A. Multiple isoforms of APP exist, typically distinguished by the number of amino acids in the particular isoform. Generally, most isoforms of APP are approximately 100 kD in molecular weight. Isoforms of APP include, for example, APP770 (which also contains a sequence homologous to the Kunitz family of serine protease inhibitors and a sequence homologous to the MRC OX-2 antigen), APP751 (the most abundant APP isoform in non-neuronal tissues), APP714, APP695 (the most abundant form in the brain), L-APP752, L-APP733, L-APP696, L-APP677, APP563 and APP365. All of the above-mentioned isoforms of APP, with the exception of APP563 and APP365, are transmembrane proteins that contain a single membrane-spanning domain and a long N-terminal extracellular (about two-thirds of the protein) and C- terminal cytoplasmic regions. APP563 and APP365 lack a transmembrane domain and are secreted. Examples of amino acid sequences for some of the APP isoforms are provided in SEQ ID NOs: 2 (APP770), 28 (APP751) and 30 (APP695). In addition, several mutations of the APP gene demonstrated in families with AD and other amyloidosis-associated diseases yield APP forms with varying amino acid sequences. Mutations of APP include those that result in a Val to Gly substitution at position 717 (V717G) of APP770 (the "London variant"), the "Swedish variant" double mutation at amino acid positions 670 and 671, with reference to the APP770 isoform, or positions 595 and 596, with reference to the APP695 isoform, in which a lysine is substituted with an asparagine and a methionine is substituted with a leucine, respectively, and a mutation at position 693 of the APP770 isoform that is associated with hereditary cerebral hemorrhage with amyloidsis-Dutch type (HCHWA-D). Unless a specific isoform is specified, APP when used herein generally refers to any and all isoforms of APP. As used herein, "cleavage" when used with reference to a substrate, e.g., a protein, polypeptide, peptide, or fragment(s) thereof, refers to an alteration in the substrate structure. The alteration can be one resulting from, for example, an alteration, elimination or reduction of one or more interactions between elements within the substrate. In one example, if the substrate is a protein (polypeptide or peptide), cleavage of the substrate can be the degradation of the protein by a loss of one or more amino acids from the protein. The protein substrate, may, for example, be degraded into two or more fragments, each of which contains less than all the amino acids that the substrate contained. For instance, the processing of a larger precursor protein to yield a smaller mature protein can involve protein cleavage. Such cleavage can be, for example, the result of the hydrolysis of one or more peptide bonds in the protein. Thus, cleavage includes proteolytic cleavage of protein substrates. An alteration of a substrate structure due to cleavage (e.g. , the particular one or fragments generated upon cleavage of a protein substrate) can provide information relating to the types of compositions (e.g., protease or proteolytic enzymes) and/or conditions or activities to which the substrate has been exposed.

As used herein, "processing" with reference to a protein, polypeptide or peptide refers to any post-translational modifications or alterations of the protein, polypeptide or peptide, such as may occur in maturation, degradation and/or clearance of such a molecule in a cell, and/or any post-translational packaging or transport of such a molecule through a pathway or process, such as a secretory pathway, uptake/internalization process, exo- or endocytosis, sequestration (e.g., into a vesicle or endosome or lysosome) and clearance, h one example of processing, a protein, polypeptide or peptide can undergo cleavage, for example, to yield an active peptide from a larger inactive precursor protein, to liberate a functional fragment or peptide, such as a signaling peptide, or to degrade/digest a protein, polypeptide or peptide.

As used herein, "portion" and "fragment" are used interchangeably with reference to a protein, polypeptide or peptide and refer to a protein, polypeptide or peptide with a primary structure that is less than or smaller than that of the protein, polypeptide or peptide of which it is a portion or fragment. For example, a fragment or portion of a protein can be a peptide generated upon cleavage of a larger precursor protein.

As used herein, "amyloid-jS peptide" or "A/3" refers to a peptide such as (a) a peptide that results from processing or cleavage of an APP and that is amyloidogenic, (b) one of the peptide constituents of /3-amyloid plaques, (c) the 43-amino acid sequence set forth in SEQ ID NO: 4 or a fragment or portion thereof, and including substantially homologous sequences and/or (d) a fragment or portion of a peptide as set forth in (a) or (b). A|8 can also be referred to as /3AP, A/3P or /3A4. A/3 peptides derived from proteolysis of APP generally are -4.2 kD proteins and are typically 39 to 43 amino acids in length (see, e.g., SEQ ED NO: 4 showing the 43-amino acid sequence of an A/3 peptide), depending on the carboxy-terminal end-point, which exhibits heterogeneity. However, A/3 peptides containing less than 39 amino acids, e.g., A/339, A/338, A/337 and A/334, also can occur. A/3 peptides can be produced in an amyloidogenic APP processing pathway in which APP is cleaved by /3-secretase (BACE) and one or more γ-secretase activities. A/3 peptides include those that begin at position 672 of APP770 (see SEQ ID NO: 2). Generally, as used herein, "Aβ peptide" includes any and all A/3 peptides, unless the amino acid residues are specified, such as, for example, 1-42 (A/342), 1-40 (A/340), 1- 39 (A/339), 1-38 (A/338), 1-37 (Aj837), 1-34 (Aj834) and others. As used herein "at least one Aβ peptide" refers to one or more species or sequence of amino acids of Aβ. For example, at least one Aβ peptide can be Aβ42, Aβ40, Aβ39, Aβ38, Aβ34, and combinations therof.

As used herein, "form of Aβ" or "Aβ form" refers to the conformational state of Aβ, for example monomers, oligomers such as dimers, trimers, pentamers, low molecular weight and high molecular weight oligomers of Aβ. Forms of Aβ also include aggregates, fibrils, tangles, and soluble Aβ. As used herein, "low molecular weight forms of Aβ" refers to monomers and low molecular weight oligomers of Aβ, including oligomers containing from about two to about 10 molecules of Aβ. As used herein, "high molecular weight forms of Aβ" refers to high molecular weight forms of Aβ such as aggregates of 50 or more Aβ molecules. As used herein, "Aβ misregulation" refers to altered, abnormal or impaired Aβ regulation. For example, Aβ misregulation can be imbalances or disturbances in intracellular and/or secreted levels such as may result from altered Aβ production, clearance or degradation in a cell. As used herein, "cellular" or "cell-associated" with reference to a molecule, such as, for example, a protein or peptide, refers to a molecule that is located within a cell (e.g., in the cytoplasm or an intracellular organelle or vesicle) and/or at least partially associated with or in a cell membrane (e.g., the plasma membrane or an intracellular membrane). As used herein, "low-density lipoprotein receptor-related protein (LRP)" refers to a protein homologous to LRPs, which have been identified and described for a number of species, including several mammalian species. An example of an amino acid sequence of an LRP is provided in SEQ ED NO: 10. LRP proteins, which are discussed below in more detail, generally are cell surface receptors that bind and internalize a number of diverse extracellular ligands, including apolipoprotein E (apoE), cβ-macroglobulin

(c_2M), APP, tissue-type plasminogen activator (tPA) and lactoferrin, for degradation by lysosomes. LRP expression is widespread; however, it is most highly expressed in the liver, brain and placenta. LRP is a member of the low-density lipoprotein receptor (LDLR) family. The extracellular region of receptors in this family contains several structural modules which include ligand-binding repeats of -40 amino acids (including six cysteine residues forming three disulfide bonds), epidermal growth factor (EGF) precursor repeats (each also containing six cysteine residues), and modules with a consensus tetrapeptide (YWTD). In addition to these modules, these receptors contain a single transmembrane domain and a relatively short cytoplasmic tail with endocytosis signals and elements for interaction with cytoplasmic adaptor and scaffold proteins (e.g., Dab, FE65, c-jun N-terminal kinase interacting proteins (JEPs) and postsynaptic density protein PSD-95) for mediating signal transduction.

As used herein, a "composition of low-density lipoprotein receptor-related protein (LRP)" refers to the make-up of LRP. The LRP may be LRP that is present anywhere, for example, in an analysis mixture, including an assay medium in which an analysis is performed, an extracellular medium, or a cell membrane, lysate or extract. For example, a composition of LRP refers to the overall combination of any intact LRP protein(s), fragments thereof, sizes thereof, ratios and amounts thereof.

As used herein, "presenilin" refers to a protein homologous to the presenilin 1 (PS 1) or presenilin 2 (PS2) proteins, and/or fragment(s) thereof, that have been identified and described for a number of species, including several mammalian species. Presenilins show a high degree of conservation between species, particularly of the hydrophobic structure. Examples of amino acid sequences of PS1 and PS2 proteins are provided in SEQ ID NOs: 6 and 8, respectively and in PCT Application Publication No. WO96/34099. Presenilin proteins generally are polytopic membrane proteins that can possess two or more aspartic acid residues within adjacent predicted transmembrane segments. Many presenilins possess protease-associated domains and are involved in a catalytic complex having catalytic activity. Presenilins can undergo proteolytic processing which can generate fragments, such as, for example, an ~35-kD N-terminal fragment and an -20-25 kD C-terminal fragment. In vivo, the majority of detectable presenilin appears in the form of N- and C-terminal fragments that are tightly regulated and form a stable complex after processing. Thus, as used herein, "presenilin" refers to any full-length presenilin protein, presenilin proteins encoded by allelic and splice variants, and any fragments thereof, including biologically active fragments and functional units.

As used herein, "presenilin activity" or "presenilin-dependent activity" refers to an activity, such as a biological event or process, that is directly or indirectly influenced by a presenilin protein. An activity can be, for example, any biological, chemical, biochemical or molecular activity, including, but not limited to, interaction between molecules, such as binding between a protein or peptide and another molecule, a chemical reaction, e.g., hydrolysis, and a cellular event, e.g., secretion, endocytosis, signaling, molecular trafficking. A presenilin-dependent activity is influenced by a presenilin in such a way that the activity differs in the presence and absence of a presenilin. The difference can be, for example, a modification or alteration in the activity or a complete or near complete elimination of the activity, hi a particular example, a presenilin-dependent activity is an enzymatic activity. One such presenilin-dependent enzymatic activity is a presenilin-dependent proteolytic processing of APP, e.g., γ- secretase cleavage of APP. Other presenilin-dependent enzymatic activities include, but are not limited to, cleavage of LRP, Notch, E-cadherin and Erb-B4. As used herein, "presenilin substrate," "substrate for presenilin activity" and/or

"substrate for presenilin-dependent enzyme activity" refers to a peptide, polypeptide, protein or fragment(s) thereof that is altered (e.g., proteolytically processed, at least in part) in a presenilin-dependent manner. Thus, for example, in the case of a presenilin substrate that is altered by proteolytic processing of the substrate, if presenilin is absent, or presenilin activity is inhibited or reduced, the proteolytic processing of the presenilin substrate is altered, for example by an alteration in the levels and/or composition of fragments generated from the substrate, relative to the proteolytic processing of the substrate that occurs in the presence of normal (e.g., wild-type) presenilin activity. Generally, a presenilin substrate can contain about one transmembrane domain, an ectodomain that is released or shed into the extracellular medium, and/or an intracellular domain. Exemplary presenilin substrates include, but are not limited to APP, LRP, Notch, TrkB, APLP2, hlrelo; E-cadherin and Erb-B4.

As used herein, "C-terminal fragment (CTF)" refers to a fragment of a protein that results from cleavage of the protein by a presenilin-dependent activity. For example, an LRP-CTF refers to a C-terminal fragment of LRP. When an LRP composition is assessed, for example, it can be evaluated to determine if any fragment(s) indicative of presenilin-dependent cleavage (or altered presenilin-dependent cleavage) of LRP is/are present and/or the level of any such fragment(s) produced. A presenilin-dependent cleavage described herein occurs within the C-terminal portion of LRP and within the β chain. Thus, a presenilin-dependent cleavage of LRP can be one, for example, that occurs in the C-terminal portion of LRP at a position C-terminal to amino acid position 3925 of SEQ ED NO: 10 (or of the amino acid sequence provided as GenBank Accession No. Q07954). The presenilin-dependent cleavage of LRP can be one that occurs within the sequence of the last approximately 580, 500, 450, 400, 350, 300, 250, 200, 150, 100, 50, 25, or less amino acids of LRP. The presenilin-dependent cleavage can be one that occurs C-terminal to the extracellular portion of the β chain (i.e., approximately amino acids 3944-4420 of SEQ ED NO: 10 or of the amino acid sequence provided as GenBank Accession No. Q07954); thus, C-terminal to amino acid 4420 of SEQ ED NO: 10. The presenilin-dependent cleavage of LRP can be one that occurs near or within the region of the LRP protein that extends from a point located intracellularly and adjacent to the cytoplasmic face of the cell membrane to a point located within the cell membrane. Such a presenilin-dependent cleavage of LRP can be one that generates a soluble intracellular peptide containing the extreme C-terminus of LRP and a membrane-associated peptide containing amino acid sequence of the transmembrane region of LRP, particularly the more C-terminal region of the transmembrane segment of LRP. Such fragments of LRP can be referred to as LRP-CTFs. Any LRP fragments generated by such presenilin- dependent activities have a molecular weight that is less than that of the β chain of LRP (β chain molecular weight is approximately 85-90 kD, or approximately 67 kD after deglycosylation with N-glycosidase F) and are encompassed by the term LRP-CTFs. Similarly, characteristic C-terminal fragments of APP are produced upon exposure to an a presenilin-dependent activity.

As used herein, "normal" with reference to a protein refers to a protein which performs its usual or normal physiological role and which is not causative of a disease or pathogenic condition. A normal gene or coding sequence is also one that is not causative of a disease or pathogenic condition and may encode a normal protein. The term normal is generally synonymous with wild-type. For any given gene, or corresponding protein, a number of normal allelic variants may exist, none of which is associated with the development of a pathogenic condition or disease.

As used herein, "mutant" with reference to a protein refers to a protein which does not perform it usual or normal physiological role, e.g. , it may be dysfunctional, and which can be associated with a disease or pathogenic state. A mutant gene generally is one that contains an alteration relative to a normal or wild-type gene such that it has altered function (e.g., regulation or encoding of a mutant protein).

As used herein, "assess" and variations thereof refer to any type of evaluation, determination, observation, identification, detection, characterization and measurement, whether quantitative, qualitative, comparative or relative.

As used herein, "determining the level of, "assessing the level of and variations thereof with reference to a substance, such as, for example a peptide, protein or fragment thereof, can be determining the presence or absence of the substance and/or making a more quantitative assessment of level or amount of the substance.

As used herein, the term "polypeptide" is used interchangeably with the term "protein" and includes peptides of 2 or more amino acids. A polypeptide can be a single polypeptide chain, or to two or more polypeptide chains that are held together by non- covalent forces, by disulfide cross-links, or by other linkers (e.g. peptide linkers). Thus, a single heavy or light chain of an antibody, or an antibody fragment containing all or part of both heavy and light chains of an antibody, no matter how the chains are associated or joined, are exemplary molecules that are included within the term "a polypeptide." A polypeptide can contain non-proteinaceous components, such as sugars, lipids, detectable labels or therapeutic moieties. A polypeptide can be derivatized by chemical or enzymatic modifications (e.g. by replacement of hydrogen by an alkyl, acyl, or amino group; esterifϊcation of a carboxyl group with a suitable alkyl or aryl moiety; alkylation of a hydroxyl group to form an ether derivative; phosphorylation or dephosphorylation of a serine, threonine or tyrosine residue; or N- or O-linked glycosylation) or can contain substitutions of an L-configuration amino acid with a D- configuration counterpart.

As used herein, the term "chimeric polypeptide" refers to a polypeptide that contains amino acid residues from derived from two or more polypeptides or from one polypeptide but joined in different order from the original polypeptide. For example, a chimeric polypeptide can contain residues from related polypeptides from two or more species (e.g. CDR sequences from a mouse immunoglobulin (Ig), and a scaffold portion from a human Ig; or variable region residues from a mouse Ig, and constant region residues from a human Ig). A chimeric polypeptide also can contain residues from two or more unrelated polypeptides from the same or different species (e.g. CDR sequences from an Ig, and scaffold sequences from a lipocalin or Fn3 polypeptide). As used herein, "antibody" refers to an immunoglobulin, whether natural or partially or wholly synthetically produced, including any derivative thereof that retains the specific binding ability of the antibody. Hence antibody includes any protein having a binding domain that is homologous or substantially homologous to an immunoglobulin binding domain. Antibodies include members of any immunoglobulin chains, including IgG, IgM, IgA, IgD and IgE. As used herein, the term "antibody" includes, but is not limited to, polyclonal antibodies, affinity-purified polyclonal antibodies, monoclonal antibodies, antibody fragments and antigen-binding fragments. Genetically engineered intact antibodies or fragments, such as chimeric antibodies, Fv fragments, single chain antibodies and the like, as well as synthetic antigen-binding peptides and polypeptides, are also included.

As used herein, "antibody fragment" refers to any derivative of an antibody that is less than full-length, retaining at least a portion of the full-length antibody's specific binding ability. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab)₂, single-chain Fvs (scFv), FV, dsFv diabody and Fd fragments. The fragment can include multiple chains linked together, such as by disulfide bridges. An antibody fragment generally contains at least about 50 amino acids and typically at least 200 amino acids.

As used herein, an "Fv antibody fragment" is composed of one variable heavy domain (VH) and one variable light domain linked by noncovalent interactions. As used herein, a "dsFV" refers to an Fv with an engineered intermolecular disulfide bond, which stabilizes the VH-V pair.

As used herein, an "F(ab)₂ fragment" is an antibody fragment that results from digestion of an immunoglobulin with pepsin at pH 4.0-4.5; it can be recombinantly expressed to produce the equivalent fragment. As used herein, "Fab fragments" are antibody fragments that result from digestion of an immunoglobulin with papain; they can be recombinantly expressed to produce the equivalent fragment.

As used herein, "scFvs" refer to antibody fragments that contain a variable light chain (VL) and variable heavy chain (VH) covalently connected by a polypeptide linker in any order. The linker is of a length such that the two variable domains are bridged without substantial interference. Included linkers are (Gly-Ser)_n residues with some Glu or Lys residues dispersed throughout to increase solubility.

As used herein, "diabodies" are dimeric scFv; diabodies typically have shorter peptide linkers than scFvs, and they generally dimerize. As used herein, the term "complementarity determining region" or "CDR" (also known as a "hypervariable region") refers to a region of an Ig molecule that varies greatly in amino acid sequence relative to flanking Ig sequences. The length and conformation of CDRs vary among Igs, but generally CDRs form short loops supported by a sandwich of two antiparallel beta-sheets. Three CDRs, designated CDR-Ll, CDR-L2 and CDR- L3, are present in the variable region of an immunoglobulin light chain, and three CDRs, designated CDR-Hl, CDR-H2 and CDR-H3, are present in the variable region of an immunoglobulin heavy chain. Each CDR generally contains at least one, and often several, amino acids residues that make contact with antigen, but all six CDRs are not necessarily required to maintain the binding specificity of an antibody. As used herein, a "scaffold" refers to any structure that forms a conformationally stable structural support, or framework, which is able to display one or more sequences of amino acids (e.g. CDRs, a variable region, a binding domain) in a localized surface region. A scaffold can be a naturally occurring polypeptide or polypeptide "fold" (a structural motif), or can have one or more modifications, such as additions, deletions or substitutions of amino acids, relative to a naturally occurring polypeptide or fold.

Exemplary modifications to a polypeptide that render it suitable for use as a scaffold include but are not limited to, deletions of those regions that form binding loops in the naturally-occurring molecule (e.g. deletions of the naturally-occurring CDRs); deletions of those regions that are unnecessary for structural integrity of the fold; substitutions of amino acids that flank the loop regions with residues that improve the properties of the polypeptide (such as improved affinity, specificity, or solubility; reduced immunogenicity, etc.); addition of detectable sequences, such as epitope tags. A scaffold can be derived from a polypeptide of any species (or of more than one species), such as a human, other mammal, other vertebrate, invertebrate, plant, bacteria or virus. A scaffold can also be a solid support, such as a membranes, filters, chips, slides, wafers, fibers, magnetic or nonmagnetic beads, gels, tubing, plates, polymers, microparticles and capillaries, which is able to display one or more amino acid sequences (e.g. CDRs) in a localized surface region.

As used herein, the term "human polypeptide scaffold" refers to a polypeptide scaffold that is derived from a human polypeptide or has been engineered to resemble a human polypeptide. An example of a human polypeptide scaffold is a human antibody scaffold, which is used in a humanized antibody.

As used herein, the term "antibody scaffold" refers to a scaffold of an antibody that contains all or part of an immunoglobulin. Exemplary antibody scaffolds include whole antibodies, and fragments thereof, such as Fv fragments (which can or can not contain an introduced disulfide bond), Fab fragments, Fab' fragments, F(ab')₂ fragments, and single-chain scFv fragments. Antibody scaffolds also include all or part of an Ig heavy chain variable region, and all or part of an Ig light chain variable region.

As used herein, the term "clearance domain" refers to a domain that directly or indirectly mediates enhanced clearance of a polypeptide from the circulation. Thus, a polypeptide described herein as containing a "clearance domain" will have a shorter half- life in the circulation, alone and/or when bound to Aβ, than a polypeptide without such a domain.

As used herein, an "Aβ antibody" refers to an immunoglobulin, whether natural or partially or wholly synthetically produced, including any derivative thereof that is specifically reactive with at least one Aβ.

As used herein, an "Aβ binding protein" refers to a polypeptide, peptide or protein that is specifically reactive with at least one Aβ peptide. An Aβ binding protein can be an Aβ antibody or fragment(s) thereof. Aβ proteins also include chimeric polypeptides. For example, an Aβ binding protein can be a chimeric polypeptide that has the ability to bind Aβ displayed in a scaffold. An Aβ binding protein can also be derived de novo by screening for peptides, polypeptides and proteins that have the ability to bind at least one Aβ.

As used herein, "grafting" with respect to polypeptides refers to the construction of a chimeric polypeptide by covalently joining a peptide, protein or domain of a protein to a scaffold.

As used herein, "operatively linked" (or, sequences that are in "operative association") indicates that the recited nucleotide sequences are positioned such that there is a functional relationship between the sequences in the context of transcription. For example, an Aβ binding protein nucleotide sequence, a promoter sequence and a reporter sequence can be in operative association if transcription of the reporter nucleic acid sequence can occur under control of the promoter sequence as modulated by the effect of the Aβ binding protein nucleotide sequence. When the Aβ binding protein nucleotide sequence comprises the promoter, the sequences can be in operative association if transcription of the reporter nucleic acid sequence can occur under control of the Aβ binding protein nucleotide sequence. Two sequences that are "operatively linked" are not necessarily contiguous.

As used herein, an "expression construct" refers to a nucleotide sequence with the capacity to express an mRNA or protein. Generally, expression constructs have a sequence of nucleotides encoding the mRNA and/or protein to be expressed, operatively linked to a promoter sequence.

As used herein a "detectable moiety" refers to a molecule that can be detected by visible, enzymatic, physical or chemical means. Detectable moieties include, but are not limited to, reporter genes or fragments thereof, enzymes or portions thereof and radiolabels. Exemplary detectable moieties include fluorescent proteins such as green, red and blue fluorescent proteins, β-galactosidase, alkaline phosphatase and radiolabels such as I, I, Bi, mTc, In, Y, and P. Detectable moieties also include moieties that can be detected physical means such as detection of molecular weight by mass spectrometry and tags that can be detected such as a His₆ tag for metal binding or an epitope tag for antibody recognition.

As used herein, "humanized antibodies" refer to antibodies that are modified to include human sequences of amino acids so that administration to a human does not provoke an immune response, or provokes a milder immune response than a non- humanized antibody. Methods for preparation of such antibodies are known. For example, to produce such antibodies, the encoding nucleic acid in the hybridoma or other prokaryotic or eukaryotic cell, such as an E. coli or a CHO cell, that expresses the monoclonal antibody is altered by recombinant nucleic acid techniques to express an antibody in which the amino acid composition is based on human antibodies.

As used herein, "specifically reactive," "specificity," "selectivity," "selective" and variations thereof in the context of an antibody binding an antigen refers to the degree of affinity an antibody has for a target antigen and the degree of discrimination between the target antigen and other, chemically similar structures. Antibodies and proteins, such as Aβ binding proteins, are determined to be specifically reactive if: 1) they exhibit a threshold level of binding affinity, and/or 2) they do not significantly cross-react with related polypeptide molecules. Antibodies and Aβ binding proteins herein are determined to be specifically reactive if they bind the target epitope with an affinity constant in the range of about 10 1/mole to 10 1/mole, generally about 10 to 10 1/mole. In one embodiment, an antibody or Aβ binding protein is determined to be specifically reactive if it binds the target epitope with an affinity constant of at least about 10 1/mol, or at least about 10 1/mol. In a particular embodiment, an antibody or Aβ binding protein is determined to be specifically reactive if it binds the target epitope with an affinity constant of at least about 2 10 1/mol, or at least about 3 x 10 1/mol, or at least about 4 x 10 1/mol. The binding affinity of an antibody and an Aβ binding protein can be readily determined by one of skill in the art (Scatchard (1949) Ann. N. Y. Acad. Sci. 51: 660-672).

Selectivity of an antibody and an Aβ binding protein can refer to the degree of recognition of an antibody or Aβ binding protein for an antigen relative to other, particularly related, peptides or proteins. Selectivity or selectively reactive is considered a measure of the functional ability of an antibody to discriminate between the target antigen and other, chemically similar structures. In one aspect, selectivity of an antibody for a particular antigen relative to another peptide or protein can be determined by comparing the binding affinities of the antibody for the antigen and the other peptide. If the binding affinity (e.g., as represented by an affinity constant) for the antigen is, for example, 1000-fold higher than that for the other peptide, the antibody can be said to be 1000-fold more selective or selectively reactive, for the antigen relative to the other peptide.

As used herein, "bind preferentially" refers to the affinity of an Aβ binding protein, such as an Aβ antibody, for one antigen (such as an Aβ peptide or form) relative to another. For example, an Aβ binding protein can preferentially bind one Aβ form relative to another Aβ form, such as preferentially binding low molecular weight forms of Aβ relative to high molecular weight forms of Aβ. In one embodiment, an Aβ binding protein binds preferentially to a particular Aβ form relative to another Aβ form if the Aβ binding protein binds the particular Aβ form with at least 2-fold higher affinity as compared with binding to the other Aβ form. In another embodiment, an Aβ binding protein binds preferentially to a particular Aβ form relative to another Aβ form if the Aβ binding protein binds the particular Aβ form with at least 5 -fold, 10-fold or more, including 20-fold and 100-fold higher affinity as compared with binding to the other Aβ form. In another embodiment, an Aβ binding protein binds preferentially to a particular Aβ peptide or form relative to another Aβ peptide or form if the binding of the Aβ binding protein to the particular Aβ peptide or form can be detected in an immuno assay, such as western blot or ELISA assay, but the binding of the Aβ binding protein to another Aβ peptide or form is substantially less in the same or a similar assay. As used herein, "modulation" with reference to Aβ levels refers to any alteration or adjustment in cellular and/or extracellular or secreted Aβ, including, but not limited to, alteration of Aβ concentration in the cytoplasm, cellular membranes, extracellular medium and/or intracellular organelles, e.g., endoplasmic reticulum, endosome and lysosome, and any alteration of the production, clearance, and/or degradation of Aβ.

As used herein, "agent that modulates Aβ levels" refers to any substance that can modulate Aβ levels. Examples of agents include, but are not limited to, small organic molecules, amino acids, peptides, polypeptides, nucleotides, nucleic acids, polynucleotides, carbohydrates, lipids, lipoproteins, glycoproteins, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof. Agents may be obtained from a wide variety of sources including libraries of synthetic or natural compounds. Libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraries. Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, acidification, etc. to produce structural analogs.

As used herein, "test agent", in the context of methods for identifying agents that modulate Aβ levels, refers to any substance that is being evaluated as a possible agent that modulates Aβ levels.

As used herein, "amelioration" refers to an improvement in a disease or condition or at least a partial relief of symptoms associated with a disease or condition.

As used herein, "substantially unchanged" or "without substantially altering or affecting" and variations thereof are used with reference to a particular composition, activity and/or process that is not a target for modulation. These expressions refer to the state (which includes amount or level) of the non-target composition, activity and/or process under specified differing conditions. A composition may be, for example, a particular protein or peptide, such as an Aβ peptide, or a fragment or peptide generated by cleavage of a protein, such as a presenilin substrate. An activity or process may be, for example, the cleavage or processing of a protein such as a presenilin substrate. Differing conditions include any physical, chemical, environmental or other conditions in which the composition, activity and/or process occurs. For example, differing conditions can be in the presence and absence of a test agent or agent that modulates a target composition, activity or process. A non-target composition, activity and/or process is substantially unchanged or is not substantially altered or affected if any variation in the composition, activity and/or process that occurs under specified differing conditions is an acceptable variation. Those of skill in the art can identify acceptable variation. For example, acceptable variation generally can be any alteration in the composition, activity and/or process (including, e.g., increase or decrease in the amount or level) that is less than or relatively minimal in comparison to the variation in a target composition, process or activity under the specified differing conditions, or that is not associated with an undesired effect. An undesired effect can be, for example, an adverse effect on a biological composition, cell, tissue, system or organism including or containing the cell or composition. Undesired effects include, for example, deleterious alterations in any aspect of cell function, decreased cell viability and cell death. Acceptable variation can also be any alteration in the composition, activity and/or process that is inconsequential (or without significant consequence) to an overall or ultimate downstream function in which the composition, activity and/or process is involved. Thus, in a particular example of a peptide that is not a target for modulation, substantially unchanged with respect to the levels of such a non-target peptide in the presence and absence of an agent being tested as a possible modulator of a target peptide means that there is no change, or an acceptable variation, in the level of the non-target peptide in the presence of the agent compared to in the absence of the agent. Acceptable variation in a non-target composition, activity and/or process (including levels or amounts) may be different for different compositions, activities and processes, and in the context of different sets of specified differing conditions. In some particular instances, acceptable variation can range from equal to or less than about 40, 30, 20, or 10% variation when compared under differing conditions, e.g., in the presence and absence of a test agent. It should be understood that this definition of "substantially unchanged" or "without substantially altering or affecting" applies and is used with reference to a composition, activity and/or process that is not a target for modulation. In contrast, any variation (and particularly a statistically significant variation) in a composition, activity and/or process that is a target for modulation in the presence and absence of a test agent can be a sufficient modulation. As used herein, "avidity" refers to the functional affinity or combining strength of an antibody with its antigen and is related to both the affinity of the reaction between the epitopes and paratopes, and the valences or recognition sites of the antibody and antigen.

As used herein, "selective modulation of Aβ levels" refers to the modulation of the levels of one or more forms of Aβ, wherein one or more other specified compositions or specified activities, processes or mechanisms are substantially unchanged, or without substantially altering or affecting one or more other specified compositions or specified activities, processes or mechanisms. For example, selective modulation of an Aβ peptide can be relative to one or more other related polypeptide molecules (e.g., other Aβ peptides) in which the level of a particular Aβ peptide is modulated without substantially altering the levels of one or more other Aβ peptides. In another example, selective modulation of an Aβ peptide can be relative to the processing of a presenilin substrate other than APP, in which Aβ levels are modulated without substantially altering the cleavage of the presenilin substrate that is other than APP.

As used herein, "related peptide molecules" refers to any peptide molecules with chemically similar structures, any peptides molecules that undergo similar processing by the same or similar enzymes, any peptide molecules derived from the same or similar precursor peptide molecule, and/or any peptide molecules that have the same or similar activities and/or functions.

As used herein, "treatment" means any manner in which the symptoms of a conditions, disorder or disease are ameliorated or otherwise beneficially altered. Treatment also encompasses any pharmaceutical use of the compositions herein. As used herein, a "combination" refers to any association between two or among more items.

As used herein, an "agent identified by the screening methods provided herein for identifying candidate agents for the treatment and/or prevention of a disease or disorder" refers to any compound that is a candidate for use as a therapeutic or as lead compound for design of a therapeutic. Such compounds can be small molecules, including small organic molecules, peptides, peptide mimetics, antisense molecules or dsRNA, such as RNAi, antibodies, fragments of antibodies, recombinant antibodies and other such compound which can serve as drug candidate or lead compound.

As used herein, a "peptidomimetic" is a compound that mimics the conformation and certain stereochemical features of the biologically active form of a particular peptide. hi general, peptidomimetics are designed to mimic certain desirable properties of a compound, but not the undesirable properties, such as flexibility, that lead to a loss of a biologically active conformation and bond breakdown. Peptidomimetics may be prepared from biologically active compounds by replacing certain groups or bonds that contribute to the undesirable properties with bioisosteres. Bioisosteres are known to those of skill in the art. For example the methylene bioisostere CH₂S has been used as an amide replacement in enkephalin analogs (see, s_^ ., Spatola (1983) pp. 267-357 in Chemistry and Biochemistry of Amino Acids, Peptides, and Proteins, Weistein, Ed. volume 7, Marcel Dekker, New York). Morphine, which can be administered orally, is a compound that is a peptidomimetic of the peptide endorphin. For purposes herein, cyclic peptides are included among pepidomimetics.

As used herein, "production by recombinant means by using recombinant DNA methods" means the use of the well known methods of molecular biology for expressing proteins encoded by cloned DNA. As used herein, "heterologous" or "foreign" with reference to nucleic acids, cDNA, DNA and RNA are used interchangeably and refer to nucleic acid, DNA or RNA that does not occur naturally as part of the genome in which it is present or which is found in a location(s) or in an amount in the genome that differs from that in which it occurs in nature. It can be nucleic acid that has been exogenously introduced into the cell. Thus, heterologous nucleic acid is nucleic acid not normally found in the host genome in an identical context. Examples of heterologous nucleic acids include, but are not limited to, DNA that encodes a gene product or gene product(s) of interest, introduced, for example, for purposes of gene therapy or for production of an encoded protein. Other examples of heterologous DNA include, but are not limited to, DNA that encodes a selectable marker, DNA that encodes therapeutically effective substances, such as anti-cancer agents, enzymes and hormones, and DNA that encodes other types of proteins, such as antibodies.

As used herein, "expression" refers to the process by which nucleic acid, e.g., DNA, is transcribed into mRNA and translated into peptides, polypeptides, or proteins. If the nucleic acid is derived from genomic DNA, expression may, if an appropriate eukaryotic host cell or organism is selected, include splicing of the mRNA.

As used herein, "vector" or "plasmid" refers to discrete elements that are used to introduce heterologous nucleic acids into cells. Typically, vectors are used to transfer heterologous nucleic acids into cells for either expression of the heterologous nucleic acid or for replication of the heterologous nucleic acid. Selection and use of such vectors andplasmids are well within the level of skill of the art.

As used herein, "transformation" or "transfection" refers to the process by which nucleic acids are introduced into cells. Transfection refers to the taking up of exogenous nucleic acid, e.g., an expression vector, by a host cell whether or not any coding sequences are in fact expressed. Numerous methods of transfection are known to the ordinarily skilled artisan. Successful transfection is generally recognized by detection of the presence of the heterologous nucleic acid within the transfected cell, such as, for example, any visualization of the heterologous nucleic acid or any indication of the operation of a vector within the host cell. As used herein, "injection" refers to the microinjection (use of a small syringe) of nucleic acid into a cell.

As used herein, the amino acids, which occur in the various amino acid sequences appearing herein, are identified according to their well-known, three-letter or one-letter abbreviations (see Table 1). The nucleotides, which occur in the various DNA fragments, are designated with the standard single-letter designations used routinely in the art.

As used herein, "amino acid residue" refers to an amino acid formed upon chemical digestion (hydrolysis) of a polypeptide at its peptide linkages. The amino acid residues described herein are preferably in the "L" isomeric form. However, residues in the "D" isomeric form can be substituted for any L-amino acid residue, as long as the desired functional property is retained by the polypeptide. NH₂ refers to the free amino group present at the amino terminus of a polypeptide. COOH refers to the free carboxy group present at the carboxyl terminus of a polypeptide. In keeping with standard polypeptide nomenclature described inJ Biol. Chem., 243:3552-59 (1969) and adopted at 37 C.F.R. § § 1.821 - 1.822, abbreviations for amino acid residues are shown in Table 1:

It should be noted that all amino acid residue sequences represented herein by formulae have a left to right orientation in the conventional direction of amino-terminus to carboxyl-terminus. In addition, the phrase "amino acid residue" is broadly defined to include the amino acids listed in the Table of Correspondence and modified and unusual amino acids, such as those referred to in 37 C.F.R. § § 1.821-1.822, and incorporated herein by reference. Furthermore, it should be noted that a dash at the beginning or end of an amino acid residue sequence indicates a peptide bond to a further sequence of one or more amino acid residues or to an amino-terminal group such as NH₂ or to a carboxyl-terminal group such as COOH.

In a peptide or protein, suitable conservative substitutions of amino acids are known to those of skill in this art and may be made generally without altering the biological activity of the resulting molecule. Those of skill in this art recognize that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., Watson et al. Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin/Cummings Pub. co., p.224).

Such substitutions may be made in accordance with those set forth in TABLE 2 as follows:

Table 2

Original residue Conservative substitution

Ala (A) Gly; Ser

Arg (R) Lys

Asn (N) Gin; His

Cys (C) Ser

Gln (Q) Asn

Glu (E) Asp

Gly (G) Ala; Pro

His (H) Asn; Gin

He (I) Leu; Val

Leu (L) He; Val

Lys (K) Arg; Gin; Glu Met (M) Leu; Tyr; lie

Phe (F) Met; Leu; Tyr

Ser (S) Thr

Thr (T) Ser

Trp (W) Tyr

Tyr (Y) Trp; Phe

Val (V) He; Leu

Other substitutions are also permissible and may be determined empirically or in accord with known conservative substitutions.

As used herein, all assays and procedures, such as hybridization reactions and antibody-antigen reactions, unless otherwise specified, are conducted under conditions recognized by those of skill in the art as standard conditions.

As used herein: "stringency of hybridization" in determining percentage mismatch is as follows:

1) high stringency: 0.1 x SSPE, 0.1% SDS, 65°C 2) medium stringency: 0.2 x SSPE, 0.1% SDS, 50°C

3) low stringency: 1.0 x SSPE, 0.1% SDS, 50°C

Those of skill in this art know that the washing step selects for stable hybrids and also know the ingredients of SSPE (see, e_^g., Sambrook, E.F. Fritsch, T. Maniatis, in: Molecular Cloning A Laboratory Mann al, Cold Spring Harbor Laboratory Press (1989), vol. 3, p. B.13, see, also, numerous catalogs that describe commonly used laboratory solutions). SSPE is pH 7.4 phosphate- buffered 0.18 NaCl. Further, those of skill in the art recognize that the stability of hybrids is determined by T_m, which is a function of the sodium ion concentration and temperature (T_m = 81.5° C-16.6(logιo[Na ]) + 0.41(%G+C)-600/l)), so that the only parameters in the wash conditions critical to hybrid stability are sodium ion concentration in the SSPE (or SSC) and temperature.

It is understood that equivalent stringencies can be achieved using alternative buffers, salts and temperatures. By way of example and not limitation, procedures using conditions of low stringency are as follows (see also Shilo and Weinberg, Proc. Natl. Acad. Sci. USA, 2R:6789-6792 (1981)): Filters containing DNA are pretreated for 6 hours at 40°C in a solution containing 35% formamide, 5X SSC, 50 mM Tris-HCI (pH 7.5), 5 mM EDTA, 0.1% PVP, 0.1% Ficoll^®, 1% BSA, and 500 μg/ml denatured salmon sperm DNA ( 1 OX SSC is 1.5 M sodium chloride, and 0.15 M sodium citrate, adjusted to apH of7). Hybridizations are carried out in the same solution with the following modifications: 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 μg/ml salmon sperm DNA, 10% (wt/vol) dextran sulfate, and 5-20 X 10 cpm P-labeled probe is used. Filters are incubated in hybridization mixture for 18-20 hours at 40°C, and then washed for 1.5 hours at 55°C in a solution containing 2X SSC, 25 mM Tris-HCI (pH 7.4), mM EDTA, and 0.1% SDS. The wash solution is replaced with fresh solution and incubated an additional 1.5 hours at 60°C. Filters are blotted dry and exposed for autoradiography. If necessary, filters are washed for a third time at 65-68°C and reexposed to film. Other conditions of low stringency which can be used are well known in the art (e.g., as employed for cross-species hybridizations). By way of example and not way of limitation, procedures using conditions of moderate stringency is provided. For example, but not limited to, procedures using such conditions of moderate stringency are as follows: Filters containing DNA are pretreated for 6 hours at 55°C in a solution containing 6X SSC, 5X Denhart's solution, 0.5% SDS and 100 μg/ml denatured salmon sperm DNA. Hybridizations are carried out in the same solution and 5-20 X 10⁶ cpm ³²P-labeled probe is used. Filters are incubated in hybridization mixture for 18-20 hours at 55°C, and then washed twice for 30 minutes at 60°C in a solution containing IX SSC and 0.1% SDS. Filters are blotted dry and exposed for autoradiography. Other conditions of moderate stringency which can be used are well-known in the art. Washing of filters is done at 37°C for 1 hour in a solution containing 2X SSC, 0.1% SDS.

By way of example and not way of limitation, procedures using conditions of high stringency are as follows: Prehybridization of filters containing DNA is carried out for 8 hours to overnight at 65°C in buffer composed of 6X SSC, 50 mM Tris-HCI (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 μg/ml denatured salmon sperm DNA. Filters are hybridized for 48 hours at 65°C in prehybridization mixture containing 100 μg/ml denatured salmon sperm DNA and 5-20 X 10 cpm of

P-labeled probe. Washing of filters is done at 37°C for 1 hour in a solution containing 2X SSC, 0.01% PVP, 0.01% Ficoll, and 0.01% BSA. This is followed by a wash in 0.1X SSC at 50°C for 45 minutes before autoradiography. Other conditions of high stringency which can be used are well known in the art.

As used herein, "substantially identical to a product" means sufficiently similar so that the property of interest is sufficiently unchanged so that the substantially identical product can be used in place of the product.

As used herein, "isolated" when used with reference to a composition such as an antibody or portion or fragment thereof or to a protein means that such composition is in a state that is not identical to that as it may occur in nature, if it occurs in nature. Such an isolated composition typically has been manipulated or altered from its naturally occurring state in some way by the hand of man.

As used herein, "substantially pure" means sufficiently homogeneous to appear free of readily detectable impurities as determined by standard methods of analysis, such as thin layer chromatography (TLC), gel electrophoresis and high performance liquid chromatography (HPLC), used by those of skill in the art to assess such purity, or sufficiently pure such that further purification would not detectably alter the physical and chemical properties, such as enzymatic and biological activities, of the substance. Methods for purification of the compounds to produce substantially chemically pure compounds are known to those of skill in the art. A substantially chemically pure compound can, however, be a mixture of stereoisomers or isomers. In such instances, further purification might increase the specific activity of the compound.

As used herein, "target cell" refers to a cell that contains a target molecule of interest, for example, an APP and/or Aβ peptide(s).

As used herein, "test substance" refers to a chemically defined compound (e.g., organic molecules, inorganic molecules, organic/inorganic molecules, proteins, peptides, nucleic acids, oligonucleotides, lipids, polysaccharides, saccharides, or hybrids among these molecules such as glycoproteins) or mixtures of compounds (e.g., a library of test compounds, natural extracts or culture supematants) whose effect on a target of interest, e.g., Aβ peptides and/or levels thereof in a sample, is sought to be determined by, for example, methods and assays provided herein.

As used herein, the terms "a therapeutic agent," "therapeutic regimen," "radioprotectant," "chemotherapeutic" mean conventional drugs and drug therapies, including antibodies, which are known to those skilled in the art. Radiotherapeutic agents are well known in the art.

As used herein, by "homologous" (with respect to nucleic acid and/or amino acid sequences) means about greater than or equal to 25% sequence homology, typically greater than or equal to 25%, 40%, 60%, 70%, 80%, 85%, 90% or 95% sequence homology; the precise percentage can be specified if necessary. For purposes herein the terms "homology" and "identity" are often used interchangeably, unless otherwise indicated. In general, for determination of the percentage homology or identity, sequences are aligned so that the highest order match is obtained (see, e.g. : Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.W., ed., Academic

Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A.M., and Griffin, H.G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; Carillo et al. (1988) SIAMJ Applied Math 48: 1073). By sequence homology, the number of conserved amino acids are determined by standard alignment algorithms programs, and are used with default gap penalties established by each supplier. Substantially homologous nucleic acid molecules would hybridize typically at moderate stringency or at high stringency all along the length of the nucleic acid of interest. Also contemplated are nucleic acid molecules that contain degenerate codons in place of codons in the hybridizing nucleic acid molecule.

Whether any two nucleic acid molecules have nucleotide sequences that are at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% "identical" or "homologous" can be determined using known computer algorithms such as the "FAST A" program, using for example, the default parameters as in Pearson et al. (1988) Proc. Natl. Acad. Sci. USA S5:2444 (other programs include the GCG program package (Devereux, J., et al, Nucleic Acids Research 12(I):3 7 (1984)), BLASTP, BLASTN, FASTA (Atschul, S.F., et al, JMolec Biol 275:403 (1990); Guide to Huge Computers, Martin J. Bishop, ed., Academic Press, San Diego, 1994, and Carillo et al. (1988) SIAMJ Applied Math 48: 1073). For example, the BLAST function of the National Center for Biotechnology Information database can be used to determine identity. Other commercially or publicly available programs include, DNAStar "MegAlign" program (Madison, WI) and the University of Wisconsin Genetics Computer Group (UWG) "Gap" program (Madison WT). Percent homology or identity of proteins and/or nucleic acid molecules can be determined, for example, by comparing sequence information using a GAP computer program (e.g., Needleman et al. (1970) J. Mol Biol. 48:443, as revised by Smith and Waterman ((1981) Adv. Appl. Math. 2:482). Briefly, the GAP program defines similarity as the number of aligned symbols (i.e., nucleotides or amino acids) which are similar, divided by the total number of symbols in the shorter of the two sequences. Default parameters for the GAP program can include: (1) a unary comparison matrix (containing a value of 1 for identities and 0 for non-identities) and the weighted comparison matrix of Gribskov et al. (1986) Nucl. Acids Res. 14:6745, as described by Schwartz and Dayhoff, eds., ATLAS OF PROTEIN SEQUENCE AND STRUCTURE, National Biomedical Research Foundation, pp. 353-358 (1979); (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap; and (3) no penalty for end gaps.

Therefore, as used herein, the term "identity" or "homology" represents a comparison between a test and a reference polypeptide or polynucleotide. As used herein, the term at least "90% identical to" refers to percent identities from 90 to 99.99 relative to the reference nucleic acid or amino acid sequences. Identity at a level of 90% or more is indicative of the fact that, assuming for exemplification purposes a test and reference polypeptide length of 100 amino acids are compared. No more than 10% (i.e., 10 out of 100) amino acids in the test polypeptide differs from that of the reference polypeptides. Similar comparisons can be made between a test and reference polynucleotides. Such differences can be represented as point mutations randomly distributed over the entire length of an amino acid sequence or they can be clustered in one or more locations of varying length up to the maximum allowable, e.g. 10/100 amino acid difference (approximately 90% identity). Differences are defined as nucleic acid or amino acid substitutions, or deletions. At the level of homologies or identities above about 85-90%, the result should be independent of the program and gap parameters set; such high levels of identity can be assessed readily, often without relying on software.

As used herein, "primer" refers to an oligonucleotide containing two or more deoxyribonucleotides or ribonucleotides, generally more than three, from which synthesis of a primer extension product can be initiated. Experimental conditions conducive to synthesis include the presence of nucleoside triphosphates and an agent for polymerization and extension, such as DNA polymerase, and a suitable buffer, temperature and pH.

As used herein, "animals" include any animal, such as, but are not limited to, goats, cows, deer, sheep, rodents, pigs and humans. Non-human animals, exclude humans as the contemplated animal.

As used herein, the term "subject" is used interchangeably with the term "individual" and includes mammals, such as humans. B. Pathogenesis of Alzheimer's Disease

Neuropathologically, AD is characterized by massive neuronal cell loss in certain brain areas, and by the deposition of proteinaceous material in the brains of AD patients. These deposits are the neurofibrillary tangles and the β-amyloid plaques. The major protein component of the β-amyloid plaque is the Aβ peptide which is derived from processing of amyloid precursor protein (APP). Increased accumulation of Aβ peptide has been postulated to be a causal factor in the pathogenesis of AD. Supportive evidence for the causal role of Aβ in AD can be found in patients with Down's syndrome, who often develop AD-like symptoms and pathology after age 40. Down's syndrome patients produce elevated APP presumably due to an additional copy of chromosome 21 and exhibit AD-like amyloid plaques prior to the onset of other AD symptoms, suggesting that increased amyloid accumulation is an initial event (Giaccone G. et al, (1989) Neurosci Lett 97:232-8). Additional evidence implicating accumulation of Aβ peptides in AD comes from various recently identified mutations accounting for certain types of inherited AD. For example, alterations in APP processing have been linked to a subset of familial AD patients (FAD) with autosomal dominant mutations in APP (Goate, A. et al., (1991) Nature 349:704-6; Citron, M. et al., (1992) 360:672-4), presenilin 1 (Sherrington, R. et al., (1995) Nature 375:754-60), and presenilin 2 (Levy-Lehad, E., et al., (1995) Science 269:970-3). FAD individuals comprise 10% of all AD cases and generally exhibit symptoms of the disease much earlier than sporadic AD patients. For example, a double mutation of amino acids 670 and 671 of APP from Lys-Met to Asn- Lys, respectively, immediately upstream of the β-cleavage site of Aβ ("Swedish" mutation or APPΔNL) results in a 5-8-fold increase in the formation of Aβ by cells (Citron, M. et al., (1992) 360:672-4). The fact that such alterations are sufficient to cause AD-like pathology is supported by studies which show that transgenic mice overexpressing APPΔ_NL (Hsiao, K., et al., (1996) Science 274:99-102) produce higher levels of Aβ prior to the exhibition of other AD pathological features such as abnormal phosphorylation of cytoskeletal tau, microgliosis, reactive astrocytosis, reduced levels of synaptic marker proteins and memory deficits. 1. Aβ production

Aβ peptides are derived from processing of an amyloid precursor protein (APP). Although there are several isoforms of APP, forms that contain a single-transmembrane protein have an approximately 590-680 amino acid long extracellular amino-terminal domain and an approximately 55 amino acid cytoplasmic tail which contains intracellular trafficking signals. Within APP, the Aβ peptide sequence is located partially on the extracellular side of the membrane and extends partially into the transmembrane region. Positions 29-42 on the Aβ peptide lie entirely within the putative transmembrane region and are hydrophobic in nature (Miller et al. (1993) Arch. Biochem. Biophys 307:41-52). mRNA generated from the APP gene on chromosome 21 undergoes alternative splicing to yield about 10 possible isoforms, three of which (the 695, 751, and 770 amino acid isoforms; see SEQ ED NOs: 30, 28 and 2, respectively, for exemplary amino acid sequences) predominate in the brain. APP₆ s is the shortest of the three isoforms and is produced mainly in neurons. Alternatively, APP₇₅ι, which contains a Kunitz-protease inhibitor (KPI) domain, and APP₇₇o, which contains both the KPI domain and an MRC- OX2 antigen domain, are found mostly in non-neuronal glial cells. All three isoforms share the same Aβ, transmembrane, and intracellular domains and are thus all potentially amyloidogenic. APP is trafficked through the constitutive secretory pathway, where it undergoes post-translational processing including a variety of proteolytic cleavage events. APP can undergo proteolytic processing via two pathways: an amyloidogenic pathway and a non- amyloidogenic pathway. In the non-amyloidogenic pathway, cleavage of APP by a- secretase occurs within the Aβ domain releasing a large soluble N-terminal fragment (sAPPo;) for secretion and a non-amyloidogenic C-terminal fragment (C83) of about 10 kD. Because c-secretase cleaves within the Aβ domain, this cleavage precludes Aβ formation. Rather, the C-terminal fragment of APP generated by α-secretase cleavage is subsequently cleaved by γ-secretase within the predicted transmembrane domain to generate a 22-24 residue non-amyloidogenic peptide fragment termed p3. Alternatively, in the amyloidogenic pathway, cleavage of APP by β-secretase (BACE) occurs at the beginning of the Aβ domain defining the amino terminus of the Aβ peptide. This cleavage generates a shorter soluble N-terminus, APPβ, as well as an amyloidogenic C- terminal fragment (C99). Further cleavage of this C-terminal fragment by γ-secretase, a presenilin-dependent enzyme, generates Aβ. Cleavage by distinct γ-secretase activities and/or multiple γ-secretases results in

C-terminal heterogeneity of Aβ, generating fragments of various lengths. For example, Aβ40 and Aβ42, which contain 40 and 42 amino acids, respectively (see, e.g., SEQ ED NO: 4; amino acids 1-40 and 1-42), are thought to be cleaved by a cysteine protease and a serine protease, respectively (Figueiredό-Pereira et al. (1999) J. Neurochem. 72(4) : 1417-22) . Thus, selective modulation of the production of a particular form of Aβ should be possible by targeting appropriate enzymes.

The predominant forms of Aβ found in plaques are the Aβ40 and Aβ42 variants. Aβ42 accumulates primarily intracellularly, representing only 5-15% of the total Aβ secreted by most cell lines (Wang, et al, (2001) Neurobiology of Aging 23:213-223). Published immunohistochemical studies have demonstrated that in brains of individuals harboring FAD-linked mutations in APP (Val to He at codon 717), Aβ42 is deposited early and selectively in the cerebral cortex. This holds true in numerous studies with transgenic mice and in FAD patients harboring mutations in presenilin genes known to increase Aβ42 formation (relative to Aβ40). In the AD cerebral cortex, virtually all AD plaques are Aβ42 immunopositive while only approximately one third are Aβ40 immunopositive. In fact, diffuse amyloid plaques, representing the earliest stage of amyloid deposition, are almost exclusively composed of Aβ42 (Iwatsubo et al. (1994) Neuron 13: 45053; Borchelt et al. (1997) Neuron 19: 939). i vitro experiments have demonstrated that Aβ42 polymerizes faster than Aβ40, suggesting that the carboxy terminus of Aβ determines the aggregation potential, and therefore, is one of the critical determinants for the rate of amyloid fibril formation (Parvathy, et al, (2001) Arch Neurol. 58: 2025-2032). Aβ42 has also been shown to dramatically enhance precipitation of Aβ40 in vitro. Therefore, the Aβ42 species of amyloid peptide is a primary target in the development of therapeutics for the treatment of neurodegenerative disease characterized by Aβ plaque formation. Aβ42 accumulation predominantly affects neurons in the cerebral cortex and hippocampus of AD brains prior to the appearance of amyloid plaques. Neurons burdened with excessive Aβ42 can lose function and eventually undergo lysis, resulting in local dispersal of their cytoplasmic contents. Production of Aβ can occur at several distinct locations along the secretory pathway. APP produced in the endoplasmic reticulum (ER) transits to the Golgi, where it is post-translationally modified via N- and O-linked glycosylation and tyrosine sulfation before vesicular transport to the cell surface. Cell surface APP is then reinternalized via endocytosis into the endosomal/lysosomal system where it may be degraded. Cleavage of APP to form Aβ can occur in at least three sites along this pathway. The endosomal-lysosomal system may contribute minor amounts of secreted Aβ, particularly in non-neuronal cells. The trans-Golgi network (TGN) is the major site of intracellular Aβ40 production in neurons and in non-neuronal cells transfected with mutant APP. In addition, either the TGN or post-Golgi vesicles are responsible for the production of secreted Aβ in neurons. Finally, the ER is a site for the production of Aβ42. Aβ42 produced in the ER is found in an intracellular stable insoluble pool. The proteosome may aid in the degradation of these ER-generated APP fragments (Skovronsky (2000) Biochemistry 39(4): 810-7). Due to the organdie-specific differences in the generation and clearance/degradation of Aβ peptides, it is possible to selectively modulate the production, clearance and/or degradation of a particular form of Aβ by targeting appropriate γ-secretases and/or degradative enzymes.

Presenilins, multitransmembrane proteins localized predominantly to the ER and Golgi, play a crucial role in APP processing. Presenilin-1 (PS-1) was first identified as an early onset gene in Alzheimer's disease and is believed to be a critical component of the enzyme complex which cleaves the amyloid precursor protein (APP) at the γ- secretase site to produce Aβ. Over 40 dominant point mutations in PS-1 (chromosome 14) and PS-2 (chromosome 1) as well as one splice site mutation in PS-1 have been associated with familial AD (FAD) phenotypes (see, e.g., Van Gassen et al. (2000) Neurobiol Dis. 7:135-151; Hardy (1997) Trends Neurosci. 20:154-159; and Cruts and Van Broeckhoven (1998) Ann. Med. 30:560-565). Thus, presenilins are involved in the carboxy-terminal cleavage of APP in both normal and pathological states. Involvement of presenilin has also been shown in the cleavage of additional membrane proteins such as Notch, Erb-B4 (Lee et al. 2002, J. Biol. Chem. 277(8):6318-23), and E-cadherin (Marambaud et al. 2002, EMBO J. 27f<5 :1948-56). Presenilins may play a general role in intramembrane cleavage and, thus, may likely have additional substrates yet to be reported.

2. Aβ degradation/clearance

The accumulation of Aβ42 in the brain clearly depends on the production levels of the amyloid peptide, however numerous other factors also contribute significantly to brain Aβ42 levels. Some of these factors are Aβ42 proteolytic degradation, receptor- mediated clearance, non-receptor-mediated clearance, and/or aggregation/fibrillogenesis. Therefore, defects in pathways for Aβ degradation and clearance could underlie some or many cases of familial and sporadic AD as well as other diseases and disorders characterized by misregulation of Aβ. Understanding how Aβ degradation and clearance is regulated in the cerebral cortex has implications for both the pathogenesis and the treatment of such diseases and disorders. Agents that affect any of these pathways/mechanisms can be useful as therapeutic drugs.

Metabolic labeling studies in living mice show that newly generated Aβ is very rapidly turned over in the brain (Savage et al, (1998), J. Neurosci 75:1743-1752), suggesting that Aβ-degradation proteases help regulate its levels. There are numerous proteases in the brain that could potentially participate in Aβ turnover, and there is evidence that several enzymes may contribute to the degradation of Aβ peptides in brain tissue including insulin-degrading enzyme (EDE), neprilysin, plasmin, uPA/tPA, endothelin converting enzyme-1, and matrix metalloproteinase-9 (Selkoe j. (2001) Neuron 32:177-180). EDE has been shown to degrade insulin, glucagon, atrial naturetic peptide, calcitonin, TGF-o and amylin, among other small peptides of diverse sequence. EDE is believed to have little dependence on sequence specificity but recognizes a conformation that is prone to conversion to a β-pleated sheet structure. Such a property is concurrent with its propensity to degrade several peptides that undergo concentration dependent formation of amyloid fibrils (e.g., insulin, ANF, amylin, calcitonin, and Aβ). It believed that the motif recognized by EDE is not the β-pleated sheet region per se but a conformation of the monomer in a pre-amyloid state. IDE occurs principally in a soluble form in the cytoplasm. However, a form of IDE can be labeled on the cell surface, including in neurons, and is also present on intracellular membranes (Vekrellis et al. (2000) J. Neurosci. 20: 1657-1665). The existence of a membrane-anchored form of the protease suggests that it could help regulate insulin signaling at the plasma membrane and could also participate in the degradation of both soluble and membrane-associated forms of Aβ.

Neprilysin is a member of the neutral endopeptidase family of membrane- anchored proteases found on the cell surface. Neprilysin has been implicated in the degradation of Aβ peptides (Iwata et al. , (2000) Nat. Med. 6: 143-150; Carson and

Turner, (2002) J. Neurochem 81(1): 1-8), mediating the degradation of predominantly insoluble forms of Aβ. In addition, it has been shown that steady state levels of endogenous Aβ are elevated in the brains of young neprilysin-deficient mice (Iwata et α/.(2001) Science 292: 1550-1552). The rise, while highly significant, was not large, and plaque formation was not observed. Thus, it is believed that other proteases, including additional members of the neutral endopeptidase family, may function to degrade Aβ. The plasmin proteolytic cascade, known to be crucial for fibrinolysis and cell migration, has been implicated in Aβ clearance as well. In this cascade, either of two activators of plasmin, tissue-type plasminogen activator (tPA) or urokinase-type plasminogen activator (uPA), can be post-translationally activated by binding to fibrin and other substrates, hi vitro studies have suggested that Aβ aggregates can substitute for fibrin aggregates in activating tPA. In the nervous system, plasminogen, tPA and uPA are expressed in neurons, and tPA is also synthesized by microglia. In vitro assays have indicated that pure plasmin can proteolyze monomeric Aβ and fibrillar Aβ at a considerably lower efficiency.

Another protease expressed in brain that has been evaluated for its ability to degrade Aβ is endothelin converting enzyme- 1 (ECE-1) (Eckman et al. (2001) J. Biol. Chem. 276: 24540-24548). This integral membrane zinc metalloprotease, with its active site located in the lumen and extracellularly, can cleave the endothelin precursors and several other biologically active peptides, including bradykinin, substance P, and the oxidized insulin B chain. Cellular overexpression of ECE-1 leads to a marked reduction in the levels of naturally secreted Aβ40 and Aβ42 peptides in Chinese hamster ovary cells. The purified enzyme directly proteolyzed both synthetic peptides in vitro. Other purified proteases that have been reported to digest synthetic Aβ peptides under in vitro conditions include matrix metalloproteinase-9 and cathepsin D.

In addition, several cell surface receptors have been implicated in Aβ clearance, including the scavenger receptor A (Paresce et al, (1996) Neuron 7:553-565), the receptor for advanced glycation endproducts (RAGE) (Yan et al., (1996) Nature 382: 685-691), and the low-density lipoprotein receptor-related protein-1 and -2 (LRP-1 and LRP-2) (Narita et al, (1997) J. Neurochem. 69: 1904-1911; Shibata et al, (2000) J. Clin. Invest. 106(12): 1489-99; Kang et α/., (2000) J. Clin. Invest. 106(9): 1159-66; Ulery and Strickland, (2000) J. Clin. Invest. 106(15): 1077-9; Hammad et al, (1997) J. Biol. Chem. 272(30): 18644-9). Scavenger receptor binding to Aβ has been shown to facilitate the uptake of Aβ by microglia. Microglia are immune system cells associated with Alzheimer's disease plaques containing Aβ. These cells facilitate phagocytosis of amyloid fibrils into the endosomal/lysosomal system where they may subsequently be degraded by acid hydrolases in late endosomes and lysosomes (Selkoe (2001) Neuron 32: 177-180). The scavenger receptors expressed by microglia appear to play a significant role in this clearance process and, thus may be useful targets for the identification of agents that modulate Aβ levels.

Binding of Aβ to neuronal RAGE induces activation of nuclear factor KB (NF- KB), which drives expression of macrophage-colony stimulating factor (M-CSF). M- CSF signals microglia from distant sites, drawing them toward loci of neuronal perturbation and inducing cell activation, including increased proliferation, and enhanced expression of microglial scavenger receptors and apoE. Such activation may lead to increased clearance of Aβ through microglial phagocytic pathways.

LRP is a multifunctional receptor with four distinct ligand binding domains and at least 14 identified ligands, including apolipoprotein E (apoE), apoJ, cβ-macroglobulin (o2M), and lactoferrin. LRP is involved in receptor-mediated endocytosis, directing ligands to degradation via the late endosome and lysosome. Aβ has been found to bind several LRP ligands including apoE (Holtzman, (2001) J. Mol Neurosci. 17(2): 147-55), apoJ (Hammad et al, (1997) J. Biol. Chem. 272(30): 18644-9), and activated cfiM (α2M*) (Qiu et al, (1999) J. Neurochem 73f4):1393-8). Such ligand interactions, and specifically the binding of Aβ to o£2M*, are believed to facilitate Aβ clearance through an LRP-mediated endocytic pathway. Identification of agents which modulate LRP and/or components of LRP-mediated clearance pathways provides an attractive approach for therapeutic intervention.

The proteosome has also been implicated in the degradation of ER-generated APP fragments, specifically Aβ42 (Skovronsky (2000) Biochemistry 39f4):810-7). General phagocytic mechanisms and up-regulation of genes in response to inflammatory stimuli are also reported to enhance Aβ clearance, i addition, metal chelators, such as clioquinol (Cherny et al. (2001) Neuron 30:655-61), are believed to play a role in dissolving plaques and/or preventing Aβ aggregation. 3. Reduction of Aβ accumulation Based on the strong correlation between Aβ accumulation, neuronal loss and AD, a reduction in Aβ accumulation should result in decreased plaque formation and minimize neuronal cell death. There are, however, numerous mechanisms and activities which may influence brain Aβ levels, and these mechanisms can influence many other important cellular functions and processes. For example, production of an intracellular C-terminal fragment (CTF) of APP resulting from γ-secretase cleavage between amino acids 49 and 50, close to the cytoplasmic side of the transmembrane domain, is believed to play a role in signal transduction (Pinnix, I et al. (2001) J. Biol. Chem 27(5:481-487; Sasτre, M. et al. (2001) EMBO Reports 2(9j:835-41; Gu, Y et al. (2001) J. Biol Chem. 276(38): 35235-8; Cao, X and Sudhof, T.C. (2001) Science 293:115-120). Inhibition of such cleavage may result in unwanted side affects. It is, therefore, important when seeking agents for altering Aβ levels to identify agents that act specifically on the Aβ endpoint with minimal disruption of other, often overlapping, cellular pathways and processes. Due to the high degree of regulation of and organelle-specific differences in the generation, clearance, and degradation of the various Aβ peptides, identification of agents that target appropriate production enzymes, degradative enzymes, and/or related proteins and receptors involved in Aβ production and clearance pathways should make possible modulation of the production, clearance and/or degradation of one or more Aβ peptides without substantially affecting other cellular compositions, processes and activities. One approach to treating diseases associated with Aβ-based amyloidosis, such as

Alzheimer's disease, is aimed at reducing Aβ peptide production by targeting presenilin function. However, because presenilin and presenilin-dependent activities affect substrates other than APP, non-specific modulation (such as, for example, inhibition) of presenilin and/or presenilin-dependent mechanisms can result in unwanted side effects. Furthermore, because γ-secretase generates normal non-amyloidogenic peptides, such as p3 and APP CTF, non-specific modulation of γ-secretase may be undesirable. In addition, because release of Aβ peptides is a normal event in virtually every cell, it may be desirable in some instances to maintain or even elevate levels of particular Aβ peptides. There is a need for agents that modulate the levels of one or more Aβ peptides of cells and tissues (intracellular, extracellular, and/or membrane-bound Aβ), for example, by modulating compositions (e.g., proteases and proteins, such as proteins on which protease activities depend, including presenilins), mechanisms and/or activities involved in Aβ peptide formation and persistence in cells and/or extracellular medium without substantially affecting (or with only limited or minimal effect on) compositions, mechanisms and/or activities that are not significantly involved in Aβ peptide formation and persistence. There is particularly a need for agents that modulate the levels of Aβ42 peptide in cells and/or extracellular medium without substantially affecting (or with only limited or minimal effect on) compositions, mechanisms, processes and/or activities that are not significantly involved in Aβ42 peptide generation and persistence in cells and/or extracellular medium. Such agents have numerous uses. For example, such agents can be used in elucidating the precise elements and pathways involved in Aβ peptide formation, degradation and clearance in cells. Furthermore, such agents are candidates for the prevention and/or treatment of diseases and disorders involving amyloidosis, such as, for example, AD. Such agents can provide therapeutic and/or prophylactic benefit with limited-to-no potential side effects that can result from non-specific modulation of Aβ peptide processing and/or clearance.

Provided herein are methods of identifying agents that modulate the levels (including, e.g., cellular and/or extracellular) of one or more Aβ peptides. In particular embodiments, the methods can be used to identify agents that modulate the levels of

Aβ42 (including cellular and/or extracellular). In further embodiments, the methods can be used to identity agents that selectively modulate the levels of Aβ42 (including cellular and/or extracellular).

In another embodiment, the methods can be used to identify agents that modulate Aβ peptide levels (and, in particular, Aβ42 levels) without substantially affecting (or with limited, minimal or inconsequential effect on) compositions, mechanisms, processes and/or activities that are not significantly involved in the generation, degradation and/or clearance of one or more Aβ peptides. A composition, mechanism, process or activity that is not significantly involved in the generation, degradation and/or clearance of an Aβ peptide can be, for example, one that has minimal effect on the generation, degradation and/or clearance of an Aβ peptide. Thus, for example, if the generation, degradation and/or clearance of an Aβ peptide does not differ substantially in the presence and absence of a particular composition, mechanism, process or activity, then the composition, mechanism, process or activity may not be significantly involved in the generation, degradation and/or clearance of an Aβ peptide. In a particular embodiment, the method involves a step of identifying an agent that modulates the levels (including e.g., cellular and/or extracellular) of one or more Aβ peptides without substantially altering the substrate-processing activity of presenilin. The method can involve a step of identifying an agent that modulates the levels of one or more Aβ peptides without substantially altering the cleavage of a presenilin substrate, or portion(s) thereof, that is other than APP. In a further embodiment, the presenilin substrate is LRP. In another embodiment, the method involves a step of identifying an agent that modulates the levels (including, e.g., cellular and/or extracellular) of one or two Aβ peptides, without substantially altering the levels of one or more other Aβ peptides. In a particular embodiment, an agent that modulates the levels of Aβ42 only, or Aβ39 only, or Aβ42 and Aβ39 only, without substantially altering the levels of one or more other Aβ peptides, is identified. The agent can be, for example, one that modulates the levels of Aβ42 and/or Aβ39 without substantially altering the levels of Aβ40.

Also provided herein are methods of modulating Aβ peptide levels (including, e.g., cellular and/or extracellular Aβ). hi one embodiment, the method includes a step of contacting a sample, for example, a cell, with an agent that modulates the level of one or more Aβ peptides, in particular, Aβ42 and/or Aβ39, without substantially affecting or altering the level of one or more different Aβ peptides. For example, the method can include a step of contacting a sample, for example, a cell, with an agent that modulates Aβ42 and/or Aβ39 levels without substantially altering the levels of Aβ40. In another embodiment, the methods include a step of contacting a sample (e.g., a cell) with an agent that modulates the level of one or more Aβ peptides, particularly Aβ42, without substantially affecting a non- APP substrate-processing activity of presenilin. The methods can include a step of contacting a sample with an agent that modulates the level of one or more Aβ peptides without substantially affecting the cleavage and/or processing of a presenilin substrate other than APP. In a particular embodiment, the presenilin substrate is LRP.

Further provided herein is an antibody that selectively recognizes Aβ42 without substantially binding to other Aβ peptides. The antibody has numerous uses and provides specific advantages as compared to other antibodies. For example, the antibody can be used in methods of identifying agents that modulate Aβ42 levels without substantially affecting the level of other Aβ peptides. The antibody can further be used in methods of detecting Aβ42 in a sample for any purpose, including but not limited to methods of diagnosis of diseases and disorders involving amyloidosis, for example, AD. Also provided herein are compositions and methods for assessing presenilin activity and/or presenilin-dependent activity. In one embodiment, the methods involved determining the presence or absence and/or level of one or more fragments of LRP and/or the composition of LRP in a sample for which presenilin activity is being assessed. The methods can be used in methods for identifying or screening for agents that modulate presenilin and/or presenilin-dependent activity that are also provided herein. As described herein, presenilins are proteins that are involved in the processing of a number of proteins with various functions and activities, including not only APP but LRP. Because presenilins are involved in diverse reactions with a variety of substrates, it is desirable to identify agents that affect presenilin activity and presenilin-dependent mechanisms. A method provided herein for identifying agents that modulate presenilin and/or presenilin-dependent mechanisms is based on the finding described herein that LRP is a substrate that is processed in a presenilin-dependent mechanism. In one embodiment, the method includes a step of comparing the levels and/or composition of LRP C-terminal fragments in samples containing presenilin that have been contacted with a test agent and samples containing presenilin that have not been contacted with test agent. The methods for identifying an agent that modulates presenilin activity can be applied to methods for identifying candidate agents for the treatment or prophylaxis of a disease or disorder associated with altered presenilin. One embodiment of these methods includes steps of contacting a sample containing LRP and an altered presenilin that is associated with altered LRP processing with a test agent and identifying a candidate agent that restores LRP processing to that which occurs in the presence of a presenilin that is not associated with altered processing of LRP.

C. Methods of Assessing Presenilin and/or Presenilin-Dependent Activity

Presenilins are transmembrane proteins localized predominantly in the ER and Golgi. Included among the presenilin proteins are the homologous presenilin-1 (PS1) and presenilin-2 (PS2) proteins (see SEQ ID NO: 6 for an amino acid sequence of a PS 1 protein and SEQ ED NO: 8 for an amino acid sequence of a PS2 protein). Although the presenilin proteins alone may not have an enzymatic activity, they appear to play an essential role in the proteolytic processing of a variety of proteins, including APP (particularly the γ-secretase cleavage of APP) and in the trafficking and maturation of various cellular proteins (referred to herein collectively as substrates for presenilin activity and/or presenilin-dependent enzyme activity), including, but not limited to Notch, TrkB, APLP2, hlrelo E-cadherin and Erb-B4. With respect to processing of APP, it appears that presenilin participates intimately as part of a catalytic complex by which γ-secretase mediates an intramembranous proteolysis of APP. Two transmembrane aspartate residues (D257 and D385 in PS1; D263 and D366 in PS2) are individually critical for presenilin-associated γ-secretase activity as well as presenilin endoproteolysis.

Inherited mutations in the genes encoding presenilins- 1 and -2 account for up to 40% of the early onset cases of familial Alzheimer's Disease (FAD). FAD-associated mutations in PS1 and PS2 give rise to an increased accumulation of Aβ42 in AD patients and transfected cell lines and transgenic animals expressing FAD mutant forms of PS1 or PS2.

Because presenilins and presenilin-dependent activities play a key, yet mechanistically unresolved, role in the cleavage of numerous proteins involved in a variety of processes (some.of which are associated with diseases such as Alzheimer's Disease), there is a need for compositions and methods that can be used in assessing presenilin activity. For example, assessment of presenilin activity using such compositions and methods can greatly facilitate the elucidation of the mechanisms of protein processing in normal and disease states and the determination of the number, specificities, regulation and potential overlap of the proteolytic activities that function in the cleavage of an array of transmembrane proteins. Furthermore, compositions and methods for the assessment of presenilin activity may also be used in screening of agents that specifically modulate various presenilin-dependent enzyme activities. Such agents may also be of use in elucidating the mechanisms of protein processing in normal and disease states. In addition, such agents can be candidate agents for the prevention and/or treatment of diseases associated with altered proteolytic processing of cellular proteins, such as, for example, diseases involving amyloidosis, including AD.

Provided herein are compositions and methods for assessing presenilin activity and/or presenilin-dependent activity. In one embodiment, the methods involve determining the level of one or more fragments of LRP and/or the composition of LRP in the presence of a sample for which presenilin activity is being assessed. Determining the level can be determining the presence or absence of one or more fragments as well as making a more quantitative assessment of amount of the fragment. The methods are based on the finding described and demonstrated herein that the low density lipoprotein receptor-related protein (LRP) is processed by a presenilin-dependent enzyme activity. In a particular method for assessing presenilin activity, the level of a fragment of LRP that is from a C-terminal portion of LRP is determined, such as, for example, an approximately 20 kD-fragment. Also provided is a method of identifying agents that modulate presenilin activity and/or presenilin-dependent activity which involve comparing, in the presence and absence of test agents, the level of one or more fragments of LRP and/or composition of LRP in the presence of a presenilin activity, hi a particular embodiment, the level of a fragment of LRP that is from a C-terminal portion of LRP is determined, such as, for example, an approximately 20 kD-fragment. Determining the level for any of these methods can be determining the presence or absence of one or more fragments as well as making a more quantitative assessment of amount of the fragment.

Further provided is a method for identifying candidate agents for the treatment and/or prevention of a disease or disorder, such as a disease or disorder associated with altered presenilin function or activity, which includes a step of comparing, in the presence and absence of test agents, the level of one or more fragments of LRP and/or composition of LRP in the presence of a presenilin encoded by a mutant or polymorphic nucleic acid. In a particular embodiment, the level of a fragment of LRP that is from a C-terminal portion of LRP is determined, such as, for example, an approximately 20 kD- fragment. In particular embodiments, the disease or disorder is associated with amyloidosis, for example, Alzheimer's disease. The mutant nucleic acid can be, for example, one that encodes a presenilin that is linked to Alzheimer's disease. For example, the mutant nucleic acid may encode any one or more of the at least 60 mutations in human PS1 and the at least two mutations in human PS2 that have been genetically linked to early onset familial Alzheimer's disease (FAD) (see, e.g., Van Gassen et al. (2000) Neurobiol. Dis. 7:135-151; Checler (1999) IUBMB Life 48:33-39; St. George-Hyslop (2000) Biol. Psychiatry 47:183-199; Steiner et al. (1999) Eur. Arch. Psychiatry Clin. Neurosci. 249:266-270). Included among such mutations are the PS2 FAD mutation N141I (Volga German FAD mutant) and the PS1 FAD mutation M146L. Additional methods of assessing presenilin activity and/or presenilin-dependent enzyme activity involve determining the levels and/or compositions of fragments of other presenilin substrates. Presenilin substrates are peptides, polypeptides, proteins or fragments thereof that are proteolytically processed, at least in part, in a presenilin- dependent manner. Thus, if presenilin is absent, or presenilin activity is inhibited or reduced, the proteolytic processing of a presenilin substrate is altered, for example by an alteration in the levels and/or composition of fragments generated from the substrate, relative to the proteolytic processing of the substrate that occurs in the presence of normal (e.g., wild-type) presenilin activity. Generally, a presenilin substrate can contain about one transmembrane domain, an ectodomain that is released or shed into the extracellular medium, and/or an intracellular domain. Typically, processing of a presenilin substrate includes an initial cleavage of the substrate (typically by a metalloprotease) at a site located in the extracellular domain of the substrate to release an ectodomain of the substrate, followed by presenilin-mediated cleavage of the remaining membrane-bound portion of the substrate to yield an intracellular fragment, which may be translocated to the nucleus of a cell. 1. LRP Assay a. LRP

Low density lipoprotein receptor-related protein (LRP) is a cell surface receptor that binds and internalizes a number of diverse extracellular ligands, including apolipoprotein E (apoE), cβ-macroglobulin (o_2M), APP, tissue-type plasminogen activator (tPA) and lactoferrin, for degradation by lysosomes. LRP expression is widespread; however, it is most highly expressed in the liver, brain and placenta. With respect to its expression in the brain, LRP is primarily a neuronal receptor expressed in the cortex and hippocampus and is also expressed in activated astrocytes, glia and microglia. Mature LRP is a heterodimer containing an N-terminal 515 kD extracellular subunit ( chain) and a C-terminal 85 kD membrane-anchored subunit (β chain) which are non-covalently associated. The mature receptor is generated by proteolytic cleavage of a 600 kD precursor polypeptide in a trans-Golgi compartment in a process that involves the endoproteinase furin. The amino acid sequence of the LRP precursor polypeptide is provided in SEQ ED NO: 10 (see also GenBank Accession No. Q07954), and DNA encoding the polypeptide is provided in SEQ ID NO: 9. Proteolytic processing of precursor LRP to yield the mature receptor occurs at amino acid position 3925 C- terminal to the tetrabasic amino acid sequence RHRR. LRP is anchored in the plasma membrane by a single transmembrane domain, and its cytoplasmic tail includes two copies of the internalization signal NPXY. Additionally, LRP undergoes another proteolytic processing step at the cell surface which involves a metalloproteinase (Quinn et al. (1999) Exp. Cell. Res. 257:433- 441). This processing results in "shedding" from the cell surface of a portion of LRP containing the chain (an ~500-kD soluble polypeptide) that is noncovalently associated with a truncated β chain (the extracellular portion of the β chain (i.e., amino acids 3944- 4420 of SEQ ID NO: 10; Mr =~67 kD or Mr =~55 kD after deglycosylation withN- glycosidase F).

LRP is a member of the low-density lipoprotein receptor (LDLR) family. The extracellular region of receptors in this family contains several structural modules which include ligand-binding repeats of -40 amino acids (including six cysteine residues forming three disulfide bonds), epidermal growth factor (EGF) precursor repeats (each also containing six cysteine residues), and modules with a consensus tetrapeptide (YWTD). In addition to these modules, these receptors contain a single transmembrane domain and a relatively short cytoplasmic tail with endocytosis signals and elements for interaction with cytoplasmic adaptor and scaffold proteins (e.g., Dab, FE65, c-jun N- terminal kinase interacting proteins (JIPs) and postsynaptic density protein PSD-95) for mediating signal transduction.

LRP may have a significant role in the pathogenesis of AD. Several LRP ligands, including apoE, lactoferrin and o2M, bind Aβ. Such ligand interactions are believed to facilitate Aβ clearance through an LRP-mediated endocytic pathway (Qiu et al. (1999) J. Neurochem. 73:1393-8). LRP levels are reduced in AD and in τransgenic mice expressing presenilin and cells fransfected with presenilin-encoding DNA. Furthermore, transgenic mice overexpressing the M146L or L286V presenilin-1 mutations associated with AD reportedly have decreased levels of LRP expression in certain neuronal populations. LRP also interacts with APP via adaptor proteins, such as FE65. hi addition, genetic association studies indicate that the LRP gene may be a susceptibility locus for late-onset AD. b. LRP is processed by a presenilin-dependent activity As described and demonstrated herein (see the EXAMPLES), LRP is processed by a presenilin-dependent enzyme activity. LRP processing was analyzed in cell lines expressing defective (i.e., loss of function) PSl proteins encoded by nucleic acid lacking exons 1 and 2 (see, e.g., GenBank Accession No. L76518 for sequences of exons 1 and 2) of the PSl -encoding DNA or nucleic acid coding for an alanine instead of an aspartic acid residue at amino acid 385 (D385A), which is essential to PSl function. These cells had been generated by transfecting mouse neuroblastoma (N2a) cells (see, e.g., ATCC, Rockville, MD), which express endogenous LRP, with nucleic acid encoding wild-type human APP695 and nucleic acid encoding human PS-1 (wild-type, D385A mutant, or exon 1 and 2 deletion). It was discovered that LRP processing is altered in the cells expressing defective PSl proteins relative to cell lines expressing normal wild-type PSl. Specifically, an ~20-kD peptide was detected in an immunoassay of lysates of cells that had been fransfected with mutant PSl -encoding DNA that was not detected (or detected at much lower levels) in lysates of cells that had been fransfected with wild-type PS 1 - encoding DNA. The detection antibody (R9377) was one generated against the carboxyl- terminal 13 amino acids of human LRP. Because the ~20-kD peptide from a C-terminal portion of LRP, which contains an epitope recognized by an antibody generated against the C-terminal 13-amino acids of LRP, was absent or only barely detectable in lysates of cells expressing a wild-type PSl, but present at readily detectable levels in lysates of cells that contain mutant PSl protein, it appears that a PSl -dependent activity cleaves LRP in such a way as to eliminate an amino acid sequence in a C-terminal region of LRP that is recognized by C-terminus-reactive antibody. The processing of APP and Notch, two substrates for presenilin-dependent processing activity, was also analyzed in these cells, in addition to the analysis of LRP processing. Analogous results, in which particular C-terminal fragments of APP and Notch were detected in lysates of PSl mutant cells but not in lysates of wild-type PSl cells, were obtained in analyses of APP and Notch processing. Thus, the results revealed a concordance of the activity of PS-1 with the three substrates. The similar findings support a conclusion of a presenilin- dependent cleavage of LRP. It was also found that the LRP β chain alone is sufficient for processing by PS-1, and that trafficking to the plasma membrane is a necessary event for the normal processing of LRP by the PS-1 active complex.

As also described in the EXAMPLES, in the presence of the γ-secretase inhibitor DAPT (N-[N-(3,5,-difluorophenacetyl)-L-alanyl]-S-phenylglycine-t-butyl ester), an accumulation of an approximately 20 kD fragment of LRP that is from a C-terminus portion of LRP is observed. The fragment is one that is recognized and bound by a polyclonal antibody (e.g., antibody R9377 as described in the EXAMPLES) generated against a carboxyl-terminal peptide (the carboxyl-terminal 13 amino acids) of human LRP (C-GRGPEDEIGDPLA) with N-terminal cysteine added for conjugation to ovalbumin. The accumulation of the ~20-kD fragment from a C-terminal portion of LRP parallels the accumulation of APP C-terminal fragments (CTFs). This finding indicates that LRP fragment accumulation is a measure of presenilin/γ-secretase activity. Advantages of using LRP fragment analysis in a method for assessing presenilin activity include: (1) LRP is highly expressed in adult brain, (2) the analysis is easily amenable to testing in vivo samples, (3) endogenous LRP is expressed at sufficient levels in cell culture models such that transfection the cells with nucleic acid encoding LRP in order to increase expression levels for detection is not necessary, and (4) LRP appears to have a significant role in the pathogenesis of AD. Furthermore, LRP has been shown to have a potentially significant role in the clearance of Aβ as described above. c. Methods of modulating LRP

Because of the involvement of LRP in critical cellular processes, including, but not limited to, signal transduction and receptor-mediated endocytosis, and in mechanisms associated with Alzheimer's disease, there is a need for methods and compositions that can be used in modulating LRP. LRP modulation can be any alteration of LRP, including, but not limited to, any alteration in the processing, structure, function (including, for example ligand-binding) and/or activity (including, for example, signal transduction and receptor-mediated endocytosis) of LRP. Modulation of LRP has numerous uses. For example, the ability to modulate LRP can greatly facilitate the elucidation and detailed characterization of the mechanisms involved in signal transduction and receptor-mediated endocytosis. Furthermore, modulation of LRP has applications in the treatment and prophylaxis of diseases of signal transduction and endocytosis, as well as AD.

As described and demonstrated herein, LRP is processed by a presenilin- dependent enzyme activity. The processing of LRP can have significant effects on its structure, function and activity.

Provided herein are methods for modulating LRP. The LRP can be in a sample that has been selected for LRP modulation. Such samples include, but are not limited to, cells, tissues, organisms, lysates, extracts and membrane preparations of cells and cell- free samples containing LRP, including, for example, extracellular medium, tissue and body fluids. In one embodiment, the methods involve altering the structure, function and or activity of a presenilin (and/or fragments thereof) in a sample containing LRP, and/or fragment(s) thereof, and a presenilin, and/or fragment(s) thereof, whereby the LRP is modulated. The structure, function and/or activity of a presenilin can be altered in a number of ways which can vary depending in large part on the sample. For example, the function and activity of presenilins (particularly functions and activities relating to interaction of presenilin with other molecules) can be altered by contacting presenilin with antibodies, and/or fragment(s) thereof, that bind presenilin, particularly antibodies that bind to presenilin in such a way as to impede or eliminate the ability of presenilin to interact with binding partners. If the sample is a cell, the function and/or activity of presenilin in the cell can be altered, for example, by enhancing, increasing, reducing or eliminating the expression of the presenilin. Methods are known in the art for transferring nucleic acids encoding presenilin into cells and for reducing or eliminating the expression of functional proteins, such as presenilin, in cells (e.g., gene knock-out, antisense RNA and RNA interference techniques). hi another embodiment, the methods involve contacting a sample containing an LRP, and/or fragment(s) thereof, and presenilin, and/or fragment(s) thereof, with an agent that modulates presenilin or presenilin-dependent activities. The sample is one that has been selected for LRP modulation. An agent that modulates presenilin or presenilin- dependent activities can be identified using methods provided and described herein. d. Assessment of presenilin activity based on LRP In a method for assessing presenilin activity provided herein, the level of one or more fragments of LRP and/or the composition of LRP is determined for a sample for which presenilin activity is being assessed. Examples of a sample for which presenilin activity is being assessed include, but are not limited to, a cell that expresses presenilin, a lysate or extract of a cell that expresses presenilin, or membranes prepared from a cell that expresses presenilin. The cell can endogenously express presenilin and/or express heterologous presenilin. LRP can be added to the sample or can be expressed endogenously and/or heterologously by the cell. In a particular embodiment, the method includes assessing presenilin activity of a cell by evaluating the level (which includes determining the presence or absence of) of a fragment from a C-terminal portion of LRP in a cell lysate.

To assess presenilin activity in these methods, the processing of LRP is evaluated. In evaluating LRP processing, the composition of LRP can be evaluated. The composition of LRP refers to the make-up of any LRP that is present anywhere in the analysis mixture, including the assay medium in which the analysis is being performed, an extracellular medium, or a cell membrane, lysate or extract. Thus, in one embodiment, the structure of any LRP present can be evaluated to, for example, determine whether LRP is intact or has been processed and appears as a fragment or fragments of sizes smaller than the intact LRP molecule or than either one or both of the intact chains of LRP. hi evaluating LRP processing, the levels (including the presence or absence) of one or more LRP fragments can be determined.

In particular, the LRP composition is evaluated to determine if any fragment(s) indicative of presenilin-dependent cleavage or altered presenilin-dependent cleavage of LRP are present and/or the level of any such fragment(s). A presenilin-dependent cleavage described herein occurs within the C-terminal portion of LRP and within the β chain. Thus, a presenilin-dependent cleavage of LRP can be one that occurs in the C- terminal portion of LRP at a position C-terminal to amino acid position 3925 of SEQ ID NO: 10 (or of the amino acid sequence provided as GenBank Accession No. Q07954). The presenilin-dependent cleavage of LRP can be one that occurs within the sequence of the last approximately 580, 500, 450, 400, 350, 300, 250, 200, 150, 100, 50, 25, or less amino acids of LRP. The presenilin-dependent cleavage can be one that occurs C- terminal to the extracellular portion of the β chain (i.e., approximately amino acids 3944- 4420 of SEQ ID NO: 10 or of the amino acid sequence provided as GenBank Accession No. Q07954); thus, C-terminal to about amino acid 4420 of SEQ ID NO: 10. The presenilin-dependent cleavage of LRP can be one that occurs near or within the region of the LRP protein that extends from a point located intracellularly and adjacent to the cytoplasmic face of the cell membrane to a point located within the cell membrane. Such a presenilin-dependent cleavage of LRP can be one that generates a soluble intracellular peptide, containing the extreme C-terminus of LRP, and a membrane-associated peptide containing amino acid sequence of the transmembrane region of LRP, particularly the more C-terminal region of the transmembrane segment of LRP. Any LRP fragments generated by such presenilin-dependent activities have a molecular weight that is less than that of the β chain of LRP (β chain molecular weight is approximately 85-90 kD, or approximately 67 kD after deglycosylation with N-glycosidase F). hi particular embodiments, an LRP fragment generated by such presenilin-dependent activities has a molecular weight that is less than that of the extracellular portion of the β chain of LRP (the extracellular portion of the β chain molecular weight is approximately 67 kD, or approximately 55 kD after deglycosylation with N-glycosidase F). Thus, an LRP fragment generated by a presenilin-dependent cleavage can have a molecular weight that is, for example, less than about 85 kD, 80 kD, 75 kD, 70 kD, 65 kD, 60 kD, 55 kD, 50 kD, 45 kD, 40 kD, 35 kD, 30 kD, 25 kD, 20 kD, 15 kD or 10 kD or less. LRP fragments that are particularly indicative of a presenilin-dependent cleavage have a molecular weight that is less than about 15 kD, 13 kD, 12 kD, 10 kD or 5 kD. In a particular embodiment of a method for assessing presenilin activity provided herein, LRP processing in a sample for which presenilin activity is being assessed can be evaluated by evaluating the LRP composition to determine if any fragment(s) indicative of altered presenilin-dependent cleavage of LRP are present and/or the level of any such fragment(s). Altered presenilin activity can be, for example, an increase, reduction or elimination of presenilin activity. In a particular embodiment of this method, the presence or absence and/or the level of an LRP fragment that is cleaved in the presence of a presenilin-dependent activity (e.g., presenilin-dependent γ-secretase activity), and thus absent (or present at low levels) in the presence of the presenilin-dependent activity, but that can be detected intact when the presenilin-dependent activity is altered (such that it is reduced or eliminated) is assessed. One such fragment indicative of altered presenilin-dependent cleavage has a molecular weight of between about 25 kD and 15 kD, and, in particular, about 20 kD. The fragment can be one that is cleaved in the presence of a presenilin-dependent activity in such a way as to eliminate an amino acid sequence in a C-terminal region of LRP that is recognized by C-terminus-reactive antibody (i.e., the cleavage in the presence of a presenilin-dependent activity eliminates an epitope in the fragment that is recognized by an antibody generated against the C- terminal 13-amino acids of LRP). The LRP fragment can be one that contains an amino acid sequence located within the sequence of amino acids 4420-4544 of SEQ ED NO: 10. As described herein above, LRP is also cleaved by activities that are not presenilin dependent. Specifically, in one cleavage that is not a presenilin-dependent/γ- secretase activity, mature LRP, i.e., separate, but noncovalently associated, (N-terminal 515 kD extracellular subunit) and β (C-terminal 85 kD membrane-anchored subunit) chains, is generated by proteolytic cleavage at amino acid position 3925 (C-terminal to the tetrabasic amino acid sequence RHRR) of the 600-kD precursor polypeptide (see SEQ ID NO: 10 and GenBank Accession No. Q07954) in a process that involves the endoproteinase furin. Another cleavage of LRP that is not presenilin-dependent is the metalloproteinase-mediated proteolytic processing at the cell surface which results in "shedding" from the cell surface of a portion of LRP containing the chain (an ~500-kD soluble polypeptide) that is noncovalently associated with a truncated β chain (the extracellular portion of the β chain (i.e., amino acids 3944-4420 of SEQ ED NO: 10; Mr =~67 kD or Mr =~55 kD after deglycosylation with N-glycosidase F). A fragment such as these that does not result from a presenilin-dependent cleavage generally is not alone indicative of presenilin activity.

In methods for assessing presenilin activity provided herein that include a step of determining the level of one or more fragments of LRP and/or LRP composition, LRP protein and/or fragments thereof can be detected and/or measured by any method known to those of skill in the art for measuring protein level or by any method described herein. In a particular embodiment of the method, LRP protein or a peptide fragment thereof is detected by immunoassay. For example, an LRP fragment from a C-terminal portion of LRP is visualized by immunoblotting of cell lysates with the anti-LRP polyclonal antibody (R9377) prepared to the carboxyl-terminal 13 amino acid peptide of LRP (C- GRGPEDEIGDPLA) as described in the EXAMPLES. e. Methods for identifying or screening for agents that modulate presenilin activity Methods for assessing presenilin activity provided herein can be applied to the identification of or screening for agents that modulate presenilin activity. One method provided herein for identifying or screening for agents that modulate presenilin activity includes steps of contacting a sample containing a presenilin and a lipoprotein receptor- related protein (LRP) and/or portion(s) or fragment(s) thereof with a test agent and identifying an agent that alters the processing and/or cleavage of an LRP or fragment thereof.

A sample that can be used in the methods of identifying an agent that modulates presenilin activity can be any composition (e.g., a biological or physiological composition) that includes a source of presenilin and a source of LRP and/or portion(s) thereof. Examples of samples include, but are not limited to, a cell, a cell extract or lysate, a cellular membrane and a cell-free medium.

(1) Presenilin and LRP (and/or portion(s) thereof) Sources of presenilin and LRP include, but are not limited to: a cell that expresses endogenous or heterologous presenilin and/or LRP; a cell that expresses a recombinant portion(s) or fragments) of presenilin and/or LRP; lysates, extracts, or membrane fractions of any such cells; presenilin, LRP, or a portion thereof, that is isolated from such cells; and synthetic presenilin or LRP protein or synthetic proteins that represent a portion of presenilin or LRP.

Compositions, and methods of making compositions, that are sources of presenilin, LRP, and portion(s) thereof, are described herein and known in the art. For example, cells that endogenously express presenilin and/or LRP are known in the art as are nucleic acids encoding presenilin (see, e.g., SEQ ED NOs: 5 and 7) and LRP (see, e.g., SEQ ED NO: 9) that can be used to express the encoded proteins in cells. Methods of preparing lysates, extracts and membrane fractions of such cells are also described herein and known in the art, as are synthetic methods for generating proteins and peptides and preparatory methods of isolating proteins and peptides.

(2) Identifying an agent that alters the processing and/or cleavage of LRP (or portion(s) thereof)

In general, the step of identifying an agent that alters the processing and/or cleavage of LRP (or portion(s) thereof) can involve a comparison of the cleavage and/or processing of LRP (and/or portion(s) thereof) in a sample that has been contacted with the test agent (i.e., test sample) and in a sample that has not been contacted with the test agent (i.e., control sample). If the cleavage and/or processing of LRP (and/or portion(s) thereof) in the test and control samples differs, then the agent is identified as one that modulates presenilin activity. For example, processing of LRP and/or the level of a particular fragment of LRP in the test and control samples may differ by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or more than 75%. The control sample can be the same physical sample as the test sample or a different sample. When the control and test samples are the same, the control is the sample in the absence of test agent.

The processing or cleavage of an LRP or fragment(s) thereof can be assessed by determining the presence or absence and/or level of one or more fragments of LRP and/or the composition of LRP using, for example, materials and methods described herein. Thus, the LRP composition can be evaluated to determine if any fragment(s) indicative of presenilin-dependent cleavage of LRP or altered presenilin-dependent cleavage of LRP are present and/or the level of any such fragments. Such fragments and compositions are described herein. In a particular embodiment, the processing or cleavage of an LRP or fragment(s) thereof is assessed by determining the presence or absence and/or level of an LRP fragment that is cleaved in the presence of a presenilin- dependent activity (presenilin-dependent γ-secretase activity), and thus absent (or present at low levels) in the presence of the presenilin-dependent activity, but that can be detected intact when the presenilin-dependent activity is altered (such that it is eliminated or reduced). In one embodiment, the presence or absence and/or level of an LRP fragment having a molecular weight of between about 25 kD and 15 kD, and, in particular, about 20 kD, is assessed. Typically, the ~ 20 kD fragment is one that is present when an LRP is not cleaved by a presenilin-dependent activity, such as one that occurs in the presence of an inhibitor of a presenilin-dependent activity such as DAPT. In a particular embodiment, the fragment is from a C-terminal portion of LRP, i.e., a CTF. The LRP fragment can be one that contains an amino acid sequence located within the sequence of amino acids 4420-4544 of SEQ ED NO: 10. In a further embodiment, the fragment is one that is recognized by an antibody generated against C-terminal amino acids (e.g., the C-terminal 13 amino acids) of LRP, such as, for example, the polyclonal antibody R9377 described herein.

The methods for identifying an agent that modulates presenilin activity as provided and described herein can be applied to the identification of candidate agents for the treatment or prophylaxis of a disease associated with an altered presenilin. A particular embodiment of this method includes steps of contacting a sample containing a lipoprotein receptor-related protein (LRP), and/or fragment(s) thereof, and an altered presenilin, and/or fragment(s) thereof, that is associated with an altered processing of LRP with a test agent and identifying a candidate agent that restores LRP processing substantially to the processing that occurs in the presence of a presenilin, and/or fragment(s) thereof, that is not associated with an altered processing of LRP. The altered presenilin, and/or fragments) thereof, can be one that has an altered function or activity. Altered presenilins include, for example, a presenilin and or fragment(s) thereof containing a mutation and/or encoded by a polymorphic nucleic acid that contains a mutation. Thus, the altered presenilin and/or fragment(s) thereof, can be one that is altered relative to a wild-type presenilin. Typically, a wild-type protein, such as, for example, a presenilin protein, can be one that is encoded by a predominant allele in a population or any allele that is not associated with disease or a pathogenic condition. A wild-type presenilin can be one that occurs in an organism that exhibits normal presenilin-dependent LRP processing patterns. The altered presenilin can be, for example, one that is encoded by a nucleic acid linked to Alzheimer's disease. For example, the nucleic acid may include any one or more of the at least 60 mutations in human PS 1 and the at least two mutations in human PS2 that have been genetically linked to early onset familial Alzheimer's disease (FAD). Exemplary presenilins with altered activity include FAD-associated mutant forms of PSl and PS2 that give rise to an increased accumulation of Aβ42 in AD patients and fransfected cell lines and transgenic animals in which they are expressed. Included among such mutations are the PS2 FAD mutation N141I (Volga German FAD mutant) and the PSl FAD mutation M146L. Examples of diseases associated with an altered presenilin for which the methods provided herein can be used to identify candidate therapeutic or prophylactic agents include, but are not limited to, amyloidosis-associated diseases and neurodegenerative diseases. In a particular aspect, the disease is Alzheimer's Disease.

The sample used in the methods can be any sample, including samples described herein for the methods of identifying agents that modulate presenilin activity. For example, a sample can contain cell(s), tissue, a cell or tissue lysate or extract, a body fluid, a cell membrane or composition containing cell membranes and a cell-free extract or other cell-free sample. In a particular embodiment, the sample includes a cell that contains the presenilin and LRP. In general, the step of identifying a candidate agent that restores LRP processing to the processing that occurs in the presence of a presenilin that is not associated with an altered processing of LRP can involve a comparison of the cleavage and/or processing of LRP (and/or portion(s) thereof) in a sample that has been contacted with the test agent (i.e., test sample) and in a sample that has not been contacted with the test agent (i.e., control sample). If the cleavage and/or processing of LRP (and/or portion(s) thereof) in the test and control samples differs, then the test agent is identified as a candidate agent for the treatment and/or prophylaxis of a disease associated with an altered presenilin. For example, processing of LRP and/or the level of a particular fragment of LRP in the test and control samples may differ by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%>, 45%>, 50%), 55%, 60%, 65%, 70%, 75% or more than 75%. Additionally or alternatively, the cleavage and/or processing of LRP in the test sample can be compared to that in a positive control sample. An example of a positive control is a sample containing LRP (and/or portion(s) thereof) and a presenilin that is not associated with an altered processing of LRP (or an unaltered or wild-type presenilin). In comparing the test sample to the positive control, a test agent is identified as a candidate agent for the treatment and/or prophylaxis of a disease if the cleavage and/or processing of LRP (and/or portion(s) thereof) in the test and positive confrol samples is substantially similar. LRP cleavage and/or processing in the test and positive control samples could be substantially similar if the LRP processing and/or cleavage in the test sample is more similar to that in the positive control sample than that in the control sample that contains the altered presenilin and that was not contacted with the test agent.

The cleavage and/or processing of LRP (and/or portion(s) thereof) in a sample can be assessed, for example, using any of the methods and compositions provided and described herein. Assessing cleavage and/or processing of LRP can provide an assessment of presenilin activity. The processing and/or cleavage of an LRP can be assessed by determining the presence or absence and/or level of one or more fragments of LRP and/or the composition of LRP. In a particular embodiment, the processing or cleavage of the LRP or fragment(s) thereof is assessed by determining the presence or absence and/or level of an LRP fragment that has a molecular weight of between about 25 kD and 15 kD, and, in particular, about 20 kD. The fragment can be one that is contained within a transmembrane region of LRP and/or binds with an antibody generated against a C-terminal amino acid sequence of an LRP, such as, for example, a sequence of about the C-terminal 13 amino acids of an LRP. The LRP fragment can be one that contains an amino acid sequence located within the sequence of amino acids 4420-4544 of SEQ ID NO: 10. The fragment can be one that is present when an LRP is not cleaved by a presenilin-dependent activity, for example, as may occur in the presence of an inhibitor of a presenilin-dependent activity such as, for example, DAPT. In a further embodiment, the fragment is one that is recognized by an antibody generated against C-terminal amino acids (e.g., the C-terminal 13 amino acids) of LRP, such as, for example, the polyclonal antibody R9377 described herein. 2. Notch NICD Assay

Notch is a single transmembrane domain cell surface receptor that facilitates many cell fate decisions during development, including neurogenesis. Although its function in mature cells is unclear, its presence in adult mammalian brain has been demonstrated, although at significantly lower levels than in embryonic brain (Berezovska et al, 1998, J. Neuropathol Exp Neurol 57(8):738-45). In addition, a potential role in adult brain including neurite extension has been suggested (Berezovska et al, 1999, Brain Res. Mol Brain Res. 69(2):273-80). Notch, as well as APP, has been found to form stable complexes with PSl in fransfected mammalian cells (Xia, W. et al, 1997, Proc. Natl. Acad. Sci. 94:8208-8213; Ray, W.J., et al, 1999, Proc. Natl, Acad. Sci. 96:3263-3268).

Notch is synthesized as a 300 kDa precursor molecule, full-length notch (FLN), and undergoes at least three different proteolytic processing events during maturation and signal transduction. The amino acid sequence of the notch precursor polypeptide is provided in SEQ ID NO: 32, and DNA encoding the polypeptide is provided in SEQ ID NO: 31. In the frans-Golgi network lumen, FLN is cleaved by the protease Furin at a site in the extracellular domain. This cleavage generates two fragments that remain associated during transport to the cell surface forming a heterodimeric receptor at the cell surface. Ligand binding to the receptor triggers an additional cleavage of the extracellular region of the C-terminal domain shortening the extracellular region to 12 amino acids. A third presenilin-dependent proteolytic cleavage event occurs within the transmembrane domain and releases the nuclear intracellular carboxyl domain (NICD). A presenilin-dependent cleavage of Notch has been shown between residues G1743 and V1744 (SEQ ED NO: 32 or the amino acid sequence provided as GenBank Accession No. AF308602). NICD translocates to the nucleus and activates transcription of target genes that influence crucial cell fate decisions during development and particularly haematopoiesis.

In a method for assessing presenilin activity provided herein, the level of one or more fragments of Notch and/or the composition of Notch is determined for a sample for which presenilin activity is being assessed. Examples of a sample for which presenilin activity is being assessed include, but are not limited to, a cell that expresses presenilin, a lysate or extract of a cell that expresses presenilin, or membranes prepared from a cell that expresses presenilin. The cell can endogenously express presenilin and/or express heterologous presenilin. Notch can be added to the sample or can be expressed endogenously and/or heterologously by the cell. In a particular embodiment, the method includes assessing presenilin activity of a cell by evaluating the level (which includes determining the presence or absence of) of a fragment from a C-terminal portion of Notch in a cell lysate.

To assess presenilin activity in these methods, the processing of Notch is evaluated. In evaluating Notch processing, the composition of Notch can be evaluated. The composition of Notch refers to the make-up of any Notch that is present anywhere in the analysis mixture, including the assay medium in which the analysis is being performed, an extracellular medium, or a cell membrane, lysate or extract. Thus, in one embodiment, the structure of any Notch present can be evaluated to, for example, determine whether Notch is intact or has been processed and appears as a fragment or fragments of sizes smaller than the intact Notch molecule. In evaluating Notch processing, the levels, and/or the presence or absence, of one or more Notch fragments can be determined.

In particular, the Notch composition is evaluated to determine if any fragment(s) indicative of presenilin-dependent cleavage (or altered presenilin-dependent cleavage) of Notch are present and/or the level of any such fragment(s). A presenilin-dependent cleavage described herein occurs within the C-terminal portion of Notch. Thus, a presenilin-dependent cleavage of Notch can be one that occurs in the C-terminal portion of Notch at a position C-terminal to amino acid position 1743 of SEQ ID NO: 32 (or of the amino acid sequence provided as GenBank Accession No. AF308602). The presenilin-dependent cleavage of Notch can be one that occurs within the sequence of the last approximately 850, 815, 800, 750, 700, 750, 700, 650, 600, 650, 600, 550, 500, 450, 400, 350, 300, 250, 200, 150, 100, 50, 25, or less amino acids of Notch. The presenilin- dependent cleavage can be one that occurs C-terminal to the extracellular portion of Notch (i.e., C-terminal to amino acid 1727 of SEQ ED NO: 32 or of the amino acid sequence provided as GenBank Accession No. AF308602). The presenilin-dependent cleavage of Notch can be one that occurs near or within the region of the Notch protein that extends from a point located intracellularly and adjacent to the cytoplasmic face of the cell membrane to a point located within the cell membrane. Such a presenilin- dependent cleavage of Notch can be one that generates a soluble intracellular peptide containing the extreme C-terminus of Notch and a membrane-associated peptide containing amino acid sequence of the transmembrane region of Notch, particularly the more C-terminal region of the transmembrane segment of Notch. A Notch fragment generated by a presenilin-dependent cleavage can be one that can be detected by a reagent that binds to or recognizes an amino acid sequence from a C-terminal portion of Notch.

As described herein above, Notch is also cleaved by activities that are not presenilin dependent. Specifically, in one cleavage that is not a presenilin-dependent/γ- secretase activity, Notch is cleaved at the cell surface which results in "shedding" from the cell surface of a portion of the extracellular segment of Notch. A fragment such as this that does not result from a presenilin-dependent cleavage generally is not alone indicative of presenilin activity.

In particular embodiments, Notch processing by PSl/γ-secretase can be assessed by determining the levels and/or presence or absence of the Notch ICD peptide and/or the Notch membrane-associated peptide that result from presenilin-dependent cleavage of Notch. In addition, the level, presence or absence of a Notch fragment that occurs in the absence of presenilin-dependent cleavage of Notch can be determined. Notch peptide levels can be measured by any method known to those of skill in the art for measuring protein level or by any method described herein. In a particular embodiment of the method, Notch peptide levels are measured by immunoassay. Anti-Notch peptide antibodies for use in such immunoassays can be obtained by the methods described herein or known to those of skill in the art. For example, Myc-tagged Notch derivatives maybe used and detected with monoclonal anti-Myc antibodies (i.e., 9E10 from ATCC) (Schroeter et al, (1998) Nature 39: 382-386; Song et al, (1999) Proc. Natl Acad. Sci. 96: 6959-6963) or V5 antibody epitope tagged Notch derivatives may be used and detected with anti-V5 antibody as described in the EXAMPLES. 3. E-cadherin assay

E-cadherin controls a wide array of cellular behaviors including cell-cell adhesion, differentiation and tissue development. Presenilin has been shown to form complexes with the cadherin/catenin adhesion system resulting in cleavage and release of the E-cadherin intracellular domain and disassembly of adherens junctions (Baki et al. 2001, Proc. Natl. Acad. Sci. 9S(5 :2381-2386; Marambaud et al. 2002, EMBO J. 21 (8): 1948-56). The amino acid sequence encoding a full-length human E-cadherin polypeptide is provided in SEQ ED NO: 34, and DNA encoding the polypeptide is provided in SEQ ID NO: 33. A presenilin- 1 -dependent γ-secretase cleavage stimulated by apoptosis or calcium influx occurs between human E-cadherin residues Leu731 and Arg732 at the membrane-cytoplasm interface. The PSl/γ-secretase system cleaves both the full-length E-cadherin and a transmembrane C-terminal fragment, derived from a metalloproteinase cleavage after the E-cadherin ectodomain residue Pro700, approximately seven residues upsfream of the fransmembrane domain (i.e., amino acids 708-731 of SEQ ED NO: 34 or of the amino acid sequence provided as GenBank Accession No. NP_004351). Metalloproteinase cleavage of the N-terminus of full-length E-cadherin produces a 38 kDA fragment (E-Cad/CTFl) that binds both β-catenin and PSl . Full-length E-cadherin and E-Cad/CTFl are found only in the membrane and cytoskeletal (Triton X-100-insoluble) fraction. Cleavage by PSl/γ-secretase defines the N-terminal region of a 33 kDa fragment (E-Cad/CTF2 or E-Cad intracellular carboxyl domain (ICD)) that binds only β-catenin. A PSl/γ-secretase cleavage of E-cadherin has been shown between residues Leu731 and Arg732 (SEQ ED NO: 34 or the amino acid sequence provided as GenBank Accession No. NP_004351) at the interface of the membrane with the cytoplasm (Marambaud et «/. 2002, EMBOJ. 21 (8):l948-56). E-Cad ICD localizes in the membrane and in the soluble cytosol. Cleavage of E-cadherin by caspase-3 between residues 750 and 751 has also been reported (Steinhusen et al. (2001) J. Biol. Chem., 276:4972-4980). The PSl/γ-secretase cleavage dissociates E-cadherins, β-catenin and α-catenin from the cytoskeleton, thus promoting disassembly of the E- cadherin-catenin adhesion complex. Furthermore, this cleavage releases the cytoplasmic E-cadherin intracellular carboxyl domain (ICD) to the cytosol and increases the levels of soluble β- and catenins. Thus, the PSl/γ-secretase system stimulates disassembly of the E-cadherin-catenin complex and increases the cytosolic pool of β-catenin, a key regulator of the Wnt signaling pathway involved in cell proliferation. In a method for assessing presenilin activity provided herein, the level of one or more fragments of E-cadherin and/or the composition of E-cadherin is determined for a sample for which presenilin activity is being assessed (examples of which are described herein). E-cadherin can be added to the sample or, if the sample is a cell sample, E- cadherin can be expressed endogenously and/or heterologously by the cell. In a particular embodiment, the method includes assessing presenilin activity of a cell by evaluating the level and/or presence or absence of a fragment from a C-terminal portion of E-cadherin in a cell lysate.

To assess presenilin activity in these methods, the processing of E-cadherin is evaluated. In evaluating E-cadherin processing, the composition of E-cadherin can be evaluated. The composition of E-cadherin refers to the make-up of any E-cadherin that is present anywhere in the analysis mixture, including the assay medium in which the analysis is being performed, an extracellular medium, or a cell membrane, lysate or extract. Thus, in one embodiment, the structure of any E-cadherin present can be evaluated to, for example, determine whether E-cadherin is intact or has been processed and appears as a fragment or fragments of sizes smaller than the intact E-cadherin molecule. In evaluating E-cadherin processing, the levels and/or presence or absence of one or more E-cadherin fragments can be determined.

In particular, the E-cadherin composition is evaluated to determine if any fragment(s) indicative of presenilin-dependent cleavage (or altered presenilin-dependent cleavage) of E-cadherin are present and/or the level of any such fragment(s). A presenilin-dependent cleavage described herein occurs within the C-terminal portion of E-cadherin. Thus, a presenilin-dependent cleavage of E-cadherin can be one that occurs in the C-terminal portion of E-cadherin at a position C-terminal to amino acid position 731 of SEQ ED NO: 34 (or of the amino acid sequence provided as GenBank Accession No. NP_004351). The presenilin-dependent cleavage of E-cadherin can be one that occurs within the sequence of the last approximately 151, 150, 100, 50, 25, or less amino acids of E-cadherin. The presenilin-dependent cleavage can be one that occurs C- terminal to the extracellular portion of E-cadherin (i.e., C-terminal to amino acid 707 of SEQ ID NO: 34 or of the amino acid sequence provided as GenBank Accession No. NP_004351). The presenilin-dependent cleavage of E-cadherin can be one that occurs near or within the region of the E-cadherin protein that extends from a point located intracellularly and adjacent to the cytoplasmic face of the cell membrane to a point located within the cell membrane. Such a presenilin-dependent cleavage of E-cadherin can be one that generates a soluble intracellular peptide containing the extreme C- terminus of E-cadherin and a membrane-associated peptide containing amino acid sequence of the transmembrane region of E-cadherin, particularly the more C-terminal region of the transmembrane segment of E-cadherin. Any E-cadherin fragments generated by such presenilin-dependent activities would have a molecular weight that is less than that of the E-Cad/CTFl fragment produced by metalloproteinase cleavage of the N-terminus of full-length E-cadherin (E-Cad/CTF 1 molecular weight is approximately 38 kDa). Also, because caspase-3 can cleave a portion of the fragment produced by presenilin dependent cleavage, the molecular weight of such a fragment may be further reduced. Thus, an E-cadherin fragment generated by a presenilin-dependent cleavage can have a molecular weight that is, for example, less than about 40 kD, 35 kD, 30 kD, 25 kD, 20 kD, 15 kD or 10 kD or less. In a particular embodiment, an E-cadherin fragment generated by a presenilin-dependent cleavage has a molecular weight of less than about 35 kD or that is about 33 kD. An E-cadherin fragment generated by a presenilin-dependent cleavage can be one that can be detected by a reagent that binds to or recognizes an amino acid sequence from a C-terminal portion of E-cadherin. As described herein above, E-cadherin is also cleaved by activities that are not presenilin dependent. Specifically, in one cleavage that is not a presenilin-dependent/γ- secretase activity, full-length E-cadherin is cleaved by a metalloproteinase at the cell surface which results in "shedding" from the cell surface of a portion of the extracellular segment of E-cadherin (i.e., amino acids N-terminal of amino acid 701 of SEQ ED NO: 34 or of the amino acid sequence provided as GenBank Accession No. NP_004351). A fragment such as this that does not result from a presenilin-dependent cleavage is not alone indicative of presenilin activity.

In particular embodiments E-cadherin processing by PSl /γ-secretase can be determined by measuring the levels of the E-cadherin ICD peptide and/or the E-cadherin CTF1 peptide. In addition, the level, presence or absence of a Notch fragment that occurs in the absence of presenilin-dependent cleavage of Notch can be determined. For example, inhibition of the PSl/γ-secretase processing of E-cadherin may result in the accumulation of the CTF1 peptide and/or a decrease in the level of the ICD peptide. E- cadherin peptide levels can be measured by any method known to those of skill in the art for measuring protein level or by any method described herein. For example, levels of E- cadherin peptides may be measured by immunoassay using anti-E-Cad/CTFl or anti-E- Cad ICD antibodies. Antibodies for use in such immunoassays can be obtained by the methods described herein or known to those of skill in the art such as those described by Marambaud et al. (EMBOJ. (2002) 21(8): 1948-56).. 4. Erb-B4 assay Erb-B4 is a type I membrane receptor tyrosine kinase, which belongs to the epidermal growth receptor family and mediates response to multiple growth factors, including neuregulins. Erb-B4 has been implicated in many important biological and pathological processes, such as cardiovascular, mammary gland, and neuronal development, as well as malignancy and heart disease. The amino acid sequence of the -180 kDa full-length Erb-B4 polypeptide is provided in SEQ ED NO: 36, and DNA encoding the polypeptide is provided in SEQ ED NO: 35. Constitutive ectodomain shedding of full-length Erb-B4 by a metalloprotease yields an -80 kDa membrane- associated C-terminal fragment (B4-CTF) and a ~120 kDa ectodomain N-terminal fragment that is released into the exfracellular medium. B4-CTF is further cleaved by a presenilin dependent γ-secretase releasing the soluble intracellular domain of Erb-B4 ICD which translocates to the nucleus and may participate in activation of gene transcription. The Erb-B4 ICD is believed to be -80 kDa and contain a tyrosine kinase domain. Cleavage has been shown to occur at conserved residue Val673 on the C- terminal side of the fransmembrane domain (residues 649-675 of amino acid SEQ ED NO: 36). This cleavage site is topologically similar to the γ-secretase cleavage site in Notch and cleavage of APP at conserved residue Val49.

In a method for assessing presenilin activity provided herein, the level of one or more fragments of Erb-B4 and/or the composition of Erb-B4 is determined for a sample for which presenilin activity is being assessed. Examples of a sample for which presenilin activity is being assessed are described herein. Erb-B4 can be added to the sample or can be expressed endogenously and/or heterologously by a cell in the sample. In a particular embodiment, the method includes assessing presenilin activity of a cell by evaluating the level and/or presence or absence of a fragment from a C-terminal portion of Erb-B4 in a cell lysate.

To assess presenilin activity in these methods, the processing of Erb-B4 is evaluated. In evaluating Erb-B4 processing, the composition of Erb-B4 can be evaluated. The composition of Erb-B4 refers to the make-up of any Erb-B4 that is present anywhere in the analysis mixture, including the assay medium in which the analysis is being performed, an extracellular medium, or a cell membrane, lysate or exfract. Thus, in one embodiment, the structure of any Erb-B4 present can be evaluated to, for example, determine whether Erb-B4 is intact or has been processed and appears as a fragment or fragments of sizes smaller than the intact Erb-B4 molecule. In evaluating Erb-B4 processing, the levels and/or presence or absence of one or more Erb-B4 fragments can be determined.

The Erb-B4 composition can be evaluated to determine if any fragment(s) indicative of presenilin-dependent cleavage (or altered presenilin-dependent cleavage) of Erb-B4 are present and/or the level of any such fragment(s). A presenilin-dependent cleavage described herein occurs within the C-terminal portion of Erb-B4. Thus, a presenilin-dependent cleavage of Erb-B4 can be one that occurs in the C-terminal portion of Erb-B4 at a position C-terminal to Val673 of SEQ ED NO: 36 (or of the amino acid sequence provided as GenBank Accession No. AAB59446). The presenilin-dependent cleavage of Erb-B4 can be one that occurs within the sequence of the last approximately 650, 600, 550, 500, 450, 400, 350, 300, 250, 200, 150, 100, 50, 25, or less amino acids of Erb-B4. The presenilin-dependent cleavage can be one that occurs C-terminal to the extracellular portion of Erb-B4 (i.e., C-terminal to amino acid 648 of SEQ ED NO: 36 or of the amino acid sequence provided as GenBank Accession No. AAB59446). The presenilin-dependent cleavage of Erb-B4 can be one that occurs near or within the region of the Erb-B4 protein that extends from a point located infracellularly and adjacent to the cytoplasmic face of the cell membrane to a point located within the cell membrane. Such a presenilin-dependent cleavage of Erb-B4 can be one that generates a soluble intracellular peptide containing the extreme C-terminus of Erb-B4 and a membrane- associated peptide containing amino acid sequence of the transmembrane region of Erb- B4, particularly the more C-terminal region of the fransmembrane segment of Erb-B4. Any Erb-B4 fragments generated by such presenilin-dependent activities would have a molecular weight that is less than that of the -180 kDa full-length Erb-B4 polypeptide minus the -120 kDa ectodomain N-terminal fragment that is released into the extracellular medium upon metalloproteinase cleavage. Thus, an E-cadherin fragment generated by a presenilin-dependent cleavage can have a molecular weight that is, for example, less than about 100 kD, 90 kD, 80 kD, 70 kD, 60 kD, 50 kD, 40 kD, 30 kD, 20 kD, 15 kD or 10 kD or less. In a particular embodiment, an Erb-B4 fragment generated by a presenilin-dependent cleavage has a molecular weight of less than about 90 kD or that is about 80 kD. An Erb-B4 fragment generated by a presenilin-dependent cleavage can be one that can be detected by a reagent that binds to or recognizes an amino acid sequence from a C-terminal portion of Erb-b4.

As described herein above, Erb-B4 is also cleaved by activities that are not presenilin dependent. Specifically, in one cleavage that is not a presenilin-dependent/γ- secretase activity, full-length Erb-B4 is cleaved by a metalloproteinase at the cell surface which results in "shedding" from the cell surface of a portion of the extracellular segment of E-cadherin (i.e., amino acids N-terminal of amino acid 648 of SEQ ED NO: 36 or of the amino acid sequence provided as GenBank Accession No. AAB59446). A fragment such as this that does not result from a presenilin-dependent cleavage generally is not alone indicative of presenilin activity.

In particular embodiments, Erb-B4 processing by PSl /γ-secretase can be assessed by determining the levels and/or presence or absence of the Erb-B4 ICD peptide and/or the Erb-B4 membrane-associated peptide. In addition, the level, presence or absence of an Erb-B4 fragment that occurs in the absence of presenilin-dependent cleavage of Notch can be determined. Erb-B4 peptide levels can be measured by any method known to those of skill in the art for measuring protein level or by any method described herein. In a particular embodiment of the method, Erb-B4 peptide levels are measured by immunoassay. Anti-Erb-B4 peptide antibodies for use in such immunoassays can be obtained by the methods described herein or known to those of skill in the art. For example, polyclonal antibodies to the carboxyl terminus (residues 1291-1308) can be purchased (Santa Cruz Biotechnology, Inc.). Other antibodies to Erb-B4 peptides have also been described (see, e.g., Ni, et al, (2001) Science 294:2179-2181).

D. Methods of Identifying or Screening for Agents that Modulate Aβ Levels

Methods, and compositions for use therein, are provided for identifying or screening for agents that modulate the levels of one or more Aβ peptides in a sample. The sample may be any sample, such as described herein, and may be reflective of, e.g, cellular and/or extracellular Aβ levels. In a particular embodiment, the methods can be used to identify agents that modulate the levels of Aβ42, including cellular and/or extracellular Aβ42. In another embodiment, the methods can be used to identify an agent that selectively modulates the level of one or more Aβ peptides, such as, for example, Aβ42, including cellular and/or exfracellular peptides. For example, in one embodiment, the method includes a step of identifying an agent that selectively modulates the level of one or two Aβ peptides relative to one or more other Aβ peptides. hi a particular embodiment, an agent that selectively modulates the levels of Aβ42 only or of Aβ42 and Aβ39 only, relative to other Aβ peptides (including, e.g., Aβ40, Aβ38 and/or Aβ43), is identified. In another embodiment, the methods can be used to identify agents that modulate

Aβ peptide levels (and, in particular, Aβ42 levels) without substantially affecting (or with limited, minimal or inconsequential effect on) compositions, mechanisms, processes and/or activities that are not significantly involved in the generation, degradation and/or clearance of one or more Aβ peptides. In a particular embodiment, the method involves a step of identifying an agent that modulates the levels (including cellular and/or exfracellular) of one or more Aβ peptides without substantially altering the cleavage of a presenilin substrate, or portion thereof, that is not APP. hi a further embodiment, the presenilin substrate is LRP. Included among the agents that can be identified using the methods provided herein are agents that modulate Aβ levels, for example, by modulating compositions (e.g., proteases and proteins, such as proteins on which protease activities depend, including presenilins), mechanisms and/or activities involved in Aβ peptide formation, degradation and/or clearance in cells and/or extracellular medium without substantially affecting (or with only limited, minimal or inconsequential effect on) compositions, mechanisms and/or activities that are not significantly involved in Aβ peptide formation and persistence.

Agents identified by the methods provided herein have a variety of uses. For example, such agents can be used in elucidating the particular elements and pathways involved in Aβ peptide formation, degradation and clearance in cells. Such agents may be used to assess proteolytic processing in cells and to characterize enzyme and protein interactions that facilitate and/or inhibit such processing. Proteolytic processing events include, but are not limited to, those involved in the production and/or degradation of Aβ peptides. For example, agents identified by the methods may be used to identify and/or characterize regulatory molecules including, but not limited to, proteases that produce or degrade Aβ peptides and proteins involved in the activation or inhibition of such proteases. In addition, because release of Aβ peptides is a normal event in virtually every cell, the agents identified herein can be used to further characterize the role of such peptides in biochemical pathways and/or normal cellular processes. The agents identified by the methods provided herein may also serve as candidate agents for the treatment and/or prevention of disorders and diseases characterized by and/or involving inappropriate levels or misregulation of Aβ. Such diseases and disorders include any disease or disorder involving misregulation of Aβ production, clearance, and/or degradation. Exemplary disease and disorders include neurodegenerative diseases and disorders, such as, but not limited to, Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease, amylofrophic lateral sclerosis (ALS), Down's syndrome, Hereditary Cerebral Hemorrhage with Amyloidosis-Dutch Type (HCHWA-D), and advanced aging of the brain. Such agents can provide therapeutic and/or preventative benefit with limited-to-no potential side effects that can result from non-specific modulation of Aβ peptide processing.

The methods provided herein for identifying or screening for agents that modulate Aβ levels can be used to identify agents that modulate cell and/or cellular membrane (i.e., referred to herein as cellular) Aβ levels and/or extracellular Aβ levels. In general, the methods include steps of contacting a sample containing amyloid precursor protein (APP), and/or portion(s) thereof (e.g., one or more Aβ peptides), with a test agent and identifying an agent that alters the Aβ peptide-producing cleavage of the APP, the processing of the APP, the processing of Aβ and/or the levels of one or more Aβ peptides in the sample.

The step of identifying an agent that alters the Aβ peptide-producing cleavage or processing of the APP (and/or portion(s) thereof), the processing of Aβ and/or the levels of one or more Aβ peptides in the sample can be carried out in a number of ways. In general, the identification step can involve a comparison of the cleavage or processing of APP (and/or portion(s) thereof), processing of Aβ and/or the Aβ levels of a sample that has been contacted with the test agent (i.e., test sample) and of a sample that has not been contacted with the test agent (i.e., control sample). If the Aβ-producing cleavage or processing of APP (and/or portion(s) thereof), the processing of Aβ and/or the Aβ levels of the test and control samples differ, then the agent is identified as one that modulates the level of one or more Aβ peptides. The confrol sample can be the same physical sample as the test sample or a different sample. When the control and test samples are the same, the confrol is the sample in the absence of test agent. Assessing the cleavage and processing of APP (and/or portion(s) thereof), the processing of Aβ, and the Aβ levels of a sample can be conducted in a number of ways such as described herein or known in the art. In a particular method for assessing the Aβ42 level of a sample, a monoclonal antibody provided herein that selectively binds Aβ42 relative to other Aβ peptides is used to in an immunoassay for the detection and/or quantitation of Aβ42.

The methods provided herein for identifying or screening for agents that modulate Aβ levels can also include identifying an agent that alters the Aβ peptide-producing cleavage or processing of the APP (and/or portion(s) thereof), the processing of Aβ and/or the levels of one or more Aβ peptides in the sample without substantially altering the cleavage of a presenilin substrate, or portion thereof, other than APP. In these methods, a sample containing a source of a presenilin subsfrate (or a portion thereof) other than APP is contacted with the test agent. The sample may be the same as the sample containing APP (and/or portion(s) thereof) or can be a different sample. The process of identifying an agent that alters the Aβ peptide-producing cleavage or processing of the APP (and/or portion(s) thereof), the processing of Aβ and/or the levels of one or more Aβ peptides in a sample can be carried out in a number of ways as described herein. In addition, the process of further identifying an agent that also does not substantially alter the cleavage of a presenilin subsfrate (other than APP), or portion thereof, can be carried out in a number of ways, hi general, this process can involve a comparison of the presenilin-dependent cleavage and/or processing of a presenilin subsfrate (or portion thereof) other than APP and/or the levels of a peptide fragment or fragments of the presenilin substrate that is other than APP of a sample that has been contacted with the test agent (i.e., test sample) and of a sample that has not been contacted with the test agent (i.e., control sample). If the presenilin-dependent cleavage or processing of the presenilin subsfrate that is other than APP and/or the substrate fragment(s) levels of the test and confrol samples do not differ substantially, then the agent is identified as one that alters the level of one or more Aβ peptides without substantially altering the cleavage of the presenilin substrate, or portion thereof, that is other than APP. The control sample can be the same physical sample as the test sample or a different sample. When the confrol and test samples are the same, the control is the sample in the absence of test agent. 1. Samples for use in methods of identifying Aβ-modulating agents

A sample that can be used in the methods of identifying an agent that modulates the Aβ levels can be any composition (e.g., a biological or physiological composition) that includes a source of APP, and/or portion(s) thereof, or a source of one or more Aβ peptides including, but not limited to, a cell, a cell extract or lysate, a cellular membrane and a cell-free medium. When the sample contains a source of APP, it generally also contains a source of enzymatic and/or other activity that provides for processing of APP, and, in particular, Aβ peptide-producing cleavage activity. When a sample is one for use in methods that include a step of identifying an agent that alters the processing, such as degradation, of Aβ, and thus contains a source of Aβ peptides, it generally also contains a source of enzymatic and/or other activity that provides for processing of Aβ (e.g. , a catabolic activity that degrades Aβ). a. APP or portion(s) thereof The APP, and/or portion(s) thereof, provided by the source contained within the sample is generally any APP (and/or portion(s) thereof) that include(s) the Aβ peptide domains within its amino acid sequence. Aβ peptides include, but are not limited to, (1) a peptide that results from processing or cleavage of an APP and that is amyloidogenic, (2) one of the peptide constituents of β-amyloid plaques, (3) a fragment or portion of the 43-amino acid sequence set forth in SEQ ED NO: 4 and (4) a fragment or portion of a peptide as set forth in (1) or (2). Aβ peptides derived from proteolysis of APP, or degradation of Aβ, generally are typically 39 to 43 amino acids in length (see, e.g. , SEQ ED NO: 4 showing the 43-amino acid sequence of an Aβ peptide), depending on the carboxy-terminal end-point, which exhibits heterogeneity. However, Aβ peptides containing less than 39 amino acids, e.g., Aβ39, Aβ38, Aβ37 and Aβ34, also may occur. Aβ peptides include those that begin at position 672 of APP770 (see SEQ ED NO: 2). Isoforms of APP that contain an Aβ domain include APP770, APP751, APP714,

APP695, L-APP752, L-APP733, L-APP696 and L-APP697. APP can be an APP of any species. In particular embodiments, the APP is a mammalian APP, such as, for example, a rodent or human APP.

In methods of identifying or screening for agents that modulate Aβ levels that include a step of identifying an agent that alters the Aβ peptide-producing cleavage of APP, the sample can contain a source of APP that can be cleaved or modified to yield one or more Aβ peptides. In methods that include a step of identifying an agent that alters the processing, such as degradation, of Aβ, the sample generally contains a source of Aβ peptides. Such a source can be, for example, synthetic, recombinant or isolated Aβ peptides, or a source of APP that can be cleaved or modified to yield one or more Aβ peptides. In methods that include a step of identifying an agent that alters the processing of APP, the sample can contain a source of APP that can undergo processing. In methods that include a step of identifying an agent that alters the level of one or more Aβ peptides, the sample generally contains a source of Aβ peptides. Such a source can be, for example, synthetic, recombinant or isolated Aβ peptides, or a source of APP that can be cleaved or modified to yield one or more Aβ peptides.

Sources of APP, or a portion thereof, include, but are not limited to: a cell that expresses endogenous or heterologous APP; a cell that expresses a recombinant portion(s) or fragment(s) of APP; lysates, extracts, or membrane fractions of any such cells; APP, or a portion thereof, that is isolated from such cells; and synthetic APP protein or synthetic proteins that represent a portion of APP.

Sources of Aβ peptides include, but are not limited to: a cell that expresses endogenous or heterologous APP and enzymatic activities that cleave APP to yield Aβ peptides (e.g., β- and γ-secretases); a cell that expresses recombinant Aβ peptides; lysates, exfracts, or membrane fractions of any such cells; Aβ peptides that are isolated from such cells; synthetic or isolated APP that is degraded to yield Aβ peptides; and synthetic Aβ peptides.

Compositions, and methods of making compositions, that are sources of APP, portion(s) thereof, and Aβ peptides are described herein and known in the art. For example, cells that endogenously express APP and/or Aβ peptides are known in the art as are nucleic acids encoding APP (or portion(s) thereof) and/or Aβ peptides (see, e.g. , SEQ ID NOs: 1, 3, 27 and 29) that can be used to express the encoded proteins in cells. Methods of preparing lysates, extracts and membrane fractions of such cells are also described herein and known in the art, as are synthetic methods for generating proteins and peptides and preparatory methods of isolating proteins and peptides. b. Sources of activities that provide for processing of APP and/or Aβ peptides

Sources of activities that provide for cleavage or processing of APP (or portion(s) thereof) and/or Aβ peptides include, but are not limited to: a cell that expresses endogenous or heterologous molecules that give rise to the activities; lysates, exfracts, or membrane fractions of any such cells; molecules that give rise to the activities that are isolated from such cells; and synthetic molecules that give rise to the activities. Molecules that can be involved in activities that provide for cleavage or processing of APP or Aβ include, but are not limited to, secretases, including -, β- and γ-secretase, presenilins, including PSl and PS2, insulin-degrading enzyme (EDE), neprilysin, plasmin, uPA/f A, endothelin converting enzyme- 1, and matrix metalloproteinase-9 (see, e.g., Selkoe (2001) Neuron 32:177-180; Vekrellis et al. (2000) J. Neurosci. 20:1657-1665; Iwata et al. (2000) Nat. Med. <5:143-150; Carson and Turner (2002) J Neurochem 81(1): 1-8; and Eckman et al. (2001) J. Biol. Chem. 276: 24540- 24548). Such molecules can be from any species. In particular embodiments, the molecule is a mammalian molecule, such as, for example, a rodent or human molecule. c. Conditions that enhance Aβ production

When a sample is one for use in methods that include a step of identifying an agent that alters the processing of Aβ and/or the levels of one or more Aβ peptides, it is generally desirable for the sample to contain a readily detectable amount of Aβ peptide. To enhance Aβ production in a sample containing an Aβ-producing source, one or more modulatory molecules or compounds that provide for increased Aβ levels through increased Aβ production or decreased Aβ clearance can be included in the sample. For example, a modulatory molecule may function to activate β-secretase and/or γ-secretase contained within the sample for increased processing of APP into Aβ peptides.

Alternatively, a modulatory molecule may function to inhibit one or more Aβ-degrading proteases leading to decreased clearance of Aβ peptides. Exemplary modulatory molecules of this kind may include, but are not limited to serine protease inhibitors such as cd-antichymotrypsin (Mucke et al. (2000) An. J. Pathol 157: 2003-2010; Nilsson et al. (2001) J. Neurosci. 27:1444-1451). In addition, the protease inhibitor thiorphan which is known to inhibit several proteases, has been shown to induce plaque formation in rats (Iwata et al. (2000) Nat. Med. 6: 143-150). d. Medium

A sample medium can be any medium in which APP, portion(s) thereof, and/or Aβ peptides can exist. Examples of sample medium include, but are not limited to, cells, cell lysates, exfracts and membranes, and cell-free medium.

(1) Cells

(a) General features of cells

Although any cell may be used in the methods, cells that are particularly suitable are those that exhibit APP and/or Aβ peptide synthesis and processing and/or those in which Aβ levels and/or processing may readily be assessed. If a cell has an APP processing and/or cleavage activity but does not express APP (or expresses APP at only low or undetectable levels), nucleic acid encoding APP can be introduced into the cells, and vice versa. If a cell has an Aβ catabolic activity (i.e., an activity that degrades one or more forms of Aβ) but does not express Aβ (or expresses only low levels of Aβ or only particular forms of Aβ), nucleic acid encoding one or more Aβ peptides can be introduced into the cells, and vice versa. Cells that express enzymatic and/or other activities involved in APP and/or Aβ processing can also be used in conjunction with another or separate source of APP and/or Aβ peptides in the sample. Thus, fransfected or recombinant cells, as well as cells that endogenously express desired proteins and/or activities, can be used in the methods of identifying agents that modulate Aβ levels.

In particular examples, cells used in samples for the methods of identifying agents that modulate Aβ levels are eukaryotic cells. In a further example, the cells can be mammalian cells. Mammalian cells include, but are not limited to, rodent (e.g., mouse, rat and hamster), primate, monkey, dog, bovine, rabbit and human cells. In particular embodiments of the methods, the sample includes a mammalian cell, such as, for example, a rodent or human cell, that expresses endogenous and/or heterologous APP (or a portion(s) thereof) and/or Aβ, and the activity or activities for processing and cleavage of APP and/or Aβ. Cells may also be cells of in vivo or in vtvo-derived samples, including body fluids, such as but not limited to, serum, blood, saliva, cerebral spinal fluid, synovial fluid and interstitial fluids, urine, sweat and other such fluids and secretions.

Another feature of cells that are particularly suitable for use in the screening and identification methods is amenability to fransfection/fransformation with heterologous nucleic acid and amenability to gene expression alteration. A number of techniques for the introduction of heterologous nucleic acid into cells and for altering gene expression in cells are known in the art and described herein. The relative ease with which these techniques may be applied to a cell to effect recombinant expression of a heterologous nucleic acid, or reduction, alteration or elimination of one or more genes in the cell is a consideration in selection of cells for use in the methods provided herein. Amenability to gene expression alteration and analysis of Aβ may be considerations, for example, when screening agents in AD model systems (as described herein).

(b) Cells that exhibit APP and/or Aβ production Exemplary cells that exhibit APP and/or Aβ production include, but are not limited to, primary cell cultures, typically neuronal cell cultures. Primary cells from any organism that exhibits APP and/or Aβ production and/or processing may be used. Examples include mixed fetal guinea pig brain cells (Beck (2000) Neuroscience 95:243- 254). Primary cell cultures are harvested from a mammal and cultured using standard techniques and include cortical neural cells, microglia, glia, astrocytes, and the like. Briefly, neural tissue including but not limited to the brain of a mammal expressing or diagnosed with AD symptoms is harvested, and optionally subjected to enzymatic digestion to ease the separation of cells. The cells can be mechanically separated as well. Cells can also be enriched by type or characteristic using standard techniques. Primary culture cells, typically neural tissue, can be induced to express Aβ in response to growth factors, cytokines, hormones, or transcription pathway activators. Thus, suitable cells include cells capable of expressing Aβ in response to an Aβ-inducing agent. An Aβ- inducing agent means any substance that causes and/or enhances the expression of APP or Aβ and includes, but is not limited, to growth factors including but not limited to TGF, TGF-β, PDGF, and EGF; cytokines, hormones or a combination thereof.

Totipotent, pluripotent, or other cells that are not terminally differentiated can be induced to express neuronal characteristics including the production of Aβ peptides. Exemplary non-terminally differentiated cells include embryonic stem cells, adult stem cells, mesenchymal stem cells, bone marrow stem cells, adipose tissue stem cells, and neuronal stem cells. These non-terminally differentiated cells can be induced to express Aβ when exposed to growth factors, cytokines, morphogenetic factors, or tissue specific inducing media. Thus, cells that can be used in the methods of identifying or screening for agents that modulate Aβ levels include non-terminally differentiated cells induced to express Aβ. The non-terminally differentiated cells can be of any lineage, endoderm, mesoderm, or ectoderm or a combination thereof.

Other cells that express APP and/or Aβ include immortalized cell lines fransfected or transformed with exogenous nucleic acids encoding APP, Aβ, a precursor, or fragment thereof. For example, US Patent. No. 5,538,845, incorporated by reference, describes the transfection of Chinese hamster ovary (CHO) cells and 293 human embryonic kidney (HEK) cell line, ATCC accession number CRL-1573, with cDNA encoding the 695, 751, and 770 amino acid isoforms of APP. Mouse neuroblastoma cells (e.g., N2A cells; ATCC accession number CCL-131) are another example of cells that can be fransfected with nucleic acid encoding APP, a portion(s) thereof or Aβ. Any of these cells can be cofransfected, if necessary, with vectors comprising nucleic acid sequences encoding β-secretase, γ-secretase and/or presenilin for the processing of APP to generate Aβ peptides.

Additionally, SH-SY5 Y cells, a human neuroblastoma cell line that secretes Aβ into the culture medium without βAPP transfection, can be used. This cell line is available from ECACC European Collection of Cell Cultures, CAMR Centre for Applied Microbiology & Research Porton Down, Salisbury, Wiltshire (UK) SP4 OJG UK under accession number 94030304.

Cells fransfected with nucleic acid constructs can express APP and/or Aβ peptides using standard expression vectors. Expression can be, for example, constitutive or induced. (2) Cell lysates, extracts and membranes and cell-free medium

Biological compositions that can be used as samples in the methods of identifying or screening for agents that modulate Aβ levels include, but are not limited to, purified or partially purified enzyme preparations, conditioned medium from cultured cells, cellular exfracts and cell lysates. Cell lysates can be generated using methods described herein

(see, e.g., Example 8) and/or known in the art. For example, cell lysates can be prepared from cells able to process APP into Aβ and/or able to catabolize Aβ. Alternatively, appropriate APP processing or catabolic enzymes may be incubated with cell lysates devoid of such activity. (3) In vivo systems

In addition, as described below, in vivo organism systems can also be used in methods of identifying Aβ-modulating agents. The organism can be one that produces endogenous APP and/or Aβ peptides and processing and cleavage activities or a transgenic organism (non-human) that has been generated to express heterologous APP and/or Aβ peptides and/or processing and cleavage activities. Organisms include, but are not limited to, mammals (e.g., rodents) salmon (Maldonado et al. (2000) Brain Res.

858:237-251), and invertebrate animals, for example, Drosophila and C. elegans (see, e.g., Link (2001) Meek Ageing Dev. 722:1639-1649).

For example, in methods of identifying agents that modulate Aβ levels, an organism can be contacted with a test agent and the levels of Aβ in any sample from the organism, e.g., tissue, plasma, CSF and brain, can be compared between treated and untreated organisms. Plasma and CSF can be obtained from an organism using standard methods. For example, plasma can be obtained from blood by centrifugation, CSF can be isolated using standard methods, and brain tissue can be obtained from sacrificed organisms. The organism can be contacted with a test agent in various ways. For example, the test agent can be dissolved in a suitable vehicle and administered orally or by injection. The test agent also can be administered as a component of drinking water or feed.

2. Identification of agents that modulate Aβ levels Cellular and extracellular Aβ levels, and the degree of Aβ accumulation, are dependent on Aβ production, through APP cleavage and processing, as well as on Aβ catabolism, degradation and clearance. A method for identifying or screening for agents that modulate Aβ levels can include steps of contacting a sample containing APP (and/or portion(s) thereof) with a test agent and identifying an agent that alters any one or more aspects of Aβ production and/or Aβ catabolism. Thus, the method can include a step of identifying an agent that alters the Aβ peptide-producing cleavage of APP, the processing of APP, the processing of Aβ and/or the levels of one or more Aβ peptides in a sample. a. Assessment of Aβ peptide-producing cleavage of APP and APP processing Any of the methods for identifying or screening for agents that modulate Aβ levels can include a step of assessing Aβ peptide-producing cleavage of APP and APP processing of a sample. For samples that contain a source of APP and of an APP- processing activity, a variety of methods are provided for assessment of Aβ peptide- producing cleavage of APP and APP processing. In a particular embodiment measurement of Aβ levels of the sample (as described in detail below) can provide a method for assessing Aβ peptide-producing cleavage of APP and APP processing. In other embodiments, measurement of APP fragments levels in a sample other than Aβ can be used as a means for assessing Aβ peptide-producing cleavage of APP and APP processing. In other embodiments, measurement of the activity of one or more enzymes in the sample can be used to assess Aβ peptide-producing cleavage of APP and APP processing. The one or more enzymes are enzymes that participate in either the amyloidogenic or non-amyloidogenic APP cleavage pathways.

As described herein, APP can undergo proteolytic processing via two pathways: an amyloidogenic pathway and a non-amyloidogenic pathway. In the non-amyloidogenic pathway, cleavage of APP by α-secretase occurs at position 16 within the Aβ domain releasing the large N-terminal secreted ectodomain of APP ending at the α-secretase cleavage site (sAPPo) and a non-amyloidogenic C-terminal fragment of about 10 kD (C83; the 83-amino acid carboxyl tail of APP). Because osecretase cleaves within the Aβ domain, this cleavage precludes Aβ formation. Rather, the C-terminal fragment of APP generated by α-secretase cleavage is subsequently cleaved by γ-secretase within the predicted fransmembrane domain to generate a 22-24 residue non-amyloidogenic peptide fragment termed p3.

Alternatively, in the amyloidogenic pathway, cleavage of APP by β-secretase (BACE) occurs at the beginning of the Aβ domain defining the amino terminus of the Aβ peptide. This cleavage generates a shorter soluble N-terminus, APPβ, as well as an amyloidogenic C-terminal fragment (C99), the 99-amino acid C-terminal fragment that contains the fransmembrane and cytoplasmic domains of APP. Further cleavage of this C-terminal fragment by γ-secretase, a presenilin-dependent enzyme, generates Aβ.

The activity of β-secretase versus α-secretase and, thus, the proportion of APP processed by these enzymes will affect the amount of Aβ produced. Swedish APP mutations have been mapped to the β-secretase cleavage site in APP and favor β secretase cleavage of APP. Thus, cells expressing these mutations secrete increased amounts of Aβ and decreased amounts of p3 as compared with cells expressing wild-type APP. In contrast to the Swedish mutation, which increases β-secretase cleavage, activation of protein kinase C (PKC) by phorbol 12-myristate 13 -acetate (PMA) has been shown to favor α-secretase cleavage at the expense of β-secretase cleavage (Skovronsky et al, (2000) J. Bio. Chem. 275: 2568-2575) indicating that PKC-regulated c-secretase competes directly with β-secretase for cleavage of APP. Furthermore, changes in levels of APP-CTFs have been shown to mirror changes seen in sAPPβ and sAPPα (e.g., increased levels of Aβ or decreased levels of p3 can be indicated by an increase in sAPPβ or by a similar decrease in sAPPα)

Since β-secretase activity may be limited by the availability of APP, then increased cleavage of APP by other secretases could decrease β-secretase cleavage of APP and hence Aβ production. Also, by the same reasoning, decreased cleavage of APP by other secretases could increase β-secretase cleavage of APP leading to increased Aβ production. It can, therefore, generally be assumed that an alteration in the non- amyloidogenic pathway will result in a similar but opposite alteration in the amyloidogenic pathway. Thus, agents that modulate enzymes or the regulation of enzymes in either the amyloidogenic or non-amyloidogenic pathway can modulate levels of Aβ. As a result, peptide-producing cleavage of APP and APP processing may be assessed by measuring the activity of such enzymes. Assessment of the activity of such enzymes can provide information about peptide-producing cleavage of APP and APP fragment production pattern (i.e., the types and amounts of APP peptide fragments produced by APP fragment production enzymes). Alternatively, assessment of peptide fragments (particularly non-Aβ peptide fragments) produced in both pathways (APP fragment production patterns) can provide information about the activities of enzymes in the pathways and peptide producing cleavage of APP. In a particular embodiment, Aβ peptide-producing cleavage of APP can be assessed by monitoring the activity of enzymes and/or the cleavage of APP by enzymes of the non-amyloidogenic pathway, specifically α-secretase activity and/or the levels of fragments generated by cn-secretase activity including sAPPα, C83 and p3 peptide fragments. Likewise, agents that alter the Aβ peptide-producing cleavage of APP and APP processing may be screened for by monitoring enzyme activities and/or fragmentation patterns in the presence and absence of test agents. b. Assessment of Aβ processing Any of the methods for identifying or screening for agents that modulate Aβ levels can include a step of assessing Aβ processing of a sample. For samples that contain a source of APP and an APP-processing activity, methods such as those described above can provided for assessment of Aβ processing. For samples that contain a source of Aβ and of an Aβ degradation activity, a variety of methods are provided for assessment of Aβ processing. In a particular embodiment measurement of Aβ levels of the sample (as described in detail below) can provide a method for assessing Aβ processing. In other embodiments, measurement of the activity of one or more degradation and/or clearance pathways and/or degradation fragment patterns in the sample can be used to assess Aβ processing. The one or more pathways include, but are not limited to, proteolytic degradation, receptor-mediated clearance, non-receptor- mediated clearance, and/or aggregation/fibrillogenesis. Defects in pathways for Aβ degradation and clearance can lead to an alteration in the levels of Aβ and, therefore, could underlie some or many cases of amyloidosis and other neurodegenerative disease such familial and sporadic AD as well as other diseases and disorders characterized by misregulation of Aβ. Aβ processing may, therefore, be assessed by monitoring enzyme activities involved in the degradation and clearance of Aβ. In addition, fragmentation patterns of Aβ produced upon cleavage by degradative enzymes may be used to assess Aβ processing. There are numerous proteases in the brain that could potentially participate in Aβ turnover, and there is evidence that several enzymes may contribute to the degradation of Aβ peptides in brain tissue including insulin-degrading enzyme (EDE), neprilysin, plasmin, uPA/tPA, endothelin converting enzyme- 1, and matrix metalloproteinase-9 (Selkoe J. (2001) Neuron 32:177-180). Similarly, agents that alter Aβ processing may be screened for by monitoring the activity of one or more enzymes involved in the degradation and/or clearance of Aβ and/or fragmentation patterns of resulting degradation products in the presence and absence of test agents. c. Assessment of Aβ levels Any of the methods for identifying or screening for agents that modulate Aβ levels can include a step of assessing Aβ levels of a sample. For samples that contain a source of APP and of an APP-processing activity, an assessment of Aβ levels of the sample can provide a method for assessing Aβ peptide-producing cleavage of APP and for assessing APP processing. For samples that contain a source of Aβ peptides and of Aβ catabolic activity, an assessment of Aβ levels of the sample can provide a method for assessing processing of Aβ. For samples that contain a source of APP, Aβ, APP- processing activity, and a source of Aβ catabolic activity, an assessment of Aβ levels of the sample can provide a method for assessing the overall balance of Aβ peptide- producing cleavage of APP, APP processing and Aβ processing.

In assessing the Aβ levels of a sample, the total Aβ (i.e., all forms of Aβ) level can be assessed in an indiscriminant determination of the Aβ level of a sample, or the level of one or more specific forms of Aβ can be assessed. In one embodiment of the methods, the level of Aβ42, Aβ40, Aβ39 and/or Aβ38 is assessed. In a particular embodiment, the level of Aβ42 is assessed.

Methods and compositions for indiscriminant assessment of total Aβ levels and for selective assessment of a particular Aβ peptide are provided herein. In a method provided herein for indiscriminant assessment of total Aβ levels, the sample, or portion thereof, is contacted with an antibody that binds to forms of Aβ that contain amino acids 1-12 of SEQ ID NO: 4. Also provided is an antibody that binds to forms of Aβ that contain amino acids 1-12 of SEQ ED NO: 4. In a method provided herein for the selective assessment of Aβ42 levels, the sample, or portion thereof, is contacted with an antibody that selectively binds to Aβ42 (e.g., the sequence of amino acids 1-42 of SEQ ED NO: 4) relative to other forms of Aβ. Also provided is an antibody, and portions thereof, that selectively bind to Aβ42 relative to other forms of Aβ.

The Aβ levels of a sample or any portion(s) thereof may be assessed in the methods. For example, if the sample is a cell-free medium or culture medium, the Aβ levels of the medium can be assessed. If the sample is a cell sample, the Aβ levels of the exfracellular medium (e.g., secreted Aβ) of the sample and/or the cellular (e.g., intracellular and/or membrane-associated Aβ) Aβ levels can be assessed. To assess the cellular Aβ levels, lysates, exfracts, and/or membranes of the cells can be analyzed for Aβ protein. If the sample is an organism, then the cellular, tissue, and/or secreted Aβ levels can be assessed. For example, secreted Aβ levels could be assessed in fluids of the organism, such as, for example, any bodily fluids. Levels of secreted Aβ may be monitored, for example, by the methods described in Example 6. Preparation of whole cell lysates and membrane fractions are well known to those of skill in the art. Cell lysates may be obtained for instance by the method described in Example 8 for the identification of LRP-CTFs.

(1) Procedures for assessing Aβ levels

Assessment of the Aβ level of a sample or portion(s) thereof can be conducted using methods described herein or any method known in the art for detecting the presence of and/or measuring the level or amount of a peptide or protein in a sample. For example, immunological detection techniques employing binding substances such as antibodies, antibody fragments, recombinant antibodies, and the like, can be used. Detection of Aβ peptide can be carried out using any standard antibody-based assays. Exemplary immunoassays are described in detail, for example, in Antibodies: A Laboratory Manual, Harlow and Lane (Eds.), Cold Spring Harbor Laboratory Press, 1988. Representative examples of such assays include, for example, concurrent immunoelecfrophoresis, radioimmunoassay, immunoprecipitation, western hybridization, and enzyme-linked immunosorbent assays (ELISA), inhibition or competition assay, and sandwich assay. Suitable immunological methods employing a single antibody are also contemplated, for example, radioimmunoassay using an antibody specific for a particular form of Aβ, or single antibody ELISA methods.

Mass spectrometry and elecfrophoretic analysis of at least partially purified Aβ peptides are also techniques that can be used to detect and quantitate Aβ. In addition, the levels of different forms of Aβ can be quantified using known methods such as, for example, using internal standards and/or calibration curves generated by performing the assay with known amounts of standards.

(2) Immunological methods for Aβ detection

Aβ peptides, which can differ by only a single amino acid, can be fairly similar in molecular weight. Therefore, methods, such as immunological methods, that are based in detecting properties of Aβ peptides that can be more distinctive than molecular weight (at least when using standard and relatively inexpensive laboratory reagents and equipment) can be well-suited for assessing the level of a particular Aβ peptide. Methods and compositions for use in immunoassays for Aβ peptides in general are described herein. Compositions and methods for detecting Aβ peptides that contain the sequence of amino acids 1-12 of SEQ ED NO: 4, or a portion of this sequence, are provided herein. The compositions and methods are based on the generation of antibodies against a peptide having the amino acid sequence of amino acids 1-12 of SEQ ED NO: 4. In a particular embodiment, the antibody is B436, or a fragment thereof (see Examples 2 and 4). Because most Aβ peptides contain such a sequence, these compositions and methods are particularly useful in assessing the total Aβ content of a sample and in detecting most forms of Aβ.

More particularly, compositions and methods for detecting Aβ42 or assessing the Aβ42 content of a sample are provided herein. The compositions and methods are based on the development of an antibody that selectively binds Aβ42 relative to other Aβ peptides. In a particular embodiment, the antibody is A387, or a fragment thereof (see Examples 1 and 4).

(a) Antibody preparation Antibodies specific for Aβ may be prepared against a suitable antigen or hapten comprising the desired target epitope. The target epitope may include any number of amino acids within any portion of an Aβ amino acid sequence. SEQ ED NO: 4 provides the amino acid sequence of a 43-amino acid form of a human Aβ (Aβ43). Shorter forms of human Aβ peptides include, but are not limited to, those having the amino acid sequence of amino acids 1-42, 1-40, 1-39, 1-38, 1-37 and 1-34 of SEQ D NO: 4. Typically, the target epitope will include at least 2 contiguous residues and may include more than 6 contiguous residues within any portion of the Aβ amino acid sequence. The target epitope may include a sequence of amino acids from the amino terminus typically any of amino acids 1-13, the junction region typically containing any of the amino acids residues 13-26 and the carboxy terminus typically containing any of the amino acid residues 33-42.

A target epitope composed of such peptide fragments may be prepared, for example, from mammals such as humans, monkeys, rats and mice by methods which are known to those of skill in the art, and may also be purified natural samples which are commercially available. Partial peptides can be obtained by hydrolyzing longer forms of Aβ successively from the N-terminus and/or the C-terminus with exoproteases such as aminopeptidase and carboxypeptidase or mixtures thereof or various endopeptidases or mixtures thereof.

Synthetic peptides may be prepared by methods known in the art including solid phase synthesis methods and liquid phase synthesis methods. Examples of such synthesis methods include methods described in Merrifield, (1963) J. Am. Chem. Soc. §5:2149-2156; Bodanszky and Ondetti, Peptide Synthesis, Interscience Publishers, New York (1966); and Schroder and Lubke, The Peptide, Academic Press, New York, (1965). For example, when the Aβ peptides are synthesized by solid methods, any resins known in the art as insoluble resins (such as chloromethyl resins and 4- oxymethylphenylacetamidomethyl resins) are used for a successive condensation of protected amino acids to the C-terminal sides of the Aβ synthetic peptides according to usual methods. The protective groups are removed by hydrogen fluoride treatment, followed by purification by methods which are known in the art, such as high performance liquid chromatography. Thus, the desired Aβ peptides can be obtained. N-protected amino acids can be produced by the methods of protecting the a- amino-groups with Boc groups; further, for example, the hydroxyl groups of serine and threonine with Bzl groups; the ω-carboxylic acid groups of glutamic acid and aspartic acid with OBzl groups; the e -amino group of lysine with a Cl-Z group; the guanido group of arginine with a Tos group; and the imidazole group of histidine with a Bom group. Once a sufficient quantity of peptide hapten has been obtained, it may be conjugated to a suitable immunogenic carrier. Natural polymer carriers can be used as immunogenic carriers and include, for example, albumin, thyroglobulin, hemoglobin, keyhole limpet hemocyanin, or other suitable protein carriers, as generally described in Hudson and Hay, Practical Immunology, Blackwell Scientific Publications, Oxford, Chapter 1.3, 1980. Examples of synthetic polymer carriers that can be used include various latexes of polymers or copolymers such as amino acid polymers, styrene polymers, acrylic polymers, vinyl polymers and propylene polymers. An exemplary immunogenic carrier utilized in the Examples provided herein is ovalbumin. Since Aβ peptides aggregate easily, insolubilized Aβ haptens can also be directly immunized without the use of a carrier. hi addition, various condensing agents can be used for coupling of the haptens and the carriers. Examples of the condensation agents include diazonium compounds such as bis-diazotized benzidine which crosslinks tyrosine, histidine and tryptophan; dialdehyde compounds such as glutaraldehyde which crosslinks amino groups together; diisocyanate compounds such as toluene-2,4-diisocyanate; dimaleimide compounds such as N,N'-o-phenylenedimaleimide which crosslinks thiol groups together; maleimide active ester compounds which crosslink amino groups and thiol groups; and carbodiimide compounds crosslinking amino groups and carboxyl groups. When amino groups are crosslinked together, there is another way in which an active ester reagent (for example, SPDP) having a dithiopyridyl group is reacted with one amino acid, followed by reduction to introduce a thiol group, whereas a maleimide group is introduced into the other amino group by the use of a maleimide active ester reagent, and then, both can be reacted with each other. Examples of such methods for crosslinking haptens with immunogenic carriers can be found, for example, in U.S. Patent Nos. 4,140,662 and 4,486,344.

Haptens can be used alone or together with carriers and diluents to produce antibodies specific for the desired epitope by in vitro or in vivo techniques. In vitro techniques involve exposure of lymphocytes to the immunogens, while in vivo techniques require the injection of the immunogens into a suitable vertebrate host. Suitable vertebrate hosts are non-human, including, for example, monkeys, dogs, guinea pigs, mice, rats, rabbits, sheep, goats, and chickens. Immunogens are delivered to the animal according to a predetermined schedule, and the animals are periodically bled, with successive bleeds having improved titer and specificity. The immunogens can be delivered to any antibody-producible site, for example, by intramuscular, intraperitoneal, subcutaneous and intravenous injections. Adjuvant may also be employed to enhance antibody production. Adjuvants may provide for sustained release of the injected immunogen, serve as a vehicle to help deliver the immunogen to the spleen and/or lymph nodes, and/or work to activate the various cells involved in the immune response, either directly or indirectly. Adjuvants may include, for example, Freund's Complete Adjuvant, Freund's Incomplete Adjuvant, Montanide ISA Adjuvants (Seppic, Paris, France), Ribi's Adjuvants (Ribi Immuno Chem Research, Inc., Hamilton, MT), Hunter's TiterMax (CytRx Corp., Norcross, GA), Aluminum Salt Adjuvants, nitrocellulose-adsorbed protein, encapsulated antigens (such as liposome-enfrapped antigen, nondegradable ethylene-vinyl acetate copolymer (EVAc)-enfrapped antigen, and degradable polymer- entrapped antigen), and Gerbu Adjuvant (Gerbu Biotechnik GmbH, Gaiberg, Germany/C-C Biotech, Poway, CA).

Antibody producing cells can be obtained by hyperimmunizing a host animal, such as a mouse, with the desired immunogen by the methods described herein. The host is then killed, usually several days after the final immunization, the spleen and/or lymph nodes cells collected, and the cells immortalized resulting in anti-Aβ monoclonal antibody-producing hybridomas. Immortalization may be carried out by any method known to those of skill in the art or provided herein. Methods of immortalization may include, for example, fusion with a myeloma cell fusion partner (Kohler and Milstein (1975) Nature 256:495-497), EBV transformation, and transformation with bare DNA, e.g., oncogenes or retroviruses, or any other method which provides for stable maintenance of the cell line and production of monoclonal antibodies such as those described in Antibodies: A Laboratory Manual, Harlow and Lane, eds., Cold Spring Harbor Laboratory, 1988. An exemplary immortalization method utilized in the Examples provided herein is the fusion of mouse spleen cells with mouse thymocytes. Hybridomas can then be cloned and screened for avidity. Antibody avidity is the functional affinity or combining sfrength of an antibody with its antigen and is related to both the affinity of the reaction between the epitopes and paratopes, and the valences or recognition sites of the antibody and antigen. Avidity can be viewed as the total binding sfrength of all of an antibody's binding sites together. Affinity of an antibody reflects the goodness of fit of an antigenic determinant to a single antigen-binding site and is independent of the number of sites. Methods of assaying for antibody binding affinity are well known to those of skill in the art. Affinity or binding strength is generally expressed as the affinity constant (K). The affinity constant, alternatively called an association constant (Ka), can be determined by measuring the concentration of free antigen required to fill half of the antigen-binding sites on the antibody. The reciprocal of the antigen concentration that produces half-maximal binding is equal to the affinity constant of the antibody for the antigen. The affinity constant can be determined by measuring the association or dissociation constant for an antibody. Association and dissociation constants can be determined, for example, using a competition ELISA. The degree of recognition of an antibody for an antigen is related to the selectivity

(or specificity) of an antibody. Selectivity is considered a measure of the functional ability of an antibody to discriminate between the target antigen and other, chemically similar structures. Methods of assaying for antibody binding selectivity are well known to those of skill in the art. Selectivity can be determined, for example, by comparing the binding affinity of the antibody for the target antigen with the binding affinity of the antibody for other chemically similar molecules. Positive clones producing antibodies with high affinity and selectivity for specific Aβ peptides of interest can thus be chosen. The desired monoclonal antibodies can be produced by injecting the hybridoma cells selected for their ability to produce high avidity antibodies into mice or by growing them in culture. With in vivo production, hybridoma cells are injected infraperitoneally into syngeneic animals, such as, for example, BALB/c mice or SCED mice, and ascites fluid obtained and purified. In addition, a primer or adjuvant may be used, such as, for example, pristane (2,6,10,14-teframethyl pentadecane) or incomplete Freund's adjuvant to suppress the immune system so that the growth of the hybridoma cells is not strongly impaired, and to prohibit toxic irritation which may lead to peritonitis and the secretion of serous fluid. Purification may be carried out using standard antibody purification techniques, such as, for example, affinity chromatography using Protein A or Protein G. i. Aβ42-selective antibody

Particular embodiments of the methods provided herein for identifying or screening for agents that modulate Aβ levels include a step of identifying an agent that modulates the level of Aβ42 in a sample. In one embodiment, the step involves identifying an agent that selectively modulates the level of Aβ42 in a sample relative to Aβ40 and/or increasing the level of Aβ39. Thus, the practice of some of the methods provided herein involves the ability to detect a particular species of Aβ, such as Aβ42, and to distinguish it from other species (e.g., from other Aβ forms that do not contain the "42" carboxy terminus, such as Aβ40). Antibodies and fragments thereof selective or specific for Aβ42 are provided herein. Also provided are isolated antibodies selective or specific for Aβ42. Further provided are amino acid sequences and proteins that portions of the antibodies. Also provided are isolated proteins that are portions of the antibodies. In a particular embodiment, the antibody is a mouse antibody. In a particular embodiment, the antibody is a monoclonal antibody, such as, for example, a mouse monoclonal antibody. In one embodiment, the Aβ42-selective antibody is one generated against a peptide based on a mammalian Aβ amino acid sequence, including, for example, a human Aβ amino acid sequence. In a particular embodiment, the Aβ42- selective antibody is an IgG. In one embodiment, the antibody type is IgG2a kappa. The Aβ42-selective antibodies provided herein bind Aβ42 with minimal to no binding of other Aβ forms, e.g., Aβl-40, Aβl-11, 1-28, 1-38, and 1-39). In a particular embodiment, the Aβ42-selective antibody has at least 100-fold, 200-fold, 300-fold, 400- fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1000-fold or more specificity or selectivity for Aβ42 relative to other forms of Aβ, and, in particular Aβ40. In one embodiment, the Aβ42-selective antibody has at least about 1000-fold specificity or selectivity for Aβ42 relative to Aβ40. The antibodies selective for Aβ42 provided herein have a high affinity for binding to Aβ42. In a particular embodiment, the antibody has an c c affinity constant for binding to Aβ42 of at least about 10 1/mol, 2 x 10 1/mol, 3 x 10 1/mol, 4 x 10⁵ 1/mol, 5 x 10⁵ Vmol, 6 x 10⁵1/mol, 7 x 10⁵1/mol, 8 x 10⁵ 1/mol, 9 x 10⁵ 1/mol, 10 1/mol, 2 x 10 1/mol, 3 x 10 1/mol or 4 x 10 1/mol or more. In one embodiment, the antibody has an affinity constant for binding to Aβ42 of at least about 4 x 10 1/mol. In a particular embodiment, the Aβ42-selective antibody has an affinity constant for binding to Aβ42 of at least about 4 x 10 1/mol and at least about 1000-fold specificity or selectivity for Aβ42 relative to Aβ40.

In one embodiment, an antibody or portion or fragment thereof provided herein contains a sequence of amino acids as set forth in SEQ ED NO: 12. In a particular embodiment, an antibody or portion or fragment thereof contains a sequence of amino acids set forth as amino acids 1-107 or 1-95 of SEQ ED NO: 12. In another embodiment, an antibody or portion or fragment thereof contains a light chain variable region containing the sequence of amino acids 1-95 of SEQ ED NO: 12. In a further embodiment, an antibody or portion or fragment thereof contains a light chain variable region containing the sequence of amino acids selected from 1-50, 1-60, 1-70, 1-80, 1-90, 1-91, 1-92, 1-93, or 1-94 of SEQ ED NO: 12. In a particular embodiment, the light chain is a kappa light chain. In another embodiment, the antibody or portion or fragment thereof that contains a sequence of amino acids set forth as amino acids 1-95 of SEQ ED NO: 12 further contains a joining (J) region. In a particular embodiment, the J region is a Jkappa region. The J region can be one from any species, including but not limited to mammals, such as, for example, a primate, a rodent, or a human. In one embodiment, the J region contains a sequence of amino acids set forth as amino acids 96-107 as set forth in SEQ ED NO: 12. In one embodiment, an antibody or portion or fragment thereof contains a sequence of amino acids set forth as amino acids 1-95 of SEQ ED NO: 12 and a sequence of amino acids of a constant (C) region, such as, for example, a light chain C region. The C region can be one from any species, including but not limited to mammals, such as, for example, a primate, a rodent, or a human. In a particular embodiment, the C region is a Ckappa region. For example, the C region can contain the sequence of amino acids set forth in SEQ ED NOs: 63, 65 or 81. In a particular embodiment the antibody or fragment thereof contains a sequence of amino acids as set forth in SEQ ED NO: 97. hi one embodiment, an antibody or portion or fragment thereof provided herein contains a sequence of amino acids set forth in SEQ ED NO: 14. In a particular embodiment, an antibody or portion or fragment thereof contains a sequence of amino acids set forth as amino acids 1-118 or 1-97 of SEQ ED NO: 14. In one embodiment, an antibody or portion or fragment thereof contains a heavy chain variable region containing the sequence of amino acids 1-97 of SEQ ED NO: 14. In a further embodiment, an antibody or portion or fragment thereof contains a heavy chain variable region containing the sequence of amino acids selected from 1-50, 1-60, 1-70, 1-80, 1-90, 1-91, 1-92, 1-93, 1-94, 1-95, or 1-96 of SEQ ED NO: 14. In a particular embodiment, the heavy chain is a γ heavy chain. In one embodiment, the antibody is an IgG_2a. hi another embodiment, the antibody or portion or fragment thereof that contains a sequence of amino acids set forth as amino acids 1-97 of SEQ ED NO: 14 further contains a diversity and joining ("DJ") region. The DJ region can be one from any species, including but not limited to mammals, such as, for example, a primate, a rodent, or a human, hi a particular embodiment, the DJ region is a heavy chain D J region, such as a D J_γ region. In one embodiment, the DJ region contains a sequence of amino acids set forth as amino acids 98-118 as set forth in SEQ ED NO: 14. hi one embodiment, an antibody or portion or fragment thereof contains a sequence of amino acids set forth as amino acids 1-97 of SEQ ED NO: 14 and a sequence of amino acids of a constant (C) region, such as, for example, a heavy chain C region. The C region can be one from any species, including but not limited to mammals, such as, for example, a primate, a rodent, or a human. In a particular embodiment, the C region is a C_γ region. For example, the C region can contain the sequence of amino acids set forth in SEQ ED NO: 69, 71, 83, 85 or 87. In a particular embodiment the antibody or fragment thereof contains a sequence of amino acids as set forth in SEQ ED NO: 98. In one embodiment, an antibody or portion or fragment thereof contains the sequence of amino acids set forth in SEQ ED NO: 12 (or amino acids 1-107 or 1-95 of SEQ ID NO: 12) and the sequence of amino acids set forth in SEQ ED NO: 14 (or amino acids 1-118 or 1-97 of SEQ D NO: 14). hi another embodiment, an antibody or portion or fragment thereof contains the sequence of amino acids set forth in SEQ ID NO: 12 (or amino acids 1-107 or 1-95 of SEQ ED NO: 12) and the sequence of amino acids set forth in SEQ ED NO: 14 (or amino acids 1-118 or 1-97 of SEQ ED NO: 14) and further contains amino acid sequence of one or more J and/or DJ regions. For example, the J region can be a light and or heavy chain J region. The J and/or DJ region(s) can be from any species, including, but not limited to mammals, such as, for example, primates, rodents and humans. Exemplary J regions include, but are not limited to, a Jkappa region (e.g., such as one containing a sequence of amino acids 96-107 as set forth in SEQ ED NO: 12) and/or a heavy chain DJ region, such as a DJ₇ region (e.g., such as one containing a sequence of amino acids 98-118 as set forth in SEQ ED NO: 14). Other exemplary J regions include, but are not limited to, a light chain J region (e.g., such as one containing a sequence of amino acids set forth in SEQ ED NO: 46, 48, 50, 52, 54, 55, 57, 59, 61, 73, 75, 77, or 79 and/or a heavy chain J region (e.g., such as one containing a sequence of amino acids set forth in SEQ ED NO: 67, 89, or 91). In another embodiment, an antibody or portion or fragment thereof contains the sequence of amino acids set forth in SEQ ID NO: 12 (or amino acids 1-107 or 1-95 of SEQ ED NO: 12) and the sequence of amino acids set forth in SEQ ED NO: 14 (or amino acids 1-118 or 1-97 of SEQ ED NO: 14) and further contains amino acid sequence of one or more constant regions. For example, the constant region can be a light and/or heavy chain constant region. The C region(s) can be from any species, including, but not limited to mammals, such as, for example, primates, rodents and humans. Exemplary constant regions include, but are not limited to, a Cka_PPa region. Exemplary light chain constant regions may contain a sequence of amino acids set forth in SEQ ED NO: 63, 65 or 81. Exemplary constant regions may also include, but are not limited to, a C_γ region. Exemplary heavy chain constant regions may contain a sequence of amino acids set forth in SEQ ED NO: 69, 71, 83, 85 or 87. In a particular embodiment of any of the antibodies, the antibody or portion or fragment thereof is an IgG_2a type.

Also provided are derivatives and modified immunoglobulins that have the capacity to bind to Aβ. In a particular embodiment, such molecules include fragments, such as Fab' or Fab'2 produced, for example, by the proteolytic cleavage of the mAb. Such molecules may also include single-chain immunoglobulins producible, for example, via recombinant means, such as Fv, scFv. Portions or fragments of antibodies include fragments that contain at least a portion of the antigen-binding region of the antibody. The portion of the antigen-binding region can be one that binds to the same antigenic determinant as the antibody with an affinity of at least about 1%, 5%, 10%, 15%, 20%, 25%, 50%), 60%, 70%, 75%>, 80%, 90% or 100% of the affinity of the entire antibody. In particular embodiments, such fragments can be combined with one another (e.g., to form a diabody) or with other antibody fragments or receptor ligands to form "chimeric" binding molecules. Significantly, such chimeric molecules may contain substituents capable of binding to different epitopes of the same molecule (e.g., two different Aβ epitopes). Whole antibodies molecules are large proteins, -150 kDa in size, made up of four chains, two heavy chains (-50 kDa each), and two light chains (-25 kDa each). The domains responsible for targeting specifically foreign entities is called the Fv domains. The Fv domain contains a portion of a heavy chain domain (HFv) and a light domain (LFv). Fv's are not produced by the body but can be engineered. An scFv fragment is an entity very similar to the Fv fragment, except the heavy and light chains are connected via a linker sequence. A dimer of scFv fragments is called a diabody. Fab fragments contain portions of heavy and light domains that are chemically linked. Fab fragments can be prepared from the parent antibody, by simple enzymatic hydrolysis. Thus, a "portion or fragment" of antibody refers to any of these aforementioned antibody fragments as well as to any fragment or portion of an antibody that retains an at least 100-fold, 200-fold, 300-fold, 400-fold, 500-fold up to 1000-fold selectivity for Aβ42 relative to other Aβ peptides, and particularly relative to Aβ40.

Also provided herein are nucleic acids encoding an antibody or a portion or fragment thereof. Further provided are isolated nucleic acids containing nucleotide sequences encoding portions of the antibodies. In a particular embodiment, the antibody is a mouse antibody. In one embodiment, a nucleic acid contains a sequence of nucleotides that encodes the amino acid sequence set forth in SEQ ED NO: 12. In a particular embodiment, a nucleic acid contains a sequence of nucleotides that encodes the amino acid sequence set forth as amino acids 1-107 or 1-95 of SEQ ED NO: 12. In one embodiment, a nucleic acid contains a sequence of nucleotides that encodes a light chain variable region containing the sequence of amino acids 1-95 of SEQ ED NO: 12. In a further embodiment, a nucleic acid contains a sequence of nucleotides that encodes a light chain variable region containing the sequence of amino acids selected from 1-50, 1- 60, 1-70, 1-80, 1-90, 1-91, 1-92, 1-93, or 1-94 of SEQ ED NO: 12. In a particular embodiment, the light chain is a kappa chain. In another embodiment, a nucleic acid contains a sequence of nucleotides that encodes the sequence of amino acids 1-95 of SEQ ED NO: 12 and a J region. The J region can be from any species, including but not limited to mammals, such as, for example, primates, rodents and humans. For example, the J can contain the sequence of amino acids set forth in SEQ ED NO: 46, 48, 50, 52, 54, 55, 57, 59, 61, 73, 75, 77 or 79. In a particular embodiment, the J region contains a sequence of amino acids 96-107 as set forth in SEQ ED NO: 12. In another embodiment, a nucleic acid contains a sequence of nucleotides that encodes the sequence of amino acids 1-95 of SEQ ED NO: 12 and a constant (C) region. The C region can be from any species, including but not limited to mammals, such as, for example, primates, rodents and humans, hi particular embodiments, the C region is a light chain C region. For example, the C region can be a kappa light chain constant sequence. In one example, the C region can contain the sequence of amino acids set forth in SEQ ED NO: 63, 65 or 81. hi another embodiment, a nucleic acid can further contain, in addition to a sequence of nucleotides encoding a C region, a sequence of nucleotides encoding a variable region such as those described herein above. In a particular embodiment, a nucleic acid provided herein contains the sequence of nucleotides set forth in SEQ ED NO: 11 or the sequence of nucleotides 1-285 set forth in SEQ ED NO: 11.

In another embodiment, a nucleic acid contains a sequence of nucleotides that encodes the amino acid sequence set forth in SEQ ED NO: 14. In a particular embodiment, a nucleic acid contains a sequence of nucleotides that encodes the amino acid sequence set forth as amino acids 1-118 or 1-97 of SEQ ED NO: 14. fri one embodiment, a nucleic acid contains a sequence of nucleotides that encodes a heavy chain variable region containing the sequence of amino acids 1-97 of SEQ ED NO: 14. In a further embodiment, a nucleic acid contains a sequence of nucleotides that encodes a heavy chain variable region containing the sequence of amino acids selected from 1-50, 1-60, 1-70, 1-80, 1-90, 1-91, 1-92, 1-93, 1-94, 1-95, or 1-96 of SEQ ED NO: 14. hi a particular embodiment, the heavy chain is an IgG_2a heavy chain. In another embodiment, a nucleic acid contains a sequence of nucleotides that encodes the sequence of amino acids 1-97 of SEQ ED NO: 14 and a DJ region. The DJ region can be from any species, including but not limited to mammals, such as, for example, primates, rodents and humans. For example, the DJ can contain the sequence of amino acids set forth in SEQ ED NO: 67, 89 or 91. In a particular embodiment, the DJ region contains a sequence of amino acids corresponding to 98 through 118 as set forth in SEQ ED NO: 14. In another embodiment, a nucleic acid contains a sequence of nucleotides that encodes the sequence of amino acids 1-97 of SEQ ED NO: 14 and one or more a constant (C) regions. The C region can be from any species, including but not limited to mammals, such as, for example, primates, rodents and humans. In particular embodiments, the C region is a heavy chain C region. For example, the C region can be a heavy chain C_γ region. For example, the C region can contain the sequence of amino acids set forth in SEQ ED NO: 69, 71, 83, 85 or 87. In a particular example, the C region can be an IgG_2a heavy chain constant sequence. In another embodiment, a nucleic acid can further contain, in addition to a sequence of nucleotides encoding a C region, a sequence of nucleotides encoding a variable region such as those described herein above. In a particular embodiment, a nucleic acid provided herein contains the sequence of nucleotides set forth in SEQ ED NO: 13 or the sequence of nucleotides 1-291 of SEQ ED NO: 13. Nucleic acid constructs, including, for example, plasmids and expression vectors, are also provided herein. In one embodiment of a nucleic acid construct provided herein, the nucleic acid contains a sequence of nucleotides encoding the sequence of amino acids set forth in SEQ ED NO: 12 (or amino acids 1-107 or 1-95 of SEQ ID NO: 12) and the sequence of amino acids set forth in SEQ ED NO: 14 (or amino acids 1-118 or 1-97 of SEQ ED NO: 14). In a another embodiment, a nucleic acid provided herein contains the sequence of nucleotides set forth in SEQ ED NO: 11 and SEQ ED NO: 13. hi another embodiment, a nucleic acid provided herein contains the sequence of nucleotides set forth in SEQ ED NO: 11 and SEQ ED NO: 13 or a sequence of nucleotides encoding the sequence of amino acids set forth in SEQ ED NO: 12 (or amino acids 1-107 or 1-95 of SEQ ED NO: 12) and a sequence of nucleotides encoding the sequence of amino acids set forth in SEQ ED NO: 14 (or amino acids 1-118 or 1-97 of SEQ ED NO: 14) and further contains one or more sequences of nucleotides encoding one or more of the following amino acid sequences: a J region, e.g., a light or a heavy chain J region, including, for example, a kappa light chain J region and a γ heavy chain J region, and a C region, e.g., a light chain or heavy chain constant region, including, for example, a kappa light chain constant region, and a γ heavy chain C region, such as an IgG_2a heavy chain constant region. These regions can be from any species, including, but not limited to, mammals, such as, for example, primates, rodents and humans. Exemplary amino acid sequences of such regions can be any of those described herein above or known in the art. Antibodies selective or specific for Aβ42 can be made by immunizing an animal (e.g., a mouse) with a peptide that contains a sequence of amino acids within the sequence of Aβl-42 (such as human Aβl-42; see, e.g., SEQ ED NO: 4 amino acids 1-42) that includes amino acids C-terminal to amino acid 40 of Aβ. In a particular example, to minimize the likelihood of cross-reactivity of a generated antibody with the predominant Aβ40 species, a minimal peptidyl sequence of C-MVGGWIA was used to immunize animals, which represents the Aβ35-42 region (e.g., amino acids 35-42 of Aβ; see amino acids 35-42 of SEQ ED NO: 4). An N-terminal cysteine can be added for conjugation to an immunogenic carrier such as, for example, ovalbumin as described in Example 1. In a particular embodiment, an Aβ42-selective antibody provided herein is the monoclonal antibody A387 (described in detail in Examples 1 and 4). Antibody A387 demonsfrates very high affinity for Aβ42 with a measured affinity constant of >4 X 10⁶1/mol. Furthermore, A387 has at least 1000-fold specificity for binding to Aβ42 versus Aβ40. Additionally, this antibody was shown to be highly selective for Aβ42 versus other AB peptides. When tested by ELISA methods, the A387 antibody showed no reactivity to Aβl-11, 1-28, 1-38, and 1-39 peptides. The exceptionally high affinity and selectivity of the Aβ42-selective antibodies provided herein makes them a highly effective tool for detecting and quantitatively measuring Aβ42 and distinguishing this form of Aβ from other Aβ forms. Additionally, the Aβ42-selective antibodies provided herein are particularly useful for specifically assaying samples that contain detergents (such as Triton X-100, CHAPS, SHAPSO, Tween-2, and the like) or metal chelators (EDTA, EGTA, and the like) for Aβ42.

Antibodies provided herein can also be produced using recombinant DNA methods. For example, the recombinant production of immunoglobulin molecules, including humanized antibodies are described in U.S. Pat. Nos. 4,816,397 (Boss et al.), 4,816,567 (Cabilly et al.) U.K. patent GB 2,188,638 (Winter et al), and U.K. patent GB 2,209,757. Techniques for the recombinant expression of immunoglobulins, including humanized immunoglobulins, can also be found, among other places in Goeddel et al, Gene Expression Technology Methods in Enzymology Vol. 185 Academic Press (1991), and Borreback, Antibody Engineering, W. H. Freeman (1992). Additional information concerning the generation, design and expression of recombinant antibodies can be found in Mayforth, Designing Antibodies, Academic Press, San Diego (1993).

The host cell used to express the recombinant antibodies provided herein may be either a bacterial cell, such as Escherichia coli, or a eukaryotic cell, such as a Chinese hamster ovary cell. The choice of expression vector is dependent upon the choice of host cell, and may be selected so as to have the desired expression and regulatory characteristics in the selected host cell. The general methods for construction of the vector, transfection of cells to produce the host cell, culture of cells to produce the antibody are all well known in the art. Likewise, once produced, the recombinant antibodies may be purified by standard procedures of the art, including cross-flow filtration, ammonium sulphate precipitation, affinity column chromatography, gel electrophoresis and the like.

Antibodies can be made by constructing a vector containing a nucleic acid encoding a V region. Exemplary V regions include any of those described herein. The V region can be fused with a J region. The J region can be, for example, a light chain J region or a heavy chain J region, including, for example, a kappa light chain J region and a γ heavy chain J region. These regions can be from any species, including, but not limited to, mammals, such as, for example, primates, rodents and humans. Exemplary J regions include, but are not limited to, a Jkappa region (e.g., such as one containing a sequence of amino acids 96-107 as set forth in SEQ ED NO: 12 or a sequence of amino acids 101-112 as set forth in SEQ ID NO: 16) and or a heavy chain DJ region, such as a DJ_γ region (e.g., such as one containing a sequence of amino acids 98-118 as set forth in SEQ ID NO: 14 or a sequence of amino acids 99-114 as set forth in SEQ ED NO: 18). Other exemplary J regions include, but are not limited to, human and mouse light chain J regions (e.g., such as the ones containing a sequence of amino acids set forth in SEQ ED NOS. 73, 75, 77 or 79, and SEQ ED NOS. 46, 48, 50, 52, 54, 55, 57, 59 or 61 respectively) and human and mouse heavy chain J region (e.g., such as the ones containing a sequence of amino acids set forth in SEQ ED NOS. 89 or 91, and SEQ ED NO. 67 respectively). In constructing the vector the nucleic acid encoding the V and J regions can further be fused with nucleic acid encoding a C region. The C region can be, for example, a light chain C region or a heavy chain C region, including, for example, a kappa light chain constant region, and a γ heavy chain C region, such as an IgG_2a heavy chain constant region. These regions can be from any species, including, but not limited to, mammals, such as, for example, primates, rodents and humans. For example, mouse and human light chain C regions may contain a sequence of nucleotides that encodes the amino acid sequence set forth in SEQ ED NO. 63 or 65 and SEQ ED NO 81, respectively. Mouse and human heavy chain C regions may contain a sequence of nucleotides that encodes the amino acid sequence set forth in SEQ ED NO. 69 or 71 and SEQ ED NO 83, 85 or 87, respectively.

In certain embodiments, the recombinant antibodies provided herein may comprise a complete antibody molecule having full length heavy and light chains, or any fragment thereof, such as the Fab or (Fab')₂ fragments, a heavy chain and light chain dimer, or any minimal fragment thereof such as a Fv, an SCA (single chain antibody), and the like, specific for the particular Aβ antigen molecule.

The term humanized immunoglobulin or humanized antibody refers to an immunoglobulin comprising portions of immunoglobulins of different origin, wherein at least one portion is of human origin. Accordingly, provided herein are humanized immunoglobulins which bind to a mammalian Aβ peptide (e.g., human Aβ42 or Aβ40), said immunoglobulin comprising an antigen-binding region of nonhuman origin (e.g., rodent) and at least a portion of an immunoglobulin of human origin (e.g., a human framework region, a human constant region or portion thereof). For example, the humanized antibody can comprise portions derived from an immunoglobulin of nonhuman origin with the requisite specificity, such as a mouse, and from immunoglobulin sequences of human origin (e.g., a chimeric immunoglobulin), joined together chemically by conventional techniques (e.g., synthetic) or prepared as a contiguous polypeptide using genetic engineering techniques (e.g., DNA encoding the protein portions of the chimeric antibody can be expressed to produce a contiguous polypeptide chain).

Another example of the humanized immunoglobulins provided herein is an immunoglobulin containing one or more immunoglobulin chains comprising a CDR of nonhuman origin (e.g., one or more CDRs derived from an antibody of nonhuman origin) and a framework region derived from a light and/or heavy chain of human origin (e.g., CDR-grafted antibodies with or without framework changes), hi one embodiment, the humanized immunoglobulins can compete with murine A387 or B436 monoclonal antibodies for binding to the respective human Aβ peptides. In a particular embodiment, the antigen-binding region of the humanized immunoglobulin (a) is derived from A387 monoclonal antibody (e.g., as in a humanized immunoglobulin comprising CDR1, CDR2 and CDR3 of the A387 light chain and CDR1, CDR2 and CDR3 of the A387 heavy chain) or (b) is derived from B436 monoclonal antibody (e.g., as in a humanized immunoglobulin comprising CDR1, CDR2 and CDR3 of the B436 light chain and CDR1 , CDR2 and CDR3 of the B436 heavy chain). Chimeric or CDR-grafted single chain antibodies are also encompassed by the term humanized immunoglobulin.

As set forth above, such humanized immunoglobulins can be produced using synthetic and/or recombinant nucleic acids to prepare genes (e.g., cDNA) encoding the desired humanized chain. For example, nucleic acid (e.g., DNA) sequences coding for humanized variable regions can be constructed using PCR mutagenesis methods to alter DNA sequences encoding a human or humanized chain, such as a DNA template from a previously humanized variable region (see e.g., Kamman, M., et al., Nucl. Acids Res., 17: 5404 (1989)); Sato, K., et al., Cancer Research, 53: 851-856 (1993); Daugherty, B. L. et al., Nucleic Acids Res., 19(9). 2471-2476 (1991); and Lewis, A. P. and J. S. Crowe, Gene, 101 : 297-302 (1991)). Using these or other suitable methods, variants can also be readily produced. In one embodiment, cloned variable regions can be mutagenized, and sequences encoding variants with the desired specificity can be selected (e.g., from a phage library; see e.g., Krebber et al., U.S. Pat. No. 5,514,548; Hoogenboom et al., WO 93/06213, published Apr. 1, 1993)). In certain embodiments, the humanized antibodies provided herein may comprise a complete antibody molecule having full length heavy and light chains, or any fragment thereof, such as the Fab or (Fab')₂ fragments, a heavy chain and light chain dimer, or any minimal fragment thereof such as a Fv, an SCA (single chain antibody), and the like, specific for the particular Aβ antigen molecule. In one embodiment, an antibody or portion or fragment thereof provided herein contains a sequence of amino acids as set forth in SEQ ED NO: 12 and/or SEQ ID NO: 14 (or portions thereof such as amino acids 1-95 of SEQ ED NO: 12 and/or amino acids 1-97 of SEQ ED NO: 14) or modifications thereof that retain the antigen-binding properties of an antibody containing one or both of these sequences of amino acids. Such modifications can be determined empirically and include, for example, conservative amino acid substitutions as well as deletions and additions of residues that do not substantially alter the antigen-binding properties. Determination of residues that do not substantially alter antigen binding properties can be accomplished empirically, such as by systematic replacement of each residue in the polypeptide with another amino acid, such as alanine, serine or glycine, and testing of the resulting polypeptide for its ability to bind to the antigen compared to the unmodified polypeptide. Those that retain at least 1, 10, 25, or 50% of the binding affinity compared to the unmodified polypeptide or that have an affinity constant of at least 10⁶ are identified. Also polypeptides that include a portion of SEQ ED NO: 12, 14, 16, or 18 and retain such ability and modification thereof are included.

Also provided herein are methods for detecting Aβ42 and/or measuring Aβ42 levels or determining the Aβ42 content of a sample. The methods use antibodies provided herein. In one embodiment, the method includes steps of contacting a sample with an antibody or portion or fragment thereof provided herein and determining if the antibody (or portion or fragment thereof) forms any complexes with or binds to any molecules in the sample. The contacting can be performed under conditions whereby the antibody (or portion or fragment thereof) binds to or forms a complex with Aβ. In a particular embodiment, the antibody is selective for Aβ42 relative to other forms of Aβ, including Aβl-11, 1-28, 1-38, 1-39 and 1-40. In one embodiment, the antibody is selective for Aβ42 relative to Aβ40. In other embodiments, the antibody or portion or fragment thereof is any one of the compositions as set forth herein above or described anywhere herein, including the Examples.

Specific immunoassay-related techniques and procedures that may be used in the methods for detecting Aβ42 and/or measuring Aβ42 levels or determining the Aβ42 content of a sample are described herein or known in the art. Any such procedures may be employed in the methods. Exemplary formats include, but are not limited to, ELISA, sandwich assays, competitive immunoassays, radioimmunoassays, Western blots and indirect immunofluorescent assays. In a particular embodiment of the methods for detecting Aβ42 and/or measuring Aβ42 levels or determining the Aβ42 content of a sample provided herein, an Aβ42-selective antibody or portion or fragment thereof provided herein is contacted with the sample, and binding between the antibody (or portion or fragment thereof) and any protein or peptide in the sample is assessed in a sandwich assay, as described herein. ii. Aβl-12 antibody Antibodies that react substantially similarly to any Aβ peptide which contains an amino-terminal sequence substantially as set forth in the sequence of amino acids 1-12 of SEQ ID NO: 4 are also provided herein. Also provided are isolated proteins that are portions of the antibodies. Included among such antibodies are antibodies referred to herein as Aβl-12 antibodies. Such antibodies can be used, for example, in immunoassays to detect all forms of Aβ (total Aβ), or at least all forms of Aβ containing the amino- terminus as set forth in amino acids 1-12 of SEQ ED NO: 4. Such antibodies can also be used in conjunction with antibodies that are selective for a particular type or types of Aβ, e.g., Aβ42 (including Aβ42-selective antibodies provided herein), for example, to determine the ratio of Aβ42 to total Aβ in a sample. Antibodies that react substantially similarly to any Aβ peptide can also be used as capture or detection antibodies in conjunction with selective antibodies in sandwich immunoassays to detect a particular form of Aβ, e.g., Aβ42. Such methods using the Aβl-12 antibodies and Aβ42-selective antibodies provided herein are described herein.

In one embodiment, an antibody or portion or fragment thereof provided herein contains a sequence of amino acids as set forth in SEQ ED NO: 16. In a particular embodiment, an antibody or portion or fragment thereof contains a sequence of amino acids set forth as amino acids 1-112 or 1 to 100 of SEQ ED NO: 16. In one embodiment, an antibody or portion or fragment thereof contains a light chain variable region containing the sequence of amino acids 1-100 of SEQ ED NO: 16. In a further embodiment, an antibody or portion or fragment thereof contains a light chain variable region containing the sequence of amino acids selected from 1-50, 1-60, 1-70, 1-80, 1-90, 1-95, 1-96, 1-97, 1-98, or 1-99 of SEQ ED NO: 16. In a particular embodiment, the light chain is a kappa light chain. In another embodiment, the antibody or portion or fragment thereof that contains a sequence of amino acids set forth as amino acids 1-100 of SEQ ED NO: 16 further contains a joining (J) region. In a particular embodiment, the J region is a Jkappa region. The J region can be one from any species, including but not limited to mammals, such as, for example, a primate, a rodent, or a human. In one embodiment, the J region contains a sequence of amino acids set forth as amino acids 101-112 as set forth in SEQ ED NO: 16. In one embodiment, an antibody or portion or fragment thereof contains a sequence of amino acids set forth as amino acids 1-100 of SEQ ED NO: 16 and a sequence of amino acids of a constant (C) region, such as, for example, a light chain C region. The C region can be one from any species, including but not limited to mammals, such as, for example, a primate, a rodent, or a human. In a particular embodiment, the C region is a Ck pp region. For example, the C region can contain the sequence of amino acids set forth in SEQ ED NOs: 63, 65 or 81. hi a particular embodiment the antibody or fragment thereof contains a sequence of amino acids as set forth in SEQ ED NO: 99.

In one embodiment, an antibody or portion or fragment thereof provided herein contains a sequence of amino acids set forth in SEQ ID NO: 18. hi a particular embodiment, an antibody or portion or fragment thereof contains a sequence of amino acids set forth as amino acids 1 -114 or 1 -98 of SEQ ED NO: 18. In one embodiment, an antibody or portion or fragment thereof contains a heavy chain variable region containing the sequence of amino acids 1-98 of SEQ ID NO: 18. In a further embodiment, an antibody or portion or fragment thereof contains a heavy chain variable region containing the sequence of amino acids selected from 1-50, 1-60, 1-70, 1-80, 1-90, 1-91, 1-92, 1-93, 1-94, 1-95, 1-96, or 1-97 of SEQ ED NO: 18. In a particular embodiment, the heavy chain is a γ heavy chain. In one embodiment, the antibody is an IgG_2a. In another embodiment, the antibody or portion or fragment thereof that contains a sequence of amino acids set forth as amino acids 1-98 of SEQ ED NO: 18 further contains a diversity and joining (DJ) region. The DJ region can be one from any species, including but not limited to mammals, such as, for example, a primate, a rodent, or a human. In a particular embodiment, the DJ region is a heavy chain DJ region, such as a DJ₇ region. In one embodiment, the DJ region contains a sequence of amino acids set forth as amino acids 99-114 as set forth in SEQ ED NO: 18. In one embodiment, an antibody or portion or fragment thereof contains a sequence of amino acids set forth as amino acids 1 -98 of SEQ ED NO: 18 and a sequence of amino acids of a constant (C) region, such as, for example, a heavy chain C region. The C region can be one from any species, including but not limited to mammals, such as, for example, a primate, a rodent, or a human. In a particular embodiment, the C region is a C₇ region. For example, the C region can contain the sequence of amino acids set forth in SEQ ED NO: 69, 71, 83, 85 or 87. In a particular embodiment the antibody or fragment thereof contains a sequence of amino acids as set forth in SEQ ED NO: 100

In one embodiment, an antibody or portion or fragment thereof contains the sequence of amino acids set forth in SEQ ED NO: 16 (or amino acids 1-112 or 1-100 of SEQ ED NO: 16) and the sequence of amino acids set forth in SEQ ED NO: 18 (or amino acids 1 - 114 or 1 -98 of SEQ ED NO : 18) . hi another embodiment, an antibody or portion or fragment thereof contains the sequence of amino acids set forth in SEQ ID NO: 16 (or amino acids 1-112 or 1-100 of SEQ ED NO: 16) and the sequence of amino acids set forth in SEQ ED NO: 18 (or amino acids 1-114 or 1-98 of SEQ ED NO: 18) and further contains amino acid sequence of one or more J regions. For example, the J region can be a light and/or heavy chain J region. The J and/or DJ region(s) can be from any species, including, but not limited to mammals, such as, for example, primates, rodents and humans. Exemplary J regions include, but are not limited to, a Jka pa region (e.g., such as one containing a sequence of amino acids 101-112 as set forth in SEQ ED NO: 16) and/or a heavy chain DJ region, such as a DJ_γ region (e.g., such as one containing a sequence of amino acids 99-114 as set forth in SEQ ED NO: 18). Other exemplary J regions include, but are not limited to, a light chain J region (e.g., such as one containing a sequence of amino acids set forth in SEQ ED NO: 46, 48, 50, 52, 54, 55, 57, 59, 61, 73, 75, 77 , or 79) and or a heavy chain J region (e.g., such as one containing a sequence of amino acids set forth in SEQ ED NO: 67, 89 or 91). In another embodiment, an antibody or portion or fragment thereof contains the sequence of amino acids set forth in SEQ ED NO: 16 (or amino acids 1-112 or 1-100 of SEQ ED NO: 16) and the sequence of amino acids set forth in SEQ ED NO: 18 (or amino acids 1-114 or 1-98 of SEQ ID NO: 18) and further contains amino acid sequence of one or more constant regions. For example, the constant region can be a light and/or heavy chain constant region. The C region(s) can be from any species, including, but not limited to mammals, such as, for example, primates, rodents and humans. Exemplary constant regions include, but are not limited to, a Ckappa region. Exemplary light chain constant regions may contain a sequence of amino acids set forth in SEQ ED NO: 63, 65 or 81. Exemplary constant regions may also include, but are not limited to, a C₇ region. Exemplary heavy chain constant regions may contain a sequence of amino acids set forth in SEQ ED NO: 69, 71, 83, 85 or 87. In a particular embodiment of any of the antibodies, the antibody or portion or fragment thereof is an IgG_2a type.

In one embodiment, an antibody or portion or fragment thereof provided herein contains a sequence of amino acids as set forth in SEQ ED SEQ ED NO: 16 and/or SEQ ED NO: 18 (or portions thereof such as amino acids 1-100 of SEQ ED NO: 16 and 1-98 of SEQ ID NO: 18) or modifications thereof that retain the antigen-binding properties of an antibody containing one or both of these sequences of amino acids. Such modifications can be determined empirically and include, for example, conservative amino acid substitutions as well as deletions and additions of residues that do not substantially alter the antigen-binding properties. Determination of residues that do not substantially alter antigen binding properties can be accomplished empirically, such as by systematic replacement of each residue in the polypeptide with another amino acid, such as alanine, serine or glycine, and testing of the resulting polypeptide for its ability to bind to the antigen compared to the unmodified polypeptide. Those that retain at least 1, 10, 25, or 50% of the binding affinity compared to the unmodified pol peptide or that have an affinity constant of at least 10 are identified. Also polypeptides that include a portion of SEQ ID NO: 16 or 18 and retain such ability and modification thereof are included. Also provided herein are nucleic acids encoding an antibody or a portion or fragment thereof. Further provided are isolated nucleic acids containing nucleotide sequences encoding portions of antibodies. In a particular embodiment, the antibody is a mouse antibody. In one embodiment, a nucleic acid contains a sequence of nucleotides that encodes the amino acid sequence set forth in SEQ ID NO: 16. In a particular embodiment, a nucleic acid contains a sequence of nucleotides that encodes the amino acid sequence set forth as amino acids 1-112 or 1-100 of SEQ ED NO: 16. hi one embodiment, a nucleic acid contains a sequence of nucleotides that encodes a light chain variable region containing the sequence of amino acids 1-100 of SEQ ED NO: 16. In a further embodiment, a nucleic acid contains a sequence of nucleotides that encodes a light chain variable region containing the sequence of amino acids selected from 1-50, 1- 60, 1-70, 1-80, 1-90, 1-95, 1-96, 1-97, 1-98, or 1-99 of SEQ ED NO: 16. In a particular embodiment, the light chain is a kappa chain. In another embodiment, a nucleic acid contains a sequence of nucleotides that encodes the sequence of amino acids 1-100 of SEQ ID NO: 16 and a J region. The J region can be from any species, including but not limited to mammals, such as, for example, primates, rodents and humans. For example, the J can contain the sequence of amino acids set forth in SEQ ED NO: 46, 48, 50, 52, 54, 55, 57, 59, 61, 73, 75, 77 or 79. In a particular embodiment, the J region contains a sequence of amino acids from 101 to 112 as set forth in SEQ ED NO: 16. In another embodiment, a nucleic acid contains a sequence of nucleotides that encodes the sequence of amino acids 1-100 of SEQ ED NO: 16 and a constant (C) region. The C region can be from any species, including but not limited to mammals, such as, for example, primates, rodents and humans. In particular embodiments, the C region is a light chain C region. For example, the C region can be a kappa light chain constant sequence. In one example, the C region can contain the sequence of amino acids set forth in SEQ ID NO: 63, 65 or 81. hi another embodiment, a nucleic acid can further contain, in addition to a sequence of nucleotides encoding a C region, a sequence of nucleotides encoding a variable region such as those described herein above. In a particular embodiment, a nucleic acid provided herein contains the sequence of nucleotides set forth in SEQ ED NO: 15 or the sequence of nucleotides 1-300 set forth in SEQ ED NO: 15.

In another embodiment, a nucleic acid contains a sequence of nucleotides that encodes the amino acid sequence set forth in SEQ ED NO: 18. In a particular embodiment, a nucleic acid contains a sequence of nucleotides that encodes the amino acid sequence set forth as amino acids 1-114 or 1-98 of SEQ ED NO: 18. hi one embodiment, a nucleic acid contains a sequence of nucleotides that encodes a heavy chain variable region containing the sequence of amino acids 1 -98 of SEQ ED NO: 18. h a further embodiment, a nucleic acid contains a sequence of nucleotides that encodes a heavy chain variable region containing the sequence of amino acids selected from 1-50, 1-60, 1-70, 1-80, 1-90, 1-91, 1-92, 1-93, 1-94, 1-95, 1-96, or 1-97 of SEQ ED NO: 18. In a particular embodiment, the heavy chain is an IgG_2a heavy chain. In another embodiment, a nucleic acid contains a sequence of nucleotides that encodes the sequence of amino acids 1 -98 of SEQ ED NO: 18 and a DJ region. The DJ region can be from any species, including but not limited to mammals, such as, for example, primates, rodents and humans. For example, the DJ can contain the sequence of amino acids set forth in SEQ ID NO: 67, 89 or 91. In a particular embodiment, the DJ region contains a sequence of amino acids from 99 to 114 as set forth in SEQ ED NO: 18. In another embodiment, a nucleic acid contains a sequence of nucleotides that encodes the sequence of amino acids 1-98 of SEQ ED NO: 18 and one or more a constant (C) regions. The C region can be from any species, including but not limited to mammals, such as, for example, primates, rodents and humans. In particular embodiments, the C region is a heavy chain C region. For example, the C region can be a heavy chain C₇ region. For example, the C region can contain the sequence of amino acids set forth in SEQ ED NO: 69, 71, 83, 85 or 87. In a particular example, the C region can be an IgG_2a heavy chain constant sequence. In another embodiment, a nucleic acid can further contain, in addition to a sequence of nucleotides encoding a C region, a sequence of nucleotides encoding a variable region such as those described herein above. In a particular embodiment, a nucleic acid provided herein contains the sequence of nucleotides set forth in SEQ ED NO: 17 or the sequence of nucleotides 1-294 set forth in SEQ ED NO:17.

Nucleic acid constructs, including, for example, plasmids and expression vectors, are also provided herein. In one embodiment of a nucleic acid construct provided herein, the nucleic acid contains a sequence of nucleotides encoding the sequence of amino acids set forth in SEQ ED NO : 16 (or amino acids 1 - 112 or 1 - 100 of SEQ ED NO : 16) and the sequence of amino acids set forth in SEQ ED NO: 18 (or amino acids 1-114 or 1-98 of SEQ ED NO: 18). In a another embodiment, a nucleic acid provided herein contains the sequence of nucleotides set forth in SEQ ED NO: 15 and SEQ ED NO: 17. hi another embodiment, a nucleic acid provided herein contains the sequence of nucleotides set forth in SEQ ED NO: 15 and SEQ ED NO: 17 or a sequence of nucleotides encoding the sequence of amino acids set forth in SEQ ED NO: 16 (or amino acids 1-112 or 1-100 of SEQ ID NO: 16) and a sequence of nucleotides encoding the sequence of amino acids set forth in SEQ ED NO: 18 (or amino acids 1-114 or 1-98 of SEQ ED NO: 18) and further contains one or more sequences of nucleotides encoding one or more of the following amino acid sequences: a J region, e.g. , a light or a heavy chain J region, including, for example, a kappa light chain J region and a γ heavy chain J region, and a C region, e.g., a light chain or heavy chain constant region, including, for example, a kappa light chain constant region, and a γ heavy chain C region, such as an IgG_2a heavy chain constant region. These regions can be from any species, including, but not limited to, mammals, such as, for example, primates, rodents and humans. Exemplary amino acid sequences of such regions can be any of those described herein above or known in the art.

Antibodies that bind substantially similarly to any Aβ peptide which contains an amino-terminal sequence substantially as set forth in the sequence of amino acids 1-12 of SEQ ED NO: 4 can be generated using animal immunization or recombinant DNA protocols described herein or known in the art. For example, such an antibody provided herein, referred to as B436 (see EXAMPLES 2 and 4), was generated by designing a peptide immunogen having the sequence DAEFRHDSGYEV-C that represents the Aβl- 12 region. The resulting murine monoclonal antibody was determined to have high titer for both Aβ40 and Aβ42 peptides. iii. Aβ40 antibody

Antibodies that bind Aβ40 (e.g., a form of Aβ containing the sequence of amino acids 1-40 of SEQ ED NO: 4) were also generated for use in methods described herein. For example, such antibodies can be used in particular embodiments of the methods for identifying agents that modulate Aβ42 levels. In these embodiments, which are described herein, a sample containing APP and/or a portion(s) thereof is contacted with a test agent and an agent is identified that selectively modulates Aβ42 levels relative to Aβ40 levels. In a particular method, the Aβ42 levels of a sample are assessed using an Aβ42-selective antibody, such as provided and described herein, and the Aβ40 levels are assessed using an antibody that binds Aβ40. An Aβ40 antibody was produced using animal immunization protocols as described herein. The Aβ40 antibody was prepared using the same protocol as described herein for production of antibody A387 (an Aβ42-selective antibody) production except that the peptide C-AΠGLMVGGW (the sequence of amino acids 30-40 of SEQ ED NO: 4) was used to conjugate to ovalbumin and immunize mice. Subsequent titering was performed as described for the Aβ42-selective antibody.

(b) Aβ Assays Immunoassays for detecting protein are well known to those of skill in the art. Exemplary immunoassay formats include ELISA, competitive immunoassays, radioimmunoassays, Western blots, indirect immunofluorescent assays, in vivo expression or immunization protocols with purified protein preparations. In general, an immunoassay to detect a protein or peptide involves contacting a cell-based or cell-free sample with the antibody of interest and incubating for a period of time sufficient to allow binding of antibody to the epitope, usually at least about 10 minutes. Detection of immunocomplex formation is well known in the art and may be achieved by methods generally based upon the detection of a label or marker, such as any of the radioactive, fluorescent, luminous, biological or enzymatic tags. Examples of the radioisotopes include 1251, 13 II, 3H and 14C. Enzymatic tags that are stable and have a high specific activity are particularly suited for these methods. Examples of enzymatic tags include β- galactosidase, β-glucosidase, alkaline phosphatase, peroxidase, and malate dehydrogenase. Examples of fluorescent tags include fluorescamine and fluorescem isothiocyanate. Luminous tags include, for example, luminol, luminol derivatives, luciferin and lucigenin. Labels are well known to those skilled in the art (see U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241, each incorporated herein by reference). A primary antibody may be directly labeled with radioisotopes, enzymes, fluorescers, chemiluminescers, or other labels for direct detection. Alternatively, a secondary binding ligand such as a second antibody or a biotin/avidin ligand-binding arrangement may be used. The secondary ligand or reagent may be useful for amplifying the signal. Such reagents are well known in the art. For example, the primary antibody may be conjugated to biotin, with horseradish peroxidase-conjugated avidin added as a second stage reagent. Final detection uses a substrate that undergoes a color change in the presence of the peroxidase. The absence or presence of antibody binding may be determined by various methods, including flow cytometry of dissociated cells, microscopy, radiography, scintillation counting, luminometer, etc. Detection and measurement of Aβ peptides can involve the use of a two-site or

"sandwich" assay employing two antibodies, one antibody capable of distinguishing an Aβ peptide (e.g, Aβ42) from other Aβ peptides that might be found in the sample and a second antibody. One of the antibodies serves to capture the antigen while the other is used to detect the captured antigen or the antibody-antigen complex. Thus, for example, an antibody that is selective for a particular Aβ (e.g. , Aβ42) can be used as a capture antibody while an antibody that binds Aβ peptides either non-selectively or selectively, is used as a detection antibody. The first and second antibody reactions may be conducted simultaneously or sequentially. The detection antibody is conjugated to a detectable label as described above. In a particular embodiment, the detectable label is an enzymatic tag. In a further embodiment, the label is alkaline phosphatase and the presence or absence of antibody binding is determined by luminescence of a substrate that undergoes a color change in the presence of alkaline phosphatase, such as, for example, CDP-Star chemiluminescence subsfrate (Tropix, Inc.). Further, in the sandwich immunoassay methods, the Aβ selective antibodies or the antibodies used for labeling are not necessarily of one kind, but two or more kinds of antibodies may be used as mixtures for the purpose of enhancing the measuring sensitivity.

In an example of a method for detecting or measuring Aβ by the sandwich technique, the anti-Aβ antibody used in the first reaction can be reactive to a portion(s) of the Aβ peptide that is different from the portion(s) that the antibody used in the second reaction recognizes. For example, when the antibody used in the first reaction recognizes a partial peptide on the C-terminal portion of Aβ, the antibody used in the second reaction generally is one that recognizes a partial peptide other than the partial peptide on the C-terminal portion (for example, a partial peptide on the N-terminal portion of Aβ). In a particular embodiment of a method for detecting Aβ42 and/or measuring Aβ42 levels or determining the Aβ42 content of a sample provided herein, an Aβ42- selective antibody or portion or fragment thereof provided herein is used as the antibody of the first reaction in the sandwich assay (primary antibody). For example, the Aβ42- selective antibody, or portion or fragment thereof, can be one that contains the sequence of amino acids 1-95 of SEQ ED NO: 12 and/or the sequence of amino acids 1-97 of SEQ ED NO: 14. The secondary antibody can be any antibody that recognizes an epitope within the Aβ42 peptide. In one embodiment, the secondary antibody reacts with a portion(s) of Aβ42 that is different than the site(s) at which the primary antibody reacts. In a particular embodiment, the antibody of the second reaction in the sandwich assay (secondary antibody) is reactive with an N-terminal portion of Aβ42. The secondary antibody (or portion or fragment thereof) can be one that is reactive to more than one species of Aβ and can be reactive with most if not all forms of Aβ. In a particular embodiment, the secondary antibody (or portion or fragment thereof) is reactive with Aβ peptides containing amino acids 1-12 of SEQ ED NO: 4. For example, the secondary antibody, or portion or fragment thereof, can be one that contains the sequence of amino acids 1-100 of SEQ ED NO: 16 and/or the sequence of amino acids 1-98 of SEQ ED NO: 18. The secondary antibody can be used as the detection antibody and can be conjugated to a detectable label. In a particular embodiment, the detectable label is alkaline phosphatase and the presence or absence of antibody binding is determined by luminescence of a substrate that undergoes a color change in the presence of alkaline phosphatase, such as, for example, CDP-Star chemiluminescence subsfrate (Tropix, Inc.). When a method for detecting Aβ42 and/or measuring Aβ42 levels or determining the Aβ42 content of a sample as provided herein is used as method for assessing the Aβ42 levels in a step of identifying an agent that selectively modulates Aβ42, it may be combined with a method for detecting and/or measuring Aβ40 in a sample, as described herein. In such methods for identifying agents that selectively modulate Aβ42 levels, the Aβ42 level of one or more samples is assessed to identify an agent that modulate Aβ42 levels, and the Aβ40 level of one or more samples is assessed to identify those Aβ42- modulating agents that do not alter Aβ40 levels. One method for detecting and/or measuring Aβ40 in a sample for use in these methods is the above-described sandwich assay wherein an Aβ40-selective antibody (or portion or fragment thereof) is substituted for an Aβ42-selective antibody or portion or fragment thereof as the primary antibody. In a particular embodiment, the Aβ40-selective antibody is one that recognizes amino acids 30-40 of Aβ (for example, amino acids 30-40 of SEQ ED NO: 4), such as is described herein. For example, an Aβ40-selective antibody can be prepared by immunizing animals with the peptidyl sequence representing Aβ30-40 region, as described herein. Sandwich ELISA-based assays such as these for use in methods for detecting Aβ42 and/or measuring Aβ42 levels or determining the Aβ42 content of a sample as provided herein can be performed in a microtiter plate format wherein the primary antibody is coated into the wells of the plate and the sample is added to the wells. After washing, the secondary antibody (which can be conjugated to a label such as alkaline phosphatase) is added to the wells which are washed prior to adding a subsfrate, e.g., a chemiluminescent subsfrate, for detection of bound Aβ42. Such methods provide a large linear range, such as, for example, about 75-2000 pg/well, high dynamic range, e.g., about 3-30 fold over background in linear range (signal :noise), low sensitivity limit, such as, for example, less than about 20 pg/well, and selectivity for Aβ42, e.g., at least about 1000-fold selectivity for Aβ42 over other Aβ peptides, making the method highly amenable to high-throughput screening for agents that modulate Aβ42 levels.

Smaller Aβ peptides, for example, Aβ peptides having a C-terminal end that terminates before amino acid 40 (see, e.g., the sequence of amino acids 1-40 of SEQ ED NO: 4) may also be detected in the methods provided herein. In particular embodiments, these peptides are measured by their mass, size, and/or charge. For example, peptides may be immunoprecipitated with an antibody reactive to the amino-terminal end of Aβ. For example, the anti-Aβl-12 antibody described herein may be used for immunoprecipitation of these peptides. Immunoprecipitated peptides may then be identified by any method known to those of skill in the art including, for example, elecfrophoresis and mass spectrometry. In a particular embodiment, cells expressing wild-type APP are treated with test agent or vehicle control for 18 h. Media is collected and immunoprecipitated using an anti-Aβl-12:Sepharose column for 4 h. Bound peptides are eluted with 0.1% TFA 50%> acetonitrile and spotted onto NP2 CHIPS. Mass spectrometer analysis is performed on a PBS II Protein Chip Reader (Ciphergen). Data may be normalized to an internal standard, such as A i-π that is spiked into the media prior to the immunoprecipitation.

All assays and procedures, including antibody-antigen reactions, generally can be conducted under conditions recognized by those of skill in the art as standard conditions. d. Alterations of APP cleavage or processing, Aβ processing or

Aβ levels

Methods for identifying or screening for agents that modulate Aβ levels can include a step of identifying an agent that alters cleavage (particularly the Aβ peptide- producing cleavage) of APP (and/or portion(s) thereof), processing or APP (and/or portion(s) thereof), Aβ processing and/or Aβ levels of a sample. The step of identifying an agent that alters such parameters that can affect Aβ levels typically involves making assessments of one or more of the parameters. As described herein, there are a number of ways in which APP cleavage, APP processing, Aβ processing and the Aβ levels of a sample can be assessed, including, but not limited to, immunoassays for detection and/or quantitation of one or more peptides, proteins and/or fragments thereof that are reflective of these parameters. A step of identifying an agent that alters one or more of these parameters can thus involve assessment of one or more of the parameters and a determination as to whether the parameter(s) is altered under a condition of the presence of the agent. Determining if APP cleavage (particularly the Aβ peptide-producing cleavage),

APP processing, Aβ processing and/or Aβ levels of a sample is altered by a test agent can involve comparing one or more of these parameters in the presence and absence of the test agent. Thus, in general, the agent identification step can involve a comparison of the cleavage (particularly the Aβ peptide-producing cleavage) of APP (and or portion(s) thereof), processing or APP (and/or portion(s) thereof), Aβ processing and or Aβ levels of a sample that has been contacted with a test agent (i.e., test sample) and of a sample that has not been contacted with the test agent (i.e., confrol sample). If the Aβ-producing cleavage or processing of APP (and/or portion(s) thereof), the processing of Aβ and/or the Aβ levels of the test and control samples differ, then the agent is identified as one that modulates the level of one or more Aβ peptides.

(1) Contacting sample with test agent A sample for use in the methods of identifying or screening for agents that modulate Aβ levels as described herein can be maintained under conditions in which APP (and/or portion(s) thereof, including Aβ) can undergo cleavage and/or processing (e.g. , catabolism, degradation). If the sample is a test sample, it is contacted with a test agent. If the sample is a control sample, it can be one that is not contacted with test agent. Generally, a control sample is substantially similar to the test sample and maintained under substantially similar conditions as the test sample, but is not contacted with test agent. A confrol sample can be the same physical sample as the test sample (e.g. , prior to addition of test agent) or can be a different sample.

Depending on the type of control (e.g., reference confrol, negative control or positive control), a control sample may be manipulated in various ways. For example, if a control sample is a vehicle control, it may be contacted with a "vehicle", such as a medium, or element thereof, in which the test agent is contained, but that lacks the test agent. Examples of such "vehicles" include suspension, solubilizing reagents, emulsifiers, and compositions that generally serve to facilitate retention and administration of a test agent. In a particular example, a vehicle control can be DMSO. A positive confrol can be a sample that has been treated using known processes/compositions to achieve an effect that is desired by a test agent that is a "positive" identified as one that modulates Aβ levels. Thus, for example, if the methods are conducted with the specific purpose of identifying an agent that reduces one or more Aβ levels, then a positive confrol sample could be one that is treated with an agent known to reduce Aβ levels. One particular example is an APP-containing sample that has been contacted with a β- and/or γ-secretase inhibitor, such as, for example, DAPT. Test samples can be treated with a range of doses or concentrations of the test agent or with only a single concentration of agent. When a range of different test agent concentrations is used in contacting a plurality of samples in parallel and compared to the magnitude of any effect each different concentration may have on the parameter(s) (e.g., Aβ level of samples) being assessed (e.g., a dose-response study), a more detailed analysis and profile of the test agent can be made. For example, it may be possible to determine values such as EC50 or IC50 for the test agent to estimate the potency of an agent. The methods provided herein allow for the identification of very potent Aβ- modulating agents. Particular embodiments of the methods provide for the identification of agents with an EC50 or IC50 for modulating (e.g., increasing or decreasing) Aβ levels of 100 μM, 75 μM, 50 μM, 40 μM, 30 μM, 25 μM, 20 μM, 15 μM, or 10 μM or lower. In a particular embodiment of the methods, agents are identified that have an EC50 or IC50 for modulating (e.g, increasing or decreasing) Aβ levels of less than about 25 μM. In a further embodiment of the methods, agents are identified having an EC50 or IC50 of less than about 20 μM. hi one particular embodiment, agents are identified that have such values for an IC50 for reducing the levels of Aβ42. Generally, such a more detailed analysis is conducted after a test agent has been identified as one that alters one or more of the parameters at a threshold or test concentration, such as is typically done in a high- throughput screening of test agents. Threshold or test concentrations can be, for example, about 1 μM, 2 μM, 5 μM, 10 μM, 15 μM, 20 μM, 25 μM, 30 μM, 35 μM, 40 μM, 45 μM, 50 μM, 75 μM, 100 μM or more. In a one example, the threshold or test concentration can be less than about 50 μM, 40 μM, 35 μM, 30 μM, 25 μM, 20 μM, 15 μM or 10 μM. In a particular example, the threshold or test concentration can be less than or equal to about 35 μM, 30 μM, 25 μM, 20 μM, 15 μM or 10 μM. Generally, by screening at a lower test concentration, the agents identified as modulators of Aβ may tend to be more potent than if they had been identified at a higher test concentration. A sample for use in the methods of identifying or screening for agents that modulate Aβ levels as described herein can be maintained under conditions in which APP (and/or portion(s) thereof, including Aβ) can undergo cleavage and/or processing (e.g., catabolism, degradation) for an appropriate amount of time prior to being used in the methods of identifying Aβ-modulating agents and after being contacted with test agent. Such time periods can be empirically determined and generally are such to allow for detectable levels of APP cleavage or processing and/or Aβ formation or processing to occur. Similarly, the sample is contacted with test agent for an appropriate amount of time or range of time periods. Typically, when the methods are being practiced in a high- throughput screening format, a single time period of contacting is used. In one example, the time period can be on the order of minutes to hours depending, in part, on the type of sample, e.g., intact cells or cell-free medium.

In one particular example of a method for identifying an agent that modulates Aβ levels, cell cultures capable of APP expression and processing (e.g., CHO cells fransfected with DNA encoding human APP695 and human PSl) are plated in the wells of a microtiter plate and allowed to adhere for about 24 hours. The separate samples in the wells were then either treated or not treated with a test agent (-30 μM). Samples treated with DMSO vehicle (0.12%) alone were a negative control. Samples treated with 1 μM DAPT for 18 hours were used as a positive control. Supernatant removed from the wells was analyzed in a sandwich ELISA to assess the level of Aβ42 in each sample. The ELISA was conducted in a microtiter plate format using an Aβ42-selective monoclonal antibody provided herein (antibody A387) as a capture antibody which was incubated with supernatant for 1 hour. After washing of the plate, the wells were incubated for 2 hours with a detection antibody generated against an Aβl-12 peptide, as described herein, and conjugated to alkaline phosphatase. A chemiluminescence substrate was added to the wells and, after 30 minutes, the luminescence was quantified to assess and compare Aβ42 levels of the test and confrol samples in order to determine any differences and identify agents that modulate Aβ (and in particular Aβ42) levels. (2) Evaluating alterations In conducting the methods of identifying an agent that modulates Aβ levels, a way in which an agent can be identified is by identifying an agent that alters the cleavage or processing of APP (and/or portion(s) thereof), the processing of Aβ and/or Aβ levels. An alteration can be, for example, any detectable difference in the cleavage or processing of APP (and/or portion(s) thereof), the processing of Aβ and/or Aβ levels of a sample that has been contacted with a test agent as compared to a sample that has not been contacted with the test agent. Methods for assessing the cleavage or processing of APP (and/or portion(s) thereof), the processing of Aβ and Aβ levels are described and provided herein and additional assessment methods are known in the art. Thus, any difference in any one or more of these processes or compositions as detected in the assessment of test and control samples can be an alteration by which an Aβ-modulating agent can be identified.

The extent of the difference can vary depending on a variety of factors, including, for example, the particular parameter being assessed and compared, the assessment method used and the conditions under which the assessment was conducted, the concentration of the test agent used as well as other factors. Thus, for example, the difference may be an about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 or more than 75% difference in the assessed parameter, e.g., a level or amount of composition, activity or processing, when compared under differing conditions, e.g., in the presence and absence of a test agent. In one example of a particular high-throughput format of the methods, a test agent was identified as an agent that modulates Aβ42 levels if there was a greater than about 50% difference in the Aβ42 levels of test and confrol samples. In a particular example, a test agent was identified as an agent that reduces Aβ42 levels if the Aβ42 level of a test sample was more than about 50% lower than the Aβ42 level of a control sample. e. Assessment of selectivity of Aβ-modulating agents

Cellular and exfracellular Aβ levels are governed by numerous mechanisms and activities involved in Aβ synthesis through APP processing and in Aβ catabolism, degradation and clearance. These mechanisms include multiple components, such as, for example, enzymes and facilitator proteins, many of which have multiple substrates and/or multiple, closely related protein family members. In addition, some of the enzymes, e.g., γ-secretase, may function as a part of a complex that includes a number of other proteases. Although any of these components and mechanisms, individually or in combination, are potential targets for modulation in order to ultimately modulate Aβ levels, modulation of these targets may also affect other processes (i.e., other than the processing of APP and/or Aβ) and the levels of other molecules due to the multiplicity of component function and relatedness and interaction of some components to non- component molecules. Modulation of Aβ levels that also involves modulation of other cellular processes and elements, i.e., non-specific modulation of Aβ levels, can result in undesired side effects. Methods of identifying agents that more specifically or selectively modulate Aβ levels are provided herein. The methods can be used to identify agents that selectively modulate the levels of one or more Aβ peptides without substantially affecting compositions and mechanisms that are not significantly involved in the generation, degradation and/or clearance of one or more Aβ peptides.

Using the methods provided herein, agents identified as Aβ-modulating agents can also be profiled with respect to the specificity or selectivity of their modulation.

(1) Assessment of Aβ peptide selectivity Cleavage of APP to generate Aβ yields a number of Aβ peptides that can differ at the C-terminus, e.g., Aβl-43, Aβl-42, Aβl-40, and others. The C-terminal heterogeneity is the result of cleavage by distinct activities of γ-secretase and/or multiple γ-secretases. An agent that modulates the levels of all or most or more than one or two Aβ peptides may be non-selectively modulating components and mechanisms involved in processes other than the generation or degradation of Aβ in addition to modulating components and mechanisms of Aβ synthesis and degradation. Agents that selectively modulate the levels of one or two Aβ peptides, or a particular subset of Aβ peptides, are less likely to affect other compositions, activities and mechanisms and are therefore desired. Agents that selectively modulate the level of Aβ42 are of particular interest because Aβ42 is one of the predominant forms found in amyloid plaques, and is deposited early and selectively in the cerebral cortex of brains of individuals harboring some FAD-linked mutations. Aβ42 formation is also selectively elevated in some FAD-linked mutations. Methods are provided herein for identifying or screening for an agent that selectively modulates Aβ levels. In one embodiment, the method identifies agents that alter the level of a particular form or forms of Aβ to a greater extent than they alter the levels of one or more other forms of Aβ. In a particular embodiment, such agents alter the level of a particular form or forms of Aβ without substantially affecting or altering the level of one or more other Aβ peptides. In one embodiment of the methods, an agent that selectively modulates Aβ42 levels is identified. The agent can, for instance, selectively modulate Aβ42 levels relative to Aβ40 levels and/or the levels of all or most of the other forms of Aβ. In a particular embodiment, the agent modulates the levels of Aβ42 and Aβ39 relative to Aβ40 levels and/or the levels of all or most of the other forms of Aβ. hi a particular embodiment of the method, compounds are identified that selectively modulate Aβ peptides having a C-terminal end that terminates before amino acid 40. In a particular embodiment compounds are identified that selectively modulate the level of Aβ39. In a particular embodiment, the methods identify an agent that selectively increases Aβ39 levels, hi another embodiment the methods identify an agent that selectively decreases Aβ39 levels.

In general, the methods of identifying or screening for an agent that selectively modulates the level of an Aβ peptide relative to one or more other Aβ peptides includes steps of contacting a sample containing APP and/or a portion(s) thereof (including, for example one or more Aβ peptides) with a test agent and identifying an agent that alters the level of an Aβ peptide to a greater extent than it alters the level of one or more other Aβ peptides. The process of identifying an agent that modulates the level of an Aβ peptide in a sample can be carried out in a number of ways as described herein. For example, the Aβ peptide level in a sample that has been contacted with a test agent (test sample) can be compared with the Aβ peptide level in a sample that has not been contacted with the test agent (confrol sample). If the Aβ peptide levels in the two samples differ, then the agent is identified as one that modulates the level of the Aβ peptide. Methods for assessing the level of a particular Aβ peptide in a sample are described herein or known in the art. Such methods include, but are not limited to, immunoassays employing peptide-specific antibodies, mass spectrometry and elecfrophoretic analyses.

In a particular embodiment of the methods for identifying or screening for an agent that selectively modulates the level of an Aβ peptide, the Aβ42 levels of samples are assessed by contacting a sample with an antibody (or portion or fragment thereof) that selectively binds to Aβ42 (e.g., the sequence of amino acids 1-42 of SEQ ED NO: 4). In one embodiment, the antibody is any one of the Aβ42-selective antibodies provided herein, such as, for example, an antibody that contains the sequence of amino acids 1 to about 95, 94, 93, 92, 91, 90, 85, 80, 70, 60 or 50 of SEQ ED NO: 12 and/or the sequence of amino acids 1 to about 97, 96, 95, 94, 93, 92, 91, 90, 85, 80, 70, 60 or 50 of SEQ ED NO: 14. In a particular embodiment, the Aβ42-selective antibody used in assessing the Aβ42 levels of samples is antibody A387 provided herein. In another embodiment of the methods for identifying or screening for an agent that selectively modulates the level of an Aβ peptide, the Aβ39 levels of samples are assessed by contacting a sample with an antibody (or portion or fragment thereof) that selectively binds to Aβ39 (e.g., the sequence of amino acids 1-39 of SEQ ED NO: 4) or by mass specfrometric analysis of the samples. Antibodies selective for Aβ39 can be prepared using methods described herein, hi particular methods, an agent that modulates Aβ42 levels or Aβ39 levels or that modulates both Aβ42 and Aβ39 levels is identified. In a particular method, an agent that reduces Aβ42 levels and/or increases Aβ39 levels is identified.

The process of further identifying an agent that alters the level of one or more other Aβ peptides to a lesser extent than it alters a particular Aβ peptide or that does not substantially alter the level of the one or more other Aβ peptides can also be carried out in a number of ways. In general, this process can involve a comparison of the level of one or more other Aβ peptides in a sample that has been contacted with the agent (test sample) with that of a sample that has not been contacted with the agent (confrol sample). If the difference in the levels of the one or more other Aβ peptides in the test and control samples is less than the difference in the levels of the particular modulated Aβ peptide in test and control samples, or if the levels of the one or more other Aβ peptides in the test and control samples do not differ substantially (or are substantially unchanged), then the agent is identified as one that selectively modulates the level of an Aβ peptide. The skilled artisancan select appropriate concentrations of test agents at which to make such comparisons. For example, the comparison can be made at or near the EC50 or IC50 concentration for the modulation of the target Aβ peptide. If the method is for identifying an agent that selectively modulates the level of an Aβ peptide relative to only one other Aβ peptide, then the process of assessing the extent to which an agent may alter the levels of the one other Aβ peptide can involve an assessment of the levels of the one other peptide in test and confrol samples using an antibody selective for the one other peptide. If the method is for identifying an agent that selectively modulates the level of an Aβ peptide relative to most or all other Aβ peptides, then the process of assessing the extent to which an agent can alter the levels of most or all other Aβ peptides can involve an assessment of the levels of all Aβ peptides in test and confrol samples using an antibody that recognizes most or all forms of Aβ. If the ratio of the level of the modulated Aβ peptide to the level of all Aβ peptides differs in the confrol and test samples, then the agent is identified as one that selectively modulates the level of the modulated Aβ peptide relative to most or all other Aβ peptides. In a particular embodiment of such a method, the antibody that recognizes most or all forms of Aβ in a sample is one that binds to Aβl-12 (e.g., the sequence of amino acids 1-12 of SEQ ED NO: 4). In one embodiment, the antibody is one that is provided herein, such as an antibody that contains the sequence of amino acids 1 to about 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 85, 80, 70, 60 or 50 of SEQ ID NO: 16 and/or the sequence of amino acids 1 to about 98, 97, 96, 95, 94, 93, 92, 91, 90, 85, 80, 70, 60 or 50 of SEQ ED NO: 18. In a particular embodiment, the Aβl-12 antibody used in assessing the Aβ peptide levels of samples is antibody B436 provided herein.

In method of identifying an agent that selectively modulates the level of an Aβ peptide relative to one or more other Aβ peptides, the identification of the Aβ- modulating agent and the determination as to what extent, if any, the agent alters the level of one or more other Aβ peptides can be conducted sequentially or simultaneously. For example, an agent that modulates the levels of an Aβ peptide can be identified by a difference in the levels of the Aβ peptide in samples contacted with the agent (test sample) and samples not contacted with the agent (confrol samples). That agent can then be separately evaluated for its effects on the levels of one or more other Aβ peptides by comparing the levels of the one or more other Aβ peptides in samples contacted with the agent and not contacted with the agent. Alternatively, the levels of the particular Aβ peptide to be modulated and the levels of the one or more other Aβ peptides in a test sample can be assessed and compared to the levels of the particular Aβ peptide to be modulated and the levels of the one or more other Aβ peptides in a control sample simultaneously to, in one step, identify an agent that selectively modulates the level of an Aβ peptide. hi another method for identifying an agent that selectively modulates the level of an Aβ peptide relative to one or more other Aβ peptides, the test agent is one that is already known to modulate the level of one or more particular Aβ peptides. Thus, in one embodiment of this method, a sample containing APP or portion(s) thereof is contacted with a test agent that modulates the level of an Aβ peptide, and a test agent is identified as an agent that selectively modulates Aβ levels if the test agent does not substantially alter the level of one or more Aβ peptides other than the Aβ peptide that is modulated by the test agent. As described herein, the agent that modulates the level of an Aβ peptide that is used in this method can be one that was identified by a process involving contacting a sample containing APP or portion(s) thereof with a test agent and identifying an agent that alters the cleavage of APP that produces one or more Aβ peptides, the processing of APP and/or the level of one or more Aβ peptides. An agent that selectively modulates the levels of an Aβ peptide relative to one or more other Aβ peptides can alter the levels of the selectively modulated Aβ peptide(s) to a greater extent than it alters the levels of the one or more other Aβ peptides (i.e., the peptides that are not targeted for modulation). The extent to which the agent alters the levels of the particular Aβ peptide is generally significantly greater than the extent to which the agent alters the levels of one or more other Aβ peptides; that is, the greater extent of modulation is reproducible and not merely within the level of experimental error or variation. The modulation of a particular Aβ peptide by the agent can be identified by a detectable difference in the levels of the Aβ peptide in samples contacted with the agent (test sample) and samples not contacted with the agent (control samples). The agent is one that selectively modulates the levels of the particular Aβ peptide if any difference (including, for example, absolute and/or percentage difference) in the levels of one or more other Aβ peptides in samples contacted with the agent and samples not contacted with the agent is less than the difference (including, for example, absolute and/or percentage difference) in the levels of the particular Aβ peptide in test and control samples. In particular embodiments, the extent to which the agent alters the levels of one or more other Aβ peptides (i.e., the peptides that are not targeted for modulation) is less than about 40%, 35%, 30%, 25%, or 20%. In one embodiment, the extent to which the agent alters the levels of one or more other Aβ peptides (i.e., the peptides that are not targeted for modulation) is less than 20%. hi a particular embodiment of the methods of identifying an agent that selectively modulates the level of an Aβ peptide, an agent is identified that modulates the level of an Aβ peptide without substantially altering the levels of one or more other Aβ peptides. Any modulation of the level of the one or more other Aβ peptides (i.e., the peptides that are not targeted for modulation) that is not a substantial alteration is one that is generally not associated with any significant undesired or adverse consequence in a biological context, such as, for example, in a cell, cell medium, tissue or organism.

(2) Assessment of presenilin substrate selectivity An agent that modulates Aβ levels may act by modulating any one or more of the numerous mechanisms and activities, and components thereof, involved in Aβ synthesis through APP processing and in Aβ catabolism, degradation and clearance. One activity involved in the generation of Aβ is the presenilin/γ-secretase that participates in the processing and cleavage of APP. Any non-specific modulation of this activity could possibly effect other mechanisms in addition to APP cleavage due to the multiplicity of substrates and mechanisms with which presenilin and γ-secretase are involved. Such non-specific actions of an Aβ-modulating agent could result in undesired and adverse side effects of the modulation process.

Agents that more specifically or selectively modulate Aβ levels can be identified using methods provided herein that involve identifying agents that modulate Aβ levels without substantially altering or affecting non- APP substrate cleaving/processing activity of presenilin. These methods can involve the methods of assessing presenilin and/or presenilin-dependent activity provided and described herein.

One method provided herein for identifying or screening for agents that selectively modulate Aβ levels includes steps of contacting a sample containing a presenilin subsfrate, and/or portion(s) thereof, other than APP with a test agent that modulates Aβ levels and identifying a test agent as an agent that selectively modulates Aβ levels if the agent does not substantially alter the cleavage and/or processing (in particular, the presenilin-dependent cleavage and/or processing) of the presenilin substrate, and/or portion(s) thereof, that is other than APP. The sample used in this method can contain presenilin. The agent that modulates Aβ levels that is used in this method can be any agent known to modulate Aβ levels. The agent can, for example, be one that is identified by a method described herein which involves contacting a sample containing APP and/or portion(s) thereof with a test agent and identifying an agent that alters the cleavage of APP that produces one or more Aβ peptides, the processing of APP and or the level of one or more Aβ peptides. The step of identifying an agent that does not substantially alter the cleavage and/or processing of the presenilin substrate, or portion(s) thereof, that is other than APP can be carried out in a number of ways. In general, this process can involve a comparison of the cleavage and/or processing (in particular, the presenilin-dependent cleavage and/or processing) of a presenilin substrate (and/or portion(s) thereof) other than APP, and/or the levels of a peptide fragment(s) of the presenilin substrate, in a sample that has been contacted with the test agent (i.e., test sample) and in a sample that has not been contacted with the test agent (i.e., confrol sample). If the cleavage and/or processing of the presenilin subsfrate that is other than APP, and or the substrate fragment(s) levels, of the test and confrol samples do not differ substantially, then the agent is identified as one that alters the level of one or more Aβ peptides without substantially altering the cleavage of the presenilin substrate, or portion(s) thereof, that is other than APP. The confrol sample can be the same physical sample as the test sample or a different sample. When the confrol and test samples are the same, the confrol is the sample in the absence of test agent. hi a particular embodiment, the cleavage and/or processing of the presenilin substrate that is other than APP, and/or the subsfrate fragment(s) levels, of the test sample can be compared to that of a positive control sample. A positive control sample can be a sample that has been contacted with a known modulator of presenilin or presenilin-dependent activity. In one example, the known modulator is an inhibitor of presenilin or presenilin-dependent activity. A particular example is DAPT, which is an inhibitor of presenilin-dependent γ-secretase activity. When a positive control is used, an agent is identified as one that alters the level of one or more Aβ peptides without substantially altering the cleavage of the presenilin subsfrate, if the cleavage and/or processing of the presenilin subsfrate and/or the substrate fragments) levels of the test sample differ significantly and/or substantially from that of the positive control sample. For example, a substrate fragment(s) level of the test sample can differ from that of the positive control such that the test sample levels are less than about 40%>, 35%, 30%, or 20% of the positive control sample levels. In a particular embodiment the test sample levels can be less than or equal to about 20% of the confrol sample levels. In one such embodiment, the positive control sample is one that has been contacted with DAPT (with a presenilin subsfrate fragment level set as 100%) and the test sample levels of the fragment are less than or equal to about 20% of the positive control sample levels.

In a particular embodiment, the agent that modulates Aβ levels that is used in the method is one that modulates the levels of Aβ42. In a further embodiment, the agent can be one that selectively modulates the levels of Aβ42 relative to Aβ40 levels and/or the levels of all or most of the other forms of Aβ. In a particular embodiment, the agent modulates the levels of Aβ42 and Aβ39 relative to Aβ40 levels and/or the levels of all or most of the other forms of Aβ. i one embodiment, the agent reduces Aβ42 levels and/or increases Aβ39 levels. Thus, using the agent identification and screening methods provided herein in combination, it is possible to identify agents that reduce Aβ42 levels without substantially altering the levels of Aβ40 or the non- APP subsfrate cleavage/processing activity of presenilin (i.e., with an inhibitory profile (Aβ42(+), Aβ40(-), presenilin (-))).

Another method provided herein for identifying or screening for an agent that selectively modulates Aβ levels includes steps of contacting a sample containing APP and/or a portion(s) thereof (including, for example one or more Aβ peptides) and a presenilin substrate, and/or portion(s) thereof, that is other than APP with a test agent and identifying an agent that alters the Aβ peptide-producing cleavage or processing of the APP (and/or portion(s) thereof), the processing of Aβ and/or the levels of one or more Aβ peptides without substantially altering the cleavage (in particular, the presenilin- dependent cleavage) of the presenilin substrate, or portion thereof, that is not APP. The sample used in this method can contain presenilin. The process of identifying an agent that alters the cleavage of APP that produces one or more Aβ peptides, the processing of APP or Aβ, and/or the level of one or more Aβ peptides can be carried out in a number of ways as described herein.

The process of further identifying an agent that does not substantially alter the cleavage of a presenilin substrate (other than APP), or portion(s) thereof, can also be carried out in a number of ways, as described herein, hi general, this process can involve a comparison of the cleavage and/or processing (in particular, the presenilin-dependent cleavage and/or processing) of a presenilin substrate (or portion(s) thereof) other than APP, and/or the levels of a peptide fragment(s) of the presenilin subsfrate, in a sample that has been contacted with the test agent (i.e., test sample) and in a sample that has not been contacted with the test agent (i.e., confrol sample). If the cleavage and/or processing of the presenilin substrate that is other than APP and/or the subsfrate fragment(s) levels of the test and control samples do not differ substantially, then the agent is identified as one that alters the level of one or more Aβ peptides without substantially altering the cleavage and/or processing of the presenilin substrate, or portion(s) thereof, that is other than APP. The control sample can be the same physical sample as the test sample or a different sample. When the control and test samples are the same, the confrol is the sample in the absence of test agent.

In a particular embodiment of these methods, the cleavage and/or processing of the presenilin substrate that is other than APP, and/or the substrate fragment(s) levels, of the test sample can be compared to that of a positive confrol sample. A positive confrol sample can be a sample that has been contacted with a known modulator of presenilin or presenilin-dependent activity, hi one example, the known modulator is an inhibitor of presenilin or presenilin-dependent activity. A particular example is DAPT, which is an inhibitor of presenilin-dependent γ-secretase activity. When a positive confrol is used, an agent is identified as one that alters the level of one or more Aβ peptides without substantially altering the cleavage of the presenilin subsfrate, if the cleavage and/or processing of the presenilin substrate and/or the substrate fragment(s) levels of the test sample differ significantly and/or substantially from that of the positive control sample. For example, a subsfrate fragment(s) level of the test sample can differ from that of the positive confrol such that the test sample levels are less than about 40%, 35%, 30%, or 20%) of the positive confrol sample levels. In a particular embodiment the test sample levels can be less than or equal to about 20% of the confrol sample levels. In one such embodiment, the positive confrol sample is one that has been contacted with DAPT (with a presenilin substrate fragment level set as 100%) and the test sample levels of the fragment are less than or equal to about 20% of the positive control sample levels. hi one method of identifying an agent that selectively modulates Aβ levels, the identification of the Aβ-modulating agent and the determination as to whether the agent alters the cleavage and/or processing of a presenilin subsfrate (other than APP), and/or portion(s) thereof, can be conducted sequentially or simultaneously. For example, when conducting the processes sequentially, an agent that modulates Aβ levels can be identified by a difference in the Aβ-producing cleavage of APP, the processing of APP or Aβ, and/or the level of one or more Aβ peptides in samples contacted with the agent (test sample) and samples not contacted with the agent (control sample). The identified agent can then be separately evaluated for its effect on presenilin subsfrate cleavage by comparing the cleavage and/or processing (in particular, presenilin-dependent cleavage and/or processing) of the presenilin subsfrate and/or the levels of a peptide fragment or fragments of the presenilin substrate in samples contacted with the test agent and not contacted with the test agent, hi this sequential method, the sample used in the identification of the Aβ-modulating agent can be of the same or different type relative to the sample used in the determination as to whether the agent alters the cleavage of a presenilin substrate. If the same type of sample is used, it can contain APP (and/or portion(s) thereof) and a presenilin subsfrate (and/or portion(s) thereof) other than APP. The sample can also contain presenilin. If different types of samples are used, the sample used in the identification of the Aβ-modulating agent can contain APP and/or portion(s) thereof, and the sample used in the determination of alteration in the cleavage of the presenilin subsfrate can contain a presenilin subsfrate (and/or portion(s) thereof) other than APP. The sample may also contain presenilin. Alternatively, in a simultaneously performed method, a test sample containing APP (and/or portion(s) thereof) and a presenilin substrate (and/or portion(s) thereof) other than APP can be contacted with a test agent, and the Aβ-producing cleavage of APP, the processing of APP and/or Aβ, and/or the level of one or more Aβ peptides can be assessed for the test sample, as can presenilin substrate cleavage be assessed for the same test sample. The sample may also contain presenilin. The Aβ peptide-producing cleavage or processing of APP, processing of Aβ and/or levels of Aβ peptides in the test sample, as well as the presenilin subsfrate cleavage of the test sample, can be compared to that of a confrol sample in one step to identify an agent that modulates Aβ levels without substantially altering the cleavage of a presenilin substrate (or portion(s) thereof). In particular embodiments of any of the methods, a step in the method can be identifying an agent that modulates Aβ42 levels without substantially altering the cleavage and/or processing of a presenilin subsfrate that is other than APP. The step can include identifying an agent that modulates Aβ42 levels relative to Aβ40 levels and/or the levels of all or most of the other forms of Aβ. In one embodiment, the step can include identifying an agent that reduces Aβ42 levels and/or increases Aβ39 levels.

With respect to any of the methods for identifying agents that modulate Aβ levels without substantially altering or affecting the cleavage and/or processing of a presenilin subsfrate that is other than APP, the identified agents either do not alter the cleavage and/or processing (in particular the presenilin-dependent cleavage and/or processing) of a presenilin substrate, or alter it in a way that it is substantially unchanged. Such alterations can be determined in a number of ways. For example, an alteration of the cleavage and/or processing of the presenilin substrate that is not substantial can be one that generally is not associated with any significant undesired or adverse consequence in a biological context, such as, for example, in a cell, cell medium, tissue or organism. An alteration of the cleavage and/or processing of the presenilin subsfrate that is not substantial can also be one that is assessed as a difference in the processing and/or cleavage of the subsfrate, or the levels of a fragment(s) of the presenilin substrate, in test and confrol samples that is less than about 40%, 35%, 30%, 25% or 20%>. In a particular embodiment of the methods, an alteration that is not substantial can be one that is assessed as a difference in the processing and/or cleavage of the subsfrate, or the levels of a fragment(s) of the presenilin substrate, in test and control samples that is less than or equal to about 20%.

In the methods for identifying agents that modulate Aβ levels without substantially altering or affecting the cleavage and/or processing of a presenilin subsfrate that is other than APP, the presenilin substrate can be, for example, a peptide, polypeptide, protein or fragment(s) thereof that is altered (e.g., proteolytically processed, at least in part) in a presenilin-dependent manner. Thus, for example, in the case of a presenilin subsfrate that is altered by proteolytic processing of the subsfrate, if presenilin is absent, or presenilin activity is inhibited or reduced, the proteolytic processing of the presenilin subsfrate is altered, for example by an alteration in the levels and/or composition of fragments generated from the substrate, relative to the proteolytic processing of the substrate that occurs in the presence of normal (e.g., wild-type) presenilin activity. Exemplary presenilin substrates include, but are not limited to LRP, Notch, TrkB, APLP2, hlrel , E-cadherin and Erb-B4.

Thus, in particular embodiments of the methods for identifying agents that modulate Aβ levels without substantially altering or affecting the cleavage and/or processing of a presenilin subsfrate that is other than APP, agents are identified that modulate the levels of one or more Aβ peptides, such as Aβ42, without substantially altering or affecting the cleavage and/or processing (in particular, the presenilin- dependent cleavage and/or processing) of Notch, LRP, E-cadherin, Erb-B4, TrkB, APLP2 and/or hlrelo;. Such methods can involve, for example, comparing the levels in test and control samples of Notch nuclear intracellular carboxyl domain (NICD), LRP carboxy terminal fragments (CTFs), E-cadherin intracellular carboxyl domain (ICD), and/or Erb-B4 infracellular carboxyl domain (ICD). In a particular embodiment of the method, the levels of one or more LRP fragments, e.g., LRP-CTFs, in test and confrol samples are compared. The processing and processing patterns of these presenilin substrates, and characteristic fragments that can be generated therefrom, are described herein. In a particular embodiment of the methods provided herein for identifying an agent that modulates Aβ levels without substantially altering the processing and/or cleavage of LRP, the process identifying an agent that does not substantially alter the cleavage of LRP can involve a comparison of the cleavage and/or processing of LRP, and/or the levels of a peptide fragment(s) of LRP, in a sample that has been contacted with the test agent (i.e., test sample) and in a sample that has not been contacted with the test agent (i.e., confrol sample). The processing or cleavage of an LRP or fragment(s) thereof can be assessed by determining the presence or absence and/or level of one or more fragments of LRP and/or the composition of LRP using, for example, materials and methods described herein. Thus, the LRP composition can be evaluated to determine if any fragment(s) indicative of presenilin-dependent cleavage of LRP or altered presenilin- dependent cleavage of LRP are present and/or the level of any such fragments. Such fragments and compositions are described herein.

In one embodiment, the processing or cleavage of an LRP or fragment(s) thereof is assessed by determining the presence or absence and/or level of an LRP fragment that is cleaved in the presence of a presenilin-dependent activity (presenilin-dependent γ- secretase activity), and thus absent (or present at low levels) in the presence of the presenilin-dependent activity, but that can be detected intact when the presenilin- dependent activity is altered (such that it is eliminated or reduced). In one embodiment, the presence or absence and/or level of an LRP fragment having a molecular weight of between about 25 kD and 15 kD, and, in particular, about 20 kD, is assessed. Typically, the - 20 kD fragment is one that is present when an LRP is not cleaved by a presenilin- dependent activity, such as one that occurs in the presence of an inhibitor of a presenilin- dependent activity such as DAPT. In a particular embodiment, the fragment is from a C- terminal portion of LRP, i.e., a CTF. The LRP fragment can be one that contains an amino acid sequence located within the sequence of amino acids 4420-4544 of SEQ ED NO: 10. hi a further embodiment, the fragment is one that is recognized by an antibody generated against C-terminal amino acids (e.g., the C-terminal 13 amino acids) of LRP, such as, for example, the polyclonal antibody R9377 described herein.

In a particular embodiment of the methods provided herein for identifying agents that modulate Aβ levels without substantially altering or affecting the cleavage and/or processing of a presenilin substrate that is other than APP, agents that have been identified as agents that reduce Aβ42 levels (e.g., by >50%> at, e.g., 30 μM; see, for example, EXAMPLE 6) are tested for any effects on presenilin-dependent substrate processing activity by assessing the cleavage and/or processing of LRP in the presence of the Aβ42-reducing agent (test sample) and comparing it to negative and positive control samples. In this particular embodiment, LRP processing is assessed by determining the presence or absence, and, if present, the level of an -20 kDa fragment from a C-terminal portion of LRP. The fragment can be detected, for example, using an antibody generated against the C-terminal 13 amino acids of LRP. An Aβ42-reducing agent is selected as one that does not substantially alter the cleavage and/or processing of LRP if the level of the -20 kDa fragment of LRP in a sample that had been contacted with the agent (e.g., at 30 μM) is less than about 20% of that in a positive confrol sample in which presenilin- dependent γ-secretase activity has been inhibited (e.g., using DAPT at -1 μM or 1 mM). In other embodiments of the methods for identifying or screening for agents that modulate Aβ levels without substantially altering or affecting the cleavage and/or processing of a presenilin substrate that is other than APP, agents are identified that modulate the levels of one or more Aβ peptides, such as Aβ42, without substantially altering or affecting the cleavage and/or processing (in particular, the presenilin- dependent cleavage and/or processing) of Notch, E-cadherin, Erb-B4, TrkB, APLP2 and/or hire let. In particular embodiments, these methods can involve, for example, comparing the levels (and/or presence or absence) in test and confrol samples of one or more fragments of Notch, E-cadherin and/or Erb-B4 (as well as LRP) or portion(s) thereof. The methods that involved assessing processing of Notch, E-cadherin or Erb-B4 can be conducted, for example, in a manner similar to that described herein for methods that involve assessing LRP processing. Because alteration, such as, for example, inhibition, of the presenilin-dependent cleavage of Notch can result in adverse side affects including, for example, immunodeficiency and anaemia, one embodiment of the methods described herein includes screening for Aβ-modulating agents that do not substantially alter Notch cleavage and/or processing (in particular, presenilin-dependent processing). Furthermore, non-specific modulation of presenilin and/or presenilin- dependent activity may affect E-cadherin and/or Erb-B4 processing resulting in adverse side affects and, therefore, in particular embodiments of the methods described herein, agents are identified that modulate Aβ levels without substantially altering or affecting E- cadherin and/or Erb-B4 processing. In particular embodiments, the method can involve identifying agents that modulate Aβ levels without substantially altering the cleavage and/or processing of one or more or all of LRP, Notch, E-cadherin and Erb-B4.

(3) Assessment of carboxy-terminal fragments of APP and APPAICD

In addition, other parameters of APP processing may be monitored to determine if the cellular pathway is being altered by an Aβ modulating agent in a way that may result in adverse side effects. For example, an agent that inhibits γ-secretase may cause the accumulation of high amounts of the carboxy terminal fragment species of APP cleaved by a.- or β-secretase. Such fragments may be neurotoxic at high levels. Accumulation of these fragments or the N-terminal fragments produced by - or β-secretase can be determined by immunoassaying cell lysates with an appropriate antibody prepared to such peptides.

One method provided herein for identifying or screening for agents that selectively modulate Aβ levels includes steps of contacting a sample containing APP, or portion(s) thereof and ct- and/or β-secretase activity with a test agent that modulates Aβ levels and identifying a test agent as an agent that selectively modulates Aβ levels if the agent does not substantially alter the level or composition of fragments produced by - or β-secretase. The agent that modulates Aβ levels that is used in this method can be any agent known to modulate Aβ levels. The agent can, for example, be one that is identified by a method described herein which involves contacting a sample containing APP or portion(s) thereof with a test agent and identifying an agent that alters the cleavage of APP that produces one or more Aβ peptides, the processing of APP and/or the level of one or more Aβ peptides.

The step of identifying an agent that does not substantially alter the level or composition of fragments produced by - or β-secretase can be carried out in a number of ways. In general, this process can involve a comparison of the - and/or β-secretase cleavage of APP (or portion thereof) that has been contacted with the test agent (i.e., test sample) and of a sample that has not been contacted with the test agent (i.e., confrol sample). If the level or composition of fragments produced by α- and/or β-secretase cleavage of APP (or portion thereof) in the test and confrol samples do not differ substantially, then the agent is identified as one that alters the level of one or more Aβ peptides without substantially altering the - and/or β-secretase cleavage of APP. The control sample can be the same physical sample as the test sample or a different sample. When the control and test samples are the same, the control is the sample in the absence of test agent. In a particular embodiment, the agent that modulates Aβ levels that is used in the method is one that modulates the levels of Aβ42. In a further embodiment, the agent can be one that selectively modulates the levels of Aβ42 relative to Aβ40 levels and/or the levels of all or most of the other forms of Aβ. In a particular embodiment, the agent modulates the levels of Aβ42 and Aβ39 relative to Aβ40 levels and/or the levels of all or most of the other forms of Aβ. In one embodiment, the agent reduces Aβ42 levels and/or increases Aβ39 levels.

Another method provided herein for identifying or screening for an agent that selectively modulates Aβ levels includes steps of contacting a sample containing APP and/or a portion(s) thereof (including, for example one or more Aβ peptides) and - and/or β-secretase activity with a test agent and identifying an agent that alters the Aβ peptide-producing cleavage or processing of the APP (and/or portion(s) thereof), the processing of Aβ and/or the levels of one or more Aβ peptides without substantially altering the - and/or β-secretase cleavage of APP. The process of identifying an agent that selectively modulates one or more Aβ peptides can be carried out in a number of ways as described herein.

The process of further identifying an agent that does not substantially alter the level or composition of fragments produced by - or β-secretase cleavage of APP, can also be carried out in a number of ways, as described herein. In general, this process can involve a comparison of the - and/or β-secretase cleavage of APP (or portion thereof) that has been contacted with the test agent (i.e., test sample) and of a sample that has not been contacted with the test agent (i.e., control sample). If the level or composition of fragments produced by - and/or β-secretase cleavage of APP (or portion thereof) in the test and control samples do not differ substantially, then the agent is identified as one that alters the level of one or more Aβ peptides without substantially altering the a- and/or β- secretase cleavage of APP. The confrol sample can be the same physical sample as the test sample or a different sample. When the confrol and test samples are the same, the control is the sample in the absence of test agent.

In this method of identifying an agent that selectively modulates Aβ levels, the identification of the Aβ-modulating agent and the determination as to whether the agent alters the levels and/or composition of - and/or β-secretase cleavage of APP, or portion thereof, can be conducted sequentially or simultaneously. For example, an agent that modulates Aβ levels can be identified by a difference in the processing of APP or Aβ, and/or the level of one or more Aβ peptides in samples contacted with the agent (test sample) and samples not contacted with the agent (confrol sample). The identified agent can then be separately evaluated for its effects on α- and/or β-secretase cleavage of APP by comparing the - and/or β-secretase cleavage of APP (or portion thereof) in a sample that has been contacted with the test agent (i.e., test sample) and in a sample that has not been contacted with the test agent (i.e., control sample). In this sequential method, the sample used in the identification of the Aβ-modulating agent can be of the same or different type relative to the sample used in the determination as to whether the agent alters the - and or β secretase cleavage of APP. If the same sample is used, it can contain APP (and/or portion(s) thereof) and an - and/or β-secretase. If different types of samples are used, the sample used in the identification of the Aβ-modulating agent can contain APP and/or portion(s) thereof, and the sample used in the determination of alteration in the a- and/or β-secretase cleavage of APP can contain APP and/or portion(s) thereof, and an - and/or β-secretase.

Alternatively, in a simultaneously performed method, a test sample containing APP (and/or portion(s) thereof), and an o;- and/or β-secretase can be contacted with a test agent and the cleavage of APP that produces one or more Aβ peptides, the processing of APP or Aβ, and/or the level of one or more Aβ peptides can be assessed for the test sample, as can - and/or β-secretase cleavage of APP be assessed for the same test sample. The Aβ peptide-producing cleavage or processing of APP, processing of Aβ and/or levels of Aβ peptides of the test sample, as well as the - and/or β-secretase cleavage of APP of the test sample, can be compared to that of a confrol sample in one step to identify an agent that modulates Aβ levels without substantially altering the level or composition of fragments produced by - and/or β-secretase cleavage of APP (or portion thereof).

In the embodiments of the methods provided herein for identifying agents that modulate Aβ levels without substantially altering or affecting the level or composition of fragments produced by c- and/or β-secretase cleavage of APP (or portion thereof), the fragments produced by a- and/or β-secretase cleavage of APP (or portion thereof) can be detected by any methods known in the art or described herein, for example, using an antibody generated against the amino acids of sAPPα, C83, p3, sAPPβ, or C99.

Further studies have demonstrated the production of an infracellular CTF of APP resulting from γ-secretase cleavage, which, in analogy to NICD, is referred to as AICD (APP intracellular domain) (Pinnix, I et al. (2001) J. Biol. Chem 276:481-487; Sasfre, M. et al. (2001) EMBO Reports 2(9):835-4\; Gu, Y et al. (2001) J. Biol Chem. 276(38): 35235-8). Sequencing has revealed that its N-terminus does not correspond to the expected γ-secretase cleavage after amino acids 40 or 42 of the Aβ domain. Instead, cleavage occurs between amino acids 49 and 50, close to the cytoplasmic side of the fransmembrane domain. Amino acids 49 and 50 of the Aβ domain correspond to amino acids 720 and 721 of the full length APP protein (see e.g., amino acids 720 and 721 of SEQ ID NOs. 2 and 28). This cleavage is reminiscent of the S3 cleavage of Notch and may thus indicate an analogous function of AICD in signal transduction. Indeed, the cytoplasmic fragment of APP has been shown to form a franscriptionally active complex with Fe65, and Tip60 (Cao, X and Sudhof, T.C. (2001) Science 293:115-120). Inhibition of such cleavage may result in unwanted side affects. Thus, in a particular embodiment, a fragment of APP having an N-terminal end that terminates after amino acid 49 of the Aβ domain (close to the cytoplasmic side of the fransmembrane domain) is substantially unchanged in the presence of a test agent when compared to that in the absence of the test agent.

One method provided herein for identifying or screening for agents that selectively modulate Aβ levels includes steps of contacting a sample containing APP, or portion(s) thereof and γ-secretase activity with a test agent that modulates Aβ levels and identifying a test agent as an agent that selectively modulates Aβ levels if the agent does not substantially alter the level or composition of fragments of APP having an N-terminal end that terminates after amino acid 49 of the Aβ domain. The agent that modulates Aβ levels that is used in this method can be any agent known to modulate Aβ levels. The agent can, for example, be one that is identified by a method described herein which involves contacting a sample containing APP or portion(s) thereof with a test agent and identifying an agent that alters the cleavage of APP that produces one or more Aβ peptides, the processing of APP and/or the level of one or more Aβ peptides.

The step of identifying an agent that does not substantially alter the level or composition of fragments of APP having an N-terminal end that terminates after amino acid 49 of the Aβ domain can be carried out in a number of ways. In general, this process can involve a comparison of the γ-secretase cleavage of APP (or portion thereof) that has been contacted with the test agent (i.e., test sample) and of a sample that has not been contacted with the test agent (i.e., confrol sample). If the level or composition of fragments produced by γ-secretase cleavage of APP (or portion thereof) with an N- terminal end that terminates after amino acid 49 of the Aβ domain in the test and confrol samples do not differ substantially, then the agent is identified as one that alters the level of one or more Aβ peptides without substantially altering the level or composition of fragments of APP with an N-terminal end that terminates after amino acid 49 of the Aβ domain. The confrol sample can be the same physical sample as the test sample or a different sample. When the confrol and test samples are the same, the control is the sample in the absence of test agent.

In a particular embodiment, the agent that modulates Aβ levels that is used in the method is one that modulates the levels of Aβ42. In a further embodiment, the agent can be one that selectively modulates the levels of Aβ42 relative to Aβ40 levels and/or the levels of all or most of the other forms of Aβ. In a particular embodiment, the agent modulates the levels of Aβ42 and Aβ39 relative to Aβ40 levels and/or the levels of all or most of the other forms of Aβ. In one embodiment, the agent reduces Aβ42 levels and/or increases Aβ39 levels.

Another method provided herein for identifying or screening for an agent that selectively modulates Aβ levels includes steps of contacting a sample containing APP and/or a portion(s) thereof (including, for example one or more Aβ peptides) and γ- secretase activity with a test agent and identifying an agent that alters the Aβ peptide- producing cleavage or processing of the APP (and/or portion(s) thereof), the processing of Aβ and/or the levels of one or more Aβ peptides without substantially altering the level or composition of fragments of APP having an N-terminal end that terminates after amino acid 49 of the Aβ domain. The process of identifying an agent that selectively modulates one or more Aβ peptides can be carried out in a number of ways as described herein.

The process of further identifying an agent that does not substantially alter the level or composition of fragments of APP having an N-terminal end that terminates after amino acid 49 of the Aβ domain, can also be carried out in a number of ways, as described herein. In general, this process can involve a comparison of the γ-secretase cleavage of APP (or portion thereof) that has been contacted with the test agent (i.e., test sample) and of a sample that has not been contacted with the test agent (i.e., control sample). If the level or composition of fragments of APP having an N-terminal end that terminates after amino acid 49 of the Aβ domain in the test and control samples do not differ substantially, then the agent is identified as one that alters the level of one or more Aβ peptides without substantially altering the level or composition of fragments of APP having an N-terminal end that terminates after amino acid 49 of the Aβ domain. The control sample can be the same physical sample as the test sample or a different sample. When the confrol and test samples are the same, the confrol is the sample in the absence of test agent.

In this method of identifying an agent that selectively modulates Aβ levels, the identification of the Aβ-modulating agent and the determination as to whether the agent alters the levels and/or composition of fragments of APP having an N-terminal end that terminates after amino acid 49 of the Aβ domain, or portion thereof, can be conducted sequentially or simultaneously. For example, an agent that modulates Aβ levels can be identified by a difference in the processing of APP or Aβ, and/or the level of one or more Aβ peptides in samples contacted with the agent (test sample) and samples not contacted with the agent (control sample). The identified agent can then be separately evaluated for its effects on fragments of APP having an N-terminal end that terminates after amino acid 49 of the Aβ domain by comparing the fragments of APP having an N-terminal end that terminates after amino acid 49 of the Aβ domain (or portion thereof) in a sample that has been contacted with the test agent (i.e., test sample) and in a sample that has not been contacted with the test agent (i.e., confrol sample). In this sequential method, the sample used in the identification of the Aβ-modulating agent can be of the same or different type relative to the sample used in the determination as to whether the agent alters the level and/or composition of fragments of APP having an N-terminal end that terminates after amino acid 49 of the Aβ domain. If the same sample is used, it can contain APP (and/or portion(s) thereof) and an γ-secretase. If different types of samples are used, the sample used in the identification of the Aβ-modulating agent can contain APP and/or portion(s) thereof, and the sample used in the determination of alteration in the fragments of APP having an N-terminal end that terminates after amino acid 49 of the Aβ domain can contain APP and/or portion(s) thereof, and a γ-secretase activity. Alternatively, in a simultaneously performed method, a test sample containing

APP (and/or portion(s) thereof), and a γ-secretase can be contacted with a test agent and the cleavage of APP that produces one or more Aβ peptides, the processing of APP or Aβ, and/or the level of one or more Aβ peptides can be assessed for the test sample, as can fragments of APP having an N-terminal end that terminates after amino acid 49 of the Aβ domain be assessed for the same test sample. The Aβ peptide-producing cleavage or processing of APP, processing of Aβ and/or levels of Aβ peptides of the test sample, as well as the fragments of APP having an N-terminal end that terminates after amino acid 49 of the Aβ domain, can be compared to that of a confrol sample in one step to identify an agent that modulates Aβ levels without substantially altering the level or composition of fragments of APP having an N-terminal end that terminates after amino acid 49 of the Aβ domain (or portion thereof).

In the embodiments of the methods provided herein for identifying agents that modulate Aβ levels without substantially altering or affecting the level or composition of fragments of APP having an N-terminal end that terminates after amino acid 49 of the Aβ domain, the APP fragments having an N-terminal end that terminates after amino acid 49 of the Aβ domain can be detected by any methods known in the art or described herein, for example, using an antibody generated against the C-terminal amino acids of APP. The C-terminal amino acids may include any amino acid C-terminal to amino acid 49 of the Aβ domain or any amino acid C-terminal to amino acid 720 of full length APP. E. Systems

There are a number of kits, combinations and systems that can be used in performing the various methods provided herein. Such methods include methods for assessing presenilin activity, methods for identifying candidate agents for treatment or prophylaxis of a disease or disorder associated with an altered presenilin, methods for identifying or screening for agents that modulate Aβ levels and methods for identifying or screening for agents for treatment or prophylaxis of a disease or disorder characterized by and/or associated with altered Aβ levels and/or processing of APP, including for example, diseases associated with amyloidosis.

Kits, combinations and systems are also provided herein. Such kits, combinations and/or systems can include, for example, a cell(s) (and/or lysates, exfracts, medium and membranes from the cell(s)) exhibiting APP (altered and/or wild-type as well as portion(s) of APP) expression and processing, one or more presenilins (altered and/or wild-type as well as portion(s) of presenilins) expression and processing, and/or one or more presenilin substrates (altered and/or wild-type as well as portion(s) of presenilin substrates), including, for example, LRP, Notch, E-cadherin and Erb-B4. The cells of the system can be isolated cells or cell cultures that endogenously express such protein(s) or can recombinantly express such proteins as described above with respect to the methods for identifying agents. Systems in which the cells recombinantly express the proteins can be such that the cells are isolated cells or cell cultures or are contained within an animal, in particular, a non-human animal, e.g., a non-human mammal. Many examples of such cells are described herein and known in the art.

The kits, combinations and/or systems provided herein can include antibodies and/or fragment(s) thereof specifically reactive to particular Aβ peptides. For example, a system can include antibodies specifically reactive to Aβ42 versus one or more other Aβ peptides, and in particular, Aβ40. Aβ42 selective-antibodies are provided herein. Such antibodies can be made by the methods described herein, including, for example, by immunization with a peptidyl sequence of MVGGWIA, and by recombinant methods. One such antibody (and/or fragment(s) thereof) includes the sequence of amino acids 1- 95 of SEQ ED NO: 12 and/or 1-97 of SEQ ED NO: 14. A kit, combination or system can include cells that produce any such antibody (and/or fragment(s) thereof). For example, such a cell could contain nucleic acid containing the sequence of nucleotides set forth as nucleotides 1-285 of SEQ ED NO: 11 and/or the sequence of nucleotides set forth as nucleotides 1-291 ofSEQ ED NO: 13.

The kits, combinations and/or systems provided herein can include detection antibodies (and/or fragment(s) thereof) designed to be reactive to more than one species of Aβ. In one example, the antibodies that are reactive to a sequence on the N-terminus of Aβ, such as, for example amino acids 1-12 of Aβ. Such antibodies (and/or fragment(s) thereof) are provided herein and include antibodies containing one or both of the amino acids 1-100 of SEQ ED NO: 16 and 1-98 of SEQ ED NO: 18. A kit, combination or system can include cells that produce any such antibody (and/or fragment(s) thereof). For example, such a cell could contain nucleic acid containing the sequence of nucleotides set forth as nucleotides 1-300 of SEQ ID NO: 15 and/or the sequence of nucleotides set forth as nucleotides 1-294 of SEQ ED NO: 17. The detection antibody is generally conjugated to a detectable label, such as, for example alkaline phosphatase, and the presence or absence of antibody binding can be determined by luminescence of a subsfrate that is detected by a change in light emitted in the presence of alkaline phosphatase, such as, for example, CDP-Star chemiluminescence substrate (Tropix, Inc.).

One system provided herein can be used, for example, in assessing presenilin activity. In a particular embodiment, the system includes a source of presenilin activity, a source of LRP (and/or portion(s) thereof) protein and a reagent for determining LRP protein composition. In one embodiment, the source of presenilin activity can be, for example, a standard or control used in a method of assessing presenilin activity. In another embodiment, the source of presenilin activity can be the activity that is being assessed. An example of a reagent for determining LRP protein composition is an antibody (and/or fragment(s) thereof) that recognizes a fragment of LRP generated by a presenilin-dependent activity, e.g., presenilin-dependent γ-secretase or a LRP fragment that occurs in the absence of such activity. Such fragments include LRP-CTF, and, in particular an - 20 kD fragment of LRP. In one embodiment, the system includes an anti- LRP antibody prepared to the carboxyl-terminal 13 amino acid peptide of LRP (C- GRGPEDEIGDPLA). In a particular embodiment, the system includes the anti-LRP polyclonal antibody (R9377) described herein (see, e.g, the EXAMPLES). Some systems can also contain sources of other presenilin substrates, e.g., Notch, Erb-B4 and E-cadherin) and reagents, such as antibodies and/or fragment(s) thereof, that are reactive to Notch intracellular domain (NICD), E-cadherin infracellular domain, or Erb-B4 infracellular domain.

One embodiment of a system or kit for use in identifying agents that modulate Aβ levels provided herein contains a reagent for assessing cleavage of APP that produces one or more Aβ peptides, APP processing, Aβ processing and/or Aβ levels and a reagent for assessing cleavage and/or processing (in particular, presenilin-dependent processing) of a presenilin subsfrate. In a particular embodiment, the presenilin substrate is LRP and/or portion(s) thereof. Such reagents are described and provided herein. For example, reagents for assessing Aβ levels include antibodies and/or fragments thereof such as antibodies that specifically react with Aβ42, for example an antibody or fragment(s) thereof containing the sequence of amino acids 1-95 of SEQ ED NO: 12 and/or 1-97 of

SEQ ID NO: 14. Another example of an antibody that can be used in assessing Aβ levels is an antibody that recognizes most or all forms of Aβ. One example is an antibody (and/or fragment(s) thereof) containing one or both of the amino acids 1-100 of SEQ ED NO: 16 and 1-98 of SEQ ED NO: 18. An example of a reagent for determining LRP protein composition in assessing LRP cleavage and/or processing is an antibody (and/or fragment(s) thereof) that recognizes a fragment of LRP generated by a presenilin- dependent activity, e.g., presenilin-dependent γ-secretase or a LRP fragment that occurs in the absence of such activity. Such fragments include LRP-CTF, and, in particular an - 20 kD fragment of LRP. In one embodiment, the system includes an anti-LRP antibody prepared to the carboxyl-terminal 13 amino acid peptide of LRP (C-

GRGPEDEIGDPLA). In a particular embodiment, the system includes the anti-LRP polyclonal antibody (R9377) described herein (see, e.g., the EXAMPLES). Some systems can also contain reagents such as antibodies and/or fragment(s) thereof that are reactive to Notch intracellular domain (NICD), E-cadherin infracellular domain, or Erb- B4 infracellular domain.

F. Methods of Identifying Agents for the Treatment of a Disease or Disorder

Provided herein are methods for identifying candidate agents for the freatment or prophylaxis of diseases and disorders associated with or characterized by altered APP processing, Aβ production, catabolism, processing and/or levels. Disease models are a valuable tool for the discovery and testing of treatment agents. Such disease models may be cellular or organismal and may be produced by methods known to those of skill in the art and described herein.

1. Cell models

Cell models for the identification and testing of agents for the freatment of diseases and disorders characterized by altered Aβ peptide levels are provided herein. Suitable cell lines include human and animal cell lines, such as the 293 human kidney cell line, neuroglioma cell lines, neuroblastoma cell lines, HeLa cells, primary endothelial cells, primary fibroblasts or lymphoblasts, primary mixed brain cells (including neurons, astrocytes, and neuroglia), Chinese hamster ovary (CHO) cells, and the like.

In a particular embodiment, mixed brain cell cultures from fransgenic mice (e.g., Tg2576 fransgenic mice) are provided. Such primary cultures can mimic an in vivo system more closely than engineered cell lines. Primary mixed brain cultures can be established by any method known to those of skill in the art or described herein. Generally, primary mixed brain cultures can be produced by dissecting 17 day old mouse embryos utilizing a stereo scope, obtaining brain tissue and dissociating with papain, then culturing cells by standard procedures for primary neuronal cultures.

Primary cell cultures can be obtained from any host, in a particular embodiment, a non-human host, including but not limited mice, rabbits, monkeys, apes, etc. which naturally express APP or any one or combination of isoforms or fragments of APP. The primary cultures can comprise cells that express wild type versions or isoforms of APP or mutant versions. The cells can over express the protein as well.

Alternatively, engineered cell lines may be used. Cells may contain recombinant DNA that when expressed, result in altered production, degradation or clearance of Aβ peptides or altered expression of APP, such as by replacing or modifying the promoter region or other regulatory region of the endogenous gene. Such a cell can by produced by introduction of heterologous or homologous nucleic acid into the cell using methods known in the art and described herein. In a particular embodiment, the cell is a recombinant cell that expresses the protein(s) as heterologous protein(s). Such cells may overexpress or mis-express the heterologous protein(s). For example, a recombinant cell may be one that endogenously expresses the protein(s) and also has been fransfected with additional copies of nucleic acid encoding the protein(s). Alternatively, the host cell used in the generating the recombinant cell may be one that endogenously expresses little to none of the protein(s) of interest or one in which such proteins have been eliminated (e.g. , through gene knock-out methods or by inhibition with an agent that does not inhibit the activity of the heterologous protein(s)). In a particular embodiment, cell lines capable of expressing APP variants with altered Aβ peptide levels are provided. Such variants can include those having one or several amino acid substitutions directly amino-terminal ofthe Aβ cleavage site. For example, APP DNA bearing a double mutation (Lys - >Asn and Met ->Leu ) found in a Swedish FAD family produce approximately six- to-eight fold more Aβ than cells expressing normal APP. Exemplary clones and vectors for APP include but are not limited ATCC accession numbers 40305, 40347, 78397, 78510, 78510D, 86195.

Cells or less differentiated precursor cells may be stably or transiently fransfected with purified or recombinant protein(s) in vifro or in an organism. In vifro fransfection is followed by cell expansion through culturing prior to use. Cells from a known cell line are preferred, such as from neuroblastoma SH-SY5Y cells, pheochromocytoma PC 12 cells, neuroblastoma SK-N-BE(2)C cells, human SK-N-MC neuroblastoma cells, SMS- KCNR cells, human LAN-5 neuroblastoma cells, human GI-CA-N neuroblastoma cells, human neuroblastoma cells, mouse Neuro 2a (N2A) neuroblastoma cells and/or human EVER 32 neuroblastoma cells. Exemplary cell lines include human embryonic kidney 293 (HEK 293) ATCC accession number CRL-1573, CHO (including CHO and CHO- Kl(accession number CCL-61)), LTK^", N2A (accession number CCL-131), H6, and HGB. The generation, maintenance and use of such cell lines is well known. Suitable cells include mammalian cell lines, typically human cell lines that are commercially available for example from the American Type Tissue Culture Collection (ATCC), Rockville, Maryland, 20852. Exemplary cells include CHO cells expressing human APP751 from a vector containing the gene encoding APP751, human mutant APPP751 (V717F) from a vector containing a gene encoding APP751 (V717F), or a combination thereof and can be cultured in standard cell culture media supplemented with 10% fetal calf serum and optionally with antibiotics and fungicides such as 100 U/mL penicillin/streptomycin. Other suitable cells include human neuroglioma cells HS683 that express APP695, APP751, APP770 or a combination thereof from a vector containing a gene encoding for the respective protein or partial protein. Additionally, a human neuroblastoma cell line SH-SY5Y described in T. Yamazaki and Y. Ihara (1998) Neurobiology of Aging 19:S77-S79 or other cell that secretes large amounts of Aβ into the medium without Aβ fransfection can also be used.

An exemplary transformed human embryonic kidney cell line is the human 293 cell line, ATCC accession number CRL-1573. Other suitable cells include CRL-1721 and CCL-92 and those listed in the catalogue from the Indiana Alzheimer Disease Center National Cell Repository of Indiana University - Purdue University Indianapolis, 425 University Blvd., Indianapolis, IN 46202-5143, which is incorporated by reference herein in its entirety.

Additionally, primary cell cultures, immortalized cell lines, or stem cells (embryonic or adult) induced to express Aβ proteins or peptides can be used. In one embodiment, cells that are not terminally differentiated can be induced to express neuronal characteristics. Such cells can be induced for example by exposing them to a growth factor, cyotokine, hormone, neural inducing media or combination thereof. 2. Animal models Animal models for the identification and testing of agents for the freatment of diseases and disorders characterized by altered Aβ peptide levels are provided herein. Transgenic animal models and animals, such as rodents, including mice and rats, cows, chickens, pigs, goats, sheep, monkeys, including gorillas, and other primates, are provided herein, hi particular, fransgenic non-human animals that contain recombinant DNA that when expressed, result in altered production, degradation and/or clearance of Aβ peptides or altered expression of APP, such as by replacing or modifying the promoter region or other regulatory region of the endogenous gene are provided. Such an animal can by produced by promoting recombination between endogenous nucleic acid and an exogenous gene of interest that could be over-expressed or mis-expressed, such as by expression under a strong promoter, via homologous or other recombination event. Transgenic animals can be produced by introducing the nucleic acid using any know method of delivery, including, but not limited to, microinjection, lipofection and other modes of gene delivery into a germline cell or somatic cells, such as an embryonic stem cell. Typically the nucleic acid is introduced into a cell, such as an embryonic stem cell (ES), followed by injecting the ES cells into a blastocyst, and implanting the blastocyst into a foster mother, which is followed by the birth of a fransgenic animal. Generally introduction of a heterologous nucleic acid molecule into a chromosome of the animal occurs by a recombination between the heterologous nucleic acid of interest and endogenous nucleic acid. The heterologous nucleic acid can be targeted to a specific chromosome.

In some instances, knockout animals can be produced. Such an animal can be initially produced by promoting homologous recombination between an gene of interest in its chromosome and the corresponding exogenous gene of interest that has been rendered biologically inactive (typically by insertion of a heterologous sequence, e.g., an antibiotic resistance gene). In one embodiment, this homologous recombination is performed by transforming embryo-derived stem (ES) cells with a vector containing the insertionally inactivated gene of interest, such that homologous recombination occurs, followed by injecting the ES cells into a blastocyst, and implanting the blastocyst into a foster mother, followed by the birth of the chimeric animal ("knockout animal") in which a gene of interest has been inactivated (see Capecchi, Science 244.: 1288-1292 (1989)). The chimeric animal can be bred to produce homozygous knockout animals, which can then be used to produce additional knockout animals. Knockout animals include, but are not limited to, mice, hamsters, sheep, pigs, cattle, and other non-human mammals. For example, a knockout mouse is produced. The resulting animals can serve as models of specific diseases that are the result of or exhibit altered-expression of a polypeptide involved in neurodegenerative disorders. Such knockout animals can be used as animal models of such diseases e.g. , to screen for or test molecules for the ability to treat or prevent such diseases or disorders.

Other types of fransgenic animals also can be produced, including those that over- express a polypeptide involved in neurodegenerative disorders. Such animals include "knock-in" animals that are animals in which the normal gene is replaced by a variant, such a mutant, an over-expressed form, or other form. For example, one species', such as a rodent's endogenous gene can be replaced by the gene from an other species, such as from a human. Animals also can be produced by non-homologous recombination into other sites in a chromosome; including animals that have a plurality of integration events. After production of the first generation fransgenic animal, a chimeric animal can be bred to produce additional animals with over-expressed or mis-expressed polypeptides involved in neurodegenerative disorders. Such animals include, but are not limited to, mice, hamsters, sheep, pigs, cattle and other non-human mammals. The resulting animals can serve as models of specific diseases that are the result of or exhibit over- expression or mis-expression of a polypeptide involved in neurodegenerative disorders. Such animals can be used as animal models of such diseases e.g., to screen for or test molecules for the ability to treat or prevent such diseases or disorders. In a specific embodiment, a mouse with over-expressed or mis-expressed APP is produced. One useful non-human animal model harbors a copy of an expressible transgene sequence which encodes the Swedish mutation of APP (Asp595-leu596). US Patent Nos. 5,612,486 and 5,850,003, incorporated herein by reference, disclose a fransgenic rodent having a diploid genome comprising a fransgene encoding a heterologous APP polypeptide having the Swedish mutation wherein the amino acid residues at positions corresponding to positions 595 and 596 in human APP695 are asparagine and leucine, respectively. The fransgene is expressed to produce a human APP polypeptide having the Swedish mutation. The polypeptide is processed in a sufficient amount to be detectable in a brain homogenate of the transgenic rodent. The sequence generally is expressed in cells which normally express the naturally-occurring endogenous APP gene (if present). Murine and hamster models are suitable for this use. Such fransgenes typically comprise a Swedish mutation APP expression cassette, in which a linked promoter and, preferably, an enhancer drive expression of structural sequences encoding a heterologous APP polypeptide comprising the Swedish mutation.

Other suitable animal models include the fransgenic mouse disclosed in US Patent No. 5,387,742. This fransgenic mouse contains a DNA sequence with a nerve tissue specific promoter and a DNA sequence which encodes a β-amyloid precursor protein selected from the group consisting of A751 and A770. The promoter and DNA sequence which encodes the precursor protein are operatively linked to each other and integrated in the genome of the mouse and expressed to form β-amyloid protein deposits in the brain of the mouse.

Still other fransgenic animal models for the identification and testing of agents for the freatment of disease and disorders characterized by altered Aβ peptide levels include those described in US Patent Nos. 5,811,633; 6,037,521; 6,184,435; 6,187,992; 6,211,428; and 6,340,783, all of which are incorporated by reference, fransgenic mouse models Tg 2576; APPSWE mouse, K670N, M671 L, and other models including

APP(V717F), APP(K670N, M671L and V717F), PS-1 M146L, PS-1 M146V, APPSWE + PS A246E (reviewed by Emilien, et al, (2000) Arch. Neuro. 57: 176-81).

3. Evaluation of models and identification and testing of agents for the treatment of diseases and disorders Cell and animal models of diseases and disorders involving Aβ misregulation described herein have a number of uses. For example, by evaluating the cellular or organismal phenotypes associated with the altered expression of proteins involved in Aβ regulation in the cells/organisms and correlating such phenotypes with specific cellular molecules and processes, the disease/disorder models can be used in elucidating the mechanisms underlying Aβ misregulation in a cell as well as in dissecting processes and pathways involved in Aβ regulation. In addition, by evaluating the effects of test agents or candidate therapeutic agents on Aβ levels and the phenotypic manifestations of the model cells/organisms, the models can be used in screening agents and testing candidate agents for the freatment of diseases and disorders that involve Aβ misregulation. In the methods for identifying agents for the freatment or prophylaxis of a disease or disorder, any sample containing an altered test protein, and/or portion(s) thereof, and APP, and/or portion(s) thereof, wherein the altered protein is associated with altered Aβ42 production, catabolism, processing and/or Aβ42 levels may be used. Such samples can include, for example any cell, cell extract, cell model, organism or animal model described herein. The cell, organism or animal may be one that contains an altered APP, APP processing activity, or Aβ processing activity and/or expresses altered Aβ levels such as, for example, the cell and animal models described above. The altered APP, APP processing activity, Aβ processing activity, or Aβ level can be one that is altered relative to a wild-type. Typically, a wild-type protein, such as, for example, APP, APP processing enzyme or Aβ processing enzyme can be one that is encoded by a predominant allele in a population or any allele that is not associated with disease or a pathogenic condition. A wild-type APP, APP processing enzyme or Aβ processing enzyme can be one that occurs in an organism that exhibits normal APP and/or Aβ processing patterns. The altered APP, APP processing enzyme or Aβ processing enzyme can be a mutant or can be, for example, one that is encoded by a nucleic acid linked to Alzheimer's disease. For example, the altered enzyme activity may include any one or more of the at least 60 mutations in human PSl and the at least two mutations in human PS2 that have been genetically linked to early onset familial Alzheimer's disease (FAD). Exemplary presenilins with altered activity include FAD-associated mutant forms of PSl and PS2 that give rise to an increased accumulation of Aβ42 in AD patients and fransfected cell lines and fransgenic animals in which they are expressed. Included among such mutations are the PS2 FAD mutation N141I (Volga German FAD mutant) and the PSl FAD mutation M146L. Examples of diseases associated with an altered APP, APP processing activity, Aβ, and/or Aβ processing activity for which the methods provided herein can be used to identify candidate therapeutic or prophylactic agents include, but are not limited to, amyloidosis-associated diseases and neurodegenerative diseases. In a particular aspect, the disease is Alzheimer's Disease.

In one method for testing an agent for use in the freatment of a disease or disorder, the test agent is one that is already known to modulate the level of one or more particular Aβ peptides. Thus, in one embodiment of this method, a disease model is contacted with a test agent that modulates the level of an Aβ peptide, and a test agent is identified as an agent for the treatment of a disease or disorder if the test agent at least partially reverses or reduces, ameliorates or eliminates a disease frait or phenotype exhibited by a model cell or organism, or that tends to restore APP processing and/or Aβ processing or levels to compensate for disease-associated abnormalities in Aβ levels. In general, the step of identifying a test agent that at least partially reverses or reduces, ameliorates or eliminates a disease trait or phenotype exhibited by a model cell or organism, or that tends to restore APP processing and/or Aβ processing or levels can involve a comparison of the disease frait or phenotype and/or APP processing and/or Aβ processing or levels in a model that has been contacted with the test agent (i.e., test model) and in a model that has not been contacted with the test agent (i.e., confrol model). If the disease frait or phenotype and/or APP processing and/or Aβ processing or levels in the test and control models differs, then the test agent is identified as a candidate agent for the treatment and/or prophylaxis of a disease or disorder. In such an embodiment, both the test and confrol model express the disease frait or phenotype in the absence of the test agent, hi another embodiment, the control model or sample is a wild type model or sample. In such an embodiment, the step of identifying a candidate agent includes comparing the disease trait or phenotype and/or Aβ production, catabolism, processing and or Aβ levels in a test sample that has been contacted with the test agent and a positive confrol sample and identifying an agent as a candidate agent Aβ production, catabolism, processing and/or Aβ levels if Aβ production, catabolism, processing and/or Aβ levels in the test and confrol samples is substantially similar

The agent that modulates the level of an Aβ peptide that is used in this method can be one that was identified by any of the processes described herein. For example, the agent may be one that was identified by a process involving contacting a sample containing APP or portion(s) thereof with a test agent and identifying an agent that alters the cleavage of APP that produces one or more Aβ peptides, the processing of APP and/or the level of one or more Aβ peptides.

The agent that modulates the level of an Aβ peptide that is used in this method can be one that was identified as having a particular selectivity. Methods of assessing the selectivity of an Aβ modulating agent are provided herein. In a particular embodiment the agent that selectively modulates Aβ levels can be one that does not substantially alter the level of one or more Aβ peptides other than the Aβ peptide that is modulated by the test agent. In a particular embodiment, the agent that modulates Aβ levels that is used in the method is one that modulates the levels of Aβ42. In a further embodiment, the agent can be one that selectively modulates the levels of Aβ42 relative to Aβ40 levels and/or the levels of all or most of the other forms of Aβ. In a particular embodiment, the agent modulates the levels of Aβ42 and Aβ39 relative to Aβ40 levels and/or the levels of all or most of the other forms of Aβ. In one embodiment, the agent reduces Aβ42 levels and/or increases Aβ39 levels, hi a particular embodiment, the agent that reduces Aβ42 levels does not substantially alter the levels of non- APP subsfrate cleavage/processing activity of presenilin, such as LRP and/or other substrates provided herein. In anther embodiment the agent that reduces Aβ42 levels does not substantially alter the levels of Aβ40 or the non- APP subsfrate cleavage/processing activity of presenilin. In other embodiments, agents that have not previously been screen for their ability to modulate the level of one or more particular Aβ peptides may be screened in cellular and organismal disease model systems. An agent can be identified as an agent that alters the cleavage of APP that produces one or more Aβ peptides, the processing of APP or Aβ, and/or the level of one or more Aβ peptides. hi one embodiment, an alteration results in the restoration of APP processing and/or Aβ processing or levels to compensate for disease-associated abnormalities in Aβ levels. At the same time, the agent can be identified as an agent that at least partially reverses or reduces, ameliorates or eliminates a disease trait or phenotype exhibited by a model cell or organism. The process of identifying an alteration in APP processing, Aβ processing and Aβ levels can be carried out in a number of ways as described herein.

The selectivity of the agent may also be assessed in the disease model system. Any methods of assessing the selectivity of an Aβ modulating agent provided herein may be used. In a particular embodiment the agent that selectively modulates Aβ levels does not substantially alter the level of one or more Aβ peptides other than the Aβ peptide that is modulated by the test agent. In a particular embodiment, the agent that modulates Aβ levels that is used in the method is one that modulates the levels of Aβ42. In a further embodiment, the agent can be one that selectively modulates the levels of Aβ42 relative to Aβ40 levels and/or the levels of all or most of the other forms of Aβ. In a particular embodiment, the agent modulates the levels of Aβ42 and Aβ39 relative to Aβ40 levels and/or the levels of all or most of the other forms of Aβ. In one embodiment, the agent reduces Aβ42 levels and/or increases Aβ39 levels. The modulation of a particular Aβ peptide by the agent can be identified by any of the methods described herein. In general, the modulation of a particular Aβ peptide by the agent can be identified by a detectable difference in the levels of the Aβ peptide in the model cell or organism contacted with the agent (test model) and model cells or organisms not contacted with the agent (confrol models). The agent is one that selectively modulates the levels of the particular Aβ peptide if any difference (including, for example, absolute and/or percentage difference) in the levels of one or more other Aβ peptides in model contacted with the agent and model not contacted with the agent is less than the difference (including, for example, absolute and/or percentage difference) in the levels of the particular Aβ peptide in test and confrol models. In particular embodiments, the extent to which the agent alters the levels of one or more other Aβ peptides (i.e., the peptides that are not targeted for modulation) is less than about 40%>, 35%>, 30%, 25%, or 20%. In one embodiment, the extent to which the agent alters the levels of one or more other Aβ peptides (i.e., the peptides that are not targeted for modulation) is less than 20%. Any modulation of the level of the one or more other Aβ peptides (i.e., the peptides that are not targeted for modulation) that is not a substantial alteration is one that is generally not associated with any significant undesired or adverse consequence in the model cell or organism.

In a particular embodiment, agents that more specifically or selectively modulate Aβ levels can be identified in a disease model using methods provided herein that involve identifying agents that modulate Aβ levels without substantially altering or affecting non-APP substrate cleaving/processing activity of presenilin. In one embodiment, the agent that reduces Aβ42 levels does not substantially alter the levels of non-APP subsfrate cleavage/processing activity of presenilin, such as LRP and/or other substrates provided herein. In anther embodiment the agent that reduces Aβ42 levels does not substantially alter the levels of Aβ40 or the non-APP subsfrate cleavage/processing activity of presenilin. The process of further identifying an agent that does not substantially alter the cleavage of a presenilin subsfrate (other than APP), or portion(s) thereof, can be carried out by any of the methods described herein. In general, this process can involve a comparison of the cleavage and/or processing (in particular, the presenilin-dependent cleavage and/or processing) of a presenilin substrate (or portion(s) thereof) other than APP, and/or the levels of a peptide fragment(s) of the presenilin substrate, in a model cell or cells within a model organism that has been contacted with the test agent (i.e., test model) and in a model cell or cells within a model organism that has not been contacted with the test agent (i.e., confrol model). If the cleavage and/or processing of the presenilin subsfrate that is other than APP and/or the subsfrate fragment(s) levels of the test and confrol samples do not differ substantially, then the agent is identified as one that alters the level of one or more Aβ peptides without substantially altering the cleavage and/or processing of the presenilin substrate, or portion(s) thereof, that is other than APP. The control model can be the same physical model as the test model or a different model. When the confrol and test models are the same, the confrol is the model in the absence of test agent. G. Methods for Treating or Preventing Diseases or Disorders

Methods provided herein for identifying or screening for agents that modulate Aβ levels and for candidate agents for the treatment or prophylaxis of disease are useful in the discovery of particular agents for treating diseases and disorders involving or characterized by altered Aβ production, catabolism, processing and/or levels. Such diseases include, but are not limited to, diseases involving or associated with amyloidosis and neurodegenerative diseases. One example of such a disease is Alzheimer's disease. Provided herein are methods for treating or preventing diseases and disorders involving or characterized by altered Aβ production, catabolism, processing and/or levels. The methods are particularly suitable for the freatment or prevention of disease because they are designed to selectively modulate Aβ levels, and in particular, the level of Aβ42 and/or Aβ39, in order to avoid possible side-effects that non-specific modulation of Aβ can be associated with as described herein. Such methods can include a step of administering to a subject having such a disease or disorder or predisposed to such a disease or disorder an agent that modulates the cleavage of APP that produces one or more Aβ peptides, the processing of APP, the processing of Aβ and/or the level of one or more Aβ peptides. In one embodiment of the methods, the agent being administered is one that modulates the cleavage of APP that produces one or more Aβ peptides, the processing of APP, the processing of Aβ and/or the level of one or more Aβ peptides such that Aβ42 levels are modulated. The level of Aβ42 can be modulated to a greater extent than the level of one or more other Aβ peptides, in particular, Aβ40, is modulated, or without substantially altering the level of one or more other Aβ peptides, in particular Aβ40. In a particular embodiment, Aβ42 levels are reduced. h another embodiment, the agent being administered is one that modulates the cleavage of APP that produces one or more Aβ peptides, the processing of APP, the processing of Aβ and/or the level of one or more Aβ peptides such that Aβ39 levels are modulated. The level of Aβ39 can be modulated to a greater extent than the level of one or more other Aβ peptides, in particular, Aβ40, or without substantially altering the level of one or more other Aβ peptides, in particular Aβ40. In a particular embodiment, the agent increases the level of Aβ39. The agent can be one that modulates the levels of Aβ42 and Aβ39 to a greater extent than the level of one or more other Aβ peptides, in particular, Aβ40, or without substantially altering the levels of one or more other Aβ peptides, such as, for example, Aβ40. In one embodiment, the levels of Aβ42 are reduced and the levels of Aβ39 are increased.

In another embodiment of the methods, the agent being administered is one that modulates the cleavage of APP that produces one or more Aβ peptides, the processing of APP, the processing of Aβ and/or the level of one or more Aβ peptides without substantially altering one or more presenilin-dependent activities other than the presenilin-dependent processing of APP. In a further embodiment, the agent being administered is one that modulates the cleavage of APP that produces one or more Aβ peptides, the processing of APP, the processing of Aβ and/or the level of one or more Aβ peptides without substantially altering the cleavage and/or processing of a presenilin substrate and/or portion(s) thereof (e.g., Notch, E-cadherin, Erb-B4, and portion(s) thereof) that is other than APP. In another embodiment, the agent being administered is one that modulates the cleavage of APP that produces one or more Aβ peptides, the processing of APP, the processing of Aβ and/or the level of one or more Aβ peptides without substantially altering the cleavage and/or processing of LRP and/or portion(s) thereof. In particular of these embodiments, the levels of Aβ42 and/or Aβ39 are modulated, such as, for example, as follows: the levels of Aβ42 and/or Aβ39 are modulated to a greater extent than the levels of other Aβ peptides, such as, e.g., Aβ40; the levels of Aβ42 and/or Aβ39 are modulated without substantially altering the level of one or more other Aβ peptides, such as, e.g., Aβ40. In particular embodiments of these methods, the level of Aβ42 is reduced and/or the level of Aβ39 is increased. H. Methods of Modulating Aβ

Provided herein are methods for modulating Aβ levels. In a particular embodiment, the methods are for selectively modulating Aβ levels. The methods can be practiced to modulate Aβ levels in any sample. Examples of samples in which Aβ levels may be modulated include, but are not limited to, cells, tissues, organisms, lysates, exfracts and membrane preparations of cells and cell-free samples, such as, for example, samples containing APP and/or portion(s) thereof.

Modulation of Aβ can be, for example, any alteration or adjustment that results in a change in Aβ levels, including but not limited to, alteration of Aβ levels in the cell cytoplasm, intracellular organelles, cell membranes, exfracellular medium, tissue, body fluid and/or levels of secreted Aβ. Modulation of Aβ can involve an alteration in APP (and/or portion(s) thereof) cleavage or processing, Aβ cleavage or processing and/or any combination thereof. Altered APP cleavage or processing and/or altered Aβ cleavage or processing maybe the result of an alteration in any cell, organelle, enzyme, protein, and/or factor that facilitates or participates in APP cleavage or processing and/or Aβ cleavage or processing. Cells, organelles, enzymes, proteins and factors that facilitate or participate in APP cleavage or processing and/or Aβ cleavage or processing may include, but are not limited to microglial cells, proteases, such as secretases, including , β, and γ- secretases, peptidases, presenilins, degratory enzymes, including insulin-degrading enzyme (EDE), neprilysin, plasmin, uPA/tPA, endothelin converting enzyme-1, matrix metalloproteinase-9, and proteosome, cell surface receptors, including scavenger receptor A, the receptor for advanced glycation endproducts (RAGE), and the low-density lipoprotein receptor-related protein (LRP). Modulation of Aβ can also involve an alteration in receptor-mediated clearance and/or uptake into organelles capable of processing Aβ for degradation, including, for example, endosomes and lysosomes. Modulation of Aβ levels may thus involve modulating the level, functioning and/or activity of one or more cells, organelles, enzymes, proteins, and/or factors involved in modulating Aβ production, catabolism, processing and/or clearance.

Modulation of Aβ levels can be, for example, a complete or nearly complete elimination of the production of one or more forms of Aβ, a reduction in the production of one or more forms of Aβ, or an increase in the production of one or more forms of Aβ. A modulation of Aβ can also be an increase in clearance and/or degradation of one or more forms of Aβ, or a decrease in the clearance and/or degradation of one or more forms of Aβ. Modulation of Aβ can further be an alteration in the levels of different Aβ peptides relative to one another or to the total Aβ. Thus, for example, the ratio of a particular Aβ peptide to the total Aβ in a sample can be altered in modulation of Aβ. A modulation of Aβ can also be an increase in one or more forms of Aβ concurrent with a decrease in one or more other forms of Aβ.

In particular methods for modulating Aβ provided herein, the cleavage of APP that produces one or more Aβ peptides, the processing of APP, the processing of Aβ and/or the levels of Aβ is/are modulated in a manner such that Aβ levels are modulated while avoiding substantial or significant alterations in other processes, activities, mechanisms and/or compositions that are not necessary to modulate in order to modulate Aβ levels. Such modulation can be a selective or specific modulation of Aβ levels. In one embodiment, the method selectively modulates the level of particular Aβ peptides, for example one Aβ peptide or two Aβ peptides. In a particular embodiment, the method includes a step of modulating the cleavage of APP that produces one or more Aβ peptides, the processing of APP, the processing of Aβ and/or the levels of Aβ such that the level of Aβ42 is modulated to a greater extent than the level of one or more other Aβ peptides, such as, e.g., Aβ40 (or an Aβ peptide having a C-terminal end that terminates before amino acid 40, or an Aβ with an N-terminus cleaved after amino acid 49 (close to the cytoplasmic side of the transmembrane domain)) is modulated. The level of Aβ42 can be modulated without substantially altering the level of one or more other Aβ peptides, such as, e.g., Aβ40. In a particular embodiment of these methods, the level of Aβ42 is reduced; in other embodiments, level of Aβ42 is increased. In another particular embodiment, the level of Aβ39 (or the level of one or more Aβ peptides having a C-terminal end that terminates before amino acid 40) is to a greater extent than the level of one or more other Aβ peptides, such as, e.g., Aβ40, is modulated. The level of Aβ39 can be modulated without substantially altering the level of one or more other Aβ peptides, such as, e.g., Aβ40. In a particular embodiment of these methods, the level of Aβ39 is increased; in other embodiments, level of Aβ39 is reduced. In particular embodiments of any of these methods, the level of the particular Aβ peptide, such as Aβ42 or Aβ39, can be changed by greater than or equal to about 50%. hi one embodiment, Aβ42 levels of the sample are reduced by greater than or equal to about 50%.

In another embodiment of the methods for modulating Aβ levels, the cleavage of APP that produces one or more Aβ peptides, the processing of APP, the processing of Aβ and/or the levels of Aβ such that the level of Aβ42 and the level of Aβ39 are modulated to a greater extent than the level of one or more other Aβ peptides, such as, for example, Aβ40. hi a further embodiment, the level of Aβ42 and the level of Aβ39 are modulated without substantially altering the level of one or more other Aβ peptides, such as, for example, Aβ40. hi a particular embodiment of these methods, the level of Aβ42 is reduced and the level of Aβ39 is increased.

In particular embodiments of these methods, the sample contains APP and/or portion(s) thereof. Samples that can be used include, but are not limited to, a cell, tissue, organism, cell or tissue lysate, cell or tissue exfract, body fluid, cell membrane or composition containing cell membranes, and a cell-free extract or other cell-free sample. In a particular embodiment, the sample contains a cell, including, for example, a eukaryotic cell such as a mammalian cell. Particular examples of mammalian cells include rodent or human cells. In particular embodiments, the Aβ is cellular and/or extracellular Aβ. hi other embodiments of the methods for modulating Aβ levels, the method includes a step of modulating the cleavage of APP that produces one or more Aβ peptides, the processing of APP, the processing of Aβ and/or the level of one or more Aβ peptides without substantially altering (a) one or more presenilin-dependent activities other than the presenilin-dependent processing of APP, (b) the cleavage and/or processing of a presenilin subsfrate and/or portion(s) thereof that is other than APP and/or (c) the cleavage and/or processing of LRP and/or portion(s) thereof. In particular embodiments of these methods, the levels of Aβ42 are modulated. For example, the levels of Aβ42 may be modulated to a greater extent than the levels of other Aβ peptides, such as, for example, Aβ40. The levels of Aβ42 may be modulated without substantially altering the level of one or more other Aβ peptides, such as, e.g., Aβ40. In any of these embodiments, the level of Aβ42 can be reduced or increased, hi particular embodiments of these methods, the levels of Aβ39 are modulated. For example, the levels of Aβ39 may be modulated to a greater extent than the levels of other Aβ peptides, such as, for example, Aβ40. The levels of Aβ39 may be modulated without substantially altering the level of one or more other Aβ peptides, such as, e.g., Aβ40. In any of these embodiments, the level of Aβ39 can be reduced or increased. In further embodiments, the levels of Aβ42 and Aβ39 are modulated. For example, the levels of Aβ42 and Aβ39 can be modulated to a greater extent than the levels of other Aβ peptides, such as, e.g., Aβ40. The levels of Aβ42 and Aβ39 levels can be modulated without substantially altering the level of one or more other Aβ peptides, such as, e.g. , Aβ40. hi particular embodiments, the level of Aβ42 is reduced and/or the level of Aβ39 is increased. In other embodiments, the level of Aβ42 is increased. In other embodiments, the level of Aβ39 is decreased. In particular embodiments of any of these methods, the level of the particular Aβ peptide, such as Aβ42 or Aβ39, can be changed by greater than or equal to about 50%. In one embodiment, Aβ42 levels of the sample are reduced by greater than or equal to about 50%.

The sample used in these methods can be any sample, such as those described herein. For example, the sample can contain a cell, a tissue, an organism, a cell or tissue lysate, a cell or tissue exfract, a body fluid, a cell membrane or composition containing cell membranes, and/or a cell-free exfract or other cell-free sample. The sample can contain presenilin (and/or portion(s) thereof), APP (and/or portion(s) thereof), and/or one or more presenilin substrates (and/or portion(s) thereof). In particular embodiments, the sample contains one or more of: LRP, Notch, E-cadherin, TrkB, APLP2, Mrelα, Erb-B4, portion(s) of LRP, portion(s) of Notch, portion(s) of E-cadherin, portion(s) of TrkB, portion(s) of APLP2, portion(s) of hire lo and portion(s) of Erb-B4. In particular embodiments, the sample contains a cell, such as, for example, a eukaryotic cell, including, for example, a mammalian cell. Particular examples of mammalian cells include rodent and human cells. In any of the methods, the Aβ can be cellular and/or exfracellular Aβ. hi particular embodiments of the methods for modulating Aβ levels that include a step of modulating the cleavage of APP that produces one or more Aβ peptides, the processing of APP, the processing of Aβ and/or the level of one or more Aβ peptides without substantially altering the cleavage and/or processing of a presenilin subsfrate and/or portion(s) thereof that is other than APP, the presenilin subsfrate and/or portion(s) thereof, can be one or more of the following: Notch, E-cadherin, Erb-B4, and portions of Notch, E-cadherin and Erb-B4. In such embodiments, the modulation can be such that the levels of an infracellular carboxyl domain fragment of Notch, E-cadherin and/or Erb- B4 are substantially unchanged. In particular embodiments of the methods for modulating Aβ levels that include a step of modulating the cleavage of APP that produces one or more Aβ peptides, the processing of APP, the processing of Aβ and/or the level of one or more Aβ peptides without substantially altering the cleavage and/or processing of LRP and/or portion(s) thereof, the modulation can be such that the level and/or presence or absence of one or more fragments of LRP (and/or a portion(s) thereof) is substantially unchanged. In one embodiment the presence, absence and/or level of an -20 kD fragment of LRP is substantially unchanged. The fragment can be one that (a) contains an amino acid sequence that is contained within a fransmembrane region of LRP, (b) binds with an antibody generated against a C-terminal amino acid sequence of an LRP (e.g. , the C- terminal 13 amino acids of an LRP), (c) contains an amino acid sequence located within the sequence of amino acids from about amino acid 4420 to about amino acid 4544 of SEQ ID NO: 10, (d) is present when an LRP is not cleaved by a presenilin-dependent activity, and/or (e) occurs in the presence of an inhibitor (e.g., DAPT) of a presenilin- dependent activity. In one embodiment, the modulation can be such that the level and/or presence or absence of one or more C-terminal fragments (CTF) of LRP (and/or a portion(s) thereof) is substantially unchanged. hi any of the methods for modulating Aβ levels provided herein, the modulating can be effected by any method, including, but not limited to, contacting a sample with an agent that modulates the cleavage of APP that produces one or more Aβ peptides, the processing of APP, the processing of Aβ and/or the levels of Aβ such that the level of one or more Aβ peptides, such as, for example, Aβ42, is modulated as described herein. An agent may be, for example, any agent identified using the methods provided herein for identifying agents that modulate Aβ. Agents include those that modulate the level, functioning and/or activity of one or more proteins involved in modulating Aβ. Proteins involved in modulating Aβ can be, for example, APP processing enzymes, Aβ processing enzyme, receptors or modulatory proteins thereof. In particular examples, the concentration of the agent is less than or equal to about 35 μM, 30 μM, 25 μM, 20 μM, 15 μM or 10 μM. For example, the concentration of agent is less than or equal to about 30 μM. In one embodiment, the agent reduces Aβ42 levels with an ICso of about 25 μM or less or about 20 μM or less.

I. Antibodies and Proteins that bind Aβ

Provided herein are antibodies and methods of preparing antibodies which are specifically reactive with Aβ. Also provided are proteins engineered to bind Aβ. Such antibodies and Aβ binding proteins can be used in applications such as, but not limited to, diagnostic purposes, research purposes, and in freatment of Aβ-related diseases and conditions. For example, Aβ binding proteins can be used as reagents for the assays and kits described herein for the detection of the modulation or processing of APP. Antibodies and antibody fragments described herein for use in immunological detection of Aβ, such as those used in assays to monitor APP processing and modulation, can also be used in other applications such as diagnostic purposes, research purposes, and in freatment of Aβ-related diseases and conditions. Aβ binding proteins including Aβ antibodies can also be used as candidate agents as described herein for modulating Aβ levels. 1. Aβ Antibodies

Aβ antibodies provided herein are specifically reactive with Aβ. hi one embodiment, antibodies which are specifically reactive with Aβ recognize the N-terminal region of Aβ. Antibodies which recognize the N-terminal region of Aβ can be prepared by immunizing a host animal with a peptide containing the sequence of the N-terminal region of Aβ. For example, a peptide containing the sequence of amino acids 1-12 of SEQ ID NO: 4 or a fragment thereof is used to immunize mice and generate monoclonal antibodies as described herein or by method known in the art. An exemplary antibody is the Aβ antibody Aβl-12, referred to herein as B436.

In another embodiment, antibodies are prepared which recognize only a particular Aβ or a selective number of Aβ peptides. Antibodies can be prepared by immunizing a host animal such as a mouse with portions of Aβ specific for the species of interest. For example, as described herein, antibodies can be generated which recognize only Aβ42 with minimal or no binding to other Aβ peptides, such as Aβ40. An Aβ antibody selective for Aβ42 can have at least about 100-fold, 200-fold, 300-fold, 400-fold, 500- fold, 600-fold, 700-fold, 800-fold, 900-fold, 1000-fold or more specificity or selectivity for Aβ42 relative to other forms of Aβ, such as Aβ40. hi addition, the antibody can have an affinity constant for binding to Aβ42 of at least about 10 1/mol, 2 x 10 1/mol, 3 x 10 1/mol, 4 x 10⁵ 1/mol, 5 x 10⁵ 1/mol, 6 x 10⁵ 1/mol, 7 x 10⁵ 1 mol, 8 x 10⁵ 1/mol, 9 x 10⁵ 1/mol, 10 1/mol, 2 x 10 1/mol, 3 x 10 1/mol or 4 x 10 1/mol or more. An exemplary antibody is the Aβ antibody selective for Aβ42, referred to herein as A387.

Aβ antibodies can be produced which recognize some or all forms of Aβ, for example Aβ in soluble form, such as in low molecular weight forms, in plaques and in neurofibrillary tangles. Aβ antibodies can also be produced which recognize only a specific Aβ, for example Aβ42, and in some cases are also specifically reactive with specific forms of Aβ, for example Aβ42 in soluble form such as Aβ42 in plasma and Aβ42 in low molecular weight forms. Aβ antibodies which recognize only specific Aβ peptides, and/or are specifically reactive with specific forms of Aβ can be used to ascertain the form(s) and types of Aβ peptides in a sample, for purposes of diagnosis, such as in methods described herein or known in the art. Such Aβ antibodies can be used for treatment for example, where a predominant form and/or Aβ peptide is associated with an Aβ-related condition or the modulation of a form and/or a particular Aβ is effective for freatment.

Antibodies can be prepared using a variety of methods well-known in the art. For example, as described herein, a target epitope such a peptide, peptide fragment or synthetic peptide may be prepared and used to immunize a host animal. As further described herein, monoclonal antibodies can be prepared, cell lines producing monoclonal antibodies can be isolated and the nucleic acid sequence encoding the monoclonal antibodies as well as the amino acid sequence of the antibodies can be obtained. An antibody can be any derivative of an immunoglobulin. Aβ antibodies include antibodies that are less than full-length, e.g. antibody fragments, retaining at least a portion of the full-length antibody's specific binding ability. Examples of such antibodies include, but are not limited to, Fab, Fab', F(ab)₂, single-chain Fvs (scFv), Fv, dsFv and diabody fragments. Antibodies can include multiple chains linked together, such as by disulfide bridges. Antibodies can be prepared enzymatically and by recombinant DNA technology.

(a) Fab and F(ab)₂ fragments

Fab fragments are antibody fragments that can be produced from digestion of an immunoglobulin with papain. A Fab fragment contains a complete light chain paired with the variable region and the CHI region of the heavy chain. Recombinant means such as expression in a host cell, synthetic production or in vitro expression systems can also be used to produce Fab fragments of similar or equivalent structure to Fab fragments produced by enzymatic digestion .

Fab fragments can be generated which are specifically reactive with Aβ or with particular Aβ peptides. In one embodiment, an Fab recognizes all or most Aβ peptides. For example, an Fab is produced which recognizes the N-terminal amino acids of Aβ such as an Fab generated from the antibody B436 or an Fab produced using the sequence or a portion of the sequence of the B436 antibody.. In another embodiment, an Fab is specifically reactive with a specific Aβ, for example, Aβ42. Fab fragments can be produced by enzymatic means. For example, an Fab can be generated from Aβ antibodies such as A387 and/or B436 by isolating immunoglobulin from antibody producing cells, such as described in the examples herein or by methods known in the art. Fab antibodies are generated by cleaving the A387 and/or B436 immunoglobulin molecules with papain. In another embodiment, Fab molecules are generated from Aβ antibodies such as

A387 and/or B436 by recombinant means using the sequences of the light and heavy chain variable regions and mimicking the papain cleavage by constructing the polypeptides of the heavy and light chain variable domains to have the same or similar (within 1 or more amino acids in length difference) amino acid sequences. For example, A387 Fab molecules can be constructed containing the amino acid sequences or a portion thereof, of SEQ ED NOs: 12 and 14. In one aspect of the embodiment, nucleic acid molecules containing the sequence or a portion thereof of SEQ ED NO: 11 and/or 13 are used to construct an Fab antibody. B436 Fab molecules can be constructed containing the amino acid sequences or a portion thereof, of SEQ ED NOs: 16 and 18. In one aspect of the embodiment, nucleic acid molecules containing the sequence or a portion thereof of SEQ ED NO: 15 and/or 17 are used to construct an Fab antibody.

An F(ab)₂ fragment is an antibody fragment that can be produced from digestion of an immunoglobulin with pepsin at pH 4.0-4.5. An F(ab)₂ fragment contains both light chains associated with the variable regions and the CHI regions of the two heavy chains. Disulfide bridges link the two antigen binding arms of the F(ab)₂ fragment. Recombinant means such as expression in a host cell, synthetic production or in vitro expression systems can also be used to produce F(ab)₂ fragments of similar or equivalent structure to F(ab)₂ fragments produced by enzymatic digestion.

F(ab)₂ fragments can be produced which are specifically reactive with Aβ and/or specific Aβ peptides. In one embodiment, an F(ab)₂ fragment recognizes the N-terminal amino acids of Aβ such as an F(ab)₂ from the antibody B436 or an F(ab)₂ produced using the sequence or a portion of the sequence of the B436 antibody. In another embodiment, an F(ab)₂ is specifically reactive with a specific Aβ, for example, Aβ42. For example, an F(ab)₂ is generated from the antibody A387 or an F(ab)₂ is produced using the sequence or a portion of the sequence of the A387 antibody.

F(ab)₂ fragments can be produced by enzymatic means. For example, an F(ab)₂ can be generated from Aβ antibodies such as A387 and/or B436 by isolating immunoglobulin from antibody producing cells, such as described in the examples herein or by methods known in the art. F(ab)₂ antibodies are generated by cleaving the A387 and/or B436 immunoglobulin molecules with pepsin.

In another embodiment, F(ab)₂ molecules are generated from Aβ antibodies such as A387 and/or B436 by recombinant means using the sequences of the light and heavy chain variable regions and mimicking the pepsin cleavage by constructing the polypeptides of the heavy and light chains to have the same or similar (within 1 or more amino acids in length difference) amino acid sequences. For example, A387 F(ab)₂ molecules can be constructed containing the amino acid sequences or a portion thereof, of SEQ ED NOs: 12 and 14. In one aspect of the embodiment, nucleic acid molecules containing the sequence or a portion thereof of SEQ ED NO : 11 and/or 13 are used to construct an F(ab)₂ antibody. B436 F(ab)₂ molecules can be constructed containing the amino acid sequences or a portion thereof, of SEQ ED NOs: 16 and 18. In one aspect of the embodiment, nucleic acid molecules containing the sequence or a portion thereof of SEQ ED NO: 15 and/or 17 are used to construct an F(ab)₂ antibody.

(b) Fv and dsFv fragments

An Fv antibody fragment is composed of one variable heavy domain (VH) and one variable light domain linked by noncovalent interactions. Fv fragments can be generated by recombinant DNA technology produce the variable domains of the heavy and light chains, for example in a host cell, or by synthetic means. In one embodiment, an Fv fragment is generated from the A387 by recombinant means using nucleotide sequences encoding the heavy chain and light chain variable domains set forth in SEQ ED NO: 12 and 14. In one aspect of the embodiment, nucleic acid molecules containing the sequence or a portion thereof of SEQ ED NO: 11 and/or 13 are used to construct an Fv fragment.

In another embodiment, Fv fragments are generated by recombinant means using nucleotide sequences encoding the heavy chain and light chain variable domains set forth in SEQ ED NO: 16 and 18. h one aspect of the embodiment, nucleic acid molecules containing the sequence or a portion thereof of SEQ ED NO: 15 and/or 17 are used to construct an Fv fragment.

A dsFV refers to an Fv with an engineered intermolecular disulfide bond, which stabilizes the VH-VL pair. Chain dissociation may be prevented by introducing Cys residues at appropriate locations into the framework of VH and VL in order to form a disulphide crosslink (Glockshuber et al, 1990; Reiter et al, 1996). dsFv molecules can be generated by recombinant means to produce dsFv antibodies from A387 and/or B436. For example, cysteines can be engineered into the sequence of the heavy and light chains to provide a disulfide bond between them. dsFvs can then be generated by enzymatic or by recombinant means .

(c) ScFvs and diabodies scFvs refer to antibody fragments that contain a variable light chain (VL) and variable heavy chain (VH) covalently connected by a polypeptide linker in any order. The linker is of a length such that the two variable domains are bridged without substantial interference. Included linkers are (Gly-Ser)_n residues with some Glu or Lys residues dispersed throughout to increase solubility. scFvs are generated by recombinant means and may be produced synthetically, in vivo, such as by expression in a host cell or fransgenic organism, or using in vitro systems known in the art. scFvs can be advantageous because of the smaller size. scFvs can be generated which are specifically reactive with to Aβ or to specific

Aβ peptides. In one embodiment, an scFv is produced which recognizes the N-terminal region of Aβ. For example, an scFv is generated using the sequence of the antibody B436 or a portion thereof, such as the sequence comprising the variable regions of the heavy and light chain of B436. A linker region is used such as those described herein or known in the art to join the variable regions. In another embodiment, an scFvs is generated which recognizes specific Aβ peptide, for example, an scFV which are specifically reactive with Aβ42. In one embodiment, an scFv is generated using the sequence of the antibody A387 or a portion thereof, such as the sequence comprising the variable regions of the heavy and light chain of A387. A linker region is used such as those described herein or known in the art to join the variable regions. For example, an scFv is generated containing the sequence of amino acids or a portion thereof of SEQ ED NO: 12 and/or 14. In another embodiment, an scFv is generated using the sequence of the antibody B436 or a portion thereof, such as the sequence comprising the variable regions of the heavy and light chain of B436. A linker region is used such as those described herein or known in the art to join the variable regions. For example, an scFv is generated containing the sequence of amino acids or a portion thereof of SEQ ED NO: 16 and/or 18. (d) Complementarity-determining Regions (CDRs) Complementarity-determining regions (CDRs) (also referred to as hypervariable regions) refer to regions of an immunoglobulin molecule that vary greatly in amino acid sequence relative to flanking Ig sequences. The length and conformation of CDRs vary among Igs, but generally CDRs form short loops supported by a sandwich of two anti- parallel beta-sheets within the variable regions of the antibody. Three CDRs, designated CDR-Ll, CDR-L2 and CDR-L3, are present in the variable region of an immunoglobulin light chain, and three CDRs, designated CDR-Hl, CDR-H2 and CDR-H3, are present in the variable region of an immunoglobulin heavy chain. Each CDR generally contains at least one, and often several, amino acids residues that make contact with antigen, but all six CDRs are not necessarily required to maintain the binding specificity of an antibody.

Several definitions of CDRs are commonly in use, and CDRs identified according to the different definitions generally overlap, but may differ slightly in their boundaries. The Kabat CDR definition is based on sequence variability among immunoglobulins. The Chothia CDR definition is based on the location of structural loop regions. The AbM CDR definition is a compromise between the Kabat and Chothia definitions used by Oxford Molecular' s AbM antibody modeling software. The contact CDR definition is based on a comparison of the available complex crystal structures.

Taking into account these alternative CDR definitions, some general principles have been devised to identify CDRs based on a given amino acid sequence are shown in Table 3 (see, for example, www.bioinf.org.iik/abs/)-

Table 3

Applying these principles to the antibody sequences disclosed herein, exemplary CDR sequences of the A387 and B436 antibodies can be defined as shown in Table 4.

Table 4

Thus, as used herein, a "CDR of antibody A387" refers to a sequence of amino acids that is a) the same as one of the amino acid sequences set forth in rows 2-7 of Table 4; b) a fragment of SEQ ED NO: 12 or 14 with N- and/or C-terminal boundaries that differ by no more than about 4, 3, 2 or 1 amino acids relative thereto; or c) is at least 60%, 65%, 70%, 80%, 85%, 90%, 95% or more identical to a) or b). A CDR of antibody A387 also includes substitutions within the amino acid sequences of the CDRs set forth in rows 2-7 of Table 4 that when substituted into an A387 antibody do not substantially alter the binding affinity or selectivity of the antibody as compared with the unmodified A387 antibody. Such substitutions can be conservative amino acid substitutions (for example, conservative amino acid changes set forth in Table 2). Generally such substitutions can be for example, 1 amino acid change or 2 amino acid changes within a CDR sequence set forth in rows 2-7 of Table 4. As used herein, a "CDR of antibody B436" refers to a sequence of amino acids that is a) the same as one of the amino acid sequences set forth in rows 8-13 of Table 4 b) a fragment of SEQ ED NO: 16 or 18 with N- and/or C-terminal boundaries that differ by no more than about 4, 3, 2 or 1 amino acids relative thereto; or c) is at least 60%, 65%, 70%, 80%, 85%, 90%, 95% or more identical to a) or b). A CDR of antibody B436 also includes substitutions within the amino acid sequences of the CDRs set forth in rows 8- 13 of Table 4 that when substituted into a B436 antibody do not substantially alter the binding affinity or selectivity of the antibody as compared with the unmodified B436 antibody. Such substitutions can be conservative amino acid substitutions (for example, conservative amino acid changes set forth in Table 2). Generally such substitutions can be for example, 1 amino acid change or2 amino acid changes within a CDR sequence set forth in rows 8-13 of Table 4.

One or more, up to all of the CDRs of an Aβ antibody can be used to bind Aβ or a specific form of Aβ. The CDRs may be produced by recombinant means such as produced synthetically, in vivo, such as by expression in a host cell or fransgenic organism, or using in vitro systems known in the art. CDRs may be produced as isolated sequences or may comprise a portion of a larger molecule such as an immunoglobulin, an Fab, F(ab)₂, an scFv , diabody or a chimeric polypeptide. Multimerization of antibody fragments or antibody domains can be used increase the avidity of such molecules for Aβ and/or specific Aβ peptides and/or forms of Aβ. Chemical means, such as by crosslinking or disulfide bond formation can be used to generate multimeric forms of antibodies. Recombinant means can also be used, for example by constructing repetitive domains or by introducing functionalities which can then be used for cross-linking or association by other means.

2. Engineering Aβ binding proteins Antibodies or regions thereof, such as CDRs, can be engineered to generate Aβ binding proteins which bind Aβ or particular peptides or forms of Aβ. For example Aβ binding proteins can be engineered to optimize the binding to Aβ and/or a particular Aβ and/or specific forms of Aβ, to optimize attributes for specific uses such as treatment or diagnostic methods, optimize attributes for production or other desirable characteristics. In one embodiment, an Aβ binding protein is generated which binds to a particular Aβ and/or binds selectively to one or more Aβ peptides. For example, an Aβ binding protein is engineered to retain substantially the same binding properties as an Aβ antibody. In one embodiment, an Aβ binding protein is engineered to retain substantially the same binding properties as the A387 antibody. In another embodiment, an Aβ binding protein is engineered to retain substantially the same binding properties as the B436 antibody.

As described herein, Aβ binding proteins can be generated which recognize only Aβ42 with minimal or no binding to other Aβ peptides, such as Aβ40. An Aβ binding protein selective for Aβ42 can have at least about 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1000-fold or more specificity or selectivity for Aβ42 relative to other forms of Aβ, such as Aβ40. In addition, the Aβ binding protein can have an affinity constant for binding to Aβ42 of at least about 10⁵ 1/mol, 2 x 10⁵ 1/mol, 3 x 10⁵ 1/mol, 4 x 10⁵ 1/mol, 5 x 10^s 1 mol, 6 x 10⁵ 1/mol, 7 x 10⁵ 1/mol, 8 x 10⁵ 1/mol, 9 x 10⁵ 1/mol, 10⁶1/mol, 2 x 10° 1/mol, 3 x 10⁶1/mol or 4 x 10⁶1/mol or more.

Aβ binding proteins can be generated for example, from portions of antibodies that recognize Aβ can be engineered into other protein scaffolds. Nucleic acid molecules encoding such portions along with nucleic acid molecules encoding scaffolds can be used to construct Aβ binding proteins including Aβ antibodies using standard molecular biology techniques known to one skilled in the art. Exemplary nucleic acid molecules include but are not limited to SEQ ID NOs. 11, 13, 15, 17, 97, 98, 99 and 100. Additionally, nucleic acid molecules can be generated by reverse translating Aβ binding protein amino acid sequences. For example, a nucleic acid sequence is derived from a portion of an Aβ antibody, such as a CDR amino acid sequence. There are a number of possible nucleic acid sequences based on the degeneracy of codons which can be used for each amino acid. However, for the purposes of constructing Aβ binding proteins, any nucleic acid sequence which encodes the amino acid sequence can be used for constructing an Aβ binding protein. Nucleic acid molecules encoding Aβ binding proteins, antibodies or portions thereof can be mutagenized to alter binding characteristics. Additional functionalities such as detectable moiety or a therapeutic moiety can be added to Aβ binding proteins and antibodies. Protein and peptide chemistry can also be used to construct Aβ binding proteins.

(a) Scaffolds A scaffold refers to a structure that forms a conformationally stable structural support, or framework, which is able to display one or more sequences of amino acids, such as a CDR, a variable region or a binding domain, in a localized surface region. A scaffold may be a naturally occurring polypeptide or polypeptide "fold" (a structural motif), or may have one or more modifications, such as additions, deletions or substitutions of amino acids, relative to a naturally-occurring polypeptide or fold. A review of protein scaffolds and their uses can be found in Skerra (2000) J. Mol Recognition 13:167-187. i. Antibody Scaffolds Immunoglobulins comprise a natural type of biomolecular scaffold. Aβ binding proteins can be engineered based on immunoglobulin molecules or portions thereof including, CDR grafting, humanized antibodies, single Ig and Ig-like scaffolds and antibody fragments such as Fvs, scFvs, Fabs, and F(ab)₂s.

Accordingly, provided herein are antibodies and antibody fragments for use as antibody scaffolds. Such scaffolds can contain the heavy and/or light chains of an immunoglobulin or portions thereof. In one embodiment, an antibody scaffold is constructed from a heavy chain. The heavy chain can be from an Aβ antibody such as from A387 or B436 or from any heavy chain known in the art. In another embodiment, an antibody scaffold is constructed from the constant region of one antibody and the variable region from an Aβ antibody. For example, the C region can contain the sequence of amino acids set forth in SEQ ED NOs: 69, 71, 83, 85 or 87 and the variable region can contain the amino acids of SEQ ED NO 14 or 18 or a portion thereof. A joining region can be used from either an Aβ antibody or from an antibody known in the art. Exemplary joining regions are described herein. In a particular embodiment, an antibody scaffold contains a variable region containing the sequence of amino acids 1-97 of SEQ ED NO:14 or 1-98 of SEQ ED NO: 18. In another embodiment, an antibody scaffold is constructed from a light chain. The light chain may be from an Aβ antibody or from any light chain known in the art. In another embodiment, an antibody scaffold is constructed from the constant region of one light chain and the variable region from an Aβ antibody. For example, the C region can contain the sequence of amino acids set forth in SEQ ED NOs: 63, 65 or 81 and the variable region can contain the amino acids of SEQ ID NO 12 or 16, or a portion thereof. A joining region can be used from either an Aβ antibody or from an antibody known in the art. Exemplary joining regions are described herein. In a particular embodiment, an antibody scaffold contains a variable region containing the sequence of amino acids 1-95 of SEQ ED NO: 14 or 1-100 of SEQ ED NO: 16. Heavy and light chains can also be constructed containing a portion of an antibody known in the art and a portion of an Aβ antibody, for example by grafting the variable domain of an Aβ heavy chain, the DJ region and a portion of the C domain to another heavy chain containing the remainder of the C domain, thereby reconstructing a heavy chain. In another example, a light chain can be constructed by the variable domain of an Aβ light chain, the J region and a portion of the C domain to another light chain containing the remainder of the C domain, thereby reconstructing a light chain

Antibody scaffolds can be constructed for Fab, F(ab)2, Fvs, dsFvs, diabodies and other antibodies by methods as described herein or known in the art. Scaffolds for antibodies can also be constructed by utilizing other antibodies known in the art and altering the binding specificity such that antibodyrecognizes Aβ. For example, the variable region or a portion thereof can be grafted onto the antibody or used to replace the equivalent region within the scaffold. Single CDR regions can be grafted and/or used for replacement as well as all of the CDR regions of the light chain and/or heavy chain or any combination thereof. Mutagenesis can also be used to alter the binding specificity of an existing antibody such that it binds Aβ. Antibody scaffolds can also be used to generate antibodies with the specificity from one antibody and the properties of another, such as reduced immunogenicity when administered in a particular animal species. Monoclonal antibodies are most often generated in non-human species, such as mice. Humanized antibodies can be generated where at least one portion of the antibody structure is of human origin. For example, a humanized antibody can be comprised of the antigen binding regions from an antibody generated in a mouse with the remainder of the antibody framework derived from a human antibody (see, for example, Hurle and Gross, Curr Opin Biotechnol. 1994 Aug;5(4):428-33). The generation of humanized antibodies includes the methods referred to in the art as CDR-grafting. Humanized antibodies can be prepared by synthetic methods or through recombinant DNA methods well known in the art.

Accordingly, provided herein are humanized antibodies which bind to Aβ. In one embodiment, one or more CDRs of an Aβ antibody is grafted onto a human antibody framework such as an Fab and scFv framework. For example, one or more of the CDRs of the Aβ antibody A387 is grafted onto a human antibody framework to create a humanized Aβ antibody. A387 CDRs can be any one or more than one of the CDRs listed in Table 4 for A387 including A387 CDR LI, L2, L3, HI, H2 and H3 in any combination. A387 CDRs also include fragments of the amino acid sequences set forth in SEQ ED NO: 12 and SEQ ED NO: 14. In another embodiment, one or more of the CDRs of the Aβ antibody B436 is grafted onto a human antibody framework. B436

CDRs can be any one or more than one of the CDRs listed in Table 4 for B436 including B436 CDR LI, L2, L3, HI, H2 and H3 in any combination. B436 CDRs also include fragments of the amino acid sequences set forth in SEQ ED NO: 16 and SEQ ED NO: 18. In one embodiment, the humanized antibodies contain the 6 CDRs of an Aβ antibody, for example, a humanized antibody with the 6 CDRs of A387. In another example, a humanized antibody contains the 6 CDRs of antibody B436.

Any human antibody framework known in the art can be used to prepare humanized antibodies. For example, a human framework can be a human scFv antibody, a human Fab fragment, a human light chain, a human heavy chain or a full immunoglobulin structure comprised of both a heavy and a light chain. Exemplary human immunoglobulin regions useful in constructing scaffolds are those such as, but not limited to, polypeptides set for the in SEQ ED NOs: 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, and 91.

Additionally, a human antibody framework may be optimized for example to improve solubility properties or increase production in a host. For example, a camelized version of a human VH domain can be constructed as a human antibody fragment or as a portion of a larger human antibody framework (see for example, Davies and Riechmann (1995) Bio/technology 13:475-479 and Davies and Riechmann (1996) Prot. Eng 9:531- 537). CDR grafting can be used to engineer Aβ binding proteins in Ig chain scaffolds such as single Ig and Ig-like scaffolds. For example, camelid antibodies are heavy chain antibodies which are devoid of light chains so that their V_H domains remain soluble without dimerization. An Aβ binding protein can be constructed, for example, by grafting one or more of the CDRs of an Aβ antibody into the camelid antibody structure. Human and murine variable domains have been described, which do not depend on the association with another domain and can be used to create a single Ig-like scaffold for an Aβ binding protein. An additional small Ig-like framework is the minibody, for example, based on the heavy chain variable domain of an antibody comprising three strands from each β-sheet and having regions that structurally correspond to CDR-Hl and CDR-H2. Minibodies also generally contain a metal-binding site and solubilizing tri-lysine motifs at the N- or C-termini (Bianchi et al. (1994) J. Mol Biol. 236:649-659). Isolated VH domains containing CDR1 and CDR2 and associated framework can also be used (Davies et al. ,(1995) Biotechnology 13 :475-479) CDR regions of an Aβ antibody such as the CDR-Hl and CDR-H2 regions from the A387 or B436 antibodies can be used to construct Aβ minibodies. An example of a single Ig-like scaffold is the fibronectin type HI domain (FN3) which constitutes a small, monomeric natural β-sandwich protein with resemblance to a trimmed Ig VH domain. It possesses seven β-sfrands with three loops connecting the strands in a pairwise fashion at one end of the β-sheet. The loops can be replaced with one or more CDRs from an Aβ antibody to create an Aβ binding protein with a fibronectin scaffold. FN3 domains are found in numerous binding proteins, such as cell adhesion molecules, cell surface hormone and cytokine receptors, chaperonins and carbohydrate-binding proteins, and generally contain seven β-strands with three loops connecting the strands in a pairwise fashion at one end of the β-sheet. An exemplary FN3 domain scaffold is derived from the tenth FN3 repeat in human fibronectin (Koide et al. (1998) J. Mol Biol. 284:1141-1151; WO 98/56915; WO 02/04523). Another example of a single Ig-like domain scaffold is the V-like domain of the human cytotoxic T-lymphocyte associated protein-4 (CTLA-4) (Nutall et al. (1999) Proteins Struct. Funct. Genet. 36:217-227). ii. Other Polypeptide Scaffolds Beyond antibody scaffolds, other proteins with suitable architecture can be used as scaffolds to create Aβ binding proteins. Many of these proteins have defined folds and loops that are appropriate for insertion or replacement with Aβ binding regions such as one or more CDRs of an Aβ antibody. A scaffold may be derived from a polypeptide of any species (or of more than one species), such as a human, other mammal, other vertebrate, invertebrate, plant, bacteria or virus or may be generated by rational design (e.g. an artificial scaffold).

Protease inhibitors generally have a binding site that comprises an exposed loop in a context of a structural framework that is specific for the inhibitor family and thus can be employed as a scaffold for a structurally constrained peptide loop Roberts et al. (1992) Proc. Natl. Acad. Sci. USA 89:2429-2433; Markland et al. (1996) Biochemistry 35:8045- 8057; McConnell and Hoess (1995) J. Mol. Biol. 250:460-470). Protease inhibitor scaffold include but are not limited to scaffolds from Bovine (or basic) pancreatic trypsin inhibitor, BPTI, the Kunitz domain of human lipoprotein-associated coagulation inhibitor (LACI-D1), human pancreatic secretory trypsin inhibitor (PSTI), bacterial serine protease inhibitor ecotin, and Tendamistat. The exposed loop may be replaced by one or more CDRs of an Aβ antibody to create an Aβ binding protein.

Helical bundle proteins can also be used as scaffolds (Braisted and Wells (1996) Proc. Natl. Acad. Sci. USA 93:5688-5692; Ku and Schultz (1995) Proc. Natl. Acad. Sci. USA 92:6552-6556). For example, an engineered single domain, called 'Z', of Staphylococcal protein A has a simple fold as a bundle of three cϋ-helices. It is highly soluble and stable against proteolysis and heat-induced unfolding. Another example is cytochrome bsβ2, with four-helix bundle proteins providing rigid framework and two loops, each connecting one pair of the α-helices. Artificial helical bundle scaffolds are also available. One of more CDR regions from an Aβ antibody can be grafted into the helical structure for example, into the loop regions between one or more of the helices to create an Aβ protein.

An additional scaffold is the β-barrel which is made of antiparallel β-sfrands winding around a central axis with loops connecting the strands at the open end of the resulting conical structure. For example, the β barrel framework of lipocalins (Muller and Skerra (1994) Biochemistry 33: 14126-14135) may be used such as by grafting of a domain onto the solvent-exposed outer surface of the β-barrel. One or more CDRs of an Aβ antibody can be grafted onto a lipocalin scaffold. Examples of lipocalin scaffold include but are not limited to retinol-binding protein (RBP), bilin binding protein (BBP), apolipoprotein D, tear lipocalin and β-Trace, also known as prostaglandin D synthase. Many lipocalins based on their human framework and natural presence in human body fluids are suitable both for diagnostic and therapeutic purposes.

Knottins (Le Nguyen et al, 1990) comprise a structural family defined by a small triple-stranded antiparallel β-sheet stabilized by an arrangement of disulphide bonds. Members of the knottin family include the trypsin inhibitor EETI-II from Ecballium elaterium seeds, the neuronal N-type Ca channel blocker (ω-conotoxin from the venom of the predatory cone snail Conus geographus, and the C-terminal cellulose-binding domain (CBD) of cellobiohydrolase I from the fungus T. reesei. Loop structures within the Knottins can be used for insertion of or replaced with one or more CDR sequences to form Aβ binding proteins. Other structural folds that may be suitable as scaffolds include TIM barrels, which are found, for example, in triose phosphate isomerase proteins (Altamirano et al. (2000) Nature 403:617-622); GST enzyme frameworks, pleckstrin homology domains, zinc finger domains and β-prism motifs.

Exemplary modifications to a polypeptide that may make it suitable for use as a scaffold include deletions of those regions that form binding loops in the naturally- occurring molecule (e.g. deletions of the naturally-occurring binding sites); deletions of those regions that are unnecessary for structural integrity of the fold; substitutions of amino acids that flank the loop regions with residues that improve the properties of the polypeptide (such as improved affinity, specificity, or solubility; reduced immunogenicity, etc.); addition of detectable sequences, such as epitope tags; and the like. iii. Non-polypeptide Scaffolds Aβ antibodies and Aβ binding proteins, fragments thereof, such as a CDR, can also be displayed on a scaffold such as a solid support. ^' Such scaffolds are useful in applications including but not limited to, diagnostic assays, screening assays, and cellular delivery of polypeptides.

Solid supports include but are not limited to membranes, filters, chips, slides, wafers, fibers, magnetic or nonmagnetic beads, gels, tubing, plates, polymers, microparticles and capillaries. A solid support can be composed of any material that allows for the immobilization or attachment of molecules, such that these molecules retain their desired properties, such as binding ability. Examples of materials include silica, polymeric materials or glass. Solid supports can be used to display Aβ binding proteins, antibodies and fragments thereof, for example for screening purposes, diagnostic purposes, protein purification and binding assays. Additionally, solid supports such as beads and particles can be used to deliver Aβ binding proteins and antibodies to cells, animals and subjects. Aβ binding proteins, antibodies and fragments thereof can be associated with solid supports covalently such as by chemical linkage or by non-covalent interactions such as by charge interactions, interactions with other proteins or small molecules. (b) Mutagenesis of Aβ binding regions

As described herein, Aβ binding proteins can be constructed from Aβ binding regions such as Aβ antibodies and antibody fragments including one or more CDRs. Properties of such Aβ binding proteins can be altered or optimized. For example properties such as binding affinity, binding specificity, solubility, aggregation and stability can be optimized for particular applications. Mutagenesis techniques such as site-directed mutagenesis, random mutagenesis including random mutagenesis of discrete regions of Aβ binding proteins and other methods known in the art can be used to generate variations within the Aβ binding regions, or at one or more junctions between the Aβ binding regions and the scaffold. The variants can then be screened for Aβ binding by methods such as described herein or known in the art and variants with improved binding affinities or binding affinities optimized for particular applications such as diagnostics or freatment regimes can be isolated. For example, one or more CDRs of an Aβ antibody such as the CDRs of A387 and/or B436 can be mutagenized and then the variants generated are tested for Aβ binding. Random mutagenesis or directed conservative amino acid changes can be made in one or more CDRs. The variants can also be tested for selective binding to one or more specific Aβ peptides such as binding to Aβ42, or Aβl-12. The variants can be screened to assess for their binding to specific forms of Aβ. For example, variants can be assayed for their binding to Aβ in plasma, cerebral spinal fluid (CSF), plaques, and neurofibrillary tangles as well as in low molecular weight and high molecular weight forms.

Variants can also be assessed for properties other than binding to Aβ. For example, variants can be isolated which are more soluble when produced synthetically or in a host by recombinant means. Variants can also be isolated which exhibit altered stability, for example increased stability or alternatively higher turnover. Such variants can be produced by mutagenizing regions outside the Aβ binding regions for example in the scaffold, antibody framework or other domains which are part of the Aβ binding protein. Such variants can also be produced by mutagenizing the Aβ binding regions or the entire Aβ binding protein and then screened for retention of Aβ binding as one of the criteria for selecting a variant.

(c) Clearance domains A clearance domain directly or indirectly mediates enhanced clearance of a polypeptide from the circulation. A polypeptide containing a clearance domain will have a shorter half-life in the circulation, alone and/or when bound to Aβ, than a polypeptide without such a domain. Clearance mechanisms include receptor-mediated intemalization by specialized cells, such as macrophages or macrophage precursors, endothelial cells lining the sinusoids of the liver, spleen, and bone marrow, and reticular cells of lymphatic tissue and of bone marrow. Examples of receptors that mediate clearance of polypeptides in the circulation include Fc-γreceptor(s), which bind IgG-antigen complexes; lipoprotein receptors (e.g. LDL receptor-related protein receptor (LRP), LDL receptor and VLDL receptor); scavenger receptors (e.g. LRP, LDL-receptor, SR-A, SR- BI, CD36, etc.), which bind many different classes of serum macromolecules; hyaluronan receptors, which bind matrix proteoglycans; collagen alpha-chain receptors, which bind collagen alpha-chains; mannose receptors, which bind carboxy-terminal propeptides of type I procollagen and tissue plasminogen activator; and the like. A clearance domain can thus be a ligand for a receptor that mediates clearance, such as a polypeptide or fragment thereof that binds a receptor type mentioned above.

An example of a clearance domain is a ligand for an Fc receptor. There are several Fc receptors (FcR), including FcγRI, FcγRIJ, FcγRHI, and the neonatal Fc receptor (FcRn), which bind IgG antibodies. An Fc receptor ligand can be the Fc portion of an IgG (i.e. the portion containing the carboxy termini of the two heavy (H) chains, when an antibody is cleaved with papain), or a fragment thereof that retains Fc receptor binding. The antibody portions involved in Fc receptor binding are known in the art or can be determined by receptor binding assays known in the art. For example, the lower hinge and the adjacent region of the CH2 domain of IgG Fc are involved in binding to FcγRUa, whereas the Fc CH2-CH3 interface is involved in binding to FcγRIIb and FcRn (Wines et al. (2000) J. Immunol. 164:5313-5318). Exemplary clearance domains are the Fc domain of an IgGl human or an Fc domain of antibody IgG2a mouse antibody. Another example of a clearance domain is a ligand for LRP. At least 30 molecules that bind LRP are known in the art, including, for example, APP, ApoE, alpha-2-macroglobulin, tPA, blood coagulation factors, lactoferrin, CI inhibitor, pregnancy zone protein, thrombospondins, complement C3, and the like (see Herz and Strickland (2001) J. Clin. Invest. 108:779-784). The portions of these proteins that bind LRP are known in the art, or can be determined by LRP binding assays known in the art (see, for example, U.S. Patent No. 6,472,140, which describes LRP-binding fragments of alpha-2-macroglobulin that comprise residues 1366-1392 of human alpha-2- macroglobulin). Any of these molecules, of portions thereof that bind LRP, can be used as clearance domains.

Provided herein are Aβ binding proteins containing a clearance domain. In one embodiment, an Aβ binding protein comprises an Aβ antibody and an Fc region. The Fc region may originate from the Aβ antibody or the Fc domain may be from another antibody or generated synthetically and joined to the Aβ antibody by recombinant or chemical means. In another embodiment, an Aβ binding protein comprises one or more CDRs from an Aβ antibody and additionally, an Fc clearance domain, for example an Aβ binding protein containing one or more CDRs of an Aβ antibody grafted into a scaffold and an Fc clearance domain. In yet another embodiment, an Aβ binding protein comprises a clearance domain from an LRP ligand.

(d) Additional functionalities Aβ binding proteins can be constructed which comprise additional functionalities such as a moiety for detection or purification of the Aβ binding protein, a therapeutic moiety or an additional domain such as for indirect clearance.

Detectable moieties may be associated with an Aβ binding protein by chemical or recombinant means. For example, a protein domain which can be detected by visible or enzymatic assay can be coupled to an Aβ binding protein. Example of such domains include fluorescent proteins such as green, red and blue fluorescent proteins, β- galactosidase, alkaline phosphatase and others known in the art. A radiolabel may also be coupled to an Aβ binding protein for example, I, I, Bi, mTc, In, Y, or

32 P, such as for detection, imagining, diagnostic and therapeutic purposes.

Additional functional domains can also include indirect or regulated clearance domains. For example, an Aβ binding protein can comprise a biotin moiety and a streptavadin molecule such as galactosylated sfreptavadin can be used for clearance (Govindan et al. Cancer Biother Radiopharm. 2002 Jun;17(3):307-16). 3. Characterizing Aβ antibodiesand Aβ binding proteins (a) Determination of Aβ Binding Antibodies (including antibody fragments) and Aβ binding proteins described herein can be assayed by any method known in the art for assessing binding to Aβ. Methods to assess binding include assays such as ELISA, western blotting, immunoprecipitation, two hybrid assays, phage display and others well known in the art. Binding assays can be used to ascertain if the prepared antibody or Aβ binding protein binds to Aβ. Binding assays can also be used to ascertain if the antibody or Aβ binding protein binds selectively to a particular Aβ. Aβ antibodies and binding proteins can be tested against a specific Aβ to determine which are preferentially bound. Peptides tested can include deletion variants of Aβ, including both N and C-terminal truncations of Aβ, as well as deletions within the central region of the Aβ peptide. Such peptides can be used to map the minimal amino acid sequences of Aβ recognized by an Aβ antibody or binding protein. For example, such binding assays can be used to demonstrate that the exemplary antibody A387 binds preferentiallyto Aβ42 with minimal or no binding to other Aβ peptides such as Aβl-40 and Aβl-39.

Methods known in the art can also be used to ascertain the relative binding affinity and avidity of the antibodies and Aβ binding proteins for Aβ and/or various forms of Aβ. For example, Aβ antibodies and binding proteins can be tested using binding assays such as ELISA, dot blots and immunoprecipitation with Aβ in soluble form, aggregates, low molecular weight oligomers, in plaques and neurofibrillary tangles. Such assays can be performed with isolated Aβ peptides or with samples taken from cells and tissues such as those of cell lines, animal models and subjects. Aβ can be solubilized and/or aggregated using in vitro methods such as sonication, and fibril growth in vitro (O'Nuallain et al., (2002) PNAS 99(3): 1485-1490). Additionally, chemical reagents, such as metal chelators, can be used to generate low molecular weight forms of Aβ and then used to assays to assess the reactivity of an Aβ binding protein or Aβ antibody for the low molecular weight forms of Aβ. Assays can also be used to assess binding to specific molecular weight forms of Aβ such as monomers and low molecular weight oligomers or high molecular weight oligomers and aggregates. For example gel filtration and native gels can be used to assess the relative molecular weight or size of Aβ recognized by an Aβ antibody or Aβ binding protein. Western blotting and immunoprecipitation can also be used to assess selectivity of Aβ binding proteins and antibodies for a particular Aβ. For example, as described in Example 9, Aβ can be treated with the metal chelator bathocuprione (BC) and then reacted with an Aβ antibody or Aβ binding protein in subsequent immunoassays. Such assays can be used to screen Aβ antibodies and Aβ binding proteins to isolate those specific for binding Aβ and particular Aβ peptides in a specific form or which bind only to a particular Aβ in a specific form. In one embodiment, antibodies are isolated which bind only to Aβ in low molecular weight forms. In another embodiment, antibodies are isolated which bind to Aβ42 and preferentially bind Aβ42 in low molecular weight forms. An exemplary antibody which binds selectively to Aβ42 and to Aβ42 preferentially in low molecular weight forms is the antibody A387.

(b) Clearance properties Aβ antibodies and Aβ binding proteins can be assessed for their rate of clearance from the circulation using in vivo pharmacokinetic assays and/or in vitro assays that sufficiently correlate with in vivo results. Such assays are well known in the art (see, for example, Shargel and Yu (1999) "Applied Biopharmaceutics and Pharmacokinetics," 4 ed., McGraw-Hill/Appleton & Lange). For example, suitable assays can assess the half- life of the binding protein or antibody, and/or of bound Aβ, in cell-culture medium or blood; the uptake of the binding protein or antibody, and/or of bound Aβ, by a cell, tissue or organ; the intracellular or exfracellular accumulation of degradation products of the binding protein or antibody; and the like.

In one type of in vivo assay, a detectably labeled (e.g. radiolabeled) Aβ binding protein or antibody is administered to a subject, and the decreasing level of label in the circulation, or the increasing level of label in the urine or liver, is monitored to assess the rate of clearance of the Aβ binding protein or antibody from the circulation. In another type of in vivo assay, an unlabeled Aβ binding protein or antibody is injected to a subject, and at various times after dosing, plasma is collected. Various assays can then performed to determine the concentration of administered protein remaining in the circulation. For example, an ELISA assay can be performed, using suitable capture reagents (e.g. Aβ) and detection reagents (e.g. a labeled secondary antibody). Alternatively, a radioimmunoassay (RIA) can be performed, in which the plasma Aβ binding protein or antibody competes for binding of radiolabeled Aβ binding protein or antibody to a suitable secondary reagent. hi one type of in vitro assay, the uptake of detectably labeled Aβ binding protein or antibody from the culture medium by cells having receptors for the clearance domain is assessed. For example, if the clearance domain is a ligand for an Fc receptor, the cells can be macrophages. If the clearance domain is a ligand for LRP, because of the ubiquitous nature of LRP, the cells can be of essentially any tissue origin, such as hepatocytes and fibroblasts. After a suitable incubation period, cells are washed and the amount of infracellular label measured. (c) Purification

Aβ antibody and Aβ binding protein purification may be carried out using standard protein purification techniques. Exemplary methods include ion exchange chromatography, HPLC, and affinity chromatography. Affinity chromatography using Protein A or Protein G. can be used to purify Aβ antibodies and Aβ binding proteins with antibody scaffolds. Affinity chromatography with Aβ peptides can be used to purify proteins which bind Aβ. Aβ antibodies and binding proteins can be generated with purification tags, such as a His₆ tag for metal binding, to facilitate purification. Such tags can be designed to be cleaved after the affinity purification step to produce purified Aβ antibodies and binding proteins. Purification can be assessed by standard methods known in the art such as electrophoresis and staining and mass spectrometry. 4. Expression of Aβ binding proteins

Numerous techniques are known in the art for the design of constructs to express Aβ binding proteins including Aβ antibodies and/or portions thereof. Expression constructs can be used for expression, for example, in vitro or in vivo, in cells, exfracts, tissues or whole organisms. Such constructs are useful for assessing properties of Aβ binding proteins. Additionally, expression constructs are useful in the production of cell lines and fransgenic organisms expressing Aβ binding proteins, including those used in screening methods described herein and known in the art. a. Vectors and Constructs A vector will generally contain elements useful for cloning and/or expression of inserted nucleic acid molecules, such as an origin of replication compatible with the intended host cells; promoter, enhancer and/or other regulatory sequences, which can provide for constitutive, inducible or cell type-specific RNA transcription; transcription termination and RNA processing signals, such as a polyadenylation signal; one or more selectable markers compatible with the intended host cells (e.g. a neomycin or hygromycin resistance gene, useful for selecting stable or transient transfectants in mammalian cells, or an ampicillin or teteacycline resistance gene, useful for selecting fransformants in prokaryotic cells); and versatile multiple cloning sites for inserting nucleic acid molecules of interest. The choice of particular elements to include in a vector will depend on factors such as the intended host cells, the insert size, whether expression of the inserted sequence is desired, the desired copy number of the vector, the desired selection system, and the like. Vectors suitable for use in cloning and expression applications include, for example, viral vectors such as a bacteriophage, adenovirus, adeno-associated virus, herpes simplex virus, vaccinia virus, baculovirus and refrovirus; cosmids or Escherichia coli-deήved , Bacillus subtilis-άeήved and yeast-derived plasmids; bacterial artificial chromosome vectors (BACs) and yeast artificial chromosome vectors (YACs). Such vectors and their uses are well known in the art.

Nucleotide sequences that can be used to express proteins generally contain one or more transcriptional regulatory sequences (e.g. promoters, enhancers, terminators and the like) in operative association with the expressed sequence (e.g. an Aβ binding protein or portion thereof). Promoters for gene expression regulation include, for example, promoters for genes derived from viruses (e.g., cytomegalo virus (CMV), Moloney murine leukemia virus (MMLV), JC virus, rous sarcoma virus (RSV), simian virus SV40, mouse mammary tumor virus (MMTV), etc.), promoters for prokaryotic expression such as T3 and T7 promoters, and promoters for genes derived from various mammals (e.g., humans, rabbits, dogs, cats, guinea pigs, hamsters, rats, mice etc.) and birds (e.g., chickens etc.) (e.g., genes for albumin, insulin π, erythropoietin, endothelin, osteocalcin, muscular creatine kinase, platelet-derived growth factor beta, keratins Kl, K10 and K14, collagen types I and Et, atrial natriuretic factor, dopamine beta- hydroxylase, endothelial receptor tyrosine kinase (generally abbreviated Tie2), sodium- potassium adenosine triphosphorylase (generally abbreviated Na,K-ATPase), neurofilament light chain, metallothioneins I and UA, metalloproteinase I tissue inhibitor, MHC class I antigen (generally abbreviated H-2L), smooth muscle alpha actin, polypeptide chain elongation factor 1 alpha (EF-1 alpha), beta actin, alpha and beta myosin heavy chains, myosin light chains 1 and 2, myelin base protein, serum amyloid component, myoglobin, renin etc.). hαducible promoters such as chemically inducible promoters, for example, regulated by tetracycline, or steroids such as ecdysone, estrogen, or progesterone and others known in the art, may be used for expression.

The above-mentioned vectors can have a sequence for terminating the franscription of the desired messenger RNA in the fransgenic animal (generally referred to as terminator); for example, gene expression can be manipulated using a sequence with such function contained in various genes derived from viruses, mammals and birds. The simian virus S V40 terminator and other known terminators known in the are commonly used. Additionally, for the purpose of increasing the expression of the desired gene, various other elements may be included: e.g., the splicing signal and enhancer region of each gene, a portion of the intron of a eukaryotic organism gene maybe ligated 5' upsfream of the promoter region, or between the promoter region and the translational region, or 3' downstream of the translational region as desired.

Aβ binding proteins can be expressed as a single expression construct or may be expressed as multiple expression constructs. For example, an Aβ antibody comprised of a heavy and light chain can be produced by constructing an expression construct for heavy chain expression and a second expression construct for light chain expression. The two expression constructs may be contained on the same vector or on two separate vectors. They can be integrated together into a host cell or organism or alternatively integrated at different locations. b. Cell culture production Aβ binding proteins including Aβ antibodies and fragments thereof can be expressed in cell culture as a means of producing them for use in diagnostics, research or freatment. Expression in cell culture can also be used as the basis for characterizing and testing Aβ binding proteins and for further screening assays to identify molecules which modulate or alter the interaction between Aβ binding proteins and Aβ.

Nucleic acid molecules can be introduced into host cells by various well-known fransfection methods, including electroporation, infection, calcium phosphate co- precipitation, protoplast or spheroplast fusion, lipofection, micro-injection, and DEAE- dexfran-mediated transfection (e.g. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Press, Plainview, N.Y. (1989); Ausubel et al., supra, (1999), Keown et al. (1990) Methods in Enzymology 185:527-537). Host cells can be maintained and propagated by methods known in the art (e.g. Freshney, R. I. (2000) "Culture of Animal Cells: A Manual of Basic Technique," 4th ed., Wiley-Liss). Any cell line known in the art to be suitable for protein and/or antibody production can be used to produce Aβ binding proteins. Suitable host cells include human and other mammalian cells, including primary cells and cell lines. Exemplary host cells include mammalian primary cells (e.g. cells from any tissue of human, rabbit, dog, cat, guinea pigs, hamsters, rats, mice, etc.); embryonic stem cells, fertilized eggs and embryos; myeloma cells, cells contained in, or obtained from, fransgenic animals; established mammalian cell lines, such as SY5Y, RBL, COS, CHO, HeLa, NIH3T3, HEK 293, BHKBI and Ltk^" cells, mouse monocyte macrophage P388D1, J774A-1 and PC12 cells (available from ATCC, Manassas, VA); amphibian cells, such as Xenopus embryos and oocytes; avian cells; and other vertebrate cells. Exemplary host cells also include insect cells (e.g. Drosophila), yeast cells (e.g. S. cerevisiae, S. pombe, Candida tropicalis, Hansenula polymorph or Pichia pastoris), plant cells and bacterial cells (e.g. E. colϊ).

In some cases it may be desirable to modify the expressed proteins. In vifro can be used to accomplish modifications such as glycosylation, for example galactosylation and sialylation (Raju et al. Biochemistry. 2001 Jul 31;40(30):8868-76). Alternatively, in vivo modification can be accomplished by expression in cell lines which carry out such modifications or by the engineering of cell lines to provide the appropriate modifications (Choi, et al. Proc. Natl Acad Sci U S A. 2003 Apr 29;100(9):5022-7. Epub 2003 Apr 17).

Cell lines for or characterizing and testing Aβ binding proteins and for further screening assays typically include cell lines that produce Aβ, for example primary cell cultures, typically neuronal cell cultures. Totipotent, pluripotent, or other cells that are not terminally differentiated can be induced to express neuronal characteristics including the production of Aβ peptides. Exemplary non-terminally differentiated cells include embryonic stem cells, adult stem cells, mesenchymal stem cells, bone marrow stem cells, adipose tissue stem cells, and neuronal stem cells. Additionally, cells can be engineered to express forms of Aβ of fragments thereof. Examples of such cell cultures, methods for induction of Aβ production, harvesting and culturing are described herein. Aβ binding proteins including Aβ antibodies can be added exogenously to cells expressing Aβ or expression of the Aβ binding proteins can be engineered within the same cell.

Nucleic acid encoding Aβ binding protein and Aβ antibody or portion thereof may be stably incorporated into cells or may be transiently expressed using methods known in the art. Stably fransfected cells may be prepared by fransfecting cells with an expression vector having a selectable marker gene (such as, for example, the gene for thymidine kinase, dihydrofolate reductase, neomycin resistance, and the like), and growing the fransfected cells under conditions selective for cells expressing the marker gene. Transient expression may use similar methods without selectable markers or may use viral expression such as baculovirus, vaccinia virus, adenovirus and other transient systems known in the art. Heterologous nucleic acid may be maintained in the cell as an episomal element or may be integrated into chromosomal DNA of the cell. The resulting recombinant cells may then be cultured or subcultured (or passaged, in the case of mammalian cells) from such a culture or a subculture thereof. Methods for fransfection, injection and culturing recombinant cells are known to the skilled artisan. Expression of an Aβ binding protein mRNA or protein in cells can be assessed by methods known in the art such as Northern blotting, RT-PCR, Taqman, Western Blotting, ELISA, enzymatic function of an Aβ binding protein, and binding or interaction properties of an Aβ binding protein. Methods for protein expression and purification are known in the art (see, for example, Sambrook et al. (1989) MOLECULAR CLONING: A LABORATORY MANUAL 2nd Ed. Cold Spring Harbor Laboratory Press; Ausubel et al. (1995) CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Greene Publishing Co., NY; Rosenberg, I.M. (1996) "Protein Analysis and Purification: Benchtop Techniques" Springer Verlag; and Scopes, R.K. (1994) "Protein Purification: Principles and Practice" Springer Verlag.) Biological compositions can be derived from cell lines such as but are not limited to, purified or partially purified enzyme preparations, conditioned medium from cultured cells, cellular extracts and cell lysates. Such compositions can be generated using methods described herein and/or known in the art for use in characterizing Aβ binding proteins and for further screening assays. c. Transgenic Animals Also provided herein are methods of producing fransgenic animals by introducing nucleic acid encoding an Aβ binding protein into a cell and allowing the cell to develop into a fransgenic animal. The cell may be any cell that may be used in the generation of a fransgenic animal. Such cells are known to those of skill in the art of fransgenic animal production. For example, the cell may be an embryo, zygote, oocyte, fertilized oocyte or embryonic stem cell, such as, for example, a mouse embryonic stem cell. Numerous techniques for introduction of exogenous nucleic acids into cells that will be allowed to develop into fransgenic animals are also known to those of skill in the art. Such techniques include, but are not limited to, pronuclear microinjection (see, e.g., U.S. Patent No. 4,873,191), refrovirus-mediated gene transfer into germ lines [see, e.g., Van der Putten et al. (1985) Proc. Natl. Acad. Sci. U.S.A. §2:6148-6152], gene targeting into embryonic stem cells [see, e.g., Thompson et al. (1989) Cell 5(5:313-321], elecfroporation of embryos [see, e.g., Lo (1983) Mol. Cell. Biol. 3:1803-1814], and sperm-mediated gene transfer [see, e.g, Lavifrano et al. (1989) Cell 57:717-723] [for a review of such techniques, see Gordon (1989) Int. Rev. Cytol 775:171-229]. A cell into which exogenous nucleic acid has been transferred may be introduced into a recipient female animal for development into a transgenic animal containing the exogenous nucleic acid.

Methods for making fransgenic animals using a variety of fransgenes have been described [see, e.g., Wagner et al. (1981) Proc. Nat. Acad. Sc. U.S.A. 75:5016; Stewart et al. (1982) Science 217: 1046; Constantini et al. (1981) Nature 294:92; Lacy et al. (1983) Cell 34:343; McKnight et al. (1983) Cell 34:335; Brinstar et al. (1983) Nature 306:332; Palmiter et al. (1982) Nature 300:611; Palmiter et al. (1982) Cell 29:701, and Palmiter et al. (1983) Science 222:809; Ono et al. (2001) Reproduction 722:731-736; Reggio et al. (2001) Biol. Reprod. 65:1528-1533; Park et al. (2001) Animal Reprod. Sci. (55:111-120; Zakhartchenko et al. (2001) Mol. Reprod. Dev. 60:362-369; Arat et al. (2001) Mol. Reprod. Dev. 60:20-26; Koo et al. (2001) Mol. Reprod. Dev. 55:15-20; Polejaeva and Campbell (2000) Theriogenology 53: 117-126]. Such methods are also described in U.S. Patent Nos. 6,175,057; 6,180,849 and 6,133,502, 6,271,436, 6,258,998, 6,103,523, 6,252,133. A. In vitro and Synthetic systems

Aβ antibodies and Aβ binding proteins and fragments thereof can be produced in vitro in cell-free systems (Makeyev et al. (1999) FEBS let. 444:177-180). Such systems can be useful for rapid screening of constructs and mutants to ascertain function and binding specificity. For example, expressible antibodies and binding proteins can be constructed using PCR techniques to join a T7 or other known RNA polymerase tag onto the nucleotide sequence encoding the polypeptide. In vifro franscription and translation can then be used to express the polypeptides for use in binding or other assays. Single antibodies or binding proteins or libraries of such polypeptides can be produced by such methods. Synthetic means can also be used to produce Aβ antibodies and Aβ binding proteins. For example, regions of Aβ antibodies and Aβ binding proteins can be synthesized in vifro and joined to scaffold molecules. Peptides of one or more CDRs of an Aβ antibody can be synthesized and tested for reactivity with Aβ.

J. Treatment of Disease and Disorders with Aβ binding proteins

Methods are provided herein for the use of Aβ binding proteins and Aβ antibodies in the freatment or prophylaxis of diseases involving or characterized by Aβ and/or specific Aβ forms. . Such diseases include, but are not limited to, diseases involving or associated with amyloidosis and neurodegenerative diseases. One example of such a disease is Alzheimer's disease. Genetic and biochemical evidence indicates that accumulation of Aβ is involved in the pathogenesis of Alzheimer's and further that specific forms of Aβ, such as accumulation into oligomers, aggregates and plaques, participates in the pathogenesis of the disease. Immunization with Aβ peptides as well as passive immunization with Aβ antibodies has been shown to modulate both Aβ levels and related pathogenic and behavioral effects (Holtzman et al. (2002) Adv. Drud Delivery Rev. 54:1603-1613; Dodart et al., (2002) Nature Neurosci. 5(5):452-457; Bard et al., (2003) PNAS 100(4):2023-2028; WO00/72880). The methods are suitable for the freatment or prevention of disease because they are designed to selectively modulate Aβ levels. Methods herein are also provided to modulate the level of a particular Aβ, such as Aβ42.

Methods herein can include a step of administering an Aβ binding protein or Aβ antibody to a subject having such a disease or disorder or predisposed to such a disease or disorder. In one embodiment of the methods, the Aβ binding protein or Aβ antibody being administered is one that modulates the level of one or more Aβ peptides. In one embodiment, Aβ42 levels are modulated. The level of Aβ42 can be modulated to a greater extent than the level of one or more other Aβ peptides, in particular, Aβ40, such that the level of Aβ42 is modulated, or without substantially altering the level of one or more other Aβ peptides, in particular Aβ40. In a particular embodiment, Aβ42 levels are reduced.

In one embodiment, an Aβ binding protein or Aβ antibody being administered is one that preferentially binds a specific form of Aβ such as Aβ in low molecular weight forms. In one aspect of the embodiment, the Aβ binding protein or Aβ antibody is specifically reactive with a specific Aβ, in particular Aβ42, and also preferentially binds low molecular weight forms of Aβ42. In a particular embodiment, the A387 antibody or a fragment thereof is administered. In another embodiment, an Aβ binding protein which retains the binding specificity of the Aβ antibody for low molecular weight forms of Aβ42 is administered. For example, a humanized antibody that preferentially binds low molecular weight forms of Aβ42 is administered. In one embodiment, an antibody containing the sequence of SEQ ED NO: 12 and and/or SEQ ED NO: 14, or portion thereof, is administered.

In another embodiment, an Aβ binding protein or Aβ antibody being administered is one that recognizes the N-terminal region of Aβ. In a particular embodiment, the B436 antibody, or a fragment thereof is administered. In another embodiment, an Aβ binding protein which retains the binding specificity of the B436 antibody for N-terminal region of Aβ is administered. For example, a humanized antibody which retains the binding specificity of B436 is administered. In one embodiment, an antibody containing the sequence of SEQ ED NO: 16 and/or SEQ ED NO: 18, or portion thereof, is administered. 1. Predictive assays Aβ binding assays such as those described herein and known in the art can be used to assess the reactivity of Aβ antibodies and Aβ binding proteins with Aβ. Determination of specificity, affinity, avidity as well as stability and clearance can assist in determining dosages and administration regimes. Assessment of the binding properties of Aβ antibodies and Aβ binding proteins can be ascertained for binding to specific forms of Aβ such as binding to Aβ in soluble or aggregate forms, binding of monomers, low molecular weight oligomers or high molecular weight aggregates. Assays such as those described herein for assessing binding to Aβ and specific Aβ peptides and forms of Aβ, and assays for clearance as well as additional methods known in the art can be used for assessing Aβ antibodies and binding proteins. Animal models can also be used for the assessment of Aβ antibodies and Aβ binding proteins for the treatment of diseases and disorders associated with Aβ for example with altered Aβ levels, and/or altered ratios of one or more Aβ peptides and/or forms, hi particular, non-human animals that have altered production, degradation and/or clearance of Aβ peptides or altered expression of APP can be used for such assays. Examples of such animals include fransgenic animal models and animals, such as rodents, including mice and rats, cows, chickens, pigs, goats, sheep, monkeys, including gorillas, and other primates. Exemplary animal models include animals with the Swedish mutation of APP (Asp595-leu596), disclosedin US Patent Nos. 5,612,486 and 5,850,003, the fransgenic mouse disclosed in US Patent No. 5,387,742, which expresses particular APP species that form β-amyloid protein deposits in the brain of the mouse, and TASD41 fransgenic mice, which express human APP751 cDNA containing the London (V717I) and Swedish (K670M/N671L) mutations under the control of the murine Thy-1 gene (Rockenstein et al. (2001) J. Neurosci. Res. 66:573-582). Additional fransgenic animal models include those described in US Patent Nos. 5,811,633; 6,037,521; 6,184,435; 6,187,992; 6,211,428; and 6,340,783, transgenic mouse models Tg 2576; APPSWE mouse, K670N, M671L, and other models including APP(V717F), APP(K670N, M671L and V717F), PS-1 M146L, PS-1 M146V, APPSWE + PS A246E (reviewed by Emilien, et al, (2000) Arch. Neuro. 57: 176-81).

Aβ antibodies and Aβ binding proteins can be administered, such as by injection, to animal models and the effects of such treatment assessed. For example, animals can > be injected one or more times infraperitoneally, or by other suitable route, with an Aβ antibody or Aβ binding protein. Alternatively, fransgenic expression can be used to produce an Aβ antibody or Aβ binding protein in an animal and the effects are assessed in the animal. For example, an Aβ antibody or Aβ binding protein can be expressed in a wildtype animal model and the animal is then assessed. An Aβ antibody or Aβ binding protein can also be expressed in a model animal for a disease or condition. 2. Administration of antibodies to subjects Aβ antibodies and Aβ binding proteins can be administered to subjects for prophylactic and therapeutic uses. In prophylactic applications, a composition or medicament is administered to a subject at risk for a disease or condition such as Alzheimer's disease. In therapeutic treatments, a composition or medicament is administered to a subject suspected of or already suffering from a disease or condition, such as Alzheimer's disease. An amount of the composition or medicament is administered to achieve an effectiveness of freatment. As described herein, predictive assays such as in vitro and in vivo assays, including testing in animal models can be used to determine dosages and dosage regimes for freatment.

Dosages of Aβ antibodies and Aβ binding proteins for freatment will vary depending on conditions such as the means of administration, the target site, the species of subject and physiological state of the subject and the use of the treatment (e.g. prophylactic or therapeutic). Treatment dosages are optimized for safety and effectiveness. Dosages range from 0.0001 to 100 mg/kg of subject body weight. Typically, dosages are 0.01 to 10 mg/kg. hi some cases, more than a single dose of the composition or medicament is necessary to achieve an effectiveness of freatment. For example, dosages can be daily, weekly, monthly or yearly. Dosages and dosage regimes can be determined empirically for example, by measuring the levels of Aβ, specific Aβ peptides and/or forms, and achieving a desired level of such in the subject by administering an Aβ antibody or Aβ binding protein to maintain that level. The dosages and dosage regimes can also depend on the stability of an Aβ antibody or Aβ binding protein. Stability of an Aβ binding protein or antibody can be determined by measuring levels of the protein or antibody in in vitro assays, cell based assays, in animal models and in a subject. For example, an amount of an Aβ antibody or protein can be administered to a subject and subsequent samples, such as blood, plasma or cerebral spinal fluid samples, taken from the subject over time to assess the amount remaining in the subject. In some cases an Aβ antibody or an Aβ binding protein with a detectable moiety such as a radiolabel, may be used to facilitate measurements.

Aβ antibodies and Aβ binding proteins can be administered by parenteral, topical, intravenous, oral, subcutaneous, interarterial, infracranial, intraperitoneal, intranasal and intramuscular means. Aβ antibodies and Aβ binding proteins can be administered to a particular organ or tissue, for example, by injecting directly into the organ or tissue. For example, Aβ antibodies and Aβ binding proteins can be injected directly in the cranium, into a muscle and directly into the bloodstream. For administration, Aβ antibodies and Aβ binding proteins can be formulated as a solution or suspension in a physiological diluent such as sterile water, saline, glycerol, oil or ethanol. Formulations can also be prepared as liposomes or micelles, microparticles and in formulation for sustained release. Formulations can also include surfactants, emulsifying agents, wetting agents, and pH buffering substances.

Aβ antibodies and Aβ binding proteins can also be administered in combination with other treatments, for example in combination another freatment for the disease or condition. For example, an Aβ antibody can be administered along with an agent that modulates the processing or levels of APP for freatment of Alzheimer's disease. 3. Assessment of Treatment

Methods for assessing freatment can be biochemical, physiological and/or can involve assessments of behaviors or phenotypes associated with a particular condition or disease. The effectiveness of freatment can include the effectiveness of a treatment to ameliorate symptoms such as by decreasing the severity, delaying the onset, delaying the recurrence, or decreasing the number of recurrences of symptoms or by delaying the progression of a disease or condition. Effectiveness of treatment also can include the effectiveness of a freatment to prevent a disease or condition, prevent the onset of symptoms of disease or condition. The effectiveness of ameliorating or preventing symptoms and/or the occuπence of a condition or disease can be assessed in animals, animal models and/or in subjects.

(a) Biochemical and Physiological Phenotypes Levels and forms of Aβ can be observed after to freatment to ascertain changes in the levels of Aβ, such as levels of all Aβ peptides, levels of particular Aβ peptides, such as Aβ42, and changes in the form of Aβ, for example, the level of soluble Aβ and the level in plaques.

Aβ can be assessed in plasma for example after treatment and obtaining blood at sacrifice from animals by cardiac puncture. Blood is then centrifuged to obtain plasma which can then be tested for Aβ levels and forms by assays such as described herein or known in the art. For example, Aβ levels can be assed in an ELISA assay with Aβ antibodies. Additionally, the plasma can be tested for the level of freatment agent. For example, Aβ antibodies and/or Aβ binding proteins present in the sample can be detected by biochemical and/or immunological means. Levels and forms of Aβ can also be assessed in cerebrospinal fluid in a similar manner. Aβ can also be assessed in tissues such as the brain for example, by obtaining brain tissue from each animal at sacrifice. As described in the Examples herein, homogenates of brain sections can be analyzed for Aβ levels by ELISA or by other assays described herein or known in the art to assess Aβ levels and forms. Additional dissection into cortex, hippocampus and cerebellar regions before homogenization can be used to further localize Aβ. Histopathology can also be used to assess freatment .For example as described in the Examples, brain sections can be assayed for the abundance of amyloid plaques in treated and confrol animals. In situ analysis with antibody staining can also be used to ascertain levels of Aβ and Aβ forms, for example by using Aβ antibodies which recognize Aβ and/or specific Aβ forms (Dodart et al. (2002) Nature Neurosci. 5(5): 452- 257). An Aβ antibody or Aβ binding protein with a detectable moiety can be used to detect the presence, level, stability and/or localization of the administered Aβ antibody or Aβ binding protein. For example, an initial dose of an Aβ antibody or Aβ binding protein with a detectable moiety can be administered and the level, stability and/or localization assessed to determine further dosing in the same animal or subject or to assist in predicting the dosage for additional animals or subjects to be treated. (b) Behavioral Phenotypes Behavioral phenotypes specific for an Aβ-associated condition or disease can be measured to ascertain the effect of freatment. For example, an assessment of Alzheimer's disease (AD) phenotype can refer to any visible, detectable or otherwise measurable symptom or property of an individual diagnosed with AD. Such properties include, but are not limited to, dementia, aphasia (language problems), apraxia (complex movement problems), agnosia (problems in identifying objects), progressive memory impairment, disordered cognitive function, altered behavior, including paranoia, delusions and loss of social appropriateness, progressive decline in language function, slowing of motor functions such as gait and coordination in later stages of AD, amyloid-containing plaques, which are foci of extracellular amyloid beta protein deposition, dysfrophic neurites and associated axonal and dendritic injury, microglia expressing surface antigens associated with activation (e.g., CD45 and HLA-DR), diffuse ("preamyloid") plaques and neuronal cytoplasmic inclusions such as neurofibrillary tangles containing hyperphosphorylated tau protein or Lewy bodies (containing c-synuclein). Standardized clinical criteria for the diagnosis of AD have been established by NINCDS/ADRDA (National Institute of Neurological and Communicative Disorders and Stroke/Alzheimer's Disease and Related Disorders Association) (McKhann et al. (1984) Neurology 3-^:939-944). The clinical manifestations of AD as set forth in these criteria are included within the definition of AD phenotype. For example, dementia may be established by clinical exam and documented by any of several neuropsychological tests, including the Mini Mental State Exam (MMSE) (Folstein and McHugh (1975) J. Psychiatr. Res. 72:196-198; Cockrell and Folstein (1988) Psychopharm. Bull. 24:689- 692), the Blessed Test (Blessed et al. (1968) Br. J. Psychiatry 114:797-811) and the Alzheimer's Disease Assessment Scale-Cognitive (ADAS-COG) Test (Rosen et al.

(1984) Am. J. Psychiatry 141:1356-1364; Weyer et al. (1997) Int. Psychogeriatr. 9:123- 138; and E l et al. (2000) Neuropsychobiol ^:102-107).

Tests can be developed in suitable laboratory animals to assess the effects of a freatment. For example, in AD, AD model animal can be treated and assessed. In one example, an object recognition task can be used to assess freatment. The test is based on the animal's spontaneous tendency to explore a novel object more frequently than a familiar one (Ennaceur et al. (1988) Behav. Brain Res. 31:47-59; Dodart et al. (1997) Neuroreport 8:1173-1178). Briefly, an animal such as a mouse is tested in a first trial with an object (such as a marble) and then in a second trial with the first object plus a new object (such as a die). A recognition index is calculated based on the amount of time the animal spends with each object in the second trial when both objects are present and the distance traveled toward each object.

Another example of a phenotypic test for AD is the holeboard memory task. (Dodart et al. (2002) Nature Neurosci. 5(5): 452-257). The test measures the ability of an animal to remember which holes of a holeboard have been baited with food. A food pellet is placed a hole of the board and the animal is tested in several trials over consecutive days where the same hole is baited each time. A global measure of cognitive performance is calculated from the trials based on the average number of errors made by the animal each day (based on entering holes never baited, re-entering a baited hole and not entering a baited hole).

Tests such as the object recognition task, holeboard memory task and other phenotypic assays known in the art are generally done with several animals to gather an average value. Single animals or groups of animals can undergo one or more treatments with a test agent, an Aβ binding protein, Aβ antibody, or any combination thereof and then freatment can be assessed with a phenotypic test. Control animals which have not undergone any treatments or which have undergone placebo treatments can be compared to assess the effectiveness of a particular freatment relative to no freatment or placebo controls.

The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.

EXAMPLE 1

Production of Aβ42-selective antibody (A387)

A selective Aβ42 antibody was produced by designing a peptide with the following sequence C-MNGGWIA, which represents the Aβ_35-42 region with Ν- terminal cysteine added for conjugation to ovalbumin. Swiss-Webster mice were immunized with 1 mg of the conjugated peptide followed by three boosts of 0.5 mg antigen every three weeks. Following a third boost, spleens of these mice were fused to mouse B-cells. Hybridoma cells were cloned and screened for Aβ42 selectivity by determining antibody titer to both the Aβ40 and Aβ42 peptide (AnaSpec, Inc. San Jose, CA) by ELISAs (as described below). Positive clones which had selective reactivity to the Aβ42 peptide were chosen. The cells were then injected infraperitoneally into SCID mice and ascites fluid was obtained and purified using Protein A. Titer of antibodies produced was determined by coating 50 μl of Aβ peptide (AnaSpec, Inc, San Jose, CA) in PBS (500 ng/ml) on CoStar 3590 microtiter 96-well plates. Wells were blocked with 200 μ\ of 3% BSA/PBS (Sigma, St. Louis, MO) and incubated with antibody for 1 hour at room temperature. Wells were washed three times with 200 μl of PBS/0.1% Tween- 20 (Sigma, St. Louis, MO). After washing, wells were incubated with mouse:horseradish peroxidase (HRP) secondary antibody for 1 hour at room temperature. Wells were washed three times with 200 μl of PBS/0.1% Tween-20. 50 μl TMB (3,3',5,5'- teframethylbenzidine) substrate was then added according to manufacturer's recommendations (KPL, Gaithersburg, MD) and incubated for 15 min. The reaction was stopped with 50 μl of 9.8% phosphoric acid (Milwaukee, WI) and the absorbance at 450 nm was quantitated by a Biorad 96-well plate reader. One antibody, designated A387, was found to have >1000 fold specificity for Aβ42 versus Aβ40 with a very high titer as determined in the above ELISA. Additionally, this antibody was shown to be specific for Aβ42 versus other AB peptides; AβBl-11, 1-28, 1-38, and 1-39 when tested in the above assay. Antibody A387 was subtyped and confirmed to be IgG2a kappa. This antibody was then used to develop an Aβ42 assay to quantitate Aβ42 peptide produced by cells. EXAMPLE 2

Production of Aβl-12 antibody (B436)

An antibody that recognizes the amino-terminal 1-12 amino-acid region on Aβ was produced and conjugated to alkaline phosphatase for use as a detection antibody in the Aβ42 sandwich ELISA. The Aβl-12 antibody was produced by designing a peptide with the following sequence DAEFRHDSGYEV-C that represents the Aβl-12 region with a C-terminal cysteine added for conjugation to ovalbumin. Swiss-Webster mice were immunized with 1 mg of the conjugated peptide followed by three boosts of 0.5 mg. Following a third boost, spleens of these mice were fused to mouse B-cells. Hybridoma cells were cloned and screened for Aβ reactivity. The cells were then injected infraperitoneally into SCED mice and ascites was obtained and purified using Protein A. One antibody, designated B436, was found to have high titer for both Aβ40 and Aβ42 peptides, this was a desired feature since this antibody should equally react to any Aβ peptide which contains the 1-12 amino-terminal portion of the peptide. This antibody was subtyped and confirmed to be IgG2a kappa and was further purified by affinity chromatography on an Aβl-12:Sepharose column and then conjugated to alkaline phosphatase. This antibody was then used as the detection antibody in the development of the Aβ42 assay to quantitate Aβ42 peptide produced by cells.

EXAMPLE 3 Production of LRP polyclonal antibody (R9377) for detection of LRP C-terminal fragments

A polyclonal antibody that recognizes the C-terminal region on LRP designated R9377 was prepared to the carboxyl-terminal 13 amino acid peptide (C- GRGPEDEIGDPLA) of LRP which was conjugated to ovalbumin via an amino-terminal cysteine residue incorporated into the LRP peptide. Initially, rabbits were primed with Complete Freund's adjuvant then immunized 14 days later with 1 mg of conjugated antigen and Incomplete Freund's adjuvant. Following this immunization, the rabbits received monthly boosts of antigen/Incomplete Adjuvant (0.5 mg). 14 days following the third boost, serum was collected and IgG was purified using Protein A:Sepharose. The purified antibody was used in the immunoblotting experiments described in Example 8. EXAMPLE 4

Aβ42 (A387) and Aβl-12 (B436) monoclonal antibody cDNA sequencing Protocol

(1) RNA Extraction

One confluent plate (approximately 1.5x10 cells) each of A387 and B436 A-beta mAb cell lines was harvested, pelleted, washed in IX PBS, quick-frozen, and stored at - 80°C. Using the RNeasy Mini Kit (QIAGEN #74104) according to manufacturer's protocol, the cells were lysed, homogenized by vortexing, and total RNA was extracted from half of each lysate.

(2) cDNA Synthesis First-strand cDNA synthesis was performed using the Superscript First Strand cDNA Synthesis System for RT-PCR (Invifrogen #11904-018) with antisense primers specific for Mus musculus kappa light chain and IgG_2a heavy chain sequences (GenBank accession numbers D14630 and V00765, respectively). The antisense primer sequences are as follows: light chain, 5'-GGACGCCATTTTGTCGTTCACTGCCA-3' (KappaJLCC; SEQUENCE ED NO: 22); heavy chain, 5*-

TGTTGTTTTGGCTGAGGAGACGGTGA-3' (IgG2a_HCC; SEQUENCE ED NO. 23). Duplicate reactions containing 2.5 μg A387 or B436 total RNA were prepared with or without reverse franscriptase (+RT and -RT, respectively) according to the manufacturer's protocol. (3) PCR

DNA encoding the A387 and B436 light and heavy chain variable regions were amplified by touchdown polymerase chain reaction using the Expand High Fidelity System (Roche #1732641), degenerate sense primers, and the Kappa_LCC and IgG2a_HCC antisense primers. The sense primers were designed using the sequence of 12-15 N-terminal residues from each heavy and light chain, previously obtained by N- terminal amino acid sequencing performed according to standard procedures by the Protein Core Facility at the University of Nebraska on a fee for service basis. These sequences were back-translated using Vector NTI 7 software (Informax, Inc.), reducing the level of degeneracy by applying a human codon preference table. The sense primer sequences are as follows: A387 light chain 5'-

GAYATYGTSCTSACNCAGWSBCCNGC-3* (A387_LCV1; SEQUENCE ED NO. 24) A387 heavy chain, 5'-GARGTYAAGYTBGTYGARTCYGGAGG-3' (A387_HCV1; SEQUENCE ID NO: 25); B436 light chain, 5'-GAYGTYYTBATGACYCARACYCCA- 3' (B436_LCV1; SEQUENCE ED NO: 26)); and B436 heavy chain, 5'- GARGTYATGYTBGTYGARTCYGGAGG-3' (B436_HCV1 ; SEQUENCE ED NO. 27). Reaction mixtures were prepared according to the manufacturer's protocol for each A387 or B436 +RT and -RT reaction. Amplification was performed in a Perkin-Elmer 3700 thermocycler according to the following conditions: denaturation for 2 min at 94°C; 10 cycles of 15 sec at 94°C, 1 min at 70°C-0.5°C per cycle, 1 min at 72°C; 10 cycles of 15 sec at 94°C, 1 min at 65°C, 1 min at 72°C; 25 cycles of 15 sec at 94°C, 1 min at 65°C, 1 min +5 sec/cycle at 72°C; and a final extension for 7 min at 72°C. (4) Cloning

PCR products were analyzed by gel electrophoresis on a 1% agarose gel. A major band of the approximate expected size (light chain: ~487 bp; heavy chain: -408 bp) was observed in each +RT reaction. An additional approximately 300-bp band was observed in the B436 reaction. No products were detected in the coπesponding -RT control reactions. The desired ~487-bp and ~408-bp bands were purified using the QIAquick Gel Extraction Kit (QIAGEN #28704) according to the manufacturer's protocol. The TOPO TA Cloning Kit (Invifrogen #K4600-01) was used to clone each product into vector pCR®II-TOPO and transform E. coli TOP 10 cells, according to the manufacturer's protocol. PCR analysis of transformants using T7 and SP6 primers identified 9 putative A387 light chain and 12 each putative A387 heavy chain, B436 light chain and B436 heavy chain constructs. Plasmid DNA was prepared for each of these from bacterial cultures using the QIAprep Spin Plasmid Kit (QIAGEN #27106) according to the manufacturer's protocol. (4) Sequencing

The cDNA inserts were sequenced with the ABI Prism BigDye Terminators v.3.0 Cycle Sequencing Kit (ABI #4390244) using approximately 250 ng of each plasmid and 1.6 μM each of standard T7 and SP6 primers. The manufacturer's protocol for 20 μl reactions was followed, except that the BigDye reagent was reduced to 2 μl and supplemented with 4 ul 5X Sequencing Buffer (ABI #4305603) per reaction. Reactions were purified using the CleanSEQ Kit (Agencourt #000136) according to the manufacturer's protocol then analyzed on an ABI 3700 sequencer. The results were evaluated using Sequencher software (Gene Codes Corp.). A387 light chain nucleotide sequences were obtained from seven independent clones. The identity of every nucleotide between the primer binding sites was confirmed by agreement between at least six of those sequences, with 99% of the sequence identical in all seven clones. Similarly, the identity of each nucleotide was confirmed in at least 10 of 11 A387 heavy chain clones, 11 of 12 B436 light chain clones, and 11 of 12 B436 heavy chain clones, with >99% of the sequences identical in all clones for each case. Some nucleotide sequence variability was seen in the N-terminal primer binding sites due to primer degeneracy. However, the amino acid sequences for these regions was previously determined by N-terminal amino-acid sequencing.

The nucleotide sequences obtained are provided in SEQ ID NO: 11 (A387 light chain variable region (nucleotides 1-285), J region (nucleotides 286-321) and N-terminal sequence of a constant region (nucleotides 322-478)), SEQ ID NO: 13 (A387 heavy chain variable region (nucleotides 1-291), DJ region (nucleotides 292-354) and N- terminal sequence of a constant region (nucleotides 355-366)), SEQ ID NO: 15 (B436 light chain variable region (nucleotides 1-300), J region (nucleotides 301-336) and N- terminal sequence of a constant region (nucleotides 336-493)), SEQ ID NO: 17 (B436 heavy chain variable region (nucleotides 1-294), DJ region (nucleotides 294-342) and N- terminal sequence of a constant region (nucleotides 342-354)). The nucleotide sequences (and encoded amino acid sequences) are also provided in SEQ ED NOs: 37 and 38 (A387 light chain nucleotide and amino acid sequences, respectively), 39 and 40 (A387 heavy chain nucleotide and amino acid sequences, respectively), 41 and 42 (B436 light chain nucleotide and amino acid sequences, respectively), 43 and 44 (B436 heavy chain nucleotide and amino acid sequences, respectively).

Results

Key: V-J regions are underlined. Regions determined by N-terminal amino acid sequencing are double-underlined. Regions not underlined are the N-terminal portions of the C regions.

A387 light (kappa) chain (SEQUENCE ED NO. 12)

1 nrvτ τo.ςPAτ T .svspnnsvs T.SΓ.R ASOSTS NNT.HWVOQKS HKSPRTT.TKY

51 ASQSivoTPS RFSGSGSGTF FTT.TVNSVGT KDFπiyrvπpr.Q SHSWPT FGT 101 GJXLELKRAD AAPTVSIFPP SSEQLTSGGA SWCFLNNFY PRDINVKWKI

151 DGSE QNGV

A387 heavy (IgG_2a) chain (SEQUENCE ID NO.14)

1 F.wτ.vF.snnn T.WPGGST.T T. AΓAASGFTFS NnAMSWVRQT PKTCRT.FWVAS 51 TSSVGNTWP DSVKGRFTTS RDNARNTT.YT, QMSSVRSFX)T AMYYΓAPΠY

101 VSPWFSVWGQ GTT.VTVSSAK TT

B436 light (kappa) chain (SEQUENCE ED NO. 16)

1 nvrMτnτpτ .ς τ.py.ςτ.r,r)nAS TSΓRSS NTV HSSGNTYT.EW VT.OKPGQS!P 51 T.T.rV VSNRF SGVPDRFSGS GSGTDFπ, T SRVF.AF.DT.GT Wr.FQGSHVP

101 YTFGGGT T.K IKRADAAPTV SIFPPSSEQL TSGGASVVCF L NFYPRDIN

151 VKWKIDGSER QNGV

B436 heavy (IgG_2a) chain (SEQUENCE ID NO. 18) 1 KVMT.VF.SGGn τ.wpnnsτ.τ . SΓVASGFTFS RVTMSWVROT PAK-RT.F-WVAT

51 TNFGNGNTW PnSWGRFTT SRDNARNTT.Y T. MSST.RSF.n TAMYYCTST.N

101 WAYWGQGTT.V TVSSAKTT

EXAMPLE S Aβ42 and Aβ40 Sandwich ELISAs

Sandwich ELISAs (Enzyme-Linked hnmunosorbent Assays) have been developed for specific detection of Aβ42 and Aβ40 peptides. An anti-Aβ42 selective monoclonal antibody or anti-Aβ40 selective monoclonal antibody (prepared to Aβ30-40 peptide using the same protocol as described for Aβ42 antibody production) was coated on white microtiter 96-well plates (50 μl at ~5 μg/ml) in PBS, pH 7.4. Following overnight coating at 4 °C, wells were blocked with 200 μl of 3% BSA, Fraction V (Sigma, St. Louis, MO) and incubated with Aβ peptides for 1 hour at room temperature. Wells were washed three times with 200 μl of PBS/0.1% Tween-20. After washing, wells were incubated with anti-Aβl-12 conjugated to alkaline phosphatase (-0.5 μg/ml) for 1 hour. Wells were washed three times with 200 μl of PBS/0.1% Tween-20 and CDP-Star chemiluminescence subsfrate (Tropix, Inc.) was added at 50 μl/well and incubated for 15 min. The luminescence was then quantified on an ABI luminometer. Results show a large linear range of 75-2000 pg/well, high dynamic range of 3-30 fold over background in linear range (signaknoise), low sensitivity limit <20 pg/well, and >1000-fold selectivity for Aβ42 over other Aβ peptides, making the assay highly amenable to high throughput screening.

EXAMPLE 6 AB42/Aβ40 high-throughput screening assay

A selective Aβ42/Aβ40 high throughput 384-well screen to identify compounds that do not affect Aβ40 levels has been developed. Due to the high sensitivity and selectivity of the Aβ42/Aβ40 ELISA, this assay was formatted for use in 384-well plates for high throughput screening for compounds that selectively decrease Aβ42 levels while not affecting Aβ40 levels.

Human neuroblastoma cells (SH-SY5Y) were obtained from ATCC (CRL-2266) and fransfected with human APP₇₅ι in a pcDNA.l vector containing a neomycin resistant site. Cells were selected with 400 μg/ml G418 (Gibco) and cloned by limiting dilution. Cells expressing the amyloid precursor protein (APP₇₅ι) were plated in 384-wells and allowed to adhere for 24 hours. The cells were freated with a dose-response of DAPT (a positive control inhibitor used to inhibit Aβ42 production) ranging from 1 nm to 1 μM for 18 hours. Supernatant was then removed and assayed in the Aβ42 ELISA. The ELISA was carried out by coating white microtiter 384-well plates with 25 μl of -5 μg/ml solution of Aβ42 selective monoclonal antibody (A387) in PBS. Following overnight coating at 4 °C, wells were blocked with 50 μl of 3% BSA/PBS, Fraction V (Sigma, St. Louis, MO) and incubated with cell supernatant for 1 hour at room temperature. Plates were washed three times with 50 μl of PBS/0.1% Tween-20. After washing, wells were incubated with 25 μl of anti-Aβl-12 conjugated to alkaline phosphatase (-0.5 μg/ml) for 2 hours. Wells were washed three times with 50 μl of PCS/0.1% Tween-20 and 25 μl of CDP-Star chemiluminescence subsfrate (Tropix, Inc.) was added and incubated for 30 minutes at room temperature. Luminescence was quantified on an Analyst HT. The assay was repeated with a test library of compounds. Compound concentrations were -30 μM. 1 μM DAPT was used as a positive control and DMSO vehicle alone (0.12%) was added as a negative confrol. The data showed acceptable signal to background (-7-10 fold) with the positive control wells clearly distinguishable from the vehicle controls. Data from the test library screen showed that the hit criteria of <50% of plate median (50% inhibition)) is outside the normal distribution of the data therefore, compounds showing >50% inhibition in this primary screen were chosen for further follow-up assays such as Aβ40 inhibition and cytotoxic assays. The % coefficient of variation range measured was 15-17%. Taken together, these data indicate that the assay (when performed in duplicate) has a >95% chance of identifying inhibitors.

Test compounds which show >50% inhibition for Aβ42 levels are then tested for their effects on Aβ40 levels using a similar assay except that the coat antibody is Aβ40- specific. Furthermore, compounds are assessed for cytotoxicity using Alamar Blue

(Biosource, Camarillo, CA) according to manufacturer's recommendations. Briefly, 10% Alamar Blue is added to cells after incubation of compound for 18h and incubated for 4 hours at room temperature, after which fluorescence is read on a specfrophotomer. Compounds that showed >40% cytotoxicity were eliminated as hits. The screening methods have also been performed using CHO cells containing DNA that encodes human APP₆ 5 and human PSl. The screening methods may also be performed using mouse neuroblastoma (N2a) cells expressing APP. N2a cells can be fransfected with DNA encoding APP as described in Example 8.

EXAMPLE 7 Analysis of Processing of LRP Endogenous LRP protein of N2a cells expressing human wild-type and mutant PSl was analyzed and compared. Notch and APP protein in the cells was also analyzed as a reference for PSl -dependent protein cleavage.

Stable recombinant N2a cells that had been fransfected with DNA encoding wild- type human APP695 (see, e.g. , SEQ ED NO: 30 and GenBank Accession no. Y00264) and DNA encoding either wild-type human PSl (see, e.g., SEQ ED NO: 5) or mutant human PSl were grown overnight to near 70% confluence in a 10-cm tissue-culture dish. Two mutant PSl cell lines were used: Δl,2 and D385A. The Δl,2 cell line expresses defective (i.e., loss of function) PSl proteins encoded by nucleic acid lacking exons 1 and 2 of the human PSl gene. The D385A cell line contains nucleic acid coding for an alanine instead of an aspartic acid residue at amino acid 385 (see, e.g., SEQ ED NO: 6 for amino acid sequence of a wild-type human PSl) which is essential to PSl function.

The A 1,2 cells and the D385A cells were also transiently fransfected with 2 μg of DNA encoding an amino-terminal truncated form of human NOTCHΔE containing residues 1-26 (signal sequence; see, e.g., SEQ ED NO: 31) and residues 1718-2195 (see, e.g., SEQ ED NO: 31) with the methionine 1738 mutated to valine to prevent alternative translation initiation at that site. The DNA construct also contains nucleic acid sequence encoding a carboxy-terminal V5 antibody epitope which is comprised of the 14-amino acid sequence; Gly-Lys-Pro-Ile-Pro-Asn-Pro-Leu-Leu-Gly-Leu-Asp-Ser-Thr added to the carboxy-terminal end of the Notch amino acid sequence so that a V5 antibody could be used to detect the NotchΔE or the NICD. This construct encodes a -55-60 kDa protein. Transfection was carried out using Quiagen's effectene reagent for 20 h. Cells were then plated at 1.2 x 10° cells/well in 6-well plates. After 28 h, cells were freated +/- DAPT (1 μM) and then lysed in 200 μM lysate buffer (10% 10X TBS, 0.05% Tween 20, 1% Triton X-100, and a protease inhibitor cocktail) after 19 h of treatment. Cells were centrifuged at 10,000 rpm for 5 min and the supernatant was removed. The supernatant of the lysates was then separated on 8% Tris-Glycine gels and proteins were transferred to nitrocellulose membrane. The membranes then were blocked for an hour with 10% nonfat dry milk and probed with the anti-V5 (1 :2000) primary antibody (hivifrogen, San Diego) to detect accumulation of the Notch substrate, anti-LRP antibody R9377 (as described in Example 3) to probe for LRP CTFs, and anti-APP antibody R8666 (a rabbit antibody prepared to the carboxy-terminal region; amino acids C-EVPTYKFFEQMQN conjugated to ovalbumin through the amino-terminal cysteine residue) to visualize APP CTFs. Bound antibody was detected using the ECL SuperSignal system (Pierce) after incubation with anti-rabbit horseradish peroxidase-coupled secondary antibodies (Sigma). Samples were assayed in duplicate.

In lysates of the wild-type PSl cells, an approximately 20 kDa protein fragment was observed in the presence of the PS 1 inhibitor DAPT. The fragment is one that is recognized and bound by the polyclonal antibody R9377 generated against a carboxyl- terminal peptide (the carboxyl-terminal 13 amino acids) of human LRP (C-

GRGPEDEIGDPLA) and thus is one derived from a C-terminal portion of LRP. Accumulation of this fragment was not detected in lysates of wild-type PSl cells not freated with DAPT. Because little to no protein is detected in DAPT-freated cell lysates by the R9377 antibody generated against a C-terminal peptide of LRP, but a peptide fragment is detected at significant levels by the antibody in lysates of DAPT-freated cells, it can be concluded that a PSl -dependent activity cleaves LRP in such a way as to eliminate the epitope sequence on LRP that is recognized by antibody R9377. These results are consistent with presenilin-dependent cleavage of LRP.

Similar results were obtained in the analyses of lysates of wild-type PSl cells using antibodies reactive with APP and Notch, respectively. In lysates of wild-type PSl cells freated with DAPT, two peptide fragments (-17 kDa and -14 kDa representing the C99 and C83, β- and α-secretase cleavage products, respectively) were readily detected by the anti-APP antibody R8666. In lysates of cells that were not treated with DAPT, little to no protein was detected by the R8666 antibody. In lysates of wild-type PSl cells freated with DAPT, one peptide fragment was detected by the anti-V5 antibody.

Although this fragment was also detected in lysates of wild-type PSl cells that were not treated with DAPT, the amount of the fragment detected in the lysates of the DAPT- freated cells was significantly greater than in the lysates of the untreated cells.

The results of the analyses of lysates of DAPT-freated and untreated wild-type PSl cells using anti-APP and anti-Notch- V5 fusion protein antibodies are consistent with inhibition and non-inhibition, respectively, of the PSl-dependent cleavage of these presenilin substrates (i.e., APP and Notch) at a site in a C-terminal portion of these proteins. Thus, the similar findings in the analysis of LRP protein in the cell lysates and the APP and Notch analyses supports the conclusion of a presenilin-dependent cleavage of LRP.

In the lysates of the DAPT-freated and untreated Δl,2 mutant cell line, the -20 kDa LRP CTF was equally evident at significant levels. Similarly, the same fragments were detected in the lysates of the DAPT-freated and untreated A 1,2 mutant cells by the R8666 antibody, and the anti-Notch- V5 fusion protein antibody also detected the same fragment in lysates of the freated and untreated mutant cells. The same results were obtained in analyses of the lysates of the DAPT-freated and untreated mutant D385A cells with the antibodies for the detection of LRP, APP and Notch peptides. These results obtained with cells that do not express functional PSl provide confirmation that the results observed with DAPT-freated and untreated wild-type PSl cell lysates are due to the inhibition and non-inhibition of a PS 1 -dependent activity.

Furthermore, a comparison of the very minimal levels of the peptide fragments detected in immunoassays of lysates of wild-type PSl cells that were not freated with DAPT with the significant levels of the peptide fragments detected in lysates of D385A mutant cells, indicated an approximate 40-60% loss of PSl activity in the mutant cells relative to wild-type PSl cells. Because accumulation of the -20 kDa LRP fragment in the presence of DAPT and in PSl mutant cell lines parallels the accumulation of the APP and Notch fragments, these results indicate that LRP undergoes a PSl-dependent cleavage.

EXAMPLE 8 Presenilin/γ-secretase Assays

N2a mouse neuroblastoma cells (ATCC, Rockville, MD) fransfected with APPWT (Ace. No. Y00264) were incubated with DAPT (1 μM or 1 mM) or vehicle confrol (DMSO) for 24 hours. Lysates were then prepared by first washing the cell layer three times with isotonic PBS. To each 96-well, 50 ml of lysate buffer (TBS, 1% Triton X- 100, 5 mM EDTA, 0.2% Tween-20, 10 μM leupeptin, 1 mM PMSF) was added and cells were removed by agitating with a pipet tip. Cell lysates were spun for 5 min at 10,000 rpm in a microfuge and the supernatant was collected.

The lysates were then separated on 4-20% Novex gels and probed by immunoblotting with the anti-LRP polyclonal antibody (R9377). Results showed accumulation of a 20 kDa protein in lysates of cells that had been freated with DAPT. This band represented a carboxyl-terminal fragment of LRP. This accumulation of LRP CTFs paralleled the accumulation of APP CTFs, a finding that demonsfrates that LRP is a distinct presenilin substrate and can be used to quantitate presenilin activity. The LRP assay can be used to profile test compounds that modulate Aβ levels, and, in particular Aβ42 levels (such as can be identified in the high-throughput assay; see EXAMPLE 6), with respect to possible effects on presenilin activity. In one aspect, compounds that are identified as agents that reduce Aβ42 levels (e.g., by >50% at, e.g., 30 μM; see EXAMPLE 6) are tested for any effects on presenilin activity in the LRP assay in order to identify Aβ42-reducing compounds that have minimal to no inhibitory activity with respect to presenilin-dependent LRP processing activity. Compounds were chosen that had <20% increase (at the highest tested concentration of 30 μM) of LRP-CTFs as compared to the DAPT positive confrol.

EXAMPLE 9

Characterization of binding properties of Aβ antibodies

(1) Assessment of binding to different forms of Aβ

(a) Non-reducing gel electrophoresis and immunoblotting

Methods. Aβ40 or Aβ42 peptide standards (Bachem) (250 ng in 10 μl) were mixed with 10 μl native sample buffer (Invitrogen). Samples were run on 18% Novex 10-well gels at constant voltage (150 V) using native sample buffer (Invitrogen). Novex rainbow standards (250 kd to 4 kd) were used as molecular weight controls. When the dye front reached the bottom of the gel, proteins were transferred to 0.45 micron PVDF filters (pre-wetted in methanol) at 100 mA constant cuπent in IX CAPS buffer (10 mM CAPS), 10% methanol, pH 11.0 for 90 min. Filters were blocked with TBS, 10% dry milk, pH 7.4 for 60 min at room temperature. Filters were then incubated overnight at 4°C in a solution of TBS, 3% dry milk, 0.1% Tween-20 containing primary antibody (1-5 μg/ml of A387 or B436 conjugated to biotin), followed by three five-minute washes in TBS containing 0.1% Tween-20. After washing, filters were incubated in anti-biotin peroxidase-conjugated secondary antibody (Sigma; 1:2000 in TBS, 3% dry milk, 0.1% Tween-20) for 2 h at room temperature, and washed six times for five minutes each with TBS, 0.1% Tween-20. Signals were detected with the Chemiluminescence Supersignal ECL system (Pierce).

Results. By non-denaturing gel electrophoresis and immunoblotting as described above, Aβ42 peptides run at positions consistent with various forms, including insoluble fibrils (near top of the gel), high molecular weight oligomers (> -100 kd) and low molecular weight oligomers such as pentamers (-20 kd) and dimers (-10 kd). Aβ40 peptides run at positions consistent with insoluble fibrillar and low molecular weight oligomeric forms. B436 antibody was shown to detect all forms of both Aβ40 and Aβ42 peptides. As expected, A387 did not recognize any forms of Aβ40. A387 antibody did not significantly recognize either Aβ42 fibrils or high molecular weight oligomers, and instead primarily recognized Aβ42 low molecular weight oligomers.

(b) ELISA assays in presence and absence of bathocuprine

Methods. Dynex Microfluor-2 White Flat-bottom 96-well plates were coated overnight with 2-10 μg/well of A387 antibody. Aβ42 peptide standard (Bachem) was added at concentrations ranging from 5000 pg/well to 0.08 pg/well at half log intervals, in either DMEM complete medium or DMEM complete medium containing 2 mM of the metal chelator bathocuprine, and plates incubated at room temperature for 2 h. After washing in PBS/0.1% Tween-20, alkaline phosphatase-labeled B436 antibody (-0.5 μg/ml) in 1% BSA/TBS/0.1% Tween-20 was added, and plates incubated at room temperature for 2 h. After washing, CDP-Star-Sapphire Luminescence Subsfrate (Applied Biosystems) was added and the plates incubated for 5-15 min in the dark. Signal was then detected using an ABI TR717 Luminometer.

Results. Bathocuprine has been shown to solubilize Aβ aggregates into low molecular weight oligomers (Cherny et al. (1999) J. Biol. Chem. 274:23223-23228). In the presence of bathocuprine, A387 bound more Aβ42 peptide than in the absence of bathocuprine. These results are consistent with A387 recognizing lower molecular weight oligomers of Aβ42 peptide.

(2) Assessment of antibody binding to Aβ in plasma

Methods. Mouse blood was obtained by cardiac puncture at sacrifice. Briefly, mice were sedated by standard anesthesia. Upon sedation, each mouse was placed in dorsal recumbence and a 26-gauge needle attached to a heparinized 1 cc syringe inserted into the thorax tlirough the diaphragm to an approximate depth of 2 cm. Light suction was applied to the needle and placement in the cardiac (ventricular) chamber of the mouse confirmed by blood flow to the syringe chamber. Blood was aspirated until flow ceased. To obtain plasma, blood samples from each mouse were spun at 3,000 RPM for 10 minutes, and the supernatant collected. Plasma was then frozen until analyzed. For the immunoprecipitation assay, mouse plasma was centrifuged at low speed for 5 min to remove precipitated material, and the supernatant diluted in PBS 1:2. Human Aβ40 and Aβ42 peptide standards (Bachem) were added at 300 ng/ml to the diluted plasma, and the samples incubated with 1 ml of Sepharose beads for 1 h at 4°C. Beads were precipitated, and samples divided into 1 ml aliquots. To 1 ml of sample, biotin-labeled B436 or A387 antibodies (-10 μg/ml) were added, together with 40 μl of SfreptavidimSepharose beads (Pierce), and the samples rocked overnight at 4°C. Samples were spun to pellet the beads, which were washed twice with 1 ml PBS-0.1% Tween-20. 30 μl of NuPAGE sample buffer (Invitrogen) was added, samples were boiled for 3 min, and supematants loaded onto a 10% Bis-Tris NuPAGE gel (Invitrogen) at 125V. Molecular weight standards from 185 kDa to 3 kDa were also run. After electrophoresis, proteins were fransfeπed to PVDF filters at 100 mA for 90 min. Filters were then blocked in TBS-Tween containing 5% non-fat dry milk for 2 h. Blocked filters were incubated overnight with biotin-labeled 6E10 antibody, which recognizes the N-terminus of Aβ (1 :500; Signet Laboratories), and then with HRP-labeled anti-HRP for 1 h (1 :2000; Sigma). Signal was detected following incubation with Super Signal (Pierce Chemical Co.) for 1 min.

Results. Both A387 and B436 antibodies were able to immunoprecipitate human Aβ42 spiked into mouse plasma. The detected Aβ42 had an apparent molecular weight consistent with monomeric form of the peptide.

(3) Assessment of binding to Aβ in brain

Methods. The animals used in these experiments were either C57 mice, or Tg2576 mice of three or six months of age. Tg2576 mice express human APP695 with the Swedish (Lys670Asn, Met671Leu) double mutation under the confrol of the hamster prion protein gene promoter (Hsiao et al. (1996) Science 274:99-102; U.S. Patent No. 5,877,399). Mouse brain samples were prepared at sacrifice by brain removal and knife bisection along the superior sagital sulcus from the cortical surface to the extreme ventral surface. One brain hemi-section from each animal was snap frozen in liquid nitrogen. Frozen brain hemi-sections were weighed and fransfeπed to thick-walled polyallomer centrifuge tubes. A lOx volume (w vol) of 70% formic acid was added to each sample. The samples were briefly homogenized over ice, then centrifuged @ 100,000 x g for 1 hr at 4° C. The clear supernatant between the lipid layer and pellet was collected and its volume determined. An 1 lx volume (vokvol) IM Tris Base was added to neufralize the sample to pH range - 8 - 8.5, and aliquots were frozen at -80° C until analyzed.

For A387 ELISA analysis of brain samples, A387 antibody was coated onto plates, and the ELISA assay performed essentially as described in Section (l)(b) of this Example. ELISA analysis was also performed using the Human Beta-Amyloid (Abeta) [1-42] Fluorometric ELISA Kit (Biosource, catalog # 88-344), following manufacturers' directions. The coat antibody in the Biosource Kit is a monoclonal antibody directed against the N-terminus of human Aβ. The detection antibody is a rabbit polyclonal antibody that recognizes human Aβ42, but not human Aβ40 or mouse Aβ. The rabbit antibody is detected using an anti-rabbit IgG-alkaline phosphatase conjugate and a fluorescent subsfrate. For both the Biosource and the A387 ELISA assays, Aβ42 standard curves were prepared from serial dilutions of AB42 peptide (Bachem; stored in hexafluoroisopropanol) in C57 brain homogenate.

Results. Using the Biosource Kit ELISA assay, the amount of formic acid- exfractable Aβ42 detected in brains of Tg2576 animals was not significantly different from background (C57 brains). However, using A387 antibody and the ELISA protocol described herein, the amount of formic acid-extractable Aβ42 detected in brains of Tg2576 animals was about three-fold higher than background in the linear range of the Aβ42 standard curve.

EXAMPLE 10

Method for administering Aβ monoclonal antibodies to animals and for assessing the effects of the antibodies on Aβ levels and amyloid plaques

Animals. TASD41 fransgenic mice, which express human APP751 cDNA containing the London (V717I) and Swedish (K670M/N671L) mutations under the confrol of the murine Thy-1 gene, are used. The generation and properties of these animals (line 41) are described in Rockenstein et al. (2001) J. Neurosci. Res. 66:573-582. Briefly, Rockenstein et al. showed that TASD41 mice exhibit mature plaques in the frontal cortex as early as 3-4 months of age, and by 5-7 months also exhibit plaques in the hippocampus, thalamus and olfactory region. By ulfrastructural and double immunostaining analysis, these plaques were shown to contain dystrophic neuritis immunoreactive with antibodies against APP, snynaptophysin, neurofilament and tau. As such, the TASD41 mouse is a useful animal model of Alzheimer's disease.

Administration of antibodies. TASD41 mice of either about 4 months of age, or about 8 months of age, are divided into groups of 6-8 age-matched animals. Once a week for 3-6 months, each animal receives an intraperitoneal injection of either 500 μg of A387 antibody in saline, 500 μg of B436 antibody in saline, or 500 μg of confrol IgG in saline, according to the group.

Aβ ELISA assays. At sacrifice, plasma and brain samples are prepared from each animal as described in Example 9, and ELISA assays performed according to the procedure described in Example 9. A difference in Aβ40 or Aβ42 levels, or of particular Aβ40 or Aβ42 forms, can be detected.

Histopathology. One hemi-brain from each animal is fixed by immersion in 4% paraformaldehyde in PBS (pH 7.4). A series of consecutive 40μM sagittal sections are cut using a Leica Vibratome and stored in cryoprotectant solution at -20°C. Sections are stained with Thioflavine S (which binds amyloid plaques) and with Cresyl Violet, and analyzed under fluorescent and bright field microscopy, respectively. A difference in abundance of amyloid plaques between antibody-treated and Ig-freated animals can be observed.

Since modifications will be apparent to those of skill in this art, it is intended that this invention be limited only by the scope of the appended claims.

Claims

What is claimed:

1. A polypeptide, comprising a sequence of amino acids that is selectively reactive with Aβ 42 and preferentially binds to low molecular weight forms of Aβ42.

2. The polypeptide of claim 1, comprising at least one complementarity- determining region (CDR) selected from the group consisting of CDR-Ll, CDR-L2, CDR-L3, CDR-Hl, CDR-H2 or CDR-H3 of antibody A387.

3. The polypeptide of claim 1 , comprising CDR-Ll , CDR-L2, CDR-L3, CDR-Hl , CDR-H2 and CDR-H3 of antibody A387.

4. The polypeptide of claim 1 , wherein at least one CDR is selected from the group consisting of amino acids 24-34 of SEQ ED NO:12, amino acids 50-56 of SEQ ED NO: 12, amino acids 89-97 of SEQ ED NO: 12, amino acids 26-35 of SEQ ED NO: 14, amino acids 31-35 of SEQ ED NO:14, amino acids 26-31 of SEQ ED NO:14, amino acids 50-65 of SEQ ED NO:14, amino acids 50-58 of SEQ ED NO:14, and amino acids 98-107 of SEQ ED NO: 14.

5. The polypeptide of claim 1 , comprising at least a portion of a variable domain of the light chain or the heavy chain of an Aβ antibody.

6. The polypeptide 5, wherein the variable domain is selected from the group consisting of the light chain variable domain of A387, the heavy chain variable domain of A387, a polypeptide with at least 85% identity to the light chain variable domain of A387; a polypeptide with at least 85% identity to the heavy chain variable domain of A387.

7. The polypeptide of claim 1, further comprising a scaffold.

8. The polypeptide of claim 7, wherein the scaffold is a polypeptide scaffold.

9. _. The polypeptide of claim 7, wherein the scaffold is a human polypeptide scaffold.

10. The polypeptide of claim 7, wherein the scaffold is an antibody scaffold.

11. The polypeptide of claim 10, wherein the antibody scaffold is selected from the group consisting of an Fv fragment scaffold, an Fab fragment scaffold, and a single-chain (scFv) fragment.

12. The polypeptide of claim 1, further comprising a detectable moiety.

13. The polypeptide of claim 1 , further comprising a clearance domain.

14. The polypeptide of claim 13, wherein the clearance domain is a ligand for an Fc receptor.

15. A polypeptide, comprising at least one complementarity-determining region (CDR) selected from the group consisting of CDR-Ll, CDR-L2, CDR-L3, CDR- HI, CDR-H2 or CDR-H3 of antibody A387.

16. The polypeptide of claim 15 comprising CDR-Ll , CDR-L2, CDR-L3, CDR-Hl, CDR-H2 and CDR-H3 of antibody A387.

17. The polypeptide of claim 15, wherein at least one CDR is selected from the group consisting of amino acids 24-34 of SEQ ED NO:12, amino acids 50-56 of SEQ ED NO: 12, amino acids 89-97 of SEQ ED NO: 12, amino acids 26-35 of SEQ ED NO: 14, amino acids 31-35 of SEQ ED NO:14, amino acids 26-31 of SEQ ED NO:14, amino acids 50-65 of SEQ ID NO:14, amino acids 50-58 of SEQ ED NO: 14, and amino acids 98-107 ofSEQ ED NO:14.

18. The polypeptide of any of claims 15-17 further comprising a scaffold.

19. The polypeptide of claim 18 wherein the scaffold comprises a solid support.

20. The polypeptide of claim 18 wherein the scaffold is a polypeptide scaffold.

21. The polypeptide of claim 18, wherein the scaffold is a human polypeptide scaffold.

22. The polypeptide of claim 18, wherein the scaffold is an antibody scaffold.

23. The polypeptide of claim 22, wherein the antibody scaffold is selected from the group consisting of an Fv fragment scaffold, an Fab fragment scaffold, and a single-chain (scFv) fragment.

24. The polypeptide of any of claims 15-17, wherein the polypeptide is a chimeric polypeptide.

25. The polypeptide of any of claims 15-17, wherein the polypeptide is an antibody.

26.. The polypeptide of any of claims 15-17, further comprising a clearance domain.

27. The polypeptide of claim 26, wherein the clearance domain is a ligand for an Fc receptor.

28. The polypeptide of any of claims 15-17, further comprising a detectable moiety.

29. The polypeptide of claim 16, which comprises amino acids 1-95 of SEQ

ED NO: 12, or a fragment thereof and/or comprises amino acids 1-97 of SEQ ID NO: 14, or a fragment thereof.

30. The pol peptide of claim 29, further comprising one or more joining regions.

31. The polypeptide of claim 30, wherein at least one joining region comprises amino acids 96-107 of SEQ ED NO:12 or amino acids 98-118 of SEQ ED NO: 14.

32. The polypeptide of claim 30, wherein at least one joining region comprises an amino acid sequence selected from the group consisting of SEQ ED NOS: 46, 48, 50, 52, 54, 55, 57, 59, 61, 67, 73, 75, 77, 79, 89 and 91.

33. The polypeptide of claim 29, further comprising one or more constant regions.

34. The polypeptide of claim 33, wherein the constant region is a mouse constant region.

35. The polypeptide of claim 34, wherein the constant region comprises an amino acid sequence selected from the group consisting of SEQ ED NOS:63, 65, 69 and 71.

36. The polypeptide of claim 33, wherein the constant region is a human constant region.

37. The polypeptide of claim 36, wherein the constant region comprises an amino acid sequence selected from the group consisting of SEQ ED NOS:81, 83, 85 and 87.

38. The polypeptide of claim 16 comprising the amino acid sequence of SEQ ED NO:97 and/or SEQ ED NO:98.

39. The polypeptide of any of claims 15-17, which is specifically reactive with at least one AD

40. The polypeptide of claim 39, wherein A D is A D D □

41. The polypeptide of claim 39, which binds AD D D without substantially binding other AD .

42. A polypeptide comprising at least one complementarity-determining region (CDR) selected from the group consisting of CDR-Ll, CDR-L2, CDR-L3, CDR- HI , CDR-H2 or CDR-H3 of antibody B436.

43. The polypeptide of claim 42, comprising CDR-Ll , CDR-L2, CDR-L3, CDR-Hl, CDR-H2 and CDR-H3 of antibody B436.

44. The polypeptide of claim 43, wherein at least one CDR is selected from the group consisting of amino acids 24-39 of SEQ ED NO: 16, amino acids 55-61 of SEQ ED NO: 16, amino acids 94-102 of SEQ ED NO: 16, amino acids 26-35 of SEQ ED NO: 18, amino acids 31-35 of SEQ ED NO: 18, amino acids 26-31 of SEQ ED NO: 18, amino acids 50-66 of SEQ ED NO: 18, amino acids 50-59 of SEQ ED NO: 18, and amino acids 99-103 of SEQ E NO:18.

45. The polypeptide of any of claims 42-44 further comprising a scaffold.

46. The polypeptide of any of claims 45, wherein the scaffold comprises a solid support.

47. The polypeptide of any of claims 45, wherein the scaffold is a polypeptide scaffold.

48. The polypeptide of claim 45, wherein the scaffold is a human polypeptide scaffold.

49. The polypeptide of claim 45, wherem the scaffold is an antibody scaffold.

50. The polypeptide of claim 49, wherein the antibody scaffold is selected from the group consisting of an Fv fragment scaffold, an Fab fragment scaffold, and a single-chain (scFv) fragment.

51. The polypeptide of any of claims 42-44, wherein the polypeptide is a chimeric polypeptide.

52. The polypeptide of any of claims 42-44, wherein the polypeptide is an antibody.

53. The polypeptide of any of claims 42-44, further comprising a clearance domain.

54. The polypeptide of claim 53, wherein the clearance domain is a ligand for an Fc receptor.

55. The polypeptide of any of claims 42-44, further comprising a detectable moiety.

56. The polypeptide of claim 43 , which comprises amino acids 1 - 100 of SEQ

ED NO: 16, or a fragment thereof and/or comprises amino acids 1-98 of SEQ ID NO: 18, or a fragment thereof.

57. The pol peptide of claim 56, further comprising one or more joining regions.

58. The polypeptide of claim 57 wherein at least one joining region comprises amino acids 101-112 of SEQ EDNO:16 or amino acids 99-114 of SEQ EDNO:18.

59. The polypeptide of claim 57, wherein at least one joining region comprises an amino acid sequence selected from the group consisting of SEQ ED NOS: 46, 48, 50, 52, 54, 55, 57, 59, 61, 67, 73, 75, 77, 79, 89 and 91.

60. The polypeptide of claim 56, further comprising one or more constant regions.

61. The polypeptide of claim 60, wherein the constant region is a mouse constant region.

62. The polypeptide of claim 61, wherein the constant region comprises an amino acid sequence selected from the group consisting of SEQ ED NOS:63, 65, 69 and 71.

63. The polypeptide of claim 60, wherein the constant region is a human constant region.

64. The polypeptide of claim 63, wherein the constant region comprises an amino acid sequence selected from the group consisting of SEQ ED NOS:81, 83, 85 and 87.

65. The polypeptide of claim 43, comprising the amino acid sequence of SEQ ED NO:99 and/or SEQ ED NO: 100.

66. The polypeptide of any of claims 42-44 which is specifically reactive with at least one Aβ peptide.

67. A nucleic acid molecule encoding the polypeptide of any of claims 1-41.

68. A nucleic acid molecule encoding the polypeptide of any of claims 42-66.

69. A kit, comprising the polypeptide of any of claims 1-41.

70. A kit, comprising the polypeptide of any of claims 42-66.

71. A method for assessing the presence or amount of AD in a sample, comprising: contacting the polypeptide of any of claims 1-14, 39-41 or 66 with the sample under conditions whereby a complex is formed between the polypeptide and AD, and assessing the presence or amount of the complex in the sample, and thereby determining the presence or amount of AD in the sample.

72. The method of claim 71, wherein the sample is selected from the group consisting of a cell exfract, exfracellular medium, plasma, cerebrospinal fluid and brain.

73. The method of claim 71 , wherein the presence or amount of the complex is assessed using an enzyme-linked immunosorbent assay (ELISA).

74. A method, comprising administering to a subject the polypeptide of any of claims 1-66.

75. A method of binding Aβ comprising administering to a subject the polypeptide of any of claims 1-14, 39-41 or 66 to bind AD.

76. The method of claim 74 or 75, wherein the subject has, or is at risk of developing, a disease associated with accumulation of A D .

77. The method of claim 76, wherein the disease is Alzheimer's disease.

78. A method of reducing AD level in an subject, comprising administering to the subject an effective amount of the polypeptide of any of claims 1-14, 39-41 or 66 to reduce the level of at least one ADpeptide.

79. The method of claim 78, wherein the subject has, or is at risk of developing, a disease associated with accumulation of AD.

80. The method of claim 79, wherein the disease is Alzheimer's disease.

81. The method of claim 78, wherein the level of at least one ADpeptide in blood or plasma is reduced.

82. The method of claim 78, wherein the level of at least one ADpeptide in brain is reduced.

83. A method for assessing presenilin activity, comprising: contacting a sample containing a presenilin and/or fragment(s) thereof with a lipoprotein receptor-related protein (LRP) and/or fragment(s) thereof; and assessing the processing and/or cleavage of the LRP or fragment(s) thereof.

84. A method for identifying an agent that modulates presenilin activity, comprising: contacting a sample containing a presenilin, and/or fragment(s) thereof, and a lipoprotein receptor-related protein (LRP), and/or fragment(s) thereof with a test agent; and identifying an agent that alters the processing and/or cleavage of LRP and/or fragment(s) thereof.

85. A method for identifying a candidate agent for treatment or prophylaxis of a disease associated with an altered presenilin, comprising: contacting a sample that contains an altered presenilin and/or fragment(s) thereof and a lipoprotein receptor-related protein (LRP) and/or fragment(s) thereof with a test agent, wherein the altered presenilin and/or fragment(s) thereof is associated with an altered cleavage and/or processing of LRP and or fragment(s) thereof; and identifying a candidate agent that restores LRP cleavage and/or processing to substantially that which occurs in the presence of a presenilin and/or fragment(s) thereof that is not associated with an altered cleavage and/or processing of LRP and/or fragment(s) thereof.

86. A method for modulating LRP, comprising altering the structure, function and/or activity of a presenilin, and/or fragment(s) thereof, in a sample comprising LRP, and/or fragment(s) thereof, and a presenilin, and/or fragment(s) thereof, whereby the LRP is modulated.

87. A method for modulating LRP, comprising contacting a sample comprising an LRP, and/or fragment(s) thereof, and presenilin, and/or fragment(s) thereof, with an agent that modulates the presenilin and/or fragment(s) thereof or a presenilin-dependent activity, whereby LRP is modulated.

88. A method for identifying an agent that modulates Aβ levels, comprising: comparing the levels of bound Aβ binding protein in a test sample contacted with the test agent and a confrol sample not contacted with the test agent; and identifying an agent as an agent that modulates Aβ levels if the levels of bound Aβ binding protein differ in the test and confrol samples; wherein the sample comprises APP or portion(s) thereof; and the Aβ binding protein comprises the polypeptide of any of claims 1-14,

39-41 or 66.

89. A method for identifying an agent that modulates Aβ42 levels, comprising: comparing the levels of bound Aβ binding protein in a test sample contacted with the test agent and a confrol sample not contacted with the test agent; and identifying an agent as an agent that modulates Aβ42 levels if the levels of bound Aβ binding protein differ in the test and confrol samples; wherein the sample comprises APP or portion(s) thereof; and the Aβ binding protein comprises a sequence of amino acids selected from the group consisting of amino acids 1-50, 1-60, 1-70, 1-80, 1-90, 1-91, 1-92, 1-93, 1-94, 1-95, 1-96 and 1-97 of SEQ ED NO: 12 and 1-50, 1-60, 1-70, 1-80, 1-90, 1-91, 1-92, 1- 93, 1-94, 1-95, 1-96 and 1-97 of SEQ ED NO: 14 and any amino acid sequences containing modifications of these amino acid sequences that retain the Aβ binding properties of an antibody comprising one or both of the amino acid sequences set forth as amino acids 1-95 of SEQ ED NO: 12 and amino acids 1-97 of SEQ ED NO: 14.

90. A method for identifying an agent that modulates Aβ levels, comprising: assessing a test agent that modulates Aβ42 levels to determine if it modulates the level of one or more other Aβ peptides; and identifying an agent that modulates Aβ42 levels to a greater extent than it modulates the level of one or more other Aβ peptides.

91. A method for identifying an agent that modulates Aβ levels, comprising: assessing a test agent that modulates Aβ42 levels to determine if it modulates the level of one or more other Aβ peptides; and identifying an agent that modulates Aβ42 levels and Aβ39 levels.

92. A method for identifying an agent that modulates Aβ levels, comprising: assessing a test agent that alters the cleavage of APP that produces one or more Aβ peptides, the processing of APP, the processing of Aβ and/or the level of one or more Aβ peptides to determine if it effects one or more presenilin-dependent activities other than the presenilin-dependent processing of APP or portion(s) thereof; and identifying an agent that modulates Aβ levels without substantially altering one or more presenilin-dependent activities other than the presenilin-dependent processing of APP.

93. A method for identifying an agent that modulates Aβ levels, comprising: assessing a test agent that modulates the cleavage of APP that produces one or more Aβ peptides, the processing of APP, the processing of Aβ and/or the level of one or more Aβ peptides to determine if it effects the cleavage and/or processing of a presenilin substrate and/or portion(s) thereof other than APP; and identifying an agent that modulates Aβ levels without substantially altering the cleavage and/or processing of the presenilin subsfrate and/or portion(s) thereof that is other than APP.

94. A method for identifying an agent that modulates Aβ levels, comprising: assessing a test agent that modulates the cleavage of APP that produces one or more Aβ peptides, the processing of APP, the processing of Aβ and/or the level of one or more Aβ peptides to determine if it effects the cleavage and/or processing of LRP and/or portion(s) thereof; and identifying an agent that modulates Aβ levels without substantially altering the cleavage and/or processing of LRP and/or portion(s) thereof.

95. A system for use in assessing presenilin activity, comprising: a source of presenilin activity; a source of LRP (and/or portion(s) thereof); and a reagent for determining LRP protein composition.

96. A kit comprising: a reagent for assessing cleavage of APP that produces one or more Aβ peptides, APP processing, Aβ processing and/or Aβ levels; and a reagent for assessing cleavage and/or processing of a presenilin substrate.

97 A method for identifying a candidate agent for the freatment or prophylaxis of a disease, comprising: contacting a sample that contains an altered test protein, and/or portion(s) thereof, and APP, and/or portion(s) thereof, with a test agent, wherein the altered protein is associated with altered Aβ42 production, catabolism, processing and/or Aβ42 levels; and identifying a candidate agent that restores Aβ production, catabolism, processing and/or Aβ levels to substantially that which occurs in the presence of a test protein and/or portion(s) thereof that is not associated with altered Aβ42 production, catabolism, processing and/or Aβ42 levels without substantially altering the level of one or more other Aβ peptides.

98. A method for identifying a candidate agent for the treatment or prophylaxis of a disease, comprising: contacting a sample that contains an altered test protein, and/or portion(s) thereof, and APP, and/or portion(s) thereof, with a test agent, wherein the altered protein is associated with altered Aβ production, catabolism, processing and or Aβ levels; and identifying a candidate agent that restores Aβ production, catabolism, processing and/or Aβ levels to substantially that which occurs in the presence of a test protein and/or portion(s) thereof that is not associated with altered Aβ production, catabolism, processing and/or Aβ levels without substantially altering (a) one or more presenilin-dependent activities other than the presenilin-dependent processing of APP, (b) the cleavage and/or processing of a presenilin subsfrate and/or portion(s) thereof that is other than APP and/or (c) the cleavage and/or processing of LRP and/or portion(s) thereof.

99. A method for identifying an agent that modulates Aβ levels, comprising: assessing a test agent that alters the cleavage of APP that produces one or more Aβ peptides, the processing of APP, the processing of Aβ and/or the level of one or more Aβ peptides to determine if it affects one or more presenilin-dependent activities other than the presenilin-dependent processing of APP or portion(s) thereof that produces one or more Aβ peptides; and identifying an agent that modulates Aβ levels without substantially altering one or more presenilin-dependent activities other than the presenilin-dependent processing of APP or portion(s) thereof that produces one or more Aβ peptides.

100. A method for identifying an agent that modulates Aβ levels, comprising: assessing a test agent that modulates the cleavage of APP that produces one or more Aβ peptides, the processing of APP, the processing of Aβ and/or the level of one or more Aβ peptides to determine if it affects the cleavage and/or processing of APP and/or portion(s) thereof other than the processing of APP or portion(s) thereof that produces one or more Aβ peptides; and identifying an agent that modulates Aβ levels without substantially altering the cleavage and/or processing of APP and/or portion(s) thereof other than the processing of APP or portion(s) thereof that produces one or more Aβ peptides.