WO2007056401A1 - Method for identifying modulators of osbp useful for treating alzheimer's disease - Google Patents

Abstract

Methods for identifying modulators of OSBP are described. The methods are particularly useful for identifying analytes that antagonize OSBP 's effect on processing of amyloid precursor protein to Aβ peptide and thus useful for identifying analytes that can be used for treating Alzheimer disease.

Description

TITLE OF THE INVENTION

METHOD FOR IDENTIFYING MODULATORS OF OSBP USEFUL FOR TREATING

ALZHEIMER'S DISEASE

BACKGROUND OF THE INVENTION

( 1 ) Field of the Invention

The present invention relates to methods for identifying modulators of OSBP. The methods are particularly useful for identifying analytes that antagonize OSBP 's effect on processing of amyloid precursor protein to A/3 peptide and thus useful for identifying analytes that can be used for treating Alzheimer disease.

(2) Description of Related Art

Alzheimer's disease is a common, chronic neurodegenerative disease, characterized by a progressive loss of memory and sometimes severe behavioral abnormalities, as well as an impairment of other cognitive functions that often leads to dementia and death. It ranks as the fourth leading cause of death in industrialized societies after heart disease, cancer, and stroke. The incidence of Alzheimer's disease is high, with an estimated 2.5 to 4 million patients affected in the United States and perhaps 17 to 25 million worldwide. Moreover, the number of sufferers is expected to grow as the population ages. A characteristic feature of Alzheimer's disease is the presence of large numbers of insoluble deposits, known as amyloid plaques, in the brains of those affected. Autopsies have shown that amyloid plaques are found in the brains of virtually all Alzheimer's patients and that the degree of amyloid plaque deposition often correlates with the degree of dementia (Cummings & Cotman, Lancet 326: 1524-1587 (1995)). While some opinion holds that amyloid plaques are a late stage by-product of the disease process, the consensus view is that amyloid plaques and/or soluble aggregates of amyloid peptides are more likely to be intimately, and perhaps causally, involved in Alzheimer's disease.

A variety of experimental evidence supports this view. For example, amyloid /3 (A/3) peptide, a primary component of amyloid plaques, is toxic to neurons in culture and transgenic mice that overproduce A/3 peptide in their brains show extensive deposition of Aβ into amyloid plaques as well as significant neuronal toxicity (Yankner, Science 250: 279-282 (1990); Mattson et al, J. Neurosci. 12: 379- 389 (1992); Games et al, Nature 373: 523-527 (1995); LaFerla et al, Nature Genetics 9: 21-29 (1995)). Mutations in the APP gene, leading to increased Aβ production, have been linked to heritable forms of Alzheimer's disease (Goate et al, Nature 349: 704-706 (1991); Chartier-Harlan et al, Nature 353: 844- 846 (1991); Murrel et al, Science 254: 97-99 (1991); Mullan et al, Nature Genetics 1: 345-347 (1992)). Presenilin-1 (PSl) and presenilin-2 (PS2) related familial early-onset Alzheimer's disease (FAD) shows disproportionately increased production of A/31 -42, the 42 amino acid isoform of A/3, as opposed to A/31- 40, the 40 amino acid isoform (Scheuner et al, Nature Medicine 2: 864-870 (1996)). The longer isoform of A_/8 is more prone to aggregation than the shorter isoform (Jarrett et al, Biochemistry 32: 4693-4697 (1993). Injection of the insoluble, fibrillar form of AjS into monkey brains results in the development of pathology (neuronal destruction, tau phosphorylation, microglial proliferation) that closely mimics Alzheimer's disease in humans (Geula et al, Nature Medicine 4: 827-831 (1998)). See Selkoe, J.

Neuropathol. Exp. Neurol. 53: 438-447 (1994) for a review of the evidence that amyloid plaques have a central role in Alzheimer's disease.

Aβ peptide, a 39-43 amino acid peptide derived by proteolytic cleavage of the amyloid precursor protein (APP), is the major component of amyloid plaques (Glenner and Wong, Biochem. Biophys. Res. Comm. 120: 885- 890 (1984)). APP is actually a family of polypeptides produced by alternative splicing from a single gene. Major forms of APP are known as APP695, APP751, and APP770, with the subscripts referring to the number of amino acids in each splice variant (Ponte et al., Nature 331: 525-527 (1988); Tanzi et al, Nature 331: 528-530 (1988); Kitaguchi et al, Nature 331: 530- 532(1988)). APP is a ubiquitous membrane-spanning (type 1) glycoprotein that undergoes proteolytic cleavage by at least two pathways (Selkoe, Trends Cell Biol. 8: 447-453 (1998)). In one pathway, cleavage by an enzyme known as α-secretase occurs while APP is still in the trans-Golgi secretory compartment (Kuentzel et al., Biochem. J. 295:367-378 (1993)). This cleavage by α- secretase occurs within the Aβ peptide portion of APP, thus precluding the formation of Aβ peptide. In an alternative proteolytic pathway, cleavage of the Met596-Asp597 bond (numbered according to the 695 amino acid protein) by an enzyme known as β-secretase occurs. This cleavage by β-secretase generates the N- terminus of Aβ peptide. The C-terminus is formed by cleavage by a second enzyme known as γ- secretase. The C-terminus is actually a heterogeneous collection of cleavage sites rather than a single site since γ-secretase activity occurs over a short stretch of APP amino acids rather than at a single peptide bond. Peptides of 40 or 42 amino acids in length (Aβl-40 and Aβl-42, respectively) predominate among the C-termini generated by γ-secretase. Aβl-42 peptide is more prone to aggregation than Aβl-40 peptide, the major secreted species (Jarrett et al, Biochemistry 32: 4693-4697 91993); Kuo et al, J. Biol. Chem. 271 : 4077-4081 (1996)), and its production is closely associated with the development of Alzheimer's disease (Sinha and Lieberburg, Proc. Natl. Acad. Sci. USA 96: 11049-11053 (1999)). The bond cleaved by γ-secretase appears to be situated within the transmembrane domain of APP. For a review that discusses APP and its processing, see Selkoe, Trends Cell. Biol. 8: 447-453 (1998).

While abundant evidence suggests that extracellular accumulation and deposition of Aβ peptide is a central event in the etiology of Alzheimer's disease, recent studies have also proposed that increased intracellular accumulation of Aβ peptide or amyloid containing C-terminal fragments may play a role in the pathophysiology of Alzheimer's disease. For example, over-expression of APP harboring mutations which cause familial Alzheimer's disease results in the increased intracellular accumulation of C99, the carboxy-terminal 99 amino acids of APP containing Aβ peptide, in neuronal cultures and A/342 in HEK 293 cells in neuronal cultures and Aβ42 peptide in HEK 293 cells. Moreover, evidence suggests that intra- and extracellular Aβ peptide are formed in distinct cellular pools in hippocampal neurons and that a common feature associated with two types of familial Alzheimer's disease mutations in APP ("Swedish" and "London") is an increased intracellular accumulation of A/342 peptide. Thus, based on these studies and earlier reports implicating extracellular Aβ peptide accumulation in Alzheimer's disease pathology, it appears that altered APP catabolism may be involved in disease progression.

Much interest has focused on the possibility of inhibiting the development of amyloid plaques as a means of preventing or ameliorating the symptoms of Alzheimer's disease. To that end, a promising strategy is to inhibit the activity of β- and γ- secretase, the two enzymes that together are responsible for producing Aβ. This strategy is attractive because, if the formation of amyloid plaques is a result of the deposition of Aβ is a cause of Alzheimer's disease, inhibiting the activity of one or both of the two secretases would intervene in the disease process at an early stage, before late- stage events such as inflammation or apoptosis occur. Such early stage intervention is expected to be particularly beneficial (see, for example, Citron, Molecular Medicine Today 6:392-397 (2000)).

To that end, various assays have been developed that are directed to the identification of substances that may interfere with the production of AjS peptide or its deposition into amyloid plaques. U.S. Patent No. 5,441,870 is directed to methods of monitoring the processing of APP by detecting the production of amino terminal fragments of APP. U.S. Patent No. 5,605,811 is directed to methods of identifying inhibitors of the production of amino terminal fragments of APP. U.S. Patent No. 5,593,846 is directed to methods of detecting soluble AjS by the use of binding substances such as antibodies. US Published Patent Application No. US20030200555 describes using amyloid precursor proteins with modified /3-secretase cleavage sites to monitor beta-secretase activity. Esler et al, Nature Biotechnology 15: 258-263 (1997) described an assay that monitored the deposition of Aβ peptide from solution onto a synthetic analogue of an amyloid plaque. The assay was suitable for identifying substances that could inhibit the deposition of Aβ peptide. However, this assay is not suitable for identifying substances, such as inhibitors of β- or γ-secretase, that would prevent the formation of Aβ peptide.

Various groups have cloned and sequenced cDNA encoding a protein believed to be β- secretase (Vassar et al, Science 286: 735-741 (1999); Hussain et al, MoI. Cell. Neurosci. 14: 419- 427 (1999); Yan et al, Nature 402: 533-537 (1999); Sinha et al, Nature 402: 537-540 (1999); Lin et al, Proc. Natl. Acad. Sci. USA 97: 1456-1460 (2000)). U.S. Pat. Nos. 6,828,117 and 6,737,510 disclose a β- secretase, which the inventors call aspartyl protease 2 (Asp2), variant Asp-2(a) and variant Asp-2(b), respectively, and U.S Pat. No. 6,545,127 discloses a catalytically active enzyme known as memapsin. Hong et al, Science 290: 150-153 (2000) determined the crystal structure of the protease domain of human β-secretase complexed with an eight- residue peptide-like inhibitor at 1.9 angstrom resolution. Compared to other human aspartic proteases, the active site of human β-secretase is more open and less hydrophobic, contributing to the broad substrate specificity of human β-secretase (Lin et al, Proc. Natl. Acad. Sci. USA 97: 1456-1460 (2000)). Ghosh et ah, J. Am. Chem. Soc. 122: 3522-3523 (2000) disclosed two inhibitors of β- secretase, OM99-1 and OM99-2, that are modified peptides based on the β-secretase cleavage site of the Swedish mutation of APP (SEVNL/DAEFR, with "/" indicating the site of cleavage). OM99-1 has the structure VNL* AAEF (with "L* A" indicating the uncleavable hydroxyethylene transition-state isostere of the LA peptide bond) and exhibits a Ki towards recombinant β-secretase produced in E, coli of 6.84x 10-8 M±2.72x 10-9 M. OM99-2 has the structure EVNL* AAEF (with "L* A" indicating the uncleavable hydroxyethylene transition-state isostere of the LA peptide bond) and exhibits a Ki towards recombinant β-secretase produced in E. coli of 9.58x10-9 M±2.86xl0-10 M. OM99-1 and OM99-2, as well as related substances, are described in International Patent Publication WO0100665. Currently, most drug discovery programs for Alzheimer's disease have targeted either acetylcholinesterase or the secretase proteins directly responsible for APP processing. While acetylcholinesterase inhibitors are marketed drugs for Alzheimer's disease, they have limited efficacy and do not have disease modifying properties. Secretase inhibitors, on the other hand, have been plagued either by mechanism-based toxicity (γ-secretase inhibitors) or by extreme difficulties in identifying small molecule inhibitors with appropriate pharmacokinetic properties to allow them to become drugs (BACE inhibitors). Identifying novel factors involved in APP processing would expand the range of targets for Alzheimer's disease treatments and therapy.

BRIEF SUMMARY OF THE INVENTION

The present invention provides methods for identifying modulators of OSBP. The methods are particularly useful for identifying analytes that antagonize OSBP 's effect on processing of amyloid precursor protein to Aβ peptide and thus useful for identifying analytes that can be used for treating Alzheimer disease. Therefore, in one embodiment, the present invention provides a method for screening for analytes that antagonize processing of amyloid precursor protein (APP) to A/3 peptide, comprising providing recombinant cells, which ectopically expresses OSBP and the APP; incubating the cells in a culture medium under conditions for expression of the OSBP and APP and which contains an analyte; removing the culture medium from the recombinant cells; and determining the amount of at least one processing product of APP selected from the group consisting of sAPP/3 and A/3 peptide in the medium wherein a decrease in the amount of the processing product in the medium compared to the amount of the processing product in medium from recombinant cells incubated in medium without the analyte indicates that the analyte is an antagonist of the processing of the APP to Aβ peptide.

In further aspects of the method, the recombinant cells each comprises a first nucleic acid that encodes OSBP operably linked to a first heterologous promoter and a second nucleic acid that encodes an APP operably linked to a second heterologous promoter. In preferred aspects of the present invention, the APP is APPNFEV- &¹ preferred aspects, the method includes a control which comprises providing recombinant cells that ectopically express the APP but not the OSBP.

The present invention further provides a method for screening for analytes that antagonize processing of amyloid precursor protein (APP) to amyloid β (Aβ) peptide, comprising providing recombinant cells, which ectopically express OSBP and a recombinant APP comprising APP fused to a transcription factor that when removed from the APP during processing of the APP produces an active transcription factor, and a reporter gene operably linked to a promoter inducible by the transcription factor; incubating the cells in a culture medium under conditions for expression of the OSBP and recombinant APP and which contains an analyte; and determining expression of the reporter gene wherein a decrease in expression of the reporter gene compared to expression of the reporter gene in recombinant cells in a culture medium without the analyte indicates that the analyte is an antagonist of the processing of the APP to A/3 peptide.

In further aspects of the method, the recombinant cells each comprises a first nucleic acid that encodes OSBP operably linked to a first heterologous promoter, a second nucleic acid that encodes the recombinant APP operably linked to a second heterologous promoter, and a third nucleic acid that encodes a reporter gene operably linked to promoter responsive to the transcription factor comprising the recombinant APP.

In light of the analytes that can be identified using the above methods, the present invention further provides a method for treating Alzheimer's disease in an individual which comprises providing to the individual an effective amount of an antagonist of OSBP activity.

Further still, the present invention provides a method for identifying an individual who has Alzheimer's disease or is at risk of developing Alzheimer's disease comprising obtaining a sample from the individual and measuring the amount of OSBP in the sample.

Further still, the present invention provides for the use of an antagonist of OSBP for the manufacture of a medicament for the treatment of Alzheimer' s disease.

Further still, the present invention provides for the use of an antibody specific for OSBP for the manufacture of a medicament for the treatment of Alzheimer's disease.

Further still, the present invention provides a vaccine for preventing and/or treating Alzheimer's disease in a subject, comprising an antibody raised against an antigenic amount of OSBP wherein the antibody antagonizes the processing of APP to Aβ peptide.

The term "analyte" refers to a compound, chemical, agent, composition, antibody, peptide, aptamer, nucleic acid, or the like, which can modulate the activity of OSBP.

The term "OSBP" refers to oxysterol binding protein (Official Gene Symbol OSBP, NM_002556), which is a gene from a human or another mammal having an open reading frame coding for a protein of 807 amino acids in length (SEQ ID NO:2). The term further includes mutants, variants, alleles, and polymorphs of OSBP. Where appropriate, the term further includes fusion proteins comprising all or a portion of the amino acid sequence of OSBP fused to the amino acid sequence of a heterologous peptide or polypeptide, for example, hybrid immuoglobulins comprising the amino acid sequence of OSBP or OSBP fused at its C-terminus to the N-terminus of an immunoglobulin constant region amino acid sequence (see, for example, U.S. Patent No. 5,428,130 and related patents).

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a nucleic sequence encoding the human OSBP.

Figure 2 is the amino acid sequence of the human OSBP. Figure 3 is a graph showing the Relative expression of the metabolites expressed as a percent of the mean control non-silencing siRNA value of 100.

Figure 4 shows the tissue distribution of OSBP mRNA in various human tissues.

DETAILED DESCRIPTION OF THE INVENTION One strategy for treating AD is to identify factors that predispose subjects to the disease and target them therapeutically, as such factors have been found to cause aberrant Aβ metabolism in the majority of patients (most of whom do not have mutations in APP or a secretase related gene). Genetic, epidemiological, biochemical, and pharmacological data all demonstrate that cholesterol metabolism is intimately linked to the pathogenesis of Alzheimer's disease and thus represents an opportunity to intervene in the disease. The greatest known genetic risk factor for Alzheimer's disease is the ε4 allele of the apolipoprotein E gene (APOE) (Poirier et al, Neurobiol Aging. 26: 355 (2005)). APOE encodes a secreted protein that functions as the predominant lipid carrier in the brain, as well as a major determinant of peripheral cholesterol metabolism. APOE genotype determines low-density lipoprotein (LDL)- associated cholesterol levels in humans (Menzel, H.J., et al, Arteriosclerosis, 3: 310 (1983), demonstrating that APOE protein is a critical regulator of cholesterol homeostasis. It has been observed that patients who take statins, which inhibit cholesterol synthesis and lower circulating LDL cholesterol by blocking the enzyme HMG-CoA reductase, are at reduced risk for AD later in life (Jick H., et al, Lancet, 356: 1627 (2000); Wolozin B., et al, Arch. Neurol, 57: 1439 (2000)). Statins have been found to reduce amyloid plaque burden in animal models (Fassbende, K., et al, Proc Natl Acad Sci USA, 98: 5856 (2001); Refolo, L.M., et al, Neurobiol. Pis., 8: 890 (2001)). Conversely, when animal models of AD are fed a high cholesterol diet, γ-secretase activity increased and plaque pathology worsened in their brains (Shie, F.S., et al, Neuroreport., 213: 455 (2002); Wahrle, S., et al, Neurobiol. Pis., 9: 11 (2002); Burns, M., et al, J. Neurosci., 23: 5645 (2003)). This suggests there a relationship between AD and cholesterol homeostasis. Despite such a suspected connection between cholesterol and AD, cholesterol metabolism in the brain is markedly different than in the periphery, making it difficult to determine a precise mechanism by which cholesterol affects AD pathogenesis. In contrast to all other organs, the brain synthesizes its cholesterol and receives virtually none from the circulation. Furthermore, cholesterol in the brain is mostly eliminated by conversion to 24(S)-hydroxycholesterol (24-OHC), which then diffuses across the blood-brain barrier and is converted by the liver into bile acid (reviewed in Bjorkhem, L, et al, Arterioscler. Thromb. Vase. Biol.. 24: 806 (2004). This mechanism of cholesterol elimination differs from other organs, which either rely on high-density lipoproteins for export of excess cholesterol or are able to store cholesterol in an esterified form. 24-OHC is formed by the enzyme Cyp46, a brain-selective cytochrome P450 enzyme (Lund, E.G., et al, Proc. Natl. Acad. Sci. USA, 96: 7238 (1999)). Although still unproven, several genetic studies suggest that Cyp46 is associated with a delay in the onset of AD (Wollmer, M.A., Neurobio. Aging, 24: 421 (2003); Katzov, H., et al, Hum. Mutat, 23: 358 (2004); Combarros, O., et al, Dement. Geriatr. Cogn. Disord., 18: 257 (2004); Papassotiropoulos, A., et ah, Arch. Neurol., 60:29 (2003)). Taken together the findings support a role for oxysterols in cholesterol metabolism in the brain and in Alzheimer' s disease.

Applicants have found that therapeutics targeting cholesterol and, in particular, oxysterol metabolism provide novel opportunities for the treatment of AD. In addition to the findings reported herein, this concept is supported by in vitro data suggesting that 24-OHC regulates the formation of Aβ from APP in vitro (Brown, J., et al, J. Biol. Chem., 279:34674 (2004)). Key signaling molecules for oxysterols include the nuclear receptor Liver X Receptor (LXR) (Steffensen, K.R., et al. , Diabetes, 53

Suppl 1: S36 (2004)) and the oxysterol binding proteins (OSBPs) (Olkkonen, V.M., et al, Biochem. Cell Biol., 82:87 (2004)).

More particularly, Applicants have discovered that the OSBP protein has a role in the processing of amyloid precursor protein (APP) to amyloid β (Aβ) peptide. OSBP is a key oxysterol signaling molecule (Olkkonen, V.M., et al, Biochem. Cell Biol., 82: 87 (2004)). OSBP is thought to exist in two locations within the cell depending on the availability of cholesterol (Wang, P. Y., et al, Science, 307:1472 (2005)). When cholesterol is in excess, OSBP is in a cytoplasmic complex with proteins that inhibit mitochondrial activating protein (MAP) kinase signaling. When oxysterol accumulates, then OSBP releases MAP kinase signaling. OSBP and its related proteins are also involved in the shuttling of lipids between intracellular membranes and the regulation of cellular lipid homeostasis (Olkkonen, V.M., et al, above). Thus, OSBP is a key regulator of cholesterol and oxysterol signaling and metabolism.

A defining characteristic of Alzheimer's disease (AD) is the deposition of aggregated plaques containing A/3 peptide in the brains of affected individuals. Thus, Applicants' discovery that OSBP has a role processing APP to Aβ peptide suggests that OSBP has a role in the progression of

Alzheimer's disease in an individual. Therefore, in light of this discovery, identifying molecules which target activity or expression of OSBP would be expected to lead to treatments or therapies for Alzheimer's disease. Expression or activity of OSBP may also be useful as a diagnostic marker for identifying individuals who have Alzheimer's disease or are at risk of developing Alzheimer's disease.

The deposition of aggregated plaques containing amyloid β (A/3) peptide in the brains of individuals affected with Alzheimer's disease is believed to involve the sequential cleavage of APP by two secretase-mediated cleavages to produce A/3 peptide. The first cleavage event is catalyzed by the type I transmembrane aspartyl protease BACEl. BACEl cleavage of APP at the BACE cleavage site (between amino acids 596 and 597) generates a 596 amino acid soluble N-terminal sAPP/3 fragment and a 99 amino acid C-terminal fragment (jSCTF) designated C99. Further cleavage of C99 by γ-secretase (a multicomponent membrane complex consisting of at least presenilin, nicastrin, aphl, and pen2) releases the 40 or 42 amino acid AjS peptide. An alternative, non-amyloidogenic pathway of APP cleavage is catalyzed by γ-secretase, which cleaves APP to produce a 613 amino acid soluble sAPPα N-terminal fragment and an 83 amino acid /3CTF fragment designated C83. While ongoing drug discovery efforts have focused on identifying antagonists of BACEl and 7-secretase mediated cleavage of APP, the complicated nature of Alzheimer's disease suggests that efficacious treatments and therapies for

Alzheimer's disease might comprise other targets for modulating APP processing. OSBP of the present invention is another target for which modulators (in particular, antagonists) of are expected to provide efficacious treatments or therapies for Alzheimer's disease, either alone or in combination with one or more other modulators of APP processing, for example, antagonists selected from the group consisting of BACEl and γ-secretase.

OSBP was identified by screening a siRNA library for siRNA that inhibited APP processing. As described in Example 1, a library of about 15,200 siRNA pools, each targeting a single gene, was transfected individually into recombinant cells ectopically expressing a recombinant APP (APPiSiFEV)- APPNFEV has been described in U.S. Pub. Pat. Appln. No. 20030200555, comprises isoform 1-695 and has a HA, Myc, and FLAG sequences at the amino acid position 289, an optimized /3- cleavage site comprising amino acids NFEV, and a K612V mutation. Metabolites of APPNFEV produced during APP BACEl/γ-secretase or α-secretase processing are sAPP/3 with NF at the C-terminus, EV40, and EV42 or sAPPα. EV40 and EV42 are unique A/340-like and A/342-like peptides that contain the glutamic acid and valine substitutions of APPNFEV ^aiJd sAPP|3 and sAPPα each contain the HA, FLAG, and myc sequences. sAPP/3, sAPPα, EV40, and EV42 were detected by an immunodetection method that used antibodies that were specific for the various APPNFEV metabolites. Expression levels were determined relative to a non-silencing siRNA control.

Following two rounds of screening, which consisted of a primary screen done with the entire library of siRNAs and secondary screening of about 1600 siRNAs performed in triplicate repeats, a siRNA designed to target OSBP RNA was found to consistently alter processing of APP to sAPP/3, EV40, and EV42.

The nucleic acid sequence encoding the human OSBP (SEQ ID NO:1) is shown in Figure 1 and the amino acid sequence for the human OSBP (SEQ ID NO:2) is shown in Figure 2. The mJRNA encoding OSBP was found to be expressed in many tissues, including at low levels in regions of the brain subject to Alzheimer's disease pathology (Example 2).

In light of the applicants' discovery, OSBP or modified mutants or variants thereof is useful for identifying analytes which antagonize processing of APP to produce Aβ peptide. These analytes can be used to treat patients afflicted with Alzheimer's disease. OSBP can also be used to help diagnose Alzheimer's disease by assessing genetic variability within the locus. OSBP can be used alone or in combination with acetylcholinesterase inhibitors, NMDA receptor partial agonists, secretase inhibitors, amyloid-reactive antibodies, growth hormone secretagogues, and other treatments for Alzheimer's disease.

The present invention provides methods for identifying OSBP modulators that modulate expression of OSBP by contacting OSBP with a substance that inhibits or stimulates OSBP expression and determining whether expression of OSBP polypeptide or nucleic acid molecules encoding an OSBP are modified. The present invention also provides methods for identifying modulators that antagonize OSBP' s effect on processing APP to A/3 peptide or formation of A/3-amyloid plaques in tissues where OSBP is localized or co-expressed. For example, OSBP protein can be expressed in cell lines that also express APP and the effect of the modulator on A/3 production is monitored using standard biochemical assays with A/3-specifϊc antibodies or by mass spectrophotometric techniques. Inhibitors for OSBP are identified by screening for a reduction in the release of Aβ peptide which is dependent on the presence of OSBP protein for effect. Both small molecules and larger biomolecules that antagonize OSBP-mediated processing of APP to Aβ peptide can be identified using such an assay. A method for identifying antagonists of OSBP's effect on the processing APP to A/3 peptide includes the following method which is amenable to high throughput screening. In addition, the methods disclosed in U.S. Pub. Pat. Appln. No. 20030200555 can be adapted to use in assays for identifying antagonists of OSBP activity.

A mammalian OSBP cDNA, encompassing the first through the last predicted codon contiguously, is amplified from brain total RNA with sequence-specific primers by reverse-transcription polymerase chain reaction (RT-PCR). The amplified sequence is cloned into pcDNA3.zeo or other appropriate mammalian expression vector. Fidelity of the sequence and the ability of the plasmid to encode full-length OSBP is validated by DNA sequencing of the OSBP plasmid (pcDNA_OSBP).

Commercially available mammalian expression vectors which are suitable for recombinant OSBP expression include, but are not limited to, pcDNA3.neo (Invitrogen, Carlsbad, CA), pcDNA3.1 (Invitrogen, Carlsbad, CA), ρcDNA3.1/Myc-His (Invitrogen), pCI-neo (Promega, Madison, WI), pLITMUS28, pLITMUS29, pLITMUS38 and pLITMUS39 (New England Biolabs, Beverly, MA), pcDNAI, pcDNAIamp (Invitrogen), pcDNA3 (Invitrogen), pMClneo (Stratagene, La Jolla, CA), pXTl (Stratagene), pSG5 (Stratagene), EBO-pSV2-neo (ATCC 37593) pBPV-l(8-2) (ATCC 37110), pdBPV- MMTneo (342-12) (ATCC 37224), pRSVgpt (ATCC 37199), pRSVneo (ATCC 37198), pSV2-dhfr (ATCC 37146), pUCTag (ATCC 37460), 1ZD35 (ATCC 37565), pMClneo (Stratagene), pcDNA3.1, pCR3.1 (Invitrogen, San Diego, Calif.), EBO-pSV2-neo (ATCC 37593), pCLneo (Promega), pTRE (Clontech, Palo Alto, Calif.), pVUneo, pIRESneo (Clontech, Palo Alto, Calif.), pCEP4 (Invitrogen,), pSCl 1, and pSV2-dhfr (ATCC 37146). The choice of vector will depend upon the cell type in which it is desired to express the OSBP, as well as on the level of expression desired, cotransfection with expression vectors encoding APPNFEV; ^an<^ ^{tne u}ke. Cells transfected with plasmid vector comprising APP]SIFEV₅ f°^r example the

HEK293T/APPNPEV ^{ce s use}d to detect OSBP activity in the siRNA screening experiment described in Example 1, are used as described in Example 1 with the following modifications. Cells are either cotransfected with a plasmid expression vector comprising APP]SfFEV operably linked to a heterologous promoter and a plasmid expression vector comprising the OSBP operably linked to a heterologous promoter or the HEK293T/APPNFEV cells described in Example 1 and U.S. Pub. Pat. Appln.

20030200555 are transfected with a plasmid expression vector comprising the OSBP operably linked to a heterologous promoter. The promoter comprising the plasmid expression vector can be a constitutive promoter or an inducible promoter. Preferably, the assay includes a negative control comprising the expression vector without the OSBP. After the cells have been transfected, the transfected or cotransfected cells are incubated with an analyte being tested for ability to antagonize OSBP's effect on processing of APP to A/3 peptide. The analyte is assessed for an effect on the OSBP transfected or cotransfected cells that is minimal or absent in the negative control cells. In general, the analyte is added to the cell medium the day after the transfection and the cells incubated for one to 24 hours with the analyte. In particular embodiments, the analyte is serially diluted and each dilution provided to a culture of the transfected or cotransfected cells. After the cells have been incubated with the analyte, the medium is removed from the cells and assayed for secreted sAPPα, sAPP/3, EV40, and EV42 as described in Examples 1 and 5. Briefly, the antibodies specific for each of the metabolites is used to detect the metabolites in the medium. Preferably, the cells are assessed for viability. Analytes that alter the secretion of one or more of EV40, EV42, sAPPα, or sAPP/3 in the presence of OSBP protein are considered to be modulators of OSBP and potentially useful as therapeutic agents for OSBP-related diseases. Direct inhibition or modulation of OSBP can be confirmed using binding assays using the full-length OSBP, extracellular or intracellular domain thereof or a OSBP fusion proteins comprising the intracellular or extracellular domains coupled to a C-terminal FLAG, or other, epitopes. A cell-free binding assay using full-length OSBP, extracellular or intracellular domain thereof or a OSBP fusion proteins or membranes containing the OSBP integrated therein and labeled-analyte can be performed and the amount of labeled analyte bound to the OSBP determined.

The present invention further provides a method for measuring the ability of an analyte to modulate the level of OSBP mRNA or protein in a cell. In this method, a cell that expresses OSBP is contacted with a candidate compound and the amount of OSBP mRNA or protein in the cell is determined. This determination of OSBP levels may be made using any of the above-described immunoassays or techniques disclosed herein. The cell can be any OSBP expressing cell such as cell transfected with an expression vector comprising OSBP operably linked to its native promoter or a cell taken from a brain tissue biopsy from a patient. The present invention further provides a method of determining whether an individual has an OSBP-associated disorder or a predisposition for an OSBP-associated disorder. The method includes providing a tissue or serum sample from an individual and measuring the amount of OSBP in the tissue sample. The amount of OSBP in the sample is then compared to the amount of OSBP, in a control sample. An alteration in the amount of OSBP in the sample relative to the amount of OSBP in the control sample indicates the subject has an OSBP-associated disorder. A control sample is preferably taken from a matched individual, that is, an individual of similar age, sex, or other general condition but who is not suspected of having an OSBP related disorder. In another aspect, the control sample may be taken from the subject at a time when the subject is not suspected of having a condition or disorder associated with abnormal expression of OSBP. Other methods for identifying inhibitors of OSBP can include blocking the interaction between OSBP and the enzymes involved in APP processing or trafficking using standard methodologies for analyzing protein-protein interaction such as fluorescence energy transfer or scintillation proximity assay. Surface Plasmon Resonance can be used to identify molecules that physically interact with purified or recombinant OSBP. In accordance with yet another embodiment of the present invention, there are provided antibodies having specific affinity for the OSBP or epitope thereof. The term "antibodies" is intended to be a generic term which includes polyclonal antibodies, monoclonal antibodies, Fab fragments, single VJΪ chain antibodies such as those derived from a library of camel or llama antibodies or camelized antibodies (Nuttall et ah, Curr. Pharm. Biotechnol. 1: 253-263 (2000); Muyldermans, J. Biotechnol. 74: 277-302 (2001)), and recombinant antibodies. The term "recombinant antibodies" is intended to be a generic term which includes single polypeptide chains comprising the polypeptide sequence of a whole heavy chain antibody or only the amino terminal variable domain of the single heavy chain antibody (VJJ chain polypeptides) and single polypeptide chains comprising the variable light chain domain (VL) linked to the variable heavy chain domain (VH) to provide a single recombinant polypeptide comprising the Fv region of the antibody molecule (scFv polypeptides) {see Schmiedl et at, J. Immunol. Meth. 242: 101-114 (2000); Schultz et ah, Cancer Res. 60: 6663-6669 (2000); Dϋbel et ah, J. Immunol. Meth. 178: 201-209 (1995); and in U.S. Patent No. 6,207,804 Bl to Huston et al). Construction of recombinant single VH chain or scFv polypeptides which are specific against an analyte can be obtained using currently available molecular techniques such as phage display (de Haard et ah, J. Biol. Chem. 274: 18218-18230 (1999); Saviranta et ah, Bioconiugate 9: 725-735 (1999); de Greeff et ah, Infect. Immun. 68: 3949-3955 (2000)) or polypeptide synthesis. In further embodiments, the recombinant antibodies include modifications such as polypeptides having particular amino acid residues or ligands or labels such as horseradish peroxidase, alkaline phosphatase, fluors, and the like. Further still embodiments include fusion polypeptides which comprise the above polypeptides fused to a second polypeptide such as a polypeptide comprising protein A or G.

The antibodies specific for OSBP can be produced by methods known in the art. For example, polyclonal and monoclonal antibodies can be produced by methods well known in the art, as described, for example, in Harlow and Lane, Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory Press: Cold Spring Harbor, NY (1988). The OSBP or fragments thereof can be used as imrnunogens for generating such antibodies. Alternatively, synthetic peptides can be prepared (using commercially available synthesizers) and used as immunogens. Amino acid sequences can be analyzed by methods well known in the art to determine whether they encode hydrophobic or hydrophilic domains of the corresponding polypeptide. Altered antibodies such as chimeric, humanized, CDR-grafted, or bifunctional antibodies can also be produced by methods well known in the art. Such antibodies can also be produced by hybridoma, chemical synthesis or recombinant methods described, for example, in Sambrook et ah, supra., and Harlow and Lane, supra. Both anti-peptide and anti-fusion protein antibodies can be used {see, for example, Bahouth et ah, Trends Pharmacol. Sci. 12: 338 (1991); Ausubel et ah, Current Protocols in Molecular Biology, (John Wiley and Sons, N. Y. (1989)).

Antibodies so produced can be used for the immunoaffinity or affinity chromatography purification of OSBP or OSBP/ligand or analyte complexes. The above referenced anti-OSBP antibodies can also be used to modulate the activity of OSBP in living animals, in humans, or in biological tissues isolated therefrom. Accordingly, contemplated herein are compositions comprising a carrier and an amount of an antibody having specificity for OSBP effective to block naturally occurring OSBP from binding its ligand or for effecting the processing of APP to AjS peptide. Therefore, in another aspect, the present invention further provides pharmaceutical compositions that antagonize OSBP's effect on processing of APP to A/3 peptide. Such compositions include a OSBP nucleic acid, OSBP peptide, fusion protein comprising OSBP or fragment thereof coupled to a heterologous peptide or protein or fragment thereof, an antibody specific for OSBP, nucleic acid or protein aptamers, siRNA inhibitory to OSBP mRNA, analyte that is a OSBP antagonist, or combinations thereof, and a pharmaceutically acceptable carrier or diluent. In a further still aspect, the present invention further provides a kit for in vitro diagnosis of disease by detection of OSBP in a biological sample from a patient. A lot for detecting OSBP preferably includes a primary antibody capable of binding to OSBP; and a secondary antibody conjugated to a signal-producing label, the secondary antibody being capable of binding an epitope different from, i.e., spaced from, that to which the primary antibody binds. Such antibodies can be prepared by methods well-known in the art. This kit is most suitable for carrying out a two-antibody sandwich immunoassay, e.g., two-antibody sandwich ELISA.

Using derivatives of OSBP protein or cDNA, dominant negative forms of OSBP that could interfere with OSBP-mediated APP processing to A/3 release can be identified. These derivatives could be used in gene therapy strategies or as protein-based therapies top block OSBP activity in afflicted patients. OSBP can be used to identify endogenous brain proteins that bind to OSBP using biochemical purification, genetic interaction, or other techniques common to those skilled in the art. These proteins or their derivatives can subsequently be used to inhibit OSBP activity and thus be used to treat Alzheimer's disease. Additionally, polymorphisms in the OSBP RNA or in the genomic DNA in and around OSBP could be used to diagnose patients at risk for Alzheimer's disease or to identify likely responders in clinical trials.

The following examples are intended to promote a further understanding of the present invention.

EXAMPLE l OSBP was identified in a screen of a siRNA library for modulators of APP processing.

A cell plate was prepared by plating HEK293T/APPNFEV cells to the wells of a 384- well Corning PDL-coated assay plate at a density of about 2,000 cells per well in 40 μL DMEM containing 10% fetal bovine serum (FBS) and antibiotics. The cell plate was incubated overnight at 37°C in 5% Cθ2- HEK293T/APPNFEV cells are a subclone of HEK293T cells stably transformed with the APPNFEV plasmid described in U.S. Pub. Pat. Appl. No. 20030200555. In brief, APPNFEV encodes human amyloid precursor protein (APP), isoform 1-695, modified at amino acid position 289 by an in- frame insertion of HA, Myc, and FLAG epitope amino acid sequences and at amino acid positions 595, 596, 597, and 598 by substitution of the amino acid sequence NFEV for the endogenous amino acid sequence KMDA sequence comprising the BACEl cleavage site. Thus, the BACE cleavage site is a modified BACEl cleavage site and BACEl cleaves between amino acids F and E of NFEV.

Maintenance of the plasmid within the subclone is achieved by culturing the cells in the presence of the antibiotic puromycin.

The next day, the cells in each of the wells of the cell plate were transfected with a siRNA library as follows. Oligofectamine™ (Invitrogen, Inc., Carlsbad, CA) was mixed with Opti- MEM® (Invitrogen, Inc, Carlsbad, CA) at a ratio of 1 to 40 and 20 μL of the mixture was added to each well of a different 384-well plate. To each well of the plate, 980 nL of a particular 10 μM siKNA species was added and the plate incubated for ten minutes at room temperature. Afterwards, five μL of each the siRNA/Oligofectamine/Opti-MEM® mixtures was added to a corresponding well in the cell plate containing the HEK293/APPNFEV cells. The cell plate was incubated for 24 hours at 37⁰C in 5% Cθ2- Controls were provided which contained non-silencing siRNA or a siRNA that inhibited BACEl .

On the next day, for each of the wells of the cell plate, the siRNA and

Oligofectamine/Opti-MEM® mixture was removed and replaced with 70 μL DMEM containing 10% FBS and MERCK compound A (see WO2003093252, Preparation of spirocyclic [l,2,5]thiadiazole derivatives as γ-secretase inhibitors for treatment of Alzheimer's disease, Collins et al.), a γ-secretase inhibitor given at a final concentration equal to its IC50 ∞ cell-based enzyme assays. The cell plate was incubated for 24 hours at 37°C in 5% CO2.

On the next day, for each of the wells of the cell plate, 64 μL of the medium (conditioned medium) was removed and transferred to four 384-well REMP plates in 22, 22, 10, and 10 μL aliquots for subsequent use in detecting sAPPα, EV42, EV40, sAPPβ using AlphaScreen™ (PerkinElmer, Wellesley, MA) detection technology. Viability of the cells was determined by adding 40 μL 10% AlamarBlue

(Serotec, Inc., Raleigh, NC) in DMEM containing 10% FBS to each of the wells of the cell plate with the conditioned medium removed. The cell plate was then incubated at 37⁰C for two hours. The Acquest™ Molecular Devices Corporation, Sunnyvale, CA) plate reader was used to assay fluorescence intensity (ex. 545 nm, em. 590 nm) as a means to confirm viability of the cells. Assays for detecting and measuring sAPPβ, EV42, EV40, and sAPPα were detected using antibodies as follows. In general, detection-specific volumes (8 or 0.5 μL) were transferred to a small volume detection plate (Greiner Bio-One, Monroe, NC). In the case of the smaller volume, 7.5 μL of assay medium was added for a final volume of eight μL per well. One μL of Antibody/Donor bead mixture (see below) was dispensed into the solution, and one μL antibody/Acceptor bead mixture was added. Plates were incubated in the dark for 24 hours at 4⁰C. Then the plates were read using Alpha

Quest™ (PerkinElmer, Wellesley, MA). In all protocols, the plating medium was DMEM (Invitrogen, La Jolla, CA; Cat. No. 21063-029); 10% FBS, the AlphaScreen™ buffer was 50 mM HEPES, 150 mM NaCl, 0.1% BSA, 0.1% Tween-20, pH 7.5, and the AlphaScreen ™ Protein A kit was used.

Anti-NF antibodies and anti-EV antibodies were prepared as taught in U.S. Pub. Pat. Appln. 20030200555. BACEl cleaves between amino acids F and E of the NFEV cleavage site of

APP]SfP¹EV ^to produce a sAPP/3 peptide with NF at the C-terminus and an EV40 or EV42 peptide with amino acids EV at the N-terminus. Anti-NF antibodies bind the C-terminal neoepitope NF at the C- terminus of the sAPP/3 peptide produced by BACEl cleavage of the NFEV sequence of APPNFEV- Anti-EV antibodies bind the N-terminal neoepitope EV at the N-terminus of EV40 and EV42 produced by BACElcleavage of the NFEV sequence of APPNFEV- Anti-Bio-G2-10 and anti-Bio-G2-l 1 antibodies are available from the Genetics Company, Zurich, Switzerland. Anti-Bio-G2-11 antibodies bind the neoepitope generated by the γ-secretase cleavage of Aβ or EV peptides at the 42 amino acid position. Anti-Bio-G2-10 antibodies bind the neoepitope generated by the γ-secretase cleavage of Aβ or EV peptides at the 40 amino acid position. Anti-6E10 antibodies are commercially available from Signet Laboratories, Inc., Dedham, MA. Anti-6E10 antibodies bind the epitope within amino acids 1 to 17 of the N-terminal region of the Aβ and the EV40 and EV42 peptides and also binds sAPPα because the same epitope resides in amino acids 597 to 614 of sAPPα. Bio-M2 anti-FLAG antibodies are available from Sigma-Aldrich, St. Louis, MO.

Detecting sAPPβ. An AlphaScreen™ screen assay for detecting sAPPβ-NF produced from cleavage of APP]SIFEV ^at the BACE cleavage site was performed as follows. Conditioned medium for each well was diluted 32-fold into a final volume of eight μL. As shown in Table 1, biotinylated-M2 anti-FLAG antibody, which binds the FLAG epitope of the APPNFEV; ^was captured on streptavidin- coated donor beads by incubating a mixture of the antibody and the streptavidin coated beads for one hour at room temperature in AlphaScreen™ buffer. The amount of antibody was adjusted such that the final concentration of antibody in the detection reaction was 3 nM. Anti-NF antibody was similarly captured separately on protein-A acceptor beads in AlphaScreen™ buffer and used at a final concentration of 1 nM (Table 1). The donor and acceptor beads were each used at final concentrations of 20 μg/mL.

Table 1

Detecting EV42: Conditioned medium for each well was used neat (volume eight μL). As shown in Table 2, anti-Bio-G2-l 1 antibody was captured on streptavidin-coated donor beads by incubating a mixture of the antibody and the streptavidin coated beads for one hour at room temperature in AlphaScreen™ buffer. The amount of antibody was adjusted such that the final concentration of antibody in the detection reaction was 20 nM. Anti-EV antibody was similarly captured separately on protein-A acceptor beads in AlphaScreen™ buffer and used at a final concentration of 5 nM (Table 2). The donor and acceptor beads were used at final concentrations of 20 μg/mL. Table 2

Detecting EV40: Conditioned medium for each well was diluted four-fold into a final volume eight μL. As shown in Table 3, anti-Bio-G2-10 antibody was captured on streptavidin-coated donor beads by incubating a mixture of the antibody and the streptavidin coated beads for one hour at room temperature in AlphaScreen™ buffer. The amount of antibody was adjusted such that the final concentration of antibody in the detection reaction was 20 nM. Anti-EV antibody was similarly captured separately on protein-A acceptor beads in AlphaScreen™ buffer and used at a final concentration of 5 nM. The donor and acceptor beads were used at final concentrations of 20 μg/mL.

Table 3

Detecting sAPPce Conditioned medium for each well was diluted four-fold into a final volume eight μL. As shown in Table 4, Bio-M2 anti-FLAG antibody was captured on streptavidin-coated donor beads by incubating a mixture of the antibody and the streptavidin coated beads for one hour at room temperature in AlphaScreen™ buffer. Anti-6E10 antibody acceptor beads supplied by the manufacturer (Perkin-Elmer, Inc. makes the beads and conjugates antibody 6E10 to them). Antibody 6E10 (made by Signet Laboratories, Inc., a Covance Company, Dedham, MA) were used at 30 μg/ml final concentration. The donor beads were used at final concentrations of 20 μg/mL. Table 4

About 15,200 single replicate pools of siRNAs were tested for modulation of sAPPβ, sAPPα, EV40 and EV42 by the AlphaScreen™ immunodetection method as described above. Based on the profile from this primary screen, 1,622 siRNA were chosen for an additional round of screening in triplicate. siRNAs were defined as "secretase-like" if a significant decrease in sAPPβ, EV40 and EV42 was detected as well as either no change or an increase in sAPPα.

A siRNA was identified which inhibited an mRNA having a nucleotide sequence encoding a protein which had 100% identity to the nucleotide sequence encoding OSBP. Compared to control non-silencing siRNAs (set to 100%), OSBP siRNA pool significantly decreased EV40 (56.5%), EV42 (68.4%), while increasing sAPPα (339.1%).

The results are shown schematically in Figure 3 and show that OSBP has a role in APP processing, in particular, the cleavage of APP at the BACE cleavage site, an event necessary in the processing of APP to Aβ peptide. Aβ peptide is a defining characteristic of Alzheimer's disease. Because of its role APP processing, OSBP appears to have a role in the establishment or progression of Alzheimer's disease.

EXAMPLE 2

Because OSBP appeared to have a role in APP processing to A/3 peptide and thus, a role in progression of Alzheimer's disease, expression of OSBP was examined in a variety of tissues to determine whether OSBP was expressed in the brain.

A proprietary database, the TGI Body Atlas, was used to show that the results of a microarray analysis of the expression of a majority of characterized genes, including OSBP, in the human genome in a panel of different tissues. OSBP mRNA was found to be expressed throughout the body, including in the brain and within cortical structures such as the temporal lobe, entorhinal cortex, and prefrontal cortex, all of which are subjected to amyloid Aβ deposition and Alzheimer pathology. The results are summarized in Figure 4.

The results strengthen the conclusion of the Example 1 that OSBP has a role in APP processing and thus, a role in the establishment or progression of Alzheimer's disease. EXAMPLE 3

The results of Examples 1 and 2 have shown that the OSBP has a role in the establishment or progression of Alzheimer's disease. The results suggest that analytes that antagonize OSBP activity will be useful for the treatment or therapy of Alzheimer's disease. Therefore, there is a need for assays for identifying analytes that antagonize OSBP activity, for example, inhibit binding of OSBP to cholesterol or oxysterol. The following is an assay that can be used to identify analytes that antagonize OSBP activity.

HEK293T/APPNFEV ^ceUs are transfected with a plasmid encoding the human OSBP or a homolog of the human OSBP, for example, the primate, rodent, or other mammalian OSBP, using a standard transfection protocols to produce HEK293T/APPNFEV/°SBP cells. For example,

HEK293T/APPNFEV are plated into a 6-well plate at about 500,000 cells per well in 2000 μL DMEM containing 10%FBS and the cell plate incubated at 37<>C at 5% CO2 overnight.

On the next day, a mixture of 20 μL Oligofectamine™ and 500 μL Opti-MEM® is made and incubated at room temperature for five minutes. 2000 ng pcDNA_OSBP or empty control vector in 500 μL of Opti-MEM® are added and incubated at room temperature for five minutes. Next, this mixture is added to of 1 well of a 6 well plate containing HEK293/APP]SfPΕv ^cens plated in the cell plate and the plates incubated for 48 h at 37°C in 5% CO2.

The next day, the medium is removed from each well and cells are harvested in 0.4 mL 20 mM Tris pH 8, 1 mM MgCl₂, 2 mM CaCl₂, 2 mM dithiothreitol, 5% sucrose, and protease inhibitor cocktail without EDTA (Roche). Cells are homogenized in a dounce 30-35 strokes then sonicated on ice 4-5 pulses with a probe sonicator. Protein concentration is determined by OD₂so- Lysate is aliquoted into 0.1 mg aliquots per condition. 100 nM [H]3-25-hydroxycholesterol (Perkin-Elmer) is added plus or minus analytes at varying concentration or 0.01 mM unlabeled 25-hydroxycholesterol to the lysate (unlabeled 25-hydroxycholesterol is required to determine background binding). Binding of the labeled hydroxycholesterol proceeds overnight at 20° C. Next day, 0.05 mL 5% vol/vol charcoal dextran in Tris pH 8 is added to each reaction. After incubation at 20° C for thirty minutes, with shaking every five minutes, each condition is centrifuged at 10,000 x g for ten minutes at 4°C. 0.05 ml of each reaction is added to 5 mL of scintillation fluid and counted in a liquid scintillation counter. Analytes being assayed for the ability to antagonize hydroxycholesterol binding to OSBP are identified by reduction in counts per minute.

The analytes are then assessed for an effect on the APP processing to A/3 peptide in OSBP transfected cells that is either minimal or absent in cells transfected with the vector-alone as follows. Cells are transfected as above and are treated overnight with varying concentrations of analyte. The cells are incubated at 37°C at 5% CO2 overnight. The next day, conditioned media is collected in the amount of sAPPβ, EV42, EV40, and sAPPα in the conditioned media is determined as described in Example 1. Analytes that effect a decrease in the amounts of sAPPβ , EV42, and EV40 and either an increase or no change in the amount of sAPPα are antagonists of OSBP. Viability of the cells is determined as in Example 1.

EXAMPLE 4 Analytes that alter secretion of EV40, EV42, sAPPa, or sAPPb only, or more, in the presence of OSBP are considered to be modulators of OSBP and potential therapeutic agents for treating OSBP-related diseases. The following is an assay that can be used to confirm direct inhibition or modulation of OSBP.

To confirm direct inhibition or modulation of OSBP, OSBP intracellular or extracellular domains are subcloned into expression plasmid vectors such that a fusion protein with C-terminal FLAG epitopes are encoded. These fusion proteins are purified by affinity chromatography, according to manufacturer's instructions, using an ANTI-FLAG M2 agarose resin. OSBP fusion proteins are eluted from the ANTI-FLAG column by the addition of FLAG peptide (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys) (Sigma Aldrich, St. Louis, MO) resuspended in TBS (50 mM Tris HCl pH 7.4, 150 mM NaCl) to a final concentration of lOOμg/ml. Fractions from the column are collected and concentrations of the fusion proteins determined by A280.

A PD-IO column (Amersham, Boston, MA) is used to buffer exchange all eluted fractions containing the OSBP-fusion proteins and simultaneously remove excess FLAG peptide. The FLAG-OSBP fusion proteins are then conjugated to the S series CM5 chip surface (Biacore International SA, Piscataway, NJ) using amine coupling as directed by the manufacturer. A pH scouting protocol is followed to determine the optimal pH conditions for immobilization. Immobilization is conducted at an empirically determined temperature in PBS, pH 7.4, or another similar buffer following a standard Biacore™ immobilization protocol. The reference spot on the CM5 chip (a non-immobilized surface) serves as background. A third spot on the CM5 chip is conjugated with bovine serum albumin in a similar fashion to serve as a specificity control. Interaction of the putative OSBP modulating analyte identified in the assay of Example 5 at various concentrations and OSBP are analyzed using the compound characterization wizard on the Biacore™ S51. Binding experiments are completed at 30⁰C using 50 mM Tris pH 7, 200 uM MnC12 or MgC12 (+ 5% DMSO) or a similar buffer as the running buffer. Prior to each characterization, the instrument is equilibrated three times with assay buffer. Default instructions for characterization are a contact time of 60 seconds, sample injection of 180 seconds and a baseline stabilization of 30 seconds. All solutions are added at a rate of 30 μL/min. Using the BiaEvaluation software (Biacore International AB, Uppsala, Sweden), each set of sensorgrams derived from the ligand flowing through the OSBP-conjugated sensor chip is evaluated and, if binding is observed, an affinity constant determined. EXAMPLE 5

This example describes a method for making polyclonal antibodies specific for the OSBP or particular peptide fragments or epitope thereof.

The OSBP is produced as described in Example 1 or a peptide fragment comprising a particular amino acid sequence of OSBP is synthesized and coupled to a carrier such as BSA or KLH. Antibodies are generated in New Zealand white rabbits over a 10-week period. The OSBP or peptide fragment or epitope is emulsified by mixing with an equal volume of Freund's complete adjuvant and injected into three subcutaneous dorsal sites for a total of about 0.1 mg OSBP per immunization. A booster containing about 0.1 mg OSBP or peptide fragment emulsified in an equal volume of Freund's incomplete adjuvant is administered subcutaneously two weeks later. Animals are bled from the articular artery. The blood is allowed to clot and the serum collected by centrifugation. The serum is stored at - 20OC.

For purification, the OSBP is immobilized on an activated support. Antisera is passed through the sera column and then washed. Specific antibodies are eluted via a pH gradient, collected, and stored in a borate buffer (0.125M total borate) at -0.25 mg/mL. The anti-OSBP antibody titers are determined using ELISA methodology with free CS1P5 receptor bound in solid phase (1 pg/well). Detection is obtained using biotinylated anti-rabbit IgG, HRP-SA conjugate, and ABTS.

EXAMPLE 6 This example describes a method for making monoclonal antibodies specific for OSBP.

BALB/c mice are immunized with an initial injection of about 1 μg of purified OSBP per mouse mixed 1 : 1 with Freund's complete adjuvant. After two weeks, a booster injection of about 1 μg of the antigen is injected into each mouse intravenously without adjuvant. Three days after the booster injection serum from each of the mice is checked for antibodies specific for OSBP. The spleens are removed from mice positive for antibodies specific for the OSBP and washed three times with serum-free DMEM and placed in a sterile Petri dish containing about 20 mL of DMEM containing 20% fetal bovine serum, 1 mM pyruvate, 100 units penicillin, and 100 units streptomycin. The cells are released by perfusion with a 23 gauge needle. Afterwards, the cells are pelleted by low-speed centrifugation and the cell pellet is resuspended in 5 mL 0.17 M ammonium chloride and placed on ice for several minutes. Then 5 mL of 20% bovine fetal serum is added and the cells pelleted by low-speed centrifugation. The cells are then resuspended in 10 mL DMEM and mixed with mid-log phase myeloma cells in serum-free DMEM to give a ratio of 3 : 1. The cell mixture is pelleted by low-speed centrifugation, the supernatant fraction removed, and the pellet allowed to stand for 5 minutes. Next, over a period of 1 minute, 1 mL of 50% polyethylene glycol (PEG) in 0.01 M HEPES, pH 8.1, at 37OC is added. After 1 minute incubation at 37°C, 1 mL of DMEM is added for aperiod of another 1 minute, then a third addition of DMEM is added for a further period of 1 minute. Finally, 10 mL of DMEM is added over a period of 2 minutes. Afterwards, the cells are pelleted by low-speed centrifugation and the pellet resuspended in DMEM containing 20% fetal bovine serum, 0.016 mM thymidine, 0.1 hypoxanthine, 0.5 μM aminopterin, and 10% hybridoma cloning factor (HAT medium). The cells are then plated into 96-well plates.

After 3, 5, and 7 days, half the medium in the plates is removed and replaced with fresh HAT medium. After 11 days, the hybridoma cell supernatant is screened by an ELISA assay. In this assay, 96-well plates are coated with the OSBP. One hundred μL of supernatant from each well is added to a corresponding well on a screening plate and incubated for 1 hour at room temperature. After incubation, each well is washed three times with water and 100 μL of a horseradish peroxide conjugate of goat anti-mouse IgG (H+L), A, M (1:1,500 dilution) is added to each well and incubated for 1 hour at room temperature. Afterwards, the wells are washed three times with water and the substrate

OPD/hydrogen peroxide is added and the reaction is allowed to proceed for about 15 minutes at room temperature. Then 100 μL of 1 M HCl is added to stop the reaction and the absorbance of the wells is measured at 490 nm. Cultures that have an absorbance greater than the control wells are removed to two cm2 culture dishes, with the addition of normal mouse spleen cells in HAT medium. After a further three days, the cultures are re-screened as above and those that are positive are cloned by limiting dilution. The cells in each two cm2 culture dish are counted and the cell concentration adjusted to 1 x 10$ cells per mL. The cells are diluted in complete medium and normal mouse spleen cells are added. The cells are plated in 96-well plates for each dilution. After 10 days, the cells are screened for growth. The growth positive wells are screened for antibody production; those testing positive are expanded to 2 cm2 cultures and provided with normal mouse spleen cells. This cloning procedure is repeated until stable antibody producing hybridomas are obtained. The stable hybridomas are progressively expanded to larger culture dishes to provide stocks of the cells.

Production of ascites fluid is performed by injecting intraperitoneally 0.5 mL of pristane into female mice to prime the mice for ascites production. After 10 to 60 days, 4.5 x Iθ6 cells are injected intraperitoneally into each mouse and ascites fluid is harvested between 7 and 14 days later.

While the present invention is described herein with reference to illustrated embodiments, it should be understood that the invention is not limited hereto. Those having ordinary skill in the art and access to the teachings herein will recognize additional modifications and embodiments within the scope thereof. Therefore, the present invention is limited only by the claims attached herein.

Claims

WHAT IS CLAIMED:

1. A method for screening for analytes that antagonize processing of amyloid precursor protein (APP) to A/3 peptide, comprising: (a) providing recombinant cells, which ectopically expresses OSBP and the APP;

(b) incubating the cells in a culture medium under conditions for expression of the OSBP and APP and which contains an analyte;

(c) removing the culture medium from the recombinant cells; and

(d) determining the amount of at least one processing product of APP selected from the group consisting of sAPP/3 and Aβ peptide in the medium wherein a decrease in the amount of the processing product in the medium compared to the amount of the processing product in medium from recombinant cells incubated in medium without the analyte indicates that the analyte is an antagonist of the processing of the APP to A/3 peptide.

2. The method of Claim 1 wherein the recombinant cells each comprises a first nucleic acid that encodes OSBP operably linked to a first heterologous promoter and a second nucleic acid that encodes an APP operably linked to a second heterologous promoter.

3. The method of Claim 2 wherein the APP is APPNFEV-

4. The method of Claim 1 wherein a control is provided which comprises providing recombinant cells which ectopically express the APP but not the OSBP.

5. A method for screening for analytes that antagonize processing of amyloid precursor protein (APP) to amyloid β (AjS) peptide, comprising:

(a) providing recombinant cells, which ectopically express OSBP and a recombinant APP comprising APP fused to a transcription factor that when removed from the APP during processing of the APP produces an active transcription factor, and a reporter gene operably linked to a promoter inducible by the transcription factor; (b) incubating the cells in a culture medium under conditions for expression of the

OSBP and recombinant APP and which contains an analyte; and

(c) determining expression of the reporter gene wherein a decrease in expression of the reporter gene compared to expression of the reporter gene in recombinant cells in a culture medium without the analyte indicates that the analyte is an antagonist of the processing of the APP to Aβ peptide.

6. A method for treating Alzheimer's disease in an individual comprising providing to the individual an effective amount of an antagonist of OSBP activity.

7. A method for identifying an individual who has Alzheimer's disease or is at risk of developing Alzheimer's disease comprising obtaining a sample from the individual and measuring the amount of OSBP in the sample.

8. The use of an antagonist of OSBP for the manufacture of a medicament for the treatment of Alzheimer's disease.

9. The use of an antibody specific for OSBP for the manufacture of a medicament for the treatment of Alzheimer ' s disease.

10. A vaccine for preventing and/or treating Alzheimer's disease in a subject, comprising an antibody raised against an antigenic amount of OSBP wherein the antibody antagonizes the processing of APP to Aβ peptide.