WO2007067512A2 - Procede pour identifier des modulateurs d'adprh convenant pour traiter la maladie d'alzheimer - Google Patents

Procede pour identifier des modulateurs d'adprh convenant pour traiter la maladie d'alzheimer Download PDF

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WO2007067512A2
WO2007067512A2 PCT/US2006/046331 US2006046331W WO2007067512A2 WO 2007067512 A2 WO2007067512 A2 WO 2007067512A2 US 2006046331 W US2006046331 W US 2006046331W WO 2007067512 A2 WO2007067512 A2 WO 2007067512A2
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adprh
app
disease
peptide
alzheimer
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PCT/US2006/046331
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WO2007067512A3 (fr
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John M. Majercak
William J. Ray
David J. Stone
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Merck & Co., Inc.
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Publication of WO2007067512A3 publication Critical patent/WO2007067512A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2814—Dementia; Cognitive disorders
    • G01N2800/2821—Alzheimer

Definitions

  • the present invention relates to methods for identifying modulators of ADPRH.
  • the methods are particularly useful for identifying analytes that antagonize ADPRH's effect on processing of amyloid precursor protein to A ⁇ peptide and thus useful for identifying analytes that can be used for treating Alzheimer disease.
  • 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.
  • amyloid plaques 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 correlates with the degree of dementia (Cummings and Cotman, Lancet 326: 1524-1587 (1995)). 1 VZhUe 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.
  • amyloid ⁇ (A ⁇ ) peptide a primary component of amyloid plaques
  • a ⁇ peptide is toxic to neurons in culture and transgenic mice that overproduce A ⁇ peptide in their brains show significant deposition of A ⁇ into amyloid plaques as well as significant neuronal toxicity (Yankner, Science 250: 279-282 (1990); Mattson et al., J. Neurosci. 12: 379-
  • the longer isoform of A ⁇ is more prone to aggregation than the shorter isoform (Jarrett et al, Biochemistry 32:4693-4697 (1993).
  • a ⁇ 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 is a ubiquitous membrane-spanning (type 1) glycoprotein that undergoes proteolytic cleavage by at least wo pathways (Selkoe, Trends Cell Biol. 8: 447-453 (1998)). In one pathway, cleavage by an enzyme known as tx-secretase occurs while APP is still in the tra ⁇ s-Golgi secretory.
  • the C-terminus is actually a heterogeneous collection of cleavage sites rather than a single site since ⁇ -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 predominate among the C-termini generated by ⁇ -secretase.
  • a ⁇ l-42 peptide is more prone to aggregation than A ⁇ l-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 ⁇ -secretase appears to be situated within the transmembrane domain of APP.
  • Selkoe Trends Cell. Biol. 8: 447-453 (1998).
  • a ⁇ peptide or amyloid containing C-terminal fragments may play a role in the pathophysiology of Alzheimer's disease.
  • 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 A/3 peptide, in neuronal cultures and A/342 in HEK 293 cells in neuronal cultures and A ⁇ 42 peptide in HEK 293 cells.
  • 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 A ⁇ 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 A ⁇ peptide from solution onto a synthetic analogue of an amyloid plaque.
  • the assay was suitable for identifying substances that could inhibit the deposition of AjS peptide.
  • this assay is not suitable for identifying substances, such as inhibitors of ⁇ - or ⁇ -secretase, that would prevent the formation of A ⁇ peptide.
  • 6,828,117 and 6,737,510 disclose a ⁇ - 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 ⁇ -secretase complexed with an eight- residue peptide-like inhibitor at 1.9 angstrom resolution.
  • human ⁇ -secretase Compared to other human aspartic proteases, the active site of human ⁇ -secretase is more open and less hydrophobic, contributing to the broad substrate specificity of human ⁇ -secretase (Lin et al., Proc. Natl. Acad. Sci. USA 97: 1456-1460 (2000)).
  • 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 ⁇ -secretase produced in E. coli of 6.84x10-8 M ⁇ 2.72x10-9 M.
  • CM99-2 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 ⁇ -secretase produced in E. coli of 9.58* 10-9 M ⁇ 2.86> ⁇ 10-10 M.
  • OM99-1 and OM99-2, as well as related substances, are described in International Patent Publication WO0100665.
  • 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 ( ⁇ -secretase inhibitors) or by extreme difficulties in identifying small molecule inhibitors >vith 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.
  • the present invention provides methods for identifying modulators of ADPRH.
  • the methods are particularly useful for identifying analytes that antagonize ADPRH' s effect on processing of amyloid precursor protein to AjS peptide and thus useful for identifying analytes that can be used for treating Alzheimer disease.
  • 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 ADPRH and the APP; incubating the cells in a culture medium under conditions for expression of the ADPRH 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 sAPPjS 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 A/3 peptide.
  • APP amyloid precursor protein
  • the recombinant cells each comprises a first nucleic acid that encodes ADPRH operably linked to a first heterologous promoter and a second nucleic acid that encodes an APP operably linked to a second heterologous promoter.
  • the APP is APPjsfpEV-
  • the method includes a control which comprises providing recombinant cells that ectopically express the APP but not ADPRH.
  • the present invention further provides a method for screening for analytes that antagonize processing of amyloid precursor protein (APP) to amyloid ⁇ (A/3) peptide, comprising providing recombinant cells, which ectopically express ADPRH and a recombinant APP comprising APP fused to a transcription factor that when removed from APP during processing of the APP produces an active transcription factor, and a reporter gene operably linked to a promoter inducible by the
  • the recombinant cells each comprises a first nucleic acid that encodes ADPRH 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.
  • the present invention further provides a method for treating Alzheimer's disease in an individual which comprises providing to the indhidual an effective amount of an antagonist of ADPRH activity.
  • 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 ADPRH in the sample.
  • the present invention provides for the use of an antagonist of ADPRH for the manufacture of a medicament for the treatment of Alzheimer's disease.
  • the present invention provides for the use of an antibody specific for ADPRH for the manufacture of a medicament for the treatment of Alzheimer's disease.
  • 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 ADPRH wherein the antibody antagonizes the processing of APP to A/3 peptide.
  • analyte refers to a compound, chemical, agent, composition, antibody, peptide, aptamer, nucleic acid, or the like, which can modulate the activity of ADPRH.
  • ADPRH refers to ADP-ribosylarginine hydrolase (Official Gene Symbol ADPRH, NP_001116), which is a gene from a human or another mammal having an open reading frame coding for a protein of 357 amino acids in length (SEQ DD NO:2).
  • the term further includes mutants, variants, alleles, and polymorphs of ADPRH.
  • the term further includes fusion proteins comprising all or a portion of the amino acid sequence of ADPRH fused to the amino acid sequence of a heterologous peptide or polypeptide, for example, hybrid immuoglobulins comprising the amino acid sequence of ADPRH or ADPRH fused at its C-terminus to the N-terminus of an
  • Figure 1 is a nucleic sequence encoding the human ADPRH.
  • Figure 2 is the amino acid sequence of the human ADPRH.
  • FIG. 4 shows the tissue distribution of ADPRH mRNA in various human tissues.
  • ADPRH amyloid precursor protein
  • AjS amyloid ⁇
  • ADP- ribosylation is a post Iranslational modification of proteins, in which the ADP-ribose moiety of jS-NAD is transferred to specific amino acid residues in a target protein (Takada, T., et al., J. Biol. Chem. 268 (24): 17837-17843 (1993)).
  • ADP-ribosylargim ' ne hydrolase has been cloned and purified (U.S. Pat. No. 5 ,716,816).
  • ADP-ribosylarginine hydrolases release ADP-ribose from the ADP-ribosylated proteins (Takada, et al., supra). It is believed that the ADP-ribosyltransferases and hydrolases act together to regenerate an unmodified protein, i.e. active form by releasing ADP-ribose from the enzyme. Id. The precise mechanism by which ADPRH acts to increase A ⁇ secretion is not known. However, as noted above, post-translational modification of other proteins with ADP has been shown to be a key regulatory step in several physiological processes (DiGirolam, M., FEBS J. 272(18): 4565-4575 (2005)).
  • AD Alzheimer's disease
  • a defining characteristic of Alzheimer's disease is the deposition of aggregated plaques containing A ⁇ peptide in the brains of affected individuals.
  • the applicant's discovery that ADPRH has a role processing APP to A ⁇ peptide suggests that ADPRH has a role in the progression of Alzheimer's disease in an individual. Therefore, in light of the applicants' discovery, identifying molecules which target the activity or expression of ADPRH would be expected to lead to treatments or therapies for Alzheimer's disease.
  • Expression or activity of ADPRH 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.
  • a ⁇ amyloid ⁇
  • the deposition of aggregated plaques containing amyloid ⁇ (A ⁇ ) 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 AjS 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 ((3CTF) designated C99.
  • ⁇ -secretase a multicomponent membrane complex consisting of at least presenilin, nicastrin, aphl, and pen2
  • AjS peptide an alternative, non-amyloidogenic pathway of APP cleavage is catalyzed by • y-secretase, which cleaves APP to produce a 613 amino acid soluble sAPP ⁇ N-terminal fragment and an 83 amino acid /3CTF fragment designated C83.
  • ADPRH 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 T ⁇ secretase.
  • ADI 1 RH was identified by screening a siRNA library for siRNA that inhibited APP processing.
  • a library of about 15,200 siRNA pools, each targeting a single gene was transfected individually into recombinant cells ectopically expressing a recombinant APP (APPNFEV)- APPNFEV has been described in U.S. Pub. Pat. Appl. No. 2003/0200555, comprises isoform 1-695 and has a HA, Myc, and FLAG sequences at the amino acid position 289, an optimized ⁇ - cleavage site comprising amino acids NFEV, and a K612V mutation.
  • Metabolites of APPNFEV produced during APlP BACEl/ ⁇ -secretase or ⁇ -secretase processing are sAPP / 3 with NF at the C-terminus, EV40, and EV42 or sAPPo.
  • EV40 and EV42 are unique Aj840-like and Aj842-like peptides that contain the glutamic acid and valine substitutions of APPNFEV 3 ⁇ sAPP/3 and sAPP ⁇ each contain the HA 5
  • sAPPjS, sAPPo; 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.
  • ADPRH ADPRH protein was found in the cerebrospinal fluid of rodents (Miyaoka, T., et aL, Brain Res. 746 ( 1 -2): 1 -9 (1997)) and was expressed in the brains of bovines (Moss, J., et al., J. Biol. Chem.. 267(15): 10481-10488 (1992)).
  • rodents Miyaoka, T., et aL, Brain Res. 746 ( 1 -2): 1 -9 (1997)
  • brains of bovines Moss, J., et al., J. Biol. Chem.. 267(15): 10481-10488 (1992)
  • ADPRH is expressed in CNS tissues of mammals, including presumably humans.
  • the nucleic acid sequence encoding the human ADPRH (SEQ ID NO:1) is shown in Figure 1 and the amino acid sequence for the human ADPRH (SEQ ID NO.2) is shown in Figure 2.
  • the mRNA encoding ADPRH was found to be expressed in regions of the brain subject to Alzheimer's disease pathology (Example 2).
  • ADPRH or modified mutants or variants thereof is useful for identifying analytes which antagonize processing of APP to produce A ⁇ peptide. These analytes can be used to treat patients afflicted with Alzheimer's disease.
  • ADPRH 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 ADPRH modulators that modulate expression of ADPRH by contacting ADPRH with a substance that inhibits or stimulates ADPRH expression and determining whether expression of ADPRH polypeptide or nucleic acid molecules encoding an ADPRH are modified.
  • the present invention also provides methods for identifying modulators that antagonize ADPRH' s effect on processing APP to A ⁇ peptide or formation of A/3-amyloid plaques in tissues where ADPRH is localized or co-expressed.
  • ADPRH protein can be expressed in cell lines that also express APP and the effect of the modulator on AjS production is monitored using standard biochemical assays with A/S-specific antibodies or by mass spectrophotometric techniques.
  • Inhibitors for ADPRH are identified by screening for a reduction in the release of A ⁇ peptide which is dependent on the presence of ADPRH protein for effect. Both small molecules and larger biomolecules that antagonize ADPRH-mediated processing of APP to AjS peptide can be identified using such an assay.
  • a method for identifying antagonists of ADPRH's effect on the processing APP to AjS peptide includes the following method which is amenable to high throughput screening. Ih addition, the methods disclosed in U.S. Pub. Pat. Appl. No. 2003/0200555 can be adapted to use in assays for identifying antagonists of ADPRH activity.
  • a mammalian ADPRH 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).
  • RT-PCR reverse-transcription polymerase chain reaction
  • the ADPRH cDNA can be purchased from commercial vendors such as Invitrogen (Carlsbad, CA) and Origene (Rockville, MD).
  • the cDNA 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 ADPRH is validated by DNA sequencing of the ADPRH plasmid (pcDNA_ADPRH).
  • mammalian expression vectors which are suitable for recombinant ADPRH expression include, but are not limited to, pcDNA3.neo (Invitrogen, Carlsbad, CA), pcDNA3.1 (Invitrogen, Carlsbad, CA), ⁇ cDNA3.1/Myc-His (Invitrogen), pCI-neo (Promega, Madison, WI), pLITMUS28, pL]!TMUS29, pLITMUS38 and pLITMUS39 (New England Bioloabs, 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 (3
  • HEK293T/APPNFEV cells used to detect ADPRH 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 APPNFEV operably linked to a heterologous promoter and a plasmid expression vector comprising the ADPRH operably linked to a heterologous promoter or the HEK293T/APPNFEV cells described in Example 1 and U.S. Pub. Pat. Appl. No.
  • 2003/0200555 are trarisfected with a plasmid expression vector comprising the ADPRH operably linked to a heterologous promoter.
  • the promoter comprising the plasmid expression vector can be a constitutive promoter or an inducible promoter.
  • the assay includes a negative control comprising the expression vector without the ADPRH.
  • the transfected or cotransfected cells are incubated with an analyte being tested for ability to antagonize ADPRH's effect on processing of APP to A/3 peptide.
  • the analyte is assessed for an effect on the ADPRH transfected or cotransfected cells that is minimal or absent in the negative control cells.
  • 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.
  • 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 siecreted sAPP ⁇ , sAPP/S, EV40, and EV42 as described in Examples 1 and 5.
  • the antibodies specific for each of the metabolites is used to detect the metabolites in the medium.
  • the cells are assessed for viability.
  • ADPRH analjtes that alter the secretion of one or more of EV40, EV42, sAPP ⁇ , or sAPP/3 in the presence of ADPRH protein are considered to be modulators of ADPRH and potentially useful as therapeutic agents for ADPRH-related diseases.
  • Direct inhibition or modulation of ADPRH can be confirmed using binding assays using the full-length ADPRH, extracellular or intracellular domain thereof or a ADPRH 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 ADPRH, extracellular or intracellular domain thereof or an ADPRH fusion proteins or membranes containing the ADPRH integrated therein arid labeled-analyte can be performed and the amount of labeled analyte bound to the ADPRH determined.
  • the present invention further provides a method for measuring the ability of an analyte to modulate the level of ADPRH mRNA or protein in a cell.
  • a cell that expresses ADPRH is contacted with a candidate compound and the amount of ADPRH mRNA or protein in the cell is determined.
  • This determination of ADPRH levels may be made using any of the above-described immunoassays or techniques disclosed herein.
  • the cell can be any ADPRH expressing cell such as cell transfected with an expression vector comprising ADPRH 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 a ADPRH-associated disorder or a predisposition for a ADPRH-associated disorder.
  • the method includes providing a tissue or serum sample from an individual and measuring the amount of ADPRH in the tissue sample. The amount of ADPRH in the sample is then compared to the amount of ADPRH in a control sample. An .[Iteration in the amount of ADPRH in the sample relative to the amount of ADPRH in the control sample indicates the subject has a ADPRH-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 : ⁇ ot suspected of having a ADPRH 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 ADPRH.
  • ADPRH ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇
  • antibodies having specific affinity for the ADPRH or epitope thereof are provided.
  • the term "antibodies” is intended to be a generic term which includes polyclonal antibodies, monoclonal antibodies, Fab fragments, single VJJ chain antibodies such as those derived from a library of camel or llama antibodies or camelized antibodies (Nuttall et al., Curr. Pharm. Biotechnol. 1: 253-263 (2000); Muyldermans, J. Biotechnol. 74: 277-302 (2001)), and recombinant antibodies.
  • 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 (V Jj chain polypeptides) and single polypeptide chains comprising the variable light chain domain (VL) linked to the variable heavy chain domain (VJJ) to provide a single recombinant polypeptide comprising the Fv region of the antibody molecule (scFv polypeptides) (see Schmiedl et al., J. Immunol. Mgthu 242: 101- 114 (2000); Schultz et al, CancerRes ⁇ 60: 6663-6669 (2000); Dubel et al., J. Immunol.
  • 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 al., J. Biol. Chem. 274: 18218-18230 (1999); Saviranta et al,, Bioconiugate 9: 725-735 (1999); de Greeff et al., Infect. Immun. 68: 3949-3955 (2000)) or polypeptide synthesis.
  • the recombinant antibodies include
  • polypeptides having particular amino acid residues or ligands or labels such as horseradish peroxidase, alkaline phosphatase, fluors, and the like.
  • 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 ADPRH can be produced by methods known in the art.
  • 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 ADPRH or fragments thereof can be used as immunogens for generating such antibodies.
  • 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 al., supra.., and Harlow and Lane, supra. Both anti-peptide and anti-fusion protein antibodies can be used (see, for example, Bahouth et al., Trends Pharmacol. Sci. 12: 338 (1991); Ausubel et al., Current Protocols in Molecular Biology, (John Wiley and Sons, N. Y. (1989)).
  • Antibodies so produced can be used for the immunoaffmity or affinity chromatography purification of ADPRH or ADPRH/ligand or analyte complexes.
  • the above referenced anti-ADPRH antibodies can also be used to modulate the activity of ADPRH in living animals, in humans, or in biological tissues isolated thereof- Accordingly, contemplated herein are compositions comprising a carrier and an amount of an antibody having specificity for ADPRH effective to block naturally occurring ADPRH from binding its ligand or for effecting the processing of APP to A ⁇ peptide.
  • the present invention further provides pharmaceutical compositions that antagonize ADPRH's effect on processing of APP to A ⁇ peptide.
  • Such compositions include a ADPRH nucleic acid, ADPRH peptide, fusion protein comprising ADPRH or fragment thereof coupled to a heterologous peptide or protein or fragment thereof, an antibody specific for ADPRH, nucleic acid or protein aptamers, siRNA inhibitory to ADPRH mRNA, analyte that is a ADPRH antagonist, or combinations thereof, and a pharmaceutically acceptable carrier or diluent.
  • the present invention further provides a kit for in vitro diagnosis of disease by detection of ADPRH in a biological sample from a patient.
  • a kit for detecting ADPRH preferably includes a primary antibody capable of binding to ADPRH; 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.
  • ADPRH can be used to identify endogenous brain proteins that bind to ADPRH 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 ADPRH activity and thus be used to treat Alzheimer's diisease. Additionally, polymorphisms in the ADPRH RNA or in the genomic DNA in and around ADPRH could be used to diagnose patients at risk for Alzheimer's disease or to identify likely responders in clinical trials.
  • ADPFH was identified in a screen of an 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 ⁇ L DMEM containing 10% fetal bovine serum (FBS) and antibiotics. The cell plate was incubated overnight at 37°C in 5% CO2- HEK293T/APPHFEV cells are a subclone of HEK293T cells stably transformed with the APPNFEV plasmid described in U.S. Pub. Pat. Appl. No. 2003/0200555.
  • APPNFEV encodes human amyloid precursor protein (APP), isoform 1-695, modified at amino acid position 289 by an in- frame insertion of HA.
  • the BACE cleavage site is a modified BACEl cleavage site and BACEl cleaves between amino acids F and E of NFEV.
  • the cells in each of the wells of the cell plate were transfected with a siRNA library as follows. OligofectamineTM (Thvitrogen, Inc., Carlsbad, CA) was mixed with Opti- MEM® (Ihvitrogen, Inc., Carlsbad, CA) at a ratio of 1 to 40 and 20 ⁇ L 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 ⁇ M siRNA species was added and the plate incubated for ten minutes at room temperature.
  • OligofectamineTM ThigofectamineTM
  • Opti- MEM® Ihvitrogen, Inc., Carlsbad, CA
  • siRNA/OligofectaminuTM /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 37°C in 5% C ⁇ 2- Controls were provided which contained non-silencing siRNA or a siRNA that inhibited BACEl.
  • the siRNA and a siRNA that inhibited BACEl were provided which contained non-silencing siRNA or a siRNA that inhibited BACEl.
  • OligofectamineTM/Opti-MEM® mixture was removed and replaced with 70 ⁇ L DMEM containing 10% FBS and Merck compound A (see, WO2003/093252, Preparation of spirocyclic [l,2,5]thiadiazole derivatives as ⁇ -secretase inhibitors for treatment of Alzheimer's disease, Collins et al.), a ⁇ -secretase inhibitor given at a final concentration equal to its IC50 in cell-based enzyme assays.
  • the cell plate was incubated for 24 hours at 37°C in 5% CO2.
  • Assays for detecting and measuring sAPP ⁇ , EV42, EV40, and sAPP ⁇ were detected using antibodies as follows. In general, detection-specific volumes (8 or 0.5 ⁇ L) were transferred to a Greiner 384-well white .small -volume detection plate. In the case of the smaller volume, 7.5 ⁇ L of assay medium was added for a final volume of eight ⁇ L per well. One ⁇ L of Antibody/Donor bead mixture (see below) was dispensed into the solution, and one ⁇ L antibody/Acceptor bead mixture was added. Plates were incubated in the dark for 24 hours at 4°C. Then the plates were read using AlphaQuestTM (PerkinElmer, Wellesley, MA) instrumentation. In all protocols, the plating medium was DMEM
  • the AlphaScreenTM buffer was 50 mM HEPES, 150 mM NaCl, 0.1% BSA, 0.1% Tween-20, pH 7.5, and the AlphaScreenTM Protein A kit was used.
  • Anti-NF antibodies and anti-EV antibodies were prepared as taught in U.S. Pub. Pat.
  • ⁇ -secretase cleaves between amino acids F and E of the NFEV cleavage site of APPNFEV to produce a sAPPjS 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 ⁇ -secretase cleavage of the NFEV sequence of APPNFEV-
  • Anti-EV antibodies bind the N-te ⁇ ninal neoepitope EV at the N-terminus of EV40 and EV42 produced by ⁇ -secretase cleavage of the NFEV sequence of APPNFEV- Anti-Bio-G2-10 and anti-B ⁇ o-G2-l 1 antibodies are available from the Genetics Company, Zurich, Switzerland.
  • Anti-Bio-G2-11 antibodies bind the neoepitope generated by the ⁇ -secretase cleavage of A ⁇ or EV peptides at the 42 amino acid position.
  • Anti-Bio-G2-10 antibodies bind the neoepitope generated by the 7-secretase cleavage of A ⁇ 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 A ⁇ and the EV40 and EV42 peptides and also binds sAPP ⁇ because the same epitope resides in amino acids 597 to 614 of sAPP ⁇ .
  • Bio-M2 anti-FLAG antibodies are available from Sigma-Aldrich, St. Louis, MO.
  • Detecting sAPP/3 An AlphaScreenTM assay for detecting sAPP ⁇ -NF produced from cleavage of APPNFEV at the ⁇ -secretase cleavage site was performed as follows. Conditioned medium for each well was diluted 32-fold into a final volume of eight ⁇ L. As shown in Table 1 , biotinylated-M2 anti-FLAG antibody, which binds the FLAG epitope of the APPNFEV. wa s 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 AlphaScreenTM 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 ⁇ g/mL.
  • Detecting EV42 Conditioned medium for each well was used neat (volume eight ⁇ L). As shown in Table 2, a ⁇ ti-Bio-G2-l 1 antibody was captured on streptavidin-coated donor beads by incubating a mixture of the antibody and the streptav ⁇ din coated beads for one hour at room temperature in AlphaScreenTM 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 AlphaScreenTM buffer and used at a final concentration of 5 nM (Table 2). The donor and acceptor beads were used at final concentrations of 20 ⁇ g/mL.
  • Detecting EV40 Conditioned medium for each well was diluted four-fold into a final volume eight ⁇ L. 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 AlphaScreenTM 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 ⁇ rotein-A acceptor beads in AlphaScreenTM buffer and used at a final concentration of 5 nM. The donor and acceptor beads were used at final concentrations of 20 ⁇ g/mL. Table 3
  • 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 AlphaScreenTM buffer.
  • Anti-6E10 antibody acceptor beads were obtained from the manufacturer (PerkinElmer, Inc., which 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 ⁇ g/ml final concentration. The donor beads were used at final concentrations of 20 ⁇ g/mL.
  • siRNAs were tested for modulation of sAPP ⁇ , sAPPoc, EV40 and EV42 by the AlphaScreenTM 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. An siRNA was defined as "secretase-like" if a significant decrease in sAPP ⁇ , EV40 and EV42 was detected, as well as either no change or an increase in sAPP ⁇ . An si] WA was identified which inhibited an mRNA having a nucleotide sequence encoding a protein which had 100% identity to the nucleotide sequence encoding ADPRH. Compared to control non-silencing KiRNAs (set to 100%), ADPRH siRNA pool significantly decreased EV40 (56.5%), EV42 (68.4%), while increasing sAPP ⁇ (339.1%).
  • ADPRH has a role in APP processing, in particuliir, the cleavage of APP at the ⁇ -secretase cleavage site, an event necessary in the processing of APP to J ⁇ peptide.
  • A/3 peptide is a defining characteristic of Alzheimer's disease.
  • ADPRH Because of its role API? processing, ADPRH appears to have a role in the establishment or progression of Alzheimer's disease.
  • ADPRH appeared to have a role in APP processing to AjS peptide and thus, a role in progression of Alzheimer's disease
  • expression of ADPRH was examined in a variety of tissues to determine whether ADiPRH was expressed in the brain.
  • RNA from multiple human tissue sources was subjected to microarray analysis to determine the expression levels of individual genes in various organs and tissues. Differences in hybridization intensities across tissues reflects the abundance of an RNA.
  • ADPRH rnRNA was found to be expressed in several organs, including the brain and within cortical structures which are subjected to amyloid A/3 deposition and Alzheimer pathology. The results are summarized in Figure 4. Arrows indicate brain expression levels.
  • Examples 1 and 2 have shown that the ADPRH has a role in the establishment or progressiveision of Alzheimer's disease.
  • the following is an assay that can be used to identify analytes that antagonize ADPRH activity.
  • a plasmid encoding the human ADPRH or a homolog of the human ADPRH for example, the primate, rodent, or other mammalian ADPRH
  • HEK293T/APPNFEV are plated into a 96-well plate at about 8000 cells per well in 80 ⁇ L DMEM containing 10%FBS and antibiotics and the cell plate incubated at 37°C at 5% CO2 overnight. On the next day, a mixture of 600 ⁇ L OligofectamineTM and 3000 ⁇ L Opti-MEM® is made and incubated at room temperature for five minutes. Next, 23 ⁇ L Opti-MEM® is added to each well of a 96-well mixi ⁇ g plate. 50 ng pcDNA_ADPRH and empty control vector (in 1 ⁇ L volume) are added into adjacent wells of the mixing plate in an alternating fashion. The mixing plate is incubated at room temperature for live minutes.
  • ADPRH transfected cells that is either minimal or absent in cells transfected with the vector-alone as follows. The cells are incubated at 37°C at 5% CO2 overnight.
  • conditioned media is collected the amount of sAPP ⁇ , EV42, EV40, and sAPP ⁇ in the conditioned media is determined as described in Example 1.
  • Analytes that effect a decrease in the amounts of sAPP ⁇ , EV42, and EV40 and either an increase or no change in the amount of sAPP ⁇ are antagonists of ADPRH. Viability of the cells is determined as in Example 1.
  • ADPRH 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.
  • ADPRH 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.
  • a PD-10 column (Amersham, Boston, MA) is used to buffer exchange all eluted fractions containing the ADPRH-fusion proteins and simultaneously remove excess FLAG peptide.
  • the FLAG-ADPRH fusio. ⁇ proteins are then conjugated to the S series CM5 chip surface (BiacoreTM
  • Binding experiments are completed at 30 0 C 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 ⁇ L/min. Using the
  • This example describes a method for making polyclonal antibodies specific for the ADPRH or particular peptide fragments or epitope thereof.
  • the ADPRH is produced as described in Example 1 or a peptide fragment comprising a particular amino acid sequence of ADPRH 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 ADPRH 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 ADPRH per immunization.
  • a booster containing about 0.1 mg ADPRH 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.
  • the ADPRH 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-ADPRH antibody titers are determined using ELISA methodology with free cS 1P5 receptor bound in solid phase (1 pg/well).
  • Detection is obtained using biotinylated anti-rabbit IgG, HRP-SA conjugate, and ABTS.
  • This example describes a method for making monoclonal antibodies specific for ADPRH.
  • BALB/c mice are immunized with an initial injection of about 1 ⁇ g of purified ADPRH per mouse mixed 1:1 with Freund's complete adjuvant. After two weeks, a booster injection of about 1 ⁇ g 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 ADPRH.
  • mice positive for antibodies specific for the ADPRH The spleens are removed from mice positive for antibodies specific for the ADPRH and washed three times with serum-free DMEM and placed m a ste ⁇ le 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 cent ⁇ fugation 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% bovme fetal serum is added and the cells pelleted by low-speed cent ⁇ fugation.
  • the cells are then resuspended in 10 mL DMEM and mixed with mid-log phase myeloma cells m serum-free DMEM to give a ratio of 3: 1.
  • the cell mixture is pelleted by low-speed cent ⁇ fugation, the supernatant fraction removed, and the pellet allowed to stand for 5 minutes.
  • 1 mL of 50% polyethylene glycol (PEG) in 0.01 M HEPES, pH 8.1, at 37°C is added.
  • 1 mL of DMEM is added for a pe ⁇ od of another 1 minute, then a third addition of DMEM is added for a further pe ⁇ od of 1 minute.
  • DMEM fetal bovine serum
  • hypoxanthine 0.5 ⁇ M aminopterm
  • HAT medium 10% hyb ⁇ doma cloning factor
  • the hyb ⁇ doma cell supernatant is screened by an ELISA assay.
  • 96-well plates are coated with the ADPRH.
  • One hundred ⁇ L of supernatant from each well is added to a corresponding well on a screening plate and incubated for 1 hour at room temperature.
  • each well is washed three times with water and 100 ⁇ L 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/hydr ⁇ gen peroxide is added and the reaction is allowed to proceed for about 15 minutes at room temperature. Then 100 ⁇ L 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 m 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 House spleen cells. This cloning procedure is repeated until stable antibody producing hyb ⁇ domas are obtained. The stable hybndomas are progressively expanded to larger culture dishes to provide stoctjs 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 10 ⁇ cells are injected intraperitoneally into each mouse and ascites fluid is harvested between 7 and 14 days later.

Abstract

La présente invention concerne des procédés pour identifier des modulateurs d'ADPRH. Ces procédés conviennent particulièrement pour identifier des analysats antagonistes de l'effet d'ADPRH sur la maturation de la protéine précurseur d'amyloïde en peptide AjS et donc pour identifier des analysats convenant pour traiter la maladie d'Alzheimer.
PCT/US2006/046331 2005-12-08 2006-12-04 Procede pour identifier des modulateurs d'adprh convenant pour traiter la maladie d'alzheimer WO2007067512A2 (fr)

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