US20040072236A1 - PrPSc -interacting molecules and uses thereof - Google Patents

PrPSc -interacting molecules and uses thereof Download PDF

Info

Publication number
US20040072236A1
US20040072236A1 US10/256,538 US25653802A US2004072236A1 US 20040072236 A1 US20040072236 A1 US 20040072236A1 US 25653802 A US25653802 A US 25653802A US 2004072236 A1 US2004072236 A1 US 2004072236A1
Authority
US
United States
Prior art keywords
peptide
prp
motif
yyx
biological sample
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
US10/256,538
Other languages
English (en)
Inventor
Neil Cashman
Eustache Paramithiotis
Sylvie La Boissiere
Robert Lawton
Greg Francoeur
Susan Francoeur
Lisa Estey
Marc Pinard
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Idexx Laboratories Inc
Original Assignee
Caprion Pharmaceuticals Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Caprion Pharmaceuticals Inc filed Critical Caprion Pharmaceuticals Inc
Priority to US10/256,538 priority Critical patent/US20040072236A1/en
Assigned to CAPRION PHARMACEUTICALS INC. reassignment CAPRION PHARMACEUTICALS INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: FRANCOEUR, SUSAN, CASHMAN, NEIL, LA BOISSIERE, SYLVIE, LAWTON, ROBERT, PARAMITHIOTIS, EUSTACHE, PINARD, MARC, ESTEY, LISA
Priority to PCT/US2003/030273 priority patent/WO2004029072A2/en
Priority to AU2003272695A priority patent/AU2003272695B2/en
Priority to JP2004539916A priority patent/JP4533750B2/ja
Priority to EP03754894A priority patent/EP1575989B1/en
Priority to DE60332488T priority patent/DE60332488D1/de
Priority to AT03754894T priority patent/ATE467124T1/de
Priority to CA002500120A priority patent/CA2500120A1/en
Publication of US20040072236A1 publication Critical patent/US20040072236A1/en
Priority to US11/342,208 priority patent/US7435540B2/en
Assigned to IDEXX LABORATORIES, INC. reassignment IDEXX LABORATORIES, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: CAPRION PHARMACEUTICALS, INC.
Abandoned legal-status Critical Current

Links

Images

Classifications

    • 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
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • 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
    • G01N2800/2828—Prion diseases

Definitions

  • This invention relates to molecules (for example, peptides) that bind selectively to the disease-specific abnormal isoform of the prion protein, herein generically designated PrP Sc , and not to the normal isoform of this protein, designated PrP C . These molecules are useful for detecting PrP Sc in a sample, and for purifying PrP Sc . Additionally, the invention relates to diagnostic aids for the detection of PrP Sc , pharmaceuticals that inhibit the recruitment of normal PrP C to the disease-specific PrP Sc , and methods for prion decontamination.
  • the prion diseases are a group of rapidly progressive, fatal, and untreatable neurodegenerative syndromes.
  • Human prion diseases include classical Creutzfeldt-Jakob disease (CJD), which has sporadic, iatrogenic, and familial forms. More recently, a variant CJD (vCJD) has been recognized in the United Kingdom, France, the Republic of Ireland, Hong Kong, Italy and the United States (Will et al., Lancet 347:921-25, 1996; Collinge, Lancet 354:317-23, 1999), likely derived from the consumption of cattle tissues contaminated with the agent of bovine spongiform encephalopathy (“BSE”; reviewed in Cashman, Can. Med. Assoc. J.
  • BSE bovine spongiform encephalopathy
  • Prions are the infectious agents that are associated with the transmissible spongiform encephalopathies noted above.
  • the prion diseases are neurodegenerative syndromes characterized by spongiform change (e.g., microcavitation of the brain, usually predominant in gray matter), neuronal cell loss, astrocytic proliferation disproportionate to neuronal loss, and accumulation of an abnormal amyloidogenic protein, sometimes in discrete plaques in the brain.
  • the agents that transmit these diseases differ markedly from viruses and viroids in that no chemical or physical evidence for a nucleic acid component has been reproducibly detected in infectious materials (Prusiner, Science 216: 136-144, 1982).
  • PrP Sc scrapie-associated prion protein
  • PrP Sc is the most prominent (or perhaps sole) macromolecule in preparations of prion infectivity, and appears to be at least a reliable surrogate for most prion infection.
  • PrP Sc is a conformational variant of a host-encoded cellular protein designated PrP C (Oesch et al., Cell 40:735-746, 1985), which is a glycosylphosphatidylinositol (GPI)-linked cell surface protein with a molecular mass of 33-35 kD.
  • PrP C a host-encoded cellular protein designated PrP C (Oesch et al., Cell 40:735-746, 1985), which is a glycosylphosphatidylinositol (GPI)-linked cell surface protein with a molecular mass of 33-35 kD.
  • GPI glycosylphosphatidylinositol
  • PrP C has been isolated from normal brain, and has been found to be protease-sensitive and not associated with scrapie disease-producing activity (Bolton and Bendheim Ciba Found. Symp. 135:164-177, 1988). According to the prion theory, PrP C converts into PrP Sc in a template-directed process initiated by contact with PrP Sc (Prusiner, Proc. Natl. Acad. Sci, USA 95:13363-83, 1998).
  • PrP C is an evolutionarily conserved membrane protein for which the actual biological or physiological function is unclear. Mice devoid of PrP C are viable and show no obvious signs of neurological and physical impairment (Bueler et al., Nature 356:577-582, 1992), except for an ataxic syndrome in certain PrP knockout mouse strains due to upregulation in brain of the prion homolog protein dopple (Moore et al., J Mol. Biol. 292:797-817, 1999). Prnp knockout mice are not susceptible to prion infection, underscoring the central importance of PrP C in the replication of infectivity (Bueler et al., Cell 73:1339-1347, 1993; Prusiner et al., Proc.
  • Prnp knockout mice Targeted investigations of Prnp knockout mice revealed impaired synaptic function (Collinge et al., Nature 370:295-297, 1994) and altered sleep regulation (Tobler et al., J. Neurosci. 17:1869-79, 1997). Moreover, antibody-mediated ligation of PrP C at the cell surface has been shown to depress T cell activation (Cashman et al., Cell 61:185-192, 1990; Li et al., Cell. Immunol. 207:49-58 2001), suggesting a role for the protein in immune function.
  • PrP C is present in large excess to PrP Sc in accessible peripheral tissues and organs of animals and humans afflicted with prion diseases.
  • the availability of reagents that distinguish PrP Sc from PrP C would therefore be of great value in development of a test for prion infection in blood or other tissues accessible to sampling.
  • the invention therefore relates to methods for identifying molecules (e.g., peptides) that selectively bind to PrP Sc , exploiting surface hydrophobicity, charge interactions, and beta-sheet converting potential of peptides in their affinity reactions with PrP Sc .
  • molecules e.g., peptides
  • a subset of these molecules, such as peptides are defined by amino acid sequences within the prion protein sequence itself and/or by evolutionarily conserved amino acid substitutions to these sequences.
  • Other peptides are those that bind selectively to regions of PrP Sc (such as those regions of the protein that include YYX amino acid residues) based on physicochemical interactions such as hydrophobic interactions, pi-stacking, or beta sheet interactions.
  • Peptides selectively binding PrP Sc may be constrained by cyclization, and by formation of hairpins or loops by introduction of cysteines to form disulfide bonds upon oxidation.
  • peptides can be covalently coupled to a solid substrate, such as agarose beads, magnetic beads, or ELISA plates, reacted with a sample containing PrP Sc , cleared of PrP C by washing (with or without proteinase K), so that detection of bound PrP Sc can be efficiently and specifically accomplished with a PrP Sc -specific antibody, or with a non-distinguishing antibody or molecule binding both PrP Sc and PrP C .
  • Such peptides are also useful in concentrating PrP Sc from biological samples.
  • Peptides of the invention are suitable for therapeutic, diagnostic, or decontamination purposes.
  • the molecular weight of the peptide is from about 40,000 Daltons to about 300 Daltons.
  • Other peptides may fall in a narrower range, for example, 30,000 to about 1,000 Daltons, or from about 20,000 to about 2,000 Daltons.
  • Peptides of the invention may be synthesized according to standard methods known in the art.
  • the invention is directed to a detectably-labeled peptide as described herein, the peptide preferably having a covalently attached label capable of detection.
  • the peptides are linked to a spacer having multiple side chain amines, such as poly(lysine), can be used to “amplify” the available surface functionalities.
  • Multiple antigen peptide system (“MAPS”) peptides typically consist of a branched lysine core matrix (Tam, Proc Natl Acad Sci USA 85: 5409-13, 1988; Tam and Zevala, J. Immunol Methods 124: 53-61, 1989; Tam, J. Immunol Methods 196: 17-32, 1996).
  • the branched lysine core provides a scaffolding to support multiple copies of any of the peptides described herein.
  • the invention features a method for identifying a peptide that binds to PrP Sc or a fragment thereof.
  • This method includes the steps of (a) contacting a peptide of about 200 or fewer amino acids with a PrP Sc polypeptide or fragment thereof under conditions that allow for complex formation between the peptide and PrP Sc or fragment thereof, and (b) detecting the complex, wherein the presence of the complex identifies the peptide as one which selectively binds to PrP Sc or a fragment thereof.
  • the complex is detected using ELISA, RIA, western blotting, immunoprecipitation, fluorescence polarization or flow cytometry.
  • the peptide is a fragment of PrP.
  • the peptide can range in chain length from 100 to 150 amino acids, more preferably 50 to 100 amino acids, or 25 to 50 amino acids, and most preferable 9 to 25 amino acids.
  • the peptide can also be a tripeptide, a tetrapeptide, a pentapeptide, a hexapeptide, a heptapeptide, an octapeptide, a nonapeptide, or a decapeptide.
  • the peptide can include a YYX (where X is any amino acid) or YSA motif, which can be repeated in the peptide (e.g., in tandem).
  • the YYX motif can include the amino acids YYR, YYD, YYA, and YYQ.
  • the peptide can also include a YYXXYYXYY (SEQ ID NO: 1) where X is any amino acid) motif.
  • An example of a peptide with a YYXXYYXYYY motif is the peptide YYRRYYRYY (SEQ ID NO: 2).
  • the peptide is coupled to a scaffolding agent, for example, a 4-map or an 8-map.
  • the peptide can also be covalently coupled to a detectable-agent, solid support, or carrier.
  • the invention also features a method for detecting a PrP Sc in a biological sample.
  • This method includes the steps of (a) contacting the biological sample with a peptide of about 200 or fewer amino acids which selectively binds to PrP Sc or a fragment thereof under conditions that allow for complex formation between the peptide and a PrP Sc polypeptide or fragment thereof, and (b) detecting the complex as an indication that PrP Sc is present in the biological sample.
  • the peptide does not substantially bind PrP C .
  • the peptide can be 9 to 20 amino acids in length and can include a YYX, YYXXYYXYY, or YSA motif. Preferred sequences for the YYX motif are YYR, YYD, YYA, and YYQ.
  • the peptide comprising the YYX sequence is covalently coupled to a detectable-label.
  • the peptide comprising the YYX or YSA motif can also be covalently coupled to a solid support or carrier.
  • the biological sample includes any tissue or cell, tissue or cell extract, bodily fluid or biopsy.
  • the PrP Sc is from a human, a livestock species, or a pet species.
  • the PrP Sc from the biological sample is amplified PrP Sc .
  • Preferred methods of detection include ELISA, RIA, western blotting, immunoprecipitation, fluorescence polarization, and flow cytometry.
  • the present invention also includes methods for diagnosing a prion disease such as variant Creutzfeldt-Jakob Disease, bovine spongiform encephalopathy, scrapie, transmissible spongiform encephalopathy, and chronic wasting disease.
  • This method includes the steps of (a) contacting a biological sample from a mammal with a peptide of about 200 or fewer amino acids which selectively binds to PrP Sc or a fragment thereof under conditions that allow for complex formation between the peptide and a PrP Sc polypeptide or fragment thereof and (b) detecting the complex which, if present, indicates a prion disease in the mammal.
  • the peptide does not substantially bind PrP C .
  • the peptide comprises a YYX, YSA, or YAR motif, wherein the YYX motif is selected from the group consisting of YYR, YYD, YYA, and YYQ.
  • the peptide comprising a YYX motif can optionally be covalently coupled to a detectable-agent or a solid substrate.
  • the biological sample comprise a tissue or cell, a tissue or cell extract, a bodily fluid, or a biopsy and the PrP Sc is from a human, a livestock species, or a pet species.
  • Detection of the complex is preferably achieved through the use of ELISA, RIA, western blotting, immunoprecipitation, fluorescence polarization, or flow cytometry.
  • the present invention also features methods for treating or preventing a prion disease in a mammal.
  • This method includes the steps of administering to the mammal an effective amount of a peptide of about 200 or fewer amino acids which selectively binds to PrP Sc or a fragment thereof in a pharmaceutically-acceptable carrier and under conditions that allow for complex formation between the peptide and a PrP Sc poplypeptide or fragment thereof.
  • a prion disease can be selected from the group consisting of variant Creutzfeldt-Jakob Disease, bovine spongiform encephalopathy, and scrapie.
  • the peptide does not substantially bind to PrP C .
  • the peptide preferably comprises a YYX or YSA motif, wherein the YYX motif is selected from the group consisting of YYR, YYD, YYA, and YYQ.
  • Additional features of the invention include methods of inhibiting PrP Sc in a biological sample.
  • This method comprises treating the biological sample with a peptide of about 200 or fewer amino acids which selectively binds to PrP Sc or a fragment thereof under conditions that allow for complex formation between the peptide and a PrP Sc polypeptide or fragment thereof and for a period of time sufficient to permit the formation of a complex comprising the peptide and a PrP Sc .
  • the biological sample is a bodily fluid, a tissue, or an organ.
  • the biological sample is also preferably perfused with the peptide, which, in preferred embodiments, does not substantially bind to PrP Sc .
  • the present invention also features a method for decontaminating PrP Sc from a biological sample.
  • This method includes the steps of (a) treating the biological sample with a peptide of about 200 or fewer amino acids which selectively binds to PrP Sc or a fragment thereof under conditions that allow for complex formation between the peptide and a PrP Sc polypeptide or fragment thereof and for a period of time sufficient to permit the formation of a complex comprising the peptide and PrP Sc , and (b) recovering the complex from the biological sample.
  • the biological sample is preferably a tissue, bodily fluid or an organ and is perfused with the peptide.
  • the peptide does not substantially bind PrP C .
  • the peptide comprises a YYX or YSA motif, wherein the YYX motif is selected from the group consisting of YYR, YYD, YYA, and YYQ.
  • the present invention also features a method for identifying an agent for the treatment of a prion disease selected from the group consisting of variant Cruetzfeldt-Jakob Disease, bovine spongiform encephalopathy, and scrapie.
  • the method includes the steps of (a) combining a peptide of about 200 or fewer amino acids which selectively binds to PrP Sc or a fragment thereof, PrP Sc , and an agent under conditions allowing for complex formation of the peptide and the PrP Sc , and (b) determining whether complex formation is increased or decreased in comparison to complex formation in the absence of the agent, thereby identifying an agent for treating the prion disease.
  • the agent used can decrease or increase complex formation.
  • the prion disease affects a human, a livestock species, or a pet species.
  • animals that can be affected by the prion disease include a human, a bovine, a sheep, or a goat.
  • the peptide does not substantially bind PrP C .
  • the peptide comprises a YYX or YSA motif, wherein the YYX motif is selected from the group consisting of YYR, YYD, YYA, and YYQ.
  • the present invention also features an apparatus for detecting PrP Sc in a biological sample.
  • the apparatus comprises a peptide-fixed portion where a peptide for trapping an amount of an analyte in a sample is present and in a predetermined amount.
  • the analyte is a bodily fluid.
  • the peptide comprises a YYX, YYR, YYD, or YYQ amino acid sequence and has antigenicity as a PrP Sc .
  • the peptide can also have the amino acid sequence YYRRYYRYY or YYR.
  • the peptide is composed of 18 or fewer amino acids, preferably 12 or fewer amino acids, more preferably 8 or fewer amino acids, and most preferably 5 or fewer amino acids.
  • the invention also features a method for detecting an anti-PrP Sc antibody in a biological sample.
  • This method comprises the steps of (a) contacting the biological sample with a peptide of about 200 or fewer amino acids which selectively binds to PrP Sc or a fragment thereof under conditions that allow for complex formation between the peptide and anti-PrP Sc antibody, and (b) detecting the complexes as an indication that anti-PrP Sc antibody is present in the biological sample.
  • peptide is meant a molecule comprised of a chain of amino acid residues joined by peptide (i.e., amide) bonds and includes proteins and polypeptides.
  • Peptides can be expected to possess conformational preferences and to exhibit a three-dimensional structure. Both the conformational preferences and the three-dimensional structure are typically defined by the polypeptide's primary (i.e., amino acid) sequence and/or the presence (or absence) of disulfide bonds or other covalent or non-covalent intrachain or interchain interactions.
  • Exemplary peptides of the invention are those having a chain of 200 or fewer amino acids. Other peptides range from between 3 to 100 amino acids, or 9 to 25 amino acids.
  • Exemplary peptides are obtained from PrP and typically include consensus sequences such as “YYX,” “YYR, “YYQ,” “YYD,” or “YSA.”
  • the peptide can, if desired, be linked (e.g., covalently) to a detectable label for use as an affinity probe to immobilized PrP Sc (or a PrP Sc fragment).
  • Other peptides can be random amino acid sequences that specifically bind to PrP Sc based on physicochemical interaction, for example, to regions of PrP Sc that include amino acid residues YYX.
  • the invention provides peptides that specifically bind to a PrP Sc protein and preferably bind to a native non-denatured PrP Sc protein at high affinity.
  • Preferred peptides of the invention are those that specifically bind to PrP Sc , but do not substantially bind to PrP.
  • the selective binding affinity between a peptide and PrP Sc generally falls in the range of about 1 nM to about 1 mM.
  • the selective binding is on the order of about 10 nM to about 100 ⁇ M, more preferably on the order of about 100 nM to about 10 ⁇ M, and most preferably on the order of about 100 nM to about 1 ⁇ M.
  • Systematic substitution of one or more amino acids of a consensus sequence with a D-amino acid of the same type may also be used to generate more stable peptides.
  • so-called constrained peptides comprising a consensus sequence for example those described herein, or a substantially identical consensus sequence variation may be generated by methods known in the art. For example, by adding internal cysteine residues capable of forming intramolecular disulfide bridges which cyclize the peptide as is described by Tam et al. ( Eur. J. Biochem. 267:3289-3300, 2000).
  • the common modes of cyclization also include side chain to side chain cyclization or side chain to end-group cyclization as is described by Houston et al. ( J. Peptide Res. 52:81-88, 1998). For this purpose, amino acid side chains are connected together or to the peptide backbone.
  • detectable label is meant a material, which when covalently attached to the peptides of this invention, permits detection of the peptide.
  • Suitable detectable labels include, by way of example, radioisotopes, fluorescent labels (e.g., fluorescein), enzymes, epitope tags (e.g. FLAG and Myc) and the like.
  • suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase;
  • suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin;
  • suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin;
  • an example of a luminescent material includes luminol;
  • examples of bioluminescent materials include luciferase, luciferin, and aequorin;
  • suitable radioactive material include 125 I, 131 I, 111 In or 99 Tc.
  • the particular detectable label employed is not critical. Selection of the label relative to such factors is well within the skill of the art.
  • Covalent attachment of the detectable label to the peptide is accomplished by conventional methods well known in the art. For example, when the 125I radioisotope is employed as the detectable label, covalent attachment of 125I to the peptide can be achieved by incorporating the amino acid tyrosine into the peptide and then iodating the peptide. If tyrosine is not present in the peptide, incorporation of tyrosine to the N or C terminus of the peptide can be achieved by well-known chemistry.
  • 32 P can be incorporated onto the peptide as a phosphate moiety through, for example, a hydroxyl group on the peptide using conventional chemistry.
  • Other methods for detectably-labeling a peptide of the invention are well known in the art.
  • prion diseases is meant a group of prion-mediated, rapidly progressive, fatal, and untreatable brain degenerative disorders including, without limitation, Creutzfeldt-Jakob disease (CJD), variant CJD, iatrogenic CJD, familial CJD, Kuru, Gerstmann-Straussler syndrome, and fatal familial insomnia in humans (Prusiner, Science 252:1515-1522, 1991), scrapie in sheep and goats, and spongiform encephalopathy in cattle, as well as recently described prion diseases in other ruminants and cats (see, for example, Pattison, Emerg. Infect. Dis. 4:390-394, 1998).
  • treatment of prion diseases is meant the ability to reduce, prevent, stabilize, or retard the onset of any symptom associated with prion diseases, particularly those resulting in spongiform change, neuronal cell loss, astrocytic proliferation, accumulation of PrP Sc protein, dementia, or death.
  • YYX is meant a peptide having the sequence Tyrosine-Tyrosine-X, where X is any amino acid.
  • YYR is meant a peptide having the sequence Tyrosine-Tyrosine-Arginine.
  • YYQ is meant a peptide having the sequence “Tyrosine-Tyrosine-Glutamine.”
  • YYD is meant a peptide having the sequence “Tyrosine-Tyrosine-Aspartic acid.”
  • YSA is meant a peptide having the sequence Tyrosine-Serine-Alanine.
  • YYA is meant a peptide having the sequence Tyrosine-Tyrosine-Aspartic acid.”
  • YYRRYYRYY is meant a peptide having the sequence Tyrosine-Tyrosine-Arginine-Arginine-Tyrosine-Tyrosine-Arginine-Tyrosine-Tyrosine.”
  • Amino acid residues in peptides are abbreviated as follows: Phenylalanine is Phe or F; Leucine is Leu or L; Isoleucine is Ile or I; Methionine is Met or M; Valine is Val or V; Serine is Ser or S; Proline is Pro or P; Threonine is Thr or T; Alanine is Ala or A; Tyrosine is Tyr or Y; Histidine is His or H; Glutamine is Gln or Q; Asparagine is Asn or N; Lysine is Lys or K; Asparcic Acid is Asp or D; Glutamic Acid is Glu or E; Cysteine is Cys or C; Tryptophan is Trp or W; Arginine is Arg or R; and Glycine is Gly or G.
  • a “therapeutic composition” is meant a composition appropriate for administration to an animal, for example, a mammal, such as a human, a livestock species (for example, a bovine, goat, pig, or sheep), or a pet species.
  • a “small molecule” is meant a compound with a molecular weight of less than or equal to 10,000 Daltons, preferably, less than or equal to 1000 Daltons, and, most preferably, less than or equal to 500 Daltons.
  • Exemplary solid supports useful in the methods of the invention include magnetic beads or other beads (e.g., agarose), membranes (e.g. nitrocellulose, nylon), or plate wells (e.g., ELISA, or derivatized surfaces), carriers would generally be soluble substrates, such as proteins (such as albumin or thyroglobulin) or other soluble molecules.
  • magnetic beads or other beads e.g., agarose
  • membranes e.g. nitrocellulose, nylon
  • plate wells e.g., ELISA, or derivatized surfaces
  • carriers would generally be soluble substrates, such as proteins (such as albumin or thyroglobulin) or other soluble molecules.
  • the compounds (e.g., peptides) described herein are useful for the prevention and treatment of prion diseases, for example, those mediated by PrP Sc .
  • compositions comprising one or more of the compounds described herein and a physiologically acceptable carrier.
  • These pharmaceutical compositions can be in a variety of forms including oral dosage forms, as well as inhalable powders and solutions and injectable and infusible solutions.
  • Synthetic peptides can also be used directly to treat prion diseases, and medications useful in the prevention and treatment of prion diseases can be screened by inhibition of interactions between PrP Sc and PrP Sc -binding peptides.
  • PrP Sc can be adsorbed from biological fluids and tissues to neutralize prion infectivity prior to human use or as a prion disease prophylactic in animal feed.
  • the present invention further provides methods for identifying potential new drug candidates (and potential lead compounds) and methods for determining the specificities of these compounds.
  • knowing that a peptide exhibits a selective affinity to PrP Sc enables the identification of a compound that effects the binding between these molecules, e.g., either as an agonist or as an antagonist (inhibitor) of the interaction.
  • a compound that effects the binding between these molecules e.g., either as an agonist or as an antagonist (inhibitor) of the interaction.
  • Such synthetic peptides also have utility as diagnostic reagents, and in therapy of prion diseases.
  • the invention further includes a solid support which includes a peptide that selectively binds PrP Sc in the form of a detection apparatus or device which comprises a chamber, one or more inlet ports, one or more outlet ports, and a matrix within the chamber to which the peptide is adsorbed or chemically crosslinked.
  • a peptide of the invention is adsorbed to a solid support, a solution containing PrP Sc is passed over the support, and non-binding components from the solution are removed, e.g., by washing.
  • the apparatus of the invention provides detection systems for detecting, monitoring, quantitating or analyzing PrP Sc specifically retained on the surface of the platform, in a detection chamber comprising a specific binding reagent (e.g., a peptide that specifically binds PrP Sc ).
  • Detection systems useful in the manufacture and use of the platforms of the invention include, but are not limited to, fluorescent, chemiluminescent, or radioactive measurements.
  • FIG. 1 shows that the YYRRYYRYY peptide captures PrP Sc in hamster brain lysates.
  • FIG. 2 shows that the YYRRYYRYY peptide captures PrP Sc in bovine brain lysates.
  • FIG. 3 shows that peptides related to the YYRRYYRYY peptide capture PrP Sc in mouse brain lysates.
  • FIG. 4 shows that that the YYAAYYAYY (SEQ ID NO: 3) and YSAASYASY (SEQ ID NO: 4) peptides compete with YYRRYYRYY-linked beads for capture of mouse PrP Sc .
  • PrP Sc -interacting molecules e.g., peptides
  • PrP Sc -interacting molecules will be identified that exploit general physicochemical properties of PrP Sc which distinguish it from PrP C , or that exploit the sequence- and structure-specific molecular mechanism of prion isoform interaction and/or conversion. Accordingly, the present invention provides methods for identifying compounds that bind to and inactivate PrP or otherwise behave as a prion antagonist.
  • compositions comprising an effective amount of a prion antagonist, and more particularly a compound, that is useful for treating prion diseases.
  • the present invention provides methods for the identification of one or more peptides that binds to PrP Sc or a fragment.
  • the invention provides for the rapid identification of peptides having the ability to interact with PrP Sc .
  • the invention allows for the identification of highly disparate protein sequences possessing equivalent functional activities, for example, the ability to identify peptides that bind PrP Sc using (1) peptides whose sequence or composition are not determined by the sequence or composition of PrP or (2) peptides whose sequence or composition are determined by the sequence or composition of PrP.
  • the ability to identify and isolate peptides that bind to PrP Sc or a fragment thereof will prove invaluable in bringing new compounds into prion disease drug discovery programs.
  • PrP Sc is defined as a misfolded, prion disease-associated conformational isoform of the prion protein. Natural and experimental prion infections are recognized in (but not limited to) humans, sheep, goats, elk, deer, cattle, mice, and hamsters.
  • the set of abnormal conformations (designated as, for example, PrP Sc , PrP BSE , PrP CWD , PrP CJD PrP vCJD , depending upon the species of origin and prion strain; PrP Sc herein used generically) may possess partial protease resistance and high ⁇ sheet content.
  • PrP Sc fragments are typically defined as portions of PrP Sc that are sufficiently large as to retain prion infectivity.
  • the “protein only” theory of prion infectivity posits that molecules of PrP C , a normal cell surface membrane protein, are converted to PrP Sc by a template-directed process catalyzed by the abnormal isoform.
  • Peptides of the invention are, in general, molecules having an isoform-selective affinity for PrP Sc or a fragment thereof, with little or no binding to PrP C .
  • the peptides of the invention are typically a fragment of PrP that preferably contains between 3 to 75 amino acid residues of PrP, or multimers thereof.
  • Exemplary PrP amino acid sequences deposited at SwissProt include P04156 (human), P04925 (mouse), P97895 (golden hamster), P23907 (sheep), P52113 (goat), O02841 (white-tailed deer), P79142 (American elk), P10279 (bovine), Q01880 (bovine) and O18754 (cat).
  • the peptides of this invention include multimers of PrP sequences, composed of either sequences derived directly from reported PrP sequences, or containing amino acid substitutions homologous to the native sequence, as indicated by physicochemical similarity (e.g., Bacon and Anderson, J. Mol. Biol. 191: 153-61, 1986) or likelihood of substitution in evolution (e.g., Dayhoff et al., Atlas Protein Seq. Struc. 5: 345-352, 1978).
  • sequences include, but are not limited to, sequences containing PrP repeat motifs such as “YYX,” “YYR,” “YYQ,” or “YYD.”
  • the peptides are in the form of MAPS, which are prepared according to standard methods (Tam, Proc Natl Acad Sci USA 85: 5409-13, 1988; Tam and Zevala, J. Immunol Methods 124: 53-61, 1989; Tam, J. Immunol Methods 196: 17-32, 1996), including 4-map and 8-map formats.
  • One or more N-terminal cysteines for use as a coupling moiety may be added to the peptide sequence.
  • Peptides can be produced not only by recombinant methods, but also by using chemical methods well known in the art.
  • Solid phase peptide synthesis may be carried out in a batchwise or continuous flow process which sequentially adds ⁇ -amino- and side chain-protected amino acid residues to an insoluble polymeric support via a linker group.
  • a linker group such as methylamine-derivatized polyethylene glycol is attached to poly(styrene-co-divinylbenzene) to form the support resin.
  • the amino acid residues are N-a-protected by acid labile Boc (t-butyloxycarbonyl) or base-labile Fmoc (9-fluorenylmethoxycarbonyl).
  • the carboxyl group of the protected amino acid is coupled to the amine of the linker group to anchor the residue to the solid phase support resin.
  • Trifluoroacetic acid or piperidine are used to remove the protecting group in the case of Boc or Fmoc, respectively.
  • Each additional amino acid is added to the anchored residue using a coupling agent or pre-activated amino acid derivative, and the resin is washed.
  • the full length peptide is synthesized by sequential deprotection, coupling of derivitized amino acids, and washing with dichloromethane and/or N, N-dimethylformamide. The peptide is cleaved between the peptide carboxy terminus and the linker group to yield a peptide acid or amide.
  • a peptide (which optionally is conjugated to a detectable label) or a plurality of peptides are contacted with PrP Sc to identify one or more peptides that selectively binds to PrP Sc .
  • PrP Sc a peptide (which optionally is conjugated to a detectable label) or a plurality of peptides are contacted with PrP Sc to identify one or more peptides that selectively binds to PrP Sc .
  • random, combinatorial or conformationally-constrained peptide libraries can be used as a source of peptides, which can be screened to identify peptides that bind to, for example PrP Sc .
  • Many libraries are known in the art that can be used, e.g., chemically synthesized libraries or in vitro translation based libraries.
  • Conformationally constrained libraries that can be used include but are not limited to those containing invariant cysteine residues which, in an oxidizing environment, cross-link by disulfide bonds to form cystines, modified peptides (e.g., incorporating fluorine, metals, isotopic labels, are phosphorylated, etc.), peptides containing one or more non-naturally occurring amino acids, non-peptide structures, and peptides containing a significant fraction of ⁇ -carboxyglutamic acid. Screening of peptide libraries or individual peptides for a peptide that selectively binds to PrP Sc or a fragment thereof, but not to PrP, is accomplished using any of a variety of commonly known methods.
  • the step of contacting PrP Sc or a fragment thereof with a peptide or with a plurality of polypeptides may be effected in a number of ways.
  • PrP Sc can be immobilized on a solid support and a solution of the plurality of polypeptides is contacted with the immobilized PrP Sc .
  • This procedure is similar to an affinity chromatographic process, with the affinity matrix being comprised of the immobilized PrP Sc .
  • the peptides having a selective affinity for PrP Sc are then purified by affinity selection.
  • the nature of the solid support, process for attachment of PrP Sc to the solid support, solvent, and conditions of the affinity isolation or selection procedure are carried out according to conventional methods and are well known to those of ordinary skill in the art.
  • amino acid sequence is to be determined from the polypeptide itself, one may use microsequencing techniques.
  • the sequencing technique may, if desired, include mass spectroscopy.
  • the degree of selective binding between PrP Sc (or a fragment thereof) and its interacting peptide may vary, generally falling in the range of about 1 nM to about 1 mM.
  • the selective binding is on the order of about 10 nM to about 100 ⁇ M, more preferably on the order of about 100 nM to about 10 ⁇ M, and most preferably on the order of about 100 nM to about 1 ⁇ M. Binding is said to be selective when under similar experimental conditions a peptide exhibits higher affinity for PrP Sc than for PrP C .
  • the binding affinity may be quantitatively or qualitatively assessed by any method known in the art.
  • PrP Sc The interaction of PrP Sc with a peptide may be due to physicochemical forces between the molecules unrelated to a specific sequence contained within PrP. Examples of such interactions include the following.
  • PrP Sc displays increased hydrophobicity in comparison to PrP C , probably contributing to the poor solubility and increased aggregation tendency of the abnormal isoform. Moreover, increased molecular surface hydrophobicity is observed in recombinant PrP C induced by low pH and denaturants to develop increased beta sheet content reminiscent of PrP Sc (Swietnicki et al, J. Biol. Chem. 272:27517-27520, 1997).
  • Hydrophobic moieties will bind to other hydrophobic moieties in aqueous environments, due to Van der Waals interactions induced by molecular proximity and an increase in entropy of water associated with the reduction of hydrophobic molecular surface (Lodish et al., eds., Molecular Cell Biology, Palgrave Publishers, 2000). Thus, peptides containing hydrophobic amino acids will bind selectively to PrP Sc . Hydrophobic amino acids, graded from most to least hydrophobic (Kyte and Doolittle, J. Mol. Biol.
  • PrP recombinant PrP subjected to denaturants and low pH displays more tyrosyl groups at the molecular surface than PrP C , and that mono- and polyclonal antibodies directed against the PrP repeat motif tyrosine-tyrosine-arginine bind to PrP Sc but not PrP C from a number of species (see, for example, WO/0078344).
  • PrP refolded in a disease-specific form is therefore believed to possess more solvent accessible tyrosine side chains than does PrP C .
  • Pi-stacking occurs through interaction of an electron-rich aromatic ring circumference with an electron poor aromatic ring center, which may be mediated by a displaced parallel interaction, or an edge-to-face or “T” interaction conformation (McGaughey et al, J. Biol. Chem. 273:15458-63, 1998).
  • Aromatic residues classically thought to undergo pi stacking include tyrosine, tryptophan, and phenylalanine.
  • Peptides containing aromatic amino acids will specifically interact with PrP Sc through pi-stacking with tyrosine or other aromatic residues exposed on the molecular surface of PrP Sc .
  • PrP Sc displays more surface hydrophobicity than PrP C , but charged or polar moieties can participate in specific interactions.
  • Such charged and polar moieties potentially exposed to solvent on PrP Sc at physiological solutions and pH include glycans (particularly the negatively charged sialic acid residues terminating many glycan antennae; Endo et al., Biochemistry 28:8380-8,1989) and charged amino acid side chains such as arginine, lysine, aspartate, and glutamate.
  • PrP Sc contains much more beta sheet conformation than does PrP C . It is possible that peptides could adopt a beta strand conformation which would then be “incorporated” into the beta sheet-rich structure of PrP Sc . Moreover, peptides constrained in a beta sheet conformation may constitute an affinity reagent selectively interacting with PrP Sc . Parallel and antiparallel beta sheets are stabilized by hydrogen bonding between C ⁇ O groups on one strand and NH groups on an adjacent strand. Moreover, amino acids of beta strands in proteins often alternate between hydrophobic side chains buried in the protein interior, and hydrophilic side chains at the molecular surface.
  • Sequence-constrained properties may therefore be mimicked by synthetic peptides or by structurally constrained peptides, which, in turn, may be exploited for specific affinity interactions with PrP Sc .
  • the synthetic peptide may be regarded as an exogenous beta strand for domain swapping with the relevant endogenous PrP strand.
  • 3D domain swapping has been recently recognized as a mechanism by which monomeric proteins may form multimeric assemblies (reviewed in Bennett et al, Protein Science, 4: 2455-2468, 1995).
  • the interaction of PrP Sc with a peptide may be due to physicochemical forces resulting from prion protein interaction with itself, dependent upon a specific sequence contained within PrP. It is possible that such self-self interactions drive the PrP C to PrP Sc conversion central to the propagation of prion infectivity.
  • peptides are constructed to mimic a domain of PrP that is involved in the molecular process of recognition and recruitment of PrP C by PrP Sc . These interactions can utilize a number of intermolecular forces, such as ionic interactions, hydrogen bonding, hydrophobic interactions, and pi-stacking of aromatic residues.
  • the invention particularly relates to diagnostic aids that contain a PrP motif, either as a peptide with a single motif, or tandem repeats of the motif.
  • One such motif repeated three times in the PrP sequence comprises two sequential tyrosines in association with a C-terminal arginine at two sites (YYR), and a C-terminal glutamine or aspartate at the third site (YYQ/D).
  • YYX motif sequences are conserved across a number of species including, but not limited to, bovine, man, sheep, mouse, and hamster.
  • Poly- and monoclonal antibodies directed against YYR have been shown to immunoprecipitate PrP Sc specifically, establishing that motif side chains become differentially solvent accessible in the conformational conversion of the prion protein. See, for example, WO/0078344.
  • the specific interaction of solvent exposed YYR motifs in PrP with a synthetic polyamino acid chain can therefore be exploited for selective recognition of PrP Sc in a biological sample.
  • the invention relates to short synthetic prion peptides (e.g., three to ten amino acids or four to twelve amino acids, inclusive) including amino acid side chains which are differentially exposed to solvent in PrP Sc but not PrP C .
  • Critical amino acid residues participating in this interaction can be identified and a specific artificial sequence (peptide) can be constructed to selectively bind PrP Sc and not PrP C .
  • motifs participating in the conversion of PrP C to PrP Sc are to be found in the medical and biological literature (see, for example, Horiuchi et al., J. Biol. Chem. 276:15489-97, 2001).
  • amino acids from several groups often possess the potential to interact with proteins by several intermolecular forces.
  • tryptophan may interact by means of hydrophobic and pi-stacking interactions
  • tyrosine may interact by aromatic pi-stacking and hydrogen bonding involving its hydroxyl group.
  • another example of the invention is interaction with PrP Sc via a peptide modeled on the prion protein sequence, but with evolutionarily conserved amino acid substitutions.
  • the beads were then washed with wash buffer (PBS, 2% Igepal CA630 and 2% Tween-20) three times, and resuspended in gel loading buffer.
  • the samples were run on 16% Tris Glycine gels, and blots developed with monoclonal 6H4 and goat anti-mouse IgG-HRP conjugate.
  • This repeat motif derived from the prion protein amino acid sequence precipitation of the prion protein, PrP Sc derived from scrapie infected mouse brain, is observed, but not the normal isoform, PrP C derived from normal mouse brain (FIG. 1).
  • the samples were run on 4-12% NuPage MES gels, and blots developed with monoclonal 6H4 and goat anti-mouse IgG-HRP conjugate.
  • the lane assignments are as follows: Lane 1, YYR 10-mer and 10 ⁇ g normal lysate; lane 2, YYR 10-mer and 10 ⁇ g BSE lysate; lane 3, YAR 10-mer and 10 ⁇ g normal lysate; lane 4, YAR 10-mer and 10 ⁇ g BSE lysate; lane 5, YYR 10-mer and 20 ⁇ g BSE lysate; lane 6, YYR 10-mer and 10 ⁇ g BSE lysate; lane 7, YYR 10-mer and 5 ⁇ g BSE lysate; lane 6, YYR 10-mer and 1 ⁇ g BSE lysate.
  • Biotinylated versions of the (YAR) 3 peptide are effective capture reagents when coupled to streptavidin magnetic beads.
  • Other peptide sequences that have been evaluated and shown to selectively bind PrP Sc in this format include, YARYARYAR, YRAARYRAY, bovine PrP (158-183) and bovine PrP (130-147).
  • Peptides negative for PrP Sc selective capture include NHSTHNTGH (SEQ ID NO: 7), DRYYWYFDV (SEQ ID NO: 8) and DEAYYKGWFAY (SEQ ID NO: 9).
  • PrP Sc The specificity of the peptide: PrP Sc was also studied in the following competition experiments. To demonstrate that the YYAAYYAYY and YSAASYASY (SEQ ID NO: 10) peptides compete with YYRRYYRYY beads for the capture of mouse PrP Sc , 100 ⁇ l of tosyl-activated magnetic beads coated with cys-YYRRYYRYY were incubated over night at 4° C. with 500 ⁇ g/ml free peptide in IP binding buffer. Fifty ⁇ g of normal (N) or scrapie (Sc) mouse brain lysate was added to each sample, and the beads were then incubated for 2.5 hours at room temperature.
  • N normal
  • Sc scrapie
  • peptides described herein may be used, for example, for the following diagnostic, therapeutic, vaccination, and decontamination purposes, as well as for screening for novel compounds that can be utilized to diagnose or combat prion diseases or decontaminate prion samples.
  • the peptides disclosed herein find diagnostic use generally in the detection or monitoring of prion diseases.
  • the YYRRYYRYY peptide may be used to monitor the presence or absence of PrP Sc in a biological sample (e.g., a tissue biopsy, a cell, or fluid) using standard and/or amplified detection assays.
  • a biological sample e.g., a tissue biopsy, a cell, or fluid
  • assays and methods may involve direct detection of PrP Sc , and are particularly suited for screening large amounts of samples for the presence of PrP Sc .
  • any of the peptides described herein may be detectably-labeled to measure peptide: PrP complex formation.
  • any appropriate label which may be directly or indirectly visualized may be utilized in these detection assays including, without limitation, any epitope tag, radioactive, fluorescent, chromogenic (e.g., alkaline phosphatase or horseradish peroxidase), or chemiluminescent label, or a hapten (for example, digoxigenin or biotin) which may be visualized using a labeled, hapten-specific antibody or other binding partner (e.g., avidin).
  • PrP Sc may be readily detected at the cell surface (e.g., a leukocyte) using standard flow cytometry methods such as those described herein. Samples found to contain increased levels of labeled complex compared to appropriate control samples are taken as indicating the presence of PrP Sc , and are thus indicative of a prion-related disease.
  • cell surface e.g., a leukocyte
  • flow cytometry methods such as those described herein. Samples found to contain increased levels of labeled complex compared to appropriate control samples are taken as indicating the presence of PrP Sc , and are thus indicative of a prion-related disease.
  • novel compounds useful for diagnosing prion disease may be identified using the peptides of the invention.
  • combinatorial chemical libraries or small molecule libraries are screened to identify compounds having the ability to inhibit the binding interaction of one or more of the peptides described herein according to standard methods.
  • Such libraries may be derived from natural products, synthetic (or semi-synthetic) extracts, or chemical libraries according to methods known in the art.
  • natural compound sources include, but are not limited to, plant, fungal, prokaryotic, or animal sources, as well as modification of existing compounds.
  • Numerous methods are also available for generating random or directed synthesis (e.g., semi-synthesis or total synthesis) of any number of chemical compounds, including, but not limited to, saccharide-, lipid-, peptide-, and nucleic acid-based compounds, i.e. aptamers.
  • Synthetic compound libraries may be obtained commercially or may be produced according to methods known in the art.
  • any library or compound is readily modified using standard chemical, physical, or biochemical methods.
  • high affinity competitors Compounds that inhibit binding of a peptide at lowest concentration are referred to as “high affinity competitors” and are useful in the diagnostic methods of the invention. Such high affinity competitors that mimic the activity of the peptide are subsequently tested for efficient recognition and binding of PrP Sc . Once identified, high affinity competitors may be coupled to solid substrates (for example, ELISA wells or beads) for use in the capture phase of virtually any diagnostic test for prion infection or, alternatively, in blocking format assays.
  • solid substrates for example, ELISA wells or beads
  • Peptides of the invention and mixtures and combinations thereof are also useful as active components of vaccines capable of inducing a prophylactic or therapeutic immune response against prion diseases in a host susceptible to and/or harboring infection.
  • Routes of administration, antigen doses, number and frequency of injections will vary from species to species and may parallel those currently being used in the clinic and/or experimentally to provide immunity or therapy against other infectious diseases or cancer.
  • the vaccines are pharmaceutically acceptable compositions containing the peptide of this invention, its analogues or mixtures or combinations thereof, in an amount effective in the mammal, including a human, treated with that composition to raise immunity sufficient to protect the treated mammal from prion infection for a period of time.
  • PrP Sc -specific immunity prompted by immunization with peptides that include YYX amino acid residues (or YYR, YYD, or YYQ amino acid residues) or related compounds are useful to favor the degradation of PrP Sc or alleviate manifestations of the disease without affecting the expression or function of PrP C in the brain and other tissues, resulting in improvement of clinical status in clinically symptomatic humans with prion disease.
  • a vaccine may be peptide-based, nucleic acid-based, bacterial- or viral-based vaccines. More specifically, with regard to peptide vaccines, peptides corresponding to the PrP Sc -specific epitope or a functional derivatives thereof can be utilized as a prophylactic or therapeutic vaccine in a number of ways, including: 1) as monomers or multimers of the same sequence, 2) combined contiguously or non-contiguously with additional sequences that may facilitate aggregation, promote presentation or processing of the epitope (e.g., class I/II targeting sequences) and/or additional antibody, T helper or CTL epitopes to increase the immunogenicity of the PrP Sc -specific epitope as a means to enhance efficacy of the vaccine, 3) chemically modified or conjugated to agents that would increase the immunogenicity or delivery of the vaccine (e.g., fatty acid or acyl chains, KL
  • nucleic acid delivery agents e.g. polymer-, lipid-, peptide-based, degradable particles, microemulsions, VPLs, attenuated bacterial or viral vectors
  • Attenuated or killed bacterial or viral vectors can be used to deliver either the antigen or DNA/RNA that codes for the expression of the antigen. These can also be used as a means to load cells with antigen ex vivo.
  • Vaccines are prepared according to standard methods known in the art, and will be readily applicable to any new or improved method for vaccine production.
  • PrP Sc can be adsorbed from biological fluids and tissues to neutralize prion infectivity prior to human use or as a prion disease prophylactic in animal feed using the peptides disclosed herein.
  • the invention features methods for decontaminating PrP Sc from a biological sample.
  • the method involves the steps of: (a) treating the biological sample with the peptide (or a fragment or analog thereof), the treatment permitting PrP Sc complex formation with the peptide; and (b) recovering the PrP complex from the biological sample.
  • a decontamination procedure may also involve the use of perfusing a biological sample with peptide (or a fragment or analog thereof) coupled to biotin, hapten or epitope tag, such as FLAG or Myc, for the removal via streptavidin, or antibody affinity column chromatography or direct inactivation of PrP Sc by binding.
  • the methods and compositions described herein are useful for the decontamination of biological samples that are known or suspected of being contaminated with a prion, e.g. intended for transplantation.
  • biological samples may be incubated with a peptide of the invention, and the complexes removed using standard methods.
  • a peptide of the invention may be incubated with biological samples to complex with, and thereby inhibit the infectivity of prion.
  • the invention further features a method of treating or preventing a prion disease in an animal (for example, a human, a bovine, sheep, pig, goat, dog, or cat).
  • the method involves administering to the animal a therapeutically effective amount of PrP peptide identified according to the methods disclosed herein that blocks the conversion of PrP C to PrP Sc , inhibits PrP Sc :PrP aggregate formation, or blocks the recruitment of PrP C to PrP Sc .
  • the invention features a pharmaceutical preparation for the therapy and prevention of prion diseases comprising a PrP peptide of the invention or structurally related compounds, for example, compounds which exploit the PrP Sc -specific exposure of peptides including amino acid residues YYX (or YYR, YYD, or YYQ amino acid residues) can be rationally designed or obtained from combinatorial libraries which mimic the interaction of a YYX containing peptide with anti-YYX peptides. These compounds are useful in prion diagnostics or as therapies for prion diseases.
  • the peptides of the invention can be provided in the form of pharmaceutically acceptable salts.
  • Examples of preferred salts are those with therapeutically acceptable organic acids, e.g., acetic, lactic, maleic, citric, malic, ascorbic, succinic, benzoic, salicylic, methanesulfonic, toluenesulfonic, or pamoic acid, as well as polymeric acids such as tannic acid or carboxymethyl cellulose, and salts with inorganic acids such as the hydrohalic acids, e.g., hydrochloric acid, sulfuric acid, or phosphoric acid.
  • organic acids e.g., acetic, lactic, maleic, citric, malic, ascorbic, succinic, benzoic, salicylic, methanesulfonic, toluenesulfonic, or pamoic acid
  • polymeric acids such as tannic acid or carboxymethyl cellulose
  • salts with inorganic acids such as the hydrohalic acids, e.g., hydrochloric acid, sulfuric acid, or phosphoric
  • any of the peptides of the invention may be administered to a mammal, particularly a human, in one of the traditional modes (e.g., orally, parenterally, transdermally, or transmucosally), in a sustained release formulation using a biodegradable biocompatible polymer, or by using micelles, gels, and liposomes.
  • YYR epitope(s) may block the conversion reaction.
  • direct chemical modification of critical residues such as enzymatic lysis of tyrosine rings, or covalent derivatization of tyrosine rings with bulky substitutions, may also disrupt the PrP C to PrP Sc conversion reaction.
  • such compounds may be identified using the antibodies of the invention. Accordingly, combinatorial libraries or small molecule libraries or both (infra) are screened to identify compounds having the ability to inhibit the binding interaction of one or more of the peptides described herein according to standard methods. Compounds that inhibit binding of such molecules are useful in the therapeutic methods of the invention. Once identified, such compounds are tested for their ability to combat prion diseases in any appropriate model system.
  • test antagonist e.g., a peptide described herein
  • an animal known to develop such a disease e.g., Chandler, Lancet 6:1378-1379, 1961; Eklund et al., J. Infectious Disease 117:15-22, 1967; Field, Brit. J. Exp. Path. 8:129-239, 1969.
  • An appropriate animal for example, a mouse or hamster
  • test compound may be administered to an animal which has previously been injected with a prion agent or, alternatively, the test compound may be tested for its ability to neutralize a prion agent by pre-incubating the prion and the compound and injecting the prion/compound mixture into the test animal.
  • a molecule e.g., an antagonist as described above
  • anti-prion therapeutic e.g., an antagonist as described above
  • An anti-prion therapeutic according to the invention may be administered with a pharmaceutically-acceptable diluent, carrier, or excipient, in unit dosage form.
  • a pharmaceutically-acceptable diluent, carrier, or excipient for example, conventional pharmaceutical practice may be employed to provide suitable formulations or compositions to administer such anti-prion therapeutics to animals suffering from or presymptomatic for a prion disease, or at risk for developing a prion disease.
  • Any appropriate route of administration can be employed, for example, parenteral, intravenous, subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intracapsular, intraspinal, intracisternal, intraperitoneal, intranasal, aerosol, or oral administration.
  • Formulations for parenteral administration can, for example, contain excipients, sterile water, or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated napthalenes.
  • Biocompatible, biodegradable lactide polymer, lactide/glycolide, or polyoxyethylene-polyoxypropylene copolymers can be used to control the release of the compounds.
  • Other potentially useful parenteral delivery systems for anti-prion therapeutic compounds include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes.
  • Formulations for inhalation can contain excipients, for example, lactose, or can be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or can be oily solutions for administration in the form of nasal drops, or as a gel.
  • excipients for example, lactose
  • aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate
  • the methods of the present invention may be used to reduce or prevent the disorders described herein in any animal, for example, humans, domestic pets, zoo animals (such as tigers, exotic ruminants, and nonhuman primates), or livestock. Where a non-human animal is treated, the anti-prion therapeutic employed is preferably specific for that species.
  • the invention features therapeutic and diagnostic compounds identified according to any of the aforementioned methods.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Immunology (AREA)
  • Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Neurosurgery (AREA)
  • Neurology (AREA)
  • General Health & Medical Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • Physics & Mathematics (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Organic Chemistry (AREA)
  • Cell Biology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Microbiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Food Science & Technology (AREA)
  • Veterinary Medicine (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Hospice & Palliative Care (AREA)
  • Psychiatry (AREA)
  • Peptides Or Proteins (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
US10/256,538 2002-09-27 2002-09-27 PrPSc -interacting molecules and uses thereof Abandoned US20040072236A1 (en)

Priority Applications (9)

Application Number Priority Date Filing Date Title
US10/256,538 US20040072236A1 (en) 2002-09-27 2002-09-27 PrPSc -interacting molecules and uses thereof
PCT/US2003/030273 WO2004029072A2 (en) 2002-09-27 2003-09-25 PrPsc -INTERACTING MOLECULES AND USES THEREOF
CA002500120A CA2500120A1 (en) 2002-09-27 2003-09-26 Prpsc -interacting molecules and uses thereof
EP03754894A EP1575989B1 (en) 2002-09-27 2003-09-26 PrP sp sc /sp -INTERACTING MOLECULES AND USES THEREOF
JP2004539916A JP4533750B2 (ja) 2002-09-27 2003-09-26 ＰｒＰＳｃ相互作用分子とその用途
AU2003272695A AU2003272695B2 (en) 2002-09-27 2003-09-26 PrPsc -Interacting molecules and uses thereof
DE60332488T DE60332488D1 (de) 2002-09-27 2003-09-26 MIT PrP sp sc/sp WECHSELWIRKENDE MOLEKÜLE UND DEREN VERWENDUNG
AT03754894T ATE467124T1 (de) 2002-09-27 2003-09-26 Mit prp sp sc/sp wechselwirkende moleküle und deren verwendung
US11/342,208 US7435540B2 (en) 2002-09-27 2006-01-27 PrPSc-selective peptides

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
US10/256,538 US20040072236A1 (en) 2002-09-27 2002-09-27 PrPSc -interacting molecules and uses thereof

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US11/342,208 Division US7435540B2 (en) 2002-09-27 2006-01-27 PrPSc-selective peptides

Publications (1)

Publication Number Publication Date
US20040072236A1 true US20040072236A1 (en) 2004-04-15

Family

ID=32041771

Family Applications (2)

Application Number Title Priority Date Filing Date
US10/256,538 Abandoned US20040072236A1 (en) 2002-09-27 2002-09-27 PrPSc -interacting molecules and uses thereof
US11/342,208 Expired - Fee Related US7435540B2 (en) 2002-09-27 2006-01-27 PrPSc-selective peptides

Family Applications After (1)

Application Number Title Priority Date Filing Date
US11/342,208 Expired - Fee Related US7435540B2 (en) 2002-09-27 2006-01-27 PrPSc-selective peptides

Country Status (7)

Country Link
US (2) US20040072236A1 (enrdf_load_stackoverflow)
EP (1) EP1575989B1 (enrdf_load_stackoverflow)
JP (1) JP4533750B2 (enrdf_load_stackoverflow)
AT (1) ATE467124T1 (enrdf_load_stackoverflow)
CA (1) CA2500120A1 (enrdf_load_stackoverflow)
DE (1) DE60332488D1 (enrdf_load_stackoverflow)
WO (1) WO2004029072A2 (enrdf_load_stackoverflow)

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060116594A1 (en) * 2004-11-30 2006-06-01 Cardiac Pacemakers, Inc. Method and apparatus for arrhythmia classification using atrial signal mapping
US20070087972A1 (en) * 2005-09-09 2007-04-19 David Peretz Prion-specific peptoid reagents
US20080026364A1 (en) * 2006-07-28 2008-01-31 Idexx Laboratories, Inc. Identification of leucocytes bearing diagnostic markers for transmissible spongiform encephalopathies
US20080095706A1 (en) * 2006-07-28 2008-04-24 Adlyfe, Inc. Peptide probes for diagnostics and therapeutics
US20080171341A1 (en) * 2001-05-31 2008-07-17 Adlyfe, Inc. Detection of conformationally altered proteins and prions
US7435540B2 (en) 2002-09-27 2008-10-14 Idexx Corporation PrPSc-selective peptides
US20090061462A1 (en) * 2003-08-13 2009-03-05 Michelitsch Melissa D Prion-specific peptide reagents
US20090130774A1 (en) * 2005-01-13 2009-05-21 David Peretz Elisa assays using prion-specific peptide reagents
US20090191571A1 (en) * 2005-01-13 2009-07-30 Melissa Michelitsch Isolation and Detection of Pathogenic Prions
US20090280125A1 (en) * 2008-04-25 2009-11-12 Scott Napper Prion epitopes and methods of use thereof
US20110081660A1 (en) * 2005-02-15 2011-04-07 Adlyfe, Inc. Method for Detecting Misfolded Proteins and Prions
US20110189692A1 (en) * 2008-04-30 2011-08-04 Novartis Ag Assay for pathogenic conformers

Families Citing this family (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7041807B1 (en) 1999-06-23 2006-05-09 Caprion Pharmaceuticals, Inc. Antibodies to a YYX epitope of a mammalian prion protein
CA2448981C (en) 2001-05-31 2014-03-18 Arete Associates Misfolded protein sensor method
AU2004229535A1 (en) * 2003-04-14 2004-10-28 Pathogen Removal And Diagnostic Technologies, Inc. Method for identifying ligands specific for structural isoforms of proteins
NZ580256A (en) 2003-08-13 2011-07-29 Novartis Vaccines & Diagnostic Prion-specific peptide reagents comprising the sequence KKRPKPGG
US20060057671A1 (en) * 2004-09-10 2006-03-16 Orser Cindy S Immobilized probes and methods of detecting conformationally altered prion proteins
JP4769925B2 (ja) * 2006-03-15 2011-09-07 国立大学法人東北大学 異常型プリオン蛋白質の濃縮方法、および除去方法
CA2675039A1 (en) * 2007-01-12 2008-07-17 Alicon Ag Method for removing prion protein
US20120009209A1 (en) * 2010-06-10 2012-01-12 The Regents Of The University Of California Enrichment and purification of infectious prion proteins
MX2013002870A (es) * 2010-09-13 2013-06-18 Abbvie Inc Ensayo de deteccion residual de anticuerpo monoclonal altamente sensible.

Citations (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4806627A (en) * 1987-05-29 1989-02-21 Research Foundation Of Mental Hygiene Inc. Hybrid cell lines producing monoclonal antibodies dircted against scrapie-associated fibril proteins
US5597725A (en) * 1992-04-17 1997-01-28 Doheny Eye Institute Cadherin-specific antibodies and hybridoma cell lines
US5639634A (en) * 1992-04-17 1997-06-17 Doheny Eye Institute Cadherin polynucleotides
US5643781A (en) * 1992-12-29 1997-07-01 Doheny Eye Institute DNA encoding protocadherin-42
US5646250A (en) * 1992-04-17 1997-07-08 Doheny Eye Institute Cadherin polypeptides
US5679530A (en) * 1995-04-12 1997-10-21 Ludwig Institute For Cancer Research Isolated protein having molecular weight of from about 55 kilodaltons to about 65 kilodaltons (SDS-page) which binds to prion protein
US5708143A (en) * 1992-12-29 1998-01-13 Doheny Eye Institute Protocadherin materials and methods
US5750361A (en) * 1995-11-02 1998-05-12 The Regents Of The University Of California Formation and use of prion protein (PRP) complexes
US5773572A (en) * 1991-12-03 1998-06-30 Proteus Molecular Design Limited Fragments of prion proteins
US5798228A (en) * 1984-12-18 1998-08-25 Boehringer Ingelheim International Gmbh Recombinant production of dog and horse type I interferons
US5846533A (en) * 1995-09-14 1998-12-08 The Regents Of The University Of California Antibodies specific for native PrPSc
US5891641A (en) * 1997-02-21 1999-04-06 The Regents Of The University Of California Assay for disease related conformation of a protein
US6355610B2 (en) * 1998-05-12 2002-03-12 The United States Of America As Represented By The Department Of Health & Human Services Inhibitors of formation of protease resistant prion protein
US6451541B1 (en) * 1996-05-14 2002-09-17 Ernst-Ludwig Winnacker Chaperones capable of binding to prion proteins and distinguishing the isoforms PrPc and PrPsc
US6462171B1 (en) * 1995-06-07 2002-10-08 New York University Peptides and pharmaceutical compositions thereof for treatment of disorders or diseases associated with abnormal protein folding into amyloid or amyloid-like deposits

Family Cites Families (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA1339465C (en) 1986-04-30 1997-09-16 Richard J. Massey Electrochemiluminescent assays
SU1529119A1 (ru) 1987-04-20 1989-12-15 Белорусский научно-исследовательский институт эпидемиологии и микробиологии Способ определени нейровирусов в спинномозговой жидкости
WO1992006220A1 (en) 1990-10-05 1992-04-16 President And Fellows Of Harvard College Detection and isolation of ligands
DE69129760T2 (de) 1990-10-30 1999-03-25 La Jolla Cancer Research Foundation, La Jolla, Calif. T-cadherin-bindungsmolekül
WO1993023432A1 (en) 1992-05-15 1993-11-25 New York University Soluble prion polypeptides, and methods for detecting and purifying thereof
US5276059A (en) 1992-07-10 1994-01-04 The United States Of America As Represented By The Department Of Health And Human Services Inhibition of diseases associated with amyloid formation
BR9709900A (pt) * 1996-05-29 2000-01-25 Univ Mcgill Proteìnas de ligacão de proteìnas de prìons e usos das mesmas.
EP0861900A1 (en) 1997-02-21 1998-09-02 Erziehungsdirektion Of The Canton Zurich Immunological detection of prions
DE19741607A1 (de) 1997-09-20 1999-03-25 Prionics Ag Synthetische Polypeptide zur Diagnose und Therapie von Prionerkrankungen
US6165784A (en) 1997-10-14 2000-12-26 The United States Of America As Represented By The Secretary Of Agriculture Antibodies for the detection of prion protein as an indication of transmissible spongiform encephalopathies
ATE244889T1 (de) 1998-02-20 2003-07-15 Univ California Assay für spezifische stämme von mit mehreren krankheiten zusammenhängenden proteinkonformationen
GB2348203B (en) * 1998-11-04 2002-06-19 Imp College Innovations Ltd Solube beta-forms of prion proteins, methods of preparation and use
HK1048955A1 (zh) * 1999-06-23 2003-04-25 Idexx Laboratories, Inc. 朊病毒蛋白肽及其应用
AU5778400A (en) * 1999-06-29 2001-01-31 Caprion Pharmaceuticals, Inc. Prion protein binding proteins and uses thereof
JP2003530554A (ja) * 2000-04-05 2003-10-14 ブイ．アイ．テクノロジーズ，インコーポレイテッド プリオン結合ペプチドリガンドおよび同一物を使用する方法
DE10230141B4 (de) * 2002-07-04 2004-07-15 Priontype Gmbh Verfahren und Kit zur Anreicherung und zum Nachweis von veränderten Prion-Proteinen (PrPSc)
US20040072236A1 (en) 2002-09-27 2004-04-15 Neil Cashman PrPSc -interacting molecules and uses thereof

Patent Citations (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5798228A (en) * 1984-12-18 1998-08-25 Boehringer Ingelheim International Gmbh Recombinant production of dog and horse type I interferons
US4806627A (en) * 1987-05-29 1989-02-21 Research Foundation Of Mental Hygiene Inc. Hybrid cell lines producing monoclonal antibodies dircted against scrapie-associated fibril proteins
US5773572A (en) * 1991-12-03 1998-06-30 Proteus Molecular Design Limited Fragments of prion proteins
US5597725A (en) * 1992-04-17 1997-01-28 Doheny Eye Institute Cadherin-specific antibodies and hybridoma cell lines
US5639634A (en) * 1992-04-17 1997-06-17 Doheny Eye Institute Cadherin polynucleotides
US5646250A (en) * 1992-04-17 1997-07-08 Doheny Eye Institute Cadherin polypeptides
US5891706A (en) * 1992-12-29 1999-04-06 Doheny Eye Institute Human protocadherin-43 antibodies
US5708143A (en) * 1992-12-29 1998-01-13 Doheny Eye Institute Protocadherin materials and methods
US5663300A (en) * 1992-12-29 1997-09-02 Doheny Eye Institute Protocadherin-42
US5643781A (en) * 1992-12-29 1997-07-01 Doheny Eye Institute DNA encoding protocadherin-42
US5679530A (en) * 1995-04-12 1997-10-21 Ludwig Institute For Cancer Research Isolated protein having molecular weight of from about 55 kilodaltons to about 65 kilodaltons (SDS-page) which binds to prion protein
US6462171B1 (en) * 1995-06-07 2002-10-08 New York University Peptides and pharmaceutical compositions thereof for treatment of disorders or diseases associated with abnormal protein folding into amyloid or amyloid-like deposits
US5846533A (en) * 1995-09-14 1998-12-08 The Regents Of The University Of California Antibodies specific for native PrPSc
US5750361A (en) * 1995-11-02 1998-05-12 The Regents Of The University Of California Formation and use of prion protein (PRP) complexes
US6451541B1 (en) * 1996-05-14 2002-09-17 Ernst-Ludwig Winnacker Chaperones capable of binding to prion proteins and distinguishing the isoforms PrPc and PrPsc
US5891641A (en) * 1997-02-21 1999-04-06 The Regents Of The University Of California Assay for disease related conformation of a protein
US6355610B2 (en) * 1998-05-12 2002-03-12 The United States Of America As Represented By The Department Of Health & Human Services Inhibitors of formation of protease resistant prion protein

Cited By (19)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9638702B2 (en) 2001-05-31 2017-05-02 System Of Systems Analytics, Inc. Detection of conformationally altered proteins
US20080171341A1 (en) * 2001-05-31 2008-07-17 Adlyfe, Inc. Detection of conformationally altered proteins and prions
US7435540B2 (en) 2002-09-27 2008-10-14 Idexx Corporation PrPSc-selective peptides
US20090061462A1 (en) * 2003-08-13 2009-03-05 Michelitsch Melissa D Prion-specific peptide reagents
US20060116594A1 (en) * 2004-11-30 2006-06-01 Cardiac Pacemakers, Inc. Method and apparatus for arrhythmia classification using atrial signal mapping
US20090191571A1 (en) * 2005-01-13 2009-07-30 Melissa Michelitsch Isolation and Detection of Pathogenic Prions
US20090130774A1 (en) * 2005-01-13 2009-05-21 David Peretz Elisa assays using prion-specific peptide reagents
US8372593B2 (en) 2005-02-15 2013-02-12 Adlyfe, Inc. Method for detecting misfolded proteins and prions
US20110081660A1 (en) * 2005-02-15 2011-04-07 Adlyfe, Inc. Method for Detecting Misfolded Proteins and Prions
US7834144B2 (en) 2005-09-09 2010-11-16 Novartis Ag Prion-specific peptoid reagents
US20070087972A1 (en) * 2005-09-09 2007-04-19 David Peretz Prion-specific peptoid reagents
US7601506B2 (en) 2006-07-28 2009-10-13 Idexx Laboratories, Inc. Identification of leucocytes bearing diagnostic markers for transmissible spongiform encephalopathies
US20080095706A1 (en) * 2006-07-28 2008-04-24 Adlyfe, Inc. Peptide probes for diagnostics and therapeutics
US8673579B2 (en) * 2006-07-28 2014-03-18 Adlyfe, Inc. Peptide probes for diagnostics and therapeutics
US20080026364A1 (en) * 2006-07-28 2008-01-31 Idexx Laboratories, Inc. Identification of leucocytes bearing diagnostic markers for transmissible spongiform encephalopathies
US20090280125A1 (en) * 2008-04-25 2009-11-12 Scott Napper Prion epitopes and methods of use thereof
US9056918B2 (en) * 2008-04-25 2015-06-16 University Of Saskatchewan Prion epitopes and methods of use thereof
US9376476B2 (en) 2008-04-25 2016-06-28 University Of Saskatchewan Prion epitopes and methods of use thereof
US20110189692A1 (en) * 2008-04-30 2011-08-04 Novartis Ag Assay for pathogenic conformers

Also Published As

Publication number Publication date
WO2004029072A2 (en) 2004-04-08
US20060183156A1 (en) 2006-08-17
US7435540B2 (en) 2008-10-14
ATE467124T1 (de) 2010-05-15
AU2003272695A1 (en) 2004-04-19
EP1575989A4 (en) 2007-05-16
WO2004029072A3 (en) 2005-12-15
CA2500120A1 (en) 2004-04-08
EP1575989A2 (en) 2005-09-21
JP4533750B2 (ja) 2010-09-01
EP1575989B1 (en) 2010-05-05
JP2006507484A (ja) 2006-03-02
DE60332488D1 (de) 2010-06-17

Similar Documents

Publication Publication Date Title
US7435540B2 (en) PrPSc-selective peptides
EP1931695B1 (en) Prion-specific peptoid reagents
AU2010315133B2 (en) Positively charged species as binding reagents in the separation of protein aggregates from monomers
JP5162250B2 (ja) プリオン特異的ペプチド試薬を使用するｅｌｉｓａアッセイ
US20040186273A1 (en) Prion-binding ligands and methods of using same
CA2377648A1 (en) Prion protein peptides and uses thereof
JP2008527382A5 (enrdf_load_stackoverflow)
EP1554305B1 (en) Prion protein-binding peptide sequences
AU2003272695B2 (en) PrPsc -Interacting molecules and uses thereof
US20090191571A1 (en) Isolation and Detection of Pathogenic Prions
JP2010266448A (ja) アレルギー疾患の判定方法及びアレルギー疾患の判定キット
US8895697B2 (en) Isolated monophosphorylated peptide derived from human alpha-enolase useful for diagnosis and treatment of pancreatic adenocarcinoma, antibodies directed against the said monophosphorylated peptide, and uses thereof
ES2324919T3 (es) Peptidos para la discriminacion de priones.
HK1163124A (en) Prion-specific peptoid reagents
MX2008003147A (en) Prion-specific peptoid reagents
HK1137764B (en) Peptides for discrimination of prions

Legal Events

Date Code Title Description
AS Assignment

Owner name: CAPRION PHARMACEUTICALS INC., CANADA

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:CASHMAN, NEIL;PARAMITHIOTIS, EUSTACHE;LA BOISSIERE, SYLVIE;AND OTHERS;REEL/FRAME:014092/0751;SIGNING DATES FROM 20030116 TO 20030422

STCB Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION

AS Assignment

Owner name: IDEXX LABORATORIES, INC., MAINE

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:CAPRION PHARMACEUTICALS, INC.;REEL/FRAME:018164/0349

Effective date: 20060821