WO2002000713A1 - Activite de liaison au prion dans le serum et dans le plasma designes comme plasminogene et fibrinogene - Google Patents

Activite de liaison au prion dans le serum et dans le plasma designes comme plasminogene et fibrinogene Download PDF

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Publication number
WO2002000713A1
WO2002000713A1 PCT/EP2001/003481 EP0103481W WO0200713A1 WO 2002000713 A1 WO2002000713 A1 WO 2002000713A1 EP 0103481 W EP0103481 W EP 0103481W WO 0200713 A1 WO0200713 A1 WO 0200713A1
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prp
prion
factor
prpsc
plasminogen
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PCT/EP2001/003481
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English (en)
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Adriano Aguzzi
Michael Boris Fischer
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Universität Zürich
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Priority claimed from PCT/IB2000/000849 external-priority patent/WO2001023425A1/fr
Application filed by Universität Zürich filed Critical Universität Zürich
Priority to EP01923701A priority Critical patent/EP1294759A1/fr
Priority to AU2001250406A priority patent/AU2001250406A1/en
Priority to BR0111975-3A priority patent/BR0111975A/pt
Priority to CA002413742A priority patent/CA2413742A1/fr
Priority to JP2002505835A priority patent/JP2004501626A/ja
Priority to US09/840,341 priority patent/US20020004586A1/en
Publication of WO2002000713A1 publication Critical patent/WO2002000713A1/fr

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    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A61K38/482—Serine endopeptidases (3.4.21)
    • A61K38/484—Plasmin (3.4.21.7)
    • 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
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
    • G01N2333/968—Plasmin, i.e. fibrinolysin
    • 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

  • the present invention concerns a method and agents to detect transmissible spongifor encephalopathies as well as agents for the prevention and treatment of respective infections.
  • prions transmissible spongiform encephalopathies
  • PrP ⁇ C an ancient organ system in which histopathological damage and its clinical sequelae can be demonstrated as a consequence of infection with prions.
  • the only organ system in which histopathological damage and its clinical sequelae can be demonstrated as a consequence of infection with prions is the nervous system (Brandner et al . , 1996).
  • prions herewith operationally defined as the infectious agents causing transmissible spongiform encephalopathies, can colonize organs other than the central and peripheral nervous system, and can be demonstrated in extracerebral compartments (Aguzzi et al . , 1997).
  • the problem of which organ systems can harbour infectivity is further complicated by the existence of prion strains.
  • prions can come in various different flavors, each one of which has its specific preferences with regard to the host range which is infectible and also to the type of cells in which it replicates (Aguzzi, 1998).
  • BSE prions seem to be largely confined to the neural compartment of cows, even after oral exposure (Wells et al . , 1998).
  • a very accurate study of the pathogenesis of experimental BSE in cows upon feeding 100 grams of infected brain has disclosed that there is only a short and transient period during which infectivity can be demonstrated in the terminal ileum (Wells et al . , 1998).
  • BSE prions can only be shown in brain, spinal cord, and dorsal root ganglia. The exact localization of BSE in the terminal ileum is not known.
  • a first phase or neuroinvasion seems to be widespread colonization of the immune system. This colonization can be visualized by homogenizing spleen, lymph nodes, tonsils, and also appendix, and injecting the homogenates into suitable experimental animals. The dilution of the homogenates at which 50% of the experimental animals become sick, contains one ID50 of the infectious agent in each inoculum.
  • the second phase of neuroinvasion seems to be dependent upon a compartment which cannot be replaced by adoptive bone marrow transfer (Blattler et al . , 1997) and which may be represented by the peripheral nervous system and/or the follicular dendritic cells resistant to germinal center of secondary lymphatic organs. It appears that this second compartment necessitates the expression of normal prion protein in order to support neuroinvasion (Blattler et al. , 1997) .
  • Neuroinvasion is dependent on a functional immune system, and immunodeficient mice do not develop disease after inoculation with a moderate dose of the agent (Fraser et al . , 1996; Kitamoto et al . , 1991; Lasmezas et al . , 1996; O'Rourke et al . , 1994).
  • One crucial component of the immune system necessary for neuroinvasion has been traced to the physical presence of terminally mature B- lymphocytes . To date, it is not clear whether B cells are required because they bind physically prions and carry them to sites of neuroinvasion, or whether B cells produce factors, or induce processes, which are indirectly responsible for facilitating neuroinvasion (Klein et al . , 1997).
  • B-lymphocytes secreting lymphotoxin for the maturation of follicular dendritic cells, and the fact that follicular dendritic cells accumulate large amounts of scr'apie prions in experimental situations, it is plausible to speculate that the main function of B-lymphocytes in the aforementioned process consists in allowing FDCs to mature.
  • FDC follicular dendritic cells
  • SCID severe combined immuno deficient mice
  • nvCJD may be much moreußlymphoinvasive than its sporadic counterpart.
  • nvCJD prions can be easily detected in lymphatic organs such as tonsils and appendix (Hill et al . , 1999; Hill et al . , 1997; Hilton et al . , 1998), a fact that was previously demonstrated to be true for scrapie (Schreuder et al . , 1997; Schreuder et al .
  • splenic lymphocytes of experimentally inoculated mice can be infected with prions (Raeber et al . , 1999). Although prion infectivity of circulating lymphocytes appear to be at least two logs lower than that detected in splenic lymphocytes (Raeber et al . , 1999), the possibility that circulating lymphocytes may be in equilibrium with their splenic siblings call for cautionary measures.
  • leukodepletion has been advocated, but at present there is no certainty about its efficacy, and even whether the presently available technologies for leukoreduction are necessary and/or sufficient for decreasing the threat to blood supply that derives from nvCJD.
  • lysis of cells may lead to contamination of non-particulate fractions and, in the absence of appropriate measures of removal, of stable blood products.
  • infectious prions are likely to consist solely of the PrP ⁇ c protein, which has exactly the same amino acid structure as the normal cellular protein PrP ⁇ .
  • PrP ⁇ c is the only known surrogate marker for prion infectivity: this latter statement is likely to be agreeable upon by both the proponents of the protein-only hypothesis and by those who still believe that the infectious agent is a virus.
  • PrP c and PrP Sc differ in a number of physical properties, it appears to be extremely difficult to develop immunological reagents which reliably differentiate between these two isoforms. Only one monoclonal antibody has been described to react with PrpSc but not with PrP ⁇ , and its practical usefulness remains to be demonstrated since fourteen months after its publication no follow-up studies have appeared and even the company which developed this reagent in the first place does not appear to use it in its in-house screening assay for BSE prions .
  • the hitherto best method for the detection of prions is by performing Western blot analysis with homogenized brain tissue that has been digested with proteinase K (PK) .
  • PK proteinase K
  • the digestion is necessary since for Western blot analysis the secondary structure is broken up so that no difference is found any more between cellular prions (PrP ⁇ ) and pathological prions (PrpS°) , however, while Prp is readily digested by PK under specified conditions, PrP ⁇ c is only degraded to relatively large fragments called prp27-30_
  • Still another object of the present invention are agents specifically recognizing rpSc and/or p p27-30_ Still another object of the present invention are solid phase materials such as e.g. magnetic beads carrying such agents and composition comprising same.
  • compositions comprising such agents for purifying body fluids and sterilization of surgical and diagnostic instruments .
  • Still another object is to provide an improved method for diagnosing transmissible spongiform encephalopathies (TSE) and means therefor.
  • TSE transmissible spongiform encephalopathies
  • p r pSc m ans a prion protein with a confirmation which differs from the "normal" confirmation of PrP c in healthy organisms which do not show or develop any signs of TSE.
  • a “prion binding site which selectively interacts with PrP ⁇ c but not with PrP c” means a molecule or part of a molecule which can bind to PrpSc but fails to bind to PrP c .
  • Such a binding site can be provided e.g. by a low molecular organic compound, a peptide or protein as well as by antigen binding sites of antibodies, wherein said term “antibodies” inter alia, comprises conventional antibodies, scFv-fragments (Fab) and (Fab 2 ) fragments.
  • binding site reacts at least twofold, preferably at least fivefold, preferably at least tenfold, stronger with the prion protein in the PrpSc confirmation than with the prion protein in the PrP c confirmation.
  • the binding site shows at least the selectivity for rpSc as shown by plasminogen.
  • a factor with prion binding activity means a compound which can bind to a prion protein and carries the selective prion binding site of the invention.
  • the factor can be a low molecular compound but preferably is a peptide of at least 10 amino acids length or a protein, which, however, can carry further nonprotein residues such as carbohydrate residues or lipid residues.
  • Said factor can be e.g. of animal or human origin or synthetic.
  • Selective prion binding site as contained in plasminogen means a binding site as provided by a peptide or protein having all or part of the amino acid sequence as contained in a plasminogen of animal or human origin which selectively interacts with PrP Sc but not with PrP c .
  • Derivative of plasminogen plasminogen means a peptide or protein which carries at least one amino acid addition, substitution or deletion compared to the naturally occurring plasminogen or fragment thereof but still capable of selectively interacting with PrpSc and not with PrP c . Such derivatives can easily be prepared, e.g. by site directed mutagenesis of the nucleic acid encoding the naturally occurring plasminogen or by peptide synthesis .
  • Fraction of plasminogen means a part of a naturally occurring plasminogen which part is capable of selectively interacting with PrP ⁇ c and not with PrP c .
  • Carboxy terminus of PrP ⁇ c means the first ... amino acids from the carboxy terminus of a prion protein.
  • Ligand means a compound which selectively binds to the complex of PrP Sc with the binding factor according to the invention but does not interact with free p r pSc or free factor alone.
  • a ligand can be e.g. an antibody or another receptor type protein.
  • the present invention inter alia relates to a method for the concentration of PrpSc or digestion products thereof, wherein a body fluid or fluidized organ is treated with solid phase material, the material at least partly carries a prion binding site which selectively interacts with PrP Sc but not with PrP c .
  • the selectivity of a given binding site can easily be determined as further described herein below. The sufficient selectivity is shown, e.g., by plasminogen.
  • fluidized organ means tissue derived from an organ and solubilized by mechanical procedures, sonication, or other procedures in order to bring into solution or suspension a significant proportion of its constituents.
  • the fluidized organ is a homogenized tissue preferably of the central nervous system, preferably homogenized brain tissue.
  • the body fluid such as blood, plasma, serum, urine, or lymph is treated with a proteinase, preferably with proteinase K (PK) .
  • a proteinase preferably with proteinase K (PK) .
  • the selective prion binding site is contained in a factor with prion binding activity (PrPB) .
  • PrPB protein with prion binding activity
  • Such factor is preferably a peptide or protein. In case of the peptide or protein, it is of sufficient length to allow stable binding to PrP Sc or the digestion product thereof. A sufficient binding stability (or affinity) is shown, e.g. by plasminogen.
  • the solid phase material carries a PrPB as can be found in blood serum or blood plasma, preferably as contained in fraction II of the ammonium sulfate precipitation of serum or plasma.
  • the solid phase material carries a PrPB as can be found in plasminogen, fibrinogen or in plasma fraction I or ammonium sulfate precipitation.
  • the PrPB is selected from plasminogen, fibrinogen, sPrPBII and P PrPBII or fragments thereof which fragments show the same selectivity as the complete protein.
  • the present invention further relates to a method for the detection and optionally quantification of p r pSc or digestion products thereof which method comprises the step of selectively binding PrP Sc or the digestion product thereof to a prion binding site as defined above.
  • the prion binding site is contained in a factor with prion binding activity.
  • the factor is selected from factors as contained in blood serum, blood plasma, serum or plasma fractions II of ammonium sulfate precipitation, plasma fraction I of ammonium sulfate precipitation or as provided by plasminogen, fibrinogen, sPrPBII and pPrPBII or fragments thereof which show a similar selectivity as the complete protein.
  • the detection of the produced complex between p r pSc and the prion binding site can be done by any method currently applicable to the detection of such protein complexes, e.g., by any type of affinity assays or in particular immunoassays as, e.g., described in "The Immunoassay Handbook" of David Wild, Second Edition ISPN0- 33-72306-6, Nature Publishing Group. In order to achieve high sensitivity, a fluorescence detection method is preferred.
  • the present invention further provides for the first time a composition comprising a PrpSc or digestion product thereof and a material carrying a prion binding site which selectively interacts with PrP Sc but not with PrP c .
  • the prion binding site is contained in a factor with prion binding activity, preferably selected from a factor as contained in blood serum, blood plasma, serum or plasma fraction II of ammonium sulfate precipitation, plasma fraction I of ammonium sulfate precipitation or as contained in plasminogen, fibrinogen, sPrPBII and pPrPBII.
  • the present invention further provides a solid phase material which carries a prion binding site of the invention, which binding site is preferably contained in a factor with prion binding activity which factor is preferably a factor selected from a factor as contained in plasma serum, blood plasma, serum or plasma fraction II of ammonium sulfate precipitation, plasma fraction I of ammonium sulfate precipitation or as contained in plasminogen, fibrinogen, sPrPBII and pPrPBII.
  • the present invention further provides a protein complex which comprises a PrpSc and a factor with a prion binding site which selectively interacts with PrpSc but not PrP c , preferably selective PrP ⁇ c a binding site as contained in plasminogen.
  • the present invention further provides a test kit for the detection of pathological prion protein such as Prp c , in body fluids or organs such as blood, urine, cerebrospinal fluid, brain tissue, lymph nodes, tonsils
  • kit comprises a factor with a prion binding site according to the invention and the factor is preferably selected from a factor as contained in blood serum, blood plasma, serum or plasma fraction II of ammonium sulfate precipitation, plasma fraction I of ammonium sulfate precipitation or as contained in plasminogen, fibrinogen, sPrPBII and pPrPBII.
  • the present invention further provides an assay for the diagnosis of human transmissible human spongiform encephalopathies or prion encephalopathies of animals comprising the step of contacting the sample to be tested with a prion binding site which selectively interacts with PrpS c but not with PrP c .
  • the sample is derived from blood.
  • the tested animal or human is diagnosed positive, i.e. for running risk of developing a transmissible spongiform encephalopathy if PrpS c can be detected in the tested sample.
  • the present invention for the first time allows the detection of PrP ⁇ c in a sample which may contain also normal PrP c by using a binding factor for PrP c which contains the selective prion binding site according to the invention.
  • the carboxy terminal part of PrP ⁇ c is the target for the selective binding site. It has surprisingly been found that the carboxy terminus of PrP ⁇ c provides a binding site which allows discrimination between PrP ⁇ c and PrP c , which binding site is a preferred target for the binding sites according to the present invention.
  • the binding sites according to the present invention strongly discriminate between rP ⁇ c and PrP c of different species although it is known that sequence variations exist between prion proteins from different species. Therefore, PrP ⁇ c of different species can be detected by the use of a single prion binding site according to the invention.
  • a very much preferred binding site is the binding site as contained in human plasminogen. It was surprisingly found that the plasminogen binding site interacts with disease associated prion protein from species.
  • the findings according to the invention further suggest that a property common to rpSc of various species rather than the peculiarities private to the specific protein structure of individual PrP ⁇ c molecules are responsible for binding to the binding sites in plasminogen.
  • a carrier for use in accordance with the present invention essentially all materials can be considered that are currently available to perform biological assays for detecting a given compound in a biological sample.
  • a carrier includes magnetic beads, filter stripes, walls of microtiter plates.
  • the detection of PrP ⁇ c could be done "indirectly” in that first a complex is formed between p r pSc ano -(-he binding site according to the present invention and the so-formed complex is then selectively detected by a ligand, such as an antibody.
  • a ligand which selectively interacts with an epitope formed by the interaction of PrP ⁇ c and its binding factor is also highly selective and detects such a complex only but neither detects free PrP ⁇ c nor free factor according to the invention only.
  • kits which contain all ingredients for performing assays in order to detect PrP ⁇ c.
  • kits may contain in addition to the products according to the invention buffers, reagents for detecting a product being the result of the presence of PrpSc in a sample, the working instructions on how to reliably perform an assay.
  • a means for reducing free PrP ⁇ c is the binding of PrP ⁇ c to a binding site according to the invention, which binding site hence can form part of the pharmaceutical composition.
  • the binding site according to the invention can be used for removing PrP ⁇ c from a biological bacteria, e.g., in the form of a dialysis wherein blood of the animal to be tested is continuously brought into contact with a carrier containing the binding site of the invention.
  • the present invention hence allows for the first time a reliable method for diagnosing human transmissible spongiform encephalopathies and prion encephalopathies of animals in which method the material of the animal to be tested is brought into contact with the binding site according to the invention.
  • a body fluid such as e.g. blood, urine, cerebrospinal fluid etc., or fluidized organ, such as brain tissue, lymph nodes, tonsils etc.
  • a solid phase material such as magnetic beads (MB) whereby at least part of said material or beads, respectively, carries a prion binding site.
  • a preferred prion binding site is a factor with prion binding activity (PrPB) .
  • the method works very well with a fluidized organ, in particular homogenized tissue of central nervous system, preferably homogenized brain tissue.
  • PK proteinase K
  • Methods are, however, preferred which do not require the digestion of the starting material in order to achieve selectivity for the PrpS°.
  • Very suitable solid materials are magnetic beads since they can easily be treated with specific components of interests and easily be collected by applying a magnetic field.
  • a further preferred method of the present invention concerns the detection (and optionally quantification) of PrP ⁇ c or digestion products thereof, wherein prP ⁇ c is first concentrated as described above, optionally also with previous digestion of the fluid or fluidized organ, and then detected and optionally compared with a standard.
  • a suitable detection method is Western blot analysis. Such test may furthermore be embodied by other detection methods such as a microtiter plate format immunoassay (e.g. ELISA assay), an immunoprecipitation assay, a BIACORE assay, immunocytochemical assay, histoblot assay etc.
  • the present invention also concerns factors with prion binding activities such as sPrPBII, which is a prion binding activity in fraction II of ammonium sulfate precipitation of serum or pPrPBII which is a factor with prion binding activity in fraction II of ammoniu sulfate precipitation of normal or fresh frozen plasma.
  • sPrPBII which is a prion binding activity in fraction II of ammonium sulfate precipitation of serum
  • pPrPBII which is a factor with prion binding activity in fraction II of ammoniu sulfate precipitation of normal or fresh frozen plasma.
  • Said factors are of course subject matter of the present invention in any form, such as in isolated form, or as ingredient in a composition, e.g. in a fraction of ammonium sulfate precipitation.
  • Said factors can be obtained by concentration and/or isolation of PrPBs whereby serum or plasma is subjected to fractionated ammonium sulfate precipitation thus that a PrPB of interest is precipitated, preferably in only one fraction.
  • a further purification can be obtained by the application of further protein isolation methods.
  • the factors of the present invention are not only suitable for the detection of prions, in particular p r pSc, k u -j- they have further applications in methods for the purification and removal of pathological prion protein from body fluids and organs, such as blood, urine, cerebrospinal fluid, brain tissue, lymph nodes, tonsils etc., or for the sterilization of surgical and/or diagnostic tools, basing on the affinity of PrPB for the pathological prion protein. They are furthermore tools for a therapy regimen based on the modulation of production of PrPB for preventing the spread of prions in the body.
  • plasminogen that is also especially suitable for the purification of body fluids, e.g. blood units. Such purification may e.g. be performed by treating fluids with PrPBIp, in particular with immobilized plasminogen or plasma fractions containing same.
  • test for the detection of pathological prion protein such as p r pSc i n b 0 dy fluids or organs such as blood, urine, cerebrospinal fluid, brain tissue, lymph nodes, tonsils etc, that utilizes the specific binding properties of PrPB to pathological prion protein.
  • pathological prion protein such as p r pSc i n b 0 dy fluids or organs such as blood, urine, cerebrospinal fluid, brain tissue, lymph nodes, tonsils etc.
  • Such test can be embodied as a microtiter plate format immunoassay, e.g. ELISA assay, an immunoprecipitation assay, a BIACORE assay, immunogytochemical assay, histoblot assay etc.
  • DNA sequences specific for biosynthesis of PrPB are comprised by the present invention as well as vectors able to express such DNA sequences in suitable hosts.
  • a method for purification of PrPB by using prp27-30 as bait monoclonal and polyclonal antibodies produced in animals such as mice, rabbits, chicken etc., and directed against PrPB; single-chain Fv fragments and other types of fragments of antibodies produced in recombinant phages or in other recombinant systems, and directed against PrPB; a test predictive of susceptibility to prion diseases based on polymorphisms of PrPB, or on variations in the strength and pattern of production of PrPB; a transgenic animal, e.g.
  • mice that overproduces PrPB in brain, lymph nodes, or other organs, to be used in a bioassay for prions; a knockout animal, in particular a mouse, which is devoid of PrPB, to be used in a bioassay for prions; a production method of PrPB by expressing a DNA sequence specific for the biosynthesis of PrPB in a suitable host cell, such as bacteria, yeast, fungi, or eukaryotic cells, and by purification of PrPB from the aforementioned organisms; a use of natural or synthetic, preferably purified PrPB as a medicament for therapeutical applications in humans and animals; a vaccination of organisms with natural or synthetic PrPB, in particular plasminogen; a diagnostic assay for human and/or animal diseases resulting from abnormal production and/or metabolism of PrPB.
  • a suitable host cell such as bacteria, yeast, fungi, or eukaryotic cells
  • Figure 1 is a scheme showing the IAP method.
  • Figure 2 shows Western Blots and IAP experiments of dilution experiments, whereby lanes 1 to 6 and 10 represent usual Western Blots and lanes 7 to 9 and 11 to 13 represent immuno affinity purification (IAP) .
  • lanes 1 to 6 and 10 represent usual Western Blots and lanes 7 to 9 and 11 to 13 represent immuno affinity purification (IAP) .
  • FIG 3 is a scheme showing the prion affinity assay (PAA) method.
  • Figure 4 represents Western Blots showing positive and begative controls of the PAA.
  • Figure 5 shows the observation that beads coated with sheep anti mouse IgG Abs by DYNAL bind PrPSc but not PrP27-30. Upon preincubation with normal mouse serum PrP27-30 is also bound.
  • FIG. 6 represents Western Blots showing the results with serum proteins that are coupled to beads.
  • the * means that the coupling was performed in the presence of an excess of proteins.
  • Figure 7 shows the effect of the addition of PK-treated brain homogenate to the assay.
  • Figure 8 represents Western Blots showing the results with PrP-deficient material.
  • Figure 9 represents Western Blots showing PAA of ammonium sulfate precipitates.
  • Figure 10 represents Western Blots showing PAA of ammonium sulfate precipitates that are not covalently crosslinked to the beads.
  • Figure 11 shows the result of the PAA of the 58 fractions of human plasma that were obtained by chromatography and differential precipitation and tested for binding activity.
  • Figure 12 represents Western Blots showing the results with purified plasminogen and fibrinogen.
  • Figure 13 represents Western Blots showing the calcium dependency of the binding activity of plasminogen and fibrinogen.
  • Figure 14 represents Western Blots showing the dependency of the binding activity of plasminogen on the native state of the proteins.
  • Figure 15 represents Western Blots showing PAA of plasminogen that is not covalently crosslinked to the beads .
  • Figure 16 shows the concepts of the bioassay.
  • Figure 17 shows the results of the bioassay.
  • Figure 18 shows PrP ⁇ c binding activity of plasminogen to different species.
  • Figure 19 shows precipitation of human PrP CJD by plasminogen.
  • TSEs There are basically three diagnostic principles for TSEs: histopathological detection of the typical spongiform changes in the CNS, detection of the scrapie- specific isoform of the prion protein, and the bioassay that detects infectivity. All these methods have limitations: histopathology is not useful for preclinical diagnosis since the structural changes appear late in the incubation period. Detection of the scrapie the Western specific is form of prion protein is more sensitive but still much less sensitive than the bioassay. The bioassay can, in principle, detect as little as 1 infectious unit but can last months or even years.
  • the hitherto used Western blot technique is based on the partial protease resistance of PrP ⁇ c that allows to distinguish between PrP c and PrP Sc .
  • prp 7 ⁇ 30 _ the protease resistant core of PrP Sc - can be detected but not PrP c which is completely digested.
  • lanes 1 to 6 and 10 represent usual Western Blots and lanes 7 to 9 and 11 to 13 represent immuno affinity purification (IAP) .
  • PrnP% is material from PrP deficient mice.
  • MB are of course only used for IAP whereby 6H4 refers to MB coupled with 6H4 antibodies and - refers to uncoupled MBs.
  • PRP C refers to brain homogenate of non-infected mice and PrP Sc refers to brain homogenate of scrapie-infected mice.
  • PK refers to Proteinase K digestion whereby - refers to non digestion and + to digested homogenate. The same abbreviations are used for the following figures.
  • ionic detergent for prion analysis in homogenate, in particular of brain tissue, it is important to use in a first homogenation step low concentration of ionic detergent, followed by low speed centrifugation, preferably 500 g 30 minutes, 4°C applied twice.
  • high concentration of non-ionic detergent is used and a protein concentration of the homogenate of at most 5 mg/ml .
  • Conditions for the proteinase K digestion are preferably 50 ⁇ g/ml PK, 37 °C and at least half an hour.
  • Suitable incubation conditions for the beads with homogenate are e.g. about 1.5 hours at room temperature, whereby for low concentrations longer incubation times might be preferable.
  • the concentration step in said first attempt was carried out by adding to digested homogenate magnetic beads (MB) carrying said 6H4.
  • a digestion step it has to be performed prior to the concentration step, whereby the digestion, usually by proteinase K, has to be stopped prior to the concentration step by deactivating the proteinase e.g. with phenyl methyl sulfonyl fluoride or another agent known to the skilled person.
  • prp 7-30 can De concentrated up to amounts detectable by Western blot analysis from tissue comprising much less pathological prion protein than needed for the hitherto known tests.
  • the above described method can also be applied as prion affinity assay (PAA) by exchanging the monoclonal antibody 6H4 by other substances to be examined, for example in order to find a binding partner for Pr Sc (see Fig.3).
  • PAA prion affinity assay
  • the beads coupled to total mouse serum proteins did not show any affinity to any form of PrP. However, if the coupling of the total serum was performed in the presence of an excess of protein the beads showed the same binding to PrP 27-30 as the monoclonal antibody 6H4 (see Figure 6, lanes 4-6) whereas the beads that were coupled in the presence of an excess of albumine still did not show any affinity to any form of PrP (see Figure 6, lanes 1-3) . Though it was not possible to measure any difference of the coupling efficency of the two conditions it might be that offering an excess of proteins causes a sponge on the surface of the beads that binds PrP 27-30 .
  • PK-treated brain homogenate might enhance the binding as in the case of bound p r p 27"30 total PK-digested brain homogenate is present: the addition of PK-digested brain homogenate from wild-type C57BL/6 mice or Prnp o/ ° mice allowed to bind PrP Sc in addition to p r p 27"30 (see Figure 7, lanes 1-3) ; the addition of inactive PK had no influence on the binding activity (see Figure 7, lanes 7-9) . If coupled in the presence of an excess the activity of binding PrP 27-30 was also found in the serum of man, sheep, cow and in the serum of terminally scrapie-sick C57BL/6 mice (data not shown) .
  • PrP B serum of several species contains activities (collectively termed PrP B ) that interact specifically with the pathogenic isoform of the prion protein and that are kinetically favoured in binding to the beads.
  • PrP 27-30 could then be understood assuming that native PrP Sc present in sick mice is saturated with PrP B which might be released upon proteolytic digest.
  • PrP B partial proteolysis may expose PrP B binding sites on PrP Sc .
  • PK-treated brain homogenate allows to bind PrP ⁇ c indicates that there might be several different interactions leading to our observations .
  • PrP B activity is not only caused by the special coupling conditions, it should be possible to redesignpurify" it by fractionating mouse serum by differential ammonium sulfate precipitation. Indeed, it was possible to precipitate PrP B at an ammonium sulfate saturation below 50% whereby coupling of each fraction was performed in the presence of an excess of protein (see Figure 9) . While purified rabbit immunoglobulins against total mouse serum did not contain PrP B (data not shown) , they efficiently bound PrP 27-30 upon preincubation with full mouse serum (see Figure 10, lanes 1-3) or with proteins precipitating between 25% and 50% ammonium sulfate saturation (see Figure 10, lanes 4-6) .
  • PrP B activity is a property of one or more serum proteins independent of the covalent crosslink to the surface of the beads.
  • Ammonium sulfate fractionation worked with human serum as well (data not shown)
  • 58 fractions of human plasma were obtained by chromatography and differential precipitation and tested for binding activity to form an idea of the identity of PrP B . All fractions were not coupled in the presence of an excess of proteins. Therefore the results can directly be compared with 6H4 or mouse IgG.
  • PrP B activity of plasminogen is not dependent on the covalent crosslink to the beads by using magnetic beads coated with antibodies directed against plasminogen and preincubated with plasminogen (see Figure 15, lanes 3-4).
  • At least spPrP B does not only bind the pathogenic PrP but also infectivity.
  • the animals that were inoculated with beads that bind the pathogenic PrP did all develop the disease (see Figure 17, lanes 4,5 and 7).
  • Membranes were then developed using ECL detection reagents. Signals were recorded on film and/or quantified using a Kodak ImageStation. In all cases, plasminogen immobilized to magnetic beads captured PrpSc from each species when subjected to the precipitation assay. It has been reported that various breeds of sheep are variably susceptible to scrapie. Susceptibility was mapped to polymorphisms at codons 136, 154, 171 within the sheep Prnp gene.
  • the IAP protocol is the following: Bring the brain tissue in a 15 ml FALCON tube, put it on ice and leave it there for all steps. Add Homogenate Buffer (0.5% DOC / 0.5% NP-40 in PBS) to get 10 % (w/v) homogenate. Pass the tissue through a 18 gauge needle and a 22 gauge needle by sucking up and down for 15 times each. Centrifuge the homogenate for 30 minutes at 500 g and 4°C. Keep the supernatant. Determine the protein concentration. Centrifuge the homogenate for 30 minutes at 500 g and 4°C. Keep the supernatant.
  • the homogenate buffer If the protein concentration is higher than 10 mg/ml then bring the homogenate to a protein concentration of 10 mg/ml using the homogenate buffer. Bring the homogenate to a protein concentration of 5 mg/ml and 3% Tween 20 / 3% NP-40 all in PBS. Add to the tissue homogenate Proteinase K to get a final concentration of 50 ⁇ g/ml. Incubate for 60 minutes at 37°C. Add PMSF to get a final concentration of 5 mM. Add 0.25 volumes of IAP buffer (3% Tween 20 /3 % NP-40 in PBS) . Resuspend the magnetic beads (covered with 6H4 ) according to the protocol described below) thoroughly. Pipette out 100 ⁇ l . Remove buffer.
  • Couple the protein of interest to magnetic beads Bring 100 ⁇ g of protein into approx. 1ml of Coupling Buffer (0.1 M borate buffer pH 9.5: dissolve 6.183 g H3B03 in 800 ml distilled water, Adjust pH to 9.5 using 5 M NaOH and adjust volume to 1000 ml with distilled water; if necessary, change buffer by dialysis) . (If coupling was performed in the presence of an excess, 1 mg was used for 1 ml of coupling buffer.) Make a homogeneous suspension of the Dynabeads M-280 Tosylactivated by Dynal using a pipette and by vortexing for approximately 1 min.
  • Coupling Buffer 0.1 M borate buffer pH 9.5: dissolve 6.183 g H3B03 in 800 ml distilled water, Adjust pH to 9.5 using 5 M NaOH and adjust volume to 1000 ml with distilled water; if necessary, change buffer by dialysis
  • Sample II and III Add 1 ml of PAA Buffer (3 % NP-40 / 3 % Tween 20 in PBS) to 10 " ⁇ l of infected brain homogenate (Protein concentration 5 mg/ml; 0.5% DOC / 0.5 NP-40). Incubate Sample I and Sample II for 30 minutes at 37 °C without PK. Incubate Sample III for 30 minutes at 37 °C with PK at final concentration of 50 ⁇ g/ml (add 50 ⁇ l of PK l g/ml) . Add PMSF to all samples to get a final concentration of 5 mM (add 50 ⁇ l of 100 mM PMSF) . Resuspend the Magnetic Beads thoroughly.
  • PAA Buffer 3 % NP-40 / 3 % Tween 20 in PBS
  • this assay 6H4 As a positive control of this assay 6H4 is used and as a negative control mouse IgG or mouse albumin (see Figure 4 ) .
  • the beads coupled to total mouse serum proteins did not show any affinity to any form of PrP. However, if the coupling of the total serum was performed in the presence of an excess of protein the beads showed the same binding to PrP 27-30 as the monoclonal antibody 6H4 whereas the beads that were coupled in the presence of an excess of albumine still did not show any affinity to any form of PrP (see Figure 6) . Though it was not possible to measure any difference of the coupling efficency of the two conditions it might be that offering an excess of proteins causes a sponge on the surface of the beads that binds PrP 27-30 .
  • PK-treated brain homogenate might enhance the binding as in the case of bound PrP 27-30 total PK-digested brain homogenate is present: the addition of PK-digested brain homogenate from wild-type C57BL/6 mice or Prnp° /o mice allowed to bind PrP Sc in addition to PrP 27-30 ; the addition of inactive PK had no influence on the binding activity (see Figure 7).
  • PrP B activity is not only caused by the special couspling conditions, it should be possible to redesignpurify" it by fractionating mouse serum by differential ammonium sulfate precipitation. Indeed, it was possible to precipitate PrP B at an ammonium sulfate saturation below 50% whereby coupling of each fraction was performed in the presence of an excess of protein (see Figure 9). While purified rabbit immunoglobulins against total mouse serum did not contain PrP B (data not shown) , they efficiently bound PrP 27-30 upon preincubation with full mouse serum or with proteins precipitating between 25% and 50% ammonium sulfate saturation. Preincubation with proteins precipitating between 75% and 100% ammonium sulfate saturation did not lead to PrP B activity (see Figure 10) . This finding is important as it shows that the PrP B activity is a property of one or more serum proteins independent of the covalent crosslink to the surface of the beads .
  • PrP B selectively interacts with the pathogenic PrP but not with PrP c , interaction may be conformation-specific.
  • the assay was carried out in the presence of 6M urea the fraction containing purified plasminogen didn't bind PrP Sc nor PrP 27-30 ; under these conditions PrP Sc becomes protease-sensitive (Fig. 14).
  • Fig. 14 As the conformation of PrP Sc is thought to be responsible for the PK resistancy we conclude from this experiment that the interaction of plasminogen and PrP Sc is conformation- dependant .
  • PrP B activity of plasminogen is not dependent on the covalent crosslink to the beads by using magnetic beads coated with antibodies directed against plasminogen and preincubated with plasminogen (Fig. 15).
  • At least spPrP B does not only bind the pathogenic PrP but also infectivity.
  • the animals that were inoculated with beads that bind the pathogenic PrP did all develop the disease (Fig. 16, Fig. 17) .
  • Binding assay for determining the selectivity of a PrPSc specific binding partner using solid state-bound technologies includes e.g. microtiter plate formats, paramagnetic beads, non-magnetic beads, plasmon surface resonance, interferortietry, coincidence detection, mass spectrometry/mass spectroscopy, electrospray analysis, and combinations thereof.
  • microtiter plate formats paramagnetic beads, non-magnetic beads, plasmon surface resonance, interferortietry, coincidence detection, mass spectrometry/mass spectroscopy, electrospray analysis, and combinations thereof.
  • the peptides or protein or fragments thereof to be tested are coupled to a solid phase material: a. Use micro particles such as magnetic beads as solid phase and perform immunopreciptiation.
  • PrPSc or PrP27-30
  • PrPC are coupled to a solid phase material: a. Use micro particles such as magnetic beads as solid phase and perform immunopreciptiation.
  • Couple PrPSc or PrP27-30
  • PrPC PrP27-30
  • PrPC PrP27-30
  • PrPC PrP27-30
  • b. Use surface of a micro titer plate as solid phase and perform ELISA.
  • the prior art offers many possibilities to determine and detect certain parts of a protein which are involved in specific binding of the protein to a certain target.
  • Method to identify suitable fragments of plasminogen as PrpSc specific binding partners includes e.g. forward genetic selection using phage display, ribosomal display, bacterial protein fragment affinity assay, and combinations or derivations thereof. Accordingly, those parts of plasminogen that are involved in the specific binding to PrP ⁇ c can be determined as follows :
  • PrP-expressing tissue required for transfer of scrapie infectivity from spleen to brain. Nature 389, 69- 73.
  • PrP expression in B lymphocytes is not required for prion neuroinvasion. Nat Med 4, 1429-33.

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Abstract

L'invention concerne des procédés et des outils destinés à concentrer, à détecter et à quantifier des protéines prion pathologiques ainsi que des agents à utiliser dans ladite détection et/ou dans la prévention ou dans le traitement des maladies à prion. Ces agents sont des facteurs dotés d'activités de liaison au prion que l'on trouve dans le sérum sanguin et dans le plasma sanguin.
PCT/EP2001/003481 1999-09-28 2001-03-27 Activite de liaison au prion dans le serum et dans le plasma designes comme plasminogene et fibrinogene WO2002000713A1 (fr)

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EP01923701A EP1294759A1 (fr) 2000-06-26 2001-03-27 Activite de liaison au prion dans le serum et dans le plasma designes comme plasminogene et fibrinogene
AU2001250406A AU2001250406A1 (en) 2000-06-26 2001-03-27 Prion-binding activity in serum and plasma determined as plasminogen and fibrinogen
BR0111975-3A BR0111975A (pt) 2000-06-26 2001-03-27 Fator, composição, veìculo, ligando, kits de diagnósticos, processos para detectar uma prpsc em uma amostra, e para remover prpsc de material biológico, método para diagnosticar encefalopatias espongiformes humanas transmissìveis e encefalopatias de prìons de animais, e, uso de um fator
CA002413742A CA2413742A1 (fr) 2000-06-26 2001-03-27 Activite de liaison au prion dans le serum et dans les proteines
JP2002505835A JP2004501626A (ja) 2000-06-26 2001-03-27 血清および血漿中に見出されるプリオン結合活性を有する因子
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WO2003073106A2 (fr) * 2002-02-28 2003-09-04 Microsens Biophage Limited Liaison de formes pathologiques de proteines prion
DE10230141A1 (de) * 2002-07-04 2004-02-05 Labor Diagnostik Gmbh Leipzig Verfahren zur Anreicherung und zum Nachweis von veränderten Prion-Proteinen (PrPSc)
US7482172B2 (en) 2005-09-19 2009-01-27 Ortho-Clinical Diagnostics, Inc. Peptides for discrimination of prions
EP2085404A1 (fr) 2004-09-30 2009-08-05 Ortho-Clinical Diagnostics, Inc. Peptides pour la discrimination des prions
FR2940446A1 (fr) * 2008-12-22 2010-06-25 Lfb Biotechnologies Procede de detection d'une infection par prion
EP2503336A1 (fr) * 2011-03-21 2012-09-26 Etablissement Français du Sang Nanobilles recouvertes de plasminogène comme support direct d'amplification cyclique de la protéine prion PrPsc
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Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003073106A2 (fr) * 2002-02-28 2003-09-04 Microsens Biophage Limited Liaison de formes pathologiques de proteines prion
WO2003073106A3 (fr) * 2002-02-28 2004-01-08 Microsens Biophage Ltd Liaison de formes pathologiques de proteines prion
DE10230141A1 (de) * 2002-07-04 2004-02-05 Labor Diagnostik Gmbh Leipzig Verfahren zur Anreicherung und zum Nachweis von veränderten Prion-Proteinen (PrPSc)
DE10230141B4 (de) * 2002-07-04 2004-07-15 Priontype Gmbh Verfahren und Kit zur Anreicherung und zum Nachweis von veränderten Prion-Proteinen (PrPSc)
EP2085404A1 (fr) 2004-09-30 2009-08-05 Ortho-Clinical Diagnostics, Inc. Peptides pour la discrimination des prions
US7482172B2 (en) 2005-09-19 2009-01-27 Ortho-Clinical Diagnostics, Inc. Peptides for discrimination of prions
FR2940446A1 (fr) * 2008-12-22 2010-06-25 Lfb Biotechnologies Procede de detection d'une infection par prion
WO2010072969A1 (fr) * 2008-12-22 2010-07-01 Lfb-Biotechnologies Procede de detection d'une infection par prion
EP2503336A1 (fr) * 2011-03-21 2012-09-26 Etablissement Français du Sang Nanobilles recouvertes de plasminogène comme support direct d'amplification cyclique de la protéine prion PrPsc
FR2973114A1 (fr) * 2011-03-21 2012-09-28 Ets Francais Du Sang Nanobilles recouvertes de plasminogene comme support direct d'amplification cyclique de la proteine prion prpsc
US8859298B2 (en) 2011-03-21 2014-10-14 Etablissement Francais Du Sang Nanobeads covered with plasminogen as a direct support for cyclic amplification of the prion protein PrPSC
US10323616B2 (en) 2015-03-05 2019-06-18 Continental Automotive Gmbh Method of manufacturing an injector for injecting fluid and injector for injecting fluid

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