EP1200469A2 - Fragments de proteine prion cellulaire et methodes de diagnose et traitement demaladies liees aux prions - Google Patents

Fragments de proteine prion cellulaire et methodes de diagnose et traitement demaladies liees aux prions

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Publication number
EP1200469A2
EP1200469A2 EP00951682A EP00951682A EP1200469A2 EP 1200469 A2 EP1200469 A2 EP 1200469A2 EP 00951682 A EP00951682 A EP 00951682A EP 00951682 A EP00951682 A EP 00951682A EP 1200469 A2 EP1200469 A2 EP 1200469A2
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EP
European Patent Office
Prior art keywords
antibody
prp
cellular form
prion protein
binding agent
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.)
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Application number
EP00951682A
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German (de)
English (en)
Inventor
John MRC Prion Unit Dep.of Neurogenetics COLLINGE
Anthony Russel MRC Prion Unit CLARKE
Jonathan Peter 14 Rock Cottages WALTHO
Graham Stuart MRC Prion Unit JACKSON
Laszlo Luis Pereira MRC Prion Unit HOSSZU
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D Gen Ltd
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Imperial College Innovations Ltd
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Priority claimed from GBGB9917491.4A external-priority patent/GB9917491D0/en
Priority claimed from GBGB9917878.2A external-priority patent/GB9917878D0/en
Application filed by Imperial College Innovations Ltd filed Critical Imperial College Innovations Ltd
Publication of EP1200469A2 publication Critical patent/EP1200469A2/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • the present invention relates to prion proteins.
  • Prions are infectious pathogens that differ from bacteria, fungi, parasites, viroids, and viruses, both with respect to their structure and with respect to the diseases that they cause. Molecular biological and structural studies of prions promise to open new vistas into fundamental mechanisms of cellular regulation and homeostasis not previously appreciated. Kuru, Creutzfeldt- Jakob disease (CJD), fatal familial insomnia (FFI) and Gerstmann-Straussler-Scheinker syndrome (GSS) are all human neurodegenerative diseases that are caused by prions and are frequently transmissible to laboratory animals. Familial CJD and GSS are also genetic disorders. No effective therapy exists to prevent these fatal disorders 2 .
  • BSE Bovine spongiform encephalopathy
  • BSE Since 1986, more than 170,000 cattle have developed BSE in Great Britain. Many investigators contend that BSE, often referred to as "mad cow disease", resulted from the feeding of dietary protein supplements derived from rendered sheep offal infected with scrapie to cattle, a practice banned since 1988. It is thought that BSE will disappear with the cessation of feeding rendered meat and bone meal, as has been the case in kuru of humans, confined to the Fore region of New Guinea and once the most common cause of death among women and children. Kuru has almost disappeared with the cessation of ritualistic cannibalism.
  • PrP 50 a conformational isomer of host-derived prion protein
  • PrP 0 host-derived prion protein
  • PrP 50 is the principal or sole component of transmissible prions 2 .
  • the structure of PrP° has been determined 3 and has been found to consist predominantly of ⁇ -helices, the insolubility of PrP 50 , which is isolated from tissue in a highly aggregated state and which has a high ⁇ -sheet content, has precluded high-resolution structural analysis.
  • PrP 50 The detection of the disease-associated isoform of prion protein, PrP 50 , in brain or other tissues from patients is thought to be diagnostic of prion disease.
  • PrP To distinguish PrP 50 from its cellular precursor, PrP" 1 , requires either pre-treatment with proteinase K, which will completely digest PrF, but only removes a protease-sensitive N-terminal of PrP ⁇ or, alternatively, would require an antibody which distinguished between PrPand P ⁇ ? 50 . Only one such selective antibody (Korth C. et al. 1997 Nature 390, 74-77) has yet been reported and appears to be able to selectively immunoprecipitate PrP 80 . It is not clear as yet, however, whether this antibody offers any increase in diagnostic sensitivity over existing monoclonals.
  • PrP c located around the disulphide bond of the cellular ( ⁇ ) form of a prion protein, PrP c , is a nucleus of structure that persists in the unfolded state of the molecule. This nucleus of structure is a specific marker of PrP c and not PrP 50 .
  • the invention provides a peptide consisting of all or a fragment or variant of an amino acid sequence of a prion protein which exhibits stability when measured by hydrogen /deuterium amide exchange at pH 5.5.
  • the peptide exhibits a protection factor greater than the equilibrium constant between a native state an unfolded state; the protection factor (K ⁇ /K ⁇ t ) being determined by caculating amide exchange rate constants (Kg from a series ⁇ - ⁇ N HSQC spectra and using intrinsic amide exchange rates (K ⁇ ,).
  • a peptide of the invention consisting of all or a fragment or variant of an amino acid sequence from residue number 176 to 221 of one of the prion protein cellular form (PrP°) sequences shown in figure 5, and preferably consists of all or a fragment or variant of an amino acid sequence from residue number 179 to 218 of any one of the said PrP c sequences.
  • the invention also provides a peptide consisting of all, or a fragment or variant of an amino acid sequence of approximately ten residues which flanks the disulphide bond between Cys 179 and Cys 214 in one of the PrP c sequences shown in figure 5.
  • the disease-related isofo ⁇ n of PrP, PrP 80 is distinguished biochemically from the normal cellular isoform of the protein, PrP 0 , by its partial resistence to digestion with the enzyme proteinase K.
  • cellular form of a prion protein we include any form of a prion protein which does not exhibit partial resistance to digestion with proteinase K (PK).
  • PK proteinase K
  • PK proteinase K
  • the non-cellular form displays resistance to digestion at increased concentrations of PK eg 5 ⁇ g/ml PK or more.
  • Non-cellular forms of a prion protein referred to herein exhibit partial resistance to digestion with PK and include PrP 50 and the ⁇ -form as reported recently by Jackson et al, Science (March 1999) 283, pp 1933- 1937.
  • the invention also provides a peptide of the invention for use in medicine, preferably in the prevention, treatment and/or diagnosis of a prion disease.
  • the peptide sequence is selected from human, bovine or ovine prion proteins, more preferably human prion protein.
  • peptides of the invention include variants, fragments and fusions that have interactions or activities which are substantially the same as those of the stable core sequence of PrP c .
  • a relevant activity of the variants, fragments, or fusions of the invention is the ability to raise an antibody which binds preferentially to the cellular form of a prion protein, PrP c , rather than the non-cellular form, such as PrP 50 .
  • a "variant” will have a region which has at least 80% (preferably 85,90, 95 or 99%) sequence identity with the stable core region of cellular human PrP c sequence described herein or the corresponding region in the PrP c of other species as measured by the Bestfit Program of the Wisconsin Sequence Analysis Package, version 8 for Unix.
  • the percentage identity may be calculated by reference to a region of at least 10 amino acids (preferably at least 20, 30, 40 or 45) of the candidate variant molecule, and the most similar region of equivalent length in the native region.
  • the percent identity may be determined, for example, by comparing sequence information using the GAP computer program, version 6.0 described by Devereux et al. (Nucl. Acids res. 12:387, 1984) and available from the University of Wisconsin Genetics Computer Group (UWGCG).
  • the GAP program utilizes the alignment method of Neddleman and Wunsch (J. Mol. Biol. 48:443, 1970), as revised by Smith and Waterman (Adv. Appl. Math 2.482. 1981).
  • the preferred default parameters for the GAP program include : (1) a unary comparison matrix (containing a value of 1 for identities and 0 for non-identities) for nucleotides, and the weighted comparison matrix of Bribskov and Burgess, Nucl. Acids Res.
  • variants is intended to embrace prion protein sequence variations between species, in particular, the known variations at residue numbers 184, 186, 203, 205, 215, 219 and 220 (see Figure 5).
  • a “fragment” comprises any peptide sequence which is antigenic ie capable of reacting with antibody a PrP c specific and preferably is immunogenic ie capable of generating a PrP c specific antibody response itself.
  • the peptide sequence of the fragment will preferably comprise at least 45, preferably 6, 8, 10 or 12 or more amino acids of the native 176 to 221 PrP c sequences described herein.
  • an antibody comprising adininistering a cellular form (PrP°) of a prion protein or a peptide as defined in accordance with the earlier aspects of the invention, or a mixture of two or more of the peptides to an animal and collecting and optionally purifying the resulting antibody.
  • PrP° cellular form
  • antibody in accordance with the invention we include molecules which comprise or consist of antigen binding fragments of an antibody including Fab, Fv, ScFv and dAb. We also include agents which incorporate such fragments as portions for targetting prion molecules and/or prokaryotic or eukaryotic cells or viruses which display such molecules.
  • a monoclonal antibody capable of distinguishing between the cellular form of a prion protein, PrP c , and the non-cellular form of a prion protein (eg PrP 50 ) as defined in accordance with earlier aspects of the invention. Also provided is a hybridoma cell capable of producing such a monoclonal antibody.
  • the invention also provides a method of making a binding agent capable of binding to a cellular form of a prion protein comprising exposing a peptide of the invention to a sample whereby any binding agent can bind the peptide and collecting the binding agent so bound.
  • the binding agent is an antibody.
  • the invention also provides a binding agent, which is preferably obtainable by the above method, which binds preferentially to a cellular form of a prion protein rather than a non-cellular form.
  • the invention also provides the use of an antibody or binding agent in a method of detecting a cellular form of a prion protein comprising exposing a sample to the antibody or binding agent and detecting binding of the antibody or binding agent to the cellular form of a prion protein.
  • an antibody or binding agent of the invention in a method of detecting a non-cellular form of a prion protein comprising a first step of exposing a sample to the antibody or binding agent; a second step of exposing the sample to an agent which binds the non-cellular form of a prion protein; and detecting binding the agent to the non-cellular form.
  • the antibody or binding agent which has bound the cellular form is separated from the sample prior to the second step.
  • the antibody or binding agent in the first and/or second step is immobilised.
  • the invention also provides a method of removing a cellular form of a prion protein from a sample exposing the sample to an antibody or binding agent of the invention and separating the sample from the antibody or binding agent which has bound the cellular form.
  • the invention also provides a method of detecting antibodies in a sample, which antibodies bind preferentially to a non-cellular form of a prion protein rather than the cellular form, comprising exposing a peptide of the invention to the sample to permit binding of antibody to the peptide; and subsequently exposing the non-cellular form to the sample and detecting the binding of antibody to the non-cellular form.
  • the cellular and/or non- cellular form is immobilised before exposure to the sample.
  • the invention further provides a method of the invention for use in the prevention, treatment and/or diagnosis of a prion disease.
  • the method is for use in the diagnosis of the presence of a prion disease or a predisposition to such a disease.
  • the invention also provides a peptide of the invention, or an antibody or binding agent which binds preferentially to a cellular form of a prion protein rather than a non-cellular form of a prion protein, for use in medicine, preferably in the prevention, treatment and/or diagnosis of a prion disease.
  • the prion disease may be selected from one or more of the diseases affecting humans.
  • the prion diseases are selected from one or more of the diseases which affect domestic farm animals such as cows, sheep and goats.
  • Other prion diseases include transmissible mink encephalopathy; chronic wasting disease of mule deer and elk, bovine spongiform encephalopathy and, more recently, a whole series of new animal diseases that are thought to have arisen from their dietary exposure to the BSE agent.
  • feline spongiform encephalopathy affecting domestic cats and captive wild cats (such as cheetahs, pumas, ocelots, tigers) and spongiform encephalopathies of captive exotic ungulates (including kudu, nyala, gemsbok, eland).
  • the invention also provides a kit comprising means for carrying out the methods of the invention.
  • the invention also provides a kit useful for diagnosing a prion disease comprising a binding agent, preferably an antibody, which is capable of preferentially binding the cellular form rather than the non-cellular form, and means for detecting binding of the binding agent to the cellular form.
  • a binding agent preferably an antibody
  • the binding agent is coupled to an inert support.
  • the means for detecting binding comprises a radioactive, enzymic or fluorescent label.
  • the kit further comprises a binding agent, preferably an antibody, which is capable of binding the non-cellular form of a prion protein and means to detect binding of the binding agent to the non-cellular form.
  • the sample comprises or consists of a bodily tissue or fluid, which may be blood or a derivative of blood, ie a component such as plasma, or is derived from lymphoid tissue (such as tonsils, appendices, lymph nodes or spleen) or is cerebrospinal fluid, or faeces, urine or sputum, for example.
  • the biological sample may be a tissue sample eg a biopsy tissue sample.
  • the ratio of noncellular/cellular binding may be 45/55, 25/75, more preferably, 10/90, 5/95, 1/99 or substantially 0/100.
  • Aromatic CD measured at 290 nm is shown as open triangles, overlaid with amide CD measured at 222 nm, shown as open circles. The lines superimposed upon the data are fits to the function:
  • ⁇ N (K (N/U) .exp(m.D))/(l +K (N J) .exp(m.D))
  • the stable region runs from residues 176 to 221 and is highlighted by a box in Figure 5.
  • the open reading frame of the human PrP gene was amplified by PCR using oligonucleotide primers designed to create an unique N-terminal BamHI site and C-terminal Hindlll site for directional cloning of the fragment into the expression vector pTrcHisB (Invitrogen Corp.).
  • the primer corresponding to the N-temiinal region of PRNP to be expressed was designed to mutate a glycine at codon 90 to methionine, with the C- terminal primer replacing a methionine residue at 232 to a stop codon.
  • the ligated pTrcHisB/ R/VP construct was used to transform the E.
  • the cell pellet was resuspended in 50ml of lysis buffer (50mM Tris. Cl pH 8.0, 200mM NaCl, 0.1 % Triton X100, lO ⁇ g/ml DNase 1, lO ⁇ g/ml lysozyme) and disrupted by sonication in 1 minute bursts for a total of 5 minutes. Centrifugation at 9,600 rpm for 30 minutes pelleted all the insoluble material and the supernatant was discarded. The pellet was then washed twice by resuspension in 50ml of lysis buffer with centrifugation at 7,500 rpm for 5 minutes between each wash.
  • lysis buffer 50mM Tris. Cl pH 8.0, 200mM NaCl, 0.1 % Triton X100, lO ⁇ g/ml DNase 1, lO ⁇ g/ml lysozyme
  • Solubilisation of protein in the pellet was performed by resuspension in 50ml of 50mM Tris. Cl, 6M GuHCl, lOOmM DTT pH 8.0. Cell debris and insoluble material was removed by centrifugation at 9,600rpm for 30 minutes. The supernatant was clarified by passage through a 0.2 ⁇ m filter and loaded onto a 20ml Ni-NTA-Sepharose (Quiagen) column pre-equilibrated with 50mM Tris. Cl, 6M GuHCl pH 8.0.
  • the protein was loaded in 50mM Tris.Cl, 6M GuHCl pH 8.0, washed with ddH 2 0 + 0.1 % trifluoroacetic acid (TFA) and eluted with a linear gradient of 15% to 60% acetonitrile + 0.09% TFA.
  • Human PrP emerged as two major peaks; oxidised protein at 40% acetonitrile and a second peak containing reduced PrP eluted at 45% acetonitrile.
  • the oxidised peak fractions were pooled and neturalised by the addition of 1M Tris.Cl pH 8.0 to a final concentration of lOOmM and saturated ammonium sulphate added to a final concentration of 70% .
  • Precipitated PrP accumulated at the interface between organic and aqueous phases and was removed to a separate container.
  • the protein was solubilised in a minimal volume of 50mM Tris.Cl, 6M GuHCl pH 8.0 and then diluted rapidly to a protein concentration of lmg/ml and dialysed for 16 hours against 50mM Tris.Cl pH 8.0 with a buffer change after 8 hours.
  • the N-terminal fusion peptide was removed by addition of enterokinase at lunit/3mg protein. Cleavage was allowed to occur at 37°C for 14 hours and terminated by the addition of "protease complete" (Boehringer Mannheim Corp).
  • NMR spectra were acquired at 303K on 12 mg/ml 13 C/ 15 N-labelled sample in 20 mM sodium acetate-d 3 2mM Sodium Azide, pH 5.5 (10%(v/v) D 2 0) using Bruker DRX-500 and DRX-600 spectrometers.
  • Backbone resonances H N ; N; C ⁇ ; C ' ; C ⁇
  • Almost complete backbone assignments were determined, the exceptions being residues 164-172, which form an ill-defined loop in the mouse PrP c NMR structure 9 ' 11 .
  • C ; C ; C p chemical shifts were used to calculate the chemical shift index (CSI) profile of human PrP c 22 .
  • NMR data were processed and analysed on Silicon Graphics Workstations using Felix 97 software (MSI Corp).
  • Amide exchange was initiated by diluting the 13 C/ 15 N-labelled human PrP c sample with an equal volume of 20 nM sodium acetate-d 3 , 2 mM Sodium Azide, pH 5.5 dissolved in 100% D 2 O.
  • the sample was equilibrated at 303K for 5 mins in a Bruker DRX-600 spectrometer and amide exchange rate constants (K e determined from a series ! H- 15 N HSQC spectra. These were used to determine protection factors (K ⁇ /K ⁇ for observable amides using intrinsic exchange rates K ⁇ 23 . Acquisition of the first experiment began " 5 mins after mixing, setting a lower limit on the detection of protection factors of approximately 10.
  • the amide CD. absorption of 5 ⁇ M PrP c dissolved in 20mM sodium acetate-d 3 , 2 mM Sodium Azide, pH 5.5 55% D 2 0 was recorded in varying concentrations of GuHCl at 222 nm on a Jobin-Yvon CD6 spectropolarimeter through 1cm light path with an integration time of 120 sec.
  • the aromatic CD. absorption response of 50 ⁇ M PrP was measured at 290 nm through a 1cm light path at increasing denaturant concentration.
  • PrP° insoluble PrP 50 by recruitment of the native, cellular isoform of the prion protein
  • PrP° spongiform encephalopathies
  • the disease process can be triggered by inherited mutations in the encoding gene 2 ' 3 , by infection with tissue containing PrP 50 or by a rare spontaneous event which gives rise to sporadic cases.
  • prion proteins show that the chain can adopt a variety of folds depending on solution conditions 4 ' 5 .
  • the native, cellular form (PrP°) is capable of being switched between the largely ⁇ -helical conformation, characteristic of this state, to a variety of other structures by changing temperature, pH and/or redox conditions 4 .
  • Most of these alternative states are dominated by ⁇ -sheet and form insoluble aggregates.
  • Such observations have led to the proposal that the prion protein shows unusual conformations, enriched in ⁇ -sheet, which are in equilibrium with the native PrP c state.
  • these alternative conformers, or a subset thereof can then irreversibly associate to form amyloid rods 6 . This process is described most simply by the following scheme:
  • N is the native state
  • Al is an amyloidogenic folding intermediate
  • Am is irreversibly aggregated amyloid material.
  • formation of the prion amyloid can be promoted by mutations which increase the population of the amyloidogenic intermediate and by the introduction of stable amyloid material which can then recruit such an intermediate.
  • This type of mechanism for the conversion of PrP c to PrP 50 can, to some degree, be tested by comparing the free energy of unfolding of the PrP c protein with its local hydrogen exchange properties.
  • FIG. 1 Shown in figure 1 are equilibrium denaturation profiles of human PrP c at pH 5.5 and 55% D 2 0; a condition chosen to match the hydrogen exchange measurements described later.
  • the amide and aromatic CD profiles show that the molecule denatures in a single transition to a state that retains some degree of backbone and aromatic organisation ( " 30% of the native signal). Analysis of the amide and aromatic transitions renders values of 1.5 x 10 3 and 1.7 x 10 3 respectively for the equilibrium constant K (N/U) in the absence of denaturant.
  • K fl/ u ) and K (N/I) are greater than 1 and the 'U ⁇ - >I' equilibrium is fast (as with most proteins) then the intermediate, despite being more stable than the unfolded state, is only seen as a transient species.
  • the native state (N) may be in equilibrium with a partially unfolded form (PUF) 7 so that the system can be written:
  • PrP 50 an aggregated ⁇ -sheet form of the prion protein, is generated from folding intermediates or partially unfolded forms which are rich in ⁇ -sheet structure and capable of forming strong intermolecular interactions. This type of mechanism has recently been proposed for the formation of lysozyme amyloid 8 .
  • this observation can be interpreted in one of two ways. Firstly, if prion amyloid formation occurs by assembly of a partially folded intermediate then this intermediate must retain these ⁇ -helical regions intact. Secondly, if the formation of amyloid states requires the disruption of this core structure, then the molecule must pass through a highly unfolded state before adopting an alternative fold capable of self-assembly. Since, in all cases where secondary structure content has been assessed, the amyloid aggregate or amyloidogenic precursor states have been shown to be dorninated by ⁇ -structure, the first mechanism appears improbable. Elimination of this route leads to the conclusion that complete or near- complete unfolding must precede rearrangement to the amyloidogenic intermediate.
  • Prion protein is subjected to digestion with varying concentrations of proteinase K (BDH) at 37 °C for 1 hr. Protein is digested at a concentration of lm/ml in lOmM NaAcetate + lOmM Tris. Acetate pH 8.0. Digestion is terminated by the addition of Pefablock (Boehringer Mannheim Corp.) to a final concentration of ImM. Following the addition of Pefabloc samples are heated to 100°C for 5 mins in the presence of SDS loading buffer. Aliquots of 20 ⁇ l are subjected to SDS- PAGE and the gels stained with Coomassie brilliant blue.
  • BDH proteinase K
  • Antibodies may be produced in a number of ways.
  • the prion protein or peptide is purified.
  • the immunisation animal may be a "knock-out" mouse, with no prion protein at all.
  • the animal is normally a mouse; for polyclonal, a rabbit or goat.
  • Antibodies can also be produced by molecular biology techniques, with expression in bacterial or other heterologous host cells (Chiswell, D.J. & McCafferty, J. (1992) Phage antibodies: will new "coli-clonal” antibodies replace monoclonal antibodies?" Trends Biotechnol. 10, 80-84).
  • the purification method to be adopted will depend on the source material (serum, cell culture, bacterial expression culture, etc.) and the purpose of the purification (research, diagnostic investigation, commercial production). The major methods are as follows:
  • Ammonium sulphate precipitation The ⁇ -globulins precipitate at a lower concentration than most other proteins, and a concentration of 33% saturation is sufficient. Either dissolve in 200g ammonium sulphate per litre of serum, or add 0.5 vol of saturated ammonium sulphate. Stir for 30 minutes, then collect the ⁇ -globulin fraction by centrifugation, redissolve in an appropriate buffer, and remove excess ammonium sulphate by dialysis or gel filtration.
  • Adsorption to cation exchangers in a buffer of around pH 6 has been used successfully, with elution with a salt gradient, or even standard saline solution to allow immediate therapeutic use.
  • Staphylococcus aureus Outer coat protein known as Protein A
  • Staphylococcus aureus Outer coat protein known as Protein A
  • F c invariant region
  • Immunoglobulins Rapid isolation of antigens from cells with a staphylococcal protein A-antibody absorbent: Parameters of the interaction of antibody-antigen complexes with protein A. J Immunol. 115, 1617-1624. Protein A has been cloned, and is available in many different forms, but the most useful is as an affinity column: Protein A coupled to agarose.
  • a mixture containing immunoglobulins is passed through the column, and only the immunoglobulins adsorb. Elution is carried out by lowering the pH; different types of IgG elute at different pHs, and so some trials will be needed each time.
  • the differences in the immunoglobulins in this case are not due so much to the antibody specificity, but due to different types of F c region.
  • Each animal species produces several forms of heavy chain varying in the F c region; for instance, mouse immunoglobulins include subclasses IgG r , IgG ⁇ , and IgG 3 all of which behave differently on elution from Protein A.
  • Protein G from a Streptococcus sp., can be used. This is more satisfactory with immunoglobulins from farm animals such as sheep, goats and cattle, as well as with certain subclasses of mouse and rabbit IgGs.
  • the most specific affinity adsorbent is the antigen itself.
  • the process of purifying an antibody on an antigen adsorbent is essentially the same as purifying the antigen on an antibody adsorbent.
  • the antigen is coupled to the activated matrix, and the antibody-containing sample applied. Elution requires a process for weakening the antibody-antigen complex. This is particularly useful for purifying a specific antibody from a polyclonal mixture.
  • Monoclonal antibodies can be prepared to most antigens.
  • the antigen-binding portion may be a part of an antibody (for example a Fab fragment) or a synthetic antibody fragment (for example a single chain Fv fragment [ScFv]).
  • Suitable monoclonal antibodies to selected antigens may be prepared by known techniques, for example those disclosed in "Monoclonal Antibodies: A manual of techniques", H Zola (CRC Press, 1988) and in 'Monoclonal Hybridoma Antibodies: Techniques and Applications", J G R Hurrell (CRC Press, 1982).
  • Non-human antibodies can be "humanized” in known ways, for example by inserting the CDR regions of mouse antibodies into the framework of human antibodies.
  • variable heavy (V H ) and variable light (V domains of the antibody are involved in antigen recognition, a fact first recognised by early protease digestion experiments. Further confirmation was found by "humanisation" of rodent antibodies. Variable domains of rodent origin may be fused to constant domains of human origin such that the resultant antibody retains the antigenic specificity of the rodent parental antibody (Morrison et al (1984) Proc. Natl. Acad. Sci. USA 81, 6851-6855).
  • variable domains that antigenic specificity is conferred by variable domains and is independent of the constant domains is known from experiments involving the bacterial expression of antibody fragments, all containing one or more variable domains.
  • variable domains include Fab-like molecules (Better et al
  • ScFv molecules we mean molecules wherein the V H and V L partner domains are linked via a flexible oligopeptide.
  • antibody fragments rather than whole antibodies
  • the smaller size of the fragments may lead to improved pharmacological properties, such as better penetration of solid tissue.
  • Effector functions of whole antibodies, such as complement binding, are removed.
  • Fab, Fv, ScFv and dAb antibody fragments can all be expressed in and secreted from E. coli, thus allowing the facile production of large amounts of the said fragments.
  • a CDR-grafted antibody may be produced having at least one chain wherein the framework regions are predominantly derived from a first antibody (acceptor) and at least one CDR is derived from a second antibody (donor), the CDR-grafted antibody being capable of binding to the ⁇ -form PrP antigen.
  • the CDR-grafted chain may have two or all three CDRs derived from the donor antibody.
  • the or each CDR comprises a composite CDR comprising all the residues from the CDR and all the residues in the corresponding hypervariable region of the donor antibody.
  • At least one residue in the framework regions of the CDR- grafted chain has been altered so that it corresponds to the equivalent residue in the antibody, and the framework regions of the CDR-grafted chain are derived from a human antibody.
  • the framework regions of the CDR-grafted chain are derived from a human Ig heavy chain.
  • residue 35 in the heavy chain framework regions be altered so that it corresponds to the equivalent residue in the donor antibody.
  • At least one composite CDR comprising residues 26 to 35, 50 to 65 or 95 to 102 respectively is grafted onto the human framework. It will be appreciated in this case that residue 35 will already correspond to the equivalent residue in the donor antibody.
  • residues 23, 24 and 49 in such heavy chains correspond to the equivalent residues in the antibody. It is more preferred that residues 6, 23, 24, 48 and 49 in such heavy chains correspond to the donor antibody in equivalent residue positions. If desired, residues 71, 73 and 79 can also so correspond.
  • any one or any combination of residues 57, 58, 60, 88 and 91 may correspond to the equivalent residue in the donor antibody.
  • the heavy chain may be derived from the human KOL heavy chain. However, it may also be derived from the human NEWM or EU heavy chain.
  • the framework regions of the CDR-grafted chain may be derived from a human kappa or lambda light chain.
  • a human kappa or lambda light chain advantageously at least one composite CDR comprising residues 24 to 34, 50 to 56 or 89 to 97 respectively is grafted onto the human framework.
  • residue 49 also corresponds to the equivalent residue in the donor antibody.
  • residues 49 and 89 correspond to the equivalent residues in the donor antibody. It may also be desirable to select equivalent donor residues that form salt bridges.
  • the light chain is preferably derived from the human REI light chain. However, it may also be derived from the human EU light chain.
  • the CDR-grafted antibody comprises a light chain and a heavy chain, one or, preferably, both of which have been CDR-grafted in accordance with the principles set out above for the individual light and heavy chains.
  • the CDR-grafted antibody is a complete Ig, for example of isotype IgG,, or IgG 2 , IgG 3 or IgM.
  • one or more residues in the constant domains of the Ig may be altered in order to alter the effector functions of the constant domains.
  • the CDR-grafted antibody has an affinity for the ⁇ -form PrP antigen of between about lC ⁇ .M "1 to about 10 12 .M " ⁇ more preferably at least l&M 1 .
  • the or each CDR is derived from a mammalian antibody and preferably is derived from a murine MAb.
  • the CDR-grafted antibody is produced by use of recombinant DNA technology.
  • a further method for producing a CDR-grafted antibody comprises providing a first DNA sequence, encoding a first antibody chain in which the framework regions are predorninantly derived from a first antibody (acceptor) and at least one CDR is derived from a second antibody (acceptor), under the control of suitable upstream and downstream elements; transforming a host cell with the first DNA sequence; and culturing the transformed host cell so that a CDR-grafted antibody is produced.
  • the method further comprises: providing a second DNA sequence, encoding a second antibody chain complementary to the first chain, under the control of suitable upstream and downstream elements; and transforming the host cell with both the first and second DNA sequences.
  • the second DNA sequence encodes a second antibody chain in which the framework regions are predominantly derived from a first antibody (acceptor) and at least one CDR is derived from the second antibody (donor).
  • the first and second DNA sequences may be present on the same vector.
  • the sequences may be under the control of the same or different upstream and/or downstream elements.
  • the first and second DNA sequences may be present on different vectors.
  • a nucleotide sequence may be formed which encodes an antibody chain in which the framework regions are predorninantly derived from a first antibody (acceptor) and at least one CDR is derived from a second antibody (donor), the antibody chain being capable of forming a CDR-grafted antibody.
  • the CDR-grafted antibodies may be produced by a variety of techniques, with expression in transfected cells, such as yeast, insect, CHO or myeloma cells, being preferred. Most preferably, the host cell is a CHO host cell.
  • variable domain sequence of an antibody having the desired binding properties Suitable source cells for such DNA sequences include avian, mammalian or other vertebrate sources such as chickens, mice, rats and rabbits, and preferably mice.
  • the variable domain sequences (V H and V may be determined from heavy and light chain cDNA, synthesized from the respective mRNA by techniques generally known to the art.
  • the hypervariable regions may then be determined using the Kabat method (Wu and Kabat, J. (1970) /. Exp. Med. 132, 211).
  • the CDRs may be determined by structural analysis using X-ray crystallography or molecular modelling techniques.
  • a composite CDR may then be defined as containing all the residues in one CDR and all the residues in the corresponding hypervariable region.
  • These composite CDRs along with certain select residues from the framework region are preferably transferred as the "antigen binding sites", while the remainder of the antibody, such as the heavy and light chain constant domains and remaining framework regions, may be based on human antibodies of different classes. Constant domains may be selected to have desired effector functions appropriate to the intended use of the antibody so constructed.
  • human IgG isotypes, IgG ⁇ and IgG 3 are effective for complement fixation and cell mediated lysis.
  • IgM and IgE may be more suitable.
  • Human constant domain DNA sequences preferably in conjunction with their variable domain framework bases can be prepared in accordance with well-known procedures.
  • An example of this is CAMPATH 1H available from Glaxo Wellcome.
  • CDR-grafted antibodies which contain select alterations to the human-like framework region (in other words, outside of the CDRs of the variable domains), resulting in a CDR-grafted antibody with satisfactory binding affinity.
  • binding affinity is preferably from about lO ⁇ M "1 to about lO ⁇ .M "1 and is more preferably at least about lOlM "1 .
  • V H and/or V L gene segments may be altered by mutagenesis.
  • nucleotides coding for amino acid residues or sequences contained in the Fc portion or other areas of the antibody may be altered in like manner (see, for example, PCT/US89/00297).
  • Exemplary techniques include the addition, deletion or nonconservative substitution of a limited number of various nucleotides or the conservative substitution of many nucleotides, provided that the proper reading frame is maintained.
  • Substitutions, deletions, insertions or any subcombination may be used to arrive at a final construct. Since there are 64 possible codon sequences but only twenty known amino acids, the genetic code is degenerate in the sense that different codons may yield the same amino acid. Thus there is at least one codon for each amino acid, ie each codon yields a single amino acid and no other. It will be apparent that during translation, the proper reading frame must be maintained in order to obtain the proper amino acid sequence in the polypeptide ultimately produced.
  • exemplary techniques include oligonucleotide-mediated site-directed mutagenesis and the polymerase chain reaction.
  • Oligonucleotide site-directed mutagenesis in essence involves hybridizing an oligonucleotide coding for a desired mutation with a single strand of DNA containing the region to be mutated and using the single strand as a template for extension of the oligonucleotide to produce a strand containing the mutation. This technique, in various forms, is described in Zoller and Smith (1982) Nucl. Acids Res. 10, 6487.
  • PrP proteins or peptides are prepared in an immunogenic formulation containing suitable adjuvants and carriers and administered to the patient.
  • suitable adjuvants include Freund's complete or incomplete adjuvant, muramyl dipeptide, the "Iscoms" of EP 109 942, EP 180 564 and EP 231 039, aluminium hydroxide, saponin, DEAE-dextran, neutral oils (such as miglyol), vegetable oils (such as arachis oil), liposomes, Pluronic polyols or the Ribi adjuvant system (see, for example GB-A-2 189 141). "Pluronic" is a Registered Trade Mark.
  • cellular form binding agent we include any agent which is able to binds preferentially the cellular form rather than the non-cellular form of a prion protein.
  • the binding agent is preferably an antibody or antigen binding fragment thereof such a Fab, Fv, ScFv and Ab, but it may also be any other ligand which exhibits the preferential binding characteristic mentioned above.
  • Antigen or antibody is bound through its free amino groups to cyanogen- bromide-activated Sepharose particles.
  • Insolubilized antibody for example, can be used to pull the corresponding antigen out of solution in which it is present as one component of a complex mixture, by absorption to its surface. The unwanted material is washed away and the required ligand released from the affinity absorbent by disruption of the antigen-antibody bonds by changing the pH or adding chaotropic ions such as thiocyanate.
  • an antigen immunosorbent can be used to absorb out an antibody from a ⁇ iixture whence it can be purified by elution.
  • the potentially damaging effect of the eluting agent can be avoided by running the anti-serum down an affinity column so prepared as to have relatively weak binding for the antibody being purified; under these circumstances, the antibody is retarded in flow rate rather than being firmly bound. If a protein mixture is separated by iso-electric focusing into discrete bands, an individual band can be used to affinity purify specific antibodies from a polyclonal antiserum.
  • Affinity chromatography A column ts filled with change in pH for example. An antigen-linked affinity column Stpharose-hnked antibody. The antigen mature is poured tt purify antibody obviously. down the column. Only the antigen hinds and is released by
  • Antigen and antibody can be used for the detection of each other and a variety of immunoassay techniques have been developed in which the final read-out of the reaction involves a reagent conjugated with an appropriate label. Radiolabelling with 131 I, 125 I, is an established technique. Soluble Phase immunoassays
  • the binding of radioactively labelled antigen to a limited fixed amount of antibody can be partially inhibited by addition of unlabelled antigen and the extent of this inhibition can be used as a measure of the unlabelled material added.
  • the antibody content of a serum can be assessed by the ability to bind to antigen which has been in and immobilised by physical absorption to a plastic tube or micro-agglutination tray with multiple wells; the bound immunoglobin may then be estimated by addition of a labelled anti-Ig raised for anther species.
  • a labelled anti-Ig raised for anther species For example, a patient's serum is added to a microwell coated with antigen, the antibodies will bind to the plastic and remaining serum proteins can be readily washed away.
  • Bound antibody can be estimated by addition of 125 I-labelled purified rabbit anti IgG; after rinsing out excess unbound reagent, the radioactivity of the rube will be a measure of the antibody content of the patient's serum.
  • the distribution of antibody in different classes can obviously be determined by using specific antisera.
  • Add Ag I Add patients' serum I Add labelled anti-Ig Solid phase immunoassay for antibody. By attaching antibody to the solid phase, the system can be used to assign antigen. To reduce non-specific binding of IgG to the
  • Enzymes which give a coloured reaction product, usually in solid phase assays. Enzymes such as horse radish peroxidase and phosphatase have been widely employed. A way of amplifying the phosphatase reaction is to use NADP as a substrate to generate NAD which now acts as a coenzyme for a second enzyme system. Pyrophosphatase from E.coli provides a good conjugate because the enzyume is not present in tissues, is stable and gives a good reaction colour. Chemi-luminescent systems based on enzymes such as luciferase can also be used.
  • Vitamin biotin Conjugation with the vitamin biotin is frequently used since this can readily be detected by its reaction with enzyme-linked avidin or streptavidin to which it binds with great specificity and affinity.
  • the cloned nucleic acid is expressed fused to the coat-anchoring part of one of the phage coat proteins (typically the p3 or p ⁇ coat proteins in the case of filamentous phage), such that the foreign protein or polypeptide is displayed on the surface of the phage;
  • the phage displaying the protein or polypeptide with the desired properties is then selected (e.g. by affinity chromatography) thereby providing a genotype (linked to a phenotype) that can be sequenced, multiplied and transferred to other expression systems.
  • the foreign protein or polypeptide may be expressed using a phagemid vector (i.e. a vector comprising origins of replication derived from a phage and a plasmid) that can be packaged as a single stranded nucleic acid in a bacteriophage coat.
  • a phagemid vector i.e. a vector comprising origins of replication derived from a phage and a plasmid
  • helper phage is used to supply the functions of replication and packaging of the phagemid nucleic acid.
  • the resulting phage will express both the wild type coat protein (encoded by the helper phage) and the modified coat protein (encoded by the phagemid), whereas only the modified coat protein is expressed when a phage vector is used.
  • phage expressing a protein or peptide with a desired specificity are known in the art.
  • a widely used method is "panning", in which phage stocks displaying ligands are exposed to solid phase coupled target molecules, e.g. using affinity chromatography.
  • Alternative methods of selecting phage of interest include SAP (Selection and Amplification of Phages; as described in WO 95/16027) and SIP (Selectively-Infective Phage; EP 614989A, WO 99/07842), which employ selection based on the amplification of phages in which the displayed ligand specifically binds to a ligand binder.
  • SAP Selection and Amplification of Phages
  • SIP Selectively-Infective Phage
  • Proteins and multimeric proteins have been successfully phage-displayed as functional molecules (see EP 0349578A, EP 0527839A, EP 0589877A; Chiswell and McCafferty, 1992, Trends Biotechnol. 10, 80-84).
  • functional antibody fragments e.g. Fab, single chain Fv [scFv]
  • Fab single chain Fv
  • PrP or its peptide derivatives may be provided in an acetate buffer as described above.
  • Antigens may be physically (by creating recombinant PrP fusion proteins) or chemically coupled to suitable carrier proteins to provide additional T cell help for immunisation in PRNP +/+ mice and other rodents.
  • mice of various strains, rats, hamsters or rabbits can be inoculated subcutaneously with PrP (50-100 ⁇ g/ animal), emulsified in complete/incomplete Freunds adjuvant at 3 weekly intervals (Days).
  • anti-peptide activity can be assayed by ELISA.
  • a final intraperitoneal boost can be given and the animals killed for fusion 3 days later (day 50).
  • the animals may be bled after the final boost, and at regular subsequent intervals with or without further inoculation depending on anti-PrP titre.
  • Routine methods may be used (Galfre G., and Milstein, C. 1981 Methods in Enzymology 73, 3-46)
  • Rat fusions Y3 (210.RCY3.Ag 1.2.3)YO Galfre G., and Milstein, C.
  • Myeloma cells growing in exponential phase may be mixed with splenic single cell suspensions in appropriate ratios, washed free of serum, and then gently resuspended in a 50% polyethylene glycol solution at 37 °C followed after 1-2 minutes with increasing volumes of serum-free medium. After a further incubation in RPMI/10% foetal calf serum (RF 10 ) at 37°C for 30 minutes, the hybridomas may be washed and resuspended in HAT medium and hybridoma growth supplements, are cultured in 200 ⁇ l flat- bottomed tissue culture wells at 37 °C in 5% C0 2 enriched humidified air.
  • Recombinant PrP may be dialysed against appropriate coating buffer (pH 4-10) and adsorbed to standard ELISA plates for 30-60 minutes at 37°C prior to washing x4 in PBS/Tween 0.05% (PBST). After blocking in PBS/BSA 2% with or without additional sera, dilutions of serum are incubated in duplicate as are relevant negative and positive controls. After washing, the peroxidase conjugated anti-IgG secondary is incubated, washed and then fresh ortho-phenyl diamine (OPD) substrate added. Finally after stopping the reaction with 3M sulphuric acid the absorbance is measured at 492nm.
  • PBS/BSA PBS/Tween 0.05%
  • This may involve a staged two day procedure.
  • 50 ⁇ l of the growing cultures may be screened for anti-PrP IgG as in the ELISA described above.
  • This PrP may or may not be first digested with proteinase K to remove any PrP species. Positive wells in this assay may then be screened the following day in a dot blot assay modified from Sidle et al 1995.
  • Dot blot apparatus ELIFA, Pierce Wariner
  • Supernatants can be screened for binding to recombinant ⁇ -PrP, 1 % normal human brain homogenate and to a pool of 1 % homogenates from CJD brains containing types 1-4, thus enabling the preferential selection of PrP 80 - specific mAbs. Thus only mAbs that bind infectious prions and not PrP 0 from normal brain will be expanded.
  • culture supernantants can be screened for preferential binding to either cellular or ⁇ -PrP, or to synthetic peptides to which PrP Sc -specific mAbs may bind.
  • the 15B3 PrP Sc -specific mAb cross-reacts with human, bovine and murine PrP 50 , and its epitope has been mapped with linear synthetic peptides to three regions on the bovine PrP molecule: residues 142-148, 162-170 and 214- 226 and later two of which may not be recognised by antibodies that bind to both PrP 0 and PrP 550 (Korth C. et al. 1997 Nature 390, 74-77). These peptides are absorbed to ELISA plates with poly-lysine.
  • Immunoglobin subclass and culture supernatant Ig concentration can be measured by standard ELISA techniques.
  • the fine specificity of PrP° or PrP 50 specific mAbs can be defined either by using a gridded array of overlapping human PrP peptides (synthesised commercially by Jerino Bio Tools GMbH) or by using pools of PrP synthetic peptides (synthesised individually using standard f-moc chemistry) in the standard ELISA.
  • Measurements of the affinity of anti-PrP mAbs for their ligands can be made using surface plasmon resonance. Direct comparisons can be made of mAb binding to cellular and non-cellular-PrP molecules.
  • Flow cytometry and immunofluorescence microscopy may be used to study surface and intracellular PrF/PrP 50 expression in cell lines that express surface PrP (eg EVBV lymphoblastoid, U937, K562, HEl) and peripheral blood mononuclear cells. 15. Binding to PrP in tissue sections
  • PrP 50 The detection of the disease-associated isoform of prion protein, PrP 50 , in brain or other tissues from patients is thought to be diagnostic of prion disease.
  • PrP To distinguish PrP 50 from its cellular precursor, PrP, requires either pre-treatment with proteinase K, which will completely digest PrP°, but only removes a protease-sensitive N-terminal of PrP 50 or, alternatively, would require an antibody which distinguished between PrP°and PrP 50 . Only one such selective antibody (Korth C. et al. 1997
  • PrP c specific binding agents especially antibodies of the invention provide an important means distinguishing PrP 50 from PrP c .
  • Sandwich ELISA can be used to detect PrP 50 in body fluids eg serum or cerebropsinal fluid (CSF). This relies on using immobilised PrP Sc -specific mAbs to capture PrP 50 in solution and then using biotinylated mAbs or rabbit polyclonal antiserum with specificity for alternative PrP epitopes to detect the immobilised complexes. The same techniques can be used to detect PrP 50 in tissue homogenates.
  • the PrP c binding agents of the invention can be used in a sample pretreatment step to increase the sensitivity of such PrP 50 assays. They also allow PrP 50 binding agents which are not specific for PrP 50 to be used in the subsequent detection step.
  • tissue homogenates are placed directly on a suitable membrane and be treated with PrP c specific binding agent of the invention to remove PrP°.
  • the membrane can be incubated with anti-PrP antibodies and then such binding detected using an appropriate, labelled secondary antibody.
  • Various labelling systems involving enzymatic, fluorescent, radioisotopic or cherniluminescent methods are commonly used.
  • Standard Western blotting techniques can be used. These methods allow not only the detection of PrP, but of specific patterns of banding following proteinase K digestion. These patterns allow the recognition of distinct strains of prions and allow, for instance, the differentiation of new variant CJD from classical CJD (see Collinge et al. 1996 Nature 383, 685-690 and international PCT patent application published as WO 98/16834).
  • Diagnostic methods may be developed based on the differential affinity of anti-PrP mAbs for PrP° and PrP 50 .
  • Surface plasmon resonance is ideally suited for this purpose.
  • purified anti-PrP mAbs are immobilised and binding to solubilised PrP measured directly from tissue fluids and homogenates. Enrichment of PrP 50 by differential centrifugation or affinity purification or pretreatment with PrP c specific binding agents of the invention may be useful prior to the above assays.
  • 50 ⁇ l of the growing cultures may be screened for antibodies to a non-cellular form of PrP eg anti- ⁇ PrP IgG as in the ELISA described above.
  • This ⁇ -PrP may or may not be first treated with the PrP c specific antibodies of the invention to remove any cellular form PrP c , species.
  • Positive wells in this assay may then be screened the following day in a dot blot assay modified from Collinge et al 1995 Lancet 346:569-570.
  • Dot blot apparatus ELIFA, Pierce Wariner
  • Supernatants can be screened for binding to recombinant ⁇ -PrP, 1 % normal human brain homogenate and to a pool of 1 % homogenates from CJD brains containing types 1-4, thus enabling the preferential selection of PrP So -specific mAbs. Thus only mAbs that bind infectious prions and not PrP° from normal brain will be expanded.
  • culture supernantants can be screened for preferential binding to either cellular from or non-cellular ⁇ -PrP, or to synthetic peptides to which PrP So -specific mAbs may bind.
  • the 15B3 PrP 5c -specific mAb cross-reacts with human, bovine and murine PrP 50 , and its epitope has been mapped with linear synthetic peptides to three regions on the bovine PrP molecule: residues 142-148, 162-170 and 214- 226 and later two of which may not be recognised by antibodies that bind to both PrP° and PrP 50 (Korth C. et al. 1997 Nature 390, 74-77). These peptides are adsorbed to ELISA plates with poly-lysine.
  • PrP 50 in peripheral blood mononuclear cells (PBMC) of vCJD patients will be low and detection will depend on optimising methods for surface and intracellular detection of PrP and then identifying lymphocyte sub-populations with the highest prion load.
  • Anti- PrP mAbs can be purified and conjugated to biotin of fluorochromes for this purpose. Dual and three colour flow cytometry can be used to identify the PrP 50 bearing cell types. After surface staining by conventional techniques, intracellular PrP can be detected after fixation and permeabilisation of the cell membranes. Cellular manipulation (eg stimulation of proliferation of the pharmacological blockade of intracellular secretory or endocytic pathways) may be used to enhance PrP detection.
  • the PrP c specific binding agents of the invention may be used to increase the sensitivity of the above methods.
  • Prion disease may be diagnosed by abnormal patterns of PrP immunoreactivity on either formalin fixed, or frozen, tissue sections using established immonohistochemical detection techniques. Frozen tissue sections of whole brains (histoblots) may be treated with proteinase K or the PrP c specific binding agents of the invention and similarly exposed to antibodies to detect patterns of PrP 50 deposition which may also allow discrimination of prion strain types.
  • Prusiner S.B., Scott, M.R. , DeArmond, S,J. & Cohen, F.E. Cell 93, 337-348 (1998).

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Abstract

L'invention concerne des peptides et des liants, tels que des anticorps, et leurs utilisations, en particulier en médecine, notamment pour le traitement, la prévention et/ou le diagnostic de maladies à prion.
EP00951682A 1999-07-27 2000-07-25 Fragments de proteine prion cellulaire et methodes de diagnose et traitement demaladies liees aux prions Withdrawn EP1200469A2 (fr)

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GB9917491 1999-07-27
GBGB9917491.4A GB9917491D0 (en) 1999-07-27 1999-07-27 Biological materials and methods useful in the diagnosis and treastment of diseases
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GBGB9917878.2A GB9917878D0 (en) 1999-07-30 1999-07-30 Biological materials and methods useful in the diagnosis and treatment of diseases
PCT/GB2000/002873 WO2001007479A2 (fr) 1999-07-27 2000-07-25 Matieres vivantes et methodes utiles pour le diagnostic ou le traitement de maladies

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EP1416280A3 (fr) 1995-09-14 2005-08-10 The Regents of the University of California Anticorps spécifique de PrPsc native
US6537548B1 (en) 2000-07-27 2003-03-25 The Regents Of The University Of California Antibodies specific for ungulate PrP
US20050026165A1 (en) 2001-05-31 2005-02-03 Cindy Orser Detection of conformationally altered proteins and prions
MXPA03011000A (es) 2001-05-31 2004-02-27 Arete Associates Metodo de sensor de proteinas deformadas.
GB0227651D0 (en) * 2002-11-27 2003-01-08 Medical Res Council Prion
EP1457500A1 (fr) * 2003-03-14 2004-09-15 University of Zurich Proteinés solubles de prion hybrids et leurs utilisations en diagnostique, prévention et traítement de l'encephalopathie spongiforme transmissible
WO2005016127A2 (fr) 2003-08-13 2005-02-24 Chiron Corporation Reactifs peptidiques specifiques du prion
AU2006214463B2 (en) 2005-02-15 2012-08-30 Presympto, Inc. Method for detecting misfolded proteins and prions
KR20080048527A (ko) 2005-09-09 2008-06-02 노파르티스 아게 프리온-특이적 펩토이드 시약
EP2156181B1 (fr) 2006-07-28 2015-11-04 Adlyfe, Inc. Sondes peptidiques pour des diagnostics et des produits thérapeutiques

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EP0861900A1 (fr) * 1997-02-21 1998-09-02 Erziehungsdirektion Of The Canton Zurich Détection immunologique de prions
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