United States Patent: 6,765,088
Issued: July 20, 2004
Inventors: Korth; Carsten (San Francisco, CA); Stierli; Beat (Daenikon, CH); Stregt; Peter (Zurich, CH); Oesch; Bruno (Stilli, CH); Moser; Markus (Zurich, CH)
Assignee: Universitat Zurich (Zurich, CH)
Appl. No.: 380015
Filed: August 23, 1999
PCT Filed: February 18, 1998
PCT NO: PCT/EP98/00917
PCT PUB.NO.: WO98/37210
PCT PUB. Date: August 27, 1998
The presented invention relates to monoclonal antibodies useful in sensitive and specific immunological assays for the identification of prions in various tissues and body fluids, the production of such monoclonal antibodies by means of immunization of PrP0/0 mice by means of a new recombinant fragment of PrP and the use of the antibodies, e.g. for therapeutic and preventive treatments of humans and animals suffering from prion diseases.
SUMMARY OF THE INVENTION
Surprisingly the drawbacks of the prior art can be overcome by immunization of PrP0/0 knockout mice with highly purified recombinant PrP followed by fusion of splenocytes from these mice with myeloma cells. The resulting hybridoma cell lines are surprisingly stable and secrete highly specific antibodies to both PrP isoforms (PrPC and PrPSc) in their native as well as denatured state. The obtained antibodies are very useful for the development of highly specific immunological tests for prion diseases and other purposes.
The present invention concerns a monoclonal antibody or a fragment thereof capable of specifically binding to recombinant bovine prion protein, and native and denatured normal PrPC or disease-specific prion protein PrPSc in an antigen-antibody complex.
The present invention concerns further an antibody or a fragment thereof capable of specifically binding to the binding region (idiotype) of said antibody.
The present invention concerns further a hybridoma cell line capable of producing a monoclonal antibody capable of specifically binding to recombinant bovine prion protein, and native and denatured normal PrPC or disease-specific prion protein PrPSc in an antigen-antibody complex.
The present invention concerns further a recombinant expression vector for the expression of recombinant bovine prion protein.
The present invention concerns further a highly purified recombinant bovine prion protein, which may be in reduced or oxidized form.
The present invention concerns further a method for the production of an antibody as mentioned above, comprising culturing a hybridoma cell line as mentioned above and isolating the monoclonal antibody from the supernatant.
The present invention concerns further a method for the production of a hybridoma cell line as mentioned above, comprising administering to PrP0/0 mice (knockout mice without a functional PrP gene) an immunizing amount of recombinant prion protein as mentioned above, removing the spleen from the immunized mice, recovering sphenocytes therefrom, fusing the latter with P3X63Ag8U.1 hybridoma cells ATCC CRL 1597, growing the cells in a selection medium, screening the cells with recombinant PrP and isolating the positive cells.
The present invention concerns further a method for the production of an expression vector as mentioned above, comprising amplifying DNA from bovine genomic DNA coding for PrP by means of N- and C-terminal primers, and inserting the amplified DNA coding for PrP in the correct reading frame into an expression vector.
The present invention concerns further a method for the production of recombinant bovine prion protein comprising culturing microorganisms or cell lines with an expression vector as mentioned above in an appropriate culture medium and isolating and purifying the recombinant protein.
The present invention concerns further a test kit for the diagnosis of prion diseases.
The present invention concerns further an immunological detection procedure for the diagnosis of disease-specific prion proteins.
The present invention concerns further a pharmaceutical preparation for the therapy and prevention of prion diseases comprising a monoclonal antibody as mentioned above and pharmaceutical carrier.
The present invention concerns further a method for the therapy or prevention of prion diseases comprising administering to a patient suffering from such disease or being likely to becoming a victim of this disease a therapeutical or preventive amount of a monoclonal antibody as mentioned above.
The present invention concerns further a method for clearing biological material from prions comprising treating said material with a monoclonal antibody as mentioned above.
DETAILED DESCRIPTION OF THE INVENTION
In the following detailed description the spirit and scope of the invention will become more clearly explained and understood.
The monoclonal antibodies
A monoclonal antibody according to the invention is intended to bind to, to detect and qualitatively and quantitatively measure the presence of epitopes of prion proteins whether they are in soluble or insoluble form in various tissue specimens such as homogeneous or sections of brain, spleen, tonsils, white blood cells others and body fluids such as blood, cerebrospinal fluid saliva, urine or others. The present mABs bind to eptiopes of amino acids in a row or to epitopes of amino acids on different loops of the three-dimensional structure of native PrPs which are spatially close to each other. A particular group of the present antibodies binds only to native disease-specific PrP and not to native normal PrP.
Any known mABs which would fall under these definitions are exempted and disclaimed.
The term monoclonal antibody comprises also chimeric monoclonal antibodies having similar properties, which are derived from different animals, such as human/mouse chimeric antibodies or any other chimeric molecule comprising the antigen-binding part of the monoclonal antibody (idiotype) with other molecules such as antibody fragments of other monoclonal antibodies or enzymes.
A fragment of a monoclonal antibody comprising the binding part of the monoclonal antibody (idiotype) likewise capable of specifically binding the antigen and is termed Fab or (Fab')2 depending on whether the monoclonal antibody is digested with papain or pepsin, respectively.
A synthetic antibody or fragments thereof designed according to the amino acids or substituted homologous amino acids composing the idiotype responsible for binding the antigen. Homologous amino acids are defined as exchanges within the following five groups. 1. Small aliphatic, nonpolar or slightly poor residues: alanine, serine, threonine, glycine, proline; 2 Polar, negatively charged residues and their amides: aspartic acid, asparagine, glutamic acid, glutamine; 3. Polar, positively charged residues: histidine, arginine, lysine; 4. Large aliphatic, nonpolar residues: methionine, leucine, isoleucine, valine, cysteine; 5. Large aromatic residues: phenylalanine, tyrosine, tryptophan.
Preferred monoclonal antibodies are those named 6H4, 34C9, 15B3 which are produced by hybridoma cell lines DSM ACC2295, DSM ACC2296 and DSM ACC2998, respectively.
The antibodies and fragments thereof are essential tools for immunological detection procedures based on the binding of the prion protein to the presented monoclonal antibodies in an antigen-antibody complex. The monoclonal antibodies of the invention react with recombinant bovine PrP as well as native or denatured PrPC and PrPSc whether they are in soluble or insoluble state. The monoclonal antibodies react furtheron with PrP from different species, for example humans, hamsters, pigs, sheep, cattle and mice.
Furthermore, the present antibodies by forming an antigen-antibody complex between the presented monoclonal antibodies and the prion protein can be used to inhibit neurotoxic and infectious properties of the disease-specific prion protein.
The invention concerns further anti-idiotype antibodies which are antibodies that bind with their binding region (idiotype) to the binding region of the original monoclonal antibody. The anti-idiotype antibody mimicks features of the original antigen, in this case features of PrP. Anti-idiotype antibodies are raised as polyclonal antibodies (serum) or monoclonal antibodies from animals immunized with the preferred antibodies according to the invention. Anti-idiotype antibodies are valuable tools in detecting and blocking interactions of the original antigen (PrP), particularly interactions with receptors and can therefore be used in prevention and therapy of prion diseases.
The hybridoma cell lines
A stable hybridoma cell line according to the invention is capable of producing a monoclonal antibody as defined above over a prolonged time period of at least 6 months. Such cell lines are derived from the fusion of a spleen cell expressing the antibody derived from mice lacking a functional PrP gene, and a myeloma cell of mice providing survival of the fused cell lines.
Preferred hybridoma cell lines are DSM ATC2295, DSM ACC2296 and DSM ACC2298. The first two cell lines were deposited under the Budapest Treaty on Feb. 6, 1997 at the Deutsch Sammlung von Mikroorganisnien und Zellkulturen GmbH, Mascheroder Weg 1b, D-38124 Braunschweig, a recognized public depository for strains of microorganisms. The hybridome cell line producing mAB 15B3 was deposited Feb. 13, 1997 under number DSM ACCC2298 at the same depository.
The expression vector for recombinant bovine prion protein
An expression vector for the expression of the recombinant bovine prion protein is a DNA vector, based on the pET11a vector by Novagen comprising essential sequences for expression in the respective host, e.g. a T7-promoter and the DAN coding for the bovine prion protein from codons 25 to 242 with an additional codon ATG at the 5'-end the PrP-coding DNA and sequence for selecting, eg. the ampicillin gene, multiplication and termination.
Preferred expression vector is pbPrP3 as shown in FIG. 5.
The recombinant bovine prion protein
The present recombinant bovine prion protein consists of the amino acid sequence ID No:2. It may be unglycosylated or glycosylated.
The present recombinant bovine prion protein PrP is purified to a homogeneity of >98%. It can be present in oxidized or reduced form. In the oxidized form the single --S--S-- bridge is present whereas in the reduced form two SH groups are present instead. The amino acid sequence of the present recombinant bovine PrP is shown by ID NO 2. The protein is glycosylated if expressed in a glycosylating eukaryotic cell line, such as Chinese Hamster cells or unglycosylated if expressed in a procaryotic cell line, such as Escherichia coli. Mixtures of oxidized and reduced form are also comprised. The oxidized form has the molecular weight of 23676.8 Da and the reduced form 236886.1 Da as determined by electrospray mass spectroscopy. The present full length recombinant bovine prion protein is unique in terms of its homogeneity, since other groups in the art have reported of being unable to purify homogeneous full length recombinant prion protein of other species (Mehlhorn et al., 1996; Riek et al., 1996).
The reduced form of the recombinant PrP is particularly interesting since it has been reported to contain more .beta.-sheet secondary structures than the oxidized form (Mehlhorn et al., 1996), hence mimicking structural features of PrPSc. However, the reduced recombinant isoforms have been reported to be neither protease-resistant nor infectious (Mehlhorn et al., 1996).
A native prion protein PrP is the prion protein in a fully folded state, i.e. the three-dimensional structure is present. Only in the native, i.e. folded state PrP isoforms are different (normal native vs. disease-specific native PrP).
A denatured prion protein is the prion protein in the unfolded state. Unfolding is usually achieved by the addition of chaotropic substances such as urea or guanidinium hydrochloride. In the denatured state, both PrP isoforms are irreversibly the same, even if they have been normal native or disease-specific native before.
An antigen-antibody complex is a physical attachment of an antibody or fragment thereof with the corresponding antigen by intermolecular forces because the surfaces match in a unique way. The matching surface on the antibody is called idiotype and the surface on the antigen is called epitope.
Suitable epitopes detectable by the present antibodies are for example linear amino acid sequences having from about 3 to about 15 amino acids in a row or are completely three-dimensional ("patch") in that distant amino acid residues of the linear peptide backbone of the protein are, due to the unique folding, very close together in space to form an epitope.
The method for the production of an antibody
The present method for the production of an antibody according to the invention comprises culturing a hybridoma cell line as mentioned above and isolating the monoclonal antibody from the supernatant of the growth media.
Culturing is carried out in a flasks in HT-medium or in a cell culturing system called "technomouse" in serum-free, synthetic medium (Turbodoma medium, supplied by Messi, Zurich). In a "technomouse" hybridoma cells are cultured in a sterile chamber surrounded by a protein-impermeable membrane that is perfused by the respective medium in a constant flow rate (for example, turbomedium at 80 ml/h); antibodies are collected from the chamber with the help of a syringe at regular intervals.
Isolation of monoclonal antibodies is carried out by extraction from the supernatant by conventional biochemical methods, e.g. by use of affinity columns with the corresponding immobilized antigen or by any other method used in the art, such as gel filtration or ion exchange chromatography. In the "technomouse" supplied with serum-free turbomedium antibody concentrations and purities are achieved that need no further extracting procedures.
Chimeric antibodies and fragments thereof can be produced by genetic engineering methods, e.g. by sequencing the antibody or the desired fragment thereof and constructing DNAs coding for the chimeric antibody or the fragment thereof which DNAs are inserted into an appropriate expression vector and expressed to produce the antibody or the fragment thereof in both procaryotic or eukaryotic cell lines.
A fragment binding to a PrP epitope can be combined with a human heavy chain to produce chimeric antibodies for use in humans as therapeutic or preventive agents against a prion disease. A fragment binding to a PrP epitope can also be combined with other enzymes, proteins or molecules to give rise to chimeric molecules combining the biological functions of these, for example for targeting an enzymatic activity to a place defined by the proximity of the PrP epitope.
The method for the production of a hybridoma cell line
The present method for the production of a hybridoma cell line comprises administering to PrP0/0 mice (knockout mice without a functional PrP gene) an immunizing amount of a recombinant pure prion protein PrP, removing the spleen from the immunized mice, recovering splenocytes therefrom, fusing the latter with appropriate myeloma cells, growing the cells in a selection medium which does not support survival of the unfused cells, e.g. in HAT medium, screening the supernatants of the surviving hybridoma cells with recombinant PrP for the presence of antibodies to detect recombinant bovine PrP by an ELISA procedure and to detect native bovine PrPSc by a conformation-sensitive ELIFA procedure and isolating the positive cells. Positive hybridomas were selected and cloned twice by the limiting dilution method before the antibody was characterized and the epitope was mapped on a peptide library.
The peptide library used is commercially available from Jerini Biotools (Berlin Germany). It consists of 104 spots with peptides of 13 amino acids, whereby the sequence of each peptide overlaps with 11 amino acids of the foregoing peptide.
An immunizing amount of a recombinant bovine prion protein is from about 50 to 100 .mu.g. It is administered dissolved in an appropriate solvent, e. g, PBS and Freund's adjuvant several times, e.g. three times, subcutaneously followed by an intraperitoneal and an intravenous injection ultimately prior to spleen removal.
The PrP0/0 mice were a gift from Prof. Weissmann of the University of Zurich. They were obtained according to Bueler et al. (1992).
Appropriate myeloma cell are for example P3X63Ag8U.1 deposited and available under ATCC CRL 1597.
Recovering spleen cells and fusion conditions follow standard procedures, for esample as described by Kennett (1980).
The method for the production of an expression vector
The method for the production of an expression vector comprises inserting a DNA coding for PrP in the correct reading frame into an expression vector. One of the structures of the DNA coding for PRP is shown by SEQ ID NO:1. This DNA can be obtained by amplifying DNA from bovine genomic DNA coding for PrP by means of the N- and C-terminal primers shown by SEQ ID NO: 3 and SEQ ID NO: 4, respectively. Bovine genomic DNA is isolated from bovine kidney cells and supplied by Clonentech, U.S.A. Degenerate allelic forms of this DNA coding for the same PrP may be used. Furthermore, targeted mutations can be introduced into the PrP DNA to give rise to distinct conformational isoforms of the translated gene product.
The production of a purified recombinant bovine PrP
The production of a purified recombinant bovine PrP comprises culturing a cell line with an expression vector capable of expressing the bovine PrP in an appropriate culture medium, such as in the case of E. coli in Luria broth medium, isolating the PrP protein from the inclusion bodies by lysing the cells, e. g. with lysozym and Triton-X-100 in the case of E. coli, solubilizing the inclusion bodies with urea and and purifying the protein by conventional methods, e. g. by chromatography, for example on a anionic exchange sepharose column and C4 reverse phase HPLC column.
The oxidized form is obtained by treatment with an oxidizing agent, e. g. with Cu2 SO4, and the reduced form by treatment with a reducing agent, e. g. .beta.-mercaptoethanol, according of conventional methods. They can be separated by reverse-phase high pressure liquid chromatography.
Immunological detection procedures for the detection of prion disease
An immunological detection procedure for the detection of prion disease, especially BSE, whereby disease-specific PrPSc protein in biological material of an animal or human is detected, comprises treatment of a first probe of said material with a monoclonal antibody according to the invention and detecting the mixed PrPC /PrPSc -antibody complex, treating a second probe of said material first with proteinase K and then with the monoclonal antibody according to the invention, detecting the PrPSc -antibody complex and analyzing the results of both probes.
A specific monoclonal antibody according to the invention is able to detect PrPSc in a PrPSc -antibody complex without prior protease-digestion of the tissue specimen to be examined.
The biological material can be insoluble or soluble in buffer or body fluids. It can be derived from any part of the body, e. g. from the brain or the tissue sections, in which case it is used in form of a homogenate, or any body fluid, e. g. cerebrospinal fluid, urine, saliva or blood. In the case of body fluids, fluid-resident cells, e.g. white blood cells in the case of blood expressing PrP can be purified and analyzed either in immunohistochemistry or as a homogenate.
The detection of the PrPSc -antibody complex is carried out in particular by immunological procedures like the Western blotting, ELIFA, and various ELISA techniques such as capture ELISA.
The present immunological detection procedures allow the diagnosis of prion diseases. With the tools of the present invention, tissue sections, tissue homogenates or body fluids of prion-infected animals such as BSE-diseased cattle or humans having the CJD can be screened for the presence of the protease-resistant, disease-specific isoform of the prion protein in its native form, be it soluble or insoluble.
Tissue homogenates and body fluids are for example such as from biopsy of brain, lymph nodes, spleens, tonsils, peripheral nerves, cerebrospinal fluids, urine, platelets or white blood cells. Particular immunological procedures comprise for example, enzyme-linked immunofiltration assay (ELIFA) enzyme-linked immunoabsorbent assay (ELISA), Western blot assay, dot blot assay, immunodecoration and immunohistochemistry.
When native bovine PrPSc or any other disease-specific prion protein (e.g. ovine PrPSc or human PrPSc) has to be used in immunological assays, this can presently only successfully be achieved with the antibodies described in the present invention, since the present antibodies are the first of their art to be able to bind only native, disease-specific PrPSc.
The Test Kit for the Diagnosis of Prion Diseases
The test kit for the diagnosis of prion diseases comprises devices and materials enabling the diagnosis prion disease in biological materials, and is particularly suited for screening large amounts of samples for the presence of PrPSc. One test kit comprises in particular one or more monoclonal antibodies according to the invention, purified bovine recombinant PrP protein as mentioned above, nitrocellulose sheets, microtiter plates, or microtiter plates coated with the monoclonal antibodies according to the invention, a secondary anti-mouse antibody that is coupled with an enzyme and its substrate or any other molecular compound for a detection reaction (e.g. a peroxidase-labeled anti-mouse IgG antibody, TMB or any other peroxidase substrate), hydrogen peroxide, proteinase K, a blocking buffer, a homogenization buffer, a calibration curve and a description of how to perform the test.
Another test kit is designed in the dipstick format and is without need of radioactive tracers, enzymes or substrates and basically reduces the number of handling steps to one. The one-step procedure involves the capture of the disease-specific PrPSc with one of the antibodies according to claim 1 or 2 which are immobilized on a test strip. Captured disease-specific PrPSc are detected directly by a second antibody according to the invention, which is coupled to particular colloid particles. This specific detector complex results in the formation of coloured spots on the test strip which are visible in less than 30 minutes depending on the concentration of the test sample. The spots are a permanent record of the test result and, upon longer exposure even increase the sensitivity of the test without generating higher background.
Pharmaceutical Preparation for the Therapy and Prevention of Prior Diseases
The pharmaceutical preparation for the therapy and prevention of prion diseases in a mammal, including humans, comprises an effective amount of one or more antibodies fragments thereof or chimeric antibodies as described, produced according to the invention, eventually purified according to conventional methods, and a conventional pharmaceutical carrier. An antibody obtained may be solubilized together with the carrier in an appropriate buffer, e.g. an aqueous physiological sodium chloride solution. This may be clarified by centrifugation and used in concentrated liquid form for injection, or completely dried if desired by any of the conventional methods, such as lyophilization, spray or freeze drying, in form of a dry powder, which can be pressed into tablets, filled into capsules, or applied as a dry powder in form of a nasal spray, whereby conventional production methods are applied, and conventional pharmaceutical carriers are optionally added.
Method of Protecting a Mammal against Prion Disease
The monoclonal antibodies of the present invention bind to between spaces highly conserved regions in the PrP molecule that may have functional significance (Oesch et al. 1991). It is envisioned that blocking this binding site by the monoclonal antibodies, fragments thereof or chimeric antibodies as defined above will abolish biological effects of prions. Blocking of the infectivity of prions by occupying distinct sites on the disease-specific form of PrP is foreseen to represent a therapeutic strategy in treating prion diseases or a preventive strategy in preincubating suspected prion-infected tissue specimens with the present monoclonal antibodies. The normal form of PrP appears not to be of vital importance in the living animal because mice with a deleted PrP are viable (Bueler et al., 1992). Anti-PrP antibodies may therefore be used without side effects to neutralize prions in humans or animals.
The present invention concerns further a method for the therapy or prevention of prion disease or a disease mediated by the neurotoxic effects of prion proteins or fragments of prion proteins, comprising administering to a patient suffering from such disease or being likely to becoming a victim of this disease a therapeutical or preventive amount of a monoclonal antibody, a fragment thereof or a chimeric antibody as described above.
The method of protecting a mammal, including a human, again an infectious prion disease according to the present invention comprises administering one or a combination of the present antibodies, fragments thereof or chimeric antibodies or a pharmaceutical preparation comprising the antibodies produced by the present invention. The pharmaceutical preparation is preferably administered by injection, e.g. intrathecally (into the cerebrospinal fluid), into the blood with respective pharmaceutical agents or methods increasing the permeability of the blood-brain barrier or as a chimeric antibody, fused to, or containing additional signal sequences that allow passage through the blood-brain barrier (for review see Friden, 1994). An intranasal application of the monoclonal antibodies, fragments or chimeras thereof is also possible.
The pharmaceutical preparations have to be administered according to the judgment of the physician in amounts depending on the concentration of the antibodies comprised thereby and the route of administration so that a protective or curative effect is obtained. The amounts and method of administration are to be selected further depending upon the age and weight of the patient, the nature and severity of the infection as well as the general condition of the patient. In general it is sufficient to administer the antibodies in amounts of about 1 to 100 mg per patient in a single or in repeated doses.
Method for Clearing Biological Material from Prions
The method for clearing biological material from prions, e.g. intended for transplantation, substitution of biological material or oral consumption, comprises treating said material with one or several monoclonal antibodies according to the invention such that prions or prion proteins or fragments thereof become functionally inactivated in terms of their infectivity and/or neurotoxicity. The pharmaceutical preparations described above may be used for this purpose, whereby the pharmaceutical carrier may be replaced by a suitable other solvent in case the biological material is not intended to be used for transplantation.
The following examples serve to illustrate a particular embodiment of the invention but they should not be considered a limitation thereof. The immunological procedures outlined are made for the diagnosis of BSE in cattle, however, these procedures can also be applied for prion diseases in humans or animals such as sheep, hamster or mice.
Method for Protecting an Animal or a Human by Immunization with Recombinant PrP
The method for protecting animals or humans from infection with prions consists of an appropriate formulation of recombinant PrP of the appropriate species with an immunostimulator such as Freund's adjuvans. To protect against BSE, immunization is done with bovine PrP, against CJD human PrP is used, against sheep scrapie sheep PrP is used. In general, the PrP of the species where the the prion originated is used for immunization. Immunization induces the .beta.-lymphocytes to produce antibodies reacting with PrPSc. Such antibodies (of which 15B3 is a prototype) will be present in bood and lymphatic tissue and thereby bind and neutralize prions infecting a human or animal through peripheral pathways such as through skin lesions for example after accidental puncture with a needle or knife in a hospital or a slaughterhouse. The amount and type of PrP to be used for immunization will be determined according to the age and weight of the human or animal as well as the source of prions.
Claim 1 of 15 Claims
What is claimed is:
1. An isolated monoclonal antibody or a fragment thereof, wherein said antibody and said fragment are capable of binding only to native disease-specific prion protein (PrPSc) and not to native normal prion protein PrPC) in an antigen-antibody complex.