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Title:  Immunological agents specific for prion protein (PRP)

United States Patent:  6,528,269

Issued:  March 4, 2003

Inventors:  Sy; Man-Sun (Shaker Heights, OH); Gambetti; Pierluigi (Shaker Heights, OH)

Assignee:  Case Western Reserve University (Cleveland, OH)

Appl. No.:  204816

Filed:  December 3, 1998

Abstract

The present invention is directed to a panel of monoclonal antibodies (Mabs) specific for murine prion protein PrPc. These Mabs can be applied to immunoblotting, cell surface immunofluorescent staining and immunohistochemistry at light and electron microscopy. Additionally, these Mabs recognize both the normal (PrPc) and protease-resistant (PrPres) isoforms of PrP. Some Mabs are species restricted, while others react with PrP from a broad range of mammals including mice, humans, monkeys, cows, sheep, squirrels and hamsters. Moreover, several of the Mabs selectively recognize different PrP glycoforms as well as the metabolic fragments of PrPc. These newly generated PrPc antibodies are useful for exploring the biology of PrPc and to establish the diagnosis of prion diseases in both humans and animals.

DETAILED DESCRIPTION OF THE INVENTION

The objective of the present invention is to provide monoclonal antibodies, or epitope binding fragments thereof, with specificity for mammalian prion protein. The antibodies or fragments thereto are suitable for use in simple and highly sensitive immunoassays for detecting prion protein.

The present invention relates, in part, to the generation of a panel of monoclonal antibodies by immunizing prion "knock out" mice with recombinant normal mouse cellular prion protein (PrPc). Spleen cells (antibody producing lymphocytes of limited life span) from the immunized mice were fused with non-producing myeloma cells (tumor lymphocytes that are "immortal") to create hybridomas. The hybridomas were then screened for the production of antibody specific to prion and the ability to multiply indefinitely in tissue culture. These hybridons were then propagated to provide a permanent and stable source for the specific monoclonal antibodies.

In particular, the present invention is directed to monoclonal antibodies to mammalian prion protein entitled 2F8, 5B2, 6H3, 8C6, 8H4 and 9 H7 and active fragments thereof. These monoclonal antibodies are produced by cell lines PrP2F8, PrP5B2, PrP6H3, PrP8C6, PrP8H4 and PrP9H7 respectively.

These monoclonal antibodies produced recognized not only human prion protein, but they also cross-reacted with prion proteins from mouse, cow, sheep and other species. These antibodies are believed to be the first panel of monoclonal antibodies that are capable of reacting with human, mouse, sheep and cow prion proteins.

The monoclonal antibodies of the invention can be used in Western blot, in immunofluorescent staining, immunohistochemistry, immunoelectron microscopy, and ELISA. A number of the monoclonal antibodies also recognize species restricted epitopes, while others react with several test animals. Western blotting of these monoclonal antibodies revealed that they recognize different epitopes on normal PrPc. The monoclonals reacted differently to the PrPc glycoisoforms and to the fragments resulting from the metabolism of PrPc. The monoclonal antibodies of the invention facilitate the investigation of the biology of PrPc, and the progression and diagnosis of prion disease in both humans and animals.

The present invention also relates to the generation of a panel of Mabs which demonstrate that PrPc glycofoams an normal peripheral blood lymphocytes are different from the glycoforms expressed in normal brain tissue, on astrocytomas or on transfected nemoblastomas. Furthermore, the activation of lymphocytes resulted in quantitative as well as qualitative changes in the expression of PrPc. There observations help explain why activated lymphocytes are more efficient in prion propagation.

I. Manufacturing Monoclonal Antibodies Specific for Prion

Generation of monoclonal antibodies which specifically react with murine PrP is facilitated by the use of a transgenic mammal (a mammal that contains some genetic material that has been experimentally transferred into it from other source or some genetic material that has been expermentally deleted) having an nonfunctional prion protein encoding gene (Prnp). Prnp is selectively knocked out by homologous recombination in the targeted mammal. It has been shown that transgenic (Prnp-/-) mice develop normally and are insusceptible to prion diseases due to the inability to form PrP (Manson, et al., Mol. Neurobiol., 8: 121-127, 1994; Bueler, et al., Nature, 356: 577,582, 1992).

A suitable quantity of PrP is required for immunization of the transgenic mammal in order to form antibodies which specifically react with PrP. One way to produce suitable quantities of recombinant PrP is by transfecting E. coli with expression vectors carrying Prnp. Preferably, the cytoplasm of E. coli is transfected because it produces the largest quantities of PrP. The vector is designed to produce a large amount of stable mRNA that translates into PrP in the transfected cell. Prnp within the expression vectors can be derived from any mammal carrying that gene. This preferred method allows for the production of large quantities of PrP (Hornemann, et al., FEBS Lett. 413: 277-281 (1997). Alternatively, any other method can be used to produce sizeable quantities of PrP.

The transgenic (Prnp-/-) mammals are then immunized with PrP in CFA and boosted with PrP repeated in IFA. Shortly thereafter, somatic cells from the immunized Prnp-/- mammal, with potential for producing antibody, are fused with myeloma cells, forming a hybridoma of two cells by conventional protocol. Somatic cells may be derived from the spleen, lymph node, and peripheral blood of transgenic mammals. Myeloma cells which may be used for the production of hybridomas include murine myeloma cell lines such as MPCII-45.6TGI.7, NSI-Ag4/1, SP2/0-Ag14, X63-Ag8.653, P3-NS-1-Ag-4-1, P3 X63Ag8U1, OF, and S194/5XX0.BU.1; rat cell lines including 210.RCY3.Agl.2.3; cell lines including U-226AR and GM1500GTGAl.2; and mouse-human heteromyeloma cell lines (Hammerling, et al. (editors), Monoclonal Antibodies and T-cell Hybridomas IN: J. L. Turk (editor) Research Monographs in Immunology, Vol. 3, Elsevier/North Holland Biomedical Press, New York (1981)).

Somatic cell-myeloma cell hybrids are plated in multiple wells with a selective medium, such as HAT medium. Selective media allow for the detection of antibodyproducing hybridomas over other undesirable fused-cell hybrids. Selective media also prevent growth of unfused myeloma cells which would otherwise continue to divide indefinitely, since myeloma cells lack genetic information necessary to generate enzymes for cell growth. B lymphocytes derived from somatic cells contain genetic information necessary for generating enzymes for cell growth but lack the "immortal" qualities of myeloma cells, and thus, last for a short time in selective media. Therefore, only those somatic cells which have successfully fused with myeloma cells grow in the selective medium. The infused cells were killed off by the HAT or selective medium.

A screening method is used to examine for potential anti-PrP antibodies derived from hybridomas grown in the multiple wells. Multiple wells are used in order to prevent individual hybridomas from overgrowing others. Screening methods used to examine for potential anti-PrP antibodies include enzyme immunoassays, radioimmunoassays, plaque assays, cytotoxicity assays, dot immunobinding assays, fluorescence activated cell sorting (FACS), and other in vitro binding assays.

Hybridomas which test positive for anti-PrP antibody are maintained in culture and may be cloned in order to produce monoclonal antibodies specific for PrP. Alternatively, desired hybridomas can be injected into a histocompatible animal of the type used to provide the somatic and myeloma cells for the original fusion. The injected animal develops tumors secreting the specific monoclonal antibody produced by the hybridoma.

The monoclonal antibodies secreted by the selected hybridoma cells are suitably purified from cell culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-SEPHAROSE hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography. FIG. 9 is a series of graphs showing the Mab 6H3 reactive epitope present on activated, but not normal, leukocytes from two different donors. The clear areas represent staining profiles with irrelevant IgG1 control antibodies and the shaded areas represent staining profiles with anti-PrP Mabs.

II. Diagnostic Uses for Monoclonal Antibodies to PrP Diseases

For diagnostic applications, the antibodies of the invention typically are labeled with a detectable moiety. The detectable moiety can be any one which is capable of producing, either directly or indirectly, a detectable signal. By way of illustration, the detectable moiety may be a radioisotope, such as technicium, 3 H, 125 I, 35 S, 32 P, or 14 C; a fluorescent or chemiluminescent compound, such as rhodamine, fluorescein isothiocyanate, or luciferin; or an enzyme, such as alkaline phosphatase, betagalactosidase or horseradish peroxidase. Any method known in the art for separately conjugating the antibody to the detectable moiety may be employed (Pain, et al., J. Immunol. Meth. 40:219, 1981).

In addition to mammalian tissue, the use of the monoclonal antibodies described herein can be extended to the screening of mammalian biological fluids for the presence of PrP. In vitro immunoserological evaluation of sera withdrawn from mammalian subjects thereby permits non-invasive diagnosis of prion diseases. For example, mammalian fluids, such as pleural fluids or the lymph, can be taken from a subject and assayed for PrP, either as released antigen or membrane bound on cells in the sample fluid, using the monoclonal antibodies in any known assay method, such as radioimmunoassays, competitive binding assays, sandwich assays, and immunoprecipitation assays.

Radioimmunassays use radioactivity in the measurement of antibody-PrP reaction. In such a method, the antibody is radio labeled and complexed with unlabeled PrP. The radiolabeled complex is then separated from unbound material by precipitation followed by centrifugation. Once the radiolabled antibody-PrP complex is separated from the unbound material, quantitation of the complex is measured by either the radiation directly or the effect that the radiolabel has on a fluorescent molecule, such as dephenyloxazole (DPO). The latter approach requires less radioactivity and is more sensitive. This approach, termed scintillation, measures the fluorescent transmission of a dye solution that has been excited by a radiolabel, such as 3 H or 32 P. The extent of binding is determined by measuring the intensity of the fluorescence released from the fluorescent particles. This method, termed scintillation proximity assay (SPA), has the advantage of being able to measure antibody-PrP receptor binding in situ without the need for washing off unbound radioactive antibody from the particles.

Competitive binding assays rely on the ability of a labeled standard (which may be PrP or an immunologically reactive portion thereof) to compete with the test sample analyte for binding with a limited amount of antibody. The amount of test sample is inversely proportional to the amount of standard that becomes bound to the monoclonal antibodies. To facilitate determining the amount of standard that becomes bound, the monoclonal antibodies generally are insolubilized before or after the competition, so that the standard and analyte that are bound to the antibodies may conveniently separate from the standard and analyte which remain unbound.

Sandwich assays involve the use of two antibodies, each capable of binding to a different immunogenic portion, or epitope, of the protein to be detected. In a sandwich assay, the test sample analyte is bound by a first antibody which is immobilized on a solid support, and thereafter a second antibody binds to the analyte, thus forming an insoluble three part complex (David & Greene, U.S. Pat. No. 4,376,110). The second antibody may itself by labeled with a detectable moiety (direct sandwich assays) or may be measured using an anti-immunoglobulin antibody that is labeled with a detectable moiety (indirect sandwich assay). For example, one type of sandwich assay is an ELISA assay, in which case the detectable moiety is an enzyme.

Typically, sandwich assays include "forward" assays in which the antibody bound to the solid phase is first contacted with the sample being tested to extract PrP from the sample by formation of a binary solid phase antibody-PrP complex. After a suitable incubation period, the solid support is washed to remove the residue of the fluid sample, including unreacted PrP, if any, and then contacted with the solution containing an unknown quantity of labelled antibody (which functions as a "reporter molecule"). After a second incubation period to permit the labelled antibody to complex with the PrP bound to the solid support through the unlabeled antibody, the solid support is washed a second time to remove the unreacted labelled antibody. This type of forward sandwich assay may be a simple "yes/no" assay to determine whether PrP or PrPres is present. Additionally, this assay may be made quantitative for comparing the signals generated by binding of PrPc and PrPres.

Other types of sandwich assays, which may also be useful with PrP, are the so-called "simultaneous" and "reverse" assays. A simultaneous assay involves a single incubation step wherein the labelled and unlabelled antibodies are both added to the sample being tested at the same time. After the incubation is completed, the solid support is washed to remove the residue of fluid sample and uncomplexed labelled antibody. The presence of labelled antibody associated with the solid support is then determined as it would be in a conventional "forward" sandwich assay.

In the "reverse" assay, stepwise addition first of a solution of labelled antibody to the fluid sample, followed by the addition of unlabelled antibody bound to a solid support after a suitable incubation period, is utilized. After a second incubation, the solid phase is washed in conventional fashion to free it of the residue of the sample being tested and the solution of unreacted labelled antibody. The determination of labelled antibody associated with a solid support is then determined as in the "simultaneous" and "forward" assays.

Other Uses for Monoclonal Antibodies to PrP Diseases

In addition to their diagnostic utility, the monoclonal antibodies of the present invention are useful for monitoring the progression of prion disease in a subject by examining the levels of PrP or PrPres in tissues on cells or serum samples over time. Changes in the levels of PrP or PrPres over time may indicate further progression of the prion disease in the subject.

The monoclonal antibodies of the present invention are potentially useful for targeting PrP or PrPres producing cells in-vivo. They can therefore be used in mammals for monitoring PrP conversion (i.e. from PrPc to PrPres). For this application, it is preferable to use either affinity purified or humanized monoclonal antibodies. The purified monoclonal antibodies can be labeled with radioactive compounds, such as radioactive iodine, and administered to a patient intravenously. After localization of the antibodies at the site of PrP accumulation, they can be detected by emission tomographical and radionuclear scanning techniques.

Moreover, the monoclonal antibodies of this invention may be utilized in purified or humanized form for therapeutic applications. For therapeutic applications, the purified or humanized monoclonal antibodies are administered to a mammal in a pharmaceutically acceptable dosage form. The monoclonal antibodies are administered intravenously as one infusion or by continuous infusion over a period of time. The monoclonal antibodies may be administered by intra-articular, oral, intramuscular, topical, inhalation, subcutaneous, intrathecal, or intrasynovial routes. In addition to the monoclonal antibodies, the dosage forms possess pharmaceutically acceptable carriers that are nontoxic and nontherapeutic, including: serum proteins, buffers, ion exchangers, water, salts, electrolytes, sodium chloride, and the like.

The appropriate dosage of monoclonal antibodies will depend on the species of mammal, severity and course of disease, response to the monoclonal antibodies, and discretion of the physician. As stated above, the monoclonal antibodies may be administered to the subject at one time or over a series of treatments.

Claim 1 of 4 Claims

Having thus described the preferred embodiments, the invention is now claimed to be:

1. A method for detecting the amount of mammalian prion protein in a sample comprising the steps of:

a) preparing a monoclonal antibody 2F8 obtained from hybridoma cell line ATCC PTA-4065 or an epitope binding fragment thereof, which is immunoreactive with prion protein;

b) bringing said monoclonal antibody or fragment thereto into contact with the sample; and

c) measuring the amount of antigen-antibody complex formed in the sample, wherein the amount of complex is directly proportional to the amount of prion protein present in the sample.
 


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