WO2005021711A2 - Approche destinee a obtenir des anticorps monoclonaux pour des proteines de surface - Google Patents

Approche destinee a obtenir des anticorps monoclonaux pour des proteines de surface Download PDF

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WO2005021711A2
WO2005021711A2 PCT/US2004/019217 US2004019217W WO2005021711A2 WO 2005021711 A2 WO2005021711 A2 WO 2005021711A2 US 2004019217 W US2004019217 W US 2004019217W WO 2005021711 A2 WO2005021711 A2 WO 2005021711A2
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prp
monoclonal antibodies
cells
antibodies
antibody
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PCT/US2004/019217
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WO2005021711A3 (fr
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Richard Rubenstein
Richard Kascsak
Jae-Il Kim
Solomon Kurizon
Robert Petersen
Man-Sun Sy
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Research Foundation For Mental Hygiene, Inc.
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Publication of WO2005021711A3 publication Critical patent/WO2005021711A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2872—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against prion molecules, e.g. CD230

Definitions

  • the present invention relates to methods for the production of monoclonal antibodies having specificity for cell surface macromolecules which are relatively conserved between species.
  • the invention relates to the production of monoclonal antibodies that bind to prion proteins (PrP).
  • PrP prion proteins
  • TSEs Transmissible spongiform encephalopathies
  • CJD Creutzfeldt-Jakob disease
  • BSE bovine spongiform encephalopathy
  • PrPc normal prion protein
  • PrPsc infectious, pathogenic form
  • the formation of PrPsc involves a conformational change in PrP c from an ⁇ -helix to a ⁇ -pleated sheet dominated structure (Pan et al., 1993).
  • PrPsc is associated with all prion diseases, and, as a result, is now recognized as a universal diagnostic marker for these neurodegenerative disorders.
  • MAbs reactive to particularly mouse PrP when attempting fusions using lymphocytes from immunized PrPo/o mice and commercially available myeloma cell lines (Rubenstein et al., 1999; Williamson et al., 1996). Williamson et al. (1996) detected serum IgG titers against mouse, hamster, human PrPs after immunization of PrPo/o mice with PrPsc from mice or hamster with scrapie to produce anti-PrP MAbs. However, they could not establish stable hybridomas cell lines secreting those Abs (Williamson et al., 1996).
  • Hybridomas and myeloma have fairly simple and defined growth requirements. However, the establishment of hybridomas which are grown at low densities, such as after fusions and during single-cell cloning, may be particularly vulnerable and/or sensitive to external factors (Harlow & David, 1998). The effect of Ab binding to PrPc under these conditions can be significant if it blocks signal transduction pathways (Li et al., 2001) which might be critical for cell activation and/or proliferation.
  • PrPc is a sialoglycoprotein and is attached to the cell membrane of mammalian cells by a C-terminal glycosylphosphatidylinositol (GPI) anchor.
  • GPI glycosylphosphatidylinositol
  • the present invention provides a novel method for producing monoclonal antibodies against a variety of mammalian antigens, and in particular, cell surface antigens that are relatively conserved between species.
  • Currently used hybridoma technologies present significant limitations for the production of monoclonal antibodies to certain proteins or macromolecules that have conserved epitopes and that are not recognized as “foreign” or “non-self, making them less immunogenic in closely related species.
  • the present invention overcomes the "self -"non-self limitation of hybridoma technology by utilizing myeloma cell lines which have significantly reduced or are completely devoid of expression of the protein of interest together with a genetically engineered mouse which also lacks expression of the protein of interest.
  • the invention relates to a method for producing monoclonal antibodies to PrP.
  • the method comprises the steps of administering a PrP of interest to a genetically engineered mouse which no longer expresses the PrP, isolating from the mouse the cells which produce antibodies to the PrP, fusing the isolated cells with myeloma cells which have significantly reduced expression, or completely lack expression, of the PrP, to produce a hybridoma which can secrete a monoclonal antibody specific for the PrP, culturing the resultant hybridoma in a medium or an animal, and isolating the monclonal antibody from the medium or animal.
  • the present invention also relates to myeloma cell lines which have significantly reduced levels of, or are devoid of, PrP c and methods for producing them.
  • myeloma cell lines are those deposited at the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, VA., 20110-2209, on [To be inserted], and designated [To be inserted], ATCC Designation No.[To be inserted].
  • ATCC American Type Culture Collection
  • the present invention relates to new monoclonal antibodies, or fragments thereof, that specifically react with PrP, particularly, the monoclonal antibodies produced by the hybridoma cell lines prepared according to the present invention.
  • Examples of such monoclonal antibodies include, but are not limited to, those produced by the hybridoma cell lines deposited with the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, VA., 20110-2209, on [To be inserted] and having ATCC Accession Nos. [To be inserted] (MoAb [To be inserted], respectively), and to binding fragments of monoclonal antibodies that specifically react with PrP as described herein, particularly, the monoclonal antibodies produced by the hybridoma cell lines hybridoma cell lines deposited with the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, VA., 20110-2209, on [To be inserted] and having ATCC Accession Nos. [To be inserted] (MoAb [To be inserted], respectively).
  • Such monoclonal antibodies, or antibody fragments may be human, or they may be derived from other mammalian species, such as rodent, hybrids thereof, chimaeric antibodies, and the like. Binding fragments of the monoclonal antibodies of the present invention include, but are not limited to, F(ab') 2 , Fab', Fv, Fd', or Fd fragments.
  • the present invention provides hybridoma cell lines, produced by the methods described herein, which produce monoclonal antibodies that specifically bind to PrP.
  • clonally derived hybridoma cell lines include the subcloned monoclonal antibody-producing hybridoma cell lines designated 01-16/6B10 and 02-11/1 F11.
  • monoclonal antibodies that recognize different conformational epitopes and/or glycosylated forms of PrP from one or more species of mammals such as 01-16/6B10 and 02-11/1 F11 , as described herein.
  • Another aspect of the present invention is to provide hybridoma cell lines deposited with the American Type Culture Collection (ATCC) on [To be inserted] and designated [To be inserted].
  • ATCC American Type Culture Collection
  • Yet another aspect of the present invention is to provide methods of using the monoclonal antibodies, or fragments thereof, of the invention for the detection, localization, or isolation of PrP and diagnosis of PrP-related diseases in mammals.
  • the monoclonal antibodies, and/or fragments thereof, of the present invention can be utilized in a broad number of assays known to those skilled in the art, including but not limited to, immunohistochemical assays, Western Blot, dot blot, immunoprecipitation, sandwich and competition immunoassays and/or immunoaffinity chromatography.
  • Yet another aspect of the present invention is to provide methods of treating PrP-related diseases in a mammals using the monoclonal antibodies described herein, by administering one or more of the monoclonal antibodies, for example, either alone, or together with others of the antibodies in a cocktail, or one or more binding fragments thereof, or administering a mixture of intact antibody(ies) and binding fragment(s) thereof, in an amount sufficient to achieve a therapeutic effect.
  • the antibodies can be administered prophylactically or may be administered to a subject already infected by the disease.
  • the appropriate dosage of monoclonal antibodies will vary depending on such factors as the species of mammal and severity of the disease.
  • a detectable label such as a fluorophore, a chromophore, a radionuclide, or any other agent, for use in diagnostic, therapeutic, imaging, and screening compounds, for example.
  • the present invention is directed to a method for localizing PrP in a subject by administering one or more of the monoclonal antibodies described herein, or binding fragments thereof, to bind prion proteins within the subject and determining the location of the one or more monoclonal antibodies within the subject.
  • the monoclonal antibodies, or binding fragments thereof are preferably labeled with a detectable and physiologically acceptable label, such as a radionuclide.
  • the stage of the disease, and/or effectiveness of treatment can be monitored by determining the levels or changes over time of the specifically recognized prion proteins in vivo.
  • compositions comprising one or more of the monoclonal antibodies, or binding fragments, and an acceptable carrier, diluent, or excipient.
  • kits for detecting PrP comprising one or more monoclonal antibody of the invention, or binding fragments thereof, for the diagnosis and/or treatment of prion diseases.
  • Figure 1 depicts PCR analysis of PrP gene in the two myeloma cell lines. PrP gene was PCR-amplified to examine its integrity. M: DNA size marker; Lane 2: SP2/0; Lane 3, 4: normal CD-1 mice brain (positive control); Lane 5: no template (negative control).
  • Figure 2 depicts RT-PCR analysis of expression levels of PrP gene in the two myeloma cell lines.
  • Fig. 2A 1.5% agarose gel electrophoresis of RT- PCR products of PrP gene.
  • RT-PCR products were standardized by analysis of constitutive expression of ⁇ -actin gene.
  • Fig. 2B Densitometric analysis of RT-PCR products. The mean density for P3 is shown as 100%. Each value is the mean ⁇ SD. *p ⁇ 0.01 compared to P3 cells.
  • Figure 3 depicts Western Blot analysis of expression levels of PrP c in the myeloma cell lines using Mab, 7A12.
  • Lane 1 Brain homogenate from a normal CD-1 mouse
  • Lane 2 P3 cell lysate
  • Lane 3 SP2/) cell lysate
  • Lane 4 SP2/0)-ATCC cell lysate.
  • the present invention is based upon the discovery that mouse myeloma cell lines that express significantly reduced amounts of cell surface PrP can be successfully utilized as a fusion partner with lymphocytes from PrP 00 immunized knockout mice.
  • the present invention further relates to myeloma cell lines which have no detectable cell surface PrP expression. These cell lines are preferred for the generation of stable hybridoma cell lines.
  • the combination of a PrP knockout mouse and a PrP-non-expressing fusion partner for hybridoma production opens all regions and conformation of the molecule to antibody production, whether or not they are highly conserved and regardless of the species of origin. This approach leads to many new and useful monoclonal antibodies of varying specificities.
  • the principle of utilizing a myeloma cell line devoid of a cell surface protein of interest together with a mouse which lacks expression of the same protein should be applicable to any conserved protein of interest.
  • the monoclonal antibodies of the present invention are prepared by administering PrP of interest to a genetically engineered mouse that no longer expresses the PrP, isolating from the mouse the spleen cells which produce antibodies to the PrP, fusing the isolated spleen cells with myeloma cells that have significantly reduced expression, or are completely devoid of, the PrP of interest, to produce a hybridoma which can secrete a monoclonal antibody specific for the prion protein, culturing the resultant hybridoma in a medium or an animal, and isolating the monclonal antibody from the medium or animal.
  • PrPs for use in immunization protocols are described in, for example, U.S. Patent Nos. 5,846,533; 6,261,790 B1; and 6,528,269 B1 , which are herein incorporated by reference in their entirety.
  • sufficient quantities of prion proteins may be prepared by recombinant techniques well known in the art.
  • large quantities of PrP or PrP peptide fragments may be generated by introducing the DNA sequence of a PrP of interest into an appropriate expression vector for the production of large quantities of recombinant prion protein in an organism such as E. coli.
  • peptides can be synthesized from commercial sources.
  • full-length PrP So can be purified directly from the infected brains of animals or humans who died from a prion disease.
  • PrP knock-out mice may be immunized by subcutaneous and intramuscular injections. PrP knock-out mice have both alleles of the PrP gene ablated (PrP 0/0 ) (B ⁇ eler, et al., 1992). Aside from subtle changes, the PrP-deficient mice (or null mice) are indistinguishable from normal mice in their development and behavior. However, these mice do not support the replication of the infectious agent responsible for prion disease and therefore do not succumb to clinical disease. The use of PrP knock-out mice for production of monoclonal antibodies is described in U.S. Patent No. 6,528,269 B1. After allowing sufficient time to generate antibodies, the immunized PrP 00 mice are sacrificed, and antibody-producing cells are isolated and fused with a myeloma cells that have significantly reduced expression of PrP or are completely devoid of PrP.
  • the present invention circumvents the problems of tolerance and more efficiently generates panels of monoclonal antibodies capable of recognizing diverse epitopes on PrP due to the discovery that myeloma cells lines lacking significant cell surface expression of the protein of interest form stable hybridoma cell lines producing such antibodies.
  • the myeloma cell lines of the invention may be prepared as described in Example 1.
  • these myeloma cells were prepared by performing limiting dilution studies such that only one cell was present in each well of a 96-well cell culture plate. Each well from ten of these plates were analyzed by confocal microscopy using an antibody which reacts with PrP c , a cell- surface glycoprotein.
  • the cells of interest were those which showed minimal PrP c immunostaining. Those selected cells were allowed to continue growing and then tested for PrP c expression by western blotting. The levels of PrP c expression in these myeloma cells was approximately 17% of the levels found in P3 cells. The hybridoma cells generated using this myeloma cell line were not adversely affected as a result of antibody secretion. Alternatively, another method of generating a myeloma cell line expressing minimal levels or no PrP c is by disrupting the PrP gene (or other gene of interest) using the methodology of homologous recombination.
  • Non-limiting examples of myeloma cell lines which may be used for the production of the hybridomas described herein include SP2/0- PrP " deposited with the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, VA., 20110-2209, on [To be inserted] and having ATCC Accession Nos. [To be inserted], respectively).
  • Hybridomas are plated in multiple wells with an appropriate selective media, such as HAT medium.
  • Selective media allow for the detection of antibody producing hybridomas over non-antibody producing hybrids, and only those cells which have successfully fused will grow in the selective medium.
  • the antibodies may be purified using standard techniques in the art, for example, the monoclonal antibodies may be isolated and/or purified over protein A and/or protein G affinity columns. The use of either protein A or protein G is dependent on the particular isotype of the monoclonal antibody produced.
  • the present invention also provides the discovery of new monoclonal antibodies that specifically bind to PrP.
  • the methods described herein can be used to generate a large panel of novel and diverse monoclonal antibodies which are capable of cross-reacting with PrP from several species, or alternatively, will react with epitopes that are species-restricted.
  • the monoclonal antibodies produced according to the invention may cross-react with human PrP, as well as those PrP from hamster, mouse, cow, sheep and other species including, but not limited to, cat, elk, mule deer, and white-tailed deer
  • the monoclonal antibodies may react with specific conformations, fragments, or glycosylated forms (glycoisoforms) of PrP.
  • the conversion from non-infectious to infectious PrP is believed to involve conformational changes in PrP, which may in turn affect post-translational processing of the PrP molecule. Therefore, a large collection of monoclonal antibodies is essential for the diagnosis and study of prion diseases in order to develop better treatments.
  • Another embodiment of the present invention relates to monoclonal antibodies, and binding fragments or portions thereof, which recognize different glycoisoforms of PrP.
  • the present invention encompasses the deposited monoclonal antibodies, as well as antibodies, preferably monoclonal antibodies, and their binding fragments, having specificity for the above-described PrP antigens.
  • Nonlimiting examples of antibody fragments or antigen bindable fragments that bind to epitopes on the PrP antigens include the following: Fab fragments, F(ab) 2 fragments, Fab' fragments, fragments produced by F(ab) expression libraries, F(ab') 2 fragments, Fd fragments, Fd' fragments and Fv fragments.
  • the antibodies may be human, or from animals other than humans, preferably mammals, such as rat, mouse, guinea pig, rabbit, goat, cow, sheep, and pig. Preferred are mouse monoclonal antibodies and antigen-binding fragments or portions thereof. In addition, chimaeric antibodies and hybrid antibodies are embraced by the present invention.
  • the monoclonal antibodies and binding fragments thereof may be characterized as those which are 1) produced from the hybridoma cell lines deposited at the American Type Culture Collection, 10801 University Boulevard, Manassas, VA, 20110-2209 under ATCC Accession Nos. [To be inserted]; 2) antibodies that are capable of binding to the same antigenic determinant as does the monoclonal antibody produced by the hybridoma cell lines deposited at the American Type Culture Collection under ATCC Accession Nos. [To be inserted]; 3) binding fragments of the monoclonal antibodies produced by the hybridoma cell lines deposited at the American Type Culture Collection under ATCC Accession Nos.
  • the present invention further provides hybridoma cell lines produced according to the methods described herein that produce monoclonal antibodies that specifically bind to PrP antigens. Selection of hybridoma cell lines is performed by culturing the cells by single clone dilution in microtiter plates, followed by testing of the individual clone supernatants (after about two to three weeks) for the desired reactivity. Radioimunoassays, enzyme immunoassays, cytotoxicity assays, plaque assays, dot immunobinding assays, and the like, may be used.
  • Non-limiting examples of such hybridoma cell lines are those deposited with the American Type Culture Collection (ATCC) on [To be inserted] and designated [To be inserted] The hybridoma cell lines are maintained using standard techniques well known in the art.
  • ATCC American Type Culture Collection
  • Yet another embodiment of the present invention is to provide methods of using the monoclonal antibodies of the invention, or fragments thereof, in immunoassays for the detection, localization, or isolation of prion proteins and/or diagnosis of prion diseases in mammals.
  • the monoclonal antibodies, and/or fragments thereof, of the present invention can be utilized in a broad number of assays known to those skilled in the art, including but not limited to, immunohistochemical assays, Western Blot, dot blot, immunoprecipitation, sandwich and competition immunoassays and/or immunoaffinity chromatography, ELISA, immuno-PCR, immuno-RCA (rolling circle amplification).
  • the monoclonal antibodies, or binding fragments thereof, according to the present invention may be used to quantitatively or qualitatively detect the presence of PrP-specific antigens as described in mammals. This can be achieved, for example, by immunofluorescence techniques employing a fluorescently labeled antibody, coupled with light microscopic or fluorometric detection.
  • the antibodies, or binding fragments thereof, according to the present invention may additionally be employed histologically, as in immunofluorescence, immunoelectron microscopy, or non-immuno assays, for the in situ detection of PrP-specific antigen, such as for use in monitoring, diagnosing, or detection assays.
  • In situ detection may be accomplished by removing a histological specimen from a patient, and applying thereto a labeled antibody, or antibodies, or fragments thereof according to this invention.
  • the antibody, or antigen-binding fragment thereof is preferably applied by overlaying the labeled antibody or fragment onto the biological sample.
  • Immunoassay and non-immuno assays for PrP antigen, or conserved variants, or peptide fragments thereof typically comprise incubating a sample, such as a biological fluid, tissue extract, freshly harvested cells, or lysates of cells that have been incubated in cell culture, in the presence of a detectably- labeled antibody that recognizes the PrP antigen, conserved variants, or peptide fragments thereof, such as the PrP-specific monoclonal antibodies, or binding fragments thereof, of the present invention. Thereafter, the bound antibody, or binding fragment thereof, is detected by a number of techniques well known in the art.
  • the biological sample may be brought into contact with, and immobilized onto, a solid phase support or carrier, such as nitrocellulose, or other solid support or matrix, which is capable of immobilizing cells, cell particles, membranes, or soluble proteins.
  • a solid phase support or carrier such as nitrocellulose, or other solid support or matrix, which is capable of immobilizing cells, cell particles, membranes, or soluble proteins.
  • the support may then be washed with suitable buffers, followed by treatment with the detectably-labeled anti-PrP antibody.
  • the solid phase support may then be washed with buffer a second time to remove unbound antibody.
  • the amount of bound label on the solid support may then be detected by conventional means.
  • compositions are provided comprising the monoclonal antibodies, or binding fragments thereof, bound to a solid phase support, such as described herein.
  • solid phase support or carrier or matrix any support capable of binding an antigen or an antibody.
  • supports or carriers include glass, plastic, nylon wool, polystyrene, polyethylene, polypropylene, dextran, nylon, amylases, films, resins, natural and modified celluloses, polyacrylamides, agarose, alumina gels, gabbros, and magnetite.
  • the nature of the carrier can be either soluble to some extent, or insoluble for the purposes of the present invention.
  • the support material may have virtually any possible structural configuration as long as the coupled molecule is capable of binding to an antigen or antibody.
  • the support configuration may be spherical, as in a bead, cylindrical, as in the inside surface of a test tube, or the external surface of a rod.
  • the surface may be flat, such as a sheet, film, test strip, stick, and the like.
  • the solid support is preferably inert to the reaction conditions for binding and may have reactive groups, or activated groups, in order to attach the monoclonal antibody, a binding fragment, or the binding partner of the antibody.
  • the solid phase support may also be useful as a chromatographic support, such as the carbohydrate polymers Sepharose®, Sephadex®, or agarose. Indeed, a large number of such supports for binding antibody or antigen are commercially available and known to those having skill in the art.
  • the binding activity for a given anti-PrP antibody may be determined by well-known methods. Those skilled in the art will be able to determine operative and optimal assay conditions for each determination by employing routine experimentation.
  • the antibodies can be detectably labeled is by linking the antibody to an enzyme, e.g., for use in an enzyme immunoassay (EIA), (A. Voller et al., 1978, “The Enzyme Linked Immunosorbent Assay (ELISA)", Diagnostic Horizons, 2:1-7; , Microbiological Associates Quarterly Publication, Walkersville, MD; A. Voller et al., 1978, J. Clin. Pathol., 31:507-520; J.E. Butler et al., 1981, Meths.
  • EIA enzyme immunoassay
  • ELISA Enzyme Linked Immunosorbent Assay
  • EnzymoL 73:482-523; Enzyme Immunoassay, 1980, (Ed.) E. Maggio, CRC Press, Boca Raton, FL; Enzyme Immunoassay, 1981, (Eds.) E. Ishikawa et al., Kgaku Shoin, Tokyo, Japan).
  • the enzyme that is bound to the antibody reacts with an appropriate substrate, preferably a chromogenic substrate, so as to produce a chemical moiety which can be detected, for example, by spectrophotometric, fluorometric, or by visual detection means.
  • Nonlimiting examples of enzymes which can be used to detectably label the antibodies include malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase.
  • the detection can be accomplished by calorimetric methods, which employ a chromogenic substrate for the enzyme, or by visual comparison of the extent of enzymatic reaction of a substrate compared with similarly prepared standards or controls.
  • a radioimmunoassay can be used to detect PrP-specific antigens (e.g., B. Weintraub, "Principles of Radioimmunoassays", Seventh Training Course on Radioligand Techniques, The Endocrine Society, March, 1986).
  • the radioactive isotope label can be detected by using a gamma counter or a scintillation counter or by radiography.
  • the antibodies, or their antigen-binding fragments can also be labeled using a fluorescent compound.
  • a fluorescent compound When the fluorescently labeled antibody is exposed to light of the proper wavelength, its presence can then be detected due to fluorescence.
  • fluorescent labeling compounds are, without limitation, fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.
  • Detectably labeled fluorescence-emitting metals such as i52 Eu, or others of the lanthanide series, can be used to label the antibodies, or their binding fragments, for subsequent detection.
  • the metals can be coupled to the antibodies via such metal chelating groups as diethylenetriaminepentacetic acid (DTPA), or ethylenediaminetetraacetic acid (EDTA).
  • DTPA diethylenetriaminepentacetic acid
  • EDTA ethylenediaminetetraacetic acid
  • the antibodies can also be detectably labeled by coupling them to a chemiluminescent compound.
  • the presence of the chemiluminescent-tagged antibody is then determined by detecting the presence of luminescence that develops during the course of a chemical reaction.
  • chemiluminescent labeling compounds include, without limitation, luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.
  • a bioluminescent compound may be used to label the antibodies of the present invention.
  • Bioluminescence is a type of chemiluminescence found in biological systems in which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting the presence of luminescence.
  • Useful bioluminescent labeling compounds include luciferin, luciferase and aequorin.
  • Another embodiment of the present invention provides diagnostics, diagnostic methods and imaging methods for PrP-related diseases using the monoclonal antibodies and binding fragments thereof as described by the present invention.
  • the present invention is directed to a method for localizing prion proteins in a subject by administering one or more of the monoclonal antibodies described herein, or binding fragments thereof, to bind prion proteins within the subject and determining the location of the one or more monoclonal antibodies within the subject.
  • the monoclonal antibodies, or binding fragments thereof are preferably labeled with a detectable and physiologically acceptable label, such as a radionuclide.
  • the stage of the disease, and/or effectiveness of treatment can be monitored by determining the levels or changes over time of the specifically recognized prion proteins in vivo.
  • a diagnostic method comprises administering, introducing, or infusing the monoclonal antibodies or their binding fragments as described herein, with or without conjugation to a detectable moiety, such as a radioisotope.
  • a detectable moiety such as a radioisotope.
  • the antibody or binding fragment binds to PrP proteins, after which the location of the bound antibodies or fragments is detected.
  • imaging instrumentation may be used to identify the location of the PrP within the body.
  • a second detectable reagent may be administered, which locates the bound antibodies or fragments so that they can be suitable detected.
  • Yet another aspect of the present invention is to provide methods of treatment of PrP-related disease in a mammal using the monoclonal antibodies described herein, by administering one or more of the monoclonal antibodies, for example, either alone, or together with others of the antibodies in a cocktail, or one or more binding fragments thereof, or administering a mixture of intact antibody(ies) and binding fragment(s) thereof, in an amount sufficient to achieve a therapeutic effect.
  • the antibodies can be administered prophylactically or may be administered to a subject already infected by the disease.
  • the appropriate dosage of monoclonal antibodies will vary depending on such factors as the species of mammal and severity of the disease.
  • PrP monoclonal antibodies of the present invention it is possible to design therapies combining all of the characteristics described herein.
  • a therapeutic agent or agents
  • liposomes or other drug delivery systems with the monoclonal antibodies of the present invention to specifically deliver the antibodies to the area having PrP-specific antigens.
  • These systems can be produced such that they contain, in addition to monoclonal antibody, therapeutic agents, such as those described above, which would then be released at the treatment site (e.g., Wolff et al., 1984, Biochem. et Biophys. Ada, 802:259).
  • the use of a delivery system may also facilitate targeting of the monoclonal antibodies to and across the blood brain barrier. Examples of the various types of drug delivery systems are provided in more detail below.
  • the dosage ranges for the administration of the monoclonal antibodies of the invention are those large enough to produce the desired effect in which the symptoms of the PrP-related disease are ameliorated.
  • the dosage should not be so large as to cause adverse side effects, such as unwanted cross- reactions, anaphylactic reactions, and the like.
  • the dosage will vary with the age, condition, sex and extent of disease of the patient and can be determined by one of skill in the art.
  • the dosage can be adjusted by the individual physician in the event of any complication. Dosage can vary from about 0.1 mg/kg to about 2000 mg/kg, preferably about 0.1 mg/kg to about 500 mg/kg, in one or more dose administrations daily, for one or several days.
  • the monoclonal antibodies of the present invention when administered conjugated with therapeutic agents, lower dosages, comparable to those used for in vivo immunodiagnostic imaging, can be used.
  • the monoclonal antibodies of the invention can. be administered parenterally by injection or by gradual perfusion over time.
  • the monoclonal antibodies of the invention can be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity, or transdermally, alone or in combination with other agents.
  • a cocktail of different monoclonal antibodies such as a mixture of the specific monoclonal antibodies described herein, or their binding fragments, may be administered, if necessary or desired, to alleviate PrP-related disease.
  • a mixture of monoclonal antibodies, or binding fragments thereof, in a cocktail to target several PrP antigens, or different epitopes, in the mammal is an advantageous approach (See, for example, US 6,261,790).
  • the present invention assists in the diagnosis of PrP-related diseases by the identification and measurement of PrP in body fluids, such as blood, serum, plasma, sputum and the like.
  • body fluids such as blood, serum, plasma, sputum and the like.
  • the present invention provides a needed means of early diagnosis, thereby affording the opportunity for early treatment. Early detection is especially important for those PrP diseases that are difficult to diagnose in their early stages.
  • the present invention provides methods for diagnosing the presence of PrP by assaying for the appearance of PrPSc, the abnormal isoform of PrP in cells, tissues or body fluids compared with the isoform in cells, tissues, or body fluids, preferably of the same type, from normal controls.
  • the presence of PrPSc in the subject versus the normal control is associated with the presence of a PrP-related disease.
  • a positive result indicating that the subject being tested has a PrP-related disease is one in which the PrPSc antigen in or on cells, tissues or body fluid is present.
  • Normal controls those selected from any species against which the infected tissue is being compared.
  • Another embodiment of the present invention relates to pharmaceutical compositions comprising one or more monoclonal antibodies, or binding fragments thereof, according to the invention, together with a physiologically- and/or pharmaceutically-acceptable carrier, excipient, or diluent.
  • the antibodies, or binding fragments specifically recognize a PrP-epitope on one or more PrP antigens and bind to the antigens.
  • the PrP-specific antigens are further characterized as described herein and above.
  • the present invention is directed to pharmaceutical compositions comprising a monoclonal antibody, or binding fragment thereof, including the monoclonal antibodies produced from the hybridoma cell lines deposited at the American Type Culture Collection having ATCC Accession Nos. [To be inserted]; antibodies that are capable of binding to the same antigenic determinant as do the monoclonal antibodies produced by the hybridoma cell lines deposited at the American Type Culture Collection and having ATCC Accession Nos. [To be inserted]; ; binding fragments of the hybridoma cell lines deposited at the American Type Culture Collection and having ATCC Accession Nos.
  • Antibody fragments include but are not limited to F(ab') 2 fragments, F(ab) fragments, fragments produced by an F(ab) expression library, Fv fragments, Fd' fragments, or Fd fragments.
  • the antibodies or binding fragments thereof are delivered parenterally, such as by intravenous, subcutaneous, or intraperitoneal administration, e.g., injection.
  • Suitable buffers, carriers, and other components known to the art can be used in formulating a composition comprising the antibody or fragments for suitable shelf-life and compatibility with administration.
  • These substances may include ancillary agents such as buffering agents and protein stabilizing agents (e.g., polysaccharides).
  • therapeutic formulations of the antibodies, or binding fragments thereof are prepared for storage by mixing the antibodies or their binding fragments, having the desired degree of purity, with optional physiologically acceptable carriers, excipients, or stabilizers (Remington's Pharmaceutical Sciences, 17th edition, (Ed.) A. Osol, Mack Publishing Company, Easton, PA., 1985), in the form of lyophilized cake or aqueous solutions.
  • Acceptable carriers, excipients or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as Tween, Pluronics or polyethylene glycol (PEG).
  • buffers such as phosphate, citrate, and other organic acids
  • antioxidants including ascorbic acid
  • the antibodies, or binding fragments thereof also may be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (for example, hydroxymethylcellulose or gelatin-microcapsules and poly-fmethylmethacylate] microcapsules, respectively), in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules), or in macroemulsions.
  • coacervation techniques or by interfacial polymerization for example, hydroxymethylcellulose or gelatin-microcapsules and poly-fmethylmethacylate] microcapsules, respectively
  • colloidal drug delivery systems for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules
  • macroemulsions for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules
  • Antibodies or their binding fragments to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes, prior to or following lyophilization and reconstitution. The antibodies, or binding fragments thereof, ordinarily will be stored in lyophilized form or in solution.
  • Therapeutic antibody compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.
  • a sterile access port for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.
  • the route of administration of the antibodies, or binding fragments thereof, in accordance with the present invention is in accord with known methods, e.g., injection or infusion by intravenous, intraperitoneal, intramuscular, intraarterial, subcutaneous, intralesional routes, by aerosol or intranasal routes, or by sustained release systems as noted below.
  • the antibodies, or binding fragments thereof are administered continuously by infusion or by bolus injection.
  • sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the protein, which matrices are in the form of shaped articles, e.g., films, or microcapsules.
  • sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) as described by Langer et al., 1981, J. Biomed. Mater. Res., 15:167-277 and Langer, 1982, Chem. Tech., 12:98-105), or poly(vinylalcohol)], polylactides (U.S. Patent No.
  • polymers such as ethylene-vinyl acetate and lactic acid- glycolic acid enable release of molecules for over 100 days
  • certain hydrogels release proteins for shorter time periods.
  • encapsulated antibodies When encapsulated antibodies remain in the body for a long time, they may denature or aggregate as a result of exposure to moisture at 37°C, resulting in a loss of biological activity and possible changes in effectiveness. Rational strategies can be devised for antibody stabilization depending on the mechanism involved. For example, if the aggregation mechanism is discovered to be intermolecular S-S bond formation through thio- disulfide interchange, stabilization may be achieved by modifying suifhydryl residues, lyophilizing from acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer matrix compositions.
  • monoclonal antibodies or binding fragments to PrP are provided labeled with a detectable moiety, such that they may be packaged and used, for example, in kits, to diagnose or identify cells having the aforementioned antigen.
  • kits preferably contain an instruction manual for use of the kit.
  • Non-limiting examples of such labels include fluorophores such as fluorescein isothiocyanate; chromophores, radionuclides, or enzymes.
  • labeled antibodies or binding fragments may be used for the histological localization of the PrP antigen, ELISA, cell sorting, as well as other immunological techniques for detecting or quantifying the antigen, and cells bearing the antigen, for example.
  • the myeloma cell line P3-X63-Ag8.653 (Kascsak ef al., 1987)
  • SP2/0-Ag14 (Zanusso et al., 1998) (designated SP2/0) was kindly provided by Dr. Man-Sun Sy (Institute of Pathology, Case Western Reserve University School of Medicine, Ohio, USA).
  • SP2/0-Ag14 cells were also obtained from American Type Culture Collection (ATCC). For this study, these cells are designated SP2/0-ATCC.
  • All three cell lines were cultured in RPMI 1640 media supplemented with 0.6% dextrose, 2 mM glutamine, 2.5 ug/ml Fungizone, 100 U/ml penicillin, 100 U/ml streptomycin, 10 mg/ml gentamycin, 1 mM sodium pyruvate, 0.2 U/ml insulin, 0.2% chick embryo extract, and 15% controlled process serum replacement-type 3 (CPSR-3) (Sigma). Cells were maintained in a humidified incubator at 37 o C with 5% Co2.
  • PrP gene analysis was carried out using genomic DNA. DNA was isolated from approximately 510 ⁇ cells from P3 and SP2/0 myeloma cell lines by DNeasy Tissue kit (Qiagen) as per manufacturer's instructions. Genomic DNA isolated from brain of normal CD-1 mice was used as a positive control. A 0.4 ug aliquot of each DNA sample was used for PCR with primers specific for mouse PrP: 5'-GTA-CCC-ATA-ATC-AGT- GGA-ACA-AGC-CCA-GC-3' and 5'-CCT-GGG-AAT-GAA-CAA-AGG-TTT-GCT- TTC-AAC-3'.
  • DNA amplification was carried out in Taq polymerase buffer containing 2 mM MgCi2 2 mM dNTPs, 25 pmol of each primer and 1 unit of Taq polymerase (Promega) in a final volume of 25 o l.
  • the PCR products were separated on 1.5% agarose gels, and visualized with ethidium-bromide. The expected size of the PCR product was 1043 bp.
  • RNA sequences of the entire PrP protein-coding region of P3 and SP2/0 cells were analyzed to confirm their integrity.
  • RNA 50 ng
  • TRI Reagent Molecular Research Center
  • oligo(dT) primers Promega
  • the entire PrP coding sequence was amplified from the first strand cDNA using ProofStart DNA polymerase (Qiagen) and the following primers: 5'- TTA-GGA-GAG-CCA-AGC-AGA-CT-3' and 5'-CAC-GAG-AAT-GCG-AAG-GAA- CA-3'.
  • This primer pair should amplify a 840 bp cDNA, which contains the entire coding region of PrP. Both strands of the PCR products were sequenced using CEQ 2000XL (Beckman Coulter) at the Molecular Core Facility of the NYS Institute for Basic Research.
  • the nucleotide sequences of the primers for ® -actin were 5'-GTG-GGC-CGC-TCT-AGG-CAC- CAA-3' and 5'-CTC-TTT-GAT-GTC-ACG-CAC-GAT-TTC-3'.
  • the RT-PCR products were separated on 1.5% agarose gels, visualized with ethidium-bromide, and quantitated by optical densitometry.
  • soluble and membrane-associated proteins were isolated from P3, SP2/0, SP2/0-ATCC myeloma cell lines (5 X 10 6 cells each) using a nondenaturing lysis buffer containing 1% (w/v) Triton X-100, 50 mM Tris-CI (pH 7.4), 300 mM NaCI, 5 mM EDTA, 0.02% (w/v) NaN3 and protease inhibitor cocktail (Bonifacino ef al., 1999).
  • Imrnunostaining of PrP was performed using a chemiluminescent-based detection scheme involving the anti-PrP MAb 7A12 (1:10000 dilution, kindly provided by Dr. Man-Sun Sy, Institute of Pathology, Case Western University, Cleveland, Ohio) (Li ef al., 2000; Wong ef al., 2001), biotinylated goat anti-mouse IgG and alkaline phosphatase-conjugated streptavidin for signal amplification and detection.
  • immunofluorescence studies with 7A12 (1 :100 dilution) were also performed on unfixed cells as described previously (Kascsak et al., 1997).
  • SP2/0 cells which do not express PrP c were carried out by limiting dilution. SP2/0 cells were plated in ten 96 well plates at a concentration of 1 cell per well. When the cells in each well reached approximately 75% confluency, half of the sample was used for both Western blot and immunofluorescence microscopy as described above.
  • PrP gene The integrity of PrP gene of P3 and SP2/0 myeioma cell lines were analyzed by PCR. A PCR product of 1043 bp was obtained from both myeloma cell lines (Fig. 1, lane 1 & 2). This is the same size obtained from the control sample (Fig. 1, lane 3 & 4) and predicted based on the mouse PrP gene sequence. The integrity of PrP gene was investigated further by DNA sequence analysis of the entire protein-coding sequence.
  • PrP c protein The expression levels of PrP c protein were investigated by Western blot analysis (Fig. 3). Brain homogenates from normal CD-1 mice were used as a positive control for PrP c analysis (Fig. 3, lane 1). While three known PrP c glycoforms of 33, 29, 27 kDa showed relatively intense immunostaining in the cell lysates from P3 cells, only faint immunostaining was observed in SP2/0 cells (Fig. 3, lane 2 & 3, respectively). Furthermore, intermediate immunostaining of the PrP c glycoforms could be observed in SP2/0-ATCC cells (Fig. 3, lane 4).
  • SP2/0-PrP - cells which showed a marked reduction of PrP c expression in this study, may be a useful tool for obtaining hybridomas secreting anti-PrP MAbs.
  • Antigen is prepared for injection by emulsification with CytRx ( G-5).
  • PrP- knockout mice are immunized every three weeks by subcutaneous and intramuscular injections for a total of nine weeks. For five days just prior to fusion, the selected previously immunized PrP-knockout mice are boosted each day with the respective antigen used for immunization suspended in PBS (0.2 ml per mouse) (without CytRx).
  • SP2/0-PrP myeloma cells are grown in the presence of 132 ⁇ M 8-
  • Azaguanine (Sigma Chemical Co., St. Louis, MO) for one week and then expanded for one week in Explant media (see below) without 8-Azaguanine.
  • Myeloma cells are expanded in 175 cm 2 flasks. Usually four flasks (1 x 10 8 cells) are prepared per fusion. Media is saved at each refeeding as conditioned media and used for hybridoma expansion.
  • peritoneal macrophages are obtained from CD-1 mice by injection of 0.34M sucrose into the peritoneal cavity of each mouse and extraction of a cell suspension.
  • Ortho-mune lysing reagent (Ortho Clinical Diagnostics) is added to lyse the red blood cells.
  • the remaining cells are washed with Explant media and final pellet is suspended in Explant media containing 1% HAT (GIBCO) at a concentration of about 2 x 10 5 cells per ml.
  • HAT 1% HAT
  • Spleens-derived lymphocytes are washed with RPMI 1640 base medium containing 2X Penicillin/Streptomycin (Pen/Strep)( 8 mg/ml). The suspension is centrifuge at 400 xg for 5 min. at 4°C and the supernatant discarded. The pellet is suspended in 10 ml cold Ortho-mune lysing reagent, incubated for 10 min. at room temperature and centrifuged as above (400 xg, 5 min., 4°C). [0104] Mix SP2/0-PrP " myeloma cells and and spleen-derived lymphocytes at a 1:10 ratio and pellet the myeloma cell-lymphocyte mixture at 400 xg for 5 min. Incubate this myeloma cell-lymphocyte pellet with pre-warmed (37°C) PEG 3,000- 3,700 / 5% DMSO solution.
  • PEG 3,000- 3,700 / 5% DMSO solution pre-warmed (37
  • the cells are diluted to a final volume of 90 ml. with conditioned medium containing 10% Origen and 1% HAT. Plate 100 ⁇ l into 96-well plates containing the feeder layer prepared the previous day (leave outer perimeter of wells empty). The cells are incubated at 37° C in a 5% C0 2 -95% air incubator.
  • Kascsak, R.J., Fersko, R., Pulgiano, D., Rubenstein, R. & Carp, R.I. (1997). Immunodiagnosis of prion disease. Immunological Investigations 26, 259-268.

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Abstract

L'invention concerne un nouveau procédé de production d'anticorps monoclonaux contre une variété d'antigènes mammifères, et plus particulièrement, des antigènes de surface relativement conservés entre espèces. Dans un mode de réalisation spécifique, l'invention concerne la génération d'anticorps monoclonaux qui réagissent spécifiquement aux protéines de prions (PrP). Le procédé consiste à combiner une souris knock-out PrP et un partenaire de fusion à non expression de PrP pour la production d'hybridomes, ouvrant ainsi toutes les zones et la conformation de la molécule à la production d'anticorps. Cette approche conduit à plusieurs nouveaux anticorps monoclonaux, utile à diverses spécificités.
PCT/US2004/019217 2003-06-16 2004-06-16 Approche destinee a obtenir des anticorps monoclonaux pour des proteines de surface WO2005021711A2 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2186829A1 (fr) * 2008-11-14 2010-05-19 Canadian Blood Services Anticorps contre GPIbalpha
CN103044546A (zh) * 2011-11-24 2013-04-17 上海转基因研究中心 一种朊蛋白抗体及其制备方法和应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
ZANUSSO ET AL: 'Prion protein expression in different species: analysis with a panel of new mAbs' PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES USA vol. 95, July 1998, pages 8812 - 8816, XP002924868 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2186829A1 (fr) * 2008-11-14 2010-05-19 Canadian Blood Services Anticorps contre GPIbalpha
US8323652B2 (en) 2008-11-14 2012-12-04 Canadian Blood Services Antibodies against GPIbα
CN103044546A (zh) * 2011-11-24 2013-04-17 上海转基因研究中心 一种朊蛋白抗体及其制备方法和应用
CN103044546B (zh) * 2011-11-24 2015-09-30 上海转基因研究中心 一种朊蛋白抗体及其制备方法和应用

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