EP2076776A2 - Autoantigènes utilisés dans le diagnosic, le pronostic et le traitement améliorés de maladies neurologiques inflammatoires - Google Patents

Autoantigènes utilisés dans le diagnosic, le pronostic et le traitement améliorés de maladies neurologiques inflammatoires

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Publication number
EP2076776A2
EP2076776A2 EP07818849A EP07818849A EP2076776A2 EP 2076776 A2 EP2076776 A2 EP 2076776A2 EP 07818849 A EP07818849 A EP 07818849A EP 07818849 A EP07818849 A EP 07818849A EP 2076776 A2 EP2076776 A2 EP 2076776A2
Authority
EP
European Patent Office
Prior art keywords
nucleic acid
protein
peptide
antibody
derivative
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP07818849A
Other languages
German (de)
English (en)
Inventor
Ugur Sahin
Özlem TÜRECI
Alexander Zaigler
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Johannes Gutenberg Universitaet Mainz
Ganymed Pharmaceuticals GmbH
Original Assignee
Johannes Gutenberg Universitaet Mainz
Ganymed Pharmaceuticals GmbH
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Johannes Gutenberg Universitaet Mainz, Ganymed Pharmaceuticals GmbH filed Critical Johannes Gutenberg Universitaet Mainz
Publication of EP2076776A2 publication Critical patent/EP2076776A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • G01N33/56972—White blood cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/285—Demyelinating diseases; Multipel sclerosis

Definitions

  • the present invention describes autoantigens against which an immune response could be detected in patients with multiple sclerosis. These antigens are the basis for the development of methods for the diagnosis and prognosis of inflammatory neurological autoimmune diseases and for the development of therapeutic agents against inflammatory neurological autoimmune diseases.
  • the serodiagnosis of autoimmune diseases is based on the detection of autoantibodies circulating in the blood, which are specifically directed against immunogenic components (antigens) of their own proteins. These antigens are also targets for preventive and therapeutic strategies of autoimmune diseases. The knowledge of these antigens therefore allows the development of methods for the specific diagnosis and treatment of inflammatory neurological diseases.
  • Inflammatory neurological diseases and in particular multiple sclerosis are widespread diseases.
  • multiple sclerosis with a prevalence of approximately 1 in 800 in Europe and North America, is the most common neurological disorder in young adults worldwide.
  • the chronic inflammatory disease of the nervous system which occurs for the first time in patients between the ages of 15 and 40 years, leads to a demyelination of dendrites of the central nervous system (CNS). This results in progressive paralysis of the musculature, sensory deficits and mental disorders.
  • Both the clinical course and the pathology of brain disease are extremely heterogeneous (Lucchinetti et al., 2000. Ann Neurol 47, 707).
  • the disease is either chronic-progressive or relapsing-like.
  • the pathogenesis of MS lesions (Lucchinetti et al., 2000, Ann Neural 47, 707) additionally supports the importance of autoimmune processes in the course of the disease.
  • causes for the autoimmunity could eg the similarity of virus antigens with encephalitogenic antigens ("molecular mimicry") or traumatic tissue loss (Levin et al., 2002, Nat Med 8, 509, Ludewig et al., 2004, J Exp Med 200, 637)
  • the role of immune dysregulation in inflammatory neurological disorders is further supported by numerous experiments in model organisms in which "autoimmune autoimmune processes"("EAE”) can be induced by autoimmune processes ("t Hart et al.,” 2003, Curr Opinion Neural 16, 375).
  • the diagnosis of neurological autoimmune diseases and of MS in particular is currently a major problem.
  • the first symptoms such as visual or coordination disorders as well as paralysis and deafness apply to numerous neurological diseases and a differential diagnosis of autoimmune diseases is hardly possible (Schmitt, 2003, Biomed Pharmacother 57, 261).
  • Only the combination with other criteria such as the number of inflammatory brain lesions, which are obtained by means of MRI spectroscopy (magnetic resonance imaging), or the analysis of oligoclonal IgG bands in CSF finally lead to the reliable diagnosis of MS.
  • MS A disease-specific treatment of MS is not yet established.
  • the treatment of MS is currently mainly symptomatic using anti-inflammatory drugs.
  • steroids and various interferons are used (Jacobs et al., 2003, J Exp Med 343, 598, EP1289541). Basically, these drugs reduce the inflammatory immune response through toxic effects against lymphocytes, but do not prevent the further relapsing or chronic progressive course of the disease.
  • Other substances currently being tested are statins (US2002159974) and glatiramer acetate, which is said to inhibit T-cell proliferation by competition (Duda et al., 1999, J Clin Invest 105, 967, EP1467763, WO2004078145).
  • some antigen-specific approaches are currently being analyzed.
  • the object of the present invention was to provide target structures for the diagnosis, prognosis and therapy of neurological autoimmune diseases such as multiple sclerosis.
  • the object of the present invention was the identification of molecular markers that allow a differential diagnosis between neurological autoimmune diseases such as multiple sclerosis and other neurological disorders.
  • autoantibodies whose occurrence is characteristic of neurological autoimmune diseases, in particular multiple sclerosis, are detected and the respective autoantigens identified. According to the invention, it has also been possible to show that various of these autoantigens are expressed specifically in the brain.
  • the identification of the autoantigens and autoantibodies can be used diagnostically and therapeutically, with brain-specific expression of the autoantigens furthermore emphasizing an important role of the antigens and antibodies in the development and course of neurological autoimmune diseases.
  • the invention relates to methods that enable evaluation and / or prognosis of a neurological autoimmune disease.
  • the methods of the invention preferably provide information about whether a disease has been or will be due to a neurological autoimmune disease.
  • the methods of the invention allow a distinction to be made between a neurological disease other than an autoimmune disease and an autoimmune neurological disease, especially between a neurological disorder other than multiple sclerosis and multiple sclerosis.
  • the methods of the invention can also provide information about the success of a treatment of a neurological autoimmune disease.
  • a successful treatment of a neurological autoimmune disease is preferably indicated by a decrease in one or more of the autoantibodies or T lymphocytes described herein.
  • the inventive method also allow the Monitoring disease progression, wherein as the disease worsens, such as indicated by an increase in one or more of the autoantibodies or T lymphocytes described herein, it is possible to plan for a more aggressive therapy such as treatment by immunosuppression.
  • the invention relates to a method for diagnosis, prognosis and / or monitoring, i. Determining the regression, progression and / or progression of an autoimmune neurological disorder in a patient comprising detecting and / or determining the amount of antibody encoding a protein or peptide encoded by a nucleic acid selected from the group consisting of: A group consisting of: (a) a nucleic acid comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25 , 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75 , 77, 79, 81, 83, 85, 87, a part and a derivative thereof, (b) a nucleic acid which hybridizes under stringent conditions with the nucleic acid under (a), (c)
  • the protein or peptide for which the antibody is specific preferably comprises a sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 , 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74 , 76, 78, 80, 82, 84, 86, 88, a part and derivative thereof.
  • the method of the invention further comprises detecting and / or determining the amount of antibody in a biological sample isolated from a patient without the autoimmune disease and / or without a risk for the autoimmune disease.
  • the presence of the antibody and / or an increased amount of the antibody in the biological sample compared to a patient without the autoimmune disease and / or a risk for the autoimmune disease preferably indicates the presence of the autoimmune disease or a risk of developing the autoimmune disease.
  • the detection and / or the determination of the amount of the antibody is carried out with an immunoassay.
  • the detection and / or determination of the amount of antibody comprises contacting the biological sample with an agent that specifically binds to the antibody, and (ii) detecting complex formation between the agent and the antibody.
  • the agent which specifically binds to the antibody is preferably immobilized on a carrier material and / or is preferably a protein or peptide or derivative thereof which specifically binds to the antibody.
  • the protein or peptide that specifically binds to the antibody comprises a sequence encoded by a nucleic acid selected from the group consisting of: (a) a nucleic acid comprising a nucleic acid sequence selected from the group consisting of: Group consisting of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43 , 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, a part and a derivative (b) a nucleic acid which hybridizes under stringent conditions with the nucleic acid under (a), (c) a nucleic acid which is de
  • the protein or peptide that specifically binds to the antibody comprises a sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 , 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74 , 76, 78, 80, 82, 84, 86, 88, and a part thereof.
  • a derivative of the protein or peptide is preferably derived from such a protein or peptide.
  • the invention relates to a method for diagnosing, prognosing and / or monitoring an autoimmune neurological disorder in a patient comprising detecting and / or determining the amount of a T lymphocyte that is responsible for a protein or peptide derived from a nucleic acid which is selected from the group consisting of: (a) a nucleic acid comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17 , 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67 , 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, a part and a derivative thereof, (b) a nucleic acid which hybridizes under stringent conditions with the nucleic acid under (a), ( c) a nucleic acid which, with respect to the nucleic acid which,
  • the protein or peptide for which the T-lymphocyte is specific preferably comprises a sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 , 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72 , 74, 76, 78, 80, 82, 84, 86, 88, a part and derivative thereof.
  • the method according to the invention further comprises the detection and / or the determination of the amount of the T-lymphocyte in a biological sample isolated from a patient without the autoimmune disease and / or without a risk for the autoimmune disease.
  • Presence of the T lymphocyte and / or increased amount of T lymphocyte in the biological sample compared to a patient without the autoimmune disease and / or without a risk for the autoimmune disease preferably indicates the presence of the autoimmune disease or a risk for development the autoimmune disease.
  • detection and / or determination of the amount of T lymphocyte is by a lymphocyte proliferation assay.
  • the detection and / or determination of the amount of T lymphocyte comprises (i) contacting the biological sample with an agent that specifically binds to the T lymphocyte, and (ii) demonstrating complex formation between the agent and the T lymphocytes.
  • the agent which binds specifically to the T lymphocytes is preferably immobilized on a carrier material.
  • the agent that specifically binds to the T lymphocyte is a protein or peptide or derivative thereof that specifically binds to the T lymphocyte.
  • the agent that specifically binds to the T lymphocyte is a complex comprising an MHC molecule or portion thereof and a protein or peptide or derivative thereof that specifically binds to the T lymphocyte.
  • the complex is presented by a cell, such as an antigen presenting cell.
  • the protein or peptide that binds specifically to the T lymphocyte or that is encompassed by the complex that binds specifically to the T lymphocyte preferably comprises a sequence encoded by a nucleic acid selected from the group consisting of: (a) a nucleic acid comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27 , 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77 , 79, 81, 83, 85, 87, a part and a derivative thereof, (b) a nucleic acid which hybridizes under stringent conditions with the nucleic acid under (a), (c) a nucleic acid which is related to the Nucleic acid is degenerate under (a) or (b), and (d) a nucleic acid
  • the protein or peptide that specifically binds to the T lymphocyte or that is encompassed by the complex that specifically binds to the T lymphocyte comprises a sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, and a part thereof.
  • a derivative of the protein or peptide is preferably derived from such a protein or peptide.
  • the methods of the invention for monitoring a neurological autoimmune disease include determining the regression, course or onset of the disease in a sample from the patient.
  • the methods of the invention for monitoring an autoimmune neurological disorder serve to monitor a successful therapy of the autoimmune neurological disease, wherein a decrease in the level of antibodies and / or T lymphocytes detected and / or determined in accordance with the present invention is indicative of successful therapy.
  • a biological sample preferably comprises body fluid and / or body tissue, wherein the body fluid is preferably selected from serum, plasma, urine and cerebrospinal fluid.
  • the neurological autoimmune disease is preferably multiple sclerosis.
  • the invention in the above aspects relates to a method for diagnosing a neurological autoimmune disease, in particular multiple sclerosis.
  • the patient is suffering from a neurological disease, in particular an autoimmune neurological disease, and in particular has symptoms for such a disease is suspected to be a neurological disease or, in particular, being ill or suffering from an autoimmune neurological disease, or has a risk of developing a neurological disorder, in particular an autoimmune neurological disorder.
  • the biological sample isolated from a patient is compared to a comparable normal biological sample as a sample from a healthy individual.
  • the agent used for detection or determination of the amount of antibody or T lymphocyte, in particular the protein, peptide or derivative thereof, or the agent that binds to a complex between an antibody or T lymphocyte and a binding agent, in particular, an anti-immunoglobulin antibody or an antibody directed against T-lymphocytes, are preferably detectably labeled.
  • the detectable marker is a radioactive marker, fluorescent label or enzyme label.
  • the methods of the invention include detection of several of the autoantibodies and / or T lymphocytes described herein.
  • the invention relates to a kit comprising one or more agents enabling detection and / or determination of the amount of antibodies or T lymphocytes described herein in a biological sample isolated from a patient. Such agents are described herein and known to those skilled in the art.
  • the invention relates to a kit for diagnosing, prognosing and / or monitoring an autoimmune neurological disorder in a patient, comprising a protein or peptide comprising a sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6 , 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56 , 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, and a portion thereof, or a derivative of the protein or peptide.
  • the protein or peptide or derivative thereof is immobilized on a support.
  • the kit of the invention further comprises instructions for use of the kit in a method of diagnosing, prognosing, and / or monitoring an autoimmune neurological disorder in a patient, which method is preferably a method of the invention.
  • the kit further comprises a reagent for detecting binding of an antibody to the protein or peptide or derivative thereof contained therein, which reagent preferably comprises a detectably labeled binding partner for the antibody.
  • the binding partner for the antibody is preferably an anti-immunoglobulin antibody, in particular an anti-human immunoglobulin antibody coupled with a detectable marker such as an enzyme.
  • the kit according to the invention may also contain an enzyme substrate, and positive and negative controls.
  • the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising one or more components selected from the group consisting of: (i) a protein or peptide comprising a sequence selected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, and a part thereof, or a derivative of the protein or peptide, (ii) a nucleic acid expressing the protein or peptide or the derivative thereof under (i), and (iii) a host cell containing the nucleic acid under (ii).
  • the nucleic acid can be present in an expression vector.
  • the host cell will preferentially express the peptide or protein or derivative thereof.
  • a host cell contained in a pharmaceutical composition of the invention may secrete the protein or peptide or derivative thereof, express it on the surface, or may additionally express an MHC molecule which binds to the protein or peptide or derivative thereof or to a processed form thereof ,
  • the host cell expresses the MHC molecule endogenously.
  • the host cell expresses the MHC molecule and / or the protein or peptide or the derivative thereof recombinantly.
  • the host cell is non-proliferative.
  • the host cell is an antigen presenting cell.
  • a pharmaceutical composition according to the invention may comprise a pharmaceutically acceptable carrier and / or adjuvant and is preferably suitable for the treatment of a neurological autoimmune disease, in particular for the treatment of multiple sclerosis.
  • the invention further relates to a method for the treatment of an autoimmune neurological disease, in particular multiple sclerosis, comprising the administration of a pharmaceutical composition according to the invention.
  • the term "autoantigen” refers to a substance which generates an immune response, such as the production of antibodies in the animal from which it is derived is selected from the group consisting of: (a) a nucleic acid comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71 , 73, 75, 77, 79, 81, 83, 85, 87, a part and a derivative thereof, (b) a nucleic acid which hybridizes under stringent conditions with the nucleic acid under (a), (c) a nucleic acid which, with respect to the nucleic acid, is selected from (a) or ( b) is degenerate, and (d) a nucleic acid which is complementary to
  • autoantigen relates to a protein or peptide comprising a sequence selected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, a portion and derivative thereof.
  • autoantibodies refers to antibodies which are directed against an autoantigen.Autoantibodies recognize an endogenous antigen and occur, inter alia, in autoimmune diseases.Particularly, the term “autoantibodies” according to the invention relates to an antibody which is directed against an autoantigen described above and in particular specifically binds to it.
  • detection and / or determination of the amount refers to the determination of the occurrence or absence and / or the absolute and / or relative amount of the substance.
  • the term also encompasses situations in which no substance is detected, either because it is not present or its amount is below the detection limit of the detection system.
  • all methods suitable for the detection and analysis of antibodies or T lymphocytes can be used to detect and / or determine the amount thereof.
  • Possibilities for carrying out a detection and / or a determination of the amount of antibodies and T-lymphocytes in the methods according to the invention are known to the person skilled in the art.
  • any direct or indirect method can be used according to the invention for detecting antibodies.
  • the binding of the antibodies to be detected to the antigen is determined by a change in the chemical or physical properties, so that subsequent detection steps with labeled binding partners are not necessary.
  • antibodies are preferably detected in an immunoassay, preferably in a solid phase immunoassay, and with direct or indirect coupling of a binding partner.
  • the detection can be carried out in an ELISA, an RIA or a fluorescence immunoassay.
  • the implementation of these detection methods is known to the person skilled in the art.
  • any carrier capable of binding to antigen or antibody can be used.
  • Such supports include materials such as glass, polystyrene, polypropylene, polyethylene, dextran, nylon, natural or modified celluloses, polyacrylamides, agaroses, and magnetite.
  • the carrier may have any possible structural configuration, as long as the molecule bound thereto, such as antigen or antibody, is capable of binding to its binding partner. Suitable configurations include a spherical configuration, a cylindrical configuration such as the inside of a test tube, or a flat configuration such as test strips, etc.
  • antigen is bound directly or indirectly to a carrier such as polystyrene.
  • a carrier such as polystyrene.
  • antigen-bound antibodies are detected directly or indirectly by means of enzyme-coupled substances.
  • enzyme-coupled substances may be antibodies, fragments of antibodies or high affinity ligands.
  • suitable enzymes are peroxidase, alkaline phosphatase, ⁇ -galactosidase, urease or glucose oxidase.
  • the antigen is bound directly or indirectly to a carrier such as polystyrene.
  • a carrier such as polystyrene.
  • antigen-bound antibodies are detected by substances carrying a radioactive label such as 125 I.
  • These substances can be antibodies, fragments of Be antibodies or high affinity ligands.
  • the bound radioactivity can be quantified by means of a suitable measuring device.
  • the antigen-bound antibodies are detected in a fluorescence immunoassay by means of substances which carry a fluorescent label such as fluorescein isothiocyanate (FITC). These substances may be antibodies, fragments of antibodies or high affinity ligands. The bound amount of fluorescent dye is then quantified by means of a suitable measuring device.
  • FITC fluorescein isothiocyanate
  • antibodies can also be detected in an agglutination test or Geldiffusionstest. These detection methods are also known to the person skilled in the art.
  • the antigen or the antibody solutions are filled in adjacent, nearby recesses of agar or agarose plates. Diffuse the substances from their wells, forming from the wells concentration gradients. When the overlapping antigen and antibody concentrations in the gel are within certain ratios and the antibody solution contains antibodies to the antigen, visible precipitates form in the gel.
  • antigen-bearing particles such as particles of latex or polystyrene are cross-linked by antibodies.
  • the aggregates formed can be detected, for example, by turbodimetry.
  • detection or determination of the amount of a T-lymphocyte can be carried out with a cell presenting a complex between a protein, peptide or derivative thereof and an MHC molecule for which the T-lymphocyte is specific, the cell preferably having a Antigen-presenting cell is.
  • the detection or determination of the amount of a T lymphocyte is by detection of its proliferation, cytokine production and / or cytotoxic activity elicited by the specific stimulation with the complex between the protein, peptide or derivative thereof and an MHC molecule.
  • a detection or a determination of the amount of a T-lymphocyte may further by a recombinant MHC molecule or a complex of two or more MHC molecules loaded with one or more proteins, peptides or derivatives thereof.
  • the cell expresses the MHC molecule endogenously. In another embodiment, the cell expresses the MHC molecule and / or the protein, peptide or derivative thereof recombinantly.
  • the host cell is non-proliferative. In a preferred embodiment, the host cell is an antigen presenting cell.
  • a binding agent such as an antibody
  • bind preferably refers to a specific binding.
  • Specific binding means that binding to a target, such as an epitope, for which a binding agent, such as an antibody, is specific, is stronger compared to binding to another target.
  • a “stronger bond” can be characterized by a lower dissociation constant.
  • a "reference" such as a reference sample or a reference organism can be used to correlate or compare the results obtained in the methods of the invention.
  • a reference organism is a healthy organism, especially an organism that is not involved in a neurological Autoimmune disease, in particular suffering from multiple sclerosis.
  • a “reference value” can be determined empirically by measuring a sufficient number of references based on a reference.
  • a nucleic acid is preferably deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).
  • Nucleic acids according to the invention comprise genomic DNA, cDNA, mRNA, recombinantly produced and chemically synthesized molecules.
  • a nucleic acid can be present as a single-stranded or double-stranded and linear or covalently circular closed molecule.
  • isolated nucleic acid means that the nucleic acid is (i) in vitro was amplified, for example by polymerase chain reaction (PCR), (ii) was produced recombinantly by cloning, (iii) was purified, for example by cleavage and gel electrophoretic separation, or (iv) was synthesized, for example by chemical synthesis.
  • An isolated nucleic acid is a nucleic acid available for manipulation by recombinant DNA techniques.
  • a degenerate nucleic acid according to the invention is a nucleic acid which differs from a reference nucleic acid in terms of the codon sequence due to the degeneracy of the genetic code.
  • "derivative" of a nucleic acid means that single or multiple, preferably at least 2, at least 4, at least 6, and preferably up to 3, to 4, to 5, to 6 until 10, to 15 or to 20, substitutions, deletions and / or additions of nucleotides are present in the nucleic acid.
  • nucleic acid also includes a chemical derivatization of a nucleic acid on a nucleotide base, on the sugar or on the phosphate, and nucleic acids which contain non-naturally occurring nucleotides and nucleotide analogues.
  • the degree of identity between a nucleic acid and a nucleic acid which is a derivative of the former nucleic acid that hybridizes to the former nucleic acid and / or which is degenerate with respect to the former nucleic acid is at least 70%, more preferably at least 75%, at least 80% according to the invention %, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, and preferably at least 99%.
  • the degree of identity is preferably for a range of at least about 30, at least about 50, at least about 70, at least about 90, at least about 100, at least about 150, at least about 200, at least about 300, at least about 400, at least about 500, at least about 1000, or at least about 2000 consecutive nucleotides indicated.
  • the degree of identity for the entire length of the reference nucleic acid is given as the nucleic acid sequences given in the Sequence Listing.
  • One nucleic acid is "complementary" to another nucleic acid if the two sequences can hybridize to each other and form a stable duplex, preferably hybridizing under conditions allowing specific hybridization between polynucleotides (stringent conditions) Molecular Cloning: A Laboratory Manual, J.
  • Percent complementarity indicates the percentage of consecutive nucleotides in a nucleic acid that can form hydrogen bonds (e.g., by Watson-Crick pairing) with a second nucleic acid.
  • complementary nucleic acids preferably have at least 40%, in particular at least 50%, at least 60%, at least 70%, at least 80%, at least 90% and preferably at least 95%, at least 98% or at least 99% complementary nucleotides.
  • complementary nucleic acids are completely complementary, which means that all consecutive nucleotides with the same number of consecutive nucleotides will undergo hydrogen bonding in a second nucleic acid.
  • Sequence Similarity indicates the percentage of amino acids that are either identical or represent conservative amino acid substitutions.
  • Sequence identity indicates the percentage of amino acids or nucleotides that are identical between the sequences.
  • % identity shall mean a percentage of nucleotides identical between two sequences to be compared in an optimal alignment, wherein this percentage is purely statistical, the differences between the two sequences may be random and distributed throughout the sequence length, and the sequence to be compared may comprise additions or deletions relative to the reference sequence to achieve optimal alignment between two sequences. Sequence comparisons between two sequences are generally performed by comparing these sequences for optimal alignment with respect to a segment or "comparison window" to identify local regions of sequence match. The optimal alignment for a comparison can be done manually or using the local homology algorithm of Smith and Waterman, 1981, Ads App. Math. 2, 482, using the local homology algorithm of Neddleman and Wunsch, 1970, J. Mol. Biol.
  • the percent identity is obtained by determining the number of identical positions in which the sequences to be compared match, dividing this number by the positions compared, and multiplying this result by 100.
  • BLAST 2 sequences available from the website http://www.ncbi.nlm.nih.gov/blasul5l2seq/wblast2.cgi can be used.
  • Derivatives of a particular nucleic acid relate in particular to variants of the nucleic acid, in particular splice variants, isoforms and species homologues of the nucleic acid, in particular those which are naturally expressed.
  • Nucleic acids can be analyzed according to the invention in a manner known per se with respect to variants such as splice variants.
  • Splice variant analysis techniques include reverse transcription-polymerase chain reaction (RT-PCR), northern blotting, and in situ hybridization.
  • RNAse protection can also be used to identify alternatively spliced mRNAs.
  • RNAse protection involves the transcription of a Gene sequence in synthetic RNA hybridized to RNA derived, for example, from other cells. The hybridized RNA is then incubated with enzymes that recognize RNA: RNA hybrid mismatches. Fragments smaller than expected indicate the presence of alternatively spliced mRNAs. The putative alternatively spliced mRNAs can be cloned and sequenced in a manner known per se.
  • RT-PCR can also be used to identify alternatively spliced mRNAs.
  • mRNA is converted into cDNA by the enzyme reverse transcriptase in a conventional manner.
  • the entire coding sequence of the cDNA is then amplified by PCR using a forward primer located in the 3 'untranslated region and a reverse primer located in the 5' untranslated region.
  • the amplification products can be analyzed for alternative splice forms, for example, by comparing the size of the amplified products with the size of the expected product from normally spliced mRNA, for example, by agarose gel electrophoresis. Any changes in the size of the amplification products may indicate alternative splicing.
  • mutant genes can also be readily identified using the techniques for identifying alternative splice forms described above.
  • allelic forms of genes and the mRNA produced thereby, which are considered according to the invention as "mutants” can be identified.
  • nucleic acids according to the invention may be present alone or in combination with other nucleic acids which may be homologous or heterologous.
  • a nucleic acid is functionally in association with expression control sequences which may be homologous or heterologous relative to the nucleic acid, here the term “homologous” means that a nucleic acid is also naturally functionally linked to the expression control sequence and the term “ heterologous "means that a nucleic acid is not naturally functionally linked to the expression control sequence.
  • a nucleic acid preferably a transcribable and particularly a nucleic acid encoding a peptide or protein, and an expression control sequence are then "operably linked” if covalently linked such that the transcription or expression of the nucleic acid is under the control or under the influence the expression control sequence stands. If the nucleic acid is to be translated into a functional peptide or protein, in a functional connection of an expression control sequence with the coding sequence, induction of the expression control sequence leads to transcription of the coding sequence without resulting in frame shift in the coding sequence or inability of the coding sequence coding sequence comes to be translated into the desired peptide or protein.
  • expression control sequence encompasses, according to the invention, promoters, ribosome-binding sequences, enhancers and other control elements which control the transcription of a gene or the translation of mRNA
  • the expression control sequences are regulatable
  • the exact structure of expression control sequences can vary depending on the species or cell type however, generally 5'-untranscribed and 5'- and 3'-untranslated sequences involved in the initiation of transcription, such as TATA box, capping sequence, CAAT sequence and the like
  • 5'-untranscribed expression control sequences include a promoter region that includes a promoter sequence for transcriptional control of the functionally-linked nucleic acid.
  • Expression control sequences may also include enhancer sequences or upstream activator sequences ,
  • promoter refers to a nucleic acid sequence upstream (5 1 ) to the sequence to be expressed which directs expression of the sequence by providing a recognition and binding site for RNA polymerase.
  • the promoter region may include other recognition or binding sites for other factors involved in the regulation of transcription of a gene.
  • a promoter may direct transcription of a prokaryotic or eukaryotic gene.
  • a promoter may be "inducible” and initiate transcription in response to an inducer, or it may be “constitutive” if transcription is not controlled by an inducer. An inducible promoter will not expressed or only expressed to a very small extent if an induction agent is missing. In the presence of the inducer, the gene is "turned on” or the level of transcription increased. This is conventionally mediated by the binding of a specific transcription factor.
  • Preferred promoters according to the invention are, for example, promoters for SP6, T3 or T7 polymerase, human U6 RNA promoter and CMV promoter.
  • expression is used according to the invention in its most general meaning and includes the production of RNA or of RNA and protein / peptide. It also includes partial expression of nucleic acids. Furthermore, the expression can be transient or stable.
  • nucleic acid encoding a protein or peptide may be present in accordance with the invention in association with another nucleic acid encoding a peptide sequence that directs secretion of the nucleic acid-encoded protein or peptide from a host cell.
  • a nucleic acid can be present in combination with another nucleic acid which codes for a peptide sequence which brings about anchoring of the encoded protein or peptide on the cell membrane of a host cell or its compartmentalization into specific organelles of this cell.
  • a compound can be made with a nucleic acid that is a reporter gene or any "tag".
  • a nucleic acid according to the invention is present in a vector, optionally with a promoter which controls the expression of the nucleic acid.
  • the term "vector” is used in its most general meaning and includes any intermediary vehicle for a nucleic acid, which for example make it possible to introduce the nucleic acid into prokaryotic and / or eukaryotic cells and, if appropriate, to integrate it into a genome. Such vectors are preferably replicated and / or expressed in the cell. Vectors include plasmids, phagemids, bacteriophages or viral genomes.
  • plasmid refers generally to a construct of extrachromosomal genetic material, usually a circular DNA duplex, which can replicate independently of chromosomal DNA.
  • the term "host cell” refers to any cell that is transformable or transfectable with an exogenous nucleic acid, preferably DNA or RNA
  • the term "host cell” encompasses prokaryotic (eg E. coli) or eukaryotic cells (eg mammalian cells, especially cells from Human, yeast cells and insect cells). Particularly preferred are mammalian cells such as human, mouse, hamster, pig, goat and primate cells.
  • the cells can be derived from a variety of tissue types and include primary cells and cell lines.
  • the host cell is an antigen-presenting cell, wherein the term "antigen-presenting cell” according to the invention comprises dendritic cells, monocytes and macrophages
  • a nucleic acid may be present in the host cell in a single or multiple copies and in one embodiment is described in U.S. Pat the host cell expressed.
  • an expression vector may also comprise a nucleic acid sequence encoding the MHC molecule.
  • the nucleic acid sequence encoding the MHC molecule may be on the same expression vector as the nucleic acid encoding the protein or peptide, or both nucleic acids may be on different expression vectors. In the latter case, the two expression vectors can be cotransfected into a cell. If a host cell expresses neither the protein or peptide nor the MHC molecule, both nucleic acids coding therefor can be transfected into the cell either on the same expression vector or on different expression vectors. If the cell already expresses the MHC molecule, only the nucleic acid sequence encoding the protein or peptide can be transfected into the cell.
  • peptide according to the invention relates to substances containing at least two, at least 3, at least 4, at least 6, at least 8, at least 10, at least 13, at least 16, at least 20 and preferably up to 8, 10, 20, 30, 50, or 100 consecutive amino acids linked together by peptide bonds.
  • protein refers to large peptides, preferably peptides with more than 100 amino acids, but the terms “peptide” and “protein” are used herein generally used interchangeably.
  • protein or peptide is also intended to include derivatives thereof unless otherwise specified.
  • An isolated protein or peptide may be in a substantially purified state.
  • substantially purified means that the protein or peptide is substantially free of other substances present in nature or in vivo.
  • Proteins and peptides are used according to the invention, for example, the production of antibodies and can be used in immunological or diagnostic assays or as therapeutics. Proteins and peptides described according to the invention can be isolated from biological samples such as tissue or cell homogenates and can also be expressed recombinantly in a multiplicity of pro- or eukaryotic expression systems. Furthermore, according to the invention, synthesis of proteins and peptides on a solid or liquid phase can be carried out in a manner known per se.
  • proteins, peptides or derivatives thereof described herein may be used in their free or bound form for the diagnosis or treatment of patients with a neurological autoimmune disease, the proteins, peptides or derivatives thereof having in particular the ability to bind autoantibodies, neutralize and / or or selectively remove.
  • “Derivatives" of a protein or peptide or an amino acid sequence in the context of this invention include amino acid insertion variants, amino acid deletion variants and / or amino acid substitution variants.
  • Amino acid insertion variants include amino- and / or carboxy-terminal fusions, as well as insertions of single or multiple amino acids in a particular amino acid sequence.
  • amino acid sequence variants with an insertion one or more amino acid residues are introduced into a predetermined site in an amino acid sequence, although random insertion with appropriate screening of the resulting product is also possible.
  • Amino acid deletion variants are characterized by the removal of one or more amino acids from the sequence.
  • Amino acid substitution variants are characterized in that at least one residue in the sequence is removed and another residue is inserted in its place.
  • the modifications are at positions in the amino acid sequence that are not conserved between homologous proteins or peptides and / or amino acids are replaced by others with similar properties, such as hydrophobicity, hydrophilicity, electronegativity, side chain volume, and the like (conservative substitution).
  • conservative substitutions refer to the replacement of one amino acid with another, listed below in the same group as the substituted amino acid:
  • GIy is the only remainder without a side chain, giving the chain flexibility.
  • Pro has an unusual geometry that severely limits the chain. Cys can form a disulfide bridge.
  • the degree of similarity preferably identity between an amino acid sequence and an amino acid sequence that is a derivative of the former amino acid sequence, is at least 70%, more preferably at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, and preferably at least 99%.
  • the degree of identity is preferably for a range of at least about 10, at least about 30, at least about 50, at least about 70, at least about 90, at least about 100, at least about 150, at least about 200, at least about 300, at least about 400; or at least about 500 consecutive amino acids.
  • is the degree of identity for the entire length of the reference amino acid sequence is given as the amino acid sequences given in the sequence listing.
  • amino acid variants described above can readily be synthesized by known peptide synthesis techniques such as e.g. The manipulation of DNA sequences for the production of proteins and peptides with substitutions, insertions or deletions is described, for example, in Sambrook et al. (1989), by "solid-phase synthesis” (Merrifield, 1964) and similar methods or by recombinant DNA manipulation. described in detail.
  • derivatives of proteins or peptides also include single or multiple substitutions, deletions and / or additions of any molecules associated with the protein or peptide, such as carbohydrates, lipids and / or proteins or peptides "also to all functional chemical equivalents of proteins and peptides and substances which contain not only amino acid constituents but also non-amino acid constituents such as sugars and phosphate structures and also include substances which contain bonds such as ester, thioether or disulfide bonds
  • a derivative of a protein or peptide has better stability, preferably a higher in vivo half-life, than the protein or peptide from which it is derived.
  • Derivatives of a particular protein or peptide also affect post-translationally modified variants, isoforms and species homologs of the protein or peptide, particularly those that are naturally expressed.
  • a part, ie fragment, or derivative of a protein or peptide according to the invention preferably has a functional property of the protein or peptide from which it is derived.
  • Such functional properties include, for example, immunoreactivity, particularly interaction with antibodies or interaction with other peptides or proteins.
  • An important feature is the ability to complex with MHC molecules and, where appropriate, to create or prevent an immune response, for example, by stimulation or inhibition of cytotoxic or helper T cells.
  • a portion of a protein or peptide preferably comprises a sequence of at least 6, at least 8, at least 10, at least 12, at least 15, at least 20, at least 30 and preferably up to 8, up to 10, up to 12, up to 15, up to 20, up to 30 or up to 50 consecutive amino acids from the protein or peptide.
  • a part of a protein or peptide according to the invention relates to one or more epitopes of the complete peptide or protein, wherein the plurality of epitopes may be present in their natural compound or may have an artificial, ie non-naturally occurring compound, ie the epitopes may, for example an artificial linker be separated from each other.
  • part of a protein or peptide according to the invention relates to such a sequence which is a target, in particular an epitope, for an immune reaction in a patient, in particular in a patient with an autoimmune neurological disease.
  • the sequence is target for an antibody and / or T-cell mediated immune response.
  • a peptide, protein or derivative used according to the invention may also comprise a plurality of such sequences which are epitopes for antibodies or T cells.
  • a nucleic acid which codes for a protein or peptide preferably according to the invention relates to that part of the nucleic acid which encodes at least the protein or peptide and / or part of the protein or peptide as defined above.
  • a portion of a nucleic acid encoding a protein or peptide preferably relates to the portion of the nucleic acid corresponding to the open reading frame.
  • a part of a nucleic acid is that part of a nucleic acid which codes for only one or more epitopes of the protein or peptide which is encoded by the complete nucleic acid, in particular by the complete open reading frame.
  • the proteins, peptides or derivatives thereof used for therapeutic use are preferably those which inhibit binding of the autoantibodies described herein to the autoantigens described herein and / or therefore compete and / or the stimulation of T lymphocytes, the autoantigens described herein or parts thereof recognize, inhibit, and thereby protect a patient from autoimmune disease of the nervous system such as multiple sclerosis.
  • such proteins, peptides or derivatives are therapeutically useful in conjunction with the binding of T cells via their T cell receptor to the MHC / antigen complex that is responsible for initiation or Propagation of an immune recognition or an inflammatory course is necessary, interact.
  • a protein or peptide comprising an antibody and / or a T-cell epitope and administered to a patient according to the present invention may be capable of modifying the patient's response to an autoantigen, resulting in the inhibition of an autoimmune response. Therefore, according to the invention, in particular proteins, peptides or derivatives thereof are used which can compete with the autoantigens or fragments thereof for recognition by T lymphocytes or autoantibodies. Particularly preferred according to the invention are peptides which comprise or constitute a modified version of the T-cell epitope from the naturally occurring autoantigen which can bind to MHC molecules but, in contrast to the naturally occurring epitope, does not activate specific T cells.
  • the proteins, peptides or derivatives used for therapeutic use preferably compete for binding of autoantigens to antibodies and / or MHC molecules and do not trigger proliferation and / or induction of a T cell reactive with the autoantigen or portions thereof ,
  • Candidate proteins, peptides, or derivatives may be screened in a test that measures binding, particularly competitive binding to antibodies and / or MHC molecules, and / or a test measuring T-cell proliferation.
  • MHC binding peptides or “MHC-binding peptides” refers to peptides which bind to an MHC class I and / or an MHC class II molecule.
  • the binding peptides are typically 8-10 amino acids in length, although longer or shorter peptides may be effective.
  • the binding peptides are typically 10-25 amino acids long, and more preferably 13-18 amino acids long, although longer and shorter peptides may be effective.
  • an MHC-binding peptide can be administered for direct binding to MHC molecules, or an MHC-binding peptide, upon appropriate processing, especially in vivo after administration, can arise from an administered protein, peptide or derivative thereof.
  • an MHC-binding peptide can be formed by processing from an autoantigen.
  • an MHC-binding peptide is part of an administered protein, peptide or derivative of it or of a car antigen. Such cases are encompassed when, according to the invention, reference is made to proteins, peptides or derivatives used for therapeutic use, or to autoantigen-reactive T cells.
  • the ability of a peptide to bind to an antibody can be determined, for example, with one of the immunoassays described herein.
  • the ability to competitively bind to MHC molecules can be determined according to the invention, for example, with known binding assays which detect the displacement of a labeled binding molecule.
  • Autoantigens described in the invention can be used in peptide libraries, including phage display libraries, to identify and select, for example, peptide binding partners of antibodies or MHC molecules.
  • Such molecules may be used, for example, for screening assays, purification protocols, for interference with the function of the antibodies or MHC molecules, and for other purposes known to those skilled in the art.
  • Phage display can be particularly effective in identifying binding peptides.
  • a phage library (using, for example, the ml 3, fd, or lambda phage) is prepared which presents inserts of 4 to about 80 amino acid residues in length. Phage are then selected that carry inserts that bind to the target. This process can be repeated over several cycles of reselecting phages that bind to the target. Repeated laps lead to an accumulation of phages carrying certain sequences.
  • An analysis of DNA sequences may be made to identify the sequences of the expressed peptides. The smallest linear portion of the sequence that binds to the target can be determined.
  • the yeast two-hybrid system can also be used to identify peptides that bind to a target.
  • T cells are provided for testing by transformed T cell lines, such as T cell hybridomas or T cells.
  • T cell hybridomas Cells isolated from a mammal such as a human or a rodent such as a mouse. Suitable T-cell hybridomas are freely available or can be prepared in a manner known per se. T cells can be isolated from a mammal in a manner known per se; see. eg Shimonkevitz, R. et al., 1983, J. Exp. Med. 158: 303.
  • T-cells suitably express a marker that can be tested and that indicates T-cell activation or modulation of T-cell activity upon activation.
  • the mouse T cell hybridoma DOl 1.10 which expresses interleukin-2 (IL-2) upon activation, can be used.
  • IL-2 concentrations can be measured to determine if a specific peptide presented is capable of modulating the activity of this T cell hybridoma.
  • T-cells are used e.g. obtained from a T-cell hybridoma of interest or by isolation from a mammal.
  • the T cells are cultured under conditions that allow for propagation.
  • the growing T cells are brought into contact with antigen-presenting cells, which in turn have been brought into contact with a peptide to be presented or a nucleic acid coding therefor.
  • T cells are assayed for a label, e.g. the IL-2 production is measured.
  • T cells used in the assays are incubated under conditions suitable for proliferation.
  • a DOI 1 .10 T cell hybridoma is suitably incubated at about 37 ° C and 5% CO 2 in complete medium (RPMI 1640 supplemented with 10% FBS, penicillin / streptomycin, L-glutamine, and 5 x 10 -5 M 2-mercaptoethanol)
  • RPMI 1640 supplemented with 10% FBS, penicillin / streptomycin, L-glutamine, and 5 x 10 -5 M 2-mercaptoethanol
  • Serial dilutions of the peptide tested can be tested.
  • Activation signals are provided by antigen-presenting cells that have been loaded with the peptide.
  • the modulation of T cell activation can be conveniently determined by altering the proliferation of T cells as measured by known radiolabelling methods.
  • a labeled (such as tritiated) nucleotide can be introduced into a test culture medium. The introduction of such a labeled nucleotide into the DNA serves as a measure of T cell proliferation.
  • modified and substituted proteins or peptides useful in the diagnostic and therapeutic methods of the present invention can also be readily assayed for their ability to express in vitro proliferative responses of patient T cells or T cell lines or clones useful for an autoantigen, or to inhibit binding of the autoantigen to specific autoantibodies.
  • Epitopes in autoantigens against which antibodies and T cells are directed in patients with multiple sclerosis are identified through the use of truncated and / or mutagenized recombinant proteins and peptides. These peptide epitopes are tested for antigenicity upon generation of a T cell response or binding to antibodies.
  • Proteins, peptides or derivatives thereof described herein, optionally coupled to a polymer such as PEG which makes the protein, peptide or derivative tolerogenic, and / or in conjunction with an adjuvant may also be used therapeutically to create tolerance.
  • the proteins, peptides or derivatives are used in high doses.
  • the method of tolerization is described, for example, in WO 94/06828.
  • a pharmaceutical composition of the invention serves to tolerate a patient against one or more autoantigens described herein.
  • the pharmaceutical composition comprises a peptide or protein comprising an amino acid sequence corresponding to or derived from a sequence motif of an autoantigen described herein associated with an autoimmune neurological disorder described herein.
  • a peptide or protein comprising an amino acid sequence corresponding to or derived from a sequence motif of an autoantigen described herein associated with an autoimmune neurological disorder described herein.
  • such peptides or proteins bind to MHC molecules to form a complex that activates autoreactive T cells in patients with the autoimmune disease.
  • the use of such tolerance to autoimmune diseases is known and therefore need not be explained in detail.
  • Antibodies directed against the autoantigens described herein can be used to identify antigenic epitopes on the autoantigens. Once such epitopes have been identified, synthetic peptides can be produced and used, for example, as antigens in diagnostic tests or kits or for the development of therapeutics.
  • Antisera containing antibodies that specifically bind to a target can be prepared by a variety of standard methods; see. For example, “Monoclonal Antibodies: A Practical Approach” by Philip Shepherd, Christopher Dean ISBN 0-19 963722-9, “Antibodies: A Laboratory Manual” by Ed Harlow, David Lane ISBN: 0879693142 and "Using Antibodies: A Laboratory Manual: Portable It is also possible to generate affine and specific antibodies which recognize complex membrane proteins in their native form (Azorsa et al., J. Immunol., Methods 229: 35 Anderson et al., J. Immunol., 143: 1899-1904, 1989; Gardsvoll, J. Immunol., Methods: 234: 107-116, 2000). This is of particular importance for the production of antibodies which are to be used therapeutically, but also for many diagnostic applications, can be immunized with the entire protein, with extracellular partial sequences, as well as with cells that express the target molecule in physiologically folded form.
  • Monoclonal antibodies are traditionally made using Hybridoma technology (For technical details, see “Monoclonal Antibodies: A Practical Approach” by Philip Shepherd, Christopher Dean ISBN 0-19-963722-9, "Antibodies: A Laboratory Manual” by Ed Harlow, David Lane ISBN: 0879693142, "Using Antibodies: A Laboratory Manual: Portable Protocol NO by Edward Harlow, David Lane, Ed Harlow, ISBN: 0879695447).
  • the pFc 1 and Fc regions are, for example, effectors of the complement cascade but are not involved in the antigen binding.
  • an antibody from which the Fc region has been enzymatically cleaved or which has been produced without the Fc region referred to as the Fab fragment
  • Fab fragments consist of a covalently linked light chain of an antibody and part of the heavy chain of the antibody, designated Fd.
  • the Fd fragments are the major determinants of antibody specificity (a single Fd fragment can be up to ten different light chains are associated without altering the specificity of the antibody) and Fd fragments retain the ability to bind to an epitope upon isolation.
  • CDRs complementarity determining regions
  • FRs framework regions
  • Both the Fd fragment of the heavy chain and the IgG immunoglobulin light chain have four framework regions (FR1 to FR4), each separated by three complementarity determining regions (CDR1 to CDR3).
  • the CDRs and in particular the CDR3 regions, and more particularly the CDR3 heavy chain region, are largely responsible for antibody specificity.
  • non-CDR regions of a mammalian antibody can be replaced by similar regions of antibodies of the same or different specificity, while retaining the specificity for the epitope of the original antibody.
  • WO 92/04381 describes the preparation and use of mouse humanized RSV antibodies in which at least a portion of mouse FR regions have been replaced by FR regions of a human origin. Such antibodies, including fragments of intact antibodies having antigen-binding capacity, are often referred to as "chimeric" antibodies.
  • the term "antibody” also includes F (ab ') 2 , Fab, Fv and Fd fragments of antibodies, chimeric antibodies in which the Fc and / or FR and / or CDRl and / or CDR2 - and / or light chain CDR3 regions have been replaced by homologous human or non-human sequences, chimeric F (ab ') 2 fragment antibodies in which the FR and / or CDRl and / or CDR2 and / or or light chain CDR3 regions have been replaced by homologous human or non-human sequences, chimeric Fab fragment antibodies in which the FR and / or CDR1 and / or CDR2 and / or light chain CDR3 regions homologous human or non-human sequences have been replaced, and chimeric Fd fragment antibodies in which the FR and / or CDR1 and / or CDR2 regions have been replaced by homologous human or non-human sequences. also single-chain antibodies.
  • Antibodies may also be coupled to specific diagnostic agents to, for example, represent cells and tissues that express certain proteins or peptides, such as the autoantigens described herein. They may also be coupled to therapeutic agents.
  • Diagnostic agents include any label capable of: (i) providing a detectable signal, (ii) interacting with a second label to modify the detectable signal provided by the first or second label, eg, FRET (fluorescence Resonance energy transfer), (iii) to affect mobility such as electrophoretic mobility through charge, hydrophobicity, shape or other physical parameters or (iv) to provide a scavenger group, eg affinity, antibody / antigen or ionic complexation.
  • Suitable labels are structures such as fluorescent labels, luminescent labels, chromophore labels, radioisotope labels, isotope labels, preferably stable isotope labels, enzyme labels, particle labels, in particular metal particle labels,
  • Diagnostic agents include, but are not limited to, barium sulfate, iocetamic acid, iopanoic acid, calcium ipodate, sodium diatrizoate, meglumine diatrizoate, metrizamide, sodium tylopanoate and radiodiagnostics, including positron emitters such as fluorine-18 and carbon-11, gamma emitters such as Iodine-123, technetium-99m, iodine-131 and indium-111, and nuclides for nuclear magnetic resonance such as fluorine and gadolinium.
  • therapeutic agent relates to any substance that can exert a therapeutic effect.
  • MHC major histocompatibility complex
  • MHC proteins or molecules are involved in signaling between lymphocytes and antigen presenting cells in normal immune responses, binding peptides and presenting them for recognition by T cell receptors. MHC molecules bind peptides within an intracellular processing compartment and present these peptides on the surface of antigen-presenting cells for recognition by T cells.
  • the human MHC region also called HLA, is located on chromosome 6 and includes the class I and class II regions.
  • an MHC molecule is an HLA molecule.
  • patient comprises human, non-human primates or another animal, in particular mammal such as cow, horse, pig, sheep, goat, dog, cat or rodent such as mouse and rat.
  • patient is a human.
  • disease refers to a pathological condition.
  • autoimmune disease relates, according to the invention, to a disease whose cause is an excess reaction of the immune system to the body's own tissue. By mistake, the immune system recognizes the body's own tissue as a foreign body to be controlled. This leads to severe inflammatory reactions that lead to damage to the affected organs.
  • the term "neurological autoimmune disease” refers to an autoimmune disease of the nervous system.
  • inflammatory damage cascades can trigger nerve cell degeneration and associated neurological disease neurodegenerative diseases such as multiple sclerosis, stroke, Parkinson's disease and Alzheimer's disease.
  • the term "neurological autoimmune disease” refers to multiple sclerosis according to the invention
  • Multiple sclerosis is an inflammatory, demyelinating disease of the central nervous system that causes motor, sensory and cognitive deficits.
  • Multiple sclerosis arises when T lymphocytes and other cells of the immune system destroy the Inflammatory messengers block the conduction of Ranvier's ligation rings, and soluble and cellular effectors cause the breakdown of the myelin sheath leading to so-called demyelination or demyelination, leading to progressive paralysis and other neurological symptoms such as muscle tremors Deafness, itching, color blindness, double vision, blindness, loss of coordination and balance, acute paralysis and deterioration of cognitive ability.
  • the term "increased amount” preferably refers to an increase of at least 10%, in particular at least 20%, at least 50% or at least 100%
  • the amount of a substance is increased in a test object such as a biological sample with respect to a reference, even if it is detectable in the test object, but is not present in the reference and / or undetectable.
  • "Reduce” or “inhibit” herein refers to the ability to cause a decrease, such as a decrease of 20% or more, more preferably of 50% or more, and most preferably of 75% or more.
  • a biological sample can be a tissue sample, including body fluids, and / or a cellular sample, and can be obtained in a conventional manner, such as tissue biopsy, including punch biopsy, and blood, bronchial aspirate, sputum, urine, feces, or other body fluids.
  • tissue biopsy including punch biopsy, and blood, bronchial aspirate, sputum, urine, feces, or other body fluids.
  • the term "biological sample” also includes fractions of biological samples.
  • T cell and "T lymphocyte” are used interchangeably herein and include T helper cells and cytolytic T cells such as cytotoxic T cells.
  • compositions described in the invention may also be preventive, i. as vaccines, used to prevent the diseases described here.
  • nucleic acids may be either as a naked nucleic acid or in conjunction with an administration reagent.
  • administration of nucleic acids in vivo by means of target-controlled liposomes is also provided.
  • vectors derived from adenovirus (AV), adeno-associated virus (AAV), retroviruses (such as lentiviruses (LV), rhabdoviruses, murine leukemia virus), or herpesvirus, and the like can be used.
  • the tropism of the viral vectors can be modified as appropriate by pseudotyping the vectors with coat proteins or other surface antigens of other viruses, or substituting various viral capsid proteins.
  • Liposomes can aid the delivery of the nucleic acid to a particular tissue and can also increase the half-life of the nucleic acid.
  • Liposomes useful in the present invention are formed from standard vesicle-forming lipids, which generally include neutral or negatively charged phospholipids, and a sterol such as cholesterol.
  • the selection of lipids is generally determined by factors such as the desired liposome size and the half-life of the liposomes.
  • a variety of methods for producing liposomes are known; see. eg Szoka et al. (1980), Ann. Rev. Biophys. Bioeng. 9: 467; and U.S. Patents 4,235,871, 4,501,728, 4,837,028 and 5,019,369.
  • a carrier used to deliver a nucleic acid to a cell may have a bound targeting molecule.
  • a molecule such as an antibody specific for a surface membrane protein on the target cell or a ligand for a receptor on the target cell can be incorporated into or bound to the nucleic acid carrier.
  • proteins that bind to a surface membrane protein associated with the endocytosis may be incorporated into the liposome formulation to facilitate targeting and / or uptake.
  • proteins include capsid proteins or fragments thereof that are specific for a particular cell type, antibodies to proteins that are internalized, proteins that target an intracellular site, and the like.
  • compositions of the invention can be administered in pharmaceutically acceptable formulations.
  • Such preparations may ordinarily contain pharmaceutically acceptable concentrations of salts, buffers, preservatives, carriers, supplemental immunity enhancing agents such as adjuvants, CpG oligonucleotides, cytokines, chemokines, saponin, GM-CSF and / or RNA, and optionally other therapeutic agents.
  • the therapeutic agents of the present invention can be administered by any conventional route, including injection or infusion.
  • the Administration may, for example, be oral, intravenous, intraperitoneal, intramuscular, subcutaneous or transdermal.
  • Suitable methods for administering nucleic acids to cells include administering the nucleic acid to a subject by means of a gene gun, electroporation, nanoparticles, microencapsulation, and the like, or by parenteral and enteral delivery.
  • compositions of the invention are administered in effective amounts.
  • An "effective amount” refers to the amount that achieves a desired response or effect alone or with other doses.
  • the desired reaction preferably involves inhibition of the disease process. This includes slowing the progression of the disease and, in particular, interrupting or reversing the progression of the disease.
  • the desired response in treating a disease or condition may also be the delay in the onset or prevention of the onset of the disease or condition.
  • compositions of the invention will depend on the condition being treated, the severity of the disease, the individual parameters of the patient, including age, physiological condition, height and weight, duration of treatment, type of concomitant therapy (if any) specific route of administration and similar factors.
  • compositions of the invention are preferably sterile and contain an effective amount of the therapeutically active substance for the production of the desired reaction or effect.
  • compositions of the present invention administered may depend on various parameters such as mode of administration, condition of the patient, desired administration period, etc. In the event that a patient's response is insufficient at an initial dose, higher doses (or effectively higher doses achieved by a different, more localized route of administration) may be employed.
  • the pharmaceutical compositions of the invention are generally administered in pharmaceutically acceptable amounts and in pharmaceutically acceptable compositions.
  • pharmaceutically acceptable refers to a non-toxic material that does not interfere with the action of the active ingredient of the pharmaceutical composition.
  • Such preparations may usually contain salts, buffers, preservatives, carriers and optionally other therapeutic agents. When used in medicine, the salts should be pharmaceutically acceptable.
  • non-pharmaceutically acceptable salts can be used for the preparation of pharmaceutically acceptable salts thereof and are included in the invention.
  • Such pharmacologically and pharmaceutically acceptable salts include, but are not limited to, those made from the following acids: hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic, citric, formic , Malonic, succinic and the like.
  • Pharmaceutically acceptable salts may also be prepared as alkali metal or alkaline earth metal salts such as sodium, potassium or calcium salts.
  • a pharmaceutical composition of the invention may comprise a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier according to the invention relates to one or more compatible solid or liquid fillers, diluents or capsule substances which are suitable for administration, in particular to a human.
  • carrier refers to an organic or inorganic ingredient, natural or synthetic, in which the active ingredient is combined to facilitate application.
  • the components of the pharmaceutical composition according to the invention are usually such that no interaction occurs which substantially impairs the desired pharmaceutical activity.
  • the pharmaceutical compositions of the invention may contain suitable buffers such as acetic acid in a salt, citric acid in a salt, boric acid in a salt, and phosphoric acid in a salt.
  • suitable buffers such as acetic acid in a salt, citric acid in a salt, boric acid in a salt, and phosphoric acid in a salt.
  • suitable preservatives such as benzalkonium chloride, chlorobutanol, parabens and thimerosal.
  • compositions are usually presented in a unit dosage form and can be prepared in a manner known per se.
  • Pharmaceutical compositions according to the invention may be in the form of capsules, tablets, lozenges, suspensions, syrups, elixirs or as an emulsion, for example.
  • compositions suitable for parenteral administration usually comprise a sterile aqueous or non-aqueous preparation of the active ingredient, which is preferably isotonic with the blood of the recipient.
  • Compatible carriers and solvents include Ringer's solution and isotonic sodium chloride solution.
  • sterile, fixed oils are usually used as the solution or suspension medium.
  • Fig. 1 Schematic representation of the strategy for producing a brain-specific cDNA expression library.
  • Fig. 2 Immunoscreening with sera from MS patients and identification of a reactive antigen.
  • Fig. 3. Classification of the inventively identified antigens.
  • Fig. 4 Analysis of CLSTN1-specific expression.
  • Fig. 5 Analysis of ARPP19-specific expression.
  • Fig. 6 Analysis of CMTM2-specific expression.
  • Fig. 7 Analysis of CPE-specific expression.
  • Fig. 8 Analysis of LITAF-specific expression.
  • Fig. 9 Analysis of TUBGl-specific expression.
  • Fig. 10 Differential serology of selected antigens.
  • Example 1 Preparation of a brain-specific cDNA library
  • a brain-specific cDNA expression library was prepared in lambda phage (FIG. 1).
  • a complete pBluescript plasmid is contained in a lambda phage genome, combining the properties of a phage and a plasmid.
  • Human mRNA isolated from brain tissue was rewritten into methylated cDNA in a first strand synthesis using a reverse transcriptase and an oligo dT linker, at the 5 'end of which a ⁇ oi site was added. After the targeted degradation of the mRNA, the DNA was made double-stranded in a second-strand synthesis.
  • plaque-forming units The number of plaque-forming units (pfu) was adjusted so that plaque subconfluence was present (eg, -5000 pfu / 145 mm petri dish).
  • the infection mixture was plated on agar plates with tetracycline.
  • phage plaques formed on the bacterial lawn.
  • Each individual plaque represents a lambda phage clone with the nucleic acid inserted into this clone and simultaneously contains the recombinantly expressed protein encoded by the nucleic acid.
  • Nitrocellulose membranes were applied to produce replicating preparations of the recombinant proteins (plaque lift).
  • a total of approximately 1,000,000 clones were analyzed for the analysis of the brain-specific phage bank, using a total of 17 different sera. Partly pooled sera were used. Primarily identified clones were first isolated oligoclonal with the increase of adjacent non-reactive phage plaques and monoclonalized after confirmation. By PCR with the T7 / T3 standard primers of the integrated plasmid vector, the integrated human DNA was amplified from the monoclonal clones and the amplificate subsequently sequenced by standard methods. The sequences thus determined were compared via BLAST analysis with known sequences in the gene bank. The analysis isolated a total of 44 different clones that were reactive with serum from MS patients. The antigens were assigned to different groups according to their properties (Figure 3).
  • the first validation of the identified autoantigens is carried out using RNA obtained from various tissues or from tissue-specific cell lines.
  • the brain-specific expression of the autoantigens associated with the neurological diseases has a crucial importance for the subsequent therapeutic application.
  • the isolation of total RNA from native tissue samples or from cell lines is accomplished by methods that are standard in molecular biology. For example, isolation using the RNeasy Maxi kit (Qiagen, cat. No. 75162) may be done as prescribed by the manufacturer. This isolation method relies on the use of guanidinium isothiocyanate as a chaotropic reagent. Alternatively, isolation with acidic phenol may be performed (Chomczynski & Sacchi, Anal Biochem 162: 156-159, 1987). After working up the tissue by means of guanidinium isothiocyanate, the RNA is extracted with acidic phenol, then the RNA is precipitated with isopropanol and taken up in DEPC-treated water.
  • RNA 2-4 ⁇ g of the thus isolated RNA are then added e.g. rewritten by means of Superscript II (Invitrogen) according to the manufacturer's protocol in cDNA.
  • the priming of the cDNA synthesis is carried out with the aid of random hexamers (for example Roche Diagnostics) according to standard protocols of the respective manufacturer.
  • the cDNAs are amplified with primers in 30 cycles that are specific for the low-level p53 gene. Only p53 positive cDNA samples are used for the further reaction steps.
  • the target candidates will be screened by PCR or quantitative PCR (qPCR) for expression in a large set of normal tissues.
  • a DNA polymerase eg 1 U HotStarTaq DNA polymerase, Qiagen
  • the amplification mixture contains 0.3 mM dNTPs, reaction buffer (final concentration 1 ⁇ , depending on the manufacturer of the DNA polymerase) and 0.3 mM each of the gene-specific "forward" and "reverse” primers.
  • the specific primers of the target gene are, where possible, selected so that they are located in two different exons and thus genomic contaminations do not lead to false positive results.
  • the cDNA is typically incubated at 95 ° C for 15 minutes to denature the DNA and to activate the hot-start enzyme. Subsequently, the DNA in 35 cycles amplified (1 min 95 ° C, 1 min primer specific hybridization temperature (about 55- 65 ° C), 1 min 72 ° C for elongation of the amplificates). 10 ⁇ l of the PCR mixture are then applied to agarose gels and separated in the electric field. By staining with ethidium bromide the DNA in the gels is visualized and the result of the PCR is documented by a photo.
  • the expression analysis of a target gene can also be carried out by quantitative real-time PCR.
  • Various analytical systems are now available for this analysis, the most well-known being the ABI PRISM Sequence Detection System (TaqMan, Applied Biosystems), the iCycler (Biorad) and the Light Cycler (Roche Diagnostics).
  • a specific PCR approach is run in the real time devices.
  • a DNA intercalating dye e.g., ethidium bromide, CybrGreen
  • the newly synthesized DNA is visualized by specific light excitation (as reported by the dye manufacturer).
  • the entire process can be followed and a quantitative determination of the nucleic acid concentration of the target gene can be carried out.
  • the normalization of the PCR approach is carried out by measuring a "housekeeping gene" (eg 18S RNA, ß-actin)
  • Alternative strategies via fluorescence-labeled DNA probes also allow the quantitative determination of the target gene from a specific tissue sample (see TaqMan applications of the company Applied Biosystems ).
  • the cloning of the entire target gene which is necessary for the further characterization of the antigen, is carried out according to common molecular biological methods (eg in "Current Protocols in Molecular Biology", John Wiley & Sons Ltd., Wiley InterScience) for cloning or sequence analysis of the target gene this is first amplified with a DNA polymerase with "proof reading function” (eg pfu, Roche Diagnostics). The amplificate is then ligated by standard methods into a cloning vector. Positive clones are identified by sequence analysis and then characterized using prediction programs and known algorithms.
  • the antigen is purified by commercially available methods.
  • chromatographic methods have been established (Biorad, Amersham Biosciences).
  • affinity chromatography is suitable for antigen purification.
  • protein anchors such as the "His-tag", the "FLAG tag” or glutathione-S-transferase (GST) can be used (Biorad, Amersham Biosciences, Qiagen).
  • GST glutathione-S-transferase
  • the cleaning then takes place via the specific properties of the anchor molecule.
  • the affinity chromatography can also be done by means of an antigen-specific antibody.
  • protocols are found in commercial suppliers or in the literature, e.g. in the "Current Protocols of Proteinsciences” (John Wiley & Sons Ltd., Wiley Inter Science).
  • antigens found are examined for the presence of specific immune responses (antibodies) in patients with MS, as well as in non-affected control groups. This allows the determination of the antigens that are clinically relevant.
  • detection or measuring methods such as proteomic analysis based protein arrays or mostly immunological analysis methods such as ELISA, CrELISA or Western blot be used.
  • Direct serological detection using the expression system used in immunoscreening is also possible.
  • the most widely used serological detection method is the "enzyme-linked immune sorbent assay” (ELISA), which is published in a variety of different forms, in which a protein (peptide, antigen or antibody) is bound to a surface and the sample to be analyzed becomes In the next step, incubation is usually followed by another antibody coupled with a dye (eg, FITC, Cy3) or an enzyme (eg, peroxidase, alkaline phosphatase), and then depending on the coupled substance antigen detection by, for example, a color reaction or by fluorescence ELISA for the detection of various antigens are commercially available (Amersham Bioscience, Biorad, etc.), detailed protocols are described, for example, in the “Short Protocols in Molecular Biology” (Asubel, 2003, Wiley & Sons.
  • E. coli bacteria are harvested in the exponential growth phase and subconfluently infected with antigen-specific monoclonal lambda phages.
  • TOP agar and IPTG the infection batch is plated on large-area agar plates with tetracycline. The individual clones are separated from each other with the help of spacers.
  • phage plaques formed on the bacterial lawn, each plaque representing a specific lambda phage clone with the inserted nucleic acid of the antigen to be analyzed being recombinantly expressed.
  • the antigens are blotted onto a nitrocellulose membrane as in the normal immunoscreening procedure and subsequently incubated with the human sera.
  • SEROGRID Method is the parallelized analysis of many identified antigens in a trial.
  • the antigen molecules are directed to specific positions in wells or on planar surfaces such as e.g. bound to filter membranes such as nitrocellulose or modified glass surfaces.
  • the binding of the antigens can be covalently by chemical linker or non-covalently via hydrophobic van der Waals, ionic or other interactions.
  • the directional binding of the antigens to the surface can be e.g. over one day (e.g., histidine tag).
  • the spotting of the protein arrays is usually done via pin-based systems that transfer solutions in the nanoliter range. Antigen detection is often based on fluorescence.
  • One application of protein arrays is the study of antigen-antibody interactions.
  • the protein array technology can also be used as a clinical diagnostic test.
  • Antibodies can be used, for example, to characterize the peptides and / or proteins of the invention and in the diagnostic and therapeutic methods of the invention. Antibodies may recognize proteins in native and / or denatured state (Anderson et al., J. Immunol., 143: 1899-1904, 1989; Gardsvoll, J. Immunol., Methods 234: 107-116, 2000; Kayyem et al. Eur. J. Biochem 208: 1-8, 1992; Spiller et al., J. Immunol. Methods 224: 51-60, 1999).
  • Antisera containing specific antibodies that specifically bind to the target protein can be prepared by a variety of standard methods; see. For example, “Monoclonal Antibodies: A Practical Approach” by Philip Shepherd, Christopher Dean ISBN 0-19-963722-9, “Antibodies: A Laboratory Manual” by Ed Harlow, David Lane ISBN: 0879693142 and "Using Antibodies: A Laboratory Manual: Portable It is also possible to generate affine and specific antibodies which recognize complex membrane proteins in their native form (Azorsa et al., J. Immunol., Methods 229: 35 Anderson et al., J. Immunol., 143: 1899-1904, 1989; Gardsvoll, J. Immunol., Methods: 234: 107-116, 2000). This is of particular importance for the production of antibodies , which are to be used therapeutically, but also for many diagnostic applications. This can be immunized with the entire protein as well as with extracellular partial sequences.
  • One species e.g., rabbits, mice
  • a second or third immunization within a defined period of time e.g., about 2-4 weeks after the last immunization
  • the animals are bled and immune serum recovered.
  • the immune sera thus obtained contain polyclonal antibodies with which the target protein can be detected and characterized in the Western blot, by flow cytometry, immunofluorescence or immunohistochemistry.
  • the animals are usually immunized by one of four well-established methods, although other methods exist. It can be immunized with specific for the target protein peptides, the entire protein, with extracellular partial sequences of a protein that can be identified experimentally or via prediction programs. Since the prediction programs will not always work error free u.U. also worked with two domains separated by a transmembrane domain. One of the two domains must then be extracellular, which can then be confirmed experimentally (see below).
  • peptides having a length of, for example, 8-12 amino acids
  • these peptides are used for immunization.
  • 3 immunizations are performed (e.g., at a concentration of 5-100 ⁇ g / immunization).
  • the implementation of the immunization can also be done as a service by service providers.
  • immunization may be by recombinant proteins.
  • the cloned DNA of the target gene is cloned into an expression vector and the target protein analogously to the conditions of the respective manufacturer (eg Roche Diagnostics, Invitrogen, Clontech, Qiagen) cell-free in vitro, in bacteria (eg E. coli), in yeast (eg pombe), in insect cells or in mammalian cells.
  • the synthesis of the Target protein using viral expression systems possible (eg baculovirus, vaccinia virus, adenovirus). After synthesis in one of the systems, the target protein is purified. The purification is usually carried out by chromatographic methods.
  • Proteins can also be used for immunization which have a molecular anchor as a cleaning aid (eg His-Tag, Qiagen, FLAG-Tag, Roche Diagnostics, GST fusion proteins).
  • a molecular anchor eg His-Tag, Qiagen, FLAG-Tag, Roche Diagnostics, GST fusion proteins.
  • a variety of protocols are found, for example, in "Current Protocols in Molecular Biology,” John Wiley & Sons Ltd, Wiley InterScience. After purification of the target protein, immunization is performed as described above.
  • this cell line can also be used directly for the production of the specific antiserum.
  • the immunization is carried out in 1-3 injections each with about 1-5 xl O 7 cells.
  • Immunization may also be by injection of DNA (DNA immunization).
  • the target gene is first cloned into an expression vector such that the target sequence is under the control of a strong eukaryotic promoter (e.g., CMV promoter).
  • DNA e.g 1-10 ⁇ g per injection
  • a "gene gun” into strongly perfused, capillary areas of an organism (eg mouse, rabbit)
  • the transferred DNA is taken up by cells of the animal, the target gene is expressed and the animal eventually develops an immune response against the target protein (Jung et al., Mol. Cells 12: 41-49, 2001; Kasinrerk et al., Hybrid Hybridomics 21: 287-293, 2002).
  • Monoclonal antibodies are traditionally made using Hybridoma technology (For technical details, see “Monoclonal Antibodies: A Practical Approach” by Philip Shepherd, Christopher Dean ISBN 0-19-963722-9; “Antibodies: A Laboratory Manual” by Ed Harlow, David Lane ISBN: 0879693142, “Using Antibodies: A Laboratory Manual: Portable Protocol NO,” by Edward Harlow, David Lane, Ed Harlow, ISBN: 0879695447), as well as a so-called “SLAM” technology. B cells are isolated from whole blood and the cells are monoclonalized. Subsequently, the supernatant of the isolated B-cell on their Antibody specificity analyzed.
  • variable region of the antibody gene is subsequently amplified by a single-cell PCR and cloned into a suitable vector. In this way, the recovery of monoclonal antibodies is accelerated (de Wildt et al., J. Immunol., Methods 207: 61-67, 1997).
  • Example 5 Construction of a test system for the diagnosis of autoimmune diseases
  • the identified autoantigens form the basis of a diagnostic system that is specific for the diagnosis of autoimmune diseases and / or specific for the diagnosis or prognosis of multiple sclerosis.
  • the diagnosis involves detecting the presence or quantification of one or more human autoantibodies that are specific for an epitope or specific for multiple epitopes of the identified autoantigens.
  • the presence or increased concentration of one or more of these autoantibodies indicates a particular stage of MS disease or a possible more aggressive stage of the disease.
  • a test system for diagnosis can hereby be based on the use of or on the use of a combination of the identified autoantigens.
  • the test system for diagnosis is based on the use of the antigens or antibodies e.g. in immunoassays, e.g. ELISA assays or protein arrays (see Example 3).
  • the detection involves the removal of a biological sample such as e.g. Serum or CSF from MS patients.
  • Calsyntenin 1 or CLSTN1 is a gene located on chromosome 1 (Ip36.22).
  • the gene encodes a type I transmembrane protein of about 110 kDa in size (SEQ ID NO: 2).
  • the protein contains two cadherin domains and, according to homology analyzes, could be a calcium-dependent neurotransmitter.
  • CLSTN1 could be identified as an autoantigen in the autoimmune screen.
  • tissue-specific expression of CLSTN1 after the establishment of a gene-specific quantitative RT-PCR (primer pair SEQ ID NO: 89 and 90), the amount of specific transcripts in different brain regions and in other healthy tissues was analyzed.
  • CLSTNl is present in all brain regions studied Compared to the other examined tissues clearly overexpressed (fig. 4) and is therefore to be regarded as brain-specific. Expression in the tissues analyzed may be due to expression in peripheral nerve tissue.
  • the "cyclic AMP phosphoprotein 19" or ARPP-19 is a gene located on chromosome 15 (15q21) The gene encodes a probable cytoplasmic localized protein of about 12 kDa in size (SEQ ID NO: 4) Homology analyzes indicate that ARPP-19 is a phosphoprotein and belongs to the endosulphic family, suggesting that APPP-19 might be a substrate for a cAMP-dependent kinase.
  • ARPP-19 could be identified as an autoantigen in the autoimmune screen.
  • tissue-specific expression of ARPP-19 after the establishment of a gene-specific quantitative RT-PCR (primer pair SEQ ID NO: 91 and 92), the amount of specific transcripts in different brain regions and in other healthy tissues was analyzed.
  • ARPP-19 is at least 10-fold overexpressed in all examined brain regions compared to the other examined tissues (Fig. 5) and is therefore to be regarded as brain-specific. Expression in the tissues analyzed may be due to expression in peripheral nerve tissue.
  • CMTM2 (SEQ ID NO: 5) is a gene located on chromosome 16 (16q22.1). The gene encodes an integral membrane protein of about 27 kDa size (SEQ ID NO: 6). The protein belongs to the family of "chemokine-like factors" and also has significant homology to the family of signaling molecules with four transmembrane domains.
  • CMTM2 could therefore be an important molecule in cellular signaling.
  • CMTM2 could be identified as an autoantigen in the autoimmune screen
  • tissue-specific expression of CMTM2 the amount of specific transcripts in different brain regions and other healthy tissues was analyzed after the establishment of a gene-specific quantitative RT-PCR (primer pair SEQ ID NO: 93 and 94.)
  • CMTM2 is highly overexpressed in the immune-privileged testis (Fig In the other tissues examined, CMTM2 was expressed very selectively, especially in the various brain regions, so that the autoimmune response is to be regarded as brain-specific.
  • Example 9 Identification of CPE as autoantigen
  • Carboxypeptidase E or CPE is a gene located on chromosome 4 (4q32). The gene encodes a soluble protein of about 53 kDa (SEQ ID NO: 8) that is cytoplasmically located.
  • CPE is a carboxypeptidase that activates prohormones and neurotransmitters through their enzymatic function and is thus involved in the biosynthesis of these biological regulators.
  • CPE could be identified as an autoantigen in the autoimmune screen.
  • tissue-specific expression of CPE after the establishment of a gene-specific quantitative RT-PCR (primer pair SEQ ID NO: 95 and 96), the amount of specific transcripts in different brain regions and in other healthy tissues was analyzed. Quantitative RT-PCR revealed at least 5-fold overexpression in the brain compared to all other tissues examined ( Figure 7).
  • LPS-induced TNF-alpha factor or LITAF (SEQ ID NO: 9) is a gene located on chromosome 16 (16p13) The gene encodes a soluble protein of approximately 24 kDa (SEQ ID NO: 10), which is located nuclear LITAF plays an important role in the transcriptional regulation of the cytokine TNF-alpha and is associated with the neurological disorder "Charcot-Marie-Tooth” (Street, 2003. Neurology 60: 22- 26).
  • LITAF could be identified as an autoantigen in the autoimmune screen.
  • tissue-specific expression of LITAF after the establishment of a gene-specific quantitative RT-PCR (primer pair SEQ ID NO: 97 and 98), the amount of specific transcripts in different brain regions and in other healthy tissues was analyzed. An at least 2- to 5-fold overexpression in the brain could be detected compared to all other examined tissues (Fig. 8).
  • Tubulin gamma 1 or TUBGl (SEQ ID NO: 11) is a gene located on chromosome 17 (16q21). The gene encodes a soluble protein about 51 kDa in size (SEQ ID NO: 12) that is nuclear located.
  • TUBGl is a member of the tubulin Family and component of microtubules in the nucleus. The protein plays an important role in the regulation of nuclear division.
  • TUBG1 could be identified as an autoantigen in the autoimmune screen.
  • tissue-specific expression of TUBGl after the establishment of a gene-specific quantitative RT-PCR (primer pair SEQ ID NO: 99 and 100), the amount of specific transcripts in different brain regions and in other healthy tissues was analyzed. At least 2 to 5-fold overexpression in the brain could be demonstrated compared to all other tissues examined ( Figure 9).
  • Nucleosome assembly protein 1-like 3 or NAP1L3 is a gene located on chromosome X (Xq21-22) The gene encodes a soluble protein of about 58 kDa in size (SEQ ID NO: 14), which is nuclear located NAP 1L3 is a member of the "nucleosome assembly” family and plays an important role in the regulation of the nucleus.
  • Enolase 2 or ENO2 is a gene located on chromosome 12 (12ql3).
  • the gene encodes a soluble protein about 47 kDa in size (SEQ ID NO: 16) which is located cytoplasmic.
  • the ENO2 gene encodes an enzyme of the glycolytic pathway that is expressed primarily in neurons and cells of neuronal origin.
  • Example 14 Identification of autoantigens already associated with other autoimmune diseases
  • the gene SDCCAG8 (SEQ ID NO: 17) is located on chromosome 1 (lq43-44).
  • HSP90B1 (SEQ ID NO: 19) is located on chromosome 12 (12q24) and encodes a protein of about 92 kDa (SEQ ID NO: 20).
  • the gene belongs to the family of chaperones that play an important role in the genesis and transport of secreted proteins in the lumen of the ER. HSP90B1 is upregulated in myeloma.
  • the gene SAT (SEQ ID NO: 21) is located on the X chromosome (Xp22) and encodes an approximately 20 kDa protein (SEQ ID NO: 22).
  • the soluble enzyme is present in the cytoplasm as a homotetramer and performs a catalytic function in the regulation of polyamines.
  • EXOSC5 (SEQ ID NO: 23) is located on chromosome 19 (19ql3) and encodes a nuclear, approximately 25 kDa protein (SEQ ID NO: 24).
  • the enzyme has exonuclease activity and is part of the nuclear exosome. In patients with myopathies and skin diseases EX0SC5-specific autoantibodies could be detected.
  • Chromosome 20 Sequence SEQ ID NO: 25, 26; CEP63: SEQ ID NO: 27, 28; LOCl 15648: SEQ ID NO: 29, 30; Chromosome 18 sequence: SEQ ID NO: 31, 32; Chromosome 14 Sequence 1: SEQ ID NO: 33, 34; Chromosome 14 Sequence 2: SEQ ID NO: 35, 36; IQWD1: SEQ ID NO: 37, 38; C60ORF199: SEQ ID NO: 39, 40; Chromosome 22 sequence: SEQ ID NO: 41, 42; LOC400843: SEQ ID NO: 43, 44.
  • the gene product of SEQ ID NO: 28 is a soluble centrosomal localized protein of unknown function.
  • IQWD1 encodes a nuclear protein of approximately 96 kDa (SEQ ID NO: 38) with multiple WD40 domains and a nuclear translocation sequence. WD40 domains probably have a role in protein-protein interactions.
  • the gene with the SEQ ID NO: 39 encodes a protein with a size of about 48 kDa, which may be located mitochondrially and has an adenylate kinase function.
  • Example 16 Identification of other human autoantigens
  • Interferon regulatory factor 2 binding protein 2 or IRF2BP2 (SEQ ID NO: 45) is a
  • the gene located on chromosome 1 (Ip42).
  • the gene encodes a soluble protein of about 61 kDa in size (SEQ ID NO: 46), which is likely to be nuclear located.
  • IRF2BP2 The exact function of IRF2BP2 is not yet known.
  • the protein binds to the
  • the "sterol regulatory element binding factor 1" or SREBF1 (SEQ ID NO: 47) is a
  • SREBF1 Size of about 122 kDa (SEQ ID NO: 48).
  • SREBF1 has a transmembrane domain and plays a role in transcriptional regulation and in the delivery of sterol.
  • SREBFl In the non-activated state SREBFl is located in the ER, after activation there is a
  • Exportin4 or XPO4 is a gene located on chromosome 1 (13ql 1). The gene encodes a soluble protein with a size of about 130 kDa (SEQ ID NO: 49).
  • XPO4 binds to the elongation factor eIF-5A and mediates the transport of the elongation factor from the nucleus into the cytoplasm.
  • the "zinc finger protein 64" or ZFP64 (SEQ ID NO: 51) is a gene based on
  • Chromosome 1 (2Oq 13) is located.
  • the gene encodes a soluble protein about 75 kDa in size (SEQ ID NO: 52) that is nuclear located.
  • ZFP64 probably binds to DNA and has the function of a transcription factor.
  • the "formin binding binding protein 1" or FNBPl (SEQ ID NO: 53) is a gene based on
  • Chromosome 9 (9q34) is located.
  • the gene encodes a soluble protein of about 70 kDa in size (SEQ ID NO: 54), which is likely to be located cytoplasmic.
  • Protein is assigned a function in cellular growth regulation.
  • CCL4 (SEQ ID NO: 55) is a gene located on chromosome 17 (17q24).
  • Gene encodes a soluble protein about 10.5 kDa in size (SEQ ID NO: 56) that is secreted.
  • the protein binds to cytokine receptors and belongs to the family of
  • COPA (SEQ ID NO: 57) is a gene located on chromosome 1 (lq23-25). The
  • Gene encodes a soluble protein of about 138 kDa in size (SEQ ID NO: 58) is cytoplasmic. The protein is involved in the regulation of secretory
  • GHITM (SEQ ID NO: 59) is a gene located on chromosome 10 (10q23.1).
  • the gene encodes an integral membrane protein with a size of about 34 kDa (SEQ ID NO: 1]
  • NGLY1 (SEQ ID NO: 61) is a gene located on chromosome 3 (3q24.2). The
  • Gene encodes an integral membrane protein with a size of about 55 kDa (SEQ ID NO:
  • the protein is assigned a function in the degradation of misfolded proteins.
  • KTN1 (SEQ ID NO: 63) is a gene located on chromosome 14 (14q22.1). The
  • Gene encodes a membrane protein of about 156 kDa size (SEQ ID NO: 64).
  • Protein becomes a kinesin receptor and thus kinesin-driven
  • SFRSI1 (SEQ ID NO: 65) is a gene located on chromosome 1 (Iq31).
  • Gene encodes a soluble protein of about 54 kDa in size (SEQ ID NO: 66) that is likely to be nuclear located.
  • the protein becomes a function in the pre-mRNA
  • NME1-NME2 (SEQ ID NO: 67) is a gene located on chromosome 17 (17q21.3). The gene encodes a soluble protein with a size of about 17 kDa (SEQ ID NO:
  • the protein has as
  • Nucleoside diphosphate kinase has a function in the synthesis of non-ATP
  • RPS15 (SEQ ID NO: 69) is a gene located on chromosome 19 (19ql3.3). The
  • Gene encodes a soluble protein of about 17 kDa size (SEQ ID NO: 70), which is likely to be located cytoplasmic.
  • RPS 15 is a member of the "S19P" family of ribosomal proteins and plays a role in protein synthesis.
  • APC2 (SEQ ID NO: 71) is a gene located on chromosome 19 (19ql3.3). The
  • Gene encodes a soluble protein about 245 kDa in size (SEQ ID NO: 72) that is cytoplasmic, possibly co-localized with tubular structures or the Golgi protein.
  • the protein is assigned a function as a tumor suppressor.
  • GLS2 (SEQ ID NO: 73) is a gene located on chromosome 12 (12ql3).
  • Gene encodes a soluble protein of about 66 kDa in size (SEQ ID NO: 74) mitochondrial is localized.
  • the protein has an enzymatic function as
  • Glutaminase associated with the hydrolysis of glutamine.
  • TECAL8 (SEQ ID NO: 75) is a gene located on chromosome X (Xq22.1). The
  • Gene encodes a soluble protein of about 14 kDa in size (SEQ ID NO: 76).
  • the protein is assigned a function as a transcription elongation factor.
  • PPIF (SEQ ID NO: 77) is a gene located on chromosome 10 (10q22-q23). The
  • Gene encodes a protein of about 22 kDa (SEQ ID NO: 78) that is located mitochondrially.
  • the protein becomes an enzymatic function in the
  • Example 18 Serological analysis of selected autoimmune antigens

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  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Zoology (AREA)
  • Hospice & Palliative Care (AREA)
  • Psychiatry (AREA)
  • Peptides Or Proteins (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

Selon l'invention des autoanticorps, dont l'apparence est caractéristique de maladies autoimmunes neurologiques, en particulier la sclérose en plaques, sont détectés et les autoantigènes correspondants sont identifiés. De plus, plusieurs autoantigènes peuvent être exprimés de manière spécifique dans le cerveau. L'identification desdits autoantigènes et des autoanticorps est utilisée dans le diagnostic et le traitement. Une expression spécifique au cerveau des autoantigènes souligne de plus le rôle important des antigènes et des anticorps dans l'origine et le développement de maladies autoimmunes neurologiques.
EP07818849A 2006-10-11 2007-10-09 Autoantigènes utilisés dans le diagnosic, le pronostic et le traitement améliorés de maladies neurologiques inflammatoires Withdrawn EP2076776A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE102006048201A DE102006048201A1 (de) 2006-10-11 2006-10-11 Autoantigene zur verbesserten Diagnose, Prognose und Therapie von entzündlichen neurologischen Erkrankungen
PCT/EP2007/008776 WO2008043524A2 (fr) 2006-10-11 2007-10-09 Autoantigènes utilisés dans le diagnosic, le pronostic et le traitement améliorés de maladies neurologiques inflammatoires

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EP (1) EP2076776A2 (fr)
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EP2504024A2 (fr) * 2009-09-27 2012-10-03 Ruhr-Universität Bochum Méthode de thérapie et de diagnostic de morbus alzheimer
US9585380B2 (en) 2011-09-02 2017-03-07 Temple University—Of the Commonwealth System of Higher Education SSAT mRNA translation repression and activation
EP3938787B1 (fr) 2019-03-15 2025-12-31 CZ Biohub SF, LLC Auto-anticorps utilisés comme biomarqueur de l'encéphalite paranéoplasique associée au cancer testiculaire
CN114878828B (zh) * 2021-12-24 2023-03-10 天津天海新域生物科技有限公司 用于检测自身免疫性脑炎的抗体组合物及其应用

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WO2007007129A2 (fr) * 2005-07-14 2007-01-18 Universite De Geneve Methode servant a diagnostiquer des troubles associes a la deterioration cerebrale

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CA2422229A1 (fr) * 2000-09-14 2002-03-21 Universitat Zurich Proteines de liaison du calcium
US20050130245A1 (en) * 2001-09-17 2005-06-16 Syn X Pharma, Inc. Diagnosis and treatment of early pre-type-1 diabetes utilizing neuronal proteins
US20030092089A1 (en) * 2001-11-14 2003-05-15 Moscarello Mario Anthony Method for diagnosing multiple sclerosis and an assay therefore
WO2004034031A2 (fr) * 2002-10-11 2004-04-22 The Regents Of The University Of California Methode permettant de diagnostiquer et de pronostiquer la sclerose en plaques
US20050142616A1 (en) * 2003-09-30 2005-06-30 Veneta Hanson Diagnostic test for neuropsychiatric systemic lupus erythematosus
ATE551357T1 (de) * 2003-11-05 2012-04-15 Immuno Biological Lab Co Ltd Markerpeptid für die alzheimer-krankheit
US20100151483A1 (en) * 2006-07-13 2010-06-17 Cell Signaling Technology, Inc. Reagents for the detection of protein phosphorylation in signaling pathways

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WO2007007129A2 (fr) * 2005-07-14 2007-01-18 Universite De Geneve Methode servant a diagnostiquer des troubles associes a la deterioration cerebrale

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US20090252714A1 (en) 2009-10-08
WO2008043524A2 (fr) 2008-04-17
US8399210B2 (en) 2013-03-19
WO2008043524A3 (fr) 2008-09-25
DE102006048201A1 (de) 2008-04-17
WO2008043524A8 (fr) 2008-07-31

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