WO2004058972A1 - Auto-antigènes humains et leur utilisation - Google Patents

Auto-antigènes humains et leur utilisation Download PDF

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Publication number
WO2004058972A1
WO2004058972A1 PCT/EP2002/014731 EP0214731W WO2004058972A1 WO 2004058972 A1 WO2004058972 A1 WO 2004058972A1 EP 0214731 W EP0214731 W EP 0214731W WO 2004058972 A1 WO2004058972 A1 WO 2004058972A1
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WO
WIPO (PCT)
Prior art keywords
exp
seq
autoantibodies
expression product
spp
Prior art date
Application number
PCT/EP2002/014731
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German (de)
English (en)
Inventor
Hans-Jürgen Thiesen
Peter Lorenz
Original Assignee
Thiesen Hans-Juergen
Peter Lorenz
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 Thiesen Hans-Juergen, Peter Lorenz filed Critical Thiesen Hans-Juergen
Priority to EP02795271A priority Critical patent/EP1578970A1/fr
Priority to PCT/EP2002/014731 priority patent/WO2004058972A1/fr
Priority to AU2002360082A priority patent/AU2002360082A1/en
Publication of WO2004058972A1 publication Critical patent/WO2004058972A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4713—Autoimmune diseases, e.g. Insulin-dependent diabetes mellitus, multiple sclerosis, rheumathoid arthritis, systemic lupus erythematosus; Autoantigens

Definitions

  • the invention relates to human autoantigens as defined in claim 1 and their use.
  • Autoimmune diseases have a high prevalence in the population. Their treatment is thus very economically relevant. The causes of autoimmunity are unclear. To understand the individual diseases, in particular their aetiology, the knowledge of the autoantigens involved in the disease is important.
  • the detection of autoantigens is used in autoimmune diagnostics.
  • Test cells and test tissues as a basis for diagnostics.
  • the definition of new autoantigens therefore provides the opportunity to gain new insights into the aetiology and pathology of autoimmune diseases and opens up new approaches and test capabilities for autoimmune diagnostics.
  • the object of the invention is therefore the provision of such new autoantigens.
  • this object is achieved by providing the human autoantigens defined in claim 1.
  • An autoantigen in the sense of the invention is understood as meaning an antigen which reacts with the serum of a patient with a diagnosed known autoimmune disease or with diagnosed autoantibody titers.
  • autoantibodies examples include: anti-mitochondrial autoantibodies (AMA), liver-kidney microsomal (LKM) autoantibodies, anti-histidyl-tRNA synthetase autoantibodies (Jo-1), anti-nuclear membrane autoantibodies, anti-nuclear Neutrophil cytoplasmic autoantibodies (ANCA), islet cell autoantibodies (ICA), epidermal intracellular antigen autoantibodies (EICA), epidermal basement membrane autoantibodies (EBMA), anti-Golgi autoantibodies, anti-cell nuclei Autoantibodies (ANA), multiple sclerosis (MS) autoantibodies and rheumatoid arthritis (RA) autoantibodies.
  • AMA anti-mitochondrial autoantibodies
  • LLM liver-kidney microsomal
  • Jo-1 anti-histidyl-tRNA synthetase autoantibodies
  • ANCA islet cell autoantibodies
  • DNA sequences of the human autoantigens of the invention as defined in claim 1 (a) also include their complementary strands,
  • polymorphisms single-nucleotide poly orphisms (SNPs) and duplications are calculated for the allelic variants and mutants.
  • DNA sequences according to the invention are for example 95%, 90%, 85% or 80% sequence homologous to the DNA sequences defined in claim 1.
  • autoantigen DNA sequences which contain a DNA sequence according to the invention as a partial sequence are also suitable, for example at the 5'-terminus and / or or at the 3'-terminus of other nucleotides. Also suitable are autoantigen DNA sequences which are composed of a plurality of identical or different DNA sequences according to the invention, which of course may be bound to one another both directly and via spacers or linkers.
  • a protein expression library from human fetal brain in E. coli bacteria was used by Dr. med. Konrad Büssow, Max Planck Institute for Molecular Genetics in Berlin, and is available for example from the RZPD German Resource Center for Genome Research GmbH in Berlin.
  • the expression library / protein expression filter (s) used for the invention carry about 38,000 expression clones from a fetal brain cDNA library.
  • all expressed genes of the tissue are represented in the library and thus on the filter.
  • the genes are expressed in whole or in part as a fusion product of a small histidine epitope tag of E. coli bacteria.
  • the whole of the expressed proteins of fetal brain tissue is available for screening experiments such as the search for new autoantigens.
  • Each bacterial clone carries an expression clone from the library.
  • the library is redundant, i. the information for a particular protein may be present in more than one clone.
  • the number of identical clone identities also reflects the strength of the expression of a gene: the more RNA molecules, the more cDNA molecules are formed and the more clones are thus represented in the library.
  • An expression clone can in principle code for the information of entire genes, but usually a clone will contain only parts of genes. In addition, it is also clones with Give nonsense information (wrong reading frame). A amino-negative of each clone was applied a small epitope tag (His tag) using the appropriate vector so that all clones could be tested for expression with a commercially available antibody (anti-His tag). Such expression-positive clones of the library, totaling 37830 pieces, were selected and formed the starting point for the resource center protein expression filters we use.
  • the starting point for our work were the protein expression filters described above (1 set with a total of 37820 clones contains 2 filters) of the resource center and patient serums that were present in the medical faculty of the University of Rostock, in particular the Institute of Immunology. These were sera (or cerebrospinal fluid or synovial fluid) derived from patients with known autoimmune diseases or diagnosed autoantibody titers. Serum samples from 3-10 patients for each disease / diagnostic group were pooled and immunoglobulin G (G-class antibodies) isolated. Immunostaining of the protein expression filters was performed on these isolated antibody mixtures. Positive signals indicated responses of antibodies to clonal proteins. RESULTS
  • clone ID A clone ID is indicated by MPMGp ⁇ 00X12345 (p800 refers to the expression library, X stands for any letter and 12345 a combination of 5 numbers). Each clone is uniquely identified and physically stored under this name in frozen form in the Resource Center. However, the gene or gene fragment expressed in these clones is not usually known.
  • the 270 different clones were subjected to DNA sequencing and identified by comparison with databases and used to prepare an "autoantigen filter” for further screening and validation.
  • Jo-1 anti-histidyl tRNA synthetase autoantibodies
  • ANCA anti-neutrophil cytoplasmic autoantibodies
  • EICA Epidermal intracellular antigen autoantibody
  • EBMA epidermal basement membrane autoantibodies
  • DNA sequences according to the invention can be detected by conventional molecular biological methods (cf., for example, J. Sa brook, EF Fritsch, T. Maniatis, "Molecular Cloning, A Laboratory Manual", Cold Spring Harbor Laboratory Press, New York, 1989) insert optimized expression vectors, whereby the appropriate transformation / transfection, selection and cloning of prokaryotic or eukaryotic cells is possible, which are then able to synthesize the protein or polypeptide by fermentation in a position. If an overproducing cell clone is chosen, the desired protein or polypeptide can be readily produced and recovered in large quantities.
  • RNA interference which is induced by so-called small-interfering RNA (siRNA).
  • siRNAs can be easily produced with the aid of the DNA sequences according to the invention.
  • all methods known in the art are for modulation the expression applicable, for example, such as using anti-sense RNA or so-called designer zinc finger molecules. Not only the expression of the autoantigens but also the expression of the corresponding autoantibodies can be modulated in this way.
  • DNA sequences according to the invention or the amino acid sequences forming the proteins or polypeptides according to the invention can also be prepared synthetically by methods known per se, for example with commercial DNA or peptide synthesizers.
  • the identification of the expression products can be carried out using any biochemical separation and detection methods, for example by electrophoretic separation of the resulting by cell lysis protein mixture followed by Western blotting and immunodetection.
  • Electrophoretic separation is not particularly limited and may be accomplished, for example, by SDS gel electrophoresis by size or molecular weight or by isoelectric focusing or, to improve the resolution, by a combination of both techniques, i. so-called 2D electrophoresis.
  • SDS gel electrophoresis by size or molecular weight or by isoelectric focusing or, to improve the resolution, by a combination of both techniques, i. so-called 2D electrophoresis.
  • These electrophoretic separation techniques have been well established in bioanalytics for years (see, e.g., Lottspeich / Zorbas, supra) and therefore need no further discussion.
  • Subsequent Western blotting i. transferring the separated proteins to a carrier (e.g., nitrocellulose) for immobilization is also not particularly limited and may be e.g. by capillary blotting or electroblotting. These blotting techniques have been well established in bioanalytics for years (see, e.g., Lottspeich / Zorbas, supra) and, therefore, need no further discussion.
  • a carrier e.g., nitrocellulose
  • Immune detection i. the visualization of the antibody binding can be directly labeled using appropriately labeled
  • immobilization of the DNA sequences of claim 1 or the expression products or partial expression products of claim 6 or the synthetic proteins or polypeptides of claim 7 or the polyclonal or monoclonal antibodies of claim 9, 11 or 12 is desired.
  • suitable substrates or carriers as well as suitable cross-linkers, which may also be bifunctional, there are no particular restrictions. In principle, for example, the ImmobilmaschinesSysteme described in the book by Greg T. Hermanson, “Bioconjugate Techniques", Academic Press 1996, are suitable.
  • the immobilization may, but need not, be covalent.
  • autoantibody apheresis (apheresis is generally understood to be a type of blood wash for the selective removal of blood constituents) is the invention. according to autoantigens to beads (beads) are immobilized.
  • the gene segment expressed as a protein on the expression filter contains the antibody binding sites, ie epitopes, of the autoantigen.
  • the fine analysis of linear epitopes can then be carried out via short, derived from the clone sequence, synthetic peptides. Epitope mapping is important because autoantibodies have described immunodominant epitopes of autoantigens. Knowledge of autoepitope is important for the understanding of autoimmunity.
  • RNA interference is a novel method to suppress gene expression specifically at the mRNA level.
  • RNA interference can be achieved for any gene, provided its mRNA sequence is known.
  • Applications that modulate the expression of autoantigens or autoantibodies via RNAi are possible.
  • the autoantigen DNA sequences according to the invention can be used for the production of siRNA (small interfering RNA) with which such RNA interference can be induced.
  • siRNA small interfering RNA
  • This siRNA can be used to treat or prevent autoimmune diseases through RNA interference.

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Rheumatology (AREA)
  • Biochemistry (AREA)
  • Immunology (AREA)
  • Toxicology (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Hematology (AREA)
  • Rehabilitation Therapy (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Diabetes (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

L'invention concerne des auto-antigènes humains et leur utilisation, par exemple, pour déterminer la présence d'auto-anticorps, pour cartographier les épitopes d'auto-anticorps, pour réaliser l'aphérèse d'auto-anticorps, la chromatographie d'affinités ou l'électrophorèse d'affinités, pour créer une interférence ARN, par ex. par des siARN ainsi produits, ou bien pour diagnostiquer une maladie auto-immune.
PCT/EP2002/014731 2002-12-23 2002-12-23 Auto-antigènes humains et leur utilisation WO2004058972A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
EP02795271A EP1578970A1 (fr) 2002-12-23 2002-12-23 Auto-antigenes humains et leur utilisation
PCT/EP2002/014731 WO2004058972A1 (fr) 2002-12-23 2002-12-23 Auto-antigènes humains et leur utilisation
AU2002360082A AU2002360082A1 (en) 2002-12-23 2002-12-23 Human autoantigens and use thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/EP2002/014731 WO2004058972A1 (fr) 2002-12-23 2002-12-23 Auto-antigènes humains et leur utilisation

Publications (1)

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WO2004058972A1 true WO2004058972A1 (fr) 2004-07-15

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EP (1) EP1578970A1 (fr)
AU (1) AU2002360082A1 (fr)
WO (1) WO2004058972A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1859266A2 (fr) * 2005-02-24 2007-11-28 CeMines, Inc. Compositions et procedes de classification d'echantillons biologiques

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1225222A1 (fr) * 1999-10-29 2002-07-24 Mochida Pharmaceutical Co., Ltd. Nouveau gene suppresseur des tumeurs

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1225222A1 (fr) * 1999-10-29 2002-07-24 Mochida Pharmaceutical Co., Ltd. Nouveau gene suppresseur des tumeurs

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
BÜSSOW ET AL: "A METHOD FOR GLOBAL PROTEIN EXPRESSION AND ANTIBODY SCREENING ON HIGH-DENSITY FILTERS OF AN ARRAYED cDNA LIBRARY", NUCLEIC ACIDS RESEARCH, OXFORD UNIVERSITY PRESS, SURREY, GB, vol. 26, no. 21, November 1998 (1998-11-01), pages 5007 - 5008, XP002114084, ISSN: 0305-1048 *
COPE A P ET AL: "EVALUATING CANDIDATE AUTOANTIGENS IN RHEUMATOID ARTHRITIS", SPRINGER SEMINARS IN IMMUNOPATHOLOGY, SPRINGER, BERLIN, DE, vol. 20, no. 1-2, 1998, pages 23 - 39, XP001031200, ISSN: 0172-6641 *
DATABASE EMBL [online] 3 December 2000 (2000-12-03), SATTERWHITE E. ET AL.: "Homo sapiens mRNA for B-cell lymphoma/leukaemia 11B (BCL11B gene)", XP002244632, Database accession no. AJ404614 *
MOCCI S ET AL: "The role of autoantigens in autoimmune disease", CURRENT OPINION IN IMMUNOLOGY, CURRENT BIOLOGY LTD, XX, vol. 12, no. 6, 1 December 2000 (2000-12-01), pages 725 - 730, XP004257749, ISSN: 0952-7915 *
ROBINSON WILLIAM H ET AL: "Autoantigen microarrays for multiplex characterization of autoantibody responses.", NATURE MEDICINE. UNITED STATES MAR 2002, vol. 8, no. 3, March 2002 (2002-03-01), pages 295 - 301, XP002244630, ISSN: 1078-8956 *
SATTERWHITE ED ET AL.: "The BCL11 gene family: Involvement of BCL11A in lymphoid malignancies.", BLOOD, vol. 98, no. 12, 1 December 2001 (2001-12-01), pages 3413 - 3420, XP002244631 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1859266A2 (fr) * 2005-02-24 2007-11-28 CeMines, Inc. Compositions et procedes de classification d'echantillons biologiques
EP1859266A4 (fr) * 2005-02-24 2010-07-28 Cemines Inc Compositions et procedes de classification d'echantillons biologiques

Also Published As

Publication number Publication date
AU2002360082A1 (en) 2004-07-22
EP1578970A1 (fr) 2005-09-28

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