EP1578970A1 - Auto-antigenes humains et leur utilisation - Google Patents

Auto-antigenes humains et leur utilisation

Info

Publication number
EP1578970A1
EP1578970A1 EP02795271A EP02795271A EP1578970A1 EP 1578970 A1 EP1578970 A1 EP 1578970A1 EP 02795271 A EP02795271 A EP 02795271A EP 02795271 A EP02795271 A EP 02795271A EP 1578970 A1 EP1578970 A1 EP 1578970A1
Authority
EP
European Patent Office
Prior art keywords
exp
seq
autoantibodies
expression product
spp
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Withdrawn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
EP02795271A
Other languages
German (de)
English (en)
Inventor
Hans-Jürgen Thiesen
Peter Lorenz
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.)
Individual
Original Assignee
Individual
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 Individual filed Critical Individual
Publication of EP1578970A1 publication Critical patent/EP1578970A1/fr
Withdrawn legal-status Critical Current

Links

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.

Landscapes

  • 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.
EP02795271A 2002-12-23 2002-12-23 Auto-antigenes humains et leur utilisation Withdrawn EP1578970A1 (fr)

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)

Publication Number Publication Date
EP1578970A1 true EP1578970A1 (fr) 2005-09-28

Family

ID=32668685

Family Applications (1)

Application Number Title Priority Date Filing Date
EP02795271A Withdrawn EP1578970A1 (fr) 2002-12-23 2002-12-23 Auto-antigenes humains et leur utilisation

Country Status (3)

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

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2598889A1 (fr) * 2005-02-24 2006-08-31 Cemines, Inc. Compositions et procedes de classification d'echantillons biologiques

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1225222A4 (fr) * 1999-10-29 2003-07-30 Mochida Pharm Co Ltd Nouveau gene suppresseur des tumeurs

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO2004058972A1 *

Also Published As

Publication number Publication date
AU2002360082A1 (en) 2004-07-22
WO2004058972A1 (fr) 2004-07-15

Similar Documents

Publication Publication Date Title
DE69938623T2 (de) Microarrays und ihre verwendungen
Desai et al. A Drosophila receptor tyrosine phosphatase expressed in the embryonic CNS and larval optic lobes is a member of the set of proteins bearing the" HRP" carbohydrate epitope
DE69839123T2 (de) Prostattumor polynukleotid- und antigenzusammensetzungen
DE69302234T2 (de) Menschliches Prohibitin und Prohibitin kodierende DNS
EP1483389B1 (fr) Produits geniques d'expression differenciee dans les tumeurs et leur utilisation
DE60109430T2 (de) An entzündlichen darmerkrankungen beteiligte gene und deren verwendung
DE19957065B4 (de) Screening-Verfahren für Arzneistoffe
KR20200097636A (ko) 퇴행성 뇌질환 진단용 바이오마커 조성물
DE68926658T2 (de) Diagnostische gene zum nachweis von toxoplasmose
CN101099084A (zh) 作为胰岛素耐受性的靶标/标记的cd99
DE102018004759A1 (de) Diagnose einer Neuroautoimmunkrankheit
DE69725018T2 (de) Gereinigte telomerase
CN101310183A (zh) 用于脊髓损伤中抗Nogo-A抗体治疗的生物标记
DE10333406A1 (de) T-regulatorische-Zellen enthaltend Galectine zur Therapie und Diagnose von Erkrankungen
DE60113968T2 (de) Diagnose von brustkrebs unter verwendung von bcmp-7 als marker
Junaid et al. Proteomic approach for the elucidation of biological defects in autism
DE60216048T2 (de) Screeningverfahren für Peptide, die die Bindung von PP1c an Bcl-2, BCL-Xl und BCL-W Proteine inhibieren
EP0815141B1 (fr) Anticorps agissant a l'encontre d'un polypeptide fusionne comportant une partie histidine
EP1578970A1 (fr) Auto-antigenes humains et leur utilisation
DE60316161T2 (de) Neue kcnq-polypeptide und deren verwendung bei der diagnose von geisteskrankheiten
JPWO2002050269A1 (ja) アレルギー性疾患の検査方法
EP3149485B1 (fr) Procédé de diagnostic d'une maladie médiée par la voie alterne du système du complément ou d'un risque pour celle-ci
DE19964386B4 (de) Verfahren zur Herstellung von Makrophagenmigrations-Inhibitionsfaktor (MIF)
EP1436327B1 (fr) Famille de proteines ee3 et sequences d'adn correspondantes
WO2017047672A1 (fr) Procédé d'identification de protéine bioactive et protéine bioactive obtenue par ce procédé

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20050722

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR IE IT LI LU MC NL PT SE SI SK TR

AX Request for extension of the european patent

Extension state: AL LT LV MK RO

DAX Request for extension of the european patent (deleted)
STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20061023