EP1071327A1 - Fragments auto-antigenes, methodes et dosages - Google Patents

Fragments auto-antigenes, methodes et dosages

Info

Publication number
EP1071327A1
EP1071327A1 EP99919952A EP99919952A EP1071327A1 EP 1071327 A1 EP1071327 A1 EP 1071327A1 EP 99919952 A EP99919952 A EP 99919952A EP 99919952 A EP99919952 A EP 99919952A EP 1071327 A1 EP1071327 A1 EP 1071327A1
Authority
EP
European Patent Office
Prior art keywords
granzyme
autoantigenic
fragment
dna
fragments
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Withdrawn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
EP99919952A
Other languages
German (de)
English (en)
Other versions
EP1071327A4 (fr
Inventor
Nancy Thornberry
Antony Rosen
Livia Casciola-Rosen
Felipe A. Andrade
Donald Nicholson
Sophie Roy
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.)
Merck Canada Inc
Merck and Co Inc
Johns Hopkins University
Original Assignee
Merck Frosst Canada and Co
Merck and Co Inc
Johns Hopkins University
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 Merck Frosst Canada and Co, Merck and Co Inc, Johns Hopkins University filed Critical Merck Frosst Canada and Co
Publication of EP1071327A1 publication Critical patent/EP1071327A1/fr
Publication of EP1071327A4 publication Critical patent/EP1071327A4/fr
Withdrawn legal-status Critical Current

Links

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
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
    • G01N2333/95—Proteinases, i.e. endopeptidases (3.4.21-3.4.99)
    • G01N2333/964—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue
    • G01N2333/96425—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals
    • G01N2333/96427—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general
    • G01N2333/9643—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general with EC number
    • G01N2333/96433—Serine endopeptidases (3.4.21)
    • G01N2333/96436—Granzymes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/20—Screening for compounds of potential therapeutic value cell-free systems
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/24—Immunology or allergic disorders

Definitions

  • FIG. 7 Granzyme B-specific fragment of DNA-PKcs is generated in K562 cells attacked by LAK cells. Fas-negative K562 target cells were preincubated in the absence (lanes 1-3) or presence (lane 4) of lOO ⁇ M Ac-DEVD-CHO for 1 hr, followed by co-incubation for a further 4 hr at 37°C (effector:target ratio 5:1). After terminating the reactions, the following numbers of cells were electrophoresed in each gel lane: 1.7 x 106 LAK cells Qane 1); 0.34 x 106 K562 cells (lane 2); 1.7 x 106 LAK cells plus 0.34 x 106 K562 cells (lanes 3 & 4). DNA-PKcs and PARP were detected by immunoblotting; patient serum G.A. was used to detect DNA-PKcs-
  • a sample from the patient is contacted with an autoantigenic fragment having at least one terminus derived from cleavage by a granule enzyme. Detection of the presence or absence of the binding of an antibody in the sample to the autoantigenic fragment is an indication of the presence or absence of an autoimmune condition in the patient.
  • An aspect of this invention is a method of making an autoantigenic fragment from an autoantigen.
  • one isolate s cells containing at least one autoantigen and contacts the cells with a lymphocyte granule enzyme to produce a mixture containing at least one autoantigenic fragment.
  • one isolates at least one autoantigenic fragment from the mixture is a method of making an autoantigenic fragment from an autoantigen.
  • a protein or fragment thereof is considered essentially pure if it is obtained at a concentration of at least about 1000-fold higher than that found in nature.
  • a protein is sometimes referred to as partly purified if it is at least purified or isolated but it is not essentially pure.
  • a chemically synthesized protein is considered to be substantially purified when purified from its chemical precursors.
  • a purified or isolated protein can be manipulated by the skilled artisan, such as but not limited to obtaining the protein or protein fragment in quantities that afford the opportunity to generate polyclonal antibodies, monoclonal antibodies, amino acid sequencing, and peptide digestion. Therefore, the autoantigenic fragments claimed herein can be present in cell lysates or in a substantially or essentially pure form.
  • Monoclonal antibodies can be made by methods known in the art. Two different monoclonal antibodies, designated 18-2 and 25-4 (kind gifts from Dr. Tim Carter, St. Johns University, Jamaica, NY) were also used to detect DNA-PKcs by immunoblotting (see Table II).
  • Catalytic constant (kcat/km) values were calculated essentially as described (Casciola- Rosen et al, 1996). Briefly, subsaturating substrate concentrations were used in each in vitro reaction, and product appearance was assumed to be a first order process. Substrate and product bands on autoradiograms were scanned by densitometry. Several appropriate densitometry systems are available, e.g. PDI Discovery System, with Quantity One Software, Protein Databases, Inc., (Huntington Station, NY).
  • Granzyme B-mediated cleavage of NuMA generated a novel fragment migrating at 175kDa on SDS-PAGE, which was distinct from the 185kDa fragment detected after cleavage with caspase-3 (FIG. 2, lanes 7-9).
  • novel fragments of PARP migrating at 72, 62 and 42kDa were detected after incubation with granzyme B; these differed from the 89 and 24 kDa fragments generated by caspase-3-mediated cleavage of PARP (FIG. 2, lanes 10-12 and Table II).
  • Granzyme B therefore directly cleaves several of the downstream substrates of caspase-3 in vitro. In all cases, the fragments generated by granzyme B differ from those generated by caspase-3.
  • FIG. 8A Granule content- induced surface blebbing, nuclear fragmentation, formation of apoptotic bodies, and characteristic redistribution of nuclear autoantigens was prevented by Ac-DEVD-CHO (Compare FIG. 8B, 8C).
  • Ac-DEVD-CHO Compare FIG. 8B, 8C
  • a prominent diminution in the size of the nucleus was induced by granule contents in Ac-DEVD-CHO-treated cells (FIG. 8C); these nuclear changes were not observed when cells were incubated with Ac-DEVD- CHO alone.
  • the present invention also relates to polyclonal and monoclonal antibodies raised in response to the autoantigenic fragments disclosed herein.
  • An antibody is specific for an epitope of an autoantigenic fragment if one of skill in the art can use standard techniques to determine conditions under which one can detect an autoantigenic fragment in a Western Blot of a sample from cells of a tissue.
  • the blot can be of a native or denaturing gel as appropriate for the epitope.
  • An antibody is highly specific for an autoantigenic fragment epitope if no nonspecific background binding is visually detectable.
  • Hybridoma cells from antibody positive wells are cloned by a technique such as the soft agar technique of MacPherson, 1973, Soft Agar Techniques, in Tissue Culture Methods and Applications, Kruse and Paterson, Eds., Academic Press.
  • 2 ⁇ present in a sample is then contacted with the fragment to permit binding of the autoantibody to the autoantigenic fragment.
  • the presence of bound autoantibody can be detected by methods available in the art, including the use of a labeled second antibody against the antibodies from the patient.
  • the action of the granule contents or granzyme B on the cells can produce autoantigenic fragments from autoantigens present in the cells.
  • the resulting mixture can then be administered to the patient. l ⁇
  • an adjuvant be administered with the autoantigenic fragments.
  • compositions of the invention are administered to an individual in amounts sufficient to treat or diagnose disorders.
  • the effective amount can vary according to a variety of factors such as the individual's condition, weight, sex and age. Other factors include the mode of administration. The appropriate amount can be determined by a skilled physician
  • Granzyme B efficiently cleaves three caspase-3 substrates generating unique fragments not generated during any other form of cell death. To determine whether the generation of unique autoantigen fragments by granzyme B was a universal feature of autoantigens, a wide range of autoantigens were tested for cleavage by granzyme B in vitro and in vivo. It was determined that despite their diverse structure, distribution and function, >70% of the autoantigens described in systemic autoimmune diseases are efficiently cleaved by granzyme B and unique fragments are produced. In contrast, granzyme B does not generate unique fragments in all the non-autoantigen molecules tested.
  • Cytotoxic T-cell-derived granzyme B activates the apoptotic protease ICE-LAP3. Curr.Biol. 6, 897-899.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Hematology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Molecular Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Public Health (AREA)
  • Wood Science & Technology (AREA)
  • Urology & Nephrology (AREA)
  • Zoology (AREA)
  • Veterinary Medicine (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Rheumatology (AREA)
  • Pathology (AREA)
  • Transplantation (AREA)
  • Rehabilitation Therapy (AREA)
  • Cell Biology (AREA)
  • Food Science & Technology (AREA)
  • General Physics & Mathematics (AREA)
  • General Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

La présente invention concerne une méthode de production d'auto-antigènes, des compositions comprenant des fragments auto-antigènes et des méthodes d'utilisation de fragments auto-antigènes dans le traitement d'un trouble associé à une réponse auto-immune. L'invention concerne également des dosages de détection ou d'évaluation d'une réponse auto-immune.
EP99919952A 1998-04-22 1999-04-22 Fragments auto-antigenes, methodes et dosages Withdrawn EP1071327A4 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US8264398P 1998-04-22 1998-04-22
US82643P 1998-04-22
PCT/US1999/008774 WO1999053757A1 (fr) 1998-04-22 1999-04-22 Fragments auto-antigenes, methodes et dosages

Publications (2)

Publication Number Publication Date
EP1071327A1 true EP1071327A1 (fr) 2001-01-31
EP1071327A4 EP1071327A4 (fr) 2004-09-29

Family

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Family Applications (1)

Application Number Title Priority Date Filing Date
EP99919952A Withdrawn EP1071327A4 (fr) 1998-04-22 1999-04-22 Fragments auto-antigenes, methodes et dosages

Country Status (5)

Country Link
EP (1) EP1071327A4 (fr)
JP (1) JP2002511494A (fr)
AU (1) AU3755199A (fr)
CA (1) CA2329495A1 (fr)
WO (1) WO1999053757A1 (fr)

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB0126144D0 (en) * 2001-10-31 2002-01-02 Syngenta Ltd Pesticidal formulations
WO2004073739A1 (fr) * 2003-02-21 2004-09-02 Medvet Science Pty. Ltd. Procede de diagnostic et de traitement
CA2940579A1 (fr) 2014-03-13 2015-09-17 Universitaet Basel Ligands glucidiques qui se lient aux anticorps igm contre la glycoproteine associee a la myeline
AU2016323377B2 (en) 2015-09-16 2021-04-15 Universität Basel Carbohydrate ligands that bind to antibodies against glycoepitopes of glycosphingolipids

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
ANDRADE F ET AL: "Granzyme B directly and efficiently cleaves several downstream caspase substrates: implications for CTL-induced apoptosis." IMMUNITY. APR 1998, vol. 8, no. 4, 1 April 1998 (1998-04-01), pages 451-460, XP002290799 ISSN: 1074-7613 *
FROELICH CHRISTOPHER J ET AL: "Granzyme B/perforin-mediated apoptosis of Jurkat cells results in cleavage of poly(ADP-ribose) polymerase to the 89-kDa apoptotic fragment and less abundant 64-kDa fragment" BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, vol. 227, no. 3, 1996, pages 658-665, XP002290801 ISSN: 0006-291X *
See also references of WO9953757A1 *
SONG QIZHONG ET AL: "Interleukin-1-beta-converting enzyme-like protease cleaves DNA-dependent protein kinase in cytotoxic T cell killing" JOURNAL OF EXPERIMENTAL MEDICINE, vol. 184, no. 2, 1996, pages 619-626, XP002290800 ISSN: 0022-1007 *

Also Published As

Publication number Publication date
WO1999053757A1 (fr) 1999-10-28
AU3755199A (en) 1999-11-08
CA2329495A1 (fr) 1999-10-28
EP1071327A4 (fr) 2004-09-29
JP2002511494A (ja) 2002-04-16

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