WO1998029109A1 - Procedes et compositions servant a identifier des autoantigenes - Google Patents

Procedes et compositions servant a identifier des autoantigenes Download PDF

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Publication number
WO1998029109A1
WO1998029109A1 PCT/US1997/024100 US9724100W WO9829109A1 WO 1998029109 A1 WO1998029109 A1 WO 1998029109A1 US 9724100 W US9724100 W US 9724100W WO 9829109 A1 WO9829109 A1 WO 9829109A1
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protein
metal
fragments
autoantigenic
autoantigen
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PCT/US1997/024100
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WO1998029109A9 (fr
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Antony Rosen
Livia Casciola-Rosen
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Johns Hopkins University
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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid or pantothenic acid
    • A61K31/198—Alpha-amino acids, e.g. alanine or edetic acid [EDTA]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/28—Compounds containing heavy metals
    • A61K31/295—Iron group metal compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/28—Compounds containing heavy metals
    • A61K31/30—Copper compounds
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/10—Peptides having 12 to 20 amino acids
    • 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

  • the present invention generally relates to hydrolysis of certain proteins, and, more particularly, to methods and compositions for specifically cleaving autoantigens by metal-catalyzed proteolysis to form specified protein fragments.
  • the present invention particularly relates to methods for detecting and preparing the protein fragments, methods for making antibodies against the protein fragments, assays employing such protein fragments and antibodies, and methods for treating autoimmune diseases modulated by autoantigens.
  • the autoantibodies elaborated in systemic lupus erythematosus have fused attention on apoptosis as a possible setting in which cryptic structure is revealed.
  • the lupus autoantigens cluster and become concentrated in the surface blebs of apoptotic cells (L. Casciola-Rosen et al., J. Exp. Med., 179:1317-1330 (1994)), where several of these molecules are specifically cleaved by proteases of the interleukin 1 (converting enzyme (CE) family (L. Casciola-Rosen et al., J. Biol.
  • CE interleukin 1
  • Scleroderma is a disease of unknown etiology which is characterized by increased vasoreactivity, widespread tissue fibrosis and the elaboration of unique autoantibodies. Since the autoantigens recognized are not substrates for the ICE-like enzymes during apoptosis, it is likely that other mechanisms are responsible for revealing cryptic structure in this disease (L. Casciola-Rosen et al., J. Exp. Med., 182: 1625-1634 (1995)).
  • One potential mechanism that might result in the specific fragmentation of scleroderma autoantigens is suggested by the striking reversible ischemia-rep erfusion that occurs in patients with scleroderma (reviewed in (J.
  • ROS reactive oxygen species
  • D-penicillamine appears to improve the outcome patients with diffuse scleroderma, when used for extended periods (V. Steen et al., Ann. Intern. Med, 97:652-659 (1982); S. Jimenez et al., J. Rheumatol, 18:1496-1503 (1991)).
  • U.S. 5,504,090 describes a method for preventing ischemia-reperfusion injury in organ by use of a selective Al adenosine receptor antagonist.
  • Wu, U.S. 5,248,668 and, 5,047,395 discloses the use of purpurogallin and bilirubin derivatives as antioxidant agents to minimize oxyradical damage caused by ischemia-reperfusion injury.
  • Stanko, U.S. 5,480,909 relates to the use of pyruvate to inhibit free radical generation and to scavenge free radicals. Lipid peroxidation and radical formation were reduced by inositol triphosphate according to Siren, U.S. 5,019,566.
  • a diphenyl sulfone is taught as having therapeutic effectiveness against scleroderma in Goloschapov et al., U.S. 4,151,281. Aris et al., U.S. 5,310,892, describes the diagnostic use of fibrillarin to detect anti-fibrillarin antibodies in scleroderma patients.
  • the assay of the autoantigens targeted in scleroderma is an insensitive and laborious process, and several autoantigens that are potentially useful as diagnostic and prognostic markers have therefore not had utility in routine clinical practice. For example, use of immunoblotting has not been possible, due to problems with standardization and determining specificity. There is a need for technologies with greater sensitivity and specificity.
  • the present invention generally relates to methods and compositions for detecting autoantigens.
  • the present invention relates to therapies for treating or preventing various autoimmune diseases modulated by undesired fragmentation of normally tolerized autoantigenic proteins.
  • therapies have been identified that include preventing or inhibiting production of the protein fragments.
  • the invention features methods for treatment of autoimmune diseases, particularly those diseases modulated by formation of autoantigenic and immunogenic protein fragments from the tolerized autoantigens.
  • diseases include but are not limited to scleroderma, lupus, Sjogren's syndrome, polymyositis, dermatomyositis, multiple sclerosis, type I diabetes mellitus, rheumatoid arthritis, and mixed connective tissue disease (MCTD).
  • the methods involve reducing or eliminating formation of the fragments by inhibiting metal- catalyzed oxidative proteolysis of autoantigenic proteins, e.g., certain nucleic acid related proteins, that have been implicated in the development or severity of various autoimmune diseases.
  • ROS reactive oxygen species
  • Therapeutic methods of the invention can be employed to prevent or inhibit the metal-catalyzed proteolysis reactions that facilitate production of the fragments.
  • the methods generally include administering to a subject, particularly a mammal such as a primate, especially a human, a therapeutically effective amount of a compound (sometimes referred to herein as a therapeutic compound or substance) that can reduce or eliminate metal-catalyzed oxidative proteolysis of a specified autoantigen.
  • a compound sometimes referred to herein as a therapeutic compound or substance
  • an administered compound inhibits the proteolysis by at least about 10% or 25% as determined, e.g., by a standard protein fragmentation detection assay. Specific examples of such assays are described below.
  • the administered compound be capable of inhibiting production of the protein fragments by at least 30%, 40%, 50%, 70%, 80% or 90% or more as determined by a standard immunological assay.
  • assays include Ouchterlony immunodiffusion, ELISA, or other suitable immunoassay capable of detecting production of immunogenic protein fragments.
  • a particularly preferred assay is a Western immunoblot.
  • a suitable protein fragmentation detection assay can be employed to quantitate protein fragments.
  • preferred assays include one or more suitable controls such as those specified below.
  • Compounds suitable for use in accord with the treatment methods of the invention include compounds that are capable of inhibiting or eliminating metal- catalyzed oxidative proteolysis.
  • Preferred compounds inhibit metal-catalyzed production of ROS such as those reactive oxygen species produced by Fenton or Haber- Weiss related chemical reactions such as those discussed below.
  • Examples of such inhibitory compounds include metal chelators, as well as certain metals such as zinc that have been reported to inhibit the Fenton or Haber- Weiss reactions.
  • Preferred compounds exhibit an IC 50 of at least about 0.1 ⁇ M to 200 ⁇ M in a standard metal-catalyzed Fenton or Haber- Weiss reaction as defined below, more preferably an IC 50 of about l ⁇ M to 50 ⁇ M, still more preferably about 5 ⁇ M to 20 ⁇ M, and even more preferably an IC 50 of about 15 ⁇ M in the standard reaction.
  • Suitable compounds in accord with the invention are sometimes referenced as "proteolytic inhibitor compounds" or like term or phrase to denote capacity to inhibit the metal- catalyzed oxidative proteolysis reactions.
  • Suitable compounds are capable of binding an iron- or copper- catalyzed Fenton or Haber- Weiss reaction, e.g., desterroxamine, D-penicillamine, ethylenediamine tetraacetic acid (EDTA), bathocuproine disulfonate and related compounds.
  • Other suitable compounds can be readily identified by simple testing in accord with the invention, e.g., by in vitro testing of a candidate inhibitor compound relative to a suitable control for capacity to inhibit formation of autoantigenic and immunogenic protein fragments, e.g., by at least 10% or more relative to the control.
  • additional compounds capable of reducing or eliminating coordination a metal and one or more accessible amino acid groups on the autoantigen are contemplated.
  • Preferred compounds are those capable or reacting with accessible carboxylate groups, e.g., l-ethyl-3-(3-dimethlyarninopropyl) carbodiimide (ED AC).
  • ED AC accessible carboxylate groups
  • Compounds of the invention can be used to diagnose and monitor autoimmune conditions, and to assay the effectiveness of treatments for such conditions. Moreover, by preventing or inhibiting the production of such fragments, the severity or extent of injury from autoimmune diseases may be reduced.
  • the autoantigenic protein fragments may be used as tolerizing reagents in the prevention and therapy of autoimmune diseases as set forth in the detailed description.
  • the autoantigen may be topoisomerase I, the large subunit of RNA polymerase II, upstream binding factor (UBF/NOR90), or the 70kDa protein component of the Ul small nuclear ribonucleoprotein, or any antigenic peptide, and the substance may be a chelator, metal other agents that compete with the iron or copper, or other inhibitors of Fenton oxidation and metal-mediated oxidation of proteins.
  • a chelator according to the invention is less toxic and more effective than known substances such as D-penicillamine.
  • the proposed mode of action of D-penicillamine the prior art is inhibition of the Cu-requiring enzymatic reaction required for the cross-linking of collagen.
  • the studies here demonstrate that the unique fragmentation of scleroderma, autoantigens by metal-catalyzed oxidation reactions is a critical target in scleroderma, and prompts an entirely different strategy for identification of relevant agents (i.e. agents that have high nucleolar concentrations and effectively inhibit the Fenton reaction).
  • Preferred chelators include desferroxamine and bathocuproine disulfonate.
  • Additional particular treatment methods include administering a therapeutically effective amount of one or more autoantigenic and immunogenic protein fragments of the invention over a period of time sufficient to substantially or completely tolerize the patient to an autoimmune disease. Further, the methods can be used prophylactically to prevent onset of an autoimmune disease in a subject susceptible or believed to be susceptible to such a disease.
  • the autoantigenic and immunogenic protein fragments are produced in accord with the present invention, ie. by facilitating metal-catalyzed oxidative proteolysis in vivo or in vitro.
  • the invention further relates to methods of producing autoantigenic and immunogenic protein fragments of normally tolerized autoantigens by metal- catalyzed oxidative proteolysis reactions as generally described above.
  • the fragments are produced in the presence of metals which are sometimes referred to herein as "Fenton metals" to denote capacity to facilitate oxidative proteolysis in accord with Fenton or Haber-Weiss type chemical reactions discussed below. As noted above, these chemical reactions produce ROS and focus same to specific autoantigen sites resulting in autoantigen cleavage.
  • Fenton metals include iron and copper.
  • a Fenton metal is preferably used as a suitable salt as specified below.
  • certain autoantigens from scleroderma patients and patients with other autoimmune diseases have in common a susceptibility to metal-dependant ROS- mediated cleavage of the proteins into fragments. These antigenic protein fragments are believed to be related to those which are the targets of the immune system in these patients.
  • the invention also facilitates easy identification and quantitation of autoantibodies recognizing these autoantigens (such as with scleroderma: topoisomerase I, RNA polymerase II, Ul-70kDa and NOR90).
  • the invention is useful for early and specific diagnosis of auto immune patients, such as scleroderma patients, as well as the serial evaluation of disease severity to guide therapy. Diagnosis of other autoimmune diseases is readily accomplished according to the invention, by cleaving autoantigens particular to those diseases via metal- mediated oxidation, and determining whether a patient's serum cross-reacts with the autoantigenic protein fragments.
  • the field of diagnosis and treatment of autoimmune diseases is crowded and has not provided much success. Antioxidants have proved to be of limited therapeutic use. There is a pressing need for new technologies.
  • This invention succeeds where previous efforts of specifically and repeatably generating antigenic epitopes from autoantigens have failed.
  • By cleaving autoantigens according to the invention cryptic antigenic sites are revealed.
  • the use of chemical proteolysis to achieve this result was not previously known or suggested.
  • the advantages presented by the fragments produced according to the invention could not have been appreciated before this invention.
  • the method of metal-mediated protein cleavage also has wide-ranging applications in protein biochemistry, and could be readily adapted for use on an industrial scale as an alternative to enzymatic proteolysis.
  • the autoantigenic and immunogenic protein fragments of the present invention usually include one or more cryptic epitopes revealed by the metal- catalyzed oxidative proteolysis and which are typically immunologically detectable.
  • Molecular weights the fragments are influenced by several parameters including desired efficiency or completeness of a metal-catalyzed oxidative proteolysis reaction. As will be discussed below, the molecular weight is particularly effected by the number and location of metal binding sites in the autoantigen. Generally, most protein fragments will have a molecular weight in the range of between about lOkDa to 500kDa, more typically 30kDa to 250kDa as determined by standard molecular sizing techniques such as chromatography and electrophoresis. See the discussion and examples below.
  • the invention provides advantages that can be exploited in the understanding, diagnosis and therapy of autoimmune diseases. Such advantages include use of autoantibodies, e.g., as probes for analyzing initial stimulation of the immune system in different autoimmune diseases. Additionally, protein fragmentation is an efficient method to initiate an immune response to self-molecules. In particular, autoantigenic protein fragments produced in accord with the invention can be used to raise antibodies, particularly commercially.
  • the fragments can also be used in diagnostic assays to detect certain autoimmune diseases, e.g., scleroderma, MCTD, lupus, Sjogren's syndrome, polymyositis, dermatomyositis, multiple sclerosis, type I diabetes mellitus, rheumatoid arthritis and others.
  • autoimmune diseases e.g., scleroderma, MCTD, lupus, Sjogren's syndrome, polymyositis, dermatomyositis, multiple sclerosis, type I diabetes mellitus, rheumatoid arthritis and others.
  • This invention applies to nearly any autoimmune disease where autoantigenic fragments are revealed by a metal catalyzed reaction.
  • Scleroderma diffuse and limited, including the CREST variant
  • MCTD are two examples of such diseases.
  • experimental evidence shows that similar principles apply in the case of rheumatoid arthritis, and in fibrosing alveolitis spectrum of lung disease.
  • the invention also relates to a composition useful in reducing injury from scleroderma comprising an effective amount of a substance that inhibits the fragmentation of autoantigens into peptides in the presence of iron or copper and reactive oxygen species. Administration may be intravenous or otherwise as indicated below.
  • the antigenic protein fragments of the invention have defined molecular weight. Additionally, such fragments cross-react with serum from a patient for the autoimmune disease in question, and have varying degrees of immunogenicity in the sense of eliciting an immunogenic response in an individual without the autoimmune disease. For example, with scleroderma, RNA polymerase has greater specificity of binding and is more antigenic than some of the other antigenic protein fragments produced according to the invention.
  • autoantigenic and immunogenic protein fragments of normally tolerized antigens may be produced in the presence of specified metals and ROS.
  • a method scleroderma autoantigens according to the invention comprises obtaining a protein selected from the group consisting of topoisomerase I, the large subunit of RNA polymerase II, upstream binding factor (UBF/NOR90), and the 70kDa protein component of the Ul small nuclear ribonucleoprotein, and fragmenting the protein in the presence of iron or copper and reactive oxygen species.
  • a protein selected from the group consisting of topoisomerase I, the large subunit of RNA polymerase II, upstream binding factor (UBF/NOR90), and the 70kDa protein component of the Ul small nuclear ribonucleoprotein and fragmenting the protein in the presence of iron or copper and reactive oxygen species.
  • Scleroderma autoantigen fragments according to the invention may be topoisomerase I fragments migrating at 95kDa, and species migrating between 65kDa and 90kDa; fragments of the large subunit of RNA polymerase II migrating at 190, 160 and 140kDa, and fragments migrating at 200, 180, 170 and 130kDa; fragments of the upstream binding factor (UBF/NOR90) migrating 70kDa and 90kDa; and fragments of 70kDa protein component of the Ul small nuclear ribonucleoprotein (Ul-70kDa) migrating between 33 and 3kDa.
  • Yet another method of making antigenic protein fragments in accord with the invention comprises reacting autoantigens for the disease in the presence of a metal and reactive oxygen species selected to specifically cleave the protein into antigenic fragments with epitopes.
  • the cleavage into fragments according to the invention is specific and reproducible, and is distinct from dissociation into subunits, modification of side chains, and protein degradation, all of which are common reaction pathways for proteins.
  • More particular examples according to the invention involve fragmenting autoantigens by adding iron at a concentration of between about 1 ⁇ M and about
  • the method may include adding ascorbate, preferably in a concentration of between 1.7mM and 17mM.
  • the autoantigens may be scleroderma autoantigens.
  • topoisomerase I can be fragmented into a major fragment of 95kDa, and several minor, discrete species migrating between 65kDa and 90kDa.
  • the large subunit of RNA polymerase II (a protein doublet of 220 and 240kDa) generated major species of 190, 160 and 140kDa, as well as several minor species of 200, 180, 170 and 130kDa.
  • Upstream binding factor (UBF/NOR90) (a doublet of 90 and lOOkDa) yielded fragments between 70kDa and 90kDa.
  • suitable methods may involve a Cu-catalyzed oxidation system capable of fragmenting topoisomerase I, RNA polymerase II large subunit, and Ul- 70kDa, using about lO ⁇ M to about 500 ⁇ M Cu, preferably about 20 ⁇ M to about 1 OO ⁇ M; and about 0. ImM to about 5mM H 2 O 2 , preferably about ImM to about 2mM, to initiate oxygen radical production.
  • the reaction may take place in vitro, in cellular lysates, in cell culture, or in vivo. With intact cells, fragmentation may be t o accomplished by prolonged incubation with concentrations of extracellular Cu that are unable to induce protein fragmentation in cell lysates.
  • Another embodiment of the invention is a screening assay for a drug with anti- scleroderma or other effectiveness against another auto immune disease activity, comprising:
  • detection assays generally involve steps associated with detection of metal-catalyzed oxidative proteolysis in suitable cells or cell lysates. For example, one such assay generally includes the following steps 1) through 4):
  • a screening assay of the present invention can effectively measure the capacity of the known or candidate inhibitor compound to decrease production of the autoantigenic and immunogenic protein fragments. Measurements conducted in accord with the screening assays involve, e.g., quantitation of protein fragments by a suitable protein fragment detection assay such as those specified immunological assays described below. A suitable assay can be conducted with nearly any
  • suitable cells include immortalized cells such as HeLa cells and certain epithelial cells such as specified keratinocytes described in the examples that follow. Certain primary cells are also suitable for use in the assays.
  • Screening assays according to the invention can be adapted in accordance with intended use.
  • nearly any disclosed screening assay can be modified as desired to detect compounds capable of inhibiting metal-catalyzed oxidative proteolysis of autoantigens.
  • a lysate can be prepared from the cells and one or more than one of the Fenton metals can be added to the lysate in an amount sufficient to support metal-catalyzed oxidative proteolysis.
  • Addition of the metal can be performed before, during or after administration of the known or candidate inhibitor compound specified in step 2), above. In most cases however, it will be preferred to add the known or inhibitor compound prior to adding the metal to the lysate.
  • a known or candidate inhibitor compound tested in any of the disclosed assays can be employed as a sole active agent or in combination with other agents including other known inhibitors of metal-catalyzed oxidative proteolysis to be tested, particularly those proteolytic reactions accompanied by Fenton or Haber-Weiss type chemical reactions.
  • the in vitro assays are performed with a suitable control assay usually including the same test conditions as in the steps and discussion above, but without adding the known or candidate inhibitor compound to the medium or cell lysate.
  • a candidate inhibitor compound can be identified as exhibiting a desired activity by providing at least about 10% greater inhibitory activity relative to the control; more preferably at least about 20% greater activity relative to the control assay; and still more preferably at least about 30%, 40%, 50%, 60%, 70%, 80%, 100%, 150%, or 200% greater activity relative to the control.
  • the inhibitory activity can be measured by a variety of methods including quantitation of autoantigenic and immunogenic protein fragments produced from autoantigens. Alternatively, inhibitory activity can be measured by monitoring production of ROS according to conventional techniques.
  • in vitro methods for reducing fragmentation of autoantigens comprising administering agents to reduce O 2 levels.
  • Another method of reducing fragmentation is to reduce the availability of one or more Fenton metals such as iron or copper, preferably by adding a chelator such as ImM desferroxamine, ImM D-penicillamine, or ImM EDTA.
  • the chelator may be 200 ⁇ M bathocuproine disulfonate (a Cu(I)-specific chelator).
  • Adding zinc in a concentration up to about 300 ⁇ M also inhibits fragmentation, as does chemical modification of the accessible carboxylates by l-ethyl-3-(3-dimethylaminopropyl) carbodiimide (ED AC) prior to metal-mediated oxidation.
  • ED AC l-ethyl-3-(3-dimethylaminopropyl) carbodiimide
  • Other metals and competing compounds may be used to inhibit fragmentation.
  • Figure 1 is a representation of an immunoblot illustrating several scleroderma autoantigens uniquely fragmented by Fe/ascorbate or Cu/H 2 O 2 oxidation reactions.
  • HeLa lysates were prepared as described in the Methods section, and metal-catalyzed oxidation reactions were performed by adding the following: no additions (lane 1), 1.7mM ascorbate (lane 2), ImM H 2 O 2 (lane 3), lOO ⁇ M Fe(II)SO (lane 4), lOO ⁇ M Fe(II)SO 4 + 1.7mM ascorbate (lane 5), lOO ⁇ M Fe(II)SO 4 + ImM H 2 O 2 (lane 6), lOO ⁇ M Cu(II)SO 4 (lane 7), lOO ⁇ M Cu(II)SO + 1.7mM ascorbate (lane 8) and lOO ⁇ M Cu(II)SO 4 + ImM H 2 O 2 (lane 8). Samples were immunoblotted with the sera denoted
  • Figures 2 A and 2B are representations of polyacrylamide gels showing that fragmentation of scleroderma autoantigens by oxidation is a highly specific event.
  • FIGs. 2A and 2B equal protein amounts of control HeLa lysates (lanes 1 & 3), and HeLa lysates incubated with lOO ⁇ M Fe + 1.7mM ascorbate (lanes 2 & 4) were electrophoresed.
  • Figures 3A, 3B, and 3C are representations of immunoblots showing that metal chelators, zinc or ED AC strongly inhibit or abolish fragmentation of the scleroderma autoantigens induced by both Fe/ascorbate and Cu/H 2 O 2 .
  • the following additions were made to equal protein amounts of HeLa cell lysate, prior to immunoblotting with the indicated antibodies:
  • FIG. 3B Increasing concentrations of ZnCl (0-300 ⁇ M) were added to HeLa lysates prior to addition of lOO ⁇ M Fe + 1.7mM ascorbate.
  • the IC 50 value for inhibition of Fe/ascorbate- induced fragmentation of topoisomerase I or RNA polymerase II large subunit was 30-50 ⁇ M.
  • Figure 3C HeLa lysates were incubated in the absence (-) or presence (+) of
  • Figure 4 is a representation of an immunoblot illustrating that topoisomerase I is fragmented in intact keratinocytes chronically exposed to 20 ⁇ M Cu.
  • Confluent monolayers of human foreskin keratinocytes were cultured for 18 hours (lanes 3 & 4) or 2 hours (lanes 5 & 6) in keratinocyte growth medium supplemented with 20 ⁇ M
  • Figures 5A and 5B are photomicrographs showing that metal ions and topoisomerase I are concentrated in punctate intranucleolar structures.
  • Figure 5 A HeLa cells were fixed in 1% lead acetate, and subsequently stained with 1% ammonium sulfide. Bright field microscopy demonstrates intense staining of punctate intranucleolar structures (nucleolini).
  • HeLa cells were stained with a monospecific human serum recognizing topoisomerase I and FITC-goat anti-human IgG, and were examined by confocal fluorescence microscopy. Diffuse nuclear staining as well as punctate intranucleolar structures (nucleolini) are seen. Similar results were obtained with 6 other monospecific topoisomerase I sera. Nucleolini were never stained when similar experiments were performed using sera obtained from healthy individuals, or from Ro/La-positive lupus patients. Bar, lO ⁇ M. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
  • the present invention features therapeutic methods for treatment or prevention of autoimmune disorders modulated by metal-catalyzed oxidative proteolysis of autoantigens.
  • Such treatment methods generally include administering a therapeutically effective amount of a composition of the invention to a subject, preferably a patient in need of such treatment.
  • therapeutic methods of the invention comprise administration of a therapeutically effective amount of a proteolytic inhibitor compound to a subject in need of such treatment, such as a mammal, and particularly a primate such as a human.
  • Additional treatment methods of the invention include administration of an effective amount of one or more autoantigenic and immunogenic protein fragment as defined herein to a subject, particularly a mammal such as a human in need of such treatment for an indication disclosed herein.
  • Preferred fragments are those that are capable of substantially or completely tolerizing the mammal to the autoantigen or protein fragment as determined by standard immunological assays such as those disclosed below.
  • Typical subjects include mammals suffering from or susceptible to an autoimmune disorder such as those specified below. Examples of suitable
  • I S immunological assays include Western immunoblot assays involving detection of patient autoantibodies against specified autoantigens.
  • metal-catalyzed oxidative proteolysis is used herein to denote specific cleavage of at least one autoantigen by a Fenton or Haber-Weiss type chemical reaction.
  • the proteolysis can be achieved in vitro or in vivo.
  • the metal will be iron or copper. See the examples which follow.
  • cleavage site-specific hydrolysis of an autoantigen or protein fragment disclosed herein by metal-catalyzed oxidative proteolysis.
  • Site specific cleavage of the autoantigen or protein fragment can be readily detected and quantified if desired by, e.g., Western immunoblotting, ELISA, radioimmunoassay (RIA), antigen capture type assays, multiple-antibody sandwich type assays or other suitable immunoassays known in the field.
  • proteolytic inhibitor compounds can be employed in the present treatment methods. Simple testing, e.g., in a standard in vitro assay as defined above, can readily identify suitable proteolytic inhibitor compounds.
  • Preferred proteolytic inhibitor compounds include those capable of eliminating or reducing metal-catalyzed oxidative proteolysis. Specific examples of such compounds include zinc and certain metal chelators capable of forming coordination complexes with metals and particularly with iron and copper.
  • zinc and metal chelators such as desterroxamine, D-penicillamine, ethylenediamine tetraacetic acid (EDTA), bathocuproine disulfonate, or related compounds.
  • a treatment compound can be administered to a subject by one or a combination of strategies.
  • a proteolytic inhibitor compound can be administered as a prophylactic to prevent the onset of or reduce the severity of a targeted condition.
  • a proteolytic inhibitor compound can be administered during the course of a targeted condition.
  • a treatment compound can include one or more proteolytic inhibitor compounds or one or more autoantigenic protein fragments according to the invention.
  • the treatment compound can be administered to a subject, either alone or in combination with one or more other therapeutic compounds (agents), as a pharmaceutical composition in mixture with conventional excipient, i.e. l b pharmaceutically acceptable organic or inorganic carrier substances suitable for parenteral, enteral or intranasal application which do not deleteriously react with the active compounds and are not deleterious to the recipient thereof.
  • Suitable pharmaceutically acceptable carriers include but are not limited to water, salt solutions, alcohol, vegetable oils, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, perfume oil, fatty acid monoglycerides and diglycerides, petroethral fatty acid esters, hydroxymethyl- cellulose, polyvinylpyrrolidone, etc.
  • the pharmaceutical preparations can be sterilized and if desired mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously react with the active compounds.
  • auxiliary agents e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously react with the active compounds.
  • compositions may be prepared for use in parenteral administration, particularly in the form of liquid solutions or suspensions; for oral administration, particularly in the form of tablets or capsules; intranasally, particularly in the form of powders, nasal drops, or aerosols; vaginally; topically e.g. in the form of a cream; rectally e.g. as a suppository; etc.
  • the pharmaceutical agents may be conveniently administered in unit dosage form and may be prepared by any of the methods well known in the pharmaceutical arts, e.g., as described in Remington 's Pharmaceutical Sciences (Mack Pub. Co., Easton, PA, 1980).
  • Formulations for parenteral administration may contain as common excipients such as sterile water or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, hydrogenated naphthalenes and the like.
  • biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylene copolymers may be useful excipients to control the release of certain proteolytic inhibitor compounds.
  • parenteral delivery systems include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes.
  • Formulations for inhalation administration contain as excipients, for example, lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or oily solutions for administration in the form of nasal drops, or as a gel to be applied intranasally.
  • Formulations for parenteral administration may also include glycocholate for buccal administration, me hoxysalicylate for rectal administration, or citric acid for vaginal administration.
  • Other delivery systems will administer the therapeutic agent(s) directly at a selected body site, e.g. by use of stents.
  • a proteolytic inhibitor compound or autoantigenic fragment can be employed in the present treatment methods as the sole active pharmaceutical agent or can be used in combination with other active ingredients, e.g., one or more recognized immunomodulatory, anti-inflammatory or analgesic compounds such as cyclosporin, cortisol, or aspirin, respectively.
  • concentration of one or more treatment compounds in a therapeutic composition will vary depending upon a number of factors, including the dosage of the compound to be administered, the chemical characteristics (e.g., hydrophobicity) of the composition employed, and the intended mode and route of administration.
  • one or more than one of the compounds may be provided in an aqueous physiological buffer solution containing about 0.1 to 10% w/v of a compound for parenteral administration.
  • the fragment may be chemically modified, e.g., by modification of C- and/or N-terminii according to conventional methods to increase half-life.
  • Suitable dose ranges may include from about l ⁇ g/kg to about lOOmg/kg of body weight per day.
  • Therapeutic compounds of the invention are suitably administered in a protonated and water-soluble form, e.g., as a pharmaceutically acceptable salt, typically an acid addition salt such as an inorganic acid addition salt, e.g., a hydrochloride, sulfate, or phosphate salt, or as an organic acid addition salt such as an acetate, maleate, fumarate, tartrate, or citrate salt.
  • a pharmaceutically acceptable salt typically an acid addition salt such as an inorganic acid addition salt, e.g., a hydrochloride, sulfate, or phosphate salt, or as an organic acid addition salt such as an acetate, maleate, fumarate, tartrate, or citrate salt.
  • Pharmaceutically acceptable salts of therapeutic compounds of the invention also can include metal salts, particularly alkali metal salts such as a sodium salt or potassium salt; alkaline earth metal salts such as a magnesium or calcium salt; ammonium salts such an ammonium or tetramethyl ammonium salt; or an amino acid addition salts such as a lysine, glycine, or phenylalanine salt.
  • metal salts particularly alkali metal salts such as a sodium salt or potassium salt
  • alkaline earth metal salts such as a magnesium or calcium salt
  • ammonium salts such an ammonium or tetramethyl ammonium salt
  • an amino acid addition salts such as a lysine, glycine, or phenylalanine salt.
  • therapeutic compounds include novel chelators, agents that bind to metal-binding sites, and peptides for tolerizing strategies.
  • agents which decrease fragmentation of antigens in vitro and in perturbed tissues of patients with autoimmune diseases including scleroderma.
  • Two broad groups of agents are envisioned: a. those that chelate metals, particularly those capable of catalyzing a
  • Fenton reaction b. those that compete for the binding site of these metals in nucleolini, and thereby prevent Fenton chemistry from being catalyzed at that site.
  • compositions of the present invention i.e., antigenic protein fragments, and inhibitors of protein fragmentation such as chelators and compounds that compete with the metal catalysts, may be made into pharmaceutical compositions with appropriate pharmaceutically acceptable carriers or diluents.
  • pharmaceutical compositions may be formulated into preparations including, but not limited to, solid, semi-solid, liquid, or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, and aerosols, or other suitable administration formats.
  • a pharmaceutically-acceptable form should be employed which does not ineffectuate the compositions of the present invention.
  • the compositions may be used alone or in appropriate association, as well as in combination with, other pharmaceutically-active compounds.
  • compositions of the present invention can be delivered via various routes and to various sites in an animal body to achieve a particular effect.
  • Local or system delivery can be accomplished by administration comprising application or instillation of the formulation into body cavities, inhalation, or insufflation of an aerosol, or by parenteral introduction, comprising intramuscular, intravenous, peritoneal, subcutaneous intradermal, as well as topical administration.
  • the antigenic protein fragments are administered intravenously without adjuvants.
  • the inhibitors of protein fragmentation such as chelators are effectively administered systemically, with the resulting effect in the intracellular environment.
  • the "therapeutically effective amount" of the composition is such as to produce the desired effect in a host which can be monitored using several end-points known to those skilled in the art.
  • one desired effect might comprise tolerization towards self-antigens, and another would inhibition of the autoimmune disease.
  • Such effects could be monitored in terms of a therapeutic effect, e.g., alleviation of some symptom associated with the disease being treated, or further evidence of lack of auto immunity, or particularized assays.
  • each active agent included in the compositions employed the examples described herein provide general guidance of the range of each component to utilized by the practitioner upon optimizing the method of the present invention for practice either in vitro or in vivo. Moreover, such ranges by no means preclude use of a higher or lower amount of a component, as might be warranted in a particular application.
  • the actual dose and schedule may vary depending on whether the compositions are administered in combination with other pharmaceutical compositions, or depending on interindividual differences in pharmacokinetics, drug disposition, and metabolism. Similarly, amounts may vary for in vitro applications. One skilled in the art can easily make any necessary adjustments in accordance with the necessities of the particular situation.
  • the invention also relates to methods for inducing fragmentation of autoantigens as initiators of autoimmunity.
  • the process reveals immunocryptic epitopes in self-antigens which may initiate the autoimmune response.
  • scleroderma is a systemic sclerosis which may affect a patient's entire body. Its symptoms may be related to a vascular lesion, ischemia reperfiision, which is associated with an autoimmune response.
  • the reversible ischemia- reperfusion which characterizes scleroderma is associated with reactive oxygen species which can induce autoantigen fragmentation.
  • the methods and products of the invention provide unique antigenic protein fragments. They are antigenic in the sense that they bind with antibodies, but also in the sense that they are immunogenic, and can trigger the autoantibody response in autoimmune diseases. Immunogenicity is a property of substances (immunogens or antigens) that can induce a detectable immune response, which can be humoral, cellular, or both.
  • the therapeutic methods of the invention include altering concentrations of metals. Abnormal metal status is a potential pathogenic principle in this disease, and can also be used as a diagnostic or therapeutic tool.
  • the fragments can be used as tolerizing agents in therapy of autoimmune diseases.
  • treatment is intended to encompass administration of compounds according to the invention propylactically to prevent or suppress an undesired condition, and therapeutic administration to eliminate or reduce the extent or symptoms of the condition.
  • Treatment according to the invention may be for a human or an animal having a disease in need of such treatment, or it may include application in vitro to a cell culture or extracellular media. Treatment may be by systemic administration to a patient or locally to an affected site.
  • This invention relates to the mechanism involved in the breakdown of self tolerance to induce autoimmune responses to specific autoantigens, a subject that has
  • Autoimmunity is the process of developing an immune response to self antigens.
  • Autoimmune diseases may involve the generation of autoantibodies and/or the development of autoreactive T lymphocytes.
  • Autoimmune diseases comprise a varied list of diseases, which possess a myriad of clinical manifestations. Different autoantigens and pathogens are associated with specific autoimmune diseases.
  • Autoantigens may involve intracellular antigens, such as NA, small nuclear ribonucleoproteins, and intracellular proteins; membrane receptors, such as thyrotropin, acetylcholine receptor, insulin receptor; cell membranes, such as red blood cells, lymphocytes, neutrophils, platelets, muscle, and islet cells; plasma proteins, such as immunoglobulins, complement, clotting factors; and hormones, such as insulin, thyroid hormones, glucagon, and intrinsic factor.
  • intracellular antigens such as NA, small nuclear ribonucleoproteins, and intracellular proteins
  • membrane receptors such as thyrotropin, acetylcholine receptor, insulin receptor
  • cell membranes such as red blood cells, lymphocytes, neutrophils, platelets, muscle, and islet cells
  • plasma proteins such as immunoglobulins, complement, clotting factors
  • hormones such as insulin, thyroid hormones, glucagon, and intrinsic factor.
  • autoimmune disease includes scleroderma, multiple sclerosis, myasthenia gravis, polymyositis, graft-versus-host disease, graft rejection, raves disease, Addison's disease, autoimmune uveoretinitis, pemphigus vulgaris, primary biliary cirrhosis, rheumatoid arthritis, psoriasis, atopic dermatitis, osteoarthritis, Hashimoto's thyroiditis, primary myxoedema, thyrotoxicosis, pernicious anemia, autoimmune atrophic gastritis, insulin-dependent diabetes mellitus, Goodpasture's syndrome, pemphigoid, sympathetic opthalmia, phacogenic uveitis, autoimmune haemolytic anemia, idiopathic thrombocytopenic purpura, idiopathic leucopenia, active chronic hepatitis, cryptogenic
  • the methods and materials of the invention apply best to those autoimmune diseases in which autoantigens are cleaved to antigenic fragments by oxidative proteolysis such as Fenton chemistry or otherwise. These include rheumatoid arthritis, pulmonary fibrosis, myositis, primary biliary cirrhosis, and autoimmune thyroid disease. Experimental work confirms the suitability of oxidative proteolysis for these diseases. Other diseases, such as lupus and Sjogren's syndrome are believed to involve enzymatic proteolysis in their natural progression and so are less susceptible to the techniques of this invention.
  • Diagnosis of an autoimmune disease according to the invention may include identification of antibodies to the revealed epitopes of the autoantigen fragments produced by metal-mediated oxidative proteolysis in vivo or in vitro.
  • autoantigenic and immunogenic protein fragments can be made in accord with the invention and then used in a conventional immunological assay to identify autoantibodies in patient sera. Specific binding between the sera and one or more of the protein fragments is indicative of the autoimmune disease. See the examples which follow.
  • antibodies of the invention are particularly useful as diagnostic agents for detecting autoantigenic and immunogenic protein fragments in biological samples such as those obtained from a subject such as a mammal and particularly a primate such as a human patient.
  • the antibodies can be prepared by recognized immunological techniques and are typically generated to a purified sample of a desired protein fragment.
  • a substantially pure autoantigenic and immunogenic protein fragment produced by a method of the present invention is particularly preferred.
  • Such antibodies also can be generated from an immunogenic peptide that comprises one or more cryptic epitopes of the protein fragment. As noted above, such cryptic epitopes are typically not exhibited by autoantigens.
  • Monoclonal antibodies are generally preferred, although polyclonal antibodies also can be employed in the present methods.
  • antibodies can be prepared by immunizing a mammal with a purified sample of a desired autoantigenic and immunogenic protein fragment or an immunogenic peptide as discussed above, alone or complexed with a carrier.
  • Suitable mammals include typical laboratory animals such as sheep, goats, rabbits, guinea pigs, rats and mice. Rats and mice, especially mice, are preferred for obtaining monoclonal antibodies.
  • the antigen can be administered to the mammal by any of a number of suitable routes such as subcutaneous, intraperitoneal, intravenous, intramuscular or intracutaneous injection.
  • the optimal immunizing interval, immunizing dose, etc. can vary within relatively wide ranges and can be determined empirically based on this disclosure.
  • Typical procedures involve injection of the antigen several times over a number of months.
  • Antibodies are collected from serum of the immunized animal by standard techniques and screened to find specified antibodies.
  • Monoclonal antibodies can be produced in cells which produce antibodies and those cells used to generate monoclonal antibodies by using standard fusion techniques for forming hybridoma cells. See G. Kohler, et al., Nature, 256:456
  • monoclonal antibodies can be produced from cells by the method of Huse, et al., Science, 256:1275 (1989).
  • One suitable protocol provides for intraperitoneal immunization of a mouse with a composition comprising a substantially pure autoantigenic and immunogenic fragment produced from topoisomerase I (see Example 1 below) over a period of about two to seven months. Spleen cells then can be removed from the immunized mouse. Sera from the immunized mouse is assayed for titers of antibodies specific for the protein fragment prior to excision of spleen cells.
  • the excised mouse spleen cells are then fused to an appropriate homogenic or heterogenic (preferably homogenic) lymphoid cell line having a marker such as hypoxanthine-guanine phosphoribosyltransferase deficiency (HGPR ) or thymidine kinase deficiency (TK " ).
  • HGPR hypoxanthine-guanine phosphoribosyltransferase deficiency
  • TK thymidine kinase deficiency
  • Myeloma cells and spleen cells are mixed together, e.g. at a ratio of about 1 to 4 myeloma cells to spleen cells.
  • the cells can be fused by the polyethylene glycol (PEG) method. See G. Kohler, et al., Nature, supra. The thus cloned hybridoma is grown in a culture
  • Hybridomas grown after the fusion procedure, are screened such as by radioimmunoassay or enzyme immunoassay for secretion of antibodies that bind specifically to the purified protein fragment.
  • an ELISA is employed for the screen.
  • Hybridomas that show positive results upon such screening can be expanded and cloned by limiting dilution method.
  • Further screens are preferably performed to select antibodies that can bind to the protein fragment in solution as well as in a human fluid sample.
  • the isolated antibodies can be further purified by any suitable immunological technique including affinity chromatography.
  • chimeric antibody derivatives e.g. antibody molecules that combine a non-human animal variable region and a human constant region
  • a variety of types of such chimeric antibodies can be prepared, including e.g. by producing human variable region chimeras, in which parts of the variable regions, especially conserved regions of the antigen-binding domain, are of human origin and only the hypervariable regions are of non-human origin. See also discussions of humanized chimeric antibodies and methods of producing same in S.
  • the molecular weight of the antibodies of the invention will have a molecular weight of between approximately 20 to 150kDa. Such molecular weights can be readily are determined by molecular sizing methods such as SDS-PAGE gel electrophoresis followed by protein staining or Western blot analysis.
  • Antibody of the invention refers to whole immunoglobulin as well immunologically active fragments which bind a desired autoantigenic and immunogenic protein fragment.
  • the immunoglobulins and immunologically active fragments thereof include an antibody binding site (i.e., a cryptic epitope capable of specifically binding the antibody).
  • Exemplary antibody fragments include, for example, Fab, F(v), Fab', F(ab') 2 fragments, "half molecules" derived by reducing the disulfide bonds of immunoglobulins, single chain
  • the antibody or immunologically active fragment thereof may be of animal (e.g., a rodent such as a mouse or a rat), or chimeric form (see Morrison et al., PNAS, 81:6851 (1984); Jones et al., Nature, pp. 321, 522 (1986)).
  • Single chain antibodies of the invention can be preferred.
  • an antibody of the invention is meant an antibody which is capable of forming an immune complex with an autoantigenic and immunogenic protein fragment as described herein.
  • the antibody is capable of specifically binding cryptic epitopes but is not capable of specifically binding the autoantigen that gave rise to the peptide fragment as determined by, e.g., Western blotting, ELISA, RIA, or other suitable protein binding assays known in the field.
  • antibodies and reactive sera disclosed herein are capable of binding a specified autoantigen and autoantigenic and immunogenic protein fragments produced from same. Such antibodies and reactive sera are useful for a variety of applications in accord including detecting the protein fragments. See e.g., Fig. 1.
  • Preferred antibodies of the invention are typically monoclonal and specifically bind a cryptic epitope predominant to a desired autoantigenic and immunogenic protein fragment produced as disclosed herein. Indeed, preferred antibodies of the invention bind to the protein fragment at least about 2, 5, 10, 25, 50, 100 or more times greater than the binding affinity to the autoantigen from which the protein fragment was produced. Preferred antibodies of the invention do not substantially bind the autoantigen as determined by any suitable immunological assay described herein.
  • antibodies of the invention can be used to detect presence or to quantitate if desired, one or more autoantigenic and immunogenic protein fragments produced by metal-catalyzed oxidative proteolysis.
  • Preferred detection methods include suitable immunological assays such as a Western immunoblot.
  • Particularly preferred detection methods include Western immunoblotting followed by conventional densitometery to quantitate presence of the protein fragments in suitable control and experimental samples.
  • Antibodies of the invention can also be employed in accord with standard immunological practice either in vivo or in situ to detect production of autoantigenic and immunogenic protein fragments in cells, tissues and organs. For example, such antibodies are particularly useful in the subcellular identification of the protein fragments in nucleolini.
  • polypeptide refers to any polymer consisting essentially of any of the 20 amino acids regardless of its size.
  • protein is often used in reference to relatively large proteins, and “peptide” is often used in reference to small polypeptides, use of these terms in the field often overlaps.
  • polypeptide refers generally to proteins, polypeptides, and peptides unless otherwise noted.
  • the autoantigenic and immunogenic protein fragments of the present invention can be separated and purified by appropriate combination of known techniques. These methods include, for example, methods utilizing solubility such as salt precipitation and solvent precipitation, methods utilizing the difference in molecular weight such as dialysis, ultra-filtration, gel-filtration, and SDS- polyacrylamide gel electrophoresis, methods utilizing a difference in electrical charge such as ion-exchange column chromatography, methods utilizing specific affinity such as affinity chromatograph, methods utilizing a difference in hydrophobicity such as reverse-phase high performance liquid chromatograph and methods utilizing a difference in isoelectric point, such as isoelectric focusing electrophoresis, metal affinity columns such as Ni-NTA.
  • solubility such as salt precipitation and solvent precipitation
  • methods utilizing the difference in molecular weight such as dialysis, ultra-filtration, gel-filtration, and SDS- polyacrylamide gel electrophoresis
  • methods utilizing a difference in electrical charge such as ion-exchange column
  • the autoantigenic and immunogenic protein fragments of the present invention be substantially pure. That is, the protein fragments have been isolated from cell substituents that naturally accompany it so that the fragments are present preferably in at least 80% or 90% to 95% homogeneity (w/w). Suitable protein fragments having at least 98 to 99% homogeneity (w/w) are most preferred for many pharmaceutical, clinical and research applications.
  • the fragments should be substantially free of contaminants for therapeutic applications.
  • the protein fragments can be used therapeutically, or in performing in vitro or in vivo assays as disclosed herein.
  • Substantial purity can be determined by a variety of standard techniques such as chromatography and gel electrophoresis.
  • various assays in accord with the invention can be used to detect or test known or candidate compounds for therapeutic capacity to inhibit metal- catalyzed oxidative proteolysis of a desired autoantigen, e.g., topoisomerase I.
  • exemplary assays generally involve monitoring Fenton or Haber-Weiss type chemical reactions to produce one or more autoantigenic and immunogenic protein fragments from the autoantigen.
  • One preferred assay is a standard metal-catalyzed Fenton reaction involving the following steps a) through e): a) preparing a lysate from about 1 X 10 6 or less confluent cultured cells such as HeLa cells and adding to the cell lysate: 1) a known or candidate compound in an amount between about 0.1 ⁇ M to 1 OO ⁇ M and 2) at least one Fenton metal salt such as ferrous sulfate in an amount between about 0.1 ⁇ M to lOO ⁇ M, preferably one such Fenton metal salt; b) incubating the HeLa cell lysate for a time sufficient to allow the metal- catalyzed oxidative proteolysis, e.g., for about 30 minutes, and typically followed by centrifugation to remove debris; c) separating the lysate, e.g., by SDS-PAGE gel electrophoresis, and transferring the separated lysate to a solid support suitable for performing a standard Western immunoblot,
  • the antibody is an autoimmune antibody (autoantibody) capable of specifically binding autoantigenic fragments of specified nucleic acid binding proteins such as topoisomerase I.
  • the antibody or reactive sera may also bind other autoantigens such as those described herein.
  • Methods for preparing monospecific patient autoimmune sera to topoisomerase I (and other autoantigens) are known in the field and are discussed below.
  • a standard metal-catalyzed Fenton reaction or other similar phrase includes one or more of the steps a) through e) above. It will be apparent from the discussion and examples that follow that the standard Fenton reaction can be readily modified to suit intended use. For example, the iron salt in the standard Fenton reaction can be substituted with one or more copper salts as described below. Other modifications include adding specified amounts of ascorbate or hydrogen peroxide. Additionally, the standard Fenton reaction described above can accomadate use of antibodies (or reactive sera ) against a variety of autoantigens, e.g., RNA polymerase II large subunit, UBF/NOR90, or Ul-70kDa. See the examples which follow.
  • autoantigens e.g., RNA polymerase II large subunit, UBF/NOR90, or Ul-70kDa. See the examples which follow.
  • Metal chelator, ED AC and zinc competition experiments Metal- catalyzed oxidation reactions were performed on HeLa lysates essentially as described above, with the following modifications. Experiments performed in the presence of ImM desferroxamine, 200 ⁇ M bathocuproine disulfonate or ImM D-penicillamine were carried out by adding the chelators immediately prior to addition of the Fe or Cu. ZnCl 2 competition reactions, and those performed in the presence of 5mM ED AC, were executed by adding these reagents to HeLa lysates 5mM prior to adding Fe/ascorbate.
  • cells were fixed in 4% paraformaldehyde (5 minutes, 4°C), permeabilized in acetone (30 seconds, 4°C), and stained using a standard monospecific patient serum recognizing topoisomerase I (diluted 1 : 160 in PBS). Bound antibodies were visualized with FITC-conjugated goat anti-human F(ab')2 (Organon Tekitika Corp./Cappel, Durham, NC). Coverslips were mounted with Permafluor (Lipshaw, Pittsburgh, PA), and viewed on a scanning confocal microscope system (model MRC 600, Bio-Rad Laboratories).
  • topoisomerase I After incubating in vitro for 30 minutes in the absence of added metal, topoisomerase I remained stable, and migrated as a single lOOkDa species ( Figure 1, lane 1). In these metal-free conditions, addition of either ascorbate or H 2 O 2 failed to generate any fragments ( Figure 1, lanes 2 & 3). Addition of lOO ⁇ M Fe induced the specific fragmentation of topoisomerase I, generating a major fragment of 95kDa, and several minor, discrete species migrating between
  • RNA polymerase II and topoisomerase I have been found to decrease in O 2 -depleted lysates. O 2 -depletion was performed as described Materials and Methods, prior to adding lOO ⁇ M Fe and 1.7mM ascorbate. Equal protein
  • the site-specific nature of metal-catalyzed oxidation reactions has previously indicated that these reactions are "caged" processes in which amino acid residues at metal-binding sites are specific targets of highly reactive free radical species generated at that site during a Fenton reaction (B. Halliwell et al., Arch. Biochem. Biophys., 246:501-514 (1986); E. Stadtman, Ann. Rev. Biochem., 62:797-821 (1993); T. Rana et al., J. Am. Chem.
  • topoisomerase I contains a single highly charged (mixed) tract containing 72 residues
  • NOR90/UBF contains 2 highly negatively-charged tracts of 21 and 28 residues
  • Ul-70kDa) a contains 2 highly charged (mixed) tracts containing 89 and 48 mixed residues
  • the large subunit of RNA polymerase II contains 3 highly charged (mixed) tracts of 25, 17 and 12 residues.
  • topoisomerase I Only intact topoisomerase I was detected in control keratinocytes ( Figure 4, lane 1), and H 2 O 2 treatment alone did not result in any autoantigen fragmentation ( Figure 4, lane 2). Incubation with 20 ⁇ M Cu overnight did not affect cell morphology or viability, and did not produce any fragmentation of topoisomerase I in the absence of added H 2 O 2 ( Figure 4, lane 3). In contrast, addition of H O after overnight Cu loading resulted in the marked fragmentation of topoisomerase I into the predominant Cu-characteristic band of 95kDa ( Figure 4, lane 4).
  • nucleolus The most likely subcellular site in which these increased metal concentrations occur is the nucleolus, since the scleroderma autoantigens are enriched in nucleoli (reviewed in (E. Tan, Cell, 67:841-842 (1991)), and several histochemical studies have demonstrated that the nucleolus has the unusual capacity to concentrate metal ions, including silver, zinc, cobalt and lead (C. Tandler, J. Histochem. Cytochem., 4:331-340 (1956); E. Pihl, J. Microscopie, 7:509-520 (1968); G. Studzinski, J. Histochem. Cytochem., 13:365-375 (1965); C. Tandler et al., J. Cell Biol, 41:91-108 (1969)).
  • Tan, Cell, 67:841-842 (1991)), Figure 5B), where they co-localize with sites of high-affinity metal binding (G. Studzinski, J. Histochem. Cytochem., 13:365-375 (1965); G. Studzinski et al., Stain. Technol, 39:397-401 (1964)), and Figure 5A), is therefore of great interest. It suggests that these antigens are unified both by their capacity to bind metals and their location at sites containing potentially increased local concentrations of redox active metals.
  • a therapeutic application of the invention therefore preferably involves delivering fragmentation inhibitors to the nucleoli.

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Abstract

On peut diviser des autoantigènes présentant des régions immunocryptiques au niveau de zones particulières en présence de métaux, tels que du fer ou du cuivre et des espèces d'oxygène réactif, afin d'obtenir des fragments de protéines antigéniques utiles pour diagnostiquer des maladies auto-immunes. On peut utiliser des substances jouant un rôle dans le processus de fragmentation afin de traiter des maladies auto-immunes et utiliser ces fragments afin d'augmenter la tolérance des malades. Cette protéolyse enzymatique peut être mise en application dans des domaines très vastes en tant qu'outil biochimique.
PCT/US1997/024100 1996-12-30 1997-12-30 Procedes et compositions servant a identifier des autoantigenes WO1998029109A1 (fr)

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US8163764B2 (en) 2002-04-26 2012-04-24 Asan Laboratories Company (Cayman) Limited Skincare methods
US8846039B2 (en) 2002-04-26 2014-09-30 Asan Laboratories Company (Cayman), Limited Method for ameliorating pruritus
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US8946295B2 (en) 2002-07-25 2015-02-03 Sunny Pharmtech Inc. Histone hyperacetylating agents for promoting wound healing and preventing scar formation
US9370524B2 (en) 2002-07-25 2016-06-21 Sunny Pharmatech Inc. Method for reducing radiation-induced normal tissue damage
WO2005005458A1 (fr) * 2003-07-09 2005-01-20 Valtion Teknillinen Tutkimuskeskus Methode de clivage de proteines

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