EP0815223A1 - Autoantigene specifique a la dermatomyosite - Google Patents

Autoantigene specifique a la dermatomyosite

Info

Publication number
EP0815223A1
EP0815223A1 EP96905719A EP96905719A EP0815223A1 EP 0815223 A1 EP0815223 A1 EP 0815223A1 EP 96905719 A EP96905719 A EP 96905719A EP 96905719 A EP96905719 A EP 96905719A EP 0815223 A1 EP0815223 A1 EP 0815223A1
Authority
EP
European Patent Office
Prior art keywords
dna
protein
rmi
antigen
serum
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Withdrawn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
EP96905719A
Other languages
German (de)
English (en)
Inventor
Hans Peter Seelig
Manfred Renz
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.)
Privates Institut fuer Imonologie und Molekulargenetik GmbH
Original Assignee
Privates Institut fuer Imonologie und Molekulargenetik GmbH
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Privates Institut fuer Imonologie und Molekulargenetik GmbH filed Critical Privates Institut fuer Imonologie und Molekulargenetik GmbH
Publication of EP0815223A1 publication Critical patent/EP0815223A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4713—Autoimmune diseases, e.g. Insulin-dependent diabetes mellitus, multiple sclerosis, rheumathoid arthritis, systemic lupus erythematosus; Autoantigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to a dermatomyositis-specific autoantigen, such a DNA coding and a method for producing such and its use.
  • Autoimmune diseases are characterized by the emergence of autoantibodies, i.e. Antibodies that are directed against components of your own organism. Autoantibodies can induce damage to the organism, an organ or an organ component and thereby trigger serious life-threatening diseases. Such a disease arises through various pathomechanisms, such as through neutralization of antigens, e.g. Hormones, by blocking or stimulating a receptor for biologically active substances, e.g. by autoantibodies against the receptor of the thyroid stimulating hormone in hyperthyroidism, by binding to certain cell or tissue structures with induction of a complement-mediated inflammation, e.g. Anti-car body glomerulonephritis against glomerular basement membranes. Tissue damage can also be induced by cell-mediated mechanisms (autoantibody-dependent cellular cytotoxicity) or by localized and systemic immune complex deposits after the autoantibodies have bound to soluble antigens.
  • pathomechanisms such as through neutralization of antigens, e.g. Hormone
  • rheumatic diseases especially inflammatory ones Rheumatic diseases, to which collagen diseases are attributed
  • Rheumatic diseases are characterized by the appearance of numerous autoantibodies.
  • antigens of the cell nucleus such as with double-stranded DNA, single-stranded DNA, RNA, histones, non-histone proteins, ribonucleoproteins, chromosome-associated antigens, for example centromeres or spindle apparatus, or with antigens which are only present in certain Phases of the cell cycle, for example cyclin, are expressed.
  • the above autoantibodies are found in diseases such as lupus erythematosus, Sjögren's syndrome, mixed collagenosis, polymyositis, scleroderma, CREST syndrome, Wegener's granulomatosis and dermatomyositis. They are mostly detected by reacting with nuclear extracts from calf or rabbit thymocytes. However, a differential diagnosis of these diseases, especially dermatomyositis, is not possible. However, this is a prerequisite for the choice of therapy.
  • the present invention is therefore based on the object of providing a means by which dermatomyositis can be detected by differential diagnosis.
  • the invention thus relates to a dermatomyositis-specific autoantigen which comprises the amino acid sequence of FIG. 2 or a functional derivative or fragment thereof.
  • the expression "functional derivative or fragment” encompasses any derivative or fragment of the amino acid sequence of FIG. 2 against which an autoantibody can be formed.
  • the expression relates to epitopes in the amino acid sequence which can act as antigens.
  • the amino acid sequence of FIG. 2 can have additions, substitutions and / or deletions of one or more amino acids, which also applies to the functional ones Derivatives or fragments thereof apply.
  • Another object of the invention is a nucleic acid coding for the above autoantigen.
  • This can be an RNA or a DNA.
  • the latter can e.g. be a genomic DNA or a cDNA.
  • a DNA is preferred which comprises the following:
  • hybridizing DNA indicates a DNA which hybridizes with a DNA of (a) under customary conditions, in particular at 20 ° C. below the melting point of the DNA.
  • the DNA of FIG. 2 was deposited with the DSM (German Collection of Microorganisms and Cell Cultures) as E. coli Hi-fl-213 under DSM 9800 on March 13, 1995.
  • DSM German Collection of Microorganisms and Cell Cultures
  • Mi-2 DNA a DNA according to the invention or an autoantigen encoded by it is referred to as "Mi-2 DNA” (“Mi-2 cDNA”) or "Mi-2 antigen”.
  • Mi-2 cDNA a DNA according to the invention or an autoantigen encoded by it
  • anti-Mi-2 positive a DNA according to the invention or an autoantigen encoded by it
  • Mi-2 indicates the patient's serum that reacted for the first time with nuclear extracts from calf or rabbit thymocytes.
  • Mi-2 cDNA it is expedient to screen a lambda phage expression library with the above patient sera. Positive clones can then be sequenced and used for further screening if necessary.
  • the production of a Mi-2 cDNA according to the invention is described in Examples 1-2. Their structural elucidation and that of the Mi-2 anti- gens are described in Examples 3-5.
  • a Mi-2 cDNA can be present in a vector or expression vector.
  • examples of such are known to the person skilled in the art.
  • these are e.g. pGEMEX, pUC derivatives, pGEX-2T and pET3b, the latter being preferred.
  • yeast e.g. to call pY100 and Ycpadl
  • animal cells e.g. pKCR, pEFBOS, cDM8 and pCEV4 must be specified.
  • suitable cells for expressing a Mi-2 cDNA present in an expression vector include the E. coli strains HB101, DH1, x1776, JM101, JM 109, and BL21, the latter being preferred, the yeast strain Saccharomyces cerevisiae and the animal cells L, 3T3, FM3A, CHO, COS, Vero and HeLa.
  • Mi-2 cDNA has to be inserted into an expression vector. He is also aware that this DNA can be inserted in conjunction with a DNA coding for another protein or peptide, so that the Mi-2 cDNA can be expressed in the form of a fusion protein.
  • rMi-2 antigen recombinantly produced Mi-2 antigen (hereinafter referred to as rMi-2 antigen), which may also be a fusion protein, is therefore also a subject of the invention.
  • Autoantigens according to the invention are distinguished by the fact that they recognize dermatomyositis-specific autoantibodies. They are therefore suitable for the differential diagnosis of collagen diseases, in particular dermatomyositis. Such a diagnosis can be made in conventional detection methods, in particular in a Western blot, an ELISA, an immunoprecipitation or an immunofluorescence.
  • the autoantigens according to the invention if appropriate , be labeled or be used in combination with labeled antibodies directed against them.
  • autoantigens according to the invention can be present in a kit.
  • This can contain the autoantigens, as stated in the above form, together with conventional additives, such as buffers and carrier material.
  • conventional additives such as buffers and carrier material.
  • autoantigens according to the invention are also suitable for therapeutic purposes.
  • they can be used to remove autoantibodies from the circulation of dermatomyositis patients by affinity chromatography. Removal of dermatomyositis-specific autoantibody-producing lymphocytes by coupling the autoantigens according to the invention to cell toxins is also contemplated.
  • nucleic acids according to the invention are suitable for diagnostic purposes of all kinds.
  • Figure 1 shows the cloning and composition of the 218 kD Mi-2 cDNA.
  • the upper part of the figure shows a restriction map with a scale in kb.
  • the horizontal lines represent isolated clones; the clones sequenced in both mixtures are indicated by dots.
  • the two small clones in the lower left corner were obtained according to the RACE protocol (cf. Froh ⁇ mann, MA et al., Proc. Natl. Acad. Sei. USA 85 (1 988), 8998-9002).
  • nt 738-4980 1: nt 738-4980; 2: nt 89-1047; 3: nt 242-4991; 4: nt 2020-4970; 5: nt 2293-4977; 6: nt 3313-4977; 7: nt 3709-4977; 8: nt 3755-6244; 9: nt 3932-6328; 10: nt 4056-6244; 1 1: nt -89 - -38; 1 2: nt -79 - -38,
  • Fig. 2 shows the nucleotide sequence and coding amino acid sequence of the
  • the framed sequences show the seven helicase-specific motifs (I, IA, II, IM, IV, V, and VI).
  • the four underlined regions contain eleven potential core target sequences, ten of which are located in three N-terminal regions (region a contains 3 motifs; region b 4 motifs and region C 3 motifs).
  • the dotted line (nt 490-520) shows a collection of glutamic acid and aspartic acid residues, which may interact electrostatically with chromatin (histones),
  • FIG. 3 shows a Northern blot analysis of the 218 kD Mi-2 gene expression in FIG.
  • HEp-2 cells RNA samples were separated using formaldehyde agarose gel electrophoresis according to their size and hybridized with 3 P-labeled Mi-2 cDNA with high stringency. Lane A: 5 ⁇ total RNA from HEp-2 cells; Lane B: 0.3 ⁇ g of HEp-2 cells poly (A) + RNA. The size of the band in the autoradiogram is approximately 6.8 kb,
  • NT potential core target sequences
  • CB potential chromatin binding region
  • I - VI Helicase-specific domains.
  • the thick horizontal line in the lower part of the figure shows a recombinant protein (rMi-2), which was used for immunological studies. A scale with the positions of the amino acids is shown at the top,
  • Figure 5 shows the localization of the 218 kD Mi-2 gene with fluorescence in situ
  • Hybridization on the short arm of human chromosome 12 (12p 13) (arrows), 6 shows immunoblots of SDS-PAGE separated (A) recombinant mi
  • 2-polypeptide (rMi-2) and (B) core proteins of HEp-2 cells which reacted with human and rabbit sera and affinity-purified anti-rMi-2-lg.
  • Lanes AI, B1 Human non-reactive control serum.
  • Lanes A2, B2 Human anti-Mi-2 positive serum No. 1 (Table 1) recognizes the rMi-2 with 55 kD (lane A2) as well as the natural 235 kD protein (lane B2) and an additional second natural nucleus ⁇ 80 kD protein.
  • Lanes A3, B3 Affinity-purified antibodies of serum No. 1 (human anti-rMi-2-Ig) react both with the rMi-2 used for affinity purification and with the natural 235 kD nuclear protein.
  • Lane A4, B4 Non-reactive rabbit preimmune serum.
  • Lanes A5 and B5 After immunization with rMi-2, the rabbit serum recognizes the recombinant antigen (rMi-2) and the natural 235 kD protein.
  • Lanes A6 and B6 Affinity-purified rabbit antibodies (rabbit anti-rMi-2-Ig) also react with both proteins, the recombinant antigen and the natural 235 kD protein.
  • Lane C (control) nuclear proteins of HEp-2 cells react with a variety of human autoantibodies,
  • FIG. 7 shows immunoblots of nuclear (N), mitochondrial (Mt), microsomal (Ms) and cytoplasmic (S-100, S) fractions (80 ⁇ g protein / cm) from HEp-2 cells which are associated with affinity purified rabbit anti-rMi-2 antibody reacted.
  • FIG. 8b shows an immunoprecipitation of proteins of a HEp-2 cell lysate with the DM patient serum No. 1 (P1), with rMi-2 protein affinity-purified antibody of serum No. 1 (P1 alg), affinity-purified ⁇ tem rabbit antibody (Ra alg), rabbit IgG (Ra Ig), rabbit immune serum (Ra IS) and rabbit pre-immune serum (Ra pIS).
  • P1 alg affinity-purified ⁇ tem rabbit antibody
  • Ra Ig rabbit IgG
  • Ra IS rabbit immune serum
  • Ra pIS rabbit pre-immune serum
  • Rabbit anti-Mi-2 serum This immunofluorescence shows a MACHINES SHOW ⁇ che nuclear fluorescence.
  • A1 Human anti-Mi-2 positive serum (No. 3, Table 1 ).
  • A2 Serum from patient No. 9, to whom anti-mitochondrial antibodies have been added, shows nuclear and mitochondrial fluorescence.
  • A3 The mixed serum shown in A2 was affinity-cleaned with rMi-2, which led to an exclusive nuclear fluorescence.
  • B1 Rabbit preimmune serum.
  • B2 A rabbit serum after the second booster with rMi-2 antigen showed intense nuclear fluorescence.
  • B3 The affinity-purified rabbit anti-M2 antibodies stained the nucleus,
  • Mi-2 antibodies Wells of microtiter plates were coated with 0.8 ⁇ g rMi-2 antigen and blocked with BSA. ODs of serial dilutions of anti-Mi-2 positive sera and a control serum are shown. Dilutions of 1: 200 were chosen for testing human sera, and
  • Fig. 1 1 shows the detection of anti-Mi-2 antibodies with the recombinant protein ELISA. Dashed line: border region (OD 0.5-0.6). The circled numbers indicate the number of negative sera tested with ODs between 0.05 and 0.5.
  • DM dermatomyositis
  • SLE systemic lupus erythematosus
  • RA rheumatoid arthritis
  • ANA positive sera Sera that were sent in for ANA analysis and reacted positively with HEp-2 cells (titer 1: ⁇ 160). Controls: sera from healthy individuals.
  • the IgG fraction of a human anti-Mi-2 positive serum was isolated by DEAE-Sepharose chromatography and used to screen approximately 400,000 PIaques of an oligo (dT) -primed HeLa cDNA expression library ( ⁇ UNIZAPXR, Stratagene, Heidelberg) used. After repeated purification, a positive clone was obtained and sequenced. Two 40 m ⁇ f ⁇ oligonucleotides, which were complementary to the two ends of the inert of this clone (4.3 kb), were used after radioactive labeling to screen the same library again in order to obtain further cDNAs in the 5 'and 3' direction.
  • AAATAAA sequence motif very similar to the polyadenylation consensus signal (AATAAA).
  • RNA and poly (A) + RNA were isolated from a human Zeil line, separated by size electrophoretically in a formaldehyde agarose gel and hybridized with radioactively labeled Mi-2 cDNA after transfer to a membrane.
  • the autoradiogram (Fig. 3) shows a signal at about 6.8 kb. This experiment demonstrates that the Mi-2 mRNA is large enough to contain the 5.7 kb Mi-2 cDNA.
  • Example 5 Example 5:
  • a fragment of the Mi-2 cDNA (nt 242-4991) was labeled with biotin-dUTP and used for in situ hybridization on human metaphase chromosomes.
  • Hybridized DNA was detected with avidin (Jackson Immuno Research Laboratories, West Grove, PA USA) labeled with fluorescein isothiocyanate and an Axiophot photo microscope (Zeiss, Oberkochen). Images were digitized with a CCD camera (Photometrics, Tuscon, AZ, USA) and processed with an Apple computer system. Specific fluorescence signals were observed on the distal side of the short arm of chromosome 12 (12p13). Twelve out of eight checked metaphase chromosomes showed specific signals in both homologues).
  • a segment in the central part of the Mi-2 cDNA (nt 151 1 -2998) (FIGS. 1 and 2) was terminated at the 5 'end with a Ndel interface and at the 3' end with six histidine codons and a BamHI Provide the interface and after ligation into the bacterial expression vector pET3b in E. coli BL21 (DE3) pLysS (see Studier, FW et al., Methods Enzymol. 185 (1990), 60-89). After the culture had been grown to an OD ⁇ oo of 0.5, it was induced with 1 mM isopropyl-yff-D-thiogalactopyranoside, cultivated for a further 8 hours and harvested.
  • the cell pellet was suspended in 10 mM Tris-HCl (pH 8.0), 0.1 M sodium phosphate and 8 M urea and the insoluble material, which contained the recombinant protein, was obtained by centrifugation, and in 8 M urea, 6 M Guanidinium-HCI dissolved and obtained using Ni + chelate affinity chromatography (Quiagen).
  • the yield was about 200 mg of recombinant protein (rMi-2) per one liter of culture.
  • the size of the Mi-2 antigen as estimated in the SDS-PAGE corresponded to the derived molecular weight of 55 kD.
  • Microtiter plates (Immunolon, Dynatech, Denkendorf) were incubated with 100 ⁇ l per well with a solution consisting of 8 ⁇ g / ml rMi-2 antigen and 0.1 M sodium carbonate (pH 9.6) for 16 hours at 4 ° C. and then with 300 / I blocked per well with a solution consisting of PBS and 0.1% BSA for 16 hours at 4 ° C.
  • Antibody screening was carried out with 200 / I serum per well (serum dilutions: 1: 300 in PBS, 0.01% Tween and 0.1% BSA) performed during a 30 minute incubation at 37 ° C. After a washing step (5 ⁇ PBS, 0.01% Tween, 30 min.
  • the wells were each treated with 20O; l of a 1: 20000 diluted peroxidase-labeled rabbit anti-human-IgG F (ab ') 2 in PBS, 0.01% Tween and 0.5% BSA and incubated for 30 min at 37 ° C. Bound antibodies were visualized with OPD and the enzyme reaction was carried out with 50 / I 4 MH 2 SO 4 depending stopped well. The absorbance was measured at 492/620 nm bichromatic. this assay 1 368 human sera were tested (Fig. 10).
  • the average OD value was a Kon ⁇ troll group of 189 healthy persons ⁇ 0.25 _ 0.08: ODs between 0.5 and 0.6 were evaluated as borderline, ODs> 0.6 as positive
  • the antibody concentrations (ODs) of the twelve anti-Mi-2 positive dermatomyositis patients are shown in FIG 1. With the exception of one serum, which was negative in the ELISA but positive in the immunoblot assay, all patient sera showed OD values that were significantly above the limit.

Abstract

La présente invention concerne un autoantigène spécifique à la dermatomyosite, un ADN codant cet autoantigène, un procédé de préparation de cet antigène et son utilisation.
EP96905719A 1995-03-15 1996-03-08 Autoantigene specifique a la dermatomyosite Withdrawn EP0815223A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE19509279A DE19509279C1 (de) 1995-03-15 1995-03-15 Dermatomyositis-spezifisches Autoantigen
DE19509279 1995-03-15
PCT/DE1996/000444 WO1996028547A1 (fr) 1995-03-15 1996-03-08 Autoantigene specifique a la dermatomyosite

Publications (1)

Publication Number Publication Date
EP0815223A1 true EP0815223A1 (fr) 1998-01-07

Family

ID=7756673

Family Applications (1)

Application Number Title Priority Date Filing Date
EP96905719A Withdrawn EP0815223A1 (fr) 1995-03-15 1996-03-08 Autoantigene specifique a la dermatomyosite

Country Status (5)

Country Link
US (3) US6329517B1 (fr)
EP (1) EP0815223A1 (fr)
JP (1) JPH11503604A (fr)
DE (1) DE19509279C1 (fr)
WO (1) WO1996028547A1 (fr)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP5191544B2 (ja) * 2008-09-01 2013-05-08 学校法人慶應義塾 皮膚筋炎の検出方法および診断キット
CN111849930B (zh) * 2020-08-18 2022-01-25 珠海丽珠试剂股份有限公司 Mi-2重组抗原及其制备方法和应用

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU8544591A (en) 1990-09-07 1992-03-30 Board Of Regents Of The University Of Oklahoma, The Antigens associated with polymyositis and with dermatomyositis

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9628547A1 *

Also Published As

Publication number Publication date
JPH11503604A (ja) 1999-03-30
US6500923B1 (en) 2002-12-31
US6329517B1 (en) 2001-12-11
DE19509279C1 (de) 1996-05-15
WO1996028547A1 (fr) 1996-09-19
US6440679B1 (en) 2002-08-27

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