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WO1997019178A2 - Autoantigenic metal proteases and methods of diagnosing autoimmune diseases - Google Patents

Autoantigenic metal proteases and methods of diagnosing autoimmune diseases Download PDF

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Publication number
WO1997019178A2
WO1997019178A2 PCT/DE1996/002094 DE9602094W WO9719178A2 WO 1997019178 A2 WO1997019178 A2 WO 1997019178A2 DE 9602094 W DE9602094 W DE 9602094W WO 9719178 A2 WO9719178 A2 WO 9719178A2
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Prior art keywords
polypeptide
nucleic acid
autoantigenic
acid sequence
autoimmune diseases
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Application number
PCT/DE1996/002094
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German (de)
French (fr)
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WO1997019178A3 (en
Inventor
Ulrich Krawinkel
Simon Mauch
Radislav Sedlacek
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Universität Konstanz
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Publication of WO1997019178A2 publication Critical patent/WO1997019178A2/en
Publication of WO1997019178A3 publication Critical patent/WO1997019178A3/en

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    • 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
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4713—Autoimmune diseases, e.g. Insulin-dependent diabetes mellitus, multiple sclerosis, rheumathoid arthritis, systemic lupus erythematosus; Autoantigens

Definitions

  • the present invention relates to nucleic acids which contain at least one nucleic acid sequence coding for a polypeptide, the polypeptide containing at least one autoantigenic determinant originating from a metal protease, the polypeptide itself and antibodies directed against the polypeptide and specific directed against the polypeptide Inhibitors. Furthermore, the present invention relates to methods and kits for the detection of autoantigenic metal proteases and autoimmune diseases such as rheumatoid arthritis
  • the present invention is based on the object ⁇ determine a new system for diagnosing autoimmune diseases, which overcomes the disadvantages of the examination methods presently used in the prior art and insbe ⁇ sondere overall eicrnet for screening for autoimmune diseases
  • This object is achieved by the embodiments of the present invention which are characterized in the claims .
  • a first subject of the invention relates to a nucleic acid which contains at least one nucleic acid sequence coding for a polypeptide, the polypeptide comprising at least one autoantigenic determinant derived from a metal protease.
  • nucleic acid and nucleic acid sequence mean natural or semisynthetic or synthetic or modified nucleic acid molecules made from deoxyribonucleotides and / or ribonucleotides and / or modified nucleotides.
  • polypeptide encompasses naturally occurring polypeptides and recombinant polypeptides.
  • Naturally occurring polypeptides according to the invention comprise autoantigenic metal proteases per se or autoantigenic sections or fragments thereof.
  • the metal proteases are so-called membrane proteins, i.e. they adhere to the cell surface membrane in such a way that the functional parts of the protein face outwards and can optionally be cleaved off proteolytically.
  • Metal proteases preferably originate from macrophages and related cell types and occur, for example, in the inner skin of the joint, Afembrana synovialis, from mammals.
  • Recombinant polypeptides refer to a construct made using molecular biological techniques, which is based on the natural DNA of the original genome or the natural DNA modified with a foreign DNA sequence and can be recombined, e.g. with plasmids, and can be replicated and expressed in a suitable host system.
  • autoantigenic determinant or epitope means that one or more linear or conformational regions of the Metal proteases are recognized by one's own immune system by so-called autoantibodies.
  • autoantibodies The presence of autoantibodies is regarded as an indication that a protein of one's own cells, for example in the macrophages of the synovial membrane, is expressed quantitatively and qualitatively abnormally as a result of a disease.
  • the possible cause of joint destruction in rheumatoid arthritis is assumed to be that an as yet unidentified infectious agent stimulates joint macrophages for the increased expression of proteases which release further potential autoantigens through proteolysis of joint tissue (cf. Opdenakker et al., 1994, Immunol.
  • the nucleic acid comprises the nucleic acid sequence shown in FIG. 1 or sections or fragments thereof. In a particularly preferred embodiment of the present invention, the nucleic acid comprises the sequence section 1110-2206 of the nucleic acid sequence shown in FIG. 1.
  • Another object of the present invention is a vector which contains the nucleic acid according to the invention defined above for the expression of the recombinant polypeptide in prokaryotic or eukaryotic host cells.
  • the vector according to the invention can preferably contain suitable regulatory elements, such as promoters, enhancers, termination sequences.
  • the vector according to the invention can for example be an expression vector or a vector for preferably stable integration of the nucleic acid according to the invention into the genetic material of a host cell.
  • a suitable Expressi ⁇ onssystem includes, for example, the vector pET-24 and E. coli BRMDE3) or BL21 (DE3), or for expression in eukaryonti ⁇ 's cells the vector pcDNA3 and cos cells or CHO cells.
  • Another object of the present invention is a host cell which contains the nucleic acid according to the invention or the vector according to the invention.
  • Suitable host cells are for example prokaryotes such as E. coli or eukaryotic host cells such as cos or CHO. In principle, any commercially available pro- or eukaryotic expression system can be used.
  • the nucleic acid according to the invention is either stably integrated in the genetic material of the host cell or the vector according to the invention contains suitable regulatory areas for replication, transcription and / or translation in vivo and / or in vitro, ie also in the cell-free system.
  • polypeptide itself, which is encoded by the nucleic acid sequence defined above, it being possible for the nucleic acid sequence to be degenerate in accordance with the genetic code.
  • the polypeptide according to the invention can be modified in vitro by post-translational reactions in vivo or by chemical and / or enzymatic methods known in the prior art.
  • the polypeptide comprises the amino acid sequence shown in FIG. 2 or sections or fragments thereof, for example the expression product encoded by sequence section 1110-2206 of the nucleic acid sequence shown in FIG.
  • nucleic acid sequence according to the invention, the vector according to the invention and the polypeptide according to the invention can be produced by methods known in the prior art (cf. Ausubel et al., 1991, Current Protocols in Molecular Biology, Wiley Interscience, New York, N.Y.).
  • Another object of the present invention is a po- lyklonaler, preferably monospecific, or monoclonal antibody against the above-defined polypeptide ge ⁇ is directed.
  • the antibody of the invention can be produced by methods known in the art (see Coligan et al., 1993, Current Protocols in Immunology, Wiley Interscience, New York, NY).
  • the present invention further relates to a method for the detection of autoimmune diseases, in particular rheumatoid arthritis (RA) and other autoimmune forms of arthritis such as SLE (systemic lupus erythematosus), MCTD (mixed collagenosis) and PSS (progressive scleroderma), in which either the above defined polypeptide or the above defined antibody with serum from a mammal are subjected to an enzyme immunoassay, preferably an ELISA.
  • RA rheumatoid arthritis
  • SLE systemic lupus erythematosus
  • MCTD mixed collagenosis
  • PSS progressive scleroderma
  • the detection of autoantibodies against metal proteases in the ELISA has the advantages of being non-invasive and considerably less expensive than other diagnostic methods for rheumatoid arthritis, such as X-ray examination or joint puncture.
  • autoantibodies can already be detectable if imaging methods do not yet indicate damage to the joint.
  • specific inhibitors of the metal protease can be used in pharmaceutical formulations for therapy in the joint of a rheumatism patient.
  • Another object of the present invention is a diagnostic kit for the detection of an autoimmune disease, which contains at least either the above-defined polypeptide or the above-defined antibody.
  • FIG. 2 (A) is the 508 AS long primary sequence of the metal protease MPRS, which is encoded by the DNA sequence shown in FIG. 1 (molecular weight, calculated from the 57.4 kDa cDNA sequence).
  • FIG. 2 (B) shows structural properties of this metal protease.
  • FIG. 3 is a Coomassie blue-colored SDS polyacrylamide gel with recombinant MPRS isolated from mononuclear blood cells and its RASED fragment, which comes from an RA patient
  • FIG. 5 shows a homology comparison of the metal protease according to the invention shown in FIG. 2 with previously known metal proteases.
  • Bold amino acids mark strongly conserved areas.
  • the domains typical for metal proteases hydrophobic signal peptide, cleavable propeptide, catalytic domain with Zn 2+ site, "Hmge” region and Hemopexm-like domain
  • MMP-1 membrane type Ma ⁇ trix metalloprotemase
  • the isolation of mRNA from synovial membrane cells of an RA patient is carried out according to methods known in the prior art, and an expression cDNA library is produced using the lambda Zap II expression vector (from Stratagene).
  • the screening (screening) of the cDNA library is carried out with purified immunoglobulin G from the RA patient.
  • a primary clone "Rased” which comprises a fragment of 1106 bp of the metal protease (MPRS) mRNA, was obtained from the cDNA library (3 ⁇ 10 5 clones / ⁇ g mRNA), this fragment contains an autoantigenic epitope based on the screening with immunoglobulin G of the RA patient.
  • MPRS metal protease
  • MPRS mRNA is detected in peripheral mononuclear blood cells via reverse transcription / polymerase chain reaction.
  • the isolation of the mRNA from peripheral mononuclear blood cells and the production of a cDNA bank with lambda Zap II is carried out as described above.
  • the cDNA library is screened with a labeled DNA probe based on the RASED clone.
  • the database comparison shown in FIG. 5 shows at the protein level between 30 and 35% identity (with a similarity of over 50%) to known metal proteases, the catalytic domain and the domain structure typical of metal proteases being present.
  • the expression and purification of MPRS from E. coli is carried out with pET24 and with N-terminal oligo-histidine "tag".
  • the expression and purification of RASED from E. coli is performed with pGEX 1 as a fusion protein with glutamine-S-transferase.
  • the peptide constructs thus obtained were electrophoretically separated using an SDS polyacrylamide gel and the separated peptide constructs were detected by Coomas blue staining of the SDS polyacrylamide gel; see. Figure 3 '.-

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Abstract

The invention concerns nucleic acids which contain at least one nucleic acid sequence coding for a polypeptide, the polypeptide containing at least one autoantigenic determinant originating from a metallic protease. The invention also concerns the polypeptide itself, antibodies directed at the polypeptide and specific inhibitors directed at the polypeptide. The invention further concerns methods and kits for detecting autoantigenic metallic proteases and autoimmune diseases, such as rheumatoid arthritis.

Description

AUTOANTIGENE METALLPROTEASEN UND VERFAHREN ZUR DIAGNOSE VON AUTOIM MUNKRANKHEΠΈN AUTOANTIGENTAL METAL PROTEASES AND METHOD FOR DIAGNOSIS OF AUTOIM MUNKRANKHEΠΈN

Die vorliegende Erfindung betrifft Nukleinsäuren, die minde¬ stens eine für em Polypeptid kodierende Nukleinsauresequenz enthalten, wobei das Polypeptid mindestens eine aus einer Me¬ tallprotease stammende autoantigene Determinante enthält, das Polypeptid selbst sowie gegen das Polypeptid gerichtete Anti¬ körper und gegen das Polypeptid gerichtete spezifische Inhibi¬ toren. Ferner betrifft die vorliegende Erfindung Verfahren und Kits zum Nachweis von autoantigenen Metallproteasen und von Au- toimmunkrankheiten wie rheumatoide ArthritisThe present invention relates to nucleic acids which contain at least one nucleic acid sequence coding for a polypeptide, the polypeptide containing at least one autoantigenic determinant originating from a metal protease, the polypeptide itself and antibodies directed against the polypeptide and specific directed against the polypeptide Inhibitors. Furthermore, the present invention relates to methods and kits for the detection of autoantigenic metal proteases and autoimmune diseases such as rheumatoid arthritis

Trotz umfangreicher Untersuchungen konnten bisher Autoimmun¬ krankheiten und insbesondere deren Etiologie noch nicht aufge¬ klart werden, was natürlich die therapeutische Behandlung sowie die Diagnosemόglichkeiten stark enschränkt bzw. nachteilig be¬ einflußt. Beispielsweise erfordert die gegenwärtige Diagnose von rheumatoider Arthritis unter anderem eine Rontgenun- tersuchung, m gravierenden Fällen sogar eine Gelenkpunktion, was für den Patienten äußerst belastend bzw schmerzhaft sein kann. Ferner ist mit den im Stand der Technik bekannten Diagnosemoglichkeiten eine Frύdiagnose von beispielsweise rheu¬ matoider Arthritis und damit verbunden die Möglichkeit einer entsprechend frühzeitigen therapeutischen Behandlung meistens nicht gegeben (vgl. Amett, F.C. et al . (1987) Arthritis Rheum 31 315) .Despite extensive investigations, it has not yet been possible to elucidate autoimmune diseases and, in particular, their etiology, which of course severely limits or adversely affects the therapeutic treatment and the diagnostic options. For example, the current diagnosis of rheumatoid arthritis requires, among other things, an X-ray examination, in serious cases even a joint puncture, which can be extremely stressful or painful for the patient. Further, with the known in the art Diagnosemoglichkeiten a Frύdiagnose of example rheu ¬ matoider arthritis and the associated possibility of a correspondingly early therapeutic treatment usually not given (see FIG. Amett, FC et al. (1987) Arthritis Rheum 31 315).

Somit liegt der vorliegenden Erfindung die Aufgabe zugrunde, ein neues System zur Diagnose von Autoimmunkrankheiten bereit¬ zustellen, welches die Nachteile der gegenwartig im Stand der Technik verwendeten Untersuchungsmethoden beseitigt und insbe¬ sondere für Vorsorgeuntersuchungen auf Autoimmunkrankheiten ge- eicrnet ist Diese Aufgabe wird durch die in den Patentansprüchen gekenn¬ zeichneten Ausführungsformen der vorliegenden Erfindung gelöst. Thus, the present invention is based on the object ¬ determine a new system for diagnosing autoimmune diseases, which overcomes the disadvantages of the examination methods presently used in the prior art and insbe¬ sondere overall eicrnet for screening for autoimmune diseases This object is achieved by the embodiments of the present invention which are characterized in the claims .

Ein erster erfindungsgemäßer Gegenstand betrifft eine Nu¬ kleinsäure, die mindestens eine für ein Polypeptid kodierende Nukleinsäuresequenz enthält, wobei das Polypeptid mindestens eine aus einer Metallprotease stammende autoantigene Determi¬ nante umfaßt .A first subject of the invention relates to a nucleic acid which contains at least one nucleic acid sequence coding for a polypeptide, the polypeptide comprising at least one autoantigenic determinant derived from a metal protease.

Die Begriffe "Nukleinsäure" und "Nukleinsäuresequenz" bedeuten natürliche oder halbsynthetische oder synthetische oder modifi¬ zierte Nukleinsäuremoleküle aus Desoxyribonukleotiden und/oder Ribonukleotiden und/oder modifizierten Nukleotiden.The terms “nucleic acid” and “nucleic acid sequence” mean natural or semisynthetic or synthetic or modified nucleic acid molecules made from deoxyribonucleotides and / or ribonucleotides and / or modified nucleotides.

Der Begriff "Polypeptid" umfaßt natürlich vorkommende Polypep¬ tide und rekombinante Polypeptide. Natürlich vorkommende Poly¬ peptide umfassen erfindungsgemäß autoantigene Metallproteasen per se oder autoantigene Abschnitte bzw. Fragmente davon. In einer bevorzugten Ausführungsform der vorliegenden Erfindung sind die Metallproteasen sogenannte Membranproteine, d.h. sie haften so in der Zelloberflächenmembran, daß die funktionellen Teile des Proteins nach außen gewandt sind und gegebenenfalls proteolytisch abgespalten werden können. Metallproteasen stam¬ men vorzugsweise aus Makrophagen und verwandten Zelltypen und kommen beispielsweise in der Gelenkinnenhaut, Afembrana synovia - lis, von Säugern vor. Rekombinante Polypeptide bezeichnen ein mit molekularbiologischen Techniken hergestelltes Konstrukt, dem die natürliche DNA des originalen Genoms bzw. die natürli¬ che DNA modifiziert mit einer fremden DNA-Sequenz zugrunde liegt und rekombiniert werden kann, z.B. mit Plasmiden, und in einem geeigneten Wirtssystem repliziert und exprimiert werden kann.The term “polypeptide” encompasses naturally occurring polypeptides and recombinant polypeptides. Naturally occurring polypeptides according to the invention comprise autoantigenic metal proteases per se or autoantigenic sections or fragments thereof. In a preferred embodiment of the present invention, the metal proteases are so-called membrane proteins, i.e. they adhere to the cell surface membrane in such a way that the functional parts of the protein face outwards and can optionally be cleaved off proteolytically. Metal proteases preferably originate from macrophages and related cell types and occur, for example, in the inner skin of the joint, Afembrana synovialis, from mammals. Recombinant polypeptides refer to a construct made using molecular biological techniques, which is based on the natural DNA of the original genome or the natural DNA modified with a foreign DNA sequence and can be recombined, e.g. with plasmids, and can be replicated and expressed in a suitable host system.

Der Begriff "autoantigene Determinante bzw. Epitop" bedeutet, daß ein oder mehrere lineare oder konformationelle Bereiche der Metallprotease vom eigenen Immunsystem durch sogenannte Autoan¬ tikörper erkannt werden. Das Vorkommen von Autoantikörpern wird als Indiz dafür gewertet, daß ein Protein der eigenen Zellen, z.B. in den Makrophagen der Synovialmembran, als Folge einer Krankheit quantitativ und qualitativ abnormal ausgeprägt wird. Beispielsweise wird als mögliche Ursache der Gelenkzerstörung bei rheumatoider Arthritis angenommen, daß ein bisher nicht identifiziertes infektiöses Agens Gelenkmakrophagen zur ver¬ mehrten Expression von Proteasen anregt, die durch Proteolyse von Gelenkgewebe weitere potentielle Autoantigene freisetzen (vgl. Opdenakker et al . , 1994, Immunol. Today 15: 103; Wicks et al., 1994, Immunol. Today 15:553) . In einer bevorzugten Ausführungsform der vorliegenden Erfindung umfaßt die Nuklein¬ säure die in Figur 1 gezeigte Nukleinsäuresequenz oder Ab¬ schnitte bzw. Fragmente davon. In einer besonders bevorzugten Ausführungsform der vorliegenden Erfindung umfaßt die Nuklein¬ säure den Sequenzabschnitt 1110-2206 der in Figur 1 gezeigten Nukleinsäuresequenz.The term "autoantigenic determinant or epitope" means that one or more linear or conformational regions of the Metal proteases are recognized by one's own immune system by so-called autoantibodies. The presence of autoantibodies is regarded as an indication that a protein of one's own cells, for example in the macrophages of the synovial membrane, is expressed quantitatively and qualitatively abnormally as a result of a disease. For example, the possible cause of joint destruction in rheumatoid arthritis is assumed to be that an as yet unidentified infectious agent stimulates joint macrophages for the increased expression of proteases which release further potential autoantigens through proteolysis of joint tissue (cf. Opdenakker et al., 1994, Immunol. Today 15: 103; Wicks et al., 1994, Immunol. Today 15: 553). In a preferred embodiment of the present invention, the nucleic acid comprises the nucleic acid sequence shown in FIG. 1 or sections or fragments thereof. In a particularly preferred embodiment of the present invention, the nucleic acid comprises the sequence section 1110-2206 of the nucleic acid sequence shown in FIG. 1.

Ein weiterer Gegenstand der vorliegenden Erfindung ist ein Vek¬ tor, der die vorstehend definierte, erfindungsgemäße Nuk¬ leinsäure zur Expression des rekombinanten Polypeptids in pro- karyotischen oder eukaryotischen Wirtszellen enthält. Der er¬ findungsgemäße Vektor kann vorzugsweise geeignete regulato- rische Elemente, wie Promotoren, Enhancer, Terminationssequen- zen, enthalten. Der erfindungsgemäße Vektor kann beispielsweise ein Expressionsvektor oder ein Vektor zur vorzugsweise stabilen Integration der erfindungsgemäßen Nukleinsäure in das geneti¬ sche Material einer Wirtszelle sein. Ein geeignetes Expressi¬ onssystem umfaßt beispielsweise den Vektor pET-24 und E. coli BRMDE3) oder BL21(DE3) , oder für die Expression in eukaryonti¬ schen Zellen den Vektor pcDNA3 und cos-Zellen oder CHO-Zellen.Another object of the present invention is a vector which contains the nucleic acid according to the invention defined above for the expression of the recombinant polypeptide in prokaryotic or eukaryotic host cells. The vector according to the invention can preferably contain suitable regulatory elements, such as promoters, enhancers, termination sequences. The vector according to the invention can for example be an expression vector or a vector for preferably stable integration of the nucleic acid according to the invention into the genetic material of a host cell. A suitable Expressi ¬ onssystem includes, for example, the vector pET-24 and E. coli BRMDE3) or BL21 (DE3), or for expression in eukaryonti¬'s cells the vector pcDNA3 and cos cells or CHO cells.

Ein weiterer Gegenstand der vorliegenden Erfindung ist eine Wirtszelle, welche die erfindungsgemäße Nukleinsäure oder den erfindungsgemäßen Vektor enthält. Geeignete Wirtszellen sind beispielsweise Prokaryonten, wie E. coli , oder eukaryotische Wirtszellen wie cos oder CHO. Im Prinzip kann jedes im Handel erhältliche pro- oder eukaryontische Expressionssystem verwen¬ det werden. Für eine ausreichende Expressionsrate ist die erfindungsgemäße Nukleinsäure entweder im genetischen Material der Wirtszelle stabil integriert oder der erfindungsgemäße Vek¬ tor enthält geeignete regulatorische Bereiche zur Replikation, Transkription und/oder Translation in vivo und/oder in vi tro, d.h. auch im zellfreien System.Another object of the present invention is a host cell which contains the nucleic acid according to the invention or the vector according to the invention. Suitable host cells are for example prokaryotes such as E. coli or eukaryotic host cells such as cos or CHO. In principle, any commercially available pro- or eukaryotic expression system can be used. For a sufficient expression rate, the nucleic acid according to the invention is either stably integrated in the genetic material of the host cell or the vector according to the invention contains suitable regulatory areas for replication, transcription and / or translation in vivo and / or in vitro, ie also in the cell-free system.

Ein weiterer erfindungsgemäßer Gegenstand ist das Polypeptid selbst, das von der vorstehend definierten Nukleinsäuresequenz kodiert wird, wobei die Nukleinsäuresequenz entsprechend dem genetischen Code degeneriert sein kann. Das erfindungsgemäße Polypeptid kann durch post-translationale Reaktionen in vivo oder durch im Stand der Technik bekannte chemische und/oder en- zymatische Methoden in vi tro modifiziert sein. In einer be¬ vorzugten Ausführungsform der vorliegenden Erfindung umfaßt das Polypeptid die in Figur 2 gezeigte Aminosäuresequenz oder Ab¬ schnitte bzw. Fragmente davon, beispielsweise das vom Se¬ quenzabschnitt 1110-2206 der in Figur 1 gezeigten Nukleinsäure¬ sequenz kodierte Expressionsprodukt.Another object of the invention is the polypeptide itself, which is encoded by the nucleic acid sequence defined above, it being possible for the nucleic acid sequence to be degenerate in accordance with the genetic code. The polypeptide according to the invention can be modified in vitro by post-translational reactions in vivo or by chemical and / or enzymatic methods known in the prior art. In a preferred embodiment of the present invention, the polypeptide comprises the amino acid sequence shown in FIG. 2 or sections or fragments thereof, for example the expression product encoded by sequence section 1110-2206 of the nucleic acid sequence shown in FIG.

Die erfindungsgemäße Nukleinsäuresequenz, der erfindungsgemaße Vektor sowie das erfindungsgemäße Polypeptid können durch im Stand der Technik bekannte Verfahren hergestellt werden (vgl. Ausubel et al. , 1991, Current Protocols in Molecular Biology, Wiley Interscience, New York, N.Y.) .The nucleic acid sequence according to the invention, the vector according to the invention and the polypeptide according to the invention can be produced by methods known in the prior art (cf. Ausubel et al., 1991, Current Protocols in Molecular Biology, Wiley Interscience, New York, N.Y.).

Ein weiterer Gegenstand der vorliegenden Erfindung ist ein po- lyklonaler, vorzugsweise monospezifischer, oder monoklonaler Antikörper, der gegen das vorstehend definierte Polypeptid ge¬ richtet ist. Der erfindungsgemäße Antikörper kann durch im Stand der Technik bekannte Verfahren hergestellt werden (vgl. Coligan et al., 1993, Current Protocols in Immunology, Wiley Interscience, New York, N.Y.) . Ein weiterer Gegenstand der vorliegenden Erfindung betrifft ein Verfahren zum Nachweis von Autoimmunkrankheiten, insbesondere von rheumatoider Arthritis (RA) und anderer autoimmuner Arthri¬ tisformen wie SLE (Systemischer Lupus Erythematodes) , MCTD (Mischkollagenose) und PSS (Progressive Sklerodermie) , worin entweder das vorstehend definierte Polypeptid oder der vorste¬ hend definierte Antikörper mit Serum eines Säugers einem Enzym- immunoassay, vorzugsweise einem ELISA, unterworfen werden.Another object of the present invention is a po- lyklonaler, preferably monospecific, or monoclonal antibody against the above-defined polypeptide ge ¬ is directed. The antibody of the invention can be produced by methods known in the art (see Coligan et al., 1993, Current Protocols in Immunology, Wiley Interscience, New York, NY). The present invention further relates to a method for the detection of autoimmune diseases, in particular rheumatoid arthritis (RA) and other autoimmune forms of arthritis such as SLE (systemic lupus erythematosus), MCTD (mixed collagenosis) and PSS (progressive scleroderma), in which either the above defined polypeptide or the above defined antibody with serum from a mammal are subjected to an enzyme immunoassay, preferably an ELISA.

Beispielsweise hat der Nachweis von Autoantikörpern gegen Me¬ tallproteasen im ELISA gegenüber anderen Diagnoseverfahren bei rheumatoider Arthritis, wie Röntgenuntersuchung oder Gelenk¬ punktion, die Vorteile, nicht-invasiv und erheblich kostengün¬ stiger zu sein. Außerdem können Autoantikörper aufgrund der Empfindlichkeit des Immunsystems auf bereits geringe Mengen Au¬ toantigen schon nachweisbar sein, wenn bildgebende Verfahren noch keine Beschädigung des Gelenkes anzeigen. Es ist auch mög¬ lich, daß spezifische Inhibitoren der Metallprotease in pharma¬ zeutischen Formulierungen zur Therapie im Gelenk eines Rheuma¬ patienten verwendet werden können.For example, the detection of autoantibodies against metal proteases in the ELISA has the advantages of being non-invasive and considerably less expensive than other diagnostic methods for rheumatoid arthritis, such as X-ray examination or joint puncture. In addition, due to the sensitivity of the immune system to already small amounts of autoantigens, autoantibodies can already be detectable if imaging methods do not yet indicate damage to the joint. It is also possible that specific inhibitors of the metal protease can be used in pharmaceutical formulations for therapy in the joint of a rheumatism patient.

Ein weiterer Gegenstand der vorliegenden Erfindung ist ein dia¬ gnostischer Kit zum Nachweis einer Autoimmunkrankheit, der mindestens entweder das vorstehend definierte Polypeptid oder den vorstehend definierten Antikörper enthält.Another object of the present invention is a diagnostic kit for the detection of an autoimmune disease, which contains at least either the above-defined polypeptide or the above-defined antibody.

Fiemr 1 zeigt die 3222 bp lange DNA-Sequenz, die für die Me¬ tallprotease MPRS aus der Synovialmembran eines Rheumapatienten kodiert (Herkunft: Synovialmembran eines RA-Patienten; Eigen¬ schaften der Sequenz: Translationsstart ATG an pos . 82 mit charakteristischer eukaryontischer Startsequenz, Translations- stop TGA an pos. 1606, autoantigener Bereich zwischen pos. 1110 und pos. 2206 (kursiv) ; 88 % Identität zur repetitiven alu J- Subfamilie im untranslatierten Bereich von pos. 1480-1590) . Figur 2 (A) ist die 508 AS lange Primarsequenz der Metallpro¬ tease MPRS, die von der in Figur 1 gezeigten DNA-Sequenz ko¬ diert wird (Molekulargewicht, errechnet aus cDNA-Sequenz 57,4 kDa) . Figur 2 (B) zeigt strukturelle Eigenschaften dieser Me¬ tallprotease .Fiemr 1, the 3222 bp long DNA sequence encoding the Me ¬ tallprotease MPRS from the synovial membrane of patients with rheumatoid arthritis shows (origin: synovial membrane of RA patients; self ¬ properties of the sequence: translation start ATG at pos 82 eukaryotic with characteristic start sequence. Translation stop TGA at item 1606, autoantigenic area between item 1110 and item 2206 (italics); 88% identity to the repetitive alu J subfamily in the untranslated area from item 1480-1590 ) . FIG. 2 (A) is the 508 AS long primary sequence of the metal protease MPRS, which is encoded by the DNA sequence shown in FIG. 1 (molecular weight, calculated from the 57.4 kDa cDNA sequence). FIG. 2 (B) shows structural properties of this metal protease.

Figur 3 ist em Coomassie Blau-gefarbtes SDS-Polyacrylamidgel mit aus mononuclearen Blutzellen isolierter rekombinanter MPRS und ihrem RASED-Fragment, das von einer RA-Patientin stammtFIG. 3 is a Coomassie blue-colored SDS polyacrylamide gel with recombinant MPRS isolated from mononuclear blood cells and its RASED fragment, which comes from an RA patient

Figur 4 zeigt das Ergebnis emer Immunprazipitation der zell- frei translatierten MPRS durch Seren von Patienten (RA = Rheumatoide Arthritis, Patientenserum, c = Kontroll-Xeno- serum, Nl, N2 = "Normalserum", human, als Kontrolle, MCTD = Mischkollagenose, Patientenserum, SLE = Systemischer Lu¬ pus Erythematodes, Patientenserum) .FIG. 4 shows the result of an immunoprecipitation of the cell-free translated MPRS by sera from patients (RA = rheumatoid arthritis, patient serum, c = control xenoserum, Nl, N2 = "normal serum", human, as control, MCTD = mixed collagenosis, Patient serum, SLE = systemic lump erythematosus, patient serum).

Figur 5 zeigt einen Homologie-Vergleich der erfindungsgemaßen, in Figur 2 gezeigten Metallprotease mit bisher bekannten Me¬ tallproteasen Fett hervorgehobene Aminosäuren markieren stark konservierte Bereiche. Die für Metallproteasen typischen Domä¬ nen (hydrophobes Signalpeptid, abspaltbares Propeptid, kataly¬ tische Domäne mit Zn2+-Bιndestelle, "Hmge"Regιon und Hemopexm ähnliche Domäne) sind aus der Abbildung zu entnehmen (vgl Bir- kedal-Hansen, H. et al . (1993) Crit Rev Oral Biol Med ) Die MTMMP-Sequenz stammt aus der "genbank"-Datenbank (NCBI, USA) , alle anderen Sequenzen smd aus der Proteindatenbank "Swissprot" entnommen (STR01 = Stromyelιsm-1 Precursor (MMP- 3) , STR02 = Stromyelιsιn-2 Precursor (MMP-10) , STR03 = Stromyelιsm-3 Precursor (MMP-11) , 92KD = 92 kD Type IV Collagenase Precursor (MMP-9) ; 72KD = 72 kD Type IV Collagenase Precursor (MMP-2) ; MME = Macrophage Metalloelastase Precursor (MMP-12) , NEUCOLL = Neutrophil Collagenase Precursor (MMP-8) , INTCOLL = Interstital Collagenase Precursor (MMP-1) , PUMP- 1 = Matrylism Precursor (MMP-7) ; MTMMP-1 = Membrane Type Ma¬ trix Metalloprotemase) . Durch das nachfolgende Beispiel wird die vorliegende Erfindung näher erläutert.FIG. 5 shows a homology comparison of the metal protease according to the invention shown in FIG. 2 with previously known metal proteases. Bold amino acids mark strongly conserved areas. The domains typical for metal proteases (hydrophobic signal peptide, cleavable propeptide, catalytic domain with Zn 2+ site, "Hmge" region and Hemopexm-like domain) can be seen in the figure (see Birkedal-Hansen, H. et al. (1993) Crit Rev Oral Biol Med) The MTMMP sequence comes from the "genbank" database (NCBI, USA), all other sequences are taken from the protein database "Swissprot" (STR01 = Stromyelιsm-1 Precursor (MMP - 3), STR02 = Stromyelιsιn-2 precursor (MMP-10), STR03 = Stromyelιs-3 precursor (MMP-11), 92KD = 92 kD Type IV collagenase precursor (MMP-9); 72KD = 72 kD Type IV collagenase precursor (MMP-2); MME = Macrophage Metalloelastase Precursor (MMP-12), NEUCOLL = Neutrophil Collagenase Precursor (MMP-8), INTCOLL = Interstital Collagenase Precursor (MMP-1), PUMP- 1 = Matrylism Precursor (MMP-7) ; MTMMP-1 = membrane type Ma ¬ trix metalloprotemase). The present invention is explained in more detail by the following example.

1. Isolierung einer für eine autoantigene Determinante kodie¬ rende Nukleinsäuresequenz (RASED)1. Isolation of a nucleic acid sequence (RASED) coding for an autoantigenic determinant

Die Isolation von mRNA aus Synovialmembran-Zellen einer RA- Patientin erfolgt nach im Stand der Technik bekannten Ver¬ fahren und eine Expressions-cDNA-Bank wird mit dem Expressi¬ onsvektor lambda Zap II (Fa. Stratagene) hergestellt. Das Absuchen ("Screening") der cDNA-Bank wird mit gereinigtem Immunglobulin G der RA-Patientin durchgeführt.The isolation of mRNA from synovial membrane cells of an RA patient is carried out according to methods known in the prior art, and an expression cDNA library is produced using the lambda Zap II expression vector (from Stratagene). The screening (screening) of the cDNA library is carried out with purified immunoglobulin G from the RA patient.

Mit dem vorstehend beschriebenen Verfahren wurde ein primä¬ rer Klon "Rased", der ein Fragment von 1106 bp der Metall¬ protease (MPRS) -mRNA umfaßt, aus der cDNA-Bank (3xl05 Klone/μg mRNA) erhalten, wobei dieses Fragment aufgrund des Screenings mit Immunglobulin G der RA-Patientin ein autoan- tigenes Epitop enthält.With the method described above, a primary clone "Rased", which comprises a fragment of 1106 bp of the metal protease (MPRS) mRNA, was obtained from the cDNA library (3 × 10 5 clones / μg mRNA), this fragment contains an autoantigenic epitope based on the screening with immunoglobulin G of the RA patient.

2. Isolierung und Charakterisierung einer für eine als Autoan¬ tigen wirkenden Meteallorotease (MPRS) kodierende Nuklein¬ säureseguenz2. Isolation and characterization of a nucleic acid sequence coding for an autoantigenic meteallorotease (MPRS)

MPRS-mRNA wird in peripheren mononuclearen Blutzellen über reverse Transkription/Polymerasekettenreaktion nachgewiesen. Die Isolation der mRNA aus peripheren mononuclearen Blutzel¬ len und die Herstellung einer cDNA-Bank mit lambda Zap II wird wie vorstehend beschrieben durchgeführt. Das Screening der cDNA-Bank wird mit einer markierten, auf dem RASED-Klon basierenden DNA-Sonde durchgeführt.MPRS mRNA is detected in peripheral mononuclear blood cells via reverse transcription / polymerase chain reaction. The isolation of the mRNA from peripheral mononuclear blood cells and the production of a cDNA bank with lambda Zap II is carried out as described above. The cDNA library is screened with a labeled DNA probe based on the RASED clone.

Mit dem vorstehend beschriebenen Verfahren wurde ein des vollständiger cDNA-Klon der MPRS erhalten, dessen vollstän¬ dige Sequenzierung die in Figur 1 gezeigte DNA-Sequenz und die davon abgeleitete, in Figur 2 gezeigte Aminosäuresequenz ergab.With the above-described method of a complete cDNA clone was obtained the MPRS whose completeness, ¬ ended sequencing the DNA sequence shown in Figure 1 and the deduced amino acid sequence shown in Figure 2 resulted.

Der in Figur 5 gezeigte Datenbankvergleich zeigt auf Pro¬ teinebene zwischen 30 und 35 % Identität (bei einer Ähnlich¬ keit von über 50 %) zu bekannten Metallproteasen, wobei die katalytische Domäne sowie die für Metallproteasen typische Domänenstruktur vorhanden sind.The database comparison shown in FIG. 5 shows at the protein level between 30 and 35% identity (with a similarity of over 50%) to known metal proteases, the catalytic domain and the domain structure typical of metal proteases being present.

3. Gelchromatographische Analyse der Metallprotease MPRS und deren Fragment RASED3. Gel chromatographic analysis of the metal protease MPRS and its fragment RASED

Die Expression und Reinigung von MPRS aus E. coli wird mit pET24 und mit N-terminalem oligo-Histidin- "tag" durchge¬ führt. Die Expression und Reinigung von RASED aus E . coli wird mit pGEX 1 als Fusionsprotein mit Glutamin-S-Trans- ferase durchgeführt. Die so erhaltenen Peptidkonstrukte wur¬ den über ein SDS-Polyacrylamidgel elektrophoretisch aufge¬ trennt und die aufgetrennten Peptidkonstrukte durch Coomas- sie Blau-Färbung des SDS-Polyacrylamidgels nachgewiesen; vgl. Figur 3'.-The expression and purification of MPRS from E. coli is carried out with pET24 and with N-terminal oligo-histidine "tag". The expression and purification of RASED from E. coli is performed with pGEX 1 as a fusion protein with glutamine-S-transferase. The peptide constructs thus obtained were electrophoretically separated using an SDS polyacrylamide gel and the separated peptide constructs were detected by Coomas blue staining of the SDS polyacrylamide gel; see. Figure 3 '.-

4. Immunologische Analyse der Metallprotease MPRS mittels Im- munpräzioitation4. Immunological analysis of the metal protease MPRS using immune precioitation

Die zellfreie Transkription/Translation von MPRS erfolgt in Retikulozytenlysaten. Die Immunpräzipitation der zellfrei translatierten MPRS wird mit Seren von Patienten durchge¬ führt; vgl. Figur 4.Cell-free transcription / translation of MPRS takes place in reticulocyte lysates. Immunoprecipitation of cell-free translated MPRS is leading with sera from patients Runaway ¬; see. Figure 4.

Das in Figur 4 zusammengefaßte Ergebnis zeigt deutlich, daß MPRS als starkes Autoantigen bei Patienten mit rheumatoider Arthritis und Systemischer Lupus Erythematodes wirkt. The result summarized in FIG. 4 clearly shows that MPRS acts as a strong autoantigen in patients with rheumatoid arthritis and systemic lupus erythematosus.

Claims

Patentansprüche claims 1. Nukleinsäure, enthaltend mindestens eine für ein Polypeptid kodierende Nukleinsäuresequenz, wobei das Polypeptid minde¬ stens eine aus einer Metallprotease stammende autoantigene Determinante umfaßt .1. Nucleic acid containing at least one nucleic acid sequence coding for a polypeptide, the polypeptide comprising at least one autoantigenic determinant derived from a metal protease. 2. Nukleinsäure nach Anspruch 1, wobei die Metallprotease ein Merαbranprotein ist .2. Nucleic acid according to claim 1, wherein the metal protease is a membrane protein. 3. Nukleinsäure nach Anspruch 1 oder 2, wobei die Me¬ tallprotease aus der Gelenkinnenhaut von Säugern stammt.3. Nucleic acid according to claim 1 or 2, wherein the metal protease comes from the inner skin of mammals. 4. Nukleinsäure nach einem der Ansprüche 1 bis 3, umfassend die in Figur 1 gezeigte Nukleinsäuresequenz oder Abschnitte davon.4. Nucleic acid according to one of claims 1 to 3, comprising the nucleic acid sequence shown in Figure 1 or portions thereof. 5. Vektor, enthaltend die Nukleinsäure von einem der Ansprüche 1 bis 4.5. Vector containing the nucleic acid of one of claims 1 to 4. 6. Wirtszelle, enthaltend die Nukleinsäure von einem der An¬ sprüche 1 bis 4 oder den Vektor von Anspruch 5.6. Host cell containing the nucleic acid of one of claims 1 to 4 or the vector of claim 5. 7. Polypeptid, enthaltend mindestens eine aus einer Metall¬ protease stammende autoantigene Determinante.7. Polypeptide containing at least one autoantigenic determinant derived from a metal protease. 8. Polypeptid nach Anspruch 7, umfassend die in Figur 2 ge¬ zeigte Aminosäuresequenz oder Abschnitte davon.8. The polypeptide according to claim 7, comprising the amino acid sequence shown in FIG. 2 or portions thereof. 9. Antikörper, der gegen das Polypeptid von Anspruch 7 oder 8 gerichtet ist . 9. Antibody directed against the polypeptide of claim 7 or 8. 10. Verfahren zum Nachweis einer Autoimmunkrankheit, worin10. A method for detecting an autoimmune disease, wherein (a) das Polypeptid von Anspruch 7 oder 8, oder(a) the polypeptide of claim 7 or 8, or (b) der Antikörper von Anspruch 9 mit Serum eines Säugers einem Enzymimmunoassay unterworfen wird.(b) the antibody of claim 9 is subjected to an enzyme immunoassay with a mammalian serum. 11. Kit zum Nachweis einer Autoimmunkrankheit, enthaltend11. Kit for the detection of an autoimmune disease containing (a) das Polypeptid von Anspruch 7 oder 8, oder(a) the polypeptide of claim 7 or 8, or (b) den Antikörper von Anspruch 9.(b) the antibody of claim 9. 12. Verwendung des Polypeptids von Anspruch 7 oder 8 oder des Antikörpers von Anspruch 9 zum Nachweis von Au¬ toimmunkrankheiten .12. Use of the polypeptide of claim 7 or 8 or the antibody of claim 9 for the detection of autoimmune diseases. 13. Verwendung nach Anspruch 12, wobei die Autoimmunkrankheiten rheumatoide Arthritis, Systemische Lupus Erythematosus, Mischkollagenose und Progressive Sklerodermie umfassen. 13. Use according to claim 12, wherein the autoimmune diseases include rheumatoid arthritis, systemic lupus erythematosus, mixed collagenosis and progressive scleroderma.
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Cited By (2)

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Publication number Priority date Publication date Assignee Title
WO1997040157A1 (en) * 1996-04-25 1997-10-30 Takeda Chemical Industries, Ltd. Matrix metalloprotease
WO1998040475A1 (en) * 1997-03-11 1998-09-17 Abbott Laboratories Human matrix metalloprotease gene, proteins encoded therefrom and methods of using same

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH085920B2 (en) * 1991-01-21 1996-01-24 富士薬品工業株式会社 Anti-human stromlysin monoclonal antibody and enzyme immunoassay
DE69333167T2 (en) * 1992-10-29 2004-06-09 Bayer Corp. DIAGNOSTIC TEST SPECIFIC FOR THE LATENT MATRIX METALLOPROTEINASE NO. 9
EP0685557B1 (en) * 1993-11-30 1998-08-12 Fuji Yakuhin Kogyo Kabushiki Kaisha Novel metalloprotease and dna coding for the same
EP0750672B1 (en) * 1994-03-17 2001-11-28 Max-Delbrück-Centrum Für Molekulare Medizin Dna sequences for matrix metalloproteases, their production and use

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997040157A1 (en) * 1996-04-25 1997-10-30 Takeda Chemical Industries, Ltd. Matrix metalloprotease
US6566116B1 (en) 1996-04-25 2003-05-20 Takeda Chemical Industries, Ltd. Matrix metalloprotease
US7001997B2 (en) * 1996-04-25 2006-02-21 Takeda Chemical Industries, Ltd. Proteins, their production and use
WO1998040475A1 (en) * 1997-03-11 1998-09-17 Abbott Laboratories Human matrix metalloprotease gene, proteins encoded therefrom and methods of using same
US6399371B1 (en) 1997-03-11 2002-06-04 Abbott Laboratories Human matrix metalloprotease gene, proteins encoded therefrom and methods of using same

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