EP0941336A2 - Papilloma viruses, agents for detecting the same and for treating diseases caused by such viruses - Google Patents

Papilloma viruses, agents for detecting the same and for treating diseases caused by such viruses

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Publication number
EP0941336A2
EP0941336A2 EP97949918A EP97949918A EP0941336A2 EP 0941336 A2 EP0941336 A2 EP 0941336A2 EP 97949918 A EP97949918 A EP 97949918A EP 97949918 A EP97949918 A EP 97949918A EP 0941336 A2 EP0941336 A2 EP 0941336A2
Authority
EP
European Patent Office
Prior art keywords
dna
papillomavirus
protein
virus
genome
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.)
Ceased
Application number
EP97949918A
Other languages
German (de)
French (fr)
Inventor
Ethel-Michele De Villiers-Zur Hausen
Harald Zur Hausen
Donna Lavergne
Claire Benton
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.)
Deutsches Krebsforschungszentrum DKFZ
Original Assignee
Deutsches Krebsforschungszentrum DKFZ
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Publication of EP0941336A2 publication Critical patent/EP0941336A2/en
Ceased 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
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • 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
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • 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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/20011—Papillomaviridae
    • C12N2710/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/01—DNA viruses
    • G01N2333/025—Papovaviridae, e.g. papillomavirus, polyomavirus, SV40, BK virus, JC virus

Definitions

  • the invention relates to a DNA which codes for a peptide of a papillomavirus main capsid protein or a papillomavirus genome. Furthermore, the invention relates to proteins encoded by the papilloma virus genome and antibodies directed against them, and to their use in diagnosis, therapy and vaccination.
  • HP viruses Human papilloma viruses
  • benign e.g. Warts
  • genital condylomas e.g. genital condylomas
  • malignancies e.g. Carcinomas of the skin and uterus
  • epithelial neoplasms cf.
  • Papilloma viruses have an icosahedral capsid without a shell, in which a circular, double-stranded DNA molecule of approximately 7900 bp is present.
  • the capsid comprises a major capsid protein (L1) and a minor capsid protein (2). Both proteins, coexpressed or L1 expressed alone, lead to the formation of virus-like particles in vitro (cf. Kirnbauer, R. et al., Journal of Virology, (1 993), pages 6929-6936).
  • Papilloma viruses cannot be propagated in monolayer cell culture. It is therefore extremely difficult to characterize them, and the detection of papilloma viruses already creates considerable problems. This is particularly true for papilloma viruses in skin carcinomas.
  • the object of the present invention is therefore to provide an agent with which papilloma viruses, in particular in carcinomas of the skin, are can be pointed.
  • a means should also be provided to treat these papillomaviruses therapeutically.
  • the invention thus relates to a DNA coding for a peptide of a papillomavirus main capsid protein (L1), the peptide representing the amino acid sequence of FIGS. 1, 2, 3, 4, 5, 6 , Fig. 7, Fig. 8, Fig. 9, Fig. 10 or Fig. 1 1 or one of them by one or more amino acids different amino acid sequence.
  • L1 papillomavirus main capsid protein
  • Another object of the invention is a DNA coding for a peptide of a papillomavirus main capsid protein, the DNA being the base sequence of FIG. 1, FIG. 2, FIG. 3, FIG. 4, FIG. 5, FIG. 6, FIG 7, 8, 9, 10 or
  • Fig. 1 1 or one of them by one or more base pairs different base sequence comprises.
  • FIG. 2 shows the base sequence and the amino acid sequence derived therefrom of a DNA coding for a peptide of L1 of a papilloma virus. This DNA was deposited as plasmid DL20 at DSM under DSM 1 1 1 81 on 1 September 7, 1 996.
  • FIG. 3 shows the base sequence and the amino acid sequence derived therefrom of a DNA coding for a peptide from L1 of a papilloma virus. This DNA was deposited as plasmid DL27 at DSM under DSM 1 1 1 82 on 1 September 7, 996.
  • FIG. 4 shows the base sequence and the amino acid sequence derived therefrom of a DNA coding for a peptide from L1 of a papilloma virus. This DNA was deposited as plasmid DL35 at DSM under DSM 1 1 1 83 on 1 September 7, 996.
  • FIG. 5 shows the base sequence and the amino acid sequence derived therefrom of a DNA coding for a peptide of L1 of a papilloma virus. This DNA was deposited as plasmid DL40 with the DSM under DSM 1184 on September 17th, 996.
  • FIG. 6 shows the base sequence and the amino acid sequence derived therefrom of a DNA coding for a peptide of L1 of a papilloma virus. This DNA was deposited as plasmid DL78 at the DSM under DSM 1 1 1 85 on 1 September 7, 996.
  • FIG. 7 shows the base sequence and the amino acid sequence derived therefrom of a DNA coding for a peptide from L1 of a papilloma virus. This DNA was deposited as plasmid DL82 at DSM under DSM 1 1 1 86 on 1 September 7, 996.
  • FIG. 8 shows the base sequence and the amino acid sequence derived therefrom of a DNA coding for a peptide from L1 of a papilloma virus. This DNA was deposited as plasmid DL83 at DSM under DSM 1 1 1 87 on 1 September 7, 996.
  • FIG. 9 shows the base sequence and the amino acid sequence derived therefrom of a DNA coding for a peptide of L1 of a papilloma virus. This DNA was deposited as plasmid DL84 with the DSM under DSM 1 1 1 88 on 1 September 7, 996.
  • FIG. 10 shows the base sequence and the amino acid sequence derived therefrom of a DNA coding for a peptide of L1 of a papilloma virus. That DNA was deposited as plasmid DL 100 at DSM under DSM 1 1 1 89 on 1 September 7, 996.
  • Fig. 1 1 shows the base sequence and the amino acid sequence derived therefrom of a DNA coding for a peptide from L1 of a papilloma virus. This DNA was deposited as plasmid HR22 at the DSM under DSM 1 1 1 90 on 1 September 7, 996.
  • the above DNA was compared to the DNA of known papilloma viruses. Sequence homology studies were carried out. A homology that is less than 90% shows a DNA according to the invention as a new HP virus.
  • the DNAs according to the invention have the following sequence homologies with known papilloma viruses:
  • the above DNA can be present in a vector or expression vector.
  • examples of such are known to the person skilled in the art.
  • these are, for example, pGEMEX, pUC derivatives, pGEM-T and pGEX-2T.
  • yeast for example, pY100 and Ycpad 1 are mentioned, while for expression in animal cells, for example pKCR, pEF-BOS, cDM8 and pCEV4.
  • suitable cells in order to express the above DNA present in an expression vector.
  • suitable cells include the E. coli strains HB1 01, DH 1, x1 776, JM 101, JM 1 09 and XL1 -Blue, the yeast
  • Connection with a DNA coding for another protein or peptide can be inserted so that the above DNA can be expressed in the form of a fusion protein.
  • papilloma virus genome comprising the above DNA.
  • the term "papilloma virus genome” also includes an incomplete genome, i.e. Fragments of a papilloma virus genome comprising the above DNA. This can e.g. a DNA coding for L1 or a part thereof.
  • a common method can be used to provide the above papillomavirus genome.
  • a method comprising the following method steps is favorable:
  • epithelial neoplasm encompasses any neoplasms of epithelial tissue in humans and animals. Examples of such neoplasms are warts, condylomas in the genital area and carcinomas of the skin. The latter are preferably used in the present case to isolate the above papillomavirus genome.
  • vector includes any vector suitable for cloning chromosomal or extrachromosomal DNA. Examples of such vectors are
  • Cosmids such as pWE1 5 and Super Cos1
  • phages such as ⁇ -phages, e.g. IZAP Expressvector, ⁇ ZAPII Vector and ⁇ gt10 Vector.
  • ⁇ phages are preferably used.
  • the above vectors are known and are available from Stratagene.
  • Papillomavirus genomes according to the invention can be integrated in chromosomal DNA or extrachromosomal. Methods are known to the person skilled in the art to clarify this. He also knows how to find the optimal restriction enzymes for cloning the papillomavirus genomes. It will be based on genomes of known papilloma viruses. In particular, the person skilled in the art will observe the aforementioned HP viruses accordingly.
  • a papilloma virus genome designated DL1 7-G is described by way of example.
  • the entire DNA is isolated from a biopsy of a squamous cell carcinoma, cleaved with BamHI and electrophoretically separated in an agarose gel.
  • the agarose gel is then subjected to a blotting process, whereby the DNA is transferred onto a nitrocellulose membrane. will wear.
  • This is used in a hybridization process in which the DNA from FIG. 1, possibly in combination with a DNA from HP virus 65, is used as the labeled sample. Hybridization with the papilloma virus DNA present in the total DNA is obtained.
  • the above total DNA cleaved with BamHI is cloned in a ⁇ phage.
  • the corresponding clones i.e. the clones containing the papillomavirus DNA are identified by hybridization with the DNA from FIG. 1, possibly in combination with a DNA from the HP virus 65.
  • the insert of these clones is then subjected to further cloning in a plasmid vector, whereby a clone is obtained which contains the papillomavirus genome DL1 7-G. The genome is confirmed by sequencing.
  • papillomavirus genomes are provided. They are named according to the DNAs used for their preparation, with: DL20-G, DL27-G, DL35-G, DL40-G, DL78-G, DL82-G, DL83-G, DL84-G, DL1 00-G or HR22-G.
  • Another object of the invention is a protein encoded by the above papillomavirus genome.
  • a protein is e.g. a major capsid
  • the above protein is produced in a conventional manner.
  • the production of L1 and L2 of the papilloma virus genome DL1 7-G is described as an example.
  • the HP virus 65 related to the DNA of FIG. 1 is used. From this the complete sequence and the location of individual DNA coding for proteins
  • DL1 7-G-L2-DNA Denoted DL1 7-G-L2-DNA.
  • the DNA coding for L1 or L2 is inserted into an expression vector. Examples of such for E. coli, yeast and animal cells are mentioned above.
  • the vector pGEX-2T for expression in E. coli (cf. Kirnbauer, R. et al., Supra).
  • Bacculovirus or vaccinia virus system called.
  • Expression vectors that can be used for this are, for example, pEV mod. and pSynwtVI " for the bacculovirus system (cf. Kirnbauer, R. et al., supra).
  • vectors with the vaccinia virus are" early "(p7. 5 k) or” late "(Psynth to call p1 1 K) promoter (cf. Hagensee, M., E. et al., Journal of Virology (1 993), pages 31 5-322.)
  • the bacculovirus system is preferred DNA coding for L2 or L2 in pEV mod.
  • pEVmod.-DL1 7-G-L1 or pEVmod.-DL1 7-G-L2 is obtained.
  • a particle comprises an L1 protein
  • an L1 protein in the latter case it contains an L1 protein as well as an L1 protein.
  • a virus-like particle of the latter case is also obtained by inserting the above DL1 7-G-L1 and DL1 7-G-L2 DNAs together into the expression vector pSynwtVI " and the pSynwtVI DLI 7-G-L1 / L2 obtained is used for the infection of SF-9 insect cells
  • the above virus-like particles are cleaned in a conventional manner and are also an object of the invention.
  • Another object of the invention is against an above protein or virus-like particle of directed antibodies.
  • Such is produced in the usual way. It is described by way of example for the production of an antibody which is directed against a virus-like particle comprising L1 of DL1 7-G.
  • the virus-like particle BALB / c mice is injected subcutaneously. This injection is repeated every 3 weeks. About 2 weeks after the last injection, the serum containing the antibody is isolated and tested in the usual way.
  • the antibody is a monoclonal antibody.
  • Spleen cells removed and fused with myeloma cells in the usual way are also carried out according to known methods.
  • the present invention makes it possible to detect papilloma viruses, in particular in carcinomas of the skin.
  • the DNA according to the invention can be used as such or encompassed by a further DNA.
  • the latter can also be a papilloma virus gome or part of it.
  • the present invention also enables the provision of previously unknown papilloma viruses. These are found particularly in carcinomas of the
  • the invention provides proteins and virus-like particles which are due to these papillomaviruses. Antibodies are also provided which are directed against these proteins or particles.
  • the present invention thus enables diagnostic and therapeutic
  • the present invention thus represents a breakthrough in the field of papilloma virus research.
  • Example 1 Identification of the papilloma virus genome DL17-G
  • the total DNA is isolated from the biopsy of a squamous cell carcinoma of an immunosuppressed person. 1 0 / vg of this DNA are cleaved with the restriction enzyme BamHI and electrophoresed in a 0.5% agarose gel. At the same time, 10 ⁇ g of the above DNA, which has not been cleaved, are also separated.
  • the agarose gel is subjected to a blotting process, whereby the DNA from the agarose gel is transferred to a nitrocellulose membrane. This is used in a hybridization process in which the above DNA from FIG. 1 is used in combination with HP virus 65 DNA as a p 32 -labeled sample. Hybridization with the blotted DNA is obtained.
  • the biopsy DNA obtained from Example 1 is cleaved with the restriction enzyme BamHI.
  • the fragments obtained are used in a ligase reaction in which the HZAP Express vector, which has been cleaved and dephosphorylated with BamHI, is also present.
  • the recombinant DNA molecules obtained in this way are packaged in bacteriophages and used to infect bacteria.
  • the ZAP Express Vector Kit offered by Stratagene is used for these process steps.
  • the phage plaques obtained are then subjected to a hybridization process in which the p 32 -labeled DNA from FIG. 1 used in Example 1 in combination with p 32 -labeled HP virus 65- DNA is used. Hybridization with corresponding phage plaques is obtained.
  • the BamHI fragments of DL1 7-G are isolated from these and used together with a BamHI-split, dephosphorylated plasmid vector, pBluescript, in a further ligase reaction.
  • the recombinant obtained are packaged in bacteriophages and used to infect bacteria.
  • the ZAP Express Vector Kit offered by Stratagene is used for these process steps.
  • the phage plaques obtained are then subjected to a hybridization process in which the p 32 -labeled
  • DNA molecules are used to transform bacteria, E. coli XL1 -Blue.
  • a bacterial clone containing the papillomavirus genome DL1 7-G is identified by restriction cleavage or hybridization with the above DNA samples.
  • the plasmid of this bacterial clone is designated pBlue-DL1 7-G.

Abstract

A DNA is disclosed that codes for a peptide of a papilloma virus main capsid protein or for a papilloma virus genome. Also disclosed are proteins coded by the papilloma virus genome and antibodies against the same, as well as their use in diagnosis, therapy and vaccination.

Description

"Papillomviren, Mittel zu deren Nachweis sowie zur Therapie von durch sie verursachten Erkrankungen""Papilloma viruses, means for their detection and for the therapy of diseases caused by them"
Die Erfindung betrifft eine DNA, die für ein Peptid eines Papillomvirus-Hauptcap- sid-Proteins bzw. ein Papillomvirus-Genom codiert. Desweiteren betrifft die Erfindung durch das Papillomvirus-Genom codierte Proteine und gegen sie gerichtete Antikörper sowie deren Verwendung in Diagnose, Therapie und Vakzinierung.The invention relates to a DNA which codes for a peptide of a papillomavirus main capsid protein or a papillomavirus genome. Furthermore, the invention relates to proteins encoded by the papilloma virus genome and antibodies directed against them, and to their use in diagnosis, therapy and vaccination.
Es ist bekannt, daß Papillomviren das Epithelgewebe von Mensch und Tier infizieren. Human-Papillomviren (nachstehend mit HP-Viren bezeichnet) finden sich in benignen, z.B. Warzen, Kondylome im Genitalbereich, und malignen, z.B. Karzinome der Haut und der Gebärmutter, epithelialen Neoplasmen (vgl. zurPapillomaviruses are known to infect human and animal epithelial tissue. Human papilloma viruses (hereinafter referred to as HP viruses) are found in benign, e.g. Warts, genital condylomas, and malignancies, e.g. Carcinomas of the skin and uterus, epithelial neoplasms (cf.
Hausen, H. , Biochimica et Biophysica Acta (BBA) 1 288, ( 1 996), Seiten 55-78) . Auch werden HP-Viren für die Entwicklung maligner Tumoren im Oropharyn- gealbereich in Betracht gezogen (vgl. zur Hausen, H., Curr. Top. Microbiol. Immunol. 78, ( 1 977), Seiten 1 -30).Hausen, H., Biochimica et Biophysica Acta (BBA) 1 288, (1 996), pages 55-78). HP viruses are also being considered for the development of malignant tumors in the oropharyngeal area (cf. Zur Hausen, H., Curr. Top. Microbiol. Immunol. 78, (1 977), pages 1-30).
Papillomviren weisen ein ikosaedrisches Capsid ohne Hülle auf, in dem ein zirkuläres, doppelsträngiges DNA-Molekül von etwa 7900 bp vorliegt. Das Capsid umfaßt ein Hauptcapsid-Protein (L1 ) und ein Nebencapsid-Protein ( 2) . Beide Proteine, coexprimiert oder L1 alleine exprimiert, führen in vitro zur Aus- bildung von Virus-ähnlichen Partikeln (vgl. Kirnbauer, R. et al., Journal of Virolo- gy, ( 1 993) , Seiten 6929-6936) .Papilloma viruses have an icosahedral capsid without a shell, in which a circular, double-stranded DNA molecule of approximately 7900 bp is present. The capsid comprises a major capsid protein (L1) and a minor capsid protein (2). Both proteins, coexpressed or L1 expressed alone, lead to the formation of virus-like particles in vitro (cf. Kirnbauer, R. et al., Journal of Virology, (1 993), pages 6929-6936).
Papillomviren lassen sich nicht in Monolayer-Zellkultur vermehren. Ihre Charakterisierung ist daher äußerst schwierig, wobei bereits der Nachweis von Papil- lomviren erhebliche Probleme schafft. Dies trifft insbesondere für Papillomviren in Karzinomen der Haut zu.Papilloma viruses cannot be propagated in monolayer cell culture. It is therefore extremely difficult to characterize them, and the detection of papilloma viruses already creates considerable problems. This is particularly true for papilloma viruses in skin carcinomas.
Der vorliegenden Erfindung liegt somit die Aufgabe zugrunde, ein Mittel bereitzustellen, mit dem Papillomviren, insbesondere in Karzinomen der Haut, nach- gewiesen werden können. Ferner sollte ein Mittel bereitgestellt werden, um gegen diese Papillomviren therapeutisch vorgehen zu können.The object of the present invention is therefore to provide an agent with which papilloma viruses, in particular in carcinomas of the skin, are can be pointed. A means should also be provided to treat these papillomaviruses therapeutically.
Erfindungsgemäß wird dies durch die Bereitstellung der Gegenstände in den Patentansprüchen erreicht.According to the invention, this is achieved by the provision of the subject matter in the claims.
Gegenstand der Erfindung ist somit eine für ein Peptid eines Papillomvirus- Hauptcapsid-Proteins (L1 ) codierende DNA, wobei das Peptid die Aminosäuresequenz von Fig. 1 , Fig. 2, Fig. 3, Fig. 4, Fig. 5, Fig. 6, Fig. 7, Fig. 8, Fig. 9, Fig. 10 oder Fig. 1 1 oder eine davon durch ein oder mehrere Aminosäuren unterschiedliche Aminosäuresequenz umfaßt.The invention thus relates to a DNA coding for a peptide of a papillomavirus main capsid protein (L1), the peptide representing the amino acid sequence of FIGS. 1, 2, 3, 4, 5, 6 , Fig. 7, Fig. 8, Fig. 9, Fig. 10 or Fig. 1 1 or one of them by one or more amino acids different amino acid sequence.
Ein weiterer Gegenstand der Erfindung ist eine für ein Peptid eines Papillomvirus- Hauptcapsid-Proteins codierende DNA, wobei die DNA die Basensequenz von Fig. 1 , Fig. 2, Fig. 3, Fig. 4, Fig. 5, Fig. 6, Fig. 7, Fig. 8, Fig. 9, Fig. 1 0 oderAnother object of the invention is a DNA coding for a peptide of a papillomavirus main capsid protein, the DNA being the base sequence of FIG. 1, FIG. 2, FIG. 3, FIG. 4, FIG. 5, FIG. 6, FIG 7, 8, 9, 10 or
Fig. 1 1 oder eine davon durch ein oder mehrere Basenpaare unterschiedliche Basensequenz umfaßt.Fig. 1 1 or one of them by one or more base pairs different base sequence comprises.
Fig. 1 zeigt die Basensequenz und die davon abgeleitete Aminosäuresequenz einer für ein Peptid von L1 eines Papillomvirus codierenden DNA. Diese DNA wurde als Plasmid DL1 7 bei der DSM (Deutsche Sammlung von Mikroorganismen und Zellkulturen) unter DSM 1 1 1 80 am 1 7. Sept. 1 996 hinterlegt.1 shows the base sequence and the amino acid sequence derived therefrom of a DNA coding for a peptide of L1 of a papilloma virus. This DNA was deposited as plasmid DL1 7 at the DSM (German Collection of Microorganisms and Cell Cultures) under DSM 1 1 1 80 on 1 September 7, 996.
Fig. 2 zeigt die Basensequenz und die davon abgeleitete Aminosäuresequenz einer für ein Peptid von L1 eines Papillomvirus codierenden DNA. Diese DNA wurde als Plasmid DL20 bei der DSM unter DSM 1 1 1 81 am 1 7. Sept. 1 996 hinterlegt.FIG. 2 shows the base sequence and the amino acid sequence derived therefrom of a DNA coding for a peptide of L1 of a papilloma virus. This DNA was deposited as plasmid DL20 at DSM under DSM 1 1 1 81 on 1 September 7, 1 996.
Fig. 3 zeigt die Basensequenz und die davon abgeleitete Aminosäuresequenz einer für ein Peptid von L1 eines Papillomvirus codierenden DNA. Diese DNA wurde als Plasmid DL27 bei der DSM unter DSM 1 1 1 82 am 1 7. Sept. 1 996 hinterlegt. Fig. 4 zeigt die Basensequenz und die davon abgeleitete Aminosäuresequenz einer für ein Peptid von L1 eines Papillomvirus codierenden DNA. Diese DNA wurde als Plasmid DL35 bei der DSM unter DSM 1 1 1 83 am 1 7. Sept. 1 996 hinterlegt.FIG. 3 shows the base sequence and the amino acid sequence derived therefrom of a DNA coding for a peptide from L1 of a papilloma virus. This DNA was deposited as plasmid DL27 at DSM under DSM 1 1 1 82 on 1 September 7, 996. FIG. 4 shows the base sequence and the amino acid sequence derived therefrom of a DNA coding for a peptide from L1 of a papilloma virus. This DNA was deposited as plasmid DL35 at DSM under DSM 1 1 1 83 on 1 September 7, 996.
Fig. 5 zeigt die Basensequenz und die davon abgeleitete Aminosäuresequenz einer für ein Peptid von L1 eines Papillomvirus codierenden DNA. Diese DNA wurde als Plasmid DL40 bei der DSM unter DSM 1 1 184 am 17. Sept. 1 996 hinterlegt.FIG. 5 shows the base sequence and the amino acid sequence derived therefrom of a DNA coding for a peptide of L1 of a papilloma virus. This DNA was deposited as plasmid DL40 with the DSM under DSM 1184 on September 17th, 996.
Fig. 6 zeigt die Basensequenz und die davon abgeleitete Aminosäuresequenz einer für ein Peptid von L1 eines Papillomvirus codierenden DNA. Diese DNA wurde als Plasmid DL78 bei der DSM unter DSM 1 1 1 85 am 1 7. Sept. 1 996 hinterlegt.FIG. 6 shows the base sequence and the amino acid sequence derived therefrom of a DNA coding for a peptide of L1 of a papilloma virus. This DNA was deposited as plasmid DL78 at the DSM under DSM 1 1 1 85 on 1 September 7, 996.
Fig. 7 zeigt die Basensequenz und die davon abgeleitete Aminosäuresequenz einer für ein Peptid von L1 eines Papillomvirus codierenden DNA. Diese DNA wurde als Plasmid DL82 bei der DSM unter DSM 1 1 1 86 am 1 7. Sept. 1 996 hinterlegt.FIG. 7 shows the base sequence and the amino acid sequence derived therefrom of a DNA coding for a peptide from L1 of a papilloma virus. This DNA was deposited as plasmid DL82 at DSM under DSM 1 1 1 86 on 1 September 7, 996.
Fig. 8 zeigt die Basensequenz und die davon abgeleitete Aminosäuresequenz einer für ein Peptid von L1 eines Papillomvirus codierenden DNA. Diese DNA wurde als Plasmid DL83 bei der DSM unter DSM 1 1 1 87 am 1 7. Sept. 1 996 hinterlegt.FIG. 8 shows the base sequence and the amino acid sequence derived therefrom of a DNA coding for a peptide from L1 of a papilloma virus. This DNA was deposited as plasmid DL83 at DSM under DSM 1 1 1 87 on 1 September 7, 996.
Fig. 9 zeigt die Basensequenz und die davon abgeleitete Aminosäuresequenz einer für ein Peptid von L1 eines Papillomvirus codierenden DNA. Diese DNA wurde als Plasmid DL84 bei der DSM unter DSM 1 1 1 88 am 1 7. Sept. 1 996 hinterlegt.9 shows the base sequence and the amino acid sequence derived therefrom of a DNA coding for a peptide of L1 of a papilloma virus. This DNA was deposited as plasmid DL84 with the DSM under DSM 1 1 1 88 on 1 September 7, 996.
Fig. 10 zeigt die Basensequenz und die davon abgeleitete Aminosäuresequenz einer für ein Peptid von L1 eines Papillomvirus codierenden DNA. Diese DNA wurde als Plasmid DL 100 bei der DSM unter DSM 1 1 1 89 am 1 7. Sept. 1 996 hinterlegt.10 shows the base sequence and the amino acid sequence derived therefrom of a DNA coding for a peptide of L1 of a papilloma virus. That DNA was deposited as plasmid DL 100 at DSM under DSM 1 1 1 89 on 1 September 7, 996.
Fig. 1 1 zeigt die Basensequenz und die davon abgeleitete Aminosäuresequenz einer für ein Peptid von L1 eines Papillomvirus codierenden DNA. Diese DNA wurde als Plasmid HR22 bei der DSM unter DSM 1 1 1 90 am 1 7. Sept. 1 996 hinterlegt.Fig. 1 1 shows the base sequence and the amino acid sequence derived therefrom of a DNA coding for a peptide from L1 of a papilloma virus. This DNA was deposited as plasmid HR22 at the DSM under DSM 1 1 1 90 on 1 September 7, 996.
Vorstehende DNA wurde mit der DNA bekannter Papillomviren verglichen. Es wurden Sequenzhomologie-Studien durchgeführt. Eine Homologie, die weniger als 90 % beträgt, weist eine erfindungsgemäße DNA als neues HP-Virus aus. Die erfindungsgemäßen DNAs weisen zu bekannten Papillomviren folgende Sequenzhomologien auf:The above DNA was compared to the DNA of known papilloma viruses. Sequence homology studies were carried out. A homology that is less than 90% shows a DNA according to the invention as a new HP virus. The DNAs according to the invention have the following sequence homologies with known papilloma viruses:
DNA von Fig. 1 67 % zu HP-Virus 65DNA of Fig. 1 67% to HP virus 65
DNA von Fig. 2 62 % zu HP-Virus 1 7DNA of Fig. 2 62% to HP virus 17
DNA von Fig. 3 78 % zu HP-Virus 65DNA of Figure 3 78% to HP virus 65
DNA von Fig. 4DNA of Fig. 4
DNA von Fig. 5 86 % zu HP-Virus 10DNA of Fig. 5 86% to HP virus 10
DNA von Fig. 6 86 % zu HP-Virus 10DNA of Fig. 6 86% to HP virus 10
DNA von Fig. 7 62 % zu HP-Virus 8DNA of Fig. 7 62% to HP virus 8
DNA von Fig. 8 66 % zu HP-Virus 65DNA of Fig. 8 66% to HP virus 65
DNA von Fig. 9 64 % zu HP-Virus 65DNA of Fig. 9 64% to HP virus 65
DNA von Fig. 1 0: 75 % zu HP-Virus 1 5DNA from FIG. 10: 75% to HP virus 15
DNA von Fig. 1 1 : 81 % zu HP-Virus 22 bzw. 23DNA of Fig. 1 1: 81% to HP virus 22 and 23 respectively
Erfindungsgemäß kann vorstehende DNA in einem Vektor bzw. Expressionsvektor vorliegen. Beispiele solcher sind dem Fachmann bekannt. Im Falle eines Expressionsvektors für E. coli sind dies z.B. pGEMEX, pUC-Derivate, pGEM-T und pGEX-2T. Für die Expression in Hefe sind z.B. pY100 und Ycpad l zu nennen, während für die Expression in tierischen Zellen z.B. pKCR, pEF-BOS, cDM8 und pCEV4, anzugeben sind.According to the invention, the above DNA can be present in a vector or expression vector. Examples of such are known to the person skilled in the art. In the case of an expression vector for E. coli, these are, for example, pGEMEX, pUC derivatives, pGEM-T and pGEX-2T. For expression in yeast, for example, pY100 and Ycpad 1 are mentioned, while for expression in animal cells, for example pKCR, pEF-BOS, cDM8 and pCEV4.
Der Fachmann kennt geeignete Zellen, um vorstehende, in einem Expressionsvektor vorliegende DNA zu exprimieren. Beispiele solcher Zellen umfassen die E.coli-Stämme HB1 01 , DH 1 , x1 776, JM 101 , JM 1 09 und XL1 -Blue, den Hefe-The person skilled in the art knows suitable cells in order to express the above DNA present in an expression vector. Examples of such cells include the E. coli strains HB1 01, DH 1, x1 776, JM 101, JM 1 09 and XL1 -Blue, the yeast
Stamm Saccharomyces cerevisiae und die tierischen Zellen L, NH-3T3, FM3A, CHO, COS, Vero, und Heia.Strain Saccharomyces cerevisiae and the animal cells L, NH-3T3, FM3A, CHO, COS, Vero, and Heia.
Der Fachmann weiß, in welcher Weise vorstehende DNA in einen Expressions- vektor inseriert werden muß. Ihm ist auch bekannt, daß vorstehende DNA inThe person skilled in the art knows in what way the above DNA has to be inserted into an expression vector. He is also aware that the above DNA in
Verbindung mit einer für ein anderes Protein bzw. Peptid codierenden DNA inseriert werden kann, so daß vorstehende DNA in Form eines Fusionsproteins exprimiert werden kann.Connection with a DNA coding for another protein or peptide can be inserted so that the above DNA can be expressed in the form of a fusion protein.
Ein weiterer Gegenstand der Erfindung ist ein Papillomvirus-Genom, das vorstehende DNA umfaßt. Der Ausdruck "Papillomvirus-Genom" umfaßt auch ein unvollständiges Genom, d.h. Fragmente eines Papillomvirus-Genoms, die vorstehende DNA umfassen. Dies kann z.B. eine für L1 codierende DNA oder ein Teil davon sein.Another object of the invention is a papilloma virus genome comprising the above DNA. The term "papilloma virus genome" also includes an incomplete genome, i.e. Fragments of a papilloma virus genome comprising the above DNA. This can e.g. a DNA coding for L1 or a part thereof.
Zur Bereitstellung vorstehenden Papillomvirus-Genoms kann ein übliches Verfahren verwendet werden. Günstig ist ein Verfahren, das folgende Verfahrensschritte umfaßt:A common method can be used to provide the above papillomavirus genome. A method comprising the following method steps is favorable:
(a) Isolierung der Gesamt-DNA aus einer Biopsie epithelialen Neoplasmas,(a) isolation of the total DNA from a biopsy of epithelial neoplasm,
(b) Hybridisierung der Gesamt-DNA von (a) mit vorstehender DNA, wodurch ein in der Gesamt-DNA von (a) enthaltenes Papillomvirus-Genom nachgewiesen wird, und(b) hybridizing the total DNA of (a) with the above DNA, thereby detecting a papillomavirus genome contained in the total DNA of (a), and
(c) Klonierung der das Papillomvirus-Genom enthaltenden Gesamt-DNA von (a) in einem Vektor, und gegebenenfalls Subklonierung des erhaltenen Klons, wobei sämtliche Verfahrensschritte üblicher DNA-Rekombinationstechnik entstammen.(c) cloning the total DNA of (a) containing the papillomavirus genome in a vector, and optionally subcloning the resultant Clones, whereby all process steps originate from conventional DNA recombination technology.
Hinsichtlich der Isolierung, Hybridisierung und Klonierung von Zeil-DNA wird ergänzend auf Sambrook et al. , Molecular Cloning, A Laboratory Manual, zweiteWith regard to the isolation, hybridization and cloning of Zeil DNA, Sambrook et al. , Molecular Cloning, A Laboratory Manual, second
Ausgabe, Cold Spring Harbor Laboratory ( 1 989) verwiesen.Edition, Cold Spring Harbor Laboratory (1,989).
Der Ausdruck "epitheliales Neoplasma" umfaßt jegliche Neoplasmen des Epithelgewebes bei Mensch und Tier. Beispiele solcher Neoplasmen sind Warzen, Kondylome im Genitalbereich und Karzinome der Haut. Letztere werden vorliegend bevorzugt verwendet, um vorstehendes Papillomvirus-Genom zu isolieren.The term "epithelial neoplasm" encompasses any neoplasms of epithelial tissue in humans and animals. Examples of such neoplasms are warts, condylomas in the genital area and carcinomas of the skin. The latter are preferably used in the present case to isolate the above papillomavirus genome.
Der Ausdruck "Vektor" umfaßt jegliche zur Klonierung von chromosomaler bzw. extrachromosomaler DNA geeignete Vektoren. Beispiele solcher Vektoren sindThe term "vector" includes any vector suitable for cloning chromosomal or extrachromosomal DNA. Examples of such vectors are
Cosmide, wie pWE1 5 und Super Cos1 , und Phagen, wie Λ-Phagen, z.B. IZAP Expressvector, ΛZAPII Vector und Λgt10 Vektor. Vorliegend werden Λ-Phagen bevorzugt verwendet. Vorstehende Vektoren sind bekannt und bei der Firma Stratagene erhältlich.Cosmids such as pWE1 5 and Super Cos1, and phages such as Λ-phages, e.g. IZAP Expressvector, ΛZAPII Vector and Λgt10 Vector. In the present case, Λ phages are preferably used. The above vectors are known and are available from Stratagene.
Erfindungsgemäße Papillomvirus-Genome können integriert in chromosomaler DNA oder extrachromosomal vorliegen. Dem Fachmann sind Verfahren bekannt, dies abzuklären. Auch weiß er um Verfahren, die zur Klonierung der Papillomvirus-Genome optimalen Restriktionsenzyme herauszufinden. Er wird sich an Genomen bekannter Papillomviren orientieren. Insbesondere wird der Fachmann die vorstehend genannten HP-Viren entsprechend beachten.Papillomavirus genomes according to the invention can be integrated in chromosomal DNA or extrachromosomal. Methods are known to the person skilled in the art to clarify this. He also knows how to find the optimal restriction enzymes for cloning the papillomavirus genomes. It will be based on genomes of known papilloma viruses. In particular, the person skilled in the art will observe the aforementioned HP viruses accordingly.
Beispielhaft wird die Bereitstellung eines mit DL1 7-G bezeichneten Papillomvirus- Genoms beschrieben. Hierzu wird die Gesamt-DNA aus einer Biopsie eines plattenepithelialen Karzinoms isoliert, mit BamHI gespalten und in einem Agaro- segel elektrophoretisch aufgetrennt. Das Agarosegel wird danach einem Blotting- Verfahren unterzogen, wodurch die DNA auf eine Nitrozellulosemembran über- tragen wird. Diese wird in ein Hybridisierungsverfahren eingesetzt, in dem die DNA von Fig. 1 , ggfs. in Kombination mit einer DNA von HP-Virus 65 als markierte Probe verwendet wird. Es wird eine Hybridisierung mit der in der Gesamt- DNA vorliegenden Papillomvirus-DNA erhalten.The provision of a papilloma virus genome designated DL1 7-G is described by way of example. For this purpose, the entire DNA is isolated from a biopsy of a squamous cell carcinoma, cleaved with BamHI and electrophoretically separated in an agarose gel. The agarose gel is then subjected to a blotting process, whereby the DNA is transferred onto a nitrocellulose membrane. will wear. This is used in a hybridization process in which the DNA from FIG. 1, possibly in combination with a DNA from HP virus 65, is used as the labeled sample. Hybridization with the papilloma virus DNA present in the total DNA is obtained.
Im weiteren wird vorstehende mit BamHI gespaltene Gesamt-DNA in einem λ- Phagen kloniert. Die entsprechenden Klone, d.h. die die Papillomvirus-DNA enthaltenden Klone, werden durch Hybridisierung mit der DNA von Fig. 1 , ggfs. in Kombination mit einer DNA des HP-Virus 65 identifiziert. Das Insert dieser Klone wird dann einer weiteren Klonierung in einem Plasmid-Vektor unterzogen, wodurch ein Klon erhalten wird, der das Papillomvirus-Genom DL1 7-G enthält. Das Genom wird durch Sequenzierung bestätigt.In addition, the above total DNA cleaved with BamHI is cloned in a λ phage. The corresponding clones, i.e. the clones containing the papillomavirus DNA are identified by hybridization with the DNA from FIG. 1, possibly in combination with a DNA from the HP virus 65. The insert of these clones is then subjected to further cloning in a plasmid vector, whereby a clone is obtained which contains the papillomavirus genome DL1 7-G. The genome is confirmed by sequencing.
In analoger Weise werden weitere Papillomvirus-Genome bereitgestellt. Sie werden entsprechend der zu ihrer Bereitstellung verwendeten DNAs bezeichnet, mit: DL20-G, DL27-G, DL35-G, DL40-G, DL78-G, DL82-G, DL83-G, DL84-G, DL1 00-G bzw. HR22-G.Analogously, further papillomavirus genomes are provided. They are named according to the DNAs used for their preparation, with: DL20-G, DL27-G, DL35-G, DL40-G, DL78-G, DL82-G, DL83-G, DL84-G, DL1 00-G or HR22-G.
Ein weiterer Gegenstand der Erfindung ist ein Protein, das durch vorstehendes Papillomvirus-Genom codiert wird. Ein solches Protein ist z.B. ein Hauptcapsid-Another object of the invention is a protein encoded by the above papillomavirus genome. Such a protein is e.g. a major capsid
Protein (L1 ) oder ein Nebencapsidprotein (L2) . Die Herstellung eines vorstehenden Proteins erfolgt in üblicher Weise. Beispielhaft wird die Herstellung von L1 bzw. L2 des Papillomvirus-Genoms DL1 7-G beschrieben. Hierzu wird das zu der DNA von Fig. 1 verwandte HP-Virus 65 herangezogen. Von diesem ist die vollständige Sequenz und die Lage einzelner für Proteine codierender DNA-Protein (L1) or a minor capsid protein (L2). The above protein is produced in a conventional manner. The production of L1 and L2 of the papilloma virus genome DL1 7-G is described as an example. For this purpose, the HP virus 65 related to the DNA of FIG. 1 is used. From this the complete sequence and the location of individual DNA coding for proteins
Bereiche bekannt. Durch parallele Restriktionsspaltungen beider Genome und anschließender Hybridisierung mit verschiedenen, die L1 bzw. L2 codierende DNA betreffenden Fragmenten werden diese DNAs auf dem Papillomvirus- Genom DL1 7-G identifiziert. Sie werden durch Sequenzierung bestätigt. Die für L1 codierende DNA wird mit DL1 7-G-L1 -DNA und die für L2 codierende DNA mitKnown areas. These DNAs are identified on the papillomavirus genome DL1 7-G by parallel restriction cleavages of both genomes and subsequent hybridization with different fragments relating to the L1 or L2 coding DNA. They are confirmed by sequencing. The DNA coding for L1 is with DL1 7-G-L1 DNA and the DNA coding for L2 with
DL1 7-G-L2-DNA bezeichnet. Im weiteren wird die für L1 bzw. L2 codierende DNA in einen Expressionsvektor inseriert. Beispiele eines solchen für E. coli, Hefe und tierische Zellen sind vorstehend genannt. Insbesondere wird für die Expression in E. coli auf den Vektor pGEX-2T verwiesen (vgl. Kirnbauer, R. et al., supra) . Nach Insertion der DL1 7-G-L1 - bzw. DL1 7-G-L2-DNA wird pGEX-2T-DL1 7-G-L1 bzw. pGEX-2T-Denoted DL1 7-G-L2-DNA. Furthermore, the DNA coding for L1 or L2 is inserted into an expression vector. Examples of such for E. coli, yeast and animal cells are mentioned above. In particular, reference is made to the vector pGEX-2T for expression in E. coli (cf. Kirnbauer, R. et al., Supra). After insertion of the DL1 7-G-L1 - or DL1 7-G-L2-DNA, pGEX-2T-DL1 7-G-L1 or pGEX-2T-
DL1 7-G-L2 erhalten. Diese Expressionsvektoren exprimieren nach Transformation von E. coli ein Glutathion S-Transferase-L1 - bzw. Glutathion S-Transferase- L2-Fusionsprotein. Die Reinigung dieser Proteine erfolgt in üblicher Weise.Obtained DL1 7-G-L2. After transformation of E. coli, these expression vectors express a glutathione S-transferase-L1 or glutathione S-transferase-L2 fusion protein. These proteins are purified in the usual way.
Für eine weitere Expression vorstehender L1 bzw. L2 codierender DNA wird dasFor a further expression of the above coding L1 or L2 DNA, the
Bacculovirus- bzw. Vacciniavirus-System genannt. Hierfür verwendbare Expressionsvektoren sind z.B. pEV mod. und pSynwtVI" für das Bacculovirus- System (vgl. Kirnbauer, R. et al. , supra) . Für das Vacciniavirus-System sind insbesondere Vektoren mit dem Vacciniavirus " early " (p7. 5 k)- bzw . "late"(Psynth, p1 1 K)-Promotor zu nennen (vgl. Hagensee, M., E. et al., Journal of Virology ( 1 993), Seiten 31 5-322) . Vorliegend wird das Bacculovirus-System bevorzugt. Nach Insertion vorstehender L1 bzw. L2 codierender DNA in pEV mod. wird pEVmod.-DL1 7-G-L1 bzw. pEVmod.-DL1 7-G-L2 erhalten.Bacculovirus or vaccinia virus system called. Expression vectors that can be used for this are, for example, pEV mod. and pSynwtVI " for the bacculovirus system (cf. Kirnbauer, R. et al., supra). For the vaccinia virus system, vectors with the vaccinia virus are" early "(p7. 5 k) or" late "(Psynth to call p1 1 K) promoter (cf. Hagensee, M., E. et al., Journal of Virology (1 993), pages 31 5-322.) In the present case, the bacculovirus system is preferred DNA coding for L2 or L2 in pEV mod. pEVmod.-DL1 7-G-L1 or pEVmod.-DL1 7-G-L2 is obtained.
Der erstere Expressionsvektor alleine bzw. beide Expressionsvektoren zusammen führen nach Infektion von SF-9 Insektenzellen zur Ausbildung von Virus-ähnlichen Partikeln. Im ersteren Fall umfaßt ein solches Partikel ein L1 -Protein, während es im letzteren Fall neben einem L1 - auch ein L2-Protein enthält.The former expression vector alone or both expression vectors together lead to the formation of virus-like particles after infection of SF-9 insect cells. In the former case such a particle comprises an L1 protein, while in the latter case it contains an L1 protein as well as an L1 protein.
Ein Virus-ähnliches Partikel letzteren Falls wird auch erhalten, indem die vorstehenden DL1 7-G-L1 - und DL1 7-G-L2-DNAs gemeinsam in den Expressionsvektor pSynwtVI" inseriert werden und das erhaltene pSynwtVI DLI 7-G-L1 /L2 zur Infektion von SF-9 Insektenzellen verwendet wird. Die Reinigung vorstehender Virus-ähnlicher Partikel erfolgt in üblicher Weise. Sie stellen auch einen Gegenstand der Erfindung dar.A virus-like particle of the latter case is also obtained by inserting the above DL1 7-G-L1 and DL1 7-G-L2 DNAs together into the expression vector pSynwtVI " and the pSynwtVI DLI 7-G-L1 / L2 obtained is used for the infection of SF-9 insect cells The above virus-like particles are cleaned in a conventional manner and are also an object of the invention.
Ein weiterer Gegenstand der Erfindung ist ein gegen ein vorstehendes Protein bzw. Virus-ähnliches Partikel gerichteter Antikörper. Die Herstellung eines solchen erfolgt in üblicher Weise. Beispielhaft wird es für die Herstellung eines Antikörpers beschrieben, der gegen ein L1 von DL1 7-G umfassendes Virusähnliches Partikel gerichtet ist. Hierzu wird das Virus-ähnliche Partikel BALB/c- Mäusen subcutan injiziert. Diese Injektion wird im Abstand von jeweils 3 Wochen wiederholt. Etwa 2 Wochen nach der letzten Injektion wird das den Antikörper enthaltende Serum isoliert und in üblicher Weise getestet.Another object of the invention is against an above protein or virus-like particle of directed antibodies. Such is produced in the usual way. It is described by way of example for the production of an antibody which is directed against a virus-like particle comprising L1 of DL1 7-G. For this purpose, the virus-like particle BALB / c mice is injected subcutaneously. This injection is repeated every 3 weeks. About 2 weeks after the last injection, the serum containing the antibody is isolated and tested in the usual way.
In bevorzugter Ausführungsform ist der Antikörper ein monoklonaler Antikörper. Zu seiner Herstellung werden nach vorstehender vierten Injektion den MäusenIn a preferred embodiment, the antibody is a monoclonal antibody. After the fourth injection above, the mice are produced
Milzzellen entnommen und diese in üblicher Weise mit Myelomzellen fusioniert. Die weitere Klonierung erfolgt ebenso nach bekannten Verfahren.Spleen cells removed and fused with myeloma cells in the usual way. The further cloning is also carried out according to known methods.
Mit der vorliegenden Erfindung wird es ermöglicht, Papillomviren, insbesondere in Karzinomen der Haut, nachzuweisen. Hierzu kann die erfindungsgemäße DNA als solche oder von einer weiteren DNA umfaßt eingesetzt werden. Letztere kann auch ein Papillomvirus-Gom oder ein Teil davon sein.The present invention makes it possible to detect papilloma viruses, in particular in carcinomas of the skin. For this purpose, the DNA according to the invention can be used as such or encompassed by a further DNA. The latter can also be a papilloma virus gome or part of it.
Die vorliegende Erfindung ermöglicht ferner die Bereitstellung von bisher nicht gekannten Papillomviren. Diese finden sich insbesondere in Karzinomen derThe present invention also enables the provision of previously unknown papilloma viruses. These are found particularly in carcinomas of the
Haut. Desweiteren liefert die Erfindung Proteine und Virus-ähnliche Partikel, die auf diese Papillomviren zurückgehen. Darüberhinaus werden Antikörper bereitgestellt, die gegen diese Proteine bzw. Partikel gerichtet sind.Skin. Furthermore, the invention provides proteins and virus-like particles which are due to these papillomaviruses. Antibodies are also provided which are directed against these proteins or particles.
Die vorliegende Erfindung ermöglicht es also, diagnostische und therapeutischeThe present invention thus enables diagnostic and therapeutic
Maßnahmen bei Papillomvirus-Erkrankungen zu ergreifen. Darüberhinaus liefert sie die Möglichkeit, eine Vakzine gegen Papillomvirus-Infektionen aufzubauen. Die vorliegende Erfindung stellt somit einen Durchbruch auf dem Gebiet der Papillomvirus-Forschung dar.Take action on papilloma virus diseases. In addition, it provides the opportunity to build a vaccine against papillomavirus infections. The present invention thus represents a breakthrough in the field of papilloma virus research.
Die Erfindung wird durch die Beispiele erläutert. Beispiel 1 : Identifizierung des Papillomvirus-Genoms DL17-GThe invention is illustrated by the examples. Example 1: Identification of the papilloma virus genome DL17-G
Aus der Biopsie eines plattenepithelialen Karzinoms einer immun- supprimierten Person wird die Gesamt-DNA isoliert. 1 0/vg dieser DNA werden mit dem Restriktionsenzym BamHI gespalten und in einem 0,5 % Agarosegel elektrophoretisch aufgetrennt. Gleichzeitig werden auch 10μg vorstehender DNA aufgetrennt, die nicht gespalten worden ist. Das Agarosegel wird einem Blotting-Ver- fahren unterzogen, wodurch die DNA aus dem Agarosegel auf eine Nitrozellulosemembran übertragen wird. Diese wird in ein Hybridisierungsverfahren eingesetzt, in dem die vorstehende DNA von Fig. 1 in Kombination mit HP-Virus-65 DNA als p32-markierte Probe verwendet wird. Es wird eine Hybridisierung mit der geblotteten DNA erhalten.The total DNA is isolated from the biopsy of a squamous cell carcinoma of an immunosuppressed person. 1 0 / vg of this DNA are cleaved with the restriction enzyme BamHI and electrophoresed in a 0.5% agarose gel. At the same time, 10μg of the above DNA, which has not been cleaved, are also separated. The agarose gel is subjected to a blotting process, whereby the DNA from the agarose gel is transferred to a nitrocellulose membrane. This is used in a hybridization process in which the above DNA from FIG. 1 is used in combination with HP virus 65 DNA as a p 32 -labeled sample. Hybridization with the blotted DNA is obtained.
Vorstehende Verfahren sind dem Fachmann auf dem Gebiet der DNA-Rekombinationstechnik bekannt. Ergänzend wird auf Sambrook et al., supra verwiesen.The above methods are known to those skilled in the field of DNA recombination technology. In addition, reference is made to Sambrook et al., Supra.
Beispiel 2: Klonierung des Papillomvirus-Genoms DL17-GExample 2: Cloning of the papilloma virus genome DL17-G
Die aus Beispiel 1 erhaltene Biopsie-DNA wird mit dem Restriktionsenzym BamHI gespalten. Die erhaltenen Fragmente werden in eine Ligasereaktion eingesetzt, in der ebenfalls der mit BamHI gespaltene und dephosphorylierte Vektor ΛZAP Express vorliegt.The biopsy DNA obtained from Example 1 is cleaved with the restriction enzyme BamHI. The fragments obtained are used in a ligase reaction in which the HZAP Express vector, which has been cleaved and dephosphorylated with BamHI, is also present.
Die hierbei erhaltenen rekombinanten DNA-Moleküle werden in Bakteriophagen verpackt und diese zur Infektion von Bakterien verwendet. Für diese Verfahrensschritte wird der von der Firma Stratagene angebotene ZAP Express Vektor Kit verwendet. Die erhaltenen Phagenplaques werden dann einem Hybridisierungsverfahren unterzogen, in dem die in Beispiel 1 verwendete p32-markier- te DNA von Fig. 1 in Kombination mit p32-markierter HP-Virus-65- DNA verwendet wird. Es wird eine Hybridisierung mit entsprechenden Phagenplaques erhalten. Aus diesen werden die BamHI-Frag- mente von DL1 7-G isoliert und zusammen mit einem BamHI-gespal- tenen, dephosphorylierten Plasmid-Vektor, pBluescript, in eine weitere Ligasereaktion eingesetzt. Die erhaltenen rekombinantenThe recombinant DNA molecules obtained in this way are packaged in bacteriophages and used to infect bacteria. The ZAP Express Vector Kit offered by Stratagene is used for these process steps. The phage plaques obtained are then subjected to a hybridization process in which the p 32 -labeled DNA from FIG. 1 used in Example 1 in combination with p 32 -labeled HP virus 65- DNA is used. Hybridization with corresponding phage plaques is obtained. The BamHI fragments of DL1 7-G are isolated from these and used together with a BamHI-split, dephosphorylated plasmid vector, pBluescript, in a further ligase reaction. The recombinant obtained
DNA-Moleküle werden zur Transformation von Bakterien, E. coli XL1 -Blue, verwendet. Durch Restriktionsspaltungen bzw. Hybridisierung mit vorstehenden DNA-Proben wird ein das Papillomvirus- Genom DL1 7-G enthaltender Bakterienklon identifiziert. Das Plas- mid dieses Bakterienklons wird mit pBlue-DL1 7-G bezeichnet. DNA molecules are used to transform bacteria, E. coli XL1 -Blue. A bacterial clone containing the papillomavirus genome DL1 7-G is identified by restriction cleavage or hybridization with the above DNA samples. The plasmid of this bacterial clone is designated pBlue-DL1 7-G.

Claims

Patentansprüche claims
1 . DNA, codierend für ein Peptid eines Papillomvirus-Hauptcapsid-Proteins, wobei das Peptid die Aminosäuresequenz von Fig. 1 , Fig. 2, Fig. 3, Fig. 4, Fig. 5, Fig. 6. Fig. 7, Fig. 8, Fig. 9, Fig. 1 0 oder Fig. 1 1 oder eine davon durch ein oder mehrere Aminosäuren unterschiedliche Aminosäuresequenz umfaßt, und wobei die DNA durch folgende Verfahrensschritte erhältlich ist:1 . DNA coding for a peptide of a papillomavirus main capsid protein, the peptide having the amino acid sequence of FIGS. 1, 2, 3, 4, 5, 6, 7, 8 , FIG. 9, FIG. 1 0 or FIG. 1 1 or one of these amino acid sequences different from one or more amino acids, and the DNA being obtainable by the following process steps:
(a) Isolierung der Gesamt-DNA aus einer Biopsie epithelialen Neoplas- mas,(a) isolation of the total DNA from a biopsy of epithelial neoplasm,
(b) Hybridisierung der Gesamt-DNA von (a) mit einer DNA von Fig. 1 , Fig. 2, Fig. 3, Fig. 4, Fig. 5, Fig. 6, Fig. 7, Fig. 8, Fig. 9, Fig. 1 0 oder Fig. 1 1 , wodurch ein in der Gesamt-DNA von (a) enthaltenes Papillomvirus-Genom nachgewiesen wird, und(b) Hybridization of the total DNA of (a) with a DNA from FIGS. 1, 2, 3, 4, 5, 6, 7, 8, 8 9, 10 or 11, whereby a papillomavirus genome contained in the total DNA of (a) is detected, and
(c) Klonierung der das Papillomvirus-Genom enthaltenden Gesamt-DNA von (a) in einem Vektor sowie Sequenzierung des Klons.(c) cloning the total DNA of (a) containing the papillomavirus genome in a vector and sequencing the clone.
2. DNA nach Anspruch 1 , dadurch gekennzeichnet, daß die für das Peptid des Papillomvirus-Hauptcapsid-Proteins codierende DNA die Basensequenz von Fig. 1 , Fig. 2, Fig. 3, Fig. 4, Fig. 5, Fig. 6. Fig. 7, Fig. 8, Fig. 9, Fig.2. DNA according to claim 1, characterized in that the DNA coding for the peptide of the papillomavirus main capsid protein has the base sequence of Fig. 1, Fig. 2, Fig. 3, Fig. 4, Fig. 5, Fig. 6. Fig. 7, Fig. 8, Fig. 9, Fig.
10 oder 1 1 oder eine davon durch ein oder mehrere Basenpaare unter- schiedliche Basensequenz umfaßt, und wobei die DNA durch folgende10 or 1 1 or one of them by one or more base pairs different base sequence, and wherein the DNA by the following
Verfahrensschritte erhältlich ist:Process steps are available:
(a) Isolierung der Gesamt-DNA aus einer Biopsie epithelialen Neoplas- mas, (b) Hybridisierung der Gesamt-DNA von (a) mit einer DNA von Fig. 1 ,(a) isolation of the total DNA from a biopsy of epithelial neoplasm, (b) hybridization of the total DNA from (a) with a DNA from FIG. 1,
Fig. 2, Fig. 3, Fig. 4, Fig. 5, Fig. 6, Fig. 7, Fig. 8, Fig. 9, Fig. 10 oder Fig. 1 1 , wodurch ein in der Gesamt-DNA von (a) enthaltendes Papillomvirus-Genom nachgewiesen wird, und (c) Klonierung derdas Papillomvirus-Genom enthaltenden Gesamt-DNA von (a) in einem Vektor sowie Sequenzierung des Klons.Fig. 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11, whereby a in the total DNA of ( a) containing Papillomavirus genome is detected, and (c) cloning the total DNA of the papillomavirus genome from (a) in a vector and sequencing the clone.
3. DNA nach Anspruch 1 oder 2, dadurch gekennzeichnet, daß die DNA ein3. DNA according to claim 1 or 2, characterized in that the DNA
Papillomvirus-Genom umfaßt.Papilloma virus genome.
4. Protein, codiert durch das Papillomvirus-Genom nach Anspruch 3.4. Protein encoded by the papilloma virus genome according to claim 3.
5. Protein nach Anspruch 4, dadurch gekennzeichnet, daß das Papillomvirus-5. Protein according to claim 4, characterized in that the papilloma virus
Hauptcapsid-Protein als Virus-ähnliches Partikel vorliegt.Main capsid protein is present as a virus-like particle.
6. Protein nach Anspruch 5, dadurch gekennzeichnet, daß das Virus-ähnliche Partikel auch ein Papillomvirus-Nebencapsid-Protein enthält.6. Protein according to claim 5, characterized in that the virus-like particle also contains a papilloma virus secondary capsid protein.
7. Expressionsvektor, umfassend eine für das Protein nach Anspruch 4 codierende DNA.7. Expression vector comprising a DNA coding for the protein according to claim 4.
8. Transformante, enthaltend den Expressionsvektor nach Anspruch 7.8. transformant containing the expression vector according to claim 7.
9. Verfahren zur Herstellung des Proteins nach Anspruch 4, umfassend die Kultivierung der Transformante nach Anspruch 8 unter geeigneten Bedingungen.9. A method for producing the protein of claim 4, comprising culturing the transformant of claim 8 under suitable conditions.
10. Antikörper, gerichtet gegen das Protein nach einem der Ansprüche 4-6.10. Antibody directed against the protein according to any one of claims 4-6.
1 1 . Verwendung der DNA nach einem der Ansprüche 1 -3 als Reagens zur Diagnose.1 1. Use of the DNA according to one of claims 1 -3 as a reagent for diagnosis.
1 2. Verwendung des Proteins nach einem der Ansprüche 4-6 als Reagens zur Diagnose, Therapie und/oder Vakzinierung. 1 2. Use of the protein according to any one of claims 4-6 as a reagent for diagnosis, therapy and / or vaccination.
1 3. Verwendung nach Anspruch 1 1 oder 1 2, wobei die Diagnose Papillomvirus-Infektionen bzw. -Erkrankungen betrifft.1 3. Use according to claim 1 1 or 1 2, wherein the diagnosis relates to papillomavirus infections or diseases.
14. Verwendung nach Anspruch 1 2, wobei die Therapie und/oder Vakzinierung Papillomvirus-Infektionen bzw. -Erkrankungen betrifft. 14. Use according to claim 1 2, wherein the therapy and / or vaccination relates to papillomavirus infections or diseases.
EP97949918A 1996-11-26 1997-11-12 Papilloma viruses, agents for detecting the same and for treating diseases caused by such viruses Ceased EP0941336A2 (en)

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DE19648962A DE19648962C1 (en) 1996-11-26 1996-11-26 DNA encoding peptide(s) from papilloma virus major capsid protein
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PCT/DE1997/002659 WO1998023752A2 (en) 1996-11-26 1997-11-12 Papilloma viruses, agents for detecting the same and for treating diseases caused by such viruses

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DE19712541C1 (en) * 1997-03-25 1998-11-05 Deutsches Krebsforsch Papilloma viruses, means for their detection and for the therapy of diseases caused by them
DE19735118C1 (en) 1997-08-13 1998-08-13 Deutsches Krebsforsch Papilloma virus DNA
DE19925235A1 (en) 1999-06-01 2000-12-07 Medigene Ag Cytotoxic T cell epitopes of the papillomavirus L1 protein and their use in diagnostics and therapy
DE19925199A1 (en) 1999-06-01 2000-12-07 Medigene Ag Cytotoxic T cell epitopes of the papillomavirus L1 protein and their use in diagnostics and therapy
WO2005036456A2 (en) * 2003-05-12 2005-04-21 Princeton University Method and apparatus for foreground segmentation of video sequences
CA2799205A1 (en) 2010-05-25 2011-12-01 Qiagen Gaithersburg, Inc. Fast results hybrid capture assay and associated strategically-truncated probes

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DE4415743C2 (en) * 1994-05-04 1996-10-10 Deutsches Krebsforsch Papillomaviruses, means for their detection and for the therapy of diseases caused by them
DE19526386C1 (en) * 1995-07-19 1997-01-02 Deutsches Krebsforsch Papillomaviruses, means for their detection and for the therapy of diseases caused by them

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