EP0707652A1 - Dna coding for a peptide of a papilloma virus main capside protein and use thereof - Google Patents

Dna coding for a peptide of a papilloma virus main capside protein and use thereof

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Publication number
EP0707652A1
EP0707652A1 EP95919396A EP95919396A EP0707652A1 EP 0707652 A1 EP0707652 A1 EP 0707652A1 EP 95919396 A EP95919396 A EP 95919396A EP 95919396 A EP95919396 A EP 95919396A EP 0707652 A1 EP0707652 A1 EP 0707652A1
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EP
European Patent Office
Prior art keywords
dna
virus
protein
papilloma virus
peptide
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.)
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Application number
EP95919396A
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German (de)
French (fr)
Inventor
Vladimir Shamanin
Ethel-Michele De Villiers-Zur Hausen
Irene London Hospital Medical College LEIGH
Harald Zur Hausen
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Deutsches Krebsforschungszentrum DKFZ
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Deutsches Krebsforschungszentrum DKFZ
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Publication of EP0707652A1 publication Critical patent/EP0707652A1/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
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • 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
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • 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
    • C12N2710/20021—Viruses as such, e.g. new isolates, mutants or their genomic sequences
    • 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
    • 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
    • C12N2710/20023—Virus like particles [VLP]
    • 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
    • C12N2710/20061—Methods of inactivation or attenuation

Definitions

  • DNA coding for a peptide of a Papi ⁇ omvirus-Hauptcapsld protein DNA coding for a peptide of a Papi ⁇ omvirus-Hauptcapsld protein and its uses
  • HP viruses Human papilloma viruses
  • benign e.g. Warts, genital condylomas, and malignancies, e.g. Carcinomas of the skin and uterus, epithelial neoplasms (cf.zzy Hausen, H., Cancer Research 49 (1,989), pages 4677-4681).
  • HP viruses are also considered for the development of malignant tumors of the respiratory tract (cf. Kir Hausen, H., Cancer Research 36 (1 976), page 530).
  • HP viruses are considered to be at least jointly responsible for the development of squamous carcinomas of the lungs (cf. Syrjänen, K.J., Lung 1 58 (1 980), pages 1 31 -142).
  • 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 (L2). Both proteins, coexpressed or L1 expressed alone, lead to the formation of virus-like particles in vitro (cf. Kirnbauer, R. et al., Journal of
  • Papilloma viruses cannot be propagated in monolayer cell culture. Their characterization is therefore extremely difficult, with the detection of papilloma viruses already creating considerable problems. This is particularly true for papilomaviruses in skin carcinomas. So far, no reliable proof of this and therefore no targeted action against it is possible.
  • the present invention is therefore based on the object of an agent to provide, with which papilloma viruses, especially in carcinomas of the skin, can be detected.
  • 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 at least a part of the amino acid sequence or the amino acid sequence of FIG. 1,
  • amino acid sequence indicates that the amino acid sequence of the individual figures can also have a variation of one or more amino acids.
  • Another object of the invention is a DNA coding for a peptide of a papilloma virus main capsid protein, the DNA at least a part of the base sequence or the base sequence of FIG. 1, 2, 3, 4, 4 5, 6, 7, 8 or 9.
  • the expression "at least part of the base sequence” indicates that the base sequence of the individual figures can also have a variation of one or more base pairs.
  • 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. That DNA was deposited as Plasmjd CR148-59 with DSM under DSM 91 34 on April 1st, 994.
  • 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 VS40-7 at DSM under DSM 91 35 on April 1, 994.
  • 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 VS20-4 at DSM under DSM 91 36 on April 1, 994.
  • 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 VS102-4 at DSM under DSM 91 37 on April 1, 994.
  • 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 VS73-1 at DSM under DSM 91 38 on April 1, 994.
  • 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 VS42-1 at DSM under DSM 91 39 on April 1, 994.
  • 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 VS92-1 with the DSM under DSM 9140 on April 1, 994.
  • FIG. 9 shows the B.ase 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 VS75-3 with the DSM under DSM 9141 on April 2nd, 994.
  • the above DNA has the following sequence homology to 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.
  • E. coli these are, for example, pGEMEX, pUC derivatives and pGEX-2T.
  • yeast for example, pY100 and Ycpad 1 are to be mentioned, while for expression in animal cells, for example, pKCR, pEF-BOS, cDM8 and pCEV4 are to be mentioned.
  • suitable cells in order to express the above DNA present in an expression vector examples of such cells include the E.
  • the person skilled in the art knows how the above DNA has to be inserted into an expression vector. He is also aware that the above DNA is inserted in conjunction with a DNA coding for another protein or peptide can, so that the above DNA can be expressed in the form of a fusion protein.
  • papilloma virus genome which comprises the above DNA.
  • the term "papilloma virus genome” also includes an incomplete genome, i.e. Fragments of a papillomavirus genome which comprise the above DNA. This can e.g. a DNA coding for L1 or a part thereof.
  • a method comprising the following method steps can be used to provide the above papillomavirus genome:
  • 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 papilloma virus 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, and phages, such as ⁇ -phages, for example ⁇ ZAP Expressvector, ⁇ ZAPII Vector and ⁇ gt10 vector. In the present case, ⁇ 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 about procedures for cloning the
  • Papilloma virus genomes to find out optimal restriction enzymes. 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 papillomavirus genome designated VS93-1 -G is described by way of example.
  • the total 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 to a nitrocellulose membrane.
  • This is used in a hybridization process in which the DNA from FIG. 1, possibly in combination with a DNA from HP virus 29, is used as the labeled sample. Hybridization with the papilloma virus DNA present in the total DNA is obtained.
  • ⁇ phage cloned 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 29. 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
  • VS93-1 -G contains. The genome is confirmed by sequencing. Analogously, further papillomavirus genomes are provided. They are designated in accordance with the DNAs used for their preparation, with: CR148-59-G, VS40-7-G, VS20-4-G, VS102-4-G, VS73-1 -G, VS42- 1 -G , VS92-1 -G or VS75-3-G.
  • Another object of the invention is a protein encoded by the above papillomavirus genome.
  • a protein is e.g. a major capsid protein (L1) or a minor capsid protein (L2).
  • L1 or L2 of the papilloma virus genome VS93-1 -G is described as an example.
  • the HP virus 29 related to the DNA of FIG. 1 is used.
  • the complete sequence and the position of individual DNA regions coding for proteins are known from this. By parallel restriction cleavage of both genomes and subsequent hybridization with different fragments relating to L1 or L2 coding DNA, these DNAs are on the
  • Papilloma virus genome VS93-1 -G identified. They are confirmed by sequencing.
  • the DNA coding for L1 is designated VS93-1 -G-L1-DNA and the DNA coding for L2 with VS93-1 -G-L2-DNA.
  • the DNA coding for L1 or L2 is inserted into an expression vector.
  • an expression vector examples of such for E. coli, yeast and animal cells are mentioned above.
  • vector pGEX-2T for expression in E. coli (cf. Kirnbauer, R. et al., Supra).
  • pGEX-2T-VS93-1 - G-L1 or pGEX-2T-VS93-1 -G-L2 is obtained.
  • these expression vectors express a glutathione S-transferase-L1 or glutathione S-transferase-L2 fusion protein. These proteins are cleaned in the usual way.
  • the bacculovirus or vaccinia virus system is called for a further expression of the above coding L1 or L2 DNA.
  • Expression vectors that can be used for this are, for example, pEV mod. and pSynwtVI " for the bacculovirus System (see Kirnbauer, R. et al., Supra).
  • vectors with the vaccinia virus "early” (p7.5k) or "late” (Psynth, p1 1 K) promoter are to be mentioned in particular (cf. Hagensee, M., E. et al., Journal of Virology (1,993), pages 31 5-322).
  • the bacculovirus system is preferred. Coding L1 or L2 after insertion
  • DNA in pEV mod. pEVmod.-VS93-1 -G-L1 or pEVmod.-VS93-1 -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 VS93-1 -G-L1 and VS93-1 -G-L2 DNAs together into the expression vector pSynwtVI " and the resulting pSynwtVI " VS93-1 -G -L1 / L2 is used to infect SF-9 insect cells.
  • the above virus-like particles are cleaned in the usual way. They also represent an object of the invention.
  • Another object of the invention is an antibody directed against an above protein or virus-like particle. Such is produced in the usual way. It is described by way of example for the production of an antibody which comprises an L1 of VS93-1 -G
  • Virus-like particle is targeted.
  • 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.
  • the fourth injection above is used to manufacture it Spleen cells were taken from mice and fused with myeloma cells in the usual way. The further cloning is also carried out according to known methods.
  • the present invention makes it possible to detect papilloma viruses, especially in carcinomas of the skin.
  • the invention makes it possible to detect papilloma viruses, especially in carcinomas of the skin.
  • DNA as such or encompassed by a further DNA can be used.
  • 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 makes it possible to take diagnostic and therapeutic measures in papillomavirus 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.
  • the total DNA is isolated from the biopsy WV-8495 of a squamous cell carcinoma of an immunosuppressed person. 10 / vg of this DNA are digested 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 result of
  • Electrophoresis is shown in Figure 10. From this it follows that in the non-cleaved DNA a DNA molecule in the for an extrachromosomal DNA typical form, ie "supercoiled molecule” or "open circular molecule". This DNA molecule is split into two fragments by BamHI.
  • the above agarose gel is subjected to a blotting process, whereby the DNA is transferred from the agarose gel to a nitrocellulose membrane. This is used in a hybridization process in which the above DNA from FIG.
  • the DNA of the biopsy WV-8495 obtained from Example 1 is cleaved with the restriction enzyme BamHI.
  • the fragments obtained are used in a ligase reaction in which the ⁇ ZAP Express vector which is cleaved and dephosphorylated with BamHI is 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 is used in combination with p 32 -labeled HP virus 29 DNA. Hybridization with corresponding phage plaques is obtained.

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Abstract

The invention relates to DNA which codes for a peptide of a papilloma virus main capside protein. In addition, it concerns a papilloma virus genome containing such DNA. It also concerns proteins and virus-like particles coded by the papilloma virus genome, as well as antibodies directed against them and the use of those antibodies in diagnosis, treatment and vaccination.

Description

Für ein Peptid eines Papiπomvirus-Hauptcapsld-Proteins codierende DNA und dessen Vervendungen DNA coding for a peptide of a Papiπomvirus-Hauptcapsld protein and its uses
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. zur Hausen, H., Cancer Research 49 ( 1 989), Seiten 4677-4681 ). Auch werden HP-Viren für die Entwicklung maligner Tumoren des Respirations¬ trakts in Betracht gezogen (vgl. zur Hausen, H., Cancer Research 36 (1 976), Seite 530). Desweiteren werden HP-Viren für die Entwicklung squamöser Karzinome der Lunge als zumindest mitverantwortlich angesehen (vgl. Syrjänen, K.J., Lung 1 58 ( 1 980), Seiten 1 31 -142).Papillomaviruses 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. zur Hausen, H., Cancer Research 49 (1,989), pages 4677-4681). HP viruses are also considered for the development of malignant tumors of the respiratory tract (cf. zur Hausen, H., Cancer Research 36 (1 976), page 530). Furthermore, HP viruses are considered to be at least jointly responsible for the development of squamous carcinomas of the lungs (cf. Syrjänen, K.J., Lung 1 58 (1 980), pages 1 31 -142).
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 (L2). Beide Proteine, coexprimiert oder L1 alleine exprimiert, führen in vitro zur Ausbildung von Virus-ähnlichen Partikeln (vgl. Kirnbauer, R. et al., Journal ofPapilloma 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 (L2). 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), Seiten 6929-6936).Virology, (1 993), pages 6929-6936).
Papillomviren lassen sich nicht in Monolayer-Zellkultur vermehren. Ihre Cha¬ rakterisierung ist daher äußerst schwierig, wobei bereits der Nachweis von Papillomviren erhebliche Probleme schafft. Dies trifft insbesondere für Papil¬ lomviren in Karzinomen der Haut zu. Hier ist bisher kein verläßlicher Nachweis dieser und damit kein gezieltes Vorgehen gegen sie möglich.Papilloma viruses cannot be propagated in monolayer cell culture. Their characterization is therefore extremely difficult, with the detection of papilloma viruses already creating considerable problems. This is particularly true for papilomaviruses in skin carcinomas. So far, no reliable proof of this and therefore no targeted action against it is possible.
Der vorliegenden Erfindung liegt somit die Aufgabe zugrunde, ein Mittel bereitzustellen, mit dem Papillomviren, insbesondere in Karzinomen der Haut, nachgewiesen werden können. Ferner sollte ein Mittel bereitgestellt werden, um gegen diese Papillomviren therapeutisch vorgehen zu können.The present invention is therefore based on the object of an agent to provide, with which papilloma viruses, especially in carcinomas of the skin, can be detected. A means should also be provided to treat these papillomaviruses therapeutically.
Erfindungsgemäß wird dies durch die Bereitstellung der Gegenstände in denAccording to the invention, this is achieved by providing the objects in the
Patentansprüchen erreicht.Claims achieved.
Gegenstand der Erfindung ist somit eine für ein Peptid eines Papillomvirus- Hauptcapsid-Proteins (L1 ) codierende DNA, wobei das Peptid mindestens einen Teil der Aminosäuresequenz oder die Aminosäuresequenz von Fig. 1 ,The invention thus relates to a DNA coding for a peptide of a papillomavirus main capsid protein (L1), the peptide at least a part of the amino acid sequence or the amino acid sequence of FIG. 1,
Fig. 2, Fig. 3, Fig. 4, Fig. 5, Fig. 6, Fig. 7, Fig. 8 oder Fig. 9 umfaßt.2, 3, 4, 5, 6, 7, 8 or 9.
Der Ausdruck "mindestens einen Teil der Aminosäuresequenz" weist darauf hin, daß die Aminosäuresequenz der einzelnen Figuren auch eine Variation von ein oder mehreren Aminosäuren aufweisen kann.The expression "at least part of the amino acid sequence" indicates that the amino acid sequence of the individual figures can also have a variation of one or more amino acids.
Ein weiterer Gegenstand der Erfindung ist eine für ein Peptid eines Papillomvi- rus-Hauptcapsid-Proteins codierende DNA, wobei die DNA mindestens einen Teil der Basensequenz oder die Basensequenz von Fig. 1 , Fig. 2, Fig. 3, Fig. 4, Fig. 5, Fig. 6, Fig. 7, Fig. 8 oder Fig. 9 umfaßt.Another object of the invention is a DNA coding for a peptide of a papilloma virus main capsid protein, the DNA at least a part of the base sequence or the base sequence of FIG. 1, 2, 3, 4, 4 5, 6, 7, 8 or 9.
Der Ausdruck "mindestens einen Teil der Basensequenz" weist darauf hin, daß die Basensequenz der einzelnen Figuren auch eine Variation von ein oder mehreren Basenpaaren aufweisen kann.The expression "at least part of the base sequence" indicates that the base sequence of the individual figures can also have a variation of one or more base pairs.
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 VS93-1 bei der DSM (Deutsche Sammlung von Mikroorga¬ nismen und Zellkulturen) unter DSM 91 33 am 1 2. April 1 994 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 VS93-1 at the DSM (German Collection of Microorganisms and Cell Cultures) under DSM 91 33 on April 1, 994.
Fig. 2 zeigt die Basensequenz und die davon abgeleitete Aminosäuresequenz einer für ein Peptid von L1 eines Papillomvirus codierenden DNA. Diese DNA wurde als Plasmjd CR148-59 bei der DSM unter DSM 91 34 am 1 2. April 1 994 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. That DNA was deposited as Plasmjd CR148-59 with DSM under DSM 91 34 on April 1st, 994.
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 VS40-7 bei der DSM unter DSM 91 35 am 1 2. April 1 994 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 VS40-7 at DSM under DSM 91 35 on April 1, 994.
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 VS20-4 bei der DSM unter DSM 91 36 am 1 2. April 1 994 hinterlegt.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 VS20-4 at DSM under DSM 91 36 on April 1, 994.
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 VS102-4 bei der DSM unter DSM 91 37 am 1 2. April 1 994 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 VS102-4 at DSM under DSM 91 37 on April 1, 994.
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 VS73-1 bei der DSM unter DSM 91 38 am 1 2. April 1 994 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 VS73-1 at DSM under DSM 91 38 on April 1, 994.
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 VS42-1 bei der DSM unter DSM 91 39 am 1 2. April 1 994 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 VS42-1 at DSM under DSM 91 39 on April 1, 994.
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 VS92-1 bei der DSM unter DSM 9140 am 1 2. April 1 994 hinterlegt. Fig. 9 zeigt die B.asensequenz und die davon abgeleitete Aminosäuresequenz einer für ein Peptid von L1 eines Papillomvirus codierenden DNA. Diese DNA wurde als Plasmid VS75-3 bei der DSM unter DSM 9141 am 1 2. April 1 994 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 VS92-1 with the DSM under DSM 9140 on April 1, 994. FIG. 9 shows the B.ase 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 VS75-3 with the DSM under DSM 9141 on April 2nd, 994.
Vorstehende DNA weist zu bekannten Papillomviren folgende Sequenzhomo¬ logie auf:The above DNA has the following sequence homology to known papilloma viruses:
DNA von Fig. 1 82.7 % zu HP-Virus 29 DNA von Fig. 2 75 % zu HP-Virus 49 DNA von Fig. 3 78.5 % zu HP-Virus 49 DNA von Fig. 4 75.6 % zu HP-Virus 25 DNA von Fig. 5 79 % zu HP-Virus 1 7 DNA von Fig. 6 73.6 % zu HP-Virus 17 DNA von Fig. 7 73, 1 % zu HP-Virus 1 5 DNA von Fig. 8 82.8 % zu HP-Virus 1 5 DNA von Fig. 9: 75.7 % zu HP-Virus 12DNA from FIG. 1 82.7% to HP virus 29 DNA from FIG. 2 75% to HP virus 49 DNA from FIG. 3 78.5% to HP virus 49 DNA from FIG. 4 75.6% to HP virus 25 DNA from 5 79% to HP virus 1 7 DNA from FIG. 6 73.6% to HP virus 17 DNA from FIG. 7 73, 1% to HP virus 1 5 DNA from FIG. 8 82.8% to HP virus 1 5 DNA from FIG. 9: 75.7% to HP virus 12
Erfindungsgemäß kann vorstehende DNA in einem Vektor bzw. Expressions¬ vektor vorliegen. Beispiele solcher sind dem Fachmann bekannt. Im Falle eines Expressionsvektors für E. coli sind dies z.B. pGEMEX, pUC-Derivate 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. Der Fachmann kennt geeignete Zellen, um vorstehende, in einem Expressionsvektor vorliegende DNA zu exprimieren. Beispiele solcher Zellen umfassen die E.coli-Stämme HB101 , DH1 , x1 776, JM101 und JM 109, den Hefe-Stamm Saccharomyces cerevisiae und die tierischen Zellen L, 3T3, FM3A, CHO, COS, Vero, und Heia. Der Fachmann weiß, in welcher Weise vorstehende DNA in einen Expressionsvektor inseriert werden muß. Ihm ist auch bekannt, daß vorstehende 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.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 and pGEX-2T. For expression in yeast, for example, pY100 and Ycpad 1 are to be mentioned, while for expression in animal cells, for example, pKCR, pEF-BOS, cDM8 and pCEV4 are to be mentioned. 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 HB101, DH1, x1 776, JM101 and JM 109, the yeast strain Saccharomyces cerevisiae and the animal cells L, 3T3, FM3A, CHO, COS, Vero, and Heia. The person skilled in the art knows how the above DNA has to be inserted into an expression vector. He is also aware that the above DNA is inserted in conjunction with a DNA coding for another protein or peptide can, 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 vor- stehende DNA umfaßt. Der Ausdruck "Papillomvirus-Genom" umfaßt auch ein unvollständiges Genom, d.h. Fragmente eines Papillomvirus-Genoms, die vor¬ stehende 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 which comprises the above DNA. The term "papilloma virus genome" also includes an incomplete genome, i.e. Fragments of a papillomavirus genome which comprise the above DNA. This can e.g. a DNA coding for L1 or a part thereof.
Zur Bereitstellung vorstehenden Papillomvirus-Genoms kann ein Verfahren verwendet werden, das folgende Verfahrensschritte umfaßt:A method comprising the following method steps can be used to provide the above papillomavirus genome:
(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, wo¬ durch ein in der Gesamt-DNA von (a) enthaltenes Papillomvirus-Genom nachgewiesen wird, und(b) hybridization of the total DNA of (a) with the above DNA, whereby a papilloma virus 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, und gegebenenfalls Subklonierung des erhalte¬ nen Klons, wobei sämtliche Verfahrensschritte üblicher DNA-Rekom¬ binationstechnik entstammen.(c) Cloning of the total DNA from (a) containing the papillomavirus genome in a vector, and optionally subcloning of the clone obtained, all process steps coming 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, zweite Ausgabe, Cold Spring Harbor Laboratory ( 1 989) verwiesen.With regard to the isolation, hybridization and cloning of Zeil DNA, reference is also made to Sambrook et al., Molecular Cloning, A Laboratory Manual, second 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. Der Ausdruck "Vektor" umfaßt jegliche zur Klonierung von chromosomaler bzw. extrachromosomaler DNA geeignete Vektoren. Beispiele solcher Vekto¬ ren sind Cosmide, wie pWE1 5 und Super Cos1 , und Phagen, wie Λ-Phagen, z.B. ΛZAP Expressvector, ΛZAPII Vector und Λgt10 Vektor. Vorliegend wer- den Λ-Phagen bevorzugt verwendet. Vorstehende Vektoren sind bekannt und bei der Firma Stratagene erhältlich.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 papilloma virus genome. The term "vector" includes any vector suitable for cloning chromosomal or extrachromosomal DNA. Examples of such vectors are cosmids, such as pWE1 5 and Super Cos1, and phages, such as Λ-phages, for example ΛZAP 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 derPapillomavirus 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 about procedures for cloning the
Papillomvirus-Genome optimalen Restriktionsenzyme herauszufinden. Er wird sich an Genomen bekannter Papillomviren orientieren. Insbesondere wird der Fachmann die vorstehend genannten HP-Viren entsprechend beachten.Papilloma virus genomes to find out optimal restriction enzymes. 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 VS93-1 -G bezeichneten Papil¬ lomvirus-Genoms beschrieben. Hierzu wird die Gesamt-DNA aus einer Biopsie eines plattenepithelialen Karzinoms isoliert, mit BamHI gespalten und in einem Agarosegel elektrophoretisch aufgetrennt. Das Agarosegel wird danach einem Blotting-Verfahren unterzogen, wodurch die DNA auf eine Nitrozellulosemem- bran übertragen wird. Diese wird in ein Hybridisierungsverfahren eingesetzt, in dem die DNA von Fig. 1 , ggfs. in Kombination mit einer DNA von HP- Virus 29 als markierte Probe verwendet wird. Es wird eine Hybridisierung mit der in der Gesamt-DNA vorliegenden Papillomvirus-DNA erhalten.The provision of a papillomavirus genome designated VS93-1 -G is described by way of example. For this purpose, the total 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 to a nitrocellulose membrane. This is used in a hybridization process in which the DNA from FIG. 1, possibly in combination with a DNA from HP virus 29, 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 einemIn addition, the above-mentioned total DNA cleaved with BamHI is combined in one
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 29 identifiziert. Das Insert dieser Klone wird dann einer weiteren Klonierung in einem Plasmid-Vektor unterzogen, wodurch ein Klon erhalten wird, der das Papillomvirus-GenomΛ phage cloned. 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 29. 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
VS93-1 -G enthält. Das Genom wird durch Sequenzierung bestätigt. In analoger Weise werden weitere Papillomvirus-Genome bereitgestellt. Sie werden entsprechend der zu ihrer Bereitstellung verwendeten DNAs bezeich¬ net, mit: CR148-59-G, VS40-7-G, VS20-4-G, VS102-4-G, VS73-1 -G, VS42- 1 -G, VS92-1 -G bzw. VS75-3-G.VS93-1 -G contains. The genome is confirmed by sequencing. Analogously, further papillomavirus genomes are provided. They are designated in accordance with the DNAs used for their preparation, with: CR148-59-G, VS40-7-G, VS20-4-G, VS102-4-G, VS73-1 -G, VS42- 1 -G , VS92-1 -G or VS75-3-G.
Ein weiterer Gegenstand der Erfindung ist ein Protein, das durch vorstehendes Papillomvirus-Genom codiert wird. Ein solches Protein ist z.B. ein Hauptcap- sid-Protein (L1 ) oder ein Nebencapsidprotein (L2). Die Herstellung eines vor¬ stehenden Proteins erfolgt in üblicher weise. Beispielhaft wird die Herstellung von L1 bzw. L2 des Papillomvirus-Genoms VS93-1 -G beschrieben. Hierzu wird das zu der DNA von Fig. 1 verwandte HP-Virus 29 herangezogen. Von diesem ist die vollständige Sequenz und die Lage einzelner für Proteine codie¬ render DNA-Bereiche bekannt. Durch parallele Restriktionsspaltungen beider Genome und anschließender Hybridisierung mit verschiedenen, die L1 bzw. L2 codierende DNA betreffenden Fragmenten werden diese DNAs auf demAnother object of the invention is a protein encoded by the above papillomavirus genome. Such a protein is e.g. a major capsid protein (L1) or a minor capsid protein (L2). The above protein is produced in the usual way. The production of L1 or L2 of the papilloma virus genome VS93-1 -G is described as an example. For this purpose, the HP virus 29 related to the DNA of FIG. 1 is used. The complete sequence and the position of individual DNA regions coding for proteins are known from this. By parallel restriction cleavage of both genomes and subsequent hybridization with different fragments relating to L1 or L2 coding DNA, these DNAs are on the
Papillomvirus-Genom VS93-1 -G identifiziert. Sie werden durch Sequenzierung bestätigt. Die für L1 codierende DNA wird mit VS93-1 -G-L1 -DNA und die für L2 codierende DNA mit VS93-1 -G-L2-DNA bezeichnet.Papilloma virus genome VS93-1 -G identified. They are confirmed by sequencing. The DNA coding for L1 is designated VS93-1 -G-L1-DNA and the DNA coding for L2 with VS93-1 -G-L2-DNA.
Im weiteren wird die für L1 bzw. L2 codierende DNA in einen Expressions¬ vektor inseriert. Beispiele eines solchen für E. coli, Hefe und tierische Zellen sind vorstehend genannt. Ergänzend hierzu wird für die Expression in E. coli auf den Vektor pGEX-2T verwiesen (vgl. Kirnbauer, R. et al., supra) . Nach Insertion der VS93-1 -G-L1 - bzw. VS93-1 -G-L2-DNA wird pGEX-2T-VS93-1 - G-L1 bzw. pGEX-2T-VS93-1 -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 Protei¬ ne erfolgt in üblicher Weise.In addition, 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 addition, reference is made to the vector pGEX-2T for expression in E. coli (cf. Kirnbauer, R. et al., Supra). After insertion of the VS93-1 -G-L1 - or VS93-1 -G-L2-DNA, pGEX-2T-VS93-1 - G-L1 or pGEX-2T-VS93-1 -G-L2 is obtained. After transformation of E. coli, these expression vectors express a glutathione S-transferase-L1 or glutathione S-transferase-L2 fusion protein. These proteins are cleaned in the usual way.
Für eine weitere Expression vorstehender L1 bzw. L2 codierender DNA wird das 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" (p7.5k)- 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 Bacculovi- rus-System bevorzugt. Nach Insertion vorstehender L1 bzw. L2 codierenderThe bacculovirus or vaccinia virus system is called for a further expression of the above coding L1 or L2 DNA. Expression vectors that can be used for this are, for example, pEV mod. and pSynwtVI " for the bacculovirus System (see Kirnbauer, R. et al., Supra). For the vaccinia virus system, vectors with the vaccinia virus "early" (p7.5k) or "late" (Psynth, p1 1 K) promoter are to be mentioned in particular (cf. Hagensee, M., E. et al., Journal of Virology (1,993), pages 31 5-322). In the present case, the bacculovirus system is preferred. Coding L1 or L2 after insertion
DNA in pEV mod. wird pEVmod.-VS93-1 -G-L1 bzw. pEVmod.-VS93-1 -G-L2 erhalten.DNA in pEV mod. pEVmod.-VS93-1 -G-L1 or pEVmod.-VS93-1 -G-L2 is obtained.
Der erstere Expressionvektor alleine bzw. beide Expressionsvektoren zusam- men führen nach Infektion von SF-9 Insektenzellen zur Ausbildung von Virus¬ ähnlichen Partikeln. Im ersteren Fall umfaßt ein solches Partikel ein L1 -Pro¬ tein, während es im letzteren Fall neben einem L1 - auch ein L2-Protein ent¬ hält.The first 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 VS93-1 -G-L1 - und VS93-1 -G-L2-DNAs gemeinsam in den Expressionsvektor pSynwtVI" inseriert werden und das erhaltene pSynwtVI" VS93-1 -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 VS93-1 -G-L1 and VS93-1 -G-L2 DNAs together into the expression vector pSynwtVI " and the resulting pSynwtVI " VS93-1 -G -L1 / L2 is used to infect SF-9 insect cells. The above virus-like particles are cleaned in the usual way. They also represent 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 VS93-1 -G umfassendesAnother object of the invention is an antibody directed against an above protein or virus-like particle. Such is produced in the usual way. It is described by way of example for the production of an antibody which comprises an L1 of VS93-1 -G
Virus-ähnliches 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.Virus-like particle is targeted. 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 Antikör¬ per. Zu seiner Herstellung werden nach vorstehender vierten Injektion den Mäusen Milzzellen entnommen und diese in üblicher weise mit Myelomzellen fusioniert. Die weitere Klonierung erfolgt ebenso nach bekannten Verfahren.In a preferred embodiment, the antibody is a monoclonal antibody. The fourth injection above is used to manufacture it Spleen cells were taken from mice 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, insbesonde- re in Karzinomen der Haut, nachzuweisen. Hierzu kann die erfindungsgemäßeThe present invention makes it possible to detect papilloma viruses, especially in carcinomas of the skin. For this purpose, the invention
DNA als solche oder von einer weiteren DNA umfaßt eingesetzt werden. Letztere kann auch ein Papillomvirus-Gom oder ein Teil davon sein.DNA as such or encompassed by a further DNA can be used. 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 therapeuti¬ sche Maßnahmen bei Papillomvirus-Erkrankungenzu 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.The present invention thus makes it possible to take diagnostic and therapeutic measures in papillomavirus 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.The invention is illustrated by the examples.
Beispiel 1 : Identifizierung des Papillomvirus-Genoms VS93-1 GExample 1: Identification of the Papilloma Virus Genome VS93-1 G
Aus der Biopsie WV-8495 eines plattenepithelialen Karzinoms einer immunsupprimierten Person wird die Gesamt-DNA isoliert. 10/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 Ergebnis derThe total DNA is isolated from the biopsy WV-8495 of a squamous cell carcinoma of an immunosuppressed person. 10 / vg of this DNA are digested 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 result of
Elektrophorese ist in der Figur 10 gezeigt. Aus dieser geht her¬ vor, daß in der nicht-gespaltenen DNA ein DNA-Molekül in der für eine extrachromosomale DNA typischen Form, d.h. "super- coiled molecule" bzw. "open circular molecule" vorliegt. Dieses DNA-Molekül wird durch BamHI in zwei Fragmente gespalten.Electrophoresis is shown in Figure 10. From this it follows that in the non-cleaved DNA a DNA molecule in the for an extrachromosomal DNA typical form, ie "supercoiled molecule" or "open circular molecule". This DNA molecule is split into two fragments by BamHI.
Vorstehendes Agarosegel wird einem Blotting-Verfahren unter¬ zogen, wodurch die DNA aus dem Agarosegel auf eine Nitrozel¬ lulosemembran übertragen wird. Diese wird in ein Hybridisie- rungsverfahren eingesetzt, in dem die vorstehende DNA von Fig.The above agarose gel is subjected to a blotting process, whereby the DNA is transferred from the agarose gel to a nitrocellulose membrane. This is used in a hybridization process in which the above DNA from FIG.
1 in Kombination mit HP-Virus-29 DNA als p32-markierte Probe verwendet wird. Es wird eine Hybridisierung mit vorstehendem DNA-Molekül erhalten.1 in combination with HP virus 29 DNA is used as a p 32 -labeled sample. Hybridization with the above DNA molecule is obtained.
Vorstehende Verfahren sind dem Fachmann auf dem Gebiet der DNA-Rekombinationstechnik bekannt. Ergänzend wird auf Sam- brook 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 VS93-1-GExample 2: Cloning of the papilloma virus genome VS93-1-G
Die aus Beispiel 1 erhaltene DNA der Biopsie WV-8495 wird mit dem Restriktionsenzym BamHI gespalten. Die erhaltenen Frag¬ mente werden in eine Ligasereaktion eingesetzt, in der der mit BamHI gespaltene und dephosphorylierte Vektor ΛZAP Express vorliegt. 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 ver¬ wendet. Die erhaltenen Phagenplaques werden dann einem Hybridisierungsverfahren unterzogen, in dem die in Beispiel 1 verwendete p32-markierte DNA von Fig. 1 in Kombination mit p32-markierter HP-Virus-29-DNA verwendet wird. Es wird eine Hybridisierung mit entsprechenden Phagenplaques erhalten. Aus diesen werden die beiden BamHI-Fragmente von VS93-1 -G isoliert und zusammen mit einem BamHI-gespaltenen, de- phosphorylierten Plasmid-Vektor, d.h. pBluescript, in eine weite¬ re Ligasereaktion eingesetzt. Die erhaltenen rekombinanten DNA-Moleküle werden zur Transformation von Bakterien, d.h. E. coli XI1 -Blue, verwendet. Durch Restriktionsspaltungen bzw. Hybridisierung mit vorstehenden DNA-Proben wird ein das Papil¬ lomvirus-Genom VS93-1 -G enthaltender Bakterienklon identifi¬ ziert. Das Plasmid dieses Bakterienklons wird mit pBlue-VS93-1 - G bezeichnet. The DNA of the biopsy WV-8495 obtained from Example 1 is cleaved with the restriction enzyme BamHI. The fragments obtained are used in a ligase reaction in which the ΛZAP Express vector which is cleaved and dephosphorylated with BamHI is 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 is used in combination with p 32 -labeled HP virus 29 DNA. Hybridization with corresponding phage plaques is obtained. Out the two BamHI fragments of VS93-1 -G are isolated and used together with a BamHI-cleaved, dephosphorylated plasmid vector, ie pBluescript, in a further ligase reaction. The recombinant DNA molecules obtained are used for the transformation of bacteria, ie E. coli XI1 -Blue. A bacterial clone containing the papillomavirus genome VS93-1 -G is identified by restriction cleavage or hybridization with the above DNA samples. The plasmid of this bacterial clone is designated pBlue-VS93-1-G.

Claims

Patentansprüche Claims
DNA, codierend für ein Peptid eines Papillomvirus-Hauptcapsid-Pro- teins, wobei das Peptid mindestens einen Teil der Aminosäuresequenz von Fig. 1 , Fig. 2, Fig. 3, Fig. 4, Fig. 5, Fig. 6. Fig. 7, Fig. 8 oder Fig. 9 umfaßt.DNA coding for a peptide of a papillomavirus main capsid protein, wherein the peptide comprises at least a part of the amino acid sequence of FIGS. 1, 2, 3, 4, 5, 6. 7, 8 or 9.
DNA nach Anspruch 1 , dadurch gekennzeichnet, daß das Paptid die Aminosäuresequenz von Fig. 1 , Fig. DNA according to claim 1, characterized in that the paptid has the amino acid sequence of Fig. 1, Fig.
2, Fig. 3, Fig. 4, Fig. 5, Fig. 6. Fig. 7, Fig. 8 oder Fig. 9 umfaßt.2, 3, 4, 5, 6, 7, 8 or 9.
3. DNA nach Anspruch 1 oder 2, dadurch gekennzeichnet, daß die für das3. DNA according to claim 1 or 2, characterized in that for the
Peptid des Papillomvirus-Hauptcapsid-Proteins codierende DNA minde¬ stens einen Teil der Basensequenz von Fig. 1 , Fig. 2, Fig. 3, Fig. 4, Fig. 5, Fig. 6. Fig. 7, Fig. 8 oder Fig. 9 umfaßt.DNA encoding the peptide of the papillomavirus main capsid protein has at least part of the base sequence from FIG. 1, FIG. 2, FIG. 3, FIG. 4, FIG. 5, FIG. 6, FIG. 7, FIG. 8 or FIG 9 includes.
4. DNA nach einem der Ansprüche 1 -3, 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 oder Fig. 9 umfaßt.4. DNA according to one of claims 1 -3, characterized in that the DNA coding for the peptide of the papillomavirus main capsid protein comprises the base sequence of Fig. 1, Fig. 2, Fig. 3, Fig. 4, Fig. 5, 6, 7, 8 or 9.
5. Papillomvirus-Genom mit der DNA nach einem der Ansprüche 1 bis 4.5. papillomavirus genome with the DNA according to any one of claims 1 to 4.
6. Verfahren zur Bereitstellung des Papillomvirus-Genoms nach Anspruch 5, umfassend die folgenden Verfahrensschritte:6. A method for providing the papillomavirus genome according to claim 5, comprising the following method steps:
(a) Isolierung der Gesamt-DNA aus einer Biopsie epithelialen Neo- plasmas, (b) Hybridierung der Gesamt-DNA von (a) mit einer DNA nach einem der Ansprüche 1 -4, wodurch ein in der Gesamt-DNA von (a) enthaltenes Papillomvirus-Genom nachgewiesen wird, und(a) isolation of the total DNA from a biopsy of epithelial neoplasm, (b) hybridizing the total DNA of (a) with a DNA according to any one of claims 1-4, whereby a papilloma virus genome contained in the total DNA of (a) is detected, and
(c) Klonierung der das Papillomvirus-Genom enthaltenden Gesamt-(c) Cloning of the Total Papilloma Virus Genome Containing
DNA von (a) in einem Vektor, und gegebenenfalls Subklonierung des erhaltenen Klons, wobei sämtliche Verfahrensschritte übli¬ cher DNA-Rekombinationtechnik entstammen.DNA from (a) in a vector, and optionally subcloning of the clone obtained, all process steps coming from conventional DNA recombination technology.
7. Protein, codiert durch das Papillomvirus-Genom nach Anspruch 5.7. Protein encoded by the papilloma virus genome according to claim 5.
8. Protein nach Anspruch 7, dadurch gekennzeichnet, daß es ein Papil- lomvirus-Hauptcapsid-Protein ist.8. Protein according to claim 7, characterized in that it is a papilloma virus main capsid protein.
9. Protein nach Anspruch 7, dadurch gekennzeichnet, daß es ein Papil- lomvirus-Nebencapsid-Protein ist.9. Protein according to claim 7, characterized in that it is a papilloma virus secondary capsid protein.
1 0. Expressionsvektor, umfassend eine für das Protein nach einem der Ansprüche 7-9 codierende DNA.1 0. Expression vector comprising a DNA coding for the protein according to any one of claims 7-9.
1 1 . Transformante, enthaltend den Expressionsvektor nach Anspruch 1 0.1 1. Transformant containing the expression vector according to claim 1 0.
1 2. Verfahren zur Herstellung des Proteins nach einem der Ansprüche 7-9, umfassend die Kultivierung der Transformante nach Anspruch 1 1 unter geeigneten Bedingungen.1 2. A method for producing the protein according to any one of claims 7-9, comprising culturing the transformant according to claim 1 1 under suitable conditions.
1 3. Virus-ähnliches Partikel, umfassend das Papillomvirus-Hauptcapsid- Protein nach Anspruch 8.3. A virus-like particle comprising the papilloma virus main capsid protein according to claim 8.
14. Virus-ähnliches Partikel nach Anspruch 1 3, umfassend zusätzlich das14. Virus-like particle according to claim 1 3, additionally comprising the
Nebencapsid-Protein nach Anspruch 9. Secondary capsid protein according to claim 9.
1 5. Antikörper, gerichtet gegen das Protein nach einem der Ansprüche 7-9.1 5. Antibody directed against the protein according to any one of claims 7-9.
1 6. Antikörper, gerichtet gegen das Virus-ähnliche Partikel nach Anspruch 13 oder 14.1 6. Antibody directed against the virus-like particle according to claim 13 or 14.
1 7. Verwendung der DNA nach einem der Ansprüche 1 -5 als Reagens zur Diagnose.1 7. Use of the DNA according to one of claims 1 -5 as a reagent for diagnosis.
1 8. Verwendung des Proteins nach einem der Ansprüche 7-9 als Reagens zur Diagnose, Therapie und/oder Vakzinierung.1 8. Use of the protein according to any one of claims 7-9 as a reagent for diagnosis, therapy and / or vaccination.
1 9. Verwendung des Virus-ähnlichen Partikels nach Anspruch 1 3 der 14 als Reagens zur Diagnose, Therapie und/oder Vakzinierung.1 9. Use of the virus-like particle according to claim 1 3 of 14 as a reagent for diagnosis, therapy and / or vaccination.
20. Verwendung des Antikörpers nach Anspruch 1 5 oder 1 6 als Reagens zur Diagnose und/oder Therapie. 20. Use of the antibody according to claim 1 5 or 1 6 as a reagent for diagnosis and / or therapy.
EP95919396A 1994-05-04 1995-05-04 Dna coding for a peptide of a papilloma virus main capside protein and use thereof Ceased EP0707652A1 (en)

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DE19526386C1 (en) * 1995-07-19 1997-01-02 Deutsches Krebsforsch Papillomaviruses, means for their detection and for the therapy of diseases caused by them
DE19648962C1 (en) 1996-11-26 1998-02-26 Deutsches Krebsforsch DNA encoding peptide(s) from papilloma virus major capsid protein
DE19735118C1 (en) * 1997-08-13 1998-08-13 Deutsches Krebsforsch Papilloma virus DNA
DE19840263C1 (en) * 1998-09-03 2000-05-25 Deutsches Krebsforsch Papillomaviruses, means for their detection and for the therapy of diseases caused by them
EP1169480A4 (en) * 1999-04-14 2005-02-02 Musc Found For Res Dev Tissue-specific and pathogen-specific toxic agents and ribozymes
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
DE10137102A1 (en) * 2001-07-30 2003-02-27 Deutsches Krebsforsch Polyvalent vaccine against diseases caused by papillomaviruses, processes for their production and their use
JP2005537028A (en) * 2002-06-26 2005-12-08 ザ　ペン　ステート　リサーチ　ファウンデーション Methods and materials for treating human papillomavirus infections
JP6153866B2 (en) 2010-05-25 2017-06-28 キアゲン ガイサーズバーグ アイエヌシー． Rapid hybrid capture assay and associated strategically cleaved probe

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