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WO1996011267A1 - Dna coding for a zinc finger protein, a zinc finger protein and the use thereof - Google Patents

Dna coding for a zinc finger protein, a zinc finger protein and the use thereof Download PDF

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Publication number
WO1996011267A1
WO1996011267A1 PCT/DE1995/001390 DE9501390W WO9611267A1 WO 1996011267 A1 WO1996011267 A1 WO 1996011267A1 DE 9501390 W DE9501390 W DE 9501390W WO 9611267 A1 WO9611267 A1 WO 9611267A1
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Prior art keywords
dna
zinc finger
protein
finger protein
nucleotides
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Application number
PCT/DE1995/001390
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German (de)
French (fr)
Inventor
Elisabeth Schwarz
Matthias Bartelmann
Marie-Stella Reuter
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Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts
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Application filed by Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts filed Critical Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts
Priority to JP8512260A priority Critical patent/JPH10506789A/en
Priority to EP95935327A priority patent/EP0784680A1/en
Publication of WO1996011267A1 publication Critical patent/WO1996011267A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • Zinc finger DNA, protein and their use relates to a DNA coding for a zinc finger protein and such a protein.
  • the invention further relates to the use of the DNA and the protein.
  • the conversion of normal cells into tumor cells takes place in several steps, which involve changes in cellular genes or the acquisition of viral oncogenes. Changes in cellular genes include the activation of proto-oncogenes by mutation, gene amplification, overexpression or chromosome translocations as well as the inactivation of tumor suppressor genes by mutation or deletion.
  • genes the changes of which are important in the development of tumors, have so far been identified (cf. review article: Bishop, J.M., Cell 64 (1 991), 235-248).
  • the products of such genes have different functions. They work e.g. as transcription regulators, as links in different signal transduction chains, such as growth factors, growth factor receptors or protein kinases, or as activators or inhibitors of cell division.
  • transcription regulators are zinc finger proteins. These proteins have characteristic structures containing two cysteine and two histidine residues which are positioned relative to one another in such a way that they bind a zinc atom. Zinc finger proteins are DNA-binding proteins. An example of zinc finger proteins is the product of the Wilms tumor suppressor gene WT1, the loss of which plays an important role in the development of Wilms kidney tumors.
  • the present invention is therefore based on the object of providing a means with which tumor diagnosis and therapy can be carried out more comprehensively.
  • the invention thus relates to a DNA coding for a zinc finger protein, which comprises:
  • hybridizing DNA indicates a DNA which hybridizes with a DNA of (a) under customary conditions, in particular at 20 ° C. below the melting point of the DNA.
  • FIG. 1 shows the insert of the cDNA clone COS AP4-E1.
  • This clone comes from a cDNA library of the cervical carcinoma cell line ME 180 (cf. Reuter, M.S. et al., J. Virol. 65 (1991), 5564-5568).
  • This cell line contains the human papillomavirus 68-DNA (HPV 68-DNA) stably integrated.
  • the clone COS AP4-E1 contains HPV 68 DNA between nucleotides 1-21. It also contains cellular sequences between nucleotides 22-1476, these being exon sequences of a zinc finger gene between nucleotides 446-1476.
  • the exon sequences code for 4 zinc fingers zinc finger 1: 1130-1 191, zinc finger 2: 1214-1275, zinc finger 3: 1298-1360, zinc finger 4: 1382-1432.
  • the clone COS AP4-E1 was deposited with the DSM (German Collection of Microorganisms and Cell Cultures under DSM 9450 on September 30, 1994).
  • the above insert DNA can be part of a DNA coding for a complete zinc finger protein.
  • Such a DNA and the zinc finger protein encoded by it are thus also the subject of the invention.
  • the insert DNA from COS AP4-E1 in particular the DNA between nucleotides 446-1476
  • a cDNA library from blood cells or HeLa cells can also be used.
  • the tissues and cells mentioned contain sufficient RNA transcripts that hybridize with the insert DNA from COS AP4-E1, in particular the DNA between nucleotides 446-1476 (cf. FIG. 2). Clones obtained are subjected to sequencing and, starting from the insert DNA of COS AP4-E1, in particular the DNA between nucleotides 446-1476, the DNA coding for a zinc finger protein above is determined.
  • the insert DNA from COS AP4-E1 in particular the DNA between nucleotides 446-1476, and very particularly the DNA between nucleotides 1240-1476, is suitable for identifying a genomic DNA coding for the above zinc finger protein.
  • the corresponding DNA from COS AP4-E1 e.g. the DNA between the nucleotides 1240-1476, used as a probe for hybridizing a genomic DNA library.
  • Such can be created from the tissues and cells mentioned above.
  • the clone COS 1 APM is obtained. Its insert DNA contains in a 5.5 kb Sacl fragment the probe used, e.g. DNA corresponding to DNA between nucleotides 1240-1476. In Figures 3 and 4 this DNA from COS 1 APM is presented between nucleotides 256-492.
  • the clone COS 1 APM was deposited with the DSM under DSM 9462 on October 7, 1994. The invention thus also relates to a genomic DNA encoding the above zinc finger protein and the protein encoded by it.
  • the DNA coding for the zinc finger protein can be present in a vector or expression vector.
  • a vector or expression vector examples of such are known to the person skilled in the art.
  • these are, for example, pGE-MEX, pUC derivatives and pGEX-2T.
  • yeast for example, pY100 and Ycpadl should be mentioned, while for expression in animal cells, for example, pKCR, pEF- BOS, cDM8 and pCEV4 must be specified.
  • 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 cDNA can be expressed in E. coli, yeast or animal cells, whereas the above genomic DNA is only to be expressed in animal cells. Furthermore, the person skilled in the art knows the conditions for cultivating transformed or transfected cells. He is also familiar with methods of isolating and purifying the expressed zinc finger protein. An above, recombinantly produced zinc finger protein is therefore also the subject of the invention.
  • Another object of the invention is an antibody directed against an above zinc finger protein.
  • Such is produced in the usual way.
  • the zinc finger protein is injected subcutaneously into BALB / c mice. 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 are removed from the mice after the fourth injection above and these are fused with myeloma cells in the usual way. The further cloning is also carried out according to known methods.
  • the present invention provides a previously unknown zinc finger DNA and a zinc finger protein encoded by it.
  • the DNA according to the invention is suitable as a probe for diagnostic purposes.
  • it can be used for gene therapy in an expression vector known to those skilled in the art.
  • the protein according to the invention is also suitable for therapeutic purposes. For this purpose, it can be administered in a customary pharmaceutical composition.
  • the present invention further provides antibodies directed against the above protein. These are ideal for diagnostic purposes.
  • the present invention thus provides new means for the diagnosis and therapy of diseases related to the DNA according to the invention and the protein encoded by them, in particular tumor diseases.
  • FIG. 2 shows the hybridization of polyA + RNA from different cells with the DNA between nucleotides 1240-1476 of COS AP4-E1
  • FIG. 3 shows a partial sequence of the genomic insert DNA of the clone COS 1
  • FIG. 4 shows the comparison of the DNA between nucleotides 1240-1476 of clone COS AP4-E1 and the DNA between nucleotides 256-
  • the invention is illustrated by the following example.
  • a ⁇ cDNA library created from HeLa cells is subjected to a conventional hybridization process.
  • the phage plaques obtained by infection of the bacteria are incubated with the 32 p-labeled DNA insert of the clone COS AP4-E1, in particular the DNA between the nucleotides 1240-1476 (cf. FIG. 1). Hybridization with individual phage plaques is obtained.
  • the phage DNA is isolated from these and cleaved with EcoRI.
  • the fragments obtained are separated electrophoretically in a 0.5% agarose gel.
  • the agarose gel is subjected to a conventional blotting process, whereby the DNA from the agarose gel is transferred to a nitrocellulose membrane.

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Abstract

The invention concerns DNA coding for a zinc finger protein and a protein of this type. The invention further concerns the use of the DNA and the protein in diagnosis, therapy or gene therapy of tumors. The DNA coding for the zinc finger protein is an insert of the cDNA clone COS AP4-E1. This clone comes from a cDNA library of the cervix carcinoma cell line ME180, which contains the human papilloma virus 68 DNA, and has four zinc fingers in its exon sequences.

Description

Zinkf inqer-DNA, -Protein und ihre Verwendung Die Erfindung betrifft eine für ein Zinkfinger-Protein codierende DNA und ein solches Protein. Ferner betrifft die Erfindung die Verwendung der DNA und des Proteins. Zinc finger DNA, protein and their use The invention relates to a DNA coding for a zinc finger protein and such a protein. The invention further relates to the use of the DNA and the protein.
Die Umwandlung von normalen Zellen in Tumorzellen vollzieht sich in mehreren Teilschritten, bei denen es zu Veränderungen zellulärer Gene oder zum Erwerb viraler Onkogene kommt. Veränderungen zellulärer Gene umfassen die Aktivierung von Proto-Onkogenen durch Mutation, Genamplifikation, Überexpression oder Chromosomen-Translokationen sowie die Inaktivierung von Tumorsuppressorgenen durch Mutation oder Deletion.The conversion of normal cells into tumor cells takes place in several steps, which involve changes in cellular genes or the acquisition of viral oncogenes. Changes in cellular genes include the activation of proto-oncogenes by mutation, gene amplification, overexpression or chromosome translocations as well as the inactivation of tumor suppressor genes by mutation or deletion.
Verschiedene Gene, deren Veränderungen bei der Tumorentstehung von Bedeu¬ tung sind, konnten bislang identifiziert werden (vgl. Übersichtsartikel: Bishop, J.M., Cell 64 ( 1 991 ), 235-248). Die Produkte solcher Gene haben verschiedene Funktionen. Sie wirken z.B. als Transkriptionsregulatoren, als Glieder unterschiedli¬ cher Signaltransduktionsketten, wie Wachstumsfaktoren, Wachstumsfaktorrezep¬ toren oder Proteinkinasen, bzw. als Aktivatoren oder Inhibitoren der Zellteilung.Various genes, the changes of which are important in the development of tumors, have so far been identified (cf. review article: Bishop, J.M., Cell 64 (1 991), 235-248). The products of such genes have different functions. They work e.g. as transcription regulators, as links in different signal transduction chains, such as growth factors, growth factor receptors or protein kinases, or as activators or inhibitors of cell division.
Beispiele von Transkriptionsregulatoren sind Zinkfinger-Proteine. Diese Proteine besitzen charakteristische, zwei Cystein- und zwei Histidinreste enthaltende Strukturen, die so zueinander positioniert sind, daß sie ein Zinkatom binden. Zinkfinger-Proteine sind DNA-bindende Proteine. Ein Beispiel für Zinkfinger-Proteine ist das Produkt des Wilms-Tumorsuppressorgen WT1 , dessen Verlust bei der Entstehung von Wilms-Nierentumoren eine wesentliche Rolle spielt.Examples of transcription regulators are zinc finger proteins. These proteins have characteristic structures containing two cysteine and two histidine residues which are positioned relative to one another in such a way that they bind a zinc atom. Zinc finger proteins are DNA-binding proteins. An example of zinc finger proteins is the product of the Wilms tumor suppressor gene WT1, the loss of which plays an important role in the development of Wilms kidney tumors.
Bis heute sind noch nicht alle an Tumorentstehung und -Wachstum beteiligten Gene bzw. Genprodukte bekannt. Eine Tumordiagnose bzw. -therapie ist daher bisher nur begrenzt möglich.To date, not all genes or gene products involved in tumor development and growth are known. Tumor diagnosis and therapy has therefore only been possible to a limited extent.
Der vorliegenden Erfindung liegt somit die Aufgabe zugrunde, ein Mittel bereitzu¬ stellen, mit dem Tumordiagnose bzw. -therapie umfassender betrieben werden kann.The present invention is therefore based on the object of providing a means with which tumor diagnosis and therapy can be carried out more comprehensively.
ERSÄTZBLAT Erfindungsgemäß wird dies durch die Gegenstände in den Patentansprüchen erreicht.REPLACEMENT BLADE According to the invention, this is achieved by the subject matter in the claims.
Gegenstand der Erfindung ist somit eine für ein Zinkfinger-Protein codierende DNA, die umfaßt:The invention thus relates to a DNA coding for a zinc finger protein, which comprises:
(a) die DNA der Nukleotide 446-1476 von Fig. 1 oder einen Teil davon,(a) the DNA of nucleotides 446-1476 of FIG. 1 or a part thereof,
(b) eine mit der DNA von (a) hybridisierende DNA, oder(b) a DNA hybridizing with the DNA of (a), or
(c) eine mit der DNA von (a) oder (b) über den degenerierten genetischen Code verwandte DNA.(c) a DNA related to the DNA of (a) or (b) via the degenerate genetic code.
Der Ausdruck "hybridisierende DNA" weist auf eine DNA hin, die unter üblichen Bedingungen, insbesondere bei 20°C unter dem Schmelzpunkt der DNA, mit einer DNA von (a) hybridisiert.The term "hybridizing DNA" indicates a DNA which hybridizes with a DNA of (a) under customary conditions, in particular at 20 ° C. below the melting point of the DNA.
Fig. 1 zeigt das Insert des cDNA Klons COS AP4-E1. Dieser Klon entstammt einer cDNA-Bibliothek der Cervixcarcinomzellinie ME 180 (vgl. Reuter, M.S. et al., J. Virol. 65 (1991 ), 5564-5568). Diese Zellinie enthält die Human Papillomvirus 68- DNA (HPV 68-DNA) stabil integriert. Der Klon COS AP4-E1 enthält zwischen den Nukleotiden 1-21 HPV 68-DNA. Ferner enthält er zwischen den Nukleotiden 22- 1476 zelluläre Sequenzen, wobei diese zwischen den Nukleotiden 446-1476 Exon- Sequenzen eines Zinkfinger-Gens sind. Die Exon-Sequenzen codieren für 4 Zinkfin¬ ger: Zinkfinger 1 :1130-1 191 , Zinkfinger 2: 1214-1275, Zinkfinger 3: 1298-1360, Zinkfinger 4: 1382-1432. Der Klon COS AP4-E1 wurde bei der DSM (Deutsche Sammlung von Mikroorganismen und Zellkulturen unter DSM 9450 am 30. Sep¬ tember 1994 hinterlegt.1 shows the insert of the cDNA clone COS AP4-E1. This clone comes from a cDNA library of the cervical carcinoma cell line ME 180 (cf. Reuter, M.S. et al., J. Virol. 65 (1991), 5564-5568). This cell line contains the human papillomavirus 68-DNA (HPV 68-DNA) stably integrated. The clone COS AP4-E1 contains HPV 68 DNA between nucleotides 1-21. It also contains cellular sequences between nucleotides 22-1476, these being exon sequences of a zinc finger gene between nucleotides 446-1476. The exon sequences code for 4 zinc fingers: zinc finger 1: 1130-1 191, zinc finger 2: 1214-1275, zinc finger 3: 1298-1360, zinc finger 4: 1382-1432. The clone COS AP4-E1 was deposited with the DSM (German Collection of Microorganisms and Cell Cultures under DSM 9450 on September 30, 1994).
Vorstehende Insert-DNA, insbesondere zwischen den Nukleotiden 446-1476, kann Teil einer für ein vollständiges Zinkfinger-Protein codierenden DNA sein. Eine solche DNA und das durch sie codierte Zinkfinger-Protein sind somit auch Gegen¬ stand der Erfindung.The above insert DNA, especially between nucleotides 446-1476, can be part of a DNA coding for a complete zinc finger protein. Such a DNA and the zinc finger protein encoded by it are thus also the subject of the invention.
Die Bereitstellung einer für vorstehendes Zinkfinger-Protein codierenden DNA erfolgt in üblicher Weise. Beispielhaft wird die Insert-DNA von COS AP4-E1 , insbesondere die DNA zwischen den Nukleotiden 446-1476, als Sonde zur Hybri¬ disierung einer allgemein erhältlichen cDNA-Bibliothek aus Leber, Gehirn, Plazenta oder Muskel, vorzugsweise Leber, verwendet. Auch kann eine cDNA-Bibliothek aus Blutzellen oder HeLa-Zellen verwendet werden. Die genannten Gewebe und Zellen enthalten ausreichend RNA-Transkripte, die mit der Insert-DNA von COS AP4-E1 , insbesondere der DNA zwischen den Nukleotiden 446-1476 hybridisieren (vgl. Fig. 2). Erhaltene Klone werden einer Sequenzierung unterzogen und ausgehend von der Insert-DNA von COS AP4-E1 , insbesondere der DNA zwischen den Nukleotiden 446-1476, wird die ein vorstehendes Zinkfinger-Protein codierende DNA bestimmt.The provision of a DNA encoding the above zinc finger protein takes place in the usual way. For example, the insert DNA from COS AP4-E1, in particular the DNA between nucleotides 446-1476, is used as a probe for hybridizing a generally available cDNA library from the liver, brain, placenta or muscle, preferably liver. A cDNA library from blood cells or HeLa cells can also be used. The tissues and cells mentioned contain sufficient RNA transcripts that hybridize with the insert DNA from COS AP4-E1, in particular the DNA between nucleotides 446-1476 (cf. FIG. 2). Clones obtained are subjected to sequencing and, starting from the insert DNA of COS AP4-E1, in particular the DNA between nucleotides 446-1476, the DNA coding for a zinc finger protein above is determined.
Des weiteren eignet sich die Insert-DNA von COS AP4-E1 , insbesondere die DNA zwischen den Nukleotiden 446-1476, und ganz besonders die DNA zwischen den Nukleotiden 1240-1476, zur Identifizierung einer genomischen für vorstehendes Zinkfinger-Protein codierenden DNA. Hierzu wird die entsprechende DNA von COS AP4-E1 , z.B. die DNA zwischen den Nukleotiden 1240-1476, als Sonde zur Hybri¬ disierung einer genomischen DNA-Bibliothek verwendet. Eine solche kann aus den vorstehend genannten Geweben und Zellen erstellt sein.Furthermore, the insert DNA from COS AP4-E1, in particular the DNA between nucleotides 446-1476, and very particularly the DNA between nucleotides 1240-1476, is suitable for identifying a genomic DNA coding for the above zinc finger protein. For this, the corresponding DNA from COS AP4-E1, e.g. the DNA between the nucleotides 1240-1476, used as a probe for hybridizing a genomic DNA library. Such can be created from the tissues and cells mentioned above.
Erfindungsgemäß wird der Klon COS 1 APM erhalten. Seine Insert-DNA enthält in einem 5,5 kb großen Sacl-Fragment die zur verwendeten Sonde, z.B. der DNA zwischen den Nukleotiden 1240-1476, entsprechende DNA. In den Figuren 3 und 4 ist diese DNA von COS 1 APM zwischen den Nukleotiden 256-492 präsentiert. Der Klon COS 1 APM wurde bei der DSM unter DSM 9462 am 07.10.1994 hinter¬ legt. Gegenstand der Erfindung ist somit auch eine genomische, das vorstehende Zinkfinger-Protein codierende DNA sowie das durch sie codierte Protein.According to the invention, the clone COS 1 APM is obtained. Its insert DNA contains in a 5.5 kb Sacl fragment the probe used, e.g. DNA corresponding to DNA between nucleotides 1240-1476. In Figures 3 and 4 this DNA from COS 1 APM is presented between nucleotides 256-492. The clone COS 1 APM was deposited with the DSM under DSM 9462 on October 7, 1994. The invention thus also relates to a genomic DNA encoding the above zinc finger protein and the protein encoded by it.
Erfindungsgemäß kann vorstehende für das Zinkfinger-Protein codierende DNA in einem Vektor bzw. Expressionsvektor vorliegen. Beispiele solcher sind dem Fach¬ mann bekannt. Im Falle eines Expressionsvektors für E. coli sind dies z.B. pGE- MEX, pUC-Derivate und pGEX-2T. Für die Expression in Hefe sind z.B. pY100 und Ycpadl 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 , x1776, JM101 und JM 109, den Hefe-Stamm Saccharomyces cerevisiae und die tierischen Zellen L, 3T3, FM3A, CHO, COS, Vero, und HeLa. Der Fachmann weiß, in welcher Weise vorstehende DNA in einen Expressionsvektor inseriert werden muß. Ihm ist auch bekannt, daß vorstehende cDNA in E.coli, Hefe oder tierischen Zellen exprimiert werden kann, während vorstehende genomische DNA nur in tierischen Zellen zu exprimieren ist. Des weiteren kennt der Fachmann Bedingungen, transformierte bzw. transfizierte Zellen zu kultivieren. Auch sind ihm Verfahren bekannt, das exprimierte Zinkfinger-Protein zu isolieren und zu reinigen. Ein vorstehendes, rekombinant hergestelltes Zinkfinger-Protein ist somit auch Gegenstand der Erfin¬ dung.According to the invention, the DNA coding for the zinc finger protein 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, pGE-MEX, pUC derivatives and pGEX-2T. For expression in yeast, for example, pY100 and Ycpadl should be mentioned, while for expression in animal cells, for example, pKCR, pEF- BOS, cDM8 and pCEV4 must be specified. 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, x1776, JM101 and JM 109, the yeast strain Saccharomyces cerevisiae and the animal cells L, 3T3, FM3A, CHO, COS, Vero, and HeLa. 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 cDNA can be expressed in E. coli, yeast or animal cells, whereas the above genomic DNA is only to be expressed in animal cells. Furthermore, the person skilled in the art knows the conditions for cultivating transformed or transfected cells. He is also familiar with methods of isolating and purifying the expressed zinc finger protein. An above, recombinantly produced zinc finger protein is therefore also the subject of the invention.
Ein weiterer Gegenstand der Erfindung ist ein gegen ein vorstehendes Zinkfinger- Protein gerichteter Antikörper. Die Herstellung eines solchen erfolgt in üblicher Weise. Beispielsweise wird hierzu das Zinkfinger-Protein in BALB/c-Mäuse sub- cutan 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 an antibody directed against an above zinc finger protein. Such is produced in the usual way. For example, the zinc finger protein is injected subcutaneously into BALB / c mice. 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 Mäusen Milzzel¬ len 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. To produce it, spleen cells are removed from the mice after the fourth injection above and these are fused with myeloma cells in the usual way. The further cloning is also carried out according to known methods.
Die vorliegende Erfindung stellt eine bisher nicht gekannte Zinkfinger-DNA sowie ein durch sie codiertes Zinkfinger-Protein bereit. Die erfindungsgemäße DNA eignet sich als Sonde für diagnostische Zwecke. Darüberhinaus kann sie in einem dem Fachmann bekannten Expressionsvektor zur Gentherapie eingesetzt werden. Das erfindungsgemäße Protein eignet sich ebenfalls für therapeutische Zwecke. Hierzu kann es in einer üblichen pharmazeutischen Zusammensetzung verabreicht werden. Des weiteren stellt die vorliegende Erfindung gegen das vorstehende Protein gerichtete Antikörper bereit. Diese eignen sich bestens zu diagnostischen Zwek- ken.The present invention provides a previously unknown zinc finger DNA and a zinc finger protein encoded by it. The DNA according to the invention is suitable as a probe for diagnostic purposes. In addition, it can be used for gene therapy in an expression vector known to those skilled in the art. The protein according to the invention is also suitable for therapeutic purposes. For this purpose, it can be administered in a customary pharmaceutical composition. The present invention further provides antibodies directed against the above protein. These are ideal for diagnostic purposes.
Somit liefert die vorliegende Erfindung neue Mittel zur Diagnose und Therapie von mit der erfindungsgemäßen DNA und dem durch sie codierten Protein zusammen¬ hängenden Erkrankungen, insbesondere von Tumorerkrankungen.The present invention thus provides new means for the diagnosis and therapy of diseases related to the DNA according to the invention and the protein encoded by them, in particular tumor diseases.
Kurze Beschreibung der Zeichnung:Brief description of the drawing:
Fig. 1 zeigt die Insert-cDNA des Klons COS AP4-E1 ,1 shows the insert cDNA of the clone COS AP4-E1,
Fig. 2 zeigt die Hybridisierung von PolyA+RNA aus verschiedenen Zellen mit der DNA zwischen den Nukleotiden 1240-1476 von COS AP4-E1 , Fig. 3 zeigt eine Teilsequenz der genomischen Insert-DNA des Klons COS 1FIG. 2 shows the hybridization of polyA + RNA from different cells with the DNA between nucleotides 1240-1476 of COS AP4-E1, FIG. 3 shows a partial sequence of the genomic insert DNA of the clone COS 1
APM, und Fig. 4 zeigt den Vergleich der DNA zwischen den Nukleotiden 1240-1476 von Klon COS AP4-E1 und der DNA zwischen den Nukleotiden 256-APM, and FIG. 4 shows the comparison of the DNA between nucleotides 1240-1476 of clone COS AP4-E1 and the DNA between nucleotides 256-
492 von Klon COS 1 APM.492 from clone COS 1 APM.
Die Erfindung wird durch das folgende Beispiel erläutert.The invention is illustrated by the following example.
Beispiel: Herstellung einer erfindungsgemäßen Zinkfinger-DNAExample: Production of a zinc finger DNA according to the invention
Eine aus HeLa-Zellen erstellte λ cDNA-Bibliothek wird einem üblichen Hybridis- ierungsverfahren unterzogen. Hierzu werden die durch Infektion der Bakterien erhaltenen Phagenplaques mit dem 32p-markierten DNA-Insert des Klons COS AP4- E1 , insbesondere der DNA zwischen den Nukleotiden 1240-1476 (vgl. Fig. 1 ), inkubiert. Es wird eine Hybridisierung mit einzelnen Phagenplaques erhalten. Aus diesen wird die Phagen-DNA isoliert und mit EcoRI gespalten. Die erhaltenen Fragmente werden in einem 0,5%igen Agarosegel elektrophoretisch aufgetrennt. Das Agarosegel wird einem üblichen Blotting-Verfahren unterzogen, wodurch die DNA aus dem Agarosegel auf eine Nitrozellulosemembran übertragen wird. Diese wird in ein Hybridisierungsverfahren eingesetzt, in dem die entsprechende DNA des DNA-Inserts von Klon COS AP4-E1 als 3 p-markierte Probe verwendet wird. Es wird eine Hybridisierung mit einzelnen DNA-Fragmenten erhalten. Diese werden isoliert und in einem mit EcoRI gespaltenen, dephosphorylierten Plasmid-Vektor, z.B. pBluescript, kloniert. Einzelne Klone werden analysiert, und durch Restriktions¬ spaltungen sowie Sequenzierung wird ein eine erfindungsgemäße Zinkfinger-DNA enthaltender Klon identifiziert. A λ cDNA library created from HeLa cells is subjected to a conventional hybridization process. For this purpose, the phage plaques obtained by infection of the bacteria are incubated with the 32 p-labeled DNA insert of the clone COS AP4-E1, in particular the DNA between the nucleotides 1240-1476 (cf. FIG. 1). Hybridization with individual phage plaques is obtained. The phage DNA is isolated from these and cleaved with EcoRI. The fragments obtained are separated electrophoretically in a 0.5% agarose gel. The agarose gel is subjected to a conventional 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 corresponding DNA of the DNA insert from clone COS AP4-E1 is used as a 3 p-labeled sample. Hybridization with individual DNA fragments is obtained. These are isolated and cloned in a dephosphorylated plasmid vector, for example pBluescript, which has been digested with EcoRI. Individual clones are analyzed, and a clone containing a zinc finger DNA according to the invention is identified by restriction cleavage and sequencing.

Claims

Patentansprüche claims
1. DNA, codierend für ein Zinkfinger-Protein, wobei die DNA umfaßt:1. DNA coding for a zinc finger protein, the DNA comprising:
(a) die DNA der Nukleotide 446 - 1476 von Fig. 1 oder einen Teil davon,(a) the DNA of nucleotides 446-1476 of FIG. 1 or a part thereof,
(b) eine mit der DNA von (a) hybridisierende DNA, oder(b) a DNA hybridizing with the DNA of (a), or
(c) eine mit der DNA von (a) oder (b) über den degenerierten genetischen Code verwandte DNA.(c) a DNA related to the DNA of (a) or (b) via the degenerate genetic code.
2. DNA nach Anspruch 1 , nämlich die DNA der Nukleotide 256 - 492 von Fig. 3.2. DNA according to claim 1, namely the DNA of nucleotides 256-492 of FIG. 3.
3. Protein, codiert durch die DNA nach Anspruch 1 oder 2.3. Protein encoded by the DNA of claim 1 or 2.
4. Expressionsplasmid, umfassend eine für das Protein nach Anspruch 3 codie¬ rende DNA.4. Expression plasmid comprising a DNA coding for the protein according to claim 3.
5. Transformante, enthaltend den Expressionsplasmid nach Anspruch 4.5. Transformant containing the expression plasmid according to claim 4.
6. Verfahren zur Herstellung des Proteins nach Anspruch 3, umfassend die Kultivierung der Transformante nach Anspruch 5 unter geeigneten Bedin¬ gungen.6. A method for producing the protein according to claim 3, comprising culturing the transformant according to claim 5 under suitable conditions.
7. Verwendung der DNA nach Anspruch 1 oder 2 als Reagens zur Diagnose und/oder Therapie.7. Use of the DNA according to claim 1 or 2 as a reagent for diagnosis and / or therapy.
8. Verwendung des Proteins nach Anspruch 3 als Reagens zur Therapie. 8. Use of the protein according to claim 3 as a reagent for therapy.
PCT/DE1995/001390 1994-10-07 1995-10-06 Dna coding for a zinc finger protein, a zinc finger protein and the use thereof WO1996011267A1 (en)

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