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WO1998001539A1 - Conditional immortalisation process for human tumour cells for producing a vaccine - Google Patents

Conditional immortalisation process for human tumour cells for producing a vaccine Download PDF

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Publication number
WO1998001539A1
WO1998001539A1 PCT/DE1997/001406 DE9701406W WO9801539A1 WO 1998001539 A1 WO1998001539 A1 WO 1998001539A1 DE 9701406 W DE9701406 W DE 9701406W WO 9801539 A1 WO9801539 A1 WO 9801539A1
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cells
construct
retrovirus construct
retrovirus
vaccine
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PCT/DE1997/001406
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German (de)
French (fr)
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Thomas Blankenstein
Liang-Ping Li
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Max-Delbrück-Centrum für Molekulare Medizin
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Priority to EP97932733A priority Critical patent/EP0910626A1/en
Publication of WO1998001539A1 publication Critical patent/WO1998001539A1/en

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    • 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
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/515—Animal cells
    • A61K2039/5156—Animal cells expressing foreign proteins
    • 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
    • C12N2510/00—Genetically modified cells
    • C12N2510/04—Immortalised cells

Definitions

  • the invention relates to a conditional immortalization process for human tumor cells for producing a vaccine. It also relates to a general process for the production of cell lines from primary cell cultures, e.g. B. from T cells or from dendritic cells. Areas of application of the invention are medicine and the pharmaceutical industry.
  • the invention has the aim of producing cell lines from primary cell cultures, e.g. B. of T cells, dendritic cells or tumor cells.
  • the special task is to genetically engineer an autologous tumor cell vaccine and to produce reproducible cell cultures from tumor biopsies.
  • the conditional immortalization process for cell lines from primary cell cultures is characterized in that T cells, dendritic cells or tumor cells are treated with a retrovirus construct which effects the immortalization of the cells, followed by cultivation and the cells obtained are used before using them second retrovirus construct, with which the inserted first retrovirus construct is cut out.
  • An important embodiment of the invention is that tumor cells made from patient material are treated with a retrovirus construct which effects the immortalization of the tumor cells, then cultivated and the cells obtained are used before they are used to produce a vaccine with a second retrovirus construct with which the inserted first retrovirus construct is cut out, treated.
  • the first retrovirus construct consists of the genes for the herpes simplex thymidine kinase, the large T gene from SV 40 as immortalizing agent, the CMV-Promoto, Gan ⁇ yclovir as a negative selection marker and the hygromycin gene as a positive selection marker, flanked by signal sequences (LOX) for the recombinase CRE.
  • LOX signal sequences
  • the second retrovirus construct for cutting out the inserted construct contains the bacteriophage recombinase CRE under LTR control and the puromycin gene under SV 40 promoter control.
  • Both vectors have different selection markers. Infection with the large T virus leads the tumor cells through the "crisis" of adaptation to cell culture conditions. With the second virus construct, these cells can be infected at any time in order to remove the first construct. Cancyclovir can be used to select cells that fail to successfully remove the first construct (in which the TK and large T genes are deleted). This kills all cells that still contain the hygromycin gene.
  • the two retrovirus constructs to be constructed are shown in Fig. 1.
  • the first is based on the vector HyTK-CMV.
  • the vector contains a fusion gene from hygromycin and herpes simplex thymidine kinase (HyTK) gene under "long terminal repeat" (LTR) promoter control. Cells infected with the virus therefore have a positive (hygromycin) and a negative (gancyclovir) selection marker.
  • 3 of the HyTK gene is a comparatively strong promoter from the cytomegalovirus. The large T gene from SV40 is cloned behind these.
  • the large T gene is amplified by the polymerase chain reaction (PCR), the 3 'primer and a 34 base pair long sequence being extended, which corresponds to the LOX recognition sequence for the CRE recombinase and is filled in during the PCR reaction.
  • PCR polymerase chain reaction
  • a second LOX sequence is cloned as a double-stranded primer pair 5 * from the HyTK gene.
  • the result is a vector which has a LOX sequence as a substrate for the CRE recombinase to the left and right of the HyTK and large T genes.
  • the second retrovirus construct is based on the pBABE-Puro.
  • the gene for bacteriophage recombinase CRE is cloned behind the 5 LTR in a cloning interface.
  • the CRE recombinase can perform the deletion of the sequence between two LOX recognition sequences in a LOX sequence-specific manner.
  • the vector additionally contains the puromycin gene under SV40 promoter control as a selection marker. Both vectors are converted into virus particles by transfection into suitable packaging cell lines. The sequences of the large T region and the LOX regions are verified by sequence analysis. 2nd Time-limited expression of the large T gene in primary human tumor cells
  • Different tumor material is surgically obtained, mechanically / enzymatically processed into single cell suspensions, infected with the HyTK-LOX-CMV-IT virus or control virus, plus and minus selection markers (hygromycin) cultivated and the growth kinetics determined. Possibly. the infection is preceded by an enrichment of the tumor cells, for example by separation with antibodies which are specific for tumor-associated antigens, in order to prevent virus-infected cells (e.g. fibroblasts) which contaminate the tumor material from overgrowing the tumor cells.
  • virus-infected cells e.g. fibroblasts
  • Evidence that tumor cells have really been immortalized can be demonstrated by staining with antibodies that recognize tumor-associated antigens and by PCR for certain oncogenes.
  • the frequency with which long-term cultures are obtained in a large T-dependent manner is determined as a function of the tumor cell type.
  • the cell lines are infected with the virus BABE-CRE-Puro and selected for puromycin resistance.
  • the CRE recombinase carries out a LOX-specific recombination of the HyTK-LOX-CMV-IT virus, in which the HyTK fusion gene and the large T gene are deleted.
  • the phenotype of the tumor cells should be as follows in the sequence of the experiment: Puro *, Hygro 8 , GCV ", G418" 1.
  • Tumor cells from which a cell culture is created from a tumor biopsy, usually have a limited lifespan of 10-20 passages.
  • a cell culture is created from a breast cancer biopsy and the cells are either infected with the lar ⁇ e T retrovirus or left untreated.
  • the infection with the large T retrovirus leads to a continuous proliferation of the cells over several cell culture passages.
  • the number of uninfected cells decreased successively in culture, and no cell culture could be established. This proves that cell lines can be isolated from the tumor biopsy using the large T virus.
  • NIH3T3 cells are first infected with the large T retrovirus and the Hygro "cells are selected. Then the cells are infected with the CRE recombinase retrovirus and the Puro" cells are selected. Then GCV is used to select against all cells in which no recombination in which large T and thymidine kinase genes have been deleted has taken place. The cells are then stained with a large T-specific antibody. The table shows that after infection with the large T virus, virtually all cells have one show strong core coloring. After infection with the CRE recombinase virus and GCV selection, no staining with the large T antibody was detectable.
  • Retrovirus vector HyTK-LOX-CMV-IT contains the recognition sequences of the bacteriophage recombinase CRE (LOX), a fusion gene which codes for hygromycin and the herpes virus thymidine kinase (HyTK) and the large T region from SV40 (IT) under CMV- Promoter Control (CMV).
  • Retrovirus vector BABE-CRE-Puro contains the CRE recombinase under LTR control and the puromycin gene under SV40 promoter control. Not drawn to scale.
  • a cell suspension from a breast cancer biopsy was created and either infected with the retrovirus HyTK-LOX-CMV-IT (1) or left untreated (O). The infection occurred in the second week.
  • the arrows indicate the times at which the confluent cell culture was thinned 1:10.

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Abstract

The invention aims at constructing an autologous tumour cell vaccine by genetic engineering and its object is to produce for that purpose reproducible cell cultures from tumour biopsy products. The new conditional immortalisation process for human tumour cells useful for producing a vaccine is characterised in that tumour cells extracted from patients are treated with a retrovirus construct which causes the immortalisation of the tumour cells, which are then cultivated. The thus obtained cells are then treated before being used to produce a vaccine with a second retrovirus construct which cuts out from the cell the first inserted retrovirus construct. This process is in general also suitable for producing cell lines from primary cell cultures, for example from T-cells or dendritic cells. The invention has applications in the medical field and the pharmaceutical industry.

Description

Konditionales Immortalisationsverfahren für humane Tumorzellen zur Herstellung einer VakzineConditional immortalization procedure for human tumor cells for the production of a vaccine
Beschreibungdescription
Die Erfindung betrifft ein konditionales Immortalisierungs- verfahren für humane Tumorzellen zur Herstellung einer Vakzine. Es betrifft ferner ein generelles Verfahren zur Herstellung von Zellinien aus primären Zellkulturen, z. B. von T-Zellen oder von dendritischen Zellen. Anwendungsgebiete der Erfindung sind die Medizin und die pharmazeutische Industrie.The invention relates to a conditional immortalization process for human tumor cells for producing a vaccine. It also relates to a general process for the production of cell lines from primary cell cultures, e.g. B. from T cells or from dendritic cells. Areas of application of the invention are medicine and the pharmaceutical industry.
Internationaler Stand der Wissenschaft und Technik.International state of science and technology.
Es gibt bereits einige Vorschläge für gentechnisch hergestellte Tumorzellvakzine(OS DE 44 31 401), ihre Entwicklung basiert auf der Annahme, daß das Iromunsysteπ. gegen Tumorzellen aktiviert werden kann. Die Etablierung tumorspezifischer T-Zellen, die Klonierung erster tumorassoziierter Antigene und der Befund, daß die Sekretion eines einzelnen Zytokins durch Tumorzellen ausreicht, deren selektive Zerstörung zu induzieren, sprechen für die Möglichkeit einer tumorspezifischen Immunaktivierung. Da die molekulare Struktur der meisten Tumorantigene noch unbekannt ist und "gemeinsame" Tumorantigene bislang eher eine Seltenheit sind, muß eine spezifische Vakzine auf autologen Tumorzellen basieren. Für die Durchführung klinischer Vakzinierungsstudien ist die Transfektion autologer Tumorzellen daher ein kritischer Parameter. In den USA sind bereits mehrere klinische Studien angelaufen, die genetisch modifizierte autologe Tumorzellen als Vakzine überprüfen. Jaffee et al. (Cancer Res. 1993, 53: 2221-2226) und Yannelli et al. (J. Immunother. 1993, 161: 77 - 90) haben gezeigt, daß es bei ca. 30 % der Patienten möglich war, Zellkulturen aus operativ gewonnenem Tumormaterial für einen ausreichenden Zeitraum zu etablieren, um in diesen Zellen Zytokine (GM-CSF, TNF bzw. IL2) zu exprimieren. Dies wurde für Nierenzell-, Ovarial-, Pankreas-, Kolon-, Mamma- und Bronchial-karzinome und für Melanome gezeigt.There are already some proposals for genetically engineered tumor cell vaccines (OS DE 44 31 401), their development is based on the assumption that the Iromunsysteπ. can be activated against tumor cells. The establishment of tumor-specific T cells, the cloning of first tumor-associated antigens and the finding that the secretion of a single cytokine by tumor cells is sufficient to induce their selective destruction speak for the possibility of tumor-specific immune activation. Since the molecular structure of most tumor antigens is still unknown and "common" tumor antigens have so far been a rarity, a specific vaccine must be based on autologous tumor cells. The transfection of autologous tumor cells is therefore a critical parameter for conducting clinical vaccination studies. Several clinical trials have already started in the USA, which are testing genetically modified autologous tumor cells as vaccines. Jaffee et al. (Cancer Res. 1993, 53: 2221-2226) and Yannelli et al. (J. Immunother. 1993, 161: 77-90) have shown that it was possible in approx. 30% of the patients to establish cell cultures from surgically obtained tumor material for a sufficient period of time in order to be able to cytokines (GM-CSF, TNF or IL2) to express. This was for kidney cell, Ovarian, pancreatic, colon, breast and bronchial carcinoma and shown for melanoma.
Einschränkend ist hierzu zu sagen, daß 30 % Erfolgsrate immer noch unbefriedigend ist. Das "Nadelöhr" bei dem Versuch, eine autologe Tumorzellvakzine gentechnisch zu konstruieren, besteht also nach wie vor in der geringen Reproduzierbarkeit, Zellkulturen aus Tumorbiopsaten anzulegen.One limitation is that 30% success rate is still unsatisfactory. The "bottleneck" in trying to genetically engineer an autologous tumor cell vaccine is still the low reproducibility of creating cell cultures from tumor biopsies.
Die Erfindung hat das Ziel, die Herstellung von Zellinien aus primären Zellkulturen, z. B. von T-Zellen, von dendritischen Zellen oder von Tumorzellen, zu ermöglichen. Die spezielle Aufgabe besteht darin, gentechnisch eine autologe Tumorzellvakzine zu konstruieren und dazu reproduzierbare Zellkulturen aus Tumorbiopsaten herzustellen.The invention has the aim of producing cell lines from primary cell cultures, e.g. B. of T cells, dendritic cells or tumor cells. The special task is to genetically engineer an autologous tumor cell vaccine and to produce reproducible cell cultures from tumor biopsies.
Die Erfindung wird gemäß den Ansprüchen realisiert. Das konditionale Immortalisationsverfahren für Zellinien aus primären Zellkulturen ist dadurch gekennzeichnet, daß T- Zellen, dendritische Zellen oder Tumorzellen mit einem Retrovirus-konstrukt, das die Immortalisierung der Zellen bewirkt, behandelt werden, danach eine Kultivierung erfolgt und die erhaltenen Zellen vor ihrer Verwendung mit einem zweiten Retroviruskonstrukt, mit dem das inserierte erste Retroviruskonstrukt herausgeschnitten wird, behandelt werden. Eine wichtige Ausführungsform der Erfindung liegt darin, daß Tumorzellen aus Patientenmaterial mit einem Retroviruskonstrukt, das die Immortalisierung der Tumorzellen bewirkt, behandelt werden, danach eine Kultivierung erfolgt und die erhaltenen Zellen vor ihrer Verwendung zur Herstellung einer Vakzine mit einem zweiten Retroviruskonstrukt, mit dem das inserierte erste Retroviruskonstrukt herausgeschnitten wird, behandelt werden.The invention is implemented according to the claims. The conditional immortalization process for cell lines from primary cell cultures is characterized in that T cells, dendritic cells or tumor cells are treated with a retrovirus construct which effects the immortalization of the cells, followed by cultivation and the cells obtained are used before using them second retrovirus construct, with which the inserted first retrovirus construct is cut out. An important embodiment of the invention is that tumor cells made from patient material are treated with a retrovirus construct which effects the immortalization of the tumor cells, then cultivated and the cells obtained are used before they are used to produce a vaccine with a second retrovirus construct with which the inserted first retrovirus construct is cut out, treated.
Das erste Retroviruskonstrukt besteht aus den Genen für die Herpes Simplex-Thymidinkinase , dem large T-Gen aus SV 40 als immortalisierendem Agens, dem CMV-Promoto , Ganσyclovir als negativem Selektionsmarker und dem Hygromycin-Gen als positivem Selektionsmarker, flankiert von Signal-Sequenzen (LOX) für die Rekombinase CRE.The first retrovirus construct consists of the genes for the herpes simplex thymidine kinase, the large T gene from SV 40 as immortalizing agent, the CMV-Promoto, Ganσyclovir as a negative selection marker and the hygromycin gene as a positive selection marker, flanked by signal sequences (LOX) for the recombinase CRE.
Das zweite Retroviruskonstrukt zum Herausschneiden des inserierten Konstrukts enthält die Bakteriophagen-Rekombinase CRE unter LTR-Kontrolle und das Puromycingen unter SV 40- Promotor-Kontrolle.The second retrovirus construct for cutting out the inserted construct contains the bacteriophage recombinase CRE under LTR control and the puromycin gene under SV 40 promoter control.
Beide Konstrukte sind in Abb. 1 dargestellt.Both constructs are shown in Fig. 1.
Beide Vektoren tragen unterschiedliche Selektionsmarker. Durch Infektion mit dem large T-Virus werden die Tumorzellen durch die "Krise" der Adaptation an Zellkulturbedingungen geführt. Mit dem zweiten Viruskonstrukt können diese Zellen zwecks Entfernung des ersten Konstrukts zu jedem beliebigen Zeitpunkt infiziert werden. Gegen Zellen, die keine erfolgreiche Entfernung des 1. Konstrukts gelingt (bei der TK- und und large T-Gene deletiert werden) , kann mit Gancyclovir selektioniert werden. Damit werden alle Zellen abgetötet, die noch das Hygromycin-Gen in sich tragen.Both vectors have different selection markers. Infection with the large T virus leads the tumor cells through the "crisis" of adaptation to cell culture conditions. With the second virus construct, these cells can be infected at any time in order to remove the first construct. Cancyclovir can be used to select cells that fail to successfully remove the first construct (in which the TK and large T genes are deleted). This kills all cells that still contain the hygromycin gene.
Mit der Erfindung wird erreicht, daß reproduzierbar Zellkulturen aus Tumorbiopsaten angelegt werden können und daß die "Krise" humaner Tumorzellen in Kultur überwunden werden kann. Man erreicht damit, in ihrer Antigenität unveränderte Tumorzellen als Basis für die Konstruktion genmodifizierter autologer Tumorzellen in die Hand zu bekommen. Als Zelltypen sind u. a. geeignet: das Oesophagus- und Bronchialkarzinom, Magen- und Kolonkarzinom sowie Weichgewebstumoren.With the invention it is achieved that reproducible cell cultures can be created from tumor biopsies and that the "crisis" of human tumor cells can be overcome in culture. It is thus possible to get hold of unchanged tumor cells as the basis for the construction of genetically modified autologous tumor cells. As cell types u. a. suitable: esophageal and bronchial carcinoma, gastric and colon carcinoma as well as soft tissue tumors.
Die Erfindung soll nachfolgend durch Ausführungsbeispiele näher erläutert werden. AusführungsbeispieleThe invention will be explained in more detail below by means of exemplary embodiments. Embodiments
1. Konstruktion eines CRE/LOX-abhängigen Immortalisa- tionsvirus-systems1. Construction of a CRE / LOX-dependent immortalization virus system
Die beiden aufzubauenden Retroviruskonstrukte sind in Abb. 1 dargestellt. Das erste basiert auf dem Vektor HyTK-CMV. Der Vektor enthält ein Fusionsgen aus Hygromycin- und Herpes- Simplex-Thymidinkinase (HyTK)-Gen unter "long terminal repeat" (LTR)-Promotorkontrolle. Mit dem Virus infizierte Zellen haben daher einen positiven (Hygromycin) und einen negativen (Gancyclovir) Selektionsmarker. 3 vom HyTK-Gen liegt ein vergleichsweise starker Promotor aus dem Cytomegalovirus. Hinter diesen wird das large T-Gen aus SV40 kloniert. Das large T-Gen wird durch die Polymerasekettenreaktion (PCR) amplifiziert, wobei der 3 'Primer und eine 34-Basenpaar-lange Sequenz verlängert ist, die der LOX-Erkennungssequenz für die CRE-Rekombinase entspricht und während der PCR-Reaktion aufgefüllt wird. In einem zweiten Schritt wird eine zweite LOX-Sequenz als doppelsträngiges Primerpaar 5* vom HyTK-Gen aus gesehen kloniert. Das Ergebnis ist ein Vektor, der links und rechts vom HyTK- und large T-Gen je eine LOX-Sequenz als Substrat für die CRE-Rekombinase aufweist. Das zweite Retroviruskonstrukt basiert auf der pBABE-Puro. In diesen Vektor wird in eine Klonierungsschnittstelle das Gen für Bakteriophagen-Rekombinase CRE hinter das 5 LTR kloniert. Die CRE-Rekombinase kann in einer LOX-Sequenz-spezifischen Weise die Deletion der Sequenz zwischen zwei LOX-Erkennungssequenzen durchführen. Der Vektor enthält zusätzlich das Puromycin-Gen unter SV40-Promotorkontrolle als Selektionsmarker. Beide Vektoren werden durch Transfektion in geeignete Verpackungszellinien in Viruspartikel konvertiert. Die Sequenzen des large T-Bereichs und der LOX-Regionen werden durch Sequenzanalyse verifiziert. 2. Zeitlich begrenzte Expression des large T-Gens in primären humanen TumorzellenThe two retrovirus constructs to be constructed are shown in Fig. 1. The first is based on the vector HyTK-CMV. The vector contains a fusion gene from hygromycin and herpes simplex thymidine kinase (HyTK) gene under "long terminal repeat" (LTR) promoter control. Cells infected with the virus therefore have a positive (hygromycin) and a negative (gancyclovir) selection marker. 3 of the HyTK gene is a comparatively strong promoter from the cytomegalovirus. The large T gene from SV40 is cloned behind these. The large T gene is amplified by the polymerase chain reaction (PCR), the 3 'primer and a 34 base pair long sequence being extended, which corresponds to the LOX recognition sequence for the CRE recombinase and is filled in during the PCR reaction. In a second step, a second LOX sequence is cloned as a double-stranded primer pair 5 * from the HyTK gene. The result is a vector which has a LOX sequence as a substrate for the CRE recombinase to the left and right of the HyTK and large T genes. The second retrovirus construct is based on the pBABE-Puro. In this vector, the gene for bacteriophage recombinase CRE is cloned behind the 5 LTR in a cloning interface. The CRE recombinase can perform the deletion of the sequence between two LOX recognition sequences in a LOX sequence-specific manner. The vector additionally contains the puromycin gene under SV40 promoter control as a selection marker. Both vectors are converted into virus particles by transfection into suitable packaging cell lines. The sequences of the large T region and the LOX regions are verified by sequence analysis. 2nd Time-limited expression of the large T gene in primary human tumor cells
Unterschiedliches Tumormaterial wird operativ gewonnen, mechanisch/enzymatisch zu Einzelzell-suspensionen aufgearbeitet, mit dem HyTK-LOX-CMV-lT-Virus bzw. Kontrollvirus infiziert, plus und minus Selektionsmarker (Hygromycin) kultiviert und die Wachstumskinetik bestimmt. Ggf. wird der Infektion eine Anreicherung der Tumorzellen, z.B. durch Separation mit Antikörpern, die spezifisch für tumorassoziierte Antigene sind, vorangestellt, um zu verhindern, daß virusinfizierte Zellen (z. B. Fibroblasten) , die das Tumor-material kontaminieren, die Tumor2ellen überwachsen. Der Nachweis, daß wirklich Tumorzellen immortalisiert wurden, kann durch Färbung mit Antikörpern, die tumorassoziierte Antigene erkennen, und durch PCR für bestimmte Onkogene geführt werden. Außerdem wird die Frequenz, mit der in large T-abhängiger Weise Langzeitkulturen gewonnen werden, in Abhängigkeit vom Tumorzelltyp bestimmt. Nach unterschiedlich vielen Passagen werden die Zellinien mit dem Virus BABE-CRE-Puro infiziert und auf Puromycinresistenz selektioniert. In den mit beiden Viren infizierten Zellen führt die CRE-Rekombinase eine LOX- spezifische Rekombination des HyTK-LOX-CMV-IT-Virus durch, bei der das HyTK-Fusionsgen und das large T-Gen deletiert werden. Der Phänotyp der Tumorzellen sollte in der Abfolge des Experiments wie folgt sein: Puro*, Hygro8, GCV", G418" 1.VirusDifferent tumor material is surgically obtained, mechanically / enzymatically processed into single cell suspensions, infected with the HyTK-LOX-CMV-IT virus or control virus, plus and minus selection markers (hygromycin) cultivated and the growth kinetics determined. Possibly. the infection is preceded by an enrichment of the tumor cells, for example by separation with antibodies which are specific for tumor-associated antigens, in order to prevent virus-infected cells (e.g. fibroblasts) which contaminate the tumor material from overgrowing the tumor cells. Evidence that tumor cells have really been immortalized can be demonstrated by staining with antibodies that recognize tumor-associated antigens and by PCR for certain oncogenes. In addition, the frequency with which long-term cultures are obtained in a large T-dependent manner is determined as a function of the tumor cell type. After different numbers of passages, the cell lines are infected with the virus BABE-CRE-Puro and selected for puromycin resistance. In the cells infected with both viruses, the CRE recombinase carries out a LOX-specific recombination of the HyTK-LOX-CMV-IT virus, in which the HyTK fusion gene and the large T gene are deleted. The phenotype of the tumor cells should be as follows in the sequence of the experiment: Puro *, Hygro 8 , GCV ", G418" 1.Virus
Ruroa, Hygro», GCV3, G418" 2.Virus - Puro", Hygros, GCR«, G418s. Entsprechend diesem Schema wird anhand der Bestimmung der Wachstumskinetik und der sequentiellen Selektion der Zellen mit Puromycin, Gancyclovir und Hygromycin annäherungsweise bestimmt, mit welcher Frequenz nach Infektion mit dem 2. Virus spezifische Rekombination durchgeführt wird (Roro" vs. GCV*) . Die Frequenz wird zusätzlich genau dadurch bestimmt, daß die Zellen nach Infektion direkt in Gegenwart von Puromycin kloniert werden und anschließend prozentual GCVR-Zellen bestimmt werden. Ferner wird sichergestellt, daß alle Zellen, die keine spezifische Rekombination durchgeführt haben, eliminiert sind (GCV* und Gegenkontrolle Hygro"), d.h. nur large T-negative Zellen in der Kultur übrigbleiben. Dies wird molekular durch PCR mit large T-spezifischen Primern analysiert.Ruro a , Hygro » , GCV 3 , G418" 2nd Virus - Puro ", Hygro s , GCR « , G418 s . According to this scheme, the determination of the growth kinetics and the sequential selection of the cells with puromycin, gancyclovir and hygromycin approximately determines the frequency with which specific recombination is carried out after infection with the 2nd virus (Roro "vs. GCV *). The frequency becomes additionally exactly determined that the cells after infection directly in the presence of puromycin are cloned and GCV R cells are then determined as a percentage. Furthermore, it is ensured that all cells which have not carried out a specific recombination are eliminated (GCV * and counter-control hygro "), ie only large T-negative cells remain in the culture. This is analyzed molecularly by PCR with large T-specific primers .
3. Herstellung einer Ma makarzinomzellinie aus dem Tumorbiopsat3. Production of a macarcinoma cell line from the tumor biopsy
Tumorzellen, von denen aus einem Tumorbiopsat eine Zellkultur angelegt wird, haben in der Regel eine begrenzte Lebensspanne von 10-20 Passagen. Aus einem Mammakarzinombiopsat wird eine Zellkultur angelegt, und die Zellen entweder mit dem larαe T- Retrovirus infiziert oder unbehandelt gelassen. Wie aus Abb.2 zu sehen ist, führt die Infektion mit den large T-Retrovirus zu einer kontinuierlichen Proliferation der Zellen über mehrere Zellkulturpassagen. Die nicht-infizierten Zellen nahmen an Zahl sukzessive in Kultur ab, und es ließ sich keine Zellkultur etablieren. Dies beweist, daß sich mit Hilfe des large T-Virus Zellinien aus dem Tumorbiopsat isolieren lassen.Tumor cells, from which a cell culture is created from a tumor biopsy, usually have a limited lifespan of 10-20 passages. A cell culture is created from a breast cancer biopsy and the cells are either infected with the larαe T retrovirus or left untreated. As can be seen from Fig. 2, the infection with the large T retrovirus leads to a continuous proliferation of the cells over several cell culture passages. The number of uninfected cells decreased successively in culture, and no cell culture could be established. This proves that cell lines can be isolated from the tumor biopsy using the large T virus.
4. Deletion des large T-Gens durch den CRE-Rekombinase- Retrovirus4. Deletion of the large T gene by the CRE recombinase retrovirus
NIH3T3-Zellen werden zunächst mit dem large T-Retrovirus infiziert und die Hygro"-Zellen selektioniert. Anschließend werden die Zellen mit dem CRE-Rekombinase-Retrovi rus infiziert, und die Puro"-Zellen selektioniert. Dann wird mit GCV gegen alle Zellen selektioniert, bei denen keine Rekombination, bei der large T- und Thymidinkinase-Gene deletiert wurden, stattgefunden hat. Daraufhin werden die Zellen mit einem large T-spezifischen Antikörper angefärbt. Die Tabelle zeigt, daß nach Infektion mit dem large T-Virus quasi alle Zellen eine starke Kernfärbung aufweisen. Nach Infektion mit dem CRE- Rekombinase-Virus und GCV-Selektion war keine Färbung mit dem large T-Antikörper mehr nachweisbar.NIH3T3 cells are first infected with the large T retrovirus and the Hygro "cells are selected. Then the cells are infected with the CRE recombinase retrovirus and the Puro" cells are selected. Then GCV is used to select against all cells in which no recombination in which large T and thymidine kinase genes have been deleted has taken place. The cells are then stained with a large T-specific antibody. The table shows that after infection with the large T virus, virtually all cells have one show strong core coloring. After infection with the CRE recombinase virus and GCV selection, no staining with the large T antibody was detectable.
% der Zellen, die sich mit einem large T-spezifischen Ak anfärben lassen% of cells that can be stained with a large T-specific Ab
NIH3T3 infiziert mit Retrovirus 1 100 %NIH3T3 infected with retrovirus 1 100%
NIH3T3-Zellen infiziert mit Rv 1 + 2 nachNIH3T3 cells infected with Rv 1 + 2 after
GCV-Selektion 0 % GCV selection 0%
Legende zu den Abbildungen:Legend for the pictures:
Abb 1.Fig 1.
Zwei-Schritt konditionales Immortalisationssystem für primäre humane Tumorzellen. Retrovirusvektor HyTK-LOX-CMV-lT enthält die Erkennungssequenzen der Bakteriophagen-Reko binase CRE (LOX) , ein Fusionsgen, welches für Hygromycin und die Herpes Virus Thymidinkinase kodiert (HyTK) und den large T-Bereich aus SV40 (IT) unter CMV-Promotorkontrolle (CMV). Retrovirusvektor BABE-CRE-Puro enthält die CRE-Rekombinase unter LTR-Kontrolle und das Puromycingen unter SV40- Promotorkontrolle. Nicht maßstabsgerecht gezeichnet.Two-step conditional immortalization system for primary human tumor cells. Retrovirus vector HyTK-LOX-CMV-IT contains the recognition sequences of the bacteriophage recombinase CRE (LOX), a fusion gene which codes for hygromycin and the herpes virus thymidine kinase (HyTK) and the large T region from SV40 (IT) under CMV- Promoter Control (CMV). Retrovirus vector BABE-CRE-Puro contains the CRE recombinase under LTR control and the puromycin gene under SV40 promoter control. Not drawn to scale.
Abb 2.Fig 2.
Eine Zellsuspension aus einem Mammakarzinombiopsat wurde angelegt und entweder mit dem Retrovirus HyTK-LOX-CMV-lT infiziert (1) oder unbehandelt gelassen (O). Die Infektion erfolgte in der zweiten Woche. Die Pfeile geben die Zeitpunkte an, an denen die konfluente Zellkultur 1 : 10 ausgedünnt wurde. A cell suspension from a breast cancer biopsy was created and either infected with the retrovirus HyTK-LOX-CMV-IT (1) or left untreated (O). The infection occurred in the second week. The arrows indicate the times at which the confluent cell culture was thinned 1:10.

Claims

Patentansprüche claims
1. Konditionales Immortalisationsverfahren für Zellinien aus primären Zellkulturen, dadurch gekennzeichnet, daß T-Zellen, dendritische Zellen oder Tumorzellen mit einem Retroviruskonstrukt, das die Immortalisierung der Zellen bewirkt, behandelt werden, danach eine Kultivierung erfolgt und die erhaltenen Zellen vor ihrer Verwendung mit einem zweiten Retroviruskonstrukt, mit dem das inserierte erste Retroviruskonstrukt herausgeschnitten wird, behandelt werden.1. Conditional immortalization process for cell lines from primary cell cultures, characterized in that T cells, dendritic cells or tumor cells are treated with a retrovirus construct which effects the immortalization of the cells, followed by cultivation and the cells obtained before their use with a second Retrovirus construct, with which the inserted first retrovirus construct is cut out.
2. Konditionales Immortalisationsverfahren nach Anspruch 1 für humane Tumorzellen zur Herstellung einer Vakzine, dadurch gekennzeichnet, daß Tumorzellen aus Patientenmaterial mit einem Retroviruskonstrukt, das die Immortalisierung der Tumorzellen bewirkt, behandelt werden, danach eine Kultivierung erfolgt und die erhaltenen Zellen vor ihrer Verwendung zur Herstellung einer Vakzine mit einem zweiten Retroviruskonstrukt, mit dem das inserierte erste Retroviruskonstrukt herausgeschnitten wird, behandelt werden.2. Conditional immortalization method according to claim 1 for human tumor cells for the production of a vaccine, characterized in that tumor cells from patient material are treated with a retrovirus construct which brings about the immortalization of the tumor cells, followed by cultivation and the cells obtained before they are used to produce a Vaccines are treated with a second retrovirus construct, with which the inserted first retrovirus construct is cut out.
3. Retroviruskonstrukt zur Immortalisierung von Tumorzellen nach Anspruch 1 und 2, bestehend aus den Genen für die Herpes Simplex-Thymidinkinase, dem large T-Gen aus SV 40 als immortalisierendes Agens, dem CMV-Promotor, Gancyclovir als negativem Selektionsmarker und dem Hygromycin-Gen als positivem Selektionsmarker, flankiert von Signal-Sequenzen (LOX) für die Rekombinase CRE.3. retrovirus construct for immortalizing tumor cells according to claim 1 and 2, consisting of the genes for the herpes simplex thymidine kinase, the large T gene from SV 40 as an immortalizing agent, the CMV promoter, Gancyclovir as a negative selection marker and the hygromycin gene as a positive selection marker, flanked by signal sequences (LOX) for the recombinase CRE.
4. Retroviruskonstrukt zum Herausschneiden des inserierten Konstrukts nach Anspruch 1 und 2, enthaltend die Bakteriophagen-Rekombinase CRE unter LTR-Kontrolle und das Puromycingen unter SV 40-Promotor-Kontrolle. 4. retrovirus construct for cutting out the inserted construct according to claim 1 and 2, containing the bacteriophage recombinase CRE under LTR control and the puromycin gene under SV 40 promoter control.
PCT/DE1997/001406 1996-07-04 1997-07-03 Conditional immortalisation process for human tumour cells for producing a vaccine WO1998001539A1 (en)

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