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WO1998001539A1 - Procede d'immortalisation conditionnelle de cellules tumorales humaines servant a preparer un vaccin - Google Patents

Procede d'immortalisation conditionnelle de cellules tumorales humaines servant a preparer un vaccin 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
Authority
WO
WIPO (PCT)
Prior art keywords
cells
construct
retrovirus construct
retrovirus
vaccine
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/DE1997/001406
Other languages
German (de)
English (en)
Inventor
Thomas Blankenstein
Liang-Ping Li
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Max Delbrueck Centrum fuer Molekulare in der Helmholtz Gemeinschaft
Original Assignee
Max Delbrueck Centrum fuer Molekulare in der Helmholtz Gemeinschaft
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Max Delbrueck Centrum fuer Molekulare in der Helmholtz Gemeinschaft filed Critical Max Delbrueck Centrum fuer Molekulare in der Helmholtz Gemeinschaft
Priority to EP97932733A priority Critical patent/EP0910626A1/fr
Publication of WO1998001539A1 publication Critical patent/WO1998001539A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • 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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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Immunology (AREA)
  • Medicinal Chemistry (AREA)
  • Microbiology (AREA)
  • Mycology (AREA)
  • Oncology (AREA)
  • Chemical & Material Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

L'invention vise à synthétiser par génie génétique un vaccin autologue à base de cellules tumorales et son objet est de produire à cet effet des cultures reproductibles de cellules à partir de produits de biopsie de tumeurs. Ce nouveau procédé d'immortalisation conditionnelle de cellules tumorales humaines servant à préparer un vaccin se caractérise en ce que l'on traite des cellules tumorales prélevées sur un patient avec un produit rétroviral de synthèse qui entraîne l'immortalisation des cellules tumorales. On cultive ensuite ces cellules et on traite les cellules ainsi obtenues, avant leur utilisation pour produire un vaccin, avec un deuxième produit rétroviral de synthèse qui extrait de la cellule le premier produit rétroviral de synthèse. Ce procédé convient aussi en général pour produire des lignées de cellules à partir de cultures primaires de cellules, par exemple de cellules T ou de cellules dendritiques. L'invention a des applications dans les domaines de la médecine et de l'industrie pharmaceutique.
PCT/DE1997/001406 1996-07-04 1997-07-03 Procede d'immortalisation conditionnelle de cellules tumorales humaines servant a preparer un vaccin Ceased WO1998001539A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP97932733A EP0910626A1 (fr) 1996-07-04 1997-07-03 Procede d'immortalisation conditionnelle de cellules tumorales humaines servant a preparer un vaccin

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE19626830A DE19626830A1 (de) 1996-07-04 1996-07-04 Konditionales Immortalisationsverfahren für humane Tumorzellen zur Herstellung einer Vakzine
DE19626830.3 1996-07-04

Publications (1)

Publication Number Publication Date
WO1998001539A1 true WO1998001539A1 (fr) 1998-01-15

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Application Number Title Priority Date Filing Date
PCT/DE1997/001406 Ceased WO1998001539A1 (fr) 1996-07-04 1997-07-03 Procede d'immortalisation conditionnelle de cellules tumorales humaines servant a preparer un vaccin

Country Status (3)

Country Link
EP (1) EP0910626A1 (fr)
DE (1) DE19626830A1 (fr)
WO (1) WO1998001539A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001011030A3 (fr) * 1999-08-10 2001-05-31 Develogen Ag Procede et moyen permettant d'induire la multiplication cellulaire de cellules au repos
EP0955360A3 (fr) * 1998-04-09 2003-01-02 GSF-Forschungszentrum für Umwelt und Gesundheit GmbH Procédé d' immortalization de cellules au moyen de cellules auxiliaires conditionellement transformées
JP2008228685A (ja) * 2007-03-22 2008-10-02 Gunma Univ レトロウイルス産生用ベクター

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995029994A1 (fr) * 1994-04-28 1995-11-09 Michigan State University Lignees cellulaires prostatiques humaines immortalisees par un virus hybride derive de l'adenovirus 12 et du virus simien 40 (ad12/sv40)
WO1996005866A2 (fr) * 1994-08-24 1996-02-29 Max-Delbrück-Centrum für Molekulare Medizin Vaccin vivant utilise pour traiter des maladies tumorales
GB2294946A (en) * 1994-11-08 1996-05-15 Bradley Michael John Stringer Preparation of human cell-lines of fully-differentiated cells of specific tissue type

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995029994A1 (fr) * 1994-04-28 1995-11-09 Michigan State University Lignees cellulaires prostatiques humaines immortalisees par un virus hybride derive de l'adenovirus 12 et du virus simien 40 (ad12/sv40)
WO1996005866A2 (fr) * 1994-08-24 1996-02-29 Max-Delbrück-Centrum für Molekulare Medizin Vaccin vivant utilise pour traiter des maladies tumorales
GB2294946A (en) * 1994-11-08 1996-05-15 Bradley Michael John Stringer Preparation of human cell-lines of fully-differentiated cells of specific tissue type

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BERGEMANN J ET AL: "EXCISION OF SPECIFIC DNA-SEQUENCES FROM INTEGRATED RETROVIRAL VECTORS VIA SITE-SPECIFIC RECOMBINATION", NUCLEIC ACIDS RESEARCH, vol. 23, no. 21, 11 November 1995 (1995-11-11), pages 4451 - 4456, XP000644463 *
CHOULIKA A ET AL: "TRANSFER OF SINGLE GENE-CONTAINING LONG TERMINAL REPEATS INTO THE GENOME OF MAMMALIAN CELLS BY A RETROVIRAL VECTOR CARRYING THE CRE GENE AND THE LOXP SITE", JOURNAL OF VIROLOGY, vol. 70, no. 3, March 1996 (1996-03-01), pages 1792 - 1798, XP000616291 *
PANTEL K ET AL: "ESTABLISHMENT OF MICROMETASTATIC CARCINOMA CELL LINES: A NOVEL SOURCE OF TUMOR CELL VACCINES", JOURNAL OF THE NATIONAL CANCER INSTITUTE, vol. 87, no. 15, 2 August 1995 (1995-08-02), pages 1162 - 1168, XP000611729 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0955360A3 (fr) * 1998-04-09 2003-01-02 GSF-Forschungszentrum für Umwelt und Gesundheit GmbH Procédé d' immortalization de cellules au moyen de cellules auxiliaires conditionellement transformées
WO2001011030A3 (fr) * 1999-08-10 2001-05-31 Develogen Ag Procede et moyen permettant d'induire la multiplication cellulaire de cellules au repos
JP2008228685A (ja) * 2007-03-22 2008-10-02 Gunma Univ レトロウイルス産生用ベクター

Also Published As

Publication number Publication date
EP0910626A1 (fr) 1999-04-28
DE19626830A1 (de) 1998-01-08

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