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 PDFInfo
- 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
Links
- 210000004881 tumor cell Anatomy 0.000 title claims abstract description 33
- 229960005486 vaccine Drugs 0.000 title claims abstract description 10
- 238000000034 method Methods 0.000 title claims abstract description 8
- 230000008569 process Effects 0.000 title claims abstract description 6
- 210000004027 cell Anatomy 0.000 claims abstract description 41
- 241001430294 unidentified retrovirus Species 0.000 claims abstract description 30
- 238000004113 cell culture Methods 0.000 claims abstract description 16
- 210000001744 T-lymphocyte Anatomy 0.000 claims abstract description 6
- 210000004443 dendritic cell Anatomy 0.000 claims abstract description 5
- 108090000623 proteins and genes Proteins 0.000 claims description 16
- RXWNCPJZOCPEPQ-NVWDDTSBSA-N puromycin Chemical compound C1=CC(OC)=CC=C1C[C@H](N)C(=O)N[C@H]1[C@@H](O)[C@H](N2C3=NC=NC(=C3N=C2)N(C)C)O[C@@H]1CO RXWNCPJZOCPEPQ-NVWDDTSBSA-N 0.000 claims description 14
- IRSCQMHQWWYFCW-UHFFFAOYSA-N ganciclovir Chemical compound O=C1NC(N)=NC2=C1N=CN2COC(CO)CO IRSCQMHQWWYFCW-UHFFFAOYSA-N 0.000 claims description 11
- YQYJSBFKSSDGFO-UHFFFAOYSA-N Epihygromycin Natural products OC1C(O)C(C(=O)C)OC1OC(C(=C1)O)=CC=C1C=C(C)C(=O)NC1C(O)C(O)C2OCOC2C1O YQYJSBFKSSDGFO-UHFFFAOYSA-N 0.000 claims description 8
- 101150072261 large T gene Proteins 0.000 claims description 7
- 229950010131 puromycin Drugs 0.000 claims description 7
- 101710200251 Recombinase cre Proteins 0.000 claims description 6
- 239000003550 marker Substances 0.000 claims description 6
- 108020004440 Thymidine kinase Proteins 0.000 claims description 5
- 239000000463 material Substances 0.000 claims description 5
- 102000006601 Thymidine Kinase Human genes 0.000 claims description 4
- 238000004519 manufacturing process Methods 0.000 claims description 4
- 241001515965 unidentified phage Species 0.000 claims description 4
- 208000009889 Herpes Simplex Diseases 0.000 claims description 3
- 230000000694 effects Effects 0.000 claims description 3
- 229960002963 ganciclovir Drugs 0.000 claims description 3
- 108010076504 Protein Sorting Signals Proteins 0.000 claims description 2
- 239000003795 chemical substances by application Substances 0.000 claims description 2
- 238000005520 cutting process Methods 0.000 claims description 2
- 206010028980 Neoplasm Diseases 0.000 abstract description 18
- 238000001574 biopsy Methods 0.000 abstract description 9
- 229940030156 cell vaccine Drugs 0.000 abstract 1
- 238000010353 genetic engineering Methods 0.000 abstract 1
- 241000700605 Viruses Species 0.000 description 16
- 101100289792 Squirrel monkey polyomavirus large T gene Proteins 0.000 description 15
- 239000013598 vector Substances 0.000 description 9
- 108010051219 Cre recombinase Proteins 0.000 description 8
- 208000015181 infectious disease Diseases 0.000 description 8
- 239000000427 antigen Substances 0.000 description 5
- 108091007433 antigens Proteins 0.000 description 5
- 102000036639 antigens Human genes 0.000 description 5
- 238000003752 polymerase chain reaction Methods 0.000 description 5
- 230000006798 recombination Effects 0.000 description 4
- 238000005215 recombination Methods 0.000 description 4
- 206010006187 Breast cancer Diseases 0.000 description 3
- 208000026310 Breast neoplasm Diseases 0.000 description 3
- 230000004927 fusion Effects 0.000 description 3
- 206010006417 Bronchial carcinoma Diseases 0.000 description 2
- 206010009944 Colon cancer Diseases 0.000 description 2
- 108090000695 Cytokines Proteins 0.000 description 2
- 102000004127 Cytokines Human genes 0.000 description 2
- 229940030547 autologous tumor cell vaccine Drugs 0.000 description 2
- 208000003362 bronchogenic carcinoma Diseases 0.000 description 2
- 239000006285 cell suspension Substances 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 238000012217 deletion Methods 0.000 description 2
- 230000037430 deletion Effects 0.000 description 2
- 230000001419 dependent effect Effects 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- 238000001890 transfection Methods 0.000 description 2
- 208000017897 Carcinoma of esophagus Diseases 0.000 description 1
- 241000701022 Cytomegalovirus Species 0.000 description 1
- 102000004457 Granulocyte-Macrophage Colony-Stimulating Factor Human genes 0.000 description 1
- 108010017213 Granulocyte-Macrophage Colony-Stimulating Factor Proteins 0.000 description 1
- 206010030155 Oesophageal carcinoma Diseases 0.000 description 1
- 108700020796 Oncogene Proteins 0.000 description 1
- 102000043276 Oncogene Human genes 0.000 description 1
- 206010033128 Ovarian cancer Diseases 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- 206010068771 Soft tissue neoplasm Diseases 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 210000000481 breast Anatomy 0.000 description 1
- 201000008275 breast carcinoma Diseases 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 210000001072 colon Anatomy 0.000 description 1
- 238000004040 coloring Methods 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 201000005619 esophageal carcinoma Diseases 0.000 description 1
- 208000021045 exocrine pancreatic carcinoma Diseases 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 210000002950 fibroblast Anatomy 0.000 description 1
- 206010017758 gastric cancer Diseases 0.000 description 1
- 208000010749 gastric carcinoma Diseases 0.000 description 1
- 230000002496 gastric effect Effects 0.000 description 1
- 230000008570 general process Effects 0.000 description 1
- 230000005934 immune activation Effects 0.000 description 1
- 210000003292 kidney cell Anatomy 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 201000001441 melanoma Diseases 0.000 description 1
- 230000002611 ovarian Effects 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 208000008443 pancreatic carcinoma Diseases 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 201000000498 stomach carcinoma Diseases 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 229940030325 tumor cell vaccine Drugs 0.000 description 1
- 241001529453 unidentified herpesvirus Species 0.000 description 1
- 238000002255 vaccination Methods 0.000 description 1
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.
Landscapes
- 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.
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 |
Family
ID=7798830
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/DE1997/001406 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)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0955360A2 (fr) * | 1998-04-09 | 1999-11-10 | 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 |
Citations (3)
| 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 |
-
1996
- 1996-07-04 DE DE19626830A patent/DE19626830A1/de not_active Withdrawn
-
1997
- 1997-07-03 WO PCT/DE1997/001406 patent/WO1998001539A1/fr not_active Application Discontinuation
- 1997-07-03 EP EP97932733A patent/EP0910626A1/fr not_active Withdrawn
Patent Citations (3)
| 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)
| 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 (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0955360A2 (fr) * | 1998-04-09 | 1999-11-10 | GSF-Forschungszentrum für Umwelt und Gesundheit GmbH | Procédé d' immortalization de cellules au moyen de cellules auxiliaires conditionellement transformées |
| 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 |
|---|---|
| DE19626830A1 (de) | 1998-01-08 |
| EP0910626A1 (fr) | 1999-04-28 |
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