WO2001074403A1 - Combination of p53 gene and e1b-deleted p53 gene - Google Patents
Combination of p53 gene and e1b-deleted p53 gene Download PDFInfo
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- A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
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Definitions
- the p53 gene is a very popular molecule in modern medicine, with extensive research being done to characterise the gene and numerous scientific articles written on the subject.
- P53 is a 53 kilodalton phosphoprotein involved in several important mechanisms, such as cell cycle regulation, cell differentiation, DNA synthesis and repair, and apoptosis. It is a transcription factor that binds as a tetramer to a DNA sequence composed of two head-to-head repeats.
- the p53 gene is the most common target for genetic alteration across all types of human cancers; it is a tumour suppressor gene, but in its mutated form it seems to act also as an oncogene.
- the p53 gene is located on the short arm of chromosome 17 and contains 11 exons interrupted by 10 introns. In mammals its organization is similar, with the exception of the rat in which intron 6 is missing.
- the messenger RNA codes for a 394 amino acid protein, which is expressed in all tissues in a relatively low level.
- the gene is very rich in G/C (guaninexytosine), which implies that the gene evolves under the base compositional restraints. In about one quarter of all p53 mutations in human cancers are transitions at the CpG nucleotides.
- Onyx-015 is an attenuated ElB-deleted adenovirus which does not produce a 55kD protein normally formed by the wild-type adeno virus.
- the protein inactivates wild-type p53 inside a host cell during infection. In this way the virus replicates and causes cytolysis of the infected cells.
- the ElB-deleted variant fails to inactivate the p53, no viral replication can take place inside the cell, and an abortive infection results. If a cell lacks p53, or has a mutant, non-functioning p53, the 55kD protein is not required to inactivate p53 and the virus will replicate and produce cytolysis. This is the rationale for using Onyx-015 in gene therapy protocols. However, it is of no use in cancer cells that are expressing wild-type p53.
- this present invention provides a method of enhancing and increasing the cytotoxic effects of gene therapy with adenoviral vectors.
- the status of p53 expression within cancer cells is of considerable clinical significance because a large number of cancers do not express mutant p53 or are p53 deleted. Such cancers do not respond well to current gene therapy regimens. Furthermore, most cancers have a mixture of cells, some expressing mutant or deleted p53, and others expressing wild-type p53. Thus, gene therapy protocols which are aimed at mutant p53 will be only partially successful.
- the ElB-55kD protein inactivates wild-type p53 inside a host cell during infection. In this way the virus replicates and causes cytolysis of the infected cells.
- the EIB-deleted adenovirus variant fails to inactivate the p53 in cells expressing wild type p53.
- the 55kD protein is not required to inactivate p53 and therefore Ad EIB deleted virus will replicate and produce cytolysis. This is the rationale for using Ad BIB deleted (Onyx-015) in gene therapy protocols. However, it is of no use in cancer cells that are expressing wild-type p53.
- This invention shows the combination of an adenovirus vector expressing wild- type p53 and an adenovirus expressing ElB-deleted p53 is effective in killing cancer cells that express mutant or deleted p53 and also cells expressing wild-type p53 and at very low concentration
- the invention describes a new therapeutic
- This invention allows for the combination of a combination of an adenovirus vector expressing wild-type p53 and an adenovirus expressing ElB-deleted p53 This combination is effective in killing cancer cells that express mutant or deleted
- the invention allows a new therapeutic approach to cancer treatment, based on the simultaneous use of both viruses for the treatment of cancer in humans and animals.
- RESULTS HepG2 hepatoma cells, which express wild type p53, PLC PRF/5 hepatoma cells, which express mutant p53 (codon 249), Hep3B hepatoma cells, which are p53- null, Nasal septum fibroblast cells, Chang liver and human hepatocytes, which all express wild-type p53, were grown in Dulbecco's modified Eagle medium (DMEM, GIBCO/BRL) supplemented with 10% foetal bovine serum at 37°C an 10% CO 2 .
- DMEM Dulbecco's modified Eagle medium
- AD EIB deleted contains an 827-bp deletion in the region encoding the 55-kD protein in combination with a stop codon to ensure that a truncated 55-kDA product cannot be expressed.
- cytotoxicity assays 10 3 cells were fed to each well of a 96- well plate 24 hours before infection. Cells were infected with AD EIB deleted or Ap53 at MOI 1, 10, 100 in a total volume of 200 ⁇ l DMEM + 2% FBS. After incubation at 37°C for two days, cells were washed with PBS and infected with different concentrations of Ad EIB deleted or Ad-p53 for another two days. At day 4, cytotoxicity effect was determined by colorimetric assay as described by Shekan et al., (1990). Briefly, cells were fixed in 10% trichloracetic acid for 60 mins. at 4°C.
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Abstract
The present invention uses the combination of the wild-type p53 gene and the E1B-deleted p53 gene which, hitherto, had not been considered scientifically justified because the over-expression of p53 would inhibit the AdE1B-deleted virus replication and, therefore, the killing of the cells. The combination has been shown to be more effective in killing cancer cells than the individual components used separately. It has been claimed in the medical literature that the Adenovirus vector, ONYX-015, which has a deletion in the E1B region, can selectively replicate in, and kill, human cancer cells that lack functional p53, but is non-cytotoxic to cells that have an intact (wild-type) p53. Recent evidence has contradicted this claim and suggested that ONYX-015 can replicate in cancer cells with wild-type p53 and that cells lacking p53 function are resistant to adenovirus-induced cell death. Functional p53 is deficient in over half of human cancers and its absence is thought to be a major factor in the pathogenesis of many malignant conditions. Many of the current cancer gene therapy protocols are using either wild-type p53 (Adwtp53) or the E1B-deleted variant (AdE1B), and usually only include patients with p53 mutations. However, these studies have only shown limited benefit, with many patients having stable disease, but few showing tumour regression. To improve the efficacy of gene therapy for cancer it is important to determine the true significance of the p53 status in cancer cells.
Description
COMBINATION OF P53 GENE AND E1B-DELETED P53 GENE
The p53 gene is a very popular molecule in modern medicine, with extensive research being done to characterise the gene and numerous scientific articles written on the subject. P53 is a 53 kilodalton phosphoprotein involved in several important mechanisms, such as cell cycle regulation, cell differentiation, DNA synthesis and repair, and apoptosis. It is a transcription factor that binds as a tetramer to a DNA sequence composed of two head-to-head repeats. The p53 gene is the most common target for genetic alteration across all types of human cancers; it is a tumour suppressor gene, but in its mutated form it seems to act also as an oncogene. The p53 gene is located on the short arm of chromosome 17 and contains 11 exons interrupted by 10 introns. In mammals its organization is similar, with the exception of the rat in which intron 6 is missing. In human, the messenger RNA codes for a 394 amino acid protein, which is expressed in all tissues in a relatively low level. The gene is very rich in G/C (guaninexytosine), which implies that the gene evolves under the base compositional restraints. In about one quarter of all p53 mutations in human cancers are transitions at the CpG nucleotides. Onyx-015 is an attenuated ElB-deleted adenovirus which does not produce a
55kD protein normally formed by the wild-type adeno virus. The protein inactivates wild-type p53 inside a host cell during infection. In this way the virus replicates and causes cytolysis of the infected cells. The ElB-deleted variant fails to inactivate the p53, no viral replication can take place inside the cell, and an abortive infection results. If a cell lacks p53, or has a mutant, non-functioning p53, the 55kD protein is not required to inactivate p53 and the virus will replicate and produce cytolysis. This is the rationale for using Onyx-015 in gene therapy protocols. However, it is of no use in cancer cells that are expressing wild-type p53.
Alternative gene therapy strategies have used adenoviral vectors expressing wild- type p53. This is based on studies that showed that transfection of tumour cells with plasmid DNA expressing wild-type p53 or infection with retrovirus expressing wild-type p53 was cytotoxic to cells expressing mutant p53. Thus, the existing scientific evidence shows that gene therapy with either wild- type p53 or with the ElB-deleted p53 is active only against tumors that express the mutant or depleted p53 gene. This present invention is unique in that it combines the wild-type p53 adeno virus and the El B deleted adenovirus and that this combination is cytotoxic to tumour cells that express wild-type p53 and to those with mutant p53. Thus, this present invention provides a method of enhancing and increasing the cytotoxic effects of gene therapy with adenoviral vectors. The status of p53 expression within cancer cells is of considerable clinical significance because a large number of cancers do not express mutant p53 or are
p53 deleted. Such cancers do not respond well to current gene therapy regimens. Furthermore, most cancers have a mixture of cells, some expressing mutant or deleted p53, and others expressing wild-type p53. Thus, gene therapy protocols which are aimed at mutant p53 will be only partially successful.
In most wild type adenoviruses the ElB-55kD protein inactivates wild-type p53 inside a host cell during infection. In this way the virus replicates and causes cytolysis of the infected cells. The EIB-deleted adenovirus variant fails to inactivate the p53 in cells expressing wild type p53. In cancer cells with a mutant or a nonfunctioning p53, the 55kD protein is not required to inactivate p53 and therefore Ad EIB deleted virus will replicate and produce cytolysis. This is the rationale for using Ad BIB deleted (Onyx-015) in gene therapy protocols. However, it is of no use in cancer cells that are expressing wild-type p53. Alternative gene therapy strategies have used adenoviral vectors expressing wild type p53. This is based on studies that showed that transfection of tumour cells with plasmid DNA expressing wild-type p53 or infection with retrovirus expressing wild-type p53 was cytotoxic to cells expressing mutant p53. Thus, the existing scientific evidence shows that both gene therapy approaches, with either wild-type p53 or with the EIB deleted p53, are active only against tumours that express the mutant or depleted p53 gene.
The status of p53 expression within cancer cells is of considerable clinical significance because a large number of cancers do not express mutant p53 and have no p53 deletion. Such cancers do not respond well to current gene therapy regimens. Furthermore, most cancers have a mixture of cells, some expressing
mutant or deleted p53, and others expressing wild-type p53 Tumours are heterogeneous in nature Thus, gene therapy protocols which are aimed at mutant p53 will be only partially successful
In this study a series of hepatocellular cancer cell lines were transfected with Adwtp53 and it produced a cytotoxic effect in the Hep3B cells (deleted p53), partially cytotoxic in the PLC/PRF/5 cells (mutated p53), but there was no inhibition of HepG2 cells (wild-type p53) The doses of Adenovirus used were large In these tumour cell lines the use of Adwtp53 was only effective in those cells expressing mutant or deleted p53 The same cell line types, as above, were transfected, in separate experiments, with AdEIB The ElB-deleted adenovirus selectively inhibited Hep3B (deleted p53) and PLC (mutated p53), but agam failed to inhibit HepG2 (wild-type p53) The doses of Adenovirus used were also large
In a third series of expeπments, a combination of Adwtp53 and AdE IB-deleted virus was used to transfect the hepatocellular cancer cell lines The combination lead to major cytotoxicity to all cancer cell lines, irrespective of the p53 status In addition, when all three cell lines were grown together, mimicking the cell mixture typically expected in human cancers, there was a complete cytotoxic effect, with total cell death Furthemore, the cytotoxic effect was seen with a viral concentration less than that required for each virus individually even at 1 pfu/cell concentration
This invention shows the combination of an adenovirus vector expressing wild- type p53 and an adenovirus expressing ElB-deleted p53 is effective in killing
cancer cells that express mutant or deleted p53 and also cells expressing wild-type p53 and at very low concentration The invention describes a new therapeutic
90 approach, based on the simultaneous use of an adeno-associated virus competent in replication with an adeno-associated virus encoding p53 A series of hepatocellular cancer cell lines were transfected with Adwtp53 and it produced a cytotoxic effect m the Hep3B cells (deleted p53), partially cytotoxic in the PLVC cells (mutated ρ53), but there was no inhibition of HepG2 cells (wild
95 type p53). The doses used were not excessive. In these tumour cell lines the use of wtp53 was only effective in those cells expressing mutant or deleted p53. The same cell line types, as above, were transfected, in separate experiments, with AdEIB. The ElB-deleted adenovirus selectively inhibited Hep3B (deleted p53) and PLC (mutated p53), but failed to inhibit HepG2 (wild-type p53).
100 In a third series of experiments, a combination of Adwtp53 and AdElB-deleted virus was used to transfect the hepatocellular cancer cell lines. The combination led to complete cell death in all cell lines, irrespective of the p53 status. In addition, when all three cell lines were grown together, mimicking the cell mixture typically expected in human cancers, there was a complete cytotoxic
105 effect, with total cell death. Furthermore, the cytotoxic effect was seen with a viral concentration 2 log less than that required for each virus individually. This invention allows for the combination of a combination of an adenovirus vector expressing wild-type p53 and an adenovirus expressing ElB-deleted p53 This combination is effective in killing cancer cells that express mutant or deleted
110 p53 and also cells expressing wild-type p53 Therefore, the invention allows a
new therapeutic approach to cancer treatment, based on the simultaneous use of both viruses for the treatment of cancer in humans and animals.
METHODS HepG2 hepatoma cells, which express wild type p53, PLC PRF/5 hepatoma cells, which express mutant p53 (codon 249), Hep3B hepatoma cells, which are p53- null, Nasal septum fibroblast cells, Chang liver and human hepatocytes, which all express wild-type p53, were grown in Dulbecco's modified Eagle medium (DMEM, GIBCO/BRL) supplemented with 10% foetal bovine serum at 37°C an 10% CO2.
AD EIB deleted contains an 827-bp deletion in the region encoding the 55-kD protein in combination with a stop codon to ensure that a truncated 55-kDA product cannot be expressed.
In cytotoxicity assays 103 cells were fed to each well of a 96- well plate 24 hours before infection. Cells were infected with AD EIB deleted or Ap53 at MOI 1, 10, 100 in a total volume of 200μl DMEM + 2% FBS. After incubation at 37°C for two days, cells were washed with PBS and infected with different concentrations of Ad EIB deleted or Ad-p53 for another two days. At day 4, cytotoxicity effect was determined by colorimetric assay as described by Shekan et al., (1990). Briefly, cells were fixed in 10% trichloracetic acid for 60 mins. at 4°C. Fixed cells were stained with lOOμl sulphorhodamine B or 10 mins., rinsed five times with 1% acetic acid and the dye solubilised for 1 hour with 200μl unbuffered Tris base. The relative survival of cells was calculated by comparing the OD 560nm
readings of the infected cells with the readings of the uninfected cells, using a plate reader.
RESULTS In the experiments to study the cytotoxic effect induced by EIB deleted, at MOI 100 approximately 40% of Hep3B cells (p53-null) were killed by Ad EIB deleted, whereas less than 20% of HepG2 (wt-p53) or PLC PRF/5 (mut-p53) cells were killed (Figure 1).
At MOI 1, a combination of Ad-p53 and Ad EIB deleted caused death of 73% of Hep3B cells, 60% of HepG2 and PLC/PRF/5 cells, but only 30% Of fibroblasts (Figures 2 & 3). The cell death rate is significantly greater (p<0.01) for the combination of EIB deleted and Ad-p53 than for Ad-p53 alone. Tables 1 & 2 show that Ad EIB deleted, Ad Null and Ad EIB are not toxic to HepG2 cell lines at 1 an 10 pfus/cell concentrations. However, when Ad EIB deleted and Ad p53 were mixed together the combination was highly cytotoxic, even at concentrations as low as 1 pfu/cell.
Claims
CLAIMS A combination of wild-type p53 gene and the ElB-deleted p53 gene given simultaneously to produce a cytotoxic effect in human and animal cancer cells. A combination of wild-type p53 gene and the ElB-deleted p53 gene given sequentially to produce a cytotoxic effect in human and animal cancer cells. A combination of wild-type p53 gene and any adeno-associated virus competent in replication given either simultaneously or sequentially to produce a cytotoxic effect in human and animal cancer cells.
A combination of wild-type p53 and the ElB-deleted p53 given sequentially or simultaneously, as in 1&2, using an adenovirus vector to transport the combination of genes.
A combination of wild-type p53 and an adeno-associated virus competent in replication given sequentially or simultaneously, as in 3 above, using an adenovirus vector to transport the combination of genes.
A combination of wild-type p53 and an adeno-associated virus competent in replication given sequentially or simultaneously, as in 3 above, using an liposomal vector to transport the combination of genes.
A combination of wild-type p53 and the ElB-deleted p53 given sequentially or simultaneously, as in 1&2 above, using a liposomal vector to transport the combination of genes.
A combination of wild-type p53 and the ElB-deleted p53 given sequentially or simultaneously, as in 1&2, using any vector carrying the El A gene to transport the combination of genes.
A combination of wild-type p53 and an adeno-associated virus competent in replication given sequentially or simultaneously, as 3,5 & 6 above in which the adeno-associated virus competent in replication has the El A gene or encodes for the El A depleted gene A combination of wild-type p53 and the ElB-deleted p53 given sequentially or simultaneously, as in 1&2, using a vector, as in 3,4,&5, administered by the intravenous route A combination of wild-type p53 and the ElB-deleted p53 given sequentially or simultaneously, as in 1&2, using a vector, as m 3,4,&5, administered by the oral (cochleate) route A combination of wild-type p53 and the ElB-deleted p53 given sequentially or simultaneously, as in 1&2, using a vector, as in 3,4,&5, administered by the mtrapeπtoneal route A combination of wild-type p53 and the EIB-deleted p53 given sequentially or simultaneously, as in 1&2, using a vector, as in 3,4,&5, administered directly into the tumour A combination of wild-type p53 and the ElB-deleted p53 given sequentially or simultaneously, as in 1&2, using a vector, as in 3,4,&5, administered by the mtra- arteπal route A combination of wild-type p53 and the ElB-deleted p53 given sequentially or simultaneously, as in 1&2, using a vector, as in 3,4,&5, administered by any of the routes described in 6,7,8,9,&10, and used to kill human and animal cancer cells expressing a mutant p53 gene
A combination of wild-type p53 and the ElB-deleted p53 given sequentially or simultaneously, as in 1&2, using a vector, as in 3,4,&5, administered by any of the routes described in 6,7,8,9,&10, and used to kill human and animal cancer cells with a deleted p53 gene. A combination of wild-type p53 and the ElB-deleted p53 given sequentially or simultaneously, as in 1&2, using a vector, as in 3,4,&5, administered by any of the routes described in 6,7,8,9,&10, and used to kill human and animal cancer cells expressing the wild-type p53 gene. A combination of wild-type p53 and the ElB-deleted p53 given sequentially or simultaneously, as in 1&2, using a vector, as in 3,4,&5, administered by any of the routes described in 6,7,8,9,&10, and used to kill human and animal cancer cells expressing a combination of forms and expressions of p53 gene, including, but not exclusively, a combination of the forms and expressions described in 11, 12. &13. A method of using the invention whereby the adenovirus ElB-deleted form is used first and wild-type p53 is added to the vector A method of using the invention, as in 1& 2, whereby plasmid p53 and the gene encoding Rl A are used in combination or sequentially. A method of using the invention, as in 1,2, 1 1,12,13, & 14, and administered in ways described in Claims 6-10, whereby plasmid wild-type p53 and the gene encoding El A are used in combination or sequentially with the El A gene either as a plasmid or in a vector.
A combination of wild-type p53 and the ElB-deleted p53 given sequentially or simultaneously, as in 1,2, 1 1 ,12,13, & 14, and administered in ways described in Claims 6-10, using a retrovirus vector to transport the combination of genes or the genes sequentially. A combination of wild-type p53 and the ElB-deleted p53 given sequentially or simultaneously, as in 1,2, 1 1,12,13, & 14, and administered in ways described in Claims 6-10, using a Herpes virus vector to transport the combination of genes or the genes sequentially. A combination of wild-type p53 and the ElB-deleted p53 given sequentially or simultaneously, as in 1,2, 11,12,13, & 14, and administered in ways described in Claims 6-10, using fusion proteins to transport the combination of genes or the genes sequentially.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PCT/GB2000/001266 WO2001074403A1 (en) | 2000-04-04 | 2000-04-04 | Combination of p53 gene and e1b-deleted p53 gene |
| AU39769/00A AU3976900A (en) | 2000-04-04 | 2000-04-04 | Combination of p53 gene and e1b-deleted p53 gene |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PCT/GB2000/001266 WO2001074403A1 (en) | 2000-04-04 | 2000-04-04 | Combination of p53 gene and e1b-deleted p53 gene |
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| Publication Number | Publication Date |
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| WO2001074403A1 true WO2001074403A1 (en) | 2001-10-11 |
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| PCT/GB2000/001266 WO2001074403A1 (en) | 2000-04-04 | 2000-04-04 | Combination of p53 gene and e1b-deleted p53 gene |
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| AU (1) | AU3976900A (en) |
| WO (1) | WO2001074403A1 (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1327688A1 (en) * | 2002-01-14 | 2003-07-16 | Vereniging Voor Christelijk Wetenschappelijk Onderwijs | Adenoviruses with enhanced lytic potency |
| EP1409653A4 (en) * | 2001-07-23 | 2006-05-03 | Onyx Pharma Inc | Viral mutants that selectively replicate in targeted human cancer cells |
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| WO1997030732A1 (en) * | 1996-02-22 | 1997-08-28 | Onyx Pharmaceuticals, Inc. | Liposomal-viral dna complexes for treating disease |
| WO1999059604A1 (en) * | 1998-05-15 | 1999-11-25 | Onyx Pharmaceuticals, Inc. | Adenovirus-chemotherapeutic combination for treating cancer |
| GB2342042A (en) * | 1998-09-30 | 2000-04-05 | Christopher Barry Wood | Combination gene therapy using the p53 gene |
-
2000
- 2000-04-04 WO PCT/GB2000/001266 patent/WO2001074403A1/en active Application Filing
- 2000-04-04 AU AU39769/00A patent/AU3976900A/en not_active Abandoned
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| WO1997030732A1 (en) * | 1996-02-22 | 1997-08-28 | Onyx Pharmaceuticals, Inc. | Liposomal-viral dna complexes for treating disease |
| WO1999059604A1 (en) * | 1998-05-15 | 1999-11-25 | Onyx Pharmaceuticals, Inc. | Adenovirus-chemotherapeutic combination for treating cancer |
| GB2342042A (en) * | 1998-09-30 | 2000-04-05 | Christopher Barry Wood | Combination gene therapy using the p53 gene |
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| ROTHMANN T ET AL: "Replication of ONYX-015, a potential anticancer adenovirus, is independent of p53 status in tumor cells", JOURNAL OF VIROLOGY, December 1998 (1998-12-01), XP002139587 * |
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| ZHAO J ET AL: "p53 enhanced the antitumoral efficacy of E1B 55-Kilodalton mutant adenovirus on hepatocellular carcinoma cell line.", GUT, vol. 44, no. SUPPL. 1, April 1999 (1999-04-01), British Society of Gastroenterology Annual Meeting;Glasgow, Scotland, UK; March 23-25, 1999, pages A87, XP000971890, ISSN: 0017-5749 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1409653A4 (en) * | 2001-07-23 | 2006-05-03 | Onyx Pharma Inc | Viral mutants that selectively replicate in targeted human cancer cells |
| EP1327688A1 (en) * | 2002-01-14 | 2003-07-16 | Vereniging Voor Christelijk Wetenschappelijk Onderwijs | Adenoviruses with enhanced lytic potency |
| WO2003057892A3 (en) * | 2002-01-14 | 2004-02-26 | Vereniging Voor Christelijk Wetenschappelijk Onderwijs | Viruses with enhanced lytic potency |
| US8052965B2 (en) | 2002-01-14 | 2011-11-08 | Vereniging Voor Christelijk Hoger Onderwijs, Wetenschappelijk Onderzoek En Patientenzorg | Viruses with enhanced lytic potency |
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| Publication number | Publication date |
|---|---|
| AU3976900A (en) | 2001-10-15 |
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