JP5580043B2 - Radiosensitization enhancer - Google Patents

Description

  The present invention relates to a radiosensitizing enhancer comprising a recombinant virus in which a human telomerase promoter, an E1A gene, an IRES sequence, and an E1B gene are integrated in the genome.

Despite advances in diagnostic methods and clinical management, cancer remains the leading cause of death worldwide (Parkin DM et al. CA Cancer J Clin. 2005; 55: 74-108, Jema A, et al. Cancer). 2004 1; 101: 3-27). Current therapies for advanced cancer include surgical excision, radiation therapy, and cytotoxic chemotherapy, which can be combined to improve the survival rate of cancer patients. However, many cancer patients are forced to progress because they often have intrinsic or acquired resistance to these therapies (Vokes EE, et al. Clin Cancer Res. 2005; 11: 5045s-5050s, Milas L, et al. Head Neck. 2003; 25: 152-67).
Radiotherapy and chemotherapy are problematic because they cannot target only cancer cells. Therefore, there is a need for anticancer agents that selectively damage only tumor cells but not normal cells.
In such circumstances, viruses that selectively grow in a tumor cell-specific manner provide a new treatment for cancer. This virus is designed to induce tumor cell thawing after selective growth in only tumor tissue (Kirn D, et al. Nat. Med., 7: 781-787, 2001, Hawkins LK, et al. Lancet Oncol. 2002; 3: 17-26). Effective targeting of cancer cells requires a tissue or cell specific promoter that is expressed in a variety of cancers but not in normal cells.
Telomerase is a ribonucleoprotein complex involved to fully replicate the chromosome ends (Blackburn EH. Nature (Lond.) 1991; 350: 569-573). Telomerase is active in more than 85% of human cancers (Kim NW, et al. Science 1994; 266: 2011-2015) but is active only in proliferating cells in normal somatic cells (Shay JW, et al Curr). Opin Oncol 1996; 8: 66-71). Activation of telomerase is thought to be an important step in carcinogenesis, and its activity is closely related to the expression of human telomerase reverse transcriptase (hTERT) (Nakayama J, et al. Nat Genet 1998; 18 : 65-68).
Since the hTERT promoter is activated only in tumor cells having telomerase activity, it enables selective expression of viral genes downstream thereof in tumor cells, leading to selective viral growth.
We constructed an attenuated adenovirus type 5 vector (referred to as OBP-301 or telomerisin) in which the hTERT promoter sequence drives expression of the E1A and E1B genes linked to an internal ribosome binding site (IRES) (Kawashima) T, et al. Clin Cancer Res 2004; 10: 285-292). Telomerisin proliferated efficiently in human cancer cells and induced significant cell damage in human cancer cell lines. On the other hand, in normal human cells having no telomerase activity, not only cell damage but also proliferation was strongly suppressed.
OBP-301 monotherapy is under clinical development based on excellent results in preclinical studies, but in order to obtain good therapeutic results, multidisciplinary studies to promote antitumor effects in vivo Treatment is important. In fact, many clinical trials for oncolytic viruses have been performed in combination with chemotherapy or radiation therapy (Kuri FR, et al. Nat Med 2000; 6: 879-885, Reid T, et al. Cancer Res 2002; 62: 6070-6079, Hecht JR, et al. Clin Cancer Res 2003; 9: 555-561, Galanis E, et al. Gene Ther 2005; 12: 437-445).
Ionizing radiation primarily targets DNA molecules and induces double-strand breaks (DSB) (Kim CH, et al. J Pharmacol Exp Ther. 2002; 303: 753-9). Double strand breaks can be repaired by conventional molecular processes such as non-homologous end joining (NHEJ), homologous recombination (Chill D, et al. Front Biosci. 2006; 11: 1958-76).
DNA-dependent protein kinase (DNA-PK) is a nuclear protein having serine / threonine kinase activity and an essential component in the NHEJ pathway. DNA-PK contains the catalytic subunit DNA-PKcs and the regulatory subunit Ku autoantigen (Ku70 / 80) (Gottlieb ™, et al. Cell. 1993; 72: 131-42, Jeggo PA. Mutat. Res. 1997; 384: 1-14). Cell viability for induction of DSB is highly dependent on DNA repair by NHEJ (Wang H, et al. Nucleic Acids Res. 2001; 29: 1653-60). If the DNA is not repaired, DNA damage can cause chromosome loss and cell death (Dikomey E, et al. Int J Radiat Biol. 1998; 73: 269-78). Therefore, DNA-PK may be important for human tumors to acquire resistance to radiation therapy and genotoxic chemotherapy. Indeed, tumor cells deficient in the expression of either DNA-PKcs or Ku show significant radiosensitivity (Lees-Miller SP, et al. Science. 1995; 267: 1183-5) and DNA-PK Target inhibition increases the sensitivity of drug-resistant cancer cells and promotes cytotoxicity (Kim CH, et al. J Pharmacol Exp Ther. 2002; 303: 753-9). In addition, Ku70 / 80 translocates to the nucleus and physically interacts with DNA-PKcs during DSB repair (Koike M, et al. J Cell Sci. 1999; 112: 4031-9, Koike M, et al. Oncogene. 1999; 18: 7495-505, Koike M, et al. Biochem Biophys Res Commun. 2000; 276: 1105-11).

An object of the present invention is to provide a radiosensitization enhancer that enables effective radiotherapy of tumors.
As a result of intensive studies to solve the above problems, the present inventor has found that an attenuated adenovirus type 5 vector (referred to as OBP-301 or telomerisin) containing the hTERT promoter, IRES, E1A and E1B genes is sensitive to cancer radiation. The inventors have found that it is possible to increase and enhance the therapeutic effect of radiation, and have completed the present invention.
That is, the present invention is as follows.
(1) A radiosensitization enhancer comprising a recombinant virus in which a replication cassette comprising a human telomerase promoter, E1A gene, IRES sequence and E1B gene in this order is incorporated in the E1 region of the viral genome.
(2) Human telomerase promoter, E1A gene, IRES sequence, and replication cassette containing E1B gene in this order are incorporated into the E1 region of the viral genome, and a gene encoding a marker protein and a promoter capable of controlling the expression of the gene A radiosensitization enhancer comprising a recombinant virus in which a label cassette comprising is incorporated into the E3 region of the viral genome.
(3) A replication cassette comprising a human telomerase promoter, an E1A gene, an IRES sequence, and an E1B gene in this order, which is administered at least one time selected from before irradiation, after irradiation and during irradiation. An antitumor agent for combined use with radiation, comprising a recombinant virus incorporated into the E1 region of the viral genome.
(4) A replication cassette containing a human telomerase promoter, an E1A gene, an IRES sequence, and an E1B gene in this order, which is administered at least one time selected from before, after and during irradiation. Incorporated in the E1 region of the virus genome, and a recombinant virus comprising a recombinant virus in which a marker cassette containing a gene encoding a marker protein and a promoter capable of controlling the expression of the gene is incorporated in the E3 region of the virus genome Antitumor agent.
(5) A set in which a replication cassette containing a human telomerase promoter, an E1A gene, an IRES sequence, and an E1B gene in this order is incorporated in the E1 region of the viral genome in the production of the radiosensitizing enhancer or the antitumor agent for combined use with radiation. Use of replacement viruses.
(6) a replication cassette containing a human telomerase promoter, an E1A gene, an IRES sequence, and an E1B gene in this order in the production of the radiosensitizing enhancer or the antitumor agent for combined use with radiation is incorporated in the E1 region of the viral genome; Use of a recombinant virus in which a marker cassette comprising a gene encoding a marker protein and a promoter capable of controlling the expression of the gene is incorporated in the E3 region of the virus genome.
(7) A therapeutic agent for a radiation resistant tumor, comprising the recombinant virus.
In the present invention, the sensitization enhancer or antitumor agent can increase the sensitivity of a tumor or cancer to radiation. Examples of the promoter of human telomerase include the hTERT promoter, and examples of the promoter capable of controlling the expression of the gene encoding the labeled protein include the cytomegalovirus promoter or the hTERT promoter, and the oncogenic and tumor suppressor promoters. Examples of the labeled protein include GFP (Green Fluorescence Protein). The virus is preferably an adenovirus.
According to the present invention, when a tumor or cancer cell is infected with a recombinant virus of the present invention such as OBP-301 in cancer radiotherapy, the virus can increase the sensitivity of tumor cells to radiation. In addition, when a recombinant virus of the present invention such as OBP-301 is infected after irradiation of tumor or cancer cells in advance, the virus can effectively kill tumor cells. Furthermore, the recombinant virus of the present invention can effectively kill tumors or cancer cells that are resistant to radiation and anticancer agents. Therefore, the virus used in the present invention can exhibit a remarkable antitumor effect by increasing the sensitivity of radiation, and is extremely useful for combination therapy with radiation.

FIG. 1A is a diagram showing the in vitro antitumor effect of a combination of telomerase-specific oncolytic adenovirus and radiation in human cancer cells. Cytotoxic activity is demonstrated in a dose dependent or dose dependent manner with OBP-301 infection and ionizing radiation.
FIG. 1B is a graph showing the in vitro antitumor effect of a combination of telomerase-specific oncolytic adenovirus and radiation in human cancer cells. Radiosensitization enhancing effect is shown by infection of human cancer cells with OBP-301.
FIG. 1C is a diagram showing the in vitro antitumor effect of telomerase-specific oncolytic adenovirus in combination with radiation in human cancer cells. The figure shows a phase contrast image (magnification x100) of human cancer cells after OBP-301 and / or radiation treatment.
FIG. 1D is a diagram showing the in vitro antitumor effect of a combination of telomerase-specific oncolytic adenovirus and radiation in human cancer cells. The figure shows in vitro growth and spread (magnification x100) of virus in human cancer cells.
FIG. 2A is a diagram showing an evaluation of the oncolytic adenovirus growth in irradiated human cancer cells. The results of flow cytometric analysis of CAR expression in human cancer cells are shown.
FIG. 2B shows an assessment of oncolytic adenovirus growth in irradiated human cancer cells. Shown is the expression of adenovirus E1A protein following OBP-301 infection in irradiated human cancer cells.
Upper panel: 45 kD adenovirus E1A protein, lower panel: 42 kD actin as a loading control.
FIG. 2C shows an assessment of oncolytic adenovirus growth in irradiated human cancer cells. The growth efficiency of OBP-301 in irradiated human cancer cells is shown.
FIG. 3A shows the effect of ionizing radiation on the infectivity of adenovirus in human cancer cells. GFP fluorescence expression after infection of non-proliferating GFP-expressing adenoviral vector (Ad-GFP) in irradiated human cancer cells is shown.
Microscopic observation in bright field (upper panel), and GFP expression 48 hours after treatment (lower panel) (magnification × 200).
FIG. 3B shows the effect of ionizing radiation on the infectivity of adenovirus in human cancer cells. Shown are the results of flow cytometry analysis of the level of GFP expression after Ad-GFP infection in irradiated A431 and A549 cells.
FIG. 3C shows the effect of ionizing radiation on the infectivity of adenovirus in human cancer cells. The comparative analysis results of the infection efficiency of OBP-301 in A431, A549 and TE8 cells are shown.
FIG. 4A shows the effect of OBP-301 infection on the expression and localization of DNA-PK protein complexes in human cancer cells. The expression of DNA-PKcs in A549 cells after OBP-301 infection is shown.
Upper panel: 350 kD adenovirus E1A protein, lower panel: 42 kD actin as a loading control.
FIG. 4B shows the effect of OBP-301 infection on the expression and localization of DNA-PK protein complexes in human cancer cells. Intracellular localization of Ku protein in A549 cells infected with OBP-301 is shown.
Ku protein is shown in green and PI-stained DNA is shown in red (magnification x1000).
FIG. 5A is a diagram showing the antitumor effect of intratumoral injection of OBP-301 and local irradiation on tumors when xenografted A549 was established in the flank of nu / nu mice. ^* P <0.05, ^** p <0.01 (Student's t test).
FIG. 5B is a diagram showing the antitumor effect of intratumoral injection of OBP-301 and local irradiation on tumors when xenografted A549 was transplanted into the flank of nu / nu mice.
Shown are representative gross findings of the tumor 37 days after treatment.
FIG. 5C is a diagram showing the antitumor effect of intratumoral injection of OBP-301 and local irradiation on tumors when xenografted A549 was established in the flank of nu / nu mice. The results of hematoxylin and eosin staining of tumor parafilm sections are shown (magnification × 200 (upper panel), × 400 (lower panel)).
FIG. 6 is a diagram showing an outline of a procedure for producing a radiation resistant strain.
FIG. 7 is a diagram showing the results of analyzing the ratio of CD133 positive cells in the parent strain and the radiation resistant strain by flow cytometry.
a: It is a figure which shows the result of having analyzed the presence of CD133 positive cell in a parent strain and a radiation resistant strain by the flow cytometry using CD133-APC antibody.
b: A graph showing the ratio of CD133 positive cells in the parent strain and the radiation resistant strain.
FIG. 8 is a diagram showing the results of analyzing the purity of sorted cell groups by flow cytometry after sorting CD133-positive cells and CD133-negative cells in a radiation resistant strain by FACS.
FIG. 9 is a diagram showing the results of analyzing the self-replication ability (repopulation / self-renewability) of CD133 positive cells and CD133 negative cells by flow cytometry.
FIG. 10 is a diagram showing the results of analyzing the self-replication ability of CD133 positive cells and CD133 negative cells in the presence of leukemia inhibitory factor by flow cytometry in the presence of leukemia inhibitory factor.
FIG. 11 is a diagram showing the undifferentiated sphere formation of CD133 positive cells.
FIG. 12 is a diagram showing the results of analyzing the ratio of CD133 positive cells and CD133 negative cells contained in the sphere by flow cytometry.
FIG. 13 is a diagram showing the results of analysis of anti-cancer drug (5-FU) resistance of CD133 positive cells and CD133 negative cells by XTT assay. Statistical significance (p <0.05) is indicated with an asterisk.
FIG. 14 is a diagram showing the results of analysis of anti-cancer drug (cisplatin) resistance of CD133 positive cells and CD133 negative cells by XTT assay. Statistical significance (p <0.05) is indicated with an asterisk.
FIG. 15 is a diagram showing the results of analyzing the resistance of anti-cancer agent (paclitaxel) in CD133 positive cells and CD133 negative cells by XTT assay. Statistical significance (p <0.05) is indicated with an asterisk.
FIG. 16 is a diagram showing the results of analyzing the radioresistance of CD133 positive cells and CD133 negative cells by XTT assay. Statistical significance (p <0.05) is indicated with an asterisk.
FIG. 17 shows the results of analyzing the antitumor effect of telomerisin on CD133 positive cells and CD133 negative cells by XTT assay.
FIG. 18 is a diagram showing a comparison of hTERT expression levels in CD133 positive cells and CD133 negative cells.

Hereinafter, the present invention will be described in detail. The following embodiment is an example for explaining the present invention, and is not intended to limit the present invention to this embodiment alone. The present invention can be implemented in various forms without departing from the gist thereof.
It should be noted that all documents cited in the present specification, as well as published publications, patent gazettes, and other patent documents are incorporated herein by reference. In addition, the present specification includes the description and the contents of the Japanese patent application (Japanese Patent Application No. 2007-11985), which is the basis of the priority claim of the present application filed on April 27, 2007.
1. Overview
Oncolytic adenoviruses have been previously described to kill tumor cells through a different mechanism than standard cancer treatments and synergistic interactions with common treatment modalities. However, the molecular mechanism behind the synergistic effects of combination therapy has not been fully elucidated.
Therefore, the present inventors constructed a telomerase-specific growth adenovirus (referred to as OBP-301 or telomerisin) having a human telomerase reverse transcriptase (hTERT) promoter capable of regulating virus growth, and synergistic combination therapy. The effect was examined.
As an embodiment of the present invention, as shown in the Examples, the antitumor effect of OBP-301 infection and ionizing radiation combined use was evaluated in vitro by XTT assay. Adenovirus infectivity and intracellular proliferation were assessed by quantitative real-time PCR. The effects of OBP-301 on the expression of DNA protein kinase (PK) catalytic subunit (DNA-PKcs) and the nuclear translocation of Ku70 / 80 were examined by Western blotting and immunofluorescence staining, respectively. In addition, the therapeutic effect on the growth of human subcutaneous tumors implanted in nu / nu mice was compared by macroscopic observation and histochemical staining.
As a result, when OBP-301 was infected after irradiation, significant in vitro cell damage was induced. Irradiation did not affect the intracellular growth rate of OBP-301, but promoted cytotoxicity by OBP-301. Moreover, when OBP-301 was infected after irradiation, DNA-PKcs expression decreased in A549 which is a human lung cancer cell. Ku70 / 80 protein accumulated in the nucleus of A549 cells after ionizing radiation, but accumulated in the cytoplasm after infection with OBP-301. When combined with intracellular injection of OBP-301 and local radiation, synergistically significant therapeutic effect was obtained in nu / nu mice xenografted with A549 tumor, and the effect was even greater than when each was treated alone It was expensive.
From the above, the present inventors, when ionizing radiation irradiation and OBP-301 infection are used in combination, increase the infectivity of the virus by ionizing radiation irradiation, inhibit DNA repair by infection with OBP-301, As a result, they found that both increase the sensitivity of tumor cells. This combination therapy promoted anti-tumor effects both in vitro and in vivo.
Furthermore, as a result of intensive studies focusing on CD133, which is a specific marker of cancer stem cells that brings about malignant tumor or radiation resistance, the present inventors have found that the virus of the present invention is resistant to radiation and anticancer drugs. It was found to have an antitumor effect on CD133 positive cells having
2. Recombinant virus of the present invention
The recombinant virus of the present invention refers to a virus in which a polynucleotide containing a human telomerase promoter, an E1A gene, an IRES sequence, and an E1B gene in this order is incorporated into the genome. The virus to be used is not particularly limited, but adenovirus is preferable from the viewpoint of safety and the like. Among adenoviruses, type 5 adenovirus is particularly preferable from the viewpoint of ease of use.
In the recombinant virus used in the present invention, the E1A gene, the IRES sequence and the E1B gene are driven by a human telomerase promoter. Since the expression of telomerase is extremely high in tumor cells as compared with normal cells, the telomerase promoter is activated in tumor cells containing telomerase, and thus the recombinant virus included in the present invention grows. As described above, the recombinant virus contained in the pharmaceutical composition of the present invention does not grow on normal cells but grows only on tumor cells, so that it is propagated and replicated specifically for cancer cells and telomerase. . As a result, in the tumor cells, cell damage due to virus growth occurs, whereby the recombinant virus used in the present invention can specifically kill the tumor cells. Such a recombinant virus can be used as a radiosensitization enhancer or an antitumor agent for combined use with radiation in the present invention.
The “telomerase promoter” determines the transcription start site of telomerase and directly regulates its frequency. Telomerase is an enzyme that maintains telomere length by antagonizing shortening during replication of eukaryotic chromosomes. The type of telomerase promoter is not particularly limited as long as it is a suitable promoter that can correspond to the virus used for expression of the target gene. For example, human telomerase reverse transcription An enzyme (hTERT) promoter is preferred. hTERT is a region of 1.4 kbp upstream of its 5 ′ end, and many transcription factor binding sequences have been confirmed, and this region is considered to be the hTERT promoter. Among them, the sequence of 181 bp upstream of the translation start site is downstream. It is an important core region for gene expression. In the present invention, any core region can be used as long as it contains this core region. However, it is preferable to use an upstream sequence of about 378 bp completely including this core region as the hTERT promoter. This sequence of about 378 bp has been confirmed to have the same gene expression efficiency as compared to the case of the 181 bp core region alone. The nucleotide sequence of the 455 bp long hTERT promoter is shown in SEQ ID NO: 1. The base sequence of the 181 bp core region is shown in SEQ ID NO: 5.
The hTERT promoter is a DNA comprising a nucleotide sequence complementary to the DNA comprising the nucleotide sequence of SEQ ID NO: 1 or the core region (SEQ ID NO: 5) as well as the nucleotide sequence of the core region (SEQ ID NO: 5). A nucleotide sequence of a nucleotide that hybridizes under stringent conditions and has activity as an hTERT promoter is also included. Such a nucleotide is a known hybridization method such as colony hybridization, plaque hybridization, Southern blotting, etc. using a polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 1 or the nucleotide sequence of the core region, or a fragment thereof as a probe. Can be obtained from a cDNA library and a genomic library. For a method for preparing a cDNA library, “Molecular Cloning, A Laboratory Manual 2nd ed.” (Cold Spring Harbor Press (1989)) can be referred to. Commercially available cDNA libraries and genomic libraries may also be used. Examples of stringent conditions in the above hybridization include conditions of 1 × SSC to 2 × SSC, 0.1% to 0.5% SDS, and 42 ° C. to 68 ° C., and more specifically, 60 to An example is a condition in which pre-hybridization is performed at 68 ° C. for 30 minutes or more, followed by washing 4 to 6 times in 2 × SSC, 0.1% SDS at room temperature for 5 to 15 minutes. For the detailed procedure of the hybridization method, “Molecular Cloning, A Laboratory Manual 2nd ed.” (Cold Spring Harbor Press (1989); in particular, Section 9.47-9.58) and the like can be referred to. In addition, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 98% or more, or 99% or more homology with the nucleotide sequence shown in SEQ ID NO: 1 or the core region A nucleotide base sequence having nucleotides and having activity as an hTERT promoter can also be used.
In the present invention, the E1A gene, the IRES sequence, and the E1B gene are included in this order when the IRES sequence inserted between the E1A gene and the E1B gene is used when the virus infects the host cell. This is because the proliferation ability is increased. The E1A gene and the E1B gene are genes included in the E1 gene. The E1 gene is an early gene (early: E) and an early gene (late: L) related to DNA replication of the virus. One is a protein that is involved in the regulation of transcription of the viral genome. The E1A protein encoded by the E1A gene activates the transcription of genes (E1B, E2, E4, etc.) necessary for the production of infectable viruses. The E1B protein encoded by the E1B gene promotes viral replication by helping the late gene (L gene) mRNA accumulate in the cytoplasm of the infected host cell and inhibit host cell protein synthesis. . The base sequences of E1A gene and E1B gene are shown in SEQ ID NO: 2 and SEQ ID NO: 3, respectively.
E1A and E1B hybridize under stringent conditions with DNA consisting of a base sequence complementary to the DNA consisting of SEQ ID NO: 2 and SEQ ID NO: 3 in addition to the base sequences shown in SEQ ID NO: 2 and SEQ ID NO: 3, respectively. And a base sequence encoding a protein having activity as E1A and E1B, respectively. Such a base sequence is a known hybridization method such as colony hybridization, plaque hybridization, Southern blotting, etc., using a polynucleotide comprising the base sequences represented by SEQ ID NO: 2 and SEQ ID NO: 3 or a fragment thereof as a probe. Can be obtained from a cDNA library and a genomic library. The method for preparing the cDNA library and the stringent conditions are the same as described above. Further, it has a homology of 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 98% or more, or 99% or more with the base sequence shown in SEQ ID NO: 2 or 3. In addition, nucleotide base sequences having activity as E1A and E1B can also be used.
IRES (Internal Ribosome Entry Site) is a protein synthesis initiation signal specific to the Picornaviridae family and is thought to play a role as a ribosome binding site because it has a sequence complementary to the 3 'end of 18S ribosomal RNA. It has been. It is known that mRNA derived from the virus of the Picornaviridae family is translated through this sequence. The translation efficiency from the IRES sequence is high, and protein synthesis is performed independent of the cap structure even in the middle of mRNA. Therefore, in this virus, both the E1A gene and the E1B gene downstream of the IRES sequence are independently translated by the human telomerase promoter. When IRES is used, the expression of the telomerase promoter is controlled independently of the E1A gene and the E1B gene. Therefore, compared to the case where either the E1A gene or the E1B gene is controlled by the telomerase promoter, the virus growth is increased. More strictly, it can be limited to cells having telomerase activity. The IRES sequence is shown in SEQ ID NO: 4.
In addition to the nucleotide sequence shown in SEQ ID NO: 4, IRES is a protein that hybridizes under stringent conditions with DNA consisting of a complementary nucleotide sequence to DNA consisting of SEQ ID NO: 4 and has activity as an IRES A nucleotide sequence encoding is also included. Such a nucleotide sequence can be obtained by using a polynucleotide library comprising the nucleotide sequence represented by SEQ ID NO: 4 or a fragment thereof as a probe by a known hybridization method such as colony hybridization, plaque hybridization, Southern blotting, etc. And can be obtained from a genomic library. The method for preparing the cDNA library and the stringent conditions are the same as described above. Further, it has 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 98% or more or 99% or more homology with the nucleotide sequence shown in SEQ ID NO: 4, and IRES Nucleotide base sequences having activity as can also be used.
In the present invention, the human telomerase promoter is located upstream of the E1 gene. This is because proliferation can be promoted in cells having telomerase activity.
The gene contained in the recombinant virus of the present invention can be obtained by ordinary genetic engineering techniques. For example, a nucleic acid synthesis method using a DNA synthesizer generally used as a genetic engineering technique can be used. In addition, after isolating or synthesizing a gene sequence as a template, a primer method specific to each gene is designed, and the gene sequence is amplified using a PCR apparatus (Current Protocols in Molecular Biology, John Wiley & Sons (1987) Section 6.1-6.4) or gene amplification methods using cloning vectors can be used. The above method is described in Molecular cloning 2nd Edt. According to Cold Spring Harbor Laboratory Press (1989), it can be easily performed by those skilled in the art. A known method can be used to purify the obtained PCR product. For example, a method using ethidium bromide, a method using SYBR Green I (Molecular probes), a method using agarose gel by GENECLEAN (Funakoshi), QIAGEN (QIAGEN), a method using DEAE-cellulose filter paper, a freeze & squeeze method, There is a method using a dialysis tube. When an agarose gel is used, it is subjected to agarose gel electrophoresis, and a DNA fragment is cut out from the agarose gel and purified. If necessary, it can be confirmed that the expected gene is obtained by a conventional sequencing method. For example, a dideoxynucleotide chain termination method (Sanger et al. (1977) Proc. Natl. Acad. Sci. USA 74: 5463) can be used. It is also possible to analyze the sequence using an appropriate DNA sequencer (for example, ABI PRISM (Applied Biosystems)).
Thereafter, the genes obtained as described above are ligated in a predetermined order. First, each of the above genes is cleaved with a known restriction enzyme or the like, and the cleaved DNA fragment of the gene is inserted into a known vector according to a known method and ligated. Known vectors include encephalomyocarditis virus (ECMV) IRES (ribosome binding site inside mRNA), and can translate two open reading frames (ORFs) from one kind of mRNA. In addition to pIRES vectors, plasmids derived from E. coli (pCR4, pCR2, pCR2.1, pBR322, pBR325, pUC12, pUC13, etc.), plasmids derived from Bacillus subtilis (pUB110, pTP5, pC194, etc.), yeast-derived plasmids (pSH19, pSH15, etc.) ), Bacteriophage such as λ phage, animal viruses such as retrovirus, vaccinia virus, baculovirus, etc., and pA1-11, pXT1, pRc / CMV, pRc / RSV, pcDNAI / Neo Domo used. In the case of the present invention, when a pIRES vector is used, a recombinant gene containing “human telomerase promoter, E1A gene, IRES sequence and E1B gene” in this order is prepared by sequentially inserting necessary genes into the multicloning site. This is preferable. DNA ligase can be used for DNA ligation. For example, electroporation method, liposome method, spheroplast method, lithium acetate method or the like can be used for incorporation of the recombinant gene into a virus.
In the present invention, the case where hTERT is used as human telomerase will be specifically described below.
E1A gene by performing RT-PCR and / or DNA-PCR using primers such as E1A-S, E1A-AS, E1B-S, E1B-AS from cells expressing E1 gene such as 293 cells The E1B gene is amplified and, if necessary, the sequence is confirmed using a known method such as TA cloning, and then the E1A and E1B DNA fragments can be excised with a known restriction enzyme.
Next, the gene consisting of hTERT-E1A-IRES-E1B used in the present invention is placed in a known vector (eg, pIRES) in the order of “E1A-IRES-E1B”, and the multiple cloning sites, etc. It can produce by inserting using. Subsequently, the hTERT promoter sequence excised with restriction enzymes such as MluI and BglIII can be inserted upstream of E1A.
If necessary, the cytomegalovirus (CMV) promoter contained in a known vector such as pShuttle is removed with an appropriate restriction enzyme such as MfeI or NheI, and the restriction enzyme NheI or NotI is removed from that site from phTERT-E1A-IRES-E1B. The cut out sequence can be inserted. Thus, an adenovirus incorporating a cassette consisting of hTERT-E1A-IRES-E1B used in the present invention is particularly referred to as “telomerisin” or “OBP-301”. By expressing the E1 gene necessary for the growth of adenovirus under the control of the hTERT promoter, the virus can be propagated specifically in cancer cells.
In order to infect a cell with the recombinant virus, for example, it can be infected by the following method. First, cells such as human colon cancer cells SW620, human lung cancer cells A549 and H1299 are seeded on a culture plate containing an appropriate culture solution, and cultured at 37 ° C. in the presence of carbon dioxide gas. As the culture solution, DMEM, MEM, RPMI-1640 or the like generally used for animal cell culture is adopted, and serum, antibiotics, vitamins and the like can be added as necessary. The cultured cells are infected by inoculating a certain amount of the virus, for example, 0.1 to 10 MOI (multiplicity of infection), preferably 0.1 to 1 MOI. MOI refers to the ratio of the amount of virus (infectious unit) to the number of cells when a certain amount of cultured cells is infected with a certain amount of virus particles, and is used as an index when a cell is infected with a virus.
In order to confirm viral growth, virus-infected cells can be collected, DNA can be extracted, and quantitative analysis can be performed by performing real-time PCR using a primer targeting an appropriate gene of the present virus.
3. Telomerisin-GFP
In the present invention, a replication cassette containing a human telomerase promoter, an E1A gene, an IRES sequence, and an E1B gene in this order is incorporated into the E1 region of the viral genome, and a gene encoding a marker protein and the expression of the gene can be controlled. A recombinant virus in which a labeled cassette containing a promoter is incorporated into the E3 region of the viral genome is provided as a radiosensitization enhancer or an antitumor agent for combined use with radiation. The labeling cassette includes a promoter and a gene encoding a labeling protein. Examples of the promoter include a CMV (cytomegalovirus) promoter (SEQ ID NO: 6) and an hTERT promoter (SEQ ID NO: 1). Alternatively, carcinogenesis and tumor suppressor promoters can be used. The “oncogenic and tumor suppressor promoter” means a promoter of a factor whose expression is enhanced or attenuated in cancer cells, and examples thereof include a p16 promoter and a DR5 promoter.
These promoters drive the gene encoding the labeled protein.
In the present invention, a recombinant virus that uses GFP (Green fluorescence protein) as a gene encoding a labeled protein is referred to as “telomerisin-GFP”.
The recombinant gene contained in the telomerisin-GFP labeling cassette can be obtained by ordinary genetic engineering techniques. For example, a nucleic acid synthesis method using a DNA synthesizer generally used as a genetic engineering technique can be used. In addition, after isolating or synthesizing a gene sequence as a template, a primer method specific to each gene is designed, and the gene sequence is amplified using a PCR apparatus (Current Protocols in Molecular Biology, John Wiley & Sons (1987) Section 6.1-6.4) or gene amplification methods using cloning vectors can be used. The above method is based on Molecular cloning 2 ^nd Edt. According to Cold Spring Harbor Laboratory Press (1989) and the like, it can be easily performed by those skilled in the art. A known method can be used to purify the obtained PCR product. For example, a method using ethidium bromide, a method using SYBR Green I (Molecular probes), a method using agarose gel by GENECLEAN (Funakoshi), QIAGEN (QIAGEN), a method using DEAE-cellulose filter paper, a freeze & squeeze method, There is a method using a dialysis tube. When an agarose gel is used, it is subjected to agarose gel electrophoresis, and a DNA fragment is cut out from the agarose gel and purified. If necessary, it can be confirmed that the expected gene is obtained by a conventional sequencing method. For example, a dideoxynucleotide chain termination method (Sanger et al. (1977) Proc. Natl. Acad. Sci. USA 74: 5463) can be used. It is also possible to analyze the sequence using an appropriate DNA sequencer (for example, ABI PRISM (Applied Biosystems)), and then cleave the gene obtained as described above with a known restriction enzyme or the like. Then, the recombinant gene is designed so that the gene fragment encoding the promoter is arranged upstream of the gene fragment encoding the labeled protein so that the cut DNA fragment of the gene can be driven. At this time, the plasmid such as shuttle plasmid pHM11 can be used as a plasmid, and both genes are ligated using DNA ligase and inserted into a vector to prepare a labeled cassette recombinant gene.
Known vectors include pShuttle vectors, E. coli-derived plasmids (pCR4, pCR2, pCR2.1, pBR322, pBR325, pUC12, pUC13, etc.), Bacillus subtilis-derived plasmids (pUB110, pTP5, pC194, etc.), yeast-derived plasmids ( pSH19, pSH15, etc.), bacteriophages such as λ phage, animal viruses such as retrovirus, vaccinia virus, baculovirus, etc., as well as pA1-11, pXT1, pRc / CMV, pRc / RSV, pcDNAI / Neo, etc. .
Thereafter, the recombinant gene containing the replication cassette and the labeling cassette is excised using an appropriate restriction enzyme and inserted into an appropriate viral expression vector, whereby a recombinant virus can be produced. Examples of the viral expression vector include adenovirus, retrovirus, vaccinia virus, baculovirus, etc. As mentioned above, adenovirus, particularly type 5 adenovirus is preferred. For incorporation of the cassette into the virus, for example, an electroporation method, a liposome method, a spheroplast method, a lithium acetate method, or the like can be used.
In the present invention, specifically, CMV-EGFP-SV40P (A) derived from pEGFP-N1 (CLONTECH) was inserted into shuttle plasmid pHM11, and the Csp451 fragment of this plasmid was incorporated into phTERT-E1A-IRES-E1B. A telomerisin-GFP recombinant gene can be obtained by inserting into the ClaI site of the pShuttle vector.
In the present invention, specifically, a necessary sequence is excised from a recombinant gene prepared as described above using a known restriction enzyme, and a commercially available kit such as Adeno-X Expression System (CLONTECH) is used. It can be inserted into viral DNA such as -X viral DNA (the resulting product is referred to as "AdenoX-hAIB").
Then, this AdenoX-hAIB is linearized with a known restriction enzyme and then transfected into cultured cells such as 293 cells, whereby an infectious recombinant virus can be produced.
4). Radiosensitization enhancer
In the present specification, enhancement of radiosensitization is either one of enhancing the effect of radiotherapy and increasing the sensitivity of the tumor to radiation so that the tumor can be effectively killed when used in combination with radiation. Or both. Therefore, the use mode of the radiosensitization enhancer of the present invention became the form administered to the tumor before irradiation, the form administered to the tumor during radiation irradiation, the form administered to the tumor after radiation irradiation, and the radiation resistance. It includes any form administered to the tumor and includes combinations of these forms. As such a drug, telomerisin or telomerisin-GFP is used in the present invention. Examples of “radiation” include ionizing radiation, radiation generated from particle beams and radioisotopes, such as X-rays, γ-rays, α-rays, β-rays, electron beams, proton beams, and neutron beams. Is a line. Examples of organisms to be treated include mammals such as humans, mice, rats, hamsters, guinea pigs, rabbits, cats, dogs, pigs, cows, horses, goats, monkeys, or a part thereof. It is not limited to these.
In the present invention, all kinds of tumors can be used as a tumor to be radiosensitized. For example, solid cancer in head and neck, stomach, large intestine, lung, liver, prostate, pancreas, esophagus, bladder, gallbladder / bile duct, breast, uterus, thyroid, ovary, etc., or leukemia, lymphoma, sarcoma, mesenchymal tumor, etc. It is valid. It is also effective for basal cell carcinoma of the skin and cancer stem cells. Most of the tumor cells and cancer stem cells derived from human tissues show an increase in telomerase activity, and the recombinant virus of the present invention can generally act on tumor cells whose proliferation has been activated by such telomerase activity.
Recent research has revealed that some types of tumors have cells called cancer stem cells. It is known that cancer stem cells have a tumor remodeling ability, malignant tumors, and bring resistance to radiation and anticancer drugs. In addition, cancer stem cells are known to express a specific marker, CD133, on the cell surface, and it has been clarified that they can be distinguished from other tumor cells.
In the present invention, the tumor cells to be subjected to radiosensitization include those having radiation resistance or anticancer drug resistance.
As described above, telomerase expression is extremely high in tumor cells compared to normal cells. Therefore, the recombinant virus of the present invention does not grow on normal cells but grows only on tumor cells, and as a result, tumor cells can be specifically killed. And since the sensitivity with respect to the radiation of a tumor cell increases by the infection of a virus, a tumor can be killed more effectively.
In general, the irradiation amount when irradiating with radiation is considered to be 60 to 70 Gy as a standard irradiation amount, but can be appropriately changed depending on the irradiation site. In addition, it is possible to appropriately select a dose range from the upper and lower dose ranges. The recombinant virus of the present invention increases the sensitivity of tumors to radiation and thus requires less than the dose used in general radiation therapy for cancer.
5. Pharmaceutical composition
The pharmaceutical composition containing the recombinant virus of the present invention can be used as an antitumor agent for use in combination with irradiation, or as a therapeutic agent for a tumor that has become resistant to radiation, in addition to the above-described radiosensitizing enhancer. .
This is because the mechanism of cell death that telomerisin exerts on tumor cells is different from the mechanism of apoptosis caused by conventional anticancer drugs, and telomerisin is treated with a substance that has antitumor effects. This is achieved by the property of telomerisin that even if it is applied, it can proliferate and replicate in the cell without any influence.
The pharmaceutical composition of the present invention can be applied to the affected area as it is, or any known method, for example, injection such as intravenous, intramuscular, intraperitoneal or subcutaneous, inhalation from the nasal cavity, oral cavity or lung, oral administration, It can also be introduced into a living body (target cell or organ) by intravascular administration using a catheter or the like. Furthermore, the recombinant virus contained in the pharmaceutical composition of the present invention can be used in combination with other substances having other antitumor effects (other antitumor agents). In this case, the recombinant virus and the other antitumor agent can be administered at the same time, or can be introduced into the living body by a method in which one is administered and the other is administered after a lapse of a certain time.
Further, for example, it may be used as it is after being easily handled by a method such as freezing, or it may be used as it is, or a known pharmaceutically acceptable carrier such as an excipient, a bulking agent, a binder or a lubricant, a known additive (buffer) Agents, tonicity agents, chelating agents, coloring agents, preservatives, fragrances, flavoring agents, sweetening agents, etc.).
The pharmaceutical composition of the present invention includes tablets, capsules, powders, granules, pills, solutions, syrups and other oral administration agents, injections, external preparations, suppositories, ophthalmic preparations and the like. Depending on the form, it can be administered orally or parenterally. Preferably, local injection into muscle, abdominal cavity, etc. (particularly intratumoral injection), intravenous injection, and the like are exemplified.
The dosage is appropriately selected according to the type of active ingredient, administration route, administration subject, patient age, body weight, sex, symptom, and other conditions. 10 ⁶ -10 ¹¹ About PFU (place forming units), preferably 10 ⁹ -10 ¹¹ It may be about PFU and can be administered once a day or divided into several times. Further, when using the virus of the present invention, by using a known immunosuppressive agent or the like, the immunity of the living body can be suppressed and the virus can be easily infected.
Here, in the present invention, “combination” means a combination of administration of the recombinant virus (pharmaceutical composition) of the present invention and irradiation, but the administration time of the virus of the present invention is the time of irradiation. Regardless, before irradiation, after irradiation, or during irradiation, it is possible to arbitrarily combine these periods. In the case of administration after irradiation, the administration time is immediately after irradiation within 48 hours, preferably immediately after within 24 hours. However, the pharmaceutical composition of the present invention can also be administered to a patient who cannot obtain a therapeutic effect by irradiation (for example, a patient having a tumor that is resistant to irradiation). In this case, the administration time of the recombinant virus of the present invention is not limited, and can be administered even if a considerable amount of time or days have passed since the previous irradiation time. Moreover, when administering before irradiation, the administration time is 24 to 96 hours before irradiation, Preferably it is 48 to 72 hours before. Furthermore, the treatment interval and the number of treatments between irradiation and pharmaceutical composition administration can be appropriately set according to the size of the tumor and the clinical symptoms of the patient. For example, it is possible to irradiate radiation daily for 2 to 7 days (preferably 5 days) while adjusting the total dose, and to administer the pharmaceutical composition several times during the irradiation period.
The pharmaceutical composition of the present invention is considered to have a very low possibility of causing side effects for the following reasons, and can be said to be a very safe preparation.
(1) Normal somatic cells have little telomerase activity, and floating cells such as hematopoietic cells are less susceptible to infection with the virus of the present invention.
(2) Since the virus of the present invention has a proliferation ability, it can be used at a lower concentration than a non-proliferative virus used in normal gene therapy.
(3) Even when the virus of the present invention is excessively administered, the antiviral action is exerted by the normal immune action in vivo.
(4) Since the sensitivity of irradiation can be increased, the antitumor effect can be obtained even if the radiation dose and / or the virus dose of the present invention is reduced.
Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.

The present inventors examined the radiosensitizing effect on the expression of DNA-PKcs and the intracellular localization of Ku70 / 80 in human cancer cells when the tumor cells were infected with oncolytic adenovirus. In addition, the present inventors examined whether adenovirus uptake in target cells is changed by ionizing radiation. Furthermore, the therapeutic effect of oncolytic virus therapy when combined with radiotherapy was examined both in vitro and in vivo.
1. Materials and methods
(1) Cell lines and cell culture
A549, which is a human non-small cell lung cancer (NSCLC) cell line, and A431, a human epidermoid cancer cell line, are Dulbecco's modified Eagle medium containing Nutrient Mixture (Ham's F-12) supplemented with 10% fetal calf serum (FCS). DMEM). Another human NSCLC cell line, H1299, and human esophageal cancer cell line, TE8, are 10% FCS, 25 mM N-2-hydroxyethylpiperazine-N′-ethanesulfonic acid (HEPES), 100 units / ml penicillin, and 100 μg. The cells were grown in a normal manner by monolayer culture in RPMI 1640 medium supplemented with / ml streptomycin.
(2) Recombinant adenovirus
Recombinant adenoviral vector, OBP-301 (telomerici), which selectively grows in a tumor-specific manner has been previously constructed and characterized (Kawashima T, et al. Clin Cancer Res 2004; 10: 285-292). Taki M, et al. Oncogene.2005; 24: 3130-40, Umeoka T, et al. Cancer Res.2004; 64: 6259-65, Watanabe T, et al.Exp Cell Res.2006; 312: 256- 65). In this example, selective proliferative OBP-401 (Fujiwara T, et al. Int J Cancer. 2006; 119: 432, which contains a cDNA encoding green fluorescent protein (GFP) under the control of the cytomegalovirus promoter. -40, Kishimoto H, et al. Nature Med (in press), 2006.), and a non-proliferating E1-deleted adenoviral vector (Ad-GFP) containing cDNA encoding GFP (Umeoka T, et al. Cancer Res. 2004; 64: 6259-65) was also used. These viruses are CsCl ₂ After purification by step gradient ultracentrifugation, CsCl ₂ Purified by linear gradient ultracentrifugation. Viral and infectious titers were measured by plaque assay using 293 cells.
(3) Cell viability measurement
A 96-well plate was seeded with human tumor cells at 1000 cells / well and exposed to radiation 12 hours later. Thereafter, an XTT (3 ′-[1- (phenylaminocarbonyl) -3,4-tetrazolium] -bis (4-methoxy-6-nitro) benzenesulfonate sodium hydrate) assay was performed. The cells were irradiated once with a predetermined dose of γ-rays (0 to 25 Gy) using MBR-1520R (Hitachi Medical Co., Tokyo, Japan). Immediately after the irradiation, the cells were infected with OBP-301 having a predetermined MOI (multiplicity of effect). Cell viability was treated with Cell Proliferation Kit II (Roche Molecular Biochemicals, Indianapolis, IN) according to the protocol provided by the manufacturer and measured after 5 days.
(4) Fluorescence microscope observation
After infection with Ad-GFP or OBP-401, the expression of the GFP gene in human cancer cells was evaluated using an Eclipse TS-100 fluorescence microscope (Nikon, Tokyo), and photographs were taken (magnification: × 200). .
(5) Flow cytometry
2 × 10 for flow cytometric analysis ⁵ Cells were labeled with anti-Coxacky adenovirus receptor (CAR) mouse monoclonal antibody (RmcB; Upstate Biotechnology, Lake Placid, NY) for 30 minutes at 4 ° C. and fluorescein isothiocyanate (FITC) -conjugated rabbit anti-mouse IgG Subsequent antibodies (Zymed Laboratories Inc., San Francisco, Calif.) Were added and incubated and analyzed using a FACSCalibur flow cytometer (Becton Dickinson, Mountain View, Calif.) And CELL Quest software. The window on the screen was set to exclude dead cells and debris. Expression of only the GFP gene was also evaluated by FACScallibur.
(6) Quantitative real-time PCR assay
The cell is 25cm ² 5x10 in flask ⁴ And the cells were irradiated 48 hours later. Cells were exposed to mock or a predetermined dose of gamma radiation and then infected with 1.0 MOI of OBP-301 for 2 hours. After removing the virus, the cells were incubated at 37 ° C. and treated with trypsin after 2, 24, 48, and 72 hours, respectively, and collected for intracellular proliferation analysis.
DNA was extracted using QIAamp DNA Mini Kit (Qiagen Inc., Valencia, Calif.), And quantitative real-time PCR assay was performed on the E1A gene using LightCycler instrument (Roche Molecular Biochemicals). The sequence of the specific primer used for E1A is as follows.
In amplification by PCR, after a denaturation step of 95 ° C. and 600 seconds, denaturation at 95 ° C., 10 seconds, 58 ° C., 15 seconds of annealing, and 72 ° C., 8 seconds of extension reaction were performed as 40 cycles. . Data was analyzed using LightCycler software (Roche Molecular Biochemicals). Values normalized by dividing by the value of untreated cells are shown for each sample.
(7) Western blot analysis
In this example, primary antibodies against E1A (PharMingen International, San Diego, Calif.), Primary antibodies against DNA-PKcs (BD Transduction Laboratories, Lexington, KY), and peroxidase-conjugated secondary antibodies (Amersham, IL) used. The cells were washed twice with cold phosphate buffered saline (PBS) and then recovered, followed by proteinase inhibitor (0.1 mM phenylmethylsulfonyl fluoride, 0.5 mM Na ₃ VO ₄ ) -Containing lysis buffer [10 mM Tris (pH 7.5), 150 mM NaCl, 50 mM NaF, 1 mM ethylenediaminetetraacetic acid (EDTA), 10% glycerol, 0.5% NP40]. The cell lysate was allowed to stand on ice for 20 minutes and then centrifuged at 14,000 rpm at 4 ° C. for 10 minutes. The supernatant was used as a whole cell extract. Protein concentration was measured using the Bio-Rad method (Bio-Rad, Richmond, CA). An equal amount of protein (50 μg) was boiled for 5 minutes and electrophoresed under reducing conditions using a 6-12.5% (w / v) polyacrylamide gel. The protein was transferred to a Hybond-polyvinylidene fluoride (PVDF) transfer membrane (Amersham Life Science, Buckinghamshire, UK), incubated with the primary antibody, and further incubated with a peroxidase-conjugated secondary antibody. The peroxidase activity of the bound antibody was detected using an Amersham ECL chemistry sequence system (Amersham, Tokyo).
(8) Immunofluorescence staining
Semi-confluent cells were treated on tissue culture chamber slides (Nunc, Inc., Naperville, IL), then fixed with acetone and washed 3 times with PBS. The slides were then incubated with a primary antibody against Ku70 / 80 (BD PharMingen, San Jose, Calif.) At 37 ° C. for 1 hour. The slides were washed three times with PBS and then incubated with a secondary antibody FITC-conjugated goat anti-mouse antibody (Immunotech, Co., Marseille, France) at 37 ° C. for 20 minutes. Further, the slide was washed three times with PBS, then stained with propidium iodide (PI) (Molecular Probes, Eugene, OR), and a buffered glycerol solution was mounted and used for observation with a fluorescence microscope.
(9) In vivo human tumor model
Human NSCLC cells A549 (2 × 10 ⁶ Cells / mouse) were injected subcutaneously into the flank of 5-6 week old female BALB / c nu / nu mice and allowed to grow to a diameter of approximately 5 mm. At that time, mice were randomly assigned to 4 groups and each mouse was irradiated every 3 days from day 1 at a dose of 3 Gy / tumor. Immediately after irradiation, 50 μl of OBP-301-containing solution (1 dose: 1 × 10 ⁸ Tumors were injected with PFU / individual) or PBS. The vertical diameter of each tumor was measured every 3 days, and the tumor volume was calculated using the following formula.
Tumor volume (mm ³ ) = A × b ² × 0.5
In the formula, a is the longest diameter, b is the shortest diameter, and 0.5 is a constant for calculating an elliptical volume. The experimental protocol was approved by the Okayama University Animal Experiment Ethics Committee.
(10) Statistical analysis
All data are expressed as mean ± SD. About the difference between groups, the statistical significance difference was examined using the Student's t test. A P value of less than 0.05 means a statistically significant difference.
2. result
(1) Anti-tumor effect in vitro by combined use of OBP-301 and radiation on human cancer cell lines
In order to examine the possibility of interaction between OBP-301 and ionizing radiation in vitro, the present inventors firstly prepared four types of human cancer cell lines derived from different organs (lung [A549, H1299], esophagus). [TE8] and epidermis [A431]) were evaluated for the effects of various doses or doses. Cells were irradiated with a single dose of γ-ray, then infected with either mock or OBP-301, and cell viability was assessed by XTT assay 5 days after treatment. In order to optimize the experimental conditions, the concentration of OBP-301 that caused 0-100% cell damage when OBP-301 was used alone was selected.
A representative dose or dose response curve is shown in FIG. 1A. By combining radiation, the cytotoxic activity of OBP-301 increased in a dose- or dose-dependent manner in all cell lines used in the experiment. In FIG. 1A, data is shown as the ratio of viable cells to mock treated cells and is shown as the mean ± SD of triplicate test.
Cell viability of A431 and A549 cells was assessed 5 days after treatment. In A431 cells, 10 MOI of OBP-301 infection resulted in a 5.7-fold increase in the cytotoxicity of ionizing radiation (in mock and OBP-301 infected cells, LD ₅₀ = 4.0, 0.7 Gy). In A549 cells, infection with 1 MOI of OBP-301 resulted in a 4-fold increase in the cytotoxicity of ionizing radiation (in mock and OBP-301 infected cells, LD ₅₀ = 2.4, 0.6 Gy) (FIG. 1B). In contrast, infection with nonproliferative Ad-GFP had no effect on radiation therapy. In FIG. 1B, the radiation dose response curve is shown as the ratio of mock to treated cells and is shown as the mean ± SD of triplicate tests. Representative results from three independent experiments are shown.
A431 and TE8 cells were subjected to 1 Gy irradiation, 10 MOI OBP-301 infection, or a combination of both (OBP-301 infection after irradiation), and cell morphology was assessed 5 days after treatment. In the case of phase contrast microscopy, A431 cells and TE8 cells were shown to proliferate on day 5 until they became subconfluent when only a single irradiation of 1 Gy radiation was performed. On the other hand, when radiation was combined with 10 MOI OBP-301 infection, a rapid decline in cell viability due to extensive cell death, characterized by swollen and floating cells, was observed (FIG. 1C). OBP-301 infection alone showed an effect, but ionizing radiation clearly promotes the cytotoxic effect of OBP-301, and the combined use of OBP-301 infection and gamma irradiation is a marked cytotoxicity in both A431 and TE8. Induced effect.
The inventors also visually confirmed the growth and spread of the virus after the combined use of oncolytic virus therapy and radiation therapy. OBP-401 is advantageous in that virus distribution can be monitored by GFP fluorescence. A549 cells were irradiated with 2 Gy, then infected with OBP-401 at 0.1 MOI, and observed with a fluorescence microscope 5 days after the treatment. Although most cells led to cell death, GFP expression is strong and persistent, indicating viral growth and spread to surrounding tumor cells. GFP expression was also detected in A549 cells infected with OBP-401 when combined with gamma irradiation (FIG. 1D). These results indicate that the combined use of oncolytic virus therapy and radiation therapy promotes anti-tumor effects in vitro.
(2) Effect of radiation on virus growth of OBP-301 in human cancer cells
We used flow cytometry to measure the effect of radiation on the expression of CAR (anti-coxsackie adenovirus receptor) on the cell surface. A431 and A549 cells were irradiated at a dose of 1 or 10 Gy and analyzed by flow cytometry 24 hours later. Cells were incubated with anti-CAR monoclonal antibody and then detected with FITC-labeled goat anti-mouse IgG secondary antibody. FITC-bound isotype-matched normal mouse IgG1 was used as a control in all experiments. The proportion of CAR positive cells was 100% in A431 cells and 99% in A549 cells, and their expression levels were maintained after low dose (1 Gy) and high dose (10 Gy) gamma irradiation. (FIG. 2A).
The present inventors examined the effect of radiation on virus growth of OBP-301 by Western blot analysis. Specifically, A431 and A549 cells were treated with mock or irradiated with 1 or 2 Gy of radiation to infect 10 or 1 MOI of OBP-301 and collected at a predetermined time point. In addition, 293 cells were used as a positive control. Whole cell lysate was subjected to gel electrophoresis, transfer to PVDF membrane, and Western blot of E1A protein.
As a result of Western blot analysis, it was shown that infection of OBP-301 induces an easily detectable level of E1A protein expression in A431 and A549 cells even after γ-irradiation (FIG. 5). 2B). These results indicated that the hTERT promoter is transcriptionally active in irradiated tumor cells.
Furthermore, the present inventors examined the effect of ionizing radiation on the propagation of the child virus by quantitative real-time PCR analysis. A431, TE8, and A549 cells were irradiated with a dose of 1 Gy (A431, TE8) or 2 Gy (A549) and then infected with 1.0 MOI of OBP-301 for 2 hours. Cells were harvested at predetermined time points for 72 hours after infection and the extracted DNA was assayed. The number of intracellular viral copies of OBP-301 increased by 4 to 6 orders of magnitude over time when irradiated with γ rays and when not irradiated (FIG. 2C). A plateau was reached approximately 48 hours after infection. These results indicate that ionizing radiation does not interfere with the growth of OBP-301.
(3) Effects of radiation on adenovirus infection in human cancer cells
The infection efficiency of adenovirus agents varies greatly depending not only on external stimuli but also on the cell type since adenoviruses have a complex cell entry process. Therefore, the present inventors determined whether or not the infectivity of adenovirus to human cancer cell lines can be improved by irradiation, using a non-proliferative adenovirus vector (Ad-GFP) expressing the GFP gene. We examined using.
Human cancer cells A431 and A549 were irradiated with different radiation doses (1-10 Gy) and then infected with 50 MOI and 10 MOI of Ad-GFP, respectively. After 48 hours, GFP expression was assessed by fluorescence microscopy. GFP expression is observed in a portion of A431 and A549 cells that have not been irradiated, but higher levels of GFP expression are observed in A431 and A549 cells exposed to gamma radiation, and the fluorescence intensity is dose dependent. Increased (FIG. 3A). Moreover, in both A431 and A549 cells, it was shown that the expression level of GFP increases with an increase in radiation dose (FIG. 3B). Specifically, the mean fluorescence intensity (MFI) of the cells treated as described above was analyzed by flow cytometry. The MFI value of non-irradiated Ad-GFP infected cells was taken as 1.0, and the relative MFI value of irradiated cells was calculated as a ratio to the MFI value of non-irradiated cells. It is noteworthy that the increase in relative MFI value after gamma irradiation is dose dependent. This indicates that adenovirus uptake in human cancer cells is increased by ionizing radiation.
To assess whether radiation increases the infectivity of OBP-301, A431, A549, and TE8 cells are 1.0 MOI after γ-irradiation at a dose of 1 Gy (A431 and TE8) or 2 Gy (A549). Of OBP-301. According to quantitative real-time PCR analysis performed using DNA extracted 2 hours after infection, the amount of E1A DNA after OBP-301 infection in irradiated cells was 1.8 to more than that of cells not irradiated with radiation. It was shown to be 2.5 times higher (FIG. 3C). Therefore, it was shown that irradiation with ionizing radiation before OBP-301 infection increases virus uptake and enhances oncolytic activity.
(4) Effect of OBP-301 infection on repair of radiation-induced DNA damage in human cancer cells
To investigate the effect of OBP-301 infection on the repair of ionizing radiation-induced DNA DSB, we used irradiated and non-irradiated OBP-301 infected A549 cells. The expression of DNA-PKcs, a DNA repair protein, was examined by Western blot analysis. Specifically, A549 cells were treated with mock or irradiated with 2 Gy radiation, infected with mock or infected with 1 MOI of OBP-301, and the cells were collected 12 hours after the treatment. Whole cell lysates were electrophoresed and Western blotted for DNA-PKcs. DNA-PKcs protein expression was quantified by densitometric scanning using NIH image software and normalized using the actin signal. As a result, the expression of DNA-PKcs decreased more than 10 times after 12 hours by infection with 1.0 MOI of OBP-301. Exposure to 2 Gy ionizing radiation after infection with OBP-301 did not affect the decrease in the expression level of DNA-PKcs (FIG. 4A). Similar results were observed in A431 cells. This indicates that OBP-301 infection inhibits repair of DNA damage.
Ku protein plays an important role in DNA-PKcs-dependent DSB repair, and suppression of Ku protein function by antisense may increase tumor sensitivity to ionizing radiation (Li GC, et al. Cancer Res. 2003; 63: 3268-74.). In addition, the Ku protein complex (composed of Ku70 and Ku80) has been reported to independently migrate from the cytoplasm to the nucleus (Koike M, et al. J Cell Sci. 1999; 112: 4031- 9, Koike M, et al. Oncogene. 1999; 18: 7495-505, Koike M, et al. Biochem Biophys Res Commun. 2000; 276: 1105-11).
Therefore, the present inventors examined whether infection with OBP-301 affects the intracellular localization of Ku70 / 80 in A549 cells. Specifically, A549 cells were treated with mock or irradiated with 2 Gy radiation, infected with mock or infected with 1 MOI of OBP-301, then fixed 12 hours after treatment, and anti-Ku70 / Ku80 antibody (Ku70 And an antibody that detects both Ku80 subunits). Cells were also stained with PI.
Indirect immunofluorescence staining with anti-Ku70 / Ku80 antibodies showed strong and extensive staining in the nucleus and cytoplasm under unstimulated conditions. Irradiation with a dose of 2 Gy strongly induced Ku staining in the nucleus and, conversely, contributed to a decrease in cytoplasmic Ku expression. In contrast, infection with OBP-301 increased cytoplasmic Ku staining. This indicates that OBP-301 infection blocks nuclear translocation of Ku protein. Furthermore, even when OBP-301 was infected before irradiation with ionizing radiation, the transfer of Ku to the nucleus was blocked, and Ku expression was maintained in the cytoplasm (FIG. 4B).
(5) Anticancer activity by combined use of intra-tumor injection of OBP-301 and irradiation in human tumor xenografts
The present inventors evaluated the therapeutic effect in vivo with respect to human cell A549 by combined use of OBP-301 and radiation. A549 cells (2 × 10 ⁶ Cells / dose) were transplanted into the hin / flank of nu / nu mice as xenografts. Mice with palpable 7-12 mm diameter A549 tumors were irradiated with 3 Gy local radiation and 10 ⁸ Either PFU OBP-301 or PBS was injected intratumorally in 3 cycles every 2 days from day 1. PBS was used as a control and 6 mice were used as a group. In order to prevent damage to adenoviral DNA, irradiation was performed prior to injection of OBP-301. Tumor growth was significantly suppressed by either intratumoral administration or irradiation of OBP-301 compared to tumors treated with mock 42 days after the start of treatment (p <0.05). When OBP-301 and radiation were used in combination, tumors were more prominent and significantly more significant when compared to mice treated with either method (p <0.05) and mock treated tumors (p <0.01). Proliferation was inhibited (Figure 5A). In FIG. 5A, tumor growth was shown by mean tumor volume ± SEM.
Tumors treated with OBP-301 and local radiation in combination were clearly smaller compared to tumors in mice from other cohorts 37 days after treatment, indicating that the combined treatment is extremely effective. (FIG. 5B). In contrast, when non-proliferative adenovirus was injected intratumorally, no effect on the growth of A549 tumor was observed regardless of the combined use of irradiation. Three cycles of either local irradiation or OBP-301 infection were performed, and histopathological analysis was performed using sections of the tumor excised 10 days later. As a result, when OBP-301 was infected, the tissue was clearly regressed, showing a decrease in tumor cell density compared to untreated tumors. The combined treatment of OBP-301 injection and radiation resulted in extensive tissue destruction and further decreased tumor cell density. Furthermore, in cytolytic changes induced by the combination therapy, replacement with a vitrified acellular stroma (hyalinized acellular stroma) was observed (FIG. 5C). In addition, extensive virus distribution in tumors was confirmed by immunohistochemical staining of adenovirus hexon protein.
(6) Consideration
The present inventors examined the mutual sensitization enhancing effect of OBP-301 infection and ionizing radiation, and found an anticancer effect by combined use in vitro and in vivo.
Oncolytic virus therapy is a field that has the potential to grow further in the future, based on an understanding of the molecular aspects of human cancer and the development of technology for genetic modification of the viral genome. However, oncolytic virus therapy itself rarely kills tumors in preclinical animal models or clinical studies, despite high virus titers persisting in the target tumor.
Viruses cause tumor lysis by a different mechanism than that by current treatment methods. Thus, the oncolytic virus therapy of the present invention provides a useful multidisciplinary clinical approach through a synergistic mechanism of action. OBP-301, a telomerase-specific oncolytic adenovirus, is a novel biological agent that can efficiently kill a variety of human cancer cells by telomerase expression activity in most human cancers.
The infection efficiency of adenoviral agents derived from human adenovirus serotype 5 varies greatly depending on the expression of CAR (Wickham TJ, et al. Cell. 1993; 73: 309-19). Indeed, we have found that OBP-301 has no effect on some tumors that express only low levels of CAR (Taki M, et al. Oncogene. 2005; 24: 3130-40). And upregulation of CAR in tumor cells treated with the histone deacetylase (HDAC) inhibitor FR901228 increases intracellular viral growth and thereby promotes synergistic anti-tumor effects (Watabebe T, et al. Exp Cell Res. 2006; 312: 256-65) have been shown previously. In contrast to HDAC inhibitors, we have found that irradiation does not significantly increase CAR expression in all target cell lines used in the study. In addition, radiation therapy did not change CAR expression in malignant glioma cells in vitro despite showing the combined effect with the infection of ONYX-015, an E1B55 kDa-deficient oncolytic adenovirus, in vivo ( Geoerger B, et al. Br J Cancer. 2003; 89: 577-84). Interestingly, however, it was confirmed not only with OBP-301 itself but also with non-proliferative Ad-GFP that adenovirus infectivity increases after irradiation with ionizing radiation. Viral DNA levels immediately after 2 hours of infection with OBP-301 were clearly higher in irradiated cells than in untreated cells.
These results show that CARP-independent cell invasion by OBP-301 is possible even when irradiated prior to viral infection, thereby overcoming the CAR-dependent limitations of adenoviral infection.
OBP-301 was efficiently taken up by irradiating tumor cells, but the amount of proliferation did not change in the three types of cell lines regardless of the presence or absence of irradiation. This indicates that irradiation with radiation prior to infection does not change the basic viral growth cycle.
Our data showed significant down-regulation in the expression of DNA-PKcs protein after infection with OBP-301. As previously reported, one possible explanation is that the adenovirus E4 product inhibits DNA-PK-dependent DSB repair by forming a physical complex with DNA-PK. (Boyer J, et al. Virology. 1999; 263: 307-12).
The DNA DSB promotes the interaction of the Ku complex with DNA-PK and ensures more efficient repair of DNA damage so that the Ku protein translocates to the nucleus in response to ionizing radiation. The inventors have found that infection with OBP-301 inhibits this nuclear translocation and causes an increase in Ku expression in the cytoplasm. Therefore, it can be said that tumor cells infected with OBP-301 further increase the sensitivity to ionizing radiation.
Our in vitro studies show that OBP-301 infection and ionizing radiation can increase the sensitivity of human tumor cells to each other, thereby increasing the effectiveness of combination therapy. Ionizing radiation causes rapid DNA DSB and promotes viral uptake. On the other hand, infection with OBP-301 requires time for the virus to grow, induce its cytotoxic effects, and increase the sensitivity of the cells to radiation. Therefore, in actual clinical application, intra-tumor injection of OBP-301 after external-beam radiotherapy would be appropriate as a treatment plan to maximize the combined effect.
Because patients often receive radiation treatment daily from Monday to Friday, OBP-301 can grow and spread in tumor foci between treatments following intratumoral administration. Become. Our in vitro experiments showed that relatively low doses of OBP-301 (1-10 MOI) significantly promoted the cytotoxic activity of ionizing radiation in various human tumor cell lines. Furthermore, infection and proliferation of GFP-expressing oncolytic virus (OBP-401) was observed in tumor cells that survived a single treatment when observed with a fluorescence microscope. This indicates that repeated treatment is more effective at killing tumor cells previously sensitized to radiation by viral infection. This assumption could be confirmed in an in vivo xenograft model.
The combination of OBP-301 intratumoral injection and local irradiation in alternating three cycles at daily intervals clearly inhibited the growth of xenografted A549 tumors over a long period of time. Histological analysis also showed that the combined use of OBP-301 and radiation had a greater effect on tumor destruction than performing each method alone.
There are several explanations for the excellent antitumor activity in vivo shown in our experiments. First, as described above, repeated cycles of radiotherapy and viral therapy can increase the sensitivity of tumor cells to both treatments, thereby obtaining synergistic anti-tumor activity. Second, OBP-301 inhibits vascular supply by killing endothelial cells that show enhanced proliferation in irradiated tumors with high telomerase activity (Tsai JH, et al. Cancer Biol Ther. 2005). 4: 1395-1400). Also, local radiation itself can attack vascular endothelial cells at the tumor site, while preventing locally injected OBP-301 from moving into the bloodstream. Indeed, ionizing radiation inhibits endothelial cell proliferation, tube formation, migration, and clonogenic survival (Abdolahi A, et al. Cancer Res. 2003; 63: 8890-8.).
Viral therapy and radiation therapy may also target tumor cells in different parts of the tumor by distinct mechanisms. For example, when the tumor is hypoxic, the tumor cells become more resistant to ionizing radiation (Hockel M, et al. J Natl Cancer Inst. 2001; 93: 266-76.) And are localized in the tumor area. Cells can survive and grow under hypoxic conditions. For this reason, how to kill tumor cells under hypoxic conditions that are resistant to ionizing radiation has become a clinical issue.
On the other hand, hypoxia induces the transcriptional activity of the hTERT gene promoter by hypoxia inducible factor 1α (HIF-1α) (Nishi H, et al. Mol Cell Biol. 2004; 24: 6076-83, Anderson CJ, et al. Oncogene.2006; 25: 61-9), it can be inferred that OBP-301 proliferates even under hypoxic conditions and efficiently kills tumor cells. Thus, hTERT-specific oncolytic virus therapy is effective for killing surviving tumor cells after local radiation therapy.
Another advantage of this combination therapy is the low likelihood of cross-resistance because these therapies function by different cytotoxic mechanisms.
In summary, our data indicate that OBP-301 infection and ionizing radiation improve each other's biological effects and thereby enhance each other, greatly enhancing anti-tumor activity in in vivo preclinical models. Indicates to promote.

Recent research has revealed that some types of tumors have cells called cancer stem cells. A cancer stem cell is a tumor cell having characteristics similar to the self-renewal mode of a normal stem cell, has a tumor remodeling ability, and malignantizes a tumor. And it is known that radiation resistance and anticancer drug resistance are brought about.
Therefore, in order to effectively carry out tumor radiation therapy and chemotherapy, it is required to kill cancer stem cells having radiation resistance and anticancer drug resistance.
Cancer stem cells express CD133, a specific marker, on the cell surface.
Therefore, the present inventors focused on the fact that cancer stem cells having radiation resistance and anticancer drug resistance are CD133-positive, and the virus of the present invention is anti-CD133-positive against CD133-positive cells isolated from a cell group of radiation-resistant strains. It was examined whether or not it showed a tumor effect.
1. Preparation of Cell Line Human gastric cancer cells (MKN45) (hereinafter also referred to as “parent line”) were cultured in RPMI 1640 medium (SIGMA) for 7 days under conditions of 37 ° C. and 5% CO 2, and irradiated with 2 Gy ionizing radiation, Thereafter, a recovery period of 14 days (period in which the cell population re-growth) was provided. The irradiation with ionizing radiation and the setting of the recovery period were repeated three times (total 6 Gy irradiation). As a result, an ionizing radiation resistant strain (MKN45R3) was obtained (FIG. 6).
The ratio of CD133 positive cells in the obtained radiation resistant strain (MKN45R3) and parent strain (MKN45) was analyzed by flow cytometry using CD133-APC antibody (Miltenyi Biotec) and FACS (Becton-Dickson), respectively.
As a result, the ratio of CD133 positive cells in the parent cell group averaged about 2%, and the ratio of CD133 positive cells in the radiation resistant cell group averaged about 22%. From this result, it was found that the ratio of CD133 positive cells was significantly increased in the cell group of the radiation resistant strain (FIG. 7).
CD133 negative cells (CD133−) and CD133 positive cells (CD133 +) were sorted from the cell group of the radiation resistant strain using FACS, respectively. As a result, CD133 negative cells could be sorted with a purity of 97.5%, and CD133 positive cells could be sorted with a purity of 76.5% (FIG. 8).
Sorted CD133 negative cells and CD133 positive cells were used in subsequent experiments.
2. Self-replication ability of CD133 positive cells The self-replication ability (repopulation / self-renewability) of the sorted CD133 negative cells and CD133 positive cells obtained in (1) was examined by flow cytometry.
Specifically, the CD133-negative cell group in which the majority of CD133-negative cells and the CD133-positive cell group in which the majority of CD133-positive cells occupy immediately after sorting the cell groups of the radioresistant strains by FACS, respectively, promotes differentiation. The cells were cultured in RPMI 1640 medium (SIGMA) supplemented with 10% bovine serum, a factor, at 37 ° C. and 5% CO 2 for 2 weeks. In this process, CD133-negative cells and CD133-positive cells contained in cells immediately after sorting, cells after 1 week of culture, and cells after 2 weeks of culture were quantified using FACS (FIG. 9).
As a result, the cells included in the CD133 positive cell group in which the majority of CD133 positive cells are differentiated and proliferated into CD133 positive cells and CD133 negative cells, but the cells in the CD133 negative cell group in which the majority of CD133 negative cells are included. Grew mainly as CD133 negative cells.
Next, the self-replication ability of CD133 positive cells in the presence of leukemia inhibitory factor, which is a differentiation inhibitory factor, was examined by flow cytometry.
Specifically, the CD133-negative cell group in which the majority of CD133-negative cells and the CD133-positive cell group in which the majority of CD133-positive cells occupy immediately after sorting the cell groups of the radioresistant strains by FACS, respectively, promotes differentiation. Cultured in RPMI 1640 medium supplemented with 10% bovine serum as a factor and leukemia inhibitory factor (10 ng / ml) as a differentiation inhibitor under conditions of 37 ° C. and 5% CO 2 for 1 week. As a result, in the CD133-positive cell group in which the majority of CD133-positive cells are immediately after sorting, differentiation from CD133-positive cells to CD133-negative cells is suppressed, and the ratio of CD133-positive cells in the CD133-positive cell group is the leukemia suppression. It rose compared with the case where a factor was not added (FIG. 10).
3. Formation of undifferentiated spheres of CD133-positive cells A sphere is said to be a colony of cells formed when proliferating undifferentiated in a state where stem cells are not adhered in a serum-free medium and are floating. Therefore, in this section, whether or not the CD133-positive cell group in which the sorted CD133-positive cells occupy the majority forms spheres was examined, and further, the cells contained in the spheres were examined.
After sorting and separating CD133 positive cells and CD133 negative cells by FACS, RPMI 1640 medium (SIGMA) without bovine serum and containing epidermal growth factor (Invitrogen) and basic fibroblast growth factor (Invitrogen) The cells were grown for 2 weeks under conditions of 37 ° C. and 5% CO 2. As a result, the CD133-positive cell group in which the majority of CD133-positive cells occupy the majority formed spheres easily, but the CD133-negative cell group in which the majority of CD133-negative cells accounted for showed fibroblast-like proliferation and formed almost spheres. Did not (Figure 11).
In addition, when the spheres were analyzed by flow cytometry using the CD133 antibody, the cells of the spheres obtained by culturing from the CD133 positive cell group in which the majority of the CD133 positive cells occupy both CD133 positive cells and CD133 negative cells. (Figure 12 right panel). On the other hand, cells cultured from the CD133 negative cell group in which CD133 negative cells occupy the majority contained only CD133 negative cells (FIG. 12 left panel). This result indicates that CD133-positive cells have a self-replicating ability.
4). Examination of anti-cancer drug resistance and radiation resistance of CD133 positive cells and CD133 negative cells, and examination of anti-tumor effect of telomerisin (OBP-301) A CD133 positive cell group and a CD133 negative cell group sorted by FACS were 96-well culture plate The cells were seeded at 5 × 10 ² cells per well and cultured under conditions of 37 ° C. and 5% CO 2 for 24 hours.
As shown below, administration of a chemical anticancer agent, irradiation with ionizing radiation, or telomelysin infection was performed, and cell viability was measured by XTT assay after 7 days.
(1) Examination of anticancer drug resistance of CD133 positive cells and CD133 negative cells The above cultured cells were treated with 5-FU, cisplatin (CDDP) and paclitaxel at different concentrations, and the survival rate was measured by XTT assay after 7 days. . Drug resistance was calculated by the following formula.
(Absorbance of drug administration group) / (Absorbance of untreated group) × 100%
The results of treating CD133 positive cells and CD133 negative cells with 5-FU, cisplatin (CDDP), and paclitaxel are shown in FIG. 13, FIG. 14, and FIG. 15, respectively. The experiment was repeated three times with similar results. Statistical significance (p <0.05) is indicated with an asterisk.
As a result of the experiment, CD133-positive cells showed high drug resistance against chemical anticancer agents.
(2) Examination of radiation resistance of CD133 positive cells and CD133 negative cells The cultured cells were irradiated with 3 to 10 Gy of radiation, and the survival rate was measured by XTT assay after 7 days. The ionizing radiation resistance was calculated by the following formula.
(Absorbance of cells after irradiation) / (Absorbance of untreated cells) × 100%
The results are shown in FIG. CD133 positive cells were highly resistant to ionizing radiation.
The experiment was repeated three times with similar results. Statistical significance (p <0.05) is indicated with an asterisk.
(3) Anti-tumor effect of telomerisin on cancer stem cells 3 MOI to 40 MOI of telomerisin was administered to the above-mentioned radiation resistant cancer stem cells, and 7 days later, the cell viability was examined by XTT assay.
The resistance was calculated by the following formula.
(Absorbance of telomerisin administration group / absorbance of untreated group) × 100
The results of the antitumor effect of telomerisin are shown in FIG. Telomerisin has been shown to effectively kill both CD133-positive and CD133-negative cells in a dose-dependent manner.
5. Comparison of hTERT activity Cancer stem cells have characteristics such as self-renewal ability, colony formation ability, pluripotency, and tumor formation ability. From the characteristics of self-renewal ability and pluripotency, cancer stem cells may exhibit high telomerase activity and are expected to exert the antitumor effect of telomerisin.
In order to examine the possibility that telomerase expression is different between CD133-positive cells and CD133-negative cells, mRNA levels of human telomerase reverse transcriptase (hTERT), telomerase catalytic subunit, and limiting factor of telomerase activity were measured and compared.
CD133 positive cells and CD133 negative cells were cultured, and each total RNA was extracted using RNeasy Micro kit (Qiagen). The extracted total RNA was treated with DNase and subsequently reverse transcribed with random hexamer and MuLV reverse transcriptase (Applied Biosystems). Real-time PCR was performed using SYBR Green Real-Time Core Reagents (Applied Biosystems). 2 μl of diluted cDNA (obtained using 100 ng of total RNA as a template) was added per 18 μl of the PCR reaction solution. Expression levels were normalized by the housekeeping gene h-PBGD (Roche).
As a result, as shown in FIG. 18, the expression level of hTERT in CD133 positive cells was higher than that in CD133 negative cells.
According to the present example, it was revealed that tumor cells having radiation resistance can be killed by infecting tumor cells after irradiation with telomerisin (OBP-301). This indicates that the drug of the present invention remarkably enhances the effect of radiation therapy, and also indicates that the drug of the present invention has a remarkable effect as an antitumor agent for use in combination with radiation. is there.

  According to the present invention, when a tumor or cancer cell is infected with OBP-301 in radiation therapy for cancer, the sensitivity of the tumor cell to radiation can be increased. Further, when OBP-301 is infected after previously irradiating a tumor or cancer cell with radiation, the tumor cell can be effectively killed. Furthermore, the present invention can effectively kill tumors or cancer cells that are resistant to radiation or anticancer agents. Therefore, the virus used in the present invention can exhibit a remarkable antitumor effect by increasing the sensitivity of radiation, and is extremely useful for combination therapy with radiation.

SEQ ID NO: 7: synthetic DNA
Sequence number 8: Synthetic DNA
[Sequence Listing]

Claims (17)

Promoter of the human telomerase, tumors E1A gene, replication cassette comprising an IRES sequence and an E1B gene in this order comprises a recombinant virus incorporated into the E1 region of the viral genome, have acquired resistance to radiation or anticancer by irradiation or anticancer drugs Or an antitumor agent for cancer. A label comprising a replication cassette containing a human telomerase promoter, an E1A gene, an IRES sequence, and an E1B gene in this order in the E1 region of the viral genome, and a gene encoding a marker protein and a promoter capable of controlling the expression of the gene cassette comprises a recombinant virus incorporated into the E3 region of the viral genome, the anti-tumor agent for acquired tumor or cancer resistance to radiation or anticancer drugs by irradiation or anticancer drugs. The antitumor agent according to claim 1 or 2, wherein the recombinant virus is administered to a patient at a dose of 10 ⁶ to 10 ¹¹ PFU / day.   The antitumor agent according to claim 1 or 2, wherein the human telomerase promoter is an hTERT promoter.   The antitumor agent according to claim 2, wherein the labeled protein is GFP.   The antitumor agent according to claim 2, wherein the promoter capable of controlling the expression of the gene encoding the labeled protein is a cytomegalovirus promoter or an hTERT promoter.   The antitumor agent according to claim 1 or 2, wherein the virus is an adenovirus.   The antitumor agent according to claim 2, wherein the promoter capable of controlling the expression of the gene encoding the labeled protein is a carcinogenesis and tumor suppressor promoter.   The antitumor agent according to claim 1 or 2, wherein the tumor or cancer is a cancer stem cell.   The antitumor agent according to claim 1 or 2, wherein the tumor or cancer is CD133 positive.   The production of the agent according to any one of claims 1, 3, 4, 7, 9, and 10, wherein the replication cassette containing a human telomerase promoter, an E1A gene, an IRES sequence, and an E1B gene in this order is E1 of the viral genome. Use of recombinant virus integrated into the region.   In the manufacture of the agent according to any one of claims 2 to 10, a replication cassette comprising a human telomerase promoter, an E1A gene, an IRES sequence, and an E1B gene in this order is incorporated into the E1 region of the viral genome, and labeled Use of a recombinant virus in which a marker cassette comprising a gene encoding a protein and a promoter capable of controlling the expression of the gene is incorporated in the E3 region of the viral genome.   Use according to claim 11 or 12, wherein the human telomerase promoter is the hTERT promoter.   Use according to claim 12, wherein the labeled protein is GFP.   The use according to claim 12, wherein the promoter capable of controlling the expression of the gene encoding the labeled protein is a cytomegalovirus promoter or an hTERT promoter.   Use according to claim 11 or 12, wherein the virus is an adenovirus.   The use according to claim 12, wherein the promoter capable of controlling the expression of the gene encoding the labeled protein is an oncogenic and tumor suppressor promoter.