WO2008065726A1

WO2008065726A1 - Telomelysin-containing agent for breaking antitumor tolerance

Info

Publication number: WO2008065726A1
Application number: PCT/JP2006/324316
Authority: WO
Inventors: Toshiyoshi Fujiwara; Noriaki Tanaka; Yasuo Urata
Original assignee: Oncolys Biopharma Inc.
Priority date: 2006-11-29
Filing date: 2006-11-29
Publication date: 2008-06-05

Abstract

It is intended to provide an agent for breaking antitumor tolerance. This agent for breaking antitumor tolerance is characterized by containing a recombinant virus carrying a polynucleotide, which has a human telomerase promoter, an E1A gene, an IRES sequence and an E1B gene in this order, having been integrated thereinto.

Description

· · Telomeraicin-containing anti-tumor drug resistance overcoming technology field

The present invention relates to an anti-tumor drug resistance overcome agent comprising a recombinant virus in which a polynucleotide comprising a human telomerase promoter, an E1A gene, an IRES sequence and an E1B gene in this order is incorporated. Background art

In chemotherapy for cancer, there is a big problem of overcoming resistance to anticancer drugs. Cancer cells acquire resistance to anticancer drugs through various molecular mechanisms, such as transporter activation and receptor kinase changes (Bush JA, Li G. Cancer chemoresistance: the relationship between p53 and multidrugtransporters. Int J Cancer 98:, 323 · 330, 2002.) Although research on anticancer drug resistance has been conducted, there is no solution yet. Cell death due to anticancer drugs is apoptosis, and resistance to anticancer drugs is apoptotic resistance (Schmitt CA, Lowe SW. Apoptosis and chemoresistance in transgenic cancer models. J Mol Med 80: 137-146, 2002.), In recent years, restricted-proliferative viruses have been researched and developed as new therapies for these diseases. They are thought to have a completely different mechanism of action from anticancer drugs, and are often used in clinical trials centered in the United States. The present inventor has previously developed a restricted growth type adenovirus preparation using the TERT promoter (Kawashima T, Kagawa S, Kobayashi N, Shirakiya Y, Umeoka T, Terais i F, Taki M, Kyo S, Tanaka N, Fujiwara T. Telomerase-specific replication- selective

virotherapy for human cancer. Clin Cancer Res 10: 285-292, 2004.). This adenovirus was named telomerisin. Cell death by telomerisin is a different mechanism of action from apoptosis. In the combination experiment of telomerisin and the microtubule agent docetaxel, each drug exhibited an independent antitumor effect, and as a result, a combination effect independent of each other was observed in vivo. Disclosure of the invention

An object of the present invention is to provide an anti-tumor drug resistance overcome agent comprising a recombinant virus in which a polynucleotide comprising a human telomerase promoter, an E1A gene, an IRES sequence, and an E1B gene in this order is incorporated.

The present inventor has intensively studied to solve the above problems. And, it was found that telomerisin showed an extremely excellent cell killing effect against antitumor agent-resistant tumor cells, and the present invention was completed. That is, the present invention is as follows.

(1) An anti-tumor agent resistance-resolving agent comprising a recombinant virus incorporating a polynucleotide comprising a human telomerase promoter, an E1A gene, an IRES sequence, and an E1B gene in this order. .

In the above anti-tumor agent fertility overcomer, the promoter for human telomerase is, for example, hTERT. Also, the virus is preferably adenovirus, for example. Antitumor agents are, for example, platinum preparations, microtubule inhibitory activators, alkylating activators, antimetabolite activators, anticancer antibiotics and topoisomerase inhibitory activators as well as their pharmacologically acceptable It is possible to use at least one selected from the group consisting of salts. Specific examples of antitumor agents include docetaxel, .paclitaxel, cisplatin, psulfan, methotrexate, fluorouracil, tegafur, bleomycin, adriamycin, mitomycin C, vinorelbine and irinotecan and their pharmacologically acceptable conditions. And at least one selected from the group consisting of salts. The tumor is, for example, lung cancer, colon cancer, stomach cancer, breast cancer, esophageal cancer, head and neck cancer, liver cancer, pancreas cancer, gallbladder or bile duct cancer, prostate cancer, bladder cancer, cervical cancer, thyroid cancer, And at least one selected from the group consisting of ovarian cancer, leukemia, lymphoma, sarcoma, and mesenchymal tumor.

(2) a recombinant virus incorporating a polynucleotide containing a human telomerase promoter, E1A gene, IRES sequence and E1B gene in this order; A pharmaceutical composition for combination therapy of a tumor that has acquired antitumor drug resistance, comprising a substance having a use.

In the above pharmaceutical composition, the substance having an antitumor action is, for example, 'docetaxel, pinolerubin, irinotecan and histone deacetylase inhibitory activity inhibitor and their pharmacological (group consisting of this acceptable salt). At least one selected from '.

(3) A tumor that has acquired resistance to an antitumor agent, comprising administering to a mammal a recombinant virus incorporating a polynucleotide comprising a human telomerase promoter, an E1A gene, an IRES sequence, and an E1B gene in this order. A method to suppress cell proliferation.

(4) Recombinant virus in which a polynucleotide containing a human telomerase promoter, _E1A gene, IRES sequence and E1B gene in this order is incorporated, and a substance having antitumor activity or pharmacologically acceptable A method of inhibiting the growth of tumor cells that have acquired resistance to an antitumor agent, comprising administering to a mammal in combination with a salt.

(.5) Acquiring antitumor drug resistance characterized by administering to a mammal a recombinant virus incorporating a polynucleotide containing a human telomerase promoter, E1A gene, IRES sequence, and E1B gene in this order. How to treat a tumor.

In the methods (3) to (5) above, an example of a promoter for human telomerase is hTERT. The virus is preferably an adenovirus. Antitumor agents include, for example, platinum preparations, microtubule inhibitory activators, alkylating activators, antimetabolite activators, anticancer antibiotics and tobodies.Somerase inhibitor activators and their pharmacologically acceptable And at least one selected from the group consisting of possible salts. Specific examples of antineoplastic agents include docetaxel, paclitaxel, cisplatin, psnorephan, methotrexate, funoleolauracinole, tegafunore, preomesin, adriamycin, mitomycin C, vinorelbine and irinotecan and their pharmacologically acceptable conditions. And at least one selected from the group consisting of possible salts. Tumors include, for example, lung cancer, colon cancer, stomach cancer, breast cancer, esophageal cancer, head and neck cancer, liver cancer, knee cancer, gallbladder or bile duct cancer, prostate cancer, bladder cancer, cervical cancer, thyroid And at least one selected from the group consisting of adenocarcinoma, ovarian cancer, leukemia, lymphoma, sarcoma, and mesenchymal tumor.

In the method of (4) above, the substance having an antitumor action is, for example, a group consisting of docetaxenole, pinorelbine, irinotecan and histone deacetylase inhibitory activity inhibitors and pharmacologically acceptable salts thereof. At least one selected from. Brief Description of Drawings

FIG. 1 shows the structure of OBP-301 (telomerisin) and OBP-401. Figure 2 shows the characterization of drug resistance sublines. a) CDDP, b) Parkitaxel, —

FIG. 3 is a diagram in which anticancer agents were administered to various cancer cell resistant strains and the antitumor effect was observed with a microscope. ,

Fig. 4 is a diagram showing the results of an experiment in which cell death was performed.

Fig. 5 shows the analysis of MDR1 protein expression by Western blot.

Fig. 6 shows the analysis of CAE expression using flow cytometry. FIG. 7 shows the results of an XTT assay performed after infecting a cancer cell resistant strain with OBP-301. 'a) Series A431,' b) Series DU145

FIG. 8 shows the results of an experiment in which a cancer cell resistant strain was infected with OBP-301 and then subjected to XTT assembly over time. a) A431 series, b) DU145 series

Fig. 9 shows the results of CBB staining after infection of a cancer cell resistant strain with OBP-301.

FIG. 10 is a view observed with a microscope after infecting a cancer cell resistant strain with OBP-301 and OBP-401.

FIG. 11 is a view observed with a microscope after infecting a cancer cell resistant strain with OBP-301 and OBP-401. Quantitative real time PCR was used to angularly analyze the intracellular growth of OBP-301. Figure 12 shows the results of an experiment analyzing the intracellular growth of OBP-301 using quantitative real-time PCR. a) A431 series, b) DU145 series Best mode for carrying out the invention

The present invention relates to an anti-tumor drug resistance overcome agent comprising a recombinant virus in which a polynucleotide comprising a human telomerase promoter, an E1A gene, an IRES sequence and an E1B gene in this order is incorporated. The present invention will be described in detail below, but the scope of the present invention is not limited to these explanations, and the examples other than the following examples are appropriately modified and implemented within a range not impairing the gist of the present invention. obtain. It should be noted that all publications cited in the present specification, for example, prior art documents, and publications, patent gazettes and other patent documents, are incorporated herein by reference.

1. Summary of the present invention

The present invention relates to an anti-tumor agent resistance overcomer that exerts a cell-killing effect on tumor cells that have acquired anti-tumor agent resistance. Anti-tumor drug resistance refers to the fact that the effect of a tumor cell gradually acquires resistance to these anti-tumor drugs when the treatment with the same anti-tumor drug is repeated. Various molecular mechanisms such as transporter activation and receptor kinase changes are involved in the mechanism by which tumor cells acquire antitumor drug resistance. In addition, since cell death by antitumor agents is apoptosis, resistance to antitumor agents can be said to be apoptosis resistance. When anti-tumor drug resistance occurs, it is generally necessary to change the treatment policy by replacing the anti-tumor drug used so far with other drugs. Even if the treatment used is used, drug resistance may occur again, so changes in antitumor agents are not always effective. By the way, the recombinant virus contained in the antitumor agent resistance overcomer of the present invention starts to proliferate when the telomerase promoter is expressed. Here, since the expression of telomerase is extremely high in tumor cells as compared with normal cells, the recombinant virus has a feature that it does not grow in normal cells but grows only in tumor cells. Furthermore, the mechanism of cell death that this recombinant virus exerts on tumor cells is different from the mechanism of apoptosis by conventional anti-tumor agents, so that tumor cells are treated with anti-tumor agents. Even if it is controlled, it can proliferate and replicate in the cell without any influence. As a result, as described above, even when resistance to an antitumor agent is acquired in a tumor cell, the tumor cell can be specifically killed. Thus, as a result of repeated use of antitumor agents, tumor cells that have acquired antitumor agent resistance and no longer have a cytocidal effect are administered with the recombinant virus included in the present invention. It can be said that “resistance to anti-tumor agents can be overcome”. Therefore, the recombinant virus included in the present invention is useful as an anti-tumor drug resistance overcomer.

2. Recombinant virus of the present invention '

The recombinant virus used in the present invention refers to a virus in which a polynucleotide comprising a human telomerase promoter, an E1A gene, an IRES sequence, and an E.1B gene in this order is incorporated into a genome. The virus to be used is not particularly limited, but an adenovirus is preferable from the viewpoint of safety. 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 the human telomerase promoter. Since the expression of telomerase is extremely high in tumor cells as compared with normal cysts, the telomerase promoter is expressed in tumor cells containing telomerase, whereby the recombinant virus included in the present invention grows. As a result, cell damage due to virus growth occurs in the tumor cells, and the recombinant virus used in the present invention can specifically kill the tumor cells.

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 such telomerase promoter is not particularly limited, but for example, a promoter of human telomerase reverse transcriptase QiTERT) is preferable. hTERT is a 1.4 kbp region upstream of its 5 'end, and many transcription factor binding sequences have been confirmed, and this region is thought to be the hTERT promoter. Among them, the 181 bp sequence upstream of the translation start site is downstream. It is an important core region for gene expression. In the present invention, this core area As long as it contains a region, it can be used without limitation, but it is preferable to use an upstream sequence of about 378 bp completely including this core region as the hTERT promoter. It has been confirmed that the gene expression efficiency of this sequence of about 378 bp is equivalent to that of the 181 bp core region alone. The base sequence of such hTERT is not shown in SEQ ID NO: 1.

hTERT hybridizes under stringent conditions with DNA consisting of a complementary base sequence to DNA consisting of the base sequence shown in SEQ ID NO: 1 in addition to the base sequence shown in SEQ ID NO: 1, and has hTERT activity Nucleotide base sequences are also included. Examples of T stringent conditions for the above hybridization include lxSSC to 2xSSC, 0.1 to 0.5% SDS, and 42 to 68 ° C, and more specifically. For example, after pre-hybridization at ~ 68 ° C for 30 minutes or more, washing in 2xSSC, 0.1% SDS at room temperature for 5 to 15 minutes is performed 4 to 6 times. .

In the present invention, the Έ1Α gene, the IRES sequence and the E1B gene are included in this order because the IRES sequence inserted between the E1A gene and the E1B gene is used. This is because the proliferation ability is increased. The £ 1 gene and 18 genes are included in the E1 gene. The E1 gene is one of the early gene (early: E) and late gene (late: L) related to DNA replication of the virus. It codes for a protein involved in the control of transcription of the viral genome. The E1A protein encoded by the E1A gene activates transcription of genes (E1B, E2, E4, etc.) necessary for the production of infectious 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. To do. 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 are stringent with DNA consisting of a base sequence complementary to the DNA consisting of the base sequences shown in 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 that hybridizes under various conditions and has E1A and E1B activities, respectively. Up Examples of stringent conditions in the hybridization include, for example, lxSSC to 2xSSC, 0.1 to 0.5% SDS, and 42 to 68. More specifically, after prehybridization at 60 to 68 ° C for 30 minutes or more, wash in 2x SSC, 0.1% SDS at room temperature for 5 to 15 minutes. The conditions for performing 6 times are mentioned.

IRESdnternal Ribosome Entry Site) is a protein synthesis initiation signal specific to the Picornaviridae family, and since it has a sequence complementary to the 3 'end of 18S ribosomal RNA, it is thought to serve as a ribosome binding site. It has been. It is known that mRNA derived from the Picornaviridae virus is translated via this sequence. The translation efficiency from the IRES sequence is high, and protein synthesis is performed even in the middle of mRNA independent of the cap structure. Therefore, in this virus, both the E 1 A gene and the E 1B gene downstream of the IRES sequence are independently translated by the promoter of human telomerase. When IRES is used, the expression of the telomerase promoter is controlled independently by 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, virus growth Can be more strictly limited to cells having telomerase activity. The IRES sequence is shown in SEQ ID NO: 4.

In addition, IRES is highly stringent under stringent conditions with DNA consisting of a base sequence complementary to the DNA consisting of the base sequence shown in SEQ ID NO: 4, with the help of the base sequence shown in SEQ ID NO: 4. In addition, a base sequence encoding a protein having IRES activity is also included. Examples of stringent conditions in the above hybridization include lxSSC to 2xSSC, 0.1 to 0.5% 'SDS, and 42 to 68 ° C, and more specifically, 30 to 60 to 68 ° C. After prehybridization for at least 5 minutes, the conditions include washing in 2xSSC and 0.1% SDS at room temperature for 5 to 15 minutes, 4 to 6 times.

In the present invention, the promoter of human telomerase 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. The case where hTERT is used as a human telomerase is described below.

By performing RT-PCR and / or DNA-PCR from cells expressing the E1 gene, such as 293 cells, using primers such as E1A-S, El A-AS, E1B-S, and E1B-AS After amplifying the E1A gene and E1B gene and confirming the sequence using a known method such as TA cloning, if necessary, the E 1 A and E 1B DNA fragments can be excised with a known restriction enzyme.

Next, E1A-IRES-E1B can be inserted into a known vector (eg, pIRES), and then the hTERT promoter sequence excised with a restriction enzyme such as Mul or Bgllll can be inserted upstream of> E1A.

If necessary, remove the Cytogallovirus (CMV) promoter contained in a known vector such as pShuttle with restriction enzymes such as Mfel and Nhel, and use the restriction enzymes Nhel and Notl from phTERT-ElA-IRES-ElB. The cut out sequence can be inserted. Thus, in the present invention, a virus incorporating the cassette consisting of hTERT-ElA-IRES-ΕΙΒ used in the present invention (FIG. 1a) is referred to as “telomerisin” or “Telomelysin”. By expressing the E1 gene required for adenovirus growth under the control of the hTERT promoter, the virus can be propagated specifically for cancer cells. '

In order to infect cells with a recombinant virus, for example, the following method can be used. First, cells such as human colon cancer cell SW620, human lung cancer cell A549, H1299, etc. are plated on a culture plate containing an appropriate culture medium and cultured at 37 ° C in the presence of carbon dioxide. 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.:! To 10 MOI, preferably 0.1 to 1 MOI (multiplicity of infection). MOI is the ratio between the amount of virus (infectious unit) and the number of cells when a certain amount of cultured cells are infected with a certain amount of virus particles, and is used as an index for infecting cells with viruses. To confirm virus growth, collect virus-infected cells, extract DNA, and perform quantitative analysis by performing real-time PCR using a primer that targets the appropriate gene of the virus. be able to. 3. Antitumor agents

Antitumor agents refer to drugs that have the effect of inhibiting the growth and proliferation of tumor cells (cancer cells) that become tumor masses as a result of repeated cell division due to disorder of the body's regulatory mechanism and excessive cell proliferation. Also included are drugs that inhibit growth by inhibiting nucleic acid synthesis in cancer cells or inhibiting metabolism. Specifically, the following alkylation activators, antimetabolite activators, antibiotics, microtubule inhibitor activators, platinum preparations, topoisomerase inhibitor activators, etc. Absent. These antitumor agents may form pharmacologically acceptable salts.

(i) Alkylation activator: This preparation has the effect of introducing an alkyl group into the nucleic acid protein of cancer cells to cause cell damage. For example, carbo, kon, psulfan (mustard drug), dimustine ( Nitrosourea)).

(ii) Antimetabolite active agent: This preparation has the action of antagonizing enzymes in the metabolic process and inhibiting cell synthesis, such as methotrexate (folic acid), mercaptopurine, (purine), Examples include cytarabine (pyrimidine), fluorouracil, tegafur, and carmofur.

(iii) Antibiotic: Examples include anti-cancer activity, actinomycin D, bleomycin, adriamycin, mitomycin C and the like.

(iv) Microtubule inhibitory active agent: This preparation acts on microtubules and exhibits an antitumor effect. For example, docetaxel, paclitaxel (taxane), vinorelbine, pinklistin, vinblastine (alkaloid) Can be mentioned. (v) Platinum preparation: This preparation has an action of inhibiting DNA synthesis by constituting intra-DNA chain or inter-chain bond or DNA protein bond, and examples thereof include cisplatin, alpoplatin, nedaplatin and the like. .

(vi) Topoisomerase inhibitor: Irinotecan (topoisomerase I inhibitor), podophyllotoxin derivative (tobosomerase II inhibitor), which inhibits topoisomerase. What is topoisomerase?

It is an enzyme that catalyzes a reaction that temporarily cuts DNA and changes the linking number of the DNA strand. In particular, among the antitumor agents described above, the antitumor agent in the present invention is preferably a microtubule inhibitory activity '"herbal agent and a platinum preparation, and more preferably docetaxel and cisbratin, but is not limited thereto. It is not a thing.

Docetaxel is classified as a taxane among the above microtubule inhibitors and is mainly applied to the treatment of breast cancer and non-small cell lung cancer. Docetaxel is obtained as a semi-synthetic product from Sewyichii needle extract, which promotes polymerization with tubulin and suppresses microtubule depolymerization as well as microtubule formation and stops cell mitosis. It has a function.

Cisbratin (CDDP) is mainly applied to the treatment of testicular tumor, bladder cancer, renal pelvic and ureteral tumor, prostate cancer, ovarian cancer, 'head and neck cancer, lung cancer,' esophageal cancer, cervical cancer. The types of tumor (cancer) cells to which the antitumor agent resistance-resolving agent of the present invention acts are not limited, and all types of tumor cells can be used. For example, solid cancer in the head and neck, stomach, large intestine, lung, liver, prostate, sputum, esophagus, bladder, gallbladder or bile duct, breast, uterus, thyroid, ovary, etc., or leukemia, lymphoma, sarcoma, mesenchymal tumor This is effective. Most of the tumor cells derived from chick tissue show an increase in telomerase activity, and the antitumor agent resistance overcomer of the present invention generally acts on tumor cells whose proliferation has been activated by such telomerase activity. sell. Particularly effective for large intestine, lung, stomach, esophagus, liver, prostate, etc.

As the pharmacologically acceptable salts of the above-mentioned antitumor agents, inorganic acid salts (for example, hydrochloride, hydrobromide, sulfate, nitrate, perchlorate, phosphate), organic acid salts (for example, vinegar Acid salt, trifluoroacetate, maleate, oxalate, tartrate, malonate, succinate, fumarate, sulfonate, malate, etc.), organic sulfonate (eg, methanesulfonate Salts, trifluoromethanesulfonate, ethanesulfonate, benzenesulfonate, toluenesulfonate, etc.), amino acid salts (eg aspartate, glutamate, etc.), quaternary amine salts, alkali metal salts (eg, Sodium salt, strong salt, etc.), alkaline earth metal salt (eg, magnesium salt, calcium salt, etc.).

4. Antitumor drug resistance overcomer

(1) The antitumor agent resistance overcomer of the present invention is characterized in that it comprises a recombinant virus in which a polynucleotide containing a human telomerase promoter, an E 1A gene, an IRES sequence, and an E1B gene in this order is incorporated.

This is because the mechanism of action of cell death by telomerisin on tumor cells is different from the mechanism of action of apoptosis caused by conventional anticancer drugs. This is achieved by the nature of telomerisin, such that even when treated, the cells can proliferate and replicate in the cells without any influence.

The anti-tumor agent resistance overcomer of the present invention can be applied to the affected area as it is, or any known method, for example, injection such as vein, muscle, intraperitoneal or subcutaneous, or nasal cavity, oral cavity or It can also be introduced into a living body (target cells or organs) by inhalation from the lung, oral administration, intravascular administration using a catheter or the like.

In addition, it can be used as it is after handling or squeezing by a method such as freezing, or it can be used as it is, or a known pharmaceutically acceptable carrier such as an excipient, a bulking agent, a binder, a lubricant, etc. (Including buffering agents, tonicity agents, chelating agents, coloring agents, preservatives, fragrances, flavoring agents, sweetening agents, etc.) and the like.

The anti-tumor agent resistance overcomer of the present invention includes tablets, capsules, powders, granules, pills, liquids, syrups and other oral administration agents, injections, external preparations, suppositories, eye drops, etc. Depending on the dosage form, it can be administered orally or parenterally. Preferably, local injection into muscles, abdominal cavity, etc., injection into veins, etc. are exemplified. The dose is appropriately selected according to the type of active ingredient, administration route, administration subject, patient age, body weight, sex, symptom and other conditions. The daily dose of the virus included in the present invention is 10 ⁶ to about lOnPFl plaque forming units), preferably about 10 ⁹ to lOuPFU, can be administered once a day, or can be divided into several times. Further, when using the virus of the present invention, it is possible to suppress the immunity of the living body by using a known immunosuppressive agent or the like so that the virus can be easily infected.

(2) Pharmaceutical composition for combination therapy ''

If a recombinant virus incorporating a polynucleotide containing the human telomerase promoter, E1A gene, IRES sequence and ひ 1Β gene in this order in combination with a substance having an antitumor activity is used in combination, the antitumor substance The therapeutic effect on tumors (cancers) that have acquired resistance to cancer is restored. Therefore, the virus of the present invention can be used as a pharmaceutical composition in combination with at least one substance having antitumor activity.

The substance having an antitumor action of the present invention refers to the growth and proliferation of tumor cells (cancer cells) that become tumor masses as a result of cell proliferation that is disrupted due to disorder of the body's regulatory mechanism and cell overgrowth. Substances that have the action of suppressing cancer, including substances that suppress the nucleic acid synthesis of cancer cells or inhibit metabolism by inhibiting metabolism. Specific examples include the following.

(i) Substances having alkylation activity: Carbocon, psulfan, dimustine, etc.

(ii) Substances with antimetabolite activity: methotrexate, mercaptopurine, cytarabine, funoleorula Λ ^, tegafunore, canolemofunole, etc.

(iii) Antibiotics: actinomycin D, bleomycin, adriamycin, mitomycin C, etc.

(iv) Substances having microtubule inhibitory activity: docetaxel, paclitaxel, pinolenolevin, pinklistin, vinblastine, etc. (v) Substances having topoisomerase inhibitory activity: irinotecan, podophyllotoxin derivatives, etc. The substances having antitumor activity include pharmaceutically acceptable salts. Examples of such salts include the salts described above.

The dose of the substance having an antitumor activity or a pharmaceutically acceptable salt thereof included in the present invention also depends on the type of active ingredient, administration route, administration subject, patient age, body weight, sex, symptom and other conditions. It is selected appropriately. For example, in the case of docetaxel, usually, once a day in adults, 60mg / m ² of body surface area over a period of over one hour more than ¹ intravenous drip infusion at 3-4 week intervals. The dose may be adjusted according to the symptoms. However, the highest dose once a 70mg / m ^2.

In the case of vinorelbine, for example, 2.0 to 2.5 mg / m ² can be intravenously administered intravenously at weekly intervals. In the case of irinotecan, for example, irinotecan hydrochloride is usually administered to an adult once a day, 150 mg / m ² intravenously 3-4 times at 2-week intervals, and the drug is withdrawn for at least 3 weeks. This can be repeated as one course.

Thus, the combined use of the recombinant virus contained in the antitumor agent regimen overcomer of the present invention and the substance having an antitumor action can suppress the growth of tumor cells. The mode of combination is not limited to the mode in which the recombinant virus and the substance having an antitumor effect are mixed and administered at the same time, and the mode in which either one is administered first and the other is administered later is also possible. included. As long as both a recombinant virus and an antitumor substance are contained in one treatment schedule, they are included in the “combination” in the present invention. Specifically, the following treatment methods are mentioned, for example. (i) A substance having antitumor activity can be administered after telomerisin is administered. From experiments using cultured cells, it is clear that a remarkable combined effect is observed by this method. (ii) Telomerisin may be administered intratumorally and at the same time a substance having antitumor activity may be administered systemically. In animal experiments, a clear concomitant effect is recognized by simultaneous administration as described above.

(iii) It is also possible to administer telomerisin after administering the substance having antitumor activity. Administration of substances with anti-tumor activity may be short-term or long-term.

(3) The antitumor agent resistance overcomer 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.

(i) Normal somatic cells have almost no living lipase activity, and floating cells such as hematopoietic cells are less susceptible to infection with the virus of the present invention.

(ii) Since the virus of the present invention has a proliferative ability, it can be used at a lower concentration than a non-proliferative virus used in normal gene therapy.

(iii) Even when the virus of the present invention is excessively administered, the antiviral action is exerted by the normal immune action in the living body.

(4) The antitumor action of the antitumor agent resistance overcomer of the present invention can be examined by examining as follows. ''.

In order to analyze the antitumor effect of the antitumor drug resistance overcomer of the present invention on cancer cells, for example, an XTT assay can be performed.

XTT (2,3 bis [2-Methoxy 4-nitro-5-sulfpphenyl] -2H-tetrazolium-5-carboxyanilide inner salt) assay measures the activity of living cells by dehydrogenase of mitochondria in living cells This is a suitable method for monitoring cytotoxicity in vitro. The following is a detailed explanation of the XTT Atsay.

The XTT solution shows a yellow color when it does not contain phenol red or when dissolved in a balanced salt solution. When this XTT solution comes into contact with viable cells, the mitochondrial dehydrogenase of the viable cells cleaves the tetrazolium ring of XTT and becomes soluble in aqueous solution. Colored formazan crystals are produced. In fact, since the biological reduction of XTT is insufficient, an electron conjugate substance such as phenazine methosulfate (PMS) is often added to the reaction solution as a reduction accelerator. Then, the cytotoxicity of the desired substance can be measured by measuring the resulting orange solution by the colorimetric method by utilizing the change in the amount of formazan produced by increasing or decreasing the number of cells. In the analysis of the anti-tumor drug resistance overcomer of the present invention, the recombinant virus of the present invention is infected at an appropriate concentration (MOI: multiplicity of infection) to various cancer cells cultured in vitro. After culturing the cells under appropriate conditions, various concentrations of antitumor agents are administered to the virus-infected cell culture. After virus infection, after culturing for an appropriate period, the above XTT assembly can be performed to analyze the antitumor effect.

Adenoviruses infect and enter cells via the Coxsackie virus adenovirus receptor (CAR), but the efficiency of virus infection is reduced in cancer cells that are negative for CAE. Thus, the more CAR expression in cancer cells, the easier adenovirus enters the cancer cells. Whether or not CAR is expressed in tumor cells may be determined by analyzing CAE expression using flow cytometry after culturing various tumor cells under appropriate conditions. The anticancer drug FR901228 (Fujisawa Pharmaceutical Co., Ltd. (currently Astellas Pharma Inc.)) can enhance the expression of CAR in cancer cells. Therefore, when FR901228 is used in combination with the recombinant adenovirus of the present invention, In addition to the anti-tumor action of FR901228, it is preferable in that it also has the action of improving the infection efficiency of the combined recombinant adenovirus in tumor cells.

Flow cytometry is a method of measuring the size of individual cells, DNA content, etc. in a cell population by measuring the fluorescence emitted from individual particles by flowing a cell suspension at high speed. Flow cytometry not only analyzes the differences in the relative size, shape, and internal structure of each cell, but also measures fluorescence intensity and type of fluorescence by fluorescent labeling to identify cells. And the abundance ratio of various cells constituting the cell group can be analyzed in a short time.

Since the cell membrane cannot maintain its state due to cell death, the nucleus is easily stained with PI (propidium iodide) described later. Therefore, the amount of DNA and the cell cycle can be determined from the result of nuclear staining with PI. The effect of the recombinant virus in the anti-tumor drug resistance overcoming agent of the present invention on the target cell cycle is determined by PI using the flow cytometry described above to stain the nucleus. Can be analyzed. Tumor cells are considered to be highly malignant when the proportions of S phase and G2 / M phase in the cell cycle are high. Therefore, it can be said that an antitumor effect is observed if a result that suppresses the ratio of G2 / M phase is obtained.

The proliferation of the recombinant virus in the target cell of the antitumor agent resistance overcomer of the present invention can be measured, for example, using quantitative real-time PCR as follows. In other words, after culturing the recombinant virus with an anti-tumor agent for an appropriate period, collect the virus-infected cells, extract the DNA, and use real-time PCR with primers that target the appropriate gene of the virus. By performing the above, it is possible to quantitatively analyze an appropriate gene possessed by the virus. At this time, for example, if a gene encoding a fluorescent substance is incorporated into the recombinant virus of the present invention, cells in which the virus has been propagated are irradiated with excitation light to give a predetermined fluorescence (for example, in the case of GFP). Emits green fluorescence), allowing virus growth in cells to be visualized. For example, when a virus-infected cell is observed under a fluorescence microscope, GFP fluorescence expression is seen in the cell. It is also possible to observe virus-infected cells over time by observing the expression of GFP fluorescence over time using a CCD camera. If such a recombinant virus is administered in vivo, Cells can also be labeled and detected in real time in the body. In the following, the present invention will be further described by way of examples. However, the present invention is not limited to these examples.

Example 1

RT-PCR was performed under the following conditions using RNA extracted from human fetal kidney cells 293 cells using a known method and the following specific primers (E1A-S, El A-AS). The bp E1A gene was amplified. Each primer sequence:

E1A-S: 5'-aca ccg gga ctg aaa atg ag-3 '

E l A- AS: 5'-cac agg ttt aca cct tat ggc-3 '(SEQ ID NO: 6) PCR solution composition: lxPCR' buffer

0.2 mM for each dNTP

5 mM MgCl ₂

2.5U AmpHTaq Gold

0.2 μΜ per primer, reaction condition: 95 ° C for 10 minutes ―

95 ° C 1 minute → 56 ° C 1 minute → 72 ° C 1.5 minutes (x32 cycle) 72 ° C 7 minutes.

DNA-PCR was performed from the DNA extracted from 293 cells using the following primers (E 1B-S, E 1B-AS) at 4 ° C for 5 min. To amplify the l, 822 bp E 1B gene . The PCR solution composition and reaction conditions (cycle, temperature) were the same as for the E1A gene. Each primer sequence: '

E1B-S: 5'-ctg acc tea tgg agg ctt gg-3 '(SEQ ID NO: 7)

E1B-AS: 5 'gcc cac aca ttt cag tac ctc-3' (SEQ ID NO: 8) TA Cloning of each PCR product (TA Cloning Kit Dual Promoter;

After confirming the sequence, DNA fragments of 911 bp (ElA) and 1836 bp (ElB) were cut out with the restriction enzyme EcoRI.

E1A was inserted into the Mul cleavage site of pIRES Vector I (CLONTECH), and E1B was inserted into the Sail site in the forward direction (E1A-IRES-E1B). A 455 bp hTERT promoter sequence excised with restriction enzymes Mul and Bglll was inserted into the Xhol site upstream of E 1A of E 1A IRES-E 1B in the forward direction (phTERT-E 1A- IRES-E 1B)

The cytomegalovirus (CMV) promoter contained in the pShuttle vector was removed by treatment with restriction enzymes Mfel and Nhel, and a 3828 bp sequence excised from phTERT-ElA-IRES-ELB with restriction enzymes Nhel and Notl was inserted at that site. (PSh- AIB)

A 4,38 lbp sequence was excised from pSh-hAIB using restriction enzymes I-Ceul and Pl-Scel and inserted into Adeno'X Viral DNA of Adeno-X Expression System (CLONTECH) † (AdenoX hAIB) AdenoX-hAIB After linearization by treatment with the restriction enzyme Pacl, 293 cells were transfected to produce an infectious recombinant adenovirus (OBP-301; Telomelysin; Telomelysin). Figure la shows a schematic diagram of the duplicate cassette incorporated into telomerisin.

Furthermore, pEGFP-l (CLONTECH) was cleaved with Agel / Nhel, smoothed with a talenau fragment, and self-ligated (pEGFP-N2).

This pEGFP-N2 was cleaved with Nsil / Aflll, blunted with T4 DNA polymerase, and Bglll site was prepared using Bglll linker. This Bglll fragment was inserted into the BamHI site of pHMl l (pHMl l-EGFP-N2)

Furthermore, the Csp45I fragment of pHMll-EGFP-N2 was inserted into the Clal site of the pShuttle vector (pSh-hAIB) incorporating phTERT E1A-IRES-E1B.

A 4381 bp sequence was excised from the prepared recombinant gene using restriction enzymes I-Ceul and PI-SceI and inserted into Adeno—X Viral DNA of Adeno—X Expression System (CLONTECH) (AdenoX—hAIB) AdenoX—hAIB After linearization by Pacl treatment, 293 cells were transfected to produce infectious recombinant adenovirus (hereinafter referred to as “OBP-401”) (Fig. Lb). When the gft) gene expressed by the CMV promoter is inserted into the E3 region, cancer cells can be visualized as the virus grows by fluorescent coloration of the gene product.

Example 2 <Confirmation of anti-tumor resistance to CDDP and paclitaxel>

Human squamous cell carcinoma A431 cells and A431 / CDDP1, which has acquired resistance

CD431 (cisplatin) is administered to A431 / CDDP2 cells, human prostate cancer cells DU145 cells, and clone 2, 3, and 4 cells that have acquired resistance are administered paclitaxel, and how much antitumor resistance each cell has Confirmed whether to win. Specifically, cells are seeded at a density of 1,000 cells / well in a 96-well plate, and CDDP 0, 0.1, 1, 3, 10, 30, 50, ΙΟΟμΜ or paclitaxel 0, 0.1, 1, 2, 5, 10, 50, 100 ηΜ was added. On the third day after infection, a kit (Roche Diagnostics) was used. Specifically, the culture solution is removed from the well, and the reaction solution containing the sputum reagent is prepared and added and cultured. About 4 hours later, the absorbance was measured with a microtiter plate (ELISA) reader, the number of viable cells was calculated, and the antitumor effect was confirmed.

As a result, the relative resistance of the parent strain was about 3-4 times higher in the A431 system and about 6-7 times higher in the DU145 system (Fig. 2).

Moreover, A431 and CDDP resistance A431 strain, the DU145 and paclitaxel-resistant DU145 strain was CDDP 10 _mu Micromax respectively, paclitaxel ΙΟηΜ infected. The specimen was observed with a microscope on the third day after infection. As a result, a marked antitumor effect was observed in both parental strains, but resistance was confirmed in resistant strains (Fig. 3). In Fig. 3a, A431 / P is the parent strain, A431 / CDDP1 and A431 / CDDP2 are resistant strains. In Fig. 3b, Dul45 / P is the parent strain and clones 2-4 are resistant strains. '

A431 and CDDP resistant strains, DU145 and paclitaxel resistant strains were infected with CDDP and paclitaxel, respectively, and CBB staining was performed 7 days after infection. As a result, both the parent strains showed a marked antitumor effect, but the resistant strains were also resistant to high concentrations of anticancer drugs (Fig. 4).

Next, in order to investigate the molecular mechanism of anti-tumor resistance, the expression of multidrug resistance gene (MDR1) in each resistant cell was analyzed. Specifically, a protein fraction is extracted from each cancer cell, electrophoresed in a gel, and then transferred to a membrane. The membrane was reacted with an anti-MDR1 antibody, and the expression of the MDR1 protein was compared by coloring the antibody. As a result, both A431 and DU145 MDR1 expression was hardly observed in the cell lines, and it was considered that resistance was not acquired by MDR1 expression (Fig. 5).

The expression of the Coxsaki virus-adenovirus receptor (CAR) required for adenovirus entry into cells was confirmed using flow cytometry.

As a result, significant CAR expression was observed in all cell lines used in the experiment (Fig. 6).

Example 3

<OBP-301 and OBP-401 anti-tumor effect on anti-tumor drug resistant cells>

96-well plates were seeded with A431 and DU145 cells at 1,000 cells / tool, and OBP-301 was infected with mock, 0.01, 0.1, 1, 5, 10, 20 MOI. On the fifth day after infection with OBP-301, an XTT assay was performed. As a result, although a slight resistance was observed with A431 / P, a marked cell killing effect was observed in almost all cell lines. In DU145, both the parental strain and the resistant strain showed a marked cell killing effect by OBP-301 (Fig. 7, Table 1).

In addition, XTT assay was performed on days 1, 2, 3, 5, and 7 after OBP-301 infection. As a result, although a slight resistance was observed with A431 / P, rapid cell-killing effects were observed on days 1 to 3 in all resistant cell lines. In DU145, OBP-301 showed a rapid cell killing effect in both the parent and resistant strains (Fig. 8). Then, 24 cells were seeded at 10,000 cells / well and infected with OBP-301 at mock, 0.01, 0.1, 1, 10 and 50 MOI. Seven days after infection, Coomassie Brilliant Blue (CBB) staining was performed and the antitumor effect was examined. As a result, A431 / P was slightly resistant, but a resistant cell killing effect was observed in the resistant strain. In DU145, both the parent strain and the resistant strain showed a significant cell killing effect by OBP-301 (Fig. 9). table 1

Characterization of drug resistance sublines

IC50 (MOI) OBP-301 IC50 (MOI) OBP-301

A431 / P 7.14 DU145 / P 0.81, clone2 0.71

A431 / CDDP1 6.57

clone3 0.67

A431 / CDDP2 3.92 clone4 0.62

A431 and CDDP resistant strains were infected with OBP-301 and OBP-401 at 10 MOI for 2 hours. 5 days after infection Morphological changes and fluorescence expression were observed with an fc fluorescence inverted microscope. As a result, in the bright field, although it was slightly resistant to A431 / P, a marked cell killing effect was observed in the resistant strain. In addition, when the fluorescence of OBP-401 was observed, strong fluorescence was observed in all cell lines, indicating that intracellular proliferation was sufficient (FIG. 10). '

DU145 and paclitaxel resistant strains were infected with OBP-301 and OBP-401 at 0.1 MOI for 2 hours, and observed with a microscope on the fifth day after infection. As a result, in the light field, a marked cell killing effect was observed in all parental and resistant strains. As for fluorescence, strong fluorescence was observed in all cell lines, indicating that intracellular proliferation was sufficient (Fig. 11).

Quantitative real-time: ABP and 301 were infected with OBP-301 at 0.1 MOI or 1 MOI for 2 hours using PCR. Cells were collected at 2, 24, 48, and 72 hours after infection, DNA was extracted, and viral growth was quantitatively analyzed by real-time PCR.

El A primer sequence: Forward: 5 'CCT GTG TCT AGA GAA TGC AA-3' (SEQ ID NO: 9) Reverse: 5'-ACA GCT CAA GTC CAA AGG TT-3 '(SEQ ID NO: 10)

PCR solution composition lxLC FastStart DNA Master SYBR Green I

3 mM MgCl ₂

For each primer 0.5 / z M Reaction condition 95 ° C 10 min

95 ° C 10 seconds → 60 ° C 15 seconds' 72 ° C 8 seconds (χ40 cycle)

70 ° C 15 seconds

As a result, it grew rapidly 24 hours after infection, and reached its peak value at about 48 hours. In addition, the proliferation efficiency was remarkable in all cell lines (Fig. 12). Industrial applicability

INDUSTRIAL APPLICABILITY According to the present invention, there is provided an antitumor agent resistance overcome agent comprising a recombinant virus in which a polynucleotide comprising a human telomerase promoter, an E1A gene, an IRES sequence and an E1B gene in this order is incorporated. The anti-tumor drug resistance overcomer of the present invention is useful in that it can be applied as a new therapeutic strategy for anti-tumor drug resistant tumors. Sequence listing free text

Sequence number 5: Primer

Sequence number 6: Primer

Sequence number 7: Primer

Sequence number 8: Primer

Sequence number 9: Primer

Sequence number 10: Primer

Claims

Scope of claim Antitumor drug resistance-resolving agent comprising a recombinant virus in which a polynucleotide comprising a human telomerase promoter, an E1A gene, an IRES sequence, and an E1B gene in that order is incorporated. '

The antitumor agent resistance overcomer according to claim 1, wherein the promoter of human telomerase is hTERT.

3. The antitumor agent resistance overcomer according to claim 1 or 2, wherein the virus is an adenovirus. Antitumor agents include platinum preparations, microtubule inhibitory activators, alkylating activators, antimetabolite activators, anticancer antibiotics and topoisomerase inhibitory activators and their pharmacologically acceptable salts. The antitumor agent resistance overcomer according to any one of claims 1 to 3, which is at least one selected from the group consisting of:

Antitumor agents are docetaxel, paclitaxel, cisplatin, psulfan, methotrexate, fluorouracil, tegafur, bleomycin, adriamycin, mitomycin C, vinorelbine and irinotecan and their pharmacologically acceptable The antitumor agent resistance overcomer according to any one of claims 1 to 3, which is at least one selected from the group consisting of salts.

Tumor is lung cancer, colon cancer, stomach cancer, breast cancer, esophageal cancer, head and neck cancer, liver cancer, pancreas cancer, gallbladder or bile duct cancer: prostate cancer, bladder cancer, cervical cancer, thyroid cancer, ovarian cancer, leukemia, lymphoma 6. The antitumor agent resistance overcome agent according to any one of claims 1 to 5, which is at least one selected from the group consisting of sarcoma and mesenchymal tumor.

A combination of a recombinant virus incorporating a polynucleotide containing the human telomerase promoter, E1A gene, IRES sequence, and E1B gene in this order, and a tumor that has acquired antitumor drug resistance, including a substance having antitumor activity A pharmaceutical composition for therapy.

The substance having an antitumor activity is at least one selected from the group consisting of docetaxel, vinorelbine, irinotecan, and histone deacetylase inhibitory activity inhibitor and pharmacologically acceptable salts thereof. 8. A pharmaceutical composition according to item 7.

9. A tumor that has acquired anti-tumor drug resistance, characterized by administering to a mammal a recombinant virus incorporating a polynucleotide comprising a human telomerase promoter, an E1A gene, an IRES sequence, and an E1B genetic sequence in this order. A method of inhibiting cell growth.

10. Recombinant virus in which a polynucleotide comprising a human telomerase promoter, E1A gene, IRES sequence and E1B gene in this order is incorporated, and an antitumor substance or a pharmacologically acceptable product thereof A method for inhibiting the growth of tumor cells that have acquired antitumor drug resistance, comprising administering to a mammal in combination with a salt. ,

1 1. Anti-tumor drug resistance characterized by administering a recombinant virus containing a polynucleotide containing a human telomerase promoter, E1A gene, IRES sequence, and E1B gene in this order to a mammal. How to treat a tumor that has acquired.

1 2. The method according to any one of claims 9 to 11, wherein the promoter of human telomerase is hTERT.

13. The method according to any one of claims 9 to 12, wherein the virus is an adenovirus.

1 4. Antitumor agents are platinum preparations, microtubule inhibitory activators, alkylating activators, antimetabolite activators, anticancer antibiotics and toposomerase activators and their pharmacologically acceptable salts. The method according to any one of claims 9 to 13, wherein the method is at least one selected from the group consisting of:

1 5. Antitumor agents are docetaxel, paclitaxel, cisplatin, psulfan, methotrexate, funoleorula / les, tegafur, bleomycin, adriamycin, mitomycin C, vinorelbine and irinotecan and their pharmacologically acceptable The method according to any one of claims 9 to 13, wherein the method is at least one selected from the group consisting of salts.

1 6. The tumor is lung cancer, colon cancer, stomach cancer, breast cancer, esophageal cancer, head and neck cancer, liver cancer, pancreas cancer, gallbladder or bile duct cancer, prostate cancer, bladder cancer, cervical cancer, thyroid cancer, ovarian cancer, The method according to any one of claims 9 to 15, which is at least one selected from the group consisting of leukemia, lymphoma, sarcoma, and mesenchymal tumor.