JP2002218975A - Attenuated simple herpesvirus - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain an attenuated simple herpesvirus effective for cancer treatment and a composition for cancer treatment containing the same. SOLUTION: This attenuated simple herpesvirus is incorporated with an extraneous suicide gene.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to an attenuated herpes simplex virus effective for treating cancer and a composition for treating cancer containing the same.

[0002]

2. Description of the Related Art Generally, surgical resection, chemotherapy, radiation therapy and the like are performed as treatments for cancer. However, there are some cancers in which these treatments do not provide a sufficient effect. For example, a satisfactory prognosis has not yet been obtained for advanced pancreatic cancer and advanced ovarian cancer. In particular, prognosis by surgical resection cannot be expected for advanced pancreatic cancer or advanced ovarian cancer with peritoneal dissemination.

For this reason, gene therapy has been attempted as a new treatment method for cancer. As gene therapy for cancer, (1) a method of suppressing oncogenes using antisense or ribozyme, or a method of suppressing tumor cell growth by introducing a tumor suppressor gene, (2) a metabolic toxic gene (suicide) Gene) to drive tumor cells to suicide, (3) a method to enhance anti-tumor immunity by the transgene, and (4) a bone marrow stem cell using a multidrug resistance gene to improve the effect of chemotherapy. Methods for protection are being studied.

As the method (2), there is known a method using thymidine kinase of herpes simplex virus as a suicide gene. When ganciclovir is administered after the introduction of the herpes simplex virus thymidine kinase into the cancer cells, the ganciclovir is phosphorylated by the herpes simplex virus thymidine kinase to become an active form, thereby inhibiting the DNA polymerase of the cancer cells. Therefore, by introducing thymidine kinase of herpes simplex virus into cancer cells, the growth of cancer cells can be suppressed, and the cancer cells can be killed.

[0005] Since herpes simplex virus is a pathogenic virus, the wild-type herpes virus cannot be directly used for treating cancer. For this reason, it has been studied to attenuate herpes simplex virus and use it for the treatment of cancer (WO
96/39841).

[0006]

SUMMARY OF THE INVENTION In view of the above circumstances, an object of the present invention is to provide an attenuated herpes simplex virus effective for treating cancer and a composition for treating cancer containing the same. .

[0007]

SUMMARY OF THE INVENTION The present invention is an attenuated herpes simplex virus into which an exogenous suicide gene has been incorporated. In the present specification, the gene or DNA nucleotide sequence has a range that does not affect its function.
Also included are deletions, substitutions, modifications, insertions, and the like in a portion of the DNA base sequence. Hereinafter, the present invention will be described in detail.

The attenuated herpes simplex virus of the present invention (hereinafter, also referred to as attenuated HSV) is characterized in that an exogenous suicide gene is incorporated therein. By incorporating the exogenous suicide gene, the attenuated HS of the present invention
V can efficiently convert prodrugs to the active form, inhibit the growth of cancer cells, and even kill them. The attenuated HSV of the present invention may be one produced using HSV-1 or one produced using HSV-2.

The exogenous suicide gene is not particularly limited as long as it can convert prodrug into an active form. Examples thereof include cytosine deaminase gene,
Escherichia coli gpt gene, Escherichia coli deoD gene, carboxylesterase gene and the like can be mentioned. Among them, the carboxylesterase gene is preferred.

[0010] Carboxylesterase can convert the prodrugs taxol and camptothecin into active forms. Infecting cancer cells with attenuated HSV into which the carboxyesterase gene can be expressed so that the infected cancer cells become selectively highly sensitive to anticancer drugs such as taxol and camptothecin, and activated. The given drug can inhibit the growth of the surrounding cancer cells, and can exhibit a high cancer therapeutic effect. In the present specification, “to be incorporated so as to be capable of being expressed” means, for example, that a target gene is incorporated downstream of the promoter sequence as exemplified above.

The carboxyesterase gene is not particularly restricted but includes, for example, those derived from humans, mice, rats and rabbits. Among them, rabbit-derived carboxyesterase is preferable because of its high activity.

The attenuated HSV of the present invention has one or more accessory genes inactivated,
Those having cancer cell selectivity are preferred. As the attenuated HSV of the present invention, one in which one or more accessory genes are inactivated and has a cancer cell selectivity is used, so that the attenuated HSV of the present invention can proliferate in cancer cells in a superior manner. Therefore, the exogenous suicide gene can be efficiently expressed in the target cancer cell, and the anticancer agent can be selectively acted on. In the present specification, "having cancer cell selectivity" refers to the growth rate in normal cells,
It means that the growth rate in cancer cells is superior.

The accessory genes include RL
1, RL2, ORFP, ORFO, RL3, UL2, U
L3, UL4, UL7, UL10, UL11, UL1
2, UL13, UL16, UL20, UL20.5, U
L21, UL24, UL39, UL40, UL41, U
L43, UL43.5, UL44, UL45, UL4
6, UL47, UL50, UL51, UL55, UL5
6, US1, US1.5, US2, US3, US4, U
S5, US7, US8, US8.5, US9, US1
0, US11 and US12.

[0014] The attenuated HSV of the present invention preferably has cancer cell selectivity so that it can proliferate predominantly in cancer cells. Attenuated HSV in which one or more accessory genes have been inactivated include, for example, UL3
9 or UL40 are inactivated, and among them, those in which UL39 or UL40 and one or more other accessory genes are inactivated are preferable.

Attenuated HSV in which UL39 or UL40 has been inactivated is encoded by this gene, and
The ribonucleotide reductase required for A synthesis must be host dependent. For this reason, UL39 or UL
Inoculation of a cancer patient with attenuated HSV, in which 40 is inactivated, allows it to grow predominantly in actively growing cancer cells. The growth of cancer cells infected with such attenuated HSV is suppressed by cytotoxicity of various proteins possessed by the virus, and the cancer cells may even die.

Inactivation of one or more other accessory genes, in addition to UL39 or UL40, can maintain superior growth in cancer cells and further attenuate host virulence. be able to.

Attenuated HSV in which UL39 or UL40 and one or more accessory genes are inactivated include, for example, UL39 (or UL4).
0), attenuated HSV with UL55 and UL56 inactivated, UL39 (or UL40) and attenuated HSV with UL2 inactivated, UL39 (or UL4).
0) and attenuated HSV, U3 in which US3 is inactivated
Attenuated HSV in which L39 (or UL40), UL2 and US3 are inactivated can be mentioned.

Attenuated HSV in which UL39 (or UL40), UL55 and UL56 are inactivated, and UL3
Attenuated HSV in which 9 (or UL40) and UL2 are inactivated have even higher cancer cell selectivity than attenuated HSV in which only UL39 (or UL40) is inactivated. On the other hand, UL39 (or UL40) and U
Attenuated HSV in which S3 is inactivated or UL39
(Or UL40), the attenuated HSV in which UL2 and US3 are inactivated can have a function of causing apoptosis of cancer cells in addition to high cancer cell selectivity.

The method for inactivating the accessory gene is not particularly limited. For example, insertion of another sequence,
Known methods such as excision of a part of the sequence, modification of the base, and substitution of the base can be mentioned.

The attenuated HSV of the present invention can be prepared, for example, by using an HSV vector in which a promoter sequence and a terminator sequence are incorporated and an accessory gene is inactivated, and the exogenous suicide gene described above is downstream of the promoter sequence. It can be obtained by insertion.

The promoter is not particularly limited as long as it functions as a promoter in tumor cells, but a tumor-specific promoter and an HSV-derived promoter are preferable. Examples of the tumor-specific promoter include oncofetal protein promoters such as CEA and AFP; tyrosinase promoter, albumin promoter, and stress-inducible GRP78 / Bip promoter. Examples of the HSV-derived promoter include, for example, HSV early protein ICP.
8 promoter, HSV UL39 promoter and the like. The HSV vector may further have an enhancer.

The attenuated HSV of the present invention can be used for treating cancer. The cancer is not particularly limited, and examples thereof include astrocytoma, oligodendrogliomas, meningioma, neurofibromas, glioblastomas, ependymomas, schwannomas, neurofibrosarcomas, and nerve medulla Blastoma, melanoma, pancreatic cancer, prostate cancer, breast cancer, lung cancer, colon cancer, gastric cancer, fibrosarcoma, squamous cell carcinoma, neuroectodermal cell carcinoma, thyroid tumor, pituitary tumor, lymphoma, liver cancer, mesothelioma And epidermoid carcinoma. Among them, it is suitably used for those having peritoneal dissemination, such as progressive pancreatic cancer and ovarian cancer, in which surgical resection and chemotherapy are difficult.

The inoculated form of the attenuated HSV of the present invention is not particularly limited, and known forms can be used.
Usually, a patient is inoculated as an injection consisting of an aqueous solution or suspension. The injections also include infusions, nutrient replenishers, and the like.

The above-mentioned injection may contain commonly used additives in addition to the attenuated HSV of the present invention. The additives are not particularly limited, and include, for example, water, salt, dextrose, glycerol, ethanol, propylene glycol, polyethylene glycol, vegetable oils, organic esters such as ethyl oleic acid, wetting agents, emulsifiers, p
Examples include an H buffer, an adjuvant, and an immunostimulant that enhances the effect of attenuated HSV. The injection may be a solid dissolved or suspended in a solution immediately before injection. A cancer treatment composition containing the attenuated HSV of the present invention, such as the above-mentioned injection, is also one of the present invention.

The attenuated HSV of the present invention can exhibit a certain anticancer effect by cytotoxicity of various proteins possessed by the virus, even if the exogenous suicide gene is removed. Among them, those capable of expressing thymidine kinase can activate ganciclovir, so that attenuated HSV expressing thymidine kinase can be used.
Is given to cancer patients and given ganciclovir,
The activated ganciclovir inhibits the DNA polymerase of the cancer cell and suppresses the elongation reaction of the DNA chain, so that the growth of the cancer cell is suppressed.

[0026] An attenuated herpes simplex virus having one or more accessory genes inactivated and excluding an exogenous suicide gene and having cancer cell selectivity is also an aspect of the present invention. One. Here, as the accessory gene, the same gene as described above can be used. A virus vector comprising the attenuated HSV of the present invention is also one of the present invention.

[0027]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples.

Example 1 <Preparation of attenuated HSV> (1) Preparation of UL39-inactivated HSV (hrR3) A DNA fragment containing the UL39 gene of HSV-1 was cloned into a plasmid pBluescript (Stratagene) and opened. The lacZ cassette (HSV UL3
9 promoter at the 5 'end, the lacZ gene of E. coli and the polyadenylation sign of SV40.
DNA into which DNA is inserted downstream of the DNA)
Was prepared. The DNA and the infectious HSV DNA prepared from the infected cells were transfected into cells, and three days later, the produced virus was collected. Among them, plaques stained blue in the presence of X-gal were collected, and plaque cloning was further performed three times.
o Proliferated in cells and used as stock. For this mutant, Southern blot, PCR, Western
Blots and the like were performed to confirm that UL39 was inactivated.

(2) Preparation of UL39, UL55 and UL56-Inactivated HSV (ReHF) There is a strain HF derived from HSV-1 in which the region containing the UL55 and UL56 genes is deleted. This strain is significantly less virulent in intraperitoneal inoculation in mice than the wild-type strain,
In addition, cell fusion occurs in infected cells. The UL39-deficient virus and the HF strain were mixedly infected into Vero cells,
Cell fusion from the offspring virus and X-ga
In the presence of l, a virus that produced a blue plaque was collected. After cloning, PCR and Western blot
By t, it was confirmed that UL39, UL55, and UL56 were defective.

(3) UL39-inactivated HSV (hrR
3) Preparation of strain in which carboxyelastase was incorporated into 3) DNA was prepared by inserting a human carboxyesterase gene having a promoter of human cytomegalovirus immediate-early gene into a lacZ gene (open reading frame). This DNA and the infectious DNA derived from UL39 inactivated virus having lacZ were
ro cells were transfected and the produced virus was collected. A virus that forms a colorless plaque in the presence of X-gal was collected, plaque-cloned, and used as a primary stock. For each clone obtained as a primary stock, an infected cell specimen was prepared, and a fluorescent antibody method was performed using a tag antibody, and a secondary stock was prepared for a positive clone. The induction of carboxylesterase activity in infected cells was confirmed for the finally obtained clone.

Example 2 <Cancer treatment using attenuated HSV> 1 × 10 ⁷ human ovarian cancer-derived SW1990 was intraperitoneally injected into 10 nude mice (female, 6 weeks old) in each group under ether anesthesia. It was injected and transplanted near the pancreas. On day 7 and day 12 after transplantation, hrR3 or ReHF was intraperitoneally injected into two groups, each at 1 × 10 ⁷ PFU / animal. 10 days after hrR3 or ReHF inoculation (ie, 17 and 22 days after transplantation)
, 0.4 mg / 1 ganciclovir was administered to each of the hrR3 inoculated group and the ReHF inoculated group. The survival rate of each group was observed up to 60 days after transplantation, and the results are shown in FIG.

From the results shown in FIG. 1, hrR3 and R
It became clear that inoculation with eHF prolonged the survival time of nude mice. Furthermore, it was revealed that the administration of ganciclovir further prolonged the survival period. Above all, the survival rate of the group administered with ReHF + ganciclovir was high.

Example 3 <Comparison of Attenuated HSV with Known Anticancer Agents> One human ovarian cancer-derived SW1990 was injected intraperitoneally into 10 nude mice (female, 6 weeks old) in each group under ether anesthesia. The cells were implanted in the vicinity of the pancreas by × 10 ⁷ and transplanted. On day 7 and day 12 after transplantation, hrR3 or R
eHF was injected at 5 × 10 ⁷ PFU / animal, or taxol was 0.4 mg / l as a known anticancer drug.
Animals. The survival rate of each group was observed up to 60 days after transplantation, and the results are shown in FIG.

From the results shown in FIG. 2, hrR3 and R
It was found that the group inoculated with eHF had a longer survival time than the group administered taxol.

[0035]

According to the present invention having the above-described constitution, it is possible to provide an attenuated herpes simplex virus effective for treating cancer and a composition for treating cancer containing the same.

[Brief description of the drawings]

FIG. 1 is a graph showing the effect of cancer treatment by inoculating the attenuated HSV of the present invention.

FIG. 2 is a graph comparing the cancer treatment effects of the attenuated HSV of the present invention and a known anticancer agent.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) // (C12N 15/09 (C12N 7/04 C12R 1:92) C12R 1:92) (C12N 7/04 C12N 15/00 A C12R 1:92) C12R 1:92) (72) Inventor Akihiro Nami 1-16-1 Omoyama, Tenpaku-ku, Nagoya-shi Takara Mansion Yokotominami 101 F-term (reference) 4B024 AA01 BA80 CA04 DA02 EA04 FA02 HA17 4B065 AA90X AA95X AA95Y AB01 BA02 BA14 CA44 CA45 4C084 AA13 NA05 NA14 ZB261 4C087 AA01 AA02 BC83 NA05 NA14 ZB26

Claims (9)

[Claims] 1. An attenuated herpes simplex virus characterized by incorporating an exogenous suicide gene. 2. An attenuated herpes simplex virus comprising a carboxylesterase gene incorporated therein. 3. The attenuated herpes simplex virus according to claim 1, wherein one or two or more accessory genes are inactivated and have cancer cell selectivity. 4. The attenuated herpes simplex virus according to claim 1, wherein UL39 or UL40 and one or more accessory genes are inactivated. 5. The attenuated herpes simplex virus according to claim 1 or 2, wherein UL39 or UL40, UL55 and UL56 are inactivated. 6. UL39 or UL40, UL2 and / or UL40.
Or the attenuated herpes simplex virus according to claim 1 or 2, wherein US3 is inactivated. 7. The attenuated herpes simplex virus according to claim 1, 2, 3, 4, 5, or 6, which is used for treating cancer. 8. The attenuated herpes simplex virus according to claim 7, wherein the cancer has peritoneal dissemination. 9. A composition for treating cancer, comprising the attenuated herpes simplex virus according to claim 1, 2, 3, 4, 5, 6, 7, or 8.