KR20150129639A - Method for production of naturally occurring vaccinia virus obtained by subculture various type of vaccinia virus with cancer cells derived from patient - Google Patents

Abstract

The present invention relates to a novel vaccinia virus HBB301 and an anti-cancer composition comprising the vaccinia virus as an active ingredient. The novel vaccinia virus HBB301 according to the present invention reduces toxicity against a host animal when compared to a wild-type strain, has an excellent effect on improving anti-cancer activity and thus can be helpfully used as an anti-cancer composition.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing naturally occurring anticancer viruses obtained by culturing a vaccine virus from a patient with a carcinoma isolated from a patient,

The present invention relates to a novel vaccinia virus HBB301 and a method for producing the same.

The first example of a medical use of a virus is the development of a vaccine against naturally occurring poxviruses for the prevention of smallpox. Until the development of a preventive vaccine using vaccinia virus, smallpox was the deadliest disease in human history, causing death at 10% for thousands of years and permanent damage to the skin at 50%. In the 1700s Edward Zenner first showed that a smallpox virus could become a vaccine that suppresses the onset of smallpox when it grows in cattle (vaccinia virus), producing cross-immunity with smallpox viruses, without lethal effects on humans . Since then, it has been named the vaccinia virus, and in 1979 the World Health Organization (WHO) became the occasion to declare that the smallpox disease was conquered.

As a result of recent molecular biologic developments, the genetic comparison between smallpox virus and vaccinia virus has shown that the two viruses are genetically very similar and that the vaccinia virus is the first virulent virus produced by a selective evolutionary method It was found to be a virus antivirus. Based on this principle, techniques for selectively evolving attenuated viruses in vitro have been used to develop preventive vaccines such as polio, measles, and mumps. The basic principle of the vaccine using the attenuated virus is that when the pathogenic virus is isolated from human cells and continuously cultured in xenogeneic cells such as monkeys, the virus selectively accumulating in the xenogeneic cells is selectively accumulated, Although the proliferation is good in xenogeneic cells, attenuated viruses are produced in human cells, and these attenuated viruses do not cause disease in the human body, but cause wild-type pathogenic viruses and cross-immune functions, .

In addition to the development of molecular biology technologies, various types of viruses that have been used as vaccines have been genetically engineered to develop anticancer drugs. David Kirn et al. Developed ONYX-015, which was used as an anticancer drug by using tumor-selective replication ability of adenovirus variants, and conducted clinical phase II studies. However, the anticancer effect of the primary tumor-killer adenovirus was not sufficient to be used clinically, and it was reported that the repeated administration of the tumor-associated adenovirus was rapidly eliminated by the host immune system and the anti-cancer effect was decreased. Despite these limitations, it can be seen that the therapeutic efficacy of the tumor-killer adenovirus principle has been commercialized in China, reflecting the expectation of the development of tumor-killer viruses with improved clinical efficacy. In recent years, ONYX-015 has been reported to have successfully developed a more potent anticancer virus through selective evolution, and clinical trials are currently being conducted in the US using the improved virus.

On the other hand, the tumor-killing virus derived from Vaccinia virus is superior to the adenovirus in terms of anticancer function. Currently, the multinational clinical phase 2 is completed in the US, Canada, and Korea for liver cancer, colon cancer and skin cancer patients. It is. However, when repeated administration of blood vessels occurs, the anticancer efficacy is deteriorated due to the formation of neutralizing antibodies, so that there is a limit in continuous administration, and development of more advanced tumor-specific viruses is urgently required.

Accordingly, the present inventors have conducted a study to develop a novel vaccinia virus which solves the above problems and has improved anticancer effect, and as a result, it has been found that human-derived gallbladder cancer cells are infected with wild vaccinia virus WR (Western Reserve) The new vaccinia virus HBB301, which was prepared by infecting the NYCBH (strain of the New York City Board of Health) and the recombinant vaccinia virus SLJ496 at a high dose, has a reduced toxicity to the host animal, And thus the present invention has been completed.

Korean Patent Registration No. 101179166

It is an object of the present invention to provide a novel vaccinia virus HBB301 and a method for producing the same.

In order to achieve the above object, the present invention provides a novel vaccinia virus HBB301 derived from vaccinia virus and a method for producing the same.

The novel vaccinia virus HBB301 according to the present invention has an excellent effect of reducing toxicity to host animals and improving anticancer activity compared with wild type strains, and thus can be usefully used as an anticancer composition.

FIG. 1 is a diagram illustrating a process for producing a new vaccinia virus according to the present invention.
FIG. 2 is a graph showing the IC ₅₀ of recombinant Vaccinia virus (3 mix P4) according to the present invention on human gallbladder cancer cells.
FIG. 3 is a graph showing the IC ₅₀ of recombinant Vaccinia virus (3 mix P4) according to the present invention on human ovarian cancer or lung cancer cells.
4 is a diagram showing the toxicity of HBB301 according to the present invention in a respiratory infected animal model.
5 to 7 are graphs showing antitumor activity of HBB301 according to the present invention in various cancer cell lines.
FIG. 8 and FIG. 9 are diagrams showing the results of comparison of nucleotide sequences of HBB301 and parent virus according to the present invention.

Hereinafter, the present invention will be described in detail.

The present invention provides a novel vaccinia virus HBB301.

The novel vaccinia virus HBB301 according to the present invention can be obtained through the following steps.

First, recombinant vaccinia virus SLJ496 obtained from wild-type vaccinia virus WR (Western Reserve) strain, NYCBH (New York City Board of Health) strain, and rabbit tumor model was added to human-derived gallbladder cancer cells isolated from gallbladder cancer patients, It is infected at a high dose so that it is at least three times that of the cultured cell line. When the above three viruses are simultaneously infected, mutation virus induction through mutual recombination between each vaccinia virus is more effective than infection with a single vaccinia virus. A recombinant virus that predominantly proliferates in the gallbladder cancer cells is identified and then isolated, and gallbladder cancer cells obtained from another gallbladder cancer patient are infected with the recombinant virus. The above procedure is repeated three times to finally obtain a recombinant virus. The human-derived gallium carcinoma cell line HuCCT-1 is infected with the recombinant virus, and the infected cell line is harvested in about 48 hours to recover the mutant virus HBB301, which is proliferated in the cytoplasm.

It was confirmed that the mutant virus HBB301 obtained through the above process was a novel virus having different genetic information from the parent virus, and deposited this on KCTC (Korean Collection for Type Culture) on Apr. 2, 2013 (accession number: KCTC 12391BP ).

The novel vaccinia virus HBB301 according to the present invention has an excellent effect of reducing toxicity to host animals and improving anticancer activity compared with wild type strains.

Accordingly, the novel vaccinia virus HBB301 according to the present invention can be used for medicines and health functional foods for prevention, improvement or treatment of cancer.

In addition, the present invention provides a composition for preventing or treating cancer comprising the vaccinia virus HBB301 as an active ingredient.

The composition includes both a pharmaceutical composition or a food composition.

The cancer may be, but not limited to, gallbladder cancer, colon cancer, pancreatic cancer, leukemia, ovarian cancer, stomach cancer, lung cancer, breast cancer, liver cancer, bronchial cancer, nasopharyngeal cancer, laryngeal cancer, skin cancer, colon cancer and cervical cancer.

The pharmaceutical compositions of the present invention may further comprise suitable carriers, excipients and diluents conventionally used in the manufacture of pharmaceutical compositions. In addition, it can be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, oral preparations such as syrups and aerosols, external preparations, suppositories and sterilized injection solutions according to a conventional method. Suitable formulations known in the art are preferably those as disclosed in Remington ' s Pharmaceutical Science, recently, Mack Publishing Company, Easton PA. Examples of carriers, excipients and diluents which may be included include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, Polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose, lactose, Gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, solutions, emulsions, syrups and the like. Various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included in addition to water and liquid paraffin, which are simple diluents commonly used. have. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories.

Administration in the present invention means providing the subject invention with a composition of the invention in any suitable manner.

The preferred dosage of the pharmaceutical composition of the present invention varies depending on the condition and body weight of the individual, the degree of disease, the drug form, the route of administration and the period of time, and can be appropriately selected by those skilled in the art. However, for the desired effect, the daily dose of the virus of the present invention may be administered in an amount of 10 ⁵ to 10 ⁹ pfu / kg per day. The dose is not intended to limit the scope of the invention in any way.

The pharmaceutical composition of the present invention may be administered to a subject in various routes. All modes of administration may be expected, and parenteral administration is preferred and may be administered using, for example, intravenous, intraperitoneal, intramuscular, subcutaneous or topical administration.

The composition of the present invention may further contain one or more known active ingredients having an effect of preventing or treating cancer.

The composition of the present invention can be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy and biological response modifiers for the prevention or treatment of cancer.

In addition,

(a) isolating recombinant Vaccinia virus that infects two or more Vaccinia viruses in cancer cells, cultivates and proliferates them, and then propagates in the cancer cells;

(b) culturing and propagating the isolated recombinant Vaccinia virus after infecting another cancer cell; And

(c) separating the recombinant vaccinia virus that is proliferating in the cancer cells of the step (b).

The vaccinia viruses to be infected in the step (a) may be selected from the group consisting of vaccinia virus WR (Western Reserve), vaccinia virus Copenhagen strain, vaccinia virus Wyeth strain, vaccinia virus NYCBH And the recombinant vaccinia virus SLJ496. Preferably, the strain is selected from the group consisting of Western Reserve virus (WR), New York City Board of Health (NYCBH) strain, Recombinant vaccinia virus SLJ496. As described above, when two or more kinds of vaccinia viruses are simultaneously infected to cancer cells, mutagenesis of vaccinia viruses through recombination between respective vaccinia viruses is more effective than infection with a single species vaccinia virus, It is possible to produce a novel vaccinia virus having excellent anticancer activity compared to the virus.

Although the method is not limited thereto, steps (b) and (c) may be repeated one or more times, preferably 2 to 100 times, more preferably 2 to 20 times, in order to induce mutation of Vaccinia virus You can repeat this.

The cancer cells may be selected from the group consisting of gallbladder cancer, colon cancer, pancreatic cancer, leukemia, ovarian cancer, gastric cancer, lung cancer, breast cancer, liver cancer, bronchial cancer, nasopharyngeal cancer, laryngeal cancer, skin cancer, colon cancer and cervical cancer May be a cell derived from the above cancer, preferably a gallbladder cancer cell.

The novel vaccinia virus is preferably HBB301 deposited with Accession No. KCTC12391BP.

The novel vaccinia virus obtained by the above method has an excellent effect of reducing the toxicity to the host animal and improving the anticancer activity as compared with the wild strain which is the parent virus.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the examples.

Example  1. According to the invention New  transition Vaccinia  Production of viruses

In order to obtain gallbladder cancer cells isolated from cancerous tissues obtained from patients with gallbladder cancer, cancer tissues were finely cut with scissors, and then subjected to a mesh to separate single cells and treated with RPMI 1640 medium supplemented with 10% fetal bovine serum And then cultured in a CO ₂ incubator maintained at 37 ° C for 24 hours. The recombinant virus SLJ496 obtained from the vaccinia virus strain WR and the NYCBH strain and the rabbit tumor model, respectively, which were distributed from the American Cell Line Bank (ATCC) to the above cells, , And cultured for 72 hours. The recombinant virus (3mix P1), which predominantly proliferates in gallbladder cancer cells, was then infected with 1 MOI in gallbladder cancer cells isolated from other gallbladder cancer patients and cultured for 72 hours to obtain a second recombinant virus (3mix P2) Respectively. The above procedure was repeated one more time to finally obtain a third recombinant virus (3 mix P3). (HuCCT-1) purchased from Korean Cell Line Bank (KLCB) was infected with the recombinant virus (3 mix P3) to select early mutant strains capable of proliferating in human gallbladder cancer cells, and then 10% And cultured in RPMI 1640 medium supplemented with fetal serum. The infected cell line was harvested within 48 hours after infection and exposed to the virus in the cytoplasm through three freezing / thawing procedures. Cell debris was removed by centrifugation at 6000 rpm, and the mutant virus in the supernatant (3mix P4 ). The mutant virus recovered through the above procedure was used as a seed for 2-fold growth after determining the amount of virus through the titration assay. Mutant viruses propagated throughout the fourth passage were used to compare the potency with the parent virus, and single plaque isolation was performed to obtain one pure recombinant virus. The mutant virus obtained through the above procedure was named HBB301, and a specific experimental procedure was shown in Fig.

Example  2. Wild Vaccinia  Viruses and mutations Vaccinia  IC of virus ₅₀  compare

In order to confirm whether the mutant Vaccinia virus obtained in Example 1 showed an anti-cancer effect, the following experiment was conducted. After treatment of three types of parental (backbone) virus (WR strain, NYCBH strain, SLJ496) or 3mix P4 recombinant virus obtained in Example 1 on a human-derived gallium carcinoma cell line (HuCCT-1) Respectively. The results are shown in Fig.

As shown in Figure 2, IC ₅₀ of the WR strain is 1.865 MOI, IC ₅₀ of the NYCBH strain 7.686 MOI, IC ₅₀ of SLJ496 is 2.977 MOI, while, IC ₅₀ of 3mix P4 recombinant vaccinia virus according to the invention 0.182 MOI. As a result, it was confirmed that 3mixP4 recombinant Vaccinia virus according to the present invention showed excellent effect of inhibiting the growth of human gallbladder cancer cells, even though the concentration of Vaccinia virus was 10 times or more lower than that of the wild-type vaccinia virus.

In order to confirm the anticancer effect of the mutant Vaccinia virus obtained in Example 1 on other cancer cells derived from human, IC ₅₀ was compared between ovarian cancer cell A2780 and lung cancer cell A549. The results are shown in Fig.

As shown in FIG. 3, the 3mix P4 recombinant Vaccinia virus according to the present invention was found to inhibit the growth of ovarian cancer and lung cancer cells at a concentration lower than 5 times that of wild Vaccinia virus.

Example  3. Comparison of toxicity in respiratory tract infected animal models

HB5301 obtained in Example 1 and Western Reserve virus (parent virus) were administered to c57bl / 6 mice at 8 weeks of age by respiration at a concentration of 10 ⁴ , 10 ⁵ , 10 ⁶ , and 10 ⁷ pfu, respectively. Then, the change in body weight and the survival rate were observed for 2 weeks, and the toxicity of the virus was confirmed. The results are shown in Fig.

As shown in FIG. 4, when the same dose of 10 ⁶ pfu was administered, weight loss was observed due to viral toxicity in the Western Reserve treatment group as a parent virus for 7 weeks, whereas the HBB301 treatment group of the present invention showed normal weight Respectively. It was confirmed that HBB301 had less toxicity than the parent virus.

Example  4. Comparative analysis of antitumor activity

HBB301 obtained in Example 1 and Western Reserve virus of the parent virus were separately inoculated into various tumor cell lines inoculated into a 96-well plate, followed by further incubation for 2 to 3 days to confirm the death of virus-induced cancer cells. Viable cell counts were determined by the manufacturer's recommended dose of CCK-8 reagent, which produces a chromophore that absorbs at 450 nm by the reducing enzyme present in the cytoplasm. Wyeth tk-virus, an attenuated virus prepared by recombining the TK gene in Western Reserve viruses and Wyeths virus, the parent viruses, was used. The results are shown in Fig. 5 to Fig.

As shown in FIG. 5 and FIG. 6, HBB301 of the present invention was found to have a higher anticancer activity than the wyeth tk-virus in the BXPC-3 pancreatic cancer cell line and HCT-116, HCT-8 and HCT-15 colon cancer cell lines .

Further, as shown in FIG. 7, HBB301 of the present invention showed remarkably superior anticancer activity in the OVCAR3 ovarian cancer cell line as compared to the wyeth tk- and WR virus strains .

Example  5. HBB301  Identify the base sequence

In order to confirm the nucleotide sequence of HBB301 obtained in Example 1, DNA was isolated from the amplified virus, cut into 1 to 2 kb units by physical force, and then cloned into a plasmid. The nucleotide sequence of about 1000 clones randomly selected was analyzed to determine a single nucleotide sequence of ~ 180,000 nucleotides. In addition, a PCR primer capable of specifically binding and amplifying the inverted repeat sites at both ends of the virus was designed, and amplified fragments of about 10 kb in length except for 174 bp at the ends of the virus, Respectively.

In order to compare the nucleotide sequences of the HBB301 of the present invention and the Western Reserve virus of the parent virus, nucleotide sequences of all genomes were compared except 174 bp at both ends of the virus. The results are shown in Fig.

As shown in FIG. 8, HBB301 of the present invention was found to show the following differences with the parent virus.

1) Insertion of 54 bp into the 2937 bp region and the 191774 bp region of the noncoding region at both 5 'and 3' ends.

2) Insert 9 bp in VACWR011, the first heat of the 5 'terminal (3 amino acid inserted protein mutation protein).

3) Approximately 300 bp disappear at the 5 'end.

Therefore, it was confirmed that HBB301 according to the present invention is a novel virus having different genetic information from Western Reserve virus, which is a parent virus, and deposited on KCTC (Korean Collection for Type Culture) on Apr. 2, 2013 No. KCTC 12391BP).

Hereinafter, formulation examples of the composition according to the present invention will be described, but the present invention is not intended to be limited thereto but is specifically described.

Formulation example  One. Sanje  Produce

HBB301 10 ² to 10 ¹² pfu

Lactose 1g

The above components were mixed and packed in airtight bags to prepare powders.

Formulation example  2. Preparation of tablets

HBB301 10 ² to 10 ¹² pfu

Corn starch 100 mg

Lactose 100mg

Magnesium stearate 2 mg

After mixing the above components, tablets were prepared by tableting according to a conventional method for producing tablets.

Formulation example  3. Preparation of Injection

HBB301 10 ² to 10 ¹² pfu

Na ₂ HPO _{4 12} H ₂ O 26 mg

2974 mg of distilled water

After mixing the above components, an injection was prepared according to the usual production method.

Korea Biotechnology Research Institute KCTC12391BP 20130402

Claims (4)

(a) a cancer cell is infected with a vaccine strain of WR (Western Reserve), a vaccine strain of NYCBH (The New York City Board of Health) and a recombinant vaccinia virus SLJ496 strain, cultured and proliferated, Isolating the recombinant vaccinia virus that proliferates in the host cell;
(b) culturing and propagating the recombinant vaccinia virus isolated in step (a) after infection with different cancer cells; And
(c) separating the recombinant Vaccinia virus that propagates in the cancer cells of step (b). The method of claim 1, wherein the cancer cells are derived from gall bladder cancer. 2. The method according to claim 1, wherein the step (b) and (c) are repeated 2 to 100 times. The method according to claim 1, wherein the novel vaccinia virus is vaccinia virus HBB301 deposited with Accession No. KCTC12391BP.

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