WO2023128672A1 - Novel vaccinia virus variant with increased extracellular enveloped virus production - Google Patents

Novel vaccinia virus variant with increased extracellular enveloped virus production Download PDF

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WO2023128672A1
WO2023128672A1 PCT/KR2022/021646 KR2022021646W WO2023128672A1 WO 2023128672 A1 WO2023128672 A1 WO 2023128672A1 KR 2022021646 W KR2022021646 W KR 2022021646W WO 2023128672 A1 WO2023128672 A1 WO 2023128672A1
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virus
vaccinia virus
cells
present
vaccinia
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PCT/KR2022/021646
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Korean (ko)
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손우찬
이지영
손호선
김태희
고수민
이장미
강지연
정다솜
임희선
이동현
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재단법인 아산사회복지재단
울산대학교 산학협력단
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Priority claimed from KR1020220187984A external-priority patent/KR20230101747A/en
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Publication of WO2023128672A1 publication Critical patent/WO2023128672A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/76—Viruses; Subviral particles; Bacteriophages
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
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    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof

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  • the present invention relates to novel vaccinia virus variants with increased extravesicular virus productivity.
  • Vaccinia virus is a membranous virus with a linear DNA genome of about 190 kb and has been used as an ideal expression vector since the early 1980s. The reason for this is that it is possible to stably insert foreign DNA of 25 kb or more, and since replication is not performed in the cytoplasm, gene expression is possible without interference of the host genome, has a relatively high level of protein synthesis, and complete post-translational-modification (post-translational modification) is possible. -translational modification) occurs.
  • vaccinia virus is being actively studied for use as a recombinant vaccine or anticancer virus due to its usefulness.
  • Vaccinia virus infects and spreads to cells through four types of particles, and each type plays a different role from infection to spread based on its characteristics.
  • the four types of viral particles are intracellular mature virus (IMV), intracellular enveloped virus (IEV), cell associated virus (CEV), and extracellular enveloped virus. ; EEV).
  • IMV intracellular mature virus
  • IEV intracellular enveloped virus
  • CEV cell associated virus
  • EEV extracellular enveloped virus.
  • EEV extra-enveloped virus
  • EEV extra-enveloped virus
  • the extravesicular virus (EEV) of vaccinia virus usually has a low composition ratio of 1-2% of the total virus generated, but is responsible for systemic dissemination and has resistance to the complement-mediated immune system.
  • the extra-enveloped virus plays a biologically important role in the survival and spread of the vaccinia virus itself, such as having relatively resistance to neutralizing antibodies compared to the intracellular mature virus. Therefore, it is known that the production of extravesicular virus of vaccinia virus and the ability to form comet plaques, which are considered important for the spread of vaccinia virus from cell to cell, are related.
  • the inventors of the present invention conducted intensive research to secure a vaccinia virus that has excellent productivity of extravesicular virus (EEV), which is related to intratumoral propagation of the virus, and maintains infectivity so as to be advantageous for use as an anticancer virus vector.
  • EEV extravesicular virus
  • IHD strain ATCC VR-156
  • isolates vaccinia virus mutants that form comet plaques by repeating passage by infecting cells only with the extra-enveloped virus in the supernatant.
  • an object of the present invention is to provide a novel vaccinia virus mutant strain.
  • Another object of the present invention is to provide a method for producing a novel vaccinia virus according to the present invention.
  • the present invention provides a novel vaccinia virus deposited under accession number KCTC 15195BP, characterized by increased production of extracellular enveloped virus (EEV).
  • EEV extracellular enveloped virus
  • the vaccinia virus may be an IHD strain, but is not limited thereto.
  • the vaccinia virus is C2L, C1L, N1L, N2L, M1L, M2L, K1L, K2L, K3L, K4L, K5L, K6L, K7R, F1L, F2L, F3L, F4L, F5L,
  • One or more genes selected from the group consisting of A52R, A53R, A55R, and A56R may be deleted, but are not limited thereto.
  • the vaccinia virus may form comet plaque, but is not limited thereto.
  • the present invention comprises the steps of (a) infecting cells with vaccinia virus and culturing the infected cells;
  • step (b) infecting other cells with vaccinia virus with the supernatant of step (a), culturing the infected cells, isolating only the supernatant, and repeating step (b);
  • step (c) a method for preparing a novel vaccinia virus according to the present invention, comprising the step of isolating the vaccinia virus proliferating from the cells infected in step (b).
  • the step (b) may be repeated 2 to 20 times, but is not limited thereto.
  • the vaccinia virus may be an IHD strain, but is not limited thereto.
  • the present invention provides a pharmaceutical composition for preventing or treating cancer comprising the vaccinia virus according to the present invention as an active ingredient.
  • the vaccinia virus of the present invention increases the productivity of the extravesicular virus related to the virus propagation ability and can enhance the spread of the virus in the tumor, thereby maximizing the oncolysis potential of the vaccinia virus, thereby achieving a significantly higher anticancer effect than the conventional vaccinia virus. expected to show
  • FIG. 1 is a view showing the results of plaque assay for each passage after subculture in order to isolate a novel vaccinia virus according to an embodiment of the present invention.
  • FIGS. 2a to 5b show the results of next-generation sequencing analysis with wild-type vaccinia virus in order to verify genetic mutations of vaccinia virus according to an embodiment of the present invention
  • FIGS. 2a to 2c and 3a to 5b 3E is a result of next-generation sequencing analysis of the left arm region of a vaccinia virus isolate according to an embodiment of the present invention
  • FIGS. 4A to 4C and 5A to 5B are diagrams showing the results of next-generation sequencing analysis of the A52R to A56R gene region.
  • 2A to 2C show one figure as a whole, and each figure is positioned from left to right in one figure in the order of FIGS. 2A, 2B, and 2C.
  • Figures 3a to 3e represent one figure as a whole, and each figure is positioned from left to right in one figure in the order of Figures 3a, 3b, 3c, 3d, and 3e.
  • 4A to 4C show one figure as a whole, and each figure is positioned from left to right in one figure in the order of Figs. 4A, 4B, and 4C.
  • Figures 5a and 5b show one figure as a whole, and in one figure, Fig. 5a is located on the left and Fig. 5b is located on the right.
  • FIG. 6 is a diagram showing the results of measuring the titers of vaccinia virus and wild-type vaccinia virus according to an embodiment of the present invention.
  • the inventors of the present invention conducted intensive research to secure a vaccinia virus that has excellent productivity of extravesicular virus (EEV), which is related to intratumoral propagation of the virus, and maintains infectivity so as to be advantageous for use as an anticancer virus vector.
  • EEV extravesicular virus
  • IHD strain ATCC VR-156
  • isolates vaccinia virus mutants that form comet plaques by repeating passage by infecting cells only with the extra-enveloped virus in the supernatant.
  • the present invention provides a novel vaccinia virus mutant strain deposited under accession number KCTC 15195BP, characterized by increased production of extracellular enveloped virus (EEV).
  • EEV extracellular enveloped virus
  • novel vaccinia virus according to the present invention can be obtained through the following steps:
  • Vero cells were spread on a 6-well plate at 8 ⁇ 10 5 per well and cultured overnight . Dilute it in the medium at 10 -5 and dispense 1 mL each. After 48 hours of incubation, all the medium in each well was sampled, centrifuged at 3600 rpm for 5 minutes, and only the supernatant was separated and used as the virus to be infected in the next passage.
  • the vaccinia virus according to the present invention can be obtained by isolating the virus after finding it.
  • the vaccinia virus according to the present invention obtained through the above process is a novel virus with different genetic information from the parent virus (wild-type IHD strain vaccinia virus), which was published in KCTC (Korean Collection for Type Culture) in November 2022. It was deposited on the 15th (accession number: KCTC 15195B).
  • vaccinia virus is a large, complex enveloped virus that has a linear double-stranded DNA gene of about 190 kbp and encodes about 200 genes.
  • the role of vaccinia virus as a vaccine to eradicate smallpox is well known. After the eradication of smallpox, scientists have been studying the use of vaccinia virus as a tool to transfer genes into biological tissues (gene therapy and genetic engineering).
  • Vaccinia viruses are unique among DNA viruses because they replicate only in the cytoplasm of the host cell. Thus, a large genome is required to encode viral enzymes and proteins necessary for viral DNA replication.
  • IMV intracellular mature virus
  • IEV intracellular enveloped virus
  • CEV cell associated virus
  • EEV extracellular enveloped virus
  • the strain of the vaccinia virus may be Western Reserve, Copenhagen, Wyeth, or IHD, but is preferably an IHD strain.
  • the vaccinia virus may be natural or genetically modified. According to one embodiment of the present invention, it may preferably be ATCC's VR-156 IHD strain, but is not limited thereto.
  • the vaccinia virus is C2L, C1L, N1L, N2L, M1L, M2L, K1L, K2L, K3L, K4L, K5L, K6L, K7R, F1L, F2L, F3L, F4L, F5L, A52R, A53R,
  • One or more genes selected from the group consisting of A55R and A56R may be deleted, but are not limited thereto.
  • the vaccinia virus is the C2L, C1L, N1L, N2L, M1L, M2L, K1L, K2L, K3L, K4L, K5L, K6L, K7R, F1L, F2L, F3L, F4L, and It may be characterized by deletion of about 15 kb in the left arm region containing the F5L gene.
  • the vaccinia virus may be characterized in that A52R, A53R, A55R, and A56R gene deletions of about 3.7 kb in length from the A52R to A56R gene location occur.
  • the vaccinia virus may form a comet plaque, but is not limited thereto.
  • plaque means a comet-shaped plaque
  • the formation of comet plaque of vaccinia virus may represent the formation and propagation of an extravesicular virus that plays an important role in systemic propagation.
  • the present invention comprises the steps of (a) infecting cells with vaccinia virus and culturing the infected cells;
  • step (b) infecting other cells with vaccinia virus with the supernatant of step (a), culturing the infected cells, isolating only the supernatant, and repeating step (b);
  • step (c) isolating the proliferating vaccinia virus from the infected cells of step (b);
  • the supernatant contains the extravesicular virus of the vaccinia virus, thereby infecting the cells with the vaccinia virus.
  • step (b) another cell (second cell) distinct from the cell (first cell) of step (a) is infected with vaccinia virus with the supernatant of step (a) and cultured. Then, only the supernatant of the second cell is separated, and another cell (third cell) is infected with vaccinia virus using the supernatant separated from the second cell, cultured, and then only the supernatant of the third cell After separation, it means repeating a series of processes such as infecting and culturing another cell (fourth cell) using the supernatant separated from the third cell. That is, step (b) means subculture using the culture supernatant of vaccinia virus-infected cells.
  • cells used in the vaccinia virus production method may be cells of the same type or different types, and the cells may be animal cultured cells.
  • the animal cultured cells may be generally used animal cultured cells such as CHO cells, HEK cells, COS cells, 3T3 cells, myeloma cells, BHK cells, HeLa cells, Vero cells, etc. According to the embodiment, Vero cells, but any cell known to be used for infection or propagation of vaccinia virus may be used without limitation.
  • the step (b) is 2 to 20 times, 2 to 18 times, 2 to 16 times, 2 to 14 times, 2 to 12 times, 2 to 10 times, 2 to 10 times 8 times, 2 to 6 times, 2 to 4 times, 2 to 3 times, 3 to 18 times, 3 to 16 times, 3 to 14 times, 3 to 12 times, 3 to 10 times , 3 to 8 times, 3 to 6 times, 3 to 4 times, 4 to 5 times, 5 to 9 times, 9 to 15 times, 2 times, 3 times, 4 times, 5 times, 6 times It may be repeated 7 times, 8 times, 9 times, 10 times, 11 times, 12 times, 13 times, 14 times, or 15 times, and according to an embodiment of the present invention, the back time of step (b) There is no limit to the number of repetitions of step (b) as long as comet plaques are formed in cells infected with Nia virus.
  • the vaccinia virus may be an IHD strain, and according to an embodiment of the present invention, it may preferably be ATCC's VR-156 IHD strain, but is not limited thereto.
  • the present invention may provide a pharmaceutical composition for preventing or treating cancer comprising the vaccinia virus according to the present invention as an active ingredient.
  • the vaccinia virus is characterized in that the production of extravesicular virus (EEV), which plays an important role in systemic dissemination, is improved, and thus the efficiency of anticancer treatment can be improved by maximizing the oncolytic potential of the vaccinia virus.
  • EEV extravesicular virus
  • the novel vaccinia virus can be used for the treatment and prevention of hyperproliferative diseases such as cancer.
  • Examples contemplated for treatment include gallbladder cancer, colorectal cancer, pancreatic cancer, leukemia, ovarian cancer, stomach cancer, lung cancer, breast cancer, liver cancer, bronchial cancer, nasopharynx cancer, laryngeal cancer, skin cancer, colon cancer, cervical cancer, head and neck cancer, prostate cancer, kidney cancer, bone cancer, testicular cancer, gastrointestinal cancer, lymphoma, precancerous lesions in the lung, melanoma, sarcoma, bladder cancer, and any other cancer or tumor that can be treated.
  • the effective amount of the pharmaceutical composition is defined as sufficient to induce tumor killing or destruction or lysis of cancer cells as well as slowly inhibiting or reducing the growth of tumors or their size, and in any case eradication of tumors include
  • the route of administration of the composition of the present invention will vary depending on the location and nature of the lesion, for example intradermal, transdermal, parenteral, intravenous, intramuscular, intranasal, subcutaneous, regional (e.g. , in areas proximal to the tumor, particularly through the tumor's blood vessels or adjacent blood vessels), intratracheal, intraperitoneal, intraarterial, intravesical, intratumoral, inhalation, perfusion, lavage, and oral administration and formulation.
  • Intratumoral injection or direct injection into tumor blood vessels is particularly contemplated for isolated, accessible solid tumors.
  • Topical, regional or systemic administration may also be appropriate.
  • the volume administered will be between about 4 and 10 ml, and for tumors smaller than 4 cm, a volume of between about 1 and 3 ml will be used.
  • Multiple infusions delivered in a single dose include volumes from about 0.1 to about 0.5 ml.
  • Viral particles can advantageously be contacted via multiple infusion administration to the tumor spaced about 1 cm apart.
  • the present invention is used prior to surgery to allow for resection of inoperable tumor subjects.
  • Continuous administration may also be applied where appropriate, for example by inserting a catheter into a tumor or tumor blood vessel.
  • Such continuous perfusion can be from about 1 to 2 hours, up to about 2 to 6 hours, up to about 6 to 12 hours, up to about 12 to 24 hours, up to about 1 to 2 days, up to about 1 to 2 weeks, or Can be done over a longer period of time.
  • the dosage of the composition via continuous perfusion will be equivalent to that given via single or multiple infusions, adjusted over time while perfusion occurs.
  • limb perfusion may be used to administer the compositions of the present invention, particularly in the treatment of melanoma and sarcoma.
  • Vero African green monkey kidney epithelial cell line
  • ATCC American Type Culture Collection
  • AA antibiotic-antimycotic
  • Vaccinia virus (Vaccinia virus) and IHD strain used in this experiment were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). The virus was propagated using Vero cells as host cells. The virus used was an infectious substance corresponding to Biosafety level 2, and the experiment was performed in a facility with appropriate safety facilities. The purchased virus was infected with 2 ⁇ 10 6 Vero cells spread on a 75T plaster, and after 48 hours, the supernatant was sampled and centrifuged to remove cell debris before use.
  • Example 3 Isolation of vaccinia virus with increased production of extracellular enveloped virus (EEV)
  • Vero cells were laid on a 6-well plate at 8 ⁇ 10 5 per well and cultured overnight. All of the medium was removed, and the vaccinia virus prepared in Example 2 was diluted in the medium at 10 0 to 10 -5 and dispensed by 1 mL. After culturing for 48 hours, the culture medium of each well was sampled, centrifuged at 3600 rpm for 5 minutes, and only the supernatant was separated and used as a virus to be infected in the next passage. This passage process was repeated 14 times. Cells sampled after 48 hours of virus infection were stained with crystal violet to confirm plaques caused by virus infection. Medium obtained from wells showing plaques that could be counted without killing all the cells in the wells was used as a virus to be infected in the next passage.
  • Crystal violet dye was prepared with 0.15% crystal violet, 8% formaldehyde, and 5% ethanol in distilled water. 1 mL of crystal violet solution was dispensed to the cells from which the medium was removed, and the cells were allowed to stand at room temperature for 5 minutes. After removing the dye by aspiration, the plate was dried to confirm the number and shape of plaques.
  • a single viral plaque was isolated through the agarose overlay method.
  • the isolated viral plaques were cultured in 10 175T flasks in large quantities and then concentrated using an ultra-high-speed centrifuge.
  • PK digestion was performed to remove non-specific protein components of the concentrated high-concentration virus sample
  • viral gDNA was extracted using a phenol/chloroform method.
  • the extracted DNA was from Macrogen Inc. (Korea)'s Whole Genome Sequencing service using the next-generation sequencing (NGS) technique was used to verify the genetic mutation of the wild-type virus and the isolated virus mutant.
  • NGS next-generation sequencing
  • Vero cells 8 ⁇ 10 5 Vero cells were seeded in a 6-well plate and cultured overnight at 37 °C, 5% CO 2 , under humid conditions, and then wild-type IHD virus and passaged virus (P14) were added to the cells at an MOI of 10. Infected and cultured for 30 minutes in a 37 ° C incubator. The virus solution was removed by aspiration, washed three times with PBS, and cultured for 24 hours by supplying 1 mL of a new medium. After 24 hours, only the supernatant was obtained and impurities were removed and used as an extracellular virus (Extra) sample.
  • Extra extracellular virus
  • Viral titers were determined by the TCID 50 assay, one of the infectivity assays. 4.2 ⁇ 10 3 Vero cells were suspended in growth medium, dispensed into each well of a 96-well plate, and incubated overnight at 37°C, 5% CO 2 , under humid conditions. A serial dilution of 1:10 of the original virus sample, for example, from 10 -2 to 10 -8 of the original virus sample was prepared, and all virus samples were vigorously vortexed until immediately before transferring 50 ⁇ l of virus to each well. The cell plate was cultured for 4 days in a 37°C, CO 2 incubator, and then CPE (Cytopathic effect) was observed.
  • CPE Cytopathic effect
  • the ratio of the virus outside the envelope of the passaged virus (P14) was higher than that of the wild-type IHD virus.
  • the vaccinia virus of the present invention increases the productivity of the extravesicular virus related to the virus propagation ability and can enhance the spread of the virus in the tumor, thereby maximizing the oncolysis potential of the vaccinia virus, thereby achieving a significantly higher anticancer effect than the conventional vaccinia virus. As can be shown, there is industrial applicability.

Abstract

The present invention relates to a vaccinia virus with increased extracellular enveloped virus production, and the like. The vaccinia virus of the present invention improves intratumoral virus spread by increasing the production of extracellular enveloped virus related to viral transmission, thereby maximizing the oncolytic potential of vaccinia virus, and thus is expected to exhibit a significantly high anticancer effect compared to conventional vaccinia viruses.

Description

외피외 바이러스 생산이 증가한 신규한 백시니아 바이러스 변이주Novel vaccinia virus mutants with increased extravesicular virus production
본 발명은 외피외 바이러스 생산성이 증가한 신규한 백시니아 바이러스 변이종에 관한 것이다. The present invention relates to novel vaccinia virus variants with increased extravesicular virus productivity.
본 발명은 2021년 12월 29일에 출원된 대한민국 특허출원 제10-2021-0191795호 및 2022년 12월 28일 출원된 대한민국 특허출원 제10-2022-0187984호 에 기초한 우선권을 주장하며, 상기 출원들의 명세서 및 도면에 개시된 모든 내용은 본 출원에 원용된다.The present invention claims priority based on Korean Patent Application No. 10-2021-0191795 filed on December 29, 2021 and Korean Patent Application No. 10-2022-0187984 filed on December 28, 2022, All contents disclosed in their specifications and drawings are incorporated in this application.
백시니아 바이러스는 약 190 kb 선형의 DNA 게놈을 가진 막성 바이러스로, 1980년대 초부터 이상적인 발현 벡터로 쓰이게 되었다. 그 이유는 25 kb 이상의 외부 DNA를 안정하게 삽입이 가능하고, 세포질에서 복제가 이루어지지 때문에 숙주 게놈의 간섭 없이 유전자 발현이 가능하고 비교적 높은 단백질 합성 수준을 보유하고 있으며, 완전한 번역후-변형 (post-translational modification)이 일어나기 때문이다. 발현 벡터로서 뿐만 아니라, 백시니아 바이러스는 이러한 유용성으로 인하여 유전자 재조합 백신 또는 항암바이러스로 사용하기 위한 연구가 활발히 진행되고 있다.Vaccinia virus is a membranous virus with a linear DNA genome of about 190 kb and has been used as an ideal expression vector since the early 1980s. The reason for this is that it is possible to stably insert foreign DNA of 25 kb or more, and since replication is not performed in the cytoplasm, gene expression is possible without interference of the host genome, has a relatively high level of protein synthesis, and complete post-translational-modification (post-translational modification) is possible. -translational modification) occurs. In addition to being an expression vector, vaccinia virus is being actively studied for use as a recombinant vaccine or anticancer virus due to its usefulness.
백시니아 바이러스는 4가지 형태의 입자를 통해 세포에 감염 및 전파되며 각각의 형태가 가진 특징을 바탕으로 감염에서 전파까지 다른 역할을 맡고 있다. 4가지 바이러스 입자의 형태는 세포내 성숙 바이러스(Intracellular Mature virus ; IMV), 세포내 피막성 바이러스(Intracellular enveloped virus ; IEV), 세포성 바이러스(Cell associated virus ; CEV), 외피외 바이러스(Extracellular enveloped virus ; EEV)이다. 백시니아 바이러스는 세포에 감염된 후 세포질에서 복제 및 조립되어 한 겹의 막으로 둘러싸인 세포 내 성숙 바이러스(IMV)를 생성한다. 다수의 세포내 성숙 바이러스는 세포 용해를 통해 세포 밖으로 전파되지만, 일부 세포내 성숙 바이러스는 소포체 혹은 골지체로부터 유래한 성분으로 포장되어 두 겹의 막을 가진 세포내 피막성 바이러스(IEV)를 형성한다. 세포내 피막성 바이러스(IEV)가 미세소관에 의해 세포표면으로 이동한 뒤, 세포내 피막성 바이러스의 바깥막과 세포막이 융합하여 세포성 바이러스(CEV)를 형성하며, 이 세포성 바이러스가 액틴 중합화(Polymerization of actin)에 의해 세포 표면에서 떨어져 배양액으로 분비된 것이 외피외 바이러스(EEV)이다. 위의 4가지 형태 중 세포성 바이러스(CEV)는 세포 간 감염에서 중요한 역할을 담당하며, 외피외 바이러스(EEV)는 바이러스의 전신 전파에 중요한 역할을 맡고 있다. Vaccinia virus infects and spreads to cells through four types of particles, and each type plays a different role from infection to spread based on its characteristics. The four types of viral particles are intracellular mature virus (IMV), intracellular enveloped virus (IEV), cell associated virus (CEV), and extracellular enveloped virus. ; EEV). After vaccinia virus infects a cell, it replicates and assembles in the cytoplasm to produce an intracellular mature virus (IMV) surrounded by a single layer of membrane. Many intracellular mature viruses propagate out of the cell by cell lysis, but some mature intracellular viruses are packaged with components derived from the endoplasmic reticulum or Golgi apparatus to form intracellular enveloped viruses (IEV) with a double membrane. After the intracellular enveloped virus (IEV) moves to the cell surface by microtubules, the outer membrane of the intracellular enveloped virus and the cell membrane fuse to form a cellular virus (CEV), which polymerizes actin. EEV is an extra-enveloped virus (EEV) that is separated from the cell surface by polymerization of actin and secreted into the culture medium. Among the above four types, cellular virus (CEV) plays an important role in cell-to-cell infection, and extravesicular virus (EEV) plays an important role in systemic transmission of the virus.
한편, 보통 백시니아 바이러스의 외피외 바이러스(EEV)는 생성되는 전체 바이러스 중 1-2%로 구성비가 낮지만, 전신 전파 담당, 보체 매개 면역시스템에 대한 저항성을 가진다. 또한 외피외 바이러스는 세포내 성숙 바이러스와 비교 시, 상대적으로 중화항체에 대한 저항성을 가지는 등 백시니아 바이러스 자체의 생존 및 확산에 생물학적으로 중요한 역할을 담당하고 있다. 따라서, 백시니아 바이러스의 외피외 바이러스의 생산과 백시니아 바이러스가 세포에서 세포로 퍼지는데 중요한 것으로 간주되는 comet plaque의 형성 능력은 관련이 있는 것으로 알려져 있다.On the other hand, the extravesicular virus (EEV) of vaccinia virus usually has a low composition ratio of 1-2% of the total virus generated, but is responsible for systemic dissemination and has resistance to the complement-mediated immune system. In addition, the extra-enveloped virus plays a biologically important role in the survival and spread of the vaccinia virus itself, such as having relatively resistance to neutralizing antibodies compared to the intracellular mature virus. Therefore, it is known that the production of extravesicular virus of vaccinia virus and the ability to form comet plaques, which are considered important for the spread of vaccinia virus from cell to cell, are related.
본 발명의 발명자들은 바이러스의 종양 내 전파력과 관계있는 외피외 바이러스(EEV) 생산성이 우수하면서 항암 바이러스 벡터로서 사용하기에도 유리하도록 감염력이 유지되는 백시니아 바이러스를 확보하고자 예의 연구한 결과, 외피외 바이러스 생산과 관련 있는 comet plaque를 형성하지 않던 IHD strain (ATCC VR-156)의 상층액 내 외피외 바이러스만 세포에 감염시키는 방식으로 계대(passage)를 반복함으로써 comet plaque를 형성하는 백시니아 바이러스 변이주를 분리하여 본 발명을 완성하였다.The inventors of the present invention conducted intensive research to secure a vaccinia virus that has excellent productivity of extravesicular virus (EEV), which is related to intratumoral propagation of the virus, and maintains infectivity so as to be advantageous for use as an anticancer virus vector. IHD strain (ATCC VR-156), which did not form comet plaques related to production, isolates vaccinia virus mutants that form comet plaques by repeating passage by infecting cells only with the extra-enveloped virus in the supernatant. Thus, the present invention was completed.
따라서, 본 발명의 목적은 신규한 백시니아 바이러스 변이주를 제공하는 것이다.Accordingly, an object of the present invention is to provide a novel vaccinia virus mutant strain.
본 발명의 다른 목적은 본 발명에 따른 신규한 백시니아 바이러스의 제조방법을 제공하는 것이다.Another object of the present invention is to provide a method for producing a novel vaccinia virus according to the present invention.
그러나, 본 발명이 이루고자 하는 기술적 과제는 이상에서 언급한 과제에 제한되지 않으며, 언급되지 않은 또 다른 과제들은 아래의 기재로부터 본 발명이 속하는 기술 분야의 통상의 지식을 가진 자에게 명확하게 이해될 수 있을 것이다.However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems not mentioned can be clearly understood by those skilled in the art from the description below. There will be.
상기 목적을 달성하기 위하여, 본 발명은 기탁번호 KCTC 15195BP로 수탁된 외피외 바이러스(extracellular enveloped virus; EEV) 생산이 증가한 것을 특징으로 하는 신규한 백시니아 바이러스를 제공한다.In order to achieve the above object, the present invention provides a novel vaccinia virus deposited under accession number KCTC 15195BP, characterized by increased production of extracellular enveloped virus (EEV).
본 발명의 일 실시예에 따르면, 상기 백시니아 바이러스는 IHD 스트레인(strain)일 수 있으나, 이에 제한되지 않는다.According to one embodiment of the present invention, the vaccinia virus may be an IHD strain, but is not limited thereto.
본 발명의 다른 실시예에 따르면, 상기 백시니아 바이러스는 C2L, C1L, N1L, N2L, M1L, M2L, K1L, K2L, K3L, K4L, K5L, K6L, K7R, F1L, F2L, F3L, F4L, F5L, A52R, A53R, A55R, 및 A56R로 이루어진 군으로부터 선택된 하나 이상의 유전자가 결실된 것일 수 있으나, 이에 제한되지 않는다.According to another embodiment of the present invention, the vaccinia virus is C2L, C1L, N1L, N2L, M1L, M2L, K1L, K2L, K3L, K4L, K5L, K6L, K7R, F1L, F2L, F3L, F4L, F5L, One or more genes selected from the group consisting of A52R, A53R, A55R, and A56R may be deleted, but are not limited thereto.
본 발명의 또 다른 실시예에 따르면, 상기 백시니아 바이러스는 코멧 플라크(comet plaque)를 형성할 수 있으나, 이에 제한되지 않는다. According to another embodiment of the present invention, the vaccinia virus may form comet plaque, but is not limited thereto.
또한, 본 발명은 (a) 세포에 백시니아 바이러스를 감염시키고 감염된 세포를 배양하는 단계;In addition, the present invention comprises the steps of (a) infecting cells with vaccinia virus and culturing the infected cells;
(b) 상기 단계 (a) 의 상층액으로 다른 세포를 백시니아 바이러스에 감염시키고 감염된 세포를 배양하며, 상층액만을 분리하여 (b) 단계를 반복 수행하는 단계; 및(b) infecting other cells with vaccinia virus with the supernatant of step (a), culturing the infected cells, isolating only the supernatant, and repeating step (b); and
(c) 상기 단계 (b) 의 감염된 세포에서 증식하는 백시니아 바이러스를 분리하는 단계를 포함하는 본 발명에 따른 신규한 백시니아 바이러스의 제조방법을 제공한다.(c) a method for preparing a novel vaccinia virus according to the present invention, comprising the step of isolating the vaccinia virus proliferating from the cells infected in step (b).
본 발명의 일 실시예에 따르면, 상기 단계 (b) 는 2회 내지 20회 반복할 수 있으나, 이에 제한되지 않는다.According to one embodiment of the present invention, the step (b) may be repeated 2 to 20 times, but is not limited thereto.
본 발명의 다른 실시예에 따르면, 상기 백시니아 바이러스는 IHD 스트레인(strain)일 수 있으나, 이에 제한되지 않는다.According to another embodiment of the present invention, the vaccinia virus may be an IHD strain, but is not limited thereto.
또한, 본 발명은 본 발명에 따른 백시니아 바이러스를 유효성분으로 포함하는 암 예방 또는 치료용 약학적 조성물을 제공한다.In addition, the present invention provides a pharmaceutical composition for preventing or treating cancer comprising the vaccinia virus according to the present invention as an active ingredient.
본 발명의 백시니아 바이러스는 바이러스 전파력과 관련된 외피외 바이러스 생산성이 증가되어 종양 내 바이러스 확산을 향상시킬 수 있어 백시니아 바이러스의 종양 용해 가능성을 극대화함으로써 종래의 백시니아 바이러스보다 유의적으로 높은 항암 효과를 나타낼 것으로 기대된다.The vaccinia virus of the present invention increases the productivity of the extravesicular virus related to the virus propagation ability and can enhance the spread of the virus in the tumor, thereby maximizing the oncolysis potential of the vaccinia virus, thereby achieving a significantly higher anticancer effect than the conventional vaccinia virus. expected to show
도 1은 본 발명의 일 실시예에 따라 신규한 백시니아 바이러스를 분리하기 위하여 계대 배양한 뒤 각 계대별로 플라크 분석(Plaque assay)을 실시한 결과를 나타낸 도면이다.1 is a view showing the results of plaque assay for each passage after subculture in order to isolate a novel vaccinia virus according to an embodiment of the present invention.
[규칙 제91조에 의한 정정 07.04.2023]
도 2a 내지 도 5b는 본 발명의 일 실시예에 따른 백시니아 바이러스의 유전체 변이를 검증하기 위하여 야생형 백시니아 바이러스와 차세대염기서열분석을 실시한 결과를 나타낸 것으로, 도 2a 내지 도 2c 및 도 3a 내지 도 3e는 본 발명의 일 실시예 따른 백시니아 바이러스 분리주의 left arm 영역의 차세대염기서열분석 결과, 도 4a 내지 도 4c 및 도 5a 내지 도 5b는 A52R 부터 A56R 유전자 영역의 차세대염기서열분석 결과를 나타낸 도면이다. 도 2a 내지 도 2c는 전체로서 하나의 도면을 나타내며, 각 도면은 하나의 도면에서 좌측에서 우측으로 도 2a, 도 2b, 및 도 2c의 순서로 위치한다. 도 3a 내지 도 3e는 전체로서 하나의 도면을 나타내며, 각 도면은 하나의 도면에서 좌측에서 우측으로 도 3a, 도 3b, 도3c, 도 3d, 및 도 3e의 순서로 위치한다. 도 4a 내지 도 4c는 전체로서 하나의 도면을 나타내며, 각 도면은 하나의 도면에서 좌측에서 우측으로 도4a, 도 4b, 및 도 4c의 순서로 위치한다. 도 5a 및 도 5b는 전체로서하나의 도면을 나타내며, 하나의 도면에서 도 5a는 좌측, 도 5b는 우측에 위치한다.[Correction under Rule 91 07.04.2023]
2a to 5b show the results of next-generation sequencing analysis with wild-type vaccinia virus in order to verify genetic mutations of vaccinia virus according to an embodiment of the present invention, FIGS. 2a to 2c and 3a to 5b 3E is a result of next-generation sequencing analysis of the left arm region of a vaccinia virus isolate according to an embodiment of the present invention, and FIGS. 4A to 4C and 5A to 5B are diagrams showing the results of next-generation sequencing analysis of the A52R to A56R gene region. am. 2A to 2C show one figure as a whole, and each figure is positioned from left to right in one figure in the order of FIGS. 2A, 2B, and 2C. Figures 3a to 3e represent one figure as a whole, and each figure is positioned from left to right in one figure in the order of Figures 3a, 3b, 3c, 3d, and 3e. 4A to 4C show one figure as a whole, and each figure is positioned from left to right in one figure in the order of Figs. 4A, 4B, and 4C. Figures 5a and 5b show one figure as a whole, and in one figure, Fig. 5a is located on the left and Fig. 5b is located on the right.
도 6은 본 발명의 일 실시예에 따른 백시니아 바이러스와 야생형 백시니아 바이러스의 역가를 측정한 결과를 나타낸 도면이다.6 is a diagram showing the results of measuring the titers of vaccinia virus and wild-type vaccinia virus according to an embodiment of the present invention.
본 발명의 발명자들은 바이러스의 종양 내 전파력과 관계있는 외피외 바이러스(EEV) 생산성이 우수하면서 항암 바이러스 벡터로서 사용하기에도 유리하도록 감염력이 유지되는 백시니아 바이러스를 확보하고자 예의 연구한 결과, 외피외 바이러스 생산과 관련 있는 comet plaque를 형성하지 않던 IHD strain (ATCC VR-156) 의 상층액 내 외피외 바이러스만 세포에 감염시키는 방식으로 계대(passage)를 반복함으로써 comet plaque를 형성하는 백시니아 바이러스 변이주를 분리하여 본 발명을 완성하였다.The inventors of the present invention conducted intensive research to secure a vaccinia virus that has excellent productivity of extravesicular virus (EEV), which is related to intratumoral propagation of the virus, and maintains infectivity so as to be advantageous for use as an anticancer virus vector. IHD strain (ATCC VR-156), which did not form comet plaques related to production, isolates vaccinia virus mutants that form comet plaques by repeating passage by infecting cells only with the extra-enveloped virus in the supernatant. Thus, the present invention was completed.
이하, 본 발명에 대해 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 발명은 기탁번호 KCTC 15195BP로 수탁된 외피외 바이러스(extracellular enveloped virus; EEV) 생산이 증가한 것을 특징으로 하는 신규한 백시니아 바이러스 변이주를 제공한다.The present invention provides a novel vaccinia virus mutant strain deposited under accession number KCTC 15195BP, characterized by increased production of extracellular enveloped virus (EEV).
본 발명에 따른 신규한 백시니아 바이러스는 하기와 같은 단계를 통해 수득될 수 있다:The novel vaccinia virus according to the present invention can be obtained through the following steps:
야생형 백시니아 바이러스(ATCC VR-156)를 세포에 감염시키기 전 6-웰 플레이트에 Vero 세포를 웰당 8×105개씩 깔고 overnight 정치 배양한 뒤, 배지를 모두 제거하고 야생형 백시니아 바이러스를 100 ~ 10-5로 배지에 희석하여 1 mL씩 분주한다. 48시간 배양 후 각 웰의 배지를 모두 샘플링하여 3600 rpm, 5분 원심분리하여 상층액만 분리하여 다음 계대에 감염시킬 바이러스로 사용하고, 이러한 계대 과정을 2-20회 반복하여 comet plaque가 형성된 백시니아 바이러스를 찾아낸 후 이를 분리함으로써 본 발명에 따른 백시니아 바이러스를 수득할 수 있다.Before infecting the cells with wild-type vaccinia virus (ATCC VR-156), Vero cells were spread on a 6-well plate at 8 × 10 5 per well and cultured overnight . Dilute it in the medium at 10 -5 and dispense 1 mL each. After 48 hours of incubation, all the medium in each well was sampled, centrifuged at 3600 rpm for 5 minutes, and only the supernatant was separated and used as the virus to be infected in the next passage. The vaccinia virus according to the present invention can be obtained by isolating the virus after finding it.
상기 과정을 통해 수득한 본 발명에 따른 백시니아 바이러스는 모 바이러스(야생형 IHD strain 백시니아 바이러스)와는 유전정보가 상이한 신규한 바이러스임을 확인하였으며, 이를 KCTC (Korean Collection for Type Culture)에 2022년 11월 15일자로 수탁하였다 (기탁번호 : KCTC 15195B).It was confirmed that the vaccinia virus according to the present invention obtained through the above process is a novel virus with different genetic information from the parent virus (wild-type IHD strain vaccinia virus), which was published in KCTC (Korean Collection for Type Culture) in November 2022. It was deposited on the 15th (accession number: KCTC 15195B).
본 발명에서 “백시니아 바이러스”란 약 190kbp의 선형 이중 나선 DNA 유전자를 가지고 있으며, 약 200개 유전자를 코딩하는 크고, 복잡한 외피성 바이러스이다. 천연두를 근절시킨 백신으로서의 백시니아 바이러스의 역할은 주지되어 있다. 천연두의 근절 후, 과학자들은 생물학적 조직 내로 유전자를 전달하기 위한 도구로서의 백시니아 바이러스의 사용을 연구하여 왔다 (유전자 치료 및 유전 공학). 백시니아 바이러스는 DNA 바이러스 중에서도 특이한데, 이는 단지 숙주 세포의 세포질에서만 복제하기 때문이다. 따라서 바이러스 DNA 복제를 위해 필요한 바이러스 효소 및 단백질을 코딩하기 위해서는 큰 게놈이 요구된다. 복제 과정 동안, 백시니아는 이들의 외막에서와는 상이한 몇몇 감염 형태인 세포내 성숙 바이러스(Intracellular Mature virus; IMV), 세포내 피막성 바이러스(Intracellular enveloped virus; IEV), 세포성 바이러스(Cell associated virus; CEV) 및 외피외 바이러스(Extracellular enveloped virus; EEV)를 생산한다. IMV는 가장 풍부한 감염 형태이며 접해있는 이웃세포의 감염에 관여하는 것으로 생각된다. 한편, CEV는 세포와 세포의 분포에 일정한 역할을 하는 것으로 생각되며, EEV는 숙주 기관 내의 긴 범위 전파에 중요한 것으로 생각된다. In the present invention, "vaccinia virus" is a large, complex enveloped virus that has a linear double-stranded DNA gene of about 190 kbp and encodes about 200 genes. The role of vaccinia virus as a vaccine to eradicate smallpox is well known. After the eradication of smallpox, scientists have been studying the use of vaccinia virus as a tool to transfer genes into biological tissues (gene therapy and genetic engineering). Vaccinia viruses are unique among DNA viruses because they replicate only in the cytoplasm of the host cell. Thus, a large genome is required to encode viral enzymes and proteins necessary for viral DNA replication. During the process of replication, vaccinia develops several forms of infection that differ from those in their outer membrane: intracellular mature virus (IMV), intracellular enveloped virus (IEV), and cell associated virus (CEV). ) and extracellular enveloped virus (EEV). IMV is the most abundant form of infection and is thought to be involved in infection of adjacent cells. On the other hand, CEVs are thought to play a role in cells and their distribution, and EEVs are thought to be important for long-range dissemination within host organs.
본 발명에 있어서, 상기 백시니아 바이러스는 종(strain)은 웨스턴 리저브(Western Reserve), 코펜하겐(Copenhagen), 와이에쓰(Wyeth), IHD일 수 있으나, 바람직하게는 IHD 스트레인(strain)일 수 있다. 상기 백시니아 바이러스는 자연형 또는 유전적으로 변형된 것일 수도 있다. 본 발명의 일 실시예에 따르면, 바람직하게는 ATCC사의 VR-156 IHD 스트레인일 수 있으나, 이에 제한되지 않는다.In the present invention, the strain of the vaccinia virus may be Western Reserve, Copenhagen, Wyeth, or IHD, but is preferably an IHD strain. The vaccinia virus may be natural or genetically modified. According to one embodiment of the present invention, it may preferably be ATCC's VR-156 IHD strain, but is not limited thereto.
본 발명에 있어서, 상기 백시니아 바이러스는 C2L, C1L, N1L, N2L, M1L, M2L, K1L, K2L, K3L, K4L, K5L, K6L, K7R, F1L, F2L, F3L, F4L, F5L, A52R, A53R, A55R, 및 A56R로 이루어진 군으로부터 선택된 하나 이상의 유전자가 결실될 수 있으나, 이에 제한되지 않는다. In the present invention, the vaccinia virus is C2L, C1L, N1L, N2L, M1L, M2L, K1L, K2L, K3L, K4L, K5L, K6L, K7R, F1L, F2L, F3L, F4L, F5L, A52R, A53R, One or more genes selected from the group consisting of A55R and A56R may be deleted, but are not limited thereto.
본 발명의 일 실시예에 따르면, 상기 백시니아 바이러스는 상기 C2L, C1L, N1L, N2L, M1L, M2L, K1L, K2L, K3L, K4L, K5L, K6L, K7R, F1L, F2L, F3L, F4L, 및 F5L 유전자가 포함된 left arm 영역에서 약 15kb가 결실된 것을 특징으로 할 수 있다. 또한, 본 발명의 일 실시예에 따르면, 상기 백시니아 바이러스는 A52R부터 A56R 유전자 위치까지 약 3.7kb 정도 길이의 A52R, A53R, A55R, A56R 유전자 결실이 발생한 것을 특징으로 할 수 있다.According to one embodiment of the present invention, the vaccinia virus is the C2L, C1L, N1L, N2L, M1L, M2L, K1L, K2L, K3L, K4L, K5L, K6L, K7R, F1L, F2L, F3L, F4L, and It may be characterized by deletion of about 15 kb in the left arm region containing the F5L gene. In addition, according to one embodiment of the present invention, the vaccinia virus may be characterized in that A52R, A53R, A55R, and A56R gene deletions of about 3.7 kb in length from the A52R to A56R gene location occur.
본 발명에 있어서, 상기 백시니아 바이러스는 코멧 플라크(comet plaque)를 형성할 수 있으나, 이에 제한되지 않는다.In the present invention, the vaccinia virus may form a comet plaque, but is not limited thereto.
본 발명에서 “코멧 플라크(comet plaque)”란 혜성 모양의 플라크를 의미하는 것으로, 백시니아 바이러스의 코멧 플라크 형성은 전신 전파에 중요한 역할을 담당하는 외피외 바이러스의 형성과 전파를 나타내는 것일 수 있다.In the present invention, “comet plaque” means a comet-shaped plaque, and the formation of comet plaque of vaccinia virus may represent the formation and propagation of an extravesicular virus that plays an important role in systemic propagation.
또한, 본 발명은 (a) 세포에 백시니아 바이러스를 감염시키고 감염된 세포를 배양하는 단계;In addition, the present invention comprises the steps of (a) infecting cells with vaccinia virus and culturing the infected cells;
(b) 상기 단계 (a) 의 상층액으로 다른 세포를 백시니아 바이러스에 감염시키고 감염된 세포를 배양하며, 상층액만을 분리하여 (b) 단계를 반복 수행하는 단계; 및(b) infecting other cells with vaccinia virus with the supernatant of step (a), culturing the infected cells, isolating only the supernatant, and repeating step (b); and
(c) 상기 단계 (b) 의 감염된 세포에서 증식하는 백시니아 바이러스를 분리하는 단계를 포함하는 본 발명에 따른 신규한 백시니아 바이러스의 제조방법을 제공하며, 본 발명의 일 실시예에 따르면, 상기 상층액에는 백시니아 바이러스의 외피외 바이러스가 포함되어 있어, 이에 의해 백시니아 바이러스에 세포가 감염되는 것을 특징으로 한다.(c) isolating the proliferating vaccinia virus from the infected cells of step (b); The supernatant contains the extravesicular virus of the vaccinia virus, thereby infecting the cells with the vaccinia virus.
구체적으로, 상기 (b) 단계는 상기 단계 (a)의 상층액으로 상기 단계 (a)의 세포(제1세포)와 구별되는 다른 세포(제2세포)를 백시니아 바이러스에 감염시키고, 배양한 후, 제2세포의 상층액만을 분리한 뒤, 제2세포에서 분리한 상층액을 이용하여 또 다른 세포(제3세포)를 백시니아 바이러스에 감염시키고 배양한 후, 제3세포의 상층액만을 분리한 뒤, 제3세포에서 분리한 상층액을 이용하여 또 다른 세포(제4세포)를 감염시키고 배양하는 것과 같이 일련의 과정을 반복 수행하는 것을 의미한다. 즉, 상기 (b) 단계는 백시니아 바이러스 감염된 세포의 배양 상층액을 이용하여 계대 배양하는 것을 의미한다.Specifically, in step (b), another cell (second cell) distinct from the cell (first cell) of step (a) is infected with vaccinia virus with the supernatant of step (a) and cultured. Then, only the supernatant of the second cell is separated, and another cell (third cell) is infected with vaccinia virus using the supernatant separated from the second cell, cultured, and then only the supernatant of the third cell After separation, it means repeating a series of processes such as infecting and culturing another cell (fourth cell) using the supernatant separated from the third cell. That is, step (b) means subculture using the culture supernatant of vaccinia virus-infected cells.
본 발명에 있어서, 상기 백시니아 바이러스의 제조방법에서 사용하는 세포는 동일한 종류 또는 상이한 종류의 세포를 사용할 수 있으며, 상기 세포는 동물 배양세포일 수 있다. 상기 동물 배양 세포는 예를 들면, CHO 세포, HEK 세포, COS 세포, 3T3 세포, 미엘로마 세포, BHK 세포, HeLa 세포, Vero 세포 등 일반적으로 사용되고 있는 동물 배양 세포를 사용할 수 있으며, 본 발명의 일 실시예에 따르면 Vero 세포이나, 백시니아 바이러스의 감염 또는 증식에 사용되는 것으로 알려진 세포라면 제한없이 사용될 수 있다.In the present invention, cells used in the vaccinia virus production method may be cells of the same type or different types, and the cells may be animal cultured cells. The animal cultured cells may be generally used animal cultured cells such as CHO cells, HEK cells, COS cells, 3T3 cells, myeloma cells, BHK cells, HeLa cells, Vero cells, etc. According to the embodiment, Vero cells, but any cell known to be used for infection or propagation of vaccinia virus may be used without limitation.
본 발명에 있어서, 상기 단계 (b) 는 2회 내지 20회, 2회 내지 18회, 2회 내지 16회, 2회 내지 14회, 2회 내지 12회, 2회 내지 10회, 2회 내지 8회, 2회 내지 6회, 2회 내지 4회, 2회 내지 3회, 3회 내지 18회, 3회 내지 16회, 3회 내지 14회, 3회 내지 12회, 3회 내지 10회, 3회 내지 8회, 3회 내지 6회, 3회 내지 4회, 4회 내지 5회, 5회 내지 9회, 9회 내지 15회, 2회, 3회, 4회, 5회, 6회, 7회, 8회, 9회, 10회, 11회, 12회, 13회, 14회, 또는 15회 반복할 수 있으며, 본 발명의 일 실시예에 따르면, 상기 단계 (b)의 백시니아 바이러스에 감염된 세포에서 코멧 플라크가 형성된다면 상기 단계 (b)의 반복 수행 횟수에는 제한이 없다.In the present invention, the step (b) is 2 to 20 times, 2 to 18 times, 2 to 16 times, 2 to 14 times, 2 to 12 times, 2 to 10 times, 2 to 10 times 8 times, 2 to 6 times, 2 to 4 times, 2 to 3 times, 3 to 18 times, 3 to 16 times, 3 to 14 times, 3 to 12 times, 3 to 10 times , 3 to 8 times, 3 to 6 times, 3 to 4 times, 4 to 5 times, 5 to 9 times, 9 to 15 times, 2 times, 3 times, 4 times, 5 times, 6 times It may be repeated 7 times, 8 times, 9 times, 10 times, 11 times, 12 times, 13 times, 14 times, or 15 times, and according to an embodiment of the present invention, the back time of step (b) There is no limit to the number of repetitions of step (b) as long as comet plaques are formed in cells infected with Nia virus.
본 발명에 있어서, 상기 백시니아 바이러스는 IHD 스트레인(strain)일 수 있으며, 본 발명의 일 실시예에 따르면, 바람직하게는 ATCC사의 VR-156 IHD 스트레인일 수 있으나, 이에 제한되지 않는다.In the present invention, the vaccinia virus may be an IHD strain, and according to an embodiment of the present invention, it may preferably be ATCC's VR-156 IHD strain, but is not limited thereto.
또한, 본 발명은 본 발명에 따른 백시니아 바이러스를 유효성분으로 포함하는 암 예방 또는 치료용 약학적 조성물을 제공할 수 있다.In addition, the present invention may provide a pharmaceutical composition for preventing or treating cancer comprising the vaccinia virus according to the present invention as an active ingredient.
상기 백시니아 바이러스는 전신 전파에 중요한 역할을 하는 외피외 바이러스(EEV) 생산이 향상된 것을 특징으로 하는 바, 백시니아 바이러스의 종양 용해 가능성을 극대화시켜 항암 치료의 효율을 증진시킬 수 있으므로 본 발명에 따른 신규한 백시니아 바이러스는 암과 같은 과증식성 질병의 치료 및 예방에 사용될 수 있다. 치료에 고려되는 예로는 담낭암, 대장암, 췌장암, 백혈병, 난소암, 위암, 폐암, 유방암, 간암, 기관지암, 비인두암, 후두암, 피부암, 결장암, 자궁경부암, 두경부암, 전립선암, 신장암, 골암, 고환암, 위장관암, 림프종, 폐에서의 전암 병변, 흑색종, 육종, 방광암 및 임의의 다른 암 또는 치료될 수 있는 종양이 포함되나, 이에 제한되는 것은 아니다.The vaccinia virus is characterized in that the production of extravesicular virus (EEV), which plays an important role in systemic dissemination, is improved, and thus the efficiency of anticancer treatment can be improved by maximizing the oncolytic potential of the vaccinia virus. The novel vaccinia virus can be used for the treatment and prevention of hyperproliferative diseases such as cancer. Examples contemplated for treatment include gallbladder cancer, colorectal cancer, pancreatic cancer, leukemia, ovarian cancer, stomach cancer, lung cancer, breast cancer, liver cancer, bronchial cancer, nasopharynx cancer, laryngeal cancer, skin cancer, colon cancer, cervical cancer, head and neck cancer, prostate cancer, kidney cancer, bone cancer, testicular cancer, gastrointestinal cancer, lymphoma, precancerous lesions in the lung, melanoma, sarcoma, bladder cancer, and any other cancer or tumor that can be treated.
본 발명에서 약학적 조성물의 유효량이란 종양의 성장 또는 이의 크기를 서서히 억제 또는 감소시키는 것뿐 아니라, 암 세포의 종양 살상 또는 파괴 또는 용해를 유도하기에 충분한 것으로서 정의되며 임의의 경우에는 종양의 근절을 포함한다.In the present invention, the effective amount of the pharmaceutical composition is defined as sufficient to induce tumor killing or destruction or lysis of cancer cells as well as slowly inhibiting or reducing the growth of tumors or their size, and in any case eradication of tumors include
본 발명의 조성물의 투여 경로는 병변의 위치 및 성질에 따라 다양할 것이며, 예를 들어, 피 내, 경피(transdermal), 비경구, 정맥 내, 근육 내, 비 내, 피하, 구역 (예를 들어, 종양에의 근접 구역, 특히 종양의 혈관 또는 인접한 혈관을 통함), 기관 내, 복막 내, 동맥 내, 방광 내, 종양 내, 흡입, 관류, 세척, 및 경구 투여 및 제제를 포함한다.The route of administration of the composition of the present invention will vary depending on the location and nature of the lesion, for example intradermal, transdermal, parenteral, intravenous, intramuscular, intranasal, subcutaneous, regional (e.g. , in areas proximal to the tumor, particularly through the tumor's blood vessels or adjacent blood vessels), intratracheal, intraperitoneal, intraarterial, intravesical, intratumoral, inhalation, perfusion, lavage, and oral administration and formulation.
종양 내 주입 또는 종양 혈관으로의 직접 주입은 특히 분리된, 접근가능한 고체 종양에 대해 고려된다. 국소적이거나, 구역적이거나 또는 전신적 투여 또한 적절할 수 있다. 4 cm 초과의 종양에 있어, 투여되는 부피는 약 4 내지 10 ml일 것이며, 4 cm 미만의 종양에 있어서는, 약 1 내지 3 ml의 부피가 사용될 것이다. 단일 투여량으로 전달되는 다중 주입은 약 0.1 내지 약 0.5 ml 부피를 포함한다. 바이러스 입자는 약 1 cm 간격으로 떨어져 있는 종양에의 다중 주입 투여를 통해 유리하게 접촉될 수 있다. 수술적 개입의 경우에, 본 발명은 수술 전에 사용되어 수술 불가능한 종양 대상의 절제를 가능하게 한다. 예를 들어, 종양 또는 종양 혈관 내로 카테터를 삽입함으로써 계속적 투여 또한 적절한 곳에 적용될 수 있다. 이러한 계속적 관류는 치료 시작 후 약 1 내지 2 시간에서부터, 약 2 내지 6 시간까지, 약 6 내지 12 시간까지, 약 12 내지 24 시간까지, 약 1 내지 2 일까지, 약 1 내지 2 주까지, 또는 더 긴 시간 동안 행해질 수 있다. 일반적으로, 계속적 관류를 통한 조성물의 투여량은 관류가 일어나는 동안의 시간에 걸쳐 조절되는, 단일 또는 다중 주입을 통해 주어지는 것과 균등할 것이다. 또한 특히 흑색종 및 육종의 치료에서의 본 발명의 조성물을 투여하는데 사지 관류를 사용할 수 있음을 고려한다.Intratumoral injection or direct injection into tumor blood vessels is particularly contemplated for isolated, accessible solid tumors. Topical, regional or systemic administration may also be appropriate. For tumors larger than 4 cm, the volume administered will be between about 4 and 10 ml, and for tumors smaller than 4 cm, a volume of between about 1 and 3 ml will be used. Multiple infusions delivered in a single dose include volumes from about 0.1 to about 0.5 ml. Viral particles can advantageously be contacted via multiple infusion administration to the tumor spaced about 1 cm apart. In the case of surgical intervention, the present invention is used prior to surgery to allow for resection of inoperable tumor subjects. Continuous administration may also be applied where appropriate, for example by inserting a catheter into a tumor or tumor blood vessel. Such continuous perfusion can be from about 1 to 2 hours, up to about 2 to 6 hours, up to about 6 to 12 hours, up to about 12 to 24 hours, up to about 1 to 2 days, up to about 1 to 2 weeks, or Can be done over a longer period of time. Generally, the dosage of the composition via continuous perfusion will be equivalent to that given via single or multiple infusions, adjusted over time while perfusion occurs. It is also contemplated that limb perfusion may be used to administer the compositions of the present invention, particularly in the treatment of melanoma and sarcoma.
이하, 본 발명의 이해를 돕기 위하여 바람직한 실시예를 제시한다. 그러나 하기의 실시예는 본 발명을 보다 쉽게 이해하기 위하여 제공되는 것일 뿐, 하기 실시예에 의해 본 발명의 내용이 한정되는 것은 아니다.Hereinafter, a preferred embodiment is presented to aid understanding of the present invention. However, the following examples are provided to more easily understand the present invention, and the content of the present invention is not limited by the following examples.
[실시예][Example]
실시예 1. 세포 배양Example 1. Cell culture
Vero (African green monkey kidney epithelial cell line)은 American Type Culture Collection (ATCC, Manassas, VA, USA)에서 구입하였다. 세포는 2% FBS (fetal bovine serum)와 1% AA (antibiotic-antimycotic)을 함유한 DMEM 배지를 이용하여 유지하였다. 세포는 37℃, 5% CO2, 습윤하 조건에서 배양하였다.Vero (African green monkey kidney epithelial cell line) was purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were maintained using DMEM medium containing 2% FBS (fetal bovine serum) and 1% AA (antibiotic-antimycotic). Cells were cultured at 37°C, 5% CO 2 , under humid conditions.
실시예 2. 바이러스 확보Example 2. Virus acquisition
본 실험에서 사용한 Vaccinia virus(백시니아 바이러스), IHD strain은 American Type Culture Collection (ATCC, Manassas, VA, USA)에서 구입하였다. 바이러스는 Vero 세포를 숙주세포로 이용하여 증식시켰다. 사용한 바이러스는 Biosafety level 2에 해당하는 감염성 물질로, 적절한 안전설비가 확보된 시설에서 실험을 수행하였다. 구매한 바이러스를 75T 플라스트에 깔은 2×106개 Vero 세포에 감염시키고 48시간 후에 상층액을 샘플링하고 원심분리하여 세포 부스러기를 제거한 후 사용하였다.Vaccinia virus (Vaccinia virus) and IHD strain used in this experiment were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). The virus was propagated using Vero cells as host cells. The virus used was an infectious substance corresponding to Biosafety level 2, and the experiment was performed in a facility with appropriate safety facilities. The purchased virus was infected with 2×10 6 Vero cells spread on a 75T plaster, and after 48 hours, the supernatant was sampled and centrifuged to remove cell debris before use.
실시예 3. 외피외 바이러스(extracellular enveloped virus; EEV) 생산이 증가한 백시니아 바이러스 분리Example 3. Isolation of vaccinia virus with increased production of extracellular enveloped virus (EEV)
세포에 감염시키기 전 6-웰 플레이트에 Vero 세포를 웰당 8×105개씩 깔고 overnight 정치 배양하였다. 배지를 모두 제거하고 상기 실시예 2에서 준비한 백시니아 바이러스를 100 ~ 10-5로 배지에 희석하여 1 mL씩 분주하였다. 48시간 배양 후 각 웰의 배지를 모두 샘플링하여 3600 rpm, 5분 원심분리하여 상층액만 분리하여 다음 계대에 감염시킬 바이러스로 사용하였다. 이러한 계대 과정을 14회 반복하였다. 바이러스 감염 48시간 후 배지를 샘플링하고 난 세포는 Crystal violet으로 염색하여 바이러스 감염에 의해 발생한 플라크(plaque)를 확인하였다. 웰 안의 세포를 모두 죽이지 않고 계수 가능한 정도의 플라크를 보이는 웰에서 얻은 배지를 다음 계대에 감염시킬 바이러스로 사용하였다.Before infecting the cells, Vero cells were laid on a 6-well plate at 8×10 5 per well and cultured overnight. All of the medium was removed, and the vaccinia virus prepared in Example 2 was diluted in the medium at 10 0 to 10 -5 and dispensed by 1 mL. After culturing for 48 hours, the culture medium of each well was sampled, centrifuged at 3600 rpm for 5 minutes, and only the supernatant was separated and used as a virus to be infected in the next passage. This passage process was repeated 14 times. Cells sampled after 48 hours of virus infection were stained with crystal violet to confirm plaques caused by virus infection. Medium obtained from wells showing plaques that could be counted without killing all the cells in the wells was used as a virus to be infected in the next passage.
실시예 4. 플라크 분석(Plaque assay)Example 4. Plaque assay
계대 과정 중 이전 차수에서 배지를 샘플링하고 난 세포는 Crystal violet 용액으로 염색하여 바이러스 감염에 의해 발생한 플라크(plaque)를 확인하였다. Crystal violet 염색제는 증류수에 0.15% crystal violet, 8% formaldehyde, 5% ethanol로 조제하여 사용하였다. 배지를 제거한 세포에 crystal violet 용액을 1 mL 분주하고 5분간 상온에서 대기하였다. 염색제를 흡인하여 제거한 후 플레이트를 건조시켜 플라크의 수와 모양을 확인하였다.During the passaging process, the medium was sampled in the previous round, and the cells were stained with a crystal violet solution to confirm plaques caused by viral infection. Crystal violet dye was prepared with 0.15% crystal violet, 8% formaldehyde, and 5% ethanol in distilled water. 1 mL of crystal violet solution was dispensed to the cells from which the medium was removed, and the cells were allowed to stand at room temperature for 5 minutes. After removing the dye by aspiration, the plate was dried to confirm the number and shape of plaques.
도 1에 나타낸 바와 같이, 각 계대별 바이러스 플라크의 모양을 확인한 결과, 야생형(original) IHD strain은 원형의 작은 플라크를 형성하는 반면 P3부터 혜성 모양의 comet plaque가 관찰되었으며, P3 이후의 모든 계대에서 혜성 모양의 플라크가 관찰되는 것을 확인할 수 있었다.As shown in Figure 1, as a result of confirming the shape of the viral plaque for each passage, the wild type (original) IHD strain formed a small round plaque, whereas a comet-shaped comet plaque was observed from P3, and at all passages after P3 It was confirmed that comet-shaped plaques were observed.
실시예 5. 차세대염기서열분석(Next-Generation Sequencing; NGS)Example 5. Next-Generation Sequencing (NGS)
야생형 백시니아 바이러스 IHD 균주와 14회 계대를 거친 바이러스를 Vero 세포에 감염시킨 후 Agarose overlay 방법을 통해 단일 바이러스 플라크를 분리하였다. 분리한 바이러스 플라크를 175T 플라스크 10개에 대량으로 배양한 다음 초고속원심기를 이용하여 농축하였다. 농축된 고농도의 바이러스 샘플의 비특이적인 단백질 성분을 제거하기 위해 PK digestion을 진행한 후, Phenol/Chloroform 방식으로 바이러스 gDNA를 추출하였다. 추출한 DNA는 Macrogen Inc. (Korea)의 차세대염기서열분석(NGS) 기법을 이용한 Whole Genome Sequencing 서비스를 통해 야생형 바이러스와 분리한 바이러스 변이주의 유전체 변이를 검증하였다.After infecting Vero cells with wild-type vaccinia virus IHD strain and virus passaged 14 times, a single viral plaque was isolated through the agarose overlay method. The isolated viral plaques were cultured in 10 175T flasks in large quantities and then concentrated using an ultra-high-speed centrifuge. After PK digestion was performed to remove non-specific protein components of the concentrated high-concentration virus sample, viral gDNA was extracted using a phenol/chloroform method. The extracted DNA was from Macrogen Inc. (Korea)'s Whole Genome Sequencing service using the next-generation sequencing (NGS) technique was used to verify the genetic mutation of the wild-type virus and the isolated virus mutant.
그 결과, 도 2 내지 3b에 나타낸 바와 같이, left arm의 약 15kb가 삭제된 것을 확인할 수 있었으며, 유전자에서 삭제된 유전자 영역은 C2L, C1L, N1L, N2L, M1L, M2L, K1L, K2L, K3L, K4L, K5L, K6L, K7R, F1L, F2L, F3L, F4L, 및 F5L에 해당하였다.As a result, as shown in Figures 2 to 3b, it was confirmed that about 15 kb of the left arm was deleted, and the deleted gene regions in the gene were C2L, C1L, N1L, N2L, M1L, M2L, K1L, K2L, K3L, K4L, K5L, K6L, K7R, F1L, F2L, F3L, F4L, and F5L.
또한, 도 4 내지 5b에 나타낸 바와 같이, 백시니아 바이러스의 EEV를 세포에서 반복 계대할 경우, 야생형 IHD strain 바이러스와 다르게 A52R부터 A56R 유전자 위치에 약 3.7kb 정도 길이의 유전자 deletion이 발생한 것을 확인할 수 있었다. 유전자에서 삭제된 유전자 영역은 A52R, A53R, A55R, A56R와 같으며, 삭제된 유전자 영역의 기능은 표 1에 나타내었다.In addition, as shown in FIGS. 4 to 5B, when the vaccinia virus EEV was repeatedly passaged in cells, it was confirmed that a gene deletion of about 3.7 kb in length occurred at the A52R to A56R gene location, unlike the wild-type IHD strain virus. . Gene regions deleted from the gene are the same as A52R, A53R, A55R, and A56R, and the functions of the deleted gene regions are shown in Table 1.
Figure PCTKR2022021646-appb-img-000001
Figure PCTKR2022021646-appb-img-000001
실시예 6. TCIDExample 6. TCID 5050 (Median Tissue Culture Infectious Dose) assay(Median Tissue Culture Infectious Dose) assay
8×105개의 Vero 세포를 6-웰 플레이트에 분주하여 37 ℃, 5% CO2, 습윤하 조건에서 밤새 배양한 후, 세포에 야생형 IHD 바이러스 및 계대를 진행한 바이러스(P14)를 10 MOI로 감염시켜 37℃ 인큐베이터에서 30분간 배양하였다. 바이러스 용액을 흡인하여 제거하고 PBS로 3회 세척 후 1 mL의 새 배지를 공급하여 24시간 동안 배양하였다. 24시간 후 상층액만 얻어 불순물을 제거한 것을 세포 외 바이러스(Extra) 샘플로 이용하였다. 모든 배지를 제거한 웰에 1 mL의 새 배지를 넣은 후 cell scraper로 세포를 플레이트 바닥에서 탈락시킨 후 Freezing & Thawing을 3회 진행한 샘플을 세포 내 바이러스(Intra) 샘플로 이용하였다. 8 × 10 5 Vero cells were seeded in a 6-well plate and cultured overnight at 37 °C, 5% CO 2 , under humid conditions, and then wild-type IHD virus and passaged virus (P14) were added to the cells at an MOI of 10. Infected and cultured for 30 minutes in a 37 ° C incubator. The virus solution was removed by aspiration, washed three times with PBS, and cultured for 24 hours by supplying 1 mL of a new medium. After 24 hours, only the supernatant was obtained and impurities were removed and used as an extracellular virus (Extra) sample. After adding 1 mL of new medium to the well from which all medium was removed, the cells were removed from the bottom of the plate with a cell scraper, and the sample subjected to Freezing & Thawing three times was used as an intracellular virus (Intra) sample.
바이러스 역가는 감염성 분석 중 하나인 TCID50 분석으로 결정하였다. 4.2×103개의 Vero 세포를 성장배지에 현탁하여 96-웰 플레이트의 각 웰에 분주하고 37℃, 5% CO2, 습윤하 조건에서 밤새 배양하였다. 바이러스 샘플 원본의 1:10으로 예를 들어, 바이러스 원본의 10-2에서 10-8까지 일련의 희석을 준비하였으며, 모든 바이러스 샘플은 각 웰에 50 μl의 바이러스를 옮기기 직전까지 강하게 vortexing 하였다. 세포 플레이트는 37℃, CO2 인큐베이터에서 4일간 배양한 후 CPE (Cytopathic effect)를 관찰하였다. CPE가 3개의 개별 판독값에 대해 희석당 동일하게 나타날 때 endpoint를 결정하였으며, Spearman-Karber 방법 (Ramakrishnan, 2016)에 기초하여 역가를 계산하였다. EEV의 비율(%)은 Intra 및 extra 바이러스 역가의 합에 대한 extra 바이러스의 역가를 백분율로 계산하였다.Viral titers were determined by the TCID 50 assay, one of the infectivity assays. 4.2×10 3 Vero cells were suspended in growth medium, dispensed into each well of a 96-well plate, and incubated overnight at 37°C, 5% CO 2 , under humid conditions. A serial dilution of 1:10 of the original virus sample, for example, from 10 -2 to 10 -8 of the original virus sample was prepared, and all virus samples were vigorously vortexed until immediately before transferring 50 μl of virus to each well. The cell plate was cultured for 4 days in a 37°C, CO 2 incubator, and then CPE (Cytopathic effect) was observed. An endpoint was determined when CPE was the same per dilution for three separate readings, and titers were calculated based on the Spearman-Karber method (Ramakrishnan, 2016). The ratio (%) of EEV was calculated as a percentage of extra virus titers relative to the sum of intra and extra virus titers.
역가 측정 결과, 표 2에 나타낸 바와 같이, 계대를 진행한 바이러스(P14)의 외피외 바이러스 비율이 야생형 IHD 바이러스보다 높게 나타났다.As a result of the titer measurement, as shown in Table 2, the ratio of the virus outside the envelope of the passaged virus (P14) was higher than that of the wild-type IHD virus.
Figure PCTKR2022021646-appb-img-000002
Figure PCTKR2022021646-appb-img-000002
나아가, 도 6에 나타낸 바와 같이, 계대를 진행한 바이러스의 세포 내 및 세포 외 바이러스 역가가 야생형 IHD 바이러스보다 약 10배 증가하여 전체적으로 외피외 바이러스 생산성이 향상된 것을 확인할 수 있었다.Furthermore, as shown in FIG. 6, it was confirmed that the intracellular and extracellular viral titers of the passaged virus increased about 10-fold compared to the wild-type IHD virus, indicating that the overall productivity of the extravesicular virus was improved.
전술한 본 발명의 설명은 예시를 위한 것이며, 본 발명이 속하는 기술분야의 통상의 지식을 가진 자는 본 발명의 기술적 사상이나 필수적인 특징을 변경하지 않고서 다른 구체적인 형태로 쉽게 변형이 가능하다는 것을 이해할 수 있을 것이다. 그러므로 이상에서 기술한 실시예들은 모든 면에서 예시적인 것이며 한정적이 아닌 것으로 이해해야만 한다.The above description of the present invention is for illustrative purposes, and those skilled in the art can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, the embodiments described above should be understood as illustrative in all respects and not limiting.
본 발명의 백시니아 바이러스는 바이러스 전파력과 관련된 외피외 바이러스 생산성이 증가되어 종양 내 바이러스 확산을 향상시킬 수 있어 백시니아 바이러스의 종양 용해 가능성을 극대화함으로써 종래의 백시니아 바이러스보다 유의적으로 높은 항암 효과를 나타낼 수 있는바 산업상 이용가능성이 있다.The vaccinia virus of the present invention increases the productivity of the extravesicular virus related to the virus propagation ability and can enhance the spread of the virus in the tumor, thereby maximizing the oncolysis potential of the vaccinia virus, thereby achieving a significantly higher anticancer effect than the conventional vaccinia virus. As can be shown, there is industrial applicability.
Figure PCTKR2022021646-appb-img-000003
Figure PCTKR2022021646-appb-img-000003

Claims (7)

  1. 기탁번호 KCTC 15195BP로 수탁된 외피외 바이러스(extracellular enveloped virus; EEV) 생산이 증가한 것을 특징으로 하는 신규한 백시니아 바이러스.A novel vaccinia virus characterized by increased production of extracellular enveloped virus (EEV) deposited under accession number KCTC 15195BP.
  2. 제1항에 있어서,According to claim 1,
    상기 백시니아 바이러스는 IHD 스트레인(strain)인 것을 특징으로 하는, 백시니아 바이러스.Characterized in that the vaccinia virus is an IHD strain, vaccinia virus.
  3. 제1항에 있어서,According to claim 1,
    상기 백시니아 바이러스는 C2L, C1L, N1L, N2L, M1L, M2L, K1L, K2L, K3L, K4L, K5L, K6L, K7R, F1L, F2L, F3L, F4L, F5L, A52R, A53R, A55R, 및 A56R로 이루어진 군으로부터 선택된 하나 이상의 유전자가 결실된 것을 특징으로 하는, 백시니아 바이러스.The vaccinia virus is C2L, C1L, N1L, N2L, M1L, M2L, K1L, K2L, K3L, K4L, K5L, K6L, K7R, F1L, F2L, F3L, F4L, F5L, A52R, A53R, A55R, and A56R. A vaccinia virus, characterized in that one or more genes selected from the group consisting of are deleted.
  4. 제1항에 있어서,According to claim 1,
    상기 백시니아 바이러스는 코멧 플라크(comet plaque)를 형성하는 것을 특징으로 하는, 백시니아 바이러스.Vaccinia virus, characterized in that the vaccinia virus forms a comet plaque (comet plaque).
  5. (a) 세포에 백시니아 바이러스를 감염시키고 감염된 세포를 배양하는 단계;(a) infecting cells with vaccinia virus and culturing the infected cells;
    (b) 상기 단계 (a) 의 상층액으로 다른 세포를 백시니아 바이러스에 감염시키고 감염된 세포를 배양하며, 상층액만을 분리하여 (b) 단계를 반복 수행하는 단계; 및(b) infecting other cells with vaccinia virus with the supernatant of step (a), culturing the infected cells, isolating only the supernatant, and repeating step (b); and
    (c) 상기 단계 (b) 의 감염된 세포에서 증식하는 백시니아 바이러스를 분리하는 단계를 포함하는 제1항의 신규한 백시니아 바이러스의 제조방법.(c) isolating vaccinia virus proliferating from the cells infected in step (b);
  6. 제5항에 있어서,According to claim 5,
    상기 단계 (b) 는 2회 내지 20회 반복하는 것을 특징으로 하는, 신규한 백시니아 바이러스의 제조방법.The method for producing a novel vaccinia virus, characterized in that step (b) is repeated 2 to 20 times.
  7. 제5항에 있어서,According to claim 5,
    상기 백시니아 바이러스는 IHD 스트레인(strain)인 것을 특징으로 하는, 신규한 백시니아 바이러스의 제조방법.The method for producing a novel vaccinia virus, characterized in that the vaccinia virus is an IHD strain.
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