WO2023106839A1 - Virus de la vaccine recombiné exprimant l'il-12 et son utilisation - Google Patents

Virus de la vaccine recombiné exprimant l'il-12 et son utilisation Download PDF

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WO2023106839A1
WO2023106839A1 PCT/KR2022/019840 KR2022019840W WO2023106839A1 WO 2023106839 A1 WO2023106839 A1 WO 2023106839A1 KR 2022019840 W KR2022019840 W KR 2022019840W WO 2023106839 A1 WO2023106839 A1 WO 2023106839A1
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cancer
vaccinia virus
recombinant vaccinia
virus
gene
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PCT/KR2022/019840
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English (en)
Korean (ko)
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손우찬
김태희
이지영
손호선
고수민
이장미
강지연
정다솜
임희선
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재단법인 아산사회복지재단
울산대학교 산학협력단
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Priority claimed from KR1020220169132A external-priority patent/KR20230086610A/ko
Application filed by 재단법인 아산사회복지재단, 울산대학교 산학협력단 filed Critical 재단법인 아산사회복지재단
Publication of WO2023106839A1 publication Critical patent/WO2023106839A1/fr

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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
    • A61K35/768—Oncolytic viruses not provided for in groups A61K35/761 - A61K35/766
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof

Definitions

  • the present invention relates to a cancer therapeutic agent using a recombinant virus, and more particularly, to a recombinant vaccinia virus having an interleukin-12 (IL-12) gene and a B8R gene deletion, and a use thereof.
  • IL-12 interleukin-12
  • Oncolytic virus which is one of the treatments that has recently attracted attention in the pharmaceutical industry, does not proliferate in normal cells, but proliferates specifically in cancer cells and destroys cancer cells.
  • the four main mechanisms of oncolytic viruses are oncolysis by direct infection, release of tumor-associated antigen (TAA) that contributes to the activation of anti-tumor immune cells, promotion of host immunity by expression of foreign genes genetically inserted into recombinant viruses, and And as a mechanism to prevent cancer recurrence due to B-cell and T-cell immune memory, in addition to directly attacking cancer cells, it promotes the body's adaptive immune response (Immune stimulation) and specifically infects and destroys tumor vascular endothelial cells. It is known to inhibit angiogenesis.
  • TAA tumor-associated antigen
  • the inventors of the present invention found that when the B8R gene of vaccinia virus is substituted with the interleukin-12 gene, immunity due to increased IFN- ⁇ activity and IL-12 activity The present invention was completed by confirming that anti-tumor immunity is remarkably increased by synergistic effect in .
  • an object of the present invention is a recombinant vaccinia virus comprising an interleukin-12 (IL-12) gene, characterized in that the interleukin-12 gene is inserted into the B8R region of the vaccinia virus gene. to provide vaccinia virus.
  • IL-12 interleukin-12
  • Another object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer comprising the recombinant vaccinia virus as an active ingredient.
  • Another object of the present invention is to provide a composition for enhancing anti-tumor immunity comprising the recombinant vaccinia virus as an active ingredient.
  • Another object of the present invention is to provide a kit for preventing or treating cancer comprising the recombinant virus as an active ingredient.
  • Another object of the present invention is to provide a method for preparing the recombinant virus.
  • the present invention is a recombinant vaccinia virus comprising an interleukin-12 (IL-12) gene, characterized in that the interleukin-12 gene is inserted into the B8R region of the vaccinia virus gene To provide a recombinant vaccinia virus.
  • IL-12 interleukin-12
  • the interleukin-12 (IL-12) gene may consist of the nucleotide sequence of SEQ ID NO: 9, but is not limited thereto.
  • the vaccinia virus gene B8R may consist of the nucleotide sequence of SEQ ID NO: 11, but is not limited thereto.
  • the recombinant vaccinia virus may have a vaccinia virus gene B8R region deleted, but is not limited thereto.
  • the vaccinia virus is Western Reserve (WR), New York Vaccinia Virus (NYVAC), Wyeth (The New York City Board of Health; NYCBOH), LC16m8, Lister , Copenhagen, Tian Tan, USSR, Tashkent, Evans, and International Health Division (IHD) vaccinia virus strain.
  • it may be an International Health Division (IHD) vaccinia virus species, but is not limited thereto.
  • the present invention provides a composition for preventing or treating cancer comprising the recombinant vaccinia virus as an active ingredient.
  • the present invention provides a composition for enhancing anti-tumor immunity comprising the recombinant vaccinia virus as an active ingredient.
  • the present invention provides a kit for preventing or treating cancer comprising the recombinant vaccinia virus as an active ingredient.
  • the present invention may provide a food composition containing the recombinant vaccinia virus as an active ingredient, and the food composition may be a health functional food composition.
  • the present invention provides a method for preventing or treating cancer comprising administering the recombinant vaccinia virus to a subject in need thereof.
  • the present invention provides a use of the recombinant vaccinia virus for preventing or treating cancer.
  • the present invention provides a use of the recombinant vaccinia virus for the preparation of a cancer therapeutic agent.
  • the present invention provides a method for enhancing anti-tumor immunity comprising administering the recombinant vaccinia virus to a subject in need thereof.
  • the present invention provides a use of the recombinant vaccinia virus for enhancing antitumor immunity.
  • the present invention provides a use of the recombinant vaccinia virus for preparing an anti-tumor immunity enhancer.
  • the cancer is squamous cell carcinoma, glioma, lung cancer, adenocarcinoma of the lung, peritoneal cancer, skin cancer, eye cancer, rectal cancer, perianal cancer, esophageal cancer, small intestine cancer, endocrine adenocarcinoma, parathyroid cancer, adenocarcinoma Renal cancer, osteosarcoma, soft tissue sarcoma, urethral cancer, blood cancer, liver cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial cancer, uterine cancer, salivary gland cancer, kidney cancer, prostate It may be at least one selected from the group consisting of cancer, vulvar cancer, thyroid cancer, head and neck cancer, oral cancer, tongue cancer, brain cancer, malignant melanoma, and stromal tumor, but is not limited thereto.
  • the composition may satisfy at least one characteristic selected from the group consisting of, but is not limited thereto.
  • the composition may enhance anti-tumor immunity, but is not limited thereto.
  • the present invention includes the steps of inserting the interleukin-12 (IL-12) gene into a delivery vector; and inserting the transfer vector into the vaccinia virus.
  • IL-12 interleukin-12
  • the present invention relates to a recombinant vaccinia virus expressing IL-12 and a use thereof, wherein the recombinant vaccinia virus in which the B8R gene is deleted from the vaccinia virus and the IL-12 gene is inserted at the site of the deleted B8R is IFN- ⁇
  • the recombinant vaccinia virus in which the B8R gene is deleted from the vaccinia virus and the IL-12 gene is inserted at the site of the deleted B8R is IFN- ⁇
  • FIG. 1 is a schematic diagram showing a cloning method of plasmid pB8R(-)mCherry-mIL12.
  • FIGS. 2a and 2b are schematic diagrams of recombinant plasmids pSP72-B8R and pB8R(-)mCherry-mIL12.
  • FIG. 3 is a view showing the results of confirming the plaque of the recombinant vaccinia virus (B8R(-)mCherry-mIL12 virus) of the present invention isolated through a single plaque isolation process (A: Bright-field microscopy, ⁇ 100/B: Fluorescence microscopy (Red), ⁇ 100).
  • Figure 4 shows the expression of IL-12 protein by performing Western blot analysis on recombinant vaccinia virus-treated cells and media for characterization of the recombinant vaccinia virus (B8R(-)mCherry-mIL12 virus) of the present invention. This is a diagram showing the result.
  • FIG. 5 is a diagram showing the results of comparing the cytotoxicity of the recombinant vaccinia virus (B8R(-)mCherry-mIL12 virus) of the present invention in Vero cells, MC38 cells and LLC1 cells.
  • Figure 6 shows the results of comparing the cytotoxicity of IHD virus (parental virus) to which no genetic manipulation was applied and the recombinant vaccinia virus (B8R(-)mCherry-mIL12 virus) of the present invention on mouse pancreatic cancer cells (Pan02). It is a drawing
  • Figure 7 shows the results of comparison of cytotoxicity against mouse melanoma cells (B16F10) of IHD virus (parental virus) without genetic manipulation and the recombinant vaccinia virus (B8R(-)mCherry-mIL12 virus) of the present invention. is the drawing shown.
  • FIG. 8a to 8e confirm the anti-tumor effect of the recombinant vaccinia virus (B8R(-)mCherry-mIL12 virus) of the present invention in a mouse pancreatic cancer model.
  • FIG. 8c is a result of measuring the tumor volume
  • FIG. 8d is a result of measuring the tumor weight
  • FIG. 8e is a view showing a tumor extracted from a mouse pancreatic cancer model.
  • FIG. 9 is a view showing the results of confirming T cell activity after treatment with the recombinant vaccinia virus (B8R(-)mCherry-mIL12 virus) of the present invention using spleen tissue extracted from a mouse pancreatic cancer model.
  • 10A to 10C show the results of analyzing the number of tumor-infiltrating lymphocytes by performing immunostaining on tumor tissue extracted from a mouse pancreatic cancer model after treatment with the recombinant vaccinia virus (B8R(-)mCherry-mIL12 virus) of the present invention.
  • 10A is a diagram showing the analysis result of a CD3 immunostaining slide
  • FIG. 10B is a diagram showing the analysis result of a CD8 immunostaining slide
  • FIG. 10C is a diagram showing the analysis result of a CD4 immunostaining slide.
  • FIG. 11a to 11e confirm the anti-tumor effect of the recombinant vaccinia virus (B8R(-)mCherry-mIL12 virus) of the present invention in a mouse melanoma model.
  • FIG. 11c is a result of measuring the tumor volume
  • FIG. 11d is a result of measuring the tumor weight
  • FIG. 11e is a diagram showing a tumor extracted from a mouse melanoma model.
  • FIG. 12 is a view showing the results of confirming T cell activity after treatment with the recombinant vaccinia virus (B8R(-)mCherry-mIL12 virus) of the present invention using spleen tissue extracted from a mouse melanoma model.
  • FIG. 13 is a diagram showing the results of confirming the distribution of infiltrated cytotoxic T cells in tumors by performing CD8 immunofluorescence staining on tumor tissue extracted from a mouse melanoma model.
  • FIG. 14a to 14d confirm the anti-tumor effect of the recombinant vaccinia virus (B8R(-)mCherry-mIL12 virus) of the present invention in a hamster melanoma model.
  • FIG. 14c is a result of measuring the tumor weight
  • FIG. 14d is a view showing a tumor extracted from a hamster melanoma model.
  • FIG. 15 is a view showing the results of confirming T cell activity after treatment with the recombinant vaccinia virus (B8R(-)mCherry-mIL12 virus) of the present invention using spleen tissue extracted from a hamster melanoma model.
  • 16a to 16c show the results of analyzing the number of tumor-infiltrating lymphocytes by performing immunostaining on tumor tissue extracted from a hamster melanoma model after treatment with the recombinant vaccinia virus (B8R(-)mCherry-mIL12 virus) of the present invention
  • 16a is a view showing the analysis result of the CD3 immunostaining slide
  • FIG. 16b is the analysis result of the CD8 immunostaining slide
  • FIG. 16c is the analysis result of the CD4 immunostaining slide.
  • 17 is a diagram schematically showing the anti-tumor mechanism of the recombinant vaccinia virus (B8R(-)mCherry-mIL12 virus) of the present invention.
  • FIG. 18 is a schematic diagram of the recombinant vaccinia virus (B8R(-)mCherry-mIL12 virus) of the present invention.
  • the present invention relates to a recombinant vaccinia virus containing an interleukin-12 (IL-12) gene and a use thereof, wherein when the B8R gene of the vaccinia virus is replaced with the IL-12 gene, IFN- ⁇ activity And it was completed by confirming that an increase in IL-12 activity exhibits a synergistic effect on immunity and enhances anti-tumor immunity to exhibit a remarkable anti-cancer effect.
  • IL-12 interleukin-12
  • the present invention is a recombinant vaccinia virus containing an interleukin-12 (IL-12) gene,
  • the recombinant vaccinia virus is characterized in that the interleukin-12 gene is inserted into the B8R region of the vaccinia virus gene.
  • vaccina virus also called cowpox virus
  • cowpox virus is one of the most widely studied oncolytic viruses, which proliferates in the cytoplasm and has an envelope with 200 kb double-stranded DNA as its genome. It is a virus that belongs to poxvirus.
  • Vaccinia virus transcribes and translates 200 kb of double-stranded DNA to express a total of 250 genes, and has a 200 kb linear DNA genome that allows deletion of multiple genes and insertion of up to 40 kb of transgenes, enabling gene therapy and It is widely used as a transportation tool, and since it proliferates depending on the EGFR/RAS pathway, which is activated in epithelial cancer, it has an innate tumor specificity and has the advantage of low risk of carcinogenicity.
  • the vaccinia virus is Western Reserve (WR), New York Vaccinia Virus (NYVAC), Wyeth (The New York City Board of Health; NYCBOH), LC16m8, Lister, Copenhagen ), Tian Tan, USSR, Tashkent, Evans, International Health Division (IHD) vaccinia virus strain, but is not limited thereto.
  • IHD strain vaccinia virus was used.
  • “recombinant vaccinia virus” means a vaccinia virus in which one or more foreign genes are inserted (introduced) into the genome.
  • the position in the genome where the foreign gene is inserted is not limited, but preferably, the recombinant vaccinia virus according to the present invention is characterized in that the IL-12 gene is inserted into the B8R region of the vaccinia virus gene, more preferably, The recombinant vaccinia virus is characterized in that the B8R gene of the vaccinia virus is substituted with the IL-12 gene.
  • the vaccinia virus to be genetically modified in the present invention may be wild-type, or may have some sequences deleted to enhance infection efficiency, replication function, anticancer effect, and the like.
  • B8R is a gene of vaccinia virus that encodes a secretory interferon gamma (IFN- ⁇ ) binding protein, and the interferon gamma binding protein inhibits the interferon gamma activity of the host by blocking the binding of interferon gamma. It is characterized by delaying antiviral immunity.
  • the recombinant vaccinia virus according to the present invention is deficient in the vaccinia virus gene B8R region and thus does not produce interferon gamma binding protein, so that it can inhibit T lymphocytes and natural killer cells (NK cells) in the host. It is characterized by increasing interferon gamma activity involved in activating and improving humoral immunity.
  • the B8R gene may consist of the nucleotide sequence represented by SEQ ID NO: 11, but is not limited thereto, and variants of the nucleotide sequence are included within the scope of the present invention.
  • the nucleic acid molecule of the nucleotide sequence represented by SEQ ID NO: 11 of the present invention is a functional equivalent of the nucleic acid molecule constituting it, for example, a part of the nucleotide sequence of the nucleic acid molecule is deleted, substituted (substitution) or inserted (insertion) Although modified by , it is a concept that includes variants that can functionally have the same action as a nucleic acid molecule.
  • the B8R gene has 70% or more, more preferably 80% or more, still more preferably 90% or more, and most preferably 95% or more sequence homology with the nucleotide sequence represented by SEQ ID NO: 11, respectively. sequence may be included. For example, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85 %, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence homology It includes a polynucleotide having.
  • the “% of sequence homology” for polynucleotides is determined by comparing two optimally aligned sequences with a comparison region, wherein a portion of the polynucleotide sequence in the comparison region is a reference sequence (addition or deletion) for the optimal alignment of the two sequences. may include additions or deletions (i.e., gaps) compared to (not including).
  • the B8R gene may be characterized in that it encodes a protein having the amino acid sequence of SEQ ID NO: 12.
  • interferon gamma IFN-g or IFN- ⁇
  • IFN-g or IFN- ⁇ IFN-g or IFN- ⁇
  • deletion used in relation to a viral genetic sequence herein has the meaning of including a complete deletion of the corresponding sequence as well as a partial deletion of the sequence.
  • the expression of the functional protein of the gene does not occur or is inhibited by the deletion of the sequence.
  • IL-12 interleukin-12
  • IL-12 is one of the most effective and promising anticancer cytokines.
  • IL-12 is a heterodimeric protein comprising 40 kDa and 35 kDa subunits linked by disulfide bonds and is produced by activated macrophages, monocytes, dendritic cells and activated B lymphocytes.
  • IL-12 can enhance T-helper 1 cell immunity, increase cellular T-lymphocyte cytotoxicity, and inhibit angiogenesis.
  • IL-12 activates IFN- ⁇ production of T cells and NK cells.
  • Local expression of IL-12 sensitizes tumor cells to T-cell mediated cytotoxicity, resulting in inhibition of tumor growth and establishment of systemic immunity.
  • the IL-12 gene may be a mouse IL-12 gene (mIL-12).
  • the IL-12 gene may consist of the nucleotide sequence represented by SEQ ID NO: 9, but is not limited thereto, and variants of the nucleotide sequence are included within the scope of the present invention.
  • the nucleic acid molecule of the nucleotide sequence represented by SEQ ID NO: 9 of the present invention is a functional equivalent of the nucleic acid molecule constituting it, for example, a part of the nucleotide sequence of the nucleic acid molecule is deleted, substituted (substitution) or inserted (insertion) Although modified by , it is a concept that includes variants that can functionally have the same action as a nucleic acid molecule.
  • the IL-12 gene has a sequence homology of at least 70%, more preferably at least 80%, even more preferably at least 90%, and most preferably at least 95% with the nucleotide sequence represented by SEQ ID NO: 9.
  • the branch may include a nucleotide sequence.
  • the "% sequence homology" for polynucleotides is determined by comparing two optimally aligned sequences with a region of comparison, wherein a portion of the polynucleotide sequence in the region of comparison is a reference sequence for the optimal alignment of the two sequences (additional or not including deletions) compared to additions or deletions (i.e., gaps).
  • the IL-12 gene may be characterized in that it encodes a protein having the amino acid sequence of SEQ ID NO: 10.
  • the IL-12 gene can be introduced into the B8R gene region in the genome of vaccinia virus using a vector, and according to an embodiment of the present invention, the recombinant vaccinia virus according to the present invention uses a vector It is characterized in that the IL-12 gene is expressed without B8R gene expression by replacing the B8R gene with the IL-12 gene.
  • the genome modification method of the recombinant vaccinia virus according to the present invention is not limited and can be prepared using known genetic recombination techniques.
  • the recombinant vaccinia virus according to the present invention may have a foreign gene (ie, IL-12 gene) inserted into the genome through homologous endjoining.
  • Homologous end joining is one of the methods for repairing double-strand breaks in DNA. It is characterized by synthesizing the cut DNA using a homologous sequence as a template during DNA repair, and non-homologous ends in which the cut DNA strands are connected as they are. It is distinct from non-homologous endjoining.
  • cells are infected with wild-type vaccinia virus, and then a vector containing a gene to be inserted into the virus genome is introduced into the cell to convert the vaccinia virus genome into the cell. Modification (i.e. insertion of a gene) can be induced to occur.
  • the introduction of the vector into the cells and the infection of the wild-type vaccinia virus are not limited in order and can be performed simultaneously or sequentially.
  • vectors into cells i.e., transfection or transfection of vectors
  • introduction of vectors into cells is accomplished by a variety of techniques commonly used to introduce exogenous nucleic acids (DNA or RNA) into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate Precipitation method, DEAE-dextran transfection or lipofection may be performed.
  • the recombinant vaccinia virus according to the present invention may further include a marker gene for selecting transformants.
  • the marker gene may be included without limitation as long as it is simultaneously expressed with the IL-12 gene inserted into the genome of the vaccinia virus to impart a phenotype enabling identification of the virus or transformant.
  • the recombinant vaccinia virus according to the present invention may include a fluorescent protein coding gene as a marker gene.
  • Fluorescent protein refers to a protein that emits light of a specific wavelength to the outside in the process of being activated by absorbing light of a specific wavelength and then returning to a normal state, thereby emitting light in a unique color.
  • the fluorescent protein examples include a green fluorescent protein (GFP) coding gene, a red fluorescent protein (RFP) protein coding gene, and an mCherry coding gene.
  • the recombinant vaccinia virus according to the present invention may further include an mCherry coding gene (eg, SEQ ID NO: 13) as a marker gene.
  • the location of the marker gene in the genome is not limited, but may preferably be present in the B8R gene region, more preferably upstream or downstream of the IL-12 gene inserted into the genome of the recombinant vaccinia virus. ) can be located.
  • the present invention provides a pharmaceutical composition for preventing or treating cancer comprising the recombinant vaccinia virus according to the present invention as an active ingredient.
  • active ingredient means a component that exhibits the desired activity alone or can exhibit the desired activity together with a carrier having no activity itself.
  • cancer is characterized by uncontrolled cell growth, which results in the formation of a cell mass called a tumor, infiltrating surrounding tissues and, in severe cases, metastasizing to other organs in the body.
  • cancer may be solid cancer or blood cancer, and non-limiting examples thereof include squamous cell carcinoma, glioma, lung cancer, lung adenocarcinoma, peritoneal cancer, skin cancer, eye cancer, rectal cancer, perianal cancer, esophageal cancer, and small intestine.
  • cancer endocrine cancer, parathyroid cancer, adrenal cancer, osteosarcoma, soft tissue sarcoma, urethral cancer, blood cancer, liver cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial cancer, It may be selected from the group consisting of uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, head and neck cancer, oral cancer, tongue cancer, brain cancer, malignant melanoma, and stromal tumor.
  • the blood cancer may be leukemia, lymphoma, multiple myeloma, and the like.
  • the content of the recombinant vaccinia virus in the composition of the present invention can be appropriately adjusted according to the symptoms of the disease, the degree of progression of the symptoms, the condition of the patient, etc., for example, 0.0001 to 99.9% by weight, or 0.001 to 50% by weight based on the total weight of the composition. It may be % by weight, but is not limited thereto.
  • the content ratio is a value based on the dry amount after removing the solvent.
  • the pharmaceutical composition according to the present invention may further include suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions.
  • the excipient may be, for example, one or more selected from the group consisting of a diluent, a binder, a disintegrant, a lubricant, an adsorbent, a moisturizer, a film-coating material, and a controlled release additive.
  • compositions according to the present invention are powders, granules, sustained-release granules, enteric granules, solutions, eye drops, elsilic agents, emulsions, suspensions, spirits, troches, perfumes, and limonadese, respectively, according to conventional methods.
  • tablets, sustained-release tablets, enteric tablets, sublingual tablets, hard capsules, soft capsules, sustained-release capsules, enteric capsules, pills, tinctures, soft extracts, dry extracts, fluid extracts, injections, capsules, perfusate It can be formulated and used in the form of an external agent such as a warning agent, lotion, pasta agent, spray, inhalant, patch, sterile injection solution, or aerosol, and the external agent is a cream, gel, patch, spray, ointment, warning agent , lotion, liniment, pasta, or cataplasma may have formulations such as the like.
  • Carriers, excipients and diluents that may be included in the pharmaceutical composition according to the present invention include lactose, dextrose, sucrose, oligosaccharide, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.
  • composition according to the present invention When formulated, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.
  • the pharmaceutical composition according to the present invention may be formulated so that the recombinant vaccinia virus contained in the composition is bioavailable when administered into a subject. After administration, vaccinia virus present in tumor tissue, blood, and other tissues can be monitored through a detection method such as ELISA.
  • composition according to the present invention is administered in a pharmaceutically effective amount.
  • pharmaceutically effective amount means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level is the type of patient's disease, severity, activity of the drug, It may be determined according to factors including sensitivity to the drug, administration time, route of administration and excretion rate, duration of treatment, drugs used concurrently, and other factors well known in the medical field.
  • the pharmaceutical composition according to the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered single or multiple times. Considering all of the above factors, it is important to administer an amount that can obtain the maximum effect with the minimum amount without side effects, which can be easily determined by a person skilled in the art to which the present invention belongs.
  • the pharmaceutical composition according to the present invention may be administered once or twice or more repeatedly to a subject in need thereof, and may be repeatedly administered until the tumor is reduced or disappears.
  • the pharmaceutical composition of the present invention can be administered to a subject by various routes. All modes of administration can be envisaged, eg oral administration, subcutaneous injection, intraperitoneal administration, intravenous injection, intramuscular injection, paraspinal space (intrathecal) injection, sublingual administration, buccal administration, intrarectal insertion, vaginal It can be administered by intraoral insertion, ocular administration, otic administration, nasal administration, inhalation, spraying through the mouth or nose, dermal administration, transdermal administration, and the like.
  • the recombinant vaccinia virus according to the present invention may be administered intratumorally through direct or intravenous administration, and an appropriate administration method may be selected depending on the state and type of tumor.
  • the pharmaceutical composition of the present invention is determined according to the type of drug as an active ingredient together with various related factors such as the disease to be treated, the route of administration, the age, sex, weight and severity of the disease of the patient.
  • the present invention provides a method for preventing or treating cancer comprising administering the recombinant vaccinia virus to a subject in need thereof.
  • the present invention provides a use of the recombinant vaccinia virus for preventing or treating cancer.
  • the present invention provides a use of the recombinant vaccinia virus for the preparation of a cancer therapeutic agent.
  • “individual” means a subject in need of treatment of a disease, and more specifically, a human or non-human primate, mouse, rat, dog, cat, horse, cow, etc. of mammals.
  • administration means providing a given composition of the present invention to a subject by any suitable method.
  • prevention refers to any action that suppresses or delays the onset of a desired disease
  • treatment means that the desired disease and its resulting metabolic abnormality are improved or improved by administration of the pharmaceutical composition according to the present invention. All actions that are advantageously altered are meant, and “improvement” means any action that reduces a parameter related to a target disease, for example, the severity of a symptom, by administration of the composition according to the present invention.
  • the recombinant vaccinia virus according to the present invention expresses the IL-12 protein (see Example 3).
  • the recombinant vaccinia virus according to the present invention showed superior tumor killing ability compared to the parent virus (see Example 5).
  • composition according to the present invention may satisfy one or more properties selected from the group consisting of:
  • composition according to the present invention can enhance anti-tumor immunity.
  • the present invention provides a composition for enhancing anti-tumor immunity comprising the recombinant vaccinia virus according to the present invention as an active ingredient.
  • the present invention provides a method for enhancing anti-tumor immunity comprising administering the recombinant vaccinia virus to a subject in need thereof.
  • the present invention provides a use of the recombinant vaccinia virus for enhancing antitumor immunity.
  • the present invention provides a use of the recombinant vaccinia virus for preparing an anti-tumor immunity enhancer.
  • anti-tumor includes death of cancer cells, reduction in viability of cancer cells, inhibition or delay of pathological symptoms of cancer due to inhibition of cancer cell proliferation, inhibition of cancer metastasis, and inhibition of cancer recurrence. it means to
  • composition for enhancing antitumor immunity of the present invention may be used in combination or mixing with any approved anticancer agent.
  • anticancer agent includes any anticancer agent that exhibits cytotoxicity to cancer cells, such as a metabolic antagonist, an alkylating agent, a topoisomerase antagonist, a microtubule antagonist, a plant-derived alkaloid, any cytokine drug, any antibody It includes pharmaceuticals, any immune checkpoint inhibitor medicines, any cell therapy (car-T cell therapy, car-NK cell therapy) medicines, and the like.
  • taxol nitrogen mustard, imatinib, oxaliplatin, gefitinib, bortezomib, sunitinib, carboplatin, cisplatin, rituximab, erlotinib, sorafenib, IL-2 drugs, INF- ⁇ drugs, INF - ⁇ drugs, Trastuzumab, Blinatumomab, Ipilimumab, Pepbrolizumab, Nivolizumab, Atezolizumab, Durvalumab, Bevacizumab, Cetuximab, Tisagenrelucel ( Tisagenlecleucel (Kymria), Axicabtagene Ciloleucel (Yescata), etc. may be exemplified, and in addition to these exemplified anticancer agents, other anticancer agents known in the art are used in combination with the composition of the present invention without limitation or may be used in combination.
  • the present invention may provide a food composition for prevention or improvement containing the recombinant vaccinia virus according to the present invention as an active ingredient, and the food composition may be a health functional food composition.
  • the recombinant vaccinia virus according to the present invention When the recombinant vaccinia virus according to the present invention is used as a food additive, the recombinant vaccinia virus may be added as it is or used together with other foods or food ingredients, and may be appropriately used according to a conventional method.
  • the mixing amount of the active ingredient may be appropriately determined according to the purpose of use (prevention, health or therapeutic treatment).
  • the recombinant vaccinia virus of the present invention may be added in an amount of 15% by weight or less, or 10% by weight or less based on the raw material.
  • the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount greater than the above range.
  • Examples of foods to which the above substances can be added include meat, sausages, bread, chocolates, candies, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice creams, various soups, beverages, tea, drinks, There are alcoholic beverages and vitamin complexes, and includes all health functional foods in a conventional sense.
  • the health beverage composition according to the present invention may contain various flavoring agents or natural carbohydrates as additional components, like conventional beverages.
  • the aforementioned natural carbohydrates are monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrins and cyclodextrins, and sugar alcohols such as xylitol, sorbitol and erythritol.
  • natural sweeteners such as thaumatin and stevia extract, or synthetic sweeteners such as saccharin and aspartame may be used.
  • the proportion of the natural carbohydrate is generally about 0.01-0.20 g, or about 0.04-0.10 g per 100 mL of the composition of the present invention.
  • the composition of the present invention contains various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, A carbonation agent used in carbonated beverages and the like may be contained.
  • the composition of the present invention may contain fruit flesh for preparing natural fruit juice, fruit juice beverages and vegetable beverages. These components may be used independently or in combination. The ratio of these additives is not critical, but is generally selected in the range of 0.01-0.20 parts by weight per 100 parts by weight of the composition of the present invention.
  • health functional food is the same term as food for special health use (FoSHU), and refers to foods with high medical and medical effects that are processed to efficiently display bioregulatory functions in addition to nutritional supply.
  • the food may be prepared in various forms such as tablets, capsules, powders, granules, liquids, and pills in order to obtain useful effects for preventing or improving cancer.
  • the health functional food of the present invention can be prepared by a method commonly used in the art, and can be prepared by adding raw materials and components commonly added in the art during the preparation.
  • unlike general drugs there is an advantage in that there is no side effect that may occur when taking a drug for a long time by using food as a raw material, and it can be excellent in portability.
  • the present invention provides a kit for preventing or treating cancer comprising the recombinant vaccinia virus according to the present invention as an active ingredient.
  • the kit may further include an immuno-anticancer agent.
  • the kit means a combination of substances or devices that can be used to prevent or treat cancer, and may be in a form capable of preparing, storing, or administering the recombinant vaccinia virus according to the present invention, and the specific form is not limited. . Therefore, the kit according to the present invention may include, without limitation, those known in the art as components of kits for the treatment of specific diseases as well as recombinant vaccinia virus and immuno-anticancer agents.
  • the present invention includes the steps of inserting the interleukin-12 (IL-12) gene into a delivery vector; and inserting the transfer vector into the vaccinia virus.
  • IL-12 interleukin-12
  • pB8R(-)mCherry-mIL12 was used as the delivery vector, but is not limited thereto.
  • vector can be used as a carrier nucleic acid molecule into which an exogenous nucleotide sequence can be inserted for introduction into a cell where the vector can be replicated.
  • a nucleotide sequence may be "exogenous,” meaning that it is external to the cell into which the vector is being introduced, or that the sequence is homologous to a sequence within the cell, but the nucleic acid is located at a location within the host cell where the sequence is not normally found.
  • Vectors may include plasmids, cosmids, viruses (bacteriophages, animal viruses and plant viruses) and yeast artificial chromosomes (eg YAC).
  • Inserting the gene of interest into the vector may be performed by a method selected from all genetic recombination methods known in the art, and inserting the vector into the vaccinia virus may be performed by any transfection method known in the art. It may be performed by a method selected from among.
  • the term "combination thereof" included in the expression of the Markush form means one or more mixtures or combinations selected from the group consisting of the components described in the expression of the Markush form, It means including one or more selected from the group consisting of components.
  • the plasmid vector for the production of recombinant vaccinia virus is derived from vaccinia virus for insertion by recombination reaction at the B8R locus in the pSP72 plasmid having an origin of replication for growth in E. coli and an antibiotic ampicillin resistance gene.
  • a homologous sequence was inserted, and additionally, a foreign gene, mCherry fluorescent protein, and a mouse single-chain IL-12 gene were inserted in combination with an appropriate promoter.
  • pB8R(-)mCherry-mIL12 was prepared by constructing a plasmid in which the B8R gene was deleted and inserting the mCherry-mIL12 transgene. The overall steps of the plasmid cloning method are shown in Figure 1.
  • pSP72 a commercial plasmid
  • primers pSP72-R (SEQ ID NO: 1) and pSP72-F (SEQ ID NO: 2) (Phusion flash high-fidelity 70 PCR Master mix, F548S) described in Table 1 below. was amplified through PCR reaction using (Step1).
  • the B8R locus was also amplified through a PCR reaction using IHD virus genomic DNA as a template using the primers SP72-B8R_Left-F (SEQ ID NO: 3) and SP72-B8R_Right-R (SEQ ID NO: 4) described in Table 1 below (Step2 ).
  • the sP72 PCR product and the B8R locus PCR product were assembled to construct the SP72-B8R plasmid using an overlap cloner kit (EBK1011, Elpis Bio.) (Step3).
  • the SP72-B8R plasmid sequence was DNA sequenced by Cosmogenetech (Korea) using primers Seq-B8Rp-F (SEQ ID NO: 7), Seq-B8R-R (SEQ ID NO: 8), and commercial primers T7 and SP6 described in Table 1 below. It was verified through a genome sequencing service.
  • sequences without the B8R gene were amplified by PCR (Step4).
  • the sequence lacking the B8R locus was amplified with the primers ApaI-B8Rminus_Left-R (SEQ ID NO: 5) and XhoI-B8R_Right-F (SEQ ID NO: 6) described in Table 1 below.
  • the synthesized gene product was digested with the enzymes ApaI and XhoI (Step5).
  • the amplified B8Rminus sequence was assembled with the gene product synthesized by overlap cloner (Step6).
  • the validated plasmid was transformed into appropriate E. coli cells, plated on LB Agar plates containing ampicillin, and cultured overnight at 37°C to form colonies. Colony PCR was performed on pB8R(-)mCherry-mIL12 E.coli colonies using the primers Seq-B8Rp-F (SEQ ID NO: 7) and Seq-B8R-R (SEQ ID NO: 8) described in Table 1, respectively. Colonies with the expected band size were inoculated into 10 mL Ampicillin broth containing LB Broth medium and cultured overnight in a shaking incubator at 37 ° C. Plasmid DNA Purification Kit Mini (CMP0112, Cosmogenetech, Korea) was used to obtain purified plasmids.
  • the final product, pB8R(-)mCherry-mIL12 was prepared by Cosmogenetech using primers Seq-B8Rp-F (SEQ ID NO: 7) and Seq-B8R-R (SEQ ID NO: 8), and commercial primers T7 and SP6 described in Table 1 above. Korea) was verified through a DNA sequencing service.
  • Vaccinia virus IHD strain (VR-156) was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). The recombinant vaccinia virus was transformed by transfecting the plasmid transfer vector into Vero cells infected with the IHD virus (parental virus) (jetPRIME, #101000015, Polyplus, USA). Agarose overlay method was continuously performed to isolate single recombinant plaques until all plaques expressed red fluorescence.
  • recombinant vaccinia virus was amplified in Vero cells and purified by 30% sucrose gradient ultra-centrifugation (13,700 rpm, 4°C 1hr 20min, Acel 9, Decel 9).
  • Viral titers were determined by the TCID 50 assay, one of the infectivity assays. 4.2 ⁇ 10 3 Vero cells were prepared in growth medium in each well of a 96-well plate and cultured 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 -3 to 10 -7 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 plates were moved back to a 37°C, CO 2 incubator to monitor cytopathic effect (CPE) for 3 to 4 days. 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).
  • Vero cells seeded in 6-well plates with 1 mL of appropriate media were infected with viruses (parent virus, and pB8R(-)mCherry-mIL12 virus) for 36 hours.
  • the medium was carefully transferred to a 1.5 mL e-tube and centrifuged at 13,000 rpm for 5 minutes, and the supernatant was transferred to a new 1.5 mL e-tube, and 10 mM PMSF (phenylmethylsulfonyl fluoride) was added and stored at 20 ° C.
  • the remaining cell pellet was scraped into 1 mL PBS and centrifuged at 4,000 rpm for 3 minutes. The supernatant was carefully removed and the pellet was incubated in 200 uL PRO-PREP solution for a minimum of 30 min and stored at 20 °C.
  • Cells seeded in 96-well plates were infected with viruses at different concentrations (0.001, 0.01, 0.1, 0.5, 1, 5, 10, 50, 100, and 500 MOI) for 72 hours.
  • 10 uL of CCK-8 (Dojindo, Kumamoto, Japan) solution was added to each well of the plate. Plates were incubated at 37 for 2 hours.
  • Cell viability was analyzed with a TECAN 2000 spectrophotometer (Spark, Mannedorf, Switzerland) at a wavelength of 450 nm.
  • mice Female C57BL/6 mice (7 weeks old) were purchased from Orient Bio (Korea) and female Syrian golden hamsters were purchased from Jabaio (Korea). All animals were treated according to the guidelines of the Institutional Animal Care and Use Committee (IACUC) of Asan Medical Center. After purchase, a one-week acclimatization period was taken before starting drug treatment or tumorigenesis. Animals were housed under standardized conditions with controlled temperature and humidity and a 12/12-hour light/dark cycle, with free access to food and water.
  • IACUC Institutional Animal Care and Use Committee
  • mice were subcutaneously injected into the right flank with 5 ⁇ 10 5 Pan02 and B16F10 cells.
  • the mice were randomly divided into three groups, and mice in each group were treated with PBS (negative control), 1 ⁇ 10 8 TCID 50 of IHD virus, and 1 ⁇ 10 8 TCID 50 of B8R.
  • (-)mCherry-mIL12 virus was injected intratumorally once. Tumor size was measured every 2-3 days with a Vernier caliper, and tumor volume was calculated as [(width) 2 ⁇ length] ⁇ 0.5. Mice were euthanized when tumors reached 16 mm in any dimension or the experiment was terminated.
  • hamsters were subcutaneously injected into the right flank with 1 ⁇ 10 7 RPMI 1846 cells.
  • the hamsters were randomly divided into three groups, and the hamsters in each group were treated with PBS (negative control), 1 ⁇ 10 8 TCID 50 of IHD virus, and 1 ⁇ 10 8 TCID 50 of B8R.
  • (-)mCherry-mIL12 virus was injected intratumorally once.
  • Hamsters were euthanized by ending the experiment before the tumor volume reached 10000 mm 3 .
  • ELISpot plates were prepared, washed 4 times with sterile PBS (200 ul/well), and conditioned for at least 30 minutes with the same medium containing 10% serum as used for the cells.
  • a single isolated splenocyte was seeded into each well (mouse, 5.0 ⁇ 10 5 splenocytes; hamster, 2.5 ⁇ 10 5 splenocytes) and a stimulus (tumor cell) was added at a ratio of 5:1. Plates were incubated in a humidified incubator at 37° C. with 5% CO 2 for about 18 hours.
  • the detection antibody (mouse, R4-6A2-biotin; hamster, MTH29-biotin) was diluted to 1 ug/ml in PBS containing 0.5% FBS (PBS-0.5% FBS), and 100 ⁇ l was added to each well to plate the plate. Incubated for 2 hours at room temperature. Plates were washed 3 times with PBS. Streptavidin-ALP was diluted (1:1000) in PBS-0.5% FBS, and 100 ul was added to each well.
  • Plates were incubated for 1 hour at room temperature. Plates were washed 3 times with PBS. A ready-to-use substrate solution (BCIP/NBT-plus) was filtered through a 0.45 um filter and added to each well in an amount of 100 ul. Plates were washed extensively with tap water and then dried. Plates were inspected and spots counted using an ELISpot reader.
  • BCIP/NBT-plus ready-to-use substrate solution
  • Tumor and spleen tissues of each group were collected and fixed in 10% neutral formalin. Tissues were minced, dehydrated using a Shandon Excelsior ES tissue processor (Thermo Fisher Scientific), embedded in paraffin, and longitudinally embedded in paraffin blocks using an EG1150H paraffin-embedding station (Leica Biosystems, Wetzlar, Germany). Staining was performed by sectioning paraffin blocks at 3 ⁇ m.
  • Immunohistochemistry of sections was performed using an automated slide preparation system (Benchmark XT; Ventana Medical Systems Inc., Arlington, AZ). Deparaffinization, epitope retrieval, and immunostaining were performed according to the manufacturer's instructions. Epitope retrieval was performed using a cell conditioning solution. For immunostaining, BMK UltraView Universal DAB Detection Kit (catalog no. 670-501) was used. Tumor and spleen sections were prepared for 36 min at 37°C for CD3 (1:400, ab16669, Abcam, Cambridge, UK), CD4 (1:1000, ab183685, Abcam, Cambridge, UK), CD8 (1:2000, ab217344, Abcam , Cambridge) antibody was reacted.
  • CD3 1:400, ab16669, Abcam, Cambridge, UK
  • CD4 (1:1000, ab183685, Abcam, Cambridge, UK
  • CD8 (1:2000, ab217344, Abcam , Cambridge
  • UltraMap Anti-rabbit HRP was used as secondary antibody for 16 min at 37°C. Positive signals were amplified using UltraView DAB and UltraView Copper, and sections were counterstained with hematoxylin and bluing reagent. Histological examination of the tumor and spleen was performed by an experienced pathologist, and scanned images at 400 times magnification of the stained slides were obtained using a Motic Easy Scan slide scanner.
  • Tumor and spleen tissues of each group were collected and fixed in 10% neutral formalin. Tissues were minced, dehydrated using a Shandon Excelsior ES tissue processor (Thermo Fisher Scientific), embedded in paraffin, and longitudinally embedded in paraffin blocks using an EG1150H paraffin-embedding station (Leica Biosystems, Wetzlar, Germany). Paraffin blocks were sectioned at 3 ⁇ m and used for staining.
  • a plasmid (pSP72-B8R) containing the SP72-B8R locus was first prepared, and then the final product, pB8R(-)mCherry-mIL12, was successfully prepared.
  • a schematic diagram of the prepared plasmid is shown in Figures 2a and 2b. The base sequence was verified by confirming the expected band size of the PCR product after gel electrophoresis and genome sequencing service using several primers.
  • the B8R(-)mCherry-mIL12 virus was prepared through homologous recombination during transfection, which means that the viral B8R gene region was converted into the same sequence in the constructed plasmid vector. After recombination, as shown in Figure 3, the enhanced red fluorescent protein (mCherry; GenBank: BAP87017.1) gene was inserted into the viral gene to allow cells infected with the recombinant virus to express red fluorescence.
  • CCK cell counting kit
  • a cell counting kit (CCK) assay was performed on a mouse pancreatic cancer cell line (Pan02) and a mouse melanoma cell line (B16F10).
  • CCK cell counting kit
  • the B8R(-)mCherry-mIL12 virus showed better tumor killing ability than the IHD virus.
  • Example 6 In mouse pancreatic cancer model in vivo Confirmation of anti-tumor effect
  • the mouse pancreatic cancer cell line Pan02 was maintained in 10% FBS, 1% AA (Antibiotic-Antimycotic) RPMI-1640 medium while being statically cultured. After harvesting and counting the cells, a mixture of 5 ⁇ 10 5 Pan02 cells and 50 ⁇ L of Matrigel was subcutaneously injected into the right flank of a 6-week-old C57BL/6 mouse after hair removal. When the tumor was formed, the size of the major axis and the minor axis were measured and observed with a caliper, and the volume of the tumor was calculated using the formula a 2 b/2 (a: length of the minor axis, b: length of the major axis).
  • mice When the average tumor size reached 100 mm 3 , the mice were randomly divided into 3 groups, and the mice in each group were inoculated with PBS (negative control), 1 ⁇ 10 8 TCID 50 of the parent virus, and 1 ⁇ 10 8 TCID 50 of the parental virus.
  • B8R(-)mCherry-mIL12 virus was administered intratumorally as a single dose of 50 ⁇ L. The size of the tumor and the weight of the animal were measured every 2-3 days, and the animal was sacrificed on the 9th day based on the day of virus administration (day 0), and the tumor and spleen were removed.
  • the animal's body weight was measured.
  • FIG. 8B as a result of calculating the relative weight of the spleen to the body weight, it was confirmed that the B8R(-)mCherry-mIL12 virus-treated group increased about 2-fold compared to the negative control group (PBS-administered group) and the parent virus-administered group. , which means that immune boosting was induced by B8R(-)mCherry-mIL12 virus treatment.
  • tumor tissue from all animals was distinctly isolated from the right flank.
  • FIG. 8D when the weight of the isolated tumor was measured, a significant weight reduction was confirmed in the B8R(-)mCherry-mIL12 virus-administered group, and as shown in FIG. 8E, the size of the tumor was significantly reduced visually.
  • Spleen tissues were obtained after sacrifice of C57BL/6 mice transplanted with mouse pancreatic cancer cell line Pan02 tumors.
  • the spleen tissue from which the surrounding fat was removed was ground and separated into single cells, washed and counted to obtain spleen cells for each animal.
  • 5 510 splenocytes for each animal were dispensed, and 1 ⁇ 10 5 Pan02 was added as a stimulus.
  • the test was performed using a minimum of 3 wells for each animal. Splenocytes and stimulants were incubated at 37° C. for 18 hours, washed, and treated with secondary antibodies to develop spots formed by IFN- ⁇ secreted from splenocytes. The number of spots on the stained plate was counted using AID EliSpot Reader.
  • a tumor tissue obtained by sacrificing a C57BL/6 mouse transplanted with a mouse pancreatic cancer cell line Pan02 tumor was fixed in 10% neutral buffered formalin and then processed into a paraffin-embedded block. Paraffin blocks were cut to a thickness of 3 ⁇ m and attached to New Silane III-coated slides. Immunohistochemical staining using T cell marker (CD3, CD4, CD8) antibodies was performed on the slides thus obtained. The slides were subjected to deparaffinization and antigen retrieval, and reacted with primary antibodies diluted in appropriate concentrations, respectively, and reacted with secondary antibodies suitable for the primary antibody host animal, and then developed with DAB. T cells infiltrated into the tumor were stained with brown staining on the cell membrane.
  • a 400-fold magnified scanned image of the stained slide was obtained using a Motic Easy Scan slide scanner.
  • the tumor area and the number of infiltrated T cells in the tumor were analyzed using the QuPath-0.3.2 program, and the number of infiltrated T cells per unit (tumor) area was calculated.
  • Example 7 In a mouse melanoma model in vivo Confirmation of anti-tumor effect
  • Mouse malignant melanoma cell line B16F10 was maintained in 10% FBS, 1% AA (Antibiotic-Antimycotic) RPMI-1640 medium while being statically cultured. After harvesting and counting the cells, a mixture of 5 ⁇ 10 5 B16F10 cells and 50 ⁇ L of Matrigel was subcutaneously injected into the right flank of 6-week-old C57BL/6 mice. When the tumor was formed, the size of the major axis and the minor axis were measured and observed with a caliper, and the volume of the tumor was calculated using the formula a 2 b/2 (a: length of the minor axis, b: length of the major axis).
  • mice When the average tumor size reached 100 mm 3 , the mice were randomly divided into 3 groups, and the mice in each group were inoculated with PBS (negative control), 1 ⁇ 10 8 TCID 50 of the parent virus, and 1 ⁇ 10 8 TCID 50 of the parental virus.
  • B8R(-)mCherry-mIL12 virus was administered intratumorally as a single dose of 50 ⁇ L. The size of the tumor and the body weight of the animal were measured every 2-3 days, and the tumor was removed by sacrificing the animal on the 9th day based on the day of virus administration (day 0).
  • the animal's body weight was measured.
  • FIG. 11B as a result of calculating the relative weight of the spleen to the body weight, it was confirmed that the B8R(-)mCherry-mIL12 virus-treated group increased compared to the negative control group (PBS-administered group) and the parent virus-administered group, This means that immune boosting was induced by B8R(-)mCherry-mIL12 virus treatment.
  • tumor tissue from all animals was distinctly isolated from the right flank.
  • FIG. 11D when the weight of the isolated tumor was measured, a significant weight reduction was confirmed in the B8R(-)mCherry-mIL12 virus-administered group, and as shown in FIG. 11E, the size of the tumor was significantly reduced visually.
  • Spleen tissues were obtained after sacrifice of C57BL/6 mice transplanted with mouse malignant melanoma cell line B16F10 tumors.
  • the spleen tissue from which the surrounding fat was removed was ground and separated into single cells, washed and counted to obtain spleen cells for each animal.
  • 5 ⁇ 10 5 splenocytes for each animal were dispensed in a mouse IFN- ⁇ ELISpot 96-well plate, and 1 ⁇ 10 5 B16F10 was added as a stimulus.
  • the test was performed using a minimum of 3 wells for each animal. Splenocytes and stimulants were incubated at 37° C. for 18 hours, washed, and treated with secondary antibodies to develop spots formed by IFN- ⁇ secreted from splenocytes. The number of spots on the stained plate was counted using AID EliSpot Reader.
  • Tumor and spleen tissues of each group were collected and fixed in 10% neutral formalin. Tissues were minced, dehydrated using a Shandon Excelsior ES tissue processor (Thermo Fisher Scientific), embedded in paraffin, and longitudinally embedded in paraffin blocks using an EG1150H paraffin-embedding station (Leica Biosystems, Wetzlar, Germany). Paraffin blocks were sectioned at 3 ⁇ m and used for staining.
  • Example 8 In a hamster melanoma model in vivo Confirmation of anti-tumor effect
  • RPMI 1846 The hamster malignant melanoma cell line RPMI 1846 was maintained in 10% FBS, 1% AA (Antibiotic-Antimycotic) McCoy's 5A medium in static culture. After harvesting and counting the cells, a mixture of 1 ⁇ 10 7 RPMI 1846 cells and 100 ⁇ L of Matrigel was subcutaneously injected into the right flank of a 6-week-old Syrian hamster. When the tumor was formed, the size of the major axis and the minor axis were measured and observed with a caliper, and the volume of the tumor was calculated using the formula a 2 b/2 (a: length of the minor axis, b: length of the major axis).
  • the hamsters were randomly divided into 3 groups, and the hamsters in each group were treated with PBS (negative control), 1 ⁇ 10 8 TCID 50 parental virus, and 1 ⁇ 10 8 TCID 50 B8R (-)mCherry-mIL12 virus was administered intratumorally once at a volume of 100 ⁇ L.
  • the size of the tumor and the weight of the animal were measured every 2-3 days, and the tumor was removed by sacrificing the animal on the 10th day based on the day of virus administration (day 0).
  • the animal's body weight was measured.
  • the tumor volume was measured from day 0 to day 10
  • the B8R(-)mCherry-mIL12 virus-treated group showed significant tumor suppression.
  • tumor tissue from all animals was distinctly isolated from the right flank.
  • FIG. 14c when the weight of the isolated tumor was measured, a significant weight reduction was confirmed in the B8R(-)mCherry-mIL12 virus-administered group, and as shown in FIG. 14d, the size of the tumor was significantly reduced visually.
  • Spleen tissues were obtained after sacrifice of Syrian hamsters transplanted with hamster malignant melanoma cell line RPMI-1846 tumors.
  • the spleen tissue from which the surrounding fat was removed was ground and separated into single cells, washed and counted to obtain spleen cells for each animal.
  • IFN- ⁇ ELISpot 96-well plate 2.5 ⁇ 10 5 splenocytes for each animal were dispensed, and 5 ⁇ 10 4 RPMI-1846 was added as a stimulus. The test was performed using a minimum of 3 wells for each animal. Splenocytes and stimulants were incubated at 37° C. for 18 hours, washed, and treated with secondary antibodies to develop spots formed by IFN- ⁇ secreted from splenocytes. The number of spots on the stained plate was counted using AID EliSpot Reader.
  • Hamster malignant melanoma cell line RPMI 1846 Tumor tissues obtained by sacrificing Syrian golden hamsters transplanted with tumors were fixed in 10% neutral buffered formalin, processed, and made into paraffin-embedded blocks. Paraffin blocks were cut to a thickness of 3 ⁇ m and attached to New Silane III-coated slides. Immunohistochemical staining using T cell marker (CD3, CD4, CD8) antibodies was performed on the slides thus obtained. The slides were subjected to deparaffinization and antigen retrieval, and reacted with primary antibodies diluted in appropriate concentrations, respectively, and reacted with secondary antibodies suitable for the primary antibody host animal, and then developed with DAB. T cells infiltrated into the tumor were stained with brown staining on the cell membrane.
  • T cell marker CD3, CD4, CD8
  • a 400-fold magnified scanned image of the stained slide was obtained using a Motic Easy Scan slide scanner.
  • the tumor area and the number of infiltrated T cells in the tumor were analyzed using the QuPath-0.3.2 program, and the number of infiltrated T cells per unit (tumor) area was calculated.
  • the part where necrosis occurred due to hypoxic conditions and the antitumor effect of the virus was excluded from the analysis.
  • FIGS. 16A to 16C it was confirmed that significantly more cytotoxic T cells infiltrated the tumors extracted from the B8R(-)mCherry-mIL12 virus-administered group compared to the negative control group and the parent virus-administered group.
  • CD4-positive helper T cells were most infiltrated in the parent virus-administered group, but the degree of difference was not large compared to the B8R(-)mCherry-mIL12 virus-administered group.
  • the immune enhancement in the virus-treated group was due to highly activated T cells, in particular, T cells expressing CD4 and CD8, and this immune enhancement was due to IFN- ⁇ activity and IL-12 activity. It was judged to be a synergistic effect. However, in the results of the hamster allotransplantation model, it was judged that the IL-12 gene sequence inserted into the virus was mouse-derived, and therefore showed a relatively less immune enhancing effect compared to mice.
  • the present invention relates to a recombinant vaccinia virus expressing an interleukin-12 gene and a use thereof, wherein the recombinant vaccinia virus in which the B8R gene is deleted and the interleukin-12 gene is inserted at the site of the deleted B8R has IFN- ⁇ activity and IL Increased activity of -12 can exhibit a synergistic effect in immunity and enhance anti-tumor immunity, so it can be usefully applied to the prevention, improvement, or treatment of cancer, which has industrial applicability. there is.

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Abstract

La présente invention concerne un virus de la vaccine recombiné exprimant l'IL-12 et son utilisation. Le virus de la vaccine recombiné ayant un gène B8R supprimé et un gène IL-12 inséré dans le site du B8R supprimé augmente l'activité IFN-γ et l'activité IL-12 pour posséder un effet de synergie immunologique, résultant en une amélioration de l'immunité anti-tumorale et devrait donc être utilisé avantageusement pour prévenir, atténuer ou traiter le cancer.
PCT/KR2022/019840 2021-12-07 2022-12-07 Virus de la vaccine recombiné exprimant l'il-12 et son utilisation WO2023106839A1 (fr)

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KR20210174188 2021-12-07
KR10-2021-0174188 2021-12-07
KR1020220169132A KR20230086610A (ko) 2021-12-07 2022-12-06 Il-12를 발현하는 재조합 백시니아 바이러스 및 이의 용도
KR10-2022-0169132 2022-12-06

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