Anticancer viruses co-expressing CD55 and CD59

Abstract

The present invention relates to an anticancer virus that co-expresses the complement regulatory proteins CD55 and CD59. The anticancer virus of the present invention maintains its efficacy even after intravenous injection, making it suitable for the treatment of various solid carcinomas and metastatic cancers in addition to superficial solid cancers. Furthermore, by expressing complement regulatory proteins on the viral surface, the anticancer virus of the present invention acquires resistance to attack by the endogenous complement system, is stable in the blood, and maintains stable anticancer activity upon intravenous injection, thereby reducing the viral dosage and minimizing the side effects of anticancer drugs. Therefore, the anticancer virus of the present invention can be useful for the prevention or treatment of cancer.

Description

[Technical Field]

[0001] The present invention relates to an anti-cancer virus that co-expresses the complement regulatory proteins CD55 and CD59. [Background technology]

[0002] Cancer is a major cause of death worldwide and places a huge burden on individuals and society. Therefore, the importance of developing innovative anti-cancer therapeutic agents is increasing day by day. Accordingly, various approaches based on advances in molecular biology are being attempted.

[0003] Oncolytic viruses, an anti-cancer treatment that has recently been gaining attention, are replicable and infectious viruses that are used to treat cancer by inserting specific genes into wild-type or attenuated viruses. These genetically engineered anti-cancer viruses replicate and then spread throughout tissues, lysing cells, with viral spread occurring selectively within cancer cells and the blood vessels surrounding them. A representative anti-cancer virus is Imlygic (talimogene laherparepvec), developed by Amgen, a US company, which was approved by the US Food and Drug Administration (FDA) in October 2015 as a melanoma treatment.

[0004] Meanwhile, intravenous injection of drugs is one of the most popular forms of administration due to its ease of administration and its convenient and easy method for rapidly delivering drugs that cannot be administered orally. However, anticancer virus preparations have the limitation that their efficacy is reduced when administered intravenously because they are rapidly eliminated by the body's immune system upon intravenous injection. Therefore, anticancer virus preparations are generally injected directly into target tumors rather than intravenously to increase the probability of the virus reaching the tumor.

[0005] The intratumoral injection method is generally effective for easily accessible superficial cancers such as melanoma, breast cancer, and head and neck cancer, but for carcinomas such as deep-seated solid cancers, the administration of an effective dose depends on the technical ability of the doctor performing the injection. In addition, the intratumoral injection procedure is very invasive, so it is not as easy to use as a repeated treatment method as intravenous injection.

[0006] Despite the advantages of intravenous injection, it is difficult to administer anti-cancer viruses via intravenous injection because foreign substances (i.e., viruses) administered into the bloodstream are gradually eliminated by the body's defense mechanisms, reducing the activity of the anti-cancer viruses. In other words, when a virus travels through the body through the bloodstream, the body recognizes it as a foreign substance and activates the innate immune system to neutralize and eliminate it.

[0007] When the human body is exposed to foreign bacteria or viruses, the complement system, the innate immune system's first line of defense, is first activated. Complements are proteins present in the blood that surround the surface of foreign microorganisms and facilitate phagocytosis by macrophages and neutrophils. Specifically, when a virus penetrates the bloodstream, proteins on the virus surface first bind to complement, forming agglutinations, activating the complement. Activated complement surrounds the virus surface, facilitating phagocytosis by phagocytes (opsonization), and lyses the virus by creating holes in the virus surface. The lysed virus debris is then completely removed by macrophages through phagocytosis.

[0008] To prevent damage to surrounding normal cells caused by excessive activation of the complement system, the human body expresses complement regulatory proteins such as CD55, CD46, and CD59 on the cell surface to regulate the action of excessive complement.

[0009] A portion of anticancer viruses (approximately 5-20%) that replicate within host cells are characterized by their "extracellular enveloped virus (EEV)" form, which is produced within the cell membrane of the infected cell. These EEVs are known to avoid complement activation and have a long survival period in the blood. This mechanism is known to be due to complement regulatory proteins, such as CD55, CD46, and CD59, expressed on the cell membrane of the host cell surrounding the virus. For example, most anticancer vaccinia viruses produced are in the form of "intracellular mature virus (IMV)," which do not express complement regulatory proteins on their surface and therefore have their activity significantly reduced by complement. Therefore, research and development is needed to improve the anticancer activity of anticancer viruses when administered intravenously. Summary of the Invention [Problem to be solved by the invention]

[0010] An object of the present invention is to provide a novel anticancer virus, specifically an anticancer virus that co-expresses CD55 and CD59.

[0011] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer, which comprises the anticancer virus as an active ingredient.

[0012] It is yet another object of the present invention to provide a genetic construct for insertion into an anti-cancer virus, comprising all or part of the genes encoding CD55 and CD59, operably linked to a promoter for expression.

[0013] A further object of the present invention is to provide an anti-cancer adjuvant containing the anti-cancer virus as an active ingredient.

[0014] It is yet another object of the present invention to provide a method for preventing or treating cancer, which comprises the step of administering to an individual a composition containing the anticancer virus as an active ingredient.

[0015] Yet another object of the present invention is to provide use of the anticancer virus or a composition containing the same for the prevention or treatment of cancer.

[0016] It is yet another object of the present invention to provide use of the anticancer virus or a composition containing the same for the manufacture of a medicament for the prevention or treatment of cancer.

[0017] The technical problems to be achieved based on the technical ideas of the invention disclosed in this specification are not limited to the problems to be solved above, and other problems not mentioned will be clearly understood by those skilled in the art from the following description. [Means for solving the problem]

[0018] This will be explained in more detail as follows. Meanwhile, each description and embodiment disclosed in this application can also be applied to each of the other descriptions and embodiments. In other words, all combinations of various elements disclosed in this application belong to the category of this application. Furthermore, the specific description described below is not considered to limit the category of this application.

[0019] In one embodiment for achieving the above-mentioned object of the present invention, the present invention provides an anti-cancer virus that co-expresses CD55 and CD59.

[0020] In particular, in the present invention, as an example, the transmembrane domain of the membrane protein of the anticancer virus itself is linked to the CD55 gene, and the membrane protein of the anticancer virus itself is linked to the CD59 gene so that the CD55 and CD59 proteins are expressed on the surface of the "intracellular mature virus" (IMV). This not only provides resistance to the human complement system and maintains stable anticancer activity upon intravenous injection, but also reduces the dose of the anticancer virus, minimizing side effects from treatment.

[0021] The term "oncolytic vaccinia virus" as used herein may also be referred to as "oncolytic virus," which includes recombinant viruses engineered through the deletion of all or part of endogenous genes or the introduction of foreign genes. Such oncolytic viruses may be vaccinia virus, adenovirus, herpes simplex virus, retrovirus, reovirus, Newcastle disease virus, coxsackievirus, enterovirus, or herpes virus.

[0022] In the present invention, the anticancer virus may be one in which the expression of the thymidine kinase gene is suppressed.

[0023] The term "thymidine kinase (TK)" as used herein refers to an enzyme involved in the biosynthesis of nucleotides. The thymidine kinase encoded by the TK gene can bind the phosphate at the gamma (γ) position of ATP to thymidine to produce nucleotides that constitute viral DNA. The TK may have a sequence such as, but is not limited to, GenBank: AAR17937.1 or AY313847.1. Specifically, the TK or its gene may have the amino acid sequence of GenBank: AAR17937.1 or the nucleotide sequence of GenBank: AY313847.1, but is not limited to these. Furthermore, the TK or its gene may have approximately 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more homology with the amino acid sequence of GenBank: AAR17937.1 or the nucleotide sequence of GenBank: AY313847.1.

[0024] The term "complement regulatory protein" as used herein refers to a protein that effectively regulates the complement activation pathway in vivo. Complement regulatory proteins are broadly classified into serotype (water-soluble) regulatory proteins and membrane-bound regulatory proteins. Serotype regulatory proteins include C4b-binding protein, factor H, SGP120, and properdin (P), while membrane-bound regulatory proteins include CRI (CD35), CR2 (CD21), CR3 (CD11b / CD18), CR4 (CD11c / CD18), DAF (CD55), membrane cofactor proteins (MCP, CD46), and CD59. Among these, only P acts to enhance complement activation, while the others act to attenuate it. The complement regulatory protein contained in the recombinant vaccinia virus of the present invention may be CD35, CD21, CD18, CD55, CD46, or CD59, specifically, but not limited to, CD55 and / or CD59.

[0025] Specifically, the CD55 gene may consist of or be a part of the nucleotide sequence of Genbank: NM_000574.3, and may have about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more homology to the nucleotide sequence of NCBI Reference Sequence: NM_000574.3. More specifically, the CD55 may consist of the amino acid sequence of SEQ ID NO: 1 or the nucleotide sequence of SEQ ID NO: 2.

[0026] Specifically, the CD59 gene may consist of or be a part of the nucleotide sequence of Genbank: NM_203331.3, and may have about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more homology to the nucleotide sequence of NCBI Reference Sequence: NM_203331.3. More specifically, the CD59 may consist of the amino acid sequence of SEQ ID NO: 3 or the nucleotide sequence of SEQ ID NO: 4.

[0027] As used herein, the term "transmembrane domain" is also called a transmembrane region or a transmembrane region, and refers to a region that penetrates and crosses the lipid bilayer of a membrane protein.

[0028] The anti-cancer virus of the present invention may further comprise a gene encoding an anti-cancer virus membrane protein or a transmembrane domain thereof.

[0029] In the present invention, for example, CD55 and / or CD59 may be expressed separately from the gene encoding the transmembrane domain, or may be expressed in a linked fusion form.

[0030] In the present invention, for example, the gene encoding the anticancer virus membrane protein may be linked to the gene encoding CD59 via a linker. More specifically, the linker may be (G4S)3 consisting of the amino acid sequence of SEQ ID NO: 7 or the nucleotide sequence of SEQ ID NO: 8, but is not limited thereto.

[0031] In the present invention, the vaccinia virus membrane protein may be H3L, A27L, D8L, A16L, F9L, G9R, L1R, A9L, A13L, A21L, A28L, E10R, G3L, H2R, 12L, J5L, L5R, or O3L, and may include all or part of the above membrane protein sequences, examples of which are shown in Table 1 below.

[0032] [Table 1-1]

[0033] [Table 1-2]

[0034] In this regard, CD55 and / or CD59 and the anticancer virus protein or its transmembrane domain may be engineered to contain all or part of the gene through genetic recombination. Specifically, among the anticancer viruses, the transmembrane domain of the vaccinia virus membrane protein may be composed of, but is not limited to, the amino acid sequence of SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, or SEQ ID NO: 21, or the nucleotide sequence of SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, or SEQ ID NO: 22.

[0035] In the present invention, gene expression suppression or gene inactivation refers to the deletion of a part or all of a gene, or the insertion of a foreign gene into a gene, resulting in the gene not being expressed or only a part of the gene being expressed, and the activity of the protein encoded by the gene not being exhibited. The gene deletion and foreign gene insertion methods can be performed using methods well known in the art. For example, foreign gene insertion methods can be performed using the methods disclosed in "Molecular Cloning, A Laboratory Manual, Second Edition (2003)" by J. Sambrook, E.F. Fritsch, and T. Maniatis, Cold Spring Harbor Laboratory Press; "Virology Methods Manual (1996)" edited by Brian W.J. Mahy and Hiliar O. Kangro, Academic Press; and "Expression of Genes by Vaccinia Virus Vectors. Current Protocols in Molecular Biology, Chapter 16 (1998)" published by John Wiley and Sons. Specifically, the expression of the thymidine kinase gene may be suppressed when a foreign gene is inserted into a part or the whole of the thymidine kinase J2R region. TMForeign genes were inserted using a PCR Cloning Kit (Solgent, Korea, Cat. No. SOT01-K020). CD55 and / or CD59 contained in the anticancer virus of the present invention can be expressed under the control of, but not limited to, the late-early VACV p7.5 promoter, the vaccinia synthetic early-late promoter (pSEL), the vaccinia synthetic late promoter (pSL), the vaccinia variant H5 (mH5) promoter, the vaccinia short synthetic early-late pS promoter, the pLEO160 promoter, the pLEO38 promoter, the pLate promoter, the pC11R promoter, the pF11L promoter, the psFJ1-10 synthetic early promoter, the pHyb synthetic early promoter, any native vaccinia early promoter, or a late-early optimized (LEO) promoter. For example, the complement regulatory protein CD55 or CD59 can be expressed under the control of the pLEO160 promoter or the pLEO38 promoter. More specifically, the CD55 can be expressed under the control of the pLEO160 promoter of SEQ ID NO:5, and the CD59 can be expressed under the control of the pLEO38 promoter of SEQ ID NO:6.

[0036] The anticancer virus of the present invention may further contain a gene capable of enhancing the efficacy of cancer treatment. Such genes may include, but are not limited to, anticancer therapeutic genes, various immune regulatory factors, factors that degrade structures such as intratumor fibrous substances, etc., such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-14, IL-15, IL-17, IL-18, IL-21, IL-23, IL-24, interferon-α, interferon-β, interferon-γ, CCL3, CCL5, CXCR4, CXCL9, CXCL10, CXCL11, and interleukin superagonists (IL-2 superagonist, IL-15 superagonist). The therapeutic agent may further include, but is not limited to, TGF-β blockade, TLR-2 agonist, TLR-3 agonist, TLR-7 agonist, STAT-3 inhibitor, PTENα, p53, p63, p73, adenosine deaminase-2, cancer-specific antigen, cancer-associated antigen, hyaluronidase, collagenase, protease, and the like.

[0037] In the present invention, anti-cancer viruses include vaccinia virus, adenovirus, herpes simplex virus, retrovirus, reovirus, Newcastle disease virus, coxsackievirus, enterovirus, herpes virus, etc., and examples of vaccinia viruses include, but are not limited to, Western Reserve (WR), New York Vaccinia Virus (NYVAC), Wyeth, LC16m8, Lister, Copenhagen, Tian Tan, USSR, TashKent, Evans, IHD-J (International Health Division-J), and IHD-W (International Health Division-White) strains.

[0038] In another aspect to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating cancer, which comprises the above anticancer virus as an active ingredient.

[0039] The term "active ingredient" as used herein means an ingredient that exhibits a desired activity alone or that can exhibit an activity together with a carrier that is inactive by itself.

[0040] The term "cancer (or tumor)" as used herein does not distinguish between primary cancer and metastatic cancer, but includes all cancers. In the present invention, the cancer may be a solid cancer or a blood cancer. The solid cancer may be, but is not limited to, any cancer selected from the group consisting of lung cancer, colon cancer, prostate cancer, thyroid cancer, breast cancer, brain cancer, head and neck cancer, esophageal cancer, skin cancer, thymus cancer, stomach cancer, colon cancer, liver cancer, ovarian cancer, uterine cancer, bladder cancer, rectal cancer, gallbladder cancer, biliary tract cancer, pancreatic cancer, kidney cancer, osteosarcoma, sarcoma, chondrosarcoma, and combinations thereof. The blood cancer may be, but is not limited to, any cancer selected from the group consisting of lymphoma, leukemia, multiple myeloma, and combinations thereof.

[0041] The term "metastatic cancer" as used herein refers to cancer that develops when cancer cells leave the primary organ, migrate to other organs, and grow there. The metastatic cancer may include, but is not limited to, both direct invasion of surrounding organs by cancer tissue growing from the primary cancer, and distant metastasis to other distant organs via blood vessels or lymphatic vessels.

[0042] The term "prevention" as used herein means any action that suppresses or delays the onset of cancer through administration of the pharmaceutical composition of the present invention, and the term "treatment" as used herein means any action that improves, alleviates, or beneficially changes cancer through administration of the pharmaceutical composition of the present invention.

[0043] The specific dosage of the pharmaceutical composition containing the anti-cancer virus of the present invention can be selected by a person skilled in the art depending on factors such as the formulation method, the patient's condition and weight, the patient's sex, age, the degree of disease, the drug form, the route and period of administration, the excretion rate, and the reaction sensitivity, and the dosage and frequency do not limit the scope of the present invention in any way. Specifically, the pharmaceutical composition of the present invention can be administered in a dose of 10 5 to about 10 13 These may include, but are not limited to, pfu (plaque forming units) of anti-cancer viruses.

[0044] The term "pharmaceutical composition" as used herein refers to a substance prepared for the purpose of preventing or treating a disease, and can be formulated into various forms according to conventional methods for use. For example, depending on the route of administration, the composition can be formulated into oral preparations such as powders, granules, tablets, capsules, suspensions, emulsions, and syrups, and can also be formulated into topical preparations and sterile injectable solutions. Specifically, the administration route can be any appropriate route, including topical, oral, intravenous, intramuscular, and direct absorption through mucosal tissues. Two or more routes can also be used in combination. An example of a combination of two or more routes is a combination of two or more drugs formulated according to the administration route, such as when one drug is administered primarily via the intravenous route and another drug is administered secondarily via the topical route.

[0045] The pharmaceutical compositions of the present invention may be for intratumoral, intravascular, intramuscular, or intraperitoneal administration, by way of example only, for intravenous or arterial administration.

[0046] The pharmaceutical compositions of the present invention can be prepared in the form of pharmaceutical compositions for treating or preventing cancer, further comprising suitable carriers, excipients, or diluents commonly used in the preparation of pharmaceutical compositions, including non-naturally occurring carriers. Specifically, the pharmaceutical compositions can be formulated into oral preparations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, topical preparations, suppositories, and sterile injectable solutions, using conventional methods. Examples of carriers, excipients, and diluents that can be included in the pharmaceutical compositions of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil. When formulated, they are prepared using commonly used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid formulations for oral administration include tablets, pills, powders, granules, capsules, and the like. These solid formulations are prepared by mixing at least one or more excipients, such as starch, calcium carbonate, sucrose or lactose, and gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Oral liquid formulations include suspensions, oral solutions, emulsions, syrups, and the like. In addition to commonly used simple diluents such as water and liquid paraffin, various excipients, such as wetting agents, sweeteners, flavoring agents, and preservatives, are also used. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations, and suppositories. As non-aqueous solvents and suspensions, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, and the like can be used.As a base for a suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin, etc. can be used.

[0047] Specific formulations of pharmaceutical compositions are known in the art, and reference can be made to, for example, Remington's Pharmaceutical Sciences (19th ed., 1995), which is incorporated herein by reference.

[0048] The pharmaceutical compositions of the present invention can be administered to mammals such as rats, dogs, cats, cows, horses, pigs, and humans by various routes, with humans being preferred. All methods of administration are contemplated, including but not limited to oral, intravenous, intraarterial, intramuscular, or subcutaneous injection.

[0049] For example, the anticancer virus of the present invention is resistant to the human complement system and maintains stable anticancer activity upon intravenous administration. It also maximizes therapeutic efficacy by reducing the dose of the anticancer virus, allowing it to exhibit sufficient therapeutic efficacy even when administered intravenously.

[0050] In another aspect to achieve the above-mentioned object, the present invention provides a gene construct for insertion into an anti-cancer virus, which comprises all or part of the genes encoding CD55 and CD59, operably linked to a promoter for expression, and a vector containing the same.

[0051] In the present invention, the gene structure (or gene construct) can be inserted into the inactivated thymidine kinase gene region of an anti-cancer virus.

[0052] In the present invention, the inactivated thymidine kinase gene region includes a deletion of all or part of the thymidine kinase gene.

[0053] In the present invention, the gene structure is located between the J1R region and the J3R region in the anticancer virus.

[0054] In the present invention, the gene construct may include, but is not limited to, those exemplified in FIG. 1, and may additionally include various promoters and regulatory sequences for regulating gene expression in an operably linked form.

[0055] The terms "transmembrane domain", "CD55", and "CD59" of the present invention are as defined above.

[0056] In the present invention, the gene construct is intended to be inserted into all or part of the thymidine kinase of vaccinia virus, specifically, into all or part of the thymidine kinase J2R region.

[0057] In another embodiment to achieve the above object, the present invention provides an anti-cancer adjuvant comprising the above recombinant vaccinia virus as an active ingredient.

[0058] The anticancer adjuvant refers to any form of anticancer drug intended to enhance the anticancer effect of an anticancer drug or to suppress or ameliorate the side effects of an anticancer drug. The anticancer adjuvant of the present invention can be administered in combination with various types of anticancer drugs or anticancer adjuvants, and even if the anticancer drug is administered at a lower dose than the usual dose of the anticancer drug during combined administration, it can exhibit the same level of anticancer therapeutic effect, thereby enabling safer anticancer treatment.

[0059] The anticancer adjuvant may be administered via any common route as long as it can reach the target tissue. The anticancer adjuvant of the present invention may be administered intraperitoneally, intravenously, intramuscularly, subcutaneously, intrapulmonary, or intrarectally depending on the purpose, but is not limited thereto. In addition, the anticancer adjuvant may be administered via any device that can transport the active substance to the target cells.

[0060] The anti-cancer adjuvant of the present invention can be preferably formulated as an anti-cancer adjuvant by containing one or more pharmaceutically acceptable carriers in addition to the active ingredient for administration.The carriers, excipients or diluents that can be contained in the anti-cancer adjuvant of the present invention include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0061] The anticancer adjuvant of the present invention may be a formulation for parenteral administration, and the description of the formulation replaces the description of the formulation of the pharmaceutical composition.

[0062] Furthermore, any anticancer adjuvant disclosed in this technical field can be applied to the present invention without any particular limitations.

[0063] In one embodiment of the present invention, the anti-cancer agent may be one or more selected from the group consisting of a chemotherapeutic agent, a biological therapeutic agent, a radiation therapy, an immunotherapy, a hormonal therapeutic agent, an anti-vascular therapeutic agent, a cryotherapy agent, and a toxin therapeutic agent, but is not limited thereto.

[0064] In another embodiment to achieve the above object, the present invention provides a method for preventing or treating cancer, which comprises administering to an individual a composition containing the recombinant vaccinia virus as an active ingredient.

[0065] The terms "cancer," "prevention," and "treatment" of the present invention are as defined above.

[0066] The individual may be a mammal, specifically, but not limited to, a human, cow, sheep, goat, horse, pig, dog, cat, rabbit, rat, mouse, fish, bird, etc.

[0067] The composition containing the recombinant vaccinia virus as an active ingredient can be appropriately administered by a skilled artisan depending on the patient's age, sex, weight, severity of symptoms, and administration route, and can be administered once or several times a day, and can be repeatedly administered at an appropriate interval.

[0068] The dosage of the composition containing the recombinant vaccinia virus as an active ingredient varies depending on the condition and weight of the individual, the degree of disease, the drug form, the administration route and period, and can be appropriately selected by a person skilled in the art. 5 to about 10 13 The pfu (plaque forming units) can be administered and can include various values or ranges between the above ranges.

[0069] In the method for treating cancer of the present invention, the composition may be administered via any common route as long as it can reach the target tissue. The composition of the present invention may be administered via routes such as oral administration and rectal administration, but is not limited thereto, and may also be administered via other routes depending on the purpose.

[0070] In the present invention, the cancer treatment method includes a treatment method through the combined administration of a conventionally known anticancer agent and anticancer therapy.

[0071] To achieve the above object, another aspect of the present invention provides use of the anticancer virus or a composition containing the same for the prevention or treatment of cancer.

[0072] In order to achieve the above object, another aspect of the present invention provides use of the anticancer virus or a composition containing the same for the manufacture of a medicament for preventing or treating cancer. [Effects of the Invention]

[0073] The anticancer virus of the present invention maintains its efficacy even when administered intravenously, making it suitable for the treatment of various solid carcinomas, hematological cancers, and metastatic cancers in addition to superficial solid cancers. Furthermore, the anticancer virus of the present invention expresses a complement regulatory protein, making it resistant to the body's complement system. This allows it to maintain stable anticancer activity, particularly when administered intravenously, thereby reducing the dosage of the virus and minimizing side effects of anticancer drugs. Therefore, the anticancer virus of the present invention can be useful for the prevention or treatment of cancer. [Brief explanation of the drawings]

[0074] [Figure 1] FIG. 1 is a diagram illustrating the recombinant vaccinia virus, an anti-cancer virus of the present invention, in which the TK gene has been deleted and the complement regulatory proteins CD55 and CD59 have been introduced. [Figure 2] FIG. 2 shows the results of Western blotting to confirm whether the recombinant vaccinia virus of the present invention (SJ-640) expresses CD55 and CD59. [Figure 3] FIG. 3 shows the results of immunofluorescence staining to determine whether or not CD55 is expressed in osteosarcoma cells infected with the recombinant vaccinia virus (SJ-640) of the present invention. [Figure 4] FIG. 4 shows the results of immunofluorescence staining to determine whether or not CD59 is expressed in osteosarcoma cells infected with the recombinant vaccinia virus (SJ-640) of the present invention. [Figure 5] FIG. 5 shows the results of confirming the expression of CD55 and CD59 in the recombinant vaccinia virus of the present invention (SJ-640) using a transmission electron microscope. [Figure 6] FIG. 6 shows the results of confirming the stability of the recombinant vaccinia virus of the present invention (SJ-640) in serum using 20% active human serum. [Figure 7] Figure 7 shows the results of confirming the antitumor efficacy of SJ-640 through changes in tumor volume in an MDA-MB-231 breast cancer xenograft model. [Figure 8]Figure 8 shows the results of examining changes in body weight over time after administration of SJ-610 and SJ-640 to an MDA-MB-231 breast cancer xenograft model. DETAILED DESCRIPTION OF THE INVENTION

[0075] The present invention will be described in more detail with reference to the following examples, which are merely illustrative of the present invention and are not intended to limit the scope of the present invention.

[0076] The present inventors constructed vectors into which the human CD55 and human CD59 genes were inserted, produced recombinant vaccinia viruses in which expression of the genes was induced by homologous recombination between the vectors and vaccinia viruses, and confirmed their properties as anticancer substances.

[0077] Production example: Production of recombinant vaccinia virus SJ-640 that lacks TK and expresses CD55 and CD59 As shown in Figure 1, the J2R region in the thymidine kinase domain was completely deleted from the vaccinia virus Wire strain, and CD55 and CD59 were inserted in its place. To express CD55 in the viral envelope, a gene derived from hCD55 with a deleted GPI anchor region was fused to the transmembrane domain of a vaccinia virus membrane protein, and pLEO160 was used as the promoter for expression. To express CD59 in the viral envelope, a gene derived from hCD59 with a deleted GPI anchor region was linked to the vaccinia virus membrane protein using a linker, and pLEO38 was used as the promoter for expression. The specific process is as follows.

[0078] 1. Construction of plasmid vectors expressing hCD55 and hCD59 genes To delete the J2R region of vaccinia virus, T-blunt TMThe J1R and J3R genes, which are the left and right flanking regions of the J2R region, were cloned into a vector (Solgent) using the NEBuilder® HiFi DNA Assembly Cloning Kit (NEW ENGLAND BioLabs, Catalog No. E2621). J1R and J3R DNA were prepared by PCR amplification of the corresponding regions of JX-594 (Pexastimogene Devacirepvec, Pexa-Vec). The J1R region is identical to the region of amino acids 1 to 154 of J1R (Protein ID=AAR17936.1) of the vaccinia virus Acamins 2000 strain, except that amino acid 118 is replaced by alanine with serine. J3R (Protein ID=AAR17938.1) is the region of amino acids 1 to 145. The hCD55 gene (GenBank: NM_000574.3), a transmembrane domain region of the vaccinia virus membrane protein H3L, the hCD59 gene (GenBank: NM_20331.3), a (G4S)3 linker (SEQ ID NO: 8) linking the hCD59 gene to the vaccinia virus membrane protein, and a gene encoding the vaccinia virus membrane protein A27L were cloned into this vector to construct a shuttle vector. The amino acid and nucleotide sequences of hCD55 and hCD59 are shown in SEQ ID NOs: 1 and 2, respectively, and the amino acid and nucleotide sequences of hCD59 are shown in SEQ ID NOs: 3 and 4, respectively.

[0079] hCD55 was PCR amplified and cloned into the pCMV3 plasmid (Sinobio, Catalog No. HG10101-UT). hCD55 (GenBank: NM_000574.3) uses the region from amino acids 1 to 352, with the GPI anchor region deleted from the entire amino acid sequence. The promoter for expression is pLEO160 (ttttattttttttttttggaatataaatatccggtaaaattgaaaaaatatacactaattagcgtctcgtttcagacgctagccggtaccccgggttcgaaatcgataagcttggatccggagagctcccaacctcgaggaattcggtaccccgggttcgaaatcgataagcttggatccggagagctcccaacctcgagctagctcgag, SEQ ID NO: 5, synthesized by Macrogen). The 3' end of the hCD55 gene was linked to the transmembrane domain region of vaccinia virus H3L so that CD55 was expressed on the viral envelope membrane. Information on the transmembrane domain region of H3L used and the base sequence are shown in Tables 2 and 3.

[0080] [Table 2]

[0081] [Table 3]

[0082] hCD59 was PCR amplified from cDNA of human cervical cancer cells, HeLa. hCD59 (GenBank: NM_203331.3) lacks the GPI anchor region of the entire amino acid sequence, and the region between amino acids 1 and 101 was used. The promoter for expression was pLEO38 (ttttattttttttttttggaatataaatatccggtaaaattgaaaaaatatacactaattagcgtctcgtttcagacgctagctcgag, SEQ ID NO: 6, synthesized by Macrogen). The 3' end of the hCD59 gene was linked to the vaccinia virus A27L gene via a (G4S)3 linker, allowing CD59 to be expressed on the viral envelope. The sequences of A27L and the linker used are shown in Table 4.

[0083] [Table 4]

[0084] 2. Production of SJ-640 Recombinant Vaccinia Virus by Homologous Recombination The SJ-640 recombinant vaccinia virus was produced by homologous recombination of the wild-type vaccinia virus strain Wire with a pre-constructed plasmid vector in which expression of the hCD55 and hCD59 genes was induced.

[0085] The wild-type Wire strain was prepared by inserting the J2R region gene of the Western Reserve (WR) vaccinia virus strain (ATCC, Catalog No. VR-1354) into the inactivated J2R region (TK region) of JX-594 (Pexastimogene Devacirepvec, Pexa-Vec). The J2R region gene of the Western Reserve strain (Protein ID=YP_232976.1) corresponds to amino acids 1 to 178. The J2R region gene of the Western Reserve strain was amplified by PCR, and the wild-type Wire strain was constructed by homologous recombination of this PCR product with JX-594.

[0086] To recombine SJ-640, 143B osteosarcoma cells (Creative Bioarray) were infected with the wild-type WYAS strain and transfected with a plasmid vector expressing hCD55 and hCD59. To select recombinant SJ-640 virus plaques lacking the TK domain, 143B osteosarcoma cells were infected with vaccinia virus and transfected with the plasmid vector. The cell lysates were treated with 5-Bromo-2'-deoxyuridine (BrdU), a TK deletion selection reagent, and then cultured. The selected recombinant virus plaques were then passaged twice more in 143B cells to obtain the purified single clone SJ-640. The nucleotide sequence of the recombination site of SJ-640 was finally confirmed by sequencing.

[0087] Example 1: Confirmation of CD55 and CD59 expression in SJ-640 The recombinant vaccinia virus SJ-640 prepared in the above Preparation Example was lysed, and protein expression was confirmed by Western blot analysis. A virus (JX-594) that does not express CD55 and CD59 was used as a control.

[0088] Specifically, HeLa cells were infected with recombinant vaccinia virus SJ-640 and grown for approximately 26 hours. The infected cells were centrifuged to remove the culture medium, and then lysed with cell lysis buffer. The cell lysate was treated with Benzonase to remove cellular DNA and filtered through a cellulose acetate filter to remove cellular debris. The filtrate was concentrated using 36% sucrose cushion centrifugation.

[0089] To isolate proteins present in the purified virus, the centrifuged virus pellet was dissolved in RIPA lysis buffer (Thermo Scientific, Catalog No. 89900), and the amount of protein was quantified using a BCA protein assay (Thermo Scientific, Catalog No. 23227). Protein from each virus was diluted with loading buffer (Biosesang, Catalog No. S2002) to prepare a loading amount of 2.5 μg or 5 μg per lane, and electrophoresis was performed on an SDS-PAGE gel (BIO-RAD, Catalog No. 4561083). The electrophoresed gel was then transferred to a nitrocellulose membrane (BIO-RAD, Catalog No. 1704270). For detection of the target proteins, the membrane was blocked with 5% skim milk (Difco, Catalog No. 232100) for 1 hour and then treated with anti-hCD55 antibody (SantaCruz, Catalog No. sc-51733) or anti-hCD59 antibody (SantaCruz, Catalog No. sc-133171) overnight at 4°C. The next day, the membrane was washed 3-4 times with 1x TBST and treated with HRP-conjugated secondary antibody, followed by hydrogen peroxide and luminol substrate treatment. Chemiluminescence was detected using a detector.

[0090] As a result, as shown in FIG. 2, it was confirmed that CD55 (approximately 70 to 75 kDa) and CD59 (approximately 35 kDa) were expressed in SJ-640.

[0091] In addition, osteosarcoma cells (U-2 OS) were infected with the recombinant vaccinia virus SJ-640 produced in the above Production Example, and 10 hours later, the presence or absence of CD55 and CD59 expression was confirmed by immunofluorescence staining. As a negative control, SJ-610 cells, which lack TK and do not express CD55 or CD59, were used.

[0092] Specifically, U-2 OS cells were infected with recombinant vaccinia virus SJ-640 or the negative control SJ-610. 10 hours postinfection, the infected cells were fixed with 4% paraformaldehyde. The fixed infected cells were then permeabilized with 0.1% Triton X-100 and blocked with 3% bovine serum albumin. For immunofluorescent staining of the target proteins, the cells were incubated overnight at 4°C with anti-hCD55 antibody (Invitrogen, Catalog No. MA1-82066), anti-hCD59 antibody (Invitrogen, Catalog No. MA1-19133), or anti-vaccinia virus A27 antibody (Abcam, Catalog No. ab35219). The next day, the primary antibodies were washed with PBS, and the cells were treated with secondary antibodies conjugated to AlexaFluor488 or AlexaFluor594, which bind to each primary antibody, and 4'6-diamidino-2-phenylindole (DAPI). The expression of vaccinia virus A27 protein, CD55, CD59, and DAPI in infected cells was observed by confocal microscopy.

[0093] As a result, we confirmed that CD55 was expressed in cells infected with the SJ-640 virus, as shown in Figure 3, and that CD59 was expressed in cells infected with the SJ-640 virus, as shown in Figure 4. No expression of CD55 or CD59 was observed in SJ-610, which was used as a control.

[0094] In addition, antibodies specific to CD55 or CD59 were bound to the recombinant vaccinia virus SJ-640 prepared in the above preparation example, and then immunogold labeling was performed to observe the presence or absence, expression location, and distribution of CD55 and CD59.

[0095] To achieve this, recombinant vaccinia virus SJ-640 was infected into HeLa cells and grown for approximately 28-30 hours. The infected cells were then centrifuged to remove the culture medium, and cell lysis buffer was added to lyse the cells. The cell lysate was treated with Benzonase to remove cellular DNA and filtered through a cellulose acetate filter to remove cellular debris. The filtrate was concentrated using 36% sucrose cushion centrifugation. The concentrated virus was then passed through ion-exchange membrane chromatography to adsorb only the virus and remove impurities, after which the virus was eluted and finally purified.

[0096] Purified virus was adsorbed onto nickel grids and blocked with 3% bovine serum albumin. Anti-hCD55 antibody (SantaCruz, Catalog No. sc-51733) or anti-hCD59 antibody (Invitrogen, Catalog No. MA1-19133), which binds to the target protein, was incubated for 2 hours at room temperature. After washing with PBS, the cells were incubated with a secondary antibody conjugated to 12 nm gold colloid particles (JacksonImmuno, Catalog No. 115-205-166) for 2 hours at room temperature. Samples prepared for transmission electron microscopy were negatively stained to assess the expression of gold-labeled CD55 and CD59.

[0097] As a result, expression of CD55 and CD59 was confirmed in SJ-640, and it was confirmed that they were distributed in large quantities on the outer membrane of mature virions (MV) (Fig. 5). Expression of CD55 and CD59 was not observed in JX-594, which was used as a control virus.

[0098] Example 2: Confirmation of viral stability in serum of SJ-640 To determine whether the SJ-640 virus maintains its activity in serum, its stability in serum was measured.

[0099] Specifically, U-2 OS osteosarcoma cells were plated in 12-well tissue culture plates at 2.5 x 10 cells per well. 5The cells were seeded with 12-well plates and cultured overnight. The next day, when the cells reached nearly 100% confluency, recombinant vaccinia virus SJ-640 was diluted in serum-free DMEM medium to approximately 80–100 PFU per well. Normal human serum was added to the diluted virus at 20% of the total volume, and the diluted virus was then applied to plates containing U-2 OS cells for 2 hours. Serum-free DMEM medium was used instead of normal human serum as a control. After 2 hours, the virus was removed from the 12-well plates and treated with 1.5% carboxymethylcellulose solution. The 12-well plates were then cultured at 37°C for 72 hours, and the plaques formed were stained with 0.1% crystal violet and counted. The number of plaques formed in the serum-free medium was set at 100%, and the number of plaques formed by the recombinant virus cultured with 20% normal human serum was calculated. The stability of the recombinant virus SJ-640 in serum was compared with that of the negative control, JX-594.

[0100] As a result, as shown in Figure 6, in the case of the control JX-549, the viral activity was reduced to 30%, while the activity of the SJ-640 virus of the present invention was confirmed to be maintained at up to 87%.

[0101] Example 3: Evaluation of antitumor efficacy in a breast cancer xenograft model MDA-MB-231 human breast cancer cells were transplanted into NSG immunodeficient mice to establish a xenograft mouse tumor model. 3 Once this was reached, SJ-640 or the control SJ-610 was administered at 1 x 10 6 A single intravenous administration of pfu was administered, and the tumor growth inhibitory effects were compared.

[0102] Tumor size was measured two to three times a week using calipers along the long and short axes in all groups. Tumor size was calculated by dividing the tumor size by two. The mice were weighed every time the tumor size was measured. Tumors between 1,500 and 2,000 mm were removed. 3 When this was reached, the experiment was terminated.

[0103] As a result, as shown in Figure 7, the recombinant vaccinia virus of the present invention, SJ-640, showed a tumor growth inhibitory effect of 52.9% compared to the untreated control group (PBS), while the tumor growth inhibitory effect of SJ-610 was 23.6%, confirming that the recombinant vaccinia virus of the present invention, SJ-640, has a significantly superior antitumor effect.

[0104] Furthermore, as shown in FIG. 8, there was no statistically significant difference in the weight change of mice in all experimental groups, and no clinical toxicity was observed.

[0105] From the above description, those skilled in the art to which the present invention pertains will understand that the present invention can be embodied in other specific forms without changing the technical spirit or essential characteristics thereof. In this regard, it should be understood that the above-described embodiments are illustrative in all respects and are not limiting. The scope of the present invention should be interpreted as including all modifications and variations derived from the meaning and scope of the following claims, as well as equivalent concepts, rather than the above detailed description.

Claims

1. An anti-cancer virus that co-expresses CD55 and CD59.

2. The anticancer virus according to claim 1 , wherein the expression of a thymidine kinase gene is suppressed.

3. The anticancer virus according to claim 2, wherein the expression of the thymidine kinase gene is suppressed by a partial or complete deletion of the gene or by an insertion of a foreign gene into the gene.

4. The anticancer virus according to claim 2, wherein a foreign gene is inserted into a part or all of the thymidine kinase J2R region.

5. The anti-cancer virus according to claim 1, wherein the CD55 is composed of the amino acid sequence of SEQ ID NO:

1.

6. The anticancer virus according to claim 1, wherein the CD59 comprises the amino acid sequence of SEQ ID NO:

3.

7. The anti-cancer virus according to claim 1, further comprising a gene encoding an anti-cancer virus membrane protein or its transmembrane domain.

8. The anticancer virus according to claim 7 , wherein the gene encoding the anticancer virus membrane protein is linked to the gene encoding CD59 via a linker.

9. The anticancer virus according to claim 8, wherein the linker is composed of the amino acid sequence of SEQ ID NO:

7.

10. The anticancer virus according to claim 7 , wherein a gene encoding a transmembrane domain of the anticancer virus membrane protein is fused with a gene encoding CD55.

11. The anticancer virus of claim 7, wherein the membrane protein of the anticancer virus is H3L, A27L, D8L, A16L, F9L, G9R, L1R, A9L, A13L, A21L, A28L, E10R, G3L, H2R, 12L, J5L, L5R, or O3L.

12. The anticancer virus of claim 1, wherein CD55 and CD59 are expressed under the control of the late-early VACV p7.5 promoter, the vaccinia synthetic early-late promoter (pSEL), the vaccinia synthetic late promoter (pSL), the vaccinia mutant H5 (mH5) promoter, the vaccinia short synthetic early-late pS promoter, the pLEO160 promoter, the pLEO38 promoter, the pLate promoter, the pC11R promoter, the pF11L promoter, the psFJ1-10 synthetic early promoter, the pHyb synthetic early promoter, any native vaccinia early promoter, or the late-early optimized (LEO) promoter, respectively.

13. The anti-cancer virus of claim 1 , wherein the anti-cancer virus is a vaccinia virus, an adenovirus, a herpes simplex virus, a retrovirus, a reovirus, a Newcastle disease virus, a coxsackie virus, an enterovirus, or a herpes virus.

14. 14. The anticancer virus of claim 13, wherein the vaccinia virus is a Western Reserve (WR), New York Vaccinia Virus (NYVAC), Wyeth, LC16m8, Lister, Copenhagen, Tian Tan, USSR, TashKent, Evans, International Health Division-J (IHD-J), or International Health Division-White (IHD-W) strain.

15. A pharmaceutical composition for preventing or treating cancer, comprising the anticancer virus according to any one of claims 1 to 14 as an active ingredient.

16. 16. The pharmaceutical composition of claim 15, wherein the cancer is a solid cancer or a blood cancer.

17. 17. The pharmaceutical composition of claim 16, wherein the solid cancer is any one selected from the group consisting of lung cancer, colorectal cancer, prostate cancer, thyroid cancer, breast cancer, brain cancer, head and neck cancer, esophageal cancer, skin cancer, thymus cancer, stomach cancer, colon cancer, liver cancer, ovarian cancer, uterine cancer, bladder cancer, rectal cancer, gallbladder cancer, biliary tract cancer, pancreatic cancer, kidney cancer, osteosarcoma, sarcoma, chondrosarcoma, and combinations thereof.

18. 17. The pharmaceutical composition of claim 16, wherein the blood cancer is any one selected from the group consisting of lymphoma, leukemia, multiple myeloma, and combinations thereof.

19. 16. The pharmaceutical composition of claim 15, wherein the composition is for intratumoral, intravascular, intramuscular, or intraperitoneal administration.

20. 16. The pharmaceutical composition of claim 15, wherein the composition is for intravenous or intraarterial administration.

21. A gene construct for insertion into an anti-cancer virus, comprising all or part of the genes encoding CD55 and CD59, operably linked to a promoter for expression.

22. The gene construct according to claim 21, wherein the gene construct is intended to be inserted into an inactivated thymidine kinase gene region of an anti-cancer virus.

23. An anticancer adjuvant comprising the anticancer virus according to any one of claims 1 to 14 as an active ingredient.

24. A method for preventing or treating cancer, comprising the step of administering to an individual a composition comprising the anticancer virus according to any one of claims 1 to 14 as an active ingredient.

25. 15. Use of the anticancer virus according to any one of claims 1 to 14 or a composition comprising the same for the prevention or treatment of cancer.

26. 15. Use of the anticancer virus according to any one of claims 1 to 14 or a composition comprising the same for the manufacture of a medicament for the prevention or treatment of cancer.

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