Chimeric Zika virus anti-cancer vaccine obtained by passage of breast cancer cells

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating breast cancer, containing, as an active ingredient, a chimeric Zika virus obtained by passage of breast cancer cells.

Description

Technical Field

[0001] The present invention relates to a pharmaceutical composition for preventing or treating cancer, which contains a chimeric Zika virus obtained by passage of breast cancer cells as an active ingredient. Background Art

[0002] Breast cancer is the most common cancer in women and the second leading cause of death. Risk factors for breast cancer include race, age, and mutations in the tumor suppressor genes BRCA-1, BRCA-2, and p53. Alcohol consumption, a high-fat diet, physical inactivity, exogenous postmenopausal hormones, and ionizing radiation also increase the risk of cancer.

[0003] Common treatments for cancer, including cancer, include chemotherapy, radiotherapy, and surgery. However, except for some early-stage cancers, cancer is difficult to completely remove by surgery or has side effects due to the cytotoxicity of chemotherapy or radiotherapy. Among the various technologies that complement this, the strategy of attacking and killing only cancer cells is one of the most promising technologies. Among others, "oncolytic viruses", which are viruses that infect and destroy cancer cells based on the virus's inherent or artificially added cancer cell selection ability, are a cancer treatment technology that has long attracted much attention.

[0004] In other words, an oncolytic virus is a virus with conditional replication ability, which selectively proliferates and kills tumor cells in tumor cells. During the 1950s to 1970s, the oncolytic effect of the virus was demonstrated by using adenovirus or mumps virus research, but oncolytic viruses were rarely paid attention to in decades due to their temporary efficacy and toxic side effects. However, after developing some viruses that are safer than previous viruses, such as thymidine kinase (TK) defective variants of herpes simplex virus-1 (HSV-1) and dl1520 (ONYX-015), dl1520 is a selectively proliferated adenovirus mutation in p53 defective tumor cells in the 1990s, and the research progress of oncolytic viruses is rapid (Jagus R et al., Int. J. Biochem. Cell Biol., (1999) 31: 123-138; Bischoff JR et al., Science, (1996) 274 (5286): 373-376). So far, about 80 or more types of oncolytic viruses have been developed, which are expected to become promising tools for cancer treatment.

[0005] Zika virus belongs to the Flaviviridae family, which includes dengue virus, yellow fever virus, West Nile virus, Japanese encephalitis virus and tick-borne encephalitis virus. Among them, yellow fever virus has been developed and used as a vaccine for a long time, and a live attenuated vaccine was first developed and commercialized around 1930. Summary of the invention

[0006] [Technical issues]

[0007] There are currently no studies using Zika virus developed specifically for breast cancer.

[0008] [Technical solution]

[0009] The object of the present invention is to provide a pharmaceutical composition for preventing or treating breast cancer, which has a chimeric Zika virus as an active ingredient.

[0010] Another object of the present invention is to provide a method for preparing the composition.

[0011] Another object of the present invention is to provide a chimeric Zika virus mutant strain obtained using a chimeric gene of an attenuated yellow fever virus strain and a Zika virus.

[0012] [Beneficial Effects]

[0013] The present invention can prepare an anti-cancer vaccine that is not only highly effective in treating breast cancer but also has good safety by using breast cancer cells to subculture a chimeric Zika virus (cZIKV) with anti-cancer effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The construction of a chimeric ZIKA virus is shown, in which the major antigen gene of the yellow fever genome is replaced by the major antigen genome of the Zika virus;

[0015] Figure 2 is a schematic diagram showing the passaging process;

[0016] Figure 3 is a schematic diagram illustrating the viral ToxGlo assay;

[0017] Figure 4 The results of in vivo attenuation test to verify the safety of cZIKV are shown;

[0018] Figure 5 The experimental design using Balb / c nude mice (T cell-deficient mice) is shown;

[0019] Figure 6 The results of testing the infectivity of cZIKV on five normal cell lines are shown;

[0020] Figure 7 Differences in oncolytic virus infectivity depending on breast cancer subtype are shown;

[0021] Figure 8 Results comparing the infectivity of the passaged oncolytic virus (adapted virus, Bx61) and the parental virus (original virus, cZIKV) are shown;

[0022] Fig. 9 Results of examining the cytotoxicity of passaged oncolytic virus (adapted virus, Bx61) and parental virus (original virus, cZIKV) are shown;

[0023] Fig.10 is a graph showing changes in body weight and body temperature of an oncolytic virus administration group and a control group in a xenograft experiment using MDAMB231, which is highly sensitive to oncolytic viruses;

[0024] Fig.11 is a graph showing changes in tumor size in an oncolytic virus administration group and a control group in a xenograft experiment using MDAMB231, which is highly sensitive to oncolytic viruses;

[0025] Fig.12 The results of tumor histopathological examination of the oncolytic virus administration group and the control group in the xenograft experiment using MDAMB231 are shown. MDAMB231 is highly sensitive to oncolytic viruses;

[0026] Fig.13 is a graph showing changes in body weight and body temperature of an oncolytic virus-administered group and a control group in a xenograft experiment using BT474, which is hardly susceptible to oncolytic viruses;

[0027] Fig.14 is a graph showing changes in tumor size in an oncolytic virus-administered group and a control group in a xenograft experiment using BT474, which is hardly susceptible to oncolytic viruses;

[0028] Fig.15 The results of tumor histopathological examination of the oncolytic virus-administered group and the control group in a xenograft experiment using BT474 were shown. BT474 was almost insensitive to oncolytic viruses.

[0029] Fig.16 is a graph showing changes in body weight and body temperature of an oncolytic virus-administered group and a control group in a xenograft experiment using an Hs578T cell line without oncolytic virus CPE;

[0030] Fig.17 is a graph showing changes in tumor size between an oncolytic virus administration group and a control group in a xenograft experiment using an Hs578T cell line, which does not have CPE of an oncolytic virus;

[0031] Fig.18 The results of tumor histopathological examination of the oncolytic virus administration group and the control group in a xenograft experiment using the Hs578T cell line are shown. The Hs578T cell line does not have CPE of the oncolytic virus;

[0032] Fig.19Histopathological results for examining the safety of treatment with oncolytic viruses are shown; Fig. 20 The results of infectivity examination using IFA (immunofluorescence assay) according to the number of passages are shown. For reference, cZIKV: original virus before passage, MD19 represents the virus passaged 19 times in MDAMB231, B×32 represents the virus passaged 32 times in mixed cells, and B×61 represents the virus passaged 61 times in mixed cells, and the number of passages increases from left to right.

[0033] Best Mode for Carrying Out the Invention

[0034] One aspect of the present invention is a pharmaceutical composition for preventing or treating breast cancer.

[0035] In a specific embodiment, the composition is a pharmaceutical composition for preventing or treating breast cancer, which contains a chimeric Zika virus as an active ingredient, wherein the chimeric Zika virus is formed by introducing a Zika virus antigen gene into a gene of an attenuated yellow fever virus containing SEQ ID NO: 1.

[0036] According to the composition of the aforementioned specific embodiment, the Zika virus antigen gene includes i) a gene encoding pre-membrane protein (prM) and ii) a gene encoding envelope protein (E).

[0037] The composition according to any of the preceding specific embodiments, wherein the Zika virus antigen gene comprises i) a gene encoding a pre-membrane (prM) protein of SEQ ID NO:3 or a gene having 90% or higher identity with SEQ ID NO:3; and ii) a gene encoding an envelope (E) protein of SEQ ID NO:4 or a gene having 90% or higher identity with SEQ ID NO:4.

[0038] The composition according to any one of the preceding specific embodiments, wherein the chimeric Zika virus has the ability to specifically inhibit breast cancer cells through serial passaging.

[0039] The composition according to any of the preceding specific embodiments, wherein the chimeric Zika virus is obtained by at least 19 passages in MDAMB-231 cells and at least 61 continuous passages in a mixed cell consisting of HCC1954, BT474, HCC70, BT20, BT549 and HS578T.

[0040] The composition according to any of the preceding specific embodiments, wherein the chimeric Zika virus comprises a polypeptide described by the amino acid sequence of SEQ ID NO:8, or a polypeptide having 90% or higher identity with SEQ ID NO:8.

[0041] According to any of the foregoing specific embodiments, in the chimeric Zika virus, based on the sequence shown in SEQ ID NO:7, the amino acid corresponding to any one of positions 30, 60, 122, 227, 427, 497, 608, 657, 690, 1464, 1843, 2052, 2108, 2139, 2150, 2350, 2404, 2416, 2461 and 3077 is replaced by another amino acid.

[0042] According to any one of the foregoing specific embodiments, the chimeric Zika virus may include a gene encoding a yellow fever virus capsid protein and a gene encoding a non-structural protein, and the capsid (C) protein of the yellow fever virus includes a polypeptide described by the amino acid sequence of SEQ ID NO: 2 or a polypeptide having 90% or higher identity with SEQ ID NO: 2.

[0043] The composition according to any of the aforementioned specific embodiments, wherein in the chimeric Zika virus, based on the sequence shown in SEQ ID NO: 2, the amino acids corresponding to positions 30 and 60 are replaced by other amino acids.

[0044] The composition according to any of the preceding specific embodiments, wherein the pre-membrane (prM) protein of the chimeric Zika virus comprises a polypeptide described by the amino acid sequence of SEQ ID NO:3, or a polypeptide having 90% or greater identity with SEQ ID NO:3.

[0045] The composition according to any of the aforementioned specific embodiments, wherein in the chimeric Zika virus, based on the sequence shown in SEQ ID NO: 3, the amino acids corresponding to positions 122 and 227 are replaced by other amino acids.

[0046] The composition according to any of the preceding specific embodiments, wherein the envelope (E) protein of the chimeric Zika virus comprises a polypeptide described by the amino acid sequence of SEQ ID NO:4, or a polypeptide having 90% or higher identity with SEQ ID NO:4.

[0047] The composition according to any of the aforementioned specific embodiments, wherein in the chimeric Zika virus, based on the sequence shown in SEQ ID NO: 4, the amino acid corresponding to any one of positions 427, 497, 608, 657 and 690 is replaced by another amino acid.

[0048] The composition according to any of the preceding embodiments, wherein the non-structural protein comprises a polypeptide having an amino acid sequence of SEQ ID NO:5, or a polypeptide having 90% or higher identity with SEQ ID NO:5.

[0049] The composition according to any of the foregoing specific embodiments, wherein in the chimeric Zika virus, based on the sequence shown in SEQ ID NO:5, the amino acids corresponding to positions 1464, 1843, 2052, 2108, 2139, 2150, 2350, 2404, 2416, 2461 and 3077 are replaced by other amino acids.

[0050] The composition according to any one of the preceding specific embodiments, wherein the composition is administered intratumorally, intramuscularly, intraperitoneally or intravenously.

[0051] Another aspect of the present invention is a method for preparing a pharmaceutical composition for preventing or treating breast cancer.

[0052] In a specific embodiment, the preparation method includes: (a) continuously passaging the chimeric Zika virus in MDAMB-231 cells for at least 19 times to obtain a virus culture containing cell-adapted virus; (b) continuously passaging the culture containing cell-adapted virus obtained in step (a) for at least 61 times in a mixed cell composed of HCC1954, BT474, HCC70, BT20, BT549 and HS578T to obtain a virus culture containing cell-adapted virus; and (c) selecting and isolating a chimeric Zika strain from the virus culture containing cell-adapted virus obtained in step (b) by evaluating the ability of the chimeric Zika virus to specifically inhibit breast cancer cells.

[0053] Another aspect of the invention is a chimeric nucleic acid molecule.

[0054] In a specific embodiment, the chimeric nucleic acid molecule consists of the following: i) a gene encoding a signal peptide, wherein the signal peptide is located upstream of a gene encoding a pre-membrane (prM) protein, wherein the protein includes a polypeptide of SEQ ID NO: 6 or a polypeptide having 90% or higher identity with SEQ ID NO: 6; ii) a gene encoding a pre-membrane (prM) protein, wherein the protein includes a polypeptide of SEQ ID NO: 3 or a polypeptide having 90% or higher identity with SEQ ID NO: 3; or iii) a gene encoding an envelope (E) protein consisting of a polypeptide of SEQ ID NO: 4 of Zika virus or a polypeptide having 90% or higher identity with SEQ ID NO: 4, which is inserted into i) a region of a gene encoding a signal peptide located upstream of a gene encoding a pre-membrane (prM) protein; ii) a region of a gene encoding a pre-membrane (prM) protein; and iii) a region of a gene encoding an envelope (E) protein of a nucleic acid molecule of a yellow fever virus 17D vaccine strain, wherein part or all of the nucleic acid constituting Zika virus gene i), ii) or iii) is replaced, deleted or inserted.

[0055] Another aspect of the invention is a chimeric Zika virus mutant strain.

[0056] In a specific embodiment, the mutant strain is obtained by serially subculturing the chimeric nucleic acid molecule in breast cancer cells to increase the fitness of the cancer cells.

[0057] The present invention is specifically explained as follows. Meanwhile, each description and each embodiment disclosed in the present invention may also be applied to other descriptions and other embodiments, respectively. In other words, all combinations of the various elements disclosed in the present invention fall within the scope of the present invention. In addition, the scope of the present invention should not be considered to be limited by the following specific description. In addition, those skilled in the art will recognize, or be able to use no more than routine experiments to determine many equivalents to the specific embodiments of the present invention described herein. In addition, these equivalents are intended to be included in the present invention.

[0058] One aspect of the present invention provides a pharmaceutical composition for preventing or treating breast cancer, which contains a chimeric Zika virus as an active ingredient. Specifically, a pharmaceutical composition for preventing or treating cancer is provided as an active ingredient, which contains a chimeric Zika virus formed by introducing a Zika virus antigen gene into a gene of an attenuated yellow fever virus containing SEQ ID NO: 1.

[0059] The term "chimera" in the present invention may refer to the situation where cells with different genetic characteristics coexist in one organism, and in particular, in the present invention, refers to the situation where the genetic information (nucleic acid) of different individuals is inserted into the genetic skeleton of one individual to cause new genetic variation. Chimera may be used interchangeably with "chimerism", "Zika chimera", "chimeric Zika virus" or "cZIKV".

[0060] Zika virus consists of a structure including structural proteins and non-structural proteins. Specifically, structural proteins include capsid protein (C), pre-membrane protein (precursor membrane protein; prM) and envelope protein (E), and non-structural proteins include non-structural proteins NS1 to NS5. For example, the "Zika chimera" or "chimeric Zika virus" of the present invention refers to a recipient flavivirus, in which the genetic backbone is modified by exchanging at least the envelope (E) protein, or the pre-membrane (pr-M) and envelope (E) protein sequences of the recipient flavivirus with the corresponding Zika virus sequences. Specifically, the genetic backbone of the recipient flavivirus can be modified by exchanging the base sequences encoding the pr-M and E proteins of the recipient flavivirus with the corresponding sequences of the Zika virus. The recipient flavivirus may be attenuated. The recipient flavivirus may be a yellow fever (YF) virus, in particular a yellow fever virus 17D vaccine strain.

[0061] In the present invention, "chimeric Zika virus" may refer to a form in which the yellow fever virus is used as a backbone and the gene capable of inducing Zika virus immune response is inserted into the yellow fever virus nucleic acid molecule.

[0062] Examples of other attenuated YF strains that can be used include, but are not limited to, attenuated YF17D, YF17DD, and YF17D204. Virus. Specifically, the YF 17D strain can be used as a backbone. For example, the YF 17D backbone genome sequence can be composed of SEQ ID NO:1.

[0063] As an embodiment according to any one of the aforementioned embodiments, the Zika virus antigen gene may include i) a gene encoding a pre-membrane protein (prM) and ii) a gene encoding an envelope protein (E).

[0064] The chimeric Zika virus is a virus in which a foreign gene is introduced into the genome of an attenuated yellow fever virus, and may be a virus in which i) the pre-membrane (prM) protein region and ii) the envelope (E) protein region of the attenuated yellow fever virus are replaced by those derived from the i) pre-membrane (prM) protein region and ii) the envelope (E) protein region of the wild-type Zika virus, but is not limited thereto.

[0065] In an embodiment of the present invention, the transmembrane protein region of the 17D YF virus can be maintained as the transwall protein region of the chimeric Zika virus, and the pre-membrane (prM) and envelope (E) proteins derived from wild-type Zika virus can be provided as those proteins of the chimeric Zika virus. Specifically, the pre-membrane protein (prM) of the chimeric Zika virus interacts with the envelope protein (E), and may include the entire pre-membrane protein, including transmembrane proteins. In the present invention, the term pre-membrane (prM) can be used interchangeably with terms such as precursor membrane protein.

[0066] The pre-membrane (prM) protein of the chimeric Zika virus may include a polypeptide described by the amino acid sequence of SEQ ID NO: 3, or have 90% or greater identity thereto.

[0067] The envelope (E) protein is a protein located on the outermost surface of flaviviruses (including Zika virus) and is known to be a protein that plays a key role in infection by attaching to host cells. The envelope (E) protein may include the entire envelope protein including transmembrane proteins. The envelope (E) protein may include a polypeptide described in the amino acid sequence of SEQ ID NO: 4 or having 90% or higher identity thereto.

[0068] In the pre-membrane (prM) protein of the chimeric Zika virus, the amino acids at positions 122 and 227 corresponding to the sequence shown in SEQ ID NO: 3 based on the sequence number (SEQ ID NO) can be replaced by other amino acids, and the amino acids at any of positions 427, 497, 608, 657 and 690 corresponding to the sequence shown in SEQ ID NO: 4 based on SEQ ID NO in the envelope (E) protein can be replaced by another amino acid, but the pre-membrane (prM) protein and the envelope (E) protein are not limited thereto.

[0069] As an embodiment of any of the aforementioned embodiments, in the present invention, the chimeric Zika virus may include a gene encoding a yellow fever virus capsid (C) protein and a gene encoding a non-structural protein. Specifically, the capsid (C) protein of the chimeric Zika virus may be a capsid protein of a yellow fever virus, and may include a polypeptide described by the amino acid sequence of SEQ ID NO: 2 or a polypeptide having 90% or higher identity thereto.

[0070] In the capsid (C) protein of the chimeric Zika virus, the amino acids at positions 30 and 60 corresponding to the sequence shown in SEQ ID NO: 2 based on SEQ ID NO can be replaced by other amino acids, but the capsid (C) protein is not limited thereto.

[0071] Specifically, the non-structural protein of the chimeric Zika virus can be a non-structural protein of yellow fever virus, and can include a polypeptide described by the amino acid sequence of SEQ ID NO: 5 or a polypeptide having 90% or higher identity thereto.

[0072] In the non-structural protein of the chimeric Zika virus, the amino acids at positions 1464, 1843, 2052, 2108, 2139, 2150, 2350, 2404, 2416, 2461 and 3077 corresponding to the sequence shown in SEQ ID NO: 5 based on SEQ ID NO can be replaced by other amino acids, but the non-structural protein is not limited thereto.

[0073] For example, an amino acid sequence having 90% or more identity to an amino acid sequence of SEQ ID NO: 2, 3, 4 or 5 of the present invention can be an amino acid sequence having at least 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 96.26% or more, 97% or more, 97.5% or more, 97.7% or more, 97.8% or more, 98% or more, 98.5% or more, 98.7% or more, 98.8% or more, 99% or more, 99.5% or more, 99.7% or more, 99.8% or more, or more, and less than 100% homology or identity to SEQ ID NO: 2, 3, 4 or 5 of the present invention. It goes without saying that proteins having an amino acid sequence in which part of the sequence is deleted, modified, replaced or added are also included in the scope of the protein to be attenuated in the present invention, as long as the amino acid sequence has the same or corresponding homology or identity as the capsid (C) protein, pre-membrane (prM) protein, envelope (E) protein and / or non-structural protein, and exhibits the same or corresponding activity as the capsid protein, membrane protein, envelope protein and / or non-structural protein.

[0074] The capsid (C) protein, pre-membrane (prM) protein, envelope (E) protein or non-structural protein may have a polypeptide described by the amino acid sequence of SEQ ID NO: 2, 3, 4 or 5 or an amino acid sequence having 90% or higher identity thereto, or may consist of or substantially consist of the polypeptide. It may also include meaningless sequence additions (i.e., adding a sequence that does not change the function of the protein to the N-terminus and / or C-terminus of the amino acid sequence) or natural mutations, silent mutations or conservative substitutions before or after the amino acid sequence of SEQ ID NO: 2, 3, 4 or 5, or a case having 90% or higher identity thereto.

[0075] As an embodiment according to any of the foregoing embodiments, for the purpose of the present invention, the chimeric Zika virus includes i) a gene encoding a pre-membrane (prM) protein and ii) a gene encoding an envelope (E) protein gene and a capsid (C) protein gene and a non-structural protein gene derived from a Zika virus and a yellow fever virus, and may include a polypeptide described by the amino acid sequence of SEQ ID NO: 7 or a polypeptide having 90% or more identity thereto. The amino acid at any one of positions 30, 60, 122, 227, 427, 497, 608, 657, 690, 1464, 1843, 2052, 2108, 2139, 2150, 2350, 2404, 2416, 2461 and 3077 corresponding to the sequence shown in SEQ ID NO: 7 based on SEQ ID NO may be replaced by another amino acid, but the chimeric Zika virus is not limited thereto.

[0076] As an embodiment according to any of the foregoing embodiments, the passaged chimeric Zika virus may include a polypeptide described by the amino acid sequence of SEQ ID NO: 8, or a polypeptide having 90% or more identity thereto. The chimeric Zika virus may include a polynucleotide encoding the amino acid sequence of SEQ ID NO: 8, or having 90% or more identity thereto. The chimeric Zika virus may, for example, consist of SEQ ID NO: 9.

[0077] "Conservative substitutions" refer to the replacement of one amino acid with another amino acid having similar structure and / or chemical properties. Such amino acid substitutions may generally occur based on similarities in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathicity of the residues. Typically, conservative substitutions may have little or no effect on the activity of a protein or polypeptide.

[0078] In this application, the term "homology" or "identity" refers to the degree of similarity between two given amino acid sequences or base sequences, which can be expressed as a percentage. The two terms homology and identity are often used interchangeably.

[0079] The sequence homology or identity of conservative polynucleotides or polypeptides is determined by standard comparison algorithms, and the default gap penalty established by the program used can be used together with this. In practice, homologous or identical sequences can hybridize in whole or part of the sequence under medium or high stringency conditions usually. It goes without saying that hybridization also includes hybridization of polynucleotides containing common codons or codons that consider codon degeneracy in polynucleotides.

[0080] For example, known computer algorithms, such as the "FASTA" program, can be used to determine whether any two polynucleotide or polypeptide sequences have homology, similarity or identity using default parameters, such as the parameters in Pearson et al. (1988) [Proc. Natl. Acad. Sci. USA 85]: 2444. Homology, similarity or identity can be measured using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453) as implemented by the Needleman program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277) (version 5.0.0 or higher), including the GCG program package (Devereux, J. et al., Nucleic Acids Research 12:387 (1984)), BLASTP, BLASTN, FASTA (Atschul, [S.] [F.,] [et al., J MOLEC Biol 215]:403 (1990); Guide to Huge Computers, Martin J. Bishop, [ED.,] Academic Press, San Diego, 1994, and [CARILLO ETA / .] (1988) SIAM J Applied Math 48: 1073). For example, BLAST or ClustalW of the National Center for Biotechnology Information Database can be used to determine homology, similarity or identity.

[0081] Homology, similarity or identity of polynucleotides or polypeptides can be determined by comparing sequence information, for example, using the GAP computer program, such as Needleman et al. (1970), J Mol Biol. 48: 443, for example, as reported by Smith and Waterman, Adv. Appl Math (1981) 2: 482. In summary, in the GAP program, homology, similarity or identity can be defined as the value obtained by dividing the number of similarly arranged symbols (i.e., nucleotides or amino acids) by the total number of symbols in the shorter of the two sequences. Default parameters for the GAP program may include (1) a binary comparison matrix (containing identity values ​​of 1 and non-identity values ​​of 0) and the weighted comparison matrix of Gribskov et al. (1986) Nucl. Acids Res. 14:6745 (or the EDNAFULL (EMBOSS version of NCBINUC4.4) substitution matrix disclosed by Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation, pp. 353-358 (1979)); (2) a penalty of 3.0 for each gap and an additional penalty of 0.10 for each symbol in each gap (or a gap opening penalty of 10 and a gap extension penalty of 0.5); and (3) no penalty for end gaps.

[0082] In the present invention, the term "corresponding to" refers to an amino acid residue or nucleotide residue at a listed position in a polypeptide or polynucleotide, or an amino acid residue or nucleotide residue that is similar, identical, or homologous to the listed residue in the polypeptide or polynucleotide. Identifying an amino acid or nucleotide at a corresponding position may involve determining a specific amino acid or nucleotide of a sequence that references a specific sequence. As used herein, a "corresponding region" generally refers to a similar or corresponding position in a related protein or reference protein.

[0083] For example, any amino acid sequence can be aligned with SEQ ID NO: 1, 2, 3, 4 or 5, and based on this, each amino acid residue of the amino acid sequence can be numbered by reference to the amino acid residue number corresponding to the amino acid residue of SEQ ID NO: 1, 2, 3, 4 or 5. For example, the sequence alignment algorithm described in the present invention can identify the position of an amino acid or the position where a modification (such as a substitution, insertion or deletion) occurs by comparing a sequence to a query sequence (also referred to as a "reference sequence").

[0084] For such alignment, for example, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453), the Needlemman program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), etc. can be used, but the alignment is not limited thereto, and any sequence alignment program, pairwise sequence comparison algorithm, etc. known in the art can be appropriately used.

[0085] As any one of the embodiments in the aforementioned embodiments, the chimeric Zika virus of the present invention can have the ability to specifically inhibit breast cancer cells by continuous passage. For example, the chimeric Zika virus can be obtained by at least 19 passages in MDAMB-231 cells and at least 61 continuous passages in the mixed cells composed of HCC1954, BT474, HCC70, BT20, BT549 and HS578T, but is not limited thereto. According to the type of the targeted breast cancer cell line, the passage order and / or passage number can be adjusted.

[0086] The chimeric Zika virus of the present invention is an oncolytic virus, which exhibits low infection and proliferation characteristics in normal cells, but relatively high infection and proliferation in cancer cells. Using this principle, the chimeric Zika virus can be used for treatment to minimize damage to normal cells and selectively kill only cancer cells.

[0087] In the examples of the present invention, the presence or absence of infectivity was examined using primary cells of brain, testis, skin, breast, and lung, and as a result, no infectivity was observed in normal cells.

[0088] In an embodiment of the present invention, chimeric Zika virus was passaged in breast cancer cells, and the results showed that the initial chimeric Zika strain (cZIKV) was weakly infective to cancer cells, but as the number of passages increased, the infectivity to breast cancer cells with lower initial susceptibility (such as BT20 and BT474) increased.

[0089] Specifically, it has been found that by subculturing the chimeric Zika virus in breast cancer cells, the adaptation to breast cancer cells is increased, and the chimeric Zika strain does not affect normal tissue cells without mutations, but selectively infects four different subtypes of breast cancer cells, which are classified according to the presence or absence of estrogen receptor, progesterone receptor and HER2 genes, and can inhibit tumors, thereby completing the present invention.

[0090] The pharmaceutical composition of the present invention may further contain a "pharmaceutically acceptable salt". "Pharmaceutically acceptable salt" refers to a salt used in medicine in which a cation and anion are bound by electrostatic attraction, and may generally be a metal salt, a salt with an organic base, a salt with an inorganic acid, a salt with an organic acid, a salt with a basic or acidic amino acid, etc. For example, the metal salt may be an alkali metal salt (sodium salt, potassium salt, etc.), an alkaline earth metal salt (calcium salt, magnesium salt, barium salt, etc.), an aluminum salt, etc.; the salt with an organic base may be a salt of triethylamine, pyridine, picoline, 2,6-lutidine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, N,N-dibenzylethylenediamine, etc.; the salt with an inorganic acid may be a salt of hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid, etc.; the salt with an organic acid may be a salt of formic acid, acetic acid, trifluoroacetic acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, etc.; the salt with a basic amino acid may be a salt of arginine, lysine, ornithine, etc.; the salt with an acidic amino acid may be a salt of aspartic acid, glutamic acid, etc.

[0091] The term "treatment" of the present invention refers to clinical intervention that changes the natural course of the individual or cell to be treated, which can be performed or prevented when the clinical pathological condition progresses. The desired therapeutic effect includes preventing the occurrence or recurrence of the disease, alleviating symptoms, reducing all direct or indirect pathological consequences of the disease, preventing metastasis, slowing the progression of the disease, alleviating or temporarily alleviating the disease state, improving the course of the disease or improving the prognosis. In the present invention, the treatment includes all effects of improving the course of breast cancer by administering the composition. The term "prevention" of the present invention refers to all effects of inhibiting or delaying the onset of breast cancer by administering the composition of the present invention.

[0092] In an embodiment of the present invention, three cells with different viral sensitivities were used to examine the anticancer effect, and it was found that the body temperature and weight of the oncolytic virus administration group did not change, and the tumor size decreased and gradually disappeared. Histopathological examination of the tumor also showed that central necrosis was observed in the tumor of the treatment group. It can be seen that breast cancer can be effectively treated by administering a pharmaceutical composition for the prevention or treatment of breast cancer containing the chimeric Zika virus of the present invention as an active ingredient.

[0093] The pharmaceutical composition of the present invention may additionally contain suitable carriers, excipients or diluents commonly used to prepare pharmaceutical compositions. Compositions containing pharmaceutically acceptable carriers may be various oral or parenteral preparations. In the case of a composition forming a preparation, the preparation may be prepared using a diluent or excipient, such as a filler, a bulking agent, a binder, a wetting agent, a disintegrant and a commonly used surfactant. Oral solid preparations may include tablets, pills, powders, granules, capsules, etc. These solid preparations may be prepared by mixing one or more compounds with 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 may also be used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions and syrups, which may contain various excipients, such as wetting agents, sweeteners, flavoring agents and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. Parenteral preparations may include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations and suppositories. As non-aqueous solvents and suspending agents, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, etc. can be used. As bases for suppositories, Witepsol, polyethylene glycol, Tween 61, cocoa butter, lauric protein fat, glycerin gelatin, etc. can be used.

[0094] The pharmaceutical composition of the present invention may have any formulation selected from, but not limited to, tablets, pills, powders, granules, capsules, suspensions, oral solutions, emulsions, syrups, sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations and suppositories.

[0095] The pharmaceutical composition of the present invention can be administered to a subject by any appropriate method. As a route of administration, the pharmaceutical composition can be administered by various routes, whether orally or by injection, as long as it can reach the target tissue.

[0096] In the present invention, the term "subject" refers to all animals, such as rats and livestock, including humans who have or are likely to have breast cancer. As a specific example, the subject may be a mammal, including a human.

[0097] The pharmaceutical composition can be appropriately administered to the subject by conventional methods, administration routes and dosages used in the art according to purpose or need. Examples of administration routes include parenteral, subcutaneous, intraperitoneal, intrapulmonary and intranasal administration, and parenteral injections include intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Appropriate dosage and number of administrations can be selected according to methods known in the art. The amount of the pharmaceutical composition of the present invention actually administered and the number of administrations can be appropriately determined by various factors, such as the type of symptoms to be treated, administration routes, sex, health status, diet, age and weight of the subject and the severity of the disease.

[0098] As an embodiment according to any one of the preceding embodiments, the composition of the present invention is an attenuated virus vaccine having a chimeric Zika virus structure and thus may have high stability.

[0099] The term "attenuated" of the present invention refers to, but is not limited to, lack of replication and proliferation capabilities in normal cells, and includes all meanings known in the art.

[0100] In the examples of the present invention, YF17D (yellow fever 17D) and ZIKA showed replication ability in mice, but the oncolytic virus, which is a chimeric form of these two viruses, showed almost no replication and proliferation ability, verifying its safety.

[0101] It can be seen from this that by administering the pharmaceutical composition containing the chimeric Zika virus of the present invention as an active ingredient, not only can breast cancer be effectively treated, but the pharmaceutical composition is also very safe.

[0102] Another aspect of the present invention provides a method for preparing a composition for preventing or treating breast cancer. Specifically, the method may include (a) continuously passage the chimeric Zika virus in MDAMB-231 cells for at least 19 times to obtain a virus culture containing cell-adapted viruses; (b) continuously passage the culture containing the cell-adapted virus obtained in step (a) in a mixed cell composed of HCC1954, BT474, HCC70, BT20, BT549 and HS578T for at least 61 times to obtain a virus culture containing cell-adapted viruses; and (c) selecting and isolating chimeric Zika viruses from the virus culture containing cell-adapted viruses obtained in step (b) by evaluating the ability to specifically inhibit breast cancer cells.

[0103] Another aspect of the present invention provides a chimeric nucleic acid molecule consisting of: i) a gene encoding a signal peptide, wherein the signal peptide is located upstream of a gene encoding a pre-membrane (prM) protein, wherein the protein includes a polypeptide of SEQ ID NO: 6 or a polypeptide having 90% or higher identity with SEQ ID NO: 6; ii) a gene encoding a pre-membrane (prM) protein, wherein the protein includes a polypeptide of SEQ ID NO: 3 or a polypeptide having 90% or higher identity with SEQ ID NO: 3; or iii) a gene encoding an envelope (E) protein consisting of a polypeptide of SEQ ID NO: 4 of Zika virus or a polypeptide having 90% or higher identity with SEQ ID NO: 4, which is inserted into i) a region of a gene encoding a signal peptide located upstream of a gene encoding a pre-membrane (prM) protein; ii) a gene encoding a pre-membrane (prM) protein; and iii) a gene encoding an envelope (E) protein of a nucleic acid molecule of a yellow fever virus 17D vaccine strain, wherein part or all of the nucleic acid constituting the Zika virus gene i), ii) or iii) is replaced, deleted or inserted.

[0104] Another aspect of the present invention provides a chimeric Zika virus mutant strain obtained by serial subculturing of a chimeric nucleic acid molecule in breast cancer cells so as to have increased cancer cell adaptability.

[0105] The term "variant" in the present invention refers to a polypeptide in which one or more amino acids are conservatively replaced and / or modified so that the amino acid sequence is different from the variant before the variation, but the function or property is maintained. Such variants can generally be identified by modifying one or more amino acids in the amino acid sequence of the polypeptide and evaluating the properties of the modified polypeptide. In other words, the ability of the variant may be increased, unchanged or decreased compared to the polypeptide before the variation. Some variants may include variants in which one or more parts (such as an N-terminal leader sequence or a transmembrane domain) are deleted. Other variants may include variants in which certain parts are missing from the N and / or C-terminus of the mature protein. The term "variant" can be used interchangeably with terms such as modification, modified polypeptide, modified protein, mutant, mutant protein and divergence, and is not limited thereto, as long as it is a term used to indicate variation. For the purposes of the present invention, in a variant, any of positions 30, 60, 122, 227, 427, 497, 608, 657, 690, 1464, 1843, 2052, 2108, 2139, 2150, 2350, 2404, 2416, 2461 and 3077 corresponding to the amino acid of SEQ ID NO: 7 or the amino acid sequence of SEQ ID NO: 7 may be replaced by another amino acid.

[0106] Variants may include deletions or additions of amino acids that have minimal effects on the properties and secondary structure of the polypeptide. For example, a signal (or leader) sequence that participates in protein co-translation or post-translational translocation may be combined with the N-terminus of the variant. The variant may be combined with other sequences or connectors for identification, purification or synthesis. DETAILED DESCRIPTION

[0107] Hereinafter, the present invention will be described in more detail with reference to Examples. These Examples are intended only to illustrate the present invention, and the scope of the present invention should not be construed as being limited by these Examples.

[0108] Experimental Example 1. Construction of chimeric Zika virus (cZIKV)

[0109] 1-1. Preparation of Zika virus

[0110] ATCC VR-1843 wild-type Zika virus. The sequence can be easily obtained through GenBank accession number: KU365778.1 (virus strain; BeH819015).

[0111] 1-2. Preparation of Yellow Fever Vaccine Strain and 17D Virus

[0112] Yellow fever vaccine strain, 17D strain virus. 17D virus is easily accessible through GenBank No: X03700.1.

[0113] 1-3. Design and synthesis of chimeric Zika virus genes

[0114] Chimeric Zika virus with yellow fever genome as backbone was selected as a candidate for anticancer vaccine. By replacing the major antigen gene of yellow fever genome with the major antigen gene of Zika virus, a chimeric Zika virus ( Figure 1 ).

[0115] The specific experimental method is the same as the method disclosed in Korean Patent Application No. 10-2019-0127605 filed by the inventor of the present invention with the Korean Intellectual Property Office on May 3, 2019.

[0116] Experimental Example 2. Infectivity test of chimeric Zika virus (cZIKV) on normal cells (5 types) and various cancer cells (5 types)

[0117] 2-1. Preparation of normal cells and cancer cell lines

[0118] To test the infectivity of Zika virus in normal cell lines, five normal cell lines (brain, testis, skin, breast, and lung) were purchased and cultured in medium appropriate for the cell lines. In addition, cell lines representing five high-incidence cancers were purchased and cultured in medium appropriate for the cell lines.

[0119] 2-2. Preparation of 8 cancer subtypes of breast cancer cell lines and detection of cZIKV infectivity to cancer cells

[0120] To test the infectivity of Zika virus to breast cancer subtypes, eight human breast cancer cell lines were purchased (Korean Cell Line Bank and ATCC) and cultured using appropriate media.

[0121] The above cultured cells were seeded in a 6-well plate at 5E5 (5105) cells / well. The next day, the prepared cells were carefully washed once with a medium without FBS to prevent cell detachment. The medium was removed, and all cells were treated with 1 MOI of cZIKV, followed by adsorption in a 37°C incubator for 1 hour. The infected virus culture was removed, 3 mL of the appropriate medium was added to each cell, and the cells were cultured in a 37°C incubator for 3 days.

[0122] Remove the culture, fix the cells with a 100% methanol solution at -20°C at a rate of 2 mL / well, and let the cells stand at room temperature for 1 hour. Then, remove the fixative solution and wash three times with DPBS. Dilute the primary antibody (anti-flavivirus group antigen antibody, clone D1-4G2-4-15, mouse IgG2) with DPBS at 1:3000, use it at 0.5 mL / well, and react on a shaker at room temperature for 1 hour. Wash three times with DPBS. Dilute the secondary antibody (anti-mouse Alexa488) at 1:3000, use it at 0.5 mL / well, and react on a shaker at room temperature for 1 hour. At this time, cover the plate with foil to block light. Wash three times with DPBS. Use a fluorescence microscope in a dark room to observe the degree of fluorescence brightness, or use a Cytation Reader for imaging analysis.

[0123] Experimental Example 3. Adaptive Passaging of Oncolytic Zika Virus

[0124] 3-1.MDAMB231 Adaptation

[0125] MDAMB231 was seeded in a 6-well plate at a rate of 3E5 cells / well. The next day, cells reaching 80% confluence were infected with cZIKV at 1 MOI. After 1 hour, the infection solution was removed and 3 mL of DMEM (10% FBS, 1% P / S) medium was added. Cytopathic effect can be observed after 2 days. On the third day, the culture medium was found to be light yellow and the cells were scraped off. The cell lysate and culture were transferred to a 15 mL test tube and centrifuged at 3000 rpm for 5 minutes. The supernatant was taken and divided into 0.5 mL portions in a freezing bottle, defined as the 1st generation. The passage was repeated 19 times in the same manner as the above process (however, since titration could not be performed each time, the amount of virus used for reinfection was set to 1 mL). Afterwards, the separated viruses were stored at -80°C.

[0126] 3-2. Adaptability of mixed breast cancer cells

[0127] Each of six types of breast cancer cell lines (HCC1954, BT474, HCC70, BT20, BT549 and Hs578T) was inoculated at 1E5 cells / well in one hole of a 6-well plate (6E5 cells / well in total). The next day, cells reaching 80% confluence were infected with 1MOI of MDAMB231-p19 virus. The subsequent method was the same as that in Experimental Example 2-2, and passage was repeated 61 times (labeled virus Bx61) ( Figure 2 ).

[0128] 3-3. Identification using cancer cell-specific adapted viruses

[0129] In order to compare the infectivity of the passaged cZIKV and Bx61, the infectivity test performed in Experimental Example 2-2 was performed again.

[0130] To examine the differences in oncolytic activity of the cell lines, a CPE (cytopathic effect) assay was performed and compared using cell viability. At this point, the ToxGlo assay kit was used to measure cell viability. Six types of cells (Vero, MCF7, MDAMB231, BT20, Hs578T, and HMEc) were seeded at 2E4 cells / well in a white / transparent bottom 96-well plate and treated with two types of viruses (cZIKV and Bx61) at MOIs of 0.1, 1, and 5 (treated with culture medium as a negative control). At 5 DPI after treatment with the virus, the cell culture medium was removed. With the lights of the BSC turned off and in the dark (to prevent the substrate from being decomposed by light), as described above. Figure 3 As shown, mix the ATP detection substrate and detection buffer. Pour 100 μL of the mixed solution into the well and let the well stand for at least 10 minutes in a light-shielded state. At this time, carefully check for bubbles and compare the luminescence level using a luminescence detector.

[0131] 3-4. q-PCR method for quantification of oncolytic Zika virus

[0132] Viral RNA was isolated and purified from 200 μL of Zika virus solution using a viral RNA purification kit (PureLink Viral RNA / DNA Mini Kit). For analysis, a q-PCR assay kit (Zika virus polyprotein gene genesig advanced kit) and a reverse transcriptase master mix kit (genesig OasigTM Lyophied one step or precisionTM one step 2X qRT-PCR master mix) were mixed with the sample for preparation.

[0133] Under the conditions shown in Table 1 below, the prepared standard samples and virus samples were reacted using a QuantStudio 3-qPCR device.

[0134] [Table 1]

[0135] qPCR reaction conditions

[0136]

[0137] 3-5. Oncolytic Zika virus full-length sequence analysis method

[0138] Viral RNA was extracted from 200 μL of viral solution. Viral RNA was prepared using the PureLinkTM viral RNA / DNA mini kit (final elution was set to 25 μL). In the case of oncolytic Zika virus, the concentration of the viral solution used for RNA separation was 1E7 PFU / mL. The synthesis of cDNA and gene-specific PCR from the extracted viral RNA was completed in one step using a kit (SuperScript one-step RT-PCR). At this point, RT-PCR and PCR were performed on four overlapping fragment genes (about 2.5 to 3 kb in length) containing the full-length Zika virus gene. Primers are shown in Table 2 below, with cZik1-F and cZik1-R (2498) used for fragment 1, cZik2-F and c Zik2-R (5385) used for fragment 2, cZik3-F and c Zik3-R (8205) used for fragment 3, and cZik4-F and cZik4-R used for fragment 4. PCR was performed under the conditions in Table 3 below, and electrophoresis was performed.

[0139] [Table 2]

[0140] RT-PCR primer sequences

[0141]

[0142]

[0143]

[0144] [Table 3]

[0145] RT-PCR conditions

[0146] Components Volume (μL) 2X reaction mixture 25 Template RNA 5 Forward primer (10 pmole / μL) 1 Reverse primer (10 pmole / μL) 1 <![CDATA[SuperScript TM IIIRT / Platinum TM TaqMix]]> 1 RNase-free water 17 total 50

[0147] Experimental Example 4. Safety Verification of Chimeric Zika Virus (cZIKV)

[0148] The cZIKV according to the present invention is an attenuated virus vaccine having a chimeric virus structure, and is intended to verify the safety of cZIKV compared with the two viruses contained in the cZIKV construct.

[0149] Specifically, 7-week-old Balb / c female mice were purchased and used for experiments after an adaptation period of about one week. Purified samples for animal experiments were prepared at a concentration of cZIKV (4105 pfu / ml) (5 groups, 5 animals each - Group 1: VP-SFM 400 μl, Group 2: 1E2, Group 3: 1E3, Group 4: 1E4, Group 5: 1E5).

[0150] Some injection samples were prepared by mixing with an equal volume of adjuvant (aluminum hydroxide) (vortexing for 1 hour). The injection method was intravenous injection, in which the injection sample was injected into the mouse hind limb muscle using an insulin syringe. Infection was performed once, and the weight of the mice was measured every day. For each group, 100 μl of blood was collected through the orbital vein within 10 days after infection. Whole blood was collected on the 28th day. The collected blood samples were combined and 100 μl was used. The collected blood was reacted at room temperature for about 1 to 2 hours to coagulate the blood. After the blood coagulated, the test tube was transferred to ice and the reaction was carried out for 1 hour. The reaction was carried out at 13000 rpm and 4 ℃ Centrifuge for 15 minutes. Collect as much The serum was centrifuged again at 13000 rpm. After 1 minute, the upper serum was transferred to a new tube and stored frozen at -20°C. The stored serum was thawed and q-PCR was performed. Q-PCR was performed in the same manner as the above method.

[0151] The results showed that when RNA was isolated from isolated serum and tested by q-PCR, no viral RNA was detected even when cells were treated with different doses of cZIKV. Figure 4 As shown, YF17D (yellow fever 17D) and ZIKA showed replication ability in Balb / c, but the oncolytic virus, which is a chimeric form of these two viruses, showed almost no replication or proliferation ability, verifying its safety.

[0152] Meanwhile, in previous studies, ZIKV and YF 17D (yellow fever 17D) showed replication ability in Balb / c. Specifically, it was reported that wild-type ZIKV (PRVABC59; a low-passage ZIKV isolate from Puerto Rico) proliferated to 1E6 PFU / ml on the third day after challenge, and YF 17D proliferated at high levels (5E5 PFU / ml) in serum 9 days after inoculation (Nature. 2016Aug 25; 536(7617): 474-478, Microbiol Spectr. 2022Oct 26; 10(5): e0224622.).

[0153] Through the above results, it was confirmed that oncolytic virus is a virus that has been proven to be safe compared with other viruses (YF and ZIKA).

[0154] Experimental Example 5. Using three cell lines with different virus sensitivities to test animal efficacy

[0155] 5-1. Preparation of cell lines

[0156] A vial of cell line (MDAMB231) purchased from the Korean cell line bank was thawed and passaged using RPMI1640 (FBS10%, P / S1%) medium. Then, 3E6 cells were inoculated in a 175T flask and cultured in an incubator at 37°C and 5% CO2. When 100% fusion was reached, it was washed with PBS, 2.5% trypsin-EDTA was added to separate the cells, centrifuged (1000rpm, 5 minutes), the supernatant was discarded, and a new culture medium was used to obtain a cell suspension. Animal cells were prepared on the day of injection. After checking the viability using a microscope, the cells and Matrigel were mixed at 1:1, and finally the E7 cells were injected into the animal.

[0157] 5-2. Cell line transplantation

[0158] After the adaptation period in the breeding environment, one day before the cell line transplantation, the back of the mouse was shaved with an electric razor. When the cell line was prepared, it was resuspended for homogenization and immediately administered to the animal. When the cell line was transplanted, the left and right sides of the animal were disinfected with 70% alcohol, and the cell line was injected subcutaneously at E7 cells / 0.1mL / site using a syringe equipped with a 26-gauge needle, and the injection was performed on both sides of the back of the mouse.

[0159] 5-3. Animal Experiment Design

[0160] As shown in Table 4 ( Figure 5 ) to conduct the experiment.

[0161] [Table 4]

[0162]

[0163] Specifically, healthy animals were selected during the mouse adaptation period, cell line inoculation was performed, and then when the tumor size at the cell line transplantation site reached approximately 50 to 100 mm 3 At this time, the mice were sorted and grouped. At this time, the mice were separated so that the tumor size of each group was as evenly distributed as possible. The tumor size reached an average of 100mm 3The time was designated as the start day of administration (Day 0). After that, oncolytic virus (test substance) or PBS (control group) was injected into the tumor four times at intervals of three days. For intratumoral administration, animals were fixed by dorsal skin fixation and intratumoral administration was performed using an insulin syringe equipped with a No. 31 needle. During administration and observation, the type, date of occurrence and severity of general symptoms including death were observed once a day, and the condition of each subject was recorded. Subjects with worsening general symptoms were isolated, and dying and dead animals were treated as planned autopsy animals. Body weight and temperature were measured on the day of grouping, and then three times a week for four weeks. After the start of test substance administration (Day 0), tumor size measurements were performed three times a week for a total of 4 weeks. The long and short axes of the tumor were measured using a caliper, and the tumor size was calculated using the following formula.

[0164] Tumor size = ab2 / 2 (a: long axis length, b: short axis length)

[0165] On the day of autopsy (day 29), all surviving animals were anesthetized, and then once anesthesia was confirmed, the animals were opened and blood was collected from the posterior vena cava with a syringe. Subsequently, the abdominal aorta and posterior aorta were removed to cause bleeding / death. Thereafter, tumors were collected from all subjects, and spleens, brains, ovaries, and livers were collected from the three subjects in each group with the smallest tumor size compared to the control group. The harvested tumors and organs were gently washed with PBS, and the water therein was carefully removed with gauze, and then the tumors and organs were photographed and weighed. The autopsy date was adjusted appropriately according to the size of the tumor. One subject (three subjects in total) was selected from each control group (G2, G4, and G6), and two subjects (six subjects in total) with the smallest tumor size compared to the control group were selected from each treatment group (G1, G3, and G5), and tumors, ovaries, spleens, brains, and livers were removed from them, and then the right tumor tissue and half of the tissue were fixed in 10% neutral buffered formalin.

[0166] The left tumor and 100 mg of the remaining tissue were homogenized in 0.5 mL PBS (4 ° C to minimize heat generation) (for tissues less than 100 mg, PBS equivalent to 5 times the weight of the tissue was used for homogenization). Thereafter, the homogenized tissue was centrifuged at 4 ° C and 13000 rpm for 10 minutes, and the supernatant was collected and placed in two test tubes labeled with 500 μL animal numbers and stored in an ultra-low temperature freezer (about -70 ° C). The stored homogenized tissue was used to extract viral RNA and measure the number of viruses.

[0167] Example 1. Testing of the infectivity of chimeric Zika virus (cZIKV) on normal cells (5 types) and various cancers (5 types)

[0168] Primary cells from organs known to be susceptible to chimeric Zika virus infection (brain, testis, skin, breast, and lung) were purchased and examined for infectivity. To confirm the infectivity assessment, immunofluorescence assays were performed after virus infection. As a result, chimeric Zika virus showed no infectivity in five normal cell types ( Figure 6 ).

[0169] Example 2. Detection of infectivity according to breast cancer subtype

[0170] Breast cancer subtypes are divided into four types based on the presence or absence of estrogen receptors, progesterone receptors, and HER2 receptors. The four subtypes are Luminal A, Luminal B, HER2, and triple negative. The triple negative (basal-like) subtype lacks all three receptors and is known to have the worst prognosis.

[0171] Cell lines corresponding to LuminalA, Luminal B, HER2, and triple negative were prepared according to the four subtypes, and the infectivity of oncolytic viruses to these cell lines was examined. Immunofluorescence assay (IFA) was performed, and the results showed that the oncolytic virus infected all triple negative subtypes (MDAMB231 and BT20), Luminal a subtype (MCF7), and Luminal B subtype (BT474) ( Figure 7 ).

[0172] Example 3. Adaptability of improving oncolytic virus infectivity and characteristics of oncolytic viruses

[0173] In order to obtain a kind of widely effective virus that can infect a variety of cancer cells, oncolytic virus is repeatedly passed down in cancer cells.First, wherein oncolytic virus shows strong infectivity and CPE is passed down for 19 generations or more of MDAMB231 cell line (MDA19).Afterwards, MDA19 virus is further passed down in cell mixture.The cell mixture is composed of oncolytic virus showing low infectivity or no infectivity and CPE cell line, and is passed down for 61 times (Bx61) or more times.

[0174] When the chimeric Zika virus was passaged in breast cancer cells, it was found that the initial chimeric Zika strain (cZIKV) had a weak level of infectivity to cancer cells, but as the number of passages increased, the infectivity to breast cancer cells (such as BT20 and BT474) increased. Specifically, Fig. 20 As shown in Figure 2, when the infectivity was examined by passaging, it was found that the infectivity of the virus to specific cells (BT20 and BT474) increased with the increase in the number of passages ( Fig. 20 ).

[0175] 3-1. Comparison of infectivity

[0176] To characterize the passaged oncolytic virus (adapted virus Bx61), an experiment was performed to compare the infectivity and CPE of the oncolytic virus with the parental virus (original virus cZIKV). To compare the infectivity, the expression level of AXL, the receptor used by Zika virus for infection, was also studied and the correlation is illustrated in the figure.

[0177] Results, such as Figure 8 As shown, the infectivity of MDAMB231 and MCF7 cell lines is very high, the infectivity of BT20 and BT474 is low, and the infectivity of the remaining cell lines is almost undetectable. There is no correlation between the expression rate of AXL and infectivity.

[0178] 3-2. ToxGlo assay for cytotoxicity

[0179] To examine whether oncolytic viruses infect cell lines and exhibit cytotoxicity, ToxGlo assays were performed on six types of cell lines as shown.

[0180] Vero cells, known as an existing production cell line, two cell lines that are susceptible to infection (MCF7 and MDAMB231), a cell line that is barely infected (BT20), an uninfected cell line (Hs578T) and normal cells (HMEc) were tested.

[0181] Results, such as Fig. 9 As shown, when Vero cells and MCF7 and MDAMB231 cells were infected with viruses, CPE was strongly expressed in these cells. In these cells, it was found that when cells were infected with adapted viruses, CPE was about 10% higher than that of the parental virus. On the other hand, in cell lines and normal cells that were barely or not infected with viruses, there was no significant change in cell line survival due to viral infection. In normal cells, cell proliferation actually increased.

[0182] 3-3. Full-length sequence analysis of MDA19 and Bx61 viruses after passage

[0183] In order to check whether there are any mutations in the passaged MDA19 and Bx61 viruses, full-length sequence analysis was performed. The results are shown in Table 5 below. In the case of MDA19, a total of 27 amino acid mutations occurred, of which the total number of amino acid mutations in the prM-E protein as the infection site was 8. In the case of Bx61, a total of 20 amino acid mutations occurred, of which the total number of amino acid mutations in the prM-E protein was 7.

[0184] [Table 5]

[0185]

[0186]

[0187] Example 4. Animal efficacy experiments on three cells with different virus sensitivity

[0188] The following animal efficacy experiments were conducted using the oncolytic virus Bx61.

[0189] 4-1. Xenograft experiments using the MDAMB231 cell line, which is highly sensitive to oncolytic viruses

[0190] To evaluate the anticancer effect of oncolytic viruses, the MDAMB231 cell line was transplanted into Balb / c nude mice lacking T cells. After constructing a xenograft model by subcutaneously transplanting cells on both sides, the oncolytic virus was injected into the tumor, and then the mice were observed for 28 days. As a result of observing general symptoms, no subjects died during the experiment, and no abnormal symptoms caused by taking oncolytic viruses were observed. During the trial, there was no significant change in body weight or body temperature in the oncolytic virus administration group or the control group ( Fig.13 ).

[0191] As a result of measuring tumor size, the tumor size of the treatment group (G1) was significantly smaller than that of the control group (G2) from the 7th day to the 28th day after the start of oncolytic virus administration. It was found that the tumor size decreased from the second administration of oncolytic virus, and the tumor gradually disappeared ( Fig.11 ).

[0192] As a result of tumor histopathological examination, central necrosis was observed in the tumors of the oncolytic virus-treated group, and inflammatory cell infiltration and fibrosis were also observed. On the other hand, in the case of tumor tissues of the control group, tumor cells remained at the edge and formed tumors ( Fig.12 ).

[0193] 4-2. Xenograft experiments using BT474 cell line with low sensitivity to oncolytic viruses

[0194] To evaluate the anticancer effects of oncolytic viruses, the BT474 cell line was transplanted into Balb / c nude mice lacking T cells. After constructing a xenograft model by subcutaneously transplanting cells on both sides, the oncolytic virus was injected into the tumor, and then the mice were observed for 28 days. As a result of observing general symptoms, no subjects died during the experiment, and no abnormal symptoms caused by taking oncolytic viruses were observed. During the trial, there was no significant change in body weight or body temperature in the oncolytic virus-administered group or the control group, and the weight of the treated group was actually about 2g higher on average ( Fig.13 ).

[0195] As a result of measuring tumor size, the tumor size of the treatment group (G3) was significantly smaller than that of the control group (G4) from the 4th day to the 28th day after the start of oncolytic virus administration. It was found that the tumor size began to decrease from the second administration of oncolytic virus, and compared with the control group, as the tumor size decreased, the difference in tumor size between the groups gradually increased ( Fig.14 ).

[0196] As a result of tumor histopathological examination, central necrosis was observed in the tumors of the oncolytic virus-treated group, and inflammatory cell infiltration and fibrosis were also observed. On the other hand, in the case of tumor tissues of the control group, tumor cells remained at the edge and formed tumors ( Fig.15 ).

[0197] 4-3. Xenograft experiments using Hs578T (a cell line without CPE to oncolytic viruses)

[0198] To evaluate the anticancer effect of oncolytic viruses, the Hs578T cell line was transplanted into Balb / c nude mice lacking T cells. After constructing a xenograft model by subcutaneously transplanting cells on both sides, the oncolytic virus was injected into the tumor, and then the mice were observed for 28 days. As a result of observing general symptoms, no subjects died during the experiment, and no abnormal symptoms caused by taking oncolytic viruses were observed. During the trial, there was no significant change in body weight or body temperature in the oncolytic virus administration group or the control group ( Fig.16 ).

[0199] As a result of measuring tumor size, the tumor size of the treatment group (G5) was significantly smaller than that of the control group (G6) from the 6th day to the 32nd day after the start of oncolytic virus administration. It was found that compared with the control group, starting from the second administration of oncolytic virus, the growth rate of tumor size was significantly reduced, and the difference in tumor size between the groups increased ( Fig.17 ).

[0200] As a result of tumor histopathological examination, a small number of tumor cells were observed in the tumors of the oncolytic virus-treated group, and fibrosis was also observed. This indicates that after the administration of oncolytic virus, part of the tumor disappeared or was replaced by fibrous tissue, indicating that oncolytic virus affected the inhibition of tumor cell proliferation ( Fig.18 ).

[0201] Example 5. Verification of the safety of oncolytic virus therapy by histopathological results

[0202] Histopathological examination of major organs was performed. H&E staining was performed to select major organs that were sensitive to oncolytic viruses and to determine whether the selected major organs had lesions. As a result, no abnormalities were observed in the brain and ovaries of all groups ( Fig.19). Extramedullary hematopoiesis was observed in all spleen slides and some liver slides, but these lesions were common background lesions in mice. Therefore, the safety of the oncolytic virus was confirmed because no inflammatory response and abnormal pathology were observed in major organs.

[0203] According to the above description, those skilled in the art to which the present invention belongs will understand that the present invention can be implemented in other specific forms without changing its technical ideas or basic features. In this regard, it should be understood that the above embodiments are illustrative in all aspects, but not restrictive. The scope of the present invention is defined by the appended claims, rather than by the preceding description thereof, and all changes or modifications derived from the meaning and scope of the claims and their equivalent concepts should be interpreted as being included in the scope of protection of the present invention.

Claims

1. A pharmaceutical composition for preventing or treating breast cancer, comprising a chimeric Zika virus as an active ingredient, wherein the chimeric Zika virus is formed by introducing a Zika virus antigen gene into a gene of an attenuated yellow fever virus containing SEQ ID NO:

1.

2. The composition of claim 1, wherein the Zika virus antigen genes include i) a gene encoding a pre-membrane protein (prM) and ii) a gene encoding an envelope protein (E).

3. A composition according to claim 1, wherein the Zika virus antigen genes include i) a gene encoding a pre-membrane (prM) protein of SEQ ID NO: 3 or a gene having 90% or higher identity with SEQ ID NO: 3; and ii) a gene encoding an envelope (E) protein of SEQ ID NO: 4 or a gene having 90% or higher identity with SEQ ID NO:

4.

4. The composition according to claim 1, wherein the chimeric Zika virus has the ability to specifically inhibit breast cancer cells through serial passaging.

5. The composition of claim 4, wherein the chimeric Zika virus is obtained by at least 19 passages in MDAMB-231 cells and at least 61 consecutive passages in a mixed cell consisting of HCC1954, BT474, HCC70, BT20, BT549 and HS578T.

6. A composition according to claim 5, wherein in the chimeric Zika virus, based on the sequence shown in SEQ ID NO:7, the amino acid corresponding to any of positions 30, 60, 122, 227, 427, 497, 608, 657, 690, 1464, 1843, 2052, 2108, 2139, 2150, 2350, 2404, 2416, 2461 and 3077 is replaced by another amino acid.

7. The composition of claim 5, wherein the chimeric Zika virus comprises a polypeptide described by the amino acid sequence of SEQ ID NO: 8, or a polypeptide having 90% or greater identity with SEQ ID NO:

8.

8. The composition of claim 5, wherein the chimeric Zika virus comprises a gene encoding a yellow fever virus capsid protein and a gene encoding a non-structural protein.

9. The composition of claim 8, wherein the capsid (C) protein comprises a polypeptide having an amino acid sequence of SEQ ID NO: 2, or a polypeptide having 90% or higher identity with SEQ ID NO:

2.

10. The composition according to claim 9, wherein in the chimeric Zika virus, based on the sequence shown in SEQ ID NO: 2, the amino acids corresponding to positions 30 and 60 are replaced by other amino acids.

11. The composition of claim 5, wherein the pre-membrane (prM) protein of the chimeric Zika virus comprises a polypeptide described by the amino acid sequence of SEQ ID NO: 3, or a polypeptide having 90% or greater identity with SEQ ID NO:

3.

12. The composition according to claim 11, wherein in the chimeric Zika virus, based on the sequence shown in SEQ ID NO: 3, the amino acids corresponding to positions 122 and 227 are replaced by other amino acids.

13. The composition of claim 5, wherein the envelope (E) protein of the chimeric Zika virus comprises a polypeptide described by the amino acid sequence of SEQ ID NO: 4, or a polypeptide having 90% or greater identity with SEQ ID NO:

4.

14. The composition according to claim 13, wherein in the chimeric Zika virus, based on the sequence shown in SEQ ID NO: 4, the amino acid corresponding to any one of positions 427, 497, 608, 657 and 690 is replaced by another amino acid.

15. The composition of claim 8, wherein the non-structural protein comprises a polypeptide having an amino acid sequence of SEQ ID NO:5, or a polypeptide having 90% or higher identity with SEQ ID NO:

5.

16. The composition of claim 15, wherein in the chimeric Zika virus, based on the sequence shown in SEQ ID NO:5, the amino acids corresponding to positions 1464, 1843, 2052, 2108, 2139, 2150, 2350, 2404, 2416, 2461 and 3077 are replaced by other amino acids.

17. The composition of claim 1, wherein the composition is administered intratumorally, intramuscularly, intraperitoneally or intravenously.

18. A method for preparing a composition for preventing or treating breast cancer, the method comprising: (a) serially passage the chimeric Zika virus in MDAMB-231 cells for at least 19 times to obtain a virus culture containing cell-adapted virus; (b) subculturing the culture containing the cell-adapted virus obtained in step (a) in a mixed cell consisting of HCC1954, BT474, HCC70, BT20, BT549 and HS578T for at least 61 consecutive times to obtain a virus culture containing the cell-adapted virus; and (c) selecting and isolating chimeric Zika viruses from the virus culture containing the cell-adapted viruses obtained in step (b) by evaluating the ability of the chimeric Zika viruses to specifically inhibit breast cancer cells.

19. A chimeric nucleic acid molecule consisting of: i) a gene encoding a signal peptide upstream of a gene encoding a pre-membrane (prM) protein comprising a polypeptide of SEQ ID NO:6 or a polypeptide having 90% or greater identity to SEQ ID NO:6; ii) a gene encoding a pre-membrane (prM) protein comprising a polypeptide of SEQ ID NO:3 or a polypeptide having 90% or greater identity to SEQ ID NO:3; or iii) a gene encoding an envelope (E) protein consisting of a polypeptide of SEQ ID NO:4 of Zika virus or a polypeptide having 90% or greater identity to SEQ ID NO:4, inserted into the following regions to replace the Yellow Fever Virus 17D vaccine strain gene: i) a region of a gene encoding a signal peptide, said signal peptide being located upstream of a gene encoding a pre-membrane (prM) protein; ii) a region of a gene encoding a pre-membrane (prM) protein; and iii) a region of a gene encoding an envelope (E) protein of a nucleic acid molecule of a yellow fever virus 17D vaccine strain, wherein part or all of the nucleic acid constituting Zika virus gene i), ii) or iii) is replaced, deleted or inserted.

20. A chimeric Zika virus mutant strain obtained by serially subculturing the chimeric nucleic acid molecule of claim 19 in breast cancer cells so as to have increased cancer cell adaptability.

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