Pharmaceutical composition for treating tumor or suppressing metastasis

Abstract

To provide a method for effective treatment of tumor or suppression of its metastasis.SOLUTION: The above problem can be solved by a pharmaceutical composition for treating tumor or suppressing its metastasis, comprising, as an active ingredient, a nucleic acid encoding β1,3-galactosyltransferase V or a vector incorporating the nucleic acid encoding β1,3-galactosyltransferase V.SELECTED DRAWING: Figure 5

Description

Technical Field

[0001] The present invention relates to a pharmaceutical composition for treating tumors or suppressing metastasis. The present invention also relates to a nucleic acid encoding β1,3-galactosyltransferase V (hereinafter sometimes referred to as B3GALT5) or a vector containing the same, a method for determining the effectiveness of a pharmaceutical composition for treating tumors or suppressing metastasis, a method for reducing the proliferation ability or metastasis ability of tumor cells, a method for treating tumors or suppressing metastasis, a nucleic acid or vector for use in a method for treating tumors or suppressing metastasis in a subject, and the use of a nucleic acid or vector in the manufacture of a medicament for treating tumors or suppressing metastasis.

Background Art

[0002] In Japan, the annual incidence of gastric cancer is approximately 130,000, and the annual number of deaths is approximately 50,000, making it the third most frequent type of cancer. In particular, when metastasis occurs and resection is not possible, it is a cancer with a very poor prognosis. There is a need for an effective therapeutic agent for tumors that cause metastasis, such as gastric cancer.

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Non-Patent Documents

[0004]

Non-Patent Document 1

Non-Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0005] An object of the present invention is to provide an effective treatment method for tumors that cause metastasis or a method for suppressing metastasis.

Means for Solving the Problems

[0006] B3GALT5 is a kind of glycosyltransferase. B3GALT5 is an important enzyme involved in the synthesis of type I sugar chains that constitute the backbone of Lewis blood group antigens and cancer-related sugar chain antigens having a Galβ1-3GlcNAc structure (Patent Document 1). In recent years, it has been suggested that high expression of B3GALT5 may be an independent predictor of recurrence after surgery and survival after surgery in hepatocellular carcinoma (Non-Patent Document 1). In addition, it has been suggested that high expression of B3GALT5 may deteriorate the clinical outcome of breast cancer and contribute to tumor progression and metastasis (Non-Patent Document 2). The present inventor has intensively studied an effective therapeutic agent for tumors or a method for suppressing metastasis. As a result, surprisingly, it has been found that a nucleic acid encoding B3GALT5 can efficiently treat tumors or efficiently suppress tumor metastasis. The present invention is based on such findings. Therefore, the present invention relates to the following. [1] A pharmaceutical composition for treating tumors or suppressing metastasis, comprising as an active ingredient a nucleic acid encoding β1,3-galactosyltransferase V or a vector incorporated with a nucleic acid encoding β1,3-galactosyltransferase V. [2] The pharmaceutical composition according to [1], wherein the tumor comprises tumor cells in which the expression level of β1,3-galactosyltransferase V is decreased as compared with normal cells. [3] The pharmaceutical composition for treating or suppressing metastasis of a tumor according to [1] or [2], wherein the vector is a viral vector. [4] The pharmaceutical composition according to any one of [1] to [3], which is for suppressing tumor growth or metastasis. [5] The pharmaceutical composition according to [4], which is for suppressing the growth of gastric cancer or suppressing metastasis from gastric cancer. [6] The pharmaceutical composition according to any one of [1] to [5], which is for suppressing peritoneal seeding. [7] The pharmaceutical composition according to any one of [1] to [6], wherein the nucleic acid encoding β1,3-galactosyltransferase V is a nucleic acid having any one of the base sequences selected from the group consisting of the following (a) to (d): (a) The base sequence represented by SEQ ID NO: 1, (b) A base sequence in which 1 to several bases are deleted, substituted or added in the base sequence represented by SEQ ID NO: 1, (c) A base sequence having 90% or more identity with the base sequence represented by SEQ ID NO: 1, and (d) A base sequence capable of hybridizing under stringent conditions with a nucleic acid consisting of a base sequence complementary to the nucleic acid consisting of the base sequence represented by SEQ ID NO: 1, Here, when the nucleic acid encoding β1,3-galactosyltransferase V is a nucleic acid having any one of the base sequences selected from the group consisting of (b) to (d), the protein encoded by the nucleic acid having any one of the base sequences selected from the group consisting of (b) to (d) has β1,3-galactosyltransferase V activity. [8] Measuring the expression level of β1,3-galactosyltransferase V in tumor cells collected from a subject Comparing the expression level with a reference value A method for determining the efficacy of a pharmaceutical composition for treating tumors or suppressing metastasis, comprising as an active ingredient a nucleic acid encoding β1,3-galactosyltransferase V or a vector incorporated with a nucleic acid encoding β1,3-galactosyltransferase V. [9] A nucleic acid encoding β1,3-galactosyltransferase V or a vector containing a nucleic acid encoding β1,3-galactosyltransferase V for use in the production of a pharmaceutical composition for treating tumors or suppressing metastasis.

[10] A method for reducing the proliferation ability or metastasis ability of tumor cells, comprising introducing a nucleic acid encoding β1,3-galactosyltransferase V into tumor cells A method comprising the above.

[11] A method for treating tumors or suppressing metastasis, comprising administering to a subject in need of administration a vector containing an effective amount of a nucleic acid encoding β1,3-galactosyltransferase V or an effective amount of a nucleic acid encoding β1,3-galactosyltransferase V.

[12] A nucleic acid encoding β1,3-galactosyltransferase V or a vector containing a nucleic acid encoding β1,3-galactosyltransferase V for use in a method for treating tumors or suppressing metastasis in a subject.

[13] Use of a nucleic acid encoding β1,3-galactosyltransferase V or a vector containing a nucleic acid encoding β1,3-galactosyltransferase V in the production of a medicament for treating tumors or suppressing metastasis. [Advantages of the Invention]

[0007] According to the present invention, tumors can be treated efficiently. According to the present invention, tumor metastasis can be suppressed efficiently. [Brief Description of the Drawings]

[0008]

Figure 1

Figure 2

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Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Modes for Carrying Out the Invention

[0009] The present invention will be described in detail below. In this specification, unless otherwise specified, for numerical values A and B, the notation "A to B" shall be equivalent to "A or more and B or less". That is, the notation "A to B" includes the numerical values A and B at both ends. When a unit is attached only to the numerical value B in such notation, the unit shall also be applicable to the numerical value A. In this specification, a pharmaceutical composition for treating or suppressing metastasis of a tumor, which contains a nucleic acid encoding B3GALT5 or a vector incorporated with a nucleic acid encoding B3GALT5 as an active ingredient, may be referred to as the pharmaceutical composition of the present invention. In this specification, the matters described about the pharmaceutical composition of the present invention, for example, the definition of terms, embodiments, etc., are also applicable to other aspects, that is, a nucleic acid encoding β1,3-galactosyltransferase V or a vector containing the same, a method for determining the effectiveness of a pharmaceutical composition for treating or suppressing metastasis of a tumor, a method for reducing the proliferation ability or metastasis ability of tumor cells, a method for treating or suppressing metastasis of a tumor, a nucleic acid or vector for use in a method for treating or suppressing metastasis of a tumor in a subject, and the use of a nucleic acid or vector in the manufacture of a medicament for treating or suppressing metastasis of a tumor. Also, the matters described about aspects other than the pharmaceutical composition of the present invention (for example, a method for determining the effectiveness of the pharmaceutical composition for treating or suppressing metastasis of a tumor of the present invention) are also applicable to other aspects including the pharmaceutical composition of the present invention.

[0010] 《Pharmaceutical Composition》 (Nucleic acid encoding β1,3-galactosyltransferase V) B3GALT5 is a kind of type I chain-like carbohydrate synthase in mammals. B3GALT5 is an enzyme having the highest synthetic activity of the type 1 carbohydrate antigen (Galb1-3GlcNAcb1-3-R) among the β1,3-galactosyltransferase family. B3GALT5 is an enzyme having an activity of transferring galactose to the core 3 type of O-linked carbohydrate chain. Also, B3GALT5 is an enzyme having an activity of synthesizing SSEA-3 (Galβ1-3GalNAcβ1-3Galβ1-4Galβ1-4Glcβ1-1Cer). The nucleic acid encoding β1,3-galactosyltransferase V can be an isolated nucleic acid.

[0011] B3GALT5 does not necessarily have to be represented by the full-length amino acid sequence of B3GALT5 (SEQ ID NO: 1) as long as its activity is retained, and it may be represented by a part of the full-length amino acid sequence. SEQ ID NO: 1 is as follows.

[0012] [Table 1]

[0013] In the pharmaceutical composition of the present invention, the nucleic acid encoding B3GALT5 may be derived from any species, but is preferably derived from a human. In the pharmaceutical composition of the present invention, the nucleic acid encoding B3GALT5 can be a nucleic acid having any one of the base sequences selected from the group consisting of the following (a) to (d): (a) The base sequence represented by SEQ ID NO: 2, (b) A base sequence in which one to several bases are deleted, substituted or added in the base sequence represented by SEQ ID NO: 2, (c) A base sequence having an identity of 80% or more, preferably 85% or more, more preferably 90% or more, still more preferably 95% or more, and most preferably 99% or more with the base sequence represented by SEQ ID NO: 2, and (d) A base sequence capable of hybridizing under stringent conditions with a nucleic acid consisting of a base sequence complementary to the nucleic acid consisting of the base sequence represented by SEQ ID NO: 2. Here, when the nucleic acid encoding B3GALT5 is a nucleic acid having any one of the base sequences selected from the group consisting of the above (b) to (d), the protein encoded by the nucleic acid having any one of the base sequences selected from the group consisting of the above (b) to (d) shall have β1,3-galactosyltransferase V activity. Further, when the nucleic acid encoding B3GALT5 is a nucleic acid having any one of the base sequences selected from the group consisting of the above (b) to (d), preferably, when the nucleic acid is introduced into gastric cancer cells using a viral vector and expressed, the proliferation ability of the gastric cancer cells into which the nucleic acid is introduced, more preferably, the proliferation ability and the metastasis ability are significantly decreased as compared with the gastric cancer cells into which the nucleic acid is not introduced. Whether the proliferation ability of the gastric cancer cells into which the nucleic acid is introduced is significantly decreased can be verified by the method described in Example 1-3, "Proliferation ability of B3GALT5 gene-introduced cell lines". "Significantly" decreasing the proliferation ability of the gastric cancer cells into which the nucleic acid is introduced specifically means that the cell doubling time of the gastric cancer cells into which the nucleic acid is introduced is, for example, 1.10 times or more, preferably 1.15 times or more, more preferably 1.20 times or more, and still more preferably 1.22 times or more as compared with the cell doubling time of the gastric cancer cells into which the nucleic acid is not introduced. Whether the metastasis ability of the gastric cancer cells into which the nucleic acid is introduced is significantly decreased can be verified by the method described in Example 2. "Significantly" decreasing the metastasis ability of the gastric cancer cells into which the nucleic acid is introduced specifically means that at least one of the average tumor weight of the cancer cells metastasized by peritoneal seeding, the number of tumors with a size of 1 to 2 mm, and the number of tumors with a size of 5 mm or more of the gastric cancer cells into which the nucleic acid is introduced is significantly smaller than the weight of the cancer cells metastasized by peritoneal seeding of the gastric cancer cells into which the nucleic acid is not introduced. SEQ ID NO: 2 is as follows.

[0014]

Table 2

[0015] Here, the number of bases that may be deleted, substituted, or added is preferably 1 to 30, more preferably 1 to 15, particularly preferably 1 to 10, and most preferably 1 to 5.

[0016] In the pharmaceutical composition of the present invention, the nucleic acid encoding B3GALT5 may be a nucleic acid consisting only of the nucleotide sequence represented by SEQ ID NO: 2. Also, in the present invention, the nucleic acid encoding B3GALT5 may be a homolog or ortholog of the nucleic acid consisting only of the nucleotide sequence represented by SEQ ID NO: 2.

[0017] As used herein, "stringent conditions" include, for example, the methods described in Molecular Cloning - A LABORATORY MANUAL THIRD EDITION (Sambrook et al., Cold Spring Harbor Laboratory Press). For example, hybridization can be carried out by incubating for several hours to overnight at 55°C or higher and 70°C or lower in a hybridization buffer consisting of 5×SSC (composition of 20×SSC: 3M sodium chloride, 0.3M citric acid solution, pH 7.0), 0.1% by weight N-lauroyl sarcosine, 0.02% by weight SDS, 2% by weight blocking reagent for nucleic acid hybridization, and 50% formamide. The washing buffer used for washing after incubation is preferably a 1×SSC solution containing 0.1% by weight SDS, more preferably a 0.1×SSC solution containing 0.1% by weight SDS.

[0018] In the pharmaceutical composition of the present invention, the nucleic acid encoding B3GALT5 can be, for example, a nucleic acid encoding a protein consisting of a sequence containing any one amino acid sequence selected from the group consisting of the following (e) to (g): (e) the amino acid sequence represented by SEQ ID NO: 1 (f) an amino acid sequence having 80% or more, preferably 85% or more, more preferably 90% or more, still more preferably 95% or more, and most preferably 99% or more identity to the amino acid sequence represented by SEQ ID NO: 1, (g) an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 1. Here, when the nucleic acid encoding B3GALT5 is a nucleic acid encoding a protein consisting of a sequence containing the amino acid sequence of (f) or (g), the protein shall have β1,3-galactosyltransferase V activity. Also, when the nucleic acid encoding B3GALT5 is a nucleic acid encoding a protein consisting of a sequence containing the amino acid sequence of (f) or (g), preferably, when the nucleic acid is introduced into gastric cancer cells using a viral vector and expressed, compared with gastric cancer cells not introduced with the nucleic acid, the proliferation ability, more preferably the proliferation ability and metastasis ability, of the gastric cancer cells introduced with the nucleic acid are significantly reduced.

[0019] Here, as the number of amino acids that may be deleted, substituted or added, 1 to 15 are preferable, of which 1 to 10 are more preferable, and 1 to 5 are particularly preferable.

[0020] In the pharmaceutical composition of the present invention, the nucleic acid encoding B3GALT5 may be a nucleic acid encoding a protein consisting only of the amino acid sequence of (e) above.

[0021] In the pharmaceutical composition of the present invention, when the nucleic acid encoding B3GALT5 is a nucleic acid having any one base sequence selected from the group consisting of (b) to (d), or a nucleic acid encoding a protein consisting of a sequence containing the amino acid sequence of (f) or (g), the nucleic acid encoding B3GALT5 can further have any one or more of the following functions (i) and (ii). · When the nucleic acid encoding B3GALT5 is introduced into gastric cancer cells and expressed, compared with the gastric cancer cell NUGC4 not introduced with the nucleic acid, the gastric cancer cells introduced with the nucleic acid (i) It shows a decrease in the expression of pAKT protein (phosphorylated AKT protein). "Showing a decrease in expression" means that the expression level becomes, for example, 0.75 times or less, preferably 0.7 times or less, more preferably 0.65 times or less, and even more preferably 0.6 times or less. (ii) It shows a decrease in the expression of galectin-4 protein. In the pharmaceutical composition of the present invention, it is preferable that the gastric cancer cells into which the nucleic acid has been introduced show both (i) and (ii).

[0022] As long as the nucleic acid encoding B3GALT5 can produce B3GALT5 in the target cells, either DNA or RNA can be selected in consideration of the method of introducing it into the subject. The DNA or RNA encoding B3GALT5 may be in any form as long as it can produce B3GALT5 in the target cells. For example, it can be single-stranded or double-stranded and may have modifications. For example, when the nucleic acid is RNA, pseudouridine may be used. The DNA may be cDNA, and the RNA may be RNA excluding the region corresponding to the intron. The nucleic acid encoding B3GALT5 can be prepared based on conventionally known methods.

[0023] In the pharmaceutical composition of the present invention, the nucleic acid encoding B3GALT5 can be introduced by any known method in order to enable its expression in the subject. Such methods include, for example, the lipofection method, the electroporation method, the microinjection method, the calcium phosphate method, the method using lipid nanoparticles, and the method using a vector. Among such methods, the lipofection method, the method using lipid nanoparticles, and the method using a vector are preferable, and the method using a vector is more preferable.

[0024] (Lipofection method) The lipofection method is a method in which nucleic acid is encapsulated inside vesicles of liposomes, and then the nucleic acid-containing liposomes are brought into contact with the cell membrane to pass through the cell membrane. Liposomes preferably have a positive charge.

[0025] (Lipid nanoparticles) Lipid nanoparticles are particles mainly composed of lipids and encapsulating nucleic acid for introducing the nucleic acid into target cells. Lipid nanoparticles are also called LNPs. The type of lipid is not particularly limited, and a plurality of lipids may be included as long as the effects of the present invention can be obtained.

[0026] (Vector) In the pharmaceutical composition of the present invention, the nucleic acid encoding B3GALT5 is preferably incorporated into an arbitrary vector. The vector is not particularly limited as long as it is a vector that can express a protein in cultured cells, particularly in cultured cells derived from the subject to be introduced (mainly mammals including humans), or in cells in an individual of the subject to be introduced (mainly mammals including humans), and may be a plasmid or a viral vector. It is more preferable to use a viral vector as the vector. As used herein, a "viral vector" is a vector having a nucleic acid derived from a virus, and by incorporating a specific gene into this nucleic acid, the gene can be expressed after being introduced into a cell.

[0027] Examples of plasmid vectors include, but are not limited to, pVL1392, pBK-CMV, pZeoSV, pcDNA3, pcDNA3.1, pVC0396, and pRP(Exp)-CMV, etc.

[0028] The viral vector may be either a DNA viral vector or an RNA viral vector. Specific viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, herpes viral vectors, retroviral vectors, poxviral vectors, baculoviral vectors, rabies viral vectors, bovine papillomavirus vectors, lentiviral vectors, vaccinia viral vectors, cytomegalovirus vectors, polyomavirus vectors, or Sendai virus vectors. In addition to the nucleic acid encoding B3GALT5, the viral vector may also include a drug resistance gene for selection (e.g., Puromycin resistance gene), a gene encoding a fluorescent protein for selection and visualization (e.g., a gene encoding GFP), a promoter for controlling the transcription of the B3GALT5 gene, a terminator sequence for terminating the transcription of the B3GALT5 gene, and a Kozak consensus sequence, etc. Examples of the promoter include EF1α promoter, SRα promoter, SV40 promoter, LTR promoter, CMV (cytomegalovirus) promoter, and HSV-tk promoter.

[0029] In the viral vector that can be included in the pharmaceutical composition of the present invention, a promoter sequence for controlling the transcription of the B3GALT5 gene is linked upstream (5' side) of the nucleotide sequence encoding B3GALT5, and a terminator sequence for terminating the transcription of the B.GALT5 gene is linked downstream (3' side) of the nucleotide sequence encoding B3GALT5, which is preferable. Any known method can be adopted for introducing the nucleic acid encoding B3GALT5 into a vector, preferably a viral vector.

[0030] (tumor) The pharmaceutical composition of the present invention is used for the treatment of tumors. The site of tumor occurrence is not particularly limited. The pharmaceutical composition of the present invention is used for suppressing tumor metastasis. "Metastasis" means that a primary tumor causes a tumor to occur at a location different from the location where the primary tumor occurred. When the pharmaceutical composition of the present invention is used for suppressing tumor metastasis, it may be used for suppressing hematogenous metastasis, lymphatic metastasis, invasion, or disseminated metastasis, but can be particularly used for suppressing peritoneal seeding of tumors.

[0031] The tumors that are the target diseases of the pharmaceutical composition of the present invention can specifically be leukemias such as acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), and chronic lymphocytic leukemia (CLL); lymphomas such as Hodgkin lymphoma, non-Hodgkin lymphoma, and multiple myeloma; sarcomas; skin cancer; melanoma; bladder cancer; brain cancer; breast cancer; endometrial cancer; ovarian cancer; prostate cancer; lung cancer; colorectal cancer; cervical cancer; liver cancer; head and neck cancer; esophageal cancer; pancreatic cancer; kidney cancer; adrenal cancer; stomach cancer; testicular cancer; gallbladder cancer; biliary tract cancer; thyroid cancer; thymic cancer; bone tumors; brain tumors; gliomas; astrocytomas; or glioblastomas. In the present specification, the tumor is preferably stomach cancer. The tumors that are the target diseases of the pharmaceutical composition of the present invention may not include hepatocellular carcinoma and breast cancer.

[0032] The pharmaceutical composition of the present invention contains a nucleic acid encoding B3GALT5 as an active ingredient. Therefore, it is presumed to be particularly effective for treating or suppressing the metastasis of tumors with a reduced expression level of B3GALT5 compared to normal cells. As reported in Non-Patent Document 1 mentioned above, it has been suggested that high expression of B3GALT5 may be associated with a poor prognosis of hepatocellular carcinoma. As reported in Non-Patent Document 2 mentioned above, it has been suggested that high expression of B3GALT5 deteriorates the clinical outcome of breast cancer and may contribute to tumor progression and metastasis. In addition, in Non-Patent Document 2, when the breast cancer cell line AS-B634 in which B3GALT5 was silenced was injected into the mammary gland of NSG mice, the tumor growth rate was significantly suppressed compared to the control, the tumor size was decreased, and the number of metastatic lesions in the lungs of the mice was also suppressed. It is surprising that a nucleic acid encoding B3GALT5 can be used as an active ingredient of a pharmaceutical composition for treating or suppressing the metastasis of tumors.

[0033] As used herein, "treatment" means prevention, improvement of symptoms, suppression of the growth of tumors or tumor cells (including delay in the onset and progression of tumors), and improvement of prognosis in tumors, preferably metastatic tumors. As used herein, "treatment" can particularly mean suppression of the growth of tumors or tumor cells (preferably gastric cancer) (including delay in the onset and progression of tumors). As used herein, "suppression of metastasis" means prevention or improvement of tumor metastasis, preferably disseminated metastasis of tumors, more preferably peritoneal seeding of tumors, and even more preferably peritoneal seeding metastasis from gastric cancer (including delay in the occurrence of metastasis and reduction in the amount of tumors developing after metastasis), and improvement of prognosis after metastasis. As used herein, "suppression of metastasis" can particularly mean delay in the occurrence of metastasis (preferably peritoneal seeding), and reduction in the weight and number of tumors developing after metastasis. The pharmaceutical composition of the present invention can be used for treating tumors and suppressing metastasis, for suppressing the growth and metastasis of tumors, or for suppressing the growth of gastric cancer and peritoneal seeding from gastric cancer.

[0034] (Dosage form, administration method) There are no particular restrictions on the administration method of the pharmaceutical composition of the present invention. For example, depending on the dosage form of the pharmaceutical composition, the type of disease, and the condition of the patient, etc., either local administration or systemic administration can be selected. For example, in local administration, the pharmaceutical composition of the present invention can be administered by directly injecting it into a desired site (for example, a site where a tumor exists and / or a site where metastasis should be suppressed). When it is desired to suppress peritoneal seeding, the pharmaceutical composition of the present invention can be administered intraperitoneally. For the above injection, injections, specifically, conventionally known techniques such as subcutaneous injection, intramuscular injection, intradermal injection, intravenous injection, arterial injection, etc. can be appropriately used. Also, in systemic administration (for example, oral administration, intraperitoneal administration, administration into the blood, etc.), it is preferable to appropriately apply conventionally known drug delivery techniques so that the active ingredient of the medicine is stably and efficiently delivered to a desired site (for example, a tumor site).

[0035] There are no particular restrictions on the dosage form of the pharmaceutical composition of the present invention, and it can be appropriately selected according to the desired administration method. Examples of the dosage form of the pharmaceutical composition of the present invention include oral solid preparations (tablets, coated tablets, granules, powders, capsules, etc.), oral liquid preparations (oral solutions, syrups, elixirs, etc.), injections (solutions, suspensions, solid preparations for in-use dissolution, etc.), ointments, patches, gels, creams, external powders, sprays, inhalation powders, etc. Injections are preferred. In addition, as other components other than the active ingredient, desired pharmaceutical additives such as excipients, binders, disintegrants, lubricants, coloring agents, and flavoring and odor-masking agents can be included.

[0036] When the pharmaceutical composition of the present invention is an injection, the pharmaceutical composition of the present invention can be formulated according to a normal formulation recipe using a vehicle such as distilled water for injection. Examples of the aqueous solution for injection include isotonic solutions containing physiological saline, glucose, or other adjuvants (for example, D-sorbitol, D-mannose, D-mannitol, sodium chloride, etc.). Further, the above-mentioned aqueous solution for injection may be used in combination with a suitable solubilizing agent (for example, alcohol (specifically, ethanol), polyalcohol (for example, propylene glycol, polyethylene glycol, etc.)), or a nonionic surfactant (for example, polysorbate 80 (TM), HCO-50, etc.).

[0037] The dosage of the pharmaceutical composition of the present invention is not particularly limited and can be appropriately selected according to the age, weight, desired degree of effect, etc. of the patient who is the administration subject. The dosage of the pharmaceutical composition of the present invention can be an effective amount (therapeutically effective amount). The effective amount (therapeutically effective amount) can be, for example, 1 μg to 10 mg / kg body weight as the amount of the nucleic acid encoding B3GALT5 per day of administration to an adult. Also, the number of administrations of the pharmaceutical composition is not particularly limited and can be appropriately selected according to the age, weight, desired degree of effect, etc. of the patient who is the administration subject.

[0038] (Administration subject) The subject to which the pharmaceutical composition of the present invention is administered can be a subject who already has a tumor or a subject who may have a tumor in the future. Further, it can be administered to a subject who already has a tumor for the treatment of the tumor, or it can be administered to a subject who already has a tumor for suppressing metastasis to other sites of the tumor. The pharmaceutical composition of the present invention can also be administered to a subject who already has gastric cancer for suppressing peritoneal dissemination from gastric cancer. The subject to whom the pharmaceutical composition of the present invention is administered may be a subject in whom the expression level of B3GALT5 in the tumor cells has been measured, or may be a subject in whom the expression level of B3GALT5 in the tumor cells has not been measured. The subject to whom the pharmaceutical composition of the present invention is administered is preferably a subject having tumor cells in which the expression level of β1,3-galactosyltransferase V is decreased as compared with normal cells. The person who measures the expression level of B3GALT5 in normal cells and / or tumor cells and the person who administers the pharmaceutical composition of the present invention may be the same person or different persons.

[0039] 《Method for Determining the Efficacy of a Pharmaceutical Composition for Treating or Suppressing Tumor Metastasis》 The method for determining the efficacy of the pharmaceutical composition for treating or suppressing tumor metastasis of the present invention includes measuring the expression level of the B3GALT5 gene in tumor cells collected from a subject. The tumor cells collected from the subject may be tumor cells collected from experimental tumor cells or tumor cells collected from a living body. The tumor cells collected from the subject are preferably tumor cells collected from a human living body, and more preferably tumor cells collected from a human living body that may have gastric cancer. The tumor cells collected from the subject can be in vitro or in vivo tumor cells.

[0040] As the measurement of the expression level of the B3GALT5 gene, for example, methods well-known in the art such as immunoassays (e.g., immunostaining, ELISA), molecular or biochemical assays (quantitative PCR, colorimetric quantitative assay), and analytical methods (e.g., mass spectrometry) can be used.

[0041] The method for determining the effectiveness of the pharmaceutical composition for treating or suppressing metastasis of tumors of the present invention includes comparing the measured expression level of the B3GALT5 gene with a reference value. The reference value can be appropriately set in consideration of the type of tumor cells collected from the subject and the method for measuring the expression level. When the tumor cells collected from the subject are tumor cells collected from a human body, the reference value is preferably the expression level of the B3GALT5 gene in normal cells (for example, normal gastric cells if the cancer cells are gastric cancer cells) present in the tissue where the tumor cells were present, collected from the same subject.

[0042] The method for determining the effectiveness of the pharmaceutical composition for treating or suppressing metastasis of tumors of the present invention can include a step of determining that the pharmaceutical composition of the present invention is effective when the expression level of the B3GALT5 gene in the tumor cells collected from the subject is lower than the reference value. The method for determining the effectiveness of the pharmaceutical composition for treating or suppressing metastasis of tumors of the present invention can include a step of determining that the pharmaceutical composition of the present invention is not effective when the expression level of the B3GALT5 gene in the tumor cells collected from the subject has no difference from the reference value or is higher than the reference value.

[0043] The method for determining the effectiveness of the pharmaceutical composition for treating or suppressing metastasis of tumors of the present invention can further include administering the pharmaceutical composition of the present invention to a subject determined to be effective.

[0044] Also, the method for determining the effectiveness of the pharmaceutical composition for treating or suppressing metastasis of tumors of the present invention can include steps such as pretreating the tumor cells collected from the subject, as necessary. The method for determining the effectiveness of the pharmaceutical composition for treating or suppressing metastasis of tumors of the present invention can use tumor cells in vitro or in vivo.

[0045] 《Method for Reducing the Proliferation Ability or Metastasis Ability of Tumor Cells》 In the method for reducing the proliferation ability or metastasis ability of tumor cells of the present invention, the tumor cells may be in-vivo cells or in-vitro cells. The tumor cells may be cells of any of the above-mentioned tumors, but are preferably tumor cells with a reduced expression level of β1,3-galactosyltransferase V, and more preferably gastric cancer cells. Also, within the range that does not impair the effects of the present invention, it may include any other steps, for example, steps such as pretreatment of tumor cells.

[0046] (Action) The reason why the pharmaceutical composition of the present invention enables the treatment or suppression of metastasis of tumors has not been examined in detail. However, the reason why the pharmaceutical composition of the present invention enables the treatment or suppression of metastasis of tumors can be presumed as follows. However, the present invention is not limited by the following presumption. It is considered that the change in the sugar chain structure caused by B3GALT5 in glycolipids, which are components of the cell membrane, strongly affects the expression and function of galectin-4. And finally, it is considered to lead to the suppression of the proliferation ability and metastasis ability of cancer cells. Galectin-4 binds to a group of molecules related to the malignancy of cancer cells (such as cMET, CD44, etc.) through a sugar-binding domain. As a result, the localization of galectin-4 on the cell surface is stabilized, and it is considered that a complex sugar chain (glycoprotein or glycolipid) lattice is formed by galectin-4. It is presumed that the lateral diffusion of receptors present in this lattice is restricted, thereby increasing the threshold for receptor aggregation and information transmission. On the other hand, an increase in the B3GALT5 gene suppresses the expression of galectin-4 protein and weakens the dimerization and association state of a group of molecules related to cancer malignancy, thereby inferring that the signal information transmission into the cell is weakened. Specifically, it is considered that the activation of receptors such as cMET and EGFR is reduced due to the decreased binding of galectin-4, and the phosphorylation of AKT is attenuated, thereby suppressing the growth or metastasis of cancer.

Example

[0047] Hereinafter, the present invention will be specifically described by way of examples, but these do not limit the scope of the present invention.

[0048] 《Example 1》 Acquisition and Characterization of B3GALT5 Gene-Stably Expressing Cells (1-1. Acquisition of B3GALT5 Gene-Transfected Cell Lines) In this example, cells overexpressing the B3GALT5 gene were obtained from gastric cancer cells (NUGC4 cells). NUGC4, derived from poorly differentiated signet-ring cell carcinoma, was purchased from the RIKEN BioResource Research Center and cultured in RPMI1640 medium (FUJIFILM Wako Pure Chemical Corporation) supplemented with 10% FCS (fetal calf serum) and Glutamax (Thermo Fisher Scientific). The preparation of the B3GALT5 gene expression vector was performed using the contract service of VectorBuilder. Using pRP-hB3GALT5-EGFR / Puro-CMV containing the Puromycin resistance gene and the fluorescent protein GFP, Lipofetamine (registered trademark) 3000 Reagent (Thermo Fisher Scientific) was used to introduce the gene into NUGC4 cells according to the manufacturer's manual. B3GALT5-expressing cells were enriched by adding Puromycin to the medium, and further colonies were picked up to select B3GALT5-expressing cells. Three stable expression strains with different expressions of B3GALT5 (low-expression strain L1, high-expression strain H1, high-expression strain H2) were selected. The same operation was performed using pRP-EGFR / Puro-CMV to establish a control strain C1. The nucleotide sequence of the introduced B3GALT5 is the nucleotide sequence represented by SEQ ID NO: 2. The nucleotide sequence of the B3GALT5 gene expression vector is the nucleotide sequence represented by the following SEQ ID NO: 3.

[0049]

Table 3-1

[0050]

Table 3-2

[0051] (1-2. Measurement of B3GALT5 mRNA Expression Level in B3GALT5 Gene-Transfected Cell Lines) Total RNA was extracted from each clone using the RNAeasy mini kit (Qiagen). Using the extracted RNA, cDNA was synthesized with ReverTra Ace (registered trademark) qPCR RT Master Mix with gDNA Remover (TOYOBO). Real-time PCR was performed using this as a template. PCR amplification was carried out using THUNDERBIRD (registered trademark) SYBR (registered trademark) qPCR Mix (TOYOBO), and after performing primary denaturation at 95°C for 30 seconds with a PCR apparatus (Bio Rad CFX 96, Bio-Rad), amplification was repeated for 40 cycles at 95°C (5 seconds) and 60°C (30 seconds). Finally, after holding at 0.5°C intervals from 65°C to 95°C for 5 seconds, a melting curve was obtained by repeating data acquisition. The primer sequences used in this experiment are shown below. Quantitative analysis of the relative mRNA expression level was performed using the ΔΔCt method. At the same time, the expression of GAPDH was measured by real-time PCR and corrected by its expression level. The results are shown in Figure 1.

[0052] Primer Sequences B3GALT5: forward 5’-AAGCTCCCAGATACAGACTGC-3’ (SEQ ID NO: 4) reverse 5’-TGGTCCACCTCTTTCGTTTCC-3’ (SEQ ID NO: 5) GAPDH: forward 5’-CTCAGACGGCAGGTCAGGTCCAC-3’ (SEQ ID NO: 6) reverse 5’-CCACCCATGGCAAATTCCATGGC-3’ (SEQ ID NO: 7)

[0053] (1-3. Proliferation Ability of B3GALT5 Gene-Transfected Cell Lines) The growth ability of the B3GALT5 gene-introduced cell line was evaluated by the cell doubling time. The doubling time was calculated by inputting the initial cell number (N1), the cell number at the time of collection (N2), and the culture time (t) into the calculation formula [R = (3.32 log N2 / N1) / t, doubling time = 1 / R].

[0054]

Table 4

[0055] As shown in Table 4, the cell doubling time of the B3GALT5 gene-introduced strain was slightly longer than that of the control strain C1, and its growth ability was slightly decreased. The p-value (t-test) of the B3GALT5 gene-introduced strain relative to the control strain C1 showed p < 0.005, indicating a statistically significant difference. Also, as shown in the growth curve of Figure 2, the growth ability of the B3GALT5 stable expression strains (H1, H2) was decreased.

[0056] (1-4. Analysis of the expression of sugar chain antigens by flow cytometry) Since the introduction of the B3GALT5 gene was expected to enhance the expression of lacto-series sugar chain antigens, the change in the expression of Lewis A antigen in the B3GALT5 gene-introduced cell line was examined by flow cytometry. Each cell (control strain C1, low-expression strain L1, high-expression strain H1, high-expression strain H2) seeded at 3 × 10 6 cells / well in a 10-cm culture dish the previous day was recovered from the culture dish by trypsin treatment. After washing with PBS, each cell was reacted with various antibodies and further reacted with a fluorescently labeled secondary antibody that binds to the antibody. Each cell was reacted with an antibody 7LE Mouse mAb (Santa cruz Biotechnology) that recognizes the Lewis A antigen, and then further reacted with a fluorescently labeled secondary antibody that binds to the antibody to confirm the expression level of the target antigen. Subsequently, the expression level of the target antigen was confirmed using a detection device, Cell Lab Quanta SC (Beckman Coulter). As a result, as shown in Fig. 3, almost no expression was observed in the control strain C1 and the low-expression strain L1, but the expression of the Lewis A antigen was detected in the high-expression strains H1 and H2.

[0057] 《Example 2》Analysis Using a Mouse Peritoneal Seeding Model It was analyzed using a mouse model whether peritoneal seeding was suppressed in the B3GALT5 gene-introduced strain. Immunodeficient mice (BALBc nu / nu, male, 6 weeks old) were transplanted intraperitoneally with the control strain C1, the high-expression strain H1, and the high-expression strain H2, and after breeding for 40 days, laparotomy was performed and the tumor weight was measured. Three mice were used in each group, and each cell was transplanted intraperitoneally at 1×10 6 cells / individual in 200 - 300 μL of RPMI1640 medium. As shown in Fig. 4, a large number of tumors were observed in the peritoneal cavity in the control strain C1-administered group. Specifically, the average tumor weight was 375 ± 170 g, and 50 or more tumors with a size of 1 - 2 mm were confirmed. Eight or more tumors with a size of 5 mm were also confirmed. On the other hand, no tumors were observed in the peritoneal cavity in the mice of the B3GALT5 stable expression strain-administered group. Fig. 5 shows the results of measuring the tumor weight at autopsy 40 days later. From the above, it was found that peritoneal seeding was suppressed by the high expression of the B3GALT5 gene.

[0058] 《Example 3》Search for Molecules Whose Expression Changes Due to Stable Expression of B3GALT5 In this example, molecules whose state changes due to the stable expression of B3GALT5 were searched for by Western blot. Specifically, the expression of cMET, AKT, galectin-4, and GAPDH in the control strain C1, the low-expression strain L1, the high-expression strain H1, the high-expression strain H2, and NUGC4 cells (parent strain) was examined by the Western blot method. The day before, 1×10 6Each cell (control strain C1, low-expression strain L1, high-expression strain H1, high-expression strain H2, NUGC4 cells (parent strain)) seeded at [number] cells / well was washed with PBS, and then the cells were treated with cell lysis buffer RIPA buffer. After sonication, centrifugation was performed to obtain an intracellular protein lysate. The protein concentration was measured, and an amount equivalent to 10 μg of protein was mixed with SDS sample buffer and heat-treated at 100 °C for 5 minutes. Then, it was added to a 5-20% gradient SDS-PAGE gel and electrophoresis was performed in the presence of SDS. Intracellular proteins were transferred from the gel after electrophoresis to a PVDF membrane. After blocking the membrane with a 3% BSA solution, it was reacted with various antibodies described below, and further reacted with a peroxidase-labeled secondary antibody that binds to the antibody. Then, using a chemiluminescent substrate for peroxidase, the expression level of the target protein was confirmed using a ChemiDoc XRS+ system (Bio-Rad), a chemiluminescent detection device. The results are shown in Figure 6.

[0059] Various antibodies: cMET antibody (D1C2) XP (registered trademark) (Cell Signaling Technology), Rabbit mAb, pMET antibody (Y1234 / 1235) (D26) XP (registered trademark) Rabbit mAb (Cell Signaling Technology), pAKT (S473) antibody (D9E) XP (registered trademark) Rabbit mAb (Cell Signaling Technology), AKT (pan) antibody (C67E7) Rabbit mAb (Cell Signaling Technology), galectin-4 antibody, GAPDH antibody Peroxidase Conjugated Mouse mAb (Wako)

[0060] Note: The number in "[number]" in the translation of is missing in the original text. It should be filled with the actual number of cells per well for a complete and accurate translation.As shown in Fig. 6, the expression of cMET was slightly decreased in the B3GALT5 stable expression strains H1 and H2. Accordingly, phosphorylated MET also slightly decreased. In addition, the phosphorylation of the downstream signal AKT (S473) also significantly decreased, and the expression of pAKT in the high expression strains H1 and H2 relative to the parental strain and the low expression strain L1 showed a p value (*) (t-test) of p < 0.05, indicating a statistically significant difference. Specifically, based on the expression level of the control strain C1, the expression level of pAKT (S473) in the low expression strain L1 was 0.75-fold, 0.41-fold in the high expression strain H1, and 0.48-fold in the high expression strain H2.

[0061] Furthermore, as shown in Fig. 7, in the B3GALT5 high expression strains H1 and H2, the expression of galectin-4 protein, which is known as an animal lectin that recognizes sugar chains, also decreased. The p values (*) (t-test) of the B3GALT5 high expression strains H1 and H2 relative to the parental strain, the control strain C1, and the low expression strain L1 showed p < 0.05, indicating a statistically significant difference. Specifically, based on the expression level of the control strain C1, the expression level of galectin-4 in the low expression strain L1 was equivalent, 0.65-fold in the high expression strain H1, and 0.76-fold in the high expression strain H2.

[0062] From these results, it became clear that the expression of the B3GALT5 gene in the high expression strains H1 and H2 was related to the activation of cMET, the activation of the downstream signal AKT, and the regulation of the expression of galectin-4, compared with the parental strain, the control strain C1, and the low expression strain L1.

[0063] 《Example 4》Measurement of the expression levels of B3GALT5 protein in normal gastric tissues and several types of gastric cancer tissues Using paraffin sections of fixed tissues purchased from TissueArray.com, the expression of B3GALT5 protein in normal gastric tissue sections and cancer tissue sections was examined by immunohistochemical staining. For the deparaffinization process, low-toxicity Fast solve (Falma Co., Ltd.) was used as a substitute for xylene. First, the sections were immersed in the Fast solve tank 4 times for 5 minutes each, then in the absolute ethanol tank 4 times for 3 minutes each, and finally, after being immersed in the 70% ethanol tank for 3 minutes and then in the distilled water layer, the deparaffinization process was carried out. Subsequently, the sections were immersed in 0.01M citrate buffer pH6.0 and heat-treated in an autoclave at 120°C for 20 minutes. After the pressure had sufficiently decreased, the container was taken out. The sections were slowly cooled while immersed in the buffer and then washed with PBS for antigen activation treatment. Thereafter, they were immersed in a 0.3% hydrogen peroxide methanol solution for 15 minutes to suppress the activity of endogenous peroxidase. Antibody staining using the anti-B3GALT5 antibody (HPA054684, Sigma-Aldrich) was performed manually using the VECTASTAIN Elite ABC Rabbit IgG Kit (PK-6101, VECTOR). The anti-B3GALT5 antibody was diluted with 1% BSA to a concentration of 0.67 μg / mL and then added onto the sections and reacted overnight at 4°C. The color development reaction was performed manually using ImmPACT DAB (registered trademark) (SK-4105, VECTOR). After confirming the color development and washing the sections with distilled water, a Hematoxylin solution (Sigma-Aldrich) was added for counterstaining. After staining, they were thoroughly washed with running water for 15 minutes. After thoroughly removing the water, they were immersed in isopropanol 2 times for 2 minutes each, VectaMount Express Mounting Medium was added, and they were sealed with a cover glass. After natural solidification, they were observed under a microscope. The obtained images are shown in Figure 8. In normal gastric tissue, the expression of B3GALT5 protein was observed in glandular cells. On the other hand, in cancer tissue sections, strong staining of B3GALT5 protein was not observed, and non-specific color development was observed in muscle tissue. Therefore, it was shown that the expression of B3GALT5 protein decreases upon carcinogenesis.

Industrial Applicability

[0064] According to the present invention, an effective treatment method for tumors or a method for suppressing metastasis can be provided.

Claims

1. A pharmaceutical composition for treating tumors or inhibiting metastasis, comprising, as an active ingredient, a nucleic acid encoding β1,3-galactosyltransferase V or a vector incorporating a nucleic acid encoding β1,3-galactosyltransferase V.

2. The pharmaceutical composition according to claim 1, wherein the tumor comprises tumor cells in which the expression level of β1,3-galactosyltransferase V is reduced compared to normal cells.

3. The pharmaceutical composition for treating tumors or inhibiting metastasis according to claim 1 or 2, wherein the vector is a viral vector.

4. The pharmaceutical composition according to claim 1 or 2, which is used to inhibit tumor growth or metastasis.

5. The pharmaceutical composition according to claim 4, which is used to inhibit the growth of stomach cancer or inhibit metastasis from stomach cancer.

6. The pharmaceutical composition according to claim 1 or 2, which is for suppressing peritoneal dissemination.

7. The pharmaceutical composition according to claim 1 or 2, wherein the nucleic acid encoding β1,3-galactosyltransferase V is a nucleic acid having any one of the base sequences selected from the group consisting of the following (a) to (d): (a) a base sequence represented by SEQ ID NO: 1; (b) a base sequence in which one to several bases are deleted, substituted or added in the base sequence represented by SEQ ID NO: 1; (c) a base sequence having an identity of 90% or more with the base sequence represented by SEQ ID NO: 1; and (d) a base sequence capable of hybridizing under stringent conditions with a nucleic acid consisting of a base sequence complementary to a nucleic acid consisting of the base sequence represented by SEQ ID NO: 1; Here, when the nucleic acid encoding β1,3-galactosyltransferase V is a nucleic acid having any one of the base sequences selected from the group consisting of (b) to (d), a protein encoded by the nucleic acid having any one of the base sequences selected from the group consisting of (b) to (d) is considered to have β1,3-galactosyltransferase V activity.

8. Measuring the expression level of β1,3-galactosyltransferase V in tumor cells collected from the subject; comparing the expression level with a reference value; A method for determining the effectiveness of a pharmaceutical composition for treating tumors or inhibiting metastasis, which comprises, as an active ingredient, a nucleic acid encoding β1,3-galactosyltransferase V or a vector incorporating a nucleic acid encoding β1,3-galactosyltransferase V, comprising:

9. A nucleic acid encoding β1,3-galactosyltransferase V or a vector comprising a nucleic acid encoding β1,3-galactosyltransferase V, for use in producing a pharmaceutical composition for treating tumors or inhibiting metastasis.

10. 1. An in vitro method for reducing the proliferative or metastatic potential of a tumor cell, comprising: Introducing a nucleic acid encoding β1,3-galactosyltransferase V into tumor cells A method comprising:

11. A nucleic acid encoding β1,3-galactosyltransferase V or a vector comprising a nucleic acid encoding β1,3-galactosyltransferase V for use in a method for treating or inhibiting metastasis of a tumor in a subject.

12. Use of a nucleic acid encoding β1,3-galactosyltransferase V or a vector comprising a nucleic acid encoding β1,3-galactosyltransferase V in the manufacture of a medicament for treating tumors or suppressing metastasis.

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