Antitumor Agents

Abstract

The present invention makes it possible to provide an antitumor agent capable of suppressing the proliferation of cancer cells without affecting normal cells. Use is made of an antitumor agent that comprises a substance inhibiting the binding between CSN5 and CDK2.

Description

[Technical field]

[0001] The present invention relates to an antitumor agent. [Background technology]

[0002] In recent years, the number of deaths from cancer has been increasing, so the development of cancer treatments has become a social priority. Cancer treatments include surgery, drug therapy using anticancer drugs, and radiation therapy, and these treatments are used alone or in combination. Drug therapy is particularly effective for patients who cannot undergo surgery, those with recurrent cancer, and those who are undergoing surgery before or after surgery.

[0003] Drug therapy uses drugs that attack cancer cells, such as "cytotoxic anticancer drugs," "endocrine therapy drugs," and "molecularly targeted drugs." "Molecularly targeted drugs" are drugs that target specific molecules, and are currently being actively developed, with the hope of discovering new target molecules.

[0004] CSN5 (COP9 signalosome complex subunit 5) is one of the proteins that form the COP9 signalosome complex, and forms the active center of the COP9 signalosome complex. It is known that (i) CSN5 is overexpressed in various cancers, (ii) there are two types of complexes containing the CSN5 protein, a "large complex" and a "small complex", (iii) the "small complex" containing the CSN5 protein is specifically expressed in cancer cells, and (iv) the CSN5 protein binds to various proteins (e.g., CDK2, etc.) (see, for example, Non-Patent Documents 1 and 2).

[0005] In addition, since CSN5 is known to be associated with cancer, a technique has been disclosed for treating cell proliferation disorders such as cancer by silencing CSN5 gene expression using interference RNA (e.g., Patent Document 1). [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Special Publication No. 2011-516094 [Non-patent literature]

[0007] [Non-Patent Document 1] Lingaraju GM et al., “Crystal structure of the human COP9 signalosome.” Nature, 2014 Aug 14, 512(7513), 161-165 [Non-Patent Document 2] Yoshida A et al., “CSN5 specifically interacts with CDK2 and controls senescence in a cytoplasmic cyclin E-mediated manner.” Scientific Reports, 2013, 3, 1054, 1-11 Summary of the Invention [Problem to be solved by the invention]

[0008] In the technology of Patent Document 1 described above, the expression of the CSN5 protein is entirely suppressed. However, of the two types of complexes containing the CSN5 protein, the "large complex" is also expressed in normal cells and is considered to be an essential complex for the survival of the living body. Therefore, inhibiting the formation of the "large complex" will cause harmful side effects in the living body. Therefore, there is a demand for the development of a new antitumor agent that targets CSN5 and does not inhibit the formation of the "large complex" containing the CSN5 protein necessary for survival.

[0009] An object of one aspect of the present invention is to provide an antitumor agent that targets CSN5 and is capable of suppressing the proliferation of cancer cells while minimizing effects on normal cells. [Means for solving the problem]

[0010] In order to solve the above problems, the inventors have discovered the following new findings, which have led to the completion of the present invention: (i) the CDK2-binding region in the CSN5 protein has been identified; (ii) the binding between CSN5 and CDK2 can be specifically inhibited by using a polypeptide containing the CDK2-binding region; (iii) by inhibiting the binding between the CSN5 protein and CDK2, it is possible to inhibit only the formation of a "small complex" that is specifically expressed in cancer cells, while not inhibiting the formation of the "large complex," one of two types of complexes containing the CSN5 protein, which is essential for the survival of normal cells; and (iv) as a result, it is possible to specifically inhibit the proliferation of cancer cells while minimizing the effects on normal cells.

[0011] That is, the present invention includes the following configurations.

[0012] [1] An antitumor agent comprising an expression vector comprising any one of the polynucleotides selected from the group consisting of the following (a) to (e), and / or any one of the polypeptides selected from the group consisting of the following (f) to (i): (a) a polynucleotide encoding a polypeptide comprising an amino acid sequence represented by the 233rd to 252nd amino acid residues counting from the amino terminus of the amino acid sequence represented by SEQ ID NO:1; (b) a polynucleotide encoding a polypeptide comprising an amino acid sequence in which one or several amino acid residues have been substituted, deleted, inserted and / or added in the amino acid sequence depicted at positions 233 to 252 from the amino terminus of the amino acid sequence shown in SEQ ID NO:1, and having an activity of binding to CDK2; (c) a polynucleotide encoding a polypeptide comprising an amino acid sequence having 90% or more identity to the amino acid sequence represented by the 233rd to 252nd amino acids counting from the amino terminus of the amino acid sequence represented by SEQ ID NO:1 and having an activity of binding to CDK2; (d) a polynucleotide comprising a nucleotide sequence represented by the 697th to 756th bases counting from the 5' end of the nucleotide sequence represented by SEQ ID NO:2; (e) a polynucleotide that hybridizes under stringent conditions to a polynucleotide having a nucleotide sequence complementary to any one of the polynucleotides (a) to (d) and encodes a polypeptide having an activity that binds to CDK2; (f) a polypeptide comprising an amino acid sequence represented by the 233rd to 252nd amino acid residues counting from the amino terminus of the amino acid sequence represented by SEQ ID NO:1; (g) a polypeptide comprising an amino acid sequence in which one or several amino acid residues are substituted, deleted, inserted and / or added in the amino acid sequence set forth at positions 233 to 252 from the amino terminal of the amino acid sequence set forth in SEQ ID NO: 1, and having an activity of binding to CDK2; (h) A polypeptide comprising an amino acid sequence having 90% or more identity to the amino acid sequence located at positions 233 to 252 from the amino terminus of the amino acid sequence shown in SEQ ID NO:1, and having activity of binding to CDK2.

[0013] [2] The antitumor agent according to [1], comprising an expression vector comprising any one of the polynucleotides selected from the group consisting of the following (a') to (e') and / or any one of the polypeptides selected from the group consisting of the following (f') to (i'): (a') a polynucleotide encoding a polypeptide comprising an amino acid sequence represented by the 199th to 265th amino acids counting from the amino terminus of the amino acid sequence represented by SEQ ID NO:1; (b') a polynucleotide encoding a polypeptide comprising an amino acid sequence in which one or several amino acid residues have been substituted, deleted, inserted and / or added in the amino acid sequence set forth at positions 199 to 265 from the amino terminal of the amino acid sequence set forth in SEQ ID NO:1, and having an activity of binding to CDK2; (c') a polynucleotide encoding a polypeptide comprising an amino acid sequence having 90% or more identity to the amino acid sequence represented by the 199th to 265th amino acids counting from the amino terminal of the amino acid sequence represented by SEQ ID NO:1 and having an activity of binding to CDK2; (d') a polynucleotide comprising a nucleotide sequence set forth at positions 595 to 795 from the 5' end of the nucleotide sequence shown in SEQ ID NO: 2; (e') a polynucleotide that hybridizes under stringent conditions to a polynucleotide having a nucleotide sequence complementary to any one of the polynucleotides (a') to (d') and encodes a polypeptide having an activity that binds to CDK2; (f') a polypeptide comprising an amino acid sequence represented by the 199th to 265th amino acid residues counting from the amino terminus of the amino acid sequence represented by SEQ ID NO:1; (g') a polypeptide comprising an amino acid sequence in which one or several amino acid residues have been substituted, deleted, inserted and / or added in the amino acid sequence set forth at positions 199 to 265 from the amino terminal of the amino acid sequence shown in SEQ ID NO: 1, and having an activity of binding to CDK2; (h') A polypeptide comprising an amino acid sequence having 90% or more identity to the amino acid sequence located at positions 199 to 265 from the amino terminus of the amino acid sequence shown in SEQ ID NO:1, and having the activity of binding to CDK2.

[0014] [3] The antitumor agent according to [1] or [2], wherein the polypeptide is encoded by a polynucleotide contained in the expression vector, and the polypeptide further comprises a membrane-permeable peptide.

[0015] [4] An antitumor agent comprising a substance that inhibits the binding between CSN5 and CDK2. Effect of the Invention

[0016] According to one aspect of the present invention, it is possible to realize an antitumor agent that can suppress the proliferation of cancer cells without affecting normal cells. [Brief description of the drawings]

[0017] [Figure 1] FIG. 1 shows the amino acid regions within CSN5 that bind to CDK2. [Diagram 2]These are the experimental results of each fragment of the CSN5 peptide using the GST pull-down method. [Diagram 3] This shows the results of Western blotting of immunoprecipitates from extracts of cells into which a CSN5 peptide expression vector was introduced. [Figure 4] This shows the results of Western blotting of CSN complexes separated by electrophoresis. [Diagram 5] 1 shows optical microscope photographs showing the results of cell growth into which a CSN5 peptide expression vector has been introduced. [Figure 6] 1 is a graph showing the size of tumors in nude mice into which cells into which a CSN5 peptide expression vector was introduced were subcutaneously transplanted. [Figure 7] 1 is a graph showing the results of proliferation of human leukemia cells into which a CSN5 peptide expression vector has been introduced. [Figure 8] (A) Western blotting results for cells to which CSN5 peptide was added. (B) A graph showing the relationship between the amount of peptide added to cells and the number of live cells. (C) A graph showing the relationship between the amount of peptide added to cells and the number of dead cells. (D) A graph showing the time course of the number of live cells in cells to which peptide was added. [Figure 9] 1 shows images showing the growth results of cells into which expression vectors for each CSN5 peptide were introduced. [Figure 10] 1 shows images showing the growth results of cells into which expression vectors for each CSN5 peptide were introduced. [Figure 11] 1 is a graph showing the results of proliferation of cells into which a zinc-inducible expression vector for CSN5(233-252) was introduced. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] An embodiment of the present invention is described below, but the present invention is not limited thereto. The present invention is not limited to each of the configurations described below, and various modifications are possible within the scope of the claims. The embodiments and examples obtained by appropriately combining the technical means disclosed in the different embodiments and examples are also included in the technical scope of the present invention. In addition, all of the documents described in this specification are incorporated herein by reference. In this specification, when a numerical range is described as "A to B," the description means "A or more and B or less." In this specification, when simply referring to "CSN5," the gene and / or protein are intended, and when the "CSN5 protein" is explicitly stated, only the protein is intended.

[0019] 1. Antitumor Agents An antitumor agent according to one embodiment of the present invention comprises a substance that inhibits the binding of CSN5 to CDK2 (more specifically, a substance that inhibits the binding of CSN5 to CDK2 and inhibits the formation of a cancer-specific CSN5 small complex). The substance is not limited, and may be an expression vector, a polynucleotide, a polymeric compound, or a low molecular weight compound.

[0020] The above-mentioned substance not only inhibits the binding between CSN5 and CDK2, but may also inhibit the binding between CSN5 and a protein other than CDK2 (for example, CBR1, CBR3, PRDX1, PRDX3, IKK1, PKM2, NDUFV1, or MDH2).

[0021] More specifically, an antitumor agent according to one embodiment of the present invention comprises an expression vector comprising any one of the polynucleotides selected from the group consisting of the following (a) to (e), and / or any one of the polypeptides selected from the group consisting of the following (f) to (i): (a) a polynucleotide encoding a polypeptide comprising an amino acid sequence represented by the 233rd to 252nd amino acid residues counting from the amino terminus of the amino acid sequence represented by SEQ ID NO:1 (or a polypeptide consisting of said amino acid sequence); (b) a polynucleotide encoding a polypeptide comprising (or consisting of) an amino acid sequence in which one or several amino acid residues have been substituted, deleted, inserted and / or added in the amino acid sequence set forth at positions 233 to 252 from the amino terminus of the amino acid sequence set forth in SEQ ID NO:1, and having an activity of binding to CDK2; (c) a polynucleotide encoding a polypeptide comprising (or consisting of) an amino acid sequence having 90% or more identity to the amino acid sequence depicted at positions 233 to 252 from the amino terminus of the amino acid sequence shown in SEQ ID NO:1 and having an activity of binding to CDK2; (d) a polynucleotide comprising a nucleotide sequence from the 697th to 756th positions counting from the 5' end of the nucleotide sequence shown in SEQ ID NO: 2 (or a polynucleotide consisting of said nucleotide sequence); (e) a polynucleotide that hybridizes under stringent conditions to a polynucleotide having a nucleotide sequence complementary to any one of the polynucleotides (a) to (d) and encodes a polypeptide having an activity that binds to CDK2; (f) a polypeptide comprising an amino acid sequence set forth at positions 233 to 252 from the amino terminus of the amino acid sequence set forth in SEQ ID NO:1 (or a polypeptide consisting of said amino acid sequence); (g) a polypeptide comprising (or consisting of) an amino acid sequence in which one or several amino acid residues have been substituted, deleted, inserted and / or added in the amino acid sequence set forth at positions 233 to 252 counting from the amino terminus of the amino acid sequence shown in SEQ ID NO:1, and having an activity of binding to CDK2; (h) A polypeptide comprising (or consisting of) an amino acid sequence having 90% or more identity to the amino acid sequence located at positions 233 to 252 from the amino terminus of the amino acid sequence shown in SEQ ID NO:1, and having the activity of binding to CDK2.

[0022] More specifically, an antitumor agent according to one embodiment of the present invention comprises an expression vector comprising any one of the polynucleotides selected from the group consisting of the following (a') to (e') and / or any one of the polypeptides selected from the group consisting of the following (f') to (i'): (a') a polynucleotide encoding a polypeptide comprising an amino acid sequence represented by the 199th to 265th amino acid residues counting from the amino terminus of the amino acid sequence represented by SEQ ID NO:1 (or a polypeptide consisting of said amino acid sequence); (b') a polynucleotide encoding a polypeptide comprising (or consisting of) an amino acid sequence in which one or several amino acid residues have been substituted, deleted, inserted and / or added in the amino acid sequence set forth at positions 199 to 265 from the amino terminus of the amino acid sequence shown in SEQ ID NO:1, and which has an activity of binding to CDK2; (c') a polynucleotide encoding a polypeptide comprising (or consisting of) an amino acid sequence having 90% or more identity with the amino acid sequence depicted at positions 199 to 265 from the amino terminal of the amino acid sequence shown in SEQ ID NO:1 and having an activity of binding to CDK2; (d') a polynucleotide comprising a nucleotide sequence set forth at positions 595 to 795 from the 5' end of the nucleotide sequence shown in SEQ ID NO: 2 (or a polynucleotide consisting of said nucleotide sequence); (e') a polynucleotide that hybridizes under stringent conditions to a polynucleotide having a nucleotide sequence complementary to any one of the polynucleotides (a') to (d') and encodes a polypeptide having an activity that binds to CDK2; (f') a polypeptide comprising an amino acid sequence set forth at positions 199 to 265 from the amino terminus of the amino acid sequence set forth in SEQ ID NO:1 (or a polypeptide consisting of said amino acid sequence); (g') a polypeptide comprising (or consisting of) an amino acid sequence in which one or several amino acid residues have been substituted, deleted, inserted and / or added in the amino acid sequence set forth at positions 199 to 265 from the amino terminal of the amino acid sequence shown in SEQ ID NO: 1, and having an activity of binding to CDK2; (h') A polypeptide comprising (or consisting of) an amino acid sequence having 90% or more identity to the amino acid sequence located at positions 199 to 265 from the amino terminus of the amino acid sequence shown in SEQ ID NO:1, and having the activity of binding to CDK2.

[0023] The above SEQ ID NO:1 corresponds to the full-length amino acid sequence of the CSN5 protein, while the above SEQ ID NO:2 corresponds to the full-length nucleotide sequence of the CSN5 cDNA.

[0024] According to the above configuration, the proliferation of cancer cells can be suppressed without affecting normal cells.

[0025] The number of amino acids constituting the polypeptide, which is an active ingredient of the present antitumor agent, and the number of amino acids constituting the polypeptide encoded by the polynucleotide contained in the expression vector, which is an active ingredient of the present antitumor agent, are not particularly limited. From the viewpoints of (i) the polypeptide easily maintaining a three-dimensional structure required for exerting physiological activity, (ii) the polypeptide being easy to handle, and / or (iii) the polypeptide being easily transferred to a desired tissue in the body, the number of amino acids constituting the polypeptide is preferably 20 to 100, 20 to 90, 20 to 80, 20 to 70, 20 to 60, or 20 to 50. The more amino acids constituting the polypeptide, the more the above-mentioned effect (i) tends to increase. On the other hand, the fewer the number of amino acids constituting the polypeptide, the more the above-mentioned effects (ii) and (iii) tend to increase. The number of amino acids may be determined while taking into consideration the balance of these effects.

[0026] With regard to the above (b), (g), (b') and (g'), "one or several amino acid residues have been substituted, deleted, inserted and / or added" means that a number of amino acids (preferably 10 or less, more preferably 9 or less, more preferably 8 or less, more preferably 7 or less, more preferably 6 or less, more preferably 5 or less, more preferably 4 or less, more preferably 3 or less, more preferably 2 or less, and most preferably 1 or less) that can be substituted, deleted, inserted and / or added by a known method for producing mutant proteins such as site-directed mutagenesis. Note that the position in the amino acid sequence where one or several amino acid residues are substituted, deleted, inserted and / or added is not limited.

[0027] Regarding the above (c), (h), (c') and (h'), the identity of amino acid sequences can be determined, for example, by using the BLASTX program (Altschul et al. J. Mol. Biol., 215: 403-410, 1990). The program is based on the BLAST algorithm by Karlin and Altschul (Proc. Natl. Acad. Sci. USA, 87: 2264-2268, 1990, Proc. Natl. Acad.Sci. USA, 90: 5873-5877, 1993). When analyzing amino acid sequences using BLASTX, the parameters are, for example, score=50 and wordlength=3. Furthermore, when analyzing amino acid sequences using the Gapped BLAST program, it can be performed as described in Altschul et al. (Nucleic Acids Res. 25: 3389-3402, 1997). Specific techniques for these analysis methods are known. In order to optimally align the nucleotide sequences or amino acid sequences to be compared, additions or deletions (e.g., gaps, etc.) may be allowed.

[0028] With respect to (c), (h), (c') and (h') above, the amino acid sequence identity is preferably 91% or more, more preferably 92% or more, more preferably 93% or more, more preferably 94% or more, more preferably 95% or more, more preferably 96% or more, more preferably 97% or more, more preferably 98% or more, more preferably 99% or more, and most preferably 100%.

[0029] With regard to the above (e) and (e'), "stringent conditions" refer to conditions under which a double-stranded polynucleotide specific to the so-called base sequence is formed and a non-specific double-stranded polynucleotide is not formed. In other words, it can be said to be conditions under which highly homologous nucleic acids hybridize with each other at a temperature within a range of 15°C, preferably 10°C, and more preferably 5°C lower than the melting temperature (Tm value) of a perfectly matched hybrid.

[0030] For example, 0.25M Na 2 HPO 4 Hybridization is performed for 16 to 24 hours in a buffer solution consisting of 1 mM EDTA, 7% SDS, pH 7.2, 1 mM EDTA, and 1× Denhardt's solution at a temperature of 60 to 68° C., preferably 65° C., and more preferably 68° C., and then in a buffer consisting of 20 mM Na 2 HPO 4 The conditions include washing twice for 15 minutes in a buffer solution consisting of pH 7.2, 1% SDS, and 1 mM EDTA at a temperature of 60 to 68°C, preferably 65°C, and more preferably 68°C.

[0031] In another example, prehybridization is performed overnight at 42°C in a hybridization solution containing 25% formamide, or more stringent conditions such as 50% formamide, 4xSSC (sodium chloride / sodium citrate), 50mM Hepes pH7.0, 10x Denhardt's solution, and 20μg / mL denatured salmon sperm DNA, followed by addition of a labeled probe and incubation overnight at 42°C for hybridization. The washing solution and temperature conditions for subsequent washing can be about "1xSSC, 0.1%SDS, 37°C", more stringent conditions such as "0.5xSSC, 0.1%SDS, 42°C", and even more stringent conditions such as "0.2xSSC, 0.1%SDS, 65°C". Thus, the more stringent the washing conditions for hybridization, the more specific the hybridization. However, the above combination of SSC, SDS and temperature conditions is merely an example, and a person skilled in the art can realize a similar stringency to the above by appropriately combining the above or other factors that determine the stringency of hybridization (e.g., probe concentration, probe length, hybridization reaction time, etc.) This is described, for example, in Sambrook et al., Molecular Cloning, A Laboratory Manual, 3rd Ed., Cold Spring Harbor Laboratory (2001), etc.

[0032] Regarding the above (b), (c), (e), (g), (h), (b'), (c'), (e'), (g') and (h'), whether or not the polypeptide has the activity of binding to CDK2 can be determined according to a known method (for example, the GST pull-down method described in the Examples below). For example, first, beads to which a fusion protein of a polypeptide and GST has been added are mixed with CDK2 protein, and then the beads are collected by centrifugation and the supernatant is removed. Next, the beads are washed with a desired washing solution, and then the beads are collected by centrifugation and the supernatant is removed. Next, proteins are extracted from the collected beads, and the extract is subjected to electrophoresis, after which the developed proteins are transferred to a membrane for Western blotting. Finally, the CDK2 transferred to the membrane is detected using an anti-CDK2 antibody. If the CDK2 transferred to the membrane is detected, it can be determined that the polypeptide has the activity of binding to CDK2.

[0033] With regard to (d) above, SEQ ID NO: 1 corresponds to the full-length amino acid sequence of the CSN5 protein, and SEQ ID NO: 2 corresponds to the full-length nucleotide sequence of the CSN5 cDNA. Therefore, a polynucleotide containing the nucleotide sequence represented by the 697th to 756th positions counting from the 5'-end of the nucleotide sequence represented by SEQ ID NO: 2 corresponds to the amino acid sequence represented by the 233rd to 252nd positions counting from the amino terminus of the amino acid sequence represented by SEQ ID NO: 1.

[0034] With regard to (d') above, SEQ ID NO: 1 corresponds to the full-length amino acid sequence of the CSN5 protein, and SEQ ID NO: 2 corresponds to the full-length nucleotide sequence of the CSN5 cDNA. Therefore, a polynucleotide containing the nucleotide sequence represented by SEQ ID NO: 2 at positions 595 to 795 from the 5'-terminus corresponds to the amino acid sequence represented by SEQ ID NO: 1 at positions 199 to 265 from the amino terminus.

[0035] The polypeptide, which is the active ingredient of the present antitumor agent, and the polypeptide encoded by the polynucleotide contained in the expression vector, which is the active ingredient of the present antitumor agent, preferably further contain a cell penetrating peptide (CPP). With this configuration, the polypeptide can easily enter cells, thereby enhancing the efficacy of the antitumor agent.

[0036] The form of the polypeptide containing a membrane-permeable peptide is not particularly limited. For example, the polypeptide may be (i) a fusion protein of a polypeptide that is a medicinal ingredient and a membrane-permeable peptide, (ii) a polypeptide that is a medicinal ingredient and a membrane-permeable peptide are bonded to the polypeptide via any crosslinking agent, or (iii) a polypeptide that is a medicinal ingredient and a membrane-permeable peptide are bonded directly to the polypeptide.

[0037] In the case of the above-mentioned form (i), the membrane-permeable peptide may be located at the amino terminal side of the polypeptide, which is the medicinal ingredient, or may be located at the carboxyl terminal side of the polypeptide, which is the medicinal ingredient. From the viewpoint of facilitating the transfer of the medicinal ingredient into cells, it is preferable that the membrane-permeable peptide is located at the amino terminal side of the polypeptide, which is the medicinal ingredient.

[0038] The membrane-permeable peptide is not limited, and any known membrane-permeable peptide can be used. For example, a membrane-permeable peptide possessed by a known gene (e.g., antennapedia (43-58) [derived from Drosophila], TAT (48-60) [derived from human immunodeficiency virus], or Polyarginin R9 [synthetic peptide]) can be used. Among these membrane-permeable peptides, Antennapedia and Polyarginin (R9) are preferred from the viewpoint of the abundance of research findings.

[0039] The expression vector used in the antitumor agent according to one embodiment of the present invention is not limited, and any known expression vector can be used. Examples of the expression vector include non-replicating virus vectors (e.g., retrovirus vectors, adenovirus vectors, lentivirus vectors, adeno-associated virus vectors, Sendai virus vectors), replicating virus vectors (e.g., oncolytic virus vectors), non-viral vectors (e.g., plasmids, liposomes encapsulating naked DNA), and bacterial vectors.

[0040] The amount of the active ingredient (specifically, the substance that inhibits the binding between CSN5 and CDK2, the expression vector, and the polypeptide) contained in the antitumor agent according to one embodiment of the present invention is not particularly limited, but may be 0.001% by weight to 100% by weight, 0.01% by weight to 100% by weight, 0.1% by weight to 100% by weight, 0.1% by weight to 95% by weight, 0.1% by weight to 90% by weight, 0.1% by weight to 80% by weight, 0.1% by weight to 70% by weight, 0.1% by weight to 60% by weight, 0.1% by weight to 50% by weight, 0.1% by weight to 40% by weight, 0.1% by weight to 30% by weight, 0.1% by weight to 20% by weight, or 0.1% by weight to 10% by weight, preferably, when the entire antitumor agent is taken as 100% by weight.

[0041] The antitumor agent according to one embodiment of the present invention may contain components other than the active ingredient. The components other than the active ingredient may be any pharma- ceutically acceptable components, such as a buffer, a pH adjuster, an isotonicity agent, a preservative, an excipient, a carrier, a diluent, a solvent, a solubilizer, a stabilizer, an antioxidant, a high molecular weight polymer, a filler, a binder, a surfactant, and a stabilizer.

[0042] The buffering agent may include phosphoric acid, phosphates, boric acid, borates, citric acid, citrates, acetic acid, acetates, carbonic acid, carbonates, tartaric acid, tartrates, ε-aminocaproic acid, and trometamol. The phosphates may include sodium phosphate, sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium phosphate, potassium dihydrogen phosphate, and dipotassium hydrogen phosphate. The borates may include borax, sodium borate, and potassium borate. The citrates may include sodium citrate, disodium citrate, and trisodium citrate. The acetates may include sodium acetate and potassium acetate. The carbonates may include sodium carbonate and sodium bicarbonate. The tartrates may include sodium tartrate and potassium tartrate.

[0043] Examples of the pH adjuster include hydrochloric acid, phosphoric acid, citric acid, acetic acid, sodium hydroxide, and potassium hydroxide.

[0044] The isotonicity agent includes ionic isotonicity agents (sodium chloride, potassium chloride, calcium chloride, magnesium chloride, etc.) and non-ionic isotonicity agents (glycerin, propylene glycol, sorbitol, mannitol, etc.).

[0045] Examples of the preservative include benzalkonium chloride, benzalkonium bromide, benzethonium chloride, sorbic acid, potassium sorbate, methyl parahydroxybenzoate, propyl parahydroxybenzoate, and chlorobutanol.

[0046] The antioxidants include ascorbic acid, tocopherol, dibutylhydroxytoluene, butylhydroxyanisole, sodium erythorbate, propyl gallate, and sodium sulfite.

[0047] Examples of the high molecular weight polymer include methyl cellulose, ethyl cellulose, hydroxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxyethyl methyl cellulose, hydroxypropyl methyl cellulose, carboxymethyl cellulose, sodium carboxymethyl cellulose, hydroxypropyl methyl cellulose acetate succinate, hydroxypropyl methyl cellulose phthalate, carboxymethyl ethyl cellulose, cellulose acetate phthalate, polyvinylpyrrolidone, polyvinyl alcohol, carboxyvinyl polymer, and polyethylene glycol.

[0048] The carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil and the like.

[0049] The solvents and solubilizing agents include glycerin, DMSO, DMA, N-methylpyrrolidone, ethanol, benzyl alcohol, isopropyl alcohol, polyethylene glycols of various molecular weights (such as PEG300 and PEG400), and propylene glycol.

[0050] Such surfactants include nonionic, anionic, cationic, zwitterionic, polymeric, and amphoteric surfactants.

[0051] The stabilizers include fatty acids, fatty alcohols, alcohols, long chain fatty acid esters, long chain ethers, hydrophilic derivatives of fatty acids, polyvinylpyrrolidones, polyvinyl ethers, polyvinyl alcohols, hydrocarbons, hydrophobic polymers, hygroscopic polymers, and amide analogs.

[0052] The amount of ingredients other than the active ingredient contained in the antitumor agent according to one embodiment of the present invention is not particularly limited, but preferably, when the entire antitumor agent is taken as 100% by weight, the amount may be 0% by weight to 99.999% by weight, 0% by weight to 99.99% by weight, 0% by weight to 99.9% by weight, 5% by weight to 99.9% by weight, 10% by weight to 99.9% by weight, 20% by weight to 99.9% by weight, 30% by weight to 99.9% by weight, 40% by weight to 99.9% by weight, 50% by weight to 99.9% by weight, 60% by weight to 99.9% by weight, 70% by weight to 99.9% by weight, 80% by weight to 99.9% by weight, or 90% by weight to 99.9% by weight.

[0053] The administration form of the antitumor agent according to one embodiment of the present invention is not particularly limited, but examples thereof include oral agents, parenteral agents, and topical agents.

[0054] The tumor to which the antitumor agent according to one embodiment of the present invention is administered is not limited, and examples of such tumors include rhabdomyosarcoma, breast cancer, lymphoma, squamous cell carcinoma (e.g., oral squamous cell carcinoma, laryngeal squamous cell carcinoma), ovarian cancer, melanoma, neuroblastoma, lung cancer (lung adenocarcinoma), pancreatic cancer, liver cancer (hepatocellular carcinoma), thyroid cancer (medullary thyroid carcinoma), leukemia (chronic myeloid leukemia), lung cancer (non-small cell lung cancer), colon cancer, and gastric cancer.

[0055] [2. Other] The present invention can also be configured as follows.

[0056] [1] A method for treating a tumor (or a method for treating a tumor), comprising the step of administering to a subject (e.g., a mammal, a non-human mammal, a primate, an artiodactyl, a perissodactyl, a rodent, a lagomorph, or a carnivore) an expression vector comprising any of the polynucleotides selected from the group consisting of (a) to (e) above, and / or an antitumor agent comprising any of the polypeptides selected from the group consisting of (f) to (i) above.

[0057] [2] A method for treating a tumor (or a method for treating a tumor) according to [1], comprising the step of administering to a subject an expression vector comprising any one of the polynucleotides selected from the group consisting of (a') to (e') above, and / or an antitumor agent comprising any one of the polypeptides selected from the group consisting of (f') to (i') above.

[0058] [3] The method for treating a tumor (or the method for treating a tumor) described in [1] or [2], wherein the polypeptide encoded by the polynucleotide contained in the expression vector or the polypeptide further comprises a membrane-permeable peptide.

[0059] [4] A method for treating a tumor (or a method for treating a tumor), comprising the step of administering an antitumor agent containing a substance that inhibits the binding between CSN5 and CDK2 to a subject (e.g., a mammal, a non-human mammal, a primate, an artiodactyl, a perissodactyl, a rodent, a lagomorph, or a carnivore). [5] Use of an expression vector comprising any one of the polynucleotides selected from the group consisting of (a) to (e) above, and / or any one of the polypeptides selected from the group consisting of (f) to (i) above, for producing an antitumor agent. [6] Use of an expression vector comprising any one of the polynucleotides selected from the group consisting of (a') to (e') above, and / or any one of the polypeptides selected from the group consisting of (f') to (i') above, for producing an antitumor agent. [7] The use according to [5] or [6], wherein the polypeptide encoded by the polynucleotide contained in the expression vector or the polypeptide further comprises a membrane-permeable peptide. [8] Use of a substance that inhibits the binding of CSN5 and CDK2 for the production of an antitumor agent. EXAMPLES

[0060] 1. Materials and Methods Unless otherwise stated, the materials and methods used in the experiments are as follows.

[0061] [Escherichia coli] E. coli DH5α:F-,Φ80dlacZΔM15,Δ(lacZYA-argF)U169,deoR,recA1,endA1,hsdR17(rK-,mK+),phoA,supE44,λ-,thi-1,gyrA96,relA1 E. coli BL21:F-,ompT,hsdSB(rB-,mB-),gal,dcm.

[0062] [vector] The following plasmids were constructed according to known procedures: pGEX-6P-2 (purchased from GE Healthcare Life Sciences) pGEX-6P-2-CSN5 Full length (expresses the 1-334 aa region counted from the amino terminus of CSN5) pGEX-6P-2-CSN5 N-terminal (expresses the region 1-198 aa from the amino terminus of CSN5) pGEX-6P-2-CSN5 C-terminal (expresses the region 199-334 aa from the amino terminus of CSN5) pGEX-6P-2-CSN5 N-mutant (expresses the 1-26 aa region from the amino terminus of CSN5) pGEX-6P-2-CSN5 N-mutant (expresses the 1-53 aa region from the amino terminus of CSN5) pGEX-6P-2-CSN5 N-mutant (expresses the 1-86 aa region from the amino terminus of CSN5) pGEX-6P-2-CSN5 N-mutant (expresses the 1-106 aa region from the amino terminus of CSN5) pGEX-6P-2-CSN5 N-mutant (expresses the region 1-178 aa from the amino terminus of CSN5) pGEX-6P-2-CSN5 N-mutant (expresses the region 53-106 aa from the amino terminus of CSN5) pGEX-6P-2-CSN5 N-mutant (expresses the region 59-74 aa from the amino terminus of CSN5) pGEX-6P-2-CSN5 C-mutant (expresses the region 199-265 aa from the amino terminus of CSN5) pGEX-6P-2-CSN5 C-mutant (expresses the region 252-334 aa from the amino terminus of CSN5) pGEX-6P-2-CSN5 C-mutant (expresses the region 285-334 aa from the amino terminus of CSN5) pGEX-6P-2-CSN5 C-mutant (expresses the region 233-252 aa counting from the amino terminus of CSN5) pGEX-6P-2-CDK2 Full length (expresses the 1-298 aa region from the amino terminus of CDK2) pGEX-6P-2-Antennapedia (Antp) (expressing the membrane-permeable peptide of Antennapedia) pGEX-6P-2-Antp-CSN5pI (expresses a fusion protein of Antennapedia's membrane-permeable peptide and CSN5 peptide (199-265)) MT-CSN5(HA233-252) (expressing CSN5 peptide (233-252)) GFP-neo-marker vector [Cell line] HEK293T cells (Human embryonic kidney 293T, a cell line derived from human embryonic kidney) NIH3T3 cells (mouse fibroblasts) U87-mg cells (derived from human glioblastoma) K562 cells (human leukemia-derived cell line).

[0063] [antibody] Anti-CSN5 Rabbit polyclonal antibodies (prepared by the present inventors) Anti-HA Mouse monoclonal antibodies to HA peptide epitopes (manufactured by Sigma) Protein A Horseradish Peroxidase labeled (GE Heathcare Bioscience) Anti-Mouse IgG, HRP-Linked Whole Ab Sheep (GE Heathcare Bioscience) Alexa Fluor 594 goat anti-mouse / rabbit IgG (Invitrogen) Anti-Glutathion S-transferase (GST) rabbit polyclonal antibody (manufactured by Santa Cruz) Anti-γtubulin mouse polyclonal antibody (Sigma).

[0064] [Cloning] PCR primers (Forward primer-tctagagctccagccatgctctcc (SEQ ID NO: 3), Reverse primer-aagcttagcggctcacttcagggt (SEQ ID NO: 4)), PCR reaction solution (ultrapure water, 10x PCR buffer, dNTP mix, MDH2 forward primer and reverse primer, K562 cDNA, rTaq), and the template K562 cDNA library DNA were mixed and PCR was performed to amplify MDH2 cDNA. In the PCR, (i) the above-mentioned mixture was maintained at 94°C for 3 minutes, (ii) the mixture was subjected to an amplification reaction in which the reaction cycle consisting of a denaturation reaction at 94°C for 15 seconds, an annealing reaction at 62°C for 30 seconds, and an extension reaction at 72°C for 1 minute was repeated for 25 cycles, and (iii) the mixture after the amplification reaction was maintained at 72°C for 7 minutes.

[0065] The reaction product was then subjected to agarose gel electrophoresis, and 1 kb of MDH2 cDNA was excised from the agarose gel. The MDH2 cDNA was inserted into the pGEM-T easy vector, and the vector was introduced into E. coli DH5α, which was then plated on an LB plate (containing 50 μg / ml ampicillin). The vector was extracted from the transformant that appeared on the LB plate, and the sequence of the MDH2 cDNA inserted into the vector was confirmed.

[0066] [Cell culture] The cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% FBS (Fetal Bovine Serum), 80 units / ml penicillin, and 100 μg / ml streptomycin at 37°C and 5% CO 2 Cells (HEK293T cells, NIH3T3 cells) were cultured in an incubator under atmospheric conditions.

[0067] [Calcium phosphate transformation] 4.5 μg of plasmid DNA in 250 mM CaCl 2 The cells were suspended in an equal volume of 2xBBS (50 mM BES (N,N-Bis(2-hydroxyethyl)-2-aminoethanesulfonic Acid), 280 mM NaCl, 1.5 mM Na 2 HPO 4 , pH 7.10) was added and mixed, and then left to stand at room temperature for 15 minutes to prepare a transformation solution. After adding the transformation solution to cells cultured at 10% density on a 35 mm dish, the cells were cultured for 14 hours. After confirming GFP fluorescence, the cells were washed with PBS and further cultured in growth medium for 48 hours.

[0068] [Protein extraction from cells] After washing the cells (HEK293T cells) once with PBS (Phosphate-Buffered Saline), the cells were added with 100KIU / μL aprotinin, 1 mM PMSF (Phenylmethylsulfonyl Fluoride), 0.1 mM NAF (Sodium Fluoride), 0.1 mM Na 3 VO 4 EBC buffer (50 mM Tris-HCl (pH 8.0), 120 mM NaCl, 1 mM EDTA, 0.5% NP40) containing sodium orthovanadate and 10 mM β-glycerophosphate was added. After the cells were detached from the culture dish using a scraper, the cells were collected in a 1.5 mL tube. The collected cells were centrifuged to collect the soluble fraction, which was used as a cell extract.

[0069] [Induction of GST fusion protein expression] The transformed E. coli was suspended in LB liquid medium (containing 50 μg / ml ampicillin) and precultured overnight at 37°C. The precultured LB liquid medium was added to new LB liquid medium at 1% by volume, and cultured with shaking at 37°C until the OD value reached 1.0 or higher. 1M Isopropyl-β-thiogalactopyranoside (IPTG) was added to the LB liquid medium during the shaking culture at a final concentration of 0.35 mM, and the culture was continued for another 1.5 hours. After the culture, the E. coli was collected. The collected E. coli was subjected to SDS-PAGE and CBB staining to confirm the expression of the desired protein.

[0070] [Purification of GST fusion proteins] E. coli in which expression of GST fusion protein had been induced was suspended in 1 mL of NETN buffer (100 mM NaCl, 1 mM EDTA, 20 mM Tris-HCl (pH 8.0), 0.5% SDS, NP40) and disrupted using an ultrasonic disrupter. After disruption, the disrupted solution was centrifuged to collect the supernatant, and 10 μL of Gultathione beads (Glutathione Sepharose 4B) (GE Healthcare) was added to the supernatant and mixed at 4°C for 2 hours. The Gultathione beads were then washed three times using ETN buffer (100 mM NaCl, 1 mM EDTA, 20 mM Tris-HCl (pH 8.0)). Finally, the glutathione beads were washed once with NETN buffer or DETN buffer (100 mM NaCl, 1 mM EDTA, 20 mM Tris-HCl (pH 8.0), 0.5% deoxycholic acid) to obtain purified GST fusion proteins.

[0071] [GST pull-down method] After adding a washing solution to the beads to which the GST fusion protein was added, the beads were centrifuged, the supernatant was removed, and the beads were washed. Then, a predetermined amount of sample solution (HEL293T cell-derived extract) was added to the beads, and the beads were contacted with the sample solution for 2 hours at room temperature while performing tapping treatment every 10 minutes. Then, the beads were washed with EBC buffer (50 mM Tris-HCl (pH 8.0), 120 mM NaCl, 1 mM EDTA, 0.5% NP40), and the beads were added with 2×SDS sample buffer (80 mM Tris-HCl (pH 6.8), 2% SDS, 20% Glycerol, 0.1% Bromophenol Blue, 5% 2-Mercaptoethanol) to extract proteins from the beads.

[0072] [SDS-PAGE] The sample prepared by the GST pull-down method was heated to 98°C for 5 minutes. After preparing a separation gel (12% acrylamide gel solution, 0.3M Tris-HCl (pH 8.8), 0.1% SDS (sodium dodecylsulfate), 0.04% APS (ammonium persulfate), 0.06% TEMED (tetramethylethylenediamine)), a concentration gel (4% acrylamide solution, 0.1M Tris-HCl (pH 6.8), 0.1% SDS, 1% APS, 0.13% TEMED) was prepared on the separation gel to prepare a gel for SDS-PAGE. After filling the electrophoretic layer with SDS electrophoresis buffer (25mM Tris, 1.4% Glycine, 0.1% SDS), the sample was applied to the gel for SDS-PAGE and electrophoresis was performed at 10mA for about 3 hours.

[0073] [CBB staining] After SDS-PAGE, the gel was immersed in a staining solution (0.25% Coomassie Brilliant Blue, 40% methanol, 10% acetic acid) and stained at room temperature, and then the gel was immersed in a destaining solution (40% methanol, 10% acetic acid) and destained.

[0074] [Western blotting] The gel after SDS-PAGE and a PVDF (Polyvinylidene fluoride) membrane that had been hydrophilized with 100% methanol for 1 minute were immersed in transfer buffer (25 mM Tris, 192 mM glycine, 20% methanol) for 10 minutes. The gel and PVDF membrane immersed in the transfer buffer were sandwiched between filter paper, and the proteins were transferred from the gel to the PVDF membrane at 120 mA for 2 hours. After the transfer, the PVDF membrane was subjected to a blocking treatment with 5% bovineserum albumin (BSA) at 4°C for 2 hours. The PVDF membrane was reacted with a primary antibody diluted with BSA. After washing the PVDF membrane, the PVDF membrane was subjected to a blocking treatment with 2.5% skimmilk. The PVDF membrane was reacted with a secondary antibody diluted with skim milk. After washing the PVDF membrane, the desired protein was detected using an ECL kit (GE Healthcare Bioscience) and an X-ray film.

[0075] [Immunostaining] 25 nM Mitotracker (Invitrogen) was added to the cultured cells (transformed NIH3T3 cells), and the cells were incubated at 37°C for 45 minutes in CO 2 The staining reaction was carried out in an incubator. After that, the cells were washed with PBS, and then a 4% paraformaldehyde solution prepared using PBS was added to the cells, and the cells were fixed for 10 minutes. After washing the cells with PBS, chilled 100% methanol was added to the cells, and the cells were permeabilized at -20°C for 10 minutes. After washing the cells with PBS, blocking was carried out for 30 minutes using 5% BSA. After removing the BSA, a primary antibody solution was added and left to stand for 1 hour. After that, the cells were washed with PBS, and after blocking with 5% BSA, a secondary antibody solution was added and left to stand for 30 minutes. After that, the cells were washed with PBS, and a 5% BSA solution (containing 1 μg / mL Hoechist) was added, and the cells were mounted on a slide glass. After drying prevention treatment with nail polish, the cells were observed with a confocal laser scanning microscope (ZEISS LSM 700).

[0076] [Colocalization analysis] Using the image analysis program Fiji

[44] , the images were divided into three colors, red, green, and blue, by the Split Channel, and two colors were set to measure colocalization. The images were enclosed based on the size of the cells to be measured, and the enclosed areas were set as the measurement ranges (Regions of interest (ROI)). Colocalization of the two colors per cell was quantified by calculating the Pearson correlation coefficient using the Coloc 2 plug-in.

[0077] [Statistical processing] Colocalization data were analyzed using the Mann-Whitney U test, and differences in data were considered statistically significant at P<0.01 and P<0.05.

[0078] [Test 1. Identification of amino acid region] To identify the amino acid region in CSN5 to which CDK2 binds, we prepared fusion proteins of GST and various fragments of CSN5. After confirming the amount of protein by SDS-PAGE gel electrophoresis and CBB staining, we used the GST pull-down method to identify the amino acid region in CSN5 to which CDK2 binds. As CDK2, we used a fusion protein of GFP and CDK2.

[0079] The results are shown in Figures 1A to 1D and Figure 2. Figures 1A to 1D show that a region including amino acid regions 59-74 and 233-252 of CSN5 can bind to CDK2. Figure 2 shows a list of amino acid regions in CSN5 to which CDK2 binds. In Figure 2, "○" indicates an amino acid region in CSN5 to which CDK2 binds, and "×" indicates an amino acid region in CSN5 to which CDK2 does not bind.

[0080] [Test 2. Observation of the complex in cells] A vector expressing a fusion protein of the 199-265 amino acid region of CSN5 and an HA tag was introduced into HEK293T cells. The cells were cultured for 48 hours, and the protein was extracted. The extract was subjected to immunoprecipitation with anti-HA or anti-CSN5 antibodies, and the complex was detected by Western blotting.

[0081] The results are shown in Figure 3. In Figure 3, A, immunoprecipitation and Western blotting were performed with an anti-HA antibody, and in B, with an anti-CSN5 antibody. In addition, in C, an anti-HA antibody or an anti-CSN antibody was used for immunoprecipitation, and an anti-CDK2 antibody was used for Western blotting. In addition, NRS means a negative control. From Figure 3, it can be seen that a complex between CSN5 and CDK2 is formed in HEK293T cells. Also, from Figure 3, it was found that (i) the CSN5(199-265) peptide significantly reduces the complex between CSN5 and CDK2, and (ii) the CSN5(199-265) peptide significantly increases the complex between CSN5(199-265) peptide and CDK2.

[0082] [Test 3. Effect of CSN5 peptide on the complex] A CSN5(199-265) peptide expression vector was introduced into HEK293T cells, and after 48 hours of culture, protein was extracted. The extracted protein was subjected to SDS non-denaturing gel electrophoresis to separate the large complex containing CSN5 (CSN complex) and the small complex containing CSN5 (CSN small complex). The large complex containing CSN5 and the small complex containing CSN5 were then detected by Western blotting using an anti-CSN5 antibody.

[0083] The results are shown in Figure 4. Figure 4A shows that when the CSN5 peptide was expressed, the small complex was not observed. Therefore, it was shown that the CSN5 peptide specifically inhibits only the formation of the CSN5 small complex. Furthermore, Figure 4B shows that the introduction of the CSN5(199-265) peptide does not affect the expression levels of other proteins (e.g., Clu1, Clu2, γ-tubulin) or Nedd8 conjugation. Since these proteins are targets of the COP9 signalosome (large complex), the fact that the expression levels of these proteins did not change indicates that nonspecific growth inhibition was not occurring, including in normal cells.

[0084] [Test 4. Effect of CSN5(199-265) peptide on cancer cells] U87-MG (human glioblastoma-derived) cells and NIH3T3 (mouse fibroblast) cells were transfected with (i) a neoGFP marker vector, (ii) a combination of the neoGFP marker vector and CSN5(199-265) peptide expression vector, or (iii) a combination of the neoGFP marker vector and a control peptide vector. The cells transfected with the vectors were cultured for 4 days and then selectively cultured in the presence of G418 for an additional 7 days. Cell proliferation and cell morphology were confirmed by microscopic observation.

[0085] The results are shown in Figure 5. As shown in Figure 5, human cancer cells, U87-MG, were unable to grow in the presence of the CSN5(199-265) peptide. On the other hand, normal cells, NIH3T3, grew in the presence of the CSN5(199-265) peptide, and no abnormalities were observed in the cell morphology. Therefore, it was demonstrated that the CSN5(199-265) peptide specifically inhibits the growth of cancer cells.

[0086] [Test 5. Effect of CSN5(199-265) peptide on tumor cells] NIH3T3 cells were transfected with (i) an expression vector for activated Ras, (ii) a combination of an expression vector for activated Ras and a peptide expression vector for CSN5 1-52, (iii) a combination of an expression vector for activated Ras and a peptide expression vector for CSN5 199-265, or (iii) a GFP expression vector. Activated Ras has the ability to cause cells to become cancerous. These cells were subcutaneously transplanted into nude mice. The size of the tumor formed by the transplanted cells was then measured over time.

[0087] The results are shown in Figure 6. The horizontal axis of the graph in Figure 6 is the number of days, and the vertical axis is the tumor volume (mm 3 6. It can be seen from Fig. 6 that tumors do not grow in the individuals into which the CSN5(199-265) peptide expression vector has been introduced. This demonstrates that the CSN5(199-265) peptide has the effect of suppressing the growth of tumor cells.

[0088] [Test 6. Effect of CSN5(199-265) peptide on human leukemia cells] (i) neoGFP marker vector, (ii) combination of neoGFP marker vector and CSN5(199-265) expression vector, or (iii) combination of neoGFP marker vector and control peptide expression vector were introduced into K562 human leukemia-derived cell line by electroporation. After 5 days of culture, GFP-expressing cells were sorted by cell sorter, and the number of cells was measured by colocalization analysis after 1 and 2 days.

[0089] The results are shown in Figure 7. The horizontal axis of the graph in Figure 7 is the number of days, and the vertical axis is the number of cells (×10 4 7) represents the number of cells in the leukemia cell line. As can be seen from FIG. 7, the introduction of the CSN5(199-265) peptide inhibited the proliferation of K562 cells, which are derived from human leukocytes. This indicates that the CSN5(199-265) peptide is also effective in inhibiting the proliferation of leukemia cells.

[0090] [Test 7. Direct addition of peptides to HEK293T cells] PBS, GST, GST-Antp, and GST-Antp-CSN5(199-265) were incubated with HEK293T cells (5 × 10 3 2 μL of each was added to the 100 μL of each of the 100 μL of ...

[0091] After 72 hours of culture, the cell extract was collected. The collected cell extract was subjected to Western blotting using anti-GST antibody and anti-γ-tublin antibody to detect proteins. The extract of the cells to which GST-Antp was added was diluted 10-fold before use.

[0092] The detection results are shown in Figure 8 A. Figure 8 A shows that each cell extract contained the added peptide.

[0093] [Test 8. Effect of direct addition of peptides on cell proliferation] The peptides used in Test 7 were each added at 2 μM to HEK293T cells. Cells were also prepared by adding GST-Antp-CSN5(199-265) at 1 μM or 0.5 μM. After culturing these cells for 24 hours, 48 ​​hours, or 72 hours, the cells were harvested and stained with trypan blue to measure the number of live and dead cells.

[0094] The measurement results are shown in Figures 8B to 8D. Note that Figure 8C shows only the results for cells to which 2 μM of GST-Antp-CSN5 (199-265) was added. From Figure 8B, it was found that cell proliferation after 72 hours was suppressed in a concentration-dependent manner of GST-Antp-CSN5 (199-265). On the other hand, in Figure 8C, no statistically significant difference was observed in the number of dead cells after 72 hours. Also, from Figure 8D, it was found that proliferation of cells to which GST-Antp-CSN5 (199-265) was added was suppressed after 72 hours. Therefore, it was shown that GST-Antp-CSN5 (199-265) has the effect of suppressing proliferation of cells expressing CSN5 and CDK2.

[0095] [Test 9. CSN5 transfection into U87-MG cells] The neo-GFP marker vector and expression vectors expressing the 1-25, 1-52, 199-265, or 233-252 fragments of CSN5 bound to an anti-HA antibody tag were introduced into U87-MG cells (derived from human glioblastoma) by electroporation. For comparison, cells carrying only the neo-GFP marker vector were also prepared. The introduced cells were cultured in the presence of G418 for one week, and then the cell proliferation and morphology were observed under an optical microscope.

[0096] The results are shown in Figure 9. The left side (PC) of the upper drawing in Figure 9 is a cell that has not been transfected with a neo-GFP marker vector, and the right side is a cell that has been transfected with a GFP marker vector. Figure 9 shows that the cells transfected with 199-265 and 233-252 of CSN5 did not proliferate and showed no abnormalities in cell morphology. This shows that the proliferation of human cancer cells is suppressed under the expression of CSN5(199-265) or CSN5(233-252).

[0097] [Test 10. Immunostaining of CSN5-transfected cells] After culturing each of the cells prepared in Test 9 for a specified period of time, immunostaining was performed using α anti-HA antibody.

[0098] The results are shown in Figure 10. Figure 10 shows that the cells expressing GFP also express the desired peptide, and that the cells into which CSN5 199-265 and 233-252 have been introduced do not proliferate and have no abnormalities in cell morphology. Thus, as in Figure 9, it was shown that the proliferation of human cancer cells is suppressed under the expression of CSN5(199-265) or CSN5(233-252).

[0099] [Test 11. Introduction of zinc-inducible expression vector] K562 cells were transfected with the neo marker vector and the CSN5(233-252) peptide expression MT vector, in which expression of CSN5(233-252) is induced by zinc, by electroporation. The transfected cells were selectively cultured in the presence or absence of zinc in the presence of G418 for 4 weeks. Clonal cell lines were then isolated and their proliferation rates were examined.

[0100] The results are shown in Figure 11. In Figure 11, K means the control, and MT clone means the cloned cell line. Figure 11 shows that cell proliferation was suppressed only when the CSN5 (233-252) peptide was expressed. This shows that the proliferation of human leukemia cells was suppressed by the expression of the CSN5 (233-252) peptide. [Industrial Applicability]

[0101] The present invention can be utilized in the treatment or therapy of cancer.

Claims

1. An antitumor agent comprising an expression vector comprising any one of the polynucleotides selected from the group consisting of the following (a) to (e), and / or any one of the polypeptides selected from the group consisting of the following (f) to (i): (a) a polynucleotide encoding a polypeptide consisting of only the amino acid sequence represented by the 233rd to 252nd amino acid residues counting from the amino terminus of the amino acid sequence represented by SEQ ID NO:1; (b) a polynucleotide encoding a polypeptide consisting of an amino acid sequence in which one or several amino acid residues have been substituted, deleted, inserted and / or added in the amino acid sequence depicted in SEQ ID NO:1 at positions 233 to 252 counting from the amino terminus, and having an activity of binding to CDK2; (c) a polynucleotide encoding a polypeptide consisting of an amino acid sequence having 90% or more identity with the amino acid sequence represented by the 233rd to 252nd amino acid residues counting from the amino terminal of the amino acid sequence represented by SEQ ID NO:1 and having an activity of binding to CDK2; (d) a polynucleotide consisting of only the nucleotide sequence represented by the 697th to 756th bases counting from the 5' end of the nucleotide sequence represented by SEQ ID NO:2; (e) a polynucleotide that hybridizes under stringent conditions to a polynucleotide having a nucleotide sequence complementary to any one of the polynucleotides (a) to (d) and encodes a polypeptide having an activity of binding to CDK2; (f) a polypeptide consisting of only the amino acid sequence represented by the 233rd to 252nd amino acid residues counting from the amino terminus of the amino acid sequence represented by SEQ ID NO:1; (g) a polypeptide consisting of an amino acid sequence in which one or several amino acid residues have been substituted, deleted, inserted and / or added in the amino acid sequence depicted in SEQ ID NO:1 at positions 233 to 252 counting from the amino terminal thereof, and having an activity of binding to CDK2; (h) A polypeptide consisting of an amino acid sequence having 90% or more identity with the amino acid sequence represented by the 233rd to 252nd amino acid residues counting from the amino terminus of the amino acid sequence represented by SEQ ID NO:1, and having the activity of binding to CDK2.

2. The antitumor agent according to claim 1, comprising an expression vector comprising any one of the polynucleotides selected from the group consisting of the following (a') to (e') and / or any one of the polypeptides selected from the group consisting of the following (f') to (i'): (a') a polynucleotide encoding a polypeptide consisting of only the amino acid sequence represented by the 199th to 265th amino acid residues counting from the amino terminal of the amino acid sequence represented by SEQ ID NO:1; (b') a polynucleotide encoding a polypeptide consisting of an amino acid sequence in which one or several amino acid residues have been substituted, deleted, inserted and / or added in the amino acid sequence depicted in SEQ ID NO:1 at positions 199 to 265 counting from the amino terminal thereof, and having an activity of binding to CDK2; (c') a polynucleotide encoding a polypeptide consisting of an amino acid sequence having 90% or more identity with the amino acid sequence represented by the 199th to 265th amino acids counting from the amino terminal of the amino acid sequence represented by SEQ ID NO:1, and having an activity of binding to CDK2; (d') a polynucleotide consisting of the nucleotide sequence represented by the 595th to 795th bases counting from the 5' end of the nucleotide sequence represented by SEQ ID NO:2; (e') a polynucleotide which hybridizes under stringent conditions to a polynucleotide having a nucleotide sequence complementary to any one of the polynucleotides (a') to (d') and encodes a polypeptide having an activity of binding to CDK2; (f') a polypeptide consisting of only the amino acid sequence represented by the 199th to 265th amino acid residues counting from the amino terminal of the amino acid sequence represented by SEQ ID NO:1; (g') a polypeptide consisting of an amino acid sequence in which one or several amino acid residues have been substituted, deleted, inserted and / or added in the amino acid sequence represented by SEQ ID NO:1 at positions 199 to 265 counting from the amino terminal thereof, and having an activity of binding to CDK2; (h') A polypeptide consisting of an amino acid sequence having 90% or more identity with the amino acid sequence represented by the 199th to 265th amino acids counting from the amino terminus of the amino acid sequence shown in SEQ ID NO:1, and having the activity of binding to CDK2.

3. The antitumor agent according to claim 1 or 2, wherein the polypeptide encoded by the polynucleotide contained in the expression vector further comprises a membrane-permeable peptide.

Images