JP5397692B2 - Malignant melanoma antigen expression increasing agent and use thereof - Google Patents

Description

  The present invention relates to an agent for increasing expression of malignant melanoma antigen and use thereof.

In recent years, immunotherapy for malignant tumors including melanoma has been frequently performed as an adjuvant therapy. However, the clinical effect of immunotherapy is not so high, and researches for increasing the response rate are being actively conducted. Most of these studies are aimed at increasing immunity, especially the ability to attack lymphocytes and dendritic cells.
By the way, it is considered that the decrease in the expression or disappearance of cancer antigen, which is called “cancer immune escape phenomenon”, is related to the fact that the clinical effect does not increase so much. Cancer antigens are necessary for dendritic cells to activate lymphocytes against cancer and serve as landmarks when lymphocytes attack cancer cells. If the cancer antigen disappears, lymphocytes will not be able to attack the cancer and immunotherapy will not be effective. Therefore, it is considered that the effect of immunotherapy can be enhanced by increasing the expression of cancer antigen. Techniques and some drugs for this concept are shown in Patent Documents 1 and 2 and Non-Patent Documents 1 and 2.
International Publication No. 2004/087941 Pamphlet International Publication No. 2004/019886 Pamphlet Kono M et al. Mol Cancer Res. 2006 Oct; 4 (10): 779-92. Dunn IS et al. J Immunol. 2007 Aug: 179 (4): 2134-42.

The substances shown in Patent Document 1 and Non-Patent Document 1 are MAP kinase inhibitors that have not been used as pharmaceuticals, and their clinical application requires large-scale clinical trials. In the methods disclosed in Patent Document 2 and Non-Patent Document 2, interferon β is used. Currently, interferon beta is actually used to treat melanoma.
In view of the above background, an object of the present invention is to provide an effective means for treating melanoma.

The inventors of the present invention have repeatedly studied for the purpose of finding a novel substance that promotes the increased expression of an antigen (melanoma antigen) in melanoma cells. Specifically, an evaluation system using melanoma cells was constructed, and various drugs / compounds were screened using it. As a result, 11 types of drugs / compounds were found to promote the increased expression of melanoma antigen. Among the drugs / compounds that have been confirmed to be effective, substances already used as anticancer agents and the like are included, and early clinical application of them is expected. As a result of conducting screening with a wide range of subjects, it was found that nine more drugs / compounds promote increased melanoma antigen expression. As a result of further investigation, important and interesting findings were obtained regarding the mechanism that causes the increased expression of melanoma antigen.
The present invention is based on the above results and has the following configuration.
[1] Methotrexate, paclitaxel, daunorubicin hydrochloride (Daunorubicin, HCl), doxorubicin hydrochloride (Doxorubicin, HCl), actinomycin D, camptothecin, etoposide, etoposide (Cytochalasin D), 3-ATA (3-amino-9-thio (10H) -acridone), Aphidicolin, Bisindolylmaleimide I, Cytosine Arabinoside, Curcumin ), 2'3'-dideoxythymidine triphosphate, 2'3'-dideoxythymidine 5'-triphosphate, dipyrimidine dithymidylic acid, Pyridoxal 5'-phosphate hydrate, epigallo Catechin gallate ((-)-Epigallocatechin gallate), vidarabine, resveratrol and ritocor It is selected from the group consisting of acid (Lithocholic acid) comprising a compound, increased expression agent for malignant melanoma antigens.
[2] Malignant melanoma antigen consisting of a compound selected from the group consisting of paclitaxel, daunorubicin hydrochloride (Daunorubicin, HCl), doxorubicin hydrochloride (Doxorubicin, HCl), camptothecin and aphidicolin Expression-enhancing agent.
[3] Compound selected from the group consisting of arabinonucleoside (Cytosine Arabinoside), epigallocatechin gallate ((-)-Epigallocatechin gallate), vidarabine, resveratrol and lithocholic acid An agent for increasing the expression of malignant melanoma antigen.
[4] A malignant melanoma antigen expression increasing agent comprising a compound having a topoisomerase inhibitory action.
[5] A malignant melanoma antigen expression increasing agent comprising a compound having a DNA polymerase inhibitory action and an RNA polymerase inhibitory action.
[6] An agent for increasing the expression of malignant melanoma antigen, comprising a compound that suppresses the expression of a DNA polymerase gene or suppresses the action of a DNA polymerase.
[7] The agent for increasing expression of malignant melanoma antigen according to [6], wherein the compound is a compound selected from the group consisting of the following (a) to (f):
(a) siRNA targeting the DNA polymerase gene;
(b) a nucleic acid construct that generates siRNA targeting the DNA polymerase gene in the cell;
(c) an antisense nucleic acid that targets a transcript of a DNA polymerase gene;
(d) a ribozyme that targets a transcript of a DNA polymerase gene;
(e) a nucleic acid construct that generates a dominant negative mutant of the protein encoded by the DNA polymerase gene in the cell;
(f) A dominant negative mutant of the protein encoded by the DNA polymerase gene.
[8] The malignant melanoma antigen expression-enhancing agent according to [6] or [7], wherein the DNA polymerase gene is a DNA polymerase δ1 gene, a DNA polymerase δ2 gene, a DNA polymerase δ3 gene or a DNA polymerase δ4 gene.
[9] A medicament for treating malignant melanoma, comprising as an active ingredient the expression-enhancing agent according to any one of [1] to [8].
[10] The medicament for treating malignant melanoma according to [9], which is a medicament used for immunotherapy.
[11] The medicament for treating malignant melanoma according to [9], which is a medicament used for immunotherapy combined with one or more therapies selected from the group consisting of surgical resection, chemotherapy, and radiation therapy .
[12] Immunotherapy from cancer vaccine therapy, cytokine therapy, activated autolymphocyte therapy (CAT therapy), cytotoxic T lymphocyte transfer therapy (CTL therapy), and tumor tissue infiltrating T lymphocyte therapy (TIL therapy) The medicament for treating malignant melanoma according to [10] or [11], which is any treatment method selected from the group consisting of:

Comparison of melanoma antigen MART1 expression increasing activity of 11 drugs / compounds. Evaluation was performed in a test system using SK-MEL28 cells. Comparison of melanoma antigen gp100 expression increasing activity of 11 drugs / compounds. Evaluation was performed in a test system using SK-MEL28 cells. Comparison of melanoma antigen TYR expression increasing activity of 11 drugs / compounds. Evaluation was performed in a test system using SK-MEL28 cells. Comparison of melanoma antigen TRP1 expression increasing activity of 11 drugs / compounds. Evaluation was performed in a test system using SK-MEL28 cells. The test result of the test system using SK-MEL28 cells. Comparison of melanoma antigen MART1 expression increasing activity of 11 drugs / compounds. Evaluation was performed in a test system using A375 cells. Comparison of melanoma antigen gp100 expression increasing activity of 11 drugs / compounds. Evaluation was performed in a test system using A375 cells. Comparison of melanoma antigen TYR expression increasing activity of 11 drugs / compounds. Evaluation was performed in a test system using A375 cells. Comparison of melanoma antigen TRP1 expression increasing activity of 11 drugs / compounds. Evaluation was performed in a test system using A375 cells. Test results of test system using A375 cells. Comparison of activity to increase expression of various melanoma antigens of 9 drugs / compounds (test system using SK-MEL28 cells). Comparison of activity to increase expression of various melanoma antigens of 9 drugs / compounds (test system using A375 cells). Results of experiments using tumor-bearing mice. The effects of the three drugs Aphidicolin, methotrexate and doxorubicin were compared. Results of experiments using tumor-bearing mice. The expression level of melanoma antigen when methotrexate was administered for 3 days is shown. Enhanced expression of tumor histocompatibility complex (MHC) class I. For various drugs that promote the increased expression of melanoma antigen, the effect of enhancing expression of MHC class I (protein level) was compared using A375 cells. Results of RNAi targeting the DNA polymerase δ1 (POLD1) gene (quantitative PCR). It can be seen that inhibition of POLD1 gene expression increases mRNA expression of melanoma antigens MART1 and gp100. Results of RNAi targeting the DNA polymerase δ2 (POLD2) gene (quantitative PCR). Inhibition of POLD2 gene expression increases mRNA expression of melanoma antigens MART1, TYR, and TRP1. Results of RNAi targeting the DNA polymerase δ3 (POLD3) gene (quantitative PCR). Inhibition of POLD3 gene expression increases mRNA expression of melanoma antigens MART1, gp100, TYR, and TRP1. Results of RNAi targeting the DNA polymerase δ4 (POLD4) gene (quantitative PCR). It can be seen that the expression of melanoma antigen TYR mRNA is increased by inhibition of POLD4 gene expression. Results of RNAi targeting the DNA polymerase δ1 (POLD1) gene (FCM). Increased expression of melanoma antigen MART1 due to inhibition of POLD1 gene expression was confirmed at the protein level.

(Malignant melanoma antigen expression increasing agent)
The first aspect of the present invention relates to an agent for increasing the expression of malignant melanoma (hereinafter also referred to as “melanoma”) antigen. “Malignant melanoma antigen” (that is, melanoma antigen) refers to a protein or peptide expressed in melanoma that is recognized by T lymphocytes, melanocyte-specific protein, cancer expressed in various cancer cells and normal testis cells − Testis antigens, peptides with amino acid mutations derived from cancer cell mutations, etc. So far, MART1, gp100, TYR (tyrosinase), TRP1, TRP2, MAGE1, MAGE2, MAGE3, MAGE6, NY-ESO-1, MUM1, etc. are known as melanoma antigens. The “malignant melanoma antigen expression-enhancing agent” refers to a drug that increases the expression level of malignant melanoma antigen in cells. The melanoma antigen targeted by the expression enhancer of the present invention is not particularly limited, but is preferably MART1, gp100, TYR (tyrosinase) or TRP1. In addition, the expression increasing agent of the present invention may target two or more melanoma antigens.

The expression-enhancing agent of the present invention includes methotrexate, paclitaxel, daunorubicin hydrochloride (Daunorubicin, HCl), doxorubicin hydrochloride (Doxorubicin, HCl), actinomycin D, camptothecin, and etoposide (Etoposide). ), Cytochalasin D, 3-ATA (3-amino-9-thio (10H) -acridone), Aphidicolin, Bisindolelylmaleimide I, Arabinonucleoside (Cytosine Arabinoside) , Ara-C), curcumin, 2'3'-dideoxythymidine triphosphate (2'3'-dideoxythymidine 5'-triphosphate, ddTTP), thymidine dimer (dipyrimidine dithymidylic acid, pTpT), pyridoxal Phosphoric acid (Pyridoxal 5′-phosphate hydrate), epigallocatechin gallate ((-)-Epigallocatechin gallate, EGCG), vidarabine, Consisting Veratororu (resveratrol) and the compound being selected from the group consisting of lithocholic acid (Lithocholic acid). As shown in Examples described later, all of these compounds alone promoted the increased expression of melanoma antigen. Among these, paclitaxel, daunorubicin hydrochloride, doxorubicin hydrochloride (Doxorubicin, HCl), camptothecin and aphidicolin showed strong activity in both the test system using SKMEL28 cells and the test system using A375 cells (Examples described later). And particularly preferred compounds. In addition, arabinonucleoside, epigallocatechin gallate, vidarabine, resveratrol and lithocholic acid also show high activity and are particularly preferred compounds.
As explained below, many of the compounds correspond to topoisomerase inhibitors. Based on this fact, one embodiment of the present invention provides an agent for increasing the expression of malignant melanoma antigen, comprising a compound having a topoisomerase inhibitory action.
On the other hand, arabinonucleoside, curcumin, 2'3'-dideoxythymidine triphosphate, thymidine dimer, pyridoxal phosphate, epigallocatechin gallate, vidarabine, resveratrol and lithocholic acid all inhibit DNA / RNA polymerase It is a drug known as a drug. Thus, one embodiment of the present invention provides a malignant melanoma antigen expression-enhancing agent comprising a compound having a DNA polymerase inhibitory action and an RNA polymerase inhibitory action.

  Methotrexate is an analog of folic acid that inhibits dihydrofolate reductase (DHFR), which produces active folic acid necessary for nucleic acid synthesis, and inhibits DNA synthesis. It also inhibits DNA synthesis by inhibiting folate-dependent enzymes such as DHFR and thymidylate synthase. Methotrexate causes cell growth inhibition and apoptosis and exhibits antitumor activity.

  Paclitaxel is an antitumor agent isolated from yew skin and binds to β-tubulin to promote association (inhibits depolymerization). This stops the tumor cells in the G2 / M phase of the cell cycle, inhibits division and causes apoptosis.

  Daunorubicin hydrochloride is an antitumor agent and a topoisomerase I and II inhibitor. Inhibits tumor cell growth by inhibiting DNA synthesis and DNA-dependent RNA synthesis.

  Doxorubicin hydrochloride is an antitumor antibiotic. A topoisomerase II inhibitor that stabilizes the covalent complex of topoisomerase II and DNA in the state of DNA strand breakage by topoisomerase II to inhibit DNA synthesis and RNA synthesis. It also binds to DNA in tumor cells by specific intercalation and prevents DNA cleavage during cell division.

  Actinomycin D is an anti-tumor antibiotic and inhibits DNA-dependent RNA polymerase by forming a complex with DNA via deoxyguanosine residues. At high concentrations, it exhibits an inhibitory effect on DNA polymerase. It also acts as a competitive inhibitor of serine proteases.

  Camptothecin is an alkaloid contained in Nothapodytes foetida and Camptotheca acuminata, and exhibits high antitumor activity and a broad antitumor spectrum. It is a DNA topoisomerase I inhibitor.

  Etoposide is a topoisomerase II inhibitor that stabilizes the covalent complex of topoisomerase II and DNA and inhibits DNA synthesis in the state of DNA strand breakage by topoisomerase II.

  Cytochalasin D is a polymerization inhibitor of actin filaments and suppresses cytokinesis.

  3-ATA is a polyanionic polyaromatic compound that is used as a potent inhibitor of cellular processes that depend on the formation of protein-nucleic acid complexes. In addition, 3-ATA has an activity of strongly inhibiting DNA topoisomerase II and acts as a potent inhibitor of angiogenesis.

  Aphidicolin is a DNA polymerase inhibitor. Specific inhibitors of polymerases α, δ, and ε that arrest cells in the G1 phase of the cell cycle.

  Bisindole maleimide I inhibits protein kinase C (PKC).

  Arabinonucleoside is a kind of antineoplastic agent (anticancer agent) and is a first-line drug for acute myeloid leukemia. It is also a pyrimidine analog and inhibits polymerase in an antagonistic manner with dCTP.

  Curcumin is a yellow pigment of turmeric (turmeric, Curcuma longa), a spice of curry, and is a kind of polyphenol. Curcumin non-antagonistically inhibits the binding of polymerase to the template DNA and substrate dNTPs.

  2'3'-dideoxythymidine triphosphate is incorporated into the DNA strand instead of dTTP to stop the polymerase elongation reaction.

  A thymidine dimer is a dimer that is generated when a chemical bond is formed between thymine bases when DNA is irradiated with ultraviolet rays or the like. Although it causes mutation, the living body has an excision repair mechanism specific to thymidine dimer.

  Pyridoxal phosphate binds non-antagonistically with the template DNA and antagonistically with the substrate dNTP and inhibits the polymerase. It inhibits polymerases from all animals, but strongly inhibits α and ε in particular.

  Epigallocatechin gallate is a component that is abundant in green tea. It binds and inhibits antagonisticly with the template DNA and non-antagonistically with the substrate dNTP. Inhibits all animal-derived polymerases, but strongly inhibits α and λ in particular. Epigallocatechin gallate is also a COX, NO, oxidative stress inhibitor, and telomerase inhibitor.

  Vidarabine is phosphorylated in the cell to form the corresponding nucleotide, and inhibits viral DNA polymerase activity to suppress DNA elongation, thereby exhibiting an antiviral effect.

  Resveratrol is a kind of polyphenol that is contained in grape skin and red wine. Binds to polymerase α and inhibits non-antagonistically with substrate dNTPs. Inhibits polymerases α, δ, and λ, but not β.

Lithocholic acid is a type of bile acid and is also present in trace amounts in the human body. It binds to the N-terminal 8 kDa domain of polymerase β. It does not bind to the 31 kDa catalytic domain. Inhibits all animal-derived polymerases but strongly inhibits β.
In addition, all the above compounds are marketed and can be obtained easily.

As shown in Examples described later, it was found that suppression of DNA polymerase gene expression is effective as a means for promoting an increase in the expression of melanoma antigen. Based on this knowledge, one embodiment of the present invention provides an agent for increasing the expression of malignant melanoma antigen, comprising a compound that suppresses the expression of a DNA polymerase gene. On the other hand, according to the above findings, it can be said that suppression of the action of DNA polymerase is also effective as a means for promoting the increased expression of melanoma antigen. Therefore, in another aspect of the present invention, there is provided an agent for increasing the expression of malignant melanoma antigen, comprising a compound that suppresses the action of DNA polymerase. Examples of the compounds constituting the present invention are as follows. Inhibiting the expression of the DNA polymerase gene means suppressing the expression process of the DNA polymerase gene (including transcription, post-transcriptional regulation, translation, and post-translational regulation). In addition, “expression suppression” in the present invention may be either transient suppression or constant suppression.
(a) siRNA targeting DNA polymerase gene
(b) Nucleic acid construct that generates siRNA targeting DNA polymerase gene in cells
(c) Antisense nucleic acid targeting the transcript of the DNA polymerase gene
(d) Ribozymes that target DNA polymerase gene transcripts
(e) a nucleic acid construct that generates a dominant negative mutant of a protein encoded by a DNA polymerase gene in a cell
(f) Dominant negative mutant of the protein encoded by the DNA polymerase gene

  Among (a) to (f) above, (a) to (d) correspond to “compounds that suppress the expression of the DNA polymerase gene”, and (e) and (f) above “suppress the action of the DNA polymerase. Corresponding to “Compound to be”.

  There are more than 15 types of DNA polymerase, and each DNA polymerase is a complex of several subunits. The compound of the present invention suppresses the expression of a gene encoding any subunit of the target DNA polymerase, or suppresses the intended effect, ie, malignant melanoma antigen, by suppressing the function of the subunit. Demonstrates the effect of increasing expression.

  The above (a) and (b) are compounds used for expression suppression by so-called RNAi (RNA interference). In other words, according to the expression enhancer of the present invention comprising the compound (a) or (b) as an active ingredient, the expression of DNA polymerase can be suppressed by RNAi. RNAi is a sequence-specific post-transcriptional gene repression process that can occur in eukaryotic cells. In RNAi for mammalian cells, a short double-stranded RNA (siRNA) having a sequence corresponding to the sequence of the target mRNA is used. Usually, siRNA is 21-23 base pairs. Mammalian cells are known to have two pathways (sequence specific pathway and sequence non-specific pathway) that are affected by double-stranded RNA (dsRNA). In sequence-specific pathways, relatively long dsRNAs are split into short interfering RNAs (ie siRNAs). On the other hand, a sequence non-specific pathway is considered to be caused by an arbitrary dsRNA regardless of the sequence as long as it has a predetermined length or more. In this pathway, dsRNA becomes an active form, namely PKR, which stops all protein synthesis by phosphorylating the translation initiation factor eIF2 and 2 ', 5' oligoadenyl, which is involved in the synthesis of RNAase L activation molecules. Acid synthase is activated. To minimize the progression of this non-specific pathway, it is preferable to use double stranded RNA (siRNA) shorter than about 30 base pairs (Hunter et al. (1975) J Biol Chem 250: 409-17 Manche et al. (1992) Mol Cell Biol 12: 5239-48; Minks et al. (1979) J Biol Chem 254: 10180-3; and Elbashir et al. (2001) Nature 411: 494-8. Wanna)

  In order to generate target-specific RNAi, a siRNA composed of a sense RNA homologous to a part of the mRNA sequence of the target gene and an antisense RNA complementary thereto is introduced into the cell or expressed in the cell. Just do it. The above (a) is a compound corresponding to the former method, and similarly the above (b) is a compound corresponding to the latter method.

  The siRNA targeting the target gene (in the present invention, the DNA polymerase gene) is usually a hybrid of a sense RNA consisting of a sequence homologous to a continuous region in the mRNA sequence of the gene and an antisense RNA consisting of its complementary sequence. Soy double-stranded RNA. The length of the “continuous region” here is usually 15 to 30 bases, preferably 18 to 23 bases, more preferably 19 to 21 bases.

  It is known that double-stranded RNA having an overhang of several bases at the end exhibits a high RNAi effect. Therefore, in the present invention, it is preferable to employ siRNA having such a structure. The length of the base forming the overhang is not particularly limited, but is preferably 2 bases (for example, TT, UU).

  You may decide to use siRNA which consists of modified RNA. Examples of modifications herein include phosphorothioation and the use of modified bases (eg, fluorescently labeled bases).

  siRNA can be designed and prepared by conventional methods. In designing siRNA, a sequence unique to a target sequence (continuous sequence) is usually used. Note that programs and algorithms for selecting an appropriate target sequence have been developed.

  The “nucleic acid construct that generates siRNA in a cell” in (b) above refers to a nucleic acid molecule that, when introduced into a cell, produces a desired siRNA (siRNA that causes RNAi against a target gene) by a process in the cell. . One example of the nucleic acid construct is shRNA (short hairpin RNA). shRNA has a structure (hairpin structure) in which a sense RNA and an antisense RNA are linked via a loop structure part, and the loop structure part is cleaved in a cell to form a double-stranded siRNA, resulting in an RNAi effect. The length of the loop structure is not particularly limited, but is usually 3 to 23 bases.

  Another example of a nucleic acid construct is a vector that can express a desired siRNA. Such vectors include those that express shRNA that is converted to siRNA by a later process (inserted with a sequence encoding shRNA) (referred to as stem loop type or short hairpin type), sense RNA and antisense RNA. Are vectors that are expressed separately (referred to as tandem type). Those skilled in the art can prepare these vectors according to conventional methods (Brummelkamp TR et al. (2002) Science 296: 550-553; Lee NS et al. (2001) Nature Biotechnology 19: 500-505; Miyagishi M & Taira K (2002) Nature Biotechnology 19: 497-500; Paddison PJ et al. (2002) Proc. Natl. Acad. Sci. USA 99: 1443-1448; Paul CP et al. (2002) Nature Biotechnology 19: 505-508; Sui G et al. (2002) Proc Natl Acad Sci USA 99 (8): 5515-5520; Paddison PJ et al. (2002) Genes Dev. 16: 948-958 etc.). Currently, various RNAi vectors are available. You may decide to construct | assemble the vector of this invention using such a well-known vector. In this case, after preparing an insert DNA encoding a desired RNA (for example, shRNA), it is inserted into a cloning site of the vector to obtain an RNAi expression vector.

  The origin and structure of the vector are not limited as long as it has a function of generating siRNA that exerts RNAi action on the target gene in the cell. Accordingly, various viral vectors (adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, lentivirus vectors, herpes virus vectors, Sendai virus vectors, etc.), non-viral vectors (liposomes, positively charged liposomes, etc.) and the like can be used. it can. Examples of promoters that can be used in the vector are U6 promoter, H1 promoter, and tRNA promoter. These promoters are RNA polymerase III type promoters, and high expression efficiency can be expected.

  The above (c) is a compound used for expression suppression by an antisense method. In other words, according to the agent of the present invention containing the compound (c) as an active ingredient, the expression of the DNA polymerase gene can be suppressed by the antisense method. In the case of performing expression inhibition by an antisense method, for example, an antisense construct that generates RNA complementary to an intrinsic part of mRNA of a DNA polymerase gene when transcribed in a target cell is used. Such an antisense construct is introduced into a target cell, for example, in the form of an expression plasmid. On the other hand, as an antisense construct, an oligonucleotide probe that hybridizes with an mRNA / or genomic DNA sequence of a DNA polymerase gene and inhibits its expression when introduced into a target cell can also be employed. As such an oligonucleotide probe, one that is resistant to endogenous nucleases such as exonuclease and / or endonuclease is preferably used. When a DNA molecule is used as an antisense nucleic acid, oligodeoxyribonucleotide derived from a region containing a translation start site (for example, a region of −10 to +10) of mRNA of a DNA polymerase gene is preferable.

  The complementarity between the antisense nucleic acid and the target nucleic acid is preferably strict, but there may be some mismatch. The ability of an antisense nucleic acid to hybridize to a target nucleic acid generally depends on both the degree of complementarity and the length of both nucleic acids. Usually, the longer the antisense nucleic acid used, the more stable duplexes (or triplexes) can be formed with the target nucleic acid, even if the number of mismatches is large. One skilled in the art can ascertain the degree of acceptable mismatch using standard techniques.

  The antisense nucleic acid may be DNA, RNA, or a chimeric mixture thereof, or a derivative or modified form thereof. Moreover, it may be single-stranded or double-stranded. By modifying the base moiety, sugar moiety, or phosphate skeleton moiety, the stability, hybridization ability, etc. of the antisense nucleic acid can be improved. In addition, antisense nucleic acids can be used to promote cell membrane transport (eg, Letsinger et al., 1989, Proc. Natl. Acad. Sci. USA 86: 6553-6556; Lemaitre et al., 1987, Proc. Natl. Acad. Sci. 84: 648-652; PCT Publication No. W088 / 09810, published December 15, 1988) or substances that enhance affinity for specific cells may be added.

  The antisense nucleic acid can be synthesized by a conventional method, for example, using a commercially available automatic DNA synthesizer (for example, Applied Biosystems). For example, Stein et al. (1988), Nucl. Acids Res. 16: 3209 and Sarin et al., (1988), Proc. Natl. Acad. Sci. USA 85: 7448- 7451 etc. can be referred to.

  Strong promoters such as pol II and pol III can be used to enhance the action of antisense nucleic acids in target cells. That is, when a construct containing an antisense nucleic acid arranged under the control of such a promoter is introduced into a target cell, a sufficient amount of the antisense nucleic acid can be transcribed by the action of the promoter.

  Expression of the antisense nucleic acid can be performed by any promoter (inducible promoter or constitutive promoter) known to function in mammalian cells (preferably human cells). For example, the SV40 early promoter region (Bernoist and Chambon, 1981, Nature 290: 304-310), the promoter from the 3 ′ end region of Rous sarcoma virus (Yamamoto et al., 1980, Cell 22: 787-797), herpes zoster thymidine A promoter such as a kinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. USA 78: 1441-1445) can be used.

  In one embodiment of the present invention, expression suppression by a ribozyme is used (in the case of the compound (d) above). The target mRNA can be destroyed using a ribozyme that cleaves the mRNA with a site-specific recognition sequence, but preferably a hammerhead ribozyme is used. For the construction method of the hammerhead ribozyme, for example, Haseloff and Gerlach, 1988, Nature, 334: 585-591 can be referred to.

  As in the case of using the antisense method, for example, for the purpose of improving stability and target ability, a ribozyme may be constructed using a modified oligonucleotide. In order to generate an effective amount of a ribozyme in a target cell, for example, a nucleic acid construct in which DNA encoding the ribozyme is placed under the control of a strong promoter (for example, pol II or pol III) can be used. preferable.

  The compounds (e) and (f) suppress the action of the target (DNA polymerase in the present invention) using dominant negative mutation. The dominant negative mutant refers to a substance whose function (activity) is reduced by introduction of a mutation as compared to a normal protein (in the present invention, a subunit constituting a DNA polymerase). Therefore, it is not limited to those completely losing its function, but is included in dominant negative mutants whose function is partially impaired. The dominant negative mutant can indirectly suppress the function of the normal protein by competing with the corresponding normal protein. In order to obtain a dominant negative mutant, a mutation may be introduced into a nucleic acid encoding a normal protein so that the function of the protein is impaired by deletion, substitution, addition or the like of one or more amino acids. Mutation can be introduced by a conventional method such as a method using a PCR method. In addition, a commercially available kit for introducing a mutation may be used.

  In a preferred embodiment of the present invention, DNA polymerase δ is targeted. In this embodiment, DNA polymerase δ constituent genes, that is, DNA polymerase δ1 gene (hereinafter also referred to as “POLD1 gene”), DNA polymerase δ2 gene (hereinafter also referred to as “POLD2 gene”), DNA polymerase δ3 gene (hereinafter referred to as “POLD2 gene”) , Also referred to as “POLD3 gene”) or a compound that suppresses the expression of DNA polymerase δ4 gene (hereinafter also referred to as “POLD4 gene”), or suppresses the action of DNA polymerase δ1, DNA polymerase δ2, DNA polymerase δ3, or DNA polymerase δ4 Is used. The POLD1 gene sequence (SEQ ID NO: 1), POLD2 gene sequence (SEQ ID NO: 2), POLD3 gene sequence (SEQ ID NO: 3) and POLD4 gene sequence (SEQ ID NO: 4) are shown in the Sequence Listing.

An example of an siRNA sequence targeting the POLD1 gene and the like is shown below (for details, refer to the examples described later).
SiRNA targeting the POLD1 gene: SEQ ID NOS: 5 and 6
SiRNA targeting the POLD2 gene: SEQ ID NOs: 7 and 8
SiRNA targeting the POLD3 gene: SEQ ID NOs: 9, 10
SiRNA targeting the POLD4 gene: SEQ ID NOS: 11 and 12

(Medicine for malignant melanoma)
The second aspect of the present invention relates to the use of the above expression enhancer. In this aspect, a medicament for treating malignant melanoma (hereinafter referred to as “medicament of the present invention”) containing the above-described expression-enhancing agent as an active ingredient is provided. The medicament of the present invention is typically applied to a subject suffering from melanoma for the purpose of disappearance or reduction of a tumor, improvement of symptoms, and the like. However, the medicament of the present invention may be applied to preventive treatment such as recurrence prevention.

  The medicament of the present invention is used for immunotherapy. More specifically, it is used to enhance the effect of immunotherapy on melanoma. “Immunotherapy” refers to a treatment method utilizing the immune response of a living body. Typical immunotherapy includes cancer vaccine therapy (including dendritic cell therapy (DC therapy)), cytokine therapy, activated autolymphocyte therapy (CAT therapy), cytotoxic T lymphocyte transfer therapy (CTL therapy), Tumor tissue infiltrating T lymphocyte therapy (TIL therapy) is known. The medicament of the present invention can be applied to any of these immunotherapy. A therapeutic method using an antibody specific to melanoma cells (typically a melanoma antigen-specific antibody) is one of immunotherapy, and the medicament of the present invention can also be applied to the therapeutic method.

  Here, the “cancer vaccine therapy” is an active immunotherapy in which a cancer antigen (peptide, protein, etc.) is directly or is taken into an immunocompetent cell and then inoculated (administered) to a patient to activate the immune system. Those that use dendritic cells as immunocompetent cells are called dendritic cell (DC) therapy. In dendritic cell therapy, dendritic cells that have been trained to recognize cancer cells as foreign substances are returned to the body to induce immunity. In cancer vaccine therapy, tumor antigens (natural or recombinant) or partial peptides thereof (natural or recombinant), tumor cell lysates, tumor antigen HLA-binding peptides are used as substances for inducing or activating immunity. (Natural type or recombinant type) is used.

  “Cytokine therapy” is a treatment method in which a cytokine, which is a substance that activates an immune response, is administered to a living body. Interferon (IFN), interleukin 2 (IL-2), etc. are frequently used.

  “Activated self-lymphocyte therapy (CAT therapy)” is a treatment method in which T lymphocytes that play a central role in immune responses are proliferated and activated in vitro and then returned to the body. For activation of T lymphocytes, anti-CD3 antibody, interleukin 2 (IL-2) and the like are used.

  “Cytotoxic T lymphocyte transfer therapy (CTL therapy)” is a treatment method in which cytotoxic T lymphocytes that have acquired a specific ability to attack a target are transferred to a living body.

  In invasive cancer, lymphocytes (tumor tissue infiltrating T lymphocytes) having the ability to recognize cancer tissues as foreign substances are present in pleural ascites and cancer tissues. A treatment method in which the lymphocytes are proliferated and activated in vitro and then returned to the living body is called “tumor tissue infiltrating T lymphocyte therapy (TIL therapy)”.

In the immunotherapy using the medicament of the present invention, the administration timing of the medicament of the present invention is not particularly limited. That is, the medicament of the present invention is administered before or after the main treatment of immunotherapy or simultaneously with the treatment. “Main treatment of immunotherapy” refers to a core operation performed on a living body in order to elicit or activate an immune response. For example, in the case of vaccination, which is one of cancer vaccine therapies, the operation of inoculating a living body with a vaccine corresponds to “main treatment of immunotherapy”. Similarly, in the case of activated autolymphocyte therapy, the operation of transferring activated lymphocytes to the living body corresponds to “main treatment of immunotherapy”, and in the case of cytotoxic T lymphocyte transfer therapy, In contrast, the operation of transferring cytotoxic T lymphocytes corresponds to “a main treatment of immunotherapy”.
You may decide to administer the pharmaceutical of this invention in multiple times. The timing of each administration when performing multiple administrations is also not particularly limited.

  The medicament of the present invention can be applied not only to immunotherapy performed alone but also to immunotherapy combined with one or more therapies selected from the group consisting of surgical excision, chemotherapy, and radiation therapy. In a preferred embodiment, the medicament of the present invention is applied to immunotherapy combined with chemotherapy and / or radiation therapy. In addition, the chemical | medical agent used for "chemotherapy" here is not specifically limited. Drugs used for melanoma chemotherapy include dacarbazine, lomustine, melphalan, thiotepa, cisplatin, paclitaxel, tamoxifen, vincristine, simulfacin, adrimycin, vintedine, vinblastine, nimustine hydrochloride, vincristine, vindesine sulfate, sorafenib, celecoxib, Terozolomide is known. It is also attempted to use a plurality of drugs such as DAV therapy (dacarbazine, nimustine hydrochloride, vincristine), DAV-feron therapy (dacarbazine, nimustine hydrochloride, vincristine, interferon β), CDV therapy (cisplatin, dacarbazine, vincristine). ing.

  The pharmaceutical preparation of the present invention can be prepared according to a conventional method. In the case of formulating, other pharmaceutically acceptable ingredients (for example, carriers, excipients, disintegrants, buffers, emulsifiers, suspending agents, soothing agents, stabilizers, preservatives, preservatives, physiological Saline solution and the like). As the excipient, lactose, starch, sorbitol, D-mannitol, sucrose and the like can be used. As the disintegrant, starch, carboxymethylcellulose, calcium carbonate and the like can be used. Phosphate, citrate, acetate, etc. can be used as the buffer. As the emulsifier, gum arabic, sodium alginate, tragacanth and the like can be used. As the suspending agent, glyceryl monostearate, aluminum monostearate, methyl cellulose, carboxymethyl cellulose, hydroxymethyl cellulose, sodium lauryl sulfate and the like can be used. As the soothing agent, benzyl alcohol, chlorobutanol, sorbitol and the like can be used. As the stabilizer, propylene glycol, ascorbic acid or the like can be used. As preservatives, phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, methylparaben, and the like can be used. As preservatives, benzalkonium chloride, paraoxybenzoic acid, chlorobutanol and the like can be used.

The dosage form for formulation is not particularly limited. Examples of dosage forms are tablets, powders, fine granules, granules, capsules, syrups, injections, external preparations, and suppositories.
The medicament of the present invention contains an active ingredient in an amount necessary for obtaining an expected therapeutic effect (or preventive effect) (ie, a therapeutically effective amount). The amount of the active ingredient in the medicament of the present invention generally varies depending on the dosage form, but the amount of the active ingredient is set, for example, within the range of about 0.1% by weight to about 95% by weight so as to achieve a desired dose.

The medicament of the present invention is administered to the subject by oral administration or parenteral administration (intravenous, intraarterial, subcutaneous, intradermal, intramuscular or intraperitoneal injection, transdermal, nasal, transmucosal, etc.) depending on the dosage form. Applied. These administration routes are not mutually exclusive, and two or more arbitrarily selected can be used in combination (for example, intravenous injection or the like is performed simultaneously with oral administration or after a predetermined time has elapsed). The “subject” here is not particularly limited, and includes humans and non-human mammals (including pet animals, domestic animals, laboratory animals. Specifically, for example, mice, rats, guinea pigs, hamsters, monkeys, cows, pigs, goats. , Sheep, dogs, cats, chickens, quails, etc.). In a preferred embodiment, the medicament of the present invention is applied to humans. For example, oral, intravenous, intradermal, subcutaneous, intralymph node, intramuscular, intraperitoneal, transdermal, transmucosal and the like can be mentioned.
The dosage of the medicament of the present invention is set so as to obtain the expected therapeutic effect. In setting a therapeutically effective dose and administration schedule, the patient's symptoms, age, sex, weight, and the like are generally considered. A person skilled in the art can consider these matters and perform a preliminary experiment as necessary to set an appropriate dose.

(Treatment of melanoma)
The present invention further provides a method for treating melanoma. Here, “therapeutic method of melanoma” includes preventive treatment such as prevention of recurrence of melanoma. In the treatment method of the present invention, the medicament of the present invention is administered to a patient in order to enhance the therapeutic effect of immunotherapy.

  Note that matters not specifically mentioned in the present specification (conditions, operation methods, etc.) may follow conventional methods, such as Molecular Cloning (Third Edition, Cold Spring Harbor Laboratory Press, New York), Current protocols in molecular biology (edited by Frederick M. Ausubel et al., 1987), Current protocols in Immunology, John Wiley & Sons Inc, and the like.

<Search for substances that increase melanoma antigen expression>
In order to find a novel substance that promotes the increased expression of melanoma antigen, an evaluation system using melanoma cells was constructed, and various drugs / compounds were screened using it. As a result, the activity of 11 selected compounds was evaluated.

1. Materials and Experimental Methods (1) Drugs / Compounds and Reagents Bisindolelylmaleimide I and Etoposide (VP-16) are Calbiochem, Paclitaxel, Daunorubicin hydrochloride (Daunorubicin, HCl), Doxorubicin hydrochloride (Doxorubicin) HCl), Actinomycin D, Camptothecin, Methotrexate, Cytochalasin D, Aphidicolin are Wako Pure Chemicals, Aurintricarboxylic Acid (3-ATA) are ALEXIS U0126 was obtained from cell signaling Technologies. Melan-A antibody recognizing Melan-A / MART1 was obtained from Nova Castra, HMB45 antibody recognizing gp100 was obtained from Dako Cytomation, and tyrosinase (TYR) antibody and TRP1 antibody were obtained from Santa Cruz Biotechnology.

(2) Cells and culture method In order to confirm the effects of each drug / compound, human melanoma cells SK-MEL28 (RIKEN BioResource Center) and A375 (DS Pharma Biomedical Co., Ltd.) were used. SK-MEL28 is a cell with positive expression of Melan-A / MART1 and gp100, and A375 is a cell with negative expression of Melan-A / MART1 and gp100. For the culture of each cell, A375 is DMEM (Sigma-Aldrich) containing 10% fetal bovine serum (FBS, manufactured by Equintec-Bio Inc.), SK-MEL28 is 10% fetal bovine serum (FBS, MEM (manufactured by Sigma-Aldrich) containing Equintec-Bio Inc.) was used as a growth culture solution, and this was performed at 37 ° C. in an incubator containing carbon dioxide gas at a rate of 5% in air.

(3) Drug Assay Using Cells 5 × 10 ⁵ human melanoma cells (SK-MEL28, A375) are implanted in a 6-well cell culture plate (trademark: Falcon, manufactured by BD Biosciences), and cultured for 24 hours. Drug / compound was added and cultured for 72 hours.
The final concentration of drug / compound addition is as follows. U0126 used as a positive control was 10 μM, methotrexate (1 μM), paclitaxel (100 nM), daunorubicin hydrochloride (100 nM), doxorubicin hydrochloride (100 nM), actinomycin D (10 nM), camptothecin (100 nM), etoposide (VP-16) ( 1 μM), cytochalasin D (100 nM), 3-ATA (10 μM), aphidicolin (1 μM), bisindole maleimide I (10 μM).

(4) Flow cytometer (FCM) analysis In order to measure the melanoma antigen in the cytoplasm of melanoma cells, the cells cultured with the addition of a drug / compound were treated with trypsin-EDTA solution (Sigma-Aldrich). Cells collected, centrifuged and pelleted are fixed with 1 mL of 37 ° C., 2% formaldehyde for 10 minutes and chilled on ice for 2 minutes. 9 mL of ice-cooled 100% ethanol is added thereto, and the mixture is ice-cooled for 30 minutes. After washing twice with 4% FBS-containing PBS, 4% FBS-containing PBS is added again and left at room temperature for 10 minutes. Antibodies (primary antibodies) against the respective melanoma antigens are added to the pelleted cells again and left at room temperature for 45 minutes. After washing with PBS containing 4% FBS, add FITC-labeled goat anti-mouse IgG antibody corresponding to each primary antibody or FITC-labeled rabbit anti-goat IgG antibody (ZYMED LABORATORIES INC.) As a secondary antibody, and allow to warm to room temperature for 30 minutes. Put. Finally, wash twice with PBS containing 4% FBS.
The FITC fluorescence intensity of this cell sample was measured with a flow cytometer FACS Calibur (BD Biosciences), and the mean channel fluorescence of each drug / compound-added cell sample was calculated with CellQuest software (BD Biosciences). The relative increase rate was calculated compared to that of cells cultured without addition.

2. Results The results of the flow cytometer analysis are shown in FIGS. In the test system using SK-MEL28 cells, paclitaxel, daunorubicin hydrochloride, doxorubicin hydrochloride, camptothecin, cytochalasin D, aphidicolin and bisindolemaleimide I were found to have activity to increase MART1 expression (FIG. 1). The activity of aphidicolin is extremely high, surpassing the positive control U0126. Moreover, the activity of daunorubicin hydrochloride, doxorubicin hydrochloride and cytochalasin D is very high.

  On the other hand, methotrexate, paclitaxel, daunorubicin hydrochloride, doxorubicin hydrochloride, camptothecin, etoposide, cytochalasin D, 3-ATA, aphidicolin, and bisindole maleimide I were found to have increased gp100 expression (FIG. 2). Among them, the activities of paclitaxel, etoposide and cytochalasin D are very high. In addition, the activities of aphidicolin and bisindolemaleimide I are the second highest.

  Regarding melanoma antigen TYR, methotrexate, paclitaxel, daunorubicin hydrochloride, doxorubicin hydrochloride, actinomycin D, camptothecin and aphidicolin were found to have increased expression activity (FIG. 3). Among them, the activities of paclitaxel, doxorubicin hydrochloride, camptothecin and aphidicolin are extremely high.

  As for melanoma antigen TRP1, methotrexate, paclitaxel, daunorubicin hydrochloride, doxorubicin hydrochloride, actinomycin D, camptothecin, etoposide, cytochalasin D and aphidicolin were found to have increased expression activity (FIG. 4). Among them, the activities of paclitaxel and camptothecin are extremely high. Moreover, the activity of daunorubicin hydrochloride, doxorubicin hydrochloride, actinomycin D and aphidicolin is the second highest.

  On the other hand, in the test system using A375 cells, MART1 expression increasing activity was observed in methotrexate, paclitaxel, daunorubicin hydrochloride, doxorubicin hydrochloride, actinomycin D, camptothecin, etoposide, and aphidicolin (FIG. 6). Among them, the activities of daunorubicin hydrochloride, camptothecin and aphidicolin are extremely high.

  Regarding melanoma antigen gp100, methotrexate, daunorubicin hydrochloride, doxorubicin hydrochloride, actinomycin D, camptothecin, etoposide, cytochalasin D, 3-ATA and aphidicolin were found to have increased expression activity (FIG. 7). Among them, methotrexate, daunorubicin hydrochloride, doxorubicin hydrochloride, camptothecin, etoposide, and aphidicolin have extremely high activities.

  Regarding melanoma antigen TYR, methotrexate, paclitaxel, daunorubicin hydrochloride, doxorubicin hydrochloride, actinomycin D, camptothecin, etoposide, cytochalasin D, aphidicolin and bisindole maleimide I were recognized (FIG. 8). Above all, the activities of camptothecin and aphidicolin are exceptionally high. Also, the activities of methotrexate, paclitaxel, daunorubicin hydrochloride, actinomycin D and etoposide are very high.

  For melanoma antigen TRP1, all drugs (methotrexate, paclitaxel, daunorubicin hydrochloride, doxorubicin hydrochloride, actinomycin D, camptothecin, etoposide, cytochalasin D, 3-ATA, aphidicolin and bisindole maleimide I were recognized. (Figure 9) The activity of aphidicolin is exceptionally high, and the activities of methotrexate, paclitaxel, daunorubicin hydrochloride, actinomycin D, camptothecin and etoposide are also very high.

  From FIG. 5 collectively showing the test results of the test system using SK-MEL28 cells and FIG. 10 collectively showing the test results of the test system using A375 cells, paclitaxel, daunorubicin hydrochloride, doxorubicin hydrochloride, camptothecin, and aphidi Choline is a particularly effective drug.

  As described above, it has been clarified that 11 types of drugs / compounds promote increased expression of melanoma antigen. Methotrexate, paclitaxel, daunorubicin hydrochloride, doxorubicin hydrochloride, actinomycin D, camptothecin, and etoposide have already been used in humans as drugs and are expected to be clinically applicable at an early stage.

<Additional search for substances that increase melanoma antigen expression>
Drugs / compounds were screened with the goal of further discovering new substances that promote increased expression of melanoma antigens. Nine compounds selected as a result of screening were evaluated for activity.

1. Materials and Experimental Methods (1) Drugs / Compounds and Reagents Arabinoside (Cytosine Arabinoside, Ara-C), Curcumin, Pyridoxal 5'-phosphate hydrate, Epigallocatechin Galate ((-)) -Epigallocatechin gallate, EGCG), vidarabine, resveratrol, lithocholic acid, Sigma-Aldrich, 2'3'-dideoxythymidine triphosphate (2'3'- dideoxythymidine 5'-triphosphate, ddTTP) was obtained from JENA BIOSCIENCE, and thymidine dimer (dipyrimidine dithymidylic acid, pTpT) was obtained from MIDLAND CERTIFIED REAGENT. Melan-A antibody recognizing Melan-A / MART1 was obtained from Nova Castra, HMB45 antibody recognizing gp100 was obtained from Dako Cytomation, tyrosinase (TYR) antibody and TRP1 antibody from Santa Cruz Biotechnology.

(2) Cells and culture method In order to confirm the effects of each drug / compound, human melanoma cells SK-MEL28 (RIKEN BioResource Center) and A375 (DS Pharma Biomedical Co., Ltd.) were used. SK-MEL28 is a cell with positive expression of Melan-A / MART1 and gp100, and A375 is a cell with negative expression of Melan-A / MART1 and gp100. For the culture of each cell, A375 is DMEM (Sigma-Aldrich) containing 10% fetal bovine serum (FBS, manufactured by Equintec-Bio Inc.), SK-MEL28 is 10% fetal bovine serum (FBS, MEM (manufactured by Sigma-Aldrich) containing Equintec-Bio Inc.) was used as a growth culture solution, and this was performed at 37 ° C. in an incubator containing carbon dioxide gas at a rate of 5% in air.

(3) Drug assay using cells 1 × 10 ⁵ human melanoma cells (SK-MEL28, A375) were implanted in a 12-well cell culture plate (trademark: Falcon, manufactured by BD Biosciences), and cultured for 24 hours. Drug / compound was added and cultured for 72 hours.
The final concentration of drug / compound addition is as follows. Arabinonucleosides (100 μM for A375, 50 μM only when measuring tyrosinase for SK-MEL28, 20 μM for others), curcumin (all 0.2 μM), pyridoxal phosphate (all 500 μM), epigallocatechin gallate (all 100 μM), vidarabine (300 μM for A375, 60 μM for SK-MEL28), resveratrol (20 μM for A375, 50 μM for SKMEL28), lithocholic acid (100 μM for A375, 20 μM for SK-MEL28), 2'3'-dideoxy Thymidine triphosphate (2 μM only when measuring TRP-1 in A375 cells, otherwise 10 μM), dipyrimidine thymidine diphosphate (both 100 μM).

(4) Flow cytometer (FCM) analysis In order to measure the melanoma antigen in the cytoplasm of melanoma cells, the cells cultured with the addition of a drug / compound were treated with trypsin-EDTA solution (Sigma-Aldrich). Cells collected, centrifuged and pelleted are fixed with 1 mL of 37 ° C., 2% formaldehyde for 10 minutes and chilled on ice for 2 minutes. 9 mL of ice-cooled 100% ethanol is added thereto, and the mixture is ice-cooled for 30 minutes. After washing twice with 4% FBS-containing PBS, 4% FBS-containing PBS is added again and left at room temperature for 10 minutes. Antibodies (primary antibodies) against the respective melanoma antigens are added to the pelleted cells again and left at room temperature for 45 minutes. After washing with PBS containing 4% FBS, add FITC-labeled goat anti-mouse IgG antibody corresponding to each primary antibody or FITC-labeled rabbit anti-goat IgG antibody (ZYMED LABORATORIES INC.) As a secondary antibody, and allow to warm to room temperature for 30 minutes. Put. Finally, wash twice with PBS containing 4% FBS.
The FITC fluorescence intensity of this cell sample was measured with a flow cytometer FACS Calibur (BD Biosciences), and the mean channel fluorescence of each drug / compound-added cell sample was calculated with CellQuest software (BD Biosciences). The relative increase rate was calculated compared to that of cells cultured without addition.

2. Results The results of flow cytometer analysis are shown in FIGS. In the test system using SK-MEL28 cells, arabinonucleoside, 2'3'-dideoxythymidine triphosphate, thymidine dimer, epigallocatechin gallate, vidarabine and resveratrol were observed to have increased MART1 expression ( FIG. 11). The activity of arabinonucleosides is particularly high.

  On the other hand, arabinonucleoside, epigallocatechin gallate, and vidarabine showed gp100 expression increasing activity (FIG. 11). The activity of arabinonucleosides is particularly high.

  As for the melanoma antigen TRP1, arabinonucleoside, vidarabine, resveratrol and lithocholic acid were found to have a very high expression increasing activity (FIG. 11).

  As for the melanoma antigen TYR, arabinonucleoside, vidarabine and resveratrol were found to have increased expression activity (FIG. 11). Above all, the activity of vidarabine is extremely high.

  On the other hand, in the test system using A375 cells, arabinonucleoside, 2'3'-dideoxythymidine triphosphate, thymidine dimer, pyridoxal phosphate, epigallocatechin gallate, vidarabine, resveratrol and lithocholic acid MART1 expression increasing activity was observed (FIG. 12). The activities of arabinonucleoside and epigallocatechin gallate are high, and the activities of vidarabine and resveratrol are extremely high.

  As for melanoma antigen gp100, arabinonucleoside, 2'3'-dideoxythymidine triphosphate, thymidine dimer, pyridoxal phosphate, epigallocatechin gallate, vidarabine, resveratrol, and lithocholic acid (FIG. 12). Among them, the activities of arabino nucleoside, vidarabine and resveratrol are very high.

  As for melanoma antigen TRP1, arabinonucleoside, curcumin, 2'3'-dideoxythymidine triphosphate, epigallocatechin gallate and vidarabine were found to have increased expression activity (FIG. 12). Among them, the activities of arabino nucleoside and epigallocatechin gallate are very high.

  For melanoma antigen TYR, arabinonucleoside, curcumin, 2'3'-dideoxythymidine triphosphate, thymidine dimer, epigallocatechin gallate, vidarabine and lithocholic acid were found to have increased expression activity (FIG. 12). The activity of arabinonucleosides is extremely high.

  In summary, arabinonucleoside, epigallocatechin gallate, vidarabine, resveratrol and lithocholic acid are particularly effective drugs.

  As described above, it was revealed that nine types of drugs / compounds promote the increased expression of melanoma antigen. Here, arabinonucleoside, pyridoxal phosphate, and vidarabine have already been used as drugs in humans. On the other hand, curcumin, epigallocatechin gallate and resveratrol are food ingredients. Therefore, it is expected that these drugs / compounds can be clinically applied at an early stage.

<Effect confirmation experiment in vivo using cancer-bearing mice>
The in vivo effects of affilicholine, methotrexate and doxorubicin hydrochloride (the effect of increasing the expression of melanoma antigen) were examined using animal models.

1. Method
When B16 melanoma cells 2x10 ⁶ are injected subcutaneously into the back of B6 mice, tumor nodules are formed in about one week. Each drug or DMSO (control) was intraperitoneally administered to the tumor-bearing B6 mice once a day for 5 days, and tumor nodules were taken out 2 days after the final administration day. RNA was extracted from tumor cells in the nodule, and the expression levels of Mart1, gp100, and Dct, which are melanoma antigens and mouse genes corresponding to human MART1, gp100, and DCT genes, were examined. Three mice per test group were used (n = 3). Methotrexate was administered once a day for 3 days, and an experiment was also conducted using a short-term protocol in which nodules were collected the day after the last administration.

  Quantitative PCR was performed according to the following procedure. RNA was extracted using Rneasy mini kit (QIAGEN). From the extracted RNA, cDNA was prepared using Taqman Reverse Transcription Reagents (Applied Biosystems). Using this cDNA as a sample, primers and probes (both manufactured by EGT Japan) that specifically detect target genes (mouse Mart1, mouse gp100 and mouse Dct gene, mouse β-actin gene), and qPCR Mastermix Low ROX (EUROGENTEC Quantitative PCR by the Taqman method was performed with a real-time quantitative PCR system MX3000p (Stratagene). Compare the amount of mRNA of mouse Mart1, mouse gp100, and mouse Dct in the sample with each drug added to that of cells cultured without the addition of the drug / compound. -Calculated with pro software (Stratagene).

2. result
When administered for 5 days, an increase in melanoma antigen was observed for all of aphidicolin, methotrexate, and doxorubicin hydrochloride (FIG. 13). It was found that administration of doxorubicin hydrochloride for 5 days puts a burden on the mice. In the case of methotrexate, melanoma antigen expression was significantly increased even after administration for 3 days (FIG. 14). However, the expression level of melanoma antigen is higher in 5 days than in 3 days.

<Examination of expression enhancement effect of tumor tissue compatible gene complex class I molecule>
Tumor histocompatibility complex (MHC) is an indispensable molecule in presenting melanoma antigens. When MHC is decreased in melanoma cells, even if melanoma antigens are expressed abundantly in the cells, they are not presented on the surface of melanoma cells, and the efficiency of tumor cell recognition by lymphocytes does not increase. Therefore, in order to improve the success rate of immunotherapy, it is said that enhancing expression of MHC is also very important. Cytotoxic lymphocytes recognize both antigens presented on the surface of tumor cells and MHC class I molecules together and damage tumor cells, so MHC class I expression is particularly important in melanoma immunotherapy. Therefore, the effects and effects on the expression of MHC class I were examined for each drug that was found to have an effect of promoting the increased expression of melanoma antigen.

1. Method
A375 melanoma cells were treated with drugs in the same manner as before, and the increased expression of MHC class I in melanoma cells was confirmed at the protein level by cytoplasmic surface staining flow cytometer (FCM). In the FCM analysis, only the amount of MHC class I protein that was actually expressed on the cell surface and worked for antigen presentation was measured by cytoplasmic surface staining. FCM analysis of cytoplasmic surface staining was performed according to the following procedure. The cells were collected with PBS containing 5 mM EDTA and 1% Bovine Serum Albumin (BSA) (EDTA and BSA were manufactured by Sigma-Aldrich, PBS is a trademark: GIBCO, manufactured by Invitrogen), and pelleted by cooling at 4 ° C. After washing the cells with PBS, PBS is added again as a cell suspension, PE-labeled mouse anti-human MHC class I antibody (Santa Cruz Biotechnology) is added thereto, and the mixture is allowed to stand on ice in the dark for 30 minutes. After washing with 1% formaldehyde, the mixture was allowed to stand at room temperature in the dark for 10 minutes, washed with PBS and subjected to FCM analysis. The PE fluorescence intensity of this cell sample was measured with a flow cytometer FACS Calibur (BD Biosciences), and the mean channel fluorescence of each drug / compound-added cell sample was calculated with CellQuest software (BD Biosciences). The relative increase rate was calculated compared to that of cells cultured without addition.

2. Results At the protein level (FIG. 15), MHC class I expression enhancing effect was recognized for each drug.

3. Discussion Combined with the results of the above animal experiments, it can be said that each drug used in the experiment is useful as an adjuvant for immunotherapy from the two viewpoints of enhanced expression of melanoma antigen and enhanced expression of MHC.

<Investigation of the mechanism that causes the increased expression of melanoma antigen>
The increase in the expression of the melanoma antigen by the DNA polymerase inhibitor was examined as described above, and some useful drugs were clarified. By the way, more than 15 types of DNA polymerases are known. For some of the DNA polymerase inhibitors, it has been clarified which polymerase exerts the inhibitory action. Among DNA polymerase inhibitors that have been shown to promote increased expression of melanoma antigens, aphidicolin, arabinonucleoside (AraC), resveratrol, vidarabine (AraA), etc. are DNA polymerases α, β, γ, It is known to act on δ and ε. Therefore, in order to examine in detail the mechanism for the increased expression of melanoma antigen, it was investigated whether the same effect could be obtained by suppressing the gene expression of these DNA polymerases in melanoma cells.

1. Method As a representative example, a case where DNA polymerase δ is targeted will be described. 1 μg siRNA (shown below) for subunit DNA polymerase δ1, 2, 3 and 4 (POLD1-4) genes constituting DNA polymerase δ was electroporated to 1 × 10 ⁶ human melanoma cells ( SK-MEL28, A375). These were seeded in 6-well cell culture plates (trademark: Falcon, manufactured by BD Biosciences) and cultured for 48 or 72 hours. The increase in the expression of the melanoma antigen was examined by quantitative PCR at the messenger RNA level (for details, <Experiment for confirming effect in vivo using cancer-bearing mice>, see 1. Method column). For cytoplasmic staining flow cytometer (FCM) for POLD1 siRNA-introduced samples (for details, see <Additional Search for Substances that Promote Increased Melanoma Antigen Expression>, 1. Materials and Experimental Methods, (4)) ), The increase in the expression of MART1 and gp100 was examined at the protein level. In quantitative PCR, primers and probes (all manufactured by EGT, Japan) were used to specifically detect MART1, gp100, tyrosinase (TYR), tyrosinase-related gene 1 (TRP1) gene and human β-actin gene.

(SiRNA against NM_002691 (POLD1 gene, SEQ ID NO: 1))
5'-GCACAUGGGUGACCUGCAAdTdT-3 '(SEQ ID NO: 5 (sequence excluding oligo dT part))
3'-dTdTCGUGUACCCACUGGACGUU-5 '(SEQ ID NO: 6 (sequence excluding oligo dT part))
(SiRNA against NM_006230.1 (POLD2 gene, SEQ ID NO: 2))
5'-UGAGACCCUUCCUGGAGAAdTdT-3 '(SEQ ID NO: 7 (sequence excluding oligo dT part))
3'-dTdTACUCUGGGAAGGACCUCUU-5 '(SEQ ID NO: 8 (sequence excluding oligo dT part))
(SiRNA against NM_006591.1 (POLD3 gene, SEQ ID NO: 3))
5'-GCAUCCAUGUGUACAGCAUdTdT-3 '(SEQ ID NO: 9 (sequence excluding oligo dT part))
3'-dTdTCGUAGGUACACAUGUCGUA-5 '(SEQ ID NO: 10 (sequence excluding oligo dT part))
(SiRNA against NM_021173.2 (POLD4 gene, SEQ ID NO: 4))
5'-GAGGUGUGGCAGGUGCUGAdTdT-3 '(SEQ ID NO: 11 (sequence excluding oligo dT part))
3'-dTdTCUCCACACCGUCCACGACU-5 '(SEQ ID NO: 12 (sequence excluding oligo dT part))

2. result
It was suggested that the effect of increasing the expression of the melanoma antigen can be obtained by suppressing the expression of the constituent genes of DNA polymerase δ (FIGS. 16 to 20). Even when DNA polymerases α, β, and ε were targeted, increased expression of melanoma antigen was observed (results not shown).

  As described above, it was shown that the suppression of the expression of the DNA polymerase gene promotes the increase in the expression of the melanoma antigen.

  According to the expression increasing agent of the present invention, the effect of immunotherapy can be enhanced by increasing the expression level of an antigen (melanoma antigen) in melanoma cells. Thus, the present invention is useful in melanoma immunotherapy. The expression-enhancing agent of the present invention can be used to prevent recurrence of melanoma.

The present invention is not limited to the description of the embodiments and examples of the invention described above. Various modifications may be included in the present invention as long as those skilled in the art can easily conceive without departing from the description of the scope of claims.
The contents of papers, published patent gazettes, patent gazettes, and the like specified in this specification are incorporated by reference in their entirety.

Claims (3)

Cytochalasin D, 3-ATA (3-amino-9-thio (10H) -acridone), Aphidicolin, 2'3'-dideoxythymidine triphosphate (2'3'-dideoxythymidine) A compound selected from the group consisting of 5'-triphosphate, dipyrimidine dithymidylic acid, pyridoxal 5'-phosphate hydrate, and lithocholic acid , or the following (a ) To (f), that is,
(a) siRNA targeting DNA polymerase δ1 gene, DNA polymerase δ2 gene, DNA polymerase δ3 gene or DNA polymerase δ4 gene;
(b) a nucleic acid construct that generates siRNA targeting a DNA polymerase δ1 gene, a DNA polymerase δ2 gene, a DNA polymerase δ3 gene or a DNA polymerase δ4 gene in a cell;
(c) an antisense nucleic acid targeting a transcription product of DNA polymerase δ1 gene, DNA polymerase δ2 gene, DNA polymerase δ3 gene or DNA polymerase δ4 gene;
(d) a ribozyme targeting a transcript of the DNA polymerase δ1 gene, DNA polymerase δ2 gene, DNA polymerase δ3 gene or DNA polymerase δ4 gene;
(e) a nucleic acid construct that generates intracellularly a dominant negative mutant of a protein encoded by a DNA polymerase δ1 gene, a DNA polymerase δ2 gene, a DNA polymerase δ3 gene or a DNA polymerase δ4 gene;
(f) a dominant negative mutant of the protein encoded by DNA polymerase δ1 gene, DNA polymerase δ2 gene, DNA polymerase δ3 gene or DNA polymerase δ4 gene,
A medicament for the treatment of malignant melanoma, comprising as an active ingredient a malignant melanoma antigen expression-enhancing agent comprising a compound selected from the group consisting of The medicament for treating malignant melanoma according to claim 1 , which is a medicament used for immunotherapy combined with one or more therapies selected from the group consisting of surgical resection, chemotherapy, and radiation therapy. From the group where immunotherapy consists of cancer vaccine therapy, cytokine therapy, activated autolymphocyte therapy (CAT therapy), cytotoxic T lymphocyte transfer therapy (CTL therapy), and tumor tissue infiltrating T lymphocyte therapy (TIL therapy) The medicament for the treatment of malignant melanoma according to claim 1 or 2 , which is any treatment method selected.