JP2018070563A - Anticancer activity enhancer and cancer treatment support method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an anticancer activity enhancer that enhances a cancer cell proliferation inhibitory effect of an anticancer agent, and a cancer treatment support method that determines a synergetic effect of the anticancer activity enhancer and the anticancer agent.SOLUTION: An anticancer activity enhancer is used in combination with an anticancer agent, which is 5-fluorouracil or cisplatin, to enhance its anticancer activity, and contains: a polynucleotide encoding interferon λ4; interferon λ4; or an agonist for a receptor with interferon λ4 as a ligand.SELECTED DRAWING: None

Description

  The present invention relates to an anticancer action enhancer that enhances the growth inhibitory effect on cancer cells possessed by an anticancer agent, and a cancer treatment support method for determining the effect of a combination of the anticancer action enhancer and an anticancer agent.

  Interferon λ4 (sometimes referred to as IFNλ4 or IFNL4) is induced due to a frame shift caused by a polymorphism existing upstream of the interferon λ3 gene (sometimes referred to as IFNL3 or IL28B) (Non-patent Document 1). : Nat Genet. 2013 Feb; 45 (2): 164-71). The polymorphism existing upstream of the IL28B gene is specifically referred to as ss469415590 and can take TT or ΔG. Specifically, the above-described frame shift is caused by ss469415590 [ΔG], resulting in a gene encoding IFNL4.

  It is known that ss469415590 is in high linkage disequilibrium with rs12979860, a genetic marker that shows a strong association with the removal of hepatitis C virus (HCV). However, at present, sufficient analysis has not been made on the functional role, biological and clinical significance of interferon λ4.

  In addition, a plurality of SNPs that are strongly related to the therapeutic effect of pegylated interferon / ribavirin (PEG-IFN / RBV) combination therapy are known around the IL28B gene (Non-Patent Document 2: Tanaka Y, et al). ., Nat Genet. 41; 1105-9, 2009). In other words, patients with a minor allele (TG or GG) of rs8099917 (TT / TG / GG), which is a representative SNP at the IL28B locus, are significantly more PEG-IFN than those with a major allele (TT). / RBV combination therapy is known to be ineffective. Moreover, it has been reported that the major allele (CC) group of rs12979860 has a rapid virus reduction by the PEG-IFN / RBV combination treatment compared with the minor allele group (Non-patent Document 3: Thompson AJ, et al., Gastroenterology. 139; 120-9, 2010).

  On the other hand, a genotype analysis of a patient with hepatoma with chronic hepatitis C who had undergone hepatectomy or radiofrequency ablation showed a significant difference between the genotype of rs8099917 and the recurrence rate. It has been reported that there was a correlation (Non-patent Document 4: Hodo et al., Clin. Cancer. Res. April 2013, 19 (7), pp1827-1837). Specifically, patients with an rs8099917 major allele (TT) have been shown to have a significantly higher recurrence rate of hepatocellular carcinoma compared to patients with an rs8099917 minor allele (TG or GG).

Ludmila Prokunina-Olsson et al., Nat Genet. 2013 Feb; 45 (2): 164-71 Tanaka Y, et al., Nat Genet. 41; 1105-9, 2009 Thompson AJ, et al., Gastroenterology. 139; 120-9, 2010 Hodo et al., Clin. Cancer. Res. April 2013, 19 (7), pp1827-1837

  As described above, although the clinical significance of the polymorphism existing around the IL28B gene is being elucidated, it encodes IFNL4 induced by the polymorphism as described in Non-Patent Document 1. However, the functional role and clinical significance of these genes remained unknown.

  Therefore, in view of the above-mentioned circumstances, the present invention clarifies the functional significance of IFNL4, and aims to provide a novel use of the IFNL4, a polynucleotide encoding IFNL4, and an agonist for a receptor having IFNL4 as a ligand. To do.

  As a result of intensive studies by the present inventors to achieve the above-mentioned object, as a functional significance of IFNL4, the function of enhancing the growth inhibitory effect on cancer cells possessed by an anticancer agent was found, and the present invention was completed. It came.

The present invention includes the following.
(1) An anticancer agent potentiator for an anticancer agent comprising, as an active ingredient, a polynucleotide encoding interferon λ4; interferon λ4; or an agonist for a receptor having interferon λ4 as a ligand.
(2) The anticancer activity enhancer according to (1), wherein the interferon λ4 comprises the amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence having 90% or more identity to the amino acid sequence.
(3) The anticancer activity enhancer according to (1), wherein the anticancer agent is 5-fluorouracil or cisplatin.
(4) The anticancer activity enhancer according to (1), wherein the dose is increased or decreased based on the expression level of the interferon λ4 gene.
(5) The anticancer activity enhancer according to (1), wherein the dose is increased or decreased based on whether the polymorphism defined by ss469415590 in the IL-28B gene encoding interferon λ3 is a major allele or a minor allele.
(6) The anticancer activity enhancer according to (1), wherein the administration target patient is a cancer patient with a low expression level of interferon λ4 gene.
(7) The anticancer activity enhancer according to (1), wherein the subject patient is a cancer patient whose polymorphism defined by ss469415590 in the IL-28B gene encoding interferon λ3 is a major allele.
(8) a step of measuring the expression of the interferon λ4 gene in the subject using a means capable of specifically detecting the expression of the interferon λ4 gene, and an anticancer agent based on the expression level of the interferon λ4 gene in the subject ( 1)-(7) A cancer treatment support method for determining a combined effect with the anticancer activity enhancer according to any one of the above.
(9) The above means has a pair of primers that specifically amplify the transcription product of the interferon λ4 gene, and a probe that specifically hybridizes to the nucleic acid amplified by the pair of primers. (8) The cancer treatment support method according to (8).
(10) A pharmaceutical composition comprising a combination of the anticancer activity enhancer according to any one of (1) to (7) above and an anticancer agent.
(11) The pharmaceutical composition according to (10), wherein the anticancer agent is 5-fluorouracil or cisplatin.
(12) A pharmaceutical composition used in combination with an anticancer agent, comprising the anticancer activity enhancer according to any one of (1) to (7) above.
(13) The pharmaceutical composition according to (12), wherein the anticancer agent is 5-fluorouracil or cisplatin.

  The anticancer activity enhancer according to the present invention can enhance the growth inhibitory effect on cancer cells by the anticancer agent. Therefore, according to the anticancer action enhancer according to the present invention, an excellent therapeutic effect of the anticancer agent can be expected. Moreover, according to the anticancer action enhancer which concerns on this invention, the dosage of an anticancer agent can be reduced, maintaining a medicinal effect, and the side effect by an anticancer agent can be suppressed.

  On the other hand, in the cancer treatment support method according to the present invention, in cancer treatment using an anticancer agent, the therapeutic effect of the anticancer agent is predicted, and the application of the anticancer action enhancing agent that enhances the growth inhibitory effect on the cancer cells by the anticancer agent is determined. be able to. Therefore, according to the cancer treatment support method of the present invention, it is possible to provide an optimal treatment policy for patients who need cancer treatment.

It is a characteristic view which shows the relationship between the genotype of IL28B and the success rate of hepatic arterial injection chemotherapy. It is a characteristic figure which compared the total survival rate about the genotype of IL28B about the liver arterial injection chemotherapy example. FIG. 6 is a characteristic diagram showing the results of univariate analysis and multivariate analysis using IL28B genotype and the like as variables. It is a characteristic diagram which shows the result of having measured ISG induction activity and anti-HCV activity about p179 (interferon (lambda) 4) etc. which can be expressed with the minor allele of IL28B. It is a characteristic view which shows the result of having measured ISG induction activity about various IFN. It is a characteristic view which shows the result of having measured anti- HCV activity about various IFN. It is a characteristic figure which shows the result of having measured the cell viability when IFNL4 and an anticancer agent are used together. FIG. 6 is a characteristic diagram showing the results of measuring cell viability when IFNL4 is used in combination with 5FU at various concentrations. It is a characteristic view which shows the result of having measured the caspase 3/7 activity when using various IFN and 5FU together. It is a characteristic view which shows the result of having measured the caspase 3/7 activity when using various IFN and CDDP together. FIG. 4 is a characteristic diagram showing that a primer and a probe for specifically detecting IFNL4 expression were designed. It is a characteristic figure which shows having detected the expression of IFNL4 specifically. It is a characteristic view which shows the result of having detected various IFN recombinant proteins. It is a characteristic view which shows the result of having detected various IFN recombinant proteins by Western blot.

  The anticancer activity enhancer according to the present invention has a function of enhancing the anticancer activity of an anticancer agent having a growth inhibitory effect on cancer cells, that is, the growth inhibitory effect on cancer cells. The anticancer activity enhancer comprises a polynucleotide encoding interferon λ4 (abbreviated as IFNλ4 or IFNL4); interferon λ4; or an agonist for a receptor having interferon λ4 as a ligand as an active ingredient.

<Interferon λ4>
Interferon λ4 (IFNλ4 or IFNL4) is an interferon λ3 gene (IFNL3 gene, IL28B gene) as disclosed in Ludmila Prokunina-Olsson et al., Nat Genet. 2013 Feb; 45 (2): 164-71. It is a polymorphism located upstream, and is encoded by a gene created by a polymorphism defined by ssID number (Submitted SNP ID number): ss469415590. The polymorphism defined in ss469415590 is a single nucleotide insertion / deletion type (Indel type) polymorphism, the major allele is TT, and the minor allele is ΔG (the 5 ′ T in the major allele is deleted) A polymorphism (rs67272382 or rs11322783) and a polymorphism (rs74597329) in which 3'-side T in the major allele is G). When the polymorphism specified by ss469415590 is a minor allele (ΔG), a frame shift occurs and interferon λ4 consisting of 179 amino acid sequences is generated.

  Type III interferons (IFNL1, IFNL2, and IFNL3) have highly conserved helices AF that are involved in interaction with the receptor. As disclosed in Ludmila Prokunina-Olsson et al., Nat Genet. 2013 Feb; 45 (2): 164-71, interferon λ4 is IFNL3 in helix A and helix F interacting with a receptor known as IL28R1. And most similar. On the other hand, interferon λ4 differs from IFNL3 in helix D, which interacts with the second strand of the interferon λ receptor complex known as IL10R2.

  The nucleotide sequence of the coding region encoding interferon λ4 and the amino acid sequence of interferon λ4 are shown in SEQ ID NOs: 1 and 2, respectively. For example, the amino acid sequence and base sequence of interferon λ4 can be obtained from a known database storing gene-related information such as the GenBank database. As for interferon λ4, its mRNA sequence is stored as ACCESSION number: JN806234, and stored in the GenBank database as Protein ID: AFQ38559.1.

  However, in the present invention, interferon λ4 is not limited to those defined in SEQ ID NOS: 1 and 2. For example, the receptor having an amino acid sequence in which one or more amino acids are substituted, deleted and / or added in the amino acid sequence shown in SEQ ID NO: 2 and interferon λ4 consisting of the amino acid sequence of SEQ ID NO: 2 interacts It may be a protein having an activity of interacting with the protein. Here, the term “plurality” refers to 2 to 20 amino acids, preferably 2 to 15 amino acids, more preferably 2 to 10 amino acids, and 2 to 5 amino acids. More preferred is an amino acid.

  Further, the interferon λ4 is not limited to the amino acid sequence shown in SEQ ID NO: 2, and for example, 70% or more sequence identity with respect to the amino acid sequence shown in SEQ ID NO: 2, preferably 80% or more sequence identity, Interferon λ4 having an amino acid sequence preferably having 90% or more sequence identity, more preferably 95% or more sequence identity, or more preferably 97% or more sequence identity, and comprising the amino acid sequence of SEQ ID NO: 2. It may be a protein having an activity of interacting with a receptor with which it interacts. Here, the value of sequence identity can be calculated as the ratio of amino acid residues that are completely matched by performing pairwise alignment with the amino acid sequence of SEQ ID NO: 2 (179).

  Furthermore, the polynucleotide encoding interferon λ4 is not limited to the one consisting of the base sequence shown in SEQ ID NO: 1, but is a polynucleotide that hybridizes under stringent conditions to the complementary strand of the base sequence shown in SEQ ID NO: 1. It may be a nucleotide that encodes a protein having an activity of interacting with a receptor with which interferon λ4 consisting of the amino acid sequence of SEQ ID NO: 2 interacts. The stringent condition is, for example, usually 42 ° C., 2 × SSC, 0.1% SDS, preferably 50 ° C., 2 × SSC, 0.1% SDS, and more preferably The conditions are 65 ° C., 0.1 × SSC, and 0.1% SDS, but are not particularly limited to these conditions. Factors affecting the stringency of hybridization include a plurality of factors such as temperature and salt concentration, and those skilled in the art can realize optimum stringency by appropriately selecting these factors. Hybridization can be performed according to the method described in, for example, Molecular Cloning: A laboratory Manual, 4th Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.

  For the interaction between interferon λ4 consisting of the amino acid sequence of SEQ ID NO: 2 and the receptor, for example, the EMBO Journal (2013) 32, 3055-3065 can be referred to. A receptor having a protein having an amino acid sequence different from SEQ ID NO: 2 or a protein encoded by a polynucleotide having a nucleotide sequence other than the nucleotide sequence shown in SEQ ID NO: 1 interacts with interferon λ4 having an amino acid sequence of SEQ ID NO: 2 Whether it has an activity to interact with can be examined with reference to this paper.

  By the way, although interferon (lambda) 4 may be an amino acid sequence different from the amino acid sequence of sequence number 2 as mentioned above, it is preferable that the area | region involved in interaction with a receptor is preserve | saved. That is, in the amino acid sequence of SEQ ID NO: 2, the regions corresponding to helices A to F are preferably conserved. In other words, the amino acid residue to be substituted, deleted and / or added is not particularly limited, but is preferably in a region other than helices A to F in the amino acid sequence of SEQ ID NO: 2.

  Helix A corresponds to positions 40 to 54 in the amino acid sequence of SEQ ID NO: 2, helix B corresponds to positions 66 to 69 in the amino acid sequence of SEQ ID NO: 2, and helix C corresponds to the amino acid sequence of SEQ ID NO: 2. Corresponds to positions 78 to 85, helix D corresponds to positions 105 to 113 in the amino acid sequence of SEQ ID NO: 2, helix E corresponds to positions 131 to 145 in the amino acid sequence of SEQ ID NO: 2, and helix F is It corresponds to the 148th to 169th positions in the amino acid sequence of SEQ ID NO: 2.

<Polynucleotide>
The anticancer activity enhancer according to the present invention can be prepared such that the polynucleotide encoding interferon λ4 described above is incorporated into a vector and can be expressed in a target tissue (eg, liver) using the vector. The vector is not particularly limited, and various vectors that can be used for humans can be used. As the vector, either a viral vector or a non-viral vector may be used.

  Of these, it is preferable to use a viral vector. As a viral vector, an adeno-associated virus (AAV) vector, a lentiviral vector, a herpes virus vector, and the like can be used. Of these, an AAV vector is preferable. AAV vectors are particularly preferably used because they are highly safe and can expect relatively long-term gene expression. Adeno-associated viruses include type 1 AAV virus (AAV1), type 2 AAV virus (AAV2), type 3 AAV virus (AAV3), type 4 AAV virus (AAV4), type 5 AAV virus (AAV5), type 6 AAV virus (AAV6), type 7 AAV virus (AAV7), type 8 AAV virus (AAV8) and the like are known, and any of them can be used for the production of an AAV vector.

  In particular, the optimum serotype of the AAV vector varies depending on the target organ or tissue in which the polynucleotide encoding interferon λ4 is expressed. Therefore, it is desirable to select a serotype as appropriate according to the organ or tissue in which the polynucleotide is expressed. For example, when the organ that expresses the polynucleotide is a liver, it is preferable to use an AAV8 vector.

  The anticancer activity enhancer according to the present invention is not particularly limited, but when an AAV vector is used, a recombinant vector can be prepared according to the following method. Hereinafter, the case where an AAV vector is used as a preferred embodiment of the present invention will be specifically described. However, the scope of the present invention is not limited to the case where an AAV vector is used. Inverted terminal repeat (ITR) sequences present at both ends of the type 8 AAV (AAV3) virus genome by inserting the above-mentioned polynucleotide encoding interferon λ4 between the cytomegalovirus (CMV) promoter region and the SV40 polyA region A vector plasmid is prepared by incorporating it into the plasmid. The above-mentioned vector plasmid, packaging plasmid (plasmid for expressing Rep, which is a non-structural protein of AAV, and VP, which is a capsid protein), and helper plasmid (base sequence derived from adenovirus, E2A, E4, and VA regions) AAV vector containing a polynucleotide encoding interferon λ4 can be prepared by transfecting human kidney-derived HEK293 cells.

<Protein>
Moreover, when the active ingredient is the above-described interferon λ4 (protein), the anticancer activity enhancer according to the present invention can be adjusted according to a conventional method. That is, the above protein can be obtained based on the amino acid sequence of SEQ ID NO: 2. Such proteins can be produced in prokaryotic or eukaryotic host cells by expression of a polynucleotide encoding the amino acid sequence. Alternatively, such proteins can be synthesized by chemical methods.

  Here, when the protein is synthesized by a chemical method, the carboxy terminus and / or amino terminus can be modified. Examples of modifications include acetylation and amidation. Further modifications can include amino terminal modifications such as acylation or alkylation (eg methylation), carboxy terminal modifications such as amidation, and other terminal modifications such as cyclization. By making these modifications, it is possible to improve the stability of the synthesized protein, improve the pharmacological action, improve the resistance to serum proteases, acquire the desired pharmacokinetic properties, Chemical, biochemical and pharmaceutical properties can be obtained.

  Specifically, in order to obtain the above protein, a polynucleotide encoding the target protein may be introduced into an expression vector and expressed in a host cell. Alternatively, the protein can be obtained using a cell-free system (cell-free system).

  The expression vector is not particularly limited, and any vector can be used as long as it can be replicated in a host cell such as a plasmid, phage, or virus. The vector contains a replication origin, a selectable marker, and a promoter, and may contain an enhancer, a transcription termination sequence (terminator), a ribosome binding site, a polyadenylation signal, and the like as necessary.

  Introduction of a polynucleotide into a vector can be performed by a known method. The vector preferably contains a polylinker with various restriction sites therein or contains a single restriction site. A specific restriction site in the vector can be cleaved with a specific restriction enzyme, and the polynucleotide can be inserted into the cleavage site. An expression vector containing the polynucleotide can be used for transformation of an appropriate host cell to express and produce the protein or partial fragment in the host cell.

  Examples of host cells include bacterial cells such as E. coli, Streptomyces, Bacillus subtilis, fungal cells, baker's yeast, yeast cells, insect cells, mammalian cells, and the like.

  Transformation can be performed by known methods such as calcium chloride, calcium phosphate, DEAE-dextran mediated transfection, electroporation, lipofection and the like.

  The obtained protein can be separated and purified by various separation and purification methods. For example, ammonium sulfate precipitation, gel filtration, ion exchange chromatography, affinity chromatography and the like can be used alone or in appropriate combination. In this case, when the expression product is expressed as a fusion protein with GST or the like, it can be purified by utilizing the properties of the protein fused with the target protein. For example, when expressed as a fusion protein with GST, since GST has an affinity for glutathione, it can be efficiently purified by affinity chromatography using a column in which glutathione is bound to a carrier. In addition, when expressed as a fusion protein with a histidine tag, a protein having a histidine tag binds to the chelate column and can be purified using the chelate column.

<Agonist>
On the other hand, as described above, interferon λ4 is involved in signal transduction through IL-10Rα and IL-28Rβ, which are receptors as ligands. As shown in the examples below for details, by enhancing the expression of interferon λ4, the cancer cell proliferation inhibitory effect by the anticancer agent can be enhanced, the progression of cancer can be suppressed, or cancer can be suppressed. Since it can be improved, an agonist that interacts with the receptor of interferon λ4 can also enhance the cancer cell growth inhibitory effect by the anticancer agent, suppress the progression of cancer, or improve the cancer be able to.

  Here, an agonist means a substance having a function of interacting with IL-10Rα and IL-28Rβ, which are receptors for interferon λ4, and activating signal transduction via IL-10Rα and IL-28Rβ. . Examples of the substance include, but are not limited to, polypeptides, oligopeptides, nucleic acids, low molecular compounds, and the like.

<Anticancer agent>
The anticancer activity enhancer according to the present invention enhances the cancer cell proliferation inhibitory effect of the anticancer agent.
Here, although it does not specifically limit as an anticancer agent, A chemotherapeutic agent, a hormonal therapeutic agent, an immunotherapeutic agent, a molecular target drug, etc. can be mentioned. That is, the anticancer activity enhancer according to the present invention can be used in combination with one or more drugs selected from these (hereinafter sometimes abbreviated as concomitant drugs). These active ingredients (drugs) may be low molecular compounds, high molecular proteins, polypeptides, antibodies, or vaccines. Two or more active ingredients (drugs) may be mixed and used at an appropriate ratio.

  Examples of the “chemotherapeutic agent” include anticancer agents such as alkylating agents, platinum preparations, antimetabolites, topoisomerase inhibitors, anticancer antibiotics, plant-derived anticancer agents. Examples of the “alkylating agent” include nitrogen mustard, nitrogen mustard hydrochloride-N-oxide, chlorambutyl, cyclophosphamide, ifosfamide, thiotepa, carbocon, improsulfan tosylate, busulfan, nimustine hydrochloride, mitoblonitol, Faran, dacarbazine, ranimustine, sodium estramustine phosphate, triethylenemelamine, carmustine, lomustine, streptozocin, piprobroman, etoglucid, altretamine, ambmustine, dibrospdium hydrochloride, fotemustine, predonimustine, pumitepa, ribomustine, temosofredomestre , Trophosphamide, dinostatin styramer, adzeresin, systemustin, biselecin, etc. That. Examples of the “platinum preparation” include carboplatin, cisplatin, miboplatin, nedaplatin, oxaliplatin and the like. Examples of the “antimetabolite” include, for example, mercaptopurine, 6-mercaptopurine riboside, thioinosine, methotrexate, enocitabine, cytarabine, cytarabine okphosphat, ancitabine hydrochloride, 5-FU drugs (eg, fluorouracil, tegafur, UFT, Doxyfluridine, carmofur, garocitabine, emiteful, etc.), aminopterin, leucovorin calcium, tabloid, butosine, folinate calcium, levofolinate calcium, cladribine, fludarabine, gemcitabine, hydroxycarbamide, pentostatin, pyritrexim, idoxyuridine, mitoguanzone, thiazoline azofumuthone Etc. Examples of the “topoisomerase inhibitor” include topoisomerase I inhibitors (eg, irinotecan, topotecan etc.) and topoisomerase II inhibitors (eg, sobuzoxane etc.). Examples of the “anticancer antibiotic” include anthracycline anticancer drugs (doxorubicin hydrochloride, daunorubicin hydrochloride, aclarubicin hydrochloride, pirarubicin hydrochloride, epirubicin hydrochloride, etc.), actinomycin D, actinomycin C, mitomycin C, chromomycin A3 Bleomycin hydrochloride, bleomycin sulfate, pepromycin sulfate, neocalcinostatin, misramycin, sarcomomycin, carcinophylline, mitotane, zorubicin hydrochloride, mitoxantrone hydrochloride, idarubicin hydrochloride, and the like. Examples of “plant-derived anticancer agents” include vinca alkaloid anticancer drugs (vinblastine sulfate, vincristine sulfate, vindesine sulfate, etc.), taxane anticancer drugs (paclitaxel, docetaxel, etc.), etoposide, etoposide phosphate, teniposide, vinorelbine, etc. Is mentioned.

  Examples of the “hormone therapeutic agent” include corticosteroids (eg, dexamethasone, prednisolone, betamethasone, triamcinolone, etc.), and prednisolone is particularly preferable.

  Examples of the “immunotherapeutic agent (BRM)” include picibanil, krestin, schizophyllan, lentinan, ubenimex, interferon, interleukin, macrophage colony stimulating factor, granulocyte colony stimulating factor, lymphotoxin, BCG vaccine, corynebacterium parvum, And levamisole, polysaccharide K, and procodazole.

  `` Molecular targeted drugs '' include, for example, ibritumomab tiuxetane, gefitinib, tamibarotene, tretinoin, panitumumab, lapatinib, trastuzumab, rituximab, imatinib, gemtuzumab ozogamibine, bortezimab, cetuximab, cetuximab, cetuximab Examples include sorafenib, dasatinib, panitumbab, pegaptanib, batalanib, ranibitumab, SU-6668, SU-11248, neobastat, vandetanib, temsirolimus, everolimus, sirolimus and the like. In addition to these molecular targeted drugs, human epidermal growth factor receptor 2 inhibitor, epidermal growth factor receptor inhibitor, Bcr-Abl tyrosine kinase inhibitor, epidermal growth factor tyrosine kinase inhibitor, mTOR inhibitor, blood vessel Inhibitors targeting angiogenesis such as endothelial growth factor receptor 2 inhibitor (α-VEGFR-2 antibody), various tyrosine kinase inhibitors such as MAP kinase inhibitor, inhibitors targeting cytokines, proteasome inhibitors In addition, molecular target drugs such as antibody-anticancer drug combinations can also be included.

  The anticancer agents as described above are applied to the following cancer treatments, for example. Specific cancers include lung cancer (eg, non-small cell lung cancer), melanoma (eg, metastatic malignant melanoma), renal cancer (eg, clear cell carcinoma), prostate cancer (eg, hormone refractory prostate adenocarcinoma) Colon cancer (eg colon cancer), breast cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, skin or intraorbital malignant melanoma, uterine cancer, ovarian cancer, anal cancer, gastric cancer, testicular cancer, fallopian carcinoma Endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, non-Hodgkin's lymphoma (eg, diffuse large B cell lymphoma, follicular lymphoma), esophageal cancer, small intestine cancer, endocrine cancer, Thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, hepatocellular carcinoma, acute myeloid leukemia, chronic myelogenous leukemia, acute lymphoblastic white Hematological disease, chronic or acute leukemia including chronic lymphocytic leukemia, childhood solid cancer, lymphocytic lymphoma, bladder cancer, renal or ureteral cancer, renal pelvic carcinoma, central nervous system (CNS) tumor (eg glioblastoma) ), Primary CNS lymphoma, tumor angiogenesis, spinal tumor, brain stem glioma, pituitary adenoma, Kaposi sarcoma, squamous cell carcinoma, squamous cell carcinoma, T cell lymphoma, environmentally induced cancer including asbestos-induced cancer .

  As an example, chemotherapy for hepatocellular carcinoma includes treatment with sorafenib as systemic chemotherapy for unresectable hepatocellular carcinoma of Child-Pugh classification A, hepatic arterial infusion chemotherapy combined with systemic administration of interferon and hepatic arterial infusion of cisplatin, Examples include 5-FU hepatic arterial infusion chemotherapy combined with interferon and cisplatin-only hepatic arterial infusion chemotherapy.

<Formulation, administration>
The anticancer activity enhancer according to the present invention contains, as described above, a polynucleotide, protein, a partial fragment thereof, or an agonist as an active ingredient. In addition to these active ingredients, any carrier, for example, a pharmaceutically acceptable agent is included. A carrier may be included.

  As a pharmaceutically acceptable carrier, for example, a pharmaceutically acceptable carrier may be included. Pharmaceutically acceptable carrier means a pharmaceutically acceptable substance, composition or vehicle, such as a liquid or solid excipient, diluent, lubricant, or solvent that encapsulates the substance. . Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject to be administered. Specific examples of pharmaceutically acceptable carriers include, for example, sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; celluloses such as cellulose, sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; Tragacanth; malt; gelatin; talc; excipients such as cacao butter, suppository wax; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, soybean oil; glycols such as propylene glycol; glycerin, sorbitol Polyols such as mannitol and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; physiological saline; Le solution; ethyl alcohol; polyesters, polyanhydrides such as polycarbonate, although the like, not particularly limited. Those skilled in the art can appropriately select these carriers.

  In order to promote the introduction of the expression vector into the cell, the anticancer activity enhancer according to the present invention can further contain a nucleic acid introduction reagent. When the anticancer activity enhancer according to the present invention includes a viral vector, particularly a retroviral vector, retronectin, fibronectin, polybrene, or the like can be used as a gene introduction reagent. Further, when the anticancer activity enhancer according to the present invention includes a plasmid vector, lipofectin, lipofectamine, DOGS (transfectam), DOPE, DOTAP, DDAB, DHDAB, HDAB, polybrene, or poly (ethylene) Cationic lipids such as imine) (PEI) can be used.

  Preparations suitable for oral administration include liquids, capsules, sachets, tablets, suspensions, emulsions and the like. Formulations suitable for parenteral administration (eg, subcutaneous injection, intramuscular injection, local infusion, intraperitoneal administration, etc.) include aqueous and non-aqueous isotonic sterile injection solutions, which include antioxidants Further, a buffer solution, an antibacterial agent, an isotonic agent and the like may be contained. Aqueous and non-aqueous sterile suspensions are also included, which may contain suspending agents, solubilizers, thickeners, stabilizers, preservatives and the like. The preparation can be enclosed in a container in unit doses or multiple doses like ampoules and vials. Alternatively, the active ingredient and a pharmaceutically acceptable carrier can be lyophilized and stored in a state that may be dissolved or suspended in a suitable sterile vehicle immediately before use.

  In the pharmaceutical composition, the content of the expression vector capable of expressing the polypeptide is, for example, about 0.1 to 100% by weight of the whole pharmaceutical composition.

  The dosage of the anticancer activity enhancer according to the present invention includes the type of anticancer agent used together, the dosage of the anticancer agent, the activity and type of the active ingredient, the severity of the disease, the species of animal to be administered, the administration Although it varies depending on the drug acceptability, body weight, age, etc. of the subject, it is generally about 0.001 to about 500 mg / kg as an active ingredient amount per day for an adult.

  The anticancer activity enhancer according to the present invention and the above-described anticancer agent may be administered as one preparation, or may be used as separate preparations. When used as separate preparations, for example, a combination of preparations having different administration routes such as an injection and an oral preparation may be used, or both may be the same administration route as an injection or oral preparation. In addition, the anticancer activity enhancer according to the present invention and the above-described anticancer agent may be administered simultaneously, or one of them may be administered first and the other may be administered later. That is, the anticancer activity enhancer according to the present invention may be administered before the anticancer agent described above, may be administered simultaneously, or may be after the start of administration of the anticancer agent.

  In particular, the anticancer activity enhancer according to the present invention is preferably administered simultaneously with the administration of the anticancer agent described above, or is administered for a certain period of time prior to the administration of the anticancer agent described above. Specifically, it is preferable to administer the anticancer activity enhancer according to the present invention from 2 weeks before the start of the above-described anticancer agent administration, and more preferably from 1 week before the start of the above-described anticancer agent administration. Preferably, it is more preferable to administer from 3 days before the start of administration of the anticancer agent described above. By administering the anticancer activity enhancer according to the present invention prior to the administration of the anticancer agent described above, it is possible to effectively exert the cancer cell proliferation inhibitory effect of the anticancer agent described above, and the cancer treatment effect of the anticancer agent is optimal. Can be pulled out.

  In addition, the “combination of the anticancer activity enhancer according to the present invention and the above-described anticancer agent” in the present specification is not limited to the case where the preparation is integrated as a drug, and the preparation is an anticancer action enhancer. Even if it is provided alone, as long as it is used in combination with the anticancer agent for the purpose of enhancing the growth inhibitory effect of cancer cells by the anticancer agent described above, the “anticancer agent potentiating agent according to the present invention” has been described above. It is included in the concept of “combination with anticancer agents”.

  By the way, as described above, interferon λ4 is highly likely to be endogenously expressed when the polymorphism defined by ss469415590 is a minor allele (ΔG).

  Therefore, the anticancer activity enhancer according to the present invention can increase or decrease the dose based on the expression of the interferon λ4 gene. More specifically, for patients who do not express the interferon λ4 gene or have a low expression level, the dose of the anticancer activity enhancer according to the present invention is relatively increased, and the expression level of the interferon λ4 gene is increased. For high patients, the dose of the anticancer activity enhancer according to the present invention can be relatively reduced. Alternatively, the anticancer activity enhancer according to the present invention can be administered to cancer patients in which the interferon λ4 gene is not expressed or the expression level is low.

  In addition, the anticancer activity enhancer according to the present invention can increase or decrease the dose based on whether the polymorphism defined in ss469415590 is a major allele or a minor allele. More specifically, for patients whose polymorphism defined by ss469415590 is a major allele, the dosage of the anticancer activity enhancer according to the present invention is relatively large, and the polymorphism defined by ss469415590 is minor. For allelic patients, the dose of the anticancer activity enhancer according to the present invention can be relatively reduced. Alternatively, the anticancer activity enhancer according to the present invention may be administered to cancer patients whose polymorphism defined by ss469415590 is a major allele.

<Cancer treatment support method>
As described above, the anticancer activity enhancer according to the present invention enhances the cancer cell growth inhibitory effect of the above-described anticancer agent, but the patient does not express the interferon λ4 gene or has a low expression level of the gene. The anticancer action enhancing effect can be expected more. Therefore, it is possible to realize a cancer treatment support method for determining the combined effect of the anticancer activity enhancer and the anticancer agent according to the present invention based on the expression of the interferon λ4 gene.

  In this cancer treatment support method, first, the expression of the interferon λ4 gene in a subject is measured. Here, the subject is synonymous with a biological sample collected from a cancer patient or the like, and examples thereof include tissues, cells, body fluids, urine, and protein extracts derived from other biological samples. Here, body fluid refers to blood, lymph fluid, tissue fluid (interstitial fluid, intercellular fluid, interstitial fluid), body cavity fluid, serous cavity fluid, pleural effusion, ascites, pericardial fluid, cerebrospinal fluid (spinal fluid), joint fluid ( Synovial fluid), aqueous humor (aqueous humor), digestive fluid, pancreatic juice, intestinal fluid, semen and amniotic fluid. Further, the subject may be any one or a plurality of protein extracts derived from tissues, cells, body fluids, urine, and other biological samples. The tissue includes a part of the tissue obtained during surgery performed for the purpose of treating cancer-affected patients, a part of tissue collected by biopsy from a subject suspected of cancer, and a patient with cancer. It means to include a part of tissue collected by biopsy or the like for the purpose of discriminating between primary and metastatic.

  Specifically, in order to measure the expression of the interferon λ4 gene, for example, the amount of mRNA for the gene to be measured may be measured, or the amount of protein that is a product of the gene to be measured may be measured.

  As a method for measuring the mRNA level of the interferon λ4 gene, a known detection method for gene expression can be used. For example, Northern blotting method, real-time-PCR method, RT-PCR method, hybridization method, DNA array method and the like can be used to detect the amount of mRNA of the gene to be measured. In addition, a polynucleotide having a DNA sequence that hybridizes under stringent conditions to the interferon λ4 gene gene can be used as a probe. Detection of the amount of mRNA of the gene to be measured using the probe can be appropriately performed using a known method. For example, when a probe is prepared, a label such as a fluorescent label is appropriately given to the probe, and this is hybridized with mRNA isolated from a biological sample collected from a judgment subject (or cDNA synthesized from mRNA). To do. Thereafter, the amount of mRNA of the gene to be measured can be detected by measuring the fluorescence intensity derived from the hybridized probe. In addition, as a probe, it can also fix and use for support bodies, such as a glass bead and a glass substrate. That is, it can also be used in the form of a microarray or DNA chip in which probes prepared for the gene (or genes) to be measured are immobilized on a support. The support is not particularly limited as long as the polynucleotide can be immobilized, and may have any shape or material. In general, examples of the support include inorganic materials such as glass plates, silicon wafers, and resins, nitrocellulose as a natural polymer material, and nylon as a synthetic polymer material.

  The polynucleotide to be immobilized may be a synthetic oligonucleotide. It is also possible to introduce a nucleic acid derivative capable of fluorescent labeling onto the synthetic oligonucleotide sequence. In addition, a so-called Affymetrix-type DNA chip technique capable of synthesizing a target oligonucleotide on a support can also be used. Furthermore, it is also possible to fix by spotting on a so-called 3D-Gene type columnar surface in which the support has a three-dimensional structure.

  “Hybridize under stringent conditions” means, for example, 1 × SSC (0.15 M NaCl, 0.015 M sodium citrate), 0.1% SDS (Sodium dodecyl sulfate) at 42 ° C. It means that hybridization is maintained even by a washing treatment at 42 ° C. with a buffer solution containing In addition to the above temperature conditions, there are various factors that influence the stringency of hybridization. Those skilled in the art can combine various elements to obtain a string equivalent to the above-described hybridization stringency. It is possible to realize a genie.

  The probe and primer set for quantitatively detecting mRNA and cDNA derived from the interferon λ4 gene are not particularly limited. For example, the probe and primer set shown in the examples described later can be used.

  On the other hand, as a method for measuring the amount of protein that is a product of the interferon λ4 gene, a known protein detection method can be used. Specifically, various methods using an antibody against interferon λ4 can be applied.

  As long as interferon λ4 is used as an antigen and binds to the antigen, the antibody is not particularly limited, and a mouse antibody, a rat antibody, a rabbit antibody, a sheep antibody, or the like can be used as appropriate. The antibody may be a polyclonal antibody or a monoclonal antibody, but a monoclonal antibody is preferable in that a homogeneous antibody can be stably produced. Polyclonal antibodies and monoclonal antibodies can be prepared by methods well known to those skilled in the art.

  A hybridoma producing a monoclonal antibody can be basically produced using a known technique as follows. That is, a desired antigen or a cell expressing the desired antigen is used as a sensitizing antigen and immunized according to a normal immunization method, and the resulting immune cell is fused with a known parent cell by a normal cell fusion method. And can be prepared by screening monoclonal antibody-producing cells (hybridomas) by a normal screening method. The hybridoma can be produced, for example, according to the method of Milstein et al. (Kohler. G. and Milstein, C., Methods Enzymol. (1981) 73: 3-46).

  Here, when producing monoclonal antibodies, interferon λ4 can be used as an antigen, and cells expressing a fragment of interferon λ4 can be used as an antigen. These proteins or fragments of the proteins can be obtained, for example, by the method described in Molecular Cloning: A Laboratory Manual 2nd Edition, Vol. 1-3, Sambrook, J. et al., Cold Spring Harber Laboratory Press, New York, 1989. Accordingly, it can be easily obtained by those skilled in the art. Cells expressing these proteins or fragments of the proteins were also described in Molecular Cloning: A Laboratory Manual, 2nd edition, volume 1-3, Sambrook, J. et al., Cold Spring Harber Laboratory Press publication New York 1989. According to the method, it can be easily obtained by those skilled in the art.

  The resulting monoclonal antibody can be used to quantify the protein to be measured for enzyme-linked immunosorbent assay (ELISA), enzyme immunodot assay, radioimmunoassay, agglutination-based assay, or other well-known immunoassay methods. Can be used as a test reagent. The monoclonal antibody is preferably labeled. When labeling, for example, an enzyme, a fluorescent substance, a chemiluminescent substance, a radioactive substance, or a staining substance known in the art can be used as the labeling compound.

  The support may be any material that can immobilize proteins, for example. In general, a glass plate, a silicon wafer, an inorganic material such as a resin, a natural polymer material nitrocellulose, a synthetic polymer material nylon, polystyrene, or the like can be used.

  Next, in the cancer treatment support method, the combined effect of the anticancer agent and the anticancer agent according to the present invention is determined based on the expression level of the interferon λ4 gene in the subject. That is, when the expression level of the interferon λ4 gene in the subject is low, the combined effect can be expected to be high. As described above, in the cancer treatment support method, it is possible to estimate with high accuracy whether or not the anti-cancer agent has an effect of suppressing the growth of cancer cells when the anti-cancer activity enhancer according to the present invention is used for a predetermined patient. it can. Therefore, by applying this cancer treatment support method, it is possible to provide an effective treatment for cancer using the anticancer activity enhancer according to the present invention.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, the technical scope of this invention is not limited to a following example.

[Example 1]
In this example, the success rate when hepatic arterial infusion chemotherapy was performed using the CDDP + 5FU + IFNα regimen in 109 hepatocellular carcinoma cases with Child-Pugh classification A or B and no extrahepatic lesions. IL28B gene polymorphism major type (rs8099917 major allele (TT)) and minor type (rs8099917 minor allele (TG or GG)) were compared.

  The results are shown in FIG. In addition, as shown in FIG. 1, the sum of complete response (CR) and partial response (PR) was made into the response group, and the sum of stability (SD) and progression (PD) was made into the non-response group. As can be seen from Fig. 1, the response rate (CR + PR) is 27/87 (31%) for the IL28B gene polymorphism major type, and 12/19 (63%) for the minor type. High (p = 0.0097).

  In addition, the overall survival rate of 109 hepatic arterial infusion chemotherapy patients was compared between the major and minor types of IL28B gene polymorphism. The results of survival curve analysis by Kaplan-Meyer analysis are shown in FIG. In FIG. 2, the vertical axis indicates the overall survival rate, and the horizontal axis indicates the overall survival period (OS). As shown in FIG. 2, the IL28B minor type significantly increased the overall survival rate.

  Furthermore, clinical factors (univariate and multivariate analysis) contributing to overall survival by Cox regression analysis were examined. The results are shown in FIG. As shown in FIG. 3, univariate analysis revealed that clinical factors contributing to overall survival were related to gender, IL28B gene polymorphism, presence of major vascular lesions, and AFP values. Multivariate analysis revealed that the IL28B genotype, the presence or absence of major vascular lesions, and AFP values were independent prognostic factors.

  By the way, Ludmila Prokunina-Olsson et al., Nat Genet. 2013 Feb; 45 (2): 164-71 discloses that the expression of IFNλ4 is related to HCV clearance. In this example, the expression levels of interferon-responsive gene (MX1 gene in this example) and hepatitis C virus RNA (HCV) when the expression product of IL28B gene containing IFNλ4 was overexpressed were verified.

First, HCV RNA (H77 strain) 1ig was introduced into well-differentiated human liver cancer-derived cell line Huh7 cells (12 well plate, 1 × 10 ⁵ cells) using TranIT-mRNA Transfection Kit (Mirus). Next, after 24 hours, p107 expression plasmid, p124 expression plasmid, p131 expression plasmid, p143 expression plasmid disclosed in Ludmila Prokunina-Olsson et al., Nat Genet. 2013 Feb; 45 (2): 164-71, 500 ng of p170 expression plasmid or p179 expression plasmid was introduced using FuGENE6 Transfection regent (Promega). A cell line introduced with each of these plasmids alone, a cell line introduced in combination (a cell line introduced with p107 and p179, a cell line introduced with p107 and p131, a cell line introduced with p179 and p131, p107, cell lines into which p131 and p179 were introduced). Note that p179 is IFNλ4.

  After 48 hours, RNA was extracted using High Pure RNA Isolarion Kit (Rocho), and after reverse transcription, the expression levels of MX1 and HCV-RNA were quantified by real-time PCR. At the same time, proteins were extracted using RIPA lysis buffer, and the expression of phosphorylated STAT1, STAT1 and β-actin was evaluated by Western blotting.

  MX1 probes and primer sets used for real-time PCR were purchased from ABI (Hs00895608_m1). HCV-RNA probes and primer sets are Forward primer 5'-CGGGAGAGCCATAGTGG-3 '(SEQ ID NO: 3), Reverse primer 5'-AGTACCACAAGGCCTTTCG-3' (SEQ ID NO: 4), Probe 5'-CTGCGGAACCGGTGAGTACAC-3 '(FAM) (SEQ ID NO: 5) was custom-ordered and used. As antibodies for Western blotting, Phospho-Stat1 (Tyr701) (58D6) Rabbit mAb, Stat1 Antibody Rabbit mAb, β-Actin Antibody Rabbit mAb, Anti-rabbit IgG, and HRP-linked Antibody (Cell Signaling) were used.

  The result of real-time PCR and the result of Western blot are shown in FIG. As shown in FIG. 4, in HCV-infected Huh7 cells overexpressing p179 (IFNλ4), it was revealed that ISG-inducible gene expression was increased and the expression level of HCV-RNA decreased. In contrast, in HCV-infected Huh7 cells overexpressing p107, p124, p131, p143, and p170, the expression level of the ISG-inducible gene was not increased, and the expression level of HCV-RNA did not change. Furthermore, it was revealed that the expression level of phosphorylated STAT1 increased in HCV-infected Huh7 cells overexpressing p179 (IFNλ4). From the above results, it became clear that only the overexpression of IFNλ4 exhibits ISG-induced anti-HCV activity.

Further, in this example, ISGλ4 was examined for ISG-induced anti-HCV activity in comparison with other IFNs. First, expression plasmids for expressing recombinant proteins each incorporating a FLAG-tag sequence at the C-terminus of IFNA1, IL28B, and IFNL4 were constructed. Specifically, the following primers were designed for IFNA1 (IFNα1), IL28B (IFNλ3) and IFNL4 (IFNλ4) and constructed by inserting various IFN-FLAG-tag-attached PCR fragments into the pcDNA3.4 vector (pcDNA3. 4-IFNA1-FLAG, pcDNA3.4-IL28B-FLAG and pcDNA3.4-IFNL4-FLAG).
IFNA1 Forward primer 5'-ATGGCCTCGCCCTTTGC-3 '(SEQ ID NO: 6)
IFNA1 Reverse primer 5'-TCACTTGTCATCGTCATCCTTGTAGTCTTCCTTCCTCCTTAATC-3 '(SEQ ID NO: 7)
IL28B Forward primer 5'-ATGACCGGGGACTGCATG-3 '(SEQ ID NO: 8)
IL28B Reverse primer 5'-TCACTTGTCATCGTCATCCTTGTAGTCGACACACAGGTCCCCG-3 '(SEQ ID NO: 9)
IFNL4 Forward primer 5'-ATGCGGCCGAGTGTCTG-3 '(SEQ ID NO: 10)
IFNL4 Reverse primer 5'-TCACTTGTCATCGTCATCCTTGTAGTCGAGGCAAGGCCCAGAG-3 '(SEQ ID NO: 11)

Next, 500 ng of various IFN-FLAG expression plasmids were introduced into Huh7 cells (12 well plate, 1 × 10 ⁵ cells) using FuGENE6 Transfection regent (Promega). 24 hours later, HCV (isolated from H77 strain) was infected with MOI = 5. 72 hours later, RNA was extracted using High Pure RNA Isolarion Kit (Rocho), and after reverse transcription, MX1 gene and HCV-RNA were quantified by real-time PCR.

  The result of measuring the expression level of the MX1 gene by real-time PCR is shown in FIG. 5, and the result of quantifying HCV-RNA by real-time PCR is shown in FIG. As shown in FIG. 5, in the system in which Huh7 cells introduced with IFNλ4 were infected with HCV, the expression of the MX1 gene, which is an ISG-inducible gene, was increased in the same manner as when IFNα1 was introduced. Further, as shown in FIG. 6, in the system in which Huh7 cells introduced with IFNλ4 were infected with HCV, it was clarified that the expression level of HCV-RNA was reduced in the same manner as when IFNα1 was introduced. From the above results, it was revealed that overexpression of IFNλ4 exhibits ISG-induced anti-HCV activity equivalent to IFNα1.

[Example 2]
In this example, the antitumor effect when IFNλ4 and an anticancer agent were used in combination was verified.
First, 500 ng of IFNA1-FLAG expression plasmid or 500 ng of IFNL4-FLAG expression plasmid prepared in Example 1 was introduced into Huh7.5 cells (12 well plate, 1 × 10 ⁵ cells) using FuGENE6 Transfection regent (Promega). . After 24 hours, 5FU was treated with 10 ig / ml. This time was defined as Day0 (D0). Thereafter, cell proliferation ability was evaluated using Cell Counting Kit-8 (DOJINDO) at D0, D1, D2, D3 and D4.

  The results are shown in FIG. As shown in FIG. 7, HuF7.5 cells expressing IFNλ4 showed a more prominent cell growth inhibitory effect due to 5FU compared to Huh7.5 cells and Huh7.5 cells expressing IFNα1. Therefore, it became clear that IFNλ4 exhibits an action of enhancing the antitumor effect by 5FU.

In addition, 500 ng of IFNL4-FLAG expression plasmid was introduced into Huh7.5 cells (12 well plate, 1 × 10 ⁵ cells) and HCV-infected Huh7.5 cells (see Example 1) using FuGENE6 Transfection regent (Promega). After 24 hours, 5FU was treated with 0, 0.05, 0.1, 1, 5 or 10 ig / ml. After 72 hours, cell proliferation ability was evaluated using Cell Counting Kit-8 (DOJINDO). At the same time, proteins were extracted using RIPA lysis buffer, and expression of phosphorylated ATF2, phosphorylated cJun, HCV-core and a-actin was evaluated by Western blotting. Antibodies for Western blotting are Phospho-ATF-2 (Thr71) (11G2) Rabbit mAb, Phospho-c-Jun (Ser63) (54B3) Rabbit mAb, β-Actin Antibody Rabbit mAb, Anti-rabbit IgG, HRP-linked Antibody Anti-mouse IgG, HRP-linked Antibody (Cell Signaling), and Anti-Hepatitis C Virus Core 1b antibody [C7-50] (abcam) were used.

  The evaluation results of cell proliferation ability and the results of Western blotting are shown in FIG. As shown in FIG. 8, HuF7.5 cells that express IFNλ4 and HCV-infected Huh7.5 cells that express IFNλ4 showed a more prominent cell growth inhibitory effect due to 5FU. In particular, Huh7.5 cells expressing IFNλ4 were found to have a more prominent effect on cell proliferation by 5FU compared to HCV-infected Huh7.5 cells expressing IFNλ4. From the results of Western blotting, it was found that the expression levels of phosphorylated ATF2 and phosphorylated cJUN increased in Huh7.5 cells expressing IFNλ4 and HCV-infected Huh7.5 cells expressing IFNλ4. From this result, in the Huh7.5 cells expressing IFNλ4 and the HCV-infected Huh7.5 cells expressing IFNλ4, the apoptosis activity via the ATF2-cJUN pathway is enhanced. It became clear.

Furthermore, various IFN-FLAG expression plasmids (pcDNA3.4-IFNA1-FLAG, pcDNA3.4-IL28B-FLAG and pcDNA3.4-IFNL4) prepared in Example 1 were used in Huh7 cells (12 well plate, 1 × 10 ⁵ cells). -FLAG) 500 ng was introduced using FuGENE6 Transfection regent (Promega). After 24 hours, 5FU was treated with 10 ig / ml and CDDP was treated with 10 ig / ml. After 48 hours, Caspase activity was evaluated using Caspase-Glo ™ 3/7 Assay kit (Promega).

  The result of measuring caspase activity when 5FU treatment was performed is shown in FIG. 9, and the result of measuring caspase activity when CDDP treatment was performed is shown in FIG. As shown in FIGS. 9 and 10, in both 5FU treatment and CDDP treatment, Huh7.5 cells expressing IFNλ4 are compared with Huh7.5 cells expressing IFNα1 and Huh7.5 cells expressing IFNλ3. As a result, it was revealed that caspase activity was high and apoptosis was strongly induced.

Example 3
In this example, a measurement system for specifically detecting IFNλ4 was established. That is, based on the nucleotide sequence of the coding region encoding IFNλ4 shown in FIG. 11, and the same nucleotide sequences relating to p107, p124, p131, p143 and p170, primers and probe sets for real-time PCR capable of specifically detecting IFNλ4 are prepared. Designed. FIG. 11 shows the position of the primer set (forward primer and reverse primer) and the position of the probe.
Forward primer 5'-CTCCGCGGCCATCGT-3 (SEQ ID NO: 12)
Reverse primer 5'-AGACCACGCTGGCTTTGC-3 (SEQ ID NO: 13)
Probe 5'-TGCCTTGAGCTGGCA-3 '(FAM) (SEQ ID NO: 14)

In addition, it was confirmed whether the primers and plasmids designed in this example could specifically detect IFNλ4. That is, png expression plasmid, p124 expression plasmid, p131 expression plasmid, p143 expression plasmid, p170 expression plasmid or p179 (IFNλ4) expression plasmid 500 ng prepared in Example 1 was transferred to Huh7 cells (12 well plate, 1 × 10 ⁵ cells). Each was introduced using FuGENE6 Transfection regent (Promega). After 72 hours, RNA was extracted using High Pure RNA Isolarion Kit (Rocho), and after reverse transcription, IFNλ4 was quantified by real-time PCR using the above probe and primer set.

  The results are shown in FIG. As shown in FIG. 12, when the above primer set and probe were used, the PCR amplified fragment could be quantitatively measured only with Huh7 cells into which the p179 (IFNλ4) expression plasmid was introduced.

Example 4
In this example, as described above, a system for purifying IFNλ4 having apoptosis-inducing activity via the ATF2-cJUN pathway as a recombinant protein was established.

  First, for IFNα1, IL28B (IFNλ3) and IFNL4, genes expressing recombinant IFN proteins each having a DDDDK tag added to the C-terminal side were synthesized and introduced into a pcDNA3.4 vector (invitorgen). The prepared plasmid was transfected into ExpiCHO cells (invitorgen) using ExpiFectamine (invitorgen), respectively, and various recombinant IFN proteins were transiently expressed. Recovered IFN protein expression was confirmed by SDS-PAGE and western blotting of the collected medium and further measurement of IFN activity. Thereafter, the recombinant IFN protein was purified using DDDDK tag antibody-bound beads (MBL).

Next, Huh7 cells (12 well plate, 1 × 10 ⁵ cells) were each treated with various IFN recombinant proteins, 24 hours later, RNA was extracted using High Pure RNA Isolarion Kit (Rocho), and after reverse transcription The expression of IFIT1 (Interferon Induced Protein With Tetratricopeptide Repeats 1) was quantified by real-time PCR. In addition, proteins were extracted using RIPA lysis buffer, and expression of phosphorylated ATF2, phosphorylated cJun and β-actin was evaluated in the same manner as in Example 2 by Western blotting.

  The results of quantifying IFIT1 expression by real-time PCR are shown in FIG. 13, and the results of Western blotting are shown in FIG. As shown in FIG. 13, it was revealed that various recombinant IFN proteins including the recombinant protein of IFNλ4 prepared in this example had interferon activity. In addition, it was revealed that the recombinant protein of IFNλ4 purified in this example retained the function of activating the ATF2-cJUN pathway.

Claims (13)

A polynucleotide encoding interferon λ4;
Interferon λ4; or an anticancer enhancer of an anticancer agent comprising an agonist for a receptor having interferon λ4 as a ligand as an active ingredient.   The anti-cancer activity enhancer according to claim 1, wherein the interferon λ4 is composed of the amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence having 90% or more identity to the amino acid sequence.   The anticancer activity enhancer according to claim 1, wherein the anticancer agent is 5-fluorouracil or cisplatin.   The anticancer activity enhancer according to claim 1, wherein the dose is increased or decreased based on the expression level of the interferon λ4 gene.   The anticancer activity enhancer according to claim 1, wherein the dose is increased or decreased based on whether the polymorphism defined by ss469415590 in the IL-28B gene encoding interferon λ3 is a major allele or a minor allele.   The anticancer effect enhancer according to claim 1, wherein the administration target patient is a cancer patient with a low expression level of interferon λ4 gene.   The anticancer activity enhancer according to claim 1, wherein the administration target patient is a cancer patient whose polymorphism defined by ss469415590 in the IL-28B gene encoding interferon λ3 is a major allele. Measuring the expression of the interferon λ4 gene in the subject using a means capable of specifically detecting the expression of the interferon λ4 gene;
A cancer treatment support method for determining a combined effect of an anticancer agent and an anticancer activity enhancer according to any one of claims 1 to 7, based on an expression level of an interferon λ4 gene in a subject.   The means includes a pair of primers that specifically amplify a transcription product of the interferon λ4 gene, and a probe that specifically hybridizes to a nucleic acid amplified by the pair of primers. Item 9. The cancer treatment support method according to Item 8.   The pharmaceutical composition formed by combining the anticancer action enhancer as described in any one of Claims 1-7, and an anticancer agent.   The pharmaceutical composition according to claim 10, wherein the anticancer agent is 5-fluorouracil or cisplatin.   The pharmaceutical composition used together with an anticancer agent containing the anticancer action enhancer as described in any one of Claims 1-7.   The pharmaceutical composition according to claim 12, wherein the anticancer agent is 5-fluorouracil or cisplatin.

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