Composition for treating cancer

Abstract

Provided is a novel therapeutic composition effective against cancer, particularly immune-resistant cancer.　Provided is a composition for use in a method for treating or preventing cancer. The composition contains interleukin 12 (IL-12) or messenger ribonucleic acid (mRNA) comprising a nucleotide sequence encoding IL-12. The method comprises administrating, to a subject, a combination of (a) IL-12 or mRNA comprising a nucleotide sequence encoding IL-12, (b) one or more chemotherapeutic agents or molecular targeting agents, and (c) one or more immune checkpoint inhibitors.

Description

Cancer treatment composition

[0001] The present invention relates to compositions for treating cancer, particularly immune-resistant cancer.

[0002] mRNA-based cancer therapies have advanced rapidly in recent years, and it has been reported that intratumoral administration of cytokine-encoding mRNA leads to significant tumor reduction in animal models (Patent Document 1). Intratumoral administration of cytokine mRNA is known to exert a strong effect on cancerous lesions, and its effectiveness in clinical application is anticipated. These past research results have mainly focused on so-called "hot" tumors, which are rich in immune cells including dendritic cells (DCs) and T lymphocytes, and which respond well to immune checkpoint inhibitors (ICIs). In contrast, sufficient tumor reduction effects have yet to be found for the treatment of so-called "cold" tumors with low activity of DCs, etc.

[0003] Pancreatic ductal adenocarcinoma (PDAC) is known as a malignant tumor with an extremely poor prognosis and a high mortality rate. Despite advances in multidisciplinary therapy including surgery, chemotherapy, radiotherapy, and immunotherapy, its five-year survival rate remains low at approximately 10% (Non-Patent Literature 1). Peritoneal metastasis is observed in 25-50% of PDACA patients, regardless of whether surgical intervention or systemic treatment is performed (Non-Patent Literature 2). PDACA is characterized by an immune-resistant tumor microenvironment, and peritoneal metastasis (peritoneal dissemination) in particular is one of the factors contributing to the treatment resistance of PDACA. It is known to cause symptoms such as abdominal distension and pain, significantly impairing the patient's quality of life. Peritoneal dissemination exhibits particularly "cold" immunological characteristics, and compared to liver or lung metastases, it is associated with CD8 ＋ It is known that there is little T cell infiltration (Non-Patent Literature 3). Currently, there is no effective treatment for PDAC accompanied by peritoneal dissemination, and the development of an effective treatment is urgently needed.

[0004] In the treatment of PDA with peritoneal dissemination, combination therapy with ICI and chemotherapy has not shown sufficient efficacy (Non-Patent Literature 4 and 5). As next-generation immunotherapies for PDA, whole-cell immunomodulators including allogeneic pancreatic cancer cell algenpantscel-L (Non-Patent Literature 6) and the CD40 agonist antibody sotigalimab have been reported (Non-Patent Literature 7), but these therapies target antigen-presenting cells such as dendritic cells (DCs). However, it has been reported that a deficiency of DCs in PDA leads to impaired immune surveillance (Non-Patent Literature 8).

[0005] Special table 2020-509016 publication

[0006] Siegel, RL, et al., Cancer J. Clin. Vol. 73, pp. 17-48 (2023)Morizane, C., et al., Pancreas, Vol. 40, pp. 415-421 (2011)Sasaki, T., et al., Int. J. Clin. Oncol., Vol. 27, pp. 948-957 (2022)O'Reilly, EM, et al., JAMA Oncol., Vol. 5, pp. 1431-1438 (2019)Renouf, DJ, et al., Nat. Commun., Vol. 13, p.5020 (2022)Hewitt, DB, et al., Ann. Surg., Vol. 275, pp. 45-53 (2022)Padron, LJ, et al., Nat. Med., Vol. 28, pp. 1167-1177 (2022)Hegde, S., et al., Cancer Cell, Vol. 37, pp. 289-307, e289 (2020)

[0007] The present invention aims to provide a novel therapeutic composition effective against cancer, particularly immune-resistant cancer.

[0008] This specification provides the following inventions: (1) A composition for use in a method for treating or preventing cancer, the composition comprising interleukin-12 (IL-12) or messenger ribonucleic acid (mRNA) comprising a nucleotide sequence encoding IL-12, wherein the method comprises co-administering (a) IL-12 or mRNA comprising a nucleotide sequence encoding IL-12; (b) one or more chemotherapeutic agents or molecularly targeted drugs; and (c) one or more immune checkpoint inhibitors; the composition; (2) The composition according to (1), comprising mRNA encoding IL-12; (3) The composition according to (1) or (2), wherein the method further comprises co-administering (d) interferon-alpha (IFN-α) or mRNA comprising a nucleotide sequence encoding IFN-α; (4) The composition according to (3), further comprising IFN-α or mRNA encoding IFN-α; (5) The composition according to (4), comprising mRNA encoding IFN-α. (6) The composition according to any one of (1) to (5), wherein the cancer is an immune-resistant cancer. (7) The composition according to (6), wherein the immune-resistant cancer is pancreatic ductal adenocarcinoma (PDAC) or peritoneal dissemination of PDAC. (8) The composition according to (6), wherein the immune-resistant cancer is scirrhous gastric cancer or peritoneal dissemination of scirrhous gastric cancer. (9) The composition according to any one of (1) to (8), wherein the chemotherapeutic agent or molecular targeted drug is selected from oxaliplatin, lenvatinib, and combinations thereof. (10) The composition according to any one of (1) to (9), wherein the immune checkpoint inhibitor is one or more immune checkpoint inhibitors selected from the group consisting of anti-PD-1 antibody, anti-PD-L1 antibody, and anti-CTLA-4 antibody. (11) The composition according to (10), wherein the administration of the immune checkpoint inhibitor is the administration of anti-PD-1 antibody and / or anti-PD-L1 antibody and anti-CTLA-4 antibody.(12) The composition according to any one of (1) to (11), wherein the IL-12 is a protein comprising a subunit having the amino acid sequence described in SEQ ID NO: 7 or an amino acid sequence having 80% or more sequence identity with the amino acid sequence described in SEQ ID NO: 7, and a subunit having the amino acid sequence described in SEQ ID NO: 8 or an amino acid sequence having 80% or more sequence identity with the amino acid sequence described in SEQ ID NO: 8. (13) The composition according to any one of (3) to (12), wherein the IFN-α is a protein having the amino acid sequence described in any one of SEQ ID NOs: 1 to 3 or an amino acid sequence having 80% or more sequence identity with the amino acid sequence described in any one of SEQ ID NOs: 1 to 3. (14) The composition according to any one of (1) to (13), for intratumor administration. (15) A composition for use in a method for treating or preventing cancer, wherein the composition comprises one or more chemotherapeutic agents or molecular targeted drugs, and the method comprises co-administering (a) IL-12 or mRNA containing a nucleotide sequence encoding IL-12; (b) one or more chemotherapeutic agents or molecular targeted drugs; and (c) one or more immune checkpoint inhibitors to a target. (16) The composition according to (15), wherein the method further comprises co-administration of (d) IFN-α or mRNA containing a nucleotide sequence encoding IFN-α. (17) A composition for use in a method for treating or preventing cancer, wherein the composition comprises one or more immune checkpoint inhibitors, and the method comprises co-administration of (a) IL-12 or mRNA containing a nucleotide sequence encoding IL-12; (b) one or more chemotherapeutic agents or molecularly targeted drugs; and (c) one or more immune checkpoint inhibitors. (18) The composition according to (17), wherein the method further comprises co-administration of (d) IFN-α or mRNA containing a nucleotide sequence encoding IFN-α.(19) A kit for use in a method of treating or preventing cancer, comprising two or more of the following: (i) a composition comprising IL-12 or mRNA comprising a nucleotide sequence encoding IL-12; (ii) a composition comprising one or more chemotherapeutic agents or molecularly targeted drugs; (iii) a composition comprising an anti-PD-1 antibody and / or an anti-PD-L1 antibody; and (iv) a composition comprising an anti-CTLA-4 antibody. (20) The kit according to (19), wherein the composition of (i) further comprises IFN-α or mRNA comprising a nucleotide sequence encoding IFN-α. (21) A kit for treating or preventing cancer, comprising two or more of the following: (i') a composition comprising IL-12 or mRNA comprising a nucleotide sequence encoding IL-12; (ii) a composition comprising one or more chemotherapeutic agents or molecularly targeted drugs; (iii) a composition comprising an anti-PD-1 antibody and / or an anti-PD-L1 antibody; and (iv) a composition comprising an anti-CTLA-4 antibody. (22) The kit according to (21), further comprising: (ii') an mRNA comprising IFN-α or a nucleotide sequence encoding IFN-α. (23) A composition for use in a method for treating or preventing cancer, wherein the composition comprises IFN-α or an mRNA comprising a nucleotide sequence encoding IFN-α, and the method comprises co-administering: (a) IL-12 or an mRNA comprising a nucleotide sequence encoding IL-12; (b) one or more chemotherapeutic agents; and (c) one or more immune checkpoint inhibitors; and (d) an mRNA comprising IFN-α or an mRNA comprising a nucleotide sequence encoding IFN-α. (24) A method for treating or preventing cancer, comprising co-administering: (a) IL-12 or an mRNA comprising a nucleotide sequence encoding IL-12; (b) one or more chemotherapeutic agents or molecular targeted drugs; and (c) one or more immune checkpoint inhibitors. (25) The method of (24), further comprising administering (d) IFN-α or mRNA containing a nucleotide sequence encoding IFN-α to the target. This specification incorporates the disclosures of Japanese Patent Application No. 2025-016064, which forms the basis of the priority of this application.

[0009] According to the present invention, it is possible to provide a therapeutic composition effective against cancer, particularly immune-resistant cancer, thereby providing a novel therapeutic method that is effective even against cancers that were previously difficult to treat.

[0010] This is a schematic diagram showing the immune response cycle against tumors. This is a schematic diagram showing the creation of subcutaneous tumor and peritoneal metastatic tumor model mice and the schedule for drug administration. These are the survival curves (Kaplan-Meier curves) for each pancreatic cancer model mouse group in Example 2. The "***" in the figure indicates that P < 0.001 was observed in the log-rank test comparing the group administered five drugs (oxaliplatin, anti-PD-1 antibody, anti-CTLA-4 antibody, IFNα-mRNA, and IL12-mRNA) (group 5A) with the other groups. This is a graph showing the change in tumor volume over time for each mouse group in Example 2. The "**" in the figure indicates that P < 0.01 was observed in the Tukey-Kramer test. Error bars indicate the standard error (n = 12). These are box plots showing the number, total volume, and total weight of intraperitoneal tumors for each mouse group in Example 2. Figure 5A shows the number of tumors, Figure 5B shows the total volume, and Figure 5C shows the total weight. In the figures, "**" and "***" indicate that the Tukey-Kramer test yielded P < 0.01 and P < 0.001, respectively. These graphs show the area of ​​dendritic cells (CD11c-positive cells) in subcutaneous tumors and peritoneal metastatic tumors for each mouse group in Example 3. Figure 6A shows the area of ​​dendritic cells in subcutaneous tumors, and Figure 6B shows the area of ​​dendritic cells in peritoneal metastatic tumors. In the figures, "**" indicates that the Tukey-Kramer test yielded P < 0.01. Error bars indicate the standard error (n = 6). These graphs show the area of ​​CD8-positive cells in subcutaneous tumors and peritoneal metastatic tumors for each mouse group in Example 3. Figure 7A shows the area of ​​CD8-positive cells in subcutaneous tumors, and Figure 7B shows the area of ​​CD8-positive cells in peritoneal metastatic tumors. In the figures, "**" indicates that the Tukey-Kramer test yielded P < 0.01. Error bars indicate the standard error (n = 6). Figure 8A shows the area of ​​HMGB1-positive cells in subcutaneous tumors and peritoneal metastatic tumors in each mouse group in Example 4, and Figure 8D shows the color intensity of HMGB1 in immunohistochemistry.Error bars indicate the standard error (n=6). This is a continuation of Figure 8-1. This is a graph showing the area of ​​calreticulin-positive cells in subcutaneous tumors and peritoneal metastatic tumors for each mouse group in Example 4, and a box plot showing the color intensity of calreticulin in immunohistochemistry. Figure 9A shows the area of ​​calreticulin-positive cells in subcutaneous tumors, Figure 9B shows the color intensity of calreticulin in immunohistochemistry of subcutaneous tumors, Figure 9C shows the area of ​​calreticulin-positive cells in peritoneal metastatic tumors, and Figure 9D shows the color intensity of calreticulin in immunohistochemistry of peritoneal metastatic tumors. In the figures, "***" indicates that P < 0.001 in the Tukey-Kramer test. Error bars indicate the standard error (n=6). This is a continuation of Figure 9-1. This is a graph showing the area of ​​Ki67-positive cells in subcutaneous tumors and peritoneal metastatic tumors for each mouse group in Example 4. Figure 10A shows the area of ​​Ki67-positive cells in subcutaneous tumors, and Figure 10B shows the area of ​​Ki167-positive cells in peritoneal metastatic tumors. In the figures, "***" indicates that P < 0.001 in the Tukey-Kramer test. Error bars indicate the standard error (n = 6). CD8 in the peripheral blood of mice administered each drug in Example 5. ＋ This is T cell flow cytometry. The CD8 percentage in peripheral blood cells of each mouse group, as counted by flow cytometry in Example 5, is... ＋ This graph shows the proportion of T cells and TEM cells. Figure 12A shows the proportion of CD8+ T cells, and Figure 12B shows the proportion of TEM cells. In the figures, "**" and "***" indicate that the Tukey-Kramer test yielded P < 0.01 and P < 0.001, respectively. Error bars indicate the standard error (n = 4). CD8 in the peritoneal lavage fluid of mice administered each drug in Example 5. ＋ This is T cell flow cytometry. The CD8 content of cells in the peritoneal lavage fluid of each mouse group, as counted by flow cytometry in Example 5, is... ＋These are graphs showing the proportions of T cells and TEM cells. Figure 14A shows the proportion of CD8+ T cells, and Figure 14B shows the proportion of TEM cells. In the figures, "**" indicates that P < 0.01 in the Tukey-Kramer test. Error bars indicate the standard error (n = 4). These are graphs showing the area of ​​GZMB-positive cells in subcutaneous tumors and peritoneal metastatic tumors for each mouse group in Example 6. Figure 15A shows the area of ​​GZMB-positive cells in subcutaneous tumors, and Figure 15B shows the area of ​​GZMB-positive cells in peritoneal metastatic tumors. In the figures, "***" indicates that P < 0.001 in the Tukey-Kramer test. Error bars indicate the standard error (n = 6). These are graphs showing the area of ​​IFN-γ-positive cells in subcutaneous tumors and peritoneal metastatic tumors for each mouse group in Example 6. Figure 16A shows the area of ​​IFN-γ-positive cells in subcutaneous tumors, and Figure 16B shows the area of ​​IFN-γ-positive cells in peritoneal metastatic tumors. In the figures, "***" indicates that P < 0.001 in the Tukey-Kramer test. Error bars indicate the standard error (n = 6). This is a graph showing the change in tumor volume over time for each mouse group in Example 7. In the figures, "*" indicates that P < 0.05 in the Tukey-Kramer test. Error bars indicate the standard error (n = 6). These are box plots showing the number, total volume, and total weight of intraperitoneal tumors for each mouse group in Example 7. Figure 18A shows the number of tumors, Figure 18B shows the total volume, and Figure 18C shows the total weight. In the figures, "**" and "***" indicate that P < 0.01 and P < 0.001, respectively, in the Tukey-Kramer test. Flow cytometry of cells in the peripheral blood of 5A and 5A+FTY720 mice in Example 7. Lymphocytes and CD8 cells in the peripheral blood of the 5A and 5A+FTY720 mouse groups in Example 7. ＋ This graph shows the proportion of T cells and TEM cells. Figure 20A shows lymphocytes, and Figure 20B shows CD8 cells. ＋Figure 20C shows the proportion of T cells and TEM cells. In the figure, "***" indicates that P < 0.001 in the t-test. Error bars indicate the standard error (n = 6). This is a heatmap of V-J pairs of T cell receptors (TCRs) in tumor cells in Example 8. Figure 21A shows the heatmap of V-J pairs of TCRs in tumors after Control therapy, and Figure 21B shows the heatmap of V-J pairs of TCRs in tumors after 5A therapy. These are the survival curves (Kaplan-Meier curves) for each mouse group in Example 9. Figure 22A shows the survival curves for the Control, Luc-mRNA + ICI, IL12-mRNA + ICI, IL12-mRNA + ICI + 5-FU, IL12-mRNA + ICI + Paclitaxel, IL12-mRNA + ICI + Oxaliplatin, and IL12-mRNA + ICI + Lenvatinib groups. Figure 22B shows the survival curves for the Control, All, -αCTLA-4, -αPD-1, -IL-12-mRNA, and -Lenvatinib groups. The graphs show the time course of tumor volume for each mouse group in Example 9. Figure 23A shows the time course of subcutaneous tumor volume on the side injected with mRNA, and Figure 23B shows the time course of subcutaneous tumor volume on the side not injected with mRNA. Error bars indicate the standard error. The graphs show the time course of tumor volume for each mouse group in Example 10. Figure 24A shows the time course of subcutaneous tumor volume on the side injected with mRNA, and Figure 24B shows the time course of subcutaneous tumor volume on the side not injected with mRNA. Error bars indicate the standard error. In the figures, "*" indicates that P < 0.05 in the Tukey-Kramer test.

[0011] [1] Overview and Definitions (1) Overview Figure 1 shows an overview of the cancer immune cycle (referencing Chen DS and Mellman I., Immunity, Vol. 39, pp.1-10 (2013)). The cancer immune cycle is divided into seven stages. First, in the first stage, antigens are released from dead cancer cells. Dendritic cells (DCs) take up the released antigens (second stage), and after reaching the lymph nodes, they present the antigens to T cells and become activated (third stage). The activated T cells are delivered to the tumor by the bloodstream (fourth stage). T cells infiltrate the tumor (fifth stage), recognize and bind to cancer cells (sixth stage). Cancer cells are damaged by T cells (seventh stage), leading to the first stage. The first stage, i.e., the release of cancer cell antigens, is promoted by chemotherapy, radiotherapy, and targeted therapy. The second stage, antigen presentation by DCs, is promoted by IFN-α, GM-CSF, and CD40 agonists. The third stage, T cell activation, is promoted by IL-2 and IL-12 and suppressed by CTLA-4, CD137, OX40, and CD27. The fifth stage, T cell infiltration into tumors, is suppressed by VEGF. The seventh stage, cancer cell damage, is suppressed by PD-L1, PD-1, and IDO. Many existing cancer treatments are known to be involved in promoting one or more stages of this cancer immune cycle.

[0012] Pancreatic ductal adenocarcinoma (PDAC), and especially PDAC peritoneal dissemination, are immune-resistant tumors, or in other words, "cold" tumors, that do not show significant activation of dendritic cells (DCs) or T cells. It is known that even if one stage of the cancer immune cycle is activated, the effect is very limited.

[0013] The inventors hypothesized that activating multiple stages of the cancer immune cycle would allow the cancer immune cycle to function even in immune-resistant tumors, thereby promoting tumor suppression. They then verified this hypothesis using a mouse model of peritoneal dissemination in drug-resistant cancer. As a result, they found that the following combination of anticancer agents suppressed drug-resistant cancer and its peritoneal dissemination: (a) IL-12 or mRNA containing a nucleotide sequence encoding IL-12; (b) one or more chemotherapeutic agents; and (c) one or more immune checkpoint inhibitors.

[0014] Furthermore, in particular, in a mouse model of peritoneal dissemination of PDAC, it was found that PDAC and its peritoneal dissemination were suppressed by combining the following anticancer drugs in addition to (a) to (c) above: (d) IFN-α or mRNA containing a nucleotide sequence encoding IFN-α.

[0015] More specifically, we found that the following combination of five anticancer drugs suppresses PDAC and PDAC peritoneal dissemination: (a') mRNA containing a nucleotide sequence encoding IL-12; (b') oxaliplatin; (c') anti-PD-1 antibody or anti-PD-L1 antibody; (c'') anti-CTLA-4 antibody; and (d') mRNA containing a nucleotide sequence encoding IFN-α; IFN-α mainly contributes to the second stage of the cancer immune cycle shown in Figure 1. IL-12 mainly contributes to the third stage of the immune cycle. Oxaliplatin directly attacks cancer cells and mainly contributes to the first stage of the immune cycle. Anti-PD-1 antibody or anti-PD-L1 antibody inhibits the binding of PD-1 and PD-L1, which suppresses the attack of cytotoxic T cells on cancer cells, and mainly contributes to the seventh stage of the immune cycle. Anti-CTLA-4 antibodies inhibit the suppression of T cell activation by dendritic cells due to CTLA-4 expression on the surface of T cells. This primarily contributes to the third stage of the immune cycle. By simultaneously promoting multiple stages of the immune cycle in this way, they exhibit a strong inhibitory effect even against immune-resistant cancers.

[0016] Furthermore, the inventors have found that a combination of the following four anticancer drugs suppresses scirrhous gastric cancer and peritoneal dissemination of scirrhous gastric cancer: (a') mRNA containing a nucleotide sequence encoding IL-12; (b'') lenvatinib; (c') anti-PD-1 antibody or anti-PD-L1 antibody; and (c'') anti-CTLA-4 antibody.

[0017] The inventors have found that the following combination of two anticancer drugs suppresses hepatocellular carcinoma and immune-abnormal liver cancer: (a) IL-12 or mRNA containing a nucleotide sequence encoding IL-12; and (c) one or more immune checkpoint inhibitors.

[0018] More specifically, we found that the following combination of two anticancer drugs suppresses the above-mentioned hepatocellular carcinoma: (a') mRNA containing a nucleotide sequence encoding IL-12; and (c-1) an anti-PD-1 antibody or an anti-PD-L1 antibody.

[0019] (2) Definitions In this specification, “composition” means “pharmaceutical composition” unless otherwise specified. A pharmaceutical composition is a composition used to treat and / or prevent a target disease, disorder, abnormality, etc.

[0020] In this specification, "subject" refers to an animal, preferably a mammal. Mammals refer to animals belonging to the class Mammalia, subphylum Vertebrata, phylum Chordata, including humans and non-humans, such as humans, primates including chimpanzees, pet animals such as dogs and cats, domesticated animals such as cattle, pigs, horses, sheep and goats, rodents such as mice and rats, and mammals kept in zoos. In this specification, the subject is preferably a human.

[0021] In this specification, "sequence identity" of an amino acid sequence or base sequence refers to a value that can be determined with or without introducing gaps using a protein search system or gene search system such as BLAST (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi).

[0022] In this specification, "cancer" is also referred to as a malignant tumor and encompasses all malignant diseases such as sarcomas, carcinomas, and lymphomas. For example, it includes solid tumors or hematological malignancies, and also includes head and neck cancers, colon cancers, rectal cancers, pancreatic cancers, gastric cancers, bladder cancers, breast cancers, hepatocellular carcinomas, lung cancers, medulloblastomas, atypical teratomas / rhabdoid tumors, leukemias, lymphomas, myelomas, esophageal cancers, biliary tract cancers, ovarian cancers, uterine cancers, kidney cancers, prostate cancers, and pleural tumors. Furthermore, it encompasses all malignant tumors regardless of the stage or pathology of the cancer. In this specification, the cancers targeted for treatment are preferably immune-resistant cancers that are difficult to cure with conventional cancer treatments. It is also preferable that the cancers are rapidly progressing and metastatic. In this specification, "immune-resistant cancer" refers to so-called "cold" cancers in which dendritic cell activation is difficult to induce by cancer cell antigens induced by chemotherapy, radiotherapy, etc., or in which the cancer immune cycle is difficult to activate. In particular, many peritoneal disseminations of PDAC are known to be immune-resistant. For example, cancers that are more suitable targets for treatment include pancreatic ductal adenocarcinoma (PDAC), hepatocellular carcinoma, gastric cancer, biliary tract cancer, and colorectal cancer. Gastric cancer, in particular, refers to treatment-resistant gastric cancers such as scirrhous gastric cancer. Hepatocellular carcinoma, in this context, refers to immune-abnormal liver cancer.

[0023] In this specification, "treatment or prevention" includes suppressing the disease, preventing its onset, alleviating one or more symptoms of the disease, curing the disease, and preventing the recurrence of the disease. "Treatment or prevention of cancer" includes reducing the size of tumors in the subject, preventing recurrence of cancer in remission, or preventing cancer metastasis. The effectiveness of treatment or prevention can be evaluated by qualitative and quantitative changes in biomarkers, imaging findings, etc.

[0024] In this specification, "intratumoral administration" refers to directly administering a pharmaceutical composition (therapeutic agent) into a tumor. For example, intratumoral administration is carried out by injecting the therapeutic agent at any position of the tumor. Depending on the site, it may be possible to administer directly from outside the body. However, for tumors deep within the body, such as in the case of pancreatic cancer, it can be administered directly via the gastric wall using an upper gastrointestinal endoscope.

[0025] (3) mRNA In this specification, "messenger ribonucleic acid (mRNA)" is an RNA produced during the process of synthesizing a protein from genomic information stored in a living body. It replicates (transcribes) the base sequence from the genome and translates it into an amino acid sequence on a ribosome to be used for expressing the target protein.

[0026] The base sequence of mRNA may be a sequence optimized for codons for expression in the human body. Any known method may be used as the codon optimization method. Codon optimization is carried out, for example, to enhance the stability of mRNA by adjusting the GC content or to reduce the secondary structure part; to reduce tandem repeat codons or the continuity of the same base; to adjust the transcription and translation control regions; to insert or remove protein transport sequences; to remove or add post-translational modification sites (e.g., glycosylation sites) within the encoded protein; to add, remove, or shuffle protein domains; to insert or delete restriction enzyme recognition sites; to modify the ribosome binding site and the mRNA degradation site; to adjust the translation speed so that various domains of the protein are properly folded, etc. Codon optimization tools, algorithms, and services are known in the art. Non-limiting examples include the services of GeneArt (Life Technologies), DNA2.0 (Menlo Park), and / or an independent method, all of which can be used. In particular, the open reading frame (ORF) sequence encoding IFN-α is preferably optimized using an optimization algorithm.

[0027] In this specification, the mRNA may have a 5' cap structure and / or a 3' poly(A) tail. In particular, it preferably has a 5' cap structure. By having a 5' cap structure, it is possible to produce a protein without being immediately degraded after being taken up into cells of a subject (e.g., a human). As the 5' cap of the mRNA, any known chemical RNA cap analog can be used. For example, 3'-O-Me-m7G(5')ppp(5')G (ARCA cap), G(5')ppp(5')A; G(5')ppp(5')G; m7G(5')ppp(5')A; m7G(5')ppp(5')G, etc. can be used. The 3' poly(A) tail is a stretch of adenine nucleotides added to the 3' end of the transcribed mRNA. The length of the poly(A) tail is not particularly limited and can be appropriately changed depending on the structure of the mRNA and the subject. For example, it can contain 20 to 500, particularly 100 to 200 adenines.

[0028] The mRNA may have untranslated regions on the 5' end side and the 3' end side. As the 5'-UTR sequence, for example, together with the optimized "Kozak sequence" for increasing translation efficiency, the 5'-UTR sequence of human alpha-globin mRNA may be used. As the 3'-UTR sequence, in order to increase the amount of protein produced and ensure the long-term maintenance of the mRNA, a combination of two sequence elements (FI element) derived from the "amino terminal enhancer of split" (AES) mRNA (referred to as F) and mitochondrial encoded 12S ribosomal RNA (referred to as I) arranged between the coding sequence and the poly(A) tail may be used (see WO2017 / 060314). Alternatively, the 3'-UTR may be the two repeated 3'-UTRs of human beta-globin mRNA.

[0029] In this specification, the ORF region of mRNA may be unmodified or chemically modified. The nucleosides constituting mRNA include modified nucleotides or nucleosides. Such modified nucleotides and nucleosides may be naturally occurring modified nucleotides and nucleosides or modified nucleotides and nucleosides that do not exist in nature. Such modifications include modifications of known nucleotides and / or nucleosides at the sugar, backbone, or nucleic acid base portion.

[0030] In this specification, examples of modified nucleic acid bases of mRNA include 5-methoxymethyluridine, 5-methylthiouridine, 1-methoxymethylpseudridine, 5-methylcytidine, and / or 5-methoxycytidine. A polyribonucleotide may contain at least two combinations of the above modified bases.

[0031] It is preferable that mRNA having these stabilizing elements and / or modifications, when transfected into host cells, has stability for 12 to 18 hours, or more than 18 hours, for example, 12, 18, 24, 36, 48, 60, 72 hours, or more than 72 hours, and is expressible by host cells.

[0032] (4) Lipid Nanoparticles (LNPs) In this specification, mRNA is preferably encapsulated in lipid nanoparticles (LNPs) in order to enhance molecular stability and improve the efficiency of uptake into cells. In particular, it is preferable to encapsulate one mRNA molecule in one LNP.

[0033] Any LNP known to be used in mRNA preparations can be used. For example, an LNP with a composition similar to that used in commercially available vaccines listed in Table 1 of Schoenmaker L., et al., Int. J. Pharm. 601(2021) 120586 (e.g., "BNT162b2; Community (Pfizer-BioTech mRNA vaccine)") can be used. Such an LNP may contain, for example, 20-60 mol% of ionizable cationic lipids, 5-25 mol% of non-cationic lipids (e.g., neutral lipids), 25-55 mol% of sterols or steroids, and 0.5-15 mol% of polymer conjugate lipids (e.g., PEG-modified lipids). The ionizable cationic lipid can be selected from the group consisting of, for example, 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-nonano-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate. The sterol or steroid may be, for example, cholesterol. The neutral lipid may be, for example, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). The polymer conjugate lipid may be, for example, PEG2000 DMG.

[0034] LNPs can be prepared, for example, by referring to the descriptions in International Publication No. 2017 / 070626, International Publication No. 2018 / 170347, International Publication No. 2021 / 159040, and International Publication No. 2021 / 213945.

[0035] (5) Interferon-alpha (IFN-α) Interferon-alpha (IFN-α) is known to exert antitumor activity by directly inhibiting the cell cycle progression of cancer cells and inducing apoptosis, thereby suppressing tumor growth. Furthermore, IFN-α is known to stimulate antigen presentation by dendritic cells (DCs), and to activate and mature DCs, thereby promoting the differentiation of effector T cells.

[0036] In this specification, "interferon-α (IFN-α)" encompasses all subtypes, including IFN-α2, IFN-α8, IFN-α10, IFN-α1, IFN-α21, IFN-α5, IFN-α14, IFN-α17, IFN-α7, IFN-α6, IFN-α4, and IFN-α16. IFN-α is preferably human-derived IFN-α. Furthermore, IFN-α is preferably an amino acid sequence having 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity with the amino acid sequence described in SEQ ID NO: 1, or an amino acid sequence having 80% or more, 85% or more, 90% or more, or 91% or more sequence identity with the amino acid sequence described in SEQ ID NO: 2 (Human IFN-α4), or an amino acid sequence having 80% or more, 85% or more, 90% or more, or 91% or more sequence identity with the amino acid sequence described in SEQ ID NO: 2. The above refers to an amino acid sequence having 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity with the amino acid sequence described in Sequence ID No. 3 (Human IFN-α10) or the amino acid sequence described in Sequence ID No. 3, or an amino acid sequence having 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity with the amino acid sequence described in Sequence ID No. 3. In this specification, IFN-α is characterized by having the above amino acid sequence and having DC activation function.

[0037] In this specification, "nucleotide sequence encoding IFN-α" is preferably an amino acid sequence described in SEQ ID NO: 1 or a nucleotide sequence encoding an amino acid sequence having 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more or 99% or more sequence identity with the amino acid sequence described in SEQ ID NO: 2 or the amino acid sequence described in SEQ ID NO: 2. A nucleotide sequence that represents an amino acid sequence having sequence identity of 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, or an amino acid sequence that represents an amino acid sequence that has sequence identity of 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more with the amino acid sequence described in Sequence ID No. 3.More specifically, the "nucleotide sequence encoding IFN-α" is preferably a nucleotide sequence having 70% or more, 75% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94%, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity with the nucleotide sequence encoding human IFN-α4 or the nucleotide sequence encoding IFN-α4. This refers to a nucleotide sequence having 2% or more, 83% or more, 84% or more, 85% or more, 86% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% stripe, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity with the nucleotide sequence described in Sequence ID No. 6 (the nucleotide sequence encoding human IFN-α10) or the nucleotide sequence described in Sequence ID No. 6, and having 70% or more, 75% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% stripe, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity with the nucleotide sequence described in Sequence ID No. 6. The nucleotide sequence encoding IFN-α may be a sequence that has been further codon-optimized for administration to humans. Table 1 shows the sequence of sequence numbers 1 to 6.

[0038]

[0039] (6) Interleukin-12 (IL-12) Interleukin-12 (IL-12) is a cytokine produced in response to malignant tumors and promotes innate and adaptive immunity through the activation of T cells and NK cells. IL-12 stimulates T cells to promote a strong IFN-γ response. It also enhances the production of pro-inflammatory cytokines. This greatly contributes to the priming and activation of T cells. Furthermore, IL-12 enhances the cancer immune response by suppressing or reprogramming immunosuppressive cells, including tumor-associated macrophages and bone marrow-derived suppressor T cells.

[0040] In this specification, "interleukin-12 (IL-12)" refers to a cytokine produced by macrophages, dendritic cells, etc., and is a protein having a structure of a heterodimer of a p35 subunit and a p40 subunit, or a homodimer of a p40 subunit. IL-12 is preferably human-derived IL-12, and is preferably a heterodimer. Furthermore, IL-12 preferably refers to a protein comprising a p35 subunit having an amino acid sequence that has 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity with the amino acid sequence described in SEQ ID NO: 7, and a p40 subunit having an amino acid sequence that has 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity with the amino acid sequence described in SEQ ID NO: 8. In this specification, IL-12 is characterized by having the above amino acid sequence and having T cell activation function.

[0041] In this specification, "nucleotide sequence encoding IL-12" preferably refers to the amino acid sequence described in SEQ ID NO: 8 or a nucleotide sequence encoding an amino acid sequence (p35 subunit) having 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity with the amino acid sequence described in SEQ ID NO: 8, and a nucleotide sequence encoding an amino acid sequence (p40 subunit) having 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity with the amino acid sequence described in SEQ ID NO: 9. More specifically, the "nucleotide sequence encoding IL-12" is preferably the nucleotide sequence described in Sequence ID No. 9 (the nucleotide sequence encoding the p35 subunit) or the nucleotide sequence described in Sequence ID No. 9 with a distribution of 70% or more, 75% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94%, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. This refers to a nucleotide sequence having sequence identity, and a nucleotide sequence having 70% or more, 75% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94%, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity with the nucleotide sequence described in Sequence ID No. 10. Table 2 shows the sequences of Sequence ID Nos. 7 to 10.

[0042]

[0043] (7) Chemotherapy agents or molecularly targeted drugs In this specification, "chemotherapy agent" refers to chemically synthesized compounds that have antitumor activity. While not particularly limited, examples include alkylating agents (such as cyclophosphamide), platinum preparations (such as oxaliplatin and cisplatin), antimetabolites (such as 5-fluorouracil), and plant alkaloids (such as vincristine, docetaxel, and paclitaxel). In this specification, "molecularly targeted drug" refers to a therapeutic agent that specifically recognizes and attacks only molecules such as proteins and genes involved in the proliferation and metastasis of tumor cells. Many commercially available drugs are known, including imatinib, gefitinib, and lenvatinib. Antibody-drug conjugates (ADCs) are also included in the definition of "chemotherapy agents or molecularly targeted drugs" as defined herein. Many types of chemotherapeutic agents or molecularly targeted drugs are commercially available as pharmaceutical compositions (pharmaceuticals), and any of them may be used. In particular, those that have the property of releasing cancer cell antigens outside the tumor and strongly inducing immunogenic cell death (ICD) are preferably used.

[0044] The chemotherapeutic agents or molecularly targeted drugs used are not particularly limited, but depending on the type of tumor being treated, chemotherapeutic agents or molecularly targeted drugs known to be effective in treatment can be used. When the tumor being treated is PDAC, the inventors have found that the chemotherapeutic agent oxaliplatin is particularly useful in enhancing the combined effect. Oxaliplatin is a compound having the structure of the following formula (I), and is usually administered by intravenous drip infusion.

[0045] When the tumor to be treated is scirrhous gastric cancer, the inventors have found that the molecularly targeted drug lenvatinib is particularly useful in enhancing the combined effect.

[0046] (8) Immune checkpoint inhibitors In this specification, "immune checkpoint inhibitor (ICI)" refers to a drug that blocks PD-1 (Programmed cell Death 1), PD-L1 (Programmed cell Death 1-Ligand 1), CTLA-4 (Cytotoxic T-lymphocyte Antigen-4), etc., which have the function of suppressing T cells' attack on cancer cells. Preferably, the "immune checkpoint inhibitor (ICI)" is one or more ICIs selected from the group consisting of anti-PD-1 antibody, anti-PD-L1 antibody, and anti-CTLA-4 antibody. More preferably, the "immune checkpoint inhibitor (ICI)" is administered in combination with an anti-PD-1 antibody and / or an anti-PD-L1 antibody and an anti-CTLA-4 antibody. PD-1 is expressed on the surface of T cells, and it is known that when PD-1 binds to PD-L1 on the surface of cancer cells, the activity of T cells is suppressed. Anti-PD-1 antibody or anti-PD-L1 antibody inhibits the suppression of T cell activation by blocking the binding of PD-1 and PD-L1. CTLA-4 is expressed on the surface of T cells and binds to B7, which is expressed on dendritic cells. T cell activation occurs when antigens presented on the surface of dendritic cells bind to the TCR on T cells, and when B7 expressed on the surface of dendritic cells binds to CD28 on the surface of T cells. However, when CTLA-4 is expressed and binds to B7, the binding of B7 to CD28 is inhibited, thereby suppressing T cell activation. Anti-CTLA-4 antibodies block CTLA-4 on the surface of T cells, thereby suppressing the inhibition of binding between B7 and CD28, and thereby promoting T cell activation.

[0047] Pharmaceutical compositions (pharmaceuticals) containing anti-PD-1 antibodies include nivolumab (trade name Opdivo) and pembrolizumab (trade name Keytruda), among others. Pharmaceutical compositions containing anti-PD-L1 antibodies include atezolizumab (trade name Tecentriq), avelumab (trade name Bavencio), and durvalumab (trade name Imfinzi), among others, among others. These commercially available pharmaceutical compositions may be used as anti-PD-1 antibodies and / or anti-PD-L1 antibodies.

[0048] Pharmaceutical compositions containing anti-CTLA-4 antibodies include ipilimumab (trade name Yervoy) and tremelimumab (trade name Ijud). These commercially available pharmaceutical compositions may be used as the anti-CTLA-4 antibody.

[0049] In this specification, unless otherwise specified, all mixing ratios are expressed as weight ratios. Furthermore, unless otherwise specified, all concentrations expressed as "%" represent weight percent.

[0050] [2] Compositions comprising cytokines or mRNA encoding cytokines. One aspect of the present invention is a pharmaceutical composition comprising a cytokine, specifically IL-12, or mRNA encoding it. Furthermore, the composition of this aspect is a composition for use in a method of treating or preventing cancer, wherein the composition comprises IL-12 or mRNA comprising a nucleotide sequence encoding IL-12, and the method comprises the co-administration of (a) IL-12 or mRNA comprising a nucleotide sequence encoding IL-12; (b) one or more chemotherapeutic agents; and (c) one or more immune checkpoint inhibitors.

[0051] The above method may further include (d) co-administration targeting IFN-α or mRNA encoding IFN-α.

[0052] The compositions of this embodiment may further contain (d) IFN-α or mRNA encoding IFN-α, insofar as they are applicable to the combined administration of (a) to (c) described above. In particular, when treating highly resistant tumors such as PDAC, it is preferable to include both (a) and (d) as long as they do not negatively affect the therapeutic effect. Herein, it is not excluded to include the cytokine itself (one or a mixture of two types) and mRNA encoding the cytokine (one or a mixture of two types) in a single composition, but considering the ease of formulation, suitability of the administration route, etc., it is preferable to use a composition that contains only either the cytokine itself or mRNA.

[0053] Pharmaceutical compositions containing IFN-α are currently marketed as treatments for chronic myeloid leukemia, hairy cell leukemia, and other diseases, and can be used in combination therapy. However, existing pharmaceutical compositions are administered systemically by subcutaneous or intramuscular infusion, but the proteins may be degraded or modified before reaching the tumor, potentially resulting in insufficient efficacy relative to the dosage. Therefore, it is preferable to include mRNA for generating IFN-α within cells as the component in the composition, rather than IFN-α itself. Similarly, for IL-12, it is preferable to include mRNA for generating IL-12 rather than IL-12 itself. In either case, while cytokines are highly effective, they are also known to carry a risk of serious adverse events, so intratumoral administration rather than systemic administration is preferable.

[0054] [2-1] Composition containing mRNA encoding IL-12 (IL-12 mRNA) Hereinafter, embodiments of pharmaceutical compositions containing mRNA rather than the cytokine itself will be described as examples of compositions of this embodiment, but the scope of the present invention is not intended to be limited to each embodiment.

[0055] The first embodiment of the composition of this embodiment is a composition comprising IL-12 mRNA as an active ingredient. The composition of this embodiment is a composition for use in combination with the following therapeutic agents: (i'') a composition comprising IFN-α mRNA; (ii) a composition comprising one or more chemotherapeutic agents; (iii) a composition comprising an anti-PD-1 antibody and / or an anti-PD-L1 antibody; and (iv) a composition comprising an anti-CTLA-4 antibody.

[0056] (1) mRNA IL-12 mRNA preferably has a base sequence encoding an amino acid sequence (p35 subunit) that has 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more or 99% or more sequence identity with the amino acid sequence described in Sequence ID No. 7, and a base sequence encoding an amino acid sequence (p40 subunit) that has 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more or 99% or more sequence identity with the amino acid sequence described in Sequence ID No. 8. Furthermore, the nucleotide sequence encoding IL-12 is preferably the nucleotide sequence described in Sequence ID No. 9 or a nucleotide sequence having 70% or more, 75% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity with the nucleotide sequence described in Sequence ID No. 9 (encoding the p35 subunit). It is preferable that the nucleotide sequence is the nucleotide sequence described in Sequence ID No. 10 or has 70% or more, 75% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94%, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity with the nucleotide sequence described in Sequence ID No. 10 (the nucleotide sequence encoding the p40 subunit). The nucleotide sequence encoding IL-12 may be the above sequence further codon-optimized for administration to humans.

[0057] The nucleotide sequences encoding the p35 subunit and the nucleotide sequences encoding the p40 subunit may be linked together using a linker or the like and present on the same mRNA molecule, or they may exist as separate mRNA molecules. The nucleotide sequence encoding IFN-α may be a sequence further codon-optimized for administration to humans.

[0058] It is preferable that the IL-12 mRNA is encapsulated in an LNP. If the nucleotide sequence encoding the p35 subunit and the nucleotide sequence encoding the p40 subunit are present on separate mRNA molecules, i.e., if there are two types of mRNA for IL-12, one LNP may contain both types of mRNA, or multiple LNPs, each containing one type of mRNA, may be mixed in the composition. The composition of this embodiment may contain other mRNAs, in which case one LNP may contain IL-12 and other mRNAs, or they may be encapsulated in separate LNPs and mixed in the composition. The amount of mRNA encapsulated in the LNP, the encapsulation method, etc., are the same as in previously reported methods.

[0059] The amount of mRNA contained in the composition of this embodiment can be, for example, 1 ng to 1 μg / μL, 10 to 700 ng / μL, 100 to 500 ng / μL, or 150 to 400 ng / μL. Alternatively, if other mRNA is included, the amount of Total mRNA can be, for example, 1 ng to 1 μg / μL, 10 to 700 ng / μL, 100 to 500 ng / μL, or 150 to 400 ng / μL.

[0060] (2) Other components of the composition The composition of this embodiment may contain, in addition to mRNA or mRNA encapsulated in lipid nanoparticles, a pharmaceutically acceptable carrier as necessary. The term "pharmaceutically acceptable carrier" here includes diluents, excipients, or other pharmaceutically acceptable carriers. In this embodiment, the pharmaceutically acceptable excipient can be appropriately designed according to its dosage form.

[0061] The dosage form of the composition of this embodiment is not particularly limited as long as it can maintain the effects of mRNA or mRNA encapsulated in lipid nanoparticles and other additional components, but it is especially preferable to use a dosage form suitable for intratumoral administration. Examples of dosage forms for intratumoral administration include injectable preparations. As for the injectable preparation, any existing form may be used, such as a sterilized liquid injectable preparation or a lyophilized injectable preparation that is dissolved or suspended in sterile water or the like immediately before use. Furthermore, the composition of this embodiment may be in a form suitable for transport and storage in a frozen state.

[0062] In the case of injectable formulations, the composition of this embodiment is a suspension, solution, or emulsion in an oily or aqueous solvent, and may contain formulation agents such as suspending agents, stabilizers, and / or dispersants. Suitable solvents for injectable formulations are not particularly limited, but include lipophilic solvents and fatty oils, such as sesame oil, synthetic fatty acid esters, such as ethyl oleate or triglycerides, and liposomes. The aqueous suspension for injection may contain a substance that increases the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. Furthermore, if transported and stored in a frozen state, it may contain a cryoprotective agent, such as trehalose or sucrose. The injectable formulation may also contain an appropriate stabilizer and an agent that increases the solubility of the drug in order to enable the preparation of high-concentration solutions.

[0063] The injectable formulation is provided in unit dosage form, for example, in a single-dose ampoule or in a multi-dose container. The composition of this embodiment contains RNA or a combination of RNA in a therapeutically effective amount. A therapeutically effective amount, as used herein, means an amount sufficient to treat or prevent cancer in a subject.

[0064] (3) Method of administration The compositions of this embodiment are used for a method of treating or preventing a target cancer. The compositions of this embodiment are not particularly limited, but (i'') when administering a composition containing IFN-α mRNA, it is preferable to administer it in the same dosage form as the composition. The compositions of this embodiment may be administered simultaneously with a composition containing IFN-α mRNA, separately, or alone. Furthermore, (ii) a composition containing one or more chemotherapeutic agents, (iii) a composition containing an anti-PD-1 antibody and / or an anti-PD-L1 antibody, and (iv) a composition containing an anti-CTLA-4 antibody may each be administered in the same dosage form as the compositions of this embodiment, or in a different dosage form, as long as the effect of the active ingredient is not impaired and no serious adverse events occur. They may also be administered simultaneously with or separately from the compositions of this embodiment.

[0065] The administration method of the composition of this embodiment is not particularly limited, but intratumoral administration is preferred. The number of administrations is not particularly limited, but can be 1 to 10 times, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times. The administration interval is not particularly limited, but can be about 3 to 4 months.

[0066] The composition of this embodiment is administered to the subject in an amount sufficient to treat or prevent cancer and that does not usually cause serious side effects. RNA encoding IL-12 is translated in vivo to produce an antigen, which then promotes T cell activation in the subject. The amount of RNA administered is preferably such that the therapeutic or preventive effect is obtained and that serious side effects do not usually occur. For example, a single dose for an adult human may be about 0.1 μg to 1 mg.

[0067] [2-2] Compositions comprising IL-12 mRNA and mRNA encoding IFN-α (IFN-α mRNA) A second embodiment of the composition of this embodiment is a composition comprising IFN-α mRNA and IL-12 mRNA as active ingredients. The composition of this embodiment is a composition for use in combination with the following therapeutic agents: (ii) a composition comprising one or more chemotherapeutic agents; (iii) a composition comprising an anti-PD-1 antibody and / or an anti-PD-L1 antibody; and (iv) a composition comprising an anti-CTLA-4 antibody.

[0068] (1) IL-12 mRNA The composition of this embodiment includes both IL-12 mRNA and IFN-α mRNA. As IL-12 mRNA, the IFN-α mRNA described in item [2-1](1) can be used. (2) IFN-α mRNA Preferably, the IFN-α mRNA is a base sequence that encodes an amino acid sequence that has 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more or 99% or more sequence identity with the amino acid sequence described in SEQ ID NO: 1. Furthermore, it is preferable to have a nucleotide sequence that has 70% or more, 75% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94%, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity with the nucleotide sequence described in Sequence ID No. 4. The nucleotide sequence encoding IFN-α may be a sequence that has been further codon-optimized for administration to humans.

[0069] It is preferable that IL-12 mRNA and IFN-α mRNA are encapsulated in LNPs. The three types of mRNA—IL-12, mRNA encoding the p35 subunit of IFN-α mRNA, and mRNA encoding the p40 subunit—may all be contained in a single LNP, or multiple LNPs, each containing one type of mRNA, may be mixed in the composition. Alternatively, two types of mRNA and one type of mRNA may be contained in separate LNPs. The composition of this embodiment may also contain other mRNAs. In this case, one LNP may contain IL-12 mRNA and / or IFN-α mRNA and other mRNAs, or they may be encapsulated in separate LNPs and mixed in the composition. The amount of mRNA encapsulated in the LNP, the encapsulation method, etc., are the same as in previously reported methods.

[0070] The total amount of IL-12 mRNA and IFN-α mRNA contained in the composition of this embodiment can be, for example, 1 ng to 1 μg / μL, 10 to 700 ng / μL, 100 to 500 ng / μL, or 150 to 400 ng / μL. Alternatively, if other mRNAs are included, the amount of Total mRNA can be, for example, 1 ng to 1 μg / μL, 10 to 700 ng / μL, 100 to 500 ng / μL, or 150 to 400 ng / μL.

[0071] (3) Other components of the composition The components of the composition of this embodiment other than mRNA are the same as those of the composition of the first embodiment, i.e., as described in section [2-1](2), unless otherwise specified.

[0072] (4) Method of administration The compositions of this embodiment are used for a method of treating or preventing a target cancer. In this embodiment, (ii) a composition comprising one or more chemotherapeutic agents, (iii) a composition comprising an anti-PD-1 antibody and / or an anti-PD-L1 antibody, and (iv) a composition comprising an anti-CTLA-4 antibody may be administered in the same dosage form as the compositions of this embodiment, or in a different dosage form, as long as the effect of the active ingredient is not impaired and no serious adverse events occur. They may also be administered simultaneously with the compositions of this embodiment or separately.

[0073] The administration method of the composition of this embodiment is not particularly limited, but intratumoral administration is preferred. The number of administrations is not particularly limited, but can be 1 to 10 times, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times. The administration interval is not particularly limited, but can be about 3 to 4 months.

[0074] The composition of this embodiment is administered to the subject in an amount sufficient to treat or prevent cancer and that does not usually cause serious side effects. The amount of RNA administered is preferably such that the therapeutic or preventive effect is obtained and that serious side effects do not usually occur. For example, a single dose for an adult human may be approximately 0.1 μg to 1 mg.

[0075] [2-3] Compositions comprising mRNA encoding IFN-α (IFN-α mRNA) The Specified further provides compositions comprising IFN-α mRNA as an active ingredient. The compositions are for use in combination with the following therapeutic agents: (i') a composition comprising mRNA comprising a nucleotide sequence encoding IL-12; (ii) a composition comprising one or more chemotherapeutic agents; (iii) a composition comprising an anti-PD-1 antibody and / or an anti-PD-L1 antibody; and (iv) a composition comprising an anti-CTLA-4 antibody.

[0076] (1) mRNA The composition contains IFN-α mRNA. As IFN-α mRNA, the IFN-α mRNA described in item [2-2](2) can be used.

[0077] (2) Other components of the composition The composition may, in addition to mRNA or mRNA encapsulated in lipid nanoparticles, optionally contain a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" here includes diluents, excipients, or other pharmaceutically acceptable carriers. In this embodiment, the pharmaceutically acceptable excipient can be appropriately designed according to its dosage form.

[0078] The aforementioned dosage form is not particularly limited as long as it can maintain the effects of mRNA or mRNA encapsulated in lipid nanoparticles and other additional components, but it is especially preferable to use a dosage form suitable for intratumoral administration. Examples of dosage forms for intratumoral administration include injectable preparations. As for the injectable preparation, any existing form may be used, such as a sterilized liquid injectable preparation or a lyophilized injectable preparation that is dissolved or suspended in sterile water or the like immediately before use. Furthermore, the composition may be in a form suitable for transport and storage in a frozen state.

[0079] In the case of injectable formulations, the composition is a suspension, solution, or emulsion in an oily or aqueous solvent and may contain formulation agents such as suspending agents, stabilizers, and / or dispersants. Suitable solvents for injectable formulations are not particularly limited, but include lipophilic solvents and fatty oils, such as sesame oil, synthetic fatty acid esters, such as ethyl oleate or triglycerides, and liposomes. Aqueous suspension injections may contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. Furthermore, if transported and stored in a frozen state, cryoprotective agents such as trehalose or sucrose may be included. Injectable formulations may also contain appropriate stabilizers and agents that increase the solubility of the drug in order to enable the preparation of high-concentration solutions.

[0080] The injectable formulation is provided in unit dosage form, for example, in a single-dose ampoule or in a multi-dose container. The composition contains RNA or a combination of RNA in a therapeutically effective amount. A therapeutically effective amount, as defined herein, means an amount sufficient to treat or prevent cancer in a subject.

[0081] (3) Method of administration The composition is used for a method of treating or preventing the target cancer. The composition is not particularly limited, but it is preferable to administer it in the same dosage form as the composition containing (i') mRNA encoding IL-12. The composition may be administered simultaneously with the composition containing mRNA encoding IL-12, or separately. Furthermore, (ii) a composition containing one or more chemotherapeutic agents, (iii) a composition containing an anti-PD-1 antibody and / or an anti-PD-L1 antibody, and (iv) a composition containing an anti-CTLA-4 antibody may each be administered in the same dosage form as the aforementioned composition, or in a different dosage form, as long as the effect of the active ingredient is not impaired and no serious adverse events occur. They may also be administered simultaneously with the aforementioned compositions or separately.

[0082] The administration method of the composition is not particularly limited, but intratumoral administration is preferred. The number of administrations is not particularly limited, but can be 1 to 10 times, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times. The administration interval is not particularly limited, but can be about 3 to 4 months.

[0083] The composition is administered to the subject in an amount sufficient to treat or prevent cancer and that does not usually cause serious side effects. The RNA encoding IFN-α is translated in vivo to produce an antigen, which then promotes the activation of dendritic cells (DCs) in the subject. The amount of RNA administered is preferably such that the therapeutic or preventive effect is obtained and that serious side effects do not usually occur. For example, a single dose for an adult human may be about 0.1 μg to 1 mg.

[0084] [3] Compositions comprising chemotherapeutic agents or molecularly targeted drugs Another aspect of the present invention is a pharmaceutical composition comprising one or more chemotherapeutic agents or molecularly targeted drugs. The composition of this aspect is a composition for use in a method of treating or preventing cancer, comprising one or more chemotherapeutic agents or molecularly targeted drugs, wherein the method comprises co-administration of (a) interleukin 12 (IL-12) or mRNA containing a nucleotide sequence encoding IL-12; (b) one or more chemotherapeutic agents or molecularly targeted drugs; and (c) one or more immune checkpoint inhibitors.

[0085] The above method may further include (d) co-administration targeting IFN-α or mRNA encoding IFN-α.

[0086] The compositions of this embodiment may use any chemotherapeutic agent or molecular targeted drug effective for the treatment or prevention of cancer, as long as they are applicable to the combination administration described in (a) to (c) or (a) to (d) above. The chemotherapeutic agent or molecular targeted drug used may be a single agent, but multiple chemotherapeutic agents or molecular targeted drugs may be used in combination, provided that the therapeutic or preventive effect is not impaired and no serious adverse events usually occur.

[0087] The chemotherapeutic agent is not particularly limited as long as it contributes to the release of cancer antigens from tumors, but those that induce ICD can be preferably used. Chemotherapeutic agents that induce ICD can be selected, for example, by adding them to commercially available cancer cells in vitro and evaluating whether the expression of markers such as High Mobility Group Box-1 (HMGB1) and calreticulin is promoted.

[0088] When the tumor to be treated is PDAC, oxaliplatin can be preferably used as the chemotherapeutic agent. As a composition containing oxaliplatin, a commercially available pharmaceutical product may be used, or it may be separately prepared for the combined use of the above (a) to (c) or (a) to (d). When using a commercially available oxaliplatin, its usage and dosage can be as described in the attached document. When the subject is an adult human, administration can be carried out by the following Method A or Method B. As oxaliplatin, 85 mg / m ２ (body surface area) is intravenously infused over 2 hours once a day and drug withdrawal is carried out for at least 13 days. This is repeated as one cycle (Method A). As oxaliplatin, 130 mg / m ２ (body surface area) is intravenously infused over 2 hours once a day and drug withdrawal is carried out for at least 20 days. This is repeated as one cycle. Alternatively, when a high antitumor effect is observed in the above (a) to (c) or (a) to (d), the oxaliplatin dosage may be reduced.

[0089] When the chemotherapeutic agent is oxaliplatin, the administration form is preferably intravenous drip. Alternatively, it may be administered into the tumor simultaneously with the above composition containing mRNA or by mixing the chemotherapeutic agent with the composition containing mRNA.

[0090] When the target tumor is scirrhous gastric cancer, lenvatinib can be suitably used as a molecularly targeted drug. A commercially available drug may be used as the composition containing lenvatinib, or it may be prepared separately for the combination of (a) to (c) or (a) to (d) above. When using commercially available lenvatinib, the dosage and administration may be as described in the package insert. For adult humans, 24 mg or 20 mg is administered orally once daily. Alternatively, if a high antitumor effect is observed in (a) to (c) or (a) to (d) above, the lenvatinib dose may be reduced.

[0091] The composition of this embodiment may contain, in addition to the chemotherapeutic agent or molecular targeted drug, a pharmaceutically acceptable carrier as needed. The dosage form of the composition of this embodiment is not particularly limited as long as it can maintain the effects of the chemotherapeutic agent or molecular targeted drug and other additional components, but it is preferable to use a dosage form suitable for intravenous injection (infusion) or oral administration, for example. An example of a dosage form for intratumor administration is an injectable preparation. As the injectable preparation, any existing form may be used, such as a sterilized liquid injectable preparation or a lyophilized injectable preparation that is dissolved or suspended in sterile water or the like immediately before use.

[0092] In the case of injectable formulations, the compositions of this embodiment are suspensions, solutions, or emulsions in oily or aqueous solvents and may contain formulation agents such as suspending agents, stabilizers, and / or dispersants. Suitable solvents for injectable formulations are not particularly limited, but include lipophilic solvents and fatty oils, such as sesame oil, synthetic fatty acid esters, such as ethyl oleate or triglycerides, and liposomes. Aqueous suspensions for injection may contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. Injectable formulations may also contain appropriate stabilizers and agents that increase the solubility of the drug in order to enable the preparation of high-concentration solutions.

[0093] The injectable formulation is provided in unit dosage form, for example, in a single-dose ampoule or in a multi-dose container. The composition according to this embodiment contains a therapeutically effective dose of a chemotherapeutic agent. The therapeutically effective dose, as used herein, refers to an amount sufficient to treat or prevent cancer in a subject.

[0094] For oral administration, excipients, binders, disintegrants, fillers, emulsifiers, flow additive modifiers, lubricants, etc., can be used as carriers. Examples of excipients include sugars such as monosaccharides, disaccharides, cyclodextrins, and polysaccharides (more specifically, but not limited to, glucose, sucrose, lactose, raffinose, mannitol, sorbitol, inositol, dextrin, maltodextrin, starch, and cellulose), metal salts (e.g., sodium chloride, sodium phosphate or calcium phosphate, calcium sulfate, magnesium sulfate, calcium carbonate), citric acid, tartaric acid, glycine, low, medium, and high molecular weight polyethylene glycol (PEG), Pluronic®, kaolin, silicic acid, or combinations thereof.

[0095] Examples of binders include starch paste made from corn, wheat, rice, or potato starch, simple syrup, glucose solution, gelatin, tragacanth, methylcellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose, shellac, and / or polyvinylpyrrolidone.

[0096] Examples of disintegrants include the aforementioned starch, lactose, carboxymethyl starch, cross-linked polyvinylpyrrolidone, agar, laminaran powder, sodium bicarbonate, calcium carbonate, alginic acid or sodium alginate, polyoxyethylene sorbitan fatty acid ester, sodium lauryl sulfate, monoglyceride stearate, or salts thereof.

[0097] Examples of fillers include the aforementioned sugars and / or calcium phosphate (for example, tricalcium phosphate or calcium hydrogen phosphate).

[0098] Examples of emulsifiers include sorbitan fatty acid esters, glycerin fatty acid esters, sucrose fatty acid esters, and propylene glycol fatty acid esters.

[0099] Examples of fluid additive regulators and lubricants include silicates, talc, stearates, or polyethylene glycol.

[0100] Such carriers are primarily used to facilitate dosage form formation and maintain the dosage form and pharmacological effect, and should be used as appropriate as needed. In addition to the above-mentioned additives, if necessary, flavoring agents, solubilizers, suspending agents, diluents, surfactants, stabilizers, absorption enhancers, bulking agents, humectants, adsorbents, disintegration inhibitors, coating agents, colorants, preservatives, antioxidants, fragrances, flavoring agents, sweeteners, buffering agents, etc. may also be included.

[0101] The dosage form of an orally administered drug is not particularly limited as long as it does not inactivate the active ingredient, and may be, for example, a liquid, solid, or semi-solid. Specific dosage forms include, for example, liquids, powders, granules, tablets, capsules, sublingual preparations, lozenges, etc.

[0102] [4] Compositions comprising immune checkpoint inhibitors (ICIs) Another aspect of the present invention is a pharmaceutical composition comprising one or more ICIs. The composition of this aspect is a composition for use in a method of treating or preventing cancer, comprising one or more ICIs, wherein the method comprises co-administration of (a) interleukin 12 (IL-12) or mRNA comprising a nucleotide sequence encoding IL-12; (b) one or more chemotherapeutic agents; and (c) one or more ICIs.

[0103] The above method may further include (d) co-administration targeting IFN-α or mRNA encoding IFN-α.

[0104] Here, it is preferable that the one or more ICIs in (c) above be a combination of two or more ICIs with different mechanisms of action. For example, it is preferable to use a combination of an anti-PD-1 antibody and / or an anti-PD-L1 antibody and an anti-CTLA-4 antibody. In this case, both the anti-PD-1 antibody and the anti-PD-L1 antibody may be used, or only one of them may be used. The following describes a form in which either an anti-PD-1 antibody or an anti-PD-L1 antibody is used with an anti-CTLA-4 antibody, but this is not intended to limit the scope of the present invention.

[0105] The ICI is preferably administered by intravenous infusion. Alternatively, it may be administered intratumorally simultaneously with the mRNA-containing composition, or mixed with the mRNA-containing composition.

[0106] As the ICI, commercially available pharmaceuticals may be used as appropriate. Alternatively, (a) and (b) above, or (a), (b) and (d), may be separately prepared in a form suitable for use in combination with an anti-PD-1 antibody or an anti-PD-L1 antibody and an anti-CTLA-4 antibody. As a method for preparing each ICI, for example, the methods described in International Publication No. 2015 / 153639 and International Publication No. 2013 / 79174 may be used (incorporated by reference).

[0107] [4-1] Compositions containing anti-PD-1 antibody or anti-PD-L1 antibody The first embodiment of this embodiment is a composition containing an anti-PD-1 antibody or an anti-PD-L1 antibody. When using a commercially available anti-PD-1 antibody (e.g., nivolumab) or anti-PD-L1 antibody (e.g., pembrolizumab), the dosage and administration may be as described in the package insert. For example, when using an anti-PD-1 antibody in adult human subjects, 240 mg is administered by intravenous infusion every two weeks or 480 mg is administered by intravenous infusion over 30 minutes every three weeks or 400 mg is administered by intravenous infusion over 30 minutes every six weeks. Alternatively, if a high antitumor effect is observed by the combination of (a) to (d) above, the ICI dose may be reduced.

[0108] [4-2] Composition containing an anti-CTLA-4 antibody The second embodiment of this embodiment is a composition containing an anti-CTLA-4 antibody. When using a commercially available anti-CTLA-4 antibody (e.g., ipilimumab), the dosage and administration may be as described in the package insert. For example, when used in adult human subjects, 3 mg / kg (body weight) is administered by intravenous infusion four times at 3-week intervals. Alternatively, if a high antitumor effect is observed by the combination of (a) to (d) above, the ICI dose may be reduced.

[0109] [4-3] Composition comprising an anti-PD-1 antibody or anti-PD-L1 antibody and an anti-CTLA-4 antibody The third embodiment of this embodiment is a composition comprising an anti-PD-1 antibody or anti-PD-L1 antibody and an anti-CTLA-4 antibody. The composition of this embodiment contains two types of ICI in one composition. The content of each ICI may be set in accordance with the usage and dosage of commercially available products, and the dosage of each ICI may be reduced if the combined effect is high.

[0110] [5] Kits Another aspect of the present invention is a kit for use in a method for treating or preventing cancer. More specifically, the kit of this aspect is a kit comprising two or more compositions from among the compositions described in sections [2] to [4] above.

[0111] [5-1] First Embodiment The kit of the first embodiment of this aspect comprises two or more of the following: (i) a composition comprising interleukin 12 (IL-12) or mRNA comprising a nucleotide sequence encoding IL-12; (ii) a composition comprising one or more chemotherapeutic agents or molecularly targeted drugs; (iii) a composition comprising an anti-PD-1 antibody and / or an anti-PD-L1 antibody; and (iv) a composition comprising an anti-CTLA-4 antibody.

[0112] In the kit of this embodiment, the composition of (i) may include IFN-α or mRNA containing a nucleotide sequence encoding IFN-α. IFN-α or mRNA containing a nucleotide sequence encoding IFN-α is particularly effective in treating PDAC.

[0113] The cytokines or mRNA, other components, and dosage forms included in the composition of (i) above are as described in section [2] unless otherwise specified. The chemotherapeutic agents, other components, and dosage forms included in the composition of (ii) above are as described in section [3] unless otherwise specified. The ICIs included in the compositions of (iii) and (iv) above, and their dosage forms, are as described in section [4]. The kit of this embodiment may comprise all of the compositions of (i) to (iv). Alternatively, the kit of this embodiment may comprise, for example, two or three of the compositions of (i) to (iv) and be used in combination with a commercially available product.

[0114] The kit of this embodiment may include, in addition to two or more of the compositions (i) to (iv), a package insert (PI) describing the usage, dosage, etc., of the combination therapy.

[0115] [5-2] Second Embodiment The kit of the second embodiment of this aspect comprises two or more of the following: (i) a composition comprising IL-12 or mRNA comprising a nucleotide sequence encoding IL-12; (ii) a composition comprising one or more chemotherapeutic agents or molecularly targeted drugs; (iii) a composition comprising an anti-PD-1 antibody and / or an anti-PD-L1 antibody; and (iv) a composition comprising an anti-CTLA-4 antibody.

[0116] The kit of this embodiment may further comprise (i') a composition comprising IFN-α or mRNA comprising a nucleotide sequence encoding IFN-α; The composition comprising IFN-α or mRNA comprising a nucleotide sequence encoding IFN-α is particularly effective for the treatment of PDAC.

[0117] The cytokines or mRNA, other components, and dosage forms included in the compositions (i) and (i') above are as described in section [2] unless otherwise specified. The chemotherapeutic agents or molecular targeted drugs, other components, and dosage forms included in the composition (ii) above are as described in section [3] unless otherwise specified. The ICIs included in the compositions (iii) and (iv) above, and their dosage forms, are as described in section [4]. The kit of this embodiment may comprise all of the compositions (i) to (iv). Alternatively, the kit of this embodiment may comprise, for example, two or three of the compositions (i) to (iv) and be used in combination with a commercially available product.

[0118] The kit of this embodiment may include, in addition to two or more of the compositions (i) to (iv), a package insert (PI) describing the usage, dosage, etc., of the combination therapy.

[0119] [5-3] Third Embodiment The kit of the third embodiment of this aspect comprises at least: (i) a composition comprising IL-12 or mRNA comprising a nucleotide sequence encoding IL-12; and (i') a composition comprising IFN-α or mRNA comprising a nucleotide sequence encoding IFN-α.

[0120] The kit of this embodiment may optionally further comprise at least one of the following compositions: (ii) a composition comprising one or more chemotherapeutic agents or molecularly targeted drugs; (iii) a composition comprising an anti-PD-1 antibody and / or an anti-PD-L1 antibody; and (iv) a composition comprising an anti-CTLA-4 antibody.

[0121] The cytokines or mRNA, other components, and dosage forms included in the compositions (i) and (i') above are as described in section [2] unless otherwise specified. The chemotherapeutic agents or molecular targeted drugs, other components, and dosage forms included in the composition (ii) above are as described in section [3] unless otherwise specified. The ICIs included in the compositions (iii) and (iv) above, and their dosage forms, are as described in section [4]. The kit of this embodiment may comprise all of the compositions (i) to (iv). Alternatively, the kit of this embodiment may comprise, for example, two or three of the compositions (i) to (iv) and be used in combination with a commercially available product.

[0122] The kit of this embodiment may include, in addition to two or more of the compositions (i) to (iv), a package insert (PI) describing the usage, dosage, etc., of the combination therapy.

[0123] [6] Method for treating or preventing cancer Another aspect of the present invention is a method for treating or preventing cancer. The method of this aspect is a method for treating or preventing cancer, comprising administering in combination to (a) IL-12 or mRNA containing a nucleotide sequence encoding IL-12; (b) one or more chemotherapeutic agents; and (c) one or more immune checkpoint inhibitors.

[0124] The method of this embodiment may further include co-administration of (d) IFN-α or mRNA containing a nucleotide sequence encoding IFN-α.

[0125] (a) The specific structure, administration method, dosage, etc. of IL-12 or mRNA containing a nucleotide sequence encoding IL-12 shall be as described in section [2-1] or [2-2] unless otherwise specified. (b) The administration method, dosage, etc. of one or more chemotherapeutic agents shall be as described in section [3] unless otherwise specified. (c) The administration method, dosage, etc. of one or more immune checkpoint inhibitors shall be as described in section [4] unless otherwise specified. (d) The specific structure, administration method, dosage, etc. of IFN-α or mRNA containing a nucleotide sequence encoding IFN-α shall be as described in section [2-2] or [2-3] unless otherwise specified.

[0126] The method of this embodiment is particularly preferred for application to immune-resistant cancers. In the method of this embodiment, it is preferable to produce an anti-cancer effect, including reducing the size of tumors in the target, preventing recurrence of cancer in remission, or preventing cancer metastasis.

[0127] The method of this embodiment is not particularly limited, but may be carried out in combination with other anti-cancer treatments. Examples of other anti-cancer treatments include radiotherapy, additional chemotherapy, local therapy, and endovascular therapy. Other anti-cancer treatments can be selected according to the patient's cancer progression rate, presence or absence of complications, medical history, etc.

[0128] [7] Methods for treating or preventing PDAC and PDAC peritoneal dissemination. The Specified herein provides methods for treating or preventing PDAC and PDAC peritoneal dissemination. The methods include administering at least five of the following agents: (a) IL-12 or mRNA containing a nucleotide sequence encoding IL-12, preferably mRNA containing a nucleotide sequence encoding IL-12; (b) a chemotherapeutic agent or molecularly targeted drug, preferably oxaliplatin; (c') an anti-PD-1 antibody or an anti-PD-L1 antibody; (c'') an anti-CTLA-4 antibody; and (d) IFN-α or mRNA containing a nucleotide sequence encoding IFN-α, preferably mRNA containing a nucleotide sequence encoding IFN-α.

[0129] [8] Methods for treating or preventing scirrhous gastric cancer and scirrhous gastric cancer peritoneal dissemination. The Specified herein provides methods for treating or preventing scirrhous gastric cancer and scirrhous gastric cancer peritoneal dissemination. The method comprises administering at least four of the following agents: (a) IL-12 or mRNA containing a nucleotide sequence encoding IL-12, preferably mRNA containing a nucleotide sequence encoding IL-12; (b) a chemotherapeutic agent or molecularly targeted drug, preferably lenvatinib; (c') an anti-PD-1 antibody or an anti-PD-L1 antibody; and (c'') an anti-CTLA-4 antibody.

[0130] [9] Methods for treating or preventing hepatocellular carcinoma. This specification provides methods for treating or preventing hepatocellular carcinoma. The methods include administering at least two of the following agents: (a) mRNA containing IL-12 or a nucleotide sequence encoding IL-12, preferably mRNA containing a nucleotide sequence encoding IL-12; and (c') an anti-PD-1 antibody or an anti-PD-L1 antibody.

[0131] The hepatocellular carcinoma to which the above method is applied is preferably an immune-abnormal liver carcinoma. More specifically, it is preferably an antigen-presenting abnormal liver carcinoma, and even more specifically, an MHC class I abnormal liver carcinoma.

[0132] All patent and non-patent document disclosures cited herein are incorporated herein by reference as a whole.

[0133] The following examples illustrate the present invention in more detail, but the scope of the present invention is not intended to be limited to the scope of these examples.

[0134] [Example 1] Construction of subcutaneous and peritoneal tumor animal models showing the tumor immune microenvironment of highly treatment-resistant pancreatic ductal adenocarcinoma (PDAC) cells (1) Tumor cell line: Professor David Tuveson of Cold Spring Harbor Laboratory Cancer Center provided the C57BL / 6 strain of Pdx1-Cre ＋We received the FC1245 cell line isolated from the PDAC tissue of KrasLSL-G12D / + and Trp53LSL-R172H / + mice. This cell line has been reported to have lower immunogenicity and higher tumorigenic potential compared to other PDAC cell lines (Roy, I., et al., Cancer Res. Vol. 75, pp. 3529-3542 (2015), Carbone, C., et al., J. Immunother. Cancer Vol. 9 (2021)). The FC1245 cell line was cultured in DMEM medium (Fujifilm Wako Pure Chemical Industries) supplemented with 5% fetal bovine serum (FBS), and 5% CO2. ２ The samples were maintained in a humidified incubator at 37°C and recovered using a 0.05% trypsin / 0.03% EDTA solution (Fujifilm Wako Pure Chemical Industries).

[0135] (2) Construction of a mouse tumor model Male C57BL / 6 mice aged 4-6 weeks were housed in a temperature-controlled environment with access to sterile water and food as needed, under a 12-hour light / dark cycle. After acclimatizing all mice for at least 3 days, the FC1245 cell line prepared in (1) was transplanted. The mice were anesthetized with 2.5% isoflurane before transplantation. As shown in Figure 2, a total of 1 × 10¹⁶ cells were placed in 100 μL of PBS or 100 μL of Matrigel (BD Biosciences). ５ The cells were suspended and subcutaneously transplanted into C57BL / 6 mice on day 0. Furthermore, on day 3, 1 x 10 cells were added to 200 μL of PBS. ６ Individual cells were suspended and inoculated into the peritoneal cavity to create a peritoneal dissemination model.

[0136] (3) Drug administration Each model mouse was administered gemcitabine, anti-PD-1 antibody, anti-CTLA-4 antibody, or gemcitabine + anti-PD-1 antibody (combined). Gemcitabine was administered intraperitoneally at an initial dose of 25 mg / kg on day 5 after intraperitoneal tumor transplantation, followed by additional doses of 25 mg / kg every 7 days. Anti-PD-1 antibody (αPD-1; J43; BioXCell) or anti-CTLA-4 antibody (αCTLA-4; 9D9; BioXCell) was administered intraperitoneally at a dose of 100 μg / head on days 5 and 11.

[0137] Model mice 17 days after intraperitoneal tumor transplantation were surgically opened, and the volume of tumors within the peritoneal cavity was visually compared (results not shown). In all drug administration models, the presence of tumors throughout the entire peritoneal cavity was confirmed. This confirmed the construction of a tumor model that exhibits resistance to existing antitumor therapies.

[0138] [Example 2] Verification of the antitumor effect of five-drug combination therapy in subcutaneous and peritoneal tumors (1) Preparation of mRNA therapeutic agents Sequences containing codon-optimized protein coding regions of mouse IFN-α11 (RefSeq: NM_008333.2), IL-12α (RefSeq: NM_001159424.2), and IL-12β (RefSeq: NM_001303244.1) were inserted into a pUC57 plasmid DNA (pDNA) vector, positioned downstream of the T7 promoter sequence. The obtained pDNA was amplified by PCR using a primer set containing a reverse primer with an 80 nt poly-T sequence to prepare template DNA. Next, mRNA was prepared by in vitro transcription using the mMage mMachine T7 kit (Thermo Fisher Scientific) and by adding Cap (Anti-Reverse capping analog (ARCA)). ALC-315-based lipid nanoparticles (LNPs) were prepared as previously reported (Tockary, TA, et al. Proc. Natl. Acad. Sci. USA, Vol. 120, e2214320120 (2023)), and the above mRNA was introduced to prepare cytokine mRNA-LNPs. Table 3 shows the nucleotide sequences (SEQ ID NOs. 11-13) of each mRNA actually used.

[0139]

[0140] (2) Drug Administration The model mice prepared in Example 1 (1) and (2) were administered drugs. An overview of the drug administration schedule is shown in Figure 2. Cytokine mRNA-LNP was prepared in a volume of 50 μL and injected directly into the subcutaneous tumors on days 5, 8, 11, and 14. Oxaliplatin (Tokyo Chemical Industries, Ltd., Tokyo, Japan) was administered intraperitoneally as a single dose of 2 mg / kg on day 5. Both anti-PD-1 antibody (αPD-1; J43; BioXCell) and anti-CTLA-4 antibody (αCTLA-4; 9D9; BioXCell) were administered intraperitoneally at a dose of 100 μg / head on days 5 and 11. In addition to the above, we created individuals in which no drugs were administered (Control), individuals in which only oxaliplatin administration was omitted (-Ox), individuals in which only IFN-α-mRNA administration was omitted (-IFN-α-mRNA), individuals in which only IL-12-mRNA administration was omitted (-IL-12-mRNA), individuals in which only anti-PD-1 antibody administration was omitted (-αPD-1), and individuals in which only anti-CTLA-4 antibody administration was omitted (-αCTLA-4).

[0141] (3) Measurement of body weight, tumor volume, and number, volume, and weight of peritoneal metastatic tumors Body weight and tumor volume were measured at least twice a week for each individual treated with the drug. Tumor volume was calculated using the following formula (I): A × B × (A + B) / 2 … (I) [wherein A and B are the longitudinal diameter and the perpendicular diameter, respectively] On day 17, all individuals underwent laparotomy, and all tumors with a long axis exceeding 2 mm in the abdominal cavity were collected. The number of tumors, total volume, and total weight were measured.

[0142] Figure 3 shows the survival curves for each drug administration condition, and Figure 4 shows the change in tumor volume over time for each drug administration condition. In the negative control group, all individuals died within 21 days, whereas in the group treated with all five drugs (5A), more than half were still alive at day 21, and some were still alive after 30 days. On the other hand, in the group that did not receive even one of the five drugs, there was almost no extension of survival time compared to the negative control group. Regarding tumor volume, it was confirmed that the increase in tumor volume was significantly suppressed in the group treated with all five drugs compared to the other groups (p < 0.01).

[0143] Figure 5 shows the number, total volume, and total weight of intraperitoneal tumors. Figure 5A shows the number of tumors, Figure 5B shows the total volume, and Figure 5C shows the total weight. In all cases, individuals treated with all five drugs (5A) showed significantly suppression compared to individuals treated with one drug omitted (P < 0.01).

[0144] [Example 3] Verification of dendritic cell and T lymphocyte infiltration into tumors with the combined use of five drugs. We verified whether the combined use of five drugs promoted the infiltration of dendritic cells and T lymphocytes into tumors.

[0145] (1) Preparation of luciferase mRNA-LNPs Luciferase mRNA-LNPs were prepared using the method described in Example 2(1), except that the mRNA sequence used was replaced from cytokine mRNA to luciferase mRNA (SEQ ID NO: 14, Table 4).

[0146]

[0147] (2) Drug Administration The model mice prepared in Example 1 (1) and (2) were administered drugs. Individuals administered with five drugs (5A), individuals not administered any drugs (Control), individuals administered with oxaliplatin only (Ox), individuals administered with oxaliplatin + anti-PD-1 antibody + anti-CTLA-4 antibody + luciferase mRNA-LNP (Ox + ICI + Luc-mRNA), and individuals administered with oxaliplatin + anti-PD-1 antibody + anti-CTLA-4 antibody + cytokine mRNA-LNP (Ox + ICI + IFN-α / IL-12-mRNA (also referred to as 5A (combined use of five drugs))) were created. Itokine mRNA-LNP or luciferase mRNA-LNP as a negative control was prepared in 50 μL volumes and injected directly into the subcutaneous tumors on days 5, 8, 11, and 14. Oxaliplatin was administered intraperitoneally as a single dose of 2 mg / kg on day 5. Both anti-PD-1 antibody (αPD-1; J43; BioXCell) and anti-CTLA-4 antibody (αCTLA-4; 9D9; BioXCell) were administered intraperitoneally at a dose of 100 μg / head on days 5 and 11.

[0148] (3) Immunohistochemical Analysis Subcutaneous and peritoneal metastatic tumors were excised from drug-treated mice, fixed overnight in Mildform 20N (Fujifilm Wako Pure Chemical Industries), embedded in paraffin, and sectioned to a thickness of 4 μm. The sections were immersed in sodium citrate buffer (pH 6.0) and then incubated overnight at 4°C in the presence of anti-CD11c monoclonal antibody (No. 97585, Cell Signaling Technology) or anti-CD8 monoclonal antibody (No. 98941, Cell Signaling Technology) as the primary antibody. After washing the sections, they were incubated in the presence of HRP-labeled anti-mouse IgG antibody or anti-rabbit IgG antibody (Nichirei Bioscience Co., Ltd.), washed again, and colored using diaminobenzidine (Fujifilm Wako Pure Chemical Industries). Next, the nuclei were stained with hematoxylin. The number of cells and the area of ​​the stained region after staining were quantified and evaluated using image analysis with Fiji (ImageJ) software.

[0149] Figure 6 shows the proportion of CD11c in subcutaneous tumors and peritoneal metastatic tumors. ＋ Figure 6A shows the area of ​​dendritic cells, and Figure 6B shows the degree of dendritic cell invasion in subcutaneous tumors, while Figure 6B shows the degree of dendritic cell invasion in peritoneal metastatic tumors. In both subcutaneous tumors and peritoneal metastatic tumors, individuals treated with the five-drug combination (5A) showed significantly greater dendritic cell invasion compared to other individuals. On the other hand, oxaliplatin and oxaliplatin in combination with immune checkpoint inhibitors did not show any effect in promoting dendritic cell invasion.

[0150] Figure 7 shows the proportion of CD8 in subcutaneous tumors and peritoneal metastatic tumors. ＋ This shows the area of ​​T cells. Figure 7A shows CD8 cells in a subcutaneous tumor. ＋ T cell infiltration; Figure 7B shows CD8 cells in a peritoneal metastatic tumor. ＋ This indicates the degree of T cell infiltration. In both subcutaneous tumors and peritoneal metastatic tumors, individuals treated with five-drug combination therapy (5A) showed significantly higher CD8 counts compared to other individuals. ＋ T cell infiltration was confirmed. On the other hand, neither oxaliplatin nor oxaliplatin combined with immune checkpoint suppression therapy showed any effect in promoting dendritic cell infiltration.

[0151] These results indicate that the combination of five drugs transforms the immune response to tumors in a "cold" state into a "hot" state.

[0152] [Example 4] Enhancement of immunological cell death (ICD) by five-drug combination therapy To verify whether five-drug combination therapy promotes the cancer immune cycle, the effect of five-drug combination therapy on ICD, the first stage of the cancer immune cycle, was investigated. High Mobility Group Box 1 (HMGB1) and calreticulin were detected in tumors as markers for ICD. Meanwhile, Ki67 was detected as a marker of tumor cell proliferation.

[0153] (1) Similar to drug administration example 3(2), mice were prepared to receive various drugs.

[0154] (2) Immunohistochemical analysis: Except for using an anti-HMGB1 antibody (No. 6893, Cell Signaling Technology), an anti-calreticulin antibody (No. ab2907, Abcam), or an anti-Ki67 antibody (No. 9129, Cell Signaling Technology) as the primary antibody, tumor tissue sections were immunohistochemically stained and image analyzed using the same method as in Example 3(3).

[0155] Figure 8 shows the area and color intensity of HMGB-positive cells in subcutaneous tumors and peritoneal metastatic tumors. Figure 8A shows the area (%) occupied by HMGB1-positive cells in subcutaneous tumors, and Figure 8 shows the color intensity of HMB1-positive cells in subcutaneous tumors. Figure 8C shows the area (%) occupied by HMGB1-positive cells in peritoneal metastatic tumors, and Figure 8D shows the color intensity of HMB1-positive cells in peritoneal metastatic tumors. While oxaliplatin administration increased the proportion of HMGB1-positive cells, it was confirmed that the combination of five drugs significantly increased the number of HMGB1-positive cells (P < 0.001).

[0156] Figure 9 shows the area and color intensity of calreticulin-positive cells in subcutaneous tumors and peritoneal metastatic tumors. Figure 9A shows the area (%) occupied by calreticulin-positive cells in subcutaneous tumors, and Figure 9B shows the color intensity of calreticulin-positive cells in subcutaneous tumors. Figure 9C shows the area (%) occupied by calreticulin-positive cells in peritoneal metastatic tumors, and Figure 9D shows the color intensity of calreticulin-positive cells in peritoneal metastatic tumors. In subcutaneous tumors, no increase in calreticulin-positive cells was observed with oxaliplatin administration or with the combined use of oxaliplatin and immune checkpoint suppressors, but a significant increase in calreticulin-positive cells was observed with the combined use of five drugs (P < 0.001). In peritoneal metastatic tumors, an increase in calreticulin-positive cells was observed with oxaliplatin administration and with the combined use of oxaliplatin and immune checkpoint suppressors. Furthermore, a significant increase in calreticulin-positive cells was confirmed with the combined use of five drugs (P < 0.001).

[0157] The results shown in Figures 8 and 9 indicate that the combination of five drugs resulted in the expression of HMGB1 and calreticulin within the tumor, confirming that ICD was enhanced and the cancer immune cycle was promoted.

[0158] Figure 10 shows the area of ​​Ki67-positive cells in subcutaneous tumors and peritoneal metastatic tumors. Figure 10A shows the area (%) of Ki67-positive cells in subcutaneous tumors, and Figure 10B shows the area (%) of Ki67-positive cells in peritoneal metastatic tumors. In subcutaneous tumors, oxaliplatin administration and oxaliplatin combined with immune checkpoint suppressors did not reduce the number of Ki67-positive cells, but a significant reduction in calreticulin-positive cells was observed with five-drug combination therapy (P < 0.001). In peritoneal metastatic tumors, oxaliplatin administration and oxaliplatin combined with immune checkpoint suppressors reduced the number of Ki67-positive cells, and further, a significant reduction in Ki67-positive cells was confirmed with five-drug combination therapy (P < 0.001). The results in Figure 10 clearly show that five-drug combination therapy significantly reduces the proliferation of tumor cells.

[0159] [Example 5] Percentage of effector memory T cells after five-drug combination therapy Peripheral blood and intratumor CD8 in mice after five-drug combination therapy ＋ We investigated the proportion of effector memory T cells (TEM cells) within a given cell.

[0160] (1) Similar to drug administration example 3(2), mice were prepared to receive various drugs.

[0161] (2) Flow cytometry of peripheral blood Peripheral blood was collected 17 days after subcutaneous tumor transplantation into C57BL / 6 mice. After lysing the red blood cells with erythrocyte lysis buffer (pluriSelect), the remaining cells were neutralized in a culture medium containing FBS and filtered into a 5 mL tube through a 35 μm Falcon® cell strainer (Corning). The cells were incubated in the dark at 4°C for 30 minutes in the presence of APC-labeled anti-CD8 antibody (53-6.7; BioLegend), PE-labeled anti-CD44 antibody (IM7; BioLegend), and FITC-labeled anti-CD62L antibody (MEL-14; BioLegend). After washing the cells, the fluorescence intensity was measured using a FACSlyric flow cytometer (BD Biosciences).

[0162] Figure 11 shows the peripheral blood CD8 levels of mice administered with each drug. ＋ Flow cytometry of T cells is shown. Figure 12 shows each CD44 Ｈｉｇｈ CD62L Ｌｏｗ The results of counting the number of cells in the cell (effector memory T cell) fraction are shown. Oxaliplatin administration alone did not result in proliferation of effector memory T cells, but the combination of oxaliplatin and an immune checkpoint suppressor resulted in a significant increase in effector memory T cells compared to the control group (P < 0.01). Furthermore, the combination of five drugs resulted in a significant increase in effector memory T cells compared to the other groups (P < 0.001).

[0163] (3) Flow cytometry of peritoneal lavage fluid C57BL / 6 mice were laparotomyed 17 days after subcutaneous tumor transplantation, the peritoneum was washed with culture medium containing FBS, and the peritoneal lavage fluid was collected. The peritoneal lavage fluid was neutralized and filtered into a 5 mL tube through a 35 μm Falcon® cell strainer (Corning). Cells were incubated in the dark at 4°C for 30 minutes in the presence of APC-labeled anti-CD8 antibody (53-6.7; BioLegend), FITC-labeled anti-CD44 antibody (IM7; BioLegend), and FITC-labeled anti-CD62L antibody (MEL-14; BioLegend). After washing the cells, the fluorescence intensity was measured using a FACSlyric flow cytometer (BD Biosciences).

[0164] Figure 13 shows CD8 in the peritoneal lavage fluid of mice administered with each drug. ＋ The flow cytometry of T cells is shown. Figure 14 shows each CD8 ＋ T cells, CD44 Ｈｉｇｈ CD62L Ｌｏｗ The results of counting the number of cells are shown. Figure 14A is CD8 ＋ T cell, Figure 14B is CD44 Ｈｉｇｈ CD62L Ｌｏｗ The cell counting results are shown. Compared to oxaliplatin monotherapy, mice treated with a combination of oxaliplatin and ICI showed a higher CD8 count. ＋ ;CD44 ＋ CD62L Ｌｏｗ We demonstrated a significant enhancement in the proportion of T cells (TEM cells). No increase in TEM cells was observed with oxaliplatin administration or oxaliplatin plus immune checkpoint suppression therapy. On the other hand, intraperitoneal CD8 ＋ T cell counts increased after oxaliplatin treatment and its combination with ICI. In contrast, 5A therapy increased CD8 ＋ Not only did it increase the number of T cells, but it also increased the proportion of TEM cells.

[0165] [Example 6] Infiltrating CD8 with Five-Drug Combination Therapy ＋Increased Granzyme B and Interferon-γ Expression in T Cells: We evaluated the cytotoxicity of CD8+ T cells in tumors under five-drug combination therapy. As a sample of cytotoxicity, we investigated the expression of Granzyme B (GZMB) and Interferon-γ (IFN-γ) in T cells.

[0166] (1) Similar to drug administration example 3(2), mice were prepared to receive various drugs.

[0167] (2) Immunohistochemical analysis: Except for using an anti-GZMB antibody (No. 46890, Cell Signaling Technology) or an anti-IFN-γ antibody (No. MAB485, R&D System) as the primary antibody, the tumor tissue sections were immunohistochemically stained and image analyzed using the same method as in Example 3(3).

[0168] Figure 15 shows the area of ​​GZMB-positive cells in subcutaneous tumors and peritoneal metastatic tumors. Figure 15A shows the area (%) occupied by GZMB-positive cells in subcutaneous tumors, and Figure 15B shows the area (%) occupied by GZMB-positive cells in peritoneal metastatic tumors. It was shown that oxaliplatin + ICI combination therapy significantly enhanced intratumoral GZMB expression compared to the control group. Furthermore, it was shown that five-drug combination therapy significantly enhanced intratumoral GZMB expression.

[0169] Figure 16 shows the area of ​​IFN-γ-positive cells in subcutaneous tumors and peritoneal metastatic tumors. Figure 16A shows the area (%) occupied by IFN-γ-positive cells in subcutaneous tumors, and Figure 16B shows the area (%) occupied by IFN-γ-positive cells in intraperitoneal tumors. Oxaliplatin administration and oxaliplatin + ICI combination therapy did not increase IFN-γ expression in tumors, but combination therapy with five drugs significantly enhanced IFN-γ expression in tumors.

[0170] These results indicate that combination therapy with five drugs enhances the cytotoxicity of T cells that have infiltrated the tumor.

[0171] [Example 7] Inhibition of the peritoneal dissemination suppression effect of five-drug combination therapy by concomitant use of FTY72 We hypothesized that the effect of five-drug combination therapy in suppressing tumor peritoneal dissemination occurs because the five drugs activate immune cells in the subcutaneous tumor, which then reach the peritoneum. To test this hypothesis, we administered FTY720, a sphingosine-1-phosphate receptor agonist that inhibits lymphocyte release from lymph nodes, together with the five drugs to evaluate whether it weakens the effect of the five-drug combination therapy.

[0172] (1) Drug administration: The model mice prepared in Example 1 (2) were administered drugs. Cytokine mRNA-LNP was prepared in a volume of 50 μL and injected directly into the subcutaneous tumors on days 5, 8, 11, and 14. Oxaliplatin was administered intraperitoneally as a single dose of 2 mg / kg on day 5. Both anti-PD-1 antibody and anti-CTLA-4 antibody were administered intraperitoneally at a dose of 100 μg / head on days 5 and 11. FTY720 (Tokyo Chemical Industries, Ltd.) was administered orally at a dose of 25 μg / head on day 3, and then orally at a dose of 5 μg / head daily thereafter. In addition to the above, individuals without drug administration (Control) and individuals receiving only the five-drug combination (5A) were created.

[0173] The tumor volume of each individual was measured at least twice a week. On day 17, all mice were laparotomed, and all tumors with a long axis exceeding 2 mm in the abdominal cavity were collected. The number, total volume, and total weight of these tumors were then measured.

[0174] Figure 17 shows the changes in tumor volume over time in the group administered with five drugs (5A) and the group administered with five drugs and FTY720 (5A + FTY720). In both 5A and 5A + FTY720, the increase in tumor volume was significantly suppressed compared to Control (p < 0.05), and furthermore, 5A significantly suppressed the increase in tumor volume compared to 5A + FTY720 (p < 0.05).

[0175] Figure 18 shows the number, total volume, and total weight of intraperitoneal tumors in 5A and 5A + FTY720. Figure 18A shows the number of tumors, Figure 18B shows the total volume, and Figure 18C shows the total weight. In all cases, it was confirmed that the number of tumors, which was suppressed in 5A, significantly increased when FTY720 was added (P < 0.01 for the number and total weight of tumors; P < 0.001 for the total volume).

[0176] (2) Flow cytometry analysis Peripheral blood was collected 17 days after subcutaneous tumor transplantation into C57BL / 6 mice. After lysing the red blood cells with erythrocyte lysis buffer (pluriSelect), the remaining cells were neutralized in a culture medium containing FBS and filtered into a 5 mL tube through a 35 μm Falcon® cell strainer (Corning). The cells were incubated in the dark at 4°C for 30 minutes in the presence of PerCP-labeled anti-TCRβ chain antibody (H57-597; BioLegend), APC-labeled anti-CD8α antibody (53-6.7; BioLegend), FITC-labeled anti-CD44 antibody (IM7; BioLegend), and FITC-labeled anti-CD62L antibody (MEL-14; BioLegend). After washing the cells, the fluorescence intensity was measured using a FACSlyric flow cytometer (BD Biosciences).

[0177] Figure 19 shows flow cytometry results of peripheral blood from 5A individuals and 5A+FTY720 individuals. Figure 20 shows the percentage of lymphocytes and CD8 cells counted by flow cytometry. ＋ Figures 20A and 20B show the percentage of T cells and TEM cells. Figure 20A shows the lymphocyte percentage (%), and Figure 20B shows CD8 ＋ Figure 20C shows the percentage of T cells (%) and TEM cells (%). It was confirmed that the migration of all immune cells into the peripheral blood was suppressed by FTY720. These results support the hypothesis that the suppression of tumor peritoneal metastasis by 5A is due to the activation of immune cells and their migration to the peritoneum.

[0178] [Example 8] T-cell receptor repertoire analysis by five-drug combination therapy. PDAC model mice were prepared under the conditions described in Example 1, and mRNA therapeutic agents were prepared under the conditions described in Example 2. The five drugs were administered to the PDAC model mice under the conditions described in Example 2, and RNA was extracted from the subcutaneous tumor tissue of each drug-administered individual 17 days after subcutaneous transplantation. The V and J regions were amplified, and TCR repertoire analysis was performed. The data was trimmed using TrimGalore software, and the TRA and TRB repertoires were extracted using MiXCR software. V-J pairs were displayed in Manhattan plots, heatmaps, and circus plots. Figure 21 shows the heatmaps of V-J pairs. Figure 21A shows the Control group, and Figure 21B shows the heatmaps of V-J pairs in tumors after five-drug combination therapy. Compared to the Control group, the group after five-drug combination therapy showed increased diversity in each plot.

[0179] [Example 9] Effects of drug administration on a peritoneal tumor animal model showing the tumor immune microenvironment of scirrhous gastric cancer cells (1) Tumor cell line A G9C cell line was constructed by knocking out the Cdh1 gene using the CRISPR / Cas9 system on a GIF9 cell line isolated from the normal gastric mucosa of C57BL / 6 strain p53-null mice established in the Department of Molecular Oncology, Tokyo Medical and Dental University (now Tokyo University of Science) (Fukamachi, H., et al. Biochem Biophys Res Commun. Vol. 321, pp. 58-64 (2004)). The G9C cell line was cultured in DMEM medium (Fujifilm Wako Pure Chemical Industries) with 10% fetal bovine serum (FBS) added, and 5% CO2 ２ The samples were maintained in a humidified incubator at 37°C and recovered using a 0.05% trypsin / 0.03% EDTA solution (Fujifilm Wako Pure Chemical Industries).

[0180] (2) Construction of a mouse tumor model Male C57BL / 6 mice aged 4-6 weeks were housed in a temperature-controlled environment with access to sterile water and food as needed, under a 12-hour light / dark cycle. After acclimatizing all mice for at least 3 days, the G9C cell line prepared in (1) was transplanted. The mice were anesthetized with 2.5% isoflurane before transplantation. As shown in Figure 2, a total of 1 × 10¹⁶ cells were placed in 100 μL of PBS or 100 μL of Matrigel (BD Biosciences). 6 The cells were suspended and subcutaneously transplanted into C57BL / 6 mice on day 0. Furthermore, on day 5, 1 x 10 cells were added to 200 μL of PBS. 6 Individual cells were suspended and inoculated into the peritoneal cavity to create a peritoneal dissemination model.

[0181] (3) Drug administration The model mice prepared in Example 8 (1) and (2) were administered drugs. Cytokine mRNA-LNP or luciferase mRNA-LNP as a negative control was prepared in a volume of 50 μL and injected directly into the subcutaneous tumors on days 12, 15, 18, and 21. 5-FU (Tokyo Chemical Industries, Ltd., Tokyo, Japan, 100 mg / kg), paclitaxel (Tokyo Chemical Industries, Ltd., Tokyo, Japan, 10 mg / kg), and oxaliplatin (Tokyo Chemical Industries, Ltd., Tokyo, Japan, 10 mg / kg) were administered intraperitoneally on days 12, 19, 26, and 33. Lenvatinib (Biosynth, Berkshire, United Kingdom) was administered orally daily from day 12. Both anti-PD-1 antibody (αPD-1; J43; BioXCell) and anti-CTLA-4 antibody (αCTLA-4; 9D9; BioXCell) were administered intraperitoneally at a dose of 100 μg / head on days 12, 19, 26, and 33. The above combinations were used to identify individuals without drug administration (Control), individuals administered luciferase mRNA-LNP + anti-CTLA-4 + anti-PD-1 antibody (Luc-mRNA + ICI), individuals administered IL-12-mRNA-LNP + anti-CTLA-4 + anti-PD-1 antibody (IL12-mRNA + ICI), individuals administered IL-12-mRNA-LNP + anti-CTLA-4 + anti-PD-1 antibody + 5-FU (IL12-mRNA + ICI + 5-FU), and IL-12-mRNA-L We created individuals administered with NP + anti-CTLA-4 + anti-PD-1 antibody + paclitaxel (IL12-mRNA + ICI + Paclitaxel), individuals administered with IL-12-mRNA-LNP + anti-CTLA-4 + anti-PD-1 antibody + oxaliplatin (IL12-mRNA + ICI + Oxaliplatin), and individuals administered with IL-12-mRNA-LNP + anti-CTLA-4 + anti-PD-1 antibody + lenvatinib (IL12-mRNA + ICI + Lenvatinib).Furthermore, we created individuals that received no drugs (Control), individuals that received IL-12-mRNA-LNP + anti-CTLA-4 + anti-PD-1 antibody + lenvatinib (All), individuals in which only the administration of anti-CTLA-4 antibody was omitted (-αCTLA-4), individuals in which only the administration of anti-PD-1 antibody was omitted (-αPD-1), individuals in which only the administration of IL-12-mRNA was omitted (-IL-12-mRNA), and individuals in which only the administration of lenvatinib was omitted (-Lenvatinib).

[0182] Figure 22 shows the survival curves (Kaplan-Meier curves) for each mouse group, and Figure 23 shows the changes in tumor volume over time for each mouse group. Figure 22A shows the survival curves for the Control, Luc-mRNA+ICI, IL12-mRNA+ICI, IL12-mRNA+ICI+5-FU, IL12-mRNA+ICI+Paclitaxel, IL12-mRNA+ICI+Oxaliplatin, and IL12-mRNA+ICI+Lenvatinib groups. Figure 22B shows the survival curves for the Control, All, -αCTLA-4, -αPD-1, -IL-12-mRNA, and -Lenvatinib groups. Figure 23A shows the changes in tumor volume over time for subcutaneous tumors on the side where mRNA was injected, and Figure 23B shows the changes in tumor volume over time for subcutaneous tumors on the side where mRNA was not injected. The IL12-mRNA + ICI group had better overall survival than the Control group and the Luc + ICI group. When anticancer drugs and molecular targeted drugs used in the treatment of gastric cancer, such as 5-FU, paclitaxel, oxaliplatin, and lenvatinib, were added to IL12-mRNA + ICI, the IL12-mRNA + ICI + lenvatinib group had better overall survival than the other groups. To verify that all four drugs (IL12-mRNA + ICI + lenvatinib) are essential, regimens were administered with one drug omitted from each. As a result, when IL12-mRNA and lenvatinib were omitted, the inhibitory effect on subcutaneous tumor growth on both the side where mRNA was injected and the side where it was not was reduced. Furthermore, when any of the drugs were omitted, overall survival worsened.

[0183] [Example 10] Effects of drug administration on a subcutaneous tumor animal model showing the tumor immune microenvironment of MHC class I abnormal liver cancer (1) Tumor cell line Professor Dan G. Duda of Massachusetts General Hospital, Harvard University provided the liver cancer cell line RIL175 (Kapanadze, T., et al., J Hepatol. Vol. 59, pp. 1007-1013 (2013)), which was created from C57BL / 6 strain mice and contained p53-null, HrasG12V. Cells in which the B2m gene was knocked out of RIL175 (RIL175-B2m-KO) were constructed in the Department of Molecular Oncology, Tokyo Medical and Dental University (now Tokyo University of Science) using the CRISPR / Cas9 system. RIL175-B2m-KO cell lines were cultured in DMEM medium (Fujifilm Wako Pure Chemical Industries) supplemented with 10% fetal bovine serum (FBS), maintained in a humidified incubator at 5% CO2 and 37°C, and harvested using a 0.05% trypsin / 0.03% EDTA solution (Fujifilm Wako Pure Chemical Industries).

[0184] (2) Construction of a mouse tumor model Male C57BL / 6 mice aged 4-6 weeks were housed in a temperature-controlled environment with access to sterile water and food as needed, under a 12-hour light / dark cycle. After acclimatizing all mice for at least 3 days, the RIL175-B2m-KO cell line prepared in (1) was transplanted. The mice were anesthetized with 2.5% isoflurane before transplantation. As shown in Figure 2, a total of 1 × 10¹⁶ cells were placed in 100 μL of PBS or 100 μL of Matrigel (BD Biosciences). ６ The cells were suspended and subcutaneously transplanted to both sides of the back of C57BL / 6 mice on day 0.

[0185] (3) Drug administration The model mice prepared in Example 9 (1) and (2) were administered drugs. Cytokine mRNA-LNP or luciferase mRNA-LNP as a negative control was prepared in a volume of 50 μL and injected directly into the subcutaneous tumor on the right back on days 5, 8, 11 and 14. Both anti-PD-1 antibody (αPD-1; J43; BioXCell) and anti-CTLA-4 antibody (αCTLA-4; 9D9; BioXCell) were administered intraperitoneally at a dose of 100 μg / head on days 5 and 11. By combining the above, we created individuals that received no drug administration (Control), individuals administered IFN-α + IL-12-mRNA-LNP + anti-CTLA-4 + anti-PD-1 antibody (All), individuals in which only the administration of anti-CTLA-4 antibody was omitted (-αCTLA-4), individuals in which only the administration of anti-PD-1 antibody was omitted (-αPD-1), individuals in which only the administration of IFN-α-mRNA was omitted (-IFN-α-mRNA), and individuals in which only the administration of IL-12-mRNA was omitted (-IL-12-mRNA).

[0186] Figure 24 shows the changes in tumor volume over time for each mouse group. Figure 24A shows the changes in tumor volume over time for subcutaneous tumors on the side injected with mRNA, and Figure 24B shows the changes in tumor volume over time for subcutaneous tumors on the side not injected with mRNA. In particular, in individuals where IL-12-mRNA was omitted, it was confirmed that the inhibitory effect on subcutaneous tumor growth was attenuated in both the side injected with mRNA and the side not injected. In tumors administered with IL-12-mRNA alone, dendritic cells were relatively abundant and PD-1-positive T cells were increased, while Foxp3-positive regulatory T cells (Treg) were not increased (results not shown). From these results, it was considered that the IL-12-mRNA + αPD-1 regimen with the addition of anti-PD-1 antibody was optimal among IFNα-mRNA (dendritic cell activation), anti-PD-1 antibody (improvement of PD-1-positive T cell exhaustion), and anti-CTLA-4 antibody (inhibition of Treg cells). All publications, patents, and patent applications cited herein shall be incorporated herein by direct reference.

Claims

1. A composition for use in a method for treating or preventing cancer, wherein the composition comprises interleukin-12 (IL-12) or messenger ribonucleic acid (mRNA) containing a base sequence encoding IL-12, and the method comprises co-administering (a) IL-12 or mRNA containing a base sequence encoding IL-12; (b) one or more chemotherapeutic agents or molecularly targeted drugs; and (c) one or more immune checkpoint inhibitors to a target.

2. The composition according to claim 1, comprising mRNA encoding IL-12.

3. The composition according to claim 1, wherein the method further comprises co-administering (d) interferon-α (IFN-α) or mRNA containing a nucleotide sequence encoding IFN-α to the target.

4. The composition according to claim 3, further comprising IFN-α or mRNA encoding IFN-α.

5. The composition according to claim 4, comprising mRNA encoding IFN-α.

6. The composition according to claim 1, wherein the cancer is an immune-resistant cancer.

7. The composition according to claim 6, wherein the immune-resistant cancer is pancreatic ductal adenocarcinoma (PDAC) or peritoneal dissemination of PDAC.

8. The composition according to claim 6, wherein the immune-resistant cancer is scirrhous gastric cancer or peritoneal dissemination of scirrhous gastric cancer.

9. The composition according to claim 1, wherein the chemotherapeutic agent or molecular targeted drug is selected from oxaliplatin, lenvatinib, and combinations thereof.

10. The composition according to claim 1, wherein the immune checkpoint inhibitor is one or more immune checkpoint inhibitors selected from the group consisting of anti-PD-1 antibody, anti-PD-L1 antibody, and anti-CTLA-4 antibody.

11. The composition according to claim 10, wherein the administration of the immune checkpoint inhibitor is the administration of an anti-PD-1 antibody and / or an anti-PD-L1 antibody and an anti-CTLA-4 antibody.

12. The composition according to claim 1, wherein IL-12 is a protein comprising a subunit having the amino acid sequence described in SEQ ID NO: 7 or an amino acid sequence having 80% or more sequence identity with the amino acid sequence described in SEQ ID NO: 7, and a subunit having the amino acid sequence described in SEQ ID NO: 8 or an amino acid sequence having 80% or more sequence identity with the amino acid sequence described in SEQ ID NO:

8.

13. The composition according to claim 3, wherein IFN-α is a protein having an amino acid sequence described in any of SEQ ID NOs: 1 to 3 or an amino acid sequence having 80% or more sequence identity with the amino acid sequence described in any of SEQ ID NOs: 1 to 3.

14. The composition according to claim 1, for intratumor administration.

15. A composition for use in a method for treating or preventing cancer, wherein the composition comprises one or more chemotherapeutic agents or molecularly targeted drugs, and the method comprises co-administering (a) IL-12 or mRNA containing a nucleotide sequence encoding IL-12; (b) one or more chemotherapeutic agents or molecularly targeted drugs; and (c) one or more immune checkpoint inhibitors to a target.

16. The composition according to claim 15, wherein the method further comprises co-administering (d) IFN-α or mRNA containing a base sequence encoding IFN-α to the target.

17. A composition for use in a method for treating or preventing cancer, wherein the composition comprises one or more immune checkpoint inhibitors, and the method comprises co-administering (a) IL-12 or mRNA containing a nucleotide sequence encoding IL-12; (b) one or more chemotherapeutic agents or molecularly targeted drugs; and (c) one or more immune checkpoint inhibitors to a target.

18. The composition according to claim 17, wherein the method further comprises co-administering (d) IFN-α or mRNA containing a base sequence encoding IFN-α to the target.

19. A kit for use in a method of treating or preventing cancer, comprising: (i) a composition comprising IL-12 or mRNA comprising a nucleotide sequence encoding IL-12; (ii) a composition comprising one or more chemotherapeutic agents or molecularly targeted drugs; (iii) a composition comprising an anti-PD-1 antibody and / or an anti-PD-L1 antibody; and (iv) a composition comprising an anti-CTLA-4 antibody; and two or more of the above.

20. The kit according to claim 19, wherein the composition of (i) further comprises IFN-α or mRNA containing a base sequence encoding IFN-α.

21. A kit for treating or preventing cancer, comprising two or more of the following: (i) a composition comprising IL-12 or mRNA comprising a nucleotide sequence encoding IL-12; (ii) a composition comprising one or more chemotherapeutic agents or molecularly targeted drugs; (iii) a composition comprising an anti-PD-1 antibody and / or an anti-PD-L1 antibody; and (iv) a composition comprising an anti-CTLA-4 antibody.

22. The kit according to claim 21, further comprising (i'') an mRNA comprising IFN-α or a base sequence encoding IFN-α.

23. A composition for use in a method for treating or preventing cancer, wherein the composition comprises IFN-α or mRNA containing a nucleotide sequence encoding IFN-α, and the method comprises co-administering (a) IL-12 or mRNA containing a nucleotide sequence encoding IL-12; (b) one or more chemotherapeutic agents; and (c) one or more immune checkpoint inhibitors; and (d) IFN-α or mRNA containing a nucleotide sequence encoding IFN-α to a target.

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