anticancer drugs

Abstract

The present invention relates to agents, uses, and methods for cancer, which are designed to promote anti-tumor effects against a variety of different cancers. Exemplary synergistic therapies include compositions of combinations of active agents, including antisense agents for inhibiting or suppressing the expression of TGF-β2, checkpoint inhibitors, and interleukin immunotherapeutic agents. One or more biomarkers, including IRF5 and ITGAM, can be used to select subjects who will benefit from the agents, uses, and methods. The agents can be used together with chemotherapy and other standard therapies. TIFF2025535604000013.tif107161

Description

[Technical Field]

[0001] Sequence Listing This application was filed on November 6, 2023, and contains a Sequence Listing submitted electronically as file ST.26, named 018988-005WO1_SL.xml, and is 120,186 bytes in size.

[0002] Technical Field The present invention describes agents, uses, and methods for treating or ameliorating cancer symptoms in human or animal subjects. The agents are designed to promote anti-tumor effects against a variety of different cancers. Exemplary synergistic therapies include combinations of various active agents, including agents for inhibiting or suppressing TGF-β2 expression, checkpoint inhibitors, and interleukin immunotherapeutics. One or more biomarkers, including IRF5 and ITGAM, can be used to select subjects who will benefit from the agents, uses, or methods. The treatments can be used in combination with chemotherapy, radiation therapy, and other standard therapies. [Background technology]

[0003] background Cancer is a complex disease that involves multiple different cellular pathways. This complexity has made it difficult to identify effective therapeutic strategies that can have antitumor effects across a wide range of cancers.

[0004] Shortcomings of conventional therapies include a lack of efficacy against various cancers.

[0005] Further drawbacks of conventional therapies include significant unwanted side effects, such as the death of normal cells in addition to killing cancer cells.

[0006] A further drawback of anti-cancer drugs is their high toxicity at the required levels of therapeutic administration.

[0007] What are needed are methods, agents and uses for cancer disease that increase efficacy and reduce toxicity and unwanted side effects.

[0008] For example, what is needed are compositions, uses, or methods that combine different agents that have significant anti-tumor and cancer immunotherapy effects and can reduce side effects and adverse health effects. Improved guidance for such compositions is needed, using appropriate biomarkers to select synergistic compositions.

[0009] There is an urgent need for new methods, agents, and uses that combine cancer immunotherapy strategies with strategies of direct antitumor attack to treat various cancers.

[0010] For example, what is needed are therapeutic compositions that combine T cell and immunotherapy of cancer with potent anti-cancer agents. Summary of the Invention

[0011] overview The present invention provides methods for treating or ameliorating cancer symptoms in human or animal subjects using pharmaceutical compositions designed to promote anti-tumor effects against a variety of different cancers. The synergistic pharmaceutical therapy of the present invention involves the use of a potent direct anti-tumor agent together with a cancer immunotherapeutic agent. Cancer immunotherapeutic agents can include checkpoint inhibitors and protein immunotherapeutics. The methods, agents, and uses of the present invention can combine cancer immunotherapy strategies with direct anti-tumor attack strategies to treat a variety of cancers.

[0012] In some aspects, the methods and treatment strategies of the present invention can increase the effectiveness and reduce toxic side effects and adverse health effects in cancer treatment.

[0013] In a further aspect, the methods and treatment strategies of the present invention can use appropriate biomarkers to select synergistic effects of compositions to improve guidance of therapy.

[0014] Exemplary synergistic pharmaceutical therapy includes a combination of various active agents, including agents for inhibiting or suppressing the expression of TGF-β2, checkpoint inhibitors, and interleukin immunotherapy agents.One or more biomarkers, including IRF5 and ITGAM, can be used to select subjects who will benefit from the method, agent, or use.The composition can be used in combination with chemotherapy and other standard therapies.

[0015] Aspects of the present invention include the following.

[0016] An antisense agent for inhibiting or suppressing the expression of TGF-β2 in combination with a checkpoint inhibitor for use in treating or ameliorating symptoms of cancer in a human subject or animal.

[0017] Use of an antisense agent for inhibiting or suppressing the expression of TGF-β2 in the preparation of a medicament for treating or ameliorating symptoms of cancer in a human subject or animal in combination with a checkpoint inhibitor.

[0018] 1. A method for treating or ameliorating symptoms of cancer in a human or animal subject in need thereof, comprising: administering to a subject a therapeutically effective amount of an antisense agent for inhibiting or suppressing expression of TGF-β2; administering a therapeutically effective amount of a checkpoint inhibitor to a subject A method comprising:

[0019] The above agents in combination with an interleukin immunotherapeutic agent.

[0020] Use of the above in combination with an interleukin immunotherapeutic agent.

[0021] The above method, comprising administering to the subject a therapeutically effective amount of an interleukin immunotherapeutic agent.

[0022] The above agents, uses or methods, wherein the agent for inhibiting or suppressing expression of TGF-β2, the checkpoint inhibitor, and the interleukin immunotherapeutic agent are administered in parallel, simultaneously, sequentially, or temporally separately.

[0023] The above agents, uses or methods, wherein the agent for inhibiting or suppressing the expression of TGF-β2, the checkpoint inhibitor, and the interleukin immunotherapeutic agent are administered separately or in a mixed formulation by injection or infusion.

[0024] Any of the above agents, uses or methods, wherein the cancer is pancreatic cancer, melanoma, skin cancer, lung cancer, breast cancer, prostate cancer, colorectal cancer, kidney cancer, gastric cancer, ovarian cancer, cervical cancer, liver cancer, or multiple myeloma.

[0025] The agent, use, or method described above, wherein the agent for inhibiting or suppressing the expression of TGF-β2 is a TGF-β2-specific antisense oligonucleotide that is complementary to a TGF-β2 transcript and is 15 to 30 nucleotides in length.

[0026] The agent, use, or method described above, wherein the agent for inhibiting or suppressing the expression of TGF-β2 is a TGF-β2-specific antisense oligonucleotide that is complementary to the RNA precursor, mRNA precursor, or mRNA of TGF-β2 and is 18 to 21 nucleotides in length.

[0027] The agent, use, or method described above, wherein the agent for inhibiting or suppressing the expression of TGF-β2 is one or more TGF-β2-specific antisense oligonucleotides shown in Table 1 that are complementary to the transcript of TGF-β2, as well as chemically modified variants thereof, LNA variants thereof, gapmer variants thereof, and any combination or pool thereof.

[0028] The agent, use or method above, wherein the TGF-β2 specific antisense oligonucleotide has no more than one or two mismatches compared to the target human TGF-β2.

[0029] The agent, use or method as described above, wherein the TGF-β2 specific antisense oligonucleotide reduces TGF-β2 transcript levels by at least 60%, or at least 70%, or at least 80%, or at least 90%.

[0030] The above agent, use or method, wherein the TGF-β2 specific antisense oligonucleotide reduces any TGF-β1 transcript levels and any TGF-β3 transcript levels by less than 10%, or less than 5%, or less than 1%.

[0031] The above agent, use, or method, wherein the TGF-β2-specific antisense oligonucleotide has one or more nucleotides chemically modified as a phosphorothioate internucleoside linkage, a methoxypropylphosphonate internucleoside linkage, an aminophosphoro linkage to a morpholino group, a 2'-OMe ribose group, a 2'-MOE methoxyethyl ribose group, a 2'-4' constrained methoxyethyl bicyclic ribose group, a 2'-4' constrained ethyl bicyclic ribose group, an LNA ribose group, a 2'-F ribose group, or a 5-methylcytosine base.

[0032] An agent, use or method as described above, wherein the antisense agent is conjugated to polyethylene glycol, a lipid, or triantenarry N-acetyl-galactosamine.

[0033] The above agents, uses, or methods, wherein each agent comprises a carrier which is sterile water for injection, saline, isotonic saline, or a combination thereof, which may be the same or different for each agent.

[0034] An agent, use or method as described above, wherein the agent is substantially free of excipients.

[0035] An agent, use, or method as described above, wherein the agent is stable for at least 14 days at 37°C in a carrier that is substantially free of excipients.

[0036] The above agent, use or method, wherein the checkpoint inhibitor is an inhibitor of PD-1.

[0037] Any of the above agents, uses or methods wherein the checkpoint inhibitor is pembrolizumab, nivolumab, cemiplimab, spartalizumab, atezolizumab, avelumab, or durvalumab.

[0038] The above agent, use or method, wherein the interleukin immunotherapeutic agent is natural IL-2, high dose IL-2, recombinant IL-2, or aldesleukin.

[0039] The agent, use, or method described above, comprising selecting a subject who will benefit from the agent, use, or method based on the level of one or more biomarkers: TGF-β2, IL-2, CD19, IRF5, ITGAM, and combinations thereof.

[0040] The agent, use or method as described above, wherein the one or more biomarkers is IRF5 and the subject is selected if expression of IRF5 is at a level above the median.

[0041] The agent, use or method as described above, wherein the one or more biomarkers is ITGAM, and the subject is selected if the expression of ITGAM is at a level above the median.

[0042] The above agent, use or method, wherein the subject after administration or use has a reduced level of TGF-β2 compared to before administration or use.

[0043] The above agent, use, or method, wherein the subject after administration or use has an increased level of IRF5 compared to before administration or use.

[0044] The above agent, use, or method, wherein the subject after administration or use has a reduced level of ITGAM compared to before administration or use.

[0045] The agent, use or method as described above, which comprises administering a therapeutically effective amount of an expression product of IRF5 or ITGAM to a subject.

[0046] The agent, use or method as described above, wherein the expression product is an mRNA, a polypeptide, a protein, or a fragment thereof, or a combination thereof.

[0047] Any of the above agents, uses or methods, wherein the administration or use reduces mortality at 6, 12, 18, 24, 30 or 36 months.

[0048] Any of the above agents, uses or methods, wherein the administration or use improves overall survival at 6, 12, 18, 24, 30 or 36 months.

[0049] The above agents, uses or methods in combination with any one or more pharmaceutical agents including targeted anti-cancer agents, cancer growth blockers, EGFR inhibitors, and combinations thereof.

[0050] The above agent, use, or method in combination with any one or more medicaments selected from bevacizumab, everolimus, velzutifan, dabrafenib, trametinib, and combinations thereof.

[0051] The above agent, use, or method in combination with any one or more pharmaceutical agents that are cancer growth inhibitors selected from angiogenesis inhibitors, histone deacetylase inhibitors, hedgehog blockers, mTOR inhibitors, p53 inhibitors, PARP inhibitors, proteasome inhibitors, tyrosine kinase inhibitors, and combinations thereof.

[0052] Any of the above agents, uses, or methods in combination with any one or more medicaments which are EGFR inhibitors selected from erlotinib, gefitinib, afatinib, osimertinib, dacomitinib, and combinations thereof.

[0053] Any of the above agents, uses or methods in combination with a chemotherapeutic agent.

[0054] The above agents, uses or methods in combination with radiation therapy or electric field therapy.

[0055] An antisense agent for inhibiting or suppressing the expression of TGF-β2 in combination with an interleukin immunotherapeutic agent for use in treating or ameliorating symptoms of cancer in a human subject or animal.

[0056] 20. Use of an antisense agent for inhibiting or suppressing the expression of TGF-β2 in the preparation of a medicament for treating or ameliorating symptoms of cancer in a human subject or animal in combination with an interleukin immunotherapeutic agent.

[0057] 1. A method for treating or ameliorating symptoms of cancer in a human or animal subject in need thereof, comprising: administering to a subject a therapeutically effective amount of an antisense agent for inhibiting or suppressing expression of TGF-β2; administering a therapeutically effective amount of an interleukin immunotherapeutic agent to the subject; A method comprising:

[0058] The above agent, use or method, wherein the agent for inhibiting or suppressing expression of TGF-β2 and the interleukin immunotherapeutic agent are administered in parallel, simultaneously, sequentially or temporally separately.

[0059] The agent, use or method as described above, wherein the agent for inhibiting or suppressing the expression of TGF-β2 and the interleukin immunotherapeutic agent are administered by injection or infusion, separately or in a mixed formulation.

[0060] Any of the above agents, uses or methods, wherein the cancer is pancreatic cancer, melanoma, skin cancer, lung cancer, breast cancer, prostate cancer, colorectal cancer, kidney cancer, gastric cancer, ovarian cancer, cervical cancer, liver cancer, or multiple myeloma.

[0061] The agent, use, or method described above, wherein the agent for inhibiting or suppressing the expression of TGF-β2 is a TGF-β2-specific antisense oligonucleotide that is complementary to a TGF-β2 transcript and is 15 to 30 nucleotides in length.

[0062] The agent, use, or method described above, wherein the agent for inhibiting or suppressing the expression of TGF-β2 is a TGF-β2-specific antisense oligonucleotide that is complementary to the RNA precursor, mRNA precursor, or mRNA of TGF-β2 and is 18 to 21 nucleotides in length.

[0063] The agent, use, or method described above, wherein the agent for inhibiting or suppressing the expression of TGF-β2 is one or more TGF-β2-specific antisense oligonucleotides shown in Table 1, which are complementary to a TGF-β2 transcript.

[0064] The above agent, use, or method, wherein the TGF-β2-specific antisense oligonucleotide has one or more nucleotides chemically modified as a phosphorothioate internucleoside linkage, a methoxypropylphosphonate internucleoside linkage, an aminophosphoro linkage to a morpholino group, a 2'-OMe ribose group, a 2'-MOE methoxyethyl ribose group, a 2'-4' constrained methoxyethyl bicyclic ribose group, a 2'-4' constrained ethyl bicyclic ribose group, an LNA ribose group, a 2'-F ribose group, or a 5-methylcytosine base.

[0065] An agent, use or method as described above, wherein the antisense agent is conjugated to polyethylene glycol, a lipid, or a triple-stranded N-acetyl-galactosamine.

[0066] The above agents, uses, or methods, wherein each agent comprises a carrier which is sterile water for injection, saline, isotonic saline, or a combination thereof, which may be the same or different for each agent.

[0067] An agent, use or method as described above, wherein the agent is substantially free of excipients.

[0068] An agent, use, or method as described above, wherein the agent is stable for at least 14 days at 37°C in a carrier that is substantially free of excipients.

[0069] The above agent, use or method, wherein the interleukin immunotherapeutic agent is natural IL-2, high dose IL-2, recombinant IL-2, or aldesleukin.

[0070] Any of the above agents, uses or methods, wherein the administration or use reduces mortality at 6, 12, 18, 24, 30 or 36 months.

[0071] An agent, use or method as described above, wherein the administration or use improves survival at 6, 12, 18, 24, 30 or 36 months.

[0072] The above agents, uses or methods in combination with any one or more pharmaceutical agents including targeted anti-cancer agents, cancer growth blockers, EGFR inhibitors, and combinations thereof.

[0073] The above agent, use, or method in combination with any one or more medicaments selected from bevacizumab, everolimus, velzutifan, dabrafenib, trametinib, and combinations thereof.

[0074] The above agent, use, or method in combination with any one or more pharmaceutical agents that are cancer growth inhibitors selected from angiogenesis inhibitors, histone deacetylase inhibitors, hedgehog blockers, mTOR inhibitors, p53 inhibitors, PARP inhibitors, proteasome inhibitors, tyrosine kinase inhibitors, and combinations thereof.

[0075] Any of the above agents, uses, or methods in combination with any one or more medicaments which are EGFR inhibitors selected from erlotinib, gefitinib, afatinib, osimertinib, dacomitinib, and combinations thereof.

[0076] Any of the above agents, uses or methods in combination with a chemotherapeutic agent.

[0077] The above agents, uses or methods in combination with radiation therapy or electric field therapy.

[0078] The agent, use, or method as described above, comprising selecting a subject who will benefit from the agent, use, or method based on the level of one or more biomarkers of TGF-β2, IRF5, ITGAM, and combinations thereof.

[0079] The agent, use, or method as described above, comprising selecting a subject who will benefit from the agent, use, or method based on the level of one or more biomarkers of TGF-β2, IRF5, ITGAM, neoantigens, mutational load, macrophages, and combinations thereof.

[0080] The agent, use or method as described above, wherein the one or more biomarkers is IRF5 and the subject is selected if expression of IRF5 is at a level below the median.

[0081] The agent, use or method as described above, wherein the one or more biomarkers are tumor-associated macrophages, and the subject is selected if tumor-associated macrophages are below average.

[0082] The agent, use or method as described above, wherein the one or more biomarkers is tumor neoantigen mutational burden, and the subject is selected if the neoantigen tumor burden is below average.

[0083] The agent, use, or method described above, which comprises administering to a subject a therapeutically effective amount of an agent for inhibiting or suppressing the expression of ITGAM or IRF5.

[0084] The agent, use, or method described above, wherein the agent for inhibiting or suppressing the expression of ITGAM or IRF5 is an antisense oligonucleotide targeting ITGAM or IRF5, respectively.

[0085] 1. A kit for treating or ameliorating symptoms of cancer, comprising: a therapeutically effective amount of an antisense agent for inhibiting or suppressing the expression of TGF-β2; and Therapeutically effective amounts of checkpoint inhibitors Includes a kit.

[0086] The above kit, comprising a therapeutically effective amount of an interleukin immunotherapeutic agent.

[0087] The above kits, wherein the cancer is pancreatic cancer, melanoma, skin cancer, lung cancer, breast cancer, prostate cancer, colorectal cancer, kidney cancer, stomach cancer, ovarian cancer, cervical cancer, liver cancer, thymic cancer, or multiple myeloma.

[0088] The above kit, wherein the agent for inhibiting or suppressing the expression of TGF-β2 is a TGF-β2-specific antisense oligonucleotide that is complementary to a TGF-β2 transcription product and has a length of 15 to 30 nucleotides.

[0089] The above kit, wherein the agent for inhibiting or suppressing the expression of TGF-β2 is a TGF-β2-specific antisense oligonucleotide that is complementary to a TGF-β2 RNA precursor, mRNA precursor, or mRNA and is 18 to 21 nucleotides in length.

[0090] The above kit, wherein the agent for inhibiting or suppressing the expression of TGF-β2 is one or more TGF-β2-specific antisense oligonucleotides shown in Table 1, which are complementary to a TGF-β2 transcription product.

[0091] The above kit, wherein the TGF-β2-specific antisense oligonucleotide has one or two or less mismatches compared to the target human TGF-β2.

[0092] The above kit, wherein the TGF-β2 specific antisense oligonucleotide reduces TGF-β2 transcript levels by at least 60%, or at least 70%, or at least 80%, or at least 90%.

[0093] The above kit, wherein the TGF-β2 specific antisense oligonucleotide reduces any TGF-β1 transcript levels and any TGF-β3 transcript levels by less than 10%, or less than 5%, or less than 1%.

[0094] The above kit, wherein the TGF-β2-specific antisense oligonucleotide has one or more nucleotides chemically modified as a phosphorothioate internucleoside linkage, a methoxypropylphosphonate internucleoside linkage, an aminophosphoro linkage to a morpholino group, a 2'-OMe ribose group, a 2'-MOE methoxyethyl ribose group, a 2'-4' constrained methoxyethyl bicyclic ribose group, a 2'-4' constrained ethyl bicyclic ribose group, an LNA ribose group, a 2'-F ribose group, or a 5-methylcytosine base.

[0095] The above kit, wherein the antisense agent is conjugated to polyethylene glycol, a lipid, or a triple-stranded N-acetyl-galactosamine.

[0096] The above kit, wherein each agent comprises a carrier which is sterile water for injection, saline, isotonic saline, or a combination thereof, which may be the same or different for each agent.

[0097] The above kit, wherein the agent is substantially free of excipients.

[0098] The above kit, wherein the agent is stable for at least 14 days at 37°C in a carrier that is substantially free of excipients. [Brief explanation of the drawings]

[0099] [Figure 1] Figure 1 shows the Kaplan-Meier overall survival chart for one such clinical efficacy study. Figure 1 demonstrates that the use of PD-1 inhibitors improved survival in patients with high IL-2 levels, which can be achieved with IL-2 immunotherapy (left panel), and low TGF-β2 levels, which can be achieved with antisense TGF-β2 inhibitors (right panel). These clinical data support the rationale for the therapeutic combination of antisense TGF-β2 inhibitors, PD-1 checkpoint inhibitors, and IL-2 immunotherapy to improve overall survival in melanoma patients.

[0100] [Figure 2] Figure 2 shows that the use of PD-1 inhibitors strongly suggests improved survival at higher IL-2 / TGF-β2 ratios, which can be achieved with IL-2 immunotherapy and antisense TGF-β2 inhibitors (left panel). These clinical data provide a rationale for the therapeutic combination of antisense TGF-β2 inhibitors, PD-1 checkpoint inhibitors, and IL-2 immunotherapy to improve overall survival in melanoma patients.

[0101] [Figure 3] Figure 3 shows data on the use of ITGAM as a biomarker. Figure 3 (upper left and right panels) shows Kaplan-Meier overall survival charts for melanoma patients in this study. Figure 3 (lower left and right panels) shows Kaplan-Meier overall survival charts for pancreatic cancer (PDAC) patients in this study in terms of TGF-β2 expression.

[0102] [Figure 4] Figure 4 shows data on the use of ITGAM as a biomarker. Figure 4 (left and right panels) shows Kaplan-Meier overall survival charts for this study of pancreatic cancer patients (PDAC) in terms of IL-2 expression.

[0103] [Figure 5] Figure 5 shows data on the use of CD8A as a biomarker. Figure 5 (upper left and right panels) shows Kaplan-Meier overall survival charts for this study of melanoma patients. Figure 5 (lower left and right panels) shows Kaplan-Meier overall survival charts for this study of pancreatic cancer (PDAC) patients in terms of TGF-β2 expression.

[0104] [Figure 6] Figure 6 shows data on the use of CD8A as a biomarker. Figure 6 (left and right panels) shows Kaplan-Meier overall survival charts for this study of pancreatic cancer patients (PDAC) in terms of IL-2 expression.

[0105] [Figure 7] Figure 7 shows data on the use of CD4 as a biomarker. Figure 3 (upper left and right panels) shows Kaplan-Meier overall survival charts for this study of melanoma patients. Figure 7 (lower left and right panels) shows Kaplan-Meier overall survival charts for this study of pancreatic cancer (PDAC) patients in terms of TGF-β2 expression.

[0106] [Figure 8] Figure 8 shows data regarding the use of CD4 as a biomarker. Figure 8 (left and right panels) shows Kaplan-Meier overall survival charts for this study of pancreatic cancer patients (PDAC) in terms of IL-2 expression.

[0107] [Figure 9] Figure 9 shows data on the use of ITGAX as a biomarker. Figure 9 (upper left and right panels) shows Kaplan-Meier overall survival charts for this study of melanoma patients. Figure 9 (lower left and right panels) shows Kaplan-Meier overall survival charts for this study of pancreatic cancer (PDAC) patients in terms of TGF-β2 expression.

[0108] [Figure 10]Figure 10 shows data on the use of ITGAX as a biomarker. Figure 10 (left and right panels) shows Kaplan-Meier overall survival charts for this study of pancreatic cancer patients (PDAC) in terms of IL-2 expression.

[0109] [Figure 11] Figure 11 shows data on the use of CD19 as a biomarker. Figure 11 (upper left and right panels) shows Kaplan-Meier overall survival charts for this study of melanoma patients. Figure 11 (lower left and right panels) shows Kaplan-Meier overall survival charts for this study of pancreatic cancer patients (PDAC) in terms of TGF-β2 expression.

[0110] [Figure 12] Figure 12 shows data regarding the use of CD19 as a biomarker. Figure 12 (left and right panels) shows Kaplan-Meier overall survival charts for this study of pancreatic cancer patients (PDAC) in terms of IL-2 expression.

[0111] [Figure 13] Figure 13 shows data on the use of IRF5 as a biomarker. Figure 13 (upper left and right panels) shows Kaplan-Meier overall survival charts for this study of melanoma patients. Figure 13 (lower left and right panels) shows Kaplan-Meier overall survival charts for this study of pancreatic cancer (PDAC) patients in terms of TGF-β2 expression.

[0112] [Figure 14] Figure 14 shows data regarding the use of IRF5 as a biomarker. Figure 14 (left and right panels) shows Kaplan-Meier overall survival charts for this study of pancreatic cancer patients (PDAC) in terms of IL-2 expression.

[0113] [Figure 15]Figure 15 shows data on the use of NOS2 as a biomarker. Figure 15 (upper left and right panels) shows Kaplan-Meier overall survival charts for this study of melanoma patients. Figure 15 (lower left and right panels) shows Kaplan-Meier overall survival charts for this study of pancreatic cancer patients (PDAC) in terms of TGF-β2 expression.

[0114] [Figure 16] Figure 16 shows data regarding the use of NOS2 as a biomarker. Figure 16 (left and right panels) shows Kaplan-Meier overall survival charts for this study of pancreatic cancer patients (PDAC) in terms of IL-2 expression.

[0115] [Figure 17] Figure 17 shows data on the use of CD163 as a biomarker. Figure 17 (upper left and right panels) shows Kaplan-Meier overall survival charts for this study of melanoma patients. Figure 17 (lower left and right panels) shows Kaplan-Meier overall survival charts for this study of pancreatic cancer patients (PDAC) in terms of TGF-β2 expression.

[0116] [Figure 18] Figure 18 shows data regarding the use of CD163 as a biomarker. Figure 18 (left and right panels) shows Kaplan-Meier overall survival charts for this study of pancreatic cancer patients (PDAC) in terms of IL-2 expression.

[0117] [Figure 19]Figure 19 shows the results of this clinical study on overall survival of melanoma patients. Figure 19 shows a Kaplan-Meier overall survival chart for such a clinical efficacy study. Using various immune cell indicators, namely, basophils, B cells, eosinophils, and M0 macrophages, and further stratification based on Th1 helper cells, Figure 19 shows that in all cases, high levels of IL-2 significantly prolonged survival when using PD-1 checkpoint inhibitors. M0 macrophages were a critical factor.

[0118] [Figure 20] Figure 20 shows the results of this clinical study on overall survival of melanoma patients. Figure 20 shows a Kaplan-Meier overall survival chart for such a clinical efficacy study. Figure 20 shows that with PD-1 checkpoint inhibitors, and further stratification based on various immune cell indicators, namely, basophils, B cells, eosinophils, and M0 macrophages, and Th1 helper cells, high levels of IL-2 significantly prolonged survival in all cases. Figure 20 (upper right panel and lower panel) shows comparative baseline results.

[0119] [Figure 21] Figure 21 shows the results of this clinical study on overall survival for pancreatic cancer patients. Figure 21 shows a Kaplan-Meier overall survival chart for such a clinical efficacy study. Figure 21 (upper left panel) shows that for patients with low M2 tumor-associated macrophages, high tumor mRNA levels of TGF-β2 significantly shortened survival. The log-rank P value in Figure 21 (upper left panel) indicates a high significance of improved survival with the use of therapeutic antisense TGF-β2 inhibitors based on this clinical study and condition. Patients whose tumors had a high TGF-β2 expression range survived for only 15 months compared with 73 months for patients whose tumors had a low TGF-β2 expression range.

[0120] [Figure 22]Figure 22 shows the Kaplan-Meier overall survival chart for the clinical efficacy study of combination cancer therapy.

[0121] [Figure 23] Figure 23 shows the Kaplan-Meier overall survival chart for the clinical efficacy study of combination cancer therapy.

[0122] [Figure 24] Figure 24 shows the Kaplan-Meier overall survival chart for the clinical efficacy study of combination cancer therapy.

[0123] [Figure 25] Figure 25 shows Kaplan-Meier overall survival charts for a clinical efficacy study of combination cancer therapy.

[0124] [Figure 26] Figure 26 shows Kaplan-Meier overall survival charts for a clinical efficacy study of combination cancer therapy.

[0125] [Figure 27] Figure 27 shows Kaplan-Meier overall survival charts for a clinical efficacy study of combination cancer therapy.

[0126] [Figure 28] Figure 28 shows Kaplan-Meier overall survival charts for a clinical efficacy study of combination cancer therapy.

[0127] [Figure 29] Figure 29 shows Kaplan-Meier overall survival charts for a clinical efficacy study of combination cancer therapy.

[0128] [Figure 30] Figure 30 shows Kaplan-Meier overall survival charts for a clinical efficacy study of combination cancer therapy.

[0129] [Figure 31]Figure 31 shows Kaplan-Meier overall survival charts for a clinical efficacy study of combination cancer therapy.

[0130] [Figure 32] Figure 32 shows the Kaplan-Meier overall survival chart for the clinical efficacy study of combination cancer therapy.

[0131] [Figure 33] Figure 33 shows Kaplan-Meier overall survival charts for a clinical efficacy study of combination cancer therapy.

[0132] [Figure 34] Figure 34 shows Kaplan-Meier overall survival charts for a clinical efficacy study of combination cancer therapy.

[0133] [Figure 35] Figure 35 shows Kaplan-Meier overall survival charts for a clinical efficacy study of combination cancer therapy.

[0134] [Figure 36] Figure 36 shows Kaplan-Meier overall survival charts for a clinical efficacy study of combination cancer therapy.

[0135] [Figure 37] Figure 37 shows Kaplan-Meier overall survival charts for a clinical efficacy study of combination cancer therapy.

[0136] [Figure 38] Figure 38 shows Kaplan-Meier overall survival charts for a clinical efficacy study of combination cancer therapy.

[0137] [Figure 39] Figure 39 shows Kaplan-Meier overall survival charts for a clinical efficacy study of combination cancer therapy.

[0138] [Figure 40]Figure 40 shows Kaplan-Meier overall survival charts for a study of clinical efficacy of combination cancer therapy.

[0139] [Figure 41] Figure 41 shows Kaplan-Meier overall survival charts for a clinical efficacy study of combination cancer therapy.

[0140] [Figure 42] Figure 42 shows Kaplan-Meier overall survival charts for a clinical efficacy study of combination cancer therapy.

[0141] [Figure 43] Figure 43 shows Kaplan-Meier overall survival charts for a clinical efficacy study of combination cancer therapy.

[0142] [Figure 44] Figure 44 shows Kaplan-Meier overall survival charts for a study of clinical efficacy of combination cancer therapy.

[0143] [Figure 45] Figure 45 shows Kaplan-Meier overall survival charts for a clinical efficacy study of combination cancer therapy. DETAILED DESCRIPTION OF THE INVENTION

[0144] Detailed Description of the Disclosure The present invention relates to methods, compositions, agents, and their therapeutic uses for treating or ameliorating the symptoms of cancer in human or animal subjects using pharmaceutical compositions designed to promote anti-tumor effects against a variety of different cancers.

[0145] Exemplary synergistic pharmaceutical therapies include compositions combining various active agents, including agents for inhibiting or suppressing the expression of TGF-β2, checkpoint inhibitors, and interleukin immunotherapeutic agents.

[0146] Aspects of the present invention include methods, agents, and their therapeutic uses for treating or ameliorating symptoms of neoplastic disease, where the agents may be administered concurrently, simultaneously, sequentially, or temporally separately.

[0147] In certain embodiments, highly stable formulations of one or more anti-TGF-β2 agents can be used in combination with one or more immunotherapeutic agents for neoplastic diseases, where the anti-TGF-β2 agents and immunotherapeutic agents are used in parallel, simultaneously, sequentially, or temporally separated.

[0148] In some embodiments, one or more biomarkers, including IRF5 and ITGAM, can be used to select subjects who will benefit from the method, agent, or use. The composition can be used in combination with chemotherapy and other standard therapies.

[0149] In some embodiments, the present invention contemplates biomarker-guided combinations of immunotherapeutics, including checkpoint inhibitors, with TGF-β2 inhibitors.

[0150] Embodiments of the present invention encompass methods, agents, and uses for immunotherapeutics, including checkpoint inhibitors in combination with TGF-β2 inhibitors, that are guided by biomarkers. These embodiments recognize that overexpression of TGF-β2 is a useful indicator for avoiding a cascade of downstream effects and poor outcomes in neoplastic disease.

[0151] The present invention can use the detection of TGF-β2 biomarker as a guide for selecting subjects for treatment with immunotherapeutic agents, including checkpoint inhibitors combined with TGF-β2 inhibitors.Because the antisense oligonucleotide of the present invention, such as OT-101, targets and inhibits TGF-β2, the selection of subjects for treatment with TGF-β2 biomarkers can advantageously lead to improved outcomes.

[0152] More particularly, the present invention can utilize TGF-beta2 as a biomarker, which is surprisingly superior to TGF-beta-1 or TGF-beta-3 for outcome in neoplastic disease. TGF-beta2 as a biomarker predicts improved outcome, whereas TGF-beta-1 or TGF-beta-3 do not and may indicate a poorer outcome.

[0153] In some embodiments, the combination of a TGF-β2 antisense inhibitor and a PD-1 checkpoint inhibitor can be surprisingly effective, hi certain embodiments, the combination of a TGF-β2 antisense inhibitor and a PD-1 checkpoint inhibitor is surprisingly effective because the combination of a TGF-β2 antisense inhibitor and a PD-L1 checkpoint inhibitor is not.

[0154] Embodiments of the present invention take advantage of these facts to provide methods, agents, or uses for tumor diseases by selecting subjects using TGF-β2 biomarkers, and subjects are selected if they have elevated expression of TGF-β2.

[0155] In some embodiments, a method for treating or ameliorating symptoms of cancer in a human subject or animal subject in need thereof can comprise, in combination, administering to the subject a therapeutically sufficient amount of a pharmaceutical composition comprising an agent for inhibiting or suppressing expression of TGF-β2, and administering to the subject a therapeutically sufficient amount of a pharmaceutical composition comprising a checkpoint inhibitor, and administering to the subject a therapeutically sufficient amount of a pharmaceutical composition comprising an interleukin immunotherapeutic agent, wherein the subject is selected using a TGF-β2 biomarker, and the subject is selected if expression of TGF-β2 is elevated.

[0156] In a further aspect, the agents for inhibiting or suppressing the expression of TGF-β2 in combination with checkpoint inhibitors and interleukin immunotherapeutics for use in treating or ameliorating symptoms of cancer in a human subject or animal can be used in neoplastic diseases by selecting subjects using a TGF-β2 biomarker, wherein the subject has elevated expression of TGF-β2.

[0157] The therapy of the present invention may be applied to pancreatic cancer, melanoma, skin cancer, lung cancer, breast cancer, prostate cancer, colorectal cancer, kidney cancer, gastric cancer, ovarian cancer, cervical cancer, liver cancer, or multiple myeloma.

[0158] As used herein, the term "agent" can refer to one or more active compounds, a combination of active compounds, or a composition comprising one or more active compounds and a carrier, and / or a solvent, and / or any number of excipients. In some embodiments, the composition can be a pharmaceutical composition. In certain embodiments, the composition can be a pharmaceutical composition comprising a therapeutically effective amount of one or more active compounds. Some examples of excipients are listed in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa. 1975, and Liberman, HA and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, NY, 1980. Methods for determining the therapeutically effective amount of a compound are known in the art.

[0159] Anti-cancer agents and methods Many cancers, such as pancreatic cancer (PDAC), melanoma, etc., cause patients to express high levels of TGF-β2. Agents for inhibiting or suppressing the expression of TGF-β2 can be effective in treating these types of cancer. For example, patients with pancreatic cancer (PDAC) have an overall survival time that is more than doubled from 15 months for patients with high TGF-β2 to 37 months for patients with low TGF-β2.

[0160] The present invention includes methods for treating or ameliorating symptoms of cancer in a human or animal subject in need thereof by administering to the subject a therapeutically sufficient amount of a pharmaceutical composition comprising an agent for inhibiting or suppressing the expression of TGF-β2; administering to the subject a therapeutically sufficient amount of a pharmaceutical composition comprising a checkpoint inhibitor; and administering to the subject a therapeutically sufficient amount of a pharmaceutical composition comprising an interleukin immunotherapeutic agent.

[0161] In certain embodiments, the present invention includes an agent for inhibiting or suppressing the expression of TGF-β2 in combination with a checkpoint inhibitor for use in treating or ameliorating symptoms of cancer in a human subject or animal.

[0162] In a further aspect, the invention includes an agent for inhibiting or suppressing the expression of TGF-β2 in combination with an interleukin immunotherapeutic agent for use in treating or ameliorating symptoms of cancer in a human subject or animal.

[0163] In an additional aspect, the invention includes an agent for inhibiting or suppressing the expression of TGF-β2 in combination with a checkpoint inhibitor and an interleukin immunotherapeutic agent for use in treating or ameliorating symptoms of cancer in a human subject or animal.

[0164] The present invention further contemplates the use of a composition comprising an agent for inhibiting or suppressing the expression of TGF-β2 in the preparation of a medicament for treating or ameliorating symptoms of cancer in a human subject or animal in combination with a checkpoint inhibitor and / or an interleukin immunotherapeutic agent.

[0165] Therapies of the present invention using one or more agents to inhibit or suppress the expression of TGF-β2 can be applied to pancreatic cancer, melanoma, skin cancer, lung cancer, breast cancer, prostate cancer, colorectal cancer, kidney cancer, gastric cancer, ovarian cancer, cervical cancer, liver cancer, or multiple myeloma.

[0166] Examples of agents for inhibiting or suppressing the expression of TGF-β2 include antisense agents.

[0167] Human TGF-β2-specific antisense oligodeoxynucleotide agent Antisense oligonucleotides (ASOs) can be single-stranded deoxyribonucleotides and can be complementary to mRNA targets. Antisense therapy can downregulate molecular targets by inducing RNase H endonuclease activity, which cleaves RNA-DNA heteroduplexes and significantly reduces the translation of target genes. Other ASO mechanisms include inhibiting 5' cap formation, altering splicing processes such as splice switching, and steric hindrance of ribosomal activity.

[0168] Antisense therapeutic strategies can utilize single-stranded DNA oligonucleotides that inhibit protein production by mediating catalytic degradation of target mRNA or by binding to the site on mRNA that is required for translation.Antisense oligonucleotides can be designed to target the RNA genome of viruses or viral transcription products.Antisense oligonucleotides can provide an approach to identify potential targets and therefore represent potential therapeutic agents.

[0169] Antisense oligonucleotides are small synthetic fragments of single-stranded DNA that can be 15-30 nucleotides in length. ASOs can specifically bind to complementary DNA / RNA sequences through Watson-Crick hybridization. Once bound to the target RNA, they can inhibit the translation process either by inducing cleavage mechanisms or by inhibiting mRNA maturation. ASOs can selectively inhibit gene expression with specificity. Chemical modifications of DNA or RNA can be used to enhance stability.

[0170] For example, modifications can be introduced into the phosphodiester bond, sugar ring, and backbone. ASO antiviral agents can block the translation process either by (i) cleavage of mRNA mediated by ribonuclease H (RNAse H) or RNase P, or (ii) steric (non-binding) blocking of enzymes involved in the translation of target genes. Human TGF-β2-specific phosphorothioate antisense oligodeoxynucleotides, such as OT-101, AP 12009 travedelsen SEQ ID NO:8, can be used to reduce TGF-β2 protein levels in malignant tumors and slow disease progression.

[0171] Antisense oligodeoxynucleotides are short strings of DNA designed to downregulate gene expression by interfering with the translation of specific encoded proteins at the mRNA level. OT-101 is a synthetic 18-mer phosphorothioate oligodeoxynucleotide (S-ODN) in which all 3'-5' linkages are modified to phosphorothioate. The molecular formula is C 177 H 208 N 60 Na 17 O 94 P 17 S 17 and has a molecular weight of 6,143 g / mol. OT-101 was designed to be complementary to a specific sequence of human TGF-β2 mRNA after gene expression.

[0172] Antisense oligodeoxynucleotides are short strings of DNA designed to downregulate gene expression by interfering with the translation of specific encoded proteins at the mRNA level. For example, SEQ ID NO:8 (OT-101) is a synthetic 18-mer phosphorothioate oligodeoxynucleotide (S-ODN) in which the non-bridging oxygen of each phosphate moiety is replaced by a sulfur atom. OT-101 is complementary to the specific sequence of human TGF-β2 mRNA derived from gene expression. OT-101 can be an RNA therapeutic agent designed to suppress the immunosuppressive effect of TGF-β2 and reduce the level of TGF-β2 in malignant tumors, in order to treat or alleviate cancer symptoms or delay the progression of the disease.

[0173] The target TGF-β2 mRNA can be the NCBI reference sequence: NM_003238.3, which is 5,882 bp in length. The target region of the TGF-β2 mRNA can be the protein-coding sequence from 1,369 to 2,613 of the reference.

[0174] Examples of agents of the present disclosure for inhibiting or suppressing the expression of TGF-β2 include TGF-β2-specific antisense oligonucleotides shown in SEQ ID NOs: 1 to 136 in Table 1. Table 1. TGF-β2-specific antisense oligonucleotides TIFF2025535604000002.tif23147TIFF2025535604000003.tif235147TIFF20255356040 00004.tif235147TIFF2025535604000005.tif235147TIFF2025535604000006.tif90147

[0175] The sequences in Table 1 can be chemically modified to provide active variants thereof, LNA variants thereof, and gapmer variants thereof, as known in the art. The sequences in Table 1 can be used in any combination, for example, pooled combinations, as active agents.

[0176] It will be appreciated that additional antisense oligonucleotides can be constructed based on the gene sequence of TGF-β2.

[0177] In some embodiments, the TGF-β2-specific antisense oligonucleotides of the present invention can have no more than one or two mismatches compared to the target human TGF-β2.

[0178] In certain embodiments, the TGF-β2-specific antisense oligonucleotides of the invention are capable of reducing TGF-β2 transcript levels by at least 60%, or at least 70%, or at least 80%, or at least 90%.

[0179] In additional embodiments, the TGF-β2-specific antisense oligonucleotides of the present invention may be selective for TGF-β2 and may reduce any TGF-β1 transcript levels and any TGF-β3 transcript levels by less than 10%, or less than 5%, or less than 1%.

[0180] In further embodiments, the therapeutically effective amount of an antisense agent for inhibiting or suppressing expression of TGF-β2 can be 0.1 to 3000 mg per day, or 1 to 1000 mg per day, or 2 to 500 mg per day, or 2 to 200 mg per day.

[0181] In certain embodiments, a formulation of an antisense agent for inhibiting or suppressing expression of TGF-β2 can have a concentration of 0.05 to 50 μM, or 0.1 to 25 μM, or 0.1 to 10 μM, or 0.1 to 7.5 μM, or 0.1 to 5 μM.

[0182] In certain embodiments, methods for using antisense agents to inhibit or suppress expression of TGF-β2 include administering a dose of 1 to 1000 mg / m 2 / day, or 1-500 mg / m 2 / day, or 1-250 mg / m 2 / day, or 1-100 mg / m 2 / day, or 1-50 mg / m 2 An effective dosage of 1 / day can be used. The average human body surface area is approximately 1.6 to 1.9 m 2 It could be.

[0183] In additional embodiments, methods for using antisense agents to inhibit or suppress expression of TGF-β2 include administering doses of 0.05 to 40 mg / kg / day, or 0.1 to 30 mg / kg / day, or 0.2 to 20 mg / m 2 / day, or 0.3-10 mg / m 2 / day, or 0.5-5 mg / m 2 An effective dosage of 100 mg / day can be used. The average human body weight can be about 60 kg.

[0184] In certain embodiments, the agent of the present disclosure for inhibiting or suppressing the expression of TGF-β2 can be prepared from a lyophilized powder of the agent.

[0185] In some examples and embodiments, the agent may be a TGF-β2-specific antisense oligonucleotide selected from SEQ ID NOs: 1-136, and may be administered or used by injection or infusion at a dose of 4 μl / min at a dose level of 10 μM on days 1-7, or at a dose of 20 μM on days 1-7, or at a dose of 40 μM on days 1-7, or at a dose of 80 μM on days 1-7. In some embodiments, the agent may be a chemically modified TGF-β2-specific antisense oligonucleotide selected from SEQ ID NOs: 9-136, and may be administered or used by injection or infusion at a dose of 4 μl / min at a dose level of 10 μM on days 1-7, or at a dose of 20 μM on days 1-7, or at a dose of 40 μM on days 1-7, or at a dose of 80 μM on days 1-7.

[0186] In certain embodiments, OT-101 can be supplied as a sterile lyophilizate for solution prior to administration in 20R glass vials at 250 mg / vial. The lyophilizate can be aseptically reconstituted in sterile, preservative-free, isotonic NaCl solution. OT-101 solution can be administered every 14 days using a portable pump system as a continuous IV infusion on days 4-7, according to a 4-day on, 10-day off schedule. Schedules may also include 7-day on / 7-day off and 4-day on / 10-day off scheduling. Doses may be 40, 80, 160, 140, 190, 250, or 330 mg.

[0187] In certain embodiments, the agent can be a TGF-β2 gene sequence-specific antisense oligonucleotide selected from SEQ ID NOs: 1-136, administered at 40, 80, 160, 140, 190, 250, 330 mg / m on days 1-7. 2 or at doses of 40, 80, 160, 140, 190, 250, or 330 mg / m on days 1–4. 2 It may be administered or used by injection or infusion at a dose of

[0188] In some examples and embodiments, the agent may be a TGF-β2 gene sequence-specific antisense oligonucleotide selected from TSEQ ID NOs: 1-136 and may be administered or used by injection or infusion at a dose of 4 μl / min or 2-8 μl / min, at a dose level of 2 μM on days 1-7, or at a dose of 4 μM on days 1-7, or at a dose of 8 μM on days 1-7, or at a dose of 10 μM on days 1-7.

[0189] In some embodiments, the agent may be a TGF-β2 gene sequence-specific antisense oligonucleotide selected from SEQ ID NOs:9-136 and may be administered or used by injection or infusion at a dose of 4 μl / min or 2-8 μl / min, at a dose level of 2 μM on days 1-7, or at a dose of 4 μM on days 1-7, or at a dose of 8 μM on days 1-7, or at a dose of 10 μM on days 1-7.

[0190] A therapeutically effective amount can also be determined by routine experimentation, for example, by monitoring the subject's response to administration of the agent and adjusting the dosage. See, e.g., Remington, The Science and Practice of Pharmacy (Gennaro ed. 20th edition) (2000).

[0191] Embodiments of the invention that involve the administration or use of compositions of agents can ameliorate or suppress symptoms caused by TGF-β2-induced proteins.

[0192] Embodiments of the present invention further include pharmaceutical compositions for inhibiting or suppressing TGF-β2 expression or treating or ameliorating symptoms of cancer in humans or animals. The pharmaceutical compositions can include a TGF-β2 inhibitor, a pharmaceutically acceptable salt form, ester, polymorph, or stereoisomer thereof, or any combination thereof, and a carrier. The TGF-β2 inhibitor can be selected from TGF-β2-specific antisense oligonucleotides SEQ ID NOs: 1-136, or SEQ ID NOs: 9-136, and chemically modified variants thereof. The carrier can be sterile water for injection, saline, isotonic saline, or a combination thereof.

[0193] Importantly, the composition of the present disclosure may be substantially free of excipients.The composition of the present invention that is substantially free of excipients has been found to be surprisingly stable in a carrier.In some embodiments, the composition may be stable in a carrier at 37°C for at least 14 days, or at least 21 days, or at least 28 days.

[0194] In additional embodiments, pharmaceutical compositions for injection can contain less than 1% by weight of excipients, or less than 0.5% by weight of excipients, or less than 0.1% by weight of excipients.

[0195] The agents of the present disclosure can be diluted to formulate into a mixture for administration by injection in components such as an intravenous bag, syringe, and tubing, as is known in the art. Such formulations can include multiple agents and excipients.

[0196] Aspects of the present invention further contemplate treatment modalities in which the compositions of the present invention are administered or utilized in conjunction with standard treatments for a disease. Examples of additional pharmaceutical agents that may be administered or utilized in combination with the compositions of the present invention include anti-inflammatory agents, anti-inflammatory steroids, piperiquine, pyronaridine, curcumin, frankincense, remdesivir, Sompraz D, Zifi CV / Zac D, CCM, Broclear, Budamet, Rapitus, Montek LC, low molecular weight heparin, prednisolone, paracetamol, vitamin B complex, vitamin C, pantoprozol, doxycycline, ivermectin, zinc, Foracort Rotacaps inhalation, ceftriaxone injection, paracetamol tablets, Fragmin injection, Covifor tablets, azithromycin, dexamethasone injection, ondansetron injection, multivitamin tablets, ascorbic acid tablets, calcium carbonate tablets, and zinc sulfate tablets.

[0197] Some TGF-β2 specific antisense oligonucleotide agents are set out in US 9,963,703, US 9,758,786 and US 8,476,246.

[0198] For example, the API travedelsen (OT-101) is a synthetic 18-mer S-ODN composed of the bases adenine (A), thymine (T), guanine (G), and cytosine (C), with all 3'-5' linkages modified to phosphorothioates. This sulfur modification makes the drug more resistant to degradation, resulting in increased stability in vitro and in vivo. Its primary molecular structure, i.e., nucleotide sequence, was designed to be complementary to a specific sequence in human transforming growth factor-beta 2 (TGF-β2) mRNA. This sequence and related sequences can be used to achieve optimal antisense efficacy in vitro and in vivo due to their excellent chemical and structural properties, biological activity, and specificity.

[0199] The chemical structure, exemplary phosphorothioate moiety (CAG), and physical properties of travedersen are shown in Table 2. Table 2. Chemical and physical properties of travedelsen (OT-101) TIFF2025535604000007.tif83162

[0200] The investigational medicinal product can be supplied as a sterile lyophilizate for solution for infusion in a 50 mL glass vial (primary container) containing 7.37 mg of travedersen (intratumoral treatment) and a 20R glass vial (primary container) containing 250 mg of travedersen (intravenous treatment), respectively. The finished drug product may contain no excipients. The glass vial can be used parenterally. The glass vial can be sealed with a sterile rubber stopper suitable for lyophilization. The stopper can be sealed with a crimped capsule containing a colored flip-off cap. For clinical use, each vial can be provided in a white-colored folding box to protect the vial from exposure to light and damage during transport. Both the glass vial and folding box can be labeled according to local requirements. The primary and secondary containers of the closure system can meet international quality standards for packaging of sterile solid drug products for injection.

[0201] The kit can provide OT-101 as a lyophilized powder in different aliquots in 50 mL glass vials, specifying the total volume (mL) after dissolution and the resulting concentration (μM).

[0202] The kit can provide OT-101 as a lyophilized powder in 20 mL glass vials in different aliquots; the calculated amount of OT-101 per patient and treatment cycle can be dissolved in a total volume of 85 mL of isotonic saline. The CADD ambulatory infusion pump can provide measured drug therapy to patients in hospital and outpatient settings. It can be used for therapies requiring a continuous rate of infusion. When administering drugs via the CADD pump, the use of central venous access and a Luer lock connector with a split valve septum is recommended. Drug doses can be concentrated into small volumes. Required materials include a pump (Smiths Medical CADD SOLIS VIP), a flow stop, a clamp, and a yellow drug cassette reservoir with a 100 mL female Luer, a male Luer, a clamp, a CADD extension set with a 0.2 micron air removal filter, and an integrated anti-siphon valve with a male Luer.

[0203] The present invention further provides a kit containing 250 mg of one or more TGF-β2-specific antisense oligonucleotides selected from SEQ ID NOs: 1-136 in lyophilized powder form in vials. The kit may include appropriate vials and all necessary components of an application system, i.e., syringes, tubing, and filters. The OT-101 lyophilized powder can be dissolved in isotonic (0.9%) aqueous sodium chloride solution prior to use.

[0204] Checkpoint inhibitors As mentioned herein, checkpoint inhibitors known in the art are immune checkpoint inhibitors. Checkpoint inhibitors are immunotherapeutic drugs that block checkpoint proteins from binding to their partner proteins. This prevents the "off" signal from being sent, thereby enabling T cells to kill cancer cells. More specifically, checkpoint proteins such as PD-1 on T cells block immune responses. The binding of PD-L1 to PD-1 prevents T cells from killing tumor cells. Therefore, blocking the binding of PD-L1 to PD-1 with immune checkpoint inhibitors can enable T cells to kill tumor cells. The immune system is essentially reactivated, allowing T cells to attack cancer cells.

[0205] In some embodiments, the checkpoint inhibitors of the present disclosure can be inhibitors of CTLA-4, PD-1, or PD-L1.

[0206] In certain embodiments, the checkpoint inhibitors of the present disclosure can be inhibitors of PD-1.

[0207] In certain aspects, the checkpoint inhibitor of the present disclosure can be pembrolizumab.

[0208] In certain aspects, the checkpoint inhibitor of the present disclosure can be pembrolizumab, nivolumab, cemiplimab, spartalizumab, atezolizumab, avelumab, or durvalumab.

[0209] Without wishing to be bound by theory, PD-1 receptor-ligand interactions may be a major pathway by which tumors hijack immune regulation. Under healthy conditions, the normal function of PD-1, expressed on the cell surface of activated T cells, is to downmodulate unwanted or excessive immune responses, including autoimmune reactions. Following T cell stimulation, PD-1 recruits the tyrosine phosphatases SHP-1 and SHP-2 to the immunoreceptor tyrosine-based switch motif within its cytoplasmic tail, resulting in the dephosphorylation of effector molecules involved in the CD3 T cell signaling cascade, such as CD3 zeta (CD3ζ), protein kinase C-theta (PKCθ), and zeta chain-associated protein kinase (ZAP70).

[0210] IL-2 immunotherapy In certain embodiments, the interleukin immunotherapeutic agent of the present disclosure can be natural or synthetic IL-2, high-dose IL-2, recombinant IL-2, or aldesleukin.

[0211] Immunotherapeutic agents can have anti-cancer effects by targeting the tumor microenvironment and activating the immune response against cancer cells. For example, interleukin-2 (IL-2) activates natural killer (NK) cells and cytotoxic CD8 + It can promote the activation of T lymphocytes.Anti-tumor immune responses may require T helper 1 (Th1) and other tumor cell killing activities.

[0212] Anticancer Agents and Therapeutic Combinations An embodiment of the present invention includes the combination of a TGF-β2 specific inhibitor that does not inhibit or only minimally inhibits the closely related TGF-β1 and TGF-β3 isoforms with a PD-1 checkpoint inhibitor.

[0213] In certain embodiments, the present invention provides therapeutic combinations of one or more antisense TGF-β2 inhibitors and a PD-1 checkpoint inhibitor.

[0214] Therapeutic aspects of the invention using one or more agents to inhibit or suppress expression of TGF-β2 in combination with a PD-1 immune checkpoint inhibitor may be applicable to pancreatic cancer, melanoma, skin cancer, lung cancer, breast cancer, prostate cancer, colorectal cancer, kidney cancer, gastric cancer, ovarian cancer, cervical cancer, liver cancer, or multiple myeloma.

[0215] Additional embodiments of the present invention include therapeutic combinations of a TGF-β2 inhibitor, an IL-2 immunotherapeutic agent, and a PD-1 checkpoint inhibitor.

[0216] In certain embodiments, the present invention provides therapeutic combinations of one or more antisense TGF-β2 inhibitors, an IL-2 immunotherapeutic agent, and a PD-1 checkpoint inhibitor.

[0217] Therapeutic aspects of the invention using one or more agents to inhibit or suppress TGF-β2 expression in combination with a PD-1 immune checkpoint inhibitor and an IL-2 immunotherapeutic agent may be applicable to pancreatic cancer, melanoma, skin cancer, lung cancer, breast cancer, prostate cancer, colorectal cancer, kidney cancer, gastric cancer, ovarian cancer, cervical cancer, liver cancer, or multiple myeloma.

[0218] The unexpectedly advantageous synergistic effects of a therapeutic combination of an antisense TGF-β2 inhibitor, an IL-2 immunotherapeutic agent, and a checkpoint inhibitor for cancer treatment can be significantly amplified when the checkpoint inhibitor is a PD-1 checkpoint inhibitor.

[0219] In the treatment of cancer patients, the use of a therapeutic combination of a PD-1 checkpoint inhibitor, an antisense TGF-β2 inhibitor, and an IL-2 immunotherapeutic agent can significantly extend overall survival. The overall survival (OS) of such patients can be more than doubled. In some embodiments, the therapeutic combination of an antisense TGF-β2 inhibitor, an IL-2 immunotherapeutic agent, and a PD-1 checkpoint inhibitor can provide unexpectedly advantageous synergistic effects based on clinical data.

[0220] Synergistic effects of therapeutic combinations Embodiments of the present invention can provide synergy for the therapeutic combination of agents for inhibiting or suppressing TGF-β2 expression with PD-1 checkpoint inhibitors and interleukin immunotherapeutic agents to treat or ameliorate symptoms of cancer.

[0221] In some embodiments, the anti-cancer use of a combination of an antisense agent for inhibiting or suppressing TGF-β2 expression, a PD-1 checkpoint inhibitor, and an IL-2 immunotherapeutic agent may be particularly effective for patients with high levels of tumor-associated monocytes and / or tumor-associated macrophages. Thus, high levels of tumor-associated monocytes and / or tumor-associated macrophages can be used as a biomarker to select patients who are likely to benefit from the combination therapy. This synergistic effect may be strongest in combination therapy with a PD-1 checkpoint inhibitor compared to CTLA4 or PD-L1 specific checkpoint inhibitors.

[0222] Without wishing to be bound by theory, it is believed that PD-1 is present in M2-type tumor-associated macrophages, and antisense agents for inhibiting or suppressing TGF-β2 expression are effective in repolarizing M2 and promoting anti-tumor effects. Therefore, when used in combination with IL-2, these two agents may function synergistically against the same target. TGF-β2 may play a central role in programming M1-type tumor-associated macrophages, which can exhibit anti-tumor effects. In some aspects of the present invention, inhibition or suppression of TGF-β2 by antisense agents can have an anti-tumor effect. Antisense agents may be effective in reprogramming tumor-associated macrophages to promote M1-type tumor-associated macrophages. Such reprogramming, particularly when combined with agents that counteract high TGF-β2 levels, may be effective in actively reducing and / or eliminating cancer tumors.

[0223] To ensure unbiased and reasonable stratification, the median of the population data is defined as the cutoff. For each gene of interest, the median expression level is calculated across all samples. The median is the middle value of the list of numbers sorted in ascending or descending order, and is used because it is less affected by outliers than the mean. Then, samples are stratified into two groups based on whether the expression level of a specific gene is above or below the median. This creates a "high expression" group and a "low expression" group.

[0224] As used herein, "immunogenically hot tumor" refers to a type of cancer that provokes a strong response from a patient's immune system. Conversely, a "cold" tumor is less immunogenic, meaning that it does not elicit a strong immune response.

[0225] In some embodiments, the agents, uses or methods of the invention may be applied to immunogenic cold pancreatic cancer or immunogenic hot melanoma.

[0226] In some aspects, the agents, uses or methods of the present invention may be applied to cancers that are between immunogenic cold and immunogenic hot, which may be skin cancer, lung cancer, breast cancer, prostate cancer, colorectal cancer, kidney cancer, stomach cancer, ovarian cancer, cervical cancer, liver cancer or multiple myeloma.

[0227] Numbered aspects of the present invention include the following.

[0228] 1) An antisense agent for inhibiting or suppressing the expression of TGF-β2 in combination with a checkpoint inhibitor for use in treating or ameliorating symptoms of cancer in a human subject or animal.

[0229] 2) Use of an antisense agent for inhibiting or suppressing the expression of TGF-β2 in the preparation of a medicament for treating or ameliorating symptoms of cancer in a human subject or animal in combination with a checkpoint inhibitor.

[0230] 3) A method for treating or ameliorating symptoms of cancer in a human or animal subject in need thereof, comprising: administering to the subject a therapeutically effective amount of an antisense agent for inhibiting or suppressing expression of TGF-β2; administering to said subject a therapeutically effective amount of a checkpoint inhibitor. A method comprising:

[0231] 4) The agent of embodiment 1 in combination with an interleukin immunotherapeutic agent.

[0232] 5) The use of embodiment 2 in combination with an interleukin immunotherapeutic agent.

[0233] 6) The method of embodiment 3, comprising administering to said subject a therapeutically effective amount of an interleukin immunotherapeutic agent.

[0234] 7) The agent, use, or method of any of aspects 1 to 6, wherein the agent for inhibiting or suppressing expression of TGF-β2, the checkpoint inhibitor, and the interleukin immunotherapeutic agent are administered in parallel, simultaneously, sequentially, or temporally separately.

[0235] 8) The agent, use, or method of any of aspects 1 to 7, wherein the agent for inhibiting or suppressing expression of TGF-β2, the checkpoint inhibitor, and the interleukin immunotherapeutic agent are administered by injection or infusion, either separately or in a combined formulation.

[0236] 9) The agent, use, or method of any one of aspects 1 to 8, wherein the cancer is pancreatic cancer, melanoma, skin cancer, lung cancer, breast cancer, prostate cancer, colorectal cancer, kidney cancer, gastric cancer, ovarian cancer, cervical cancer, liver cancer, or multiple myeloma.

[0237] 10) The agent, use, or method of any one of aspects 1 to 9, wherein the agent for inhibiting or suppressing expression of TGF-β2 is a TGF-β2-specific antisense oligonucleotide that is complementary to a TGF-β2 transcription product and has a length of 15 to 30 nucleotides.

[0238] 11) The agent, use, or method of any one of aspects 1 to 10, wherein the agent for inhibiting or suppressing expression of TGF-β2 is a TGF-β2-specific antisense oligonucleotide that is complementary to a TGF-β2 precursor RNA, precursor mRNA, or mRNA and is 18 to 21 nucleotides in length.

[0239] 12) The agent, use, or method of any of aspects 1 to 11, wherein the agent for inhibiting or suppressing expression of TGF-β2 is one or more TGF-β2-specific antisense oligonucleotides shown in Table 1 that are complementary to a TGF-β2 transcript, as well as chemically modified variants thereof, LNA variants thereof, gapmer variants thereof, and any combination or pool thereof.

[0240] 13) The agent, use, or method of any one of aspects 1 to 12, wherein the TGF-β2-specific antisense oligonucleotide has no more than one or two mismatches compared to the target human TGF-β2.

[0241] 14) The agent, use, or method of any of aspects 1 to 13, wherein the TGF-β2-specific antisense oligonucleotide reduces TGF-β2 transcript levels by at least 60%, or at least 70%, or at least 80%, or at least 90%.

[0242] 15) The agent, use, or method of any of aspects 1 to 14, wherein the TGF-β2-specific antisense oligonucleotide reduces any TGF-β1 transcript levels and any TGF-β3 transcript levels by less than 10%, or less than 5%, or less than 1%.

[0243] 16) The agent, use, or method of any one of aspects 1 to 15, wherein the TGF-β2-specific antisense oligonucleotide has one or more nucleotides chemically modified as a phosphorothioate internucleoside linkage, a methoxypropylphosphonate internucleoside linkage, an aminophosphoro linkage to a morpholino group, a 2'-OMe ribose group, a 2'-MOE methoxyethyl ribose group, a 2'-4' constrained methoxyethyl bicyclic ribose group, a 2'-4' constrained ethyl bicyclic ribose group, an LNA ribose group, a 2'-F ribose group, or a 5-methylcytosine base.

[0244] 17) The agent, use, or method of any of embodiments 1-16, wherein the antisense agent is conjugated to polyethylene glycol, a lipid, or triantenarry N-acetyl-galactosamine.

[0245] 18) The agent, use, or method of any of aspects 1-17, wherein each agent comprises a carrier that is sterile water for injection, saline, isotonic saline, or a combination thereof, which may be the same or different for each agent.

[0246] 19) The agent, use, or method of any one of aspects 1 to 18, wherein the agent is substantially free of excipients.

[0247] 20) The agent, use, or method of any one of aspects 1 to 19, wherein the agent is stable for at least 14 days at 37°C in a carrier that is substantially free of excipients.

[0248] 21) The agent, use, or method of any one of aspects 1 to 20, wherein the checkpoint inhibitor is an inhibitor of PD-1.

[0249] 22) The agent, use, or method of any one of aspects 1 to 21, wherein the checkpoint inhibitor is pembrolizumab, nivolumab, cemiplimab, spartalizumab, atezolizumab, avelumab, or durvalumab.

[0250] 23) The agent, use, or method of any one of aspects 1 to 22, wherein the interleukin immunotherapeutic agent is native IL-2, high-dose IL-2, recombinant IL-2, or aldesleukin.

[0251] 24) The agent, use, or method of any of embodiments 1 to 23, comprising selecting a subject that will benefit from said agent, use, or method based on levels of one or more biomarkers: TGF-β2, IL-2, CD19, IRF5, ITGAM, and combinations thereof.

[0252] 25) The agent, use, or method of any one of aspects 1 to 24, wherein the one or more biomarkers is IRF5 and the subject is selected if IRF5 expression is at a level above the median.

[0253] 26) The agent, use, or method of any one of aspects 1 to 25, wherein the one or more biomarkers are ITGAM and the subject is selected if expression of ITGAM is above the median level.

[0254] 27) The agent, use, or method of any one of aspects 1 to 26, wherein the subject after said administration or use has a reduced level of TGF-β2 compared to before said administration or use.

[0255] 28) The agent, use, or method of any one of aspects 1 to 27, wherein the subject after said administration or use has an increased level of IRF5 compared to before said administration or use.

[0256] 29) The agent, use, or method of any one of aspects 1 to 28, wherein the subject after said administration or use has a reduced level of ITGAM compared to before said administration or use.

[0257] 30) The agent, use, or method of any one of aspects 1 to 29, comprising administering to the subject a therapeutically effective amount of an expression product of IRF5 or ITGAM.

[0258] 31) The agent, use, or method of any one of aspects 1 to 30, wherein the expression product is an mRNA, a polypeptide, a protein, or a fragment thereof, or a combination thereof.

[0259] 32) The agent, use, or method of any one of aspects 1 to 31, wherein said administration or use reduces mortality at 6, 12, 18, 24, 30, or 36 months.

[0260] 33) The agent, use, or method of any one of aspects 1 to 32, wherein said administration or use improves overall survival at 6, 12, 18, 24, 30, or 36 months.

[0261] 34) The agent, use, or method of any of aspects 1-33 in combination with any one or more medicaments including targeted anti-cancer agents, cancer growth blocking agents, EGFR inhibitors, and combinations thereof.

[0262] 35) The agent, use, or method of any one of aspects 1 to 34 in combination with any one or more pharmaceutical agents selected from bevacizumab, everolimus, velzutifan, dabrafenib, trametinib, and combinations thereof.

[0263] 36) The agent, use, or method of any of aspects 1-35 in combination with any one or more pharmaceutical agents that are cancer growth inhibitors selected from angiogenesis inhibitors, histone deacetylase inhibitors, hedgehog blockers, mTOR inhibitors, p53 inhibitors, PARP inhibitors, proteasome inhibitors, tyrosine kinase inhibitors, and combinations thereof.

[0264] 37) The agent, use, or method of any of aspects 1-36 in combination with any one or more pharmaceutical agents that are EGFR inhibitors selected from erlotinib, gefitinib, afatinib, osimertinib, dacomitinib, and combinations thereof.

[0265] 38) The agent, use, or method of any one of aspects 1 to 37 in combination with a chemotherapeutic agent.

[0266] 39) The agent, use, or method of any of aspects 1 to 38 in combination with radiation therapy or electric field therapy.

[0267] 40) An antisense agent for inhibiting or suppressing the expression of TGF-β2 in combination with an interleukin immunotherapeutic agent for use in treating or ameliorating symptoms of cancer in a human subject or animal.

[0268] 41) Use of an antisense agent for inhibiting or suppressing the expression of TGF-β2 in the preparation of a medicament for treating or ameliorating symptoms of cancer in a human subject or animal in combination with an interleukin immunotherapeutic agent.

[0269] 42) A method for treating or ameliorating symptoms of cancer in a human or animal subject in need thereof, comprising: administering to the subject a therapeutically effective amount of an antisense agent for inhibiting or suppressing expression of TGF-β2; administering to said subject a therapeutically effective amount of an interleukin immunotherapeutic agent. A method comprising:

[0270] 43) The agent, use, or method of any of aspects 40 to 42, wherein the agent for inhibiting or suppressing expression of TGF-β2 and the interleukin immunotherapeutic agent are administered in parallel, simultaneously, sequentially, or temporally separately.

[0271] 44) The agent, use, or method of any one of aspects 40 to 43, wherein the agent for inhibiting or suppressing expression of TGF-β2 and the interleukin immunotherapeutic agent are administered by injection or infusion, either separately or in a combined formulation.

[0272] 45) The agent, use, or method of any one of aspects 40 to 44, wherein the cancer is pancreatic cancer, melanoma, skin cancer, lung cancer, breast cancer, prostate cancer, colorectal cancer, kidney cancer, gastric cancer, ovarian cancer, cervical cancer, liver cancer, or multiple myeloma.

[0273] 46) The agent, use, or method of any one of aspects 40 to 45, wherein the agent for inhibiting or suppressing expression of TGF-β2 is a TGF-β2-specific antisense oligonucleotide that is complementary to a TGF-β2 transcript and is 15 to 30 nucleotides in length.

[0274] 47) The agent, use, or method of any of aspects 40 to 46, wherein the agent for inhibiting or suppressing expression of TGF-β2 is a TGF-β2-specific antisense oligonucleotide that is complementary to a TGF-β2 precursor RNA, precursor mRNA, or mRNA and is 18 to 21 nucleotides in length.

[0275] 48) The agent, use, or method of any one of aspects 40 to 47, wherein the agent for inhibiting or suppressing expression of TGF-β2 is one or more TGF-β2-specific antisense oligonucleotides shown in Table 1 that are complementary to a TGF-β2 transcription product.

[0276] 49) The agent, use, or method of any of aspects 40-48, wherein the TGF-β2-specific antisense oligonucleotide has one or more nucleotides chemically modified as a phosphorothioate internucleoside linkage, a methoxypropylphosphonate internucleoside linkage, an aminophosphoro linkage to a morpholino group, a 2'-OMe ribose group, a 2'-MOE methoxyethyl ribose group, a 2'-4' constrained methoxyethyl bicyclic ribose group, a 2'-4' constrained ethyl bicyclic ribose group, an LNA ribose group, a 2'-F ribose group, or a 5-methylcytosine base.

[0277] 50) The agent, use, or method of any of embodiments 40-49, wherein the antisense agent is conjugated to polyethylene glycol, a lipid, or a triple-arm N-acetyl-galactosamine.

[0278] 51) The agent, use, or method of any of aspects 40-50, wherein each agent comprises a carrier that is sterile water for injection, saline, isotonic saline, or a combination thereof, which may be the same or different for each agent.

[0279] 52) The agent, use, or method of any one of aspects 40-51, wherein the agent is substantially free of excipients.

[0280] 53) The agent, use, or method of any of aspects 40-52, wherein the agent is stable for at least 14 days at 37°C in a carrier that is substantially free of excipients.

[0281] 54) The agent, use, or method of any one of aspects 40 to 53, wherein the interleukin immunotherapeutic agent is native IL-2, high-dose IL-2, recombinant IL-2, or aldesleukin.

[0282] 55) The agent, use, or method of any of aspects 40-54, wherein the administration or use reduces mortality at 6, 12, 18, 24, 30, or 36 months.

[0283] 56) The agent, use, or method of any one of aspects 40 to 55, wherein the administration or use improves survival at 6, 12, 18, 24, 30, or 36 months.

[0284] 57) The agent, use, or method of any of aspects 40-56 in combination with any one or more medicaments including targeted anti-cancer agents, cancer growth blocking agents, EGFR inhibitors, and combinations thereof.

[0285] 58) The agent, use, or method of any of aspects 40 to 57, in combination with any one or more pharmaceutical agents selected from bevacizumab, everolimus, velzutifan, dabrafenib, trametinib, and combinations thereof.

[0286] 59) The agent, use, or method of any of aspects 40-58 in combination with any one or more pharmaceutical agents that are cancer growth inhibitors selected from angiogenesis inhibitors, histone deacetylase inhibitors, hedgehog blockers, mTOR inhibitors, p53 inhibitors, PARP inhibitors, proteasome inhibitors, tyrosine kinase inhibitors, and combinations thereof.

[0287] 60) The agent, use, or method of any of aspects 40-59 in combination with any one or more pharmaceutical agents that are EGFR inhibitors selected from erlotinib, gefitinib, afatinib, osimertinib, dacomitinib, and combinations thereof.

[0288] 61) The agent, use, or method of any of aspects 40-60 in combination with a chemotherapeutic agent.

[0289] 62) The agent, use, or method of any of aspects 40-61 in combination with radiation therapy or electric field therapy.

[0290] 63) The agent, use, or method of any of embodiments 40-62, comprising selecting a subject who will benefit from said agent, use, or method based on the level of one or more biomarkers: TGF-β2, IRF5, ITGAM, and combinations thereof.

[0291] 64) The agent, use, or method of any of aspects 40-63, comprising selecting a subject who will benefit from said agent, use, or method based on levels of one or more biomarkers: TGF-β2, IRF5, ITGAM, neoantigens, mutational load, macrophage, and combinations thereof.

[0292] 65) The agent, use, or method of any one of aspects 40 to 64, wherein the one or more biomarkers is IRF5 and the subject is selected if expression of IRF5 is below the median level.

[0293] 66) The agent, use, or method of any one of aspects 40 to 65, wherein the one or more biomarkers are tumor-associated macrophages, and the subject is selected if tumor-associated macrophages are below average.

[0294] 67) The agent, use, or method of any one of aspects 40 to 66, wherein the one or more biomarkers is tumor neoantigen mutational burden, and the subject is selected if the neoantigen tumor burden is below average.

[0295] 68) The agent, use, or method of any of aspects 40 to 67, comprising administering to the subject a therapeutically effective amount of an agent for inhibiting or suppressing expression of ITGAM or IRF5.

[0296] 69) The agent, use, or method of any one of aspects 40 to 68, wherein the agent for inhibiting or suppressing the expression of ITGAM or IRF5 is an antisense oligonucleotide targeting ITGAM or IRF5, respectively.

[0297] 70) A kit for treating or ameliorating symptoms of cancer, comprising: a therapeutically effective amount of an antisense agent for inhibiting or suppressing the expression of TGF-β2; and Therapeutically effective amounts of checkpoint inhibitors Includes a kit.

[0298] 71) The kit of embodiment 70, comprising a therapeutically effective amount of an interleukin immunotherapeutic agent.

[0299] 72) The kit of any one of aspects 70 to 71, wherein the cancer is pancreatic cancer, melanoma, skin cancer, lung cancer, breast cancer, prostate cancer, colorectal cancer, kidney cancer, gastric cancer, ovarian cancer, cervical cancer, liver cancer, thymic cancer, or multiple myeloma.

[0300] 73) The kit of any one of aspects 70 to 72, wherein the agent for inhibiting or suppressing expression of TGF-β2 is a TGF-β2-specific antisense oligonucleotide that is complementary to a TGF-β2 transcription product and has a length of 15 to 30 nucleotides.

[0301] 74) The kit of any one of aspects 70 to 73, wherein the agent for inhibiting or suppressing expression of TGF-β2 is a TGF-β2-specific antisense oligonucleotide that is complementary to a TGF-β2 precursor RNA, precursor mRNA, or mRNA and is 18 to 21 nucleotides in length.

[0302] 75) The kit of any one of aspects 70 to 74, wherein the agent for inhibiting or suppressing expression of TGF-β2 is one or more TGF-β2-specific antisense oligonucleotides shown in Table 1 that are complementary to a TGF-β2 transcription product.

[0303] 76) The kit of any of aspects 70 to 75, wherein the TGF-β2-specific antisense oligonucleotide has no more than one or two mismatches compared to the target human TGF-β2.

[0304] 77) The kit of any of aspects 70-76, wherein the TGF-β2-specific antisense oligonucleotide reduces TGF-β2 transcript levels by at least 60%, or at least 70%, or at least 80%, or at least 90%.

[0305] 78) The kit of any of aspects 70-77, wherein the TGF-β2-specific antisense oligonucleotide reduces any TGF-β1 transcript levels and any TGF-β3 transcript levels by less than 10%, or less than 5%, or less than 1%.

[0306] 79) The kit of any of aspects 70-78, wherein the TGF-β2-specific antisense oligonucleotide has one or more nucleotides chemically modified as a phosphorothioate internucleoside linkage, a methoxypropylphosphonate internucleoside linkage, an aminophosphoro linkage to a morpholino group, a 2'-OMe ribose group, a 2'-MOE methoxyethyl ribose group, a 2'-4' constrained methoxyethyl bicyclic ribose group, a 2'-4' constrained ethyl bicyclic ribose group, an LNA ribose group, a 2'-F ribose group, or a 5-methylcytosine base.

[0307] 80) The kit of any of aspects 70-79, wherein the antisense agent is conjugated to polyethylene glycol, a lipid, or a triple-arm N-acetyl-galactosamine.

[0308] 81) The kit of any of aspects 70-80, wherein each agent comprises a carrier that is sterile water for injection, saline, isotonic saline, or a combination thereof, which may be the same or different for each agent.

[0309] 82) The kit of any one of aspects 70-81, wherein the agent is substantially free of excipients.

[0310] 83) The kit of any of aspects 70-82, wherein the agent is stable for at least 14 days at 37°C in a carrier that is substantially free of excipients.

[0311] All publications, including patents, published patent applications, and non-patent publications, and the sequence listing, referred to in this description are each expressly incorporated herein by reference in their entirety for all purposes.

[0312] Although the foregoing disclosure has been described in detail by way of example for purposes of clarity of understanding, it will be apparent to those skilled in the art that certain changes and modifications are included in this disclosure and can be made without undue experimentation within the scope of the appended claims, which are presented by way of example and not limitation. The present invention includes all such additional embodiments, equivalents, and modifications. The present invention includes any combination or mixture of the features, materials, elements, or limitations of the various exemplary components, examples, and claimed embodiments.

[0313] It is emphasized herein that, according to common practice, the features of the drawings are of any scale and are intended to cover similar features which may be arbitrarily enlarged or reduced. [Example]

[0314] Example 1. A clinical study was conducted to understand the overall survival of melanoma patients with a therapeutic combination of an antisense TGF-β2 inhibitor, an IL-2 immunotherapy agent, and a PD-1 checkpoint inhibitor. Clinical results were obtained from 423 patients with a diagnosis of melanoma cancer (see KM plotter for immunotherapy, available at https: / / kmplot.com / analysis / index.php?p=service&cancer=immunotherapy).

[0315] Figure 1 shows the results of this clinical study on overall survival for melanoma patients. Figure 1 also shows a Kaplan-Meier overall survival chart for this clinical efficacy study. Figure 1 demonstrates that the use of PD-1 inhibitors improved survival in the high IL2 (left panel), which can be achieved with IL-2 immunotherapy, and in the low TGF-β2 (right panel), which can be achieved with antisense TGF-β2 inhibitors. These clinical data support the rationale for the therapeutic combination of antisense TGF-β2 inhibitors, PD-1 checkpoint inhibitors, and IL-2 immunotherapy to improve overall survival in melanoma patients. The log-rank P value in Figure 1 indicates a high degree of significance for the improved survival observed with this therapeutic combination based on this clinical study.

[0316] Figure 2 shows the results of a clinical study on overall survival for melanoma patients. Figure 2 shows a Kaplan-Meier overall survival chart for such a clinical efficacy study. Figure 2 shows that the use of PD-1 inhibitors strongly improved survival when the IL-2 / TGF-β2 ratio, which can be achieved with IL-2 immunotherapy and antisense TGF-β2 inhibitors, was higher (left panel). These clinical data provide a rationale for the therapeutic combination of antisense TGF-β2 inhibitors, PD-1 checkpoint inhibitors, and IL-2 immunotherapy to improve overall survival in melanoma patients. The log-rank P value in Figure 2 (left panel) indicates a high degree of significance for the improved survival observed with this therapeutic combination based on this clinical study.

[0317] The clinical data in Figure 2 demonstrate that a high IL-2 / TGF-β2 ratio, corresponding to high IL-2 and low TGF-β2, is a strong driving factor for survival in melanoma patients. A high IL-2 / TGF-β2 ratio dramatically increased survival in the top quartile of melanoma patients from 6.7 months to 15 months (p=5.4e-06) (Figure 2, left panel). Thus, these clinical data provide evidence for the remarkable synergistic effect of the therapeutic combination of an antisense TGF-β2 inhibitor, a PD-1 checkpoint inhibitor, and an IL-2 immunotherapy agent to improve overall survival in melanoma patients.

[0318] Example 2. A clinical study was conducted to understand overall survival in melanoma (N=162) and pancreatic cancer (N=88) patients using a therapeutic combination of an antisense TGF-β2 inhibitor, an IL-2 immunotherapy agent, and a PD-1 checkpoint inhibitor. (See KM Plotter for immunotherapy, available at https: / / kmplot.com / analysis / index.php?p=service&cancer=immunotherapy) and pancreatic cancer (N=177) (KM Plotter pan-cancer RNA-seq, available at https: / / kmplot.com / analysis / index.php?p=service&cancer=pancancer_rnaseq).

[0319] Figures 3-18 show the results of this clinical study regarding overall survival for melanoma patients treated with PD-1 therapeutics and for untreated pancreatic cancer patients. Figures 3-18 show Kaplan-Meier overall survival charts for such clinical efficacy studies, covering the two extremes of hot immunogenic melanoma tumors and cold non-immunogenic pancreatic tumors.

[0320] Figures 3, 5, 7, 9, 11, 13, 15, and 17 (upper left and right panels) show Kaplan-Meier overall survival charts for melanoma patients in this study. Figures 3, 5, 7, 9, 11, 13, 15, and 17 (lower left and right panels) show Kaplan-Meier overall survival charts for pancreatic cancer (PDAC) patients in this study, measured in terms of TGF-β2 expression. Figures 4, 6, 8, 10, 12, 14, 16, and 18 (left and right panels) show Kaplan-Meier overall survival charts for pancreatic cancer (PDAC) patients in this study, measured in terms of IL-2 expression.

[0321] Figure 3 (upper left panel) shows that low TGF-β2 and high ITGAM levels were associated with improved survival in melanoma patients treated with PD-1 inhibitors. These data suggest that high ITGAM levels are a useful biomarker for overall patient survival and can be used to select patients likely to benefit from the therapeutic combination of antisense TGF-β2 inhibitors and PD-1 checkpoint inhibitors. Figure 3 (upper right panel) shows that low ITGAM levels are not considered favorable under these conditions.

[0322] Figure 3 (lower left and right panels) shows that neither low nor high ITGAM is considered a favorable indicator for the use of PD-1 inhibitors in pancreatic cancer patients (PDAC).

[0323] Figure 4 (left panel) shows that low TGF-β2 and high ITGAM levels were associated with improved survival in patients with pancreatic cancer (PDAC) treated with PD-1 inhibitors. A log-rank value of P = 0.034 indicates significance (<0.05), but lower P values ​​are preferred. These data suggest that high ITGAM levels are a useful biomarker for overall patient survival when treated with a therapeutic combination of antisense TGF-β2 inhibitors and PD-1 checkpoint inhibitors and can be used to select patients likely to benefit. Figure 4 (right panel) shows that low ITGAM levels are not considered a favorable indicator under these conditions.

[0324] Figure 5 (upper left panel) shows that neither low nor high CD8A appears to be a favorable indicator for the use of PD-1 inhibitors in melanoma patients.

[0325] Figure 5 (lower left and right panels) shows that neither low nor high CD8A levels appear to be favorable indicators for the use of PD-1 inhibitors in pancreatic cancer patients (PDAC).

[0326] Similar analysis of the data in Figures 3-18 leads to conclusions summarized in Table 3. Table 3. Biomarkers with favorable significance for melanoma and PDAC TIFF2025535604000008.tif38170

[0327] The data can be summarized for melanoma treated with PD-1 as follows: CD19 and IRF5 were highly significant for TGF-β2-induced improved overall survival outcomes compared to melanoma treated with PD-1 alone.

[0328] The data can be summarized for PDAC as follows: IRF5-low and CD19-low were significant for improved outcome in TGF-β2-induced overall survival. With the exception of ITGAM, the impact of these biomarkers on IL-2-induced overall survival in PDAC was not significant.

[0329] Thus, the first finding of this clinical study was that ITGAM, CD19, IRF5, and CD163 are useful biomarkers for overall survival in melanoma patients treated with the therapeutic combination of an antisense TGF-β2 inhibitor and a PD-1 checkpoint inhibitor.

[0330] The second finding of this clinical study was that IRF5 is a useful biomarker for overall survival in patients with pancreatic cancer (PDAC) when therapeutic antisense TGF-β2-specific inhibitors are used.

[0331] The third finding of this clinical study was that ITGAM may be a useful biomarker for overall survival in patients with pancreatic cancer (PDAC) when using the therapeutic combination of IL-2 therapy and PD-1 checkpoint inhibitors.

[0332] Another finding of this clinical study was that IRF5 is particularly useful as a biomarker for overall survival in melanoma patients when therapeutic antisense TGF-β2-specific inhibitors are used.

[0333] Another finding of this clinical study was that IRF5 is particularly useful as a biomarker for overall survival in patients with pancreatic cancer (PDAC) when using the therapeutic combination of an antisense TGF-β2 inhibitor and a PD-1 checkpoint inhibitor.

[0334] This clinical study involves the following cell types:

[0335] B cells = CD19

[0336] CD8+ T cells = CD8A

[0337] CD4 + T cells = CD4

[0338] M1 macrophages = NOS2 or IRF5

[0339] M2 macrophages = CD163

[0340] Neutrophil=ITGAM

[0341] Dendritic cells = ITGAX

[0342] Example 3. A clinical study was conducted to understand the overall survival of patients with pancreatic cancer (PDAC) using a therapeutic combination of an antisense TGF-β2 inhibitor, an IL-2 immunotherapy agent, and a PD-1 checkpoint inhibitor.

[0343] We conducted a study to understand overall survival in melanoma (see KM Plotter for Immunotherapy, available at https: / / kmplot.com / analysis / index.php?p=service&cancer=immunotherapy) and pancreatic cancer (N=177) (KM Plotter pan-cancer RNA-seq, https: / / kmplot.com / analysis / index.php?p=service&cancer=pancancer_rnaseq) treated with immunotherapy (N=423).

[0344] Figures 19-20 show the results of this clinical study regarding overall survival of patients with PDAC. Figures 19-20 show Kaplan-Meier overall survival charts for such a clinical efficacy study.

[0345] Figures 19-20 show that IL-2-induced survival was further stratified using various immune cell indicators, namely, basophils, B cells, eosinophils, and MO macrophages, with Th1 helper cells as the basis for the analysis. High levels of IL-2 significantly prolonged survival in all cases. MO macrophages were a highly significant factor. Figures 19-20 show that the log-rank P values ​​indicated significance for improved survival when using IL-2 immunotherapy in combination with PD-1 checkpoint inhibitors based on this clinical study and condition. Figure 20 (upper right panel and lower panel) shows comparative baseline results.

[0346] This clinical study in pancreatic cancer (PDAC) demonstrated the synergistic effect of low macrophages on IL-2 immunotherapy in PDAC.

[0347] Example 4. A clinical study was conducted to understand the overall survival of pancreatic cancer patients with therapeutic antisense TGF-β2 inhibitors. Clinical results were obtained from 80-108 patients with a diagnosis of pancreatic cancer (cBioPortal for Cancer Genomics).

[0348] Figure 21 shows the results of this clinical study on overall survival for pancreatic cancer patients. Figure 21 shows a Kaplan-Meier overall survival chart for such a clinical efficacy study. Figure 21 (upper left panel) shows that for patients with low tumor-associated macrophages, high tumor mRNA levels of TGF-β2 significantly shortened survival. The log-rank P value in Figure 21 (upper left panel) indicates a high significance of improved survival with the use of therapeutic antisense TGF-β2 inhibitors based on this clinical study and condition. Patients whose tumors had a high range of TGF-β2 expression survived for only 15 months compared with 73 months for patients whose tumors had a low range of TGF-β2 expression.

[0349] Figure 21 (upper right panel) shows that for patients with low tumor-associated macrophages and low mutational burden (neoantigens), high tumor mRNA levels of TGF-β2 significantly shortened survival. The log-rank P value in Figure 21 (upper right panel) indicates a high significance of improved survival with the use of therapeutic antisense TGF-β2 inhibitors based on this clinical study and condition. Patients whose tumors had high TGF-β2 expression in the low TGF-β2 expression range survived for only 15 months, compared with 73 months for patients whose tumors had low TGF-β2 expression in the low range. Only TGF-β2 was shown to have an effect on survival. Neither TGF-β1 nor TGF-β3 had an effect on survival (log-rank P, see Figure 21 (lower left and lower right panels, respectively)).

[0350] Thus, this clinical study demonstrated that tumor mRNA levels of TGF-β2, low tumor-associated macrophages, and low mutational burden (neoantigens) are biomarkers for improved overall survival in pancreatic cancer patients with therapeutic antisense TGF-β2 inhibitors. These biomarkers can be used to select patients who will benefit from treatment with TGF-β2 inhibitors.

[0351] Example 5. A clinical study was conducted to understand the overall survival of cancer patients using a therapeutic combination of an antisense TGF-β2 inhibitor, an IL-2 immunotherapy agent, and a checkpoint inhibitor. Clinical results were obtained for 1,045 patients with cancer diagnoses (cBioPortal for Cancer Genomics). Clinical results were obtained for a variety of cancer diagnoses, including bladder (N=73), esophageal adenocarcinoma (N=103), glioblastoma (N=28), hepatocellular carcinoma (N=22), HNSCC (N=5), melanoma (N=423), NSCLC (N=21), NSCL (N=22), and urothelial (N=348).

[0352] Figures 22-30 show the results of this clinical study on overall survival of cancer patients. Figures 22-30 show Kaplan-Meier overall survival charts for such a clinical efficacy study.

[0353] Figure 22 shows that high levels of IL-2 significantly extended survival across all tumor types when using any checkpoint inhibitor (PD-1, PD-L1, or CTLA-4). The log-rank P-values ​​in Figure 22 indicate the significance of improved survival with IL-2 immunotherapy based on this clinical study and setting. Patients whose tumors had a high range of IL-2 expression survived for 18 months compared to 14 months for patients whose tumors had a low range of TGF-β2 expression.

[0354] Figure 23 shows that low levels of TGF-β2 slightly improved survival when using checkpoint inhibitors: patients in the low TGF-β2 expression range survived for 18 months compared to 15 months for patients in the high TGF-β2 expression range.

[0355] Importantly, Figure 24 shows that the combination of high levels of IL-2 and low levels of TGF-β2 significantly extended survival when using checkpoint inhibitors. Figure 24 shows overall survival stratified by IL2 / TGFB2 expression ratio. The log-rank P value in Figure 24 indicates the high significance of the improved survival when using the therapeutic combination of an antisense TGF-β2 inhibitor, an IL-2 immunotherapy, and a checkpoint inhibitor based on this clinical study and condition. Patients in the high IL2 / TGFB2 expression range survived for 20 months, compared with only 14 months for patients in the low IL2 / TGFB2 expression range. This clinical data demonstrates the synergistic effect of the therapeutic combination of an antisense TGF-β2 inhibitor, an IL-2 immunotherapy, and a checkpoint inhibitor.

[0356] The synergistic effect of the therapeutic combination of an antisense TGF-β2 inhibitor, an IL-2 immunotherapeutic agent, and a checkpoint inhibitor was found to be significantly increased individually for melanoma. For 423 melanoma patients, Figure 25 shows that high levels of IL-2 significantly prolonged survival when using checkpoint inhibitors. Patients in the high IL-2 expression range survived for 33 months compared to 21 months for patients in the low IL-2 expression range.

[0357] For 423 melanoma patients, Figure 26 shows that low levels of TGF-β2 significantly prolonged survival when using checkpoint inhibitors: patients in the low TGF-β2 expression range survived for 29 months compared to 18 months for patients in the high TGF-β2 expression range.

[0358] Importantly, Figure 27 shows that the combination of high levels of IL-2 and low levels of TGF-β2 significantly extended survival in 423 melanoma patients when using checkpoint inhibitors. Figure 24 shows overall survival stratified by the IL2 / TGFB2 expression ratio. The log-rank P value in Figure 24 indicates an unexpectedly high significance for the improved survival achieved with the therapeutic combination of an antisense TGF-β2 inhibitor, an IL-2 immunotherapy, and a checkpoint inhibitor, based on this clinical study and condition. Patients in the high IL2 / TGFB2 expression range survived for 20 months, compared with only 14 months for patients in the low IL2 / TGFB2 expression range. This clinical data demonstrates the synergistic effect of the therapeutic combination of an antisense TGF-β2 inhibitor, an IL-2 immunotherapy, and a checkpoint inhibitor.

[0359] In cancer patients, the synergistic effect of the therapeutic combination of antisense TGF-β2 inhibitors and IL-2 immunotherapeutic agents with checkpoint inhibitors was found to be significantly increased individually with respect to PD-1 checkpoint inhibitors.

[0360] For all cancer patients, Figure 28 shows that high levels of IL-2 significantly prolonged survival when using PD-1 checkpoint inhibitors: patients in the high IL-2 expression range survived 28 months compared to 17 months for patients in the low IL-2 expression range.

[0361] For cancer patients, Figure 29 shows that low levels of TGF-β2 significantly prolonged survival when using PD-1 checkpoint inhibitors: patients in the low TGF-β2 expression range survived 28 months compared to 16 months for patients in the high TGF-β2 expression range.

[0362] Importantly, Figure 30 shows that the combination of high levels of IL-2 and low levels of TGF-β2 significantly extended survival in cancer patients when using a PD-1 checkpoint inhibitor. Figure 30 shows overall survival stratified by the IL2 / TGFB2 expression ratio. The log-rank P value in Figure 30 indicates an unexpectedly high significance for the improved survival observed when using the therapeutic combination of an antisense TGF-β2 inhibitor, an IL-2 immunotherapy, and a PD-1 checkpoint inhibitor, based on this clinical study and condition. Patients in the high IL2 / TGFB2 expression range survived for 31 months compared with 14 months for patients in the low IL2 / TGFB2 expression range. This clinical data demonstrates the synergistic effect of the therapeutic combination of an antisense TGF-β2 inhibitor, an IL-2 immunotherapy, and a PD-1 checkpoint inhibitor.

[0363] Example 6. A clinical study was conducted to understand overall survival in cancer patients using a therapeutic combination of an antisense TGF-β2 inhibitor and an IL-2 immunotherapeutic agent and a checkpoint inhibitor. (A clinical study was conducted to understand overall survival in immunotherapy-treated melanoma (N=423) (see KM Plotter for Immunotherapy, available at https: / / kmplot.com / analysis / index.php?p=service&cancer=immunotherapy) and pancreatic cancer (N=177) (KM Plotter pan-cancer RNA-seq, https: / / kmplot.com / analysis / index.php?p=service&cancer=pancancer_rnaseq).

[0364] This clinical study demonstrated that overall survival was not improved when the therapeutic combination of an antisense TGF-β2 inhibitor, an IL-2 immunotherapeutic agent, and a checkpoint inhibitor was used, when the checkpoint inhibitor was CTLA-4 or PD-L1. Thus, in other examples herein, the improvement in overall survival when the therapeutic combination of an antisense TGF-β2 inhibitor, an IL-2 immunotherapeutic agent, and a PD-1 checkpoint inhibitor was surprisingly favorable. The data are detailed below.

[0365] Figures 31-33 show the results for three stratification biomarkers, namely, PDCD1, CD274, and IL-2 / TGF-β2 ratio, for any checkpoint inhibitor (PD-1, CTLA-4, or PD-L1) and all tumor types (N=976). High expression levels of each biomarker demonstrated improved overall survival.

[0366] Figures 34-36 show the results of three stratification biomarkers, namely, PDCD1, CD274, and IL-2 / TGF-β2 ratio, for PD-L1 checkpoint inhibitors and all tumor types (N=955). With the exception of IL-2 / TGF-β2, each biomarker showed improved overall survival at high expression levels. Therefore, PD-L1 was not the preferred checkpoint inhibitor.

[0367] Figures 37-39 show the results of three stratification biomarkers, namely, PDCD1, CD274, and IL-2 / TGF-β2 ratio, for CTLA-4 checkpoint inhibitors and all tumor types (N=121). Only the CD274 biomarker showed a slight improvement in overall survival at high expression levels. Therefore, CTLA-4 was not the preferred checkpoint inhibitor.

[0368] Figures 40-42 show the results of three stratification biomarkers, namely, PDCD1, CD274, and IL-2 / TGF-β2 ratio, in response to either checkpoint inhibitor in melanoma patients (N=397). High expression levels of each biomarker showed improved overall survival.

[0369] Figures 43-45 show the results of three stratification biomarkers, namely, PDCD1, CD274, and IL-2 / TGF-β2 ratio, for CTLA-4 checkpoint inhibitors in melanoma patients (N=112). With the exception of IL-2 / TGF-β2, each biomarker showed a slight improvement in overall survival at high expression levels. Therefore, CTLA-4 was not the preferred checkpoint inhibitor.

Claims

1. A pharmaceutical for use in a method for treating or ameliorating symptoms of cancer in a human subject or animal, the pharmaceutical comprising an antisense agent for inhibiting or suppressing expression of TGF-β2, and the agent for inhibiting or suppressing expression of TGF-β2 is used in combination with a checkpoint inhibitor.

2. Use of an antisense agent for inhibiting or suppressing the expression of TGF-β2 in the preparation of a medicament for use in a method for treating or ameliorating symptoms of cancer in a human subject or animal, wherein the agent for inhibiting or suppressing the expression of TGF-β2 is used in combination with a checkpoint inhibitor.

3. The pharmaceutical according to claim 1, wherein the agent for inhibiting or suppressing the expression of TGF-β2 is used in combination with an interleukin immunotherapeutic agent.

4. The use according to claim 2, wherein the agent for inhibiting or suppressing the expression of TGF-β2 is used in combination with an interleukin immunotherapeutic agent.

5. The pharmaceutical or use according to any one of claims 1 to 4, wherein the agent for inhibiting or suppressing the expression of TGF-β2, the checkpoint inhibitor, and the interleukin immunotherapeutic agent are administered in parallel, simultaneously, sequentially, or temporally separately.

6. The pharmaceutical, or use, or method according to any one of claims 1 to 4, wherein the agent for inhibiting or suppressing the expression of TGF-β2, the checkpoint inhibitor, and the interleukin immunotherapeutic agent are administered by injection or infusion, separately or in a mixed formulation.

7. The pharmaceutical or use according to any one of claims 1 to 4, wherein the cancer is pancreatic cancer, melanoma, skin cancer, lung cancer, breast cancer, prostate cancer, colorectal cancer, kidney cancer, gastric cancer, ovarian cancer, cervical cancer, liver cancer, or multiple myeloma.

8. The pharmaceutical or use according to any one of claims 1 to 4, wherein the agent for inhibiting or suppressing the expression of TGF-β2 is a TGF-β2-specific antisense oligonucleotide that is complementary to a TGF-β2 transcription product and is 15 to 30 nucleotides in length.

9. The pharmaceutical or use according to any one of claims 1 to 4, wherein the agent for inhibiting or suppressing the expression of TGF-β2 is a TGF-β2-specific antisense oligonucleotide that is complementary to a TGF-β2 RNA precursor, mRNA precursor, or mRNA and is 18 to 21 nucleotides in length.

10. The agent for inhibiting or suppressing the expression of TGF-β2 is one or more TGF-β2-specific antisense oligonucleotides shown below (Table 1) that are complementary to the TGF-β2 transcript:

5. The medicament or use according to any one of claims 1 to 4, wherein the medicament or use is selected from the group consisting of: a nucleotide sequence of 1 to 4, ...

11. The pharmaceutical or use according to claim 10, wherein the TGF-β2-specific antisense oligonucleotide has one or two or fewer mismatches compared to the target human TGF-β2.

12. The pharmaceutical or use according to claim 10, wherein the TGF-β2-specific antisense oligonucleotide reduces TGF-β2 transcript levels by at least 60%, or at least 70%, or at least 80%, or at least 90%.

13. The pharmaceutical or use of claim 10, wherein the TGF-β2-specific antisense oligonucleotide reduces any TGF-β1 transcript level and any TGF-β3 transcript level by less than 10%, or less than 5%, or less than 1%.

14. The pharmaceutical or use of claim 10, wherein the TGF-β2-specific antisense oligonucleotide has one or more nucleotides chemically modified as a phosphorothioate internucleoside linkage, a methoxypropylphosphonate internucleoside linkage, an aminophosphoro linkage to a morpholino group, a 2'-OMe ribose group, a 2'-MOE methoxyethyl ribose group, a 2'-4' constrained methoxyethyl bicyclic ribose group, a 2'-4' constrained ethyl bicyclic ribose group, an LNA ribose group, a 2'-F ribose group, or a 5-methylcytosine base.

15. 11. The method or use of claim 10, wherein the antisense agent is conjugated to polyethylene glycol, a lipid, or triantenarry N-acetyl-galactosamine.

16. The pharmaceutical composition or use according to any one of claims 1 to 4, wherein each agent comprises a carrier which may be the same or different for each agent, and which is sterile water for injection, saline, isotonic saline, or a combination thereof.

17. The medicament or use according to any one of claims 1 to 4, wherein the agent is substantially free of excipients.

18. 5. The medicament or use according to any one of claims 1 to 4, wherein the agent is stable for at least 14 days at 37°C in a carrier that is substantially free of excipients.

19. The pharmaceutical or use according to any one of claims 1 to 4, wherein the checkpoint inhibitor is an inhibitor of PD-1.

20. The pharmaceutical or use according to any one of claims 1 to 4, wherein the checkpoint inhibitor is pembrolizumab, nivolumab, cemiplimab, spartalizumab, atezolizumab, avelumab, or durvalumab.

21. The pharmaceutical or use according to any one of claims 1 to 4, wherein the interleukin immunotherapeutic agent is natural IL-2, high-dose IL-2, recombinant IL-2, or aldesleukin.

22. The medicament or use according to any one of claims 1 to 4, wherein subjects who will benefit from the medicament or use are selected based on the levels of one or more biomarkers: TGF-β2, IL-2, CD19, IRF5, ITGAM, and combinations thereof.

23. 23. The medicament or use according to claim 22, wherein the one or more biomarkers are IRF5 and the subject is selected if expression of IRF5 is at a level above the median.

24. The pharmaceutical or use according to claim 22, wherein the one or more biomarkers are ITGAM and the subject is selected if expression of ITGAM is at a level above the median.

25. The medicament or use according to any one of claims 1 to 4, wherein the subject after use of the medicament has a reduced level of TGF-β2 compared to before use.

26. 5. The medicament or use according to any one of claims 1 to 4, wherein the subject after use of the medicament has an increased level of IRF5 compared to before use.

27. The medicament or use according to any one of claims 1 to 4, wherein the subject after use of the medicament has a reduced level of ITGAM compared to before use.

28. A pharmaceutical or use described in any one of claims 1 to 4, wherein the agent for inhibiting or suppressing the expression of TGF-β2 is used in combination with a therapeutically effective amount of an expression product of IRF5 or ITGAM.

29. 29. The pharmaceutical or use according to claim 28, wherein the expression product is an mRNA, a polypeptide, a protein, or a fragment thereof, or a combination thereof.

30. 5. The medicament or use according to any one of claims 1 to 4, wherein the use of said medicament reduces mortality at 6, 12, 18, 24, 30 or 36 months.

31. 5. The medicament or use according to any one of claims 1 to 4, wherein the use of said medicament improves overall survival at 6, 12, 18, 24, 30, or 36 months.

32. A pharmaceutical or use described in any one of claims 1 to 4, wherein the agent for inhibiting or suppressing the expression of TGF-β2 is used in combination with any one or more pharmaceuticals including targeted anticancer agents, cancer growth blocking agents, EGFR inhibitors, and combinations thereof.

33. A pharmaceutical or use described in any one of claims 1 to 4, wherein the agent for inhibiting or suppressing the expression of TGF-β2 is used in combination with any one or more pharmaceuticals selected from bevacizumab, everolimus, velzutifan, dabrafenib, trametinib, and combinations thereof.

34. A pharmaceutical or use according to any one of claims 1 to 4, wherein the agent for inhibiting or suppressing the expression of TGF-β2 is used in combination with one or more pharmaceuticals that are cancer growth inhibitors selected from angiogenesis inhibitors, histone deacetylase inhibitors, hedgehog inhibitors, mTOR inhibitors, p53 inhibitors, PARP inhibitors, proteasome inhibitors, tyrosine kinase inhibitors, and combinations thereof.

35. A pharmaceutical or use described in any one of claims 1 to 4, wherein the agent for inhibiting or suppressing the expression of TGF-β2 is used in combination with one or more pharmaceuticals that are EGFR inhibitors selected from erlotinib, gefitinib, afatinib, osimertinib, dacomitinib, and combinations thereof.

36. A pharmaceutical or use according to any one of claims 1 to 4, wherein the agent for inhibiting or suppressing the expression of TGF-β2 is used in combination with a chemotherapeutic agent.

37. A pharmaceutical or use according to any one of claims 1 to 4, wherein the agent for inhibiting or suppressing the expression of TGF-β2 is used in combination with radiation therapy or electric field therapy.

38. A pharmaceutical for use in a method for treating or ameliorating symptoms of cancer in a human subject or animal, the pharmaceutical comprising an antisense agent for inhibiting or suppressing the expression of TGF-β2, and the agent for inhibiting or suppressing the expression of TGF-β2 is used in combination with an interleukin immunotherapeutic agent.

39. Use of an antisense agent for inhibiting or suppressing the expression of TGF-β2 in the preparation of a medicament for use in a method for treating or ameliorating symptoms of cancer in a human subject or animal, wherein the agent for inhibiting or suppressing the expression of TGF-β2 is used in combination with an interleukin immunotherapy agent.

40. The pharmaceutical or use of claim 38 or 39, wherein the agent for inhibiting or suppressing the expression of TGF-β2 and the interleukin immunotherapeutic agent are administered in parallel, simultaneously, sequentially, or separately in time.

41. 40. The pharmaceutical or use according to claim 38 or 39, wherein the agent for inhibiting or suppressing the expression of TGF-β2 and the interleukin immunotherapeutic agent are administered by injection or infusion, separately or in a mixed formulation.

42. 40. The pharmaceutical or use according to claim 38 or 39, wherein the cancer is pancreatic cancer, melanoma, skin cancer, lung cancer, breast cancer, prostate cancer, colorectal cancer, kidney cancer, gastric cancer, ovarian cancer, cervical cancer, liver cancer, or multiple myeloma.

43. The pharmaceutical or use according to claim 38 or 39, wherein the agent for inhibiting or suppressing the expression of TGF-β2 is a TGF-β2-specific antisense oligonucleotide that is complementary to a TGF-β2 transcription product and is 15 to 30 nucleotides in length.

44. The pharmaceutical or use according to claim 38 or 39, wherein the agent for inhibiting or suppressing the expression of TGF-β2 is a TGF-β2-specific antisense oligonucleotide that is complementary to a TGF-β2 RNA precursor, mRNA precursor, or mRNA and is 18 to 21 nucleotides in length.

45. The pharmaceutical or use according to claim 38 or 39, wherein the agent for inhibiting or suppressing the expression of TGF-β2 is one or more TGF-β2-specific antisense oligonucleotides shown in Table 1, which are complementary to a TGF-β2 transcription product.

46. 46. ​​The pharmaceutical or use of claim 45, wherein the TGF-β2-specific antisense oligonucleotide has one or more nucleotides chemically modified as a phosphorothioate internucleoside linkage, a methoxypropylphosphonate internucleoside linkage, an aminophosphoro linkage to a morpholino group, a 2'-OMe ribose group, a 2'-MOE methoxyethyl ribose group, a 2'-4' constrained methoxyethyl bicyclic ribose group, a 2'-4' constrained ethyl bicyclic ribose group, an LNA ribose group, a 2'-F ribose group, or a 5-methylcytosine base.

47. 46. ​​The method or use of claim 45, wherein the antisense agent is conjugated to polyethylene glycol, a lipid, or a triple-stranded N-acetyl-galactosamine.

48. 40. The medicament or use of claim 38 or 39, wherein each agent comprises a carrier which is sterile water for injection, saline, isotonic saline, or a combination thereof, which may be the same or different for each agent.

49. 40. The medicament or use of claim 38 or 39, wherein the agent is substantially free of excipients.

50. 40. The medicament or use according to claim 38 or 39, wherein the agent is stable for at least 14 days at 37°C in a carrier that is substantially free of excipients.

51. 40. The pharmaceutical or use according to claim 38 or 39, wherein the interleukin immunotherapeutic agent is natural IL-2, high-dose IL-2, recombinant IL-2, or aldesleukin.

52. 40. The medicament or use of claim 38 or 39, wherein the use of the medicament reduces mortality at 6, 12, 18, 24, 30, or 36 months.

53. 40. The medicament or use of claim 38 or 39, wherein the use of the medicament improves survival at 6, 12, 18, 24, 30, or 36 months.

54. A pharmaceutical or use as described in claim 38 or 39, wherein the agent for inhibiting or suppressing the expression of TGF-β2 is used in combination with any one or more pharmaceuticals including targeted anticancer agents, cancer growth blocking agents, EGFR inhibitors, and combinations thereof.

55. A pharmaceutical or use as described in claim 38 or 39, wherein the agent for inhibiting or suppressing the expression of TGF-β2 is used in combination with any one or more pharmaceuticals selected from bevacizumab, everolimus, velzutifan, dabrafenib, trametinib, and combinations thereof.

56. A pharmaceutical or use as described in claim 38 or 39, wherein the agent for inhibiting or suppressing the expression of TGF-β2 is used in combination with one or more pharmaceuticals that are cancer growth inhibitors selected from angiogenesis inhibitors, histone deacetylase inhibitors, hedgehog inhibitors, mTOR inhibitors, p53 inhibitors, PARP inhibitors, proteasome inhibitors, tyrosine kinase inhibitors, and combinations thereof.

57. A pharmaceutical or use as described in claim 38 or 39, wherein the agent for inhibiting or suppressing the expression of TGF-β2 is used in combination with one or more pharmaceuticals that are EGFR inhibitors selected from erlotinib, gefitinib, afatinib, osimertinib, dacomitinib, and combinations thereof.

58. A pharmaceutical or use according to claim 38 or 39, wherein the agent for inhibiting or suppressing the expression of TGF-β2 is used in combination with a chemotherapeutic agent.

59. A pharmaceutical or use according to claim 38 or 39, wherein the agent for inhibiting or suppressing the expression of TGF-β2 is used in combination with radiation therapy or electric field therapy.

60. 40. The pharmaceutical or use of claim 38 or 39, wherein subjects who will benefit from the pharmaceutical or use are selected based on the levels of one or more biomarkers of TGF-β2, IRF5, ITGAM, and combinations thereof.

61. 40. The medicament or use of claim 38 or 39, wherein subjects who will benefit from the medicament or use are selected based on the levels of one or more biomarkers: TGF-β2, IRF5, ITGAM, neoantigens, mutational load, macrophages, and combinations thereof.

62. 62. The medicament or use of claim 61, wherein the one or more biomarkers is IRF5 and the subject is selected if expression of IRF5 is at a level below the median.

63. 62. The medicament or use of claim 61, wherein the one or more biomarkers are tumor-associated macrophages, and the subject is selected if tumor-associated macrophages are below average.

64. 62. The medicament or use of claim 61, wherein the one or more biomarkers is tumor neoantigen mutational burden, and the subject is selected if the neoantigen tumor burden is below average.

65. A pharmaceutical or use described in claim 38 or 39, wherein the agent for inhibiting or suppressing the expression of TGF-β2 is used in combination with a therapeutically effective amount of an agent for inhibiting or suppressing the expression of ITGAM or IRF5.

66. The pharmaceutical or use according to claim 65, wherein the agent for inhibiting or suppressing the expression of ITGAM or IRF5 is an antisense oligonucleotide targeting ITGAM or IRF5, respectively.

67. 1. A kit for treating or ameliorating symptoms of cancer, comprising: a therapeutically effective amount of an antisense agent for inhibiting or suppressing the expression of TGF-β2; and Therapeutically effective amounts of checkpoint inhibitors Includes a kit.

68. 68. The kit of claim 67, comprising a therapeutically effective amount of an interleukin immunotherapeutic agent.

69. 68. The kit of claim 67, wherein the cancer is pancreatic cancer, melanoma, skin cancer, lung cancer, breast cancer, prostate cancer, colorectal cancer, kidney cancer, gastric cancer, ovarian cancer, cervical cancer, liver cancer, thymic cancer, or multiple myeloma.

70. The kit of claim 67, wherein the agent for inhibiting or suppressing the expression of TGF-β2 is a TGF-β2-specific antisense oligonucleotide that is complementary to a transcription product of TGF-β2 and is 15 to 30 nucleotides in length.

71. The kit of claim 67, wherein the agent for inhibiting or suppressing the expression of TGF-β2 is a TGF-β2-specific antisense oligonucleotide that is complementary to a TGF-β2 precursor RNA, precursor mRNA, or mRNA and is 18 to 21 nucleotides in length.

72. 68. The kit of claim 67, wherein the agent for inhibiting or suppressing the expression of TGF-β2 is one or more TGF-β2-specific antisense oligonucleotides shown in Table 1 that are complementary to a TGF-β2 transcript.

73. 68. The kit of claim 67, wherein the TGF-β2-specific antisense oligonucleotide has no more than one or two mismatches compared to the target human TGF-β2.

74. 68. The kit of claim 67, wherein the TGF-β2-specific antisense oligonucleotide reduces TGF-β2 transcript levels by at least 60%, or at least 70%, or at least 80%, or at least 90%.

75. 68. The kit of claim 67, wherein the TGF-β2-specific antisense oligonucleotide reduces any TGF-β1 transcript level and any TGF-β3 transcript level by less than 10%, or less than 5%, or less than 1%.

76. 68. The kit of claim 67, wherein the TGF-β2-specific antisense oligonucleotide has one or more nucleotides chemically modified as a phosphorothioate internucleoside linkage, a methoxypropylphosphonate internucleoside linkage, an aminophosphoro linkage to a morpholino group, a 2'-OMe ribose group, a 2'-MOE methoxyethyl ribose group, a 2'-4' constrained methoxyethyl bicyclic ribose group, a 2'-4' constrained ethyl bicyclic ribose group, an LNA ribose group, a 2'-F ribose group, or a 5-methylcytosine base.

77. 68. The kit of claim 67, wherein said antisense agent is conjugated to polyethylene glycol, a lipid, or a triple-stranded N-acetyl-galactosamine.

78. 68. The kit of claim 67, wherein each agent comprises a carrier that is sterile water for injection, saline, isotonic saline, or a combination thereof, which can be the same or different for each agent.

79. 68. The kit of claim 67, wherein the agent is substantially free of excipients.

80. 68. The kit of claim 67, wherein the agent is stable for at least 14 days at 37°C in a carrier that is substantially free of excipients.

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