Anti-cancer agent

Abstract

The present invention relates to cancer agents, uses and methods designed to enhance anti-tumor effects against a variety of different cancers. Exemplary synergistic therapies include compositions of active agent combinations including an antisense agent for inhibiting or suppressing TGF-beta 2 expression, a checkpoint inhibitor, and an interleukin immunotherapeutic agent. One or more biomarkers, including IRF5 and ITGAM, can be used to screen subjects that benefit from these agents, uses, and methods. The agents may be used in combination with chemotherapy and other standard therapies.

Description

[0001] Sequence Listing

[0002] This application includes a sequence listing submitted electronically in the form of an ST.26 file, created on November 6, 2023, named 018988-005WO1_SL.xml, and of 120,186 bytes in size. Technical Field

[0003] The present invention describes agents, uses and methods for treating or improving symptoms of cancer in human or animal subjects. The agents are designed to enhance the anti-tumor effects on a variety of different cancers. Exemplary synergistic therapies include various active agent combinations, 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 screen for subjects who benefit from the agents, uses or methods. The therapy can be used in combination with chemotherapy, radiotherapy and other standard therapies. Background Art

[0004] Cancer is a complex pathology involving multiple distinct cellular pathways. Due to this complexity, identifying effective therapeutic strategies that can exert anti-tumor effects against various cancers has been difficult.

[0005] Disadvantages of conventional therapies include a lack of efficacy against many cancers.

[0006] Other drawbacks of conventional therapies include severe adverse side effects, such as killing healthy cells in addition to cancer cells.

[0007] Other disadvantages of anticancer agents include high toxicity at levels required for therapeutic administration.

[0008] There is a need for methods, agents, and uses for cancer diseases that improve therapeutic efficacy and reduce toxicity and adverse side effects.

[0009] For example, what is needed are compositions, uses or methods of combining different agents that have significant anti-tumor and cancer immunotherapy effects while reducing side effects and adverse health effects. It is necessary to use appropriate biomarkers to select combinations with synergistic effects to improve the guidance of such compositions.

[0010] There is an urgent need for new approaches, agents, and uses that combine cancer immunotherapy strategies with direct anti-tumor attack strategies to treat various cancers.

[0011] For example, what is needed are therapeutic compositions that combine cancer T cell and immunotherapy with potent anticancer agents. Summary of the Invention

[0012] The present invention provides methods for treating or ameliorating cancer symptoms in human or animal subjects using pharmaceutical compositions designed to enhance anti-tumor effects against a variety of different cancers. The synergistic drug therapies of the present invention involve the use of potent direct anti-tumor agents with cancer immunotherapeutics. Cancer immunotherapeutics 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 for the treatment of various cancers.

[0013] In some embodiments, the methods and treatment strategies of the present invention can improve the efficacy of cancer treatments and reduce toxic side effects and adverse health effects.

[0014] In further embodiments, the methods and treatment strategies of the present invention can improve the guidance of therapy by using appropriate biomarkers to select for synergistic effects of compositions.

[0015] Exemplary synergistic drug therapies include compositions of various active agent combinations, 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 screen for subjects who benefit from the methods, agents, or uses. The compositions can be used in combination with chemotherapy and other standard therapies.

[0016] Embodiments of the present invention include the following:

[0017] Antisense agents for inhibiting or suppressing TGF-β2 expression are used in combination with checkpoint inhibitors to treat or ameliorate symptoms of cancer in human subjects or animals.

[0018] Use of an antisense agent for inhibiting or suppressing TGF-β2 expression in the preparation of a medicament for treating or ameliorating cancer symptoms in a human subject or animal in combination with a checkpoint inhibitor.

[0019] A method for treating or ameliorating symptoms of cancer in a human or animal subject in need thereof, the method comprising:

[0020] administering to the subject a therapeutically effective amount of an antisense agent for inhibiting or suppressing TGF-β2 expression;

[0021] A therapeutically effective amount of a checkpoint inhibitor is administered to the subject.

[0022] The above-mentioned agent is combined with an interleukin immunotherapy agent.

[0023] The above use is combined with an interleukin immunotherapy agent.

[0024] The above method comprises administering to the subject a therapeutically effective amount of an interleukin immunotherapy agent.

[0025] The above-mentioned medicament, use or method, wherein the agent for inhibiting or suppressing TGF-β2 expression, the checkpoint inhibitor and the interleukin immunotherapy agent are administered concurrently, simultaneously, sequentially or separately in time.

[0026] The above-mentioned medicament, use or method, wherein the agent for inhibiting or suppressing TGF-β2 expression, the checkpoint inhibitor and the interleukin immunotherapy agent are administered alone or in the form of a combined preparation by injection or infusion.

[0027] The above-mentioned medicament, use or method, 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.

[0028] The above-mentioned agent, use or method, wherein the agent for inhibiting or suppressing TGF-β2 expression is a TGF-β2-specific antisense oligonucleotide complementary to TGF-β2 transcript and having a length of 15-30 nucleotides.

[0029] The above-mentioned agent, use or method, wherein the agent for inhibiting or suppressing TGF-β2 expression is a TGF-β2-specific antisense oligonucleotide complementary to TGF-β2 preRNA, pre-mRNA or mRNA and having a length of 18-21 nucleotides.

[0030] The above-mentioned agent, use or method, wherein the agent for inhibiting or suppressing TGF-β2 expression is one or more TGF-β2-specific antisense oligonucleotides complementary to TGF-β2 transcripts shown in Table 1, and chemically modified variants thereof, LNA variants thereof, gapmer variants thereof, and any combination or collection thereof.

[0031] The above-mentioned agent, use or method, wherein the TGF-β2-specific antisense oligonucleotide has no more than one or two mismatches compared to the target human TGF-β2.

[0032] The above-mentioned agent, use or method, wherein the TGF-β2-specific antisense oligonucleotide reduces the level of TGF-β2 transcript by at least 60%, or at least 70%, or at least 80%, or at least 90%.

[0033] The above-mentioned medicament, use or method, wherein the TGF-β2-specific antisense oligonucleotide reduces the level of any TGF-β1 transcript and the level of any TGF-β3 transcript by less than 10%, or less than 5%, or less than 1%.

[0034] The above-mentioned agent, use or method, wherein the TGF-β2-specific antisense oligonucleotide has one or more nucleotides chemically modified to be phosphorothioate internucleoside linkages, methoxypropylphosphonate internucleoside linkages, or phosphoramidite linkages linked 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.

[0035] The above-mentioned agent, use or method, wherein the antisense agent is conjugated to polyethylene glycol, lipid or tri-branched N-acetylgalactosamine.

[0036] In the above-mentioned medicament, use or method, each medicament comprises a carrier of sterile water for injection, saline, isotonic saline or a combination thereof, and the carrier of each medicament may be the same or different.

[0037] The above-mentioned medicament, use or method, wherein the medicament is substantially free of excipients.

[0038] The above-mentioned medicament, use or method, wherein the medicament is stable for at least 14 days at 37°C in a carrier substantially free of excipients.

[0039] The above-mentioned agent, use or method, wherein the checkpoint inhibitor is an inhibitor of PD-1.

[0040] The above-mentioned agent, use or method, wherein the checkpoint inhibitor is pembrolizumab, nivolumab, cemiplimab, spartalizumab, atezolizumab, avelumab or durvalumab.

[0041] The above-mentioned medicament, use or method, wherein the interleukin immunotherapy agent is natural IL-2, high-dose IL-2, recombinant IL-2 or aldesleukin.

[0042] The above-mentioned agents, uses or methods include selecting subjects who will benefit from the agents, uses or methods based on the levels of one or more biomarkers TGF-β2, IL-2, CD19, IRF5, ITGAM and combinations thereof.

[0043] The above-mentioned medicament, use or method, wherein the one or more biomarkers is IRF5, and the subject is selected when the expression level of IRF5 is above the median.

[0044] The above-mentioned medicament, use or method, wherein the one or more biomarkers is ITGAM, and the subject is selected when the expression level of ITGAM is above the median.

[0045] The above-mentioned medicament, use or method, wherein the TGF-β2 level of the subject is reduced after administration or use compared to before administration or use.

[0046] The above-mentioned medicament, use or method, wherein the level of IRF5 in the subject is increased after administration or use compared to before administration or use.

[0047] The above-mentioned medicament, use or method, wherein the level of ITGAM in the subject is reduced after administration or use compared to before administration or use.

[0048] The above-mentioned medicament, use or method comprises administering a therapeutically effective amount of an expression product of IRF5 or ITGAM to a subject.

[0049] The above-mentioned agent, use or method, wherein the expression product is mRNA, polypeptide, protein or fragment thereof, or a combination thereof.

[0050] The above-mentioned medicament, use or method, wherein the administration or use reduces mortality at 6, 12, 18, 24, 30 or 36 months.

[0051] The above-mentioned medicament, use or method, wherein the administration or use increases the overall survival rate at 6, 12, 18, 24, 30 or 36 months.

[0052] The above-mentioned agents, uses or methods are combined with any one or more drugs including targeted cancer drugs, cancer growth inhibitors, EGFR inhibitors and combinations thereof.

[0053] The above-mentioned agent, use or method is combined with any one or more drugs selected from bevacizumab, everolimus, belzutifan, dabrafenib, trametinib and combinations thereof.

[0054] The above-mentioned agents, uses or methods are combined with any one or more drugs, which are cancer growth inhibitors selected from angiogenesis inhibitors, histone deacetylase inhibitors, hedgehog factor blockers, mTOR inhibitors, p53 inhibitors, PARP inhibitors, proteasome inhibitors, tyrosine kinase inhibitors and combinations thereof.

[0055] The above-mentioned agents, uses or methods are combined with any one or more drugs, wherein the drug is an EGFR inhibitor selected from erlotinib, gefitinib, afatinib, osimertinib, dacomitinib and combinations thereof.

[0056] The above-mentioned agents, uses or methods are combined with chemotherapy drugs.

[0057] The above-mentioned medicament, use or method is combined with radiation therapy or electric field therapy.

[0058] Antisense agents for inhibiting or suppressing TGF-β2 expression are used in combination with interleukin immunotherapeutics to treat or ameliorate cancer symptoms in human subjects or animals.

[0059] Use of an antisense agent for inhibiting or suppressing TGF-β2 expression in the preparation of a medicament for treating or ameliorating cancer symptoms in a human subject or animal in combination with an interleukin immunotherapy agent.

[0060] A method for treating or ameliorating symptoms of cancer in a human or animal subject in need thereof, the method comprising:

[0061] administering to the subject a therapeutically effective amount of an antisense agent for inhibiting or suppressing TGF-β2 expression;

[0062] A therapeutically effective amount of an interleukin immunotherapeutic agent is administered to the subject.

[0063] The above-mentioned medicament, use or method, wherein the agent for inhibiting or suppressing TGF-β2 expression and the interleukin immunotherapy agent are administered concurrently, simultaneously, sequentially or separately.

[0064] The above-mentioned medicament, use or method, wherein the agent for inhibiting or suppressing TGF-β2 expression and the interleukin immunotherapy agent are administered separately by injection or infusion or in the form of a combined preparation.

[0065] The above-mentioned medicament, use or method, 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.

[0066] The above-mentioned agent, use or method, wherein the agent for inhibiting or suppressing TGF-β2 expression is a TGF-β2-specific antisense oligonucleotide complementary to TGF-β2 transcript and having a length of 15-30 nucleotides.

[0067] The above-mentioned agent, use or method, wherein the agent for inhibiting or suppressing TGF-β2 expression is a TGF-β2-specific antisense oligonucleotide complementary to TGF-β2 preRNA, pre-mRNA or mRNA and having a length of 18-21 nucleotides.

[0068] The above-mentioned agent, use or method, wherein the agent for inhibiting or suppressing TGF-β2 expression is one or more TGF-β2-specific antisense oligonucleotides complementary to TGF-β2 transcripts shown in Table 1.

[0069] The above-mentioned agent, use or method, wherein the TGF-β2-specific antisense oligonucleotide has one or more nucleotides chemically modified to be phosphorothioate internucleoside linkages, methoxypropylphosphonate internucleoside linkages, or phosphoramidite linkages linked 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.

[0070] The above-mentioned agent, use or method, wherein the antisense agent is conjugated to polyethylene glycol, lipid or tri-branched N-acetylgalactosamine.

[0071] In the above-mentioned medicament, use or method, each medicament comprises a carrier of sterile water for injection, saline, isotonic saline or a combination thereof, and the carrier of each medicament may be the same or different.

[0072] The above-mentioned medicament, use or method, wherein the medicament is substantially free of excipients.

[0073] The above-mentioned medicament, use or method, wherein the medicament is stable for at least 14 days at 37°C in a carrier substantially free of excipients.

[0074] The above-mentioned medicament, use or method, wherein the interleukin immunotherapy agent is natural IL-2, high-dose IL-2, recombinant IL-2 or aldesleukin.

[0075] The above-mentioned medicament, use or method, wherein the administration or use reduces mortality at 6, 12, 18, 24, 30 or 36 months.

[0076] The above-mentioned medicament, use or method, wherein the administration or use increases the survival rate at 6, 12, 18, 24, 30 or 36 months.

[0077] The above-mentioned agents, uses or methods are combined with any one or more drugs including targeted cancer drugs, cancer growth inhibitors, EGFR inhibitors and combinations thereof.

[0078] The above-mentioned medicament, use or method is combined with any one or more drugs selected from bevacizumab, everolimus, bezitufan, dabrafenib, trametinib and combinations thereof.

[0079] The above-mentioned agents, uses or methods are combined with any one or more drugs, which are cancer growth inhibitors selected from angiogenesis inhibitors, histone deacetylase inhibitors, hedgehog factor blockers, mTOR inhibitors, p53 inhibitors, PARP inhibitors, proteasome inhibitors, tyrosine kinase inhibitors and combinations thereof.

[0080] The above-mentioned agents, uses or methods are combined with any one or more drugs, wherein the drug is an EGFR inhibitor selected from erlotinib, gefitinib, afatinib, osimertinib, dacomitinib and combinations thereof.

[0081] The above-mentioned agents, uses or methods are combined with chemotherapy drugs.

[0082] The above-mentioned medicament, use or method is combined with radiation therapy or electric field therapy.

[0083] The above-mentioned agents, uses or methods include selecting a subject that will benefit from the agents, uses or methods based on the levels of one or more biomarkers TGF-β2, IRF5, ITGAM and combinations thereof.

[0084] The above-mentioned agents, uses or methods include selecting subjects who benefit from the agents, uses or methods based on the levels of one or more biomarkers TGF-β2, IRF5, ITGAM, new antigens, mutation load, macrophages and combinations thereof.

[0085] The above-mentioned medicament, use or method, wherein the one or more biomarkers is IRF5, and the subject is selected when the expression level of IRF5 is below the median.

[0086] The above-mentioned medicament, use or method, wherein the one or more biomarkers are tumor-associated macrophages, and the subject is selected when the tumor-associated macrophages are below average.

[0087] The above-mentioned medicament, use or method, wherein the one or more biomarkers is tumor neoantigen mutation burden, and the subject is selected when the neoantigen tumor burden is below average.

[0088] The above-mentioned agent, use or method comprises administering to a subject a therapeutically effective amount of an agent for inhibiting or suppressing the expression of ITGAM or IRF5.

[0089] The above-mentioned agent, use or method, wherein the agent for inhibiting or suppressing the expression of ITGAM or IRF5 is an antisense oligonucleotide targeting ITGAM or IRF5, respectively.

[0090] A kit for treating or improving cancer symptoms, comprising:

[0091] a therapeutically effective amount of an antisense agent for inhibiting or suppressing TGF-β2 expression; and

[0092] A therapeutically effective amount of a checkpoint inhibitor.

[0093] The above kit contains a therapeutically effective amount of interleukin immunotherapy agent.

[0094] The above kit, 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.

[0095] In the above kit, the agent for inhibiting or suppressing TGF-β2 expression is a TGF-β2 specific antisense oligonucleotide complementary to TGF-β2 transcript and having a length of 15-30 nucleotides.

[0096] In the above kit, the agent for inhibiting or suppressing TGF-β2 expression is a TGF-β2 specific antisense oligonucleotide complementary to TGF-β2 pre-RNA, pre-mRNA or mRNA and having a length of 18-21 nucleotides.

[0097] In the above kit, the agent for inhibiting or suppressing TGF-β2 expression is one or more TGF-β2 specific antisense oligonucleotides complementary to TGF-β2 transcripts shown in Table 1.

[0098] The above kit, wherein the TGF-β2 specific antisense oligonucleotide has no more than one or two mismatches compared to the target human TGF-β2.

[0099] The above kit, wherein the TGF-β2 specific antisense oligonucleotide reduces the level of TGF-β2 transcript by at least 60%, or at least 70%, or at least 80%, or at least 90%.

[0100] The above kit, wherein the TGF-β2 specific antisense oligonucleotide reduces the level of any TGF-β1 transcript and the level of any TGF-β3 transcript by less than 10%, or less than 5%, or less than 1%.

[0101] The above-mentioned kit, wherein the TGF-β2 specific antisense oligonucleotide has one or more nucleotides chemically modified into a phosphorothioate internucleoside linkage, a methoxypropylphosphonate internucleoside linkage, or an aminophosphoric acid linkage connected 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.

[0102] The above kit, wherein the antisense agent is conjugated to polyethylene glycol, lipid or tri-branched N-acetylgalactosamine.

[0103] In the above-mentioned kit, each medicament comprises a carrier of sterile water for injection, saline, isotonic saline or a combination thereof, and the carrier of each medicament may be the same or different.

[0104] The above kit, wherein the pharmaceutical agent is substantially free of excipients.

[0105] The above kit, wherein the agent is stable at 37° C. for at least 14 days in a carrier that is substantially free of excipients.

[0106] BRIEF DESCRIPTION OF THE DRAWINGS

[0107] Figure 1 Kaplan-Meier overall survival plots used to investigate such clinical effects are shown. Figure 1 The results showed that after the use of PD-1 inhibitors, the survival of patients with high IL2 (left panel) (which can be provided by IL-2 immunotherapy) and patients with low TGF-β2 (right panel) (which can be provided by antisense TGF-β2 inhibitors) was improved. These clinical data provide a basis for the therapeutic combination of antisense TGF-β2 inhibitors, PD-1 checkpoint inhibitors and IL-2 immunotherapy to improve the overall survival of melanoma patients.

[0108] Figure 2 The results show that a high IL-2 / TGF-β2 ratio significantly improves survival after PD-1 inhibition (left panel), which can be provided by IL-2 immunotherapy and antisense TGF-β2 inhibitors. These clinical data provide a basis for the therapeutic combination of antisense TGF-β2 inhibitors, PD-1 checkpoint inhibitors, and IL-2 immunotherapy to improve overall survival in melanoma patients.

[0109] Figure 3 Data are shown for the use of ITGAM as a biomarker. Figure 3 (Upper left and right panels) Shown are Kaplan-Meier overall survival plots for melanoma patients in this study. Figure 3(Lower left and right panels) Kaplan-Meier overall survival plots of pancreatic adenocarcinoma (PDAC) patients in this study are shown in terms of IL-2 expression.

[0110] Figure 4 Data are shown for the use of ITGAM as a biomarker. Figure 4 (Left and right) Kaplan-Meier overall survival plots of pancreatic cancer (PDAC) patients in this study are shown in terms of IL-2 expression.

[0111] Figure 5 Data are shown for the use of CD8A as a biomarker. Figure 5 (Upper left and right panels) Shown are Kaplan-Meier overall survival plots for melanoma patients in this study. Figure 5 (Lower left and right panels) Kaplan-Meier overall survival plots of pancreatic adenocarcinoma (PDAC) patients in this study are shown in terms of TGF-β2 expression.

[0112] Figure 6 Data are shown for the use of CD8A as a biomarker. Figure 6 (Left and right) Kaplan-Meier overall survival plots of pancreatic cancer (PDAC) patients in this study are shown in terms of IL-2 expression.

[0113] Figure 7 Data are shown for the use of CD4 as a biomarker. Figure 3 (Upper left and right panels) Shown are Kaplan-Meier overall survival plots for melanoma patients in this study. Figure 7 (Lower left and right panels) Kaplan-Meier overall survival plots of pancreatic adenocarcinoma (PDAC) patients in this study are shown in terms of TGF-β2 expression.

[0114] Figure 8 Data are shown for the use of CD4 as a biomarker. Figure 8 (Left and right) Kaplan-Meier overall survival plots of pancreatic cancer (PDAC) patients in this study are shown in terms of IL-2 expression.

[0115] Figure 9 Data are shown for the use of ITGAX as a biomarker. Figure 9 (Upper left and right panels) Shown are Kaplan-Meier overall survival plots for melanoma patients in this study. Figure 9 (Lower left and right panels) Kaplan-Meier overall survival plots of pancreatic adenocarcinoma (PDAC) patients in this study are shown in terms of TGF-β2 expression.

[0116] Figure 10Data are shown for the use of ITGAX as a biomarker. Figure 10 (Left and right) Kaplan-Meier overall survival plots of pancreatic cancer (PDAC) patients in this study are shown in terms of IL-2 expression.

[0117] Figure 11 Data are shown for the use of CD19 as a biomarker. Figure 11 (Upper left and right panels) Shown are Kaplan-Meier overall survival plots for melanoma patients in this study. Figure 11 (Lower left and right panels) Kaplan-Meier overall survival plots of pancreatic adenocarcinoma (PDAC) patients in this study are shown in terms of TGF-β2 expression.

[0118] Figure 12 Data are shown for the use of CD19 as a biomarker. Figure 12 (Left and right) Kaplan-Meier overall survival plots of pancreatic cancer (PDAC) patients in this study are shown in terms of IL-2 expression.

[0119] Figure 13 Data are shown for the use of IRF5 as a biomarker. Figure 13 (Upper left and right panels) Shown are Kaplan-Meier overall survival plots for melanoma patients in this study. Figure 13 (Lower left and right panels) Kaplan-Meier overall survival plots of pancreatic adenocarcinoma (PDAC) patients in this study are shown in terms of TGF-β2 expression.

[0120] Figure 14 Data are shown for the use of IRF5 as a biomarker. Figure 14 (Left and right) Kaplan-Meier overall survival plots of pancreatic cancer (PDAC) patients in this study are shown in terms of IL-2 expression.

[0121] Figure 15 Data are shown for the use of NOS2 as a biomarker. Figure 15 (Upper left and right panels) Shown are Kaplan-Meier overall survival plots for melanoma patients in this study. Figure 15 (Lower left and right panels) Kaplan-Meier overall survival plots of pancreatic adenocarcinoma (PDAC) patients in this study are shown in terms of TGF-β2 expression.

[0122] Figure 16 Data are shown for the use of NOS2 as a biomarker. Figure 16 (Left and right) Kaplan-Meier overall survival plots of pancreatic cancer (PDAC) patients in this study are shown in terms of IL-2 expression.

[0123] Figure 17 Data are shown for the use of CD163 as a biomarker. Figure 17 (Upper left and right) Shows the Kaplan-Meier overall survival plots for melanoma patients in this study. Figure 17 (Lower left and right panels) Kaplan-Meier overall survival plots of pancreatic adenocarcinoma (PDAC) patients in this study are shown in terms of TGF-β2 expression.

[0124] Figure 18 Data are shown for the use of CD163 as a biomarker. Figure 18 (Left and right) Kaplan-Meier overall survival plots of pancreatic cancer (PDAC) patients in this study are shown in terms of IL-2 expression.

[0125] Figure 19 Shows the results of this clinical study on overall survival of melanoma patients. Figure 19 Kaplan-Meier overall survival plots for such clinical efficacy studies are shown. Figure 19 They showed that after treatment with a PD-1 checkpoint inhibitor and additional stratification based on various immune cell markers (i.e., basophils, B cells, eosinophils, M0 macrophages, and Th1 helper cells), high levels of IL-2 significantly improved survival in all cases. M0 macrophages were the highly significant factor.

[0126] Figure 20 Shows the results of this clinical study on overall survival of melanoma patients. Figure 20 Kaplan-Meier overall survival plots for such clinical efficacy studies are shown. Figure 20 They showed that after treatment with a PD-1 checkpoint inhibitor and additional stratification based on various immune cell markers (i.e., basophils, B cells, eosinophils, M0 macrophages, and Th1 helper cells), high levels of IL-2 significantly improved survival in all cases. Figure 20 (Upper and lower right panels) show comparative baseline results.

[0127] Figure 21 Shows the results of this clinical study on overall survival of patients with pancreatic cancer. Figure 21 Kaplan-Meier overall survival plots for such clinical efficacy studies are shown. Figure 21 (Upper left panel) shows that high tumor mRNA levels of TGF-β2 significantly reduced survival in patients with low M2 tumor-associated macrophages. Figure 21The log-rank P value in (upper left panel) indicates that, based on this clinical study and the conditions, the improvement in survival with therapeutic antisense TGF-β2 inhibitors was highly significant. Patients with high tumor TGF-β2 expression had a survival of only 15 months, whereas patients in the low range of tumor TGF-β2 expression had a survival of 73 months.

[0128] Figure 22 Shown is a Kaplan-Meier plot of overall survival for a clinical efficacy study of combined cancer therapy.

[0129] Figure 23 Shown is a Kaplan-Meier plot of overall survival for a clinical efficacy study of combined cancer therapy.

[0130] Figure 24 Shown is a Kaplan-Meier plot of overall survival for a clinical efficacy study of combined cancer therapy.

[0131] Figure 25 Shown is a Kaplan-Meier plot of overall survival for a clinical efficacy study of combined cancer therapy.

[0132] Figure 26 Shown is a Kaplan-Meier plot of overall survival for a clinical efficacy study of combined cancer therapy.

[0133] Figure 27 Shown is a Kaplan-Meier plot of overall survival for a clinical efficacy study of combined cancer therapy.

[0134] Figure 28 Shown is a Kaplan-Meier plot of overall survival for a clinical efficacy study of combined cancer therapy.

[0135] Figure 29 Shown is a Kaplan-Meier plot of overall survival for a clinical efficacy study of combined cancer therapy.

[0136] Figure 30 Shown is a Kaplan-Meier plot of overall survival for a clinical efficacy study of combined cancer therapy.

[0137] Figure 31 Shown is a Kaplan-Meier plot of overall survival for a clinical efficacy study of combined cancer therapy.

[0138] Figure 32 Shown is a Kaplan-Meier plot of overall survival for a clinical efficacy study of combined cancer therapy.

[0139] Figure 33Shown is a Kaplan-Meier plot of overall survival for a clinical efficacy study of combined cancer therapy.

[0140] Figure 34 Shown is a Kaplan-Meier plot of overall survival for a clinical efficacy study of combined cancer therapy.

[0141] Figure 35 Shown is a Kaplan-Meier plot of overall survival for a clinical efficacy study of combined cancer therapy.

[0142] Figure 36 Shown is a Kaplan-Meier plot of overall survival for a clinical efficacy study of combined cancer therapy.

[0143] Figure 37 Shown is a Kaplan-Meier plot of overall survival for a clinical efficacy study of combined cancer therapy.

[0144] Figure 38 Shown is a Kaplan-Meier plot of overall survival for a clinical efficacy study of combined cancer therapy.

[0145] Figure 39 Shown is a Kaplan-Meier plot of overall survival for a clinical efficacy study of combined cancer therapy.

[0146] Figure 40 Shown is a Kaplan-Meier plot of overall survival for a clinical efficacy study of combined cancer therapy.

[0147] Figure 41 Shown is a Kaplan-Meier plot of overall survival for a clinical efficacy study of combined cancer therapy.

[0148] Figure 42 Shown is a Kaplan-Meier plot of overall survival for a clinical efficacy study of combined cancer therapy.

[0149] Figure 43 Shown is a Kaplan-Meier plot of overall survival for a clinical efficacy study of combined cancer therapy.

[0150] Figure 44 Shown is a Kaplan-Meier plot of overall survival for a clinical efficacy study of combined cancer therapy.

[0151] Figure 45 Shown is a Kaplan-Meier plot of overall survival for a clinical efficacy study of combined cancer therapy.

[0152] Detailed Description of the Disclosure

[0153] The present invention relates to methods, compositions, medicaments and therapeutic uses thereof for treating or ameliorating symptoms of cancer in human or animal subjects using pharmaceutical compositions designed to promote anti-tumor effects against a range of different cancers.

[0154] Exemplary synergistic drug therapies include compositions of various active agent combinations, including agents for inhibiting or suppressing TGF-β2 expression, checkpoint inhibitors, and interleukin immunotherapeutics.

[0155] Embodiments of the present invention include methods, agents, and therapeutic uses thereof for treating or ameliorating symptoms of neoplastic diseases, wherein the agents may be administered concurrently, simultaneously, sequentially, or separately in time.

[0156] 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 to treat neoplastic diseases, wherein the anti-TGF-β2 agent and the immunotherapeutic agent are used concurrently, simultaneously, sequentially, or separately in time.

[0157] In some embodiments, one or more biomarkers including IRF5 and ITGAM can be used to screen for subjects who will benefit from the methods, agents, or uses. The compositions can be used in combination with chemotherapy and other standard therapies.

[0158] In some embodiments, the present invention relates to a biomarker-guided combination of immunotherapeutics comprising a checkpoint inhibitor and a TGF-β2 inhibitor.

[0159] Embodiments of the present invention relate to methods, agents, and uses of immunotherapeutic agents comprising a combination of a checkpoint inhibitor and a TGF-β2 inhibitor and guided by biomarkers. These embodiments recognize that overexpression of TGF-β2 is a useful indicator for avoiding the cascade of downstream effects and poor prognosis in neoplastic diseases.

[0160] The present invention can use the detection of TGF-β2 biomarkers as a guide to select subjects for treatment with immunotherapeutic agents that include a combination of a checkpoint inhibitor and a TGF-β2 inhibitor. Because the antisense oligonucleotides of the present invention (e.g., OT-101) target and inhibit TGF-β2, using TGF-β2 biomarkers to select subjects for treatment advantageously provides improved results.

[0161] More specifically, the present invention utilizes TGF-β2 as a biomarker that surprisingly outperforms TGF-β-1 or TGF-β-3 in terms of tumor disease outcome. TGF-β2 as a biomarker is predictive of improved outcome, whereas TGF-β-1 or TGF-β-3 are not and may indicate a poorer outcome.

[0162] In some embodiments, the combination of a TGF-β2 antisense inhibitor and a PD-1 checkpoint inhibitor can have surprising efficacy. In certain embodiments, the combination of a TGF-β2 antisense inhibitor and a PD-1 checkpoint inhibitor has surprising efficacy, as the combination of a TGF-β2 antisense inhibitor and a PD-L1 checkpoint inhibitor does not.

[0163] Embodiments of the present invention take advantage of these facts to provide methods, agents or uses for tumor diseases by selecting subjects using the TGF-β2 biomarker, wherein the subjects are selected when TGF-β2 expression is elevated.

[0164] In some embodiments, a method for treating or ameliorating a symptom of cancer in a human or animal subject in need thereof may comprise administering to the subject a therapeutically sufficient amount of a pharmaceutical composition comprising an agent for inhibiting or suppressing TGF-β2 expression, 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 immunotherapy agent, wherein the subject is selected using a TGF-β2 biomarker, and the subject is selected when TGF-β2 expression is elevated.

[0165] In a further embodiment, a combination of an agent for inhibiting or suppressing TGF-β2 expression with a checkpoint inhibitor and an interleukin immunotherapy agent for treating or ameliorating cancer symptoms in a human subject or animal can be used for tumor diseases by selecting subjects using a TGF-β2 biomarker, wherein the subject is selected when TGF-β2 expression is elevated.

[0166] The therapy of the present invention 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.

[0167] As used herein, the term medicament may 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 may be a pharmaceutical composition. In certain embodiments, the composition may be a pharmaceutical composition comprising a therapeutically effective amount of one or more active compounds. Some examples of excipients are given 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 a therapeutically effective amount of a compound are known in the art.

[0168] Anticancer agents and methods

[0169] Many cancers, such as pancreatic adenocarcinoma (PDAC) and melanoma, are associated with elevated levels of TGF-β2. Agents that inhibit or suppress TGF-β2 expression can be effective in treating these cancer types. For example, overall survival in pancreatic adenocarcinoma (PDAC) patients has more than doubled, from 15 months in patients with high TGF-β2 expression to 37 months in patients with low TGF-β2 expression.

[0170] 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 TGF-β2 expression; 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 immunotherapy agent.

[0171] In certain embodiments, the present invention includes a combination of an agent for inhibiting or suppressing TGF-β2 expression and a checkpoint inhibitor for treating or ameliorating symptoms of cancer in a human subject or animal.

[0172] In a further embodiment, the present invention includes a combination of an agent for inhibiting or suppressing TGF-β2 expression and an interleukin immunotherapy agent for treating or ameliorating symptoms of cancer in a human subject or animal.

[0173] In additional embodiments, the present invention includes a combination of an agent for inhibiting or suppressing TGF-β2 expression with a checkpoint inhibitor and an interleukin immunotherapy agent for treating or ameliorating symptoms of cancer in a human subject or animal.

[0174] The present invention also relates to the use of a composition comprising an agent for inhibiting or suppressing TGF-β2 expression in the preparation of a medicament for treating or improving cancer symptoms in human subjects or animals in combination with a checkpoint inhibitor and / or an interleukin immunotherapy agent.

[0175] Therapies of the present invention using one or more agents for inhibiting or suppressing TGF-β2 expression 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.

[0176] Examples of agents for inhibiting or suppressing TGF-β2 expression include antisense agents.

[0177] Human TGF-β2 specific antisense oligodeoxynucleotide

[0178] Antisense oligonucleotides (ASOs) are single-stranded deoxyribonucleotides that are complementary to mRNA targets. Antisense therapies can downregulate molecular targets by inducing RNase H endonuclease activity, which cleaves RNA-DNA heteroduplexes, thereby significantly reducing translation of the target gene. Other ASO mechanisms may include inhibition of 5' cap formation, alterations in splicing processes (e.g., splice switching), and steric hindrance of ribosomal activity.

[0179] Antisense therapeutic strategies utilize single-stranded DNA oligonucleotides (ssDNA oligonucleotides) that inhibit protein production by mediating the catalytic degradation of target mRNAs or by binding to sites on mRNA required for translation. Antisense oligonucleotides can be designed to target either the viral RNA genome or viral transcripts. Antisense oligonucleotides offer a means of identifying potential targets and therefore represent potential therapeutic agents.

[0180] Antisense oligonucleotides are small, synthetic, single-stranded DNA fragments that can be 15-30 nucleotides in length. ASOs can specifically bind to complementary DNA / RNA sequences via Watson-Crick hybridization and, once bound to the target RNA, inhibit translation by inducing cleavage or by inhibiting mRNA maturation. ASOs can selectively inhibit specific gene expression. Chemical modification of DNA or RNA can be used to improve stability.

[0181] For example, modifications can be introduced at phosphodiester bonds, sugar rings, and backbones. ASO antivirals can block translation by: (i) ribonuclease H (RNAse H) or RNase P-mediated mRNA cleavage, or (ii) steric (non-bonded) blocking of enzymes involved in target gene translation. Human TGF-β2-specific phosphorothioate antisense oligodeoxynucleotides, such as OT-101 (AP 12009 Trabedersen SEQ ID NO: 8), can be used to reduce TGF-β2 protein levels in malignant tumors and slow disease progression.

[0182] Antisense oligodeoxynucleotides are short strings of DNA designed to downregulate gene expression by interfering with the translation of specific protein-coding mRNA. OT-101 is a synthetic 18-mer phosphorothioate oligodeoxynucleotide (S-ODN) in which all 3'-5' bonds are modified to phosphorothioate. Its molecular formula is C 177 H 208 N 60 Na 17 O 94 P 17 S 17 , with a molecular weight of 6,143 g / mol. OT-101 is designed to be complementary to a specific sequence in human TGF-β2 mRNA after gene expression.

[0183] 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 one non-bridging oxygen of each phosphate moiety is replaced by a sulfur atom. OT-101 is complementary to a specific sequence of human TGF-β2 mRNA from genetic expression. OT-101 can be used as an RNA therapeutic designed to eliminate the immunosuppressive effects of TGF-β2 and reduce TGF-β2 levels in malignant tumors, thereby treating or ameliorating cancer symptoms, or delaying disease progression.

[0184] The target TGF-β2 mRNA can be the NCBI reference sequence: NM_003238.3, which has a sequence length of 5,882 bp. The target region of the TGF-β2 mRNA can be the protein coding sequence of reference 1,369 to 2,613.

[0185] Examples of the agent for inhibiting or suppressing TGF-β2 expression disclosed herein include TGF-β2-specific antisense oligonucleotides shown in SEQ ID NOs: 1-136 in Table 1.

[0186] Table 1: TGF-β2 specific antisense oligonucleotides

[0187]

[0188]

[0189]

[0190]

[0191]

[0192]

[0193]

[0194] 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 as active agents in any combination, such as a pooled combination.

[0195] It will be appreciated that additional antisense oligonucleotides can be constructed based on the TGF-β2 gene sequence.

[0196] In some embodiments, the TGF-β2-specific antisense oligonucleotides of the present invention may have no more than one or two mismatches compared to the target human TGF-β2.

[0197] In certain embodiments, the TGF-β2-specific antisense oligonucleotides of the invention can reduce TGF-β2 transcript levels by at least 60%, at least 70%, at least 80%, or at least 90%.

[0198] In further embodiments, the TGF-β2-specific antisense oligonucleotides of the invention may be selective for TGF-β2 and reduce any TGF-β1 transcript levels and any TGF-β3 transcript levels by less than 10%, less than 5%, or less than 1%.

[0199] In further embodiments, a therapeutically effective amount of an antisense agent for inhibiting or suppressing TGF-β2 expression 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.

[0200] In certain embodiments, antisense formulations used to inhibit or suppress TGF-β2 expression may 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.

[0201] In certain embodiments, methods of inhibiting or suppressing TGF-β2 expression using antisense agents may use 1 to 1000 mg / m 2 / day, or 1 to 500 mg / m 2 / day, or 1 to 250 mg / m 2 / day, or 1 to 100 mg / m 2 / day, or 1 to 50 mg / m 2 The average human body surface area can be about 1.6 to 1.9 m 2 .

[0202] In other embodiments, methods of inhibiting or suppressing TGF-β2 expression using antisense agents may use 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 to 10 mg / m 2 / day, or 0.5 to 5 mg / m 2 The average human body weight may be about 60 kg.

[0203] In certain embodiments, the agent for inhibiting or suppressing TGF-β2 expression of the present disclosure can be prepared from a lyophilized powder of the agent.

[0204] In some examples and embodiments, the agent may be a TGF-β2 specific antisense oligonucleotide selected from SEQ ID NOs: 1-136 and is administered or used by injection or infusion at a dose of 4 μl / min at a dose level of 10 μM on days 1 to 7, or 20 μM on days 1 to 7, or 40 μM on days 1 to 7, or 80 μM on days 1 to 7. In some embodiments, the agent may be a TGF-β2 specific antisense oligonucleotide selected from SEQ ID NOs: 9-136 that is chemically modified and is administered or used by injection or infusion at a dose of 4 μl / min at a dose level of 10 μM on days 1 to 7, or 20 μM on days 1 to 7, or 40 μM on days 1 to 7, or 80 μM on days 1 to 7.

[0205] In certain embodiments, OT-101 can be provided as a sterile lyophilisate in 20R glass vials, 250 mg per bottle, to prepare a solution before administration. The lyophilisate can be aseptically reconstituted in a sterile, preservative-free isotonic sodium chloride solution. The OT-101 solution can be administered continuously intravenously every 14 days using a portable pump system, administered on days 4-7 according to a 4-day dosing and 10-day withdrawal regimen. The regimen can be 7 days of dosing / 7 days of withdrawal, and 4 days of dosing / 10 days of withdrawal. The dosage can be 40, 80, 160, 140, 190, 250, or 330 mg.

[0206] In certain embodiments, the agent can be a TGF-β2 gene sequence-specific antisense oligonucleotide selected from SEQ ID NO: 1-136, and is administered or used by injection or infusion at a dose of 40, 80, 160, 140, 190, 250, 330 mg / m 2 (on days 1 to 7), or at doses of 40, 80, 160, 140, 190, 250, or 330 mg / m 2 (On days 1 to 4).

[0207] In some examples and embodiments, the agent can be a TGF-β2 gene sequence-specific antisense oligonucleotide selected from SEQ ID NOs: 1-136 and is 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 to 7, or 4 μM on days 1 to 7, or 8 μM on days 1 to 7, or 10 μM on days 1 to 7.

[0208] In some embodiments, the agent can be a TGF-β2 gene sequence-specific antisense oligonucleotide selected from SEQ ID NO: 9-136 and is 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 to 7, or 4 μM on days 1 to 7, or 8 μM on days 1 to 7, or 10 μM on days 1 to 7.

[0209] The therapeutically effective amount can also be determined using routine experimentation, for example, by monitoring the subject's response to drug administration and adjusting the dosage. For example, see Remington, The Science and Practice of Pharmacy (Gennaroed. 20th edition) (2000).

[0210] Embodiments of the present invention involving the administration or use of compositions of agents can ameliorate or suppress symptoms caused by TGF-β2-induced proteins.

[0211] Embodiments of the present invention also include pharmaceutical compositions for inhibiting or suppressing TGF-β2 expression, or for treating or ameliorating symptoms of cancer in humans or animals. The pharmaceutical composition may comprise a TGF-β2 inhibitor, a pharmaceutically acceptable salt form, ester, polymorph or stereoisomer thereof, and any combination thereof, and a carrier. The TGF-β2 inhibitor may be selected from the group consisting of TGF-β2-specific antisense oligonucleotides SEQ ID NOs: 1-136 or SEQ ID NOs: 9-136, and chemically modified variants thereof. The carrier may be sterile water for injection, physiological saline, isotonic saline, or a combination thereof.

[0212] Importantly, the compositions of the present disclosure can be substantially free of excipients. It has been found that the substantially excipient-free compositions of the present invention are surprisingly stable in a carrier. In some embodiments, the compositions can be stable in a carrier at 37° C. for at least 14 days, or at least 21 days, or at least 28 days.

[0213] In other embodiments, the pharmaceutical composition for infusion may 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.

[0214] The pharmaceutical agents of the present disclosure can be diluted and formulated into mixtures for administration by infusion in assemblies, such as intravenous infusion bags, syringes, and tubing known in the art.Such formulations can contain multiple pharmaceutical agents and excipients.

[0215]

[00146] Embodiments of the present invention further contemplate treatment modalities wherein the compositions of the present invention are administered or used in conjunction with standard of care therapies for a disease. Examples of other drugs that can be administered or used in combination with the compositions of the present invention include anti-inflammatory drugs, anti-inflammatory steroids, piperiquine, pyronaridine, curcumin, frankincense, remdesivir, Sompraz D, Zifi CV / Zac D, CCM, Broclear, budamate, Rapitus, Montek LC, low molecular weight heparin, prednisolone, paracetamol, vitamin B complex, vitamin C, pantoprazole, doxycycline, ivermectin, zinc, Foracort Rotacaps inhaler, injection ceftriaxone, paracetamol tablets, injection fragment protein Fragmin), Covifor Tablet, Azithromycin, Dexamethasone Injection, Ondansetron Injection, Multivitamin Tablet, Ascorbic Acid Tablet, Calcium Carbonate Tablet, and Zinc Sulfate Tablet.

[0216] Some TGF-β2-specific antisense oligonucleotide agents are disclosed in US 9,963, US 9,758,786 and US 8,476,246.

[0217] For example, the API trabedersen (OT-101) is a synthetic 18-mer S-ODN composed of adenine (A), thymine (T), guanine (G), and cytosine (C) bases, in which all 3'-5' bonds are modified to phosphorothioates. This sulfur modification makes the drug more resistant to degradation, thereby improving its stability in vitro and in vivo. Its primary molecular structure, namely the nucleotide sequence, is designed to be complementary to a specific sequence in the human transforming growth factor-β2 (TGF-β2) mRNA. This sequence and related sequences can leverage their excellent chemical and structural properties, biological activity, and specificity to achieve optimal antisense effects in vitro and in vivo.

[0218] Table 2 shows the chemical structure, examples of phosphorothioate moieties (CAG), and physical properties of trabedersen.

[0219] Table 2: Chemical and physical properties of Trabedersen (OT-101)

[0220]

[0221]

[0222] The investigational drug product is available as a sterile lyophilizate in 50 mL glass vials (primary container) containing 7.37 mg of trabedersen for intratumoral therapy and 20R glass vials (primary container) containing 250 mg of trabedersen for intravenous therapy, for preparation as a solution for infusion. The finished drug product may be excipient-free. The glass vials are suitable for parenteral administration. The glass vials are sealed with a sterile rubber stopper suitable for lyophilization. This stopper can be sealed with a crimped capsule with a colored flip-top cap. For clinical use, each vial is placed in a white folding box to protect the vial from light exposure and damage during transportation. Both the glass vial and the folding box can be labeled according to local requirements. The primary and secondary containers of the closure system meet international quality standards for sterile solid pharmaceutical packaging for injection.

[0223] The kit provides varying quantities of OT-101 lyophilized powder in 50 mL glass vials, specifying the total volume (in mL) and resulting concentration (in μM) after reconstitution.

[0224] The kit provides varying quantities of OT-101 lyophilized powder in 20 mL glass vials, and the calculated amount of OT-101 for each patient and treatment cycle is dissolved in a total volume of 85 mL of isotonic saline solution. The CADD portable infusion pump provides metered-dose medication for both inpatient and outpatient settings. It can be used for treatments requiring a continuous infusion rate. When administering medication via the CADD pump, central venous access and a Luer lock connector with a split-valve septum are recommended. Drug doses can be concentrated into small volumes. Required materials include a pump (Smiths Medical CADD SOLIS VIP), a yellow pillbox reservoir with a flow stop, clamp, and 100 mL female Luer connector, a CADD extension kit with a male Luer connector, clamp, 0.2-micron air elimination filter, and an integral anti-siphon valve with a male Luer connector.

[0225] The present invention also provides a kit comprising a lyophilized powder in vials, each containing 250 mg of each of one or more TGF-β2-specific antisense oligonucleotides selected from SEQ ID NOs: 1-136. The kit can include suitable vials and all necessary components of an application system, such as a syringe, catheter, and filter. The OT-101 lyophilized powder can be dissolved in an isotonic (0.9%) aqueous sodium chloride solution prior to use.

[0226] Checkpoint inhibitors

[0227] As referred to herein, checkpoint inhibitors known in the art are immune checkpoint inhibitors. Checkpoint inhibitors are immunotherapy drugs that block the binding of checkpoint proteins to their partner proteins. This prevents the "off" signal from being sent, thereby preventing T cells from killing cancer cells. More specifically, checkpoint proteins, such as PD-1 on T cells, can keep the immune response in check. The binding of PD-L1 to PD-1 prevents T cells from killing tumor cells. Therefore, using immune checkpoint inhibitors to block the binding of PD-L1 to PD-1 can enable T cells to kill tumor cells. The immune system is essentially turned back on, allowing T cells to attack cancer cells.

[0228] In some embodiments, the checkpoint inhibitors of the present disclosure may be inhibitors of CTLA-4, PD-1, or PD-L1.

[0229] In certain embodiments, the checkpoint inhibitors of the present disclosure may be inhibitors of PD-1.

[0230] In certain embodiments, a checkpoint inhibitor of the present disclosure may be pembrolizumab.

[0231] In certain embodiments, a checkpoint inhibitor of the present disclosure may be pembrolizumab, nivolumab, cemiplimab, spartalizumab, atezolizumab, avelumab, or durvalumab.

[0232] Without wishing to be bound by theory, the PD-1 receptor-ligand interaction may be a major pathway hijacked by tumors to suppress immune control. The normal function of PD-1, expressed on the surface of activated T cells in a healthy state, is to downregulate unnecessary or excessive immune responses, including autoimmune responses. Upon 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, leading to dephosphorylation of effector molecules involved in the CD3 T cell signaling cascade, such as CD3 zeta (CD3ζ), protein kinase C-theta (PKCθ), and ζ chain-associated protein kinase (ZAP70).

[0233] IL-2 immunotherapy

[0234] In certain embodiments, the interleukin immunotherapeutic of the present disclosure may be natural or synthetic IL-2, high-dose IL-2, recombinant IL-2, or aldesleukin.

[0235] Immunotherapeutics have anti-cancer effects because they can target the tumor microenvironment and activate immune responses against cancer cells. For example, interleukin-2 (IL-2) can promote the activation of natural killer (NK) cells and cytotoxic CD8 + Activation of T lymphocytes. Anti-tumor immune responses can involve the killing activity of helper T cells 1 (Th1) and other tumor cells.

[0236] Anticancer agents and treatment combinations

[0237] Embodiments of the present invention include combinations of TGF-β2-specific inhibitors with zero to minimal inhibition of the closely related TGF-β1 and TGF-β3 isoforms, and PD-1 checkpoint inhibitors.

[0238] In certain embodiments, the present invention provides therapeutic combinations of one or more antisense TGF-β2 inhibitors and a PD-1 checkpoint inhibitor.

[0239] Therapeutic embodiments of the present invention using a combination of one or more agents for inhibiting or suppressing TGF-β2 expression with a PD-1 immune checkpoint inhibitor 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.

[0240] Other embodiments of the invention include therapeutic combinations of a TGF-β2 inhibitor, an IL-2 immunotherapeutic agent, and a PD-1 checkpoint inhibitor.

[0241] In certain embodiments, the present invention provides therapeutic combinations of one or more antisense TGF-β2 inhibitors, IL-2 immunotherapeutics, and PD-1 checkpoint inhibitors.

[0242] The therapeutic embodiments of the present invention using one or more agents for inhibiting or suppressing TGF-β2 expression in combination with a PD-1 immune checkpoint inhibitor and an IL-2 immunotherapeutic agent 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.

[0243] The unexpected beneficial synergistic effects of therapeutic combinations of antisense TGF-β2 inhibitors, IL-2 immunotherapeutics, and checkpoint inhibitors for cancer treatment can be significantly increased when the checkpoint inhibitor is a PD-1 checkpoint inhibitor.

[0244] In the treatment of cancer patients, the use of a combination of a PD-1 checkpoint inhibitor, an antisense TGF-β2 inhibitor, and an IL-2 immunotherapeutic agent can significantly improve overall survival. The overall survival (OS) of such patients can be more than doubled. In some embodiments, the combination of an antisense TGF-β2 inhibitor, an IL-2 immunotherapeutic agent, and a PD-1 checkpoint inhibitor can provide an unexpected synergistic effect (based on clinical data).

[0245] Synergistic effects of therapeutic combinations

[0246] Embodiments of the present invention may provide synergistic effects of therapeutic combinations of agents that inhibit or suppress TGF-β2 expression with PD-1 checkpoint inhibitors and interleukin immunotherapeutics for treating or ameliorating cancer symptoms.

[0247] In some embodiments, the combination of an antisense agent that inhibits or suppresses TGF-β2 expression, a PD-1 checkpoint inhibitor, and an IL-2 immunotherapy agent for anti-cancer purposes is particularly effective for patients with high levels of tumor-associated monocytes and / or tumor-associated macrophages. Therefore, high levels of tumor-associated monocytes and / or tumor-associated macrophages can be used as biomarkers to screen patients who may benefit from combination therapy. Compared with CTLA4 or PD-L1 specific checkpoint inhibitors, the synergistic effect of combination therapy using PD-1 checkpoint inhibitors may be the strongest.

[0248] Without wishing to be bound by theory, since PD-1 is present in M2 tumor-associated macrophages, and antisense agents that inhibit or suppress the expression of TGF-β2 have the effect of repolarizing M2 and promoting anti-tumor effects, it appears that these two agents, when used in combination with IL-2, can act synergistically on the same target. TGF-β2 can play a central role in the programming of M1 tumor-associated macrophages, which can exhibit anti-tumor effects. In some aspects of the invention, inhibiting or suppressing TGF-β2 with antisense agents can produce anti-tumor effects. Antisense agents can have the effect of reprogramming to promote M1 tumor-associated macrophages. This reprogramming can have the effect of actively reducing and / or eliminating cancer tumors, especially when used in combination with agents that are inhibited by high TGF-β2.

[0249] To achieve effective stratification without any bias, the median of the population data was defined as the cutoff value. For each gene of interest, the median expression level across all samples was calculated. The median is the middle value in an ascending or descending list of numbers and is used because it is less affected by outliers than the mean. The samples were then stratified into two groups based on whether the expression level of a particular gene was above or below the median. This created a "high expression" group and a "low expression" group.

[0250] As used herein, "immunogenically hot tumors" refer to cancer types that are able to elicit a strong response from a patient's immune system. In contrast, "cold" tumors are less immunogenic, meaning they do not elicit a strong immune response.

[0251] In some embodiments, the agents, uses or methods of the present invention may be applied to immunogenic cold pancreatic cancer or immunogenic hot melanoma.

[0252] In some embodiments, the agents, uses or methods of the present invention can be applied to cancers that are between immunogenic cold and immunogenic hot, which can be skin cancer, lung cancer, breast cancer, prostate cancer, colorectal cancer, kidney cancer, gastric cancer, ovarian cancer, cervical cancer, liver cancer or multiple myeloma.

[0253] Numbered embodiments of the present invention include the following:

[0254] 1) An antisense agent for inhibiting or suppressing TGF-β2 expression in combination with a checkpoint inhibitor for treating or ameliorating cancer symptoms in human subjects or animals.

[0255] 2) Use of an antisense agent for inhibiting or suppressing TGF-β2 expression in the preparation of a medicament for treating or ameliorating cancer symptoms in human subjects or animals in combination with a checkpoint inhibitor.

[0256] 3) A method for treating or ameliorating cancer symptoms in a human or animal subject in need thereof, the method comprising:

[0257] administering to the subject a therapeutically effective amount of an antisense agent for inhibiting or suppressing TGF-β2 expression;

[0258] A therapeutically effective amount of a checkpoint inhibitor is administered to the subject.

[0259] 4) The agent according to embodiment 1, in combination with an interleukin immunotherapy agent.

[0260] 5) The use according to embodiment 2, in combination with an interleukin immunotherapy agent.

[0261] 6) The method of embodiment 3, comprising administering to the subject a therapeutically effective amount of an interleukin immunotherapeutic agent.

[0262] 7) The agent, use or method of any one of embodiments 1-6, wherein the agent for inhibiting or suppressing TGF-β2 expression, the checkpoint inhibitor and the interleukin immunotherapy agent are administered concurrently, simultaneously, sequentially or separately in time.

[0263] 8) The agent, use or method of any one of embodiments 1-7, wherein the agent that inhibits or suppresses TGF-β2 expression, the checkpoint inhibitor and the interleukin immunotherapy agent are administered alone or in a combined preparation by injection or infusion.

[0264] 9) The agent, use or method of any one of embodiments 1-8, 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.

[0265] 10) The agent, use or method according to any one of embodiments 1 to 9, wherein the agent for inhibiting or suppressing TGF-β2 expression is a TGF-β2-specific antisense oligonucleotide complementary to the TGF-β2 transcript and having a length of 15-30 nucleotides.

[0266] 11) The agent, use or method according to any one of embodiments 1 to 10, wherein the agent for inhibiting or suppressing TGF-β2 expression is a TGF-β2-specific antisense oligonucleotide that is complementary to TGF-β2 pre-RNA, pre-mRNA or mRNA and has a length of 18-21 nucleotides.

[0267] 12) The agent, use or method of any one of embodiments 1-11, wherein the agent for inhibiting or suppressing TGF-β2 expression is one or more TGF-β2-specific antisense oligonucleotides complementary to TGF-β2 transcripts shown in Table 1, and chemically modified variants thereof, LNA variants thereof, gapmer variants thereof, and any combination or collection thereof.

[0268] 13) The agent, use or method according to any one of embodiments 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.

[0269] 14) The agent, use or method according to any one of embodiments 1-13, wherein the TGF-β2 specific antisense oligonucleotide reduces the level of TGF-β2 transcript by at least 60%, or at least 70%, or at least 80%, or at least 90%.

[0270] 15) The agent, use or method according to any one of embodiments 1-14, wherein the TGF-β2 specific antisense oligonucleotide reduces the level of any TGF-β1 transcript and the level of any TGF-β3 transcript by less than 10%, or less than 5%, or less than 1%.

[0271] 16) The agent, use or method of any one of embodiments 1-15, wherein the TGF-β2 specific antisense oligonucleotide has one or more nucleotides chemically modified to be phosphorothioate internucleoside linkages, methoxypropylphosphonate internucleoside linkages, or phosphoramidite linkages linked 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.

[0272] 17) The agent, use or method of any one of embodiments 1-16, wherein the antisense agent is conjugated to polyethylene glycol, a lipid or a tri-branched N-acetylgalactosamine.

[0273] 18) The medicament, use or method according to any one of embodiments 1 to 17, wherein each medicament comprises a carrier of sterile water for injection, saline, isotonic saline or a combination thereof, and the carrier of each medicament can be the same or different.

[0274] 19) The medicament, use or method according to any one of embodiments 1-18, wherein the medicament is substantially free of excipients.

[0275] 20) The medicament, use or method according to any one of embodiments 1-19, wherein the medicament is stable at 37°C for at least 14 days in a carrier substantially free of excipients.

[0276] 21) The agent, use or method of any one of embodiments 1-20, wherein the checkpoint inhibitor is an inhibitor of PD-1.

[0277] 22) The agent, use or method of any one of embodiments 1-21, wherein the checkpoint inhibitor is pembrolizumab, nivolumab, cemiplizumab, spartazimab, atezolizumab, avelumab or durvalumab.

[0278] 23) The agent, use or method of any one of embodiments 1-22, wherein the interleukin immunotherapeutic agent is natural IL-2, high-dose IL-2, recombinant IL-2 or aldesleukin.

[0279] 24) The agent, use or method of any one of embodiments 1-23, comprising selecting a subject who would 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.

[0280] 25) The agent, use or method of any one of embodiments 1-24, wherein the one or more biomarkers is IRF5, and the subject is selected when the expression level of IRF5 is above the median.

[0281] 26) The agent, use or method of any one of embodiments 1-25, wherein the one or more biomarkers is ITGAM, and the subject is selected when the expression level of ITGAM is above the median.

[0282] 27) The agent, use or method of any one of embodiments 1-26, wherein the subject has reduced levels of TGF-β2 after administration or use compared to before administration or use.

[0283] 28) The agent, use or method of any one of embodiments 1-27, wherein the subject has increased IRF5 levels after administration or use compared to before administration or use.

[0284] 29) The agent, use or method of any one of embodiments 1-28, wherein the subject has reduced ITGAM levels after administration or use compared to before administration or use.

[0285] 30) The agent, use or method of any one of embodiments 1-29, comprising administering to a subject a therapeutically effective amount of an IRF5 or ITGAM expression product.

[0286] 31) The agent, use or method according to any one of embodiments 1-30, wherein the expression product is mRNA, polypeptide, protein or fragment thereof, or a combination thereof.

[0287] 32) The agent, use or method of any one of embodiments 1-31, wherein said administration or use reduces mortality at 6, 12, 18, 24, 30 or 36 months.

[0288] 33) The agent, use or method of any one of embodiments 1-32, wherein the administration or use increases overall survival at 6, 12, 18, 24, 30 or 36 months.

[0289] 34) The agent, use or method of any one of embodiments 1-33, in combination with one or more drugs comprising targeted cancer drugs, cancer growth blockers, EGFR inhibitors and combinations thereof.

[0290] 35) The agent, use or method of any one of embodiments 1-34, in combination with one or more drugs selected from bevacizumab, everolimus, bevacizumab, dabrafenib, trametinib and combinations thereof.

[0291] 36) The agent, use or method of any one of embodiments 1-35, in combination with any one or more drugs, which are cancer growth inhibitors selected from angiogenesis inhibitors, histone deacetylase inhibitors, hedgehog factor blockers, mTOR inhibitors, p53 inhibitors, PARP inhibitors, proteasome inhibitors, tyrosine kinase inhibitors and combinations thereof.

[0292] 37) The agent, use or method of any one of embodiments 1-36, in combination with any one or more drugs, wherein the drug is an EGFR inhibitor selected from erlotinib, gefitinib, afatinib, osimertinib, dacomitinib and a combination thereof.

[0293] 38) The agent, use or method according to any one of embodiments 1-37, in combination with a chemotherapeutic drug.

[0294] 39) The agent, use or method of any one of embodiments 1-38, in combination with radiation therapy or electric field therapy.

[0295] 40) An antisense agent for inhibiting or suppressing TGF-β2 expression is used in combination with an interleukin immunotherapy agent for treating or ameliorating cancer symptoms in human subjects or animals.

[0296] 41) Use of an antisense agent for inhibiting or suppressing TGF-β2 expression in the preparation of a medicament for use in combination with an interleukin immunotherapy agent to treat or improve cancer symptoms in human subjects or animals.

[0297] 42) A method for treating or ameliorating cancer symptoms in a human or animal subject in need thereof, the method comprising:

[0298] administering to the subject a therapeutically effective amount of an antisense agent for inhibiting or suppressing TGF-β2 expression;

[0299] A therapeutically effective amount of an interleukin immunotherapeutic agent is administered to the subject.

[0300] 43) The agent, use or method according to any one of embodiments 40-42, wherein the agent for inhibiting or suppressing TGF-β2 expression and the interleukin immunotherapy agent are administered concurrently, simultaneously, sequentially or separately in time.

[0301] 44) The agent, use or method according to any one of embodiments 40-43, wherein the agent for inhibiting or suppressing TGF-β2 expression and the interleukin immunotherapy agent are administered separately by injection or infusion or in the form of a combined preparation.

[0302] 45) The agent, use or method of any one of embodiments 40-44, 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.

[0303] 46) The agent, use or method of any one of embodiments 40-45, wherein the agent for inhibiting or suppressing TGF-β2 expression is a TGF-β2-specific antisense oligonucleotide that is complementary to the TGF-β2 transcript and has a length of 15-30 nucleotides.

[0304] 47) The agent, use or method of any one of embodiments 40-46, wherein the agent for inhibiting or suppressing TGF-β2 expression is a TGF-β2-specific antisense oligonucleotide that is complementary to TGF-β2 pre-RNA, pre-mRNA or mRNA and has a length of 18-21 nucleotides.

[0305] 48) The agent, use or method of any one of embodiments 40-47, wherein the agent for inhibiting or suppressing TGF-β2 expression is one or more TGF-β2-specific antisense oligonucleotides shown in Table 1 that are complementary to the TGF-β2 transcript.

[0306] 49) The agent, use or method of any one of embodiments 40-48, wherein the TGF-β2 specific antisense oligonucleotide has one or more nucleotides chemically modified to be phosphorothioate internucleoside linkages, methoxypropylphosphonate internucleoside linkages, or aminophosphoric acid linkages linked 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] 50) The agent, use or method of any one of embodiments 40-49, wherein the antisense agent is conjugated to polyethylene glycol, a lipid or a tri-branched N-acetylgalactosamine.

[0308] 51) The agent, use or method according to any one of embodiments 40-50, wherein each agent comprises a carrier of sterile water for injection, saline, isotonic saline or a combination thereof, and the carrier of each agent can be the same or different.

[0309] 52) The medicament, use or method of any one of embodiments 40-51, wherein the medicament is substantially free of excipients.

[0310] 53) The medicament, use or method according to any one of embodiments 40-52, wherein the medicament is stable at 37°C for at least 14 days in a carrier substantially free of excipients.

[0311] 54) The agent, use or method of any one of embodiments 40-53, wherein the interleukin immunotherapeutic agent is natural IL-2, high-dose IL-2, recombinant IL-2 or aldesleukin.

[0312] 55) The medicament, use or method of any one of embodiments 40-54, wherein said administration or use reduces mortality at 6, 12, 18, 24, 30 or 36 months.

[0313] 56) The agent, use or method of any one of embodiments 40-55, wherein said administration or use increases survival at 6, 12, 18, 24, 30 or 36 months.

[0314] 57) The agent, use or method of any one of embodiments 40-56, in combination with any one or more drugs comprising targeted cancer drugs, cancer growth blockers, EGFR inhibitors and combinations thereof.

[0315] 58) The agent, use or method according to any one of embodiments 40-57, in combination with any one or more drugs selected from bevacizumab, everolimus, bezitufen, dabrafenib, trametinib and combinations thereof.

[0316] 59) The agent, use or method of any one of embodiments 40-58, in combination with any one or more drugs, which are cancer growth inhibitors selected from angiogenesis inhibitors, histone deacetylase inhibitors, hedgehog factor blockers, mTOR inhibitors, p53 inhibitors, PARP inhibitors, proteasome inhibitors, tyrosine kinase inhibitors and combinations thereof.

[0317] 60) The agent, use or method of any one of embodiments 40-59, in combination with any one or more drugs, wherein the drug is an EGFR inhibitor selected from erlotinib, gefitinib, afatinib, osimertinib, dacomitinib and a combination thereof.

[0318] 61) The agent, use or method according to any one of embodiments 40-60, in combination with a chemotherapeutic drug.

[0319] 62) The agent, use or method of any one of embodiments 40-61, in combination with radiation therapy or electric field therapy.

[0320] 63) The agent, use or method of any one of embodiments 40-62, comprising selecting a subject who would benefit from the agent, use or method based on the level of one or more biomarkers TGF-β2, IRF5, ITGAM and combinations thereof.

[0321] 64) The agent, use or method of any one of embodiments 40-63, comprising selecting a subject who would benefit from the agent, use or method based on the levels of one or more biomarkers TGF-β2, IRF5, ITGAM, neoantigens, mutation load, macrophages and combinations thereof.

[0322] 65) The agent, use or method of any one of embodiments 40-64, wherein the one or more biomarkers is IRF5 and the subject is selected when the expression level of IRF5 is below the median.

[0323] 66) The agent, use or method of any one of embodiments 40-65, wherein the one or more biomarkers are tumor associated macrophages, and the subject is selected when the tumor associated macrophages are below average.

[0324] 67) The agent, use or method of any one of embodiments 40-66, wherein one or more biomarkers is tumor neoantigen mutation burden, and the subject is selected when the neoantigen tumor burden is below average.

[0325] 68) The agent, use or method of any one of embodiments 40-67, comprising administering to a subject a therapeutically effective amount of an agent for inhibiting or suppressing the expression of ITGAM or IRF5.

[0326] 69) The agent, use or method of any one of embodiments 40-68, wherein the agent for inhibiting or suppressing the expression of ITGAM or IRF5 is an antisense oligonucleotide targeting ITGAM or IRF5, respectively.

[0327] 70) A kit for treating or improving cancer symptoms, comprising:

[0328] a therapeutically effective amount of an antisense agent for inhibiting or suppressing TGF-β2 expression; and

[0329] A therapeutically effective amount of a checkpoint inhibitor.

[0330] 71) The kit of embodiment 70, comprising a therapeutically effective amount of an interleukin immunotherapeutic agent.

[0331] 72) The kit according to any one of embodiments 70-71, 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.

[0332] 73) The kit according to any one of embodiments 70-72, wherein the agent for inhibiting or suppressing TGF-β2 expression is a TGF-β2 specific antisense oligonucleotide complementary to the TGF-β2 transcript and having a length of 15-30 nucleotides.

[0333] 74) The kit according to any one of embodiments 70-73, wherein the agent for inhibiting or suppressing TGF-β2 expression is a TGF-β2-specific antisense oligonucleotide complementary to TGF-β2 pre-RNA, pre-mRNA or mRNA and having a length of 18-21 nucleotides.

[0334] 75) The kit according to any one of embodiments 70-74, wherein the agent for inhibiting or suppressing TGF-β2 expression is one or more TGF-β2 specific antisense oligonucleotides complementary to TGF-β2 transcripts as shown in Table 1.

[0335] 76) The kit according to any one of embodiments 70-75, wherein the TGF-β2 specific antisense oligonucleotide has no more than one or two mismatches compared to the target human TGF-β2.

[0336] 77) The kit according to any one of embodiments 70-76, wherein the TGF-β2 specific antisense oligonucleotide reduces the level of TGF-β2 transcript by at least 60%, or at least 70%, or at least 80%, or at least 90%.

[0337] 78) The kit according to any one of embodiments 70-77, wherein the TGF-β2 specific antisense oligonucleotide reduces the level of any TGF-β1 transcript and the level of any TGF-β3 transcript by less than 10%, or less than 5%, or less than 1%.

[0338] 79) The kit of any one of embodiments 70-78, wherein the TGF-β2 specific antisense oligonucleotide has one or more nucleotides chemically modified to be phosphorothioate internucleoside linkages, methoxypropylphosphonate internucleoside linkages, aminophosphoric acid linkages linked 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.

[0339] 80) The kit of any one of embodiments 70-79, wherein the antisense agent is conjugated to polyethylene glycol, a lipid or a tri-branched N-acetylgalactosamine.

[0340] 81) The kit according to any one of embodiments 70-80, wherein each agent comprises a carrier of sterile water for injection, saline, isotonic saline or a combination thereof, and the carrier of each agent can be the same or different.

[0341] 82) The kit according to any one of embodiments 70-81, wherein the agent is substantially free of excipients.

[0342] 83) The kit according to any one of embodiments 70-82, wherein the agent is stable at 37°C for at least 14 days in a carrier substantially free of excipients.

[0343] All publications mentioned in this specification, including patents, patent application publications, and non-patent publications, as well as the Sequence Listing, are expressly incorporated herein by reference in their entirety for all purposes.

[0344] Although the foregoing disclosure has been described in detail through examples for clarity of understanding, it will be understood by those skilled in the art that the present disclosure encompasses certain changes and modifications and can be implemented within the scope of the appended claims without undue experimentation, and that these examples are intended to be illustrative and not limiting. The present invention encompasses all such additional embodiments, equivalents, and modifications. The present invention encompasses any combination or mixture of the features, materials, elements, or limitations of the various illustrative components, embodiments, and embodiments claimed.

[0345] It is emphasized that, in accordance with common practice, the features in the drawings are of arbitrary scale and are intended to cover similar features that may be arbitrarily enlarged or reduced in size. Example

[0346] Example 1

[0347] A clinical study was conducted to investigate the overall survival of melanoma patients treated with a combination of antisense TGF-β2 inhibitors, IL-2 immunotherapy agents, and PD-1 checkpoint inhibitors. The study obtained clinical results of 423 patients diagnosed with melanoma (for immunotherapy, see KM plotter, website

[0348] https: / / kmplot.com / analysis / index.php?p=service&cancer=immunotherapy).

[0349] Figure 1 Shown are the results of this clinical study of overall survival in patients with melanoma. Figure 1 Kaplan-Meier overall survival plots for such clinical efficacy studies are shown. Figure 1 The results show that after the use of PD-1 inhibitors, the survival of patients with high IL2 (left panel) is improved, which can be provided by IL-2 immunotherapy agents; the survival of patients with low TGF-β2 (right panel) is improved, which can be provided by antisense TGF-β2 inhibitors. These clinical data provide a basis for the combination of antisense TGF-β2 inhibitors, PD-1 checkpoint inhibitors and IL-2 immunotherapy agents to improve the overall survival of patients with melanoma. Figure 1 The log-rank P value in indicates that based on this clinical study, the use of this treatment combination has a highly significant effect on improving survival.

[0350] Figure 2 Shows the results of a clinical study on overall survival of patients with melanoma. Figure 2 Kaplan-Meier overall survival plots for such clinical efficacy studies are shown. Figure 2 The results show that after the use of PD-1 inhibitors, a higher IL-2 / TGF-β2 ratio (left panel) clearly indicates a significant improvement in survival, which can be provided by IL-2 immunotherapy agents and antisense TGF-β2 inhibitors. These clinical data provide a basis for the treatment combination of antisense TGF-β2 inhibitors, PD-1 checkpoint inhibitors and IL-2 immunotherapy agents to improve the overall survival of melanoma patients. Figure 2 The log-rank P value in (left panel) indicates that the use of this treatment combination has a highly significant effect on improving survival based on this clinical study.

[0351] Figure 2 Clinical data from the study showed that a high IL-2 / TGF-β2 ratio (corresponding to high IL2 and low TGF-β2) is a powerful driver of survival in melanoma patients. Under a high IL-2 / TGF-β2 ratio, the survival of melanoma patients in the upper quartile surprisingly extended from 6.7 months to 15 months (p = 5.4e-06) ( Figure 2 , left). Thus, these clinical data provide evidence that the therapeutic combination of an antisense TGF-β2 inhibitor, a PD-1 checkpoint inhibitor, and an IL-2 immunotherapy agent has an unexpected synergistic effect in improving overall survival in patients with melanoma.

[0352] Example 2

[0353] A clinical study was conducted to investigate overall survival in patients with melanoma (N=162) and pancreatic cancer (N=88) who received a combination of an antisense TGF-β2 inhibitor, an IL-2 immunotherapy drug, and a PD-1 checkpoint inhibitor (for immunotherapy, see KM Plotter, 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_rnase q).

[0354] Figure 3-18 The results of this clinical study, which evaluated the overall survival of melanoma patients treated with PD-1 therapy and pancreatic cancer patients who did not receive treatment, were presented. Figure 3-18 Kaplan-Meier overall survival plots from such a clinical efficacy study are shown, covering two extremes: hot immunogenic melanoma and cold nonimmunogenic pancreatic tumors.

[0355] Figure 3 、 5 , 7, 9, 11, 13, 15, and 17 (upper left and upper right panels) show the Kaplan-Meier overall survival plots of melanoma patients in this study. Figure 3 、 5 , 7, 9, 11, 13, 15, and 17 (lower left and lower right panels) show the Kaplan-Meier overall survival plots of pancreatic cancer patients (PDAC) in this study in terms of TGF-β2 expression. Figure 4 、 6 , 8, 10, 12, 14, 16, and 18 (left and right) show the Kaplan-Meier overall survival plots of pancreatic cancer patients (PDAC) in this study in terms of IL-2 expression.

[0356] Figure 3(Top left) shows that low TGF-β2 and high ITGAM indicate improved survival in melanoma patients treated with PD-1 inhibitors. These data suggest that high ITGAM is a useful biomarker for overall survival in patients receiving the combination of antisense TGF-β2 inhibitors and PD-1 checkpoint inhibitors and can be used to identify patients who are likely to benefit. Figure 3 (Top right) shows that low ITGAM is not a preferred indicator in these cases.

[0357] Figure 3 (Lower left, lower right) show that neither low nor high ITGAM is a preferred indicator for pancreatic cancer patients (PDAC) using PD-1 inhibitors.

[0358] Figure 4 (Left panel) shows that for pancreatic cancer patients (PDAC) using PD-1 inhibitors, low TGF-β2 and high ITGAM indicate improved survival. The log-rank P value = 0.034 indicates significance (<0.05), although even lower P values ​​are preferred. These data suggest that high ITGAM is a useful biomarker for overall survival in patients receiving a combination of antisense TGF-β2 inhibitors and PD-1 checkpoint inhibitors and can be used to screen patients who may benefit. Figure 4 (Right panel) shows that low ITGAM is not a preferred indicator in these cases.

[0359] Figure 5 (Top left) shows that neither low nor high CD8A is a preferred indicator for melanoma patients taking PD-1 inhibitors.

[0360] Figure 5 (Lower left, lower right) show that neither low nor high CD8A is a preferred indicator for pancreatic cancer patients (PDAC) using PD-1 inhibitors.

[0361] Through Figure 3-18 The data were analyzed similarly and the conclusions are summarized in Table 3.

[0362] Table 3: Prioritized biomarkers for melanoma and PDAC

[0363]

[0364]

[0365] The data for melanoma treated with PD-1 can be summarized as follows: CD19 and IRF5 were highly significant for TGF-β2-driven improved overall survival outcomes compared with melanoma treated with PD-1 alone.

[0366] The data for PDAC can be summarized as follows: low IRF5 and low CD19 were significantly associated with improved overall survival outcomes in TGF-β2-driven studies. With the exception of ITGAM, these biomarkers had no significant impact on overall survival in IL-2-driven studies in PDAC.

[0367] Therefore, the first finding of this clinical study is that ITGAM, CD19, IRF5, and CD163 are useful biomarkers for overall survival in melanoma patients receiving the treatment combination of an antisense TGF-β2 inhibitor and a PD-1 checkpoint inhibitor.

[0368] A second finding from this clinical study was that IRF5 is a useful biomarker for overall survival in pancreatic adenocarcinoma (PDAC) patients receiving therapeutic antisense TGF-β2-specific inhibitors.

[0369] A third finding of this clinical study is that ITGAM may be a useful biomarker for overall survival in pancreatic cancer (PDAC) patients receiving a treatment combination of IL-2 therapy and a PD-1 checkpoint inhibitor.

[0370] Another finding from this clinical study was that IRF5 was particularly useful as a biomarker for overall survival in melanoma patients receiving therapeutic antisense TGF-β2-specific inhibitors.

[0371] Another finding from this clinical study was that IRF5 was particularly useful as a biomarker for overall survival in pancreatic cancer (PDAC) patients receiving a combination of antisense TGF-β2 inhibitors and PD-1 checkpoint inhibitors.

[0372] The following cell types were involved in this clinical study:

[0373] B cells = CD19

[0374] CD8 + T cells = CD8A

[0375] CD4 + T cells = CD4

[0376] M1 macrophages = NOS2 or IRF5

[0377] M2 macrophages = CD163

[0378] Neutrophils = ITGAM

[0379] Dendritic cells = ITGAX

[0380] Example 3

[0381] To investigate the overall survival of patients with pancreatic adenocarcinoma (PDAC) receiving a combination of an antisense TGF-β2 inhibitor, an IL-2 immunotherapy agent, and a PD-1 checkpoint inhibitor.

[0382] The purpose of this study was to investigate overall survival in patients with melanoma (N=423) and pancreatic cancer (N=177) treated with immunotherapy (KM Plotter Pan-cancer RNA-seq, https: / / kmplot.com / analysis / index.php?p=service&cancer=immunotherapy).

[0383] Figure 19-20 Shown are the results of this clinical study on overall survival of PDAC patients. Figure 19-20 Kaplan-Meier overall survival plots for such clinical efficacy studies are shown.

[0384] Figure 19-20 They showed that for IL-2-driven survival, and when additionally stratified by various immune cell markers (i.e., basophils, B cells, eosinophils, M0 macrophages, and Th1 helper cells), high IL-2 levels significantly improved survival across all cases. M0 macrophages were a highly significant factor. Figure 19-20 The results showed that based on the clinical study and conditions, the log-rank P value indicated that the combination of IL-2 immunotherapy and PD-1 checkpoint inhibitors had a significant effect on improving survival. Figure 20 (Upper and lower right panels) show comparative baseline results.

[0385] This pancreatic adenocarcinoma (PDAC) clinical study demonstrated that low macrophages synergistically interacted with IL-2 immunotherapy in PDAC.

[0386] Example 4

[0387] To investigate the overall survival of pancreatic cancer patients treated with antisense TGF-β2 inhibitors, a clinical study was conducted. Clinical outcomes were obtained from 80 to 108 patients diagnosed with pancreatic cancer (cBioPortalFor Cancer Genomics).

[0388] Figure 21 Shows the results of this clinical study on overall survival of patients with pancreatic cancer. Figure 21Kaplan-Meier overall survival plots for such clinical efficacy studies are shown. Figure 21 (Upper left panel) shows that high tumor mRNA levels of TGF-β2 significantly reduced survival in patients with low levels of tumor-associated macrophages. Figure 21 The log-rank P value in (upper left panel) indicates that, based on the clinical study and conditions, the improvement in survival with therapeutic antisense TGF-β2 inhibitors was significant. Patients with high tumor TGF-β2 expression had a survival of only 15 months, whereas patients with low tumor TGF-β2 expression had a survival of 73 months.

[0389] Figure 21 (Top right) shows that for patients with low tumor-associated macrophages and low mutational burden (neoantigens), high tumor mRNA levels of TGF-β2 significantly reduced survival. Figure 21 The log-rank P values ​​in the upper right panel indicate that, based on the clinical study and conditions, the improvement in survival with a therapeutic antisense TGF-β2 inhibitor was significant. Patients whose tumors expressed high levels of TGF-β2 had a survival of only 15 months, whereas those whose tumors expressed low levels of TGF-β2 had a survival of 73 months. This suggests that only TGF-β2 had an effect on survival. Neither TGF-β1 nor TGF-β3 had an effect on survival (see Figure 21 The log-rank P in (lower left and lower right figures, respectively).

[0390] Therefore, this clinical study shows that tumor mRNA levels of TGF-β2, low tumor-associated macrophages, and low mutational burden (neoantigens) are biomarkers of improved overall survival in pancreatic cancer patients receiving therapeutic antisense TGF-β2 inhibitors. These biomarkers can be used to select patients who benefit from treatment with TGF-β2 inhibitors.

[0391] Example 5

[0392] To understand the overall survival of cancer patients receiving a combination of antisense TGF-β2 inhibitors, IL-2 immunotherapies, and checkpoint inhibitors, a clinical study was conducted. Clinical outcomes were obtained from 1,045 patients diagnosed with cancer (cBioPortal For Cancer Genomics). Clinical outcomes covered a variety of cancer diagnoses, including bladder cancer (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 carcinoma (N=348).

[0393] Figure 22-30Shows the results of this clinical study on overall survival of cancer patients. Figure 22-30 Kaplan-Meier overall survival plots for such clinical efficacy studies are shown.

[0394] Figure 22 The results showed that high levels of IL-2 significantly prolonged survival when using any checkpoint inhibitor (PD-1, PD-L1, or CTLA-4) for all tumor types. Figure 22 The log-rank P value in the data suggests that, based on this clinical study and the conditions, the use of IL-2 immunotherapy significantly improved survival. Patients with high IL-2 expression had a survival of 18 months, while patients with low tumor TGF-β2 expression had a survival of 14 months.

[0395] Figure 23 The study showed that low levels of TGF-β2 slightly improved survival when using checkpoint inhibitors. Patients with low TGF-β2 expression had a survival of 18 months, while those with high TGF-β2 expression had a survival of 15 months.

[0396] Importantly, Figure 24 showed that low TGF-β2 levels and high IL-2 levels significantly improved survival when using checkpoint inhibitors. Figure 24 Overall survival stratified based on the IL2 / TGFB2 expression ratio is shown. Figure 24 The log-rank P value in the data indicates that, based on the clinical study and conditions, the combination of an antisense TGF-β2 inhibitor, an IL-2 immunotherapy, and a checkpoint inhibitor significantly improved survival. Patients with high IL2 / TGFB2 expression had a survival of 20 months, while those with low IL2 / TGFB2 expression had a survival of only 14 months. This clinical data demonstrates that the combination of an antisense TGF-β2 inhibitor, an IL-2 immunotherapy, and a checkpoint inhibitor has a synergistic effect.

[0397] The study found that the synergistic effect of the therapeutic combination of an antisense TGF-β2 inhibitor, an IL-2 immunotherapy agent, and a checkpoint inhibitor was uniquely and significantly enhanced for melanoma. Figure 25 The study showed that for 423 melanoma patients, high levels of IL-2 significantly improved survival after checkpoint inhibitors were used. Patients with high IL-2 expression had a survival of 33 months, while those with low IL-2 expression had a survival of 21 months.

[0398] Figure 26The study showed that low levels of TGF-β2 significantly prolonged survival in 423 melanoma patients treated with checkpoint inhibitors, with patients with low TGF-β2 expression living for 29 months, compared to 18 months for those with high TGF-β2 expression.

[0399] Importantly, Figure 27 showed that among 423 melanoma patients, low levels of TGF-β2 and high levels of IL-2 significantly improved survival when using checkpoint inhibitors. Figure 24 Overall survival stratified based on the IL2 / TGFB2 expression ratio is shown. Figure 24 The log-rank P value in the data indicates that the combination of an antisense TGF-β2 inhibitor, an IL-2 immunotherapy, and a checkpoint inhibitor has an unexpectedly high significance for improving survival, given the clinical study and conditions. Patients with high IL2 / TGFB2 expression had a survival of 20 months, while patients with low IL2 / TGFB2 expression had a survival of only 14 months. This clinical data suggests that the combination of an antisense TGF-β2 inhibitor, an IL-2 immunotherapy, and a checkpoint inhibitor has a synergistic effect.

[0400] The study found that the synergistic effect of the therapeutic combination of an antisense TGF-β2 inhibitor, an IL-2 immunotherapy agent, and a checkpoint inhibitor was uniquely and significantly enhanced with PD-1 checkpoint inhibitors in cancer patients.

[0401] Figure 28 The study showed that for all cancer patients, high levels of IL-2 significantly prolonged survival after treatment with PD-1 checkpoint inhibitors. Patients with high IL-2 expression had a survival of 28 months, while those with low IL-2 expression had a survival of 17 months.

[0402] For cancer patients, Figure 29 The study showed that low levels of TGF-β2 significantly prolonged survival after treatment with PD-1 checkpoint inhibitors, with patients with low TGF-β2 expression living for 28 months, compared to 16 months for those with high TGF-β2 expression.

[0403] Importantly, Figure 30 showed that in cancer patients, low levels of TGF-β2 and high levels of IL-2 significantly improved survival when using PD-1 checkpoint inhibitors. Figure 30 Overall survival stratified based on the IL2 / TGFB2 expression ratio is shown. Figure 30The log-rank P value in the data indicates that the combination of an antisense TGF-β2 inhibitor, an IL-2 immunotherapy, and a PD-1 checkpoint inhibitor had an unexpectedly high significance for improving survival, given the clinical study and conditions. Patients with high IL2 / TGFB2 expression had a survival of 31 months, while those with low IL2 / TGFB2 expression had a survival of 14 months. These clinical data suggest that the combination of an antisense TGF-β2 inhibitor, an IL-2 immunotherapy, and a PD-1 checkpoint inhibitor has a synergistic effect.

[0404] Example 6

[0405] To investigate the overall survival of cancer patients receiving a combination of an antisense TGF-β2 inhibitor, an IL-2 immunotherapy agent, and a checkpoint inhibitor, a clinical study was conducted. A clinical study was conducted to investigate the overall survival of melanoma patients (N=423) treated with immunotherapy (for immunotherapy, see KM plotter, URL: https: / / kmplot.com / analysis / index.php?p=service&cancer=immunotherapy) and pancreatic cancer patients (N=177) (KMPlotter Pan-cancer RNA-seq, URL: https: / / kmplot.com / analysis / index.php?p=service&cancer=pancancer_rnase q).

[0406] This clinical study showed that when the checkpoint inhibitors were CTLA-4 or PD-L1, the combination of antisense TGF-β2 inhibitors, IL-2 immunotherapy agents, and checkpoint inhibitors did not improve overall survival. Therefore, in other examples herein, the combination of antisense TGF-β2 inhibitors, IL-2 immunotherapy agents, and PD-1 checkpoint inhibitors showed a surprising advantage in improving overall survival. The data details are as follows.

[0407] Figures 31-33 Results for three stratified biomarkers (i.e., 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) are shown. Each biomarker showed improved overall survival at high expression levels.

[0408] Figures 34-36Results for three stratified biomarkers (i.e., PDCD1, CD274, and IL-2 / TGF-β2 ratio) for PD-1 checkpoint inhibitors and all tumor types (N=955) are shown. Except for IL-2 / TGF-β2, each biomarker showed improved overall survival at high expression levels. Therefore, PD-L1 is not a preferred checkpoint inhibitor.

[0409] Figures 37-39 Results for CTLA-4 checkpoint inhibitors and all tumor types using three stratified biomarkers (i.e., PDCD1, CD274, and IL-2 / TGF-β2 ratio) are shown (N=121). Only the CD274 biomarker showed a slightly improved overall survival at high expression levels. Therefore, CTLA-4 is not a preferred checkpoint inhibitor.

[0410] Figures 40-42 Results for three stratification biomarkers (i.e., PDCD1, CD274, and IL-2 / TGF-β2 ratio) in melanoma patients for any checkpoint inhibitor (N=397) are shown. Each biomarker showed improved overall survival at high expression levels.

[0411] Figures 43-45 Results of three stratified biomarkers (i.e., PDCD1, CD274, and IL-2 / TGF-β2 ratio) for CTLA-4 checkpoint inhibitors in melanoma patients (N=112) are shown. Except for IL-2 / TGF-β2, each biomarker showed slightly improved overall survival at high expression levels. Therefore, CTLA-4 is not a preferred checkpoint inhibitor.

Claims

1. An antisense agent for inhibiting or suppressing TGF-β2 expression in combination with a checkpoint inhibitor for treating or ameliorating cancer symptoms in human subjects or animals.

2. Use of an antisense agent for inhibiting or suppressing TGF-β2 expression in the preparation of a medicament for use in combination with a checkpoint inhibitor to treat or improve cancer symptoms in human subjects or animals.

3. A method for treating or ameliorating a symptom of cancer in a human or animal subject in need thereof, the method comprising: administering to the subject a therapeutically effective amount of an antisense agent for inhibiting or suppressing TGF-β2 expression; A therapeutically effective amount of a checkpoint inhibitor is administered to the subject. The agent according to claim 1 , which is combined with an interleukin immunotherapy agent. The use according to claim 2 , which is combined with an interleukin immunotherapy agent.

6. The method of claim 3, comprising administering to the subject a therapeutically effective amount of an interleukin immunotherapeutic agent.

7. The agent, use or method according to any one of claims 1 to 6, wherein The agent for inhibiting or suppressing TGF-β2 expression, the checkpoint inhibitor, and the interleukin immunotherapy agent are administered simultaneously, concurrently, sequentially, or separately in time.

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

9. The medicament, use or method of any one of claims 1 to 6, 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.

10. The agent, use or method according to any one of claims 1 to 6, wherein the agent for inhibiting or suppressing TGF-β2 expression is a TGF-β2-specific antisense oligonucleotide complementary to the TGF-β2 transcript and having a length of 15-30 nucleotides.

11. The agent, use or method according to any one of claims 1 to 6, wherein the agent for inhibiting or suppressing TGF-β2 expression is a TGF-β2-specific antisense oligonucleotide complementary to TGF-β2 pre-RNA, pre-mRNA or mRNA and having a length of 18-21 nucleotides.

12. The agent, use or method according to any one of claims 1 to 6, wherein the agent for inhibiting or suppressing TGF-β2 expression is one or more TGF-β2-specific antisense oligonucleotides complementary to TGF-β2 transcripts as shown below (Table 1): and chemically modified variants thereof, LNA variants thereof, gapmer variants thereof, and any combination or collection thereof.

13. The medicament, use or method of claim 12, wherein the TGF-β2 specific antisense oligonucleotide has no more than one or two mismatches compared to the target human TGF-β2.

14. The medicament, use or method of claim 12, 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%.

15. The medicament, use or method of claim 12, 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%.

16. The medicament, use or method according to claim 12, wherein the TGF-β2 specific antisense oligonucleotide has one or more nucleotides chemically modified to be phosphorothioate internucleoside linkages, methoxypropylphosphonate internucleoside linkages, or phosphoramidite linkages 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.

17. The medicament, use or method of claim 12, wherein the antisense agent is conjugated to polyethylene glycol, a lipid or tri-branched N-acetylgalactosamine.

18. The medicament, use or method according to any one of claims 1 to 6, wherein each medicament comprises a carrier of sterile water for injection, saline, isotonic saline or a combination thereof, and the carrier of each medicament may be the same or different.

19. The medicament, use or method of any one of claims 1 to 6, wherein the medicament is substantially free of excipients.

20. The medicament, use or method of any one of claims 1 to 6, wherein the medicament is stable at 37°C for at least 14 days in a carrier that is substantially free of excipients.

21. The agent, use or method of any one of claims 1-6, wherein the checkpoint inhibitor is an inhibitor of PD-1.

22. The medicament, use or method of any one of claims 1-6, wherein the checkpoint inhibitor is pembrolizumab, nivolumab, cemiplizumab, spartazimab, atezolizumab, avelumab or durvalumab.

23. The medicament, use or method of any one of claims 1-6, wherein the interleukin immunotherapeutic agent is natural IL-2, high-dose IL-2, recombinant IL-2 or aldesleukin.

24. The agent, use or method of any one of claims 1 to 6, 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.

25. The medicament, use or method of claim 24, wherein the one or more biomarkers is IRF5 and the subject is selected when the expression level of IRF5 is above the median.

26. The medicament, use or method of claim 24, wherein the one or more biomarkers is ITGAM and the subject is selected when the expression level of ITGAM is above the median.

27. The agent, use or method according to any one of claims 1 to 6, wherein The subject's TGF-β2 level is reduced after administration or use compared to before administration or use.

28. The agent, use or method according to any one of claims 1 to 6, wherein The level of IRF5 in the subject is increased after administration or use compared to before administration or use.

29. The agent, use or method according to any one of claims 1 to 6, wherein The subject's ITGAM level is reduced after administration or use compared to before administration or use.

30. The medicament, use or method according to any one of claims 1 to 6, comprising administering to a subject a therapeutically effective amount of an expression product of IRF5 or ITGAM.

31. The medicament, use or method of claim 30, wherein the expression product is mRNA, a polypeptide, a protein or a fragment thereof, or a combination thereof.

32. The medicament, use or method of any one of claims 1 to 6, wherein the administration or use reduces mortality at 6, 12, 18, 24, 30 or 36 months.

33. The medicament, use or method of any one of claims 1-6, wherein the administration or use increases overall survival at 6, 12, 18, 24, 30 or 36 months.

34. The medicament, use or method according to any one of claims 1 to 6, in combination with any one or more drugs comprising targeted cancer drugs, cancer growth retardants, EGFR inhibitors and combinations thereof.

35. The medicament, use or method according to any one of claims 1 to 6, in combination with any one or more drugs selected from bevacizumab, everolimus, bezutifan, dabrafenib, trametinib and combinations thereof.

36. The medicament, use or method according to any one of claims 1 to 6, in combination with any one or more drugs which are cancer growth retardants selected from the group consisting of angiogenesis inhibitors, histone deacetylase inhibitors, hedgehog blockers, mTOR inhibitors, p53 inhibitors, PARP inhibitors, proteasome inhibitors, tyrosine kinase inhibitors and combinations thereof.

37. The medicament, use or method according to any one of claims 1 to 6, in combination with any one or more drugs that are EGFR inhibitors selected from erlotinib, gefitinib, afatinib, osimertinib, dacomitinib and combinations thereof.

38. The medicament, use or method according to any one of claims 1 to 6, in combination with a chemotherapeutic drug.

39. The medicament, use or method of any one of claims 1 to 6, in combination with radiation therapy or therapeutic electric fields.

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

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

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

43. The medicament, use or method according to any one of claims 40 to 42, wherein the agent for inhibiting or suppressing TGF-β2 expression and the interleukin immunotherapy agent are administered concurrently, simultaneously, sequentially or separately in time.

44. The medicament, use or method according to any one of claims 40 to 42, wherein the agent for inhibiting or suppressing TGF-β2 expression and the interleukin immunotherapy agent are administered separately or as a combined preparation by injection or infusion.

45. The medicament, use or method of any one of claims 40-42, 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.

46. ​​The agent, use or method according to any one of claims 40 to 42, wherein the agent for inhibiting or suppressing TGF-β2 expression is a TGF-β2 specific antisense oligonucleotide complementary to the TGF-β2 transcript and having a length of 15 to 30 nucleotides.

47. The agent, use or method according to any one of claims 40 to 42, wherein the agent for inhibiting or suppressing TGF-β2 expression is a TGF-β2-specific antisense oligonucleotide complementary to TGF-β2 pre-RNA, pre-mRNA or mRNA and having a length of 18-21 nucleotides.

48. The agent, use or method according to any one of claims 40 to 42, wherein the agent for inhibiting or suppressing TGF-β2 expression is one or more TGF-β2 specific antisense oligonucleotides shown in Table 1 that are complementary to TGF-β2 transcripts.

49. The medicament, use or method of claim 48, wherein the TGF-β2-specific antisense oligonucleotide has one or more nucleotides chemically modified to be phosphorothioate internucleoside linkages, methoxypropylphosphonate internucleoside linkages, or phosphoramidite linkages 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.

50. The medicament, use or method of claim 48, wherein the antisense agent is conjugated to polyethylene glycol, a lipid or tri-branched N-acetylgalactosamine.

51. The medicament, use or method according to any one of claims 40 to 42, wherein each medicament comprises a carrier of sterile water for injection, saline, isotonic saline or a combination thereof, and the carrier of each medicament may be the same or different.

52. The medicament, use or method of any one of claims 40-42, wherein the medicament is substantially free of excipients.

53. The medicament, use or method of any one of claims 40-42, wherein the medicament is stable at 37°C for at least 14 days in a carrier that is substantially free of excipients.

54. The medicament, use or method of any one of claims 40-42, wherein the interleukin immunotherapeutic agent is natural IL-2, high-dose IL-2, recombinant IL-2 or aldesleukin.

55. The medicament, use or method of any one of claims 40-42, wherein the administration or use reduces mortality at 6, 12, 18, 24, 30 or 36 months.

56. The medicament, use or method of any one of claims 40-42, wherein the administration or use increases survival at 6, 12, 18, 24, 30 or 36 months.

57. The medicament, use or method according to any one of claims 40 to 42, in combination with any one or more drugs comprising targeted cancer drugs, cancer growth retarders, EGFR inhibitors and combinations thereof.

58. The medicament, use or method according to any one of claims 40 to 42, in combination with any one or more drugs selected from bevacizumab, everolimus, bezutifan, dabrafenib, trametinib and combinations thereof.

59. The medicament, use or method according to any one of claims 40 to 42, in combination with any one or more drugs which are cancer growth retardants selected from the group consisting of angiogenesis inhibitors, histone deacetylase inhibitors, hedgehog blockers, mTOR inhibitors, p53 inhibitors, PARP inhibitors, proteasome inhibitors, tyrosine kinase inhibitors and combinations thereof.

60. The medicament, use or method according to any one of claims 40 to 42, in combination with any one or more drugs that are EGFR inhibitors selected from erlotinib, gefitinib, afatinib, osimertinib, dacomitinib and combinations thereof.

61. The medicament, use or method according to any one of claims 40-42, in combination with a chemotherapeutic drug.

62. The medicament, use or method of any one of claims 40-42, in combination with radiation therapy or therapeutic electric fields.

63. The medicament, use or method of any one of claims 40-42, comprising selecting a subject who would benefit from the medicament, use or method based on the level of one or more biomarkers TGF-β2, IRF5, ITGAM and combinations thereof.

64. The agent, use or method of any one of claims 40-42, comprising selecting a subject who would benefit from the agent, use or method based on the level of one or more biomarkers TGF-β2, IRF5, ITGAM, neoantigens, mutational load, macrophages, and combinations thereof.

65. The medicament, use or method of claim 64, wherein the one or more biomarkers is IRF5 and the subject is selected when the expression level of IRF5 is below the median.

66. The medicament, use or method of claim 64, wherein the one or more biomarkers are tumor associated macrophages and the subject is selected when tumor associated macrophages are below average.

67. The medicament, use or method of claim 64, wherein the one or more biomarkers is tumor neoantigen mutation burden, and the subject is selected when the neoantigen tumor burden is below average.

68. The medicament, use or method of any one of claims 40-42, comprising administering to a subject a therapeutically effective amount of an agent for inhibiting or suppressing expression of ITGAM or IRF5.

69. The agent, use or method of claim 68, wherein the agent for inhibiting or suppressing the expression of ITGAM or IRF5 is an antisense oligonucleotide targeting ITGAM or IRF5, respectively.

70. A kit for treating or ameliorating cancer symptoms, the kit comprising: A therapeutically effective amount of an antisense agent for inhibiting or suppressing TGF-β2 expression; as well as A therapeutically effective amount of a checkpoint inhibitor.

71. The kit of claim 70 comprising a therapeutically effective amount of an interleukin immunotherapeutic agent.

72. The kit of claim 70, 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.

73. The kit of claim 70, wherein the agent for inhibiting or suppressing TGF-β2 expression is a TGF-β2-specific antisense oligonucleotide complementary to the TGF-β2 transcript and having a length of 15-30 nucleotides.

74. The kit of claim 70, wherein the agent for inhibiting or suppressing TGF-β2 expression is a TGF-β2-specific antisense oligonucleotide complementary to TGF-β2 pre-RNA, pre-mRNA or mRNA and having a length of 18-21 nucleotides.

75. The kit of claim 70, wherein the agent for inhibiting or suppressing TGF-β2 expression is one or more TGF-β2-specific antisense oligonucleotides complementary to TGF-β2 transcripts shown in Table 1.

76. The kit of claim 70, wherein the TGF-β2-specific antisense oligonucleotide has no more than one or two mismatches compared to the target human TGF-β2.

77. The kit of claim 70, 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%.

78. The kit of claim 70, 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%.

79. The kit of claim 70, wherein the TGF-β2-specific antisense oligonucleotide has one or more nucleotides chemically modified to have a phosphorothioate internucleoside linkage, a methoxypropylphosphonate internucleoside linkage, or a phosphoramidite linkage linked 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.

80. The kit of claim 70, wherein the antisense agent is conjugated to polyethylene glycol, a lipid, or tri-branched N-acetylgalactosamine.

81. The kit according to claim 70, wherein each agent comprises a carrier of sterile water for injection, saline, isotonic saline or a combination thereof, and the carrier of each agent can be the same or different.

82. The kit of claim 70, wherein the reagents are substantially free of excipients.

83. The kit of claim 70, wherein the agent is stable in a carrier substantially free of excipients at 37°C for at least 14 days.

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