mTOR Drugs for Cancer

Abstract

The present invention relates to methods, agents, and uses for treating cancer. More specifically, the present invention discloses methods, agents, and uses for inhibiting and / or suppressing mTOR, which can lead to improved clinical outcomes for cancer. The present invention provides stable formulations of novel mTOR agonists, including antisense oligonucleotide structures, and also provides combinations with other agents and other therapies for treating cancer. The present invention includes an aspect for selecting subjects or subpopulations that will benefit from mTOR agonists for cancer. TIFF2026505108000005.tif143141

Description

[Technical Field]

[0001] Sequence Listing This application contains a Sequence Listing that has been submitted electronically as an ST.26 file, created on January 26, 2024, entitled "018988-009WO1_SL.xml," and is 18,396 bytes in size.

[0002] Technical Field The present invention relates to the method, agent and use for treating cancer.More specifically, the present invention discloses the method, agent and use for inhibiting or suppressing mTOR, which leads to the improvement of clinical outcomes related to cancer.The present invention provides the stable formulation of novel mTOR agonists, including antisense oligonucleotide compositions, and also includes the combination with other therapies for treating cancer.The present invention also includes a biomarker method for selecting subjects who may benefit from the use of mTOR inhibitors and mTOR therapy. [Background technology]

[0003] background The activity of mTOR has been found to be dysregulated in several types of cancer, including breast cancer, prostate cancer, lung cancer, melanoma, bladder cancer, and brain tumor.In some cases, mTOR may be abnormally activated, which may increase tumor growth and metastasis.It is believed that excessive activation of mTOR signaling may cause tumor initiation and development.In addition, the overexpression of mTOR downstream effectors is also correlated with poor cancer prognosis.

[0004] Several cancer treatments have been developed using mTOR inhibitors, which are natural products of Streptomyces species or derivatives of natural macrocyclic lactones. The therapeutic strategy is to reduce the overexpression of the mTOR complex in tumors.

[0005] However, mTOR inhibitors involve drawbacks and problems of complex feedback loops, which may impair their effectiveness and / or contribute to the progression of cancer.

[0006] In another example, immune checkpoint inhibitors have significantly advanced oncology treatments and have become first- or second-line treatments for many cancers. However, immune checkpoint inhibitors have drawbacks and problems, such as a lack of efficacy in more than half of patients with some cancers.

[0007] What is needed are new agents and methods for inhibiting mTOR. New structures and compositions for inhibiting mTOR can provide new therapeutic approaches. Furthermore, combinations with known mTOR inhibitors can also provide new therapeutic approaches.

[0008] There is an urgent need for methods to select subjects who may benefit from the use of mTOR inhibitors and mTOR therapy. What is needed is a method that uses biomarkers to identify subjects who may benefit from mTOR therapy. Summary of the Invention

[0009] overview The present invention relates to methods, agents, and uses relating to mTOR therapy for treating cancer.

[0010] The present invention includes novel agents and methods for inhibiting mTOR. The novel structures and compositions of the present disclosure for inhibiting mTOR can provide novel therapeutic methods.

[0011] In some embodiments, the novel agents of the present disclosure that inhibit mTOR can be combined with additional mTOR inhibitors to provide novel therapeutic approaches.

[0012] The present invention includes methods for selecting subjects who may benefit from the use of mTOR agonists and mTOR therapy. A variety of specific biomarkers can be used to identify subjects who may benefit from mTOR therapy.

[0013] Aspects of the present invention include the following.

[0014] A method for treating or ameliorating the symptoms of cancer in a human or animal subject in need thereof, comprising the steps of: administering to the subject a therapeutically sufficient amount of a pharmaceutical composition comprising an agent for inhibiting mTOR.

[0015] Agents for inhibiting mTOR to treat or ameliorate the symptoms of cancer in a human or animal subject in need thereof.

[0016] Use of a composition comprising an agent for inhibiting mTOR in the preparation of a medicament for treating or ameliorating the symptoms of cancer in a human or animal subject in need thereof.

[0017] The method, agent, or use as described above, wherein the cancer is sarcoma, lung cancer, ovarian cancer, or pancreatic cancer.

[0018] Any of the methods, agents or uses described above, comprising using one or more biomarkers to select subjects who will benefit from the method, agent or use.

[0019] The method, agent, or use as described above, wherein the one or more biomarkers are levels of tumor mutational burden (TMB), levels of tumor neo-antigens, levels of tumor-associated immune cells, or a combination thereof.

[0020] The method, agent, or use described above, wherein the one or more biomarkers are levels of basophil cells, levels of B cells, levels of T cells, levels of helper T cells (Th), levels of eosinophil cells, levels of macrophage cells, levels of mesenchymal stem cells, or a combination thereof, determined in the tumor microenvironment.

[0021] The method, agent, or use described above, wherein the one or more biomarkers are levels of CD4+ cells, levels of memory T cells, levels of CD8+ cells, levels of natural killer T cells, levels of regulatory T cells, levels of type 1 helper T cells (Th1), levels of type 2 helper T cells (Th2), or a combination thereof, determined in the tumor microenvironment.

[0022] The above-mentioned method, agent, or use, wherein the cancer is a sarcoma and the one or more biomarkers are levels of tumor mutational burden (TMB), levels of tumor neoantigens, levels of mesenchymal stem cells determined in the tumor microenvironment, levels of type 1 helper T cells (Th) determined in the tumor microenvironment, or a combination thereof.

[0023] The method, agent, or use described above, wherein the cancer is lung squamous cell carcinoma and the one or more biomarkers are levels of CD4+ cells determined in the tumor microenvironment, levels of memory T cells determined in the tumor microenvironment, levels of eosinophil cells determined in the tumor microenvironment, or a combination thereof.

[0024] The method, agent, or use described above, wherein the cancer is pancreatic cancer and the one or more biomarkers are levels of CD8+ cells determined in the tumor microenvironment, levels of tumor mutational burden (TMB), or a combination thereof.

[0025] The method, agent, or use described above, wherein the cancer is ovarian cancer and the one or more biomarkers are levels of natural killer T cells determined in the tumor microenvironment, levels of tumor neo-antigens determined in the tumor microenvironment, or a combination thereof.

[0026] The method, agent or use as described above, wherein the agent for suppressing or inhibiting mTOR is an mTOR-specific antisense oligonucleotide.

[0027] The method, agent, or use described above, wherein the agent for inhibiting mTOR is an mTOR-specific antisense oligonucleotide that is complementary to the mTOR transcript and is 15 to 30 nucleotides in length, or an mTOR-specific antisense oligonucleotide that is complementary to the mTOR pre-RNA, pre-mRNA, or mRNA and is 18 to 21 nucleotides in length.

[0028] The method, agent, or use as described above, wherein the agent for suppressing or inhibiting mTOR is an mTOR-specific antisense oligonucleotide selected from Table 1, as well as chemically modified variants thereof, LNA variants thereof, gapmer variants thereof, and any combination thereof or any pool thereof.

[0029] An agent, use, or method as described above, wherein the mTOR-specific antisense oligonucleotide has only one or two mismatches compared to the target human mTOR.

[0030] An agent, use, or method as described above, wherein the mTOR-specific antisense oligonucleotide reduces the level of mTOR transcript by at least 60%, or at least 70%, or at least 80%, or at least 90%.

[0031] The above-mentioned agent, use, or method, wherein the mTOR-specific antisense oligonucleotide has one or more nucleotides chemically modified as a phosphorothioate internucleoside linkage, a methoxypropylphosphonate internucleoside linkage, an aminophosphoro linkage with 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-methylcytodine base.

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

[0033] The method, agent or use as described above, comprising an antisense oligonucleotide in a carrier that is sterile water for injection, saline, isotonic saline, or a combination thereof.

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

[0035] The method, agent or use as described above, wherein the antisense oligonucleotide is stable in the carrier at 37°C for at least 14 days.

[0036] The method, agent, or use described above, wherein the administration or use of the composition is in combination with a standard of care for cancer, wherein the standard of care comprises chemotherapy or radiation therapy, and wherein the administration or use of the composition is performed in the absence of an immune checkpoint inhibitor.

[0037] Any of the above methods, agents or uses, wherein the administration or use reduces mortality at 6 months, 12 months, 18 months, 24 months, 30 months or 36 months.

[0038] Any of the above methods, agents or uses, wherein the administration or use increases survival at 6 months, 12 months, 18 months, 24 months, 30 months or 36 months.

[0039] A method for treating or ameliorating the symptoms of cancer in a human or animal subject in need thereof, comprising the steps of: administering to the subject a therapeutically sufficient amount of a pharmaceutical composition comprising an mTOR-specific antisense oligonucleotide in combination with an agent for inhibiting mTOR.

[0040] The aforementioned method, wherein the cancer is sarcoma, lung cancer, ovarian cancer, or pancreatic cancer.

[0041] The method as described above, comprising using one or more biomarkers to select subjects who will benefit from said method.

[0042] The aforementioned method, wherein the one or more biomarkers are levels of tumor mutational burden (TMB), levels of tumor neo-antigens, levels of tumor-associated immune cells, or a combination thereof.

[0043] The aforementioned method, wherein the one or more biomarkers are a level of basophil cells determined in the tumor microenvironment, a level of B cells determined in the tumor microenvironment, a level of T cells determined in the tumor microenvironment, a level of helper T cells (Th) determined in the tumor microenvironment, a level of eosinophil cells determined in the tumor microenvironment, a level of macrophage cells determined in the tumor microenvironment, a level of mesenchymal stem cells determined in the tumor microenvironment, or a combination thereof.

[0044] The aforementioned method, wherein the one or more biomarkers are levels of CD4+ cells determined in the tumor microenvironment, levels of memory T cells determined in the tumor microenvironment, levels of CD8+ cells determined in the tumor microenvironment, levels of natural killer T cells determined in the tumor microenvironment, levels of regulatory T cells determined in the tumor microenvironment, levels of type 1 helper T cells (Th1) determined in the tumor microenvironment, levels of type 2 helper T cells (Th2) determined in the tumor microenvironment, or a combination thereof.

[0045] The aforementioned method, wherein the cancer is a sarcoma and the one or more biomarkers are a level of tumor mutational burden (TMB), a level of tumor neoantigens, a level of mesenchymal stem cells determined in the tumor microenvironment, a level of type 1 helper T cells (Th) determined in the tumor microenvironment, or a combination thereof.

[0046] The aforementioned method, wherein the cancer is lung squamous cell carcinoma and the one or more biomarkers are a level of CD4+ cells determined in the tumor microenvironment, a level of memory T cells determined in the tumor microenvironment, a level of eosinophil cells determined in the tumor microenvironment, or a combination thereof.

[0047] The aforementioned method, wherein the cancer is pancreatic cancer and the one or more biomarkers are a level of CD8+ cells determined in the tumor microenvironment, a level of tumor mutational burden (TMB), or a combination thereof.

[0048] The aforementioned method, wherein the cancer is ovarian cancer and the one or more biomarkers are levels of natural killer T cells determined in the tumor microenvironment, levels of tumor neo-antigens determined in the tumor microenvironment, or a combination thereof.

[0049] The above method, wherein the mTOR-specific antisense oligonucleotide is any of Table 1, and chemically modified variants thereof, LNA variants thereof, gapmer variants thereof, and any combination thereof or any pool thereof.

[0050] The above method, wherein the mTOR-specific antisense oligonucleotide has only one or two mismatches compared to the target human mTOR.

[0051] The above method, wherein the mTOR-specific antisense oligonucleotide reduces the level of mTOR transcript by at least 60%, or at least 70%, or at least 80%, or at least 90%.

[0052] The above method, wherein the mTOR-specific antisense oligonucleotide has one or more nucleotides chemically modified as a phosphorothioate internucleoside linkage, a methoxypropylphosphonate internucleoside linkage, an aminophosphoro linkage with 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-methylcytodine base.

[0053] The above method, wherein the antisense oligonucleotide is conjugated to polyethylene glycol, a lipid, or triantenarry N-acetylgalactosamine.

[0054] The above method, wherein the antisense oligonucleotide is contained in a carrier which is sterile water for injection, saline, isotonic saline, or a combination thereof, and the antisense oligonucleotide is stable in the carrier at 37°C for at least 14 days.

[0055] The method as described above, comprising an antisense oligonucleotide that is substantially free of excipients.

[0056] The aforementioned method, wherein the agent for inhibiting mTOR is rapamycin, everolimus, temsirolimus, sirolimus, deforolimus, ridaforolimus, zotarolimus, torkinib, samotricisib, omipalisib, apitolisib, bistusertib, dactolisib, jedatolicib, voxtalisib, chrysophanic, or a combination thereof.

[0057] Agents for inhibiting mTOR include 1-[4-[4-(1-oxopropyl)-1-piperazinyl]-3-(trifluoromethyl)phenyl]-9-(3-quinolinyl)-benzo[h]-1,6-naphthyridin-2(1H)-one, 3-[4-(4-morpholinylpyrido[3',2':4,5]furo[3,2-d]pyrimidin-2-yl]phenol hydrochloride, N-[4-[4-(4-morpholinyl)-1-[1-(3-pyridinylmethyl)-4-piperidinyl]-1H-pyrazolo[3,4-d]pyrimidin-6-yl]phenyl] -carbamic acid methyl ester dihydrochloride, 2,4-difluoro-N-[2-methoxy-5-[4-(4-pyridazinyl)-6-quinolinyl]-3-pyridinyl]benzenesulfonamide (omipalisib), 5-chloro-N-(2-chloro-4-nitrophenyl)-2-hydroxybenzamide (niclosamide), or a combination thereof.

[0058] The aforementioned method, wherein the administering is in combination with a standard treatment for the cancer, wherein the standard treatment comprises chemotherapy or radiation therapy, and wherein the administering is performed in the absence of an immune checkpoint inhibitor.

[0059] The above method, wherein the mTOR-specific antisense oligonucleotide and the agent for inhibiting mTOR are each administered contemporaneously, simultaneously, sequentially, or at separate times.

[0060] The method as described above, wherein the administration reduces mortality at 6 months, 12 months, 18 months, 24 months, 30 months, or 36 months.

[0061] The above method, wherein the administration increases survival at 6 months, 12 months, 18 months, 24 months, 30 months, or 36 months. [Brief explanation of the drawings]

[0062] [Figure 1] Figure 1 shows the highly significant improvement in overall survival for sarcoma patients who had reduced mTOR. [Figure 2] Figure 2 shows a highly significant improvement in overall survival for sarcoma patients in the lowest mTOR quartile. [Figure 3] Figure 3 shows a highly significant improvement in overall survival for sarcoma patients with mTOR below the median. [Figure 4] Figure 4 shows a highly significant improvement in overall survival for patients with squamous cell lung cancer who had reduced mTOR when CD4+ memory T cells were expanded above the median as measured in the tumor microenvironment. [Figure 5] FIG. 5 shows a highly significant improvement in overall survival for squamous cell lung cancer patients who had below-median eosinophils as measured in the tumor microenvironment. [Figure 6] Figure 6 shows a highly significant improvement in overall survival for pancreatic ductal adenocarcinoma patients with reduced mTOR when CD8+ T cells were expanded above the median as measured in the tumor microenvironment. [Figure 7] FIG. 7 shows a highly significant improvement in overall survival for pancreatic ductal adenocarcinoma patients whose tumor mutational burden had decreased below the median. [Figure 8] FIG. 8 shows a highly significant improvement in overall survival for pancreatic ductal adenocarcinoma patients whose neoantigen load was elevated above the median. [Figure 9] Figure 9 shows a highly significant improvement in overall survival for sarcoma patients whose tumor mutational burden had decreased below the median. [Figure 10] FIG. 10 shows a highly significant improvement in overall survival for sarcoma patients whose neoantigen load dropped below the median. [Figure 11] FIG. 11 shows a highly significant improvement in overall survival for sarcoma patients whose mesenchymal stem cells were elevated above the median. [Figure 12] FIG. 12 shows a highly significant improvement in overall survival for sarcoma patients who had increased type 1 helper T cells above the median. [Figure 13] FIG. 13 shows a highly significant improvement in overall survival for sarcoma patients who had increased T helper type 1 cells and mesenchymal stem cells above the median. [Figure 14] Figure 14 shows a highly significant improvement in overall survival for sarcoma patients who had above-median increases in type 1 helper T cells and mesenchymal stem cells, but below-median decreases in tumor mutational burden. [Figure 15] FIG. 15 shows a highly significant improvement in overall survival for sarcoma patients who had above-median increases in type 1 helper T cells but below-median decreases in neoantigen load. DETAILED DESCRIPTION OF THE INVENTION

[0063] Detailed Description of the Disclosure The present invention relates to methods, agents, and uses relating to mTOR therapy for use against cancer.

[0064] The present invention provides methods, agents, and uses for treating cancer. Some aspects of the present invention disclose methods, agents, and uses for inhibiting or suppressing mTOR, which can lead to improved clinical outcomes for cancer. The present invention provides stable formulations of novel mTOR agonists, including antisense oligonucleotide compositions, and also includes combinations with other therapies for treating cancer. The present invention also includes biomarker methods for selecting subjects who may benefit from the use of mTOR inhibitors and mTOR therapy.

[0065] The present invention includes novel agents and methods for inhibiting mTOR.The novel structures and compositions of the present disclosure for inhibiting mTOR can provide novel therapeutic methods.The novel structures of the present invention for inhibiting mTOR include antisense oligonucleotides and their chemically modified structures.

[0066] In some embodiments, novel antisense agents can be combined with known mTOR inhibitors to provide novel therapeutic approaches.

[0067] The present invention comprises the method for selecting the subject who can benefit from the use of mTOR inhibitor and mTOR therapy.A variety of specific biomarkers can be used to identify the subject who can benefit from mTOR therapy.In some embodiments, specific biomarkers and biomarker combinations can be used to select the patient or subpopulation who will benefit from mTOR therapy.

[0068] In some aspects, the treatments disclosed herein may be specific for sarcomas determined by their association with tumor microenvironment components Th1, MSC, and genetic mutation burden. For example, the treatments include methods, agents, and uses of mTOR inhibitors for sarcomas, particularly sarcomas with reduced genetic mutation burden, increased T helper 1 (Th1), and increased mesenchymal stem cells (MSC).

[0069] Components of the tumor microenvironment may be determined by means known in the art, including analysis of fresh frozen and FFPE tissue samples by various techniques, microarray screening, analysis of immune cells and subtypes by various techniques, as well as genomic stability, TMB, expression profiling, and various NGS techniques.

[0070] In a further aspect, the present disclosure provides novel therapeutic agents as mTOR inhibitors, including mTOR-specific antisense oligonucleotides.

[0071] In some embodiments, the present disclosure provides mTOR activator, which can be used as various compositions, including injection preparation and nanoparticle preparation.The mTOR activator of the present disclosure can be used through various routes, including intravenous, subcutaneous, intramuscular, intrathecal and intracranial routes.In some embodiments, the activator can be encapsulated in nanoparticles, and the size of the nanoparticles is less than about 100 nm, or less than about 80 nm, or less than about 50 nm.

[0072] In some embodiments, the mTOR agonists of the present disclosure may be used via continuous intracranial, intrathecal, or intracerebroventricular infusion.

[0073] Certain embodiments of the present invention include agents for suppressing or inhibiting mTOR, including mTOR-specific antisense oligonucleotides.

[0074] The present invention includes a method for treating or ameliorating the 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 suppressing or inhibiting mTOR.

[0075] A further embodiment provides agents for suppressing or inhibiting mTOR for treating or ameliorating symptoms of cancer in a human or animal subject in need thereof.

[0076] In certain embodiments, the present invention provides the use of a composition comprising an agent for suppressing or inhibiting mTOR in the preparation of a medicament for treating or ameliorating symptoms of cancer in a human or animal subject in need thereof.

[0077] The treatment methods of the present invention may have efficacy against a variety of cancers, including sarcoma, lung cancer, ovarian cancer, pancreatic cancer, and similar lesions.

[0078] Further aspects of the present disclosure include methods, agents, and uses that utilize one or more biomarkers to select subjects who will benefit from treatment with or use of the agent.

[0079] In some embodiments, biomarkers may include levels of tumor mutational burden (TMB), levels of tumor neo-antigens, levels of tumor-associated immune cells, and combinations thereof.

[0080] Certain embodiments relate directly to biomarkers including levels of basophil cells, levels of B cells, levels of T cells, levels of helper T cells (Th), levels of eosinophil cells, levels of macrophage cells, levels of mesenchymal stem cells, and / or combinations thereof.

[0081] Further embodiments directly utilize biomarkers, such as levels of CD4+ cells, levels of memory T cells, levels of CD8+ cells, levels of natural killer T cells, levels of regulatory T cells, levels of type 1 helper T cells (Th1), levels of type 2 helper T cells (Th2), or combinations thereof, where the levels may be determined in the tumor environment or tumor microenvironment.

[0082] Certain embodiments of the present invention further contemplate novel treatments and agents for sarcoma by selecting patients using biomarkers, such as tumor mutational burden (TMB), levels of tumor neoantigens, mesenchymal stem cells, type 1 helper T cells (Th1), or combinations thereof, which may be determined in the tumor environment or tumor microenvironment.

[0083] In some embodiments, the methods, agents, and uses of the present invention can provide efficacy against lung squamous cell carcinoma, where biomarkers, such as levels of CD4+ cells, levels of memory T cells, levels of eosinophil cells, or a combination thereof, can be determined to select subpopulations that will benefit from the therapy.

[0084] In a further aspect, the present disclosure provides methods, agents, and uses for pancreatic cancer, wherein a biomarker, such as the level of CD8+ cells, the level of tumor mutation burden (TMB), or a combination thereof, is determined to describe and deliver the benefit of the therapy to selected patients.

[0085] In further embodiments, some methods, agents, and uses of the present invention act against ovarian cancer, where biomarkers, such as levels of natural killer T cells, levels of tumor neoantigens, or a combination thereof, can be determined to select subjects who will benefit from the methods, agents, and uses.

[0086] The present invention also encompasses an agent for suppressing or inhibiting mTOR, and the agent comprises mTOR-specific antisense oligonucleotide.The mTOR-specific antisense oligonucleotide can be chemically modified, including LNA variant and gapmer variant.Method and formulation can include pooling mTOR antisense oligonucleotide, and can also include combining antisense oligonucleotide with other agents for inhibiting mTOR.In some embodiments, antisense oligonucleotide can be used in combination with other agents for inhibiting mTOR, and each agent can be administered simultaneously, simultaneously, consecutively, or at different times.

[0087] The formulation of the present disclosure comprises providing one or more agents for suppressing or inhibiting mTOR in a carrier that is sterile water for injection, saline, isotonic saline, or their combinations.In some embodiments, the formulation can be substantially free of excipients.The formulation of the present disclosure can also be stable in such carrier at 37 ℃ for at least 14 days.

[0088] Embodiments of the present invention further include the use or administration of compositions or formulations of agents for suppressing or inhibiting mTOR in combination with standard of care treatments for cancer, where the standard of care may include chemotherapy or radiation therapy.

[0089] Embodiments of the present invention further include the use or administration of compositions or formulations of agents to suppress and / or inhibit mTOR, which may reduce mortality in selected patients at 6 months, 12 months, 18 months, 24 months, 30 months, or 36 months.

[0090] Embodiments of the present invention further include the use or administration of compositions or formulations of agents to suppress and / or inhibit mTOR, which may increase survival rates in selected patients at 6 months, 12 months, 18 months, 24 months, 30 months, or 36 months.

[0091] The present disclosure provides compositions or formulations of agents for suppressing and / or inhibiting mTOR for use as single agents, where subjects may also be selected using biomarkers.

[0092] The compositions or formulations of the invention may be prepared and / or used in the absence of immune checkpoint inhibitors. The therapeutic methods of the invention may be practiced in the absence of immune checkpoint inhibitors.

[0093] Embodiments of the present invention include compositions of mTOR antisense oligonucleotides for use in the absence of immune checkpoint inhibitors.

[0094] More Treatments for Cancer Additional therapeutic methods for cancer of the present disclosure include methods for treating or ameliorating the symptoms of cancer in a human or animal subject in need thereof by selecting a subject that will benefit from the method based on one or more biomarkers and administering a therapeutically sufficient amount of a pharmaceutical composition comprising a combination based on an agent for suppressing mTOR and an agent for inhibiting mTOR.

[0095] Certain embodiments provide combinations based on agents for suppressing mTOR and agents for inhibiting mTOR for treating or ameliorating symptoms of cancer in a human or animal subject in need thereof, wherein subjects who would benefit from the method may be selected based on one or more biomarkers.

[0096] In some embodiments, an agent for suppressing mTOR and an agent for inhibiting mTOR may be used in combination, where each agent may be administered contemporaneously, simultaneously, sequentially, or at separate times.

[0097] In certain embodiments, the disclosure includes a composition comprising a combination of an agent for suppressing mTOR and an agent for inhibiting mTOR in the preparation of a medicament for treating or ameliorating symptoms of cancer in a human or animal subject in need thereof, wherein the subject to benefit from the method is selected based on one or more biomarkers.

[0098] The method, agent, or use as described above, wherein the cancer is sarcoma, lung cancer, ovarian cancer, or pancreatic cancer.

[0099] In such embodiments, biomarkers that can be determined include the level of tumor mutation burden (TMB), the level of tumor neoantigens, the level of tumor-associated immune cells, or a combination thereof. In some embodiments, the biomarkers that can be determined are the level of basophil cells, the level of B cells, the level of T cells, the level of helper T cells (Th), the level of eosinophil cells, the level of macrophage cells, the level of mesenchymal stem cells, or a combination thereof. In some embodiments, biomarkers that can be used include the level of CD4+ cells, the level of memory T cells, the level of CD8+ cells, the level of natural killer T cells, the level of regulatory T cells, the level of type 1 helper T cells (Th1), the level of type 2 helper T cells (Th2), or a combination thereof.

[0100] For treatment with the formulation for sarcoma, biomarkers that may be determined include levels of tumor mutational burden (TMB), levels of tumor neoantigens, levels of mesenchymal stem cells, levels of type 1 T helper cells (Th1), or a combination thereof.

[0101] For treatment with the formulation for lung squamous cell carcinoma, biomarkers that may be determined include levels of CD4+ cells, levels of memory T cells, levels of eosinophil cells, or a combination thereof.

[0102] For treatment with the formulation for pancreatic cancer, biomarkers that may be determined include levels of CD8+ cells, levels of tumor mutational burden (TMB), or a combination thereof.

[0103] For treatment with the formulation for ovarian cancer, biomarkers that may be determined include levels of natural killer T cells, levels of tumor neoantigens, or a combination thereof.

[0104] Examples of agents for inhibiting mTOR include rapamycin, everolimus, temsirolimus, sirolimus, deforolimus, ridaforolimus, zotarolimus, tolquinib, samotricisib, omipalisib, apitolisib, bistusertib, dactolisib, jedatolisib, voxtalisib, chrysophanic, and combinations thereof.

[0105] Examples of agents for inhibiting mTOR include 1-[4-[4-(1-oxopropyl)-1-piperazinyl]-3-(trifluoromethyl)phenyl]-9-(3-quinolinyl)-benzo[h]-1,6-naphthyridin-2(1H)-one, 3-[4-(4-morpholinylpyrido[3',2':4,5]furo[3,2-d]pyrimidin-2-yl]phenol hydrochloride, N-[4-[4-(4-morpholinyl)-1-[1-(3-pyridinylmethyl)methyl]- [4-(4-pyridazinyl)-6-quinolinyl]-3-pyridinyl]benzenesulfonamide (omipalisib), 5-chloro-N-(2-chloro-4-nitrophenyl)-2-hydroxybenzamide (niclosamide), or a combination thereof.

[0106] The cancer treatment methods of the present invention may be combined with standard treatments for the cancer, examples of which include chemotherapy and radiation therapy.

[0107] Certain embodiments of the present invention, which involve the use or administration of compositions or formulations of agents to suppress and / or inhibit mTOR, may reduce mortality in selected patients at 6 months, 12 months, 18 months, 24 months, 30 months, or 36 months.

[0108] Certain embodiments of the present invention, which involve the use or administration of compositions or formulations of agents to suppress and / or inhibit mTOR, may increase survival rates in selected patients at 6 months, 12 months, 18 months, 24 months, 30 months, or 36 months.

[0109] The present disclosure includes a method for treating or ameliorating symptoms of cancer in a human or animal subject in need thereof, the method comprising selecting a subject to benefit from the method based on one or more biomarkers, and administering in the subject a therapeutically sufficient amount of a pharmaceutical composition comprising an agent for inhibiting mTOR in the absence of an immune checkpoint inhibitor.

[0110] The compositions or formulations of the invention may be prepared and / or used in the absence of immune checkpoint inhibitors. The therapeutic methods of the invention may be practiced in the absence of immune checkpoint inhibitors.

[0111] Embodiments of the present invention include compositions of mTOR antisense oligonucleotides for use in the absence of immune checkpoint inhibitors.

[0112] The present invention includes the use of a composition comprising an agent for inhibiting mTOR in the preparation of a medicament for treating or ameliorating the symptoms of cancer in a human or animal subject in need thereof, wherein subjects who would benefit from the method may be selected based on one or more biomarkers.

[0113] mTOR-specific antisense oligonucleotides Antisense oligonucleotide (ASO) can be a single-stranded deoxyribonucleotide, which can be complementary to mRNA target.Antisense therapy can downregulate molecular target, which can be achieved by inducing the endonuclease activity of RNase H, which cuts RNA-DNA heteroduplex, thereby significantly reducing the translation of target gene.Other mechanisms of ASO can include inhibiting 5' cap formation, altering splicing process, such as splice switching, and steric hindrance in ribosome activity.

[0114] Antisense therapeutic strategy can be to utilize single-stranded DNA oligonucleotide, which mediates the catalytic degradation of target mRNA or binds to the site on mRNA that is required for translation, thereby inhibiting the production of protein.Antisense oligonucleotide can provide an approach to identify promising targets, and therefore is a promising therapeutic drug.

[0115] Antisense oligonucleotides can be short synthetic fragments of single-stranded DNA, which can be 15 to 30 nucleotides in length. ASOs can specifically bind to complementary DNA / RNA sequences through Watson-Crick hybridization, and upon binding to target RNA, can inhibit the translation process by either inducing cleavage mechanisms or inhibiting mRNA maturation. ASOs can selectively inhibit gene expression with specificity. Chemical modifications of DNA or RNA can be used to increase stability.

[0116] For example, modifications may be introduced into the phosphodiester bond, into the sugar ring, and into the backbone.

[0117] Phosphorothioate antisense oligodeoxynucleotides specific for human mTOR can be used to suppress or inhibit mTOR.

[0118] For example, the reference sequences for mTOR are provided as NG_033239.1 and NM_004958.4.

[0119] Antisense oligodeoxynucleotides are short stretches of DNA that can be designed to downregulate gene expression by interfering with the translation of a specific protein encoded by mRNA. In some embodiments, all 3'-5' linkages can be phosphorothioate modified.

[0120] In some embodiments, the agent for inhibiting mTOR can be an mTOR-specific antisense oligonucleotide that is complementary to the mTOR transcript and is 15-30 nucleotides in length.

[0121] In certain embodiments, the agent for inhibiting mTOR can be an mTOR-specific antisense oligonucleotide that is complementary to mTOR pre-RNA, pre-mRNA, or mRNA and is 18-21 nucleotides in length.

[0122] Examples of agents of the present disclosure for suppressing or inhibiting mTOR include mTOR-specific antisense oligonucleotides set forth in SEQ ID NOs:1-20 of Table 1, as well as chemically modified variants thereof, LNA variants thereof, gapmer variants thereof, and pools thereof and combinations thereof.

[0123] Table 1. mTOR-specific antisense oligonucleotides TIFF2026505108000002.tif129128

[0124] The sequences of Table 1 may be chemically modified as known in the art to provide active variants thereof, LNA variants thereof, and gapmer variants thereof. The sequences of Table 1 may be used as any combination of active agents, such as pooled combinations.

[0125] For example, synthetic mTOR-specific antisense oligonucleotides of the present disclosure may be phosphorothioate modified at one or all of the 3'-5' linkages.

[0126] It is understood that additional antisense oligonucleotides can be constructed based on the gene sequence of mTOR. In some embodiments, certain criteria for selecting target sites of mTOR can be used as follows: 40% <= GC, % <= 60%; No GGGG in the target sequence; average probability of not pairing with the nucleotide at the target site >= 0.5; For each peak in the accessibility profile that exceeds the probability threshold of 0.5, all sites targeting that peak may be ranked by their average unpairing probability, and up to n sites may be selected for each peak, where n is determined by the maximum value ([peak width / site length], 2).

[0127] In some embodiments, the mTOR-specific antisense oligonucleotides of the present invention may have only one or two mismatches compared to the target human mTOR.

[0128] In certain embodiments, the mTOR-specific antisense oligonucleotides of the invention may reduce the level of mTOR transcript by at least 60%, or at least 70%, or at least 80%, or at least 90%.

[0129] In some aspects, mTOR-specific antisense oligonucleotides may have one or more nucleotides chemically modified as phosphorothioate internucleoside linkages, methoxypropylphosphonate internucleoside linkages, aminophosphoro linkages with morpholino groups, 2'-OMe ribose groups, 2'-MOE methoxyethyl ribose groups, 2'-4' constrained methoxyethyl bicyclic ribose groups, 2'-4' constrained ethyl bicyclic ribose groups, LNA ribose groups, 2'-F ribose groups, or 5-methylcytodine bases.

[0130] In certain embodiments, the antisense oligonucleotides may be conjugated to polyethylene glycol, lipids, or triantenarry N-acetylgalactosamine.

[0131] Methods and Compositions for Cancer The agents of the present invention can be used to treat or ameliorate the symptoms of cancer in a human or animal subject in need thereof. The agents can be prepared as pharmaceutical compositions for injection.

[0132] The pharmaceutical composition for injection may contain an agent for inhibiting or suppressing mTOR and may be administered to a subject in a therapeutically sufficient amount.

[0133] Examples of mTOR inhibitors include antisense oligonucleotides, and pharmaceutically acceptable salt forms, esters, and variants thereof.

[0134] The pharmaceutical agents or active substances of the present disclosure may be dissolved or suspended in a physiological solvent or any other suitable solvent. The agents may be in the form of the free base of the compound, or in the form of a salt, hydrate, ester, amide, enantiomer, isomer, tautomer, polymorph, prodrug, or derivative of the compound. The above-mentioned agents and combinations thereof may be used in the devices, methods, kits, combinations, and compositions described herein.

[0135] The compositions of the present invention may include compounds formulated as injectable preparations for injection into a subject, such as aqueous solutions or suspensions of the compounds suitable for intravenous delivery. When preparing compositions for injection, particularly compositions for intravenous delivery, the continuous phase illustratively comprises an aqueous solution of a tonicity adjusting agent, buffered to a pH of, for example, less than 7, or buffered to a pH of, for example, less than 6. Tonicity adjusting agents include, for example, sodium chloride, glucose, mannitol, trehalose, glycerol, or other pharmaceutical agents that make the osmotic pressure of the formulation isotonic with blood.

[0136] The systems of the present invention may include preservatives added to the formulation, including benzalkonium chloride, propylparaben, butylparaben, chlorobutanol, benzyl alcohol, phenol, sodium benzoate, or EDTA.

[0137] The composition of the present disclosure may comprise a pharmaceutically acceptable carrier.The carrier material that can be used to prepare the composition of the present invention is any of the excipients commonly used in pharmacy, and the carrier material should be selected based on the compatibility with the drug and the release profile characteristics of the desired dosage form.

[0138] The compositions of the present invention may contain excipients, such as those described in "Remington's Pharmaceutical Sciences", Mack Publishing Co., Easton, Pa. 1975, and Liberman, H.A. and Lachman, L., Eds., "Pharmaceutical Dosage Forms", Marcel Decker, New York, NY, 1980.

[0139] Antisense formulation Antisense oligonucleotides may be supplied in various amounts as lyophilized powder in 50 mL glass vials. Each vial may be identified by the following: investigational product name, study number, dosing group, mode of administration, amount of ASO contained (in mg), total volume after dissolution (in mL) and resulting concentration (in μM), sponsor name, manufacturer name, batch number, vial number, storage temperature, and expiration date. Drugs may be provided in sealed units packaged separately for each concentration. The package may include the appropriate vial and all necessary components of the application system (i.e., the injection system). The lyophilized ASO powder may be dissolved in isotonic (0.9%) aqueous sodium chloride solution prior to use.

[0140] In some examples and embodiments, the agent may be an mTOR-specific antisense oligonucleotide selected from SEQ ID NOs: 1-20 and chemically modified variants thereof, and the agent may be administered or used by infusion at a dose of 4 μl / min at a dose level of 10 μM on days 1-7, or at a dose of 20 μM on days 1-7, or at a dose of 40 μM on days 1-7, or at a dose of 80 μM on days 1-7.

[0141] In some examples and embodiments, the agent may be an mTOR-specific antisense oligonucleotide selected from SEQ ID NOs: 1-20 and chemically modified variants thereof, and the agent may be administered or used by infusion at a dose level of 2 μM on days 1-7, or at a dose of 4 μM on days 1-7, or at a dose of 8 μM on days 1-7, or at a dose of 10 μM on days 1-7, at a dose of 4 μl / min or at a dose of 2-8 μl / min.

[0142] In some embodiments, the agent may be an mTOR-specific antisense oligonucleotide selected from SEQ ID NOs: 1-20 and chemically modified variants thereof, and the agent may be administered by injection at a concentration of 61.43 mg / ml (10 μM), 1 mg / ml, 7.35 mg / ml, 15 mg / ml, or 18.23 mg / ml.

[0143] In a further embodiment, the agent may be an mTOR-specific antisense oligonucleotide selected from SEQ ID NOs: 1-20 and chemically modified variants thereof, and the agent may be administered or used by infusion, either alone, in combination with a drug compatible with the formulation, or in combination with standard of care.

[0144] The active substance of the present disclosure can be a pharmaceutically acceptable salt, salt polymorph, ester, or isomer, and one or more pharmaceutically acceptable excipients.The excipient can include any one or more pharmaceutically acceptable excipients selected from diluents, stabilizers, disintegrants, and anti-caking agents.In some embodiments, the excipient can include any one or more of microcrystalline cellulose, polysorbate 80, crospovidone, croscarmellose sodium, and magnesium stearate.

[0145] The pharmaceutical composition may include an mTOR inhibitor and a carrier, which may be sterile water for injection, saline, isotonic saline, or a combination thereof.

[0146] Importantly, the composition of the present disclosure may be substantially free of excipients.Surprisingly, the composition of the present invention that is substantially free of excipients can be stable in carrier.In some embodiments, the composition can be stable in carrier at 37 ℃ for at least 14 days, or at least 21 days, or at least 28 days.

[0147] In further embodiments, a pharmaceutical composition for injection 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.

[0148] Certain aspects of the present invention further contemplate treatment modalities in which the compositions of the present invention are administered or utilized in combination with standard treatments for a disease.

[0149] In further embodiments, the therapeutically effective amount of an antisense agent for inhibiting or suppressing expression of mTOR 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.

[0150] In certain embodiments, a formulation of an antisense agent for inhibiting or suppressing expression of mTOR 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.

[0151] In certain embodiments, methods for using antisense agents to inhibit or suppress expression of mTOR include administering a dose of 1 to 1000 mg / m 2 / day, or 1–500 mg / m 2 / day, or 1–250 mg / m 2 / day, or 1–100 mg / m 2 / day, or 1–50 mg / m 2 An effective dose of 100 mg / day may be used. The average human body surface area is approximately 1.6 to 1.9 m 2 It could be.

[0152] In further embodiments, the method for using antisense agents to inhibit or suppress expression of mTOR comprises administering a dose of 0.05 to 40 mg / kg / day, or 0.1 to 30 mg / kg / day, or 0.2 to 20 mg / m 2 / day, or 0.3–10 mg / m 2 / day, or 0.5–5 mg / m 2 An effective dose of 100 mg / day may be used. The average human body weight may be about 60 kg.

[0153] In certain embodiments, the agents of the present disclosure for inhibiting or suppressing the expression of mTOR can be prepared from a lyophilized powder of the agent.

[0154] In some examples and embodiments, the agent may be an mTOR-specific antisense oligonucleotide selected from SEQ ID NOs: 1-20 and chemically modified variants thereof, and the agent may be administered or used by injection or infusion at a dose of 4 μl / min at a dose level of 10 μM on days 1-7, or at a dose of 20 μM on days 1-7, or at a dose of 40 μM on days 1-7, or at a dose of 80 μM on days 1-7.

[0155] In some embodiments, an mTOR-specific antisense oligonucleotide selected from SEQ ID NOs: 1-20 and their chemically modified variants may be supplied as a sterile lyophilizate in a 20R glass vial at 250 mg / vial, ready to be dissolved prior to administration. The lyophilizate may be aseptically reconstituted in a preservative-free sterile isotonic NaCl solution. The antisense oligonucleotide solution may be administered every 14 days using a portable pump system as a continuous IV infusion on days 4-7, following a 4-day on, 10-day off schedule. Schedules may include 7 days on / 7 days off and 4 days on / 10 days off. Doses may be 40 mg, 80 mg, 160 mg, 140 mg, 190 mg, 250 mg, or 330 mg.

[0156] In some embodiments, the agent may be an antisense oligonucleotide specific for the gene sequence of mTOR selected from SEQ ID NOs: 1-20 and chemically modified variants thereof, and the agent is administered at a dose of 40 mg / m on days 1-7. 2 , 80 mg / m 2 , 160 mg / m 2 , 140 mg / m 2 , 190 mg / m 2 , 250 mg / m 2 , 330 mg / m 2 or 40 mg / m on days 1-4 2 , 80 mg / m 2 , 160 mg / m 2 , 140 mg / m 2 , 190 mg / m 2 , 250 mg / m 2 , 330 mg / m 2 may be administered or used by injection or infusion at a dose of

[0157] In some examples and embodiments, the agent may be an antisense oligonucleotide specific to the gene sequence of mTOR selected from SEQ ID NOs: 1-20 and chemically modified variants thereof, and the agent may be administered or used by injection or infusion at a dose level of 2 μM on days 1-7, or at a dose of 4 μM on days 1-7, or at a dose of 8 μM on days 1-7, or at a dose of 10 μM on days 1-7, at a dose of 4 μl / min or at a dose of 2-8 μl / min.

[0158] As used herein, the term chemically modified variants can refer to LNA variants and gapmer variants.The antisense agents of the present disclosure can be used by pooling in formulations, or can be used in any combination.

[0159] Numbered aspects of the present invention may include: 1) 1. A method for treating or ameliorating the symptoms of cancer in a human or animal subject in need thereof, comprising: administering to the subject a therapeutically sufficient amount of a pharmaceutical composition comprising an agent for inhibiting mTOR. The method comprising: 2) Agents for inhibiting mTOR to treat or ameliorate the symptoms of cancer in a human or animal subject in need thereof. 3) Use of a composition comprising an agent for inhibiting mTOR in the preparation of a medicament for treating or ameliorating the symptoms of cancer in a human or animal subject in need thereof. 4) The method, agent, or use of any of embodiments 1-3, wherein the cancer is sarcoma, lung cancer, ovarian cancer, or pancreatic cancer. 5) The method, agent, or use of any of embodiments 1-4, comprising using one or more biomarkers to select subjects who will benefit from said method, agent, or use. 6) The method, agent, or use of any of embodiments 1-5, wherein the one or more biomarkers are levels of tumor mutational burden (TMB), levels of tumor neo-antigens, levels of tumor-associated immune cells, or a combination thereof. 7) The method, agent, or use of any of embodiments 1-6, wherein the one or more biomarkers are levels of basophil cells, levels of B cells, levels of T cells, levels of helper T cells (Th), levels of eosinophil cells, levels of macrophage cells, levels of mesenchymal stem cells, or a combination thereof, determined in the tumor microenvironment. 8) The method, agent, or use of any of embodiments 1-7, wherein the one or more biomarkers is levels of CD4+ cells, levels of memory T cells, levels of CD8+ cells, levels of natural killer T cells, levels of regulatory T cells, levels of type 1 helper T cells (Th1), levels of type 2 helper T cells (Th2), or a combination thereof, determined in the tumor microenvironment. 9) The method, agent, or use of any of embodiments 1-8, wherein the cancer is a sarcoma and the one or more biomarkers are levels of tumor mutational burden (TMB), levels of tumor neo-antigens, levels of mesenchymal stem cells determined in the tumor microenvironment, levels of type 1 helper T cells (Th) determined in the tumor microenvironment, or a combination thereof. 10)

[0023] The method, agent, or use of any of embodiments 1-9, wherein the cancer is lung squamous cell carcinoma and the one or more biomarkers is the level of CD4+ cells determined in the tumor microenvironment, the level of memory T cells determined in the tumor microenvironment, the level of eosinophil cells determined in the tumor microenvironment, or a combination thereof. 11) 11. The method, agent, or use of any of embodiments 1-10, wherein the cancer is pancreatic cancer and the one or more biomarkers are levels of CD8+ cells determined in the tumor microenvironment, levels of tumor mutational burden (TMB), or a combination thereof. 12) 12. The method, agent, or use of any of embodiments 1-11, wherein the cancer is ovarian cancer and the one or more biomarkers are levels of natural killer T cells determined in the tumor microenvironment, levels of tumor neo-antigens determined in the tumor microenvironment, or a combination thereof. 13) Agents that inhibit mTOR an mTOR-specific antisense oligonucleotide complementary to the mTOR transcript and 15-30 nucleotides in length; or an mTOR-specific antisense oligonucleotide that is complementary to mTOR pre-RNA, pre-mRNA, or mRNA and is 18 to 21 nucleotides in length; The method, agent, or use of any of embodiments 1 to 12. 14) 14. The method, agent, or use of any of embodiments 1-13, wherein the agent for inhibiting mTOR is an mTOR-specific antisense oligonucleotide comprising any of the 20 nucleotide long sequences of Table 1, and chemically modified variants thereof, LNA variants thereof, gapmer variants thereof, and pools thereof and combinations thereof. 15) 15. The agent, use, or method of any of embodiments 1-14, wherein the mTOR-specific antisense oligonucleotide has no more than one or two mismatches compared to the target human mTOR. 16) 16. The agent, use, or method of any of embodiments 1-15, wherein the mTOR-specific antisense oligonucleotide reduces levels of mTOR transcript by at least 60%, or at least 70%, or at least 80%, or at least 90%. 17) 17. The agent, use, or method of any of embodiments 1-16, wherein the mTOR-specific antisense oligonucleotide has one or more nucleotides chemically modified as phosphorothioate internucleoside linkages, methoxypropylphosphonate internucleoside linkages, aminophosphoro linkages with morpholino groups, 2'-OMe ribose groups, 2'-MOE methoxyethyl ribose groups, 2'-4' constrained methoxyethyl bicyclic ribose groups, 2'-4' constrained ethyl bicyclic ribose groups, LNA ribose groups, 2'-F ribose groups, or 5-methylcytodine bases. 18) 18. The agent, use, or method of any of embodiments 1-17, wherein the antisense oligonucleotide is conjugated to polyethylene glycol, a lipid, or triantennary N-acetylgalactosamine. 19) 19. The method, agent, or use of any of embodiments 1-18, comprising an antisense oligonucleotide in a carrier that is sterile water for injection, saline, isotonic saline, or a combination thereof. 20) 20. The method, agent, or use of any of embodiments 1-19, wherein the antisense oligonucleotide is substantially free of excipients. twenty one) 21. The method, agent, or use of any of embodiments 1-20, wherein the antisense oligonucleotide is stable in the carrier at 37° C. for at least 14 days. twenty two) 22. The method, agent, or use of any of embodiments 1-21, wherein the administration or use of the composition is in combination with a standard of care for the cancer, wherein the standard of care comprises chemotherapy or radiation therapy, and wherein the administration or use of the composition is performed in the absence of an immune checkpoint inhibitor. twenty three) 23. The method, agent, or use of any of embodiments 1-22, wherein the administration or use reduces mortality at 6 months, 12 months, 18 months, 24 months, 30 months, or 36 months. twenty four) 24. The method, agent, or use of any of embodiments 1-23, wherein the administration or use increases survival at 6 months, 12 months, 18 months, 24 months, 30 months, or 36 months. twenty five) 1. A method for treating or ameliorating the symptoms of cancer in a human or animal subject in need thereof, comprising: administering to said subject a therapeutically sufficient amount of a pharmaceutical composition comprising an mTOR-specific antisense oligonucleotide in combination with an agent for inhibiting mTOR. A method comprising: 26) The method of embodiment 25, wherein the cancer is sarcoma, lung cancer, ovarian cancer, or pancreatic cancer. 27) The method of any of embodiments 25-26, comprising using one or more biomarkers to select subjects who will benefit from said method. 28) The method of any of embodiments 25-27, wherein the one or more biomarkers are levels of tumor mutational burden (TMB), levels of tumor neo-antigens, levels of tumor-associated immune cells, or a combination thereof. 29) 29. The method of any of embodiments 25-28, wherein the one or more biomarkers are a level of basophil cells determined in the tumor microenvironment, a level of B cells determined in the tumor microenvironment, a level of T cells determined in the tumor microenvironment, a level of helper T cells (Th) determined in the tumor microenvironment, a level of eosinophil cells determined in the tumor microenvironment, a level of macrophage cells determined in the tumor microenvironment, a level of mesenchymal stem cells determined in the tumor microenvironment, or a combination thereof. 30) 30. The method of any of embodiments 25-29, wherein the one or more biomarkers are levels of CD4+ cells determined in the tumor microenvironment, levels of memory T cells determined in the tumor microenvironment, levels of CD8+ cells determined in the tumor microenvironment, levels of natural killer T cells determined in the tumor microenvironment, levels of regulatory T cells determined in the tumor microenvironment, levels of type 1 helper T cells (Th1) determined in the tumor microenvironment, levels of type 2 helper T cells (Th2) determined in the tumor microenvironment, or a combination thereof. 31)

[0039] The method of any of aspects 25-30, wherein the cancer is a sarcoma and the one or more biomarkers are levels of tumor mutational burden (TMB), levels of tumor neoantigens, levels of mesenchymal stem cells determined in the tumor microenvironment, levels of type 1 helper T cells (Th) determined in the tumor microenvironment, or a combination thereof. 32) 32. The method of any of embodiments 25-31, wherein the cancer is lung squamous cell carcinoma and the one or more biomarkers are a level of CD4+ cells determined in the tumor microenvironment, a level of memory T cells determined in the tumor microenvironment, a level of eosinophil cells determined in the tumor microenvironment, or a combination thereof. 33) The method of any of aspects 25-32, wherein the cancer is pancreatic cancer and the one or more biomarkers are a level of CD8+ cells determined in the tumor microenvironment, a level of tumor mutation burden (TMB), or a combination thereof. 34) 34. The method of any of aspects 25-33, wherein the cancer is ovarian cancer and the one or more biomarkers are levels of natural killer T cells determined in the tumor microenvironment, levels of tumor neo-antigens determined in the tumor microenvironment, or a combination thereof. 35) mTOR-specific antisense oligonucleotides is complementary to the mTOR transcript and is 15 to 30 nucleotides in length; or complementary to mTOR pre-RNA, pre-mRNA, or mRNA and 18 to 21 nucleotides in length; The method of any one of aspects 25 to 34. 36) 36. The method of any of embodiments 25-35, wherein the mTOR-specific antisense oligonucleotide comprises any of the 20 nucleotide sequences of Table 1, as well as chemically modified variants thereof, LNA variants thereof, gapmer variants thereof, and pools thereof and combinations thereof. 37) The method of any of embodiments 25-36, wherein the mTOR-specific antisense oligonucleotide has only one or two mismatches compared to the target human mTOR. 38) The method of any of embodiments 25-37, wherein the mTOR-specific antisense oligonucleotide reduces the level of mTOR transcript by at least 60%, or at least 70%, or at least 80%, or at least 90%. 39) 39. The method of any of embodiments 25-38, wherein the mTOR-specific antisense oligonucleotide has one or more nucleotides chemically modified as a phosphorothioate internucleoside linkage, a methoxypropylphosphonate internucleoside linkage, an aminophosphoro linkage with 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-methylcytodine base. 40) 40. The method of any of embodiments 25 to 39, wherein the antisense oligonucleotide is conjugated to polyethylene glycol, a lipid, or triantennary N-acetylgalactosamine. 41) an antisense oligonucleotide in a carrier that is sterile water for injection, saline, isotonic saline, or a combination thereof; and the antisense oligonucleotide is stable in the carrier at 37°C for at least 14 days; The method of any one of aspects 25 to 40. 42) 42. The method of any of embodiments 25-41, comprising the antisense oligonucleotide substantially free of excipients. 43) 43. The method of any of embodiments 25-42, wherein the agent for inhibiting mTOR is rapamycin, everolimus, temsirolimus, sirolimus, deforolimus, ridaforolimus, zotarolimus, torkinib, samotricisib, omipalisib, apitolisib, bistusertib, dactolisib, jedatolicib, voxtalisib, chrysophanic, or a combination thereof. 44) Agents for inhibiting mTOR include 1-[4-[4-(1-oxopropyl)-1-piperazinyl]-3-(trifluoromethyl)phenyl]-9-(3-quinolinyl)-benzo[h]-1,6-naphthyridin-2(1H)-one, 3-[4-(4-morpholinylpyrido[3',2':4,5]furo[3,2-d]pyrimidin-2-yl]phenol hydrochloride, N-[4-[4-(4-morpholinyl)-1-[1-(3-pyridinylmethyl)-4-piperazinyl]-4- ... 44. The method of any of aspects 25-43, wherein the compound selected from the group consisting of 5-(4-pyridazinyl)-1H-pyrazolo[3,4-d]pyrimidin-6-yl]phenyl]carbamic acid methyl ester dihydrochloride, 2,4-difluoro-N-[2-methoxy-5-[4-(4-pyridazinyl)-6-quinolinyl]-3-pyridinyl]benzenesulfonamide (omipalisib), 5-chloro-N-(2-chloro-4-nitrophenyl)-2-hydroxybenzamide (niclosamide), or a combination thereof. 45) The method of any of embodiments 25 to 44, wherein the administering is in combination with a standard of care for the cancer, wherein the standard of care comprises chemotherapy or radiation therapy, and wherein the administering is performed in the absence of an immune checkpoint inhibitor. 46) The method of any of embodiments 25-45, wherein the mTOR-specific antisense oligonucleotide and the agent for inhibiting mTOR are administered contemporaneously, simultaneously, sequentially, or at separate times, respectively. 47) The method of any of embodiments 25-46, wherein the administration reduces mortality at 6 months, 12 months, 18 months, 24 months, 30 months, or 36 months. 48) The method of any of embodiments 25-47, wherein the administration increases survival at 6 months, 12 months, 18 months, 24 months, 30 months, or 36 months.

[0160] All publications referenced in this description, including patents, published patent applications, and non-patent publications, together with the Sequence Listing, are each expressly incorporated herein by reference in their entirety for all purposes.

[0161] While the foregoing disclosure, which is presented for purposes of illustration and not limitation, has been described in detail by way of example for purposes of clarity of understanding, it will be understood by those skilled in the art that certain changes and modifications are encompassed by the disclosure and that such changes and modifications may be made within the scope of the appended claims without undue experimentation. The present invention includes all such additional embodiments, equivalents, and modifications. The present invention includes any combination or mixture of the features, materials, components, or limitations of the various exemplary components, examples, and claimed embodiments.

[0162] It is emphasized herein that the features in the drawings are, according to convention, to any scale and are intended to cover similar features that may be arbitrarily enlarged or reduced. [Example]

[0163] Example 1. The disclosed methods and agents for inhibiting mTOR can be used against sarcomas.

[0164] A clinical outcome study of 259 sarcoma patients with various types of sarcoma was conducted, which showed improved overall survival (OS) with reduced mTOR expression. This improvement was quite surprising, as comparative data from over 7,200 other cancer patients with a wide range of cancers showed no such improvement with reduced mTOR expression. The patients were treated in the absence of immune checkpoint inhibitors.

[0165] A Kaplan-Meier plotter was used to calculate patient survival and determine the association between improved overall survival and reduced mTOR expression in sarcoma. Part of the raw data is available in: Kovacs, "Transcriptomic datasets of cancer patients treated with immune-checkpoint inhibitors: a systematic review", J. Transl. Med., 2022, May 31, Vol. 20(1), pp. 249.

[0166] The results of the sarcoma study are shown in Figure 1, which demonstrates a highly significant (log-rank P = 0.0025) improvement in overall survival for sarcoma patients with reduced mTOR expression. The improvement in overall survival observed with reduced mTOR expression was significant, ranging from 55 months in the high mTOR cohort to 87 months in the low mTOR cohort. Sarcoma subtypes in this study included all stages, genders, races, grades, mutational burdens, and neoantigen burdens. The probe expression range was 345–4010. This study did not restrict or exclude immune cell components of the tumor microenvironment.

[0167] Further clinical outcomes of 3,711 sarcoma patients with various sarcoma types were studied, and this also showed improved overall survival (OS) with decreased mTOR expression. In this study, cBioPortal for Cancer Genomics was used to calculate patient survival rates and determine the association between improved OS and decreased mTOR expression in sarcomas. Sarcoma types included: soft tissue sarcoma (1,987), bone cancer (529), gastrointestinal stromal tumor (395), uterine sarcoma (341), sarcoma (255), endometrial cancer (136), nerve sheath tumor (64), and soft tissue cancer (4). Patients were treated in the absence of immune checkpoint inhibitors.

[0168] The results of this further study for sarcoma are shown in Figure 2, which shows a highly significant (log-rank P value = 0.0121) improvement in overall survival for 504 sarcoma patients in the lowest mTOR quartile. The improvement in overall survival found with decreased mTOR expression ranged from 47 months in quartile D, with the highest mTOR expression, to 85 months in cohort A, with the lowest mTOR expression.

[0169] Similar results were found for the same data using median mTOR expression analysis, as shown in Figure 3. Figure 3 shows a highly significant (log-rank P = 0.0119) improvement in overall survival for 504 sarcoma patients with mTOR expression below the median. The improvement in overall survival observed with reduced mTOR expression was significant, from 54 months for patients with mTOR expression above the median to 85 months for patients with mTOR expression below the median. This surprising improvement in overall survival with reduced mTOR was independent of sarcoma type, as both the above- and below-median mTOR expression groups contained approximately equal numbers and similar types of sarcomas. This surprising improvement in overall survival with reduced mTOR was also independent of the anatomical location of the sarcoma, as both the above- and below-median mTOR expression groups contained approximately equal numbers of sarcomas from similar anatomical regions.

[0170] Example 2. The disclosed methods and agents for suppressing and inhibiting mTOR can be used for lung squamous cell carcinoma, and biomarkers are used to select patients who will benefit from the methods and / or agents. For lung squamous cell carcinoma, the important biomarkers are the level of CD4+ memory T cells determined in the tumor microenvironment and the level of eosinophil cells determined in the tumor microenvironment.

[0171] A clinical outcome study was conducted on patients with squamous cell lung cancer, which showed improved overall survival (OS) with reduced mTOR expression when CD4+ memory T cells were enriched in the tumor microenvironment. This improvement was quite surprising, as data from over 900 other cancer patients with a wide range of cancers showed no such improvement with reduced mTOR expression when patients were not selected using biomarkers.

[0172] A Kaplan-Meier Plotter was used to calculate patient survival and determine the association between improved overall survival and reduced mTOR expression in lung squamous cell carcinoma, where biomarkers were used to select patients who would benefit from the method and / or agent.

[0173] The results of this study on squamous cell lung carcinoma are shown in Figure 4. Figure 4 demonstrates a highly significant (log-rank P = 0.0105) improvement in overall survival for patients with increased CD4+ memory T cells above the median level as measured in the tumor microenvironment, associated with decreased mTOR expression. In the presence of increased CD4+ memory T cells, the improvement in overall survival observed with decreased mTOR expression was significant, with a hazard ratio of 2.17; patients with increased CD4+ memory T cells essentially had twice the survival rate. Carcinoma subtypes in this study included all tumors by stage, gender, race, grade, mutational burden, and neoantigen burden. The probe expression range was 437–4839.

[0174] Further clinical outcome studies on patients with squamous cell lung cancer showed improved overall survival (OS) with reduced mTOR expression when eosinophils were present in the tumor microenvironment. This improvement was quite surprising, as data from over 900 patients with a wide range of other cancers showed no such improvement with reduced mTOR expression when patients were not selected using biomarkers.

[0175] A Kaplan-Meier Plotter was used to calculate patient survival and determine the association between improved overall survival and reduced mTOR expression in lung squamous cell carcinoma, where biomarkers were used to select patients who would benefit from the method and / or agent.

[0176] The results of the study on squamous cell lung carcinoma are shown in Figure 5. Figure 5 shows a highly significant (log-rank P = 0.0157) improvement in overall survival for patients with reduced eosinophils below the median level as measured in the tumor microenvironment. The improvement in overall survival observed with reduced mTOR expression in the presence of reduced eosinophils was significant, with a hazard ratio of 2.29; patients with reduced eosinophils essentially had twice the survival rate. Carcinoma subtypes in this study included all stages, sexes, races, grades, mutational burden, and neoantigen burden. The probe expression range was 437–4839.

[0177] Example 3. The disclosed methods and agents for suppressing and inhibiting mTOR can be used for pancreatic cancer, and biomarkers are used to select patients who will benefit from the methods and / or agents.The important biomarkers for pancreatic cancer are the level of CD8+ cells determined in the tumor microenvironment and the level of tumor mutation burden (TMB).

[0178] A clinical outcome study of pancreatic cancer patients showed improved overall survival (OS) with reduced mTOR expression in patients with enriched CD8+ T cells in the tumor microenvironment. The improvement in OS observed for the highest quartile of patients with enriched CD8+ T cells and with reduced mTOR expression was significant, ranging from 16 months in the high mTOR cohort to 24 months in the low mTOR cohort. This improvement was particularly surprising, as data from over 900 other cancer patients with a wide range of cancers did not demonstrate such improvements with reduced mTOR expression when patients were not selected using biomarkers.

[0179] A Kaplan-Meier Plotter was used to calculate patient survival and determine the association between improved overall survival and reduced mTOR expression in pancreatic ductal adenocarcinoma, where biomarkers were used to select patients who would benefit from the method and / or agent.

[0180] The results of the study on pancreatic ductal adenocarcinoma are shown in Figure 6. Figure 6 shows a highly significant (log-rank P = 0.024) improvement in overall survival for patients with reduced mTOR expression who had increased CD8+ T cells above the median level as measured in the tumor microenvironment. In the presence of increased CD8+ T cells, the improvement in overall survival observed with decreased mTOR expression was significant, with a hazard ratio of 3.89, meaning that the CD8+ T cell-enhanced group had a nearly fourfold increased survival rate. Cancer subtypes in this study included all stages, sex, race, grade, mutational burden, and neoantigen burden. The probe expression range was 731–2164.

[0181] Further clinical outcome studies on patients with pancreatic ductal adenocarcinoma showed improved overall survival (OS) with reduced mTOR expression when tumor mutation burden was also reduced. The improvement in OS observed for the highest quartile of the study group with reduced mTOR expression when tumor mutation burden was also reduced was significant, ranging from 17 months in the high mTOR cohort to 35 months in the low mTOR cohort. This improvement was quite surprising, as data from over 900 other cancer patients with a wide range of cancers had not shown such improvement with reduced mTOR expression when patients were not selected using biomarkers.

[0182] A Kaplan-Meier Plotter was used to calculate patient survival and determine the association between improved overall survival and reduced mTOR expression in pancreatic ductal adenocarcinoma, where biomarkers were used to select patients who would benefit from the method and / or agent.

[0183] The results of the study on pancreatic ductal adenocarcinoma are shown in Figure 7. Figure 7 shows a highly significant (log-rank P = 0.02) improvement in overall survival for patients whose tumor mutational burden was reduced below the median. The improvement in overall survival observed with reduced mTOR expression was significant in the presence of reduced tumor mutational burden, with a hazard ratio of 3.97, meaning that patients with reduced tumor mutational burden had a nearly fourfold increased survival rate. Cancer subtypes in this study included all stages, genders, races, grades, and neoantigen burdens. The probe expression range was 287–2231.

[0184] Example 4. The disclosed methods and agents for suppressing and inhibiting mTOR can be used for ovarian cancer, with biomarkers being used to select patients who will benefit from the methods and / or agents. The key biomarker for ovarian cancer was the level of neoantigen load.

[0185] A clinical outcome study was conducted on ovarian cancer patients, which showed improved overall survival (OS) with decreased mTOR expression in patients with elevated neoantigen load. The improvement in OS observed with decreased mTOR expression in patients with elevated neoantigen load above the median ranged significantly from 40 months in the high mTOR cohort to 55 months in the low mTOR cohort. This improvement was quite surprising, as data from over 900 other cancer patients with a wide range of cancers did not demonstrate such improvement with decreased mTOR expression when patients were not selected using biomarkers.

[0186] A Kaplan-Meier Plotter was used to calculate patient survival and determine the association between improved overall survival and reduced mTOR expression in ovarian cancer, where biomarkers were used to select patients who would benefit from the method and / or agent.

[0187] The results of the ovarian cancer study are shown in Figure 8. Figure 8 shows a highly significant (log-rank P = 0.045) improvement in overall survival for patients with elevated neoantigen load above the median. The improvement in overall survival observed with decreased mTOR expression in the presence of elevated neoantigen load was significant, with a hazard ratio of 1.66, meaning that patients with decreased neoantigen load had a nearly 70% increased survival rate. Cancer subtypes in this study included all stages, genders, races, grades, and mutational burdens. The probe expression range was 304–5797. This study did not restrict or exclude immune cell components of the tumor microenvironment.

[0188] Example 5. The disclosed methods and agents for suppressing and inhibiting mTOR can be used for sarcoma, with biomarkers being used to select patients who will benefit from the methods and / or agents. An important biomarker for sarcoma was the level of tumor mutational burden.

[0189] A clinical outcome study of sarcoma patients showed improved overall survival (OS) with reduced mTOR expression when tumor mutation burden was reduced. When tumor mutation burden was reduced below the median, the improvement in OS observed for the highest quartile with reduced mTOR expression was significant, from 23 months in the high mTOR cohort to 66 months in the low mTOR cohort—a nearly three-fold increase in OS. This improvement was quite surprising, as data from over 900 other cancer patients with a wide range of cancers had not shown such improvements with reduced mTOR expression when patients were not selected using biomarkers.

[0190] A Kaplan-Meier Plotter was used to calculate patient survival and determine the association between improved overall survival and reduced mTOR expression in this subtype of sarcoma, with biomarkers used to select patients who would benefit from the method and / or agent.

[0191] The results of this study on this subtype of sarcoma are shown in Figure 9, which demonstrates a highly significant (log-rank P = 0.00092) improvement in overall survival for patients whose tumor mutational burden was reduced below the median. The improvement in overall survival observed with reduced mTOR expression in the presence of reduced tumor mutational burden was significant, with a hazard ratio of 3.15, meaning that patients with reduced tumor mutational burden had a more than three-fold increased chance of survival. The cancer subtypes in this study included all stages, genders, races, grades, and neoantigen burdens. The probe expression range was 429–3103. This study did not restrict or exclude immune cell components of the tumor microenvironment.

[0192] Example 6. The disclosed methods and agents for suppressing and inhibiting mTOR can be used for sarcoma, with biomarkers being used to select patients who will benefit from the methods and / or agents. The key biomarker for sarcoma was the level of neoantigen load.

[0193] A clinical outcome study of sarcoma patients was conducted, which showed improved overall survival (OS) with reduced mTOR expression when neoantigen levels were reduced. The improvement in OS observed for the highest quartile of mTOR expression with reduced neoantigen levels below the median ranged from 19 months in the high mTOR cohort to 36 months in the low mTOR cohort, a nearly two-fold increase in OS. This improvement was quite surprising, as data from over 900 other cancer patients with a wide range of cancers did not demonstrate such improvements with reduced mTOR expression when patients were not selected using biomarkers.

[0194] A Kaplan-Meier Plotter was used to calculate patient survival and determine the association between improved overall survival and reduced mTOR expression in this subtype of sarcoma, with biomarkers used to select patients who would benefit from the method and / or agent.

[0195] The results of this study on this subtype of sarcoma are shown in Figure 10. Figure 10 shows a highly significant (log-rank P = 0.0032) improvement in overall survival for patients whose neoantigen load was below the median. The improvement in overall survival observed with reduced mTOR expression in the presence of reduced neoantigen load was significant, with a hazard ratio of 2.05, meaning that patients with reduced neoantigen load had a more than two-fold increased survival rate. The cancer subtypes in this study included all stages, genders, races, grades, and mutational burdens. The probe expression range was 345–4010. This study did not restrict or exclude immune cell components of the tumor microenvironment.

[0196] Example 7. The disclosed methods and agents for suppressing and inhibiting mTOR can be used for sarcoma, with biomarkers being used to select patients who will benefit from the methods and / or agents. For sarcoma, the key biomarker was the level of mesenchymal stem cells in the tumor microenvironment.

[0197] A clinical outcome study was conducted on sarcoma patients, which showed improved overall survival (OS) with reduced mTOR expression when mesenchymal stem cell levels were elevated. The improvement in median OS observed with reduced mTOR expression when mesenchymal stem cells were elevated above the median range was significant, from 39 months in the high mTOR cohort to 86 months in the low mTOR cohort, a more than two-fold increase in OS. This improvement was quite surprising, as data from over 900 other cancer patients with a wide range of cancers did not demonstrate such improvement with reduced mTOR expression when patients were not selected using biomarkers.

[0198] A Kaplan-Meier Plotter was used to calculate patient survival and determine the association between improved overall survival and reduced mTOR expression in this subtype of sarcoma, with biomarkers used to select patients who would benefit from the method and / or agent.

[0199] The results of this study on this subtype of sarcoma are shown in Figure 11, which shows a highly significant (log-rank P = 0.0048) improvement in overall survival for patients whose mesenchymal stem cell levels were elevated above the median. The improvement in overall survival observed with decreased mTOR expression in the presence of elevated mesenchymal stem cells was significant, with a hazard ratio of 1.99; patients with decreased neoantigen levels had a two-fold increased survival rate. Cancer subtypes in this study included all stages, sexes, races, grades, mutational burdens, and neoantigen levels. The probe expression range was 345–3147.

[0200] Example 8. The disclosed methods and agents for suppressing and inhibiting mTOR can be used for sarcoma, with biomarkers being used to select patients who will benefit from the methods and / or agents. For sarcoma, the key biomarker was the level of type 1 helper T cells in the tumor microenvironment.

[0201] A clinical outcome study of sarcoma patients was conducted, which showed improved overall survival (OS) with reduced mTOR expression in patients with elevated levels of type 1 helper T cells. The improvement in median OS observed with reduced mTOR expression in patients with elevated type 1 helper T cells above the median was significant, from 28 months in the high-mTOR cohort to 86 months in the low-mTOR cohort—a more than three-fold increase in OS. This improvement was quite surprising, as data from over 900 other cancer patients with a wide range of cancers did not demonstrate such improvements with reduced mTOR expression when patients were not selected using biomarkers.

[0202] A Kaplan-Meier Plotter was used to calculate patient survival and determine the association between improved overall survival and reduced mTOR expression in this subtype of sarcoma, with biomarkers used to select patients who would benefit from the method and / or agent.

[0203] The results of this study on this subtype of sarcoma are shown in Figure 12, which shows a highly significant (log-rank P = 0.0037) improvement in overall survival for patients whose type 1 helper T cells were elevated above the median. The improvement in median overall survival observed with decreased mTOR expression in the presence of type 1 helper T cells was significant, with a hazard ratio of 2.28, i.e., patients with elevated type 1 helper T cells had a more than two-fold increased survival rate. Cancer subtypes in this study included all stages, genders, races, grades, mutational burdens, and neoantigen burdens. The probe expression range was 345–3522.

[0204] Example 9. The disclosed methods and agents for suppressing and inhibiting mTOR can be used for sarcoma, with biomarkers being used to select patients who will benefit from the methods and / or agents. Important biomarkers for sarcoma were the level of type 1 helper T cells in the tumor microenvironment and the level of mesenchymal stem cells in the tumor microenvironment.

[0205] A clinical outcome study of sarcoma patients showed improved overall survival (OS) with reduced mTOR expression when type 1 helper T cell and mesenchymal stem cell levels were elevated. The improvement in median OS observed with reduced mTOR expression when type 1 helper T cell and mesenchymal stem cell levels were elevated above the median range was significant, from 12 months in the high-mTOR cohort to 33 months in the low-mTOR cohort—a nearly three-fold increase in OS. This improvement was quite surprising, as data from over 900 other cancer patients with a wide range of cancers did not demonstrate such improvements with reduced mTOR expression when patients were not selected using biomarkers.

[0206] A Kaplan-Meier Plotter was used to calculate patient survival and determine the association between improved overall survival and reduced mTOR expression in this subtype of sarcoma, with biomarkers used to select patients who would benefit from the method and / or agent.

[0207] The results of this study of sarcoma subtypes are shown in Figure 13. Figure 13 shows a highly significant (log-rank P = 0.0025) improvement in overall survival for patients with elevated type 1 T helper cells and mesenchymal stem cells above the median. The improvement in median overall survival observed with decreased mTOR expression in the presence of elevated type 1 T helper cells and mesenchymal stem cells was significant, with a hazard ratio of 2.63; that is, patients with elevated type 1 T helper cells and mesenchymal stem cells had a more than two-fold increased survival rate. Cancer subtypes in this study included all stages, sexes, races, grades, mutation burdens, and neoantigen burdens. The probe expression range was 345–3103.

[0208] Example 10. The disclosed methods and agents for suppressing and inhibiting mTOR can be used for sarcoma, with biomarkers being used to select patients who will benefit from the methods and / or agents. For sarcoma, the key biomarkers are the level of type 1 helper T cells in the tumor microenvironment, the level of mesenchymal stem cells in the tumor microenvironment, and tumor gene mutation burden.

[0209] A clinical outcomes study of sarcoma patients showed improved overall survival (OS) with reduced mTOR expression in patients with reduced tumor mutation burden, increased levels of type 1 helper T cells, and increased levels of mesenchymal stem cells. In patients with increased type 1 helper T cells and mesenchymal stem cells above the median but reduced tumor mutation burden, the improvement in median OS with reduced mTOR expression was significant, from 12 months in the high-mTOR cohort to 49 months in the low-mTOR cohort—a more than fourfold increase in OS. This improvement was particularly surprising, as data from over 900 other cancer patients with a wide range of cancers had not shown such improvements with reduced mTOR expression when patients were not selected using biomarkers.

[0210] A Kaplan-Meier Plotter was used to calculate patient survival and determine the association between improved overall survival and reduced mTOR expression in this subtype of sarcoma, with biomarkers used to select patients who would benefit from the method and / or agent.

[0211] The results of this study of this sarcoma subtype are shown in Figure 14. Figure 14 shows a highly significant (log-rank P = 0.00073) improvement in overall survival for patients with elevated type 1 T helper cells and mesenchymal stem cells (MSCs) above the median but with reduced tumor mutation burden (below the median). In the presence of elevated type 1 T helper cells and MSCs but with reduced tumor mutation burden, the improvement in median overall survival observed with reduced mTOR expression was significant, with a hazard ratio of 5.73; that is, patients with elevated type 1 T helper cells and MSCs and reduced tumor mutation burden had an almost six-fold increased survival rate. Cancer subtypes in this study included all stages, sexes, races, grades, and neoantigen burdens. The probe expression range was 429–3103.

[0212] Example 11. The disclosed methods and agents for suppressing and inhibiting mTOR can be used for sarcoma, with biomarkers being used to select patients who will benefit from the methods and / or agents. Important biomarkers for sarcoma were the level of type 1 helper T cells and neoantigen load in the tumor microenvironment.

[0213] A clinical outcome study of sarcoma patients showed improved overall survival (OS) with reduced mTOR expression in patients with reduced neoantigen load and increased levels of type 1 helper T cells. In patients with elevated type 1 helper T cells above the median but reduced neoantigen load, the improvement in median OS observed with reduced mTOR expression was significant, from 12 months in the high mTOR cohort to 30 months in the low mTOR cohort—a more than two-fold increase in OS. This improvement was quite surprising, as data from over 900 other cancer patients with a wide range of cancers had not shown such improvements with reduced mTOR expression when patients were not selected using biomarkers.

[0214] A Kaplan-Meier Plotter was used to calculate patient survival and determine the association between improved overall survival and reduced mTOR expression in this subtype of sarcoma, with biomarkers used to select patients who would benefit from the method and / or agent.

[0215] The results of this study of sarcoma subtypes are shown in Figure 15. Figure 15 shows a highly significant (log-rank P = 0.00078) improvement in overall survival for patients with elevated type 1 T helper cells above the median but with reduced neoantigen levels below the median. In the presence of elevated type 1 T helper cells and reduced neoantigen levels, the improvement in median overall survival observed with reduced mTOR expression was significant, with a hazard ratio of 2.95; patients with elevated type 1 T helper cells and reduced neoantigen levels had a nearly threefold increased survival rate. Cancer subtypes in this study included all stages, sexes, races, grades, and mutational burdens. The probe expression range was 345–3522.

Claims

1. 1. A method for treating or ameliorating the symptoms of cancer in a human or animal subject in need thereof, comprising: administering to the subject a therapeutically sufficient amount of a pharmaceutical composition comprising an agent for inhibiting mTOR. The method comprising:

2. Agents for inhibiting mTOR to treat or ameliorate the symptoms of cancer in a human or animal subject in need thereof.

3. Use of a composition comprising an agent for inhibiting mTOR in the preparation of a medicament for treating or ameliorating the symptoms of cancer in a human or animal subject in need thereof.

4. 4. The method, agent or use of any one of claims 1 to 3, wherein the cancer is sarcoma, lung cancer, ovarian cancer or pancreatic cancer.

5. 5. The method, agent or use of any one of claims 1 to 4, comprising the use of one or more biomarkers to select subjects who will benefit from said method, agent or use.

6. 6. The method, agent or use of claim 5, wherein the one or more biomarkers are levels of tumor mutational burden (TMB), levels of tumor neo-antigens, levels of tumor-associated immune cells, or a combination thereof.

7. 6. The method, agent or use of claim 5, wherein the one or more biomarkers are levels of basophil cells, levels of B cells, levels of T cells, levels of helper T cells (Th), levels of eosinophil cells, levels of macrophage cells, levels of mesenchymal stem cells, or a combination thereof, determined in the tumor microenvironment.

8. 6. The method, agent or use of claim 5, wherein the one or more biomarkers are levels of CD4+ cells, levels of memory T cells, levels of CD8+ cells, levels of natural killer T cells, levels of regulatory T cells, levels of type 1 helper T cells (Th1), levels of type 2 helper T cells (Th2), or a combination thereof, determined in the tumor microenvironment.

9. 6. The method, agent, or use of claim 5, wherein the cancer is a sarcoma and the one or more biomarkers are levels of tumor mutational burden (TMB), levels of tumor neoantigens, levels of mesenchymal stem cells determined in the tumor microenvironment, levels of type 1 helper T cells (Th) determined in the tumor microenvironment, or a combination thereof.

10. 6. The method, agent, or use of claim 5, wherein the cancer is lung squamous cell carcinoma and the one or more biomarkers are levels of CD4+ cells determined in the tumor microenvironment, levels of memory T cells determined in the tumor microenvironment, levels of eosinophil cells determined in the tumor microenvironment, or a combination thereof.

11. 6. The method, agent, or use of claim 5, wherein the cancer is pancreatic cancer and the one or more biomarkers are levels of CD8+ cells determined in the tumor microenvironment, levels of tumor mutational burden (TMB), or a combination thereof.

12. 6. The method, agent, or use of claim 5, wherein the cancer is ovarian cancer and the one or more biomarkers are levels of natural killer T cells determined in the tumor microenvironment, levels of tumor neo-antigens determined in the tumor microenvironment, or a combination thereof.

13. Agents that inhibit mTOR an mTOR-specific antisense oligonucleotide that is complementary to the mTOR transcript and is 15-30 nucleotides in length; or an mTOR-specific antisense oligonucleotide that is complementary to mTOR pre-RNA, pre-mRNA, or mRNA and is 18 to 21 nucleotides in length; 13. A method, agent or use according to any one of claims 1 to 12.

14. 2. The agent for inhibiting mTOR comprises a 20 nucleotide sequence of the following table: and any of their chemically modified variants, their LNA variants, their gapmer variants, and pools thereof and combinations thereof.

15. 15. The agent, use, or method of claim 14, wherein the mTOR-specific antisense oligonucleotide has no more than one or two mismatches compared to the target human mTOR.

16. 15. The agent, use, or method of claim 14, wherein the mTOR-specific antisense oligonucleotide reduces the level of mTOR transcript by at least 60%, or at least 70%, or at least 80%, or at least 90%.

17. 15. The agent, use, or method of claim 14, wherein the mTOR-specific antisense oligonucleotide has one or more nucleotides chemically modified as a phosphorothioate internucleoside linkage, a methoxypropylphosphonate internucleoside linkage, an aminophosphoro linkage with 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-methylcytodine base.

18. 15. The agent, use, or method of claim 14, wherein the antisense oligonucleotide is conjugated to polyethylene glycol, a lipid, or triantennary N-acetylgalactosamine.

19. 19. The method, agent or use of any one of claims 15 to 18, comprising the antisense oligonucleotide in a carrier that is sterile water for injection, saline, isotonic saline, or a combination thereof.

20. 20. The method, agent or use of any one of claims 15 to 19, wherein the antisense oligonucleotide is substantially free of excipients.

21. 21. The method, agent or use of any one of claims 15 to 20, wherein the antisense oligonucleotide is stable in the carrier at 37°C for at least 14 days.

22. 22. The method, agent or use of any one of claims 1 to 21, wherein the administration or use of the composition is in combination with a standard of care for cancer, the standard of care comprising chemotherapy or radiation therapy, and wherein the administration or use of the composition is carried out in the absence of an immune checkpoint inhibitor.

23. 23. The method, agent or use of any one of claims 1 to 22, wherein the administration or use reduces mortality at 6 months, 12 months, 18 months, 24 months, 30 months or 36 months.

24. 23. The method, agent or use of any one of claims 1 to 22, wherein the administration or use increases survival at 6 months, 12 months, 18 months, 24 months, 30 months or 36 months.

25. 1. A method for treating or ameliorating the symptoms of cancer in a human or animal subject in need thereof, comprising: administering to said subject a therapeutically sufficient amount of a pharmaceutical composition comprising an mTOR-specific antisense oligonucleotide in combination with an agent for inhibiting mTOR. A method comprising:

26. 26. The method of claim 25, wherein the cancer is sarcoma, lung cancer, ovarian cancer, or pancreatic cancer.

27. 27. The method of any one of claims 25 to 26, comprising using one or more biomarkers to select subjects who will benefit from said method.

28. 28. The method of claim 27, wherein the one or more biomarkers are a level of tumor mutational burden (TMB), a level of tumor neo-antigens, a level of tumor-associated immune cells, or a combination thereof.

29. 28. The method of claim 27, wherein the one or more biomarkers are a level of basophil cells determined in the tumor microenvironment, a level of B cells determined in the tumor microenvironment, a level of T cells determined in the tumor microenvironment, a level of helper T cells (Th) determined in the tumor microenvironment, a level of eosinophil cells determined in the tumor microenvironment, a level of macrophage cells determined in the tumor microenvironment, a level of mesenchymal stem cells determined in the tumor microenvironment, or a combination thereof.

30. 28. The method of claim 27, wherein the one or more biomarkers are levels of CD4+ cells determined in the tumor microenvironment, levels of memory T cells determined in the tumor microenvironment, levels of CD8+ cells determined in the tumor microenvironment, levels of natural killer T cells determined in the tumor microenvironment, levels of regulatory T cells determined in the tumor microenvironment, levels of type 1 helper T cells (Th1) determined in the tumor microenvironment, levels of type 2 helper T cells (Th2) determined in the tumor microenvironment, or a combination thereof.

31. 28. The method of claim 27, wherein the cancer is a sarcoma and the one or more biomarkers are a level of tumor mutational burden (TMB), a level of tumor neoantigens, a level of mesenchymal stem cells determined in the tumor microenvironment, a level of type 1 helper T cells (Th) determined in the tumor microenvironment, or a combination thereof.

32. 28. The method of claim 27, wherein the cancer is lung squamous cell carcinoma and the one or more biomarkers are levels of CD4+ cells determined in the tumor microenvironment, levels of memory T cells determined in the tumor microenvironment, levels of eosinophil cells determined in the tumor microenvironment, or a combination thereof.

33. 28. The method of claim 27, wherein the cancer is pancreatic cancer and the one or more biomarkers are levels of CD8+ cells determined in the tumor microenvironment, levels of tumor mutational burden (TMB), or a combination thereof.

34. 28. The method of claim 27, wherein the cancer is ovarian cancer and the one or more biomarkers are levels of natural killer T cells determined in the tumor microenvironment, levels of tumor neo-antigens determined in the tumor microenvironment, or a combination thereof.

35. mTOR-specific antisense oligonucleotides is complementary to the mTOR transcript and is 15-30 nucleotides in length; or complementary to mTOR pre-RNA, pre-mRNA, or mRNA and 18 to 21 nucleotides in length; 35. The method of any one of claims 25 to 34.

36. The mTOR-specific antisense oligonucleotides have the 20 nucleotide sequences of the following table: and any of their chemically modified variants, their LNA variants, their gapmer variants, and pools thereof and combinations thereof.

37. The method of any one of claims 35 to 36, wherein the mTOR-specific antisense oligonucleotide has only one or two mismatches compared to the target human mTOR.

38. 37. The method of any one of claims 35 to 36, wherein the mTOR-specific antisense oligonucleotide reduces the level of mTOR transcript by at least 60%, or at least 70%, or at least 80%, or at least 90%.

39. 37. The method of any one of claims 35-36, wherein the mTOR-specific antisense oligonucleotide has one or more nucleotides chemically modified as a phosphorothioate internucleoside linkage, a methoxypropylphosphonate internucleoside linkage, an aminophosphoro linkage with 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-methylcytodine base.

40. 37. The method of any one of claims 35 to 36, wherein the antisense oligonucleotide is conjugated to polyethylene glycol, a lipid, or triantennary N-acetylgalactosamine.

41. an antisense oligonucleotide in a carrier that is sterile water for injection, saline, isotonic saline, or a combination thereof; and the antisense oligonucleotide is stable in the carrier at 37°C for at least 14 days; 37. The method according to any one of claims 35 to 36.

42. 37. The method of any one of claims 35-36, comprising an antisense oligonucleotide substantially free of excipients.

43. 43. The method of any one of claims 25-42, wherein the agent for inhibiting mTOR is rapamycin, everolimus, temsirolimus, sirolimus, deforolimus, ridaforolimus, zotarolimus, torkinib, samotricisib, omipalisib, apitolisib, bistusertib, dactolisib, jedatolicib, voxtalisib, chrysophanic, or a combination thereof.

44. Agents for inhibiting mTOR include 1-[4-[4-(1-oxopropyl)-1-piperazinyl]-3-(trifluoromethyl)phenyl]-9-(3-quinolinyl)-benzo[h]-1,6-naphthyridin-2(1H)-one, 3-[4-(4-morpholinylpyrido[3',2':4,5]furo[3,2-d]pyrimidin-2-yl]phenol hydrochloride, N-[4-[4-(4-morpholinyl)-1-[1-(3-pyridinylmethyl)-4-piperidinyl]-1H-pyrazolo[3,4-d]pyrimidin-6-yl]phenol hydrochloride, and 1-[4-[4-(4-morpholinyl)-1-[1-(3-pyridinylmethyl)-4-piperidinyl]-1H-pyrazolo[3,4-d]pyrimidin-6-yl]-2-methyl-4-methyl-2-methyl-3-methyl-4-methyl ...

43. The method of any one of claims 25 to 42, wherein the compound is selected from the group consisting of 5-(4-(4-pyridazinyl)-6-quinolinyl)-3-pyridinyl)benzolsulfonamide, 2,4-difluoro-N-[2-methoxy-5-[4-(4-pyridazinyl)-6-quinolinyl]-3-pyridinyl]benzenesulfonamide (omipalisib), 5-chloro-N-(2-chloro-4-nitrophenyl)-2-hydroxybenzamide (niclosamide), or a combination thereof.

45. 45. The method of any one of claims 25-44, wherein the administering is in combination with a standard of care for the cancer, wherein the standard of care comprises chemotherapy or radiation therapy, and wherein the administering is performed in the absence of an immune checkpoint inhibitor.

46. 46. ​​The method of any one of claims 25 to 45, wherein the mTOR-specific antisense oligonucleotide and the agent for inhibiting mTOR are administered contemporaneously, simultaneously, sequentially, or at separate times, respectively.

47. 47. The method of any one of claims 25-46, wherein the administration reduces mortality at 6 months, 12 months, 18 months, 24 months, 30 months, or 36 months.

48. 47. The method of any one of claims 25-46, wherein administering increases survival at 6 months, 12 months, 18 months, 24 months, 30 months, or 36 months.

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