Cancer therapeutic agents employing antisense nucleic acids and interferon-gamma

Abstract

The present invention relates to agents, compositions, and methods for treating or ameliorating symptoms of cancer. Exemplary synergistic therapies include the use of antisense oligonucleotide agents to suppress expression of TGF-β2, alone and in combination with interferon-gamma. One or more biomarkers can be used to select subjects for treatment.

Description

[0001] sequence list

[0002] This application includes a sequence list electronically submitted in the form of an ST.26 file, created on August 27, 2024, named 018988-013WO1_SL.xml, with a size of 120,204 bytes. Technical Field

[0003] This invention relates to pharmaceutical agents, compositions, and methods for treating or improving cancer symptoms. Exemplary synergistic therapies include active agents, alone or in combination with interferon-γ, used to suppress TGF-β2 expression. One or more biomarkers may be used to select subjects for treatment. Background Technology

[0004] Cancer is a complex pathology involving multiple different cellular pathways. Due to this complexity, many anticancer drugs have limited or partial therapeutic efficacy.

[0005] Disadvantages of conventional therapies include a lack of efficacy, as measured by overall survival. In some cases, surgical resection is not possible due to the anatomical location of the tumor.

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

[0007] Other drawbacks of anticancer drugs include high toxicity at the required therapeutic dose levels. In some cases, monotherapy may not be a viable strategy due to the presence of the therapeutic target and the potential for variations in its levels.

[0008] What is needed are agents, compositions, and methods for treating cancer to increase efficacy and reduce toxicity and adverse side effects. Therapies involving combinations of active agents can improve treatment outcomes. Furthermore, guidance from biomarkers can be a powerful tool for improving treatments.

[0009] There is a need for therapeutic compositions of different agents to provide significant antitumor and cancer immunotherapy effects, while improving efficacy, reducing side effects, and minimizing adverse health impacts. Improved guidance on the use of such compositions requires the use of appropriate biomarkers to select agents and compositions for synergistic effects. Invention Overview

[0011] This invention provides pharmaceutical agents, compositions, and methods for treating cancer or improving cancer symptoms. The synergistic drug therapies of this invention include the use of potent antitumor agents, alone or in combination with interferon-gamma. The cancer treatment approaches disclosed herein include selecting patients and cancers for therapy based on biomarkers and disease variants to improve outcomes.

[0012] Embodiments of the present invention cover the suppression of high levels of TGF-β2 expression (which can be done in combination with interferon-γ) and the selection of patients based on certain biomarker levels.

[0013] Further embodiments of the present invention include individually suppressing high levels of TGF-β2 expression and selecting patients based on biomarkers.

[0014] In some embodiments, the methods and treatment strategies of the present invention may include the use of appropriate biomarkers to select for synergistic effects of compositions and agents, thereby providing enhanced guidance for successful patient outcomes.

[0015] In some implementations, selecting cancer patients with elevated IFNGR2 levels can improve clinical outcomes.

[0016] The embodiments of the present invention include the following: An agent for suppressing TGF-β2 expression, in combination with interferon-γ, for treating cancer in subjects in need or improving cancer symptoms in subjects in need, wherein the subjects may be selected as subjects having (a) decreased JAK1 and STAT1, and (b) increased TGF-β2 and IFNGR2.

[0017] A method for treating cancer in a subject in need or improving cancer symptoms in a subject in need, wherein the method may include: Subjects who may have (a) decreased JAK1 and STAT1, or both, and (b) increased TGF-β2 and IFNGR2 were selected; Application of a composition containing an agent for suppressing TGF-β2 expression; and Administer a composition containing interferon-γ.

[0018] A composition comprising an agent for suppressing TGF-β2 expression and a pharmaceutically acceptable carrier, wherein the composition is used in combination with interferon-γ to prepare a medicament for treating a subject’s cancer or improving the subject’s cancer symptoms, wherein the subject may be selected as a subject having (a) decreased JAK1 and STAT1, and (b) increased TGF-β2 and IFNGR2.

[0019] The above-described pharmaceutical agents, methods, or compositions may contain brain cancer or spinal cord cancer, glioma, glioblastoma, diffuse intrinsic pontine glioma (DIPG), diffuse midline glioma (DMG), diffuse hemispheric glioma, or leptomeningeal or brain metastases, or wherein the cells of said cancer exhibit somatic mutations containing H2-K27M, H3-K27M, or H3-G34 genomic variants.

[0020] The above-described agents, methods, or compositions, wherein the agents used to suppress TGF-β2 expression and interferon-γ can be administered in parallel, simultaneously, sequentially, or separately in time.

[0021] The above-described pharmaceutical agents, methods, or compositions, wherein the compositions and pharmaceutical agents can be administered by infusion or injection.

[0022] The above-described pharmaceutical agents, methods, or compositions may have reduced mRNA levels of one or both of JAK1 and STAT1, which are lower than the median in a control group of subjects with the same cancer, and elevated levels of TGF-β2 and IFNGR2, which are higher than the median in a control group of subjects with the same cancer.

[0023] The agents, methods or compositions described above, wherein the agents used to suppress TGF-β2 expression may be selected from Table 1 or Table 2, and their chemically modified variants, their LNA variants, their gapmer variants, and any combination or collection thereof.

[0024] The above-mentioned agents, methods, or compositions, wherein the agent used to inhibit or suppress TGF-β2 expression can be C G G C A T G T C T A T T T T G T A SEQ ID NO:137 (OT-101) or CGGCATGTCTATTTTGTASEQ ID NO:1.

[0025] The above-mentioned pharmaceutical agents, methods, or compositions may contain interferon-γ, which may be recombinant human interferon-γ.

[0026] The above-mentioned pharmaceutical agents, methods, or compositions may include a carrier, wherein the carrier is sterile water for injection, saline, isotonic saline, or a combination thereof.

[0027] The above-described pharmaceutical agents, methods, or compositions may be substantially free of excipients.

[0028] The above-described pharmaceutical agents, methods, or compositions, wherein the composition is stable in a carrier at 37°C for at least 14 days.

[0029] The above-described pharmaceutical agents, methods, or compositions, wherein the subject may have reduced TGF-β2 expression after administration or use.

[0030] The above-described agents, methods, or compositions, wherein the administration or use thereof can reduce mortality at 6, 12, 18, 24, 30, or 36 months.

[0031] The above-described agents, methods, or compositions, wherein the administration or use can improve survival rates at 6, 12, 18, 24, 30, or 36 months.

[0032] The above-described pharmaceutical agents, methods, or compositions, wherein the administration or use of the composition may be combined with standard care treatment for cancer, wherein the standard care treatment includes chemotherapy or radiation therapy.

[0033] An agent for suppressing TGF-β2 expression, used to treat cancer in a subject in need or to improve cancer symptoms in a subject in need, wherein the subject may be selected as a subject having (a) elevated IFNGR2 and (b) elevated TGF-β2.

[0034] A method for treating cancer in a subject in need or improving cancer symptoms in a subject in need, wherein the method may include: Subjects with (a) elevated IFNGR2 and (b) elevated TGF-β2 were selected; and Administer a composition containing an agent for suppressing TGF-β2 expression.

[0035] A composition comprising an agent for suppressing TGF-β2 expression and a pharmaceutically acceptable carrier, for use in preparing a medicament for treating a subject’s cancer or improving the subject’s cancer symptoms, wherein the subject may be selected as a subject having (a) elevated IFNGR2 and (b) elevated TGF-β2.

[0036] The above-described pharmaceutical agents, methods, or compositions, wherein the cancer may be brain cancer or spinal cord cancer, glioma, glioblastoma, diffuse endophytic pontine glioma (DIPG), diffuse midline glioma (DMG), diffuse hemispheric glioma, or leptomeningeal or brain metastases, or wherein the cells of the cancer exhibit somatic mutations containing H2-K27M, H3-K27M, or H3-G34 genomic variants.

[0037] The above-mentioned pharmaceutical agents, methods, or compositions may be administered by infusion or injection.

[0038] The above-mentioned drugs, methods, or compositions may include subjects with elevated IFNGR2 mRNA levels below the median of control subjects with the same cancer and elevated TGF-β2 mRNA levels above the median of control subjects with the same cancer.

[0039] The above-described agents, methods, or compositions, wherein the agents used to suppress TGF-β2 expression are selected from Table 1 or Table 2, and their chemically modified variants, LNA variants, gapmer variants, and any combination or collection thereof.

[0040] The above methods, agents, or compositions, wherein the agent used to inhibit or suppress TGF-β2 expression can be C G G C A T G T C T A T T T T G T A SEQ ID NO:137 (OT-101) or CGGCATGTCTATTTTGTASEQ ID NO:1.

[0041] The above-described pharmaceutical agents, methods, or compositions may include a carrier, wherein the carrier is sterile water for injection, saline, isotonic saline, or a combination thereof.

[0042] The above-described pharmaceutical agents, methods, or compositions, wherein the compositions may be substantially free of excipients.

[0043] The above-mentioned pharmaceutical agents, methods, or compositions, wherein the composition is stable in a carrier at 37°C for at least 14 days.

[0044] The above-mentioned drugs, methods, or compositions may result in reduced TGF-β2 expression in the subject after administration.

[0045] The above-described agents, methods, or compositions, wherein the administration of which reduces mortality at 6, 12, 18, 24, 30, or 36 months.

[0046] The above-described agents, methods, or compositions, wherein the administration of which can improve survival rates at 6, 12, 18, 24, 30, or 36 months.

[0047] The above-described pharmaceutical agents, methods, or compositions, wherein the administration of said composition may be combined with standard care treatment for cancer, said standard care treatment including chemotherapy or radiation therapy.

[0048] Brief description of the attached figures

[0049] Figure 1 The study showed upregulation of IFNGR2 mRNA expression in brain tumor samples. Compared with normal pontine tissue, the IFNGR2 mRNA level in DMG samples was significantly increased (1.58-fold increase; p = 5.5 × 10⁻⁶). -4 JAK1 and STAT1 decreased.

[0050] Figure 2 The Kaplan-Meier overall survival chart is shown in the clinical outcome study of glioma patients. Figure 2 The results showed that when TGF-β2 levels were high (indicating high disease activity), high levels of IFNGR2 resulted in a surprisingly improved overall survival (log-rank P = 0.012). This example demonstrates that the IFNGR2 biomarker leads to surprisingly improved outcomes in the treatment of brain cancer.

[0051] Figure 3 The Kaplan-Meier overall survival chart is shown in the clinical outcome study of glioma patients. Figure 3 The results showed that when TGF-β2 levels were high (indicating high disease activity), higher levels of IFNGR2 resulted in a surprisingly improved overall survival (top curve, log-rank p < 0.001). This example demonstrates the efficacy of the IFNGR2 biomarker in leading to surprisingly improved outcomes in the treatment of brain cancer.

[0052] Figure 4 The Cox proportional hazards measure obtained in a clinical outcome study of glioma patients is shown. Figure 4 The results showed that when an interaction term was allowed between TGF-β2 and IFNGR2 levels for clinical outcomes in gliomas, TGF-β2 was largely independent of IFNGR2.

[0053] Figure 5 The Kaplan-Meier overall survival chart is shown in the clinical outcome study of glioma patients. Figure 5 The results showed that a significant improvement in overall survival was observed for suppressed low levels of TGF-β2.

[0054] Figure 6 The Kaplan-Meier overall survival chart is shown in the clinical outcome study of glioma patients. Figure 6 The results showed a surprising improvement in overall survival at relatively high JAK1 levels (top curve, log-rank p < 0.001). This example demonstrates the efficacy of the JAK1 biomarker in leading to surprisingly improved outcomes in the treatment of brain cancer.

[0055] Figure 7 The Kaplan-Meier overall survival chart is shown in the clinical outcome study of glioma patients. Figure 7 The results showed a surprisingly improved overall survival for relatively high levels of STAT1 (top curve, log-rank p < 0.001). This example demonstrates the efficacy of the STAT1 biomarker in leading to surprisingly improved outcomes in the treatment of brain cancer. Invention Details

[0057] This invention relates to pharmaceutical agents, compositions, and methods for treating cancer or improving cancer symptoms. Exemplary synergistic therapies include active agents for suppressing TGF-β2 expression, which can be used alone or in combination with interferon-γ. One or more biomarkers can be used to select subjects for treatment.

[0058] In some implementations, one or more biomarkers may be used to select subjects for the methods, compositions, or uses. The methods, compositions, and compositions may be used in combination with chemotherapy and standard care therapies for the same cancer.

[0059] Embodiments of this invention encompass the combination of inhibiting high levels of TGF-β2 expression with interferon-γ, and selecting patients with low levels of certain biomarkers. High levels of intratumoral TGF-β2 mRNA expression in cancer patients are strongly associated with poor survival; therefore, suppressing TGF-β2 expression has been shown to have therapeutic effects. As demonstrated by overall survival, suppressing high levels of TGF-β2 expression in combination with interferon-γ and selecting patients with some levels of one or more biomarkers, IFNGR2, JAK1, and STAT1 mRNA expression, can surprisingly improve treatment outcomes.

[0060] A further embodiment of the invention includes suppressing high levels of TGF-β2 expression in combination with selection of patients with a certain level of a biomarker. High levels of intratumoral TGF-β2 mRNA expression in cancer patients are strongly associated with poor survival, and suppressing TGF-β2 expression has been shown to have a therapeutic effect. As demonstrated by overall survival, the combination of suppressing high levels of TGF-β2 expression with selection of patients with high levels of the biomarker IFNGR2 mRNA expression can surprisingly improve treatment outcomes.

[0061] In some implementations, selecting cancer patients with high IFNGR2 expression can improve clinical outcomes. Suppressing high TGF-β2 expression in patients with high IFNGR2 expression can improve clinical outcomes. In some aspects, subjects with both (a) elevated IFNGR2 and (b) elevated TGF-β2 can be selected for treatment.

[0062] In a further embodiment, for patients with low levels of either JAK1 or STAT1, suppression of high levels of TGF-β2 in combination with IFN-γ administration can improve survival outcomes. IFN-γ can activate interferon-γ receptors in a variety of immune cells and activate Janus kinases JAK1 and JAK2, as well as signal transducers and transcriptional activators STAT1 and STAT3 pathways. In some embodiments, interferon-γ can be delivered to the tumor microenvironment. IFN-γ can be administered for treatment via a variety of routes and delivery systems known in the art. In some aspects, subjects with (a) decreased levels of one or more of JAK1 and STAT1 and (b) elevated levels of TGF-β2 and IFNGR2 can be selected for treatment.

[0063] In some embodiments, the cancer to be treated may include any one of brain cancer or spinal cord cancer, glioma, glioblastoma, diffuse endophytic pontine glioma (DIPG), diffuse midline glioma (DMG), diffuse hemispheric glioma, or leptomeningeal or brain metastases, or said cancer cells exhibiting somatic mutations including H3-K27M, H2-K27M, or H3-G34 genomic variants.

[0064] In some embodiments, the cancer of this disclosure to be treated may have cells exhibiting somatic mutations containing genomic variants of H3-K27M, H2-K27M, or H3-G34.

[0065] In a further embodiment, the cancer of this disclosure to be treated may have cells exhibiting somatic mutations containing the H3-K27M genomic variant.

[0066] In some embodiments, the cancer to be treated may include diffuse midline glioma having cells exhibiting somatic mutations containing the H3-K27M genomic variant.

[0067] In some implementations, diagnostic, genomic variant, or biomarker transcript levels can be obtained or measured by methods and / or facilities known and / or compiled in the art, including next-generation sequencing, RNA-Seq, whole-exome sequencing, tissue biopsy, liquid biopsy, and radiological methods.

[0068] As used herein, the terms patient and subject are used interchangeably. Subjects requiring cancer therapies as described herein may be human or animal subjects.

[0069] As used in this article, the terms TGF-β2, TGFB2, and TGF-B2 are synonyms.

[0070] As used herein, the term pharmaceutical agent can refer to one or more active compounds, a combination of active compounds, or a composition comprising one or more active compounds and a carrier, and / or a solvent, and / or any number of excipients. In some embodiments, the composition may be a pharmaceutical composition. In some embodiments, the composition may be a pharmaceutical composition containing a therapeutically effective amount of one or more active compounds. The formulation of the active agent can be determined by those skilled in the art. Some examples of excipients are given in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa. 1975, and Liberman, HA and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, NY, 1980. Methods for determining a therapeutically effective amount of a compound are known in the art.

[0071] Anticancer methods and compositions

[0072] This invention includes methods for treating cancer in subjects of need or improving cancer symptoms in subjects of need. The methods may include selecting subjects having (a) decreased JAK1 and STAT1, or (b) increased TGF-β2 and IFNGR2; administering a composition comprising an agent for suppressing TGF-β2 expression; and administering a composition comprising interferon-γ. Improved overall survival in cancer patients can be achieved even when the tumor microenvironment may be immunologically “cold.”

[0073] The present invention further includes methods for treating brain or spinal cord cancer in a subject of need, or for improving symptoms of brain or spinal cord cancer in a subject of need. The methods may include selecting a subject with elevated TGF-β2 and IFNGR2, and administering a composition comprising an agent for suppressing TGF-β2 expression.

[0074] In some embodiments, the methods disclosed herein for treating cancer or improving cancer symptoms may include one or more steps for selecting subjects with elevated TGF-β2 expression and a certain level of guiding biomarkers for treatment. Improved overall survival in cancer patients can be achieved even when the tumor microenvironment may be immunologically cold.

[0075] In a further embodiment, the method disclosed herein for treating cancer or improving cancer symptoms may include one or more steps of selecting subjects with elevated TGF-β2 and IFNGR2 expression for treatment.

[0076] In another respect, the combined drugs can be administered in parallel, simultaneously, sequentially, or separately.

[0077] Human TGF-β2 specific phosphate thioester antisense oligodeoxynucleotide

[0078] Antisense oligonucleotides (ASOs) can be single-stranded deoxyribonucleotides that are complementary to mRNA targets. Antisense therapy can downregulate molecular targets, which can be achieved by inducing RNase H endonuclease activity, which cleaves RNA-DNA heteroduplexes, thereby significantly reducing the translation of target genes. Other ASO mechanisms may include inhibiting 5' cap formation, altering splicing processes (e.g., splice transition), and steric hindrance of ribosome activity.

[0079] Antisense therapeutic strategies can utilize single-stranded DNA oligonucleotides, which inhibit protein production by mediating the catalytic degradation of target mRNA or by binding to sites on mRNA required for translation. Antisense oligonucleotides can be engineered to target viral RNA genomes or viral transcripts. Antisense oligonucleotides can provide a method for identifying potential targets and therefore represent potential therapeutic agents.

[0080] Antisense oligonucleotides (ASOs) can be small synthetic fragments of single-stranded DNA, ranging in length from 15 to 30 nucleotides. ASOs specifically bind to complementary DNA / RNA sequences via Watson-Crick hybridization, and once bound to the target RNA, they inhibit translation through cleavage induction or by suppressing mRNA maturation. ASOs can selectively suppress gene expression with specificity. Stability can be increased through chemical modifications to DNA or RNA.

[0081] For example, modifications can be introduced into the phosphodiester bond, sugar ring, and backbone. ASO antiviral agents can block translation by (i) ribonuclease H (RNase H) or RNase P-mediated mRNA cleavage; or (ii) by spatially (non-bonded) blocking of enzymes involved in the translation of target genes. Human TGF-β2-specific phosphate thioester antisense oligodeoxynucleotides (OT-101; AP 12009; Trabedersen), hereinafter referred to as OT-101 or AP12009, are designed to reduce the level of TGF-β2 protein in malignant gliomas and thus delay disease progression.

[0082] Antisense oligodeoxynucleotides are short segments of DNA designed to downregulate gene expression by interfering with the translation of specific encoded proteins at the mRNA level. OT-101 is a synthetic 18-meric phosphate-thioester oligodeoxynucleotide (S-ODN) in which all 3'-5' links are modified to phosphate-thioester. Its molecular formula is C1. 177 H 208 N 60 Na 17 O 94 P 17 S 17 It has a molecular weight of 6,143 g / mol. OT-101 is designed to be complementary to a specific sequence of human TGF-β2 mRNA after gene expression.

[0083] OT-101 is available as a lyophilized powder in three different amounts in 50 mL glass vials. Each vial is labeled with the name of the investigational product, trial number, dosing group, route of administration, amount of OT-101 contained (in mg), total volume after dissolution (in mL), and concentration obtained (in μM), sponsor name, manufacturer name, batch number, vial number, storage temperature, and expiry date. The investigational drug can be supplied in closed units, each concentration individually packaged. The packaging may contain the appropriate vial and all necessary components of the application system (i.e., syringe, tubing, and filter). Before use, dissolve the OT-101 lyophilized powder in an isotonic (0.9%) aqueous sodium chloride solution.

[0084] Examples of agents disclosed herein for inhibiting or suppressing TGF-β2 expression include the TGF-β2-specific antisense oligonucleotides given in Table 1, SEQ ID NO: 1-136.

[0085] Table 1: TGF-β2-specific antisense oligonucleotides

[0086] The sequences in Table 1 can be chemically modified to provide their active variants, their LNA variants, and their gapmer variants known in the art. The sequences in Table 1 can be used as active agents in any combination, such as aggregated combinations.

[0087] It will be understood that other antisense oligonucleotides disclosed herein can be constructed based on the TGF-β2 gene sequence.

[0088] In some embodiments, the agent of the antisense sequence can be a gapmer formed by adding 1 to 5 protected ribonucleotides to each flanking position of the phosphate thioester deoxynucleotide sequence in Table 1. For example, the ribonucleotides can be protected with 2'-OMe, 2'-OEt, or 2'-O-MOE substituents, or with LNA, cMOE, or cEt bridges and phosphate thioester linkages.

[0089] In some implementations, the antisense sequence agent can be nMn RNA (2'-OMe). -DNA -RNA(2'-OMe) The gapmer is defined as n = 3-7 and M = 6-12. In some implementations, the gapmer can be a 3-10-3 or 5-10-5 LNA. -DNA -LNA or cEt -DNA -cEt gapmer (Indicates thiophosphate linkage).

[0090] Examples of agents disclosed herein for inhibiting or suppressing TGF-β2 expression include the TGF-β2-specific phosphate thioester antisense oligonucleotides based on the sequences in Table 1, given in Table 2 as SEQ ID NO:137-144.

[0091] Table 2: TGF-β2-specific phosphate thioester antisense oligonucleotides

[0092] Embodiments of the present invention also include pharmaceutical compositions for inhibiting or suppressing TGF-β expression or for treating human or animal cancers or alleviating symptoms of human or animal cancers. The pharmaceutical composition may contain a TGF-β suppressor, a pharmaceutically acceptable salt form thereof, an ester, a polymorph or stereoisomer thereof, or any combination thereof, and a transporter. The TGF-β suppressor may be selected from a TGF-β2-specific antisense oligonucleotide. The transporter may be sterile water for injection, saline, isotonic saline, or a combination thereof.

[0093] Importantly, the compositions disclosed herein are substantially excipient-free. The substantially excipient-free compositions of the present invention have been found to exhibit surprising stability in a carrier. In some embodiments, the compositions are stable in a carrier at 37°C for at least 14 days, at least 21 days, or at least 28 days.

[0094] In other embodiments, the pharmaceutical composition for infusion may contain less than 1% by weight, or less than 0.5% by weight, or less than 0.1% by weight of excipients.

[0095] OT-101 antisense oligonucleotide

[0096] API Tribedsen / OT-101 is a synthetic 18-mer S-ODN composed of adenine (A), thymine (T), guanine (G), and cytosine (C) bases, with all 3'-5' links modified to thiophosphate esters. This thiophosphate ester modification enhances the drug's resistance to degradation, thereby improving its stability in vitro and in vivo. Its molecular structure (nucleotide sequence) is engineered to be complementary to a specific sequence of human transforming growth factor-β2 (TGF-β2) mRNA. This sequence was selected from relevant molecules due to its excellent chemical and structural properties, biological activity, and specificity, achieving optimal antisense effects in vitro and in vivo.

[0097] Table 3 shows the chemical structure of trobedsen, the exemplary thiophosphate moiety (CAG), and its physical characteristics.

[0098] Table 3: Chemical and physical characteristics of Tribedsen

[0099] This IMP is available as a sterile lyophilized infusion solution in 50H glass vials (master container) containing 7.37 mg trobedsen (intratumoral therapy) and 20R glass vials (master container) containing 250 mg trobedsen (intravenous therapy). The finished drug product may be excipient-free. These glass vials are commonly used for parenteral medications. Sterile rubber stoppers suitable for lyophilization can seal the glass vials. The stoppers can be sealed with crimped capsules with colored flip-tops. For clinical use, each vial is provided in a white folding box to protect it from light exposure and damage during transport. Labeling of both the glass vials and folding boxes can be done according to local requirements. Both the master and secondary containers of this closed system meet international quality standards for the packaging of sterile solid pharmaceutical products for injection.

[0100] Biomarkers for brain and spinal cancer

[0101] The mRNA levels of biomarkers transforming growth factor β2 (TGF-β2) and interferon gamma receptor 2 (IFNGR2) have biomarker potential in highly aggressive cancers. The expression levels of IFNGR2 and TGF-β2 can be upregulated in cancer compared to normal tissues.

[0102] In some implementations, the agent used to suppress TGF-β2 expression may be used in combination with interferon-γ to treat cancer in a subject in need or to improve cancer symptoms in a subject in need, wherein the subject is selected as a subject having (a) decreased JAK1 and STAT1 or both and (b) increased TGF-β2 and IFNGR2.

[0103] In a further embodiment, a method for treating cancer in a subject in need or improving cancer symptoms in a subject in need may include the following steps: selecting a subject having (a) decreased JAK1 and STAT1 or both and (b) increased TGF-β2 and IFNGR2; administering a composition comprising an agent for suppressing TGF-β2 expression; and administering a composition comprising interferon-γ.

[0104] Embodiments of the present invention also cover compositions comprising an agent for suppressing TGF-β2 expression and a pharmaceutically acceptable carrier, which, in combination with interferon-γ, are used to prepare a medicament for treating a subject’s cancer or improving a subject’s cancer symptoms, wherein the subject is selected as a subject having (a) decreased JAK1 and STAT1 or both and (b) increased TGF-β2 and IFNGR2.

[0105] Improved overall survival can be achieved in cancer patients even when the tumor microenvironment may be immunologically cold.

[0106] Multivariate Cox proportional hazards can be used to suggest that TGF-β2 may be a prognostic indicator for cancer. Survival outcomes in cancer patients may indicate that eliminating the expression of target mRNAs (e.g., TGF-β2) in an immunologically cold tumor microenvironment could be a therapeutic approach for cancer patients.

[0107] Furthermore, the inclusion of interferon-γ (IFN-γ) to stimulate and activate JAK1 and STAT1 in anti-tumor APC cells present in the tumor microenvironment can enhance the effect of blocking target mRNA expression (such as blocking TGF-β2).

[0108] For example, diffuse midline glioma (DMG) is a malignant primary glioma that occurs in all age groups, but is more common in children, accounting for 10-20% of all central nervous system tumors and nearly one-third of high-grade gliomas in pediatric patients.

[0109] Our understanding of DMG has significantly improved due to advancements in biopsy techniques. For example, epigenetic studies have identified H3K27 alterations common in DMG and similar midline gliomas in the thalamus, spine, and brainstem. These alterations can include mutations in H3.3, H3.1, and H3.2, as well as EZHIP gene overexpression and EGFR mutants. One type of diffuse high-grade glioma is characterized by altered H3 K27. Other types of gliomas include H3 wild-type, IDH (isocitrate dehydrogenase) wild-type, and infantile gliomas.

[0110] Because of the tumor's anatomical location in the pons, adjacent to the brain nuclei involved in vital autonomic functions, conventional surgical resection may not be feasible.

[0111] Standard care management for DMG includes fractionated radiation therapy over six weeks, with a total dose of 54 Gy. Cytotoxic chemotherapy drugs include temozolomide, gemcitabine, capecitabine, targeted therapies such as tyrosine kinase inhibitors, and stem cell transplantation. However, overall, none have shown a significant improvement in overall survival. Other therapies include adoptive immunotherapy toxicity, vaccines, oncolytic virus therapy, and immune checkpoint inhibitors.

[0112] Embodiments of the present invention include therapeutic guidance using molecules derived from the brainstem.

[0113] In some implementations, TGF-β2 suppression can be used in cancer therapy to reduce immunosuppression and immune escape, as well as tumor progression. Suppressing TGF-β2 can relieve immunosuppression in tumors. Activating certain elements of the tumor microenvironment with interferon-γ (IFN-γ) can promote the synthesis of pro-inflammatory cytokines, enhance phagocytosis, and increase tumor antigen presentation, thereby improving anti-tumor responses.

[0114] In some implementations, TGF-β2 suppression can be combined with IFN-γ administration for tumor therapy and may provide enhanced outcomes for cancer patients.

[0115] In a further implementation, TGF-β2 may be effective for overall survival and progression-free survival in brain cancer. Cancer patients eligible for treatment may exhibit a combination of high levels of tumor TGF-β2 mRNA and elevated levels of mRNA encoding the IFN-γ receptor. Alternatively, cancer patients eligible for treatment may exhibit a combination of high levels of tumor TGF-β2 mRNA and reduced levels of mRNA encoding IFN-γ signaling molecules.

[0116] In other implementations, therapies can be used to improve survival in patients with low levels of downstream interferon signaling markers and high levels of TGF-β2 mRNA. Therapies targeting elevated TGF-β2 levels can also provide enhanced antitumor response and overall survival.

[0117] In further embodiments, biomarkers that may be used include tumor mutation burden (TMB) levels, tumor neoantigen levels, tumor-associated immune cell levels, or combinations thereof. In some embodiments, biomarkers that may be used are levels of the following cells: basophils, B cells, T cells, T helper cells (Th), eosinophils, macrophages, mesenchymal stem cells, or combinations thereof. In some embodiments, biomarkers that may be used include CD4+ cells, memory T cells, CD8+ cells, natural killer T cells, regulatory T cells, type 1 T helper cells (Th1), type 2 T helper cells (Th2), or combinations thereof.

[0118] In some implementations, one or more components of the tumor microenvironment can be identified by means known in the art, including analysis of fresh, frozen and FFPE tissue samples using a variety of techniques, microarray screening, immune cell and subtype analysis using a variety of techniques, as well as genomic stability, TMB, expression profiling analysis and various next-generation sequencing (NGS) technologies.

[0119] Surfactant

[0120] Antisense oligonucleotides can be supplied in varying quantities in glass vials as lyophilized powder. The lyophilized antisense oligonucleotide powder can be dissolved in an isotonic (0.9%) sodium chloride solution before use.

[0121] In some embodiments and implementations, the antisense oligonucleotide agent may be administered or used by infusion at a dose level of 10 µM, or at a dose of 20 µM, or at a dose of 40 µM, or at a dose of 80 µM on days 1 through 7 at a dose of 4 µl / min.

[0122] In some embodiments and implementations, the antisense oligonucleotide agent can be administered or used by infusion at a dose level of 2 µM on days 1 to 7, or at a dose level of 4 µM on days 1 to 7, or at a dose level of 8 µM on days 1 to 7, or at a dose level of 10 µM on days 1 to 7 at a dose level of 4 µl / min or 2-8 µl / min.

[0123] In some implementations, the antisense oligonucleotide agent can be administered by injection at concentrations of 61.43 mg / ml (10 µM), 1 mg / ml, 7.35 mg / ml, 15 mg / ml, or 18.23 mg / ml.

[0124] In a further implementation, the antisense oligonucleotide agent may be administered or used by infusion alone, in combination with a formulation-compatible drug, or in combination with standard care therapy.

[0125] The pharmaceutical agents disclosed herein may be pharmaceutically acceptable salts, polymorphs of salts, esters or isomers, and one or more pharmaceutically acceptable excipients. Excipients may include any one or more pharmaceutically acceptable excipients selected from diluents, stabilizers, disintegrants, and anti-caking agents. In some embodiments, excipients may include any one or more of microcrystalline cellulose, polysorbate 80, crospovidone, croscarmellose sodium, and magnesium stearate.

[0126] The pharmaceutical composition may contain an active agent and a pharmaceutically acceptable carrier. The carrier may be sterile water for injection, saline, isotonic saline, or a combination thereof.

[0127] Importantly, the compositions disclosed herein are substantially excipient-free. The substantially excipient-free compositions of the present invention can exhibit surprising stability in a carrier. In some embodiments, the compositions are stable in a carrier at 37°C for at least 14 days, at least 21 days, or at least 28 days.

[0128] In other embodiments, the pharmaceutical composition for infusion may contain less than 1% by weight, or less than 0.5% by weight, or less than 0.1% by weight of excipients.

[0129] Embodiments of the present invention further cover treatment methods in which the compositions of the present invention are administered or used in combination with standard care therapies for the disease.

[0130] In a further implementation, the therapeutically effective dose of the antisense agent may 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.

[0131] In some embodiments, the formulation of the antisense agent 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.

[0132] In some embodiments, the method of using antisense agents may employ effective doses of 1 to 1000 mg / m² / day, or 1 to 500 mg / m² / day, or 1 to 250 mg / m² / day, or 1 to 100 mg / m² / day, or 1 to 50 mg / m² / day. The average human body surface area may be approximately 1.6 to 1.9 m².

[0133] In other embodiments, the method of using the antisense agent may employ an effective dose of 0.05 to 40 mg / kg / day, or 0.1 to 30 mg / kg / day, or 0.2 to 20 mg / m² / day, or 0.3 to 10 mg / m² / day, or 0.5 to 5 mg / m² / day. The average human body weight may be approximately 60 kg.

[0134] In some embodiments, the pharmaceutical agent disclosed herein may be prepared from a lyophilized powder of the pharmaceutical agent.

[0135] In some embodiments and implementations, the agent may be administered or used by injection or infusion at a dose level of 10 µM, or at a dose of 20 µM, or at a dose of 40 µM, or at a dose of 80 µM at a dose of 4 µl / min on days 1 to 7.

[0136] In some embodiments, the antisense oligonucleotide can be supplied in 250 mg / vial in 20R glass vials as a sterile lyophilized powder for the solution prior to administration. The lyophilized product can be aseptically reconstituted in a sterile, preservative-free isotonic NaCl solution. The antisense oligonucleotide solution can be administered as a continuous intravenous infusion using a portable pump system on days 4–7 of a 4-day dosing, 10-day stop-dose schedule every 14 days. Schedules can be 7-day dosing / 7-day stop-dose or 4-day dosing / 10-day stop-dose schedules. Doses can be 40, 80, 160, 140, 190, 250, or 330 mg.

[0137] In some embodiments, the agent may be administered or used by injection or infusion at doses of 40, 80, 160, 140, 190, 250, 330 mg / m² on days 1 to 7, or at doses of 40, 80, 160, 140, 190, 250, 330 mg / m² on days 1 to 4.

[0138] In some embodiments and implementations, the agent may be administered or used by injection or infusion at a dose level of 2 µM, or at a dose of 4 µM, or at a dose of 8 µM, or at a dose of 10 µM on days 1 through 7, at a dose of 4 µl / min or 2-8 µl / min.

[0139] As used herein, the term chemical modification may refer to LNA variants and gapmer variants. The antisense agents of this disclosure may be used in combination or aggregated in formulations.

[0140] Methods and compositions for cancer

[0141] The pharmaceutical agents of the present invention can be used to treat cancer in human subjects or animals in need or to improve cancer symptoms in human subjects or animals in need. The pharmaceutical agents can be prepared into pharmaceutical compositions for injection or infusion.

[0142] The pharmaceutical composition for injection or infusion can be administered to the subject in a therapeutically adequate amount.

[0143] Methods and compositions for administering interferon-γ are known in the art. Human IFN-γ can be produced recombinantly and can be vector-free. Interferon-γ is a dimerizing soluble cytokine and the sole member of the type II interferon class. IFN-γ has been used for a wide range of clinical indications. Interferon-γ is a regulator of the immune response and is signaled via the Janus-activated kinase (JAK)-signal transduction and activating transcription factor (STAT) pathway. IFN-γ is the primary interferon produced by lymphocytes stimulated by mitogens or antigens. See Entrez Gene ID 3458 (human).

[0144] The pharmaceutical preparations or active substances disclosed herein are soluble or suspended in physiological solvents or any other suitable solvents. The preparations may be in the form of a free base, or salt, hydrate, ester, amide, enantiomer, isomer, tautomer, polymorph, prodrug, or derivative of these compounds. The above-described preparations and combinations thereof can be used in the apparatus, methods, kits, combinations, and compositions described herein.

[0145] The compositions of the present invention may contain a compound formulated as an injectable preparation for infusion into a subject, such as an aqueous solution or suspension of a compound suitable for intravenous delivery. When preparing compositions for injection, particularly for intravenous delivery, exemplary, the continuous phase comprises an aqueous solution of an isotonic adjuster buffered to, for example, a pH below 7.4, or, for example, a pH below 7 or below 6.6. Isotonic adjusters include, for example, sodium chloride, glucose, mannitol, trehalose, glycerol, or other pharmaceutical agents that make the osmotic pressure of the preparation isotonic with that of blood.

[0146] The system of the present invention may contain preservatives added to the formulation. Preservatives include benzalkonium chloride, propylparaben, butylparaben, chlorobutanol, benzyl alcohol, phenol, sodium benzoate, or EDTA.

[0147] The compositions disclosed herein may contain pharmaceutically acceptable carriers. The carrier material that can be used to prepare the compositions of the present invention is any of those excipients commonly used in pharmaceutical science, and should be selected based on compatibility with the pharmaceutical formulation and the release profile properties of the desired dosage form.

[0148] The compositions of the present invention may contain excipients, such as those described in the following literature: Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa. 1975; and Liberman, HA and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, NY, 1980.

[0149] The numbered embodiments of the present invention include the following: (1) An agent for suppressing TGF-β2 expression, in combination with interferon-γ, for treating cancer in a subject in need or improving cancer symptoms in a subject in need, wherein the subject is selected as a subject having (a) decreased JAK1 and STAT1 or both and (b) increased TGF-β2 and IFNGR2.

[0150] (2) A method for treating cancer in a subject in need or improving cancer symptoms in a subject in need, said method comprising: Subjects were selected who had (a) decreased JAK1 and STAT1 or both and (b) increased TGF-β2 and IFNGR2; Application of a composition containing an agent for suppressing TGF-β2 expression; and Administer a composition containing interferon-γ.

[0151] (3) A composition comprising an agent for suppressing TGF-β2 expression and a pharmaceutically acceptable carrier, wherein the composition is used in combination with interferon-γ to prepare a medicament for treating a subject’s cancer or improving the subject’s cancer symptoms, wherein the subject is selected as a subject having (a) decreased JAK1 and STAT1 or both and (b) increased TGF-β2 and IFNGR2.

[0152] (4) The agent, method or composition according to any one of embodiments 1-3, wherein the cancer is brain cancer or spinal cord cancer, glioma, glioblastoma, diffuse endophytic pontine glioma (DIPG), diffuse midline glioma (DMG), diffuse hemispheric glioma, or leptomeningeal or brain metastasis, or wherein the cells of the cancer exhibit somatic mutations containing H2-K27M, H3-K27M or H3-G34 genomic variants.

[0153] (5) The agent, method or composition according to any one of embodiments 1-4, wherein the agent for suppressing TGF-β2 expression and the interferon-γ are administered in parallel, simultaneously, sequentially or separately in time.

[0154] (6) The pharmaceutical agent, method or composition according to any one of embodiments 1-5, wherein the composition and pharmaceutical agent are administered by infusion or injection.

[0155] (7) The pharmaceutical agent, method or composition according to any one of embodiments 1-6, wherein one or both of JAK1 and STAT1 have reduced mRNA levels below the median of control subjects with the same cancer, and elevated TGF-β2 and IFNGR2 above the median of control subjects with the same cancer.

[0156] (8) The agent, method or composition according to any one of embodiments 1-7, wherein the agent for suppressing TGF-β2 expression is selected from Table 1 or Table 2, and its chemically modified variants, its LNA variants, its gapmer variants and any combination or collection thereof.

[0157] (9) The agent, method, or composition according to any one of embodiments 1-8, wherein the agent for inhibiting or suppressing TGF-β2 expression is C G G C A T G T C T A T T T T G T A SEQ ID NO:137 (OT-101) or CGGCATGTCTATTTTGTA SEQ ID NO:1.

[0158] (10) The pharmaceutical preparation, method or composition according to any one of embodiments 1-9, wherein the interferon-γ is human recombinant interferon-γ.

[0159] (11) A pharmaceutical agent, method or composition according to any one of embodiments 1-10, wherein the pharmaceutical agent or composition comprises a carrier, said carrier being sterile water for injection, saline, isotonic saline or a combination thereof.

[0160] (12) The pharmaceutical agent, method or composition according to any one of embodiments 1-11, wherein the pharmaceutical agent or composition is substantially free of excipients.

[0161] (13) The pharmaceutical agent, method or composition according to any one of embodiments 1-12, wherein the composition is stable in a carrier at 37°C for at least 14 days.

[0162] (14) The pharmaceutical preparation, method or composition according to any one of embodiments 1-13, wherein the subject has reduced TGF-β2 expression after administration or use.

[0163] (15) The agent, method or composition according to any one of embodiments 1-14, wherein the administration or use reduces mortality at 6, 12, 18, 24, 30 or 36 months.

[0164] (16) The agent, method or composition according to any one of embodiments 1-15, wherein the administration or use improves survival at 6, 12, 18, 24, 30 or 36 months.

[0165] (17) A pharmaceutical agent, method or composition according to any one of embodiments 1-16, wherein the administration or use of the composition is combined with standard care treatment for cancer, wherein the standard care treatment includes chemotherapy or radiation therapy.

[0166] (18) An agent for suppressing TGF-β2 expression, used to treat cancer in a subject in need or to improve cancer symptoms in a subject in need, wherein the subject is selected to have (a) elevated IFNGR2 and (b) elevated TGF-β2.

[0167] (19) A method for treating cancer in a subject in need or improving cancer symptoms in a subject in need, said method comprising: Subjects with (a) elevated IFNGR2 and (b) elevated TGF-β2 were selected; and Administer a composition containing an agent for suppressing TGF-β2 expression.

[0168] (20) A composition comprising an agent for suppressing TGF-β2 expression and a pharmaceutically acceptable carrier, for use in preparing a medicament for treating a subject’s cancer or improving the subject’s cancer symptoms, wherein the subject is selected as a subject having (a) elevated IFNGR2 and (b) elevated TGF-β2.

[0169] (21) The pharmaceutical agent, method or composition according to any one of embodiments 18-20, wherein the cancer is brain cancer or spinal cord cancer, glioma, glioblastoma, diffuse endophytic pontine glioma (DIPG), diffuse midline glioma (DMG), diffuse hemispheric glioma, or leptomeningeal or brain metastasis, or wherein the cells of the cancer exhibit somatic mutations comprising H2-K27M, H3-K27M or H3-G34 genomic variants.

[0170] (22) A pharmaceutical agent, method or composition according to any one of embodiments 18-21, wherein the composition or pharmaceutical agent is administered by infusion or injection.

[0171] (23) The pharmaceutical preparation, method or composition according to any one of embodiments 18-22, wherein the subject has elevated IFNGR2 with a lower mRNA level than the median of a control subject group with the same cancer; and elevated TGF-β2 with a higher mRNA level than the median of a control subject group with the same cancer.

[0172] (24) The agent, method or composition according to any one of embodiments 18-23, wherein the agent for suppressing TGF-β2 expression is selected from Table 1 or Table 2, and its chemically modified variants, its LNA variants, its gapmer variants and any combination or collection thereof.

[0173] (25) The method, agent, or composition according to any one of embodiments 18-24, wherein the agent for inhibiting or suppressing TGF-β2 expression is C G G C A T G T C T A T T T T G T A SEQ ID NO:137 (OT-101) or CGGCATGTCTATTTTGTA SEQ ID NO:1.

[0174] (26) A pharmaceutical preparation, method or composition according to any one of embodiments 18-25, wherein the composition comprises a carrier, the carrier being sterile water for injection, saline, isotonic saline or a combination thereof.

[0175] (27) A pharmaceutical preparation, method or composition according to any one of embodiments 18-26, wherein the composition is substantially free of excipients.

[0176] (28) The pharmaceutical agent, method or composition according to any one of embodiments 18-27, wherein the composition is stable in a carrier at 37°C for at least 14 days.

[0177] (29) The pharmaceutical preparation, method or composition according to any one of embodiments 18-28, wherein the subject has reduced TGF-β2 expression after administration.

[0178] (30) The agent, method or composition according to any one of embodiments 18-29, wherein the administration reduces mortality at 6, 12, 18, 24, 30 or 36 months.

[0179] (31) The agent, method or composition according to any one of embodiments 18-30, wherein the administration improves survival at 6, 12, 18, 24, 30 or 36 months.

[0180] (32) The agent, method or composition according to any one of embodiments 18-31, wherein the application of the composition is combined with standard care treatment for cancer, wherein the standard care treatment includes chemotherapy or radiation therapy.

[0181] All publications mentioned in this specification, including patent, patent application publications and non-patent publications and serial lists, are expressly incorporated herein by reference in their entirety for all purposes.

[0182] Although the foregoing disclosure has been described in detail by way of example for purposes of clarity, it will be apparent to those skilled in the art that this disclosure includes certain changes and modifications and can be practiced within the scope of the appended claims without excessive experimentation, the claims being presented in an exemplary and not restrictive manner. The invention includes all such additional embodiments, equivalents, and modifications. The invention includes any combination or mixture of various exemplary components, examples, and features, materials, elements, or limitations of the claimed embodiments.

[0183] The nomenclature of the pharmaceuticals, compounds and structures disclosed herein is intended to cover all possible isomers, stereoisomers, diastereomers, enantiomers and / or optical isomers of the particular structure, including any mixtures thereof (racemic or other forms).

[0184] Example

[0185] Example 1. This example shows a surprising increase in IFNGR2 mRNA in brain cancer.

[0186] Figure 1The upregulation of IFNGR2 mRNA expression was demonstrated in pbDMG tumor samples. The mRNA expression levels (log2-converted TPM) of IFNGR2, JAK1, and STAT1 in primary tumor samples from pbDMG patients located in the pons / brainstem were examined, including molecular subtype classifications: DMG, H3K27M (n = 23); DMG, H3K27M and TP53 (n = 8); HGG, H3 wild-type (n = 2); HGG, H3 wild-type and TP53 (n = 1); HGG, unclassified (n = 10); and 1 undetermined. The mRNA expression levels (log2 TPM) of IFNGR2 (n = 45), JAK1 (n = 45), and STAT1 (n = 45) in DMG samples were compared with those in normal pontine samples from 29 pontine regions. Expression in these 29 normal pontine regions was determined by averaging TPM values ​​from 2–8 independent samples / regions from 21 subjects. Bar plots show a comparison of mean mRNA expression levels (dark gray bars) in tumor specimens from DMG patients with mean expression levels (light gray bars) in normal pontine samples. The statistical significance of differences in mRNA expression levels (expressed as log2-transformed TPM values) was assessed using a two-way ANOVA with linear contrast and FDR-adjusted p-values.

[0187] Compared with normal pontine tissue, the IFNGR2 mRNA level in DMG samples was significantly increased (1.58-fold increase; p = 5.5 × 10⁻⁶). -4 ).

[0188] JAK1 decreased, and STAT1 decreased significantly, which is consistent with the dysregulation of the IFN signaling pathway.

[0189] Figure 1 The results showed that IFNGR2 mRNA levels were significantly upregulated in pbDMG patients compared to normal pontine tissue (an increase of 1.58-fold; p = 5.5 × 10⁻⁶). -4 Examination of STAT1 expression revealed significantly lower levels in pbDMG tumors compared to normal brainstem / pons tissue (p = 0.0029). These clinical facts demonstrate the role of these biomarkers in pbDMG tumors and in IFN signaling dysregulation.

[0190] Example 2. This example demonstrates that administration of exogenous IFN-γ to IFN-γ-deprived DMG tumors increases STAT1 and JAK1 levels. The combination of exogenous IFN-γ with TGFB2 suppression achieved using an antisense agent such as OT-101 yielded a surprisingly improved outcome in the treatment of brain cancer.

[0191] In studies of clinical outcomes in pediatric DMG patients, IFN-γ was not expressed in three-quarters of patients, and its maximum expression was less than 1 per million transcripts, with the upper quartile expression at 0.64 TPM. This is a very low expression level. Despite the relative absence of IFN-γ, its receptor IFNGR2 was surprisingly upregulated.

[0192] Cancer patients with high TGF-β2 and surprisingly upregulated IFNGR2 were selected and treated with a combination of exogenous IFN-γ and TGF-β2 suppression, which resulted in a surprising improvement in outcomes.

[0193] Unwilling to be bound by theory, exogenous IFN-γ therapy resulted in increased expression of downstream STAT1 and JAK1. Therefore, administration of exogenous IFN-γ to IFN-γ-deprived DMG tumors achieved improved outcomes.

[0194] Cancer patients with high TGF-β2, surprisingly upregulated IFNGR2, and low JAK1 or low STAT1 were selected and treated with a combination of exogenous IFN-γ and TGF-β2 suppression, which achieved surprisingly improved outcomes.

[0195]

[0196] Univariate Cox (upper quartile of high expression) for IFN-γ: The hazard ratio (HR) for the high IFNG group was determined (HR (95% CI range) = 1.04 (0.53–2.04); P = 0.909).

[0197] Multivariate Cox regression analysis using TGF-B2 (median cutoff) and IFNG (upper quartile cutoff): The hazard ratio (HR) for the high TGF-β2 patient group was determined (HR (95% CI range) = 1.04 (0.46–2.34); P = 0.933).

[0198] The hazard ratio (HR) for the high IFNG group was determined (HR (95% CI range) = 0.94 (0.35–2.52); P = 0.907).

[0199] The HR for age as a covariate was determined (HR (95% CI range) = 1.02 (0.95-1.08); P = 0.624).

[0200] The HR of the interaction item was determined (HR (95% CI range) = 1.2 (0.31-4.67); P = 0.793).

[0201] Example 3. This example demonstrates that the IFNGR2 biomarker led to a surprising improvement in the outcome of brain cancer treatment.

[0202] Figure 2 The Kaplan-Meier overall survival chart is shown in the clinical outcome study of glioma patients. Figure 2 The results showed that when TGF-β2 levels were high (indicating high disease activity), a surprising improvement in overall survival was found with high IFNGR2 levels (log-rank P = 0.012).

[0203] Figure 2 The results showed that pbDMG patients with high TGF-β2 levels and low IFNGR2 mRNA expression levels had significantly shorter overall survival (OS) than those with high IFNGR2 mRNA expression levels. RNA sequencing-based mRNA expression data (cBioPortal) were analyzed in 45 patients diagnosed with pbDMG. Percentiles of TGF-β2 and IFNGR2 expression in the 45 pbDMG patients were calculated using TPM indices determined by RSEM. Four patient groups were then formed based on TGF-β2 and IFNGR2 expression levels: (A) Both are expressed at low levels, with TGFB2 and IFNGR2 being low (below the 50th percentile expression levels of both TGF-β2 and IFNGR2). (B) Combination of high and low expression levels of both: low TGFB2 / high IFNGR2; (C) High TGFB2 / Low IFNGR2, and (D) Both expression levels were high, with TGFB2 being high and IFNGR2 being high, exceeding or equaling the 50th percentile expression levels of both TGF-β2 and IFNGR2.

[0204] The overall survival (OS) curves were then compared between these groups to assess the survival impact of the combined TGF-β2 and IFNGR2 levels. The recorded patient group survival times are as follows: The median survival for patients with low TGFB2 / low IFNGR2 was 10 months (95% CI range 8 to NA, 9 events). The median survival for patients with low TGFB2 / high IFNGR2 was 13 months (95% CI range 10 to NA, 11 events). The median survival for patients with high TGFB2 / low IFNGR2 was 7 months (95% CI range 5 to NA, 13 events); and The median survival for patients with high TGFB2 / high IFNGR2 was 15 months (95% CI range 7 to NA, 10 events).

[0205] The examination of pairwise log-rank differences in OS time showed that there was a significant difference between the low TGFB2 / high IFNGR2 patient group and the high TGFB2 / low IFNGR2 patient group (p = 0.009) and between the high TGFB2 / low IFNGR2 patient group and the high TGFB2 / high IFNGR2 patient group (p = 0.012).

[0206] In pbDMG patients with increased IFNGR2 expression, inhibition of TGF-β2 expression was associated with improved overall survival (OS). Selecting DMG cancer patients with increased IFNGR2 expression and inhibiting TGF-β2 expression resulted in a surprisingly improved OS outcome.

[0207] The study investigated the effects of TGF-β2 and IFNGR2 expression levels on overall survival in four groups of pbDMG patients. Low mRNA expression levels were compared for both TGF-β2 and IFNGR2 (low TGFB2 / low IFNGR2; below the 50th percentile expression levels of both TGF-β2 and IFNGR2), combinations of high and low TGF-β2 and IFNGR2 expression levels (low TGFB2 / high IFNGR2 and high TGFB2 / low IFNGR2), and high expression of both TGF-β2 and IFNGR2 (high TGFB2 / high IFNGR2; above or equal to the 50th percentile expression levels of both TGF-β2 and IFNGR2).

[0208] Pairwise examination of median overall survival (OS) time revealed significant differences between the low TGFB2 / high IFNGR2 group (median = 13 months (95% CI = 10–NA months)) and the high TGFB2 / low IFNGR2 group (median = 7 months (95% CI = 5–NA months)) (p = 0.009), and between the high TGFB2 / low IFNGR2 group and the high TGFB2 / high IFNGR2 group (median = 15 months (95% CI = 7–NA months)) (p = 0.012). These results suggest that high levels of IFNGR2, at high TGF-β2 mRNA expression levels, confer a significant survival benefit.

[0209] However, pbDMG patients with low levels of IFNGR2 and high levels of TGFB1 or TGFB3 did not exhibit worse OS time, suggesting that the prognostic impact of TGF-β2 in the context of low IFNGR2 expression is surprising and specific to TGFB2.

[0210] Example 4. This example demonstrates that the IFNGR2 biomarker led to a surprising improvement in the outcome of brain cancer treatment.

[0211] Figure 3 The Kaplan-Meier overall survival chart is shown in the clinical outcome study of glioma patients. Figure 3 The results showed that relatively high IFNGR2 levels improved overall survival (top curve, log-rank p < 0.001). Furthermore, suppressing high TGF-β2 levels (indicating high disease activity) resulted in a surprisingly improved OS (top curve). Selecting patients with relatively high IFNGR2 levels and suppressing high TGF-β2 levels resulted in a surprisingly improved overall survival, with the median time nearly doubling. Because these effects were largely independent, the improved survival outcome demonstrates an unexpected synergistic effect of achieving an outcome that could not be achieved by either feature alone.

[0212] Figure 3 The study showed that among the 45 patients, 13 patients had high levels of TGF-β2 and low levels of IFNGR2 (lower curve). Their median overall survival was 7 months, which contrasted significantly with the remaining 32 patients (median overall survival of 13 months (95% CI = 10–15 months)). The survival outcome between the two groups showed a significant difference, with a log-rank chi-square value of 13.5 and a p-value of 2.3 × 10⁻⁶. -4 This indicates that it has a significant prognostic impact on patients with pbDMG.

[0213] Clinical metadata and RNA sequencing-based mRNA expression data (cBioPortal) of 45 patients diagnosed with pbDMG were analyzed. Percentiles of TGF-β2 and IFNGR2 expression in the 45 pbDMG patients were calculated using TPM indices determined by RSEM. Two patient groups were then formed based on TGF-β2 and IFNGR2 expression levels: High expression of TGF-β2 mRNA and low expression of IFNGR2 (high TGFB2 / low IFNGR2; expression level above or equal to the 50th percentile of TGF-β2 and below the 50th percentile of IFNGR2 (n = 13)); and "The remaining patients" (n = 32; patients from the combined groups of low TGFB2 / high IFNGR2, low TGFB2 / low IFNGR2 and high TGFB2 / high IFNGR2).

[0214] Overall survival (OS) curves were then compared between these groups to assess the survival impact of the combination of TGF-β2 and IFNGR2 levels. In the subset of patients with high TGF-β2 / low IFNGR2, based on glioma grade and mutation status, 8 were classified as DMG / H3K27M, 1 as DMG / H3K27M / TP53, 1 as HGG / H3 wild-type / IDH wild-type / TP53 mutation, 2 as HGG / unclassified, and 1 as NA. In the remaining subset of patients, based on glioma grade and mutation status, 15 were classified as DMG / H3K27M, 7 as DMG / H3K27M / TP53, 2 as HGG / H3 wild-type / IDH wild-type, and 8 as HGG / unclassified.

[0215] In the subset of patients with high TGFB2 / low IFNGR2, the mean (±SEM) and median (range) log2-TPM mRNA expression values ​​of IFNGR2 were 4.7 ± 0.1 and 4.9 (3.7–5.4), respectively. For the “remaining patients” subset, the corresponding values ​​were 5.6 ± 0.1 and 5.7 (3.2–6.9), respectively.

[0216] In the subset of patients with high TGFB2 / low IFNGR2, the mean (±SEM) and median (range) log2-TPM mRNA expression values ​​of TGF-β2 were 5.3 ± 0.3 and 5.2 (4.1–7), respectively. For the “remaining patients” subset, the corresponding values ​​were 3.5 ± 0.3 and 3.3 (0.6–5.7), respectively.

[0217] In the group of 13 patients with high TGFB2 / low IFNGR2, the median overall survival was 7 months (95% CI = 5–NA months; 13 events). In contrast, the median overall survival in the remaining group of 32 patients was 13 months (95% CI: 10–15, 30 events). Therefore, a statistically significant difference in survival was observed between the two groups, with a log-rank chi-square value of 13.5 and a p-value of 2.3 × 10⁻⁶. -4 .

[0218] Example 5. Figure 4 The Cox proportional hazards measure obtained in a clinical outcome study of glioma patients is shown. Figure 4The results show that when an interaction term is allowed between TGF-B2 and IFNGR2 levels for clinical outcomes in gliomas, TGF-B2 is essentially independent of IFNGR2. Because they are independent, the improved survival outcomes described above demonstrate an unexpected synergistic effect in achieving outcomes that would not be achievable by using either feature alone.

[0219] Figure 4 The results showed that in a multivariate Cox proportional hazards model that considered age and the interaction between TGF-B2 and IFNGR2, pbDMG patients with high TGF-B2 mRNA expression levels exhibited a significantly increased hazard ratio. Multivariate analysis was used to determine the potential impact of TGF-B2 and IFNGR2 levels on overall survival (OS) by adjusting for age using a multivariate Cox proportional hazards model and comparing models with and without the TGF-B2 and IFNGR2 interaction. Clinical metadata and mRNA expression data (cBioPortal) of 45 patients diagnosed with pbDMG were analyzed.

[0220] Both models included (i) TGF-β mRNA expression level as a categorical variable comparing high and low TGF-β mRNA expression levels (50% cutoff of the TPM range); (ii) IFNGR2 mRNA expression level as a categorical variable comparing high and low IFNGR2 mRNA expression levels (50% cutoff of the TPM range); and (iii) age as a linear covariate. Forest plots were used to visualize the hazard ratios of the Cox proportional hazards model for OS outcomes.

[0221] The effects of incorporating the interaction term as the fourth parameter (TGF-B2 x IFNGR2) in the Cox proportional hazards model were analyzed to compare the independent effects of TGF-B2 and IFNGR2 in models with and without the interaction term.

[0222] The hazard ratio (HR) results for the non-interaction term model showed no significant increase in HR in the high TGFB2 group (HR (95% CI range) = 1.29 (0.66–2.49); p = 0.457). However, the HR was significantly decreased in the high IFNGR2 group (HR (95% CI range) = 0.38 (0.19–0.75); p = 0.006). Furthermore, the HR for age as a linear covariate was not significantly increased (HR (95% CI range) = 1.03 (0.97–1.1); p = 0.353).

[0223] Modeling the interaction between IFNGR2 and TGF-β2 revealed a significantly increased hazard ratio in the high TGFB2 group (HR (95% CI range) = 2.88 (1.12–7.39); p = 0.028). However, the HR was not significantly reduced in the high IFNGR2 group (HR (95% CI range) = 0.8 (0.32–1.99); p = 0.635), nor was the HR for age as a linear covariate significantly reduced (HR (95% CI range) = 1.06 (0.98–1.14); p = 0.157). The results also showed a significant effect of the interaction term (HR (95% CI range) = 0.17 (0.04–0.72); p = 0.015).

[0224] Overall, the decrease in HR observed in the high IFNGR2 group was not associated with TGF-β2 levels, and the inclusion of an interaction term in the model showed an increase in HR in the high TGFB2 group, independent of IFNGR2 levels and the interaction between TGF-β2 and IFNGR2. The effect of IFNGR2 was captured in the interaction term.

[0225] exist Figure 4 middle, This indicates that p < 0.05. This indicates that p < 0.01.

[0226] When controlling for age and IFNGR2 levels, amplified TGF-β2 levels were an independent negative prognostic indicator for OS.

[0227] Using a Cox proportional hazards model, we investigated the effects of TGF-β2 and IFNGR2 levels in a multivariate context, including age as a linear control. This model, without interaction terms, showed no significant increase in the risk of death (HR) in the high TGF-β2 group (HR (95% CI range) = 1.29 (0.66–2.49); p = 0.457). However, the HR was significantly decreased in the high IFNGR2 group (HR (95% CI range) = 0.38 (0.19–0.75); p = 0.006), indicating an overall pro-survival benefit from high IFNGR2 levels.

[0228] Including the interaction term in the Cox proportional hazards model yielded a more complex effect, revealing a significantly increased hazard ratio in the high TGFB2 patient group (HR (95% CI range) = 2.88 (1.12–7.39); p = 0.028), independent of IFNGR2 level and age. Furthermore, the significant pro-survival effect of high IFNGR2 levels observed in the model without the interaction term was now captured in the significant effect of the IFNGR2 x TGF-β2 interaction term (HR (95% CI range) = 0.17 (0.04–0.72); p = 0.015). These parameters were analyzed in a Cox proportional hazards regression model that incorporated the interaction term of a combination of high and low TGF-β2 mRNA expression groups as parameters, in the context of low and high IFNGR2 mRNA expression in patient groups. The median overall survival (OS) in the low TGFB2 group was 11 months, which was higher than the upper 95% confidence level (upper 95% confidence interval = 10 months) in the high TGFB2 group.

[0229] Example 6. This example demonstrates that suppressing TGF-β2 expression achieved a surprising improvement in the outcome of brain cancer treatment. Figure 5 The Kaplan-Meier overall survival chart is shown in the clinical outcome study of glioma patients.

[0230] Figure 5 The study showed that pbDMG patients with suppressed low levels of TGF-B2 mRNA expression exhibited significantly longer overall survival (OS). A multivariate Cox proportional hazards model considering age and the interaction between TGF-B2 and IFNGR2 levels was used. Survival proportions were calculated based on parameters in a Cox proportional hazards regression model that incorporated the interaction term between high and low TGF-B2 mRNA expression groups in the context of the low IFNGR2 mRNA expression group. Clinical metadata and mRNA expression data from 45 patients diagnosed with pbDMG were analyzed (cBioPortal).

[0231] Survival curves were plotted for 45 pbDMG patients by comparing the median OS time between the high TGFB2 group and the low TGFB2 group in patients expressing low levels of IFNGR2.

[0232] In the context of low IFNGR2 expression, low levels of TGF-β2 resulted in a more favorable overall survival (OS) time, with a median OS of 11 months in the low TGFB2 group, which is greater than the upper limit of the 95% confidence interval (upper limit of 95% confidence interval = 10 months) in the high TGFB2 group.

[0233] Example 7. This example demonstrates that by suppressing TGFB2 expression, the JAK1 biomarker led to a surprising improvement in the outcome of brain cancer treatment. Figure 6 The Kaplan-Meier overall survival chart is shown in the clinical outcome study of glioma patients. Figure 6 The results showed that increased JAK1 levels surprisingly improved overall survival (top curve, log-rank p < 0.001), as observed in the “remaining patients” group.

[0234] Furthermore, suppressing high TGF-β2 levels (indicating high disease activity) resulted in a surprisingly improved overall survival (OS) (top curve). Selecting patients with relatively high JAK1 levels and suppressing high TGF-β2 levels yielded a surprisingly improved overall survival, with the median time nearly doubling. Because these effects are largely independent, the improved survival outcome suggests an unexpected synergistic effect of achieving an outcome that would not be achievable by either feature alone.

[0235] Figure 6 The results showed that, for the "remaining patients," pbDMG patients with relatively low TGF-β2 levels and relatively high JAK1 mRNA expression levels exhibited significantly longer overall survival (OS). Clinical metadata and RNA sequencing-based mRNA expression data (cBioPortal) of 45 patients diagnosed with pbDMG were analyzed. Percentiles of TGF-β2 and JAK1 expression in the 45 pbDMG patients were calculated using TPM indices determined by RSEM. Patients were then divided into two groups based on their TGF-β2 and JAK1 expression levels: High expression of TGF-β2 mRNA and low expression of JAK1 (TGFB2 high / JAK1 low; expression level above or equal to the 50th percentile of TGF-β2 and below the 50th percentile of JAK1 (n = 11)); and The remaining patients (n=34; patients from the combined groups of low TGFB2 / high JAK1, low TGFB2 / low JAK1, and high TGFB2 / high JAK1).

[0236] In the high TGFB2 / low JAK1 subset and the “remaining” subset, 8 and 15 patients, respectively, were classified as DMG / H3K27M; 1 and 7 patients, respectively, were classified as DMG / H3K27M / TP53; and 2 and 8 patients, respectively, were classified as HGG / unclassified. Furthermore, in the remaining group of patients, based on glioma grade and mutation status, 2 patients were classified as having the HGG / H3 wild-type / IDH wild-type gene, 1 patient was classified as having the HGG / H3 wild-type / IDH wild-type / TP53 mutation, and 1 patient was classified as NA.

[0237] In the subset of patients with high TGFB2 / low JAK1, the mean (±SEM) and median (range) log2-TPM mRNA expression values ​​of JAK1 were 5.3 ± 0.1 and 5.4 (4.7–5.5), respectively. For the “remaining” subset, the mean (±SEM) and median (range) were 5.6 ± 0.1 and 5.7 (3.8–6.5), respectively.

[0238] In the subset of patients with high TGFB2 / low JAK1, the mean (±SEM) and median (range) log2-TPM mRNA expression values ​​of TGF-β2 were 5.1 ± 0.2 and 5.2 (4.2–6.7), respectively. For the “remaining” subset, the mean (±SEM) and median (range) were 3.6 ± 0.3 and 3.4 (0.6–7), respectively.

[0239] The overall survival (OS) curves were then compared between these groups to assess the survival impact of the combination of TGF-β2 and JAK1 levels. In the high TGF-β2 / low JAK1 group, the median OS was 5 months (95% CI: 4–NA; events = 11) in 11 patients, while in the remaining groups, the median OS was 13 months (95% CI: 9–15; events = 32) in 34 patients. This difference in survival outcomes between the two groups was surprisingly significant (log-rank chi-square = 13.5; p = 2.4 × 10⁻⁶). -4 ).

[0240] The effects of JAK1 and STAT1 (downstream signaling molecules activated by IFNGR2) mRNA expression levels on the combination of TGF-B2 were investigated to assess the biochemical significance of the observed interaction between TGF-B2 and IFNGR2. OS curves were then compared between two patient groups (high TGFB2 / low JAK1 versus the rest) to assess the survival impact of the TGF-B2 and JAK1 level combination.

[0241] Figure 6 The study showed that among the 45 patients, the median overall survival was 5 months (95% CI: 4–NA; events = 11) in the high TGFB2 / low JAK1 group, while the median overall survival was 13 months (95% CI: 9–15; events = 32) in the remaining 34 patients. This survival difference was found to be statistically significant (log-rank chi-square value = 13.5; p = 2.4 × 10⁻⁶). -4Similarly, the median overall survival was 7 months (95% CI: 4–NA; events = 11) in the 11 patients in the high TGFB2 / low STAT1 group, while the median overall survival was 13 months (95% CI: 8–15; events = 32) in the remaining 34 patients.

[0242] Example 8. This example demonstrates that the STAT1 biomarker led to a surprising improvement in the outcome of brain cancer treatment. Figure 7 The Kaplan-Meier overall survival chart is shown in the clinical outcome study of glioma patients. Figure 7 The results showed that for relatively high levels of STAT1, overall survival was improved (top curve, log-rank p < 0.001), as was observed primarily in the “remaining patients” group.

[0243] Figure 7 The results showed that, in the "remaining patients" group, pbDMG patients with relatively low TGF-B2 levels and relatively high STAT1 mRNA expression levels exhibited significantly longer overall survival (OS), with the median time almost doubling. Clinical metadata and RNA sequencing-based mRNA expression data (cBioPortal) of 45 patients diagnosed with pbDMG were analyzed. Percentiles of TGF-B2 and STAT1 expression in the 45 pbDMG patients were calculated using TPM indices determined by RSEM. Two patient groups were then formed based on TGF-B2 and STAT1 expression levels: High expression of TGF-β2 mRNA and low expression of STAT1 (TGFB2 high / STAT1 low; expression level above or equal to the 50th percentile of TGF-β2 and below the 50th percentile of STAT1 (n = 11)); and The remaining patients (n = 34; patients from the combined groups of low TGFB2 / high STAT1, low TGFB2 / low STAT1, and high TGFB2 / high STAT1).

[0244] In the subset of patients with high TGFB2 / low STAT1, 6 were classified as DMG with H3K27M mutation, 1 as DMG with both H3K27M and TP53 mutations, 1 as HGG carrying both H3 wild-type and IDH wild-type genes, 2 as HGG without mutations, and 1 as NA. In the remaining subset of patients, 17 were classified as DMG with H3K27M mutation, 7 as DMG with both H3K27M and TP53 mutations, 2 as HGG carrying both H3 wild-type and IDH wild-type genes, and 8 as HGG without mutations.

[0245] In the subset of patients with high TGFB2 / low STAT1, the mean and median log2-TPM mRNA expression values ​​of STAT1 were 4.3 ± 0.2 and 4.4 (3.4–5.1), respectively. For the “remaining” subset of patients, these values ​​were 5.4 ± 0.2 and 5.4 (3.1–8), respectively.

[0246] For the patient subset with high TGFB2 / low STAT1, the mean and median log2-TPM mRNA expression values ​​were 5 ± 0.2 and 5 (4.1–6.7), respectively. For the “remaining” patient subset, these values ​​were 3.7 ± 0.3 and 3.4 (0.6–7), respectively.

[0247] Then, the overall survival (OS) curves were compared among these groups to assess the survival impact of the combination of TGF-β2 and STAT1 levels.

[0248] In the high TGFB2 / low STAT1 group, the median overall survival (OS) was 7 months in 11 patients (95% CI: 4–NA; number of events = 11), while in the other groups, the median OS was 13 months in 34 patients (95% CI: 8–15; number of events = 32). The difference in survival outcomes between these groups was surprisingly significant (log-rank chi-square value = 10.3; p = 0.0014).

[0249] Example 9. The measurements of this disclosure include comparing mRNA expression levels in brain cancer tissue with those in normal brain tissue from the same anatomical location.

[0250] Transforming growth factor receptor ligands TGFB1, TGFB2, and TGFB3; IFN-γ receptor and downstream signaling molecules IFNGR2, JAK1, and STAT1; and mRNA transcript expression levels from brain tissue RNA-seq experiments were obtained from Human Protein Atlas version 23.0. Human tissues were anatomically dissected and analyzed using transcriptomics and normal tissue mRNA samples extracted from frozen tissue sections. After sequencing, alignment, and quantification of extracted nuclear RNA, genes were annotated using the Ensembl database version 109. TPM (transcriptome) expression values ​​were compiled only from the pontine region by filtering through the "tissue group" annotation in the accompanying description file. This data file includes the average gene expression levels of 29 pontine regions filtered using the keyword "pons," which retrieved values ​​for the following regions: "ventral anterior cochlear nucleus," "dorsolateral cochlear nucleus," "dorsolateral tegmental nucleus," "dorsolateral tegmental region," "Kolliker-Fuse nucleus," "lateral lemniscus nucleus," "lateral parabrachial nucleus," "lateral vestibular nucleus," "locus coli nucleus," "medial olivary nucleus," "medial parabrachial nucleus," "medial periolournal nucleus," "motor facial nucleus," "motor trigeminal nucleus," "trapeziosome nucleus," "paramedian reticular nucleus," "pontine nucleus," "pontine raphe nucleus," "ventroposterior cochlear nucleus," "subsensory trigeminal nucleus," "caudate pontine reticular nucleus," "oral pontine reticular nucleus," "reticular tegmental nucleus," "oral spinal trigeminal nucleus," "inferior locus coli," "superior olivary nucleus," "superior vestibular nucleus," "ventral periolournal nucleus," and "ventrolateral tegmental region, A5 NE cell group."

[0251] Gene mRNA expression levels from pediatric cancer genomics (cBioPortal) were obtained using reported RNAseq TPM values ​​and compared with mRNA expression levels in pbDMG tumors. The data array of mRNA expression values ​​for each gene was normalized to “transcripts per million” (TPM) for gene abundance values ​​calculated using RSEM alignment algorithms. Data were compiled by the Open Pediatric Brain Tumor Atlas (OpenPBTA) and Pediatric Brain Tumor Atlas (PBTA, provisional) consortium.

[0252] By applying a two-way ANOVA model, TPM expression values ​​obtained from 29 pontine regions of the brain were filtered using annotations under "Tissue Group" in the description file to compare with TPM expression values ​​from 45 pbDMG patients to identify differentially expressed genes. Log2-transformed TPM values ​​of genes (TGFB1, TGFB2, TGFB3, JAK1, STAT1, and IFNGR2) and tissues (29 normal pontine tissues; 45 brainstem / pontine specimens from pbDMG patients) were included as fixed factors, along with an interaction term to investigate gene-level effects between normal and pbDMG tissues (gene x tissue). For each gene, we compared between normal pontine and pbDMG samples, and then determined significance by adjusting p-values ​​using the false discovery rate algorithm provided in the R package (FDR correction for all pairs in Model 1 and a prevent-at-the-gene design in Model 2). The calculations were performed using the `multcomp_1.4-17` and `emmeans_1.7.0` packages running in R (version 4.1.2), with RStudio as the frontend (RStudio 2021.09.0+351 "Ghost Orchid" version). The bar charts were constructed using the `ggplot2_3.3.5` R package.

[0253] Example 10. The characteristics of pbDMG patients with low-grade gliomas and high-grade gliomas were experimentally compared, and a subset of patients was stratified relative to mRNA expression levels.

[0254] Clinical and RNA sequencing data from 45 patients with pbDMG were analyzed to stratify patients based on mRNA expression levels. Diagnosis of pbDMG was primarily determined using radiological methods, which revealed borderless, diffuse, expansive, high-signal lesions in the pons of all 45 patients, extending into other areas of the brainstem. Of these patients, 44 had designated lesions originating from the pons, and one patient carried the DMG / H3K27M mutation in the brainstem / medulla oblongata region.

[0255] Upon review of clinical data files, 31 out of 45 patients were reported to have diffuse midline gliomas with the H3K27M mutation (DMG / H3K27M; termed "diffuse midline glioma with H3 K27 alteration" according to the WHO 2021 classification, which designates these tumors as grade 4). Thirteen tumors were designated as WHO grade 3 / 4 high-grade gliomas (HGG), and one tumor was undesignated (NA). Two HGG patients were reported to carry both H3 and IDH wild-type genes, and one patient carried both H3 wild-type and IDH wild-type genes, as well as a TP53 mutation.

[0256] mRNA expression values ​​were determined from 22 deceased patients: 1 at diagnosis, 20 from initial CNS tumors, and 2 from patients with disease progression. Patient characteristics of these 45 pbDMG patients were compared with those of 171 pediatric high-grade gliomas and 404 low-grade gliomas obtained from the PBTA database (cBioPortal), and compiled by the Open Pediatric Brain Tumor Atlas (OpenPBTA) and the Pediatric Brain Tumor Atlas (PBTA, provisional) consortium.

[0257] The age distribution (median = 7 years; range = 2–18 years), genomic alteration fraction (median = 0.16; range = 0–0.81)), and mutation count (median = 23; range = 2–499) of these 45 pbDMG patients fell within the distribution range observed in HGG and LGG patients. The impact of gene-level mRNA expression on overall survival (OS) was determined based on patient-level data. Current treatment strategies for pbDMG patients have been established, including standard radiation therapy followed by specialized therapy using FDA-approved targeted drugs. Treatment was guided by gene expression analysis, whole-exome sequencing, and biomarkers. Percentiles of TGFB2, JAK1, STAT1, and IFNGR2 expression in the 45 pbDMG patients were calculated using TPM indices. Then, based on the expression levels of TGF-β2 and IFNGR2, four patient groups were formed: both highly expressed (high TGFB2 / high IFNGR2; above or equal to the 50th percentile expression levels of both TGF-β2 and IFNGR2); both low expressed (low TGFB2 / low IFNGR2; below the 50th percentile expression levels of both TGF-β2 and IFNGR2); and a combination of high and low expression levels of both (high TGFB2 / low IFNGR2 and low TGFB2 / high IFNGR2).

[0258] Example 11. An experimental comparison of overall survival (OS) outcomes in pbDMG patients stratified relative to TGF-β2 and IFNGR2 / JAK1 / STAT1 mRNA expression levels was performed.

[0259] OS curves were compared between groups to assess the survival impact of combinations of four stratified groups with TGF-β2 and IFNGR2 levels. The effects of high TGF-β2 / low IFNGR2, high TGF-β2 / low JAK1, and high TGF-β2 / low STAT1 on OS were determined relative to the remaining patients to analyze the impact of the IFNGR2 / JAK1 / STAT1 axis on these patients' survival. OS outcomes were compared among subsets of patients using the Kaplan-Meier (KM) method, and statistical significance was tested using the log-rank chi-square test and the following R-based software packages: survival_3.2-13, survminer_0.4.9, and survMisc_0.5.5. Graphical representations of treatment outcomes were visualized using the following plotting packages implemented in R: dplyr_1.0.7, ggplot2_3.3.5, and ggthemes_4.2.4. After multiple comparison correction for the four groups (six comparisons) using the Benjamini-Hochberg method, a p-value less than 0.05 was considered significant.

[0260] Example 12. Multivariate analysis was performed on the overall survival (OS) outcomes of pbDMG patients stratified relative to TGF-B2 and IFNGR2 mRNA expression levels and controlling for age and the interaction between TGF-B2 and IFNGR2.

[0261] To determine the impact of TGF-B2 and IFNGR2 levels on overall survival (OS), a multivariate analysis was performed using a Cox proportional hazards model, controlling for age and the interaction between TGF-B2 and IFNGR2. Briefly, the model included (i) TGF-B2 mRNA expression levels as a categorical variable, comparing high vs. low TGF-B2 mRNA expression levels at a 50% cutoff value; (ii) IFNGR2 mRNA expression levels as a categorical variable, comparing high vs. low IFNGR2 mRNA expression levels (50% cutoff value); and (iii) age, implemented in R (survival_3.2-13 running in R version 4.1.2). The hazard ratios of the Cox proportional hazards model for OS outcomes were visualized using a forest plot (survminer_0.4.9 running in R version 4.1.2 (November 1, 2021)). Life table hazard ratios (HRs) were estimated using exponential regression coefficients from Cox proportional hazards analysis performed in R (survival_3.2-13 running in R version 4.1.2). The effects of incorporating the interaction term as a fourth parameter (TGF-B2 x IFNGR2) in the Cox proportional hazards model were investigated to compare the independent effects of TGF-B2 and IFNGR2 in models with and without the interaction term. To visualize the survival rates at any given time for combinations of high and low TGF-B2 mRNA expression groups in the context of high and low IFNGR2 mRNA expression groups from the interaction model, we plotted and computed changes in baseline OS curves for 45 pbDMG patients based on fitted hazard functions. In these comparisons, median OS was compared between patients in the high TGFB2 and low TGFB2 expression groups in those expressing high or low IFNGR2. Significant interaction effects from the interaction model indicated a difference in OS between the high and low TGFB2 groups based on IFNGR2 levels.

[0262] Example 13. Downregulation of antitumor antigen-presenting cell (APC) biomarkers in pbDMG tumors.

[0263] The expression of CD14, CD163, and ITGAX mRNAs was significantly reduced in pbDMG tumors, by 1.64-fold (p = 0.037), 1.75-fold (p = 0.019), and 3.33-fold (p < 0.0001), respectively. CD86 mRNA expression in pbDMG patients showed a non-significant 1.42-fold reduction compared to normal brainstem / pons tissue (p = 0.14). The effects of high levels of TGF-β2 combined with low levels of these antigen-presenting cell markers in pbDMG patients were investigated. Patients with a combination of low levels of CD14 and CD163 expression and high levels of TGF-β2 exhibited worse overall survival (OS), and these markers were also expressed at lower levels in pbDMG tumors compared to normal brainstem / pons tissue. In the high TGFB2 / low CD14 group, the median overall survival (OS) of 14 patients was 7.5 months (95% CI: 5-12; number of events = 14), which was significantly shorter than the median OS of 13 months (95% CI: 8-15; number of events = 29) of the other 31 patients (log-rank p-value = 0.007). In the high TGFB2 / low CD163 group, the median OS of 9 patients was 7 months (95% CI: 5-NA; number of events = 9), while the median OS of the other 36 patients was 11 months (95% CI: 8-15; number of events = 34; log-rank p-value = 0.014).

[0264] In the high TGFB2 / low CD86 group, the median overall survival (OS) of 13 patients was 7 months (95% CI: 5-NA; events = 13), which was significantly shorter than the overall survival of 13 months (95% CI: 8-15; events = 30) of the other 32 patients (log-rank p = 0.001). In the high TGFB2 / low ITGAX group, the median OS of 9 patients was 7 months (95% CI: 5-NA; events = 9), which was shorter than the median overall survival of 11 months (95% CI: 8-14; events = 34) of the other 36 patients, but the difference was not statistically significant (p = 0.885).

[0265] Example 14. Amplified expression of TGF-β2 relative to TGFB1 and TGFB3 mRNA in pbDMG patients and normal pontine tissue.

[0266] The applicant found that, compared with normal pontine tissue, brainstem / pontine tissue from pbDMG patients showed selective upregulation of TGF-β2 and downregulation of TGFB1 and TGFB3, with TGFB1, TGFB2, and TGFB3 mRNA expression being significantly decreased by 2.84-fold, increased by 1.51-fold, and decreased by 4.08-fold, respectively (p < 0.0001, 0.002, and < 0.0001, respectively). The expression profiles of TGFB ligands were significantly different in pbDMG brainstem / pontine and normal pontine tissues; in pbDMG samples, TGF-β2 mRNA expression was significantly higher than TGFB1 mRNA expression (1.54-fold increase; p = 4.1 × 10⁻⁶). -4 The expression of TGFB1 and TGFB3 mRNA was significantly increased (2.25-fold increase; p < 0.0001), indicating a specific upregulation of the TGF-B2 subtype in pbDMG tumor tissue. When comparing TGFB1 and TGF-B2 mRNA in normal pons tissue, TGF-B2 expression was found to be significantly lower, decreasing by 2.78-fold (p < 0.0001). Similarly, when comparing TGF-B2 and TGFB3 mRNA expression, TGF-B2 mRNA levels showed a highly significant 2.74-fold decrease (p < 0.0001).

Claims

1. An agent for suppressing TGF-β2 expression, which, in combination with interferon-γ, is used to treat cancer in a subject in need or to improve cancer symptoms in a subject in need, wherein the subject is selected as a subject having (a) decreased JAK1 and STAT1 or both and (b) increased TGF-β2 and IFNGR2.

2. A method for treating cancer in a subject in need or improving cancer symptoms in a subject in need, the method comprising: Subjects were selected who had (a) decreased JAK1 and STAT1 or both and (b) increased TGF-β2 and IFNGR2; Application of a composition containing an agent for suppressing TGF-β2 expression; and Administer a composition containing interferon-γ.

3. A composition comprising an agent for suppressing TGF-β2 expression and a pharmaceutically acceptable carrier, which, in combination with interferon-γ, is used to prepare a medicament for treating a subject's cancer or improving the subject's cancer symptoms, wherein the subject is selected as a subject having (a) decreased JAK1 and STAT1 or both and (b) increased TGF-β2 and IFNGR2.

4. The pharmaceutical agent, method, or composition according to any one of claims 1-3, wherein the cancer is brain cancer or spinal cord cancer, glioma, glioblastoma, diffuse endophytic pontine glioma (DIPG), diffuse midline glioma (DMG), diffuse hemispheric glioma, or leptomeningeal or brain metastases, or wherein the cells of the cancer exhibit somatic mutations comprising H2-K27M, H3-K27M, or H3-G34 genomic variants.

5. The agent, method, or composition according to any one of claims 1-3, wherein the agent for suppressing TGF-β2 expression and the interferon-γ are administered in parallel, simultaneously, sequentially, or separately in time.

6. The pharmaceutical agent, method, or composition according to any one of claims 1-3, wherein the composition and pharmaceutical agent are administered by infusion or injection.

7. The pharmaceutical agent, method, or composition according to any one of claims 1-3, wherein one or both of JAK1 and STAT1 have reduced mRNA levels below the median in a control group of subjects with the same cancer; and elevated TGF-β2 and IFNGR2 mRNA levels above the median in a control group of subjects with the same cancer.

8. The agent, method, or composition according to any one of claims 1-3, wherein the agent for suppressing TGF-β2 expression is selected from Table 1 or Table 2, and its chemically modified variants, its LNA variants, its gapmer variants, and any combination or collection thereof.

9. The agent, method, or composition according to any one of claims 1-3, wherein the agent for inhibiting or suppressing TGF-β2 expression is C. G G C A T G T C T A T T T T G T A SEQ ID NO:137 (OT-101) or CGGCATGTCTATTTTGTA SEQ ID NO:

1.

10. The pharmaceutical preparation, method, or composition according to any one of claims 1-3, wherein the interferon-γ is recombinant human interferon-γ.

11. The pharmaceutical preparation, method, or composition according to any one of claims 1-3, wherein the pharmaceutical preparation or composition comprises a carrier, said carrier being sterile water for injection, saline, isotonic saline, or a combination thereof.

12. The pharmaceutical agent, method, or composition according to any one of claims 1-3, wherein the pharmaceutical agent or composition is substantially free of excipients.

13. The pharmaceutical preparation, method, or composition according to any one of claims 1-3, wherein the composition is stable in a carrier at 37°C for at least 14 days.

14. The pharmaceutical preparation, method, or composition according to any one of claims 1-3, wherein the subject has reduced TGF-β2 expression after administration or use.

15. The agent, method, or composition according to any one of claims 1-3, wherein the administration or use reduces mortality at 6, 12, 18, 24, 30, or 36 months.

16. The agent, method, or composition according to any one of claims 1-3, wherein the administration or use improves survival rates at 6, 12, 18, 24, 30, or 36 months.

17. The pharmaceutical agent, method, or composition according to any one of claims 1-3, wherein the administration or use of the composition is in combination with standard care treatment for cancer, wherein the standard care treatment includes chemotherapy or radiation therapy.

18. A drug agent for suppressing TGF-β2 expression, used to treat cancer in a subject in need or to improve cancer symptoms in a subject in need, wherein the subject is selected as a subject having (a) elevated IFNGR2 and (b) elevated TGF-β2.

19. A method for treating cancer in a subject in need or improving cancer symptoms in a subject in need, the method comprising: Subjects with (a) elevated IFNGR2 and (b) elevated TGF-β2 were selected; as well as Administer a composition containing an agent for suppressing TGF-β2 expression.

20. A composition comprising an agent for suppressing TGF-β2 expression and a pharmaceutically acceptable carrier, for use in preparing a medicament for treating a subject's cancer or improving the subject's cancer symptoms, wherein the subject is selected as a subject having (a) elevated IFNGR2 and (b) elevated TGF-β2.

21. The pharmaceutical agent, method, or composition according to any one of claims 18-20, wherein the cancer is brain cancer or spinal cord cancer, glioma, glioblastoma, diffuse endophytic pontine glioma (DIPG), diffuse midline glioma (DMG), diffuse hemispheric glioma, or leptomeningeal or brain metastases, or wherein the cells of the cancer exhibit somatic mutations comprising H2-K27M, H3-K27M, or H3-G34 genomic variants.

22. The pharmaceutical agent, method, or composition according to any one of claims 18-20, wherein the composition or pharmaceutical agent is administered by infusion or injection.

23. The pharmaceutical agent, method, or composition according to any one of claims 18-20, wherein the subject has elevated IFNGR2 with a mRNA level lower than the median of a control group of subjects with the same cancer; and elevated TGF-β2 with a mRNA level higher than the median of a control group of subjects with the same cancer.

24. The agent, method, or composition according to any one of claims 18-20, wherein the agent for suppressing TGF-β2 expression is selected from Table 1 or Table 2, and its chemically modified variants, its LNA variants, its gapmer variants, and any combination or collection thereof.

25. The method, agent, or composition according to any one of claims 18-20, wherein the agent for inhibiting or suppressing TGF-β2 expression is C. G G C A T G T C T A T T T T G T A SEQ ID NO:137 (OT-101) or CGGCATGTCTATTTTGTA SEQ ID NO:

1.

26. The pharmaceutical preparation, method, or composition according to any one of claims 18-20, wherein the composition comprises a carrier, said carrier being sterile water for injection, saline, isotonic saline, or a combination thereof.

27. The pharmaceutical preparation, method, or composition according to any one of claims 18-20, wherein the composition is substantially free of excipients.

28. The pharmaceutical preparation, method, or composition according to any one of claims 18-20, wherein the composition is stable in a carrier at 37°C for at least 14 days.

29. The pharmaceutical preparation, method, or composition according to any one of claims 18-20, wherein the subject has reduced TGF-β2 expression after administration.

30. The agent, method, or composition according to any one of claims 18-20, wherein the administration reduces mortality at 6, 12, 18, 24, 30, or 36 months.

31. The agent, method, or composition according to any one of claims 18-20, wherein the administration improves survival rates at 6, 12, 18, 24, 30, or 36 months.

32. The pharmaceutical agent, method, or composition according to any one of claims 18-20, wherein the application of the composition is combined with standard care treatment for cancer, wherein the standard care treatment includes chemotherapy or radiation therapy.

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